Ivins J K, Penning T M
Cancer Res. 1987 Feb 1;47(3):680-4.
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 microCi/nmol, the assay provides a 5000-fold increase in sensitivity over the existing spectrophotometric method. The radiochemical assay was validated by comparing the Km and Vmax 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 (Km = 0.77 +/- 0.11 mM, Vmax = 2.14 +/- 0.13 nmol/min/mg protein) and determined spectrophotometrically (Km = 0.96 +/- 0.10 mM, Vmax = 6.31 +/- 0.50 nmol/min/mg protein). Using the radiochemical method, dihydrodiol dehydrogenase activity was detected in extrahepatic sites of polycyclic aromatic hydro-carbon metabolism: lung greater than small intestine greater than testis greater than bladder greater than 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.
二氢二醇脱氢酶（EC 1.3.1.20）催化（-）-7R，8R-二羟基-二氢苯并（a）芘和（+）-7S，8S-二羟基-二氢苯并（a）芘的NADP +依赖性氧化，这两种物质都是强效的近致癌物（Smithgall、Harvey和Penning，《生物化学杂志》，261:6184 - 6191，1986）。以苯二氢二醇[（+）-反式-1,2-二羟基-3,5-环己二烯]作为这些反应的模型底物，通过测量吡啶核苷酸吸光度的变化，可以在大鼠肝脏胞液中测定二氢二醇脱氢酶。该方法缺乏检测肝外组织中该酶的灵敏度。在此，我们描述了一种用于二氢二醇脱氢酶的灵敏放射化学测定法，其中苯二氢二醇氧化为邻苯二酚与儿茶酚-O-甲基转移酶（EC 2.1.1.6）催化的O-甲基化反应相偶联。通过这种方式，氧化步骤中形成的邻苯二酚可以使用S-腺苷[甲基-3H]甲硫氨酸作为甲基供体进行放射性标记。生成的氚化产物愈创木酚很容易萃取到甲苯中，并通过闪烁计数进行定量。使用比活度为0.1微居里/纳摩尔的S-腺苷[甲基-3H]甲硫氨酸，该测定法的灵敏度比现有的分光光度法提高了5000倍。通过比较从大鼠肝脏胞液的40 - 75%硫酸铵分级分离物中获得的Km和Vmax值与分光光度法测定的值，验证了该放射化学测定法。放射化学测定值（Km = 0.77 ± 0.11 mM，Vmax = 2.14 ± 0.13纳摩尔/分钟/毫克蛋白质）与分光光度法测定值（Km = 0.96 ± 0.10 mM，Vmax = 6.31 ± 0.50纳摩尔/分钟/毫克蛋白质）之间有密切的一致性。使用放射化学方法，在多环芳烃代谢的肝外部位检测到了二氢二醇脱氢酶活性：肺＞小肠＞睾丸＞膀胱＞前列腺。比活性变化超过50倍（0.866 - 0.017纳摩尔/分钟/毫克蛋白质），并且与器官对多环芳烃诱导的化学致癌作用的易感性没有严格的负相关。主要参与反式二氢二醇氧化的四个组织（肝脏、肺、小肠和睾丸）含有二氢二醇脱氢酶，其受到吲哚美辛的强烈抑制，其中两个组织（肝脏和小肠）含有对醋酸6-甲氧基孕酮抑制敏感的脱氢酶。这些观察结果表明，吲哚美辛和醋酸6-甲氧基孕酮可能会阻止反式二氢二醇近致癌物在参与其进一步代谢和活化的主要组织中的氧化。
