Summary of Work: The CYP enzymes metabolize drugs and xenobiotics, and polymorphism in some of these enzymes accounts for variability in metabolism in man. We have identified five defects in CYP2C19 responsible for poor metabolizers (PMS) of the anticonvulsant drug mephenytoin and the antiulcer drug omeprazole and developed genetic tests for two of these polymorphisms. Genetic tests developed in our laboratory identify about 100% of Oriental and about 96% of Caucasian PMS in clinical studies. A mutation in the initiation codon of certain poor metabolizers prevents translation. A new mutation in the heme binding region has been identified which prevents expression oth eh protein in a cDNA expression system. An amino acid mutation in exon 3 was identified in a poor metabolizer of mephenytoin and is being expressed in cDNA expression systems to verify whether it is a defective allele. Additional studies are directed toward identifying additional defects in CYP2C19 and developing and improving the accuracy of genetic testing. A defective allele of CYP2C9 affects its affinity for substrates such as the antidiabetic drug tolbutamide and the anticancer drug cyclophosphamide and affects metabolism of tolbutamide and warfarin in vivo. Sequencing of cDNAs of CYP2Cs have identified four new CYP2Cs in the mouse, a common laboratory animal for toxicity and carcinogenicity tests. These mouse P450s are being expressed in cDNA expression systems and their comparison of metabolism of drugs and endogenous compounds such as arachidonic acid is being compared with that of the human forms. A new canine CYP2C was identified which has been shown to be polymorphic and may be responsible for variability in metabolism of certain compounds in the dog, a common laboratory test animal for drugs. cDNA expression and site-directed mutagenesis studies are being used to identify amino acids which are important in the substrate specificity oof the CYP2C subfamily. Structure-activity studies of human CYP2Cs have identified areas in SRS 2 and 4 as important in metabolism of omeprazole, warfarin, an anticoagulant, and diclofenac, an anti-inflammatory drug. Studies are directed toward identifying key amino acids in the human P450s which confer specificity for particular drugs and environmental chemicals. cDNA expression studies are directed toward identifying which CYP2C enzymes are involved in pesticide metabolism, and metabolism of the arachidonic acid and other substrates. Such studies are also attempting collaboratively to identify regions of the P450 model involved in suicide activation of substrates such as tienilic acid and the formation of autoimmune antibodies in humans. These studies will help identify which portions of the P450 molecule permit their high degree of specificity and selectivity toward certain drugs and xenobiotics, and models will help predict which addtional compounds will be metabolized by these P450 enzymes.