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4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific lung carcinogen which may play an important role as a cause of lung cancer in smokers. NNK is extensively metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which like NNK is a potent pulmonary carcinogen. NNAL in turn is glucuronidated, and both NNAL and its glucuronides are excreted in human urine. Previous studies have clearly demonstrated the presence in human urine of 4-(methylnitrosamino)-1-(3-pyridyl)-1-(O-beta-D-glucopyranuronosyl)butane (NNAL-O-Gluc), but did not exclude the presence of 4-(methylnitrosamino)-1-(3-pyridyl-N-beta-D-glucopyranuronosyl)-1-butanolonium inner salt (NNAL-N-Gluc). In this study, we quantified NNAL, NNAL-N-Gluc, and NNAL-O-Gluc in the urine of smokers, snuff-dippers, and people who used the oral tobacco product "toombak". The presence of NNAL-N-Gluc in the urine of toombak users was confirmed by LC-ESI-MS/MS. In smokers' urine, NNAL-N-Gluc, NNAL-O-Gluc, and NNAL comprised (mean +/- SD) 26.5 +/- 6.2, 32.1 +/- 17.6, and 41.4 +/- 16.6%, respectively, of total NNAL. In snuff-dippers' urine, the corresponding figures were 13.6 +/- 5.1, 46.6 +/- 11.7, and 36.6 +/- 9.3%. NNAL-N-Gluc comprised 50 +/- 25% of total glucuronidated NNAL in smokers and 24 +/- 12% in snuff-dippers. This difference was significant (P = 0.01), suggesting that smoking induces glucuronidation of NNAL. The results of this study demonstrate that NNAL-N-Gluc contributes substantially to NNAL-glucuronides in human urine. These results are important for a clearer understanding of mechanisms of detoxification of NNK in humans.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke and is considered to be a causative agent for several tobacco-related cancers. Glucuronidation of the major metabolite of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), has been implicated as an important mechanism for NNK detoxification. To characterize NNAL metabolism by N-glucuronidation in humans, high-pressure liquid chromatography was used to detect glucuronide conjugates of NNAL formed in human liver microsomes in vitro. In addition to peaks corresponding to the O-glucuronides of NNAL (NNAL-O-Gluc), a second series of peaks were observed in human liver microsomes that were identified by liquid chromatography-mass spectrometry to be NNAL N-glucuronides (NNAL-N-Gluc). Microsomes prepared from liver specimens from individual subjects (n ‫؍‬ 42) exhibited substantial variability in the levels of NNAL-N-Gluc (49-fold variability) and NNAL-O-Gluc (49-fold variability) formed in vitro. This variability was likely not due to differences in tissue quality, as substantial variability (5-fold) was also observed in the ratio of NNAL-N-Gluc/NNAL-O-Gluc formation, with a mean ratio of 1.7 in the 42 specimens. Liver microsomes from smokers (n ‫؍‬ 14) exhibited no significant difference in the levels of either NNAL-N-Gluc or NNAL-O-Gluc formation, or in the ratio of NNAL-N-Gluc/NNAL-O-Gluc formation, as compared with liver microsomes from never smokers (n ‫؍‬ 28). Overexpressed UDP-glucuronosyltransferase (UGT) 1A4 exhibited significant levels of N-glucuronidating activity (V max /K m ‫؍‬ 3.11 l ⅐ min ؊1 ⅐ g ؊1 ) in vitro; no NNAL-N-glucuronide formation was detected for the 11 other overexpressed UGT enzymes tested in these studies. These results demonstrate the importance of Nglucuronidation in the metabolism of NNAL and the role of UGT1A4 in this pathway.The nicotine-derived tobacco-specific nitrosamine NNK 1 is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke (Hecht and Hoffmann, 1989;Hecht, 1998). NNK levels in tobacco smoke are 3 to 15 times higher than the levels of another major potent carcinogen in tobacco smoke, benzo[a]pyrene (Adams et al., 1987). NNK induces predominantly lung adenocarcinomas in rodents independent of the route of administration (Hecht, 1998). In the Fischer 344 rat, NNK induces pancreatic tumors (Rivenson et al., 1988) and, when applied together with the related tobacco-specific nitrosamine, NЈ-nitrosonornicotine, oral cavity tumors (Hecht et al., 1986). The cumulative dose of 1.8 mg of NNK/kg of body weight required to produce lung tumors in rodents (Belinsky et al., 1990) is similar to the cumulative lifetime dose of 1.6 mg of NNK/kg of body weight for the average American smoking two packs of cigarettes a day for 40 years (Hecht and Hoffmann, 1989;Hecht, 1998). NNK is therefore...

Liquid chromatography coupled with mass spectrometry (LC-MS) is the method of choice for analysis of covalent modification of DNA. DNA adductomics is an extension of this approach allowing for the screening for both known and unknown DNA adducts. In the research reported here, a new high-resolution/accurate mass MSn methodology has been developed representing an important advance for the investigation of in vivo biological samples and for the assessment of DNA damage from various human exposures. The methodology was tested and optimized using a mixture of 18 DNA adducts representing a range of biologically relevant modifications on all four bases and using DNA from liver tissue of mice exposed to the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In the latter experiment, previously characterized adducts, both expected and unexpected, were observed.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolized to enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), found in the urine of virtually all people exposed to tobacco products. We assessed the carcinogenicity in male F-344 rats of (R)-NNAL (5 ppm in drinking water), (S)-NNAL (5 ppm), NNK (5 ppm) and racemic NNAL (10 ppm) and analyzed DNA adduct formation in lung and pancreas of these rats after 10, 30, 50 and 70 weeks of treatment. All test compounds induced a high incidence of lung tumors, both adenomas and carcinomas. NNK and racemic NNAL were most potent; (R)-NNAL and (S)-NNAL had equivalent activity. Metastasis was observed from primary pulmonary carcinomas to the pancreas, particularly in the racemic NNAL group. DNA adducts analyzed were O (2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O (2)-POB-dThd), 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine(7-POB-Gua),O (6)-[4-(3-pyridyl)-4-oxobut-1-yl]deoxyguanosine(O (6)-POB-dGuo),the 4-(3-pyridyl)-4-hydroxybut-1-yl(PHB)adductsO (2)-PHB-dThd and 7-PHB-Gua, O (6)-methylguanine (O (6)-Me-Gua) and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing adducts. Adduct levels significantly decreased with time in the lungs of rats treated with NNK. Pulmonary POB-DNA adducts and O (6)-Me-Gua were similar in rats treated with NNK and (S)-NNAL; both were significantly greater than in the (R)-NNAL rats. In contrast, pulmonary PHB-DNA adduct levels were greatest in the rats treated with (R)-NNAL. Total pulmonary DNA adduct levels were similar in (S)-NNAL and (R)-NNAL rats. Similar trends were observed for DNA adducts in the pancreas, but adduct levels were significantly lower than in the lung. The results of this study clearly demonstrate the potent pulmonary carcinogenicity of both enantiomers of NNAL in rats and provide important new information regarding DNA damage by these compounds in lung and pancreas.