Longitudinal Study of Urinary Phenanthrene Metabolite Ratios: Effect of Smoking on the Diol Epoxide Pathway Stephen S. Hecht, Menglan Chen, Andrea Yoder, Joni Jensen, Dorothy Hatsukami, Chap Le, and Steven G. Carmella The Cancer Center and Transdisciplinary Tobacco Use Research Center, University of Minnesota, Minneapolis, Minnesota Abstract We have proposed that urinary phenanthrene metabolites could be used in a carcinogen metabolite phenotyping approach to identify individuals who may be susceptible to cancer induction by polycyclic aromatic hydrocarbons (PAH). In support of this proposal, we have developed methods for quantitation of r -1,t -2,3,c -4-tetrahydroxy- 1,2,3,4-tetrahydrophenanthrene (PheT) and phenanthrols (HOPhe) in human urine. PheT is the end product of the diol epoxide metabolic activation pathway of PAH, whereas HOPhe are considered as detoxification products. In this study, we investigated the longitudinal consistency of these metabolites over time in smokers and nonsmokers and compared their levels. Twelve smokers and 10 non- smokers provided urine samples daily for 7 days, then weekly for 6 weeks. Levels of PheT, HOPhe, and PheT/ HOPhe ratios were relatively constant in most individuals, with mean coefficients of variation ranging from 29.3% to 45.7%. There were no significant changes over time in levels of the metabolites or in ratios. These results indicate that a single urine sample should be sufficient when comparing phenanthrene metabolites in different groups. PheT/HOPhe ratios were significantly higher in smokers than in nonsmokers, showing that smoking induces the diol epoxide metabolic activation pathway of phenan- threne. This finding is consistent with previous studies indicating that inducibility of PAH metabolism contributes to cancer risk in smokers. (Cancer Epidemiol Biomarkers Prev 2005;14(12):2969 – 74) Introduction Polycyclic aromatic hydrocarbons (PAH) are ‘‘reasonably anticipated to be human carcinogens’’ (1). Abundant evidence supports the role of PAHs as important causes of lung cancer and, perhaps, other cancers in smokers (2, 3). Many PAHs are potent locally acting carcinogens, and fractions of cigarette smoke condensate enriched in these compounds are tumori- genic. DNA adducts of benzo(a )pyrene (BaP), a prototypical PAH, have been identified in the lungs of smokers, and the spectrum of mutations seen in the p53 gene isolated from lung tumors is similar to that induced by PAH and their diol epoxide metabolites (4). PAHs are also believed to be causative agents for cancers of the lung in coke production workers and cancer of the skin in workers exposed to coal tars, shale oil, and soot (5, 6). PAHs require metabolic activation to exert their carcino- genic effects (7). The principal route of metabolic activation of BaP that results in DNA adduct formation in human tissues proceeds by way of anti -7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a ]pyrene (BPDE; Fig. 1; refs. 8-10). A size- able body of evidence supports this metabolic activation pathway as the major one for many other PAHs as well (9). There are also other mechanisms of BaP metabolic activation, but the evidence that these contribute to DNA adduct formation in humans is presently more limited (11, 12). Competing with PAH metabolic activation are a variety of detoxification pathways, including direct hydroxylation to form phenols, conjugation of epoxides and diol epoxides with glutathione, and glucuronidation of dihydrodiols (7). Multi- ple enzymes are involved in the metabolic activation and detoxification of PAH. Cytochromes P450 and epoxide hydrolase are involved in both activation and detoxification, whereas glutathione S -transferases and UDP-glucuronosyl- transferases are involved mainly in detoxification (7). Many studies have investigated the role of polymorphisms in these enzymes as modifiers of cancer risk in people exposed to PAHs. The results of these studies have been somewhat inconsistent, although certain genotype variant combinations may lead to higher risk (13-16). Our goal has been to develop a carcinogen metabolite phenotyping approach, which would capture all genetic and environmental influences on PAH metabolism by actually measuring their metabolites in urine. We initiated this work by developing a method for analysis in human urine of r -7,t -8,9,c -10-tetrahydroxy-7,8,9,10-tetrahy- drobenzo[a ]pyrene (Fig. 1), but the levels of this metabolite were so low that the method would not be practical for application in epidemiologic studies (17). Therefore, we turned our attention to phenanthrene (Fig. 1), the simplest PAH with a bay region, a feature closely associated with carcinogenicity. Phenanthrene occurs in higher concentrations in the environment than does BaP, and its metabolites are more plentiful in urine (18). The metabolites of higher molecular weight PAHs are excreted mainly in feces, detracting from their use as biomarkers. Although phenan- threne is not considered to be carcinogenic, its pathways of metabolism are similar to those of BaP and other PAHs. Thus, as illustrated in Fig. 1, phenanthrene is metabolized to a diol epoxide in a manner similar to BaP (9, 19). Phenanthrene is also converted to phenanthrols (HOPhe) metabolically (20). The end product of the diol epoxide pathway is r -1,t -2,3,c -4- tetrahydroxy-1,2,3,4-tetrahydrophenanthrene (PheT; ref. 21). We propose that a ratio of PheT (as a marker of metabolic activation) to HOPhe (as a marker of detoxification) would be characteristic of a given individual’s ability to metabolically activate or detoxify PAHs. One major goal in this study was 2969 Cancer Epidemiol Biomarkers Prev 2005;14(12). December 2005 Received 6/1/05; revised 9/9/05; accepted 9/27/05. Grant support: National Cancer Institute grants CA-92025 and DA-013333 and ACS grant RP-00-138 (S.S. Hecht). Mass spectrometry and statistics were carried out in the core facilities of The Cancer Center, University of Minnesota, supported in part by National Cancer Institute grant CA-77598. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: S.S. Hecht is an American Cancer Society research professor. Requests for reprints: Stephen S. Hecht, The Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street Southeast, Minneapolis, MN 55455. Phone: 612-624-7604; Fax: 612-626-5135. E-mail: hecht002@umn.edu Copyright D 2005 American Association for Cancer Research. doi:10.1158/1055-9965.EPI-05-0396 Research. on September 4, 2021. © 2005 American Association for Cancer cebp.aacrjournals.org Downloaded from