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.