Evidence for endogenous formation of tobacco-specific nitrosamines in rats treated with tobacco alkaloids and sodium nitrite

Carcinogenic tobacco-specific nitrosamines are present in tobacco productsand are believed to play a significant role in human cancers associatedwith tobacco use. Additional amounts of tobacco-specific nitrosamines couldbe formed endogenously. We tested this hypothesis by treating rats withnicotine and sodium nitrite and analyzing their urine. Initially, wetreated groups of rats with (S)-nicotine (60 micromol/kg) and NaNO2 (180micromol/kg), (S)-nicotine alone, NaNO2 alone or4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 12 nmol/kg) by gavagetwice daily for 4 days. We collected urine and analyzed for two metabolitesof NNK; 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanol and its glucuronide.We did not detect these metabolites in the urine of rats treated withnicotine alone or nicotine plus NaNO2, indicating that endogenousconversion of nicotine to NNK did not occur. However, the urine did containN'- nitrosonornicotine (NNN), N'-nitrosoanabasine (NAB) and N'-nitrosoanatabine (NAT). Analysis of the (S)-nicotine used in thisexperiment demonstrated that it contained trace amounts of nornicotine,anabasine and anatabine. In a second experiment, we used an identicalprotocol to compare the endogenous nitrosation of this (S)-nicotine withthat of synthetic (R,S)-nicotine, which did not contain detectable amountsof nornicotine, anabasine or anatabine. NNN (0.53 x 10(-3)% of nicotinedose), NAB (0.68%) and NAT (2.1%) were detected in the urine of the ratstreated with the (S)-nicotine and NaNO2. NNN (0.47 x 10(- 3)% of dose), butnot NAB or NAT, was present in the urine of the rats treated with synthetic(R,S)-nicotine and NaNO2. NNN probably formed via nitrosation ofmetabolically formed nornicotine. These results demonstrate for the firsttime that endogenous formation of tobacco- specific nitrosamines occurs inrats treated with tobacco alkaloids and NaNO2. The potential significanceof the results with respect to nitrosamine formation in people who usetobacco products or nicotine replacement therapy is discussed.