Metabolites of the Polycyclic Aromatic Hydrocarbon Phenanthrene in the Urine of Cigarette Smokers from Five Ethnic Groups with Differing Risks for Lung Cancer.

Results from the Multiethnic Cohort Study demonstrated significant differences in lung cancer risk among cigarette smokers from five different ethnic/racial groups. For the same number of cigarettes smoked, and particularly among light smokers, African Americans and Native Hawaiians had the highest risk for lung cancer, Whites had intermediate risk, while Latinos and Japanese Americans had the lowest risk. We analyzed urine samples from 331-709 participants from each ethnic group in this study for metabolites of phenanthrene, a surrogate for carcinogenic polycyclic aromatic hydrocarbon exposure. Consistent with their lung cancer risk and our previous studies of several other carcinogens and toxicants of cigarette smoke, African Americans had significantly (p<0.0001) higher median levels of the two phenanthrene metabolites 3-hydroxyphenanthrene (3-PheOH, 0.931 pmol/ml) and phenanthrene tetraol (PheT, 1.13 pmol/ml) than Whites (3-PheOH, 0.697 pmol/ml; PheT, 0.853 pmol/ml) while Japanese-Americans had significantly (p = 0.002) lower levels of 3-PheOH (0.621 pmol/ml) than Whites. PheT levels (0.838 pmol/ml) in Japanese-Americans were not different from those of Whites. These results are mainly consistent with the lung cancer risk of these three groups, but the results for Native Hawaiians and Latinos were more complex. We also carried out a genome wide association study in search of factors that could influence PheT and 3-PheOH levels. Deletion of GSTT1 explained 2.2% of the variability in PheT, while the strongest association, rs5751777 (p = 1.8x10-62) in the GSTT2 gene, explained 7.7% of the variability in PheT. These GWAS results suggested a possible protective effect of lower GSTT1 copy number variants on the diol epoxide pathway, which was an unexpected result. Collectively, the results of this study provide further evidence that different patterns of cigarette smoking are responsible for the higher lung cancer risk of African Americans than of Whites and the lower lung cancer risk of Japanese Americans, while other factors appear to be involved in the differing risks of Native Hawaiians and Latinos.

High throughput liquid and gas chromatography-tandem mass spectrometry assays for tobacco-specific nitrosamine and polycyclic aromatic hydrocarbon metabolites associated with lung cancer in smokers.

We developed and applied high throughput liquid and gas chromatography-tandem mass spectrometry (LC-MS/MS and GC-MS/MS) methods for the cigarette smoking-associated biomarkers 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT), which are urinary metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the polycyclic aromatic hydrocarbon phenanthrene. NNAL and PheT levels have been linked to lung cancer in previous studies of smokers. Confirmation of these relationships will require further molecular epidemiology studies, necessitating improved methodology applicable to large numbers of small urine samples. Furthermore, NNAL is excreted in urine either unconjugated or as an N- or O-glucuronide, but little data are available on the amounts of each in urine. For the high throughput analysis of NNAL, 3 aliquots were processed from each urine sample, one for the analysis of free NNAL, one for free NNAL plus NNAL-N-Gluc, and one for total NNAL (the sum of free NNAL, NNAL-N-Gluc, and NNAL-O-Gluc). Ninety-six well plate technology was used for sample enrichment by supported liquid extraction plates, mixed mode reverse-phase/cation exchange solid-phase extraction, and LC-MS/MS analysis. For the analysis of PheT, the urine samples were cleaned up by solid-phase extraction on styrene-divinylbenzene sorbent, silylated, and analyzed by GC-MS/MS, both in 96-well format. The methods were validated analytically with respect to accuracy and precision, and applied in an ongoing molecular epidemiology study of smokers. The amount of total NNAL in smokers' urine was (mean ± SD) 1.65 ± 2.13 pmol/mL (N = 2641). Free NNAL, NNAL-N-Gluc, and NNAL-O-Gluc represented (mean ± SD) 31 ± 11%, 22 ± 14%, and 48 ± 15% of total NNAL, respectively. The amount of PheT in smokers' urine was (mean ± SD) 1.43 ± 2.16 pmol/mL (N = 2613). The methodology described here should be widely applicable in future studies of tobacco use and cancer.