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Radiochemical Detection of Dihydrodiol Dehydrogenase: Distribution of the Enzyme in Male Sprague-Dawley Rat Tissues and Its Sensitivity to Inhibition by Indomethacin and 6-Medroxyprogesterone Acetate¹

Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Dihydrodiol dehydrogenase (EC 1.3.1.20) catalyzes the NADP⁺-dependent oxidation of (-)-7R,8R-dihydroxy-dihydro-benzo(a)pyrene and (+)-7S,8S-dihydroxy-dihydro-benzo(a)pyrene, which are potent proximate carcinogens (Smithgall, Harvey, and Penning, J. Biol. Chem., 261: 6184–6191, 1986). Using benzenedihydrodiol [(+)-trans-1,2-dihydroxy-3,5-cyclohexadiene] as a model substrate for these reactions, dihydrodiol dehydrogenase can be assayed in rat liver cytosol by measuring the change in absorbance of the pyridine nucleotide. This method lacks the sensitivity to detect the enzyme in extrahepatic tissues. Here we describe a sensitive radiochemical assay for dihydrodiol dehydrogenase in which the oxidation of benzenedihydrodiol to pyrocatechol is coupled to O-methylation catalyzed by catechol-O-methyltransferase (EC 2.1.1.6). In this manner the pyrocatechol formed in the oxidation step can be radiolabeled using S-adenosyl[methyl-³H]methionine as methyl donor. The resulting tritiated product, guaiacol, is readily extracted into toluene and quantified by scintillation counting. Using S-adenosyl[methyl-³H]methionine at a specific activity of 0.1 µCi/nmol, the assay provides a 5000-fold increase in sensitivity over the existing spectrophotometric method. The radiochemical assay was validated by comparing the K_m and V_max values obtained for the 40-75% (NH₄)₂SO₄ fraction of rat liver cytosol with those measured spectrophotometrically. There was close agreement between values determined radiochemically (K_m = 0.77 ± 0.11 mM, V_max = 2.14 ± 0.13 nmol/min/mg protein) and values determined spectrophotometrically (K_m = 0.96 ± 0.10 mM, V_max = 6.31 ± 0.50 nmol/min/mg protein). Using the radiochemical method, dihydrodiol dehydrogenase activity was detected in extrahepatic sites of polycyclic aromatic hydrocarbon metabolism: lung > small intestine > testis > bladder > prostate. Specific activities varied over 50-fold (0.866-0.017 nmol/min/mg protein) and did not show a strict inverse correlation with organ susceptibility to PAH-induced chemical carcinogenesis. Four tissues predominantly concerned with trans-dihydrodiol oxidation (liver, lung, small intestine, and testis) contain dihydrodiol dehydrogenase which is potently inhibited by indomethacin, two of these tissues (liver and small intestine) contain dehydrogenase sensitive to inhibition by 6-medroxyprogesterone acetate. These observations suggest that indomethacin and 6-medroxyprogesterone acetate may prevent the oxidation of trans-dihydrodiol proximate carcinogens in major tissues involved in their further metabolism and activation.

¹ These studies were supported by NIH Grant CA-39504, a Biomedical Research Support Grant (2-SO7-RR-07083-20-sub 23) awarded to the University of Pennsylvania by the Division of Research Resources, NIH, and a Grant-In-Aid from the American Heart Association, South Eastern Pennsylvania Affiliate.

² Supported by NIH Training Grant GM-07229.

³ To whom requests for reprints should be addressed, at University of Pennsylvania, School of Medicine, Department of Pharmacology/G3, 34th and Hamilton Walk, Philadelphia, PA 19104.

Received 5/16/86. Revised 9/22/86. Accepted 10/31/86.