The general objective of these ongoing studies is to provide a quantitative assessment of biochemical steps that may contribute to the modification of cellular DNA resulting from the metabolism of polycyclic aromatic hydrocarbons (PAH). Unidentified forms of cytochrome P-450 in uninduced rat liver dominate PAH activation. These forms will be purified, and polyclonal and monoclonal antibodies will be developed to quantitate their levels in liver and extrahepatic tissues and their contribution to PAH activation. PAH metabolism in uninduced rat liver requires unidentified forms that may also contribute to PAH activation in extrahepatic tissues. High affinity binding to P-450c by benzo(a)pyrene and certain phenolic metabolites contrasts with far weaker binding by BP-7,8-dihydrodiol. This has a major impact on the formation of the ultimate electrophile, (+)anti-BP-7,8- dihydrodiol 9,10-oxide (BPDE). Proposed experiments will evaluate the generality of this selectivity to other PAH and forms of P-450 and will determine the implications of these interactions upon the kinetics of activation of PAH. This is of great importance to understanding the actions of initiation inhibitors and co-carcinogens. Generation of superoxide radicals during PAH metabolism will be investigated (from uncoupled turnover at P-450 or quinone redox cycling?). These studies relate to the possible role of superoxide in promotion. PAH dihydrodiol oxides are generated in cell membranes and bind readily to serum lipoproteins. The effects of individual fatty acids, lipids, and lipoproteins on the reactivity of anti-BPDE will be quantitated with respect to hydrolysis, glutathione conjugation, DNA modification, and reaction with the anti-carcinogenic agent ellagic acid. Activation and detoxication of PAH in hepatocytes will be analyzed with respect to product inhibition of BP metabolism, the relative contributions of reactive electrophiles and superoxide to glutathione depletion, and the distribution of BPDE inside and outside the cells. In mammary cells, we will determine the characteristics of P-450 involvement in PAH metabolism and the effects of metabolism on GSH homeostasis. Finally, the mouse embryo 10 T1/2 cell transformation system will be developed for use with a liposomal activation system containing purified cytochrome P-450 and other purified microsomal enzymes in order to ask more specific questions about metabolic contributions to transformation.
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