Short Communication Comparative Levels of O-Methylguanine, Pyridyloxobutyl-, and Pyridylhydroxybutyl-DNA Adducts in Lung and Liver of Rats Treated Chronically with the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in rats and may be a cause of lung cancer in smokers. NNK is metabolized by cytochromes P450 to intermediates that react with DNA forming methyl, pyridyloxobutyl (POB), and pyridylhydroxybutyl (PHB) adducts, which are critical in carcinogenesis. The methyl adduct O-methylguanine (O-methyl-G) has miscoding properties, but there are no reports on levels of this adduct in rats treated chronically with NNK in the drinking water, nor has its levels been compared with those of POBand PHB-DNA adducts. We used liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring to quantify O-methyl-G in lung and liver DNA of rats treated with a carcinogenic dose of 10 ppm of NNK in the drinking water and sacrificed after 1, 2, 5, 10, 16, and 20 weeks. The maximal level of O-methyl-G in lung DNA, 2550 263 fmol/mg DNA, was reached at 5 weeks and was significantly greater (P < 0.05) at that point than all other adducts (measured previously) except O-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine. Overall levels of O-methyl-G in lung were intermediate between those of total POBand PHB-DNA adducts. In liver, the wave of O-methyl-G peaked at 2 weeks while that of total POBDNA adducts peaked at 10 weeks, and levels of total PHB-DNA adducts were low throughout. The results of this study demonstrate that substantial amounts of O-methyl-G are formed at various time points in lung and liver DNA of rats treated chronically with NNK, supporting its role in carcinogenesis. Lung cancer is the leading cause of cancer death in the world, killing approximately 3000 people every day (International Agency for Research on Cancer, 2004). Cigarette smoking causes approximately 90% of lung cancer (International Agency for Research on Cancer, 2004). Although there are multiple pulmonary carcinogens in cigarette smoke, one of the most potent in animal models is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Hecht, 1998, 2008). NNK induces adenoma and adenocarcinoma of the lung in rats, mice, hamsters, and ferrets, independent of the route of administration (Hecht, 1998). It is particularly effective in the rat, in which chronic treatment with 1 ppm in the drinking water for 2 years caused a significant incidence of lung tumors (Rivenson et al., 1988). The major mechanism by which NNK initiates the carcinogenic process is cytochrome P450 (P450)-mediated -hydroxylation to give intermediates 1 and 2 (Fig. 1) (Hecht, 1998). Multiple P450s including rat P450 2A3, human P450 2A13, mouse P450 2A5, and others are involved in this process, which results in the formation of pyridyloxobutyl (POB)-DNA adducts from intermediate 3 and methyl-DNA adducts from intermediate 4 (Jalas et al., 2005; Hecht, 2008). Similar pathways act upon the NNK metabolite NNAL to produce pyridylhydroxybutyl (PHB)-DNA adducts as well as methyl adducts (Upadhyaya et al., 2008). The POB-DNA adducts are O-[4-(3-pyridyl)-4-oxobut-1-yl]-2 -deoxyguanosine (O-POB-dG), 7-[4-(3-pyridyl)-4-oxobut-1-yl]-2 -deoxyguanosine (7-POB-dG), O-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O-POB-T), and O-[4-(3-pyridyl)-4oxobut-1-yl]-2 -deoxycytidine (O-POB-dC). [7-POB-dG and O-POB-dC have been quantified as the corresponding bases 7-[4-(3pyridyl)-4-oxobut-1-yl]guanine (7-POB-G) and O-[4-(3-pyridyl)-4oxobut-1-yl]cytosine (O-POB-C).] The PHB-DNA adducts are the corresponding carbonyl-reduced forms, and the methyl-DNA adducts are O-methylguanine (O-methyl-G), 7-methyl-G, and Omethylthymidine (O-methyl-T). Among these, O-methyl-G, Omethyl-T, O-POB-dG, and O-POB-T are known to have miscoding properties (Loechler et al., 1984; Altshuler et al., 1996; Delaney and Essigmann, 2001; Pauly et al., 2002; Sharma et al., 2008). Although the DNA pyridyloxobutylation and methylation pathways of NNK metabolic activation have been well characterized for years, only This work was supported in part by the National Institutes of Health National Cancer Institute [Grants CA81301, CA77598]. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.109.027078. ABBREVIATIONS: NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; P450, cytochrome P450; POB, pyridyloxobutyl; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; PHB, pyridylhydroxybutyl; O-POB-dG, O-[4-(3-pyridyl)-4-oxobut-1-yl]-2 -deoxyguanosine; 7-POB-dG, 7-[4-(3pyridyl)-4-oxobut-1-yl]-2 -deoxyguanosine; O-POB-T, O-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine; O-POB-dC, O-[4-(3-pyridyl)-4-oxobut-1-yl]2 -deoxycytidine; O-POB-C, O-[4-(3-pyridyl)-4-oxobut-1-yl]cytosine; 7-POB-G, 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine; O-methyl-G, Omethylguanine; O-methyl-T, O-methylthymidine; LC-ESI-MS/MS-SRM, liquid chromatography-electrospray ionization-tandem mass spectrometryselected reaction monitoring; MS, mass spectrometry; AGT, O-alkylguanine-DNA-alkyltransferase. 0090-9556/09/3706-1147–1151$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 6 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 27078/3477030 DMD 37:1147–1151, 2009 Printed in U.S.A. 1147 at A PE T Jornals on A ril 3, 2017 dm d.aspurnals.org D ow nladed from