This study shows that levels of PheT in urine samples of smokers collected years before cancer diagnosis are independently and statistically significantly associated with the risk of developing lung cancer after adjustment for self-reported smoking intensity and duration and urinary total NNAL and total cotinine. This is the first study to show a significant relationship between lung cancer in smokers and a specific biomarker of PAH uptake and metabolism.
Strong evidence supports a major role for PAH as a primary cause of lung cancer in smokers (22
). Carcinogenic PAHs in cigarette smoke include BaP, benz[a
]anthracene, methylchrysenes, benzofluoranthenes, indeno[1,2,3-cd
]pyrene, dibenz[a, h
]anthracene, and several others. Certain PAHs, including some of those in cigarette smoke, are potent locally acting carcinogens that induce lung tumors in rodents (7
An important metabolic activation pathway of BaP and other carcinogenic PAHs has been described previously (24
). In the case of BaP, cytochrome P450s, including P450s 1A1 and 1B1, which are expressed in the lungs of smokers, catalyze the first step to BaP-7,8-epoxide (25
). This is hydrated with catalysis by microsomal epoxide hydrolase yielding BaP-7,8-diol, which is then oxidized by P450s and other enzymes to BaP-7,8-diol-9,10-epoxide (BPDE; refs. 24
). The latter reacts easily with DNA, producing a major adduct at the N2
position of deoxyguanosine (29
). BPDE also reacts with H2
O yielding trans, anti-BaP-tetraol, a good indicator of the extent of metabolic activation of BaP by this pathway. We have developed a method to quantify trans, anti-BaP-tetraol in human urine (31
). The amounts are quite low and likely impractical for a study such as the one reported here. However, we did observe a significant correlation between levels of trans, anti-BaP-tetraol and PheT in the urine of smokers (33
). Measurement of PheT is well suited for large-scale epidemiologic studies. Phenanthrene is the simplest PAH with a bay region, a feature closely associated with carcinogenicity (7
), although phenanthrene is generally regarded as noncarcinogenic (34
). The metabolism of phenanthrene by the diol epoxide pathway closely parallels that of BaP (35
). In this study, we measured urinary PheT by an analytically validated assay (17
). Among smokers, urinary PheT increased with increasing number of cigarettes smoked per day. More importantly, urinary PheT was independently associated with the risk of developing lung cancer in smokers even after controlling for self-reported number of cigarettes smoked per day, number of years of smoking, urinary total cotinine (an objective biomarker for nicotine uptake), and urinary total NNAL, a metabolite of the tobacco smoke carcinogen, NNK. Only 1 previous epidemiologic study examined the association between PheT and lung cancer risk in a U.S. population and found a 21% higher level of PheT in serum of smokers who developed lung cancer than smokers who remained free of lung cancer (8
). This difference was not statistically significant, possibly as a result of the small study sample size (100 cases and 100 controls).
PAHs are formed during the incomplete combustion of organic material. Human exposure to PAH can come from active or passive tobacco smoking; PheT and other PAH biomarkers are significantly elevated in smokers (7
). There are also diffuse environmental sources. Non tobacco sources of PAH exposure include industrial and urban air pollution and diet (7
). High occupational exposure can occur during the conversion of coal to coke and coal tar and during the processing and use of products derived from coal tar (7
). Many previous studies showed significantly elevated levels of urinary 1-hydroxypyrene, a widely used biomarker of PAH exposure, in the urine of coke-oven workers and others with occupational exposures to PAH (7
). Chinese nonsmokers regularly exposed to smoke from burning coal exhibited elevated urinary levels of specific PAH including BaP (38
); they were also found to experience significantly elevated risk of lung cancer (39
). Another substantial source of exposure to PAH is consumption of grilled or smoked foods or foods grown in PAH-polluted environments (7
). Given the ubiquitous nature of PAH in foods, the estimation of their dietary consumption is challenging. In this study, we examined but failed to establish any statistical association between intake frequencies of specific foods and urinary levels of PheT (data not shown). The biomarker approach of this study, which quantified PheT in urine, offers an objective surrogate measure of PAH exposure from all sources, including those that await identification. A statistically significant positive association between urinary PheT and lung cancer risk independent of smoking history and urinary total cotinine and total NNAL shows the advantage of this biomarker approach for assessment of PAH exposure plus metabolic activation in smokers.
In the same cohort, we previously reported a statistically significant positive association between urinary total NNAL and lung cancer risk in smokers (9
). In this analysis with an additional 325 cases and 346 controls, we confirmed this positive association. The positive association did not differ between the former and current data sets. Further adjustment for urinary PheT did not materially alter the positive association between total NNAL and lung cancer risk, suggesting that total NNAL is an independent risk factor for lung cancer. When all 3 urinary biomarkers were examined simultaneously together with smoking intensity and duration, the association of urinary total NNAL with lung cancer risk was weaker than those for total cotinine and PheT. The weaker association of urinary total NNAL could be due, at least in part, to the relatively low levels of NNK in the smoke of cigarettes used in Shanghai and consequently low levels of urinary total NNAL in this study population (40
). The geometric mean of urinary total NNAL among controls of this study was 0.20 pmol/mg creatinine, significantly lower than that of smokers in Singapore (0.66 pmol/mg creatinine) and in the United States (1.36 pmol/mg creatinine; refs. 9
The results of this study showing a strong positive association between total cotinine level and lung cancer risk confirmed the findings of our previous report as well as those of others (9
). Cotinine is a major metabolite of nicotine. Nicotine is the major addictive substance in tobacco smoke, but it is not carcinogenic. However, our findings have shown that urinary levels of total cotinine are informative as an independent and objective measure of in vivo
exposure to cigarette smoke in general. The independent association between urinary total cotinine and lung cancer risk after adjustment for urinary total NNAL and PheT supports the notion that compounds in tobacco smoke other than NNK and PAH also play a role in the development of lung cancer in smokers. Besides NNK and PAH, multiple established carcinogens have been identified in cigarette smoke, which also has tumor promoting, cocarcinogenic, and inflammatory properties.
One of the strengths of this study is that urinary biomarkers including PheT, total NNAL, and total cotinine were 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. Another strength is the simultaneous measurement of multiple urinary biomarkers representing the uptake and metabolism of PAH, NNK, and nicotine in individual study subjects, thus allowing for the examination of the biomarkers’ independent and joint effects on lung cancer risk. The large sample size of the study provided definitive results on the association between these urinary biomarkers and lung cancer risk in male smokers from Shanghai, China.
A potential limitation of this study is that urine samples were collected only once, at baseline, from all subjects. However, our previous study of 70 smokers who smoked 10 or more cigarettes per day over a 1-year period, with sampling every other month, showed relatively constant levels of PheT, total NNAL, and total cotinine, with estimated intraclass correlation coefficients of 76% for total NNAL and 58% for total cotinine in urine, and 56% for PheT in plasma (42
). Thus, single measurements from urine somewhat adequately represented individual mean exposure over time. The intraindividual variation in urinary biomarkers measured over a wide time period would diminish the observed association between these urinary biomarkers and lung cancer risk. Therefore, this study with a single assessment at baseline would lead to the underestimation of the true effect of these urinary biomarkers on risk of lung cancer if such intraindividual variation had not existed or multiple measurements of urinary biomarkers at different time points had been used.
In summary, using prospectively collected urine samples from participants of the Shanghai Cohort Study, we showed statistically significant, independent, dose-dependent associations between urinary concentrations of PheT, total NNAL, and total cotinine, biomarkers of uptake and metabolism of PAH, NNK, and nicotine, respectively, and increased risk of lung cancer among smokers with comparable smoking histories. These 3 noninvasive biomarkers with as-yet-to-be identified other biomarkers of tobacco smoke exposure and metabolism potentially can be used to develop an individual-based, predictive model for lung cancer risk in smokers.