Bioquímica y Biología Molecular

Livers from aryl hydrocarbon receptor-null mice showed a 3-fold increase in retinoids and a 65% decrease in retinoic acid metabolism. Levels of expression of the retinoic acid 4-hydroxylase, P45ORAI, did not change, whereas cytochrome P4501A2 levels were lower in the null mouse, as shown earlier; however, this enzyme was found not to be active toward retlnolc acid. These data suggest that aryl hydrocarbon receptor controls retlnolc acid catabolism, through modulation of an unidentified target gene. Aldehyde dehydrogenases 1 and 2 were down-regulated markedly in the aryl hydrocarbon receptor-deficient mouse liver. 2,3,7,8-Tetrachlo rodlbenzo-p-dloxin Induced cytochrome P4501A2 but not the aldehyde dehydrogenases in wild-type mice, suggesting that aryl hydrocarbon re ceptor Is not Involved directly In the down-regulation of this gene. Trans glutalninase II, a retinoic acid-responsive gene product, was Increased 2-fold, consistent with the liver fibrosis phenotype observed In the null mice. These findings suggest a molecular connection between xenoblotlc activated receptor signaling and retinoid homeostasis.

Background: Transcriptional networks in cancer are deeply misregulated. Retrotransposons and other repetitive elements occupy more than 40% of the size of mammalian genomes, and their epigenetically silenced status have been recently reported to be lost in several cancer stages. Besides, their sequences are potential targets for transcription factors (TFs) regulation. In spite of this, little is known about how transcriptional networks are modulated by the presence of repetitive elements.

and cell-type specificity. ᭧ 1996 Academic Press, Inc. activated transcription factor that regulates the expression of several drug-metabolizing enzymes and has been implicated in immunosuppression, teratogenesis, cell-specific hyperplasia, and certain types of malignancies and toxicities. In order to examine tis-Exposure to polycyclic or halogenated aromatic hysue-specific regulation of the mouse Ah receptor gene drocarbon ligands of the Ah receptor (AHR) 2 causes (Ahr), we studied chimeric deletion constructs, connumerous deleterious environmental and health effects taining the Ahr 5 flanking region and the firefly lucif-(1-3). Ligand activation of the AHR, a basic regionerase reporter gene (Luc). Transient transfection helix-loop-helix (bHLH) transcription factor, proassays were performed in five established mouse cell motes its dimerization with ARNT, a second bHLH prolines: Hepa-1c1c7 (derived from hepatoma), JB6-C1 41tein, and translocation of the complex to the nucleus 5a (epidermis), MLE-12 (lung epithelium), F9 (em-(4-6). AHR/ARNT heterodimers recognize specific bryonal carcinoma), and NIH/3T3 (fibroblasts). Treat-DNA sequences (termed AhREs, DREs, or XREs) ment of the cell lines included: dioxin (2,3,7,8-tetrawithin the regulatory regions of the cytochromes P450 chlorodibenzo-p-dioxin), retinoic acid (RA), cyclic CYP1A1 and CYP1A2 genes and of a number of other adenosine 3:5-monophosphate (cAMP), or 12-O-tetradioxin-responsive genes, thereby activating transcripdecanoylphorbol 13-acetate (TPA). Expression levels tion (7, 8). Induction of Phase I and Phase II drugof Luc varied widely from one untreated cell line to metabolizing enzymes results in activation and/or metanother, this finding was also confirmed by measureabolic detoxification of genotoxic ligands such as ments of AHR mRNA steady-state levels. In all cell lines benzo[a]pyrene.

Studies have suggested that phenobarbital (PB) induces members of the CYP1A subfamily by both transcriptional and post-transcriptional mechanisms. Using the AhrϪ/Ϫ mice, we examined the induction of CYP1A1 and CYP1A2 by PB. CYP1A2 mRNA and protein were induced by PB in the null mice, suggesting that CYP1A2 is regulated by PB by a mechanism independent of the aryl hydrocarbon receptor (AHR). In contrast, the regulation of CYP1A1 is highly dependent on the AHR. BIOCHEM PHARMACOL 55;2:235-238, 1998.

maintenance of the immature state of human dendritic cells Aryl hydrocarbon receptor contributes to the MEK/ERK-dependent http://www.bloodjournal.org/content/121/15/e108.full.html Updated information and services can be found at: (5229 articles) Immunobiology (132 articles) e-Blood Articles on similar topics can be found in the following Blood collections http://www.

Aims Dihydropyrimidine dehydrogenase (DPD) catalyses the reduction of pyrimidines, including the anticancer agent 5-fluorouracil (5FU). Impaired 5FU degradation, through low DPD activity, has led to severe, life-threatening or fatal toxicity after administration of 5FU. Complete DPD deficiency is associated with the inherited metabolic disease thymine uraciluria. Several mutations in the gene encoding DPD have recently been identified, but the phenotype-genotype concordance of these alterations in the general population has not been reported. Methods Mononuclear cells were isolated from whole blood and DPD activity was determined after ex vivo incubation with 14 C-5FU followed by h.p.l.c. analysis of 5FU metabolites. Analysis of mutations in the DPD gene at an exon splice site, codons 534, 543, and 732, and a deletion at base 1897 (DC1897) were performed in 30 subjects with the lowest and 30 subjects with the highest enzyme activity using PCR-RFLP. Results DPD activity was measured in 226 Caucasian subjects and was highly variable (range 19.1-401.4 pmol min −1 mg −1 protein). Mutations were frequently observed at codons 543 (allele frequency 28%), 732 (allele frequency 5.8%), and 534 (allele frequency 0.8%), but were not associated with low DPD activity. There were no splice site or DC1897 mutations found in this population.

Hemoglobin adducts and lung DNA adducts of 4-ABP are found in tobacco smokers. In vitro metabolism studies with human and rat liver microsomes have shown that CYP1A2 is primarily responsible for catalyzing N-hydroxylation, the initial step in the metabolic activation of 4-ABP. To determine whether this P450 is a rate limiting pathway for hepatocarcinogenesis, CYP1A2-null mice were analyzed at 16 months of age and were compared with wild-type mice in their response to 4-ABP using the neonatal mouse bioassay and two different doses of the carcinogen. Overall differences in incidences of hepatocellular adenoma, carcinoma and preneoplastic foci were not significant between either genotypes or 4-ABP doses used, whereas small, but significant, differences were found for specific types of foci. These results suggest that while CYP1A2 levels may not be rate limiting for 4-ABP metabolism to produce tumors and foci, it may modulate the induction process of some types of liver foci in either a positive or negative manner. In vitro studies using CYP1A2-null and wild-type mouse liver microsomes revealed that CYP1A2 is not the sole P450 required for 4-ABP N-hydroxylation and that another, yet to be identified, P450 is likely to be involved.

The transcription factor aryl hydrocarbon receptor (AhR) has relevant functions in cell proliferation. Interestingly, the AhR can either promote or inhibit proliferation depending on the cell phenotype. Although recent data reveal potential pathways for AhR signaling in cell proliferation, the mechanisms that regulate its activity in tumor cells remain unknown. Here, we have analyzed promoter hypermethylation as a potential mechanism controlling AhR expression in human tumor cells. AhR promoter CpG methylation was sporadic in a panel of 19 tumor cell lines except for the chronic myeloid leukemia (CML) K562 and the acute lymphoblastic leukemia (ALL) REH. When compared with normal lymphocytes, REH had very low constitutive AhR expression that could be attributed to promoter hypermethylation since treatment with the DNA demethylating agent 5-aza-2 0 -deoxycitidine (AZA) significantly increased AhR mRNA and protein. These results in leukemia-derived cell lines were further confirmed in primary ALL, where 33% of the patients (7/21) had AhR promoter hypermethylation. Chromatin immunoprecipitation (ChIP) showed that methylation impaired binding of the transcription factor Sp1 to the AhR promoter, thus providing a mechanism for AhR downregulation in REH cells. Therefore, promoter hypermethylation represents a novel epigenetic mechanism downregulating AhR activity in hematological malignancies such as ALL.

is the most abundant of the heterocyclic amines found in cooked meat. Based on in vitro studies with rats and humans, CYP1A2 is believed to be the primary enzyme responsible for N 2 -hydroxylation, the initial step in the metabolic activation of PhIP. To determine whether CYP1A2 is the primary P450 responsible for metabolic activation of PhIP in mice that leads to tumor formation, neonatal Cyp1a2-null and wild-type mice were treated with 11 (low dose) and~22 (high dose) mg/kg PhIP at days 8 and 15, corresponding cumulatively to 600 and 1200 nmol PhIP, and analyzed at 19-21 months of age. Three major induced tumors were found; lymphomas and tumors in lung and liver. The incidence of lymphoma was higher in Cyp1a2-null females than wild-type females treated with low dose (600 nmol) PhIP whereas no significant differences were observed in other treatment groups of mice. Overall differences in incidences of lung adenoma/adenocarcinoma were in general not consistent among sexes, genotypes and PhIP doses used, although reduced incidences of lung tumors were found in Cyp1a2-null males with low dose (600 nmol) and null females with high dose (1200 nmol) PhIP. Higher incidences of hepatocellular adenoma were observed in Cyp1a2-null female and male mice as compared with wild-type mice. In vitro studies using Cyp1a2-null and wild-type mouse liver microsomes revealed that CYP1A2 is the major enzyme required for PhIP N 2 -hydroxylation in mouse, the initial metabolic activation of PhIP that is thought to lead to tumor formation. These in vivo and in vitro results suggest that although the metabolic activation of PhIP is carried out primarily by CYP1A2, an unknown pathway unrelated to CYP1A2 appears to be Abbreviations: 4-ABP, 4-aminobiphenyl; AHR, aryl hydrocarbon receptor; APMSF, 4-amidinophenylmethanesulfonyl fluoride; DMSO, dimethylsulfoxide; MRM, multiple reactions monitoring; N 2 -OH-PhIP, N 2 -hydroxy-PhIP; 4Ј-OH-PhIP, 4Ј-hydroxy-PhIP; P450s, cytochromes P450; PhIP, 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine.

Nitric oxide (NO) is responsible for cytochrome P450 (CYP450) loss during isolation and cytokine treatment of primary rat hepatocytes. As P450s mediate the metabolism of toxic chemicals, their inhibition could compromise the cells competence to eliminate toxins, a condition potentially relevant in neurological diseases involving constitutive activation of nitric oxide synthase (NOS) and NO overproduction. Here, we have investigated the correlation between NO accumulation and CYP1A2 down-regulation during maturation of mouse cerebellar granule cells (CGC). As neurons matured in culture, the inducible levels of CYP1A2 protein and catalytic activity decreased to almost undetectable values. In parallel, a signi®cant increase in NO concentration was observed. Neuronal NOS remained constitutively active during maturation, thus contributing to NO accumulation. The NOS inhibitor L-NAME, restored CYP1A2 catalytic activity up to 9 days in vitro, supporting a role for NO in the inhibition process. Maturation was also followed by increased NMDA receptor activity and intracellular Ca 2 concentration. We suggest that maintained NOS activity during CGC maturation could lead to NO accumulation and to decreased CYP1A2 inducibility. Increased NMDA receptor activity and Ca 2 entry could contribute to this process. Thus, neurodegeneration could diminish the induction of speci®c P450s and impair the metabolism of foreign and/or endogenous chemicals in the CNS.

The aryl hydrocarbon (dioxin) receptor (AhR) has been studied for several decades largely because of its critical role in xenobiotic-induced toxicity and carcinogenesis. Albeit this is a major issue in basic and clinical research, an increasing number of investigators are turning their efforts to try to understand the physiology of the AhR under normal cellular conditions. This is an exciting area that covers cell proliferation and differentiation, endogenous mechanisms of activation, gene regulation, tumor development and cell motility and migration, among others. In this review, we will attempt to summarize the studies supporting the implication of the AhR in those endogenous cellular processes.

Access the most recent version at doi: 1996 10: 60-69 Genes Dev. S Kimura, Y Hara, T Pineau, et al. forebrain, and pituitary. essential for the organogenesis of the thyroid, lung, ventral The T/ebp null mouse: thyroid-specific enhancer-binding protein is References http://genesdev.cshlp.org/content/10/1/60#related-urls Article cited in: http://genesdev.cshlp.org/content/10/1/60.

Dihydropyrimidine dehydrogenase (DPD) catabolizes endogenous pyrimidines and pyrimidine-based antimetabolite drugs. A deficiency in human DPD is associated with congenital thymine-uraciluria in pediatric patients and severe 5-fluorouracil toxicity in cancer patients. The dihydropyrimidine dehydrogenase gene (DPYD) was isolated, and its physical map and exon-intron organization were determined by analysis of P1, PAC, BAC, and YAC clones. The DPYD gene was found to contain 23 exons ranging in size from 69 bp (exon 15) to 961 bp (exon 23). A physical map derived from a YAC clone indicated that DPYD is at least 950 kb in length with 3 kb of coding sequence and an average intron size of about 43 kb. The previously reported 5 donor splice site mutation present in pediatric thymine-uraciluria and cancer patients can now be assigned to exon 14. All 23 exons were sequenced from a series of human DNA samples, and three point mutations were identified in three racial groups as G1601A (exon 13, Ser534Asn), A1627G (exon 13, Ile543Val), and G2194A (exon 18, Val732Ile). These studies, which have established that the DPYD gene is unusually large, lay a framework for uncovering new mutations that are responsible for thymine-uraciluria and toxicity to fluoropyrimidine drugs.

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens in laboratory animals and are suspected human carcinogens. The PAHs are bioactivated by cytochrome P450 (CYP)1A1/ 1B1 enzymes to reactive intermediates that bind to DNA, a critical step in the initiation of carcinogenesis. The Ah receptor (AHR) plays a critical role in the induction of CYP1 enzymes (i.e., CYP1A1, 1A2 and 1B1) by PAHs such as benzo[a]pyrene (BP) and 3-methylcholanthrene (MC). In our investigation, we tested the hypothesis that AHR-null animals are less susceptible to PAH-induced DNA adduct formation than wild-type animals. Wild-type [AHR (؉/؉)] mice or mice lacking the gene for the AHR were treated with a single dose (100 mol/kg) of BP or MC, and hepatic DNA adducts were analyzed by 32 P-postlabeling. BP induced multiple hepatic DNA adducts in wild-type as well as AHR-null animals, suggesting the existence of AHR-independent mechanisms for BP metabolic activation. On the other hand, DNA adduct formation was markedly suppressed in AHRnull animals exposed to MC, although the major MC-DNA adduct was produced in these animals. Hepatic activities and apoprotein contents of 7-ethoxyresorufin O-deethylase (EROD) (CYP1A1) and 7-methoxyresorufin O-demethylase (MROD) (CYP1A2) activities were markedly induced by BP and MC in the wild-type, but not, in AHR-null animals. CYP1B1 expression was also induced, albeit to a lesser extent by the PAH MC, but not BP, in the wild-type animals. In conclusion, these results demonstrate the existence of AHRand CYP1A1-independent mechanisms of PAH metabolic activation in mouse liver, a phenomenon that may have important implications for PAH-mediated carcinogenesis.