DRUG METABOLIZING ENZYMES IN ANIMAL MODELS AND HUMAN TISSUE

CYP酶包括代谢的单加氧酶系统

The CYP enzymes comprise the monooxygenase system which metabolizes foreign chemicals. We are studying the CYP2C subfamily which includes a P450 which is polymorphic in man and metabolizes the drug S- mephenytoin. We used a yeast cDNA expression system to express CYP2C8, 2C9, 2C18, and 2C19 and several allelic variants and compared their ability of these recombinant proteins to metabolize warfarin, and tolbutamide. 2C9 was the principal enzyme metabolizing tolbutamide, but only 2C18 has thus far been found to metabolize S-mephenytoin but turnover numbers for mephenytoin were somewhat low. We are extending the S-mephenytoin metabolism with a more rigorous study of optimum incubation conditions. To identify which of these enzymes is involved in the polymorphism, we are attempting to develop specific antibodies to all four gene products to compare metabolism with protein levels. The recombinant proteins are being expressed in bacteria prior to antibody production. The complete gene for 2C18 and all of 2C9 except for exon 6 have been isolated from human liver and >1kb of upstram region and all exons and intron junctions sequenced. We sequenced the upstream region of 2C18 from a high and poor metabolizer but found no sequence differences. Upstream regions of 2C18 and 2C9 have been expressed using a luciferase reporter gene construct. The relative promotor abilities of 2C9 and 2C18 appear consistent with the greater expression of 2C9 in human liver. Potential liver-specific sites, glucocorticoid responsive elements, a possible inhibitory region, and an element which may infer phenobarbital inducibility have been identified. Future studies will address the genetic defect(s) which affect metabolism of S- mephenytoin and other 2C substrates in humans. We are also examing variabilities in expression of CYP1A2 and CYP1A1 in human liver. PCR analysis will correlate expression of these genes with smoking history and exposure to dioxins. Dose response data for the induction of CYP1A1 AND CYP1A2 are being performed in human and rat liver slices.