Carbonyl reduction of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in cytosol of mouse liver and lung

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-buta none (NNK) is a strong lung carcinogen in all species tested. To elicit its tumorigenic effects, NNK requires metabolic activation which is supposed t o occur via alpha-hydroxylation by cytochrome P450 enzymes. Carbonyl reduct ion to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) followed by glu curonosylation is considered to be the main detoxification pathway in human s. Therefore, NNK carbonyl reducing activity is crucial for NNK inactivatio n since it initiates the final excretion of this lung carcinogen. Until the present work, studies on NNK metabolism have focused exclusively on micros omal fractions, and several cytochrome P450 enzymes have been shown to be i nvolved in alpha-hydroxylation of NNK. In addition, 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD 1) which is located in the endoplasmic re ticulum of the cell has been identified to catalyze the carbonyl reduction of NNK in microsomes. In this study, we provide evidence that carbonyl redu ction of NNK does also take place in the cytosolic fraction of mouse liver and lung, and that cytosolic carbonyl reductase contributes to the detoxifi cation of NNK. At a fixed substrate concentration of 1 mM NNK, the specific activity of cytosolic NNAL formation amounts to 72% (liver) and 28% (lung) compared with that in the respective microsomal fractions. Although consid erable NNK carbonyl reduction occurred with NADH, the preferred cosubstrate in cytosol is either NADPH or an NADPH-regenerating system. Due to the inh ibitor sensitivity to menadione, ethacrynic acid, dicoumarol and quercitrin , it is concluded that carbonyl reductase (EC 1.1.1.184) is mainly responsi ble for NNAL formation in liver and lung cytosol. The expression of cytosol ic carbonyl reductase and microsomal 11 beta-HSD 1 was established on the m RNA level by reverse transcription-PCR in both liver and lung. Enzyme kinet ic studies revealed a nonsaturable Michaelis-Menten kinetic of NNK carbonyl reduction in cytosol. Possibly some other cytosolic NNK carbonyl reducing enzymes are also involved in NNAL formation. In conclusion, this is the fir st report to show that carbonyl reduction of NNK does occur in cytosol. Fur ther studies with purified enzyme preparations are needed to explore the de tailed contribution of the cytosolic enzymes participating in the final eli mination of this lung carcinogen. (C) 1999 Elsevier Science Ireland Ltd. Al l rights reserved.