Stereospecific Metabolism of the Tobacco-Specific Nitrosamine, NNAL

Abstract

Among the most potent carcinogens in tobacco are the tobacco-specific nitrosamines (TSNAs), with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) the most abundant as well as the most potent. NNK is extensively metabolized to the equally carcinogenic 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Of the two NNAL enantiomers, (S)-NNAL appears to be preferentially glucuronidated and excreted in humans, but also exhibits higher stereoselective tissue retention in mice and humans and has been shown to be more carcinogenic in mice than its (R)- counterpart. Due to the differential carcinogenic potential of the two NNAL enantiomers, it is increasingly important to know which UGT enzyme targets the specific NNAL enantiomers for glucuronidation. To examine this, a chiral separation method was developed to isolate entiomerically pure (S)- and (R)-NNAL. Comparison of NNAL glucuronide (Gluc) peaks formed in reactions of UGT2B7-, UGT2B17-, UGT1A9-, and UGT2B10-over-expressing cell microsomes with pure NNAL enantiomers showed large differences in kinetics for (S)- versus (R)-NNAL, indicating varying levels of enantiomeric preference for each enzyme. UGT2B17 preferentially formed (R)-NNAL-O-Gluc and UGT2B7 preferentially formed (S)-NNAL-O-Gluc. When human liver microsomes (HLM) were independently incubated with each NNAL enantiomer, the ratio of (R)-NNAL-O-Gluc to (S)-NNAL-O-Gluc formation in HLM from subjects exhibiting the homozygous deletion UGT2B17 (*2/*2) genotype was significantly lower (p=0.012) than HLM from wild-type (*1/*1) subjects. There was a significant trend (p=0.015) towards decreased (R)-NNAL-O-Gluc:(S)-NNAL-O-Gluc ratio with increasing numbers of the UGT2B17*2 deletion allele. These data demonstrate that variations in the expression or activity of specific UGTs may affect individual susceptibility to cancers induced by specific NNAL enantiomers.

Association between Glucuronidation Genotypes and Urinary NNAL Metabolic Phenotypes in Smokers

“…D 4 -NNAL was kindly provided by Shantu Amin (Penn State University). D 4 -NNAL- N -Gluc and D 4 -NNAL- O -Gluc were biochemically synthesized by incubating D 4 -NNAL with UDPGA and bovine liver microsomes, prepared as previously described (21, 26), for 2 h at 37°C, and then purified by high-performance liquid chromatrography (HPLC).…”

“…D 4 -NNAL- N -Gluc and D 4 -NNAL- O -Gluc were synthesized by using Bovine liver microsomes (5 mg protein/ml reaction) in glucuronidation reaction containing D 4 -NNAL and UDPGA and then collected and purified by HPLC as previously described (26). ( S )-NNAL- O -Gluc and ( R )-NNAL- O -Gluc standards were synthesized by human liver microsomes (5 mg protein/ml reaction) in glucuronidation reaction containing racemic NNAL (26) and UDPGA, and were collected and purified by UPLC method described below with the mass spectrometer disconnected.…”

“…( S )-NNAL- O -Gluc and ( R )-NNAL- O -Gluc standards were synthesized by human liver microsomes (5 mg protein/ml reaction) in glucuronidation reaction containing racemic NNAL (26) and UDPGA, and were collected and purified by UPLC method described below with the mass spectrometer disconnected.…”

“…Studies of NNAL glucuronidation in vitro have shown that UGTs 2B7 and 2B17 exhibit preferential glucuronidation activity for ( S )- and ( R )-NNAL, respectively, while UGT1A9 is relatively non-stereospecific (26). Common functional variants are known to exist for UGTs 2B7 and 2B17.…”

“…Common functional variants are known to exist for UGTs 2B7 and 2B17. A polymorphic whole-gene deletion of the UGT2B17 gene has been shown to be associated with significant decreases in the formation of total NNAL- O -Gluc and ( R )-NNAL- O -Gluc in HLM (24, 26) and with increased risk for lung adenocarcinoma in women (27). While a missense polymorphism at codon 268 (His>Tyr) of the UGT2B7 gene was associated with a 1.4-fold decrease in total NNAL- O -Gluc formation activity in HLM (28), no studies have yet been performed examining these polymorphisms in vivo in smokers.…”