Preferential Glutathione Conjugation of a Reverse Diol Epoxide Compared to a Bay Region Diol Epoxide of Phenanthrene in Human Hepatocytes: Relevance to Molecular Epidemiology Studies of Glutathione-S-Transferase Polymorphisms and Cancer

Abstract

Bay region diol epoxides are recognized ultimate carcinogens of polycyclic aromatic hydrocarbons (PAH), and in vitro studies have demonstrated that they can be detoxified by conjugation with glutathione, leading to the widely investigated hypothesis that individuals with low activity forms of glutathione-S-transferases are at higher risk of PAH induced cancer, a hypothesis that has found at most weak support in molecular epidemiology studies. A weakness in this hypothesis was that the mercapturic acids resulting from conjugation of PAH bay region diol epoxides had never been identified in human urine. We recently analyzed smokers' urine for mercapturic acids derived from phenanthrene, the simplest PAH with a bay region. The only phenanthrene diol epoxide-derived mercapturic acid in smokers' urine was produced from the reverse diol epoxide, antiphenanthrene-3,4-diol-1,2-epoxide (11), not the bay region diol epoxide, anti-phenanthrene-1,2-diol-3,4-epoxide (10), which does not support the hypothesis noted above. In this study, we extended these results by examining the conjugation of phenanthrene metabolites with glutathione in human hepatocytes. We identified the mercapturic acid N-acetyl-S- (r-4,t-2,3-trihydroxy-1,2,3,4-tetrahydroc-1-phenanthryl)-L-cysteine (14a), (0.33-35.9 pmol/mL at 10 µM 8, 24h incubation, N = 10) in all incubations with phenanthrene-3,4-diol (8) and the corresponding diol epoxide 11, but no mercapturic acids were detected in incubations with phenanthrene-1,2-diol (7) and only trace amounts were observed in incubations with the corresponding bay region diol epoxide 10. Taken together with our previous results, these studies clearly demonstrate that glutathione conjugation of a reverse diol epoxide of phenanthrene is favored over conjugation of a bay region diol epoxide. Since reverse diol epoxides of PAH are generally weakly or non-mutagenic/carcinogenic, these results, if generalizable to other PAH, do not support the widely held assumption that glutathione-S-transferases are important in the detoxification of PAH in humans.

Preferential Glutathione Conjugation of a Reverse Diol Epoxide Compared with a Bay Region Diol Epoxide of Benzo[a]pyrene in Human Hepatocytes

“…The mixture was stirred for 2 h, and the isomers were separated by high-performance liquid chromatography (HPLC) and collected using a 250 ϫ 10 mm, 10, C18 Vydac 210TP column (Separations Group, Hesperia, CA) eluted at 3 ml/min with 30 mM NH 4 HCO 3 (60%) and 40% CH 3 OH, overall yield, 1.6 mg, 45. (Hecht et al, 2008(Hecht et al, , 2009). The mercapturic acid metabolites illustrated are produced by normal metabolic processing of initially formed GSH conjugates by successive action of ␥-glutamyltranspeptidase, cysteinylglycine dipeptidase, and cysteine S-conjugate N-acetyltransferase.…”

“…In brief, freshly isolated hepatocytes were plated onto 12-well plates (7 ϫ 10 5 cells/well) and overlaid with Matrigel 24 to 48 h after attachment. Cells were shipped overnight on cold preservation media, and, upon receipt, media were replaced with serum-free Williams' E media (without phenol red) and supplements as described previously (Hecht et al, 2009). Cells were allowed to recover from shipping for 10 h at 37°C in an atmosphere containing 5% CO 2 .…”

“…Cell viability was assessed at 24 h by trypan blue exclusion staining. Expression of GSTM1 and GSTA1 and measurement of GST activity by conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) were carried out as described previously (Hecht et al, 2009).…”

“…Reverse diol epoxides are not generally associated with strong mutagenicity or carcinogenicity, although there are some exceptions (Conney, 1982;Glatt et al, 1993). In further studies, we investigated the metabolism of Phe-1,2-diol, Phe-3,4-diol, Phe-1,2-D-3,4-E, and Phe-3,4-D-1,2-E in human hepatocytes (Hecht et al, 2009). Consistent with the results of our analyses of human urine, we found that GSH conjugation of the reverse diol epoxide Phe-3,4-D-1,2-E was strongly favored over conjugation of the bay region diol epoxide Phe-1,2-D-3,4-E (Hecht et al, 2009).…”

“…In further studies, we investigated the metabolism of Phe-1,2-diol, Phe-3,4-diol, Phe-1,2-D-3,4-E, and Phe-3,4-D-1,2-E in human hepatocytes (Hecht et al, 2009). Consistent with the results of our analyses of human urine, we found that GSH conjugation of the reverse diol epoxide Phe-3,4-D-1,2-E was strongly favored over conjugation of the bay region diol epoxide Phe-1,2-D-3,4-E (Hecht et al, 2009). Taken together, these results were quite surprising because they seemed to contradict the hypothesis that carcinogenic bay region diol epoxides, such as Phe-1,2-D-3,4-E and BPDE, were detoxified by GST-catalyzed conjugation.…”