title: Characterization of dihydrodiol dehydrogenase in rat H-4IIe hepatoma cells.
abstract: Dihydrodiol dehydrogenase (EC 1.3.1.20) catalyzes the NADP+-dependent oxidation of a variety of trans-dihydrodiol proximate carcinogens, a reaction that may suppress their carcinogenicity. Using benzenedihydrodiol [(+)-trans-1,2-dihydroxy-3,5-cyclohexadiene] as a substrate, this enzyme can be detected spectrophotometrically in rat H-4IIe hepatoma cells with a specific activity similar to that observed in rat liver cytosol. The hepatoma cell enzyme is potently inhibited by 6-medroxy-progesterone acetate (IC50 = 38 nM) and indomethacin (IC50 = 3.5 microM). These cells contain 3 alpha-hydroxysteroid dehydrogenase which is also sensitive to inhibition by the same two drugs. Chromatofocusing of hepatoma cell lysates indicates that both dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activities coelute with a pI = 5.8. Western blot analysis of hepatoma cell lysates, using rabbit anti-rat 3 alpha-hydroxy-steroid/dihydrodiol dehydrogenase serum detects a single immunoreactive species with a Mr 34,000. Using this antiserum it was possible to immunotitrate both these enzyme activities in H-4IIe lysates. Exposure of confluent cells to either 10 microM benz[a]anthracene or 10 microM dexamethasone, which are known inducers in H-4IIe cells of aryl-hydrocarbon hydroxylase and tyrosine aminotransferase respectively, failed to elevate dihydrodiol dehydrogenase activity. The following agents also failed to induce dihydrodiol dehydrogenase activity: phenobarbital, ethoxyquin, phenolic anti-oxidants, testosterone, estradiol-17 beta, and growth hormone. Since the hepatoma cell enzyme has properties in common with the purified rat liver enzyme (which is identical to 3 alpha-hydroxysteroid dehydrogenase) including, Mr, pI, immunoreactivity, and sensitivity to drug inhibition, this cell line represents a useful system for studying the role of dihydrodiol dehydrogenase in the further metabolism of trans-dihydrodiols. Interestingly, the enzyme does not appear to be under the control of known inducers of phase I and phase II drug metabolizing enzymes.
authors.affiliation: Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
authors.forename: A L
authors.initials: AL
authors.lastname: Buller
authors.forename: R B
authors.initials: RB
authors.lastname: Sharp
authors.forename: T M
authors.initials: TM
authors.lastname: Penning
mesh_terms: D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001711:Biotransformation; D015153:Blotting, Western; D002853:Chromatography, Liquid; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism; D007213:Indomethacin / Q000494:pharmacology; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008525:Medroxyprogesterone / Q000494:pharmacology; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011083:Polycyclic Compounds / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics / Q000633:toxicity; D051381:Rats; D014407:Tumor Cells, Cultured
chemical_list: D011083:Polycyclic Compounds; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D006913:Hydroxysteroid Dehydrogenases; C023125:dihydrodiol dehydrogenases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C116788:cis-1,2-dihydro-1,2-dihydroxynaphthalene dehydrogenase; D008525:Medroxyprogesterone; D007213:Indomethacin