We have identified new genetic polymorphisms in the CYP family in humans which cause variability in the way humans metabolize clinical drugs, environmental chemicals, and endogenous chemicals. These polymorphisms can alter human susceptibility of humans to chemical exposure and to disease. We have identified polymorphisms in human CYP2C9 which metabolizes ~15% of clinically important drugs including the anticonvlsant phenytoin, the antidiabetic drug tolbutamide and glipizide, the anticoagulant warfarin, and numerous nonsteroidal antiinflammatory drugs (NSAIDs) used in the treatment of arthritis. We have identified many variant alleles of CYP2C9 in Caucasians and African Americans; this year we also identified five new genetic polymorphisms in CYP2C9 which produce amino acid changes in CYP2C9 in Asians (in a warfarin therapy study). Using site-directed mutagenesis and a bacterial cDNA expression system, we expressed the recombinant human enzymes and assessed their catalytic activity toward the antidiabetic drug tolbutamide. One variant produced a a premature stop codon (a null variant), while two of other the six variants (R125H) and T299A were markedly defective in metabolizing tolbutamide in vitro and thus also predicted to be defective in vivo. New rapid pyrosequencing genotyping tests have been developed for known or putative defective alleles of human CYP2C9 alleles (*2, *3, *5, *6, 10, and 11 variants) found in Caucasians and African Americans and are being used in a prospective study in patients on warfarin therapy, to determine whether these alleles predict which individuals are susceptible to serious adverse bleeding effects in patients on warfarin therapy. In addition we found new polymorphisms in human CYP26A1 which is the priniciple enzyme in the embryo which protects from excess vitamin A synthesis. We discovered 12 mutations in human CYP26A1 including changes three which produce amino acid changes. Two of these alleles were predicted to be defective in the metabolism of retinoids based on expression of the recombinant proteins in a mammalian cell line. The CYP3A subfamily metabolizes also approximately 40% of all known drugs and many pesticides. We discovered a new allele CYP3A5*11 which is markedly deficient in metabolizing the calcium channel blocker drug nifedipine. Genotyping tests for CYP3A4 and CYP3A5 polymorphisms, identified the defective CYP3A5*3 allele in a poor metabolizer of methylprednisolone.? Factors controlling transcriptional regulation of the CYP enzymes also alter expression of the human CYP2Cs and can produce tolerance or drug-drug interactions. 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), GR (glucocorticoid receptor), and liver-enriched receptors. CYP2C8 was shown to be inducible in human hepatocytes by a variety of drugs and herbal remedies such as paclitaxel, rifampicin phenobarbital, phenytoin, hyperforin (in the Herbal remedy St John?s Wort and Citco. CYP2C8 drug induction occurs via a single distal CAR/PXR site at ~8.8 kb upstream. The murine CYP2Cs including their promoters are possible models for studying function and regulation of the human CYP2Cs and knockout mice indicate that cyp2c37 and cyp2c29 are induced via primarily via CAR receptors, rather than PXR; in contrast humans appear to be induced via both receptors. Quantitative PCR detected the presence of CYP2C9 and CYP2C8 in human aorta and cardiovascular tissues where their metabolites appear to be involved in vasodilatation and cardiac contractility.
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