Received for publication October 24, 2007.
Revised January 28, 2008.
Accepted for publication January 28, 2008.

Glucuronidation of tobacco-specific nitrosamines by UGT2B10

Abstract

NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) 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-glucuronosyltranferases (UGTs) responsible for the N-glucuronidation of the TSNAs N'-nitrosonornicotine, N'-nitrosoanabasine and N'-nitrosoanatabine was performed. Utilizing HEK293 cells over-expressing UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, and 2B17, only UGTs 1A4 and 2B10 exhibited N-glucuronidating activity against these TSNAs. The K_M's for UGT2B10 were 15-22-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 3.6-27-fold higher than UGT1A4 against the three TSNAs as determined by V_max/K_M after normalization by levels of UGT2B10 versus UGT1A4 mRNA. Similarly high levels of activity were also observed for UGT2B10 against a fourth TSNA, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), exhibiting a 6.3-fold lower K_M and 3-fold higher normalized V_max/K_M than that observed for UGT1A4. Real-time PCR analysis demonstrated 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.

Key words: carcinogen metabolism, glucuronidation, hepatic elimination, HPLC, lung cancer, UDP glucuronyltransferases