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Characterization of dihydrodiol dehydrogenase in rat H-4IIe hepatoma cells
A L Buller1, R B Sharp, T M Penning
1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
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.
Insights
Dihydrodiol dehydrogenase, an enzyme that may reduce carcinogen activity, was studied in rat hepatoma cells. This enzyme shares properties with the rat liver enzyme and is not induced by common drug metabolizing enzyme inducers.
Area of Science:
- Biochemistry
- Enzymology
- Carcinogenesis Research
Background:
- Dihydrodiol dehydrogenase (EC 1.3.1.20) oxidizes trans-dihydrodiol proximate carcinogens, potentially suppressing carcinogenicity.
- Rat H-4IIe hepatoma cells were investigated as a model system for studying this enzyme's activity and characteristics.
Purpose of the Study:
- To characterize dihydrodiol dehydrogenase activity in rat H-4IIe hepatoma cells.
- To compare the properties of the hepatoma cell enzyme with the purified rat liver enzyme.
- To investigate the regulation of dihydrodiol dehydrogenase activity in response to various agents.
Main Methods:
- Spectrophotometric detection of enzyme activity using benzenedihydrodiol as a substrate.
- Inhibition studies with 6-medroxy-progesterone acetate and indomethacin.
- Chromatofocusing for pI determination, Western blot analysis for molecular weight and immunoreactivity, and immunotitration.
- Enzyme induction studies using known inducers of drug metabolizing enzymes.
Main Results:
- Dihydrodiol dehydrogenase activity in H-4IIe cells was similar to rat liver cytosol and sensitive to 6-medroxy-progesterone acetate and indomethacin.
- The enzyme coeluted with 3 alpha-hydroxysteroid dehydrogenase at pI 5.8, showed a Mr of 34,000, and was immunoreactive with anti-3 alpha-hydroxysteroid/dihydrodiol dehydrogenase serum.
- Neither benz[a]anthracene nor dexamethasone, nor other tested agents, induced dihydrodiol dehydrogenase activity in the hepatoma cells.
Conclusions:
- Rat H-4IIe hepatoma cells possess dihydrodiol dehydrogenase activity with properties mirroring the purified rat liver enzyme, making them a valuable model.
- The enzyme's characteristics, including pI, Mr, immunoreactivity, and drug sensitivity, suggest it is identical to 3 alpha-hydroxysteroid dehydrogenase.
- Dihydrodiol dehydrogenase activity in these cells is not regulated by common inducers of phase I and phase II drug metabolizing enzymes.