Most chemical carcinogens, including polycyclic aromatic hydrocarbons (PAH), require metabolic activation to interact with cellular macromolecules, the presumed initial step in carcinogenesis. Activation of PAH has been envisioned to be catalyzed by monooxygenase enzymes. Recently, however, experimental results have begun to suggest that activation of PAH and other chemical carcinogens may occur by multiple mechanisms, and peroxidases, especially prostaglandin endoperoxide synthetase (PES), may also play a role in certain target tissues. Cytochrome P-450 catalyzes both one-electron and two-electron oxidations, while peroxidases catalyze the former. Increasing lines of evidence, including preliminary observation of benzo[a]pyrene (BP) - DNA adducts in mouse skin which can be formed by both one-electron and two-electron oxidation, suggest that multiple mechanisms of PAH activation occur and are significant in carcinogenesis. Working from the hypothesis that covalent binding of PAH to DNA is the first essential step in carcinogenesis, analysis of PAH-DNA adducts provides evidence to identify mechanisms of activation of PAH and the enzymes which catalyze them. Activation of PAH to form ultimate vicinal bay region diol epoxide metabolites, which bind to DNA, can be readily distinguished from activation by one-electron oxidation because the structures of the resulting PAH-DNA adducts are different and the two types of adducts behave very differently on high pressure liquid chromatography. The research proposed here will compare DNA adducts formed by radiolabeled BP in vivo in two target tissues, mouse skin and rat mammary gland, with the adducts formed in vitro with nuclei and/or microsomes from these two tissues using three different co-factors, NADPH, cumene hydroperoxide and arachidonic acid. Adducts will be identified by comparison with model adducts synthesized in vitro chemically or enzymatically in sufficient quantity for complete structural analysis. These studies are expected to provide considerable insight into the mechanisms of activation of BP in these two target tissues for PAH carcinogenesis. The results will also help us to identify the enzymes catalyzing these processes.
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