Drug Metabolizing Enzymes In Humans And Animal Models

Environmental Health Sciences

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Abstract

We have identified new genetic polymorphisms in the CYP family in humans which are responsible for introducing variability in the way humans metabolize specific drugs and environmental chemicals and thus alter susceptibility of humans to these chemicals. Our laboratory has made substantial contributions in identifying polymorphisms in cytochrome P450 enzymes. Most progress has been made in the CYP2C subfamily. These include CYP2C9 and CYP2C19. CYP2C9 metabolizes numerous clinically important drugs including phenytoin, tolbutamide, warfarin, glipizide and numerous nonsteroidal antiinflammatory drugs (NSAIDs). By resequencing racially diverse populations, we have identified 35 single nucleotide polymorphisms (SNPs) in CYP2C9 including 6 alleles with new coding changes alleles of CYP2C9 several of which appear to be defective. These alleles have been expressed as recombinant proteins in an E. coli expression system, and their catalytic activity toward tolbutamide assessed. The newly discovered CYP2C9 alleles include the amino acid changes L19I (2C9*7), R150H (2C9*8), H251R (2C9*9), E272G (2C9*10), R335W (2C9*11) and P489S (2C9*12). The R335W (CYP2C9*11) allele is very defective in its metabolism of tolbutamide (90% decrease). Of six additional alleles discovered in Asians, Two are null (producing no protein) and two are catalytically defective. Most of these new defective alleles of CYP2C9 were found in African-Americans or Asian. The CYP3A subfamily metabolizes approximately 40% of all known drugs and many pesticides. We discovered a new allele CYP3A4*17 is 99% deficient in metabolizing the calcium channel blocker drug nifedipine. This allele is linked to the deective CYP3A5*3 in certain Caucasian ethnic minorities from Eastern Europe, Turkey and the US. Genotyping tests have been developed for the new alleles and pharmacogenetic studies of a poor metabolizer of methlprednisolone completed. Factors controling transcriptional regulation of the CYP enzymes are critical to regulation of the CYP2Cs and produce drug-drug interactions. There is evidence in the literature that the human CYP2Cs may be upregulated by prior exposure to drugs, but the mechanism has been unknown. Our work focuses on the promoter regions of the human CYP2C genes as well as their murine counterparts and their transcriptional regulation by the nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), GR (glucocorticoid receptor), RXR (retinoid X receptor) and other receptors. We have shown that CYP2C9 is upregulated in HepG2 cells by drugs which are hPXR ligands. Two CAR/PXR sites are present in the CYP2C9 promoter. The proximal site is essential while the upstream site appears to contribute to full induction. Drugs and herbal remedies such as rifampicin, St John's Wort and phenobarbital appear to upregulate primarily via human PXR (hPXR). Hepatic nuclear factor 4 alpha sites have been discovered in CYP2C9 which appear to be necessary for CAR activation and for PXR mediated induction via rifampicin. CYP2C8 is inducible by a variety of drugs and herbal remedies such as paclitaxel, rifampicin, phenobarbital, phenytoin, hyperforin and Citco (a CAR agonist). Some of these compounds are PXR agonists while others are CAR agonists, or act with both receptors. CYP2C8 activation by CAR/PXR appears to be confirmed by a single distal 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 cyp2c29 is induced via CAR receptors. We have discovered new murine CYP2Cs, including a new CYP2C44 which stereospecifically metabolize arachidonic acid, is found in kidney and may be involved in vasodilation in this tissue. Quantitative PCR shows this P450 is more abundant than liver than kidney (10X). Quantitative PCR is assessing CYP2C9 and CYP2C8 in human aorta and heart where their metabolites may also be involved in vasodilation.