Papers by georges de sousa
Par notre environnement, nous sommes continuellement exposés à des mélanges de produits chimiques... more Par notre environnement, nous sommes continuellement exposés à des mélanges de produits chimiques. Répondre à la question de cet impact sur notre santé et celle de l'environnement reste un challenge, l'évaluation des risques des produits chimiques à des fins réglementaires reposant principalement sur l'évaluation des composés pris individuellement. Si des méthodologies et les démarches pour évaluer les risques liés à l'exposition combinée à de multiples produits chimiques ont été élaborées pour différents secteurs réglementaires, il n'existe pas d'approche harmonisée et cohérente pour l'évaluation et la gestion de ces risques.
Food and Chemical Toxicology, 2020
Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk asses... more Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compoundspecific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.
Food and Chemical Toxicology, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Chemical Research in Toxicology, 2018
Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and ... more Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects, and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RARα and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment.
Archives of Cardiovascular Diseases Supplements, 2015
Evaluation of ROS generation and mitochondrial respiration in atrial samples from diabetic and no... more Evaluation of ROS generation and mitochondrial respiration in atrial samples from diabetic and non-diabetic coronary patients
Toxicology Letters, 2014
International audienc
Transplantation Proceedings, 2009
Objective. To investigate the possible antiapoptotic prosurvival role of the pregnane X receptor ... more Objective. To investigate the possible antiapoptotic prosurvival role of the pregnane X receptor (PXR) in hepatic ischemia-reperfusion injury in rats using clotrimazole (CTZ), a strong PXR transactivator. Materials and Methods. Male Sprague-Dawley rats were divided into 3 groups of 6 each: sham-treated, control, and CTZ-treated animals. Control and CTZ-treated animals were subjected to 30 minutes of normothermic ischemia of the whole liver followed by 6 hours of reperfusion. The animals were then killed, and the liver was excised and blood samples collected. Results. Clotrimazole induced a significant increase in expression of the CYP3A gene, indicating PXR transactivation, whereas expression of the antiapoptotic Bcl-xL gene was not increased. Serum concentrations of aspartate aminotransaminase and alanine aminotransaminase were lower in CTZ-treated animals than in control animals (difference not significant). Levels of poly(adenosine diphosphate-ribose) polymerase, a caspase-3 substrate, remained significantly higher in the CTZ-treated group compared with controls (P Ͻ .05). Clotrimazole increased the expression of phospho-p 44/42 extracellular signal-regulated kinase 1,2 (P Ͻ .05). The gene expression of the heat shock proteins 27, 70 and 90 was significantly lower in CTZ-treated animals than in controls (P Ͻ .05). Conclusion. Clotrimazole-mediated PXR transactivation protects the liver against ischemiareperfusion apoptosis in rats. Phospho-p 44/42 extracellular signal-regulated kinase 1,2 is activated, whereas gene expression of heat shock proteins 27, 70, and 90 is downregulated by induction of PXR.
Toxicology, 2009
Lindane, a persistent organochlorine pesticide, is recognized as a major public health concern be... more Lindane, a persistent organochlorine pesticide, is recognized as a major public health concern because of its potential toxic effects on human health. Despite observations pointing to the toxicity of lindane, mechanisms underlying its deleterious effects in liver have yet to be understood. In this study, we investigated the effects of lindane on autophagic, apoptotic and necrotic cell death in primary cultured rat hepatocytes. We found that lindane deregulated the autophagic process as demonstrated by (1) the formation of enlarged acidic vesicles labeled with LC3, Rab7 and LAMP1 (specific markers of autophagic vacuole maturation), (2) the conversion of LC3-I (the cytosolic form) into LC3-II (membrane bound), (3) the deregulation of the Beclin 1 protein expression and (4) the enhanced formation of the Bcl-xL/Beclin 1 complex. Lindane induced vacuolization together with the inhibition of spontaneous and intrinsic apoptosis. This disruption of cell suicide was linked to Bcl-xL up-regulation, Bax down-expression, prevention of cytochrome c release, and inhibition of caspase-9 and-3 activities. Lindane-induced disruption of apoptosis and autophagy occurred in parallel with necrosis induction in rat hepatocytes. In consequence, we proposed that lindane toxicity in primary rat hepatocytes could be jointly attributed to the disruption of autophagic process, the inhibition of apoptotic cell death and the induction of necrosis. These events account, at least in part, for the involvement of both cytotoxic and carcinogenic signaling pathways in the action of lindane in the liver.
Polycyclic Aromatic Compounds, 1999
We developed a new method, which allowed black seabream hepatocytes to be maintained in primary-c... more We developed a new method, which allowed black seabream hepatocytes to be maintained in primary-culture for several days. This method was shown to be suitable for studying sea fish CYPs expression and regulation following xenobiotic treatment. After isolation, hepatocytes were directly mixed with a type I collagen gel and culture medium, seeded in 6-well tissue culture plates (2.10 cells/35 mm
Liver International, 2010
Background/Aims: Bile acids damage the liver, essentially by inducing hepatocyte apoptosis. Clini... more Background/Aims: Bile acids damage the liver, essentially by inducing hepatocyte apoptosis. Clinical studies have shown that several activators of the pregnane X receptor (PXR) may induce the remission of cholestasis. However, the molecular mechanisms involved in this beneficial effect remain unclear. We analysed the effect of an activator of PXR, clotrimazole (CLO), on the apoptosis induced by bile acids in primary cultures of rat hepatocytes. Methods: Rat hepatocytes were isolated by collagenase perfusion of the liver. Then, cells were pretreated with CLO for 24 h, after which they were exposed to deoxycholic and glycochenodeoxycholic acids (DCA, GCDCA). Apoptosis and necrosis were monitored morphologically and biochemically using cytotoxicity assays, phase-contrast microscopy, Annexin V/propidium iodide staining and evaluations of lactate dehydrogenase release. The activation of caspases and the proteolysis of their substrates were analysed by enzyme assays and Western blot. The signal transductions involved in the protective effect of the PXR activation were analysed by assessing the phosphorylation status of kinases belonging to the ERK, Akt and p38 pathways and by analysing proand anti-apoptotic proteins. Results: CLO protected rat hepatocytes against DCA-and GCDCA-induced apoptosis, preventing morphological aspects of this process (membrane blebbing, nuclear and chromatin condensation and DNA breakdown). This effect was attributable, at least partly, to caspases inhibition, Bcl-xL induction, the activation of ERK and Akt signalling and p38 inhibition. Conclusion: This study provides the description of the cytoprotective effect of PXR activation against bile acid-induced apoptosis and highlights molecular pathways that could be targeted in the treatment of cholestasis.
Food and Chemical Toxicology, 2012
Persistent organic pollutants (POPs) are a group of organic or chemicals that adversely affect hu... more Persistent organic pollutants (POPs) are a group of organic or chemicals that adversely affect human health and are persistent in the environment. These highly toxic compounds include industrial chemicals, pesticides such as organochlorines, and unwanted wastes such as dioxins. Although studies have described the general toxicity effects of organochlorine pesticides, the mechanisms underlying its potential carcinogenic effects in the liver are not well understood. In this study, we analyzed the effect of three organochlorine pesticides (dichlorodiphenyltrichloroethane, heptachlore and endosulfan) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the epithelial to mesenchymal transition (EMT) in primary cultured human hepatocytes. We found that these compounds modified the hepatocyte phenotype, inducing cell spread, formation of lamellipodia structures and reorganization of the actin cytoskeleton in stress fibers. These morphological alterations were accompanied by disruption of cell-cell junctions, E-cadherin repression and albumin down-regulation. Interestingly, these characteristic features of dedifferentiating hepatocytes were correlated with the gain of expression of various mesenchymal genes, including vimentin, fibronectin and its receptor ITGA5. These various results show that organochlorines and TCDD accelerate cultured human hepatocyte dedifferentiation and EMT processes. These events could account, at least in part, for the carcionogenic and/or fibrogenic activities of these POPs.
Food and Chemical Toxicology, 2011
Although many studies of lindane toxicity have been carried out, we still know little about the u... more Although many studies of lindane toxicity have been carried out, we still know little about the underlying molecular mechanisms. We used a microarray specifically designed for studies of the hepatotoxic effects of xenobiotics to evaluate the effects of lindane on specific gene expression in primary cultured rat hepatocytes. These genes were assigned to detoxication processes (CYP3A4, Gsta2, CYP4A1), cell signalling pathways and apoptosis (Eif2b3, Eif2b4, PKC). In this study, we demonstrate that lindane up-regulates PKC by increasing oxidative stress. TEMPO (a well known free radical scavenger) and Ro 31-8220 (an inhibitor of classical PKCs) prevented the inhibition of spontaneous and intrinsic apoptosis pathway (characterised by Bcl-xL induction, Bax down-regulation, caspases inhibition) and the induction of necrosis by lindane in rat hepatocytes. Thus, these findings indicate that several dependent key signalling pathways, including detoxification, apoptosis, PKC activity and redox status maintenance, contribute to lindane-induced toxicity in primary cultured rat hepatocytes. This may account more clearly for the acute and chronic effects of lindane in vivo, with the induction of cell death and tumour promotion, respectively.
Cryobiology, 1992
In drug metabolism studies, isolated and cultured human hepatocytes provide a useful model for ov... more In drug metabolism studies, isolated and cultured human hepatocytes provide a useful model for overcoming the difficulty of extrapolating from animal data. In vitro studies with human hepatocytes are scarce because of the lack of livers and suitable methods of storage. After developing a new method for cryopreservation of human hepatocytes, we evaluated the effects of deep freezing storage on their viability, morphology, and functional and toxicological capabilities in classical culture conditions. Freshly isolated human hepatocytes were cryopreserved in medium containing 10% Me2SO and 20% fetal calf serum, using a Nicool ST20 programmable freezer (-1.9 degrees C/min for 18 min and -30 degrees C/min for 4 min). Cells were stored in liquid nitrogen. Viability of thawed human hepatocytes was 50-65% as assessed by erythrosin exclusion test prior to purification on a Percoll density gradient. Morphological criteria showed that thawed human hepatocytes require an adaptation period to the medium after seeding. Functional assessments showed that human hepatocytes which survive freezing and thawing preserve their protein synthesis capabilities and are able to secrete a specific protein, anionic peptidic fraction, which is involved in the hepatic uptake of bile-destined cholesterol. We then studied Midazolam biotransformation to test metabolic functions, and erythromycin toxicity by Neutral Red test (cell viability) and 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide test (cell metabolism). All of these experiments indicated that thawed human hepatocytes should be used 38 h after seeding for optimum recovery of their functions: membrane integrity, protein synthesis, and stabilization of drug metabolism enzymes.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1999
Microsomal cytochrome P450-dependent lauric acid hydroxylase activities were characterized in liv... more Microsomal cytochrome P450-dependent lauric acid hydroxylase activities were characterized in liver, kidney, and intestinal mucosa of the sea bass (Dicentrarchus labrax). Microsomes from these organs generated (omega-1)-hydroxylauric acid and a mixture of positional isomers including (omega)-, (omega-2)-, (omega-3)- and (omega-4)-hydroxylauric acids, which were identified by RP-HPLC and GC-MS analysis. Peroxisome proliferators, such as clofibrate and especially di(2-ethylhexyl) phthalate, increased kidney microsomal lauric acid hydroxylase activities. The synthesis of 11-hydroxylauric acid was enhanced 5.3-fold in kidney microsomes. Liver microsomal lauric acid hydroxylase activities were weakly affected and no significant induction was found in small intestine microsomes from clofibrate or di(2-ethylhexyl) phthalate-treated fish. The differences in lauric acid metabolisation and the tissue-specific induction by peroxisome proliferators suggest the involvement of several P450s in this reaction. Incubations of liver and kidney microsomes with lauric acid analogues (11- or 10-dodecynoic acids) resulted in a time- and concentration-dependent loss of lauric acid hydroxylase activities. The induction of these activities in fish by phthalates, which are widely-distributed environmental pollutants, may be taken into consideration for the development of new biomarkers.
Chemico-Biological Interactions, 1999
Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes fro... more Ethical, economic and technical reasons hinder regular supply of freshly isolated hepatocytes from higher mammals such as monkey for preclinical evaluation of drugs. Hence, we aimed at developing optimal and reproducible protocols to cryopreserve and thaw parenchymal liver cells from this major toxicological species. Before the routine use of these protocols,
Cell Biology and Toxicology, 1996
The study was designed to investigate the effects of phenobarbital (PB), 3-methylcholanthrene (3-... more The study was designed to investigate the effects of phenobarbital (PB), 3-methylcholanthrene (3-MC), and oltipraz (OPZ), a synthetic derivative of 1,2-dithiole-3-thione, on the levels of cytochrome P450 1A1/2 and gluthathione transferase (GST) mRNAs in both fresh and cryopreserved human, monkey, and dog hepatocytes in primary culture. GST alpha mRNAs were demonstrated in liver parenchymal cells from the three species: after 4 days of culture, their basal levels were decreased, but were strongly higher in PB- and OPZ-treated cells from the three species. In contrast 3-MC was mostly effective on human hepatocytes. The increased levels of GST alpha mRNAs in the presence of PB or OPZ were not observed in all cell populations. GST mu mRNAs, which were detected in both dog and monkey hepatocytes, were induced only in the presence of OPZ. GST pi mRNAs were expressed in dog hepatocytes but did not respond to any of the inducers. In all cases, similar effects were observed in fresh and thawed hepatocytes. Similarly, CYP1A1/2 transcripts were induced by 3-MC in both fresh and cryopreserved cells from the three species but also after OPZ treatment for monkey hepatocytes. These findings demonstrate that enzymes which play a major role in bioactivation/detoxication of xenobiotics remain expressed and inducible in hepatocytes from various species after cryopreservation and thawing.
Biochemical Pharmacology, 1999
Malaria remains the most prevalent infectious disease of tropical and subtropical areas of the wo... more Malaria remains the most prevalent infectious disease of tropical and subtropical areas of the world. It represents a crucial problem in public health care, affecting 750 million people annually, of whom at least two million die. Various antimalarials currently used were studied for their capability to induce expression of the cytochrome P450 1A1 (CYP1A1) gene, an enzyme that plays an important role in the activation of xenobiotics to genotoxic derivatives. Studies on human hepatocytes and HepG2 cell lines showed that primaquine was capable of dose dependently increasing both the ethoxyresorufin-O-deethylase activity and CYP1A1 mRNAs, suggesting a transcriptional activation of this gene. Moreover, alpha-naphthoflavone, a partial aryl hydrocarbon receptor (AhR) antagonist, and 8-methoxypsoralen, which interferes with the binding of activated AhR to the xenobiotic responsive element, were shown to suppress CYP1A1 induction when added to the cultures. However, neither primaquine nor its metabolites were able to displace [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin from AhR in competitive binding studies using 9S-enriched fractions of human cytosol. These data, together with the induction of CYP1A1 promoter-directed chloramphenicol acetyl transferase gene expression, suggest that CYP1A1 induction involves the participation of the AhR but not a direct primaquine-receptor interaction. This supports the notion that an alternative ligand-independent mechanism has to be considered. Given the pharmaco-toxicological significance of CYP1A1 induction, these findings may have important implications in the treatment of malaria with primaquine and new analogs.
Biochemical Pharmacology, 1994
The level and number of CYP2E1 gene transcripts were investigated by northern blot analysis in va... more The level and number of CYP2E1 gene transcripts were investigated by northern blot analysis in various human adult tissues including liver, lung, placenta, skin and neurinoma. Three transcripts of 1.8, 2.6 and 4 Kb were expressed in a tissue-specific manner. The origin of the various transcripts was studied and showed that both 4 and 2.6 Kb mRNAs contained sequences from the 3' non-translated region of the gene and that the 4 Kb also contained region localized in the 5' non-translated region. Furthermore, it clearly appeared that a catalytically active CYP2E1 enzyme (as proved by NDMA demethylase activity) was only detected in tissues expressing the 1.8 Kb. The human CYP2E1 was also identified through immunohistochemical techniques. Finally, we observed a relation between the hypomethylation of the human CYP2E1 gene and the hypoexpression of the corresponding protein.
Biochemical Journal, 2001
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Papers by georges de sousa