11Beta-hydroxysteroid dehydrogenase responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mouse lung microsomes.
The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in laboratory animals and is most likely involved in the etiology of tobacco smoke-induced lung cancer. To exert its carcinogenic potential, NNK must be metabolically activated by alpha-hydroxylation at either the methyl or methylene carbons adjacent to the N-nitroso group. The main detoxification pathway of NNK involves carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol followed by glucuronosylation at the hydroxy moiety produced by carbonyl reduction. Whereas there has been great success in the identification of cytochrome P450 species catalyzing NNK activation, the enzyme responsible for NNK carbonyl reduction has been searched for since 1980. In previous investigations, we succeeded in identifying the NNK carbonyl reducing enzyme in mouse liver microsomes as being 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD 1; EC 1.1.1.146), an enzyme that is physiologically involved in glucocorticoid oxidoreduction. In this study, the expression of 11beta-HSD 1 was established on the mRNA (reverse transcription-PCR) and protein (immunoblot) levels. Kinetics of glucocorticoid oxidoreduction were determined with corticosterone and dehydrocorticosterone as substrates for oxidation and reduction, respectively. The apparent Vmax (135.8 versus 48.1 pmol/min/mg of protein) and Km (6.8 versus 35.8 microM) values were much in favor for corticosterone oxidation compared to dehydrocorticosterone reduction. NNK carbonyl reduction displayed an apparent Vmax of 655 pmol/min/mg of protein and a Km of 629 microM. Interestingly, the intrinsic clearance (Vmax/Km ratio) of NNK carbonyl reduction (1.04) corresponds roughly to that of glucocorticoid reduction (1.34). The physiological glucocorticoid substrates of 11beta-HSD 1 (corticosterone and dehydrocorticosterone) and the selective 11beta-HSD 1 inhibitor glycyrrhetinic acid turned out to be strong inhibitors of NNK carbonyl reduction, displaying Ki values of 37.8, 21.3, and 10.9 microM, respectively. Affinity-purified antibodies specific for mouse liver 11beta-HSD 1 inhibited NNK carbonyl reduction in a concentration-dependent manner. For example, at the highest antibody concentration (5 microg of protein), 11beta-HSD 1 activity was decreased to a residual 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol formation of only 7.9% compared to the uninhibited control, thus conclusively demonstrating NNK carbonyl reduction to be mediated by 11beta-HSD 1 in mouse lung microsomes. Evidence is provided in the present study that 11beta-HSD 1 is expressed in mouse lung and that it functions as NNK carbonyl reductase in mouse lung microsomes. These findings may have potentially important implications for smokers who express low levels of 11beta-HSD 1/NNK carbonyl reductase and/or are concurrently being exposed to 11beta-HSD 1 modulators.
Maser
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Department of Pharmacology and Toxicology, School of Medicine, Philipps-University of Marburg, Germany.
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000287:Administration, Topical; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D005260:Female; D005938:Glucocorticoids / Q000378:metabolism / Q000494:pharmacology; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D006913:Hydroxysteroid Dehydrogenases / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D008168:Lung / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D009602:Nitrosamines / Q000378:metabolism*; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D005938:Glucocorticoids; D009602:Nitrosamines; D012333:RNA, Messenger; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D006034:Glycyrrhetinic Acid