We report occasional significant increases in urinary biomarkers of the carcinogen NNN in some users of nicotine gum or lozenge, as compared to baseline smoking levels in the same subjects. We made these observations in the course of analyzing data from two separate studies designed to monitor changes in urinary biomarkers of a number of tobacco carcinogens in people who stopped smoking. Our findings suggest that significant amounts of NNN are formed occasionally in some users of oral NRT products, most likely via endogenous nitrosation of nornicotine that is metabolically formed from nicotine or originally present in NRT products. Given the carcinogenicity of NNN, this presents a possible cancer risk in long-term users.
In 13 out of 34 nicotine gum or lozenge users from both studies, total NNN at one or more time points after smoking cessation was comparable to, or considerably higher than, the baseline levels. Significant decreases in urinary total NNAL, exhaled CO, and urinary anatabine in these subjects confirmed their abstinence from tobacco products. NNN intake from NRT products could potentially contribute to the increase in urinary total NNN in our NRT users. Since these studies were not designed to specifically investigate the possible endogenous formation of NNN in users of oral NRT products, we did not analyze NNN in the nicotine gum or nicotine lozenges that were given to our subjects. However, our previous study demonstrated that NNN is virtually absent in this category of NRT products (1
). Another potential contributor to the measured high total NNN levels could be artefactual NNN formation in the urine after its collection or during sample preparation, via nitrosation of nornicotine present in the urine. The lack of increase in total NNN in urine samples incubated with an excess amount of nornicotine, as compared to non-treated aliquots from the same urine sample, does not support this hypothesis. Moreover, nitrate and nitrite levels measured in selected urine samples (Table S3 of the Supporting Information
) did not correlate with total NNN levels in the same samples. Given the design of the studies, we were not able to test urine samples for the presence of bacteria. However, it is unlikely that bacteria-mediated artefactual NNN formation occurred exclusively in the urine of oral NRT users: for most of the subjects who chose to use nicotine patch as a smoking cessation aid, urinary total NNN at all post-quit time points was less than 37% of their mean baseline levels ( and Table S1
Only one patch user demonstrated a sudden large increase in urinary total NNN 17.9 pmol/24h at day 28 of nicotine patch use, compared to 4.3 pmol/24h at baseline (subject P7, Table S1
). This increase coincided with an increase in urinary nitrate, suggesting an overall increase in nitrosation potential at this time point. Subjects O2 and O6 (Table S1
) stood out among oral NRT users. In subject O2, after 7 days of smoking cessation and oral NRT use, urinary total NNN was 700 times higher than baseline. This increase was not accompanied by an increase in either urinary nitrate or nitrite. Subject O6, who at several time-points after smoking cessation had ~ 30 times higher urinary total NNN than at baseline, also had elevated urinary total NNAL at the same time points, while anatabine was not detected. This is the first indication that NNK also can be formed endogenously in humans. The sporadic nature of high total NNN concentrations observed here most likely results from the multiple factors which influence endogenous nitrosation including different dietary catalysts and inhibitors of nitrosation, timing of their consumption, and infections. There are also indications that the extent of endogenous nitrosation in humans might be dependent on variations in the atmospheric concentrations of NO2
An interesting observation is that the QuitNic study participants with a sharp decline in urinary total NNN after smoking cessation also had lower average baseline total NNN levels when compared to the subjects whose urinary total NNN levels during nicotine lozenge use indicate endogenous nitrosation. These results suggest that some smokers, in addition to their exposure to NNN from cigarette smoke, probably form NNN endogenously, depending on host factors and/or dietary habits.
In subjects who did not have increases in urinary total NNN after smoking cessation, the levels of this biomarker dropped to 11% of the baseline value 3 days after quitting (p=0.015) (). It took an average of 4 weeks for total NNAL to decrease to the same 11% of the baseline value. These results support the idea of NNAL retention in the body, followed by slow release and reconversion to NNK, which, in turn, is again metabolized to NNAL. The decrease in urinary NNAL upon NRT use observed for most of the subjects in this study is consistent with previous studies (8
Total NNN and total NNAL as % of mean baseline levels in eight POB subjects (7 used nicotine patch and 1 used oral NRT) who did not have increased urinary NNN excretion during NRT use.
Major limitations of this investigation include the fact that neither of the two studies was designed to investigate endogenous formation of NNN in NRT users, and the lack of information on NNN in the NRT products. We also lacked of control group in which subjects did not use any NRT product after smoking cessation.
Despite these limitations, the presence of significant amounts of NNN in the urine of some oral NRT users is an alarming sign, especially in view of the reported increased use of NRT products and their over-the-counter availability (16
). In attempts to quit smoking, nicotine gum is one of the most frequently used NRT products (18
), and some former smokers use these products for prolonged periods of time (19
). These people, if susceptible to endogenous formation of NNN, can be continuously exposed to relatively high levels of this strong carcinogen and may eventually develop cancer.
In summary, we observed that significant amounts of NNN are excreted occasionally in some users of oral NRT, endogenous formation of this carcinogen being the most likely source. This presents a possible risk of cancer in long term users. Additional studies are urgently needed to understand the factors affecting endogenous NNN formation, and to develop preventive measures. The feasibility of preventing endogenous NNN formation in oral NRT users is supported by the sporadic nature of the increases in urinary total NNN, the significant reduction in urinary total NNN in some oral NRT users after smoking cessation, and the overall knowledge of the major factors affecting endogenous nitrosation in humans.