Abstract
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is an important tobacco-specific nitrosamine (TSNA) in the etiology of tobacco-related cancers, and N-glucuronidation is an important mechanism of NNAL detoxification. In the present study, an analysis of the UDP-glucuronosyltransferases (UGTs) responsible for the N-glucuronidation of the TSNAs N′-nitrosonornicotine, N′-nitrosoanabasine, and N′-nitrosoanatabine was performed. Using human embryonic kidney 293 cells overexpressing UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and UGT2B17, only UGT1A4 and UGT2B10 exhibited N-glucuronidating activity against these TSNAs. The KMs for UGT2B10 were 15 to 22-fold lower than those of UGT1A4 against the three TSNAs and were similar to those observed for microsomes prepared from human liver specimens. The overall activity of UGT2B10 was 3.6 to 27-fold higher than UGT1A4 against the three TSNAs as determined by Vmax/KM after normalization by levels of UGT2B10 versus UGT1A4 mRNA. Similarly high levels of activity were also observed for UGT2B10 against a fourth TSNA, NNAL, exhibiting a 6.3-fold lower KM and 3-fold higher normalized Vmax/KM than that observed for UGT1A4. Real-time polymerase chain reaction analysis showed that UGT2B10 was expressed at a level that, on average, was 26% higher than that observed for UGT1A4 in a screening of normal liver tissue specimens from 20 individual subjects. These data suggest that UGT2B10 is likely the most active UGT isoform in human liver for the N-glucuronidation of TSNAs.
Footnotes
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These studies were supported by Public Health Service Grants P01-CA68384 (P.L.), R01-DE13158 (P.L.), and R01-CA75074 (T.E.S.) from the National Institutes of Health and two formula grants under the Pennsylvania Department of Health's Health Research Formula Funding Program (State of PA, Act 2001-77, part of the PA Tobacco Settlement Legislation; P.L., T.E.S.).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.019406.
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ABBREVIATIONS: TSNA, tobacco-specific nitrosamine; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNN, N′-nitrosonornicotine; NAB, N′-nitrosoanabasine; NAT, N′-nitrosoanatabine; UGT, UDP glucuronosyltransferase; NNAL-Gluc, glucuronide conjugate of NNAL; HLM, human liver microsome; UDPGA, UDP-glucuronic acid; HPLC, high-performance liquid chromatography; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney.
- Received October 23, 2007.
- Accepted January 28, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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