Drug Metabolizing Enzymes In Humans And Animal Models

Environmental Health Sciences

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Abstract

Summary: [unreadable] We have identified new genetic polymorphisms in CYP2C9 in humans which cause variability in the way humans metabolize clinical drugs, environmental chemicals, and endogenous chemicals. These polymorphisms can increase risk of toxicity of drugs and environmental chemicals to humans. human CYP2C9 metabolizes 15% of clinically important drugs including the anticoagulant warfarin, anticonvlsant phenytoin, the antidiabetic drug tolbutamide and glipizide, and numerous nonsteroidal antiinflammatory drugs (NSAIDs), and endogenous chemicals involved in control of vasodilation and other physiological processes. In previous studies we have identified over 14 alleles of CYP2C9, at lest half of which have been shown to be defective in vivo and/or predicted to be defective based on studies of the recombinant proteins produced in bacteria and studied in vitro. We have developed new rapid pyrosequencing genotyping tests for known or putative defective alleles of human CYP2C9 alleles (*2, *3, *5, *6, 10, and 11 variants) which occur in Caucasians and African Americans. These were used in a translational study: a large prospective study of 500 Caucasian and African American patients on warfarin therapy. The presence of variant alleles of CYP2C9 conferred a 5-fold increase in risk for major hemorrhage before stabilization of dose, and a 2-fold risk even after stabilization of dose (Limdi et al). A lower dose of warfarin was also required in individuals with aberrant CYP2C9 alleles. A new defective allele was also found in an African American who had a major hemorrhage. A second collaborative study addressed the effect of CYP2C8 polymorphisms on metabolism of the antimalarial amodiaquine in vivo and in vitro. Recombinant mutant CYP2C8 alleles had decreased metabolism of amodiaquine in vitro. Amodiaquine is used for malaria in Africa. There was no effect of CYP2C9 genotype on efficacy in Sub-Saharan Africans since the metabolite is also active; however there is a possibility of increased toxicity in poor metabolizers since the metabolite is not readily activated to a reactive metabolite. In addition we found 12 new polymorphisms in human CYP26A1 which is the principle enzyme in the embryo which protects from excess vitamin A synthesis. Three mutations in human CYP26A1 produced amino acid changes . At least two of these variant alleles containing amino acid changes are predicted to be defective in the metabolism of retinoids based on studies in our laboratory of the recombinant proteins expressed in a mammalian kidney cell line (Cos-1). Other mutations may afffect splicing of the human proteini. The human CYP3A subfamily metabolizes approximately 40% of all drugs and many pesticides, and one new human defective allele CYP3A5*11 has been identified by sequencing. This allele was expressed as a recombinant protein in bacteria and found to be deficient in metabolizing the calcium channel blocker drug nifedipine . Studies in human vascular studies identified the presence of CYP2C9 >CYP2J2> CYP2C8 mRNA and protein in human aorta, coronary arteries by quantitative PCR and Western blotting while CYP2C8 and CYP2J2 were highest in human heart where their endogenous metabolites of arachidonic acid are known to be involved in vasodilatation and cardiac contractility (Delozier et al) . These were seen in heart muscle and endothelia of blood vessels in the heart. CYP2C9 is particularly high in cornary blood vessels and aorta where it produces metabolites of arachidonic acid which affect vasodilation, while CYP2C8 is present in heart and arachidonic acid metabolites are believed to affect contractility. There was a high degree of variability of the CYP2Cs in vascular tissue and heart in different individuals which could affect disease processes such as hypertension.[unreadable] Retinoic acid receptor-related orphan receptors (ROR) alpha, beta, and gamma are differentially expressed in liver, kidney, lung, muscle, brown fat, thymus, and brain and are involved in the regulation of many physiological processes such as immune function, brain development, circadian rhythm, and lipid metabolism. ROR and are expressed in liver, and hepatic genes such as human apolipoprotein genes have been previously shown to be transcriptionally regulated by ROR. We examined whether the human CYP2C promoters are also transactivated by RORs in HepG2 cells. Overexpression of ROR and ROR in HepG2 cells significantly increased luciferase activity of a 3kb CYP2C8 promoter construct, but not that of CYP2C9 or CYP2C19 promoter constructs. CYP2C8, which metabolizes both endogenous compounds and clinical drugs such as arachidonic acid, retinoic acid, paclitaxel, and certain antidiabetic drugs. We have identified potential ROR responsive elements (RE) and mutational analysis has identified the crucial site. These data support a role of RORs in regulation of CYP2C8 expression in liver and possibly other extrahepatic tissues.[unreadable] Transcriptional regulation of the CYP enzymes also alters expression of the human CYP2Cs and can produce tolerance or drug-drug interactions. In liver and intestines the CYP2Cs can be increased >2-8 fold by prior administration of drugs, producing higher metabolism in exposed individuals. Our new work focuses on the promoter regions of the human CYP2C genes by the nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), and liver-enriched receptors such as HNF4 alpha. HNF4 sites in the promoter have been shown to enhance inducibility and HNF4 and CAR activate the CYP2C9 promoter synergistically. We theorize that various coactivators may be involved in forming a bridge between the distal CAR site and the proximal HNF4 site in the CYP2C9 promoter. Results of yeast two hybrid screens identified known coactivators PGC-1 as well as at several previously unidentified interacting cofactors. We have overexpressed CAR and HNF4 in adenoviral vectors and performed pull downs with GST-HNF4 and GST-CAR. We identified CAR in the GST-HNF4 complex from nuclear extracts over expressing CAR both by Western blotting and by mass spectrometric analysis. Other cofactors such as PGC-1 were also identified in the complex. When these coregulators were translated in vitro, they could also be pulled down with HNF4-GST and identified by Western blotting. Finally, using promoter assays, the synergy between HNF4 and CAR or PXR in the presence of their respective ligands could be blocked by adenoviral constructs to siRNAs for a putative coregulator which had been found in the complex. In chromatin immunoprecipitation (ChIP) assays antibodies to CAR pulled down both the CAR binding site and the HNF4 binding site. Antibodies to HNF4 pulled down the HNF4 site in cells overexpressing HNF4 but only a small amount of the CAR site. Antibodies to various coregulators pulled down the CAR site and the HNF4 site under different conditions. Future studies in human hepatocytes will address to what extent the liver-specific HNF4 is required for CAR and PXR mediated induction of CYP2C9 by compounds such as taxol, rifampicin, and Citco. Using murine models we examined transcriptional activation of the CYP2Cs(2C37 and 2C29) promoters by phenobarbital and phenytoin as well as induction of CYP2C37 and CYP2C29 mRNA and protein using knockout mice for CAR and the PXR receptor. Studies indicate that the nuclear receptor CAR (not PXR) mediates the action of pPhenobarbital and phenytoin in inducing CYP2C29 and CYP2C37 protein and mRNA and activate their respective promoters in luciferase assays in a murine model. Moreover the prototype PXR agonist PCN, 5-pregnen-3-ol-20-one-16-carbonitrile do not induce any of the murine CYP2Cs tested.