Most chemica1 carcinogens require metabolic activation to intermediates which react with DNA to form adducts and perhaps initiate cancer. Carcinogenic polycyclic aromatic hydrocarbons (PAH) are metabolized by two main pathways, monooxygenation and one-electron oxidation, forming reactive diol-epoxides or radical cations, respectively, as ultimate intermediates. The objectives of this grant are to continue elucidating the benzo(a)pyrene (BP)- DNA adducts formed by one-electron oxidation, to begin investigating their significance, and to examine the hypothesis that one mechanism by which cytochrome P-450 activates PAH is cooxidation in reactions supported by hydroperoxides formed by lipid peroxidation in the cell. To accomplish this we will 1) Identify BP-DNA adducts formed chemical1y and enzymically in vitro and in topically-treated mice and mammary-treated rats using monoclonal antibodies raised against BP-deoxyguanosine adducts synthesized electrochemically. 2) Examine the stable and labile DNA adducts formed by reaction of BP with a 229 base-pair fragment of pBR322 DNA by a) analyzing single-strand breaks in DNA reacted with BP radical cation perchlorate or treated with BP in the horseradish peroxidase system, b) analyzing alkali-labile 1esions in the BP-treated DNAs, and c) digesting BP-treated DNAs, using exonuclease III to identify the sites of stable adducts. 3) Examine the ability of cytochrome P-450 to cooxidize BP in reactions supported by hydroperoxides of various chain lengths by a) determining and comparing the profiles of BP metabolites and levels of binding of BP to DNA, b) determining the inhibitory effects of trapping agents on the binding of BP to DNA, c) identifying the BP- DNA adducts formed with activation by rat liver microsomal cytochrome P-450 with cumene hydroperoxide, and d) identifying the BP-cysteine adducts formed with hydroperoxidesupported cytochrome P-450. These experiments are expected to demonstrate the presence of both stable and labile BP-DNA addvcts formed by one-electron oxidation in target tissues and identify sites of adduct formation in DNA sequences. In addition, we will determine the ability of bio1ogically relevant hydroperoxides to support cytochrome P-450- mediated activation of BP.
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