Analysis of human urine for pyridine-N-oxide metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific lung carcinogen.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in rodents and is believed to be a causative factor for lung cancer in smokers. NNK also may be involved in oral cancer etiology in users of smokeless tobacco products. Pyridine-N-oxidation of NNK and its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), produces NNK-N-oxide and NNAL-N-oxide, respectively, which are detoxification products of NNK metabolism and are excreted in the urine of rodents and primates. Our goal is to develop a panel of urinary biomarkers to assess the metabolic activation and detoxification of NNK in humans. In this study, we developed methodology to analyze human urine for NNK-N-oxide and NNAL-N-oxide. The key step in the method was conversion of the N-oxides to NNK and NNAL by treatment with Proteus mirabilis. The resulting samples were then analyzed essentially by methods that we have described previously. 4-(Methylnitrosamino)-4-(3-pyridyl-N-oxide)-1-butanol (iso-NNAL-N-oxide) was used as internal standard. Levels of NNAL-N-oxide in smokers' urine ranged from 0.06 to 1.4 pmol/mg creatinine, mean +/- SD 0.53 +/- 0.36 pmol/mg creatinine. Its presence was confirmed by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry. NNK-N-oxide was not detected in smokers' urine. Levels of NNAL-N-oxide in the urine of smokeless tobacco users ranged from 0.02 to 1.2 pmol/mg creatinine, mean +/- SD 0.41 +/- 0.35 pmol/mg creatinine. The amounts of NNAL-N-oxide in urine were less than 20% of those of [4-(methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc) and were approximately 50% as great as those of free NNAL. These results demonstrate that pyridine-N-oxidation is a relatively minor detoxification pathway of NNK and NNAL in humans. The method was applied to analysis of urine from 11 smokers who consumed a diet containing watercress. In an earlier study (S.S. Hecht et al., Cancer Epidemiol., Biomarkers & Prev., 4: 877-884, 1995), we showed that consumption of watercress, a source of phenethyl isothiocyanate (PEITC), caused an increase in urinary excretion of NNAL plus NNAL-Gluc. This was attributed to inhibition of alpha-hydroxylation of NNK by PEITC, as seen in rodents in which PEITC also inhibits the pulmonary carcinogenicity of NNK. However, PEITC also could have inhibited pyridine-N-oxidation of NNK and NNAL. The urine of these smokers was analyzed for NNAL-N-oxide. The results demonstrated that watercress consumption had no effect on levels of NNAL-N-oxide in urine, supporting the conclusion that PEITC does inhibit the metabolic activation of NNK in humans.