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, CYP2C19, and CYP2C8. CYP2C9 metabolizes numerous clinically important drugs including phenytoin, tolbutamide, warfarin, glipizide and numerous nonsteroidal antiinflammatory drugs (NSAIDs). By resequencing CYP2C9 in clinically known poor metabolizers and racially diverse populations, we have identified a total of 8 alleles of CYP2C9 many of which are defective. Several alleles have been shown to affect drug metabolism and toxicity (sometimes requiring hospitalization) in humans in clinical studies. Sequencing DNA from diverse racial populations have identified 35 single nucleotide polymorphisms (SNPs) in the promoter, exons, and intron-exon juctions of CYP2C9 including 6 alleles with new coding changes. These alleles have been expressed as recombinant enzymes in an E. coli expression system, and their catalytic activity toward tolbutamide examined. 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 CYP2C9*11 allele is markedly defective in its ability to metabolize tolbutamide (90% ). Clinical studies will address these alleles in the future. New alleles have been discovered in Asians. Many new defective alleles of CYP2C9 and CYP2C19 were prevalent in African-American or African populations, suggesting that these populations have not received sufficient study in the past. The CYP3A subfamily metabolizes approximately 40% of all known drugs and many pesticides. One new allele CYP3A4*17 is 99% deficient in metabolizing nifedipine a calcium channel blocker drug. This allele appears to be linked to CYP3A5*3 in certain Caucasian ethnic minorities from Eastern Europe, Turkey and the US. Genotyping tests have been developed for the new alleles. 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. We are investigating the CYP2C subfamily to elucidate how nuclear receptors control gene expression by binding specific elements within gene regulatory regions. Our work focuses on the promoter regions of the human CYP2C9, CYP2C19, and CYP2C8, as well as murine cyp2c29 and their regulation by the nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), GR (glucocorticoid receptor), RXR (retinoid X receptor), and the transcription factor C/EBP (CCAAT enhancer binding protein). We have shown that CYP2C9 is upregulated in HepG2 cells by drugs in cells transfected with hPXR. Two sites are present in the CYP2C9 promoter and one in the CYP2C19 promoter. Drugs and herbal remedies such as rifampicin, St John?s Wort and phenobarbital appear to upregulate via human PXR (HPXR) rather than human CAR. CYP2C9 is upregulated ~2-4 fold in HepG2 cells while CYP2C19 is upregulated only two fold. This is consistent with in vivo data in man. Hepatic nuclear factor 4 sites have been discovered in CYP2C9. CYP2C19 is structurally related to CYP2C9, but CYP2C19 is expressed at much lower levels than CYP2C19 in human liver, and appears less inducible. We identified only one CAR/PXR binding site in the CYP2C19 promoter versus two in the CYP2C9 promoter. CYP2C8 appears to contain at least one far upstream site and one more proximal site. The involvement of hPXR and hCAR in induction of this enzyme is being examined In contrast, in the mouse, CAR knockout studies and promoter constructs show that CYP2C29 is upregulated primarily by mouse CAR. The availability of CAR/PXR knockout mice enables us to examine whether drugs induce the CYP2Cs via CAR or alternate mechanisms. Thee murine CYP2Cs including their promoters are possible models for studying function and regulation of the human CYP2Cs. 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). model system. A total of 15 murine genes and pseudogenes have been discovered with different substrate specificity toward arachidonic acid. One metabolizes vitamin A and is deficient in the Ah receptor knockout model, causing hepatic vitamin A toxicity. A knockout mouse for CYP2C29 has been produced in collaborative studies and the physiological consequences will be addressed
NIH ExPorter
NIH ExPorter