This application will continue with a systematic study of the role of dihydrodiol dehydrogenase in the detoxification of proximate and ultimate carcinogens of polycyclic aromatic hydrocarbons (PAH). The research program includes, chemical, biochemical, cellular and human studies designed to provide information on the role of this enzyme in PAH metabolism. We have shown that the homogeneous dihydrodiol dehydrogenase from rat liver cytosol oxidizes a wide variety of non-K-region trans-dihydrodiols of carcinogenic PAH to the corresponding ortho-quinones which can be trapped as mercaptoethanol adducts. To characterize this alternative pathway of proximate carcinogen metabolism, the reactivity of non-K- region ortho-quinone products towards cellular nucelophiles (e.g., cysteine and glutathione) will be examined, the influence of bay-region methylation on reactivity will be determined, and structures of glutathione adducts will be elucidated. To determine whether glutathione conjugation is enzyme catalyzed, the ability of the ortho-quinone products to act as substrates for six glutathione-S-transferases purified from rat liver cytosol will be assessed. The involvement of dihydrodiol dehydrogenase in ultimate carcinogen metabolism will be examined by testing (+)-anti-diol-epoxides of naphthalene, phenanthrene, chrysene, 5-methylchrysene and benzo[a]pyrene as substrates. Rat hepatoma cells (H-4IIe) known to contain dihydrodiol dehydrogenase will be incubated with the 7,8-trans-dihydrodiol of benzo[a]pyrene and if possible the glutathione adduct of the 7,8-ortho-quinone of benzo[a]pyrene will be identified as a cellular metabolite. The metabolism of the 7,8-trans-dihydrodiol of benzo[a]pyrene will be studied in H-4IIe cells in the presence of known inhibitors of dihydrodiol dehydrogenase [e.g., indomethacin, 6-medroxyprogesterone acetate and the suicide substrate 1-(4'-nitrophenyl)-2-propen-1-o1]. If the dehydrogenase plays a significant role in PAH metabolism enzyme inhibition should result is an increase in diol-epoxides of benzo[a]pyrene (detected as tetraols and DNA adducts). Human liver hepatoma (Hep-G2) and hamster embryo cells will also be screened for the drug sensitive dihydrodiol dehydrogenase. If present the experiments described for H-4IIe cells will be repeated. To determine whether dihydrodiol dehydrogenase plays a role in PAH metabolism in humans, the enzyme will be detected in human liver samples by enzyme assay and Western blot analysis. Activities will be resolved by chromatography and isozymes which oxidize the 7,8-trans-dihydrodiol of benzo[a]pyrene will be identified. The mRNA species coding for the human enzyme will be identified by Northern analysis using the cDNA for the rat liver enzyme.
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