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11ß-Hydroxysteroid Dehydrogenase Responsible for Carbonyl Reduction of the Tobacco-specific Nitrosamine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone in Mouse Lung Microsomes¹

Department of Pharmacology and Toxicology, School of Medicine, Philipps-University of Marburg, D-35033 Marburg, Germany

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 -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 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD 1; EC 1.1.1.146), an enzyme that is physiologically involved in glucocorticoid oxidoreduction. In this study, the expression of 11ß-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 V_max (135.8 versus 48.1 pmol/min/mg of protein) and K_m (6.8 versus 35.8 µM) values were much in favor for corticosterone oxidation compared to dehydrocorticosterone reduction. NNK carbonyl reduction displayed an apparent V_max of 655 pmol/min/mg of protein and a K_m of 629 µM. Interestingly, the intrinsic clearance (V_max/K_m ratio) of NNK carbonyl reduction (1.04) corresponds roughly to that of glucocorticoid reduction (1.34). The physiological glucocorticoid substrates of 11ß-HSD 1 (corticosterone and dehydrocorticosterone) and the selective 11ß-HSD 1 inhibitor glycyrrhetinic acid turned out to be strong inhibitors of NNK carbonyl reduction, displaying K_i values of 37.8, 21.3, and 10.9 µM, respectively. Affinity-purified antibodies specific for mouse liver 11ß-HSD 1 inhibited NNK carbonyl reduction in a concentration-dependent manner. For example, at the highest antibody concentration (5 µg of protein), 11ß-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 11ß-HSD 1 in mouse lung microsomes. Evidence is provided in the present study that 11ß-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 11ß-HSD 1/NNK carbonyl reductase and/or are concurrently being exposed to 11ß-HSD 1 modulators.

¹ This study was supported by grants from the Stiftung Verhalten und Umwelt (Verum; Munich, Germany) and the European Commission (BIO4-97-2123).

² To whom requests for reprints should be addressed, at Department of Pharmacology and Toxicology, School of Medicine, Philipps-University of Marburg, Karl-von-Frisch-Strasse 1, D-35033 Marburg, Germany. Phone: 49-0-6421-28-5465; Fax: 49-0-6421-28-5600.

Received 12/ 9/97. Accepted 5/18/98.

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