CHARACTERIZATION OF HUMAN P450S AND THEIR GENES

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The long-term goals of this project include: 1) The development of methods to assess P450 expression in humans to determine the roles of these enzymes in safe drug therapy and susceptibility to environmentally- based diseases such as cancer and mental disorders. 2) To develop convenient recombinant human P450-based systems that can be used in drug development and carcinogenesis research. The expression and catalytic activities of human P450s expressed in lung and liver tissues are being directly examined. The human lung was found to contain several P450 forms, the most abundant being CYP4B1. CYP2B7 and CYP2F1, expressed in human lung, are also found at lower levels in liver tissue. In lungs from tobacco smokers, CYP1A1 is found. A large degree of inter- individual variability in levels of these mRNAs were found, but no clear evidence for a polymorphism emerged from these data. The CYP2D6 P450 is responsible for a major genetic polymorphism in drug oxidation. A number of null CYP2D6 alleles have been characterized by direct DNA sequencing of genes from subjects deficient in CYP2D6 catalytic activities. These account for the primary defect in up to 10% of Caucasians. Two variant alleles, CYP2D6C and CYP2D6J, were found that have amino acid differences from the wild-type CYP2D6 enzyme, resulting in lower catalytic rates. CYP2D6J was found to be very frequent in Asians, thus accounting for the slower metabolic rates of this ethnic group for metabolism of many clinically used drugs. PCR tests have been developed for most defective CYP2D6 alleles that can be used to diagnose deficient metabolizers. Studies have also revealed a potential polymorphism in the CYP2C9 gene that encodes a P450 responsible for metabolism for warfarin. Several human P450s have been expressed into catalytically-active enzymes using baculovirus and vaccinia virus. A locus of P450 genes found on the long arm of chromosome 19 were cloned on a contiguous segment of over 275 kbp. P450 genes in the CYP2A, CYP2B, and CYP2F gene subfamilies were localized by restriction mapping and Southern blotting and these are currently being completely sequenced.