Cytochrome P450s, the principal enzymes of foreign compound metabolisms, oxidize and inactivate most of the clinically used drugs so that they can be more easily eliminated from the body. P450s also activate chemical carcinogens to highly reactive electrophiles capable of binding to DNA and mutating genes. A large degree of species differences exist for P450 genes, particularly between rodents and humans. These differences are at the regulatory and catalytic level and are most prevalent between genes in the various CYP2 subfamilies. The CYP2A, CYP2B and CYP2C subfamilies are particularly complex with many members. cDNAs encoding two different human CYP2A and CYP2B P450s and four CYP2C P450s have been identified in humans. Since numerous rodent P450s within each of these subfamilies have been identified and the presence of multiple human CYP2A, CYP2B and CYP2C genes are suggested by Southern blotting analysis, we believe that other human P450s within these subfamilies have yet to be identified. We therefore developed strategies using polymerase chain reactions (PCR) to find and isolate new human P450s. CYP2CI8 was identified by PCR and its CDNA was cloned and completely sequenced. The CDNA was expressed into a protein with a relative molecular weight of 49,000. We have not, however, identified a favorable substrate for this enzyme. A PCR-based assay was developed to quantify levels of CYP2C8, CYP2C9 and CYP2CI8 MRNA. Studies are in progress to determine the catalytic specificities of human P450s using CDNA expression. Metabolism of the anticoagulant warfarin was studied using vaccinia virus-expressed P450s. Metabolism of carcinogens to mutagenic metabolites was analyzed by use of the lymphoblastoid CDNA expression system.
