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

Abstract

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a strong lung carcinogen in all species tested. To elicit its tumorigenic effects, NNK requires metabolic activation which is supposed to occur via α-hydroxylation by cytochrome P450 enzymes. Carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) followed by glucuronosylation is considered to be the main detoxification pathway in humans. Therefore, NNK carbonyl reducing activity is crucial for NNK inactivation since it initiates the final excretion of this lung carcinogen. Until the present work, studies on NNK metabolism have focused exclusively on microsomal fractions, and several cytochrome P450 enzymes have been shown to be involved in α-hydroxylation of NNK. In addition, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD 1) which is located in the endoplasmic reticulum of the cell has been identified to catalyze the carbonyl reduction of NNK in microsomes. In this study, we provide evidence that carbonyl reduction of NNK does also take place in the cytosolic fraction of mouse liver and lung, and that cytosolic carbonyl reductase contributes to the detoxification 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 considerable NNK carbonyl reduction occurred with NADH, the preferred cosubstrate in cytosol is either NADPH or an NADPH-regenerating system. Due to the inhibitor sensitivity to menadione, ethacrynic acid, dicoumarol and quercitrin, it is concluded that carbonyl reductase (EC 1.1.1.184) is mainly responsible for NNAL formation in liver and lung cytosol. The expression of cytosolic carbonyl reductase and microsomal 11β-HSD 1 was established on the mRNA level by reverse transcription-PCR in both liver and lung. Enzyme kinetic 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 first report to show that carbonyl reduction of NNK does occur in cytosol. Further studies with purified enzyme preparations are needed to explore the detailed contribution of the cytosolic enzymes participating in the final elimination of this lung carcinogen.