Tobacco-specific nitrosamines (TSNAs) are believed to be important carcinogens in smoking related cancers. Among the TSNAs present in both tobacco smoke and smokeless tobacco products are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which has been linked with the induction of lung cancer, the major procarcinogenic metabolite of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), N’-nirosonornicotine (NNN), which has been linked with the induction of esophageal cancer, the weak carcinogen, N’-nitrosoanabasine (NAB), and N’-nitrosoanatabine (NAT), which is not considered carcinogenic. NNN, NAB, NAT and their pyridine-N-glucuronides have been identified in smoker’s urine. Although studies characterizing the glucuronidation of NNAL have been previously performed, studies examining the UDP-glucuronosyltranferases (UGTs) responsible for the glucuronidation of these additional TSNAs have not yet been undertaken. In the present study, we determined glucuronidation activity against these TSNAs by human liver microsomes (HLMs) as well as by human individual UGTs. HLMs exhibited high activity against NNN, NAT, NAB, and modest activity against NNK. By screening individual over-expressed UGTs, we identified four UGTs that exhibited activity against NNN, NAT, NAB, or NNK: UGTs 1A3, 1A4, 2B4, and 2B10. No glucuronidation activity was observed for UGTs 1A1, 1A6, 1A7, 1A8, 1A10, 2B7, 2B11, 2B15, 2B17 and 2B28 against any of these four nitrosamines. Among the four active UGTs, the hepatic UGT1A4 showed the highest activity and was the only one to glucuronidate all four nitrosamines. Kinetic analysis of UGT1A4-over-expressing cell homogenates demonstrated that the Km of UGT1A4 against NAT and NAB was 5.8 mM and 4.4 mM, respectively, values that are similar to that observed previously for UGT1A4 against NNAL. These data are consistent with an important role for UGT1A4 in the glucuronidation of these TSNAs in vivo.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]