GC-MS analysis of phenoxy herbicide residues from surface waters.

An International Journal for the Rapid Publication of Short Reports on all Aspects of Toxicology Especially Mechanisms of Toxicity Volume 144 (2003) Supplement 1 Abstracts of the 41st Congress of the European Societies of Toxicology EUROTOX 2003 Florence, Italy September 28 - October 1, 2003 # 2003 Elsevier Ireland Ltd. All rights reserved. This journal and the individual contributions contained in it are protected under copyright by Elsevier Ireland Ltd., and the following terms and conditions apply to their use: Photocopying Single photocopies of single articles may be made for personal use as allowed by national copyright laws. Permission of the publisher and payment of a fee is required for all other photocopying, including multiple or systematic copying, copying for advertising or promotional purposes, resale, and all forms of document delivery. Special rates are available for educational institutions that wish to make photocopies for non-profit educational classroom use. 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Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. Although all advertising material is expected to conform to ethical (medical) standards, inclusion in this publication does not constitute a guarantee or endorsement of the quality or value of such product or of the claims made of it by its manufacturer. Advertising information Advertising orders and enquiries can be sent to: USA, Canada and South America: Mr Tino DeCarlo, The Advertising Department, Elsevier Science Inc., 360 Park Avenue South, New York, NY 10010-1710, USA; phone: (/1) (212) 633 3815; fax: (/1) (212) 633 3820; e-mail: [email protected]. Japan: The Advertising Department, Elsevier Science K.K., 9-15 Higashi-Azabu 1-chome, Minato-ku, Tokyo 106-0044, Japan; phone: (/81) (3) 5561 5033; fax: (/81) (3) 5561 5047. Europe and ROW: Commercial Sales Department, Elsevier Science Ltd., The Boulevard, Langford Lane, Kidlington, Oxford OX5 1G8, UK; phone (/44) 1865 843016; fax: (/44) 1865 843976; e-mail: [email protected]. k The paper used in this publication meets the requirements of ANSI NISO Z39.48-1992 (Permanence of Paper). ELSEVIER Toxicology Letters 144 (2003) Supplement 1 www.elsevier.com/locate/toxlet ABSTRACT BOOK (Continuing Education Courses, Invited Lectures and Posters) Sunday September 28, 2003 Abstracts Continuing Education Courses 1– 4 5– 9 10–13 14–16 CEC 1 CEC 2 CEC 3 CEC 4 Molecular immunotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Safety testing of cosmetic ingredients and cosmetic formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . Exposure models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . “Omics” in toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s1 s2 s3 s4 Monday September 29, 2003 Abstracts Invited Lectures 17–20 21 22–25 26–29 30–33 34–37 38–41 42–45 46–48 49–54 S1 PL S2 W1 S3 W2 W3 W4 S4 W5 Omics in drug development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gerhard Zbinden Memorial Lecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Developmental immunotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fine particle exposure and adverse health effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem cells in toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alteration of signal transduction in neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Are toxicologists communicating risk effectively? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . In vitro methods in toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Risk assessment in food: general principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Safety evaluation of flavourings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Assessment of exposure to flavouring agents and natural flavour mixtures . . . . . . . . . . . . . . . . . 2. Thresholds in safety evaluation of flavouring agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s7 s8 s8 s9 s11 s12 s13 s14 s15 s16 s16 s17 Tuesday September 30, 2003 Abstracts Invited Lectures 55–58 59–62 63–65 66–71 72 73–79 80–84 S5 S6 S7 S8 KNL W6 S9 Molecular epidemiology in occupational toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Apoptosis and cell regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Risk assessment in food: examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunotoxicology and immunopathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Key Note Plenary Lecture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . State-of-the-Art of the Pesticide European Revision: Council Directive 91/414 . . . . . . . . . . . . . . . Genetic susceptibility towards genotoxic agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s17 s19 s20 s21 s22 s23 s25 Wednesday October 1, 2003 Abstracts Invited Lectures 85–87 88–92 93–95 RT W7 W8 Is toxicology protocol adequate for drug evaluation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuclear receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jet fuel toxicity and exposure issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s26 s27 s28 iv Contents Monday September 29, 2003 Abstracts Poster Sessions Group A 96–114 115–144 145–148 149–180 181–184 185–194 195–196 197–222 223–225 226–250 251–272 273–333 334–349 350–356 357–379 380–392 393–421 P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 P17 Allergy sensitisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immunotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ocular and skin toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alternative methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantitative structure–activity relationships (QSAR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicogenomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stem cells in toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Food safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gastrointestinal toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Natural toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Drug toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiovascular toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kidney toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Liver toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lung toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reproductive and developmental toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s29 s34 s42 s43 s52 s53 s56 s56 s63 s64 s70 s75 s91 s95 s97 s103 s107 Tuesday September 30, 2003 Abstracts Poster Sessions Group B 422–424 425–444 445–449 450–454 455–459 460–475 476–478 479–503 504–541 542–558 559–574 575–586 587–611 612–622 623–627 628–652 653–676 677–694 695–701 702–723 724–728 P18 P19 P20 P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 P31 P32 P33 P34 P35 P36 P37 P38 Author Index Mechanisms of cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signal transduction in toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Behavioural toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heavy metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biotransformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomarkers and exposure assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Occupational toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dioxins and halogenated hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ecotoxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Endocrine disrupters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Environmental pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulatory toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Risk assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Toxicodynamics/toxicokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s114 s115 s120 s121 s123 s124 s128 s129 s135 s145 s150 s154 s157 s164 s167 s168 s175 s182 s186 s188 s194 ............................................................................................ s197 s1 Continuing Education Courses CEC 1 Molecular immunotoxicology 1 SIGNAL TRANSDUCTION PATHWAY AS TARGET OF IMMUNOTOXICITY E. Corsini. Laboratory of Toxicology, Department of Pharmacological Sciences, University of Milan, Milan, Italy Immunotoxicity can result from exposure to a wide variety of unrelated chemicals. For the vast majority, these compounds directly interact with immunocompetent cells, interfering with signal transduction and resulting in alteration in the status and/or functionality of the immune system. There are examples of immunotoxic compounds interfering with all basic signal transduction pathways. Examples relative to the effects of heavy metals, dioxins, drugs, pesticides and cannabinoids will be presented. Experimental evidence suggests that reactive oxygen species (ROS) may also be important mediators of cellular injury, either as a result of macromolecular damage or by interfering with extracellular and intracellular regulatory processes. ROS production is transient and may act as a modifier of key signaling enzymes through reversible oxidation of critical thiols: ROS have been implicated in a variety of responses from transcriptional activation to cell proliferation and apoptosis. Increased or prolonged free radical action can overwhelm ROS defense mechanisms, contributing to disease and toxicity. ROS by activating NF-κB and AP-1, two important transcription factors, influence the expression of many early response genes involved in inflammation, immune activation and carcinogenesis. Among immunotoxic compounds acting through ROS generation particulate matters and organotin compounds will be discussed. The characterization of specific interference with cell signaling induced by immunotoxicants leads to a better understanding of their molecular mechanism of action. The identification of the mechanism of immunotoxic action of environmental contaminants (pesticides, metals, solvents, etc.), will contribute to rendering more reliable species-species extrapolation, and evaluation of its relevance in the risk assessment for human beings. Overall, it is the understanding of the mechanisms by which xenobiotics alter adaptive and natural immune responses that might shed light on the etiology of environmental and occupational immune diseases. 2 TOXICOGENOMICS OF SUBCHRONIC HEXACHLOROBENZENE EXPOSURE OF BROWN NORWAY RATS mesenteric lymph nodes (MLN), liver, blood, kidney and thymus were collected, total RNA was extracted and analyzed using rat RGU-34A expression probe arrays (Affymetrix). Cluster analysis showed clustering of immunological organs, while Principal Component Analysis showed that the effects on the transcript abundance was clearly dose-related. Significant (p<0.001) changes in the spleen and MLN included genes associated with granulocytes, chemokines, cytokines, immunoglobulins, and genes involved in drug metabolism and acute phase responses. In the liver significant changes included cytochrome P450 genes, genes related to estrogen and porphyrin metabolism, but also to immunological parameters. In the blood MHCII and T cell markers were altered. Genes affected in the kidney included cytokines, complement components but also cytochrome P450. As expected the thymus was only weakly affected since it is not a target organ. In conclusion, the toxicogenomics approach revealed several expected changes that are in line with the known (immuno)toxicological effects but also revealed novel ones, which may suggest additional (immuno)toxic effects of HCB exposure and/or provide more insight into the mechanism of HCB immunotoxicity. 3 MOLECULAR BASIS AND IMPLICATIONS OF FUNCTIONAL SUBPOPULATIONS OF T LYMPHOCYTES – THE TYPE 1/TYPE 2 PARADIGM I. Kimber. Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK The quality of adaptive immune response is determined in large part by the differential development of functional subpopulations of T lymphocytes; both CD4+ T helper (Th) cells and CD8+ T cytotoxic (Tc) cells. Many phenotypes have been identified, but in both instances the most polarised functional subpopulations have been designated type 1 (Th1 and Tc1) and type 2 (Th2 and Tc2). The operational significance of functional subsets of T lymphocytes in the context of host resistance is that they provide a mechanism whereby the adaptive immune system is able to tailor specific responses to address effectively varying antigenic challenges. The quality of immune response required to provide effective resistance to a viral pathogen is different from the type of response needed for immunity to, for instance, a multicellular parasite. In addition to protective immunity, functional subpopulations of T lymphocytes are influential in immunopathological processes, including allergy and autoimmunity. Attention here will focus on the relevance of Th and Tc subsets for the development of allergic sensitisation to chemicals and proteins. 4 CYTOKINE POLYMORPHISMS IN SILICOSIS AND OTHER CHRONIC INFLAMMATORY DISEASES J.H. Harleman 1 , F. Staedtler 1 , J. Ezendam 2,3,4 , J. Pennings 3 , R.J. Vandebriel 3 , R. Pieters 2 , J.G. Vos 3,4 . 1 Novartis Pharma AG, Basel, Switzerland, 2 IRAS-Immunotoxicology, Utrecht University, Utrecht, The Netherlands; 3 National Institute for Public Health and the Environment, Bilthoven, The Netherland; 4 Division of Pathology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands B. Yucesoy 1,2 , M.I. Luster 1 . 1 National Institute for Occupational Safety and Health, Health Effects Laboratory Division, Toxicology and Molecular Biology Branch, Morgantown, West Virginia, 26505, 2 Ankara University, Faculty of Pharmacy, Department of Toxicology, 06100, Ankara, Turkey Hexachlorobenzene (HCB) is an environmental pollutant with pronounced (immuno)toxic effects in man and rat. Reported adverse effects are hepatic porphyria and immunological alterations. The Brown Norway (BN) rat is very susceptible to HCB-induced immunotoxicology. Oral exposure to HCB induces enlargement of liver, spleen and lymph nodes, increased serum IgM, IgG and IgE levels, and inflammatory skin and lung lesions. The mechanism of HCB-induced immunopathology is not yet fully understood. Previous work has shown that both T cells and macrophages seem to play an important role. To gain more insight into the molecular mechanisms of HCB-induced toxicity, transcript profiling by means of high-density DNA microarrays was selected as experimental approach in the present study. BN rats were exposed for four weeks to a diet supplemented with 0, 150 or 450 mg/kg HCB. Spleen, Silicosis is a complex multifactorial disease caused by the pulmonary response to inhaled crystalline silica. Increased accumulation and local proliferation of macrophages coinciding with the chronic secretion of inflammatory and fibrotic mediators are important events involved in chronic inflammation and fibrosis. Experimental animal and clinical studies reveal that silica-exposed macrophages produce cytokines and growth factors (e.g. TNF-α, IL-1, TGF-β, PDGF) which play a crucial role in the onset, progression and termination of the inflammation and fibrotic lesions. Genes that encode cytokines are subject to mutations which quantitavely affect their production and modify the severity of inflammatory response. To assess the role of IL-1 and TNF-α polymorphisms in silicosis, we examined their frequencies in ex-miners with moderate and severe silicosis and miners with no lung disease. The odds ratio of disease for carriers of s2 Continuing Education Course CEC 2. Safety testing of cosmetic ingredients and cosmetic formulations TNF-238 was markedly higher for severe silicosis (4.0). Regardless of disease severity, the odds ratio of disease for carriers of the IL-1RA +2018 or TNF-308 variants were elevated. In addition, several significant gene-gene and gene-environment interactions were observed. Recently a number of single nucleotide polymorphisms have been identified in cytokine genes and their frequencies are associated with certain chronic inflammatory diseases including, asthma, Alzheimer’s disease, periodontitis and, contact dermatitis. Association studies between cytokine gene polymorphisms and chronic inflammatory diseases will not only discover gene-gene and geneenvironment interactions that might affect genetic susceptibility but also will improve the pharmacogenetic and diagnostic interventions. variation in this type of study was investigated in 10 European laboratories. The test materials were benzoic acid, caffeine and testosterone, representing a range of different physical-chemical properties. All participating laboratories performed their studies according to a general study protocol in which the experimental design and parameters were defined (see table 1). Each laboratory performed at least three independent experiments for each test chemical. No attempt was made to standardise the diffusion cell equipment and both static and flow through cells were used. Seven laboratories used radiolabeled compounds and performed the analysis of their samples in house (receptor fluid and samples for mass balance determination). The analysis of the samples from the three laboratories using non-radiolabeled compounds was performed centrally (receptor fluid only). Table 1: Standardised experimental conditions CEC 2 Safety testing of cosmetic ingredients and cosmetic formulations 5 SAFETY ASSESSMENT FOR CONSUMER AEROSOL SPRAYS Philip Carthew. Safety and Environmental Assurance Centre, Unilever Colworth Laboratory, Colworth House, Sharnbrook, Bedfordshire, MK44 1LQ, UK The safety assessment of aerosol sprays used by consumers has largely concentrated on the safety of the propellant gases used in products. The resins used in hairspray products have received little peer review, or debate in the published literature, despite their widespread use, both in hairdressing salons, and the home. As many of the resins used in products can be regarded as ‘poorly soluble particles’, their persistence and potential to cause inflammation in the lung needs to be carefully considered. The safety assessment for these resins currently involves determining the type of lung pathology that can be caused in animal inhalation exposure studies, usually of 90 days duration, with or without recovery periods, subsequent to exposure. The nature of the pathologies induced by such sub-chronic exposures are then established, along with the No Observable Effect Level (NOEL) for the effects regarded as adverse. The likely human consumer exposure can be determined by techniques which model the simulated exposure under ‘in use’ conditions. From these values it is then possible to derive the likely safety factors for human exposure. An important part of the safety assessment process is to recognise the intrinsic differences between rodents and man, in terms of the respirable doses that each species experiences, during inhalation exposures. Interspecies scaling factors become necessary, when comparing the exposure doses experienced by rats, compared to man. This is because of basic differences between species in lung clearance rates, and the alveolar area in the lungs exposed to the test material. Differences in the toxicodynamic component of the uncertainty factor used for interspecies extrapolation, in the risk assessment process, is also important in deciding the relative safety factor required for ensuring safe consumer exposure to resins in aerosol products. 6 ROBUSTNESS OF IN VITRO PERCUTANEOUS ABSORPTION STUDIES - AN INTERNATIONAL EVALUATION J.J.M. van de Sandt 1 , W.J.M. Maas 1 , J.A. van Burgsteden 1 , P. Sartorelli 2 , L. Montomoli 2 , F. Larese 3 , J.-P. Payan 4 , J.C. Limasset 4 , P. Carmichael 5 , S. Kenyon 5 , E. Robinson 6 , I. Dick 6 , J.B. Nielsen 7 , K.-H. Schaller 8 , G. Korinth 8 , S. Geh 8 , S. Cage 9 , S. Wilkinson 10 , F.M. Williams 10 . 1 TNO Nutrition and Food Research, Zeist, The Netherlands, 2 Istituto di Medicina del Lavoro, Siena, Italy, 3 Università di Trieste, Trieste, Italy, 4 Institut National de Recherche et de Sécurité, Vandoeuvre Cedex, France, 5 Imperial College School of Medicine, London, UnK, 6 Health & Safety Laboratory, Sheffield, UK, 7 University of Southern Denmark, Odense, Denmark, 8 University of Erlangen-Nurenberg, Erlangen, Germany, 9 Huntingdon Life Science Ltd, Eye, UK, 10 University of Newcastle, Newcastle upon Tyne, UK In order to obtain a better insight into the robustness of in vitro percutaneous absorption methodology, the intra-and interlaboratory Dose test compound Concentration test compound Exposure time Collection of receptor fluid samples Number of replicates Skin membrane Receptor fluid 100 :g/cm2 4.0 mg/ml in ethanol/water (1:1, v/v) 12 h 1, 2, 4, 8, 24 h after application 7 membranes per compound per experiment Frozen human skin (< 1.0 mm) Frozen rat skin (SD rats of ca 4 weeks old) Saline (benzoic acid, caffeine); Saline + 5% BSA, pH 7.4 (testosterone) The ranking of the three compounds was equal for all laboratories: benzoic acid > caffeine > testosterone. Rat skin was more permeable to caffeine, but not to benzoic acid and testosterone. The intralaboratory variation (coefficient of variation of 3–5 experiments) was 15%, 30% and 45% for benzoic acid, caffeine and testosterone, respectively. The variation between laboratories was between 10-fold and 26-fold fold. This variation could to a large extent be attributed to the differences in thickness of the skin membranes used in the different laboratories. Especially the absorption of the lipophilic testosterone was found higher in split-thickness skin (0.3 – 0.4 mm) compared to full-thickness skin (0.7 – 1.0 mm). Each laboratory used different human skin samples, and, despite that experiments were performed with skin from minimally 3 donors, this is also a source of variation to be considered. The project was financially supported by the European Commission (5th framework programme, project QLRT-2000-00196). 7 EU COSMETIC DIRECTIVE AND REQUIREMENTS FOR TOXICOLOGICAL EVALUATION OF COSMETIC INGREDIENTS Vera Rogiers. Department of Toxicology, Vrije Universiteit Brussel, Brussels, Belgium The safety assessment of cosmetics in the EU is based on three important articles, present in Council Directive 76/768/EEC amended by the 6th Amendment, and on the 7th Amendment. Article 2 requires safety for human health and places the full responsibility on the manufacturer, the first importer in the EU or marketer. Article 7 underlines that the safety of cosmetics is based on the safety of the ingredients, namely their chemical structure, toxicological profile and exposure. Article 4 states that safety for human health must be guaranteed without animal tests carried out on EU territory.- The 7th Amendment allows 6 years for obtaining validated alternatives to replace all safety tests with the exception of repeated exposure tests, for which 10 years are provided. In the safety evaluation of cosmetic ingredients two distinct channels are operative: (i) the safety evaluation of colouring agents, preservatives and UV-filters, taken up in Annexes IV, VI and VII of the Directive, respectively, and of ingredients restricted in concentration and application (Annex III) is done by the SCCNFP (Scientific Committee on Cosmetics and Non-Food Products) on the basis of raw material dossiers provided by the industry; (ii) for all other ingredients, the safety evaluation is done by a safety assessor in the context of a cosmetic product dossier. Indeed, the 6th Amendment requires a technical information file for all finished cosmetic products coming on the EU market. It must cover both safety and efficacy. Continuing Education Course CEC 3. Exposure models In both cases, the evaluation procedure follows the general structure for risk assessment of actives (drugs, pesticides, ...) namely hazard determination, dose-response assessment, exposure assessment and risk characterisation. Useful guidance is taken up in the so-called “Notes of Guidance for Testing of Cosmetic Ingredients for their Safety Evaluation" (SCCNFP/0321/00 final). 8 SAFETY ASSESSMENT OF OXIDATIVE HAIR DYE PRODUCTS - THE CHALLENGE OF EVALUATING REACTIVE MIXTURES Verner Schuh. Product Safety, Wella AG, Darmstadt, Germany Abstract not received. 9 ALLERGIC CONTACT DERMATITIS: CHEMICAL AND METABOLIC MECHANISMS AND SENSITIZATION RISK ASSESSMENT Camilla K. Pease. Safety & Environmental Assurance Centre (SEAC), Unilever Colworth Laboratory, Sharnbrook, Bedford. UK. MK44 1LQ. UK Many types of local and systemic toxicities can arise following skin contact with xenobiotics. Allergic contact dermatitis (ACD) is the clinical definition of a chemical-specific, type IV delayed hypersensitivity skin reaction that becomes manifest 48–72h following contact with a small molecule skin sensitizer. It has been estimated that the condition affects a significant proportion (up to 5%) of the world’s population. It is important to assess the sensitization hazard of new chemical entities (NCEs) and to establish the relative potencies of known sensitizers in order to minimise and manage the risks of human exposure via skin contact with sensitizing compounds. At present, in vivo methods (most recently the murine local lymph node assay (LLNA)) are used to assess sensitization hazard and potency of NCEs and no in vitro assays exist. The European Union together with Industry share the aim of developing novel in vitro/in silico alternative testing strategies for sensitization hazard and potency assessments. To achieve this aim, it is vital to understand the pivotal mechanisms underlying skin sensitisation at the cellular and molecular levels. In this presentation, some of the current understanding of molecular mechanisms will be explored, for certain sensitizing chemicals, through illustrations of reaction chemistries, structure activity relationships, absorption, distribution and metabolism (ADM) properties, together with some elements of immunorecognition at the molecular level. CEC 3 Exposure models 10 THE MONTE CARLO PROJECT FOR STOCHASTIC MODELLING OF HUMAN EXPOSURE TO FOOD CHEMICALS AND NUTRIENTS: AIM AND FUTURE DEVELOPMENT s3 soup, ketchup, pizza etc.) using an advanced recipe search facility. Equally, plant foods can be re-calculated back to raw agricultural commodities e.g. bread to wheat, cider to apples etc. Data output is extremely detailed. If x simulations are carried out for y iterations, any individual computed value can be examined thus yielding true transparency. Existing databases were re-structured or new databases were created where an estimate of ‘true’ intake of food chemicals could be ascertained for pesticides, nutrients, additives and sweeteners. The reference value for the deterministic approach to exposure estimates was the Step 2 approach where the chemical is assumed be present if legally permitted and at the maximum legal limit. In order for a probabilistic exposure to be valid, the value had to lie between the ‘true’ and the deterministic value. Satisfactorily validated models were developed for additives, sweeteners and pesticides. 11 ASSESSMENT OF EXPOSURE TO FOOD CHEMICALS: METHODOLOGICAL ASPECTS FOR THE COLLECTION AND USE OF DATA ON FOOD INTAKE AND CHEMICAL CONCENTRATION C. Leclercq. INRAN (National Research Institute for Food and Nutrition), Via Ardeatina 546, 00178 Rome, Italy Ideally, the intake of a food chemical can be assessed by combining data on concentration in all food products with data on their consumption. However, the information currently available in the European Union present many limitations when used for this purpose. A significant source of uncertainty in the estimation of food chemicals intake is introduced by the difficulty in matching the food descriptions for which occurrence data are available with the food descriptions existing in food consumption databases. For the assessment of exposure to food additives and pesticides, intake is therefore commonly estimated using methods based on highly conservative assumptions, in order to prevent consumers from being exposed to high intakes of certain food chemicals. These methods are useful and economical screening tools for identifying those substances for which acceptable levels of intake may be exceeded, they are not suitable for predicting actual exposure since they are designed to cover the worst case scenario. Refined techniques are currently available and may be used to estimate, as accurately as possible, the intake of food additives (based on brand level food consumption records and additive concentration data) and pesticide residues (based on duplicate diet studies). However, the food products market changes very rapidly in relation to both product formulation and consumer preferences and these kind of estimates are rarely repeatable because the databases generated and used to calculate them require an extraordinary expenditure of time and resources. In this context, the application of probabilistic modeling could be a feasible and cheaper alternative to evaluate the intake of a food chemical because of a reduced need for expensive primary data collection. Within the Montecarlo project, refined databases have been created for the purpose of estimating as accurately as possible ‘true’ chemical intakes in order to assess the validity of probabilistic models. 12 STOCHASTIC MODELLING APPLIED TO THE ESTIMATION OF INTENSE SWEETENERS INTAKE Michael J. Gibney. Institute of European Food Studies Trinity College, Dublin, Ireland D. Arcella. National Research Institute for Food and Nutrition, Rome, Italy Monte Carlo is the acronym for a multi-centre research project funded under the EU 5th Framework Programme for the development and validation of probabilistic models for food chemical exposure estimates. The process began with the development of conceptual models, which were effectively a framework for describing the qualitative and quantitative elements of the models. On the basis of the conceptual models appropriate software code was written and linked to existing software, which enables users to access the program via the Internet. The program was linked to food consumption databases in a way which allowed it accept many different styles of databas4e architecture. The software has the capacity to link to high performance computing. All data can be entered as distribution functions or as raw data. Food categories can be created to encompass primary foods or dishes (tomato The three EU directives which fixed Maximum Permitted Levels (MPL) for intense sweeteners for all Member States also include the general obligation to establish national systems for monitoring the intake of food additives, in order to evaluate their use safety. In the EU the most commonly used methods for the assessment of exposure to these substances follow a deterministic approach based on conservative assumptions but over the past years, to get a more realistic view of exposure to food chemicals, risk managers are getting more interested in the probabilistic approach. Within the EU funded “Montecarlo” project, stochastic models of exposure to different chemical substances from the diet and a computer software were developed and validated. With respect to intense sweeteners different probabilistic models aimed at estimating the intake of saccharin from table-top sweeteners and cyclamate s4 Continuing Education Course CEC 4. “Omics” in toxicology from soft drinks by Italian teenagers were tested. Data on food consumption and concentration of intense sweeteners were collected and used to validate such models. A food frequency questionnaire designed to identify adolescents who were high consumers of sugar free soft drinks and/or of table top sweeteners was filled in by 3,982 teenagers living in the District of Rome. Moreover 362 subjects participated in a detailed food survey by recording, at brand level, all foods and beverages ingested over 12 days. Producers were asked to provide the intense sweeteners concentration of sugar-free products. Results showed that consumer behaviour with respect to brands has an impact on exposure assessments. Only probabilistic models that took into account indicators of correlation between eating events (i.e. market share and brand loyalty) provided an acceptable intake estimate for higher consumers. This example demonstrates that when chemicals intake is estimated by means of a probabilistic model it is important to consider significant correlations and dependencies in order to avoid an underestimation of exposure. 13 STOCHASTIC MODELLING APPLIED TO DIETARY PESTICIDE RESIDUES EXPOSURE ASSESSMENTS Jesús A. Ocio, Gobierno Vasco. Departamento de Sanidad. Dirección de Salud Pública. C Donostia-San Sebastián 1. 01010 Vitoria-Gasteiz, Spain The applications of dietary pesticide residue exposure assessments are (i) the registration of new pesticides and (ii) the risk characterisation of those already in the market. Initially, in the risk assessments, only chronic exposure was estimated by deterministic approaches. The studies performed in UK at the beginning of the nineties discovered the importance of the unit-to-unit variability of the pesticide residues and their influence on short-term exposure. This discovery led to the development of conservative approaches for acute exposure assessments. Computer developments and the increasing availability of pesticide residue data have allowed the use of probabilistic approaches to assess acute exposure. Stochastic modelling provides more realistic intake estimates and information on their variability and uncertainty. As part of the Monte Carlo project, a probabilistic conceptual model to estimate dietary pesticide residue intake was designed and a purpose built software program was developed. Both, the model and the software were validated. Food surveys are the main source of food consumption data for the model. For each eating event, the model back transforms the food items into Raw Agricultural Commodities (RAC), since the monitoring programmes measure the pesticide residues in RACs. Then a concentration of pesticide residue is assigned to the RAC. When there are no data or the residues are below the limit of reporting, the model gives several options that allow characterising the associated uncertainty. This concentration is then adjusted for the unit-to-unit variability of the pesticide residues and the effects of processing (washing, peeling, cooking, etc.) The RAC consumption is multiplied by the adjusted residue concentration and divided by the bodyweight of the appropriate subject to obtain the pesticide intake for that particular eating event. Then, the sum of the pesticide intake of all eating events provides the total intake for the subject. The application allows setting the number of subjects and multiple simulations with or without bootstrapping. CEC 4 “Omics” in toxicology 14 GENOMIC TECHNOLOGIES AND APPROACHES IN TOXICOLOGY Peter G. Lord. Drug Evaluation, Johnson & Johnson Pharmaceutical Research & Development LLC, 1000 Route 202, PO Box 300, Raritan, NJ 08869, USA Genomic responses are a fundamental feature of the adaptation of cells to changes in their environment. It follows, then, that cellular responses to a xenobiotic include changes in the expression of a number of genes. Until a few years ago the technology to study gene expression was relatively slow and laborious. The development of arrayed gene platforms has led to the ability to monitor the expression of many (tens to thousands) genes in parallel. Coupled with huge investment in the sequencing of whole genomes and the major improvements in computing power, the use of these technologies has rapidly increased our understanding of gene responses as they relate to toxic mechanisms. To introduce the genomic technologies as they are applied to toxicology, a brief history of the development of genomic techniques will be presented. This will cover gene expression analysis from the early days of the northern blot to the recent developments in gene chips and real-time RT-PCR approaches. Attention will also be given to the computational tools which are used for organising and making sense of the data. The emphasis will be on the practical capabilities, including the limitations, of the techniques. Examination of the changes in gene expression induced by toxic agents give one part of the picture as to their mechanisms of toxicity. However there is a lot of information that can be gleaned by a genomic approach. Perhaps the biggest contribution that a genomic approach can make in toxicology is in hypothesis generation. This may be in a “predictive toxicology” setting, in which compounds are screened for toxic potential. Additionally it may be in a “mechanistic toxicology” setting in which a starting point is required to formulate an investigative programme to elucidate a toxic mechanism. The concepts of each of these approaches, and how the different genomic technologies are being applied to them, will be discussed. 15 METHODOLOGICAL APPROACHES IN PROTEOMICS D.C. Thompson. Drug Safety Evaluation, Pfizer, St. Louis, MO, USA Recently there has been a tremendous increase in interest in the application of proteomics to a variety of life science disciplines, including toxicology. One of the most common applications of proteomics in the field of toxicology involves the analysis of differential protein expression in control versus toxicant-treated samples. These analyses are typically carried out with the intention of finding potential safety markers for the lesion or for studying mechanisms of toxicity. Two-dimensional polyacrylamide gel electrophoresis (2DPAGE), followed by image analysis and identification of selected proteins by mass spectrometry (MS) is the traditional approach used for these studies. The 2D-PAGE approach, however, has a number of drawbacks, including a rather limited dynamic and molecular mass range, difficulty in detecting low abundance proteins, poor resolution of highly acidic or basic proteins, and relatively large sample requirements. While notable improvements have recently been made in 2D-PAGE (e.g. narrow range immobilized pH gradient (IPG) strips, multiple fluorophores for staining, robotic sample handling), new technologies are being developed to circumvent many of these problems. These include amino acid labeling methods such as isotope-coded affinity tags (ICAT), multi-dimensional HPLC-MS techniques, and protein microarrays. High quality antibodies are being used to produce protein microarrays; however, a number of novel types of capture agents that are less problematic to work with than antibodies are also being developed for potential use in protein biochips. These capture agents include aptamers, partial-molecule imprints, modified binding proteins, antigen mimics and biochemical surfaces. Although most of these methods are at early stages of development, a currently available system utilizing surface-coated protein chips coupled with MS detection (SELDI) has shown promise in profiling protein patterns in sera from patients with specific types of cancer as well as biomarker discovery in toxicology studies. 16 METHODOLOGICAL APPROACH IN METABONOMICS Elaine Holmes. Biological Chemistry, Biomedical Sciences, Imperial College, South Kensington, London, SW7 2AZ, UK The role of chemometric analysis in analyzing and interpreting genomic, proteomic and metabonomic data has evolved rapidly in the past decade. Metabonomics provides a systems approach to measuring dynamic biochemical responses of organisms to pathological stimuli or genetic modification and operates by profiling the metabolic responses of key intermediary biochemical pathways. Continuing Education Course CEC 4. “Omics” in toxicology Metabonomic technology, coupling sophisticated analytical methods such as high resolution 1 H NMR spectroscopy or mass spectrometry with appropriate chemometric strategies, enables simultaneous measurement of a wide range of metabolites in biofluids or tissues in a dynamic manner. Such analysis has been shown to be of considerable value in providing detailed information regarding the metabolic status of an organism and in characterizing and predicting a wide range of pathological conditions. Models of site- or mechanismspecific toxicity can be constructed and combined to form predictive expert systems for toxicity screening. The complexity and inter- s5 active nature of biological systems introduce confounding variation into the metabonomic data. Various chemometric and bioinformatic strategies for optimizing the characterization and prediction of pathological conditions can be adopted in order to increase the sensitivity of metabonomic analysis by reducing the influence of confounding random and systematic noise, accommodating the presence of large dynamic range in the measurement variables and/or incorporating the temporal dependence of pathological lesions. Several strategies for data optimization are discussed here and the potential role of Metabonomics in 21st century medicine considered. s7 Invited Lectures S1 Omics in drug development 17 ments made in applying the technology are maturing and there is a determined effort to bring the full power of the technology into drug risk assessment. PROGRESS IN APPLYING GENOMICS IN DRUG DEVELOPMENT Peter G. Lord. Drug Evaluation, Johnson & Johnson Pharmaceutical Research & Development LLC, 1000 Route 202, PO Box 300, Raritan, NJ 08869, USA The introduction of arrayed gene platforms for gene expression analysis has led to much investment by drug companies, government agencies and technology providers in applying genomics-based approaches in drug development. There are two areas of drug development for which gene expression profiling has received particular attention and interest. These are “predictive” toxicology and mechanism-based risk assessment. The last few years has seen a lot of progress being made in linking the profiles of gene expression induced by drugs with their toxicities. By developing databases of expression profiles for a wide variety of toxic compounds and toxic models it has been possible to create statistical and computational methods which provide an indication of the toxic potential of a drug from the pattern of gene expression changes it elicits in in vitro or in vivo systems. The predictive toxicology field is evolving rapidly and there is much debate about the predictive power of genomic approaches. One debate is whether information from the whole genome is essential for a prediction or whether predictive power is increased by focussing on small sets of genes whose function in toxic mechanisms is known. As the knowledge base of toxicity related gene expression builds up it is becoming apparent that understanding of the toxicological relevance of specific genes is needed to guide the predictive modelling (through the use of supervised learning). It remains to be seen whether the genomics-based predictive toxicity assays provide sufficient improvements on current cell-based or biochemical assays, but there is no doubt that such an approach is highly applicable to predictive toxicity screening. In recent times, an understanding of the mechanism of toxicity of a new drug has become a major part of its risk assessment. Because gene expression is central to many responses to xenobiotics, genomic approaches lend themselves very readily to mechanistic toxicology studies. By examining changes in gene expression in cells and tissues in response to drugs it is possible to generate hypotheses as to the underlying mechanism and in some cases it is possible to evaluate hypotheses of toxic mechanism. Gene arrays representing the whole genome are generally used in hypothesis generation so as not to bias the outcome. For hypothesis testing it is possible to create a specific set of genes according to what is proposed for the mechanism. Examples of both these approaches are now appearing in the literature. There are still a number of concerns around the use of gene expression data in drug risk assessment. There are technical concerns about the sensitivity and reliability of the methods. There are also concerns about the interpretation of the data. For example, using a whole genome hypothesis generation approach may indicate some gene expression changes which are at best, uninterpretable and at worst, open to misinterpretation. To address these concerns (with particular attention to using genomic data in the regulatory environment) a consortium of academic, governmental and industrial representatives formed a committee on the use of genomics in mechanism based risk assessment coordinated by the ILSI Health and Environmental Sciences Institute (HESI). The committee’s findings have shed much light on the technical issues and have shown the relevance of the data in understanding several mechanisms of toxicity. The data are being placed in the public domain in partnership with the European Bioinformatics Institute. The committee aims to provide guidance on the application of toxicogenomics to risk assessment such that the technology can be applied in a pragmatic and realistic manner. Genomics, and more specifically toxicogenomics can no longer be regarded as “new” technology in drug development. The invest- 18 APPLICATION OF PROTEOMIC TECHNOLOGIES IN THE DRUG DEVELOPMENT PROCESS D.C. Thompson. Drug Safety Evaluation, Pfizer, St. Louis, MO, USA Proteins are the principal targets of drug discovery. Most large pharmaceutical companies now have a proteomics-oriented biotech partner or have started their own proteomics division. Common applications of proteomics in the drug development process include target identification and validation, identification of efficacy and toxicity biomarkers, and investigations into mechanisms of drug action or toxicity. For target identification and validation the emphasis is typically on finding specific proteins directly associated with a disease state. This involves identifying proteins whose expression levels or activities change in the presence of disease. These proteins may serve as potential targets for drug therapy or may be used to identify or classify patients for future clinical trials. Proteomics technologies may also help identify protein-protein interactions that may influence either the disease state or proposed therapy. The identification of efficacy and toxicity biomarkers in readily accessible biological fluids is a second major use of proteomics technology in the drug development process. Efficacy biomarkers are used to assess whether target modulation has occurred. They are used for the characterization of animal disease models and to assess the effects of lead candidates in animal models. They are also used for determining appropriate dose levels in human clinical trials and to help make critical go/no go decisions at various stages in development. Toxicity (safety) biomarkers are used to screen compounds in pre-clinical studies for target organ toxicities as well as later on in development during clinical trials. Finally, proteomics technologies play a significant role in studies on the mechanism of action or toxicity of individual drugs in a specific target tissue. Often, complimentary approaches such as metabonomics and genomics are used in conjunction with proteomics at various stages in the drug development process to create more of a unified, systems biology approach. 19 METABONOMIC APPROACHES TO THE UNDERSTANDING OF DIRECT AND IDIOSYNCRATIC DRUG TOXICITY Jeremy K. Nicholson. Biological Chemistry, Faculty of Medicine, Imperial College of Science Technology and Medicine, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ UK One of the key challenges in the post-genomic era is the establishment of functional relationships between gene expression and patho-physiological end-points in health and disease. Biosystems can be conveniently be viewed at several closely interlinked and interdependent levels of organization based on biochemistry: viz. Genomic, transcriptomic, proteomic and metabonomic levels (1). The measurement of these biofeatures by whatever analytical method generates complex multivariate data sets that require the use of chemometric modelling methods to extract latent biological information which can be related to suitable biological end-points. In order to investigate the complex series of metabolic consequences of disease processes, toxic reactions and genetic manipulations, non-selective, but specific analytical approaches for are required which are “information-rich”. A number of spectroscopic methods can be applied to generate metabolic signatures of complex biomaterials and data processed using chemometric methods to relate to particular end-points (2). However, metabolism in higher organisms is subject to many extragenomic influences which complicate and limit mathematical modelling of pathway activity (3). In this lecture the relationships between genes, proteins and metabolic pathway control will be s8 Symposium S2. Developmental immunotoxicology discussed together with the application of NMR-based metabonomic approaches to investigate toxicological and functional genomic problems and the integration of transcriptomic and metabonomic datasets. A new probabilistic modelling theory for integrative metabolism is also proopised that could help in the understanding of idiosyncratic toxicity. 20 INTEGRATING GENOMICS, PROTEOMICS AND METABONOMICS T.G. Klenø 1 , D. Baunsgaard 1 , L.L. Rønnedal 1 , S.M. Laursen 2 , T.S. Sørensen 1 . 1 Applied Trinomics, Novo Nordisk A/S, Måløv, Denmark; 2 Toxicology, Novo Nordisk A/S, Måløv, Denmark Genomics, proteomics and metabonomics are rapidly developing technologies which have given researchers new ways of gaining information of toxicity, pharmacological effects and mechanisms of drug treatments. Gene expression micro arrays allow us to study regulation of mRNAs encoded by the genome in response to a drug perturbation. Likewise, proteomics can be used to reveal changes in the expression pattern of hundreds of proteins simultaneously, including posttranslational modifications important for cell function. Information of how regulation is reflected in biochemical pathways can further be elucidated by metabonomics measuring endogenous metabolites. The three technologies are thus complementary and can be interpreted separately. When combining the data valuable information can be obtained. However, tools to handle, reduce and integrate the large amount of information obtained by the three omics techniques are required to identify correlated events caused by drug treatment. Examples are presented where the three “omics” technologies (gene expression micro arrays, proteomics and metabonomics) have been applied to studies of toxicity of hydrazine. Hydrazine is a commonly used model hepatotoxin in animal studies. A major effect of hydrazine is accumulation of fat in the liver leading to liver steatosis. Hydrazine is affecting numerous cellular processes; however, the biochemical mechanism of toxicity of hydrazine is not yet fully understood. PL Gerhard Zbinden Memorial Lecture 21 adducts from exogenous compounds, although the overall relative biological significance of each type of damage is unknown. The use of DNA and protein adduct measurements in molecular epidemiology will be illustrated. The examples will include the detection of DNA and protein damage from occupational carcinogens, environmental pollution, foodstuffs, and cigarette smoking, and the modification of DNA bases by endogenous hydroxyl radicals and methylating species. DNA AND PROTEIN ADDUCTS AS MARKERS OF GENOTOXICITY P.B. Farmer. Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, University Road, Leicester, LE1 7RH, UK Determination of the interaction products (adducts) of a carcinogen with DNA or protein indicates the amount of genotoxic material that has reached the tissue under study and provides invaluable information for molecular epidemiological studies. Protein adducts are not repaired and are considered simply as exposure monitors, but DNA adducts may also give further information with regard to the mutagenic significance of the exposure. The sensitivity and applicability of the analytical methods for the detection and quantification of carcinogen adducts has improved dramatically in recent years, and DNA damage levels as low as one adduct per 108 nucleotides can now routinely be measured. Although 32 Ppostlabelling has previously been the most sensitive technique for detection of non-radioactive carcinogen adducts with DNA, mass spectrometric approaches using LC-MS-MS are now reaching similar sensitivities. Under normal living conditions, it has been shown that human DNA and protein are exposed to a wide variety of chemicals which have the potential to modify their structures. One of the significant findings resulting from this work has been the discovery of ‘background’ modifications of DNA and protein in supposedly unexposed individuals. These background modifications arise from endogenous processes, often associated with oxidative damage. In human DNA, the extent of oxidative DNA damage is generally greater than the extent of formation of covalently-linked DNA S2 Developmental immunotoxicology 22 IMMUNOTOXICOLOGICAL CONSEQUENCES OF PERINATAL CHEMICAL EXPOSURES Henk van Loveren, Aldert Piersma. Laboratory for Toxicology, Pathology and Genetics, National Institute for Public Health and the Environment RIVM, P.O.Box 1, 3720 BA Bilthoven, The Netherlands T lymphocytes play a crucial role in immunocompetence. Maturation of T lymphocytes takes place in the thymus. During the differentiation of progenitor T cells into mature T lymphocytes the repertoire of antigen specificities is generated, and desired specificities are positively selected, while undesired specificities are deleted. During the maturation also differentiation into different subpopulations with their respective regulatory or effector functions take place. Building the repertoire of B cells does not take place in the thymus, but more systemically, including in the bone marrow. Even if such processes probably take place during the entire life, most of these processes are completed at an early stage in life. Immunocompetence starts to develop in utero, and is largely completed early in life. This is illustrated by the involution of the thymus, that progresses with progressing age. It is therefore likely that effects of exposure to immunotoxic chemicals may have important consequences especially during developmental stages, i.e. starting in utero. The immune system of the fetus and neonates is characterized by Th2 type of interleukins. It is suggested that the reason for this is to minimize the development of reactions of the fetal immune system to maternal tissue antigens in the placenta, that would be mediated by Th1 type immune responses. In the early postnatal period the immune system matures to provide a balanced Th1/Th2 state, facilitating resistance to infections, at a time when adverse reactions to maternal components is no longer an issue. It has been shown that infections and vaccinations, that may influence the Th1/Th2 balance, have an impact on the maturation of the immune system. In addition to in utero developmental stages of the immune system, the post-natal period is therefore likely to be another vulnerable period during which immunotoxic chemicals may have relatively pronounced consequences. A number of chemicals that have immunotoxic consequences if exposure takes place during developmental stages of the immune system have been identified. These include TCDD, PCB’s, HCH, heavy metals, steroid hormones and DES, and cytostatic agents. These findings have prompted further activities towards inclusion of developmental toxicity testing in guideline-based protocols. Current predictive immunotoxicity testing is mainly done in the context of general toxicity, according to OECD guideline 407. There is a number of test systems that address effects of exposure during development. This pertains to OECD 414, 415, and 416 respectively, that have all their specific design. Some of these may be adequate for addressing potential effects of immunotoxicants on the developing immune system. In OECD 414 developmental toxicity protocol, given the time of necropsy at one day before birth, inclusion of immunotoxicological parameters is not readily feasible. Although the OECD 415 and 416 generation studies allow for inclusion of immunotoxicological parameters, both these tests are already laborious. A less expensive test that does include exposure during all developmental stages of the immune system, i.e. from day 6 of gestation until weaning of the offspring is described in the draft guideline OECD 426. This test is aimed at detecting developmental neurotoxicity and includes behavioral tests at young adult age. Littermates could be used for immunotoxicity testing. Workshop W1. Fine particle exposure and adverse health effects 23 DEVELOPMENTAL IMMUNOTOXICITY DUE TO TCDD EXPOSURE Ralf Stahlmann. Institute of Clinical Pharmacology and Toxicology, Dept. of Toxicology Benjamin Franklin Medical Center, Freie Universität Berlin, Berlin, Germany The developmental immunotoxicity of TCDD has been known for almost 3 decades (Vos & Moore, 1974); experiments have been performed mainly in mice and rats. It is of interest that the immune system of rats during pre- and/or postnatal development reacts rather sensitively, which stands in contrast to adult rats which are rather resistent to dioxin-induced immunosuppression. TCDD acts on thymic epithelial cells and has the potential to induce terminal differentiation of these cells. Also, T-lymphocyte subpopulation distribution is affected as has been shown in a variety of species. Furthermore, a direct action of TCDD on bone marrow cells has been discussed as a possible mechanism for the immunotoxic effects. Smialowicz and coworkers published a series of papers dealing with the effects of TCDD on the immune system of rats during development. Relative thymus weight and thymic cellularity decreased in 19-day-old rat fetuses after maternal treatment on day 14 of pregnancy. In comparison to controls, the percentage of double positive (CD4+ CD8+ ) cells decreased and the percentage of CD8+ -thymocytes increased. It was also shown that the immunosuppressive effect of perinatal (i.e. placental and lactational) TCDD exposure of rats persisted into adulthood and that suppression of the DTH (delayed-type hypersensitivity) response represents the most sensitive biomarker for TCDD-induced developmental immunotoxicity in rats. The lowest maternal dose that produced DTH suppression in a test system using BSA (bovine serum albumin) was 0.1 µg TCDD/kg body weight. Certainly, the findings in rats are of concern because effects could be induced at comparatively low doses during development and they persisted into adulthood. 24 IMMUNOLOGICAL EFFECTS OF BACKGROUND EXPOSURE TO POLYCHLORINATED BIPHENYLS AND DIOXINS IN DUTCH PRESCHOOL CHILDREN: AN EXPLORATORY STUDY OF HEALTH EFFECTS FROM ‘NORMAL’ ENVIRONMENTAL EXPOSURE Nynke Weisglas-Kuperus 1 , Svati Patandin 1 , Guy A.M. Berbers 2 , Theo C.J. Sas 1 , Paul G.H. Mulder 3 , Pieter J.J. Sauer 1 , Herbert Hooijkaas 4 . 1 Department of Pediatrics, Division of Neonatology, Erasmus Medical Centre - Sophia Children’s Hospital, Rotterdam, The Netherlands; 2 Department of Clinical Vaccine Research, National Institute of Public Health and the Environment, Bilthoven, The Netherlands; 3 Institute of Epidemiology and Biostatistics, Erasmus University Rotterdam, The Netherlands; 4 Department of Immunology, Erasmus University and University Hospital, Rotterdam, The Netherlands Prenatal exposure to polychlorinated biphenyls (PCBs) and dioxins is associated with changes in the T-cell lymphocyte population in healthy Dutch infants. We investigated whether these changes persist into later childhood and whether background exposure to PCBs and dioxins is associated with the prevalence of infectious or allergic diseases and humoral immunity at preschool age. The total study group consisted of 207 healthy mother-infant pairs. Prenatal exposure to PCBs and dioxins was estimated by the sum of PCBs 118, 138, 153 and 180 (PCB) in maternal and cord plasma and in breast-fed infants by the dioxin, planar and mono-ortho PCB toxic equivalent (TEQ) levels in human milk. At 42 months of age, current body burden was estimated by the PCB in plasma. The prevalence of infectious and allergic diseases was assessed by parent questionnaire. Humoral immunity was measured by antibody levels for mumps, measles and rubella after primary vaccination. In a subgroup of 85 children immunological marker analyses of lymphocytes were done. Prenatal PCB exposure was associated with an increased number of lymphocytes, T-cells, and CD3CD8+ (cytotoxic), CD4+CD45RO+(memory), TcR αβ+ and CD3+HLADR+ (activated) T cells and lower antibody levels to mumps and measles at preschool age. Adjusted for confounders, prenatal PCB exposure was associated with less shortness of breath with wheeze and current PCB body burden was associated with a higher prevalence of recurrent middle ear infections and of chickenpox and s9 a lower prevalence of allergic reactions. A higher dioxin TEQ was associated with a higher prevalence of coughing, chest congestion and phlegm. We conclude that in Dutch preschool children the effects of perinatal background exposure to PCBs and dioxins persist into childhood and might be associated with a greater susceptibility to infectious diseases. Common infections acquired early in life may prevent the development of allergy and therefore PCB exposure might be associated with a lower prevalence of allergic diseases. 25 ALTERATIONS IN THE IMMUNE SYSTEM OF CHILDREN FROM MOTHERS TREATED WITH IMMUNOSUPPRESSIVE AGENTS DURING PREGNANCY R. Cimaz 1 , E. Meregalli 1 , M. Biggioggero 1 , O. Borghi 2 , A. Tincani 3 , M. Motta 3 , C. Antonioli 3 , P.L. Meroni 2 . 1 Pediatrics, ICP, Milan, Italy, 2 Istituto Auxologico and University of Milan, Italy, 3 Spedali Civili, Brescia, Italy Ideally, immunosuppressive agents should not be used during pregnancy; however in transplantation and in connective tissue diseases their use is often needed, both to protect the mother’s health and to insure a successful pregnancy. Many of these drugs cross the placental barrier and enter the fetal circulation, with a possible impact on the fetal immune system. There have been reports of transient immune suppression in babies after in utero exposure to cyclosporine A, azathioprine, and corticosteroids, particularly the fluorinated ones (dexamethasone and betamethasone, that are not inactivated by placental enzymes). Immune alterations described include lymphopenia, decreased immunoglobulin levels, and decreased survival of lymphocytes in culture. These findings, even if transient, are of importance both for the possibility of increased susceptibility to infections and for the possibility of an impaired response to vaccines. Moreover, it has been suggested that fetal exposure to immunosuppressive agents may be associated with occurrence of autoimmune disease later in life, for a disruption in development of self-tolerance by T cells, and with alterations in the Th1/Th2 cell differentiation in the developing immune system that could increase the susceptibility to atopy in genetically predisposed individuals. We have started a pilot study in order to evaluate possible immune alterations in children from mothers treated with immune suppressants for connective tissue diseases. We have up to now studied 6 babies whose mothers had been exposed to cyclosporine A (2), azathioprine (1) and dexamethasone (3) during pregnancy, and 9 babies from mothers with similar diseases but who had not been treated (controls). Complete blood count, IgA, IgG, IgM, IgG subclasses, and lymphocyte subpopulations were determined in all cases. Moreover, serum levels of anti-HBsAg and presence of autoantibodies (ANA, ENA) was also determined. Children were tested at a mean age of 7.5 months, both in study and in control group (range 1–19 months). Results were compared with agematched values. Of all parameters tested, only Hb levels and IgA levels resulted slightly lower in patients than in controls. Antibody levels to hepatitis B vaccinations were similar in the two groups, and there was no development of new autoantibodies in any case. Although our results are preliminary, we concur with the literature data that prenatal exposure to immunosuppressive drugs does not have a profound effect to the developing immune system. More data and a longer follow-up are needed to confirm these results. W1 Fine particle exposure and adverse health effects 26 FINE PARTICLE EXPOSURE AND ADVERSE HEALTH EFFECTS Norbert Englert. Federal Environmental Agency (Umweltbundesamt), Berlin, Germany Adverse health effects of exposure to particles have been described in numerous epidemiological studies. Health endpoints thoroughly studied are all cause and cause-specific mortality, and hospital s10 Workshop W1. Fine particle exposure and adverse health effects admissions. Older studies focussed on associations with PM10 (then named fine particles). During the last decade, PM2.5 was increasingly emphasized, and the term “fine particles” was restricted to this size fraction. Currently, ultrafine particles (UF, PM0.1 ) are discussed to be another important fraction which should be characterized by particle number instead of particle mass. However, data on UF exposure and health effects are still limited. The mechanisms by which particles influence human health are only poorly understood. Under discussion is the role of particle size (which size fraction is most important with respect to adverse health effects?) and particle composition (which compounds or elements are health-relevant?). The risk assessment of coarse particles (i.e. the size fraction between 2.5 and 10 µm) suffers from inconsistent findings ranging from“ irrelevant to health” to “relevant, especially to respiratory effects”. The question of causality is not yet decided. However, it is widely accepted that PM is some kind of container including components which are toxicologically relevant and others which might be seen mainly as indicators. Thus, the local mix may influence the toxicological potency of PM, and results from studies carried out in one region may not necessarily be consistent with results gained elsewhere. Recently, reanalyses of epidemiological studies performed by the Health Effects Institute (HEI) qualitatively confirmed the original results. New insight in the influence of socioeconomic factors extended the knowledge on health effects of particles. To some extent, the quantitative findings had to be adjusted. In addition, the slope of the dose response relationships from time-series analyses seems to need downward adjustment due to some problems with statistical analyses prorgammes. Nevertheless, the whole body of knowledge supports the role of PM as a type of air pollution with great influence on human health. 27 SCIENCE BASIS AND APPROACHES FOR REGULATING OCCUPATIONAL AND AMBIENT EXPOSURE TO PARTICLES U. Heinrich. Fraunhofer-Institut für Toxikologie und Experimentelle Medizin, Hannover, Germany Ambient particles consist of complex mixtures of soluble and insoluble, organic and inorganic substances emitted from various anthropogenic and natural sources accompanied by a vast amount of gaseous emissions. Depending on the climate conditions, sunlight exposure, as well as on the ambient half-life and on the gas phase surrounding the particle, chemical transformations of airborne substances occur prior to the inhalation and deposition in the respiratory tract of humans. So far, no ambient particle compound(s) or physical status of the particle have been found that are absolutely necessary for the causation of the increased risk of health damage observed in humans exposed to slightly increased ambient particle concentrations or, which if not existent would not lead to the observed increased health risk. Only the aerodynamic size fraction of the ambient particles given by the cut-off size of 2.5 or 10 µm of the particle sampler used in epidemiological studies correlate significantly - and even depending on the concentration - with health effects in humans. Epidemiological - but so far not experimental - animal studies have convincingly shown the ambient particle health effects including local effects in the respiratory tract as well as systemic effects involving the cardiovascular system and therefore particle exposure limits have to be established as preventive measures. But, the real culprit in respect of emission source and particle component(s) is so far unknown and, therefore, no well-aimed but only general dust reduction measures can be recommended. Particles emitted from high temperature processes appeared to contribute most to the observed health effects. Particle exposure at the workplace can very often relate to a specific emission source and also to a causative agent contained in this emission and a health-based threshold limit value can be evaluated quite often using the data from toxicological and mechanistic studies with cell cultures and experimental animals, as well as from observations in exposed workers. This limit values are related to only one single substance and combination effects of mixtures are only taken into account in mathematical considerations assuming additive or independent effects. A real particle effect can be observed with poorly soluble or insoluble dusts deposited in the alveolar region of the lung if the alveolar macrophages are for various reasons unable to cope with these deposits and to remove these particles from the alveolar surface adequately and timely. In this situation of the unspecific defence reaction of the lung reactive oxygen species a. o. are generated continuously. During this process of perpetuating inflammatory reaction lung tissue will be adversely affected. Oxidative DNA damage can also occur increasing the risk of neoplastic lung lesions. Based on alveolar particle clearance data on experimental animals and on humans and epidemiological data on chronic bronchitis, a particle exposure concentration can be evaluated that will not seriously disturb the alveolar particle clearance and, thus, inflammation related non-neoplastic lung diseases should not occur. But, how much particle related inflammation and oxidative DNA damage can be tolerated without increasing the risk of causing lung cancer? Is it allowed to extrapolate in this case from rat to human and is it possible to establish mechanistic-based risk-related threshold limit values for these effects of particles with low solubility? In addition, the ambient particles consist to a certain percentage of poorly soluble material, but this mass concentration in ambient air is at least two orders of magnitude lower than the general dust limit value for workplaces in Germany. The fine poorly soluble particle as carrier and depot for potential carcinogenic substances (e.g. soot) in combination with the above described particle effect may explain the increased risk of lung cancer after long-time exposure to ambient particles. 28 POSSIBLE MECHANISMS OF THE CARDIOVASCULAR EFFECTS OF INHALED PARTICLES: SYSTEMIC TRANSLOCATION AND PROTHROMBOTIC EFFECTS A. Nemmar. Laboratory of Pneumology (Lung Toxicology), K.U.Leuven, Leuven, Belgium Particulate air pollution is associated with cardiovascular morbidity and mortality. It is established that fine particles with a diameter <2.5 µm (PM2.5 ) have an important role in triggering biological responses. These particles, and particularly the ultrafine fraction (<100 nm), remain airborne for long periods of time and penetrate deeply into the respiratory tract. Exposure to PM2.5 for as little as 2 hours has been shown to increase the risk of myocardial infarction. However, a plausible explanation for these epidemiological observations is lacking. Recently, we have demonstrated that ultrafine particles are able to translocate from the lung into the systemic circulation in hamsters and humans (Nemmar et al., Circulation. 2002; 105: 411-4.). In urban areas, diesel engines are considered to be the major source of PM2.5 . We therefore evaluated the acute (1 hour) role of diesel exhaust particles (DEP) in a hamster model of peripheral vascular thrombosis induced by free-radical mediated endothelial injury, using intravenous Rose Bengal and local illumination. Pulmonary inflammation was assessed by bronchoalveolar lavage (BAL). Intratracheal doses of 5 to 500 µg of DEP per animal induced inflammation with elevation of neutrophils, total proteins and histamine in BAL. The same doses enhanced experimental arterial and venous platelet rich-thrombus formation in vivo. Blood samples taken from hamsters 30 and 60 minutes after instillation of 50 µg of DEP caused platelet activation, when analyzed in the Platelet Function Analyser (PFA-100). The direct addition of as little as 0.5 µg DEP/mL to untreated hamster blood also caused platelet aggregation (Nemmar et al., Circulation. 2003;107: 1202-8). These effects persisted at 6 h and 24 h after instillation (50 µg/animal). Preliminary data indicate that these effects can be mitigated by pretreatment with an H1-histamine receptor antagonist (diphenhydramine). Our results provide plausible mechanistic explanations for the epidemiologically established link between air pollution and acute cardiovascular effects. Supported by K.U.Leuven (OT/02/45) and FWO-Vlaanderen (G.0165.03) Symposium S3. Stem cells in toxicology 29 THE ROLE OF THE QUARTZ SURFACE IN DRIVING ITS INFLAMMOGENICITY Vicki Stone 1 , Rodger Duffin 2 , Kirsty Rollo 1 , Rebecca Jones 1 , David Brown 1 , Ken Donaldson 3 . 1 Biomedicine Research Group, School of Life Sciences, Napier University, 10 Colinton Road, Edinburgh EH10 5DT. UK. 2 Particle Research Core, Institut für Umweltmedizinische Forschung (IUF), D-40225 Düsseldorf, Germany. 3 ELEGI/Colt Laboratories, Department of Respiratory Medicine, Division of Clinical and Radiological Sciences, University of Edinburgh, Edinburgh, UK In 1997 quartz (crystalline silica) was classified by IARC as a carcinogen. As part of this classification it was recognised that quartz does not act as a carcinogen in all industries, suggesting that the quartz hazard is a variable entity. Many studies suggest that the reactivity of the quartz surface within biological systems is responsible for promoting damage and inflammation leading to disease. Modification of the quartz surface by interaction with ions, such as aluminium, may attenuate the surface reactivity, potentially inhibiting disease initiation or progression. In this study, instillation of DQ12 quartz (250 µg) into the rat lung resulted in a significant inflammation as indicated by the number of neutrophils in bronchoalveolar lavage within 18 hours of exposure (20 ± 1.5 million neutrophils compared to the control 0.1 ± 0.0 million neutrophils; p<0.001). In contrast, DQ12 pretreated with aluminium lactate completely prevented any alteration in neutrophil cell count compared to native quartz. In addition, DQ12 induced a 5-fold increase (p<0.01) in MIP-2 mRNA expression in BAL cells, an effect which was significantly (p<0.01) prevented by aluminium lactate coating. Haemolysis of human erythrocytes was used as a measure of surface reactivity. DQ12 was treated with either aluminium lactate, or a soluble extract of either coal dust or clay samples prior to incubation with the erythrocytes. The control DQ12 (5 mg/ml) induced extensive haemolysis (72±2%) which was significantly inhibited by treatment of the DQ12 with either aluminium lactate, coal dust extract, or extracts of the clays kaolin, attapulgite, or montmorillonite (p<0.001). These data suggest that aluminium lactate and aluminium containing clays can modify the reactive quartz surface, reducing its ability to either induce inflammation, or to disrupt cell membranes. Further studies will be conducted to investigate the ability of clay extracts to protect the lung against DQ12. This work was funded by The Colt Foundation. molecules of stem and precursor cells as well as of differentiated phenotypes. These data will be applicable to pharmacological and toxicological studies and basic stem cell research. 31 30 EMBRYONIC STEM CELLS AS A MODEL FOR TOXICOLOGICAL STUDIES A.M. Wobus. In Vitro Differentiation Group, Institute for Plant Genetics and Crop Plant Research, Corrensstr. 3, D-06466 Gatersleben, Germany Embryonic stem (ES) cells, pluripotent cell lines established from undifferentiated cells of early embryos, are characterized by the capacity to proliferate and to differentiate in vitro into cells of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established protocols to develop mouse ES cells into specialized cardiac, skeletal and vascular smooth muscle, neuronal, epithelial, pancreatic and hepatic cell types. Tissue-specific genes, proteins, receptors and ion channels were found to be expressed in a developmentally controlled manner during in vitro differentiation. On the basis of these findings, we used mouse ES cells to analyse early developmental processes by in vitro ‘gain-of-function’ and ‘loss-of-function’ studies. Further, the ES cell technology has been established to be used in pharmacological and embryotoxicological studies to analyse the effects of drugs or environmental factors on differentiation and cell function. Mouse ES cells were employed to generate precursor cells and specialized cell types, such as dopaminergic neurons and pancreatic β-like cells. Transcriptome profiling will further help to identify unknown genes and signalling NEURAL STEM CELLS AND CELL DEATH S. Ceccatelli 1 , C. Tamm 1 , E. Sleeper 1 , S. Orrenius 1 , E.Y. Snyder 2 . 1 Division of Toxicology and Neurotoxicology, IEM, Karolinska Institute, Stockholm Sweden, 2 Neurology and Pediatrics, Harvard Med. School, Boston, MA, USA Neural stem cells (NSC) undergo apoptotic cell death as an essential component of neural development. Here we present the results of our studies on the mechanisms by which NSC undergo cell death in response to neurotoxic insults. As experimental models we used primary culture of adult NCS from the subventricular zone of the rat brain, and the neural stem cell line C17.2 initially derived from developing mouse cerebellum. NSC undergo apoptosis in response to staurosporine (0.25µM) as well as agents inducing oxidative stress such as 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). Exposed cells demonstrate an apoptotic morphology, positive TUNEL staining and phosphatidyl serine exposure as labeled with Annexin V. Using an antibody specific for cytochrome c, we found that cells exposed to staurosporine or DMNQ exhibited diffuse fluorescence throughout the cytosol, implying a release from the mitochondria. In addition to positive immunoreactivity against the active fragment (p17) of caspase-3, the administration of the pan-caspase inhibitor, z-VADfmk (40 µM), prevents apoptosis. Both NSC and C17.2 express the Fas receptor, and pro-caspase 8, but exposure to agonistic Fas mAb (250 ng/ml) fails to induce apoptosis. Pretreatment with cycloheximide or actinomycin D does not influence the cell response to Fas mAb, suggesting that the endogenous inhibitor of caspase 8 FLICE-inhibitory protein (FLIP) is not responsible for the inhibition of the Fas/FasL pathway. Thus, it appears that the cell death Fas dependent pathway is not operative in these cells, while the mitochondrial pathway is active and caspase-3 serves as an executioner caspase in the apoptotic machinery. It is known that Fas not only induces apoptosis, but can also deliver growth stimulatory signals through activation of the extracellular-signal regulated kinase (ERK) pathway. The Fas-induced ERK phosphorylation that we detect in C17.2 cells suggests that in NSC Fas may function as a mediator of growth rather than death. 32 S3 Stem cells in toxicology s11 SIDE EFFECTS IN GENE THERAPY WITH HEMATOPOIETIC CELLS C. Baum 1,2 , Z. Li 1 , U. Modlich 1 , J. Meyer 1 , B. Schiedlmeier 1 , H. Klump 1 , G. Beutel 1 , C. von Kalle 2 , B. Fehse 3 , J. Kraunus 3 , J. Bohne 1 . 1 Experimental Cell Therapy, Dept. of Hematology and Oncology, Hannover Medical School, Hannover, Germany; 2 Dept. of Experimental Hematology, Cincinnati Children’s Hospital, Cincinnati, Ohio, USA; 3 Bone Marrow Transplantation, University-Hospital Eppendorf, Hamburg, Germany Transgenes delivered by retroviral (including lentiviral) vectors are efficiently maintained in cells with high replicative potential. Significant progress achieved in vector design and transduction technologies has resulted in the correction of severe monogenic disorders in a few clinical trials and several animal models. However, recent observations suggest that further progress will be dependent on a systematic evaluation of side effects. These could originate either from the ectopic expression of the transgene (phenotoxicity) or from genetic damage induced by the gene transfer procedure (genotoxicity). We observed leukemias in mouse models for retroviral gene marking of hematopoietic cells. In one study, insertional activation of the Evi-1 proto-oncogene by the retroviral vector was identified as a key event. Circumstantial evidence suggested signal interference induced by the expression of a truncated cell-surface receptor (dLNGFR) as a collaborating factor. A number of experiments were initiated in the meantime to investigate whether activation of a single oncogene in combination with strong replication pressure may be sufficient to generate a leukemia (in mice), without a need for transgene phenotoxicity. Similar unfortunate cooperations may underlie the secondary leukemias s12 Workshop W2. Alteration of signal transduction in neurotoxicity observed in the first clinical trial performed to correct X-linked severe combined immunodeficiency. A related challenge is to adjust transgene expression levels within defined therapeutic windows. Using a retroviral vector expressing HOXB4 to high levels, human hematopoietic stem cells could be expanded in vivo (NOD/SCID mice). However, lymphoid and myeloid differentiation was disturbed depending on the dosage of transgenic HOXB4. Similar dose issues may apply to other signal molecules and metabolic markers proposed for stem cell selection and disease correction. Based on these findings, standards for preclinical evaluation of genetic interventions may have to be revised. Insights into the pathomechanisms of gene therapeutic side effects will have immediate practical consequences for technology optimization and also provide new concepts for crucial aspects of stem cell biology and oncogenesis. 33 THE USE OF STEM CELLS IN TOXICOLOGY TO SUPPORT DRUG DISCOVERY AND DEVELOPMENT A. Rossi 1 , M.A. Thiede 2 . Research and Development, Pharmacia Corporation, 1 Italy and 2 St. Louis, MO, USA The major concern during preclinical safety evaluation of drug candidates is to extrapolate data obtained in animal models to humans. Among the in vitro systems and assays that have been developed in the last years to help this appraisal, the in vitro primary culture from human tissues has played a major role. However, the use of primary culture of human cells involves a range of ethical, safety, legal and logistical issues not always easy to overcome, especially when healthy donor tissue is requested. The increasing knowledge of stem cell biology has created great opportunity in the preclinical setting to find a different source of healthy human cell types to help improve assessment of candidate drugs. Particular attention is given to the isolation and culturing of stem cells from adult tissues since this does not create ethical issues as opposed to embryonic stem cells. Recent studies show that adult stem cells isolated from bone marrow have a large differentiation potential and, depending on the culture conditions they are able to differentiate into different cell types, including neurons, endothelial cells, hepatocytes and cardiomyocytes. The aim of this presentation is to give an overview of the possible use of the adult stem cells as a predictive tool in drug safety evaluation and screening in humans. W2 Alteration of signal transduction in neurotoxicity 34 DIFFERENT MECHANISM OF BLOCKADE OF NEUROEXOCYTOSIS BY PRESYNAPTIC NEUROTOXINS O. Rossetto, M. Rigoni, P. Caccin, C. Montecucco. Dept. of Biomedical Sciences, University of Padova, Padova, Italy Nerve terminals are specific sites of action of a very large number of toxins produced by many different organisms. Two different groups of protein neurotoxins which interfere directly with the process of neurotransmitter release are the main topic of our experimental work: 1)the clostridial neurotoxins which act inside nerves and block neurotransmitter release via their metalloproteolytic activity directed specifically on SNARE proteins, and 2)the snake presynaptic neurotoxins with phospholipase A2 activity whose site of action is still undefined and which induce the release of acethylcholine followed by impairment of synaptic functions. Botulinum neurotoxins (BoNTs, serotype A-G) and tetanus neurotoxins (TeNT) are produced by toxigenic strains of anaerobic bacteria of genus Clostridium and they are responsible for the clinical syndrome of botulism and tetanus respectively. These neurotoxins are closely similar and are composed of two polypeptide chains joined by a single interchain disulphide bond. The L chains of the clostridial neurotoxins are zinc-dependent proteases very specific for three protein components of the neuroexocytosis apparatus (called SNARE proteins), whose cleavage results in a sustained blockade of the release of neurotransmitters at the synapse. Their modes of binding, sites of action and biochemical activities will be discussed in relation to the symptoms of the diseases they cause. Snake presynaptic neurotoxins with phospholipase A2 (PLA2) activity cause a persistent blockade of neurotransmitter release from nerve terminals. The intra-peritoneal or intra-muscular injection of these neurotoxins in small mammals leads to animal death by respiratory failure due to the muscle paralysis for induction of acetylcholine release not followed by synaptic vesicles recycling. The molecular mechanism of action of these neurotoxins is still unknown. In principle, the site of PLA2 activity could be external or internal to the nerve ending. However, the available data and the fact that lipid vesicles fuse among themselves upon incubation with PLA2, are in agreement with the possibility that these neurotoxins penetrate and act inside the nerve terminal. Recent data obtained in mouse phrenic nerve-hemidiaphragm and in primary neuronal cell cultures intoxicated with these toxins will be presented. 35 INFLAMMATORY AGENT-INDUCED ALTERATIONS IN NITRIC OXIDE/CYCLIC GMP SIGNALLING IN CNS CELLS Agustina García. Instituto de Biotecnología y Biomedicina ’V. Villar Palasí’ and Departamento de Bioquímica y Biología Molecular, Universidad Autónoma de Barcelona, 08193 Bellaterra, Spain During neuroinflammation associated with trauma, infection or neurodegenerative disorders, relatively high amounts of nitric oxide (NO) can be generated after induction of NO synthase type 2 (NOS-2) in glial cells. In these conditions, NO can be in part responsible for neuroinflammatory cell damage. Compared with the large amount of information about the regulation of NOS-2 expression by inflammatory agents in brain cells, very little is known about the regulation of the metabolism of cyclic GMP (cGMP), the physiological second messenger of NO. During the last years, we have obtained evidence that different compounds capable of inducing neuroinflammatory reactions dramatically alter cGMP metabolism in CNS cells. Using primary cultures enriched in rat brain astroglia we have demonstrated that long term treatment (> 3 h) with bacterial lipopolysaccharide (LPS), proinflammatory cytokines (IL-1β, TNFα) or β-amyloid peptides (βA) which are known to induce astroglial reactivity and NOS-2 expression, also cause a decrease in the activity of the NO receptor soluble guanylyl cyclase (sGC). This effect appears to occur by two mechanisms: a decrease in the half-life of sGC protein that is NO-independent but requires transcription and protein synthesis, and a NO-dependent decrease in sGC subunit mRNA. Exposure of cells to NO donors also leads to a decrease in sGC activity that at short times (< 2 h) is reversed by glutathion, indicating that is due to sGC thiol nitrosylation, but at longer (18–20 hours) involves down-regulation of sGC protein and mRNA, in agreement with the effect of endogenously formed NO. Reduction in sGC expression is also observed in rat brain after intracerebral administration of LPS, IL-1β or βA. On the other hand, the HIV-1 coat protein gp120, decreases NO-dependent cGMP accumulation in astroglial cells by increasing the activity of cGMP phosphodiesterase. In summary, our results indicate that regulatory mechanisms operate in astroglial cells to maintain the levels of cGMP low when exposed to agents that can induce a high NO output. This work was supported by SAF 2001-2540 and SGR 2001-212 grants. 36 CYTOKINES ROLE IN NEURODEGENERATIVE EVENTS B. Viviani. Laboratory of Toxicology, Department of Pharmacological Sciences, University of Milan, Milan, Italy During the past decade, the concepts about the development of neurodegenerative diseases have been completely revised mainly because of the recognition that most neurological disorders are the consequence of a complex relationship between glia and neurons. Following an insult to the CNS, glia becomes activated and releases cytokines, some of which are neurotoxic. This is well pointed out by the neurotoxicity of the HIV-1 envelope glycoprotein gp120 and of the neurotoxicant trimethyltin (TMT). Exposure of hippocampal Workshop W3. Are toxicologists communicating risk effectively? neurons in co-culture with glia to both gp120 and TMT results in an exacerbated neural death coupled with an increased production of IL-1β and TNF-α respectively. Treatment of the co-culture with an antibody against IL-1β during gp120 exposure or against TNF-α during TMT exposure prevented neural cell death. Neural survival was also promoted inhibiting cytokines production by pre-treatment of glial cell with antioxidants. These observations underlay the central role of IL-1β and TNF-α in sustaining neurodegeneration. IL-1 β per se does not induce neuronal death, nevertheless in vivo it exacerbates various pathophysiological conditions of the CNS involving N-methyl-D-aspartate (NMDA) receptor activation. We observed that IL-1β affects NMDA-receptor function(s) in hippocampal neurons, dose-dependently enhancing both NMDAinduced [Ca2 + ]i increases and NMDA-induced neuronal death. Increased tyrosine phosphorylation of NMDA receptor was also observed after exposure to this cytokines. IL-1β may thus exacerbate neuronal death by increasing NMDA receptor function through activation of tyrosine kinases and subsequent phosphorylation. These evidences identify cytokines as pathogenic mediators of the central nervous system. The knowledge of the molecular mechanisms involved in their action will be thus instrumental in providing new means of intervention in neuronal degeneration. 37 SIGNAL TRANSDUCTION MECHANISMS INVOLVED IN THE ANTIPROLIFERATIVE EFFECTS OF ETHANOL IN GLIAL CELLS Costa 1,2 , Guizzetti 1 , Vitalone 2 . 1 Toxicology L.G. M. A. Program, University of Washington, Seattle, WA, USA, 2 Dept. of Pharmacology and Human Physiology, University of Bari, Italy In utero exposure to ethanol is deleterious to brain development, and offspring of alcohol-abusing mothers often present a syndrome, Fetal Alcohol Syndrome, characterized by facial dysmorphology, growth retardation, and central nervous system dysfunctions. The latter, which include microencephaly and mental retardation, are of most concern, as they can be considered long lasting if not irreversible. While ethanol exerts toxic effects on developing neurons, evidence is also emerging which indicates that glial cells, in particular astrocytes, are also targeted by alcohol. In particular, ethanol has been shown to inhibit proliferation of astroglial cells stimulated by some, but not all mitogens; this effect has been suggested to be involved in ethanolinduced microencephaly. Activation of the M3 subtype of cholinergic muscarinic receptors causes proliferation of astroglial cells (rat and human fetal astrocytes and human astrocytoma cells) and this effect is inhibited by relevant ethanol concentrations (25–100 mM). The signal transduction cascade activated by M3 receptors in these cells and involved in mitogenesis includes phospholipases C and D, protein kinases C epsilon and zeta, phosphatidylinositol-3 kinase, MAPK, p70S6 kinase and NF-kB. Ethanol appears to primarily target the activity of phospholipase D, causing decreased formation of phosphatidic acid, decreased activation of PKC zeta, and those downstream of p7056 kinase and NF-kB. Other signaling pathways are affected only by higher (>100 mM) ethanol concentrations. Supported in party by AA-08154 and ES-07033. W3 Are toxicologists communicating risk effectively? 38 RISK COMMUNICATION - AN OVERVIEW William Leiss. FRSC Executive-in-Residence McLaughlin Centre for Population Health Risk Assessment, University of Ottawa 1 Stewart Street, Room 308 Ottawa, ON K1N 6N5 Canada Major public controversies over the management of health and environmental risks have been ongoing since the 1970s, starting with chemicals (pesticides and dioxins) and running through risks associated with many other industrial technologies. We can find in those controversies many common features, which cut across differences in both the technologies themselves and the types of risks they engender. This understanding also enables us to propose s13 strategies to organizations to help them better respond to the public’s needs (and the public interest) when concerns over risks arise. Effective risk communication practices are among the most important responsibilities for industry and governments in this regard. Since its origins in the late 1980s, risk communication practice has achieved a better understanding both of its goals and of how to achieve them. We are now in a position to specify with some precision what the fundamental requirements of good risk communication are, and they fall into three basic areas: (1) undertaking “science translation,” (2) addressing uncertainties, and (3) dealing with the science/policy interface. Within these three areas there are a set of ten specific tasks, representing what may be called the minimum essential content requirements for every effective risk communication effort. These tasks will be itemized, discussed, and illustrated in this presentation. There will also be a presentation of an internet-based public information resource dealing with the issue of endocrine disruptors (www.emcom.ca), which is designed to show how the principles of good risk communication may be operationalized. 39 RISK COMMUNICATION: BALANCING SCIENCE, POLICY MAKING AND PUBLIC PERCEPTION Ortwin Renn. Center for Technology Assessment in Baden Württemberg, Germany Health and environmental scientists, professional risk managers and the general public strongly disagree about the seriousness of many risks. Most members of the public are concerned about longterm effects of risks, equity and fairness issues, lack of personal control, and the pace of technological diffusion into their cultural environment, whereas professional toxicologists and risk managers focus on the task to minimize the probability of adverse effects caused by a potentially hazardous agent or activity. To bridge the gap between the professional mandate and the public perception of risk, two-way-communication has to be initiated between scientists, risk managers, interest groups, and representatives of the affected public. This dialogue should serve three major functions: 1. to facilitate understanding of different risk perspectives among scientists, regulators and stakeholders as well as groups of the public; 2. to enlighten all these constituencies about different rationales for dealing with toxicological risks; 3. to develop appropriate procedures for conflict resolution. A prerequisite for a successful communication is the willingness of each group to respect the perspective of all the other participating groups and to include their concerns into the decision making process. The conference paper reviews the literature on the three main functions of risk communication: message recognition, mutual understanding and respect as a prerequisite for trust building and resolution of risk-related conflicts. The paper discusses the structure of the communication process from a descriptive and a normative point of view and draws on empirical studies about risk perception and communication. The argument will be made that risk cannot be understood as a monolithic concept that penetrates different research disciplines and risk management fields. Risk should rather be seen as a mental instrument that allows prediction of future hazards and facilitates risk reduction measures. Due to the inherent ambiguity and uncertainty of conceptualizing risk, different concepts of risk compete with each other and rely on different rationales. The main goal of risk communication is therefore integration of different concepts of risks, in particular with respect to setting priorities in risk reduction and mitigation. The author will introduce a recent initiative by the OECd Chemical Risk Group to accomplish this goal. 40 THE PUBLIC AND EFFECTIVE RISK COMMUNICATION L. Frewer. Marketing and Consumer Behaviour Group, University of Wageningen, The Netherlands Public perceptions of risk have often been dismissed on the basis of “irrationality”, and have tended to be excluded from policy processes by risk assessors and managers. However, people’s responses to s14 Workshop W4. In vitro methods in toxicology different risks are determined by psychological factors. In contrast, the technical risk estimates traditionally provided by experts do not influence people’s behaviours and responses in the same way as their risk perceptions. For example, a risk that people perceive to be involuntary in terms of their personal exposure is more threatening than one that they choose to take, even if the probability of harm is the same, or even less. Other concerns are very specific to particular hazard domains (for example, consider risk – and related - perceptions associated with transgenic organisms or endocrine disruptors). It is also important to communicate the difference between probability and variability associated with risk estimates. Risk communication with the public about a risk must take account of the actual concerns of the public (for example, potential for negative environmental impact, unintended human health effects, or vulnerable groups within the population). When the public want information about a risk, they prefer a clear message regarding risks and associated uncertainties, including the nature and extent of disagreements between different experts. Furthermore, societal priorities for risk mitigation activities may not align with those identified by expert groups. Dismissing the former as irrelevant may result increased distrust in the motives of regulators and industry, with consequences for public confidence in regulatory activities linked to public protection. Awareness and understanding of public concerns is also the basis for the development of an effective risk communication strategy, as these concerns should be explicitly addressed as part of the communication process. Examples relevant to risk communication and toxicology will be provided. 41 COMMUNICATING THE RISK OF ACRYLAMIDE IN FOOD THE LESSONS LEARNED Leif Busk. National Food Administration, Uppsala, Sweden The presentation of data demonstrating increased levels of acrylamide in a variety of heated food commodities attracted great media interest in Sweden. This was followed by media coverage at a lower level in other European countries as well as in the United States. Consumers were probably confused by diverging message from different sources. The issue of acrylamide in food is complex. For example, risk characterisation is based on animal data with inherent problems to calculate risk levels for man. The mechanism of action needs further elucidation. The difference between doses that cause carcinogenic effect in animals and those that man is exposed to is lower than for any other single chemical carcinogen in food. Acrylamide is found in a variety of commodities, many regarded as staple foods. We have been exposed to acrylamide for a very long time. Hence, communication with consumers is very difficult since the issue involves both the more ¡normal¡ questions of validity of data in addition to difficult general scientific questions, e.g. the value of animal data versus human data. The issue is further complicated by the fact that a drastic reduction of the exposure would have a profound effect on life style and be extremely costly. This restricts the realistic available management options adding to the communication problem. In Sweden the issue became even more complicated. This was due to an initially hostile media reacting on the wordings in the invitation to the press conference where the data were presented. The workshop presentation will focus on the general problems of communicating complex issues as acrylamide in foods with special emphasis on what can be learned from this specific case. W4 In vitro methods in toxicology 42 THE NEW EU CHEMICALS POLICY G. Vollmer. European Chemical Bureau, Ispra, Italy The European Commission develops for the time being the drafts for a new chemicals regulation. It includes registration, evaluation and authorization of dangerous chemicals. This new legislation will not only give more burden to industry and authorities. It will increase the evaluation of use and exposure. It will also give an enormous importance to alternative tests, which will replace the previous ones. These alternative tests will have to be carried out for some 20 000 chemicals. During the speech, the foreseen alternative methods, the request for further validation, the timetable for the implementation and the impact on industry will be outlined 43 IN VITRO METHODS IN TOXICOLOGY: SKIN IRRITATION P.A. Botham. Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, UK Several alternative in vitro methods for identifying skin irritants have been developed in the last 10 years, the most promising of which use either reconstituted human skin models or animal (e.g. pig or mouse) skin organ cultures. In 1998, the European Centre for the Validation of Alternative Methods (ECVAM) commissioned a pre-validation study of five methods which had successfully met a pre-set challenge of performance using 10 specified reference chemicals and had clearly defined protocols and prediction models. Two reconstituted human skin methods (Epiderm and EPISKIN) and one animal skin model (the mouse skin integrity function test, SIFT) performed well in Phases I and II of the study (intralaboratory reproducibility and protocol transfer) and proceeded to Phase III which assessed reproducibility and predictive ability in three independent laboratories using a set of 20 coded test chemicals (10 irritants and 10 non irritants). Intralaboratory reproducibility was again acceptable for all three methods but inter-laboratory reproducibility was acceptable only for EPISKIN. The predictive ability of all three methods was also inadequate (between 55–58% overall, with Epiderm and SIFT giving particularly high under-prediction rates and EPISKIN a high over-prediction rate). Subsequently, refinements were made to both the Epiderm and EPISKIN methods to improve their predictive ability. This work also showed that it was possible to establish a common protocol for the two methods which gave acceptable under and over-prediction rates. Modification of the statistical analysis of data from the SIFT model similarly improved its predictive ability. In 2003, ECVAM concluded that all three methods could proceed to a full validation study. This will be conducted in two phases and is scheduled for completion by the end of 2004. 44 APPLICATION OF IN SILICO TOOLS TO HUMAN HEALTH SAFETY ASSESSMENTS Joanna S. Jaworska. Procter & Gamble, Eurocor, Central Product Safety Brussels, Belgium There is a renewed interest in (Quantitative) Structure Activity Relationships ((Q)SARs) owing to various societal and political pressures such as the drive to reduce animal testing and the proposed changes in EU chemical management regulations. Applications of (Q)SARs that are considered range from decision support in early phases of product development to regulatory decision making such as in risk assessment or classification. (Q)SARs are simplified (mathematical) representations of complex chemical-biological interactions and consequently (Q)SAR predictions are potentially more uncertain than the underlying test data. This imposes limitations on the acceptable use of (Q)SAR in chemical management and decision-making. Approaches to determine the acceptability of (Q)SAR predictions have been developed in the past, but because of their breadth and generality they have not been widely applied or respected by either (Q)SAR users or developers. As a consequence, decision making on the basis of existing models must be done with care and is subject to expert opinion as there is currently no framework for QSAR use and following it lack of confidence in these predictions. In general, (Q)SARs for environmental endpoints are founded on relatively larger quantitative databases with some mechanistic understanding, whereas the ability to predict local and systemic effects in humans is compromised by a lack of data and a limited understanding of the underlying mechanisms. To date, for human health endpoints, the models are often poor because the endpoints are expressed through many different mechanisms, are receptor mediated, are multi-stage processes comprising adsorption, distribution, metabolism and excretion (ADME) and are site specific. For certain human health endpoints, biological data are also not fully Symposium S4. Risk assessment in food: general principles representative of the hazards to humans (e.g. Draize data). This currently imposes severe limitations on the successful development of (Q)SARs suitable for non-congeneric sets of compounds. Hence, developments in the human health area are focused on sharing of data and expansion of databases with emphasis on data quality evaluation. To aid these developments it will be necessary to overcome the barriers to the sharing of proprietary information. 45 VALIDATION OF ALTERNATIVE METHODS FOR DEVELOPMENTAL TOXICITY TESTING A.H. Piersma. Department of Reproductive Toxicology, Laboratory for Toxicology, Pathology and Genetics, National Institute for Public Health and the Environment RIVM, Bilthoven, The Netherlands Developmental toxicity testing according to current international guidelines involves exposure of pregnant animals, mostly rats and rabbits, and subsequent assessment of toxic effects in their fetuses. As the offspring in the late fetal stages are counted as experimental animals, these tests require relatively high numbers of animals. This situation urges research into alternative methods for developmental toxicity testing which reduce the number of animals used. Alternative methods have been developed since the early nineteeneighties. They include cell differentiation assays using either primary cell cultures or immortailized cell lines. At a higher integration level the development of organ anlagen in vitro has been employed in assays for developmental toxicity. The most complex assays in this area make use of isolated postimplantation rodent embryos which are cultured in vitro during the phase of major organogenesis. All these in vitro culture systems have proven their usefulness in studies of mechanisms of embryonic development. The possibilities for their application as toxicity screens have been investigated in various validation studies. The most elaborate validation study of embryotoxicity assays carried out to date was sponsored by ECVAM. The study included the embryonic stem cell test EST, the limb bud micromass MM, and whole embryo culture WEC. Twenty chemical compounds were carefully selected on the basis of existing in vivo developmental toxicity data and designated as none, weak or strong embryotoxicants. Chemicals were then tested in four independent laboratories. Methods were standardized between laboratories before testing. The results have shown a marked success in the reproducibility of results between laboratories. The predictivity of the test systems was determined on the basis of predefined prediction models according to the validation paradigm defined by ECVAM. Predictivity was generally good, with WEC gaining the most favourable outcome. The extrapolation of these results of a limited set of chemicals to a generalized judgment of the tests for the world of chemicals remains a matter for further discussion. The addition of metabolizing systems would increase the applicability of the tests. The results of the validation study have provided invaluable data for further scrutiny. S4 Risk assessment in food: general principles 46 HAZARD IDENTIFICATION AND CHARACTERIZATION Corrado L. Galli. Research Center on Risk Assessment, University of Milan, Milan, Italy Hazard identification for a substance detected in food involves assessment of its potential to cause adverse effects and the determination of the nature of those effects. Hazard identification also has to address the relevance of the effect to humans, and the nature, incidence and severity of the adverse effect. In the subsequent hazard characterization phase, the most sensitive and relevant adverse effect, i.e. that occurring at the lowest dose relevant to route and duration of exposure, is selected as the critical effect. Hazard may be identified from animal-based toxicology, in vitro toxicology and human observations, as well as inferred by structure-activity considerations. Animal models continue to be the main system for hazard identification of low molecular weight food chemicals The advantages of in vivo studies in animals are that a whole organism, s15 with its inter-related metabolic functions and organ system intact, is the most appropriate model for humans. Repeated dose toxicity tests, conducted in more than one species, are the core studies. It is advisable in such studies to administer relatively high doses in order to mimic the human exposure; the doses should not be exaggerated compared with human exposure to avoid artefacts. Even if, at present, in vitro tests have limited utility in predicting long-term hazard, their potential utility to obtain mechanism-derived information in order to assess the relevance to humans of observations made during the in vivo studies in animals is well recognised. If suitable information on adverse effects in humans is available, such data should take precedence over extrapolated animal data. Thus, uncertainties related to extrapolation from animal studies and from high dose to low dose are eliminated. Besides, the chemical structure and the presence of specific chemical groups may provide a structural alert to the possibility of a particular hazard. Hazard characterization has to address two main points. (a) qualitative considerations of the relevance of the health endpoints for humans and (b) quantitative considerations of the dose-response relationship and its application to the human population. Similarities and differences between animals and humans in the fate of the chemical (toxicokinetics) and in toxicodynamics, variability of the fate of the chemical in humans, mode of action and dose-response relationship for the critical effect(s) are basic points for hazard characterization. 47 RISK CHARACTERISATION A.G. Renwick. Clinical Pharmacology Group, School of Medicine, University of Southampton, Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, UK Information on hazard identification, hazard characterisation (including dose-response assessment) and exposure assessment are brought together under risk characterisation in order to provide advice to risk managers. The aims of hazard identification and characterisation are to define the adverse effects (hazards) which the substance is capable of producing, the dose-response relationship for each hazard, and the adverse effect of greatest concern (usually that occurring at the lowest exposures). Inter-species differences and human variability in sensitivity are taken into account under hazard characterisation, the output of which may be a health based guidance value, such as an acceptable daily intake (ADI) for threshold effects, or an estimate of the intake associated with a predefined level of risk for non-threshold effects. Exposure assessment aims to define average and high intakes, and has to take into account different dietary patterns, for example children. The risk characterisation, may relate to an existing exposure, for example a contaminant, or to a future exposure, for example an application for approval of a food additive or pesticide. These different exposure scenarios may affect the nature of the available data, and also the time available to resolve any database deficiencies. Consistency needs to be considered when hazard and exposure data are brought together under risk characterisation, for example the hazard characterisation data should relate to those life-stages with the highest exposures. The duration of exposure is important in risk characterisation because hazard characterisation related to life-time risk, such as the ADI, may not be relevant to short-term exposure, such as following occasional exposure to a pesticide residue. Risk characterisation has to be an iterative process in which information on hazard and exposure are matched and any discrepancy taken into account, where necessary by the generation of additional data. 48 EXPOSURE ASSESSMENT – OUTCOME OF THE EC CONCERTED ACTION ON FOOD SAFETY IN EUROPE (FOSIE) Detlef Müller 1 , Juliane Kleiner 2 , Robert Kroes 3 . 1 Procter and Gamble, Germany; 2 ILSI Europe, Brussels, Belgium; 3 Utrecht University, Institute for Risk Assessment Sciences, Utrecht, The Netherlands Exposure assessment is one of the key parts of the risk assessment process. The European Commission funded via its fifth Framework s16 Workshop W5. Safety evaluation of flavourings programme a project on Food Safety in Europe (FOSIE): Risk Assessment of Chemicals in Food and Diet. One expert group within this project carried out a thorough and comprehensive investigation on the assessment of intake from the diet. In the case of chemicals in food this is based on three major aspects: (i) how to determine quantitatively the presence of a chemical in individual foods and diets including its fate during the processes within the food production chain; (ii) how to determine the consumption patterns of the individual foods containing the relevant chemicals and (iii) how to investigate both the likelihood of consumers eating large amounts of the given foods and of the relevant chemical being present in these foods at high levels. A critical review of the techniques used for the evaluation of these three aspects has been undertaken to determine those areas where the current approaches provide a solid basis for assessments and those areas where improvements are needed. In all three areas, the limitations of the approaches currently used lead to uncertainties, which can either cause an over- or underestimation of real intakes and thus risks. The expert group recommends using worst-case scenarios by applying conservative screening methods as a first step. In cases where these relatively crude tools predict a toxicologically significant exposure more sophisticated methods are needed to provide more accurate intake estimates. W5 Safety evaluation of flavourings 49 THE SAFETY EVALUTATION OF FLAVOURS: CHALLENGES AND APPROACHES Robert L. Smith. Biological Chemistry & Molecular Toxicology, Imperial College London, United Kingdom, SW7 2AZ There is nowadays general agreement that the conventional toxicological approaches are inappropriate for the safety evaluation of food flavour materials. The reasons for this are: (a) the large number of flavour substances in commercial use; massive resources would be needed should the evaluations be conducted using conventional procedures (b) the use-levels of many flavour substances amount to a few kg/year and there is inadequate economic base to support conventional assessment (c) human levels of exposure through the food are generally very low and self-limiting (d) there is a history of exposure to many naturally occurring flavour materials through food consumption (e) the majority of flavours belong to simple chemical classes devoid of ’structural alerts for toxicity’ although there are a few exceptions to this. With these facts in mind, several governmental (e.g. U.S.FDA, Europeans S.C.F. and JECFA) and non-governmental (FEMA Expert Panel) agencies have explored and developed non-traditional paradigms for the safety evaluation of food flavours. There is now a high degree of commonality in the various approaches but there are also some differences. These approaches take advantage of the fact that the majority of flavours belong to well-defined chemical groups where there exists a reasonable homology in terms of toxicity and metabolic fate. The goal of this symposium is to explore some of these differences and, in particular, to review the different ways at evaluating exposure and also to consider the applicability of some more recently derived concepts for assessing flavour safety. The key questions to be addressed are as follows: Assessment of exposure to flavours: What are the most appropriate methodologies; are there sub-groups of exposed people that require special attention? Thresholds and their utility: Are there robust thresholds for toxicity/carcinogenicity/saturation of metabolic pathways and metabolic switching and can these be used for safety assessment purposes? How can the safety of natural flavour complexes be evaluated: There are about 400 of these materials in current use as flavours; is there a need for a new paradigm for these materials? 1. Assessment of exposure to flavouring agents and natural flavour mixtures 50 NOVEL ESTIMATES OF THE EXPOSURE TO FLAVOURING SUBSTANCES P. Cadby. Department of Product Safety and Regulatory Affairs, Firmenich SA, Geneva, Switzerland There are thousands of flavouring substances and hundreds of ways of consuming them. It is consequently impossible to carry out detailed analysis of the consumption patterns of each one, so as a result, it is necessary to find more practical and conservative methods for assessing exposure. Two studies have compared the suitability of one method: the Maximised Survey-derived Daily Intake (MSDI). In one of these studies, the MSDI-estimated intakes of 9 flavouring substances and one spice oleoresin were compared with detailed dietary intakes calculated over a 14 day period using frequency of eating, portion sizes, levels of the flavouring substance and probability of presence in a particularly food item. In a second study, the MSDI-estimated intakes of 12 flavouring substances were compared with intakes calculated using a stochastic model. This model used real levels in over 40,000 flavour formulae used in 31 different categories of food for which the intakes of males in the 16–24 year age group had been surveyed. In both of these studies, the ability of the MSDI to accurately but conservatively estimate upper percentile intakes was demonstrated. This stochastic method offers an opportunity to test whether the MSDI can also be used to estimate the intakes of the volatile constituents that are common to a number of different botanically-derived flavouring materials and food ingredients. 51 SAFETY EVALUATION OF NATURAL FLAVOUR COMPLEXES V.J. Feron 1 , T.B. Adams 2 , S. Cohen 3 , J. Doull 4 , J.I. Goodman 5 , R.L. Hall 6 , L.J. Marnett 7 , I.C. Munro 8 , P.S. Portoghese 9 , R.L. Smith 10 , W.J. Waddell 11 , B.M. Wagner 12 . 1 TNO Nutrition and Food Research, Zeist, The Netherlands, 2 Scientific Secretary of the FEMA Panel Flavor & Extract Manufacturers Association, Washington, DC, USA, 3 Nebraska Medical Center, University of Nebraska, Omaha, NB, USA, 4 Dept. of Pharmacology and Toxicology, University of Kansas Medical Center, Kansas City, KS, USA, 5 Dept. of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA, 6 Towson, MD, USA, 7 Dept. of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA,8 CanTox Health Sciences International, Mississauga, ON, Canada, 9 Dept. of Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA, 10 Div. of Biomedical Sciences, Section of Molecular Toxicology, Imperial College School of Medicine, South Kensington, London, UK, 11 Dept. of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA, 12 New York University School of Medicine, New York, B.M. Wagner Associates, Milburn, NJ, USA Natural flavour complexes (NFCs) are chemical mixtures obtained by applying physical separation methods to botanical sources such as pulp, peel, vegetables and spices. Many of the approximately 300 NFCs have a food origin. To date no scheme that allows the safety evaluation of NFCs has been developed by any agency, governmental or non-governmental. In this presentation, such a scheme, “The Naturals Paradigm”, will be discussed. The scheme is intended only for the safety evaluation of NFCs derived from higher plants to be used as flavouring substances for food and beverages. The scheme begins with a review of data on the history of dietary use of the NFC, and describes its chemical composition to ensure that the commercial product conforms to the composition limits that define the product. Next the paradigm assigns each known constituent to one of three structural classes of toxic potential, each class with its own exposure threshold of toxicological concern. Moreover, each constituent is assigned to one of some 30 congeneric groups. In subsequent steps, for each congeneric group Symposium S5. Molecular epidemiology in occupational toxicology the method determines the daily per capita intake, considers the detoxification pathways and explores the availability of toxicological data. Toxicity testing may be needed. The paradigm also addresses constituents of unknown structure. As a conservative default assumption, the unknowns are placed in the structural class of greatest toxic potential and, thus, their total intake is compared to the most conservative threshold of toxicological concern. Further analytical data or toxicity tests may be needed. Finally, the paradigm considers the possibility of additive or synergistic interactive effects among congeneric groups. With the developed strategy, the overall objective of the paradigm can be attained: that no reasonably significant risk associated with the intake of NFCs goes unevaluated. 2. Thresholds in safety evaluation of flavouring agents 52 Since 1996 the FAO/WHO Joint Expert Committee on Food Additives (JECFA) has evaluated the safety of over 1100 flavouring substances, based on a decision tree related to chemical structure and intake. Safety conclusions are based no whether the estimated intake is above or below a threshold of toxicological concern that is relevant to that compound. The decision tree includes toxicity thresholds for the three structural classes determined by the Cramer et al decision tree. A review by Munro et al of the available data on compounds in the three structural classes defined the 5th percentile of the NOAEL values from chronic and sub-chronic studies. These values were divided by the normal 100-fold uncertainty factor to derive a threshold of toxicological concern for each structural class. The daily intake thresholds were 1800 micrograms for Class I (structures of low predicted toxicity), 540 micrograms for Class II (structures that cannot be assumed to be of low toxicity) and 90 micrograms for Class III (structural features indicative of high toxicity). If the substance is predicted to be metabolised to innocuous products there is no safety concern if the intake is below the threshold, but suitable toxicity data on the compound or structural analogues are required if the intake exceeds the threshold. If the substance is not predicted to be metabolised to innocuous products, and the intake is below the appropriate threshold, safety evaluation is based on data on the compound or structural analogues, but adequate toxicity data are required on the compound if the intake exceeds the threshold. An additional threshold of 1.5 micrograms per day, which was derived from an analysis of the estimated 1 in 1,000,000 risk for chemicals in the cancer potency database, is applied for compounds for which appropriate toxicity data are not available. 53 (Generally Recognized As Safe) and structurally related compounds have been reported to be carcinogenic in rodent studies. Four of these flavors had an increasing response at two doses; three had increasing responses at 3 doses; one had increasing responses at four doses. The three compounds with three doses fit this plot with a correlation coefficient of at least 0.9; the four doses of methyleugenol fit with a correlation coefficient of 0.999983. The intercept at zero percentage tumors of these was at least several orders of magnitude greater than the estimated daily dose of these flavoring agents to individuals in the United States. This is interpreted to indicate that these flavoring agents have a clear threshold for carcinogenicity in animals that is well above the levels currently approved for use in foods; consequently, these animal studies should not be a cause for concern for carcinogenicity of these compounds in humans. Rather, the animal studies should be viewed as providing evidence for the safety of these compounds at current levels of human exposure. 54 TOXICOLOGY DATABASES AND THE TOXICITY THRESHOLD CONCEPT USED BY JECFA A.G. Renwick. Clinical Pharmacology Group, School of Medicine, University of Southampton, Biomedical Sciences Building, Bassett Crescent East, Southampton SO16 7PX, UK s17 THE IMPACT OF DOSE-DEPENDENT METABOLIC SWITCHING UPON TOXICITY: THE ALLYLBENZENE FLAVOURS METHYLEUGENOL AND ESTRAGOLE AS CASE EXAMPLES J. Caldwell. Faculty of Medicine, University of Liverpool, Liverpool L69 3GA, United Kingdom The allylbenzenes are a important group of natural food flavours, present in a large number of widely consumed foods and beverages. Estragole and methyleugenol, the two most important congeners, are hepatocarcinogenic to rodents and we have employed a mechanism-based approach in which production of the hepatotoxic and hepatocarcinogenic sulfate conjugate of the 1’-hydroxy metabolite is used to interpret the pathological changes observed in laboratory rodents. Both the qualitative and quantitative aspects of the molecular disposition of methyleugenol and estragole and their associated toxicological sequelae have been relatively well defined from mammalian studies. Their profiles of metabolism, metabolic activation, and covalent binding are clearly dose dependent. The relative importance of metabolic activation and associated covalent binding to hepatic protein and DNA is markedly less at low levels of exposure (i.e. these events are not linear with respect to dose). In particular, rodent studies suggest that these events are negligible in the dose range 1–10 mg/kg body weight, approximately 100–1000 times the anticipated human exposure to these substances. This dose range contrasts markedly with the 37.5 – 150 mg/kg/day doses of methyleugenol used in the recent NTP study. For these reasons, it is concluded that present levels of dietary exposure to methyleugenol and estragole resulting from consumption of food, mainly in spices or added as such, does not pose a significant cancer risk. Nevertheless, further studies are needed to define both the nature and implications of the dose-response curve at low levels of exposure to methyleugenol and estragole. S5 Molecular epidemiology in occupational toxicology ANALYSIS OF THRESHOLDS FOR CARCINOGENICITY William J. Waddell. Department of Pharmacology and Toxicology, University of Louisville, Louisville, Kentucky, USA Extrapolation from studies of chemical carcinogenicity in rodents at high doses to humans at the typically low doses to which we are exposed has been one of the most controversial issues in toxicology. Current approaches usually assume that there is zero tumor production only at zero dose and doses are evaluated on a linear scale. Re-evaluations of large prominent studies, e.g., the ED01 study, N-nitrosodiethylamine, unequivocally demonstrate thresholds for carcinogenicity when the dose-response curves for animal studies done at high doses are calculated according to fundamental principles of chemistry (Waddell and Bates, 1969; Rozman, et al. 1996). This requires dose to be on a logarithmic scale and percent tumors on a linear scale. Fifteen compounds approved by the FEMA (Flavor and Extract Manufacturers Association) expert panel as GRAS 55 EVALUATION OF OCCUPATIONAL EXPOSURE TO CARCINOGENS BY MOLECULAR EPIDEMIOLOGY METHODS R.J. Sram, B. Binkova, O. Beskid, P. Rossner, I. Chvatalova, E. Biros, A. Milcova, Z. Stavkova, Z. Smerhovsky. Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Prague, Czech Republic Molecular epidemiology is a new and evolving area of research combining laboratory measurements of internal dose, biologically effective dose, biologic effects, and influence of individual susceptibility with epidemiologic methodologies. City policemen represent a group occupationally exposed to increased concentrations of carcinogenic polycyclic aromatic hydrocarbons (carcPAHs). Personal exposure to carcPAHs was evaluated by personal samplers during working shift prior to collection of biological samples. During the sampling s18 Symposium S5. Molecular epidemiology in occupational toxicology period the personal exposure to carcPAHs was 12.0±11.1 ng/m3 and 6.2±3.5 ng/m3 , for exposed and control groups, respectively. The use of different biomarkers of exposure, effects and susceptibility for this type of occupational exposure was assessed. As sensitive biomarkers of exposure were evaluated metabolite 1-OH-pyrene in urine, DNA adducts by 32 P-postlabeling, p53 and p21WAF proteins, as biomarkers of effects chromosomal aberrations by conventional method and fluorescence in situ hybridization (FISH), as biomarkers of susceptibility polymorphisms of metabolic genotypes (CYP1A1-MspI, CYP1A1-Ile/Val, GSTM1, GSTP1, GSTT1, EPHX, MS, MTHFR) and DNA repair genotypes (hOOG1, XPD, XRCC1). Using FISH technique and probes for chromosomes 1 and 4 (Cambio, UK) the genomic frequency of translocations calculated as FG /100 was 1.72 and 1.24 for exposed and control groups (P<0.05), respectively. FISH assay data were associated with GSTP1, EPHX, MTHFR, hOGG1 and XPD polymorphisms. Comparing these results with our studies on occupational exposure to acrylonitrile, 1,3-butadiene and ethylbenzene in petrochemical industry, the FISH method seems to be more sensitive to determine chromosomal aberrations by the occupational exposure to carcinogens than conventional method. The potential for using molecular epidemiology data for assessing occupational exposure to carcinogens will be discussed. Supported by the grant of Czech Ministry of Environment VaV/340/2/00, and by EC grant QLK4-CT-2000–00091. 56 MONITORING OF EXPOSURE TO 1,3-BUTADIENE BY MARKERS OF DOSE, EFFECT AND SUSCEPTIBILITY S. Fustinoni 1 , L. Perbellini 2 , L. Soleo 3 , M. Manno 4 , V. Foà 1 . 1 Department of Occupational and Environmental Health, University of Milan and ICP, Milan, Italy, 2 Department of Medicine and Public Health, University of Verona, Verona, Italy, 3 Department of Internal Medicine and Public Medicine, University of Bari, Bari, Italy, 4 Department of Preventive Medical Sciences, University of Napoli Federico II, Napoli, Italy In the recent years molecular epidemiology in occupational toxicology has become an important tool in the understanding mechanism of action of dangerous substances, particularly carcinogens, and in the identification of biomarkers to be used in prevention protocols. In the present study exposure to 1,3-butadiene (BD), a chemical classified as a probable carcinogen to humans, has been investigated. BD exposure may occur in occupational settings where this monomer is produced and/or polymerized and in life environment, being BD an ubiquitous pollutant with cigarette smoke and automobile exhausts as major sources. Occupationally exposed subjects working in a polymer plant (N=42), internal non-occupationally exposed controls (N=43) and external controls (N=88) were investigated. BD exposure and effect were assessed by measuring personal exposure to BD, BD in exhaled air, blood, urine, urinary N-acetyl-S-(3,4-hydroxybutyl)-L-cysteine (MI), sister chromatid exchange (SCE) and chromosomal aberration (CA) frequency in peripheral blood lymphocytes. Polymorphism of genes involved in BD metabolism, e.g. CYP2E1, 2A6 and 1A2, GSTM1, T1 and P1 was also determined. Preliminary results showed that BD personal exposure in the investigated subjects was very low (median 1.5, 0.4 and 0.2 µg/m3 in exposed, internal and external controls, respectively), but significantly higher in the exposed compared to unexposed subjects. Blood and urinary BD showed a similar pattern (3.7, <2.6 and <2.6 ng/L, p<0.05, for blood BD and 2.4, <1.0 and <1.0, p<0.05, for urinary BD), while CA and SCE were not influenced by exposure categories. Cigarette smoke had a great impact on BD biomarkers: an increase of BD up to 10 folds was found in exhaled air, blood and urine of smokers compared to non-smokers; a significant increase in the frequency of SCE was also found. A multiple linear regression analysis correlated BD in exhaled air, in blood or in urine with personal exposure and cigarette smoking (0.72≤R≤0.77). The genetic polymorphism of the metabolic enzymes did not show any influence on the biomarkers investigated so far. In conclusion, in the present study the occupational exposure to BD was extremely limited: at this exposure level the influence of smoking habit on BD body burden significantly overcame the effect of occupational exposure. 57 ARYL-HYDROCARBON RECEPTOR-DEPENDENT PATHWAY AND TOXIC EFFECTS OF TCDD IN HUMANS: A POPULATION-BASED STUDY IN SEVESO, ITALY Andrea Baccarelli. Genetic Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Bethesda, USA and Department of Occupational and Environmental Health, University of Milan, Milan, Italy Approximately 20 years after the Seveso, Italy accident we conducted a population-based study to evaluate the impact of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) exposure on cancer using mechanistically-based biomarkers of dioxin response in humans. TCDD toxic effects are mediated by the aryl hydrocarbon receptor (AhR). We studied the AhR-dependent pathway in lymphocytes from 62 subjects randomly sampled from the highest exposed Zones and 59 subjects from the surrounding non-contaminated area, frequency matched for age, gender, and smoking. To our knowledge, this is the most comprehensive investigation to date designed to evaluate the key genes in the pathway, including AhR, aryl hydrocarbon receptor nuclear translocator (ARNT), CYP1A1, and CYP1B1 transcripts, and CYP1A1-associated 7-ethoxyresorufin O-deethylase (EROD) activity in a population heavily exposed to dioxin. Current lipid-adjusted plasma TCDD concentrations in these subjects ranged from 3.5 to 90 ng/kg (or ppt) and were negatively associated with AhR mRNA in unstimulated peripheral blood mononuclear cells (p=0.03). When mitogen-induced lymphocytes were cultured with 10nM TCDD, all AhR-dependent genes were induced 1.2 to 13-fold. In these cells, plasma TCDD was associated with decreased EROD activity. In addition, there was a strong positive correlation between AhR and CYP1A1 expression (p=0.001) and between AhR and CYP1B1 expression (p=0.006). CYP1A1 expression was also strongly correlated with EROD activity (p=0.001). Four CYP1B1 polymorphisms and related haplotypes were studied. Both CYP1B1 genotypes and haplotypes significantly affected constitutive and induced CYP1B1 expression. CYP1A1, GSTM1, and GSTT1 genotypes were not significantly associated with CYP1A1 expression and EROD activity. The analysis of the expression of dioxin-inducible genes involved in carcinogenesis may help in determining dose-response relationships for human exposure to dioxin in vivo and in assessing the variability of human response, which may indicate the presence of subjects more susceptible to disease as a result of such exposures. 58 APPLICATION OF OLD AND NEW BIOTECHNOLOGY TO CROSS-SECTIONAL STUDIES OF WORKERS EXPOSED TO KNOWN OR SUSPECTED CARCINOGENS Nathaniel Rothman. Occupational and Environmental Epidemiology Branch, Div. of Cancer Epidemiology and Genetics, NCI, NIH, Dept. of Health and Human Services, Bethesda, MD 20817, USA There are continuing questions about the carcinogenic potential of numerous chemical agents encountered in the workplace. Although there are increasingly sophisticated approaches available to screen agents of concern using in vitro and animal studies, there will always be questions about the generalizability of such results to exposed human populations. The classic epidemiologic approach. which rests primarily on cohort studies and in some instances case-control studies, can identify cancer risk only after it has occurred. Also, these study designs have some limitations with regard to access to working populations and sample size considerations (for cohort studies) and the quality of exposure assessment (for both cohort and case-control studies). The judicious use of older technologies and the availability of new approaches to assess intermediate biologic endpoints in healthy workers exposed to chemicals of concern can lead to a better understanding of the carcinogenic potential of compounds before they have caused disease. At the same time, the need to determine the relationship between these early intermediate endpoints and subsequent cancer risk remains critical. The unprecedented availability of prospective cohort studies with stored biologic samples will provide new opportunities to study these relationships. ,Evaluation of exposure-intermediate endpoint relationships in cross-sectional studies, and the exploration of intermediate endpoint-cancer relationships in cohort studies, could rapidly provide new insights into the carcinogenic potential of many workplace exposures. Symposium S6. Apoptosis and cell regulation S6 Apoptosis and cell regulation 59 MITOCHONDRIAL REGULATION OF APOPTOTIC CELL DEATH S. Orrenius. Karolinska Institutet, Stockholm, Sweden Although it has long been known that impairment of mitochondrial function may lead to ATP depletion and necrotic cell death, recent work has revealed that these organelles also play an important role in the overall regulation of apoptotic cell death by mechanisms which have been conserved through evolution. Thus, it seems that a number of death triggers target the mitochondria and stimulate their release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and other parts of the apoptotic process. Cytochrome c release is governed by the Bcl-2 family of proteins, whereas subsequent caspase activation is modulated by other proteins, including inhibitor of apoptosis proteins (IAPs) and heat shock proteins. Recent findings indicate that cytochrome c extrusion occurs by a two-step process, which is initiated by a disruption of the association of this protein with cardiolipin, the phospholipid that anchors it to the outer surface of the inner mitochondrial membrane. Release of the solubilized pool of cytochrome c into the cytosol may then occur by pore formation mediated by pro-apoptotic Bcl-2 family proteins, notably Bax and Bak, or Ca2 + -triggered mitochondrial permeability transition. Recent evidence suggests that cytochrome c release during apoptosis may in fact involve a combination of these two mechanisms. Taken together, these findings have placed the mitochondria in the focus of apoptosis research and further underlined the important function of these organelles in cell life and death. 60 ROLE OF APOPTOSIS IN LIVER TUMOR PROMOTION W. Bursch 1 , B. Grasl-Kraupp 1 , U. Wastl 1 , M. Chabicovsky 2 , W. Parzefall 1 , R. Schulte-Hermann 1 . 1 Institute of Cancer Research University of Vienna, Borschkegasse 8a, A-1090 VIENNA; 2 Igeneon Immunotherapy of Cancer AG, Brunner Strasse 69, A-1230 Vienna, Austria Cancer risk assessment of chemical substances is largely based upon life-time bioassays with mice and rats. Among the species routinely used for this purpose, some mouse strains (B6C3F1, C3H/He) exhibit a high, others (C57BL/6J) a low (“resistent”) susceptibility to spontaneous and chemically induced hepatocarcinogenesis. As apoptosis may serve as defense mechanism against cancer development we investigated whether failure of apoptosis might be concurrently causative for the high cancer susceptibility in B6C3F1 mice. First, in a series of short-term in vivo experiments (7–14 days), mouse liver growth (C3H/He, C57BL/6J, B6C3F1, NMRI) was induced by administration of phenobarbital (PB; 750 ppm via the food) or nafenopin (NAF; 500 ppm via the food), cessation of PB and NAF treatment served to initiate liver involution. Liver weight, DNA and protein content, DNA synthesis, mitotic rates, hepatocyte ploidy, apoptotic activity were studied as endpoints. Secondly, in a long-term study liver carcinogenesis was initiated by a single dose of N-nitrosodiethylamine (NDEA, 90 mg/kg b.w.) to 5 weeks old C57Bl/6J, C3H/He and B6C3F1 mice. After two weeks, mice received either standard diet or a diet containing phenobarbital (PB, 90 mg/kg b.w) for 20, 40, 52, 76 and 92 weeks. Quantitative histological analysis of cell proliferation and apoptosis in normal liver tissue and (pre)neoplastic tissue was performed on H&E stained liver sections. Our short term studies revealed that PB/NAF-induced mouse liver growth essentially is due to cell enlargement (hypertrophy). A moderate DNA increase exclusively was brought about by increased nuclear and cellular ploidy. Furthermore, in all strains of mice under study liver regression upon cessation of PB/NAF treatment was not associated with a significant increase in histologically detectable apoptoses; this observation was confirmed by biochemical analysis of liver DNA content. Taken together, no difference among these mouse strains with respect to the occurrence of apoptosis was detected. The long-term study confirmed and extended previous findings by others: PB exhibited a tumor promoting effect in all strains s19 of mice. Remarkably, C57BL/6J mice often labelled “resistant” to cancer formation demonstrated a quantitative rather than a qualitative difference in the development of liver (pre)neoplasia as compared to cancer susceptible mouse strains. Furthermore, quantitative analysis of apoptosis in normal and (pre)neoplastic liver tissue all the three strains of mice revealed no clue to explain their different cancer susceptibility. Rather, cell proliferation seems to be the prevailing determinant of tumor promotion in the liver of C3H/He-, C57BL/6Jand B6C3F1-mice. Finally, the short-term experiments strongly suggests that mouse liver differs profoundly from rat liver in respect to apoptosis control. This conclusion is supported by in vivo as well as cell culture studies revealing that mouse hepatocytes are much less sensitive to the pro-apoptotic action of TGF-β1 as compared to rat hepatocytes. Likewise, the long-term experiment provided evidence that apoptosis may play a quantitatively different role for tumor promotion in mouse and rat liver. 61 REGULATION OF APOPTOSIS BY PEROXISOME PROLIFERATORS Ruth A. Roberts 1 , Cecile Michel 1 , Eric Boitier 1 , Jean-Charles Gautier 1 , Beth Coyle 2 , Caroline Freathy 2 , Kelvin Cain 2 . 1 Aventis Pharma, Centre de Recherche de Paris, 94403 Vitry-sur-Seine, France and 2 MRC Toxicology Unit, Leicester, UK Peroxisome proliferators (PPs) constitute a large and chemically diverse family of nongenotoxic rodent hepatocarcinogens that activate activate the PP activated receptor α (PPARα) an. This family includes fibrate hypolipidaemic drugs such as bezafibrate and gemfibrozil, given to patients at risk of heart disease to lower blood cholesterol and restore lipid balance. Also, the PP class includes chemicals of environmental and industrial significance such as the plasticizer di-(2-ethylhexyl)phthalate (DEHP). In order to investigate the hypothesis that PPs elicit their carcinogenic effects through the suppression of apoptosis, we established an in vitro assay for apoptosis using both primary rat hepatocytes and the FaO rat hepatoma cell line. Apoptosis was induced by transforming growth factor β 1 (TGF β 1), the physiological negative regulator of liver growth. In this system, PPs could suppress both spontaneous and TGF β 1-induced apoptosis. In order to understand the mechanisms of this regulation of apoptosis, we conducted microarray analysis followed by pathway-specific gene clustering in TGF β 1 treated cells. Cluster analysis identified several phases of gene response starting with genes encoding for the extracellular matrix and cytoskeleton and later the stress response genes. These data suggest that the regulation of these genes by TGF β 1 may represent the primary mechanism by which TGF β 1 induces apoptosis. The ability of PPs to impact on this TGF β 1 pathway remains to be determined. To approach this question, we have developed a technique using laser capture microdissection of PP-induced liver foci coupled with array analysis. This will be used to determine those genes that are altered by PP exposure to regulate apoptosis, ultimately leading to tumors. 62 TUMOR PROMOTERS AS INHIBITORS OF APOPTOSIS IN RAT HEPATOCYTES D. Schrenk. Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Kaiserslautern, Germany Multistage carcinogenesis in rat liver is widely used as an experimental model for the study of the critical events in tumor promotion. After an initial treatment with a genotoxic liver carcinogen (‘initiation’), subsequent application of certain non-genotoxic agents can lead to the clonal expansion of putative preneoplastic cells (‘promotion’). Obviously, the expansion of these clones is correlated with an increased occurrence of benign and malignant liver tumors at later time points. Since both proliferation and apoptosis were reported to be enhanced in putative preneoplastic liver foci, inhibition of apoptosis was suggested to play a critical role in tumor promotion. In rat hepatocytes in primary culture, 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) or phenobarbital inhibit apoptosis initiated by treatment of s20 Symposium S7. Risk assessment in food: examples the cultures with UV irradiation. With both agents, no suppression of basal apoptosis was observed. The suppression of apoptosis with TCDD coincided with an attenuated increase of the tumor suppressor protein p53 observed upon UV irradiation. Furthermore, TCDD treatment resulted in a marked hyperphosphorylation of p53. The fact that almost identical concentration-response curves were obtained for the phosphorylation of p53 and the induction of cytochrome P450(CYP)1A-catalyzed 7-ethoxyresorufin O-deethylase (EROD) activity indicates that p53 phosphorylation after TCDD treatment is mediated by the aryl hydrocarbon receptor (AhR) signalling cascade. With tumor-promoting ‘non-dioxinlike’ polychlorinated biphenyls inhibition of UV-induced apoptosis was also observed. A comparative study investigating the effects of various concentrations did not reveal, however, a clear correlation between the suppression of apoptosis and the induction of CYP2B-catalyzed 7-pentoxyresorufin O-dealkylase (PROD) activity. In summary, inhibition of UV-induced apoptosis with liver tumor promoters is observed in rat hepatocytes in culture. Hyperphosphorylation of key proteins of apoptosis including p53 seems to play a critical role in this effect. S7 Risk assessment in food: examples 63 PROCESSING-RELATED CHANGES IN FOOD A. Tritscher. Department of Quality and Safety, Nestlé Research Center, Lausanne, Switzerland Many types of food processing techniques have been employed throughout human history, e.g. heat treatment, freezing, acidification, hydrolysis, fermentation, salting etc. The main purpose of these treatments is to ensure the microbiological and chemical safety of products and to increase storage times, but also to improve texture and flavour. The growing consumer demand for healthy and nutritious, convenient food, which is as fresh and ’low-processed’ as possible, is a key driver for new developments in food processing. In the case of ’Novel Processes’, according to EU legislation, the effect of the process on the product has to be carefully evaluated, if possible in comparison with traditional processing techniques. As example for an approved novel process the high pressure pasteurization of fruit preparations is explained. A number of scientific studies were conducted to prove that a given product treated by this new process is as safe as the conventionally processed product. Although designed for beneficial purposes, several processing techniques may also lead to formation of potentially harmful compounds in the foods. One example is the formation of 3monochloropropanediol (3-MCPD) in a variety of industrially and domestically produced foods in the presence of fat and chloride (salt). MCPD is considered a non-genotoxic carcinogen, and the EU Scientific Committee on Food has established a tolerable daily intake (TDI). Highest levels of MCPD have been detected in hydrolyzed vegetable protein and soy sauce and regulatory limits have been established for these products. The second example is the formation of acrylamide in certain high heat-treated foods. This new finding has raised considerable concern, since acrylamide is considered a genotoxic carcinogen. Acrylamide is also known to cause neurotoxicity in humans (occupationally), and reproductive toxicity in experimental animals. Neurotoxicity and reproductive toxicity may only occur at higher exposure levels, however dose-response relationships and mechanistic information regarding carcinogenicity are lacking, thus precluding the assessment of health risk from acrylamide exposure through food. State-of-the-art knowledge on acrylamide will be reviewed. 64 ASSESSING HUMAN SAFETY OF FOODS PRODUCED BY BIOTECHNOLOGY Trish Malarkey. Syngenta Biotechnology Inc. Research Triangle Park, North Carolina, USA Biotechnology was used in the first generation of so-called ‘GM’ crops to provide growers with complimentary and sometimes al- ternative crop management solutions to pesticides. Selected host genes or genes identified from other plants or non-plant sources are modified or transferred to a crop plant. The new or altered protein expression resulting from these modifications confer on the plant a desired physiological trait, such as resistance to particular herbicides or insect pests. Second generation modifications provide traits such as enhanced nutritional or health-promoting characteristics that are of benefit to consumers. The following are the commonly raised concerns about possible implications for human health. • Inherent toxicity of the novel gene and their products • The potential to express novel antigenic proteins or alter levels of existing protein allergens. • The potential for unintended effects resulting from alterations of host metabolic pathways or over expression of inherently toxic or pharmacologically active substances. • The potential for nutrient composition in the new food occur differing significantly from a conventional counterpart. Foods produced using biotechnology are subjected to far greater levels of scrutiny than foods produced by traditional plant breeding techniques. The accepted analytical, nutritional and toxicological methods employed to support this scrutiny and to assess and assure that a ‘GM’ food is a safe and nutritious as its ’non-GM’ counterpart will be discussed. The challenges associated with identifying unintended effects in whole GM foods and the promise new (proteonomics/genomic) technologies offer opposite traditional toxicity testing paradigms will be critically appraised. 65 CRITERIA FOR RISK ASSESSMENT OF BOTANICAL FOOD SUPPLEMENTS R. Walker. School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, United Kingdom The increasing use of botanical food supplements with perceived health benefits has raised concerns among scientific and regulatory communities. There have been occasional cases of intoxication from such products as a result of misuse, misidentification of the botanical species or contamination with extraneous plants. Consequently, risk assessment of botanical products requires that they are adequately specified with regard to identity and composition. The sources of botanical supplements vary from staple food plants to herbals used in traditional medicine and the supplement may consist of the whole plant, extracts thereof or more purified components and this variability poses problems in adopting a generic approach to their risk assessment. Factors that determine the nature and extent of toxicological testing required to characterize hazard and assess possible risks include: nature and complexity of the supplement, prior knowledge of human consumption of the product or its source material, the likely magnitude of human exposure and its nutritional impact, and the intended physiological/beneficial effects. Generally, for herbs or complex extracts, it is not possible to make a risk assessment on the basis of a single active component as there may be more than one of toxicological significance and matrix effects also may affect factors such as bioavailability. Nevertheless, studies on single components may be useful in elucidating potential interactions. Because the consumption of botanical supplements is intended to produce physiological effects, there may be a need to distinguish a No Observed Effect Level from a No Observed Adverse Effect Level and the margin of exposure between that required to produce the desired effect and the upper safe level may be smaller than is customarily adopted for food additives and contaminants. In this regard, human efficacy studies including observations for possible adverse side effects may help in determining the adequacy of the margin of exposure. In relation to these considerations, a decision tree developed at a Workshop organized by ILSI Europe in May, 2002 will be presented to assist in determining the extent of data requirements based on the nature of the product Symposium S8. Immunotoxicology and immunopathology S8 Immunotoxicology and immunopathology 66 Workshop Immunotoxicology and Immunopathology: “Introduction & Summary” Paul-Georg Germann 1 , Jacques Descotes 2 . 1 Preclinical Safety, Novartis Pharma AG, Basel, Switzerland, and 2 Univ of Lyon, France During the last years, accumulating evidence occurred that potential immunotoxicity might be associated with significant increase in incidence and severity of clinical side effects. The Scope of this IFSTP/ESTP Workshop “Immune-Toxicology and ImmunePathology” is to present different aspects, views and issues of the current state of the art of this important field, also by giving specific examples. The awareness of immunotoxicology has led to an intensified and controverse discussion within the different Guideline Committees to address this important field adequately in preclinical studies. In his talk, titled “Status of Implementation and Impact of Guidelines on Immunotoxicology and -pathology”, Dr G.Bode will address these current discrepancies between the different Guidelines. Specialised animal models and modified study designs in preclinical immunotoxicology studies are of increasing importance. Their difficulties especially their limitations in interpretation are the topic of Dr.J.Descotes presentation: “Importance of Immunotoxicology on Safety Assessment: A Toxicologists View.” Dr.D.Germolec is contributing the toxicological pathologists‘ view in her talk about “Improving the Sensitivity and Predictability of Current Testing Strategies for Immunotoxicology.” Pathologists are playing an increasing role with their interpretation of immunotoxic effects. An international pathology working group report with its results is pointing to the usefulness of extended pathological contributions. The value of immunization studies, including analysis of antibody formation under immunosuppression, is presented and discussed in the two case studies from Dr.D.Roman & Dr.P.Ulrich. Finally, in the panel discussion we will try to figure out future development trends in the assessment of new drugs as well as open and emerging issues in immunotoxicology. The result of this discussion will be also included in the full Summary of the Workshop. 67 STATUS OF IMPLEMENTATION AND IMPACT OF GUIDELINES ON IMMUNOTOXICOLOGY AND PATHOLOGY G. Bode. Institut fur Patologie und Toxicologie, Altana AG, Hamburg, Germany The importance of immunotoxicology including immunopathology has increased in the past years. Regulatory agencies, together with the pharmaceutical industry have worked on drafting guidelines in order to improve nonclinical immunotoxicity testing and to support safety in humans during clinical trials and post marketing. According to the FDA (CDER), immunotoxicity is defined as immunosuppression, antigenicity, hypersensitivity, autoimmunity or adverse immunostimulation. In principle, this definition is widely accepted. Today, we are confronted with several guidelines and drafts on immunotoxicology. In the EU the recommendation on how to investigate immuntoxic potential is incorporated in a recent guideline dealing with repeated dose toxicity studies (Note for Guidance on repeated dose toxicity, 3Bs2a). The FDA and the EU guideline differ considerably in their intent and strategy (case by case decision in FDA guideline vs. fix test program in EMEA Guideline). A harmonization is needed to overcome this dilemma. First attempts to nominate immunotoxicology as a new ICH topic in 2001 failed, because FDA and US Pharmaceutical industry considered this issue as immature for harmonization. In the mean time, a number of scientific workshops and discussions took place and a growing amount of new data is available. A new initiative was started in February 2003 in Chiba/Japan at the ICH steering committee meeting. A questionnaire (a Japanese initiative) has been distributed worldwide and is currently being analyzed to evaluate the predictive power of existing immuntoxicological investigations for the safety in humans. All this information and activity has reopened the discussion and strengthened the intention to cooperate globally on a harmonized new concept. This concept will trigger new studies, will lead to s21 a thorough assessment of the results and their reproducibility and finally will improve our testing strategy. At the end of this process we will be able to improve preclinical assessment of immunotoxicity, to facilitate clinical trails and to accelerate the reviewing process. The presentation will provide information on progress, differences and possible solutions. 68 IMPORTANCE OF IMMUNOTOXICITY IN SAFETY ASSESSMENT: A TOXICOLOGIST’S VIEW J. Descotes. Poison Centre, E.Herriot Hospital, Lyon, France Clinical data accumulated during the past decades unequivocally demonstrate that immunotoxicity can be associated with significant morbidity. Immunotoxic effects are best divided into four distinct categories, namely immunosuppression, immunostimulation, hypersensitivity and autoimmunity. Each category is associated with a specific set of potential adverse health consequences, the prediction of which should be made using different strategies. Even though concern has primarily been on immunosuppression, hypersensitivity and increasingly immunostimulation are key issues, especially with drugs. Histology of the lymphoid organs is not considered sufficient to reliably predict immunosuppression. Thus, at least one immune function assay, primarily an antibody response assay, is recommended. It is unsure that function assays used to predict immunosuppression are applicable to predict immunostimulation. Specific assays, e.g. cytokine release assays, can be helpful in some circumstances. With the exception of contact sensitization or highly reactive chemicals, current models are seldom valid to predict the risk of hypersensitivity. Finally, autoimmunity cannot be predicted in most instances. A critical issue is the timing and design of preclinical immunotoxicity studies. Although no guidelines require evaluation of drug immunotoxicity prior to clinical trials, it is logical to perform some immunotoxicity studies early in safety evaluation. Inclusion of an antibody response assay in safety pharmacology can be considered. Additional testing could be decided case by case depending on these early results, possible histology changes, and the structure and pharmacological properties of the drug. For immunotoxicity risk assessment, host resistance assays, e.g. experimental infections (immunosuppression) or autoimmunity models (immunostimulation) may be needed. Finally, as preclinical immunotoxicity studies will be performed routinely to meet recent regulatory requirements, a wealth of unexpected immune changes is likely to be seen. It is therefore essential that some of the selected endpoints in animal studies can also be investigated in clinical trials. 69 IMPROVING THE SENSITIVITY AND PREDICTABILITY OF CURRENT TESTING STRATEGIES FOR IMMUNOTOXICITY D. Germolec, A. Nyska, C. Frieke Kuper, M. Luster, C. Portier, M. Kashon, Russell Helms, Vera Kommineni, Michael Holsapple, Keith Johnson, R. Maronpot. USA The identification of chemicals that have the potential to cause injury to the immune system is of considerable public health significance, as alterations in immune function can lead to increased incidence of hypersensitivity and autoimmune disorders, infectious diseases or neoplasias. Experimental animal data collected over the past 20 years using standardized testing panels have provided a database from which the sensitivity and predictability of a variety of tests commonly used for the screening of chemicals for immunotoxicity has been evaluated (Luster et al., 1992). These results have been used as guidelines for risk assessment in immunotoxicity and have been the basis for a number of regulatory activities. A working group was established to design and implement a study to evaluate the sensitivity and predictability of extended histopathology as an indicator of immunotoxicity, as compared with the National Toxicology Program’s functional testing battery. Standardized slide sets from thymus; spleen and mesenteric lymph nodes were generated for 11 chemicals, which had previously been evaluated for their immunotoxicity using functional tests. This group of chemicals included two negative compounds, one immunostimulatory compound and eight compounds that had been shown to have immunosuppressive effects. A working group meeting to standardize s22 KNL. Key Note Plenary Lecture the parameters to be evaluated was convened. A semiquantitative assessment was adopted in order to estimate the histopathological changes within different anatomical compartments of the lymphoid organs. The diagnostic terms for identifying and semiquantitating the histopathologic changes were those recommended by Kuper et al. (2000). The pathologists did not know the identity of the tested compounds but had received data identifying positive and negative controls from treatment groups and information regarding dosages and organ weights before commencing their microscopic evaluation; thus, no true “blind scoring” was used. Findings from each pathologist were entered into a common database and evaluated for agreement between pathologists, sensitivity of individual parameters and correlation with the “immunotoxic” call. Statistical analyses indicated that for a majority of the outcomes, there was good agreement between the pathologists. A direct comparison of the ratings for each pathologist indicated that certain individuals tended to be more conservative while others were more able to discern subtle changes. Additional analyses examined the consistency of a pathologist’s ratings in a single tissue by investigating the correlation among all the measures in the same tissue type. When data from all pathologists were combined, measures in each of the three tissues were highly correlated with the other measures from that tissue. However, when data were examined for each pathologist independently, only the thymus evaluations maintained the same high degree of correlation. For spleen, and lymph node measures, the outcomes for each pathologist were less well correlated. While overall there was good agreement between histopathology and functional tests, the antibody forming cell (AFC) assay detected immune suppression in two instances where no changes in pathology were indicated. In contrast, the AFC assay failed to detect oxymetholone as immunotoxicant, although other immune parameters, as well as extended histopathology indicated immunologic changes. These studies suggest that there is not a single most sensitive parameter for assessing damage to the immune system, but rather that a battery of functional tests combined with pathology examinations are needed to flag a compound as immunotoxic and provide data into its mechanism of action. 70 DETERMINATION OF THE EFFECT OF CALCINEURIN INHIBITORS ON THE IMMUNE SYSTEM AFTER KLH IMMUNISATION. D. Roman, P. Ulrich, G. Paul, M. Court, P. Vit, J. Kehren, A. Mahl. Novartis Pharma AG, Preclinical Safety, Basel, Switzerland The calcineurin inhibitors cyclosporin A (CsA), tacrolimus (TA) and pimecrolimus (PI) are proposed for a wide array of dermatological indications such as psoriasis and atopic dermatitis. The effect of these treatments on the immune response was investigated in this study after immunization of rats with Keyhole Limpet Hemocyanin (KLH). Male rats (10/group) were orally administered PI at 10 or 30 mg/kg/day, TA at 3 mg/kg/day or CsA at 20 mg/kg/day for 4 weeks. Control animals similarly received the vehicle only. The last 5 animals per group were immunized with KLH on day 16 and challenged on day 24. Seven days after the last injection, functionality of the immune system was investigated by detecting KLH-specific antibodies in the serum and by examination of cell infiltration at the site of the boosting injection of KLH. In addition, a correlation between functional and structural changes was established by quantification of lymphocyte sub-populations in the periphery or residing in lymphatic tissue. In KLH-immunized rats, CsA caused complete suppression of the KLH-specific IgM and IgG production, whereas only IgG production was affected by PI (30 mg/kg/day) and TA (3 mg/kg/day; more pronounced). Immunophenotyping of lymphocyte sub-populations in spleen and lymph node indicated a decrease in T lymphocytes with PI at 30 mg/kg/day, TA and CsA, whereas these changes were marginal for PI at 10 mg/kg/day. Immunophenotyping of peripheral white blood cells revealed a decrease in the absolute number of T lymphocytes with all three test items. A slight increase in absolute numbers of T lymphocytes was observed in KLHimmunized animals with PI at ≥10 mg/kg/day when compared with non-immunized animals. In conclusion, the ability of the immune system to respond to KLH was not affected with PI at 10 mg/kg/day. Only a slight effect with PI at 30 mg/kg/day was observed, whereas the inhibition of the immune system function was more pronounced with TA and most pronounced with CsA. 71 VALIDATION OF IMMUNE FUNCTION TESTING DURING A 4-WEEK ORAL TOXICITY STUDY WITH FK506. P. Ulrich, G. Paul, E. Perentes, A. Mahl, D. Roman. Novartis Pharma AG, Basel, Switzerland Assessment of the immune system’s capability to respond to antigens with the generation of specific antibodies, whilst under the influence of a test article, is required in toxicity tests according to the European guideline for repeated dose toxicity testing of medicinal products. The purpose of this study in rats was to validate methodology for the determination of Keyhole Limpet Hemocyanin (KLH)-specific antibodies under the influence of an immunologically active compound. The immunosuppressant FK506, commercially available as Prograf, was administered orally (gavage) to 5 rats/sex/group at dosages of 0.5 or 3 mg/kg/day, once daily for a period of 4 weeks. On days 14 and 22, KLH was administered subcutaneously, with an adjuvant (AluGel), to the two treated groups and a control (i.e. without FK506 treatment) approximately 1 hour following administration of FK506. Terminal investigations included haematology parameters, titration of KLH-specific antibodies in serum (ELISA), macroscopic pathology, spleen and thymus weights, immunophenotyping of splenocytes (FACS analysis) and histopathology of the lymphatic tissues. At 3 mg/kg/day a minimal reduction of subcutaneous KLH-induced granuloma formation and a moderate to marked reduction of germinal centre development (axillary lymph node and spleen) were observed. Reduced CD4+ (T-cell) counts were found in the spleen of males, consistent with a suppressed production of KLH-specific antibodies (IgG in both sexes, IgM in males only) and a higher incidence of atrophy in the periarteriolar lymphoid sheaths of males. Slight to moderate lymphopenia was present in both sexes at 3 mg/kg/day. These findings are consistent with the known pharmacological activity of FK506. In conclusion, determination of antibody titres following immunisation of rats with KLH, with concurrent exposure to a test item, appears to be a valid method in the context of the immunotoxicity evaluation required by European regulation. KNL Key Note Plenary Lecture 72 CHEMICAL WEAPONS. DOCUMENTED USE AND COMPOUNDS ON THE HORIZON Chantal Bismuth. Hôpital Fernand Widal Paris 7 Defense Councillor, France Weapons’ designers consider that the toxicity of chemical weapons, at identical charge and range, is 7 times inferior to explosives! However, health professionals, the media, and populations are highly sensitive to chemical threats, terrorism being more and more invoked. A. Some chemical agents, possibly used in military combat or against civilians are authorized. These include tear-gas in street-fights or the herbicides in guerilla warfare. B. Among the forbidden weapons, – some are incapaciting agents, neutralizing the system for several hours or days such as • agent Bz, an anticholinergic intoxicant • or psychotropic drugs: neuroleptics, narcotics, tranquilisers, anesthetics • or the ricin-derivatives, whose toxicity depends on the route of administration – others are possibly lethal agents: (1) either with a local-regional toxicity. – Suffocating gases, chloride, phosgene, used during the first world war with a 1% lethality. The gas-mask is a a correct protection – Vesicant agents (mustard gas, lewisite), whose penetration is both cutaneous and pulmonary, with a 4% lethality during the Iran-Iraq war. The gas mask is unprotective. Workshop W6. State-of-the-Art of the Pesticide European Revision: Council Directive 91/414 (2) or with systemic toxicity: – like cyanides – their volatility makes them efficient only in closed areas – or like organophosphate compounds, type tabun, sarin, soman, VX, irreversible anticholinesterase agents, with a cutaneous and pulmonary penetration. The mortality was 12 victims out of 1500 symptomatic intoxications in Tokyo in 1995 with Sarin. The medical public has available antidotes: atropine against the muscarinic syndrome, oximes as enzyme reactivators and also efficient supportive care with assisted ventilation. The interest of pyridostigmine, a preventive, reversible anticholinesterase agent, remains debated. The danger of the different agents depends largely on the munition and of its vectors: air bombs, missiles, hand grenades, rocket artillery and sprinkling devices. Some are delivered as binary munitions which release the intoxicant only after impact (with the purpose of protecting the workers of arms’ factories). The death of 117 hostages/800 during the assault of the Moscow Theatre in 2002 evokes the use of chemicals in active counterterrorism. The fact that these products were not forbidden -according to the users- by the International Convention lets suppose that they were medications of narcotic or anesthetic drug type. W6 State-of-the-Art of the Pesticide European Revision: Council Directive 91/414 73 THE EUROPEAN COMMISSION VIEW REGARDING FURTHER DEVELOPMENT OF LEGISLATION FOR PLANT PROTECTION PRODUCTS Goffredo Del Bino. Head of Division Plant Health, DG Health and Consumer Protection European Commission There are three pillars of legislation underpinning Community policy on plant protection products. All are currently under review. These are: 1. Upstream evaluation and prior assessment of risks before authorisation of plant protection products is governed by Council Directive 91/414/EEC on the placing of plant protection products on the market. A proposal to Council and Parliament to amend this is currently being drafted with adoption by the Commission foreseen in the coming year. Under the existing directive a variety of measures are already taken to ensure that all substances will have been evaluated by 2008. 2. The actual use phase of plant protection products is addressed in the Commission Communication of 12 June 2002 on the Sustainable Use of Plant Protection Products. The Communication, open for comment, foresees additional measures of both legislative and non-legislative nature to decrease the risks to health and environment associated with pesticide use. It is foreseen to bring proposals forward in 2004. 3. Downstream measures concerned with consumer protection from residues of pesticides applied to food crops is currently governed by four Council Directives on the setting, monitoring and control of pesticides residues in products of plant and animal origin. A proposal to Council and Parliament to consolidate and amend these was adopted by the Commission in March 2003 and is currently in the co-decision process. This presentation will focus mainly on the issues surrounding the preparation of a Commission Proposal to amend Directive 91/414/EEC. 74 ECCO-PEER REVIEW: DEVELOPMENT SINCE 1996 AND FUTURE PROSPECTS D.J. Flynn 1 , J.-R. Lundehn 2 . 1 Pesticides Safety Directorate, York, UK, 2 Bundesamt für Verbraucherschutz und LebensmittelsicherheitBVL, Braunschweig, Germany Plant protection products have to be evaluated and authorised in the EC-Member States since 1993 in accordance with Directive 91/414/EEC. s23 Fundamental elements of this Directive include: • a joint procedure (European Commission and Member States) for the evaluation and assessment of the active substances and the compilation of a positive list (Annex I of 91/414/EEC) • the evaluation, assessment and authorisation of the plant protection products at national level Furthermore, the evaluations and assessments are based on: • agreed data requirements (Annexes II and III) • uniform principals for evaluations and assessments (Annex VI) • harmonised procedure and guidelines (guidance and working documents) • possibility of mutual recognition under certain conditions The programme for evaluating existing active substances (which were on the market before mid 1993) involves several steps and stages, stretching over a period of up to formerly 10, and now 15 years. The programme for the evaluation of existing active substances is co-ordinated by the European Commission, since 1996 with the assistance of the ECCO-Team (European Community Co-ordination). The ECCO-Team consists of two groups; one is situated at the Pesticides Safety Directorate in York (United Kingdom) and the other at the Bundesanstalt für Verbraucherschutz und Lebensmittelsicherheit – BVL (Federal Office for Consumer Protection and Food Safety) in Braunschweig (Germany). They take care of or support the technical and administrative co-ordination of the programme for the evaluation of active substances for the European Commission. Their tasks include e.g.: • document management • organisation of peer review meetings • taking minutes • co-ordinating the development of guidance/working documents • compilation of manuals, full reports and draft review reports The programme is very successful and relies on the close and trusting co-operation of all the parties involved (applicants, Member States, the Commission services, chosen experts). Until the end of 2003, decisions will probably be able to be made on around 100 active substances. With the foundation of the European Food and Safety Authority (EFSA), ’risk assessment’ (EFSA) and ’risk management’ (Commission) have been separated. This will continue to effect the evaluation procedure, effects which are not all foreseeable at present. However, all the groups involved are of the opinion that the work which has proved so successful in the past should be continued, and that the existing network offers a suitable basis on which to continue with the evaluation of existing active substances and to complete this project successfully. PSD/York and BVL/Braunschweig are prepared to make their contribution to this as one of the bearers of the ECCO-Project. 75 PRESENT SITUATION AND ROLE OF EFSA Bruno Henning. EFSA, Bruxelles, Belgium Abstract not received. 76 STATE OF THE ART OF THE PESTICIDE EUROPEAN REVISION: COUNCIL DIRECTIVE 91/414/EEC – THE POINT OF VIEW OF INDUSTRY Bruce Julin. European Crop Protection Assoc., Brussels, Belgium Council Directive 91/414/EEC, the primary regulatory instrument covering the placing on the market of plant protection products (PPPs) within the EU, is generally acknowledged to be a very complex piece of legislation with very ambitious goals. A major review (reregistration) programme covering some 800 existing active substances was begun in 1992. Given the complexity of the Directive and its implementing legislation, there is general consensus that significant progress has been made during the past twelve years. In its July 2001 Report to the EP and Council on the status of the Review Programme, the Commission announced that it planned to bring forward proposals for amendments of Directive 91/414 in 2003. At the same time, it was decided to review the data requirements for active substances and products, as well as the evaluation and decision making criteria. s24 Workshop W6. State-of-the-Art of the Pesticide European Revision: Council Directive 91/414 Industry, being a major stakeholder in the 91/414 process, welcomes the opportunity to participate and contribute to the revision of the Framework Directive as well as its annexes. Industry is beginning to see the consequences of the implementation of the Directive in: • The loss of approx. 460 existing active substances • A reduction in the number of new active substances being introduced each year • Less support for minor crops and their uses • Consolidation/contraction of the PPP industry Industry has identified several areas where we believe the 91/414 registration process should be strengthened in order to make it more efficient and predictable. Several of these will be addressed in detail as well as some other areas which we believe could have an overall negative impact, if adopted. In addition to the Revision of 91/414, this year also marks the start-up of the European Food Safety Authority (EFSA). EFSA will, together with the Commission, play a major role in the evaluation of PPPs active substances and in the establishment of maximum residue limits (MRLs) and import tolerance for pesticides in food and feed. Industry views on the role of EFSA in the EU approval process will also be put forward. 77 SWEDISH/NORDIC INPUT REGARDING WORK RELATED TO PLANT PROTECTION PRODUCTS L. Törnqvist. National Chemicals Inspectorate, Solna, Sweden Background: A lot of efforts have been made to harmonize risk assessments and decision making in the EU review program of plant protection products during the last years. This work has been successful but resource demanding. Many issues have been solved but some need further discussions. One important issue is pesticides also being endocrine disrupters. The Swedish view is that a special strategy should be settled at least for substances also toxic to reproduction. The EU program is important to farmers and authorities in Sweden. Sweden became EU-member in 1995. Since 1987, national programs have been conducted every fifth year by the Board of Agriculture and the Chemicals Inspectorate. In order to reduce risks, co-operation between farmers, authorities, industry and scientists has become indispensable. Therefore, the results gained so far and further possibilities for reduction of risks to human health and environment is of high importance. Future: The work on amendments of 91/414/EEC has started. Some simplifications and also adaptation to present EU-policy is needed. Below inputs, important for Sweden, some also supported by other Nordic Member States, are listed: • Annex I non-inclusion criteria could be used in the initial stages of an assessment to identify pesticides that are clearly unacceptable within European Union from a health and /or environmental protection standpoint. The idea behind such criteria is to facilitate prompt and easy Annex I exclusion procedures and predictability in the outcome of the decision. The criteria should at least correspond to the criteria presently discussed in the Commission “Chemicals strategy”. • Substitution/Comparative assessment has the potential to be a valuable tool for risk reduction within the EU. It will give signals to the market/manufacturer on which risks are high and should be avoided if possible (presence of alternatives). Practicing substitution is to minimize risks while still keeping up plant protection performance at a high level. Substantial difference between the products is recommended. 78 MEDITERRANEAN COUNTRIES IMPACT Kyriaki Machera 1 , Kostas Markakis 2 . 1 Laboratory of Pesticides Toxicology, Benaki Phytopathological Institute, 7, Ekali Street, 145 61 Kifissia, Athens, Greece, 2 Hellenic Ministry of Agriculture, General Directorate of Plant Produce, Directorate of Plant Produce Protection, Department of Pesticides For the implementation of the Framework Council Directive 91/414/EEC, participation of all Member States (M.S.) in the review program of plant protection products (ppp’s) is required. In the first round of evaluation, Mediterranean countries evaluated 37 of the 90 active substances and their respective formulations. Until now, 10 of the 25 compounds, listed in Annex I to the Directive, were evaluated by Mediterranean countries (Doc. 3010/ 12- 2002). For the second round of evaluation, 63 complete dossiers have been submitted, 12 of them are allocated to the Mediterranean countries for evaluation. In addition to the above, 96 actives, not notified, are scheduled to be out of the market in July 2003. The evaluation according to the Article 5 of Directive 91/414/EEC, focuses on the ppp’s safety for the human health and the environment. The main part of safety evaluation is the risk assessment for the workers (operators and re-entry workers) and for the consumers of the agricultural products where residues of ppp’s may be present. The risk assessment requires both the hazard identification and the estimation of the degree of exposure. Although the strategy for hazard identification has to follow a uniform and harmonized approach among M.S., the strategy for the exposure assessment may differ significantly between North European and Mediterranean countries for both the workers and the consumers. Differences in climatic conditions, plant protection needs and practices, due to the variety of crops and pests and differences in food consumption, influence the levels of exposure. The current approach limitations, for the assessment of operator exposure and the Mediterranean countries’ contribution to the establishment of a more representative and reliable database, for the Southern Europe, will be presented. For the consumer’s risk assessment the identified shortcomings and recommendations for their avoidance will be presented as well. The potential rise of plant protection problems, in Mediterranean countries, due to the withdrawal from the market of a wide range of plant protection products, will be discussed. 79 THE ACCESSION COUNTRIES BENEFIT A. Stanêk. Department of Ecotoxicology, Plant Protection Product Division, State Phytosanitary Administration, Brno, The Czech Republic Ten candidate countries are expected to join EU in 2004. At EU level, associated states will have roles in evaluation of applications for listing of new active substances (NAS) in Annex I of Directive 91/414/EEC and review of existing active substances (EAS). At national level, regulatory procedures and authorisations will have to comply with EU Directives. Sale and use of plant protection products (PPP) in the Czech Republic is subject to authorisation by the State Phytosanitary Administration (SPA) in Brno (co-ordination and evaluation of efficacy, chemistry, environmental fate and ecotoxicology) and by the National Institute of Public Health (NIPH) in Prague (evaluation of toxicology, operator exposure and residues). Since 1 January 1997 PPP authorization is regulated by the Phytosanitary Care Act No. 147/1996 (as amended), which already includes certain parts of Directive 91/414/EEC (e.g. Annex II/III data). A new Act is expected in 2003/04 to implement all requirements. We are also currently identifying PPP withdrawals to comply with existing Annex I-listing decisions. Registration of new PPP with new or existing substances to the Czech Republic require a full Annex II/III data package in line with Directive 91/414/EEC. However, for most EAS not on Annex I or undergoing EU Review, such data packages may not be available. Most pre-1997 authorisations are based on poor data packages. Czech PPP re-registration after EU Review will enable all PPP to be authorised with full supporting packages. There are currently no Czech procedures for some Directive requirements. Implementation of these will be very beneficial for the regulatory authority, agrochemical industry and growers. New, more efficient systems will have to be established such that EU and National work is carried out to the required technical standards and deadlines. ‘Mutual recognition’ will decrease resource required for data evaluation and allow simpler PPP authorisation. ‘Extension of use’ will enable minor crop growers to obtain authorisations. Symposium S9. Genetic susceptibility towards genotoxic agents S9 Genetic susceptibility towards genotoxic agents 80 CYTOGENETIC BIOMARKERS AND GENETIC POLYMORPHISMS H. Norppa. Finnish Institute of Occupational Health, Helsinki, Finland Cytogenetic biomarkers have long been applied in surveillance of human genotoxic exposure and early effects of genotoxic carcinogens. Due to their wide use, it has been possible to evaluate, in international collaborative studies, if a high level of these biomarkers in peripheral lymphocytes is predictive of cancer risk. Thus far, such an association has been observed for chromosomal aberrations (CAs), but not for sister chromatid exchanges (SCEs) or micronuclei (MN). The cancer risk predictivity of CAs did not appear to be explained by tobacco smoking or occupational exposure to carcinogens, but was seen in unexposed non-smokers as well. This suggests a role for individual susceptibility factors. Genetic polymorphisms of various xenobiotic-metabolising enzymes, influencing the metabolic activation and detoxification of carcinogens, have been associated with cancer risk and some of them also appear to affect cytogenetic biomarkers. The lack of glutathione S-transferase M1 (GSTM1 null genotype) appears to be associated with increased sensitivity to genotoxicity of tobacco smoke, and GSTM1 null smokers show an increased frequency of CAs. N-acetyltransferase (NAT2) slow acetylation genotype and glutathione S-transferase T1 (GSTT1) null genotype seem to elevate the baseline level of CAs and SCEs, respectively - possibly because of reduced capacity to detoxify some wide-spread genotoxins. For some chemicals, in vitro cytogenetic studies with lymphocyte donors representing different genotypes have been able to predict a differential in vivo response. The in vitro genotoxicity of styrene and epoxide metabolites of 1,3-butadiene is modified by GSTM1 and GSTT1 genotypes - which also influence the excretion of specific mercapturic acids in humans exposed to butadiene and styrene. Polymorphisms of DNA repair and folate metabolism are expected to be of special importance in modulating genotoxic effects, but there is presently inadequate information about their possible effect on cytogenetic biomarkers. (Supported by EU QLK4–2000–00628) 81 CHROMOSOMAL ABERRATIONS AND INDIVIDUAL SENSITIVITY TOWARDS IONISING RADIATION. A. Vral, A. Baeyens, H. Thierens, L. De Ridder. Department of Anatomy, Embryology, Histology and Medical Physics, University of Ghent, Ghent, Belgium The development of biomarkers of risk or susceptibility to identify individuals who are at increased risk for the development of cancer is an important issue. Possible candidate susceptibility markers are biomarkers that measure the sensitivity of chromosomes to ionising radiation. Several studies have indeed confirmed that chromosomal radiosensititvity is a hallmark not only for a certain number of genetic disorders but also for a significant proportion of breast cancer patients. These findings which favour the application of chromosomal aberration assays, such as G2 assay and micronucleus (MN) assay, as biomarkers of susceptibility induced us to start a thorough study of the reliability and sensitivity of the G2 and micronucleus assay. Before these assays can be used for individual risk assessment it is indeed essential to know how specific, sensitive and reliable they are. For this, the G2 assay and the MN assay were performed on repeated occasions on blood samples of healthy individuals and intra- and interindividual variabilities were analysed. For the MN assay Go lymphocytes were exposed to 3.5 Gy Co γ-rays either at high dose-rate (HDR) or at low dose-rate (LDR). For the G2 assay lymphocytes were irradiated with a dose of 0.4 Gy Co γ-rays in G2 phase of the cell cycle. The repeat experiments on blood samples of the same donor revealed that the intraindividual coefficients of variation were not significantly different from the interindividual coefficients of variation in both G2 and MN assay. As the intraindividual variability determines the assay reproducibility this would indicate that the assays are not able to s25 detect real, reproducible differences in radiation sensitivity between normal individuals in the population. The repeat experiments further revealed that for some healthy donors in one or two occasions high values were obtained while at all the other time points the values were within the normal range (non-sensitive). Although a normal mean value was obtained for these healthy individuals, they could have been regarded as sensitive based on one occasional high value. We conclude that results obtained with the G2 and MN assay that are based on single measurements are insufficient to determine the susceptibility of an individual. To yield reliable conclusions, the results should be based on multiple measurements from multiple blood samples. 82 GENETIC POLYMORPHISM AND CYTOGENETIC DAMAGE IN HUMANS EXPOSED TO URBAN AIR POLLUTION P. Hrelia. Department of Pharmacology, University of Bologna, Bologna,Italy Urban air pollution is considered a major hazard to human health. Benzene, a recognized hematotoxin and leukemogen, might be responsible for adverse health effects in population exposed to high level of air pollution. The aim of this study was to investigate the relationship between benzene exposure and biomarkers of exposure (personal passive samplers, µg/m3 ), effect (micronuclei (MN)) and susceptibility (MPO, GSTP1 and GSTT1 genotypes). Samples of peripheral blood were collected from 49 policemen (32 no-smokers and 17 smokers mean age:39.53±7.14), carrying on different outdoor activities in high traffic area of Bologna, and 36 indoor workers enrolled as controls (23 no-smokers and 13 smokers, mean age: 40.13±7.22). The average benzene exposure during the work-shift was significantly higher in the traffic policemen compared to the control population (24.32±14.38 and 4.39±0.99 µg/m3 , P= 0.001). MN frequency was significantly increased in policemen as compared with controls (MN/1000 binucleated (BN) cells: 7.06±2.87 and 4.61±2.04, P=0.001). Multiple regression analysis showed MN frequency was significantly modulated by age (P= 0.001) whereas smoke habits and gender did not have any influence. All the fortynine policemen were genotyped. Preliminary results showed higher frequency of MN in subjects with GSTT1 null genotype compared with those with positive genotype (8.30±4.64 and 6.79±2.15 MN/BN); on the other hand no association was observed with any of the other selected genes. Results support the idea that long-term exposure to urban air pollution increases DNA damage and that polymorphisms analysis should be taken into account in understanding benzene-induced adverse health effects. The integration of biomarkers of exposure, effect and susceptibility in population studies may contribute significantly to the establishment of risk profiles of individuals in given exposure situations. 83 IMPACT OF PHASE I OR PHASE II ENZYME POLYMORPHISMS ON LYMPHOCYTE DNA ADDUCTS OF SUBJECTS EXPOSED TO AMBIENT AIR POLLUTION AND ENVIRONMENTAL TOBACCO SMOKE P. Georgiadis 1 , J. Topinka 2 , M. Stoikidou 3 , M. Bekyrou, K. Katsouyianni 3 , R. Sram 2 , H. Autrup 4 , A. Kyrtopoulos 1 . 1 National Hellenic Research Foundation, Athens, Greece; 2 Laboratory of Genetic Ecotoxicology, Institute of Experimental Medicine Acad. Sci. C.R. and Regional Institute of Hygiene of Central Bohemia, Prague, Czech Republic; 3 Laboratory of Hygiene and Epidemiology, University of Athens Medical School; 4 Department of Environmental Medicine, Univ. of Aarhus, Denmark The levels of bulky DNA adducts were measured by 32 P-postlabelling (nuclease P1 enrichment) in lymphocytes of 194 non-smoking technical institute students living in the city of Athens, and the rural region of Halkida, Greece, in whom personal exposure to PAH was also measured. Individuals were sampled twice, during two consecutive winter and summer periods. Genotype analysis of various polymorphic genes which encode biotransforming enzymes involved in the activation (phase I) or detoxification (phase II) of xenobiotics was performed and the effects on adduct levels was assessed. Genetic polymorphisms were examined in cytochromes P450 1A1, 1B1 and s26 Round Table RT. Is toxicology protocol adequate for drug evaluation? 2E1, in the GSTM1, GSTP1 and GSTT1 as well as in the NAT2, the NQO1, and microsomal epoxy hydrolase (mEH) genes. Subjects with the CYP1*2A mutant genotype and GSTM1 null genotype tended to have higher DNA adduct levels, an effect which was more pronounced in subjects carrying both genotypes. A similar effect was also observed with the combined CYP1A1*2A/GSTP1 (Ile/Val) and the CYP1A1*2A/mEH “slow” polymorphisms. In both cases the effect was more pronounced among subjects with higher levels of ETS exposure. Stepwise (sequential) restriction of the observations to subjects characterized by a)GSTP1, b) GSTM1, c) mEH “slow” and d) ETS exposure resulted in an increasing trend of DNA adduct levels only among individuals with at least one CYP1A1*2A mutated allele, illustrating the importance and complexity of gene-exposure and gene-gene interactions in determining the level of genotoxic damage at the level of individual. 84 DNA REPAIR, CELL CYCLE CONTROL AND RISK OF CANCER Kari Hemminki, Rajiv Kumar, Sabrina Angelini. Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany and Department of Biosciences, Karolinska Institute, Novum, 141 57 Huddinge, Sweden One of the dilemmas of the present molecular medicine field is that it is often easier to genotype rather than phenotype, in order to analyze associations to disease frequency. The data on single nucleotide polymorphisms (SNPs) in various genes, including DNA repair genes, is exploding while data on their possible functional effects is lagging behind. The ultimate functional test is to show an effect in humans in vivo. A DNA repair test has been developed, which was based on the quantification of specific UV radiation (UVR)-induced photoproducts in human skin biopsies by the 32 Ppostlabelling technique. For the determination of a repair rate, a single dose of UVR was administered on the buttocks of volunteers and the removal of damage was followed with biopsies taken at various periods of time after UV exposure, giving the individual’s DNA repair kinetics. We have used the in situ DNA repair assay to functionally test the effects of various DNA repair gene SNPs, including those in XPC, XPD, XRCC1 and XRCC3. We have also used intermediary markers, such as DNA breaks and chromosomal aberrations as markers for DNA repair defects. The RAS-RAF-MEK-ERK-MAP kinase pathways mediate the cellular response to growth signals. In melanocytes BRAF, one of the RAF family members, is involved in the mediation of cAMP-dependent growth signals. Recently, activating mutations in the BRAF gene were reported in a large proportion of melanoma cell lines, short-term culture melanomas and primary tumours. We analysed exons 1, 11 and 15 of the BRAF gene for mutations in primary and metastatic melanomas and in nevi. Mutations in exon 15 were detected in some 50% of melanomas and in over 50% of benign nevi. In most cases mutation involved ‘hot spot’ codon 599 of the BRAF gene. BRAF mutation is usually mutually exclusive with transforming mutations at codon 61 of the N-ras gene. RT Is toxicology protocol adequate for drug evaluation? 85 ADEQUATE IMMUNOTOXICITY TESTING IN DRUG DEVELOPMENT REQUIRES BIOLOGICAL CONTEXT FOR INTERPRETATION D.J. Herzyk. Immunologic Toxicology, Preclinical Safety Assessment, GlaxoSmithKline Pharm. R&D, Philadelphia, USA The immune system is extremely complex and involves interdependent functioning of the tightly regulated network of various lymphoid and other cell types interacting by cell-to-cell contact and communicating via soluble mediators such as cytokines. Modulation of the immune system can lead to either immunostimulation or immunosuppression and can be either intended or unintended. While many effects on the immune components can be found as a result of a drug treatment or chemical exposure, a true immunotoxicity occurs when such treatment results in adverse effects or defects in the immune responses. Immune-mediated disorders in patients due to immunostimulation range from the relatively minor like hay fever to more serious conditions such as anaphylaxis, autoimmune haemolytic anaemia or systemic lupus erythaematosus. At the other end of the spectrum, consequences of immunosuppression include the increased susceptibility to the development of lymphomas and infections, e.g., among patients receiving long-term (intended) immunosuppressive therapy following organ transplantation. While the mechanisms involved in systemic hypersensitivity and/or autoimmune diseases are generally poorly understood and animal models are practically non-existent, the evaluation of immunosupression is much more advanced using multiple test systems including those in animals. Expectations for toxicological endpoints in the regulatory assessment of pharmaceuticals warrant usage of validated methods and generation of robust and reproducible results. Immunotoxicological evaluation of pharmaceuticals in animal studies still falls short of meeting these criteria. More importantly, there are no criteria for qualification of a drug as an “immunotoxicant”. Often data interpretation is based on statistical analysis of observed changes in any immune parameter measured without addressing biological meaning or relationship of these effects. Not only more adequate studies but also more adequate interpretation of measured endpoints are needed in the immunotoxicity testing. 86 PHASE I CLINICAL TRIALS IN ITALY:AIMS AND INTERPRETATION OF REQUESTED PRE CLINICAL STUDIES Annarita Meneguz 1,2 , Marino Massotti 1 . 1 Laboratory of Pharmacology Istituto Superiore di Sanità, V.le Regina Elena 299, 00161-Rome, Italy; 2 National expert at the Safety Working Party of the EMEA, the European drug registration agency Before any clinical trial is carried out, results of non clinical investigations or previous human studies, should be sufficient to indicate that the drug is acceptable safe for the proposed investigation in humans. Throughout drug development, emerging animal toxicological data should be reviewed and evaluated by qualified experts to assess their implications for the safety of trial subjects. In response to such findings, future studies and, when necessary in progress studies should be appropriately modified. For Phase I clinical investigation the appropriate safety conditions are determined by extrapolation from results of pre clinical studies indicating the tolerability in animals, obtained from a drug preparation with specified characteristic of purity. For the complexity and relevance of this first evaluation assessment process, the Italian regulatory rules, from 1974 provide an authorisation process by a “ad hoc” Committee for the evaluation of safety and quality of new drugs for phase I trials, operating at the Istituto Superiore di Sanità, the National Public Health Institute, though qualified experts routinely involved in researches regarding quality and pre clinical pharmaco-toxicology. Taking into consideration the high scientific relevance of phase I clinical study representing the first check to the extrapolation of laboratory data to humans, and the tool to define the safety levels for use, this abstract will provide the activity of the Committee from 1974,in order to contribute to the currently discussion on the pre clinical protocols investigations, especially for biotechnology products, gene therapy and somatic cell therapy. The Committee has analysed more than 600 active products, the contribution concerns analysis of the quality and pre clinical pharmaco-toxicological studies divided for therapeutic class, with particular focus on recent request of gene therapy products. 87 DRUG INTERACTIONS AND ADR Phil Routledge. Department of Pharmacology, Therapeutics and Toxicology, University of Wales College of Medicine, Cardiff, Wales, UK Abstract not received. Workshop W7. Nuclear receptors W7 Nuclear receptors 88 IMMUNE MODULATION BY GLUCOCORTICOIDS: ONE RECEPTOR, MULTIPLE MODES OF ACTION Martin Göttlicher 1 , Olivier Kassel, Christine Heilbock, Sandra Schneider, Margarether Litfin, Holger M. Reichard 2 , Günther Schütz 2 , Peter Herrlich. Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-H.-Platz 1, D-76344 Eggenstein, Germany; 1 Current address: GSF-Research Center Environment and Health, Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany; [email protected]; 2 German Cancer Research Center, Heidelberg, Germany Glucocorticoids (GCs) might be considered as model ,immunotoxins’ since they potently induce apoptosis of immune cells and suppress inflammatory responses. GC effects are mediated by the glucocorticoid receptors (GCR) for which several modes of action have been described, e.g. the activation of target genes by binding as a dimer to degenerate palindromic binding sites (GREs) in target gene promoters, by binding to so-called negative regulatory elements (nGREs) and by, mostly negative, interferences with the activity of other transcription factors such as NF-κB or AP-1 without itself binding to promoter sequences (transcriptional cross-talk). To understand the mechanisms behind the multiple effects of GCs on the immune systems two approaches have been followed. The analysis of mutant mice carrying a mutant GCR that can not bind to GREs reveiled that initiation of apoptosis in thymocytes apparently requires the dimer-dependent induction of pro-apoptotic genes in thymocytes whereas initiation of apoptosis in cultured splenocytes and suppression of inflammatory responses does not require the GCR dimer. The mechanism of negative cross-talk with AP-1 or NF-κB was addressed by searching partner proteins interacting with a mutant GR that fails to activate target gene expression. One prominent cofactor was defined that is required for transcriptional cross-talk and interacts with, both, GCR and AP-1 or NF-κB. In summary, the model case of GCR demonstrates that, despite only one prominent cellular target molecule, GCs exert a multitude of cellular effects by several qualitatively distinct mechanisms of action. 89 SIGNAL TRANSDUCTION IN CELLS EXPOSED TO HYPOXIA AND/OR DIOXIN-LIKE ENVIRONMENTAL POLLUTANTS Lorenz Poellinger. Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden HIF-1alpha (hypoxia inducible factor-1alpha) and the dioxin receptor belong to a family (bHLH/PAS) of transcription factors conditionally regulated by environmental cues. Signal transduction by these gene regulatory proteins is activated by multi-step pathways in hypoxic cells and/or cells exposed to dioxin-like environmental pollutants. In the hypoxia signaling pathway the process is initiated by potent inhibition of the activity of the von Hippel-Lindau tumor suppressor protein as an ubiquitin ligase acting on HIF1alpha, followed by induced nuclear translocation of HIF-1alpha, release of the molecular chaperone hsp90, dimerisation with a partner transcription factor (Arnt), and subsequent recruitment of transcriptional coactivator proteins, unmasking e.g. angiogenic gene expression programs in entothelial cells or tumors. Evidence will also be presented for hypoxia-dependent and hypoxia-independent mechanisms of negative modulation of HIF function. In contrast to HIF-1alpha, the dioxin receptor represents a ligand-dependent transcription factor (so far the only one known outside of the steroid receptor superfamily). The hsp90 chaperone and associated cochaperone proteins play a pivotal role in repression of receptor function in the absence of xenobiotic ligand, and derepression in response to ligand. In conclusion, molecular details of these activation processes will be discussed, as well as the biological implications of the generating active forms of the dioxin receptor and/or HIF-1alpha for adaptive responses to environmental stress, cell homeostasis, and oncogenesis 90 s27 REGULATION OF CELL LIFE AND DEATH BY RETINOIDS Hinrich Gronemeyer, Lucia Altucci 1 , Nicole Clarke, Ana Jimenez-Lara, Aurelie Rossin, Emilie Voltz. Dept. of Cell Biology and Signal Transduction, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)/CNRS/INSERM/ULP Illkirch Cedex, C.U. de Strasbourg, France and 1 Diptimento di Patologia generale, Seconda Universita di Napoli (SUN), Italy In our laboratory, we are interested in deciphering the pathways and mechanisms leading to retinoic acid-induced cell death. Previously, we have demonstrated that retinoic acid treatment induced the death receptor ligand TRAIL in the NB4 acute promyelocytic leukemia (APL) cells and in APL patients’ blasts. This work also demonstrated that retinoic acid-induced TRAIL was responsible for the paracrine cell death observed with these, as well as with heterologous, cells. These findings could provide a mechanistic link between the well-known anti-tumor activity of retinoids and the action of a tumorselective death ligand, and may lead to novel approaches for cancer therapy. In order to elucidate the molecular mechanisms underlying the induction of TRAIL by retinoic acid we have cloned the promoter and undertaken in vivo and in vitro studies to determine the elements responsible for this induction, as well as the factors involved in the response. We have mapped regions that are important for the retinoic acid inducibility by DNAse I hypersensitivity, EMSAs and transfection experiments. We have further identified the mediators of the retinoid induction and confirmed the recruitment of these factor(s) by chromatin immunoprecipitation. A model for retinoic acid induction of the death receptor ligand, TRAIL, will be presented in the context of the various regulatory pathways by which retinoids and rexinoids regulate cell life and death. 91 HOW DO NATURAL AND SYNTHETIC ESTROGENS INDUCE CELL PROLIFERATION AND DIFFERENTIATION? A TRANSCRIPTIONAL VIEW OF THE UTEROTROPHIC RESPONSE G. Orphanides, J.G. Moggs, H. Tinwell, T. Spurway, I. Pate, A. Soames, I. Kimber, J. Ashby. Syngenta Central Toxicology Laboratory, Alderley Park, Cheshire, SK10 4TJ, United Kingdom Endocrine disruptors that target the estrogen receptor family of nuclear hormone receptors induce developmental and reproductive toxicity. In the female reproductive organs, estrogenic compounds control tissue growth and development by regulating the transcriptional activity of estrogen receptors α and β. However, the mechanisms by which endocrine disruptor-induced regulation of transcription promotes cellular growth and reprogramming in these organs are poorly understood. We have used gene expression profiling to examine the way in which exogenous estrogens induce uterine growth in an immature rodent model. Our data reveal that estrogens induce a tightly coordinated transcriptional program that regulates successive and interlinked cellular processes during organ growth and maturation. The gene expression profiles we have identified provide novel insights into the way in which estrogens promote increases in vascular permeability and fluid uptake, attract immune and inflammatory cells, increase mRNA and protein synthesis, suppress apoptosis, induce cell proliferation, and promote differentiation of uterine cells in preparation for embryo implantation. We will also present data that compares the mechanisms by which the natural estrogen 17β-estradiol and the phytoestrogen genistein elicit their effects in the uterus. Our analysis defines how estrogen receptor ligands coordinate a transcriptional program that results in proliferation and differentiation of a complex organ and provides a paradigm for understanding the modes of action of other toxicants that target nuclear hormone receptors. 92 CHROMATIN CHANGES IN LEUKEMIAS Pier Giuseppe Pelicci. European Institute of Oncology, Department of Experimental Oncology, 20141 Milan, Italy Recent evidence suggest that leukemia-associated fusion proteins proteins induce pathogenetically relevant changes of chromatin structure in target cells. Specifically, aberrant recruitment of differ- s28 Workshop W8. Jet fuel toxicity and exposure issues ent chromatin modifiers (histone-deacetylases, histone-methylases, DNA-methyltransferases) to target promoters induce heterochromatin formation and transcriptional silencing. The leukemic phenotype can be reverted using small inhibitory compounds which target chromatin. Recent data from my laboratory will be discussed. In particular, I will focus on the biological activities of the APL-associated PML-RAR fusion protein. W8 Jet fuel toxicity and exposure issues 93 RISK FACTORS OF JET FUEL COMBUSTION PRODUCTS I. Tesseraux. UMEG, Großoberfeld 3, D-76135 Karlsruhe, Germany Air travel is increasing and airports are being newly built or enlarged. So, concern is rising about the exposure to toxic combustion products in the population living in the vicinity of large airports. Jet fuels are well characterized regarding their physical and chemical properties. Health effects of fuel vapours and of liquid fuel are described after occupational exposure and in animal studies. Less is known about combustion products of jet fuels and exposure to those. Aircraft emissions vary with the engine type, the engine load and the fuel. Among jet aircrafts there are differences between civil and military jet engines and their fuels. Combustion of jet fuel results in CO2 , H2 O, CO, C, NOx , particles and a great number of organic compounds. Among the emitted polycyclic aromatic hydrocarbons (PAH) no compound characteristic for jet engines (tracer) could be detected so far. Jet engines do not seem to be a source of halogenated compounds or heavy metals. In contrast to vehicle emissions there are hardly any data on the toxicology of jet engine emissions. According to analyses of their chemical composition, however, they contain various toxicologically relevant compounds including carcinogenic substances. A comparison has been reported between organic compounds in the emissions of jet engines and diesel (similar to jet fuel) vehicle engines. No major differences in the composition were found. Soot and particles are released to a lesser degree by jet engines compared to diesel engines. Risk factors of jet engine fuel combustion products can only be named in context of exposure data. Using available monitoring data a risk assessment approach is demonstrated for the population living around large airports. 94 EXAMINATION OF VARIOUS BIOMARKERS MEASURING GENOTOXIC ENDPOINTS FROM BARCELONA AIRPORT PERSONNEL D. Anderson 1 , M. Pitarque 2 , A. Creus 2 , R. Marcos 2 . 1 Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, BD7 1DP, United Kingdom, 2 Grup de Mutagenesi, Departament de Genetica i de Microbiologia, Edifici Cn, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain Three different biomarkers: micronuclei (MN), sister-chromatid exchanges (SCE), and the Comet assay, were used to evaluate different kinds of genetic damage in peripheral blood lymphocytes from thirty four male workers at Barcelona airport. They were exposed to low levels of hydrocarbon and jet fuel derivatives. The control group consisted of eleven unexposed men. Ras p21 protein levels were also investigated in plasma, in order to evaluate whether this product of the ras gene could serve as a suitable potential marker of carcinogenic pollution in occupationally exposed cohorts. MN and SCE analyses failed to detect any statistically significant increase in the airport workers when compared with the controls, and in fact, the frequency of binucleated cells with MN in the exposed group was significantly lower than that obtained in the control. However, slight but significant differences in the mean comet length and genetic damage index were observed between the exposed and control groups when using the Comet assay. There were no statistically significant differences between both groups in p21 plasma levels. Smoking was shown to affect significantly both SCE and high frequency cells (HFC) in the exposed group. 95 LOCAL AND SYSTEMIC TOXICITY OF JP-8 FROM CUTANEOUS EXPOSURES J.N. McDougal. Department of Pharmacology and Toxicology, Wright State University School of Medicine, Dayton, Ohio, USA JP-8 jet fuel is a version of the commercial jet fuel, Jet A, with additives designed to enhance performance characteristics. JP-8 is a complex mixture of primarily aliphatic (but also aromatic) hydrocarbons that varies slightly in composition from batch to batch. There is potential for dermal exposure to jet fuels with personnel involved in aircraft refueling and maintenance operations. Cutaneous exposures have the potential to cause skin irritation, sensitization or skin cancer. JP-8 is recognized to be more irritating to the skin than the previous jet fuel, JP-4. JP-8 has been shown to alter skin condition and cause molecular changes in the skin of laboratory animals. The local lymph node assay has been used to assess skin sensitization potential. The mechanisms of some of these effects have been investigated in intact skin and cultured skin cells. Hydrocarbons have also been shown to cause skin cancer with repeated application to the skin. Additionally, there is concern about systemic toxicity from dermal exposures to jet fuels, such as JP-8. Assessing risks from systemic absorption of hydrocarbon components is complex because most of the components are present in the mixture in small quantities (less than one percent). The effect of the fuel as a vehicle, different rates of penetration through the skin and different target organ toxicities all complicate the assessment of the hazards of cutaneous exposures. There have been studies in rodents that show changes in the immune system with cutaneous exposures but few studies that show systemic toxicity with cutaneous exposures. The purpose of this presentation is to review studies of local and systemic toxicity and summarize the risks. s29 Poster Sessions P1 Allergy sensitisation 96 IMPROVED RESPONSIVENESS OF LANGERHANS/DENDRITIC CELLS TO ALLERGENS J. Sheasgreen, S. Ayehunie, S. Lamore, R. Lappen, K. Bellevance, M. Klausner. MatTek Corporation, 200 Homer Avenue, Ashland, MA 01721 Langerhans cells (LC) are immature dendritic cells (DC) that are highly specialized antigen-presenting cells (APC) located in the skin, mucosa, and lymphoid tissues. LC play a key role in the induction phase of contact allergenicity, and hence it may be possible to develop an in vitro LC-based assay for contact sensitization. The difficulty in harvesting and the short survival time of LC in culture has prevented researchers from widespread use of LC. Although improvements have been made, generating large number of cells remains a limiting factor and the functionality and cytokine production capacity of cells in response to different stimuli is not consistent. Here we report a new method of generating LC from CD34+ progenitor cells harvested from umbilical cord blood. The generated LC were expanded 200 fold and they expressed CD1a and HLA-DR, characteristic of LC. LC were cultured for up to 41 days with no significant changes in CD1a and HLA-DR expression. Transmission electron microscopy showed the presence of Birbeck granules, a key ultrastructural marker of LC. Upon stimulation with lipopolysaccharide and phorbol 12-myristate 13-acetate, the LC showed a reproducible (n = 4), high level of gene and protein responsiveness in terms of IL-12, MIP-1α, MIP-3α, IL-6, and TNF-α expression. The generated cells were infectable with HIV and they were able to stimulate allogeneic T cells. In conclusion, we have developed a method to harvest and culture functional LC expressing key LC markers. These cells have longer life span in culture and can be used in: 1) allergenicity, 2) viral infection, 3) antigen presentation, 4) immuno-therapeutic, and numerous other studies. 97 ANALYSIS OF THE IN VITRO ACTIVATION OF DENDRITIC-LIKE CELLS EXPOSED TO POTENTIAL SENSITIZERS Pierre Aeby 1 , Christoph Wyss 1 , Heinz Beck 1 , Carsten Goebel 2 . SA, (Research company of Wella AG) Marly, Switzerland, 2 Department of product safety, Toxicology, Wella AG, Darmstadt, Germany 1 Cosmital In vitro sensitization tests are needed to identify the relevant aspects of the complex interactions of a chemical with the different compartments of the immune system. Furthermore, they receive public interest since animal testing should be avoided whenever possible. Here we propose a new approach for an in vitro test system, considering the following essential properties of sensitizing chemicals: skin penetration, protein binding, metabolism and dendritic cell (DC) activation. Activation of immature DCs derived from peripheral blood monocytes was evaluated by flow cytometric analysis of CD86 positive cells and quantitative measurement of the mRNA expression of “classical” (interleukin-1β) as well as newly identified activation markers (Aquaporin P3) using the Lightcycler® real time PCR system. Bioavailability was evaluated by in vitro skin penetration measurement using the pig skin model and the influence of skin metabolism was estimated using selected enzymatic activities if indicated. Aromatic amine derivatives and the known sensitizer 2,4,6-trinitrobenzenesulfonic acid were tested according to that strategy and induced substantial modulation of DC activation markers reflecting their potential sensitizing properties. The irritant sodium lauryl sulfate did not trigger any relevant response. The in vitro data for DC activation and skin penetration were compared to the results of an in vivo murine local lymph node assay. We conclude that using adequate culture conditions and measurement time points, potential sensitizers can be identified due to their capacity to induce specific DC activation in vitro, when an estimation of the bioavailabilty is possible. Furthermore, we propose a concept to integrate in vitro protein binding and in vitro metabolism into a DC based in vitro test system. 98 PHENOTYPE ALTERATIONS IN CD34-DERIVED DENDRITIC CELLS AFTER EXPOSURE TO CONTACT ALLERGENS AND IRRITANTS R.L. Van Den Heuvel, A.C.A. De Smedt, G.E.R. Schoeters. Department of EnvironmentalToxicology, Vito, Mol, Belgium One of the alternatives to identify the sensitizing potency of new products is the in vitro culture of human DC. DC are highly specialized antigen-presenting cells of the immune system, which play a crucial role in the induction of allergic reactions. As immature cells, DCs are specialized in capturing and processing exogenous antigens and are weak in priming T cells. During their maturation process, DCs are characterized by different phenotypes and functions. Following maturation, there is decreased uptake, enhanced costimulatory function (up-regulation of co-stimulatory molecules) and activation of naive T cells. Immature DCs were generated in vitro from cord blood CD34+ precursors (CD34-DC). The aim of our study was to assess the phenotype of in vitro CD34-DC after 24 hour incubation with allergens (NiCl2 : 100, 300 µM; DNCB (dinitrochlorobenzene):5, 10 µM; DNFB (dinitrofluorobenzene):6, 10 µM; DNBS (dinitrobenzenesulfonicacid):1000, 1500 µM) or the irritants (SDS: 0.005, 0.001%; BC (benzalkoniumchloride): 0.003, 0.0054%). After exposure the expression of surface markers (CD86, CD83, HLA-DR) was analyzed using flowcytometry. The percentage CD86 positive cells was significantly up regulated after exposure to NiCl2 , DNCB and DNFB. DNBS, DNCB, DNFB and NiCl2 exposure resulted in a significant increase in the mean fluorescence intensity (mfi) of CD86. DNCB and NiCl2 exposure induced a significant increase in the expression of HLA-DR (both expressed as mfi or % positive cells). The percentage of CD83 positive DC increased significantly after DNCB and NiCl2 exposure. No alterations in the DC phenotype were detected after irritant exposure. Results indicate that the in vitro model using CD34-derived dendritic cells (DC), has the potency to distinguish between allergens and irritants based on altered phenotypic characteristics. 99 THE CONTACT SENSITIZER NICKEL SULFATE ACTIVATES THE TRANSCRIPTION FACTORS NF-KB AND AP-1 AND INCREASES THE EXPRESSION OF THE INDUCIBLE ISOFORM OF NITRIC OXIDE SYNTHASE IN SKIN DENDRITIC CELLS. M.T. Cruz 1,2 , A.L. Vital 1,2 , M. Gonçalo 3 , C.B. Duarte 2 , M.C. Lopes 1,2 . 1 Faculty of Pharmacy, 2 Center for Neuroscience and Cell Biology, 3 Faculty of Medicine (Department of Dermatology), University of Coimbra, 3000 Coimbra, Portugal Recent evidences suggest that nitric oxide (NO), produced by the inducible nitric oxide synthase (iNOS), is involved in skin allergic dermatitis. The contact sensitizer nickel is one of the most frequent causes of skin allergy, but the effect of this sensitizer on NO production by skin dendritic cells (DC) was never addressed before. The aim of this study was to know whether nickel sulfate (NiSO4 ) activates the transcription factors NF-kB and AP-1 and induces iNOS protein expression, in a fetal skin-derived dendritic cell line (FSDC). The results of this study showed that FSDC produces nitric oxide (NO) in response to the contact sensitizer NiSO4 and increases the expression of the iNOS protein, as evaluated by immunofluorescence and Western blot analysis. The sensitizer NiSO4 increased cytoplasmic iNOS expression to 131.9±10.3% of the control and nitrite production, as assayed by the Griess reaction, s30 Poster Session P1. Allergy sensitisation to 125.2±9.1% of the control (50 µg/ml NiSO4 ). The results of transcription factors activation, as evaluated by electrophoretic mobility shift assay (EMSA), indicated that exposure of FSDC to NiSO4 activates the NF-kB and AP-1. Together, these results indicate that NiSO4 activates the NF-kB and AP-1 and increases iNOS expression in skin dendritic cells. Supported by FCT of 3. The EC3 vaules calculated were 25.1% for eugenol, 62.1% for DHEA and 2.3% for DHEB. Collectively these data suggest that assessments of relative potency deriving from non-RI LLNA responses correlate well with evaluations based on GPMTdata. These investigationssuggest that the non RI LLNA may be of value when there is a need to avoid radioisotopes. 102 100 BISPHENOL A SHOWED NO SKIN SENSITIZING OR PHOTOALLERGIC POTENTIAL IN A MODIFIED LOCAL LYMPH NODE ASSAY IN MICE. H.-W. Vohr, H.J. Ahr, G. Stropp. Bayer Health Care, Toxicology, Aprather Weg, Wuppertal, Germany Introduction: Bisphenol A (BPA) is a chemical intermediate for the production of e.g. polycarbonates and epoxy resins. There are publications regarding the skin (photo)sensitizing properties of BPA in early animal experiments and positive patch test reactions in humans. However, most of the results those animal studies are not definitive and case reports in humans are rare. In addition, some of these assessments are based on non-validated test systems or on questionable results. Thus, the purpose of this study was to verify whether BPA had a skin sensitising or photoallergic potential by utilizing a validated test system according to established regulatory guidelines. Methods: First we used a modified Local Lymph Node Assay (LLNA), i.e. the Integrated Model for the Differentiation of Skin reactions (IMDS; 1,2), to test for skin sensitizing or irritating properties of BPA. In a second study we used the so-called UVIMDS to determine photo-reactive properties of BPA. Female outbred NMRI mice (6 animals per group) were treated with the vehicle alone (DAE433), 3%, 10% or 30% BPA on three consecutive days. 30% BPA was the maximum feasible concentration which did not show acute skin reactions during the induction phase. For the investigation of photo-reactivity, mice were irradiated with 20J UV-A/cm2 directly after each application. Results: In contrast to positive control mice neither a skin sensitizing or photoallergic nor a irritating or photoirritating potential could be determined for BPA up to the highest concentration. Conclusion: The results obtained in these validated test systems were not able to reveal any skin sensitizing or photoallergic potential of Bisphenol A. 101 ASSESSMENT OF THE SKIN SENSITIZATION POTENCY OF EUGENOL AND ITS DIMERS USING A NON-RADIOISOTOPIC MODIFICATION OF THE LOCAL LYMPH NODE ASSAY M. Takeyoshi 1 , S. Noda 1 , S. Yamazaki 2 , H. Kakishima 2 , K. Yamasaki 1 , I. Kimber 3 . 1 Chemicals Assessment Center, Chemicals Evaluation and Research Institute, Hita, Japan, 2 Cosmetic Laboratory, Kanebo Ltd., Odawara, Japan and 3 Syngenta Central Toxicology Laboratory, Macclesfield, UK The murine local lymph node assay (LLNA) is accepted as the standalone test for skin sensitization and equivalent to guinea pig prediction tests such as the guinea pig maximization test (GPMT). We have investigated further a modification of this assay (nonradioisotopic [non-RI] LLNA) which in place of tritiated thymidine to measure lymph node cell proliferation, employs incorporation of BrdU (5-bromo-2’-deoxyuridine). Using the non-RI LLNA and also the GPMT, we have examined the skin sensitizing activity of eugenol (a known human contact allergen) and it’s dimers 2,2’-dihydroxyl-3,3’-dimethoxy-5,5’-diallyl-biphenyl (DHEA), and 4,5’-diallyl-2’-hydroxy-2,3’-dimethoxy phenyl ether (DHEB). On the basis of GPMT assays eugenol, was classified as a mild skin sensitizer, DHEA as a weak skin sensitizer and DHEB as an extreme skin sensitizer. In the non-RI LLNA all chemical were found to give positive responses, insofar as each was able to provoke a stimulation index (SI) of 3 or greater at one or more test concentrations. The relative skin sensitizing potency of these chemicals was evaluated in the non-RI LLNA by derivation of an EC3 value; this being the estimated concentration of chemical required to provoke an SI THE MOUSE LOCAL LYMPH NODE ASSAY: COMPARISON OF CELLULAR PROLIFERATION RESPONSE (CPR) USING VARIOUS VEHICLES (1996–2002) E.L. Moore, L.A. Waterson, A.D. Bull. Huntingdon Life Sciences, Huntingdon, Cambridgeshire, UK The mouse local lymph node assay (LLNA) is a refined in vivo alternative to traditional guinea pig tests for assessment of potential to cause skin sensitisation. A new OECD guideline (OECD 429) recognises the LLNA as a valid alternative whilst the EPA OPPTS 870.2600 revised skin sensitisation guideline refers to the LLNA as the preferred alternative method. These guidelines provide a list of vehicles ranked in order of preference; acetone:olive oil (4:1 v/v) (AOO), dimethylformamide (DMF), propylene glycol (PPG) and dimethyl sulfoxide (DMSO). Historically the aqueous vehicles, acetone (AC) and absolute alcohol (AA), have been used for such studies. The objective of this review was to determine whether the choice of vehicle affects the background cellular proliferation response (CPR). In addition, as AOO is the preferred vehicle, assessment was made as to the consistency of the CPR over a 7-year period. The protocols followed a standard design using pooled lymph node values; animals used were 6 to 8 or 8 to 12 week old female CBA/Ca strain mice obtained from Harlan UK Ltd. The 206 studies were performed between 1996 and 2003. The CPR was quantitatively measured as disintegrations per minute (DPM) after incorporation of 3 H-methyl thymidine. Comparison of the various vehicles with AOO revealed that the mean CPR values for DMSO were higher, for DMF CPR values were generally similar, whilst for PPG, AA and AC CPR values were lower. Data for AOO over time indicated that since 1999 the background CPR showed a declining trend; parallel trends were noted for the other vehicles. In addition, AOO data for 2001 and 2002 were consistently lower than previous years. In conclusion, the analyses show that the OECD test guideline preferred vehicles AOO and DMF produce similar background CPR and over time AOO CPR values declined 103 INCLUSION OF FLOW CYTOMETRIC ENDPOINTS IN THE LOCAL LYMPH NODE ASSAY D.F. Lanham 1 , M. Wragg 2 , S. Signs 2 , D. Coleman 3 , L.A. Waterson 3 , M.G. Wing 1 . 1 Department of Experimental Biology, Huntingdon Life Sciences, Cambridgeshire, U.K., 2 The Lubrizol Corporation, Wickliffe, Ohio, U.S.A, 3 Department of Short Term Toxicology, Huntingdon Life Sciences, Cambridgeshire, UK The mouse local lymph node assay (LLNA) may be used to identify chemical contact sensitizers by measuring draining lymph node proliferation following topical exposure. However, reliance on proliferation may not distinguish true sensitizers from strong irritants. Inclusion of flow cytometry to detect changes in the cellular composition or activation status of the draining lymph nodes has been claimed to provide additional information, to assist with risk assessment. The draining lymph nodes were analysed using flow cytometry and proliferation by, 3 H-thymidine incorporation, following 3 daily topical applications of either the sensitizer oxazolone (OXZ) (0.1 w/v), the weak sensitizer hexyl cinnamic aldehyde (HCA) (50% w/v), or the irritant sodium lauryl sulphate (SLS) (25% w/v). Five days after the first application the draining lymph nodes were excised and processed for analysis by flow cytometry or 3 H-thymidine incorporation. The sensitizers OXZ and HCA gave Stimulation Indices (SI) of 64 and 22.2 respectively, with SLS giving a value of 7.5. Since the cut-off value for sensitizers is accepted to be >3, the irritant SLS could not be distinguished from HCA and thus would be classified as a sensitizer. Lymphocyte analysis demonstrated that all three chemicals resulted in a decrease in the T to B lymphocyte (T:B) Poster Session P1. Allergy sensitisation ratio compared to their vehicle controls, with a ranking OXZ < HCA < SLS. Analysis of lymphocyte activation markers revealed that B lymphocyte MHC class II and the proportion of lymph node cells co-expressing CD69+/Ia+ were elevated above the vehicle controls only for OXZ. In summary, activation marker analysis and proliferation were unable to distinguish the strong irritant from the weak sensitizer, however measurement of the T:B lymphocyte ratio was able to detect a quantitative difference between all three chemicals. Further analysis of strong irritants and weak sensitizers will be necessary to confirm the utility of this method and to establish a suitable cut-off ratio. 104 two days of treatment with Oxazolone, while transcripts were nearly undetectable after SDS administration. Furthermore, we found a time dependent change in MCP-1 and IP-10 expression. After the first application of test compounds the expression of both genes was especially triggered by SDS treatment. 24 hours after the second application we found a significiantly stronger upregulation of both genes after administration of Oxazolone, while no additional effect was observed after the second treatment with SDS. The results of the DNA expression analysis corroborates with the parameters which are commonly measured within the IMDS (e.g. cell proliferation and earthickness). Furthermore the differential expression of genes which are characteristic for the local immune network may help to establish a reliable short term in vitro assay for the differentiation between skin sensitizing compounds and irritants. EXPERIENCE IN THE ROUTINE USE OF THE LOCAL LYMPH NODE ASSAY (LLNA) B. Griffon, X. Manciaux, S. de Jouffrey, R. Forster. CIT, Evreux, France The Local Lymph Node Assay (LLNA) is an in vivo alternative to traditional Guinea pig testing methods for assessment of the potential of chemicals to cause skin sensitization. In this assay, sensitizing activity is measured using parameters of the induction phase, rather than of the elicitation phase, by quantification of the proliferative responses in draining lymph nodes after topical application of the test item. Testing procedures have been formalized in a recently approved OECD guideline (No. 429). In this guideline, skin sensitizers are defined as test items which, at one or more tested concentrations, elicit a 3-fold or greater increase in the incorporation of radioactivity (a measure of cellular proliferation) compared with vehicle controls, together with consideration of a dose-response relationship. As the method is described in the OECD guideline, the LLNA and its interpretation seem easy. However, based on our experience of over 150 individual studies performed in the last 12 months, it appears that interpretation may sometimes be problematic. We have previously shown that definitive classification of a test item as a skin sensitizer cannot be based only on the criterion of a stimulation index (SI). Integration of a quantitative measure of skin irritation (for example, by measurement of ear thickness) in the study protocol is necessary to permit unequivocal interpretation of the results since it is well established that an irritant action can give rise to “false positive” increases in cellular proliferation. More recently, in routine studies, we have been confronted by various data sets for which interpretation was problematic. For example, an SI greater than 4 at the highest tested concentration but accompanied by slight irritation? An SI exceeding 2 at the highest feasible dose-level, without evidence of dose-relationship? In our experience the LLNA is an informative and robust test but routine use requires careful attention to the interpretation of results. 105 s31 A HIGH SENSITIVE DNA ARRAY METHOD FOR THE CHARACTERIZATION AND DISCRIMINATION OF DIFFERENT LOCAL IMMUNE REACTIONS IN MICE E. Heisler, H.-J. Ahr, H.-W. Vohr. Bayer Health Care, Institute of Genetic & Molecular Toxicology, Wuppertal, Germany The modified local lymph node assay (Integrated Model for Differentiation of Skin reactions; IMDS) is a reliable and valid method to discriminate the chemical induced hypersensitivity from skin irritancy. However the different molecular and cellular mechanisms leading to an antigen dependent immune reaction on the one hand, or to an unspecific inflammatory process on the other, are very complex and still remain unclear. Therefore we used a new high sensitive DNA array method based on Planar Wave Guide Technology to investigate the gene expression in cells from the local draining lymph nodes of outbred NMRI mice after topical administration of different test substances. Oxazolone was selected as a well characterized sensitizing agent while SDS was choosen because of its specifically irritating potential. The arrays we used comprised a representative choice of seventy immune relevant genes encoding cell surface markers, cytokines, chemokines and their receptors. Our attention was especially focussed on the expression of TARK/CCL 17 and MIP-3 alpha/CCL20, because both were expressed during the first 106 CYTOKINE PROFILING OF CHEMICAL ALLERGENS: INTER-ANIMAL VARIATION R.J. Dearman, I. Kimber. Syngenta Central Toxicology Laboratory, Macclesfield, Cheshire, UK Topical exposure of BALB/c strain mice to chemical allergens stimulates cytokine secretion profiles consistent with the selective activation of divergent subpopulations of T lymphocytes. Lymph node cells (LNC) isolated after treatment with the contact allergen 2,4-dinitrochlorobenzene (DNCB) display a type 1 cytokine secretion phenotype with relatively high expression of interferon γ (IFN-γ) and interleukin (IL) 12, but low levels of the type 2 cell products IL-4, IL-5, IL-10 and IL-13. Exposure to the respiratory allergen trimellitic anhydride (TMA) results in the converse type 2 cytokine expression pattern. In order to provide for sufficient supernatant to analyze the complete panel of cytokines by enzyme-linked immunosorbant assay, lymph nodes have been pooled on an experimental group basis. Analyses of inter-experimental differences in cytokine secretion of these populations revealed that DNCB-stimulated LNC invariably expressed significantly higher levels of type 1 cytokines whereas TMA-activated LNC produced significantly higher levels of type 2 cytokines. It is now possible to measure multiple cytokines in small volumes, using flow cytometric techniques, allowing cytokine secretion patterns to be measured for individual animals. Cytokine profiles have been analyzed for DNCB- and TMA-stimulated LNC, where lymph nodes have been pooled on an individual animal basis (n=5). Very marked type 1 and type 2 cytokine secretion profiles, respectively, were observed for LNC isolated from each of the DNCB- and TMA-exposed mice. LNC derived from DNCB-treated animals produced significantly more IFN-γ (p<0.05) and IL-12 (p<0.01) than did those isolated from TMA-treated mice, whereas exposure to TMA stimulated significantly higher levels of IL-4, IL-5 and IL-10 (p<0.01). These data suggest that there is little inter-animal variation in the ability of DNCB and TMA to elicit distinct type 1 and type 2 cytokine secretion profiles and demonstrate that similar cytokine phenotypes are detected whether lymph nodes are pooled on a group basis or individual animals are assessed. 107 ALLERGIC REACTIONS TO TOPICAL CHAMOMILE M. Saeedi 1 , K. Morteza-Semnani 2 . 1 Department of Pharmaceutics, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran, 2 Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran The genus Paederus has a world-wide distribution and comprises several hundred species and is most common during May through September in Iran. There appears to be no specific treatment for the Paederus dermatitis, thus we decided to evaluate herbal gel from chamomile and myrrh on Paederus dermatitis. Chamomile and myrrh have anti-inflammatory, anti-bacterial and anti-fungal activity in Iranian herbal medicine, thus we selected these plants for treatment of Paederus dermatitis. Data collected in one research showed that the most frequent allergic reactions were due to five plants that chamomile extract had the fourth step. Ethanolic chamomile extract and the tincture of myrrh were prepared, then different gels containing chamomile (3.5%), chamomile (3.5%)-myrrh (1%) and myrrh (1% and 2%) were formulated and the best formulations were se- s32 Poster Session P1. Allergy sensitisation lected for preliminary clinical trial study in comparison with gel base. The results showed severe allergic reactions in 55% and 85% of patients which received topical gel containing chamomile (3.5%)-myrrh (1%) and chamomile (3.5%), respectively. The inflammation, erythema and itching were the main allergic reactions in investigated volunteers. These side effects were observed in 10% and 5% of patients, which received myrrh 2% and 1% topical gel, respectively. The results showed that the preparation containing 1% myrrh had the fewer side effects and the most effects on treatment of Paederus dermatitis. 108 EXPOSURE TO ENZYMES IN DETERGENTS DOES NOT LEAD TO THE DEVELOPMENT OF IgE ANTIBODY AMONG CONSUMERS. K. Sarlo 1 , B. Kirchner 1 , R. Parker 2 , E. Troyano 3 , C. Rodriguez 3 , R. Stachlewitz 4 . The Procter and Gamble Company, 1 Cincinnati, Ohio, 2 Egham, GB, 3 Brussels, Belgium and 4 Abbott Bioresearch Center, Worcester, MA Protease and amylase enzymes from Bacillus organisms have been safely used in detergent products for over 30 years. These products are used for machine and hand laundry of garments by millions of people around the world. The safety of enzymes in detergents has been supported by clinical studies and safe market history. Tests on 2,500 allergy clinic patients in the early 1970’s showed that none had IgE antibody to the enzyme used in granule detergent (Clin Exp All, 1973). A 2-year study of atopic Filipino women using enzyme detergents for hand wash showed that none developed IgE antibody (JACI, 1997). While testing the safety of new enzyme-containing body lotions, we screened 2,500 women for IgE antibody to the protease BPN’. Four women had IgE antibody that recognized BPN’ in the skin prick test (SPT) but only 1 used a detergent that contained this enzyme. None of the 4 was SPT positive to other enzymes used in detergent products. None of the 4 reported symptoms when using detergent products. A subset of women (515) was tested with other Bacillus proteases and amylases found in many detergent products; all were SPT negative to these enzymes. An analysis of SPT data from detergent plant employees showed that none of several hundred had IgE antibody to BPN’ or other enzymes prior to their work exposure. These data confirm the history that enzymes in detergents do not lead to IgE antibody. However, there is an increase use of enzymes in other products (cosmetics, spray cleaners). These uses indicate a need for industry to continue to monitor the population to ensure expanded uses of enzymes in consumer products do not lead to the risk of allergy. 109 DEVELOPMENT OF IgE ANTIBODY TO ENZYMES IN PERSONAL CARE PRODUCTS K. Sarlo 1 , J.D. Innis 1 , R. Parker 2 , G. Adamson 3 . 1 The Procter and Gamble Company, Cincinnati, Ohio, 2 Egham, GB, and 3 Avon Products, Suffern, NY Enzymes have been safely used in detergent products for over 30 years but their use in personal care products (PCP) for dry skin, shine control, etc. is relatively novel. Consumer exposures to enzymes via PCP’s are different to those from detergents; so simple extrapolation from the safe use of enzymes in detergents to PCP’s is not appropriate. The safety program for enzymes in PCP’s includes exposure assessments and robust clinical testing. Robust clinical testing is necessary to assess the potential of exposure to enzyme in a PCP to induce enzyme specific IgE. Exposure can occur via inhalation, contact with mucosal surfaces and contact with skin. Exposure of enzyme to normal skin carries a very low risk of inducing IgE. Testing with up to 1000ppm protease enzyme under occluded patch in repeat insult patch tests did not lead to positive skin prick test (SPT) responses among several hundred normal and atopic test subjects. Prospective clinical tests of enzymecontaining PCP’s were conducted to assess the risk of developing IgE antibody under product use conditions. Four of 61 test subjects using enzyme-containing body soap developed IgE antibody to the enzyme between 4 to 6 months of use of the soap in the shower (JACI, 1998). Residual enzyme left on the skin after application of prototype body lotion may aerosolize in a shower. An 18-month clinical study showed that 4 of 864 test subjects developed IgE antibody to a protease enzyme used in a body lotion. The lotion was used intermittently (5 days/month). Daily use may lead to a higher rate of subjects with IgE antibody. Neither the body soap nor the body lotion was commercialized since it was possible to generate specific IgE antibody to enzyme under typical use of these products. 110 SAFETY PROFILE OF A DISH-WASHING LIQUID CONTAINING A BPN’ PROTEASE E. Troyano 1 , D.A. Page 2 , F. Bielen 1 , K. Sarlo 2 , R. Parker 3 , C. Rodriguez 1 . The Procter and Gamble Company, 1 Brussels, Belgium, 2 Cincinnati, Ohio and 3 Egham, GB The addition of a small level of a BPN’ protease to a dish-washing liquid has been found to pose no concerns regarding the potential to cause respiratory sensitization or adverse skin effects during normal consumer use of the product. Inhalation exposures to enzymes during the normal use of a dishwashing liquid occur primarily from aerosols generated during the sink fill burst and the washing-up task. The exposures under these conditions were found to be similar to the exposures generated during traditional laundry tasks and have been assessed to pose no additional risk above those encountered in normal use of enzymecontaining laundry products. The results of a 12-month in-home use study conducted among atopic subjects further confirmed the exposure-based risk assessment: 0/157 test subjects had IgE antibody to BPN’ in the skin prick test (SPT) after 12 months on study. Since proteases can cause skin irritation, several clinical studies representative of exaggerated and/or realistic consumer exposures were conducted to confirm the skin safety profile of the product. Hand skin condition was assessed for redness and dryness by trained skin graders and dermatologists. Transepidermal water loss (TEWL) was also measured. The studies included people with normal skin, atopics, the elderly and subjects with self-assessed sensitive skin ad/or dry skin. No significant differences on clinical hand grades scores or TEWL measurements were noted among the more than 500 test subjects using enzyme- and nil enzyme-containing dishwashing liquids. The results showed no adverse skin effects due to the use of the BPN’-containing dishwashing liquid. 111 A MULTI-LABORATORY EVALUATION OF A COMMON IN VITRO PEPSIN DIGESTION ASSAY PROTOCOL USED IN ASSESSING THE SAFETY OF NOVEL PROTEINS D.J. Esdaile 4 , G.A. Bannon 1 , M. Bartels 2 , R.J. Dearman 3 , T.J. Fu 5 , C.M. Glatt 6 , N. Hadfield 3 , S.L. Hefle 7 , J.R. Heylings 3 , R.E. Goodman 1 , B. Henry 8 , C. Herouet 4 , M. Holsapple 11 , G.S. Ladics 6 , T.D. Landry 2 , S.C. MacIntosh 8 , E.A. Rice 1 , L.S. Privalle 9 , H.Y. Steiner 9 , R. Teshima 10 , K. Thomas 11 , R. van Ree 12 , M. Woolhiser 2 , J. Zawodny 8 . 1 Monsanto Co., St. Louis, MO, USA, 2 The Dow Chemical Co., Midland, MI, USA, 3 Syngenta Central Toxicology Laboratory, Alderley Park, UK, 4 Bayer CropScience, Sophia Antipolis, France, 5 U.S. Food and Drug Administration, National Center for Food Safety and Technology, Summit-Argo, IL, USA, 6 DuPont, Co., Newark, DE, USA, 7 University of Nebraska, Lincoln, NE, USA, 8 Bayer CropScience, Research Triangle Park, NC, USA, 9 Syngenta Biotechnology, Inc., Research Triangle Park, NC, USA, 10 National Institute of Health Sciences, Tokyo, Japan, 11 ILSI Health and Environmental Sciences Institute, Washington, DC, USA, 12 Sanquin Research, Amsterdam, The Netherlands Evaluation of the potential allergenicity of food proteins derived from genetically modified crop plants has conventionally involved the use of a decision tree that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no commonly accepted protocol to assess the digestibility of proteins using simulated gastric fluid, although it is a requirement for regulatory submission. Potential variations in assay parameters include: pH, pepsin purity, pepsin-to-target protein ratio, target protein purity, and method of detection. A standard set of proteins was tested in nine independent laboratories, using a common digestion protocol, with each laboratory Poster Session P1. Allergy sensitisation utilizing their normal electrophoreses and detection methods. The objective was to determine the robustness of the assay. Single batches of pepsin, and each of the following test proteins, were supplied to each laboratory: Ara h 2, β-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose disphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10 units of pepsin activity/µg test protein was selected for all tests (3:1 pepsin to protein, w:w). Assays were conducted at pH 1.2 and 2.0, at 37°C, with sampling after 0.5, 2, 5, 10, 20, 30, and 60 minutes. The reaction was stopped with NaCO3 and the protein digestibility assessed from stained gels following SDS-PAGE of digestion samples and controls. Results were relatively consistent across laboratories for the fulllength protein (91% agreement at pH1.2 and 77% agreement at pH 2.0). The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. However, the assay pH did not influence the time to disappearance of the full-length protein or protein fragments. In conclusion, these data demonstrate that this common protocol for evaluating the in vitro pepsin digestibility of proteins is robust. Furthermore, a digestion pH of 1.2 was found to be as sensitive as digestion at pH 2.0 but is more consistent between laboratories, hence pH 1.2 is recommended for use in routine pepsin digestibility testing. 112 THE INDUCTION OF ORAL TOLERANCE TO OVALBUMIN IN BALB/C STRAIN MICE. I. Kimber, C.J. Betts, H.T. Caddick, R.J. Dearman. Syngenta Central Toxicology Laboratory, Macclesfield, Cheshire, UK We have shown previously that intradermal (id) exposure of BALB/c strain mice to allergenic proteins such as peanut lectin or ovalbumin (OVA) results in the development of T helper (Th) 2 type responses culminating in the production of high titre IgE antibody. We have now investigated the influence of prior oral (gavage) exposure to OVA on the development of specific antibody responses following id administration. Groups of mice (n=5–10) were pretreated by oral gavage with 25mg OVA, 0.25mg OVA, or water alone, on day 0. Test groups were then exposed to 2% OVA via id injection into the dorsum of each ear on days 7 and 14. Control groups received OVA by gavage alone. Serum was collected on day 21 and analysed by enzymelinked immunosorbent assay or by homologous passive cutaneous anaphylaxis assay for the presence of OVA-specific IgG and IgG1, or IgE antibody, respectively. Oral exposure to OVA (25 or 0.25mg) alone did not provoke detectable IgE antibody expression, and only low titre IgG and IgG1 antibody. Intradermal administration of OVA stimulated vigorous anti-OVA IgG and IgG1 antibody production and induced relatively high titre IgE antibody (ranging from a titre of 1/16 to 1/32). Prior gavage exposure to 25mg OVA was without marked effect on IgG or IgG1 antibody responses, but completely ablated the anti-OVA IgE antibody response. Interestingly, oral pre-exposure to 0.25mg OVA resulted in somewhat increased IgG and IgG1 anti-OVA antibody titres and IgE antibody titres of between 1/32 and 1/64. These data demonstrate dose-related immunomodulating actions of oral OVA administration on IgE antibody production. 113 COMPLEXITY OF THE IMMUNE RESPONSE TO A PROTEASE AND IMPLICATIONS FOR THE SAFETY ASSESSMENT OF NOVEL ENZYME-CONTAINING PRODUCTS. E.S. Finn, A.C. Pursifull, L.C. Limardi, S. Chapoval, K. Sarlo, B.A. Kirchner, D.N. Rubingh. The Procter & Gamble Co., Cincinnati, OH Microbial enzymes have been safely used in detergent products for about 30 years. Their use is expanding in new applications such as personal care products. This may result in exposures and potential for allergy development that differ from detergent usage and, therefore, require additional safety assessment. Since these enzymes may induce IgE antibody and cause occupational allergy, understanding the immune response to such enzymes is a key step in the safety assessment of new enzyme-containing applications. s33 In studies presented here, humans and transgenic (tg) mice were evaluated for antibody responses, T cell responses and genetic susceptibility to a variant of the protease BPN’ (Y217L variant). Blood from occupationally exposed workers was tested for MHC Class II haplotype. A number of haplotypes were found among workers with IgE antibody to the BPN’ variant. The DQ8 allele in humans was statistically associated with development of IgE to the BPN’ variant (susceptible) whereas the haplotype DQ6/DR15 was associated with the absence of IgE (protection). Studies in HLA Class II tg mice also indicate that DQ8 mice generate IgG1 and IgE to the BPN’ variant following intranasal exposure. T cell epitope mapping studies in humans and DQ8 mice indicate that the BPN’ variant has a number of T cell epitopes. In humans, these epitopes vary among individuals and are most likely influenced by haplotype. T cells from humans and DQ8 mice recognize similar epitopes. Therefore, studies in humans and tg mice show that allergic responses to the BPN’ variant is complex and diverse. This increases the complexity of the safety programs needed to assure these ingredients are safe for use in personal care products. A safety program that assesses use of enzymes in new products must also include exposure assessment and robust clinical testing. 114 STATISTICAL EVALUATION OF CLASSIFICATION METHODS FOR POTENTIAL ALLERGENICITY BASED ON LOCAL ALIGNMENT D. Soeria Atmadja 1 , A. Zorzet 1 , Å. Björklund 1 , M. Gustafsson 2 , U. Hammerling 1 . 1 Division of Toxicology, National Food Administration, Uppsala, Sweden, 2 Signal and Systems Group, Uppsala University, Uppsala, Sweden Atopic allergy and other hypersensitivity reactions affect up to 20– 25% of the population in industrial nations. The prevalence of food allergy among adults within the EU is about 2% and 2–4 times higher in the paediatric population. Due to marketplace-introduction of genetically modified foods, specially designed proteins for household and skin-care use and parentally administered pharmaceutical proteins, safety assessment of allergenic potential of proteins has become a major issue. Pipeline peptides and proteins, designed for oral administration as medication, emphasises a need for more accurate assessment procedures for allergenic potential, compared to conventional schemes. Reliable prediction of the potential allergenicity of novel foods including GM foods is a significant issue within the European Commission and elsewhere. The joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology has advocated a decision-tree for the assessment of potential GM food allergenicity. Extensive testing and validation of several distinct but interrelated formats on computational classification, each based on descriptors obtained from local alignment, are described. In each case the pair of extracted FASTA3 output features was merged into vectors that fed a learning system. Key parameters of the local alignment program employed provided were tuned over a wide range, which provided valuable information on preferred settings. Three different machine learning algorithms, the k-nearest neighbour (kNN), the Linear Gaussian Classifier (LG) and the Quadratic Gaussian Classifier, were subjected to extensive evaluation as regards assortment of allergenic proteins, using the aforementioned input format. Key resources in this context are also two in-house assembled sets of amino acid sequences, one devoted to protein allergens (318 listings) and the other to counterparts carefully selected to minimise their likeliness of being associated with allergy (1007 listings). For each classification system outlined here, the assortment accuracy has been evaluated using 200 iterations of test- and training data sets. Accordingly obtained data were compared by Receiver Operating Characteristic (ROC) curve analysis. The preferred machine learning algorithms are the LG, closely followed by the kNN. The LG classifier revealed the overall best classification result when trained by a hybrid feature vector that combined the alignment score from the BLOSUM 50 substitution matrix with the alignment score from the identity matrix. To achieve a minimum allergen detection rate of 70% (with 95% confidence), the maximal rate of false alarms (classifying a non-allergenic protein as allergenic) is 12,5% (with 95% confidence). s34 Poster Session P2. Immunotoxicology P2 Immunotoxicology 115 DEVELOPMENTAL IMMUNOTOXICOLOGY (DIT) – DEVELOPMENT OF A TESTING FRAMEWORK M. Holsapple, A. Lavin. Immunotoxicology Technical Committee (ITC), ILSI Health and Enironmental Sciences Institute (HESI), Washington, DC, USA Interest in developing a guideline for the testing of DIT has been based on concerns that the developing immune system may be more sensitive than the adult system to chemical exposures, and that early exposure to a chemical may result in persistent immunosuppression. However, data comparing the developing and adult immune systems are still quite limited. Moreover, although techniques exist for assessing immune alterations in adult animals, there are currently no validated or widely accepted methods for evaluating effects on the developing immune system. To help address these issues, the ILSI HESI ITC hosted a workshop in June 2001 and a roundtable discussion in May 2003 aimed at examining possible approaches for assessing DIT. European and U.S. scientists from industry, academia, and regulatory agencies participated in both of these events. The following features were identified as key to providing a foundation for a DIT testing framework. 1. Consider incorporating methods to assess immune function as part of the already required standard developmental/reproductive toxicity studies in rats, the species of choice in spite of known developmental differences from humans. 2. Assess all critical windows of exposure at once (up to and including young adult stages) and dissect out specific windows of concern if an effect is observed. 3. Incorporate not only histopathological examination of young pups, but also immune functional testing of young adult animals, if possible. It is acknowledged that although functional assays are deemed important for assessing immunosuppression in developing animals, consensus has not yet been reached regarding what tests are most appropriate. In addition, it may be necessary to add extra animals to the testing framework in order to incorporate functional assays. In conclusion, while no specific design for a DIT protocol has been yet established, it is recognized that the above features should be incorporated in any future DIT testing framework. 116 “CELLCHIP” - A NEW TECHNOLOGY FOR IMMUNOTOXICITY SCREENING? T. Ringerike 1 , E. Ulleras 2 , G. Nilsson 2 , R. Vandebriel 3 , H. van Loveren 3 , J. Dastych 4 , M. Lovik 1 . 1 National Institute of Public Health, Division of Environmental Medicine, Oslo, Norway 2 Uppsala University, Department of Genetics and Pathology, Uppsala, Sweeden 3 National Institute of Public Health and the Environment, Laboratory for Pathology and Immunobiology, Bilthoven, The Netherlands, 4 International Institute of Molecular and Cell Biology, Laboratory of Molecular Immunology, Warsaw, Poland BACKGROUND: Until recently, most tests for immunotoxicity have been developed and validated in experimental animals. New approaches use the knowledge of cytokines, which are critical regulators of immune responses. Increasing evidence shows that at least certain types of immunotoxicity, like those leading to hypersensitivity and autoimmunity, are associated with modulation of cytokine production. The CellChip project aims at finding a new method for in vitro testing of compounds for immunotoxicity by employing changes in expression of cytokines in cell lines upon exposure. MATERIALS AND METHODS: EL4 cells (mouse lymphoblasts) were transfected with the promoter region of interleukin (IL)2 followed by the sequence for green fluorescent protein (GFP). Hence, an increased expression of cytokine will result in an increased level of GFP fluorescence. To examine depression of cytokine expression, cells were induced by incubation with 1 mikroM Ionomycin and 10 ng/mL PMA. Cells were incubated with different test compounds with already established immunotoxic potential, and GFP expression was measured by flow cytometry. Compounds were incubated with the cells for 24 hours and in 3 different concentrations. The highest dose tested was selected to result in 10% cytotoxicity, the two other doses were 10% and 1% of this dose. RESULTS: After 24 hours Cyclosporin A totally inhibited IL2 expression at all tested concentrations, while Benzocaine and Pentamidine showed a dose dependent inhibition. Finally Rapamycin seems to have inhibitory effects at the highest dose, but at the two lower doses shows a slightly increased expression of IL2 beyond the expression caused by Ionomycin/PMA. Future perspectives are to make indicator celllines, with promoter-regions from i.e. IL4, IL10, interferon (INF)γ and tumor necrosis factor (TNFα). (This project is A European Commision Sheared-cost Research projct: QLK4-CT-2000–00787). 117 ASSESSMENT OF THE NONHUMAN PRIMATE IMMUNE SYSTEM BY IMMUNOPHENOTYPING: COMPARISON OF TWO RELEVANT SPECIES WITH THE HUMAN W. Frings, G.F. Weinbauer. Covance Laboratories GmbH, Münster, Germany According to prevailing guidelines, all new drugs have to be investigated for immunotoxicity in preclinical studies. Nonhuman primates are frequently the animal model of choice for immunotoxicologic evaluation of biologics (biotechnology-derived, functional peptides) because of their high protein-homology to humans, thus obviating the problems of non-responsiveness or induction of drug-clearance by neutralizing antibodies. The cynomolgus monkey (Macaca fascicularis) and the common marmoset (Callithrix jacchus) are generally well suited and therefore often used for toxicity studies in drug development. Immunophenotyping of lymphocyte subpopulations is an accepted method for the evaluation of the immune system. We established a 4-colour immunophenotyping protocol using antihuman antibodies and investigated 75 adult cynomolgus monkeys of each sex in comparison to 10 human volunteers of each sex. In addition, we analysed the development of lymphocyte populations during the first year after birth in 5 cynomolgus monkeys. Our results indicate that identical staining protocols can be used for human and cynomolgus monkey blood, demonstrating a high comparability of the two species. The monkeys generally exhibited larger inter-species variations and had a slightly modified CD4/CD8 ratio. However, the known change in the CD4/CD8 ratio during early infant development in humans is comparable to that in cynomolgus monkeys. For reasons of availability of the test article and because some biologics only function in that species, the marmoset is another common primate model in toxicology. We obtained immunophenotyping data which prove that this species is also well suited for immunotoxicity studies, although other antibodies need to be used than for the cynomolgus monkey. For the marmoset, the CD4/CD8 ratio is comparable to that of humans whilst the ratio of T-lymphocytes to B-lymphocytes and NK-cells is slightly different. In conclusion, both the cynomolgus monkey and the marmoset appear well suited as models for determination of immunotoxicity by immunophenotyping in preclinical studies. 118 EXPLORATION OF THE PHAGOCYTIC ACTIVITY IN RATS, MONKEYS AND DOGS USING TWO HUMAN KITS F. Horand 1 , C. Cretinon 1 , F. Condevaux 1 , J. Descotes 2 . 1 MDS Pharma Services, L’Arbresle, France, 2 Poison Centre, E Herriot Hospital, Lyon, France Investigating phagocytosis is an important part of immunotoxicity evaluation since phagocytes play a crucial role in host defences against microorganisms. In this study, phagocytosis was measured by flow cytometry using 2 commercial human kits: the Phagotest® or the Bursttest® (Orpegen). These kits are used to determine the phagocytosis and the oxidative burst of leukocytes respectively, after activation by bacteria. They allow the measurement of the percentage of active cells (neutrophils + monocytes) as well as the activity per cell. The responses of rats, Cynomolgus monkeys and dogs were compared. Overall the results in monkeys and dogs were similar to those documented in man, with some variations in responses. In the Phagotest® , the proportion of phagocytic cells detected was approximately 80% in monkeys and dogs, but only 60% in rats. In the Bursttest® , two stimuli were used 1) opsonized E.coli and 2) phorbol myristate acetate (PMA). In monkeys, PMA gave response Poster Session P2. Immunotoxicology of 90% whereas E.Coli gave 70% of positive phagocytic cells, a similar difference is reported in human. In dogs and rats, the percentage of positive cells was similar with either stimuli, but only 60% of phagocytes were positive in rats compared with 80% in dogs. In addition to the percentage, both allow the mean fluorescence intensity to be calculated, estimating the number of bacteria or enzymatic activity per cell. In this study, the fluorescence intensities were not homogeneous due to sampling influences and variations in the number of bacteria used. The Phagotest® and the Bursttest® were both found to be suitable for the evaluation of phagocytosis in rats, monkeys and dogs. Monkeys and dogs, with a higher proportion of active cells, are likely to be more sensitive than the rats in this type of assessment, which confirms our previous results using chemiluminescence. 119 COMPARISON OF NK CELL ACTIVITY MEASUREMENT USING THE 51 Cr RELEASE ASSAY AND FACS SCAN ANALYSIS: EFFET OF A SINGLE IV DOSE OF ANTI-ASIALO GM1 IN THE RAT F. Condevaux 1 , J. Guichard 1 , F. Horand 1 , J. Descotes 2 . 1 MDS Pharma Services, L’Arbresle, France, 2 Poison Centre, Edouard Herriot Hospital, Lyon, France NK cell activity measurement is required by the EMEA for drug immunotoxicity evaluation. The 51 Cr release assay is the most commonly used method, but FACS analysis is an interesting alternative. It has been shown that a single intravenous dose of rabbit anti-asialo GM1 antiserum abrogates NK cell activity in rodents. Sixteen male adult Sprague Dawley rats were given 10 ml/kg of anti-asialo GM1 antiserum intravenously 3 days before sacrifice and 8 animals served as controls. Spleen cells from each animal were divided into 2 aliquots for simultaneous testing. For the 51 Cr release assay, spleen cells were incubated with 51 Cr-labelled YAC-1 murine lymphoma cells (effector-to-target-cell ratios of 200:1, 100:1 and 50:1) for 4 hours. For the FACS analysis, spleen cells were incubated with CFSE-labelled YAC-1 cells (ratio of 50:1) for 18 hours. The specific 51 Cr release was calculated from the 51 Cr released by target cells in the presence of spleen cells, the spontaneous 51 Cr release, and the maximum release in the presence of HCl 4N. The percentage of dead target cells was measured from the number of propidium iodide-positive CFSE-labelled YAC-1 cells by FACS. Both methods revealed a highly significant reduction in NK cell activity in the treated animals. The percentage of dead target cells, however, was slightly less with the FACS analysis than in the 51 Cr release assay. These results indicate a similar sensitivity of the two methods. FACS analysis offers the advantages of avoiding radioisotope use and simultaneous NK cell counting, so validation of this method is warranted as an alternative to the 51 Cr release assay. 120 CONTRASTING IMMUNOMODULATION BY MERCURIC CHLORIDE FOLLOWING ACUTE AND SUBCHRONIC EXPOSURE John Carey 1,2 , Shailendra Anoopkumar-Dukie 2 , Peter O’Keeffe 2 , Ashley Allshire 1,2 , Frank van Pelt 1,2 . 1 Environmental Research Institute; 2 Dept. Pharmacology and Therapeutics, University College, Cork, Ireland Mercury is a ubiquitous environmental xenobiotic with effects on immune system homeostasis that are not well understood. The aim of this study was to investigate modulation of critical immune parameters following acute and sub-chronic exposure to HgCl2 , using the Reporter Antigen-Popliteal Lymph Node Assay. For acute exposure, eight-week old female BALB/c mice each received a single injection containing the reporter antigens Trinitrophenyl-Ovalbumin (TNP-OVA, T cell dependent antigen) or TNP-Ficoll (T cell independent antigen) and 0–50 µg HgCl2 , in the right hind footpad. After one week the popliteal lymph node draining the site of injection was harvested. Lymph node cells were isolated, counted and analyzed for specific Antibody Forming Cells (AFC) by ELISPOT assay. The distribution of lymphocyte sub-populations was determined by flow cytometric analysis of cell surface antigens. For sub-chronic exposure, mice were orally pre-treated by gastric gavage s35 with 0–10 mg/kg/day HgCl2 for three weeks. After two weeks of oral pre-treatment, mice were injected with reporter antigen and adjuvant. Acute exposure to HgCl2 stimulated cell proliferation in the draining PLN. IgM, IgG1 and IgG2a AFC numbers were also augmented for both reporter antigens. The IgG1 sub-type predominated, indicating a Th2 type response. Consistent with ELISPOT data, analysis of cells by flow cytometry showed a clear increase in the numbers of T and B cells in the lymph nodes of mercury treated mice. These data show that HgCl2 has inherent adjuvant and immune sensitising potential. In contrast, subchronic pretreatment with HgCl2 caused a dosedependent decrease of most acute effects. For example, a 50% reduction in PLN cell proliferation was seen at 5 mg/kg/day. In addition, specific AFC are decreased by up to 80%. Therefore in this mouse model acute exposure to low micromolar HgCl2 is immunostimulatory, while sub-chronic exposure is inhibitory.Supported by the Irish Higher Education Authority (PRTLI, cycle 2) 121 BIOLOGICAL MARKERS MONITORING Pb IMMUNOTOXICITY; INTERFERENCE WITH ETHANOL E. Codorean, C. Tanase, C. Iosif, M. Neagu, G. Manda, D. Popescu, E. Raducan, M. Georgescu. Laboratory of Biochemie, Department of Pathology, Victor Babes National Institute of Pathology, Bucharest, Romania Alcoholism is frequently an additional important health problem in occupational exposure to heavy metals. In this study we investigated experimentally the interference of ethanol with Pb in generation of immunotoxic effects in rat. Some biological markers/immune parameters - lymphoid organs weight and morphology, lymphoproliferative response of splenic lymphocytes to mitogens, primary antibody response to SRBC (sheep red blood cells), delayed type hypersensitivity against BSA (bovine serum albumine) – were investigated in Wistar rats subchronically exposed to Pb, respectively to Pb+ethanol. The results indicate Pb exerting immunosuppressive effects at lower concentrations but opposite effects at higher concentration on humoral immune response and a dose-effect typed suppression on the cellular immune response and on the proliferative response to Con A (79%) and to PWM (57%). Pb immunotoxicity was sensitively but not linearly modulated by ethanol: higher variations of lymphoid organs weights (statistically significant for thymus) and morphology; additive suppression of the lymphoproliferative response to ConA (27%), but not of those to PWM (50%); higher impairement of cellular immune response but opposite effect on humoral immune response. In conclusion, the potential consequences of synergic effects of Pb and ethanol are unpredictible, their interference inducing not a simple additive immune impairement. The results indicate T lymphocytes and cellular immune response, as most sensitives to association of ethanol with Pb exposure. 122 SAFE DENTAL AMALGAM REMOVAL IN PATIENTS WITH IMMUNOTOXIC REACTIONS TO MERCURY G. Guzzi 1 , C. Minoia 2 , P. Pigatto 3 , A. Ronchi 2 , A. Gatti 2 , S. Angeleri 2 , O. Formichi 1 . 1 Italian Association Metal Research and Biocompatibility- A.I.R.M.E.B., Milan, Italy; 2 Laboratory of Environmental Hygiene and Industrial Toxicology “S.Maugeri”, Pavia, Italy; 3 Department of Dermatology and IRCCS, University of Milan, Milan, Italy Dental amalgam removal is well known to cause an high level of mercury vapour in the air in the breathing zone. This procedure consists of cutting the dental amalgam filling surfaces with a high-speed air turbine with cylindrical-shaped diamond or tungsten burr, under high stream of air-water cooled spray and continuous aspiration. Despite these safety measures the mercury vapour in the breathing zone may exceed the threshold limit value (TLV) for mercury (0.05 mg/m3 ). We evaluated a new method of dental amalgam removal which consisted of a ‘ no-touch technique’ that we called ‘lift-on technique’ because we do not touch the surfaces of the amalgam during the removal of the filling to ensure a lower mercury vapour exposure. s36 Poster Session P2. Immunotoxicology To test our hypothesis, we performed a serial measurement of the air in the breathing zone during the amalgam removal. The study was conducted from April 1998 to February 2002; we recruited 105 patients who had received diagnosis of micromercurialism. Estimation of mercury vapor during dental amalgams removal was detected by opcalite mercury vapor trap using O.S.H.A test protocol associated with FI-Hg-AAS and ICP-MS spectrometry. To measure directly the intra-oral and extra-oral mercury released from dental amalgam during the ‘lift-on technique’ we used AAS with Zeeman background correction (RA-915 Lumex). Breathing zone air concentration of mercury vapor during ‘lifton technique’ amalgam removal ranged from 0.00025 to 0.00045 mg/m3 . The background ambient mercury was stable (ranged from 0.000062 to 0.00009 mg/m3 ). By comparison, mercury vaporization during amalgam removal by standard drill-out method (ranged from 0.50 mg/m3 to 0.70 mg/m3 ) was significantly higher than ‘lift-on technique’. We next tested whether this technique would also be safe for patients with evidence of strong allergic reactions to mercury established by patch-testing with confirmed type IV reaction. We studied 12 patients with allergy to mercury after dental amalgam removal performed with ‘lift-on technique’. There was no evidence of adverse reactions after dental approach with ‘lift-on technique’ in all allergic subjects under observation. Room Hg Vapor Level before Procedure mg/m3 0.000062 – 0.00009 Hg Vapor Level During Cutting Amalgam mg/m3 Hg Vapor During “Lift-on” Technique mg/m3 0.5 – 0.7 0.00025 – 0.00045 From the results of our study, we propose a new technique for dental amalgam removal especially for individuals into high-risk subgroups: immune-susceptible (allergy to mercury compounds, oral lichen planus, angioedema, auto-immune diseases), pregnant and lactating-woman, and kidney diseases. 123 124 EFFECT OF HEPATITIS B VACCINE IN MERCURIC CHLORIDE-TREATED NZBxNZW F1 MICE G. Ravel 1 , M. Christ 1 , R. Burnett 1 , J. Descotes 2 . 1 MDS Pharma Services, L’Arbresle, France, 2 Poison Centre, Edouard Herriot Hospital, Lyon, France Vaccines are generally considered safe. However, they have been suspected of exacerbating or inducing autoimmune diseases, even though epidemiological studies have so far failed to demonstrate any causal relationship between vaccines and autoimmunity. Although the mechanisms leading to autoimmunity are not known, the role of genetic predisposition and environmental factors is widely accepted. The present study was undertaken to test the hypothesis that vaccines might lead to autoimmunity in genetically-prone mice when exposed to an autoimmunogenic chemical. Mercuric chloride was used to accelerate the spontaneous course of the autoimmune disease. Four groups (n=16) of seven weeks old female lupus-prone (NZB x NZW) F1 mice were used. Two treated groups received three intraperitoneal injections of 1 or 10 µg of a hepatitis B vaccine and 40 µg of mercuric chloride by the subcutaneous route, three time a week for 6 weeks. A comparative control group was treated with mercuric chloride only and an absolute control group was given saline by both routes. A marked increase (+29%) in serum IgG levels and a slight increase (+6%) in antinuclear autoantibody levels were seen in the mice given the high dose of vaccine. These increases were not seen in the group given HgCl2 alone, nor in the low dose group. The mean survival time, mean body weight, anti-dsDNA levels were not affected. In view of the extreme experimental conditions used, these findings cannot be directly extrapolated to man. They are, however, in keeping with the hypothesis that autoimmunity might develop following vaccination in at-risk individuals with genetic predisposition in association with chemical exposure. These results tend to support the potential value of animal models for the prediction of the risk of autoimmunity with respect to the combined influences of genetic predisposition, environmental factors and chemical exposure. IMMUNOTOXIC EFFECTS OF LEAD ACETATE IN MALE SWISS MICE I. Iavicoli 1 , G. Carelli 1 . 1 Centro di Igiene Industriale, Istituto di Medicina del Lavoro, Università Cattolica del Sacro Cuore, Rome, Italy Lead (Pb) is a ubiquitous toxic metal that can be found in all phases of inert environment and in all biological systems. In humans it can cause patho-physiological changes in many organ systems including the central nervous, renal, hematopoietic and immune systems. Previous studies have shown that Pb exerts immunotoxic effects on T lymphocytes. In our study, adult Swiss male mice were administered Pb acetate in their drinking water for six months at a moderate (40 mg/L) and at a high Pb level (400 mg/L). Controls were given drinking water without the addition of Pb acetate. During the experiment, air Pb level was determined in the environment where the mice were caged. At the end of exposure, blood Pb level was determined in all the animals to provide a biological exposure index. We also measured two type-1 cytokines (IL-2, INF-γ) and one type-2 cytokine (IL-4) in the serum and possible changes were evaluated. At 40 mg/L, we observed a blood lead level of 9.8 ± 3.3 µg/dL (mean ± sd; n=12), a significant increase in IL-4 production and a decrease of IFN-γ production compared to the controls. At 400 mg/L, the mean blood lead level was 108 ± 18 µg/dL and a further increase in IL-4 production was associated with a further decrease in IFN-γ production. The blood lead level in controls was 1.2 ± 0.3 µg/dL (mean ± sd; n=12). Our findings suggest that a moderate concentration of 40 mg/L causes a Th2 response accompanied with a suppressed Th1 response. This may indicate a very early immunotoxic effect on T cells. A high dose, 400 mg/L, produces the same effect: a further strong Th2 response that is concomitant with a suppressed Th1 response results in a greater imbalance between Th 1 and Th 2 activation. 125 CYTO- AND CHEMOKINE EXPRESSION IN THE MOUSE MACROPHAGE RAW 264.7 CELL LINE CELLS EXPOSED TO HARD- AND SOFTWOOD DUSTS J. Määttä, M.-L. Majuri, U. Andersson, H. Alenius, K. Savolainen. Finnish Institute of Occupational Health, Helsinki, Finland In addition to nasal and sino-nasal adenocarcinomas, wood dust exposure can induce many nonmalignant, mainly respiratory diseases such as allergic rhinitis, asthma, cronic bronchitis, and allergic alveolitis. To find out whether wood dust is able to influence to development of inflammatory process through macrophages, we have elucidated the effects of wood dust exposure on the cytokine and chemokine expression of mouse macrophage cell line cells (RAW 264.7). The cells were exposed to graded doses of selected hardwood (birch, beech, oak, and teak) and softwood dusts (pine and spruce). TiO2 and LPS were used as controls. The mRNA expression of major proinflammatory cytokines (IL-1β, TNF-α, and IL-6), an anti-inflammatory cytokine (IL-10), and several chemokines (MIP1α, MIP-1β, MCP-1, eotaxin-2, and RANTES) were assessed by real time PCR at several time points after wood dust exposure. TNF-α, IL-6, and MCP-1 expression was studied also at the protein level using the ELISA method. Wood dust had in general more effects on cyto- and chemokine expression than inorganic dust TiO2 . All the tested wood dusts induced TNF-α, IL-6, MIP-1α, and MIP-1β expression and inhibited IL-1β and eotaxin-2 expression. Many differences were detected in the strength of the induction or inhibition between different wood dusts. In the case of MCP-1, for example, birch, beech, pine, and spruce induced MCP-1 production but oak and teak dusts had no effect. It is of interest that oak dust, that has been previously shown to be carcinogenic, seems to be a weaker inducer of inflammatory response than the other tested wood dusts. Our results show that exposure to different wood dusts can elicit dose-dependent and different changes in the levels of inflammatory mediators in mouse macrophage cells. These findings suggest that exposure to wood dust may significantly influence development of Poster Session P2. Immunotoxicology inflammatory process in the airways by modulating the expression of proinflammatory cytokines and chemokines. 126 ROLE OF ACTIVATED NEUROTROPHIC LEUKOCYTES IN PATHOGENESIS OF DIFFERENT DISEASES A.V. Sorokina, L.P. Kovalenko, E.V. Shipaeva, T.A. Gudasheva, R.U. Ostrovskaya, S.B. Seredenin. State Zakusov’s Institute of Pharmacology RAMS, Moscow, Russia The prooxidant mechanism involved in the action of adjuvanttype immunomodulating drugs exerts positive effect during chronic infectious processes in patients with secondary immunodeficiency. Neutrophils form the forefront line of defense of an organism exposed to infectious processes, although during most non-infectious diseases the activated neurotrophils produce notable cyto- and organotoxic action. Neutrophils have a powerful aggressive potential inducing infectious (purulent) and non-infectious inflammations (during posttraumatic disorders of different genesis, vascular dementia, brain ischemia). Neutrophils are involved in middle and late phase of autoimmune, and probably allergic inflammation of different forms. During stroke there is observed the infiltration of cerebral microvessels by leukocytes, which induce remarkable perivascular edema, an enhanced adhesion of leukocytes to microvessels endothelium and brain tissue infiltration. All these events result in ischemic area enlargement. Free radical activity of neutrophilic leukocytes leads to the expression of iNOS-synthase in neutrophils, COX-2 in cellular membranes thus triggering eukozanoids synthesis, and namely PGE2 . One of valuable constutuent of multicomponent action produced by cardiotropic and psychotropic drugs is their differently oriented effect upon membranes of various cells and their mediator control. It may induce an anti-inflammatory effect or pseudoallergic response. Therefore, it appears beneficial when experimental testing the immunotoxicity of novel membrane-active substances to use the chemoluminescence for evaluating their effect on neutrophic lykocytes activated by phorbolmiristatacetate or other enhacers. It is also of value to estimate acute exudative (carragenan- and Con A-induced edema) and chronic (adjuvant arthritis) immune inflammations. After single dosage of psychotropic adaptogenic agent estriglutone (per os) and DNA-containing compound AVP (subcutaneously) a significant dose-depended increase in the inflammatory response to Con A was observed. The inflammatory action was found in 2-mercaptobenzimidasole derivatives, an antiarrhythmic agent Bradisole and selective anxiolytic Afobazol. Pronounced antioxidant and anti-inflammatory properties were shown in dipeptide nootropic Noopept. 127 EFFECT OF IL-6 AND IL-15 ON THE NO PRODUCTION AND EXPRESSION OF iNOS IN PMN AND PBMC J. Jablonski 1 , E. Jablonska 2 , W. Puzewska 2 , M. Marcinczyk 2 . of Toxicology, Medical University of Bialystok, Poland, 2 Department of Immunology, Medical University of Bialystok, Poland 1 Department Nitric oxide (NO) is a small gaseous molecule with significant bioactivity. Peripheral blood neutrophils (PMNs) are the major participants in a number of pathological conditions with involving NO. NO production by cells requires the presence of a one or more of the nitric oxide synthases (NOS). Inducible type of NOS (iNOS) is usually induced by the presence of certain inflammatory cytokines. The aim of this study was to examine the NO production and the iNOS expression by PMN and, for comparison, peripheral blood mononuclear cells (PBMC). Cells were isolated by Gradisol G gradient (1.115g/ml) from 15 healthy donors. The PMN and PBMC were suspended in the culture medium to provide 5x106 cells/ml and cultured for 4h at 37°C in humidified incubator with 5% CO2 (Nuaire™). RhIL-6 (50ng/ml, R&D Systems) and rhIL-15 (50ng/ml, R&D Systems) were tested to stimulate expression iNOS and NO production. Expression iNOS was detected by western blot analysis using mAb capable of detecting iNOS (R&D Systems). NO in the culture of cells was quantified by Griess reagent. s37 Results obtained indicate that the antibody in human unstimulated and rhIL-6 and rhIL-15-stimulated PMN and PBMC identified band of 130kD from each donors. The rhIL-6 and rhIL-15-stimulated cells expressed a little increased iNOS protein in comparison with unstimulated cells. NO production by rhIL-6 and rhIL-15-stimulated cells was higher than NO production by unstimulated cells. Although observations above do not indicate a significant influence of IL-6 and IL-15 on the iNOS expression and NO production by PMN, the regulation of these proteins by higher concentrations of IL-6 and IL-15 may be of importance for inflammatory and other reactions controlled by these cells. 128 THE IN VITRO FUNCTIONS OF NORMAL PERIPHERAL LYMPHOCYTES ARE INFLUENCED BY LOW MOLECULAR WEIGHT HEPARINES M. Neagu 1 , G. Manda 1 , M. Gaghes 1 , C. Constantin 1 , C. Tanaseanu 2 . 1 Immunology Department, Victor Babes National Institute, Bucharest, Romania, 2 Sf. Pantelimon Emergency Hospital, Bucharest, Romania Aim: Evaluation of the in vitro proliferative capacity of normal peripheral lymphocytes in presence of various commercial low molecular weight heparins (LMWH) in order establish the prevention potential of the compounds and the effects on the immune cell population. The most frequently new generation of anticoagulants used in the treatment of cadiovascular diseases were studied. Donors. Healthy volunteers (n=50), average body weight, ages 35±15, 57% females, 43% males, no medication, no clinical sings of any illness at the investigation moment and with no present or family cardiovascular symptoms were investigated. LMWH. Fraxiparine (Sanofi), Fragmin (Pharmacia&UpJohn AB), Clivarin (Knoll), Innohep (Leo Laboratories), Clexan (Bellon France) were tested in the range of 0.06–600 UAXa/ml (doses that match the therapeutical range). Methods: Peripheral cells were isolated on Hystopaque gradient centrifugation. The proliferative capacity of unstimulated or mitogen (phytohaemagglutinine M - PHA or pokeweed mitogen - PWM) stimulated lymphocytes was assessed with H3 -Td incorporation method in presence and absence of LMWH. Results: All the tested LMWH induced with various intensities an activation of the proliferative capacity of unstimulated lymphocytes. 0,6UAXa/ml Fraxiparine induced the highest activation in vitro. We found that the activation capacity of Fraxiparine was higher on the subgroup of normal subjects that displayed lower basal proliferative capacity. In the costimulation systems with PHA, Fraxiparine induced significant inhibition of the proliferation capacity of policlonaly-stimulated lymphocytes in the tested concentration range. In the costimulation systems with PWM, only 6UAXa/ml Fraxiparine significantly reduced the proliferative capacity of normal peripheral lymphocytes. No effects were registered in the mentioned systems in presence of the other mentioned LMWH. Conclusions: Fraxiparine probably hinders the mitogen binding on T- and B-lymphocytes. Due to the registered in vitro effect, LMWH can influence the proliferative capacity of normal lymphocytes and consequently alter the immune response. The study emphasize on the relation between the particularity of the normal immune cells functionality and the in vitro registered effect of LMWH. 129 RESISTANCE TO IN VITRO INFECTION WITH LEISHMANIA MAJOR IN MACROPHAGES FROM ARYL HYDROCARBON RECEPTOR-NULL MOUSE L. Vega 1 , M.I. Vázquez 1 , M. Rodríguez 2 , G. Elizondo 1 . 1 Sección Externa de Toxicología, Centro de Investigación y Estudios Avanzados-IPN. 2 Instituto Nacional de Cardiología, México D. F., México Aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor that is activated by ligand binding and dimerization with AhR nuclear translocator (ARNT) in response to xenobiotics. AhR mediates transcriptional activation of several genes encoding xenobiotic-metabolizing enzymes such as CYP450 1A1 and 1A2. s38 Poster Session P2. Immunotoxicology It also plays an important role in the development of the immune system and could have a central role in activating the immune response when it interacts with several xenobiotics. We evaluated the in vitro immune response to L. major in macrophages from AhR-null and wild-type animals. Peritoneal macrophages and spleen cells were collected from male mice 6– 10 weeks old. Spleen cells and macrophages were evaluated for 1) in vitro proliferative response to Con A or LPS by 3 [H]-T incorporation, 2) CD4 and CD8 lymphocyte subpopulations by flowcytometry, 3) Con A or LPS induced IL-12, IL-4 and IFN-γ secretion by ELISA. Macrophages were infected with L. major promastigotes and incubated for 2 h. OVA-responding macrophages were cultured with naïve spleen cells for 48 or 72 h and supernatants were collected. AhR-null animals showed an increased number of spleen cells and peritoneal macrophages, and increased Con A-induced proliferation. CD8 lymphocytes were reduced in AhR-null mice compared to wild-type animals. Basal and antigen-induced cytokines were increased in AhR-null mice. Co-culture of OVA responding macrophages with lymphocytes from wild type, heterocygous or AhR-null mice showed similar responses. The number of parasites infecting macrophages and the number of macrophages infected were lower in the AhR-null mice cells, indicating that the AhR-null macrophages were resistant to L. major infection in vitro. Based on the cytokine secretion profile, and on the parasite burden, it is evident that the AhR-null mouse showed an increased Th1 type immune response to L. major infection and a resistance to L. major infection. These data suggest that the AhR plays an important role in the switch from Th1 to Th2 type immune response or is involved in the Th2 specific activation pathway. 130 EFFECT OF PHOSPODIESTERASE INHIBITORS ON SPLENIC LYMPHOCYTES PROLIFERATION G. Dyulgerova, N. Boyadjieva. Department of Pharmacology and Toxicology, Medical Div., Medical Univ. of Sofia, Sofia, Bulgaria The role of cyclic adenosine monophosphate (cAMP) in the immune system has been the subject of investigation. We have previously shown that the phosphodiesterase isoenzymes (PDE) play an important role in regulation of cellular cAMP in immune cells. For better understanding the immune-regulatory effects of PDE3 and PDE4, we investigated the effects of Milrinone (PDE3) and Rolipram (PDE4) on splenic lymphocytes proliferation. Lymphocytes were isolated from male rats. The proliferation of lymphocytes was determined by mitogen-stimulated assay. Lypopolysaccharide (LPS) was used as a stimulating agent for cell proliferation. MTT assay was used for determination of number of splenic lymphocytes after 96 hours of treatment with Rolipram (10µmol) and Milrinone (10µmol). The results demonstrate that both Rolipram and Milrinone decreased the LPS-stimulated proliferation of cultured lymphocytes. Our data suggest that inhibitors of PDE3 and PDE4 depressed the immune functions via inhibition of mitogen-activated lymphocyte proliferation. 131 EFFECTS OF DERIVATIVES OF DITHIOCARBONIC ACID (XANTHATES) ON SOME PHAGOCYTISE CELLS S. Yanev 1 , O. Karagiozova 1 , D. Uzunova 1 , V. Hadzimitova 2 . Dept. of Drug Toxicology, Inst. of Physiology, Bulgarian Academy of Sciences, 2 Dept.Biophysics, Medical School, Sofia, Bulgaria ide and hypochlorist radicals. That effect is mainly due to xanthates antioxidant activity. In human neutrophiles xanthates potentiate the stimulated effect of sub-maximal PMA concentrations on the production of active oxygen spicies by still unknown mechanism. The NO production in LPS-stimulated rat alveolar macrophages is decreased by different xanthates. That could be due to decrease of iNOS expression as well as to direct (binding by xanthates or some of their metabolites) or indirect (increased cellular levels of superoxide radicals) effects on cellular NO. Our results suggested that the modulator effects of xanthates on some cellular functions are due not only to inhibition of phospholipase C activity but also to changes in NO/superoxide cellular equilibrium. 132 MOLECULAR MECHANISM OF ACTION OF THE FUNGICIDE MANCOZEB ON THE INHIBITION OF CYTOKINE PRODUCTION E. Corsini 1 , S. Birindelli 2 , M. Marinovich 1 , C. Colosio 2 , C.L. Galli 1 . 1 Laboratory of Toxicology, Department of Pharmacological Sciences, University of Milan, Milan, Italy; 2 ICPS, International Centre for Pesticide Safety, Busto Garolfo, Italy We have previously observed in agricultural workers exposed to the fungicide mancozeb a statistically significant decrease in lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) production (p<0.01). The purpose of this work was to establish an in vitro model reflecting in vivo data on mancozeb inhibition of LPS-induced TNF-α release and to characterize its molecular mechanism of action. The human promyelocytic cell line THP-1 was used to develop the in vitro model to study the effects of mancozeb and its main metabolite ethylenthiourea (ETU) on LPS-induced TNF-α release. Cells were treated with increasing concentrations of mancozeb or ETU for different times. TNF-α release was evaluated following LPS stimulation by specific ELISA. Cell viability was assessed by measuring lactate dehydrogenase leakage. Mancozeb, but not ETU, at non cytotoxic concentrations, induced a dose and time related inhibition of LPS-induced TNF-α release. The results obtained indicated that THP-1 cells were indeed a suitable model to study the molecular mechanism of action of mancozeb. For the characterisation of the molecular mechanism of action the effect of mancozeb on TNF-α mRNA expression was first evaluated by semi-quantitavive RT-PCR. We observed that decreased TNF-α release following mancozeb and LPS treatment was due to a pre-transcriptional event. Successively, in order to better define the molecular target of mancozeb its effect on LPS-induced NF-κB activation and reactive oxygen species (ROS) generation was characterised. Transcription factor activation was evaluated measuring by ELISA the DNA binding by specific transcription factor present in nuclear extract obtained from cells treated in the presence or absence of mancozeb following LPS stimulation. ROS generation was evaluated using a flow cytometric method using the dye dichlorofluorescein diacetate. We demonstrated that mancozeb functioning as an antioxidant causes a direct inhibition of NF-κB activation, which in turn resulted in decreased TNF-α production in monocytes. 1 Xanthates (salts of alkyl or aryl derivatives of dithiocarbonic acid, ROCS2 K) are well known metal chelators and one electron acceptors. Xanthates effects on some cellular functions are explained by inhibition of phospholipase C (D609 used as a model compound) and/or of protein kinase C. In the present study the effect of xanthates on some functions of different phagocyte cell lines was studied: NO production in stimulated or non-stimulated with LPS rat alveolar macrophages; production of active oxygen species in PMA-stimulated rat peritoneal macrophages and human blood neutrophyles. Xanthates diminished luminol-dependent chemiluminescence in PMA-stimulated rat peritoneal macrophages. The effect is different with different xanthates and is similar with other sources of superox- 133 IMMUNOMODULATORY EFFECTS OF EBDTCs ON AGRICOLTURAL WORKERS OCCUPATIONALLY EXPOSED TO THE FUNGICIDE MANCOZED S. Birindelli 1 , E. Corsini 2 , M. Marinovich 2 , C. Colosio 1 , M. Maroni 1 , C.L. Galli 2 . 1 ICPS, International Centre for Pesticide Safety, Busto Garolfo, Italy 2 Laboratory of Toxicology, Department of Pharmacological Sciences, University of Milan, Milan, Italy Literature data suggest that ethylenebisdithiocarbamate (EBDTCs) may have immunostimulatory effects in humans. The aim of this study was to investigate the effects on the immune system of agricultural workers exposed to mancozeb, an EBDTC fungicide. Twenty-six healthy subjects entered the study, 13 agricultural workers and 13 matched controls. The exposure to mancozeb was assessed Poster Session P2. Immunotoxicology through the determination of the levels of urine excretion of the main EBDTCs metabolite, ethylenthiourea (ETU). Immune functions were performed using blood samples collected before and after the work shift in the workers and only once (morning samples) in controls. The following parameters were measured: complete and differential blood count, serum immunoglobulins, complement fractions, autoantibodies, lymphocyte subpopulations. As for the functional assays, the proliferative response to mitogens and cytokine production were assessed using a whole blood assay. Agricultural workers resulted significantly exposed to mancozeb (p=0.0001 vs control group and p=0.008 vs baseline samples). As for the immune testing, the post shift assay showed a significant increase in the percentage and number of CD19 and a decrease in the percentage of CD25 positive cells in comparison with controls (p=0.016 and p=0.0001, respectively). Furthermore, mancozeb exposure resulted in increased proliferative responses, that reached statistically significance for pokeweed and PMA plus ionomycin stimulation (p=0.016 and p=0.004, respectively). Relatively to cytokine synthesis, a trend to an increase in γ-IFN, and a significant decrease in TNF-α production was observed (p=0.006). There was no evidence of any increase in clinical illness in the exposed compared with the control group. In summary, our results indicate that exposure to mancozeb has slight dose-related immunomodulatory effects and, point out the possibility to monitor and reveal immunomodifications in workers occupationally exposed to low-dose of potential immunotoxic compounds using a whole blood assay. 134 IMMUNE-MORPHOLOGICAL ASPECTS OF ORGANOPHOSPHORUS INDUCED DELAYED NEUROPATHY STUDYING. A. Pushkin, Y. Rumbal, S. Dvoretskaya, V. Petrunin. State Research Institute of Organic Chemistry and Technology (GosNIIOKhT), Moscow, Russia Within the bounds of actual problem of early diagnostics of delayed neuropathy and organophosphorus compounds (OP) activity prediction we have made an attempt to investigate the cytomorphological changes in lymphocytes from peripheral blood of rats and hens. We used tri-o-cresyl phosphate (TOCP) and 3-phenylphosphite (TPP) as modeling compounds. Peripheral blood is rather convenient and available material for in vivo continuous controlling observations and lymphocytes is rather correct object since they like neurons contain the neuropathy target esterase – organophosphorus induced delayed neuropathy (OPIDN) marker enzyme. The level of neuropathy target esterase (NTE) inhibiting in hen and human lymphocytes (in vitro) have been determined. Microscopical investigations of blood smear ware being taken at the different period of time and have shown that all our modeling compounds possess the lymphocyte harmful activity. Revealed cytopatology in lymphocytes looked like nuclei ectopy and their basophilia decreasing, cytoplasm vacuolization and swelling, forming a lot of cytoplasmic growth, changing the form of surface. The number of damaged cells also has been determined in our study. The low-doses of these substances action are the most essential results of our research. Our investigations have shown the reliable increase of number of anomalous form of lymphocytes in hen blood at least in 3 times, and in rat blood more then in 5 times. Meanwhile the problem of lymphocytes changes specificity needs more investigations, the fact of their high sensitivity to the OP action indicate the belonging of immune effectors to the delayed neurotoxicity pathogenesis. This results force us to study the possible contribution of central immunogenic mechanisms for OPIDN developing During the histological investigations of thymus and adrenal glands of rat, being subjected the action of mentioned substances definite changes also have been found. All these changes may be explained as immunopathological manifestations. There is degranulation of mast cells in thymus and significant dystrophic lesions of chromaffin cells in medullary substance of adrenal glands have been revealed. The investigations of changes in thymus mass after subcutaneous OP injection at the doses 1,84mg/kg, 0,92mg/kg and 0,092mg/kg have determined the reliable increase of this index in 24 hours. It’s quite possible that all these changes concerned s39 with plethora of capillary pool and disorder of permeability due to histamine hyper secretion by mast cells. With a view of following investigations of the role of immune component in the mechanism causing OPIDN we have started the series of experiments with hormonal suppression of T-lymphocytes proliferation, lymphokines production and macrophages activity. Moreover in our experiments we tried to effect on the secondary mediators in the latest phase of IgG mediated reactions. In order to provide all these effects we have used dexamethasone at the concentration 2,0mg per animal for 5 days subcutaneously every day, and after that rats have been exposed to the sub chronic action of TOCP during 4 days 1000,0mg/kg intra gastric every day. All this data concern the analysis of combined effect on the mass coefficient of thymus and adrenal glands of experimental animals. So that the dysfunction was being caused by dexamethasone results in reliable and significant thymus reduction and of growth of adrenal glands mass which can be increased by additional injection of TOCP. Histological research, being carried out now will show us how all these dysfunctions can provoke some lesions in nervous structures of medulla and sciatic nerve in rats. This work has been supported by ISTC project #574. 135 COMPARATIVE INVESTIGATION OF BEHAVIORAL, NEUROTOXICOLOGICAL, AND IMMUNOTOXICOLOGICAL INDICES IN DETECTION OF SUBACUTE COMBINED EXPOSURE WITH METHYL PARATHION AND PROPOXUR IN RATS L. Institoris 1 , A. Papp 1 , B.D. Banerjee 2 , O. Siroki 1 . 1 Department of Public Health, Faculty of Medicine, University of Szeged, Hungary, 2 Department of Biochemistry, University College of Medical Sciences and Guru Teg Bahadur Hospital, University of Delhi, Shahdara, Delhi 110095, India The effect of six weeks oral exposure to propoxur (PR; at doses of 0.851 and 8.51 mg/kg b.w.), methylparathion (MPT; at doses of 0.218 and 0.872 mg/kg b.w.) and their combinations was investigated in male Wistar rats. Measurement endpoints of the investigation were certain general toxicological parameters (body weight gain, organ weights), plaque forming cell (PFC) count of the spleen, open field (OF) behavior, auditory startle response (ASR) and pre-pulse inhibition (PPI), rotarod performance, somatosensory and auditory cortical evoked potentials, and peripheral nerve conduction velocity. The treated rats did not show any sign of acute intoxication during the 6 weeks exposure. The higher dose of PR, but not MPT, significantly decreased the relative liver weight. Both agents produced a significant dose-dependent increase of OF activity, with larger expression after 2 than after 6 weeks. The number of ASR responses and the ASR amplitude increased. The amplitude after PPI was increased by MPT but only minimally altered by PR and the combinations. There was a small, but with high dose PR significant, increase in the latency of the somatosensory evoked potentials. Neither of the two substances alone had any effect on the PFC response. The effect of the combination of high dose PR and low dose MPT was significantly different from that of high dose PR alone on the liver weight, on the ASR amplitude and on both PFC counts. With high dose MPT and low dose PR, no such interaction was observed. According to the results, in combined exposure the non-effective dose of MPT can influence the toxicity and/or the functional detection limit of the effective dose of PR. 136 IMMUNOTOXICITY OF ORGANOPHOSPHOROUS PESTICIDES, METHIDATHION AND PIRIMIPHOS-METHYL ON THE IMMUNE SYSTEM OF BALB/C MICE Juno H. Eom, Hyung Soo Kim, Seung Tae Chung, Jae Hyun Park, Jung Hyun Kil, Jong Kwon Lee, Hye Young Oh. Immunotoxicology Division, Department of Specialized Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration, 5 Nokbun-Dong, Eunpyung-Ku, Seoul, 122–704, South Korea Organophosphorus (OP) pesticides have largely replaced the use of organochlorine pesticides and have been widely used in agriculture and houses in recent years because of their rapid breakdown in water s40 Poster Session P2. Immunotoxicology and their low environmental persistence. As a result, consumers are directly or indirectly exposed to organophosphorus pesticides through several food groups including meat, dairy products, fruits, vegetables, dried foods, and most processed foods in which a significant amount of pesticide residues have been found. However, so far there have been few reports on the immunotoxic effects of OPs. In the present study, Balb/c mice were used to determine the immunotoxic effects of OPs, methidathion and pirimiphos-methyl. Results showed that methidathion dosage did not change significantly body weight, relative thymus and spleen weight, and thymus and spleen cellularities of Balb/c mice. Pirimiphos-methyl treatment also showed there is no significant changes in body weight, relative thymus weight, and thymus cellularity of Balb/c mice, but high dose treatment (120 mg/kg) of pirimiphos-methyl significantly decreased relative spleen weight and spleen cellularity of Balb/c mice. On the other hand, the percentages of thymocyte and splenocyte subsets, LPSproliferation response of splenocytes and ACTH concentrations in bloods were not affected by methidathion and pirimiphos-methyl exposures. However, both methidathion and pirimiphos-methyl dosages reduced ConA-stimulation response of splenocytes. The result indicate that high dose exposure of pirimiphos-methyl affects spleens of Balb/c mice, and further study will be required to assess whether the effects of pirimiphos-methyl are directly reflected on the immune function of Balb/c mice or not 137 EVALUATION OF IMMUNOTOXICITY INDUCED BY DIAZINON IN C57bl/6 MICE E. Zabihi Neishabouri 1 , Z.M. Hassan 2 , S.N. Ostad 1 . 1 Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran,2 Department of Immunology, Faculty of Medical Sciences, Tarbiat Modarres Univrsity, Tehran Iran Diazinon (DZN) as an organophosphate insecticide has been used for several years in agriculture industry and urban zones. Despite of possible risk of DZN exposure to workers in pesticide plants, and public as a residue on farm products, there is no clear investigation on its immunotoxicity potential as yet. In this study we examined the immunotoxicity of intraperitoneally administered diazinon in C57bl/6 female mice. Diazinon was administered at doses 25, 2, 0.2 mg/Kg/day for 28 days (5 injections per week). Then animals were sacrificed and observed for cellularity and histopathological changes in thymus (TM), spleen (SP), bone marrow and peripheral blood. Furthermore, humoral and cellular functional tests including Hemmaglutination (HA) titration test, IgM-Plaque Forming Colony Assay (PFC), Delayed Type Hypersensitivity (DTH) to SRBC and T-cell sub-typing (CD4/CD8) were performed. Obtained results showed that DZN at 25mg/kg/day not only could produce gross histopathological changes in TM and SP but also could suppress both humoral and cellular responses of the immune system. At medium dose (2 mg/kg/day) there were no observable changes in cellularity or histology of immune tissues except for a little change in IgM-PFC and DTH responses and spleen CD4 percentage. At the dose of 0.2 mg/Kg/day no histopathological or functional disturbances were detectable compared with the vehicle: (Sweet Almond Oil) group. It is concluded that DZN, induce immunotoxicity in C57bl/6 mice as immunosuppression at doses more than 2 mg/kg/day. The present results however indicate that under recommended Allowed Daily Intake (ADI) limit (<0.02 mg/Kg), no observable immunotoxicity effect is expected. 138 COMPARISON OF SOME IMMUNE RESPONSES OF TWO GROUPS OF WORKERS EXPOSED TO VARIOUS LEVELS OF PAHs I. Ates 1 , B. Yucesoy 1 , M. Yilmazer-Musa 2 , A. Karakaya 1 . of Toxicology, Faculty of Pharmacy, Ankara University, 06100, Tandogan, Ankara, Turkey; 2 Department of Toxicology, Faculty of Pharmacy, Gazi University, 06330, Etiler, Ankara, Turkey 1 Department Polycyclic aromatic hydrocarbons (PAHs) are well known carcinogens in humans and formed by pyrolysis or incomplete combustion of organic materials. It was suggested that immunotoxic effects of PAHs contribute to their carcinogenic potential. Although immunotoxic potential of PAHs has been well documented in animal studies, little is known about the effects in humans. The present study of asphalt and coke oven workers points to effects of PAH exposure on natural killer (NK) cell activities and T-lymphocyte proliferative responses. Analysis of 1-hydroxy pyrene (1-OHP) metabolite in urine was also performed in order to monitor exposure to PAHs and the results were compared to these obtained in healthy unexposed controls. In this study, while we found a significant increase in urinary 1-OHP excretion for coke oven workers compared to control subjects, there was a slight increase in that of asphalt workers but no statistically significant change between asphalt workers and controls we observed. Although there was no significant difference in NK cell activities, it was clearly seen that there was a statistically significant suppression of T-cell responses induced by PAH in both two groups of exposed workers. Our previous study and these data suggest that chronic exposure to PAHs may affect some immune functions in humans. This study was supported by Research Fund of Ankara University (98–03–00–07). 139 PROTECTIVE EFFECTS OF GLUTATHIONE ON 5-FLOROURACIL INDUCED MYELOSUPPRESSION IN MICE K. Takaba, N. Kimoto, T. Takeda, M. Kakuni, M. Naya, N. Kojima, T. Harada, M. Hiura, T. Hara. Toxicological Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, Yamaguchi, Japan The protective effects of glutathione (GSH) administration on myelosuppression induced by 5-fluorouracil (5-FU) were investigated in female BALB/c mice. Animals were allocated into four groups (16 mice /group). GSH was orally given at a dose of 800 mg/kg to Groups 3 and 4 for 21 consecutive days (day 0 to day 20). 5-FU was repeatedly administered at a dose of 40 mg/kg to Groups 2 and 3 for 1 week (day 7 to day 13) by gavage. Group 3 served as a combined treatment group and Group 1 as a non-treated control group. Total observation period was 3 weeks. Body weight was measured once a week. A decrease in body weight due to 5-FU treatment was observed in Groups 2 and 3 on day 14. Although the body weight in Group 2 had not increase by 1-week after cessation of 5-FU treatment, the value in Group 3 markedly recovered. Hematology, total nucleated myelocyte count and histopathology of bone marrow were carried out on day 14 and day 21. In Groups 2 and 3, these examinations showed thrombocytopenia, leukopenia, reticulocytopenia and myelosuppression on day 14. However, platelets and bone marrow were less affected in Group 3 than in Group 2. On day 21, the thrombocytopenia in Groups 2 and 3 was resolved. The myelosuppression, leukopenia and reticulocytopenia resolved in Group 3, but not in Group 2. Although simple microcytic anemia occurred delayed on day 21, it was less severe in Group 3 than in Group 2. Therefore, GSH may have preventive effects against 5-FU-induced hematopoietic toxicity, and accelerate recovery after cessation of 5-FU treatment. 140 THE IMMUNOTOXIC ACTIONS OF CYCLOSPORIN A, DEXAMETHASONE AND FUROSEMIDE IN THE RAT: A COMPARATIVE STUDY R. Forster, C. Mimouni, B. Griffon, J-M. Pavard, M. Attia. CIT, Evreux, France In this study Sprague-Dawley rats were treated with cyclosporin A (at 10, 15 and 20 mg/kg), dexamethasone (15 µg/kg) and furosemide (70 mg/kg) by oral gavage for 28 days. Hematological investigations were performed on blood samples taken at terminal sacrifice. The antibody response to the T-cell dependent antigen Keyhole Limpet Hemocyanin (KLH) was evaluated by subcutaneous administration of KLH on Day 22, followed by ELISA determination of IgM levels on blood samples taken terminal sacrifice. Lymphocyte subsets in peripheral blood were quantified by flow cytometry on samples taken at the end of the study. Organ weight analysis and histopathological examinations were performed on lymphoid organs. The methods used correspond to those proposed for the s41 Poster Session P2. Immunotoxicology evaluation of the potential toxicity to the immune system in recent European testing guidelines for new drugs. The test materials were all well tolerated clinically, and treatments with all three resulted in reductions in bodyweight gain, thus providing evidence that the selected dose-levels resulted in some general toxicity. Treatment with cyclosporin A provoked a dose-related decrease in the IgM antibody response to KLH, but treatment with dexamethasone or furosemide did not affect this parameter. Treatment with cyclosporin A resulted in a reduction in the proportion of CD4 and CD8 T-cells in rats of both sexes. Treatment with dexamethasone or furosemide did not influence T-cell populations. None of the treatments adversely affected B-cell populations. The test methods clearly permitted identification of the immunosuppressive actions of cyclosporin A. The immunosuppressive action of dexamethasone was not detected by the KLH response or lymphocyte subset analysis at the dose-level selected for this study. The results with furosemide were consistent with the view that this drug does not have immunotoxic properties. 141 TOXICITY ASSAY TO SINGLE DOSIS AND TO REPEATED DOSIS OF HUMANIZED ACMT1HT BY ENDOVENOUS ROUTE IN CENP:SPRD RATS. Y. González 1 , A. Casacó 2 , A.M. Bada 1 , D. Fuentes 1 , B. González 1 , N. Subiros 1 , M.E. Arteaga 1 , O. Hernández 1 , J. Hernández 1 . 1 Center of Experimental Toxicology (CETEX), National Center for the Laboratory Animal Breeding (CENPALAB), 2 Department of Clinical Assays, Center of Molecular Immunology, (CIM) The humanized AcM T1hT is proposed to the Reumatoid Arthritis treatment. The objective of the studies was to evaluate the toxic effects that may appear after the endovenous administration of a single and repeated doses of AcM during 14 days in Cenp:SPRD rats. In both studies, corporal weight and rectal temperature rates were evaluated. Studies were conducted following OCDE and ICH international regulations. In both studies, three groups were conformed: control, low dosis, high dosis, each consisting of 5 animals/sex. In the repeated dosis, they were analyzed, also, hematological parameters: hemoglobin, erythrocytes, hematocrit, corpuscular constant, platelets. total leukocytes, leukocytes differential count, and clinical biochemistry (alcaline phosphatase, aspartatoaminotransferasa, alaninoaminotransferasa, albumin, total protein, glucose, cholesterol, triglicerides, creatinine, uric acid. Macroscopically changes in organs and tissues and local effects of the substance in the administration site were evaluated, besides, organs weight and microscopically alterations on organs and tissues were determined. Both studies ended with 100% of survival. Animal observation did no revealed toxic signs. Corporal weight analysis did not revealed significant differences between groups. Histological study revealed a slight increase in the quantity of lymphoid follicles in the spleen of treated animals with AcMt1ht. In the application site, it was detected the presence of focal acantosis with hiperqueratois on the epidermis, as well as, occasional paraqueratosis in the dermis. It was observed a slight presence of fibroblasts in the venopunction site. Not existing a dosis-response relation, and the organs weight values are among the ones reported for this species in our institution, we consider that these differences are not related to the assay substance. We conclude that AcMt1ht administration to single and repeated dosis during 14 days in Cenp:SPRD rats do not cause alterations in the normal general condition of the animals. 142 IMMUNE FUNCTION ALTERATIONS INDUCED BY OCHRATOXIN A IN EX VIVO AND IN VITRO MODELS OF WISTAR RATS. A. López de Cerain, L. Alvarez, O. Ezpeleta, A.G. Gil. Laboratory of Toxicology, Department of Food Sciences and Toxicology, University of Navarre, Pamplona, Spain Ochratoxin A (OTA) is a mycotoxin produced by fungi of Aspergillus and Penicillium genera. The main toxic effect of ochratoxin A is nephrotoxicity. In addition, it is hepatotoxic, neurotoxic, teratogenic genotoxic and carcinogenic. OA affects the immune system function; however, the data available is contradictory and a wide dose range has been tested in different species. We have studied the effect of OTA on in vivo and in vitro models of Wistar rats. In the ex vivo study, the immune system function has been evaluated after 28 days of treatment with µg dose (50, 150, 450 µg OTA/ Kg b.w.) of the mycotoxin; the OTA concentration in each serum sample was determined by HPLC. Equivalent concentrations (0.5 and 2 µM) were tested in the in vitro study; in addition, a concentration whcih was ten times higher (20 µM) was assayed. We have evaluated different immune functions, such as: NK cell-mediated cytotoxicity, CTL-mediated cytolysis, macrophages activity, B and T lymphoproliferation, and humoral response to sheep red blood cell (SRBC). In the in vitro mode, short and long treatment times were assayed (1, 5 and 72 hours). Results of the ex vivo assays are summarized in the following table: Assay Ochratoxin A Dose B cell proliferation T cell proliferation NK cell activity CTL assay Macrophages activity Humoral response to SRBC 50 µg /kg 150 µg /kg 450 µg /kg n.s. n.s. ** * ** n.s. n.s. n.s. *** n.s. n.s. n.s. n.s. n.s. *** n.s. n.s. n.s. In both models, B cell response to lipopolysaccharide and T cell response were not modified. NK cell activity showed a dose-related decrease in the in vivo model. In vitro, this pattern of toxicity was reproduced at the equivalent concentrations; at 20 µM, a lower effect was observed. CTL activity was affected by the lowest dose of OTA, both in vivo and in vitro. Also, OTA exposure affected macrophage activity. The data obtained with both systems is highly correlated on the in vivo range of OTA concentration. 143 RESPIRATORY ALLERGY AND INFLAMMATION DUE TO AMBIENT PARTICLES (RAIAP) – A EUROPEAN-WIDE ASSESSMENT. ALLERGY SCREENING. T. Løvdal, E.C. Groeng, E. Dybing, M. Løvik. Norwegian Institute of Public Health, Oslo, Norway Differences exist in prevalence and severity of respiratory allergies throughout Europe. A main objective of the RAIAP project is to examine whether qualitative differences in particulate air pollution at different locations may in part explain the unequal distribution of respiratory illnesses. Ambient particulate matter (coarse and fine) was collected in Amsterdam, Rome, Lodz, and Oslo, during the spring, summer, and winter 2001/2002, as well as from a Dutch sea-site background location (de Zilk). In the part of the study presented here, these particles were screened for allergy adjuvant activity, measured as the primary popliteal lymph node response and secondary allergen specific IgE response. Diesel exhaust particles from NIST (SRM 1650) was included as a positive control. All fractions, with exception of a few of the coarse ones, have an adjuvant effect in the doses examined (100–200 µg per mouse). A significantly stronger IgE response with fine particles from Rome, as compared to Oslo and Lodz, was observed with particles collected in the spring season. Beyond that, no marked differences between the locations were observed. A significant increase in the allergen specific IgG2a response was observed for the fine and some of the coarse fractions, indicating a non-allergic Th1 response. It should be noticed that particulate matter from the “clean” background site de Zilk did not stand out from the other locations. (A European Commission Shared-Cost Research Project, QLK4-CT/-2000–00792.) s42 144 Poster Session P3. Ocular and skin toxicity POLYMORPHISMS OF DNA REPAIR AND BIOTRANSFORMATION GENES AND POSSIBLE RELATIONSHIPS WITH IMMUNOLOGICAL RESPONSE. M. Kuricova 1 , P. Vodicka 2 , R. Kumar 5 , L. Vodickova 3 , J. Tulinska 1 , E. Jahnova 1 , A. Liskova 1 , M. Dusinska 1 , P. Soucek 3 , L. Fuortes 4 , K. Hemminki 5 . 1 Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, 2 Institute of Experimental Medicine, Acad. Sci. Czech Republic, Prague, Czech Republic, 3 National Institute of Public Health, Prague Czech Republic, 4 University of Iowa, Department of Preventive Medicine and Environmental Health, Iowa City, IA USA, 5 German Cancer Institute, Heidelberg, FRG Many xenobiotic metabolizing enzymes exhibit polymorphisms in their expression and activities. Changes in activity, affinity or expression of these enzymes as a result can influence profile of the metabolism in the individual. It can markedly affect risk of development of serious diseases. The links between genetic polymorphisms in genes coding for biotransformation enzymes (CYP1A1, CYP2E1, CYP2E intron 6, EPHX act, EPHX4, EPHX5, GSTM1, GSTP1 and GSTT1) and DNA repair enzymes (XPD exon 23, XPG exon 15, XPC exon 15, XRCC1 exon 10 and XRCC3 exon 7) and levels of immunological biomarkers were analysed in this study. Our results show that phagocytic activity of polymorphonuclear leukocytes and monocytes is significantly affected by genetic polymorphism in CYP2E intron 6 and GSTP1. Moreover the percentage of monocytes shows relation to all examined CYP and also GSTM1 gene polymorphisms. In the case of EPHX exon 4 gene polymorphism we observed changes in expression of adhesive molecules CD62l, CD11a and CD11b on monocytes. The proliferative response of T-dependent B-lymphocytes to pockeweed mitogen is significantly different in various genetic polymorphims in EPHX exon 5 gene and GSTP1. Changes in monocyte and polymorphonuclear leukocyte count show the connection with some DNA repair enzymes (XPD23 and XRCC1 exon 10 respectively). Allelic variant in XRCC3 exon 7 did not show any interactions with the investigated immunological parameters. In adition, the polymorphism in Cyclin D gene and its relationships with immune parameters was also included in this study and we found a link with number of lymphocytes and polymorphonuclear leukocytes. The associations between various genetic polymorphisms and a wide range of immunological parameters may provide useful information about individual susceptibility of humans toward environmental/occupational agents. We ackonowledge the support of: Grant Agency of the Czech Republic (GACR 310/01/0802 and 310/03/0437) and NIEHS (5p30E05605–08) P3 Ocular and skin toxicity 145 2252 eyes of VCW and 2228 eyes of normal cases as control have been chosen. These two groups went through an optometry and ophthalmic examination. Their refractive errors were measured by autorefractometer (Topcon RM 2300). The results achieved by autorefractometer were compared between the two groups. We used t-test for our analysis. This study showed that: 1. Prevalence of refractive error in VCW group was higher than the group of normal. 2. Astigmatism was the main refractive errors in VCW groups and their prevalence was higher than the groups of normal. 3. The type of astigmatism that mentioned above was mostly Against the Rule and Oblique astigmatism. Though a With the Rule astigmatism had also a significant share in the combination. 4. Prevalence of myopic astigmatism in VCW groups was higher than the other type of astigmatisms. For interpretation of this result we need to more research in this area. 146 ETBE-RELATED EFFECTS ON RAT FIBROBLASTS I. Iavicoli, G. Carelli. Centro di Igiene Industriale, Istituto di Medicina del Lavoro, Università Cattolica del Sacro Cuore, Rome, Italy Ethyl tert-butyl ether (ETBE) is an ether oxygenate which, in the future, could be used as a gasoline additive to increase oxygen content and reduce tail-pipe emissions of pollutants. This potential increase in the use of ETBE could lead to exposure of the general population. The aim of our study was to evaluate the toxic effects of ETBE on cell proliferation and survival of normal rat fibroblasts after 48h incubation, and on apoptosis. After incubation for 48 hours, MTT test was used to observe the effects of ETBE on proliferation and survival of Rat-1 fibroblasts at concentrations of 0, 0.312, 3.12, 31.2 mM. Cytofluorimetric analysis was used to study cell cycle. Furthermore we evaluated apoptosis by quantifying cytoplasmic DNA fragments. Cell growth was dose-dependent and LD50 was observed at 3.12 mM. Exposure of fibroblasts to ETBE caused a time-dependent inhibition of cell proliferation. Concentrations of 31.2, 3.12 and 0.312 mM ETBE resulted in accumulation of cells in phase S after 24h (48%, 32% and 21%; controls 15%), decrease of cells in phase G2/M (2%, 4% and 12%; controls 10%) and in phase G1/M (50%, 64% and 67%; controls 75%). After 48 hours, a significant increase in cytosolic DNA fragments (p < 0.05) was observed at ETBE concentration of 3.12 mM. Furthermore, at LD50, the occurrence of a subdiploid peak confirmed that apoptosis. The fact that ETBE inhibits cell proliferation with an interruption in phase S confirms toxicity that is probably linked to an inhibition of DNA synthesis or to the transition from phase S to phase G2/M. The occurrence of apoptosis suggests that high doses of ETBE completely inhibit enzymatic activities. INCIDENCE OF REFRACTIVE ERRORS IN VICTIMS OF CHEMICAL WEAPONS AS A DELAY EFFECTS Khosro Jadidi 1 , Abbass Riazi 2 , Mostafa Naderi 1 , Ali Karimi 2 , Mahmod Babaei 1 , Hamid Safabaksh 1 , Momamad Manaei 2 . 1 Dept of Ophthalmology, Baghyatolla (a.s) University of medical sciences; 2 Dept of physiology and biophysics, Baghyatolla (a.s) University of medical sciences During the recent years many victims of chemical weapons (VCW) express the appearance of some degree of refractive errors, which they feel did not exist before, presumably as a long-term (delayed) effect of sulfur mustard exposure and no related study has been found. Generally myopia, hyperopia and astigmatism are the main refractive errors. Prevalence of these errors depends on several factors. Among these factors are genetic, age, job, near work, and also some geographical factors, The effect of sulfur mustard on the eye is very complexes and related to intensity of injury. Cornea is the main target in the eye for sulfur mustard. These effects are produced in early stages of contact with this material. The delayed effects are discussed here. We study about refractive errors as one of delayed effect of sulfur mustard in VCW. In this study about 147 EFFECTS OF LATANOPROST AND GLC756, A NOVEL DOPAMINE D2 AGONIST AND D1 ANTAGONIST, ON CULTURED NORMAL HUMAN DERMAL FIBROBLASTS U.W. Laengle 1 , R. Markstein 2 , D. Pralet 1 , B. Greiner 1 , D. Roman 1 . 1 Toxicology/Pathology, 2 Ophthalmics, Novartis Pharma AG, Basel, Switzerland Proliferation of subconjunctival fibroblasts plays a critical role in scarring and failure of glaucoma filtering surgery. Long-term topical glaucoma medications appear to increase fibroblast proliferation. In this study, the effects of topical antiglaucoma drugs latanoprost and GLC756, a novel dopamine D2 agonist and D1 antagonist, on cultured normal human dermal fibroblasts (NHDF) were examined. The NHDF cell line was incubated with latanoprost, prostaglandinF2α, GLC756, or 5-fluoroucracil as a positive control at concentrations of 3 and 30 µM for 6, 18, and 24 hours. Fibroblast growth was measured by 5-bromodeoxyuridine (BrdU) uptake using Laser Scanning Cytometry. Latanoprost had the potential to increase transiently the number Poster Session P4. Alternative methods of cultured NHDF positively stained with BrdU. The stimulating effect on proliferation occurred early, 6 hours after incubation. GLC756, in contrast, revealed only inhibitory effects on BrdU uptake 18 to 24 hours after incubation. The inhibitory pattern of GLC756 was similar to that of the positive control 5-fluorouracil. Latanoprost seemed to have a direct stimulating effect on the proliferation of cultured NHDF. GLC756 had a fully antiproliferative effect on the NHDF, indicating an additional potential of novel dopamine compounds for topical glaucoma medication. 148 workshops at key scientific meetings and delivered presentations, on invitation, at a number of establishments within the UK and beyond. One of the challenges encountered by researchers searching for information on how to achieve reduction without compromising the scientific validity of a study is the fact that there are few “centralised information resources” which can be used to access the necessary details of useful techniques and which are freely available. Consequently the committee plans to launch an experimental design web-site which will combine the information available from a diverse range of sources, from databases to web-sites, within one web-site that will be easily and freely accessible to researchers worldwide. THE EFFECT OF ACETAZOLAMIDE ON EYE DEVELOPMENT J. Soleimani Rad, K. Sadegi, A. Alizadeh. Depts of Anatomical Sciences & Ophthalmology, Faculty of Medicine, Medical Sciences University of Tabriz, Tabriz – Iran Acetazolamide as a carbonic anhydrase inhibitor is widely used for decreasing intraocular pressure in treatment of Glaucoma. Acetazolamide administration causes ciliary body inflammation and a transient myopia. In the present study the effect of Acetazolamide on developing eye have been investigated. For this purpose, pregnant wistar rats are divided in control and experimental groups. In the experimental group, from 6th day of pregnancy onward, rats received 70 mg Acetozolamide / day for 10 days (orally). After delivery, 1 day old and 5 days old newborns, both in control and experimental groups were sacrificed and their eyes were fixed in 10% formaldelyde and prepared for light microscopic and morphometric studies. The results show that in control group, the thickness of cornea was 2.43±0.30 mm, while in Acetazolamide injected rats it reduced to 1.68± 0.16mm. The difference between two groups were statically significant (P<0.001). Additionally, in corneal stroma, muclei of fibroblasts became round and vesicular. While, nuclei in corneal epithelium and endothelium were more condensed than those in control group. Histological studies of Lens revealed that in experimental group, Lens fibers were arranged irregulary . in the lining of ciliary process, Acetazolamide injection produced vesicular nuclei and the two layers of pigmented and nonpigmented cells were not easily distinctive. Retinal study did not show a significant changes when compared to control group. It is concluded that Acetazolamide administration during pregnancy produces minor disorders. However, further studies are needed to clarify if these changes could result in visual defect or not. P4 Alternative methods 149 s43 THE CHALLENGE: REDUCING ANIMAL USE S. Vaughan, R. Combes. FRAME (Fund for the Replacement of Animals in Medical Experiments), Russell and Burch House, 96–98 North Sherwood Street, Nottingham, NG1 4EE, UK In 1998 the FRAME (Fund for the Replacement of Animals in Medical Experiments) Reduction Committee was created to facilitate means of reducing the number of animals used in biomedical research, education and testing. The committee comprises individuals with expertise in animal welfare, alternatives, experimental design and statistical analysis. The work of the committee focuses on the promotion of the use of good experimental design and statistical analysis techniques that allow researchers to achieve reduction, without compromising the scientific validity of a study. The members of the committee have been involved in the production of a number of training materials that are designed to demonstrate how experimental design and statistical analysis techniques can be used to achieve reduction. These include the production of a book and CD-ROM, together with the compilation of a list of relevant books and published papers. The latter can be accessed via the FRAME website at www.frame.org.uk. A list of software and web-sites has been compiled, which includes details of useful scientific papers that are freely accessible on-line, and will soon be added to the FRAME web-site. The members of the committee have organised a number of 150 SAFETY TESTS FOR COSMETIC FORMULATIONS: POSITIONING, CORRELATIONS AND PREDICTIVITY OF IN VITRO AND IN VIVO DATA E. Camel, J.P. Guillot. Institut d’Expertise Clinique (I.E.C.), 88, Boulevard des Belges, 69006 Lyon, France We are presenting the data obtained in-house, on nearly 6500 cosmetics tested between 1997 et 2002 with Single Patch tests, 3800 with In Use tests and 2700 with in vitro tests, with 4 different aims. Firstly, we compared the results obtained with different methodologies: tolerance was judged very good for 43% of the tested products with the in vitro Het Cam test (n = 2700), for 31% of the products with Human Single Patch test (n = 6500) and for only 13% of the products with Human In Use test (n = 3800). Respectively with these 3 methods, tolerance was judged good for 41%, 55% and 76% of the products, medium for 14%, 9% and 8% of the products, and bad for 2%, 5% and 3% of the products. Secondly, we compared the results obtained according to the type of products: 19±7% of irritative and discomfort reactions were reported for the whole tested products (n = 3800) during Human In Use tests, 18±4% for day creams, 23±6% for anti-wrinkles and 30±8% for skin care masks. Thirdly, we exposed the correlations which can exist between in vitro and in vivo tests (concordance test of Kappa): Het Cam (in vitro) versus Draize test (in vivo) gave 0% of discordant, 80% of close and 20% of concordant results (n = 122 rinse-off products), as Human Single Patch test versus Human In Use test gave 19% of discordant, 0% of close and 81% of concordant results (n = 665 products). Finally we compared the tolerance data obtained in Causasian and Asian volunteers: index of Primary Cutaneous Irritation (P.C.I.) was found to be statistically higher (p < 0.05; Wilcoxon test; n = 30 products tested each in 20 to 50 subjects) in Japanese (P.C.I. = 0.19 for non rinse-off and 0.21 for rinse-off products) versus Singaporean (P.C.I. = 0.10 for rinse-off and non rinse-off products) and Caucasian volunteers (P.C.I. = 0.13 for non rinse-off and 0.14 for rinse-off products). These results show the low predictivity between and within the quoted in vitro and Human tests, and the large discrepancy between safety data depending upon, in particular, cosmetic formulations and racial origin. 151 SKIN IRRITATION - EVALUATION OF MECHANISMS: DESCRIPTION OF AN IL-1α THRESHOLD T. Welss, K. Schröder. VTB-Skin Biochemistry, Henkel KGaA Skin irritation is one of the most common adverse reaction in humans. In vivo it is defined as a locally arising, non-immunogenic inflammatory reaction characterized by erythema, oedema, heat and pain. These characteristics are signs of an inflammatory reaction and are the ultimate physiological manifestation of a complex chain of biochemical, cellular, vascular and neural responses following the initial irritation stimulus. Even though considerable attention has been invested in attempts to understand the underlying mechanism(s), to date, the molecular and cellular responses following contact with irritants are still poorly understood. The aim of this study is to evaluate cellular mechanisms of skin irritation in vitro, to detect new markers of irritation and to compare various in vitro models. Here we investigated the outstanding role of IL-1α in the onset of irritation. Therefore, we set up two different experiments. s44 Poster Session P4. Alternative methods In order to investigate the role of IL-1 α in vitro, at first we treated Skinethic™and EpiDerm™epidermis models systemically with increasing concentrations of recombinant human IL-1α (rhIL1α), monoclonal antibodies against IL-1α, IL-1ra or combination of these. Second, we applied three anionic surfactants (SDS, LES, LSS) topically to investigate the inflammatory network in acute irritation. Subsequently, we examined in both experiments the release of various cytokines (e.g. IL-6, IL-8, TNF-α, PGE2 and IL-1α) using ELISA techniques. In the first experimental set-up, using Skinethic™, we detected a specific and dose-dependent release of IL-8 and TNF-α with enhanced release upwards a threshold at 500–1000 pg/ml rhIL-1 α. EpiDerm™ released IL-8, PGE2 and TNF-α, but using these markers we could not differentiate the individual treatments. Both epidermis models did not secrete detectable amounts of IL-6. In the second experimental set up, we determined a dose- and substancedepending release of IL-1 α in both models (SDS>LES>LSS). Using EpiDerm™ we did not detect a clear relation between the treatments and the release of IL-8. For Skinethic™ models we detected an IL-1 α/IL-8-threshold relationship: only those models released IL-8, in which topical application induced the release of IL-1 α greater than the described threshold. Using Skinethic™, these results suggest that an acute irritant stimulus, which does not lead to the release of the here-described IL-1 α threshold concentration will not trigger the secretion of IL-8. 152 A TIERED STRATEGY FOR IN VITRO PHOTOTOXICITY TESTING A. King, P. Jones. Safety and Environmental Assurance Centre, Unilever Colworth, Sharnbrook, Bedford, MK44 1LQ Where substances are intended for use in products, which are either applied to (or come into contact with) the skin, an assessment of potential phototoxic hazard is required. This may be carried out using a tiered strategy involving in vitro phototoxicity assays. The initial assay measures the UV/visible absorption spectrum of a test material to identify absorption at relevant wavelengths and therefore potential for photoreactivity to sunlight. This is followed by the in vitro 3T3 cell neutral red uptake phototoxicity test (3T3 NRU PT). The 3T3 NRU PT is a validated test of phototoxic potential which has been shown to identify both photoirritant and photoallergenic chemicals. To further evaluate the potential phototoxic effects on human skin, a confirmatory phototoxicity assay using a 3-D human skin model may then be conducted. This assay allows the application of test materials and formulations to the air-exposed surface, thus mimicking the in vivo situation and allowing potency comparisons with benchmark materials. This is particularly useful where inconclusive results are obtained in the 3T3 NRU PT. This report compares results for materials tested in a human skin model assay with the original 3T3 NRU PT predictions of phototoxic hazard, to gain insight into the practical use of these assays. Materials showing clear phototoxicity in the 3T3 NRU PT are generally confirmed as potentially phototoxic in a human skin model. In this case an assessment of potency may also be carried out by comparison with a known phototoxicant. Materials of border-line activity may either be confirmed as phototoxic or be less active (non-phototoxic) towards a human skin model. In the latter case this suggests that the material is unlikely to be a human phototoxicant, because a human skin model is more sensitive than human skin in vivo. These results illustrate the value (in a tiered strategy) of using human skin models to help interpret the results of initial in vitro phototoxicity testing using the 3T3 NRU PT. the in vitro EpiDerm™ and EPISKIN™ corrosivity tests. These tests are designed to predict and classify the skin corrosivity potential of a test material by assessing its effect on a reconstituted 3-dimensional (3-D) human skin model, and are based on the fact that corrosive chemicals show cytotoxic effects following short-term application to the epidermal stratum corneum. Both the EpiDerm™ and EPISKIN™ tests distinguish between corrosives and non-corrosives, but the prediction model for the EPISKIN™ test also assigns EU risk phrases or UN packing groups to corrosive materials. Test protocols differ in terms of number of replicates, incubation times and detailed MTT assay methodology, and the cultures are also physically different. EpiDerm™ tissues are larger (surface area 0.63cm2 ) than EPISKIN™ (0.38cm2 ), but the latter have a larger housing, using different culture plates and culture medium volumes (12-well plate/2.2ml medium per well for EPISKIN™ and 6-well plate/0.9ml medium per well for EpiDerm™). EPISKIN™ cultures also have to be removed from their housing for extraction of reduced MTT, instead of simply placing the whole culture in a well of extractant. The in-house performance of the EpiDerm™ and EPISKIN™ corrosivity tests was assessed and compared using materials of known corrosive potential (six corrosives and six non-corrosives). Both the EpiDerm™ and EPISKIN™ tests correctly identified the known corrosives and non-corrosives; however, the identification of corrosive labelling/packing groups by the EPISKIN™ test in this study was not reliable. Much larger numbers of corrosive chemicals would need to be tested to assess the in-house use of EPISKIN™ for assigning corrosive class correctly. There were therefore no differences between the two tests in terms of predictive ability in this study. 154 J.J. Hoffmann 1 , E. Heisler 2 , P. Peters 1 , S. Karpinski 1 , H.-W. Vohr 2 . 1 CellSystems Biotechnologie Vertrieb GmbH, St. Katharinen, Germany, 2 Bayer Health Care, Institute of Genetic & Molecular Toxicology, Wuppertal, Germany Modern pharmacotoxicology and dermatology research implies the development of in vitro models with high reproducibility and comparability to the native situation. In this study we present data obtained by the use of “Advanced Skin Test 2000” (AST2000, CellSystems Biotechnologie Vertrieb GmbH, St. Katharinen, Germany) a reconstructed human skin consisting of a dermal layer with fibroblasts overlayed by an epidermal layer with proliferating, differentiating and cornified keratinocytes which shows a high comparability to normal human skin. The major focus of this work was to determine the phototoxicity properties of several substances, e. g. promethazin or chlorpromazin, in the absence or presence of UVA light. If unradiated most of the tested substances provoked none or weak effects to the skin model while in the presence of UVA light it showed increased histological damages (H & E staining) and a significant reduction of cell viability (conversion of MTT (3-[4,5 – dimethylthiazol-2-yl] – 2,5 – diphenyltetrazolium bromide)). Furthermore the tissue reacted with an increased release of inflammation mediators such as IL-8 and PgE2 as determined by standard ELISA and Cytometric Bead Array (CBA). We conclude from these results, that the full thickness reconstructed skin (AST2000) is a usefull tool for correct classifications of substances concerning their phototoxic properties. 155 153 ASSESSMENT OF THE IN-HOUSE PERFORMANCE OF THE EPIDERM™ AND EPISKIN™ IN VITRO CORROSIVITY TESTS DETERMINATION OF PHOTOTOXICITY PROPERTIES OF DIFFERENT COMPOUNDS USING A FULL THICKNESS SKIN MODEL (AST-2000) ADVANCED SKIN TEST 2000 (AST-2000) AS A POTENT IN VITRO TOOL FOR THE CHARACTERIZATION OF SKIN REACTIONS BY PROTEIN FINGERPRINTING P. Jones, A. King. Safety and Environmental Assurance Centre, Unilever Colworth, Sharnbrook, Bedford, MK44 1LQ E. Heisler 1 , J.J. Hoffmann 2 , P. Peters 2 , H.J. Ahr 1 , H.-W. Vohr 1 . 1 Bayer Health Care, Institute of Genetic & Molecular Toxicology, Wuppertal, Germany, 2 CellSystems Biotechnologie Vertrieb GmbH, St. Katharinen, Germany Determination of the labelling requirements for certain products and ingredients requires information on their skin corrosivity potential. This information may now be obtained by using an in vitro skin corrosivity test. There are two validated commercial tests available, Different validation studies (COLIPA, ECVAM, OECD) revealed encouraging data concerning in vitro reconstructed human skin models in toxicological research. In cytotoxicity testings based on these skin models, comprehensive results were obtained, which highly correlate Poster Session P4. Alternative methods to those from well established in vivo methods. Therefore, artificial skin models provide fundamental advantages in comparison to single cell culture testings. In our present studies we focussed our research on the full thickness skin model AST-2000 (Advanced Skin Test). In contrast to reconstructed epidermal models, Advanced Skin Test additionally consists of a dermal layer with fibroblasts overlayed by an epidermal layer with proliferating, differentiating and cornified keratinocytes and, therefore, provides an architecture which is comparable to normal human in vivo skin. In an in-house validation study using topical application of irritating or corrosive compounds we found that all tested substances were correctly classified by AST-2000. Furthermore, supernatants were analyzed with respect to the induced production of immunomodulating molecules including proinflammatory cytokines as well as chemokines and matrix metalloproteases which are related to inflammation, tissue damage and wound healing in vivo. In this context also substances with a specifically sensitizing potential were tested. The overall results reveal that we are now able to show a characteristical immunorelevant fingerprint of a potent in vitro engeneered skin substitute according to the group of applied substances. We conclude from these results that AST-2000 is capable to classify test substances regarding their cytotoxic potential. Furthermore, AST-2000 provides a broad range of immunotypic parameters for the characterization of different skin reactions, because it presents the opportunity to evaluate important cell cell interactions between epidermal keratinocytes and dermal fibroblasts in vitro. Therefore, AST-2000 may help to discriminate between irritation, corrosion and sensitization. 156 RESULTS OF A SKIN IRRITATION VALIDATION STUDY WITH TOPICAL ACNE FORMULATIONS Joanna Harvey 1 , Marina Cappadoro 2 , Bart De Wever 2 , Adrian Davis 1 , Stuart Freeman 1 . 1 GlaxoSmithKline Consumer Healthcare R&D, Weybridge, Surrey UK. 2 SkinEthic Laboratories, Nice, France Facial acne of mild to moderate severity can be treated with numerous topical products which are considered safe, effective and convenient to use. Adverse effects associated with their use have prompted a need to develop formulations with improved efficacy/adverse effect profiles. This study characterises the Skin Ethic Reconstituted Human Epidermis (RHE) model as a predictor of skin toxicity. The irritation potential of thirteen clinically important acne products (retinoids [tretinoin, tazarotene and adapalene], benzoyl peroxide (BP), lactic acid, azelaic acid in both cream and gel formulations) were assessed in the model at 24 and 72 hours. Compounds were selected based on availability of high quality clinical data within the scientific literature. Cutaneous toxicity was assessed by cell viability, release of inflammatory mediators (IL-1α and IL-8) and histological examination. By several measures, the relative order of tolerance in vitro correlated well with clinical data for the majority of actives. All retinoid products were irritant in vitro. Adapalene was better tolerated in vitro than tretinoin or tazarotene; an effect mimicked in the clinic. Clear differences between formulations were generally not noted, however water based BP was well tolerated in vitro but alcohol based BP was not; a trend observed in the clinic. Differences were notable for azelaic and lactic acid; both were highly toxic in vitro, which does not concur with clinical data. These products are acidic and associated with stinging following application. Both act via the stratum corneum and given the age of the RHE constructs suggests an increased sensitivity to these products requiring adjusted contact time or concentrations. In conclusion, this model offers a promising alternative in selecting formulations with improved tolerability in dermatological drug development. 157 USE OF PROTEOMIC TECHNOLOGIES FOR DISCOVERY OF NEW MARKERS OF SKIN IRRITATION S.T. Fletcher 1 , V.A. Baker 2 . 1 SEAC, Unilever, Bedfordshire, UK, 2 CuDoS Cellular Development Systems Ltd, Nottingham, UK The development of predictive in vitro test systems for assessing skin irritation relies on a greater understanding of the mechanistic s45 basis of the human skin irritation response. Recent progress in the development of proteomic technologies means that tools for the investigation of important biochemical events in the processes of skin irritation and the discovery of potential new markers are now available. This study was designed to profile and identify proteins involved in the skin irritation response, following exposure of a reconstructed human skin model (EpiDerm™ (MatTek)) to a range of skin irritants: sodium lauryl sulphate (SLS), benzalkonium chloride (BKC), nonanoic acid (NAA) and phenol. EpiDerm™ cultures were treated in triplicate with non-cytotoxic doses of the skin irritants (0.1mg/ml SLS, 0.01% (v/v) BKC, 0.025% (v/v) NAA and 0.25% (v/v) phenol) as determined by MTT assay and histological examination, for exposure times ranging from 15min to 24hours. Proteomics was performed using SELDI-TOF mass spectrometry to investigate the protein expression profiles following exposure to skin irritants. A number of proteins (MW 6–52kDa) were found to be differentially expressed when profiled (analysis up to 300kDa) using a range of chromatographic ProteinChip™ arrays (Ciphergen). For example, proteins of MW 9.9 and 12.9kDa exhibited upregulation following exposure to skin irritants at the majority of time points tested, while some protein changes were specific to certain chemicals. In addition, differential phosphorylation (80Da mass shift) patterns correlating with exposure to skin irritants were also identified. Principal Components Analysis and hierarchical clustering enabled identification of potential markers of interest. 2D-gel electrophoresis was also performed in combination with MALDI-TOF mass spectrometry. A number of proteins including heat shock proteins, metallothioneins, calmodulin-like skin protein and involucrin demonstrated differential expression/post-translational modification following exposure to skin irritants. In conclusion, these results demonstrate the potential of proteomic technologies to investigate the differential regulation of proteins in response to skin irritants, some of which could represent potential new in vitro markers of skin irritation. 158 EPIDERM™FULL THICKNESS (EPIDERM-FT), A DERMAL-EPIDERMAL SKIN MODEL WITH A FULLY DEVELOPED BASEMENT MEMBRANE. P. Hayden, J. Kubilus, B. Burnham, G. Jackson, J. Sheasgreen, Mitch Klausner. MatTek Corp., Ashland MA Paracrine signaling between dermal fibroblasts (FB) and epidermal keratinocytes (KC) is believed to modulate skin responses during contact irritant or allergic reactions. Dermal FB also play an important role in photo-aging and photo-damage, wound healing and cancer progression (1–4). To enable in vitro study of these and other dermal phenomena in which FB-KC interactions are important, a full thickness skin model composed of a FB-containing dermis/KCcontaining epidermis was developed. Normal human epidermal KC and dermal FB were cultured to produce highly differentiated full-thickness tissues extending wall-to-wall in cell culture inserts. Histologic examination of the tissue shows a collagen dermis populated by numerous viable FB and an epidermis consisting of stratified KC including basal, spinous, granular and stratum corneum components. The ultrastructure of the dermal/epidermal junction was examined by transmission electron microscopy. A well-developed basement membrane was evident. Hemidesmosomes were observed at the basal membranes of KC, with associated tonofilaments extending into the cytoplasm. Well-defined, continuous lamina lucida and lamina densa and fine anchoring filaments were present beneath the basal KC. Anchoring fibrils with characteristic striated structure connected the lamina densa to the underlying collagen matrix. Tissue responses to ultraviolet irradiation (UVR) were also evaluated. Twenty-four hours after irradiation with 40 J/cm 2 of UVR, tissues were examined histologically, and culture media was assayed for pro-MMP-1 secretion by ELISA. Irradiation produced numerous sunburn cells and disruption of basal KC organization compared to control tissues. Also, pro-MMP-1 secretion was significantly increased compared to controls. EpiDerm-FT overcomes shortcomings of previous models in terms of providing a wall to wall tissue as well as appropriate in vivolike basement membrane development. These attributes will enable s46 Poster Session P4. Alternative methods more realistic in vitro toxicological studies of dermal/epidermal phenomena. 159 STRONG REPRODUCIBILITY FOR 50 CHEMICALS TESTED FOR THEIR IN VITRO SKIN IRRITATION POTENTIAL ON SKINETHIC RECONSTITUTED HUMAN EPIDERMIS USING MULTIPLE END POINT ANALYSIS. M. Cappadoro, C. Tornier, B. De Wever, M. Rosdy. SkinEthic Laboratories, Nice, France Development of in vitro tests on Reconstituted Human Epidermis (RHE) allows improving our knowledge on epidermal sensitivity to chemicals since they provide very reproducible results using an experimental design that is faster, cheaper and more convenient that the Rabbit Draize test and the Human Patch Test (HPT). 20 chemicals chosen by ECVAM (Zuang et al, 2002) and 30 additional chemicals evaluated in a human patch test (Basketter et al, 1999) were applied for 4 hours at 37° C on 4 different batches of SkinEthic RHE. An in vitro patch test protocol was compared to a direct topical application protocol. Multiple Endpoint Analysis (MEA) including tissue viability (MTT conversion), histology and IL-1alpha release measurement was performed. A strong inter-batch reproducibility of the individual endpoints in the MEA was observed. Moreover, both the in vitro patch test and the direct application protocols produced very similar results. Products that induced either significant loss of tissue viability, tissue necrosis, or significant release of IL-1alpha were classified as irritants. Results for the 50 test compounds are discussed in comparison with available human and rabbit skin irritation data. 161 M. Engelke, J. Patzke, S. Tykhonova, M. Zorn-Kruppa. Institute for Biophysics, Department of Physics and Electrical Engineering, University of Bremen, Bremen, Germany Validation studies of in vitro alternatives to the Draize rabbit eye test have revealed that cytotoxicity tests of simple corneal cell cultures are only valid in combination with organ cultured tests from animals for the prediction of potential ocular irritancy. The use of a human corneal model may close the gap between human and animal on the one hand, and between monolayer cell cultures and complex tissue on the other hand. Based on SV40 transformed human corneal cell lines we have reconstructed a 3-dim in vitro cornea which consist of an epithelial multilayer, an endothelial monolayer and keratocytes embedded in a collagen matrix. Histological cross sections of these engineered corneas resemble human corneas in tissue structure. The work that remains to be done is to characterise the model with respect to its epithelial barrier function and its permeability. It has to be shown, if the corneal model is suitable for toxicological test, and if it responses to factors that cause cornea irritancy in a manner comparable to the Draize test or other tests system. We also present the possibilities - and problems - to visualise and to quantify toxic effects of selected compounds on this corneal model. The toxic effects were determined from the cell viability using the LIVE-DEAD-assay in Confocal Fluorescence Scanning Microscopy. This work was supported by ZEBET (Bundesinstitut für Risikoforschung); project number 40100085. 162 160 INDUCTION OF CYTOCHROME P450 ENZYME ACTIVITY BY UVB AND XENOBIOTICS IN NORMAL HUMAN KERATINOCYTES G. Bertazzoni 1 , L. Benassi 1 , C. Magnoni 1 , M. Caselli 2 , S. Seidenari 1 . 1 Department of Dermatology, University of Modena and Reggio Emilia, Italy, 2 Department of Chemistry, University of Modena and Reggio Emilia, Italy The function of the skin is to provide a barrier for protection against the external environment. Relatively little is known about the overall role of CYP450 in the metabolism of xenobiotics or endogenous cellular compounds in the skin. The aim of this study was to analyse the expression and induction of several drug metabolizing enzymes involved in either phase I or phase II reactions, in proliferating human keratinocytes after exposure to UVB radiation and to three classical cytochrome inducers such as: β-naphthoflavone (BNF), 3-methylcholanthrene (MC), phenobarbital (PB). We investigated 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin Odepenthylase activities (PROD). Normal human keratinocytes were cultivated with mitomycin-treated 3T3 cells in Dulbecco’s modified Eagle’s medium/Ham’s F12. At confluency cells were incubated with inducers or irradiated with different doses of UVB. The microsomal fraction was studied by western blot analysis. The MC-induced EROD activity was up to 4 fold higher when compared with BNF induced activity. UVB exposure resulted in a dose-dependent (25– 75mJ) and time dependent (6–24h) induction of CYP450 1A1. Immunoblotting assay showed expression for CYP450 1B1 for both keratinocytes and melanocytes. Proadifen, an inhibitor of CYP450monooxygenase, led to a significant decrease in EROD activity. The results of the present study clearly show that irradiation with UVB is capable of modifying the activity of CYP450 isoenzymes in keratinocytes. The fase II enzymes glutathione S-transferase activity (GST) were induced by UVB and PB. These experimental findings stress the value of epidermal cell colture for pharmacotoxicological studies of topical agents used in dermatology. A HUMAN CORNEAL EQUIVALENT AS IN VITRO MODEL FOR ASSESSING OCULAR IRRITATION OCULAR PERMEATION OF TIMOLOL THROUGH EXCISED RABBIT CORNEA AND RABBIT CORNEAL EPITHELIAL CELL LAYERS: A COMPARISON S. Burgalassi, A. Brignoccoli, P. Chetoni, D. Monti, M.F. Saettone. Dept. Bioorganic Chemistry and Biopharmaceutics, University of Pisa, Pisa, Italy In vivo studies of transcorneal drug permeation involve sacrificing a large number of animals, and the same occurs when ophthalmic drug transport experiments are performed ex vivo, on isolated corneas mounted in appropriate chambers. These tests are currently criticized on the basis of ethical considerations, and development of alternative methods has been recommended. Corneal epithelial cell layers grown on a permeable support might prove a valid substitute of living corneas. In the last decade significant efforts have been dedicated to establish cell cultures as an efficient screening tool to asses drug transport processes. A number of cell lines have been characterized and are used routinely in pharmaceutical research and development. Cell cultures have many advantages over conventional techniques, including rapid assessment of data, control of the experimental design and possibility of using human rather than animal tissues. In this study we tested rabbit corneal epithelium cell cultures grown up to 15 days in air-interface conditions on specific polycarbonate membranes (Snapwell® ) as model substrate to permeation studies. The perfusion system consisted of six individual diffusion chambers, into which Snapwell devices thermostated by water-bath allowed to carry out simultaneous tests, thus improving the experimental precision. Timolol was used as model drug, and was tested alone and in combination with different permeation enhancers (benzalkonium chloride, polyethoxylated castor oil, polyoxyethylene (20) stearyl ether, sodium deoxycholate and escin). The apparent corneal permeability coefficients obtained by permeation studies through excised rabbit corneas and through the epithelial cell cultures were compared. Reconstituted corneal epithelium appeared less permeable to timolol than whole rabbit cornea. Poster Session P4. Alternative methods 163 EVALUATING THE OCULAR IRRITATION POTENTIAL OF 54 TEST ARTICLES USING THE EPIOCULAR™ HUMAN TISSUE CONSTRUCT MODEL (OCL-200) J. Sheasgreen 1 , M. Blazka 2 , J. Harbell 3 , M. Klausner 1 , P. Hayden, J. Merrill 3 , J. Kubilus 1 , C. Kloos 2 , D. Bagley 2 . 1 MatTek Corporation, Ashland, MA; 2 Colgate-Palmolive Company, Piscataway, NJ; 3 The Institute for In Vitro Sciences, Inc., Gaithersburg, MD Colgate-Palmolive is sponsoring a research program to validate the use of the EpiOcular™ Model in evaluating the eye irritation potential of surfactants. Previously, in a study that demonstrated the reliability of the EpiOcular Model, four laboratories using a formal and detailed study protocol tested 19 test materials. In the current study, two laboratories (MatTek Corp. and Institute for In Vitro Sciences, Inc.) have tested 54 test articles using the same study protocol. EpiOcular is a commercially available three-dimensional in vitro model of the human corneal epithelium composed of normal human-derived epidermal keratinocytes. Test articles included a shampoo formulation and 30 different surfactants (10-cationic; 11-anionic; 7-nonionic; 1-amphoteric; 1zwitterionic) which were liquids, powders or creams. Multiple concentrations of 11 of the surfactants were tested, to evaluate the model’s ability to predict dose-related differences in a test article’s potential for ocular irritation. Testing was conducted in compliance with FDA GLPs. The laboratories were blinded to the identities of the test articles. Test results were compared to previously published animal eye irritation studies. In terms of reliability, the results were reproducible within and between the laboratories. In terms of relevance, the EpiOcular model correctly predicted the Draize score for a majority of the samples tested. The model also correctly predicted increasing irritation potential of surfactants with increased concentrations. These data provide additional +evidence that the EpiOcular model meets the validation criteria, as defined by the Interagency Coordinating Committee on the Validation of Alternative Methods (NIH Publication No. 97–3981), for assessing the ocular irritation potential of certain classes of surfactant and surfactant-based formulations. 164 EVALUATION OF THE EFFECTS OF CHRONIC SUBCYTOTOXIC SURFACTANT EXPOSURE TO A HUMAN CORNEAL CELL LINE P.J. Wilkinson 1 , R.H. Clothier 1 . 1 FRAME Laboratories, School of Biomedical Sciences, University of Nottingham, Nottingham, United Kingdom In this study we aim to generate an in vitro model to investigate chronic corneal damage. Initially, the viability and barrier function integrity of SV40 human corneal epithelial cells (J-HCET) was monitored following chronic exposure to subcytotoxic concentrations of surfactants. Human Corneal Epithelial cells were cultured in a defined media containing non-cytotoxic concentrations of Tween 20 (T20: 25µgml−1 ), Sodium Dodecyl Sulphate (SDS: 4µgml−1 ), Benzalkonium chloride (BAK: 0.0025µgml−1 ) or Cocoamidpropylbetaine (CAPB: 6µgml−1 ). Pre-exposed and unexposed J-HCET were grown to confluency (n=8) in 96 well plate culture inserts and exposed to acute sub-lethal concentrations of T20 (150mgml−1 ), SDS (0.3mgml−1 ), BAK (0.15mgml−1 ) or CAPB (1.0mgml−1 ). Cell viability and barrier function, using the Resazurin reduction- and fluorescein leakage-assay, were determined prior to acute exposure and every 24 hour intervals over 96hrs. Acute surfactant exposure effects following pre-exposure were determined and compared statistically (repeat measures ANOVA) with non-pre-exposed cultures. Pre-exposure to BAK and SDS rendered J-HCET more susceptible (P<0.0001) to subsequent acute exposures of all classes of surfactants. CAPB pre-exposure render J-HCET more susceptible (P<0.0001) to acute CAPB and BAK exposure and moderately less susceptible (P<0.015) to acute SDS exposure. CAPB preexposure has no significant effect on subsequent acute T20 exposure. Pre-exposure to T20 reduced susceptibility to acute SDS, CAPB and BAK exposure (P<0.0001) and moderately less susceptible (P<0.025) to acute T20 exposure. Barrier function integrity was s47 lost in all combinations of acute-chronic exposure with subsequent recovery over time. Pre-exposure to BAK (cationic), SDS (anionic) and CAPB (amphoteric), render J-HCET more susceptible to acute exposures of ionic surfactants. In contrast, pre-exposure to a non-ionic surfactant (T20) renders J-HCET less susceptible to subsequent acute exposures. Modulation via the intercellular junctions following acute exposure is therefore dependent on pervious exposure history. This work was funded by a grant from the FRAME research council. 165 ACUTE TOXICITY: ALTERNATIVES TO THE LD50 TEST C. Longobardi, V. Pacelli, A. Argentino Storino. RTC, Research Toxicology Centre S.p.A., Pomezia - Rome, Italy The objectives of acute toxicity testing are to obtain information on the primary toxicological properties of chemicals, pharmaceuticals and biocidal products. This information is not only used in hazard identification and risk management during production, handling, packaging and labelling of chemical and biocidal products, but also for the identification of toxicological effects (in terms of clinical signs and lethality) of pharmaceutical products. The LD50 value, defined as statistically derived dose that is expected to cause death in 50% of the treated animals following a single administration in rats or mice has been considered, up until the end of 2001, as the basis for toxicological classification. The Fixed Dose, Toxic Class and Up and Down methods are valid alternatives to the LD50 test. However, although these methods have the advantage of using just a few animals, they only provide an estimation of the potential range of the LD50 value. The first version of these methods was adopted in 1996. Since then modifications have been made to further reduce the number of animals used (only females) and a new version of each method was adopted at the end of 2001. The previous and recent versions of these methods were applied to various products. The advantages and disadvantages, in terms of number of animals used, objectives and the time necessary to achieve them, have been analysed. The methods were compared in order to establish which was the most suitable for each type of product and to evaluate if the recent versions are an improvement on the previous methods. All three methods are suitable for the classification of chemicals. The further reduction in the number of animals and the use of females only (more sensitive sex) generally provides an estimation of the LD50 value which is towards the lower limit of the range. However, these procedures do not provide adequate toxicity data for pharmaceutical products to be used for further toxicological evaluations (i.e. maximum tolerated dose or minimal lethal dose). 166 PRELIMINARY (PHASE I) RESULTS OF A VALIDATION STUDY TO EVALUATE THE RELIABILITY AND RELEVANCE OF TWO IN VITRO CYTOTOXICITY ASSAYS FOR PREDICTING RODENT AND HUMAN ACUTE SYSTEMIC TOXICITY S. Casati 1 , J.A. Strickland 2,3 , M.W. Paris 2,3 , W.S. Stokes 2 , R.R. Tice 2,3 , J. Haseman 4 , A.P. Worth 5 , H. Raabe 6 , C. Cao 7 , R. Clothier 8 , J. Harbell 6 , R. Curren 6 , M.L. Wenk 9 , M.K. Vallant 4 , G. Mun 6 , M. Clear 6 , G.O. Moyer 6 , J. Madren-Whalley 7 , C. Krishna 7 , M. Owen 8 , N. Bourne 8 . 1 European Centre for the Validation of Alternative Methods (ECVAM) JRC, Ispra Italy, 2 NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), RTP, NC USA 27709, 3 ILS, Inc., RTP, NC USA, 4 National Institute of Environmental Health Sciences (NIEHS), RTP, NC USA, 5 European Chemicals Bureau (ECB) JRC, Ispra Italy, 6 Institute for In Vitro Sciences, Gaithersburg, MD USA, 7 U.S. Army Edgewood Chemical Biological Center, APG, MD USA, 8 Univ. of Nottingham, Nottingham, UK, 9 BioReliance Corp., Rockville, MD USA In order to assess the reliability and relevance of two in vitro basal cytotoxicity assays for predicting acute systemic toxicity in rodents and humans, NICEATM and ECVAM designed and started a joint validation study in July 2002. The 3T3 neutral red uptake (NRU) assay and the normal human keratinocyte (NHK) NRU s48 Poster Session P4. Alternative methods assay are being tested in three laboratories to assess the toxicity of seventy-two coded chemicals representative of all five Globally Harmonised System (GHS) hazard classification categories as well as the unclassified category. The Registry of Cytotoxicity (RC) Prediction Model will be applied to the new set of data to evaluate its predictive ability for rodent LD50 values whereas human acute lethal blood concentrations collected from the literature and from the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) in vivo database (MEMO) will be used to establish and evaluate the relationship between in vitro results and in vivo human acute toxicity. The three-phase design of the study allows the establishment of (1) a historical positive control database with sodium lauryl sulfate (SLS) in each laboratory (Phase Ia); (2) a two-stage protocol performance and optimisation phase with three coded chemicals (Phase Ib) and with nine coded chemicals (Phase II); and (3) a formal validation study with sixty coded chemicals (Phase III). Results obtained in Phase Ia and Phase Ib will be presented and the intraand inter-laboratory reproducibility of the data will be discussed. Supported by NIEHS contracts N01-ES-85424 and N01-ES-75408, EPA IAG DW-75–93893601–0, and European Commission contract N° 19416–2002–04 F2ED ISP GB. 167 APPROACHES TO THE MINIMISATION OF DOG USE IN THE SAFETY ASSESSMENT OF PHARMACEUTICALS: AN INDUSTRY/ANIMAL WELFARE INITIATIVE D. Smith 1 , R. Combes 2 , G. Descotes 3 , S. Dyring Jacobsen 4 , R. Hack 5 , J. Kemkowski 6 , K. Krauser 7 , L. Lammens 8 , R. Pfister 9 , B. Phillips 10 , Y. Rabemampianina 11 , S. Sparrow 12 , M. Stephan-Gueldner 13 , F. von Landenberg 14 . 1 Astra Zeneca, UK, 2 FRAME, UK, 3 Servier, France,4 Novo Nordisk, Denmark, 5 Aventis Pharma, Germany,6 Altana, Germany, 7 Schering, Germany; 8 Johnson & Johnson, Belgium; 9 Novartis Pharma AG, Switzerland; 10 RSPCA, UK; 11 Pfizer, France; 12 UK, 12 GlaxoSmithKline, UK, 14 Merck, Germany The primary non-rodent species used in toxicology is the dog. It is generally agreed that, for ethical and economic reasons, dog use should be reduced to the minimum consistent with maintaining the scientific quality of toxicology studies and ensuring human safety. Dog use in toxicology has been discussed widely, both from a scientific and ethical viewpoint, and there appears to be real potential for achieving significant reductions in the number of dogs used in pharmaceutical safety testing. An Industry/Animal Welfare Initiative was started in 2000 with the aim of evaluating and, where possible, putting into practice, scientifically valid approaches to minimise dog use in regulatory toxicology. The Steering Group categorised potential reduction approaches into three distinct areas, i) industrial co-operation/data sharing; ii) best practice in study design, and iii) assessing the need for particular studies. Progress has been made in evaluating and/or implementing approaches within the first two of these areas. One way to reduce dog use would be to establish a database of effects of vehicles and other non-active ingredients used in pharmaceutical formulations. These data are currently informally shared between a limited number of companies. Access to such a database would minimise the need for dog studies when it is intended to use a previously tested material or use it under different conditions. The possibility of establishing such a database is currently being explored. A best practice guide in aspects of study design, including appropriate group sizes, the use of control animals, single sex studies and the design of MTD studies, is currently being prepared and its current status will be presented. 168 IMPROVED PREDICTION OF THE HUMAN ACUTE TOXICITY BY THE DELAYED CYTOTOXICITY OF THE MEIC REFERENCE CHEMICALS IN Fa32 CELLS P.J. Dierickx. Instituut voor Volksgezondheid, Laboratorium Biochemische Toxikologie, Wytsmanstraat 14, B-1050 Brussel, Belgium An important accomplishment of the MEIC (Multicentre Evaluation of In vitro Cytotoxicity) study was that acute toxicity data for humans were established. The peak concentration of the approximate LC50 curve, expressed as the logM, appeared to be the best measure for human acute toxicity (Ekwall et al., ATLA 26: 571–616, 1998). Another important conclusion from the MEIC study is that in vitro systems predict the acute human toxicity better than rat or mice models. Searching for an improved in vitro/in vivo correlation, we now investigated the delayed cytotoxicity of the MEIC reference chemicals. Rat hepatoma-derived Fa32 cells were seeded at 60 000/microtiterplate well. After 24h the cells were treated with different concentrations of the test chemicals for 24h. They were then washed with PBS, and further cultured for 5 days in complete culture medium. The cytotoxicity was then measured by the neutral red uptake inhibition. The results were quantified by the NI50del, the concentration of test compound required to reduce the neutral red uptake with 50%. These values were compared with the NI50, measured immediately after 24h treatment. Six chemicals could not be tested because of solubility limitations. Delayed cytotoxicity was observed for 9 chemicals (NI50del< or =NI50). NI50del was 2–8.6x higher for 16 chemicals, and 1–2x higher for the remainder 19 chemicals. When the NI50del values were compared with the acute human toxicity, expressed as the peak concentration of the approximate LC50 curve, the correlation coefficient (r2 =0.76) was significantly higher than the one obtained previously in human hepatoma-derived Hep G2 cells (r2 =0.69), using total proteins as the endpoint. The latter was the best acute in vitro assay for the prediction of human toxicity within the MEIC study. Delayed cytotoxicty in Fa32 cells, therefore, better predicts the human acute toxicity than any of the in vitro assays of the MEIC study did. 169 ASSESSMENT OF THE IMPACT OF TOXIC EXPOSURES TO VIABILITY OF HIGHER EUKARYOTIC CELLS BY FLUORESCENT CELL METABOLIC AND CELL PROLIFERATION ASSAYS. Marleen Maras 1 , Jurgen del Favero 2 , Bart Naudts 3 , Paul Vanhummelen 4 , Erwin Witters 5 , Harry Van Onckelen 5 , Eddy Esmans 5 , Christine Van Broeckhoven 2 , Alain Vershoren 6 , Ronny Blust 1 , Wim De Coen 1 . 1 Laboratory for Ecophysiology, Biochemistry and Toxicology, University Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, 2 Department of Molecular Genetics VIB8, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, 3 Department of Mathematics and Computer Science, University of Antwerp, Middelheimlaan 1, B-2020 Antwerp, 4 VIB MicroArray Facility, UZ Gasthuisberg, Onderwijs en Navorsing, Herestraat 49, B-3000 Leuven, 5 Lab. for Plant Biochemistry and Physiology, University Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, 6 Department of Mathematics and Informatics, University Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium The lack of knowledge concerning the impact of many thousands of chemicals on human health and on the environment is a cause of growing concern. A growing number of chemicals is synthesized and wasted into terrestrial and aquatic ecosystems. For the majority of the compounds, little or no information is available concerning negative health effects. Until now, different biochemical, physiological and histological parameters of toxic stress, also called ’biomarkers’ were studied ’in vivo’ as well as ’in vitro’. While the latter assays provided valuable information, they do not allow volume screening of compounds, because they are time consuming and/or costly. Recently, new molecular biological techniques were developed that allow fast, easy and broad screening of chemicals. Microarray analysis, for instance, allows studying the expression of a large set of genes at specific time points after toxic exposures. In order to decide on relevant doses for mentioned toxic exposures, fluorescent cell metabolic and cell proliferation assays are performed: the Vybrant cytotoxicity assay (Molecular Probes) is used to measure glucose6-phosphate dehydrogenase activity in the medium above the cells as a consequence of damage to cellular membranes. Resazurin (also known as ’Alamar Blue’) is used as a dye to measure redox status of cell cultures. Intracellular esterase activity is measured using calceinAM (Live/Dead assay, Molecular Probes). Proliferation of cells is followed during one week following 24 or 48 hours toxic exposures, Poster Session P4. Alternative methods using the Cyquant dye (Molecular Probes). The information obtained from these assays will guide us to determine relevant exposure doses in hepG2 and MCF7 cells for 20 different chemicals with different modes of action. Expression profiles of exposed cells will be studied using microarray analysis (www.microarrays.be) and will allow us to select a set of clones for the construction of a ’Toxicological Custom-array’. 170 OLFACTORY MUCOSAL TOXICITY SCREENING AND MULTIVARIATE QSAR MODELLING FOR CHLORINATED BENZENE DERIVATIVES C. Carlsson 1 , M. Harju 2 , F. Bahrami 1 , T. Cantillana 3 , M. Tysklind 2 , I. Brandt 1 . 1 Department of Environmental Toxicology, Uppsala University, Norbyvägen 18A, SE-752 36, Uppsala; and 2 Department of Environmental Chemistry, University of Umeå, SE-901 87, Umeå and 3 Department of Environmental Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden The olfactory mucosa (OM) is an important target for metabolismdependent toxicity of drugs and chemicals. Several OM toxicants share a 2,6-dichlorinated benzene structure. The herbicides dichlobenil (2,6-dichlorobenzonitrile), chlorthiamid (2,6-dichlorothiobenzamide), and the environmental dichlobenil metabolite 2,6dichlorobenzamide all induce toxicity in the OM following covalent binding in the Bowman’s glands. In addition, we have shown that 2,6-dichlorophenyl methylsulphone targets the Bowman’s glands and is probably the most potent OM toxicant so far described. These findings suggest that the 2,6-positioning of substituents and an electron-withdrawing group in the primary position of the benzene ring may be an arrangement that facilitates OM toxicity. This study aimed to identify molecular and physicochemical characteristic for the 2,6-dichlorinated OM toxicants. A number of 2,6-dichlorinated benzene derivatives with various types of substituents in primary position were tested for OM toxicity in mice. In addition, some other 2,6 and 2,5-substituted benzene derivatives were examined. Two novel OM toxicants, 2,6-dichlorobenzaldehyde oxime and 2,6dichloronitrobenzene, were identified. By the use of partial least squares projection to latent structures (PLS), a preliminary QSAR model was built based on the lowest observed adverse effect levels (LOAEL) obtained combined with previously reported OM toxicity LOAELs. Physicochemical properties positively correlated to olfactory mucosal toxicity were identified as: heat of formation, molecular dipolar momentum, and the electronic properties of the substituent. Negatively correlated descriptors were variables describing the hydrophobicity of the molecule and electronic properties such as electron affinity, the energy difference in frontier molecular orbitals and the electronic charge on the primary carbon. In conclusion, this preliminary PLS model shows that a 2,6-dichlorinated benzene derivative with a large, polar, and strongly electron withdrawing substituent in the primary position has the potential of being a potent OM toxicant in mice. Further studies are needed to define the requirements on the substituents in 2,6-position 171 DEVELOPMENT OF AN IN VITRO BLOOD-BRAIN BARRIER SYSTEM T. Toimela 1 , H. Tähti 1 . 1 University of Tampere, Medical School, Tampere, Finland The blood-brain barrier (BBB) isolates the brain from systemic blood circulation. The BBB is formed by endothelial cells and partly by astrocytes. In vitro models are needed to study the permeation properties of chemicals, to evaluate their possible neurotoxic effects. There are several cell culture models for studying barrier functions, but isolation of primary endothelial cells is time-consuming and the results are usually not well reproducible and comparable. It would be of great importance if a human continuous cell line could be used to form the barrier In this study, we compared rat brain endothelial cells (RBE4) with human retinal pigment epithelial (RPE) cells for barrier functions. RPE cell line (ARPE-19) was chosen as a human barrier cell line candidate, because RPE cells form part of the natural blood-retinal barrier and are able to form tight junctions when grown in vitro s49 on porous filters. ARPE-19 cells or alternatively RBE4 cells were grown on microporous membrane filters to confluency. Human glial cells (U373MG) were grown on the opposite side of the membrane filters, because there are indications that glial cells may induce the formation of a tighter barrier. Human neuronal SH-SY5Y cells were the target cells on the bottom of the cell culture wells. In testing the barrier system, the model chemicals were inorganic and organic mercury (0.1–100 µM), and aluminium (1–1000 µM). They all show characteristic though different toxicities, and are known to be neurotoxic. Fluorescein-dextrane was used to monitor the leakage induced during the exposure. After the exposure, cytotoxicity was evaluated in all cell layers with total ATP measurement. Both of the barrier cell lines, ARPE-19 and RBE4, gave comparable results. With inorganic mercury, neuronal cytotoxicity followed the cytotoxicity in the barrier cell layer. With methyl mercury, cytotoxicity appeared in neuronal cells earlier than in barrier cells, but with aluminium there was no remarkable cytotoxicity in any layer. The results suggest that ARPE-19 cell line is a promising candidate for barrier cell line, but further characterisation is needed. 172 NEUROPATHY TARGET ESTERASE ACTIVITY REACTIVATION IN CHROMAFFIN CELL CULTURES TREATED WITH MIPAFOX. E. Quesada, M.A. Sogorb, E. Vilanova, V. Carrera. División de Toxicología, Universidad Miguel Hernández, Elche (Alicante) Spain Carboxylesterase activity, including the neuropathy target esterase (NTE), are widely distributed in many tissues, including bovine adrenal medulla and chromaffin cells. However, the biological function of these enzymes remains unclear. Some organophosphorus compounds induce a neurodegenerative syndrome related to the covalent modification of NTE. In this work, we studied some properties of carboxylesterases to complete the characterization of the model in order to use the primary cultures from bovine chromaffin cells in neurotoxicity studies. The mipafox inhibition kinetic in chromaffin cells primary cultures is studied in assays at variable time (1–120 min) with mipafox fixed concentrations. The inhibition of phenylvalerate esterase activity with mipafox at 1.5, 5, 15 and 75 µM was progressive with time. NTE activity (measured as the 40 µM paraoxon-resistant and 250 µM mipafoxsensitive esterase activity) was 9.2 ± 1.5 mU/106 (n=5) cells, which represented between 50–80% of the phenylvalerate esterase total activity (20.1 ± 1.5 mU/106 cells, n=5). This percentage was slightly lower than the NTE proportions found in the whole adrenal medulla homogenate. To study the possible reactivation of inhibited NTE, cells were preincubated with 3, 9, 15, 25 and 35 µM mipafox during 60 min, the inhibitor was removed by washing 3 time with Krebs medium. Immediately after removing the inhibitor, we observed an inhibition of 43%, 82%, 92%, 96% and 95% respectively. Forty eight hours after inhibition, the percentages of inhibition were 19%, 49%, 58%, 59%, and 63%, respectively. These results confirm a reactivation/recuperation of NTE activity between 34–57% at 48 hours. Despite the covalent inhibition of NTE, it is observed a partial recuperation of activity at 48 hours. It is unclear if this recuperation is a reactivation of inhibited activity or a resynthesis of new protein. ACKNOWLMENTS: E. Quesada is enjoining a fellowship from Spanish Ministry of Education and Culture. 173 DELAYED NEUROPATHY FORECAST: THE NEUROPATHY ESTERASE ACTIVITY IN VITRO AND IN VIVO AS A DELAYED NEUROTOXICITY OF ANTICHOLINESTERASE COMPOUNDS (PESTICIDES AND DRUGS) TEST N.V. Kokshareva, M.G. Prodanchuk, M.L. Zinovieva, O.V. Gudz. Medved’s Institute of Ecohygiena and Toxicology, Kyiv, Ukraine The mechanism of the delayed neurophaty (OPIDN) (development of ataxia, paralyses and produce axon demyelinization in 21 days after exposure of some organophosphorus (OP) compound) is not clear. The enzyme target of OPIDN is neuropathy esterase (NE), which is located in the nervous system. Activity similar to brain NE has also been found in lymphocytes of peripheral blood. The first biochemical lesion in the nervous system after OP treatment is significantly s50 Poster Session P4. Alternative methods depressed NE in nervous tissue. More than 30 substances with different structure (pesticides, drugs) in vitro and in vivo were examined. Correlation has been found between inhibition NE in human, hen, guinea-pig and rat brains, as well as in lymphocytes from peripheral human blood. Our investigation had shown that strong (at 78–92%) NE inhibition in 24 hours after treatment TOCP, aphos, oxyphosphonate in toxic doses (0.5 LD50 ) is early indication of OPIDN. OPs such as proserin, carbophos, malaoxon, hostacvik, fenitrothion ets., which have low inhibitory activity to NE (10– 32%), do not produce clinical disorders in hens. Electrophysiological investigation showed that this OP does not cause degeneration and demyelinisation in spinal cord and long peripheral nervous fibres. It was concluded that in vitro tests may be used in OPIDN predictions if OP does not undergo metabolic activation. 174 CYTOTOXIC EFFECTS OF 100 TOXIC COMPOUNDS ON HEP G2 AND HELA CELLS. Willem G.E.J. Schoonen 1 , Walter M.A. Westerink 1 , Jeroen A.D.M. de Roos 1 , Eric Débiton 2 . 1 Department of Pharmacology, Research and Development, N.V. Organon, Molenstraat 110, 5340 BH Oss, The Netherlands, 1 INSERM UMR 484, Rue Montalembert, 63005 Clermont-Ferrand, France The high attrition rate due to toxic effects of drug candidates in the development phase warrants the need for more mechanistic cellular approaches in early toxicological studies. The main drawback is that several of the toxic effects such as the formation of radical oxygen species (ROS), glutathione depletion, the induction of cell membrane damage, mitochondrial activity and cellular proliferation can not directly be related to specific biological target proteins. To get insight in these processes one has to focus on the molecular effect itself. In this study the focus is on relative sensitive fluorometric measurements and easy to handle assays. Dichlorofluorescein diacetate (DCF) can be used for the measurement of the formation of ROS molecules, while monochlorobimane (MCB) is used for the quantification of the glutathione status in a cell. The membrane stability, on the other hand, can be assessed after uptake of calcein-AM (CAM). The mitochondrial energy status of the cell is measured with Alamar Blue (AB) and cellular proliferation with Hoechst 33342 (HOE). The DCF, MCB, AB and HOE assays were carried out with human liver Hep G2 cells and the CAM assays with human cervix HeLa cells. The HeLa cells were used because of their better response noise ratio. The choice of 100 toxic compounds was initiated partly by a program on a Multicenter Evaluation for In vitro Cytotoxicity (MEIC) and partly by different and diverse categories of pharmaceutical compounds, being narcotic anelgesics, hypnotics, vasodilators, specific cellular energy blockers, cellular proliferation inhibitors, ion channel blockers, estrogens, antiestrogens, androgens, progestagens and others. The outcome of this study revealed that the same set of drugs was toxic in MCB, CAM, AB and HOE assays, while a very small selective set could only be identified for DCF. With DCF only doxorubicine, tertiair butyl hydrogen peroxide, sulfamoxole and ferrous sulphate were identified as ROS producers. With MCB, CAM, AB and HOE assays upto a level of 3.16 x 10−5 M, chlorprothixene citrate, cytarabine, dacarbazine, dixogin, doxorubicine, ellipticine, fluorouracil, methampyrone, methotrexate, Nethylmaleimide, nitropyrene, oligomycine B, papaverine, rotenone, tertiair butyl hydrogen peroxide and 3-methylcholanthrene showed a clear dose dependent effect in these assays. Further evaluation of these assays at higher toxic dosages, demonstrated toxicity effects for 56 of these 100 reference compounds in these assays. The time aspect of 24 h for MCB and CAM and 72 h for AB and HOE contributes to the slightly higher toxicity rates in the latter assays. Since the DCF assay is only a 3 h incubation assay, this explains its lower sensitivity for toxic radical formating compounds. In conclusion, 56 of the 100 tested compounds gave toxicity effects on Hep G2 and Hela cells used in this study. The results confirmed the outcome for the compounds used in the earlier MEIC study. Moreover, the toxic effects of MCB and CAM had a high correlation as well as between AB and HOE. All four assays identify more or less the same compounds as being toxic, making high throughput screening for early toxicity possible. 175 DIFFERENTIAL PROTEOME ANALYSIS OF RAT LIVER FOLLOWING TCDD EXPOSURE R. Pastorelli 1 , D. Carpi 1 , S. Tavazzi 1 , E. Fattore 1 , C. Chiabrando 1 , L. Airoldi 1 , H. Hakansson 2 , R. Fanelli 1 . 1 Department of Environmental Health Sciences, Istituto “Mario Negri”, Milano, Italy; 2 Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden This preliminary study was undertaken to investigate the extent to which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure alters liver protein expression and to determine which, if any, protein markers might be indicative of TCDD toxicity mechanisms. We have applied proteomic technologies to study changes in the levels of liver proteins of male rats treated with a single oral dose of TCDD (10ug/kg) on PND 21. Liver tissue was taken 22 days after the treatment. Liver proteins were separated by two-dimensional gel electrophoresis, followed by MALDI-TOF analysis of the tryptic peptides for protein identification. Only protein spots that changed more than 2-fold in magnitude, in the same direction (i.e. up or down) and were observed in all the triplicate gels were considered for gel excision and mass spectrometry analysis. The differential protein expression studies revealed the presence of significant derangements in about 10 proteins, following administration of TCDD. TCDD treatment modulates the expression of several enzymes related to oxidative stress, including an aldehyde dehydrogenase isoform with putative retinoid-related activity. For the first time, we reported a significant increase in the presence of 2 isoforms of the selenium binding protein 2 after TCDD treatment. The physiological role of this protein is still unknown. Interestingly, we observed a strong up-regulation of a protein related to the androgen metabolism, suggesting a putative new molecular target of endocrine disruption by TCDD. In summary, our proteomic approach allow us to have a global perspective of the many different known effects of TCDD. Most of the protein expression changes observed were consistent with the extensive literature on TCDD effects. However, some of the protein changes observed may lead to new insights into the mechanism of TCDD toxicity. 176 ENDPOINT SELECTION FOR HEPATIC CYTOTOXICITY EVALUATION USING LIVER SPHEROID MODEL Jinsheng Xu, Wendy M. Purcell. Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Bristol, UK In toxicology studies, toxic effect is indicated by assessment of endpoints. For toxicity screening purposes, few reliable endpoints are ideal and essential. Unfortunately, no single endpoint can indicate all toxic effects given assay interference by a test compound, different toxic mechanisms and method sensitivity. Therefore combined use of endpoints is necessary for robust assessment. This study investigates endpoint selection for hepatic cytotoxicity evaluation using a liver spheroid model. Liver cells were isolated from male Sprague rats and spheroids cultured by a gyrotatory-mediated method. Spheroids (6 days) were used for exposure. Diclofenac, galactosamine, isoniazid and paracetamol were selected as model hepatotoxicants. Endpoints selected include enzyme leakage (LDH and γ-GT), nutrient consumption (galactose and pyruvate) and secretion of products (albumin and urea). The results showed that galactosamine interfered with γ-GT assay, paracetamol interfered with LDH assay and isoniazid interfered with both assays. All the four selected chemicals decreased pyruvate consumption. Diclofenac, galactosamine and paracetamol significantly decreased galactose consumption but isoniazid had no effect on galactose consumption. However, isoniazid significantly affected albumin secretion even at a lower concentration. The significant decrease of albumin secretion was also seen after exposure to diclofenac and paracetamol but not apparent after exposure to galactosamine. Urea secretion was the poorest indicator among all the selected parameters and changed in an unpredictable way. Comparing all the selected endpoints, only pyruvate consumption predicted the toxicities of all the selected model toxicants and hence is the first choice for tests in this model. This study suggests Poster Session P4. Alternative methods that a test protocol should include a variety of indicators such as enzyme leakage, nutrient consumption and product secretion to provide a reliable assessment. 177 EVALUATION OF THE TOXICITY EFFECT OF ETHANOL ON CACO-2/TC7 COLON TUMOR CELL CULTURES. A. Chiusolo, G. Dal Negro. Cellular & Biochemical Laboratory, Safety Assessment Department, GlaxoSmithKline R&D, Verona The evaluation of new molecular entities by combinatorial chemistry is a well-known challenge for pharmaceutical companies. In the preclinical development, poorly-water soluble compounds are often encountered. Co-solvents, such as ethanol (EtOH), are used to increase the solubility but may have some toxic effects. The aim of the present work was to study the toxic effects of ethanol on Caco-2 cells in transport studies and also to determine its effect on monolayer integrity. Caco-2 cell line, a well-known in vitro model for the rapid screening of the intestinal drug absorption, was chosen to assess EtOH-mediated toxicity. The assessment of the cytotoxic effects of EtOH was carried out by measuring the release of Lactate Dehydrogenase and some other enzymes (Alkaline Phosphatase, Aspartate Aminotransferase, γ-Glutamyltransferase, Alanine Aminotransferase and Glutamate Dehydrogenase) in culture supernatant. The effect of EtOH on tight junctions was evaluated by measuring the apparent permeability of Lucifer Yellow and the trans-epithelial electrical resistance (TEER) as a function of EtOH concentration on Caco-2/TC7 monolayers. Furthermore, the expression of cytochrome P4502E1 involved in the solvent metabolism was investigated in untreated and in EtOH-treated Caco-2/TC7 cells. 178 VALIDATION OF AN IN VITRO MODEL FOR THE EARLY ASSESSMENT OF PHOSPHOLIPIDOSIS IN DRUG DEVELOPMENT. Alessandro Casartelli, Monica Bonato, Federica Crivellente, Illaria Masotto, Luca Vandin, Gianni Dal Negro. Cellular & Biochemical Laboratory, Safety Assessment Department, GlaxoSmithKline R&D, Verona Phospholipidosis is a term commonly used to indicate a cellular phospholipid storage disorder and characterised by an excessive accumulation of phospholipids within cells. Phospholipidosis could be induced by several mechanisms; regarding drug-induced phospholipidosis, Cationic Amphiphilic Drugs (CADs) are described to possibly induce phospholipidosis by the direct interaction of xenobiotics with intracellular phospholipids and phospholipid synthesis/metabolism impairment. In pharmaceutical research, lipid metabolism impairment and phospholipid accumulation can represent an issue; the identification of phospholipidosis-related findings in pre-clinical studies could have implications in the follow-up required in future clinical trials. Therefore, rapid and sensitive tests for the early assessment of phospholipidosis are needed in pre-clinical development. In this work, the validation of a cell culture-based model that enables the early and rapid assessment of the phospholipidogenic potential is discussed. In this method, the intracellular phospholipid content is measured after treatment with the test drug by using a human monocyte-derived cell line (U-937) and the staining with the fluorescence probe Nile Red. For the validation protocol, a panel of 36 drugs was tested; this panel included 23 positive drugs (whose phospholipid accumulation was documented by EM in different tissues/organs in the rat), 7 steatogenic drugs (causing lipid accumulations in rat livers) and 7 negative drugs (no lipid accumulation described). According to results obtained, 30 out of 36 test drugs were correctly identified (83.3%), 3 drugs were classified as false positive and 3 drugs as false negative (8.3% and 8.3% respectively). In conclusion, the U-937 method demonstrated a good sensitivity and specificity in discriminating drugs causing phospholipidosis. This method could be used as a screening tool for the rapid assessment of the phospholipidogenic potential of drugs in pharmaceutical research. 179 s51 A NEW METHODOLOGY FOR THE DETERMINATION OF PHOSPHOLIPASE ACTIVITY F.K. Ticli 1 , J.J. Franco 1 , A.M. Soares 2 , S.A. Uyemura 1 , S.V. Sampaio 1 . 1 Laboratório de Toxinologia, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto - SP. Brasil, 2 Unidade de Biotecnologia, Universidade de Ribeirão Preto, Ribeirão Preto – SP. Brasil Phospholipases have been extensively used in pharmacological investigation because of their toxic effects such as induction of inflammation, edema and myotoxicity. The importance of developing rapid and practical methods that will permit the quantitation of the inhibition of this activity by other substances is fundamental. The objective of the present study was to develop a method involving a solid culture medium to assess the phospholipase activity in egg yolk culture by measuring the halo formed by the activity in question. Phospholipase activity was determined in vitro in egg yolk culture medium (6 yolks/L) containing CaCl2 (0.56 g/L) and agar (20 g/L) according to the method described by Desnuelle et al. (Bull. Soc. Chim. Biol., v.37, p.285, 1955). Crude B. jararacussu venom (VBj) was used to determine the amount of venom to be used for the formation of a halo of a reasonable diameter. Since VBj presents high phospholipase activity, a higher concentration of other venoms is needed to obtain the same diameter. The ideal halo size was set at 10 mm and the activities were assessed with 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 µg VBj, after incubation and measurement at different time intervals (1, 2, 4, 8 and 24 h.), different incubation temperatures (25, 30 and 37°C), and different pH (4, 5, 6, 7 and 8). Analysis of the results demonstrated that the ideal VBj concentration needed to obtain a halo of 10 mm is 1 µg at the temperature of 37°C, incubation time of 24 hours, and pH 8. This is a small quantity (1 µg) compared to other methods, with pH 8 being ideal for phospholipase activity in egg yolk, since smaller diameters were obtained at other pH values using the same concentrations of VBj. 180 DEVELOPMENT OF MULTIPLEXED COMPETITIVE LUMINEX PROTEIN ASSAYS FOR TOXICITY PREDICTION SCREENS. Rhiannon Lowe 1 , Julie Byard 2 , Samuel L.L. Roberts 1 , Hayley C. Cordingley 1 . 1 GlaxoSmithKline Ware R&D, Department of Investigational Pathology and Toxicology GSK R&D, Ware, Hertfordshire UK, 2 Scientific Generics Limited, Harston Mill, Harston, Cambridge, Cambridgeshire UK The Luminex LabMAP™ system is a potentially powerful tool for use in proteomic biomarker profiling for toxicity prediction. In this poster a novel application of an indirect style of competitive immunoassay is presented which allows new assays to be developed more easily than with the standard “sandwich” format. Test assays for four proteins associated with oxidative stress (transferrin, NFκB, HSP60 and HSP70) were developed using this methodology and then multiplexed. Using a statistical experimental design, interactions between the multiplexed assays were investigated. Three of the four assays could be multiplexed with no significant interactions (transferrin and NFκB with either HSP60 or 70), but if all four assays were multiplexed HSP70 levels impacted on the HSP60 assay results. Further investigation of the effect of HSP70 on the HSP60 assay allowed development of a combined model for both proteins and the successful incorporation of the fourth assay into the multiplex. The transferrin and NFκB assays were developed further using cells treated with Hydrogen Peroxide, Menadione, DMSO, CdCl2 and Ciprofibrate. Proteins were extracted in IGEPAL extraction buffer. Increasing concentrations of Ciprofibrate resulted in a decrease in transferrin whilst Cadium Chloride caused an initial inhibition of transferrin followed by a gradual induction as concentrations were increased. NFκB was significantly induced by Hydrogen Peroxide treatment and changes with Ciprofibrate and Cadmium Chloride were also observed. These findings were re-confirmed by Western Blot. The development of these assays facilitates the future implementation of multiplexed LabMAP™ technology in predictive toxicity screens. s52 Poster Session P5. Quantitative structure–activity relationships (QSAR) P5 Quantitative structure–activity relationships (QSAR) 181 DEVELOPMENT OF A DATABASE FOR (Q)SAR IN REPEATED DOSE TOXICITY A. Bitsch, U. Wahnschaffe, N. Simetska, I. Mangelsdorf. Fraunhofer Institute of Toxicology and Experimental Medicine (ITEM), Nikolai-Fuchs-Str.1, 30625 Hannover, Germany Besides the use in pharmaceutical development, (Q)SAR approaches could gain remarkable importance in toxicology in future, i.e. for the evaluation of existing chemicals (whitepaper). Existing models vary considerably in their ability to predict different endpoints in toxicology. Furthermore, most of them do not cover the repeated dose toxicity. Therefore, within the framework of the Long Range Research Initiative of the chemical industry, we have developed a database to analyze the relationship between chemical functional groups/categories and target organs in repeated dose toxicity studies (14 days up to life-time). Content of the database: Data were taken from review documents or risk assessments in order to get a pre-screened selection of valid data. Chemicals were chosen by their structure which should be rather simple for well defined chemical categories. Inorganics, metal compounds and mixtures were excluded. Studies were selected by reliability; in general guideline studies were preferred. The database consists of three core data sets for each chemical: 1. structural features and physicochemical data (water solubility, state of aggregation, boiling point, log Pow, vapour pressure), 2. data on study design (species, number of animals, sex, application form, dose levels, study duration), 3. study results including overall NOAELs/LOAELs and all effects in all target organs with corresponding LOAELs. Standardisation and Transparency: To allow consistent queries, a high degree of standardisation in fields containing important chemical or study information is necessary. Therefore, PC data were summarized in categories and glossaries were developed for chemical category, functional groups, target organs and effects. In order to achieve a high degree of transparency, all information about a chemical is available to the user in form of a full substance report.Present status: The database consists of 212 chemicals investigated in 428 studies which resulted in a total amount of 1811 specific effects. The LOAELs cover the range from 0.08 up to 68526 mg/kg bw per day. Standard queries have been developed, which allow to analyze the influence of structural features and PC data on LOAELs, target organs and effects. First queries have shown that the database is a valuable tool. Most chemicals affect at least at high doses liver, kidney or body weight. Chemical structures which are related to other, more specific target organs or to high and low LOAELs can be identified. Future: Additional data will be added to the database and the query functions will be extended. Further, statistical analysis of query results will be performed 182 SAR AND QSAR USED IN A WEIGHT OF EVIDENCE APPROACH FOR EVALUATING THE EFFECTS ON HUMAN HEALTH OF PESTICIDES P. Crettaz. Swiss Federal Office of Public Health, Chemicals Division, 3003 Bern, Switzerland Pesticides are classified with respect to their human health effects mainly on the basis of animal studies. In vivo studies take a long time to complete and induce expensive costs. Data on close analogues and (Quantitative) Structure-Activity Relationships offer an interesting alternative for chemicals without data. They can also be used in a weight-of-evidence approach for chemicals with toxicological data. This latter application is discussed here for pesticides. We have launched a research project that aims to test the relevance and limitations of methods based on analogs and of QSAR models for human health. This project intends to provide a tool to classify pesticides more consistently, to better understand their mode of action by identifying structural alerts responsible for adverse effects and to decide if further testing is required to clarify an equivocal endpoint. A case study with dimethachlor was carried out. The software ToxSys™ has been used to find analogs to that herbicide. A list of similar compounds has been identified, which includes other chloroacetamides such as acetochlor, alachlor and metolachlor. Toxicological data have been found for these analogs and have been used to discuss the carcinogenic potential of dimethachlor. This step requires an expert judgement and is therefore of central relevance. The carcinogenicity has also been predicted by using commercial QSAR models. These models have enabled to detect structural alerts in the structure of dimethachlor and their predictions have been compared to the experimental data. The validation of these QSAR models is a central step in order to increase their acceptance by regulatory authorities. 183 PREDICTION OF ACUTE AQUATIC TOXICITY TO FISH COMPARING DIFFERENT QSAR APPROACHES A. Roncaglioni 1 , A. Colombo 1,2 , U. Maran 2 , M. Karelson 2 , E. Benfenati 1 . 1 Laboratory of Environmental Toxicology and Chemistry, Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy, 2 Department of Chemistry, University of Tartu, Tartu 51014, Estonia QSAR technique can be used to predict the risk of a substance from its chemical structure in order to fill the gap in experimental data. The present study provides an investigation and comparison among three different approaches in QSAR modelling, evaluating the possibility to obtain a better description of aquatic toxicity. We want to evaluate the improvement of performances in the developing of a QSAR model if complementary information (chemical and toxicological) is taken into account. To test this perspective we developed models considering different ways to group the compounds: based on the same chemical classes, or the same Mode Of Action (MOA) including the information available on the toxicological behaviour, or modelling all substances together without any a priori knowledge. We considered a large dataset provided by U.S-EPA Mid-continent Ecology Division containing 568 chemicals, belonging to fifteen chemical classes, and characterised by the experimental assignment to a specific MOA; the 96-hours Lethal median Concentration (LC50 -96h) for Fathead minnow (Pimephales promelas) was investigated. Several chemical descriptors were calculated with different software in order to obtain different categories of input variables capable to describe both 2D and 3D structures of the compounds (topological, geometrical, constitutional, electrostatic, hydrophobic and quantum chemical ones). Partial Least Square (PLS) method was utilised to predict LC50 96h, and internal and external validation procedures were used to validate the models developed with the different datasets. Comparison of results shows that best performances (Rcv 2 > 0,9) were obtained with the subsets based on both MOA and chemical class assignment, using no more than four chemical descriptors. On the other hand, models developed on the whole data set showed a Rcv 2 minor of 0,7 using five descriptors. We evaluated the characteristics of these three approaches evaluating performances, applicability of the approach, and availability of the complementary information required. These results emphasize the importance of developing appropriate tools for the a priori assignment to a specific QSAR model in order to obtain a sound predictive system.Acknowledgements: We acknowledge the financial contribution of the EC, IMAGETOX project (HPRN-CT-1999–00015). 184 THE COMPARATIVE USE OF QUANTITATIVE STRUCTURAL ANALYSIS RELATIONSHIPS (QSARs) AND MOLECULAR MODELLING FOR UNDERSTANDING RECEPTOR MEDIATED MECHANISMS OF TOXICITY, RECEPTOR CROSS-TALK AND IMPLICATIONS FOR ENDOCRINE DISRUPTION M.N. Jacobs 1 , C. Luscombe 2 , M. Dickins 3 , S. Hood 3 , D.F.V. Lewis 1 . 1 School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, UK, 2 Discovery Research, GlaxoSmithKline, Gunnels Wood Rd, Stevenage, UK, 3 DMPK, GlaxoSmithKline, Park Road, Ware, Herts, UK, 3 now at Pfizer, Sandwich, Kent UK We have generated a variety of quantitative structural analysis relationships (QSARs) for the xenobiotic and steroid hormone receptors Poster Session P6. Toxicogenomics AhR, human PXR, and human ERα using multivariate techniques, specific descriptors, and biological data sets. These QSARs have utilized in-house and experimentally generated data sets, collaborative (unpublished) and literature sourced data sets.These sets include biological data derived from a range of different species specific AhR containing cell lines treated with flame retardants, polybrominated diphenyl ethers (PBDEs), and chemical class specific and broad data sets (drugs, pollutants, phytochemicals, hormones) in hPXR and hERα. VolSurf descriptors were generated for each of the compound structures for which biological data was available and subsequently used to develop PLS regression based models; With the AhR model: (n=12, RMSEE=15.71), 2,3,7,8 TCDD was an outlier compared to the PBDEs, and major differences were apparent between the different cell lines, with EC50s (n=7) reflecting PBDE ligand potencies and cell line EROD values (n=7) reflecting P450 induction. For the hPXR model: (n=33, RMSEE = 22.37, R2 = 0.83) % maximum induction relative to rifampicin was used. This model was successfully internally validated using the randomization test. With the ERα model: (n=30), RMSEE=1.3 for Log relative binding affinity (RBA), two subsequent validation sets with a similar range of compounds demonstrated the applicability of the model (n=30, n=60, RMSEP=1.6). We have supported these QSARs by utilizing the crystal structures of hERα, hPXR and a homology model of AhR (based on the ERα crystal structure), to examine the mode of binding of representative compounds in the ligand binding domain (LBD) of the receptor crystal structures and model. Key commonalities and differences between the receptors LBDs and the ligands have been observed. Analyses of the Volsurf variable contributions together with observed in silico modes of ligand binding in the respective receptors, allow additional insights into receptor activation on a compound specific basis, as well as ligand promiscuity and ligand-receptor cross talk. This is of relevance to both drug design (for example in the treatment of ERα positive breast cancers and consideration of drug-xenobiotic interactions), and a better understanding of the mechanisms of pollutant toxicity and endocrine disruption. MNJ is supported by a BBSRC and GSK CASE PhD studentship. 186 s53 LIVER ENRICHED TRANSCRIPTION FACTORS AS PREDICTORS FOR TOXICITY – IMPLICATIONS FOR TOXICOGENOMIC RESEARCH J. Borlak 1 , A. Sowa 1 , M. Niehof 1 . Fraunhofer Institute for Toxicology and Experimental Medicine, 1 Drug Research and Medical Biotechnology New platform technologies offer unique opportunities to explore simultaneously an expression of thousands of genes and proteins. In conjunction with other molecular endpoints, reliable predictions of drug safety may now be feasible, at early stages of drug development. Drug induced alterations in gene and protein expression are also considered to be of critical importance in the onset and progression of organ toxicity and major efforts are on the way to determine the relevance of individual expression profiles in response to drug exposure. The observed changes in target gene/protein expression are proceeded by modulation of transcription factor protein/DNA interactions [1–3]. Examples will be given to demonstrate, on the one hand, the severe limitations in the use of tumor cell lines and other badly validated cell culture models for the prediction of tissue specific toxicity and, on the other hand, how properly designed and validated in vitro models hold promise for reliable predictions of hepatotoxicity. Results from studies with model hepatotoxins are given to provide experimental evidence. This work is supported by Grants from the Lower Saxony Ministry of Science and Culture and the German Ministry of Science and Education (BMBF). References: [1] Schrem, H., Klempnauer, J. and Borlak, J. Liver enriched transcription factors in liver function and development. Pharmacological Reviews 2002, 54: 129–158 [2] Borlak, J., Dangers, M. and Thum, T. Aroclor 1254 modulates gene expression of nuclear transcription factors: Implications for albumin gene transcription and protein synthesis in rat hepatocyte cultures. Toxicology and Applied Pharmacology 2002, 180 (in press) [3] Borlak, J. and Thum, T. Induction of nuclear transcription factors, CYP monooxygenases and GSTA2 gene expression in Aroclor 1254 treated rat hepatocyte cultures. Biochemical Pharmacology 2001, 61: 145–153 P6 Toxicogenomics 187 185 THE PREDICTIVE VALUE OF GENE EXPRESSION MICORARRAYS IN TOXICOGENOMICS Hans Gmünder, Andreas Hohn. Genedata AG, Maulbeerstrasse 46, CH-4016 Basel, Switzerland Micro array-based gene expression profiling is poised to play a central role in the field of toxicogenomics. Pinpointing the potential toxicity of unknown compounds by comparing their mechanisms of action (MOA) with those of known substances will lead to the rapid and reliable categorization of novel compounds and the prediction of potential, severe side effects at an earlier experimental stage. However, an effective and reliable use of microarray data in toxicology studies depends heavily on a well-designed strategy of streamlined and highly standardized data analysis procedures. Ultimately, the use of computational analysis in large-scale toxicological studies will result in the more cost-effective production of safer and more effective drugs. Genedata has developed an in-silico solution specifically developed to optimize gene expression data analysis for high-volume toxicological studies. Building and using a “reference compendium” is at the core of this solution. In an initial step, a collection of reliable, quality assessed mRNA signatures of relevant well-known compounds are used for building a “reference compendium”. These data are collected along with additional, vitally important biological and experimental information. In a second step, the value (or quality) of this compendium is cross validated and, if necessary, statistical analysis is used to find the characteristic genes that indicate a distinct pattern. Finally, statistical analysis can be used to classify novel, uncharacterized compounds into the “reference compendium”, allowing a prediction of their potential toxicity and their MOA DEVELOPMENT OF RAT AND FISH LIVER NUCLEOTIDE ARRAYS FOR STUDYING GENE EXPRESSION IN RESPONSE TO ESTROGENIC COMPOUNDS K. Schirmer 1 , J. Wober 2 , A. Schreer 1 , A. Caldarelli 2 , G. Vollmer 2 . 1 Junior Research Group of Molecular Animal Cell Toxicology, UFZ Centre for Environmental Research, Leipzig, Germany, 2 Department of Zoology, University of Technology Dresden, Dresden, Germany An increasing number of xenobiotic environmental contaminants and natural compounds are suspected to be capable of modulating endocrine regulation in humans as well as in animals. Effects elicited by these substances are reflected in changes in gene expression, either directly through transcriptional regulation or indirectly as part of a cellular response to chemical stress. Therefore, the assessment of gene expression is an indispensable tool for understanding mechanisms by which these substances induce potential longterm toxicological effects. Recent technological advances, allowing simultaneous characterisation of large numbers of genes, promise to aid in identifying these mechanistic effects. It is for this reason that we develop gene arrays that specifically aim at liver function in the model systems rat and rainbow trout upon exposure to estrogenic compounds. We selected liver because it is an important target organ for estradiol and other hormones as well as a site of hormone metabolism. To select genes for the organ-specific arrays, two approaches are being pursued. Firstly, genes that have previously been described or are suspected to be regulated by estrogenic compounds are being added. Secondly, differential display rtPCR is used as a tool to identify genes not described until now in the context of estrogen exposure. Regulation of novel genes is being verified by means of quantitative rtPCR. For example in the rat liver, carboanhydrase 2 and proliferation cell nuclear antigen were found to be estrogen-responsive genes and added to the targeted array. Genes s54 Poster Session P6. Toxicogenomics added specifically to the rainbow trout array are vitellogenin and zona radiata. Examples of genes common to both arrays are estrogen receptor β and cyclooxygenase 2. By our comparative approach, we hope to obtain new insights into species specific mechanisms and risks brought about by estrogenic compounds in both vertebrate species. 188 COMPARING THE TOXICITY AND POTENTIAL CARCINOGENICITY OF TWO ANTIESTROGENS. A SHORT-TERM STUDY WITH MICROARRAY ANALYSIS IN FEMALE RAT LIVER P. Hirsimäki 1 , A. Aaltonen 2 , A. Flores-Morales 3 , L. Kangas 2 , G. Norstedt 3 . 1 Turku University Central Hospital, Dept. of Pathology, BioCity, Tykistökatu 6 B 6, 20520 Turku, Finland; 2 Hormos Medical Corp, PharmaCity, Itäinen Pitkäkatu 4, 20520 Turku, Finland; 3 Dept. of Molecular Medicine and Cell and Molecular Biology, Karolinska Institutet, 17176 Stockholm, Sweden Introduction: Tamoxifen and toremifene are triphynylethyle antiestrogens used in the treatment of breast cancer. Tamoxifen is genotoxic hepatocarcinogen in the rat, whereas toremifene is not. In humans, long-term tamoxifen treatment causes an increased risk of endometrial cancer. The major difference between the genotoxicity of the two drugs is the formation of liver DNA-adducts in tamoxifen-treated rats. Differential gene expression in rat liver after administration of antiestrogens was studied by microarray analysis. Materials and methods: Equimolar doses of antiestrogens were given p.o. to six week old female Sprague-Dawley rats for 10 days. Total RNA was isolated using TRIzol Reagent. Equal amounts of total RNA from four animals in the same group was pooled and then purified from 1 mg total RNA using 35 mg oligo(dT)cellulose. The cDNA array contained approximately 4000 cDNA clones selected from the TIGR Rat Gene Index. The samples were hybridized to rat cDNA array scanned with a GMS 418 scanner (Afflymetrix). Image analysis was performed using the GenePix Pro software. Normalization was performed using Lowess normalization. The results from rat cDNA array were evaluated according to “Significance Analysis of Microarrays” (SAM). Results: SAM plot for tamoxifen vs. control revealed 445 significant genes from which 183 were positive significant (upregulated, red) q < 5%, 165 and 262 negative significant genes (down-regulated, green) q < 5% 242. Particularly the NADPHcytochrome P450 oxidoreductase and microsomal epoxide hydrolase were upregulated genes and are apparently related to tamoxifeninduced carcinogenesis. SAM plot for toremifene vs. control revealed 443 significant genes from which 129 were positive significant q < 5%, 35 and 314 negative significant genes (down-regulated, green) q < 5% 75. Conclusion: Microarray technology gives important information of the toxicity as well as of the carcinogenicity of antiestrogens in experimental conditions. 189 GENE EXPRESSION CHANGES IN RAT LIVER INDUCED BY TWO HYPOLIPIDEMIC DRUGS; A PPAR-α AGONIST AND A HMG-COA REDUCTASE INHIBITOR AND DIETHYLHEXYL PHTHALATE USING AFFYMETRIX GENECHIP® TECHNOLOGY N. Simecek, J.P. Wood, S.L. Margrett, C.J. Waterfield, H. Thakkar, J. Lyon. Department of Investigative Pathology and Toxicology, GlaxoSmithKline, Welwyn, Herts, AL6 9AR, United Kingdom Peroxisome proliferators are a diverse group of chemicals that result in increased peroxisomal β-oxidation in rodents and peroxisome proliferation, hepatomegaly and eventually liver tumours. The lipid lowering drug Fenofibrate, a well known peroxisome proliferator, lowers triglycerides via activation of the peroxisome proliferator activated-receptor-alpha (PPAR-α). This results in the lipolysis of triglyceride rich particles through activation of lipoprotein lipase and by the inhibition of VLDL triglyceride synthesis. Simvastatin belongs to another class of lipid lowering agents referred to as HMG-CoA reductase inhibitors. HMG-CoA catalyses the conversion of HMG-CoA to mevalonate which is an early and rate limiting step in cholesterol biosynthesis. The purpose of this study was to compare the underlying mechanisms by which these compounds alter β-oxidation of fatty acids and lipid metabolism in the liver and to identify pathways with the potential to produce markers of peroxisome proliferation. This was done by monitoring compound induced gene expression changes using Affymetrix GeneChip® technology. For comparative purposes the PPAR-α agonist diethylhexyl phthalate (DEHP), a plasticiser also known to cause peroxisome proliferation, was also included. Four groups, of 5 male Sprague Dawley rats were treated by oral gavage for 7 days with 0.5% (w/w) hydroxymethlycellulose and 0.1% (w/w) polysorbate 80 in phosphate buffer, pH7 (control animals), Fenofibrate, (200mg/kg/day), DEHP (1.2g/kg/day) or Simvastatin (120mg/kg/day). On day 8 livers were removed and samples taken for electron microscopy or snap frozen in liquid nitrogen. Peroxisome counts were carried out showing that Fenofibrate increased peroxisome number approximately 5 fold relative to control. DEHP and Simvastatin increased peroxisome number approximately 2.5 fold relative to control. Total RNA was isolated from the liver tissue, biotin labelled via reverse transcription and hybridised to an Affymetrix Rat Array consisting of approximately 9000 genes. Data was analysed using univariate and multivariate methods including Principal Component Analysis and Partial Least Squares Discriminant Analysis. The greatest number of significant gene changes was observed with Fenofibrate then DEHP and the least number were observed with Simvastatin. Significant gene expression changes were then imported into various pathway mapping tools where a diverse array of pathways were found to be modulated, many of which were in keeping with the known biology of these compounds. Using the results from this study, further analyses will be carried out using RT-PCR to validate significant gene changes. 190 TOXICOGENOMICS: COMPARISON OF IN VITRO AND IN VIVO MODELS USING PIQOR™ Tox cDNA MICROARRAYS Katrin Buss 1 , Tanja Hansen 2 , Stefan Tomiuk 1 , Kay Hofmann 1 , Frank Hübel 1 , Andreas Bosio 1 , Jürgen Borlak 2 . 1 memorec Stoffel GmbH, Stöckheimer Weg 1, D-50829 Cologne, Germany; 2 Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Str.1, D-30625 Hannover, Germany Early toxicology is an invaluable tool to reduce costs of drug development by discriminating troublesome compounds from further processing. The concept of toxicogenomics is to discover particular expression patterns that can be related to toxic endpoints. By comparing expression profiles caused by known toxicants versus those of approved drugs it should be possible to identify candidate genes/ patterns that forecast (liver-)toxicity of chemical entities. Together with the Fraunhofer ITEM we have started to set up the ToxSAYS™ database, which will contain expression profiles of ‘bad guys’ and of frequently prescribed drugs. Besides the topic of interspecies comparison, another challenging question is the predictivity of in vitro models for the in vivo situation. Therefore, all compounds are investigated in GLP compliant in vivo studies in rats and in vitro in rat and human primary hepatocytes (collagen sandwich cultures). The expression profiles are gained by using the PIQOR™ Tox Rat microarray which contains 1250 well defined cDNA fragments of genes covering tox-relevant processes. Three substances (galactosamine, 2-acetylaminofluorene and WY14643) mediating liver toxicity by different mechanisms were investigated in male and female rats in 28 day toxicity studies. The altered gene expression was analysed in liver, lung, heart and kidney by using PIQOR™ Tox Rat cDNA microarrays. The results are compared to expression profiles of the respective substances generated in rat primary hepatocytes. The ability to gather from in vitro studies to the in vivo situation is discussed. Poster Session P6. Toxicogenomics 191 APPLICATION OF GENE EXPRESSION TOWARD DISCOVERY OF CANDIDATE BIOMARKERS OF NEPHROTOXICITY: A COLLABORATION WITHIN THE INTERNATIONAL LIFE SCIENCES INSTITUTE CONSORTIUM ILSI/HESI Genomics Nephrotoxicity Working Group. The rapid development and evolution of genomic, proteomic, and metabonomic based technologies has led to the development of a new field of “toxicogenomics” that will allow application of these technologies to improve the efficiency of safety and risk assessments. This will be accomplished by facilitating better understanding of the mechanisms by which compound induced injury occurs coupled with the potential for identification of early, sensitive biomarkers of toxicity. Investigators from industry, government, and regulatory agencies have been participating in an industry wide collaborative effort targeted at evaluating the development and application of genomics technologies in toxicology. This effort is currently coordinated by the International Life Sciences Institute’s (ILSI) Health and Environmental Sciences Institute (HESI). Members of the ILSI HESI consortium include a large representation of pharmaceutical companies, as well as membership from other industry sectors and academic and government institutions including the FDA. One subteam of this effort has focused on use of microarray technology to elucidate mechanisms and identify candidate biomarkers of kidney toxicity. An experiment was conducted to analyze the toxicity to three nephrotoxic compounds, cisplatin, gentamicin, and puromycin. These three compounds differ in their structure and pharmacologic action and each have features that are both distinct as well as overlapping with respect to their target organ toxicity profile. Cisplatin and gentamicin primarily target the proximal tubule of the kidney, while puromycin targets the glomerular region. Sprague-Dawley rats were exposed to these compounds at several doses for collection at several time points. Microarray analysis of kidney RNA was conducted on several chip platforms at several ILSI HESI collaborator sites. One set of analyses were conducted at NIEHS on a 7K rat chip, and these data were assessed in conjunction with standard clinical markers and pathology. Principal component analysis and profile clustering confirmed separation based on relative toxicity and allowed identification of specific gene expression changes consistent with region of toxicity. In addition, several candidate biomarkers for phospholipidosis and necrosis were indicated. In one instance, comparison of puromycin-induced gene expression changes with those modulated by tubular toxicants was valuable in the identification of tubular toxicity, in addition to the more prominent glomerular toxicity that was observed histopathologically. Current efforts are focused on further validation of these markers and validation of the approach to apply renal gene expression profiling, coupled with classic toxicity markers, to reveal useful information/markers related to mechanisms of renal toxicity 192 NAPHTALENE EXPOSURE: EFFECTS ON APOPTOSIS AND GENE EXPRESSION IN HUMAN CORD BLOOD CELLS. Ilaria Malerba 1 , Cristina Diodovich 1 , Gerard Bowe 1 , Marco G. Bianchi 2 , Francesco Acquati 2 , Laura Gribaldo 1 . 1 ECVAM, Institute for Health and Consumer Protection, J.R.C., Ispra, Italy; 2 Dipartimento di Biologia Strutturale e Funzionale, Universita’ dell’Insubria-Varese, Italy Naphthalene is a compound widely used in the synthesis of a number of products, such as mothballs, grinding wheels, coal tar soaps and shampoos. The humans could be exposed to particulate and vapors of naphthalene during its production, product usage, combustion processes, in particular from vehicle exhausts, cigarette smoking or contamination of drinking water Epidemiological studies revealed that naphthalene is not genotoxic but it caused an increase in bladder cancer in workers occupationally exposed and its vapors induced respiratory epithelial adenomas and olfactory epithelial neuroblastoma in rat. Moreover, naphthalene can interfere with the correct haematopoietic process causing acute haemolytic anaemia and other haematological effects in workers exposed to naphthalene s55 vapours and in human exposed by ingestion to solid moth balls of naphthalene. In this study we evaluated the effect of naphthalene on the induction of apoptosis in cord blood cells, as well as on gene expression profiles using genomic technology and its activity on the bcl-2 related protein expression. Our data demonstrated that naphthalene (500µM) after 6hrs, 24hrs and 48hrs of exposure induced cell proliferation in the cord blood and in the CD34+ primitive cells, rendering the cells more resistant to toxicant and capable of surviving after chemical compound treatment. Western blot data revealed an over expression of BCl-2, Jun, Fos and Raf-1 proteins, which are involved in the anti-apoptotic response and in the cell growth, differentiation and development. Macroarray analysis showed that naphthalene modified the cord blood gene expression, inducing IL-8 precursor and T-cell transcription factor and decreasing the level of RNA-binding protein fus/tls and 60S ribosomal protein L6. 193 TOXICOGENOMICS ANALYSIS OF HUMAN UMBILICAL CORDS TO ESTABLISH A NEW RISK ASSESSMENT OF HUMAN FETAL EXPOSURE TO MULTIPLE CHEMICALS M. Komiyama 1,2 , D. Nishimura 1 , K. Takashima 1 , T. Adachi 3 , C. Mori 1,4 . 1 Department of Bioenvironmental Medicine, Graduate School of Medicine,Chiba University, 2 Center for Environment, Health and Field Sciences, Chiba University, Chiba, Japan, 3 Center for Research and Development of Bioresources, Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Japan, 4 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST), Kawaguchi, Japan Our previous studies using umbilical cords show that human fetuses are exposed to multiple chemicals including endocrine disrupters in Japan. Since there is anxiety that these multiple chemical exposures may cause delayed long-term effects after the fetuses are born and grow up, it is urgently necessary to establish a new evaluation method of health risk derived from exposure to multiple chemicals during fetal period. We are attempting to apply toxicogenomics analysis of human umbilical cords to the future risk assessment. In this study, we analyzed relationship between concentrations of chemicals (PCBs and organochlorine pesticides) and gene expression patterns in umbilical cords of 9 Japanese newborns. Using cultured human umbilical vein endothelial cells (HUVEC) as reference, gene expression in each umbilical cord was examined by Agilent Human 1 cDNA microarray. Out of about 15000 genes on the microarray, 1765 genes were expressed in some or all of the umbilical cords at relative intensity of <0.5 or 2< as compared to HUVEC. Cluster analysis using the 1765 genes revealed that the 9 umbilical cords were clustered into 4 groups based on expression profiles of the genes. Interestingly, the hierarchical order of the clustered umbilical cords corresponded to the order of total concentrations of a selection of chemicals with one exception. In the exceptional umbilical cord, total concentration of the chemicals was lowest, but its gene expression profile was most similar to that of the umbilical cord which exhibited the highest level of the total chemical concentration. These results suggest that gene expression profile of umbilical cord can be used for evaluation of exposure levels at fetal period. Moreover, it might be useful to detect potential high risk group, if higher exposure of fetuses to multiple chemicals means higher health risk to them. 194 ILSI HESI COMMITTEE ON THE APPLICATION OF GENOMICS TO MECHANISM BASED RISK ASSESSMENT Peter G. Lord 1 , Syril D. Pettit 2 , William D. Pennie 3 . 1 Johnson & Johnson, Raritan, New Jersey, USA, 2 ILSI/HESI, Washington DC, USA, 3 Pfizer, Groton, Connecticut, USA In mid-1999 the membership of the ILSI Health and Environmental Sciences Institute (HESI) formed a project committee to develop a collaborative scientific program to address issues, challenges and opportunities afforded by the emerging field of toxicogenomics. Experts and advisors from academia and government laboratories participate on the Committee, along with approximately 30 corpo- s56 Poster Session P8. Food safety rate member organizations from the pharmaceutical, agrochemical, chemical and consumer products industries. The committee has, since its formation, designed, conducted and analyzed numerous toxicogenomics experiments within the broad fields of hepatotoxicity, nephrotoxicity and genotoxicity. The considerable body of data generated by these programs has been instrumental in increasing our participants’ understanding of sources of biological and technical variability, the alignment of toxicant-induced transcription changes with the accepted mechanism of action of these agents (as reported in the literature), and the challenges in the consistent analysis and sharing of the voluminous datasets generated by these approaches. From a technological perspective, the Committee’s findings to date have revealed and characterized multiple potential and actual sources of variability, including expected sources of biological variability, operating procedures in the isolation and labeling of mRNA samples, non-standard settings on hardware and analysis software, microarray number and differences in gene coverage, and/or annotation across different technical platforms. The experimental programs have highlighted several important aspects of the use of genomics in risk assessment. 1) Patterns of gene expression relating to biological pathways are robust enough to allow insight into mechanism. 2) Gene expression data can provide strong information on topographic specificity. 3) Dose dependant changes can be observed. 4) Concerns around over-sensitivity of the technology (in comparison to more established toxicological assays) may be unfounded. Looking forward, the Committee has recognized the importance of standardized microarray data formats and public repository databases as the mechanism by which microarray data can be compared and interpreted by the scientific community at large. In view of this, the Committee has partnered with the European Bioinformatics Institute to develop a database to house the data generated by the Committee as part of its collaborative research. The database will be consistently annotated and integrated with other relevant information (e.g., histopathology, clinical chemistry, gross observations, etc.), employ standard controlled vocabulary, and be supported by a query and data analysis interface. Consistent with the ILSI/HESI charter, this database will ultimately be made available to the public, with final deployment anticipated in late 2003 or early 2004. P7 Stem cells in toxicology 195 CYTOCHROME P450 2E1 IS PRESENT AND ACTIVE IN HUMAN HEMATOPOIETIC STEM CELLS P. Anzenbacher 1 , E. Anzenbacherová 2 , L. Kousalová 1 , J. Vondráková 3 , I. Skoumalová 3 . 1 Institute of Pharmacology and 2 Institute of Medical Chemistry and Biochemistry, Faculty of Medicine, Palacky University, CZ-775 15 Olomouc, and 3 Department of Hematology, University Hospital, CZ-775 20 Olomouc, Czech Republic Hematopoietic stem cells as progenitor cells were shown to differentiate to different lineages (1). This opens the possibility of planned preparation of many cell lines as e.g. hepatocytes for liver transplantation. One of the most characteristic properties of hepatocytes is their ability to metabolize xenobiotics by cytochromes P450. This is why the presence of cytochromes P450 in hematopietic stem cells is followed. The cells were isolated from peripheral blood by magnetic field using particles with bound antibodies. The final preparation contained over 95% of CD34+ hematopoietic stem cells. As the preliminary study in the literature indicated a possibility that the CYP2E1 may be expressed there (2), we have focused our attention on a proof of CYP2E1 presence and activity in these cells. The stem cells were shown to express the CYP2E1 protein by Western blotting. In this work, also the presence of an active CYP2E1 protein in hematopoietic stem cells is documented by their ability to convert a typical CYP2E1 substrate using a HPLC method of Lucas et al. (3). The activity of CYP2E1 was estimated to be 3.7 pmol/mg protein/min. This is the first proof of the specific CYP2E1 activity in the hematopoietic stem cells. Acknowledgment: This work has been supported by grant from Internal Grant Agency, Czech Ministry of Health (11NL/7295–3). 196 IN VITRO PLURIPOTENCY OF HUMAN MARROW MESENCHYMAL STEM CELLS Sarah Snykers 1 , Tamara Vanhaecke 1 , Peggy Papeleu 1 , Greetje Elaut 1 , Ivan Van Riet 2 , Vera Rogiers 1 . 1 Dept. Toxicol., Vrije Universiteit Brussel, Belgium; 2 Dept. Med. Oncol.-Hematol.-Stem Cell Lab., Academical Hospital, Vrije Universiteit Brussel, Belgium The bone marrow stroma consists of a subset of non-hematopoietic cells, referred to as mesenchymal stem cells (MSC), which are capable of self-renewal as well as differentiation into several types of “mesenchymal-derived” cells including adipocytes, osteoblasts and chondrocytes. In the present study, we investigated whether these cells are also capable to differentiate in vitro into endodermal cells such as hepatocytes. Therefore, after expansion for 4 passages in fetal calf serum-containing medium, low density-MSC (CD45− , Thy+ ) were cultured on a collagen gel type I matrix in the presence of growth factors known to be critical for late embryonic liver development [fibroblast growth factor-4 (FGF-4), hepatocyte growth factor (HGF), insulin-transferrin-selenious acid (ITS) and dexamethasone (dex)]. Microscopic analysis of the cell morphology as well as specific cytological staining showed that MSC cultured in the presence of all growth factors (FGF-4, HGF, ITS and dex), simultaneously differentiated into neuron-like cells (positive Cresyl Fast Violet and Bodian staining), adipocytes (positive Sudan III and Oil-Red O staining) and osteoblasts (positive Von Kossa staining) from day 5 on. Although no cells with typical morphologic characteristics of hepatocytes were seen, periodic-acid shift staining showed an up-regulation of glycogen storage from day 9 to 22. Moreover, the expression of vimentin, an oval cell marker, and the hepatocyte specific marker cytokeratin 18 were seen by immunocytochemical staining from day 5 and day 7 on, respectively. In order to obtain more homogeneous MSC-derived cell cultures, the MSC were separately treated with each of the growth factors FGF, HGF, HGF + ITS + dex. It appeared that FGF-4 stimulated the differentiation of MSC into neuron-like cells and osteoblasts, while ITS and dexamethasone, more than HGF, induced the differentiation of MSC into adipocytes. In conclusion, our results clearly indicate that in “hepatocyte specific culture conditions”, low-density MSC differentiate into mesodermal (adipocytes and osteoblasts) as well as ectodermal cells (neuronlike cells). Also potential progenitors of hepatocytes were obtained. The culture conditions for their further differentiation into mature hepatocyte-like cells are currently being optimized. P8 Food safety 197 CANCER CHEMOPREVENTION AND ANTHOCYANINS: INDUCTION OF APOPTOSIS AND CELL DIFFERENTIATION PROMOTED BY CYANIDIN 3-O-β-GLUCOPIRANOSIDE IN TWO LEUKAEMIA CELL LINES. F. Berti 1 , C. Fimognari 1 , G. Cantelli-Forti 1 , P. Hrelia 1 . 1 Dept. of Pharmacology, University of Bologna, Bologna, Italy 3-O-β glucopyranosyde (Cy-g) is the main anthocyanin present in juice of pigmented oranges. However, little is currently known regarding the potentially chemopreventive mechanisms of anthocyanins apart from their antioxidant activity. The present study was designed to expand our knowledge about the potential chemopreventive properties of Cy-g. The range of concentrations was set through the trypan blue exclusion test, then we investigated the induction of apoptosis by Cy-g in two leukaemia cell lines (Jurkat T-cells and HL-60 cells). We therefore tested Cy-g also on the non-transformed counterpart of Jurkat T-cells which are human proliferating blood T-lymphocytes, to evaluate whether the effects of Cy-g are specific for transformed cells. Poster Session P8. Food safety Moreover, since HL-60 cell line has a pivotal role in the study of differentiation in human leukaemias, we extended our study by investigating the inducing activity of differentiation by Cy-g. In all three cell systems a gradual dose-dependent decrease in the number of viable cells was recorded, although Jurkat cells seemed to be more responsive than HL-60 and untransformed T cells. Cy-g induced apoptosis (as well as necrosis) in all three systems, but in Jurkat cells, signals of apoptosis were recorded at lower concentrations than in HL-60 and normal T cells. Membrane permeabilization as necrosis marker presented similar kinetics, in fact Cy-g was more active in Jurkat cells than in HL-60 or in non-transformed T cells. Cy-g induced dose-dependent differentiation of HL-60 cells, as determined by nitroblue tetrazolium (NBT) reduction and by the increased number of adherent cells, suggesting that Cy-g induces differentiation into granulocyte and monocyte/macrophage-like phenotype. This was confirmed by morphological analysis. These interesting biological properties should encourage further investigation into the chemopreventive and/or chemotherapeutic potential of Cy-g. Nevertheless, the activity of Cy-g in untransformed T-cells underlines the need for extensive toxicological investigation prior to any advocacy of chemopreventive dietary supplementation. 198 EVALUATION OF SAFETY OF TWO NATURAL ANTIOXIDANTS EXTRACTS ON CHROMAFFIN CELLS PROTECTION M.S. Miranda 1 , E.Q. Paredes 2 , E.S. Blasco 2 , E. Vilanova 2 . of Pharmacia of University Federal of Bahia, Brazil2 Laboratory Toxicology of Institute of Bioengineer of Universidad Miguel Hérnandez, Elche, Alicante, Spain 1 Faculty In the last years there is a large increment for natural antioxidants by consumers and several studies about natural antioxidant activity “in vitro” and “in vivo” were published There are only a few studies specifically about safety on toxicity of theses substances in relationship with concentration. For this reason nowadays there is concern and interest to evaluate the safety of these bioactive substances. In the present work, we used two natural ethanol extracts (O. crassostrea rhyzophorea and Rosemarynus officinalis), with potential antioxidant activity previously determined. The ethanol extracts were dried by nitrogen and suspended in Krebs buffer at six different concentrations and tested in chromaffin cells esterase activity behaviour. Chromaffin cells were isolated from bovine adrenal glands by collagenase digestion and cultured at 7.5 x 104 /plate for characterizing potential protection of cytotoxicity effect. The colour resulted of phenylvalerate, SDS and ferricianide reagents were photometrically measured at 510 and 660nm in Biomek 1000 automated laboratory workstation. The Krebs modification solution was used for positive control. Our earlier results demonstrated that an extract concentrations of 200 to 400 µg/mL presented higher protection of chromaffin cells behaviouer than lower or higher concentrations. Both antioxidants tested presented the same behaviour. We further intend to explore the mechanism involved in these observations. 199 DNA-DAMAGE REDUCING EFFECTS OF FLAVONOID SUPPLEMENTATION IN A HUMAN INTERVENTION STUDY L. Wilms, J. Kleinjans. Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht, The Netherlands Flavonoids are natural polyphenols, which are important constituents of fruits, vegetables, nuts, tea and red wine. Flavonoids are claimed to be able to protect against cardiovascular disease, certain forms of cancer and ageing. In this project quercetin is used as a model for the abundant group of flavonoids. Aim of this study is to determine whether a four-week period of increased quercetin intake through food enhances the anticarcinogenic defence mechanism in healthy volunteers. During a four-week period eight volunteers increased their quercetin intake through consuming one litre of a mixture of blueberry juice (50%) and apple juice (50%). Blood and 24 h urine were collected both before and after the intervention, and dietary s57 flavonoid intake was determined based on the subject’s dietary records. Quercetin levels and antioxidant capacity were determined in plasma, and quercetin metabolites in urine. Peripheral blood lymphocytes were isolated and exposed ex vivo to an effective dose of Benz(a)Pyrene (B(a)P) and hydrogen peroxide. As indices for DNA damage BPDE-DNA adducts and oxidative damage were measured by 32 P-postlabelling and COMET-assay, respectively. Preliminary results: Supplementation of quercetin containing foods appeared to have protective effects in ex vivo experiments with PBL. After 4 weeks the level of BPDE-DNA adducts upon ex vivo B(a)P administration, as well as the level of oxidative damage upon exposure to H2 O2 was decreased. Further, large interindividual differences in quercetin were observed, possibly pinpointing towards a role of polymorphisms in genes encoding for anticarcinogenic/ antioxidative defence. 200 REPEATED DOSE ORAL TOXICITY (13 WEEKS) OF 4-HYDROXY-3-METHOXY-BENZALDEHYDE (VANILLIN) TO SPRAGUE DAWLEY RATS Axel Mancebo 1 , Yoagne M. Trapero 2 , Yana González 1 , Bárbara O. González 1 , Dasha Fuentes 1 , Osvaldo Hernández 1 , Juana Hernández 1 , Consuelo González 1 , Yasnay Hernández 1 , María E. Arteaga 1 , Nelvys Subirós 1 , Ana M. Bada 1 . 1 Centro de Toxicología Experimental (CETEX), Centro Nacional para la Producción de Animales de Laboratorio (CENPALAB) 2 Centro de Biofísica Médica 4-hydroxy-3-methoxy-benzaldehyde (vanillin) it’s a well-known food additive, and it has been considered to have antimutagenic properties. In order to assess its possible toxicity to humans, it was made a repeated dose oral assay (13 weeks) in Sprague Dawley rats. Intragastric administration (gavage) was selected for animals dosing. Three dose levels were established (80, 240 and 400 mg of vanillin/Kg of body weight) and a control group was administered with the vehicle used to solve vanillin, carboxymethylcellulose 2%. Daily observations were performed, and body weight gain and food and water consumption were weekly evaluated. Blood samples were collected for hematological (red blood cell, white blood cell, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, differential leukocyte count and platelet count) and serum biochemical determinations (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, glucose, blood urea nitrogen, total protein, total cholesterol, total bilirubin, creatinin, uric acid and triglycerides). Gross necropsy was made in all animals, and histological studies of organs and tissues were performed. Organ weights were measured for thymus, adrenals, testis, ovary, heart, lung, kidney, spleen, liver and brain. Nine animals died during the assay, being of treated and control groups. There were not clinical signs of relevancy among survivors, and the behavior of the body weight gain and food and water consumption was normal for the used specie. Hematological and blood chemistry analysis showed slight differences between sampling as regard to triglycerides and alkaline phosphatase, been clearly associated to rat’s aging. Gross necropsy of animals died during the study allowed to establish the cause of the deaths, complications in intragastric administration. In the other hand, anatomopathological analysis of survivors showed nephrocalcinosis in females, the majority in control group. This finding has been previously reported in rats after oral administration of carboxymethylcellulose. It could be concluded that under experimental conditions, and according to the doses levels used, the repeated oral administration of vanillin for 13 weeks does not produce toxicity. 201 13-WEEK TOXICITY STUDY OF ESTRAGOLE ADMINISTERED BY GAVAGE TO F344/N RATS AND B6C3F1 MICE Kamal M. Abdo, Ronald Herbert, Jerry D. Johnson. NIEHS/NIH, Research Triangle Park, NC, 27709, USA; Battelle, Columbus, OH, 43201 USA Estragole [1-methyl-4-(2-propenyl)benzene] is used as a flavoring agent in foods. It is present in many spices such as basil, tarragon, anise, and bitter fennel. Estragole in corn oil was administered s58 Poster Session P8. Food safety once daily, 5 days per week for 13 weeks to groups of 10 male and 10 female F344/N rats and B6C3F1 mice at doses of 0, 37.5, 75, 150, 300, and 600 mg/kg. Additional groups of 10 male and 10 female rats were dosed similarly for 31 days and used for hematology and clinical chemistry measurements. All rats survived until the end of the study. One male and all female mice dosed with 600 mg/kg/day died or were sacrificed due to moribundity. Final mean body weights of male rats receiving estragole and final mean body weights of female rats and male and female mice dosed with 300 or 600 mg estragole/kg/day were lower than that of their respective control. Estragole also caused increases in liver weight of rats and mice. Estragole hematology and clinical chemistry related effects in rats included increased red blood cell count and decreased hemoglobin, hematocrit, mean corpuscular cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and increased serum sorbitol dehydrogenase and alanine aminotransferase activities. Lesions related to estragole administration to rats included an increased incidence of lesions in the liver, salivary glands, glandular stomach, lung, nose, testes, and kidney. Treatment related lesions were observed in the liver, nose and the glandular stomach of mice. 202 METABOLISM AND CYTOTOXIC MECHANISMS OF SALICYLALDEHYDE IN ISOLATED RAT HEPATOCYTES Hossein Niknahad 1 , Peter J. O’Brien 2 . 1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Fars, Iran, 71345; 2 Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 2S2 Salicylaldehyde (SA) is an aromatic aldehyde present in spices such as vanilla or cinnamon used as food additives and preservatives. In the present study SA was found to cause cytotoxicity towards freshly isolated rat hepatocytes with an LD50 of 0.6 mM for 2 hours. The events before plasma membrane disruption were immediate inhibition of mitochondrial respiration followed by depletion of ATP content of the cells. GSH content of hepatocytes was also partially depleted before cytotoxicity occurred. On the other hand, depleting GSH content of hepatocytes before the addition of SA increased the susceptibility of hepatocytes to SA and caused stronger inhibition of mitochondrial respiration. Increasing hepatocyte NADH levels with betahydroxybutyrate, xylitol, ethanol or lactate prevented SA-induced cytotoxicity while oxidizing NADH with acetoacetate increased its toxicity. The ATP generators fructose or dihydroxyacetone and the mitochondrial permeability transition pore inhibitor cyclosporin also prevented SA cytotoxicity, suggesting that SA is a mitochondrial toxin. Unlike other aldehydes, SA did not increase lipid peroxidation in hepatocytes, although induced some ROS formation. SA was not a good substrate for microsomal aldehyde dehydrogenase, which is important in the detoxification of aromatic aldehydes; however, SA was oxidatively detoxified by mitochondrial aldehyde dehydrogenase and inhibiting this enzyme by cyanamide or citral increased SA-induced cytotoxicity and reduced its LD50 to about 0.2 mM. On the other hand, SA was a good substrate for alcohol dehydrogenase, and salicyl alcohol formed was not cytotoxic even at a concentration nearly 10-fold higher. Inhibiting ADH with methylpyrazole increased SA cytotoxicity. SA was much more toxic than other aromatic aldehydes probably because it was poorly metabolized by aldehyde dehydrogenases while the aldehydes that were good substrates for these enzymes were less toxic than SA. This suggests that unmetabolized aldehydes could bind to critical intracellular targets, e.g. proteins, resulting in cytotoxicity. SA could also form a Schiff base with protein e-amino groups such as cytochrome c that can result in polymerization of cytochrome c and prevent its function. 203 ASSESSMENT OF EXPOSURE TO FOOD CHEMICALS, IN ROMANIA, 2001–2002 Carmen Hura 1 , I. Palamaru 1 , B.A. Hura 2 . 1 Food Toxicology Laboratory, Institute of Public Health, Iassy, Romania, 2 Electrotechnic Faculty, Technical University “Gh. Asachi” Iassy, Romania Ever since humans have become aware that health is inseparably linked to an impact and healthy environment, the control and reduction of pollution have become the focus of world wide concern. Investigations on possible health and environmental hazards involved have led many industrial countries to restrict or ban the use of chemicals (pesticides, heavy metals) and enforce the tolerance levels for the residues in food and feeds. The aim of the study was to investigate the variation of some chemical pollutants with cancer risk (nitrate/nitrite, heavy metals, pesticides residues) in some food (vegetables, meat, milk, fish, daily diets) from the Romania area, in 2001- 2002 period. The concentration of the nitrates/nitrites and the heavy metals (Cu, Cd, Pb, Mn, Zn, Ni) were investigated in 5386 food samples (vegetables (1364 samples), meat and meat products (2743 samples), milk and dairy products (952 samples), fish (55 samples), total diets (327 samples). The food were harvested from the Romania area (32 districts), in 2001- 2002 period. In the milk, vegetables, meat and total diets were analysed the metals (Cd, Pb, Zn, Cu, Ni, Mn, Fe) by absorption spectrophotometric method. The nitrate/nitrite were determined by colorimetric method and the pesticides residues by gas-chromatography. In all analysed samples these chemical pollutants were found. Generally, a wide variation between in individual samples were observed. Nitrate/nitrite contents were, generally, in normal limits and the total diets contained quantities below acceptable daily intake. The analysis of results obtained showed that in food was found the heavy metals in varied concentrations but in the admissible limits. The results give emphasis that the pesticide residues are present in all food analyzed. The determinations of chemical pollutants in food are important in environmental monitoring for the prevention, control and reduction of pollution as well as for occupational health, legal, decisions and epidemiological studies. 204 CYTOTOXIC EFFECTS OF MALACHITE GREEN IN TWO HUMAN CELL LINES I. De Angelis 1 , A. Giuliano Albo 2 , C. Nebbia 2 , A. Stammati 1 , F. Zampaglioni 1 , M. Dacasto 2 . 1 Dipartimento Ambiente e prevenzione primaria, Istituto Superiore di Sanità, Roma, Italia. 2 Dipartimento di Patologia Animale, sezione di Farmacologia e Tossicologia, Università di Torino, Italia The triphenylmethane dye malachite green (MG) is still illegally used in aquaculture as a fungicide on larvae and juvenile fish. MG is rapidly absorbed, metabolised to its reduced derivative leucobase leucomalachite green (LMG) and then excreted. Apart from the LMG residues in muscular tissues of treated animals, MG has been reported to be a tumour promoter both in vitro and in vivo. The aim of this study was to assess the in vitro toxicity of MG. Two human cell lines were used: Hep-2 cells, derived from a human larynx carcinoma and Caco-2 cells, obtained from a human colon carcinoma. A reduction of cell viability, measured as NRU uptake and TPC content, was observed in Hep-2 cells treated for 24 hrs with different concentrations of MG (0.26–3.92 µM); the IC50 values obtained were 2.14 and 2.22 µM, respectively. Besides, MG reduced the proliferation capability, measured as CFA, of Hep-2 cells treated for 24 hr with MG and then sub-cultured in absence of the compound;. the relative IC50 value was 2.04 µM. As regards the Caco-2 cell model, dose-related increasing percentages of cytotoxicity, measured by NRU, LDH leakage and MTT assays, were observed in cells exposed for 24 hrs to different MG concentrations (0.1–100 µM); the IC50 values were 13.8µM, 18.4 µM and 16.2 µM for NRU, LDH leakage and MTT, respectively. In conclusion, MG was demonstrated to be cytotoxic to both Hep-2 and Caco-2 cells, and the former seems to be more sensitive to the dye toxic effect. The assessment of LMG cytotoxicity is actually under investigation. Supported by grants from Ricerca Finalizzata Regione Piemonte. s59 Poster Session P8. Food safety 205 STRATEGY OF FOOD SAFETY IN THE REPUBLIC OF UZBEKISTAN ACCORDING TO THE INTENTION OF ENTRY TO THE WORLD TRADE ORGANIZATION A.S. Khudaiberganov. Laboratory of Toxicology, Academy of the Ministry of Internal Affairs of the Republic of Uzbekistan, Tashkent, Uzbekistan In the article the analysis of legislative basis statement on food safety and characteristics of principles and methods, which are used for toxicological evaluation of food products in Uzbekistan, are determined. It is defined that inclusion of the problems on food safety in the political order of the day of developing countries is the first step on the way of safety increasing of food products and organization of proper system of the control for the quality of food products. The necessity of harmonization the concrete legislative documents on international request and putting them according to the Agreement on sanitary and phyto-sanitary measures of World Trade Organization. It is offered the harmonization of existed methods of toxicological evaluation of food products with System GLP (Good Laboratory Practice) and criterias of safety of the Codex Alimentarius. 206 SEVERE ALLERGIC REACTIONS TO FOOD - THE NORWEGIAN NATIONAL REPORTING SYSTEM AND REGISTER M. Løvik 1 , H.G. Wiker 1 , R. Kjelkevik 2 , E. Egaas 3 , B.A. Stensby 1 , B. Gondrosen 2 . 1 Norwegian Institute of Public Health, Oslo, 2 Norwegian Food Control Authority, Oslo, and 3 Veterinary Institute, Oslo, Norway The Norwegian Institute of Public Health in collaboration with the Norwegian Food Control Authority July 1, 2000 launched a national reporting system and register for severe allergic reactions to food. Funding was provided under the Government Action Plan against asthma, allergy and indoor air-related diseases, and has later become permanent. The purpose is to obtain better information about serious allergic reactions to food in Norway. An important element is also food surveillance in relation to food labeling and allergen occurrence in presumably “safe” foods. The reporting system has three arms: a one-page reporting form, serum analysis, and food allergen analysis. Submitting doctors get back comments on the case and results of the analyses, which are all provided free of cost. About 6500 doctors with relevant practices, plus emergency wards (“first line care providers”) are contacted by mail 1–3 times a year with information material and are encouraged to report cases. Submission has steadily increased. By April 1, 2003 a total of 200 cases have been reported (70 - 80 per year) from the Norwegian population of about 4 millions. Some key findings are: no clear gender difference; a very marked peak of reported cases are their early twenties; nuts, peanuts and shellfish are the most common allergens; most serious reactions occur outside the home. No deaths have been reported so far. 207 THE NORWEGIAN FISH AND GAME STUDY DESCRIPTION OF A DIETARY SURVEY FOCUSING ON FOODS CONTAINING ENVIRONMENTAL CONTAMINANTS H.M. Meltzer 1 , C. Bergsten 2 , H. Stigum 1 , M.L. Wiborg 2 , K. Færden 2 , J. Alexander 1 . 1 Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway, 2 Norwegian Food Control Authority, Oslo, Norway In the period 1999–2002 three dietary surveys have been conducted, with focus on the consumption patterns of foods which may contain high levels of cadmium, mercury, PCB and dioxines. These contaminants are mainly found in crab, flatfishes, halibut, crustaceans, perch and pike, liver and kidney from game, and wild mushrooms. These food items are not included in national dietary surveys, but are crucial in the estimation of dietary intakes. The aim of the study is to describe the exposure distribution of such contaminants in the population. Part A of the survey encompassed a nation-wide postal qualitative food frequency questionnaire sent to ten thousand randomly chosen persons between 18 and 79 years old. The response rate was 60%. Part B was a postal semiquantitative food frequency questionnaire sent to ten thousand randomly chosen persons from specific coastal and three inland municipalities with ample supply of fish and game. On this assumption, consumption is expected to be above average. The response rate was 55%. Part C of our survey encompasses high consumers of the foods, selected from part B, where analyses of environmental contaminants in blood and urine are included. 210 persons participate. Analysis of biological material will be combined with data on levels of contaminants in different foods. Preliminary results, estimated intakes of mercury and cadmium, based on part A of the survey, are as follows Group Hg, whole population Hg, consumers only Cd, whole population Cd, consumers only Average intake, µg/day High-consumers, µg/day 4.0 12.3 15.8 35.2 18.5 38.3 49.3 92.5 This survey is the first of its kind in Norway. As far as we know, no similar studies have been conducted elsewhere. We will present further results from part A and B of the study. 208 THE DEVELOPMENT OF A FOOD FREQUENCY QUESTIONNAIRE TO ESTIMATE THE INTAKE OF FOODS, NUTRIENTS AND NON-NUTRIENTS IN THE NORWEGIAN MOTHER AND CHILD COHORT STUDY M. Haugen 1 , T. Ydersbond 2 , H.M. Meltzer 1 , J. Alexander 1 . 1 Division of Environmental Medicine, Department of Food Toxicology, Norwegian Institute of Public Health, Oslo, 2 Statistics Norway, Oslo, Norway Background: Most food frequency questionnaires are designed to estimate the common food and nutrient intake. In the Norwegian Mother and Child Cohort Study exposure to environmental contaminants, as well as non-nutrient health promoting food factors are of concern, to be able to evaluate the impact of all aspects of food intake on the health outcome of mother and child. Objectives: To develop a food frequency questionnaire allowing estimation of exposure to health promoting and toxic food factors, besides calculating the intake of nutrients. Methods: Putative health promoting food factors are e.g. flavonoids in fruit and vegetables. Hence great concern was put into capturing the intake of both raw and cooked vegetables and fruits. The analysed content of mercury, cadmium, PCB and dioxins is high in certain fishes and offal from game from certain parts of Norway. Hence questions concerning intake of fish and game were given priority in the questionnaire. To reduce the total number of questions it was decided to avoid portion sizes and to concentrate on frequency intake. Results: Besides being able to calculate intake of food and nutrients, this questionnaire will enable us to evaluate the intake of the following food factors: Food factor Flavones and flavonols Most important food contributors Fresh fruits Raw vegetables Vegetables, boiled/in casseroles Tea Isoflavonoids Soymilk, soy products, leguminous Mercury Tuna, pike, perch, halibut, crab, mussels Cadmium Crab, fish liver, liver and kidney from game, bran PCB/dioxin Fish liver, mackerel, herring, salmon, trout, crab, seagull egg, Number of questions Estimated percentage of total intake 14 12 8 2 2 8 20–30 20–30 10 0–20 80–90 ∼90 6 ∼90 14 ∼80 Conclusion: With this questionnaire, which consists of 283 questions, we presume to be able to estimate the intake of 85–90% of the flavonoids, Hg, Cd, PCBs and dioxins. Several simulation modules will be used in the analysis of intake and the impact on health outcome. s60 209 Poster Session P8. Food safety PROBABILISTIC EXPOSURE ASSESSMENT FOR ACRYLAMIDE IN FLEMISH ADOLESCENTS M. Bilau 1 , C. Matthys 1 , C. Vinkx 2 , S. De Henauw 1 , J.L. Willems 1 . 1 Ghent University, Departement of Public Health, 2 Blok A, De Pintelaan 185, B-9000 Ghent, Belgium, 2 Health, Food Chain Safety and Environment PPS, State Administrative City, B-1010 Brussels, Belgium Background: Acrylamide (AA) (CH2 =CHCONH2 ) has recently been found in a range of food items as a result of certain cooking procedures. As it is a neurotoxic agent and a probable, human carcinogen (IARC 2A), human exposure to this chemical might constitute an important public health issue. Objective: to assess the exposure to AA via food in Flemish adolescents on the basis of a probabilistic model. Methods: Data on food consumption, derived from a 7-day food record dietary survey (1997) in 341 adolescents, were combined with contamination data for following food groups: fried (potato derived) products, crisps, biscuits, breakfast cereals, beer and chocolate. Levels below the limit of determination (LOD=100 µg AA/kg product) were put equal to half LOD. As the Belgian data for bread were all below the LOD, we used data from the Netherlands, based on analyses with a lower LOD (30 µg AA/kg product). Exposure modelling was based on Monte Carlo simulations. Results: At median uncertainty, the exposure via these food items ranged from 68 ng/kgBW/day at the 5th percentile, over 229 ng/kgBW/day at the 50th percentile, to 573 ng/kgBW/day at the 95th percentile. Bread, despite its low AA content, seems relevant as source of AA exposure at the lower percentiles. At higher percentiles the contribution of french fries and crisps is more important. Conclusion: An intake assessment, based on consumption patterns in Flemish adolescents and on a limited nummber of contamination data, generated levels of AA-exposure via food in the lower range of intake, calculated for other Western-European countries. The relevance of such exposure levels for public health in terms of cancer risk remains a subject of scientific debate. 210 EFFECT OF DIFFERENT STORAGE CONDITIONS ON NITRATE/NITRITE LEVELS, MICROBIOLOGICAL QUALITY AND N-NITROSAMINES CONTENT IN POLISH EDIBLE OFFALS PROCESSED MEAT PRODUCTS K. Domańska 1 , B. Kowalski 2 . 1 Department of Poultry Diseases, 2 Laboratory of Radiological Protection and Isotopic Research, National Veterinary Research Institute, Pulawy, Poland The levels of nitrate and nitrite, the microbiological quality (total aerobic counts - TCA, pathogenic staphylococci, coliforms and E.coli), pH and the content of volatile N-nitrosamines were determined in 18 samples of edible offals processed meat products fresh and stored in different conditions e.g. 72 h at 4–80 C and 72 h at 4–80 C plus 24 h at 22–260 C. This kind of meat products is a very popular and produced and consumed in large amounts in Poland. Measurements of nitrate/nitrite and bacteria were done according to Polish Norms. The analysis of N-nitrosamines were carried out by gas chromatography (GC) coupled with thermal energy analyser (TEA). The presence of N-nitrosamines in selected samples was confirmed by mass spectrometry (GC-MS). In fresh samples nitrate and nitrite were found at the mean concentration of 18.4 mg/kg and 25.4 mg/kg, respectively. Storage for 72 h at 4–80 C increased nitrate concentration to the level of 24.8 mg/kg. However, additional storage for 24 h at 22–260 C induced decrease of nitrate concentration to the level of 17.4 mg/kg. The levels of nitrite in all studied time points were nearly the same (25.4 – 25.6 mg/kg of sample). In fresh samples TCA at the level of 4.79 log10 CFU per g, pathogenic staphylococci at the level of 2.74 log10 CFU per g and coliforms and E.coli at the level of 0.85 log10 CFU per g were found. Storage for 72 h at 4–80 C resulted in growth retardation and no changes in bacteria counts were noted. Additional storage for 24 h at 22–260 C induced further changes of microbiological quality: TCA, staphylococci and coliforms were found at the level of 5.7 log10 CFU per g, 4.63 log10 CFU per g and 1.97 log10 CFU per g, respectively. pH values increased slightly during whole period of storage. Fresh samples sporadically contained low amounts of Nnitrosodimethylamine (NDMA) (about 0.02 µg/kg). Storage for 72 h at 4–80 C influenced the appearance of NDMA and other N-nitroso compounds. In almost all samples NDMA and N-nitrosopiperidine (NPIP) were found at the mean concentrations of 2.8 µg/kg and 4.2 µg/kg, respectively. Few samples contained also low amounts N-nitrosodibutylamine (NDBA) and N-nitrosomorpholine (NMOR). Additional storage for 24 h at 22–260 C induced further changes of N-nitrosamines occurrence. As previously, almost in all meat products NDMA and NPIP were found at the mean concentrations of 1.1 µg/kg and 4.6 µg/kg respectively. In a few samples traces of NDBA were detected. No sample contained NMOR but in three samples N-nitrosopirrolidine (NPYR) was present. Some correlations between storage conditions, nitrate and nitrite levels, microbiological quality and N-nitrosamine occurence were found. 211 OCCURRENCE OF NITROSAMINES IN RUBBER BABY TEATS IN SLOVENIA L. Perharic 1 , V. Golja 1 , M. Gabrijelcic 1 , M. Seljak 1 , S. Barlow 2 . 1 Institute of Public Health of the Republic of Slovenia (IPHRS), Trubarjeva 2, 1000 Ljubljana, Slovenia, 2 8 Harrington Rd, Brighton, BN1 6RE, UK IPHRS is actively involved in monitoring and safety evaluation of consumer products and materials coming into contact with food. In 2002, excessive amounts of nitrosamines and their precursors were detected in a brand of rubber baby teats (Table 1 below). The national legal limit for nitrosamines and N-nitrosatable substances in materials coming into contact with food, to be enforced in 2004, is set at 10 µg/kg, and 100 µg/kg respectively. A supplier voluntarily withdrew dummies from sale, although about 400 had already been sold. We were asked to provide an ad hoc risk assessment in order to evaluate a possible recall. The quantitative oral risk estimate was based on the oral slope factor for NDBA given in the US EPA’s Integrated Risk Information System. An extra lifetime risk of cancer for a 4 kg baby was calculated to be 45 x 10−6 , assuming that a teat containing 1 µg/day is used for 8h/day. Our report stressed that we had not accounted for any possible additive/synergistic effects of other nitrosamines and their precursors, possible higher susceptibility of the very young to genotoxic carcinogens, and that exposure should be kept as low as practically achievable. However, considering product already purchased had a likely lifetime of only a few months together with the fact that exposure of babies to nitrosamines from other sources (diet, cigarette smoke) was unlikely, it was concluded that an overall contribution from the teats to the lifetime cancer risk was relatively low. Therefore, recall was considered unnecessary. Abstract 211 – Table 1: Concentration of nitrosamines and N-nitrosatable substances in rubber baby teats Nitrosamines Measured µg/kg* Corrected µg/kg** Calculated µg/item*** N-nitrosatable substances NDBA NDEA NDMA Other Total NDBA NDEA NDMA Other Total 47 37 0.74 <1 n.a. n.a. <1 n.a. n.a. <1**** n.a. n.a. 47 37 0.74 176 n.a. 3.5 360 n.a. 7.2 <4 n.a. n.a. < 4**** n.a. n.a. 536 389 7.8 NDBA-nitrosodibutylamine, NDEA-nitrosodiethylamine, NDMA-nitrosodimethylamine, n.a. not applicable. *Migration into artificial saliva at 40°C/24h using gas chromatography with mass selective detector. **Correction according to standard EN12868 (2002). ***Calculated per teat weighting 20 g. ****All other nitrosamines with exception of nitrosopyrrolidine (<2.5 and <10 respectively) Poster Session P8. Food safety 212 DETERMINATION OF Pb AND Cd IN SOME SERBIAN WHITE WINES BY DPSV Z. Basić 1 , V. Kilibarda 1 , S. Ražić 2 . 1 Military Medical Academy, Belgrade, 2 Faculty of Pharmacy, University of Belgrade, Serbia and Monte Negro Wine samples (6 wines) were chosen from the 3 Serbian winegrowing regions. Cadmium and lead are commonly determinated in food and beverages due to their toxicological effects. Their permitted concentrations are very low, so the methods for their determination should be very sensitive. In this paper the direct determination of cadmium and lead in six white wines was performed by differential puls stripping voltametry (DPSV).In order to investigate the possible matrix effect, the standard addition method was applied too. The alcoholic calibration solutions were used. The determination potentials were: -0.65V for Pb and -0.82V for Cd. The other experimental conditions were optimized too. In the case of red wines, the matrix effect is more pronounced. The results confirm the suitability of the proposed method for the routine simultaneously analysis of the Cd and Pb in white wines. perform rapid screening of numerous samples using relatively simple technologies. We propose a screening method based on the measurement of damages produced by the treatment on the food DNA. It employs the agarose block techniques to isolate high molecular weight DNA and a continuous field electrophoresis technique on low agarose concentration gel. The method has been standardized using cell in vitro labelled with both C14 thymidine and a DNA fluorescent probe. It can cover a dose range from few Gy to KGy changing the agarose concentration or increasing the electrophoresis voltage. The technique considers several steps: cell and nuclei extraction, agarose blocks formation, lysis, electrophoresys, fluorescent staining and evaluation. The method is rapid, reliable, and simple to perform and several samples can be prepared and analyzed. The disadvantage is that it cannot be considered specific for irradiated food, however it presents advantages over other screening methods already tested and approved by the ECC, such as the Comet Assay, the photo-induced luminescence and the direct epifluorescent filter/aerobic plate count agar technique. 215 213 FLUORIDE LEVELS IN SOFT DRINKS CONSUMED IN THE AUTONOMOUS COMMUNITY OF THE CANARY ISLANDS, SPAIN. C. Rubio 1 , M.I. Rodríguez 1 , A. Hardisson 1 , A. Burgos 2 . 1 Toxicology Department. University of La Laguna, School of Medicine, 38071 La Laguna, S/C de Tenerife, Spain.; 2 Preventive Medicine and Public Health Department. University of La Laguna, School of Medicine, 38071 La Laguna, S/C de Tenerife, Spain It is generally accepted that fluoride is an essential component of all diets and that most of the body requeriments are obtained from drinking water. A small fraction of this fluoride accumulates on the teeth and bones, and most of it is eliminated in urine and sweat. An excessive concentration of fluoride can cause fluorosis. The exposure of a certain community to fluoride is normally estimated from the levels in the public water supply. Although it is true that these levels are considered to be the main fluoride source, a more precise assessment of the total fluoride intake should take into account other sources such as food, beverages and air (1,2). The present study concentrates on fluoride provided by the intake of soft drinks. We have determined the fluoride content of the most widely consumed soft drinks in Tenerife (Canary Islands). The analytical technique used for the determination of fluoride in beers is based on the direct potentiometry method of the fluorideselective electrode (1,2). We have analysed 75 soft drinks samples. The mean concentration obtained was 0.72 mg fluoride/L (SD = 0.34). Only three samples presented a fluoride concentration over 1 mg fluoride/L, the reason for these high levels is that the water used in their manufacturing process presented a fluoride concentration between 1–1.5 mg/L (1,3). For the remaining samples, the water natural fluoride contents ranged from 0.4 to 0.7 mg/L (1). 214 A SIMPLE AND RAPID METHOD FOR SCREENING IRRADIATED FOOD B. Di Carlo, A. Maggi, O. Sapora. Dipartimento Ambiente e Salute, Istituto Superiore di Sanità, Via Regina Elena 299, 00161 Rome, Italy The directive1999/2/EC of the European Parliament and Council on the application of the laws of Member States concerning foods and food ingredients treated with ionizing radiation lays down general and technical aspects for carrying out the processes. The possible risks of deviations from good manufacturing practice in relation to irradiation were not different from those encountered by other food processing methods. For control purposes post irradiation detection methods are being tested in collaborative studies but an universal detection method has not yet been found. The already standardized methods are based on expensive and highly specialized technologies such as ESR, Gas Chromatography, Mass Spectroscopy and Luminescence. There is a need for wide spectrum methods to s61 RADIATION BIOTECHNOLOGY. INFLUENCE OF GAMMA-IRRADIATION ON STABILITY OF ORGANOCHLORINATED PESTICIDES CONTAMINATING FOOD Tatyana V. Melnikova 1 , Lyudmila P. Polyakova 1 , Gennady V. Kozmin 1 . 1 State University of Nuclear Power Engineering, Obninsk, Russia The monitoring data testify that the residual amounts of organochlorinated pesticides (OCP) in food frequently exceed limit levels. Because of application of radiation technology in a food-processing industry research of stability OCP under irradiation becomes very actual. The goal of the work was to study the OCP radiation degradation at a various doses and dose rates and to make the rating of biological activity of radiolysis products. The model solutions, consist of individual OCP and their mixtures (DDT, DDE, alpha- and gamma- isomer HCH) with concentration 0.01 - 1 ppm, were irradiated on “Issledovatel” (60 Co) and “Luch 1” (60 Co) installations at doses 0.77–55.55 kGy with a variation of dose rate from 0.0005 up to 0.14 kGy/min. In a range of doses 1–10κΓ p the increase of degradation extent on increasing of a dose of a gamma-irradiation was showed. By decreasing of a dose by order we have received values of a degradation extent at 5–15 of time below. The dependence of a degradation extent of OCP from a dose rate gamma-irradiation has complex nature. At a dose 10κΓ p the degradation extent of OCP with magnification of a dose rate at first increase, passes through a maximum, and then decreases. The degradation extent OCP in 2-propanol is higher than in hexane. For alpha-HCH it reaches 82% in hexane and 97% in 2-propanol; accordingly for gamma-HCH - 50% and 88%; DDE 52% and 88%; DDT - 66% and 77%. The basic products of decomposition which were identified in the irradiated solutions are DDE and DDD in a solution DDT, DDD - in DDE, beta- and gamma-HCH - in alpha-HCH and alpha- and beta-isomers HCH - in gamma-HCH. It was established that the degradation extent of OCP at their simultaneous presence in solutions depends on number of components in pesticides mixture and their chemical nature. As a preliminary results phitotoxic effect of radiolysis products of OCP was found. It is possible to suggest, that the negative biological effect is caused by total action of initial pesticide, radiolysis products and addition products. s62 216 Poster Session P8. Food safety NATURAL CO-OCCURRENCE OF DEOXYNIVALENOL AND OCHRATOXIN A IN BEER E.K. Tangni, Y. Larondelle. Unité de Biochimie de la Nutrition, Place Croix du Sud 2 Bte 8, Faculté d’Ingénierie biologique, agronomique et environnementale, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium Deoxynivalenol (DON) and ochratoxin A (OTA) are field and storage fungal produced toxins involved in brewing products. They may be detected along the barley-malt-beer chain. The preliminary survey presented here refers to the simultaneous occurrence of DON and OTA in domestic (20) and imported (20) beers commercialized in Belgium, the European country with the greatest diversity of beers. DON and OTA contents were assayed by means of immunoaffinity (OchraTest™ and DONTest™) clean-up and liquid chromatography. DON and OTA were found in 45% and 100% of the tested beers at the mean levels of 40.7 µg/l and 36.7 ng/l, respectively. Considering a daily intake of 0.3 l of beer per capita, these mean levels of contamination contribute in average to 20.5% and 3.7% of the estimated tolerable daily intake of DON and OTA, established at 1 µg/kg and 5 ng/kg body weight by the Joint FAO/WHO Committee on food additives and contaminants and the scientific committee on food of the European Commission, respectively. Interestingly, DON and OTA were simultaneously detected in 45 % of the tested beers with a significant positive correlation coefficient (r = +0.417, p = 0.007) found between DON and OTA levels in all tested beers, highlighting thus the possibility of the co-contamination of beers consumed in Belgium by DON and OTA. The question of the multi – contamination of beers should thus not be underestimated since interactive effects of mycotoxins may occur in the human body, even at low levels. We therefore recommend performing comprehensive studies on the deleterious effects of the natural co-occurrence of these mycotoxins. 217 AN IN VITRO INVESTIGATION ON THE VARIABLES CONTROLING THE BIOACCESSIBILITY AND ADSORPTION OF CADMIUM TO/FROM LETTUCE (LACTUCA SATIVA L. CV. OSTINATA): HELPFUL FOR PREDICTING BIOAVAILABLE CD IN FOODS? M. Waisberg 1 , W.D. Black 2 , B. Hale 1 . 1 Department of Land Resource Science, and 2 Department of Biomedical Sciences, University of Guelph, Guelph, ON This study evaluated the influence of diverse variables in the effectiveness of in vitro digestion protocols, which have been used to determine bioaccessibility of metals from the diet. The protocols consisted of two phases, gastric and intestinal, where ground lettuce was mixed respectively with pepsin (at pHs from 1 to 3) followed by the addition of pancreatin and bile extracts (pHs from 4.5 to 6.5). The percentage of solubilized metal was measured in the digestates in relation to digestion time, pH of each digestion phase and the dietary source of the metal in the diet. Because it would be convenient to add the metal to the diet before digestion instead of growing contaminated vegetables, the importance of metal incorporation in the plant in comparison to amendment through foliar spraying after harvesting was also studied. From our results we concluded that the dietary source of metal in the protocols we tested doesn’t seem to be a significant factor if we compare the group sprayed with cadmium with the group that had cadmium incorporated in it, but that time affects the quality of the digestion in different ways depending of the dietary source of cadmium. On the other hand, pH on both intestinal and gastric phases is a relevant factor and should be taken into consideration when analyzing the results from experiments using this kind of protocols. Since the intestinal phase in our experiments did not improve the quality of the digestion but actually decreased the soluble fraction of cadmium, we investigated the effect of pH on the adsorption of this metal to lettuce and found that there is an increased binding of cadmium at pHs above 3. Therefore we suggest that part of the observed response in our in vitro digestion esperiments could be explained by an increase in adsorption of metal to the plant material at higher pHs. The fact that lettuce binds cadmium in alkaline pHs similar to the pHs encoutered in human intestines suggests that this dietary component may act as a sink for cadmium, and possibly other heavy metals, therefore helping to reduce the risk posed by other diet constituents that might be contaminated (ex. wheat, rice, etc). Also, because dry lettuce is capable of binding high quantities of heavy metals it could possibly be used as a biosorbent for clean-up of metals in contaminated waters. Results from our latest in vivo experiment studying the effect of gastric pH on the absorption of cadmium will also be presented. 218 THE NECESSITY OF ADDITIVES DETERMINATION IN PLASTIC CONTAINERS S. Honary. Mazandaran University of Medical Sciences, School of Pharmacy. Sari, Iran Plastics have become of increasing importance as packages for foodstuffs and pharmaceutical dosage forms. Polymer containers mainly consist different additives e.g antioxidants, antistatics, light stabilizers and lubricants, which are mainly added in small amounts in order to alter the properties of the polymers in the desired way and/or simplify their processing. Direct contact between plastic containers and their contents could result in the migration of the low molecular weight additives from the plastices into the packaged materials, which is called migration. Since theses materials may have toxic, teratogenic, allergenic or carcinogenic effect, so it is important to determine the additives. Armid® as a lubricant, Irganox 1010, 1076, 1330, Irgafos 168 and BHT as different antioxidants were used. The samples were made by hexan extraction of Armid and antioxidants from polymer sheet in 24 hours, amount of Armid in sheets were determined by plotting its standard curve at λmax = 232 nm Chromatography for antioxidants was performed on 10 cm x 20cm silica of 60 F254 HPTLC plates and hexan – methanol (1/4) as mobile phase. Recovery study was carried out by analyses of different thickness of polymer sheet including known amount of each additive. The results show the recovery percentage of additives is dependent on polymer sheet thickness and increased as the thickness decreased in all cases for Armid. Percent of recovery was calculated 64% for thickness = 150µm, 82% for 100 µm and 98% for 20µm. 219 EFFECT OF TORTILLA PROCESSING ON FUMONISIN B1 DESTRUCTION H.A. Amra 1 , A. Aboul Enein 2 , A.A. Ragab 2 , A.M. Ayesh 1 , M.I. Mohamed 1 . 1 Food Toxicology and Contaminates Dept., National Research Center, Dokki, Cairo, Egypt; 2 Biochemistry Dept., Faculty of Agriculture, Cairo, University The study was carried out to evaluate the effect of tortilla processing on Fumonisin B1 in fermented corn. FB1 content after each process steps were determined in triplicate by HPLC. The average of percentage loss was 66.05% after alkaline cooking by calcium hydroxide, while it was 11.85, 53.94 and 29.27% after corn washing (naxitmal), cooking (mass) and tortilla bread (200 °C for 20 min) respectively. The average of initial FB1 in fermented corn and after tortilla processing were 250.6±5.65 and 24.47±3.66 mg /corn sample. The reduction of FB1 toxin was 90.23% in final product. The results indicated that the tortilla bread processing of fermented corn did not cause complete destruction of FB1 . 220 A STANDARDIZED AND RELIABLE APPROACH TO THE ASSESSMENT OF SAFETY AND EFFICACY OF NUTRACEUTICALS. F. Pizzocheri 1 , A. Conto 2 , N. Corsico 1 . 1 Pharmasafe sas, Milan – Italy, 2 Chemsafe sas Colleretto Giacosa-Italy Nutraceuticals is a general term that includes a variety of substances ranging from dietary supplements to functional foods and is extensively applied to any substance that enhances general well being. Nutraceuticals are playing an important role in therapy both by themselves and in association to conventional pharmacological treatments. In addition, these substances will be more and more used for minimizing the risk of diseases throughout the life-span. Regulatory requirements for marketing these substances are not well Poster Session P9. Gastrointestinal toxicology defined both in Europe and in USA and nutraceuticals generally fall within the novel foods and ingredient regulations. In spite of the absence of a clear legislation, FDA considers the company responsible for safety and claims of efficacy of nutraceuticals. To fulfil this requirement, a standard repeated-dose toxicity protocol according to OECD 407 Guideline (currently adopted in the safety assessment for the Notification of New Chemical Entities) can be suggested for testing simultaneously safety and efficacy. Although this study design requires a limited number of animals, it allows an accurate assessment of the possible toxic effects; in addition, this basic study design can be implemented to monitoring and evaluating in-vivo and ex-vivo, specific markers related to the activity of the test compound. Basic scientific knowledge of the substance under examination is fundamental for the identification of the markers and to address experimental studies. The markers to be evaluated can be biochemical, physiological or behavioural and should be feasible, reproducible, sensitive and specific. Criteria for the design of experimental protocols suitable for the development of new nutraceuticals will be discussed. The same approach can be also adopted for the assessment in terms of safety and efficacy of nutraceuticals already on the market. 221 THE STUDY OF ACUTE TOXICITY OF BIEBERSTEINIA MULTIFIDA TOTAL EXTRACT IN MICE S.N. Ostad 1 , H. Montazeran 1 , B. Minaee 2 , H.R. Monsef 3 . Dept. Toxicology & Pharmacology1 , Dept. Pharmacognosy2 Faculty of Pharmacy & Dept Histology Faculty of Medicine3 , University of Tehran Medical Sciences, Tehran, IRAN Several reports have been published about the pharmacological effects of Adamak (Biebersteinia M) alkaloid, vasicinone. Vasicinone is the major component of Biebersteinia total extract. The acute and delayed analgesic effects of this alkaloid has been shown by carrageenan and formalin test. In Iranian traditional medicine the total extract of this plant has been used as dermal analgesic agent for several years and there is concern about its toxicity. In this study the parenteral and dermal acute toxicity of the total extract have been tested in mice. The sample of plant was collected from Royeen in Iran followed by methanol extraction. Animals were ordered in the standard groups and classical LD50 was measured by Probit analysis. The results showed that LD50 of parenteral administration is 246.09 mg/kg which is classified as moderately toxic agent. Dermal administration of agent showed no sign of toxicity confirmed by pathological examination. 222 TOXO-PATHOLOGICAL EFFECTS INDUCED BY UREA IN BROILER CHICKS s63 P9 Gastrointestinal toxicology 223 ASSESSMENT OF GASTROTOXIC EFFECTS OF NIMESULIDE, A COX2 -SELECTIVE NSAID, AFTER SINGLE AND MULTIPLE OVERDOSAGE IN RATS S. Dobric 1 , V. Cupic 2 , Z. Milovanovic 1 , V. Jacevic 1 , R. Velev 3 , D. Bokonjic 1 . 1 National Poison Control Centre, Military Medical Academy, Belgrade, Serbia and Montenegro, 2 Faculty of Veterinary Medicine, Belgrade, Serbia and Montenegro, 3 Faculty of Veterinary Medicine, Skopje, FYR Macedonia Nimesulide is COX2 -selective non-steroidal antiinflammatory drug (NSAID) with strong antiinflammatory, analgesic and antipyretic activity. It has very low ulcerogenic potential when given in therapeutic doses. The aim of this study was to estimate its gastrotoxic effects after single and multiple administration in very high doses because scarce data have existed concerning this subject. Indomethacin, a strong NSAID with established ulcerogenic activity served as a positive control. Adult male Wistar rats (200–250 g) starving for 16–18 hours before the experiment were used. Both drugs were dissolved in DMSO and given through the orogastric tube in a single dose of 25 mg/kg once (the single dose experiment) or every day during 7 days (the multiple dose experiment). In our laboratory condition this dose is about 8 and 25 times higer than the mean antiinflammatory doses (ED-50) of indomethacin and nimesulide in rats, respectively. Four hours after administration (in the multiple dose experiment after the last dose) animals were sacrificed and the length, area and intensity score of gastric lesions were determined, as well as the pathohistological examination of their stomach. In the multiple dose experiment general condition, behavior and body weight of treated animals were recorded every day. The results demonstrated that both NSAIDs given in a single dose of 25 mg/kg once produced gastric damage in all treated animals, but it was significantly more pronounced in rats given indomethacin than nimesulide (e.g. mean intensity score of gastric lesions was 4.8±0.45 and 0.75±0.29, respectively). In the multiple dose experiment all animals in indomethacin-treated group died, while all nimesulide-treated animals survived with gastric damage even less pronounced than that in the single dose experiment. These results confirm the low ulcerogenic potential of nimesulide, even taken in overdosage, and suggest the development of resistance of gastric mucosa against its harmful effects during multiple administration. 224 TOXICITY TESTING PROTOCOL OPTIMIZATION WITH CACO-2 CELLS: A CASE-STUDY ON HEAVY METALS A.A. Sharkawy 1 , M. Mubarak 2 . 1 Department of Forensic Medicine and Toxicology, Fac. Vet. Med., Assiut Uni., Assiut; 2 Department of Pathology and Clinical Pathology, Fac. Vet. Med., Assiut Uni., Assiut, Egypt A. Stammati 1 , L. Turco 1 , F. Zucco 2 , I. De Angelis 1 . 1 Dipartimento Ambiente e prevenzione primaria, Istituto Superiore di Sanità, Roma, Italia. 2 Istituto di Neurobiologia e Medicina Molecolare, Consiglio Nazionale delle Ricerche, Roma, Italia Sixty, day-old, chicks were reared up to 3 weeks of age and then randomly divided into 4 equal groups (3 treated and one control group). Urea (46% nitrogen) was added to the feed (grower-finisher ration) of the treated birds at the levels of 1%, 3% and 5%. Feed and water were available ad libitum for all birds over the time of experiment. At days 7, 20 and 30 post exposure, 5 birds from each group were weighed, bled and sacrificed. All birds spontaneously died during the experiment were also necropsied. Hematological parameters (RBCs, WBCs counts, PCV and Hb), biochemical variables [urea, glucose, uric acid, alkaline phosphatase, ALP and lactate dehydrogenase, LDH] and body weight gain were assessed. The encountered pathological changes were described. The obtained results indicated that: (1) decrease in RBCs, WBCs counts, PCV and Hb, (2) increase in ALP, LDH, urea and uric acid, while glucose level was decreased. (3) decrease in body weight gain in all treated birds. There were significant pathological changes in kidneys, heart, liver and lungs of the treated birds. It was concluded that addition of urea to poultry feeds to replace the more expensive protein-nitrogen has serious sequences which affect the health condition and weight gain of the birds. Caco-2 cells are widely used for different applications including studies of permeability, metabolism and toxicity, but the protocols employed are rarely comparable and need standardisation. With these aims, a European inter-laboratory study has been performed to better define: i) the characteristics of parental Caco-2 cell line and of selected Caco-2 derived clones; ii) set the minimal requirements for their reliable use for different purposes and, eventually, iii) make it ready for a (pre)validation stage. In the first phase of the study, a careful choice and characterisation of Caco-2 culture conditions have been investigated using common assays representative of different Caco-2 aspects: trans-epithelial electrical resistance and mannitol permeability as barrier integrity markers and alkaline phosphates activity as differentiation one. The second phase of the project provide a further improvement and optimisation of Caco-2 model for specific applications, which for ISS group is toxicity testing. On the basis of phase 1 results, two Caco-2 clones (AQ and TC7) have been selected: cells grown on permeable filter support for 15 days, have been exposed on the apical side to different concentrations of mercury(di)chloride, for 24 hr. Toxicity determination were performed using the same s64 Poster Session P10. Natural toxins end-points of phase 1 experiments as well as neutral red release assay, a classical cytotoxicity test. All tests have been carried out either with culture medium added with 10% foetal calf serum, or with insulin-transferrin-selenium medium supplemented with defined lipids (cholesterol, palmitic acid, oleic acid). Other metals will be tested and comparison between the two different experimental conditions will be evaluated. Supported by E U, Contract ISS/ECVAM 17299–2000–12-FIED ISP IT (45%) contained between 200 and 400 µg B1 /kg. FB2 was found in 3 samples (6%), and the concentrations were 68, 109 and 3084 µg/kg. Although the year when samples were collected was extremely wet and the frequency of finding, as well as the concentration of mycotoxins is certainly not common, our results indicate that fumonisins are very frequent contaminants of corn in our country. 227 225 VALIDATION OF PEPSINOGEN MEASUREMENT IN THE STOMACH AND SERUM OF SPRAGUE DAWLEY RAT. Elena Giannotti, Luca Vandin, Gianni Dal Negro. Cellular & Biochemical Laboratory, Safety Assessment Department, GlaxoSmithKline R&D, Verona The measurement of pepsinogen A and C in serum is considered to be one of the non-invasive biochemical markers for monitoring peptic secretion and obtaining information on the gastric mucosa status. In this work, pepsinogen measurements have been used as an effective biochemical method for evaluating the effects of test drug treatment, during pre-clinical safety evaluation program. The aim of the present study was to determine the most appropriate method applicable to different species for routine use in laboratory. Human serum pepsinogen levels are usually determined by radioimmunoassay (RIA) or by enzyme-linked immunosorbent assay (ELISA). But, these methods are not easily applicable to non-human species because of lack of species-specific commercial antibodies. Other methods, for detecting protease activity, are based on the enzymatic digestion of a substrate solution. In this work, pepsinogen measurement has been performed on serum, gastric juice and gastric mucosa homogenates of Sprague Dawley rats by using casein conjugated with a quenched fluorophore, as substrate. Following casein hydrolysis by pepsin, fluorescence is produced proportionally to the amount of hydrolysed substrate. A validation program was successfully conducted in order to assess the sensitivity and the specificity of this method. In conclusion the method for measuring pepsinogen level in the stomach and serum of Sprague Dawley rats demonstrated to be a suitable tool to monitor variation of pepsinogen level, indicative of gastric changes. P10 Natural toxins 226 SCREENING FOR FUMONISINS B1 AND B2 IN CORN COLLECTED IN REPUBLIC OF CROATIA A.-M. Domijan 1 , M. Peraica 1 , R. Fuchs 1 , A. Lucić 1 , B. Radić 1 , Ž. Jurjević 2 , B. Cvjetković 2 . 1 Unit of toxicology, Institute for Medical Research and Occupational Health, Ksaverska c. 2, 10000 Zagreb, Croatia, 2 Department of Phytopathology, Faculty of Agriculture, University of Zagreb, Svetošimunska 25, 10000 Zagreb, Croatia Fumonisins are mycotoxins produced by some strains of Fusarium moulds. They cause equine leukoencephalomalacia, pulmonary edema in swine and hepatic and kidney lesions in various laboratory animals. Fumonisins are frequently found as contaminants of corn in temperate climatic zone, but the knowledge about their occurrence in our country is scarce. 49 corn samples were collected during fall 2002. from 14 counties of Republic of Croatia. Samples were purified by means of immunoaffinity clean-up procedure and the concentration of fumonisin B1 (FB1 ) and fumonisin B2 (FB2 ) were determined by HPLC method with fluorescent detection. Limit of detection for both mycotoxins was 10 µg/kg. Reproducibility of the method expressed as Relative Standard Deviation (RSD) was below 10%. FB1 was found in all analyzed samples of corn in the range of 142–1378 µg/kg, and the mean concentration was 460 µg/kg. Most of samples CYCLODEPSIPEPTIDE TOXICITY ON ISOLATED CARDIOMYOCYTES OF THE GUINEA PIG K. Kouri, M. Kamyar, A. Rapp, R. Lemmens-Gruber. Dept. of Pharmacology and Toxicology, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria Beauvericin (BEA) and Enniatin (ENN) are secondary metabolites of pathogenic fungi including the genus Fusarium, an important phytopathogen. Plant diseases due to mycotoxins are widely known, however BEA- or ENN-induced toxicity to mammalian organisms has not yet been directly proven. The action of BEA and ENN was tested on isolated ventricular myocytes of the guinea pig with the patch clamp method in the inside-out mode. Both antibiotics were found to be cation-selective potent channel-forming ionophores. In order to test the physiological significance of channel formation on the cellular ionic homeostasis, their effects were subsequently tested with fluorescence imaging. Myocytes were loaded with the ratiometric dyes FURA 2AM, SBFI and PBFI. Intracellular pH values were measured by BCECF. ENN, much like BEA, at concentrations up to 10 µM, was found to increase [Ca2+ ]i and [Na+ ]i within 10 to 15 min, the onset of action being concentration-dependent. Cellular death was preceded by rigor contraction and Ca2+ overload. Intracellular acidification developed shortly after mycotoxin application. The Ca2+ - and Na+ -overload could be reversed by extracellular application of 2 mM ATP. The results indicate that the ionophoric effects of these cyclodepsipeptide antibiotics can seriously compromise the physiological ionic balance. Thus, we further investigate if the ATP dependent reversal of the toxic effects could involve the ATP-binding cassette, which is a major natural defense of the cells against toxins as well as drugs. 228 CYTOTOXICITY, INHIBITION OF PROTEIN SYNTHESIS AND INDUCTION OF STRESS PROTEIN EXPRESSION IN HEP G2 CELL LINE IN RESPONSE TO ZEARALENONE Wafa Hassen 1 , Isabelle Baudrimont 2 , M. Moncef Ladjimi 3 , Edmond Creppy 2 , Hassen Bacha 1 . 1 Laboratoire de Recherche sur les Substances Biologiquement Compatibles, Faculté de Médecine Dentaire. Monastir 5019, Tunisia; 2 Laboratoire de Toxicologie et d’Hygiène Appliquée, UFR Sciences Pharmaceutiques, Université Victor Ségalen. Bordeaux II 33000, France; 3 Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, UMR 7631, CNRS-Université Pierre et Marie Curie, Paris, France Mycotoxins are toxic metabolites of various fungi commonly found in feed and foodstuff and can cause very serious health problems in animals as well as in humans. Zearalenone (ZEN), a mycotoxin produced by various Fusarium species has several adverse effects. Indeed, ZEN has strong estrogenic activity associated with hyperestrogenism and several physiological alterations of the reproductive tract. In addition, ZEN was shown to be hepatotoxic, nephrotoxic, haematotoxic, immunotoxic and genotoxic. The exact mechanism of ZEN toxicity has not been completely estabished as of yet. The observed strong estrogenic effect of ZEN resulting from its competition with 17β-estradiol in the binding to oestrogen receptors is generally considered to underline most toxic effects of ZEN, but estrogenic activity alone cannot explain the diverse and apparent adverse effects. The objective of the present study was to determine the involvement of others possible mechanisms in ZEN induced toxicity. Cytotoxicity, inhibition of protein synthesis as well as the presumed earlier marker of oxidative stress, expression of HSP 70, were monitored in Hep G2 cells exposed to ZEN toxicity. Our results showed that ZEN reduces cell viability, inhibits protein synthesis and induce HSP 70 in Hep G2 cell line in time Poster Session P10. Natural toxins and concentration dependant manners. We assumed that cytotoxicty and oxidative damage are additionnal mechanisms of ZEN mediated toxicity. 229 LEVELS OF OCHRATOXIN A AND IGG AGAINST CONIDIA FROM PENICILLIUM VERRUCOSUM IN BLOOD SAMPLES FROM HEALTHY FARM WORKERS AND BLOOD DONORS M.A. Skaug. Faculty of Health Studies, Hedmark University College, Elverum, Norway Ochratoxin A (OTA) is a mycotoxin frequently found in human blood and milk samples in the colder climatic zones. In addition to dietary intake, exposure to OTA may occur by inhalation of fungal conidia. The purpose of this work was to investigate the level of OTA in blood samples from farm workers and non-farm working controls, and to examine if serum levels of OTA were related to exposure to conidia of Penicillium verrucosum, the main OTA producer in temperate climates. The levels of OTA and IgG antibodies against P. verrucosum conidia were determined in blood samples from 210 Norwegian participants. The concentration of OTA was determined by ion-pair HPLC with fluorescence detection (DL 10 ng/l), and the IgG level was determined by ELISA. All serum samples contained OTA (mean 397 ng/l, range 21 – 5534 ng/l). The OTA level in serum was unrelated to farm working, gender, age, and IgG level. The mean IgG level was significantly higher among farm workers than controls. Conclusions: Farm working, or exposure to P. verrucosum, was not related to higher OTA serum levels. Exposure to OTA through inhalation of P. verrucosum conidia seems to be of minor importance compared to dietary intake. 230 OXIDATIVE DNA DAMAGE INDUCED BY THE CARCINOGENIC MYCOTOXIN OCHRATOXIN A IN VITRO AND IN VIVO H.G. Kamp 1 , R. Turesky 2 , J. Schlatter 3 , G. Eisenbrand 1 , C. Janzowski 1 . 1 Dept. of Chemistry, Div. of Food Chemistry & Environmental Toxicology, University, Kaiserslautern, Germany, 2 National Center for Toxicology Research, Jefferson, USA and 3 Swiss Federal Office of Public Health, Bern, Switzerland Ochratoxin A (OTA) is a potent nephrotoxic and nephrocarcinogenic mycotoxin, produced by Aspergillus and Penicillium strains, to which humans are widely exposed via food contamination. Its carcinogenicity has been documented in rats and mice. A genotoxic mechanism of action has been postulated. DNA adducts of OTA or its metabolites, however, have not been unambiguously identified. Free radical formation and oxidative cell damage are also suggested to be involved in OTA-mediated toxicity. In this study we investigated, whether OTA induces oxidative DNA damage in cell lines (CV-1, V79), primary rat kidney cells, and in liver and kidney of rats in vivo. DNA damage was assessed by the comet assay; oxidative DNA damage, determined by additional treatment with formamidopyrimidineDNA-glycosylase (FPG), was expressed as difference in comet tail intensity, TI%. We recently found that after 1 h incubation of OTA at high concentrations, FPG sensitive sites were induced (CV-1: 500µM, TI%: 6, n=3; V79: 2000 µM, TI%: 8, n=3). Viability (trypan blue exclusion) was not impaired under these conditions (CV-1: > 85%; V79 >75%). 24 h incubation with 1µM OTA resulted in oxidative DNA damage in both cell lines (CV-1: TI%: 5, n=2; V79: TI%: 5, n=4). Membrane integrity of attached cells, used for the comet assay, was not impaired (CV-1, 83%; V79, 90%). In primary proximal tubular cells of rat kidney, 1h incubation with 25–100 µM OTA resulted in concentration dependent increase of oxidative DNA damage (TI%: 5–10, n=2–3). In rats oxidative DNA damage was induced in the kidney after 4 week treatment with OTA (0,3 mg/kg bw, TI%: 3). In conclusion, the potency of OTA to induce (oxidative) DNA damage was considerably higher in primary cells than in cell lines. OTA also showed the potential to induce oxidative DNA damage in vivo. Acknowledgement: Grants No 00.000314 and 02.000677, Swiss Fed. Off. Publ. Health 231 s65 MODIFICATION OF TOXICITY MARKERS BY OCHRATOXIN A IN RAT HEPATOCYTES PRIMARY CULTURE AND HK-2 CELL LINE O. Ezpeleta, L. Alvarez, L. Arbillaga, A. López de Cerain. Laboratory of Toxicology, Department of Food Sciences and Toxicology, University of Navarre, Pamplona, Navarre, Spain Ochratoxin A (OTA) is a mycotoxin that is present in many types of food and feed. OTA is a secondary metabolite produced by several fungal species of the Aspergillus and Penicillium genera. It has been shown to be nephrotoxic, hepatotoxic, teratogenic, carcinogenic, and immunosuppressive. In this study, in order to investigate OTA toxicity in vitro, several toxicity markers were evaluated in rat hepatocytes primary culture (Hep) and in one human kidney cell line (HK-2). Previous to this study, cytotoxicity assays were performed in both cell types after treating cells for 24 hours with the mycotoxin. MTT, Neutral Red and Propidium Iodide methods were used as cytotoxicity assays, and the following rank of IC50 were obtained: HK-2 (7.2–49.3 µM) Hep (31.3–200 µM). For this study, three different concentrations of OTA (1, 10 and 100 µM) were added to the cells during 1, 4, 12 and 24 hours, and then, the following toxicity indicators were simultaneously evaluated: lipid peroxidation, GSH depletion, total protein decrease, LDH leakage and glucose consumption. The experimental design carried out in our study allowed processing all of the samples together at the end of the incubations, and measuring all of the parameters from the same cell culture. A dose-dependent increase of lipid peroxidation was observed in Hep cultures after. short and long-term treatments; in HK-2, this effect was only produced after 24 h of treatment. The proportion of lipoperoxidation was inversely correlated with the intracellular level of GSH. These results confirm the role of CYP 450 in the generation of radical species by OTA. LDH leakage to the culture medium was a relatively sensitive parameter because a dose-dependent increase was detected in Hep cells and in HK-2, after treatments of long duration. The other two parameters evaluated resulted much less sensitive: a clear effect was only detected after long treatments,and it was not well related to OTA concentration. 232 OCHRATOXIN A TOXICITY AND CARCINOGENICITY D. Holzhäuser, T. Delatour, M. Marin-Kuan, S. Junod, G. Guignard, D. Piguet, J. Richoz, C. Bezencon, B. Schilter, C. Cavin. QS Department, Nestle Research Center, Lausanne, Switzerland Ochratoxin A (OTA) is a mycotoxin occurring in various foods. In rodents, OTA has been identified as a kidney carcinogen although its mechanism of carcinogenicity is not understood. Furthermore, the health significance of OTA-exposure in human is not known. OTA has been claimed to produce DNA damage and research is underway to know whether it acts through direct DNA binding or through an indirect mechanism. OTA is known to induce an oxidative stress response and therefore oxidative stress may be the mechanism of DNA-damage. In the present investigation, the OTA-mediated stimulation of the stress response has been further studied in rat NRK kidney cells and primary hepatocytes. In both cell culture systems, a dose-dependent increase in the mRNA and protein expression specific for the inducible nitric oxide (iNOS) was found after 24 hours of OTA treatment. This induction was correlated to the activation of the redox sensitive transcription factors AP-1 and NF-KappaB. The toxicological relevance of the OTA-mediated iNOS induction was further studied. A dose-dependent increase in protein-bound 3-nitrotyrosine was correlated with the OTA-dependent iNOs induction. Interestingly, the OTA-mediated iNOs induction and the resulting increase in bound nitrotyrosine were prevented by the pretreatment of the cells by the chemoprotective agents coumarin and the coffee diterpene cafestol and kahweol. These preventive effects were correlated with a reduction of OTA cytotoxicity and a prevention of the OTA-mediated inhibition of the de novo protein synthesis. The present data suggest that OTA produces oxidative stress and nitric oxide which may play a significant role in OTA-toxicity. Further investigation addressing the role of iNOs induction in OTA-induced DNA damage and carcinogenicity are currently undertaken. This work was supported by the EU-Grant#QLK1-CT-2001–011614. s66 233 Poster Session P10. Natural toxins HISTOPATHOLOGICAL ALTERATIONS IN LIVER AND KIDNEY BROILERS TREATED WITH INCREASED DOSES OF OCHRATOXIN A Jelena Nedeljkovic Trailovic, Snezana Sinovec, Z. Sinovec. Faculty of Veterinary medecine, University of Belgrade, Yugoslavia; Faculty of Veterinary Medecine, Department of Nutrition, Bulevar JNA 18, 11000 Belgrade, Yugoslavia Introduction: Ochratoxins are the group of seven isocoumarin derivatives linked with an amide bond to the amino group of L
-phenylalanine and they have been reported as metabolites of six species of Penicillium and seven species of Aspergillus. One of the most toxic and abundant ochratoxins is ochratoxin A (7-carboxy5-chloro-8-hydroxy-3,4-dihydro-3R methylisocoumarin) which is a highly toxic compound commonly produced (Frisvad and Samson, 1991) as secondary metabolite by two species of fungi: Penicillium verrucosum Dierckx and Aspergillus ochraceus Wilhelm (alutaceus). It has been found that OTA primarily provokes pathomorphological alterations in kidneys (Buck and Osweiler, 1976; Uraguchi and Yamazaki, 1978; Humphreys, 1988), while other authors (Wyllie and Morehouse, 1978; Leeson et al., 1995) also describe alterations in liver and other organs. Matherials and Methods: After 14 days long preexperimental period a total of 48 broilers were submitted to the trial. Broilers were divided into three experimental groups (A, D, E) and one control group (C). During next 7 days experimental groups were offered fed contaminated with 0.5 1.0 or 1.5 ppm OTA, respectively.Sample collection. Liver and kidney samples were taken after the period of toxin administration (21st day) and the remaining birds from the control and experimental groups were normally fed and watered without toxin addition until the end of trial (42nd day) when blood and kidney samples were taken again. Results: Histopathological changes in liver semples were not detected in broilers of all experimental groups. Histopathological alterations of the kidney were noticed only in the group of broilers offered the feed with the highest amount of OTA and sacrificed immediately after the treatment period as well as in those broilers sacrificed after the withdrawal period, but they were diverse in character, intensity and extent. 234 ANTIOXIDANT PROPERTY OF ESSENTIAL OIL ISOLATED FROM BLACK COMIN SEEDS (NAGILLA SATIVA) AND CLOVE BUDS (SYZYGIUM AROMATICUM) IN RATS DURING AFLATOXICOSIS Mosaad A. Abdel-Wahhab, Soher E. Aly. Food Toxicology and Contaminants Dept. National Research Center, Dokki, Cairo, Egypt Aflatoxins (AF), a group of closely related, extremely toxic chemicals, produced by Aspergillus Spp. and can occur as natural contaminants of foods and feeds. Aflatoxins have been shown to be toxigenic, carcinogenic, mutagenic, and teratogenic to different animal species. Nagilla Sativa (N.S.) and Clove essential oil are used for the treatment of inflammatory diseases. They have antioxidant properties and act as radical sevengers. The aim of this study was to investigate the ability of these volatile oils to scavenger free radicals generated during aflatoxicosis. Sixty male rats were divided into six treatment groups including: control group and groups treated for 30 days with N-sativa and clove essential oils with or without aflatoxin. Blood samples were collected at the end of experiment period. The results indicated that treatment on the aflatoxins resulted in heamatological and biochemical changes typical to aflatoxicosis. On the other hand, treatment with N.S. or clove essential oil to the rats fed aflatoxin- contaminated diet resulted in significant protection against AF. Moreover, N. S. essential oil was found to be effective than clove essential oil in restoration of the parameters that were altered by AF in rats. 235 CORRELATION BETWEEN DNA ADDUCT FORMATION AND OTA METABOLITES IN OPPOSUM KIDNEY CELLS V. Faucet 1 , C. Lebeau 2 , M. Castegnaro 1 , A. Pfohl-Leszkowicz 1 . 1 Unit of Toxicology & Food Safety, Ecole Nationale Supérieure Agronomique Toulouse, F-31326,2 ULB-Erasme, Bruxelles, B61070, Belgium Ochratoxin A (OTA) a nephrotoxic mycotoxin, probably implicated in human Balkan Endemic Nephropathy, induces renal carcinomas in rodents and pigs. OTA induces DNA-adduct formation, but the structure of these adducts and their role in nephrotoxicity, genotoxicity and carcinogenicity has only partly been elucidated. In a in vivo study, we have demonstrated that 2-mercaptoethane sulfonate (MESNA) protect rats against nephrotoxicity but not against carcinogenicity, indicating that two different mechanisms are implicated in the nephrotoxicity and carcinogenicity induced by OTA. For a better understanding of the mechanism of OTA carcinogenicity, OTA-DNAadduct formation has been measured using the 32 P-postlabelling method after incubation of opposum kidney cells in presence of OTA alone or in presence of several compounds such MESNA or N-acetylcysteine (NAC), (another agent which, like MESNA, reduces oxidative stress by increase of free thiol in kidney), buthionine sulfoximine (BSO) (an inhibitor of glutathione-syntase), acivicin (an inhibitor of gamma glutamyl transpeptidase (GGT) and melatonin (an antioxydant). Cytotoxicity has been evaluated by MTT technic. None of these agents diminished significantly OTA cytotoxicity, even acivicin increases OTA cytotoxicity. Analysis of HPLC profiles of OTA metabolites yielded during these incubations indicated that the distribution, the quantity of metabolites and the nature of the derivatives were modulated by these agents. Some genotoxic OTA metabolites has been identified as quinone derivatives. Another important metabolite is the ring-open OTA. Some metabolites are present only when DNA adducts are observed. 236 DETERMINATION OF α-AMANITIN IN HUMAN BIOLOGICAL FLUIDS BY LIQUID CHROMATOGRAPHY – CID – MASS SPECTROMETRY AFTER SOLID PHASE EXTRACTION M. Vujovic 1 , V. Kilibarda 2 , S. Cusic 2 . 1 Laboratory of Toxicology, Institute of Forensic Medicine, Nis, Serbia and Montenegro, 2 Institute of Toxicology and Pharmacology, Military Medicine Academy, Belgrade, Serbia and Montenegro The most poisonous mushroom toxins are produced by Amanita phalloides (death cap). Alpha-amanitin is the major toxin. This toxin is also found in A. verna, A.virosa, A.ocreata, A.tenuifolia anad other Amanita spices. Indetification of the mushroom or its toxin are key in successfully treating an individual poisoned by A.phalloides, as delay in treatment may result in death from hepatorenal failure and neurological injure. This assay represent an easy, fast and specific method of determination α-amanitin in serum and urin with electrospray liquid chromatography – mass spectrometry. Mass spectras were took down after collision induction disosiation (CID) on diffrent voltages. The most suitable mass spectra was on the 40 V. The calibration curve for α-amanitin was linear, precise and exact from 5–100 µg/L. Intraassay coefficient of variation was 1,34? (n=5). LOD was 2,5 µg/L. We also examined various techniques of sample preparation like deprotonization, liquid–liquid extraction and solid phase extraction with diffrent values of pH. Conclusion was, that the SPE (pH=7) with copolymer, hydrophilic–lypophilic balance cartridges (HLB) is the optimal technique for isolation α-amanitin from biological fluids. The absolute recovery after SPE was 91,94?. This is an specific, sensitiv and reproductive method which can take important place in clinical toxicology for rapid determination of α-amanitin in serum and urin. Poster Session P10. Natural toxins 237 VALIDATION OF THE ELISA TEST FOR URINARY α-AMANITIN ANALYSIS IN HUMAN AMANITA PHALLOIDES POISONING T. Coccini, G. Randine, C. Locatelli, R. Butera, L. Manzo. Toxicology Division-Poison Control Center, IRCCS Maugeri Foundation and University of Pavia, Pavia, Italy The value of the ELISA assay for urinary α-amanitin was examined in relation to its validity as diagnostic tool in patients with mushroom (Amanita phalloides) poisoning. Linearity was assessed on pooled blank urine spiked with 0, 1, 10, 20 ng/ml α-amanitin. The equation of the calibration plot was y = -0.047x + 1.95, with r = 0.99, where y was the Log percent bound α-amanitin value and x was the α-amanitin concentration. The lower and higher concentrations of the linear range were used as lower and higher limits of quantitation. Accuracy ranged from 90% at the 10 ng/ml level (best accuracy) to 75% at the 1 ng/ml (worst case). Intra-assay precision, evaluated on replicate measurements (N = 20) on three real samples provided by our Poison Control Center resulted 16% at the 6 ng/ml level, and 15% at the 20 ng/ml level. Inter-assay precision, evaluated on real samples (three replicates on ten runs) resulted better than 28% at all concentrations evaluated. The ELISA specificity for α-amanitin was studied adding α, β-amanitin, and phalloidin (1:1:1, m/v, 1–20 ng/ml) to blank pooled urine and comparing the results with equally spiked urine containing only α-amanitin. In urines containing the three toxins the measured concentration values of α-amanitin were reduced by 50–65% as compared to those detected in urines added α-amanitin only, indicating a masking effect of both β-amanitin and phalloidin. However, when tested individually, either β-amanitin or falloidin were not measurable by this assay. Validation parameters (linearity, accuracy, precision) proved the ELISA assay for urinary α-amanitin to be a suitable diagnostic tool. However, further studies are needed to better define the relevance of the interference of mushroom components other than α-amanitin with the determination of the actual concentrations of α-amanitin in urine samples. 238 ACONITE POISONING IN WESTERN COUNTRIES: DIFFERENT CLINICAL PICTURES M.L. Colombo 1 , P.A. Moro 2 , F. Zoppi 3 , S. Primavera 4 , C. Potì 5 , F. Assisi 2 , A. Martella 1 , C. Zanardini 1 . 1 Dept. Plant Biology, Faculty of Pharmacy, University of Torino, Italy, 2 Poisoning Control Centre of Milan, Niguarda Ca’ Granda Hospital, Milano, Italy,3 Lab. Clinical Analysis, Niguarda Ca’ Granda Hospital, Milano, Italy, 4 Dept. Anaesthesiological Reanimation, Desenzano del Garda Hospital, Brescia, Italy, 5 Forensic Medicine, Mestre Hospital, Venise, Italy Aconite poisoning is far common in Asia, particularly China and Hong Kong, than in Western Countries. This may be because of the widespread use of herbal medicines containing Aconite derivatives by the Asian communities. Outside Asia, poisononing usually occurs after ingesting – as mistake - the wild plant of the Aconitum species. The clinical picture of Aconite poisoning is characterized by neurological, gastrointestinal and cardiac symptoms. Within 10 – 30 min after Aconite ingestion, the patients usually develop a tingling, burning sensation in the tongue, lips and whole mouth, gradually extending to the arms and the entire body, accompanied by a feeling of cold and of being very sick. Nausea, vomiting and diarrhea are frequent, but not constant symptoms, and various cardiac abnormalities and severe disrhythmias have been reported and may be fatal. To assess the magnitude of poisoning by herbal medicines or by plants, a survey of patients was conducted (1995 – 2002) by the Poisoning Control Center of Milan, Niguarda Ca’ Granda Hospital, Italy. In this period, 25 patients (seven in the last two years) presented clinical features of Aconite poisoning following the ingestion of roots, leaves or seeds. Described here are accidental aconitine poisoning following the ingestion of aconite mistaken for an edible grass and intentional poisoning for suicide attempt. In the patients we observed the severity of poisoning was quite variable, and sometimes the clinical picture s67 seemed disagree with the supposed elevated amount of ingested toxins. The cause of this could be related to a higher toxicity of the fresh, unprocessed plant compared with the processed Aconite roots or leaves, after cooking, decoction or conservation in olive oil, and so on. This is due to the chemical feature of Aconite alkaloids, which are di-esters of acetic and benzoic acids of the aconine base. When the raw Aconite roots or leaves were processed, the di-ester alkaloid contents were gradually reduced, coupled with the conversion of the toxic alkaloids - aconitine and related compounds – into the less poisonous benzoylaconines. 239 EFFECTS OF LAUREL (Laurus nobilis L.) LEAVES AND BERRIES ETHER OIL, PCBs AND CCl4 ON PRODUCTION OF OXYGEN RADICALS M. Popovic 1 , B. Kaurinovic 1 , T. Cebovic 2 , M. Vojinovic-Miloradov 1 . 1 Faculty of Sciences, Department of Chemistry, University of Novi Sad, Novi Sad, Serbia and Montenegro, 2 Faculty of Medicine, Department of Biochemistry, University of Novi Sad, Clinical centre Novi Sad, Novi Sad, Serbia and Montenegro Laurel (Laurel nobilis L.) is a well-known medicinal plant. In folk medicine, laurel leaves ether oil is used as carminative, excitoaromatic, nervine, as well as in perfume production. In past, laurel fruits have been used as a bitter substance and spice, but nowadays they are used only for their oil, and as a cream for hemorroides (external application). Laurel oil is used as skin irritant, most often in combination with other balms. The plant contains several classes of secondary plant products. Fruit contains about 30% of fat and up to 1% of ether oil, sugar, starch, basorin, etc. Leaves contain bitter substances and tannins, and ether oil contains mostly cineol and alpha-pinen. In this study we investigated the effect of laurel leaves and berries ether oil on production of hydroxil radicals (OH• ). Beside that, we were examining sinergistic effect of these ether oils and CCl4 and PCBs (pyralene and askarel). Production of OH• radicals was measured by the TBA test. Laurel leaves and berries ether oils increased both the production of OH• radicals, but this increase was lower as mass concentration of ether oils increased. PCBs (pyralene and askarel), used alone, increased the production of OH• radicals, but this increase was lower as mass concentration of ether oils increased. CCl4 highly increased the production of OH• radicals. Combination of laurel leaves and berries ether oil and PCBs and CCl4 decreased OH• production in comparison to PCBs and CCl4 used alone, but the production of OH• radicals was still significantly higher in comparison to control group (statistically speaking). 240 EFFECTS OF LAUREL (Laurus nobilis L.) LEAVES AND BERRIES ETHER OIL AND FULERENE DERIVATIVES ON PRODUCTION OF OXYGEN RADICALS AND LIPID PEROXIDATION OF LIPOSOMES B. Kaurinovic 1 , M. Popovic 1 , T. Cebovic 2 , A. Djordjevic 1 . 1 Faculty of Sciences, Department of Chemistry, University of Novi Sad, Novi Sad, Serbia and Montenegro, 2 Faculty of Medicine, Department of Biochemistry, University of Novi Sad, Clinical centre Novi Sad, Novi Sad, Serbia and Montenegro Laurel (Laurel nobilis L.) is a well-known medicinal plant. In folk medicine, laurel leaves ether oil is used as carminative, excitoaromatic, nervine, as well as in perfume production. In past, laurel fruits have been used as a bitter substance and spice, but nowadays they are used only for their oil, and as a cream for hemorroides (external application). Laurel oil is used as skin irritant, most often in combination with other balms. The plant contains several classes of secondary plant products. Fruit contains about 30% of fat and up to 1% of ether oil, sugar, starch, basorin, etc. Leaves contain bitter substances and tannins, and ether oil contains mostly cineol and alpha-pinen. In this study we investigated the effect of laurel leaves and berries ether oil on production of hydroxil radicals (OH• ) and lipid peroxidation of liposomes. Beside that, we were examining s68 Poster Session P10. Natural toxins sinergistic effect of these ether oils and derivative of fulerene (C60 (OOC-CHOH-CH2 -CH3 )n ). Laurel leaves and berries ether oils increased both the production of OH• radicals and intensity of lipid peroxidation, but this increase was lower as mass concentration of ether oils increased. Fulerene derivative used alone increased the production of OH• radicals and the intensity of lipid peroxidation (higher percentage than production of OH• radicals). Combination of laurel leaves and berries ether oil and fulerene derivative increased OH• production even more. Intensity of lipid peroxidation remained unchanged in combination of fulerene derivative and laurel ether oils. 241 STUDY EFFECT PEGANUM HARMALA PLANT ON SKELETAL SYSTEM M.T. Goghataei, F. Kermanian, M. Mehdizadeh. Anatomy Department, Iran University, Tehran, Iran Peganum Harmala is popular plant in traditional medicine. It used for the treatment of asthma, parasitical and microbial infections, postpartum hemorrhage and etc. In order to study the side effects of PJHL in pregnant mice such as growth retardation and abortion, we injected P.H. intraperitoneally to the pregnant mice in three doses: 6, 15,30 mg/kg/day from the first up to the lo"h day of pregnancy, then we killed the mice in 15–19" pregnancy days. After Laparatomy we extract fetuses and measured their height and weight. Then the fetuses were stained with Alizarin Red and observed with stereomicroscope for skeletal anomalies. Conclusions showed that there were no skeletal anomalies. But in comparison with control group, absorption and decrease of height and weight was observed. Since the most absorption of the fetuses was in the last days of pregnancy (18–19th), we believe that P.H. causes abortion by means of affecting on growth and development offetus. 242 MICROCYSTIN-LR CAUSES REORGANIZATION OF ACTIN FILAMENTS AND MICROTUBULES IN RABBIT WHOLE EMBRYO CULTURES M.C. Žužek 1 , M. Kosec 1 , J. Mrkun 1 , D. Šuput 3 , B. Sedmak 2 , R. Frangež 1 . 1 Veterinary faculty, Institute of physiology, pharmacology and toxicology, University of Ljubljana, Slovenia, 2 National Institute of Biology, University of Ljubljana, Slovenia, 3 Medical faculty, University of Ljubljana, Slovenia Microcystins are a group of cyclic heptapeptide hepatotoxins, produced by different cyanobacterial genera. Cyanobacteria are inhabitants of terrestrial, fresh and brackish water. Microcystin-LR (MC-LR), the most investigated microcystin, is produced by different Microcystis species. MC-LR is inhibitor of cellular protein phosphatases types 1 and 2A. The consequences of this effect are evident as alteration and redistribution of intermediate filaments, microtubules and microfilaments. The purpose of this study was to establish the in vitro effect of MC-LR on microtubules and actin filaments of whole embryo cell cultures. The embryos were harvested from super ovulated four months old female rabbits (New Zealand White) and cultivated in the presence of different final concentrations of microcystin (10 µM, 20 µM and 100 µM) for a period of 24 h. Whole embryo cell cultures were fixed, stained with fluorescent dye Rhodamine phaloidin for actin filaments and immunostained with anti-α-tubulin, mouse monoclonal antibodies and Alexa Fluor® fluorescent dye conjugate secondary antibodies for microtubules. Confocal laser microscopy was used for visualization of changes in organization of cytoskeleton. Low concentrations (10 µM and 20 µM) of MC-LR caused reorganization of microtubules and actin filaments in all whole embryo cell cultures without evident morphological changes. 100 µM MC-LR caused morphological changes which resulted in rounding of cells and loss of cell-cell adhesion, leading to detachment and dispersion of the cells. Our results confirmed that MC-LR affects actin and microtubule network distribution in whole embryo cell cultures in vitro. 243 EFFECT OF CROTAPOTIN ON THE BIOLOGICAL ACTIVITY OF D49 AND K49 BOTHROPS PLA2 E.C. Arantes 1 , A.L. Cecchini 1,2 , A.M. Soares 3 , R. Cecchini 4 , C.A. Vieira 2 , J.R. Giglio 2 . 1 Depto. Física e Química, FCFRP-USP; 2 Depto. Bioquímica e Imunologia, FMRP-USP; 3 Unidade de Biotecnologia, UNAERP; 4 Depto. Patologia Geral, Centro de Ciências Biológicas, UEL; 5 Depto. Química, FFCLRP, USP; Ribeirão Preto, SP, Brazil Myonecrosis is the most striking local effect caused by Bothrops snake venoms, while the ischaemia evoked by edema may aggravate the venom-induced lesion. These effects are partially due to phospholipase A2 toxins (PLA2 ). Some of them are catalytically active, (D49-PLA2 s), whereas others have little or no enzymatic activity due to a substitution of aspartic acid for lysine at position 49 (L49-PLA2 ), althoug they are very active as myonecrosis inducers. Crotoxin from Crotalus durissus terrificus venom is made up of two non-identical subunits: a basic PLA2 subunit and an acidic, non-toxic and non-enzymatic subunit, crotapotin, which prevents the PLA2 subunit from binding to non-specific sites, increasing its toxicity. In addition, crotapotin inhibits the edema induced by snake venoms or carrageenin, probably by interacting with PLA2 s secreted during the inflammatory process. We have investigated the effect of crotapotin on mouse paw edema and myonecrosis induced by isolated PLA2 s (D49 and K49) from three different species of Bothrops snake venom (BthTX-I and BthTX-II – B. jararacussu, PrTX-I and PrTX-III – B. pirajai and MjTX-II – B. moojeni). We also assayed the enzymatic activity of the PLA2 s in the presence and absence of crotapotin. The combination of crotapotin with PLA2 (2:1, w/w) prior to injection resulted in great reductions in edema (the thickness of the mouse paw was used as an index of edema) and myonecrosis. Crotapotin (100 µg) inhibited around 40–50% the myotoxicity caused by PLA2 s (50 µg), evidenced by decrease in creatine kinase activity (CK-UV kit from Sigma Chemical Co.). It inhibited significantly the edema induce by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%). Saline and crotapotin alone were used as controls. We could not detect any significant inhibition of D49-PLA2 phospholipase activity, indicating that the interaction with crotapotin does not change the catalytic site. Support: FAPESP, CNPq. 244 NORADRENERGIC AND NITRERGIC EFFECTS INDUCED BY Tityus serrulatus VENOM ON THE RAT ISOLATED RETRACTOR PENIS MUSCLE. E.C. Arantes 1 , J.H.G.G. Bomfim 1 , M.A.F. de Godoy 2 , J.R. Giglio 3 , A.M. de Oliveira 1 . 1 Depto. Física e Química, FCFRP-USP, 2 Depto Farmacologia, FMRP-USP, 3 Depto. Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto, SP, Brazil. The main toxins from Tityus serrulatus venom (TsV) affect voltagegated sodium channels inducing the release of neurotransmitters from sympathetic and parasympathetic nerve terminals. Some evidence regarding the participation of nitrergic response on the effects induced by TsV has been provided by functional studies on rabbit corpus cavernosum. Thus, the aim of the present study was to investigate the participation of noradrenergic and nitrergic components on the responses of rat retractor penis muscle (RPM) to TsV, fractions X, XI, XIIa, XIIb and TsTX-I. RPM was isolated and mounted under 0.6 g isotonic resting tension in 5 mL organ bath containing physiological salt solution. Contractions and relaxations were recorded as changes in the displacement (mm) from baseline and expressed as percentage of the maximum effect induced by KCl (90 mM) or of the maximum relaxant response, respectively. The relaxant responses were performed by contracting the muscle with bethanechol (100 µM), in the presence of prazosin (1 µM, 20 min) and guanetidine (30 µM, 10 min), associated or not with Nω -nitro-L-arginine methyl ester (L-NAME, 100 µM, 30 min) or tetrodotoxin (5 µM, 30 min). TsV and fractions (0.03 – 100 µg/mL) induced concentrationdependent contractile responses, while TsTX-I did not. Prazosin, guanetidine and tetrodotoxin completely abolished the contractile responses. TsV or fractions did not affect the cholinergic innervation. These results indicate that the contractile responses are due to the Poster Session P10. Natural toxins release of noradrenaline, preceded by the sodium channel activation. TsV, fractions and TsTX-I induced concentration-dependent relaxant responses in the bethanechol precontracted RPM. Tetrodotoxin and L-NAME completely abolished this response, indicating that the relaxant responses are due the NO release preceded by sodium channel activation of the nitrergic system. 245 PURIFICATION AND CHARACTERIZATION OF A METALLOPROTEASE FORM KOREA SNAKE VENOM (AGKISTRODON BREVICAUDUS) Sook-Jim Hur 1 , Hye-Jin Won 1 , Myung-Sook Lee 1 , Ji-Yun Kim 1 , Motohide Takahashi 2 , Sook-Ho Lee 1 . 1 Division of Bacterial Products, Center for Biologics Evaluation, Korea Food & Drug Administration, Korea, 2 National Institute of Infectious Disease, Japan We have purified and characterized a metalloprotease from the snake venom of Agkistrodon brevicaudus that is the most abundant snakes in Korea. We isolated and purified 68 kDa and 30 kDa proteins at pI 4.8 by Sephacryl-S200HR gel filtration chromatography, DEAE-Sepharose Fast Flow ion exchange chromatography, then rechromatographed on Superdex 75 column gel filtration column, followed by 2-D electrophoresis. The N-terminal of 30 kDa protein was IVSPP VKGNE LLEVG GYPEN MQNE (315–328) and of 68 kDa was SVGIV QDYSP INLY (399–427). Its cDNA sequence was confirmed to be Agkistrodon halys brevicaudus metalloprotease from BASTp analysis. The MHD of 68 kDa protein possessed the hemorrahagic activity of 0.17µg while hemorrhagic activity was not shown form 30 kDa protein. We suppose that the 68 kDa protein must have lost the activity after its post-translational process that gave rise to 30 kDa. Proteolytic activity appeared to be most stable in pH 7.0 and at the temperatures between 25 and 35°. The 68 kDa precursor protein was not inhibited by several serine protease inhibitors but was inhibited by EDTA, EGTA or L-cysteine. Therefore, we have concluded that the 68 kDa precursor protein with hemorrhagic activity is a metalloprotease, that can be effected by Ca2+ , Mn2+ , Sr2+ and Zn2+ ions. 246 INHIBITION OF THE MYOTOXIC AND EDEMATOGENIC ACTIVITY OF CRUDE BOTHROPS JARARACUSSU VENOM AND OF THE BthTX-I AND II TOXINS BY ROSMARINIC ACID ISOLATED FROM THE PLANT CORDIA VERBENACEA (BORAGINACEAE). F.K. Ticli 1 , A.M. Soares 2 , P.S. Pereira 2 , S.V. Sampaio 1 . Laboratório de Toxinologia, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto - SP. Brasil, 2 Unidade de Biotecnologia, Universidade de Ribeirão Preto, Ribeirão Preto - SP. Brasil 1 The venoms of snakes of the genus Bothrops are characterized by the induction of several physiopathological effects such as edema, coagulation, myotoxicity and hemorrhage. The venom of the snake B. jararacussu (VBj) is known for its high myotoxic and edematogenic activity. The two major myotoxins isolated from this venom (BthTXI and II) induce muscle necrosis, edema and cytotoxicity. Extracts of the plant C. verbenacea are used in Brazilian folk medicine as healing and anti-inflammatory agents. Swiss mice (18–22 g) were used to assess the effect of rosmarinic acid (RA) isolated from this plant against the myotoxic and edematogenic action of the venom. Myotoxicity was induced by intramuscular injection (im) of 50 µg venom or BthTX-I or II into the right gastrocnemius muscle. Three hours after injection, blood samples were collected from the tail vein into heparinized capillaries and plasma was obtained by centrifugation. Treatment was performed with a mixture of venom or myotoxin solution plus RA at the concentrations of 50 µg (1:1, w/w) and 500 µg (1:10, w/w) RA per animal. The plasma obtained was used for creatine kinase (CK) determination using the CK-UV 20 kit from Sigma. Edema was induced by subplantar injection of 50 µg venom or BthTX-I or II into the hind paw. Paw measurements were performed at different times (0.5, 1, 2, 4 and 24 h) with a low pressure pachymeter. Treatment was performed with a mixture s69 of the venom or myotoxin solution plus RA at the concentration of 175 µg (1:3, 5; w/w) per animal. RA significantly inhibited the myotoxicity and edema induced by the BthTX-I and II myotoxins at all concentrations studied, but did not inhibit the myotoxicity or the edema induced by VBj. RA alone showed specificity for the inhibition of basic myotoxic PLA2 , proving to be a potent inhibitor of these enzymes which effectively participate in the envenoming caused by this snake species. 247 THE BLOCKING ACTIVITY OF DIFFERENT TOXINS AGAINST POTASSIUM CHANNELS Kv3.4 IN RLE CELLS Mehdi Saberi 1 , Edward Rowan 2 , Alan Harvey 2 . 1 Department of Pharmacology, Faculty of Medicine, Baghiyatollah (a.g) University of Medical Sciences, Tehran, Iran, P.O. Box 19568, Tehran, Iran; 2 Department of Physiology and Pharmacology, Strathclyde University, Glasgow, Scotland Voltage-gated k+ channels play an essential role in the production of action potential activity by excitable cells. The effects of toxins on this channel are not fully known. In this study the whole-cell patch clamping technique was used to investigate the action of a series of toxins on potassium channels Kv3.4 current in RLE cloned cells for these channels. The cells were grown in Williams E medium and after 6–8 days were suitable for patch clamping. A family of currents was recorded during voltage-clamp steps to various potentials applied from a holding potential of -60mv to 80 mv in 10 mv increments. Upon depolarization, all channels opened with a sigmoidal time course, reached to peak within a few tenths of milliseconds and then slowly inactivated. Bath application of tetraethyl ammonium (TEA) showed that the current of these channels is very sensitive to TEA and inhibited completely by 3mM. Also, 3, 4-diaminopyridine (DAP) with a similar action, at concentration of 25 µM and BgK at 30µM could fully block the Kv3.4 channels and consequently the current. The HMK toxin up to 10 µM could not completely inhibit the current. But toxins such as β-bugarotoxin, corotoxin, novel toxin, DIP and DPK even in high concentrations (up to 100 mM) had not any significant effect on the current of these channels. Comparison of chemical structures of these effective agents with other reported effective toxins such as blood depressing substances (BDS I and BDS II) show no homology between them, but specially the potency of 3, 4-DAP is comparable with these toxins. Although, the sensitivity of Kv3.4 channel to TEA is less than Kv1.1 or Kv1.1 channels, but our result showed that this channel is more sensitive to 3, 4-DAP in comparison to other K channels. More investigation is necessary to find more selective and potent inhibitor of Kv3.4 channels. 248 CHANGE IN DISTRIBUTION OF TETRODOTOXIN IN THE OVARY OF A MARINE PUFFERFISH TAKIFUGU VERMICULARIS DURING SPAWNING SEASON O. Arakawa 1 , K. Okada 2 , Y. Mahmud 2 , T. Takatani 1 , K. Kawatsu 3 , Y. Hamano 3 , T. Noguchi 4 . 1 Faculty of Fisheries, Nagasaki University, Nagasaki 852–8521, Japan, 2 Graduate School of Science and Technology, Nagasaki 852–8521, Japan, 3 Department of Food Microbiology, Osaka Prefectural Institute of Public Health, Osaka 537–0025, Japan, 4 Japan Frozen Foods Inspection Corporation, Minato, Tokyo 105–0012, Japan Micro-distribution of tetrodotoxin (TTX) in the ovary of a marine pufferfish Takifugu vermicularis (“nashifugu”) was investigated by means of a monoclonal antibody-based immunohistological technique. In the year round investigation, TTX was mainly recognized at yolk vesicles of oocytes in late peri nucleus stage and yolk vesicle stage, and at yolk granules in yolk granule stage-I and II from January to April. Entering into yolk granule stage-III, however, TTX antigen-antibody complex was hardly visible in the oocytes in May when the gonadosomatic index (GSI) of the puffer was measured to be the highest. After ovulation, no TTX antigen was recognized in the liberated oocytes. But, interestingly the cross-reaction between antigen and antibody appeared again in the oocytes which were remained in the ovary and denatured in June. After July, oocytes in s70 Poster Session P11. Clinical toxicology early peri nucleus stage became dominant, where no positive reaction of immunostain was observed. On the other hand, mouse assay of the puffer tissues showed that the toxin amount of ovary begun to increase from January, reached to its maximum level in May, and remained nearly static in June, which drastically declined in July. In case of liver, the toxin amount increased from May, and reached to its maximum level in July. The results suggest that TTX antigen was probably in binding form with protein in yolk granule stage-III, showing negative reaction with anti-TTX antibody. When the unovulated oocytes were denatured, they released TTX in the blood stream of puffer, and eventually the toxin was transferred to the liver from June to July. During this transitional time, blood plasma of the puffer reasonably showed the highest toxin concentration in enzyme-linked immunosorbent assay (ELISA). 249 IMMUNOENZYMATIC VISUALIZATION OF TETRODOTOXIN (TTX) IN THE SKIN CELLS OF A PUFFERFISH TETRAODON NIGROVIRIDIS Y. Mahmud 1 , O. Arakawa 2 , A. Ichinose 3 , M.B. Tanu 2 , T. Takatani 2 , K. Tsuruda 1 , K. Kawatsu 4 , Y. Hamano 4 , T. Noguchi 5 . 1 Graduate School of Science and Technology, Nagasaki University, Japan, 2 Faculty of Fisheries, Nagasaki University, Japan, 3 Institute of Tropical Medicine, Nagasaki University, Japan, 4 Department of Food Microbiology, Osaka Prefectural Institute of Public Health, Japan, 5 Japan Frozen Foods Inspection Corporation, Tokyo, Japan To explore the physiological role and toxigenesis of tetrodotoxin (TTX), recently we have initiated a comprehensive immunohistological study on TTX-bearers by means of a highly specific monoclonal antibody, and meanwhile on the basis of this approach intra-tissue distributions of TTX in some marine pufferfish and a Japanese newt have been investigated under light microscope. In continuation, the present attempt was made to investigate the micro-distributions of TTX in the skin of a brackishwater pufferfish Tetraodon nigroviridis (“midorifugu”) under light and transmission electron microscope. In light microscopy TTX antigen was visualized as brown color at undifferentiated basal cells and succiform cells. The succiform cells of the skin in brackishwater puffer are claimed to secret TTX for biological defense. In electron microscopy, TTX was detected as black dots of immunogolds in lysosomes of the basal cells. From the results, it can be inferred that TTX (with its carrier protein) from the blood stream of puffer is accumulated by the undifferentiated basal cells of the skin through diffusion, and subsequently it is taken to the lysosomes by phagocytosis. The lysosomal enzymes degrade carrier protein as foreign invader but they possibly cannot do it with TTX since TTX is a low molecular weight substance and characteristically quite stable at acid media. It possibly exists there through binding with internal constituent(s) of lysosomes. Eventually, succiform cells of the skin accumulate TTX through mitotic proliferation of basal cells for defensive purpose. To our knowledge, this is the first experimental effort to localize the intracellular distribution of TTX. 250 HISTOPATHOLOGICAL, HEMATOBIOCHEMICAL AND URINALYSIS CHANGES IN EXPERIMENTAL OAK) Quercus brantii) POISONING IN SHEEP M. Pourjafar, A. Derakhshanfar, H. Talebanfard. School of Veterinary Medicine, Shahrekord University, Shahrekord, Iran A large part of our country(Iran) such as Zagros Mountains is covered with oak forests. In the many parts of Iran, oak ration is used for livestock because of its cheapness and accessibility, and also because of bad economic condition. Acorn contains variable amount of tannins, so that selective toxicity in livestock is not impossible. It seems that the evaluation of oak poisoning is of considerable importance. In order to determine the oak poisoning, 9 female sheep of the same breed, about one year old, weighting 40±3 Kg and were examined during 20 days in this study. These sheep were divided into 2 groups: control group (3sheep) and treatment group (6 sheep). The mean amount of acorn consumed was 2.225 kg/day (26.25% of ration). This amount was added gradually increased (from 100 g. –2.5% of ration – on 0 day to 2 kg –50% of ration – on the 20th day of the study). The ration for control group consisted of dried hay. Laboratory tests were histopathological, CBC, blood biochemistry, and urine biochemistry. The samples were taken from both groups at 0 day, 10th and 20th days and for histopathological examinations samples were taken after autopsy .The test results were analyzed with “ paired student’s test”. In this study only fibrinogen on 10th day significantly increased and the other parameters didn’t show significant changes. The Blood Urea Nitrogen (BUN), the most important parameter, which highly increases in oak poisoning, based on the other researchers results. Histopathological changes in the kidneys and liver were very mild, So it seems that the use of this amount of acorn powder for sheep didn’t cause considerable problem. So farmers can apply these amounts in short periods of time in livestock (sheep) rations instead of expensive rations (such as grain concentrates) without significant poisoning risk, if it’s amount isn’t more than about 25.26% of daily ration. In another project, we are going to detect the relation between the amount of tannins in consumed oak with hematological, biochemical, urinalysis and histopathological changes in much more long time duration. P11 Clinical toxicology 251 FATAL POISONING BY AMANITA PHALLOIDES MUSHROOMS D. Chaparoska 1 , E. Masin 2 , N. Becarovski 1 , L. Melovska 1 . Department of Toxicology1 and Department of Nephrology2 , University Clinical Center, Skopje,R.Macedonia We report a family of Amanita phalloides poisoning after picking the mushrooms. Mushrooms poisoning from the genus Amanita is a medical emergency, with Amanita phalloides being the most common species. The typical simptoms of nausea, vomiting, abdominal pain, and diarrhea are nonspecific and can be mistaken for gastroenteritis. If not adequately treated, hepatic and renal failure may ensue within several days of ingestion. In a family, 2 adults (the parents) poisoned with Amanita phalloides are described with spectrum of clinical prsentations and outcomes to complete recovery, and 2 other patients with fulminant hepatic and renal failure. A specific antidote against the amanitin toxins is not available. Ancillary drugs, including penicillin G and silibinin, were used for detoxification, correction of blood- clothing deficiencies, and hepatic protection. We describe a 2 patients of a family with confirmed Amanita phalloides poisoning treated with hemoperfusion immediately after arrival in our hospital (72 hours after ingestion). The both patients die of acute renal failure. Survival of patients depended on the amount of ingested mushroom, on the early admission of patients to dialysis centre and on the beginning of extracorporeal treatment until the period of 24 hours after acute poisoning. The results, assessed using mortality (5overall) and frequency of complications, indicate that plasmapheresis is a safe and affective treatment for Amanita phalloides poisoning but the further investigations are needed, especially involving measurements of efficacy and the efficiency of toxin removal. 252 PROTHROMBINE TIME-USEFULL PROGNOSTIC MARKER IN AMANITA PHALLOIDES POISONING L. Petkovska, Z. Pereska, Dz. Naumovski, C. Bozinovska, D. Petrovski, F. Licoska, A. Babulovska. Clinic of Toxicology and Urgent internal medicine, Clinical Centre-Skopje, Macedonia Objectives: Mushroom poisonings caused by amatoxins are mostly lethal. The main toxicological effect is toxic hepatitis. Elevation of aminotransferases and lowering of plasma prothrombine are very sensitive markers of hepatocellular damage. In order to determine Poster Session P11. Clinical toxicology correlation between initial aminotransferases and PT values with severity of poisoning we evaluated biochemical changes in 12 pts with phalloid intoxications. Methods: We retrospectively evaluated medical histories of twelve patients with phalloid poisoning. Hospitaly treated in the Clinic of Toxicology and Urgent Internal Medicine last year. We compared the initial values of aminotransferases and PT in patients divided into two groups, according to the outcome. Four pts with lethal outcome (33.3%) compared to the second group of 8 patients (66.6%) whosurvived. Results: Increased levels of transaminases were noted after the second day, but highest values were detected approximately 5-th day. In the group with lethal outcome the average value of initial AST was 2052.75 (198–5366), and of ALT was 1445.75 (277–3330). In the second group the average initial value of AST was 327 (22–661), and ALT was 801(22–2421). Changes in PT were noted earlier and average initial values of PT was 56.5 (35.5–90.2) sec. in the first group, and 18.425 (10.8–35.5) in the second group. Conclusion: Aminotransferases and PT are important markers for clinical course of phalloid poisonings, but initial values of serum aminotransferases are not predictive as much as initial values of PT, which make it more useful prognostic parameter. 253 254 s71 LATE COMPLICATIONES OF CAUSTIC INJURIES F. Licoska, C. Bozinovska, J. Naumovski, B. Pavlovski, L. Petkovska, Z. Pereska, A. Cibisev. Clinic of toxicology and Urgent Internal Medicine, Skopje, Macedonia Caustic injuries after intentional or accidental ingestiones are serious clinical problem in our everyday work. Chemical burns of oesophageal and or stomach mucous are deep and penetrating in submucosal layers giving structural changes in the acute and later in the reconvalesent period. One of the late complications of these lifetreating poisonings is stenosis in the upper gastrointestinum. Most were female our 2 years material we noted from 144 treated patients, hospitaly treated at our Clinic. Most were female (96%), the rest (48%) male. With a suicidal intention were 130, rest were accidental after the acute phase in the later course of the postcorrosive treatment, all patients were radiologicaly investigated, with a contrast x-ray. In the analyzed period 8 patients developed stenosis that needed surgical treatment. From them pyloric stenosis developed at 5, antrio-pylorico at 2, oesophageal stenosis in the distal part is 1. Showing the number of patients that developed stenosis that needed surgical help we potentiate this problem that needs a multiexpert approach in their posthospital course. A CASE REPORT OF A STRYCHNINE INDUCED ACUTE POISONING AND ITS SUCCESSFUL Abdolkarim Pajoumand 1 , Nasser Jalali 1 , Shahin Shadnia 1,2 , Mahsa Moinosadat 1 . 1 -Poisoning Ward, Loghman Hakim Hospital, Faculty of Medicine, Shaheed Beheshti University of Medical Science, Tehran-Iran; 2 -Department of Toxicology, Faculty of Pharmacy, Tehran University of Medical Science, Tehran-Iran Objectives: Strychnine was used medicinally for analgesic, cathartics, analeptics and tonic effects in human previously. Because of the highly toxicity of strychnine, it’ s products were removed from medicinal uses. Today, it is used as a rodenticide and poison for dogs and etc. Strychnine-related poisoning is relatively rare occurrence. Strychnine is too toxic and does not have any antidote for it’ s poisoning treatment and the treatment is basically symptomatic and supportive. From this view, we reported a case of strychnine poisoning and it’ s successful treatment. Case Report: The patient is a 28-year-old man who was brought to our poisoning center 2 hours after intentional ingestion of dogs’ poison. At admission time, patient had respiratory distress and was agitated. He presented with generalized tonic- clonic seizures and hyperactivity. The patient treated with Intravenous Diazepam, Sodium Bicarbonate solution, Glucose Hypertonic 50% and oral administration of Charcoal and Sorbitol. Then he admitted in poisoning ward. Patient had generalized tonic-clonic seizure again, so in order to preventing from aspiration and also for maintenance the airway the patient intubated and for seizure control he received Thiopental infusion. For prevention from acute renal failure due to rabdomiolysis, the alkaline diuresis with Intravenous administration of Normal Saline Serum, Sodium Bicarbonate Solution and Furosemide injection was performed. For identification of poison, the sample was sent to toxicology laboratory of faculty of pharmacy of Tehran University of Medical Science and tested with mandeline reagent for strychnine. The result of test was positive. Two days after admission the patient presented with fever, leukocytosis and rise of serum creatinine. The alkaline diuresis was continued but in spite of treatment the urinary output decrease from 1900CC to 250CC in 4th day of hospitalization. Dialysis was performed and fluid therapy and alkaline diuresis were continued. After 30 days of hospitalization the urinary output increased and the patient discharged by a good condition from the hospital. Conclusion: Strychnine is a competitive antagonist of glycine receptors and it is a CNS stimulant. Death results from respiratory failure and subsequent cardiac arrest, hypertermia and severs metabolic acidosis. Prognosis is good if the patient can be supported well over the first 6 to 12 hours. The rabdomiolysis and acute renal failure due to strychnine poisoning must be considered. 255 CHILD POISONING WITH CAUSTIC CORROSIVES IN CROATIA 1997–2001 I. Vidić Štrac, A. Barišin. Croatian National Institute of Public Health, Department of Health ecology, Zagreb, Croatia Household chemicals are a heterogeneous group of products that may easily come within the reach of children. In view of their affecting health directly and for requiring complex treatment, these agents pose a major problem in accidents involving children. We did a survey among the parents of pre-school age children aimed to establish whether they can recognize the potential danger of domestic caustic corrosives. Between 1997 and 2001 the number of cases reported as poisoning with caustic corrosives (T 54; ICD-10) was monitored using individual notifications on patient statistical cards, as was the correlation with toxic effects of substances chiefly non medical as to source (T 51–65, ICD-10). This data was compared with WHO/HFA programme findings. Of the 5-year total affected by some caustic-corrosive poisoning, children aged 0–6 years accounted for 43,03%. WHO indicators showed Croatia still to be reporting fewer poisonings and injuries than the EU countries. As our survey indicates, around 30% of the parents, irrespective of their educational background, were unable to recognize the warnings signs for irritants and corrosives as a potential danger in household. This revealed the need for implementation of preventive measures. 256 TREATMENT OF MERCURY OXIDE INTOXICATION WITH N-ACETYL CYSTEINE D. Pasqualatto 1 , M. Pachón 2 , D. Rodríguez 2 , M. Albarrán 2 . 1 Servicio de Información de Medicamentos y Tóxicos, Facultad de Farmacia, Universidad Central de Venezuela;2 Hospital “Dr. M. Pérez Carreño ” Caracas, Venezuela A 30 year old male, who intentionally ingested, 13 days before, an unknown amount of mercury oxide and bromazepam 90 mg; inmediatly after ingestion he presented vomiting, diarrhea, abdominal pain, and 3 days after he had headache, anuria and fever; hemodialysis was started (3 times per week). At that time, the patient was referred to our hospital, on the physical examination he had oral ulcerations (treated by cautery), anuric, stomach and duodenal ulcerations, serum creatinine 15.5 mg/dl and BUN 84. After 24 h of treatment with N-acetyl cysteine (NAC) (Mucomyst-10. Lab. Brystol-Meyers-Squibb) he starting diuresis 24 h later. Blood and urine mercury levels, 14 days after ingestion were 543.75 µg/L and 64.3 µg/L, respectively. Hemoglobin and hematocrit decreased to 6.6 and 21.3%, respectively; requiring multiple transfusions Dialysis was performed until reached normal level of serum creatinine and BUN. Renal biopsy indicated interstitial nephritis. He received NAC s72 Poster Session P11. Clinical toxicology iv during 50 days and the same doses with oral therapy during 30 days, until finding acceptable urinary mercury levels. At the present, he has been studied for high arterial blood pressure (actually treated with amlodipine). He does not have any gastrointestinal, neurological or ophthalmic signs. 257 N-ACETYLCYSTEINE IN ZINC CHLORIDE POISONING D. Pasqualatto, M.C. Fernández. Servicio de Información de Medicamentos y Tóxicos. Facultad de Farmacia, Universidad Central de Venezuela, Caracas, Venezuela An emergency department called the poison control center regarding a 2 year-old boy (12 kg) who was found playing with an empty bottle of soldering flux (zinc chloride) 2 hours before. The child has vomited several times, he had eritema on abdominal skin and he was mildly depressed (somnolence). Eight hours post-ingestion, blood zinc levels were 831 ug/100 ml (normal 50–150 ug/100 ml), he started to receive N-acetyl cysteine (NAC) (300 mg/6 hours iv). The endoscopy revealed ulcerations in stomach. He received sucralfate and omeprazole. After 3 days with NAC, blood zinc level were 158 ug/ 100 ml. 258 ADVERSE EFFECTS OF INTRAVENOUS N-ACETYL CYSTEINE IN ACETAMINOPHEN POISONING TREATMENT N. Jalali 1 , F. Taghaddosinejad 2 , H. Sanaei Zadeh 2 , Sh. Shadnia 1 . center, Loghman-Hakim Hospital, Faculty of Medicine, Shaheed-Beheshti University of Medical Science, Tehran, Iran, 2 Department of Forensic Medicine, Faculty of Medicine, Tehran University of Medical Science, Tehran, Iran 1 Poison Acetaminophen poisoning is one of the most common poisoning in the world. N-acetyl cysteine (NAC) as a antidote for acetaminophen poisoning is a glutathione precursor, enhancing glutathione synthesis and increasing acetaminophen sulfation. It is used in oral and intravenous route. This study has been done as a cross-sectional research over adverse effects of intravenous administration of NAC during one year from 2000 to 2001 in adults who had ingested toxic dose of acetaminophen and had been referred to Loghman-Hakim Hospital Poison Center. Results of this study over target population including 206 patients, show that male to female ratio was 1 to 1.6. Of these 206 cases, 130 patients developed adverse effects after intravenous administration of NAC, including nausea and vomiting in 118 cases (57.2%) and anaphylactoid reactions in 48 cases (23.3%). Among the patients who developed nausea and vomiting, 77 cases were managed with a single intravenous dose of metoclopramid and among the patients with anaphylactoid reactions, 26 cases were managed with antihistaminic and corticosteroid therapy and the others were managed by decreasing in infusion rate or temporary discontinuation of NAC administration. In spite of continuation of NAC in all of the cases, mortality was not seen. In this study, according to the mild severity of adverse effects in the most cases and ability of controlling these effects, in spite of high prevalence of adverse effects, the authors recommended that intravenous NAC can be used as the first step in management of cases who ingested toxic dose of acetaminophen, after primary supportive care in all poisoning. Urgent Internal Medicine, Clinical Centre- Skopje, after ingestion of various amount of mercury chloride. Results: All of them showed caustic lesiones in the upper gastrointestinum from whom 4 (57%) represented life-threatening upper bleeding. 5 (71%) of them developed acute renal failure. 4 cases, (57%) were dyalised, in one case it was unnecessary. Antidothal therapy was applied in two patients only. The overall lethal outcome was 3 (42%). Conclusion: Showing all the manifested clinical features, therapy and follow up of these patients, we review the broad spectre of clinical impairments, notifying the need for on-time treatment, combined antidothal, active hemodialysis treatment and first of all to define the need for treatment of all patient even if, at the beginning they are only suspected for this form of poisoning. 260 C. Locatelli, V. Petrolini, R. Butera, G. Bernareggi, S. Arrigoni, M. Carli, L. Manzo. Pavia Poison Center, IRCCS Maugeri Foundation Hospital and University of Pavia, Pavia, Italy This report describes 6 cases of thallotoxicosis caused by the use of thallium (Tl) as a homicidal agent. A 50-year-old man came to the emergency department complaining of severe abdominal pain, chest tightness, and paresthesias in lower extremities. Poisoning was suspected when similar though less severe symptoms were seen in other five persons who had shared a meal with the patient three days before. Investigations revealed consumption of red wine contaminated with Tl for criminal purpose. The severity of symptoms (Table) was correlated in all cases with the amount of wine ingested excepting a young bulimic patient (pt. n. 6) who could decontaminate herself by early vomiting. Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Wine consumed (ml, estimated) Chest tightness Early onset paresthesias Constipation Hair loss Polyneuropathy Paralysis and respiratory failure Psychiatric symptoms CLINICAL COURSE IN ACUTE PERORALY MERCURY SALTS D. Petrovski, Dz. Naumovski, Z. Pereska, L. Petkovska, A. Cibisev, A.E. Kovkarova, F. Licoska. Clinic of Toxicology and Urgent Internal Medicine, Clinic Centre- Skopje, Macedonia Objective: Mercury is naturally occuring element existing in multiple forms. Ingestion of mercury metal is usually without effect. Ingestion of inorganic salts may cause severe gastrointestinal irritation, renal failure, and death with acute lethal doses in humans ranging from 1 to 4 grams. Methods: In this matter we present the clinical material of 7 patients, hospitally treated at the Clinic of Toxicology and 300 + + + + + 170 + + + + + 80 + + + + – 80 + + +– + – 20 + + – – – 70 + + + + – – – – – – – – – – – – Neurotoxicity developed in the most severe case (pt. n. 1) as a distal, sensory-motor neuropathy accompanied by psychiatric disturbances including insomnia, anxiety, allucinatory and paranoid symptoms. Sensory and motor alterations were accentuated in the legs with intense pain, paresthesias progressive weakness, difficulty in walking and paralysis. Less severe cases showed distal sensory loss, muscle pain, signs of diminished muscle strength, and fatigability. Prussian Blue (PB) was given daily (250 mg/kg/day, p.o.) until normalisation of urinary Tl levels (0.3 mcg Tl/L). Despite PB therapy, elevated thalliuresis occurred in patients n. 1 and 2 up to 83 and 48 days post-ingestion, respectively. In these subjects, restitutio ad integrum was incomplete with electrophysiological changes persisting 21 months after exposure. In the remaining patients, complete recovery occurred within two months after poisoning. 261 259 LONG-LASTING POLYNEUROPATHY AND PSYCHIATRIC DISORDERS IN THALLIUM POISONING. STUDY OF SIX CASES INTERMEDIATE SYNDROME IN ORGANOPHOSPHOROUS POISONING, A CLINICAL STUDY TO CORRELATE IT’S PREVALENCE WITH RBC CHOLINESTERASE. A.H. Shah, A.R. Shah. New civil Hospital, Ahmedabad, India Intermediate syndrome of organophosphorous poisoning arises in the time between the acute cholinergic crisis of fasciculations, muscle weakness & delayed neuropathy. Objective: To estimate frequency of intermediate syndrome in organophosphorous poisoned patients & to examine it’s relation to RBC cholinesterase inhibition. Method: This was an 18 month prospective study in New Civil Hospital, Ahmedabad, India, which included 176 consecutive pa- Poster Session P11. Clinical toxicology tients. We divided patients in to three groups depending upon RBC cholinesterase on the presentation. (RBC cholinesterase of <500, 500–1000 and >1000). Out of 176 patients 82 (48.24%) were male and 94 (53.41%) were female. Most of the cases were suicidal attempt. Forty six (26.41%) patients developed intermediate syndrome. Twenty eight (54.90%) out of 51patients (RBC cholinesterase <500), 15 (57.69%) out of 26 patients (RBC cholinesterase 500–1000) and 3 (3.03%) out of 99 patients (RBC cholinesterase >1000) developed Intermediate syndrome. Duration of Intermediate syndrome was 5 days (RBC cholinesterase >1000), 9 days (RBC cholinesterase 500–1000) and 12 days (RBC cholinesterase <500). Most common findings were proximal muscle weakness, followed by neck flexors weakness, respiratory muscle weakness and motor cranial nerve paralysis. Time of onset was ranging from 8 to 72 hours. Conclusion: Intermediate syndrome is not rare and its’ onset and prevalence correlates with RBC cholinesterase. 262 muscles, which rapidly subsided as PAM administration was halted. There were no relapses of muscarinic symptoms, nor reduction in pChE or AChE levels in association with these neuromuscular changes. Serum pralidoxime levels ranged from 9 to 18 mcg/ml. Data suggesting a causal role of PAM in the observed neuromuscular changes include the delayed occurrence of these symptoms and their prompt resolution after drug discontinuation in all three patients. PAM neurotoxicity may be of special importance in massive OP poisoning. Pharmacokinetic studies have indicated that the distribution volume of PAM given as an antidote in patients with severe OP poisoning is about 10-times higher than in normal healthy volunteers, a condition reflecting increased drug accumulation in tissues. On this basis, PAM therapy could induce neuromuscular alterations even at moderate dosages in the presence of relatively low serum drug concentrations. This also suggests that measurement of serum PAM levels may not be sufficiently valuable in clinical setting to predict development of PAM-induced neurotoxicity. PAM neurotoxicity can be considered in differential diagnosis of neuromuscular disorders associated with OP poisoning. OTC - PROGNOSTIC PARAMETER IN ACUTE ORGANOPHOSPHATE POISONINGS Z. Pereska, L. Petkovska, Dz. Naumovski, C. Bozinovska, Gj. Pilovski, D. Petrovski, A. Babulovska. Clinic of toxicology and Urgent Internal Medicine, Clinical Center, Skopje, Maceodnia Organophosphate-based insecticides (OPI) are irreversibile inhibitors of acetylcholinesterase enzyme activity (ACHE). Clinical presentation of acute poisonings with OPI beside cholinergic syndrome includes also cardiotoxicity that indicates poor prognosis. The aim of this study is to estimate the correlation between ECG signs of myocardial repolarisation disturbances measured as QT interval prolongation corrected for HR (QTc) and a) the level of decrease of plasma (p) CHEA and b) the quantity of used antidotes (atropine) in the first 24 hours. The study include 36 patients acutely exposed to OPI, hospitaly treated at the Clinic of toxicology. The including criteria were 35–50% reduction of pCHEA levels at admission (range 1900– 3800U/l). p (CHEA) was determined by spectrofotometry method (Knedel and Butger). ECG performed at entrance was analyzed. The QTc was calculated by Bazzet’s formula. 450 msec was taken as critical value for QT prolongation for men and 470 msec for women. Patients were devided in 2 groups considering the QTc interval duration. First group (61.1% of pts) is consist of pts with prolonged QTc and second (38.4% of pts) with QTc duration under the critical values. Statistically, the correlation between the QTc interval duration and pCHEA for the first group of pts was positive and statistically significant (p< 0.05) and positive but statistically insignificant (p>0.05) in the second group. The correlation between QTc interval and the quantity of used antidotes (atropine) was statistically significant (p<0.05). Our results show that the OPI-induced cardiotoxicity is dose dependent and is not close correlated with pCHEA. The estimation of QTc interval prolongation can be prognostic parameter for the quantity of used antidotes and clinical course of these poisonings. 263 s73 SIDE EFFECTS OF ANTIDOTAL THERAPY WITH PRALIDOXIME IN HUMAN ORGANOPHOSPHATE POISONING C. Locatelli, R. Butera, V. Petrolini, A. Bove, J. Georgatos, A. Agazzi, L. Manzo. Poison Control Center, IRCCS Maugeri Foundation and University of Pavia, Pavia, Italy Although oximes are generally regarded as the antidote of choice in organophosphate (OP) poisoning, the safety profile of these agents has not been sufficiently defined. Experimental studies have indicated that large doses of pralidoxime (PAM) can induce reversible neuromuscular blockade. Similar effects in skeletal muscles are also produced by OPs as a consequence of their nicotinic action. In a study of 3 patients with severe OP poisoning, treatment with PAM given at doses up to 500 mg/hour was associated with occurrence of delayed neuromuscular changes suggestive for PAM-induced neurotoxicity. Six to 7 days after starting PAM therapy, the patients developed fasciculations and tremors involving pectoral, deltoid and respiratory 264 ELECTROCARDIOGRAPHIC CHANGES OBSERVED IN ACUTE INHALATORY IRRITATIVE POISONINGS J. Naumovski, Z. Pereska, L. Petkovska, C. Bozinovska, I. Naumov, E. Kovkarova. Clinic of Toxicology and Urgent Internal Medicine, Clinical Centre, Skopje, Macedonia Background: Irritant gases exert major effects on upper and lower respiratory tract mucosa. Typical clinical features include cough, labored breathing, and burning of eyes, nose and throat. These poisonings may cause cardiac manifestations, which are monitored less frequently. Material and methods: The study population included 67 poisoned patients, intoxicated with irritative fumes from household bleaches, detergents, and other cleaning/sanitizing agents. 57 patients (85%) were treated as outpatients. Subjective complaints and objective clinical features, as well as electrocardiography, roentgenology and laboratory findings, were recorded and analyzed. Results: At the time of presentation, electrocardiographic changes were detected in 42 patients (63%). The most frequent change observed was sinus tachycardia, which was seen in 37 (55%) of all patients. Unspecific changes of depolarization, such as ST segment and T wave changes were seen in 12 (18%) patients, while less frequent were premature beats, seen in 4 (6%) patients as supraventricular beats, and in 3 (4%) patients as ventricular ectopic beats. Some patients had single electrocardiographic changes, while some patients had a combination of 2 or more simultaneous changes. At the end of the treatment the electrocardiographic changes disappeared in all patients. Conclusion: Cleaning with sanitizing agents is a potential hazard for inhaling chlorine or ammonia gases, which, in poorly ventilated spaces, may cause intoxications. Cardiac complains are not frequent. However, it is important to monitor electrocardiographic changes in order to provide necessary treatment and prevent more serious disturbances of rhythm and conduction in older and patients with previous heart diseases. 265 ASTHMA AFTER EXPOSURE TO GLUTARALDEHYDE G.F. Desogus. Study Reports of Toxicology, Azienda USL 7 di Carbonia, Italy Epidemiological studies show a direct connection between asthma and concentration of chemical pollutants, that act as adjuvant of the immunological reactions, mediated by a first non specific infiammatory reaction. Some environmental pollutants promote the IgE-mediated allergic sensitization, for adjuvant effect and all this is a base condition because of the increased susceptibility to “react” to occupational allergens. The use of specific chemical disinfectants in hospital (particularly glutaraldehyde and formaldehyde) is linked to the appearance of allergic symtoms and also small doses can induce IgE-dependent reactions both at pulmonary and epithelial level. This work studies if occupational exposure to glutaraldehyde and formaldehyde is associated with an increase of asthma and respiratory s74 Poster Session P11. Clinical toxicology allergic pathologies. Twenty-eight exposed workers (group 1: age 42 ± 7 years, 8 males and 20 females) and thirteen non exposed (group 2: age 43 ± 7 years, 4 males and 9 females) are investigated to verify the presence of respiratory allergic pathologies correlated to the exposure of chemical disinfectants. In the first group, one worker (3,6%) is suffering from bronchial asthma and 14 workers (50%) from clinical symptoms correlated to occupational exposure of chemical disinfectants (allergy, hay fever, bronchitis), probably of poly-allergic nature, while in the second group, we haven’t noticed any specific pathologies. In particular, the pathologies reported in the exposed population are often coincident with the use of disinfectant substances in hospital and under working conditions characterized by indexes of environmental pollution. The exposed poly-allergic workers don’t show with the use of adequate procedures of job and of individual protective measures, even if there is a tendence to the normalization in coincidence with an improvement of some rino-sinu-bronchial symptoms. The difficulty of an efficacious stratification with the habits of life and the state of individual susceptibility and the short period of observation limit the statistic meaning, but these preliminary data require a widest experimentation, also with the contribution of new scientific reports about the occupational allergic pathologies. 266 COMPREHENSIVE SCREENING OF ABUSED DRUGS IN TRAFFIC ACCIDENTS AND TRAUMA VICTIMS 1 2 1 1 T. Söylemezoǧlu , B. Yücesan , Ş.Ş. Çeçen . Ankara University, Institute of Forensic Medicine, Dikimevi, Ankara, Turkey, 2 Ministry of Health, Ankara Education and Research Hospital, Dr. Nilgün Sönmez Acar Blood Bank, Cebeci, Ankara, Turkey Alcohol consumption and drug addiction, growing problem of world’s mostly industrialized and newly developing countries, are considered to be a major contributor to all types of trauma. In view of the importance of the problem and in the lack of local data the prevalence of drug abuse and alcohol consumption in trauma patients and relationship between drug use and severity of injury and demographics of each trauma patient were evaluated. Sera from 102 patients with trauma injuries, who were admitted to the emergency service were screened for blood alcohol by gas chromatography with flame ionization detector and for drugs of abuse with an enzymatic immunoassay method, CEDIA ® DAU. Patient ages ranged from 16 to 80 and above with a mean of 35. Of this population, the age-sex distribution was predominantly male (80%), and in the third decade of life (30–39 years). Motor-vehicle related injuries accounted for %52 of submitted patients. Of the 102 patients who received toxicological screening, 39.21% tested positive for alcohol+drug, whereas 35.2% of patients tested positive for alcohol only with BAC’s ranging from 25–300 mg/dl. The mean BAC for the alcohol positive patients was 143.87 mg/dl. Only 3.9% of patients were found to have drugs with abuse potential in their urine. The most common detected drugs were barbiturates and benzodiazepines, followed by opiates. Patients who were tested positive for alcohol and drug had low Glasgow coma scales and low trauma scores (7–11) with low probabibility of survival (12–71%). Our results demonstrate that while alcohol continues to be a major problem among trauma patients, drug use, especially barbiturates and benzodiazepines, in combination with alcohol may become a larger problem if spesific precautions are not taken immediately. 267 FATAL METHANOL POISONING A. Dip 1 , A. Demircan 2 , A. Keleş 2 , B. Demirel 3 , A.F. Işık 3 , T. Söylemezoǧlu 1 . 1 Inst. of Forensic Medicine, Ankara University, 2 Dept. of Emergency, Fac. of Medicine, Gazi University, Turkey Methyl alcohol is the simplest alcohol in structure, so it is used in antifreeze, solvents and dissolvents, and also as a chemical reagent in the production of many organic chemical compounds. Methanol is the poisonous type of alcohol, intoxication is fatal at high doses and this dose is variable in most cases. We present a case of methanol poisoning in a sibling who take cleansing solution with methanol ingredient as a beverage to get drunk. The woman was 60-year old, the man was 48-year-old and they are arrived to the hospital after about 18 h with various symptoms including blurred vision and unconsciousness. Metabolic acidosis had started at the arrival time with the serum pH levels of 6,83 and 6,85 respectively. When the patients had come, the blood methanol levels of the woman was 1,325 promil and the man’s was 3,965 promil. Although serum pH was turned to normal level after application of ethanol treatment and hemodialysis, two patients eventually died with extensive neuropathy. Deaths are reported at 45th and 66th hours after methanol intake. The vitrous humor sample of woman were measured as 0,530 and of man as 0,920 promil. Blood samples used in analysis were taken before and after ethanol treatment and vitrous humors were taken after death. Headspace gas chromatography was used to determine the methanol levels, propanol was used as internal standard and the results were given as promil. 268 LONG QT SYNDROME INDUCED BY OXATOMIDE OVERDOSE IN CHILDREN C. Locatelli 1 , V. Petrolini 1 , R. Butera 1 , D. Lonati 1 , S. Mannarino 2 , E. Stacul 3 , E. Fufi 3 , A. Valli 4 , P. Papa 4 , L. Manzo 1,5 . 1 Pavia Poison Center, IRCCS Fondazione Maugeri, Pavia; 2 Pediatric Cardiology Division, 3 Department of Pediatrics and 4 Laboratory of Clinical Toxicology, IRCCS Policlinico San Matteo, Pavia; 5 University of Pavia, Pavia, Italy Overdose of 2nd generation antihistamines, such as terfenadine and astemizole, has often been associated with ECG abnormalities such as prolongation of the QT interval. In this respect, no data are available on Oxatomide (Ox), an agent which is largely used in Italy (pediatric drops 2.5%) at the recommended dosage of 0.5 mg/kg bis in die. Accidental ingestion of Ox is not rare in children. From July 1998 to November 2002, we examined 12 patients (mean age: 42.4 months, range: 21 days-14 years) admitted to hospital for Ox overdose. Ten subjects had taken the drug in a single dose ranging from 1.6 to 30 mg/kg; two other patients had been repeatedly treated with Ox at doses higher than those recommended in children. Serum Ox levels measured in 8/12 patients by HPLC were 105–1300 ng/ml (normal values: 20–40 ng/ml). The plasma levels were retested till normalization in 6/8 patients. ECG was recorded on admission and before discharge with repeated recordings in the patients showing altered ECG. A total of 5/12 (41.6%) children developed QTc prolongation (447–639 msec). In this group, 2 patients had ingested a single high dose of Ox and 1 patient had repeated overdosage. In one patient showing very high serum concentrations (400 ng Ox/ml) despite moderate drug overdosage (3 mg/kg), Ox had been coadministered with erythromycin. The maximum QT prolongation was found in a 3-week old patient following ingestion of 6.9 mg/kg Ox. Four patients in the group with drug-induced ECG abnormalities showed normal QTc at discharge. These observations indicate that oxatomide poisoning can prolong QT interval in children. 269 APPLICATION OF COTININE DETERMINATION FOR EVALUATION OF EFFECTIVENESS OF BUPROPIONE THERAPY D. Zuba 1 , E. Florek 2 , W. Piekoszewski 1,3 , E. Gomółka 3 , A. Kamenczak 4 , B. Jenner 3 . 1 Institute of Forensic Research, Krakow, Poland, 2 Laboratoryof Environmental Research, Department of Toxicology, University of Medical Sciences, Poznan, Poland, 3 Department of Clinical and Industrial Toxicology, Jagiellonian University, Krakow, Poland, 4 Toxicology Clinic, Jagiellonian University, Krakow, Poland In pharmacological therapy of tobacco smoke dependence the two approaches are used, the first is nicotine replace therapy and the second is administration of new antidepressive drug bupropione (Zyban). The aim of this study was evaluation of the effectiveness of bupropione (Zyban) therapy just after cessation of bupropione administration and one year after therapy. In the clinical experiment 82 volunteers (41 female and 48 male) took part. The drug was administrated during seven weeks according producer schedule of administration. The effectiveness of therapy was checked by determination of cotinine level in urine. For cotinine determination high performance liquid chromatography was applied. Poster Session P12. Drug toxicology The mean value of cotinine concentration before therapy was 2117 ng/ml and after seven weeks the concentration dropped to 540±714 ng/ml. In the studied group in the final time of therapy 61% of patients quit smoking and 24% reduced smoking by half. After one year only 21% of patients still do not smoke and 22% smoked near half of the number of cigarettes compared with the period before therapy. 270 TOXICITY AND SEIZURE CAUSED BY INJECTION OF LIDOCAINE Ebrahim Nasiri, H. Samadae. Faculty member, Mazandaran University of medical sciences, Sari, Iran Introduction: Lidocaine as an anesthetic drug has a common use in otolaryngology and regional anesthesia. Plasma absorption of lidocaine in subcutaneous injection with adrenaline is reduced by %50, and injection of more than 10 mg /kg of body weight may lead to convulsion. Tatum in 1927, for the first time, showed the brain stimulatory property of such anesthetic drugs. There are reports indicating the prevalence injection of anesthetic drugs about one in 6000 cases, which different conditions of the patients affect it. The main factor of seizure, is the high blood concentration of the drug, which inhibits the synaptic inhibitors present on brain cortex, and it blockage would cause stimulation of brain and the result is seizure. Material and Methods: For in this study, the relevant articles were reviewed and controversial reports on the side effects, such as toxicity of anesthetic drugs were studied. This study is about a case of seizure and apnea, due to injection of lidocaine in septorhinoplasty surgery in a 20 years girl of 60kg body weight under neuroleptic analgesia15 ml of 2% lidocaine with 1/100,000 adrenaline, within 4 minutes was injected and infiltrated nose region. Results: 40 seconds after injection, the symptoms of seizure were severe and apnea was observed, treatment included 100% oxygen therapy with PPV and stopping operation, followed by intravenous injection of sodium thiopental. The patient was under complete monitoring and care, transferred Intensive Care Unite, and was under intensive care and treatment till complete recovery. Conclusion: We found that, subcutaneous and submucosal injection of lidocaine with adrenaline in high vessels of nose even with less than amount indicated in different reports, may lead to complications such as seizure and apnea. Hence complete alertness of operation team in case of local and regional application of anesthetic drug is required. 271 DRUG-INDUCED COMAS C. Bozinovska, Z. Pereska, J. Naumovski, L. Petkovska, N. Popovski, A. Babulovska. Clinic of toxicology and urgent internal medicine, Clinical center, Skopje, Macedonia Objective: The aim of the study is to evaluate frequency, sex distribution, most frequently used agents and lethality of druginduced coma (DC) Material and methods: Dates from the medical histories of patients (pts) acutely poisoned with drugs and hospitaly treated at the Clinic of toxicology were analyzed. In a 5 years period, total number of hospitaly treated poisoned pts was 1982. 1045 (52.7%) of them were drug-induced intoxications and 94 pts (8.99%) of them were in the state of coma. Within the total number of 133 pts admitted in poisoning induced-coma, drug-induced coma with 94 pts participate with 70.7%. The group of DC includes 28 males (29.7%) and 66 female (70.3%) pts. Considering the etiologic agents, patients were divided in 2 groups: first group included 41 (43.6%) one-drug poisoned pts and the second one 53 (56.3%) pts poisoned with two or more different drugs. The most frequently used drugs in the first group were benzodiazepines (18 pts or 43.9%) and in the second group it was the combination of benzodiazepines and TCA (22 pts or 41.5%). Lethal outcome had 8 pts (8.51%) in DC. In this period, DC participated with 16% in the total lethal outcomes. Conclusion: Drugs are very often etiology agents that induce comas in the comatose pts admitted at our Clinic. Benzodiazepines and TCA are more often cause of DC. Females and mixed intoxications have higher frequency of appearance. But, they are with smaller s75 lethal outcome and better prognosis than the comas induced by other agents 272 A TOXIC EVENT SURVEILLANCE SYSTEM IN THE EMERGENCY DEPARTMENT: A USEFUL TOOL TO ASSES ACUTE CHEMICAL RISK IN HUMANS A. Ferrer Dufol 1 , S. Nogué Xarau 2 , R. Royo Hernandez 1 , E. Civeira Murillo 1 , F. Vargas Marcos 3 , O. Castillo Soria 3 . And the members of the Toxic Surveillance System Program. 1 Unit of Toxicology, University Clinic Hospital, Zaragoza, Spain, 2 Toxicology Unit, Clinic Hospital, Barcelona, Spain, 3 Ministry of Healt,. Madrid Spain Objective: To maintain an updated profile of the toxic incidents caused by chemical products that reach the Emergency Departments of Spanish Hospitals, in the frame of a collaborative program developed by the Health Ministry ant the section of Clinical Toxicology of the Spanish Association of Toxicology since 1999. Methods: Data are submitted by members of the emergency department staff of the participant hospitals. The clinical data for each patient include: sex, age, symptoms, treatment and outcome and product identification, exposure cause, exposure place and exposure route. We present here the results of the first 4 years of the program. Results: We have got 19 participant hospitals, which have reported a total of 2174 cases. Admission has been required in 613 cases (28%). Mean age is 37 years. Males represent 50,8 % and females 49,2%. Reason for the exposure has been domestic accidents in 1425 cases (65,5%), suicidal in 290 cases (13,3 %), occupational 345 (15,9%), other 80 (3,7%), criminal 1 (0,05%) and unknown in 11 cases (0,5%). The main families of chemical compounds have been classified in: gases 765 cases (35,3%), caustics 669 cases (30,7%), solvents 184 cases (8,5%) and detergents 176 cases (8,1%), pesticides 247 (11,4%), metals 14 (0,6%), other 115 (5,3%). The most frequent individual agent is CO (419 cases) followed by domestic bleach (415 cases). The route of exposure has been oral in 890 cases, respiratory in 809 cases, cutaneous in 118 cases and ocular in 339 cases, some of them associated. 1887 cases have had some symptoms: neurologic 481, respiratory 490, digestive 676, cutaneous 89 and ocular 321, renal 4, cardiovascular 39. Some treatment has been used in 1828 cases: gastric decontamination in 225, cutaneous or ocular decontamination in 224, antidotes in 471, enhanced elimination in 25 and symptomatic measures in 1269 cases. Mean time in hospital has been around 39 hours. There have been 39 deaths, caused by methanol (5), paraquat and other pesticides (17), HCl (11) and CO (4). Most of the non lethal cases have had a good outcome with a few minor sequels. Conclusion: This program is useful to maintain an updated profile of poisoning by chemical products. The data show a homogeneity along the years that allows identifying the most dangerous compounds and families and contributing to develop preventive strategies to avoid the most frequent or dangerous exposures. P12 Drug toxicology 273 ASSESSMENT OF LABRASOL® /LABRAFIL® /TRANSCUTOL® (4/4/2, V/V/V) AS A VEHICLE FOR HYDROPHOBIC COMPOUNDS AFTER 4-WEEK ORAL TOXICITY STUDY IN WISTAR RATS C. Spire 1 , J.F. Lepage 2 , G. Vermeil de Conchard 1 , A. Beamonte 1 , H. Bertheux 1 , F. Goldfain-Blanc 1 , J.L. Delongeas 1 , N. Claude 2 . 1 Drug Safety Assessment, Servier, Orléans-Gidy, France, 2 Institut de Recherches Internationales Servier, Courbevoie, France Labrasol® , Labrafil® and Transcutol® are excipients used as bioavailability enhancers and solubilizers for hydrophobic compounds. Labrasol® and Labrafil® are mixtures of mono-, di- and triglycerides with mono- and diesters of polyethylene glycol and fatty acids, and Transcutol® is a diethylene glycol monoethyl ether. As the safety profile of blend Labrasol® /Labrafil® /Transcutol® (4/4/2, v/v/v) [L/L/T] is not well documented, L/L/T was tested daily for 4 weeks by oral route in Wistar rats (10 rats/sex/group) at dose volumes of 5, 10 s76 Poster Session P12. Drug toxicology and 20 ml/kg and compared to controls given 20 ml/kg of 1% (w/v) hydroxyethylcellulose in purified water. Clinical signs, bodyweight, feed and water intake, haematology, clinical chemistry, urinalysis, ophthalmology, organ weights, macroscopic and histomorphologic examinations and evaluation of hepatic CYP450, were evaluated. L/L/T was broadly well tolerated at 5 ml/kg and was lethal at 20 ml/kg. Changes in appearance and behaviour were observed from 10 ml/kg with dose-related incidence, severity and duration. Reduced feed intake were observed from 5 ml/kg (females) or 10 ml/kg (males), resulting in low bodyweights for high dose males only (-20% of controls). There was a dose-related induction of hepatic CYP 1A1/2, 2B1/2 and/or 2E1 subfamilies from 5 ml/kg, with high liver weight, centrilobular hepatocellular hypertrophy and high ALAT, triglyceride and cholesterol serum values at 20 ml/kg. Renal cell damages (granular material in glomerular spaces, crystal deposits in the medulla and tubular alterations), associated with proteinuria and calcium oxalate crystalluria, were observed at 20 ml/kg and consistent with the ethylene glycol toxicity. In addition, vacuolations in the adrenal cortex, with a sex-dependant localization, were found at the high dose-volume. According to these results, 5 ml/kg was considered as an acceptable volume for further use of L/L/T as a vehicle for hydrophobic drugs in rat toxicity studies. Higher volumes were associated with renal and adrenal changes, likely related to ethylene glycol toxicity, as well as hepatic enzyme induction. 274 IMPLICATION OF THE NITROGEN MONOXIDE SYSTEM IN ANTI-ATHEROSCLEROTIC POTENTIAL OF LACIDIPINE IN THE APOE-DEFICIENT MICE P. Cristofori 1 , A. Lanzoni 1 , D. Spagnolo 1 , V. Zantedeschi, M. Andreoli 2 , F. Crespi 2 . 1 Safety Assessment Dept. Pathology Research Centre Glaxo Smith Kline Verona, Italy; 2 Biology Dept.Psychiatry C.E.D.D.- Research Centre Glaxo Smith Kline Verona, Italy Nitrogen monoxide (NO) is a highly reactive molecule widely distributed throughout the body. Biologically NO was first characterised in 1987 as endothelial derived relaxing factor. Dihidropyridines (DHPs) such as amlodipine, lercanidipine and lacidipine, are compounds capable of vascular protection via their calcium antagonist activity. Lacidipine that is a clinically active antihypertensive calcium antagonist is also capable of vascular protection when administered (prophylactically and therapeutically) at non-sustained anti-hypertensive doses to salt sensitive Dahl-S rats or to Apo-E mice. Recent works have suggested that DHPs modulate vascular relaxation via increase in the release of nitrogen monoxide (NO). Microdialysis experiments have shown that NO metabolites (nitrites and nitrates) can be monitored in vivo in rat treated with N-methyl-D-aspartate (NMDA). By means of the electrochemical method of voltammetry applied with Nafion and ortho-phenylenediamine (oPD) coated carbon fiber micro-electrodes (mCFE, 30µm diameter, 3 mm length) used as biosensor we have recently demonstrated that NMDA stimulated release of NO can be monitored in vivo in real time in rat striatum. Furthermore, the same method has been implemented for measurement of substance P (endothelial NO synthase, eNOS, activator) stimulated release of NO in rat aortic rings. It is known that E (apoE)-deficient (apoE−/− ) mice show progressively complex and widespread lesions that closely resemble the inflammatory-fibrous plaques seen in humans. The present study investigated the anti-atherosclerotic potential of lacidipine in the apoE-deficient mice fed a Western type diet and treated for 8 weeks with either placebo or lacidipine (1, 3 or 10 mg/kg/day) given by gavage. In parallel to histological studies of putative atherosclerotic lesions in the aorta of such mice, ex vivo electrochemical analysis of voltammetric levels of NO have been performed in order to study the implication of such system involved in oxidative processes. In particular, the interaction between lacidipine and NO system was investigated. Functional studies were also performed to analyse the efficacy of lacidipine on maintaining vascular properties. 275 TOXICOLOGICAL EVALUATION OF A NOVEL ACAT INHIBITOR VULM 1457 M. Zemánek 1 , I. Sadloňová 2 , E. Ujházy 3 , M. Dubovický 3 , V. Faberová 2 , Š. Bezek 1 . 1 Slovakofarma, JSC., Hlohovec, 2 Drug Research Institute, JSC., Modra, 3 Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovakia Hypercholesterolemia, and the associated atherosclerotic process, is a primary risk factor for initiation and development of coronary heart disease. The key process of cholesteryl ester accumulation may be prevented by inhibition of acyl-CoA: cholesterol acyltransferase (ACAT) activity. The main objective of this study concerning safety evaluation was to examine toxic effects of a novel ACAT inhibitor, VULM 1457, an agent with hypocholesterolemic and antiatherosclerotic properties. Acute toxicity (mice and rats), 28-day toxicity (rats and rabbits) and reproduction/development toxicity (rats) studies were performed. Acute toxicity: Oral administration at the dose of 1000 mg/kg did not reveal any clinical symptoms of toxicity, abnormal behavior or mortality of animals. No macroscopic alterations in any of the organs and tissues inspected were found. Intraperitoneal administration at the dose of 350 mg/kg resulted in decreased mobility up to recumbent position, dyspnea and abdominal convulsions. However, these toxic symptoms disappeared within two days. 28-day toxicity: Oral administration at the dose of 600 mg/kg/day in male rats caused a significant decrease of red blood cells, followed by a decrease of hemoglobin and hematocrit. There was no comparable effect found in female rats. Oral administration at the doses 30, 150 and 300 mg/kg/day in rabbits did not exert any effect on biochemical variables determined. There was no toxic effect on adrenals. Reproduction/development toxicity: Oral administration at the doses 30, 120 and 300 mg/kg in rats did not disclose any adverse effect on conception, course of pregnancy, delivery and on development of pups studied during the first four days of lactation. Macroscopic investigation revealed no malformations in pups in either dose group. In conclusion, toxicological evaluation revealed a subtle unfavorable effect of VULM 1457 at the highest dose only in male rats. 276 SEVEN-DAY REPEATED-DOSE ORAL COMPARATIVE METABOLISM STUDY WITH THREE ANTIPSYCHOTIC AGENTS G. Szũcs, M. Albert, D. Dányi. Department of Toxicology, EGIS Pharmaceuticals Ltd., Budapest, Hungary The available literature suggests that antipsychotic agents can induce obesity in patients and impair glucose homeostasis and lipid milieu. Although the mechanisms are poorly understood, clinical experience suggests that these adverse effects are major areas of concern in the antipsychotic drug development. The reproducibility of these clinical findings in laboratory animals is understudied. The aim of our study was to investigate the influence of a new antipsychotic drug candidate (EGIS-11150) on the metabolism of Wistar rats compared with the effect of two well-known agents (risperidone and olanzapine). All three compounds were given by the oral route to groups of 10 female rats in multiples of equipotent doses on rat conditioned avoidance response (CAR) test (1-, 5-, or 25-times the ED50 for 7 days). Each compound increased the body weight gain, although statistically significant changes could only be detected in the animals dosed with 0.5 and 2.5 mg/kg risperidone (one and five times the CAR ED50 ). The highest dose levels for each compound proved to be toxic, however, there were significant differences in toxicity. Dosing with EGIS-11150 affected the body weight gain of female rats least of all. The number of lipid droplets slightly decreased in the animals given the high dose of EGIS-11150 (17.5 mg/kg), risperidone 2.5 mg/kg, and 12.5 mg/kg, and olanzapine 11,0 mg/kg at the microscopic examination. Prominent decrease in the liver fat content was only seen in the dose group olanzapine 55 mg/kg. The experimental set-up (probably due to short duration) proved not to be sufficient to demonstrate the changes seen in patients with schizophrenia at clinical chemistry. Even an opposite correlation was observed; serum glucose and lipid (triglyceride, LDL, HDL, and total cholesterol) levels decreased versus the untreated controls or “0” value. Poster Session P12. Drug toxicology 277 EFFECT OF ACUTE HEROIN ON BASAL AND ELECTRICAL VAGAL STIMULATION ACID AND PEPSIN SECRETION IN RAT Nabavizadeh Rafsanjani Fatemeh, Najafi Ali, Esmaeili Farzaneh. Department of Physiology, Kerman University of Medical Sciences Opioid peptides and their receptors are present in the majority of body tissues including gastrointestinal tract. Heroin is one of the opioid derivative that abuse increasingly today. As by now there is no study on the effect of acute heroin administration on gastric acid and pepsin secretion. This study was design to define the effect of the acute heroin on basal and electrical vagal stimulation gastric acid and pepsin secretion in rat. At the time of experiment pure heroin (0.5 mg/kg, ip) was injected. After confirming the signs of heroin effect, animals were anesthetized with sodium thiopental (60mg/kg, ip). Then tracheostomy and laparatomy were done, gastric effluents were collected by Wash out technique in 15 minute intervals and total titrable acid was measured by acid titrator (W.Germany, DIN). The pepsin content was measured by Anson method. Vagal electrical stimulation was used to stimulate the secretion of acid and pepsin. The results of this experiment showed a significant increase in basal and vagotomized state of gastric acid secretion in group that received heroin in comparison to control group. Also in comparison to control group electrical stimulation of vagus nerve increased gastric acid secretion in animals that received heroin, but this was not significant. The basal and vagally stimulated secretion of pepsin was increased in heroin group in comparison to control group, but this was not significant. This study showed that basal and stimulated gastric acid and pepsin secretions increase after heroin administration in rat. 278 GLUCOSE UPTAKE BY THE NEURONAL CELL LINES, SH-SY5Y AND U-373 MG MAY BE MODULATED BY SELECTED NEUROLOGICAL DRUGS M. Mannerström, H. Tähti. Medical School, Department of Toxicology, University of Tampere, Tampere, Finland The brain uses glucose exclusively as its source of energy. Glucose transport across the cell membrane is the first step of its utilization. Any modifications in the glucose transport capacity may have a great impact on the function of the brain. Many neurological drugs contribute to changes in glucose metabolism. However, there is not much data about their action mechanisms in detail In the present study, glucose uptake was studied in the neuronal cell lines neuroblastoma SH-SY5Y (undifferentiated, and differentiated with retinoic acid and 12-O-tetradecanoyl phorbol 13-acetate) and astrocytoma U-373 Mg. Tracer methods were used. The effect of selected neurological drugs, amtriptyline, selegiline, carbamazepin and phenytoin, on glucose uptake was evaluated in order to shed light on the possible mechanisms of how neurological drugs might affect glucose homeostasis. In addition, the aim was to evaluate whether these drugs affect cell function (glucose uptake) without affecting cell viability (causing cell death). Therefore the effect of these drugs on cell viability was studied using luminescence-based ATP-measurement. Differentiated SH-SY5Y-cells showed better glucose uptake than undifferentiated SH-SY5Y-cells. The drug concentrations used did not affect cell viability in any of the cell cultures used, as indicated by ATP-measurement. Instead, the drugs affected glucose uptake in different ways depending on factors such as differentiation of cells, drug exposure time and age of the cell culture. The tendency was that in differentiated SH-SY5Y cells the drugs mainly enhanced glucose uptake, while in undifferentiated cells more variability was detected. In U-373 Mg cells carbamazepin enhanced glucose uptake. Neuronal cell lines are sensitive and more studies are needed to optimize culturing conditions for experimental set ups designed for clarifying drug effects on different functions, such as glucose uptake. This study was supported by Tekes, the National Technology Agency 279 s77 TERATOGENIC EFFECTS OF DIAZEPAM INTAKE DURING PREGNANCY TO CLEFT PALATE & CLEFT LIP N. Takzaree, K. Yarmohammadi, A.R. Takzaree, A. Bakhtiarian. Dept. of Embryology, Tehran University of Medical Sciences, Faculty of Medicine, Tehran, Iran Diazepam is nonsedative that belongs to Benzodiazepines. It has been increasingly used recently. It should be noticed that Diazepam consumption during pregnancy might have teratogenic effects on embryo. Pregnant women use this drug in pregnancy pica, short sleeping or necessarily in psychological and neurological disease. So in this research we have studied Diazepam intake during pregnancy and its side effects leading to cleft lip, cleft palate and anopsia. In our study the virgin rats of known age weight have been selected. After being pregnant they were divided in three groups: Control group: 10 rats (injection of sterile water) First case group: 10 rats (sterile water and Diazepam 3 mg/kg/day Second case group: 10 rats (sterile water and Diazepam 8 mg/kg/day). These three groups took the drugs daily, after embryonic period pregnant rats have been killed and their embryos have been divided also in the same three groups. After being studied macroscopically the embryos were observed microscopically. This showed that some anomalies have been appeared in some cases. After analyzing there were significant differences between case and control groups (P value > 0.05). So it was proved that Diazepam is teratogen and is dangerous for pregnant women. 280 SAFETY ASSESSMENT OF CARGLUMIC ACID IN JUVENILE RATS R. Forster 1 , G. Chevalier 1 , M. Attia 1 , L. Martin 2 , M.-C. Fortun 2 . 1 CIT, Evreux, France and 2 Orphan Europe, Paris, France Carglumic acid (N-carbamoyl-L-glutamic acid) is currently the only specific drug therapy for N-acetylglutamate (NAG) synthase deficiency, a very rare congenital disorder of the urea cycle. Sufferers from this condition are unable to eliminate nitrogen which accumulates as ammonia in the blood, with consequent harmful effects, in particular for the brain, and rapid lethal course in most cases. Carglumic acid can substitute for the lacking NAG in activating the first enzyme of the urea cycle (carbamoyl phosphate synthetase) and thus re-establishing nitrogen elimination. As part of a package of regulatory studies to support registration of the pharmaceutical grade carglumic acid supplied by Orphan Europe as an orphan drug, a chronic toxicity study was performed in rats. Since childhood therapy is often required, the study included an evaluation of growth and development during the juvenile period. Carglumic acid was administered daily to rats from age 4 weeks for a period of 6 months. In addition to standard parameters (body weight, clinical observations, ophthalmology, haematology, blood biochemistry, urinalysis and histopathology) the study included evaluation of the development of bone (by DXA in vivo and ex-vivo), teeth and body length. Drug levels in plasma and urine samples were determined in order to investigate systemic exposure and elimination of the compound. On completion of 26 weeks treatment, potential effects on the immune system were assessed through histopathology of lymphoid organs and lymphocyte subset determinations. Cell proliferation in selected tissues was quantified by PCNA staining (kidneys, liver and testes). Potential effects on reproductive functions were evaluated in males (by mating trial with untreated females and seminology) and in females (estrus cycle). Treatment with carglumic acid was well tolerated at the selected dose-levels, which represents appropriate multiples of the clinical long-term treatment-levels, supporting the safety of the proposed clinical use in NAGS deficiency. 281 INFLUENCE OF VERAPAMIL ON THE ANALGESIC EFFECT AND TOXICITY OF INDOMETHACINE L. Tantcheva, E. Stoeva, M. Nikolov. Department of Drug Toxicology, Institute of Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria The effect of verapamil on the general and specific toxicity of indomethacine in mice was studied. Verapamil (Ver) in single oral s78 Poster Session P12. Drug toxicology dose (20 mg/kg) did not increased general indomethacine (Imc) toxicity (mortality rate) and decreased Imc-analgesic effect (hote plate and acetic acid test). Ver protected against the hepatotoxic effect of Imc (risen glutamate-pyruvate transaminase activity in blood plasma was normalized). Although Ver alone decreased significantly the alkaline content in the blood plasma, when it was applied in combination with Imc this parameter was normalized. The bleeding time also was not affected by the combination of Ver+Imc. Strong ulceroprotective effect of Ver was established in Imc-treated animals. The possible mechanisms of ulceroprotective effect of Ver as a modulator of Ca2+ channels, as an antistressor agent or a stimulant of prostaglandin E-synthesis were discussed. 282 EFFECTS OF NIFEDIPINE ON HISTOPATHOLOGICAL CHANGES OF KIDNEY IN DIABETIC RAT H. Mahdavinasab 1 , H. Mehrani 2 , H. Imani 1 , H. Sadrai 1 , H. Dashtnavard 1 , M. Mofid 1 , M. Ahmadian 1 . 1 Department of Anatomy, Faculty of Medicine, Baghyiatallah University, Tehran-Iran; 2 Department of Biochemistry, Faculty of Medicine, Baghyiatallah University, Tehran-Iran Diabetic nephropathy is the most common renal disease, which is complicated by another form of glumerular disease. The most characteristics lesion of diabetic glomerulonephropathy is nodular intercapillary glumerulosclerosis. In this study we designed three groups: 1) Diabetic group that diabetes was induced by sterptozotocin. 2) Diabetic + Nifedipine group that after 7 day of diabetes were orally treated daily with 40 mg/kg nifedipine for 4 month. 3) Control group were received water alone. For histopathological study all group rats were sacrificed and their kidneys were taken, fixed, dehydrated, embedded in paraffin, and sectioned at 5 micron from different area. The sections were then stained with H&E technique. The results of this study showed that internal diameter of collecting tubules were significantly decreased both in Diabetic and Diabetic + Nifedipine groups when compared to control group (respectively P<0.01 and P<0.05). Diffuse nodular glumerulosclerosis, acute tubular necrosis, intranuclear inclusions and some large and acellular nodules were also significantly increased in Diabetic and Diabetic + Nifedipine groups when compared to control group (respectively P<0.01 and P<0.05). In conclusion: 1) Diabetes could induce glumerulonephropathy in rats. 2) Nifedipine as a calcium blocker could reduce glumerulonephropathy in diabetic Rats. 283 EVALUATION OF ANTISECRETORY EFFECT OF GLYCERYL TRINITRATE IN RATS TREATED WITH CYCLOOXYGENASE NON-SELECTIVE AND SELECTIVE NSAIDs R. Velev 1 , S. Dobrić 2 , V. Ćupić 3 , Z. Milovanović 2 , D. Bokonjić 2 . 1 Faculty of Veterinary Medicine, Lazar Pop Traajkov 5–7, 1000 Skopje, Republic of Macedonia; 2 National Poison Control Center, Military Medical Academy, Belgrade, Yugoslavia and; 3 Faculty of Veterinary Medicine, Belgrade, Yugoslavia Indomethacin (IND) and nimesulide (NIM) are very potent nonsteroidal anti-inflammatory drugs (NSAIDs). The first one is cyclooxygenase (COX) non-selective NSAID, while the second belongs to the group of COX-2 selective inhibitors. Our earlier investigations showed that both drugs produced dose-related gastrotoxic effects after single administration, but they were significantly more pronounced in rats given IND than NIM. Recently it was demonstrated that nitric oxide (NO) may play an important role in gastric mucosal defense. The aim of this study was to evaluate the influence of glyceryl trinitrate (GTN), NO-generating compound, on secretion and total acid output of gastric juice in rats treated by IND and NIM. Adult male Wistar rats deprived of food for 24h and pylorus-ligated were used in the experiment. NSAIDs tested were given by gastric tube in a single dose of 25 mg/kg. GTN in gastroprotective dose of 6.25 mg/kg and 0.78 mg/kg was given p.o. or i.p. immediately after IND or NIM, respectively. In a separate group of experiments L-arginine (100 mg/kg i.p.), as an endogenous NO-donor, was given alone or concomitantly with L-NAME (1mg/kg i.p.), an inhibitor of NO-synthase, immediately after indomethacin. Four hours after administration of NSAIDs the animals were sacrificed, gastric juice was collected and its acidity was determined by titration with 0.1 N NaOH by using phenolphtaleine as an indicator. In rats given IND, but not NIM significant increase of gastric acidity was found, without significant changes in the volume of gastric juice. Treatment by GTN, regardless the route of administration normalised acidity of gastric juice without influence on its volume in rats treated with IND. L-arginine, like GTN normalised acidity of gastric juice in rats treated by IND, while L-NAME completely abolished this effect of L-arginine. Our results suggest that an increase in gastric acidity caused by IND could at least partly be responsible for its high gastrotoxic potential. Mechanism of antiulcer activity of GTN, among the other ones, could include NO-mediated processes influencing the gastric acidity. 284 BENZAMIDE-BASED TRICHOSTATIN A ANALOGUES ARE POTENT AND METABOLICALLY STABLE INHIBITORS OF HISTONE DEACETYLASE G. Elaut 1 , G. Laus 2 , P. Papeleu 1 , V. Breckx 2 , J. Van Hemel 2 , M. Erra 3 , G. Brosch 3 , S. Snykers 1 , T. Vanhaecke 1 , D. Tourwé 2 , V. Rogiers 1 . Departments of Toxicology1 and Organic Chemistry2 , Vrije Universiteit Brussel, Brussels, Belgium, 3 Department of Microbiology, University of Innsbruck, Innsbruck, Austria Histone deacetylase (HDAC) inhibitors show great therapeutic potential for the treatment of diseases characterized by dedifferentiation and aberrant proliferation of mature cells, such as cancer. This stems from their ability to convert proliferating cells to a differentiated, non-proliferating phenotype through modulation of the eukaryotic chromatin structure, affecting DNA accessibility and gene expression. A number of hydroxamate-based HDAC inhibitors have been shown to inhibit tumor growth both in vitro and in vivo, and several of them are currently in clinical trial. Little is known, however, with respect to their pharmacokinetic and toxicologic properties, which are important determinants of their further success as a drug. In this context, the use of liver-derived in vitro models can provide valuable information on both biotransformation and drug-induced toxicity. They are of great value in the early discovery stage as well as throughout the rest of the drug development process. The natural compound, Trichostatin A (TSA), was the first specific hydroxamate-based HDAC inhibitor discovered, active in the nanomolar range. Previous in vitro studies performed in our lab showed a rapid and extensive phase I biotransformation of TSA in rat hepatocyte suspensions, implying a limited in vivo efficacy. In this study, we therefore focused on the synthesis of nine benzamide-containing structural analogues of TSA. HPLC-MS and tandem MS enabled us to separate and identify the analogues and their phase I metabolites. For the quantification of their inhibitory potencies towards hepatocyte HDAC, the removal of 3 H-acetate from prelabeled histones was measured. Acute cytotoxic effects caused by the mother compounds and their metabolites were evaluated by lactate dehydrogenase leakage in the incubation medium of rat hepatocytes. Our results show that benzamide-containing analogues of TSA represent a group of easily synthesizable, metabolically stable compounds that selectively inhibit HDAC in the (low) micromolar range and show little toxicity towards well-differentiated cells. 285 PL 14736: A 4-WEEK INTRAVENOUS TOXICITY STUY IN RATS FOLLOWED BY A 4-WEEK RECOVERY PERIOD AND A 4-WEEK INTRAVENOUS TOXICITY STUDY IN DOGS M. Veljača 1 , Ž. Krnić 1 , Ž. Ferenčić 1 , M. Kolega 1 . PLIVA Pharmaceutical Industry Inc., Zagreb, Croatia PL 14736 is a synthetic pentadecapeptide that shows protective and healing activity in trinitrobenzene sulphonic acid (TNBS) model of colitis in rats. It has been developed for the treatment of ulcerative colitis. Repeated dose toxicity studies were performed in rats (Sprague Dawley Crl:CD (SD) BR) and dogs (Beagle). Groups of male and female animals received single daily doses of 0, 1, 3 or 10 mg/kg/day Poster Session P12. Drug toxicology PL 14736 by intravenous administration for 4 consecutive weeks. The 4-week toxicity study in rats was followed by a 4-week recovery period. Examination carried out during the study included clinical observations, body weight and food intake measurements, opthalmological examinations and laboratory investigations. Electrocardiograms were recorded only in dogs. The necropsy was performed at the end of the treatment periods and in the rat study at the end of the recovery period. Principal organs were weighed out and histopathological examination was performed. In the rat study a slight decrease in MCHC was confined to males of the higher dose groups. This slight decrease was also observed at the end of the recovery period. Blood chemistry revealed some slight changes, which were mainly confined to the higher dose groups and involved the following parameters: glucose (increase), AST (decrease), total protein (increase), triglycerides (increase), sodium and chloride (increase). At the end of the recovery period, high-dosed males showed minor blood chemistry changes (increases in serum levels of glucose, total cholesterol, sodium, chloride). No clinical or morphological findings that could be related to the test article administration were found at any dose. In the dog study, only slight increase in frequency and degree of perivascular acute inflammation at the injection site was seen in the high dose treated animals, compared to controls. All other changes observed at the injection sites had a comparable frequency and degree among treated and control animals. No other findings were observed in the study. In conclusion, PL 14736 given to rats and dogs by intravenous route for 4 consecutive weeks at dose levels of 1, 3 or 10 mg/kg/day was on the whole well tolerated inducing only mild changes in a few laboratory parameters, mainly confined to the higher doses in rats. 286 FLOW CYTOMETRIC MONITORING OF IMMUNE-MEDIATED TOXICITY INDUCED BY LONG-TERM ANTIGLAUCOMA TREATMENTS ON CONJUNCTIVAL EPITHELIAL CELLS USING THREE INFLAMMATORY MARKERS: HLA-DR, IL-6 AND IL-8. F. Brignole 1,3 , C. Blondin 3 , L. Bensoussan 3 , P. Hamard 3 , G. Sabeh Afaki 1 , C. Baudouin 2,3 , C. Creuzot-Garcher 4 , J.-M. Warnet 1,3 . 1 Laboratoire De Toxicologie, FacultÉ Des Sciences Pharmaceutiques Et Biologiques, UniversitÉ RenÉ Descartes Paris 5. 2 Hôpital Ambroise ParÉ, Ap-Hp, Boulogne. 3 Centre Hospitalier National D’ophtalmologie Des Quinze-Vingts, Ea 3123 Pierre Et Marie Curie University, Paris 6. 4 Centre Hospitalo-Universitaire De Dijon, France Aim: To assess the toxicity of antiglaucoma treatments using a flow cytometric analysis of three inflammatory markers: HLA-DR, IL-6 and IL-8 in conjunctival epithelial cells obtained by impression cytology (IC) from long-term treated glaucoma patients. Patients and methods: 45 patients suffering from primary openangle glaucoma and receiving topical treatments for at least one year, and 15 subjects without any ophthalmologic disease (controls) were studied. Surface HLA-DR and cytoplasmic IL-6 and IL-8 were assessed by, respectively, direct and indirect immunofluorescence techniques. Fluorescence levels were quantified using calibrated fluorescent beads. Results: The percentages of HLA-DR-positive cells were significantly higher in multitreated glaucoma patients and in those treated with preserved betablocker or preserved prostaglandin analog than in control subjects, whereas unpreserved betablockers did not significantly increase the percentage of HLA-DR-positive cells. However, the percentages of IL-6- and IL-8-positive cells as well as IL-6 and IL-8 expression levels were significantly higher in all patients than in controls, regardless of the treatment type and the presence of preservative. A significant positive correlation was found between HLA-DR and cytoplasmic IL-6 or IL-8 expressions as well as between IL-6 and IL-8 cytoplasmic expressions. Conclusions: This prospective study i) confirms an increased expression of HLA-DR in treated glaucoma patients and ii) demonstrates that antiglaucoma treatments lead to higher intracellular levels of the pro-inflammatory cytokine IL-6 and IL-8 in conjunctival epithelial cells. Benzalkonium-preserved eye drops and preserved multitherapy induce stronger inflammatory responses than did unpre- s79 served eye drops. However, further studies are needed to determine the respective inflammatory role of preservative and therapeutic molecules. Here, we have shown that flow cytometry allows the intracellular detection of pro-inflammatory cytokines in conjunctival cells obtained by impression cytology. Thus, flow cytometric analysis of inflammatory markers may offer a standardized and reliable tool for monitoring the inflammatory conjunctival status in various ocular surface disorders. 287 INHIBITION KINETICS AND EXPRESSION OF GLUTAMATE TRANSPORTERS IN RETINAL PIGMENT EPITHELIAL CELLS H. Mäenpää, H. Tähti. Medical School, University of Tampere, Finland Retinal pigment epithelial (RPE) cells form the blood-retina barrier, and their glutamate transporters are essential for retinal homeostasis. Glutamate is the main excitatory neurotransmitter in the retina. The toxicity of glutamate is connected to the dysfunction of the glutamate transporter. Our main objective was to study the expression and kinetics of glutamate transporters in the RPE cells in vitro. The second aim was to clarify the effects of tamoxifen and toremifene on the glutamate transporter. These compounds are used in the breast cancer therapy and tamoxifen has caused retinal changes as a side effect. The pig RPE culture and two human RPE cell lines, D407 and ARPE-19, were used. The cultures were solubilised in a buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS and separation of proteins was made with SDS–PAGE. Proteins were blotted onto nitrocellulose, and the binding of five known glutamate transporter antibodies was detected with ECL. Glutamate uptake inhibition was investigated by using L-[3 H]glutamate as a tracer. The cells were exposed to 0.1–5 µM tamoxifen for 7 days (western blots) and to 7.5 µM tamoxifen/ toremifene for 10 min (uptake assays). The transporter subtypes EAAT4 and EAAC-1 were found in RPE cells. EAAT4 was expressed in the cell lines only. The EAAC1 signal was stronger in the cell lines compared to the pig RPE cells. Tamoxifen did not change the EAAT4 expression. In contrast, in the kinetic analyses tamoxifen and toremifene increased the Km constant for glutamate transport, which indicates that inhibition evoked by them is competitive. Both drugs were more effective in the human RPE cell line than in the pig RPE cells. This result showed for the first time that the antioestrogens tamoxifen and toremifene hamper glutamate transport by replacing glutamate as the substrate. 288 OCULAR SURFACE TOXICITY OF PRESERVED ANTIGLAUCOMA TREATMENTS: AN EX VIVO AND IN VITRO COMPARISON OF PROSTAGLANDINS AND BETA-BLOCKERS USING CYTOFLUORIMETRIC ASSAYS F. Brignole 1,3 , P.-J. Pisella 1,2 , C. Debbasch 1 , P. Hamard 1,3 , V. Parier 2 , P. Rat 1 , Ch. Baudouin 3 , J.-M. Warnet 1 . 1 Dpt of Toxicology, Faculty of Biological and Pharmacological Sciences, Paris 5 University René Descartes, Paris, France, 2 Dpt of Ophthalmology, University Hospital of Tours, Tours, France, 3 Dpt of Ophthalmology Quinze-Vingts National Ophthalmology Hospital and Ambroise Paré Hospital, APHP, Paris Ouest, Paris, France The effects of prostaglandin analog latanoprost on conjunctival epithelium was compared with those of preserved and unpreserved beta-blocker timolol, ex vivo in impression cytology (IC) from longterm treated glaucoma patients, and in vitro using a conjunctival cell line. IC specimens were collected in long-term treated eyes: 21 with 0.02%BAC-associated latanoprost, 15 with preserved 0.5% timolol containing 0.01% BAC (timolol, BAC+) and 17 with unpreserved 0.5% timolol (timolol, BAC-). Specimens were analyzed using flow cytometry for inflammatory profile (HLA-DR and ICAM1) and mucin detection (MUC5AC). In addition, a continuous human conjunctival cell line was treated with unpreserved timolol, 0.02% BAC-containing timolol, 0.02% BAC alone and 0.02%BACcontaining latanoprost for 15 minutes. Analyses were performed immediately and after 4 and 24 hours of cell recovery in normal medium. Membrane integrity and chromatin condensation were s80 Poster Session P12. Drug toxicology assessed using microplate cold light cytofluorimetry (neutral red test and Hoechst 33342 test respectively). IC analyses showed a significant increase in HLA-DR and ICAM-1 expressions, and a significant decrease in goblet cell density in timolol, BAC+ and latanoprost groups as compared to timolol, BAC- group, with higher effects in Timolol, BAC+ group than in latanoprost group. Moreover, in cultured cells, after 15 minutes of treatment, an apoptotic phenomenon was observed with preserved timolol and latanoprost, but significantly lesser than with BAC alone (respectively p = 0.003 and p = 0.02) despite the same concentration of preservative in all solutions. Unpreserved timolol did not show any toxicity. Latanoprost appeared to be less toxic than timolol, BAC+ on the conjunctival epithelium, both ex vivo in IC specimens from glaucomatous patients and in vitro in a conjunctival cell line. These studies suggest a cytoprotective role of prostaglandin analogs against the preservative toxicity upon conjunctival cells 289 IN VITRO EFFECTS OF PRESERVED OR UNPRESERVED ANTIGLAUCOMA DRUGS ON APOPTOTIC MARKER EXPRESSION BY HUMAN TRABECULAR CELLS. F. Brignole 1,2 , P. Hamard 1,2,3 , C. Blondin 2 , C. Debbasch 1 , Ch. Baudouin 2,3 , J.-M. Warnet 1 . 1 Laboratory of Toxicology, Faculty of Pharmacological and Biological Sciences, René Descartes University, Paris 5. 2 Immuno-Ophthalmology Unit, EA3123, Pierre and Marie Curie University, Paris 6. 3 Departments of Ophthalmology, Quinze-Vingts National Hospital and Ambroise Paré Hospital, AP-HP, Paris, France Rationale: In order to determine whether drug-induced apoptosis could be involved in trabecular cell loss in glaucoma patients, we evaluated the effects of benzalkonium-preserved (BAC+) or preservative-free (BAC-) antiglaucoma medications (beta-blockers or prostaglandin analogs) on apoptotic marker expression by human trabecular cells. Methods: Normal and glaucomatous trabecular cell lines were treated for 15 minutes with the following antiglaucoma drugs in a 1/100 dilution: 0.5% timolol BAC+ or BAC-, 0.25% betaxolol suspension BAC+ or BAC-, 0.005% latanoprost BAC+, or pure 0.01% BAC. Apo2.7 expression, annexin V binding and DNA content were evaluated using flow cytometry and confocal microscopy. Results: Both normal and glaucomatous trabecular cell lines responded similarly to drug exposure. Apoptotic marker levels remained unchanged in cells treated with unpreserved betablockers compared to untreated cells. Preserved beta-blockers and prostaglandin analogs significantly increased Apo2.7 expression, while pure benzalkonium chloride induced a significant increase of the three apoptotic features. Conclusion: This study shows that none of the unpreserved beta-blockers displayed pro-apoptotic effect on trabecular cells in vitro. The mild pro-apoptotic effect of preserved antiglaucoma drugs appeared to be principally due to the presence of preservative. The strong pro-apoptotic activity of benzalkonium chloride was largely hindered by active compounds in preserved eye drops, through a mechanism that remains to be elucidated. 290 ANATOMOPATHOLOGIC CHANGES IN SPRAGUE DAWLEY RATS AFTER 14 DAYS REPEATED ADMINISTRATION OF HUMAN RECOMBINANT ERYTHROPOIETIN. D. Fuentes 1 , M. Arteaga 1 , O. Hernández 1 , A. Casacó 2 , A. Thomas 1 , Y. Rivero 1 , Y. Torres 1 , N. Subirós 1 , B. González 1 . 1 División de Toxicología y Experimentación Animal del Centro Nacional para la Producción de Animales de Laboratorio, La Habana, Cuba. 2 Departamento de Ensayos Clínicos del Centro de Inmunología Molecular, La Habana, Cuba Erythropoietin (EPO) is the principal factor responsible for the regulation of red blood cells production during steady-state conditions and for accelerating recovery of red blood cell mass following hemorrhage. EPO is dominantly produced in the kidney and the primary stimulus for increased EPO synthesis is tissue hypoxia resulting from decreased blood O2 availability. The production of this protein by recombinant DNA techniques has made possible its wide use as a therapeutic drug for several human diseases, particularly, the anemia associated with chronic renal failure. Toxicology test constitute an important tool before its use in human patients. Our objective was to determine the anatomopathologic changes in Sprague Dawley rats after 14 days repeated intravenous administration of this protein. Gross necropsy was made in all animals where they were examined the external body surface, orifices, cranial, thoracic and abdominal cavities, and all organs. The organ absolute weight and organ weight to body weight per cent ratio were determined for the liver, kidneys, heart, spleen, lungs, thymus, adrenals glands, ovary, testis and brain. We took samples of liver, kidneys, spleen, thymus, mesenteric lymphatic ganglia and administration site. These tissues were fixed in 10% neutral buffered formaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin for this microscopically examination. Gross examination and organ weight showed a significative increase spleen size in animals treated with high doses of EPO. Microscopic findings confirmed the presence of marked extramedular erythropoiesis characterized by numerous megacariocytes, and abundant erythroblasts in spleen red pulp and also around hepatic capillaries. No other tissues in any of the treated groups showed signs of toxicological lesions. The observed changes are according to biological functions of the erythropoietin We concluded that human recombinant Erithropoietin’ repeated intravenous administration in Sprague Dawley rats provoked anatomopathological changes associated with pharmacological expected action of the evaluated substance. 291 DOSE REPEATED TOXICITY STUDY OF THE HUMANIZED ANTI-EPIDERMAL GROWTH FACTOR RECEPTOR MONOCLONAL ANTIBODY h-R3 (THERACIM) BY ENDOVENOUS ROUTE IN CERCOPITHECUS AETHIOPS SABAEUS MONKEYS. A. Casacó 1 , M. Arteaga-Pérez 2 , M. Maceira 1 , O. Hernández-Sosa 2 , A. Bada-Barro 2 , A. León-Goñí 2 , R. Orpheé-Suárez 2 , A. Cuevas-Fiallo 2 , D. Moreno-Díaz 2 , P. Padro-Gutiérrez 3 , F. Baro-González 3 , V. Rodríguez-Rodríguez 3 , L. Charro-Ruiz 3 , F. Vázquez-Castro 4 , A. Ballester-Labrada 5 . 1 Centro de Inmunología Molecular, 2 Centro Nacional para la Producción de Animales de Laboratorio, 3 Centro de Neurociencias, 4 Centro de Investigaciones Médico Quirúrgicas, 5 Centro de Investigaciones Clínicas, Havana, Cuba The h-R3 monoclonal antibody is a humanized anti epidermal growth factor receptor drug proposed for the treatment of head and neck tumors of transformed cells that over-express the Epidermal Growth Factor receptor. The present study was designed to evaluate the toxicity of repeated intravenous doses of the h-R3 mAb in a relevant species demonstrated by the avidin-biotin-peroxidase immunohistochemical technique in skin biopsy samples from three Cercopithecus aethiops sabaeus monkeys (green monkeys). Additionally, eighteen green monkeys were daily intravenously treated during 14 consecutive days. Monkeys were distributed into 3 experimental groups with 3 animals of each sex in each group. Group I received saline and served as control group; group II received 2.85 mg/kg of h-R3 mAb; and group III received 11.4 mg/kg of the h-R3 mAb. During the study there were no deaths, neither pathological clinical signs, or variations in the corporal weight curve. The electroneurophysiological and blood chemistry results did not evidence alterations related to the assay substance. Areas of hematomas, hemorrhages, and inflammation, probably related with the administration procedure were observed at the administration areas of all animals, this fact could also explain the increase in the neutrophil count of all animals at the end of the study. The electrocardiography study showed that in the 14 days of the study one female monkey, from the higher dose group, shifted its cardiac axis from +600 to +1200 , this finding could be interpreted as a right ventricular elongation due to the relative high daily administered volume. It is concluded that doses up to 11.4 mg/kg of h-R3, administered during 14 consecutive days, by endovenous route, to Cercopithecus aethiops sabaeus monkeys do not produce considerable toxic effects in the studied system. Poster Session P12. Drug toxicology 292 ACUTE AND REPEATED DOSE INTRAMUSCULAR TOXICITY OF N-ACETILGM3 CANCER VACCINE IN SD RATS A. Bada 1 , A. Casacó 2 , A. Mancebo 1 , O. Hernández 1 , D. Fuentes 1 , J. Hernández 1 , N. Subirós 1 , B. González 1 , M.E. Arteaga 1 . 1 División de Toxicología y Experimentación Animal del Centro Nacional para la Producción de Animales de Laboratorio, Habana, Cuba, 2 Departamento de Ensayos Clínicos, Centro de Inmunología Molecular, Habana, Cuba GM3 is a ganglioside overexpressed in some tumors, but it is also an autoantigen present in normal mammalian tissues. We have designed a GM3-based cancer vaccine for the treatment of human breast and melanoma tumors. We carried out two studies for evaluated the toxicity of GM3 cancer vaccine: acute intramuscular toxicity and repeated dose intramuscular toxicity in SD rats. Our objective was to determine the toxicity signs manifested in rats after intramuscular administration of single dose and repeated doses during 14 days. All the rats were inspected daily for clinical signs. Body weight was measured once a week during the test. Blood samples were collected for hematological (red blood cell, white blood cell, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, differential leukocyte count and platelet count) and serum biochemical determinations (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, glucose, blood urea nitrogen, total protein, total cholesterol, total bilirubin, creatinin, uric acid and triglicerides). Gross necropsy was made in all animals. Organ weights were measured for the thymus, adrenals, testis, ovary, heart, lung, kidney, spleen, liver and brain. These tissues and the administration site and the abnormal tissues were taken and fixes in 10% formalin. These were embedded in paraffin, sectioned, and stained with hematoxilylin-eosin for histopatological examinations. There were neither death nor observable differences regarding body weight and rectal temperature; likely decrease in hemoglobin and hematocrit was observed, and the white blood cell and neutrophils was increased in the GM3 vaccine group. The levels of total proteins and albumin were significantly decreased in the treated group. All treated rats showed hardening and inflammatory reaction, characterized by cysts, fibrocytes and fibroblastes around administration site. No other tissues in any of the treated group showed signs of toxicologic lesions. In conclusion, GM3 vaccine was confirmed to have a low toxicity. GM3 vaccine may be used as an alternative anticancer therapy. 293 THE EFFECT OF ANABOLIC - ANDROGENIC STEROID ON THE BODY, TESTIS AND EPIDIDYMIS WEIGHT IN ADULT MALE RAT F. Mesbah 1 , H. Mirkhani 2 , S. Karbalaei doust 1 , S. Shokri 1 . 1 Anatomy & 2 Pharmacology Departments, Shiraz Medical School, Shiraz, Iran Anabolic-Androgenic Steroids (AAS) compounds are used by athletes, prepubescent and adolescents for improving athletic ability, appearance or muscle mass. Therefore, administration of these compounds is increased significantly. Many of athletes who use AAS believe that the side effects are neither serious nor permanent. Many of undesirable side effects on the male reproductive function have been reported, but little is known about how these compounds effect on sexual behavior and tissue of the reproductive system. The main aim of this study is to identify the effect of long term administration of AAS compounds on the body, testis and epididymis weight. Five groups of Sprague-Dawley male rats (3 month) were used. First and second groups (experimental=37) injected with 3 mg/kg and 10-mg/kg Nanderlon decanoate and third and forth groups (vehicle=24) with 3 mg/kg and 10-mg/kg peanut oil respectively, one time per week for 14 weeks, intramuscularly. Fifth group was kept under standard condition during this time. After sacrificing, the weight of body, testis and epididymis of the rats were measured. The body weight did not differ significantly between treatment and vehicle and control groups. The experimental groups showed significant differences in testis and epididymis weight with other groups. s81 The result of this study shows that the long term administration of AAS compounds causes testis and epididymis atrophy. 294 THE EFFECT OF ANABOLIC-ANDROGENIC STEROID ON THE SPERM COUNT, MOTILITY AND MORPHOLOGY IN ADULT MALE RATS S. Karbalay doust 1 , H. Mirkhani 2 , F. Mesbah 1 , S. Shokri 1 . Anatomy1 & Pharmacology2 Departments. Shiraz Medical School, Shiraz IRAN Anabolic-Androgenic Steroids (AAS) compounds are used by athletes, prepubescent and adolescents for improving athletic ability, appearance or muscle mass. Therefore, administration of these compounds is increased significantly. Many of the athletes who use AAS believe that the side effects are neither serious nor permanent. Many of undesirable side effects on the male reproductive function have been reported, but little is known about how these compounds effect sexual behavior and tissue of the reproductive system. The main aim of this study is to identify the effects of long-term administration of AAS compounds on the sperm parameters. Five groups of Sprague-Dawley male rats (3 month) were used. First and second groups (experimental=37) injected with 3mg/kg and 10 mg/kg Nanderlon decanoate and third and forth groups (vehicle=24) with 3mg and 10 mg/kg Peanut oil respectively, one time per week for 14 weeks, intramusculary. The fifth group was kept under standard condition during this time. After sacrifice, the shape, motility and number of sperms of the rats were assessed. Sperm count and motility rate were decreased in experimental groups and showed shape that is more abnormal. Change in sperm parameters differed significantly (p≤0.01) between experimental and vehicles and control groups. The results of this study show that AAS compounds affect fertility parameters. 295 RISK CRITERIA OF TOXIC EFFECTS ADVANCEMENT UNDER IMPACT OF SEVERAL HORMONAL MEDICINES M. Kudrya, I. Palagina, L. Mogilat, A. Gladkova. Laboratory of Drug Toxicology, Ukrainian Institute of Endocrine Pathology Problems, Kharkov, Ukraine Hormonal medicines are biologically active compounds. In this connection, at all stages of their pre-clinical studies and manufacturing an evaluation of their efficacy must be combined with assessment of their potential toxicity. Within the latter, an important role is played by the risk assessment of their collateral or toxic effects within the clinical and industrial conditions based on investigations over experimental animals. Objects of our study were Estrasine (E) – a derivative of the natural hormone Estronum designated for treatment of the prostate cancer and adenoma, and L-Thyroxine (L-T) used for stimulation of thyroid gland function and normalisation of thyroid hormones level. Aim of this study was to define the risk degree and criteria of E and L-T under different ways of introduction into an organism. Experiments were conducted over 250 Wistar rats. We found that laying of E onto the skin of male and female rats in dose 20 mg/kg led to a sharp decline of a body mass (P<0,05), to infringement of protein- and cholesterol-synthesizing functions of the liver, and to changes in reproduction system. Female rats showed changes in duration and structure of estrous cycle, reduction of progesterone level, inhibition of gonadotropic function of pituitary body; male rats demonstrated diminishing of the mass quotient of spermaries, seed blisters and prostate gland (P<0,05). Within the oral subchronic injection (d=500 mg/kg) of E, we recognised its major risk criteria in hemo- and hepatotoxicity. 1-fold inhalatory impact of E in concentrations 0.83; 3.6; 11.9 mg/m3 caused a decrease of the spermatozoa mobility time and decline of the seed blisters mass. L-T under the epidermal (20 applications) and oral subchronic injection (d=250 mg/kg) caused infringements in the myocardium, CNS functions, and in the liver basal metabolism. Acute inhalatory impact of L-T in concentrations 0.1; 0.56; 1.43, 5.0 mg/m3 led to intensification of the lipids peroxidation (LP), infringement of the heart function, increase of T4 level in the blood serum (by 205–587%), and augmentation of T3 content (by 1,7–2 times). s82 Poster Session P12. Drug toxicology Thus, we concluded in establishing the risk criteria of toxic impact, which include: for E – changes of the generative function, and for L-T – activation of LP and hypertoxinemia. The extent of such risk depends on the effective dose (concentration) of medicines. 296 MORPHOLOGICAL CHANGES IN MOUSE TESTS AFTER TREATMENT WITH FLUTAMIDE R. Anahara 1 , Y. Ono 1 , Y. Toyama 2 , C. Mori 1,3 . 1 Department of Bioenvironmental Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan, 2 Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba, Japan, 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, Japan Flutamide (Flu) inhibits androgen uptake and/or nuclear binding of androgen in target tissues and is used as a medicine for prostate cancer. The purpose of the present study was to investigate the effect of Flu on testes of newborn and adult mice. Newborn ICR male mice were subcutaneously injected with 0.005, 0.05, 0.5, 5, 50 or 500µg/mouse/shot of Flu on Days 2, 4, 6, 8, 10 and 12. The day of birth was regarded as Day 1. Also, ICR adult male mice were subcutaneously injected with 0.05, 0.5, 5 or 50µg/mouse/day of Flu for sequential 5 days. Control animals received vehicle (corn oil). The testes taken from newborn until adult mice (Day 7, 14, 21, 28, 35, 42, 56, 63 and 84) were processed for electron microscopy. Some newborn animals were kept until the age of 84 days, and housed with normal females for fertility test. Spermatids with deformed nuclei and/or acrosomal caps were often observed in the seminiferous epithelium of Flu-treated mice. In addition, complete or partial deletion in the ectoplasmic specialization between the Sertoli cell and spermatids was observed. These abnormalities were found at all doses. The specialization between adjoining Sertoli cells, or the blood testis barrier, was not affected. The newborn animals were shown to be fertile when they reached 84 days of age. Since similar observations were reported after treatment with β-estradiol 3-benzoate, the presence of Flu, a potent anti-androgen, may create an ‘oestrogenic environment’ in mouse testes. (This work was supported by the fund for endocrine disrupters from the Ministry of the Environment, Japan.) 297 LONG-TERM METABOLIC AND ENDOCRINE ACTIVITY CHANGES IN RESPONSE TO CLENBUTEROL IN MATURE FEMALE PIGS T. Gojmerac 1 , B. Mandić 2 , M. Koršić 3 , A. Tomašić 1 , B. Vinković 1 . 1 Croatian Veterinary Institute, Zagreb, Croatia; 2 Vuk Vrhovac Institute for Diabetes, Endocrinology and Metabolic Diseases, School of Medicine, University of Zagreb, Croatia; 3 University Department of Medicine-Rebro, Zagreb, Croatia Clenbuterol, a β2 -adrenergic agonist, has been illegally used as a repartitioning agent to improve the performance of meat-producing animals, since its abuse may cause risk for consumer health. The direct and indirect effect of clenbuterol especially depends on the mode and duration of administration, i.e. acute or long-term. In the present study, the effect of intravenously repeated administration of clenbuterol in a growth-promoting dose on the metabolic and endocrine status of mature female pigs was evaluated on the basis of biochemical findings. The growth-promoting dose of clenbuterol was administered intravenously daily for 20 days of estrous cycle to cross-bred mature female pigs (80–100 kg, n=12). Before (0) and at 30,60,90,120,180,240 and 300 min after the last drug dosage, blood samples were collected by jugular venipuncture and analyzed for serum insulin, nonesterified fatty acid (NEFA) and blood glucose concentration. Serum 17β-estradiol (17β-E) and progesterone (P) concentration were measured in the samples collected 2 times daily at 5-h intervals on the first 4 days after last drug dosage. Serum NEFA concentrations moderately increased and reached highest level, and serum insulin levels moderately decreased and reached lowest level at 30 min preprandially after the last clenbuterol dosage, but the changes did not differ significantly from those in control animals. During the same period, the blood glucose concentration reached the highest level at 90 min postprandially after the last dosage and showed no significant difference from those in control animals. Serum 17β-E and P concentrations on the days around the onset of the next expected estrous were lower but not significantly, resulting in normal occurrence of estrous in the pigs. On the basis of the data obtained, we suggest that long-term administration of clenbuterol in a growth-promoting dose to female pigs provoked only subtle metabolic and endocrine activity changes, indicating down-regulation of the specific β2 -adrenergic receptors in target tissues. 298 A STUDY OF HEPARIN EFFECTS ON HEMATOBIOCHEMICAL FACTORS AND BILIARYBILIRUBIN SECRETION IN SHEEP M. Pourjafar, K. Mostaghni. School of Veterinary Medicine, Shahrekord University, Shahrekord, Iran This study was carried out to find out the effects of heparin on hemogram, biochemical factors in serum, biliary secretion and the concentration of biliary bilirubin in sheep. In this study, 10 healthy Iranian crossbred male sheep, aged between 2–3 years and weighting 66–78 kg (71±4.06) were randomly selected. Before the experiment, blood samples were obtained in 3 consecutive days and the results of the tests for hematological, serum enzymes and serum bilirubin concentration were determined. These normal values were accepted as a control measure. In a preliminary study, to find out the abnormal effects of heparin to the above mentioned factors, heparin was administered at the rate of 107 IU/Kg b.w., IV as a loading dose, and 320 IU/KG b.w.,S.C every 12 hours for 5 consecutive days as a maintenance dose. The finding of the tests from the blood samples, during treatment periods and after that it has been noticed that the heparin did not have any significant effects on the factors mentioned above at this dose levels and also in increased dose by 1.5 times. Following anesthesia by using thiopental sodium (16 mg /Kg b.w., LV) and finding a cannula in the duodenum and the gall bladder, the volume of bile and the concentration of biliary bilirubin, were measured. The bile was collected and reinjected into the duodenum through the existing cannula, for maintaining the enterohepatic circulation throughout the experiment. Before using heparin, blood and bile samples were collected for 3 days. The samples were used for the determination of hemogram, enzymes, the concentration of bilirubin in serum, the volume of bile secretion, and the concentration of biliary bilirubin as normal values. Then, heparin was given at the rate of 214 IU/Kg b.w.LV, as a loading dose and 640 IU/Kg b.w., SC. every 12 hours as a maintenance dose for 5 consecutive days. The blood samples were taken in every hour, during treatment period and also three days following of treatment. At the same time biliary samples were taken every half an hour through the cannula within the gall bladder. Having measured the volume and sampled, the remaining bile was reinjected into the intestine through the existing cannula. The blood samples were used to determine the levels of Hb and HCT, red and white blood cell counts (and WBC differentiate counts), measurement of enzymes (AST, ALT, ALP) and bilirubin in serum. Also, from the bile samples, the concentration of bilirubin was measured. The results of the measurements prior to injection, during and after injection were analyzed by using the pair T-test with p=0.01. The released data from these experiments indicate that levels of Hb concentration and HCT and also red blood cell counts in the second day following the injection of heparin were significantly lowered in comparison with the control values (p<0.01). However, no significant changes were observed in white blood cell counts, its differentiate counts and the level of enzymes AST,ALT and ALP (p<0.01). On the other hand, a significant increase was observed in serum bilirubin concentration, biliary secretion and biliary bilirubin concentration on the second post injection day (p<0.01). From this study, in addition to the determination of treatment dose of heparin, it can be concluded that the toxic level of heparin injection can cause extravascular hemolysis in sheep. Obviously, reduction of red blood cells by the reticuloendothelial system, catabolism of heme, increased bile volume, increased concentration Poster Session P12. Drug toxicology of serum and bile bilirubin verifies the hypothesis that heparin has a potential to enhance the activity of the reticuloendothelial system. 299 TOLERANCE DEVELOPES TO ANTI-INFLAMMATORY EFFECT OF MORPHINE P. Hassanzadeh, A. Ahmadiani, M. Alebouyeh. Department of pharmacology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran,Iran.19835–355. Morphine is effective in most kinds of acute and chronic pain; as well as being antinociceptive, morphine also has anti- inflammatory effect which has been studied systematically. Development of tolerance is a characteristic feature of morphine which extends to most of its pharmacological effects. In the current study after confirming anti-inflammatory effect of morphine(7mg/kg, i.p.), against carrageenan(0.05 ml, 3% w/v, s.c.)-induced paw edema in mice, it has been tried to assess the probable occurrence of tolerance - a troublesome physiological response- to this therapeutic effect of morphine. Increasing concentrations of morphine for three days, were administered in order to study the effect of morphine chronic therapy on the process of inflammation. According to the results, morphine(7mg/kg, i.p.) has not been able to prevent carrageenan(0.05ml, 3% w/v, s.c.)induced hind paw edema, following long-term administration. So it is likely to consider that the phenomenon of tolerance extends to anti-inflammatory effect of morphine 300 A SELECTIVE COX-2 INHIBITOR, NIMESULIDE, AS GASTROENTEROPROTECTIVE AGENTS IN T-2 TOXIN POSONED RATS V. Jaćević 1 , L. Zolotarevski 2 , K. Jelić 2 , V. Kilibarda 1 , J. Dimitrijević 2 , M.P. Stojiljković 1 . 1 National Poison Control Centre, Military Medical Academy; 2 Institute for Pathology and Forensic Medicine, Military Medical Academy, Belgrade, Serbia and Montenegro T-2 mycotoxin can suppress cell-mediated and humoral immunity. Also, its cytotoxic effects such as reduced concentrations of immunoglobulins and depressed phagocytes activity of both macrophages and neutrophils. Nonsteroidal anti-inflamatory drugs (NSAIDs), nimesulide is selective COX-2 inhibitor. Cyclooxygenase (COX), the key enzyme involved in the synthesis of prostaglandins (PGs) which plays an important role in the acute inflammation. PGs are produced in the gastrointestinal tract and play an important role in the gastric defence mechanism called “mucosal defence barrier”. The aim of this study was to evaluate the gastroenteroprotective effects of nimesulide. The experiment was performed on adult female Wistar rats weighing 200–250 g. The animals were divided into four treatment-groups containing 8 rats each: (1) the control, (2) T-2 toxin (0.18 mg/kg sc), (3) nimesulide (30 mg/kg ip) and (4) T-2 toxin + nimesulide. T-2 toxin was produced in laboratory conditions from Fusarium sporotrichoides fungi. Animals were sacrificed 24h after administration. The gut paraffin sections were stained by haematoxilin and eosin (HE) and periodic acid-Schiff’s (PAS) methods. T-2 toxin produced necrosis of the gut crypt epithelium and lymphoid tissues. T-2 toxin caused necrosis of epithelial and lymphoid cells in the tunica mucosa. Majority of these ulcerations were enlarged to the tunicae muscularis. A large number of necrotic epithelial and glandular cells with mucus fluid were present in bottom of the enlarged Lieberkühn’s crypts. In all parts of the digestive wall intensive edema were found. The blood vessels were congested with thicken walls, and near them a large amount of hypereamia, hemorrhages and mononuclear cell infiltrations were present, too. Described pathohistological alterations were not presented in the gut of poisoned rats treated by nimesulide. These results imply that nimesulide, as a selective COX-2 inhibitor, afford a significant gastroenteroprotection against T-2 toxin poisoning in rats. 301 s83 EFFECT OF ROLIPRAM ON NATURAL KILLER CELLS ACTIVITY G. Dyulgerova, N. Boyadjieva. Department of Pharmacology and Toxicology, Medical Division, Medical University of Sofia, Sofia, Bulgaria The role of cyclic adenosine monophosphate (cAMP) as a second messenger in the immune system has been discussed in the literature. Elevation of intracellular cAMP has been associated with inhibition of lymphocyte activation. It is well known that natural killer cells (NK cells) are lymphocytes that are capable of destroying tumor cells and virally infected cells. We have previously reported that various drugs, modulating cAMP cellular system, play an important role in controlling NK cells activity. Several phosphodiesterase isoenzymes (PDE) have been described. PDE4 is the predominant isoenzyme expressed in the immune cells. The aim of this study is to determine the effect of PDE4 on NK cells cytolytic activity. Splenocytes were isolated from male rats and used for the determination of NK cell cytolytic activity against YAK-1 lymphoma cells in standard 51 Cr release cytolytic assay. The dose effect of Rolipram (PDE4 inhibitor) was determined. The results demonstrated that Rolipram decreased NK cell cytolytic activity in a dose-dependent manner. Our data suggested that PDE4 controls the function of NK cells. Our data also demonstrate that the down-regulation of NK cell function during treatment with PDE4 inhibitor plays a role in the inhibition of NK cells activity. 302 EFFECTS OF CAFFEINE ON INDUCTION OF APOPTOSIS IN BLOOD MONOCYTE AND ALVEOLAR MACROPHAGE CELLS M. Jafari 2 , A. Rabbani 1 . 1 Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran, 2 Department of Biochemistry, Faculty of Medicine, Baghiyatollah University, Tehran, Iran, Caffeine, a purine alkaloid, is a key component of many popular drinks, most notably tea and coffee. It has a variety of pharmacological and physiological effects. Caffeine acts as a stimulant for the central nervous, respiratory and cardiac systems and is used for the prevention and treatment of asthma and apnea of prematurity in newborn medicine. In this study, alveolar macrophages by lavage from rat lung and human blood monocytes from the peripheral blood of healthy volunteers were prepared, exposed to various concentrations of caffeine (0.05–80 mM) and then incubated for 24 hours at standard condition. The effect of caffeine was investigated by measuring percent of viable cells, superoxide anion production and DNA fragmentation. The results show that caffeine effects are highly dose dependent. Low concentrations remarkably enhance cell survival and at these concentrations, the effects of caffeine on monocyte cells were similar to macrophages. Analysis of anion superoxide production and DNA fragmentation patterns also incited no significant increase in both of the cells, suggesting that low concentrations of caffeine prevent macrophage and monocyte apoptosis. When cells were cultured in the presence of 5–20 mM of caffeine (24 hours culture), viability of the cells was reduced to the control level, while at higher concentrations of caffeine (>20 mM), a significant decrease in the cell survival was observed. On the other hand, anion superoxide production was increased, but at these concentrations, the O2- production and DNA fragmentation of macrophages was higher than blood monocytes. Also, DNA fragmentation of monocytes was similar to the control. These results suggest that pulmonary alveolar macrophages are more sensitive to caffeine concentrations than blood monocytes and at moderate concentrations (5–20 mM) induces apoptosis while at high concentrations (>20 mM) necrosis may have occurred in macrophages. The results are discussed in relation to the mechanism of cAMP. s84 303 Poster Session P12. Drug toxicology DNA DAMAGE AND REPAIR INDUCED BY PHOTOSENSITIZING DRUGS: FLUOROQUINOLONES O. Sapora, G. La Sala, B. Di Carlo, A. Maggi. Dipartimento Salute e Ambiente, Istituto Superiore di Sanità, Via Regina Elena 299, 00161 Rome, Italy The phototoxic mechanisms of two fluoroquinolones (FQ), ofloxacin (OFLX) and lomefloxacin (LFLX), have been investigated using two different human cell lines, K562 and HL60. Different DNA damages such as single (ssb) and double (dsb) strand breaks, and abasic sites have been considered. The cell have been treated following different protocols: (i) exposure 330 nm UV light, (ii) exposure to UV in the presence of FQ and (iii) exposure to UV of cells incubated for two hours with FQ and then washed and suspended in buffer without FQ. The following results have been obtained: (i) the photosensitising effect can be detected at concentrations of FQ close to that found in treated patients, (ii) is function of drug concentration and UVR dose, (iii) and is detectable also in the pre-incubated cells, (iv) the FQ’s in combination with UVR produce OH radicals, (v) DNA damages depending by a single event such as ssb are produced when the FQ is present at the time of irradiation, (vi) and are rapidly and efficiently repaired, (vii) no dsb are detected in treated cells (viii) no DNA damage is evident in pre-treated cells irradiated in the absence of FQ. The results together with that already published on membrane damage, suggest that the FQ’s phototoxic action is exerted trough the formation of oxygen reactive species, manly OH radicals. Such reactive species induced damages on DNA which can be correlate with a single event but not to damage such as dsb were two single strand scissions on the two opposite strand are required. The DNA damages are rapidly and efficiently repaired in 30–60 minutes suggesting that the DNA although damaged, is not directly involved in the phototoxic mechanisms of FQ. 304 SERUM CALCIUM, MAGNESIUM AND PHOSPHORUS LEVELS IN RATS TREATED BY SOME AMINOGLYCOSIDE, QUINOLONE ANTIBIOTICS, GLICLAZIDE, PIRETANIDE AND VERAPAMIL N.M. Abdel-Hamid 1 , F.R. Abdallah 2 , R.S. Amin 1 . 1 Biochemistry Department, Faculty of Pharmacy, El-Minia University, Egypt Biochemistry Department, Faculty of Pharmacy, Zagazig University2 , Egypt Both calcium, magnesium and phosphorus are important constituents of bone, teeth, enzymes and participate in many metabolic processes . This study was conducted mainly to stand on the possible effects of some shortly used drugs (gentamicin, norfloxacin and ofloxacin ) and long term used drugs for different pathologic conditions as gliclazide (oral hypogylcemic), piretanide (diuretic antihypertensive) and verapamil (calcium chanel blocker with antiarhythmic use) on the three aforementioned elements. Antibiotics were given for one week, while other drugs for one month to rats in human doses converted to corresponding animal doses, orally, except gentamicin which was given intramuscularly. The study revealed that serum total calcium level was significantly elevated by the three antibiotics and verapamil. Both gliclazide and piretanide significantly decreased that level. Serum magnesium level was only significantly increased by both norfloxacin and ofloxacin, while it was significantly decreased by gliclazide, piretanide and verapamil. Serum phosphorus level was significantly decreased by piretanide, while it was elevated by other tested drugs. It is obvious that both serum calcium, magnesium and phosphorus were significantly affected by both short and long term used drugs in this study. Thus, we recommend monitoring of these parameters regularly cheifly for long term drug users to manage the possible synchronus effects that can be speculated to be underlying causes of bone remodeling disorders and other neurologic or haematologic sequelae. 305 CHANGES IN CONCENTRATION OF ZINC IN URINE, SERUM, AND SALIVA AS INDICES OF GENTAMICIN NEPHROTOXICITY IN MALE WISTAR RATS M. Roohi Azizi 1 , N. Rezvan 2 , A.R. Dehpour 3 , S.Sh. Sadr 2 , A. Norouzy 2 , B. Minaee. 1 Laboratory of Clinical Neuro-Physiology, Department of Basic Sciences, Faculty of Rehabilitation, Iran University of Medical Sciences,Tehran,Iran, 2 Department of Physiology and 3 Pharmacology and Histology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran Male Wistar rats were treated with gentamicine in repeated doses (5 × 100 mg/kg) intraperitoneally. The concentrations of a trace element (zinc) in urine, serum, and pure saliva obtained from submandibular glands were determined 24 h and 20 d after the last administration of the drug. At the same time, kidneys and submandibular glands were axamined histopathologically by electron microscopy. Rats receiving gentamicine demonstrated phospholipidosis at the end of 5 d of administration. At the same time, elevated zinc levels in urine, increased zinc levels in serum, and zinc levels in saliva were observed. Twenty days after the last injection, the levels of this metal were comparable to the control group and none of the sections demonstrated phospholipidosis. Based on these results, it may be concluded that certain changes in the levels of this metal in serum and saliva could also be an indicator of acute gentamicine nephrotoxicity. 306 ISONIAZID INDUCE A HEPATIC MICROSOMAL CYTOCHROME P-450-DEPENDENT ACTIVITY IN ETHANOL CONSUMPTION RATS A. Voronina. Department of General Toxicology, Institute of Pharmacology & Toxicology, Kyiv, Ukraine In societies where alcoholic beverages are widely consumed, a large number of individuals are exposed to the effects of drugalcohol combinations. Such exposures can lead to a broad range of medical problem. Isoniazid continues to be an effective drug used for chemoprophylaxis and treatment of tuberculosis. The risk of isoniazid-associated hepatotoxicity among persons consumption alcohol. Male Wistar rats of first group were pretreated with chronic ethanol ingestion (4 months), rats of second group were treated with isoniazid at a 75 mg/kg daily for a period of 4 weeks by intragastric administration during final fourth month. Content of cytochrome P-450, p-nitrophenol hydroxylase (p-NPH) activity (a marker of cytochrome P-450 2E1) in the liver microsomes as well as alanine aminotransferase (AlAT) activity in blood serum was investigated. Chronic ethanol consumption by rats increased content of cytochrome P-450 by 1.5 fold, p-NPH activity by 2 fold above the control level. In isoniazid treated rat content of cytochrome P-450 yet more increased by 1.8 fold and p-NPH activity by 3.2 fold in the liver microsomes. Obtained results indicated that AlAT activity decline on 43% in isoniazid treated rat as compared control level. Result of studies indicated that long-term consumption of alcohol induced the hepatotoxicity of therapeutic doses of isoniazid in the rats. Induction of the cytochrome P-450 mixed function oxidizing enzyme system may cause an increase in the production of reactive intermediate metabolites. The enhanced generation the reactive intermediate metabolites of isoniazid may lead to the development of liver toxicity. 307 CONTINUOUS INTRAVENOUS INFUSION IN NEW ZEALAND WHITE RABBITS R. Cicalese, R. Sisti, J. Brightwell. RTC, Research Toxicology Centre S.p.A., Pomezia - Rome, Italy Some questions in experimental teratology, pharmacology or toxicology can only be answered when the continuous exposure to a substance to be investigated can be guaranteed. Continuous exposure will be achieved by intravenous infusion. Examples for the applicability of continuous intravenous infusion are the following: • Studies where the in vivo half life of the substance is short due to rapid metabolism (chloramphenicol) or excretion (penicillin) Poster Session P12. Drug toxicology • Studies which require steady state conditions (radioisotopes, drugs) • Preclinical testing of drugs when continuous infusion will be the route of administration to humans • Low solubility of the drugs in the vehicle • Presence of acute irritation at the injection site during bolus intravenous administration The work presented describes the preparation of animals (10 males and 10 females), the technical set-up and shows the results in terms of body weight, food consumption, clinical observations and clinical pathology investigations after at least 4 weeks of treatment. In addition, post-mortem observations and organ weights are presented. 308 DRUGMATRIX™ PROVIDES PREDICTIVE CHEMOGENOMICS SOLUTIONS FOR SAFETY ASSESSMENT IN DRUG DISCOVERY AND DEVELOPMENT Pauline Gee 1 , Richard Mitchell 1 , Leslie Browne 2 , Kyle Kolaja 2 , Kurt Jarnagin 2 , George Natsoulis 2 , Alan Roter 2 . 1 MDS Pharma Services, Bothell, Washington, USA, 2 Iconix Pharmaceuticals, Inc., Mountain View, California, USA DrugMatrix makes it possible to predict the potential toxicity and off-target effects of lead compounds in discovery, thus reducing the overall attrition rate of compounds in development. DrugMatrix is a reference database that combines the advantages of gene expression profiling with measurements in molecular pharmacology, clinical pathology and histopathology that are performed routinely in the drug discovery and development process. Together with essential observations found in the literature, this comprehensive database has been mined for unique patterns, called Drug Signatures?, which are developed through extensive mathematical analyses, including some proprietary algorithms. Novel drug candidates can be prioritized for advancement in the drug discovery process based on predictions of toxicity due to pharmacological or non-selective effects. These predictions are derived from the chemogenomic profiles of 550 compounds within DrugMatrix. DrugMatrix is populated largely with approved and unapproved drugs and is supplemented with standard toxicants and other biochemical reagents. To optimize candidate selection and stratify risk in a testing paradigm, novel drug candidates can be evaluated directly with the domains that comprise DrugMatrix, and the results may be compared to compounds in DrugMatrix. The domains of DrugMatrix include: DrugMatrixScreen?, a battery of approximately 130 in vitro molecular pharmacology bioassays; genes expression profiling measured on 10,000-probe microarrays for up to 12 tissues in rats exposed in a time and dose dependent manner; concomitant clinical observations; hematology and clinical chemistry; and histopathology in the same rats. All of these domains can be interpreted in contextual reference to reported literature observations. Such data take into consideration in vivo consequences of ADME in rats and in vitro human specificity in the molecular pharmacology assays that make it possible to derive the Drug Signature patterns that tap the full potential of the predictive power of DrugMatrix. 309 MECHANISMS OF TOXIC DAMAGE IN ORGANISM UNDER THE IMPACT OF ANTI-DIABETIC MEDICINES (ADMs) – DICARBOXYLIC ACIDS DERIVATIVES I. Palagina 1 , M. Kudrya 1 , F. Kolodub 1 , N. Ustenko 1 . 1 Laboratory of Drug Toxicology, Ukrainian Institute of Endocrine Pathology Problems, Kharkov, Ukraine An important aspect in risk and safety assessment of drugs covers study of membranes systems of the liver cells’ endoplasmatic reticulum (EPR) – the monooxygenase (MOGS), free-radical lipoperoxidation (LP) and anti-radical protection (ARPS). Analysis of these systems enables to determine key biochemical mechanisms of cells toxic damage. Aim of our study was to reveal peculiarities of changes in MOGS, LP and ARPS systems of liver microsomes which predetermine mechanisms of toxic disorder under subchronic impact of ADMs. Objects of study were the Wistar male rats. Animals received s85 Phensuccinalum (PhS) and Diacamph (Dc) – derivatives of succinic and camphoric acids resp. PhS was introduced orally dosaged 5000, 500 and 100 mg/kg, and Dc, dosaged 1000 and 100 mg/kg. Concentration of inhaled PhS levelled at (20.2±1.4) and (1.91±0.15) mg/m3 . State of MOGS, PL and ARPS was estimated with chemiluminescent and spectrophotometrical methods. We found that 15and 30-fold introduction of PhS in dose 100 mg/kg, as contrasting to the similar exposition of Dc, activated free-radical processes (FRP) in membranes of the liver EPR; the latter were stipulated by tension of the electron-transporting chain (increase of cytochromes B5 and P450 level). Increase of dose of PhS to 500 mg/kg and of Dc, to 1000 mg/kg caused a higher FRP intensity combined with a growth of capacity of metabolising MOGS already after a 5-fold introduction of drugs. After 15 and 30 introductions, both ADMs stimulated LP processes. In case of PhS, accumulation of Lipids hydroperoxides (LHP) was caused by both a primary initiation of LP with the activated forms of oxygen, and deceleration of a further oxidation of LHP. Under PhS introduction in dose 5000 mg/kg, activation of FRP and LP was even more expressed, being accompanied by inhibition of ARPS activeness. When inhaled, PhS changed parameters of FRP and LP state only at concentration of (20.2±1.4) mg/m3 . Orientation of these changes coincided with the one registered under PhS per os introduction in dose 500 mg/kg but their extent was twice smaller. Thus, we established effects of PhS and Dc impact on state of the MOGS, FRP, LP and ARPS. We concluded that intensification of LP may be considered a key biochemical process which determines early manifestations of PhS and Dc toxicity. 310 BLOCKERS OF VOLUME REGULATED Cl-CHANNELS: A NEW GROUP OF ANTI-CANCER DRUGS EXHIBITING LOW TOXICITY Jens Lichtenberg, Palle Christophersen. NeuroSearch A/S, Pederstrupvej 93, 2750 Ballerup, Denmark Volume regulated anion channels (VRAC) are important for cell volume regulation. Upon cell swelling VRAC activate and results in loss of salt (Cl− , K+ ) and water, thereby restoring cell size. Recently, VRAC has been suggested to be central in the control of cell proliferation (1), possibly indicating the need for tight volume control during the cell cyclus. In vitro, VRAC blockers arrest endothelial cells in G0/G1 and they are effective in models of in vitro angiogenesis as well (2,3). We have developed potent, non-toxic in vivo active VRAC blockers and tested them in various animal models of angiogenesis and cancer: Endovion, the most progressed compound (Phase 1 status) has shown anti-angiogenesis properties in a mouse model, producing comparable effect to standard cytotoxic compounds. Furthermore, Endovion has shown effects in various in vivo models of solid tumor growth and metastasis, showing similar efficacy as Paclitaxel. The toxicological profile is benign comprising of an increase of bilirubin caused by an inhibition of the UGT1A1 enzyme. 311 AMIFOSTINE PROTECTION AGAINST DOXORUBICIN-INDUCED RAT HEART MAST CELL ACCUMULATION S. Dobric 1 , V. Dragojevic-Simic 1 , V. Jacevic 1 , D. Bokonjic 1 , L. Zolotarevski 2 , K. Jelic 2 . 1 National Poison Control Centre, Military Medical Academy, 2 Institute for Pathology and Forensic Medicine, Military Medical Academy, Belgrade, Serbia and Montenegro Clinical use of doxorubicin (DOX), broad spectrum chemotherapeutic agent, is limited by its severe, dose-dependent cardiotoxicity. It is thought that this toxic effect is mediated mainly by highly reactive oxygen free radicals. DOX is also very powerful mast celldegranulating agent, and the releases of humoral mediators such as histamine maybe contribute to the development of cardiomyopathy. Amifostine (AMI), an agent with strong free radical scavenging properties, has already shown heart protective effects in DOX-treated rats. Our aim was to investigate the protective effects of AMI on DOX-induced changes in cardiomyocites, as well as on DOX effects s86 Poster Session P12. Drug toxicology on mast cells in epicardium, endocardium and myocardium of the rat heart. DOX was given in a dose of 1.25 mg/kg ip, 4 times per week, 4 weeks to adult male Wistar rats. Protected animals were given AMI 75 mg/kg ip each time 20 min. before DOX. The animals were sacrificed 28 days after the last dose of drugs and pathomorphological examinations of their hearts were done. The results showed that DOX significantly damaged cardiomycytes and increased the mast cells number in all examined structures of the rat heart. The increase was most prominent in the myocardium. Mast cells were accumulated all over the tissue, and were not only adjacent to small blood vessels and capillaries like in the control animals. Moreover, mast cell granules were more numerous and more densely stained in DOX-treated group comparing to the control one. AMI significantly ameliorated myocardial damage and decreased number of mast cells in all structures of the heart in DOX-treated rats. In protected rats, mast cells could also be seen everywhere in the myocardium, but they were predominantly situated around the blood vessels like in control. It could be concluded that AMI successfully protected rat hearts from DOX-induced toxicity, at least partly, by the reduction of mast cell number and, consequently, histamine release. 312 SAFETY PHARMACOLOGY STUDY OF CKD-732, A NEW FUMAGILLIN ANALOGUE ANTICANCER AGENT Eun-Joo Kim 1 , Hyun-Jin Kim 1 , Sun-Hee Do 1 , Joon-Kyum Kim 2 . 1 Korea Institute of Toxicology, KRICT, P.O.Box 107, Yuseong-gu, Daejoen, Korea, 2 Medical Research center, Jong Kun Dang Co. Kuro, Seoul, Korea The safety pharmacology core battery studies of CKD-732, a new fumagillin analogue anticancer agent, [20S-7-{2-(N-Isopropylamino) ethyl}-camptothecin·HCl], a member of the family of angiogenesis inhibitors, were conducted according to the ICH S7A guidelines in compliance with Good Laboratory Practice(GLP) Regulations. CKD732, a fumagillin anticancer agent, was developed by Jong Kun Dang Co. and possess anti-angiogenic activity in tumor treatment. In this study, the general pharmacologic properties of CKD-732 at high doses were investigated. The doses utilized in the study were selected in order to fully examine the potential pharmacologic activity of CKD-732, and therefore represent vast multiples of clinically effective doses of the drug. The drug was administered intravenously at doses of 10, 30, 40, and 50 mg/kg to mice, rats and guinea pigs. The high dosage of CKD-732 caused decreased body temperature and prolonged hexobarbital hypnosis time. However, CKD-732 demonstrated no effects on motor activity, behavioral changes, coordination and respiratory system even at high doses. 313 CYTOTOXICITY EVALUATION OF THREE PHTHALOCYANINE PHOTOSENSITISERS ON HUMAN CORNEAL EPITHELIUM CELL CULTURES D. Monti 1 , S. Burgalassi 1 , P. Chetoni 1 , G. Roncucci 2 , S. Tampucci 2 , M.F. Saettone 1 . 1 Dept. Bioorganic Chemistry and Biopharmaceutics, University of Pisa, Pisa, Italy, 2 Molteni Farmaceutici S.p.A., Firenze, Italy Photodynamic therapy (PDT) is a very promising approach for treatment of some types of cancer and other hyperproliferative diseases, as well as of selected nononcological diseases. A relatively new application consists of photoinactivation of yeast cells and bacteria. PDT involves administration of a photosensitising molecule and subsequent local irradiation: the resulting oxidative process leads to cell death and tissue necrosis. To verify the suitability of some photosensitising phthalocyanine molecules for ophthalmic applications, their cytotoxicity on human corneal epithelium (HCE) cell cultures was tested. The immortalised HCE cell line used in this study was developed by Araki and co-workers.Toxicity tests on HCE cells were carried out using a WST-1 commercially available cell proliferation reagent based on cleavage of the tetrazolium salt WST-1 by active mitochondria to produce a soluble coloured formazan salt. HCE were treated for 1 hour with appropriate concentrations (0.05 – 5.0 µM) of three phthalocyanines, then were irradiated for various time periods with red light (680 nm) using a 500 W halogen lamp at light fluences up to 22.5 J/cm2 . Twenty four hours after irradiation the cell viability was evaluated. Dark incubation of HCE cells for 1 hour with different concentration of phthalocyanines without subsequent photoactivation did not produce any toxic effect, while the toxicity was evident after irradiation. The results showed that toxicity was related to the total light fluence and to the specific activity of the photosensitising molecules. 314 NONSPECIFIC EFFECT OF NARCOTIC ANALGESICS ON THE MODELS OF CELL MEMBRANES S.M. Rogacheva 1 , P.E. Kuznetsov 2 , V.A. Zlobin 2 , G.V. Nazarov 2 , N.B. Kuznetsova 2 . 1 Department of Ecology, Saratov State Technical University, Saratov, Russia, 2 Department of Biochemistry and Biophysics, Saratov State University, Saratov, Russia The effect of narcotic analgesics is based on the ligand-receptor interaction process. The opiate receptors are located in the bilipid layer of the cell membranes. It is known that membrane lipids can modulate and regulate a reception process by means of their participation in the receptor-ligand recognition, or influence at the protein conformation. Besides, lipids may be responsible for the connection between a receptor and an enzyme system. That’s why the changes in the membrane bilipid layer may cause different metabolic displacement in the cell, in other words they may have a cytotoxic effect. It was shown earlier that some characteristics of membranes are changed by the action of opiates in accordance with their physiological activity. In this study we have investigated the effect of various opiates on the artificial bilayer lipid membranes. They were formed by using Muller’s method on the hole of the teflon pot, inserted into the thermostatic vessel with the stirring rod. The buffer solution was put into the vessel and into the pot, an opiate was dissolved only on one side of the membrane. Thus there was simulated the initial moment of opiate presence near the cell membrane. The time stability of the membrane was determined by using the optical microscope. It was found out that the membrane was destructed in the presence of definite concentrations (with the order of 10−9 – 10−4 M) of opiates. The correlation between these concentrations and opiates analgesic activity has been determined. Then the effect of morphine on liposomes and erythrocytes – cells without opiate receptors - has been investigated. The ability of morphine in concentration range of 10−2 – 10−8 M to change the ATP-ase activity and the hemolytic stability of erythrocytes and the viscosity of the liposome emulsions has been found out. The obtained data allowed us to suggest that low concentrations of the narcotic analgesics are toxic for cells because of their ability to change the condition of the membrane bilipid layer. 315 PL 14736: 14-DAY INTRACOLONIC TOXICITY STUDIES IN RATS AND DOGS Ž. Krnić 1 , M. Veljača 1 , M.K. Prinsen 2 . 1 PLIVA Pharmaceutical Industry Inc., Zagreb, Croatia, 2 Departments of General Toxicology and Analytical and Molecular Phamacology, TNO Nutrition and Food Research, Zeist, The Netherlands PL 14736 is a synthetic pentadecapeptide that shows protective and healing properties in trinitrobenzene sulphonic acid (TNBS) model of colitis in rats. It has been developed for the treatment of ulcerative colitis. Repeated dose toxicity studies were performed in rats (Wistar outbred (Crl:(WI)WU BR)) and dogs (Beagle). Groups of male and female animals received single daily doses of 0, 1, 5 or 25 mg/kg/day PL 14736 by intracolonic administration (catheter) for 14 days. Examination carried out during the study included clinical observations, body weight and food intake measurements, opthalmological examinations and laboratory investigations (haematology, blood chemistry and urinalysis). Electrocardiograms were recorded only in dogs. The necropsy was performed on day 14, the principal Poster Session P12. Drug toxicology organs were weighed out and histopathological examination was performed. In rats and in dogs, 14 days repeated intracolonic administration of PL 14736 revealed decreases in RBC’s, Hb concentration, PCV and an increase in the number of reticulocytes. These effects were probably related to the repeated blood sampling for toxicokinetic determinations. In rats, gross pathology and histopathological examination did not reveal treatment-related changes, other than lesions (inflammation with haemorrhages) at the area of the colon application site that was primarily caused by mechanical trauma of the catheter uses. The inflammation was slightly more pronounced in male PL 14736 treated groups. These lesions were not observed in the dog study. In conclusion, 14 days repeated intracolonic administration of PL 14736 at dose levels of 1, 5 or 25 mg/kg/day in rats and dogs was generally tolerated without distinct test substance related adverse effects, although a minor effect on the RBC’s, Hb concentration, PCV and the number of reticulocytes cannot be completely excluded. 316 COMPARATIVE STUDY OF DEXTROMETORPHAN ASSAY BY UV SPECTROPHOTOMETRY AND SPECTROFLUORIMETRY A. Florea 1 , D.L. Baconi 2 , A. Popa 2 , M. Ilie 2 , D. Balalau 2 . Medicines Agency, Bucharest, Romania; 2 “Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Bucharest, Romania 1 National Dextrometorphan is relatively safe even in overdose and intoxications are not very often. But severity of intoxications is increased in mixtures with other medicines (monoamine oxidase inhibitors – MAOI or serotonin re-uptake inhibitors). Possible risks of dextrometorphan abuse result in the necessity of sensible and specific analytical methods for quantifications of dextrometorphan. The aim of this paper is the evaluation of two methods of dextrometorphan assay: UV spectrophotometry and spectrofluorimetry. The UV spectrum of dextrometorphan hydrobromide aqueous acid solution exhibits a maximum of absorbance at 278 nm. Calibration curve proves the linearity of standard response in the range 10 – 80 µg/ml; the detection limit determined was 2 µg/ml. Dextrometorphan aqueous acid solution is fluorescent, with maxima at 304 nm (after excitation at 278 nm). The spectrofluorimetric calibration curve performed in the range of concentration 1 – 100 ng/ml reveals a good linearity with a very low detection limit 0.1 ng/ml. The reproductibility of the elaborated methods have been verified on plasma samples collected from patients admitted in Bucharest Emergency Hospital. The confirmation of the results of the comparative study regarding the two methods has been obtained by using HPLC methods with UV and fluorimetric detection. 317 SAFETY OF PANAX GINSENG AQUA-ACUPUNCTURES SOLUTION ON P450-DEPENDENT DRUG METABOLISM IN CULTURE OF ADULT RAT HEPATOCYTES Boo Hyeong Byun 1 , Eun-Joo Kim 2 , Sun-Hee Do 2 . 1 Dept of Oriental Medicine Kyungsan Univ., Daegu, Korea, 2 Korea Institute of Toxicology, KRICT, P.O.Box 107, Yuseong-gu, Daejoen, Korea Acupuncture is one of the main treatment mechanism in oriental medicine which works by controlling the functions of internal organs by stimulating points on the skin. Panax ginseng aquaacupuncture therapy is a new type of acupunture treatment method that incorporates acupuncture meridian theory, and herbal medication to stimulate meridian points on the body. The present study was designed to investigate on the safety of Panax ginseng aquaacupuncture solution in primary culture of adults rat hepatocytes. Primary culture of adults rat hepatocytes has been considered as an ideal model for toxicological studies because cultured hepatocytes maintained many liver-specific functions. In this research, we investigated the effects of Panax ginseng aqua-acupuncture solution (1–10 /ml) on P450-dependent drug metabolism in primary culture of adult rat hepatocytes using P450 isozyme specific enzyme activity assays. Treatment of Panax ginseng aqua-acupuncture solution did not sig- s87 nificantly affect constitutive and inducible ethoxyresorufin O deethylation (P450A1/2) and pentoxyresorufin O-dealkylase (P450B2/1). Hepatic glutathione level, glutathione-s-transferase activity levels, and albumin synthesis were not affected by treatment with Panax ginseng aqua-acupuncture solution alone. According to the results, it is suggested that hepatotoxicity was not affected by treatment with Panax ginseng aqua-acupuncture solution alone. 318 STUDY ON SINGLE DOSE TOXICITY OF PANAX GINSENG AQUA-ACUPUNCTURE SOLUTION IN RATS Boo-Hyeong Byun 1 , Bu-il Seo 1 , Soon-Jae Rhee 2 , Eun-Joo Kim 3 , Sun-Hee Do 3 . 1 Dept of Oriental Medicine Kyungsan Univ., Daegu, Korea, 2 Dept of Food and Nutrition, Dae-gu Catholic Univ., Korea, 3 Korea Institute of Toxicology, KRICT, P.O.Box 107, Yuseong-gu, Daejoen, Korea The purpose of this study is to investigate the single dose toxicity of Panax ginseng Aqua-Acupuncture solution, a herbal acupuncture in rats. Panax ginseng, a herbal acupuncture was once administered to both sexes of rats at the dose levels of 2000 mg/kg, 1000 mg/kg, 500 mg/kg, and 125 mg/kg for oral route. After single administration, clinical sign were observed every day for 14 days and body weights were measured 5 times including initial measurement on day 0 (the days of administration). When observation period was over, the animals were sacrificed and microscopic examination major organs was conducted. In addition, the histopathological profiles of these major organs were also conducted. Neither significant clinical signs nor death after administration was observed during the observation periods except for soft feces or diarrhea. No abnormal necropsy findings, changes of body weight and histopathological profiles were observed at terminal necropsy in both sexes. From these results, it is considered that LD50 of Panax ginseng Aqua-Acupuncture solution is over 2000 mg/kg in oral administration in both sexes of rats. 319 TOXICOLOGICAL STUDY ON THREE HERBAL FORMULATIONS IN IRAN M.R. Heidari, M. Hassibi, A. Mondegari. Department of toxicology and pharmacology, Kerman University of Medical Sciences, Kerman, Iran Some of medicinal herbs are marketed without enough knowledge about their toxicological properties today, but there are some reports about toxic effects of these herbal medicine. The effect of three medicinal herbs, Cassia angustifolia, Cassia obovata and Rosa damascena which used as laxative and purgative, on enzymatic liver function tests was studied in this investigation. Percolated extracts of these herbs with doses of 200, 800 and 1600 mg/kg/day were orally administrated to groups of 5 rats for 5 days; then blood serum samples of animals were used for determination of ALT, AST and ALP. Weights of animals were determinated before and after tests. Data have been shown no significant difference in ALP and AST enzymes in comparison to control group. ALT decreased in group which received C. angustifolia and R. damascena extract (p< 0.05). The animals which received extracts showed a less weight increase than control group The results of this investigation suggested that use of herbal medicine also need the toxicological studies. 320 BIOLOGICAL ACTIVITY OF ARNEBIA EUCHROMA ROYLE K. Javidnia 1 , R. Bahri Najafi 1 , A. Jafari 2 . 1 Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran, 2 Research center of Natural Resource and Animal Husbandry, Yasuje, Iran Arnebia euchroma Royle ssp. euchroma is grown wildly in Iran. It is used as anti-infective and anti-inflammatory agent in folk medicine. Ether, ethylacetate and methanolic extract of the plant were prepared. Antibacterial and antifungal activities of the extracts were studied by Disc Diffusion method. The extracts didn’t have antifungal activity, but methanolic extract showed antibactrial activity especially against S. aureus (62.5 µg/disc) and ethylacetate extract had antibacterial activity against B. subtilis (12.5 µg/disc). Cytotoxicity of three s88 Poster Session P12. Drug toxicology extracts was studied by brine shrimp method. The results showed that ethylacetate extract was most cytotoxic (LD50 = 150 µg/cc), the alcoholic extract had LD50 = 205 µg/ cc and the ether extract didn’t show cytotoxocity. 321 STUDY ON ADSORPTION OF ET-743 TO MATERIALS USED IN INTRAVENOUS RODENT INFUSION STUDIES J. Verbeeck 1 , A. Looszova 1 , T. Verhaeghe 1 , L. Diels 1 , L. Lammens 1 , R. De Coster 1 , I. Manzanares 2 , P. Aviles 2 , W. Coussement 1 . 1 Johnson and Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V., GPCD/PCDE, Beerse, Belgium 2 PharmaMar, Madrid, Spain Yondelis™ (trabectedin, ET-743) is a marine derived anti-cancer agent with a unique mode of action that is administered intravenously in low concentrations and, in rodent infusion studies, at low flow rates (∼ 5 ml/kg b.w./h). Adsorption of the compound to the infusion materials used in these studies becomes problematic. The purpose of the presented work was to determine and assess the extent of adsorption of Yondelis™ to different catheter and syringe materials and to select appropriate materials for future rodent infusion studies. LC-MS/MS was the method of choice to analyze the formulation for Yondelis™. Firstly, adsorption to different materials was tested after overnight instillation of an Yondelis™ formulation. Adsorption was slightly higher for polyethylene than for glass syringes, but remained acceptable. Polyethylene and silicone tubing showed clearly more adsorption than polyurethane tubing. Secondly, polyurethane tubing, in combination with a polyurethane catheter, was tested in a mock-up infusion setting. A polyethylene syringe was used, as adsorption to this type of syringe was acceptable. Infusion was performed using a low concentration (0.67 µg/ml, 0.75 ml/h) over 24 hours and a high concentration (3.33 µg/ml, 1.5 ml/h) over 3 hours. In the first setting, adsorption was clearly present in the beginning of the infusion but decreased (saturation). Total adsorption was between 10 to 13% which is considered acceptable. Infusing a formulation at the high concentration over 3 hours caused a total adsorption of about 9%. In vitro adsorption experiments indicate that adhesion of Yondelis™ to polyethylene and silicone infusion lines is clearly present. Yondelis™ is compatible with polyurethane infusion lines, which will be used as the material of choice in future rodent infusion studies. The impact on clinical studies is negligible since higher flow rates and/or concentrations are used in these studies. 322 in acetaminophen intoxication. Melanins are stable polyradical, contain some semiquinone radicals and accumulate the active oxygen species and electrophyl toxic compounds. Some thise properties determine the antitoxic activity of biomelan preparation. The revealed biomelan hepatoprotective activity makes it possible to be applied as an effective antidote therapy under acetaminophen toxicity. SUBSTATIONAL OF HEPATOPROTECTIVE ACTION OF BIOMELAN PREPARATION IN EXPERIMENT A. Shayakhmetova, G. Saifetdinova, V. Kovalenko, O. Voloshina. Institute of Pharmacology and Toxicology, Kyiv, Ukraine, Pharmacological Centre, Ministry of Health, Kyiv, Ukraine Hepatoprotective efficacy of a new original sorbent biomelan preparation was studied under acetaminophen toxicity using Wistar rats. Acetaminophen was administered intragastrically for two days in the dose 1.25 mg/kg. The biomelan preparation (it built on a basis of melanin from the biomass of fungal producent Cladosporidium cladosporioides) was administered intragastrically, treatment-andprophylactic in the dose 10 mg/kg. After treatment of acetaminophen the cytochrome P-450 and b5 level in liver microsomes decreased by 2 fold, the liver protein SH-groups contents and catalase activity decreased by 31% and 15%. We observed a decrease of cholesterol level by 19% and an increase of AlAT by 2.5 fold in serum of blood as compared to intact animals. It was shown biomelan preparation administration as treatment or prophylaxis significantly attenuates analgesic toxicity enhancing liver detoxication via concerving both cytochrome P-450 and b5 content. Biomelan administration prevents electrophylic acetaminophen metabolites binding with the free protein SH-groups, normalized the cytochrome P-450 and b5 level, increases enzyme antioxidant catalase activity by 2 fold as compared to acetaminophen treated rats, corrects lipid turnover as well as limits the phenomenon of hepatocytolysis. Administration of biomelan preparation to rats exerted significant normalizing influence on liver function exceeding efficacy of metionine, which is use as an antidote 323 CYTOTOXICITY AND ANTIMICROBIALBIOL ACTIVITY OF EUPHORBIA HEBECARPA BOISS R. Miri 1 , K. Javidnia 1 , A. Jafari 2 . 1 Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran, 2 Research center of Natural Resource and Animal Husbandry, Yasuje, Iran Euphorbia is a family of plants spread in all parts of the world exept in cold regions. Some reports have been shown that the genus Euphorbia has cytotoxicity and anti tumor activity. Euphorbia hebecarpa Boiss ia an endemic plant of Iran, which in literature review no reports can be found on its pharmacological activities. Four extracts (petroleum ether, ether, ethylacetate and methanolic) of the plant were prepared by maceration method. Antibacterial and antifungal activities of the extracts were studied by Disc Diffusion method. The ethylacetate and methanolic extract showed a moderate antibacterial and antifungal activity. Biological activities of four extracts were studied by brine shrimp method. The results showed that methanolic extract was most cytotoxic (LD50 = 11 µg/cc). Cytotoxic activity of methanolic extract was studied on four different cell lines by MTT assay. The methanolic extract was mostly active against K562 and vero cell lines. 324 ANALGESIC ACTIVITY OF IRANIAN HYPERICUM PERFORATUM K. Morteza-Semnani 1 , M. Mahmoudi 2 , M. Saeedi 1 , A. Javanmardi 1 . 1 Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran, 2 Department of Pharmacology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Hypericum is a genus of about 400 species, wide-spread in warm temperature areas throughout the world and well represented in the mediterranean area. Some species of this genus are used in folk medicine as anthelmintics, diuretics, on wounds, scalds and herpes. One of the most important species of this genus is Hypericum perforatum L. (Hypericaceae). The analgesic effect of Iranian Hypericum perforatum extract, a native plant of Iran, was studied using formalin, hot plate and writhing tests. The aerial parts of plant were dried in the shade and powdered. Powder was Soxhlett-extracted with methanol for 16 h., then this extract was evaporated to dryness and weighed (24 g, 24%). Just prior to use, the dried methanolic extract was dissolved in a mixture of propylene glycol and water (1:4). We have also tested the analgesic doses of extract for its effect on motor coordination by rotarod test. In the formalin test, the extract (25–250 mg/kg, i.p.) caused graded inhibition of both phases of formalin-induced pain (P<0.001). In the hot plate test, the i.p. administration of the extract at the doses of 25- 250 mg/kg significantly raised the pain threshold at an observation time of 30 min in comparison with the control group (P<0.001). In the writhing test, the extract at doses of 25 mg/kg (P<0.05), 50, 75, 100 and 150mg/kg (P<0.001) produced a significant decrease in the number of writhing in comparison with the control group. The extract, at antinociceptive doses, did not affect motor coordination of animals when assessed in the rotarod model. It seems that the extract relieved pain through both central and peripheral mechanisms. Based on the results of this study, we suggest that the anti-nociceptive effect of this extract may be attributed to inhibition of prostaglandin synthesis or release and other mediators. Poster Session P12. Drug toxicology 325 ANTI-INFLAMMATORY ACTIVITY AND ACUTE TOXICITY OF IRANIAN HYPERICUM PERFORATUM M. Mahmoudi 1 , K. Morteza-Semnani 2 , M. Saeedi 2 , A. Javanmardi 2 . 1 Department of Pharmacology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran, 2 Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran Hypericum perforatum is a medicinal plant, which has been known in traditional medicine as anti-inflammatory and healing agent. Hypericum perforatum L. was collected from the suburb of Yasuj, in the west of Iran, in June 2001. The anti-inflammatory effect of Iranian Hypericum perforatum extract, a native plant of Iran, was studied using carrageenan-induced edema in the hind paws of rats. Just prior to use, the dried methanolic extract was dissolved in a mixture of propylene glycol and water (1:4). The extract at the doses of 25, 50, 75, 100 and 150 mg/kg and indomethacin at the dose of 4 mg/kg were administered intraperitoneally (i.p.). Drugs or drugless vehicle were injected 1h before the carrageenan treatment. Paw volume was measured immediately after carrageenan injection and at 1-, 2-, 3- and 4-h intervals after the administration of the edematogenic agent using a plethysmometer. The degree of swelling induced was evaluated by the ratio a/b, where a and b are total volumes of both hind paws after and before carrageenan treatment, respectively. A ratio smaller than 1.5 after drug administration was considered as a significant inhibitory effect of the drugs. The degree of swelling (a/b) during 3h after carrageenan injection was < 1.5 at the doses of 25–150 mg/kg of the extract. Similar activity against carrageenan-induced rat paw edema was observed with Hypericum perforatum extract (100 and 150 mg/kg) and indomethacin (4 mg/kg), 3h after carrageenan injection. The degree of swelling at the doses of 100 and 150 mg/kg of the extract was no significant (P>0.05). The LD50 determined by probit test using a death percent versus doses’ log. The 72h acute LD50 value of this extract after i.p. administration in mice was 1111.47 mg/kg. 326 ISOLATION AND CHARACTERIZATION OF ROSMARINIC ACID FROM THE PLANT CORDIA VERBENACEA F.K. Ticli 1 , S.M.C. da Costa 1 , J.O. da Silva 1 , P.S. Pereira 2 , S.V. Sampaio 1 . 1 Laboratório de toxinologia, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto - SP. Brasil, 2 Unidade de Biotecnologia, Universidade de Ribeirão Preto, Ribeirão Preto - SP. Brasil Cordia verbenacea, popularly known in Brazil as “baleeira”, belongs to the family Boraginaceae. Phytochemical studies conducted on this genus have demonstrated the presence of flavonoids, glycosylated or not, rutin, phenol derivatives, and hesperidine, among others. Extracts of this plant are used in Brazilian folk medicine as healing and anti-inflammatory agents. Investigators have demonstrated that the crude extracts and artemetin isolated from this plant have an anti-inflammatory action and low toxicity in rats. The objective of the present study was to isolate a new substance with an antiinflammatory effect from the plant Cordia verbenacea. Leaves of the plant were picked at blooming time, dried in an oven with circulating air at 40°C, and ground. The ground leaves were macerated in CHCl3 for 3 days, the solution was filtered and the procedure was repeated three times using the same solvent. The residue was extracted with MeOH to obtain the methanol extract and the filtered solution was evaporated in a rotary evaporator. The MeOH extract obtained was fractionated on a Sephadex LH-20 column (35 x 2 cm) using MeOH as the mobile phase, and 3 fractions were obtained. Fraction no 3 (F3) was refractionated by HPLC through a semipreparative supelcosil C-18 column eluted with MeOH/H2 O in an elution gradient with a flow of 2 mL/min, and seven sub-fractions (CL1 to CL7) were obtained, with sub-fraction CL6 being found to be pure in CCDC. Sub-fraction CL6 was submitted to spectrometric analysis (RMN 1 H, RMN 13 C, COSY, HMBC, HMQC and IV) in order to obtain the molecular structure of this substance, which was characterized as rosmarinic acid, a polyphenol compound described for the first time as a secondary metabolite of the plant Cordia verbenacea. 327 s89 ANTI-INFLAMMATORY AND ANTIOXIDANT PROPERTIES OF C-PHYCOCYANIN. Diadelis Remirez 1 , Ricardo Gonzalez 2 , Cheyla Romay 2 , Nuris Ledon 3 . 1 National Center for the quality control of drugs and vaccines, 2 Center of ozone investigations, 3 Molecular Immunology Center Phycocyanin (Pc) is one of the major constituents of Spirulina, a microalgae used in many countries as dietary supplement whose nutritional and therapeutic value has been very well documented. In this regard antioxidant and anti-inflammatory effects have been experimentally studied to Pc, when it was evaluated as an antioxidant in vitro and it was able to scavenge alkoxyl, hydroxyl and peroxyl radicals and to react with peroxinitrite and hypochlorous acid. Pc also inhibits microsomal lipid peroxidation by Fe 2+ -ascorbic acid or the free radical initiator 2,2’ azobis (2-amidinopropane) hydrochloride. Pc has been evaluated in several models of inflammation such as zymosan-induced arthritis, acetic acid-induced colitis, skin reactions to histamine and compound 48/80 and endotoxin-treated animals. Pc was administered in a range of doses of 100–300 mg/kg 30 min before the agent which induces the injury. Pc exerted antiinflammatory effects in a dose-dependent fashion in all of these. Thus, Pc reduced edema, histamine release, myeloperoxidase activity and the levels of prostaglandin and leukotriene in the inflamed tissues. These anti-inflammatory effects of Pc can be due to its scavenging properties towards oxygen reactive species and its inhibitory effects on cyclooxygenase 2 activity and on Hi release from mast cells. Taking into account that Pc is a major constituent of microalgae Siprulina (20% of algae dry weight), it might exert therapeutic effects when it is administered alone or included in the microalgae used as dietary supplement. To our knowledge these are the first reports on the anti-inflammatory and antioxidant effects of phycocyanin. 328 DETERMINATION OF LEAD AND CADMIUM CONTENTS IN MEDICINAL HERBAL RAW MATERIALS Z. Nazari, N. Poorreza. Dept. of toxicology & Pharmacology, Pharmacy school, Ahvaz University of Medical Sciences, Ahvaz, Iran Herbal supplements are being widely used as alternatives to conventional drugs. Increased use requires that appropriate methods to evaluate both the safety and efficacy of this product be put into place. Because medicinal herbal raw materials are potential sources of exposure (via orally) there is an increasing attention to the contamination of heavy metals in these supplements. Many studies on Pb and Cd in medicinal herbal have been conducted in other countries, however little has been done in Iran. Therefore, to evaluate the lead and cadmium levels in Cichorium intybus L. and Valeriana officinalis, this study was carried out. For this purpose, 10 samples of each two herbs, were collected. An adequate amount of each milled sample was weighted into an ashing vessel, covered with a lid and dried at 110C°-120C° an oven. Then vessel was placed in a cold furnace and the temperature was set at 500C°-550C° and kept at this temperature over until white carbon free ash was obtained. Then removed and cooled. Residue was dissolved in HNO3-H2O (1+9), quantitatively transferred to 50 ml volumetric flask and diluted with water. Deionized water was used for blank. Citric acid 10% and 3 drops of bromo-cresol-green 1% in ethanol were added to aliquot volumes of sample, blank and standard solution, adjusted to ca pH 5.4 by adding NH4OH and citric acid. 5 ml APDC 2% (Ammonium pyrrolidinedithiocarbamate ) and n-butyl acetate were added to them and shake vigorously 2 min. Then organic phases were separated and the amount of Cd +2 and Pb+2 in samples were measured by Flame Atomic absorption Spectrometer. The mean values of lead in Cichorium intybusL. and Valeriana officinalis were 0.45 and 0.48 mg/kg respectively. Also the mean value of Cd in cichorium intyus and valeriana officinalis were0 0.017 and 0.021mg/kg respectively. s90 329 Poster Session P12. Drug toxicology INVESTIGATION OF SYSTEMIC TOXICITY AND LOCAL TISSUE TOLERANCE OF A NEW MICROPOROUS IMPLANT Lj. Pitic 1 , J. Markov 1 , D. Jeremic 1 , D. Djukanovic 2 . 1 Biomedical Research Center, Institute, Galenika a.d., Belgrade, Serbia and Montenegro, 2 Clinic for Peridontology and Oral Medicine, Faculty for Stomatology, University of Belgrade, Serbia and Montenegro Aldovit is a new bioceramic microporous Al trioxide implant used for filling of alveolar infrabones defects. According to ISO 10993 standard all new implant materials must undergo preclinical testing prior to investigation in humans. The aim of this paper was to study acute i.v. and short term oral toxicity (systemic safety) and local tolerance of Aldovit in prolonged contact with subcutaneous tissue.For acute i.v. toxicity study, NMRI Haan (20±1g) mice were used treated with a single intravenous dose of 50ml/kg of Aldovit extract. For short term oral toxicity study, Wistar rats (150±10g) were used given with daily oral Aldovit dose of 1g/kg for 7 consecutive days. The observation period for both toxicity tests was 14 days, with the test animals regularly examined for clinical signs of toxicity and possible mortality. At the end of the study all the animals were undergone macroscopic and histopathological organ examination. Albino guinea pigs (400±20g) were used in subcutaneous implant test. Aldovit granules were applied in a dose of 10 mg into skin pockets created in dorsal quadrants of anesthetized animals for a contact period of 2. or 12. weeks. Local tissue tolerance in the form of inflammatory reaction of different degree to tissue disintegration as the consequence of the prolonged test material and tissue contact is assessed based on histopatological examination of the treated animals and expressed in terms of gradation from slight to severe tissue reaction. The results obtained from systemic safety studies show that, there were no deaths and no manifested clinical signs of toxicity. Histopathological organ examination (heart, lungs, spleen, liver, kidneys, adrenal glands, pancreas, stomach) showed no pathological changes either. Subcutaneous connective tissue didn’t show signs of hyperemia, lymphoid cells, connective tissue cell organization and any other tissue reaction. Aldovit, applied i.v. and orally, induces no signs of systemic toxicity and can be with great certainity considered safe.Also, the local tissue tolerance of Aldovit is good at prolonged subcutaneous contact. 330 mesial defects were implanted by Al-trioxide granules, while the distal ones were filled by Interpore 200, as control. Animals were sacrificed 6 weeks, 3 and 6 months after surgery. Demineralized bone preparations were cut by cryocut (-200 C) and stained by haematoxylin-eosin. The results have shown that Aldovit produced no cytotoxic effects in direct contact with the fibroblast cells culture, in vitro, because it produced neither malformation, degeneration, spreading, nor lyses of fibroblasts cells. Histological immature connective tissue cells and fibers penetrating between the implant granules, from periphery to the center were observed. Signs of inflammation and incapsulation haven’t been noticed. On the basis of these preclinical studies it can be concluded that test material Aldovit is noncytotoxic and it is a biocompatible bone implant. So, it may be recommended for clinical study. 331 PHARMACOLOGY AND TOXICOLOGY OF OXIME REACTIVATORS OF CHOLINESTERASE C. Dishovsky. Department Experimental Toxicology, Military Medical Academy, Sofia, Bulgaria In order to improve the treatment of poisoning with highly toxic organophosphates, different laboratories have synthesized a variety of bispyridinium mono- and dioximes over the last few years. Prof. Ilze Hagedorn and her team gave new direction in the development of the reactivators of cholinesterase activity. The most well-known among them is HI-6. Others and our research so far show that at the moment HI-6 is one of the best reactivators of the acetyl cholinesterase inhibited from Soman. Our investigation has shown that HI-6 has lower acute toxicity in comparison with the oximes, such as HS-3 and HS-6, as well as the classical toxogo-nin and 2-PAM. We consider that the recovery of the neuromuscular transmission is an important mecha-nism of antidotal action of the oximes and it is not related to the cholinesterase reactivation. Our SAR studies demonstrated that the H-oxime’s molecule could be considered as consisting of two parts - the first one is responsible for the binding with choline receptor, and the second one interacts with the molecule of the organophosphate compound. The investigation of the concentration-time profile of oximes alone and after intoxication with nerve agents is important. The rate of absorption, distribution and elimination of oximes could give some explanation of their prophylactic and therapeutic action during intoxication with nerve agents. Therefore, we suggest that the monitoring of oximes’ plasma levels will improve the therapy of these intoxications. EVALUATION OF CITOTOXICITY AND BIOCOMPATIBILITY OF BIOCERAMIC MICROPOROUS IMPLANT J. Markov 1 , Lj. Pitic 1 , D. Djukanovic 2 . 1 Biomedical Research – Institute, Galenika a.d. Belgrade, Serbia and Montenegro, 2 Clinic for Periodontology and Oral Medicine, Faculty for Stomatology, University of Belgrade, Serbia and Montenegro Various microporous allolplastic implants are used in the regenerative treatment of infrabone defects in the patients with periodontal diseases. Prior the application of any new implant in humans, it is obligatory to perform peclinical tests required by ISO standard. The aim of this preclinical study was to assess cytotoxicity in vitro and biocompatibility of the alloplastic aluminium trioxide implant. Cytotoxic potential of Aldovit (Al-trioxide) implant was examined in direct contact with a cell culture, in vitro. Cells NCTC, clone L 929 (fibroblasts separated from adipose tissue of the C 3H strain mice) were cultured on the RPMI enriched medium until confuent. Fibroblasts were devided into 3 Petri dishes with addition of 2ml of fresh medium. Sterilized sample of test material Aldovit was placed in one dish. A positive control was 0.4% sterile aqueous solution of foraldehide in the second dish, while negative control was a sterile inert plastic catheter (10x10x2mm)placed into the third dish. The coded dishes where incubated for 24h at 370 C with 5% CO2 . Microscopic examination of test samples was done post-incubation. The local effects after implantation (biocompatibility) of test material were evaluated in the mandibular premolar regions of six German shepherd dogs. In the course of MWFO, arteficially created 332 STORAGE OF UNUSED MEDICATIONS IN A SAMPLE OF TEHRAN’S HOUSEHOLDS. H.R. Rasekh, F. Roshan-Zamir. Department of Pharmacology/Toxicology, Shaheed Beheshti School of Pharmacy, Tehran, Iran Since prescribed drugs might not necessarily be fully consumed, the unused portion of the medication can be stored permanently at home which might lead to possible self-medication and poisoning. In the following study, 1000 households were randomly selected in Tehran. The amount of stored (currently unused) drugs in each home was recorded using a questionnaire. The results were reported in terms of dosage forms, expiration dates, quantity, and class of drugs. It was shown that 77% of respondents had some types of medical insurance and average number of household members was 4. Antibiotics were the most stored of class drugs (8165 units, 33% of total), then analgesics and CNS drugs (6102, 24%) and followed by G.I. drugs (4421, 18%). It was found that total number of stored (unused) drugs at homes were 25,124 units from which tablet forms had the highest number (60% of stored medications). Mean number of stored drugs in each household was 25±15 units. Among the respondents, 36% thought that parentral dosage forms were the most effective form of medications, whereas, 23% selected capsules and 41% tablets as the most effective dosage form in the treatment of disease. It was also concluded that 46% of respondents have somehow obtained their medication from Poster Session P13. Cardiovascular toxicology black market, and that only 65% of respondents have completely finished their prescribed antibiotics. It was found that 66% of respondents have always received proper medication consultation from physicians, whereas, pharmacist consultation included only 46% of respondents. Antibiotic consumption is of wide importance both from an economics and health standpoint. The following study points out to some potential points of concern namely an emphasis for medication consultation in health care personnel especially pharmacists and also patient education for proper drug consumption and potential poisoning prevention 333 EFFECTS OF NALOXONE AND YOHIMBINE ON MORPHINE AND CLONIDINE ANALGESIA IN MICE I. Ebrahimi, M. Roohi Azizi, M.R. Keyhani. Laboratory of Clinical Neurophysiology, Department of Basic Sciences, Faculty of Rehabilitation, Iran University of Medical Sciences, Tehran, Iran Several studies showed that morphine is a very potent and valuable agent in decrease of pain, but produces physical dependency and its abrupt cessation can lead to a withdrawal syndrome. On the other hand, clonidine (an α 2- adrenergic receptor agonist) has been used in the treatment of hypertension, opioid detoxification, reduction of morphine withdrawal syndrome and pain management by suppression of nor- adrenergic system, although sudden cessation of it produces a similar withdrawal syndrome. On the basis of our studies, physical dependency in clonidinedependent mice was observed by precipitating an abstinence syndrome with both yohimbine (an α 2-adrenergic antagonist)and naloxone (an opioid antagonist), although in morphine-dependent animals this syndrome was observed by injection of naloxone only. It has been shown that analgesic activity of morphine and high doses of clonidine almost were equal to each other in tail immersion test and they were reversed partially by both antagonists in a dose dependent manner and produce hyperalgesia together with withdrawal symptoms. These results suggest that similar mechanisms and pathways exist between the development of dependence on the noradrenergic and opioid systems, although role of other neurotransmitter systems are probable. P13 Cardiovascular toxicology 334 EFFECTS OF ARTESUNATE, AN ANTIMALARIAL AGENT, ON hERG CHANNEL CURRENTS EXPRESSED IN CHO CELLS Eun-Joo Kim 1 , Ki-Suk Kim 1 , Sun-Hee Do 1 , Sang-Seop Han 1 , Boo-Hyeong Byun 2 . 1 Korea Institute of Toxicology, KRICT, P.O.Box 107, Yuseong-gu, Daejoen, Korea, 2 Dept of Oriental Medicine Kyungsan Univ., Daegu, Korea Prolongation of the QT interval may result in a potentially dangerous arrhythmia. The most commonly proposed mechanism for QT interval prolongation(LQT) by pharmaceuticals is inhibition of the rapid delayed rectifier potassium channel (IKr ). The LQT potency of pharmaceuticals can be effectively evaluated by examining the effect on hERG channel expressed in CHO cells, known to be equal to IKr . The objective of this study was to investigate the effect of Artesunate, an antimalarial agent, on hERG channel currents to evaluate the LQT potency. Artesunate, [(3R, 5aS, 6R, 8aS, 9R, 10R, 12R, 12aR,)-decahydro-3,6,9-trimethyl-3,12epoxy-12H-pyrano-[4,3-j]-1,2-benzodioxepin-10-yl] ester, is an antimalarial agent, available in oral, rectal and parenteral formulations, that provides a rapid clinical effect in patients with Plasmodium falciparum malaria. E-4031, the positive control substance, was used to compare the effect of Artesunate. E-4031 decreased hERG channel currents dosedependently. IC50 was 7.977 nM. However, Artesunate caused no effect on hERG channel current in the much higher concentration(100 µM) than anticipated maximal therapeutic plasma concentration(0.57 µM). 335 s91 METABOLISM IS REQUIRED FOR THE EXPRESSION OF ECSTASY-INDUCED CARDIOTOXICITY IN VITRO Márcia Carvalho 1 , Fernando Remião 1 , Nuno Milhazes 2 , Fernanda Borges 2 , Eduarda Fernandes 3 , Félix Carvalho 1 , Maria Lourdes Bastos 1 . 1 REQUIMTE, Toxicology Department, 2 CEQOFFUP, Organic Chemistry Department, 3 REQUIMTE, Physical-Chemistry Department, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha, 164, 4050/047 Porto, Portugal The metabolism of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has recently been implicated in the mechanisms underlying ecstasy-induced neurotoxicity and nephrotoxicity. However, its potential role in ecstasy-induced heart toxicity remains to be investigated. Thus, freshly isolated adult rat cardiomyocytes were used to evaluate the toxicity induced by MDMA and its major metabolites 3,4-methylenedioxyamphetamine (MDA), N-methylα-methyldopamine (N-Me-α-MeDA) and α-methyldopamine (αMeDA). The cells suspensions were incubated with these compounds in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 4 hours. The toxic effects were evaluated by measuring cellular morphology and viability, levels of reduced (GSH) and oxidized (GSSG) glutathione, and the activities of glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST). MDMA and MDA had a negligible effect in all parameters evaluated. In contrast, their catechol metabolites N-Me-α-MeDA and α-MeDA induced a concentration- and time-dependent GSH depletion that was accompanied by an initial loss of normal cell morphology followed by cell death, and significant decreases in GR, GPX and GST activities. GSH depletion was not accompanied by increase in GSSG levels, which indicates that the catechol metabolites of MDMA are being conjugated with GSH. The present study provide evidence that metabolism is required for the expression of MDMA-induced cardiotoxicity in vitro. This work was supported by a project grant from FCT (PraxisXXI/BD/20087/99). 336 THE EFFECTS OF ACUTE AND SUBACUTE LEAD ACETATE EXPOSURE ON THE RESPONSIVENESS OF ADRENERGIC SYSTEM IN RAT AORTA Ali Khoshbaten 1 , R. Dehpour 3 , G.R. Karimi 3 , S. Tabaei 2 , M.R. Zarrindast 3 , M. Fahim 4 , A. Asgari 1 , A. Noroozzadeh 1 , A. Heydari 1 . 1 Department of Physiology and Biophysics, University of Baghiyatollah (A.S.) Medical Sciences, 2 Department of Physiology, Azad University, 3 Department of Pharmacology, Tehran University of Medical Sciences, 4 Department of Physiology, Delhi University of Medical Sciences Exposure to lead causes increased blood pressure in humans and experimental animals. The precise mechanism by which lead induces hypertension remains uncertain and there are different possibilities and factors including alteration of adrenergic system responsiveness to be involved. In this study, the effect of acute and subacute (100ppm 0.01% for 28 days) lead acetate exposure on thoracic aorta were investigated. Adult male Sprague-Dawley rats (200– 250 gram) were used, thoracic aorta was removed either from the pretreated rats with lead acetate, or normal rats and then placed in cold physiological solution and cleaned off from fat and connective tissues. 2–3 mm rings of aorta were sectioned and mounted in 10ml tissue bath for measurement of isometric contraction. The tissue bath contained krebs’ solution at 37°C gassed with 95% O2 plus 5% CO2 and were renewing the buffer every 15minutes. After stabilization, the preparations were contracted twice with KCL (40mM) and the last contraction was taken as the reference value for analysis. Contraction responses were assessed by adding cumulative concentration of different agents such as Phenylephrine, Clonidine, Dopamine, Yohembin, Prazosin and SCH in presence and absence of L-NAME and Indomethacin. Although the results showed that both acute and subacute lead exposure induced significant increase in contractile responsiveness but it does not seem that the effect of lead depends on change in adrenergic responsiveness per se but other factors such as alteration in intracellular Ca++ exchange, Nitric Oxide, and cyclooxygenase pathway could be involved. s92 337 Poster Session P13. Cardiovascular toxicology TRANSCRIPTOMIC ANALYSIS OF VASCULITIS INDUCED IN RAT MESENTERIC ARTERIES BY A PHOSPHODIESTERASE INHIBITOR Stephan Chevalier 1 , Nicolas Dagues 1 , Claudia Garcia-Allen 2 , Valérie Pawlowski 1 . PFIZER Global R&D 1 Molecular and Cellular Toxicology Laboratory, Amboise, France and 2 Genetic Technologies, Sandwich1 England Vasculitis is a relatively common finding during the pre-clinical toxicity testing of drug candidates. Vasculitis has drawn increased attention since the development of drugs has been directly affected by vasculitis observed in animal toxicology studies. The rat appears to be the most sensitive species for developing vasculitis in the mesenteric tissues after exposure to phosphodiesterase (PDE) inhibitors. Despite extensive pre-clinical and clinical studies, the mechanisms of toxicity induced by chemicals or drugs leading to toxic vasculitis are largely unknown. Sprague Dawley rats were treated with a selective PDE inhibitor and several tissues, including mesenteric arteries, were collected for histopathological observations and gene expression analysis. Real-Time RT-PCR analysis of cytokine expression in mesenteric tissue was performed. The Affymetrix GeneChip technology, that allows monitoring of the expression of around 8,800 rat genes on a chip (RG_U34A), was used in order to identify potential biomarkers and to better understand the molecular mechanisms of PDE inhibitor-induced vasculitis in rats. 226 probes feature with significantly altered expression after treatment with the PDE inhibitor were identified. The genes with altered expression are involved in inflammation, oxidative stress and regulation of vascular tone. Taken together, the data analysis supports the hypothesis that the vasculitis induced in rat mesenteric arteries by the PDE inhibitor could be the consequence of haemodynamic changes due to an exacerbation of the compounds pharmacological effect. 338 DOXORUBICIN TREATMENT LEADS TO REPRESSION OF CERTAIN TRANSCRIPTION FACTORS IN RAT HEARTS: A MICROARRAY GENE EXPRESSION STUDY IN AID FOR HYPOTHESIS GENERATION J. Borlak 1 , J. Wagener 1 , T. Thum 1 . Fraunhofer Institute for Toxicology and Experimental Medicine, 1 Drug Research and Medical Biotechnology Doxorubicin (DOX) belongs to the group of anthracycline antibiotics and was identified as an effective anti-neoplastic remedy. It is mainly used for the treatment of acute lymphocytic and granulocytic leukemias, as well as breast cancers and its mechanism of action is linked to intercalating with DNA as well as fostering singleand double-strand breaks and radical oxygen DNA insult. Well known to clinicians, therapy with DOX is limited because of its cardiotoxic effects, which may result in cardiomyopathy. As of today, the underlying molecular events leading to cardiotoxicity are far from clear. In aid for hypothesis generation we employed a microarray approach to investigate cardiac gene expression upon DOX treatment. Rats were therefore treated consecutively for 4 days with DOX (10mg/kg) and hearts were removed by standard surgical procedures and shock-frozen in liquid nitrogen. 14 genes were identified to be significantly increased >1.5-fold and 44 genes were found to be repressed to <70% of controls. A major finding was the repression of some transcription factors (ATF3, JUNB, c-jun, c/EBP beta, MAX, CREM, HAND2 amongst others), as well as phosphatases (DUSP1, PTPN3) and kinases (MAPK14). On the other hand, genes coding for cell cylce (RFC2, CCNA2), extracellular matrix modulating proteins (TIMP3) and metabolism (eNOS, CYP4A8) were upregulated. Our study illustrates complex interactions of DOX with the transcriptional machinery of heart muscle cells. Specifically, the down-regulation of heart specific transcription factors may provide the rational for some of the clinical findings observed upon drug treatment, e.g. metabolic deregulation, enhanced apoptosis and cardiac remodelling. 339 DYNAMICS OF CARDIOVASCULAR EFFECTS OVER A 5-YEAR FOLLOW UP OF CS2 EXPOSED WORKERS A. Bortkiewicz, E. Gadzicka. Nofer Institute of Occupational Medicine, Lodz, Poland Ischaemic heart disease in workers exposed to carbon disulfide (CS2 ) may result from disturbances in neurovegetative regulation of the cardiovascular system Thus we launched a prospective study in order to assess neurovegetative regulation of the cardiovascular system through determination of heart rate variability (HRV) in exposed workers. A Medea-HRV (Gliwice-Poland) was used to register 512 consecutive normal cardiac cycles under resting conditions. Time domain (mean R-R and STD R-R) and frequency-domain parameters were analyzed. The power spectrum density was estimated via Fast Fourier Transformation (FFT) for the following frequency bands: very low-VLF (0.00167–0.05 Hz), low-LF (0.05–0.15 Hz), highHF (0.15–0.35 Hz), total-TPS (0.00167–0.5 Hz). The examination included 114 workers. It was carried out twice over the period of 5 years. In the first examination (I) the mean age of the examined subjects was 43±9 and in the second 48±9 years. The time of employment under exposure to CS2 differed significantly (p=0.000) and was 15.8±9.5 years in examination I and 17.7±9.1 years in examination II. We analyzed the data using t-Student and Wilcoxon matched-pairs signed-ranks test and adjusted for age, smoking habit and alcohol consumption. It was found that STD R-R was significantly lower (p=0.005) in examination I than in examination II (39.8±17.7 vs. 47.1±19.0) and was negatively correlated (p.=0.003) with the time of employment under exposure. VLF, LF and TPS were significantly higher in examination II and were, respectively: VLF 10.1±3.8 vs. 14.1±4.2, LF 20.4±7 vs. 28.1±12, TPS 66.1±7.7 vs. 73.6±13.1. The LF/HF ratio also differed significantly and was 0.96±04 in examination I and 1.3±0.6 in examination II. It was observed that the values of VLF, LF and TPS increase significantly as the time of employment under exposure increases (p=0.008, p=0.03, p=0.005). The results of the study show that neurovegetative regulation of the cardiovascular system is significantly disturbed (the prevalence of the sympathetic system) under the influence of exposure to CS2 . 340 FOLLOW-UP STUDY ON BLOOD PRESSURE IN WORKERS EXPOSED TO CARBON DISULFIDE E. Gadzicka, A. Bortkiewicz. Nofer Institute of Occupational Medicine, Lodz, Poland The aim of our study was to establish the dynamics of changes in arterial blood pressure related to exposure to carbon disulfide (CS2 ) because occupational exposure to CS2 may cause disorders in arterial blood pressure.The examination was carried out twice over the period of 5 years and included: general physical examination, and a long-term analysis of arterial blood pressure (ABP). ABP was monitored using Medilog ABP (Oxford) every half hour during the day and every hour during sleep (about 41 measurements per day). The mean systolic and diastolic blood pressure for 24 hours (BPSO, BPDO), day time activity (BPSD, BPDD) and night-time rest (BPSN, BPDN) were calculated with the Staessen’s standards of arterial blood pressure as reference values. The day-night ratios were determined for systolic (BPSD/N) and diastolic (BPDD/N) blood pressure. The normal value of BP ratio is 1.1 or more. The subjects with BP ratio<1.1 are called non-deepers. The examination was carried out in 114 workers occupationally exposed to CS2 for 18 ± 9 years. In examination I the age of the examined subjects was 43 ± 9 years and in examination II 48 ± 9 years. We analyzed the data using t-Student and Wilcoxon matched-pairs signed-ranks test. The results, adjusted for age, smoking habit and alcohol consumption, show that in examination II BPDD and BPDO were significantly higher (p=0.005 and p=0.008, respectively). The number of individuals with exceeded BPDD and BPDO limit was also significantly higher in examination II (p=0.04 and p=0.03) The BPSD/BPSN ratio decreased significantly as the time of work under exposure increased (p=0.04). It was found out that the risk (odds ratio) of non-deepers effect increased in workers with longer period of employment and was 6.7 for individuals employed for 15 years and 12.6 for those employed for 20 years. The results demonstrate Poster Session P13. Cardiovascular toxicology that exposure to CS2 may be considered a risk factor in disturbances of arterial blood pressure regulation. 341 MICROARRAY ANALYSIS REVEALS COMPLEX DEREGULATION OF GENE EXPRESSION IN HEART TISSUE UPON AROCLOR 1254 TREATMENT – IMPLICATIONS FOR CARDIOTOXICITY T. Thum 1 , J. Borlak 1 . Fraunhofer Institute for Toxicology and Experimental Medicine, 1 Drug Research and Medical Biotechnology Polychlorinated biphenyls (PCBs) are well known environmental pollutants and several reports are available to implicate PCBs in cardiovascular disease. Little is known about the effects of PCBs on gene expression in the heart. We investigated the effects of Aroclor 1254 (20mg/kg), a well known mixture of PCB isomers and congeners on gene expression in rat hearts by employing a microarray. Its design enabled a survey of gene expression and included gene coding for basic biological functions, such as detoxification, cell proliferation, tumor development, heat shock response, signal transduction, apoptosis, cell cycle regulation, metabolism and so forth.We found 10 genes to be increased >1.5-fold and 25 genes to be repressed <70% of controls. The transcription factors c-jun and serum response factor were significantly repressed upon Aroclor 1254 treatment. Further, gene expression of the vascular endothelial growth factor and the early growth response protein were repressed to 65% and 24% of controls. In contrast, genes coding for the catecholamine-degrading enzymes catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) were significantly upregulated (1,9 and 2,3-fold). Similar, transcript level of the aldehyde dehydrogenase ALDH1A1 was strongly increased upon Aroclor 1254 treatment. We additionally investigated promoters of regulated genes and identified several Ahr binding sites in basically all genes deregulated by Aroclor 1254. We suggest Ahr-ARNT to play a role in the transcriptional activation of heart specific genes upon PCB treatment and found PCBs to modulate expression of genes coding for programs of cellular differentiation and stress. Our findings in rat heart may be of importance in understanding the increase of cardiovascular disease in polluted areas. 342 CARDIOVASCULAR TOXIC EFFECTS OF ACUTE EXPOSURE TO HIGH GLUCOSE CONCENTRATIONS IN RATS C. Di Filippo 1,3 , R. Marfella 2,3 , A. Ceriello 4 , L. Berrino 1 , D. Giugliano 2,3 , A. Filippelli 1,3 , F. Rossi 1,3 , M. D’Amico 1,3 . 1 Department Experimental Medicine, 2 Department Geriatrics and Metabolic Diseases and 3 Excellence Centre for Cardiovascular Disease, Second University of Naples, Italy. 4 Department Pathology and Medicine, University of Udine, Italy Chronic hyperglycemia leads to cellular dysfunction that may become irreversible over time, a process that is termed glucose toxicity. This is mainly due to excessive intracellular glucose concentration which induces damage by increasing the production of free radicals. Cardiovascular toxic effects induced by hyperglycemia occur following acute exposure of cardiovascular specimens to high glucose concentrations (HGC). Our study showed that HGC (33.3 mmol/l) increase iNOS gene expression and nitric oxide levels in isolated rat hearts. Up-regulation of iNOS was accompanied by a marked concomitant increase of superoxide (O− 2 ) production, a condition favouring the production of peroxynitrite, a powerful pro-oxidant that mediates the toxic effects of high glucose on heart, as suggested by the detection of cell apoptosis. Increased cardiac malondialdehyde (MDA) and poly(ADP-ribose) synthetase (PARS) activity were found. Cardiovascular consequences of these biochemical alterations were QT interval prolongation, coronary perfusion pressure (CPP) increase, and heart dysfunction. Glutathione, a powerful antioxidant capable of quenching both O− 2 and peroxynitrite, when infused along with high glucose, normalized CPP and reverses cardiac QT interval prolongation, induced by high glucose. Glutathione also reduced formation of peroxynitrites into cardiac cells as evidenced by reduced levels of nitrotyrosine immunostaining into the hearts subjected to s93 HGC. Similarly, increase PARS levels and MDA activity induced by high glucose concentration were reduced by addition of glutathione to the medium. Therefore, therapeutic interventions against glucose toxicity are warranted because of the elevated markers of damage following hyperglycemia. 343 OXIDIZED LDL IS A MAJOR REGULATOR OF METABOLIC PATHWAYS IN HUMAN ENDOTHELIAL CELLS T. Thum 1 , J. Borlak 1 . Fraunhofer Institute for Toxicology and Experimental Medicine, 1 Drug Research and Medical Biotechnology, Germany Enhanced oxidation of low density lipoprotein particles (oxLDL) is an important risk factor for vascular disease and atherosclerotic plaque formation. Upon intracellular availability oxLDL causes vascular toxicity and is considered to be responsible for an altered expression of adhesion molecules, production of radical oxygen species (ROS), scavenging of nitric oxide (NO) and formation of peroxynitrates. Overall, these events impair endothelial function and regulation of vascular tonus. To further our understanding of oxLDL induced endothelial toxicity, cultures of human endothelial cells (EAhy926) were treated with ascending doses of oxLDL (10 – 100µg/ml). We used an oligonucleotide microarray to study the expression of 9614 genes (Nimblegen). The design of the microarray enabled a survey of gene expressions and included genes coding for detoxification, cell proliferation, tumor development, heat shock response, signal transduction, apoptosis, cell cycle regulation, metabolism and so forth. We found oxLDL treatment to result in >2-fold induction of 130 highly abundant expressed genes and repression of 119 genes (<30% of control). Further, 69 genes were only expressed in oxLDL treated cells, whereas 78 gene were present in controls only. We employed gene ontology to interrogate metabolic networks and found glycolytic, lipid and steroid hormone metabolism to be altered. Specifically, oxLDL treatment of cultured endothelial cells resulted in increased transcript levels of palmotylprotein thioesterase 2, sterol O-acetyl transferase, phospholipase C and A2, pyrroline-5-carboxylase amongst others. Likewise, oxLDL treatment resulted in altered arachidonic acid metabolism based on expression analysis of the cytochrome P450 monooxygenases 2C8, 2C9, CYP17 and others. This treatment also repressed gene expression of transcription factors nuclear factor 1 and HNF3alpha. Some of the altered gene expression findings were correlated with metabolic functions of the coded proteins. For instance, CYP2C mediated production of epoxyeicosatrienoic acids was repressed, as was the transcript level of the coded gene. We thus demonstrate gene expression in cultured human endothelial cells to be dramatically altered upon treatment with oxLDL. We show oxLDL to be a powerful regulator of genes coding for metabolic pathways, and particular vascular cytochrome P450 monooxygenases. Our study contributes towards an understanding of endothelial toxicity brought about by oxLDL. 344 MODULATION OF PLASMA LIPID LEVELS AFFECTS B[A]P-INDUCED DNA DAMAGE IN TISSUES OF TWO HYPERLIPIDEMIC MOUSE MODELS D.M.J. Curfs 1 , L. Beckers 1 , R.W.L. Godschalk 1,2 , M.J.J. Gijbels 3,4 , F.J. van Schooten 1 . 1 Departments of Health Risk Analysis and Toxicology, 3 Molecular Genetics and 4 Pathology, University of Maastricht, Maastricht, The Netherlands. 2 Division of Toxicology and Cancer Risk Factors, German Cancer Research Centre (DKFZ), Heidelberg, Germany To which extent modulation of plasma lipids plays a role in the uptake, transportation and distribution of lipophilic carcinogens like benzo[a]pyrene (B[a]P) is not yet clear. Therefore, we have investigated the effects of dietary modulated plasma lipids on B[a]Pinduced DNA adducts after a single oral dose of B[a]P in several organs of two hyperlipidemic mouse models. Male apoE*3-Leiden (n=22) and apoE-KO mice (n=20) were fed a high fat diet (HFC) or normal mouse chow (SRM-A) for three weeks, after which the animals were exposed to a single oral dose of 5 mg/kg.bw B[a]P and killed 4 days later. Plasma lipids were s94 Poster Session P13. Cardiovascular toxicology determined and DNA adducts were measured in aorta, heart, lung, liver, brain and stomach. The HFC diet intervention lead to a significant increase of total cholesterol and LDL cholesterol in both mouse strains, whereas a decrease of triglycerides was seen only in the apoE-KO mice. In apoE-KO mice on a normal diet, DNA adduct levels were highest in aorta (10.8±1.4 adducts/108 nucl.), followed by respectively brain (7.8±1.3), lung (3.3 ± 0.7), heart (3.1±0.6), liver (1.5±0.2) and stomach (1.2±0.2). In the apoE*3-Leiden mice adduct levels were equally high in aorta, heart and lung (4.6 ± 0.7, 5.0 ± 0.5 and 4.6 ± 0.4, respectively), followed by stomach (2.7±0.4), brain (2.3±0.2) and liver (1.7±0.2). In the apoE-KO mice, the HFC diet intervention resulted in lower adduct levels in lung (2.1±0.2), heart (1.9±0.2) and brain (2.9±0.5) as compared to the SRM-A group. In contrast, in aorta a non-significant increase of adducts were found (13.1±1.5). A similar but non-significant trend was observed in the apoE*3-Leiden mice. Multiple regression analysis showed that in aorta, DNA adduct levels were inversely related to plasma triglyceride levels (p=0.004) and additionally modulated by the apoE genotype (p<0.001). The results of the present study further support investigation into the role of dietary modulation of plasma lipids, apoE and PAH exposure on the formation of DNA adducts in chronic degenerative diseases. 345 CIGARETTE SMOKE-INDUCED ENDOTHELIAL DYSFUNCTION IN VITRO: ROLE OF THE CYCLOOXYGENASE PATHWAY R. Schleef, K. von Holt. Philip Morris Research Laboratories GmbH, Koeln, Germany Endothelial dysfunction is hypothesized to be a key mechanism by which cigarette smoke promotes vascular disease. Although animal assays for endothelial dysfunction routinely investigate the vasodilatory properties of aortic rings in vitro, information is limited on the ability of these vessels to function in the presence of tobacco smoke. In this study, we examined the effect of cigarette mainstream smoke (MS) on the contraction and relaxation of rat aortic rings in vitro. Thoracic aortas were prepared from euthanized male Wistar rats and aortic rings were suspended in Krebs buffered saline in organ bath chambers. MS from 2R4F reference cigarettes was generated on a 30port smoking machine and bubbled through phosphate buffered saline (PBS) and used within 30 minutes. Incubation of aortic rings for up to 60 minutes with smoke-bubbled (SB)-PBS (≤0.06 puff/ml) had no effect on norepinephrine-induced contraction; however, relaxation of the rings in response to acetylcholine (Ach; 10−8 - 10−4 M) was reduced in a dose-dependent manner. For example, relaxation of norepinephrine-contracted aortic rings, which were pre-incubated in vitro for 45 minutes with 0.06 puff/ml SB-PBS, was 16.8 ± 11.9% at 10−4 M Ach compared to 96.3 ± 6.4% relaxation for control rings (P<0.001; n=6). The MS-inhibition of Ach-induced relaxation was reversible and normal contraction/relaxation curves could be obtained following washing to remove the SB-PBS and/or vasoactive reaction products. MS-inhibition of Ach-mediated relaxation appeared to be mediated by a cyclooxygenase-dependent pathway because indomethacin (10 uM) was effective in normalizing the Ach-relaxation curves to levels observed for controls incubated in the presence of indomethacin (p<0.01, n=6). These data indicate that the incubation of aqueous preparations of MS with rat aorta in vitro result in the generation of a cyclooxygenase-dependent vasoconstrictor and raise the possibility that this pathway may play a role in the vasoconstricting effects of MS within certain regions of the circulatory system. 346 INHIBITION OF MACROPHAGES AND ATHEROGENESIS BY BISPHOSPHONATES P. Ylitalo, O.-M. Tuominen, A. Lepoluoto, R. Ylitalo. Department of Pharmacological Sciences, Medical School, University of Tampere, Tampere, Finland Bisphosphonates (BPs) are used for the treatment of osteolytic bone diseases. BPs inhibit the phagocyting activity of osteoclasts and macrophages. Etidronate and pamidronate also reduce the experi- mental atherogenesis. We have studied the antiatherosclerotic effects and mechanisms of BPs in rabbits and cultured macrophage-like cells. In rabbits on cholesterol diet, clodronate (25 mg/kg) intravenously twice a week for 12 weeks reduced the development of atherosclerosis. 14C-labelled clodronate, as well as etidronate and pamidronate, concentrated in the aorta of healthy and especially of atherosclerotic rabbits. BPs also accumulated in human artery in vitro. Since macrophages have a key role in atherogenesis, we also studied the effects of BPs on the accumulation of LDLderived cholesterol in phagocyting cells. We found that liposomal clodronate and etidronate inhibited the uptake and degradation of acetylated LDL in cultured macrophage-like RAW 264 cells in a concentration-dependent manner. BPs also reduced the viability of macrophages and inhibited their transformation to lipid-containing foam cells. In conclusion, BPs inhibit the development of experimental atherosclerosis, accumulate in arteries and inhibit macrophages in internalization and degradation of atherogenic LDL. Quite recently, etidronate has been found to reduce the intima-media thickening of carotid artery in man with type 2 diabetes (Koshiyama et al. 2000). 347 A VALIDATED METHOD FOR THE DETERMINATION OF EPOXYEICOSATRIENOIC ACIDS (EETS) USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY – ELECTROSPRAY IONISATION/MASS SPECTROMETRY (HPLC-ESI/MSN ) J. Borlak 1 , V. Zelinski 1 , T. Thum 1 . Fraunhofer Institute for Toxicology and Experimental Medicine, 1 Drug Research and Medical Biotechnology Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid (AA) and regulate vascular tonus. Usually, EET production in biological systems is extremely low, which demonstrates the effectiveness of these highly active molecules. This, however, necessitates highly sensitive analytical methods for EET detection and quantification. Previous methods for the determination of arachidonic acid metabolites include radio HPLC (Rifkind et al., 1995) or HPLC with fluorescence detection (Nithipatikom et al., 2000), but the latter methods do not enable identification of novel products. We developed an HPLC-ESI/MSn method for the detection of major EETs (8,9-EET, 11,12-EET and 14,15-EET) with high sensitivity and specifity. We analysed EET production in human endothelial cells (EAhy926) after incubation with 100µM AA for 60min at 37°C. Then, culture supernatant was harvested, ethanol (98%) was added (1:1) and aliquots were stored at -80°C until further measurements. In addition, metabolism experiments with microsomal membranes from human livers or CYP engineered cells (“supersomes”, CYP1A1, CYP2B6, CYP2C8, CYP2C9, CYP2C9*2 and CYP2C9*3, each 50pM) and addition of AA (100µM, 60min) were done. 500ng tridecanoic was added to 100µl of cell culture supernatant as an internal standard. Chromatographic separation of arachidonic acid metabolites was achieved on a C18 Supelco Discovery column with the dimensions 150 x 2.1 mm and a particle size of 5 µm (Sigma-Aldrich, Germany) and a Securigard ODS precolumn (4 x 2mm). Ionisation was via ion electrospray (ESI) with a nebulizer pressure of 15 psi and a dry gas temperature to 320° C, while + 3,71 kV were applied to the nebulizing capillary. We were able to detect 8,9-, 11,12-, and 14,15-EET in supernatants of endothelial cells and in microsomal extracts from human livers or supersomes. Production of all EETs followed the order CYP2C9 > CYP2C8 > CYP2C9*2 > CYP2C9*3 > CYP1A1. With human liver microsomes and endothelial cell cultures EET production was less. In conclusion, our LC-MS/MS assay enabled EETs to be detected and quantified with high specifity and sensivity. Poster Session P14. Kidney toxicology 348 DIMETHYLBENZ(A)ANTRACENE (DMBA) BINDING CORRESPONDS TO CYP1A1 EXPRESSION IN ENDOTHELIAL CELLS OF THE BLOOD-BRAIN INTERFACES EVAS FAVORIT Anna Östergren 1 , Lizette Granberg 2 , Ingvar Brandt 2 , Eva Brittebo 1 . 1 Dept. of Pharmaceutical Biosciences, Biomedical Centre, Uppsala University, Box 594, SE-751 24 Uppsala, Sweden, 2 Dept. of Environmental Toxicology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18 A, SE-752 36 Uppsala, Sweden Experimental and epidemiological studies indicate that polycyclic aromatic hydrocarbons (PAH) may be involved in the development of cardiovascular disease. Following administration of a model PAH, 3 H-dimethylbenz(a)anthracene (DMBA), to mice pretreated with the aryl hydrocarbon receptor (AhR) agonists β-naphthoflavone or 3,3’,4,4’,5-pentachlorobiphenyl, a marked irreversible binding of metabolites in endothelial cells (EC) of the choroid plexus and of veins was observed by light-microscopic autoradiography. Furthermore, a high level of irreversibly bound DMBA-metabolites occurred in EC at these sites in precision-cut mouse/rat brain slices and in excised blood-brain interfaces incubated with 3 HDMBA. Vehicle-treated mice had no 3 H-DMBA-binding at these sites following exposure in vivo or in vitro. Immunohistochemistry showed that CYP1A1 was preferentially induced in EC in the choroid plexus and in veins of AhR agonistpretreated mice. In vehicle-treated mice no expression of CYP1A1 was observed in cerebral EC. Expression of CYP1B1 was similar in AhR agonist- and vehicle-pretreated mice, with expression in smooth muscle cells (SMC) of arteries in the leptomeninges, cerebral arteries/arterioles and to a low extent in ependymal cells. We conclude that rodents express a constitutively low, but highly inducible CYP1A1 activity in EC of some of the blood-brain interfaces and that 3 H-DMBA is covalently bound at these sites both in vivo and in vitro. The results suggest a CYP1A1 catalysed bioactivation of DMBA to reactive intermediates in EC of blood-brain interfaces. The role of environmental PAHs in the etiology of cerebrovascular disease needs further consideration. P14 Kidney toxicology 350 HALLMARKS OF ION CHANNEL GENE EXPRESSION IN END-STAGE HEART FAILURE J. Borlak 1 , T. Thum 1 . Fraunhofer Institute for Toxicology and Experimental Medicine, 1 Drug Research and Medical Biotechnology Electrical conductance is greatly altered in end-stage heart failure, but little is known about the underlying events. We therefore investigated the expression of genes coding for major inward and outward ion channels, calcium binding proteins, ion receptors, ion exchangers, calcium ATPases and calcium/calmodulin dependent protein kinases in explanted hearts (n=13) of patients diagnosed with end-stage heart failure. With the exception of Kv 11.1 and Kir 3.1 and when compared with healthy controls, major sodium-, potassiumand calcium- ion channels, ion transporters and exchangers were significantly repressed, but expression of Kv 7.1, HCN4 and troponin C and I, SERCA1 and phospholamban was elevated. Hierarchical gene cluster analysis provided novel insight into regulated gene networks. Importantly, the significant induction of the transcriptional repressor m-Bop and the translational repressor NAT1 coincided with repressed cardiac gene expression. The statistically significant negative correlation between repressors and ion channels point to a mechanism of disease. We observed co-regulation of ion channels and the androgen receptor and propose a role for this receptor in ion channel regulation. Overall, the reversal of repressed ion channel gene expression in patients with implanted assist devices exemplifies the complex interactions between pressure load/stretch force and heart specific gene expression. EFFFECT OF LONG-TERM CADMIUM TREATMENT ON APOPTOSIS AND PROLIFERATION OF RAT KIDNEY CELLS A. Plewka, G. Nowaczyk, D. Plewka, M.M. Brzóska 1 , M. Kamiński, J. Moniuszko-Jakoniuk 1 . Department of Histology and Embryology, Medical University of Silesia, Katowice, 1 Department of Toxicology, Medical University of Białystok, Poland The study aimed to evaluate the effect of nutritional exposure to cadmium on the apoptosis, metallothionein (MT) distribution and proliferation of rat kidney cells. Cadmium was used at concentrations humans may be exposed to either environmentally or occupationally. Mature Wistar female rats were divided into three groups. Group 1 included control rats kept in standard conditions. Groups 2 and 3 included rats that were given cadmium chloride in their drinking water (5 mg Cd/dm3 and 50 mg Cd/dm3 , respectively) for 3-, 6-, 9and 12 months. Apoptotic cells were detected by the M30 CytoDEATH system, the proliferation rate of epithelial cells by Ki67 Antigen Kit and metallothionein distribution by monoclonal antibody to horse-MT. In this study, the level of cadmium in the kidney increased steadily to reach 10.28 µg/g tissue for the lower dose, while for the higher dose the rate of cadmium accumulation was lower (up to 63.50 µg/g tissue), which probably resulted from a decrease in the daily intake of cadmium in drinking water during the experiment. Apoptotic and regenerative processes were observed mainly in proximal convoluted tubules. Their scale is connected with the dose and time of exposition. Metallothionein accummulation in the kidney increased with the duration of exposure in the following order: convoluted tubules, collecting tubules and straight tubules. There was a positive metallothionein reaction mainly in the cytoplasm and less frequently in the nuclei of renal tubules, especially in those of collecting tubules. 351 349 s95 ULTRASTRUCTURAL FEATURES OF CELL DEATH BY HgCl2 IN RAT PROXIMAL TUBULES A. Stacchiotti 1 , A. Lavazza 2 , L. Rodella 1 , R. Rezzani 1 , R. Bianchi 1 . 1 Division of Human Anatomy, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy, 2 Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia-Romagna, Brescia, Italy HgCl2 induces acute renal failure in S3 proximal tubules in a dose-dependent manner. To our knowledge few studies deal with the involvement of necrosis vs apoptosis in this model. In the present morphological study we identified and quantified cell-death in proximal tubules of rat treated with progressive low mercury doses and its temporal appearance using a well-known nephrotoxic dose. Materials and Methods: Sprague Dawley rats were treated with a single i.p.injection of 0.25, 0.50, 1 mg/kg HgCl2 (T groups) or saline (control group) and sacrificed after 24 h. Another group was i.p.injected with 1 mg/kg HgCl2 but killed at 6h. Kidneys were fixed with glutaraldehyde/OsO4 and embedded in araldite. 200 nuclei/group were observed on semithin sections and percentages of cell-death statistically analysed. For qualitative analysis, ultrathin sections were examined under a Philips CM10 electron microscope. Results: In the T0.25-group interphasic nuclei were over than 99% like in controls. In the T0.5-group necrosis was 35%. In the T1group necrosis was only 20% at 6h and increased up to 50% at 24 h, when tubular atrophy was extensive. By contrast in the same group, early apoptosis with segregated nucleoli and mitotic arrest was 7% at 6h and 12% at 24h. Oncosis with chromatolysis was 5% in the T 0.5-group, 8% in the T1-group at 24 h, less than 3% at 6 h. Conclusions: These data suggest that HgCl2 affected rat proximal tubules by different cell-death mechanisms. Even if necrosis occured more frequently, both apoptosis and oncosis have been well characterized by ultrastructure. s96 352 Poster Session P14. Kidney toxicology THE INFLUENCE OF PYRETHROIDS ON HISTOPATHOLOGICAL CHANGES IN KIDNEYS: ANTERIOR (HAEMATOPOIETIC TISSUE) AND POSTERIOR (NEPHRON SYSTEM AND HAEMATOPOIETIC TISSUE) IN FISH. H. Lutnicka. Department of Fish Diseases and Biology, Faculty of Veterinary Medicine, Agricultural University, Lublin, Poland Pyrethroids are very toxic to fish. They interfere with ion regulation causing the increase of excretion of renal Na+ and K+ . They cause an immunosuppressive effect in fish, too. The aim of the study was to find out if they cause histopathological changes in anterior and posterior kidney of fish exposed to pyrethroids. The experiments were carried out on carp weighing 70±10 g, in static aquarial conditions, in river water. The period of fish exposure was two weeks. One group of fish was exposed to one pyrethroid, used only once. After the exposure period the fish were transferred to clean water for the next four weeks. The studied pyrethroids were: cypermethrin (0.02 µg/l), deltamethrin (0.02 µg/l), fenvalerate (0.05 µg/l) and permethrin (0.04 µg/l). The kidneys were taken out twice: at the end of the exposure and after the four-week convalescence period. The histopathological changes were observed in light and electron microscopes. The following conclusions were drawn: The vacuolization of cytoplasm and local disintegration process in the posterior kidney epithelial cells could be observed. The kidney tubules were swollen and a secretion was observed in their light. The disintegration process was observed in mitochondria of epithelial cells. The cytoplasm was rarefied and endoplasmic reticulum was fragmented. The large and local increase of the haematopoietic tissue between renal tubules of posterior kidney was observed. The disintegration process was not generall observed in the internal membrane of mitochondria in haematopoietic tissue of anterior kidney. The immature neutrophils were more mature and richer in granules than those observed in the control fish. It suggests that the granulopoiesis was stimulated. All these changes could be observed to the end of whole experiment. 353 EFFECT OF STATINS ON PROTEIN UPTAKE AND CHOLESTEROL BIOSYNTHESIS IN KIDNEY PROXIMAL TUBULE CELLS James E. Sidaway 1 , Robert G. Davidson 2 , Fergus McTaggart 2 , Terry C. Orton 1 , Robert C. Scott 1 , Graham J. Smith 2 , Nigel J. Brunskill 3 . AstraZeneca, 1 Safety Assessment and 2 Cardiovascular and Gastrointestinal Discovery, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK, 3 Department of Nephrology, Leicester General Hospital, Gwendolen Road, Leicester, LE5 4PW, UK Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase which catalyses the major rate-limiting step in the synthesis of cholesterol and non-sterols such as the isoprenoids. Although they are widely used for the therapeutic reduction of cholesterol-containing atherogenic lipoproteins, statins can also affect cellular processes (e.g. apoptosis and endocytosis) through the depletion of isoprenoids. There have been reports of proteinuria occurring in association with statin treatment and we hypothesised that this could be due to inhibition of HMG-CoA reductase in the renal proximal tubule (PT) cells leading to reduced efficiency of protein re-absorption via endocytosis from the tubular lumen. Using the PT-derived opossum kidney (OK) cell line, we measured protein uptake by incorporation of fluorescein-isothiocyanate-labelled albumin and assessed HMG-CoA reductase activity by the conversion of 14 C-acetate into cholesterol. In the presence of 10µM simvastatin albumin uptake was inhibited to 64% of control at 10 hours and to 36% by 24 hours, in the absence of cell toxicity. All statins tested inhibited albumin uptake into OK cells in a dose-dependent fashion (approximate IC50 values after 24 hours of exposure were 0.3µM for fluvastatin, 1µM for simvastatin, 6µM for atorvastatin, 30µM for rosuvastatin and 100µM for pravastatin), and this was in accordance with the lipophilicity of each statin. A similar rank order of potency was observed for inhibition of HMG-CoA reductase, although a high degree of HMG-CoA reductase inhibition (approximately 80%) was required to observe an effect on albumin uptake. The inhibitory effect of simvastatin on albumin uptake could be ameliorated by the co-addition of mevalonate (the product of HMG-CoA reductase) and by the isoprenoid geranylgeranyl pyrophosphate, but not by cholesterol. These results establish the principle that inhibition of HMG-CoA reductase in PT cells could reduce the rate of renal tubular protein re-absorption in vivo and may provide a mechanistic explanation for statin-associated proteinuria. 354 DOES NTBC MODULATE D-SERINE-INDUCED NEPHROTOXICITY? R.E. Williams, E.A. Lock. Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, UK D-serine causes selective damage to renal tubule cells in rats, the mechanism of toxicity is currently not fully understood. An elevation of plasma fumaryl acetoacetate hydrolase (FAH) has been reported at 4h post-dosing (250, 500 & 750mg/kg ip). FAH, an intracellular enzyme involved in tyrosine catabolism, is responsible for the conversion of 4-fumarylacetoacetate to acetoacetate and fumarate. To assess whether FAH is involved in the mechanism of D-serine-induced nephrotoxicity we have examined the effect of pre-treatment with NTBC. NTBC inhibits 4-hydroxyphenylpyruvate dioxygenase (HPPD) which is a key enzyme in tyrosine catabolism located upstream of FAH. Alderley Park rats were dosed with either NTBC (0.5mg/kg po) or corn oil (vehicle) then 30 minutes later they received either D-serine (250mg/kg ip) or deionised water (vehicle) resulting in 4 experimental groups (n=5 per group). Urine samples, collected at 12h intervals, were analysed by 1 H NMR spectroscopy followed by principal component analysis (PCA). Terminal blood samples (48h post-dosing) were analysed by clinical chemistry and the kidneys were taken for pathology (H&E stain). Markers of kidney toxicity monitored using 1 H NMR spectroscopy, including elevated lactate and glucose and decreased citrate and hippurate, were evident in the urine of animals treated with D-serine and NTBC+D-serine. PCA could not distinguish between the urine samples from these two groups suggesting that pre-treatment with NTBC dose not modulate D-serine-induced nephrotoxicity. Plasma urea and creatinine levels and pathological examination of the kidneys further confirmed this (creatinine (µmol/l): control, 28.6±0.9; NTBC, 28.8±0.8; *Dserine, 162.2±37.4; *D-serine+NTBC, 188.3±13.6; *P<0.001 compared to control and NTBC, one-way ANOVA). Crystals, identified as tyrosine, were observed in the urine of animals treated with NTBC+D-serine amounting to approximately 50mg over 48h. HPPD inhibition normally results in elevated plasma tyrosine and excretion of 4-hydroxyphenylpyruvate and 4-hydroxyphenyllactate, therefore these results demonstrate that the renal damage induced by D-serine markedly effects the handling of tyrosine formed as a result of HPPD inhibition. 355 THE EFFECTS OF AN INCREASE IN TIME BETWEEN SACRIFICE AND TISSUE FIXATION ON IMMUNOHISTOCHEMICAL STAINING FOR PROLIFERATING CELL NUCLEAR ANTIGEN IN THE RAT KIDNEY W. Roosen 1 , L. Lammens 1 , J. Vandenberghe 1 , M. Desmidt 1 , J. Verbeeck 1 , W. Coussement 1 . 1 Johnson and Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V., GPCD/PCDE, Beerse, Belgium The purpose of this study was to evaluate the effect of an increase in time between the sacrifice and the tissue fixation on the immunohistochemical staining for Proliferating Cell Nuclear Antigen (PCNA) in the rat kidney. PCNA is known as a marker for cell proliferation. To this purpose, 4 groups of 20 female Sprague Dawley rats each were sacrificed and an increasing time between sacrifice of the animals and fixation of kidney tissue was applied (30, 60, 120 and 180 minutes) (groups A, B, D and E, respectively). The rats were anaesthetised with pentobarbitone and killed by exsanguination Poster Session P15. Liver toxicology via the carotid artery. Histopathological changes and autolysis were examined on hematoxylin-eosin (H.E.) stained kidney tissue, while PCNA-positive nuclei were counted in immunohistochemically stained kidney slides. The total number of ‘clearly-positive’ staining renal corticotubular nuclei were counted per transverse kidney section by systematic screening of the total renal cortical surface. Nuclei were considered PCNA-positive when clear red staining of the total nucleus was observed. Autolytic changes were observed histologically in all rats. The severity grade of autolysis was clearly increased in the B, D and E groups versus group A. The increase in severity grade of autolysis was in accordance with the increase in time between sacrifice and tissue fixation. Statistically significant decreases in the number of PCNApositive renal corticotubular nuclei were observed in groups B, D and E versus group A. It was concluded that an increase in time between sacrifice and tissue fixation resulted in an increase in autolysis, and consequently in a decrease of the number of PCNA-positive staining renal corticotubular nuclei. 356 TOXIC EFFECT OF AQUEOUS COFFEE EXTRACT ON MALE RAT KIDNEY (STEREOLOGICAL STUDY) M.R. Panjehshahin, F. Dehghani, R. Dezfullian. Shiraz Medical School, Shiraz, Iran Coffee is a famous traditional drink in many countries. These plants are diuretic, cause vascular expansion and reduce free radical oxygen. There is evidence that show the effect of on kidney, for instance, low dose of coffee reduces blood urea and creatinine but coffee overdose may lead to glomerulosclerosis. In this study, the effect of high dose of coffee on rat kidney was evaluated by stereological method. For this purpose, 60 Sprague-Dawley rats 230–250 g were selected and divided randomly into 5 groups. The control group was fed with only tap water and the experimental groups were fed with different doses of aqueous extracts of coffee (0.25, 0.5, 1, 1.5 g/kg) twice daily. After 48 hours, the animals were deeply anesthetized and right kidneys were removed. The 5µm slides were prepared and stained with Hematoxyline-Eosin. From each kidney 15–17 glomeruli were selected and means of glomerular volume were estimated, according to Cavalieri principle and Point Counting method. The results revealed that low doses of coffee extraction (0.25 g/kg) led to increase in glomerular volume (0.59×106 µm3 ), but higher doses decreased these volumes (0.48×106 µm3 for 1.5g/kg). These results were significantly different from control (0.45×106 µm3 ). In conclusion high consumption of coffee decreased glomerular volume. It seems that decrease in glomerular volume leads glomerulosclerosis and reduction of Glomerular Filtration Rate. However there should be more investigation such as urea and creatinine measurement to clarify the exact mechanisms. s97 a control group. Group II: received a single oral hepatotoxic dose of acetaminophen (600 mg/kg). Group III: received 600 mg/kg acetaminophen followed by N-acetyl cysteine (NAC) (150 mg/kg) orally after 1 hour. Group IV: pretreated with zinc sulphate (20 mg/kg) orally for 7 days followed by acetaminophen and NAC in the same above-mentioned doses. Group V: pretreated with sodium selenite (100 mg/kg) orally for 7 days followed by acetaminophen and NAC. They have a hepato-protective effect against some hepatotoxic chemicals. Selenium is stored mainly in the liver. Being an integral part of the enzyme glutathione peroxidases or to its direct antioxidant effect, selenium has a main role in the protection against liver damage caused by lipid peroxides. This work was designed to detect the role of hepatic CPS-1, hepatic glutamine synthetase and serum arginase enzyme activities as markers of acetaminophen hepatotoxicity in rats. Furthermore, to evaluate the correction of these markers by using the specific acetaminophen antidote-N-acetyl cysteine alone and with addition of an antioxidant either zinc or selenium. It was found that hepatic carbamyl phosphate synthetase-1, hepatic glutamine synthetase and serum arginase activities are significantly affected by hepatotoxic doses of acetaminophen. This indicates the usefulness of these parameters as hepatotoxic indicators of acetaminophen overdose and for evaluation of treatment. The use of drug combination; zinc sulphate with NAC or sodium selenite with NAC especially the latter could be another effective alternative treatment of acetaminophen overdose in view of the possible side effects produced by NAC. 358 PROTECTIVE EFFECTS OF CALCIUM ANTAGONISTS ON ACUTE ACETAMINOPHEN HEPATOTOXICITY IN ADULT MALE ALBINO RATS Usama M. El-barrany 1 , Ashraf M.F. Kamel 1 , Ashraf M. Emara 2 . 1 Departments of Forensic Medicine & Toxicology; and 2 Histology, Faculty of Medicine, Cairo and Tanta Universities The hepatotoxicity of acetaminophen overdose depends on the metabolic activation to a toxic reactive metabolite by the hepatic mixed function oxidases. There is evidence that an increase in cytosolic calcium (Ca2+ ) is involved in acetaminophen hepatotoxicity. The effects of Ca2+ antagonists nifedipine (NF), verapamil (V) and diltiazem (DL) on acute acetaminophen hepatotoxicity were studied in adult male albino rats. The results of this study showed that NF, V and DL, administered one hour before acetaminophen, significantly decreased acetaminophen-induced hepatic damage measured by serum aminotransferases, liver weights and liver histology. Nifedipine significantly increased cytochrome P450 content, while V and DL showed no significant changes. Diltiazem significantly decreased lipid peroxidation, while NF and V showed no significant changes. Nifedipine, V and DL showed no significant changes on either hepatic reduced glutathione (GSH) or the GSH depletion provoked by the toxic dose of acetaminophen. P15 Liver toxicology 359 357 DETERMINATION OF CARBAMYLE PHOSPHATE SYNTHETASE-1, GLUTAMINE SYNTHETASE AND ARGINASE ENZYME ACTIVITIES AS MARKERS OF ACETAMINOPHEN HEPATOTOXICITY IN RATS Hoda Fouad 1 , Nadia Barghash 2 , Fairouz El-Sayed 2 , Mervat Barakat 3 . 1 Departments of Forensic Medicine and Toxicology, 2 Medical Biochemistry, and 3 Pharmacology and Drug Toxicology, Faculty of Medicine, University of Alexandria, Egypt This study was designed to detect the role of hepatic carbamyl phosphate synthetase-1 (CPS-1), hepatic glutamine synthetase and serum arginase enzyme activities in acetaminophen induced hepatotoxicity. At the same time, to evaluate to which extent these enzyme activities were modified by giving various therapeutic regimens as a treatment for acetaminophen toxicity. This study was carried out on 50 adult male albino rats divided into equal five groups: Group I: received gum and served as HEPATOTOXICITY OF NONSTEROIDAL ANTI-INFLAMMATORY DRUGS IN FRESHLY ISOLATED RAT HEPATOCYTES Akram Jamshidzadeh, Hossein Niknahad, Behrokh Eskandari, Mansooreh Mohammadi. Department of pharmacology and Toxicology, faculty of pharmacy- Shiraz University of Medical Sciences, Shiraz, Iran Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most frequently prescribed drugs. Hepatotoxicity is considered a characteristic of NSAIDs, mechanismes are not well known. In the present study we showed NSAIDs (diclofenac, acetaminophen, piroxicam, mefenamic acid, indomethacin, tolmetin, iboprofen, naproxen, salicylic acid) were toxic towards freshly isolated rat hepatocytes. Cytotoxicity was time and dose dependent for different NSAIDs. The event before cell death were formation of oxidant species, oxidation and depletion of GSH, lipid peroxidation and ATP depletion. The LC50 after 2 hour of incubation was 148, 180, 331, 325, 178, 420, 206, 395, 360 µM for diclofenac, acetaminophen, piroxicam, s98 Poster Session P15. Liver toxicology mefenamic acid, indomethacin, tolmetin, iboprofen, naproxen and salicylic acid, respectively. It is hypothesized that metabolism of NSAIDs and production of free radicals and/or resulting oxidative stress may be the cause of cytotoxicity. 360 THE PROTECTIVE EFFECT OF SILYBUM MARIANUM AND VITAMIN E ON LIVER TOXICITY INDUCED BY SODIUM VALPROATE H. Kalantari, F. Talibi. Depart. Of Pharmacology &Toxicology, Pharmacy School, Ahvaz University Of Medical Sciences Sodium valproate is an effective antiepileptic drug, which is widely used in therapy. One of its side effects is liver toxicity. The aim and purpose of this study was to find out the protective effects of Silybum marianum and vitamin E in hepatotoxicity caused by sodium valproate. In this study the crude extract of Silybum marianum in doses of 100, 200 and 800 mg/kg and vitamin E in doses of 100, 200 and 400 mg/kg were orally administered to mice one hour before the toxic dose of sodium valproate for one week. The positive control received toxic dose of sodium valproate (500 mg/kg) and negative control groups received normal saline and Seasame oil. On the 8th day, blood was taken for measurement of enzyme activities such as SGOT & SGPT and also liver tissues were taken for histopathological study. Results showed that significant hepatoprotection was observed in doses of 800 mg/kg of Silybum marianum and 400 mg/kg vitamin E. Also it has been observed that the adjuvant dose of Silybum marianum and vitamin E in doses of 100 mg/kg were synergistically effective. In conclusion, this herbal extract and vitamin E can protect liver damage caused by sodium valproate in mice. trials and has shown activity in ovarian, breast, endometrial cancer, and advanced pretreated soft tissue sarcoma. In patients, the most prevalent drug-induced toxicities are fatigue, non cumulative neutropenia and reversible transaminase increase. In order to better understand the hepatotoxic potential of Yondelis™, its safety was evaluated in repeated dose studies in rodent (Sprague-Dawley rats) and non-rodent models (Cynomolgus monkeys). Cynomolgus monkeys were chosen as the preferred non-rodent species due to the similarities in metabolic profile to that of humans. In these studies, ET-743 was administered via a 3-hour intravenous infusion every 3 weeks for 3 (rat) or 4 (monkey) cycles. After repeated administration in rats, mortality was observed at 50 µg/kg in females and at 50–75 µg/kg in males. A pronounced dose-dependent and only partially reversible hepatotoxicity (transaminase increase, inflammation, hepatocytic necrosis and bile duct proliferation, cholangitis) was noted, and toxicity was cumulative and more pronounced in female rats. The difference in gender sensitivity in rats is likely to be linked to differences in the metabolic profile, biliary excretion and/or liver retention. In Cynomolgus monkeys dosed up to 120 µg/kg, a similar toxicity profile was seen in both sexes. Hepatotoxicity was less pronounced than in rats and non-cumulative in nature (transaminases increase, hepatocellular hypertrophy, hepatocytic degeneration/necrosis and mixed inflammatory cells in the sinusoids and portal tracts). At 50µg/kg, exposure to ET-743 was however higher in monkeys (23–28 µg.h/l) than in rats (1.7–8.6 µg.h/l), without gender difference. These data suggest that the Cynomolgus monkey is a more relevant and predictive model for human Yondelis™ hepatotoxicity than the rat. 363 361 PROBIOTICS EFFECTS ON ACETAMINOPHEN-INDUCED HEPATOTOXICITY IN RAT M. Rezayat, H.R. Varmazyar, B. Djhangiurir, A. Mohammadie, M. Ghazi-Khansari. Depatment of Pharmacology School of Medicine, Tehran University of Medical Sciences Probiotics are bacteria consisting of Lactobacillus and Bifidobacterium and some fungal. Probiotics are alive microorganisms that produce some compounds such as lactic acid in natural floral of intestine. They also stimulate the immune system and combat with virulent microorganisms. It is suggested that with any disease or intoxication, the natural flora of intestine would change, results in reduction of probiotics. Probiotics have shown to decrease the hepatotoxic effect of galactosamine. Therefore, in this study the effect of probiotics on acetaminophen-induced hepatotoxicity was examined. Fourteen groups of five male Wistar rat (200–250g) were used in this study according to the following procedure: Groups 1–4 consist of 2.5% fat milk (2ml/rat) and NAC (400mg/rat) and probiotic (1,6 unit dose/rat). Groups5–14 consist of Act (750 mg/kg), 2.5% at milk + Act, NAC (100, 200, 400mg/rat) + Act. Probiotics, milk and NAC was given to the rat for a week everyday orally by gavage. Then acetaminophen was administered intraperitoneally to rat. After 24 hours liver enzymes activity (SGOT, SGPT, ALK PH, LDH) and bilirubin (total, direct) and total liver tissue glutathion were determined. The hepatotoxicity of acetaminophen has been shown to decrease significantly by probiotic (p<0.05). 362 HEPATOTOXICOLOGICAL DIFFERENCES WITH ET 743 BETWEEN SPRAGUE DAWLEY RATS AND CYNOMOLGUS MONKEYS J. Verbeeck 1 , A. Vynckier 1 , A. Looszova 1 , K. Anciaux 1 , N. Bode 1 , R. De Coster 1 , L. Lammens 1 , P. Aviles 2 , I. Manzanares 2 , W. Coussement 1 . 1 Johnson and Johnson Pharmaceutical Research and Development., GPCD/PCDE, Beerse, Belgium; 2 PharmaMar, Madrid, Spain Yondelis™ (trabectedin, ET-743) is a tetrahydroisoquinoline compound isolated from the marine ascidian Ecteinascidia turbinata. The compound is currently under clinical investigation in phase II TOXICITY EFFECTS OF VITAMIN A ON THE LIVER AND BONE IN RATS M. Taheri Moghadam, I. Rashidi. Department of Pathology, Medical University, Ahvaz, Iran In this study, effects of choronic and acute vit. A intoxication, on liver and bone tissues and serum activity of SGOT, SGPT and serum calcium in rat were evaluated, from two routes of administration. Chronic doses were 25000 and 50000 i.u. /Rat/day vit.A for 10 days and acute dose was 200000 i.u./Rat/ day vit. A for 2 days. The results of this study showed that: The activity of SGOT and SGPT and serum calcium of treated groups significantly differed from untreated control. Also enhancement of this factors in i.m. treated groups was grater than p.o. treated groups. Mild, moderate and severe liver lesions (cholestasis, fethery, degeneration, congestion and hepatitis) and bone lesions (osteoporosis and cartilage degeneration) were happened. Severity of this lesions depend on dose of drug, and in i.m. treated groups grater than p.o. treated groups. 364 HEPATOTOXIC EFFECTS OF 3,4-METHYLENEDIOXYAMPHETAMINE AND α-METHYLDOPAMINE IN FRESHLY ISOLATED RAT HEPATOCYTES Márcia Carvalho 1 , Nuno Milhazes 2 , Fernando Remião 1 , Fernanda Borges 2 , Eduarda Fernandes 3 , Terrence J. Monks 4 , Félix Carvalho 1 , Maria Lourdes Bastos 1 . 1 REQUIMTE, Toxicology Department, 2 CEQOFFUP, Organic Chemistry Department, 3 REQUIMTE, Physical-Chemistry Department, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha, 164, 4050/047 Porto, Portugal. 4 Center for Molecular and Cellular Toxicology, College of Pharmacy, University of Texas at Austin, Austin, TX 78712/1074, USA The consumption of 3,4-methylenedioxymethamphetamine (MDMA or “ecstasy”) and 3,4-methylenedioxyamphetamine (MDA or “love”) in humans has been associated with numerous reports of hepatocellular damage. MDA itself is a metabolite of MDMA. Although MDMA undergoes extensive hepatic metabolism, the role of metabo- Poster Session P15. Liver toxicology lites in MDMA-induced hepatotoxicity remains unclear. The aim of the present study was to evaluate the toxic effects of MDA and its major metabolite α-methyldopamine (α-MeDA) in freshly isolated rat hepatocytes. The cells were incubated with MDA or α-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 hours. Toxic effects were evaluated by measuring cellular viability, extent of lipid peroxidation, levels of reduced (GSH) and oxidized (GSSG) glutathione, formation of glutathione adducts, and the activities of glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione-S-transferase (GST). MDA induced GSH depletion but had a negligible effect on lipid peroxidation, cell viability or on the studied enzymes activities. On the other hand, α-MeDA (1.6 mM, 3 hours) induced a marked depletion of GSH that was accompanied by the loss of cell viability and the decrease in GR, GPX and GST activities, although no significant effect on lipid peroxidation was found. For both metabolites, GSH depletion was not accompanied by increase in GSSG levels, which suggest GSH adduct formation. In accordance, 2-(glutathion-S-yl)-αMeDA and 5-(glutathion-S-yl)-α-MeDA adducts were identified by HPLC-DAD/EC within the cells incubated with MDA or α-MeDA. These results give clear evidence that one of the early consequences of MDMA metabolism is a disruption of thiol homeostasis, which may result in loss of protein function and initiation of a cascade of events leading to cellular damage. This work was supported by a project grant from FCT (PraxisXXI/BD/20087/99). 365 THE METABOLISM OF 3,4-METHYLENEDIOXYMETHAMPHETAMINE (ECSTASY) INTO N-METHYL-α-METHYLDOPAMINE DRASTICALLY INCREASES ITS IN VITRO TOXICITY Márcia Carvalho 1 , Nuno Milhazes 2 , Fernando Remião 1 , Fernanda Borges 2 , Eduarda Fernandes 3 , Félix Carvalho 1 , Maria Lourdes Bastos 1 . 1 REQUIMTE, Toxicology Department, 2 CEQOFFUP, Organic Chemistry Department, 3 REQUIMTE, Physical-Chemistry Department, Faculty of Pharmacy, University of Porto, Rua Aníbal Cunha, 164, 4050/047 Porto, Portugal 3,4-Methylenedioxymethamphetamine (MDMA or “ecstasy”) is a well-known recreational drug of abuse. In the past decade, increasingly clinical evidence has shown the liver as a target organ for MDMA toxicity. The aim of the present study was to evaluate the toxic effects of MDMA and its major metabolite N-methylα-methyldopamine (N-Me-α-MeDA) in freshly isolated rat hepatocytes. The cells suspensions were incubated with MDMA or N-Me-α-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 hours. Toxic effects were evaluated by measuring cellular viability, levels of reduced (GSH) and oxidized (GSSG) glutathione, and the activities of glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione-S-transferase (GST). MDMA induced a concentration- and time-dependent GSH depletion but had a negligible effect on cell viability or on the studied enzymes activities. On the other hand, N-Me-α-MeDA induced a more marked depletion of GSH that was accompanied by loss of cell viability and the decrease in GR, GPX and GST activities. GSH depletion was not accompanied by increase in GSSG levels; in fact, for the highest concentrations of N-Me-α-MeDA tested a significant decrease in GSSG levels was observed, which suggest GSH adduct formation. The results obtained in this study suggest that MDMA metabolism, resulting in the formation of the highly reactive compound N-Me-αMeDA which conjugates with GSH, may be considered one of the main causes of MDMA-induced liver damage. This work was supported by a project grant from FCT (PraxisXXI/BD/20087/99). 366 s99 EARLY CHANGES IN RAT LIVER INJURED BY ORAL ADMINISTRATION OF FURAN K.C. Hickling 1 , J.M. Hitchcock 2 , J.K. Chipman 2 , J.G. Evans 1 , T.G. Hammond 1 . 1 AstraZeneca R&D Charnwood Safety Assessment, Bakewell Road Loughborough Leics, LE11 5RH; 2 School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, B15 2TT Cholangiocarcinoma (CC) is a common primary hepatic malignancy in humans. Much information concerning its development has been obtained from rodent models, particularly after administration of the non-genotoxic carcinogen furan. The initial precursor lesion is characterised by an intestinal metaplasia amongst proliferating bile ducts seen in liver parenchyma after initial hepatocyte loss. This study was designed to follow the initial temporal development of the preneoplastic changes in the liver following furan administration with an evaluation of initial injury and repair mechanisms that act to restore lost hepatocytes. An oral dose of 30 mg/kg furan was administered once per day to groups of 5 rats for 8h, 1, 3, 7 and 10 days. Furan preferentially targeted the right and caudate lobes, typically inducing an initial subcapsular and centrilobular hepatocyte necrosis and rarely a pan-lobular necrosis that incorporated the biliary epithelium of portal tracts. Over the ensuing 10 days separate mechanisms of hepatocyte and biliary replacement were detected by light microscopy and immunocytochemistry with different temporal patterns and organisation. Hepatocyte Replication - This was first observed by increased Proliferating Cell Nuclear Antigen (PCNA) immunostaining of periportal and midzonal hepatocytes at 24 hours, peaking at 3 days but remaining above control levels at subsequent time points Biliary cell proliferation – The PCNA index for cells of normal biliary ducts was raised from 8h post-furan administration. Organised extensions of biliary ductules, that were positive for OV-6, Ecadherin and β-catenin were observed proliferating radially from existing biliary ducts, these were seen to merge with hepatic plates as they approached injured central zones. Emerging ductules extended along a laminin rich matrix and were chaperoned by Desmin/GFAP positive fibroblast like cells, which expanded away from portal tracts in concert with the expanding biliary epithelium. Biliary/Oval Cell proliferation – Tortuous proliferation of poorly differentiated ducts from portal tracts into a fibronectin rich matrix of early fibrosis which bridged between adjacent portal areas and central veins, particularly between lobes adjacent to the liver capsule. These ducts were also OV-6, E-cadherin and β-catenin positive and had a raised PCNA positive cell population when compared with phenotypically normal proliferating biliary ductules. The relation with mesenchymal cells was poorly organised. Definitive metaplastic ducts with intestinal phenotype were observed by day 10 within these expanding areas. Mature biliary and metaplastic ducts had similar expression of OV-6, E-cadherin and β-catenin to that present in normal rat intestinal epithelium. In conclusion, furan primarily induces an organised proliferative response in both hepatocyte and biliary cell populations that act to repair lost centrilobular hepatocytes. The metaplastic intestinal ducts, characteristic pre-cursor lesions of cholangiocarcinoma, appear to originate from a rare tortuous proliferation of ducts that expand into a matrix rich in fibronectin. It is hypothesised that the tortuous uncontrolled nature of this reponse results from loss of the normal lobular structure following pan-lobular necrosis. 367 HEPATIC P450-DEPENDENT MONOOXYGENASES AND GLUTATHIONE LEVELS IN PARACETAMOL AND N-ACETYLCYSTEINE TREATED RATS AFTER CHRONIC OF TRICHLOROETHYLENE EXPOSITION A. Plewka 1 , G. Nowaczyk 1 , D. Plewka 1 , B. Zielińska-Psuja 2 , J. Kowalówka-Zawieja 2 , M. Kamiński 1 , J. Orłowski 2 . 1 Department of Histology & Embryology, Silesian School of Medicine, Katowice-Ligota, 2 Department of Toxicology, Karol Marcinkowski University of Medical Sciences, Poznań, Poland The study was performed on male Wistar rats. The rats were treated with of three different xenobiotics given either separately or in s100 Poster Session P15. Liver toxicology combination. The exposition was performed during the following 7 days, 6 hours for each day, with constant trichloroethylene (TRI) concentration; 50 mg per kg body weight. Paracetamol (APAP) and/or N-acetylcysteine (NAC) with single doses at end exposition of TRI were given at 250 mg/kg and 150 mg/kg body weight, respectively. The rats were decapitated 4-, 12-, 24-, 48- or 120 hours after treatment. In liver homogenate the level of glutathione (GSH) was determined, but in the microsomal fraction the activity of the cytochrome P450-dependent monooxygenase system and the CYP2E1, CYP2B1/2 and CYP1A2. TRI slightly induced cytochrome P450 and NADPH-cytochrome P450 reductase. After APAP treatment, the level of cytochrome P450 and the activity of NADPH-cytochrome P450 reductase decreased slightly, but they increased from 24 h. Initially TRI had positive effect on GSH. The increase of GSH level was observed to 48 h group. APAP transiently decreased the level of glutathione; after 12 h the level of glutathione returned to the control value. NAC had no effect on GSH over the whole period of observation. The combination of three xenobiotics had no negative effect on cytochrome P450 and NADPH-cytochrome P450 reductase. In this case, CYP2E1 and CYP2B1/2 were induced, while CYP1A2 increased initially, but it returned to the control value at 12 h. The level of glutathione approximated the control value over the whole period of observation. 368 m-DINITROBENZENE-INDUCED HEPATIC CYTOTOXICITY IN RAT LIVER SPHEROIDS Jinsheng Xu, Wendy M. Purcell. Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Bristol, UK m-Dinitrobenzene (m-DNB) is an important industrial chemical widely used as an intermediate in the chemical synthesis of rubber additives, pesticides, dyes and explosives. It is a multi-target toxicant to humans and animals and can cause testicular toxicity, hepatotoxicity and neurotoxicity. m-DNB is a substrate of a P450 NADPH-dependent reductase and 3-nitroaniline (3-NA) is its major metabolite. During this biotransformation, a reactive intermediate, 3nitrosonitrobenzene, is produced and causes cell injury. The present study investigates the response of liver spheroids to m-DNB and 3-NA to examine the activity of the P450 NADPH-dependent reductase in mature liver spheroids indirectly and the suitability of the liver spheroid model for testing this category of chemicals. Liver cells were isolated from male Sprague rats and spheroids cultured by a gyrotatory-mediated method. Mature spheroids (6 days) were exposed to m-DNB (125–1000 µM) and 3-NA (250–1000 µM) for 24 h. The results showed that m-DNB caused leakage of LDH and decreased glucose secretion and galactose comsumption in a concentration-dependent manner. m-DNB induced nitric oxide synthesis. The highest nitrite release was detected after exposure to 250 µM m-DNB. At concentrations lower than 250 µM, m-DNBinduced a concentration-dependent increase of nitrite release. At the higher concentrations (>250 µM), nitrite release decreased with the increase of m-DNB concentration. By contrast, 3-NA did not induce significant toxic effects but induced NO synthesis. The results show that m-DNB has wide range effects. NO synthase induction is one of its important effects but is unlikely to play a role in m-DNB-induced toxicity. This study indicates that liver spheroids metabolised m-DNB and cytotoxicity was apparent. Liver spheroids can be used for testing chemicals which are metabolically activated through P450 NADPH-dependent reductase. 369 THE EFFECTS OF THE SMOKE PRODUCED FROM CIGARETTES EXPOSED TO SPECIFIC PULSED ELECTROMAGNETIC FIELD ACCORDING TO NIKOLAOU TECHNOLOGY IN RATS AFTER 60-DAYS RESPIRATORY EXPOSURE M. Jokanovic 1 , P. Stukalov 2 , D. Angelopoulos 3 , T. Nikolaou 3 , V. Bajic 2 . 1 Faculty of Pharmacy, Department of Toxicology, Vojvode Stepe 450, Belgrade, Serbia and Montenegro, 2 Clinical Center of Serbia, Pasterova 2, Belgrade, Serbia and Montenegro, 3 Fenomeno Electronic Technologies, Athens, Greece The purpose of this study was to evaluate clinical, biochemical and pathophysiological effects of the smoke produced from treated and normal cigarettes in rats during 60-day exposure period. There were three experimental groups each of 15 male and 15 female rats: the first group was exposed to 16 cigarettes treated according to Nikolaou technology during 40 minutes per day by means of specially designed smoking machine; the second group was exposed in the same way to the smoke produced from normal cigarettes of the same brand, and the third group was exposed to fresh air. There were neither mortalities nor differences in clinical appearance of animals from either group. This study revealed that the growth rate of rats exposed to processed smoke was a little slower than that in rats treated with normal cigarette smoke. The increase in carboxyhemoglobin content, due to the presence of carbon monoxide in cigarette smoke, was significantly lower (p<0.05) in the group of rats treated with processed cigarette smoke than in those treated with normal smoke. There were no significant differences in other haematological parameters. Pathohistological analysis revealed a major difference between animals exposed to the cigarette smoke: while emphysema and perivascular inflammation in the lungs were expectedly observed in rats exposed to the smoke from normal cigarettes, there were no such effects in animals of either sex exposed to the smoke from cigarettes treated according to Nikolaou technology. On the basis of the results obtained in this study it can be concluded that the smoke from normal cigarette smoke induces significantly worse toxic effects in rats than the smoke processed through pulsed electromagnetic field. 370 CAN SHORT-WAVE DIATHERMY MAKE HISTOLOGICAL CHANGES IN THE RAT LIVER? H. Bhadoran, Gh. Kaka, S.H. Sadraei, H. Dashtnavard, M.H. Asadi. Departement of anatomy, Baghyatallah University of Medical Sciences, Tehran, Iran In recent years there has been increased the use of electromagnetic fields in the human life such as industry, medicine and military. The present study was designed to investigate the effects of short-wave diathermy at a frequency of 27.12 MHz on the liver rat. Female rats exposed continuously to 10 W/cm2 at 27.12 MHz radiation 30 minutes twice daily for 7 days. Total exposure time was 210 minutes for each experimental rat. Another group was desighned as control group. Finding included a considerable increase body temperature in the experimental group. The liver of rats were removed, fixed and prepared for histological studies. The paraffin sections were then stained with H&E technique. Histological finding of rat liver in experimental group showed cellular changes were sinusoidal congestion, presence of acidophylic types in liver, hydropic degeneration. Our results showed that the number of kupffer cells were increased significantly in experimental group when compared to control group (p<0.05). In conclusion, application of short-wave diathermy can increased body temperature in experimental group. Futher study needed to investigate about possible carcinogen effects of short-wave therapy. Poster Session P15. Liver toxicology 371 THE EFFECT OF FRITILLARIA IMPERIALIS BULB EXTRACT ON THE MICE LIVER H. Kooshapur 1 , H. Kalantari 2 , F. Skandari 2 . 1 Department of Pharmacognosy, 2 Department of Pharmacology and Toxicology, Ahwaz University of Medical Sciences and Health Services, Ahwaz, I.R.Iran The main objective of this study was to find out the effect of Fritillaria imperialis bulb extracts on the mice liver. Following soxhelet extraction using water and ethanol 50%, solvents were removed from both extractions under vaccum evaporator. Doses of 100, 200, 400 and 800 mg/Kg of each crude extract were administered orally to mice (test group) for 6 days. The control group received normal saline only. On the day 7, samples of mice serum were collected for the measurement of GOT and GPT activities. Histopathological examinations of the liver were carried out too. The results indicated that, all doses were toxic but, both extracts were highly toxic in the dose of 800 mg/Kg, as all mice were died in this group. All test groups indicated an increase in GOT and GPT activities and significant changes in liver histology in comparison with control group. 372 HEPATIC STIMULATOR SUBSTANCE PROTECTS AGAINST ACUTE CADMIUM-INDUCED LIVER INJURY. G.I. Panoutsopoulos 1 , K.N. Tzirogiannis 1 , M.D. Demonakou 2 , A.I. Papadopoulos 1 , R.I. Hereti 1 , V.G. Kondili 1 , G.K. Papadimas 1 , K.T. Kourenzi 1 , L.A. Euliati 1 . 1 Department of Experimental Pharmacology, Medical School, Athens University, 75 Mikras Asias St., Athens 115 27, Greece, 2 Histopathology Laboratory, Sismanoglion G.D. Hospital, Sismanogliou 1, Marousi, Attiki 151 27, Greece Cadmium is one of the most abundant toxic metals in the biosphere with detrimental effects on the majority of human and animal tissues. When administered acutely cadmium is a potent hepatotoxin. Hepatic Stimulator Substance (HSS) is a potent stimulator of liver regeneration and also exerts a protective effect on acute liver failure induced by various hepatotoxins. HSS has been extracted from neonatal and regenerating liver and identified in the hepatocyte cytosol. In the present study, we investigated the protective effect of HSS against liver injury induced by acute cadmium administration. Emphasis was placed on cadmium-induced apoptosis in hepatocytes and nonparenchymal cells and peliosis hepatitis. Cadmium (3.5 mg/kg b.w) was administered intraperitoneally and rats were randomly assigned into two groups: group I received normal saline and group II HSS (100mg protein/kg b.w.) 2h later. The animals of both groups were sacrificed at 12,16,24,48, and 60h after cadmiun administration. Liver injury was evaluated by analysis of HE-sections for necrosis, apoptosis, peliosis, mitoses, inflammatory infiltration and by the serum levels of AST and ALT. Apoptosis was also quantified by the Tunnel technique. The mitotic index, the enzymatic activity of liver Thymidine Kinase and the 3 H-thymidine incorporation into hepatic DNA were used as indices of liver regeneration. The extent of necrosis, hepatocyte apoptosis and peliosis as well as the serum levels of AST and ALT were greatly diminished by HSS administration. Nonparenchymal cell apoptosis was not quantitatively influenced by HSS administration though its time profile was altered. Liver regeneration peaked earlier in HSS treated rats. HSS administration reversed cadmium-induced necrosis, peliosis and hepatocyte apoptosis. Direct protection of hepatocytes against the metal’s toxic effect and/or ischemia due to endothelial cell injury as well as disruption of interaction between hepatocytes and nonparenchymal cells are possible mechanisms for the observed effects. 373 s101 THE ROLE OF PUTRESCINE IN ACUTE CADMIUM HEPATOTOXICITY K.N. Tzirogiannis 1 , G.I. Panoutsopoulos 1 , M.D. Demonakou 2 , C.C. Vlachos 1 , A.C. Basayiannis 1 , R.I. Hereti 1 , K.N. Alexandropoulou 1 , A.I. Papadopoulos 1 . 1 Department of Experimental Pharmacology, Medical School, Athens University, 75 Mikras Asias St., Athens 115 27, Greece, 2 Histopathology Laboratory, Sismanoglion G.D. Hospital, Sismanogliou 1, Marousi, Attiki 151 27, Greece Putrescine is detected in trace amounts in the normal liver, but its levels subsequently increase during liver regeneration. Although putrescine exhibits protective effect against acute liver injury caused by various hepatotoxins, the mechanisms by which it exerts this effect are still unkown. Cadmium is a toxic metal whose concentrations are increasing in the biosphere mainly as a result of its industrial uses. Acute cadmium administration results mainly in acute hepatotoxicity. In the present study, we investigated the protective effect of putrescine against acute cadmium liver injury, with emphasis being placed in hepatoprotection of hepatocytes and nonparenchymal cells. Rats were injected with cadmium (3.5mg/kg b.w.) intraperitoneally and divided into two groups. Group I was treated with normal saline and group II with putrescine (300µmol/kg b.w.) at 2, 5 and 8h after cadmium administration. The animals of both groups were killed at 12,16,24,48 and 60h after cadmium intoxication. The time course of liver injury was evaluated by analysis of HE-sections for necrosis, apoptosis, peliosis and inflammatory infiltration. Total apoptosis, hepatocyte apoptosis and nonparenchymal liver cell apoptosis were quantified by Tunnel assay. The serum enzyme activities of AST and ALT were also assayed. Hepatic regeneration was estimated by mitotic index, thymidine kinase activity and the rate of 3 H-thymidine incorporation into DNA. Putrescine administration profoundly decreased the extent of necrosis, apoptosis and peliosis. Hepatocyte apoptosis was also minimized, but nonparenchymal cell apoptosis was not affected although its time profile was altered. The serum levels of AST and ALT were totally reversed and close to control values in putrescinetreated rats. Liver regeneration showed a similar time profile in both groups. The above results showed that putrescine suppressed cadmiuminduced acute hepatotoxicity. Protection of hepatocytes from the toxic effect of the metal or from ischemia due to endothelial cell injury induced by cadmium are possible mechanisms for the hepatoprotective effect of putrescine. 374 INFLUENCE OF SOME DERIVATIVES OF PYRIDINE CARBOXYLIC ACIDS ON STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF ENDOPLASMIC RETICULUM MEMBRANES AND FRACTIONATED NUCLEAR CHROMATIN OF A LIVER OF EXPERIMENTAL ANIMALS UNDER POISONING WITH TETRACHLOROMETHANE AND 1,2-DICHLOROETHANE P.G. Zhminko 1 , Yu.I. Gubskiy 2 , A.N. Marchenko 2 , E.L. Levitsky 2 , N.V. Litvinova 2 , A.G. Goriushko 2 , A.V. Matvienko 3 , V.Ph. Danilenko 4 , T.N. Kurapova 2 , A.N. Velichko 2 , L.P. Babenko 2 , N.M. Kurskaya 2 . 1 Laboratory of General Toxicology, L.I. Medved’s Institute of Ecohygiene and Toxicology, Kiev, Ukraine; Departments of 1 Biochemical Pharmacology, 3 Pathomorphology, 4 Synthesis of Physiologically Active Substances, 1nstitute of Pharmacology and Toxicology, Kiev, Ukraine In the conducted studies (in vitro and in vivo) the data indicating utility of research of derivatives of pyridine carboxylic acids (PCA) as potential pharmacological products, which have pronounced antioxidant, hepatoprotective, membranostabilizing, genomoprotective and cytoprotective properties, have been acheived. It has been established on experimental models of white rats (Wistar strain) liver chemical lesion with both tetrachloromethane (1 LD50 , intraperitoneally) and 1,2-dichloroethane (1 LD50 , intraperitoneally) that injection of some PCA derivatives (in dosages from 1/50 to 1/10 LD50 ) to the animals results in increase of their survival rate in 1.2–1.9 times. It has been established by means of biochemical, physical-chemical and morphological methods that some s102 Poster Session P15. Liver toxicology representatives of these compounds have pronounced antioxidant and antiradical activity which exceeds activity of classic antioxidant alpha-tocopherol in a number of cases; an injection of some PCA derivatives to the animals, poisoned with both tetrachloromethane and 1,2-dichloroethane to a great extent promotes correction of damage of endoplasmic reticulum membranes and fractionated nuclear chromatin of hepatocytes, normalization of their physical-chemical and structural-dynamic characteristics, level of lipids peroxidation in them, and also promotes normalization of liver structural and functional characteristics on the whole. 375 GENE EXPRESSION ANALYSIS OF GALACTOSAMINE-INDUCED HEPATOTOXICITY IN-VIVO AND IN-VITRO H. Hildebrand 1 , G. Kempka 1 , H. Ellinger 1 , B. Stuart 2 , B. Wahle 2 , H.J. Ahr 1 . Bayer AG, 1 Toxicology, 42096 Wuppertal, Germany and 2 Toxicology, Stilwell (USA) D-Galactosamine is a well known model hepatotoxin inducing hepatitis. In a rat study we examined the transcriptional alterations which are induced by, and accompany the intraperitoneal administration of this compound in rats. Animals were administered the test compound at a dose of 500 mg/kg bw, dissolved in saline, whereas controls received vehicle alone. Necropsies were performed at 6h, 12h, 24h, and 48h. Histological examination of liver tissue sections was conducted on H&E stained slides. In an in-vitro experiment cultured rat hepatocytes were incubated with D-Galactosamine at 5.0 mM for different periods of time. Cytotoxicity was recorded via MTT test. RNA was isolated from tissue samples and cultured hepatocytes, and gene expression analysis was conducted using Affymetrix RG U34A microarrays. The histological findings revealed the occurrence of multifocal necrosis increasing over time, and of reactive inflammation. Gene expression analysis of liver tissues and of cultured cells demonstrated the differential expression of a multitude of genes the number of which increased over time. The analysis of individual responses showed that several common pathways were affected both in-vivo and in-vitro. However, differences were observed which might be assigned to contributions from non-parenchymal cells of the liver. Particularly, immunity and defense-related genes showed a strong up-regulation in liver tissue by D-Galactosamine whereas cultured hepatocytes were missing these signals. Thus our results help to understand to which extent an in-vitro model can reflect the responses seen in-vivo. 376 HEPATOCELLULAR INJURY IN OFFSPRINGS OF RATS UNDER LONG TERM CHOLINE-DEFICIENT DIET C. Liapi 1 , N. Kambas 1 , P. Galanopoulou 1 , S. Theocharis 1,2 . Departments of 1 Experimental Pharmacology and 2 Forensic Medicine-Toxicology, University of Athens, Medical School, Athens, Greece Choline is an essential nutrient for cellular structure and function. The effect of choline deficient (CD) diet on liver histology was investigated in lactating offsprings of female Wistar rats provided with a CD diet for 4 months (pregnancy and lactation period included). The expression of cell cycle related molecules, such as cyclin-D1, cyclin-E, Ki-67, and cyclin dependent kinase (Cdk)inhibitors p16, p21 and p27 was examined immunohistochemically. Liver injury characterized as steatohepatitis was prominent in offprings rats under CD diet (Group I), while no signs of toxicity were observed in control ones (Group II). Increased hepatocyte mitotic activity and Ki-67 expression were noted in the liver of Group I animals compared to those found in Group II (p<0.001). Statistically significant alteration in the expression of all examined cell cycle related molecules was also found in Group I animals compared to the control ones (p<0.001). In conclusion our data suggest that the use of CD by mothers exerted prolonged liver injury in the newborn rats accompanied by increased hepatocyte proliferative capacity and altered cell cycle related molecules expression. 377 NUCLEAR MAGNETIC RESONANCE DETECTION AND CHARACTERISATION OF LIVER STEATOSIS IN RATS A. Suozzi 1 , S. Davalli 1 , P. Marzola 2 , D. Benati 2 , C. Zancanaro 2 , C. Marchioro 1 , L. Marocchio 1 , P. Cristofori 1 . 1 Research Centre, GlaxoSmithKline, Verona, Italy, 2 NMR Laboratory, DSMB, University of Verona, Verona, Italy Localised nuclear magnetic resonance spectroscopy (MRS) and high-resolution (600 MHz) proton (1 H) and carbon (13 C) NMR spectroscopy were employed in order to detect and characterise in vivo and ex vivo the hepatic lipid deposits in rats treated with a cationic amphyphilic compound known to induce steatosis. Han Wistar rats were treated with 50 or 200 mg/kg/day of compound. In anaesthetised animals, MRS of the liver was made in 2 cubic voxels (5x5x5mm) at 4.7 Tesla. After necropsy histological examination and high-resolution 1 H and 13 C NMR spectroscopy were performed on liver extracts. In rats treated with 200 mg/kg/day of compound the mean ratio between water and lipid peak was significantly lower (p<0.0013) than in control rats, thereby indicating a higher liver lipid content in the former. The in vivo NMR results were accounted as a mild steatosis at histology. High resolution 1 H NMR spectroscopy of liver extracts from the same animals showed shorter acyl chains (p<0.03), lower mean unsaturation (p<0.003) and polyunsaturation (p<0.0006) in fatty acids of steatotic livers. In steatotic livers, 13 C spectra showed a decrease in the relative percentage of 20:4 (p<0.03) and 22:6 (ns) and an increase of 18:2 (p<0.03) and 18:1 (ns) fatty acids; the relative percentage of cholesterol decreased but not significantly. In rats treated with 50 mg/kg/day of compound high resolution 1 H and 13 C NMR spectroscopy confirmed the general pattern found in 200 mg/kg/day treated rats, but the differences between treated and control rats were not statistically significant. These data suggest that in vivo localised MRS is able to detect non-invasively liver steatosis in the intact animal. This technique would allow for repeated investigation of the same animal yielding data on the time-course of liver steatosis. Analysis of 1 H NMR spectra characterised liver steatosis showing that the lipid deposit of treated rats contains shorter and more saturated fatty acids in comparison to controls. In addition, in 13 C NMR spectroscopy a redistribution of the relative content of unsaturated fatty acids showing a shift to mono- and di-unsaturated fatty acids was seen in treated rats. 378 HEPATIC STIMULATOR SUBSTANCE (HSS) ADMINISTRATION MODULATES CELL CYCLE RELATED MOLECULES EXPRESSION IN AN ANIMAL MODEL OF FULMINANT HEPATIC FAILURE AND ENCEPHALOPATHY A.P. Margeli 1 , E. Manolis 2 , L. Papadimitriou 2 , J. Stamoulis 1 , G. Gribilas 1 , S. Theocharis 1 . Departments of 1 Forensic Medicine-Toxicology, Medical School, and 2 Anatomy-Histology, Nursing School, University of Athens, Athens, Greece Hepatic stimulator substance (HSS) is a liver specific growth factor, implicated in hepatocellular proliferation and hepatoprotection in cases of acute liver injury. In the present study, we examined the effect of exogenous HSS administration on liver proliferating capacity and final outcome in an experimental animal model of fulminant hepatic failure (FHF) and encephalopathy. FHF was induced in adult male Wistar rats by three consecutive intraperitoneal injections of thioacetamide (TAA) (400mg/kg of body weight), at 24 h time intervals. The animals received also intraperitoneally either a saline solution or HSS (50mg protein/kg of body weight), 2 h after the second and third TAA injections, and were killed at 6, 12 and 18 h post the last TAA injection. Serum levels of hepatic enzymes and urea, blood ammonia values, liver histology, stage of hepatic encephalopathy and survival were statistically significantly improved in TAA-intoxicated and HSS-treated rats compared to TAA-intoxicated and saline-treated ones. In addition HSS ameliorated, in a statistically significant manner, liver regenerative indices as DNA biosynthesis, thymidine kinase activity and hepatocyte mitotic activity. The immunohistochemical expression of cell cycle related molecules Cyclin -A, -B, -D1, -E, p16, p21 and p27 was also statistically significantly increased in Poster Session P16. Lung toxicity TAA-intoxicated and HSS-treated rats compared to TAA-intoxicated and saline-treated ones. In conclusion our data suggest the beneficial effect of HSS administration in this animal model of FHF and encephalopathy, by decreasing toxicity and mainly by augmenting liver proliferative capacity. The ability of HSS to modulated cell cycle related events suggest its possible use as supportive therapy, in the management of FHF. 379 PROTEOMIC INVESTIGATION INTO N-NITROSOMORPHOLINE MEDIATED CHANGES IN RAT LIVER* A. Oberemm 1 , N. Querfurth 1 , C. Meckert 1 , L. Brandenburger 1 , A. Herzig 1 , K. Kalenberg 2 , E. Krause 2 , C. Ittrich 3 , A. Kopp-Schneider 3 , J. Hellmann 4 , P. Bannasch 3 , H.-B. Richter-Reichhelm 1 , U. Gundert-Remy 1 . 1 Department of Assessment of Chemicals, Federal Institute for Risk Assessment, Berlin, Germany, 2 Institute of Molecular Pharmacology, Berlin, Germany, 3 German Cancer Research Institute, Heidelberg, Germany, 4 Institute of Toxicology,, Merck KGaA, Darmstadt, Germany Proteomic approaches are widely explored to evaluate their usefulness for detecting early toxicological endpoints and for gaining insight into the mechanisms behind the toxic response. In order to relate changes in protein expression to conventional endpoints of toxicity, a common animal model of chemical hepatocarcinogenesis was used. N-nitrosomorpholine (NNM) at 20 mg/kg body weight was applied to young adult male Wistar rats for 7 weeks to induce hepatocarcinogenesis. After 18 weeks of exposure-free period, animals were killed and left liver lobes were prepared for histopathological and proteomic investigations. Liver tissue from 5 animals of each treatment group (vehicle-control + NNM group) was analyzed. Proteins were separated using 2D electrophoresis. Gels were visualized using ruthenium II tris and ProExpress™ imaging platform. Gel images were analyzed using ProteinMine™ 2D image analysis software. Spot values were statistically evaluated by Impressionist™ 2D data analysis software. 7 upregulated and 27 downregulated spots were detected in livers of treated animals. 31 spots were found in gels of the NNM-treated group only, 17 spots were restricted to gels of the control group. Differentially expressed spots were excised from gels mechanically and proteins were identified by common MS methods. Results of histopathology demonstrated a significantly increased number of focal preneoplastic and benign neoplastic lesions in livers of treated specimens. GST-p, a common marker for neoplastic tissues, was found to be highly expressed in livers of the treatment group, as confirmed by histological staining. Other upregulated spots included L-plastin, chloride intracellular channel protein 1, elongation factor-2, P-47 and aflatoxin B1 aldehyde reductase, which is suspected to play a mechanistic role in hepatocarcinogenesis and chemoprotection in the rat. Among downregulated spots senescence marker protein-30 was found, which may cause dysregulation of Ca-dependent pathways. Among others, Transaldolase, Serotransferrin precursor, Catalase, Ubiquitin carboxyl-terminal hydrolase 14 and 3-oxo-5-beta-steroid 4-dehydrogenase were detected only in the NNM-treated group. *Funded by the German Ministry of Education and Research, Grant-No. 0312618 P16 Lung toxicity 380 COMPARATIVE INHALATION STUDY OF THE STANDARD REFERENCE CIGARETTES 1R4F AND 2R4F E. Van Miert 1 , A. Teredesai 2 , G. Schepers 2 , B. Friedrichs 2 , P. Vanscheeuwijck 1 . 1 PHILIP MORRIS Research Laboratories bvba, Belgium, 2 PHILIP MORRIS Research Laboratories GmbH, Germany While the adverse health effects of cigarette smoke are well established, experimental research is still required to understand which s103 smoke constituents might be responsible for the various effects and by what mechanisms. Since 1983, the University of Kentucky Reference Cigarette 1R4F has been used as a standard in tobacco research. Such a reference cigarette is extremely useful because it provides a basis for comparing data that have been collected in various studies and laboratories. With stocks of the 1R4F running low, an equivalent reference cigarette, the 2R4F, was produced in 2001. Since there are small differences in the smoke chemistry of the two reference cigarettes, the present study compares them on a more complex biological level. In a 90-day inhalation study, male and female Sprague Dawley rats were exposed to mainstream smoke at 75 and 150 µg total particulate matter/liter or to fresh air 6h/day, 7 days/week for 90 days. A 42-day post-inhalation period was included to investigate the reversibility of findings. Selected respiratory physiology parameters, urinary excretion of metabolites of nicotine, acrolein, and 1,3-butadiene, and carboxyhemoglobin levels in blood indicated a comparable smoke uptake. There were no remarkable differences in in-life observations. Male rats exposed to smoke from the 2R4F had a slightly lower body weight gain. Typical smoke-related changes of clinical chemistry parameters were observed to a similar extent for both cigarettes. Lung inflammation as assessed by the accumulation of neutrophils in bronchoalveolar lavage fluid was comparable, but the serum levels of the chemokineinduced-chemoattractant-1 (CINC-1, rat analogue of GRO) tended to be lower in the 2R4F groups. Histopathological evaluation revealed mainly hyperplastic and metaplastic epithelial changes in the respiratory tract, but no differences between the cigarettes. By the end of the post-inhalation period, smoke-related effects had regressed to the same degree for both cigarettes. Within the framework of this study, inhalation toxicity of the Reference Cigarette 2R4F was equivalent to that of the Reference Cigarette 1R4F. 381 CYTOCHROME P450 GENE EXPRESSION AND PROTEIN ACTIVITY IN BRONCHOALVEOLAR MACROPHAGES AND BRONCHIAL EPITHELIAL CELLS OF SMOKERS AND NON-SMOKERS T. Thum 1 , V. Erpenbeck 2 , J. Möller 1,2 , N. Krug 2 , J. Borlak 1 . Fraunhofer Institute for Toxicology and Experimental Medicine, Drug Research and Medical Biotechnology, 2 Immunology/Allergology and Clinical Inhalation 1 The lung is a major target organ for inhaled toxicants, but also an interesting route for systemic drug application. Both toxification and detoxification of drugs and xenobiotics is mediated via cytochrome P450 monooxygenases and other drug metabolising enzymes. As tobacco smoke may alter tissue specific metabolism, we investigated gene expression and protein activity of major cytochrome P450 monooxygenases, as well as phase II enzymes (UGT2A1, EH, GSTA2, GSTp1, GSTm1), interleukines and interleukine receptors in lung cells obtained from bronchoalveolar lavage (BAL) and bronchial biopsies (BB) of smokers (S, n=9) and non-smokers (NS, n=10). We also studied production of IL1beta, IL8 and TNFalpha in freshly isolated cells from BAL samples. BAL-fluid recovery did not differ between S and NS. However, the total cell count of BAL was significantly increased in S compared with NS. This was mainly due to increased numbers of alveolar macrophages and neutrophils. Gene expression of CYP1A1, CYP2C9 and CYP2S1 was significantly increased in mRNA extracts from BAL of S, whereas transcript levels of CYP2B6/7, CYP2J2 and CYP3A5 was predominant in NS. Additionally, in mRNA extracts from BB of S gene expression of CYP1A1, CYP2C9, CYP2S1 and CYP3A5 was enhanced, but in NS mRNA expression of CYP2J2 was stronger. Likewise, gene expression of GSTp1 and GSTm1 was upregulated in BAL and BB of S, whereas EH was increased in BAL of S and in BB of NS. CYP monooxygenase gene expression correlated well with protein function as evidenced by a 3-fold increased EROD activity. Testosterone metabolism was significantly decreased in BAL samples of S. Finally, we observed decreased IL1beta and TNFalpha expression in BAL of S and detected significant increased IL8 protein secretion in this group. Upregulation of IL-8 in BAL of S may be linked to repression of CYP2B6/7, CYP2J2 and CYP3A5, whereas the significant CYP1A1 gene/protein induction is likely due to activation of the aryl-hydrocarbon receptor via constituents s104 Poster Session P16. Lung toxicity of tobacco smoke. We thus demonstrate significant changes in the gene expression and protein function of pulmonaryf CYPs, phase II enzymes and interleukines in various compartments of lung tissue of S and NS. These changes should be considered when inhaled drugs are prescribed to S or NS. 382 EVALUATION OF CIGARETTE SMOKE-INDUCED INTERMEDIATE CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) BIOMARKER RESPONSES IN MICE. C.J. Obot 1 , K.M. Lee 2 , A.F. Fuciarelli 2 , R.B. Westerberg 2 , W.J. McKinney 1 . Philip Morris USA, Richmond, VA; 2 Battelle Toxicology Northwest, Richland, WA Recent studies suggest that C57BL/6 (C57) mice develop emphysema following a chronic cigarette smoke exposure while ICR mice do not. Based on this information, these two strains of mice were used to investigate acute mainstream cigarette smoke exposure-related changes in intermediate biomarkers of COPD. Male C57 and ICR mice were exposed 2-hrs nose-only to mainstream cigarette smoke of 2R4F cigarettes (0, 75, 250 and 600 µg TPM/L) or filtered air for 7 consecutive days. BAL fluid samples were collected at 2 and 12 hours post-exposure and analyzed for biomarkers of effect {LDH, protein, cell differentials, NAG, Apoptosis, KC (IL-8), TNF-α, IFNγ, IL-1β, IL-5, IL-6, IL-10, IL-13, IL-17, GM-CSF, JE, MIP-1α, RANTES, TARC, SDF-1β, GSH/GSSG, collagenase/elastase and desmosine}. At 0-hr post exposure blood samples were analyzed for biomarkers of exposure (HbCO and Nicotine) and the respiratory tract macroscopically examined. Exposure biomarkers were slightly greater in the C57 when compared to the ICR. More necrosis was observed in the nasal epithelium of exposed C57. In general exposure-related increases in BAL fluid cytokines and neutrophils were greater in the ICR mice. NAG, a marker of necrosis, was significantly increased in the BAL fluid of C57 mice at 250ug/L TPM. Cellular apoptosis was greater in the ICR mice at 600ug/L TPM. These results suggest that cellular responses to cigarette smoke exposures (e.g. apoptosis and/or necrosis) may be early determinants of COPD in mice. Furthermore, measuring specific cytokines (e.g. KC, IL-1) along with apoptosis, neutrophil numbers and early markers of cellular activation/lysis (NAG) may be good intermediate biomarkers of COPD in mice that can be used in the evaluation of smoking products designed to reduce exposure to toxic constituents. 383 INVESTIGATION OF COMBINED EFFECT OF AMOSITE AND TOBACCO SMOKE INHALATION EXPOSURE ON INFLAMMATORY PARAMETERS OF BRONCHOALVEOLAR LAVAGE Hurbánková 1 , Beňo 1 , Černá 1 , Kováčiková 1 , M. M. S. Z. P. Bobek 1 , S.A. Kyrtopoulos 2 . 1 Respiratory Toxicology, Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic; 2 Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece Fischer 344 rats inhaled amosite asbestos fibrous dusts in a noseonly inhalation device during 6 months at two dosages. Six groups were exposed to: 1) 60 mg/m3 amosite fibres for one hour every two days combined with exposure to mainstream smoke from three cigarettes daily; 2) 60 mg/m3 amosite fibres for one hour every two days; 3) 30 mg/m3 amosite fibres for one hour every two days combined with exposure to mainstream smoke from three cigarettes daily; 4) 30 mg/m3 amosite fibres for one hour every two days; 5) exposure to mainstream smoke from three cigarettes daily plus immobilization stress as animals exposed to dust; 6) immobilization stress as animals exposed to dust. The animals breathe diluted mainstream tobacco smoke at the target concentration 30 mg of total particulate matter m3 air for one hour per daily exposure requiring to burn three cigarettes. The aim of this study was to find combined effect of amosite + cigarette smoke on the selected inflammatory BAL parameters. Following parameters were investigated: number of leukocytes and alveolar macrophages - AM; differential cell count: AM, lymphocytes, polymorphonuclear leukocytes; multinuclear cells and total proteins. Conclusion: a) Inflammatory parameters were the most changed after 60mg/m3 in both groups (with or without smoking but with higher significance in group exposed to combined effect); b) Dose dependence was evident in both groups (with/without tobacco smoke but mainly after combined exposure); c) Separate exposure to smoking or to amosite did not cause so strong effect as the combined exposure. This indicates the possible synergic effect of both noxious substances. 384 RESPIRATORY SYMPTOMS AND VENTILATORY DISORDERS AMONG A GROUP OF RUBBER WORKERS A. Rajaeefard, M. Neghab. Faculty of Health, PO Box 111, Postcode 71645, Shiraz, Iran Talc, a hydrated magnesium silicate, is one of the most important hazards in the rubber industry and occupational exposure to it has been associated with chronic respiratory diseases and ventilatory disorders. The purpose of this study was to determine the prevalence of respiratory symptoms and/or ventilatory impairments among a group of rubber workers with current occupational exposure to talc dust. Standard respiratory symptom questionnaires as well as pulmonary function tests were administered to a group of 105, randomly selected, male rubber workers. Additionally, seventy five subjects underwent chest radiography. Moreover, environmental monitoring, measurement and analysis of atmospheric inhalable and respirable dust, were performed at the site. The length of exposure to talc dust (Mean ±SD) was 16.8±6.5 years. Analysis of the dust showed that it contained about fifty five percent of crystalline silica, quartz (sio2 ). Additionally, the concentrations of inhalable and respirable dust were calculated to be 37.5±22.8 and 17.8±7.8 mg/m3 , (Mean ±SD), respectively which are far beyond the current standard of 0.3 mg/m3 for inhalable and 0.1 mg/m3 for respirable fractions. The prevalence of abnormal clinical symptoms including, cough, phlegm, wheezing and dyspnea were found to be 71.4%, 69.5%, 68.6% and 73.3%, respectively. These symptoms were more frequent among workers with more than 10 years of exposure. Chest radiograms of the subjects showed about eleven percent of abnormalities including emphysematous and infiltrative changes, pleural thickening and density in the right superhilar area. Respiratory parameters of the workers were found to be significantly different from the standard reference values. Therefore, it is concluded that exposure to talc dust is associated with impaired lung function and symptoms abnormalities. 385 ULTRAFINE PARTICLES CAUSE CYTOSKELETAL DYSFUNCTIONS IN MACROPHAGES: ROLE OF INTRACELLULAR CALCIUM Vicki Stone 1 , David Brown 1 , Winfried Möller 2 , Joachim Heyder 2 . 1 Napier University, School of Life Sciences, Edinburgh EH10 5DT, Scotland, UK. 2 GSF - National Research Centre for Environment and Health, Institute for Inhalation Biology, D-82131 Munich-Gauting, Germany Increased concentrations of fine and ultrafine particles (UFP) in the ambient air are correlate with increased morbidity and mortality. The mechanisms of health effects and the fate of UFP in the lung are not known. Alveolar macrophages (AM) are the primary defence cells in the lung periphery. The phagocytic and subsequent migratory activities of AM requires an intact cytoskeleton. The purpose of this study was to investigate the effect of fine and ultrafine particles on cytoskeleton associated functions, and to study the role of intracellular calcium transients in driving any changes. Alignment of phagocytized ferromagnetic microparticles (1.8 µm diameter) in a brief magnetic field pulse was used to detect a weak magnetic cell field (cytomagnetometry). The decay of the cell field (relaxation) was monitored to determine intracellular stochastic phagosome motions. Twisting of the magnetic phagosomes yielded cytoskeletal stiffness and mechanical integrity. J774A.1 mouse macrophages or primary AM from beagle dogs were treated with either carbon UFP, diesel exhaust particles (DEP) or urban dust (UD) (100 µg/ml/million cells) for 4 hours in serum-free medium, Poster Session P16. Lung toxicity or in combination with calcium antagonists, such as verapamil (Ca2+ channel blocker), BAPTA-AM (Ca2+ chellator), nacystelin (antioxidant), or W-7 (calmodulin inhibitor). Cell viability was tested by propidium iodide (PI) exclusion. UFP caused retarded intracellular phagosome transport, increased stiffness of the cytoskeleton and decreased cell viability. These effects were inhibited by the Ca2+ channel blocker verapamil and by the calmodulin inhibitor W-7, but not by the antioxidant nacystelin. The DEP and UD were less toxic than the pure carbonaceous UFP. Similar results were obtained for the primary AM and the J774A.1 cell line. The data show that UFP can induce disruption of cytoskeletal functions and cytotoxicity in AM and that intracellular calcium plays a major role in this process. UFP induced dysfunctions in AM implies a weakening of the cellular defence in the lung with an increased risk for infections and chronic diseases. infiltrative changes (2.3%). However, no significant changes were noted in the radiograms of the control group. Similarly, the results of spirometry demonstrated statistically significant reduction in lung function parameters i.e., vital capacity (VC, p=0.002), forced vital capacity (FVC, p=0.0006), forced expiratory volume in the first second (FEV1, p=0.0006), forced expiratory flow between 25% and 75% of the FVC (FEF25-75% , p=0.0003) and peak expiratory flow (PEF, p=0.01) in exposed workers when compared with controls. In conclusions, our data provide corroborative evidence to further substantiate the contention that exposure to cement dust is associated with respiratory symptoms and functional impairments. Acknowledgements: Funding through the Shiraz University of Medical Sciences, contract no. 82–1642, supported these investigations. The authors also wish to thank Dr Dastgheib, radiologist, Shahid Faghihi Hospital, for reading and reporting the radiograms. 387 386 STUDIES OF WORK -RELATED RESPIRATORY MORBIDITY AMONG EMPLOYEES OF A CEMENT INDUSTRY IN SHIRAZ, IRAN M. Neghab, M. Kamalee Neya. School of Health, P. O. Box 111, post code 71645, Shiraz, Iran Portland cement is made from hydrated calcium silicates, aluminum oxide, magnesium oxide, iron oxide, calcium sulfate, clay, shale and sand. The mixture is crushed and calcinated at high temperature with the addition of gypsum. Cement finds numerous uses in road and building construction. Pathological conditions encountered in cement industry include diseases of the respiratory tract, digestive disorders, skin diseases, rheumatic and nervous conditions, hearing and visual disorders. Although the main hazard in cement processing is dust and respiratory tract diseases are the most important group of occupational diseases in this industry, evidence for associations between exposure to cement dust and either respiratory symptoms or functional impairment has not been so conclusive. Additionally, the potential adverse health effects of portland cement have not been extensively studied. Therefore, this study was undertaken to evaluate more thoroughly, the effects of occupational exposure to cement dust on the respiratory system. The study population consisted of a group of 88, randomly selected, male workers with current occupational exposure to cement dust and 80, healthy male office workers without present or past exposure to dust that served as the control group. The average (Mean ±SD) age (years), weight (kg), height (cm) and the duration of exposure to dust for the exposed group were 44.3±7.9, 73.8±10.7, 170.8±7.2 and 18.8±7, respectively. The corresponding values for the control group were 41.7±5.8, 76.4±11.6, 172.5±7.8 and 0±0, respectively and there was no statistically significant difference between any of these means. Subjects were interviewed and respiratory symptom questionnaires, as suggested by the American Thoracic Society (ATS, 1978), were completed for all of them. They were classified as smokers and non-smokers and underwent chest X-ray and lung function tests according to the guidelines given by the ATS, 1979. Furthermore, using standard methods, personal dust monitoring for airborne inhalable and respirable dust was carried out at nine different worksites. Moreover, X-ray diffraction (XRD) and X-ray fluorescence (XRF) were performed to determine the silica phases and the Sio2 contents of the dust samples. XRD and XRF revealed that the crystalline silica phase of the sample was Quartz and the sample contained 26.9% Sio2 . Similarly, level of exposures to inhalable and respirable cement dust were estimated to be 53.4±42.6 and 26±14.2 mg/m3 , respectively (Mean ±SD). These values exceeded the current standard of 10 mg/m3 for inhalable and 3mg/m3 for respirable dust. Exposed workers, regardless of their smoking habits, had higher prevalence of regular cough (31.81%), phlegm (26.1%), wheezing (28.4%) and shortness of breath (17%). The corresponding values for control group were 20%, 15%, 5% and 5%, respectively and the differences were statistically significant (p<0.05). These symptoms were more frequent among the smokers for both groups. Chest radiograms of exposed workers showed emphysematous changes (15.9%), old calcified granulomas (5.7%), emphysematous changes associated with inflammatory process (4.5%), evidence of chronic inflammatory process (4.5%), focusal calcification of the lungs (4.5%) and s105 TIME COURSE OF PULMONARY RESPONSES OF RATS AFTER INSTILLATION OF QUARTZ- IMPORTANCE OF SURFACE CHARACTERISTICS - C. Albrecht 1 , W. Drommer 3 , R. Schins 1 , A. Becker 1 , D. Höhr 1 , K. Unfried 2 , A. Knaapen 1 , P. Borm 1 . 1 Particle Research Core, 2 Experimental Toxicology, Institut für Umweltmedizinische Forschung (IUF), Düsseldorf, Germany; 3 Dept. of Pathology, School of Veterinary Medicine Hannover, Germany Respirable quartz is known to induce inflammation, fibrosis and cancer in the rat lung. But mechanisms are not yet fully understood. However, surface characteristics of particles seems to be an important criteria for pathological alterations. Recently we were able to show, that surface coating of quartz particles by polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL) is able to inhibit acute pulmonary response after particles instillation.To analyze the importance of particle surface characteristics, we investigated the in vivo effects of different quartz preparations in rat lungs after a single intratracheal instillation of 2mg native DQ12 quartz, or DQ12 coated with PVNO, AL or appropriate controls (saline, PVNO, AL) at six different time points up to 1 year. After quartz instillation female Wistar rats showed a marked and persistent increase in cell toxicity (LDH, total protein), inflammation (total cell number, differentials, macrophage inflammatory protein-2, β-glucuronidase) and oxidative stress response (myeloperoxidase, Trolox equivalent antioxidant capacity) measured in broncho-alveolar lavage. Lung sections demonstrated inflammation, epithelial hyperplasia (Surfactant protein C-in situ hybridization, Ki-67-immunohistochemistry) as well as fibrosis (Sirius red staining based severity score). Up to 90 days these effects could be completely blocked by surface modification with PVNO and partly using AL coating. Determination of silica burden suggests that also lung clearance is different for coated quartz particles. However, one year after instillation histopathological investigation revealed no differences between the various quartz preparations. 388 LUNG TUMOR FORMATION IN RATS AFTER INTRATRACHEAL INSTILLATION OF FINE AND ULTRAFINE PARTICLES. Paul J.A. Borm 1 , Catrin Albrecht 1 , Wolfgang Drommer 2 . 1 Particle Research Group, Institut für Umweltmedizinische Forschung (IUF), Düsseldorf, and 2 Institute for Pathology, School of Veterinary Medecine, Hannover, Germany Objectives: This study set out to investigate lung tumor response in the rat by fine and ultrafine (uF) particles of the same chemical composition, and to compare tumors induced by two different uF. Methods: Female Wistar rats (190 gr) were instilled with F/ uF TiO2 , F/uF carbon black and compared to untreated controls and rats treated with amorphous silica as a negative particle control (Aerosil) or diesel as a positive control. For each treatment group 48 rats were used and doses applied for fine particles (30, 60, 120 mg), ultrafine particles (15, 30, 60 mg), diesel (7.5, 15 and 30 mg) were based on gravimetric and volumetric overlap. Results: At 129 weeks bronchoalveolar hyperplasia, interstitial fibrosis and inflammation was visible in all groups treated with particles. In control and s106 Poster Session P16. Lung toxicity Aerosil-treated (15, 30 mg) rats no lung tumors were observed, while in PSP treated rats a dose-dependent lung tumor response was seen. At a similar mass dose (60 mg) of test particles, fine TiO2 showed significantly less malignant tumors (8%), compared to other PSP (40– 60%). Among malignant tumors bronchoalveolar adenocarcinoma (BAAC) and squamous cell carcinoma (SCC) occurred almost equally in PSP treated animals, except for diesel. A larger number of tumors was seen after uF CB in comparison to uF TiO2 , but only small differences in tumor type. Conclusions: Relating lung tumor response to surface area and volume shows that uF and fine or F induced lung tumors fit on the same line when surface is used, and on different lines when volume is used. We therefore hypothesize that uF particles in addition to a volumetric effect on macrophage overload, have additional effects that can increase or catalyze their carcinogenic outcome. In this study we used biological material of the so-called 19-dusts study originally designed by Pott and Roller 389 PM10 EFFECT ON EMAP-II EXPRESSION IN LUNG EPITHELIAL CELLS Ernesto Alfaro-Moreno 1 , Yee M. Heng 2 , Jodie Edgson 2 , Peter Symonds 2 , Alvaro Osornio-Vargas 1 , Irma Rosas 3 , J. Clifford Murray 2 . 1 Subdirección de Investigación Básica, Instituto Nacional de Cancerología, México, 2 Department of Clinical Oncology, University of Nottingham, Nottingham UK, 3 Centro de Ciencias de la Atmósfera, UNAM, México PM10 can be retained in the lung and may be linked to induction of neoplasia. These particles have been shown to induce expression of TNFα, IL-6, PGE-2, and also to cause DNA damage. We recently showed that under certain conditions tumour cells release the novel cytokine-like molecule, endothelial monocyte-activating polypeptide-II (EMAP-II), which can activate apoptosis in lymphocytes, and thus may assist tumour in evading the immune system. We hypothesised that PM10 might up-regulate the expression by lung epithelial cells of EMAP-II. In this study, we used the A549 cell line as a model of lung epithelium. We exposed A549 cells to PM10 (2.5–20µg/cm2 ) for 24hr. Using semi-quantitative RT-PCR, we detected increased expression of EMAP-II mRNA. To confirm that increases in EMAP-II mRNA were due to enhanced transcriptional activity, we transfected A549 cells with a reporter construct containing a 400bp fragment of the EMAP-II promoter region cloned upstream of the luciferase gene, and exposed these cells to PM10 for 24 hours. Exposure to PM10 significantly up-regulated luciferase reporter expression. Our results suggest that PM10 induce the expression of EMAP-II. PM10 induce DNA damage and pro-inflammatory effects, and therefore may promote the development of neoplasia. Simultaneous expression of molecules such as EMAP-II may aid this process by suppressing the immune response. 390 TRANSCRIPTIONAL REGULATION OF Nrf2-RELATED GENES BY 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) IN HUMAN BRONCHIAL EPITHELIAL CELLS H.W. Kim 1 , D.H. Cho 2 , M.H. Cho 1 . 1 Laboratory of Toxicology, College of Veterinary Medicine, Seoul National University, Seoul, Korea 2 National Center for Toxicological Research, KFDA, Seoul, Korea The nuclear related factor (Nrf2) binds to the antioxidant responsive element (ARE) and initiates the transcription of genes encoding for detoxifying enzymes and cytoprotective proteins. Rapamycin causes the phosphorylation of eIF4E binding protein 1 (4E-BP1) and releases eukaryotic translation initiation factor 4E (eIF4E) essential for cap-dependent protein translation. In this study, the effect of 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; tobaccospecific nitrosamine) on Nrf2-related genes was examined on human bronchial epithelial cells (NL-20). The AP-1, ARE, NF-kB, Nrf2 wild type, Nrf2 mutant, and Nrf2-promoter constructs were transfected into NL-20. After transfection, various concentrations of NNK with/without 50 nM rapamycin were treated on the cells. On the other hand, time-dependent studies were performed by the treatment of 100 nM of NNK with/without rapamycin. Concentration-dependent study revealed that NNK with/without rapamycin did not induced any significant changes of the transcriptional levels of NF-kB, and AP-1. However, concentration-dependent decrease of Nrf2 transcriptional level was clearly observed in both NNK alone and NNK with rapamycin. General expression level of wild-type Nrf2 was higher in NNK+rapamycin than in NNK alone. Such findings were reversed in Nrf2 mutant studies. Regardless of rapamycin pretreatment, NNK caused concentration-dependent increase of Nrf2 mutant expression while general expression level was higher in NNK alone compared to Nrf2 wild-type study. NNK with/without rapamycin caused concentration-dependent reduction of ARE transcription In time-course course study, the transcriptional level of Nrf2 wild type increased until 16 hrs and then decreased after that time in NNK with/without rapamycin. Interestingly, transcriptional level of Nrf2 mutant began to decrease after 8 hrs in NNK with/without rapamycin in time-dependent manner. The transcriptional level of ARE increasedin time-dependent manner by the treatment of NNK+rapamycin, whereas NNK alone did not cause any significant changes. Our results showed that NNK induced significant changes of Nrf2 and ARE through controlling protein translation while NNK did not cause any significant changes of AP-1 and NF-kB. Supported by BK21 391 DIFFERENTIAL EFFECTS OF TRIVALENT ANTIMONY, PENTAVALENT ANTIMONY AND ANTIMONY TRIOXIDE ON RAT ALVEOLAR MACROPHAGES IN VITRO R. Poon, I. Chu. Environmental Health Science Bureau, Health Canada, Ottawa, Canada, K1A 0L2 Humans are exposed to antimony compounds present in various forms in water, trivalent (SB3+ ) and pentavalent (SB5+ ) states in anthelmintic drugs, and inhalable particulate form as antimony trioxide (SB2 O3 ). While inhaled SB2 O3 is known to cause pneumoconiosis and upper airway inflammation and proliferation of macrophages in humans, and increased incidence of lung tumor in rats, the pulmonary effects of SB3+ and SB5+ are not known. The acute effect of SB3+ (potassium antimony tartrate), SB5+ (sodium stibogluconate) and SB2 O3 on freshly isolated alveolar macrophage (AM) was therefore studied with emphasis on cell viability (LDH release), production of reactive oxygen species (chemiluminescence), and secretion of cytokines (TNF-α and IL-6). Incubation of AM with SB3+ (0.1 mM) at 37 °C resulted in a significant release of LDH into the media at 5 but not at 3 h, indicating that SB3+ may be cytotoxic after longer term exposure. Under similar incubation conditions, SB2 O3 (0.5 mg/ml) and SB5+ (0.1 mM) were noncytotoxic. SB2 O3 (0.5 mg/ml) stimulated AM to generate chemiluminescence while SB3+ and SB5+ at up to 0.1 mM were without effect. The potency of SB2 O3 to stimulate chemiluminescene was compared with that of Zymosan A and titanium dioxide (TiO2 ), both known activators of AM, and the following ranking was observed: TiO2 > SB2 O3 > Zymosan A. However, after AM was preincubated with SB2 O3 or SB3+ for 30 min, both compounds caused a dose-dependent suppression of zymosan A-induced chemiluminescence. Incubation of AM with SB3+ significantly reduced the amount of TNF-α released into the media but SB2 O3 and SB5+ were without effect. All three compounds had no effect on the level of IL-6 in the incubation medium. The results demonstrated differential effects of antimony compounds on AM in vitro. The stimulatory effect of SB2 O3 is consistent with its know pulmonary toxicity. The cytotoxicity of SB3+ , and the suppressive effect of SB2 O3 and SB3+ on Zymosan-A induced chemiluminescence suggest that these compounds may compromise the bactericidal activities of AM. 392 C0-ADMINSTRATION OF CAPTOPRIL OR NIACIN WITH PARAQUAT IN ISOLATED RAT LUNG PERFUSION G. Nasiri, M. Honarjoo, M. Ghazi-Khansari. Dept of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Paraquat (PQ), a strong contact and non-selective weed killer,causes lung damage in man and in several species of laboratory animals. The damage is manifested at first by hemorrhage and edema and then, Poster Session P17. Reproductive and developmental toxicology at later stages, by consolidation of the lung and the development of fibrosis. Angiotensin converting enzyme inhibitors previously reported to inhibit fibrosis due to stimulation of fibroblast proliferation and collagen synthesis by non-parenchymal cells. Decrease in the levels of serum fibrosis markers and the amount of collagen in cardiac muscle, inhibition of pulmonary fibrogenesis in irradiated rats and improvement to proteinuria and glomerular lesions have been achieved by the administration of ACE inhibitors. The anti- oxidative action of captopril, an ACE inhibitor, appears to be attributable to the sulphahydryl group (SH) in the compound. Niacin also showed that to replenishing the NAD and ATP that are depleted in response to drug- induced reactive oxygen species. There are some studies that shown niacin in the drug-induced animal model of fibrosis resulted in a decreased in pulmonary fibrosis. In this study, male wistar rats weighing 250–300g were used in this experiment. Animals were divided into 6 groups. In group 1–4, lung were perfused by kerbs buffer alone (control), niacin (150uM), captopril (10uM) and PQ (600uM). Groups 5–6, after 5 mins of stabilization with Kerbs buffer, PQ (600µM) and Niacin (150µ M) or Captopril (10µ M) were added to perfusion fluid. Then, the biochemical changes in perfusion fluid of isolated rat lung were examined within 1 hr and compared to PQ alone. The results shows that captopril (10uM) and Niacin (150uM) significantly decreased PQ induced lung toxicity. LDH (lactate dehydrogenase) activity significantly decreased in treatment groups as compared to the PQ group (p<0.001). This study showed that paraquat causes an increase in lipid peroxidation and LDH activity while cause a decrease in GSH and total protein in the isolated lung perfused rat. The biochemical changes by captopril or niacin may be due to stored glutathione, which may results in reduction of LDH activity in lung tissue. This mechanism together with the prevention of lipid peroxidation by captopril may reduce the lung toxicity of PQ. P17 Reproductive and developmental toxicology 393 HDAC-INHIBITORS ARE TERATOGENIC IN THE MOUSE Francesca Di Renzo, Maria Luisa Broccia, Valentina Massa, Elena Menegola, Erminio Giavini. University of Milan- Department of Biology, 20133 Milan, Italy Drugs modulating the acetylation status of histones have been proposed recently as novel approach for the treatment of cancer. A major concern for Histone Deacetylase (HDAC)-inhibitors therapy is the expected toxicity of these compounds, because of the general involvement of HDACs in a variety of fundamental cellular processes. In support of this view there is one publication reporting malformations in embryos exposed in vitro to the HDAC-inhibitor Trichostatin A (TSA), but another paper refers of absence of embryotoxicity in mouse embryos exposed in utero to 15 µg TSA. Furthermore, Valproic Acid (VPA), a well known teratogenic agent both in human and in laboratory animals, has been shown to be able to mimic the histone deacetylase inhibitors, causing hyperacetylation of histones in cultured cells. We performed this experiment in order to compare the effects of TSA and VPA on embryonic development. Female CD mouse were treated i.p. with 2 mg/kg TSA on day 8 at 9 a.m. or 7 p.m., or with 4 mg/kg TSA on day 8 at 9 a.m (day of positive plug 0). Another group was treated with 300 mg/kg of Valproic Acid (VPA) on day 8 at 9 a.m or 7 p.m. Control females were treated with solvent. The females were killed on day 18 of gestation and their fetuses were prepared for skeletal examination after double staining with Alcyan blue and Alizarin red. TSA exposed fetuses showed axial skeleton malformations dose and time related: duplications and homeotic respecification of axial segments; there were also a few cases of exencephaly. VPA treatment produced, as expected, axial skeleton malformations. In conclusion TSA and VPA are teratogenic in mouse producing a very similar spectrum of axial skeleton defects, probably due to ana lateration of the patterning of the gene expression controlling of this region, which could be related to HDAC inhibition. 394 s107 MECHANISMS INVOLVED IN TRIAZOLE-INDUCED TERATOGENESIS: IN VITRO STUDY Valentina Massa, Maria Luisa Broccia, Francesca Di Renzo, Elena Menegola, Erminio Giavini. University of Milan- Department of Biology, 20133 Milan, Italy Triazole-derivatives are antimycotic compounds used in agricolture as well as in clinical and veterinary therapy. Literature data, confirmed by results obtained in our previous studies, showed severe alterations at the level of the branchial apparatus after triazoles in vitro exposure (hypoplasia of I and II branchial arches, fusion between I and II branchial arches). Our previous works on Fluconazole (FLUCO) showed that FLUCO exposure is able to alter the morphogenesis of the branchial apparatus modifying the rhomboencephalic neural crest cells (NCC) migration in rat embryos cultured in vitro. The hindbrain segmentation was also altered by FLUCO. The aim of the present work was to extend the study of the mechanisms to other molecules of this class to verify if different molecules of the same family have the same target. For this purpose 9.5 d.p.c. old rat embryos were exposed in vitro to Flusilazole (25 µM), Triadimefon (250 µM) and Triadimenol (125 µM). The morphology of the embryos was analysed after 48 hours of culture. Some embryos cultured for 60 hours were immunostained using antibodies anti-160 kDa neurofilament in order to evaluate the cranial nerve structures. The localisation and distribution of NCC was evaluated after 24, 30 and 48 hours of culture, using the specific immunostaining of CRAB proteins. The expression and localisation of Hox b1 and Krox 20 proteins (used as markers for the study of the correct hindbrain segmentation) was evaluated using whole mount immunostaining after 24 hours of culture. The obtained results showed very similar effects after exposure to Flusilazole, Triadimefon and Triadimenol: abnormalities at the level of the branchial apparatus; disorganisation and fusions at the level of the cranial nerves; abnormalities in the migration of NCC, not able to form 3 distinct migration stripes from the rhomboencephalon to the branchial apparatus; alteration of the hindbrain segmentation, with reduced and scattered immunolocalised stripes. The collected data suggest a common target for all the examined molecules: the observed branchial abnormalities are due to anomalous NCC migration related to an incorrect organisation and specification of the rhomboencephalon. 395 EMBRYOTOXICITIC EFFECTS OF RANITIDINE B. Nassrollazadeh, M. Lalancy, D. Onsory. Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran In this work, we tried to know something more about the embryotoxicity effects of the doses of 50, 200, 400 mg/kg/day of ranitidine of (H2 antihistaminic agent) by intraperitoneal administration on mice. The studies were performed on albino mice kept under specific conditions and a constant dark-light cycle at 24+1 C and 55+5% relative humidity. Generally, the animals were acclimatised for four weeks before mating. Two female mice at 12–14 weeks of age were placed overnight with a male of proven fertility. The day on which a vaginal plug was found, was taken as day one of pregnancy. Also the vaginal smear was prepared for further proof. Treatment of pregnant females was started from day 7 and continued up to the 15th day of gestation and then on day 18 they were necropsied for routine teratological observations. The live fetuses were weighed and inspected forgross external abnormalities under a dissecting microscope. Resorption plus dead fetuses less than 6mm of length were designated early death and dead fetuses of more than 6mm of length were consequently called late dead. The statistical study was done by student - t Test. One-third of the fetuses were fixed in bouin’s fluid to detect visceral malformations by the rasor-section technique. There was no significant difference in the frequency of late death between the control groups and the groups given raniditin. Differences were observed in the number of implantation sites except for 400mg/kg/day. Data pooled from all experimental groups clearly show that pig tail, deformed cranium, low body weight and skeleton, unshaped external car and jaw and polydactylity are the most s108 Poster Session P17. Reproductive and developmental toxicology common external abnormalities. Results of this study show the hazards of ranitidine used during early pregnancy. 396 THE EFFECTS OF A SINGLE TERATOGENIC DOSE OF VALPROIC ACID ON PLASMA ZINC CONCENTRATION IN THE FEMALES RAT P. Pasbakhsh, M. Barbarestani, F. Abolhassani, M. Abozaripour. Department of Anatomy,Tehran university of medical science,Tehran,Iran Valproic acid with good anticonvulsant activity and comparatively low central nervous sedation has become accepted as one of the most important anti epileptic drugs.For this reason it has been estimated that, in the United States, 700–1000 Pregnant women take valproic acid each year.The potential teratogenicity of valproic acid on neural tube in infants born to mothers who have taken this drug has been described and dismissed in several letters.The incidence of neural tube defect following exposure to valproic acid (VPA) during the first weeks of pregnancy can increase to 1–2%.VPA readily binds zinc. For this reason and for a relationship between zinc deficiency and neural tube defects and highest incidences of these abnormalities in areas of the world where human zinc deficiency exists we investigated the effect of VPA on plasma zinc concentration in females rat.For this purpose, we selected 12 rats in 2 groups: 1. Control (normal saline 4CC/kg-intra peritoneal). 2. Experimental (VPA 350Mg/kg-intra peritoneal). All the dams were sacrificed with cervical cutting and plasma prepared. Plasma zinc concentration was measured with flameless atomic absorption method.All statistical evaluations of data compared treatment group with control at P<0.05 levels of significance.Quantitative data were presented as a mean±SD and were analized by ANOVA. Results: plasma zinc concentration (mean±SD) in experimental group was lower than control group. the results of this study indicate that VPA reduces plasma zinc concentration. Regarding the role of zinc in embryonic development this reduced plasma zinc concentration is a possible mechanism of VPA teratogenesis. 397 MENSTRUAL FUNCTION IN FEMALE WORKERS PROFESSIONALLY EXSPOSED TO CYTOSTATIC DRUGS: MODEL OF TOXICOLOGICAL EVALUATION G.F. Desogus. Study Reports of Toxicology, Azienda USL 7 di Carbonia, Italy Toxic effects, that are linked to the use of cytostatic drugs are known both on the gonadic function and at endocrine level in professionally exposed nurses, with reported alterations in sexual hormonal secretions and adverse effects on the oogenesis and the fertility. In this research work, an experimental model in order to value the associated risk of alterations concerning the menstrual cycle in nurses that manipulate cytostatic drugs. The epidemiological study is carried out on the staff exposed to the manipulation of the cytostatic drugs, taking into consideration some strictly correlated factors (age, smoke, alcohol, diet, pregnancy) and such data must be homogeneously distributed. Data referring to alterations of the menstrual cycle must be collected and analyzed for every worker. Data include changes about the duration of the cycle, variations of the menstrual flow, specific troubles and pathologies linked to irregular ovulations and mixed disfunctions of ovarian nature or linked to the activity of the adrenal gland. In order to improve the validity of data about the menstrual cycle, a series of information must be acquired. These are linked to the hormonal dosage (hypophysial gonadotropin, prolactine, estrogens): they are valid functional index in the diagnostic of female infertility. Data referring to personal, physiological and pathological anamnesis, must be collected and analysed, with the presetting of a series of generic questions (fertility) and specific ones (pregnancy), included the etiological factors of physiological and anatomical nature, observed births, waiting time of pregnancy, spontaneous abortions and reproductive pathologies. The correlation between the data of the observed female fertility and the aspected ones will define the relations of standardized fertility (RFS): In the demographic-statistic field, the fertility will be associated to the concepts of fertilization and to the absence of correlated pathologies to the professional exposure on the nurses exposed to cytostatic drugs, normally active, with regular ovulation, with the exception of steril, pregnant and post-partum women. From the examination of the data the cases of infertility will be emphasized for non conception in the presence of sexual relations and absence of contraception and those cases of sterility for the presence of reproductive pathologies. Moreover, it will be necessary to consider some factors of individual susceptibility, studying the biochemical and hormonal variations, with the use of suitable epidemiological indicators of clinical type (fertility of couple, analysis of the menstrual cycle, malformations, reproductive troubles), in order to supplement the acquisition of specific knowledges around the mechanism linked to the environmental exposure to cytostatic drugs. 398 STUDY OF THE EFFECTS OF MATERNAL ADMINISTRATION OF MORPHINE ON THE EMBRYONIC LIVER SINUSOID AND KUPFFER CELLS Gh. Kaka 1 , H. Sahraei 2 , S.H. Sadraei 1 , H. Bahadoran 1 , H. Dashtnavard 1 . 1 Departement of anatomy; 2 Departement of Physiology, Baghyatallah University of Medical Sciences, Tehran, Iran The hazardous and adverse effects of drug consumption in pregnancy on development of embryonic tissues is an obvious fact. In the present study, the effect of morphine consumption on the structure and number of Kupffer cells and liver sinusoids in the embryos were investigated. Sexually mature female Wistar rats (w:250–300 gr) were treated with 0.1 mg/ml of morphine sulfate solution orally. Addicted female rats were then caged overnight with non-addicted male rats and the day that sperm was detected was considered day zero of pregnancy. The female rats were killed by chloroform on gestation day 17 and their emberyos were taken out rapidly. The liver of embryos were removed, fixed and prepared for histological studies. The paraffin sections were then stained with H&E technique. Quantitative computer-assisted histomorphology study were done on the Kupffer cells and sinosoidal dilatation, as well as arrengment of the hepatocytes were examined. Our results showed that the number of kupffer cells and the widening of liver sinusoids were increased significantly in experimental group when compared to control group (p<0.05). In addition, the arrengment of the hepatocytes was more irregular in experimental group than control group. The conclusion of this study revealed the hazardous and risk of morphine addiction of female rats on the development of liver of their embryos. 399 IN VIVO MICROINJECTION OF ANTISENSE MORPHOLINO OLIGOS TO PREDICT THE TERATOGENIC POTENTIAL OF NEW DRUGS: AN INITIAL VALIDATION WORK ON VEGF D. Manera, M. Longo, S. Zanoncelli, P. Meroni, K. Gunnarsson. Department of Discovery and Development Toxicology, Pharmacia S.p.a, Milan, Italy Angiogenesis is fundamental vertebrate developmental process that requires signaling by the secreted protein vascular endothelial growth factor-A (VEGF-A). VEGF-A is an important regulator of angiogenesis in humans and it is a target for anti-cancer drugs. We have used morpholino-based targeted gene knock-down technology to generate a zebrafish loosing VEGF-A function. This is done by injecting specific morpholino phosphorodiamidate oligonucleotides into the fertilized eggs, at the one- to eightcell stage. Results obtained will be presented. Concomitantly, compounds with anti VEGF activity were tested in a Zebrafish Teratogen assay which is a 72-hour whole embryo developmental toxicity screening. After fertilization, 1000-cell stage blastulae are cultured up to the larval stage of “protruding mouth”, corresponding to the end of the embryogenetic and morphogenetic periods, in the presence of the substances to be tested. Phenotype of VEGF-A knock-down zebrafishes and those obtained with anti VEGF-A compounds will be compared to evaluate Poster Session P17. Reproductive and developmental toxicology the predictivity of the model in assessing the teratogenic liability linked to the inhibition of the pharmacological target. In order to investigate the possibility of extrapolation of the results obtained in fishes to mammals, the same compounds were also tested in vitro using the mammalian the Whole Embryo Culture model. Rat embryos were explanted on Day 9.5 of pregnancy and cultured for 48 hours in a medium containing the test articles. Results obtained in mammals were then compared with those obtained in Zebrafish, and showed that effects observed in Zebrafish, mimic effects obtained in rat embryos. Therefore, we conclude that the use of knock-down target technology in Zebrafish may serve a predictive in vitro model to anticipate teratogenic potential linked to the pharmacological target of compounds. The Zebrafish teratogen assay is a reliable model for prediction of the teratogenic potential of lead compounds and product candidates with an higher throughput compared to the mammalian embryo assays. 400 MODIFIED ONE-GENERATION REPRODUCTION STUDY OF URSODEOXYCHOLIC ACID IN RATS AND ITS SUBCHRONIC TOXICITY IN THE F1 OFFSPRING V. Štětinová, V. Herout, J. Květina. Institute of Experimental Biopharmaceutics, Joint Research Center of PRO.MED.CS Praha a.s. and the Academy of Sciences of the Czech Republic, Hradec Králové, the Czech Republic Ursodeoxycholic acid (UDCA) is widely used as therapeutics agent for the treatment of hepatobiliary diseases. The aim of the study was to evaluate the safety of administration of UDCA during the pregnancy because the information concerning its reproductive toxicity is scarce. Daily p.o. dose (1000 mg/kg) was administered to mothers during pregnancy and lactation and thereafter to several groups of the F1 offspring (both sexes) for 1, 2 and 3 months while other groups of F1 offspring were treated with the vehicle (polyethylene glycol 400). Control groups received the vehicle (e.g. parental females and their offspring). Fertility, gestation, maternal toxicity, number of live foetuses per litter and their clinical status were not changed after administration of UDCA. No evidence of embryolethal or teratogenic effects of UDCA was found. In F1 generation animals treated with UDCA, the weight gain was significantly reduced during suckling period and water consumption was increased, significantly during the first 4 weeks of administration. Urine and hematological examinations were not changed. Clinical chemistry determination showed only slightly increased creatinine concentration and in several animals elevated plasma level of ALT and AST. The weight of organs and gross necropsy did not show any changes and histopathology revealed only slightly higher frequency of single necrosis of hepatocytes in comparison with the control animals. 401 DEVELOPMENTAL TOXICITY OF 1,2-DIBROMOETHANE AND 1,3-BUTADIENE AFTER INHALATION EXPOSURE OF PREGNANT RATS. Ludmila Vodicková 1 , Emil Frantík 1 , Miroslava Hornychová 1 , Pavel Vodicka 2 . 1 National Institute of Public Health, Prague, Czech Republic; 2 Institute of Experimental medicine, Academy of Sciences of the Czech Rep. Prague, Czech Rep 1,2-Dibromoethane (DBE) shows high reproductive toxicity and is classified as probably carcinogenic to humans (Group 2A -IARC 1999) similarly as 1,3-butadiene (BD). Concentrations of DBE were chosen to avoid toxicity for mothers, BD is genotoxic. Mothers were exposed to 0.5 mg/l DBE for 6 hrs or to 1 mg/l DBE for 3 hrs or to 1 mg/l BD for 6 hrs, the 10th, 11th and 12th days of pregnancy. Tree control groups exposed to clean air differed in the access to food: a) ad libitum, b) ad libitum except 6 hrs of exposure, c) food limited to the amount consumed by the DBE exposed group. Following parameters were analysed: maternal toxicity, length of pregnancy, number of living offspring and number of perinatal deaths, postnatal somatic and neurobehavioural development; last examinations were performed four weeks after weaning. Results: DBE 1 mg/l - caused transient maternal toxicity, increased number of dead foetuses, and higher level of spontaneous s109 motor activity when compared to all controls. In both exposed groups was found lower birth weight than in controls. Group exposed to 0.5 mg/l DBE showed lower exploratory activity and lower peak of night activity as well as lower index of neurobehavioural development in the 11th day of testing. In the tested concentrations, no neurobehavioral toxicity of BD was observed; DBE influenced the behaviour of offspring up to the 8th postnatal week of life, with exclusion effect of limited food intake. Acknowledgement: This study was supported by GACR 310/01/ 0802 402 NEUROTOXICOLOGICAL EFFECTS OF METHYL MERCURY ON DEVELOPING BRAIN OF ALBINO RATS: HISTOLOGICAL STUDY Eman A. Seif 1 , Amany S. Ismaeel 2 . 1 Forensic Medicine and Toxicology, 2 Histology department, Faculty of Medicine, Alexandria University, Alexandria, Egypt Mercury is considered one of the most important and widespread environmental pollutants, which poses a serious potential threat to the human health. Mercury exists in the environment in three major forms: organic, inorganic and elemental. Organic methyl mercury is the most widely distributed and toxic form of mercury. It is used extensively in agriculture as fungicide. It is also bioaccumulated in sea food and fresh water fish. The central nervous system is the critical organ for mercury exposure. The aim of the present work was to study the possible neurotoxicological effects of low-level methyl mercury exposure on developing brain of albino rats. The study was carried out on nine pregnant female albino rats. Three pregnant rats constituted the control group. The remaining six pregnant rats were given methyl mercury orally in a dose of 2 mg/kg body weight daily during late gestational period and continued for 25 days after birth (until weaning). Control and experimental rat pups were sacrificed following weaning (25 postnatal days). The whole brain was completely removed and stored in 10% formaline for subsequent histological processing. The present study revealed that there were marked histological changes in the brain of rat pups following low level methyl mercury exposure. It was concluded that methyl mercury is a potent neurotoxic substance. 403 ANALYSIS OF THE MORPHOLOGICAL CHANGES INDUCED BY INGESTION OF LEAD IN THE REPRODUCTIVE TRACT OF THE RAT. T.M. Ruiz 1 , J.L. Morán 2 , C. Morán 2 , E. González 2 , A. Handal 2 . 1 Centro de Química Benemérita Universidad Autónoma de Puebla., Puebla; México. Laboratorio de Investigaciones Biológicas2 del Instituto de Ciencias de la Benemérita Universidad Autónoma de Puebla, Puebla; México In this study were analyzed the morphological changes at tissue level produced by the toxic effect of the lead in different concentrations. The lead concentrations that were used during the experiment, was the same lead value average quantified in the blood of human population of San Nicolás de los Ranchos Puebla, Puebla; Mexico. This population is exposed to the following sources of pollution by lead: the ashes and steams of the volcano Popocatépetl (in eruption); and the crockery glassed to prepare the foods. Rats of the stock CII-ZV were exposed to different concentrations of lead acetate in the water during 90 days. It was formed a control group and three experimental groups of 8 rats each one. To each group was administered to respectively with 0.0, 0.003, 0.03 and 0.6 g/l of lead acetate. Two rats of each control and experimental groups in each one day of cycle estrous were sacrificed by decapitation. To the autopsy were extracted ovary, uterus, kidney, adrenal glands, liver and pituitary gland, those which were weighed and analyzed by histology and by confocal microscopy. In the blood was measured lead. In those rats with 0.6 g/l of lead acetate was observed an increase of follicular atresia, as well as a lead deposit within the ovary. These results constitute an important information to estimate the impact of the lead in the reproductive health. s110 404 Poster Session P17. Reproductive and developmental toxicology EFFECTS OF THE LEAD (PB) TREATMENT ON THE ONSET OF PUBERTY AND FOLLICULAR DEVELOPMENT IN FEMALE RATS. E.H. Scott 1 , A. Handall 2 , J.L. Morán 2 , C. Morán 2 . 1 Escuela de Biología, Benemérita Universidad Autónoma de Puebla, Puebla, México, 2 Laboratorio de Investigaciones Biológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, México Lead is a known reproductive toxicant in humans and experimental animals. Cases of sterility, frequent abortions and abnormal menses have been found to occur in women working in lead-based industries. This study was done to correlate Pb concentration in the blood with onset of puberty and follicular development. Twelve adult virgin female rats were divided into 4 groups of 3 each and given lead (as lead acetate in water) during gestation and lactation, in 3 doses at 0.1, 0.5, 1% and one group served as a control. Rats were provided with animal feed and water ad libitum after weaned. Female offspring from each of the four groups were monitored daily for vaginal opening (VO). When VO occurred, rats were determined to be in the first estrus and were sacrificed by decapitation the same day. Lead level in the blood (µg/dl) was found to increase in a dosedependent manner (0.1%: 92.06±6.68, 0.5%: 131.37±18.52, 1%: 271.06±52.24, and control: 2.29±0.71; P<0.0001). A difference in body weights between control and Pb-exposed groups was not observed. However, the ovary weights (mg) decreased significantly (1%: 27.6±0.3 vs control: 46.4±1.4; P<0.001); and a decrease the uterus weights (mg) was also found (1%: 102±3.8, vs control: 171.4±4.2; P<0.001). Female pups exposed to Pb during early development showed a delay in the timing of puberty as determined by the day of VO (1%: 58.75±3.68, P<0.001; 0.5%: 50.90±1.45, P<0.05; control: 45.16±1.30). In all Pb-treatment groups, the total number of healthy follicles decreased (1%: 4.66±2.33, P<0.01; 0.5%: 31.33±19.41, P<0.05; control: 89.66±3.48). Many multiple oocytes (several oocytes into a single follicle), and many oocytes with more than one nucleolus were founded. Furthermore, at high concentration, healthy preovulatory follicles were not found. The present results suggest that lead has the ability to make damage to follicular development and a capacity to induce delay in the beginning of puberty. 405 THE EFFECT OF ORAL HIGH ALUMINIUM INTAKE ON RAT SPERMATOGENESIS 406 TOXIC EFFECTS OF HYDRO ALCOHOLIC EXTRACT OF KIWI ON THE HISTOLOGICAL STRUCTURE OF THE MALE REPRODUCTIVE TISSUE T. Talaei, M.R. Panjehshahin, F. Dehghani, Z. Panahi. Department of Anatomy, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran Hormonally active agents are widespread in the environment and exist in the human diet. There is insufficient knowledge regarding the influence of phytochemical on reproductive pathophysiology of male reproduction. Kiwi is a rich source of isoflavones and flavones. These compounds are the major classes of phytoestrogens. In male, estrogen is present at low concentration in blood, but can be extraordinary high in semen and rete testis fluid. It is well known that male reproductive tissue express estrogen receptors. Therefore, the objective of this project was to determine whether kiwi extract could change the structure of male reproductive tissue. For this purpose, 40 male rats were selected and divided into 4 groups. Three experimental groups were fed with 150, 100 and 75 mg/kg of kiwi extract and control group was fed with solvent for 50 days. The rats were sacrified and their testis, ductus deferens, seminal vesicle, prostate and epididymidis were removed, fixed, processed and stained with HE and acridin orange. The specimens were studied under light and fluorescent microscopy. Histological observations were revealed some changes in testis but not in the other parts of male reproductive tissues. In the testis, some spermatocytes were become fusiform and the number of involved cells was increased dose dependently. There were many spermatocyes at the metaphase stage in 150 mg/kg and 100mg/kg treated groups and some of these mitotic figures, sperms and most of the spermatogonia and spermatocytes stained red with acridin orange which indicated denaturing of DNA strands. A few fragmented nucleuses were observed in 150mg/kg treated groups. In conclusion, it seems that kiwi extract can change the spermatocyte cytoarchitecture and has some effects on the spermatogonia and spermatozoa. It may exert dual effects on proliferating cells such as spermatogenesis linage. In low concentration, it may induce proliferation and in high concentration, it may lead to cell death and nucleus fragmentation. 407 EFFECT OF CITRULUS COLOCYNTHIS ON FERTILITY RATE AND THE NUMBER OF EMBRYO IN MOUSE I.M.D. Rashidi. Head of department & member of expert committee Dept of pathology Medical school Ahwaz medical university, Ahwaz IRAN F. Dehghani, M.R. Panjehshahin, M. Azizi, F. Mesbah, S. Karbalaedoost, T. Talaei. Department of Anatomy, Shiraz University of Medical Sciences, Shiraz, Iran Aluminium is one of the most abundant elements in the earth crust and enters to the body through drinking water,nutrients and drugs like antiacids. Aluminium poising causes wide range of disorders, including: a decrease in the release of neurotransmiters and inhibition of voltage dependent calcium channels. The role of calcium on GnRH release and its action is detected so, in this studying, the effect of high aluminium intake on rats spermatogenesis is investigated. The experiment performed in four groups, a control group and three experimental groups consumed 0.625, 1.25 and 2.5 mg aluminium per gram diet for 60 days. Epididymis and vas deferens were dissected cut and diluted with normal salin. In all groups weight of vas deferens, epididymis, testis and whole animal, sperm count per gram deferens and epididymis tissues were determined then, the testicular tissues fixed in formalin for study of histopathology. The results have shown that in experimental groups which consoumed 1.25 and 2.5 mg aluminium per gram diet, the vas deferens, epididymis, testis and animal weight were significantly decreased. In this animals the number of sperm per gram tissues from vas deferens, epididymis were reduced. The maturation arrest is seen in seminoferous duct and it haven‘t spermatogenesis. Therefore, this studying indicated that high aluminium intake in rat have an inhibiting effect on spermatogenesis and this effect is dose dependent. Citrulus colocynthis is used in traditional medicine to reduce the glucose blood level. There is some evidence that reveals it can inhibit the implantation of embryos. The objectives of this project were to search the inhibitory effects of citrulus colocynthis on implantation and reduction of fertility rate. To do this, 84 female bulb C mice were selected and divided randomly into 4 groups. Mice caged with male for one night. Observing the vaginal plaque was considered as zero day of gestation. The pregnant mice were fed with 0.03, 0.06, 0.12 mg/kg organic extract of citrulus colocynthis till 17th day of gestation. Control group was fed with solvent (olive oil). The mice sacrificed at the day 17 of gestation and the number of healthy and absorbed embryos was counted. The data analyzed statistically. The results indicated that citrulus colocynthis reduced the fertility rate in a dose dependent manner, so that, the number of pregnant females were reached to zero in the groups were fed with 0.03 and 0.06 mg/kg. It seems that the extract did not have any effect on the number of embryo in pregnant animals. It is concluded that the extract can interfere with implantation or it may has toxic effects on the preimplantation embryo. Although, to find the exact mechanism of the effect of citrulus colocynthis extract needs more investigation. Poster Session P17. Reproductive and developmental toxicology 408 MORPHOMETRIC STUDY OF ENDOMETRIAL LUMINAL EPITHELIUM IN ELECTROMAGNETIC FIELD EXPOSED RATS Leila Rowshangar, Rad J. Soleimani. Department of Histology and Embryolog, Faculty of Medicine, Tabriz medical Sciences,Tabriz-Iran It is known that electromagnetic field (EMF) as an environmental factor could produce functional and structural disorders. Endometrial luminal epithelium has an important role on blastocyst adhesion and subsequent implantation. The aim of the present study is to evaluate the effect of EMF on endometrial luminal epithelium. For this purpose, 30 Wistar rats were divided in to control and experimental groups. Rats in experimental group are exposed to 5mT EMF, produced by 50 HZ alternative current, 4 hours/day for 3 months. After the experimental period, uteri were disected apart and prepared for light microscopic studies. According to histological features, endometria in secretory phase, was only selected for morphologic and morphometric studies. In morphometric studies, Vv of nucleus to cytoplasm, mean diameter of nuclei and nuclear axial ratio were determined using photoshop software and point counting method. Statistical analysis of data was performed using Student t-test. The results showed that endometrial luminal epithelium in control group appeared high columnar and their length was 1.23±0.02 mm, while in experimental group, they looked high cobuidal with highly condensed nuclei and their height was 0.98±0.05 mm. The difference between two groups was significant (p < 0.001). Mean diameter or profile of the nuclei in control group was 0.98±0.02 and in experimental group was 0.78±0.18 and the difference was significant (p<0.01). Volume fraction (Vv) of nucleus to cytoplasm in experimental group was 1.12±0.15 which was significantly lower (p< 0.04). than in controls (1.37±0.10). Axial ratio of nuclear diameter in controls (1.45±0.11) was significantly (p<0.04) higher than the experimentals (1.35±0.39). It is concluded that EMF could produce significant morphometric changes in luminal epithelium of endometrium which may interfere with implantation. 409 INTRAUTERINE EXPOSURE TO RADIOFREQUENCY FIELDS CAN INCREASE APOPTOSIS IN BONE FORMATION OF RAT EMBRYOS (ULTRASTRUCTURE STUDY) S.H. Sadraie 1 , K. Parivar 2 , G.H. Kaka 1 , H. Bahadoran 1 , H. Dastnavard 1 , M.H. Asadi 1 . 1 Laboratory of Cell Research, Department of Anatomy, Baghyiatallah University of Medical Sciences, Tehran, I.R. Iran, 2 Department of Cell Biology, Tarbiat Moallem University, Tehran, I.R. Iran Cell death is a common and reproducible feature of the development of many mammalian tissues. Like normal development, abnormal development is also associated with increased cell death in tissues that develop abnormally after exposure to a wide variety of teratogens such as radiofrequency (RF) fields. In this study pregnant SpragueDawley rats were exposed to high intensity 27.12 MHz radiation. Cell death was evaluated in regions of diaphysis of tibia of 21day rat embryos after intrauterine exposure to RF fields. The cell death data obtained in this study correlate with previously observed malformation rates, suggesting that a quantitative relationship exists between RF-induced embryonic cell death and bone formation defects. Glutaraldehyde-formaldehyde fixed specimens were processed for transmission electron microscopy. Examination of ultrathin sections revealed various types and stages of cell death in the osteogenic zone. Typical appearance of osteocytes undergoing apoptosis with shrunken cells, within opened up lacunae, containing irregularly shaped nuclei and blocks of condensed peripheral chromatin and long thin cytoplasmic projections. We also observed cells with a nucleus filled by condensed chromatin, and some osteoclasts exhibited a large vacuole containing membranous and granular fragments of cytoplasm. Vesicular bodies and electron-dense granules which is given off the apoptotic bodies into the matrix. Extracellular vesicles packed together and surrounded by calcifying matrices. They are s111 morphologically heterogenious and contain cell debris and matrix vesicles, suggesting chondrocyte fragments and matrix vesicles may act as a scaffold for the initial calcification. Our results showed an increasing of apoptosis in bone formation zone of the tibia of experimental rat embryos. In conclusion RF induced embryotic cell death had morphological characteristics of apoptosis. It appears that RF fields expands areas of naturally occurring cell death in bone formation region of rat embryos. 410 EFFECT OF 2-ETHOXYETHANOL ON SPERMATOGENESIS IN THE EXPOSED WORKERS R.S. Wang 1 , M. Suda 1 , X. Gao 2 , B.L. Wang 2 , T. Honma 1 . 1 Department of Health Effects Research, National Institute of Industrial Health, Japan, 2 Beijing Institute of Industrial Hygiene and Occupational Diseases, Beijing, China 2-Ethoxyethanol (ethylene glycol monoethyl ether) is a common solvent used in industry and in consumer goods. This compound has been known to have toxic effects on testes and blood system in a number of species, causing oligospermia as well as some haematological abnormalities. In this study, we investigated the toxic effects of 2-ethoxyethanol exposure among the male workers in two factories manufacturing the photopolymer sensitization plate in Beijing. Some workers were exposed to high levels of ambient 2-ethoxyethanol, and others were working in places with very low level or no detectable concentration of the solvent and served as the comparison group in this study. The urinary metabolite of the compound, ethoxyacetic acid, was measured, and it was much higher in those with high level exposure than in the comparison group. Semen samples were collected from some of the workers, and sperm analyses showed that sperm count, progressive motility and percentage of sperms with normal morphology were significantly lower in the exposure group than in the comparison group. In some subjects with high level exposure, the values were out of the normal range. On the other hand, the mean concentrations of sex-related hormones in blood, testosterone, LH, FSH, prolactin and estradiol, were at the same extent in both groups. Haematological examinations showed that the red blood cell count, haemoglobin level, packed cell volume as well as white blood cell count were all lower in the group of exposure. No difference was found in platelet count between the two groups. Liver function was not found to be damaged in the exposed subjects. The SNPs of aldehyde dehydrogenase 2 were analyzed, and its effects on the metabolism and toxicity expression of 2-ethoxyethanol were demonstrated. 411 CYTOTOXICITY OF HERBICIDE ATRAZINE ON CHINESE HAMSTER OVARY CELL LINE IN MONOLAYER J. Kniewals, I. Kmetič, V. Gaurina Srček, B. Šimić, Z. Kniewald. Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) is a selective triazine herbicide, one of the most widely used herbicides in corn and other crops. Therefore the extensive use leads to its possible presence in ground water, as well as in raw and industrial food products. Even though atrazine is declared to be slightly toxic as toxicity class III, its possible ingestion via the food chain may present a risk for the reproductive process. The safety assessment of pesticides and the introducing these data into a human risk assessment evaluation requires a large number of expensive, regulated tests in different animal species. Currently a wide range of animal replacement alternative methods is available. By introducing in vitro methodology in toxicity testing, it is possible to evaluate the toxic influence of a chemical without using the great amount of experimental animals. In the present study, in order to determine cytotoxic effect of atrazine within the reproductive hormone-dependent tissues, using CHO K1 (Chinese Hamster Ovary) cell line in monolayer (CCL-61) performed the research. Cell viability and number of cells were measured by Trypane Blue exclusion method in Fuchs-Rosenthal heamocytometer, and by colorimetric assays as Kenacid Blue R binding method measured the change in total cell protein, lyzosomal s112 Poster Session P17. Reproductive and developmental toxicology activity measured by Neutral Red method and MTT assay measured the mitochondrial succinate dehydrogenase activity. The cells were maintained at 370 C in an atmosphere of 95% air and 5% CO2 in Dulbecco medium supplemented with 10% fetal calf serum. The production of biomass was in T-bottle, and the cells were separated in the early logarithmic phase of growth. The initial concentrations of CHO cells were 2.5x104 cells/mL/well on 6-multiwell plates and after 72 h it succeeded 21x105 cells/mL in the control well. Cells were seeded 16 h before the treatment with atrazine in the concentrations of 2.5–80 µg/mL/well. The number of cells in the presence of atrazine is compared with that observed in control cultures and the percent inhibition of growth calculated. The cytotoxicity was determined after 24, 48 and 72 h. The cell growth inhibition ranged after 72 h from 5 to 75% and was dose-responded. The IC20 , IC50 and IC80 were measured from the slope of % inhibition vs. log dose values. The values of IC50 for atrazine after 72 h was ∼55–60 µg/mL with all applied methods, acceptable for the quantitative determination of cytotoxic effects of atrazine on CHO K1 cell line. As the first-step in toxicity evaluation, by excluding the experiments with animals, application of cell lines from reproductive tissues like the Chinese hamster ovary enables determination of toxicity in the reproductive processes. Supported by Ministry of Science and Technology Republic of Croatia, grants 0058010 and 0058001 and by PLIVA inc. 412 IDENTIFICATION OF PESTICIDES SEX-SPECIFIC GONADAL TOXICITY N.R. Shepelskaya, M.G. Prodanchuk. Medved‘s Institute of Ecohygiene and Toxicology, Kiev, Ukraine The purpose of this study was to detect the gonadal toxicity of 30 pesticides in male and female rats and to compare each sex sensitivity. All studies were conducted using Wistar rats. Pesticides were administered five days per week for eleven weeks for males and ten weeks for females, orally by gavage in the form of aqueous emulsions.After the scheduled period of treatment (three dose levels plus controls), females and males were paired with untreated animals. All pregnant females were killed on the 20th day of pregnancy. The reproductive perfomance of treated and intact females was evaluated based on parameters as the estrous cycle normality (vaginal smear cytology), precoital interval, fertility index, mating index, conception rate, gestation index, number of ovarian corpuses luteum, implantation number, litter size, embryonic and fetal loss, pup weights, external malformations and variations.The gonadal function of males was evaluated using additionally such parameters, as a testes and epididymides weigthts, sperm number and quality (morfology, motility). Histopathological investigations of the gonads from males and females were carried out. Based on results of this experiments it was concluded that administration of 20 pesticides resulted adverse effect on the gonadal and reproductive functions of animals. The males were more sensitive comparable with females. For 7 chemicals LOELs in males were more little than in females, 10 substances demonstrated identical LOELs for both animal sexes and only for 3 pesticides LOELs for males were more high than for females. divided the rats randomly into case and control groups. The rats of the case group were feeded with water soluble OCPs from the first day. After 20th day of pregnancy the embryos were delivered by cesarian and then were studied. In female embryos some changes were seen in the external genitalia. Because of androgenic effects of progestinal hormones. Clitoris has grown more and a small scrotom like sac was seen between anus and external genitalia. In male rats undescended testis and small penis was observed. In statistical analysis, significant difference was seen between case and control groups (P value < 0.05). 414 P. Afshari, S. Sadeghi. Midwifery and Nurcsing Dept., Dezful Azad University of Medical Sciences. IRAN Several factors including chromosomal disorders, increasing maternal age and number of pregnancies, and other factors might cause abortion. One of the important factors of abortion is environmental toxin. The effect of some chemical compounds such as nitrous oxide on spontaneous abortion has been reported. To compare the abortion rates between workers of surgical room and of other wards of hospitals in Ahvaz, Iran. It was a case control study conducted on 40 workers in each of surgery and non-surgery wards. Confounding variables such as age, sex and other risk factors of abortion were matched and the prepared questionnaires were filled in for all participants. The data were analyzed by using t-Student and Mantel-Haenszel tests. The mean age of workers of surgery and non-surgery wards was 31.2 and 32 years, respectively. Abortion rates were 28 and 4% for the former and the latter, respectively; and the difference was statistically significant (P< 0.0001). The prevalence of abnormal fetus in the two groups was 9 and 4%, respectively. It seems that to be exposed by anesthesia gases could be a risk factor for abortion among the workers of surgery ward. Further investigations regarding this hypothesis is strongly recommended. 415 OCP INTAKE DURING PREGNANCY AND ITS SIDE EFFECTS LEADING TO ANOMALIES N. Takzaree, A.-R. Takzaree, K. Yarmohammady, S. Takzaree, B. Rashidi, B. Montazeri. Tehran university of medical sciences, Faculty of medicine, Dept of Anatomy, IRAN Some researchers have reported the risks of OCP intake during pregnancy. Using progestinal drugs before 20th week of gestational age because of some androgenic effects can cause male pattern anomalies in the females external genitalia. Sometimes women are not aware of pregnancy and may continue OCP intake for weeks after getting pregnant. In this research we studied the effects of OCPs containing estrogen and progestron on reproductive system differentiation. 10 female virgin rats of the same age and weight were used for our research. After the rats getting pregnant and observing vaginal plaque the day zero of pregnancy was determined. Then we A STUDY OF SHORT/LONG-TERM RESPIRATORY COMPLICATIONS IN MUSTARD GAS VICTIMS. P. Afshari. Dezful Azad University According to the BERGHOFF report, acute respiratory complications have been induced by even the short early exposure to the mustard gas (sulfur mustard) penetrating through mocusal bissues, but the prolonged lonsequences are not yet, clefined 200 chermically injured soldiers were studied (after 14 years) hmough a questionnaire. It is indicated that 65.5% of victims complained at respiratory problems within the exposure time of which 42,7% declares severe dyspnea. After 14 years, this figure has led to 83.9%. Opposing the current assumption, the severity at mustord respiratory effects is increased in the process f time. 416 413 COMPARATIVE STUDY OF ABORTION BETWEEN THE WORKERS OF SURGERY AND NON- SURGERY WARDS OF HOSPITALS IN AHVAZ, IRAN ENDOCRINE DISRUPTING CHEMICALS, EXPOSURES AND CLINICAL OUTCOMES IN 324 PREGNANCIES IN CALGARY, CANADA J. Jarrell 1 , S. Chan 2 . Department of Obstetrics and Gynecology1 and Toxicology2 , University of Calgary, 1403 29th St. Calgary Alberta Canada T2N 2T9 The presence and concentration of priority chemicals in critical tissue compartments during pregnancy is of interest due to the possibility they may play a role in the development of adverse human reproductive outcomes. To evaluate this question, a prospective longitudinal observational design was selected. 324 women were approached in the second trimester of pregnancy while attending a clinic for the determination of whether they would have a genetic amniocentesis. Only subjects who were seeking the amniocentesis for late maternal age were approached. They provided amniotic fluid, serum during the second trimester, serum at birth and breast milk postpartum. Cord Poster Session P17. Reproductive and developmental toxicology serum was also collected. The patients also completed an environmental questionnaire. The following rates of adverse outcomes were observed: premature labor 3.0%, pregnancy induced hypertension 10.5%, low birth weight 3.5%. This report summarizes the relationships of these outcomes to twenty-four PCB congeners and nineteen organochlorine pesticides from samples collected during pregnancy. The PCBs were analyzed with GC/NCIMS and GC/EIMS was used for pesticide analysis. There were no PCBs or pesticides identified in the amniotic fluid. Significant (p<0.05) findings were the increased concentrations of certain coplanar PCBs (ng/ml) among those patients experiencing pre-term labor: PCBs 118, 138, 170, 153, pregnancy induced hypertension: PCB 118 but not for birth weight <2500 g. Notably there were no differences in the levels of the remaining PCBs or specifically the pesticide DDE in the serum during pregnancy for any of these adverse outcomes. These findings, while limited by the low rate of adverse outcome in the population under review, do support the further exploration of the potential roles of toxic co-planars in outcomes of pregnancy. (Supported by Toxic Substances Research Initiative, Health Canada) 417 CIGARETTE SMOKING AND p53 EXPRESSION IN THE PLACENTAL TISSUE. P. Myllynen 1 , P. Pienimäki 1 , K. Vähäkangas 1,2 . 1 Department of Pharmacology and Toxicology, University of Oulu, Oulu, Finland,2 Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland, Various conditions including DNA damage caused by benzo(a)pyrene can lead to rapid induction of p53 protein. The aim of this study was to clarify whether smoking increases p53 expression in the placental tissue. Samples from 42 placentas were collected from uncomplicated term pregnancies. Thirty-two of the mothers reported themselves as smokers and 10 were non-smokers. Smoking status of the participants was confirmed by an assay for urinary nicotine metabolites. DNA was isolated from placental samples using a phenol extraction/ethanol precipitation method and BPDE-DNA adducts were analysed by synchronous fluorescensence spectrophotometry. The level of p53 protein was analysed using immunoblotting. The smokers and non-smokers did not differ significantly in age, gestational weeks or pregnancy number. The birth weight was 3649±474 g if the mother was a smoker and 3208±620 g if the mother was a non-smoker (p<0.05). The mean concentration of nicotine metabolites was 623±582 ng/nmol creatinine in the confirmed smokers and 8±3 ng/nmol creatinine in non-smokers (p<0.01). Ten out of 32 smokers, but none of the nonsmokers, had indications of BPDE-DNA adducts in the placental DNA. Late sampling decreased detectability of BPDE-DNA adducts. Detectable levels of p53 in the placenta were found in 50% of smokers and 40% of non-smokers. Out of ten adduct positive placentas 8 (80 %) had detectable levels of p53 protein while only 7 out of 21 (33%) samples without DNA adducts had detectable levels of p53 protein. Statistically significant correlations were found between smoking and birth weigh, smoking and urinary nicotine metabolites and duration of the gestation and birth weight but not between other variables. In conclusion DNA-adducts formed due to smoking in placental tissue probably contribute to the levels of p53 protein in the placenta although the difference was not statistically significant due to small number of studied placentas. 418 s113 STRUCTURAL CHANGES IN SELECTED MORPHOMETRIC PARAMETERS AND LEVELS OF NICOTINE AND COTININE IN PLACENTAE OF SMOKING MOTHERS. Ewa Wielgus 1 , Rafał Celiński 2 , Krzysztof Pawlicki 1 , Halina Sybirska 2 , Marcin Kamiński 1 . 1 II Department of Histology and Embriology, Silesian Medical University, Katowice, Poland; 2 Forensic Medicine Department, Silesian Medical University, Katowice, Poland Studies on the effect of smoking cigarettes by women even when they are pregnant on both microanatomic and biochemical structure of placenta show that this organ undergoes various changes. The aim of this paper was to determine concentrations of nicotine and cotinine in meconium and both central and peripheral areas of the placenta as well as to appraise the select morphometric parameters and activity of succinate dehydrogenase (SDH) in syncytiotrophoblasts. Both biomarkers were determined by HPLC - MS with use of a Finningan LCQ Duo apparatus. The meconium from neonates and placentae of healthy women, the primapara without hormonotherapy were collected to examine. This material was divided into 2 groups. Group I comprised placentae of the women being not only nonbut also non-passive smokers. In group II there were placentae of mothers smoking 20 or even more cigarettes a day. In both areas of the placentae of non-smokers, nicotine and cotinine were found and the concentrations of these biomarkers were statistically much more higher in their peripheral than central areas. The simultaneous morphometric analysis showed that in peripheral placentae of smoking mothers, the area and circumference of both villi and capilaries, the length of vasculo-syncytial membranes and also the SDH activity increased but the area of a syncytitrophoblast decreased. Simultaneously, in central placentae there was noticed the decrease in both villous area and circumference, the area of syncytiotrophoblast and SDH activity but the increase in the area and circumference of capillaries and the length of vasculo-syncytial membranes. Cumulation of the cigarette smoke biomarkers in meconium and both placental areas as well as concomitant changes in villous morphometric parameters irrespective of being adaptative or compensatory make for threat to the fetus. 419 THE EFFECT OF ENVIRONMENTAL FACTORS ON SPONTANEOUS ABORTION: A CASE CONTROL STUDY IN IRAN Sorri Nouhjah. H. School of Health, Ahwaz University of Medical Sciences, Ahwaz, Iran Objective: To evaluate the effect of some environmental factors on spontaneous abortion. Methods: This s a cross-sectional study. Data was collected from governmental and private hospital in Ahwaz 1999–2001. Cases were 133 pregnant women who were taken to obstetric and gynecology departments due to spontaneous abortion. For every case one control was selected.Controls were 133 women who were taken to hospital due to delivery. Kind of hospital, age of woman, gravida, female education, history of abortion were matched between cases and controls. Interview,filling of a questionnaire by trained questioner were instruments of data collection. Environmental factors were job of mother and her husband, smoking during pregnancy by mother, father smoking, use of drugs by mother or father, Xray exposure, unwanted pregnancy and vaginal infections. Results: Analysis of data showed that job of father,father smoking are risk factors for abortion. 420 TERATOGENIC EFFECT OF LORAZEPAM ON THE MORPHOGENESIS AND ORGANOGENESIS OF THE EYE AND PALATE K. Mehrannia, P. Pasbakhsh, T. Mehrannia. University of Tehran, Medicine Faculty, Tehran, Iran lorazepam belongs of the 1,3 dihydro. 2 keton group of 1,4 Benzodiazepine derivatives. It is drug increasingly used in our country s114 Poster Session P18. Mechanisms of cell death for relieving anxiety and tranquillizer. Lorazepam was regarded by some authors as potential teratogen inducing oral cleft (1) but there are disagreements on this matter. In this study, a group of adult wistar rats of definite age and weight were selected and exposed to 2mg/kg – 20mg/kg of lorazepam after conception (during the organogenesis between days 9 and 18) in case and control groups. The fetuses were first studied macroscopically regarding gross anomalies, and than histologically to exactly inspect the defect of tissue organogenesis. The result shows that: in the group who received 18 mg/kg and 20 mg/kg lorazepam, cleft palate and open eyelid has been seen. Above mentioned results indicate that the lorazepam has the ability to produce cranio facial malformation under the condition of these ability lorazepam may be able to affect neural crest mesenchymal components during the later part of its migration (2) Pelton et al indicated that growth factors TGFβ1 , TGFβ2 , TGFβ3 have an effect on differentiation process of epithelial cells to mesenchymal cells. It seems that lorazepam at the doses of 18 mg/kg and 20 mg/kg causes inhition of these growth factors. Our result also indicate that lorazepam has teratogenic potential in certain doses (doses of 18 mg/kg 20 mg/kg) so usage of lorzepam in the second half of fetal period has influenced development and caused irreducible malformation. 421 OCULAR ABNOMALITIES DUE TO TOXIC EFFECTS OF THE CARTHAMUS TINCTORIUS IN THE MOUSE EMBRYO S. Bahmanpour 1 , K. Javidnia 2 . 1 Department of Anatomy, School of Medicine, 2 Department of Medicinal Chemistry, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran Background: Carthamus tinctorius is one of the popular plants used as color and flavor in food industries. Safaran is used for the same purpose but it is less expensive than Carthamus tinctorius. Therefore it is used instead of Safaron in traditional medicine to cause menstrual bleeding. However, in case of pregnancy, this might have teratogenic effect on the embryo. This study was designed to investigate the probable effects of this plant on embryo. Methods: Some outbreed mice were initially kept in standard condition. Then 2 females were caged with one male. The pregnancy was confirmed by vaginal plug detection. The pregnant mice were divided into 2 groups. The experimental group was injected the aqueous extract intraperitoneally on the 7th or 8th days of pregnancy as single dose and 9th and 10th days as multidose. The experimental group was further subdivided based on the dose treatment (1mg/kg, 10mg/kg, 25mg/kg and 50mg/kg). Distilled water was injected to the control group. The uterus of the pregnant mice was opened on the day 18th of gestation, and the embryos were examined for ocular malformation. Results: The results showed teratogenic signs at higher doses like 25 mg/kg and 50 mg/kg; therefore, the ocular defects were dose depedent. Toxicity effects included: cataract, lentocorneal adhesion and eyelid defects. Conclusion: Considering the fact that the extract of Carthamus tinctorius has the embryotoxicity effects, it should be used cautiously by pregnant mothers. Further studies are required in order to investigate the mechanism involved in the molecular defects. cells grown as three-dimensional spheroids are more resistant to ionizing radiation than are the same cells grown in monolayer. However, the mechanisms at the basis of this increase in resistance are not yet completely understood. In the present study, three-dimensional spheroids composed of HT-29 cells were irradiated with 15 Gy at two different culture times: 24 h, when spheroids were loosely aggregated (early spheroids) and 72 h, when they were well-compacted (late spheroids). After 48 h of irradiation, morphological characteristics (scanning electron microscopy), spheroid growth (growth curves and cell cycle analyses) and cell death (apoptosis and necrosis) were examined. In early spheroids, evident signs of cell damage were present, whereas in late spheroids no such damage was observed. Growth curves showed that 15 Gy induced reduction in cell growth only in early spheroids. In addition, studies of cell death revealed that cells composing early spheroids prevalently died by necrosis whereas cells composing of late spheroids died essentially by apoptosis. Finally, cell cycle analyses demonstrated an accumulation of cells in the G2 /M phase of the cell cycle only in spheroids irradiated after 72 h of growth. Taken together, the data presented suggest that the three-dimensional architecture of spheroids may play a pivotal role in determining their response to ionizing radiation. In this context, since cell-cell adhesion molecules (CAMs) and specific cell cycle regulators could play a fundamental role, E-cadherin and D1 cyclin were analyzed by “western blot”. However, the preliminary results seem to indicate that these two factors are not involved in the different susceptibility of HT-29 tumor spheroids to the dose of ionizing radiation used in this study. 423 S.H. Inayat-Hussain 1 , R. Baker 2 , D. Ross 2 , L.B. Din 3 , K. Yusoff 4 , A.M. Ali 5 . 1 Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 2 School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO, USA, 3 Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Malaysia and 4 Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, Selangor and 5 Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia, Serdang Malaysia Styryllactones such as goniothalamin represent a new class of compounds with potential anti-cancer properties. In this study, we investigated the mechanisms of goniothalamin, a plant styryllactone induced apoptosis in human promyelocytic leukemia HL-60 cells. This plant extract which did not inhibit purified human topoisomerase IIα, resulted in apoptosis in HL-60 cells as assessed by the externalisation of phosphatidylserine, loss of mitochondrial transmembrane potential (ψ m ), activation of initiator caspase-9 and the executioner caspases-3 and -7. Pretreatment with the pharmacological caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) abrogated apoptosis as assessed by all of the apoptotic features in this study. In summary, our results demonstrate that goniothalamin induced apoptosis occurs via the mitochondrial pathway in a caspase dependent manner. 424 P18 Mechanisms of cell death 422 RESPONSE OF HT-29 THREE-DIMENSIONAL TUMOR SPHEROIDS TO IONIZING RADIATION. A. Ferrante 1 , G. Rainaldi 1,2 , P.L. Indovina 2,3 , M.T. Santini 1,2 . 1 Laboratorio di Ultrastrutture, Istituto Superiore di Sanità, Rome, Italy; 2 Istituto Nazionale per la Fisica della Materia,Unità di Napoli, Naples, Italy; 3 Dipartimento di Scienze Fisiche, Università di Napoli “Federico II”, Naples, Italy Cell-cell interaction and cell adhesion have been shown to influence greatly numerous cell functions including cell proliferation and cell survival itself. Numerous studies have demonstrated that the same CHARACTERIZATION OF GONIOTHALAMIN INDUCED APOPTOSIS IN HL-60 LEUKEMIA CELLS EVALUATION OF APOPTOSIS OF THE OOCYTE IN MOUSE ATRETIC FOLLICLE: A TEM STUDY F. Mesbah 1 , P.R. Hurst 2 . 1 Anatomical Department, Shiraz Medical School, Shiraz Iran; 2 Department of Anatomy and Structural Biology, Otago University, New Zealand Recent advances in the study of growth regulation at the cellular and molecular level have made it clear that growth of a tissue is the result of a tightly monitored and regulated balance between the processes of cell division and apoptosis. Programmed cell death (apoptosis) is a physiological process where by cell die in a controlled fashion in several tissues. Apoptosis, which is distinct from necrosis, has been characterized by morphological and biochemical level changes. In the ovary, more than 99% of the follicles are destined to degeneration during pre and postnatal life via apoptosis. It has been widely accepted that the apoptotic granulosa cells demonstrate chromatin Poster Session P19. Oxidative stress margination, cytoplasmic vacuolization, cytoplasmic blebbing, both cytoplasmic and nuclear condensation and fragmentation, and nucleolar segregation. Biochemical assessments have supported the assignment of apoptosis in granulosa cell. Whether, the oocyte also undergo degeneration by apoptosis has been a more controversial matter. Few ultrastructural observations of atretic oocytes have shown none typical features of apoptosis in oocyte. Therefore the main aim of this study was to define ultrastructural characteristics of degenerated oocyte in mouse atretic follicle. The ovaries of the eight weeks mice were prepared and viewed by TEM. The typical features of apoptosis were not seen in the oocytes of first and second-class follicles. The oocytes of third class of follicle were shown typical features of apoptosis accompany granolosa cells. The unique nature of oocyte relative to other cell types may be the cause for its unusual manner of cell death. Oocytes can remain arrested in meiosis for years, are surrounded by an acellular zona pellucida, are nonproliferating, and are known to rely on surrounding granulosa cells for survival. Apoptosis is an active process though to protect the rest of an organism from an aberrant cell. Meiotic oocytes may not be required to undergo apoptosis, because they pose no threat of excessive proliferation and tumor formation. P19 Oxidative stress potent toxic cocaine metabolite which is produced only in the presence of ethyl alcohol. The objective of this study is a) to enlighten the toxicity mechanism of cocaine (50 mg/kg, i.p) and b) to investigate the effect of ethanol (3 g/kg, i.p.) on its toxicity and c) compare pre- and post-niphedipine therapies by the assesments of malondialdehyde (MDA), nonprotein sulphydryl groups (NP-SH) and total sulphydryl (T-SH) levels. All tissue MDA levels in group dosed with cocaine, increased significantly whereas a decrease is observed in NP-SH and T-SH levels. After ethanol+cocaine treatment MDA levels in liver, heart (p<0.001) and brain (p<0.01) increased. The same group had lower NP-SH and T-SH levels in all tissues compared with both cocaine administered group and control group. Preand post treatment with niphedipine prevented cocaine-induced increases in MDA levels in all tissues significantly. Pre-treatment with niphedipine caused a significant elevation in NP-SH and T-SH levels only in liver (p<0.001) and heart (p<0.01). Post-niphedipine therapy showed a significant increase in NP-SH levels in liver (p<0.001) and in T-SH levels in liver, kidney, brain (p<0.001) and heart (p<0.05). Present results suggest LPO may be an alternative mechanism in cocaine toxicity. Alcohol and cocaine administration effected mainly liver and heart and moderately brain. Both pre and post treatments of niphedipine were observed to have an effective antidotal activity in cocaine-induced toxicity. However, pre-treatment of niphedipine is evaluated to have a more effective protection on heart and brain when compared to post-niphedipine treatment. 427 425 THE PROTECTIVE AND ANTIDOTAL EFFECTS OF TAURINE ON HEXAVALENT CHROMIUM-INDUCED OXIDATIVE STRESS IN MICE KIDNEY TISSUE I. Bosgelmez, G. Guvendik. Department of Toxicology, Faculty of Pharmacy, Ankara University, Tandogan, 06100, Ankara-Turkey Chromium(VI) compounds, which have extensive industrial uses, have serious toxic, carcinogenic and mutagenic effects in human and animals. It has been suggested that reactive intermediates and free radicals generated during the reduction of Cr(VI) may be responsible for Cr(VI) toxicity. In this study, the effects of pre- or post-treatment of Taurine (TAU) on Cr(VI)-induced oxidative stress in the kidney tissue of Swiss Albino mice were investigated. It was observed that single intraperitoneal treatment with K2 Cr2 O7 (20 mg Cr/kg), as Cr(VI) compound, produced significant oxidative stress in this tissue. The level of thiobarbituric acid reactive substances (TBARS) was significantly elevated as compared to the control group (p<0.001). Non-protein thiols (NPSH) level and Cu,Zn-superoxide dismutase (SOD1) and catalase (CAT) enzyme activities were reduced in the exposed group as compared to the control group (p<0.01, p<0.05, p<0.001 respectively). TAU administration (1000 mg/kg) before or after Cr(VI) exposure resulted in a significant reduction in the TBARS levels (p<0.001). While NPSH level and CAT enzyme activity were restored only by TAU pre-treatment (p<0.001, p<0.05 respectively). On the other hand, administration of the antioxidant before or after K2 Cr2 O7 caused a significant improvement in the SOD1 enzyme activity (p< 0.01). In conclusion, TAU seems to exert both protective and antidotal effects and it can be suggested that TAU treatment might be beneficial in protecting against Cr(VI)-induced oxidative stress in mice kidney tissue. 426 PROTECTIVE EFFECTS OF PRE- AND POST-ADMINISTRATION OF NIPHEDIPINE IN MICE IN COCAINE-INDUCED TOXICITY Ş.Ş. Çeçen, G. Cengiz, T. Söylemezoǧlu. Ankara Univesity, Institute of Forensic Medicine, Dikimevi, Ankara/TURKEY Cocaine is a natural alkaloid that shows its toxicity by lipid peroxidation (LPO). Increase in LPO induced by cocaine administration results in the depletion of one of the most important defence systems, gluthathione (GSH) to critical levels. As a result of oxidative metabolism of cocaine; norcocaine, N-hydroxynorcocaine and norcocaine nitroxide, a free radical is produced. Cocaethylene is another s115 ANTIOXIDANT ACTIVITY OF CAPSINOIDS A. Rosa 1 , M. Deiana 1 , V. Casu 1 , G. Corona 1 , G. Appendino 2 , M. Ballero 3 , M.A. Dessì 1 . 1 Departement of Biologia Sperimentale, Section of Patologia Sperimentale, University of Cagliari, Cittadella Universitaria, SS 554, Km 4.5, 09042 Monserrato, Cagliari, Italy, 2 Discaff, University of Piemonte Orientale, Viale Ferrucci 33, 28100 Novara, Italy; 3 Departement of Scienze Botaniche, University of Cagliari, Viale Fra Ignazio 13, 09123 Cagliari, Italy Peppers (capsicum ss.vv.) are popular as spices and vegetable foods, and are a remarkable source of antioxidants, including flavonoids, phenolic acids, carotenoids, tocopherols, and the pungent capsaicinoids, capsaicin and dihydrocapsaicin. Recently, the capsinoids, capsiate [4-hydroxy-3-methoxybenzyl (E)-8-methyl-6-nonenoate] and dihydrocapsiate [4-hydroxy-3-methoxybenzyl 8-methylnonanoate], nonpungent ester analogues of capsaicin and dihydrocapsaicin, have been obtained from the fruits of a nonpungent cultivar of Capsicum annuum l. (CH-19 Sweet) and have been shown to share the same biological activities of capsaicinoids, but are non-offensive and devoid of pungency. We have investigated the antioxidant activity of natural capsiate and dihydrocapsiate, and three synthetic analogues, vanillyl nonanoate [4-hydroxy-3-methoxybenzyl-nonanoate], and its dimers (DVN 4 and DVN 5). The ability to inhibit lipid peroxidation was investigated during both the autoxidation and the iron or edtamediated oxidation of linoleic acid at 37°C in absence of solvent, and compared to that of α-tocopherol, luteolin, and the synthetic BHT. The oxidation pattern was followed monitoring the consumption of the fatty acid and the formation of its major oxidation products, the hydroperoxy-octadeca-dienoic acid isomers (HPODEs). All tested compounds were active in these systems and none of them showed any prooxidant activity. During linoleic acid autoxidation, BHT showed the major antioxidant activity. Capsiate, DVN 4 and 5 exerted a highly significant antioxidant activity, comparable to that of luteolin and α-tocopherol, but the activity of dihydrocapsiate and vanillyl nonanoate is also remarkable. Also in the test of the iron-catalyzed oxidation of linoleic acid, BHT was the most powerful compound. The ratio of the HPODEs formed (c,t/t,t) for all tested compounds was measured to point out any hydrogen atom donating activity from the phenolic moieties. Vanillyl nonanoate, a simplified analogue of natural capsiates, was also assayed on cell cultures for cytotoxic activity and the capacity to inhibit the oxidative damage induced by FeCl3. Supported by PIC-Interreg 3. s116 428 Poster Session P19. Oxidative stress THE EFFECT OF ANTIOXIDANTS ON SOLVENT-INDUCED OXIDATIVE DAMAGES IN THE RAT BRAIN Sven Edelfors. Institute of Pharmacology, Panum Institute, University of Copenhagen, Denmark The neurotoxic effect of organic solvents may be due to oxidative stress and peroxidation of the membrane lipids caused by reactive metabolites from the CYP-enzymes. Peroxidation of the mitochondrial membrane will reduce the membrane potential and initiate the apoptotic process. One of the defense mechanisms is antioxidants. The lipophilic Vit E or tocopherol is widespread as well as the hydrophilic Vit.C or ascorbic acid. An in vitro experiment in which rat synaptosomes were incubated with 1-octanol and different antioxidants was carried out. Oxidative stress was measured as hydrogen peroxide and membrane peroxidation as malondialdehyde. Incubation in vitro with 1-octanol caused a significant increase in the synaptosomal oxidative stress. Addition of Vit.E caused a decrease in oxidative stress measured as peroxidation of membrane lipids, whereas the antioxidant effect of Vit.C was not significant. The results indicate that the antioxidant process is going on inside the lipophilic part of the membrane. 429 THE INFLUENCE OF THE SOME AMINO ACIDS ON THE ANTIOXIDANT STATUS IN THE LIVER OF RATS N.E. Petushok, D. Miskevich. Institute of Biochemistry, Grodno, Belarus Amino acids and their derivatives are known to be the natural regulators of biological reactions. Such properties can be used in regulation of metabolic processes. The aim of our work was to investigate the influence of amino acids on the state of antioxidant system. Male Wistar rats weighing 140–160 g were used. Group I consisted of healthy controls. The II-VI groups of rats fed daily intragastrically arginine (100 mg/kg), histidine (50 mg/kg), taurine (50 mg/kg), lysine (250 mg/kg), mixture of named amino acids (MIX, 450 mg/kg) and MIX with addition of ascorbic acid (Asc,15 mg/kg) respectively for 14 days. In the liver homogenates the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase/peroxidase (GR, GPO), glucose-6-phosphate dehygdrogenase (G-6-PhDH), 6-phosphogluconate dehydrogenase (6-PhDG) and concentration of reduced glutathione (GSH) and thiobarbituric acid-reactive substances (TBARS) were measured. It was found the decreasing of SOD and CAT activities in the liver of rats received the MIX. In the same group were increased the G-6-PhDG activity, enhanced NADPH production. Activation of the glutathione system leads to reduction of TBARS concentration. Treatment of rats by MIX with addition of well known antioxidant ascorbic acid activates the SOD and glutathione system (the GR and GPO activities are nearly 2-fold increased, the GSH level elevated by 70%). That caused the depletion of TBARS content. In animals of others experimental groups did not find pronounced changes. These findings suggest that the combination MIX with Asc possess the greatest positive effect on the antioxidant system in the rats’ liver. 430 SCAVENVING ACTIVITY FOR REACTIVE OXYGEN AND NITROGEN SPECIES BY NONSTEROIDAL ANTI-INFLAMMATORY INDOLE AND PYRROLE DERIVATIVE DRUGS Eduarda Fernandes, Sofia A. Toste, José L.F.C. Lima, Salette Reis. REQUIMTE/ Departamento de Química-Física, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, 164, 4050–047 Porto, Portugal Indomethacin, acemetacin, etodolac are indoleacetic non-steroidal anti-inflammatory drugs (NSAIDs), and tolmetin a pyrroleacetic derivative NSAID. These compounds block prostaglandin synthesis by non-selective inhibition of COX-1 and COX-2 (indomethacin, acemetacin, and tolmetin) or by selective inhibition for COX2 (etodolac). Although the inhibition of prostaglandin synthesis constitutes the primary mechanism of the anti-inflammatory action of these drugs, it has been suggested that the anti-inflammatory activity of NSAIDs may be also partly due to their ability to scavenge ROS and NOS as well as of inhibiting the respiratory burst of neutrofils triggered by various activator agents. The aim of the present work was to evaluate and compare the scavenging activity of the NSAIDs indomethacin, acemetacin, etodolac and tolmetin · against an array of ROS (HO· , O·− 2 , and HOCl) and RNS ( NO and ONOO− ) using in vitro systems and to clarify the possible contributions of the chemical structure of these COX inhibitors, for the scavenging activity. The results obtained in this study demonstrate that indomethacin, acemetacin, tolmetin and etodolac · · − exhibit scavenging activity against O·− 2 , HO , NO, and ONOO , being however devoided of any scavenging effect for HOCl. These effects may strongly contribute for the anti-inflammatory therapy pretended to be obtained by this compounds. The authors would like to thank FCT for financial support POCTI/FCB/47186/2002. 431 OXIDATIVE STRESS – PROTECTIVE EFFECT OF NEW PHARMACOLOGICAL PREPARATIONS DURING ACUTE NITRITE INTOXICATION O. Gonchar 1 , E. Klyuchko 2 , M. Seredenko 1 . 1 Hypoxic States Department, 2 Department of Cellular Membranology, Bogomoletz Institute of Physiology, Kiev, Ukraine Nitrite intoxication is followed by the formation of reactive oxygen species that leads to the development of oxidative stress. We studied homogenates, cytosol and mitochondria fractions of liver, heart, lungs and brain tissues of Wistar rats during acute intoxication that followed injection of sodium nitrite (6 mg/100 g of rat weight). 50 minutes after injection animals were decapitated (when maximum of methaemoglobin formation was reached). During the development of intoxication we registered an increase of lipid peroxidation (LPO) in all studied tissues, disorders in enzymatic and non-enzymatic antioxidant system activities, acidosis development, depression of electron transport and mitochondrion functions of energy synthesis. For correction of these states we used new preparations: yackton, derivative of succinic acid and splenoside – natural metabolite, nucleoside complex. After the injections of preparations an intensity of LPO decreased in all studied tissues (malonyldialdehyde content), increased an activity of antioxidant enzymes – superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase as well as increased the content of reduced glutathione and NAD/NADH rate, respectively decreased lactate/ piruvate rate in comparison with nitrite intoxication state. After splenoside injection we registered too an activation of glucose-6-phosphatedehydrogenase with slight changes in succinate dehydrogenase activity (p<0,5) that evidences about the comparative priority of pentose-phosphate pathway. After yackton administration in hypoxia conditions in mitochondria we saw an increase of succinate dehydrogenase activity (p<0,01) that leads to increase of mitochondrion functions of electron transport and energy synthesis. In conditions of nitric intoxication yackton and splenoside can promote optimization of changed metabolic processes and these substances have significant antioxidant effect. 432 ANETHOLE DITHIOLETHIONE (ADT): A NEW MODULATOR OF OXIDATIVE STRESS INDUCED BY FLUOROQUINOLONE ANTIBIOTICS IN TENDON CELLS. F. Pouzaud 1,2 , P. Rat 1,2 , M.-O. Christen 3 , M. Thevenin 2 , J.-M. Warnet 1,2 . 1 Unité de Pharmaco-Toxicologie Cellulaire,EA 3123 Paris VI, CHNO des XV/XX - 75012 Paris; 2 Laboratoire de Toxicologie, Faculté des Sciences Pharmaceutiques et Biologiques, 75270 Paris cedex 06, France; 3 Laboratoires Solvay Pharma Suresnes 92151, France Anethole dithiolethione (ADT-Sulfarlem® ) prescribed in clinic as choleretic and sialogogue was also well known as an antioxidant, radio- and chemoprotective agent. The aim of this study was to investigate and to modulate oxidative stress associated with five fluoroquinolones (FQ): Pefloxacin (PEF), Ofloxacin (OFX), Ciprofloxacin (CIP), Levofloxavin (LEV), Moxifloxacin (MOX), directly incubated with rabbit tenocyte cell line, at their human blood (10−3 –10−6 M) and infraclinical (10−7 –10−8 M) concentrations. Cell Poster Session P19. Oxidative stress viability, redox potential, reduced glutathione, and reactive oxygen species (ROS) production were assessed using neutral red, Alamar Blue, monobromobimane and H2 DCF-DA probes. Fluorescent signal has been scanned directly in 96-well microplates with high sensitivity (pg/ml), using cold light cytofluorimeter (Fluorolite 1000-Dynex™). Low toxicity appeared after 24h for all FQ, but a high significant delayed tenotoxicity was detected after 48–72h, specially for PEF, CIP and MOX. The FQ tested can be classified in two groups according to the importance of the intracellular redox potential decrease: a strong one (−60% vs control) for PEF, CIP and MOX, and a moderate one (−20% vs control) for OFX and LVX. Reduced GSH decrease seems to be associated with redox potential alteration and ROS production. The FQ-induced oxidative stress (ROS) could be modulated using anetholedithiolethione (ADT). For all FQ, ADT induced a significant oxidative stress decrease (↓ ROS). Moreover, a glutathione increase without cytotoxic effect was observed with ADT. Conclusion: Anetholedithiolethione limited in our model oxidative stress induced with by different FQ and so can be proposed as a therapeutic adjuvant to prevent tendinopathy induced by these antibiotics. 433 EFFECTS OF DIFFERENT EXTRACTS OF MISTLETOE LEAVES (VISCUM ALBUM L.) ON CCl4 -INDUCED HEPATOTOXICITY IN RATS T. Cebovic 1 , M. Popovic 2 , B. Kaurinovic 2 . 1 Department of Biochemistry, Medical Faculty, Clinical centre Novi Sad, Hajduk Veljkova 1–9, 21000 Novi Sad, Yugoslavia, 2 Institute of Chemistry, Faculty of Sciences, T. D. Obradovica 3, 21000 Novi Sad, Yugoslavia Mistletoe (Viscum album L.) is well-known as a medicine from ancient times and the earliest notes. Today it is used as a remedy. Mistletoe contains viscotoxins, phenylpropanes, lignans, flavonoids, biogenamines, polisaccharides and lectins, which have cytostatic effects and immunomodelling potential. Isolated viscotoxins show hipotensive effects. During some preliminary analysis, we concluded that mistletoe contains significant amount of flavonoids and phenolic compounds. Considering well known antioxidative properties and hepatoprotective effects of flavonoids, the effects of different extracts obtained from mistletoe leaves on some biochemical parameters in rats blood hemolysate were examined. Mistletoe extracts were prepared using successive extractions with four solvents of increasing polarity; ether (Et2 O), chloroform (CHCl3 ), ethylacetate (EtOAc), and n-butanol (nBuOH). The residue was the aqueous extract (H2 O). All five extracts were evaporated to dryness and after that dissolved in 50% ethanol to make 10% solutions to be used in the experiment. Albino “Wistar” rats of both sexes, that were used in this experiment, were devided into groups of five animals in each. Some groups have been receiving pure ethanol solutions of different fractions (Et2 O, CHCl3 , EtOAc, nBuOH and H2 O) for seven days, while some groups received carbon-tetrachloride (CCl4 ) in olive oil (1:1) on the eight day beside the mistletoe extract. Twenty four hours after intoxication with CCl4 , animals were sacrified. Liver was removed, weighed and homogenized; also, blood samples were collected. Liver homogenate and blood samples were further used for determination of the following enzymes: xanthine oxidase (XOD), catalase (Cat), peroxidase (Px), glutathione peroxidase (GSHPx), as well as reduced glutathion content (GSH) and intensity of lipid peroxidation (LPx). Results of the investigation showed some very interesting points concerning changes of activity of examined enzymes, both their increase/decrease of activity after administering pure extracts and after intoxication with CCl4 (more detailed results are going to be given during presentation). We concluded that ethanol solutions of different extracts of mistletoe leaves showed some very good antioxidative and protective properties. 434 s117 ANALYSIS OF AMINOCHROMES BY HPLC-PHOTODIODE ARRAY. ADRENOCHROME EVALUATION IN RAT BLOOD. F. Remião 1 , N. Milhazes 2,3 , F. Borges 2 , F. Carvalho 1 , M.L. Bastos 1 , F. Lemos-Amado 4 , P. Domingues 4 , A. Ferrer-Correia 4 . 1 CEQUP/Serv. Toxicologia or 2 CEQOFFUP/Serv. Química Orgânica, Fac. Farmácia, Univer. Porto, R. Aníbal Cunha, 4050/047 Porto. 3 Instit. Ciências Saúde-Norte, 4585/116 Paredes. 4 Dep. Química, Univer. Aveiro, 3810/123 Aveiro. Portugal The catecholamines oxidation process and its products may induce cardiotoxicity and neurotoxicity. Catecholamines can oxidise to aminochromes through autoxidation or by enzymatic or nonenzymatic catalysis. Although some toxic effects seem to be related with the formation of aminochromes there is still scarce information concerning the identification and evaluation of these reactive compounds in in vivo models. In this study five catecholamines were oxidised to their respective aminochromes: adrenaline to adrenochrome, noradrenaline to noradrenochrome, dopa to dopachrome, dopamine to dopaminochrome and isoproterenol to isoprenochrome. An isocratic reverse phase HPLC-Photodiode Array detection method was developed to analyse each pair catecholamine/aminochrome. The analytical system was then applied to the detection of adrenochrome in rat blood at 490 nm. Thus, adrenochrome was administered i.p. to rats and its concentration in whole blood was monitored after 5, 15 and 25 minutes. Blood treatment for adrenochrome evaluation consists of an acidification for protein precipitation followed by a rapid neutralization. The results showed a rapid decrease of adrenochrome concentration in blood after its administration. The adrenochrome present in blood was characterised by UV and tandem mass spectrometry. Thus, the development of this methodology allows a direct quantification of aminochromes in blood or tissues and the correlation of its levels with the observed toxic effects. 435 RELIABILITY OF THE LEVEL OF URINARY 8-HYDROXY – 2’ – DEOXYGUANOSINE (8-OHDG) AND ITS USEFULNESS FOR EPIDEMIOLOGICAL STUDIES R. Yoshida 1 , Y. Ogawa 1 , H. Kasai 2 . 1 National Institute of Industrial Health, Kawasaki, Japan 2 University of Occupational and Environmental Health, Kitakyusyu, Japan Measurement of urinary 8-OHdG has recently become more popular as a means of assessing oxidative stress level in the human. In this study we evaluated the reliability of urinary 8-OHdG as a biomarker for assessing the level of oxidative stress and the usefulness in epidemiological studies. To assess the reliability, samples were taken from 12 volunteers 4 times at one-hour interval and also taken from 70 volunteers 3 times at one-year interval from year 2000 to 2002. Urinary 8-OHdG levels were measured by HPLC-ECD method. Serum MDA, LPO measurements and blood chemistry tests were also performed for comparison. There was no difference in urinary 8-OHdG levels of each volunteer during 3 hours duration (P = 0.1). Correlation coefficient of the urinary 8-OHdG between 2000 and 2001 was 0.8, which was quite high compared with 0.17 of serum MDA and 0.53 of serum LPO. Considering from the view point of stability of the measurement, these results show that the level of urinary 8-OHdG is possibly more reliable oxidative stress marker than serum MDA or serum LPO. To assess the usefulness in epidemiological studies, we examined whether smoking affect the level of urinary 8-OHdG. Although there was no significant difference in cigarette number between smoker of 2000 and 2001, the mean level of urinary 8-OHdG in smokers (4.09) was significantly higher than that of non-smokers (3.25) in 2000 (P<0.05). There was also similar difference in the mean levels of urinary 8-OHdG between smokers and non-smokers in 2001. Considering all these results, the level of urinary 8-OHdG is probably one of the best oxidative stress markers when used at epidemiological studies. s118 436 Poster Session P19. Oxidative stress 32 P-HPLC ANALYSIS OF 8-OXO-2’-DEOXYGUANOSINE M. Zeisig 1 , T. Hofer 1 , J. Cadet 2 , L. Möller 1 . 1 Karolinska Institutet, Dept. of Biosciences at Novum, Unit of Analytical Toxicology, Stockholm, Sweden, 2 Département de Recherche, Fordamensole sur la Matière Condensée, SCIB/LAN, CEA/Grenoble, France Oxidative stress cause oxidative damages in the organism related to aging, inflammation and many common and severe human diseases, like cancer, cardiuovascular disease, asthma, diabetes, rheumatism and Alzheimer. A common result of oxidative stress is the oxidative DNA damage 8-oxo-2’-deoxyguanosine (8-oxo-dG), formed at a hit rate of approximately one damage per cell and second, and a steady-state level of 1–10 per 106 normal nucleotides or 1–10 ng per g cells. To be able to perform analyses on 8-oxo-dG using small samples a very sensitive method is required. 32 P-HPLC has been used to analyze several other DNA damages, including methylation and small lipofilic DNA adducts, with high sensitivity and was considered a suitable candidate for 8-oxo-dG as well. A new scheme for the analysis was developed and the 32 P-HPLC method adapted to the analysis. The method employs enzymatic hydrolyses of DNA to nucleotides, enrichment of damaged nucleotides through an reverse-phase HPLC pre-separation step, enzymatic postlabeling with radioactive [32 P]phosphate and modification of the damaged nucleotides, and reverse-phase HPLC separation from remaining normal nucleotides with online radioactivity detection. The HPLC pre-separation reduced the amount of normal nucleotides enough to allow detection of 8-oxo-dG even in the normal million-fold excess of normal nucleotides in the sample. It also reduced the risk of artefact 8-oxo-dG formation during workup and analysis which otherwise would result in a false high result. The method was applied to both standards, calf thymus DNA and human DNA. The new 32 P-HPLC method enabled more sensitive analyses of 8-oxo-dG and using smaller samples compared to other methods in use. It showed good agreement with an established method when in parallell analyzing µg amounts of DNA for 8-oxo-dG. However, the method required workup with several enzymatic steps and an HPLC pre-separation. 437 CITOTOXICITY AND OXIDATIVE STRESS INDUCED BY POLYCYCLIC AROMATIC HYDROCARBONS IN HUMAN LEUKOCYTES R. Uribe-Hernández 1 , A.J. Pérez-Zapata 2 . 1 Laboratorio de Ecotoxicología, Dirección de Medio Ambiente y Seguridad, Instituto Mexicano del Petróleo, México DF; 2 Laboratorio de Citopaotología Ambiental Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México DF The enzymatic oxidation of the polycyclic aromatic hydrocarbons (PAH) induces the production of reactive oxygen species (ROS) in human blood cells. Human leukocytes were exposed to five concentrations of the anthracene and B(a)P. The lethal citotoxicity (LC50 ) was determined through of the neutral red test for both PAH. The oxidative stress was evaluated through of the superoxide radical and hydrogen peroxide, the levels of intracellular iron and porphyrins concentrations. The LC50 obtained for the anthracene was0.228 µM and 3.13 µM for the B(a)P. As other biomarker of citotoxicity the porphyrins showed a significant increase in both compounds PAHs (P<0.01), showing a proportional relation the porphyrins with both polycyclic hydrocarbons. Respecting to the oxidative stress the levels concentrations of the intracellular free iron were four-fold more bigger than the control (P<0.05) this suggest disruption in the iron metabolism. Likewise was observed a relationship between superoxide radical and hydrogen peroxide with each one of the PAH obtaining a lineal relationship in both cases, with R= 0.98, P<0.05 for anthracene and R= 0.96, P<0.01 for B(a)P. Was observed synergic effect between of the porphyrins and PAH, in the case of anthracene showed a proportional increase of the superoxide radical for each augment in the level of porphyrins (r=0.95, P<0.01), for the B(a)P the lineal regression was R=0.89, P<0.01. We conclude that antracene was more cytotoxic than B(a)P, but that the latter induced more oxidative stress than anthracene. It is possible than both hydrocarbons induced the accumulation of porphyrins and free iron through the block of the biosynthetic pathway of prosthetic group hem. 438 THE INFLUENCE OF LPS AND ETHANOL STIMULATION ON N-NITROSODIMETHYLAMINE (NDMA) SYNTHESIS BY TRANSFECTED HEPG2 CELLS. J. Jablonski 1 , A. Holownia 2 , E. Jablonska 3 , J. Moniuszko-Jakoniuk 1 . 1 Department of Toxicology, Medical University of Bialystok, Bialystok, Poland, 2 Department of Clinical Pharmacology, Medical University of Bialystok, Bialystok, Poland, 3 Department of Immunology, Medical University of Bialystok, Bialystok, Poland NDMA is known as a cancerogenic factor and can be synthesized through nitrosation of amines by NO and O− 2 in confrontation with iNOS expression. NDMA is metabolised by CYP2E1 isoform in p-450 system. The aim of this study was to examine the ability of unstimulated and stimulated HepG2 cells to synthesize NDMA. Examinations were carried out in the culture of transfected HepG2 cells with CYP2E1 expression (E47) or transfected empty vector cells (C34). LPS (1mg/ml), ethanol (100mg/cm3 ) and inhibitor of iNOS (LNAME) (10mM) were added to the culture. The concentration of NDMA in the culture medium was detected by GC-MS method using Perkin-Elmer Turbomass gas chromatograph. The concentration of NO in the culture medium was estimated by Griess’s method. Expression of iNOS was detected through Western-blott. It was shown that HepG2 cells have the ability to synthetize NDMA. LPS stimulation has led to an increased NDMA concentration in the culture supernatants. In contrast, in the presence of L-NAME and ethanol a significant decrease in the NDMA concentration was observed. In the culture of E47 cells, in the presence of ethanol the amount of NDMA was undetectable. The concentration of NDMA in the cultures of the cells examined was associated with NO production and iNOS expression. The results obtained indicate that HepG2 cells have the ability to synthesize NDMA in a manner dependent on the NO synthesis. The lower concentration of NO in the culture of E47 cells indicates that NO links with NDMA that is metabolized by microsomal system. The lack of NDMA in the culture medium of E47 cells stimulated with ethanol suggests an enhanced metabolism of this compound through higher expression of CYP2E1 induced by ethanol. 439 THE EFFECT OF ORAL L- TYROSINE, VITAMIN E AND VITAMIN C ADMINISTRATION ON PERPHENAZINE – INDUCED CATATONIA IN RAT A. Arzi, S. Ghobishavi. Depart. Of Pharmacology & Toxicology, Pharmacy School, Ahvaz University of Medical sciences, Ahvaz, Iran A variety of medication options and medical strategies have been proposed as a mean of parkinson’s disease treatment. On the other hand, the problems of disease progression and levodopa therapy complications have become a major focus of investigation. Consequently, the further study of existing drugs and introduction of new drugs have improved the treatment of parkinson’s disease. According to Free – radical hypotheses and endogen toxins in ethiology of parkinson’s disease it seem that by reducing forming and collection of Free- radicals can decrease disease progression.In the present study, the role of oral L-Tyrosine, Vitamin E and Vitamin C administration on perphenazine induced parkinsonism in rat has been investigated. The NMRI rats were divided into eight groups (n=8) and were treated orally as a single dose with Vitamin E (250 mg/kg), Vitamin C (300mg/kg) and L-Tyrosine (1500mg/kg), Vitamin E plus Vitamin C, Vitamin E plus L-Tyrosine, Vitamin C plus L-tyrosine, Vitamin E and Vitamin C plus L-tyrosine and Normal saline as the vehicle of drugs (Control group) for two weeks, respectively. One hour after the last oral administration, perphenazine (5 mg/kg) was administered intraperitonealy and relative muscular rigidity was determined 20, 40, 60, 90, 120, 180 and 240 minutes after injections. Poster Session P19. Oxidative stress The results indicated that muscular rigidity decreased significantly (p<0.05) in groups that received L-tyrosine in comparison with Control group. The groups that received Vitamin C plus Vitamin E also showed significant decrease in muscular rigidity as compared with Control group. However the group that received Vitamin E and Vitamin C plus L-Tyrosine, showed a low degree of muscular rigidity in comparison with other groups. 440 CATECHOL-CONTINAINING ANTIOXIDANTS SHIFT OXIDATIVE DAMAGE TOWARD THIOL ARYLATION A.W. Boots 1 , G.R.M.M. Haenen 1 , G.J.M. den Hartog 1 , A. Bast 1 . 1 Department of Pharmacology and Toxicology, Faculty of Medicine, University Maastricht, PO Box 616, 6200 MD Maastricht, The Netherlands Oxygen toxicity in aerobic life forms is called oxidative stress. Reactive oxygen species (ROS) may induce the peroxidation of membrane lipids. This lipid peroxidation may result in increased membrane permeability and inactivation of membrane-bound enzymes and receptors. Further toxicity of ROS involves damage to calcium ATPase. Microsomal calcium sequestration by calcium ATPase is critically dependent on sulfhydryl groups of the enzyme. It is generally accepted that ROS are implicated in several pathophysiological processes like diabetes and chronic obstructive pulmonary disease (COPD). To prevent or treat such pathologies, antioxidant therapy has been suggested. The catechol moiety is an important antioxidant pharmacophore present in various antioxidants such as flavonoids like quercetin. Catechol-containing antioxidants e.g. quercetin are able to protect against lipid peroxidation by non-enzymatic scavenging of free radicals with their catechol moiety. During the actual antioxidant activity oxidation products like semiquinone radicals and quinones are formed. These oxidation products are able to arylate critical free thiol groups of the enzyme responsible for the GSH-dependent protection against lipid peroxidation, i.e. the free radical reductase. One of the consequences of this sulfhydryl arylation is that it may impair the endogenous defence against oxidative stress (Biochim. Biophys. Acta. 1583(2002) 279–284). One of the primary toxic effects of oxidative stress is inhibition of calcium ATPase. We found that the oxidation products of catecholcontaining antioxidants also inactivate the calcium sequestration in liver microsomes by arylation of free thiol groups in the responsible enzyme calcium ATPase. So, despite the apparent protection afforded by catechol-containing antioxidants against lipid peroxidation, the toxic effect on a final target, i.e. calcium ATPase, is similar. It is concluded that catechol containing antioxidants like quercetin shift radical damage toward sulfhydryl arylation. This should be considered in the application of antioxidants. 441 CYSTATHIONINE PATHWAY-DEPENDENT CYTOTOXICITY OF DIETHYL MALEATE AND DIAMIDE IN Fa32 AND Hep G2 CELLS P.J. Dierickx. Instituut voor Volksgezondheid, Laboratorium Biochemische Toxikologie, Wytsmanstraat 14, B-1050 Brussel, Belgium Glutathione (GSH) plays a role in many toxicologically important metabolic processes. It was previously established that L-buthionineS,R-sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine synthetase, reduces the GSH content more efficiently in rat (Fa32) than in human (Hep G2) hepatoma-derived cells. We therefore investigated whether the cystathionase inhibitor propargylglycine (PPG) could further decrease the BSO-induced GSH depletion in Hep G2 cells. The influence of the cystathionine precursors Nacetylmethionine, methionine and homocysteine on the cytotoxicity of diethyl maleate (DEM), an electrophilic substance, and diamide [1,1’-azobis(N,N-dimethylformamide)], an oxidant, was also investigated. Experiments were performed on cells seeded at 60 000/microtiterplate well. The cytotoxicity was measured by the neutral red uptake inhibition. The reduced GSH content in the cells was s119 measured fluorimetrically with monochlorobimane, and the protein content with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde. N-Acethylmethionine and homocysteine were more toxic in Fa32 cells than in Hep G2 cells. Methionine was the least toxic of the cystathionine precursors. PPG was 2.5x less toxic in Hep G2 than in Fa32 cells. PPG reduced the GSH content in both cell lines, but to a much lower extent than BSO. A further GSH decrease in Hep G2 was obtained when using a BSO+PPG combination containing relatively high concentrations of PPG. BSO diminished the toxicity of PPG. Methionine abolished the cytotoxicity of diamide in Fa32 cells but not in Hep G2 cells. Homocysteine was the most efficacious of the tested cystathionine precursors in increasing the GSH content and reducing the cytotoxicity of DEM and diamide in Fa32 and Hep G2 cells. 442 INHIBITION OF PYRIMIDINE SYNTHESIS BY LEFLUNOMIDE MODULATES THE OXIDATIVE ACTIVITY OF GRANULOCYTES IN RHEUMATOID ARTHRITIS G. Manda 1 , M. Neagu 1 , L. Toma 2 , C. Codreanu 3 . 1 Immunobiology Laboratory, Department of Immunology, “Victor Babes” National Institute, Bucharest, Romania, 2 Research Laboratory, Biotehnos, Bucharest, Romania, 3 Rheumathology Methodological Center “Dr. Ion Stoia”, Bucharest, Romania Leflunomide (Arava, Aventis) is an inhibitor of dihydroorotate dehydrogenase, recently used in the therapy of refractory rheumatoid arthritis. The inhibition of pyrimidine biosynthesis by leflunomide down-regulates lymphocyte proliferation and antibody production. In this study we investigated the effects exerted in vivo by leflunomide on the functions developed ex vivo by peripheral granulocytes (PMNs) isolated from rheumatoid arthritis patients. 20 patients with severe rheumatoid arthritis were investigated before and one month after leflunomide therapy (20 mg dayly). We investigated the evolution of the CR3 and fMLP-R-triggered oxidative activity developed ex vivo by peripheral PMNs. Intracellular production of superoxide anion was measured by the NBT reduction method, while superoxide anion release was measured by the cytochrome c reduction test. Leflunomide abnormally amplifies the intracellular oxidative activity of peripheral PMNs, but does not enhance superoxide anion release beyond normal values. Our results also reveal the modulatory effect exerted by leflunomide on the respiratory burst: initially low or normal values were intensifed, whereas high values were normalized. Leflunomide targets cellular mechanisms that underlie the intraand extracellular respiratory burst of peripheral granulocytes in rheumatoid arthritis. Persistent inhibition of pyrimidine synthesis with leflunomide sustains the oxygen-dependent microbicidal mechanisms and limits the extracellular tissue-damaging oxidative stress. Leflunomide does not raise the risk of recurrent infections due to an impaired non-specific immune response induced by therapy. It is worth noticing that pyrimidine synthesis inhibition affects both proliferating (lymphocytes) and non-proliferating cells (granulocytes). The mentioned effects exerted in vivo by leflunomide are possibly associated to other mechanisms than the inhibition of pyrimidine synthesis. 443 EFFECT OF CLOFIBRATE AND ALCOHOL TREATMENT ON ETHANOL METABOLIC ENZYMES AND LIPID PEROXIDATION IN THE RAT LIVER P.S. Pronko, T.I. Khomich, L.R. Bardina, V.I. Satanovskaya. Laboratory of Alcohol and Aldehyde Biochemistry, Institute of Biochemistry, NAS of Belarus, BLK 50, 230017 Grodno, Belarus It is known that the peroxisome proliferator clofibrate is capable of inducing the activities of some enzymes involved in ethanol metabolism (catalase, aldehyde dehydrogenase (ALDH), at the same time preventing cytochrome P4502E1 induction in the liver when added to an alcohol diet. Thus, clofibrate can change ethanol metabolism and influence alcohol related metabolic disorders. We studied the effect of clofibrate (400 mg/kg, 4 days, i.g.) and acute alcohol administration (5 g/kg, i.g) after either the vehicle or clofibrate pre-treatment of rats on the activities of ethanol and acetaldehyde s120 Poster Session P20. Signal transduction in toxicity metabolic enzymes (alcohol dehydrogenase (ADH), microsomal ethanol oxidizing system (MEOS), catalase, ALDH), enzymatic and non-enzymatic components of the antioxidant defence system and on the spontaneous and ascorbate-initiated lipid peroxidation in the liver. A significant increase in the activities of rat liver catalase and high Km ALDH and a significant decrease of MEOS activity were noticed after 4 days following the clofibrate administration. Under these conditions, ADH activity tended to decrease, whereas that of low Km ALDH remained unchanged. The administration of ethanol after the clofibrate pre-treatment did not change ADH activity, but it decreased catalase activity and did not change the ALDH level as compared to the rats treated only with clofibrate. MEOS activity was somewhat increased after the ethanol administration and did not differ from the control values. The administration of clofibrate increased the activity of glutathione peroxidase and decreased glutathione transferase activity. The ethanol administration after the clofibrate pre-treatment decreased glutathione peroxidase activity down to the level in the control (vehicle-treated) group and increased ascorbate-dependent production of malonedialdehyde. The clofibrate-induced peroxisome proliferation and increased hydrogen peroxide production may be suggested to make liver tissue more sensitive to activation of lipid peroxidation under the action of ethanol. 444 TOTAL ANTIOXIDANT STATUS IN THE PLASMA OF RATS EXPOSED TO TOBACCO SMOKE SIMULTANEOUSLY WITH RUTIN ADMINISTRATION Jagna Wrzosek 1 , Ewa Florek 1 , Ewa Ignatowicz 2 , Wojciech Piekoszewski 3,4 , Jerzy Moczko 5 , Malina Karkucińska 1 , Emilia Ślusarska 1 . 1 Department of Toxicology, 2 Department of Pharmaceutical Biochemistry, 5 Department of Statistics and Computer Sciences, University of Medical Sciences, Poznan, Poland; 3 Department of Clinical and Industrial Toxicology, University School of Medicine, Jagiellonian University, Krakow, Institute of ForensicResearch, Krakow, Poland The measure of total antioxidant status (TAS) considers the cumulative action of the antioxidants present in plasma and body fluids, thus providing an integrated parameter rather than the simple sum of measurable antioxidants. The activity of known and unknown antioxidants and their synergistic interaction is therefore assessed. The aim of this study was to evaluate the influence of rutin (one of well known flavonoids) on TAS in the plasma of rats exposed to tobacco smoke. The spectrophotometric method was used for the determination of TAS in rats’ plasma. In this method, stable radical of ABTS (2,2’-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid) was reduced by the antioxidants present in the sample. The female Wistar rats, pregnant and non-pregnant, were exposed to tobacco smoke in a concentration of 1500 mg CO/m3 of air for 3 weeks, 5 days in a week, 6 hours daily. The effectiveness of the exposure was confirmed by the amount of cotinine in urine. The rats obtained rutin every day p.o. in the dose of 40 mg/kg, for 5 weeks: 2 weeks before exposure and 3 weeks simultaneously with tobacco smoke exposure. The plasma total antioxidant status increased significantly in a group of pregnant rats (0,082 TAEC/mg Pr) as compared to nonpregnant (0,061 TAEC/mg Pr). Also in the group of pregnant tobacco smoke exposed rats we observed higher TAS (0,086 TAEC/mg Pr). The rutin administration did not cause a significant alteration of TAS as compared to the control group (0,063 TAEC/mg Pr). To conclude, pregnancy and tobacco smoke caused plasma antioxidant defence increase, however rutin administration caused no difference between control and tobacco smoke exposed pregnant rats. P20 Signal transduction in toxicity 445 DRUG INDUCED REVERSIBLE ALOPECIA AFTER THIOUREYLENES G. Bocheva. Department of Pharmacology and Toxicology, Medical University, Sofia, Bulgaria Introduction: Thyroid dysfunction may cause either Grave’s disease and toxic nodular goitre. Thioureylenes are the most used antithyroid agents as a Carbimazole, Methimazole and Thiouracil. Their mechanism of action is though inhibition of thyroperoxidase reducing iodination of thyroglobulin. Thioureylenes may cause reversible hair loss (10–20%), and other unwanted effect such as granulocytopenia (0,1–1%), skin rashes, headaches etc. Methods: The clinical parameters of thyroid dysfunction were observed. The blood levels of TSH, T3 and T4 were determined by RIA. The dermatological symptoms were examined. Results: Our results showed that overdose or intoxication with Carbimazole and Thiouracil can lead to diffuse or even total hair loss (alopecia). This type of alopecia was an early type (up to 3 weeks after administration). It is known as an anagen effluvium. Conclusions: This drug-induced alopecia is due to direct cytotoxic effect and inhibition of cellular proliferation and differentiation in hair matrix. The alopecia was reversible after the drug was stopped. 446 METALLOTHIONEIN REGULATES NFκB LEVEL AND ACTIVITY IN MURINE FIBROBLASTS J. Koropatnick, H. Butcher. London Regional Cancer Centre, University of Western Ontario, London, Ontario, Canada Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal binding protein associated with resistance to chemotherapeutic drugs, toxic metals, and protection from apoptosis. MTs are bound primarily to zinc under normal conditions, and have been proposed as mediators of intracellular zinc availability and activity of zinc-dependent and zinc-sensing proteins, including signal transduction and transcription factors. Inducing conditions that result in increased MT levels (including exposure to reactive oxygen species, chemotherapeutic anticancer drugs, and other agents mediating toxic stress) also increase the activity of the zinc-dependent anti-apoptotic transcription factor nuclear factor-κB (NFκB). We examined whether MTs regulate the level and/or activity of NF-κB. NF-κB protein levels and NF-κB-dependent luciferase reporter activity was assessed in immortalized fibroblast cell lines originating from mice with genetically-ablated MT-1 and MT-2 genes (MT-KO cells), and control mouse cells with functional MT genes (MT-WT cells). We report that the steady-state level of the NF-κB p65 subunit protein (but not p50 subunit) is significantly reduced in three clonal MT-KO cell lines compared to two MT-WT clonal cell lines. There was no difference in steady-state NF-κB p65 mRNA levels, suggesting that post-transcriptional events mediate MT-dependent elevation of NF-κB p65. Levels of mRNA encoding IκBα protein, and IκBα protein (a key regulator of NF-κB activity) were similar in MT-KO and MT-WT cells. The activity of an NFκB-responsive luciferase reporter construct transiently or stably transfected into all 5 test cell lines was also significantly reduced in MT-KO compared to WT cells. Interestingly, nuclear NF-κB p65 protein levels were not correlated with the decreased NF-κB activity in cells lacking MT-1 and MT-2, suggesting that loss of MTs resulted in decreased nuclear NFκB specific activity. These results indicate that MTs may function as positive regulators of overall cellular NF-κB level, and of specific transcriptional activity in the mammalian cell nucleus. MTs have been shown to mediate resistance to anticancer drugs and a wide range of human toxins, and the dependence of NFκB activity on MT reveals a mechanism by which this can occur. The potential for MT to regulate the availability of zinc to NFκB in order to mediate stability and transcriptional activity is under investigation. Supported by a grant from the Canadian Institutes for Health Research (CIHR) Poster Session P21. Transcription factors 447 ACUTE EXPOSURE TO POLYCYCLIC AROMATIC HYDROCARBONS AFFECTS MULTIPLE CELL SIGNALING COMPONENTS IN RAT LIVER EPITHELIAL CELL LINE M. Machala 1 , L. Blaha 1 , P. Kapplova 1 , K. Pencikova 1 , J. Neca 1 , Z. Andrysik 2 , B. Upham 3 , J. Vondracek 2,1 . 1 Veterinary Research Institute, Brno, Czech Republic; 2 Institute of Biophysics AVCR, Brno, Czech Republic; 3 Michigan State University, East Lansing, MI Exposure to various polycyclic aromatic hydrocarbons (PAHs) or non-coplanar polychlorinated biphenyls (PCBs) can lead to an acute inhibition of gap-junctional intercellular communication (GJIC) in the rat liver epithelial WB-F344 “stem-like” cells. In the present study, we show that basic unit of gap junction channels, connexin43, does not become hyperphosphorylated after treatment with PAHs/PCBs, in contrast to effects of other GJIC inhibitors such as epidermal growth factor (EGF) or phorbol ester (TPA). Only marginal or no activation of ERK1/2, p38, PI3K and SAPK/JNK was found in the WB-F3544 cell line during the first 3 hours of treatment with selected PAHs and PCBs. Using selective chemical inhibitors, we found that these kinases, as well as some other serine/threonione kinases (PKC, PKA), receptor tyrosine kinases (ErbB-1, ErbB-2 and NGFR), phospholipases A2 and phosphoinositol-specific phospholipase C, are probably not involved in the inhibition of GJIC induced by PAHs/PCBs. However, other small molecule inhibitors, including D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), RHC 801267 inhibiting DAG lipase, H-89, known to inhibit PKA or some G-protein coupled receptors (GPCR), PP2, a selective inhibitor of src tyrosine kinase, or genistein efficiently prevented GJIC inhibition. The PAHs eliciting GJIC inhibition were found to induce a release of arachidonic acid. These results seem to suggest that activation of PC-PLC and DAG lipase (followed by arachidonic acid release), and yet unidentified upstream tyrosine kinase(s) or GPCR might be involved in early intracellular events in rat liver epithelial cells following an acute exposure to some PAHs/PCBs. (Supported by the Grant Agency of the Czech Republic No. 525/03/1527). 448 CRYSTALLINE SILICA-INDUCED SIGNALLING CASCADES IN EPITHELIAL LUNG CELLS INDUCTION OF IL-8 RELEASE THROUGH MAPKS, PTKS AND SCAVENGER-LIKE RECEPTORS J. Øvrevik, M. Refsnes, P. Schwarze, R. Hetland, R. Becher, J.A. Holme, M. Låg. Norwegian Institute of Puplic Health, Oslo, Norway Inhalation of crystalline silica has been associated with a range of pulmonary diseases such as silicosis, chronic bronchitis and lung cancer. Accumulating evidence suggests that the triggering of inflammatory responses may play an important role in the development of these diseases. However, despite some notable discoveries, the molecular mechanisms involved in silica-induced inflammation remain to be elucidated. In the present study we focus on the initial events of intracellular signal transduction underlying silica-induced interleukin (IL)-8 release from the human epithelial lung cell line A549. We have shown that inhibitors of ERK1/2 and p38 pathways (PD98059 and SB202190, respectively), as well as inhibitors of EGF-receptor (AG1478) and Src-family PTKs (PP2), attenuate IL-8 release from silica-exposed A549 cells. Western blotting further suggests that crystalline silica induce activation of ERK1/2, p38, EGF-receptor and Src. Moreover, incubation with AG1478 and PP2 prior to silica-exposure inhibits phosphorylation of ERK1/2 but not p38. Interestingly, polyinosinic acid (Poly-I), a known inhibitor of scavenger receptors, attenuates IL-8 release and phosphorylation of both ERK1/2 and p38. Taken together, our findings suggest that crystalline silica-induced IL-8 release from A549 cells depend on activation of both the ERK1/2 and the p38 signalling cascades. Moreover, ERK1/2 phosphorylation seems to involve Src and EGF-receptor activation, whereas p38 seems to be activated through different mechanisms. The inhibitory effect of Poly-I suggests that interactions between silica particles and scavenger or scavenger-like receptors on the cell surface may initiate the signalling cascades leading to IL-8 release. 449 s121 AHR-ACTIVATING POLYCYCLIC AROMATIC HYDROCARBONS INDUCE A RELEASE FROM CONTACT INHIBITION OR APOPTOSIS IN RAT LIVER EPITHELIAL CELL LINE J. Vondracek 1,2 , Z. Andrysik 1 , K. Chramostova 1 , B. Vojtesek 3 , K. Soucek 1 , A. Kozubik 1 , M. Machala 2 . 1 Laboratory of Cytokinetics, Institute of Biophysics, Brno, Czech Republic, 2 Department of Chemistry and Toxicology, Veterinary Research Institute, Brno, Czech Republic, 3 Laboratory of Tumor Biology, Masaryk Memorial Cancer Institute, Brno, Czech Republic In the present study, we investigated potential effects of polycyclic aromatic hydrocarbons (PAHs) on contact inhibition in rat liver epithelial WB-F344 cells. The non-mutagens that are weak activators or non-activators of aryl hydrocarbon receptor (AhR)-mediated activity, had no effect on proliferation of confluent cells. On the other hand, relatively strong or moderate AhR ligands with relatively low mutagenic potencies, such as benzofluoranthenes, benz[a]anthracene and chrysene, were found to increase cell numbers, which corresponded to an increased percentage of cells entering S-phase. The release from contact inhibition did not correspond with an inhibition of gap junctional intercellular communication. These results were similar to effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, and indicated that a release from contact inhibition could be a potential tumor promoting effect of several PAHs. Contrary to that, strong mutagens, such as benzo[a]pyrene and dibenzo[a,l]pyrene, induced cell death in this cell line, which was associated with accumulation of p53. Apoptosis was prevented by pifithrin-alpha, an inhibitor of p53-dependent transcriptional activity. Since it has been reported that PAHs can activate various types of mitogen-activated protein kinases, effects of PAHs on their activation were investigated. It was found that both benzo[a] pyrene and dibenzo[a,l]pyrene can activate ERK1/2. Using a specific inhibitor of MEK1/2 kinases (U0126), we observed that inhibition of ERK1/2 activation partly prevented apoptosis induced by these two PAHs. These results seem to suggest that both p53 and ERK1/2 might be involved in regulation of cell death following the PAH-induced DNA damage. This work was supported by grants No. 525/01/D076 and 525/03/1527 from Grant Agency of the Czech Republic. P21 Transcription factors 450 ARYL HYDROCARBON RECEPTOR-NULL MOUSE EXACERBATES THE TH1 TYPE IMMUNE RESPONSE TO OVOALBUMIN L. Vega 1 , R.M. López 1 , M. Rodríguez 2 , G. Elizondo 1 . 1 Sección Externa de Toxicología, Centro de Investigación y Estudios Avanzados-IPN. 2 Instituto Nacional de Cardiología, México D. F., México Aryl hydrocarbon receptor (AhR) is a basic helix-loop-helix transcription factor that is activated by ligand binding and dimerization with AhR nuclear translocator (ARNT) in response to xenobiotics. AhR mediates transcriptional activation of several genes encoding xenobiotic-metabolizing enzymes such as CYP450 1A1 and 1A2. It also plays an important role in the development of the immune system and could have a central role in activating the immune response when it interacts with xenobiotics. To determine the role of AhR in the immune response, we evaluated the immune system response to ovoalbumin (OVA) immunization and challenge in the AhR-null mouse in vivo. Male mice 6–10 weeks of age were immunized by 3 separate inoculations of OVA (1 mg/Kg). Serum was collected in each time point previous to immunization. Spleen cells were collected 15 days after the last immunization and were evaluated for 1) in vitro proliferative response to OVA by 3 [H]-T incorporation, 2) CD4 and CD8 lymphocyte subpopulations by flow-cytometry, 3) OVA-induced IL-12, IL-4 and IFN-γ secretion by ELISA and, 4) serum specific IgG1, IgG2a and IgG2b levels by titer ELISA. AhR-null animals showed an increased number of spleen cells without modification of T cell subpopulations, and increased antigeninduced proliferation compared to wild-type mice. OVA-specific s122 Poster Session P21. Transcription factors IgG1 was reduced, and IgG2a and b were elevated when compared with wild-type animals. OVA-stimulation of lymphocytes induced an increased secretion of IL-12 and IFN-γ in AhR-null mice and decreased IL-4 secretion. Based on the cytokine secretion profile of cultured T cells challenged with OVA after in vivo immunization, and on the specific IgG’s produced in vivo, it is evident that the AhR-null mouse presented an increased Th1 type immune response to OVA immunization. These data suggest that the AhR plays an important role in the switch from Th1 to Th2 type immune response or is involved in the Th2 specific activation pathway. 451 THE BHLH-PAS FACTOR ARNT FUNCTIONS AS A ER CO-ACTIVATOR Elin Rydin, Ingemar Pongratz. Department of Biosciences, Karolinska Institute at Novum, SE-141 57 Huddinge, Sweden Most of the biological effects of estrogens are mediated by the estrogen receptors ERα and ERβ. These receptors regulate gene expression through binding to DNA enhancer elements and subsequently recruiting co-factors that modulate their transcriptional activity. Co-activators like SRC-1 and TIFF-2 share extensive similarity with the bHLH-PAS family of transcription factors. The PAS domain was originally characterized in the proteins PER (regulator of circadian rhythm), ARNT (obligatory heterodimerization partner of the aryl hydrocarbon receptor (AhR)) and the hypoxia inducible factor 1α (HIF-1α) and SIM (neurodevelopmental regulator). Given the sequence homology between classical co-activators like SRC-1 and TIFF-2 and ARNT, we decided to investigate the effect of ARNT on ER activity. Also, in contrast with the AhR and HIF-1α, ARNT is not conditionally regulated and has a broad range of interaction partners. We show that ARNT functions as a co-activator for ERα and ERβ transcriptional activity. These observations together with previous results, which show that ARNT is crucial for conditionally regulated bHLH-PAS proteins like the AhR or HIF-1α expand the range of cellular functions of the general dimerization partner factor ARNT to include regulation of ERα and ERβ transcriptional activity. 452 IL-1-INDUCED AP-1 ACTIVATION REQUIRES REACTIVE OXYGEN SPECIES BUT DOES NOT REGULATE iNOS EXPRESSION IN ARTICULAR CHONDROCYTES M.M. Caramona 1 , A.F. Mendes 1,2 , A.P. Carvalho 2 , M.C. Lopes 1,2 . of Pharmacy, 2 Center for Neuroscience and Cell Biology, University of Coimbra, 3000 Coimbra, Portugal 1 Faculty Activator Protein-1 (AP-1) is an important mediator of the catabolic responses induced by the pro-inflammatory cytokine interleukin-1β (IL-1), in articular chondrocytes. AP-1 has been reported to mediate, as well as to repress the expression of the inducible isoform of the nitric oxide synthase (iNOS). This study aimed at elucidating the role of AP-1 on IL-1-induced iNOS expression in articular chondrocytes. Primary confluent cultures of bovine articular chondrocytes were treated with various concentrations of hydrogen peroxide (H2 O2 ) or with IL-1 (5 ng/ml), in the presence or absence of PD 98059 (a MEK-1 inhibitor) or of diphenyleneiodonium chloride (DPI, which inhibits the production of Reactive Oxygen Species, ROS). AP-1 activation was detected using the electrophoretic mobility shift assay (EMSA). The iNOS mRNA levels were assessed by Northern blot. The results obtained show that IL-1 time- and dose-dependently induced AP-1 activation. PD 98059 (60 µM) inhibited IL-1-induced AP-1 activation, but not iNOS expression. DPI inhibited both IL1-induced AP-1 activation and iNOS expression. H2 O2 activated AP-1 in a time- and dose-dependent manner, but did not induce iNOS expression. Treatment of the chondrocyte cultures with H2 O2 and IL-1 did neither increase nor reduce the iNOS mRNA levels relatively to IL-1 alone. Induction of AP-1 by pretreatment of the cells with H2 O2 (50–300 µM), before the addition of IL-1, did neither prevent nor enhance IL-1-induced iNOS mRNA levels. These results indicate that ROS mediate IL-1-induced AP-1 activation, but that this transcription factor is neither an activator nor a repressor of the iNOS gene in articular chondrocytes. (Supported by FCT) 453 DEVELOPMENT OF A CELL BASED NFκB ASSAY Bernd Laffert, Claudia Gelli, Alexander Loa. CCS Cell Culture Service, Falkenried 88, 20251 Hamburg, Germany NFκB is a key transcription factor for the induction of numerous genes involved in inflammatory responses and inhibition of apoptosis. It also plays an important role in a broad range of human diseases resulting from aberrant activities of the immune system. Therefore, modulation or inhibition of NFκB activities is a promising approach for the treatment of inflammatory disorders and development of improved immunosuppressive therapies. Consequently, appropriate assays for discovery and validation of inflammatory or immunosuppressive drugs are crucial and of high value for the pharmaceutical industry. Recognizing these needs, CCS has developed an NFκB assay exhibiting two distinct features: i) it is a cell-based reporter assay and therefore allows monitoring any effects on the activity of NFκB within a natural physiological environment, ii) this assay is designed as ready-to-use assay, that is, the transgenic reporter cells are pre-seeded and subsequently frozen in 96-well microplates. Thus, the assay can be performed shortly after thawing the cells, and time- and money-consuming cultivation and seeding of the indicator cells is unnecessary. 454 OVEREXPRESSION OF PPARδ AND CYCLIN D1 IN DYSPLASTIC ABERRANT CRYPT FOCI AND IN ADENOMAS, BUT NOT IN HYPERPLASTIC ABERRANT CRYPT FOCI IN APC MIN/+ MICE H.K. Knutsen 1 , H.B. Ølstørn 1 , J.E. Paulsen 1 , T. Husøy 1 , I.L. Goverud 2 , E.M. Løberg 2 , K. Kristiansen 3 , J. Alexander 1 . 1 Department of Food Toxicology, Norwegian Institute of Public Health, Oslo, Norway, 2 Deptartment of Anatomy and Pathology, Ullevaal University Hospital, Oslo,Norway, 3 Deptartment of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Apc Min/+ mice, that carry a truncating mutation in one allele of the tumour suppressor gene Apc, develop numerous intestinal adenomas. Intact APC protein controls the level of the proto-oncogene βcatenin, which is involved in both cell adhesion and activation of transcription. Peroxisome Proliferator-Activated Receptor delta (PPARδ), which together with the closely related PPARα and PPARγ comprise a subgroup in the nuclear receptor family, has been reported to be a direct transcriptional target of β-catenin. Furthermore, it has been reported that absence of PPARδ in Apc Min/+ mice might reduce growth of small intestinal adenomas. Cyclin D1, which promotes G1 to S transition by activating cyclin-dependent kinases 4 and 6, is also transcriptionally activated by β-catenin. We have previously reported relationship between dysplastic aberrant crypt foci (ACF) and tumorigenesis in the Min/+ mouse colon, and have shown that β-catenin is induced in both dysplastic ACF as well as in colon adenomas. In contrast, β-catenin level was similar in normal tissue and in hyperplastic ACF, lesions that probably do not undergo neoplastic transformation. In the present study, we show that PPARδ is expressed in nuclei and cytoplasm in normal large and small intestinal crypts. The highest nuclear expression is found in the bottom of the crypts, whereas no expression is found in nuclei facing the lumen. Compared with the normal colon, PPARδ was overexpressed in dysplastic ACF and in adenomas, but not in hyperplastic ACF. Cyclin D1 is also induced in nuclei in dysplastic ACF, but not in hyperplastic ACF. Similarly, we also found that the level of PPARδ, cyclin D1 and β-catenin was increased in both small and large intestinal adenomas. Double immunofluorescence staining of PPARδ and β-catenin revealed nuclear co-localisation of both proteins only in a small proportion of epithelial cells in the adenomas. The functional consequences of PPARβ/δ induction in dysplastic ACF and small and large intestinal adenomas remain to be determined. Poster Session P22. Chemical carcinogenesis P22 Chemical carcinogenesis 455 TRUNCATION MUTATIONS IN THE TUMOUR SUPPRESSOR GENE ADENOMATOUS POLIPOSIS COLI IN INTESTINAL TUMORS OF MIN/+ MICE EXPOSED TO 2-AMINO-1-METHYL-6-PHENYLIMIDAZO[4,5-B]PYRIDINE L. Møllersen, A. Andreassen, R. Vikse, I.-L. Steffensen, A. Mikalsen, J.E. Paulsen, J. Alexander. Department of Food toxicology, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway We have previously shown that PhIP induced intestinal tumors of C57BL/6J-Min/+ (Multiple intestinal neoplasia) mice show truncation mutations in or loss of the wt Apc allele. In this study we show that increasing the dose of 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) from 10 to 50 mg/kg given as a single s. c. injection on day 3–5 after birth increased the number of intestinal tumors and reduced the frequency of loss of heterozygosity (LOH) in the Apc gene. We screened tumors apparently retaining the Apc wild type allele for truncation mutations in the first half of exon 15 from this experiment and from previous experiments with PhIP in Min/+ mice. We verified 25 mutations. Of these were 60% G→T transversions, and 16% G deletions. We were not able to identify any G deletions in GGGA runs, which previously was claimed to be a signature mutation of PhIP in Apc. Most of the mutations were located between codon 989 and 1156 corresponding to the first part of the β-catenin binding region. We also identified two Apc truncation mutations in spontaneous formed intestinal tumors from untreated mice. These mutations were a C→T transition and a T insertion, which were different from those induced by PhIP. 456 SHORT-TERM CARCINOGENICITY STUDIES OF TRIACRYLATES IN THE Tg.AC TRANSGENIC MOUSE MODEL R.S. Chhabra, M. Hejtmancik. NIEHS, Research Triangle Park, NC, USA and Battelle, Columbus, OH, USA Polyfunctional acrylates, pentaerythritol triacrylate (PETA) and trimethylolpropane triacrylate (TMPTA) are used in the manufacture of many chemical products. The carcinogenic potential of these acrylates was studied in male and female hemizygous Tg.AC transgenic mice. Five dose groups each consisting of 15 animals of both sexes were administered PETA or TMPTA dermally in acetone at doses of 0.75, 1.5, 3, 6, and 12 mg/kg for 27–28 weeks. The control groups were administered acetone only. Survival rates in test and control groups ranged from 80–100% except in the high dose levels where the lowest survival rate was 40% in the female mice exposed to PETA. Mean body weights were comparable to controls. In both PETA and TMPTA studies at the site of application, there were dose-related increases in epidermal hyperplasia, hperkeratosis, and active chronic inflammation. At the site of chemical application, squamous cell papillomas were induced in a treatment-related fashion. In some of the PETA treated groups there were a few incidences of squamous cell carcinoma. In the two high dose groups 80–100% animals had multiple papillomas. The latency period for tumor development ranged from 12 to 19 weeks. The results suggest PETA and TMPTA are likely to be dermal carcinogens in the traditional two-year bioassay as well. 457 DIFFERENT LEVELS OF PhIP-DNA ADDUCTS IN MICE DEPENDING ON EXPOSURE TIME POINT AND APC STATUS COULD NOT BE EXPLAINED BY DIFFERENCES IN CELL PROLIFERATION I.-L. Steffensen 1 , H.A.J. Schut 2 , J. Alexander 1 . 1 Department of Food Toxicology, Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway, 2 Department of Pathology, Medical College of Ohio, Toledo, Ohio, USA In this study, we gave whole litters consisting of both C57BL/6JMin/+ (Multiple intestinal neoplasia) mice having one mutated allele of the adenomatous polyposis coli (Apc) gene, and +/+ (wild-type) s123 mice, one s.c. injection of 50 mg/kg of the food mutagen 2-amino1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) on either day 12 or 36 after birth. Levels of PhIP-DNA adducts were quantitated 8h, 12h, 24h, 3 days or 7 days after exposure with 32 P-postlabelling. In Min/+ mice, the PhIP-DNA adducts levels were significantly higher after exposure on day 12 compared with day 36 for middle (47–747%) and distal (29–552%) small intestine, measured from 8h to 3 days after PhIP exposure, but not for colon and proximal small intestine. This variation in levels of PhIP-DNA adducts at different age and between different intestinal regions corresponds very closely with our previously observed variation in PhIP-induced intestinal tumors. In the liver, the PhIP-DNA adducts levels were 97–654% higher after exposure on day 12 compared with day 36, measured from 8h to 24h after exposure, however, the liver is not a target organ for PhIP in this model. In +/+ mice, the PhIP-DNA adduct levels were lower than the levels in the Min/+ mice, indicating that Apc gene status might possibly affect PhIP-induced intestinal tumorigenesis. The observed differences in PhIP-DNA adduct levels could not be explained by differences in cell proliferation, as measured by immunohistochemical detection of bromodeoxyuridine (BrdU) incorporation. 458 STYRENE TOXICITY IN HEPG2 CELLS. D. Maurici, V. Campi, I. Malerba, M. Carfi’, G. Bowe, L. Gribaldo. ECVAM, Inst. for Health and Consumer Protection, JRC, Ispra, Italy Environmental, occupational and recreational exposures to carcinogens contribute to cancer risk in humans. Styrene is one of the most important organic chemicals. It is mainly produced to prepare solid polystyrene foam, expanded polystyrene foam and styrene-butadiene rubber. It has been shown to be genotoxic after metabolic activation and to induce cytogenetic effects in many experimental systems. The toxic effect of styrene on HepG2 (wild-type p53) cells was analysed. Those cell are considered to be metabolically competent to activate different classes of mutagens into biologically active metabolites. We exposed HepG2 cell line to styrene and to its metabolite styrene oxide at subtoxic concentrations (up to 1mM and to 200 uM respectively) to analyse the expression of genes and proteins involved in apoptosis and cell cycle regulation. We performed a cDNA macroarray and found an overexpression of TGFβ2, TGFβIII receptor, confirmed by real time PCR. We also checked the expression of different TGFβs and TGFβs receptors by semi-quantitative PCR. The protein levels of Bax (pro-apoptosis) and Bclx-L (anti-apoptosis) in HepG2 cell line treated with styrene was also investigated. The endogenous level of Bclx-L increased according with the increasing concentration of the chemical. On the contrary, Bax protein level did not change in treated and untreated cells. Our data suggests that the activity on cell proliferation/cell death should be monitored as an early endpoint of exposure. Moreover, macroarray technology can be used as useful tool to evaluate gene expression profiles after exposure to subacute doses of chemicals. This approach is also used in in vivo experiments to evaluate the possibility of detection of early molecular marker of toxicity. The in vitro results should be compared to in vivo data in order to evaluate the possibility to replace animal experiments with in vitro toxicogenomics 459 PRELIMINARY DATA ON THE INVESTIGATION OF LESION INDUCED BY TWO DIFFERENT SUBCLONES OF SUIT-2 PANCREATIC CANCER CELL LINE IMPLANTED IN NUDE MICE A. Piaia 1 , C. Sorio 2 , A. Bonora 3 , A. Lanzoni 1 , S. Munaro 1 , A. Scarpa 2 , P. Cristofori 1 . 1 Safety Assessment Dept. Histopathology Unit Research Center GlaxoSmithKline Verona Italy – 2 Pathology Dept., Università di Verona, Italy; 3 Surgery Science & Gasteroenterology Dept. Università di Verona, Italy Because of the highly aggressive behavior and the metastatic properties of pancreatic cancers and the very poor outcome for patients, a better understanding of the malignant behavior of these cells may provide new directions for the treatment. In this study we tested two subclones derived from pancreatic adenocarcinoma cell line (SUIT-2). These subclones have been recently characterized by our s124 Poster Session P23. Genetic toxicology groups to have different capability to grow in soft agar and to show a different morphology/motility in culture (subclone M = motile, subclone O = non-motile). As alteration in cell motility has been associated to malignant progression, we inoculated a suspension of these cells subcutaneously in nude mice to evaluate their behavior in vivo. Both the subclones developed tumors in mice, however subclone O developed a larger tumor mass when compared to subclone M (median: 2371 mm3 vs 311 mm3 ) and showed also a lower incidence of neoplastic ascites. Another significant difference was the higher incidence and extent of necrosis within the growing subcutaneous masses of subclone M, supporting the idea that their growth depended also on different promotion of angiogenesis. To test this hypothesis we therefore examined the in vitro secretion of Vascular Endothelial Growth Factor (VEGF), a pro-angiogenetic factor known to be secreted by pancreatic carcinoma cells. We observed a 30% increase of its secretion in the subclone O in comparison with subclone M. Analysis of other angiogenetic factors in these subclones is in progress. In conclusion, in vitro differences of the two subclones (motile vs non-motile) have been confirmed in the in vivo model in which subclone M showed a tendency to develop smaller masses, more necrotic tissue and more frequent ascites in comparison with subclone O. Furthermore, these data confirm the importance of in vivo testing for cancer cell cultures to properly drive the assessment of specific biochemical markers which could be potential target for the therapeutic intervention. P23 Genetic toxicology 460 THE PROTECTIVE EFFECTS OF THYMOL AND CARVACROL AGAINST OXIDATIVE DNA DAMAGE S. Aydın 1 , A.A. Başaran 2 , N. Başaran 1 . 1 Department of Toxicology, Faculty of Pharmacy, University of Hacettepe, Ankara, Turkey, 2 Department of Pharmacognosy, Faculty of Pharmacy, University of Hacettepe, Ankara, Turkey, Phenolic phytochemicals are a large group of substances that have been regarded as possible antioxidants. However the full chemical properties and the effects of phenolic phytochemicals as antioxidants in protecting DNA against oxidative damage have not been completely examined since they have suggested to have both antioxidant and prooxidant activities. Thymol and carvacrol are naturally occuring phenolic compounds found in significant quantities in essential oil fraction of oregano and thyme which are widely used species as spices and herbal teas. In the present study the modulating effects of thymol and carvacrol against the oxidative DNA damage induced by H2 O2 in human lymphocytes was investigated by the alkaline Comet assay. The lymphocytes incubated with thymol and carvacrol alone at concentrations of 0.5, 5, 10, 25, 50, 100, 200, 500, 1000 and 2000 µM and it was found that both thymol and carvacrol at concentrations above 100 µM significantly induced DNA damage in human lymphocytes but at smaller concentrations they have not induced DNA strand breakage. When thymol and carvacrol at concentrations below 100 µM incubated with 100 µM H2 O2 a significant reduction in DNA strand breakage was observed (p<0.05). It seemed that at low concentrations thymol and carvacrol prevent the oxidative DNA damage induced by H2 O2 and can be used as an antioxidant in free radical related disorders. 461 PROTECTIVE EFFECTS OF MELATONIN ON THE IONIZING RADIATION INDUCED OXIDATIVE DNA DAMAGE IN THE RAT BRAIN Ü. Ündeǧer 1 , B. Giray 1 , A.F. Zorlu 2 , K. Öge 3 , N. Başaran 1 . 1 department Of Toxicology, Faculty Of Pharmacy, University Of Hacettepe, Ankara, Turkey, 2 Department Of Radiation Oncology, Faculty Of Medicine, University Of Hacettepe, Ankara, Turkey, 3 Department Of Neurosurgery, Faculty Of Medicine, University Of Hacettepe, Ankara, Turkey Ionizing radiation is demonstrated as one of the main exogenous sources of oxidative damage and it is reported to induce of producing OH as well as other radical species by interaction with water in cells and tissues. DNA is the most often reported molecule to be damaged by ionizing radiation. Ixonizing radiation is a wellknown cytotoxic and mutagenic agent of which the biological results are attributable to its free radical producing effects. Melatonin, a hormone produced by the pineal gland, has been shown to act as an antioxidant and have radioprotective effects. The effect of melatonin on the DNA strand breakage and lipid peroxidation induced by ionizing radiation in the rat brain were investigated in order to clarify its radioprotective ability. The dna strand breakage in rat brain exposed to 1000 cGy ionizing radiation was assessed by alkaline single cell gel electrophoresis and the lipid peroxidation was evaluated by measuring thiobarbituric acid reactive substances (TBARS) concentrations. A significant increase in DNA damage (P<0.05) and tbars concentrations (P<0.01) was found in the radiation treated rat brain. Pre-treatment of rats with intraperitoneal doses of 100 mg/kg melatonin provided a significant decrease in the DNA strand breakage and lipid peroxidation and our results indicate that melatonin protects brain cells from oxidative damage induced by ionizing radiation. • 462 DEPENDENCE OF ANTIMUTAGENIC EFFECTS OF AFOBAZOLE UPON ANTIOXIDANT SYSTEM PHENOTYPE A.K. Zhanataev 1 , A.D. Durnev 1 , S.B. Seredenin 1 . 1 State Zakusov’s Institute of Pharmacology of RAMS, Moscow, Russia The pharmacologically active 2-mercaptobenzimidazole derivatives are able to lessen the mutagenic effects of chemical prooxidants through the normalization of free radical oxidation and related formation of endogenous mutagens. Recent studies revealed the antimutagenic properties of the novel anxiolytic among 2mercaptobenzimidazole derivatives - afobazole, which also exhibits the antioxidant properties. This agent appears to be of therapeutic value for the complex treatment of pathologies accompanied by the elevation of the mutation rate due to pro/antioxidant balance disturbance and “oxidative stress” development. The major embarrassment envisaged during clinical treatment with antioxidants is that their effects are differently oriented in various patients, thus making it of great current concern to evaluate the dependence of afobazole antimutagenic activity manifestations upon the antioxidant system phenotype. The purpose of the present study was to evaluate the effects of afobazole (dose range from 1 to 100 mg/kg, per orally) in c57Bl/6 and BALB/C mice strains, which exhibit distinctive differences in main parameters characterizing the levels of antioxidant protection and free radical oxidation. Dioxidine DN (100 and 300 mg/kg) and cyclophosphamide CP (20 mg/kg) were used as mutagens since their cytogenetic effects significantly vary in mice strains tested upon the intraperitoneally administration. Three regimens of animal’s treatment were employed. The first was the co-administration of afobazole and mutagen, the second was the injection of mutagen while on the 5-day pre-treatment with afobazole, and the third combined the administration of both agents studied over 5 days. Upon single dosage afobazole produced in C57Bl/6 and BALB/C mice almost the same antimutagenic activity towards the mutagens used. In pre-treated BALB/C mice afobazole completely prevented the cytogenetic effects of DN (300 mg/kg) and maximally reduced the effects of CP by 71%. In C57Bl/6 mice the maximal antimutagenic action of afobazole made in experiments with DN and CP 72% and 45% accordingly. Similar findings were obtained with afobazole co-administered for 5 days with the mutagens tested. In BALB/C mice afobazole was found to suppress fully the mutagenic effects of DN (300 mg/kg) and diminished the CP mutation rate by 70%. Conversely, in C57Bl/6 mice the decrease in mutagens effects (DN by 55% and CP by 44%) was observed only at highest afobazole dose. Thus, it is evident from results obtained that upon pre-treatment and 5-day co-administration with mutagens, the antimutagenic effects of afobazole are most pronounced in BALB/C mice, and it may be attributed to different rates of antioxidant protection. BALB/C mice demonstrate significantly higher as compared to C57Bl/6 activity of enzymes involved in the antioxidant protection, SOD and Poster Session P23. Genetic toxicology catalase. Hence, in parallel with this high enzymes activity the treatment with an antimutagenic agent is able to prevent an abrupt shift in pro/antioxidant balance, thus giving rise to a more potent antimutagenic action of afobazole. The data obtained allow the assumption about the dependence of the antimutagenic effects exerted with the antioxidant properties on genetically determined activity of antioxidant protection system. This fact permits to purposely target as promising goal the search for predictors of individual effects produced by antimutagens or agents among other classes with antioxidant properties in genetically heterogeneous human population. comparatively slight increase on the number of MN, within the dose range of 25–100 µM. Centromers in micronuclei were detected with anti-kinetochore antibodies from CREST patients, to determinate if the induced micronuclei contain whole chromosomes or acentric fragments. Genistein induced mostly CREST negative micronuclei, i.e. MN containing only chromosomal fragments. Hence, genistein appeared as a clastogen. MN induced by high concentrations of daidzein were partly CREST(+) and CREST(-). In essence, this points to a differential genotoxicity of genistein and daidzein. 465 463 THE REPAIR OF STYRENE OXIDE-INDUCED DNA BREAKS IN XPA AND XPC CELLS COMPARED TO NORMAL HUMAN FIBROBLASTS R. Štetina 1 , R. Köhlerova 2 , P. Vodicka 3 . 1) Military Medical Academy J.E. Purkyne, Dept. of Toxicology, Hradec Kralove, 2 Charles University, Medical Faculty in Hradec Kralove, Dept. of Biochemistry, Hradec Kralove, 3 Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Dept. of Genetic and Molecular Toxicology, Prague, Czech Republic The mechanism, by which styrene-induced DNA damage is repaired, is not known precisely yet. In this study we have followed the kinetics of the repair of single strand DNA breaks (SSB) induced in human embryo lung fibroblasts, used as reference cell line, and DNA repair defective human cells. Two cell lines of Xeroderma pigmentosum cells known to be defective in the incision step of nucleotide excision repair (NER) were used: 1) XPA (XP12ROSV40) and 2) XPC (XP8CAC/SV). Cells were treated with 100 uM or 250 uM of styrene oxide for 1 hour, styrene oxide was removed and cells incubated for different periods of time to follow the DNA repair. At intervals 0, 3, 6, 12, and 24 hours after the treatment the number of SSBs was measured using the alkaline version of comet assay (single cell gel electrophoresis). SO induced comparable amount of SSBs in all types of cells tested. The kinetic of the removal of SSB from the DNA was also very similar in both normal (embryo fibroblasts) and DNA repair deficient cells (XPA and XPC). The half time of the repair was approximately 2–4 hours. Results clearly show, that the DNA breaks induced in all three types of cells with SO are repaired with the same efficiency. These results suggest that the base excision repair (BER) seems to be the most probable way of the repair for SO-induced DNA damage, while the nucleotide excision repair (NER) plays only a negligible role in the acute in vitro experiments. This study has been supported by GACR 310/01/0802 and 310/03/0437 464 MECHANISMS OF GENOTOXICITY OF THE PHYTOESTROGENS GENISTEIN AND DAIDZEIN A.L. Di Virgilio, H.M. Bolt. Institut für Arbeitphysiologie an der Universität Dortmund, Ardeystr. 67, 44139, Dortmund The presence of hormonally active phytoestrogens in the human diet has been a matter of recent concern. Apart from the hormonal (estrogenic) activities of these compounds, the question of genotoxicity has been raised for daidzein and genistein. The latter differs from daidzein only by one additional hydroxyl group. Genistein and daidzein were tested for cytotoxicity using the Neutral Red Uptake assay. Chinese hamster fibroblast V79 cells were seeded into a 96 well plate (104 cells/well in 200 µl DMEM medium) in the presence of different concentrations of the phytoestrogens. The dose range for daidzein and genistein were 25–150 µM and 5–100 µM respectively. Genistein and daidzein caused no cytotoxicity within this dose range. The micronucleus (MN) assay in V79 cells was used to study chromosomal genotoxicity. V79 cells were grown in 25 cm2 flasks. Solutions of the test compounds in DMSO (0.1% v/v) were added and incubated for 18 h. Following disaggregation with trypsine/EDTA and resuspension, cells were subjected to hypotonic conditions with 0.4% KCl and fixation. Cells were mounted on slides and stained with Acridine Orange. Genistein gave rise to a significant induction of MN at concentrations of 5–25 µM. By contrast, daidzein showed a s125 IN VIVO MICRONUCLEUS TEST FOR THE GENOTOXIC EVALUATION OF BACTIVEC AND GRISELESF A. Curbelo, A. Remigio, G. Pérez, N. Fernández, A. Bada, Y. Rivero, R. Ocaña. División de Toxicología y Experimentación Animal del Centro Nacional para la Producción de Animales de Laboratorio, Habana, Cuba BACTIVEC and GRISELESF are biolarvicide elaborate from LABIOFAM, Cuba. These products are effective for control of Dengue, Malaria, Filariasis, Encephalitis, and other mosquitotransmitted diseases. BACTIVEC contains spores and toxic crystals of the bacterium Bacillus thuringiensis strain 14 and other essential elements for the effective control of mosquito’s larvae. The active ingredients of GRISELESF are spores and endotoxic crystal of the bacterium Bacillus sphaericus strain 2362. In this work it was evaluatied the in vivo mutagenic effect of BACTIVEC and GRISELESF, by means of the bone marrow micronucleus test in Cenp:NMRI mice obtained from National Center for Breeding of Laboratories Animals (CENPALAB, Cuba). This assay is widely used as an alternative and effective assay to the chromosomal aberration test for the evaluation of genotoxic and clastogenic potential. One dose level of each product was established (BACTIVEC 2.5 x 108 UCF/mL y GRISELESF 2.8 x 108 UCF/mL), and the choosed administration route was intragastric (gavage). It was accomplished two consecutive administrations at 24 hours intervals, followed by sacrifice of animals (cervical dislocation) and sampling. Sterilized water was use as negative control, and as positive control was used intraperitoneal cyclophosphamide (40 mg/kg of body weigth). Each established group includes 5 males and 5 females, obtaining two slides of each one. Microscopic examination of slides was performed using an Opton microscope (100x), and taken into account the relation between policromatic and normocromatics erythrocytes, and the presence of micronucleus in policromatic erythrocytes. Differences between control and treated groups were assessed either considering actual counts. Factorial ANOVA (analysis of variance) was applied to asses the differences between treatments. The results of the micronucleus assay with bone marrow erythrocytes was no difference in micronucleus frequency between treated and control groups. There were no evidence of toxic effects in the erythrocytic population studied, and negative results were obtained in the citotoxicity and clastogenicity inducement (chromosome aberrations inducement). 466 INFLUENCE OF THE INDOLE COMPOUNDS ON CELLULAR LEVEL OF GLUTATHIONE AND GLUTATHIONE-S-TRANSFERASE ACTIVITY Ksenija Durgo 1 , Sanjica Jakupec 2 , Jasna Franekić Čolić 1 , Maja Osmak 2 . 1 Faculty of Food Technology and Biotecnology, Pierottijeva 6, 10000 Zagreb, Croatia; 2 Laboratory for Genotoxic Agents, Ruðer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia Cruciferous vegetables such as cauliflower, cabbage, kale, kohlrabi, turnips or broccoli, contain µg/g levels of glucosinolate glucobrassicin (indolyl methyl glucosinolate). Indole-3-carbinol is the major hydrolytic product obtained from glucobrassicin. When the plant cells are damaged by cutting or chewing, a thioglucosidase-mediated autolytic process takes place generating indole-3-carbinol, glucose and thiocyanate ion. Recent reports have shown that indole compounds can modulate cellular response when cells are exposed to variety of environmental xenobiotics. In our previous work we have shown that indole-3carbinol and cauliflower extract did not induce point mutations in s126 Poster Session P23. Genetic toxicology Salmonella typhimurium TA98 and TA100. We have also shown that indole compounds have inhibited formation of point mutations when Salmonella typhimurim TA98 and TA100 were treated with two standard mutagens 4-nitroquinoline-1-oxide (direct acting mutagen) and 2-aminoanthracene (promutagen). In this work we have examined indole-3-carbinol and cauliflower extract for their ability to influence on phase II biotransformation enzyme activities in four cell lines: human laryngeal carcinoma cells (parental HEp2 and their cisplatin resistant subline CK2 ) and human cervix cancer (parental HeLa and their cisplatin resistant subline CK). We have measured and compared cellular concentration of glutathione and enzymatic activity of total glutathione-S-transferases after 72 hours of treatment of the cells with the highest nontoxic concentrations of indole-3-carbinol and cauliflower extract. Concentrations of glutathione in four cell lines were compared with the basal level of glutathione in untreated cells. It was shown that cauliflower extract and indole-3-carbinol have caused significant increase of glutathione in both, Hep2 and HeLa, parental cells. On the contrary, cisplatin resistant cells have increased basal level of glutathione, and both drugs decreased the level of glutathione. The activity of glutathione-S-transferases were increased in parental HEp2 and HeLa cells after treatment with indole-3-carbinol and cauliflower extract. On the other hand, indole compounds caused significant decrease of glutathione-S-transferases activity in resistant cells. 467 INDUCTION OF DOMINANT LETHAL MUTATIONS IN MALE MICE BY PEREZONE G. Chamorro 1 , L. Garduño-Siliciano 1 , M. Salazar 1 . 1 Laboratorio de Toxicología Preclínica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México D.F., México Perezone (2-(1,5-dimethyl-4-hexenyl)-3-hydroxy-5-methyl-1,4-benzoquinone) is a naturally occurring sesquiterpenic benzoquinone in the roots of the genus Pereziae. It has been shown to possess hypoglycemic, antiplatelet and antiarrythmic properties. Morever, an antiimplantation effect was demonstrated in pregnant female mice. In this study as a part of an ongoing programme to obtain data on its safety, a dominant lethal test in male CFI mice was employed. The drug was given orally at doses of 0, 25, 50 and 100mg/kg for 5 days. Males were mated with untreated females following a 7-day mating schedule with three consecutive mating events. The incidence of pregnancy of female mated on days 1–7 after males were given 100mg/kg of perezone was decreased. Upon examining surgically exposed uteri and ovaries of pregnant females on gestation days 13– 15, an increased incidence of pre-implantation losses with 100mg/kg of perezone and increased incidence of post-implantation losses with 50mg/kg and 100mg/kg were observed. Semen examination of a separate group of mice showed a decreased concentration and motility of spermatozoa. The results support the conclusion that perezone is a germ cell mutagen and its effects are more pronounced during the post-meiotic stage. 468 INTERACTION WITH CYTOSKELETAL PROTEINS AND GENOTOXICITY OF NITROBENZENE AND BENZONITRILE R. Thier 1 , D. Bonacker 2 , T. Stoiber 3 , K.J. Böhm 3 , E. Unger 3 , H.M. Bolt 2 , G.H. Degen 2 . 1 School of Biomedical Sciences, University of Queensland, St. Lucia, QLD 4072, Australia, 2 Institut für Arbeitsphysiologie an der Universität Dortmund, Ardeystr. 67, D-44139 Dortmund, 3 Institut für Molekulare Biotechnologie Jena e. V., Beutenbergstr. 11, D-07745 Jena Cytoskelatal motor proteins are significant subcellular target molecules for toxic chemicals. Interactions of chemicals with structures composed of cytoskeletal proteins can possibly lead to genotoxicity on the chromosomal level. For example, micronuclei (MN) may result as a consequence of disturbed microtubule assembly and subsequent disorganised chromosome segregation during mitosis. We studied genotoxic effects of nitrobenzene and benzonitrile in V79 hamster fibroblasts using the MN assay. Nitrobenzene and benzonitrile caused MN dose-dependently. Nitrobenzene induced MN at concentrations ≥ 0.01 µM and benzonitrile at concentrations ≥ 0.05 µM. CREST analysis recorded mainly aneugenic MN for both compounds. Possible effects on tubulin assembly and kinesin-driven motility were examined in two cell-free systems, the tubulin assembly assay and the kinesin motility assay. Microtubule assembly was affected at concentrations above 1 mM nitrobenzene or 2 mM benzonitrile. Neither nitrobenzene (≤ 15 mM) nor benzonitrile (≤ 50 mM) modulated the gliding velocity of microtubules along immobilised kinesin. Our data verify a genotoxic potential for nitrobenzene and benzonitrile in vitro. They also indicate interactions with cytoskeletal motor proteins such as tubulin at higher concentrations. However, interaction of nitrobenzene or benzonitrile with tubulin seems not to be the only factor responsible for the observed formation of aneugenic MN. These studies were supported by CEFIC/LRI: CC-1FOAR-0003. 469 ALUMINUM GENOTOXICITY FOR PLANT AND ANIMALS B. Synzynys 1 , O. Kharlamova 1 , N. Bulanova 1 , E. Tjantova 2 . 1 Obninsk State University of Nuclear Power Engineering, Obninsk, Kaluga Region, Russia,2 Medical Radiological Research Center of Russian Academy of Medical Science, Obninsk, Kaluga Region, Russia Aluminum (Al) is the third most abundant element in the Earth crust (8,8%), surpassed only by oxygen and silicon. Owing to its reactivity Al quickly forms insoluble compounds, which do not penetrate into living cells and tissues and thus is practically safe for plants and animals. Acid rains and acid food release Al3 + and compounds from soils into water and kitchen utensils, where it is accessible to living organisms: humans, plants and animals. Seeds of the Elit wheat variety were germinated in Petry dishes in solutions of Al2 (SO4 )3 , K2 SO4 , Al(NO3 )3 , KNO3 , AlCl3 , KCl of different concentrations at 25°C for 48 h. Al anaphase and telophase cells were examined in each preparation and the percentage of cells with chromosome aberrations were recoded. The dependence of the yield of aberrant cells on aluminum concentration was nonlinear a followed a curve with maximum at 5·10−4 mg/ml (1 PCL for Russia for potable water) for Al2 (SO4 )3 ; 10−3 mg/ml (2 PCLs) for Al(NO3 )3 or 0,5·10−4 mg/ml for AlCl3 (0,1 PCLs). Aluminum ions induced all types of structural chromosome aberration; chromatid mutations (49,2%) was prevailed. Data on chromosome aberrations after seeds and seedlings with water boiled for 2 h demonstrated an insignificant cytogenetical effect of water boiled in an aluminum but not in an enameled pot. The same results was shown by Manna G.K. and Das R.K. (1972) for bone marrow cells of mice after injected with 0,1 M solutions of aluminum chloride at 1ml per 30 gm of body weight. The real mechanism for the induction of chromatid breaks by Al was not well understood. It is mediated by damage of membrane structure were DNA replication initiation sites are located. Recent research from the medical and toxicological field has indicated that cellular mechanisms of Al toxicity could involve interactions between Al3 + and components of the phosphoinositide signal transduction pathway that has been well characterized in animal cells and is beginning to be understood in plant cells. Perhaps Al will be demonstrated the demonic central role in plants and animals cell signaling systems. 470 CISPLATIN-INDUCED PLATINUM-DNA CROSS-LINKS IN HUMAN OVARIAN ADENOCARCINOMA CELLS (SK-OV-3) A. Movahhed 1 , S. Bahrami 1 , Y. Sanahmadi 2 , Z.S. Bahrami 3 , F.H. Shirazi 1 . 1 Department of Toxicology and Pharmacology, School of Pharmacy, Saheed Beheshti University of Medical Sciences, Tehran, Iran. 2 Day Hospital, Tehran, Iran. 3 Pasture Institute of Iran, Tehran, Iran Cisplatin is one of the most commonly used drugs in cancer chemotherapy. Cisplatin is extensively used in the treatment of different cancers, including lung and ovarian carcinoma. However, the mechanisms of cisplatin-induced cell death are not completely understood. This project has been conducted to try to further elucidate these mechanisms by determining the extent of platinum-DNA crosslinks formed after exposure of SK-OV-3 cells to cisplatin. Cisplatin cytotoxicity was assessed by the clonogenic assay. DNA damage Poster Session P23. Genetic toxicology induced by cisplatin was investigated using the comet assay as a method for the detection of interstrand cross-links. SK-OV-3 cells were grown in DMEM/F-12 media supplemented with 10% FCS. Experiments were carried out during the late logarithmic phase of growth. SK-OV-3 cells were exposed to 15 µg/ml dexamethasone for 24 hours to induce DNA double-strand breaks, followed by exposure to different concentrations of cisplatin in the range of 0.5–100 µg/ml. Cisplatin induced DNA cross-links in a dose-dependent manner. We have found a statistically direct biphasic correlation between the cisplatin dose and the extent of platinum-DNA cross-links. Cisplatin formed a rapid extensive DNA cross-links up to 1 µg/ml (slope=58.51, r2 =1), followed by a slow phase (slope=-5.27, r2 =0.94). This confirms that SK-OV-3 cells DNA contains cisplatin specific binding sites, which saturates at 1 µg/ml. A complete data analysis will be presented at the meeting. This assay may potentially be a simple and efficient method to estimate the extent of DNA cross-links caused by cisplatin or other alkylating agents. 471 COMUTAGENIC EFFECTS OF CALCIUM CHANNEL BLOCKERS IN MICE A.D. Durnev, E.V. Nesterova, S.B. Seredenin. State Zakusov’s Institute of Pharmacology of RAMS, Moscow, Russia Ferguson L.R., Baguley B.C. (1988) and Scheid W. Et al. (1991) were the first to reveal the comutagenic action of calcium channel antagonists in pro- and eukaryotic cells studied in vitro. The present investigation is aimed to evaluate the influence of different calcium channels blockers upon the clastogenic effects of dioxidine, cyclophosphamide, acrylonitril and acrylamide. The experiments were carried out in C57BL/6 and BALB/c male mice aged 1.5 – 2 months. The above strains were opted for mice distinction as to antioxidant system phenotype. Mice of the first strain proved more resistant to prooxidant loads and effects of certain mutagens, although prior testing SOD and catalase were found less active as compared to the other strain. The mutagens were administered intraperitoneally as a single dosage or throughout four days at doses as follows: 100 and 200 mg/kg of dioxidine, 10 mg/kg of cyclophosphamide and acrylonitril, 50 and 100 mg/kg of acrylamide. Calcium channels blockerslacidipine (1,4-dihydropyridine derivative) and verapamil (phenylalkylamide derivative) were administered per orally and intraperitoneally jointly with mutagens. The cytogenetic preparations of bone marrow cells were prepared 24 hrs after the last administration of tested combinations and examined following common procedure. Lacidipine at doses of doses of 0.1 and 1.0 mg/kg diminished the clastogenic effect of dioxidine (200 mg/kg) in C57BL/6 mice. At doses of 5 mg/kg, and especially at 10 mg/kg lacidipine caused statistically significant increase in the clastogenic effect of this mutagen (100 and 200 mg/kg) in mice of both strains. Verapamil at doses ranging 0.1 to 10 mg/kg increased the clastogenic effect of all four mutagens used. Verapamil showed a certain specificity of comutagenic modification in experiments with different mutagens. It was expressed depending on experiment design, i.e. duration and route of administration. The comutagenic activity of verapamil proved more pronounced following repeated administration and in certain test regimens it depended on mice strain. It is of principal concern to emphasize no differences in quantitative parameters of comutagenic effects exerted by tested calcium channel blockers. The yield of cells with chromosomal aberrations rise in 50% to 120% range under lacidipine and verapamil effect independently on used mutagen, and it was shown to vary only with the route of administration. 472 ALKALINE ELUTION TECHNIQUE AND DNA FLUOROMETRIC QUANTIFICATION: MODIFICATIONS, EVALUATION AND STATISTICS M. Goumenou, K. Machera. Laboratory of Pesticides Toxicology, Benaki Phytopathological Institute, Kifissia, Athens, Greece The DNA alkaline elution technique in combination with fluorometric measurement of the eluted DNA, has been shown to be a s127 suitable method for the determination of DNA breaks. Modifications of the alkaline elution method developed by Kohn et al and of the fluorometric method developed by Cesarone et al, targeting to the refinement of accuracy and the reduction of the time of analysis are presented. Different filter type, elution time and process for the extraction of DNA retained in the elution filter were tested. In addition, the influence of the strand number in the DNA molecule and denaturation solution (EDTA/TEA) used, as well as the influence of pH, dye concentration and incubation time, in the SS-DNA quantification under the presence of denaturation solution were studied. The modified methods as concluded from the above studies were applied on standard DNA and sample tissue DNA. The choice of the appropriate estimate for the SSBs evaluation and the possible statistical approach are also discussed. For the evaluation of the variability and the accuracy the concurrent historical control data of our laboratory were also considered. Reduction of the fluorescence intensity from the thermally denaturated DS-DNA was observed. The fluorescence was further reduced when denaturation was performed with EDTA/TEA solution. Regarding the quantification of the SS-DNA in the presence of the EDTA/TEA solution, optimum sensitivity and linearity (R2 >0.98) were observed after neutralization of the denaturated DNA solution at pH 7.0, addition of 1.5 × 10−6 M of Hoechst 33258 and 30 min incubation for the dye – DNA binding. The LOD and LOQ of SS-DNA were 0.10 µg/ml and 0.33 µg/ml respectively. Performing in vivo experiments in rat liver using both negative and positive (MNU) control groups, it is concluded that the most appropriate SSB estimates for the evaluation of SSB are the elution constant (k) and the fraction of the non-eluted DNA. Despite of the relatively high variability of the k values for the positive controls (0.294 ± 0.083, N=21) no overlapping area with the respective negative control distribution (0.037 ± 0.010, N=71) in 95% C.L. was observed. Normal distribution of k values in both cases was observed indicating parametrical statistical approach as the most appropriate one. 473 PROBLEMS OF TOXICOLOGICAL EVALUATION OF FOOD PRODUCTS CONTAINING GENETIC-MODIFIED SOURCES N. Maksudova. Department of General and Radiation Hygiene, Second Tashkent State Medical Institute, Tashkent, Republic of Uzbekistan Investigations were been conducted at Department of Toxicology of Central Scientific-Investigation Laboratory of Second Tashkent State Medical Institute during 2001–2002 with using of GLP ( Good Laboratory Practice) (R.S.813.016.5), Standards of United Expert Council for investigations on Chemistry # 423 – “Accurate toxicological evaluation – classic method”, which have been accepted on March 22, 1996. The object of investigations was genetic-modified Soya of 2001 harvest (Soya been USA). The investigations have been conducted on experimental animals (HANCREL: WIST HAN (GLX: Brl), Total quantity of rats – 66, among them 33 – female, 33 – male and laboratory mice in groups on 3 animals in each groups – Total quantity – 33 mice). The purpose of investigations is to evaluate the chronicle toxicity of effect the genetic-modified Soya on state on substances change for the posterity of experimental animals. These investigations provides with information for both of risks: evaluation of the risk and classification of the risk. On results of investigations no macroscopic changes in necroscopy have been determined and no negative clinical symptoms during the course of investigation have been received, excluding features of obesity in experimental animals in comparison with control group of experimental animals. Statistical reliable increasing of weight and appropriate infringement of lipid change in experimental animals requests more deep investigations of statement the lipid change and changes of classical approach, accepted for toxicological investigations of genetic-modified food products with passage to investigation of genetic code. Bibliography – 8, tables – 2. s128 474 Poster Session P24. Behavioural toxicology COMBINED MICRONUCEUS FORMATION AND DNA CONTENT ABBERATIONS ASSAYED BY LASER SCANNING CYTOMETRY. products and biologically significant criteria in carrying out new cigarette designs aimed at producing safer cigarettes, with a reduced or removed amount of hazardous compounds. E. Luther, M. Lee. CompuCyte Corporation, Cambridge, MA 02139 Automated laser scanning cytometric technology has the ability to precisely quantify the amount of DNA per cell and obtain cell cycle information. This cell cycle information is combined with other well features in an analysis such as the number of cells, the size of the nuclei to determine detrimental effects of test substances on the genome of cells. A more sensitive indicator for genomic damage is the in vitro micronucleus assay, where clastogenic effects of substances are indirectly assayed by quantifying the number of micronuclei in samples. This process has also been automated and is performed simultaneously with the DNA content analysis. CHO cells were grown in microtiter plates, treated with titrations of test substances known to induce micronuclei formation including mitomycin C, etoposide and cis-platinum, either with or without cytochalasin B. Plates were then analyzed on the iCyte imaging cytometer (Compucyte Corporation, Cambridge, MA). The results shown that for the substances tested, at lower dosages there was significant increase in the number of micronuclei over background values. At higher dosages, there were other indicators of genotoxic damage including blockage in the cell cycle, enlargement of nuclei (especially evident with etoposide), fragmentation of nuclei. At the highest dosages, there was evidence of necrosis including substantial cell loss in the wells, along with decreased numbers of micronuclei. Together, these illustrate that for the micronucleus assay to be effective, the cell population must be synthesizing DNA. In cases where the test substance is blocking this synthesis, the micronucleus results are not valid. Having cell cycle information available in this multiple readout assay enables determination of the micronucleus results, as well as other information regarding the test sample. 475 MUTAGENIC POTENTIAL OF SMOKE CIGARETTE COMPOUNDS: AN EVALUATION FROM LITERATURE DATA Angela Martino, Floriana Flamma, Alfredo Nunziata, Cristina Andreoli. Research Department, Eti S.p.A., Rome, Italy P24 Behavioural toxicology 476 CHRONIC ACTIVATION OF CB1 RECEPTORS FAILS TO IMPAIR SENSORIMOTOR GATING IN RODENTS. Marco Bortolato, Mauro Fà, Roberto Frau, Gian Nicola Aru, Marco Orrù, Christian Dessì, Gian Luigi Gessa. Department of Neuroscience “B.B. Brodie”, Center of Excellence “Neurobiology of dependence”, University of Cagliari, Italy According to a growing body of evidence, chronic intake of cannabis preparations entails an overall impairment of cognitive properties and predispose to psychotic disorders. Other clinical reports, however, have shown no clear causal relationship in this respect. Inasmuch as the majority of the reactions elicited by cannabis depend on the action of its main psychoactive ingredient (9 -tetrahydrocannabinol) on CB1 receptors, we tested the outcomes of a chronic exposure to WIN 55212,2, a potent and selective CB1 receptor agonist, on some basic cognitive patterns, such as startle response and informational filtering. In this perspective, we used the behavioural paradigm of prepulse inhibition (PPI) of the startle reflex, one of the most powerful and validated models for the evaluation of sensorimotor gating, whose deficit is typical in psychosis. The test was performed both on Sprague-Dawley rats and in CD1 mice, in order to assess possible inter-species variations. To avoid tolerance effects, animals were treated with a scalar dose of the above drug (1, 1.25 and 1.5 mg/Kg for each of the weeks of the treatment, respectively). At the end of every week, each animal underwent an experimental session consisting in 17 pulse-alone trials of 120 dB, interposed by 30 trials of pulse preceded by 73, 76 and 82 dB prepulse as well as 8 no stimulus trials. Comparisons were drawn with animals treated with vehicle throughout the whole period. WIN produced a reduction in startle magnitude both in rats and mice, plausibly due to both apathy and muscular relaxation caused by the activation of CB1 receptors. Interestingly, no variation in PPI was detected, neither throughout the whole treatment nor after its conclusion. These findings suggest that, contrarily to what commonly indicated, chronic activation of CB1 agonists fails to alter sensorimotor gating and informational filtering, providing the evidence that the way through which cannabis could eventually induce psychosis is not based on the activation of CB1 receptors. Cigarette smoke contains about 4800 substances, 60 of them have been reported to be tumorigenic to several rodent species. The carcinogenic compounds present in cigarette smoke are listed by Hoffmann. In this list there are several polycyclic aromatic hydrocarbons (PAHs) and nitrosamines (NAs). The present study is focused on estimate the mutagenic potential of some compounds belonging to these chemical classes: four 477 DIVERGING TRENDS OF SUBJECTIVE, AUTONOMIC, PAHs (benz(a)anthracene, benzo(a)pyrene, dibenz(a,h)anthracene, AND INFLAMMATORY RESPONSES IN YOUNG ADULTS dibenzo(a,i)pyrene) and four NAs (nitrosodimethylamine, nitrosopyWITH SELF-REPORTED MULTIPLE CHEMICAL rrolidine, nitrosonornicotine, 4-methylnitrosamino-3-pyridyl-1-butanone), SENSITIVITY (SMCS) at the concentration present in the cigarette smoke, by extrapolating the mutagenic activity in Ames test from literature data. E. Kiesswetter, C. van Thriel, M. Schäper, M. Blaszkewicz, To this aim, scientific publications, applying Ames test in TA G. Wiesmüller, A. Seeber. Institute for Occupational Physiology at 98 and TA 100 Salmonella typhimurium strains in presence of the University of Dortmund, Germany microsomial fraction S9, were selected. Young subjects with self-reported multiple chemical sensitivity The papers have been reviewed on the basis of the following criteria: compliance of experimental protocols to the guidelines (sMCS) might be a potential risk group with regard to the mancodified by international organisms (OECD, ICH, etc), publication ifestation of MCS. The aim of the study was to compare sMCS year, journal kind and research center. subjects with controls concerning their psycho-physiological beAs reported in literature, a clear dose-related mutagenic activity haviour in different experimental situations with inhalative organic was found for all compounds in TA100, while nitrosonornicotine solvent exposure. and 4-methylnitrosamino-3-pyridyl-1-butanone did not show any The participants of the experiments were selected by a questionmutagenic effect on TA98 strain. Moreover, the role of metabolic naire on Chemical and General Environmental Sensitivity (CGES) activation in modulating the mutagenic response has been highfrom a total sample of 550 young adults. Four laboratory experiments lighted. In fact, both different animal sources and hepatic inductors were carried out (12 control and 12 sMCS subjects each) with 4 (arochlor, phenobarbital etc.) influenced the magnitude of the effect hours exposures to one of the following solvent concentrations: (E1) of microsomial fraction on the mutagenic response. However, it ethyl benzene, 10 or 100 ppm, (E2) 2-butanone 10 or 190 ppm, is important to point out that the lowest dose employed in the (E3) 2-Ethlyhexanol, 1.5, 10, 20 ppm constant, (E4) 2-Ethlyhexanol, experimental designs, that in some cases did not induce an increase 1.5, 10, 20 ppm variable. Continuous or repeated measurements in the number of revertants, was approximately 1000 times higher were performed during the experiment concerning irritations (ratings than the concentration detected in cigarette smoke. These results of eye and nose irritation, substance P, eye blink rate), autonomic underline the importance to define both a testing strategy for tobacco functions (breathing and heart rate), well-being (annoyance ratings). Poster Session P25. Neurotoxicity In experiment 1 and 2 the breathing rate of sMCS subjects was generally higher. Autonomic responses (breathing and heart rate) were stronger but independent of the exposure condition. Both studies with 2-Ethlyhexanol revealed strong associations between blink rates or substance P and exposure level. However, controls and sMCS subjects did not differ remarkably. The most consistent differences between sMCS and control subjects were elevated ratings of annoyance and irritation. The experiments revealed stronger subjective and autonomic responses in sMCS subjects compared to controls. Corresponding group differences for the biomarkers of chemical irritation were not found. 478 MECHANISMS OF LEAD-MEDIATED LONG-TERM MEMORY IMAPIRMENT IN THE ADULT RAT BRAIN Adrinel Vázquez 1 , Sandra Peòa de Ortiz 1 . 1 Biology Department, University of Puerto Rico, San Juan, Puerto Rico, Box 23360, 00931–23360 The behavioral and cognitive dysfunctions caused by lead (Pb+2 ) are well documented in humans and other vertebrates. However, the molecular mechanisms of Pb+2 -induced neurotoxicity are not well understood. Our studies are aimed at determining the molecular mechanisms of Pb+2 -mediated memory impairment in the adult rat brain. For this purpose, we characterized the effects of multiple intrahippocampal microinfusions of low doses of Pb+2 in adult rats trained in a hippocampal-dependent holeboard task. Results obtained from learning measures showed that while Pb+2 did not significantly impair acquisition of the task, it did reduce long-term memory (LTM). Moreover, our results demonstrated a sigmoidal dose-response curve of variable slope that reached a significant maximum effect near the 1 nmol dose and at plateau immediately higher doses. We next examined the hypothesis that the LTM impairment observed in Pb+2 treated adult rats is due to an interference with learning-induced changes in Ca+2 /phospholipid-dependent protein kinase C (PKC) activity. Our results showed that indeed Pb+2 blocked the learning-induced activation of hippocampal PKC. Pb+2 treated rats showed significantly less PKC translocation after training than controls. Since LTM requires changes in gene expression and new protein synthesis, we propose that the Pb+2 treatment also blocks learning-related gene expression in the brain. Current studies are using cDNA microarrays to answer this question. Overall, our studies are helping us understand the molecular mechanisms of LTM impairment caused by Pb+2 . This work was supported by NIH (S.P.O. grants NIGMS-MBRS SOGGMO 8102–26S1, NINDS-SNRP U54 NS39405, A.V. STAR EPA Fellowship). P25 Neurotoxicity 479 EFFECTS OF COX-2 INHIBITOR ON SPATIAL MEMORY AND EXPRESSION OF CHOLINE ACETYLTRANSFERASE (CHAT) AND VESICULAR ACETYLCHOLINE TRANSPORTER (VACHT) PROTEINS M. Sharifzadeh, S. Khosravani. Dept. Toxicology and Pharmacology, School of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155–6451, Tehran, Iran In this study we investigated the effects of intrahippocampal infusion of celecoxib as a selective cox-2 inhibitor and indomethacin as a non-selective cox inhibitor on spatial memory in morris water maze. Rats were trained for 3 days; each day included two blocks and each block contained 4 trials. Tests were performed 48 h after surgery. intra-hippocampal infusion of indomethacin (0.01, 0.1 and 1 m/rat, bilaterally) did not show any significant effect on memory consolidation but celecoxib (0.02, 0.06, 0.1 and 0.2 m/rat, bilaterally) altered escape latency and traveled distance significantly. The maximum effect was obtained by 0.1 m of celecoxib. furthermore, immunohistochemical studies showed that the celecoxib infusion also reduced the number of labeled vacht- and chat-containing neurons in the hippocampus. These results confirmed that cox-2 is s129 probably involved in spatial memory. moreover, the memory deficit generated by the celecoxib could be partially mediated by the inhibition of choline acetyltransferase (chat) and vesicular acetylcholine transporter (vacht) expression. 480 EFFECTS OF DEVELOPMENTAL CO-EXPOSURE TO METHYLMERCURY AND PCB153 ON BRAIN CHOLINERGIC MUSCARINIC RECEPTORS IN THE RAT T. Coccini, G. Randine, L. Balloni, A.F. Castoldi, L. Manzo. Research Centre, Toxicology Division, IRCCS Maugeri Foundation and University of Pavia, Pavia, Italy Methylmercury (MeHg) and ortho-substituted PCBs (e.g., PCB153) are co-present in widely consumed food (e.g., seafood) and neurodevelopmental alterations have been suggested to ensue following in utero and lactational exposure. Evidence indicates that the cholinergic system (e.g., muscarinic receptors) is affected by both MeHg and PCBs. Therefore, interactions between these contaminants may produce synergic effects resulting in enhanced toxicity. Female rats were orally treated with 1 mg/kg/day MeHg from day 7 of pregnancy (GD7) to day 7 post-partum (PD7) and/or with 20 mg/kg/day PCB153 (GD10-GD16). At PD21 saturation binding studies were carried out on cerebral muscarinic receptors (MRs) in the offspring and in dams for comparison. In the offspring, MeHg and PCB, alone and combined, caused a trend of Bmax changes in cerebellum and cortex similar to that observed in adults. In both adult and immature cortices all treatments resulted in an increased Bmax (20–30% of control). Regarding cerebellum only MeHg augmented the number of MR sites (15% in pups and 57% in dams), whereas Bmax was reduced by 20% and 30% in PCB-treated pups and dams, respectively, and this % decrease was not modified in the co-presence of MeHg. In the hippocampus, MeHg, PCB153 and MeHg+PCB153 enhanced the density of MR in adults by 32%, 15% and 45%, respectively, while the same treatments lowered offspring Bmax by 15%, 50% and 15%, respectively. Both compounds exerted changes in MR density differing upon the rat age and the brain area considered. These effects were detectable 2–4 weeks after cessation of treatments. The mostly marked differences in the trend of Bmax changes between adults and offspring were observed in the hippocampus, a brain area highly involved in learning and memory processes. (Grants: EU QLK4-CT-2001–00186 and Italian Ministry of Health) 481 HUMAN ASTROCYTOMA CELLS UNDERGO APOPTOSIS FOLLOWING STYRENE OXIDE EXPOSURE T. Coccini 1 , A.F. Castoldi 1 , G. Randine 1 , S. Barni 2 , L. Manzo 1,2 . Research Centre, Toxicology Division, IRCCS Maugeri Foundation and University of Pavia, Pavia, Italy Styrene is one of the most important organic chemicals used in occupational setting. Styrene metabolism involves its bioactivation to styrene oxide (SO). Disturbances in neurological function have been reported in styrene-exposed workers. A recent study has demonstrated the occurrence of apoptosis involving caspase activation in neuronal cells exposed in vitro to SO (Brain Res 2002;933:12). In the present study the molecular effects of SO (0.3, 0.5 and 1 mM) were investigated on human D384 astrocytoma cells exposed to the compound for 24, 48 and 72h. These treatments produced a time-dependent decrease in the total cell number. In addition, SO at 0.5 and 1 mM caused cytotoxicity within 24h exposure, while the lowest concentration required 48 h for the effect to become manifest. Apoptosis and, to a minor extent, necrosis contributed to SO-induced cell death as assessed by nuclear morphology (fluorescence microscopy) and by cell ultrastructural changes (transmission electron microscopy). Both caspases-3 and 8 activities were increased before the onset of apoptotic nuclear signs. These results show that SO can trigger the apoptosis of astrocytoma cells via a caspase-dependent pathway, at levels of exposure comparable to those causing neuronal cell death. Activation of these proteases appears to be a critical event in the cascade leading to neuronal and glial damage induced by SO exposure. (EU grant QLK4-CT99–01356). s130 482 Poster Session P25. Neurotoxicity NEUROTOXICITY OF 3-MONOCHLOROPROPANE-1,2-DIOL IN RATS K. Kim, C. Song, Y. Park, S. Koh, J. Kim, S. Kim, Y. Kim, H. Jung, D. Cho, K. Kil. Department of General Toxicology, National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul 122–704, Korea 3-Monochloro-1,2-propanediol (3-MCPD) is a contaminant of acidhydrolyzed vegetable protein. Several reports have suggested that chronic exposure to 3-MCPD could produce neurotoxicity in vitro or neurobehavioral aspects of experimental animals. The present study further explored the in vitro neurotoxic effects of 0.1–100 µM 3-MCPD on PC12 and N18D3 cell lines. In addition, to investigate the effects of repeated ingestions of 3-MCPD on neurobehavioral impairments parameters in rats, motor activity, landing foot splay, and grip strength tests were preformed, following the treatment of 3-MCPD at doses of 10, 20, and 30 mg/kg/day for 11 weeks. We demonstrated that no significant neurotoxic effects in vitro and in neurobehavior were observed in the 3-MCPD-treated rats compared to saline-treated control rats, whereas, acrylamide, used as a positive control, induced significant increases of all neurobehavioral deficit parameters in both male and female rats. On the other hand, body weight gain was significantly decreased in high dose 3-MCPDtreated male rats as well as in acrylamide-treated rats. Taken together, these results suggest that 3-MCPD, at the dose levels of this study, does not produce in vitro neurotoxicity or neuromotor deficits. hydroxytryptamine, 5-HT) and its metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTPL) in the locus coeruleus were studied in rats using the microdialysis method. Repeated administration of tryptophan (50 mg/kg, i.p.) for 3 days caused an increase in the levels of 5-HIAA, but not 5-HT, in the locus coeruleus, while administration of ethanol (1.25 g/kg) had no effect on the levels of 5-HT and its metabolites. Simultaneous administration of twice a day with tryptophan and ethanol for 3 consecutive days produced an increase of 5-HIAA level in the locus coeruleus, but not 5-HTPL level. This may imply that the induced microsomal enzymes (CYP2E1) by repeated administration of ethanol accelerate the metabolism of tryptophan. However, a significant increase in 5HTPL level induced by concurrent administration was not observed. This may be explained by the fact that an increased 5-HIAA affects only aldehyde dehydrogenase and/or CYP2E1, which is capable of metabolizing 5-HIAL to 5-HIAA because the metabolism of 5-HIAL to 5-HTPL is not promoted by brain alcohlol dehydrogenase. In addition, a time lag in the increased 5-HIAA levels between tryptophan alone and tryptophan plus ethanol was not observed. Moreover, teeth-chattering was significantly detected in the tryptophan plus ethanol-treated rats when compared with the tryptophan-treated rats, but not in the saline-treated controls. These results may suggest that the increased levels of 5-HIAA and 5-HTPL in the locus coeruleus induced by tryptophan are potentiated by ethanol, and that these levels are partly responsible for behavioral activation. 485 483 NEUROTOXICITY AND REACTIVITY OF 1,2-DIACETYLBENZENE (1,2-DAB) WITH MOTOR AND CYTOSKELETAL PROTEINS IN RAT SPINAL CORD AND SCIATIC NERVES M.I. Sabri, S.B. Hashemi, P.S. Spencer. Center for Research on Occupational and Environmental Toxicology and Department of Neurology, Oregon Health & Science University, Portland, OR, USA Rats treated with 1,2-DAB, a gamma diketone-like aromatic hydrocarbon, develop in spinal motor neurons giant proximal axonal swellings filled with 10-nm neurofilaments (NF), a prominent early feature of neurodegeneration in amyotrophic lateral sclerosis. The underlying mechanism of NF accumulation in axonal swellings is not understood. This study investigated in vitro and in vivo the effect of 1,2-DAB or 1,3-DAB (a non-neurotoxic isomer) on motor and cytoskeletal proteins in the central and peripheral nervous system of rats. Animals were systemically treated with 20 mg/kg/day 1,2DAB, 1,3-DAB or vehicle for ten days. 1,2-DAB but not 1,3-DAB treated rats showed signs of neurotoxicity featured by blue discoloration and hind limb paralysis. Spinal cord (SC) and sciatic nerves (SN) homogenates were immunoblotted using monoclonal antibodies to kinesin, dynein, NFM and tau. Native protein bands showed 50≥75% reduction of both kinesin and NFM in SN of 1,2-DAB but not 1,3-DAB treated rats. Dynein and tau were reduced ≤50%. In SC, reduction of kinesin, NFM, dynein and tau was also detected. In vitro treatment of SC tissue with 1,2-DAB (1–10 mM) for 30 min at 370 C revealed a concentration-dependent loss of dynein > kinesin > tau. High molecular weight polymers were seen in SC treated with 5mM and 10 mM 1,2-DAB. In summary, 1,2-DAB reacts and depletes motor proteins kinesin and dynein and NFM in SN. A deficit of kinesin may cause blockade of anterograde axonal transport and thereby explains the accumulation of 10-nm NF in the proximal axons of 1,2-DAB treated animals. Supported by NIEHS grants ES10338 and ES11384, and the State of Oregon’s Worker’s Benefit Fund. 484 EFFECT OF REPEATED ADMINISTRATION OF TRYPTOPHAN AND ETHANOL ON 5-HIAA METABOLITE IN THE LOCUS COERULEUS IN RATS K. Hoshi 1 , M. Hayashi 2 , T. Bandoh 1 . 1 Department of Clinical Pharmacology, Hokkaido College of Pharmacy, Otaru, Japan, 2 Chitose City Hospital, Pharmacy, Chitose, Japan The effects of consecutive administration of tryptophan alone or in combination of tryptophan and ethanol on serotonin (5- NEUROLOGICAL AND NEUROPHYSIOLOGICAL FOLLOW-UP ON WORKERS WITH SEVERE CHRONIC EXPOSURE TO TOLUENE P. Urban 1 , E. Lukáš 1 , D. Pelclová 2 , Z. Fenclová 2 , Z. Dlasková 2 . 1 National, Institute of Public Health, Prague, 2 Department of Occupational Medicine, 1st Medical Faculty, Charles University, Prague, Czech Republic Background: Since the 1980s, we have surveyed a group of 58 rotogravure printers with a very high level of exposure to toluene. The mean airborne concentration of toluene in the plant was about 2,000 mg/m3 . The blood concentration of toluene at the end of a working shift ranged from 2–26 mg/l. The group mean of the concentration of hippuric acid in urine was about 33 mmol/l. Most of the workers experienced acoustic pseudohallucinations during the repeated episodes of acute subintoxication from toluene. The plant was closed in 1992. The objective of this study was to describe the current neurological status of the former printers and its development. Subjects: In 2003, we managed to re-examine ten workers from the original group. They underwent a comprehensive clinical and laboratory check-up. This included neurological and neurophysiological examinations, the results of which are presented here. All subjects were men, aged 56±7 yrs, duration of exposure 17±6 yrs, and the mean time elapsed since exposure cessation was 12±3.5 yrs. Results: (1) Psychoorganic syndrome and/or other signs of CNS damage were found in 8 workers; (2) EEG was abnormal in 6 workers; (3) VEP abnormality was found in 3 workers; (4) The mean Bowman Color Confusion Index was 1.36±0.33. (5) Signs of a toxic polyneuropathy were observed in 3 workers. This diagnosis was confirmed by nerve conduction studies in 2 of them. (6) There was no significant change in the health status of the workers when compared with the situation in the 1980s. Conclusions: (1) Symptoms and signs compatible with the diagnosis of a chronic toxic encephalopathy could still be found in 80 % of former rotogravure printers, about twelve years after their removal from severe long-term exposure to toluene. (2) The abnormal findings did not show any significant development over time. This suggests that CNS damage due to toluene may not be fully reversible, but does not seem to be progressive, upon cessation of exposure. (3) The few observed cases of incipient peripheral polyneuropathy were attributable to alcohol abuse rather than toluene exposure. (4) On the basis of the present re-examination, compensation for a persistent occupational disease was recommended for 4 workers. Acknowledgement: The study was supported by grants MSM J13/98 111100002, 111100005, and CEZ:L31/98:23795.001. Poster Session P25. Neurotoxicity 486 NEUROTOXIC EFFECTS OF HEXACHLOROBUTADIENE ON THE YOUNG MALE W/A RAT M.T. Boroushaki 1 , P. Grasso 2 , P.S. Goldfarb 2 . 1 Department of Pharmacology, Ghaem Hospital, Mashhad University Of Medical Sciences, Mashhad, Iran, 2 Molecular Toxicology Group, School Of Biological Sciences, University Of Surrey, Guildford, Surrey, GU2 7XH, UK Hexachlorobutadiene (HCBD), a by-product in the synthesis of perchloroethylene and trichloroethylene and a prominent environmental pollutant, is one of the most nephrotoxic chlorinated-hydrocarbon in rodents. Its organ-specific toxicity is based on a bioactivation mechanism that includes hepatic conjugation with glutathione to produce (penta-chloro, butadienyl)-glutathione (PCBG) and finally to (penta-chloro butadienyl)-cysteine (PCBC), translocation and subsequent enzymatic degradation to toxic metabolites by the enzyme C-S-lyase/GTK/KAT. In this study 28-day old male W/A rats were used. Groups of rats received HCBD 25mg /kg, ip (low dose), for 2, 3, 4 and 7 days and 100mg/kg body weight, ip (high dose), for one and two days only. Control group received corn oil, 0.1ml /kg, ip, Animals were killed, the brain removed, halved, one half fixed in formalin and embedded in paraffin for histopathology and the other half was frozen in dried-ice isopantane for enzyme assay. Sections of 5 µm were prepared and stained with hematoxylin & eosin. Light microscopic examination showed an extended damage in the choroid plexus of lateral and third ventricles in HCBD treated rats, especially in 1-day HCBD (100mg /kg) treated group, compare with control and other groups. In groups treated with low dose of HCBD there is a minor haemorrhage in lateral ventricles with pyknotic and mitotic sells in coroidal cells. GTK specific activity in high dose treated groups was lower, but in low dose treated groups was higher than control group. This study has shown that HCBD is a neurotoxin and choroid plexus in the lateral and third ventricles is the most sensitive organ that is affected. 487 EFFECT OF MEMANTINE ON THE PERMEABILITY OF THE MICE BLOOD-BRAIN BARRIER IN SOMAN POISONING B. Antonijevic 1 , M.P. Stojiljkovic 2 , D. Bokonjic 2 , M. Maksimovic 1 , M. Nedeljkovic 1 . 1 Institute of Toxicological Chemistry, Faculty of Pharmacy, University of Belgrade, 2 National Poison Control Center, Military Medical Academy, Belgrade, Serbia The role of various neurotransmitter systems in the initiation, maintenance and pathological consequences of nerve agent-induced seizures is not totally understood. Three different types of compounds involving three different neurotransmitter systems, anticholinergics, GABA agonists and NMDA antagonists, all have been shown effective in moderating the development of nerve agent-induced seizures and brain damage. The present study addressed the relationship among soman-induced seizure, its effect on the mice blood-brain barrier and protective effect of some drugs on barrier permeability. Evans blue-dye at a dose of 40 mg/kg iv was used for rapid visual assessment of cerebrovascular permeability. The dye was injected by tail vein 5 min before soman (1 LD-50 sc). Midazolam (2.5 mg/kg ip), ketamine (20 mg/kg ip) or memantine (10 mg/kg iv) were injected 5–10 min before soman. Toxicity signs were typical of cholinesterase inhibition and among others, included hypersecretion, hyperactivity, tremors and clonic-tonic cramps. All convulsing animals showed some degree of barrier penetration by Evans blue-dye. Nonconvulsing animals were free of extravasated dye. The degree of brain stain in animals treated prophylactically with memantine (non-competitive NMDA antagonist) or midazolam was significantly lower than in animals pretreated with ketamine. The results of this study provide evidence that soman-induced breach of the mice blood-brain barrier is convulsive dependent. Prevention of seizure associated with soman toxicity by the use of memantine or midazolam prevents barrier leaks. 488 s131 AGE AND GENDER RELATED DIFFERENCES IN NEUROTOXICITY OF AN ORGANOPHOSPHATE PESTICIDE – TRIAZOPHOS IN YOUNG AND ADULT RATS M. Singh 1 , S. Rishi 1 . 1 Department of Veterinary Pharmacology & Toxicology, Chaudhary Charan Singh Harayana Agricultural University, Hisar, India Pesticides are extensively used in agriculture and animal health care programmes. Though indispensable, they are known to cause mammalian toxicity. The cholinesterase inhibiting pesticides which are more widely used are known to affect the young ones more; especially their developing brain is more susceptible to their toxic effects. The present investigation was designed to study the age and gender related neurotoxicological effects of triazophos in young i.e. Post natal days 20 (pnd20) and adult i.e. Post natal days 90 (pnd90) wistar rats and to explore the correlation between neurobehavioral effects and brain and plasma acetycholinesterase (ache) activities. Young rats were found to be more sensitive to triazophos toxicity as compared to adult, which is, reflected in their median lethal dose (ld50) and maximum tolerated dose values. The ld50 values were found to be 19 mg/kg body weight in young and 35 mg/kg body weight in adult rats, whereas mtd in young rats was 16 mg/kg and 30 mg/kg body weight in adult rats through intraperitoneal route. Different doses of triazophos i.e. 40, 62.5, 80 and 100 per cent of mtds given intraperitoneally caused significant alterations in the neurobehavioral parameters. The onset and peak time of neurobehavioral effects was achieved earlier in young rats as compared to adult rats, also severity of effects was more but recovery was faster in young rats. Inhibition of brain and plasma ache activities was earlier and more intense in young rats. However, the recovery of ache activities was earlier in young rats than adults. A very high correlation was found between brain and plasma ache activities during inhibitory and recovery phases both in young and adult rats. Also a good correlation between brain and plasma ache activities and neurobehavioral changes was fond both in young and adult rats. Behavioral testing in conjunction with neuropathology is a comprehensive way of assessing the functional development and integrity of the nervous system when used in a toxicity study. Histopathological studies revealed important changes in brain viz. Congestion, hemorrhages, dilation of virchew’s space, satellitosis and microglial cell proliferation. Changes were more extensive in young as compared to adult rats. The study indicated that young rats were more sensitive to triazophos toxicity than adult rats and the neurobehavioral effects were correlated well with ache inhibition both in brain and plasma of young and adult rats. Further, the plasma ache inhibition was also significantly related to the brain ache inhibition, thus, can be taken as a good biochemical indicator of triazophos toxicity. 489 EARLY FORECASTING OF DELAYED NEUROTOXICITY DEVELOPMENT IN HENS O.A. Khodakovskaya, N.A. Vodolazskaya, L.D. Glukhova, S.I. Timofeeva, V.G. Zoryan, A.S. Polezina, S.I. Dvoretskaya, E.I. Malochkina, I.N. Golubev, V.A. Petrunin. State Research Institute of Organic Chemistry and Technology, Moscow, Russia Many organophosphorus compounds (OP) induce delayed neurotoxicity (OPIDN) in various animals and human after their single or repeated administration. DNT symptoms are observed in a certain period (1–4 weeks) after contact with OP. A problem of physiologic influence of those substances on human is extraordinarily actual because substances of this class are used in economic activity of people. Hens were selected as model animals. They reflect development of polyneuropathy in human the most correctly. In the work, complex assessment of specific effect of two OP - tri-o-cresyl phosphate (TOCP) and triphenyl phosphite (TPP) - has been carried out. For this purpose, neurotoxic esterase (NTE) activity in blood lymphocytes and platelets, cholinesterase (ChE) activity in blood plasma, and structural reconstructions in membranes of formed blood elements were determined with EPR probes after single administration to hens. High degree (more than 80%) of NTE inhibition in lymphocytes and platelets was determined after oral administration of TOCP at s132 Poster Session P25. Neurotoxicity doses of 1.0 and 1.5 g/kg and TPP at a dose of 0.65 g/kg that induced OPIDN development. In 0.5–2 hours after OP administration, cholinergic intoxication was observed in hens. Blood plasma ChE inhibition depended on dose and time after injection of substance. Relationship of NTE activity changes in lymphocytes and cellular membrane parameter changes was determined. Our investigations on NTE activity determination in lymphocytes in 30 min after neurotoxicant administration allow to forecast development of neurotoxic effects in hens. This work has been supported by ISTC project #574. 490 OXIDATIVE STRESS IN THE MECHANISM OF ORGANOPHOSPHATES NEUROTOXICITY V.D. Tonkopii. Laboratory of Toxicology, Institute of Limnology, Russian Academy of Sciences, St.Petersburg, Russia. The acute toxicity of organophosphates (OPs) in mammals is primarily due to their irreversible inhibition of acetylcholinesterase in the nervous system which leads to increased synaptic acetylcholine levels. However, the toxic effect of some OPs is not limited to inhibition of cholinesterase: following the cholinergic crisis changes in non-cholinergic neurotoxic parameters, such as specific damage to cell membranes, are observed. One of the major problems in assessing the role of lipid peroxydation in any chemical toxicity is to resolve whether this pathogenic cascade is a cause or a consequence of damage. The present study was undertaken to elucidate the relations between lipid peroxidation, OPs toxicity and delayed, long lasting, non-cholinergic changes. We studied the influence of OP intoxication on lipid peroxidation in rat cerebral hemispheres. The level of lipid peroxidation was measured as the amount of common phospholipids, peroxidate lipids and malondialdehyde (MDA) in reaction with thiobarbituric acid. The rate of reaction of conditioned reflex of active avoidance was measured. Results were compared to those with pre-treatment with atropine and reversible cholinesterase inhibitor - galanthamine alone or together with different antioxidants (α-tocopherol and oxymetacyl). OPs caused a rapid, dose-dependent increase of peroxidate lipids and MDA 15–30 days after intoxication. The level of lipid peroxidation correlated with the rate of conditioned reflex reaction. With paraoxon and sarin pre-treatment with atropine and galanthamine totally prevents the all symptoms of intoxication and changes in lipid peroxidation. Comparatively such type of prophylaxis in soman and malathion poisoned rats didn’t normalize the biochemical and physiological parameters. The protective effect of antioxidants against soman and malathion - induced lipid peroxidation was shown. Therefore soman and malathion- associated lipid peroxidation is likely to arise mainly as a primary change which may, however, play a significant role in delayed neurotoxicity and conditioned reflex activity. 491 EFFECT OF OPIOD ANTAGONIST NALTREXONE ON NOS IN ALCOHOL TREATED RATS George Z. Dimitrov, Nadka I. Boyadjieva. The opioid antagonist naltrexone is used for the treatment of alcoholism. We have previously shown that alcohol treatments altered the expression of nNOS mRNA, but not iNOS in “in vitro” experiments on cultured neuronal cells. For better understanding the mechanism of alcohol action on hypothalamus, we investigated the effects of opioid antagonist naltrexone on the expression of nNOS in alcohol treated rats. Male rats were treated with naltrexone with or without alcohol for a period of 3 weeks. The expression of nNOS was determined by immunocytochemistry. The results indicated that the longer treatments with alcohol increased the nNOS in the hypothalamus. Naltrexone application for 3 weeks antagonized the chronic effect of alcohol on nNOS. Our results suggest that opioid receptors play a role in the regulation of nNOS in hypothalamus of rats. Moreover the data presented here indicate the possible role of opioid receptors in alcohol regulated expression of nNOS in the brain. 492 STUDY OF QUANTITATIVE ABNORMALITIES AFTER ORAL ADMINISTRATION OF MORPHIN TO BALB C MICE. F. Bahrami 1 , M. Ramezani 2 , M. Lahijani 2 . 1 Department of Physiology and Biophysics, baghiyatollah University of Medical Science. Tehran Iran; 2 Department of Sience, Shaheed Beheshti University Because of their passage through placenta, in addition to their effect in adults opiate have disrupting effects on development of embryos. In this research, teratogenic effect of addiction to morphine (by oral administration) on Balb/C mice embryos, were studied. Therefore, one control(n=6) and seven experimental groups (for every dose, n=121,normal and addicted females during pregnancy, post- pregnancy, and pre and post pregnancy periods with normal and addicted males) were used. Then, three doses of morphine (0.01, 0.05&0.1 mg/ml, 3 weeks with increasing concentration of 0.1, 0.2, 0.3 & 0.4 mg/kg, in drinking water)were administrated as initial doses. After investigating embryos on day 17 of pregnancy, it was found out that addiction to morphine can cause significant decrease in the litter size (in all three doses), (p<0.05), weight of embryo (p<0.001), length of crown-crump (p<0.001, p<0.05), weight (p<0.001, P<0.05) and diameter (p<0.00) of placenta in two doses (0.01, 0.05 mg/kg). The difference in rate of abnormal embryos was also significant in all experimental group (p<0.001) studying the skeletal structures if embryo with abnormal curvature and their forelimbs (after staining with alizarin red and alcian blue) showed extra rib in thorax region an existence of small extra digit in the forelimbs without formation of cartilage or bone.So,1) morphine can create abnormalities in mice embryo,2) teratogenic effects of treatment with morphine during pregnancy is much greater than prepregnancy; also addiction in male have some effects on embryo; and 3) rate of teratogenic effects increase with lower doses of morphine. 493 SINGLE DOSE ORAL SAFETY PHARMACOLOGY STUDY IN THE WISTAR RAT: THE MODIFIED IRWIN’S TEST G. Teuns 1 , B. Verstynen 1 , A. Lampo 1 , W. Coussement 2 . Dept. of Toxicology and Pathology, 1 Drug Evaluation and 2 Full Development, Johnson & Johnson Pharmaceutical R & D, a Division of Janssen Pharmaceutica, Beerse, Belgium In a single dose safety pharmacology study performed according to the Modified Irwin’s Test, chlorpromazine hydrochloride was administered orally via gavage to male SPF Wistar rats (Hannover substrain) at single doses of 5, 20 or 80 mg base eq./kg body weight in order to evaluate the neurofunctional integrity in this animal species. Mortality, behavioural observations in the cage and during manipulation, general clinical observations, body weight and weight gain were evaluated during a 7-day observation period following oral administration. Mortality was absent at all doses tested. Neurofunctional integrity of rats was not affected after a single oral dose of chlorpromazine hydrochloride at 5 mg b.e./kg body weight and there were no adverse general observations noted. body weight and weight gain increased at a normal rate during the 7-day observation period. Dosing at 20 mg b.e./kg b.w. led to behavioural (motor-affective and sensoro-motor responses), neurologic (muscle tone, equilibrium and gait) and autonomic (eyes, hypothermia, respiratory rate) effects, which were considered drug-related. Peak effects were noted after 2 to 4 hours. There were no signs of delayed neurotoxic effects recorded on day 7 post-dosing. General clinical observations were absent during the 1-week observation period. A decrease in weight gain was recorded after 1 week. Dosing at 80 mg b.e./kg b.w. led to behavioural (motor-affective and sensoro-motor responses), neurologic (muscle tone, equilibrium and gait) and autonomic (eyes, secretion and excretion, hypothermia, respiratory rate) effects. Peak effects were noted after 2 to 4 hours. The duration of action lasted up to 24 hours after dosing. There were no signs of delayed neurotoxic effects recorded on day 7. Dosing at 80 mg b.e./kg b.w. led to a bad condition with a wet urogenital region, a crusty nose and chromodacryorrhea, to agitation and to a decrease in body weight and weight gain. Poster Session P25. Neurotoxicity The observed findings are characteristic for chlorpromazine-type major tranquillisers and thus justify the selection of the chosen dose levels. 494 TOXICITY OF LOW DOSE LOCAL ANESTHETIC IS DEMONSTRATED BY REVERSIBLE NEURITE RETRACTION, ROUNDING AND ANNEXIN V STAINING M.E. Johnson, C.B. Uhl. Mayo Clinic Anesthesiology Department; Rochester, Minnesota; USA Introduction: The local anesthetic lidocaine in spinal anesthesia at high concentrations (≥2.5%) can cause neuronal death. However, low concentration lidocaine (≤0.5%) is not toxic in in vitro assays for death, even though 0.5% causes the clinical syndrome of Transient Neurologic Symptoms. This could be because low concentration lidocaine is nontoxic, or causes reversible injury, or causes delayed apoptotic cell death that is not immediately apparent. Morphologic assay of neurons for rounding is an early and sensitive indicator of injury which we have applied to investigate the toxicity of low concentration lidocaine. Methods: ND7 neurons (derived from rat dorsal root ganglion) were exposed at 37 ° C to lidocaine 0.25% x 45 min, then to 120 min recovery without lidocaine. Phase contrast photomicrographs of the same field were acquired every 5 min with a Zeiss Axiovert 135TV inverted microscope and Axiocam digital camera. Neurons were then stained with Annexin V-FITC to detect early commitment to apoptotic cell death, and propidium iodide to detect necrotic and late apoptotic cell death. Results: Rounding was rare prior to lidocaine, and in control neurons exposed to equimolar Tris buffer or to tetrodotoxin. Lidocaine caused rounding (71±17% of neurons in 6 experiments, P<0.001), defined as rounding and shrinkage of the soma, with retraction of neurites and loss of fine structure. Rounding was frequently reversible, decreasing to 17±19% of neurons after 120 min recovery without lidocaine (P<0.01). After 120 min recovery, 29±21% of neurons were committed to apoptosis, vs. 3±6% necrosis (P<0.05). Conclusions: Morphologic rounding is a sensitive assay for reversible neuronal injury from low concentration lidocaine. Most injury is reversible. Irreversible injury is manifest primarily as apoptosis. 495 A NEW IN VITRO MODEL FOR TESTING DEVELOPMENTAL NEUROTOXICITY Ellen Fritsche, Ulrike Hübenthal, Josef Abel. Institut fürUmweltmedizinische Forschung at the Heinrich-Heine University, auf’m Hennekamp 50, 40225 Düsseldorf, Germany It is a common opinion that in vitro models are needed for testing developmental neurotoxicity. Therefore, we established a human cell culture model that consists of normal human cells and allows us to study the impact of chemicals on neural development. Normal human neural progenitor (NHNP) cells (Clonetics™) grow in neurospheres and can be kept in culture for several months. Upon growthfactor withdrawal they differentiate into neurons, astrocytes and oligodendrocytes. This cell system seemed to be an excellent model to study influences of chemicals on differentiation of these cells. Polychlorinated biphenyls (PCBs) are strongly suspected to impair fetal brain development in humans. The effects of PCBs on brain development can be at least partly attributed to endocrine disruption of the thyroid hormone system. This system is known to play an important role in oligodendrocyte differentiation. To test our hypothesis that treatment of neurospheres before differentiation influences their cellular fate, we added 1 µM PCB118 to the medium. After 1 week the cells were plated for differentiation. In addition, we treated neurospheres 1 to 2 weeks with T3, retinoic acid (RA) or T3 & RA. As expected, the T3 treated group developed 6–9 times more oligodendrocytes than the untreated controls, while the RA treated group showed none. Interestingly, cotreatment with T3 & RA abrogated the effect of T3. T3 decreased the percentages of neurons, whereas RA and T3 & RA had no significant influences on neuronal cell numbers. PCB exhibited similar effects on neural s133 differentiation than T3. The number of oligodendrocytes increased 10 fold over the vehicle controls. PCB also reduced the percentage of neurons (10%). Our preliminary findings support the theory that neurotoxic effects of PCBs are mediated through thyroid hormone disruption. In summary, this is the first time showing an effect of low dose PCB118 on oligodendrocyte differentiation in NHNP cells. 496 VALUES OF PERIPHERAL BLOOD LYMPHOCYTE MUSCARINIC RECEPTORS AND PLATELET MONOAMINE OXIDASE B ACTIVITY IN HEALTHY HUMANS T. Coccini, A.F. Castoldi, G. Randine, L. Balloni, L. Manzo. Research Centre, Toxicology Division, IRCCS Maugeri Foundation and University of Pavia, Pavia, Italy The lymphocyte cholinergic muscarinic receptors (MR) and the platelet enzyme monoamine oxidase-b (MAO) activity have been used as peripheral markers of neurotoxicity (e.g., alcohol abuse, exposure to mercury, organophosphate pesticides, styrene) and neurodegenerative and neuropsychiatric diseases. The applicability of such markers to humans requires the availability, for each parameter under study, of data defining a physiological range of values in healthy subjects. In this study platelet MAO activity and lymphocyte MR binding were measured in peripheral blood samples of 138 and 155 healthy blood donors, respectively. The control group for MAO activity included 95 men (mean age±SD 42±13 years) and 43 women (mean age 35±12 years). Women displayed a significantly higher enzyme activity than men, as the mean platelet MAO activity was 12.4±5.3 nmol/mg protein/h (range 3.1–25.8) in the first group, and 9.0±5.2 nmol/mg protein/h (range 1.9–31.9) in the latter group. The mean value of lymphocyte MR binding did not significantly differ between males (n=86; mean age 43±10 years) and females (n=69; mean age 37±10). In men it averaged 12.2±9.9 fmol/106 cells (range 1.0–37.9) and in women 10.7±9.7 fmol/106 cells (range 1.1–39.7). Altogether these data indicate (i) wide inter-individual differences in the values of platelet MAO activity and lymphocyte MR binding in healthy subjects; (ii) gender-related differences in MAO activity, but not in MR binding. The application of these assays to a cohort of 7 year-old Faroese children exposed to methylmercury and PCBs through the diet has been recently undertaken to assess whether these peripheral markers are altered by environmental exposure to these neurotoxicants. (Supported by the European Union, grant QLK4-CT-2001–00186, and by the Italian Ministry of Health). 497 EARLY DIAGNOSTICS OF DELAYED NEUROTOXICITY O.A. Khodakovskaya, N.A. Vodolazskaya, L.D. Glukhova, S.I. Timofeeva, A.S. Polezina, S.I. Dvoretskaya, E.I. Malochkina, I.N. Golubev. State Research Institute of Organic Chemistry and Technology, Moscow, Russia Complex assessment of specific effect for three organophosphorus compounds (OPC), 0,0-diisopropyl phosphorofluoridate (DFP), tri-ocresyl phosphate (TOCP), and triphenyl phosphite (TPPi), inducing delayed neurotoxicity (DNT) after a single injection to hens has been carried out. Activity of neurotoxic esterase enzyme (NTE) in blood lymphocytes and platelets, and brain, cholesterase (ChE) activity in blood plasma were determined. Intracellular calcium content in lymphocytes was determined by a fluorescent method. Structure rearrangements in membranes of blood formed elements were investigated with ESR probes. NTE activity in lymphocytes and platelets was inhibited by 100% in 30 minutes after sublingual administration of TPPi in sunflower oil at a dose of 0.65 g/kg and by 60% in 2 hours after administration of 0.5 g/kg. The most dose induced DNT. After single subcutaneous injection of DFP at doses of 0.4, 0.7, 1.0, and 1.5 mg/kg, NTE inhibition by 83.61± 1.22% in lymphocytes and platelets was noted already after 15 minutes at a lose of 0.4 mg/kg. Dose increasing to 1.0–1.5 mg/kg resulted in NTE inhibition by 95–99%. Calcium content in lymphocytes in 3 hours after administration of DFP at a dose of 1.0 mg/kg was lower s134 Poster Session P25. Neurotoxicity as compared with control, and in a day there was its considerable growth. Cholinergic intoxication was observed in 0.5–2 hours after OPC injection as well as inhibition of blood plasma ChE. ChE inhibition depended not only on injected dose but also on the itme after injection of the substance: inhibition of the enzyme increased by increasing the quantity of injected OPC and later restoration was observed. High degree of NTE inhibition was determined in lymphocytes and platelets after sublingual administration of TOCP at a dose of 1.5 g/kg: after an hour - 92.2±0.9%, after 2 hours - reduction (by 5%) as compared with 1 hour, and at a dose of 1,0 g/kg after 2 hours inhibition was 82±3.7%. Inhibition of blood plasma ChE also depends on dose and circulation time of substance in the body. Simultaneously with determination of NTE inhibition degree in lymphocytes and platelets, investigation of structural state of bood cell membranes was carried out with EPR proves - stable nitroxide radicals based on stearic acid - 5’and 16’ - doxyl derivatives controlling structural rearrangements in membrane region at a depth of 5–6Å and 20–22Å respectively. Effect of OPC under investigation was in breach of membrane structures in vital blood cells and accompanied by changes of their physicochemical properties. Interrelation of NYE enzyme activity changes in lymphocytes and cell membrane parameters was determined. Our investigations on determination of NTE activity in lymphocytes soon after injection of neurotoxicants (in 15–30 minutes) enable to forecast progress of delayed neurotoxic effects. This work has been supported by ISTC project #574. 498 IMPROVEMENTS IN ECG RECORDING IN CONSCIOUS PRIMATES J. Derrick, S. Laycock, D. Gallacher, K. Semple. Quintiles Ltd, Research Avenue South, Heriot Watt University Research Park, Riccarton, Edinburgh, EH14 4AP, UK Measurement of ECGs in preclinical studies and in particular the measurement of QT interval is of significant regulatory interest at present. A new ICH guideline is expected to introduced within the next year requiring an assessment of the effects of compounds on QT interval before first in man studies. ECGs are normally measured either as part of a regulatory toxicology study or as a safety pharmacology study where animals may be instrumented with telemetry devices. We have refined procedures for dosing and recording which enhance the quality of the data recorded in primate studies and thereby improve the power of detection of drug induced effects. During the oral dosing procedure heart rate in the primate can increase dramatically thereby affecting ECG and shortening QT interval. Whilst corrections can be made for changes in rate these are often imperfect and can affect the ability to detect an effect on QT interval. Acclimatising of cynomolgus monkeys to oral gavage dosing reduces the magnitude and duration of the dose administration-induced tachycardia. This is associated with reduced apparent stress of the animal. In telemetry studies the ECG signal quality can be poor in telemetered cynomolgus monkeys where the animals are freely moving around the cage. Reduced signal quality causes a reduction in power of detection and requires significantly longer time to be spent on analysis. We have developed a modified cage environment that markedly improves signal quality without restriction of movement of the animal. 499 INSIGHT INTO THE CAUSE AND TREATMENT OF ALZHEIMER’S DISEASE FROM A TRANSRANSGENIC MOUSE MODEL. N. Omidi 1 , P. Pasbakhsh 2 , D. German 3 . 1 Iran university of Medical Science, Dept Anatomy; 2 Tehran university of medical science,Dept Anatomy Tehran,Iran -3 The university of Texas Southwestern, Dept Psychiatry Alzheimer’s disease (AD) is a uniquely human disorder. Although the Pathogenesis of Alzhemier’s disease (AD) is not fully understood. Growing evidence indicates that the deposition of beta-amyloid (A beta) and the local reactions of Various cell types of this Protein play major roles in The development of the disease.In the Present study transgenic mice expressing mutant amyloid Precursor proteins (APPs) has been used. These mice exhibit selective neuronal death in the brain regions that are most affected in AD, suggesting that amyloid plaque formation is directly involved in AD neurons loss. Brains form 24 transgenic animals and 24 age - matched non transgenic Littermate controls (2,4 months and 1,2 years old) were examined histoPathologically. Between 2–4 months of age (n-6), no obvious Pathology was detected, however, at 4 months to 2 years of age (n=18) transgenic animals began to exhibit deposist of human AB in the hippocampus, corus collosum and cerebral cortex. These increased with age. By 2 years old many deposits (30–200µm) were seen. In the cortex amyloid plaques were associated with intense acetylcholinesterase activity and fibers surrounded by dystrophic acetylcholinesterase positive.. The major finding was reduced of cholinergic cells in the medial septum, striatum and diagonal band of Broka.These results demonstrate that over expression of APP caused, besides amyloid plaques in aged mouse brain, also are cholinergic deafferentation and cholinergic cell shrinkage. 500 AGGREGATIONS OF AMYLOID BETA-PROTEINS IN THE PRESENCE OF METAL IONS K. Yano 1 , N. Hirosawa 2 , Y. Sakamoto 2 , H. Katayama 3 , T. Moriguchi 1 . 1 Department of Chemistry, 2 Department of Biomedical Research Center, 3 Saitama Medical College, Saitama Medical School, Moroyama, Saitama, Japan Development of amyloid β-protein aggregates in the brain is the main pathological feature of Alzheimer’s disease (AD). Their major constituents are the 40 and 42 amino acid fragments [ Aβ(1–40) and Aβ(1–42)] of the beta-amyloid precursor protein. Certain metals have been proposed as risk factors for AD. Their mechanisms, however, are still unknown. Thus, we investigated the aggregation of Aβ(1–40), Aβ(1–42), and Aβ(42–1) (as a reference) in the presence of metal ions (Al3 + , Ca2 + , Cd2 + , Co2 + , Cu2 + , Fe2+ , Fe3 + , Hg2 + , Mg2 + , Mn2 + , Ni2 + , Pb2 + , Sn2 + , and Zn2 + ) by fluorometry and Fourier transform infrared spectroscopy. A mixture of an amyloid protein (2.5µg) and a metal ion (10µmol) in 20µl of HEPES buffer solution (pH 7.4) was incubated at 37° for 0, 2, 6, and 24 h. From the reaction mixture, each 5µl was placed onto a slide glass and dried on air. After washing with water and drying, the spots were treated with 0.1% thioflavin-S and measured by a chromato scanner with an excitation light at 365 nm and an emission filter of 460 nm. For infrared spectral study, each 1µl of the mixture was placed onto a CaF2 plate, and the dried spots were measured with an infrared microscope. We found the aggregations of Aβ(1–42) in the water (blank) as well as in the presence of Sn2 + and Al3 + . With Aβ(1–40), the aggregations were observed only in the presence of Hg2 + and Al3 + . No significant aggregations of Aβ(42–1) were observed in the present experimental conditions. The present results suggest that metal ions have both promotion and inhibition effects on the aggregation of Aβ proteins depending on the kinds of amyloid proteins and metal ions at each step of amyloidogenesis. 501 OXIDATIVE STRESS AS AN EXPERIMENTAL MODEL OF DEPRESSION: EFFECTS OF DUAL-ACTIVE ANTIDEPRESSANTS M. Varadinova, D. Drenska, N. Boyadjieva. Department of Pharmacology and Toxicology, Medical University, Sofia, Bulgaria It is well-known that the optimizing of antidepressant pharmacology plays an important role in the treatment of depressions. The dualacting antidepressants have been proposed. The mechanisms of antidepressant activity have been an objective in our studies. The aim of this study is to determine the effect of milnacipran on a new model of depression. Male rats were used. An experimental model of oxidative stress was developed and parameters were determined. Depressive symptoms were evaluated in animals with oxidative stress by various tests (forced-swimming, behavioural tests,etc.) Acute and chronic effects of milnacipran on depressive symptoms were tested. The results demonstrated the dose response of anti-depressive action of milnacipran. Male rats showed changes in the indications in the Poster Session P26. Heavy metals tests mentioned above. Our data suggest that oxidative stress plays an important role in depression. Moreover, for the first time in literature our results present that dual acting antidepressants inhibit both oxidative stress and depressive symptoms. 502 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ANALYSIS OF NITRITE AND NITRATE IN RAT BRAIN HOMOGENATE M. Curcic 1 , M. Djukic 1 , B. Antonijevic 1 , D. Djukic 1 . Institute of Toxicological Chemistry, Faculty of Pharmacy, Belgrade, Serbia and Montenegro Nitrite and nitrate are detectable metabolites of reactive nitrogen species. It is well known that paraquat application induced production of reactive nitrogen species via the process of enzyme activation. Therefore, activation of neuronal nitric oxide synthase is crucial event for the production of nitrate and nitrite as final products of nitric oxide metabolism. Rat brain homogenates were kept at -20°C. After the precipitation of proteins, samples were filtered and injected directly into the HPLC system: HPLC pump LKB Bromma 2150, sample loop 50 µL, UVdioda array detector LKB Bromma 2152 (214 nm). Chromatographic separation was carried out on a anion column with precolumn IC-PACK™, Anion Guard-PACK™ at room temperature. Eluent solution consisted of borate buffer/gluconate concentric, methanol, acetonitrile and water (2:12:12:74, V/V/V/V). Flow rate of the mobile phase was 1.5 ml/min. Retention times of nitrite and nitrate were 4.72 and 6.78 min., respectively. Standard curves of nitrite (16–200 nmol/mL) and nitrate (12–160 nmol/mL) were linear in these ranges (r2 =0.9989 and 0.9776, respectively). The autors of previous studies used a spectrometrical method, with Griess reagent. The method did not allow an exact differentiation of nitite and biogenic amines that are physiologically present in plasma. We have developed a sensitive and cost-effective method for direct determination of nitrite and nitrate using high performance liquid chromatography. Statistical analysis showed that presented HPLC method could be used instead of Griess spectrophotometric method for nitrite and nitrate determination in brain homogenates. 503 ETHANOL-EXTRACTED PROPOLIS ATTENUATES KAINATE-INDUCED NEUROTOXICITY VIA ADENOSINE A1 RECEPTOR IN THE RAT E.J. Shin 1 , Y.S. Choi 2 , H. Lim 2 , W.K. Jhoo 1 , M.A. Cheon 1 , S.H. Lee 1 , K.S. Kang 1 , H.C. Kim 1 , M.S. Kwon 2 . 1 Neurotoxicology Program, Section of Pharmacology. & Toxicology, College of Pharmacy, Kangwon National University & Korea Institute Of Drug Abuse, Chunchon 200–701, Korea; 2 Dept of Immunopharmacology, Faculty of Veterinary Medicine, Kangwon National University & Korea Institute of Propolis, Chunchon 200–701, Korea It is well recognized that kainic acid (KA)-induced neurotoxicity is mediated, at least in part, by oxidative stresses. Because some antioxidants block KA-induced neurotoxicity, we examined the effect of antioxidant propolis on KA-induced neurotoxicity in rats. Sprague-Dawley rats received ethanol-extracted propolis (EEP) (50, 100, 200mg/kg, p.o.) five times at twelve times intervals. KA (10mg/kg, i.p.) was injected 1 hour after last propolis treatment. Convulsing behaviors were significantly decreased in the seizing rat that pretreated with EEP in a dose-dependent manner. Malondialdehyde (MDA), protein carbonyl (CO) and glutathione (GSH) levels of rat hippocampus were examined as oxidative stress markers. MDA and CO levels were significantly increased 4 hours after KA administration. GSH levels were reduced by KA administration. Pretreatment with EEP blocked these changes in a dose-dependent manner. These neuroprotective effects of propolis were counteracted by adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine [CPT (1mg/kg, i.p.)] and adenosine A2 receptor antagonist, 3,7-dimethyl1-propargylxanthine [DMPX (1mg/kg, i.p.)]. However, CPT was more efficacious than DMPX in counteracting protective effect of propolis. Consequently, these results suggest that antioxidant properties of propolis on KA-induced neurotoxicity are mainly via s135 adenosine A1 receptor [This study was supported by BK21 project and a grant (# HMP-98-N-2–0013) of the Good Health Research and Development Project (1998) of Ministry of Health and Welfare Republic of Korea.]. P26 Heavy metals 504 INCREASED CADMIUM ABSORPTION IN ADOLESCENT GIRLS WITH LOW BODY IRON STORES E. Bárány 1 , I.A. Bergdahl 2 , L.-E. Bratteby 3 , T. Lundh 4 , G. Samuelson 3,5 , S. Skerfving 4 , A. Oskarsson 1 . 1 Dept. of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Uppsala, Sweden, 1 Occupational Medicine, Umeå University, Umeå, Sweden, 3 Clinical Physiology, University of Uppsala, Uppsala, Sweden, 4 Dept. of Occupational and Environmental Medicine, University Hospital, Lund, Sweden, 5 Dept. of Nursing, University Trollhättan Uddevalla, Vänersborg, Sweden In the gastro-intestinal tract (GI), cadmium uptake may be influenced by iron status. When the iron absorption is upregulated, as in iron deficiency, cadmium absorption may increase. In a longitudinal study, cadmium in blood (B-Cd) was analysed in addition to serum ferritin (S-fer) and soluble transferrin receptor in serum (sTfR), which are iron status indicators. The study population consisted of 234 Swedish boys and girls examined at ages 15 (15-y) and 17-y. B-Cd and S-fer correlated inversely (rs=-0.32, p<0.0005), and as expected there were positive correlations between B-Cd and sTfR (rs=0.30, p<0.0005), and between B-Cd and sTfR/S-fer (rs=0.37, p<0.0005) in nonsmoking girls at both ages. No such correlations were found in nonsmoking or smoking boys. Iron deficiency and low body iron stores were also more common among the girls. In smoking 15-y girls, B-Cd correlated inversely with S-fer (rs=-0.44, p=0.021). No other correlations were found in smoking girls. The results indicate an increased uptake of cadmium due to low iron stores in adolescent girls. This was most prominent in nonsmokers, as they are exposed to cadmium via food with absorption in the GI. Smokers are exposed to cadmium primarily through smoking and the subsequent absorption is unrelated to iron status. However, in the present study a relationship was found in 15-y smoking girls, probably because they had only been smokers for a limited period of time and their B-Cd was still significantly influenced by cadmium absorbed in the GI. 505 COMBINED EARLY TREATMENT WITH DMSA AND DTPA TO MOBILIZE CADMIUM IN RATS M. Blanuša, M. Matek Sarić, D. Jureša, M. Šarić, V.M. Varnai, K. Kostial. Mineral Metabolism Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia The influence of chelating agents: meso-2,3-dimercaptosuccinic acid (DMSA); calcium trisodium diethylenetriaminepentaacetate (DTPA) and their combination on tissue retention and distribution of cadmium (Cd) was compared in female albino rats. Special attention was given to time of chelators application after cadmium administration. After oral cadmium intubation chelators were applied either orally (DMSA) or intraperitoneally (DTPA) in various short time intervals after cadmium. The dose of cadmium chloride was 0,25 mmol/kg body weight and chelators dose was 1 mmol/kg, each. Three experiments were carried out with four treatment groups in each of them: 1) Cd (control); 2) Cd + DMSA; 3) Cd + DTPA; 4) Cd + DMSA + DTPA. Time intervals for chelator application after cadmium administration were: immediately in the first, half an hour in the second and one hour in the third experiment. Cadmium, iron, copper and zinc were measured in 24-hour urine collected after chelators application and in organs (liver, kidney and brain) at the end of each experiment. Results showed that the efficiency of cadmium removal from the body is lower when the time of chelator application is longer after cadmium administration. The two chelators differ in efficiency in mobilizing cadmium, with DMSA being more efficient than DTPA. The combined therapies of two chelators give slightly s136 Poster Session P26. Heavy metals better results of cadmium chelation. It seems that DMSA that is given orally after oral cadmium removes this element very efficiently from the gastrointestinal tract. However, DTPA which is given parenterally removes absorbed cadmium very modestly. Whenever DTPA was given to animals zinc concentration is significantly higher in kidneys and much higher in urine than in other groups. Iron and copper do not change dramatically after chelation treatment. 506 PROTECTIVE EFFECT OF IP ADMINISTERED OLIVE OIL IN ACUTE CADMIUM INTOXICATION V. Eybl, D. Kotyzova, J. Koutensky. Charles University Faculty of Medicine in Pilsen, Czech Republic Oxidative tissue damage is involved as an important factor in cadmium (Cd) toxicity and carcinogenesis. Several years ago we have demonstrated a protective effect of olive oil in acute cadmium intoxication. Its components appear to be potent antioxidants. Therefore in present experiments we have focused on the influence of olive oil on oxidative tissue damage and changes in cadmium and trace elements tissue level in Cd intoxication. Male CD mice (Anlab Prague, 25–28g b.w.) were injected sc with a single dose of cadmium chloride (0.03 mmol/kg). Commercially available extra virgin olive oil was administered ip at the dose 0.005 ml/g b.w. 24 hours before cadmium administration. The experiment was finished 24 hours after cadmium administration. Lipid peroxidation in the liver homogenates, expressed as malondialdehyde production, increased due to Cd intoxication by 31% (p<0.01) and was prevented by olive oil pretreatment. The level of GSH and catalase activity decreased due to Cd administration. Both parameters were significantly corrected (p<0.05) by olive oil pretreatment. The activity of GSH-Px was induced by Cd administration to 112% (p<0.05), no effect of olive oil was seen. Olive oil increased the level of cadmium in the liver and significantly decreased cadmium concentration in kidneys, brain and testes. The changes in essential elements level caused by Cd administration in the liver (increased zinc level) and in the testes (increased calcium and iron level, decreased magnesium and zinc level) remained unaffected by olive oil pre-treatment. In further experiment also the protective effect of olive oil in acute cadmium toxicity in male mice was approved. Experiments on orally administered olive oil in cadmium intoxication are in progress. Supported by the grant GAÈR 305/02/1231 507 CADMIUM REDUCTION OF β-CASEIN AND α-LACTALBUMIN mRNA EXPRESSION IN THE MOUSE MAMMARY TISSUE A. Oskarsson, M. Yoshioka, J. Tallkvist. Department of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Uppsala, Sweden Cadmium (Cd) is retained in the mammary tissue in rodents. The transfer of cadmium via milk is low, there is however a possibility that cadmium may exert adverse effects in the suckling offspring by affecting milk synthesis and secretion. We have recently investigated the effects of cadmium on fatty acid composition in milk (see abstract by Petersson Grawé et al) and are now reporting on cadmium effects in the mammary gland on the gene expression of milk proteins. Pregnant mice were injected subcutaneously with CdCl2 (3 mg Cd/kg body weight/day) or only vehicle as controls on three consecutive days on gestational day (GD) 14–16. RNA was prepared from the mammary glands at GD 17 and also from mammary glands of virgin mice and mice at lactation day (LD) 14 with or without cabergoline treatment. Gene expression of β-casein and α-lactalbumin was determined by real-time RT-PCR. Cadmium reduced the β-casein mRNA expression by approximately 40% as determined at GD 17. Also the expression of α-lactalbumin mRNA was reduced, although the effect was less prominent than for β-casein. The gene expression of β-casein and α-lactalbumin increased during proliferation and differentiation of the mammary gland. The expression was higher for β-casein and the increase between virgin and GD 17 was also higher for β-casein than α-lactalbumin, which had a higher increase between GD 17 and LD 14 than β-casein. Cabergoline reduced the gene expression at LD 14 for both milk proteins, but to a higher extent for β-casein. The results demonstrate that the gene expression of milk proteins can be determined by this method and the preliminary results indicate that cadmium has an effect in the mammary gland on milk synthesis. 508 FATTY ACID COMPOSITION IN MILK OF CADMIUM EXPOSED LACTATING RATS AND IN BRAIN OF THE SUCKLING OFFSPRING K. Petersson Grawé 1 , J. Pickova 2 , P.C. Dutta 2 , A. Oskarsson 1 . 1 Department of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Uppsala, Sweden, 2 Department of Food Science, Swedish University of Agricultural Sciences, Uppsala, Sweden In previous studies we have reported neurochemical and neurobehavioural effects in offspring exposed to low cadmium (Cd) levels in milk during the suckling period. Cd is retained in the mammary tissue and there may be an effect of cadmium on milk synthesis and secretion. In the present study we have investigated the effects of Cd on fatty acid composition in milk and offspring brain. Fatty acid composition was studied in rat milk at day 14 after parturition of lactating rats exposed to 0, 5 or 25 mg Cd/l via drinking water during lactation, and in the brain of their offspring at day 19 after birth. Significantly higher proportions of 16:0 and lower proportions of medium-chain fatty acids, 8:0–14:0, were observed in milk of dams in the high dose group, indicating decreased activity of thioesterase II, which is an enzyme unique to the mammary epithelial cells. There were no treatment related alterations in body weights of the pups, thus the reduced levels of medium-chain fatty acids in milk did not result in general adverse nutritional effects in the offspring. Slightly increased levels of 20:3(n-6), the fatty acid preceding arachidonic acid in the n-6 pathway, were observed in brains of pups from Cd exposed dams, compared to controls. The levels of arachidonic and docosahexaenoic acids, which are long-chain polyunsaturated fatty acids crucial for normal development of the CNS, were not altered. 509 GENOTOXIC EFFECTS OF CADMIUM CHLORIDE IN V79 CELL CULTURE Vilena Kašuba 1 , Ružica Rozgaj 1 , Ivančica Trošić 2 . 1 Mutagenesis Unit, 2 Radiobiology Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia Cadmium chloride was tested for the ability to induce genotoxic effects in V79 cell culture. DNA damage induced by different doses of cadmium chloride was observed through the frequency of micronuclei in cytochalasin B-blocked micronucleus assay and through DNA migration in the single cell gel electrophoresis (comet assay). Cells were treated with different doses of cadmium chloride (10−4 to 10−6 M) 24 hours after seeding. The effects of ascorbic acid (conc. 100 uM) on cadmium-induced DNA damage was also evaluated. The results were analyzed by one-way ANOVA. The cytochalasin B-blocked micronucleus assay showed that 10−4 to 10−6 M and vitamin C supplemented 10−6 M frequencies of MN significantly differed from the control sample, and that 10− 4 , 10−5 M and vitamin C supplemented 10−5 M samples were significantly differed from the vitamin C sample. The Comet assay showed that the tail lengths from un-supplemented and vitamin C supplemented 10−4 M sample significantly differed from the control sample; and that 10−5 M and vitamin C supplemented 10−4 M sample significantly differed from vitamin C sample. Tail moments from unsupplemented 10−4 M and vitamin C supplemented 10−6 M samples significantly differed from the vitamin C sample. Results indicate that cadmium chloride increases DNA damages as well as frequency of micronuclei. On the other hand, protective role of vitamin C was not confirmed at all tested concentrations of cadmium chloride. Poster Session P26. Heavy metals 510 BLOOD Cd-CONCENTRATION IN DAIRY CATTLE LIVING IN DIFFERENT AREA OF Cd-POLLUTION Micaela Sgorbini, Rosalba Tognetti, Marco Bizzeti, Michele Corazza. Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, Viale delle Piagge 2, 26100 Pisa, Italy Cadmium (Cd) is an important environmental pollutant in industrialised countries and it is becoming an even more widely used metal in industry for its technologically advanced properties (Elinder et al, 1981). Retention of Cd in small quantities has been demonstrated in all animal tissues, but about 50 to 75 percent of the body burden of cadmium is stored in the liver and kidneys (Lind et al., 1997). Its half-life in the body is not known exactly and it may be as long as 30 years (Lind et al. 1997; Venugal and Luckey, 1979) and there is progressive accumulation in the soft tissues, particularly in kidneys. The slow excretion and prolonged retention in tissues suggest that no homeostatic mechanism exists for cadmium (Venugal and Luckey, 1979). The aim of this study is to evaluate blood Cd-concentration in healthy dairy cattle (established by clinical examen and haematological and biochemical analysis) who live in different Cd-polluted area and to verify the possibility of using these animals as bioindicators of Cd-pollution. Materials and methods: Blood samples were obtained from 37 dairy cattle (aged between 5 and 12 years) living in three different areas of Leghorn province (Italy). These areas have been choosen in relation with the degree of cadmium-pollution and have been divided in low (group 1: 6), medium (group 2: 18) and high (group 3: 13) degree cadmium-polluted area (Scerbo et al., 1999) Blood samples for Cd evaluation have been collected from the caudal vein in lithium-heparinized vacuum tubes (Vacutainer® ) and stored at -20°C. Blood cadmium concentrations have been evaluated by electrothermal atomic absorption spectrometry after a high pressure microwave assisted oxidating digestion of the organic matrix of the samples (atomic absorption spectrometer 4100ZL with integrated platform pyrolitic graphite electrothermal furnace and A-70 autosampler - Perkin Elmer, USA). The background compensation was made with a longitudinal Zeeman effect corrector (Tsalev et al., 1995, 1996a-b; Slaveykova et al., 1997). Statistical analysis: Mean and standard error (SE) have been calculated for each group; t Student test for unpaired data has also been calculated. Different between groups has been considered statistical significative for p<0.05 and high significative for p<0.01. Results: Blood Cd concentrations ranged respectively: from 0.3 to 1.7 ppb (µg/Kg) (mean: 0.82; SE: 0.23) in low degree Cd-polluted area; from 0.1 to 2.3 ppb (mean; 0.83; SE: 0.12) in medium Cdpolluted area; from 6.00 to 568.00 ppb (mean: 131.54; SE: 43.03) in high Cd-polluted area. There is no differences in blood Cd-concentration between group 1 and 2 (p<0.95); there is an high significative difference between group 2 and 3 (p<0.01) and between 1 and 3 (p<0.01). Discussion and conclusions: Our results confirm that the relation between environmental exposure to Cd and blood concentration is evident especially in high risk areas for environmental Cd-pollution (group 3). The metal presence, then, could be an early indication of a real risk of buildup in animals living in highly industrialized surroundings. As an outcome, it could be also hypothesized the possibility of a cadmium buildup and carry from productive subjects to man, by way of the food chain (meat and milk). Furthermore it could be an opportunity to use subjects in animal husbandry as biomonitors for cadmium exposition alert in zones at environmental risk. Acknowledgments: The authors thank Mr M. Mascherpa and dott. Leonardo Lampugnani (National Research Council -CNR) for his skillful technical assistance in the cadmium analysis. 511 s137 METALLOTHIONEIN EXPRESSION IN THE MAMMARY GLANDS OF MICE – EFFECTS OF CADMIUM J. Tallkvist, M. Yoshioka, A. Oskarsson. Department of Pharmacology and Toxicology, Swedish University of Agricultural Sciences, Uppsala, Sweden Previous studies have shown that cadmium is retained in the mammary glands of rodents and that the transfer of cadmium via milk is low. The present study was undertaken to investigate whether this may be related to an onset of metallothionein (MT) synthesis in the mammary glands. Pregnant mice were injected subcutaneously with CdCl2 (3 mg Cd/kg body weight/day) for 3 consecutive days on gestational days (GD) 14–16. Mammary glands were dissected at GD 17 and either prepared for MT-immunohistochemistry or quantification of MT-I and MT-II gene expression by real-time RT-PCR. MT-I and MT-II gene expression in controls was also examined in the mammary glands of virgin as well as lactating mice on lactation day (LD) 14. The immunohistochemistry showed that cadmium treatment resulted in an induction of MT protein in the alveolar cells of the mammary glands. Control MT-I gene expression, which was generally higher than MT-II, was at the same level in both virgin and GD 17 mice, whereas an up-regulation was observed in LD 14 mice. In contrast, MT-II gene expression was at the same level at all examined time points and was thus neither up-regulated in the mammary glands during gestation nor lactation. Cadmium treatment resulted in an up-regulation of both MT-I and MT-II gene expression at GD 17. In summary, our results demonstrate that MT-I gene expression is up-regulated in the mammary glands during lactation whereas MT-II is not. Furthermore, our results indicate that both MT-I and MT-II is induced in the alveolar cells of the mammary glands by cadmium treatment. 512 THE INTERACTION BETWEEN CADMIUM CATION POLLUTANT WITH SULFUR CONTAINING LIGANDS Marcella Belcastro, Tiziana Marino, Nino Russo, Emilia Sicilia. Dipartimento di Chimica and Centro di Calcolo ad Alte Prestazioni per Elaborazioni, Parallele e Distribuite-Centro d’Eccellenza MURST, Universita’ della Calabria, I-87030 Arcavacata di Rende (CS), Italy. It is well known that cadmium cation produced by a series of technological processes acts as a dangerous pollutant in land and in water environments. Cd+2 can be removed by thiol-rich peptides, called phytochelatins (PC), enzymatically synthesized. They are characterized by a selective ability to bind metal ions through thiol groups. Among the common metals, cadmium is well known as the strongest inducer of PCs synthesis in most plants. As a starting point of our investigation on sulfur containing ligands, able to coordinate heavy metal cations, we have considered the cysteine and the 3-mercaptopropionic acid (MPA). In this communication we report the results of our theoretical investigation on the interaction between Cd2 + and MPA considering several complexation modes and stoichiometric ratios of the ion. Moreover, also water molecules in its inner coordination sphere have been included. The computations have been performed using the hybrid Becke and Lee Yang and Parr exchange-correlation functional, all electron extended basis sets for C, H, O, N and S atoms and the pseudo potential LANL2DZ for Cd. Results show that: – A single cadmium ion form a stable cyclic structure with MPA; – Cd+2 interacting with two ligands prefers the cyclic structure; – The geometry around the cation corresponds to a distorted tetrahedral; – The water molecules used to complete the Cd+2 coordination sphere are substituted by four interaction with the ligands during the optimization procedure. Work supported by MIUR through the MEMOBIOMAR project. s138 513 Poster Session P26. Heavy metals THE EFFECT OF ETHANOL ON CADMIUM INDUCED OXIDATIVE DAMAGE IN ACUTE EXPERIMENT IN MICE D. Kotyzova, V. Eybl, J. Koutensky. Charles University Faculty of Medicine in Pilsen, Czech Republic The influence of low-doses ethanol treatment on cadmium (Cd) acute intoxication was investigated relating to lipid peroxidation, antioxidant defense system and cadmium and essential element level in the tissues of mice. In the experiments a single dose of cadmium chloride (0.035 mmol/kg) was injected sc to male mice with/without co-treatment with ethanol (0.5 g/kg, ip) at 24th h before, 1st h before and 6th hour after Cd intoxication. After 24 hours the lipid peroxidation (LP), expressed as malondialdehyde production, glutathione (GSH), glutathione peroxidase (GSH-Px) and catalase (CAT) activities were evaluated in the liver homogenates of control, Cd-intoxicated, Cd+ethanol treated and ethanol treated mice. Liver, kidneys, brain and testes were examined for Cd and essential element (Ca, Mg, Zn, Cu, Fe) concentration changes. Cd-induced lipid peroxidation (148% of controls), depleted GSH level (63% of controls) and CAT activity (75% of controls) remained unaffected by ethanol co-treatment. Cd-induced activity of GSH-Px was prevented by ethanol co-treatment (p<0.05). Ethanol treatment alone enhanced lipid peroxidation (127%), however did not show any effect on antioxidant defense parameters. A significant increase of Cd concentration in the liver and decreased Cd concentration in the testes was found in Cd+ethanol treated mice compared to Cd-only treated group. The alteration in essential element concentration caused by Cd intoxication – increased Ca, Zn level in the liver; decreased Ca, Mg level in the kidneys; increased Ca, Fe level and decreased Mg, Zn, Cu level in the testes – remained unaffected by ethanol co-treatment. Ethanol treatment alone did not influence essential element level in the tissues with the exception of enhanced Cu level in the testes. Supported by the grant GAÈR 305/02/1231 514 THE INFLUENCE OF EGF ON CADMIUM INDUCED TOXICITY IN HUMAN LUNG CELLS A. Rämisch, F. Glahn, E. Stehfest, H. Foth. Institute of environmental toxicology, Martin Luther University Halle, Wittenberg, Halle/Saale, Germany Many physiological effects have been described for EGF and its receptor EGFR, for instance on proliferation, transformation and apoptosis. In many cases the additional treatment of EGF to apoptotic stimulating agents protects cells from apoptosis. Otherwise it has been described that treatment with EGF may increase the apoptotic effect of cell death inducing factors. EGF is an important growth factor to stimulate human lung tissue during explant cultures in vitro. Lung tumor cell lines and normal peripheral lung cells were cultivated with EGF (10- 250 ng/ml). The tumor cell line A549 was stimulated in cell vitality whereas H358 and H322 cell did not respond by proliferation within 24h. Human primary peripheral lung cells (PLC) showed a decrease in cell vitality (formazan formation in the MTT assay) after treatment with small levels of EGF. In cultured NHBEC and PLC EGF decreased the total protein levels of the hepatocyte growth factor receptor c-Met (scatter factor). The epithelium of the lung is prone to many environmental compounds which are taken up by particulate matter and damage the lung For instance cadmium is a well known apoptotic agent. We treated the cells with EGF (20ng/ml, additional to the basic supply of EGF in the medium) for 24h and then for 24h with different doses of cadmium (2,5 to 50µM). PLCs respond with an increased susceptibility to the toxicity of cadmium after treatment with EGF. The caspase3/7 activity also increased in EGF plus cadmium treated cells and also in EGF treated cultures compared to untreated controls. The annexin apoptosis assay showed a higher frequency of apoptotic cells in cadmium (5µM) treated cells plus EGF compared to cadmium treatment only. We have obtained evidence, that in lung epithelium EGF increases the apoptotic effect of cadmium. Supported by Philip Morris External Research 2002 Program (MLU Nr. 321003) 515 EFFECTS OF CADMIUM ON MULTIDRUG TRANSPORTERS FONCTIONALITY AND GENE EXPRESSION IN CACO-2 CELLS Elsa Le Prieur 1 , Sophie Ayrault 2 , Stéphane Orlowski 3 , Marcel Delaforge 1 , Aloïse Mabondzo 1 . 1 CEA, Service de Pharmacologie et d’Immunologie, DRM/DSV, 91191 Gif sur Yvette Cedex, France; 2 CEA, Laboratoire Pierre Sue, DRECAM/DSM, 91191 Gif sur Yvette Cedex, France; 3 CEA, Service de Biophysique des Fonctions Membranaires, DBJC/DSV, 91191 Gif sur Yvette Cedex, France Cadmium (Cd) is an ubiquitous environmental and industrial contaminant and is considered to be a potential carcinogen to human. Food intake represents the major route of exposure and the intestine plays a key role in Cd disposition since it represents the first barrier to be crossed. Then, the effects of Cd on Caco-2 cells, a human intestinal cell line that has been used widely as a representative model of mammalian intestinal absorbing cells, were investigated. Caco-2 cells were cultured on porous filters (Transwell*) to form an epithelial monolayer. These cells were exposed 4 h to 1 or 5 µm of CdCl2 and several endpoints were assayed: 1. cellular viability (MTT test), 2. membrane integrity (transepithelial electrical resistance measurements, paracellular sucrose transport), 3. transport fonctionnality of p-glycoprotein (P-gp) using digoxine as a P-gp substrate, 4. transport of Cd using inductively coupled plasma mass spectrometry methods (ICP-MS), and 5. quantitative real time pcr with specific P-gp and MRP-1 gene primers. This study shows that cellular viability and membrane integrity were not altered within 4h of 5µM Cd exposure. Digoxine transport from the basal to the apical compartment was increased in the presence of Cd. Under the same conditions, Cd induces a P-gp gene expression that can explain this increase. Otherwise, Caco-2 epithelium mediated a basal-to-apical flux of Cd, and probenecid inhibited this flux. Thus, Cd seems to be transported across Caco-2 monolayer via the MRP-1 transporter. Taken together, our findings support the possibility that the two multidrug transporters P-gp and MRP-1 may represent a mechanism by which Caco-2 cells can escape the cytotoxic effects of Cd. 516 THE EFFECT OF HEAVY METAL IONS ON THE EXPRESSION OF MRPS AND UMAT IN RELALATION TO INTRACELLULAR GLUTATHIONE LEVEL F. Glahn, A.W. Torky, H. Foth. Institute of Environmental Toxicology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany The MRP proteins (multidrug resistance associated proteins) transport a wide range of substrates from physiologic substrates to xenobiotics. Some substances are being transported as glutathionecomplexes, e.g. GSH-metal-complexes, or conjugated to glutathione (GSH). GSH is also needed for the detoxification of reactive oxygen species (ROS) by GSH-peroxidase. The toxic effect of heavy metals is in part based on the formation of ROS. The recently discovered transporter ubiquitously expressed mammalian abc-halftransporter (UMAT) is supposed to play a important role in metal ion homeostasis. therefore an up regulation of MRPS and UMAT by metals is expected. UMAT and MRP1–5 are expressed on the mRNA level in primary cultures of normal human lung tissue and in tumor cell lines. UMAT could also be determined by immuno-histochemistry in human peripheral lung cells and normal human bronchial epithelial cells. According to microscopic images UMAT is localized near the nucleus. It is expressed only in a small fraction of cells maintained under normal culture conditions. We analysed the effect of copper (15 and 25µM), mercury (2,5 and 10 µM) and arsenite (2,5 and 5µM) on peripheral lung cells and the tumor cell lines H322, H358 and A549 in 24 hour tests. In 24 hour toxicity tests on the tumor cell line H322 copper causes cytotoxic effects starting at concentrations of 25µM, mercury at concentrations of 40µM and arsenite at concentrations of 15µM. The expression of mRNA of MRP1–5 and UMAT was analysed by RT-PCR in tumor cell lines and peripheral lung cells exposed for Poster Session P26. Heavy metals 24 hours under sub-toxic conditions. The intracellular GSH-content was monitored by sensitive HPLC with fluorescence detection. Supported by Philip Morris External Research 2002 Program MLU: 321003 517 EXPRESSION OF MRP1–5 IN HUMAN LUNG CELLS CULTURE WITH AN APICAL AIR SURFACE AND ITS CORRELATION TO CADMIUM AND ZINC IN RESPECT TO THE GLUTATHIONE POOL. E. Stehfest, A.W. Torky, H. Foth. Institute of Environmental Toxicology, Martin Luther University Halle, Halle (Saale), Germany In treatment of lung cancer very often a failure of chemotherapy is observed. This phenomenon is caused by proteins such as multi drug resistance related proteins, MRP, a sub group of the ABC-binding cassette transport proteins. The expression, localization and function of the different MRP isoforms in the lung are widely unknown. At least MRP1–4 are supposed to be involved in the regulation of the glutathione pool and transport of conjugates. MRP1–7 are expressed on their mRNA level in peripheral lung cell (PLC) cultures in different quantities (real-time PCR). MRP1–5 could be determined in the cell culture qualitative by immuno-histochemistry. Trough modulation of the glutathione pool by Buthionine sulfoximine and N-acetylcysteine, the transport activity of MRP1 was modulated. Cadmium (2,5 and 5 µM) and zinc (10 – 20 µM) affected the MRP-expression in long term experiments (4 – 6 weeks). Under these conditions a clear raising (between doubling up to tripling) of the glutathione pool was found. To investigate the peripheral lung cells under more physiological conditions the cells were cultured in a alternative way: insert dishes with a semi permeable membrane provide a culture system with a wet (medium) and a dry (air) surface. These cultures were characterized by immuno-histochemistry as human lung cells and show a very different morphology than cells which were grown in plastic dishes covered with medium. Under these culture condition the lung cells develop their natural polarization. That makes it possible to localize the subcellular distribution of different mrp-isomers on their natural position and allows so to draw more profound conclusions about their specific function. Up to now we found MRP1, 3 and 5 localized in the cell membrane, especially in higher densities on cell-cell-contact sites. Research described in this abstract was supported by Philip Morris Incorporated, MLU: 328001 518 LOCALIZATION AND FUNCTION OF MRP1–5 IN HUMAN LUNG CELL CULTURES IN VITRO A.W. Torky, E. Stehfest, H. Foth. Institute of Environmental Toxicology, Martin Luther University Halle, Halle (Saale), Germany, Tumor cell resistance to cytotoxic drugs is considered one of the major obstacles to successful chemotherapy. This phenomenon can be produced by ABC–(ATP-Binding-Cassette) transpotproteins. Prominent members of this family are transport-proteins summerized in the group of the multidrug-resistance-associated proteins. Inhibition of this transport is from major interest to provide a successfull therapy of cancer patients. The different functions of the isomers are closely related to their localiztion in the cell. We detected the MRP isomers 1–5 in peripheral human lung cell cultures by immuno-histochemistry. They are expressed in different manners and mainly found on the cell membrane. MRP1 was also detected intra-cellularly. We used a recently established model of an air/liquid interface culture of human lung cells to demonstrate where the single MRPisomers are situated (apical, baso-lateral or intracellular). Preliminary studies showed baso-lateral localization of MRP1 and 5. MRP is supposed to be involved in many toxicologically relevant processes by transport of metals as glutathion complexes. We used the heavy metal nickel because of its occupational relevance and iron because of its physiological importance to investigate their effect on MRP. Nickel and Iron were tested by means of MTT-test s139 (vitality test) in order to work in concentrations below the acute cell toxicity. Lung cell cultures did not show any signs of toxicity up to nickel concentrations of 500µM and iron concentrations of 20µM in 24 hours test. We will verify how nickel (10µM) and iron (10µM) influence the expression of MRP1–5 mRNA in human lung cell cultures in short term experiments (24 hours) and long-term experiments (4 – 6 weeks) as other metals did show adaptation reaction. Supported by Philip Morris External Research 2002 Program MLU: 321003 and DFG Graduate College 519 EFFECTS OF THE LEAD ACETATE TREATMENT IN THE REPRODUCTIVE TRACT OF THE MALE RATS. M.J. Ochoa 1 , J.L. Morán 2 , C. Morán 2 , A. Handal 2 . 1 Escuela de Biología Universidad Autónoma de Puebla, Puebla, México. Laboratorio de Investigaciones Biológicas2 del Instituto de Ciencias de la Benemérita Universidad Autónoma de Puebla, Puebla, México The aim of this investigation was to analyze the effects of the lead treatment in the morphology and physiology of the male reproductive tract. Male rats of 1-month of age the stock CII-ZV. They were separated in four experimental groups of eight animal each one including the control group. To each group respectively was administered daily 0.0, 0.003, 0.03 and 0.6 g/l of lead acetate in the drinking water, during four months. The different groups were weighed and sacrificed by decapitation. To each one, were weighed and dissected the testes. Of each animal was collected the blood to analyze the lead concentration. The results showed in Sertoli cells, nuclei appeared fragmentated. Diminution in the number of the Leyding cells and differences in the arrangement of the spermatozoids into seminiferous tubules. These results suggest that increase in the lead concentration in the blood increase testicular atrophy, cellular degeneration, and reductions in the spermatic count. 520 RELATIONSHIP BETWEEN BLOOD LEAD CONCENTRATION AND FREE PROTOPORPHIRIN IN ERYTHROCYTES IN WORKERS EXPOSED TO LOW LEAD LEVELS Z. Paskalev 1 , D. Aposolova 2 , S. Pavlova 2 , D. Adjaraov 2 . 1 National Center of Radiobiology and Radiation Protection, Center of Occupational Diseases, Clinic of Toxicology, Sofia, Bulgaria In recent years the medical literature has focused its attention on biological effects resulting from low to moderate levels of Pb exposure. It is well known that elevations in PbB are associated to an increase in free protoporphyrin in erythrocytes levels (EPP). A significant correlation between these two indices can be observed the biological response (EPP). In the present study, the relationships between PbB and EPP were evaluated in 192 workers of a battery factory consisting of males with varying degrees of occupational lead poisoning. The number of workers with PbB lower than 2,1 µmol/L (Group I) was 115 workers /mean age 42, range 24 – 56 years), and PbB higher than 2,1 (umol/L) to 4,6 (umol/L) – 67 workers (Group II) (mean age 55, range 31 – 60 years). For each of the workers examined in this study we determined the PbB (umol/L) and EPP (nmol) gHb, using the method of Pionrellis. The length of exposure was from 2 to 15 years. Simultaneous measurement of free protoporphyrin in erythrocytes showed hight diagnostic sensitivity in detecting lead poisoning in occupationally exposed subjects. The results shown high interindividual variability of the EPP. In our study, we used the mean of PbB and EPP values and standard deviation for group I and group II. For each group statistically significant correlation was found between PbB concentration and EPP, expressed in the following equation: For group I: EPP (nmol/gHB) = (1,6 ± 0,8) ± (28,3 ± 8,2) PbB (µmol/L) correlation coefficient r = (0,54 ± 0,11). For group II EPP (nmol/gHG) = (2,8±0,9) ± (34,1±10,8) PbB (nmol/L) correlation coefficient = (0,54 ± 0,11). In fact, free protoporphyrin in erythrocytes levels increase exponentially when there is a sharp increase in PbB. In this study, we observed that an increase in EPP level is s140 Poster Session P26. Heavy metals associated with lead intoxication since Pb interference in Fe incorporation in heme molecules determines a gradualaccumulataion of portoporphyrin in erythrocytes. 521 EFFECT OF CHRONIC LEAD EXPOSURE ON PROAPOPTOTIC BAX AND ANTIAPOPTOTIC BCL-2 PROTEIN EXPRESSION IN RAT HIPPOCAMPUS IN VIVO A.M. Sharifi, S. Baniasadi, M. Jorjani, F. Rahimi, M. Bakhshayesh. Department of Pharmacology & Cellular and Molecular Research Center, Iran University of Medical Sciences,Tehran, Iran Despite reduction in environmental lead, chronic lead exposure still posess a public health hazard, particularly in children, with devastating effects on developing CNS. To investigate the mechanism of this neurotoxicity, young and adult rats were used to study whether exposure to low concentrations of lead could induce apoptosis in hippocampus. 2–4 and 12–14- week-old rats received lead acetate in concentration of 500 ppm for 40 days. Control animals received deionized distilled water. In lead treated groups, the Blood lead levels was increased by 3–4 folds. Light and electron microscopical study of hippocampus revealed increased apoptotic cells. Western blot analysis of Bax and Bcl-2 (pro- and antiapoptotic gene products respectively) indicated higher expression of Bax protein and no significant change in bcl-2 expression and accordingly increased the Bax/Bcl-2 ratio compared to control group, confirming the histological study. In conclusion these data suggest that neurotoxicity of chronic lead exposure in hippocampus in vivo may partly be due to facilitation of apoptosis. 522 INFLUENCE OF HEAVY METALS FROM MASS GRAVES BONES ON IDENTIFICATION BY GENOMIC DNA D. Sutlovic 1 , S. Andjelinović 1 , M. Definis Gojanović 1 , J. Pavlov 2 . of Pathology and Forensic Medicine, Split University Hospital and School of Medicine, Split, Croatia, 2 Public Health Institute of Split-Dalmatia County, Split, Croatia 1 Department The identification process of dead bodies or human remains is being conducted under different circumstances. Exhumation and war victim identification have a special connotation. Different identification methods are used depending on the case circumstance and the state of postmortem body changes. One of the methods is the identification by DNA typing from different biological samples (genotyping). Considering to the fact that every person inherits half of the genetic material from the mother and half from the father, DNA typing can verify the relationship between the examined persons. A particular problem is the isolation and DNA typing from human remains found in mass graves, that had undergone the degradation process, as well as postmortem DNA contamination with bacteria, fungi, humic acids, metals etc. This study analyzed the possible influence of metal ions on the successfulness of DNA typing of bone samples from mass graves. The study included 30 bone samples from mass graves and the 5 fresh bone samples. Successful and unsuccessful DNA typing has been determined the concentration of iron, cooper, lead and cadmium ions and their possible correlation. The influence of single metal ions the and influence of different combinations and concentrations of iron, cooper, cadmium and lead ions on DNA typing has been analyzed through in vitro experiments with fresh bone suspensions and metal ions. The results revealed that iron, cooper, lead and cadmium ions, if present in bone samples from mass graves, do not inhibit DNA amplification, while they inhibit the DNA amplification only if they are present in the amplification reaction mix. 523 GENETIC VARIABILITY OF δ-AMINOLEVULINIC ACID DEHYDRATASE (δ-ALA) AND THE WHOLE BLOOD LEAD CONCENTRATION IN NORTHEAST SPAIN. M. Torra 1,2 , C. Barrot 2 , M. Ortega 2 , A. Xifró 2 , E. Huguet 2 , J. Corbella 1,2 , M. Gené 2,3 . 1 Servei de Toxicologia, Hospital Clínic de Barcelona; 2 Escola Professional de Medicina del Treball de la Universitat de Barcelona; 3 Unitat Bàsica de Prevenció, Hospital Universitari de Bellvitge, C/ Villarroel 170, 08036 Barcelona. Spain The second enzyme of the heme biosynthetic pathway δ-aminolevulinic acid dehydratase, is a cytosolic enzyme that catalyses the condensation of two molecules of 5-aminolevulinat (ALA) to form porphobilinogen. The inhibition of ALA-D activity by lead is a sensitive indicator of exposure and has been used as a diagnostic tool. The gene that encodes ALAD exists in two polymorphic forms that may modify lead toxicokinetics, bioaccumulation and ultimately influences individual susceptibility to lead poisoning. This gene is located in chromosome 9q34, which has two codominant alleles: ALAD1 and ALAD2. The ALAD2 allele contains a G → C transversion at position 177 of the coding region, resulting in the substitution of a positive charged lysine by a neutral aspargine at amnioacid 59 concluding in a higher affinity for lead by the ALAD-2. This substitution is responsible of three different isoenzyme phenotypes: ALAD1–1, ALAD1–2 and ALAD2–2. Another mutation consisting in a T → C transversion, without translation changes, increases the gene polymorphic degree. Venous blood samples were obtained from 100 healthy subjects residing in Barcelona, Spain. Blood lead concentration was determined by means of a graphite oven atomic absorption spectrometry. The ALAD1 and ALAD2 alleles were detected by amplification of a 916bp region of genomic DNA and digested with the restriction endonucleases MspI and RsaI. The cleavage products are then analyzed on agarose gel using ethidium bromide and ultraviolet detection. To investigate the possible relation between the blood lead concentration and the ALA-D polymorphism, a model of multiple regression was applied. The preliminary results, obtained until now, seem to indicate that the presence of the ALAD2 allele only contributes to modify the toxicokinetics of lead at high exposure levels. 524 THE CHANGES IN THE PARAMETERS OF ENERGETIC STATUS OF THE STELLATE STURGEON Acipenser stellatus Palls SPERMATOZOA AFTER SHORT-TIME EXPOSURE TO LEAD IONS IN VITRO I. Baranowska-Bosiacka 1 , A. Rzemieniecki 2 , M. Rutkowska 1 , G.J. Dietrich 3 , A. Ciereszko 3 , J. Głogowski 3 , J. Domagala 2 , A.J. Hlynczak 1 . 1 Department of Biochemistry, University of Szczecin, Poland, 2 Department of Zoology, University of Szczecin, Felczaka 3a St, 70–412 Szczecin, Poland, 3 Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10–747 Olsztyn, Poland Objectives: The aim of this work was to evaluate the influence of short-time exposure to lead ions on the parameters of energetic status of stellate sturgeon (Acipenser stellatus, Palls) spermatozoa. Material: The experiment was carried out on milt samples obtained from three 5-year-old stellate surgeon males. Methods: Sperm production was stimulated by intraperitoneal injection of 18–20 µg [D-Ala6 ,Pro9 Net] m-GnRH+ 8–10 mg of metoclopramide (formulated in Ovopel). Milt was collected 48 h after injection. Spermatozoa were separated from semen plasma by centrifugation and resuspended in sperm immobilizing solution (20mM Tris-HCl buffer, pH 8,0, containing 400 mM saccharose). Then solutions of lead acetate dissolved in the immobilizing solution were added to the final lead ion concentrations: of 1, 10, 100 µg Pb/dl. Samples were incubated in standard conditions for 0 min; 1h; 4 h; 24 h. The concentrations of ATP, ADP, AMP, adenosine (Ado), GTP, GDP, GMP, guanosine (Guo), IMP, inosine (Ino), NAD, NADP, hypoxantine (Hyp) in spermatozoa were measured using HPLC. Adenylate (guanylate) energy charge- AEC(GEC) and total adenine (guanine) nucleotide-TAN(TGN) were calculated. s141 Poster Session P26. Heavy metals Results: The median purine compounds concentrations in spermatozoa (nM/106 cells) in control group were: ATP (0.08); ADP (0.04); AMP (0.01); GTP (0.07); GDP (0.03); GMP (0.01); IMP (0.09); NAD (0.03); NADP (0.02); Ado (0.003); Guo (0.002); Ino (0.006); Hyp (0.02) and the values of AEC (0.78); GEC (0.77); TAN (0.13); TGN (0.11). Incubation of the Stellate sturgeon spermatozoa with the lead ions in vitro resulted in the decrease in the ATP, GTP, NAD, NADP concentrations and decrease in the values of AEC and GEC. There was also a significant increase in purine compounds degradation products: ADP, AMP, Ado, GMP and Hyp. Conclusions: The studies concerning in vitro effects of lead ions on the purine compounds conversion in spermatozoa showed that lead decreases high energy purine nucleotides concentrations and increases the products of their catabolism. 525 MERCURY DIETARY INTAKE IN THE CANARY ISLANDS, SPAIN Rubio 1 , Hardisson 1 , 2 1 Martín-Izquierdo 1 , Suárez 2 , C. A. R.E. M.L. I.F. Gonzalez-Delgado . Toxicology Department. University of La 2 Laguna. Tenerife. Canary Islands, Spain, Legal Medicine Institute. Tenerife. Spain As food is usually the most important source of heavy metal intake, it is important to monitor the heavy metal dietary intakes (Storelli et al., 1998; Llobet et al, 1998; Vega et al., 2001; Robberecht etal., 2002). The purpose of this study is to determine the levels of Hg (AAS) in the foods and drinks of highest consumption in the Canary Islands in order to estimate the total Hg dietary intake using the last nutritional survey made in our Community (ENCA 2000). We are proving that the total Hg intake of the Canarian population (5.684 µg/Kg/day) don’t exceed the PTWI (Provisional Tolerable Weekly Intake) limit of 0.3 mg/week of total mercury or 43 µg/person/day) fixed by the FAO/WHO (WHO, 1993). We are also comparing our results with the those found for other national and international communities (Boudene, 1990; Schuhmacher et al., 1994; Cuadrado et al., 1995; Llobet et al., 1998). 526 MODIFYING EFFECT OF SPIRULINA FUSIFORMIS AGAINST MERCURY INDUCED RENAL DAMAGES IN SWISS ALBINO MICE Mukesh Kumar Sharma, Madhu Kumar, Ashok Kumar. Department of Zoology, University of Rajasthan, Jaipur-302004 (INDIA) The toxicity of mercury to animals and man is well established and this depends greatly on the form of the mercury compounds. In most animal species, including man, the kidney is the main site of deposition of inorganic mercury and target organ for its toxicity. In the present study Spirulina fusiformis (a Cyanobacterium, belongs to family-Oscillatoriaceae) has been investigated as a possible modifiers of mercury induced renal damages in Swiss albino mice. Animals were divided into four groups. (i) Control group-No treatment was given (ii) HgCl2 treated group-5.0 mg/kg b.w. HgCl2 administered as i.p. (iii) Spirulina treated group- 800 mg/kg b.w. Spirulina extract was administered orally. (iv) Combination group- Spirulina fusiformis was administered 10 days before mercuric chloride administration and continued upto 30 days after mercuric chloride administration (5.0 mg/kg b.w.). The animals were autopsied on 1,3,7,15 & 30 days and the activity of Alkaline phosphatase (ALP), Acid phosphatase (ACP), Lactate dehydrogenase (LDH) and Lipid peroxidation (LPO) were measured in kidney homogenates. The results indicated that there was a significant enhancement in LPO content, ACP activity and decrease in LDH, ACP activity after HgCl2 treatment. The animals treated with Spirulina alone did not show any significant alterations in ACP and ALP activity. However, there was a significant increase in LDH activity and decrease in LPO level observed. In combined treatment of Spirulina with HgCl2 , a significant decrease in LPO content & ACP and elevation in LDH & ALP activity was observed as compared to HgCl2 treated group. Thus the results from the present study suggest that Spirulina fusiformis can significantly modify the renal damages against mercuric chloride induced toxicity. 527 POSSIBLE INFLUENCE OF ELEMENTAL MERCURY IN PREGNANCY A.B. Kobal 1 , J. Osredkar 2 , M. Horvat 3 , M. Prezelj 2 , A. Sesek-Briski 2 , M. Krsnik 2 , D. Gibicar 3 , C. Knap 1 . 1 Idrija Mercury Mine, Idrija, Slovenia, 2 University Medical Centre Ljubljana, Clinical Institute of Clinical Chemistry and Biochemistry, Ljubljana, Slovenia, 3 Jozef Stefan Institute, Ljubljana, Slovenia Until now not many investigations of a possible influence of elemental mercury (Hg°) on pregnant women and their newborn children have been made. The purpose of the present work was to observe changes of mercury in whole blood (B-Hg) and urine, superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in erythrocytes, selenium in whole blood (B-Se), plasma (P-Se) and urine (U-Se), and ferritin in serum during pregnancy, and to evaluate the relationship between mother and child. The study included 11 mercury exposed pregnant women living in Idrija and seven non-exposed pregnant women as a control group. After cessation of Idrija Mercury Mine activity in 1994, the air mercury concentration in the town has been significantly decreased. In the last 8 years the average concentration of elemental mercury in Idrija has been between 30 and 170 ng/m3 . The blood and urine samples from mothers were taken in the first and last trimester and the cord blood was taken at the time of birth. The main conclusions indicate that (1) the Hg concentrations in blood and urine in the exposed group were not significantly higher than those in the control group; (2) the mean SOD activity from the exposed pregnant women was significantly higher than that of the control group (p < 0,01); (3) we found the mean PSe, B-Se, ferritin concentration, and GPx activity lower in the 3rd trimester of pregnancy than in the 1st trimester, but P-Se and ferritin concentrations were significantly lower (p < 0,001); (4) significant differences in SOD activities and P-Se concentrations were found between the 1st trimester of pregnancy and the cord blood samples in both groups, whereas no significant difference was found between 3rd trimester of pregnancy and cord blood samples, except in the control group, where P-Se concentrations were significantly lower (p < 0,02) and SOD activities were higher (p < 0,02); (5) the following correlations during pregnancy in the exposed group were observed: U-Se and P-Se (-0,471; p < 0,03), B-Se and ferritin concentrations (0,891; p < 0,02) and between B-Se and B-Hg (0,818; p < 0,05) in cord blood samples. The results of B-Se, P-Se and GPx are in the accordance with the results represented by Zachara et al. (1993), where decreasing of these parameters showed increased requirement for the element during pregnancy, the same applies to ferritin results. SOD demonstrates an increased activity to the oxygen radical production although no significant increase of Hg° has been noted during pregnancy or in cord blood in the both groups, which is probably due to the small number of subjects. Therefore, further studies are needed. Abstract 525 – Table: Hg dietary intake in the Canary Islands and its relation with the Fish consumption Canarias, 1998 Gran Canaria, 1998 Lanzarote, 1998 Fuerteventura, 1998 Tenerife, 1998 La Palma, 1998 La Gomera, 1998 El Hierro, 1998 Hg dietary intake (µg/día) 5,684 5,568 6,744 6,979 5,494 4,504 5,418 6,133 Daily fish consumption (g/día) 45,8 44,7 54,7 57 44,1 35,8 43,5 49,3 s142 528 Poster Session P26. Heavy metals EVALUATION OF MERCURY AND SELENIUM COMPOUNDS TOXICITY ON E. coli K802N: ANTAGONISTIC EFFECTS AND SPECIATION APPLICATIONS E.S.T. Campos, M.C.C. Batoréu, C.M.L. Carvalho. Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649–003 Lisbon, Portugal The aim of this work is to characterize the behavior ofEscherichia coli K802N (pcFF04) strain in the presence of different mercury compounds as well as to investigate the influence of different species of selenium on their toxicity. Selenium and mercury are of major importance in Food Toxicology due to their significant quantities in marine environment, i.e. in foods such as fish and shellfish. Selenium is an essential micronutrient that has been identified as a constituent of several enzymes, which reduce damage from oxygen free radicals. Furthermore, toxicological and epidemiological evidence pointed out an inverse relationship between biological selenium levels and the risk of development of several diseases. On the other hand, mercury is a toxic element responsible by several adverse effects including, nephrotoxicity, immunotoxicity and especially the teratogenicity and neurotoxicity of organic forms. Selenium has been reported as an antagonist of mercury toxicity but the mechanisms supporting that interaction are still a matter of discussion. The toxicity of mercury chloride (HgCl2 ), methylmercury (MeHg), ethylmercury (EtHg) and phenylmercury (PhHg) was evaluated in an in vitro study with E. coli. The organomercurial solutions were prepared to a concentration of 10−1 M with dimethyl sulfoxide and then diluted with bi-distilled water to prevent solvent inhibition. E. coli was harvested to BHI broth and left overnight at 37°C. The bacteria suspension was diluted in Mueller-Hinton broth to 106 cells/ml and inoculated in Elisa plates. The mercury compounds in the concentrations of 10−2 M, 10−3 M, 10−4 M and 10−5 M were then applied by micro dilution method. After 24h at 37°C the plates were observed for the determination of the minimum inhibitory concentration (MIC). Each determination was performed at least 6 times to calculate the standard deviation. The results obtained showed the lower toxicity of HgCl2 (MIC = 1.19 × 10−5 M) when compared to the organomercurial forms that displayed MICs in the range 10−7 -10−6 M. A justification for this may be the different enzymatic processes involved in the detoxification processes of the inorganic and organic species of mercury. It should be stressed that the carbon chain length correlated very well with the MIC values displayed by E. coli (R2 =0.9996). Consequently, MeHg was selected to perform the interaction studies with the selenium forms due to its higher toxicity. The compounds of selenium chosen to test their protective effect against mercury toxicity were selenite, selenate, selenomethionine (SeMet) and selenocystine (SeCys) The concentration of 10−3 M was added to the cell growth media and the inoculated plates were prepared as described above with the addition of MeHg. The ongoing work shows a positive (although moderate) effect of the selenite, selenate and SeMet against MeHg toxicity with the increasing of MIC values in the same order of magnitude. SeCys was the only exception as it fully inhibits E. coli growth at the concentration tested. The need of metal speciation in foodstuffs is emerging as different species of the same element can perform either beneficial or different toxic effects in organisms. Therefore, another application of this work would be the application of E. coli strain to a preliminary speciation process that would allow the detection of different forms of mercury and selenium. *Project POCTI/41741/ESP/2001 – Risk Assessment of Methylmercury Exposure: Integrated actions through the food chain (FCT, MCES, Portugal) *Portuguese-Spanish Integrated Actions 2001/2002 No E-13/01 529 THERATOGENIC EFFECTS OF MERCURIC CHLORIDE ON DEVELOPMENT OF EMBROYS CORTEX OF RAT M. Mehdizadeh’, T. Rastegar, M. Nobakht. Anatomy Department, Iran University, Tehran, Iran Mercuric chloride (HgC12) is a white, cristelin and poison powder which is absorbed from <L.3C and skin, and excreted from kidney and urine. This ynateriai is used in compound of laxative drugs, beauty creams and contrast material. Chronic poisoning with mercuric chloride causes sensorial and mobile behavoi’icil und mentol disorders*. Therefore 36 rates of sprague dawley were injected by mercufic Chloride (Kxperiniental groups) and normal saline solution (control groups) In eighth, ninth and tenth days of gestation intraperitunealy. I%∼enbroys were removed from uterus in 15th day of gestation and then tissue passage procedures, 5 micron slides were prepared and stained by II w E method and were studied by light yilicroscope. In microscopic studies in experimental groups cell∼ disarrangment, different urienlation of nuclei were seeiL Cell death and extracellular space were increased and concentration of eU,swere decreased. Mitosis division and diameter of cotrex were increased. Therefore results that mercuic chloride have theratogenic effects on embroy cortex and results in cell death and nervous disorders. 530 ARSENIC CONTENT IN FEMORAL HEAD SPONGIOUS BONE OF THE HABITANTS OF SOUTHERN AND CENTRAL POLAND D. Wiechuła 1 , A. Jurkiewicz 2 , J. Kwapuliński 1 , K. Loska 3 . 1 Silesian University of Medicine, Department of Toxicology, Sosnowiec, Poland; 2 Silesian University of Medicine, Department of Orthopaedics Surgery, Sosnowiec, Poland, 3 Silesian Technical University, Institute of Water and Wastewater Engineering, Gliwice, Poland This research aimed at estimating arsenic content in femoral head spongious bone. The tested people were not professionally exposed to arsenic compounds and lived in highly industrialized Polish cities: Łódź - textile, chemical and machine-building industries (n = 12), Kraków - metallurgy, electrical, chemical and machine-building industries (n = 13), and Silesian region - mining, metallurgical and chemical industries, porcelain factories (n = 13). The material used was the femur head obtained during the operation of total hip arthroplasty from patients with diagnosed coxarthrosis. The tested group comprised 29 women and 9 men. The average age was 68.0 ± 9.9, 69.15 ± 9.6 and 68.3 ± 7.3 for Silesia, Kraków and Łódź respectively. 0.2–0.3 g bone samples weighed out and put directly into Teflon vessels were mineralized applying wet microwave mineralization. Arsenic content was determined in samples applying the hydride generation technique of atomic absorption spectrophotometry (HGAAS), using the spectrophotometer SPECTRAA 880Z produced by Varian. Arsenic content in femoral head spongious bone differed with respect to the place of living of the people tested. The highest, mean arsenic content was found in femoral head spongious bone of Łódź - 0.34 µg/g and Kraków residents - 0.45 µg/g, while for Silesia residents it was lower by half and amounted to 0.18 µg/g. This difference was significant statistically. The differences of arsenic content in the femur head of men and women were not statistically significant. Neither the age nor sex of the people tested affected arsenic content in femoral head spongious bone. Poster Session P26. Heavy metals 531 FOCAL ADHESION AND CYTOSKELETON DISRUPTION BY SODIUM ARSENITE REDUCES CELL MIGRATION RATES IN MUSCLE MYOBLASTS S.L. Yancy 1 , E.A. Shelden 2 , M.J. Welsh 2 . 1 Toxicology Program, Department of Environmental Health Sciences, University of Michigan, Ann Arbor, Michigan, USA, 2 Department of Cell and Developmental Biology, Medical School, University of Michigan, Ann Arbor, Michigan, USA Sodium arsenite exposure causes numerous effects in a variety of cell types. In this study we investigated the role of sodium arsenite exposure on focal adhesion and actin cytoskeleton organization and how this affects cell motility. H9C2 muscle myoblasts were treated in with 1, 2.5 and 5 µM for 48 hours. MTT and propidium iodide analysis was used to determine that sodium arsenite concentrations were not cytototoxic. Immunofluorescence labeling of focal adhesions showed a reduced number of vinculin stained focal adhesions at 1µM and a redistribution of focal adhesions to the cell periphery at 2.5 and 5 µM. Actin stress fibers were labeled with Oregon green phalloidin and were also reduced in number at 2.5 and 5 µM sodium arsenite exposure. Western blotting analysis showed that sodium arsenite did not reduce the level of focal adhesion proteins paxillin, FAK, talin or vinculin. However, β1-integrin expression was reduced in a dosedependent manner. Since focal adhesion organization is dependent upon tyrosine phosphorylation we investigated the affect of sodium arsenite on focal adhesion protein tyrosine phosphorylation status. FAK and paxillin were immunoprecipitated and the samples analyzed by immunoblotting with a phosphotyrosine antibody. Sodium arsenite decreased the amount of phosphorylated FAK and paxillin in a dose dependent manner. Analysis of tyrosine phosphatase activity showed that sodium arsenite treatment stimulated phosphatase activity. Focal adhesion organization is crucial for cell motility and time-lapse video microscopy showed that sodium arsenite exposure reduced cell migration rates. Sodium arsenate also reduced cell size and total F-actin content in a dose dependent manner. Isolation of focal adhesions using magnetic beads coated with an RGD-peptide showed a slower recruitment of focal adhesion proteins in H9C2 cells treated with 2.5 µM sodium arsenite. These data indicate that sodium arsenite can affect focal adhesion organization and assembly and this may subsequently affect cell migration rates. 532 REGULATION OF GLUTATHIONE TURNOVER IN PORCINE AORTIC ENDOTHELIAL CELLS TREATED WITH VARIOUS ARSENIC COMPOUNDS Y.H. Cheng, L.C. Cheng, L.W. Chang, J.Y. Yeh. Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Taiwan, R.O.C Arsenic (As) toxicity was associated with vascular endothelial cell injury. It was also proved to be an anticancer drug for Acute promyelocytic leukemia. The elevation of intracellular glutathione (GSH) prevents cell damages from As-induced oxidative stress and increased As resistant in cancer cell lines. The objective of this study was to investigate the regulation of glutathione turnover in arsenic-treated porcine aortic endothelial cells (PAECs). The primary culture of PAECs was incubated with sodium arsenite (NaAsO2 ), arsenic trioxide (As2 O3 ) and sodium arsenate (Na2 HAsO4 ) up to 72 hr at 0, 1, 5, and 10µM, respectively. Intracellular GSH and GSSG, as well as the activities and mRNA expression of γglutamylcysteinyl synthetase (GCS) andγ-glutamyl transpepetidase (GGT) were measured. Na2 HAsO4 had no effect (p>.05) on the intracellular GSH content and its turnover-related enzymes. NaAsO2 and As2 O3 increased (p< 0.05) intracellular GSH and GSSG contents in PAECs. The GCS activity increased (p < 0.05) at 24 hr and the mRNA levels increased (p=0.02) at 72 hours in As2 O3 -treated PAECs. The GGT activity increased (p < 0.05) at 72 hours with the tendency of increased mRNA levels in As2 O3 treated PAECs. The GCS and GGT activities did not affect by NaAsO2 treatment, whereas the GGT mRNA levels increased (p< 0.05) at 72 hours. These results indicated that GSH modulation in PAECs by various trivalent arsenic compounds were different. The elevation of GSH induced by As2 O3 was modulated through increases of activities and mRNA expression of GCS and GGT. The GSH modulation by s143 NaAsO2 was associated with the up-regulation of GCS and GGT mRNAs. 533 MOLECULAR MECHANISMS OF ARSENIC -INDUCED CELL DEATH T. Sakurai, K. Fujiwara. Laboratory of Environmental Chemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo, Japan Inorganic arsenic are potent toxicans and carcinogens in humans. In mammals, including humans, inorganic arsenic often undergoes methylation, compounds such as dimethyarsinic acid (DMAsV ) being formed. It has been believed that the methylation of inorganic arsenic results in lowering of its general toxicity, as indicated by the increased lethal dose in vivo in 50% of a population (LD50 ), but recent some evidence indicates DMAsV is a complete carcinogen in rodents. Thus, we recently investigated the details of the in vitro cytolethality of DMAsV compared to that of the trivalent inorganic arsenic sodium arsenite, using mouse resident immune effector cells, peritoneal and alveolar macrophages, and rat TRL 1215 liver cells. Arsenite was very cytotoxic in these cells [lethal dose in vitro in 50% of a population (LC50 ) = 5 µM for macrophages and 35 µM for TRL 1215 cells after 48 hours exposure]. With arsenite, most dead cells were necrotic, and it induced marked release of an inflammatory cytokines, interleukin 1α and tumor necrosis factor-α from macrophages at cytotoxic doses. The arsenite cytolethality increased when cellular reduced glutathione (GSH) was depleted with the glutathione synthase inhibitor L-buthionine-[S,R]-sulfoximine (BSO). In contrast, DMAsV was much less cytotoxic (LC50 = 5 mM for macrophages and 1.5 mM for TRL 1215 cells) than arsenite, and most cells died through apoptosis. DMAsV suppressed the releases of inflammatory cytokines from macrophages at all doses. BSO actually decreased DMAsV -induced cell death, and ethacrynic acid, an inhibitor of glutathione S-transferase that catalyzes GSH-substrate conjugation, also suppressed DMAsV -induced apoptosis. These findings indicate that DMAsV forms a conjugate with intracellular GSH, and this becomes cytotoxic and induces apoptosis. The methylation of inorganic arsenic in mammals may play an important role in protection against both severe immunosuppression and inflammatory responses caused by inorganic arsenics. Otherwise, increased apoptosis induced by DMAsV can be associated with the development of proliferative lesions, but further study is required to define its role in DMAsV -induced neoplasia. 534 ROLE OF CELLULAR GLUTATHIONE IN LOW-LEVEL CHRONIC EXPOSURE OF INORGANIC AND ORGANIC ARSENIC-INDUCED ARSENIC TOLERANCE C. Kojima, T. Sakurai, K. Fujiwara. Laboratory of Environmental Chemistry, School of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo, Japan, In this study, we examined the effects of the low-level chronic exposure of inorganic and organic arsenic on the cellular characters in vitro using rat normal liver cells, TRL 1215 cells. We exposed arsenite (0.5, 5 µM), arsenate (0.7, 7 µM), monomethylarsonic acid (MMAsV ; 0.13, 1.3 mM), dimethylarsinic acid (DMAsV ; 70, 700 µM) or trimethylarsine oxide (TMAV O; 1, 10 mM) to TRL 1215 cells for 20 weeks. As the results, these cells acquired the tolerance of the acute arsenic cytolethality. The tolerance in chronic MMAsV exposed cells and chronic TMAV O-exposed cells were decreased by the incubation in arsenic free medium for more 8 weeks, on the other hand, the tolerance in the chronic arsenite-exposed cells, chronic arsenate-exposed cells and chronic DMAsV -exposed cells was no difference after the culture in arsenic free. The tolerance of the acute cytolethality of arsenics in these chronic arsenic-exposed cells was decreased by the depletion of cellular reduced glutathione (GSH). These chronic arsenic-exposed cells were also significantly increased the cellular glutathione S-transferase (GST) activity and cellular GSH concentration compared with the passage-matched control cells, only chronic DMAsV -exposed cells were significantly decreased the cellular GSH levels. Furthermore, cellular accumulation of arsenics was less in these chronic arsenic-exposed cells than control s144 Poster Session P26. Heavy metals cells, and the accumulated arsenics was more readily eliminated. However, the depletion of cellular GSH showed significantly the increase of arsenic accumulation and the decrease of the efflux of the accumulated arsenics in these chronic arsenic-exposed cells, especially chronic DMAsV -exposed cells. These results show that chronic inorganic and organic arsenics-exposed cells acquire arsenic tolerance and cellular GSH play important role in the tolerance. It is suggested that the low-level chronic exposure of inorganic and organic arsenic would induce the arsenic excretion mechanism depended on cellular GSH, and that the long-term stimulus of DMAsV would be associated with the induction of this mechanism. 535 THE TERATOGENIC EFFECT OF COPPER CHLORIDE (II) ON NEURAL TUBE DEVELOPMENT OF MOUSE EMBRYOS BY EMPHAZING ON CERVICAL SPINAL CORD. M. Mehdizadeh, M. Nobacht, Iran F. Mohamadzadeh. Anatomy Department, Iran University, Tehran, Iran Copper is an essential trace metal. This metal is absorbed in digestive system. Copper chloride is one of the most important of its component. The lack of copper results to structural and metabolic defects. According to recent studies,copper has toxic effect on central nervous system. In spite of a little amount of copper is neccessary for brain development, but high amount of it may causes defects in central nervouse system. In this study, we investigated the effects on mouse embryo. We divided the pregnant mice in three group. 1. Experimental 2.Control and 3. Intact. Each group had six pregnant mice. Experimental group was injected 5 mg/kg CuCl2 on 7’h, 8’" and 9’" gestational days via intraperitoneal (IP). Control group was injected distilled water on the same days. Intact group was not injected. Embryos were extruded from the uterus on 15th gestational days. Weight and number of absorbed embryos were measured. Following tissue passage and sectioning, sections were stained with H & E. According to macroscopic measurement, weight of embryos in all experimental groups was decreased significantly (P<0.05). In micrscopic observservation, cellular irregularity and gliosis were seen. In morphometric evaluations, in all groups, thickness of marginal layer of spinal cord was decreased (P<0.05). Thickness of ventricular layer in 7’h and 9’h day increased significantly, mantle layer in all group was decreased but in 9th day was significant (P<0.05). 536 THE ROLE OF ANTIOXIDANT VITAMINS ON CHROMIUM(VI) INDUCED DAMAGE B. Poljšak 1,4 , Z. Gazdag 2 , M. Pesti 2 , N. Farkas 3 , S. Plesničar 1 , P. Raspor 4 . 1 Polytechnic Nova Gorica, School of Environmental Science, Vipavska 13, 5000 Nova Gorica, Slovenia, 2 Department of General and Environmental Microbiology, Faculty of Sciences, University of Pecs, P.O.B. 266, H-7601 Pecs, Hungary, 3 Central Research Laboratory, University of Pécs, P. O. Box 266, Hungary, 4 University of Ljubljana, Biotechnical Faculty, Food Science and Technology Department, Chair of Biotechnology, Jamnikarjeva 101, 1000 Ljubljana, Slovenia The effect of antioxidant vitamins ascorbic acid (vitamin C) and Trolox (vitamin E water soluble analogue) pretreatment on chromium(VI) induced damage was investigated using yeast Saccharomyces cerevisiae as model organism. The objective of this study was to pretreat yeast cells with two antioxidant vitamins in order to increase cell tolerance against reactive chromium intermediates and reactive oxygen species (ROS) formed during chromium(VI) treatment. Intracellular oxidant formation was estimated using two fluorescence indicators dihidro-2, 7-dichlorofluorescein and dihydrorhodamine 123, respectively. The role of antioxidant pretreatment on chromium(VI) genotoxicity was determined by measuring mitotic gene conversion and reverse mutations on S. cerevisiae strain D7. Additionally, 8-OHdG a marker of oxidative damage of DNA was measured using competitive enzyme-linked immunoasorbent assay (ELISA) and hydroxyl radical generation induced by chromium(VI) reduction was measured by electron spin resonance spectroscopy in cell extract of S. cerevisiae. Furthermore, chromium content in biomass was determined by flame atomic absorption spectrometry (FAAS). Results indicate the important role of cytosol reduction capacity on increased chromium accumulation, modification of the Cr(V) formation and better ROS scavenging ability. 537 ASSESSMENT OF GENOTOXIC HAZARD POSED BY ORAL EXPOSURE TO VANADIUM PENTAVALENT: P. Leopardi 1 , E. Siniscalchi 1 , E. Cordelli 2 , P. Villani 2 , E. Veschetti 1 , R. Crebelli 1 . , 1 Istituto Superiore di Sanità, Rome, Italy, 2 CRE-ENEA Casaccia, Rome, Italy In recent years, relatively high concentrations of vanadium pentavalent (V5 + ), above the legal limit of 50 µg/L, have been measured in drinking water in a large area surrounding the volcano Etna. In vitro studies show that micromolar concentrations of V5 + can impair chromosome segregation and induce ROS-mediated DNA damage. On the other hand, scanty data, mainly concerning acute administrations, are available on in vivo effects. In order to characterize the genotoxic hazard posed by oral exposure to V5 + , micronuclei and DNA lesions detectable by comet assay have been measured in mice receiving V5 + in drinking water along a five weeks period. Groups of five male CD-1 mice were treated with a wide range of concentrations of ortho-vanadate, corresponding to approximately 0.75, 7.5, 75 and 150 mg/kg b.w. The highest dose of Na3 VO4 was the same i.g. administered in an acute preliminary experiment. Micronuclei were analysed both in blood reticulocytes at various time intervals, and in the bone marrow PCEs. Comet assay was performed on splenocytes, bone marrow and testicular cells immediately after sacrifice. Some toxicity parameters, i.e. body weight variation and reticulocytes profile, as well as individual water and food intake were also registered. Kidney, liver, spleen, bone and testis were removed at the end of treatment to evaluate vanadium uptake by atomic absorption spectrometry. After the acute administration of Na3 VO4 at 150 mg/kg b.w. both splenocytes and bone marrow cells showed only a statistical significant increase of DNA lesions by the comet assay. On the other hand, the results obtained after the five weeks assay highlight a significant increase of micronuclei in reticulocytes and bone marrow cells of animals treated with the lowest dose. No treatment related increase of DNA damage was detected by comet assay. Further work is ongoing to confirm these preliminary findings, and to investigate whether the increase of micronuclei evidenced only at the lowest dose could be imputable to the known inhibition of apoptosis caused by low doses of vanadium. This work was partially supported by ACOSET (Azienda Consorziale Servizi Etnei) 538 Hg, Mn, Ni AND Cu LEVELS IN CANNED MUSSELS (Mytilus spp.) NORMALLY CONSUMED IN TENERIFE (CANARY ISLANDS, SPAIN) Carmen Rubio 1 , Ángel Gutiérrez 2 , Gonzalo Lozano 2 , Arturo Hardisson 1 , Tomás González 3 . 1 Toxicology Department, University of La Laguna, S/C de Tenerife, Spain, 2 Animal Biology (Marine Sciences Section) Department, University of La Laguna, S/C de Tenerife, Spain, 3 Canary Islands Government Public Healht Service, S/C de Tenerife, Spain A study of the metal content (Hg, Mn, Ni and Cu) of 600 samples of canned mussels (Mytilus spp.) has been carried out. The studied brands have been those most consumed and distributed in the island of Tenerife and correspond to six different manufacturers located in Galicia, north-west coast of Iberian Peninsule. Four different commercial presentations of the mussels has been considered: Natural, Bionature, Pickled sauce, and Coquille ST. Jacques sauce (His et al., 2000; Spangenberg and Cher, 1996). Inductively coupled plasma atomic emition spectrometry was used in the determination of Mn, Ni and Cu and cold vapour atomic absorption spectrometry in the study of Hg. The obtained Hg levels are underneath the maximum level fixed for human consumption (0,5 µg/Kg w/w). The very low concentration levels of mercury were disquised by background of analytical system. The obtained Mn, Ni and Cu contens (1.43–1.80, 4.78–8.03 and 1.40–1.76 mg/Kg, respectively) have resulted to be in the same range of those values referred by other authors. Poster Session P27. Pesticides 539 ELEMENT CONTENT IN SHREW CROCIDURA RUSSULA EXPOSED TO EFFLUENTS FROM A LANDFILL A. Sánchez-Chardi, J. Nadal. Departament de Biologia Animal (Vertebrats), Facultat de Biologia, Universitat de Barcelona, Barcelona, España Landfills sites are the most common means of disposal of solid wastes in most Mediterranean countries. The liquid effluents from such wastes contain a large variety of potentially toxic compounds such as heavy metals and organic compounds. We assess the effects of effluents from the landfill at Garraf (Barcelona, NE Spain) on the bioaccumulation of elements (heavy metals and others) in wild small mammals. In the last 30 years this site has received and accumulated different kinds of wastes (approx. 400,000 t yr−1 ) from the city of Barcelona and the surrounding area (3 million inhabitants). The greater white-toothed shrew Crocidura russula (Hermann, 1780) was used as bioindicator. Our hypothesis is that waste effluents produce a direct increase in the accumulation of toxic elements in these animals, which leads indirectly to stress in the form of metabolic alteration and cell damage. From February to May, 1998, 56 adult wild shrews were collected in 2 areas: 1-Vall d’En Joan: affected by the effluents from the Garraf landfill (n=22) and 2- Olesa de Bonesvalls: A clean control site (n=34). All animals were sexed and aged. Liver and right kidney were removed from each animal, dried, and digested by nitric and perchloric acids in a clean room. Ca, S, Zn, Mg, Mn, Cu, Sr, Rb, Sn, Mo, Pb, Cr, Co, Ba, As and V were measured by Induced Coupled Plasma (ICP-MS and ICP-AES). Data were compared according to areas, sex and age by ANOVA tests. Results showed a significant increase of Ca (p=0.003), Pb (p<0.001), Cu (p<0.001) and Cr (p<0.001) in animals from the landfill area. Cr was detected only in animals from the polluted site. In addition, there was a tendency of certain elements such as S and Mn to increase in shrews from the polluted area. No differences between sexes or ages were observed. This study confirm the entrance of Pb, Cu and Cr from the landfill to the trophic chain. We conclude that the effluents from this landfill increase the accumulation of certain elements in these fauna. The greater white-toothed shrew may be a good bioindicator of the effects of low levels of heavy metal pollution in Mediterranean areas. were within the range of values reported in literature, except for Zn, which showed the highest concentrations, higher than previously reported in literature. 541 HEAVY METALS IN LIVER OF THUNNUS THYNNUS FROM THE COAST OF THE SOUTHERN TYRRHENIAN SEA P. Licata 1 , D. Trombetta 2 , M. Cristani 2 , M. Calò 1 , A. Celona 3 , F. Naccari 1 . 1 Department of Veterinary Public Health, University of Messina, Messina, Italy, 2 Department of Biologic-Pharmacy, University of Messina, Messina, Italy, 3 Aqua-Studio Research Institute, Messina, Italy Aim of the present study was to determine in bluefin tuna (Thunnus thynnus) the levels of “toxic” (As, Cd, Hg and Pb) and “essential” (Cu, Mn and Zn) metals. In particular, our investigation was made in Thunnus thynnus (161–265 cm in length and 95–300 Kg in body weight) caugh by the deep-sea fishing in “Canale di Sicilia” during April 2002 and then stalled for six months in aquaculture centres located in coastal waters of the Tyrrhenian Sea. Quantitative analysis of Cd, Cu, Mn and Pb were carried out on 20 liver samples with an atomic absorption spectrophotometer (Varian model 220/Zeeman) equipped with graphite furnace and an air-acetylene flame for Zn; Hg was determined by the cold vapor generation technique and As was analyzed by hot vapor generation technique. Our results showed concentrations of both “toxic” and “essential” metals in line with those reported in literature. Among “toxic” metals, Cd concentrations (0.07–2.17 µg/g w.w.) were higher than other metals (As, Hg and Pb); while Pb levels (0.02–0.30 µg/g w.w.) were lowest. Concentrations of As and Hg (0.06–2.99 µg/g w.w. and 0.08– 1.86 µg/g w.w.) showed intermediate values respectively. Among the “toxic” metals studied, only Hg levels were lower than those reported for various coastal areas of the Mediterranean. As regards the “essential” metals, Zn concentrations (47.9–386.9 µg/g w.w.) were higher than other metals (Cu and Mn); Cu levels were lowest (0.05–3.26 µg/g w.w.) while Mn showed intermediate values (0.98– 5.71 µg/g w.w.). Altogether, the data obtained for “essential” metals FLUCTUATION OF SOME HEAVY METALS THROUGH TWO ECOSYSTEMS S.D. Brankovic 1 , D.B. Blagojevic 2 , S. Stankovic 3 , B. Petrovic 3 . 1 Institute for protection of nature of Serbia, dept. Nis, Nis, 2 Faculty of occupational safety, Nis, 3 Institute of public health Nis, Yugoslavia Point of this research was following concentration of lead, copper, cadmium, zinc and manganese in samples (soil, plants, tissues of pheasants and rabbits) taken from hunting ground Nis (South Serbia) and hunting ground Velika Greda (Vojvodina). The first sampling point is Vinik hill, near large city- Nis where probable sources of metals are industry and traffic. The second sampling point is small town with very high level of agricultural activities. Samples were taken during December 2000 and kept frozen at -20° C. Samples were analyzed by AAS method in flame. Results show that some samples are very close or cross over maximal aloud values according to the Yugoslav laws: Nis-kidneys of rabbit 0.0923 mg/kg (Cd), Backa Palanka-Soil 11.4542 mg/kg (Cd), 492.7576 mg/kg (Mn), plants 4.0271 mg/kg (Cd), muscle of pheasant 0.2066 mg/kg (Cd), liver of rabbit 0.5304 mg/kg (Cd), muscle of rabbit 0.2871 mg/kg (Cd). Our experimental approach to this problem will provide fundamental information on how these of contaminants interact, and provide the basis for making ecologically sound decisions concerning appropriate bioremediation or mitigation strategies for contaminated field sites, as well as base for making a model for risk assessment that considers the population which use meat of these animals as food. P27 Pesticides 542 540 s145 GENOTOXICOLOGICAL AND BIOCHEMICAL HAMFULL EFFECTS FOR THE ORGANOPHOSPHOURUS PESTICIDE NUVACRONE ON ALBINO MICE AND THEIR EMBRYOS K.B. Abd el Aziz, E.M. El Nahas, M. Zahran, A. Abdel Raoof. Cell Biology Department, National Research Center, Giza, Egypt Nuvacrone, which is one from the most fast acting organophosphorous pesticides, was tested for its genotoxicological effects (chromosomal abnormalities) on adult males, pregnant females of mouse as well as their embryos. Some biochemical parameters such as changes in Nucleic acid concentrations, total protein and enzyme activity, were also studied. For conducting the study, three different doses were orally administrated (0.65; 0.25; 0.30 mg/Kg) a parallel control group was kept at the same time. Concerning the chromosomal abnormalities, the observed types were mainly in the form of gaps, breaks, centromeric attenuations, centric fusions, end to end associations, deletions and endomitosis. In contrast, polyploidy cell was the only observed type of chromosomal abnormality. All the treatments showed a highly significant increase over the control group (P<0.001) for the recorded structural abnormalities. It was also observed that the percentages of the abnormalities were dosedependent. Abnormalities in germ cells (spermatocytes) of treated males showed also a highly significant increase over the control for the 3 doses tested; also the percentages of the abnormalities were dose-dependent. The observed types of abnormalities were autosomal univalents, chains, fragments, ring and ploidy spermatocytes. Regarding biochemical studies, a significant decrease below the normal level in the concentrations of plasma proteins, DNA and RNA was observed. Cholinesterase enzyme activity was also decreased significantly. In contrast, a gradual increase in gamma–glutamyl transferase activity as the dose of pesticide increased. The results indicated that the Nuvacrone organophosphorous pesticide, has a genotoxicological and biochemical harmful effects, attention should be paid towards it. s146 543 Poster Session P27. Pesticides DOSE-DEPENDENT EFFECTS EXERTED IN VITRO BY CHLORPYRIFOS ON THE OXIDATIVE BURST OF HUMAN PERIPHERAL GRANULOCYTES D.L. Baconi 1 , M. Manda 2 , M. Neagu 2 , M. Ilie 1 , D. Balalau 1 . Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Bucharest, Romania; 2 “Victor Babes” National Institute, Bucharest, Romania 1 “Carol Chlorpyrifos is an organophosphate insecticide having a broad spectrum. Overall, relatively little is known about the immunotoxic effects of this organophosphate insecticide. Aim: The immunotoxic potential of 1–10ng/mL chlorpyrifos with respect to the oxygen-dependent cytotoxic functions developed in vitro by human peripheral granulocytes. Methods: Human granulocytes (PMNs) isolated from venous blood were in vitro activated with the bacterial tripeptide fMLP and treated with the organophoshoric compound chlorpyrifos in the dose range 1–10 ng/106 PMNs/mL. We have investigated the oxidative burst developed in vitro by PMNs, namely The intracellular generation of superoxide anion, measured by the TBN reduction test as % PMNs with intracellular oxidative activity The release of superoxide anion, measured by the cytochrome c reduction test as difference of optical densities at 550 nm and 535 nm. Cellular functions were investigated for resting and fMLPactivated PMNs, in the absence or presence of chlorpyrifos. Results: Experimental data show that chlorpyrifos exerts both inhibitory and stimulatory effects on the oxidative burst of resting PMNs in a dose-dependent manner. A negative correlation between the proportion of PMNs with intracellular oxidative burst and the intensity of radical release was emphasized. Cellular functions triggered by fMLP are less affected by chlorpyrifos, indicating the dominance of the stimulatory signals delivered by fMLP. Conclusion: Our in vitro conducted study shows that the effects exerted by chlorpyrifos on the respiratory burst of PMNs are dependent on the dose of the compound and on the cellular activation status. A marked individual variability was also noticed. Chlorpyrifos might impair the oxygen-dependent antibacterial defence mechanisms, while potentiating the extracellular oxidative stress, thus exerting harmful effects on the non-specific immune response. We highlight that, at particular doses, the investigated organophosphate compound intensifies the cytotoxic mechanisms developed by PMNs and hinders the tissue-damaging oxidative stress. This unexpected beneficial behaviour has to be further clarified. 544 METABOLISM OF CHLORPYRIFOS AND DIAZINON BY HUMAN LIVER MICROSOMES C. Sams 1 , J. Cocker 1 , M.S. Lennard 2 . 1 Health and Safety Laboratory, Broad Lane, Sheffield, UK, 2 Academic Unit of Molecular Pharmacology and Pharmacogenetics, University of Sheffield, Sheffield, UK Chlorpyrifos (CPS) and diazinon (DZ) are extensively used organophosphate (OP) insecticides. Their established toxic effects arise from cytochrome P450 (CYP)-catalysed bioactivation to the phosphate ester or ‘oxon’ (chlorpyrifos-oxon (CPO) or diazinon-oxon (DZO)), which are potent inhibitors of the enzyme acetylcholinesterase. CPS and DZ are also metabolised to non-toxic hydrolysis products (trichloropyridinol (TCP) and isopropylmethylpyrimidinol (IMP) respectively). The balance between rates of bioactivation and detoxification may be important for identifying sensitive sub-populations. This study has therefore investigated inter-individual variation in rates of biotransformation of CPS and DZ in vitro using liver microsomes prepared from individual human donors (HLM). CPS and DZ, over the concentration range 3–100µM, were incubated with HLM (n=5) and the metabolites CPO or DZO and TCP or IMP were determined by HPLC. The role of individual CYPs in this reaction was investigated using chemical inhibitors and recombinant CYPs. Both CPS and DZ biotransformation exhibited greater intrinsic clearance (CLint ; Vmax /Km ) for detoxification than for bioactivation. However, considerable variation was observed in CLint between individuals. For CPS biotransformation, mean CLint of CPO formation was 14µl/min/mg protein (range 7–24; n=5) and mean CLint of TCP production was 62µl/min/mg protein (37–101; n=5). CLint values for DZ biotransformation were 17 (15–23) for DZO production and 44 (35–62) for IMP formation. These data indicate that, generally, human liver contains higher capacity to detoxify OP than to bioactivate it. Preliminary experiments indicated that recombinant CYP2B6 exhibited the highest activity to bioactivate CPS to CPO and that CYP2C19 exhibited highest activity to detoxify CPS to TCP. Variability in activity of these CYPs may influence individual susceptibility. In particular, individuals possessing the CYP2C19 PM phenotype may be less able to detoxify CPS, and thus could be more susceptible to its toxic effects. 545 EFFECTS OF A NEUROTOXIC PESTICIDE ON VOLTAGE-DEPENDENT K+ CHANNELS OF RAT CEREBELLUM GRANULAR CELLS A. Murgia, G. Prestipino. Institute of Biophysics – CNR, via De Marini 6, 16149 Genova, Italy A class of contaminants diffused over the world is represented by neurotoxic substances, in particular organophosphate and carbamate pesticides. These are largely employed in European countries for many purposes, including agricultural, garden and even domestic pest control. Among these, the organophosphate compounds have been studied for a wide range of aims: from chemical weapon, to pest control and also medical compounds (anxiolytic, antispasmoic, regulators of eye pressure, etc.). These pesticides interfere with the status of cholinergic transmission in the nervous system of their target, and affect human health because of impairment of neuronal development, as well as of memory and psychomotor speed and affective symptoms. At long term, nervous system disorders may occur: for instance, increasing the incidence of Parkinsonism. Furthermore, neurotoxic pesticides effects are directed towards embryonic development as shown by experiments on invertebrates and vertebrates differentiation. Our work has been focused on the neurotoxin organophosphate Cidial, the commercial pesticide used in agriculture with the aim of to understand the action mechanism at molecular level, describing its effects on neuron ion channels. The pesticide has been tested on K+ currents of cerebellum granular cells in primary culture obtained from 7-day-old Wistar rat, using the patch-clamp technique in the whole-cell configuration. We have characterized the interaction of this compound on the two main components of the outward currents: a slow activating current characterized by nonactivating kinetics (Id ) and the second one characterized by fast activating and inactivating kinetics (IA ). Cidial has shown to inhibit both the components of potassium currents in the cultured neurons, and the experiments with atropine suggest that these currents are modulated by muscarinic receptors. 546 DITHIOCARBAMATE PROPINEB AND DISULFIRAM DEPOLYMERIZE ACTIN AND INCREASE ACETYLCHOLINE RELEASE IN DIFFERENTIATED PC12 CELLS S. Bartesaghi, M. Binaglia, B. Viviani, Corrado L. Galli, M. Marinovich. Department of Pharmacological Sciences, Center of Excellence on Neurodegenerative Diseases, University of Milan, Italy Propineb is mainly used in Europe as fungicide for a broad range of fruit and vegetable crops. The related dithiocarbamate (DTC) disulfiram has been used for about 50 years in alcohol aversion therapy. Neurological complications as well as distal neuropathy and muscular weakness are the prevailing symptoms in animals and humans chronically exposed to DTCs. We recently showed that propineb interferes with cholinergic transmission on longitudinal muscle + myenteric plexus preparations. This study was designed to investigate the molecular mechanisms involved in propineb and disulfiram effect. Propineb 1 pM - 100 nM induced acetylcholine (ACh) release from rat pheochromocytoma cells (PC12), treated with NGF to obtain the differentiation into sympathetic neuron-like cells. Poster Session P27. Pesticides ACh release dose-dependently increased reaching maximal effect within 1 nM propineb. Disulfiram caused a similar ACh increased release, reaching the maximal effect at 100 pM. Further increases in propineb and disulfiram concentrations caused a progressive disappearance of the effect. The propineb-induced increase in ACh release was not prevented by Ca2+ -free conditions. Treatment of PC12 cells with propineb 100 pM and 1 nM induced a significant actin depolymerization with a calcium-independent mechanism. The same effect was obtained with 10–100 pM disulfiram. Pretreatment of PC12 cells with 500 nM Jasplakinolide, a peptide that binds and stabilizes filamentous actin in vitro, prevented actin depolymerization and propineb-evoked ACh release. The data suggest that DTCs may interfere with cholinergic transmission through an impairment of cytoskeletal actin structure and consequently affecting synaptic vesicles processing. 547 THE EFFECTS OF NEUROTOXIC INSECTICIDES ON THE CENTRAL CHOLINERGICALLY-MEDIATED HYPERTENSION IN RATS Jasmina Stankovic 1 , S. Mitovanovic 1 , V.M. Varagic 2 . 1 Institute for Medical Research, Military Medical Academy, Belgrade, 2 Department of Pharmacology, Faculty of Medicine, Belgrade Physostigmine has been known to produce a central cholinergically mediated hypertension (CCMH) in rats. This effect was shown to be due to a central interaction between cholinergic and adrenergic mechanisms, which finally produce a blood pressure rise (I). Taking into account the fundamental importance of this interaction, it was decided to study the effects of three neurotoxic insecticides (lindan, malathion, permethrine) on the CCMH in rats. The blood pressure of the rats was recorded directly from the carotid artery and all the drugs were injected intravenously. It was found that malathion (80 to 160 µg/kg) produced a significant decrease of CCMH and of its duration. This finding is similar to that already earlier obtained with paraoxon. Malathion is an irreversible inhibitor of acetylcholine esterase, thus leaving no functionally competent enzyme for the action of physostigmine. Malathion by itself produced a shortlasting fall in blood pressure, but this effect in some experiments was followed by a longlasting increase. Both lindan (8 to 40 µg/kg) and permethrine (40 to 200 µg/kg) by themselves also produced a shortlasting fall in blood pressure, followed by a longer lasting hypertension in some experiments. Meanwhile, both substances produced a depression of CCMH but this effect was not related to the doses of insecticides used. Thus, all the three insecticides, in the doses used, produced a decrease in CCMH. This effect might be due to a depression of cholinergic-adrenergic interaction in the central nervous system of the rat but its precise mechanism remains to be determined. samples were collected at intervals of 10 days during the course of study to assess the apoptosis in peripheral blood mononuclear cells. At the end of the experiment, apoptosis was also assessed in spleenocytes. Apoptosis and cytotoxicity were detected by DNA gel electrophoresis, fluorescent microscopy and flow cytometry and cell viability was determined by trypan blue dye exclusion. The results tend to suggest that endosulfan may induce apoptosis in peripheral blood mononuclear cells and spleenocytes in chickens. 549 ENDOSULFAN-INDUCED APOPTOSIS IN CHICKENS M.K. Aggarwal 1 , A.K. Tiwari 2 , G.S. Rao 1 , J.K. Malik 1 . 1 Division of Pharmacology and Toxicology, 2 National Biotechnology Center, Indian Veterinary Research Institute (IVRI), Izatnagar- 243 122 (UP), INDIA Increased demand for food and fiber has led to the intensive use of pesticides in agriculture, which resulted in environmental pollution. Residues of the environmental pollutants particularly the pesticides have been reported in various edible products. The presence of these chemicals and/or their metabolites finally affect the animals, birds and humans and held responsible for various kinds of immunotoxicological alterations leading to immunosuppression, recurrent infections, vaccination failures, increased incidence of cancer and disease outbreaks. Several organochlorinated pesticides have been reported to cause immunotoxicity. Endosulfan is one of the organochlorinated pesticides that is widely used in various agricultural operations. It leaves its residues in food grains and other edible products, which may lead to health hazards in animals and human beings. The present study was carried out to study the effect of endosulfan on immune system of the chickens. Twenty one-day old chicks were randomly divided into two groups of 10 chicks each. Group I chicks were kept as control and Group II chicks were fed endosulfan @ 30 ppm in feed for a period of 60 days. Blood LAMBDA-CYHALOTHRIN’S INFLUENCE ON MEMORY PROCESSES, MOVEMENT CO-ORDINATION AND SPONTANEOUS MOVEMENT ACTIVITY IN MICE EXPOSED TO TRANSIENT OLIGEMIC BRAIN HYPOXIA IN BCCA MODEL. Barbara Nieradko, Andrzej Borz˛ecki. Department of Hygiene, Medical University in Lublin, Poland Lambda-cyhalohrin is one of synthetic pyrethroids of family of compounds with α-cyano-3-phenoxybenzyl moiety. The aim of the work was to evaluate the influence of lambda-cyhalothrin on memory processes, movement co-ordination and spontaneous movement activity in mice exposed to transient oligemic brain hypoxia in BCCA model. There were four groups of animals examined: I) sham-operated, II) after BCCA, III) sham-operated, treated with beta-cyfluthrin, and IV) after BCCA, treated with beta-cyfluthrin. Bilateral clamping of carotid arteries (BCCA) is an experimental model of transient ischemic attacks (TIAs), that occur in humans. 24 hours after the surgery, the mice had a training in the passive avoidance task. The next day the animals from group III and IV were injected with 0,1 LD50 lambda-cyhalothrin intraperitoneally. 30 minutes after administration the animals were examined in the passive avoidance task. Then, their movement co-ordination on a rota- rod was examined. After that the mice were placed in a Y maze to examine their spontaneous movement alterations and later, their spontaneous movement activity was checked. Results obtained were analysed with Anova and the post hoc tests. There is a statistically significant difference (p<0,05) in spontaneous movement activity within first 30 minutes of examination in group IV versus I and after 60 minutes in group IV vs all the others and in group II vs sham. Conclusions: 1. Memory retention is most impaired by lambdacyhalothrin in sham-operated animals whereas BCCA-procedure protects their brains from pesticide’s toxic action. 2. Lambdacyhalothrin’s effect on fresh spatial memory and spontaneous motor activity is enhanced by BCCA. 550 548 s147 INTERACTION OF ESTROGEN WITH PYRETHROID COMPOUNDS IN THE MCF-7 HUMAN BREAST CARCINOMA CELL LINE I. Kakko 1 , T. Toimela 1 , H. Tähti 1 . 1 University of Tampere, Medical School, Tampere, Finland Pyrethroids are the most widely used insecticides for indoor pest control. Their action on the central and peripheral nervous systems is well known. There is no clear indication of carcinogenic effects, except an induction of abnormal cell division in cell cultures. However, some studies show that exposure to mixtures of commercial pyrethroid compounds alters the activity of xenobiotics-metabolizing enzymes in the liver and other organs, which possibly leads to changes in the proliferation of cells. In the present study our aim was to investigate cell activity and cell proliferation after exposure to chemically different pyrethroids. The compounds used were natural pyrethrin and the two synthetic pyrethroids permethrin (no alpha cyano group) and cypermethrin (with alpha cyano group), which were studied with or without 0.1 nM estradiol treatment. The MCF-7 human breast carcinoma cells were cultured on 96-well microtiter plates. After a 7-day exposure time, cell proliferation was evaluated by determining the total ATP with a luminescence method, and the mitochondrial metabolic activity (MMA) with WST-1-test (tetrazolium reduction test). Exposure to 0.1–100 µM of pyrethrin, permethrin and cypermethrin showed dose-dependent increase in cell activity and cell s148 Poster Session P27. Pesticides proliferation in both assays. Exposure with estradiol potentiated the effect markedly. The most effective compound was cypermethrin. When estradiol was present at 0,1 µM concentration of cypermethrin, the total ATP was 350%, and MMA was 145% of control. Without estradiol, the total ATP was 124% and MMA about 100% of control. The corresponding results for 0.1 µM permethrin were 310 %/120% (total ATP with/without estradiol) and 130%/100% (MMA with/without estradiol) and those for natural pyrethrin were 330%/130% (total ATP) and 140%/100% (MMA), respectively. At 1-µM concentrations of natural pyrethrin and permethrin with estradiol, the total ATP started to decrease from the peak value, but was still very high till 25 µM. The 10-µM cypermethrin concentration was already cytotoxic. Without estradiol, permethrin and pyrethrin had no increasing effect on MMA. Our results show that pyrethroids have a clear effect on cell proliferation and viability, and that estrogen potentiates the effects of pyrethroids in human breast cancer cell cultures. 551 GENOTOXIC POTENCY OF THE COMBINATION OF CARBENDAZIM AND CAPTAN FORMULATIONS AND THEIR INTERACTION, DETERMINED BY THE IN VIVO BONE MARROW MICRONUCLEUS TEST M. Goumenou, K. Machera. Laboratory of Pesticides Toxicology, Benaki Phytopathological Institute, Kifissia, Athens, Greece Captan (CA) and carbendazim (CB) are two widely used fungicides applied as individual formulations, as a commercially available premixture or as spray tank mixtures. CB is a well-known aneugen while CA is not considered as an in vivo genotoxic substance. For the study of the in vivo genotoxic potency of the combination of CA and CB formulations (CAF, CBF) as well as their possible interaction, 2 separate experiments were performed using the bone marrow micronucleus test in rats. In both experiments, 3 dose levels of the mixture were studied (977, 1563 and 2500 mg of each active ingredient/kg b.w.). Sampling time for all test groups was 24h while for the highest dose group samples were also taken at 48h. The individual formulations were tested at 2500 mg a.i./kg b.w, at 24 and 48h sampling time. For the study of possible interaction, the dose-response curve of the CBF at the above mentioned dose levels (24h post treatment of male rats) and the respective curve for the same CBF dose levels with constant addition of the CAF at 2500 mg a.i./kg b.w were established. Regarding the MN incidence in comparison to the control group, a significant increase was observed in all CBF groups at 24 and 48h. A slight, statistically non-significant increase at 24h in CAF alone and a marginal increase in some combination groups were observed. CBF and the combination were cytotoxic at 48h while CAF was cytotoxic at both 24 and 48h. The dose-response curve for micronuclei formation following CBF administration was well fitted to the cubic model. Comparing this curve with the curve established after constant addition of 2500 mg a.i. / kg b.w. of CAF to the CBF doses, a considerable reduction of the MN incidence induced from CBF was observed. Additionally, the MN incidence from mixture groups with different composition from those that formed the above-mentioned curve was lower than every CBF dose group. Consequently, the 2 fungicide formulations seem to interact resulting in a reduction of the CBF genotoxicity. technique (AET) were applied. The MNT was performed according to the OECD protocol with minor deviations. According to several statistical tests, no statistically significant increase in MN incidence was observed in any test group, while cytotoxicity was observed from both individual formulations and their combination. No evidence of interaction was observed. The alkaline elution technique for the determination of single strand breaks (SSB) was performed in rat liver tissue according to the principles of kohn’s method, as modified and validated in our laboratory. Both paraquat and linuron formulations induced SSB in liver DNA 24h post treatment. From the established dose-response curves at 3 dose levels (20%, 40% and 80% of the respective LD50), a dose related increase of the elution constant was observed. Both curves were well fitted to the 2nd degree polynomial model with R2 > 0.99. These findings are consistent with the very limited published experimental data and the toxicological profile of both substances. Studying the genotoxic potency of the formulations’ combination, a mixture containing 40% of the LD50 of each compound was administered, assuming that this doses level for the combination approaches the MTD of the mixture. The DNA damage, caused by paraquat and linuron individually, was clearly reduced when their formulations were administered as the above mixture. Their possible interaction was evaluated according to both dose additivity and response additivity criteria. 553 J.H. Kim 1 , H.W. Park 1 , J.K. Moon 1 , H. Choi 1 , H.S. Lee 2 , K.H. Liu 3 . 1 School of Agricultural Biotechnology, Seoul National University, Suwon 441–744, Korea; 2 College of Pharmacy, Wonkwang University, Iksan, Chonbuk 570–749, Korea; 3 College of Medicine, Inje University, Busan 614–72–35, Korea Metamifop is a new herbicide developed by the Dongbu Hannong chemical, Korea. This Aryloxyphenoxy propionate is known to inhibit ACCase which is involved in the biosynthesis of fatty acid in plants. The metabolism of metamifop by human, rat and mouse microsomes was studied to compare the metabolic pathways. Nine metabolites were found in human microsomal reaction with metamifop and six were identified by LC-MS/MS. They were N-(2-Fluoro-phenyl)-2-(4-methoxy-phenoxy)-N-methylpropionamide (M1), 6-Chloro-3H-benzooxazol-2-one (M2), N-(2Fluoro-phenyl)-2-(4-hydroxy-phenoxy)-N-methyl-propionamide (M3). N-(2-Fluoro-phenyl)-2-(4-hydroxy-phenoxy)-propionamide (M4). 2[4-(6-Chloro-benzooxazol-2-yloxy)-phenoxy]-propionic acid (M5), 2-[4-(6-Chloro-benzooxazol-2-yloxy)-phenoxy]-N-(2-fluoro-phenyl)propionamide (M9). Four metabolites were identified from six metabolites from mouse microsomal reaction. They were M1, M2, M3, and M5. Unknown metabolite M8 in human microsomal reaction was also observed and a new metabolite M10 formed. Three metabolites M1, M2 and M3 were identified from six metabolites from mouse microsomal reaction. Three unknowns were M7 in human microsomal reaction and two new metabolites M11 and M12. In general, M1, M2 and M3 were common metabolites while M4 and M9 were unique for human microsomal reaction. 554 552 IN VIVO GENOTOXICITY OF THE BINARY MIXTURE OF PARAQUAT AND LINURON FORMULATIONS AND THEIR INTERACTION M. Goumenou, K. Machera. Laboratory of Pesticides Toxicology, Benaki Phytopathological Institute, Kifissia, Athens, Greece In the agricultural practice, commercial formulations of paraquat and linuron are very often applied as a binary mixture for weed control. Taking into account the lack of genotoxicity data for the whole product, as it is applied by the operators and the lack of any toxicological data for mixtures of the active ingredients, the in vivo genotoxicity of the mixture of the two plant protection products as well as their possible interaction was studied. For this purpose, the bone marrow micronucleus test (MNT) and the DNA alkaline elution IN VITRO BIOTRANSFORMATION OF A HERBICIDE METAMIFOP BY HUMAN, RAT AND MOUSE MICROSOMES ACCUMULATION OF CHLOROPHENOXY HERBICIDE RESIDUES WITHIN CEREAL TISSUES R. Krzyzanowski, B. Leszczynski, A. Bednarczyk. Department of Biochemistry, University of Podlasie, Siedlce, Poland Aminopielik D450SL and Chwastox 300 SL are the most popular herbicides used in cereal protection in Poland. However, there is no complete information on accumulation of these herbicides within the cereal tissues. In the present paper we report on accumulation of biologically active chlorophenoxy herbicides from the Aminopielik D450SL and Chwastox 300SL within tissues of the winter wheat cultivar Roma. Dichloromethane extract of the wheat seedlings was performed. The extracts were evaporated to dryness and dissolved in a small volume of methanol. Then the chlorophenoxy herbicides were analyzed using gas chromatography combined with mass spectrometry. Poster Session P27. Pesticides The chlorophenoxy herbicides showed different accumulation in case of (4-chloro-2-methylphenoxy)acetic acid (MCPA) and (2,4dichlorophenoxy)acetic acid (2,4-D). The 2,4-D was not detected within the wheat tissues at all. The highest concentration of MCPA was found within the wheat tissues 2 hours after treatment and was declined. After next twelve hours the level of the herbicides was undetectable. The MCPA accumulation within the wheat tissues was clearly related to applied doses of the studied herbicide. effects, but parameters of such effects were more expressed in the level of small doses than in high doses. Therefore we suppose that it is necessary to develop the new approaches for testing of GRP instead of traditional, which are used for all pesticides. First of all, such tests have to include more widely range of low doses and additional tests for study of target-organs. 557 555 SOLID PHASE MICROEXTRACTION OF PHENOXY HERBICIDES RESIDUES FROM AQUEOUS SAMPLES R. Krzyzanowski, B. Leszczynski. Department of Biochemistry, University of Podlasie, Siedlce, Poland Solid phase microextraction (SPME) is one of the most promising method in analyzing of the herbicide residues. However, an application of the SPME method requires determination of the proper analytical conditions. In the present paper we report on standarization of the solid phase microextraction of the phenoxy herbicides: 2-(4-chloro-2-methylphenoxy)propionic acid (MCPP), (4-chloro-2-methylphenoxy)acetic acid (MCPA), 2-(2,4dichlorophenoxy)propionic acid (2,4-DP), (2,4-dichlorophenoxy)acetic acid (2,4-D). Carbowax/PDMS fiber was used to study: time, temperature and ionic strength of the extraction and temperature and time of the herbicides desorption. The adsorption profile was studied by monitoring the area counts as a function of exposure time in the range from 5 to 30 min. The temperature and time required to completely desorption all the analytes from the coating fiber was determined for 10 min with temperature programmed from 140°C to 240°C. The obtained results showed the highest absorption of the MCPA after 20 min, the concentration of 0.0035–0.0072 µg/l and 0.0052– 0.0110 µg/l for the MCPP and MCPA, respectively. The best absorption time for the 2,4-DP and 2,4-D was after 15 min, when the concentration was 0.0007 µg/l 2,4-DP and 0.0110 µg/l of the 2,4-D. Standard condition for desorption of MCPP and MCPA was as follow: desorption temperature 180°C and desorption time 6 min, instead desorption time from 2,4-DP and 2,4-D between 4–8 min. The performed standarization procedure showed that SPME is an useful method for extraction of the phenoxy herbicide residues. 556 EVALUATION OF CARCINOGENIC ACTIVITY OF PLANTS GROWN REGULATES O. Reshavska, E. Bagliy, N. Nedopytanska. Medved’s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine The grown regulates of plants (GRP) are perspective biological active chemicals which are widely used in an agriculture to increase of productivity, to increase of disease resistance. GRP have combined such good qualities as high biological activity, low toxicity for experimental animals and low use rates. Thus, GRP considerably reduce a chemical pressure on an environment. At the same time, the remote effects of its action, including carcinogenicity is not clear until now. It may be because from one hand it is well known, that the chemical structure of many synthesized GRP looks structural similarly to natural compounds of a metabolism, from other hand, the levels of their residues in an environment very low. In Ukraine some effective GRP of different chemical classes have been synthesized and widely use in an agriculture. We have studied carcinogenic activity some of them by means of the different models and tests. Sulphonylurea derivatives (“Ellips” – chlorsulfoxym-methyl diethylethanolamine, “Krug” - sulphonylurea diethanolamine salt), N-oxid-piridines (“Ivin” – N-oxid2,6-dimethylpiridine, “Triman-1” - aqua-N-oxid-2-methylpiridine marganec-2-chloride) and amidines (“Simarp” – dichlorhydrate N(3-dimethylamino) propylthrycloracetamidine) had studied by means of micronuclear test, alkaline unwinding assay DNA in different cells, NDEA-partial hepatectomy model, lung tumors induction in strain BALBc mice assays and traditional chronic experiments in rodents. All of testing substances did not cause genotoxic and oncogenic s149 BIOCHEMICAL IDENTIFICATION OF ESFENVALERATE TOXICITY ON THE LIVER OF OREOCHROMIS NILOTICUS Elif Oruç, Nevin Üner, Tüzin Aytekin, Yusuf Sevgiler. University of Çukurova, Faculty of Arts and Science, Department of Biology, Balcalı, Adana, Turkey The aim of the present study was to determine the LC50 value and the mode of action of the endocrine disrupter pyrethroid esfenvalerate and to find out the level of liver damage caused by this insecticide and its relation with oxidative stress in Oreochromis niloticus. Fish were exposed to different concentration of the insecticide over three periods and the activity of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase together with the activities of acetylcholinesterase, sodium-potassium adenosine triphosphatase, glutamic pyruvic transaminase and the content of malondialdehyde were determined by using spectroscopic techniques. The 96 hour LC50 value of esfenvalerate was found as 5.06 ppb for the first time in the present study. At high concentrations of esfenvalerate superoxide dismutase activity increased and that of glutathione peroxidase decreased with time. Esfenvalerate did not affect the activities of catalase, acetylcholinesterase, sodiumpotassium adenosine triphosphatase, glutamic pyruvic transaminase and the content of malondialdehyde. It was determined that esfenvalerate has a high toxicity toO. niloticus. Its chronical effect in high concentration might have caused oxidative stress. It was not a neurotoxic agent for O. niloticus and did not cause liver damage. 558 TOXICOLOGICAL CRITERIA IN RISK ASSESSMENT OF NONLETHAL PESTICIDES POISONINGS N. Prodanchuk, P. Zhminko, S. Sergeyev, A. Kravchuk. L.I. Medved Institute of Ecohygiene and Toxicology, Kiev, Ukraine Nonlethal poisonings is the most frequently meeting displays of pesticides toxic action on agricultural workers. As example is an acute group poisoning of 58 workers, which was exposed of 2,4-Ddimethylammonium herbicide aerosol when neighboring field was sprayed. Initial symptoms of a poisoning were shown as headache, dizziness, weakness in extremities, nausea, vomiting, burning of a face skin and mucous membranes, in the subsequent - asthenovegetative syndrome, vegetosensory polyneuropathy of upper and lower extremities, toxic cardiomyopathy, hepatopathy. 2,4-D-dimethylammonium herbicide formulation did not cause death of laboratory animals at inhalation (investigated concentrations up to 2000 mg/m3 ) and dermal (up to 2000 mg/kg) exposure. It is established that concentration of substance in air of a breath zone at the moment of a poisoning could exceed 2,4D-dimethylammonium maximum concentration limit in working zone air (1 mg/m3 ) in 2.5 times. This concentration corresponded theoretically to a range of chronic action threshold for similar a.i. (from 10 up to 15 mg/m3 ), which is lower than an acute action threshold, at least in 2–3 times. If to take into account these data the poisoning was poorly probable, however it has taken place. The received data testify, that an estimation of pesticides formulations, even with well-known a.i., taking into account only lethal effects at inhalation and dermal exposure, does not allow to reveal functional disorder of organs and systems and to predict an opportunity of nonlethal poisonings. The outlines of toxicological experiment, which allows to reveal functional disorder of organs and systems at single and repeated exposure of pesticides at nonlethal doses level, active ingredient and pesticides formulations toxicodynamics distinctions, to establish criteria for risk assessment of acute, subacute and chronic poisoning at inhalation and dermal exposure of pesticides are offered. s150 Poster Session P28. Biotransformation P28 Biotransformation 559 MECHANISTIC STUDIES OF COVALENT HEME BINDING TO CYP4B1 Y. Zheng, B.R. Baer, M.B. Kneller, K.R. Henne, K.L. Kunze, A.E. Rettie. Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington 98195, USA CYP4B1 is largely an extrahepatic P450 isoform capable of detoxification and bioactivation of an enormous range of endogenous and exogenous substrates. Recently we found that rabbit CYP4B1, and several other members of the CYP4 family of enzymes, are covalently linked to their heme prosthetic group through an ester linkage. In the current study, we mutated two conserved CYP4 I-helix residues, E310 (to G, A, or D) and T314 (to K) in rabbit CYP4B1 to examine effects on the extent of covalent heme binding and catalysis. All mutants expressed well in insect cells. Rates of metabolism decreased in the order E310 > A310 ≫ G310 > D310 > T314K, with the A310 and G310 mutants exhibiting alterations in regioselectivity for ω-1 and ω-2 hydroxylation of lauric acid, respectively. The T314K mutant was completely devoid of activity due to formation of a nitrogenous ligand to the heme, as determined from its novel spectroscopic properties. In marked contrast to the wildtype E310 enzyme, none of these mutants bound heme covalently. Uniquely, the acid-dissociable heme obtained from the D310 mutant contained an additional 16 amu relative to heme and exhibited the same chromatographic behavior as the monohydroxyheme species released upon base treatment of the covalently linked wild-type enzyme. Expression studies with H18 2 O demonstrated incorporation of the heavy isotope from the media into the monohydroxyheme isolated from the D310 mutant at a molar ratio of approximately 0.8:1. These data demonstrate that, (i) E310 serves as the site of covalent attachment of heme to the protein backbone of rabbit CYP4B1; (ii) this I-helix glutamate residue influences substrate orientation in the active site of CYP4B1; (iii) covalent heme binding to CYP4B1 is related to enzyme catalysis; and (iv) the mechanism of covalent heme attachment most likely involves a carbocation species located on the porphyrin. 560 COMPARATIVE HEPATIC METABOLISM STUDIES OF 4-BROMO-2,5-DIMETHOXYPHENETHYLAMINE (2C-B) IN SIX SPECIES INCLUDING HUMAN USING CRYOPRESERVED HEPATOCYTES Helena Carmo 1 , Jan G. Hengstler 3 , Douwe de Boer 2 , Michael Ringel 3 , Fernando Remião 1 , Félix Carvalho 1 , Eduarda Fernandes 1 , Lesseps A. dos Reys 2 , Franz Oesch 3 , Maria de Lourdes Bastos 1 . 1 REQUIMTE, Toxicology Department, Faculty of Pharmacy, Porto University, Portugal. 2 Laboratory of Doping Control, Lisbon Sports Medicine Centre, Portugal. 3 Toxicology Institute, Mainz University, Germany 4-Bromo-2,5-dimethoxyphenethylamine (2C-B) is an amphetamineand mescaline-like designer drug that is often, but wrongly, given the general name “Ecstasy”. Its abuse has been reported in Europe and USA and it has been recently recommended for international control. Concern has been raised because little is known about its toxicity and metabolism in humans. In the present study male human, monkey (Cynomolgus), dog (Beagle), rabbit (Chinchilla), rat (Sprague-Dawley) and mouse (CD1) cryopreserved hepatocytes were incubated with 2C-B in order to identify the metabolites formed and to determine possible toxic effects using an ATP assay. The main hepatic metabolic pathways of 2C-B in humans were elucidated and (i) 4-bromo-2,5-dimethoxyphenylacetic acid, (ii) 4-bromo-2-hydroxy-5-methoxyphenylacetic acid, (iii) 4-bromo2,5-dimethoxybenzoic acid, (iv) 2-(4-Bromo-2,5-dimethoxyphenyl)ethanol, and (v) 2-(4-bromo-2-hydroxy-5-methoxyphenyl)-ethanol were identified as the main metabolites. Only minor interspecies differences in metabolism were observed. A reduction in ATP content after incubation with 1000 µM 2C-B was observed for all species, whereas 100 µM 2C-B represents a non-toxic concentration. Comparing the toxic effects of 2C-B between hepatocytes of the six examined species only minor interspecies differences were observed. However, large interindividual differences were found in one batch of human hepatocytes, thus suggesting the possible existence of human subpopulation(s) having an increased risk when exposed to this amphetamine-like substance. Acknowledgements: Helena Carmo wishes to thank FCT for financial support (PRAXIS XXI/BD/20088/99). 2C-B was a generous gift of the United Nations - Scientific Section, PDAB/DOA/UNDCP. 561 SPE EXTRACTION AND HPLC-UV DETERMINATION OF AMPHETAMINE DERIVATIVES IN HUMAN URINE BY HPLC-UV M.E. Soares, M. Carvalho, H. Carmo, F. Carvalho, M.L. Bastos. REQUIMTE, Laboratory of Toxicology, University of Porto, 4050–047 Porto, Portugal Amphetamine derivatives are a class of compounds increasingly abused. Belonging to this class of compounds are the well known d-amphetamine (AMP) and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), but also 4-bromo-2,5-dimetoxiphenethylamine (2C-B) and 4-methylthioamphetamine (4-MTA) have been more recently reported to be used for similar purposes. In spite of the clinical and forensic interest in the quantification of these compounds in biological fluids, the development of adequate techniques is not yet fully accomplished specially for 2C-B and 4-MTA. The aim of this work was to develop and validate a solid phase extraction and a HPLC method for the simultaneous quantification, in human urine, of AMP and its metabolite p-hidroxyamphetamine (p-HAMP), MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA), 2C-B and 4-MTA. After acid hydrolysis, urine was purified through an OASIS MCX column, being the drugs eluted with 5% ammonium hydroxide in methanol. The eluate was injected into a HPLC-UV system equipped with a C18 column being the eluent constituted by methanol:acetate buffer 0.05 M containing 0.1% triethylamine, pH 3.9, in a gradient mode. The detection was made at 210 nm. A coeficient of variation between 3.3% and 5.9% was obtained for the precision for all the compounds for the overall procedure. The accuracy was better than 85% for all the compounds. The limits of detection were 14.0, 5.3, 100.0, 34.0, 47.0, 33.0 ng for AMP, p-HAMP, MDMA, MDA, 2C-B and 4-MTA, respectively, in 20 µl of injected sample. The SPE procedure proporctionated a good recovery of the compounds and a very clean extract, which enabled the chromatographic separation of the six compounds without coelution of endogenous impurities. To our knowledge this is the first HPLC method enabling the simultaneous determination of these six compounds in human urine. 562 METABOLIC PATHWAYS OF BISPHENOL A IN PRECISION-CUT LIVER AND INTESTINE SLICES FROM CD1 MICE D. Zalko, J-.P. Jaeg, E. Perdu-Durand, L. Dolo, J.-P. Cravedi. UMR 1089 Xénobiotiques, INRA/ENVT, BP3, 31931 Toulouse cedex, France Bisphenol A [2,2-bis (4-Hydroxyphenyl)propane, (BPA)] is a monomer used in the manufacture of polycarbonate plastic products and resins such as those used to coat cans containing food and beverages and those found in dental sealents. The estrogenicity of BPA has been demonstrated in a variety of in vitro and in vivo assays, and this chemical is suspected to produce endocrine effects in humans. In addition, BPA transforms Syrian hamster embryo cells and forms dose-dependent DNA adducts, suggesting it is mechanistically carcinogenic. The route of exposure has been shown to be important for the toxicological effects of BPA and the absorption and metabolism of this xenoestrogen may partly account for these findings. In the present study we examined the metabolic fate of BPA in CD1 mice using a radiolabelled molecule. BPA was incubated at different concentrations with precision-cut liver, duodenum, jejunum and caecum slices. Metabolites were separated by HPLC and the peaks were monitored by on-line radioactivity detection. Identification of the biotransformation products was established on the basis of co-chromatography with authentic standards and confirmed by mass Poster Session P28. Biotransformation spectrometry when necessary. As far as BPA conjugates were concerned, enzymatic hydrolyses were performed using β-glucuronidase or aryl-sulfatase. Several metabolites, mainly glucuronide and sulfate conjugates were isolated and identified in all incubates indicating that intestine is able to metabolise this chemical, although in a limited extent as compared with hepatic tissue. Traces of BPA breakdown products were also present in the liver and intestine metabolic profiles. The conjugation of BPA within the intestinal wall, leading to inactive metabolites, may partly explain the reason why dietary BPA is much less toxic than the subcutaneous or intraperitoneal routes of exposure. 563 THE USE OF MINIPIG LIVER MICROSOMES IN THE STUDY OF INTERACTIONS BETWEEN CYTOCHROMES P450 AND METABOLITES OF BENZENE Eliška Kondrová 1,2 , Pavel Souček 2 . 1 3rd Faculty of Medicine, Charles University, Ruská 87, 100 00 Praha 10, Czech Republic, 2 Centre of Industrial Hygiene and Occupational Health, National Institute of Public Health, Šrobárova 48, 100 42 Praha 10, Czech Republic Benzene is an established human and animal carcinogen, its myelotoxic and carcinogenic effects are ascribed to its reactive metabolites. We have previously shown that some benzene metabolites (benzoquinone, hydroquinone) cause cytochrome P450 (CYP) destruction and initiate the generation of hydroxyl radicals in vitro with rat and human liver microsomes. The destruction of CYP in vivo may influence the toxicity of xenobiotics and activation of procarcinogens and it may also be considered a general marker of protein damage. Our previous results show that human liver CYP is significantly more resistant to the destructive action of NADPH-mediated CYP futile cycle than rat CYP. On the other hand, human liver CYP was significantly more sensitive to the effect of benzoquinone in comparison with rat. Rat is therefore not suitable as a model animal for testing CYP sensitivity towards NADPH-mediated oxidative stress and destruction by quinones. Further we have shown that CYP enzyme levels and marker activities in minipig and human liver are similar and therefore minipigs may serve as model animals in pharmacological and toxicological studies with substrates of human CYPs. We incubated minipig liver microsomes 60 minutes at 37°C in the presence of different concentrations of NADPH and substrates (catechol, benzoquinone, hydroquinone) and determined CYP content spectrophotometrically. We also measured the formation of hydroxyl radicals. We found that the resistance of minipig CYP to NADPHmediated CYP destruction is similar to human CYP. After incubation with substrates we observed concentration-dependent CYP destruction that was greatest with benzoquinone. However, the sensitivity of CYP to benzoquinone proved to be much lower in minipig than in human liver. Possibly, minipig liver contains higher levels of reducing enzymes which protect against benzoquinone toxicity. We found that benzoquinone was less destructive when incubated together with NADPH, which is most probably due to its enhanced reduction to hydroquinone. The formation of hydroxyl radicals by NADPH was concentration-dependent and did not correlate with CYP destruction. Results of our pilot study suggest that minipig liver microsomes are a better model system for testing of response of human microsomal enzymes to oxidative stress than rat liver. Due to limited availibility and high enzyme levels variability of human microsomes further investigation is necessary to obtain straightforward data on quinone toxicity. 564 INDUCTION OF RAT LIVER CYTOCHROME P450 ISOENZYMES CYP 1A AND CYP 2B BY SOME PESTICIDES G. Kostka, B. Wiadrowska, S. Pawlak. Department Of Environmental Toxicology, National Institute Of Hygiene, Warsaw, Poland It is well recognised that the induction of cytochrome P450 monooxygenases has toxicological consequences. For example, some hepatic s151 cytochrome P450 2B inducers have been demonstrated to be nongenotoxic hepatocarcinogens and/or liver tumour promoters. The effect of three structural analogous of DDT on relative liver weight (RLW) and the activity of hepatic CYP isoenzymes (CYP 2B and 1A) was studied. The purpose of this work was to characterise dose-response relationship of CYP 2B and 1A microsomal enzyme following exposure to nuarimol, bromopropylate and fenvalerate. Male wistar rats received these compounds for 4 days at 24 h intervals in daily oral doses of 1/10, 1/50 and 1/100 LD50. The effects of these compounds were compared with DDT used as phenobarbitaltype of monooxygenase system inducer. Positive controls for CYP 2B and 1A were obtained by treating male rats with intraperitoneal injections of phenobarbital (60 mg/kg body weight x day-1 in saline for 4 days) and 3-methylcholanthrene (25 mg/kg body weigh x day-1 in saline for 3 days). The activities of CYPs were measured as Odealkylation of 7-pentoxy- (PROD) and 7-etoxyresorufin (EROD). Thus this biochemical procedure permits to determine whether tested compounds belong to one of two main types of inducers of cytochrome P450 monooxygenase system. It was found that all compounds studied (except fenvalerate) were shown to be inducers of CYP 2B and to cause hepatomegaly. In animals treated with nuarimol and bromopropylate the metabolism of 7-pentoxyresorufin increased in a dose dependent manner. It should be noted that tested compounds induced only slight increase in O-dealkylation of 7-ethoxyresorufin (CYP 1A). These results indicate that nuarimol and bromopropylate as well as DDT show the ability to induce the phenobarbital-type of CYP in wistar rats. 565 DO MULTIPLE P450 ISOFORMS CONTRIBUTE TO DIAZINON METABOLISM IN MAN? Elaine Mutch, Faith M. Williams. The Toxicology Unit, Environmental Medicine, The Medical School, University of Newcastle, Newcastle upon Tyne. NE2 4HH. U.K Phosphorothioate pesticides (P=S) such as diazinon are activated by P450-mediated oxidative desulphuration to form the toxic oxon (P=O). Detoxication of the oxon occurs via hydrolysis by A-esterases such as PON1 or by binding of the oxon to B-esterases. The aim of this study was to determine the P450 isoforms involved in diazinon’s metabolism and the extent of hydrolysis of its oxon diazoxon, by human liver (HL) microsomes. HL microsomes or recombinant P450 isoforms were incubated (5min or 2h) with diazinon and NADPH (1mM) and formation of diazoxon (DZO) and the pyrimidinol (IHMP) metabolite was determined by reverse-phase HPLC. The rate of diazoxon (500µM) hydrolysis to IHMP by HL microsomes was also determined. Seven specific P450 marker substrates were used to characterise the HL microsomes. Diazinon (50µM and 500µM) was metabolised to diazoxon (0.134–0.692 and 0.149–0.961 nmol/min/mg protein) and IHMP (0.190–1.47 and 0.643–3.72nmol/min/mg protein) by HL microsomes (n=19). Of eight recombinant P450 isoforms, CYPs 2D6, 2C19, 3A5, 3A4 were most efficient in producing diazoxon (37.1, 28.6, 28.6, 24.3pmol/hr/pmol P450) and IHMP (79.2, 75.7, 88.3, 57.5pmol/hr/pmol P450) from diazinon (500µM). Diazoxon hydrolysis to IHMP by HL microsomal esterases varied five-fold (42.7–243.6nmol/min/mg protein). There were significant correlations between diazoxon and IHMP formation from diazinon with three CYP3A4/5 marker reactions. None of the other P450-mediated reactions correlated with diazinon metabolism. Diazoxon hydrolysis correlated significantly with IHMP formation from diazinon (50µM and 500µM). These data indicate that CYP3A4/5 is the major enzyme involved in diazinon activation and detoxication, although other isoforms may have a role when CYP3A4/5 is poorly expressed. The importance of PON1 in the further detoxication of the toxic oxon was also highlighted. Therefore, the individual’s unique CYP3A4/5 and PON1 profile may be useful in identifying persons susceptible to the toxic effects of this pesticide. s152 566 Poster Session P28. Biotransformation THE INHIBITION OF MALATHION DETOXICATION BY ISOMALATHION AND OTHER OPTs IN HUMAN LIVER MICROSOMES F.M. Buratti, E. Testai. Laboratory of Comparative Toxicology and Ecotoxicology, Biochemical Toxicology Unity, Istituto Superiore di Sanità, Rome, Italy An epidemic of malathion (MAL) poisonings occurring in Pakistan in 1976, affected 40% of spraymen and mixer working for a malaria control programme. The episode evidenced the importance of impurities, such as isomalathion (ISO), in the commercial formulations. Indeed, at the same level of exposure, about 2800 workers experienced various degree of toxic effects related to the different contents of ISO in the commercial MAL formulations. ISO is formed during MAL manufacturing (usual content in commercial products 0.02%0.2%); however these levels can significantly increase during storage, especially at warm temperature. ISO potentiates MAL toxicity by inhibiting carboxylesterase (CE), responsible for MAL detoxication. As a consequence, a higher amount of MAL is desulfurated by hepatic CYPs to the toxic metabolite malaoxon. Furthermore, operators frequently use mixed formulation or different OPTs at the same time, being concurrently exposed to different agents. We have therefore characterized the human hepatic activity of CE toward MAL in a panel of 20 human liver microsomes and the inhibitory effect of ISO, chlorpyrifos (CPF) and parathion (PAR). CE activity showed a low level of variation among individuals (4-fold). The reaction consists of two different phases with relatively low Km values (Kmappa1 =0.25–0.68µM; Kmapp2 =1.7–12.4µM), confirming for CE a good MAL detoxifying activity. ISO resulted as a potent non competitive inhibitor of MAL detoxication (Ki 0.62µM), with a higher efficiency than CPF and PAR (Ki =8.2µM and 50.2µM, respectively). However, CPF-oxon, the toxic product of CPF desulfuration, showed the highest inhibitory potency towards CE-mediated detoxication, being characterized by a Ki =22 nM. The present results evidence the importance of considering in risk assessment metabolic interactions between chemicals to which humans can be concurrently exposed. This work has been partially supported by the ISS Projects n° 2181/RI and n° 3AAS. 567 UP-REGULATION OF POLYCYCLIC AROMATIC HYDROCARBON-METABOLIZING CYTOCHROME P4501A1 HEMOPROTEINS BY 56 Fe2 O3 OR 54 Fe2 O3 FOLLOWING THE EXPOSURE OF SPRAGUE DAWLEY RATS TO BENZO(A)PYRENE-COATED ONTO 56 Fe2 O3 OR 54 Fe2 O3 PARTICLES P. Shirali 1 , B. Maunit 2 , F. Zerimech 3 , P. Gosset 4 , C. Creusy 4 , J.F. Müller 3 , G. Garçon 1 . 1 Laboratoire de Recherche en Toxicologie Industrielle et Environnementale, Université du Littoral - Côte d’Opale, Dunkerque, France; 2 Laboratoire de Spectrométrie de Masse et de Chimie Laser, Université de Metz, France; 3 Laboratoire de Biochimie et de Biologie Moléculaire, Hôpital Huriez, Lille, France; 4 Laboratoire d’Anatomie et de Cytologie Pathologique du Groupement Hospitalier de l’Institut Catholique de Lille, Faculté Libre de Médecine, Lille, France Any influence of iron in polycyclic aromatic hydrocarbon (PAH)/iron oxide mixtures on the capacity of PAHs to induce metabolizing enzymes will be one of many ways that iron oxides can affect PAH carcinogenicity. A major point will be made regarding CYP: they are hemoproteins. Hence, it will be of great interest to investigate the possible involvement of Fe2 O3 in benzo(a)pyrene (B(a)P)/Fe2 O3 mixtures on the induction of PAH-metabolizing cytochrome P4501A1 (CYP1A1) enzymes on one of the first-entry organ target, the lung. Male Sprague Dawley rats were intratracheally instilled with hematite (56 Fe2 O3 or 54 Fe2 O3 ; 3 mg), B(a)P (3 mg) or B(a)P (3 mg)-coated onto hematite (56 Fe2 O3 or 54 Fe2 O3 ) particles (3 mg). Firstly, lung mRNA expressions of cyp1a1 were carried out. Secondly, in view of the crucial role of CYP1A1 in the metabolic activation of B(a)P and its biochemical nature, protein concentrations and catalytic activities (7-ethoxyresorufin O-deethylase; EROD) of CYP1A1 were determined. Thirdly, time-of-flight laser microprobe mass spectrometry (TOF-LMMS) allowed us to determine the possi- ble incorporation of 54 Fe in B(a)P/54 Fe2 O3 iron oxide mixtures in the hem moiety of CYP1A1. Statistically significant increases in cyp1a1 mRNA expressions and protein concentrations were observed in rats exposed to B(a)P, to B(a)P-coated onto 56 Fe2 O3 particles or to B(a)Pcoated onto 54 Fe2 O3 particles, versus controls (p<0.01). Significant increases in EROD activities were seen in rats treated with B(a)P (p<0.001) or particularly with B(a)P-coated onto 56 Fe2 O3 particles or with B(a)P-coated onto 54 Fe2 O3 particles, versus controls. In addition, exposure to B(a)P/56 Fe2 O3 or 54 Fe2 O3 mixtures induced higher CYP1A1 protein concentrations and EROD activities, than B(a)P alone (p<0.01). TOF-LMMS showed that the 54 Fe/56 Fe ratio in the microsomes of B(a)P-coated onto 54 Fe2 O3 -instilled animals was 1.3 instead of the theoretical 54 Fe/56 Fe ratio of 0.063. Animal short-term exposure to B(a)P/Fe2 O3 mixtures favored dramatically the up-regulation of PAH-bioactivating CYP 1A1 enzymes in lungs, notably by interfering in its translation into functional hemoproteins. Overall, this should lead to a better understanding of the underlying mechanism involved in the enhanced carcinogenicity of B(a)P-coated onto Fe2 O3 particles in lungs. 568 TAXANES: ANTITUMOR EFFECTS AND INTERSPECIES DIFFERENCES IN METABOLISM R. Václavíková 1 , S. Horský 1 , L. Svobodová 1 , B. Otová 2 , P. Šimek 3 , I. Gut 1 . 1 National Institute of Public Health, Prague, CZ, 2 First Medical Faculty, Charles University, Prague, CZ, 3 Institute of Entomology, CAS, Èeské Budìjovice, CZ Taxanes are important recently introduced antineoplastic drugs. We have investigated cytochrome P450 - catalyzed metabolism of paclitaxel and docetaxel in rat, minipig, regular pig and human liver microsomes. In rat microsomes paclitaxel was metabolized mainly to C3’-hydroxypaclitaxel (C3’-OHP), less to C2-hydroxypaclitaxel (C2-OHP), di-hydroxypaclitaxel (di-OHP) and an unknown hydroxypaclitaxel. In minipig microsomes, this unknown hydroxypaclitaxel was the main metabolite, whereas C3’-OHP and C2-OHP were minor products and the same held true for regular pig microsomes. In human liver microsomes 6α-hydroxypaclitaxel (6α-OHP) was the main metabolite, followed by C3’-OHP, C2-OHP and two other metabolites not yet fully characterized. However, the proportion of 6α-OHP and C3’-OHP in different human microsomes varied significantly. It became obvious that despite various general similarities between human and pig metabolism reported, the profiles of paclitaxel metabolites in the studied species were different and 6α-OHP remained a uniquely human metabolite. Among different cDNA-expressed CYP enzymes (CYP1A2, 1B1, 2A6, 2C9, 2E1 and 3A4), only CYP3A4 enzyme formed C3’-OHP, C2-OHP and one unknown metabolite. In regular pig, minipig and human liver microsomes, docetaxel (DXT) was metabolized mainly to hydroxydocetaxel (OHDTX), whereas rat microsomes produced primarily diastereomeric hydroxyoxazolidinones. Human liver microsomes from different individuals formed OHDTX at different rates in relation to CYP3A4 content. Significant blood levels of paclitaxel and docetaxel, respectively, were reached after i.p.administration of the drugs to rats and maintained for at least 6 hours. Upon repeated administration, these levels tended to decrease in case of paclitaxel; it possibly participated in a lower antitumor action of paclitaxel against s.c. lymphoma. Significant levels of C3’-OHP after paclitaxel and OHDTX after docetaxel were detected in blood of the rats. Acknowledgements: This study was supported by Grant IGA NL6715–3. 569 EFFECT OF POLYVITAMIN COMPOSITION ON SOME HEPATIC P-450-DEPENDENT ENZYMES IN ISONIAZID AND RIFAMPICIN EXPOSURE RATS V. Kovalenko 1,2 , A. Voronina 1 , A. Shayakhmetova 1,2 , O. Voloshina 2 , L. Berejna 1 . 1 Institute of Pharmacology and Toxicology, 2 State Pharmacological Centre, Kyiv, Ukraine Isoniazid and rifampicin are still widely used for the treatment of tuberculosis. They may both cause liver damage after therapeutic doses are given. In this study we investigated the influence of these Poster Session P28. Biotransformation tuberculostatics on cytochrome P-450 system. The isoniazid (43 mg/kg) and rifampicin (86 mg/kg) were administered by gavage daily for 28 days to male Wistar rats (8–10 weeks old). After that the animals were sacrificed and hepatic microsomes were extracted. Hepatic microsomal p-nitrophenol hydroxylase (CYT P-450 2E1 mark), NADPH-cytochrome-P-450 reductase activity, NADPHinduced TBA-products generation and the total cytochrome P-450 concentration were analyzed. We have shown tuberculostatic-induced increasing of rat liver microsomal both p-nitrophenol hydroxylase and NADPH-cytochrome-P-450 reductase activity (170%) and rate NADPH-induced generating of TBA-products (78%) as compared with intact animals. But total cytochrome P-450 concentration was without any changes. Treatment by polyvitamin composition, that was administered per os (75 mg/kg) simultaneously with isoniazid and rifampicin decreased the activity of the above enzymes to the level of control rats. These results indicate possibility of cytochrome P-450 2E1 inhibition and consequently decreasing CYT 2E1-induced hepatotoxicity caused by combined administration of isoniazid and rifampicin. 570 EXPRESSION OF CYTOCHROMES P450 INVOLVED IN BIOTRANSFORMATION OF XENOBIOTICS IN RATS EXPOSED TO ELECTROMAGNETIC FIELDS. R. Wiaderkiewicz 1 , P. Czekaj 1 , A. Wiaderkiewicz 1 , A. Pałasz 1 , J. Karpowicz 2 , K. Gryz 2 . 1 II Department of Histology & Embryology, Medical University of Silesia, Katowice, Poland; 2 Central Institute for Labor Protection, Warszawa, Poland The results of experimental studies on the effect of electromagnetic fields on biotransforming potential of living organisms are sparse, inconsistent and even contradictory. Setting up the standard conditions of exposure and problems with monitoring them during experiment are just some of the reasons of such a situation. In the presented study we analyzed the cytochrome P450 system in rats exposed for 5 days to electric (27,12 MHz, 80V/m) and magnetic (50 Hz, 1 mT) fields which were precisely monitored during whole experiment. In the microsomal fraction of the liver the total content of cytochrome P450, cytochrome b5 and activity of their corresponding NADPH and NADH reductases was measured. The levels of CYP1A2, CYP2B1/2, CYP3A1 and CYP2E1 were analyzed by immunoblotting and expression of their corresponding genes by RT-PCR. Simultaneously, the sections of the liver were stained with H/E and histochemically for the activity of classical marker enzymes of hepatocyte function (SDH, LDH, G6Pase, ATPase Mg+2 , AcP). The results showed that both electric and magnetic fields increased slightly the total amount of cytochrome P450 and expression of CYP2B1/2 and CYP2E1. It is concluded that in the conditions of performed experiments the exposure of experimental animals to electric or magnetic fields have no effect on morphology of the liver and basic metabolic processes in hepatocytes. However, it modifies the expression of selected CYP450 isoforms what may influence the biotransforming potential of the liver. (Supported by CIOP, grant no. I.3.12.) 571 PULMONARY CYP1A1, CYP2B1/2, CYP2E1 AND CYP3A1/2 EXPRESSIONS IN OLD MALE RATS P. Czekaj, A. Wiaderkiewicz, A. Palasz, R. Wiaderkiewicz. II Department of Histology & Embryology, Medical University of Silesia, Katowice, Poland The majority of studies indicate a decrease in cytochrome P450 content and maximum induction level with age. These changes affect the catalytic activity of CYP-dependent monooxygenases and can be responsible for higher susceptibility to xenobiotics during aging. The effect of age on constitutive and inducible expression of cytochrome P450 isoforms involved in the metabolism of xenobiotics in lungs was studied in 20-, 24- and 28-months old Spraque-Dawley male rats. The following inducers were used: β-naphthoflavone (BNF; 50mg/kg b.w./3 days), phenobarbital (PB; 80mg/kg b.w./3 days), dexamethasone (Dex; 30 mg/kg/b.w./3 days) and ethanol (Et; 5% solution/3 weeks). No inductory effect of PB, BNF, Dex, and Et on the activity of NADPH-cytochrome P450 reductase s153 and NADH-cytochrom b5 reductase was observed. We found very low constitutive levels of CYP1A1, CYP2E1 and CYP3A1, and inductory changes for CYP1A1, CYP3A1 and CYP3A2 both at the transcriptional (RT-PCR) and protein level (Western blotting). There were no changes in inducible CYP2B1/B2 and CYP2E1 expressions in old rats as compared to controls. It is concluded that age only slightly modifies the effect of studied inducers on the mRNA and protein expression of P450 isoforms in rat lungs, in the studied range of 20–28 months. (Supported by KBN, grant no. 3P05D03623.) 572 EXPRESSION OF CYP1A1 AS A FUNCTION OF CELL CYCLE PROGRESSION IN THE ABSENCE OF AN EXOGENOUS INDUCER. G. Elizondo, I. Medina-Díaz, G. Ponce. Laboratory of Molecular Toxicology, Toxicology Section, CINVESTAV, México DF, México Cytochrome P450 (CYP) 1A1 has long been known to be one of the metabolic enzymes involved in activating many procarcinogens. The induction of CYP1A1 gene is mediated via ligand dependent activation of aryl hydrocarbon receptor (Ahr). Several studies suggest that the Ahr plays an important role in the cell cycle regulation. Ahr-defective Hepa 1c1c7 cells present a G1 cell cycle arrest, and a direct interaction between Ahr and phosphorylated retinoblastoma protein has also been described. However, very little is known about genes under Ahr control as a function of the cell cycle. In the present study we analyzed the CYP1A1 mRNA levels at distinct phases of the cell cycle, in absence of exogenous inducer, in murine Hepa 1c1c7 cell cultures. Hepa-1 cell cycle progression was characterized and CYP1A1 mRNA levels were determined in synchronized cell cultures at different times. CYP1A1 transcript signal was observed at 1.0 and 1.5 h after cell culture initiated, indicating that CYP1A1 transcription, in absence of exogenous inducer, occurs at G1 phase. In contrast, no signal was detected at S or G2/M phases. These results are in agreement with previous studies that link Ahr with G1 phase progression and support the existence of an endogenous Ahr ligand. On the other hand, the physiological role of CYP1A1 expression on G1 phase, other than xenobiotic metabolism, still unknown. 573 EFFECTS OF OLFACTORY TOXICANTS ON THE EXPRESSION OF CYP2A5 Anna Franzén 1 , Elena Piras 1 , Ulrika Bergström 2 , Estíbaliz L. Fernández 1 , Françoise Raffalli-Mathieu 1 , Matti Lang 1 , Eva B. Brittebo 1 . 1 Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden 2 Department of Environmental Toxicology, Uppsala University, Uppsala, Sweden Certain members of the cytochrome P450 (CYP) 2A subfamily catalyze the biotransformation of many protoxicants and procarcinogens commonly found in the environment. In this study a detailed analysis of CYP2A5 expression has been done with in situ hybridisation and immunohistochemistry in the nasal passages of mice. There was a marked expression in sustentacular cells and Bowman’s glands in the olfactory mucosa whereas in the respiratory mucosa there was no or only a weak expression. Expression also occurred in excretory ducts of sero-mucous nasal glands and in the epithelium of the nasolacrimal duct. Typical hepatic CYP2A5 inducers did not change the expression in the olfactory mucosa. The effects of the olfactory toxicant dichlobenil known to induce permanent changes in the dorsomedial part of the olfactory region, and the effects of the olfactory toxicant methimazole known to induce reversible changes, were also examined. There was a distinct expression of the enzyme in the damaged atypical epithelium in the olfactory region both four days and two weeks after administration with dichlobenil or methimazole. In contrast, four days after treatment with these toxicants there was no expression in the damaged Bowman’s glands. Two weeks after treatment with dichlobenil there was no expression of the enzyme in the fibrotic lamina propia. In contrast, two weeks after treatment with methimazole the expression of CYP2A5 in the regenerated Bowman’s glands was similar to s154 Poster Session P29. Genetic polymorphisms control. The expression of CYP2A5 in the atypical respiratory-like epithelium in the olfactory region suggests that also the damaged epithelium has the ability to biotransform chemicals. 574 DRUG METABOLISM IN INFECTED BODY: HEPATIC MONOOXYGENASE ACTIVITY AND FREE RADICAL LIPID PEROXIDATION DURING EXPERIMENTAL INFLUENZA VIRUS INFECTION L. Tantcheva 1 , V. Savov 4 , M. Mileva 5 , E. Stoeva 1 , A.S. Galabov 2 , A. Braykova 3 . 1 Institute of Phisiology, 2 Institute of Microbiology, 3 Institute of Molecular Biology, Bulgarian Academy of Sciences,4 University of Sofia “St. Kliment Ohridski”, 5 Medical University of Sofia, Sofia, Bulgaria White male mice ICR were experimentally infected with influenza virus A/Aichi/2/68(H3N2) intranasally (1.5 LD50 ). On the 5th day after virus inoculation (crucial for viral infection) a significant inhibition of drug metabolizing enzyme activity was established. In liver 9 000xg supernatant the metabolism of some monooxygenase substrates (amidopyrine, metimizole, aniline and ethylmorphine) was decreased significantly. The content of the terminal component of the microsomal system - cytochrome P-450, is reduced up to 50% and NADPH-cytochrome C reductase activity - up to 70%. High reactive oxygen species (ROS) levels (as biochemical markers for the oxidative stress) also were established. Increased levels of lipid peroxidation (LPO) products (conjugated dienes and TBA reactive substances) in various tissues and organs (blood, lungs, liver and brain) were measured. In liver the reduced concentrations of cytochrome P-450 and CCR activity correlate inversely proportionally to the increased primary and secondary LPO products (conjugated dienes/cyt P-450 r=-0.945, and MDA-TBARS/cyt P-450 r=-0.873; conjugated dienes/CCR r=-0.671 and MDA-TBARS/CCR r=-0.796). This inverse proportional dependence, with high significance (p=0.001) allow us to suggest the activation of the LPO processes during influenza virus infection, can be one of the causes for DMES inhibition. We suggest that generated LPO products modify endoplasmatic biomembranes and cyt P-450 was transformed into its inactive P-420 form. In this way the whole electron-transport chain of hepatic monooxygenases is inhibited by the influenza in different degrees. P29 Genetic polymorphisms 575 GENETIC POLYMORPHISMS OF DNA REPAIR AND BIOTRANSFORMATION GENES AND POSSIBLE LINKS WITH CHROMOSOMAL ABERRATIONS AND SINGLE-STRAND BREAKS IN DNA P. Vodička 1 , R. Kumar 2 , P. Soucek 3 , V. Haufroid 4 , R. Stetina 5 , M. Dusinska 6 , M. Kuricova 6 , M. Zamecnikova 7 , J. Buchancova 8 , H. Norppa 9 , L. Vodickova 3 , Z. Matousu 1 , K. Hemminki 2 . 1 Inst. Exper. Med., Acad. Sci. Czech Rep., Prague, Czech Republic, 2 Dept. Biosci at Novum, Karolinska Institute, Huddinge, Sweden and German Cancer Institute, Heidelberg, FRG, 3 Natl. Inst. Publ. Health, Prague, Czech Rep., 4 Catholic University of Louvain, Louvain, Belgium, 5 Purkynje Military Medical Academy, Hradec Kralove, Czech Rep., 6 Inst. Prevent. Clin. Medicine, Bratislava, Slovak Rep., 7 Natl. Inst. Publ. Health, Bratislava, Slovak Rep., 8 Dept. Occup. Med., Med. Faculty in Martin, Martin, Slovak Rep., 9 Finnish Inst. Occup. Health, Helsinki, Finland In the study we analysed the links between genetic polymorphisms in genes coding for biotransformation enzymes (CYP1A1, CYP2E1, EPHX. GSTM1, GSTP1 and GSTT1) and DNA repair enzymes (XPD, XPG, XPC, XRCC1 and XRCC3) and the levels of chromosomal aberrations and single-strand breaks in DNA in a central European population. Our results show that chromosomal aberrations are affected by the EPHX activity genotype (F=2.4, P=0.043, ANOVA) and by genetic polymorphism in XPD, exon 23 (F=3.6, P=0.028, ANOVA). Exon 23-polymorphism in the XPD gene was found as a major influencing factor on CA (F=4.2, P=0.017, MANOVA). Single-strand breaks in DNA are modulated by both CYP2E1 polymorphisms (F=5.5, P=0.026 and F=4.7, P=0.038, respectively) as well as by polymorphisms in XPD (F=4.3, P=0.023), XPG (F=4.3, P=0.024) and XRCC1 (F=3.0, P=0.064), analysed by multifactorial analysis of variance. Using MANOVA irradiationspecific DNA repair rates (representing mainly base-excision repair) are affected by XRCC1 (F=5.9, P=0.010) and XPC polymorphisms (F=4.2, P=0.046). This is a first study, which relates transient markers of genotoxic/carcinogenic effects with a large number of genetic polymorphisms in the genes coding for enzymes involved in biotransformation and DNA repair. The salient features of our results suggest relation between markers of genotoxicity and polymorphisms in genes coding for biotransformation and DNA repair enzymes. The study was supported by EU QLK4-CT-1999–01368 and GACR 310/01/0802, 310/03/0437. 576 ASSOCIATION OF DNA DAMAGE TO HUMAN PERIPHERAL LYMPHOCYTES WITH AGE AND OGG1 GENOTYPE Vanessa Gage, Emma Davis, Julian Leathart, Peter Blain, Ann Daly, Faith Williams. Toxicology & Pharmacogenetics, School of Clinical and Laboratory Sciences, University of Newcastle, Medical school, Newcastle Upon Tyne, NE2 4HH, U.K There have been conflicting reports on the influence of increasing age on DNA damage. 8-oxoguanosine glycosylase 1 (OGG1), responsible for the repair of the 8-hydroxy-2-deoxyguanosine lesion, exhibits a polymorphism leading to a ser 326 cys change. The aim of this study was to see if an association existed between age, DNA damage and OGG1 genotype. 99 elderly (65+ yr old) and 67 young (20–40 yr old), healthy, urban dwelling volunteers were recruited for the study. DNA damage was measured by the COMET assay in freshly isolated lymphocytes. Cell images were captured by confocal microscopy and analysed for DNA damage expressed as Tail Distributed Moment (TDM). OGG1 genotyping was performed on stored lymphocyte DNA by PCR followed by RFLP using the restriction enzyme Fnu4H1. The mean DNA damage in lymphocytes from the elderly was 28.8 ± 0.6 (TDM mean ± sem) and was greater than the mean for the young, 26.4 ± 0.8 (TDM mean ± sem), (unpaired t-test, P<0.05). The elderly genotype distribution for OGG1 was Ser/Ser 62% (TDM 28.7 ± 0.8), Ser/Cys 33% (TDM 28.8 ± 0.9) & Cys/Cys 5% (TDM 29.4 ±1.8). The young genotype distribution for OGG1 was Ser/Ser 51% (TDM 26 ± 0.9), Ser/Cys 42% (TDM 28 ± 1.4) & Cys/Cys 7% (TDM 25.2 ± 2.6). There were no differences in the distribution of the OGG1 genotype between the old and young volunteers (OR=1.77, p=0.10). Neither was there any difference in the distribution of DNA damage between genotype for either old or young volunteers. This study showed increased lymphocyte DNA damage with increased age that was not influenced by genotype for codon 326 of the repair enzyme OGG1. 577 THE DISTRIBUTION OF SISTER CHROMATID EXCHANGE (SCE) AND HIGH FREQUENCY CELLS (HFCS) IN LEAD EXPOSED WORKERS; INFLUENCE OF δ-AMINOLEVULINIC ACID DEHYDRATASE (ALAD) POLYMORPHISM Y. Duydu, H.S. Süzen. Ankara University, Faculty of Pharmacy, Department of Toxicology, 06100 Tandoðan, Ankara, Turkey The cytogenic responses were measured by means of sister chromatid exchange (SCE) frequencies and high frequency cells (HFCs) in peripheral blood lymphocytes from 71 storage battery manufacturing workers occupationally exposed to lead. The mean blood lead levels (BLL) of the workers were measured as 38,25 ± 8,32 µg/dl. To evaluate a lead effect, volunteers were divided into three subgroups (groups A, B and C) according to their BLLs. These constituted of the workers who had BLLs of lower than 40 µg/dl (n=19), between 40 and 50 µg/dl (n=13) and above 50 µg/dl (n=8), respectively. All workers in the subgroups have statistically higher (p<0,05, t-test) Poster Session P29. Genetic polymorphisms SCE values when compared with the control group. According to our genotype analysis of δ-aminolevulinic acid (ALA) dehydratase (ALAD), 50 workers were ALAD 1–1 and 21 workers were ALAD 1–2 carriers. In spite of the statistically insignificant difference in mean SCE/cell values between ALAD 1–1 and ALAD 1–2 carrying workers the percentage of HFC outliers was statistically (χ 2 test, p<0,05) higher in ALAD 1–1 carriers. According to this result we suggest that ALAD 1–1 carriers might be more susceptible to cytogenetic effects in lead exposure than ALAD 1–2 carriers. 578 BIOMARKERS OF SUSCEPTIBILITY AND DNA DAMAGE IN HUMANS EXPOSED TO CHRONIC LOW LEVEL OF IONIZING RADIATION S. Angelini 1,2 , R. Kumar 2 , F. Maffei 1 , F.S. Violante 3 , G. Cantelli Forti 1 , K. Hemminki 2 , P. Hrelia 1 . 1 Department of Pharmacology, University of Bologna, Bologna, Italy; 2 Department of Biosciences, Karolinska Institute, Huddinge, Sweden; 3 Occupational Health Unit, Policlinico Sant’ Orsola-Malpighi Hospital, Bologna Italy Ionizing radiation (IR) exposure damages cellular DNA in many ways, requiring the concerted action of a number of DNA repair enzymes to restore genetic integrity. Recently common polymorphisms in different DNA genes have been identified and it is possible that they may effect DNA repair capacity and thus modulate DNA damage deriving after IR exposure. In the contest of a medical surveillance program aimed at preventing cancer risk from exposure to IR, we have investigated the relationship between biomarkers of effect (micronuclei (MN)) and susceptibility (XRCC1, XRCC3 and XPD genotypes). Samples of peripheral blood were collected from hospital workers (n=21, mean age 45.7±8.6) chronically exposed to low level of IR (Equivalent dose to the whole body (Hwb): 0.9– 116.47mSv) and subjects without any work-related exposure to IR (n=20, mean age 43.6±8.2). Results indicated that overall MN frequency was significantly higher in exposed workers than in controls (P<0.05). Interestingly, Hwb did not influence the observed MN frequency and we hypothesized an influence from genotype distribution in DNA reapir genes. The XRCC1–399Gln and XRCC3–241Met variants as well the common alleles in XPD exon 10 and 23 (Asp312 and Lys751) were associated with higher MN frequencies, although the differences were not statistically significant. XRCC1–399Gln homozygotes had an average MN frequency of 8.5±4.4 compared with 7.9±2.4 in Arg homozigotes. XRCC3–241Met had an average MN frequency of 9.8±3.2 compared with 8.0±4.5 in Thr homozygotes. Regarding the XPD gene, the homozygotes Asp312 and Lys751 had an average of MN frequency of 8.14±1.57 and 9.87±3.27 respectively, compared with 7.67±3.56 and 6.80±1.17 of the correspondent variants homozygotyes. Although based on relatively small population group, our results provide evidence supporting that aminoacid variants in DNA repair genes may contribute to IR sensitivity and consequently susceptibility to cancer. Additional molecular epidemiological studies are needed to extend these findings. 579 CYP1A1, GSTM1 AND GSTT1 POLYMORPHISMS IN A TURKISH POPULATION M. Iscan, A.O. Ada, H.S. Süzen. Department of Toxicology, Faculty of Pharmacy, Ankara University, 06100 Tandoðan, Ankara, Turkey It is well known that cytochrome P-450 (CYP) enzymes are able to metabolize many xenobiotics including drugs, polycyclic aromatic hydrocarbons, pesticides and aromatic amines to reactive metabolites, which could be very toxic and/or carcinogenic, as well as endobiotics such hormones. Among them, CYP1A1 is a key enzyme in carcinogen metabolism. The glutathione S-transferases (GST)s are a family of cytosolic enzymes generally involved in the detoxication of activated, electrophilic xenobiotics. Thus genetic polymorphisms of these biotransformation enzymes are very important in the interindividual variability in xenobiotic metabolism and toxicity. In this study CYP1A1 m2, GSTM1 and GSTT1 gene polymorphisms were determined among 133 healthy individuals. On the basis of an improved polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) methodology, the frequency of CYP1A1 m2 mutation was determined. The frequencies of Ile/Ile (wild type) s155 and Ile/Val (heterozygous variant) CYP1A1 m2 genotypes were 90.23% and 9.77%, respectively. The genetic polymorphism analysis for the GSTM1 and GSTT1 genes was determined simultaneously in a single assay using a multiplex PCR approach. As an internal control exon 7 of the CYP1A1 gene was co-amplified. The prevalence of the deleted GSTM1 and GSTT1 genotypes were 51.88% and 17.29%, respectively. These results are in accordance with the reported frequencies of the CYP1A1 m2, GSTM1 and GSTT1 gene polymorphisms among Caucasian populations. 580 CYP1A1 Ile/Val GENETIC POLYMORPHISM IN HEAD & NECK CANCER PATIENTS P. Czekaj 1 , J. Adamska 2 , A. Fila 2 , U. Mazurek 2 , T. Wilczok 2 . 1 II Department of Histology & Embryology; 2 Department of Molecular Biology, Biochemistry and Biopharmacy, Medical University of Silesia, Katowice, Poland Mutation within CYP1A1 gene, exon 7 (A→G), exchanges an aminoacid at position 462 (Ile/Val), and significantly increases CYP1A1-dependent metabolic activation of polycyclic aromatic hydrocarbons. In some ethnic groups hetero-(Ile/Val) and homozygous (Val/Val) genotypic variants of CYP1A1 correlate with increased risk of cancer. To determine genetic frequencies of CYP1A1 Ile/Ile, Ile/Val and Val/Val in Caucasian population, a group of 108 healthy persons, and 49 of head&neck cancer patients, females and males, aged 18 to 81, were examined. Genomic DNA was isolated from patients’ peripheral blood leucocytes and cancer tissue. The CYP1A1 exon 7 fragment was amplified using PCR method, and then subjected to RFLP-PCR analysis with restrictive enzyme NcoI, SSCP, and DNA sequencing. RFLP analysis revealed existence of 3 polymorphic types of CYP1A1 Ile/Val both in population of healthy and ill persons. Occurrence rate of CYP1A1 heterozygotes Ile/Val (11%) and homozygotes Val/Val (2%) was similar in both populations. Val allele, associated with increased risk of neoplasm incidence, appeared twofold more often in males. The results suggest lack of association of CYP1A1 Ile/Val or Val/Val genotypes with a risk of human head&neck Ca planoepitheliale in Caucasian population. Smoking cigarettes may be the factor stimulating a development of laryngeal Ca planoepitheliale independently on CYP1A1 Ile/Val genetic polymorphism, if it is analyzed alone. 581 THE INFLUENCES OF CYP1A1, GSTM1, AND GSTT1 POLYMORPHÝSMS ON URÝNARY 1-HYDROXYPYRENE LEVELS AFTER PAH EXPOSURE. A.O. Ada 1 , M. Yilmazer-Musa 2 , S. Suzen 1 , C. Demiroglu 2 , A.E. Demirbag 3 , S. Efe 4 , Y. Alemdar 4 , S. Burgaz 2 , M. Iscan 1 . 1 University of Ankara, Faculty of Pharmacy, Dept. of Toxicology, Ankara, Turkey; 2 Gazi University, Faculty of Pharmacy, Dept. of Toxicology, Ankara, Turkey; 3 Yuksek Ihtisas Hospital, Dept. of Gastrointestinal Surgery, Ankara, Turkey; 4 Eregli Iron and Steel Works CO. Karadeniz Eregli/Turkey Polycyclic aromatic hydrocarbons (PAHs) in coke oven emissions cause a cancer risk to humans. The PAHs are activated mainly by P450s and inactivated by cytosolic glutatione S-transferases (GSTs). In the present study, the influences of genetic polymorphisms of PAH metabolizing enzymes on the urinary 1-hydroxypyrene (1-OHP) excretion, which is a biomarker of PAH exposure and shown to vary great among the individuals, have been investigated. Accordingly, the urinary 1-OHP levels and CYP1A1, GSTM1 and GSTT1 polymorphisms in 50 coke oven workers of Ereðli Iron and Steel plant and 50 controls have been detected. Urinary 1-OHP was analyzed by High Pressure Liquid Chromatography after enzymatic hydrolysis. Lymphocyte DNA was used for PCRbased genotyping. The interindividual difference in the excretion of 1-OHP was vast. The mean urinary 1-OHP level of coke-oven workers were significantly higher than that of controls. Genetic polymorphisms of CYP1A1, GSTM1 and GSTT1 either alone or in combination observed to have no influences on 1-OHP excretion in coke oven workers. In the control group, however, urinary 1OHP levels of individuals carrying the GSTT1- genotype were s156 Poster Session P29. Genetic polymorphisms significantly different from those of carrying GSTT1+ genotype. In addition, the combined GSTM1-, GSTT1- genotypes appeared to have significantly higher levels of urinary 1-OHP than those of individuals carrying GSTT1- or GSM1- genotypes, indicating their synergistic effect on the excretion of 1-OHP.The results show that urinary 1-OHP is a sensitive indicator of recent human exposure to PAHs and PAH metabolic polymorphisms may also to some extent reflect the interindividual variation in susceptibility to PAHs only in low PAH exposure. (Supported by Research Fund of Ankara University, 2001–08–03–025 and TUBÝTAK, SBAG-AYD-350) 582 583 ROLE OF PEROXISOME PROLIFERATORS-ACTIVATED RECEPTORS ALPHA AND GAMMA IN ATROPHIC AND METAPLASTIC GASTRITIS A. Sapone 1 , L. Gatta 2 , S. Trespidi 1 , D. Vaira 2 , F. Perna 2 , G.L. Biagi 1 , G. Cantelli-Forti 1 , M. Paolini 1 . 1 Department of Pharmacology and 2 1st Medical Clinic, Alma Mater Studiorum, University of Bologna, Bologna, Italy Background: Helicobacter pylori (HP) colonization leading to epithelial cell hyperproliferation within inflamed mucosa, may contribute to differences in gastric cancer risk among infected populations. Host responses that may affect the threshold for carcinogenesis include alteration of epithelial cell proliferation and apoptosis. One specific host pathway through which inflammatory mediators may influence HP-induced apoptosis is the transcription factor peroxisome proliferator-activated receptor γ (PPARγ). PPARγ and the related isoforms PPARα and PPARδ constitute a family of nuclear hormone receptors with important roles in the cell regulation. Aim: To evaluate the single or combined influence of PPARα (three) and PPARδ (four) isoforms on gastric inflammation on patients who underwent endoscopy complaining upper gastrointestinal symptoms. Methods: 312 patients (males/females:140/172; mean age 53 years SD ± 14.5 years) undergoing endoscopy during which biopsy samples were obtained have been studied. HP was evaluated and histological examination was assessed using the updated Sydney System score. Blood samples were also taken to assess single genotype for each individual using typical molecular biology techniques (PCR, restriction enzyme digestion, electrophoresis and sequencing). Results: 62.5% (95%CI: 57 to 67.7) of patients were HP positive. 59 showed endoscopic lesions (18.9%; 95%CI: 15 to 23.6), 32 had oesophagitisis (10.3%; 95%CI: 7.4 to 14.1), 11 gastric ulcers (3.5%; 95%CI: 2 to 6.2), and 22 duodenal ulcers (7.1%; 95%CI: 4.7 to 10.4). 120 patients (38.5%; 95%CI: 33.2 to 44) presented intestinal metaplasia and atrophy. 132 patients (42.3%; 95%CI: 37 to 47.9) had PPARα mutations (α∗2, α∗3, α∗4) and 130 patients (41.7%; 95%CI: 36.3 to 47.2) had PPARγ mutations (γ∗2, γ∗3) (Table 1). At univariate analysis there were no association between endoscopic or histological lesions and PPAR isoforms, except for PPARα∗4 with an OR 1.72 (95%CI: 1.01 to 2.94) for intestinal metaplasia and atrophy. However, at multivariate analysis including sex, age and HP status, the OR was not significant (p= 0.058). Conclusion: PPARs seem only weakly correlate with endoscopic or histological lesion in patients infected with HP. TENDENCY OF GENETIC PROFILE TO INFLUENCE THE CELLULAR IMMUNE RESPONSE IN WORKERS EXPOSED TO ASBESTOS AND CONTROLS M. Kuricova 1 , A. Horska 1 , M. Dusinska 1 , J. Tulinska 1 , A. Liskova 1 , E. Jahnova 1 , L. Wsolova 1 , S.A. Kyrtopoulos 2 , L. Fuortes 3 . 1 Institute of Preventive and Clinical Medicine, Bratislava, Slovak Republic, 2 Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece, 3 University of Iowa, College of Public Health, Iowa City, Iowa, USA Background: While the effects of genetic polymorphism in enzymes affecting xenobiotic metabolism on cancer risks and genotoxicity have been widely examined, studies aimed at possible modulation of immunotoxicity have been limited. Methods: Immune and hematologic parameters were examined in 61 workers exposed to asbestos fibres for an average of 23 years, and 46 town and 21 in-factory unexposed controls. Immune function assays – proliferative response of lymphocytes to mitogens and antigens, phagocytic activity and respiratory burst of neutrophils and natural killer cell activity were evaluated. Genetic polymorphism of xenobiotic metabolizing isoenzymes GSTM1, GSTP1, GSTT1, EPHX3, EPHX4, NQO1 was measured and compared with immune parameters. Results: Using multifactorial analysis of variance, a statistically significant suppression of phagocytic activity of monocytes in people with GSTM1-/+ genotype in comparison with GSTM1-/- was found in the whole population (people from asbestos plant and both controls). In the case of GSTP1 genotype, phagocytic activity of neutrophils and also leukocytes was increased in GSTP1+/+ in comparison with GSTP1-/-. The respiratory burst of neutrophils was significantly decreased in non-smokers with GSTM1-/+ genotype in comparison with GSTM1-/-. T-dependent proliferative activity of Blymphocytes was decreased in GSTP1-/+ genotypes in comparison with GSTP1-/- in the whole population. The proliferative activity of memory lymphocytes to tetanus toxoid seems to be affected by GSTM1 and NQO1 genotypes. The proliferative activity of lymphocytes in people with GSTM1-/+ genotype in comparison with GSTM1-/- was significantly increased in whole population as well as in non-smokers. On the other hand, people from both the whole cohort and the cohort of non-smokers with NQO1+/+ genotype show a significantly lower proliferative activity in comparison with genotype NQO1-/+. Conclusions: These results suggest that polymorphisms of xenobiotic metabolising isoenzymes might be responsible for interindividual differences in cellular immune responses; however there was no clear association of susceptibility of individuals to asbestosinduced immunomodulation, something which may be due to limited size of cohorts. This work was supported by the European Union (project no. QLK4–1999–01629), NIEHS Grant # 510205240 00000 and Slovak Grant Agency for Science # 04.92.11.05. 584 DEVELOPMENT OF FLUORESCENT 5’-NUCLEASE ASSAYS FOR THE ALLELIC DISCRIMINATION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN HUMAN CYTOCHROME P450 3A GENES H.W. Wilkerson 1 , S.L. Srinouanprachanh 1 , F.M. Farin 1 , J.S. McCune 2 , K.E. Thummel 2 . 1 Functional Genomics Laboratory, Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA, US, 2 Department of Pharmaceutics, University of Washington, Seattle, WA, USA Detection of single nucleotide polymorphisms (SNPs) is elemental in determining genetic variants in many large epidemiological and pharmacogenetic studies. As large-scale association studies are undertaken, reliable, rapid, cost-efficient and high-throughput genotyping methods have become increasingly critical. The cytochrome P450 3A (CYP3A) enzymes are responsible for the metabolism of many drugs, pesticides, carcinogens, steroids and other endogenous and exogenous compounds ingested from natural and artificial sources. Recently, several variant alleles of CYP3A4 and CYP3A5 genes Abstract 583 – Table PPARα∗2 1 (0.3%) (95% CI: 0.1 to 1.8) PPARγ∗2 122 (39.1%) (95% CI: 33.9 to 44.6) PPARα∗3 PPARα∗4 PPARα∗2∗4 PPARα∗3∗4 24 (7.7%) (95% CI: 37 to 47.9) 72 (23.1%) (95% CI: 18.7 to 28.1) 2 (0.6%) (95% CI: 0.2 to 2.3) 33 (10.6%) (95% CI: 7.6 to 14.5) PPARγ∗3 PPARγ∗4 PPARγ∗5 PPARγ∗2∗3 5 (1.6%) (95%CI: 0.7 to 3.7) 0 0 3 (0.1%) (95% CI: 0.3 to 2.8) Poster Session P30. Biomarkers and exposure assessment have been identified. We have developed fluorescent 5’-nuclease assays to identify the following alleles: 3A4∗1B, 3A4∗2, 3A4∗3, 3A4∗17, 3A4∗18, 3A4 G169228A (intron 10), 3A5∗2, 3A5∗3, 3A5∗6, 3A5∗7, and 3A5 K34X (exon 2). The assay takes place in a single PCR reaction and utilizes the 5’-nuclease activity of Taq DNA polymerase to cleave allele-specific probes. These probes are labeled with fluorescent reporter and quencher dyes. When both dyes are attached to the probes, the fluorescence of the reporter dye is inhibited by the quencher dye until Taq DNA polymerase cleaves the reporter dye to emit fluorescence during each cycle extension. Amplification of the allele-specific probes is then detected using an ABI Prism® 7700 Sequence Detector. Primers and probes are designed using the ABI Primer Express™ v.1.5. The validity of these genotyping assays was confirmed using DNA sequencing. This procedure is simple and provides rapid and accurate genotyping results in an efficient, cost-effective format. Supported by NIEHS Center ES-07033 and Superfund ES-04696 585 THE ROLE OF CYP2A6 POLYMORPHISM IN SMOKING BEHAVIOUR AND NICOTINE DEPENDENCE Stefania Morandi, Alfredo Nunziata, Cristina Andreoli. Research Department, Eti S.p.A, Rome, Italy Nicotine is a major constituent of tobacco smoke and is responsible for establishing and maintaining tobacco dependence. Nicotine is mainly metabolised to cotinine by the family of cytochrome P450(CYP)2A6. In the last few years, large interindividual differences in nicotine metabolism have been described. These differences result in a lower level of cotinine in plasma. In this work, we reviewed some recent studies to the aim to clarifying the role of poor nicotine metabolism in smoking behaviour and nicotine dependence. Recent papers have studied the relationship between the poor metabolism of nicotine and genetic polymorphism in the human CYP2A6 gene. Several CYP2A6 polymorphisms have been observed. The CYP2A6∗1A is the wild type. The CYP2A6∗1B arises from a gene conversion with CYP2A7 in the 3’-untranslated region. The CYP2A6∗2 allele encodes an unstable and catalytically inactive enzyme. The CYP2A6∗3 allele has gene conversion in exons 3, 6 and 8 and is inactive. CYP2A6∗4, CYP2A6∗5, CYP2A6∗6, CYP2A6∗7, CYP2A6∗8 and CYP2A6∗1×2 result in a defective or null enzyme activity. These alleles are present with a low frequency in populations. A relationship between CYP2A6 genotype and smoking habits has been proposed in many papers. There are evidences that smokers adapt their smoking behavior, such as number of cigarettes smoked, inhalation depth, volume of each puff., to maintain peripheral and central nicotine levels. It may therefore be postulated that the level of nicotine in smokers with CYP2A6 mutant form is normally higher because of impaired nicotine metabolism, and, as consequence, there is a lower intake of nicotine. In fact, it has been reported that the presence of the CYP2A6∗1 and CYP2A6∗2 alleles significantly decreases the number of cigarettes consumed by smokers, and has been suggested that individuals who carry at least one copy of either CYP2A6∗A2 or CYP2A6∗B2 may be protected against becoming tobacco dependent. 586 INDUCTION OF CYTOCHROME P450 mRNA BY TYPICAL INDUCERS IN RAT HEPATOMA CELL LINE H. Fujimura, M. Itoh, E. Dekura, N. Shimazu, M. Kurabe, C. Aruga, W. Toriumi. Exploratory Toxicology, Exploratory Toxicology & DMPK Research Laboratory, Tanabe Seiyaku Co., Ltd, Saitama, Japan Cytochrome P-450 (CYPs) play important roles in drug metabolism as phase I drug metabolizing enzymes, and induction of CYPs by a compound may have several impacts on toxicity studies in the process of drug discovery and development, i.e. liver histopathology and a decrease in exposure levels of test compounds due to selfmetabolism. The potential of compounds for CYP inductions can be evaluatedin vitro using rat primary hepatocytes, where activities of CYP isozymes decrease within a few days. H4IIE cells derived from rat hepatoma (ATCC #: CRL-1548) have been reported to continuously express CYP2B mRNA. To investigate usefulness of s157 H4IIE cells for detecting CYP induction in vitro, we compared inductions of mRNA for CYP isozymes by typical CYP inducers in H4IIE with those in rat primary hepatocytes. H4IIE cell line was purchased from ATCC and cultured in a monolayer. Rat primary hepatocytes isolated by the collagenase perfusion technique were cultured by the Matrigel™ sandwich method. Beta-naphthoflavone (10 uM), Phenobarbital (100 uM) and dexamethasone (10 uM) were used as inducers for rat major CYP isozymes, CYP1A1, 2B, and 3A1, respectively. After 48 hours exposure, mRNA for CYP1A1, 2B and 3A1 were measured by real time RTPCR (ABI PRISM 7700). In addition, expression of mRNA for PXR, a nuclear receptor regulating expression of CYP3A1, was examined. The CYP1A1, 2B and 3A1 expressions in H4IIE cells exposed to each inducer were 280, 1.5 and 65-fold higher than the basal levels, respectively. The expressions of these isozymes in rat primary hepatocytes were 80, 33 and 152-fold higher than the basal levels. In H4IIE cells, PXR mRNA was induced by dexamethasone at 6-fold higher than the basal level. These results indicate that H4IIE cells can be useful for evaluation of potentials of compounds for inducing CYPs isozymes, in particular CYP1A1 and 3A1, and also suggested that PXR may regulates the induction of CYP3A1 mRNA in the cell. At the meeting, CYP protein expression, morphology and gene expression profiles (DNA microarray) of H4IIE cells will be also presented. P30 Biomarkers and exposure assessment 587 MICRONUCLEI AND RESPONSE TO IN VITRO BLEOMYCIN CHALLENGE IN LYMPHOCYTES OF WORKERS EXPOSED TO LOW-LEVELS OF ACRYLONITRILE António Rodrigues 1,2 , Helena Borba 1 , Carla Horta 1 , José Rueff 1 . 1 Department of Genetics, Faculty of Medical Sciences, New University of Lisbon, Lisbon, Portugal; 2 University Lusófona, Lisbon, Portugal Acrylonitrile is a genotoxic industrial chemical used in the production of acrylic and modacrylic fibres, acrylonitrile-butadiene-styrene and styrene-acrylonitrile resins, adiponitrile and butadiene-acrylonitrile copolymers, and also in cigarette smoke. Occupational exposure to acrylonitrile has in the past been linked to an increase in lung cancer, leading to its classification as a probable human carcinogen, but recently this chemical has been classified as a possible human carcinogen (Group 2B). Few studies have been undertaken in exposed populations using cytogenetic markers as indicators of genetic damage. We present here results obtained in the framework of a EU funded project to ascertain the genetic damage induced by exposure to low levels of acrylonitrile in a Portuguese acrylic fibre plant, currently not exceeding 0,2 ppm. Using the standard cytokinesis blocked micronucleus assay in peripheral lymphocytes we studied 13 workers exposed to acrylonitrile at levels below 0.2 ppm and 14 controls with negligible exposure to acrylonitrile. Results show no significant difference in the number of micronucleated binucleated cells in exposed (3.8 ± 1.9) compared to controls (4.6 ± 1.6). Similarly the average number of micronuclei in exposed (4.3 ± 2.5) and controls (5.6 ± 2.7) were not significantly different. Additionally a challenge assay with bleomycin in vitro was performed in peripheral lymphocytes and the number of micronucleated binucleated cells induced in exposed workers (29 ± 10.5) was also not significantly different from controls (28.5 ± 7.1). These negative results could be due to the relatively low exposure levels of acrylonitrile in the workplace studied and also the low sensibility of the cytokinesis blocked micronucleus assay. 588 MOLECULAR TOOLS FOR THE ASSESSMENT OF GENOTOXICITY N. Prato, S. Citterio, R. Aina, A. Santagostino. Department of Environmental Science, University of Milano –Bicocca, Milan, Italy Human health damages and carcinogenic risk strictly depends on exposure to toxic substances frequently found in contaminated s158 Poster Session P30. Biomarkers and exposure assessment soil. Many xenobiotics, such as polycyclic aromatic hydrocarbons (PHAs), heavy metals, pesticides, have been recognized as being DNA damage inducers. Aim of the present study was to develop a new biomonitoring methodology for assessing soil genotoxicity using Trifolium repens L. Plants can be used as sensibility biological indicators to measure the potential contaminant toxicity. In order to investigate responsibility to known genotoxic substances Trifolium repens L. plants were exposed to soil artificially contaminated with three different heavy metal (Cr, Ni, Cd) and two different PHAs (naphthalene and benzo[a]pyrene). After two weeks DNA from root and shoot was analyzed by molecular tools to detect the DNA damage induced by these genotoxic compounds. DNA sequence alteration was revealed by amplified fragment length polymorphism (AFLP) whereas content alteration was evaluated by flow cytometry (FCM) techniques. Root and shoot dry weight as index of plant growth was also measured. Results showed that the combination of the two techniques led to efficient detection of the genotoxic effect of contaminants in soil. All of the metal treatments induced damage to the genomic sequence, except for the lowest 25 Ni mg/Kg and 5,25 Cd mg/Kg concentrations, both in root and in shoot. PHAs induced DNA alteration only in root and not in shoot, probably because these compounds were not traslocated in plant. Genotoxic effect is also early toxicity index on vegetable able to determine damage without significative difference on clover development by contrast with the classical vegetative endpoints. Work is in progress to apply the same methodology to C.elegans for detecting genotoxicity. 589 PAH-DNA ADDUCTS IN ENVIRONMENTALLY EXPOSED POPULATION IN RELATION TO METABOLIC AND DNA REPAIR GENES POLYMORPHISMS B. Binkova, Z. Smerhovsky, I. Chvatalova, E. Biros, Z. Stavkova, A. Milcova, R.J. Sram. Institute of Experimental Medicine AS CR and Health Institute of Central Bohemia, Prague, Czech Republic Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair genes polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates mutual relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N= 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N=52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by 32P-postlabeling assay. Polymorphisms of metabolizing (GSTML, GSTPL, GSTTL, EPHX, CYP1A1-MpsI) and DNA repair genes (XRCCL, XPD) were determined by PCR-based RFLP assays. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 µg/m3, PM2.5 27–38 µg/m3, c-PAHs 18–22 ng/m3; personal exposure to c-PAHs: 12.0±11.1 ng/m3 and 6.2±3.5 ng/m3, for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92±0.28 vs. 0.82±0.23 adducts/108 nucleotides, p=0.065), whereas the level of the “like” B[a]P-derived adduct was significantly higher in exposed group (0.122±0.036 vs. 0.099±0.035 adducts/108 nucleotides, p=0.003). The significant difference in both the total (P<0.05) and the “like” B[a]P-DNA adduct (P<0.01) between smokers and nonsmokers within both groups was observed. The significant positive association between DNA adduct and cotinine levels (r=0.368, P<0.001) and negative association between DNA adduct and vitamin C levels (r=-0.290, P=0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and epoxide hydroxylase gene in exon 4 as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for “like” B[a]P-DNA adduct. This study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from PAHs exposure. Supported by the grant of czech ministry of environment vav/340/2/00, and by ec grant qlk4-ct-2000 0091. 590 DNA ADDUCTS OF BENZO[A]PYRENE (B[A]P) IN WHITE BLOOD CELLS DNA OF WORKERS OCCUPATIONALLY EXPOSED TO DIFFERENT B[A]P CONCENTRATIONS B. Marczynski 1 , T. Mensing 1 , R. Preuss 2 , M. Wilhelm 3 , J. Müller 2 , C. Broding 2 , T. Merz 2 , J. Angerer 2 , F. Müller 4 , T. Brüning 1 . 1 Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin, Bochum, 2 Institut für Arbeits-, Sozial- und Umweltmedizin, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, 3 Abteilung für Hygiene, Sozial- und Umweltmedizin, Ruhr-Universität Bochum, Bochum, 4 Didier-Werke AG, Wiesbaden, Germany B[a]P is metabolized to (±)-anti- and (±)-syn-benzo[a]pyrenediolepoxide (BPDE) which can covalently bind to the DNA. anti-BPDEDNA adducts in white blood cells (WBC) of occupationally exposed workers in a fireproof material producing plant were determined and compared to the ambient B[a]P concentrations before and three months after the substitution of the binding pitch. 17 PAH-exposed workers were examined before and after the substitution of the production material. Additionally, seven workers were examined before and 16 workers after the substitution. The B[a]P-concentrations in the air were measured by personal air sampling and subsequent HPLC method (diode array detection). We used HLPC separation and fluorescence detection to determine the B[a]P-tetrol I-1 ((±)-r-7,c10,t-8,t-9-Tetrahydroxy-7,8,9,10-Tetrahydro-Benzo[a]pyren) arising after acid hydrolysis from anti-BPDE-DNA. The change in the production process led to a decrease in ambient B[a]P concentrations. For the 17 workers median concentration of B[a]P was determined to be 0.165 µg/m3 (range <0.07 - 0.54) before and <0.07 µg/m3 (range <0.07 - 16.43) three months after the substitution. In these 17 workers the rate ranged between 0.5 and 1.9 adducts/108 nucleotides before the conditions had changed and <0.5 to 0.9 adducts/108 nucleotides three months later. For all workers the median B[a]Pconcentration was 0.14 µg/m3 (range <0.07–0.85 µg/m3 ) before (n=26) and <0.07 µg/m3 (range <0.07–16.43) after the substitution (n=33). For these workers the adduct rate decreases from 0.9 adducts/108 nucleotides (0.2 – 0.9/108 ) (n=24) to <0.05 adducts/108 nucleotides (<0.5 – 3.2/108 ) (n=33) after the substitution. Although ambient B[a]P concentration and DNA adduct level were in a low range, a good correlation between the airborne B[a]P concentration and the adduct level in WBC DNA was observed. 591 EVALUATIONS OF CYP 3A 4/5 AND DNA DAMAGE IN RAT ENTEROCYTES AND IN HUMAN AND RAT LYMPHOCYTES BY FLUOROQUINOLONES. M.E. Fracasso, P. Franceschetti, D. Doria, A. Benini, E. Bertazzoni Minelli. Department of Medicine and Public Health, Section of Pharmacology, University of Verona, Verona, Italy Fluoroquinolones have been considered to be relatively well-tolerated and safe drugs, but gastrointestinal disorders and central nervous system events are the most common adverse effects. The fluoroquinolones are subjected to extensive metabolism and active metabolites can increase the risk of adverse reactions. Many interactions among drugs are due to metabolism through CYP 3A4/5 enzymes. The most expression of this emoprotein is in liver and in the small intestine. The presence of these compounds at intestinal level, at high concentration and/or for prolonged periods of time, suggested a potential risk of inducing toxic effects. On the basis of correlation with adverse reactions at skin level (phototoxicity) we evaluated a possible DNA damage in the gut and in the peripheral blood lymphocytes. The aim of this work is to assess in animals and in human subjects cellular damage (enterocytes and lymphocytes) and to verify changes in CYPs after repeated treatments. The expression of CYPs was determined by W.B. Lymphocyte and intestinal mucosa DNA damage was evaluated by Comet assay: the parameters were tail moment (TM), tail intensity (TI) and tail length (TL). Poster Session P30. Biomarkers and exposure assessment The ciprofloxacin (Cp) and moxifloxacin (Mx) oral doses were 15 mg/kg/day and 10 mg/kg/day for levofloxacin (Lv) and gemifloxacin (Gm). Preliminary results in rat indicate that the levels of CYP 1A1/2 and CYP 2B1/2 are not modified by treatments, whereas CYP 3A4/5 is increased after three and nine days (Lv>Cp>Gm>Mx). It interesting to note that the rise is overall due to the protein 3A5 (60% circa). The rat lymphocyte DNA damage is present with Gm after three and nine days. The treatment with Lv induces a significant increase in TM and TL only after the ninth dose. The rat lymphocytes do not reveal any DNA damage with Cp and Mx. The intestinal cells show cytotoxicity with Lv treatment after nine doses and with Cp after three and nine doses. Data obtained in human lymphocytes indicate that Cp (500 mg/day) induces an increase in TI and TM, only after repeated doses. Preliminary data indicate that the fluoroquinolones show interesting differences in their toxicological profile either blood or intestinal levels. Supported by grant n° 2001058114–002 (MIUR- COFIN 2001) 592 CHANGES IN LOW MOLECULAR DNA FRAGMENTATION OF WHITE BLOOD CELLS AFTER CHAMBER EXPOSURE OF WORKERS TO AROMATIC AND ALIPHATIC DIISOCYANATES B. Marczynski, R. Merget, B. Chilian, M. Korn, S. Rabstein, T. Brüning. Research Institute for Occupational Medicine, Ruhr-University of Bochum, Bürkle-de-la-Camp-Platz 1, 44789 Bochum, Germany Low molecular weight (LMW)-DNA fragmentation patterns in white blood cells (WBCs) were assessed in 16 industrial workers with work-related asthma before and after one day chamber challenges with diisocyanates [4,4’-methylenediphenyl diisocyanate (MDI, 10 workers), toluene diisocyanate (TDI, one worker) and 1,6hexamethylene diisocyanate (HDI, five workers)] in concentrations up to 30 ppb. LMW-DNA fragmentation changes were evaluated after 15 h incubation of WBCs embedded in agarose plugs in lysis buffer (pH 5.2) with or without hydrogen peroxide (H2 O2 ) at 42° C. Increased LMW-DNA fragmentation occurred in WBCs taken at 30 min or 19 h after the end of the chamber challenge in both subjects with positive and in eight of 14 subjects with negative challenges. In contrast, no change in LMW-DNA fragmentation was seen in WBCs taken at the same time intervals of 11 non-exposed controls. There was no association between changes in DNA fragmentation patterns and possible confounding factors such as age, smoking status, atopy, medication, duration of occupational exposure and period since exposure cessation. Similarities in the increase of the amounts of WBC LMW-DNA fragments following diisocyanate exposure with the DNA fragmentation after plugs lysis in buffer with H2 O2 support the hypothesis that diisocyanates exert adverse effects by changing the intracellular redox steady-state. Change in the LMW-DNA fragmentation patterns in WBCs of workers could be due to the production of H2 O2 in blood as a result of diisocyanate inhalation. We have found changes in LMW-DNA fragmentation patterns after exposure not only to aromatic MDI but also to aliphatic HDI. Thus oxidative stress due to the H2 O2 formation in white blood cells does probably not depend only on the aromatic properties of diisocyanates. 593 CARBOXYHEMOGLOBIN CONCENTRATION IN CASES OF ACUTE CARBON MONOOXIDE POISONINGS (ACMP) S.Kh. Sarmanaev, R.G. Samolova, I.E. Yamanaeva. Toxicological center, Ufa, Russia As is well known, lethality in cases of ACMP reaches up to 17.5%. According to some opinions, concentration of COHb does not have any prognostic value, whereas some authors register dependence between concentration of COHb in blood and the clinical picture of the poisoning; they assert that at COHb concentration ≥ 60% a lethal outcome ensues. Aim: Study of the prognostic value of COHb concentration and its dynamics in cases of AMCP. s159 Materials and methods: A retrospective study of medical cards of 162 patients (114 males, 48 females, mean age 42.1±1.2 years), hospitalized at Ufa Toxicological center with ACMP diagnosis in 1998–2002. The state of each patient was assessed according to the scale introduced by Persson et al (1998). Results: The study found a significant difference in COHb concentration in the blood between groups of exited and recovered patients in the first day (48.66±1.05 and 30.23±0.99 respectively; t=12.7; p<0.001). Correlational dependence between COHb concentration and the clinical assessment of severity of the poisoning according to Persson et al. (1998) was r=+0.42 (p<0.05). Results of ROC analysis: at COHb>43% at the moment of hospitalization a lethal outcome is predicted with sensitivity of 82.4%, specificity of 100% (area under the ROC–curve is 0.930±0.063). Sensitivity of the COHb measurement test becomes higher on the second day (the separation point is 20,2%, sensitivity 90.7%, specificity 100%, area under the ROC–curve is 0.960±0.049) and on the third day (sensitivity 88.0%, specificity 100%, area under the ROC–curve is 0.974±0.042). The informativeness value of the COHb decrease speed was smaller (area: 0.802±0.063, sensitivity: 57,1%). Conclusions: 1. The study found a statistically significant difference in COHb concentration in the blood between groups of exited and recovered patients (P>0.001). 2. The study registered an increase of COHb concentration depending on the degree of severity of the poisoning (r=+0.42; p<0.05). 3. The prognostic value of the test increases when studied dynamically. 594 PREPARATION AND USE OF PEPTIDE STANDARDS IN MEASUREMENTS OF 1,3-BUTADIENE HEMOGLOBIN ADDUCTS T. Anttinen-Klemetti 1 , J. Tornaeus 1 , A. Hesso 1 , K. Peltonen 2 . 1 Finnish Institute of Occupational Health, Chemistry Laboratory, 2 National Veterinary and Food Research Institute, Department of Chemistry 1,3-Butadiene is produced from petroleum as a co-product of steam cracking. BD is used as a monomer in the manufacturing of a wide range of polymers and copolymers. In 1996 BD was among the 40 most produced chemical in the USA. Approximately 30000 workers in Europe and 50000 workers in the United States are potentially exposed to BD. The primary routes of human exposure to BD are inhalation. 1,3-Butadiene (BD) is an organic, non natural product, that is classified as a probable carcinogenic to humans (Group 2A) by IARC. In environment BD is originated as a byproduct of cigarette smoke, forest fires and combustion of organic wastes, exhaust emission of fuels, emission from BD production, storage, transport and end use. 1,3-Butadiene is metabolized mainly in liver to electrophilic epoxymetabolites; monoepoxybutene (BMO), epoxybutanediol (EBD) and diepoxybutane (DEB). In reaction with N-terminal valine in globine all the epoxymetabolites forms adducts. Isomeric forms of adducts are formed and DEB has a special character to form a pyrrolidine adduct which is a result from an intra molecular ring closure. We have prepared and characterized 33 modified peptide standards, which will be used in biomonitoring purposes. These include alkylated hepta- and oktapeptide (α-globine), nona- and oktapeptide (β-globine) and corresponding deteurated analogs. The progress of reaction was monitored with HPLC, the reaction products were isolated with HPLC and characterized with LC/MS. The utilization of the standards are on going in measurements of hemoglobin adducts in animals inhalation exposed to BD. The simultaneous measurements of all adducts allow a reliable estimation of the adduct ratios formed in vivo. Acknowledgements: The Finnish Working Environment and Academy of Finland is acknowledged for financial support. s160 595 Poster Session P30. Biomarkers and exposure assessment 4-HEPTANONE IS A MAJOR METABOLITE OF THE PLASTICIZER DI(2-ETHYLHEXYL) PHTHALATE (DEHP) IN HUMANS H.G. Wahl 1,2 , T. Risler 3 , D. Luft 2 , H.M. Liebich 2 . 1 Klinikum der Philipps-Universität Marburg, Department of Clinical Chemistry and Molecular Diagnostics, 2,3 Universitätsklinikum Tübingen, 2 Department of Endocrinology and Clinical Chemistry, 3 Department of Internal Medicine III (Nephrology), Germany There is an ongoing discussion about the risk of DEHP exposure of the population with new-borns and haemodialysis patients on the high risk site. Many studies have shown that DEHP metabolites are more active with regard to cellular responses than DEHP itself. Although 4-heptanone has been shown to be a metabolite in rats this has never been tested in humans. On the other hand 4-heptanone was reported to be associated with diabetes mellitus. After establishing analytical methods for all postulated metabolites we analysed i) plasma samples from 50 patients on haemodialysis and 50 controls, ii) urine samples from 100 diabetic patients and 100 controls and iii) urine samples from ten controls exposed to DEHP. There was no significant difference of the 4-heptanone concentrations in urine between controls (128.6 ± 11.4 µg/l, mean ± SEM) and diabetics (131.2 ± 11.6 µg/l). There was no correlation between the 4-heptanone concentration in urine and plasma glucose, HbA1c or duration of diabetes in the patient group. There were significant higher 4-heptanone plasma levels (mean ± SEM) in both pre- and post-dialysis samples (n=50 each) compared to the control group (n=50): controls 10.4 ± 0.5 µg/l, pre-dialysis 95.9 ± 9.6 µg/l and post-dialysis 37.9 ± 2.7 µg/l. 23 haemodialysis patients were diabetic (HbA1c 7.3 ± 1.4, mean ± SD) and again there was no significant difference of the 4-heptanone concentration in plasma between diabetic and non diabetic patients. By infusing 1 000 ml isotonic saline from regular infusion sets, DEHP and components (e.g. MEHP) were administered intravenously to ten healthy adults. The total amount administered was estimated by GC-MS analysis of infusate aliquots. There was a significant increase in the final metabolite 4-heptanone and in the sum of all β-oxidation products (2-ethyl-3-hydroxyhexanoic acid, 2-ethyl-3-oxohexanoic acid and 4-heptanone) after the DEHP loaded infusion. Moreover, in each of the 10 individuals there was an increase of the final metabolite 4-heptanone between 302 and 2351 nmol/24h. These studies show that 4-heptanone is not associated with diabetes but is a major DEHP metabolite. 596 lates may be useful for human risk assessment and regulatory control for phthalates. (This work was supported by the Brain Korea 21 project 2003 and a grant from NITR/Korea FDA.) 597 SAFETY ASSESSMENT OF A P-PHENYLENEDIAMINE (PPD) CONTAINING PERMANENT HAIR-DYE: A [14 C]-RADIOLABELLED MASS BALANCE STUDY IN HUMAN VOLUNTEERS W.J.A. Meuling 1 , F. Hueber-Becker 2 , F. Benech-Kieffer 2 , G.J. Nohynek 3 , L. Roza 1 . 1 TNO Nutrition and Food Research, Department of Physiology, Zeist, The Netherlands, 2 L’Oréal Life Sciences Research, Skin Biolavailability Unit, Aulnay-sous-Bois Cedex France, 3 L’Oréal Life Sciences Research, Clichy Cedex, France PPD, a key primary intermediates of oxidative hair dyes, is used in hair colouring at a maximum concentration of 2%. Although hair dyes may result in considerable exposure of the human scalp, data on their potential systemic uptake are largely absent. In this study, we determined the systemic absorption of [14 C]-PPD in man during hair dyeing under typical use conditions. Exposure was monitored by measuring the radioactivity in urine, faeces and plasma, and a mass balance was established. The study was conducted according to GCP and GLP regulations. Eight healthy male volunteers participated with their written informed consent. After clipping the hair in advance to a standard length, a dark dye containing [14 C]-PPD and a developer was applied to each subject, amounting to 1.31 g ± 0.05 PPD per volunteer. After 30 minutes of exposure, the dye was rinsed-off followed by shampooing. The hair was dried, completely removed and collected. During the study, the following samples were collected: hair wash and scalp wash, cut hair, blood, urine and faeces, and all materials that came in contact with the dye. Blood samples were taken at 0, 2, 4, 6, 10, 24, 72 and 120 hours. Radioactivity recovered was 81.7 ± 2.2% in the hair wash, 0.41 ± 0.10% in the scalp wash, 13.0% ± 2.0 in hair. The total non-absorbed fraction was 95.2 ± 1.5%, the absorbed fraction was 0.54 ± 0.25% (urine: 0.5%; faeces: 0.04%). The total recovery from the study was 95.7 ± 1.5% (93.0 - 97.1%). Plasma values yielded a Cmax value of approximately 0.09 µg/ml, Tmax of 2 hours, T1/2 of 8 hours and AUC0 - 24h of 0.98 µg · ml · h−1 . In conclusion, most of the [14 C]-PPD applied was recovered (95.2%; =1.25 geq PPD) in the non-absorbed fraction. Further, the systemically absorbed amount amounted to 0.54% (7 mgeq PPD) suggesting low to negligible human systemic exposure to hair dye ingredients. HUMAN MONITORING OF PHTHALATES B.M. Lee, H.J. Koo. Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Chunchun-Dong 300, Suwon, Kyonggi-Do, South Korea Phthalate exposure levels were investigated in humans for risk assessment Using HPLC, phthalates were analyzed from sera and urine of 105 male volunteers (smokers (N)=39, nonsmokers (N)=66). In sera, 88 samples(1.54±3.1µg/Ml) were detectable for DEHP and 31 samples(1.15±1.34µg/ml) for MEHP. DEHP levels in smokers and nonsmokers were 2.48±4.36 and 0.89±1.32(µg /ml), respectively. In urine, 70 samples(1.75±2.7 µg /Ml) were detectable for DEHP and 43 samples(3±4.8 µg /M) for MEHP. DEHP levels in smokers and nonsmokers were 1.18±1.29 and 2.12±3.25(µg /ml), respectively. MEHP levels were on the other hand higher is smokers(2.80±5.5 µg /ml) than nonsmokers(2.6±4.2 µg /ml). Considering the total excretion volume of urine per day and urinary excretion rate (∼20%) of DEHP as MEHP, daily intake levels of DEHP were estimated to be 240 µg /kg/day for smokers and 225 µg /kg/day for nonsmokers, respectively. In addition, BBP, DBP and DIDP detected in sera were, 0.091±0.108(µg /Ml), 0.266±0.245(µg /Ml) and 0.523±0.377(µg /Ml) for smokers and 0.11±0.121(µg /Ml), 0.27±40.191(µg /Ml) and 1.096±0.616(µg /Ml) for nonsmokers. While in urine, 8.86±3.66(µg /Ml), 0.59±0.06(µg /Ml) and 2.04±1.15(µg /Ml) for smokers and 9.10±7.26(µg /Ml), 0.74±0.56(µg/Ml) and 2.08±2.32(µg/Ml) for nonsmokers were detected. These data suggest that human exposure levels to phtha- 598 HAIR ANALYSIS: EVALUATION OF A SIMPLE METHOD TO ASSESS CHRONIC EXPOSURE OF RATS TO THE ORGANOPHOSPHATE DIAZINON Maria Tutudaki, Andreas Tsakalof, Aristidis M. Tsatsakis. Lab of Toxicology, Medical School, University of Crete, Voutes, Heraklion, 714 09 Crete, Greece Purpose: The main purpose of the present study was to develop a sensitive analytical method for trace analysis of diazinon in a complex matrix like hair. Also to determine whether hair analysis would be a suitable method to assess chronic exposure to organophosphorus pesticides. Finally to compare the results obtained in this study to those obtained in a similar experiment using rabbits, in order to see if interspecies metabolic variation produces differences in the concentration of the pesticide measured in hair. Experimental: Sprague Dawley rats were exposed to two dose levels (6mg/kg/day and 3 mg/kg/day) of the pesticide, through their drinking water, for a period of one and a half months. Hair samples from the back of the rats were removed before commencing the experiment and at the end of the dosing period. In parallel experiments with diazinon spiked hair were carried out in order to design a simple and efficient method of extraction of the pesticide from hair. The hair was pulverized in a ball mill homogeniser, incubated in methanol at 37 0 C overnight, liquid-liquid extracted with ethyl acetate and measured by GC-MS (negative chemical Poster Session P30. Biomarkers and exposure assessment ionisation mode). The mass spectrometer was operated at the selected ion-monitoring mode and programmed for the detection of m/z 169 for diazinon, and 263 for fenthion, which was used as internal standard. Under these conditions diazinon eluted at time t=15.29 min, and fenthion eluted at time t=18.52 min. Results & Conclusions: The L.O.D. for NCI mode was 0.05 ng/mg hair as opposed to 0.1 ng/mg hair for EI mode. This enabled us to perform all analyses on the GC-MS. The concentration of diazinon in the hair of the exposed animals ranged from 0.23–0.61 ng/mg. The mean concentration of the pesticide in the hair of the low dose group was 0.24 ± 0.01 ng/mg while the mean concentration of the pesticide in the hair of the high dose group was 0.53± 0.05 ng/mg. A relationship between the administered dose and the detected pesticide concentration in hair was shown. It could be concluded that hair analysis may be used to investigate chronic exposure to the pesticide, but parameters such as differences in the metabolism must be accounted for in order to get reliable results. 599 A COMPARISON OF ENVIRONMENTAL BENZENE EXPOSURE IN SUBJECTS FROM RURAL AND URBAN POPULATIONS USING AN IMMUNOASSAY FOR THE SPECIFIC BIOMARKER S-PHENYLMERCAPTURIC ACID (S-PMA). Jacqueline Marsh 1 , Karen Houser 2 , Elizabeth Johnston 1 , Valerie Morse 2 . 1 Molecular Light Technology Research Ltd. 5 Chiltern Close, Cardiff Industrial Park, Cardiff, Wales, U.K. 2 Pembrokeshire College, Haverfordwest, Pembrokeshire, Wales, U.K Benzene is a ubiquitous environmental pollutant. Although overall environmental levels of benzene are low compared to those encountered by workers in some industrial environments, they are still of interest because of the pollutant’s known toxic and carcinogenic effects on the human haematopoietic system. Previous studies have indicated that certain urban areas have higher levels of airborne benzene than rural areas. The main sources of airborne benzene are combustion engines and processes associated with chemical manufacturing or the motor fuel industry. However, external airborne monitoring is not able to monitor all non-occupational sources of benzene exposure, such as cigarette smoke, certain foods and water. This study investigated whether there was any difference in environmental benzene exposure in non-occupationally exposed subjects from rural and urban populations. The level of benzene exposure was determined by measurement of the concentration of the specific biomarker S-PMA in urine samples, using a commercially available immunoassay. Allowances were made for the hydration status of individuals by the determination of urinary creatinine. The smoking habits of subjects were recorded since heavy smokers may have elevated levels of urinary S-PMA compared to non-smokers. The results of the study revealed that the median urinary concentration of the biomarker from the urban group was higher than that of the rural group. 600 SEVOFLURANE OCCUPATIONAL EXPOSURE MONITORING: A ROLE OF URINARY HEXAFLUOROISOPROPANOLOL F. Barbic’ 1 , M. Bagnati 2 , M. Basile 2 , V. Zanoli 1 , C. Cassani 2 , P. Porta 2 , A. Fortina 2 , L. Carettoni 1 , C. Mantovani 1 , G. Bellomo 2 . 1 Medicina del Lavoro,2 Laboratorio di Ricerche Chimico-Cliniche, Università del Piemonte Orientale- Azienda Ospedaliera “Maggiore della Carità”, Novara, Italy The increasing use of sevoflurane as anaesthetic leads to the need of finding a biological index of occupational exposure. Several studies indicate that hexafluoroisopropanolol (HFIP) is a specific sevoflurane metabolite quickly glucuronidated and excreted as HFIP-glucuronide in the urine (HFIPU). Therefore the HFIP removal kinetics and the correlation between sevoflurane occupational exposure and HFIPU are poorly understood. Aims: To evaluate, in a group of operating room workers exposed to sevoflurane, the HFIPu expressed as µg/L (A-HFIPu) or normalized for urinary creatinine as µg/g creat. (C-HFIPu). s161 To evaluate the correlation between A-HFIPu and C-HFIPu and the individual sevoflurane occupational exposure. Methods: We studied 73 healthy subjects working in 14 operating rooms of hospital “Maggiore della Carità”, Novara. Every subject underwent to an active air-sampler to evaluate the individual sevoflurane exposure (SE) (charcoal vials; flow-sampler 100–200ml/min.); for every worker the end-shift urine has been collected to evaluate HFIPu. Sevoflurane, eluted by a water-methanol mixture, and HFIP after enzymatic hydrolysis of acidified urine, were analysed and quantified by GC/MS with headspace autosampler technique. In all urine samples the creatinine concentration was also measured. Results: The HFIPu mean value in the whole group was 321±706 µg/L (range 2–5208) and 182±318 µg/g creat. (range 2–1478). The HFIPu in subjects with low sevoflurane exposure (0.093±0.095 mg/m3 ), was 3.0±0.8 µg/L (range 2.0–4.0) and 3.5±1.3 µg/g creat. (range 2.0–5.0) while the HFIPu corresponding to high sevoflurane exposure (12.35±4.53 mg/m3 ) was 1622.5±409.1 µg/L (range1079– 2072) and 983.7±373.7 µg/g creat. (range 441–1294). The correlation coefficients of SE/A-HFIPu and SE/C-HFIPu were respectively R2 =0.73 (p<0.0001) and R2 =0.87 (p<0.0001). Conclusions: In accordance with previous studies we confirmed that HFIPU is a good index of sevoflurane exposure that could be used in occupational safety programs. Moreover these preliminary results suggest that the HFIPu normalized for urinary creatinine (C-HFIPu) is an index more suitable than the A-HFIPu. 601 DEVELOPMENT OF A COMPREHENSIVE TWO-DIMENSIONAL GEL DATABASE OF RAT LIVER PROTEINS USEFUL FOR TOXICOLOGICAL HAZARD IDENTIFICATION N. Querfurth 1 , A. Oberemm 1 , C. Meckert 1 , L. Brandenburger 1 , A. Herzig 1 , K. Kalenberg 2 , Y. Lindner 2 , E. Krause 2 , H.-B. Richter-Reichhelm 1 , U. Gundert-Remy 1 . 1 Department of Assessment of Chemicals, Federal Institute for Risk Assessment, Berlin, Germany, 2 Institute of Molecular Pharmacology, Berlin, Germany After promising approaches during the past decade, the development of publicly available databases for rat liver proteins has been slowed down. Emerging proteomic technologies still depend on data generated by 2D-electrophoresis (2-DE), so we decided to develop a new 2-D electrophoretic map for male Wistar rat liver proteins in the context of a molecular toxicological joint research project*. 2-DE separation was performed using the common IPG technique developed by Goerg and coworkers. Spots were excised from 2-D gels using a spot picker. After in-gel digestion with trypsin, proteins were identified by using Peptide Mass Fingerprinting (MALDITOF) and capillary LC-MS/MS (ESI-Quad-TOF). Available protein information, i.e. protein identity, molecular weight, isoelectrical point and EC-number in Enzyme Nomenclature were combined into an HTML document, which lead to a clickable HTML protein map. Over 300 proteins were identified, among those 140 enzymes and 100 structural proteins. This map could be the basis for a publicly accessible HTML document and could provide quick information to identify protein expression patterns of toxicological relevance. Among the 300 identified proteins we found a plenty of common toxicological marker proteins. With the entire database regulated spots can be found easily and patterns of protein expression may contribute mechanistic data to assess toxic and carcinogenic effects of chemicals. *Funded by the German Ministry of Education and Research, Grant-No. 0312618 s162 602 Poster Session P30. Biomarkers and exposure assessment PROTEOMIC CHARACTERIZATION OF MOUSE BRONCHOALVEOLAR LAVAGE FLUID BY MASS SPECTROMETRY. I. METHODS. J.N. Adkins 1 , K.M. Lee 2 , N. Tolic 1 , K. Auberry 1 , R.D. Smith 1 , J.G. Pounds 1 , W.J. McKinney 3 . 1 Biological Sciences Department, Pacific Northwest National Laboratory, Richland, WA, 99352; 2 Battelle Toxicology Northwest, Richland, WA, 99352; 3 Philip Morris USA, Richmond VA Analyses of specific Bronchoalveolar lavage fluid (BALF) biomarkers have been used to evaluate acute smoke-induced lung injury. The objective of this study was to establish a procedure to identify an array of BALF proteomes as potential biomarkers for smoke-induced lung injury. Young male ICR and C57BL/6 mice were exposed via nose-only inhalation to either air or cigarette smoke (2R4F; 75, 250, or 600 µg TPM/L) for 2 h/d for 7 days. BALF was collected after 12 h post-exposure, pooled per strain/exposure, and proteins precipitated in 8% TCA, washed in acetone, and digested with trypsin. The online reversed-phase microcapillary LC with ion trap MS was used to produce tandem mass spectra. The mass spectra were analyzed by the program SEQUEST, which deemed ∼150,000 of the mass spectra analyzable to peptide identifications. Applying a typical filter for Xcorr and a 5 ppm measurement mass accuracy resulted in ∼2000 unique peptides, as confirmed by Fouier Transform Ion Cyclotron Resonance-MS. Ultimately, ∼1100 individual proteins, many identified by multiple peptides, were identified which included cytokines, growth factors and their receptors, kinases, apoptosisrelated, lung-related, some blood proteins, and ∼150 hypothetical proteins. Many proteins are identified in the BALF samples for the first time. Monitoring profiles of these novel BALF proteins will facilitate understanding the pathogenesis of pulmonary diseases. (Supported by Battelle and Phillip Morris USA). 603 FINGERNAIL CLIPPINGS AS ANALYTICAL SPECIMENS FOR DETECTION OF OPIATE AMONG HEROIN ABUSERS Hoda Fouad Abdel-salam 1 , Somia A. Madkour 1 , Ola A. Sharaky 2 , Tarek K. Molokhia 3 . 1 Forensic Medicine and Toxicology, 2 Clinical Pathology Department, and 3 Psychiatric Unit, Faculty of Medicine, University of Alexandria, Egypt Heroin (Diacetylmorphine) is commonly abused allover the world and it may be a cause of death. The aim of this study was to evaluate the use of fingernail clippings as analytical specimens alternative for hair and other body fluids (they are not available) for detection and quantitation of morphine heroin abusers morphine concentration in fingernail clippings and duration of addiction . The study included 20 consenting adult male heroin addicts, their age ranged from 21 – 45 years. They were admitted to a private hospital for withdrawal treatment. During sampling duration of addiction was recorded. Nail clippings (14.6 – 120mg) were obtained from the addicts. They stored in plastic bag at room temperature till analysis. Surface decontamination of fingernail clipping were done using sodium dodycyle sulfate (SDS), deionized water and methanol. The washed nail clippings were‘hydrolyzed and the extracts were analyzed by radio – immunoassay (RIA) for detection and quantitation of morphine. The RIA method was shown to be accurate and reproducible with almost 100% recovery of morphine and hydromorphine. The coor –A- count opiate screen is a solid – phase quantitative radio immunoassay, where in 125 I – labeled morphine competes for fixed time with opiates in the sample for antibody sites. Data were recorded and tabulated. Statistical analysis were performed using SSPS program version 6. Positive data were obtained in all cases. The level of morphine in fingernails ranged from O.28 to 6.8 ng/mg with a mean of 2.96+ 1.81 ng/mg . A significant correlation also found between morphine level in fingernails and duration of addiction where P was < 0.01. So fengernail clippings can be used as analytical specimens among other samples for detection of past opiate use. 604 ANALYSIS OF HUMAN, DOG, RAT AND MARMOSET SERUM PROTEINS BY CAPILLARY ELECTROPHORESIS. F. Crivellente 1 , M. Bonato 2 , F. Bortolotti 2 , M. Trettene 2 , L. Vandin 1 , G. Dal Negro 1 . 1 Cellular & Biochemical Laboratory, Safety Assessment Department, GlaxoSmithKline R&D, Verona; 2 Department of Medicine and Public Health, Unit of Forensic Medicine, University of Verona Capillary electrophoresis (CE) for serum protein analysis offers a new tool for the clinical laboratory organization, in a field traditionally dominated by labor intensive techniques like agarose gel electrophoresis or cellulose acetate electrophoresis. Minimal sample requirement (2 µl), complete automation and quantitative results make capillary electrophoresis a valuable technique for the analysis of proteins in humans and experimental animals. In this work, a comparison between serum protein analysis carried out by capillary electrophoresis and agarose gel electrophoresis was performed. The results obtained showed a better quality separation of serum proteins by CE in comparison with classical gel electrophoresis; peak assignation was easily carried out in CE on the basis of the similarity of the separation pattern with gel electrophoresis and by using pure standards. The direct detection of protein fractions (without staining) and easy documentation of raw data make CE an ideal technique to improve objectivity of protein analysis. 605 GLYCOCONJUGATES DISTRIBUTION PATTERN AND OXIDATIVE ENZYMES ACTIVITY AS BIOMARKERS OF POLLUANT CONTAMINATIONS IN THE MUSSEL, Mytilus galloprovincialis, FROM LAKE FARO (MESSINA, ITALY). A. Licata, S. Martella, L. Ainis, M.B. Ricca, E.R. Lauriano, C. Calabrò. Department of Animal Biology and Marine Ecology, University of Messina, Messina, Italy The presence of pollutants in acquatic environment and in vivo experiments showed that mucus overproduction in gill cheched oxidative enzymes, producing hypoxia or anoxia. In previoius investigations conducted in the in lake Faro (Sicily, Italy), pesticides compounds and heavy matals (Cd, Pb, Zn and Cu) were determined on soft tissues of the mussel, Mytilus galloprovincialis. The present study was performed on 300 samples taken in four stations (indicated as north, south, west and east) from April to November 2002. Chemical analysis showed DDE, Cd and Pb concentratrations in the south and west zones in higher concentrations than other zones. However, all samples showed levels below MRL for DDE, Cd and Pb. The aim of the present investigation was to confirm chemical results using glycoconjugates and oxidative enzymes, as biomarkers, in the mantel and gill epithelia. Morphological survey showed slight mucous cells iperplasya in both mantel and gill epithelium coming from south and west stations. In both mantel and gill epithelim of the mussels, coming from the four zones, histochemical study by biotilynated lectins (ConA, WGA, PNA, SBA and UEA-I), demonstrated that glycoconjugates distribution patterns were lack of evident differences. In gill lamellar epithelium oxydative enzymes (SDH, MDH and ICDH) activity showing the absence of respiratory suffering. Glycoconjugates, components of the surface glycocalyx in many cell types, are involved in several important functions, including cell-cell recognition and the protection from noxiosus molecules. They may be a useful tool to indagate the changes of glycoproteins and represent a reliable test for assessing stress. In conclusion this study suggested that lake Faro is without toxicological risk for both mussels and consumers. s163 Poster Session P30. Biomarkers and exposure assessment 606 NEW NON INVASIVE TESTS TO DETECT EARLY EFFECTS OF AIR POLLUTANTS ON THE RESPIRATORY EPITHELIUM: DEVELOPMENT AND APPLICATION TO SCHOOLCHILDREN LIVING IN BRUSSELS. PRELIMINARY RESULTS. K. Berthoin 1 , A. Clippe 1 , X. Dumont 1 , A. Bernard 1 . 1 Catholic University of Louvain. Faculty of Medicine-School of Public Health. Industrial Toxicology and Occupational Medicine Unit. Clos Chapelle-aux-Champs 30 bte 30 54, B-1200 Bruxelles With the support of the European Union, the Unit of Toxicology of the Catholic University of Louvain tries to develop an entirely new non invasive approach to detect early effects of air pollutants on the respiratory tract of schoolchildren living in Brussels. This approach referred to pneumoproteinemia relies on the determination in serum of proteins secreted by the lung epithelium. The immediate objective of this project is to develop and then apply sensitive immunoassay for measuring in serum one new protein, called PN-SP1 for pneumo-secretoprotein 1, recently identified by the host laboratory and specifically secreted by the lung epithelium. The best source of lung specific proteins is broncho-alveolar lavage from patients with lung dysfunction. Pooled lung lavage samples are concentrated in a Centricon® concentrator. The whole purification procedure of the native human PN-SP1 is performed with an HPLC System® from Pharmacia. This procedure requires three steps: 1. Gel filtration chromatography (Superdex 75 HR 10/ 30 column); 2. Ion-exchange chromatography (Mono Q HR 5/ 5 column); 3. Gel filtration chromatography (Superdex 75 HR 10/ 30 column). After each separation step, each fraction is tested in an fully automated immunoassay which relies on the agglutination of calibrated latex particles coated with the specific antibodies against recombinant PN-SP1. The purity of the protein is checked by electrophoresis with silver staining. A minimum of 1 mg of human native PN-SP1 is necessary for immunizing two rabbits. We will apply this new immunoassay to children recruited in different schools in Brussels. 607 MEASUREMENT OF THE FORMALDEHYDE CONCENTRATION DURING THE PERIOD OF HUMAN DISSECTION COURSE Y. Matsuno 1 , K. Ohmichi 1 , M. Koda 2 , R. Anahara 1 , E. Todaka 1,3 , H. Fukata 2 , M. Komiyama 1,3 , T. Kadota 1 , M. Ohmichi 4 , C. Mori 1 . 1 Departments of Bioenvironmental Medicine and 2 Environmental Medical Science (SRL), Graduate School of Medicine, Chiba University, 3 Center for Environment, Health and Field Sciences, Chiba University, Kashiwa, 4 Chiba City Institute of Health and Environment, Chiba, Japan Long-term exposure to relatively high levels of formaldehyde (FA) is known to increase the risk of bronchitis and cancer. However, cadavers for human dissection course are preserved in fluid containing FA, and the FA concentration level in the atmosphere of dissection room is thought to be higher than normal circumstances. Recently, medical students who appeals that they have symptoms of sickness with various chemicals which contain FA during the human dissection course are increasing. In the present study, we investigated the FA concentration level in the atmosphere of the dissection room before the course started, and the third, tenth, seventeenth day of the course, and after the course finished. The air samples were collected by detection pipe. The ceiling of the human dissection room is 3 meters high, the floor surface is 375 m2 , and there are 52 cadavers kept on the stainless steel dissection tables during the course. The air sampling was performed at four corners and near the center of the dissection room. The class held 22 times in total over two months and it lasted over 3 hours each time. The FA concentration levels in the atmosphere in the room were 0.5, 1.5, 2.0 ppm at third, tenth, seventeenth day, respectively, and the FA concentration before and after of the course were under a detection limit (0.1 ppm). Human blood samples will be also collected from the teaching staff of the course and the concentration level of FA and hormones in the serum will be discussed. 608 CREATINPHOSPHOKINASE: CRITERIA FOR ESTIMATING SEVERITY OF A CHEMICAL BURN OF THE INTESTINAL TRACT S.Kh. Sarmanaev, I.E. Yamanaeva, N.F. Valeeva. Toxicological center, Ufa, Russia Background: Estimation of severity of the intestinal tract burn is based on the analysis of its area, depth and localization (Persson et al., 1998), but the absence of a method to determine the depth of the burn in the first days limits the estimation method. Although creatinphosfokinasa (CPC) has been long used in diagnosing miocard infarction and the skeletal musculature injury, CPC is not used for estimating injuries of the smooth musculature. Aim: Determination of the diagnostic value of CRC activity for estimating the depth of a chemical burn of the intestinal tract. Materials and methods: The activity of CPC was determined by means of spectrophotometrical analysis on days 1–2, 3–7 8–14 after the hospitalization date with 40 patients who had acute corrosive poisonings (out which 13 were males). The injury of the muscular membrane of the intestinal tract was verified by endoscopically examining ulcers in the same periods. Results: A ROC analysis demonstrated that the optimal separation point is the CPC activity of 280 nmoles per liter (the area under the ROC curve = 0.859±0.076; sensitivity 87.5%; specificity 83.3%). Dynamics of CPC activity in cases of acute corrosive poisonings Agent Groups of patients No. n Depth Days of hospitalization 1–3 All corrosives I 22 No ulcers 201.7±49.3** (n=40) II 18 Ulcers 740.2±213.6 Non-resorbtive III 12 No ulcers 136.9±14.4** corrosives (n=23) IV 11 Ulcers 428.3±156.6 4–7 8–14 227.6±73.6 92.6±24.4** 459.8±131.9 440.4±236.7 138.0±17.0 84.9±43.7 701.0±147.0 263.7±73.8 *p<0.05; **p<0.01; Wilcoxon Mann-Whitney criterion; comparison with groups II and IV. We studied CPC activity with patient poisoned by corrosives, which have no resorbtive effect (see Table). The optimal separation point is 202.5 nmoles per liter (the area under the ROC curve = 0.870± 0.105; sensitivity 100.0%; specificity 83.3). Conclusions: CPC is an informative criterion for estimating the degree of injury of the intestinal tract muscular membrane in cases of corrosive poisonings. It can be used as an indication of the depth of intestinal tract injury at the time of hospitalization. 609 THE EFFECT OF WOOD DUST ON APOPTOSIS IN MURINE MACROPHAGES Lea Pylkkänen 1 , Helene Stockman-Juvala 1 , Juha Määttä 1 , Harri Alenius 1 , Kai Savolainen 1 . Finnish Institute of Occupational Health, Department of Industrial Hygiene and Toxicology, Helsinki, Finland There are data to provide evidence that wood dust may induce apoptosis and necrotic cell death in various cell types. To determine the effects of wood dust exposure on apoptosis in murine macrophages DNA fragmentation and caspase-3 activity were measured after exposure of RAW 264.7 macrophage cells to wood dusts of birch, oak, pine, and spruce. DNA fragmentation that was detected with agarose gel electrophoresis as DNA-ladders was clearly detectable after 24 and 48 hours exposure to all wood dusts studied. No fragmentation was seen after six hours exposure to any of the wood dusts studied at any concentrations. Significant dose-dependent induction of caspase-3 activity was observed after 48 hours exposure at the highest concentration (1000 µg/ml) of spruce, pine, and oak dust. The maximal induction was approximately 3-fold for pine and oak dusts and 2-fold for spruce dust as compared to the control level. The effect of birch dust on caspase-3 activity was not as evident as the other wood dusts studied. Since the preliminary results considering murine macrophages suggest that wood dust induces apoptosis, we will focus in our further studies on experiments with the simian virus-40 transformed human bronchial-epithelial cell line BEAS-2B cells. s164 610 Poster Session P31. Occupational toxicology SENSITIVITY AND SPECIFICITY OF THE TABLE-BASED METHOD OF PROGNOSIS OF OUTCOMES OF ACETIC ACID POISONINGS (AAP) I.E. Yamanaeva, S.Kh. Sarmanaev. Toxicological Center, Ufa, Russia Background: The building of scales estimating the state of a patient simplifies the problem of choice of a tactics in treating the patient. With the help of non-homogeneous consecutive procedure (a modification of Wald’s consecutive analysis) we develop a diagnostic table based on 16 features, which is used to make prognosis of outcomes in cases of acute AAP (Sarmanaev&Yamanaeva 2001). In making the prognosis, the following possible outcomes were considered: favorable, unfavorable, doubtful. Aim: Estimation of specificity and sensitivity of a prognostic table in clinical practice. Materials and methods: By means of a table-based method, we prospectively analyzed 102 patients with acute AAP, which had been hospitalized in the toxicological center of Ufa during 2001 – first half of 2002. Results: A correct prognosis was made in 94.1% of all cases, a wrong one – in 0.9% (the patient died of a complicated pneumonia on the 9th day of the disease), no prognosis was defined in 4,9% of cases. The prognostic value of the positive result of the test was 100%, of the negative result – 98.8% (excluding doubtful prognosis). The sensitivity of the tables amounted to 91.7%, specificity was 94.4% (including doubtful prognosis). The prognostic value of the test was +2.65%. Conclusions: 1. The table-based method of prognosis of outcomes in cases of acute AAP is characterized by a high sensitivity and specificity. 2. The table for prognosis of outcomes in cases of acute AAP is easy-to-use and provides a correct prognosis in 94.1% of cases. References: [1] Yamanaeva IE., Sarmrnaev SKh. Use of Wald‘s sequental test for grading the severity of acetic acid poisoning (AAP) and for predicting its outcome. Toxicol Clin Toxicol 2001;39:509. 611 TABLE-BASED METHOD OF PROGNOSIS IN CASES OF MINERAL ACIDS POISONINGS P31 Occupational toxicology 612 DETECTION OF OCCUPATIONAL BENZENE EXPOSURE IN CHEMICAL PLANT WORKERS USING AN IMMUNOASSAY FOR S-PHENYLMERCAPTURIC ACID (S-PMA) Jacqueline Marsh 1 , Richard Brown 1 , Sarka Crhova 2 , Ian Weeks 1 . 1 Molecular Light Technology Research Ltd. 5 Chiltern Close, Cardiff Industrial Park, Cardiff, CF14 5DL, Wales, UK.2 National reference laboratory for POPs, Frydek-Mistek, Czech Republic Methods that enable the biological monitoring of occupational benzene exposure are important because of the chemical’s known toxic and carcinogenic effects on the human haematopoietic system. Due to the widespread use of benzene in industry, workers in several sectors including the manufacture of plastics, chemicals, rubber and shoes are potentially at risk from occupational exposure. The identification of the specific biomarker for benzene exposure S-phenylmercapturic acid (S-PMA) has led to the development of several methods to detect its presence in urine samples from such workers. However, the majority of these methods are expensive and time-consuming, resulting in comprehensive population studies and screening programs being lengthy and prohibitively expensive. The recent development of immunoassays for S-PMA offers an affordable, rapid alternative to methods such as GCMS and HPLC. Nevertheless, immunoassay needs to demonstrate that its performance is at least comparable to more generally accepted methods such as GCMS and that it is capable of confirming exposure as identified by airborne monitoring. In this study, the S-PMA concentrations in urine samples from workers at a chemical plant were determined by both GCMS (carried out by an independent laboratory) and immunoassay. Comparison of the methods indicated that results obtained using immunoassay showed a strong correlation with those obtained by GCMS. From a total number of 49 workers, passive sampling returned levels of airborne benzene exposure in excess of 1ppm in 13 cases. In all 13 cases, the immunoassay determined that the urinary S-PMA concentration was in excess of the normal range for the assay. This illustrates that immunoassay represents a more convenient and cost effective alternative for benzene exposure biomonitoring. S.Kh. Sarmanaev, L.F. Aidarova. Toxicological Center, Ufa, Russia Background: Acute peroral poisonings by corrosives take up a considerable important place in the structure of acute poisonings (Cox, 1997, Sentzov 2000). Among all corrosive poisonings the proportion of sulfuric acid poisonings is 7.13%; by hydrochloric acid 4.7%; the lethality rate in the case of the latter reaches 15% (Sarmanaev et al. 2001). Aim: Preparation of prognostic tables to aid emergency physicians. Materials and methods: The study analyzed 150 medical cards of patients poisoned by mineral acids, that had been admitted to the toxicological center (1984–2000). We studied patients who suffered a severe degree of poisoning and those who deceased; the state of each of them was estimated according to the Persson et al scale (1998) as III and IV degrees of severity. In addition to that, we compared two groups of patients: those who required intensive care and those who did not. The processing of the data was carried out using a modified version of the Wald method (Genkin&Gubler 1978). We considered 29 diagnostic features: age, sex, presence of intention of poisoning, alcohol intoxication, dose-exposition, average arterial pressure, Altzhover index, breathing frequency, consciousness, body temperature, laboratory data, development: hemolisis, hematuria, oligouria, disphonia, shock, esophagus or stomach bleeding. The informativeness of the features was estimated according to Kullback S. (1959). Results: 1. Two types of prognostic tables were prepared: 1) for the prognosis of the outcome of the poisoning and 2) for the determination of the necessity of intensive care. The tables included only informative features. In the first case there were 14 features, in the second there were 7 of them. The rest of the features were found to be of little informative value. 2. These tables are intended for the use in clinical practice by emergency physicians. 613 CHROMOSOMAL ABERRATION (CA) AND SISTER CHROMATID EXCHANGE (SCE) FREQUENCIES IN WORKERS EXPOSED TO VERY LOW DOSES OF 1,3-BUTADIENE P. Lovreglio 1 , N. Bukvic 2 , S. Fustinoni 3 , A. Ballini 2 , I. Drago 1 , V. Foà 3 , G. Guanti 2 , L. Soleo 1 . Department of Internal Medicine and Public Medicine; 1 Section of Occupational Medicine; 2 Section of Medical Genetic, University of Bari; 3 Department of Occupational Medicine,University of Milan The aim of the study was to investigate CA and SCE frequency in peripheral blood lymphocytes of workers occupationally exposed to very low doses of 1,3-butadiene (BD). 27 workers employed at BD production or polymerisation (range of exposure 0.18 - 69.03 µg/m3 ), an internal control group (26 administrative workers of the same plant, with range of exposure 0.05 - 3.8 µg/m3 ) and an external control group (including 12 foresters) were examined. Exposure was monitored by personal samplers. A questionnaire on personal characteristics and lifestyle was administered to all workers. Cytogenetic tests were performed according to standard procedures. The mean frequency, both of SCE and CA, did not show any significative difference among the three groups. To evaluate the influence of smoking habits, all subjects were classified in no-smokers, ex-smokers and smokers. The mean frequency of SCE increased from no-smokers to smokers, with a significative difference among the groups (F=9.76; p<0.001), while no difference was found for the mean frequency of CA. A positive correlation between SCE frequency and BD personal exposure (r=0.31; p=0.027) was observed considering together exposed and internal control subjects. The number of cigarettes/day, on Poster Session P31. Occupational toxicology the contrary, was not correlated with SCE frequency. No correlation, besides, was observed between CA frequency and both the BD levels and the number of cigarettes/day. Multiple regression analysis was performed on data from exposed group and internal control group, considering as dependent variable the frequency of SCE and as independent variables all the available quantitative variables. The model resulted significative (F=4.91; p=0.011) with a relationship of the SCE frequency with the BD exposure levels (p=0.032) and the number of cigarettes/day (p=0.044). In conclusion, our results seem to suggest that, at very low environmental BD doses as those observed in this study, in addition to smoking habits, also BD exposure could influence SCE frequency. This research was cofinanced by Italian Ministry of University and Scientific Research (40%). 614 MOLECULAR BIOMARKER STUDIES IN RENAL CELL CANCER PATIENTS OCCUPATIONALLY EXPOSED TO TRICHLOROETHYLENE H.M. Bolt 1 , Hiltrud Brauch 2 , Bettina Klein 2 , G. Weirich 3 , T. Brüning 4 . 1 Institut für Arbeitsphysiologie an der Universität Dortmund (IfADo), 2 Dr. Margarethe Fischer-Bosch Institut für Klinische Pharmakologie, Stuttgart, 3 Institut für Allgemeine Pathologie und Pathologische Anatomie, Technische Universität München, 4 Berufsgenossenschaftliches Forschungsinstitut für Arbeitsmedizin (BGFA), Ruhr-Universität Bochum, Germany Background of the investigation was the frequent occurrence of extremely high occupational trichloroethylene exposures in renal cancer patients, as demonstrated in two case-control studies performed in the area of Arnsberg/Germany. This area was characterised by small enterprises of the metal industry with frequent use of trichloroethylene for degreasing purposes. The present study was to carry out molecular analyses of tumour tissues from renal cell carcinoma patients, to verify whether mutations have occurred to the von-Hippel-Lindau (VHL) tumour suppressor gene and, if so, to assay the spectrum of these mutations to identify the affected base-pairs. The study reinvestigated the cases with a renal tumour from a previous case-control study. Out of the total group of 58 cases, 17 persons were trichloroethylene-exposed, and 22 persons were not. Samples of the renal tumour tissues could be obtained from the total group and were taken for analysis. Within the non-exposed subgroup, two persons showed single VHL point mutations in the tumour tissue. There were no multiple VHL mutations in this group. By contrast, in the subgroup of trichloroethylene-exposed patients, 14 showed VHL mutations in the tumour tissue, among these one case with 4 mutations, one with three mutations, four with two mutations, seven with a single mutation. These results provide further support for the coherence between development of human renal cell cancer and high occupational exposures to trichloroethylene. 615 ORGANIC SILICON COMPOUNDS: A CHALLENGE TO TOXICOLOGICAL RISK ASSESSMENT J. Liesivuori 1,2 , J. Mäittälä 1 , S. Pennanen 1 . 1 Finnish Institute of Occupational Health, P.O.Box 93, 70701 Kuopio, Finland, 2 Department of Pharmacology and Toxicology, University of Kuopio, P.O.Box 1627, 70211 Kuopio, Finland The manufacture and application of organosilicon compounds, especially silanes, have undergone a major increase throughout the world during the last decade. This has led to an increase in the number of exposed workers in different areas of industry. Chemical structure of silanes indicate high biological reactivity. Occupational exposure studies were conducted during filament forming and the handling of coated fibres. The alkoxysilanes studied were 3-methacryloxypropyltrimethoxysilane, 3-glycidoxypropyltrimethoxysilane and 3-aminopropyltriethoxysilane. The metabolism was studied with mouse liver microsomes in vitro. The standardised mouse bioassay (ASTM E981–84) was used to evaluate the sensory irritation potency of airborne silane compounds by measuring a decrease in breathing rate due to stimulation of the trigeminal nerve endings in the nasal mucosa. s165 The silane concentrations in the air samples were below the detection limits. The mean dermal exposure to 3-glycidoxypropyltrimethoxysilane, analysed from the patch samples, was 2,800 mg/h in the forming room and 800 mg/h in the winder room of the fibreglass factory. The corresponding figures for 3- methacryloxypropyltrimethoxysilane were 3 and 9 mg/h. The RD50 values were 104 ppm for 3-aminopropyltriethoxysilane and 25 ppm for vinyltrichlorosilane having clear sensory irritation effects. Adjusting the 0.03 x RD50 approach would set acceptable occupational exposure levels for the vinyltrichlorosilane at 0.7 ppm (5 mg/m3 ), and for the gamma-aminopropyltriethoxysilane at 3.1 ppm (28 mg/m3 ). The MS studies suggested that 3-methacryloxypropyltrimethoxysilane and 3-glycidoxypropyltrimethoxysilane are metabolised in the mouse liver microsomes to several metabolites. Furthermore a 3-glycidoxypropyltrimethoxysilane glutathione conjugate was identified with HPLC-MS, suggesting that these silanes undergo intensive metabolism in mammals. The exact pathways of the metabolism, as well as their significance for toxicity, remain to be further elucidated. These results indicate that huge research work has to be undertaken for basic data to assess toxicological risks of organic silicon compounds. 616 ASSESSMENT OF GENOTOXIC EXPOSURE IN TURKISH COKE OVEN WORKERS BY CYTOGENETIC ENDPOINTS S. Burgaz 1 , C. Demiroglu 1 , M. Yilmazer-Musa 1 , A.O. Ada 2 , S. Suzen 2 , S. Efe 3 , Y. Alemdar 3 , M. Iscan 2 . 1 Department of Toxicology, Faculty of Pharmacy, Gazi University, Ankara, Turkey, 2 Department of Toxicology, Faculty of Pharmacy, University of Ankara, Ankara, Turkey, 3 Eregli Iron and Steel Works Co. Zonguldak, Turkey Polycyclic aromatic hydrocarbons (PAHs),which are carcinogenic and mutagenic to humans, are primary compounds in the coking process. Our aim was to assess whether the current exposure to PAHs of coke oven workers in a Turkish steel industry plant induced genotoxic effects. Urinary 1-hydroxypyrene (1–0HP) levels were used to monitor the internal dose, while the genotoxic exposure was assessed by monitoring micronuclei (MN) and chromosomal aberrations (CAs) in lymphocytes. Fifty coke oven workers and 50 controls were recruited in the same iron and steel works. Urinary concentrations of 1–0HP ranged from 0.05 to 14.99 (mean± SD; 1.68±2.39) µmol/mol creatinine in coke oven workers, and 0.01 to 2.68 (mean± SD; 0.34±0.41) µmol/mol creatinine in controls (p<0.01). The mean (± SD) cytochalasin-blocked MN frequencies (‰) were 13.80 (± 6.62) among the coke oven workers and 6.49 (± 3.18) among the controls, a statistically significant difference (p<0.01).The observed CAs were mainly chromatid breaks, chromatid gaps, and acentric fragments. The refrequency of aberrant cells (%) was 0.71(± 1.15) and 2.96 (± 1.99) excluding and including gaps,respectively, in exposed group, whereas in the control group, the frequency of aberrant cells (%) was 0.13(± 0.34) and 1.36 (± 1.31) excluding and including gaps, respectively. (p<0.01, excluding and including gaps). Age and smoking were not significant predictors for both cytogenetic parameters studied. It can be concluded that the cokery workers are exposed to PAHs during their occupational activities and that increased genetic damage was evident in coke oven workers, at population level, due to occupational exposure to PAHs. 617 RESPIRABLE DUST FRACTION AND CRYSTALLINE SILICA IN A CAST IRON FOUNDRY G. Miscetti, P. Garofani, R. Ceppitelli, A. Mencarelli, A. Ballerani, R. Angeloni. 1 Servizio Prevenzione e Sicurezza Ambienti di Lavoro, Area di Assisi - Azienda USL N.2, Perugia In this study we present the preliminary results of a research carried out to evaluate the respirable dust fraction and crystalline silica exposure of the workers in a cast iron foundry. It is common knowledge that one material widely used in foundry work, especially in core-making activity, is silica sand in addition with other substances (i.e. resins, organic binders, and so on) in order to fill the cores up, copying the hollow parts of the cast iron castings. s166 Poster Session P31. Occupational toxicology As the high temperatures of the production process can transform the amorphous silica in its different crystalline phases (from α-quartz to cristobalite), the study has carefully examined the composition of the raw materials by safety data sheet and the temperatures of the different production stages, this in order to address the investigation activitiy on the potentially highest-risk operations. Crystalline silica, as quartz or cristobalite, is classified as carcinogenic to humans since 1997 by IARC (group 1) with a TLV-TWA of 0.05 mg/m3 (ACGIH 2002) for respirable fraction. Data were collected following ACGIH curve (sampler: DorrOliver cyclone; flow rate: 1,7 l/m) and determination of SiO2 has been carried out by the means of XRD technique. Personal samplings were protracted for an average of 4–8 hours, in order to be representative of the whole working-day. In this preliminary investigation stage it has been observed that the mean personal exposure levels of respirable dust in the core-making area are equal to 1.5 mg/m3 (ACGIH2002), even as 95% upper confidential limits (2.2 mg/m3); anyway, in the 25% of samplings (relevant to workers of core-finishing and shell-moulding areas) the presence of crystalline silica was revealed as α-quartz, even if in concentrations lower than TLV-TWA (average = 0.039 mg/m3). 618 GENOTOXIC EFFECTS OF ANAESTHETICS R. Rozgaj, V. Kašuba. Mutagenesis Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia Chronic occupational exposure to low concentrations of anaesthetic waste gases may entail adverse health effects. Genotoxicity tests may give information about the risk before it turns into a serious health hazard. It has been shown that anaesthetics cause DNA single-strand breaks in persons administering anaesthesia. Although DNA singlestrand breaks may be reversible, a part of them will not be repaired, which leads to irreversible damages. Some damages may be seen using cytogenetic methods. The purpose of this study was to evaluate the effects of exposure to anaesthetics (nitrous oxide and halothane) in a group of 28 operating room medical workers. To do that we used the single cell alkaline gel electrophoresis assay (comet assay) and cytochalasine b blocked micronucleus test (MN). The results were compared with those of 28 control subjects who were matched for sex, age and smoking habit. The statistical significance of the results was determined using the one-way ANOVA test. The results show a significant increase in the tail length (20.82) and tail moment (0.68) as well as in the micronuclei frequency (26.93‰) in exposed subjects with respect to controls (TL=16.16; TM=0.38; MN=11.89‰). The exposure duration was significantly associated with the frequency of micronuclei, while age was significant at the P<0.01 level in the tail length. Our results support the use of genotoxicity tests in monitoring occupational exposure to anaesthetics. 619 FUNCTIONAL IMPAIRMENT OF THE OLFACTORY SYSTEM IN HUMANS AFTER EXPERIMENTAL EXPOSURE TO 2-ETHYLHEXANOL C. van Thriel, M. Schäper, E. Kiesswetter, M. Blaszkewicz, M. Kunze, A. Seeber. Leibniz Research Centre for Working Environment and Human Factors, University of Dortmund, Dortmund, Germany Unimpaired olfactory function is crucial for the identification of hazardous chemicals. Sustained solvent exposure impairs the olfactory function and a generalized impairment reducing the trigeminal sensitivity has been hypothesized. As a consequence early signs of sensory irritations (e.g., burning sensations) might be suppressed. Especially exposure peaks might have lasting effects on the chemosensory systems. In an exposure lab (≈29 cbm) two experiments with healthy male volunteers have been carried out. According to a cross-over design in experiment I (peaks) 24 subjects were exposed to 2ethylhexanol in three conditions with average concentrations of 1.5 ppm (constant), 10 ppm (hourly changing from 20 to 1.5 ppm), and 20 ppm (hourly changing from 40 to 1.5 ppm). In experiment II (constant) another group of 22 subjects was exposed to temporally constant concentrations of 1.5 ppm, 10 ppm, and 20 ppm. In both experiments 4 subjects were exposed simultaneously for 4 h and a period of two exposure-free days between two successive trials was strictly adhered to. Before and after the three conditions of both experiments odor thresholds of 2-ethylhexanol were measured by means of an olfactometer. In experiment I the odor thresholds (OTs) increased in a dose-dependent manner. The concentration for reliable detection of 2-ethylhexanol increased across the three conditions from 0.06 to 0.17 ppm, 0.08 to 0.31 ppm, and 0.09 to 0.52 ppm, respectively. In experiment II the OTs were also affected by the exposures but no dose-dependency could be confirmed. The observed increases were 0.05 to 0.11 ppm, 0.06 to 0.22 ppm, and 0.06 to 0.21 ppm. Trigeminal sensitivity was not tested functionally. However, during high conditions (20 ppm) less sensation like burning and stinging were reported by those subjects showing the strongest threshold shifts. In conclusion, these results suggested that exposure peaks might amplify olfactory impairment and that interactions with the trigeminal sensitivity, crucial for regulatory toxicology, are possible. 620 OCCUPATIONAL EXPOSURE TO SEVOFLURANE AND MALE HEPATIC FUNCTION G.F. Desogus. Study Reports of Toxicology, Azienda USL 7 di Carbonia, Italy The occupational exposure to anaesthetic causes changes in the biochemical markers and hepatic injury in exposed workers (surgeons, anesthesists, trained nurses). This work studies 1) the potential effect of sevoflurane exposure on the state of the hepatic markers (aspartate-aminotransferase and alanina-aminotransferase activity) 2) whether voluptary habits, including alcohol and smoke, can influence the haematic levels of sevoflurane. Sixty-eight sevoflurane exposed workers (group A: age 43 ± 8 years; years-old working seniority 11 ± 7 years) and twenty-one no exposed workers (group B: age 46 ± 3 years; years-old working seniority 16 ± 7 years) are examined in order to consider what is their hepatic function. The analysis of the hepatic markers don’t point out important differences between exposed and no exposed workers. The results of this study suggest that the sevoflurane doesn’t induce hepatoxicity in exposed workers of operating room. A reduced biodegradation and a quick elimination determine minimum effects at hepatocellular level in professionally exposed to sevoflurane workers. 621 THE RISK OF ALCOHOL ABUSE AND DEPENDENCE BESIDE OCCUPATIONAL STRESS IN A ISOLATED METALLURGY FACTORY D. Ionescu 1 , E.A. Pauncu 2 , C. Cristescu 3 . 1 Department of Toxicology, University of Medicine and Pharmacie, “Victor Babes” of Timisoara, Timisoara, Roumanie, 2 Medicine of Work Department, University of Medicine and Pharmacie “Victor Babes” Timisoara, Timisoara, Roumanie, 3 Department of Pharmacology, University of Medicine and Pharmacie, Victor Babes" of Timisoara, Timisoara, Roumanie The main purpose of the present study was to investigate the relationship between occupational stress and risk for alcohol disorders. The research had the following stages: 1. At baseline, all of the 1714 workers in the company (66,51% male and 33,49% female) completed standardized interviews that measured socio-demographic and occupational variables and assessed whether had met diagnostic criteria for currently active alcohol abuse-dependence syndrome with a result of 439 (25,61%) positives persons. 2. It has been applied the Cage and Audit Test finding 115 (6,70%) alcohol-dependents persons. 3. Univariate analysis suggested that male gender, age (> 35– 40 years), lower socio-economics status, cigarette smoking, family history of alcohol addiction, jobs with high physical demands and low control, occupational injury, all was found to be significantly associated with the risk alcohol abuse. 4. Multivariate analysis showed that only family and personally addictive history and jobs with high physical and mental stressors demands was statistically significantly associated with increased risk of alcohol-dependence. Poster Session P32. Dioxins and halogenated hydrocarbons A methodical survey 4 years has been achieved by interdisciplinary cooperation: psychiatry service, Medicine of work department, family physician with positives results diminution to 58 (3,38%) of alcohol-dependent persons. In conclusion, individualized education programmes and interdisciplinary coordination can perfect the management of risks alcohol abuse and occupational stress. 622 RISKS OF THE OCCUPATIONAL AND NON-OCCUPATIONAL EXPOSURE TO TALC DUST T. Rossi 1 , A. Errigo 1 , A. Baggio 2 . 1 Department of Pharmaceutical Sciences, 2 Dermatological Clinic, University of Modena and Reggio Emilia, Modena Italy Talc is a phyllosilicate formed during the breakdown and weathering of tremolite, anthophillite and serpentine rocks. It is used both for personal care and for industrial purposes, where it is used as a glident or as a lubricant. A lot of workers inhale talc dust every day with a real risk for their health. The type and quantity of associated minerals present also determine the talc’s individual properties or toxicity. The aim of our research was to collect information on the pathologies caused by occupational and non occupational exposure to talc inhalation. Clinical data outline that toxicity is independent both of the length of exposure and of sex, while the presence of contaminants as tremolite and anthophillite, which are members of the asbestos group of minerals, may represent a serious problem. A number of different lesions have been described in the lungs of persons exposed to talc (diffuse interstitial fibrosis, nodules, brochiolitis, emphysema, hypercalcemia and sometimes cancer) and this has been correlated with the presence of contaminants. People at higher risk are miners and workers employed in ceramics manufacturing or in foundry casting where talc is distributed with compressed air as a detaching agent in the casting molds [1]. A new form of talc pulmonary disease occurs in cases of intravenous drug addiction, since talc is used to hold the components of the medication together in tablet form [2]. Pathologies related to the use of talc for body care (cosmetic talc) are less frequent and are generally due to an overzealous use by adults or accidental inhalation by children [3–5]. The association between exposure to asbestiform talc and lung cancer risk is primarily based on the findings of increased cancer in workers exposed to asbestiform talc in the USA prior to the introduction of exposure control measures in 1945 [6]. Despite environmental monitoring with severe controls and laws to safeguard the health of workers, some cases of pneumoconiosis still occur, but it is hoped that the increasing knowledge of the causative agents of respiratory disease may reduce them to the minimum. P32 Dioxins and halogenated hydrocarbons 623 DIOXINS AND ENDOMETRIOSIS: PRELIMINARY RESULTS OF AN ON-GOING INVESTIGATION ON ITALIAN AND BELGIAN WOMEN Elena De Felip 1 , Anna Maria Ingelido 1 , Massimo Cardelli 1 , Maria Grazia Porpora 2 , Jacques Donnez 3 , Alessandro di Domenico 1 . 1 Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Rome, Italy, 2 Institute of Gynecological Sciences, Perinatology and Child Health, University “La Sapienza”, Rome, Italy3 , Department of Gynecology, St. Luc Hospital, Catholic University of Louvain, Brussels, Belgium Endometriosis is a gynecologic diseases affecting about 10% women in the reproductive age, frequently associated with pelvic pain and infertility. The etiology of this disease is still to be elucidated. The hypothesis of a correlation between the exposure to dioxins and dioxin-like PCBs and endometriosis is supported by experimental studies showing that exposure to these chemicals is associated with a dose-dependent increase in the incidence and severity of endometriosis in rhesus monkeys. While the results of human case-control s167 studies so far carried out are conflicting, some epidemiological observations further support the hypothesis of such a correlation. Indeed, endometriosis severity and incidence in Belgium are among the highest of the world: in this country, the general population exposure to dioxin-like compounds has been, on average, higher than in most industralised countries. The present results refer to a study preliminary to an ongoing case-control investigation on a larger population of women enrolled in the two countries, aimed to assess whether a correlation may be found between dioxin-like compounds and endometriosis. In our study, a remarkable difference between Italian and Belgian women was detected in terms of dioxin-like compound body burden (as a mean, a factor of 2–3); this result consistently correlates with the estimated dioxin daily intakes reported for the two countries (i.e., approximately 50 and 170 pgI-TE/individual for Italy and Belgium, respectively). No significant differences were found in PCDD+PCDF and dioxin-like PCB body burdens between healthy women and patients affected by endometriosis in each country, although the limited number of cases does not allow to draw a definitive conclusion. This work has been partially supported by the Italian Public Health System Project n. 0/RC. 624 THE ARYLHYDROCARBON RECEPTOR REPRESSOR (AHRR) MEDIATES ONCOSTATIN M (OSM) INDUCED DOWNREGULATION OF CYP1-FAMILY Amitabh Kohli, Sandra Wolff, Ellen Fritsche, Josef Abel. Institut für umweltmedizinische Forschung gGmbH an der Heinrich-Heine-Universität Düsseldorf, Auf’m Hennekamp 50, 40225 Düsseldorf, Germany The AhRR and its related proteins, the Arylhydrocarbon Receptor (AhR) and the Arylhydrocarbon Receptor nuclear translocator (ARNT) are important in regulation of drug metabolizing enzymes. The AhR and ARNT were shown to initiate upregulation of several xenobiotic metabolizing enzymes via binding to XRE in response to various exogenous compounds like TCDD. The AhRR gene is thought to initiate the downregulation of the respective genes. It is well known that during acute phase response several drug metabolizing enzymes like CYP1 enzymes are downregulated, however, the mechanisms involved are still unclear. Here we report that the IL-6-type cytokine OSM is able to upregulate the expression of the AhRR gene in HepG2 cells. Treatment of cells with OSM led to time and dose dependent increase of AhRR mRNA. The upregulation was transient. Treatment of HepG2 cells with 10 ng OSM/ml for 6 hours led to 7–8 fold increase of AhRR expression, thereafter mRNA levels declined and returned to control level 24 hours after treatment. The increase of the AhRR mRNA was closely followed by a decrease of CYP1A2 expression suggesting that increased sequestering of ARNT by the AhRR mediates downregulation of constitutive expression of CYP1A2. To evaluate the mechanism possibly involved in upregulation of AhRR gene we cloned a 1.9 kb fragment of the 5’-region of the human AhRR gene. The sequence data revealed no TATA and CAAT boxes within the cloned fragment but four putative bindings sites were found for SP-1. In addition, two XRE’s and several putative Eboxes could be identified. The functional importance of the indicated regulatory sequences was analyzed by reporter gene assays. 625 COXII INHIBITORS DOWN REGULATE TCDD MEDIATED CYP1A1 GENE EXPRESSION. Y.Y. Sheen 1 , K.E. Joung 1 , S.R. Bang 1 , K.N. Min 1 , J.Y. Kim 1 , M.J. Cho 1 , K.H. Chung 2 , C.K. Moon 3 . 1 Pharmacy, Ewha Womans University, Seoul, Korea, 120–750. 2 Sungkyunkwan University, Kyungkido, 440–746, Korea,3 Seoul National University, seoul, korea 151–742 We have examined the effects of COXII inhibitors and HC-toxin on the promoter activity of cyp1a1 using cyp1a1-Luciferase construct. The cyp1a1-luciferase reporter gene, was transfected into either Hepa I or MCF-7 or ZR-75–1 cells. We have measured luciferase activity and the EROD enzymatic activity of cyp1a1 in either Hepa I or MCF-7 or ZR-75–1 cells. These cell lines are rich in AhR, s168 Poster Session P33. Ecotoxicology and other nuclear receptors such as ER and PR so they are good system to study the AhR activity and also cyp1a1 gene expression regulation in terms of signal cross talks between ligand activated transcription factors. For the in vivo study, we have used C57BL/6 mice that have high level of AhR. We have examined the effect of inhibitors of COXII and histone deacetylase on the cyp1a1 activity. COX nonspecific inhibitor, aspirin pretreatment inhibited the AhR mediated EROD activity in vivo, and also inhibited the AhR mediated EROD and promoter activities in Hepa I cells in vitro. Also, COXII specific inhibitors, such as celecoxib and nimesulide inhibited the AhR mediated EROD activity and luciferase acitivity in HepaI and MCF-7 cells. Nimesulide and SB100 inhibited AhR mediated cyp1a1 mRNA level in HepaI and MCF-7 cells. Histone deacetylase inhibitor, HC-toxin stimulated AhR mediated EROD activity, cyp1a1 mRNA level and luciferase acitivity in HepaI and MCF-7 cells. HC-toxin seems to overcome the inhibitory activity of nimesulide on AhR activation based on the results of effects of concomitant treatment of nimesulide and HC-toxin on AhR mediated luciferase activity in HepaI and MCF-7 cells. The results of our study indicates that COXII might need to activate AhR by TCDD and histone deacetylation might down regulate TCDD activation of AhR in terms of CYP1A1 gene expression regulation.[Supported by the grant from Korean Institute of Environmental Science and Technology] 626 HEXACHLOROBENZENE INCREASES ORNITHINE DECARBOXYLASE ACTIVITY, FREE POLYAMINES CONTENT AND C-MYC, C-FOS AND C-JUN PROTEIN LEVELS IN RAT LIVER A.S. Randi, S. Hernández, L. Alvarez, M. Sánchez, M. Schwarcz, D.L. Kleiman de Pisarev. Department of Human Biochemistry, School of Medicine, University of Buenos Aires, Buenos Aires, Argentine Hexachlorobenzene (HCB) is one of the most widespread environmental pollutants known; it is released as a subproduct of the synthesis of other polyhalogenated aromatic compounds. Chronic exposure of laboratory animals to HCB elicits porphyria, immunosuppression, reduced levels of serum T4 , reproductive dysfunction and liver and thyroid carcinogenesis. In this study we investigated the effect of in vivo HCB treatment on protooncogenes levels (cMyc, c-Fos and c-Jun), ornithine decarboxylase (ODC) and PTK activities and free polyamine content. Our results showed that HCB (1000 mg/kg b.wt.) markedly increased ODC activity and spermine and putrescine content at 24 hours post-treatment, while at 48 hr the three polyamines (putrescine, spermidine and spermine) were elevated. These changes occured in a dose-dependent manner. Acute exposition assays (0, 6, 12, 18 and 24 hr post-treatment) showed an increase in the immunodetectable c-Myc, c-Fos and c-Jun levels at 6 hours followed by a peak in ODC activity at 18 hours (8-fold), accompanied with higher levels of putrescine and spermine. PTK activity was maximally increased at 12 hr.The elevated ODC activity at 18 hours, may be the consequence of c-Myc induction as reported by other authors. c-Fos and c-Jun protein levels remained elevated up to 10 days, probably related to the increase in polyamines. As we have previously demonstrated that HCB stimulated the EGFRProtein Tyrosine Kinase activity in rat liver, our results suggest that the growth factor receptor signal transduction pathway could be involved in the induction of the early mitogenic responses. In conclusion, we demonstrated, for the first time, that HCB stimulated the early increase of c-Myc, c-Jun and c-Fos protein levels, followed by an increase in ODC activity and polyamine content. The induction of protooncogene levels may be relevant to reports that HCB exhibits tumor promoter activity in rat liver. Although our results are obviously far from establishing a direct connection between the induction of protooncogene levels and such activity, they provide a specific direction for further analysis in this area. 627 QUANTUM-CHEMICAL AND EXPERIMENTAL MODELS OF TOXIC ACTION OF DIBENZO-P-DIOXINS AND THEIR ANALOGUES I.A. Gusarova, N.B. Kuznetsova, P.E. Kuznetsov. Department of Biochemistry and Biophysics, Biological Faculty, Saratov State University, Saratov, Russia In the previous researches the quantum-chemical modelling of behaviour of dibenzo-p-dioxines (DD) and their analogues in membrane areas and in a complex with cytochromes P-450 was carried out by Kuznetsova N.B. It was shown, that DD can be catalysts of hydrogen peroxide production not only in vivo but also in vitro. In the present work more detailed quantum-chemical and molecular-dynamic modelling of processes of generation of hydrogen peroxide in heterogeneous aqueous environments was carried out. The experimental data confirming theoretical results were discussed. In particular, the fact of hydrogen peroxide generation in these environments was confirmed. It is shown, that DD toxicity characteristics and calculated parameters describing production of hydrogen peroxide are well-correlated. The obtained experimental and theoretical results allow to suppose a new way of oxygen active forms production and DD toxic action realization. The connection of suggested molecular mechanism with existing theories of DD toxic action was discussed. P33 Ecotoxicology 628 USE OF DEFEROXAMINE ON MUCOCILIARY EPITHELIUM EXPOSED TO OXIDATIVE STRESS A. Sánchez-Chardi 1 , R.C. De Oliveira 2 , D.J.A. Lobo 2 , P.H.N. Saldiva 2 , G. Lorenzi-Filho 2 , M. Macchione 2 . 1 Departament de Biologia Animal (Vertebrats), Facultat de Biologia, Universitat de Barcelona, Barcelona, España, 2 Lboratório de Poluição Atmosférica Experimental, Departamento de Patologia, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil Air pollutants such as particulate matter (PM) are responsible for mortality and morbidity in humans. Residual oil fly ash (ROFA) from fossil combustion contains a large number of heavy metals and other elements, and is an important contributor of PM to urban environments. Moreover, iron and other metals are associated with oxidative stress in biological systems. This study assesses the effects of ROFA with high iron content on the mucociliary epithelium of the frog Rana catesbeiana (Shaw, 1802) and the protective action of Deferoxamine (DFO). DFO is a chelator used in the treatment of iron overload. Eight groups of frog palates (n = 81) were immersed in Ringer’s solution with: two concentrations of ROFA (1mg/L and 100 mg/L), two of DFO (1mM and 10mM), three concentrations of ROFA mixed with DFO (1mg/L + 1mM, 100 mg/L + 1mM and 1mg/L + 10mM) and Ringer’s solution alone (negative control). We measured the responses of mucociliary transportability (MT) and ciliary beat frequency (CBF) to treatment at 0, 30, 60 and 120 minutes. ROFA produced a significant time-dependent decrease of MT (p=0.019), whereas CBF was not affected. On the other hand, DFO increased MT (p<0.001) and CBF (p=0.031), alone or mixed with ROFA. We conclude that iron and other metals present in ROFA may affect mucociliary transport by decreasing MT. The protective action of DFO reduced the availability of metal in the epithelium, with a concomitant reduction in oxidative stress. This indicates that metals cause injury associated with oxidative stress, in a non mammalian model of mucociliary epithelium. Poster Session P33. Ecotoxicology 629 A HIGH-THROUGHPUT CELL-BASED REPORTER GENE SYSTEM FOR MEASURING CYP1A1 INDUCTION BY XENOBIOTICS IN DRINKING WATER. E. Chiesara 1 , S. Frigerio 1 , R. Fumagalli 1 , L. Marabini 1 , S. Radice 1 , M. Ferraris 1 . 1 Department of Pharmacology, Chemotherapy and Medical Toxicology “E. Trabucchi”, University of Milan, Milan, Italy We characterised an in vitro system for assessing the presence of polycyclic aromatic hydrocarbons (PAHs) or related chemicals in the drinking water distributed by the public water supply system. This in vitro bioassay, which is based on cytochrome P450 (CYP) 1A1 induction, was highly reproducible over time. On the basis of the signal-transduction mechanism of P4501A1 induction, a high-throughput reporter gene system was developed by transiently transfecting HepG2 cells with a plasmid bearing the luciferase gene under the control of specific sequences of the P4501A1 promoter. The drinking waters from the municipal water supply system of different Italian towns were sampled before disinfection, or collected directly from the tap at different points of the municipal water network. The samples were concentrated by C18 silica cartridges, and different amounts of water concentrates (equivalent to 1 L, or 0.065 L of water for each ml of culture medium) were tested for cytotoxic effects and cytochrome P4501A1 expression. The results indicate that non-cytotoxic concentrations of water seem to have different effects on P4501A1 expression and confirm the presence of undefined compounds in drinking water. The results also show that the method is sensitive in identifying CYP1A1 inducers in water, thus indicating its potential use as a complement to chemical analysis and aid in interpreting the biological data obtained from environmental studies. 630 GENOTOXICITY EVALUATION OF CHLORINATED DRINKING WATERS OBTAINED FROM SURFACE AND WATER TABLE IN METABOLICALLY COMPETENT HUMAN CELLS (HEPG2) S. Frigerio 1 , S. Radice 1 , M. Ferraris 1 , R. Fumagalli 1 , E. Chiesara 1 , L. Marabini 1 . 1 Department of Pharmacology, Chemoterapy and Medical Toxicology,University of Milan, Milan, Italy We examined DNA damage and the mutagenic effects of chlorinated drinking water concentrates in metabolically competent human HepG2 hepatoma cells using the in vitro micronucleus assay and single cell gel electrophoresis (SCGE, comet Assay). The water samples were obtained from the aqueducts of two towns with different types of water supplies: town #1 uses the surfaces of a lake, whereas town #2 is supplied from a deep water table. In the case of town #1, we also analysed a water sample taken before conditioning. Both towns use ClO2 for conditioning purposes. The samples were concentrated by adsorption on silica C18 cartridges and subsequently eluted. In this study, the samples denominated RW (raw waters, only for surface water) were obtained by concentrating the water before conditioning, the BIO samples immediately after conditioning, the A samples were taken at the point of immission into the aqueduct, and the R1 and R2 samples from two different points in the distribution that were very distant from each other. A first toxicity evaluation was made using NRU (neutral red uptake) and LDH (lactate dehydrogenase) tests, which revealed toxicity for concentrates equal to 0.5 l/ml and 0.25 l/ml of the R1 and R2 samples from town #1. No toxicity was found in the other samples at concentrations of up to 0.5 l/ml for town #1 and 1 l/ml for town #2. The short-term genotoxicity tests of the surface water samples, which were performed after 24 hours of treatment at maximal noncytotoxic doses did not reveal any variations at any point in the water aqueduct. The same was also observed for the concentrates of water from the water table. Overall, our results show that the concentrates of conditioned drinking waters from two different towns are not genotoxic to human hepatoma cells HepG2 under our experimental conditions. The cytotoxicity data highlight the importance of the supply and conditioning of aqueducts and suggest the presence of substances s169 capable of inducing toxicity by means of mechanisms other than genotoxic damage. 631 NATIONAL ASSESSMENT OF LONG-TERM USE OF PARAQUAT FOR ITS FOOD AND ENVIRONMENTAL-SAFETY IN KOREA Y.W. Kwon 1 , S.W. Park 1 , S.H. Shin 1 , J.H. Kim 2 . 1 School of Plant Science, 2 School of Agricultural Biotechnology, Seoul National University, Suwon 441–744, Korea A non-selective herbicide paraquat has long been used for weed control worldwide. Statistics shows that 8 million ha in North America and 24 million ha in Asia-Pacific were treated with paraquat in 2001. Though paraquat is well referred to as being rapidly and strongly adsorbed to soil and as environmentally safe, environmentalists often allege that paraquat residue in soil is hazardous, and soil of agricultural land will eventually be saturated with paraquat in the near future, causing irremediable disasters. This paper reports the results obtained from an extensive nationwide assessment of longterm paraquat use regarding its food and environmental safety in Korea. To date no such a thorough study has been conducted in other countries. Paraquat has been used for weed control in orchards and non-croplands of Korea since 1970. Its total consumption by 1995 amounted to 11,624 metric tons as paraquat ions, which is equivalent to twice-sprayings a year at a recommended rate on 419,910 ha for 26 years. Under this circumstance, paraquat was subjected, in 1996, to a Special Review by the National Regulatory Committee of Pesticides, Korea for its food and environmental safety. The Special Review has been concerned mainly with: 1) the present level of paraquat residues in the orchard soils and 2) in the produce of orchards, 3) biological effect of the paraquat residues on following-crops, 4) biological effect of soil-bound paraquat on following-crops, 5) plant-uptake of the soil-bound paraquat at different residue-levels, 6) capacity of agricultural soils to adsorb and biologically-inactivate paraquat, 7) effect of fertilizer practices on the release of soil-bound paraquat, 8) leaching potential of the soil-bound paraquat by heavy rainfall or intensive irrigation, 9) difference between the Korean soil and the foreign soil in strong binding capacity/strength of binding, and 10) persistence of paraquat under field conditions. National surveys and public researches have been carried out to answer the questions in 1996 and 1997. Through the studies, paraquat residues in the Korean soil have been found at an environmentally safe level, and continued use of paraquat in future in ordinary way would warrant its food and environmental safety. Paraquat residues in 60 orchard soils were averaged 8 ppm in 1996, and 7 ppm in 1997studies. Except for an extreme case of 35 ppm, most orchard plough soils (two thirds of 120 orchards) contained bound-paraquat less than 15 ppm. Paraquat was not detected with standard analyses (detection limit of 0.05 mg/kg) for crop produces such as apple, peach, pear, grape, barley or soybean grown in paraquat residual soils or the soils fortified with paraquat up to 100 ppm. Korean soil is mostly of kaolinite having CEC average ca. 10meq./100g. The strong adsorption capacity (SAC) of Korean orchard soils were averaged 231 and 276 ppm in 1996 and 1997 surveys, respectively. Within a detection limit of 1 ppb in a bioassay, none of the strongly soil-bound paraquat leached out to the environment, and was not uptaken by plants unless the residue level was over 80% of the SAC value for a given soil, even in the kaolinite-based soil that binds cations most weakly among different clay minerals. The dissipation of paraquat in the field (DT50 ) was 200 to 340 days in Korea. From this experience we suggest that a better paraquat stewardship for food and environmental safety requires determination of its SAC of soil and soil-residue level where it is continuously used. 632 THE METALLOTHIONEINS UV-SPECTRA ANALYSIS AS A TOOL FOR THE ESTIMATION OF THE METALS TOXICITY FOR FRESHWATER ANIMALS O. Stolyar, H. Falfushynska. Chemical-Biological Department, Ternopil State Pedagogical University, Ternopil, Ukraine The determination of the metal content in the freshwater organisms, their tissues or, even, in their metallothioneins (MTs) does not give the evidence of metal contamination danger for the organism. s170 Poster Session P33. Ecotoxicology This paper reports the results of correlative analysis of the UVspectra, which we observed on a freshwater fish Cyprinus carpio L. and mollusc Anodonta cygnea MTs in vivo and in vitro. The animals were exposed to Cu, Zn, Mn, Pb ions or to their mixtures in the concentrations which were approximate to its content in fresh water and to 20-fold highest ones for up 14 days. The UV-spectra were expressed as (De-Dk)/Dk (De and Dk – absorption value of experimental and control samples at the same wavelength). The correlation analysis of UV-spectra was performed. The three tipes of spectra changes were observed: looking as a bell (a), as an ascending diagonal (b) and as a diminished diagonal (c). The a-form was observed in the most of experimental cases. It accompanied with the rose of MTs content and activation of antyoxidant factors in tissue. The b-form of spectra was accompanied with the decrease of MTs content and activation of prooxidant factors in the tissue. It was revealed under the effect of high doses of metals. The c-tipe was infrequent and was accompanied with the increase of metal content in the fraction. Under the Zn effect in all cases the spectra had a “blue shift” that didn’t correlated with a, b, c-tipes. Therefore this test may be available for the specific indication of the heavy metals pollution. atomic absorption spectrophotometry utilizing a flame technique (Perkin-Elmer 305B) and a graphite furnace (Shimadzu A) was used to measure lead, chrome, nickel and cadmium concentrations. However, the hydride vapor generator was used for arsenic and mercury. Results and discussion: The averages obtained for atmospheric air concentrations are given in the following table: Elements Ca Mg Mn Zn Fe Cr Lead (µg/m3 ) (µg/m3 ) (µg/m3 ) (µg/m3 ) (µg/m3 ) (µg/m3 ) (µg/m3 ) Megrine Gabes 3.180 31.970 RE-EVALUATION OF THE HAZARDOUS EFFECT OF THE SUPERPHOSPHATE FACTORY BY-PRODUCTS AT ASSIUT GOVERNORATE, EGYPT M. Abd El- Nasser 1 , Th.A. Ibrahim 1 , A.A. Shaaban 1 , Manal M. Sayed 2 . 1 Dept. Forensic Medicine and Toxicology, Faculty of Vet. Medicine, Assiut University, Egypt, 2 Animal research Institute, Assiut laboratory The level of pollution emitted by the superphosphete factory located at Manquabad, Assiut Governorate, Egypt was debatable for long time. Five water samples as well as five feed stuffs were collected from different localities surrounding the factory. Blood, hair and organ samples were also collected from goats living in the same localities and subjected to chemical analysis for fluorine, sulpher and cadmium concentrations. This study indicated that the pollution of water as well as feed stuffs has been reduced significantly over the past few years. Gazerit El-Akrad village still has the highest concentrations of pollutants emitted from the factory in comparison to the other studied areas. In spite of the reduction of pollutants studied in this work, it still over the permissible limits recommended by the international organizations which needs more reduction of industrial wastes besides annual monitoring of the areas surrounding the factory till reaches the international safety standards. 634 LEAD, NICKEL, CADMIUM, ARSENIC, MERCURY, COPPER, ZINC, MAGNESIUM, CALCIUM, MANGANESE, IRON AND CHROMIUM CONCENTRATIONS IN ATMOSPHERIC AIR OF TWO TUNISIAN SITES F. Rouda 1 , H. Ghorbel 1 , M. Bousnina 1 , E. Helmi 2 , S. Hedhiri 1 , D. BenSalah 1 , M. Nedhif 2 , M. Amamou 1 , A. Hédhili 1 . Laboratory of Toxicology, Unity of Toxicology Research and Environment 99/UR/070,Montfleury 1008 Tunis, Tunisia, 2 Direction of Hygiene and Environmental Protection, Tunis, Tunisia Introduction: Supervision of the air quality is one of the priorities of Tunisian politic. That’s why a national air quality monitoring network has been established. Ministry of the public health has choosen for this experience two sites exposed to the industrial pollution and automobile traffic and localized in the Tunisian north (Megrine) and south (Gabes). Objective: The aim of this study is to measure some trace elements (lead, nickel, cadmium, arsenic, mercury, copper, zinc, magnesium, calcium, manganese, iron and chrome concentrations in the atmospheric air of these two regions. Materials and methods: The dust particles were collected by a special machine "PPA60" composed of nitrate cellulose filter which can select all the particles whose diameter is lower than 10µm. (PM 10). Sampling periods are about 24 hours then the samples are dispatched directly to the laboratory. Zinc, copper, magnesium, calcium, iron and manganese were determined with 0.067 0.070 0.110 0.110 0.480 0.580 0.129 0.110 0.640 0.049 Lead concentrations were compared to the Tunisian norm (≤1 µg/m3 ) and showed that the values in Megrine and Gabes are acceptable. Concerning arsenic, cadmium and mercury, the results were lower than the detection limits of the method used. Conclusion: These results do not constitute a source of preoccupation but some efforts must be agreed in order to improve the quality of the air that breathe. 635 633 10.190 5.650 ZINC AND COPPER CONCENTRATIONS IN SEAWATER AND ALGAE OF THE TUNISIAN COAST M. El Ati-Hellal 1 , H. Ghorbel 2 , M. Bousnina 2 , K. Boujlel 1 , M. Dachraoui 1 , A. Labidi 3 , M. Ndif 3 , M. Amamou 2 , A. Hédhili 2 . 1 Faculty of Sciences, Department of Chemistry, Tunis, Tunisia, 2 Laboratory of Toxicology, Unity of Toxicology Research and Environment 99/UR/070,Montfleury 1008 Tunis, Tunisia, 3 Direction of Hygiene and Environmental Protection, Tunis, Tunisia Introduction: Algae have a big power of bioaccumulation for certain heavy metals and can serve as “indicators” of chemical pollution in a littoral zone with more security than seawater. Objective: To show the interest of using algae as indicators of seawater pollution follow-up and control. Materials and Methods: Samples of algae (99) were taken in three levels of depth, (surface, at the middle - depth and in 20 cm of the bottom) in the coasts of Bizerte, Ariana and Ben Arous (Tunisia north) Sousse and Mahdia, (Tunisia center), Sfax and Medenine (Tunisia south). At the same time, samples of seawater (99) were selected in the same sites to have information about bioconcentration of heavy metals in algae. The analysis was realized by atomic absorption spectrophotometry with flame. Results and Comments: Zinc and Copper were much more concentrated in algae than in seawater. These results may confirm the hypothesis that algae is able to concentrate trace elements dissolved in water and to reflect the maritime environment state of pollution better than seawater. Bizerte’s coast presented the most elevated concentrations of these metals (202,0 ± 112,5 ppm and 17,9 ± 14,3 ppm respectively for zinc and copper). A variability inter-sectors was observed in this zone due to the local sources of industrial and urban waste. Concentrations were weaker in the other regions; they varied between 50,5 and 142,6 ppm for zinc and between 2,72 and 7,35 ppm for copper. Sousse’s region was the least polluted while Sfax’s region presented intermediate average concentrations. Accumulation of heavy metals appeared to be the same for the seven species of our study’s algae; consequently, the specie’s nature may have no impact on this property. The physico-chemical characteristics of sea might not influence algae heavy metals bioaccumulation because concentrations do not vary in a significant way according to the depth. Conclusion: The follow-up of zinc and copper pollution could be made by the analysis of these elements in algae. In fact, algae present the advantage to be accessible during all the periods of the year, easily analysable, not expensive and especially more sensitive to the amounts of chemical pollutants dissolved in water. Poster Session P33. Ecotoxicology 636 LEAD CONCENTRATIONS IN SEAWATER AND ALGAE OF THE TUNISIAN COAST M. El Ati-Hellal 1 , H. Ghorbel 2 , M. Bousnina 2 , K. Boujlel 1 , M. Dachraoui 1 , A. Labidi 3 , M. Nedhif 3 , M. Amamou 2 , A. Hédhili 2 . 1 Faculty of Sciences, Department of Chemistry, Tunis, Tunisia,2 Laboratory of Toxicology, Unity of Toxicology Research and Environment 99/UR/070,Montfleury 1008 Tunis, Tunisia, 3 Direction of Hygiene and Environmental Protection, Tunis, Tunisia Introduction: Lead is a toxic metal trace, even in very weak concentrations. It is used in different industrial processes. Most of them throw back their waste in seawater without any preliminary treatment and by there contributes to a contaminated aquatic food chain. Objective: To estimate lead accumulation in various sites of the Tunisian coast, we analysed this metal in algae and seawater. Materials and Methods: Study concerned 99 samples of algae and 99 samples of seawater, taken in different depth of the littoral zones of Bizerte, Ariana, Ben Arous, Sousse, Mahdia, Sfax and Medenine. The analysis was realized by atomic absorption spectrophotometry (oven graphite). Results: The coasts of Bizerte, Mahdia and Sfax showed relatively elevated lead concentrations in seawater; values varied between 0.118 and 0.129 ppm. Ben Arous presented the weakest average concentration (0.054 ± 0.019 ppm) while the regions of Médenine, Sousse and Ariana showed intermediate concentrations. In algae, lead contents were much weaker and it is Bizerte’s coast (especially the canal), which presented the highest contents (0.009 ± 0.016 ppm). Concentrations of lead did not present any significant difference according to the level of depth. Bizerte’s coast seems to be the most exposed zone to pollution. Strong concentrations observed as well in seawater as in algae can be attributed to local sources of industrial waste and the intensification of sea traffic (hydrocarbons derived contain some tetraethyl lead). Souse’s region is the least polluted zone; it might be due to the development of tourism and a strict regulation of pollution in this district. Concentrations recorded in the seawater are lower than Tunisian standards relative to the maritime public domain (0.5 ppm). As regards algae, no limit threshold was established until now in Tunisia. Only some fragmented works were realized without establishing standards. Concentrations observed in the Tunisian coast are much weaker than those found in certain regions of America and Africa. Conclusion: Tunisian costs could be irreparably polluted by extensive urbanization and industrialization, if a strict rule of waste draining and a continuous control of pollution’s level are not set up. 637 LEAD AND CADMIUM CONTENT OF KORBAL RICE IN NORTHERN IRAN A. Bakhtiarian, M. Gholipour, M. Ghazi-Khansari. Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Every year the entrance of factory wastes such as Shiraz Petrochemical Complex, Marvdasht sugar cube factory, and Charmineh factory, and other industrial units into the Kor and Sivand rivers and also the entrance of the Marvdasht and Zarghan city sewer system wastes into the Kor river and the use of their water in the cultivation of the rice has caused a significant increase in the lead and cadmium content of the grains of rice. To study the effect of the Kor river’s pollution on the lead and cadmium content of the grains of rice, 57 samples of 6 different types of were prepared in 19 different stations in the Korbal region and also 18 samples of 6 different types of rice, cultivated with unpolluted water, were prepared in the National Institute of Rice Research (Gilan). A comparison of the pollution level of the Korbal and Gilan rice samples shows a significant difference and indicates the significant effect of the pollution of the river on the lead and cadmium content of the Korbal rice samples. The results of the study show that the lead and cadmium content of the hybrid, prolific, and late rice sample types were greater than that of unprolific and early types, such that the amount of these two elements was highest in the Hassani type (the lead content was 0.9625 ppm and the cadmium content was 0.0793 ppm), whereas the Gasroddashti type which s171 blooms earlier and is long seeded has the lowest amount of these two elements. 638 AMMONIA AND CHLORINE IN HEAVILY POPULATED AREAS Alka Coporda, Zdravko Lovric, Franjo Plavsic. Croatian National Institute of Toxicology, Zagreb, Croatia Harmonization of the Croatian legislative with European directives on hazardous substances, and adoption of various international agreements and conventions such as APPEL program and Convention on Prevention of Transborder Transfer of Accident Sequences, have opened numerous problems related to the present situation with hazardous chemicals. One of such problems are risk facilities used for storage or use of gaseous toxins in densely populated areas. This presentation is limited to only two chemicals, chlorine and ammonia, as these are frequently found at high-risk sites. It is estimated that in Croatia, these two chemicals are found at more than 250 locations in amounts potentially risky for the population living close to the sites of their storage or usage. The problem is not only where the chemicals are found, but even more it is associated with the accident preventing measures, which as a rule are still quite inappropriate. In the Republic of Croatia, water disinfection is mostly done with chlorine, while ammonia is unavoidable in all large cooling systems (e.g., industry refrigeration plants and skating rinks). The facilities with chlorine and ammonia are generally located in densely populated areas of all Croatian cities, while the measures of accident prevention as a rule are quite poor. 639 EFFECTS OF AIR POLLUTION FROM A COAL-FIRED POWER PLANT ON INFLAMMATION IN RESPIRATORY EPITHELIUM OF WILD SMALL MAMMALS A. Sánchez Chardi 1 , M. Lemos 2 , M. Dolnhikoff 2 , P.H.N. Saldiva 2 , J. Nadal 1 . 1 Departamento de Biologia Animal (Vertebrats), Facultat de Biologia, Universitat de Barcelona, Barcelona, España, 2 Laboratório de Poluição Atmosférica Experimental (LIM05), Departamento de Patologia, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brasil We assess the effect of the air pollution from the coal-fired power plant of Cercs (Barcelona, NE Spain) on inflammation of lower respiratory airways of wild small mammals. We also evaluate the greater white-toothed shrew Crocidura russula (Mammalia, Insectivora) as a bioindicator of air pollutants under mediterranean climate. We collected 46 adult shrews in autumn 1996 and in spring 1997 in two areas: a polluted area affected by gaseous and particulate matter from the power plant (n=23), and a clean area (n=23) 20 km from the polluted area. Because the activity of the power plant is not continuous and depends on the demand for energy, shrews were collected in two separate seasons to reveal the effects of pollution at different concentrations. All specimens were sexed and aged. The concentrations of air pollutants were also monitored throughout the study. Samples of lung tissue were processed for light microscopy following conventional methods and stained by hematoxylin-eosine. Inflammatory cells were observed on the lung around the respiratory airways. Inflammation was measured on a semiquantitative scale. Each airway was scored from 0 (without inflammation) to 3 (100% of perimeter of airways occupied by inflammatory cells). Data were compared by area, season, sex and age by ANOVA tests. A significant increase (p<0.001) of inflammatory processes was observed in shrews from the polluted area ((1.78 ± 0.86) in relation to control (0.39 ± 0.20). No differences were observed between seasons, sex or age. The pollution in the area of Cercs produced a significant inflammatory response in the respiratory epithelium of wild insectivorous mammals. Males and females were equally affected, although their general health did not appear to be affected. The greater white-toothed shrew Crocidura russula was sensitive to alterations in the environment and can be considered a good bioindicator of air pollution. s172 640 Poster Session P33. Ecotoxicology TRANSPORT TOXICITY AS A CRITERION AT HYGIENIC REGULATION OF DANGEROUS GOODS L.M. Shafran 1 , G.F. Burlak 2 , D.P. Timoshina 2 , N.G. Goncharenko 2 . 1 Ukrainian Scientific and Research Institute of Transport Medicine, Odessa, 2 Ministry of Public Health, Kiev, Ukraine The process of dangerous goods (DG) transportation is connected to the increased risk of influence of toxicant chemicals and materials on safety and health of workers (pilots, seamen, dockers, railwaymen, drivers). Thus not only degree, but also time of contact with toxil products essentially differs from those for the industrial workers. For exception of probable pathological and fatal consequences on the basis of experimental (on laboratory animals) and physiological researches the concept of a transport toxicity is offered. As an example the results of the natural industrial researches on ships in long trips are given and during loading process in ports with ammonia, acrylonitrile, methanol, fertilizing (potassium chloride, phosphorites). At different stages of DG transportation analysis of air in the working zone are carried out on the contents of harmful vapors, gases and aerosols. At the seamen and dockers were determined parameters of a state of a respiratory, cardiovascular, nervous system, biochemical analysis’s of urine and peripheral blood. The researches have shown that the long continuous action on the seaman organism of migrated components of transported goods produces series of function changes, which manifestation degree correlates with a level of influence, exposure time and kind of DG. Series of physiological changes took place at concentrations of toxicants in air of below allowable norms (TLV). The dependence of observable changes on contact with transported DG is confirmed in experiences on laboratory animals (white rats), taking place on ship board during the trip. In dependence on a category of a ship premise and, accordingly, chemical exposure level a degree of biochemical and physiological changes exhibiting (the activity of lysosomal enzymes and energy metabolism, lipids peroxidation and antioxidant systems, peripheral blood) authentically differed from the control. Comparison of the researches results, which have been carried out on ship board and in laboratory on a coast, essentially differed among themselves not only on an expressiveness of changes in investigated parameters, but also certain quality features. Last, probably, is caused by complex action on an animal organism the ship factors during the trip. It produces an intoxication on a background of a general stress, which changes the reactivity and reduces fastness to the chemical factor action. The carried out researches have allowed to prove necessity of reconsideration existing TLV lowering. The conversion factors are proved. At the same time for the dockers there is no necessity for change of the hygienic norms. In these conditions, when the chemical danger grows and there can be an extreme situation down to failure, the important element of a hygienic regulation of loading DG in ports is the substantiation emergency TLV. In their basis the concept of a safety for health and life of workers, convertibility of physiological alterations as an obligatory condition of the dockers safety and vital activity is fixed. The obtained data made a basis of the methodical document determining the opportunity and conditions of safe transportation of wide DG assortment. 641 HEAVY METALS ACCUMULATION ON WOOD MOUSE (APODEMUS SYLVATICUS) AS A MARKER FOR EDAPHIC AND ATMOSPHERIC POLLUTION J. de Lapuente 1 , M. Borràs 1 , J. Nadal 2 . 1 Unitat de Toxicologia Experimental i Ecotoxicologia, Parc Científic de Barcelona, Josep Samitier 1–5, 08028 Barcelona, Spain; 2 Departament de Biologia Animal (Vertebrats), Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal, 645 08028, Barcelona, Spain Accumulation of some heavy metals (Al, Mo, Rb, Cu, Cd, Co, Pb, Cr, Ba and Hg) was investigated in liver, kidney and muscle of wood mice, Apodemus sylvaticus (Rodentia, Mammalia), taken as a sentinel species of pollution of different origins. Animals were trapped in two study sites: a location exposed to athmospheric pollution, in a partially protected zone (Collserola) within the highly industrialized metropolitan area of Barcelona, and a zone of heavy edaphic pollution caused by the leachates of the landfill of bellestar, near La Seu d’Urgell (Catalonia, north-east Spain). The wood mouse is a good prospector and integrator of environmental quality data and one of the most ubiquitous of rodents in Europe. Elements were analysed by Induction Coupled Plasma associated to mass spectrophotometry. In muscular tissue, all the analysed metals showed significantly higher levels in the animals caught in the surroundings of the Bellestar landfill, except Cu, that was higher in samples from Collserola. In liver, al was significantly higher in Collserola and Mo in Bellestar. In kidney, Mo accumulated in samples from Bellestar, while Co and Pb showed higher levels in Collserola. Our results highlight the usefulness and sensitivity of wood mouse as a sentinel species of pollution and of the bioavailability of the analysed elements in different conditions of exposure. 642 TOXICITY OF CARBOFURAN FOR AQUATIC AND TERRESTRIAL ORGANISMS M. Beklová, R. Dobšíková, J. Pikula. Department of Veterinary Ecology and Environmental Protection, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic Carbofuran, a pesticide of the carbamate group, is commonly used as an insecticide, nematocide and acaricide in agriculture throughout the world. Relatively high water solubility (0.32 g L−1 at 20°C) and low adsorption on soils and sediments enable accumulation of carbofuran in natural surface waters. Aim of this study was to evaluate the effect of the insecticidal product FURADAN 350 F containing the effective substance of carbofuran in the concentration of 360 g/l for organisms of different trophic levels in the aquatic and terrestrial environment. Toxicity for Lemna minor, Daphnia magna and Poecilia reticulata was evaluated. In terrestrial organisms toxicity was evaluated using Sinapis alba, Eisenia fetida and Coturnix coturnix japonica. Toxic effects were found in Daphnia magna. Values of 48h EC50 and 24h EC50 amounted to 19.09 µg L−1 (95% interval of confidence of 18.58 – 19.60 µg L−1 ) and 47.08 µg L−1 (95% interval of confidence of 34.75 – 59.40 µg L−1 ), respectively. In Poecilia reticulata levels of 48h LC50 and 96h LC50 amounted to 0.347 mg L−1 (95% interval of confidence of 0.301 – 0.393 mg L−1 ) and 0.225 mg L−1 (95% interval of confidence of 0.196 – 0.253 mg L−1 ), respectively. Fish manifested clinical signs of toxicity of lower concentrations by hyperaemia of gills and swimming at surface. Concentrations of 5 and 10 mg L−1 resulted within several minutes in muscular spasms, burst swimming, incoordination and subsequent mortality. The limiting test (100 mg L−1 ) of carbofuran toxicity for Lemna minor and Sinapis alba was negative. The LD50 value of carbofuran for Coturnix coturnix japonica was 2.17 mg kg−1 with the interval of confidence of 1.59–2.94 mg kg−1 . In agreement with published data, Daphnia magna was most susceptible. Employing a larger number of organisms for testing we get more pieces of information and a more reliable evaluation of the product. This research was supported by the Ministry of Education, Youth and Physical Training of the Czech Republic (CEZ: J16/98" 162700004 and FRVŠ Project No.272/2003). 643 THE INFLUENCE OF TYPE I PYRETHROIDS: CYPERMETHRIN AND DELTAMETHRIN ON ALGAE, CRUSTACEANS AND ROTIFER. H. Lutnicka. Department of Fish Diseases and Biology, Faculty of Veterinary Medicine, Agricultural University, Lublin, Poland The aim of the study was to find out if the low concentrations of cypermethrin (0.02 ppb) and deltamethrin (0.02 ppb) influence: Chlorella vulgaris, Daphnia magna, Thamnocephalus platyurus and Brachionus calyciflorus. Conventional flask test was performed with algae. The test method followed OECD Guideline. The test time was prolonging up to 14 days. Daphnia magna was used in chronic (reproduction) toxicity test according to the OECD Guideline. The organism was hatched from ephippia (Daphtoxkit T™ magna, 1996). Poster Session P33. Ecotoxicology A full range acute tests and experiments with low concentrations of tested pyrethroids were performed with Thamnocephalus platyurus (hatched from cysts), according to the Toxkit methodology (Thamnotoxkit F, 1995). Rotifer chronic (reproduction) toxicity tests were performed in full range of concentrations and with the low concentrations of tested pyrethroids. These tests were performed in standardized conditions according to the Toxkit methodology. The results were evaluated by US EPA statistical method or according to the Toxkit Standard Operational Procedure. The following conclusions were drawn: 1. The low concentration of cypermethrin or deltamethrin did not cause growth inhibition of Chlorella vulgaris after 72 h exposure time. Prolonging of the test up to 14 days caused about 13% growth inhibition. 2. No differences between mean numbers of young animals of Daphnia magna per alive parents were found for low concentrations of tested pyrethroids as compared with the control group. 3. No effect was found as an influence of low concentrations of the pyrethroids on the mortality of Thamnocecephalus platyurus in the acute 24 h tests. The acute test of cypermethrin or deltamethrin in full range of concentrations revealed LC50 /24 h values of 0.89 ppb and 1.51 ppb, respectively. 4. Low concentration of the pyrethroids influenced the reproduction of Brachionus calyciflorus reproduction test. Cypermethrin caused 2.5% stimulation while deltamethrin 2.2% inhibition of the reproduction. The full range tests for reproduction revealed EC50 /48 h value of 3828 ppb for cypermethrin while 8425 ppb for deltamethrin. 644 DEGRADATION OF ABAMECTIN IN SOIL FROM SHEEP GRAZING PASTURE L. Kolar, I. Marc, J. Kužner, V. Cerkvenik Flajs, M. Pogačnik, N. Kožuh Eržen. University of Ljubljana, Veterinary Faculty, Gerbičeva 60, 1000 Ljubljana, Slovenia The use of avermectins in veterinary medicine has been increasing rapidly in recent years. They cause a serious ecotoxicological problem, because of their specific metabolism and their action on non-target organisms. Avermectis bind strongly to faeces and have prolonged persistence with a long half-life. There is a lack of data, therefore studies on the possibility of degradation (photo and bio) for avermectins is important. The degradation of abamectin has been studied in soil from sheep grazing pasture treated with a single dose of abamectin (0.2 mg/kg b.w.). Sheep were stabled in the pasture area of 625 sq. m. They were released 6 days after the drug application. The concentration of abamectin in soil was determined from day 6 after the treatment over a time period up to 90 days. Samples were collected from the stockade pasture (approx. 100 sampling points), homogenized and stored at –20°C. After the classical extraction of abamectin from soil, extracts were cleaned-up with a solid phase extraction (Varian Bond Elut Al-N cartridges (500 mg, 3 ml)) and abamectin was determined by a high performance liquid chromatography (HPLC) with fluorescence detection (wavelength: excitation 365 nm; emission 470 nm; Supelco Supelcosil LC-8-DB column (250 x 4.6 mm id; 5 µm particle size with a Supelco guard column Supelguard LC-8-DB (20 x 4.6 mm id; 5 µm particle size; column temperature 28°C; mobile phase: methanol-acetonitrile-water ((47.5 + 47.5 + 6.0, v/v/v); flow rate 1.1 ml min−1 ). In the case of soil samples avermectins concentration decreased from 1.24 ng/g to 0.24 ng/g. The abamectin concentration in mixed soil-faeces samples decreased from 22.8 ng/g to 0.24 ng/g. The results showed the possibility of abamactin degradation under the external conditions. An environmental impact of abamectin is evident, therefore more extensive and systematic investigations are needed. 645 s173 EFFECT OF BROMADIOLONE ON BIOCHEMICAL PARAMETERS OF BLOOD PLASMA OF THE COMMON PHEASANT (PHASIANUS COLCHICUS) J. Pikula 1 , M. Beklová 1 , F. Vitula 2 . 1 Department of Veterinary Ecology and Environmental Protection, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic, 2 Game Animal Farm, Teaching Agricultural Plant, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic The aim of the study was to evaluate the effect of bromadiolone on biochemical parameters of blood plasma of the Common Pheasant (Phasianus colchicus). The effect was evaluated on the basis of the test of attractiveness and comparison of biochemical parameters in a control group and an experimental group subjected to the rodenticidal preparation containing the effective substance in the concentration of 0.005%. Test of the attractiveness of the product was done using the modified methods of BBA. Biochemical parameters of blood plasma were determined in a total of 20 adult males of the Common Pheasant. The control group of 10 males was for the whole period of the experiment fed by the commonly used feeding mixture for pheasants. The experimental group of 10 males was for three days provided by the daily ration consisting of 75% of the tested product and 25% of commonly used feeding mixture. After that, pheasants were fed by the commonly used feeding mixture again. Plasma biochemical parameters were determined in the blood collected on day 15 following the administration of the tested product. To determine the prospective differences between the control and experimental group we used the parametric t-test (independent experimental design). Homogeneity of variances was determined by the F-test prior to the t-test. In the experimental group there were significantly lower levels of glucose, creatinine and sodium as compared to the control one. On the other hand, levels of uric acid were significantly higher in the experimental group. Levels of ALP, ALT, potassium, calcium and phosphorus were comparable in both the groups, while it was not possible to evaluate levels of total proteins, bilirubin and AST using the t-test because of non-homogenous variances. Rodenticidal products on the basis of bromadiolone belong to anticoagulation preparations toxic for birds, however, due to low toxicity there were found only sporadic spots of bleeding on the musculature of the heart and pectoral muscles at autopsy. 646 EFFECTS OF HERBICIDE ATRAZINE ON SOIL BIOCHEMICAL ACTIVITY Lj. Radivojević 1 , S. Djordjević 2 , S. Gašić 1 , R. Stanković-Kalezić 1 , I. Elezović 2 . 1 Institute for Plant Protection & Environment, T. Drajzera 9, 11040 Belgrade, Serbia and Montenegro, 2 Faculty of Agriculture, Nemanjina 6, 11080 Zemun, Serbia and Montenegro Atrazine is a triazine herbicide, used worldwide since 1952 to control annual weeds in maize. In this study the effects of atrazine on soil biochemical activity (microbial biomass-C, soil respiration, relationship between soil respiration, microbial biomass-C and dehydrogenases activity) were examined. Investigation was carried out under control conditions on the silty loam soil. Atrazine was applied at three concentration: 8.0, 40.0 and 80.0 mg/kg of soil, and soil samples for determination of biochemical activity were taken 1, 7, 15, 30, 60 days after treatment. Microbial biomass-C was detected by the fumigating extraction method (Vance et al, 1982), soil respiration by the incubation method (Walter, 1952), and dehydrogenases activity by the method described by Tabatabai (1982). The highest microbial biomass-C content was found 60 days after atrazine application (8.0 mg/kg), and the lower 7, 15, and 30 days after atrazine application (concentration 40 mg/kg). The relationship between microbial biomass-C and soil respiration (qCO2 ) was increased in the period of 1 to 30 days, but after 60 days was decreased at all concentration. Dehydrogenases activity was decreased at all intervals and concentrations, compared to the control. s174 647 Poster Session P33. Ecotoxicology APPLICATIONS OF EUCARYOTIC MICROORGANISMS IN COMET ASSAY FOR IN VITRO GENOTOXICITY EVALUATION OF ENVIRONMENTAL SAMPLES Barbara Lah, Romana Marinsek-Logar. University of Ljubljana, Biotechnical faculty, Zootechnical Department, Groblje 3, 1230 Domzale, Slovenia Comet assay is a well known, rapid and sensitive method for genotoxicity testing, able to quantify DNA damage caused by genotoxic compounds, different sources of radiation and free radicals. It was originally developed for human blood cells and it is most frequently used in human monitoring. In this work the comet assay (single cell microgel electrophoresis) was adapted to an ubiquitous unicellular protozoon Tetrahymena thermophila and yeast Saccharomyces cerevisiae, which are easily and unexpensively grown in axenic laboratory cultures, to detect DNA damage caused by complex mixtures like waste waters or soil leachates. For this purpose, the original test protocol described by Singh et α. (Singh, 1988) was modified (lower concentrations of detergents in alkaline lysis buffer, reduction of electrophoresis time). Short time exposures of immobilised Tetrahymena thermophila and yeast cells to well-known genotoxicants phenol and hydrogen peroxide led to dose-dependent DNA damage with increasing concentration of genotoxicant. Further we tested the genotoxicity potential of the influent and effluent waters of the local municipal waste water treatment plant and soil leachates from intensively polluted local areas. The results of both modified protocols showed the genotoxic potential of the influent samples and the reduction of genotoxicity in effluent water. Genotoxicity of contaminated soils was proven too, correlating well with Vibrio fischeri test results. We intend to evaluate developed biotests by a selection of other genotoxic compounds. We can conclude that both tests could be used as relatively simple, sensitive and unexpensive genotoxicity tests for polluted waters and soil leachates especially with Tetrahymena cells possessing large nuclei. It is our purpose to use other eucaryotic microorganisms (like unicellular algae) for genotoxicity testing in comet assay. As eucaryotic microorganisms possess almost the same cell “machinery” as animals, plants and humans, they could be relatively successfully used instead of experimental animals for genotoxicity testing of waste waters, soil leachates and even foods. 648 THE EFFECT OF CADMIUM ON HAEMATOLOGICAL AND BIOCHEMICAL INDICES OF CARP (CYPRINUS CARPIO L.) J. Drastichová 1 , Z. Svobodová 1,2 , V. Lusková 3 , J. Máchová 2 , O. Čelechovská 1 , E. Švestková 4 . 1 University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic, 2 University of South Bohemia, Research Institute of Fish Culture and Hydrobiology, Vodňany, Czech Republic, 3 Institut of Vertebrate Biology, AS CR, Brno, Czech Republic, 4 Department of Clinical Haematology, University Children‘s Hospital Brno, Czech Republic Test of acute toxicity was carried out on carp fry throughout 96 hours. A value of 96hLC50 of cadmium chloride in carp fry was 5 – 10 mg.l−1 , that corresponds to the value of 3.07 – 6.14 mg.l−1 of cadmium. In an experimental group of carp K1–2 (12.5 mg.l−1 of cadmium chloride, period of exposition 96 hours) there were found significantly higher values of erythrocyte count (p<0.05), haemoglobin content (p<0.01) and haematocrit (p<0.01) in comparison with a control group. Total leukocyte count and absolute lymphocyte count were significantly lower (p<0.01) in experimental group. Some cytochemical investigations (PAS, Sudan, peroxidase and alkaline phosphatase reaction) of neutrophil granulocytes were performed in experimental group and compared with control ones. In experimental group of fish there occured a significant increase (p<0.01) of glucose concentration, AST, CK, LDH and phosphorus and a significant decrease (p<0.01) of calcium concentration in the blood plasma. Cadmium accumulated in tissues in the following order: kidney > liver > muscle. Cadmium acts as a significant stress factor in carp, that was manifested by remarkable changes of haematological and biochemical indices in the blood plasma. This research was supported by the Ministry of Education, Youth and Sports of the Czech Republic (FRVS Project No. 360/2002). 649 STRESS ASSESSMENT ON THE SHREW CROCIDURA RUSSULA EXPOSED TO ENVIRONMENTAL POLLUTION A. Sánchez-Chardi, M.A. Sans-Fuentes, M.J. López-Fuster, J. Nadal. Departament de Biologia Animal (Vertebrats), Facultat de Biologia, Universitat de Barcelona, Barcelona, España The aim of this study was to assess the effect of environmental pollution on developmental stability in wild populations of the greater white-toothed shrew, Crocidura russula (Herman, 1780). For this purpose fluctuating asymmetry (FA), i.e. random differences between left and right sides of a bilaterally symmetrical character, was estimated. From 1996 to 1999, 127 adult shrews were collected in three polluted (Cercs, Garraf and Doñana) and three matched control areas in Spain. Gaseous and particulate matter from a coalfired power plant affected Cercs; Garraf was exposed to liquid effluents from a landfill; and metals from a mine tailings spill affected Doñana. Heavy metals and other elements (Zn, Cu, Cr, Co, Cd, Pb, Sn, Sr, As, Mg, Mn, Tl) were analysed by ICP in the liver and kidney of all animals. To evaluate FA, thirty non-metrical traits (foramen areas) were scored on the skull and the right and left mandibles. Traits that did not show antisymmetry, correlation with character size, or directional asymmetry were used for the study. Comparisons of FA levels between animals from polluted and control areas were performed by non-parametric tests. Shrews from polluted sites showed significantly higher levels of some heavy metals (Pb, Cd, Cr, Cu, Tl) than those from the respective control sites, especially in Doñana where the increase in certain metals (Cd and Tl) was strong. Significant differences in FA levels were found only between shrews from Doñana, corresponding the highest values to those from the polluted zone. We conclude that an increase in heavy metal availability and accumulation may increase developmental instability. Further, the greater white-toothed shrew could be used as sentinel of environmental pollution. 650 ECOTOXICOLOGICAL RISK ASSESSMENT OF USELESS FORBIDDEN PESTICIDES M. Prodanchuk, L. Povyakel. Medved’s Institute of Ecohygiene and Toxycology, Kyiv, Ukraine Special alarm for the population of globe is caused with an ecological situation in connection with accumulation of chemical substances - pesticides as waste products. The way of accumulation of the forbidden pesticides has defined presence not simply dangerous, but highly or extremely dangerous substances, taking into account, that the reason of banning of their use as plant protection products were: high toxicity for warm-blooded, expressed delayed effects, stability in environment. Useless pesticides which have lost a packaging view as a result of wrong transportation or storage, and also pesticides with the expired date of use increase number of these dangerous waste products. As a result of independent storage mixes of preparations and products of disintegration and unstated structure were formed. At combined storage of these substances under influence of natural factors during toxic transformation of separate components of pesticides new more toxic substances, including gases, with all following consequences can be formed. Character of display of toxic action can be irritating, general toxic, systemic toxic, neurotoxic, cancerogenic, mutagenic. It is necessary to take into account that fact, that among the forbidden pesticides 9 are sources of persistent organic pollutants (POPS) Definition of risk for environment and health of the population, in this case, is represented as comprehension of danger of occurrence of undesirable events in the certain place and time predicted sizes of probable consequences. The risk assessment of useless and forbidden pesticides (UFPs) is complex multiphasic process. Specificity of risk assessment of UFPs is, that it is taken into account, firstly risk of separate pesticide, secondly – all weight of stored preparations, as toxic waste products and, thirdly, the account of certain ecotoxicological conditions. Risk assessment of separate pesticide undoubtedly should be carried out on parameters of toxicity on basis WHO classification by hazard. Inclusion of parameters of migration from soil in into environment media essentially strengthens the importance of this classification in this case. Poster Session P34. Endocrine disrupters Thus, the problem of risk assessment of UFPs is more profound and comes outside the limits of existing approaches of pesticides hazard estimation during their application as plant protection products. The size of this risk can considerably vary and is caused besides physical and chemical and toxicological properties of the stored preparation, natural geographical, social and economic features of concrete district. The complex approach, the account and the system analysis of many factors incorporated in the scheme for risk assessment of these waste products is required. Risk assessment of useless pesticides should be carried out with finding the way of their removal, and also developing of preventive actions with a view of maintenance of ecological safety and human health. 651 THE EFFICACY OF ACTIVATED CHARCOAL AND KLINOPTILOLITE IN PROTECTION OF ANIMALS POISONED WITH BROMADIOLONE V. Cupic 1 , S. Dobric 2 , Z. Milovanovic 2 , D. Bokonjic 2 . 1 Faculty of Veterinary Medicine, 2 National Poison Control Center, Military Medical Academy, Belgrade, Serbia and Montenegro Anticoagulant rodenticides are often the cause of accidental poisonings of domestic animals. In these cases vitamin K1 , as their causal antidote, is used. However, in every day veterinary practice it is not always available. Due to this, other supportive treatments are needed. Adsorbents, such as activated charcoal, are often used as unspecific antidotes. In this study we evaluated the efficacy of activated charcoal and klinoptilolite, one kind of zeolite with improved adsorbent capacity, in protection against acute poisoning with bromadiolone, the widely used anticoagulant rodenticide. The experiments were performed on adult Wistar rats, both sexes starving for several hours before administration of bromadiolone (1.3 LD50 , p.o.). The protected animals were given activated charcoal (5% water suspension) or klinoptilolite (20% water suspension) in doses of 1 ml/kg p.o. or 3 ml/kg p.o., immediately after bromadiolone, as well as in a dose of 3 ml/kg p.o. immediately, and 1h, 2h and 3h after bromadiolone (totally four doses of 3 ml/kg). The efficacy of adsorbents tested was estimated by means of mean lethal time (LT50 ) of protected animals in comparison with that of the control one. The results demonstrated that both adsorbents, regardless of given dosage regimen, failed to protect animals poisoned with bromadiolone. Moreover, the LT50 values of the protected groups were even lower than that of the control one. Our results suggest that activated chrarcoal and klinoptilolite could not offer any protection in animals acutely poisoned with anticoagulant rodenticides. 652 THE USE OF DUTCH MYRTHE (LEDUM PALUSTRE) AND PINE NEEDLES (PINUS SYLVESTRIS) TO DETECT THE POLYCYCLIC AROMATIC HYDROCARBONS (PAHs) AT REGIONAL SCALE M. Malawska, B. Wilkomirski. University of Warsaw, Institute of Botany, Warsaw, Poland PAH concentration and distribution has been examined in plant samples collected from different peatlands of Poland between 2000– 2002 years. Leaves from two species (Dutch myrthe and pine) were sampled at intervals through a growing season. We concluded that the influence of air concentrations was more important than meteorological conditions in determining plant content of PAHs, over a growing season. The collection of Dutch myrthe leaves and pine needles for detect of airborne PAHs is suitable technique for monitoring these compounds, however the concentration of PAHs in Dutch myrthe leaves were 2–3 times higher than in pine needles. Concentration of 4, 5 and 6-ring PAHs were positively correlated with time for both species, but there were significant differences in the PAHs profiles between species sampled from most polluted and small polluted region (Masurien and Silesia Region). This study shows that vegetation sampling can be used to show spatial differences in the atmospheric burden of a range of PAHs emission. s175 P34 Endocrine disrupters 653 EROD, UDPGT ACTIVITY AND THYROID HORMONE LEVEL AFTER IN UTERO EXPOSURE TO A LOW DOSE OF 2,2’,4,4’,5 – PENTA-BDE (PBDE 99) IN RAT OFFSPRING. I. Chahoud 1 , S. Kuriyama 1 , A.A. Fidalgo-Neto 2 , W. Wittfoht 1 . 1 Inst. of Clinical Pharmacology and Toxicology, Benjamin Franklin Medical Center, FU, Berlin, Germany, 2 Laboratory of Environmental Toxicology, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil Thyroid hormone levels, ethoxy-resorufin-O-deethylase (EROD) and uridine diphosphoglucuronyl transferase (UDPGT) activity were evaluated in offspring rats exposed in utero to a low dose of penta 2,2’,4,4’,5 –BDE (PBDE 99). The two doses were selected to be 100- and 500-fold higher than human breast milk concentration reported for women in industrialized countries. Using a dose equation previously described by our group, Wistar dams (n=10) were treated by gavage on gestation day 6 with a single dose of 60 or 300 µg PBDE 99/kg body weight or peanut oil (control). A reference control group composed of dams treated with 0.5% of the goitrogen, 6n-propyl-2- thiouracil (PTU), in drinking water (about 940 micro g PTU/kg b.w.) from GD 7 to 21 was included. Serum and liver samples obtained from dams (PND1 and PND 22) and offspring (PNDs 1, 14 and 22) were analyzed for circulating total and free thyroxin (TT4 and FT4), triiodothyronine (TT3 and FT3) and TSH and hepatic microsomal EROD and UDPGT activity, respectively. A statistically significant reduction in T4 levels was observed in PBDE–treated offspring at the end of lactation (PND 22). PTU-exposed offspring showed a decrease in T4 and TSH levels at the beginning of lactation (PND 1), which has restored to normal levels at weaning. Although, we could not detect EROD activity (due to technical limitation) in offspring at PND 1, PBDE 300-exposed males displayed significant increases in EROD activity on PND 22. UDPGT activity (marker for T4 glucuronidation) of PBDE treated offspring was increased at the beginning of lactation (PND 1), but less pronounced at weaning (PND 22). We draw three conclusions regarding low dose effects of PBDE 99 on thyroid hormone disruption: 1- pre- and postnatal (PBDE 99 passes through milk to infants) PBDE 99 exposure alter thyroid hormone status in offspring at weaning and not before; 2 - while PTU-exposed animals displayed a transient hypothyroidism, effects of PBDE were more persistent suggesting a different mechanism of disruption; 3- increased rate of T4 glucuronidation does not seem to be the main mechanism of thyroid disruption at low dose exposure. Our data demonstrate that in utero exposure to a low dose of PBDE 99 interferes with thyroid hormone homeostasis in rat offspring. Acknowledgement: Grants UBA - Forschungs-und Entwicklungsvorhaben 29965221/04 654 PITUITARY-THYROID AXIS IN THE POSTNATAL RAT OFFSPRING FOLLOWING GESTATIONAL AND LACTATIONAL EXPOSURE TO BISPHENOL A K. Kobayashi, M. Miyagawa, R.S. Wang, M. Suda, S. Sekiguchi, T. Honma. Department of Health Effects Research, National Institute of Industrial Health, Kanagawa, Japan Bisphenol A (BPA), a xenoestrogen, is very widely used in the manufacture of polycarbonate and epoxy resins. Although BPA has been reported to mimic the actions of estrogen or to affect the reproductive organs and accessory genital glands, the effects of maternal exposure to BPA on the offspring of rats still remain unclear. In the present study, we examined whether gestational and lactational exposure to BPA altered the postnatal growth and thyroid function of male and female offspring in vivo in rats. Pregnant Sprague-Dawley rats were exposed to BPA (0, 4, or 40 mg/kg/day) in corn oil once daily via oral gavage from gestation day 6 through postnatal day 20, and the control group was given the same amount of corn oil during the same period. There were no significant changes in body weight, liver weight, kidneys weight, testes weight (male), anogenital distance (AGD), or AGD indices in the BPA-exposed groups compared to the control group. Plasma concentrations of thyroid hormone (T4) and thyroid-stimulating hormone (TSH) were s176 Poster Session P34. Endocrine disrupters unaffected. No differences in the plasma T4 response to exogenous TSH stimulation occurred in all exposed groups compared to the control group. These results suggest that BPA did not produce any severe impairment in the postnatal growth and pituitary-thyroid axis of the F1 generation in rats under the present experimental conditions wherein the exposure levels were relatively high. The effects of BPA exposure are, however, still incompletely understood and further study should be carried out to confirm the toxicity of BPA during gestational and lactational period in rats. 655 ANALYSIS OF MICROARRAY DATA REVEALED THE LONG-TERM EFFECTS OF NEONATAL EXPOSURE TO GENISTEIN AND BISPHENOL A ON GENE EXPRESSION IN MICE. H. Fukata 1 , T. Adachi 2 , M. Komiyama 3,4 , K. Sakurai 5 , C. Mori 3,6 . 1 Department of Environmental Medical Science (SRL), Graduate School of Medicine, Chiba University, Chiba, Japan, 2 Center for Research and Development of Bioresources, Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Japan, 3 Department of Bioenvironmental Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan, 4 Center for Environment, Health and Field Sciences, Chiba University, Kashiwa, Japan, 5 Department of Clinical Cell Biology, Graduate School of Medicine, Chiba University, Chiba, Japan, 6 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, Japan To investigate the long-term effect of endocrine disrupting chemicals (EDCs) on gene expression in a testis and other tissues, mice were transiently exposed to bisphenol A, genistein or diethylstilbestrol for five days after birth. Gene expression levels were comprehensively measured using a cDNA microarray at 4- and 12-weeks. The analysis of data showed that (1) expression of some genes was disturbed by EDCs and the disturbance was not recovered even after 12-weeks, (2) EDCs greatly influenced newly expressing genes, (3) EDCs depending on their severity tended to inhibit testis growth, and (4) EDCs influenced not only the testis but also the spleen at 12-weeks. Statistical techniques using computer software, such as cluster analysis and principal component analysis, are powerful tools however in some cases these do not lead to meaningful results. We attempted to analyze the effect of EDCs using a DNA microarray. The data obtained was handled as a set using suitable indicator genes which showed only a little alteration on the expression level, so that new knowledge can lead the way. Our analysis showed some new approaches to the reading of microarray data. 656 EFFECT OF ALLYLISOTHIOCYANATE (SCN− ), NITRATE (NO− ) AND NITRITE (NO2 - ) ON THYROID GLAND 3 MORPHOLOGY IN RATS AND ANTIGOITROGENIC PROPERTIES OF IODINE: A QUANTITATIVE EVALUATION R.B. Kostogrys 1 , P.M. Pisulewski 1 , A. Pecio 1,2 . 1 The Agricultural University, Faculty of Food Science and Technology, Department of Human Nutrition, Krakow,Poland 2 The Jagiellonian University, The Faculty of Biology and Earth Sciences, Department of Comparative Anatomy, Krakow, Poland A two-factorial (2x4) design was used to study the effect of two iodine levels (2 and 4µg per rat) and four goitrogen treatments (control, allylisothiocyanate–6mg/100g, nitrate–300mg/100g and nitrite–25mg/100g BW) on thyroid gland morphology in rats. Forty-eight five-week old, male Wistar rats, weighing on average 95 g, were randomly allocated to eight groups (6 rats each). The rats were fed eight basal AIN’93G diets providing: I - 2µg iodine, II 2µg iodine + SCN− , III - 2µg iodine + NaNO3 , IV - 2µg iodine + NaNO2 , V - 4µg iodine, VI - 4µg iodine + SCN− , VII - 4µg iodine + NaNO3 , and VIII - 4µg iodine + NaNO2 . The feed intake was restricted to 15g per rat per day. On d 18, the rats were anaesthetised and thyroid glands were carefully excised. The glands were processed by a conventional paraffin technique, sectioned at approximately 7µm, and stained with hematoxylin and eosin for histological examination. For colloid evaluation the PAS reaction was used. The mean height (distance between basal and apical face of cell) of 100 follicle epithelial cells was measured. The analysis of the colloid area was carried out on 50 follicles, using the image analyzer coupled with camera mounted on light microscope. The animals receiving 2 µg of iodine per rat per day and the goitrogens (allylisothiocyanate, nitrate and nitrite) showed drastic changes in thyroid morphology. The height of follicular epithelial cells was significantly increased by all goitrogens in rats fed 2µg iodine (P<0,001). In addition, irregularity of follicles was evident. Marked diffuse follicular hyperplasia was apparent, and thyroidal tissue was very dense. The amount of colloid was reduced by allylisothiocyanate (P<0,01), nitrate (P=0,07) and nitrite (P<0,05). At the same time, in the rats receiving iodine at the level of 4µg per rat per day and allylisothiocyanate or nitrate, follicular epithelial cells were not affected and their histological parameters were comparable with those in the control group. Unexpectedly, in spite of iodine supplementation (4µg per rat per day), the height of follicular epithelial cells was significantly increased by nitrite (P<0,001). Nitrite produced diffuse thyroid hiperplasia, with small follicles, tall epithelium and reduced colloid (P<0,001). To conclude, dietary allylisothiocyanate, nitrate and nitrite influenced thyroid gland morphology in rats and produced thyroid hypertrophy/hiperplasia. At the same time, these adverse effects of the studied goitrogens (except nitrite) can be prevented by an extra iodine supplementation. Further studies are needed to elucidate goitrogenic effects of nitrite ions in rats. 657 IMPAIRMENT OF STEROID HORMONE METABOLISM AFTER IN UTERO EXPOSURE OF MALE MICE TO LINDANE E. Di Consiglio 1 , G. De Angelis 1 , M.E. Traina 2 , E. Urbani 2 , M. Rescia 1 , E. Testai 1 . 1 Comparative Toxicology and Ecotoxicology Lab., 2 Environmental Hygiene Lab., Istituto Superiore di Sanità, Rome, Italy The presence of environmentally persistent pollutants, as the organochlorinated pesticide lindane, has been associated to longlasting health effects including sterility, impaired development and birth defect. Direct changes in endocrine function (due to estrogen receptor binding) or indirect ones (influencing hormone metabolism) were hypothesized. The metabolism of steroid hormones is mediated by cytochrome P-450 (CYP), whose alteration may affect hormone circulating concentration, resulting in endocrine disruption. We have therefore investigated on the effects, due to in utero exposure of lindane, on possible reproductive disorders and alteration of steroid hormone metabolism, through the detection of testosterone (TST) hydroxylation and of aromatase (CYP19), the enzyme converting androgens to estrogens. Pregnant CD1 mice were treated with a daily dose of 25 mg/kg b.w. lindane or vehicle p.o. (9–16 gestational days). F1 male mice were sacrificed on postnatal days (PND): 55; 70; 100. Evaluation of reproductive endpoints as well as measurement of TST catabolism and aromatase activity in the liver were performed. Experiments were carried out in compliance with ethical provisions and authorization. Significant changes of the major reproductive endpoints (testis weight, spermatid concentration and creatine levels) were observed on PND70 with a following recover at PND100. No structural modification of the testicular tissue was histopathologically evidenced, suggesting a functional impairment. Indeed, the present results showed that lindane caused a marked modification of CYPmediated TST metabolism on PND70 F1 mice: a dramatic decrease of CYP3A4, 2C11 and 2B1 activities was observed (30–90% loss). The formation of TST metabolites, except androstenedione, showed a general recover on PND100. On the contrary, lindane did not significantly affect the activity of aromatase on PND70 F1 mice. These results suggest that the alteration of TST metabolism may be associated with the functional impairment of sperm production and maturation due to in utero lindane exposure. This work has been partially supported by the ISS Project n° 2128/RI. Poster Session P34. Endocrine disrupters 658 EFFECTS OF FLAME RETARDANTS PBDE 99 AND PCB 0N MRNA LEVELS OF ESTROGEN TARGET GENES AFTER PRENATAL EXPOSURE. R. Ceccatelli, O. Faass, I. Fleischmann, M. Conscience, M. Schlumpf, W. Lichtensteiger. Institut of Pharmakology and Toxicology, University of Zurich 190 Wintherthurerstrasse 8057 Zurich Polybrominated diphenyl ethers (PBDE) are used in large quantities as additive flame retardants in plastics and textile materials. PBDEs are persistent compounds and have been detected in wildlife and in human adipose tissue and plasma samples. Certain PCBs, a structurally related group of substances, show endocrine disrupting action in mammals. We are investigating the effects of PBDE 99 (2,2‘,4,4‘,5-PentaBDE) and a PCB mixture, Aroclor 1254, on the mRNA levels of estrogen target genes in reproductive organs of Long Evans rat by Real Time PCR, with cyclophilin as reference gene. PBDE 99 (1 or 10 mg/kg/day) or Aroclor 1254 (10mg/kg/day) was injected subcutaneosly to time-pregnant rats from gestational day 10 to 18 (vehicle control: olive oil). Prenatal exposure to PBDE 99 or Aroclor 1254 affected the development of reproductive organs as indicated by changes in organ weight of adult offspring (effects on ventral and dorsal prostate, epididymis and ovary weight with PBDE 99). The effect patterns differed between PBDE 99 and PCB mixture. These effects were accompanied by changes in mRNA levels in tissue of adult offspring, i.a., progesterone receptor mRNA (uterus) and mRNA encoding for androgen receptor and estrogen receptor α and β (ventral and dorsal prostate). Our data indicate that PBDE 99 can interfere with the development of reproductive organs and induce long term changes in gene expression patterns. 659 POSSIBLE EFFECTS OF METHOXYCHLOR ON ACTH SECRETION THROUGH DOPAMINE A. Lafuente 1 , T. Martínez-Rivas 1 , González Carracedo 1 , A.I. Esquifino 2 . 1 Laboratorio de Toxicología, Facultad de Ciencias, Universidad de Vigo, Campus de Orense, Las Lagunas, 32004-Orense, Spain. 2 Dpt. de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, Madrid, Spain Methoxychlor (MTX) is an organochlorated insecticide, currently used as a substitute for DDT. This organochloride insecticide, a oestrogen like substance, may modify the feedback mechanisms of steroids on the hypothalamus and pituitary. This work was undertaken to explore the possible effects of MTX on adrenocorticotropin hormone (ACTH) secretion and to analyze whether these effects are mediated by dopamine. Adult male Sprague-Dawley rats were administered with 25 mg/kg/day of MTX dissolved in sesame oil, for 30 days. The control group animals received sc 0.3 mL of sesame oil. The plasma ACTH levels were measured as well as dopamine (DA) content in median eminence and in anterior, mediobasal and posterior hypothalamus and in striatum. Dopamine was analyzed by high performance liquid chromatography, using electrochemical detection (HPLC.EQ) and plasma ACTH levels were analyzed by specific double antibody radioimmunoassay. The exposure to the insecticide diminished the plasma levels of ACTH (P<0.001 vs. control group). In the groups treated with MTX there were an augmentation of DA content in the anterior and posterior hypothalamus (P<0.05 vs control group) and a decrease in the median eminence (P<0.001 vs. control group), without modifying its levels in the mediobasal hypothalamus and striatum. The increase in the DA content in the anterior hypothalamus could explain the decrease of this catecholamine found in the median eminence after MTX exposure. These data suggest that MTX could inhibit the ACTH release through an increase of the DA content in the anterior hypothalamus. 660 s177 EFFECT OF ETHANOL ON PROLACTIN RELEASE IN FEMALE RATS: ROLE OF ESTROGENS T. Shumkova, N. Bojadjieva. Department of Pharmacology and Toxicology, Medical University of Sofia, Sofia, Bulgaria Alcohol drinking is known to cause hyperprolactinemia in both in humans and laboratory animals. In order to better understand the mechanisms of alcohol’s effects on pituitary, we investigated the effects of estrogens on the alcohol stimulated hyperprolactinemia. Female rats were ovariectomized (OVX) and then treated with alcohol for 3 weeks. Group of rats were treated with ethanol and 17-β-estradiol. The plasma prolactin (PRL) levels and pituitary total protein levels were determined. The result demonstrated that ethanol treatment increased the levels of PRL in OVX rats. The 17-β-estradiol treated OVX rats did not show the significant changes in the plasma PRL levels. When ethanol was combined with 17-β-estradiol, the OVX rats showed higher levels of plasma prolactin, compared with ethanol treated rats. These results suggest that estrogen plays a role in ethanol stimulated hyperprolactinemia. 661 DETERMINATION OF ORGANOCHLORINE COMPOUNDS IN ADIPOSE BREAST TISSUE FROM WOMEN WITH BREAST CANCER A.P.M. dos Santos 1 , J.P. Franco 1 , M.E. Vaz Pereira 2 , M.C.C. Batoréu 1 . 1 Laboratory of Toxicology, Faculty of Pharmacy, University of Lisbon, Av. Prof Gama Pinto, Lisbon, Portugal; 2 Service of Surgery, District Hospital of Setubal, Setubal, Portugal A recent epidemiologic study concluded that 73% of breast cancers are attributable to environmental factors. Evidence is growing that there is a connection between certain chemicals in the environment and the rising incidence of breast cancer. Several studies have shown that breast epithelial cells proliferate in response to estrogens, including environmental estrogens, and breast cancer has been correlated with cellular exposure to xenoestrogens. Xenoestrogens include several lipophilic, persistent compounds to which humans and wildlife have been exposed. Among these are a number of chlorinated organics, such as the insecticides dieldrin, DDT and its metabolites, endosulfan, chlordecone, and industrial pollutants such as some polychlorinated biphenyl (PCB) congeners and dioxins. Several of these chemicals have been shown to have estrogenic activity in vitro and/or in vivo assays. The objective of this study is to evaluate the chronic exposure to organochlorine compounds through the identification and quantification of those compounds accumulated in the adipose breast tissue from women with breast cancer. The adipose tissue samples were collected from the Service of Surgery of the Hospital of Setubal, which follows and treats women from an intensive agricultural area. Methodology: It was developed a method to extract and determine the organochlorine compounds in adipose breast tissue. A solid-liquid procedure was used for the extraction of organochlorine compounds from fat tissue: a pyrex glass column filled with Alumine Merck 90 was used for the extraction of 0.2 g of breast fat tissue dissolved in n-hexane. The eluate obtained was concentrated and dissolved again in n-hexane. After the addition of an internal standard, the extract was injected into a gas chromatographer Hewlett Packard 5890, with electron capture detector. The column installed was a HP-5. Our preliminary results indicate that the main organochlorine compounds detected in breast fat tissue are DDE (metabolite of DDT), dieldrin, endosulfan, and four PCBs congeners. Dieldrin was found in 40% of the samples analysed (0.313 – 0.429 µg/g) and was the compound with the higher concentration in tissue from women with breast cancer. In control samples, some organochlorine compounds were found nevertheless dieldrin was not detected. s178 662 Poster Session P34. Endocrine disrupters EFFECTS OF SOME PHTHALATES ON THE SELECTED ENZYME ACTIVITIES IN WISTAR RAT TESTIS B. Wiadrowska, G. Kostka, R. Bańkowski, S. Pawlak. National Institute of Hygiene, Department of Environmental Toxicology, Warsaw, Poland Phthalic acid esters (PAEs) are widely used as plasticizers in several plastic formulations and are known to be widely distributed in environment. PAEs have been found in tissues of plants and higher animals. PAEs produce reproductive dysfunction in experimental animals. The present study describes the effects of di(2ethylhexyl)phthalate [DEHP], buthyl benzyl phthalate [BBP] and di-n-buthyl phthalate [DBP] on the activity of sorbitol dehydrogenase [SDH], γ-glutamyl transpeptidase [γ-GT] and hyaluronidase considered to be markers of spermatogenesis in testis of rats. Chemicals – DEHP and DBP were purchased from Sigma Company, BBP was purchased from Aldrich Chemical Company. Animal and treatment – Young male Wistar albino rats (55±60g body weight) were housed; five per cages equipped with water supply ad libitum and were given constant access to standard laboratory diet. Environmental conditions were standard. The animals were divided into 9 groups of five animals each. PAEs were given orally at the dose DEHP, BBP - 2000mg/kg bw, and DBP 2400mg/kg bw respectively for 10 days. Body weight of control and treated animals were recorded prior to the start of treatment and every two days during the course of treatment. After the completion of treatment animals were sacrificed by cervical dislocation. The testes and epididymis were promptly removed and weight. Enzyme activities – A portion of a testis was homogenized and centrifuged. The clear supernatant fluid was used as the enzyme preparation. Biochemical observation – in all exposed groups, the activities of testicular enzymes associated with post-meiotic spermatogenic cells such as SDH and hyaluronidase, were decreased significantly. The activities of enzymes associated with pre-meiotic spermatogenic cells, Sertoli cells or interstitial cells such as γ-glutamyl transpeptidase were significantly increased. Pathemorphological observation - in all exposed groups, significant reduction in the weight of the testes were observed. The results suggest that testicular atrophy caused by DEHP, BBP and DBP is associated with an alteration in the activities of enzymes related with specific events of spermatogenesis. 663 THE EFFECTS OF NEONATAL EXPOSURE TO ENDOCRINE DISRUPTERS (EDs) IN MOUSE EPIDIDYMIS 1 1 2 3 1,4 K. Yamazaki , Y. Ono , T. Adachi , N. Seki , C. Mori , M. Komiyama 1,5 . 1 Department of Bioenvironmental Medicine, Graduate School of Medicine, Chiba University, Chiba, 2 Center for Research and Development of Bioresources, Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, 3 Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, 4 Core Research for Evolutional Science of Technology (CREST), Japan Science and Technology Corporation (JST), Kawaguchi, 5 Center for Environment, Health and Field Sciences, Chiba University, Kashiwa, Japan Epididymis is an important organ for maturation of spermatozoa, in which the sperms acquire the function of progressive motility and fertility. Recently, several studies have reported that the decrease in the number of normal sperms or in the function of fertility, and EDs have been thought to be possible cause. Epididymis deeply seems to be related to it. This study examined what effects were observed in each region of epididymis by neonatal exposure to diethylstilbestrol (DES) or 17β-estradiol (E2 ). DES or E2 (5 µg/mouse/day) was subcutaneously injected into male newborn ICR mice for 5 successive days. We examined the weight of body, testis and epididymis, and morphological changes of epididymis at 2, 4, and 8 weeks-old. The treatment with either DES or E2 reduced the body weight at 2 weeks-old comparing to control. Also, the DES treatment reduced the body weight at 8 weeks-old. The relative epididymal weight to body weight was lower than control at 4, 8 weeks-old in DES group and at 2, 4 weeks-old in E2 group. Moreover, the relative epididymal weight to testis weight was lower at 4 weeks-old in DES group. Morphological analysis of epididymis revealed that the height of epithelial cells in all regions was shorter than control in both groups and the interstitial tissue in DES group increased at 2 weeks-old. It was also observed that the height of the epithelial cells was shorter in initial segment and taller in caput than control at 4 weeks-old in both groups. Remarkable changes were not detected at 8 weeks-old in both groups. These results suggest that epididymis is very susceptible to EDs comparing to testis and the effect caused by DES was larger than the effect caused by E2 . 664 CADMIUM EXPOSURE DIFFERENTIALLY MODIFIES THE CIRCADIAN PATTERNS OF NOREPINEPHRINE AT THE MEDIAN EMINENCE AND PLASMA LH, FSH AND TESTOSTERONE LEVELS. A. Lafuente, A. González-Carracedo, A. Romero, E. Fernández-Rodríguez. Laboratorio de Toxicología, Facultad de Ciencias, Universidad de Vigo, Campus de Orense, Las Lagunas, 32004-Orense, Spain This work was designed to analyze the possible effects of cadmium exposure on time-of-day variations of norepinephrine (NE) content in median eminence and on plasma levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone in adult male rats. Adult animals were given cadmium at a dose of 25 ppm of cadmium chloride (CdCl2 ) in the drinking water for one month. Age-matched rats with access to cadmium-free water were used as controls. Significant 24-hours changes of NE content in the median eminence, plasma LH and testosterone levels occurred in control animals. Cadmium exposure induced a phase advance of the nocturnal peak of NE content that was described in the control group, to 12 h and increased its amplitude. However, the mean NE content was not changed by cadmium. Metal exposure abolished the daily pattern of plasma LH levels, although the mean levels of the hormone were not modified by cadmium. However, for testosterone, the metal increased the amplitude of its nocturnal peak and induced the appearance of another peak during the light phase at 12 h, thus increasing the mean plasma levels of this hormone. An interaction between the metal and time for NE and plasma testosterone levels was observed. These data suggest that cadmium exposure during the adulthood exerts differential effects at the median eminence, the pituitary and the testes, that may explain the changes in the 24-h pattern of plasma testosterone levels. 665 ENDOCRINE DISRUPTORS CHANGE GENE EXPRESSION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS BUT DO NOT AFFECT ON CELL PROLIFERATION D. Nishimura 1 , T. Adachi 2 , K. Sakurai 3 , H. Fukata 4 , M. Komiyama 1,5 , C. Mori 1,6 . 1 Department of Bioenvironmental Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan, 2 Center for Research and Development of Bioresources, Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Japan, 3 Department of Clinical Cell Biology, Graduate School of Medicine, Chiba University, Chiba, Japan, 4 Department of Environmental Medical Science (SRL), Graduate School of Medicine, Chiba University, Chiba, Japan, 5 Center for Environment, Health and Field Sciences, Chiba University, Kashiwa, Japan, 6 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi, Japan Many functions of blood vessel are dependent on the vascular endothelial cell. Since the vascular endothelial cell produces various substances such as cytokines, the role as an endocrine organ of vascular endothelial cell is drawing people’s attention. It has been reported that multiple chemicals are detected from serum of human umbilical vein. The purpose of the present study was to clarify the effect of endocrine disruptors on vascular endothelium function by using human umbilical vein endothelial cells (HUVEC). HUVEC (passage number = 5) were treated with 17β-estradiol, diethylstilbestrol and bisphenol A for 6, 12, 24, 48 hours at the concentration levels from 1 pM to 1 µM. Then we measured alteration of gene expression levels using real-time RT-PCR and cDNA oligonucleotide array. The results are summarized as follows. 1) Some of the gene expression levels were changed, but the cell Poster Session P34. Endocrine disrupters proliferation was not affected. 2) The change of the gene expression levels showed the vasodilatation in vitro. It was indicated that the effect by these three chemicals on vascular endothelial cells are similar to each other, although the effect-mechanisms of each chemical were not clear. (This work was supported by the fund for endocrine disrupters from the Ministry of the Environment, and the Ministry of Education, Culture, Sports, Science and Technology, Japan.) 666 ANALYSIS OF PHYTOESTROGEN WHICH ARE TRANSFERRED FROM MOTHER TO FETUS: THE EVIDENCE OF EXISTENCE OF EQUOL PRODUCER GROUP AND NON-PRODUCER GROUP IN FETUS E. Todaka 1,2 , K. Sakurai 3 , H. Miyakawa 4 , M. Uzuki 4 , H. Osada 5 , Y. Ikezuki 6 , O. Tsutsumi 6,8 , T. Iguchi 7,8 , C. Mori 1,8 . 1 Department of Bioenvironmental Medicine, Graduate School of Medicine, Chiba University, 2 Center for Environment, Health and Field Sciences, Chiba University, Kashiwa, 3 Department of Clinical Cell Biology, Graduate School of Medicine, Chiba University, 4 SRL Inc., 5 Department of Obstetrics and Gynecology, Chiba University Hospital, 6 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7 Bioenvironmental Research, Center for Integrated Bioscience, Okazaki National Research Institutes, 8 CREST Recently, increasing number of people is accepting the vegetarian like food style in North America and European countries. Phytoestrogens are estrogen-like chemicals derived from plants such as soy and alpahlpha. Although vegetables are believed to be beneficial to human health, there is a concern if it is safe when human fetus is exposed to higher level of phytoestrogens. Therefore, we investigated the feto-maternal relationship between serum concentration levels of phytoestrogens. We collected 31 maternal and newborn cord serum and measured the concentration level of genistein, daidzein, equol and coumestrol. Serum phytoestrogen concentrations were determined by liquid chromatography/tandem-mass spectrometry. Informed consent from each mother was obtained. Equol is a metabolite of daidzein and is known more estrogenic than its original daidzein. It has been reported that there are people who produce equol and who do not produce equol. In our study, equol was detected in 13 cord serum samples and 11 maternal serum samples. The serum concentration level of equol was higher in maternal than in cord serum. Genistein and daidzein were detected from almost all of the maternal and the cord serum samples. These two phytoestrogens were detected at higher level in cord serum than in maternal serum. In conclusion, we found that 1) some phytoestrogens are detected at higher level in cord serum than maternal serum, and others show opposite pattern, and 2) there are equol producer group and equol non-producer group in fetuses. It will be necessary to investigate more about the effect of phytoestrogens on fetuses. (This work was supported by the fund for endocrine disrupters from the Ministry of the Environment, Japan) 667 OECD VALIDATION OF THE HERSHBERGER ASSAY IN JAPAN: PHASE 2-DOSE RESPONSE OF METHYLTESTOSTERONE, VINCLOZOLIN AND P,P’-DDE K. Yamasaki 1 , M. Sawaki 1 , R. Ohta 2 , H. Okuda 3 , S. Katayama 4 , T. Yamada 5 , T. Ohta 6 , T. Kosaka 7 , W. Owens 8 . 1 Chemicals Evaluation and Research Institute, Oita, Japan, 2 Food Drug Safety Center, Kanagawa, Japan, 3 Japan Bioassay Research Center, Kanagawa, Japan, 4 Mitsubishi Chemical Safety Institute, Ibaraki, Japan, 5 Sumitomo Chemical Company, LTD., Osaka, Japan, 6 Panapham Labotatories Co., Ltd., Kumamoto, Japan, 7 Institute of Environmental Toxicology, Ibaraki, Japan, 8 Environmental Health and Safety Division, OECD, Paris The OECD has initiated the development of new guidelines for the screening and testing of potential endocrine disrupters. The Hershberger assay is the assays selected for validation based on the need for in vivo screening to detect androgen agonists or antagonists by measuring the response of five sex accessory organs of castrated juvenile male rats. The Phase 1 feasibility demonstration stage of s179 the Hershberger validation program has been successfully completed with single androgen agonist and antagonist as reference substances. Phase 2 validation program as employs a range of additional androgen agonists and antagonists as well as 5α-reductase inhibitors. Japanese laboratories have contributed Phase 2 validation studies of the Hershberger assay using methyltestosterone, vinclozolin and p,p-DDE. The methyltestosterone doses were 0, 0.05, 0.5, 5 and 50 mg/kg/day and those of vinclozolin and p,p’-DDE were 0, 3, 10, 30 and 100 mg/kg/day. All chemicals were orally administered for 10 consecutive days. In the antagonist version of the assay using vinclozolin and p,p’-DDE, 0.2 mg/kg/day of testosterone propionate was coadministered by subcutaneous injection. All sex accessory organs consistently responded with significant changes in weight within narrow window. Therefore, the Japanese studies support the Hershberger assay as a reliable and reproducible screening assay for the detection of androgen agonistic and antagonistic effects. 668 EFFECTS OF 4-OH-CB 107 AND 4-OH-CB 187 ON DEVELOPMENT AND ENDOCRINE STATUS OF THE RAT A.C. Gutleb 1 , C.J.K. Buitenhuis 1 , P. Cenijn 1 , J. Legler 1 , H. Lilienthal 2 , Å. Bergman 3 , A. Brouwer 1 . 1 Institute for Environmental Studies (IVM) Vrije Universiteit Amsterdam, The Netherlands, 2 Dept. Neurobehavioral Toxicology Medical Institute of Environmental Hygiene at the Heinrich Heine University Duesseldorf Germany, 3 Department of Environmental Chemistry, Stockholm University, Sweden Possible health effects from low-level exposure to chemicals that may alter the endocrine status are an issue that has attracted much attention during the last decade. The major aim of this study was to determine possible developmental neurotoxic effects and long-term consequences on reproduction and endocrine status of rats resulting from exposure to hydroxylated metabolites of polychlorinated biphenyls. Pregnant Wistar WU rats were orally dosed from GD 10–16 with the major human plasma PCB metabolites 4-OH-CB 107 (0.1, 0.5 or 5 mg/kg), 4-OH-CB 187 (0.5 or 5 mg/kg) or Aroclor 1254 (25 mg/kg). Plasma levels of total thyroxine on PND 4 were significantly decreased in females from all exposed groups. Offspring were scored for onset of hair growth, pinna detachment, bilateral eye opening, vaginal opening and balanopreputial separation and no alterations of these developmental landmarks were observed. After vaginal opening, smears were taken daily at three different time points (PND 30–70, PND 150–170, PND 210–230) throughout the experimental period of 235 days. Estrous cycle length was prolonged from PND 210 to 230 in female offspring exposed to 5 mg/kg 4-OH-CB 107, indicating earlier reproductive senescence. Strikingly, plasma estradiol concentrations in females at PND 230 were significantly increased by 256% in the 5 mg/kg 4-OH-CB 107 group. Behavioral experiments using an open field paradigm (PND 94–105) revealed a 3-fold increase in the percentage of distance moved in females exposed to 4-OH-CB 187 compared to controls in the last 2.5 min of the measurement period of 10 min (P<0.05). This work has been supported by the EU (contract no. QLK4 CT2000–00261). 669 HERSHBERGER ASSAY: DOSE RESPONSE STUDIES ON TRENBOLONE AND VINCLOZOLINE F. Krötlinger, A. Freyberger. Bayer AG, PH PD P Toxicology, D-42096 Wuppertal, Germany Under the umbrella of OECD the Hershberger assay is being validated as an in vivo rat screening assay to detect compounds with affinity for the androgen receptor and 5α-reductase inhibitors. An important component of this activity is the establishment of dose response relationships for weakly active compounds. Herein we report on studies with trenbolone (TREN), an anabolic agent with only weak activity when orally administered, and with vinclozolin (VIN), a compound that weakly lowers androgen action following metabolic activation. TREN administered by oral intubation (0 – 0.3 – 1.5 – 8 – 40 mg/kg body weight (b.w.)) for ten days to castrated rats increased the relative weights of all androgen-sensitive tissues only at the high s180 Poster Session P34. Endocrine disrupters dose. Relative weights of ventral prostate (VP), seminal vesicles (SV), gland penis (GP), levator ani and bulbocavernosus muscles (LABC), and Cowper’s glands (CG) were statistically significantly increased 1.9-, 2.7-, 1.5-, 2.2-, and 2.1-fold compared to control values. Administration of the positive control testosterone propionate (TP, 0.4 mg/kg b.w.) by subcutaneous injection strongly and highly significantly increased the relative weight of VP, SV, GP, LABC, and CP 5.6-, 11.1-, 1.6-, 2.7-, and 4.9-fold. VIN (0 - 3 - 10 - 30 - 100 mg/kg b.w.), orally administered to TP-supplemented rats (0.4 mg/kg b.w. by subcutaneous injection) for ten days dose-dependently decreased the relative weights of all androgen-sensitive tissues. Statistically significant changes were obtained at 30 mg/kg b.w. and above. At the high dose, weight reduction to 48% (VP), 44% (SV), 85% (GP), 58% (LABC), and 68% (CG) of control values was observed. Flutamide (3 mg/kg b.w.), the positive control, orally administered to TP-supplemented rats, reduced the corresponding relative tissue weights even more strongly to 33% (VP), 28% (SV), 79% (GP), 51% (LABC), and 50% (CG) of control tissue weights. Our data suggest that the Hershberger assay is suitable to screen for compounds with affinity for the androgen receptor. Further testing also including methyltestosterone, p, p’-DDE, linuron, procymidone and finasteride in a world wide effort will provide comprehensive information on the properties of the Hershberger assay. This study was performed in collaboration with OECD and was sponsored by CEFIC-EMSG. 670 A COMMON MODE OF TOXICOLOGICAL ACTIONS IN ENDOCRINE DISRUPTERS B.M. Lee, S.M. Choi. Division of Toxicology, College of Pharmacy, Sungkyunkwan University, Chunchun-Dong 300, Suwon, Kyonggi-Do, South Korea A common mode of toxicological actions was investigated for the 48 endocrine disrupting chemicals (EDCs) classified by Centers for Disease Control and Prevention (CDC). Based on a literature survey, 24 EDCs out of 48 were shown to be positive and 3 EDCs were negative, in terms of generating free radicals, and for twenty one, information was not available (NA) as to free radical generation. Therefore, generation of free radicals (measured by malondialdehyde, MDA) was examined for the remaining 21 EDCs, not reported in the literature and 3 negatives as to free radical generation. Mice were i.p. treated with each of the total 23 EDCs(except one, which is commercially unavailable) at various doses and MDA was measured in the liver. Twenty out of 23 EDCs significantly increased MDA production (p<0.05) and only three EDCs were shown to be non-peroxidative. Therefore, a total of 44 out of the 47 EDCs (94%) reported or tested was determined to be positive with respect to free radical generation. Our results indicate that 94% of the 48 EDCs generate free radicals and this feature may be useful for the investigation of a common mechanism of EDCs, method development for the screening of EDCs, and a chemoprevention strategy. (This work was supported by the brain Korea 21 project 2003 and a grant from NITR/Korea FDA.) 671 PROCYMIDONE: CHARACTERISATION OF ESTROGEN-LIKE ACTIVITY IN THE MCF-7 HUMAN BREAST CANCER CELL LINE E. Chiesara, R. Fumagalli, M. Ferraris, S. Frigerio, L. Marabini, S. Radice. Department of Pharmacology, Chemotherapy and Medical Toxicology “E. Trabucchi”, University of Milan, Via Vanvitelli 32, 20129 Milano, Italy Procymidone, a dicarboximide fungicide, is known to modify sexual differentiation in vivo and in vitro, and has been shown to induce vitellogenin synthesis in primary cultured rainbow trout hepatocytes. The aim of this study was to evaluate the estrogen-like activity of procymidone in the MCF-7 (ER+) human breast cancer cell line, using 17β-estradiol 1nM as the positive control. The estrogenic effects were assessed using a number of specific endpoints, including the mitogenic time-dependent effect measured by means of a pro- liferation assay (MTT), which can also discriminate cytotoxic and non-cytotoxic concentrations; a quantitative assay (ELISA) to measure the estrogen-regulated secretorial protein pS2; and microscopy to evaluate the development of foci which are preneoplastic cell aggregates superimposed on a monolayer background that appear in post-confluent cultures and are also characteristic of malignant transformation. Our data show that procymidone 100 µM stimulates constant cell proliferation up to the ninth day, which peaks between day 10 and day 13, and subsequently returns to baseline levels. The results clearly confirm the potent foci promoting action of procymidone 100 µM, which also induces the pS2 content of MCF-7. We conclude that procymidone has estrogen-like activity in this human cell model by means of a still unknown mechanism of action through ligand-dependent or ligand-independent activation of the ER pathway. 672 ESTABLISHMENT OF CALUX BIOASSAY SYSTEM FOR ESTROGENS AND DIOXINS Y.Y. Sheen 1 , K.E. Joung 1 , K.N. Min 1 , J.Y. Kim 1 , K. Gu 2 , S. Paik 2 , S. Hong 3 , S. Kang 3 , H. Kim 3 , S. Cho 3 . 1 College of Pharmacy, Ewha Womans University, Seoul 120–750, Korea, 2 College of Medicine Hanyang University, Seoul, 133–792, Korea 3 College of Medicine Seoul National University, Seoul, 110–744, Korea In order to establish the rapid and easy-to-perform methods using ERE-MCF-7 cells by luciferase assay. MCF-7 stable cells which are stably transfected with phERE-Luc were treated with many chemicals and then luciferase activity were determined. Estradiol (E2) and synthetic estrogen, diethylstylbesterol (DES) were induced luciferase activity in dose dependent manner ranging 20–30 folds over that of control, and their activities were blocked by Tam treatment. 29 Flavonoids and 5 curcumin derivatives were tested in this system. Their E2 equivalent concentrations (EEQs) were calculated as a concentration of E2 that resulted in the same luciferase reading of test compound from the dose response curve. And also of Kumho river of Korea showed 0.77 pM EEQ in upstrean and 7.7pM EEQ in downstream. Kum River of Korea showed 3.5pM and 1.7pM EEQ in upstream and downstream respectively. Mankyung River of Korea showed 61fM and 0.41 pM EEQ in upstream and downstream respectively. Miho Stream of Korea showed 0.2pM EEQ only in the upstream. In this study, we attempted to identify the possible association between dioxin like compounds (such as TCDD, PCDDs, PCDFs, and PCBs) and the occurrence and severity of endometriosis using CALUX (Chemically Activated LUciferase eXpression) bioassay method. We analyzed the serum levels of dioxin like compounds in the endometriosis patients (n=46) and control patients with similar symptoms (n=14). Among them, adipose tissues of 10 cases were analyzed by high resolution GC/MS for validation of CALUX bioassay. The CALUX TEQs significantly correlated with the total TEQs determine by GC/MS (r2 = 0.96). So we demonstrated that CALUX bioassay is a rapid, sensitive and quantitative assay for biomonitoring of dioxin like compounds from small volume of blood. This study showed statistically significant association between exposure to dioxin like compounds and the occurrence of endometriosis (p < 0.003). The mean TEQ of control patient was 0.144 µg TEQ/L and the mean TEQ of endometriosis patient was 0.321 µg TEQ/L. After adjusting confounding factor, we found that the higher stage of the endometriosis, the higher level of CALUX TEQ. The TEQs of endometriosis I, II, III, and IV was 0.213 µg TEQ/L, 0.284 µg TEQ/L, 0.352 µg TEQ/L, and 0.450 µg TEQ/L, respectively. (Supported by grant from KFDA of Korea) 673 EXPRESSION PATTERN AND PROMOTER ANALYSIS OF MEDAKA CHORIOGENIN AS A BIOMARKER OF ENVIRONMENTAL ESTROGEN Chulwoo Lee, Eung-Roh Park, Jin-Gyun Na, Deok-Gil Rhee, Moon-Soon Lee. Environmental Risk Research Dept., National Institute of Environmental Research (NIER), Incheon, KOREA In teleost fish, medaka (Oryzias latipes), the inner layer of egg envelope comprises two groups of subunits, designated ZI-1,2 Poster Session P34. Endocrine disrupters and ZI-3. The precursors of ZI-1,2 and ZI-3 have been named choriogenin H and choriogenin L, respectively. They are synthesized in the liver in response to estrogen, and then released into the blood stream and incorporated into the zona radiata in sexually matured female medaka. However, choriogenin is also induced in male medaka when the fish are exposed to estrogenic chemicals. In this study, full sequences of choriogenin H and L genomic DNA were identified, and measurement of choriogenin mRNA induction by estrogenic compounds was established in medaka by use of RT-PCR technique. The induction of choriogenin subunits expression by estrogenic chemicals showed a dose-dependent pattern and choriogenin L was found to be more sensitive than choriogenin H. In order to characterize the regulatory elements of choriogenin gene, we cloned and sequenced choriogenin upstream region. The estrogen receptor is a transcription factor that binds to a specific DNA sequence found in the transcriptional regulatory cis elements of estrogen-responsive genes, called estrogen responsive elements (EREs). Choriogenin was also found to have conserved sequences of ERE regions. The regulatory region of choriogenin L contains one perfect ERE palindromes (13-nucleotide inverted repeats) and two half EREs, while upstream of choriogenin H contains one imperfect ERE palindromes and two half EREs. 674 VITELLOGENIN INDUCTION BY SYNTHETIC ESTROGEN IN WILD MEDKA IN KOREA Sung-Hwan Jeon, Chulwoo Lee, Hyun-Seok Bae, Jisung Ryu, Jin-Gyun Na, Moon-Soon Lee. Environmental Risk Research Dept., National Institute of Environmental Research (NIER), Incheon, KOREA Vitellogenin (VTG), the precursor of yolk protein is directly regulated by estrogen and VTG induction is known to be a valuable biomarker for assessing exposure to estrogenic chemicals in fish. Since estrogens are the only significant stimulus for the hepatic vitellogenesis, the presence of VTG in male liver or plasma is indicative of exposure to estrogenic chemicals. There are two species of wild medaka in Korea. One is called Chinese medaka (Oryzias sinensis) which inhabits west area of Korean peninsula, while the other is called medaka which inhabits east area and has been known to be identical with Japanese medaka (Oryzias latipes). In this study ELISA (enzyme-linked immunosorbent assay) was performed to quantify VTG in wild medaka (Oryzias sinensis). We investigated the antigenic cross-reactivity and compared the VTG induction levels of Oryzias sinensis with those of Oryzias latipes (Orange-red type) using the VTG monoclonal antibody of Oryzias latipes. Both species of medaka were exposed to 17alpha-ethinylestradiol(EE2) on a same condition ranging from 25ng/L to 100ng/L for 7 days. After exposure of males to EE2, VTG expression was increased in plasma as well as in liver in a dose-depended manner. Although the monoclonal antibody was prepared against the VTG of Oryzias latipes, VTG levels in the EE2-treated Oryzias sinensis appeared to be higher than those in the EE2-treated Oryzias latipes. These results show that the cross-reactivity between these two species is remarkably high. In addition, these data suggest that wild medaka could be a sensitive and suitable model organism for vitellogenin screening of endocrine disrupting chemicals. 675 FORMATION OF ESTROGENIC METABOLITES FROM POLYCYCLIC AROMATIC HYDROCARBONS AND HALOGENATED BIPHENYLS BY CYTOCHROME P450 ACTIVITY AND DEVELOPMENT OF A PREDICTIVE COMPUTATIONAL MODEL FOR BINDING TO THE ESTROGEN RECEPTOR John H.N. Meerman, Marola M.H. van Lipzig, A.M. ter Laak, Mirjam Wamelink, Daan Geerke, Nico P.E. Vermeulen, Aldo Jongejan. Leiden/Amsterdam Center for Drug Research (LACDR), Division of Molecular Toxicology, Vrije Universiteit Amsterdam Hydroxylated metabolites of polycyclic aromatic hydrocarbons (PAH), polychloro- and polybromobiphenyls (PCBs and PBBs) structurally resemble the endogenous estrogen 17β-estradiol (E2) s181 and may act in the same manner as hormonal estrogens by binding to the estrogen receptor (ER). We found that bio-activation by rat liver microsomes with induced P4501A1 and P4501A2 activity, yields estrogenic metabolites from benzo[a]pyrene (BAP), chrysene (CHN), 2,2’-dichloro-, 4,4’-dichloro-, 2,2’-dibromo and 4,4’-dibromobiphenyl. Various mono-hydroxylated metabolites of BAP, CHN and several PBBs showed affinity for the ER (EC50 10–500 nM), induced proliferation in T47D cells and were active in a reporter gene-assay (ER-CALUX). Surprisingly, some hydroxylated PAHs induced the number of ER in mammalian cells which led to much higher maximal estrogenic effects compared to E2. The combined estrogenic effect of hydroxylated PAHs was also investigated and proved to be additive. Several hydroxylated metabolites of PBBs also inhibited estrogen sulfotransferase in the nanomolar range. Furthermore, we developed a molecular model to predict binding affinities for unknown compounds based on the crystal structure of the ERα. For the validation of our model we used known estrogens, such as E2, DES, estriol, genisteine and hydroxylated PAHs. Molecular Dynamic (MD) simulations were performed of the ER with ligands in explicit water using the AMBER 6.0 force field. Binding affinities were analysed using the linear interaction energy (LIE) approximation. We found an excellent correlation (R2 = 0.93) between calculated and experimentally determined binding affinities. Subsequently, the model was used to predict the binding affinity of ten structurally very diverse compounds such as kepone, apigenin, and daidzeine and the predicted values were all in very good agreement with experimental values. 676 SUITABILITY OF USING THE CHURB (LEUCISCUS CEPHALUS) AS A BIOINDICATOR OF SURFACE WATER POLLUTION OF XENOESTROGENIC CHEMICALS V. Žlábek, J. Kolářová, Z. Svobodová. University of South Bohemia, Research Institute of Fish Culture and Hydrobiology, Vodnany, Czech Republic, 2 University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic Induction of vitellogenin (Vtg) into male fish is an important biomarker for studying endocrine disrupting chemicals (EDCs) with estrogenic effects in surface water ecosystem. Within Czech monitoring program the salmonids (Salmonidae) were used for measuring Vtg biomarker. However salmonids are not naturally present in all localities used in monitoring. A bioindicator fish of aquatic pollution in Czech republic is chub (Leuciscus cephalus), which is a member of the cyprinid family (Cyprinidae). The aim of the study was to prove possibility of vitellogenin determination in blood plasma of chub (L. cephalus) using ELISA (Carp vitellogenin EIA kit, Biosense Laboratories, Norway) and to monitor chosen localities. Sampling of trout (Salmo trutta) and chub (L. cephalus) blood was made on the same locality (upper reach of Blanice river – Czech Republic). Both sexes were included. The vitellogenin concentration was measured in the trout blood plasma using a specific ELISA (Salmonidae) test. Based on negative results from testing on trout males the locality was considered to be unpolluted with xenoestrogens. Nonspecific ELISA (Carp) test was used for measuring vitellogenin concentration in the chub blood plasma. ELISA estimation of female blood plasma proved positive reaction of chub vitllogenin antigen with kit antibody, which was originally dedicated for carp (Cyprinus carpio) and some other cyprinids. In the chub male blood plasma vitellogenin was not proved. Our results confirmed that there were both positive and negative reactions of chub blood plasma. Chub (Leuciscus cephalus) is the next carp family fish suitable for vitellogenin concentration measuring using ELISA test. Measuring vitellogenin concentration in blood plasma of chub (L. cephalus) within monitored localities of surface water creates a new biomarker for evaluation of xenoestrogenic chemicals pollution. s182 Poster Session P35. Environmental pollutants P35 Environmental pollutants 677 MORPHOLOGICAL AND FUNCTIONAL ALTERATIONS INDUCED BY FINE ENVIRONMENTAL PARTICULATE IN CULTURED LUNG EPITHELIAL CELLS S. Meschini, A. Calcabrini, M. Marra, L. Falzano, M. Colone, B. De Berardis, L. Paoletti, G. Arancia, C. Fiorentini. Laboratory of Ultrastructures, Istituto Superiore di Sanità, Viale Regina Elena 299,00161 Rome, Italy Inhalable particulate matter (PM) constitutes an important component of air pollution deriving from the combustion of fossil fuels. Exposure to PM has been associated to an increased lung cancer risk, asthma and chronic disease in predisposed individual. This association seems to be more closely related to fine particles (particulate matter with aerodynamic diameter less than 2.5 mm – PM2.5). PM consists of an inert carbonaceous core and multiple layers of adsorbed pollutant molecules. After inhalation and deposition in the lung, the adsorbed pollutants are first released into pulmonary surfactant and then reach the pulmonary epithelial cells of airways and alveoli. In this study, we have investigated the biological effects of PM2.5 on the human lung epithelial cell line A549. Morphological analysis of cell surface and ultrastructural observations performed by electron microscopy showed that fine particles interacted with the cell surface, inducing evident alterations and, subsequently, being internalized into the cytoplasm. The architecture of the cytoskeletal components, in particular microfilaments and microtubules, was perturbed in a dose-dependent manner after PM2.5 treatment. These observations can suggest a possible involvement of the cytoskeletal components in the internalization and subsequent movement inside the cells of the particulate. Moreover, the challenge with PM2.5 provoked a slow down of the cell growth, a significant increase of cytosolic reactive oxygen species (ROS) content and the production of pro-inflammatory cytokines. In particular, exposure to PM2.5 promoted a dose- and time-dependent release of TNF-α and IL-6 in the culture medium. These data, besides confirming the induction of the inflammatory response in lung cells exposed to the inhalable particles, demonstrate that the onset of cellular changes are possibly related to the cytotoxic action exerted by the fine particulate PM2.5. This underlines the role of morphological and functional alterations in the cytotoxic action induced by inhalable particles. 678 DNA ADDUCT AND TUMOUR FORMATION IN RATS BY 3-NITROBENZANTHRONE (3-NBA); A POWERFUL MUTAGEN FOUND IN URBAN AIR. E. Nagy 1 , S. Adachi 2 , L. Möller 1 . 1 Karolinska Institutet, Dept. of Biosciences at Novum, Analytical Toxicology Unit, Stockholm, Sweden, 2 Sagami Women’s University, Dept. of Public Health, Sagamihara, Japan 3-nitrobenzanthrone (3-NBA) was isolated from diesel exhaust and airborne particles and characterised as a strong bacterial mutagen and an inducer of micronuclei in mouse peripheral reticulocytes. Additional in vitro studies have shown that one of the metabolites of 3-NBA causes mutations such as base substitutions, deletions and insertions in treated cell lines. Mutations generally occurred at sites where DNA adducts had been formed. In an in vivo study involving oral treatment of rats with 3-NBA, high levels of DNA adducts were detected in GI-tract (highest in the small intestine) and the most prominent DNA adducts were those associated with the 2’-deoxyguanosine. The study presented here was designed to evaluate the in vivo genotoxicity and tumour development owing to exposure to 3-NBA, corresponding to inhalation with a single instillation. Our findings show that DNA adducts, detected by 32 P-HPLC, are indeed formed with the highest level of adducts in the lung (main target of exposure), followed by the kidney and the liver. Six hours after exposure the DNA adducts are clearly detectable and at two days post administration the adduct levels had reached a maximum. The levels of DNA adducts were at this time on average 240, 180 and 35 DNA adduct/108 normal nucleotides in the lung, kidney and liver respectively. Histopathological examination verifies that 3-NBA induces tumours and severe damage to the exposed tissues, involving for instance hemorrhage, squamous metaplasia, squamous cell carcinomas and adenocarcinomas. This is of concern since 3-NBA is found in urban air and industrially used BA can potentially be nitrated to form 3-NBA. 679 DIFFERENTIAL DNA DAMAGE PRODUCED BY METALS FROM URBAN AIR PARTICLES (PM10 ) FROM DIFFERENT ZONES OF MEXICO CITY C.M. García-Cuellar 1 , F. Martínez-Romero 1 , Y. Sánchez-Pérez 1 , V. Calva-Treviño 1 , E. Alfaro-Moreno 1 , V. Torres-Flores 2 , I. Rosas-Pérez 3 , A.R. Osornio-Vargas 1 . 1 Instituto Nacional de Cancerología, 2 Facultad de Medicina and 3 Centro de Ciencias de la Atmósfera, UNAM. Mexico City. Mexico The DNA damage associated to urban air particulate matter (PM) components may play an important role in human disease including cancer. This damage is suggested to be importantly induced by reactive oxygen species generated by hydrogen peroxide (H2 O2 ) an inductor of hydroxyl radicals in the presence of transition metals ions. We have evidence that DNA damage is produced by Mexico City’s (MC) PM10. Particles collected in the North (industrial), Center (heavy traffic) and South (forest-urban) of the City induced DNA damage in 3T3 cells in a dose-related manner between 5–40 µg/ml (comet assay). North and Center particles had the strongest potency. To explore if the DNA damage can be induced by an oxidative mechanism, we utilize an in vitro assay exposing isolated DNA to different concentration of PMs (5, 10, 20, 40, 80 and 160 µg/ml) from North, Center and South of MC in the presence of 1 mM H2 O2 , for 1, 6, 12 and 24 h. DNA degradation was evaluated by electrophoresis. To identify if the metals of the particles have any participation in the DNA degradation, 1 mM of deferoxamine was used. We found a differential DNA degradation at 24 h. The potency to induce this damage was N<C<S. Ten µg/ml of particles from North induced a total DNA degradation, Center showed the same DNA degradation potency at 80 µg/ml and South particles at 160 µg/ml. When we use deferoxamine, the DNA degradation did not occur. Our conclusion is that the metal content of the particles is playing an important role in the production of DNA damage by an oxidative mechanism. The different metal content of the particles linked to emission source could explain the differences observed between regions. 680 BIOLOGICAL AND CHEMICAL EVALUATION OF TYRE DEBRIS ORGANIC EXTRACT M. Camatini 1 , V. Calini 1 , M. Gualtieri 1 . 1 Department of Environmental Science, University Milano-Bicocca Milan, Italy Tyre debris (TD) is one of airborne particulate matter (PM10) constituents and participates to PM10 composition up to 8–10%. PM10 correlation with increased respiratory diseases is well established, but studies on the biological toxicity of its fractions are mostly limited to diesel exhaust particles. We evaluated the biological response induced by TD organic extract on lung A549 cells. The organic fraction of TD, coming from laboratory tests, was extracted using dichloromethane in a Soxhlet equipment for 4 hours, dried and redissolved in DMSO. All biological tests were performed adding to the culture medium appropriate volumes of sterile particulate extract at increasing concentration (5 - 100 µg/ml). Cell viability was assessed with MTT on cells incubated with TD extract for 24, 48 and 72 hours. 100 µg/ml of extract, corresponding to 20 m3 of inhaled air, greatly reduced cell viability already after 24 hours treatment, while lower doses showed their cytotoxic effect after 48 hours. A clear dose dependent decrease in cell viability could be found after 72 hours. Cell proliferation showed a similar response. At non cytotoxic doses, the alkaline comet assay (V. Calini et al. Cell Biol Toxicol, 18, 369–379, 2002.) was performed to evaluate the genotoxicity of the extract after 24 hours treatment. A dose dependent increase in the percentage of damaged cells was found. Cell cycle investigations showed an increase in the percentage of cells in G0/G1 phase and a reduction of cells in S phase after 24 hours incubation. Poster Session P35. Environmental pollutants Preliminary investigations on the chemical characterization of tyre debris organic extract were carried out (M. Camatini et al. Materials characterization, 46, 271–283, 2001.). FT-IR analysis showed the presence of the polyisoprene polymer. TLC analysis excluded the presence of aromatic compounds. 681 PM10 EFFECTS ON PRO-INFLAMMATORY GENE EXPRESSION IN LUNG EPITHELIAL CELLS Ernesto Alfaro-Moreno 1 , Yee M. Heng 2 , Alvaro Osornio-Vargas 1 , Irma Rosas 3 , J. Clifford Murray 2 . 1 Subdirección de Investigación Básica, Instituto Nacional de Cancerología, México, 2 Department OF Clinical Oncology, University of Nottingham, Nottingham UK, 3 Centro de Ciencias de la Atmósfera, UNAM, México PM10 exposure is associated with a variety of biological effects. The main target cells of PM10 are thought to be epithelial cells of the lung. Our previous data suggest that exposure of endothelial cells to PM10 leads to the up-regulation of E-Selectin, and ultimately apoptosis. The aim of the present study was to evaluate the effect of PM10 particles form Mexico City at a concentration of 10 µg/cm2 . Total RNA was extracted from the cells and reversed transcribed using oligo-dT primers in the presence of 32P-CTP. The labelled single-stranded cDNA was hybridised to a commercially available macro-array of cDNAs corresponding to 112 genes associated with signal transduction pathways (Superarray). Comparison of the array pattern generated by PM10 –exposed cells with that of unexposed cells showed that PM10 induce the up-regulation of GM-CSF, leptin and E-Selectin, three genes associated with a proinflammatory state. A fourth up-regulated gene, Stra6 encodes a hypothetical protein thought to be linked to the retinoic acid signalling pathway. Our data show that PM10 induce activation of gene transcription in lung epithelial cells. Furthermore the genes activated are consistent with the generation of a phenotype associated with a pro-inflammatory state. 682 1 H-NMR STUDIES REVEAL THAT ENVIRONMENTAL FINE PARTICULATE MATTER (PM 2.5) AT VERY LOW CONCENTRATIONS ACTIVATES THE RAW 264.7 MACROPHAGE CELL LINE M.T. Santini 1,2 , G. Rainaldi 1,2 , A. Ferrante 1 , R. Romano 2,3 , S. Clemente 3 , A. Motta 4 , B. De Berardis 1 , M. Balduzzi 5 , L. Paoletti 1 , P.L. Indovina 2,3 . 1 Laboratorio di Ultrastrutture, Istituto Superiore di Sanità, Rome, Italy, 2 Istituto Nazionale per la Fisica della Materia, Unità di Napoli, Complesso Universitario Monte S. Angelo, Naples, Italy, 3 Dipartimento di Scienze Fisiche, Università di Napoli “Federico II”, Complesso Universitario Monte S. Angelo, Naples, Italy, 4 Istituto di Chimica Biomolecolare del CNR, Pozzuoli, Naples, Italy, 5 UTS Biotecnologie, Protezione dell’Ambiente e degli Ecosistemi, ENEA, Rome, Italy Because of the direct association between inhalation of airborne particulate matter (PM) and human respiratory and cardiovascular disease, it is necessary to better comprehend the complex toxicological mechanisms at the basis of tissue damage induced by these particles. One of the cell types principally involved in the body’s reaction to PM are macrophages which remove particles through phagocytosis. Since 1 H-NMR is extremely useful in monitoring, non-invasively, macrophage metabolism and since this technique has never been utilized to examine macrophage activation after exposure to PM, it was the purpose of the present study to investigate the effects of PM exposure on the RAW 264.7 stabilized macrophage cell line using 1 H-NMR spectroscopy. Particulate matter with diameter <2.5 µm (PM 2.5) was utilized because a closer association to mortality and adverse respiratory health effects has been found with this fraction than with particles of larger size. RAW 264.7 cells were exposed to three different concentrations of PM 2.5: 1 µg/ml, 0.1 µg/ml and 0.01 µg/ml and 1 H-NMR measurements were conducted on whole cells at both 500 MHz and 700 MHz as well as on perchloric acid extracts at 700 Mz. Significant variations in glutamate, inositol, taurine, choline-containing metabolites, creatine and phosphocreatine, alanine, lactate and CH2 and CH3 lipids were seen at very low concentrations of PM 2.5. Many of these changes s183 point to activation of RAW 264.7 macrophages even at doses of PM 2.5 much lower than those commonly employed in cell studies. These results are particularly significant since the same concentrations of particulate matter did not induce changes in morphology as observed by scanning electron microscopy and release of cytokines (i.e. IL-6 and TNF-α) in these cells. Therefore, 1 H-NMR spectroscopy is an extremely sensitive probe in observing, non-invasively, even the most subtle variations in macrophages after exposure to very low doses of PM 2.5. 683 ASBESTOS EXPOSURE IN GENERAL POPULATION: PRELIMINARY RESULTS FROM LIGHT, SCANNING AND TRANSMISSION ELECTRON MICROSCOPY EXAMINATIONS I. Faustinelli 1 , F. Bortolotti 2 , M. Rossetti 3 , S. Pasquetto 1 , P. Cristofori 1 , A. Lanzoni 1 , F. Tagliaro 2 . 1 Safety Assessment Dept. Histopathology Unit Research Center GlaxoSmithKline Verona Italy, 2 Dept. of Medicine and Public Health – Unit of Forensic Medicine, University of Verona, Verona Italy, 3 Pharmaceutical Development GlaxoSmithKline Verona Italy Microscopic techniques for analysis of asbestos fibres in lung tissue have provided major information in the understanding of asbestos-related diseases. Electron microscopy and elementary Xray microanalysis are diagnostic tools increasingly applied to clinical practice and medico-legal problems. The present work is intended to evaluate the ability of scanning and transmission electron microscopy to assess the low-level asbestos dust exposure in the general population under “normal” environmental pollution. To this aim the lungs of 8 subjects (aged from 5 months to 77 years) without evidence of occupational exposure were examined. Samples of lung tissue examined were taken from necropsy material of subjects who died from different causes. No pathology suggestive of asbestos exposure were recorded in the subject considered. Tissue samples stored in 10% neutral formalin were processed for light, scanning or transmission electron microscopy. For light microscopy examination samples were processed in paraffin wax, sectioned and stained with haematoxylin and eosin in order to identify histopathological changes characteristic for the presence of asbestos fibres (e.g. ferruginous bodies, fibrosis, alveolar oedema, haemorrhages etc.). At the scanning electron microscopy examination the count of asbestos fibres per gram of dry-weight was performed after chemical tissue digestion by nitric acid (14M). The characterisation of different asbestos fibre types was performed using transmission electron microscopy on ultrathin (80nm) sections processed/embedded into EPON 812 resin. Lung samples from a patient dead for mesothelioma/asbestosis related to heavy professional exposure were used as positive control. 684 HEAVY METALS IN ARTERY OF PATIENT LIVING INDUSTRIAL REGION ON SOUTH POLAND. E. Nogaj 1 , J. Kwapuliński 1 , P. Nogaj 2 , M. Olejczyk 3 . 1 Department of Toxicology Silesian Universty of Medicine in Katowice, 41–200 Sosnowiec, Jagiellońska Street 4, (+48) 32 2925541, 2 Department of Molecular Biology, Biochemistry and Biofarmacy, Silesian University of Medicine in Katowice, 41–200 Sosnowiec Narcyzów Street 1, (+48) 32 2914393; 3 Department Artery Surgery St. Barbara Hospital, Sosnowiec Recently, the development of investigation of the possibility of utilization of different biological tests as bioindicators of metals, present adequately to the exposition on a given ground, has been observed. The content of Cd, Pb, Cu, Co, Cr, Mn, Ni, Fe and Zn has been determined by the atomic-absorption spectrophotometry. The investigation showed the usefullness of femoral arteries as Cd, Pb, Cu, Co, Cr, Mn, Ni, Fe, Zn bioindicators. Moreover, the values relating to both 10, 95 percentile and geometrical mean can be used in future, as the reference values of the occurrence of metals in femoral arteries of people living in the industrial areas, which are equals (geometric mean): 12.9 µgCd/g, 20.4 µgMn/g, 22.0 µgCr/g, 36.5 µgNi/g, 52.2 µgCo/g, 53.8 µgCu/g, 63.9 µgPb/g, 686.2 µgFe/g, 1225.0 µgZn/g. s184 Poster Session P35. Environmental pollutants Abstract 684 – Table: Comparison of quotient of the geometrical mean content metals in femoral arteries of persons of studied population, in relation to the geometrical mean content of metals for the smallest content Cd and Pb Metals Group of reference Geometric mean Studied population Cd Co Cr Cu Fe Mn Ni Pb Zn 5.45 12.93 30.68 52.22 7.80 22.02 37.38 53.83 744.54 686.24 15.39 20.42 14.02 36.53 13.65 63.87 899.42 1224.96 The cross-correlation analysis revealed the characteristic correlation between the metals tested in the femoral arteries of the inhabitants of a given area. This influence of the factors tested (age, sex addiction to smoking) has been observed. On the heavy metals contents in femoral artery influence age, sex and addiction to smoking (Fig. 1–4). 685 HEALTH IMPACTS AMONG CHILDREN LIVING IN THE VICINITY OF A PETROCHEMICAL PLANT Karolina Lyubomirova. Department of Toxicology, National center of hygiene, medical ecology and nutrition, Sofia, Bulgaria An epidemiological study was conducted in the vicinity of a petrochemical plant. Air sampling in the vicinity of the plant showed pollution mainly with benzene in concentrations permanently exceeding 3–5 times TLV. Children (1–15 years old) were selected for the investigations – 713 lived in a downwind village from the plant. The rest (633) were from control villages. ISAAC questionnaire was filled in for all the children. The results showed higher prevalence (3 to 7 times) of complains connected with respiratory diseases (wheezing, asthma, night cough) among the exposed children. Skin testing with 7 common allergens showed higher prevalence of positive reaction to house dust and feather among exposed children. Spirometry performed to the same groups of children registered decreased respiratory indices FVC (42% of exposed) and FEV1 (32% of exposed) in comparison with 20% of controls with decreased FVC and 16% - with decreased FEV1. Blood counting registered leukocytosis among 22 exposed children (12,5%) in comparison with 5 controls (4,3%). Increased eosinophil account was checked among 15 of exposed (12,5%) and 8 (6,7%) controls. Determination of the serum concentration of tIgE showed the mean group value of tIgE is three times higher among the exposed. Personal analysis registered 28 exposed children (26,2%) with abnormal IgE concentration in comparison with 10 controls (10,4%). Measurement of serum concentration of Clara cells protein (in a collaboration with Prof.A.Bernard, Belgium) showed decreased values among 23 exposed children (20%) and 12 controls (9,5%). The results obtained allow suggesting a hypothesis for the potential of p-benzoquinone and hydroquinone. These metabolites of benzene could be formed in Clara cells rich of cyt P450 and affect the lung tissue. It could explain the registered increased bronchi sentisitivity to common aeroallergens and the obstruction of terminal bronchi, the decreased of serum CC16 concentration and its anti-inflammatory and protective effects. 686 A 50 HZ SINUSOIDAL MAGNETIC FIELD AFFECTS PRINCIPALLY CELL ADHESION MOLECULES (CAM’s) IN MG-63 AND SAOS-2 OSTEOSARCOMA CELL LINES M.T. Santini 1,2 , G. Rainaldi 1,2 , A. Ferrante 1 , P.L. Indovina 2,3 , P. Vecchia 4 , G. Donelli 1 . 1 Laboratorio di Ultrastrutture, Istituto Superiore di Sanità, Rome, Italy, 2 Istituto Nazionale per la Fisica della Materia, Unità di Napoli, Naples, Italy, 3 Dipartimento di Scienze Fisiche, Università di Napoli “Federico II”, Naples, Italy, 4 Laboratorio di Fisica, Istituto Superiore di Sanità, Rome, Italy Cancer cell proliferation, apoptosis, invasion and metastasis are complex phenomena in which cell adhesion molecules (CAM’s) play a pivotal role. CAM’s and their receptors mediate cell-cell and cell-matrix interactions, and also have a fundamental role in tumor growth, death, metastasis and invasion. The CAM’s principally involved in these processes are those directed against important components of the extracellular matrix (ECM) such as fibronectin, collagen, laminin, hyaluronan, heparan sulfate and elastin. While several epidemiological studies have demonstrated that extremely low frequency (ELF) sinusoidal 50–60 Hz magnetic fields of the strengths usually present in the environment due to the production and transport of electricity may have adverse effects on human health, particularly in promoting cancer, many other reports have excluded this possibility. In vitro studies using ELF fields have attempted to resolve this debate, but the data that have emerged have left many aspects still unanswered. Thus, it is apparent that further studies examining the role of ELF fields in cancer are necessary. In the present study, the effects of a sinusoidal 50 Hz magnetic field with a magnetic flux density of 0.5 mT can in the expression of CAMs in two human osteosarcoma cell lines (MG-63 and Saos2) was investigated. In particular, the expression of the VLA-2 (collagen receptor) and VLA-5 (fibronectin receptor) integrins, as well as CD44 were examined in both cell lines after these had been exposed for 7 and 14 days to a 50 Hz/0.5 mT field. Cell surface morphology (scanning electron microscopy), cell growth characteristics (growth curves and cell cycle phase distribution) and cell death (necrosis and apoptosis) were also examined. The results demonstrate that no variations in surface morphology and cell death occurred between control and exposed cells in both MG-63 and Saos-2 cells while significant changes were noted in cell growth and fibronectin and CD44 expression in MG-63 cells. 687 ANTIPROLIFERATIVE EFFECTS OF DI(2-ETHYLHEXYL) PHTHALATE (DEHP) METABOLITES 4-HEPTANONE AND 2-ETHYLHEXANOL H.G. Wahl 1,2 , P.C. Dartsch 3,4 , H.M. Liebich 2 . 1 Klinikum der Philipps-Universität Marburg, Department of Clinical Chemistry and Molecular Diagnostics, 2,4 Universitätsklinikum Tübingen, 2 Department of Clinical Chemistry, 4 Department of Occupational and Social Medicine, 3 Dartsch Scientific GmbH, Horb a.N., Germany There is great concern about the toxicity of the plasticizer di(2ethylhexyl) phthalate and its metabolites especially for risk groups such as patients on hemodialysis, critical ill patients and newborns, where DEHP and metabolites were detected in plasma and urine. Some of the proposed and shown effects in animals are carcinogenity, peroxisome proliferation, mutagenic activity, infertility and changes in lipid metabolism. Next to DEHP different metabolites, mainly its monoester MEHP, have been blamed for the toxicity. We found elevated levels of 2-ethylhexanol, 4-heptanone and 2-heptanone in breath, urine and serum from patients on hemodialysis and from patients in an intensive care unit. In a study with 10 healthy persons we recently could prove 4-heptanone to be a secondary DEHP metabolite arising from 2-ethylhexanol. Toxicity of 2-ethylhexanol and 4-heptanone was checked using adherent human cell lines of kidney (A-498) and liver (Chang liver cells). For this purpose, 5000 cells/cm2 growth area were seeded into 12-well plates and allowed to attach and spread for 24 hours. Thereafter, cells were incubated for another 24 h with 4-heptanone, 2-ethylhexanol and a mixture of both (1:1,v/v) at concentrations ranging from 0 to 500 µg/l for each compound. Cell viability was checked by counting the number of viable cells, examination of mitochondrial enzymatic activity by hydrolysis of 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT test) and lysosomal uptake of neutral red (NR test). The results clearly demonstrate a dose-dependent reduction (p<0.01) of viable and metabolically active cells within 24 hours of simultaneous incubation with equal amounts of 4-heptanone and Poster Session P35. Environmental pollutants 2-ethylhexanol (n=7). The concentration causing a 50% loss in cell viability and metabolic activity was calculated to be in the range between 300 and 500 µg/l. These concentrations have been detected in patients on hemodialysis and others exposed to DEHP. The individual metabolite testing of 2-ethylhexanol and 4-heptanone (n=3) resulted in similar values. A significant higher sensitivity of one of the two cell strains was not observed. It has to be emphasized that even the highest concentrations tested did not cause death of the cell population, but reduced the proliferative (mitotic) activity of the cells in a dose-dependent manner. 688 Malinverno 2 , Schmit 3 , Ilaria Giuseppe Bruno Mario Visca 1 . 1 Solvay Solexis S.p.A., R&D Centre, Viale 2 Lombardia, 20, 20021 Bollate, Italy, Solvay S.A., European Public Affaire, Rue du Prince Albert 33, B-1050 Bruxelles Belgium; 3 Solvay S.A. (NOH), DCRT/HSE, Rue de Ransbeek, 310, B-1120 Bruxelles, Belgium Hydrofluoropolyethers (HFPE) are a family of linear oligomeric fluorinated fluid comprising difluoromethoxy and tetrafluoroethoxy repeating units, containing two isolated hydrogen atoms in OCF2 H end groups. These fluids have been designed as low environmental impact substitutes of perfluorocarbons for a number of applications, including heat transfer, solvency and fire suppression. Several toxicological studies have been carried out, including acute and long term toxicity. For the long term studies the inhalation route has been selected due to the low boiling temperatures and high vapor pressures of these materials. Acute toxicity tests have been performed on males and females Sprague DawleyCrl:CD (SD) BR rats through oral, demal and inhalation routes. No deaths have been recorded even at the highest concentrations during the treatment period. Oral LD50 > 5000 mg/kg and dermal LD50 > 2000 mg/kg have been determined. Inhalation LC50 was found > 26411 ppm, the highest tested dose. At this level, reversible lethargy and anesthetic effect were observed. No macroscopic and histopatological abnormalities were observed and the lung to body weight ratio was within normal limits. Repeated dose inhalation was carried out for 5, 14, 28 and 90 days. On 90 day exposure study, embryo-foetal developmental effects have also been studied. The longer studies have been performed at 1000, 3300 and 10000 ppm (6h/day, 5 to 6 days/week). Both for 28 and 90 day exposure, NOAEL at 10000 ppm, NOEL at 1000 ppm were determined. The major effects were increment in urinary fluorides, increase in liver weight and centrilobular hepatocyte hypertrophy. These effects have been considered to be treatment related but not of pathological evidence. The increment in enzymes activity for the metabolism system, are typically reversibile on cessation of treatment, but sometimes this may take more than one month in rodent species. In the same way centrilobular hypertrophy disappear after treatment indicating that this effect is a typical adaptative response to the exposure of the test material and not an indication of toxicity. NOAEL for both maternal exposure and ambryo-foetal development was 10000 ppm. 689 The highest concentration of the (2,4-dichlorophenoxy)acetic acid (2,4-D) was found in Muchawka river and was about 0.0026 µg/l. The estimated concentrations of the phenoxy herbicide residues in the studied surface waters were much lower than values accepted by World Health Organization. 690 POLYCHLORINATED BIPHENYLS IN FRESHWATER FISH FROM THE ZAGREB AREA AS INDICATORS OF ENVIRONMENTAL POLLUTION Jasna Bošnir, Dinko Puntarić. Zagreb Public Health Institute, Zagreb, Croatia TOXICOLOGICAL PROFILE OF HYDROFLUOROPOLYETHERS Colombo 1 , s185 GC-MS ANALYSIS OF PHENOXY HERBICIDE RESIDUES FROM SURFACE WATERS R. Krzyzanowski, B. Leszczynski, E. Sygieniewicz. Department of Biochemistry, University of Podlasie, Siedlce, Poland Seasonal variations of phenoxy herbicide residues in surface waters of Eastern Poland were determined. The samples were collected from five sites of three local rivers: Helenka, Muchawka and Liwiec. Solid phase microextraction (SPME) that involves the partitioning of the analytes between the sample matrix and a stationary phase which is coated on a fused silica fiber, and desorption of trapped analytes into the analytical instrument was applied for extraction of the phenoxy herbicide residues. SPME extracts were separated by gas chromatography combined with mass spectrometry. Obtained results showed traces of (4-chloro-2-methylphenoxy) acetic acid (MCPA) in the studied surface waters from May to July. Aim: To determine the levels of polychlorinated biphenyls (PCB) in freshwater fish from the Zagreb area. To assess the possible variation in PCB levels in the fish from the Sava river, the main recipient of sewage and wastewater in the Republic of Croatia, in comparison with other fishing sites, as well as between two fish families living there. Material and methods: A total of 216 freshwater fish samples from 5 sites were examined: Sava river upstream from Zagreb, Sava river at Zagreb, Sava river downstream from Zagreb, Jarun Lake, and 5 ’ecologic’ fishponds from the Zagreb surroundings. Total PCB were determined by the method of gas chromatography with a detection limit of 5 µg/kg. Results: The mean total PCB level in all fish samples was 18.63 (range 0.054–66.40) µg/kg and did not exceed the allowed level of 2000 µg/kg in any of the fish samples. According to fishing site, the highest PCB levels were found in the fish from the Jarun Lake connected with the Sava river by underground waters (38.45 µg/kg), followed by the fish from the Sava river downstream from Zagreb (33.73 µg/kg) and from ’ecologic’ fishponds with no water connection with the Sava river (20.20 µg/kg). The fish of the family Ictaluridae contained several times lower total PCB levels (mean 5.59 µg/kg, range 0.054–22.41 µg/kg) than the fish of the family Cyprinidae (mean 19.54 µg/kg, range 0.054–66.40 µg/kg). Conlusions: Although the Sava river at Zagreb is the main recipient of sewage and wastewater in the Republic of Croatia, the levels of PCB were within the allowed limits in all groups of freshwater fish samples. Study results suggest the fish of the family Cyprinidae to be a good indicator of environmental pollution with PCB, because highest PCB levels were measured in the fish from the Sava river downstream from Zagreb, the location characterized by highest sewage and wastewater outlet from the Zagreb area, and only slightly lower PCB levels were found in the fish from the Jarun Lake that is connected with the Sava river by underground waters and has only one water exchange per year. 691 HAIR LEAD CONTENT OF HORSES AROUND SHIRAZ OIL AND PETROCHEMICAL INDUSTRIES M. Pourjafar, K. Badiei. School of Veterinary Medicine, Shahrekord University, Shahrekord, IRAN. Fax: 0098–381–4424412 Lead is one of the oldest metals known to man. Industrial pollution is one of the important sources of lead. Hair lead levels are diagnostically useful for lead pollution around oil and petrochemical industries. Hair samples (19 in Spring, 19 in Summer, 19 in Autumn and 19 in Winter) were obtained from horses in a radius of one to ten kilometers of Shiraz oil and petrochemical industries. Hair samples (21 in each season) which were obtained far away from these industries and not close to main roads served as control samples. Hair samples were subsequently digested and analyzed for their lead content by atomic absorption spectrophotometry. Overally, results revealed that the mean hair lead content of horses (5.27±1.7 ppm) around Shiraz oil industries (within radius of 1.5 Kilometers) were significantly higher than mean hair lead content of horses (3.12±1.4 ppm) around Shiraz petrochemical industries (p<0.05). Mean hair lead content of control cases throughout the year was 2.1±0.6 PPM Which was significantly lower than mean hair lead content of horses around oil and petrochemical industries (p<0.05). These levels show that lead pollution around Shiraz oil industries can be a serious problem and must be considered in future. s186 692 Poster Session P36. Regulatory toxicology NAPHTHOL AND 2-NAPHTHOL REMOVAL FROM WATER BY ARTHROMYCES RAMOSUS PEROXIDASE F. Naghibi 1 , K.E. Taylor 2 , J.K. Bewtra 2 , N. Biswas 2 . 1 Traditional Medicine & Materia Medica, Shaheed Beheshti University of Medical Sciences, P. O. Box 14155/ 6153, Tehran, Iran; 2 University of Windsor, Windsor, Ontario, Canada 1-naphthol and 2-naphthol, two bicyclic aromatic compounds, were subjects of an enzymatic reaction with the purpose of their complete removal from aqueous solution, since the former is the main decomposition product of the insecticide carbaryl and the latter is an intermediary product used in rubber, dye and pharmaceutical industries. Arthromyces ramosus peroxidase was used as the biological catalyst. Under optimum conditions the removal efficiency for both substrates is over 98%. These results could be achieved in buffered medium using additives, such as Triton X-100 and polyethylene glycol. 693 POLLUTION OF TAP WATER IN THE CITY OF UFA: DECADE LATER More over, the influence of total fall of chromium in this area was determinated on chromium presence in children’s blood. Variability of this element presence in children’s blood is illustrated by values of quotient of these concentrations these elements and show the eminent role of chromium presence in the air. Secondary dust emission of soil changed in range: 8.95–106.42 µgCr/g s.m. The amount of bioavailable chromium forms in soil measured 1.50 µg/g – exchangeable forms, 4.96 µg/g – organic forms, 26.98 µg/g – carbonate forms. That is the reason why in the future examination concerning presence and co-presence of metals (Cr) in blood the speciation analysis of metals present in suspended dust and in soil should be introduced as well as in total fall that probably influences the area variability of chromium content in boys and girl’s blood on the examined region with variable content of chromium in the air. P36 Regulatory toxicology 695 M.Yu. Ozerov, S.Kh. Sarmanaev, Z.S. Teregulova. Bashvodokanal, Toxicological Center (TC), Ufa, Russia Background: The effects chemical substances present in the environment produce on human health is attracting more and more attention. Unfavorable ecological situation that emerged in 1990 in Ufa (1.2 milion inhabitants) – city with highly developed petrochemical industry, was caused by a big-scale ingress of contamination in the river Ufa, which is a source of tap water for the city. Concentration of phenol in tap water was several dozen times greater than the maximal allowed limits (0.032 mg/l). 338 people required consultations at the Toxicological Center (TC), out of which 105 were hospitalized. The people complained of paroxysmal lowering of overall tonus of the organism, weakness, dryness and an unpleasnat taste in the mouth, headaches, cough, nausea, stomachaches, loose stools, etc. The conducted treatment consisted of adsorbents, hepatoprotectors, high-vitamin diet, symptomatical and dietotherapy. Case Report: During the last decade, several decisions were made, which resulted in an improvement of the ecological situation. These measures were concerned with: optimization of specialized medical service to the population of the large industrial center, enlargement TC with its rehabilitation department, the Poison Center was established, organizations providing tap water to the city worked out prevention measures. These measures were also assisted by Russian Federation regulations “On environmental protection”, sanitary regulations, issued in 1996. Conclusion: Over-year control of tap water quality in the city caused awareness of the necessity to organize drinking water supply in rural areas, where about 1.5 milion inhabitants are using self-prepared sourses of drinking water. In agreement with the SanPin requirements (1996), a Program of organization of water supply from rural areas was developed, which is also functioning in other countries. 694 CHROMIUM CONTENT IN BLOOD OF 10 YEAR-OLD CHILDREN’S DWELLING THE AREAS AS RESULT IMPACT OF SMELTER PLANT J. Kwapuliński, M. Bogunia. Department of Toxicology, Faculty of Pharmacy, Silesian University of Medicine in Katowice, Poland The problem of chromium existence in children’s blood has not been examined so far. The issue of examination of chromium existence was the population of boys (n=200) and girls (n=205) taken from 17 grammar schools in the area near smelter plants. The chromium content in children’s was determinated by AAS method using apparatus Perkin Elmer. The frequency of chromium presence in blood has the normal distribution character. The average chromium content in children’s blood is equal to 2,6 µg/dm3 . This content can be used in further examinations as reference value. Co-occurrence of chromium with other metals in blood changed regarding sex and the place of a child’s dwelling. The presence of chromium in 10 year-old children changed in function of the change of content Mg, Mn, Cd, Pb, Zn, and Ca. ACUTE INHALATION STUDIES WITH IRRITANT AEROSOLS: TECHNICAL ISSUES, INTERPRETATION OF FINDINGS AND RELEVANCE FOR RISK CHARACTERIZATION J. Pauluhn. Toxicology, Bayer HealthCare, Wuppertal, Germany Contemporary approaches for the selection of a chemical for acute inhalation testing commonly are driven by ‘likelihood of inhalation exposure’, i.e., risk based criteria, such as volatility, particle size or use patterns. If none of these criteria apply single exposure testing requirements might be waived. Despite the availability of internationally harmonized testing guidelines, the criteria prompting or waiving inhalation testing differ from one regulation to another. Current testing conventions for inhalation toxicity studies require making solid or non-volatile agents respirable to rats. This is often achieved by laboratory-specific technical solutions that may also affect the outcome of study. Conversely, internationally harmonized approaches are still lacking that would compare the results from inhalation studies with ‘contrived’ aerosols and also take into account the actual particle size of the product as it is handled and used. The focus of this paper is to consider aerosols of irritant substances eliciting their mode of action at the site of primary deposition within the respiratory tract. Assessment is based on conventional endpoints, such as mortality (LC50 ), and sublethal endpoints that included an analysis for the concentration-effect relationship of protein in bronchoalveolar lavage (BAL-protein) as a sensitive, early marker of lung edema. This analysis included whether common denominators can be found for different aerosol sizes of direct and indirect irritants, i.e., those decomposing to reactive intermediates in the lining fluids of the lung or systemically toxic substances. Collectively, these analyses demonstrate that for respiratory tract irritants both the concentration and the particle size are equally important for the outcome of test, independent whether the endpoint chosen was lethality or BAL-protein. In contrast, for systemically acting agents such a relationship cannot be established. Accordingly, for a meaningful risk characterization test results from inhalation studies with ‘contrived properties’ of irritant aerosols due to the specific techniques employed need to be compared with the properties of substance as marketed and handled. 696 PHARMA INTERMEDIATES SAFETY EVALUATION, A REDUCED TOXICOLOGY PROGRAMME Antonio Conto 1 , Fabio Pizzocheri 2 . 1 Chemsafe sas,Colleretto Giacosa (TO) Italy, 2 Pharmasafe sas, Lainate, (MI) Italy The new chemicals, historically covered by EEC 92/32 (seventh amendment of EEC 67/548), are now regulated by the EU Directive 2001/59 (August 6, 2001). The most important change from the old regulation is the possibility to access to the so called Reduced Test Package (RTP) for those chemical intermediates with limited exposure to humans; a typical condition appliable to pharma intermediates substances. Key conditions to get the RTP programme authorized: Poster Session P36. Regulatory toxicology (a) the nature of the chemical: it has to be used only as and intermediate and therefore soon transformed in another chemical entity. (b) the type of chemical plant used to transform the chemical: it must assures absence of possible exposure to humans or its significant reduction. (c) the type of marketing of the substance. Manufacturing only for two customers is allowed. Once the authorisation is delivered by Competent Authorities the testing programme is significantly reduced. No repeated dose toxicity study as well the second mutagenicity test (Chromosomal Aberration) and two ecotoxicology studies are requested. Notification dossier and Risk assessment are still requested. Risk Assessment will be performed at the end of the testing programmes by the EUSES software, correlating the hazard data arising from testing and the exposure conditions. It will give a clear indication of the possible risk for humans and environment. Further testing could be requested in order to get a best risk evaluation. Positive outcomes are expected from the application of such new approach: (a) Reduced cost for Industry; (b) Incentive to notify also for small/medium companies; (c) Increased number of chemicals known for their safety profile; (d) No loss of data. Risk assessment will help to understand when it’s the case to extend the testing programme. (e) Reduced number of laboratory animals used in research/ testing avoinding large repeated-dose studies. (f) Marketing times reduced for the notified substance (5–6 months instead of 8–9 months) 697 THE FUTURE EUROPEAN POLICY ON SAFETY OF CHEMICAL SUBSTANCES Antonio Conto 1 , Fabio Pizzocheri 2 . 1 Chemsafe sas, Colleretto Giacosa (TO), Italy, 2 Pharmasafe sas, Lainate (MI), Italy The actual policy of the European Union for Safety of chemical substances is based on two main categories: The new chemical substances regulated by EEC 92/32 and the existing chemical substances which are regulated by different European Directives with a tiered approach on the basis of priority lists of substances to be tested. While the first system works in a satisfactory way the second one is now under criticism due to the fact it will not allow ta have a great number of chemicals tested/evaluated for their risk to humans and the environment. The lack of knowledge about the impact of many chemicals on human health and the environment is a cause of concern. Increasing of concern is also due to the feeling that the current EU Chemical policy does not provide sufficient protection or at least does not give sufficient data on the majority of substances. 100.000 chemicals are currently registered to the European market at a production level of more than 10 tonnes/year and a further 20.000 are marketed at 1–10 tonnes/year. It’s therefore urgent to adopt some measures in compliance also to the Sustainable development and Precautionary Approach concepts. The White Paper (Strategy for a future chemicals policy) issued in February 2001 by the Commission, represents a tentative to introduce a new system in relation to: (a) the protection of human and the environment; (b) the maintenance and enhancement of the competitiveness of the EU chemical industry; (c) the prevention of fragmentation of the internal market; (d) the increase of transparency (access to information by the public and consumers); (e) the integration with international efforts (harmonisation of testing and classification); (f) the promotion of non-animal testing (“in vitro” methods); (g) the conformity with EU international obligations under the WTO (no barriers to trade). The white paper introduced the REACH system; a single system for new and existing chemical substances. REACH means: Registration, Evaluation and Authorisation of Chemicals. The existing substance will be gradually introduced in the system by 2012. s187 The proposed system generated a huge discussion on the following points (a) the cost of testing all substances (extimated to be 30,000 as existing chemicals) will be solely charged on chemical industry; (b) the foreseen time (deadline in 2012) is not enough to complete the programme; (c) the actual laboratory capacity worldwide in not enough to complete the programme in the due time; (d) “in vitro” toxicology methods are mostly not available at the moment; (e) particular substances with low exposure (i.e. pharma intermediates) must follow a simplyfied regulatory pathways; (f) difficulty in harmonising the safety classification at international level 698 CALCIUM CYANAMIDE –DOCUMENTATION OF POLISH MAC VALUE M. Kupczewska, S. Czerczak. The Nofer Institute of Occupational Medicine, Lodz, Poland Calcium cyanamide is a nonvolatile, noncombustible, white crystalline solid. Calcium cyanamide is a commercially used as raw material for the manufacture of calcium cyanide and dicyanamide. It is also used in the desulfurization of some types of steels. The product is used as a defoliant, fertilizer, or herbicide. Most cases of industrial calcium cyanamide poisoning involve primary skin irritation or sensitizing dermatitis. Skin irritation develops in the form of an erythematous rash over the surfaces of the body that are exposed to the substance of those body surfaces irritated by clothing or perspiration. In addition, exposed workers may develop temporary vasomotor disturbances of the upper body, with susceptibility increasing with alcohol intake. The literature on the effects of exposure to calcium cyanamide (CaCN2 ) in farmers and production workers was reviewed. 65 workers exposed to CaCN2 at levels in the range 0.23–8.36mg/m3 were examined and no evidence of damage to the skin, respiratory system, gastrointestinal tract, kidneys or nervous and circulatory systems was found. When alcohol was taken 1–7 hours after the workshift, a moderate flush reaction occurred in 6 workers and a weak reaction in 7. Calcium cyanamide is used medically for its antabuse-like effect, and the maintenance dose in adults is between 50 and 100 mg/day. Based on the human data and the therapeutic dose The Experts Group of Chemical Agent Intersectoral Commission of MAC Value in Poland, established the 8-hour MAC-TWA value of 1 mg/m3 . No STEL is recommended. This value is intended to minimize the potential for irritation of the skin and respiratory tract and for antabuse-like effects in calcium cyanamide exposed workers consuming alcoholic beverages after work hours. 699 CARCINOGENICITY ASSESSMENT OF MELPHALAN IN P53 +/- MICE A. Mosiello, A. Argentino Storino. RTC, Research Toxicology centre S.p.A., Pomezia - Rome, Italy The purpose of this study was to evaluate the potential carcinogenicity of Melphalan (a derivative of nitrogen mustard used as an antineoplastic agent) in transgenic hemizygous p53 (+/-) mice. The p53 (+/-) mouse model has been proposed as a short-term alternative to the conventional 18 to 24 month bioassay in mice. A preliminary 4 week dose range finding study was performed on wild type mice (C57BL/6) to determine doses for a subsequent short-term carcinogenicity bioassay. Several specific biomarkers were also evaluated, which could help in identifying possible mechanisms of actions. Based upon results of this study, the dose levels selected for this 26-week oral bioassay with Melphalan were 0, 0.5, 1 and 2 mg/kg/week. A positive control (Benzene, 100 mg/kg/day) and 2 groups, each with wild type mice (negative control and 2 mg/kg/week of Melphalan) were added to this study. Results of treatment-related carcinogenic or toxic effects of Melphalan as well as differences of reaction to treatment between transgenic and wild-type mice are presented. s188 700 Poster Session P37. Risk assessment TRANSPARENCY AND RELIABILITY OF CARCINOGEN RISK ASSESSMENTS – THE CASES OF TRICHLOROETHYLENE AND ACRYLAMIDE C. Rudén. Philosophy Unit, Royal Institute of Technology, Teknikringen 78, SE-100 44 Stockholm, Sweden This is a study of why risk assessors of chemicals frequently come to different conclusions, and how toxicity data are used in the risk assessment process. The results from a previous study (a detailed comparison of 29 carcinogen risk assessments made of trichloroethylene) suggest that the fact that the scientific database is constantly evolving cannot by itself explain differences in the overall conclusions of risk assessments. The data sets utilized by the trichloroethylene risk assessors were found to be surprisingly diverse and incomplete, and individual experiments were furthermore interpreted and evaluated differently by different risk assessors (Rudén, C. 2002. From Data to Decision. Doctoral thesis). In the present study the generalizability of the trichloroethylene case is addressed by applying the same method of analysis to another substance, namely acrylamide, and by comparing the results obtained for the two substances. This comparison indicates both differences and similarities between the risk assessments of these two chemicals. An example of an interesting difference is the degree of controversy about the conclusions on carcinogenic potential. The acrylamide risk assessors also come to different conclusions about this chemical’s potential to cause cancer, but the conclusions are less divergent than in the trichloroethylene example and it seems as if data availability (a time dependent factor) is a more important part of the explanation to the different overall conclusions for acrylamide compared to the case of trichloroethylene. Regarding the assessment of the carcinogenic mechanism of acrylamide (hormonal effects and/or genotoxicity) there are similarities to the trichloroethylene case in that different risk assessors required different amounts of evidence for considering a carcinogenic mechanism shown. Another similarity is the low coverage of primary data, despite the fact that the number of available studies is much lower for acrylamide than for trichloroethylene. 701 TOXICOLOGICAL DOCUMENTATION FOR MARKETING AUTHORIZATION IN THE EUROPEAN UNION AND FEDERATION OF BOSNIA AND HERZEGOVINA M. Todic, E. Kapic, N. Mulabegovic, J. Kusturica, F. Becic. Institute of Pharmacology and Toxicology, Sarajevo, Bosnia and Herzegovina The content of the application dossier in the European Union is defined in the Common Technical Document agreed in 2000. Basic information of toxicological testing of the medicinal product is included in Module 1 and 2 of the dossier. The detailed information on toxicology is in the Module 4, Nonclinical studies. Module 1, Administrative and prescribing information includes: Summary of Product Characteristics, Information about the Experts and Annex 1- Environmental risk assessment. Module 2, Quality, Nonclinical and Clinical Summaries; Nonclinical Overview and Summary (Toxicology Written and Tabulated Summaries). Module 4, Nonclinical studies; Study Reports Toxicology (Single-dose and Repeated-dose toxicity, Genotoxicity, Carcinogenicity, Reproductive and developmental toxicity, Local tolerance, Other studies) and Literature References. Legislation in the Federation consider following documentation concerning toxicology to be submitted: Documentation on pharmaco-toxicological evaluation; Safe disposal of expired drug. The differences between European and domestic rules for authorization has been and will be discussed in the future. We can conclude there is no substantial difference in the content, but regulation of the considered information in European Union is more defined and extensive. P37 Risk assessment 702 A NEW EUROPEAN POST-GRADUATE MASTER PROGRAMME IN RISK ASSESSMENT AND RISK ANALYSIS M. Maroni 1 , C.L. Galli 2 , J. Bridges 3 , R. Kroes 4 . 1 Dept. of Occupational Health, University of Milan, 2 Dept. of Pharmacological Sciences, University of Milan, 3 University of Surrey, 4 Institute for Risk Assessment Sciences, University of Utrecht Risk assessments are required, by the European Union and by individual member states, for an ever increasing range of commercial products and processes as well as for environmental contaminants. For example risk assessment will be a core activity of the new European Food Authority and will be crucial to the operation of the new EU Chemicals Policy. Also industry and the private sectors will be in increasing demand of qualified professionals to meet the risk assessment requirements necessary to develop and market new products. The process of risk assessment demands a strong scientific base which comprises: a robust, valid and transparent operational framework, sufficient relevant high quality data, and high level of expertise across a range of disciplines. Risk assessment of chemicals (e.g. human and veterinary drugs, pesticides, biocides, industrial chemicals, food additives, environmental contaminants, growth promoters, cosmetics and personal care products, materials) involves a range of disciplines, in particular: toxicology, ecotoxicology, epidemiology, pathology, environmental chemistry, pharmacokinetics, drug metabolism, statistics, information technology. In addition risk communication and risk management principles are of vital importance. Risk assessment is also required for an increasing range of biological agents (eg GMO’s) and physical agents (eg electromagnetic radiation) which demands additional disciplines, such as molecular biology, environmental microbiology, engineering, etc. In the future it is expected that there will be a need for an increased input from specialists in ethics and animal welfare and economics. To meet the demands of specifically educating young European graduates, the University of Milan, the University of Surrey and the Utrecht University have launched a common project for the creation of a new European post graduate Master in Risk Assessment and Risk Analysis, that has been endorsed by the European Commission (DG SANCO and DG Environment) and funded through the Socrates programme. The project aims at developing and testing a common curriculum that comprise two components: • a core component which is taken by every student • two or more pathways to enable students to build a specialist expertise in an area of risk assessment (for example human risk assessment, environmental risk assessment) The expected outcomes of the project are: • a common European curriculum for post graduate education in Risk Assessment and Risk Analysis • the establishment of an excellence university network across Europe that will deliver the Master • the availability of teaching materials to be exchanged among universities and be diffused through Internet • the post graduate training of 20–30 recent graduates every year • the experimentation of a distant learning approach for intensive short courses • a close co-operation between governments, industry and the academy in setting up the programme and in the identification of the expertise to be developed in order to meet the job market demands. 703 GUIDELINE FOR ASSESSING HEALTH RISK FROM DIESEL ENGINE EMISSIONS S. Czerczak, W. Szymczak. Nofer Institute of Occupational Medicine, Lodz, Poland The continuing industrial development has caused increased incidence of occupational diseases among workers exposed to various Poster Session P37. Risk assessment toxic agents. A considerable number of the occupational diseases is due to carcinogenic agents present in work environment. Therefore, it has been attempted to assess the share or contribution of the occupational factors in the process of carcinogenesis. In addition to the necessity of assessing exposure type and determining high-risk population, occupational disease prophylaxis requires a system of occupational disease prevention based on precise legal regulations. Questions of prophylactic actions are discussed in the Ordinance of the Polish Minister of Health and Social Welfare on the protection of worker health from the risks attrributable to workplace carcinogens. One provision of the Ordinance requires that the workers exposed to carcinogenic agents should receive relevant information on health risks associated with exposure to those agents. It is the employer’s duty to provide the information, and he is also obliged to determine whether or not carcinogenic agents are present in the work environment. When carcinogens are found to be present there, the employer is obliged to perform a qualitative assessment of the health risk resulting from exposure to those carcinogens. To make the task easier to the employers and relevant labour safety services, a team of experts has been order to prepare guidelines useful in assessing health risk associated with exposure to individual carcinogenic agents. Up to now, guidelines for 71 carcinogenic agents have been prepared. The reports are designed for employers, employees, and work safety monitoring services. For Diesel engine emissions the value of 8.5 × 10−3 represents the risk of cancer development during 40 year period employment under the exposure. The indicates that 8 out at 10.000 people exposed during 3 years to employment to Diesel engine emissions as particulate at 4 mg/m3 will develop lung cancer. 704 RISK ASSESSMENT OF NEW CARBOFURAN PESTICIDES FOR OPERATOR AND ENVIRONMENT Y. Chaika. L.I.Medved’s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine Toxicological evaluation of several new carbofuran pesticides was done. Carbofuran pesticides are classified to 1 hazard class (extremely hazardous) by acute peroral and inhalation toxicity according to modern pesticides’ classifications by hazard. Carbofuran is stable in soil, water and vegetating plants. Work conditions formed during use of carbofuran pesticides were examined at seed dressing factory and during seeding on field. Complex of factors forming during work was studied, including except chemical factor - dust pollution, noise and vibration controls. Subjectively workers had a number of complains. Seeds after dressing by formulation didn’t change their color. Recommendations to improve carbofuran formulation were given, application rate was decreased. pesticide’s usage was examined again after improvement. Subjectively workers had no complains. Seeds after dressing by formulation were with characteristic red color. Operator risk assessment of improved formulation was done using different existing models, including so-called German model, EURO POEM and model based on our own methodical approaches. The values of carbofuran risk were lower than acceptable level – 1, if pesticide’s application is limited by 4 hours per day and is done with appropriate operator protective bodywear (mask, gloves, suit, boots, hat). Pesticide behavior in soil was examined in laboratory, modeling natural conditions with different application rates. Investigation show that carbofuran may be classified as highly migrative from soil to water formulation. Researches of quantities of active ingredient in water, soil and air environment around seed dressing factory were provided. The results of chemical analysis allows to make conclusion, that risk of carbofuran for environment is minimal. 705 s189 IS IT POSSIBLE TO ESTABLISH A CAUSAL RELATIONSHIP BETWEEN CFS (CHRONIC FATIGUE SYNDROME) AND CHEMICAL EXPOSURES? PARACELSUS PARADIGM IMPLICATIONS. A. Ferrer-Dufol 1 , S. Nogué-Xarau 2 , E. Vilanova Gisbert 3 . 1 Toxicology Unit, University Clinic Hospital, Zaragoza, Spain, 2 Toxicology Unit, Clinic Hospital, Barcelona, Spain, 3 Toxicology Department, Elche University, Spain Paracelsus paradigm stated that there are not “non-toxic substances” and that any substance may become toxic in a dose-dependent way. His “third defense” has been considered the beginning of the scientific approach to toxicology and is the base of the strategies of prevention that assume for each substance a non-toxic level of exposure. We will discuss if some new admitted clinical pictures such as CFS implies a modification of this paradigm. CFS has been accepted as a clinical entity by CDC and defined as severe fatigue lasting more than 6 months and at least 4 of 8 signs or symptoms related to neurological, muscle-skeletal and immunologic impairment. Related for some experts to the old neurasthenic pathology, its origin has been attributed to a broad spectrum of causes: infections, trauma, stress and chemical exposure. Its relationship to chemicals has been founded on various grounds: clinical reports of CFS after acute or chronic exposure to solvents and pesticides and comorbidity with other syndromes associated to chemicals (Multiple Chemical Sensitivity, Gulf syndrome, Chemical intolerance, Sick building syndrome). On the other way there are sound arguments against: absence of any demonstrated dose-response effect, absence of relationship with any biomarker of exposure or effect, scarce documentation of exposure in most cases. A pathogenetic hypothesis has not been established. Some authors have proposed an altered sensitivity of GABAa receptor, cholinergic ways impairment and a toxicant-induced loss of tolerance to chemicals and other substances as some kind of food. We can be able to maintain the usefulness of the “third defense” admitting three different ways or response to chemical exposure: 1) toxic, implying a specific, with defined targets, and doses-dependent response, 2) allergic, less specific and mediated by the immune system; and 3) unspecific effects produced by chemicals acting as stressors in a individual-dependent way. 706 ASSESSMENT AND MODIFYING FACTORS IN RISK ASSESSMENT: THE CASE OF TRIALLATE, A NEUROTOXIC PESTICIDE F. Broeckaert 1 , A. Li 2 , D. Goldstein 2 , J. Acquavella 2 , M. Martens 1 . 1 Monsanto Europe, Brussels, Belgium, 2 Monsanto, St-Louis, Missouri, USA Triallate is a pre-emergent herbicide used on barley, lentils, peas, triticale, wheat, and canary grass (seed only) in the fall or in the spring before targeted weed species germinate. Triallate is a thiocarbamate producing a peripheral neuropathy (degeneration of nerve fibers) in experimental animals. For the risk assessment of pesticide applicators, the systemic Acceptable Operator Exposure Level (systemic AOEL) obtained from toxicology studies is compared to the absorbed dose taking in to account inhalation and dermal routes of exposure. For triallate, the systemic AOEL has been derived from a 90-day neurotoxicity study in the rat which is appropriate for an application window of a couple of weeks per year. The No Observed Effect Level (NOEL) of this study (6.4 mg/kg bw/day) has been divided by an assessment factor (AF) of 100 to turn the experimental safe dose into a human equivalent dose. This AOEL has been converted into a systemic AOEL using the fraction of the dose absorbed from the gastro-intestinal tract of the rat. Despite the fact that very sensitive techniques were used to derive the NOEL in rats, the regulatory authorities in Europe imposed a additional modifying factor (MF) of 5 on top of the AF of 100 to derive the systemic AOEL. This was imposed in 1999 unless evidence was delivered that triallate is not neurotoxic in triallate manufacturing/packaging workers. To this end, a cross-sectional neurophysiological study was conducted to evaluate the relationship s190 Poster Session P37. Risk assessment between occupational exposure to triallate and several indicators of neurological function. The results of these studies were consistent with the absence of an association between triallate exposure and neurological abnormalities. The overall analysis of the complete data set on the neurotoxicity of triallate clearly demonstrates that an assessment factor of 100 is sufficient to assess the risk to health of professional applicators who constitute one of the most exposed individuals to triallate. 707 A COMPARATIVE HEALTH RISK ASSESSMENT FOR THE GENERAL POPULATION EXPOSED TO ALACHLOR AND BENZENE IN EUROPE F. Broeckaert, M.-A. Reding1and, M.A. Martens. Monsanto Europe S.A., Brussels, Belgium Recently, alachlor (2-chloro-2’,6’-diethyl-N-methoxy-methylacetanilide), a herbicide used for weed control on corn, sunflowers and soybeans has been detected in the atmosphere of several rural and urban areas in Europe during and several weeks after its normal application time period (1–3). Levels of alachlor ranged between no detect (1/2 LOD = 0.08 ng m−3 ) and a maximum of 17.83 ng m−3 (3). The objective of this study was to determine the risks associated with short-term exposure to atmospheric alachlor based on a 28-day inhalation toxicity study in rats. Risk was expressed in term of margins of exposure (MOE), the ratio between the highest systemic level of exposure without any adverse effect in the experimental species and the absorbed dose in man derived from atmospheric monitoring data. Exposure was calculated using the maximum level of alachlor (17.83 ng/m3 ) (3), 100% pulmonary absorption and physiological breathing-activity patterns for European populations. Health risks of alachlor were compared with the risks associated with exposure to ambient levels of benzene using the most appropriate short-term toxicological study (4) and 95th percentiles of average benzene levels reported by the European Environmental Protection Agency. For the maximum level of alachlor in air, the short-term MOE by inhalation ranges between 186,951 (outdoor workers) and 434,239 (adult females). These figures are compared against the MOEs for benzene exposure in several EU countries. The risks associated with short-term respiratory exposures to alachlor are extremely low, including when food and drinking water sources are considered. 708 EPIDEMIOLOGICAL VALIDATION OF ENVIRONMENTAL CANCER RISK ASSESSMENTS: A CASE STUDY IN POPULATIONS EXPOSED TO POLYCYCLIC AROMATIC HYDROCARBONS. M. Camus 1 , A. Vyskocil 2 , C. Viau 2 . 1 Environmental Health Sciences Bureau, Health Canada, Montreal, Qc, Canada; 2 Department of Environmental and Occupational Health, University of Montreal, Montreal, Qc, Canada Can epidemiology validate local environmental cancer risk assessments (RA)? We assessed this approach in populations exposed to polycyclic aromatic hydrocarbon mixtures (PAH) in 5 aluminium smelting and 5 other communities in Quebec. Lifetime lung cancer risks predicted for these communities on the basis of PAH and benzo(a)pyrene (BaP) levels measured in the 1990s were converted to annual incidence rates among women. We estimated the time required for these predicted rates to become statistically significant in these population, using a Poisson sample size calculation. This calculation was redone assuming 10 times higher historical exposures. Using population-weighted linear regression, we estimated the exposureresponse gradient between female lung cancer rates (1989–1993) and “dispersion-adjusted” BaP exposure estimates. Risk predictions based on recent low exposure levels could not be detected statistically before 300 years. However, had pre-1970 exposures been merely 10 times higher than today, the predicted risk might be detected statistically with about 3 years of data. The linear regression model estimated an exposure-response gradient (R2 ≈ 0.8) across the 5 aluminium-smelting communities. The rates in the 5 other areas where too heterogeneous for the trend to be significant. The gradient across aluminium-smelting communities would match the BaP-based RA if pre-1970 exposures had been some 56 (95% CI 12–101) times higher than today. Although RAs seem the only way to assess health impacts of recent low levels of carcinogens in small populations, epidemiological studies may have the power to assess local impacts of past environmental exposures at least one order of magnitude higher than today. 709 EVALUATION OF FOOD RISK EXPOSURE USING EXTREME VALUE THEORY-APPLICATION TO HEAVY METALS FOR SEA PRODUCTS CONSUMERS J. Tressou 1 , P. Bertail 2 , A. Crepet 1 , M. Feinberg 3 , J.-Ch. Leblanc 4 . 1 Lab. de recherche sur la consommation,INRA, Ivry/seine, France; 2 Lab. de statistique, CREST/ENSAE, 92245 Malakoff, France; 3 Lab. de chimie analytique, INAP-G/INRA, Paris, France, 4 Direction scientifique NHSA, INRA, Paris, France This paper presents new statistical methods for evaluating food risk exposure related to some contaminants. We focused on the estimation of the probability to be exposed over the so-called provisional tolerable weekly intakes (PTWIs), when both consumption data and contamination data are independently available. For some toxic contaminants, PTWIs belongs to the exposure tail distribution, which suggests the use of Extreme Value theory to evaluate the risk. Our approach consists in modelling the exposure tail by a Pareto type distribution characterized by a Pareto index which may be seen as a measure of risk. Using propositions by Hall and Feuverger, we correct the bias of the Hill estimator to precisely estimate the risk index. We compare the results with empirical plugin methods. To illustrate our approach, we present some evaluations of risk exposure to heavy metals via sea product consumption. We also focus on the assumptions about exposure calculation such as aggregation of data or use of probabilistic calculus mode. Because of the strong impact of these assumptions, conclusions about exposure to heavy metals for consumers of sea products can not be easily summarised. As far as food risk is concerned, according to the data used in risk characterisation process, methylmercury intake via the consumption of sea products seems high for a significant part of the French population and especially for children aged 3 to 8 years old. Concerning the feasibility of our method based on tail estimation, the use of Pareto tail adjustment is nonsense if PTWI does not belong to the distribution tail, but at the opposite case it allows to quantify very low risk. Use of empirical plug-in methods is clearly relevant for very risky food contaminants. Developments are needed concerning confidence interval for such probabilities to exceed a given toxicological level. 710 RISK ASSESSMENT FOR CHROMIUM EXPOSURE STUDY OF A POPULATION LOCATED IN THE PROVINCE OF SANTA FE ARGENTINE REPUBLIC D. Gotelli 1 , M. Gotelli 1 , L. Signorini 1 , A. Lo Balbo 1 , R. Castro 2 , S. Britos 3 , C. Gotelli 1 . 1 Center for Toxicological Research, Buenos Aires; 2 Univ. of Flores, Buenos Aires; 3 Univ. of Buenos Aires Aiming to determine if the industrial activity confined within the limits of the city of Esperanza, with a population of about 30.000 inhabitants, could generate a chromium contamination capable of producing alterations to the human health and the environment, a Risk Assessment Study was implemented applying the methodologies proposed by the World Health Organization and the Agency for Toxic Substances and Disease Registry of the U.S.A. Applying the bioaccessibility criterion, chromium was quantified in the air, soil, water, food and drinks; microfauna was studied; ecotoxic tests with bioindicators were performed; the vegetal covering was studied; the evaluation of cancer and teratogenesis incidence as well as other pathologies registered in the population was analysed. Using the whole information generated, the evaluation equations and criteria specified in this methodology were applied establishing that the presence of chromium in the area does not represent a risk either for human health or the ecosystem. Poster Session P37. Risk assessment 711 COMPARISON OF THE NUTRITIONAL REQUIREMENT AND RISK ASSESSMENT FOR ESSENTIAL TRACE ELEMENTS (ETEs) BY THE INSTITUTE OF MEDICINE (IOM) AND THE USEPA: TWO CASE STUDIES – SELENIUM AND ZINC. K.A. Poirier. Kendle International, Inc., Cincinnati, Ohio, USA The IOM has recently reviewed the nutritional requirement and toxicity data and established a Tolerable Upper Intake Level (UL) for the ETEs. The UL is analogous to the USEPA’s Reference Dose (RfD), except that the UL relies on a case-by-case consideration for derivation, has more latitude for uncertainty factor (UF) application and makes recommendations based on sex, age and physiological status. The RfD is calculated as a single lifetime exposure value for all individuals. The selenium RfD is 5E-3 mg/kg-day (0.275 mg/day) based on a NOAEL of 0.015 mg/kg-day (0.853 mg/day) and a LOAEL of 0.023 mg/kg-day (1.261 mg/day) with a UF of 3 for human variability. The selenium RDA for adults is 0.055 mg. The UL for adolescents (14–18 years old) and adults is 0.4 mg/day. The current IOM recommendation for the safe maximum daily intake of selenium in individuals older than 14 years of age is approximately 45% greater than the RfD. Both the USEPA and IOM used the same human data to derive the RfD and UL, respectively, based on a critical effect of clinical selenosis in adults (average body weight of 55 kg). Yet there is considerably difference in the estimate of a safe daily upper limit of selenium intake in the adult population. The RfD for zinc is 3E-1 mg/kg-day (20 mg/day) based on a LOAEL of 60 mg/day (1.0 mg/kg-day) from a critical effect of a 47% decrease in erythrocyte superoxide dismutase in adult females (60 kg) after 10 weeks and a UF (LOAEL to NOAEL extrapolation) of 3. The zinc RDA for adults is 11 mg for males and 8 mg for females. The UL for adults is 40 mg/day, 23 mg/day for children 9–13 years of age and 34 mg/day for adolescents 14–18 years of age. The current RDA exceeds the RfD for infants and children when adjusted for body weight. The RfD process has inherent inconsistency in the paradigm. It is proposed that a creation of a RfD category specific for ETEs and a harmonization of these two approaches be undertaken in order to provide a consistent, scientifically-based recommendation for the public. 712 BENEFITS OF 24 HOUR FOOD ACCESS FOR DOGS ON TOXICOLOGY STUDIES S. Hudson, S. Wight, J. Campbell. Quintiles Ltd, Research Avenue South, Heriot Watt University Research Park, Riccarton, Edinburgh, EH14 4AP, UK Dogs on Toxicology studies are routinely housed in social groups, providing enriched environmental conditions and enhancing the general welfare of the animals. However, it is common practice to measure food consumption during the study period, usually requiring individual housing of the dogs for a specified time period. Due to the recognised benefits of maintaining the dogs in social groups, the period of time that they may be individually housed is restricted. Young dogs are often not able to consume enough diet over this period or may be more stressed and reluctant to eat, when housed individually. In these circumstances reduced food consumption may be insufficient to maintain healthy growth. Animals on toxicity studies may show inappetence due to the administered test substance that can be transient and normal appetite returns as blood levels diminish. As a consequence, problems can arise with reduction in body weight growth rates, raising welfare concerns and impacting on study data. We have addressed this problem by changing the feeding regime to a ’by dose group’ basis, allowing the dogs 24 hour access to diet. This has allowed normal social interaction, provides essential food consumption data and provides normal growth rates. A comparison has been made of average bodyweight change in individually housed animals for a 4 hour feeding period to group housed animals given 24 hour access to food. This has shown that the weight loss frequently observed at the start of the studies in the restricted feeding period animals was not observed in the 24 hour access, group housed animals. 713 s191 AN ANALYSIS OF THE NEED FOR AN ADDITIONAL TOXICOKINETIC SAFETY FACTOR FOR NEONATES N.V. Corea, A.G. Renwick. Clinical Pharmacology Group, Allergy and Inflammatory Sciences Research Division, School of Medicine, University of Southampton, Bassett Crescent East, Southampton, S016 7PX, United Kingdom The current risk assessment process for chemicals in food involves the use of default uncertainty factors to allow for inter- and intraspecies variability when converting data from an animal study into a safe level of human intake. The proposal that an additional factor of 10 should be applied for determining acceptable exposures for infants and children under the Food Quality Protection Act (1996) in the USA, implies that the early stages of human development may not be adequately protected by the normal uncertainty factors. There are limited data on the kinetics of food additives, but extensive published data for prescribed drugs in young humans (neonates, infants and children) and adults. The adequacy of the default factor has been assessed by comparing the pharmacokinetics of selected probe drugs in 10-day old and adult rats with equivalent published data in young and adult humans. Animals received a single intraperitoneal dose of 200 mg/kg chloramphenicol (which undergoes glucuronidation), 5 mg/kg caffeine, or 50 mg/kg theophylline (which are metabolised by CYP1A2). Plasma was analysed using validated HPLC methods. For each drug, the clearances (ml/min/kg) in young and adult rats were compared to equivalent published data in human neonates and adults. The ratios of the clearance in rats to the clearance in age equivalent humans were compared to the default inter-species toxicokinetic factor of 4. The default factor was exceeded slightly for chloramphenicol and caffeine when the data for 10-day old rats were compared with human neonates, but not for infants or children. These data do not support the need for an extra uncertainty factor for human neonates, infants or children in relation to toxicokinetic differences. 714 THE ADEQUACY OF THE INTERSPECIES TOXICOKINETIC (SAFETY FACTOR) USED INT EH RISK ASSESSMENT OF FOOD ADDITIVES. S.C. Tullberg, W.E. Keene, K. Walton, M. Toor, A.G. Renwick. Clinical Pharmacology Group, Allergy and Inflammatory Sciences Research Division, School of Medicine, University of Southampton, Bassett Crescent East, Southampton, S016 7PX, United Kingdom Uncertainty factors are used to extrapolate from a no-observed adverse effect level (NOAEL) in the animal test-species to a safe exposure for humans, known as the Acceptable Daily Intake, ADI. Traditionally a 100-fold factor has been used for the extrapolation comprising two 10-fold factors to allow for inter-species and intraspecies differences. Sub-division of each 10-fold factor relates to toxicokinetic or toxicodynamic differences between species and within the human population. The current work uses human and animal in vivo data to assess the adequacy of the inter-species toxicokinetic (ISTK) default factor of 4.0-fold. Kinetic parameters will be calculated following oral dosing for 4 food additives at the NOAEL, and 10 x less the NOAEL in animals, and at the ADI, and 10 x greater the ADI in humans. Results on the species differences in toxicokinetics are for thiabendazole (TBZ), propyl gallate (PG), butylated hydroxytoluene (BHT) and curcumin at the NOAEL and ADI indicate that the 4.0-fold ISTK factor is adequate for all four food additives, although the magnitude of the species difference varies for each compound. Results to date from studies with TBZ at 10 x less NOAEL (in animals) and 10 x greater than ADI (in humans) show reduced clearance in both species at the higher dose indicating that saturation of elimination occurs in both animals and humans. These results demonstrate that the value of the ISTK factor can depend on the doses selected for the comparison. These findings indicate that the default safety factor is adequate for intakes at the ADI, but that may be inadequate if intakes are above the recommended safe exposure. s192 715 Poster Session P37. Risk assessment CHOLESTEROL LOWERING VEGETABLE OIL SPREADS: RESULTS OF A POST LAUNCH MONITORING PROGRAMME L.J. Lea, P.A. Hepburn. Unilever Safety & Environmental Assurance Centre, Unilever, Colworth House, Sharnbrook, Bedfordshire, UK Phytosterol-esters (PE) have been approved under regulation (EC) No 258/97 on Novel Foods and Food Ingredients as a novel food ingredient for use in vegetable oil spreads. PE enhance the blood cholesterol lowering activity of the spread, by reducing the absorption of cholesterol from the small intestine. The premarket safety assessment of toxicological and clinical studies had established that there was a reasonable certainty of no harm resulting from consumption of PE. However, a requirement of the European Commission decision was to establish a post launch monitoring (PLM) programme to accompany the marketing of the product. The PLM scheme developed by Unilever consisted of three components: (a) Is the use as predicted/recommended? (b) Are known effects and side effects as predicted? (c) Does the product induce unknown side-effects? Market research data has shown that the target population is buying the product. Median intakes were below 20g/day, even for regular/established users which is lower than the assumptions made in the original risk assessment. The only observed side effect has been a slight reduction in the most lipophilic carotenoids. A longterm study has confirmed that an intake of 20g/day of a spread containing phytosterol-esters is not associated with a biologically significant lowering of serum carotenoids. Health-related calls made to consumer telephone care lines regarding the use of the product have been assessed centrally. Given the extensive exposure of spreads containing phytosterol-esters in Europe, only a small number of health-related calls have been received and reports to date reveal no adverse health effects associated with the product. Data collected in the market place under “real life” conditions has been helpful to confirm the validity of the initial risk assessment and as a basis to check the assumptions used to derive it. 716 ASSESSMENT FACTORS IN HEALTH RISK ASSESSMENT K. Victorin 1 , A. Falk Filipsson 1 , A. Hanberg 1 , M. Wallén 2 . 1 Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden, 2 National Chemicals Inspectorate, Stockholm, Sweden The scientific basis for default uncertainty/assessment factors has been reviewed, and the following conclusions and recommendations are drawn:Inter-species extrapolation. A species-specific factor based on allometric scaling allows for differences in basic metabolic rate (4 for rats and 7 for mice compared to humans). Regarding toxicodynamics and remaining toxicokinetic differences a distribution based on studies examining the relationship between NOAELs in mice, rats and dogs can be used. If the 95th percentile is chosen from this distribution, the corresponding factor would be 12. The resulting inter-species factor is 48 (rats) or 84 (mice).Inter-individual differences. A factor of 10–16 might be sufficient to reflect the variability between healthy adults. However, this does not include potentially sensitive subgroups of the population and genetic polymorphisms. No attempt is made to provide a factor including all risk groups.Duration of exposure. A distribution based on the ratios between NOAELs from subchronic to chronic studies can be used. If the 95th percentile is chosen, the corresponding assessment factor would be 16.Extrapolation from LOAEL to NOAEL. Dose-response modelling using the benchmark dose method is most appropriate for this purpose. Otherwise, a default factor can be used (3–10).Adequacy of the database. A poor database will need an extra assessment factor. If adequate knowledge is missing about effects on the endocrine, reproductive, immune and the nervous system, we propose an extra assessment factor for children (1–10).Derivation of an overall assessment factor. We recommend that the proposed distributions are used, although the choice of percentile is a matter of policy. If the 95th percentile is chosen (covering 95% of the substances compared), the assessment factor for inter-species and inter-individual extrapolation would be 500–800 for rat experiments. If differences in duration of exposure and other factors are included, it would result in a higher overall factor. 717 TOXICOLOGY-HYGIENIC EVALUATION AND MINIMIZATION OF THE RISK PROCESS OF LIQUIDATION THE ANTI-INFANTRY PLASTIC MINES V.G. Lishavskij, N.I. Reva. L.I.Medved’s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine For protection of the environment, sanitary-epidemiologic welfare, pollutants as introduction of chemical substances in the environment, must not induce dangerous factors and must be adequately under control. In connection with this the Toxicology-hygienic evaluation was carried out and ecological investigations were done connected with the liquidation of the anti-infantry plastic mines (AIPM). With the aim of minimization of the risk, investigations were carried out in the test and main target ranges. It were studied the main factors of risk for the environment, health of the special military people and population, that lives in the territories close to the target ranges. It was determined that the explosion AIPM gives a number of dangerous compounds and the risk of the negative influence of the products of the explosion is determined by their quantity, chemical components of the explosion matter, the case and cassette of the mine as well as the character of interaction with the objects of the environment. The atmospheric air is the must polluted with the dangerous compounds which is the risk for health of the military men, doing the work the liquidation of the AIPM. During the explosion in the atmospheric air the toxic compounds are get in as well as they are in the ground. At the distance 100 m they are dissipated as well as the fragments of liquid rubber, plastic and Aluminium residues, which are produced during the explosion the AIPM. The analysis of materials of investigation shows that in the air of working zone and the atmospheric air there are more than 20 chemical toxic compounds. The main quantity of compounds, that pollute the air is produced in time of the explosion (5 compounds), migrate from the case AIPM (13 compounds), with the particles of soil – 4. Toxicology-hygienic and ecological investigation of the conditions of liquidation AIPM allowed to determine and to value quantity the factors, which form risk for the military working people, population and the environment as well as recommend the most adequate technology for liquidation of AIPM. 718 PROBABILISTIC ASSESSMENT OF CONSUMER EXPOSURE TO INGREDIENTS USED IN HAIR CARE PRODUCTS Robert J. Safford, David Briggs, Louise N. Conway, Garrett F. Moran, Christopher R. Jones, Anita J.E. Irwin. Safety and Environmental Assurance Centre, Unilever Colworth, Sharnbrook, Beds., MK44 1LQ, UK Accurate estimation of consumer exposure is a fundamental step in the assessment of the toxicological safety of ingredients in personal care products. Traditional exposure assessments take single estimates for the variables used, often combining worst case estimates with ‘average’ values, leaving the final estimate of exposure difficult to interpret. In reality the parameters used will vary, dependant on such factors as consumer habits, which we know differ across the world, and even within single population groups. In addition to this, estimating skin exposure to, and hence penetration of ingredients used in hair care products is further complicated by the presence of hair. The model described uses a decision tree approach to account for variation in the parameters used to assess consumer exposure to ingredients in hair care products. In addition, the model accounts for the effect of hair on exposure by calculating the percentage of product that comes into contact with the skin, based on an even distribution of product over the hair, scalp and hands. The parameters used in the model are scalp, hand and hair surface areas, amount of product used, frequency of washing, body weight and skin penetration. Variation within each parameter is represented Poster Session P37. Risk assessment by taking the 10th , 50th and 90th percentile points of the distribution. The model has been written as a macro in Microsoft Excel, and provides estimates of exposure at pre-determined percentile points. Comparison of the model to the more commonly used Monte Carlo approach shows it to give comparable outputs. The output from the model provides a more realistic assessment of consumer exposure than single point estimations leading to more refined risk assessments. Input data can easily be changed to reflect different consumer habits, physical attributes and skin penetration ranges. In addition, the model is being developed further to consider different exposure scenarios for other personal care products 719 PRINCIPLES OF RISK ASSESSMENT OF COMPLEX AND COMBINED ACTION OF PESTICIDES Y. Spynu. L.I.Medved’s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine In world’s literature studying of laws of pesticides’ action is still based on research of isolated action. Meanwhile, specificity of influence of especially stable pesticides on humans consists in simultaneous pollution of many environment media and as consequence their receipt in organism by different ways. This position concerns to research of risk of pesticides’ receipt of by that or other way. Necessity of development of integrated principle to estimation of danger of multicomponent action of given pesticide, and also the combined influence of different substances on the human is obvious. Probably primary influence on human directly from source of formation, and also secondary - as a result of migration on soil - air, water and food chains. The analysis of data in literature shows, that infringements of human health are possible at low levels of influence (not exceeding hygienic standards) simultaneously by different chemicals. That’s why there is necessity of estimation of the complex and combined action of chemical substances. It is shown, that from existing types of interaction of chemical substances (synergism, potentiation, antagonism) at influence of low doses most frequently is shown the effect of summation. This principle is put into mathematical models suggested by us for calculation of risk of complex and combined action of pesticides. The classical concept of dose – time - effect is reflected with suggested models. Carried out calculations have shown, that at complex action of some small toxic and mildly stable modern pesticides the risk does not exceed 1 whereas at some persistent, highly and moderately toxic pesticides the risk is higher than 1. 720 AGRICULTURAL CHEMICAL SAFETY ASSESSMENT: A MULTI-SECTOR, INTERNATIONAL PROJECT N. Carmichael. ILSI Health and Environmental Sciences Institute (HESI) Technical Committee on Agricultural Chemical Safety Assessment, Bayer CropScience, Sophia-Antipolis, France, Washington, DC, USA Despite advances in the biological sciences in the last 20 years, as well as improved sensitivity and specificity of testing protocols, the core requirements of and rationale behind the standard toxicity testing battery for crop protection chemicals remain relatively unchanged. The ILSI HESI Technical Committee on Agricultural Chemical Safety Assessment, a multi-sector, international group of government, academia, and industry scientists, has developed a proposal for an improved testing scheme for assessing the safety of crop protection chemicals more efficiently, with fewer animals, and with fewer artifacts. The project has been developed with special emphasis on integrating metabolic and kinetic data into the safety assessment process; developing a hierarchy of study types, endpoints, and triggers to cover vulnerable life stages; developing a tiered testing framework for endpoints such as neurotoxicity, carcinogenicity, and chronic toxicity; and evaluating the range of relevant human exposure situations in the context of experimental study design. The intention is to produce a proposed approach to hazard evaluation where the studies performed are appropriate to the risk assessments for which they will be eventually employed. The proposed approach provides a sound scientific basis for determining whether a given s193 agricultural chemical poses adverse health risks in humans, taking into account the chemical’s toxicological properties and use patterns. 721 MECHANISTIC TOXICOLOGY: HOW IT CAN SUPPORT REGULATORY PACKAGES IN THE PHARMACEUTICAL DEVELOPMENT. G. Dal Negro, A. Casartelli. Cellular & Biochemical Laboratory, Safety Assessment Department, GlaxoSmithKline R&D, Verona Mechanistic toxicology describes how chemicals exert their toxic effects in biological systems. In the pharmaceutical industry, toxicology has the main role to assess the safety of new drugs. However, in regulatory toxicology, standard sets in hystopathology and clinical pathology can give answers to the question “what”, whereas very often they cannot answer the question “how”. The way a specific change occurs as a consequence of exposure to a drug can play a crucial role in the decision making process, since the involvement of a specific pathway or another can have a completely different weight from a toxicological point of view. In this presentation, a couple of examples are given, with the aim to show how the way a change occurs can influence the destiny of a new candidate. Furthermore, examples of some of the techniques that can be adopted in mechanistic toxicology are given. 722 A CRITIQUE OF THE EUROPEAN COMMISSION’S PROPOSALS FOR REGISTERING NEW & EXISTING CHEMICALS R.D. Combes. FRAME, Russell & Burch House, 96–98 North Sherwood Street, Nottingham, NG1 4EE, UK In its White Paper, Strategy for a Future Chemicals Policy, published in 2001, the European Commission (EC) proposed a REACH (Registration, Evaluation and Authorisation of CHemicals) system for both existing and new chemical substances. This is based on a top-down approach, in which the degree of toxicity information required is dictated primarily by production volume (tonnage). However, if testing is to be based on traditional methods, very large numbers of laboratory animals could be needed, causing ethical, scientific and logistical problems incompatible with the proposed testing time-schedule. The EC wishes to minimise animal use, but fails to produce a comprehensive strategy for doing so. This poster presents an overall scheme for predictive toxicity testing, whereby non-animal methods could be used in a tiered approach to provide a rapid and scientifically justified basis for the risk assessment of chemicals for their toxic effects in humans. The scheme starts with a preliminary risk assessment process (involving available information on hazard and exposure), followed by testing, based on physicochemical properties and (Q)SAR approaches. (Q)SAR analyses are used in conjunction with expert system and biokinetic modelling, and information on metabolism and identification of the principal metabolites in humans. The resulting information is then combined with production levels and patterns of use to assess potential human exposure. The nature and extent of any further testing should be based strictly on the need to fill essential information gaps in order to generate adequate risk assessments, and should rely on non-animal methods, as far as possible. The scheme also includes a feedback loop, so that new information is used to improve the predictivity of computational expert systems. In May, 2003, the EC published detailed proposals for implementing REACH, which include the creation of a European Chemicals Agency to work with Competent Authorities (CAs) in Member States and the Commission. However, these proposals do not allay many of the concerns and suggestions, previously voiced by FRAME and several other key stakeholders. In particular, there no clear coherent testing strategy and no guidance for registrants on intelligent testing to maximise the use of non-animal approaches to safety testing, based on a combination of factors for estimating exposure levels, rather than mainly on production volumes. There is also no clear programme for the development, improvement and validation of new alternative methods. This presentation explains why such measures should be introduced, together with clearer s194 Poster Session P38. Toxicodynamics/toxicokinetics guidelines for the respective roles of the Agency, Competent Authorities of EU Member States and the Commission in implementing and harmonising the REACH system. Some recommendations are made to improve the situation and the risk assessment process, including a call for the EU to actively promote the improvement and validation of (Q)SAR models and expert prediction systems, and methods for biokinetic modelling, since these offer the most realistic and economical solution to the need to test large numbers of chemicals rapidly. 723 THE APPLICATION OF PCA (PRINCIPAL COMPOUNDS ANALYSIS) AND CLUSTER ANALYSIS TO INTERPRETATION OF THE CHANGE CONCENTRATION OF HEAVY METALS IN ARTERY J. Kwapuliński 1 , E. Nogaj 1 , P. Nogaj 2 , M. Olejczyk 3 . 1 Department of Toxicology Silesian Universty of Medicine in Katowice, 41–200 Sosnowiec; 2 Department of Molecular Biology, Biochemistry and Biofarmacy, Silesian University of Medicine in Katowice; 3 Department Artery Surgery St. Barbara Hospital, Sosnowiec The obtained early results concerning of the occurrance of some elements in femoral artery are basis the analysis of influence of age, sex and addiction to smoking on the coincidence some heavy metals. To analysis were involved cluster analysis and Principal Compounds Analysis. The cluster analysis and the main factor analysis corroborated the influence of age, sex, and addiction to smoking on the incidence of Cd, Pb, Cu, Co, Cr, Mn, Ni, Fe and Zn in the femoral arteries in the examined persons. Table. The comparison of quotient of content metals related to 10 percentil in femoral arteries of persons of studied population, to value 10 percentil of reference group for the lowest concentrations of metals. Fig. Dependence between factors 1/2/3. Metals Cd Co Cr Cu Fe Mn Ni Pb Zn Group of reference 2.32 1.90 2.09 6.08 6.14 3.64 3.48 0.24 153.98 (10 percentil) Studied population 3.90 14.47 4.61 14.54 70.93 6.06 10.46 26.05 398.20 Moreover the values of content quotients of the investigated metals in the femoral arteries (obtained for 10 percentile concentration) showed the correlation of the tested elements in the femoral arteries. They also defined either physiological or toxic character of the tested metal. P38 Toxicodynamics/toxicokinetics 724 VOLUME OF DISTRIBUTION OF ETHANOL IN FEMALES AND MALES D. Zuba, W. Gubała, W. Piekoszewski. Institute of Forensic Research, Westerplatte 9, 30–031 Krakow, Poland Women have a lower prevalence of drink problems than men and they appear to become more impaired than men do after drinking equivalent amounts of alcohol, achieving higher blood alcohol concentrations even when doses are adjusted for body weight. However, women seem to eliminate significantly more alcohol per unit of lean body mass per hour than men. Speculations that gender differences in alcohol pharmacokinetics or alcohol-induced performance impairment may be caused by the menstrual cycle and variations in female sex hormones were rejected. In the study the parmacokinetic calculations based on time-concentration curves were performed in order to compare the volumes of distribution of ethanol for females and males. A group of 24 volunteers, 12 women and 12 men, participated in experiments and they consumed ethanol in the form of vodka (0.7 g of per kg of body weight for men, and 0.6 g/kg b.w. for women). Samples of venous blood were obtained through an indwelling catheter before ingestion of alcohol and then in 15 minutes intervals timed from the end of drinking. Blood alcohol concentrations were determined by means of headspace gas chromatography. The pharmacokinetic calculations were done using first-order absorption and zero-order elimination models of ethanol. The calculated apparent volumes of distribution of ethanol after oral dose were 0.78 ± 0.15 L/kg and 0.80 ± 0.24 L/kg for females and males, respectively. It showed that the mean values for both groups were very close and the difference was not statistically significant (t=0.24, p>0.1). It caused differences in shape of blood alcohol curves. Time to peak concentration for females was 1.06 ± 0.25 h and it was slightly longer in relation to males (0.87 ± 0.28 h). Both experimental and extrapolated to zero time the maximum ethanol concentrations were significantly lower in females, and amounted to 0.606 ± 0.118 and 0.785 ± 0.165 g/L (experimental) as well as 0.777 ± 0.139 and 0.951 ± 0.202 g/L (extrapolated) for females and males, respectively. The finding might be explained by change in life style and diet of the women since Widmark has created his formula. The adequacy of the coefficients equal to 0.6 for women and 0.7 for men was questioned also by other authors. Several alternative models, based on total body water volumes (TBW) and the body mass index (BMI), have been proposed in the literature. Nevertheless, the gender differences in volume of distribution were included in those models. According to the study, we suggest using the same factor equivalent to volume of distribution in back calculation of alcohol concentration. 725 CaCo-2 CELL PREDICTIVITY & ABSORPTION OF LIPOPHILIC ANALOGUES IN MAN C.K. Pease 1 , A. Priestley 2 , A.B. McEwen 3 , R.U. Pendlington 1 , D. Sanders 1 , M. York 1 , D. Griffiths 2 , S.G. Wood 3 , R.A.F. de Ligt 4 , M. Verwei 4 , A.-E. Aynaou 4 , J.J.M. van de Sandt 4 . 1 Safety and Environmental Assurance Centre (SEAC), Unilever Colworth Laboratory, Sharnbrook, BEDFORD, UK. MK44 1LQ. 2 LCG Bioscience, Bourn Hall Clinic, Bourn, Cambridge, UK. CB3 7TR. 3 Biodynamics Research Limited, Pegasus Way, Rushden, NN10 6ER. 4 TNO, Utrechtseweg 48, PO Box 360, 3700 AJ Zeist, The Netherlands In recent years, data from CaCo-2 cell systems, as fed into physiologically-based pharmacokinetic (PBPK) models, have proved to be extremely useful in vitro tools in assessing relative oral absorption properties for lead compound selection. It is envisaged that the development of novel, robust toxicokinetic and toxicodynamic models (as potential in vitro alternative testing approaches) will also require absorption data from CaCo-2 models or similar. Hydrophilic compounds are handled well in these in vitro systems. However, cosmetic and toiletry ingredients, which are typically lipophilic, may not be handled so well. The aim of this study was to interrogate the robustness and predictivity of standard two-compartment CaCo-2 cell systems and a new mono-directional ’cells-on-sheet’ system against well-characterised in vivo human data. The prototype family of lipophilic chemicals chosen were alkyl hydroxybenzoates (parabens). Unlabelled parabens analysed at 30 µM in CaCo-2 systems were methyl-, ethyl-, propyl-, butyl-, heptyl- and octyl-paraben. [14 C]-labelled propyl- and octyl parabens were analysed in vitro but were also administered orally (total parabens dose was 1%) to monitor relative absorption kinetics in vivo in man. Caffeine and mannitol were used as quality controls in the CaCo-2 studies. Recoveries of the six unlabelled parabens in the in vitro systems were variable and low to moderate (<65%) in standard CaCo-2 cell systems; in contrast for caffeine and mannitol recovery was as expected (∼100%). Improved apical to basolateral recoveries for propyl-paraben (9.3 ± 1.3 to 79.5 ± 5.5%) and octyl-parabens (8.4 ± 1.3 to 20.8 ± 4.0%) were effected by using radiolabelled material together with the ’cells-on-sheet’ system. The ‘cells on sheet’ CaCo-2 system still predicted different absorption properties for propyl-paraben (Papp 49.8 × 10−6 cm/s) and octyl-paraben (Papp 6.76 × 10−6 cm/s); in man however, these chemicals exhibited very similar rapid absorption and clearance profiles in vivo. For the development of robust kinetic models and in vitro tools of relevance to safety assessments in the consumer products industries, it will be important to define the limitations of existing CaCo-2 systems Poster Session P38. Toxicodynamics/toxicokinetics and develop in vitro oral absorption models that deal with lipophilic compounds appropriately. 726 THE RAPID GENERATION OF PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS G. Loizou, M. Spendiff, M. Penney, E. Pryde. Exposure Modelling Section, Health and Safety Laboratory, Broad Lane, Sheffield S3 7HQ, UK Physiologically based pharmacokinetic (PBPK) modelling is a powerful means of simulating the factors that determine tissue dose within any organism. The ability to correlate tissue dose with a response e.g., a health effect, has led to the increased use of PBPK models in chemical risk assessment. PBPK models are tools for integrating in vitro and in vivo mechanistic, pharmacokinetic and toxicologic information through their explicit mathematical description of important anatomical, physiological and biochemical determinants of chemical uptake, disposition and elimination. Consequently, the greater and varied input parameter demands of PBPK models, has led to them being described as ’data hungry’ and ‘resource intensive’. In order to address the latter and facilitate the more widespread use of PBPK modelling in chemical risk assessment, the Health and Safety Laboratory has developed a model equation generator (MEG) and PBPK parameter database, which permits the construction of models in minutes rather than days. Currently, the MEG is a stand-alone code generator that eliminates the need to formulate and code a set of equations. The user is engaged in a dialogue relating to the details of the physiology of the system to be modelled and the biochemistry and physicochemistry of the compound of interest. On the basis of this information, a script is produced which may be visualised in two software platforms (Berkerley Madonna and MCSim). Anatomical, physiological, biochemical and physicochemical parameters are retrieved from an electronic database, which is interrogated during use of the MEG. It is envisaged that this package would facilitate the dialogue between industry and regulator. Also, the ability to integrate in vitro data into PBPK models enhances the value, and facilitates the interpretation of many in vitro techniques proposed as alternatives to the use of animals in toxicological research. Therefore, the greater availability of such a PBPK modelling capability could potentially also lead to a marked reduction in the use of animals. 727 INFLUENCE OF HEAVY METALS UPON RETENTION OF POLONIUM-210 IN RAT J. Rencová, A. Vlková, G. Veselá. Centre of Industrial Hygiene and Occupational Diseases, National Institute of Public Health, Praha, Czech Republic In both the environment and the workplace, injurants can be taken up by the human body in combination rather than individually. For better assessment of a health hazard it is necessary to investigate the combined effect of noxious factors. To attack this problem experimental work has been directed towards the investigation of biokinetics of alpha-emitting radionuclide 210 Po as influenced by exposure to heavy metal ions. Wide distribution of 210 Po in tissues with different affinity enables to observe the reaction of a large spectrum of tissues to any pretreatment. Female rats were injected intraperitoneally with a solution of CdCl2 or Pb(CH3COO)2 (1 mg Cd2+ or 5 mg Pb2+ kg−1 body weight, according to toxicity) and after 9 or 15 h they received 210 Po nitrate (35 kBq kg−1 body weight) intravenously. Three days later, 210 Po was determined in dissolved tissue samples by the liquid scintillation method. Radioactivity was measured in the blood, spleen, liver, kidneys, brain, lungs, heart, thymus, muscle, skin, skeleton, femoral bone marrow, small and s195 large intestine. In both groups of Cd2+ pretreated rats the distribution pattern of 210 Po was similar. Mostly a small significant decrease of 210 Po in tissues was measured when compared with control rats receiving only 210 Po. A significant increase was found only in the thymus and large intestine. The total balance of 210 Po in all tissues was decreased to 87 and 95% of controls, respectively. However, distribution pattern of Pb2+ pretreated groups was different. When 210 Po was injected with a 9-h delay a large decrease of radioactivity in the blood, liver and bone marrow, and its increase in the small intestine, thymus, kidneys and skeleton were found. In the case of a 15-h delay a large decrease of 210 Po only in the blood and liver and its increase in the thymus, small and large intestine, spleen, skeleton, kidneys, muscles and skin were found. The total balance of 210 Po in the body was 72 and 81% of that in controls, respectively. Though this balance in pretreated groups partly decreased, the radiation risk from incorporated radionuclide increased in respect to its tissue redistribution. Supported by the Grant Agency of the Ministry of Health of the Czech Republic via Grant NJ6772–3/2001. 728 PREDICTION OF HUMAN VARIABILITY USING KINETIC DATA AND MONTE CARLO MODELLING FOR THE DERIVATION OF PATHWAY-RELATED UNCERTAINTY FACTORS FOR COMPOUNDS HANDLED BY MULTIPLE PATHWAYS. J.L.C.M. Dorne, A.G. Renwick. University of Southampton, Clinical Pharmacology Group, School of Medicine, Bassett Crescent East, Biomedical Sciences Building, Southampton, SO16 7PX, United Kingdom Previous studies using compounds handled extensively by a single major metabolic pathway (>60% of an oral dose) have generated pathway-related uncertainty factors to replace the general default uncertainty factor for human variability in kinetics. In the present work, the aim was to validate the use of Latin hypercube (Monte Carlo) models to predict inter-individual variability for compounds handled by multiple pathways of elimination. Seven compounds covering a wide range of monomorphic (antipyrine and paracetamol) and polymorphic pathways (codeine, diazepam, imipramine, proguanil and propranolol) were selected. For each substrate, the model was designed using quantitative metabolism data from independent studies describing the fraction of a dose handled by each pathway, with the sum of all fractions set equal a 100% of the dose, and the variability in each pathway (as lognormal distributions) derived from our published database. For all compounds, uncertainty factors were calculated from the predicted inter-individual variability from these simulations (10 simulations run for each study using 10,000 iterations) and compared with the uncertainty factors derived from the variability reported in published kinetic studies on these substrates. Overall, the results of the Latin hypercube (Monte Carlo) models have shown that it is possible to predict human variability in kinetics (and the corresponding uncertainty factors) with reasonable accuracy for compounds handled by multiple pathways. The small observed differences between published and simulated data probably arise from data limitations and inconsistencies between published studies, related to the available quantitative metabolism and kinetic data in addition to the assumptions formulated during model design and the sampling process. Any errors introduced by such mathematical approaches would be likely to be small compared with the unrecognised errors that would arise if the default uncertainty factors were to be applied inappropriately. Acknowledgements: We are grateful the Health Canada for supporting these studies. Author Index Aaltonen, A., s54 (188) Abd el Aziz, K.B., s145 (542) Abd El- Nasser, M., s170 (633) Abdallah, F.R., s84 (304) Abdel Raoof, A., s145 (542) Abdel-Hamid, N.M., s84 (304) Abdel-Wahhab, M.A., s66 (234) Abdo, K.M., s57 (201) Abel, J., s133 (495), s167 (624) Abolhassani, F., s108 (396) Aboul Enein, A., s62 (219) Abozaripour, M., s108 (396) Acquati, F., s55 (192) Acquavella, J., s189 (706) Ada, A.O., s155 (579, 581), s165 (616) Adachi, S., s182 (678) Adachi, T., s55 (193), s176 (655), s178 (663, 665) Adams, T.B., s16 (51) Adamska, J., s155 (580) Adamson, G., s32 (109) Adjaraov, D., s139 (520) Adkins, J.N., s162 (602) Aeby, P., s29 (97) Afshari, P., s112 (414, 415) Agazzi, A., s73 (263) Aggarwal, M.K., s147 (548) Ahmadian, M., s78 (282) Ahmadiani, A., s83 (299) Ahr, H.-J., s31 (105) Ahr, H.J., s30 (100), s44 (155), s102 (375) Aidarova, L.F., s164 (611) Aina, R., s157 (588) Ainis, L., s162 (605) Airoldi, L., s50 (175) Albarrán, M., s71 (256) Albert, M., s76 (276) Albrecht, C., s105 (387, 388) Alebouyeh, M., s83 (299) Alemdar, Y., s155 (581), s165 (616) Alenius, H., s36 (125), s163 (609) Alexander, J., s59 (207, 208), s122 (454), s123 (455, 457) Alexandropoulou, K.N., s101 (373) Alfaro-Moreno, E., s106 (389), s182 (679), s183 (681) Ali, A.M., s114 (423) Ali, N., s77 (277) Alizadeh, A., s43 (148) Allshire, A., s35 (120) Altucci, L., s27 (90) Alvarez, L., s41 (142), s65 (231), s168 (626) Aly, S.E., s66 (234) Amamou, M., s170 (634, 635), s171 (636) Amin, R.S., s84 (304) Amra, H.A., s62 (219) Anahara, R., s82 (296), s163 (607) Anciaux, K., s98 (362) Andjelinović, S., s140 (522) Anderson, D., s28 (94) Andersson, U., s36 (125) Andreassen, A., s123 (455) Andreoli, C., s128 (475), s157 (585) Andreoli, M., s76 (274) Andrysik, Z., s121 (447, 449) Angeleri, S., s35 (122) Angelini, S., s26 (84), s155 (578) Angeloni, R., s165 (617) Angelopoulos, D., s100 (369) Angerer, J., s158 (590) Anoopkumar-Dukie, S., s35 (120) Antonijevic, B., s131 (487), s135 (502) Antonioli, C., s9 (25) Anttinen-Klemetti, T., s159 (594) Anzenbacher, P., s56 (195) Anzenbacherová, E., s56 (195) Aposolova, D., s139 (520) Appendino, G., s115 (427) Arakawa, O., s69 (248), s70 (249) Arancia, G., s182 (677) Arantes, E.C., s68 (243, 244) Arbillaga, L., s65 (231) Arcella, D., s3 (12) Argentino Storino, A., s47 (165), s187 (699) Arrigoni, S., s72 (260) Arteaga, M., s80 (290) Arteaga, M.E., s41 (141), s57 (200), s81 (292) Arteaga-Pérez, M., s80 (291) Aruga, C., s157 (586) Arzi, A., s118 (439) Asadi, M.H., s100 (370), s111 (409) Asgari, A., s91 (336) Ashby, J., s27 (91) Assisi, F., s67 (238) Ates, I., s40 (138) Attia, M., s40 (140), s77 (280) Auberry, K., s162 (602) Autrup, H., s25 (83) Aviles, P., s88 (321), s98 (362) Aydın, S., s124 (460) Ayehunie, S., s29 (96) Ayesh, A.M., s62 (219) Aynaou, A.-E., s194 (725) Ayrault, S., s138 (515) Aytekin, T., s149 (557) Azizi, M., s110 (407) Babaei, M., s42 (145) Babenko, L.P., s101 (374) Babulovska, A., s70 (252), s73 (262), s75 (271) Baccarelli, A., s18 (57) Bacha, H., s64 (228) Baconi, D.L., s87 (316), s146 (543) Bada, A., s81 (292), s125 (465) Bada, A.M., s41 (141), s57 (200) Bada-Barro, A., s80 (291) Badiei, K., s185 (691) Bae, H.-S., s181 (674) Baer, B.R., s150 (559) Baeyens, A., s25 (81) Baggio, A., s167 (622) Bagley, D., s47 (163) Bagliy, E., s149 (556) Bagnati, M., s161 (600) Bahadoran, H., s108 (398), s111 (409) Bahmanpour, S., s114 (421) Bahrami, F., s49 (170), s132 (492) Bahrami, S., s126 (470) Bahrami, Z.S., s126 (470) Bahri Najafi, R., s87 (320) Bajic, V., s100 (369) Baker, R., s114 (423) Baker, V.A., s45 (157) Bakhshayesh, M., s140 (521) Bakhtiarian, A., s77 (279), s171 (637) Balalau, D., s87 (316), s146 (543) Balduzzi, M., s183 (682) Ballerani, A., s165 (617) Ballero, M., s115 (427) Ballester-Labrada, A., s80 (291) Ballini, A., s164 (613) Balloni, L., s129 (480), s133 (496) Bandoh, T., s130 (484) Banerjee, B.D., s39 (135) Bang, S.R., s167 (625) Baniasadi, S., s140 (521) Bańkowski, R., s178 (662) Bannasch, P., s103 (379) Bannon, G.A., s32 (111) Barakat, M., s97 (357) Baranowska-Bosiacka, I., s140 (524) Bárány, E., s135 (504) Barbarestani, M., s108 (396) Barbic’, F., s161 (600) Bardina, L.R., s119 (443) Barghash, N., s97 (357) Barišin, A., s71 (255) Barlow, S., s60 (211) Barni, S., s129 (481) Baro-González, F., s80 (291) Barrot, C., s140 (523) Bartels, M., s32 (111) Bartesaghi, S., s146 (546) Başaran, A.A., s124 (460) Başaran, N., s124 (460, 461) Basayiannis, A.C., s101 (373) Basić, Z., s61 (212) Basile, M., s161 (600) Bast, A., s119 (440) Bastos, M.L., s117 (434), s150 (561) Batoréu, M.C.C., s142 (528), s177 (661) Baudouin, C., s79 (286) Baudouin, Ch., s79 (288), s80 (289) Baudrimont, I., s64 (228) Baum, C., s11 (32) Baunsgaard, D., s8 (20) Beamonte, A., s75 (273) Becarovski, N., s70 (251) Becher, R., s121 (448) Becic, F., s188 (701) Beck, H., s29 (97) Becker, A., s105 (387) Beckers, L., s93 (344) Bednarczyk, A., s148 (554) Beklová, M., s172 (642), s173 (645) Bekyrou, M., s25 (83) Belcastro, M., s137 (512) Bellevance, K., s29 (96) Bellomo, G., s161 (600) Benassi, L., s46 (160) Benati, D., s102 (377) Benech-Kieffer, F., s160 (597) Benfenati, E., s52 (183) Benini, A., s158 (591) Beňo, M., s104 (383) BenSalah, D., s170 (634) Bensoussan, L., s79 (286) Berbers, G.A.M., s9 (24) Berejna, L., s152 (569) s198 Bergdahl, I.A., s135 (504) Bergman, Å., s179 (668) Bergsten, C., s59 (207) Bergström, U., s153 (573) Bernard, A., s163 (606) Bernareggi, G., s72 (260) Berrino, L., s93 (342) Bertail, P., s190 (709) Bertazzoni, G., s46 (160) Bertazzoni Minelli, E., s158 (591) Bertheux, H., s75 (273) Berthoin, K., s163 (606) Berti, F., s56 (197) Beskid, O., s17 (55) Betts, C.J., s33 (112) Beutel, G., s11 (32) Bewtra, J.K., s186 (692) Bezek, Š., s76 (275) Bezencon, C., s65 (232) Bhadoran, H., s100 (370) Biagi, G.L., s156 (583) Bianchi, M.G., s55 (192) Bianchi, R., s95 (351) Bielen, F., s32 (110) Biggioggero, M., s9 (25) Bilau, M., s60 (209) Binaglia, M., s146 (546) Binkova, B., s17 (55), s158 (589) Birindelli, S., s38 (132, 133) Biros, E., s17 (55), s158 (589) Bismuth, C., s22 (72) Biswas, N., s186 (692) Bitsch, A., s52 (181) Bizzeti, M., s137 (510) Björklund, Å., s33 (114) Black, W.D., s62 (217) Blagojevic, D.B., s145 (541) Blaha, L., s121 (447) Blain, P., s154 (576) Blanuša, M., s135 (505) Blasco, E.S., s57 (198) Blaszkewicz, M., s128 (477), s166 (619) Blazka, M., s47 (163) Blondin, C., s79 (286), s80 (289) Blust, R., s48 (169) Bobek, P., s104 (383) Bocheva, G., s120 (445) Bode, G., s21 (67) Bode, N., s98 (362) Bogunia, M., s186 (694) Böhm, K.J., s126 (468) Bohne, J., s11 (32) Boitier, E., s19 (61) Bojadjieva, N., s177 (660) Bokonjić, D., s78 (283) Bokonjic, D., s63 (223), s85 (311), s131 (487), s175 (651) Bolt, H.M., s125 (464), s126 (468), s165 (614) Bomfim, J.H.G.G., s68 (244) Bonacker, D., s126 (468) Bonato, M., s51 (178), s162 (604) Bonora, A., s123 (459) Boots, A.W., s119 (440) Borba, H., s157 (587) Borges, F., s91 (335), s98 (364), s99 (365), s117 (434) Borghi, O., s9 (25) Borlak, J., s53 (186), s54 (190), s92 (338), s93 (341, 343), s94 (347), s95 (349), s103 (381) Borm, P., s105 (387) Borm, P.J.A., s105 (388) Boroushaki, M.T., s131 (486) Author Index Borràs, M., s172 (641) Bortkiewicz, A., s92 (339, 340) Bortolato, M., s128 (476) Bortolotti, F., s162 (604), s183 (683) Borz˛ecki, A., s147 (549) Bosgelmez, I., s115 (425) Bosio, A., s54 (190) Bošnir, J., s185 (690) Botham, P.A., s14 (43) Boujlel, K., s170 (635), s171 (636) Bourne, N., s47 (166) Bousnina, M., s170 (634, 635), s171 (636) Bove, A., s73 (263) Bowe, G., s55 (192), s123 (458) Boyadjieva, N., s38 (130), s83 (301), s134 (501) Boyadjieva, N.I., s132 (491) Bozinovska, C., s70 (252), s71 (254), s73 (262, 264), s75 (271) Brandenburger, L., s103 (379), s161 (601) Brandt, I., s49 (170), s95 (348) Brankovic, S.D., s145 (541) Bratteby, L.-E., s135 (504) Brauch, H., s165 (614) Braykova, A., s154 (574) Breckx, V., s78 (284) Bridges, J., s188 (702) Briggs, D., s192 (718) Brightwell, J., s84 (307) Brignoccoli, A., s46 (162) Brignole, F., s79 (286, 288), s80 (289) Britos, S., s190 (710) Brittebo, E., s95 (348) Brittebo, E.B., s153 (573) Broccia, M.L., s107 (393, 394) Broding, C., s158 (590) Broeckaert, F., s189 (706), s190 (707) Brosch, G., s78 (284) Brouwer, A., s179 (668) Brown, D., s11 (29), s104 (385) Brown, R., s164 (612) Browne, L., s85 (308) Brüning, T., s158 (590), s159 (592), s165 (614) Brunskill, N.J., s96 (353) Brzóska, M.M., s95 (350) Buchancova, J., s154 (575) Buitenhuis, C.J.K., s179 (668) Bukvic, N., s164 (613) Bulanova, N., s126 (469) Bull, A.D., s30 (102) Buratti, F.M., s152 (566) Burgalassi, S., s46 (162), s86 (313) Burgaz, S., s155 (581), s165 (616) Burgos, A., s61 (213) Burlak, G.F., s172 (640) Burnett, R., s36 (124) Burnham, B., s45 (158) Bursch, W., s19 (60) Busk, L., s14 (41) Buss, K., s54 (190) Butcher, H., s120 (446) Butera, R., s67 (237), s72 (260), s73 (263), s74 (268) Byard, J., s51 (180) Byun, B.-H., s87 (317, 318), s91 (334) Caccin, P., s12 (34) Cadby, P., s16 (50) Caddick, H.T., s33 (112) Cadet, J., s118 (436) Cage, S., s2 (6) Cain, K., s19 (61) Calabrò, C., s162 (605) Calcabrini, A., s182 (677) Caldarelli, A., s53 (187) Caldwell, J., s17 (54) Calini, V., s182 (680) Calò, M., s145 (540) Calva-Treviño, V., s182 (679) Camatini, M., s182 (680) Camel, E., s43 (150) Campbell, J., s191 (712) Campi, V., s123 (458) Campos, E.S.T., s142 (528) Camus, M., s190 (708) Cantelli Forti, G., s155 (578) Cantelli-Forti, G., s56 (197), s156 (583) Cantillana, T., s49 (170) Cao, C., s47 (166) Cappadoro, M., s45 (156), s46 (159) Caramona, M.M., s122 (452) Cardelli, M., s167 (623) Carelli, G., s36 (123), s42 (146) Carettoni, L., s161 (600) Carey, J., s35 (120) Carfi’, M., s123 (458) Carli, M., s72 (260) Carlsson, C., s49 (170) Carmichael, N., s193 (720) Carmichael, P., s2 (6) Carmo, H., s150 (560, 561) Carpi, D., s50 (175) Carracedo, G., s177 (659) Carrera, V., s49 (172) Carthew, P., s2 (5) Carvalho, A.P., s122 (452) Carvalho, C.M.L., s142 (528) Carvalho, F., s91 (335), s98 (364), s99 (365), s117 (434), s150 (560, 561) Carvalho, M., s91 (335), s98 (364), s99 (365), s150 (561) Casacó, A., s41 (141), s80 (290, 291), s81 (292) Casartelli, A., s51 (178), s193 (721) Casati, S., s47 (166) Caselli, M., s46 (160) Cassani, C., s161 (600) Castegnaro, M., s66 (235) Castillo Soria, O., s75 (272) Castoldi, A.F., s129 (480, 481), s133 (496) Castro, R., s190 (710) Casu, V., s115 (427) Cavin, C., s65 (232) Cebovic, T., s67 (239, 240), s117 (433) Ceccatelli, R., s177 (658) Ceccatelli, S., s11 (31) Cecchini, A.L., s68 (243) Cecchini, R., s68 (243) Çeçen, Ş.Ş., s74 (266), s115 (426) Čelechovská, O., s174 (648) Celiński, R., s113 (418) Celona, A., s145 (540) Cengiz, G., s115 (426) Cenijn, P., s179 (668) Ceppitelli, R., s165 (617) Ceriello, A., s93 (342) Cerkvenik Flajs, V., s173 (644) Černá, S., s104 (383) Chabicovsky, M., s19 (60) Chahoud, I., s175 (653) Chaika, Y., s189 (704) Chamorro, G., s126 (467) Chan, S., s112 (416) Chang, L.W., s143 (532) Chaparoska, D., s70 (251) Chapoval, S., s33 (113) Charro-Ruiz, L., s80 (291) Author Index Cheng, L.C., s143 (532) Cheng, Y.H., s143 (532) Cheon, M.A., s135 (503) Chetoni, P., s46 (162), s86 (313) Chevalier, G., s77 (280) Chevalier, S., s92 (337) Chhabra, R.S., s123 (456) Chiabrando, C., s50 (175) Chiesara, E., s169 (629, 630), s180 (671) Chilian, B., s159 (592) Chipman, J.K., s99 (366) Chiusolo, A., s51 (177) Cho, D., s130 (482) Cho, D.H., s106 (390) Cho, M.H., s106 (390) Cho, M.J., s167 (625) Cho, S., s180 (672) Choi, H., s148 (553) Choi, S.M., s180 (670) Choi, Y.S., s135 (503) Chramostova, K., s121 (449) Christ, M., s36 (124) Christen, M.-O., s116 (432) Christophersen, P., s85 (310) Chu, I., s106 (391) Chung, K.H., s167 (625) Chung, S.T., s39 (136) Chvatalova, I., s17 (55), s158 (589) Cibisev, A., s71 (254), s72 (259) Cicalese, R., s84 (307) Ciereszko, A., s140 (524) Cimaz, R., s9 (25) Citterio, S., s157 (588) Civeira Murillo, E., s75 (272) Clarke, N., s27 (90) Claude, N., s75 (273) Clear, M., s47 (166) Clemente, S., s183 (682) Clifford Murray, J., s106 (389), s183 (681) Clippe, A., s163 (606) Clothier, R., s47 (166) Clothier, R.H., s47 (164) Coccini, T., s67 (237), s129 (480, 481), s133 (496) Cocker, J., s146 (544) Codorean, E., s35 (121) Codreanu, C., s119 (442) Cohen, S., s16 (51) Coleman, D., s30 (103) Colombo, A., s52 (183) Colombo, I., s185 (688) Colombo, M.L., s67 (238) Colone, M., s182 (677) Colosio, C., s38 (132, 133) Combes, R., s43 (149), s48 (167) Combes, R.D., s193 (722) Condevaux, F., s34 (118), s35 (119) Conscience, M., s177 (658) Constantin, C., s37 (128) Conto, A., s62 (220), s186 (696), s187 (697) Conway, L.N., s192 (718) Coporda, A., s171 (638) Corazza, M., s137 (510) Corbella, J., s140 (523) Cordelli, E., s144 (537) Cordingley, H.C., s51 (180) Corea, N.V., s191 (713) Corona, G., s115 (427) Corsico, N., s62 (220) Corsini, E., s1 (1), s38 (132, 133) Costa, L.G., s13 (37) Court, M., s22 (70) Coussement, W., s88 (321), s96 (355), s98 (362), s132 (493) Coyle, B., s19 (61) Cravedi, J.-P., s150 (562) Crebelli, R., s144 (537) Crepet, A., s190 (709) Creppy, E., s64 (228) Crespi, F., s76 (274) Cretinon, C., s34 (118) Crettaz, P., s52 (182) Creus, A., s28 (94) Creusy, C., s152 (567) Creuzot-Garcher, C., s79 (286) Crhova, S., s164 (612) Cristani, M., s145 (540) Cristescu, C., s166 (621) Cristofori, P., s76 (274), s102 (377), s123 (459), s183 (683) Crivellente, F., s51 (178), s162 (604) Cruz, M.T., s29 (99) Cuevas-Fiallo, A., s80 (291) Ćupić, V., s78 (283) Cupic, V., s63 (223), s175 (651) Curbelo, A., s125 (465) Curcic, M., s135 (502) Curfs, D.M.J., s93 (344) Curren, R., s47 (166) Cusic, S., s66 (236) Cvjetković, B., s64 (226) Czekaj, P., s153 (570, 571), s155 (580) Czerczak, S., s187 (698), s188 (703) D’Amico, M., s93 (342) da Costa, S.M.C., s89 (326) da Silva, J.O., s89 (326) Dacasto, M., s58 (204) Dachraoui, M., s170 (635), s171 (636) Dagues, N., s92 (337) Dal Negro, G., s51 (177, 178), s64 (225), s162 (604), s193 (721) Daly, A., s154 (576) Danilenko, V.Ph., s101 (374) Dányi, D., s76 (276) Dartsch, P.C., s184 (687) Dashtnavard, H., s78 (282), s100 (370), s108 (398) Dastnavard, H., s111 (409) Dastych, J., s34 (116) Davalli, S., s102 (377) Davidson, R.G., s96 (353) Davis, A., s45 (156) Davis, E., s154 (576) De Angelis, G., s176 (657) De Angelis, I., s58 (204), s63 (224) De Berardis, B., s182 (677), s183 (682) de Boer, D., s150 (560) De Coen, W., s48 (169) De Coster, R., s88 (321), s98 (362) De Felip, E., s167 (623) de Godoy, M.A.F., s68 (244) De Henauw, S., s60 (209) de Jouffrey, S., s31 (104) de Lapuente, J., s172 (641) de Ligt, R.A.F., s194 (725) de Lourdes Bastos, M., s150 (560) de Oliveira, A.M., s68 (244) De Oliveira, R.C., s168 (628) De Ridder, L., s25 (81) de Roos, J.A.D.M., s50 (174) De Smedt, A.C.A., s29 (98) De Wever, B., s45 (156), s46 (159) Dearman, R.J., s31 (106), s32 (111), s33 (112) Debbasch, C., s79 (288), s80 (289) s199 Débiton, E., s50 (174) Definis Gojanović, M., s140 (522) Degen, G.H., s126 (468) Dehghani, F., s97 (356), s110 (406, 407) Dehpour, A.R., s84 (305) Dehpour, R., s91 (336) Deiana, M., s115 (427) Dekura, E., s157 (586) Del Bino, G., s23 (73) del Favero, J., s48 (169) Delaforge, M., s138 (515) Delatour, T., s65 (232) Delongeas, J.L., s75 (273) Demirbag, A.E., s155 (581) Demircan, A., s74 (267) Demirel, B., s74 (267) Demiroglu, C., s155 (581), s165 (616) Demonakou, M.D., s101 (372, 373) den Hartog, G.J.M., s119 (440) Derakhshanfar, A., s70 (250) Derrick, J., s134 (498) Descotes, G., s48 (167) Descotes, J., s21 (66, 68), s34 (118), s35 (119), s36 (124) Desmidt, M., s96 (355) Desogus, G.F., s73 (265), s108 (397), s166 (620) Dessì, C., s128 (476) Dessì, M.A., s115 (427) Dezfullian, R., s97 (356) Di Carlo, B., s61 (214), s84 (303) Di Consiglio, E., s176 (657) di Domenico, A., s167 (623) Di Filippo, C., s93 (342) Di Renzo, F., s107 (393, 394) Di Virgilio, A.L., s125 (464) Dick, I., s2 (6) Dickins, M., s52 (184) Diels, L., s88 (321) Dierickx, P.J., s48 (168), s119 (441) Dietrich, G.J., s140 (524) Dimitrijević, J., s83 (300) Dimitrov, G.Z., s132 (491) Din, L.B., s114 (423) Diodovich, C., s55 (192) Dip, A., s74 (267) Dishovsky, C., s90 (331) Djhangiurir, B., s98 (361) Djordjevic, A., s67 (240) Djukanovic, D., s90 (329, 330) Djukic, D., s135 (502) Djukic, M., s135 (502) Dlasková, Z., s130 (485) Do, S.-H., s86 (312), s87 (317, 318), s91 (334) Dobrić, S., s78 (283) Dobric, S., s63 (223), s85 (311), s175 (651) Dobšíková, R., s172 (642) Dolnhikoff, M., s171 (639) Dolo, L., s150 (562) Domagala, J., s140 (524) Domańska, K., s60 (210) Domijan, A.-M., s64 (226) Domingues, P., s117 (434) Donaldson, K., s11 (29) Donelli, G., s184 (686) Donnez, J., s167 (623) Djordjević, S., s173 (646) Doria, D., s158 (591) Dorne, J.L.C.M., s195 (728) dos Reys, L.A., s150 (560) dos Santos, A.P.M., s177 (661) Doull, J., s16 (51) Drago, I., s164 (613) s200 Dragojevic-Simic, V., s85 (311) Drastichová, J., s174 (648) Drenska, D., s134 (501) Drommer, W., s105 (387, 388) Duarte, C.B., s29 (99) Dubovický, M., s76 (275) Duffin, R., s11 (29) Dumont, X., s163 (606) Durgo, K., s125 (466) Durnev, A.D., s124 (462), s127 (471) Dusinska, M., s42 (144), s154 (575), s156 (582) Dutta, P.C., s136 (508) Duydu, Y., s154 (577) Dvoretskaya, S., s39 (134) Dvoretskaya, S.I., s131 (489), s133 (497) Dybing, E., s41 (143) Dyring Jacobsen, S., s48 (167) Dyulgerova, G., s38 (130), s83 (301) Ebrahimi, I., s91 (333) Edelfors, S., s116 (428) Edgson, J., s106 (389) Efe, S., s155 (581), s165 (616) Egaas, E., s59 (206) Eisenbrand, G., s65 (230) El Ati-Hellal, M., s170 (635), s171 (636) El Nahas, E.M., s145 (542) El-barrany, U.M., s97 (358) El-Sayed, F., s97 (357) Elaut, G., s56 (196), s78 (284) Elezović, I., s173 (646) Elizondo, G., s37 (129), s121 (450), s153 (572) Ellinger, H., s102 (375) Emara, A.M., s97 (358) Engelke, M., s46 (161) Englert, N., s9 (26) Eom, J.H., s39 (136) Erpenbeck, V., s103 (381) Erra, M., s78 (284) Errigo, A., s167 (622) Esdaile, D.J., s32 (111) Eskandari, B., s97 (359) Esmans, E., s48 (169) Esquifino, A.I., s177 (659) Euliati, L.A., s101 (372) Evans, J.G., s99 (366) Eybl, V., s136 (506), s138 (513) Ezendam, J., s1 (2) Ezpeleta, O., s41 (142), s65 (231) Fà, M., s128 (476) Faass, O., s177 (658) Faberová, V., s76 (275) Færden, K., s59 (207) Fahim, M., s91 (336) Falfushynska, H., s169 (632) Falk Filipsson, A., s192 (716) Falzano, L., s182 (677) Fanelli, R., s50 (175) Farin, F.M., s156 (584) Farkas, N., s144 (536) Farmer, P.B., s8 (21) Farzaneh, E., s77 (277) Fattore, E., s50 (175) Faucet, V., s66 (235) Faustinelli, I., s183 (683) Fehse, B., s11 (32) Feinberg, M., s190 (709) Fenclová, Z., s130 (485) Ferenčić, Ž., s78 (285) Fernandes, E., s91 (335), s98 (364), s99 (365), s116 (430), s150 (560) Author Index Fernández, E.L., s153 (573) Fernández, M.C., s72 (257) Fernández, N., s125 (465) Fernández-Rodríguez, E., s178 (664) Feron, V.J., s16 (51) Ferrante, A., s114 (422), s183 (682), s184 (686) Ferraris, M., s169 (629, 630), s180 (671) Ferrer Dufol, A., s75 (272) Ferrer-Correia, A., s117 (434) Ferrer-Dufol, A., s189 (705) Fidalgo-Neto, A.A., s175 (653) Fila, A., s155 (580) Filippelli, A., s93 (342) Fimognari, C., s56 (197) Finn, E.S., s33 (113) Fiorentini, C., s182 (677) Flamma, F., s128 (475) Fleischmann, I., s177 (658) Fletcher, S.T., s45 (157) Florea, A., s87 (316) Florek, E., s74 (269), s120 (444) Flores-Morales, A., s54 (188) Flynn, D.J., s23 (74) Foà, V., s18 (56), s164 (613) Formichi, O., s35 (122) Forster, R., s31 (104), s40 (140), s77 (280) Fortina, A., s161 (600) Fortun, M.-C., s77 (280) Foth, H., s138 (514, 516), s139 (517, 518) Fouad, H., s97 (357) Fouad Abdel-salam, H., s162 (603) Fracasso, M.E., s158 (591) Franceschetti, P., s158 (591) Franco, J.J., s51 (179) Franco, J.P., s177 (661) Franekić Čolić, J., s125 (466) Frangež, R., s68 (242) Frantík, E., s109 (401) Franzén, A., s153 (573) Frau, R., s128 (476) Freathy, C., s19 (61) Freeman, S., s45 (156) Frewer, L., s13 (40) Freyberger, A., s179 (669) Friedrichs, B., s103 (380) Frieke Kuper, C., s21 (69) Frigerio, S., s169 (629, 630), s180 (671) Frings, W., s34 (117) Fritsche, E., s133 (495), s167 (624) Fu, T.J., s32 (111) Fuchs, R., s64 (226) Fuciarelli, A.F., s104 (382) Fuentes, D., s41 (141), s57 (200), s80 (290), s81 (292) Fufi, E., s74 (268) Fujimura, H., s157 (586) Fujiwara, K., s143 (533, 534) Fukata, H., s163 (607), s176 (655), s178 (665) Fumagalli, R., s169 (629, 630), s180 (671) Fuortes, L., s42 (144), s156 (582) Fustinoni, S., s18 (56), s164 (613) Gabrijelcic, M., s60 (211) Gadzicka, E., s92 (339, 340) Gage, V., s154 (576) Gaghes, M., s37 (128) Galabov, A.S., s154 (574) Galanopoulou, P., s102 (376) Gallacher, D., s134 (498) Galli, C.L., s15 (46), s38 (132, 133), s146 (546), s188 (702) Gao, X., s111 (410) García, A., s12 (35) Garcia-Allen, C., s92 (337) García-Cuellar, C.M., s182 (679) Garçon, G., s152 (567) Garduño-Siliciano, L., s126 (467) Garofani, P., s165 (617) Gašić, S., s173 (646) Gatta, L., s156 (583) Gatti, A., s35 (122) Gaurina Srček, V., s111 (411) Gautier, J.-C., s19 (61) Gazdag, Z., s144 (536) Gee, P., s85 (308) Geerke, D., s181 (675) Geh, S., s2 (6) Gelli, C., s122 (453) Gené, M., s140 (523) Georgatos, J., s73 (263) Georgescu, M., s35 (121) Georgiadis, P., s25 (83) German, D., s134 (499) Germann, P.-G., s20 (66) Germolec, D., s21 (69) Ghazi-Khansari, M., s98 (361), s106 (392), s171 (637) Ghobishavi, S., s118 (439) Gholipour, M., s171 (637) Ghorbel, H., s170 (634, 635), s171 (636) Giannotti, E., s64 (225) Giavini, E., s107 (393, 394) Gibicar, D., s141 (527) Gibney, M.J., s3 (10) Giglio, J.R., s68 (243, 244) Gijbels, M.J.J., s93 (344) Gil, A.G., s41 (142) Giray, B., s124 (461) Giugliano, D., s93 (342) Giuliano Albo, A., s58 (204) Gladkova, A., s81 (295) Glahn, F., s138 (514, 516) Glatt, C.M., s32 (111) Głogowski, J., s140 (524) Glukhova, L.D., s131 (489), s133 (497) Gmünder, H., s53 (185) Godschalk, R.W.L., s93 (344) Goebel, C., s29 (97) Goghataei, M.T., s68 (241) Gojmerac, T., s82 (297) Goldfain-Blanc, F., s75 (273) Goldfarb, P.S., s131 (486) Goldstein, D., s189 (706) Golja, V., s60 (211) Golubev, I.N., s131 (489), s133 (497) Gomółka, E., s74 (269) Gonçalo, M., s29 (99) Gonchar, O., s116 (431) Goncharenko, N.G., s172 (640) Gondrosen, B., s59 (206) González, B., s41 (141), s80 (290), s81 (292) González, B.O., s57 (200) González, C., s57 (200) González, E., s109 (403) Gonzalez, R., s89 (327) González, T., s144 (538) González, Y., s41 (141), s57 (200) González-Carracedo, A., s178 (664) Gonzalez-Delgado, I.F., s141 (525) Goodman, J.I., s16 (51) Goodman, R.E., s32 (111) Goriushko, A.G., s101 (374) Gosset, P., s152 (567) Gotelli, C., s190 (710) s201 Author Index Gotelli, D., s190 (710) Gotelli, M., s190 (710) Göttlicher, M., s27 (88) Goumenou, M., s127 (472), s148 (551, 552) Goverud, I.L., s122 (454) Granberg, L., s95 (348) Grasl-Kraupp, B., s19 (60) Grasso, P., s131 (486) Greiner, B., s42 (147) Gribaldo, L., s55 (192), s123 (458) Gribilas, G., s102 (378) Griffiths, D., s194 (725) Griffon, B., s31 (104), s40 (140) Groeng, E.C., s41 (143) Gronemeyer, H., s27 (90) Gryz, K., s153 (570) Gu, K., s180 (672) Gualtieri, M., s182 (680) Guanti, G., s164 (613) Gubała, W., s194 (724) Gubskiy, Yu.I., s101 (374) Gudasheva, T.A., s37 (126) Gudz, O.V., s49 (173) Guichard, J., s35 (119) Guignard, G., s65 (232) Guillot, J.P., s43 (150) Guizzetti, M., s13 (37) Gundert-Remy, U., s103 (379), s161 (601) Gunnarsson, K., s108 (399) Gusarova, I.A., s168 (627) Gustafsson, M., s33 (114) Gut, I., s152 (568) Gutiérrez, Á., s144 (538) Gutleb, A.C., s179 (668) Guvendik, G., s115 (425) Guzzi, G., s35 (122) Hack, R., s48 (167) Hadfield, N., s32 (111) Hadzimitova, V., s38 (131) Haenen, G.R.M.M., s119 (440) Hakansson, H., s50 (175) Hale, B., s62 (217) Hall, R.L., s16 (51) Hamano, Y., s69 (248), s70 (249) Hamard, P., s79 (286, 288), s80 (289) Hammerling, U., s33 (114) Hammond, T.G., s99 (366) Han, S.-S., s91 (334) Hanberg, A., s192 (716) Handal, A., s109 (403), s139 (519) Handall, A., s110 (404) Hansen, T., s54 (190) Hara, T., s40 (139) Harada, T., s40 (139) Harbell, J., s47 (163, 166) Hardisson, A., s61 (213), s141 (525), s144 (538) Harju, M., s49 (170) Harleman, J.H., s1 (2) Harvey, A., s69 (247) Harvey, J., s45 (156) Haseman, J., s47 (166) Hashemi, S.B., s130 (483) Hassan, Z.M., s40 (137) Hassanzadeh, P., s83 (299) Hassen, W., s64 (228) Hassibi, M., s87 (319) Haufroid, V., s154 (575) Haugen, M., s59 (208) Hayashi, M., s130 (484) Hayden, P., s45 (158), s47 (163) Hédhili, A., s170 (634, 635), s171 (636) Hedhiri, S., s170 (634) Hefle, S.L., s32 (111) Heidari, M.R., s87 (319) Heilbock, C., s27 (88) Heinrich, U., s10 (27) Heisler, E., s31 (105), s44 (154, 155) Hejtmancik, M., s123 (456) Hellmann, J., s103 (379) Helmi, E., s170 (634) Helms, R., s21 (69) Hemminki, K., s26 (84), s42 (144), s154 (575), s155 (578) Heng, Y.M., s106 (389), s183 (681) Hengstler, J.G., s150 (560) Henne, K.R., s150 (559) Henning, B., s23 (75) Henry, B., s32 (111) Hepburn, P.A., s192 (715) Herbert, R., s57 (201) Hereti, R.I., s101 (372, 373) Hernández, J., s41 (141), s57 (200), s81 (292) Hernández, O., s41 (141), s57 (200), s80 (290), s81 (292) Hernández, S., s168 (626) Hernández, Y., s57 (200) Hernández-Sosa, O., s80 (291) Herouet, C., s32 (111) Herout, V., s109 (400) Herrlich, P., s27 (88) Herzig, A., s103 (379), s161 (601) Herzyk, D.J., s26 (85) Hesso, A., s159 (594) Hetland, R., s121 (448) Heydari, A., s91 (336) Heyder, J., s104 (385) Heylings, J.R., s32 (111) Hickling, K.C., s99 (366) Hildebrand, H., s102 (375) Hirosawa, N., s134 (500) Hirsimäki, P., s54 (188) Hitchcock, J.M., s99 (366) Hiura, M., s40 (139) Hlynczak, A.J., s140 (524) Hofer, T., s118 (436) Hoffmann, J.J., s44 (154, 155) Hofmann, K., s54 (190) Hohn, A., s53 (185) Höhr, D., s105 (387) Holme, J.A., s121 (448) Holmes, E., s4 (16) Holownia, A., s118 (438) Holsapple, M., s21 (69), s32 (111), s34 (115) Holzhäuser, D., s65 (232) Honarjoo, M., s106 (392) Honary, S., s62 (218) Hong, S., s180 (672) Honma, T., s111 (410), s175 (654) Hood, S., s52 (184) Hooijkaas, H., s9 (24) Horand, F., s34 (118), s35 (119) Hornychová, M., s109 (401) Horska, A., s156 (582) Horský, S., s152 (568) Horta, C., s157 (587) Horvat, M., s141 (527) Hoshi, K., s130 (484) Houser, K., s161 (599) Hrelia, P., s25 (82), s56 (197), s155 (578) Hübel, F., s54 (190) Hübenthal, U., s133 (495) Hudson, S., s191 (712) Hueber-Becker, F., s160 (597) Huguet, E., s140 (523) Hur, S.-J., s69 (245) Hura, B.A., s58 (203) Hura, C., s58 (203) Hurbánková, M., s104 (383) Hurst, P.R., s114 (424) Husøy, T., s122 (454) Iavicoli, I., s36 (123), s42 (146) Ibrahim, Th.A., s170 (633) Ichinose, A., s70 (249) Ignatowicz, E., s120 (444) Iguchi, T., s179 (666) Ikezuki, Y., s179 (666) Ilie, M., s87 (316), s146 (543) ILSI/HESI Genomics Nephrotoxicity Working Group, s55 (191) Imani, H., s78 (282) Inayat-Hussain, S.H., s114 (423) Indovina, P.L., s114 (422), s183 (682), s184 (686) Ingelido, A.M., s167 (623) Innis, J.D., s32 (109) Institoris, L., s39 (135) Ionescu, D., s166 (621) Iosif, C., s35 (121) Irwin, A.J.E., s192 (718) Iscan, M., s155 (579, 581), s165 (616) Işık, A.F., s74 (267) Ismaeel, A.S., s109 (402) Itoh, M., s157 (586) Ittrich, C., s103 (379) Jablonska, E., s37 (127), s118 (438) Jablonski, J., s37 (127), s118 (438) Jaćević, V., s83 (300) Jacevic, V., s63 (223), s85 (311) Jackson, G., s45 (158) Jacobs, M.N., s52 (184) Jadidi, K., s42 (145) Jaeg, J-.P., s150 (562) Jafari, A., s87 (320), s88 (323) Jafari, M., s83 (302) Jahnova, E., s42 (144), s156 (582) Jakupec, S., s125 (466) Jalali, N., s71 (253), s72 (258) Jamshidzadeh, A., s97 (359) Janzowski, C., s65 (230) Jarnagin, K., s85 (308) Jarrell, J., s112 (416) Javanmardi, A., s88 (324), s89 (325) Javidnia, K., s87 (320), s88 (323), s114 (421) Jaworska, J.S., s14 (44) Jelić, K., s83 (300) Jelic, K., s85 (311) Jenner, B., s74 (269) Jeon, S.-H., s181 (674) Jeremic, D., s90 (329) Jhoo, W.K., s135 (503) Jimenez-Lara, A., s27 (90) Johnson, J.D., s57 (201) Johnson, K., s21 (69) Johnson, M.E., s133 (494) Johnston, E., s161 (599) Jokanovic, M., s100 (369) Jones, C.R., s192 (718) Jones, P., s44 (152, 153) Jones, R., s11 (29) Jongejan, A., s181 (675) Jorjani, M., s140 (521) Joung, K.E., s167 (625), s180 (672) Julin, B., s23 (76) s202 Jung, H., s130 (482) Junod, S., s65 (232) Jureša, D., s135 (505) Jurjević, Ž., s64 (226) Jurkiewicz, A., s142 (530) Kadota, T., s163 (607) Kaka, G.H., s111 (409) Kaka, Gh., s100 (370), s108 (398) Kakishima, H., s30 (101) Kakko, I., s147 (550) Kakuni, M., s40 (139) Kalantari, H., s98 (360), s101 (371) Kalenberg, K., s103 (379), s161 (601) Kamalee Neya, M., s105 (386) Kambas, N., s102 (376) Kamel, A.M.F., s97 (358) Kamenczak, A., s74 (269) Kamiński, M., s95 (350), s99 (367), s113 (418) Kamp, H.G., s65 (230) Kamyar, M., s64 (227) Kang, K.S., s135 (503) Kang, S., s180 (672) Kangas, L., s54 (188) Kapic, E., s188 (701) Kapplova, P., s121 (447) Karagiozova, O., s38 (131) Karakaya, A., s40 (138) Karbalaedoost, S., s110 (407) Karbalaei doust, S., s81 (293) Karbalay doust, S., s81 (294) Karelson, M., s52 (183) Karimi, A., s42 (145) Karimi, G.R., s91 (336) Karkucińska, M., s120 (444) Karpinski, S., s44 (154) Karpowicz, J., s153 (570) Kasai, H., s117 (435) Kashon, M., s21 (69) Kassel, O., s27 (88) Kašuba, V., s136 (509), s166 (618) Katayama, H., s134 (500) Katayama, S., s179 (667) Katsouyianni, K., s25 (83) Kaurinovic, B., s67 (239, 240), s117 (433) Kawatsu, K., s69 (248), s70 (249) Keene, W.E., s191 (714) Kehren, J., s22 (70) Keleş, A., s74 (267) Kemkowski, J., s48 (167) Kempka, G., s102 (375) Kenyon, S., s2 (6) Kermanian, F., s68 (241) Keyhani, M.R., s91 (333) Kharlamova, O., s126 (469) Khodakovskaya, O.A., s131 (489), s133 (497) Khomich, T.I., s119 (443) Khoshbaten, A., s91 (336) Khosravani, S., s129 (479) Khudaiberganov, A.S., s59 (205) Kiesswetter, E., s128 (477), s166 (619) Kil, J.H., s39 (136) Kil, K., s130 (482) Kilibarda, V., s61 (212), s66 (236), s83 (300) Kim, E.-J., s86 (312), s87 (317, 318), s91 (334) Kim, H.-J., s86 (312) Kim, H., s180 (672) Kim, H.C., s135 (503) Kim, H.S., s39 (136) Kim, H.W., s106 (390) Author Index Kim, J.-K., s86 (312) Kim, J.-Y., s69 (245) Kim, J., s130 (482) Kim, J.H., s148 (553), s169 (631) Kim, J.Y., s167 (625), s180 (672) Kim, K.-S., s91 (334) Kim, K., s130 (482) Kim, S., s130 (482) Kim, Y., s130 (482) Kimber, I., s1 (3), s27 (91), s30 (101), s31 (106), s33 (112) Kimoto, N., s40 (139) King, A., s44 (152, 153) Kirchner, B., s32 (108) Kirchner, B.A., s33 (113) Kjelkevik, R., s59 (206) Klausner, M., s29 (96), s45 (158), s47 (163) Kleiman de Pisarev, D.L., s168 (626) Klein, B., s165 (614) Kleiner, J., s15 (48) Kleinjans, J., s57 (199) Klenø, T.G., s8 (20) Kloos, C., s47 (163) Klump, H., s11 (32) Klyuchko, E., s116 (431) Kmetič, I., s111 (411) Knaapen, A., s105 (387) Knap, C., s141 (527) Kneller, M.B., s150 (559) Kniewald, Z., s111 (411) Kniewals, J., s111 (411) Knutsen, H.K., s122 (454) Kobal, A.B., s141 (527) Kobayashi, K., s175 (654) Koda, M., s163 (607) Koh, S., s130 (482) Köhlerova, R., s125 (463) Kohli, A., s167 (624) Kojima, C., s143 (534) Kojima, N., s40 (139) Kokshareva, N.V., s49 (173) Kolaja, K., s85 (308) Kolar, L., s173 (644) Kolářová, J., s181 (676) Kolega, M., s78 (285) Kolodub, F., s85 (309) Komiyama, M., s55 (193), s163 (607), s176 (655), s178 (663, 665) Kommineni, V., s21 (69) Kondili, V.G., s101 (372) Kondrová, E., s151 (563) Koo, H.J., s160 (596) Kooshapur, H., s101 (371) Kopp-Schneider, A., s103 (379) Korinth, G., s2 (6) Korn, M., s159 (592) Koropatnick, J., s120 (446) Koršić, M., s82 (297) Kosaka, T., s179 (667) Kosec, M., s68 (242) Kostial, K., s135 (505) Kostka, G., s151 (564), s178 (662) Kostogrys, R.B., s176 (656) Kotyzova, D., s136 (506), s138 (513) Kourenzi, K.T., s101 (372) Kouri, K., s64 (227) Kousalová, L., s56 (195) Koutensky, J., s136 (506), s138 (513) Kováčiková, Z., s104 (383) Kovalenko, L.P., s37 (126) Kovalenko, V., s88 (322), s152 (569) Kovkarova, A.E., s72 (259) Kovkarova, E., s73 (264) Kowalówka-Zawieja, J., s99 (367) Kowalski, B., s60 (210) Kozmin, G.V., s61 (215) Kozubik, A., s121 (449) Kožuh Eržen, N., s173 (644) Kraunus, J., s11 (32) Krause, E., s103 (379), s161 (601) Krauser, K., s48 (167) Kravchuk, A., s149 (558) Krishna, C., s47 (166) Kristiansen, K., s122 (454) Krnić, Ž., s78 (285), s86 (315) Kroes, R., s15 (48), s188 (702) Krötlinger, F., s179 (669) Krsnik, M., s141 (527) Krug, N., s103 (381) Krzyzanowski, R., s148 (554), s149 (555), s185 (689) Kubilus, J., s45 (158), s47 (163) Kudrya, M., s81 (295), s85 (309) Kumar, A., s141 (526) Kumar, M., s141 (526) Kumar, R., s26 (84), s42 (144), s154 (575), s155 (578) Kumar Sharma, M., s141 (526) Kunze, K.L., s150 (559) Kunze, M., s166 (619) Kupczewska, M., s187 (698) Kurabe, M., s157 (586) Kurapova, T.N., s101 (374) Kuricova, M., s42 (144), s154 (575), s156 (582) Kuriyama, S., s175 (653) Kurskaya, N.M., s101 (374) Kusturica, J., s188 (701) Kužner, J., s173 (644) Kuznetsov, P.E., s86 (314), s168 (627) Kuznetsova, N.B., s86 (314), s168 (627) Květina, J., s109 (400) Kwapuliński, J., s142 (530), s183 (684), s186 (694), s194 (723) Kwon, M.S., s135 (503) Kwon, Y.W., s169 (631) Kyrtopoulos, A., s25 (83) Kyrtopoulos, S.A., s104 (383), s156 (582) La Sala, G., s84 (303) Labidi, A., s170 (635), s171 (636) Ladics, G.S., s32 (111) Laengle, U.W., s42 (147) Laffert, B., s122 (453) Lafuente, A., s177 (659), s178 (664) Låg, M., s121 (448) Lah, B., s174 (647) Lahijani, M., s132 (492) Lalancy, M., s107 (395) Lammens, L., s48 (167), s88 (321), s96 (355), s98 (362) Lamore, S., s29 (96) Lampo, A., s132 (493) Landry, T.D., s32 (111) Lang, M., s153 (573) Lanham, D.F., s30 (103) Lanzoni, A., s76 (274), s123 (459), s183 (683) Lappen, R., s29 (96) Larese, F., s2 (6) Larondelle, Y., s62 (216) Lauriano, E.R., s162 (605) Laursen, S.M., s8 (20) Laus, G., s78 (284) Lavazza, A., s95 (351) Lavin, A., s34 (115) Author Index Laycock, S., s134 (498) Le Prieur, E., s138 (515) Lea, L.J., s192 (715) Leathart, J., s154 (576) Lebeau, C., s66 (235) Leblanc, J.-Ch., s190 (709) Leclercq, C., s3 (11) Ledon, N., s89 (327) Lee, B.M., s160 (596), s180 (670) Lee, C., s180 (673), s181 (674) Lee, H.S., s148 (553) Lee, J.K., s39 (136) Lee, K.M., s104 (382), s162 (602) Lee, M.-S., s69 (245), s180 (673), s181 (674) Lee, M., s128 (474) Lee, S.-H., s69 (245) Lee, S.H., s135 (503) Legler, J., s179 (668) Leiss, W., s13 (38) Lemmens-Gruber, R., s64 (227) Lemos, M., s171 (639) Lemos-Amado, F., s117 (434) Lennard, M.S., s146 (544) León-Goñí, A., s80 (291) Leopardi, P., s144 (537) Lepage, J.F., s75 (273) Lepoluoto, A., s94 (346) Leszczynski, B., s148 (554), s149 (555), s185 (689) Levitsky, E.L., s101 (374) Lewis, D.F.V., s52 (184) Li, A., s189 (706) Li, Z., s11 (32) Liapi, C., s102 (376) Licata, A., s162 (605) Licata, P., s145 (540) Lichtenberg, J., s85 (310) Lichtensteiger, W., s177 (658) Licoska, F., s70 (252), s71 (254), s72 (259) Liebich, H.M., s160 (595), s184 (687) Liesivuori, J., s165 (615) Lilienthal, H., s179 (668) Lim, H., s135 (503) Lima, J.L.F.C., s116 (430) Limardi, L.C., s33 (113) Limasset, J.C., s2 (6) Lindner, Y., s161 (601) Lishavskij, V.G., s192 (717) Liskova, A., s42 (144), s156 (582) Litfin, M., s27 (88) Litvinova, N.V., s101 (374) Liu, K.H., s148 (553) Lo Balbo, A., s190 (710) Loa, A., s122 (453) Løberg, E.M., s122 (454) Lobo, D.J.A., s168 (628) Locatelli, C., s67 (237), s72 (260), s73 (263), s74 (268) Lock, E.A., s96 (354) Loizou, G., s195 (726) Lonati, D., s74 (268) Longo, M., s108 (399) Longobardi, C., s47 (165) Looszova, A., s88 (321), s98 (362) Lopes, M.C., s29 (99), s122 (452) López, R.M., s121 (450) López de Cerain, A., s41 (142), s65 (231) López-Fuster, M.J., s174 (649) Lord, P.G., s4 (14), s7 (17), s55 (194) Lorenzi-Filho, G., s168 (628) Loska, K., s142 (530) Lourdes Bastos, M., s91 (335), s98 (364), s99 (365) Løvdal, T., s41 (143) Løvik, M., s41 (143), s59 (206) Lovik, M., s34 (116) Lovreglio, P., s164 (613) Lovric, Z., s171 (638) Lowe, R., s51 (180) Lozano, G., s144 (538) Lucić, A., s64 (226) Luft, D., s160 (595) Luigi Gessa, G., s128 (476) Lukáš, E., s130 (485) Lundehn, J.-R., s23 (74) Lundh, T., s135 (504) Luscombe, C., s52 (184) Lusková, V., s174 (648) Luster, M., s21 (69) Luster, M.I., s1 (4) Luther, E., s128 (474) Lutnicka, H., s96 (352), s172 (643) Lyon, J., s54 (189) Lyubomirova, K., s184 (685) Maas, W.J.M., s2 (6) Määttä, J., s36 (125), s163 (609) Mabondzo, A., s138 (515) Macchione, M., s168 (628) Maceira, M., s80 (291) Machala, M., s121 (447, 449) Machera, K., s24 (78), s127 (472), s148 (551, 552) Máchová, J., s174 (648) MacIntosh, S.C., s32 (111) Madkour, S.A., s162 (603) Madren-Whalley, J., s47 (166) Mäenpää, H., s79 (287) Maffei, F., s155 (578) Maggi, A., s61 (214), s84 (303) Magnoni, C., s46 (160) Mahdavinasab, H., s78 (282) Mahl, A., s22 (70, 71) Mahmoudi, M., s88 (324), s89 (325) Mahmud, Y., s69 (248), s70 (249) Mäittälä, J., s165 (615) Majuri, M.-L., s36 (125) Maksimovic, M., s131 (487) Maksudova, N., s127 (473) Malarkey, T., s20 (64) Malawska, M., s175 (652) Malerba, I., s55 (192), s123 (458) Malik, J.K., s147 (548) Malinverno, G., s185 (688) Malochkina, E.I., s131 (489), s133 (497) Manaei, M., s42 (145) Mancebo, A., s57 (200), s81 (292) Manciaux, X., s31 (104) Manda, G., s35 (121), s37 (128), s119 (442) Manda, M., s146 (543) Mandić, B., s82 (297) Manera, D., s108 (399) Mangelsdorf, I., s52 (181) Mannarino, S., s74 (268) Mannerström, M., s77 (278) Manno, M., s18 (56) Manolis, E., s102 (378) Mantovani, C., s161 (600) Manzanares, I., s88 (321), s98 (362) Manzo, L., s67 (237), s72 (260), s73 (263), s74 (268), s129 (480, 481), s133 (496) Marabini, L., s169 (629, 630), s180 (671) Maran, U., s52 (183) Maras, M., s48 (169) Marc, I., s173 (644) Marchenko, A.N., s101 (374) s203 Marchioro, C., s102 (377) Marcinczyk, M., s37 (127) Marcos, R., s28 (94) Marczynski, B., s158 (590), s159 (592) Marfella, R., s93 (342) Margeli, A.P., s102 (378) Margrett, S.L., s54 (189) Marin-Kuan, M., s65 (232) Marino, T., s137 (512) Marinovich, M., s38 (132, 133), s146 (546) Marinsek-Logar, R., s174 (647) Markakis, K., s24 (78) Markov, J., s90 (329, 330) Markstein, R., s42 (147) Marnett, L.J., s16 (51) Marocchio, L., s102 (377) Maroni, M., s38 (133), s188 (702) Maronpot, R., s21 (69) Marra, M., s182 (677) Marsh, J., s161 (599), s164 (612) Martella, A., s67 (238) Martella, S., s162 (605) Martens, M., s189 (706) Martens, M.A., s190 (707) Martin, L., s77 (280) Martín-Izquierdo, R.E., s141 (525) Martínez-Rivas, T., s177 (659) Martínez-Romero, F., s182 (679) Martino, A., s128 (475) Marzola, P., s102 (377) Masin, E., s70 (251) Masotto, I., s51 (178) Massa, V., s107 (393, 394) Massotti, M., s26 (86) Matek Sarić, M., s135 (505) Matousu, Z., s154 (575) Matsuno, Y., s163 (607) Matthys, C., s60 (209) Matvienko, A.V., s101 (374) Maunit, B., s152 (567) Maurici, D., s123 (458) Mazurek, U., s155 (580) McCune, J.S., s156 (584) McDougal, J.N., s28 (95) McEwen, A.B., s194 (725) McKinney, W.J., s104 (382), s162 (602) McTaggart, F., s96 (353) Meckert, C., s103 (379), s161 (601) Medina-Díaz, I., s153 (572) Meerman, J.H.N., s181 (675) Mehdizadeh’, M., s142 (529) Mehdizadeh, M., s68 (241), s144 (535) Mehrani, H., s78 (282) Mehrannia, K., s113 (420) Mehrannia, T., s113 (420) Melnikova, T.V., s61 (215) Melovska, L., s70 (251) Meltzer, H.M., s59 (207, 208) Mencarelli, A., s165 (617) Mendes, A.F., s122 (452) Menegola, E., s107 (393, 394) Meneguz, A., s26 (86) Mensing, T., s158 (590) Meregalli, E., s9 (25) Merget, R., s159 (592) Meroni, P., s108 (399) Meroni, P.L., s9 (25) Merrill, J., s47 (163) Merz, T., s158 (590) Mesbah, F., s81 (293, 294), s110 (407), s114 (424) Meschini, S., s182 (677) Meuling, W.J.A., s160 (597) Meyer, J., s11 (32) s204 Michel, C., s19 (61) Mikalsen, A., s123 (455) Milcova, A., s17 (55), s158 (589) Mileva, M., s154 (574) Milhazes, N., s91 (335), s98 (364), s99 (365), s117 (434) Milovanović, Z., s78 (283) Milovanovic, Z., s63 (223), s175 (651) Mimouni, C., s40 (140) Min, K.N., s167 (625), s180 (672) Minaee, B., s63 (221), s84 (305) Minoia, C., s35 (122) Miranda, M.S., s57 (198) Miri, R., s88 (323) Mirkhani, H., s81 (293, 294) Miscetti, G., s165 (617) Miskevich, D., s116 (429) Mitchell, R., s85 (308) Mitovanovic, S., s147 (547) Miyagawa, M., s175 (654) Miyakawa, H., s179 (666) Moczko, J., s120 (444) Modlich, U., s11 (32) Mofid, M., s78 (282) Moggs, J.G., s27 (91) Mogilat, L., s81 (295) Mohamadzadeh, I.F., s144 (535) Mohamed, M.I., s62 (219) Mohammadi, M., s97 (359) Mohammadie, A., s98 (361) Moinosadat, M., s71 (253) Möller, J., s103 (381) Möller, L., s118 (436), s182 (678) Möller, W., s104 (385) Møllersen, L., s123 (455) Molokhia, T.K., s162 (603) Moncef Ladjimi, M., s64 (228) Mondegari, A., s87 (319) Moniuszko-Jakoniuk, J., s95 (350), s118 (438) Monks, T.J., s98 (364) Monsef, H.R., s63 (221) Montazeran, H., s63 (221) Montazeri, B., s112 (413) Montecucco, C., s12 (34) Monti, D., s46 (162), s86 (313) Montomoli, L., s2 (6) Moon, C.K., s167 (625) Moon, J.K., s148 (553) Moore, E.L., s30 (102) Morán, C., s109 (403), s110 (404), s139 (519) Moran, G.F., s192 (718) Morán, J.L., s109 (403), s110 (404), s139 (519) Morandi, S., s157 (585) Moreno-Díaz, D., s80 (291) Mori, C., s55 (193), s82 (296), s163 (607), s176 (655), s178 (663, 665), s179 (666) Moriguchi, T., s134 (500) Moro, P.A., s67 (238) Morse, V., s161 (599) Morteza-Semnani, K., s31 (107), s88 (324), s89 (325) Mosiello, A., s187 (699) Mostaghni, K., s82 (298) Motta, A., s183 (682) Motta, M., s9 (25) Movahhed, A., s126 (470) Moyer, G.O., s47 (166) Mrkun, J., s68 (242) Mubarak, M., s63 (222) Mulabegovic, N., s188 (701) Mulder, P.G.H., s9 (24) Author Index Müller, D., s15 (48) Müller, F., s158 (590) Müller, J., s158 (590) Müller, J.F., s152 (567) Mun, G., s47 (166) Munaro, S., s123 (459) Munro, I.C., s16 (51) Murgia, A., s146 (545) Mutch, E., s151 (565) Myllynen, P., s113 (417) Na, J.-G., s180 (673), s181 (674) Naccari, F., s145 (540) Nadal, J., s145 (539), s171 (639), s172 (641), s174 (649) Naderi, M., s42 (145) Naghibi, F., s186 (692) Nagy, E., s182 (678) Nasiri, E., s75 (270) Nasiri, G., s106 (392) Nassrollazadeh, B., s107 (395) Natsoulis, G., s85 (308) Naudts, B., s48 (169) Naumov, I., s73 (264) Naumovski, Dz., s70 (252), s72 (259), s73 (262) Naumovski, J., s71 (254), s73 (264), s75 (271) Naya, M., s40 (139) Nazari, Z., s89 (328) Nazarov, G.V., s86 (314) Ndif, M., s170 (635) Neagu, M., s35 (121), s37 (128), s119 (442), s146 (543) Nebbia, C., s58 (204) Neca, J., s121 (447) Nedeljkovic, M., s131 (487) Nedeljkovic Trailovic, J., s66 (233) Nedhif, M., s170 (634), s171 (636) Nedopytanska, N., s149 (556) Neghab, M., s104 (384), s105 (386) Nemmar, A., s10 (28) Nesterova, E.V., s127 (471) Nicholson, J.K., s7 (19) Nicola Aru, G., s128 (476) Niehof, M., s53 (186) Nielsen, J.B., s2 (6) Nieradko, B., s147 (549) Niknahad, H., s58 (202), s97 (359) Nikolaou, T., s100 (369) Nikolov, M., s77 (281) Nilsson, G., s34 (116) Nishimura, D., s55 (193), s178 (665) Nobacht, M., s144 (535) Nobakht, M., s142 (529) Noda, S., s30 (101) Nogaj, E., s183 (684), s194 (723) Nogaj, P., s183 (684), s194 (723) Noguchi, T., s69 (248), s70 (249) Nogué Xarau, S., s75 (272) Nogué-Xarau, S., s189 (705) Nohynek, G.J., s160 (597) Noroozzadeh, A., s91 (336) Norouzy, A., s84 (305) Norppa, H., s25 (80), s154 (575) Norstedt, G., s54 (188) Nouhjah, S., s113 (419) Nowaczyk, G., s95 (350), s99 (367) Nunziata, A., s128 (475), s157 (585) Nyska, A., s21 (69) O’Brien, P.J., s58 (202) O’Keeffe, P., s35 (120) Oberemm, A., s103 (379), s161 (601) Obot, C.J., s104 (382) Ocaña, R., s125 (465) Ochoa, M.J., s139 (519) Ocio, J.A., s4 (13) Oesch, F., s150 (560) Ogawa, Y., s117 (435) Öge, K., s124 (461) Oh, H.Y., s39 (136) Ohmichi, K., s163 (607) Ohmichi, M., s163 (607) Ohta, R., s179 (667) Ohta, T., s179 (667) Okada, K., s69 (248) Okuda, H., s179 (667) Olejczyk, M., s183 (684), s194 (723) Ølstørn, H.B., s122 (454) Omidi, N., s134 (499) Ono, Y., s82 (296), s178 (663) Onsory, D., s107 (395) Orłowski, J., s99 (367) Orlowski, S., s138 (515) Orphanides, G., s27 (91) Orpheé-Suárez, R., s80 (291) Orrenius, S., s11 (31), s19 (59) Orrù, M., s128 (476) Ortega, M., s140 (523) Orton, T.C., s96 (353) Oruç, E., s149 (557) Osada, H., s179 (666) Oskarsson, A., s135 (504), s136 (507, 508), s137 (511) Osmak, M., s125 (466) Osornio-Vargas, A., s106 (389), s183 (681) Osornio-Vargas, A.R., s182 (679) Osredkar, J., s141 (527) Ostad, S.N., s40 (137), s63 (221) Östergren, A., s95 (348) Ostrovskaya, R.U., s37 (126) Otová, B., s152 (568) Øvrevik, J., s121 (448) Owen, M., s47 (166) Owens, W., s179 (667) Ozerov, M.Yu., s186 (693) Pacelli, V., s47 (165) Pachón, M., s71 (256) Padro-Gutiérrez, P., s80 (291) Page, D.A., s32 (110) Paik, S., s180 (672) Pajoumand, A., s71 (253) Palagina, I., s81 (295), s85 (309) Palamaru, I., s58 (203) Pałasz, A., s153 (570) Palasz, A., s153 (571) Panahi, Z., s110 (406) Panjehshahin, M.R., s97 (356), s110 (406, 407) Panoutsopoulos, G.I., s101 (372, 373) Paoletti, L., s182 (677), s183 (682) Paolini, M., s156 (583) Papa, P., s74 (268) Papadimas, G.K., s101 (372) Papadimitriou, L., s102 (378) Papadopoulos, A.I., s101 (372, 373) Papeleu, P., s56 (196), s78 (284) Papp, A., s39 (135) Paredes, E.Q., s57 (198) Parier, V., s79 (288) Paris, M.W., s47 (166) Parivar, K., s111 (409) Park, E.-R., s180 (673) Park, H.W., s148 (553) Park, J.H., s39 (136) Author Index Park, S.W., s169 (631) Park, Y., s130 (482) Parker, R., s32 (108, 109, 110) Parzefall, W., s19 (60) Pasbakhsh, P., s108 (396), s113 (420), s134 (499) Paskalev, Z., s139 (520) Pasqualatto, D., s71 (256), s72 (257) Pasquetto, S., s183 (683) Pastorelli, R., s50 (175) Patandin, S., s9 (24) Pate, I., s27 (91) Patzke, J., s46 (161) Paul, G., s22 (70, 71) Paulsen, J.E., s122 (454), s123 (455) Pauluhn, J., s186 (695) Pauncu, E.A., s166 (621) Pavard, J-M., s40 (140) Pavlov, J., s140 (522) Pavlova, S., s139 (520) Pavlovski, B., s71 (254) Pawlak, S., s151 (564), s178 (662) Pawlicki, K., s113 (418) Pawlowski, V., s92 (337) Payan, J.-P., s2 (6) Pease, C.K., s3 (9), s194 (725) Pecio, A., s176 (656) Pelclová, D., s130 (485) Pelicci, P.G., s27 (92) Peltonen, K., s159 (594) Pencikova, K., s121 (447) Pendlington, R.U., s194 (725) Pennanen, S., s165 (615) Penney, M., s195 (726) Pennie, W.D., s55 (194) Pennings, J., s1 (2) Peòa de Ortiz, S., s129 (478) Peraica, M., s64 (226) Perbellini, L., s18 (56) Perdu-Durand, E., s150 (562) Pereira, P.S., s69 (246), s89 (326) Perentes, E., s22 (71) Pereska, Z., s70 (252), s71 (254), s72 (259), s73 (262, 264), s75 (271) Pérez, G., s125 (465) Pérez-Zapata, A.J., s118 (437) Perharic, L., s60 (211) Perna, F., s156 (583) Pesti, M., s144 (536) Peters, P., s44 (154, 155) Petersson Grawé, K., s136 (508) Petkovska, L., s70 (252), s71 (254), s72 (259), s73 (262, 264), s75 (271) Petrolini, V., s72 (260), s73 (263), s74 (268) Petrovic, B., s145 (541) Petrovski, D., s70 (252), s72 (259), s73 (262) Petrunin, V., s39 (134) Petrunin, V.A., s131 (489) Pettit, S.D., s55 (194) Petushok, N.E., s116 (429) Pfister, R., s48 (167) Pfohl-Leszkowicz, A., s66 (235) Phillips, B., s48 (167) Piaia, A., s123 (459) Pickova, J., s136 (508) Piekoszewski, W., s74 (269), s120 (444), s194 (724) Pienimäki, P., s113 (417) Piersma, A., s8 (22) Piersma, A.H., s15 (45) Pieters, R., s1 (2) Pigatto, P., s35 (122) Piguet, D., s65 (232) Pikula, J., s172 (642), s173 (645) Pilovski, Gj., s73 (262) Piras, E., s153 (573) Pisella, P.-J., s79 (288) Pisulewski, P.M., s176 (656) Pitarque, M., s28 (94) Pitic, Lj., s90 (329, 330) Pizzocheri, F., s62 (220), s186 (696), s187 (697) Plavsic, F., s171 (638) Plesničar, S., s144 (536) Plewka, A., s95 (350), s99 (367) Plewka, D., s95 (350), s99 (367) Poellinger, L., s27 (89) Pogačnik, M., s173 (644) Poirier, K.A., s191 (711) Polezina, A.S., s131 (489), s133 (497) Poljšak, B., s144 (536) Polyakova, L.P., s61 (215) Ponce, G., s153 (572) Pongratz, I., s122 (451) Poon, R., s106 (391) Poorreza, N., s89 (328) Popa, A., s87 (316) Popescu, D., s35 (121) Popovic, M., s67 (239, 240), s117 (433) Popovski, N., s75 (271) Porpora, M.G., s167 (623) Porta, P., s161 (600) Portier, C., s21 (69) Portoghese, P.S., s16 (51) Potì, C., s67 (238) Pounds, J.G., s162 (602) Pourjafar, M., s70 (250), s82 (298), s185 (691) Pouzaud, F., s116 (432) Povyakel, L., s174 (650) Pralet, D., s42 (147) Prato, N., s157 (588) Prestipino, G., s146 (545) Preuss, R., s158 (590) Prezelj, M., s141 (527) Priestley, A., s194 (725) Primavera, S., s67 (238) Prinsen, M.K., s86 (315) Privalle, L.S., s32 (111) Prodanchuk, M., s174 (650) Prodanchuk, M.G., s49 (173), s112 (412) Prodanchuk, N., s149 (558) Pronko, P.S., s119 (443) Pryde, E., s195 (726) Puntarić, D., s185 (690) Purcell, W.M., s50 (176), s100 (368) Pursifull, A.C., s33 (113) Pushkin, A., s39 (134) Puzewska, W., s37 (127) Pylkkänen, L., s163 (609) Querfurth, N., s103 (379), s161 (601) Quesada, E., s49 (172) Raabe, H., s47 (166) Rabbani, A., s83 (302) Rabemampianina, Y., s48 (167) Rabstein, S., s159 (592) Radić, B., s64 (226) Radice, S., s169 (629, 630), s180 (671) Radivojević, Lj., s173 (646) Raducan, E., s35 (121) Raffalli-Mathieu, F., s153 (573) Rafsanjani Fatemeh, N., s77 (277) Ragab, A.A., s62 (219) s205 Rahimi, F., s140 (521) Rainaldi, G., s114 (422), s183 (682), s184 (686) Rajaeefard, A., s104 (384) Ramezani, M., s132 (492) Rämisch, A., s138 (514) Randi, A.S., s168 (626) Randine, G., s67 (237), s129 (480, 481), s133 (496) Rao, G.S., s147 (548) Rapp, A., s64 (227) Rasekh, H.R., s90 (332) Rashidi, B., s112 (413) Rashidi, I., s98 (363) Rashidi, I.M.D., s110 (405) Raspor, P., s144 (536) Rastegar, T., s142 (529) Rat, P., s79 (288), s116 (432) Ravel, G., s36 (124) Ražić, S., s61 (212) Reding1and, M.-A., s190 (707) Refsnes, M., s121 (448) Reichard, H., s27 (88) Reis, S., s116 (430) Remião, F., s91 (335), s98 (364), s99 (365), s117 (434), s150 (560) Remigio, A., s125 (465) Remirez, D., s89 (327) Rencová, J., s195 (727) Renn, O., s13 (39) Renwick, A.G., s15 (47), s17 (52), s191 (713, 714), s195 (728) Rescia, M., s176 (657) Reshavska, O., s149 (556) Rettie, A.E., s150 (559) Reva, N.I., s192 (717) Rezayat, M., s98 (361) Rezvan, N., s84 (305) Rezzani, R., s95 (351) Rhee, D.-G., s180 (673) Rhee, S.-J., s87 (318) Riazi, A., s42 (145) Ricca, M.B., s162 (605) Rice, E.A., s32 (111) Richoz, J., s65 (232) Richter-Reichhelm, H.-B., s103 (379), s161 (601) Rigoni, M., s12 (34) Ringel, M., s150 (560) Ringerike, T., s34 (116) Rishi, S., s131 (488) Risler, T., s160 (595) Rivero, Y., s80 (290), s125 (465) Roberts, R.A., s19 (61) Roberts, S.L.L., s51 (180) Robinson, E., s2 (6) Rodella, L., s95 (351) Rodrigues, A., s157 (587) Rodriguez, C., s32 (108, 110) Rodríguez, D., s71 (256) Rodríguez, M., s37 (129), s121 (450) Rodríguez, M.I., s61 (213) Rodríguez-Rodríguez, V., s80 (291) Rogacheva, S.M., s86 (314) Rogiers, V., s2 (7), s56 (196), s78 (284) Rollo, K., s11 (29) Roman, D., s22 (70, 71), s42 (147) Romano, R., s183 (682) Romay, C., s89 (327) Romero, A., s178 (664) Roncaglioni, A., s52 (183) Ronchi, A., s35 (122) Roncucci, G., s86 (313) Rønnedal, L.L., s8 (20) s206 Roohi Azizi, M., s84 (305), s91 (333) Roosen, W., s96 (355) Rosa, A., s115 (427) Rosas, I., s106 (389), s183 (681) Rosas-Pérez, I., s182 (679) Rosdy, M., s46 (159) Roshan-Zamir, F., s90 (332) Ross, D., s114 (423) Rossetti, M., s183 (683) Rossetto, O., s12 (34) Rossi, A., s12 (33) Rossi, F., s93 (342) Rossi, T., s167 (622) Rossin, A., s27 (90) Rossner, P., s17 (55) Roter, A., s85 (308) Rothman, N., s18 (58) Rouda, F., s170 (634) Routledge, P., s26 (87) Rowan, E., s69 (247) Rowshangar, L., s111 (408) Royo Hernandez, R., s75 (272) Roza, L., s160 (597) Rozgaj, R., s136 (509), s166 (618) Rubingh, D.N., s33 (113) Rubio, C., s61 (213), s141 (525), s144 (538) Rudén, C., s188 (700) Rueff, J., s157 (587) Ruiz, T.M., s109 (403) Rumbal, Y., s39 (134) Russo, N., s137 (512) Rutkowska, M., s140 (524) Rydin, E., s122 (451) Ryu, J., s181 (674) Rzemieniecki, A., s140 (524) Sabeh Afaki, G., s79 (286) Saberi, M., s69 (247) Sabri, M.I., s130 (483) Sadeghi, S., s112 (414) Sadegi, K., s43 (148) Sadloňová, I., s76 (275) Sadr, S.Sh., s84 (305) Sadraei, S.H., s100 (370), s108 (398) Sadrai, H., s78 (282) Sadraie, S.H., s111 (409) Saeedi, M., s31 (107), s88 (324), s89 (325) Saettone, M.F., s46 (162), s86 (313) Safabaksh, H., s42 (145) Safford, R.J., s192 (718) Sahraei, H., s108 (398) Saifetdinova, G., s88 (322) Sakamoto, Y., s134 (500) Sakurai, K., s176 (655), s178 (665), s179 (666) Sakurai, T., s143 (533, 534) Salazar, M., s126 (467) Saldiva, P.H.N., s168 (628), s171 (639) Samadae, H., s75 (270) Samolova, R.G., s159 (593) Sampaio, S.V., s51 (179), s69 (246), s89 (326) Sams, C., s146 (544) Samuelson, G., s135 (504) Sanaei Zadeh, H., s72 (258) Sanahmadi, Y., s126 (470) Sánchez, M., s168 (626) Sánchez-Chardi, A., s145 (539), s168 (628), s171 (639), s174 (649) Sánchez-Pérez, Y., s182 (679) Sanders, D., s194 (725) Sans-Fuentes, M.A., s174 (649) Santagostino, A., s157 (588) Author Index Santini, M.T., s114 (422), s183 (682), s184 (686) Sapone, A., s156 (583) Sapora, O., s61 (214), s84 (303) Šarić, M., s135 (505) Sarlo, K., s32 (108, 109, 110), s33 (113) Sarmanaev, S.Kh., s159 (593), s163 (608), s164 (610, 611), s186 (693) Sartorelli, P., s2 (6) Sas, T.C.J., s9 (24) Satanovskaya, V.I., s119 (443) Sauer, P.J.J., s9 (24) Savolainen, K., s36 (125), s163 (609) Savov, V., s154 (574) Sawaki, M., s179 (667) Sayed, M.M., s170 (633) Scarpa, A., s123 (459) Schaller, K.-H., s2 (6) Schäper, M., s128 (477), s166 (619) Schepers, G., s103 (380) Schiedlmeier, B., s11 (32) Schilter, B., s65 (232) Schins, R., s105 (387) Schirmer, K., s53 (187) Schlatter, J., s65 (230) Schleef, R., s94 (345) Schlumpf, M., s177 (658) Schmit, B., s185 (688) Schneider, S., s27 (88) Schoeters, G.E.R., s29 (98) Schoonen, W.G.E.J., s50 (174) Schreer, A., s53 (187) Schrenk, D., s19 (62) Schröder, K., s43 (151) Schuh, V., s3 (8) Schulte-Hermann, R., s19 (60) Schütz, G., s27 (88) Schut, H.A.J., s123 (457) Schwarcz, M., s168 (626) Schwarze, P., s121 (448) Scott, E.H., s110 (404) Scott, R.C., s96 (353) Sedmak, B., s68 (242) Seeber, A., s128 (477), s166 (619) Seidenari, S., s46 (160) Seif, E.A., s109 (402) Seki, N., s178 (663) Sekiguchi, S., s175 (654) Seljak, M., s60 (211) Semple, K., s134 (498) Seo, B.-i., s87 (318) Seredenin, S.B., s37 (126), s124 (462), s127 (471) Seredenko, M., s116 (431) Sergeyev, S., s149 (558) Sesek-Briski, A., s141 (527) Sevgiler, Y., s149 (557) Sgorbini, M., s137 (510) Shaaban, A.A., s170 (633) Shadnia, S., s71 (253) Shadnia, Sh., s72 (258) Shafran, L.M., s172 (640) Shah, A.H., s72 (261) Shah, A.R., s72 (261) Sharaky, O.A., s162 (603) Sharifi, A.M., s140 (521) Sharifzadeh, M., s129 (479) Sharkawy, A.A., s63 (222) Shayakhmetova, A., s88 (322), s152 (569) Sheasgreen, J., s29 (96), s45 (158), s47 (163) Sheen, Y.Y., s167 (625), s180 (672) Shelden, E.A., s143 (531) Shepelskaya, N.R., s112 (412) Shimazu, N., s157 (586) Shin, E.J., s135 (503) Shin, S.H., s169 (631) Shipaeva, E.V., s37 (126) Shirali, P., s152 (567) Shirazi, F.H., s126 (470) Shokri, S., s81 (293, 294) Shumkova, T., s177 (660) Sicilia, E., s137 (512) Sidaway, J.E., s96 (353) Signorini, L., s190 (710) Signs, S., s30 (103) Simecek, N., s54 (189) Šimek, P., s152 (568) Simetska, N., s52 (181) Šimić, B., s111 (411) Singh, M., s131 (488) Siniscalchi, E., s144 (537) Sinovec, S., s66 (233) Sinovec, Z., s66 (233) Siroki, O., s39 (135) Sisti, R., s84 (307) Skandari, F., s101 (371) Skaug, M.A., s65 (229) Skerfving, S., s135 (504) Skoumalová, I., s56 (195) Sleeper, E., s11 (31) Ślusarska, E., s120 (444) Smerhovsky, Z., s17 (55), s158 (589) Smith, D., s48 (167) Smith, G.J., s96 (353) Smith, R.D., s162 (602) Smith, R.L., s16 (49, 51) Snyder, E.Y., s11 (31) Snykers, S., s56 (196), s78 (284) Soames, A., s27 (91) Soares, A.M., s51 (179), s68 (243), s69 (246) Soares, M.E., s150 (561) Soeria Atmadja, D., s33 (114) Sogorb, M.A., s49 (172) Soleimani, R.J., s111 (408) Soleimani Rad, J., s43 (148) Soleo, L., s18 (56), s164 (613) Song, C., s130 (482) Sørensen, T.S., s8 (20) Sorio, C., s123 (459) Sorokina, A.V., s37 (126) Soucek, K., s121 (449) Souček, P., s151 (563) Soucek, P., s42 (144), s154 (575) Sowa, A., s53 (186) Söylemezoǧlu, T., s74 (266, 267), s115 (426) Spagnolo, D., s76 (274) Sparrow, S., s48 (167) Spencer, P.S., s130 (483) Spendiff, M., s195 (726) Spire, C., s75 (273) Spurway, T., s27 (91) Spynu, Y., s193 (719) Sram, R., s25 (83) Sram, R.J., s17 (55), s158 (589) Srinouanprachanh, S.L., s156 (584) Stacchiotti, A., s95 (351) Stachlewitz, R., s32 (108) Stacul, E., s74 (268) Staedtler, F., s1 (2) Stahlmann, R., s9 (23) Stammati, A., s58 (204), s63 (224) Stamoulis, J., s102 (378) Stanêk, A., s24 (79) Stankovic, J., s147 (547) Stankovic, S., s145 (541) s207 Author Index Stanković-Kalezić, R., s173 (646) Stavkova, Z., s17 (55), s158 (589) Steffensen, I.-L., s123 (455, 457) Stehfest, E., s138 (514), s139 (517, 518) Steiner, H.Y., s32 (111) Stensby, B.A., s59 (206) Stephan-Gueldner, M., s48 (167) Štetina, R., s125 (463) Stetina, R., s154 (575) Štětinová, V., s109 (400) Stigum, H., s59 (207) Stockman-Juvala, H., s163 (609) Stoeva, E., s77 (281), s154 (574) Stoiber, T., s126 (468) Stoikidou, M., s25 (83) Stojiljković, M.P., s83 (300) Stojiljkovic, M.P., s131 (487) Stokes, W.S., s47 (166) Stolyar, O., s169 (632) Stone, V., s11 (29), s104 (385) Strickland, J.A., s47 (166) Stropp, G., s30 (100) Stuart, B., s102 (375) Stukalov, P., s100 (369) Suárez, M.L., s141 (525) Subirós, N., s57 (200), s80 (290), s81 (292) Subiros, N., s41 (141) Suda, M., s111 (410), s175 (654) Suozzi, A., s102 (377) Šuput, D., s68 (242) Sutlovic, D., s140 (522) Süzen, H.S., s154 (577), s155 (579) Suzen, S., s155 (581), s165 (616) Švestková, E., s174 (648) Svobodová, L., s152 (568) Svobodová, Z., s174 (648), s181 (676) Sybirska, H., s113 (418) Sygieniewicz, E., s185 (689) Symonds, P., s106 (389) Synzynys, B., s126 (469) Szũcs, G., s76 (276) Szymczak, W., s188 (703) Tabaei, S., s91 (336) Taghaddosinejad, F., s72 (258) Tagliaro, F., s183 (683) Taheri Moghadam, M., s98 (363) Tähti, H., s49 (171), s77 (278), s79 (287), s147 (550) Takaba, K., s40 (139) Takahashi, M., s69 (245) Takashima, K., s55 (193) Takatani, T., s69 (248), s70 (249) Takeda, T., s40 (139) Takeyoshi, M., s30 (101) Takzaree, A.-R., s112 (413) Takzaree, A.R., s77 (279) Takzaree, N., s77 (279), s112 (413) Takzaree, S., s112 (413) Talaei, T., s110 (406, 407) Talebanfard, H., s70 (250) Talibi, F., s98 (360) Tallkvist, J., s136 (507), s137 (511) Tamm, C., s11 (31) Tampucci, S., s86 (313) Tanase, C., s35 (121) Tanaseanu, C., s37 (128) Tangni, E.K., s62 (216) Tantcheva, L., s77 (281), s154 (574) Tanu, M.B., s70 (249) Tavazzi, S., s50 (175) Taylor, K.E., s186 (692) ter Laak, A.M., s181 (675) Teredesai, A., s103 (380) Teregulova, Z.S., s186 (693) Teshima, R., s32 (111) Tesseraux, I., s28 (93) Testai, E., s152 (566), s176 (657) Teuns, G., s132 (493) Thakkar, H., s54 (189) Theocharis, S., s102 (376, 378) Thevenin, M., s116 (432) Thiede, M.A., s12 (33) Thier, R., s126 (468) Thierens, H., s25 (81) Thomas, A., s80 (290) Thomas, K., s32 (111) Thompson, D.C., s4 (15), s7 (18) Thum, T., s92 (338), s93 (341, 343), s94 (347), s95 (349), s103 (381) Thummel, K.E., s156 (584) Tice, R.R., s47 (166) Ticli, F.K., s51 (179), s69 (246), s89 (326) Timofeeva, S.I., s131 (489), s133 (497) Timoshina, D.P., s172 (640) Tincani, A., s9 (25) Tinwell, H., s27 (91) Tiwari, A.K., s147 (548) Tjantova, E., s126 (469) Todaka, E., s163 (607), s179 (666) Todic, M., s188 (701) Tognetti, R., s137 (510) Toimela, T., s49 (171), s147 (550) Tolic, N., s162 (602) Toma, L., s119 (442) Tomašić, A., s82 (297) Tomiuk, S., s54 (190) Tonkopii, V.D., s132 (490) Toor, M., s191 (714) Topinka, J., s25 (83) Toriumi, W., s157 (586) Torky, A.W., s138 (516), s139 (517, 518) Tornaeus, J., s159 (594) Tornier, C., s46 (159) Törnqvist, L., s24 (77) Torra, M., s140 (523) Torres, Y., s80 (290) Torres-Flores, V., s182 (679) Toste, S.A., s116 (430) Tourwé, D., s78 (284) Toyama, Y., s82 (296) Traina, M.E., s176 (657) Trapero, Y.M., s57 (200) Trespidi, S., s156 (583) Tressou, J., s190 (709) Trettene, M., s162 (604) Tritscher, A., s20 (63) Trombetta, D., s145 (540) Trošić, I., s136 (509) Troyano, E., s32 (108, 110) Tsakalof, A., s160 (598) Tsatsakis, A.M., s160 (598) Tsuruda, K., s70 (249) Tsutsumi, O., s179 (666) Tulinska, J., s42 (144), s156 (582) Tullberg, S.C., s191 (714) Tuominen, O.-M., s94 (346) Turco, L., s63 (224) Turesky, R., s65 (230) Tutudaki, M., s160 (598) Tykhonova, S., s46 (161) Tysklind, M., s49 (170) Tzirogiannis, K.N., s101 (372, 373) Uhl, C.B., s133 (494) Ujházy, E., s76 (275) Ulleras, E., s34 (116) Ulrich, P., s22 (70, 71) Ündeǧer, Ü., s124 (461) Üner, N., s149 (557) Unfried, K., s105 (387) Unger, E., s126 (468) Upham, B., s121 (447) Urban, P., s130 (485) Urbani, E., s176 (657) Uribe-Hernández, R., s118 (437) Ustenko, N., s85 (309) Uyemura, S.A., s51 (179) Uzuki, M., s179 (666) Uzunova, D., s38 (131) Václavíková, R., s152 (568) Vähäkangas, K., s113 (417) Vaira, D., s156 (583) Valeeva, N.F., s163 (608) Vallant, M.K., s47 (166) Valli, A., s74 (268) Van Broeckhoven, C., s48 (169) van Burgsteden, J.A., s2 (6) van de Sandt, J.J.M., s2 (6), s194 (725) Van Den Heuvel, R.L., s29 (98) Van Hemel, J., s78 (284) van Lipzig, M.M.H., s181 (675) van Loveren, H., s8 (22), s34 (116) Van Miert, E., s103 (380) Van Onckelen, H., s48 (169) van Pelt, F., s35 (120) van Ree, R., s32 (111) Van Riet, I., s56 (196) van Schooten, F.J., s93 (344) van Thriel, C., s128 (477), s166 (619) Vandebriel, R., s34 (116) Vandebriel, R.J., s1 (2) Vandenberghe, J., s96 (355) Vandin, L., s51 (178), s64 (225), s162 (604) Vanhaecke, T., s56 (196), s78 (284) Vanhummelen, P., s48 (169) Vanscheeuwijck, P., s103 (380) Varadinova, M., s134 (501) Varagic, V.M., s147 (547) Vargas Marcos, F., s75 (272) Varmazyar, H.R., s98 (361) Varnai, V.M., s135 (505) Vasco, G., s4 (13) Vaughan, S., s43 (149) Vaz Pereira, M.E., s177 (661) Vázquez, A., s129 (478) Vázquez, M.I., s37 (129) Vázquez-Castro, F., s80 (291) Vecchia, P., s184 (686) Vega, L., s37 (129), s121 (450) Velev, R., s63 (223), s78 (283) Velichko, A.N., s101 (374) Veljača, M., s78 (285), s86 (315) Verbeeck, J., s88 (321), s96 (355), s98 (362) Verhaeghe, T., s88 (321) Vermeil de Conchard, G., s75 (273) Vermeulen, N.P.E., s181 (675) Vershoren, A., s48 (169) Verstynen, B., s132 (493) Verwei, M., s194 (725) Veschetti, E., s144 (537) Veselá, G., s195 (727) Viau, C., s190 (708) Victorin, K., s192 (716) Vidić Štrac, I., s71 (255) Vieira, C.A., s68 (243) Vikse, R., s123 (455) Vilanova, E., s49 (172), s57 (198) s208 Vilanova Gisbert, E., s189 (705) Villani, P., s144 (537) Vinković, B., s82 (297) Vinkx, C., s60 (209) Violante, F.S., s155 (578) Visca, M., s185 (688) Vit, P., s22 (70) Vital, A.L., s29 (99) Vitalone, A., s13 (37) Vitula, F., s173 (645) Viviani, B., s12 (36), s146 (546) Vlachos, C.C., s101 (373) Vlková, A., s195 (727) Vodička, P., s154 (575) Vodicka, P., s42 (144), s109 (401), s125 (463) Vodicková, L., s109 (401) Vodickova, L., s42 (144), s154 (575) Vodolazskaya, N.A., s131 (489), s133 (497) Vohr, H.-W., s30 (100), s31 (105), s44 (154, 155) Vojinovic-Miloradov, M., s67 (239) Vojtesek, B., s121 (449) Vollmer, G., s14 (42), s53 (187) Voloshina, O., s88 (322), s152 (569) Voltz, E., s27 (90) von Holt, K., s94 (345) von Kalle, C., s11 (32) von Landenberg, F., s48 (167) Vondracek, J., s121 (447, 449) Vondráková, J., s56 (195) Voronina, A., s84 (306), s152 (569) Vos, J.G., s1 (2) Vral, A., s25 (81) Vujovic, M., s66 (236) Vynckier, A., s98 (362) Vyskocil, A., s190 (708) Waddell, W.J., s16 (51), s17 (53) Wagener, J., s92 (338) Wagner, B.M., s16 (51) Wahl, H.G., s160 (595), s184 (687) Wahle, B., s102 (375) Wahnschaffe, U., s52 (181) Waisberg, M., s62 (217) Walker, R., s20 (65) Wallén, M., s192 (716) Walton, K., s191 (714) Wamelink, M., s181 (675) Wang, B.L., s111 (410) Wang, R.S., s111 (410), s175 (654) Warnet, J.-M., s79 (286, 288), s80 (289), s116 (432) Author Index Wastl, U., s19 (60) Waterfield, C.J., s54 (189) Waterson, L.A., s30 (102, 103) Weeks, I., s164 (612) Weinbauer, G.F., s34 (117) Weirich, G., s165 (614) Weisglas-Kuperus, N., s9 (24) Welsh, M.J., s143 (531) Welss, T., s43 (151) Wenk, M.L., s47 (166) Westerberg, R.B., s104 (382) Westerink, W.M.A., s50 (174) Wiaderkiewicz, A., s153 (570, 571) Wiaderkiewicz, R., s153 (570, 571) Wiadrowska, B., s151 (564), s178 (662) Wiborg, M.L., s59 (207) Wiechuła, D., s142 (530) Wielgus, E., s113 (418) Wiesmüller, G., s128 (477) Wight, S., s191 (712) Wiker, H.G., s59 (206) Wilczok, T., s155 (580) Wilhelm, M., s158 (590) Wilkerson, H.W., s156 (584) Wilkinson, P.J., s47 (164) Wilkinson, S., s2 (6) Wilkomirski, B., s175 (652) Willems, J.L., s60 (209) Williams, F., s154 (576) Williams, F.M., s2 (6), s151 (565) Williams, R.E., s96 (354) Wilms, L., s57 (199) Wing, M.G., s30 (103) Witters, E., s48 (169) Wittfoht, W., s175 (653) Wober, J., s53 (187) Wobus, A.M., s11 (30) Wolff, S., s167 (624) Won, H.-J., s69 (245) Wood, J.P., s54 (189) Wood, S.G., s194 (725) Woolhiser, M., s32 (111) Worth, A.P., s47 (166) Wragg, M., s30 (103) Wrzosek, J., s120 (444) Wsolova, L., s156 (582) Wyss, C., s29 (97) Xifró, A., s140 (523) Xu, J., s50 (176), s100 (368) Yamada, T., s179 (667) Yamanaeva, I.E., s159 (593), s163 (608), s164 (610) Yamasaki, K., s30 (101), s179 (667) Yamazaki, K., s178 (663) Yamazaki, S., s30 (101) Yancy, S.L., s143 (531) Yanev, S., s38 (131) Yano, K., s134 (500) Yarmohammadi, K., s77 (279) Yarmohammady, K., s112 (413) Ydersbond, T., s59 (208) Yeh, J.Y., s143 (532) Yilmazer-Musa, M., s40 (138), s155 (581), s165 (616) Ylitalo, P., s94 (346) Ylitalo, R., s94 (346) York, M., s194 (725) Yoshida, R., s117 (435) Yoshioka, M., s136 (507), s137 (511) Yücesan, B., s74 (266) Yucesoy, B., s1 (4), s40 (138) Yusoff, K., s114 (423) Zabihi Neishabouri, E., s40 (137) Zahran, M., s145 (542) Zalko, D., s150 (562) Zamecnikova, M., s154 (575) Zampaglioni, F., s58 (204) Zanardini, C., s67 (238) Zancanaro, C., s102 (377) Zanoli, V., s161 (600) Zanoncelli, S., s108 (399) Zantedeschi, V., s76 (274) Zarrindast, M.R., s91 (336) Zawodny, J., s32 (111) Zeisig, M., s118 (436) Zelinski, V., s94 (347) Zemánek, M., s76 (275) Zerimech, F., s152 (567) Zhanataev, A.K., s124 (462) Zheng, Y., s150 (559) Zhminko, P., s149 (558) Zhminko, P.G., s101 (374) Zielińska-Psuja, B., s99 (367) Zinovieva, M.L., s49 (173) Žlábek, V., s181 (676) Zlobin, V.A., s86 (314) Zolotarevski, L., s83 (300), s85 (311) Zoppi, F., s67 (238) Zorlu, A.F., s124 (461) Zorn-Kruppa, M., s46 (161) Zoryan, V.G., s131 (489) Zorzet, A., s33 (114) Zuba, D., s74 (269), s194 (724) Zucco, F., s63 (224) Žužek, M.C., s68 (242) Toxocology Letters 144 (2003) Supplement 1 www.elsevier.com/locate/toxlet Instructions to Authors Submission of papers Only papers describing studies carried out in the most ethical manner should be submitted. Authors should ensure strict compliance with guidelines such as those published by the U.S. Public Health Service, Protection of Human Subjects (Code of Federal Regulations 45 cfr46, 1975), Laboratory Animal Welfare (NIN guide for grants and contracts, vol. 14, No. 3, 1985), Guidelines for Research Involving Recombinant DNA Molecules (USA Public Health Service, 1976) or their respective country’s equivalent regulations. The editors will not accept manuscripts describing work which violates these principles. Papers should contain a detailed explanation of experimental design, and rigorous interpretation of experimental data, with a view to elucidating biochemical mechanisms to toxicity. Isolated systems and non-mammalian studies will be considered only if leading to an understanding of mammalian toxicity. Papers describing phenomena only fall outside the journal’s scope. 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/185. Hamor, T.A., Martin, I.L., 1983. The benzodiazepines. In: Ellis, G.P., West, G.B. (Eds.), Progress in Medicinal Chemistry, vol. 20. Elsevier, Amsterdam, pp. 157
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