IDENTIFICATION AND CHARACTERIZATION OF NEW HUMAN P-450

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Chemical carcinogens are believed to be the major cause of human cancer. These compounds generally must be activated to forms, termed ultimate carcinogens, that are electrophiles capable of binding to cellular macromolecules such as proteins, RNA, and DNA. Mutation of DNA is believed to be responsible for cellular transformation. The enzymes capable of activating carcinogens are the cytochrome P-450s. Multiple forms of P-450 exist that are encoded by anywhere from 50 to 200 genes. During the past several years it has become quite evident that rodents and humans possess P-450s that can be quite distinct. Quantitative and qualitative species differences have been uncovered. In particular catalytic specificities of rodent P-450s can differ from those of human P-450s. These findings have major implications in carcinogenesis research since rodents are frequently used as experimental models for tumorigenesis mediated by chemical exposures. We have therefore focused on the characterization of human P-450s by directly cloning their cDNAs from liver and lung libraries. These are then inserted into expression vectors and used to produce cultured cells containing catalytically active P-450 enzymes. The active P-450s are then tested for their abilities to activate chemical carcinogens to DNA-binding metabolites. Two systems have been used: 1) vaccinia virus transient expression capable of producing high levels of protein for direct enzymatic and spectral analysis; ii) a human lymphoblastoid cell line stable expression system uniquely suitable for carcinogen testing.