We found that levels of nicotine in toenail samples from our study population independently predicted lung cancer risk, with a clear dose-response relation for men with higher toenail nicotine levels having a higher risk of lung cancer. Our data indicate that toenail nicotine levels reflect exposure burden of tobacco not captured entirely through reliance on reported smoking history. Established smokers tend to maintain their level of tobacco use that is driven by their addiction to nicotine. As demonstrated in controlled laboratory experiments, smokers can reduce their number of cigarettes smoked but still maintain their level of nicotine through topographic behavior of smoke-puff ratio and depth of inhalation (4
). Such higher intake of nicotine and tobacco carcinogens cannot be estimated when relying on reported number of cigarettes smoked. Similarly, for past smokers, our study demonstrates that they are still being exposed to substantial nicotine levels after they have quit, although fewer of them were exposed to the highest level of nicotine. This finding suggests that some are being exposed to low secondhand smoke levels while others are being exposed to higher levels, presumably from their fellow smokers who have not quit. It could also indicate that some of them are still occasionally smoking or that the measure of toenail nicotine reflects residual nicotine for recent quitters.
More than 10% of men with the highest levels of toenail nicotine in our study were never smokers. In a previous study (27
), never smokers exposed to heavy secondhand smoke had levels of nicotine equivalent to those of active smokers, although misclassification was much higher than in this study. Thus, previous risks of lung cancer due to smoking have likely been underestimated. The same conclusion applies to all other tobacco-related diseases for which assessment of risk has relied on questionnaire measures only.
Two recent studies determined 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine and serum of heavy smokers and found it predictive of lung cancer risk (13, 14). To our knowledge, the first study to assess biomarkers of tobacco and lung cancer risk was a case-control study from Norway by Boffetta et al. (28
), in which frozen serum samples from lung cancer cases and their matched controls were analyzed for cotinine levels. They found a significant association with cotinine but did not adjust for reported smoking history in any of their analyses; thus, it is not clear whether serum cotinine was able to predict lung cancer risk independent of reported smoking.
Another recent nested case-control study of lung cancer from the Shanghai Cohort Study assessed the ability of cotinine in urine to predict lung cancer risk and found a significant association in the unadjusted model (29
). However, when the authors adjusted for reported smoking history, the highest tertile of the urine cotinine biomarker failed to significantly predict lung cancer risk (odds ratio = 2.1, 95% CI: 0.9, 5.0; P
for trend = 0.08). Cotinine is a metabolite of nicotine and is highly variable because of interindividual metabolic and excretion differences and a short half-life of 17 hours (9
). A greater stability over time could explain the ability of the toenail nicotine biomarker to predict lung cancer independent of reported smoking history in our study.
A previous study reported on the ability of toenail nicotine levels to predict coronary heart disease among women independent of reported smoking history (31
). There was a dose-response association, and the women in the highest quintile of toenail nicotine level had a 42% increase in the risk of coronary heart disease compared with women in the lowest quintile (RR = 1.42, 95% CI: 1.33, 1.52).
Although toenail nicotine levels can add to the prediction of lung cancer risk independent of smoking history, as shown in our study, reported number of cigarettes smoked and pack-years of smoking were strongly associated with risk independent of toenail nicotine. Cumulative exposure, which is most relevant to lung cancer, may suffer less misclassification than current or period-specific average exposure. However, in our results, high intensity of smoking of 25 cigarettes or more per day predicted a higher risk of lung cancer than the highest pack-years of cumulative exposure. Pack-years does not differentiate between high-intensity shorter duration of smoking and low-intensity longer duration and may explain the lower risk.
The toenail nicotine biomarker should reflect factors such as passive smoking and depth of inhalation not captured by reported smoking habits. However, nicotine is not carcinogenic; therefore, using it as a proxy for the carcinogenic constituents of tobacco is based on the assumption that the internal dose of these constituents delivered to the lung is proportional to nicotine levels measured in nails. This assumption has been demonstrated by comparing toenail nicotine levels with toenail levels of the tobacco-specific-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; they were well correlated (r
= 0.68; P
< 0.0001) (32
Nevertheless, toenail nicotine levels represent exposure during the past year, which does not reflect the total cumulative exposure assessed by pack-years of smoking. Notably, however, the relative risk of toenail nicotine in relation to lung cancer was not substantially influenced when replacing pack-years with current smoking level in the model. In both models, there was an additional 50%–60% risk of lung cancer with each natural log unit of toenail nicotine, reflecting additional unmeasured risk that is independent of reported smoking. This finding was consistent even when limiting the analyses to smokers in multivariate analyses adjusting for pack-years.
By using toenail nicotine levels, we are assuming that they reflect usual exposure over time, with less error than existing biomarkers that reflect only a few hours or days of exposure. We have shown that toenail nicotine levels are closely correlated with smoking status 6 years prior to collection of samples (22
), despite the expected variability in smoking and tobacco exposure over this period. This finding indicates that toenails are a stable biomarker of average exposure over time. Toenail nicotine levels could also reflect the interindividual variability in nicotine metabolism in relation to lung cancer risk.
The population we studied was a group of health professionals with lower than average exposure to tobacco smoke, which could explain the inability of lower quintiles of toenail nicotine levels to significantly predict lung cancer risk. We would expect a higher predictive ability of toenail nicotine levels in other more exposed populations. Nevertheless, we were still able to demonstrate a relative risk of 10.5 for the highest quintiles in the univariate analyses and a relative risk of 3.6 in the multivariate analyses. Categories of pack-years of less than 20 were also not significantly related to lung cancer risk. It will be interesting to see the results replicated in studies from other populations.
When we stratified according to smoking status, toenail nicotine levels were still able to predict lung cancer risk. For never smokers, this would reflect passive smoking exposure. A limitation of our study is that no information was collected about passive smoking. Another source of nicotine is from nicotine replacement therapy. However, in 1987, when the toenails were collected, use of nicotine gum was negligible, and such a source is unlikely to have influenced the results of this study (33
Development of the toenail nicotine biomarker as a clinical tool to assess future risk may provide motivation for smokers to quit and for individuals heavily exposed to secondhand smoke to limit their exposure. Toenails are easily collected by participants and can be stored at room temperature for an extended period of time, making it a feasible tool for large population studies.
In conclusion, our study demonstrated that toenail nicotine levels provide a biomarker that can predict the risk of lung cancer independent of reported smoking history. Similarly, we found that smoking history predicted lung cancer risk independent of toenail nicotine level. The risk according to toenail nicotine level could be predicted for never, past, and current smokers, suggesting that previous studies that have determined risk of lung cancer from tobacco use by using only reported active smoking may have underestimated the true effects of tobacco smoke.