The cytochrome P450s comprise the principle monooxygenase system which metabolizes foreign chemicals. Some of these enzymes are polymorphic in man. We have isolate and characterized cDNAs for 8 members of the CYP2C subfamily including cDNA representing 2C8, 2C9 and three new genes (2C18, 2C17, and 2C19). A number of allelic variants were isolated. Five full length clones (representing allelic variants of 2C9, 2C18, 2Cl9) were expressed in a Cos cell expression system. Differences in metabolism of tolbutamide (primarily 2C9) and mephenytoin (2C18) were noted. Livers from humans which varied dramatically in their ability to metabolize the drug S- mephenytoin have been analyzed for 2C8, 2C9, 2C18, and 2C19 mRNAs. S-mephenytoin correlated highly with 2C18 mRNA (r = 0.95) and to a lesser extent with 2C9 (r= 0.65) suggesting that one or both of these isozymes are involved in this polymorphism. Similar methods are being established to examine the variability of human live P4501A2. A phenotypic difference in a rat P450 (2Cl3) was determined by site-directed mutagenesis to result from a single amino acid substitution in a semi-conserved region (180) which altered the half-life. Alteration of this amino acid in a human P450 by site-directed mutagenesis also decreased its half-life showing the importance of this residue. All of the full length human CYP2C cDNAs were the expressed in a yeast cDNA expression system. Metabolism was poorer than anticipated therefore, the human P450s are being purified from yeast microsomes in order to characterize their metabolism further. They have also been inserted into the AHH/TK cell line in order to characterize their ability to metabolize premutagens Preliminary data suggests that one cytochrome may metabolize aflatoxin. We ar attempting to address the cytochrome responsible for the phenotypic variation of mephenytoin metabolism in humans as well as the consequences of other phenotypic variations in humans. We are also addressing the effects of cigarette smoking an environmental chemicals on hepatic levels of another variable enzyme P4501A2.
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