DRUG METABOLIZING ENZYMES IN ANIMAL MODELS AND HUMAN TISSUE

细胞色素p450由主要的单加氧酶系统组成

The cytochrome P450s comprise the principle monooxygenase system which metabolizes foreign chemicals. Some of these enzymes are polymorphic. We are cloning and sequencing cDNAs for these P450s from individuals showing variations. We are then using cDNA expression systems such as yeast and COS-1 cells to assess the role of individual P450s to metabolize drugs and mutagens. We have prepared libraries from two human livers. No cDNA clones for P450IIC8 were identified in a human liver phenotypically low in IIC8 protein. Northern analysis and PCR analysis also indicate extremely low levels of the mRNA for this enzyme. However, a cDNA(254c) for a new human IIC P-450 was identified. We are searching for a full length clone to identify and express this enzyme. A second library was constructed from a human liver high in IIC8, and 90 essentially full length cDNA clones in the IIC subfamily were identified. Hybridization studies indicate that IIC8 represents 32% of the clones, and about 60% are identical or similar to IIC9 (either MP4 or MP8). Two variant full-length IIC9 clones were identified. Two additional cDNA clones representing variants of a new full-length IIC P-450(s) have been sequenced. Additional cDNAs are being characterized. Expression studies in yeast have compared expression of rat liver P450IIC13 and its phenotypic variant. P450IIC13 was expressed in yeast cells at a level 5-7-fold higher than its phenotypic variant containing 9 base substitutions, demonstrating that point mutations in the mRNA result in the defective expression of P450IIC13. We are presently using cDNA expression systems to express members of the human P450IIC subfamily. The ability of these human P450s to metabolize important drugs and environmental chemicals (mephenytoin, tolbutamide, and various classes of promutagens) will be analyzed. Liver tissue from approximately 25-50 humans will be analyzed for possible phenotypic variability of P450 enzymes using Northern analysis. These studies will assess the role of individual P450s in metabolic activation/deactivation of chemical mutagens and carcinogens.