EXPRESSION OF CYTOCHROME P-450S AND THEIR ROLE IN MUTAGENESIS & CARCINOGENESIS

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The cytochrome P450 superfamily of genes code for a number of enzymes which metabolize a wide array of compounds including endobiotics and xenobiotics. The ability of the cytochrome P450s to carry out such diversity of functions on a variety of substrates results from multiple forms of distinct P450s. We are primarily interested in the biochemical and molecular mechanisms of chemical carcinogenesis. To elucidate the role of P450s involved in the biotransformation of xenobiotics to toxic, mutagenic and carcinogenic forms, it is essential that the individual P450s are expressed in cells which lack endogenous activity for these enzymes. Toward this goal we have developed expression systems by which DNAs coding for individual cytochrome P450s can be delivered into mammalian cells and these engineered cell lines can be analyzed for the consequences of functional expression of cytochromes P450. Rodent cells were transduced with recombinant retroviruses containing the DNA for cytochrome P4501A2. Analysis of the clones for P4501A2 showed colinear integration of a single copy of the DNA into the chromosomal DNA of host cells and the expected P4501A2 activities. Clones constitutively expressing cytochrome P4501A2 when exposed to the food-derived carcinogenic heterocyclic amines IQ, MeIQx, and PhIP, and the aromatic amines AF and AAF each formed specific DNA adducts with the host cell DNA. The adduct formation was dependent on the duration of exposure and the dose of the carcinogen. 7,8-Benzoflavone, a known inhibitor of P4501A2 activity, blocked the formation of these adducts, indicating that metabolic activation by P4501A2 is required for the adduct formation. Analysis of clones containing DNA-adducts for possible adduct-directed mutations showed that the cells indeed suffered mutations in the hypoxanthine-guanine phosphoribosyl transferase gene and the mutant clones lacked HGPRT activity. Thus, we established a well-defined prototype mammalian cell system expressing cytochrome P4501A2 for biochemical and molecular analysis of mutagenesis induced by xenobiotics.