Abstract 4698: Preferential glutathione conjugation of a reverse diol epoxide compared to a bay region diol epoxide of benzo[a]pyrene in human hepatocytes: Relevance to glutathione-S-transferase genotyping in epidemiology studies
Authors: Upadhyaya Pramod , Hochalter J. Bradley , Balbo Silvia , Berg Jeannette Zinggeler , McIntee Edward J. , Hecht Stephen S. 
Published: February 2011

Abstract
Many studies have examined the relationship between polymorphisms in glutathione-S-transferase genes and cancer in people exposed to polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (BaP), but results to date have been modest. Missing from these studies has been an exploration of the formation of the appropriate glutathione conjugates in humans. We previously addressed this question using the three ring bay region PAH phenanthrene as a model compound and found that, in human urine, the only detectable mercapturic acids which result from metabolic degradation of glutathione conjugates were formed from a reverse diol epoxide of phenanthrene (phenanthrene-3,4-diol-1,2-epoxide), not from the bay region diol epoxide (phenanthrene-1,2-diol-3,4-epoxide) which would be typical of those associated with PAH carcinogenicity. We obtained similar results when we incubated human hepatocytes with these diol epoxides of phenanthrene. In the study reported here, we extended our investigation to BaP, a prototypic PAH carcinogen. We incubated human hepatocytes from ten donors with 10 µM racemic BaP-7,8-diol-9,10-epoxide, believed to be a major ultimate carcinogen of BaP, or with the non-carcinogenic reverse diol epoxide, racemic BaP-9,10-diol-7,8-epoxide. Incubations were carried out for 12 or 24h. We used high performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring at m/z 464→317 to analyze the incubation mixtures for the mercapturic acid products which would result from glutathione conjugation followed by normal metabolic processing of the conjugates. The standard mercapturic acids were synthesized by reaction of racemic BaP-diol epoxides with N-acetylcysteine. Major products resulting from cis- and trans- addition to each diol epoxide were characterized by proton NMR and MS. A mercapturic acid derived from phenanthrene-3,4-diol-1,2-epoxide was used as an internal standard in the hepatocyte analyses. We obtained convincing evidence for time dependent mercapturic acid formation from the non-carcinogenic reverse diol epoxide BaP-9,10-diol-7,8-epoxide in all 10 samples. Levels ranged from 0.12 to 17 pmol/ml. However, we could detect mercapturic acids from the bay region diol epoxide BaP-7,8-diol-9,10-epoxide in only 1 of 10 samples (0.05 pmol/ml). Taken together with our similar previous results of analyses of phenanthrene metabolites in human hepatocytes and human urine, the results of this study indicate that conjugation of BaP-7,8-diol-9,10-epoxide with glutathione is a minor pathway in humans, suggesting that glutathione-S-transferase genotyping is not an effective method of assessing risk of PAH induced cancer in humans, at least with respect to the diol epoxide pathway of PAH carcinogenesis.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4698.