This study demonstrates that total NNAL in urine of smokers collected years before cancer diagnosis is significantly associated with their subsequent risk of developing lung cancer. These findings corroborate the large body of laboratory data on NNK carcinogenicity in animals, further strengthening the notion that NNK in tobacco smoke is a major contributor to lung cancer in smokers.
This study also shows that among smokers with comparable smoking history, there is a close to 9-fold variation in subsequent risk of lung cancer between those with high versus low levels of urinary total NNAL and total cotinine. These findings have public health implications. Our results suggest that levels of total NNAL and total cotinine in urine are important predictors of lung cancer risk in cigarette smokers, beyond the predictive indices of smoking intensity (number of cigarettes smoked per day) and duration (number of years of regular smoking). We believe these two non-invasive biomarkers of tobacco smoke exposure can serve as the starting point of an individual-based, predictive model for lung cancer risk in a smoker. Tobacco smoke contains at least 60 established carcinogens such as polycyclic aromatic hydrocarbons (PAH), 1,3-butadiene and other volatile carcinogenes (6
). Inclusion of some of these as-yet-to-be developed biomarkers is likely to be critical in rendering the eventual risk assessment model as an useful tool in predicting the lifetime risk of lung cancer in an individual smoker.
This study demonstrates that measurements of urinary cotinine and total NNAL at a single time point in a smoker can substantially improve the predictive power of a lung cancer risk assessment model based solely on self-reported smoking history (number of cigarettes smoked per day, number of years of regular smoking). Self-reports of smoking intensity and duration are expected to be imprecise. Information on smoking behavior such as depth of inhalation and number of puffs per cigarette is difficult, if not impossible, to assess via a questionnaire-based interview. Furthermore, interview-based assessment of smoking intensity and duration does not capture inter-individual variability in metabolism of tobacco carcinogens. Therefore, it is not surprising that appropriately chosen biomarkers would significantly improve the predictive power of a lung cancer risk assessment model based solely on self-reported history of smoking habits.
NNAL is found only in the urine of people who use tobacco products or are exposed to secondhand tobacco smoke because NNK is a tobacco-specific compound, which is not present in the diet or in any other environment. NNK itself is extensively metabolized and cannot be detected in urine. NNK has been classified as carcinogenic to humans (23
Our results reveal that urinary total NNAL levels were more than 4-fold higher among smokers in Singapore than in Shanghai. One might wonder if the lower levels of total NNAL in Shanghai versus Singapore smokers were the result of degradation of NNAL in urine during storage. Shanghai samples were collected during 1986-1989 while the Singapore samples were collected during 1994-2005. We believe this is an unlikely scenario. Our experimental data have shown that total NNAL is stable for at least 4 years in urine samples stored at -20 °C. Further, mean total NNAL (0.74 pmol/mg Cr) based on samples collected during the early phase of the Singapore Study (1994-1999) was comparable to that (0.65 pmol/mg Cr) based on samples collected later (2000-2005) (P
= 0.70). We speculate that the varying levels of urinary total NNAL between smokers in Shanghai versus Singapore may stem from the considerably lower concentrations of NNK in local Chinese brands (western, imported brands were unavailable in Shanghai during 1986-1989 when subjects were recruited into the cohort) relative to the western brands used by most smokers in Singapore (24
Comparable to results of a prior study conducted in Norway (27
), the present study showed an independent, positive association between urinary total cotinine and lung cancer risk. Cotinine is a major metabolite of nicotine. Nicotine is the major additive substance in tobacco smoke but it is not carcinogenic. Therefore, the urinary level of cotinine represents a separate (i.e., aside from self reports of number of cigarettes/day) and objective measure of in vivo
exposure to nicotine and cigarette smoke. The independent association between urinary total cotinine and lung cancer risk after adjustment for urinary total NNAL and smoking history supports the notion that compounds in tobacco smoke other than NNK also play a role in the development of lung cancer in smokers. As discussed above, there are at least 60 established carcinogens in cigarette smoke including PAH and other volatile carcinogens. Strong evidence supports a major role for PAH as causes of lung cancer in smokers (4
), although data linking specific PAH biomarkers to lung cancer risk in humans are lacking.
One of the strengths of the present study is that total NNAL was measured in urine samples collected years before cancer diagnosis, thereby ruling out the possibility of a spurious association due to smoking behavior changes in lung cancer patients close to their time of clinical diagnosis. There also is remarkable consistency within our study data. A positive NNAL-lung cancer association of comparable magnitude was observed in both Shanghai and Singapore subjects despite differences in the NNK content of cigarettes smoked.
There is evidence that a single measurement of urinary NNAL closely predicts the average level of NNAL measured over a much longer time period. Our recent study of more than 50 smokers who smoked 10 or more cigarettes per day over a one year period, with sampling every other month, showed relatively constant levels of total NNAL in urine, with an overall average coefficient of variation (CV) of 27.8% (standard deviation of CV = 14.5%) (28
). This intra-individual longitudinal variation in urinary total NNAL over a one year period was relatively small compared with a 20-fold variation in total NNAL levels among those smokers (28
Dietary risk or protective factors may confound the NNAL-lung cancer association (29
). However, adjustment for urinary total isothiocyanates and serum β-cryptoxanthin, two dietary factors that are inversely related to lung cancer risk in our study population did not materially alter the association between total NNAL and lung cancer risk (13
In summary, using prospectively collected urine samples from participants of two Chinese cohorts, we demonstrated a statistically significant, dose-dependent association between urinary total NNAL, a biomarker of NNK exposure, and increased risk of lung cancer among smokers with comparable smoking histories.