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 (Vmax/Km = 3.11 μl · min–1 · 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 N-glucuronidation in the metabolism of NNAL and the role of UGT1A4 in this pathway.
Footnotes
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↵1 Abbreviations used are: NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; UGT, UDP-glucuronosyltransferase; NNAL-N-Gluc, β-N-[4-(methylnitrosamino)-1-(3-pyridyl)-1-but-1-yl]-d-glucosiduronic acid; NNAL-O-Gluc, β-O-[4-(methylnitrosamino)-1-(3-pyridyl)-1-but-1-yl]-d-glucosiduronic acid; UDPGA, UDP-glucuronic acid; HPLC, high-pressure liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry.
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These studies were supported by Public Health Service Grants DE13158 (National Institute of Dental and Craniofacial Research) to P. Lazarus, and CA68384 (National Cancer Institute; P. Lazarus, project leader; Steven Stellman, principal investigator) from the National Institutes of Health, Department of Health and Human Services.
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These studies were presented at the American Association for Cancer Research, Washington, DC, July 2003.
- Received July 31, 2003.
- Accepted September 18, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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