Short Communication COMPARATIVE METABOLISM OF THE TOBACCO-SPECIFIC NITROSAMINES 4- (METHYLNITROSAMINO)-1-(3-PYRIDYL)-1-BUTANONE AND 4-(METHYLNITROSAMINO)- 1-(3-PYRIDYL)-1-BUTANOL BY RAT CYTOCHROME P450 2A3 AND HUMAN CYTOCHROME P450 2A13

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its carbonyl-reduction product, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in rats and are presumed human lung carcinogens. NNK and NNAL are bioactivated to DNA-binding intermediates via hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e., -hydroxylation) by cytochrome P450s (P450s). Therefore, it is important to delineate which P450s are efficient catalysts of this metabolic transformation. In this study, the kinetic parameters for NNK and NNAL metabolism were determined for two extrahepatic P450s that are expressed in the lung: rat P450 2A3 and human P450 2A13. P450s 2A3 and 2A13 exhibited Vmax values for NNK 4-hydroxylation of 10.8 0.4 and 13.8 0.8 pmol min 1 pmol P450 , respectively; the corresponding Km values were 4.6 0.5 and 3.6 0.7 M. The respective Vmax values for P450 2A3and 2A13-mediated N-methyl hydroxylation of NNK were 8.2 0.3 and 4.6 0.2 pmol min 1 pmol P450 . These data indicate that P450s 2A3 and 2A13 are both efficient catalysts of the metabolic activation of NNK and are, along with mouse P450 2A5, the best catalysts of this reaction currently known. Both enzymes also catalyzed the -hydroxylation and N-oxidation of NNAL, and its oxidation to NNK. In general, Vmax/Km values for NNAL metabolism were 1 to 2 orders of magnitude lower than those for NNK metabolism, and P450 2A3 was a slightly better catalyst of NNAL metabolism than was P450 2A13. Given the exquisite sensitivity of the rat lung to NNK-induced carcinogenesis, the efficient bioactivation of NNK by rat P450 2A3, and the similar catalytic efficiency of P450s 2A3 and 2A13, P450 2A13 may be an important contributor to NNK bioactivation in the human lung. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its carbonyl-reduction product, NNAL, are potent and selective inducers of adenocarcinoma in the rat lung, and are thought to play a role in human lung cancer associated with tobacco use (Hecht, 1998). The sensitivity of the rat lung to NNKand NNAL-induced carcinogenesis is likely due, at least in part, to tissuespecific conversion of these carcinogens to electrophilic DNA-binding intermediates by cytochrome P450s (P450s) (Hecht, 1998; Ding and Kaminsky, 2003). Therefore, it is important to determine which P450s are responsible for NNK and NNAL bioactivation in animal models and humans. Both NNK and NNAL are bioactivated via P450-mediated hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e., -hydroxylation), as depicted in Fig. 1 (Hecht, 1998). Hydroxylation at the -methylene position ultimately generates OPB and lactol from NNK and NNAL, respectively, and generates a DNA-methylating agent (6) (Fig. 1) (Hecht, 1998). Hydroxylation of NNK at the -methyl carbon produces HPB and a DNApyridyloxobutylating intermediate (5), whereas the analogous reaction for NNAL generates diol, pyridyl-THF, and a pyridylhydroxybutylating species (8) (Fig. 1) (Hecht, 1998). NNK and NNAL are also oxidized at the pyridine nitrogen atom to form the respective N-oxides (Fig. 1) (Hecht, 1998). Kinetic parameters for NNK bioactivation have been reported for a variety of P450s, including human P450s 1A1, 1A2, 2A6, 2A13, 2D6, 2E1, and 3A4; rabbit P450 2A10/2A11; rat P450 2B1; and mouse P450s 2A4 and 2A5 (Hecht, 1998; Felicia et al., 2000; Su et al., 2000; Zhang et al., 2002; Jalas et al., 2003). Importantly, the P450s with the greatest catalytic efficiency for NNK -hydroxylation are members of the 2A subfamily and are expressed in the lung, for example, human P450 2A13 and mouse P450 2A5 (Felicia et al., 2000; Su et al., 2000; Zhang et al., 2002; Jalas et al., 2003). By contrast, human P450s 1A2, 2A6, and 3A4, which are predominantly expressed in the liver, are relatively poor catalysts of NNK bioactivation (Patten et al., 1996; Smith et al., 1996). Given the ability of members of the P450 2A subfamily to efficiently catalyze the metabolic activation of NNK and the sensitivity of the rat lung to NNK-induced carcinogenesis, it was important to determine the kinetic parameters for an extrahepatic rat P450, 2A3, This work was supported by National Cancer Institute Grants CA-81301 and CA-092596 (X.D.). 1 Abbreviations used are: NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; diol, 1-(3-pyridyl)-1,4-butanediol; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; P450, cytochrome P450; OPB, 4-oxo-4-(3-pyridyl)butanal; lactol, 5-hydroxy-2-(3-pyridyl)tetrahydrofuran; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; pyridyl-THF, 2-(3-pyridyl)tetrahydrofuran; HPLC, high performance liquid chromatography; Sf9, Spodoptera frugiperda. Address correspondence to: Sharon E. Murphy, Cancer Center, University of Minnesota, MMC 806, 420 Delaware St. SE, Minneapolis, MN 55455. E-mail: [email protected] 0090-9556/03/3110-1199–1202$7.00 DRUG METABOLISM AND DISPOSITION Vol. 31, No. 10 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 1185/1092620 DMD 31:1199–1202, 2003 Printed in U.S.A. 1199 at A PE T Jornals on Sptem er 8, 2017 dm d.aspurnals.org D ow nladed from