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

Abstract

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 the 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 incubated human hepatocytes from 10 donors with racemic anti-BaP-7,8-diol-9,10-epoxide (BPDE), believed to be a major ultimate carcinogen of BaP, or with the noncarcinogenic reverse diol epoxide, racemic anti-BaP-9,10-diol-7,8-epoxide (rev-BPDE). Incubations were carried out for 12 or 24 h. We used high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring at m/z 464 3 m/z 317 to analyze the incubation mixtures for the mercapturic acid products that would result from glutathione conjugation. The standard mercapturic acids were synthesized by reaction of BPDE or rev-BPDE with N-acetylcysteine. We obtained convincing evidence in human hepatocytes for mercapturic acid formation from rev-BPDE in all 10 samples, in amounts up to 17 pmol/ml. However, we could detect mercapturic acids from BPDE 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 BPDE with glutathione is a minor pathway in humans, indicating that glutathione S-transferase genotyping is not an effective method for assessing risk of PAH-induced cancer in humans, at least with respect to the diol epoxide pathway of PAH carcinogenesis.

Immediate Consequences of Cigarette Smoking: Rapid Formation of Polycyclic Aromatic Hydrocarbon Diol Epoxides

“…Addition of a carcinogenic PAH to a cigarette would be potentially hazardous and possibly unethical. Although there are similarities in the P450s that catalyze diol epoxide formation from Phe and BaP – P450s 1A1, 1A2, and 1B1 – as well as similarities in their detoxification by glutathione- S -transferases (19,31,32), there are also some potential differences in the metabolism and toxicokinetics of Phe versus other PAH that could affect the interpretation of our results. First, metabolism of Phe by the angular ring diol epoxide pathway occurs mainly via Phe reverse diol epoxide ( 5 ) in humans and to a much smaller extent by Phe bay region diol epoxide ( 2 )(Scheme 1)(15), although our unpublished data indicate that measurements of PheT and diol epoxide 2 specifically are highly correlated.…”

Metabolites of a Tobacco-Specific Lung Carcinogen in Children Exposed to Secondhand or Thirdhand Tobacco Smoke in Their Homes

“…In order to investigate the metabolism of NNA to iso -NNAL, primary human hepatocytes were purchased from Cellzdirect (St. Louis, MO), and prepared as described (20). Hepatocytes (approximately 0.12 mg protein per well) were incubated with 10 µM NNA or NNK, as a positive control.…”

Mercapturic acids: recent advances in their determination by liquid chromatography/mass spectrometry and their use in toxicant metabolism studies and in occupational and environmental exposure studies

“…The majority of mercapturate products, again, were formed by preferential GSH conjugation of revBPDE by GST (revBPDE-7Nac). Mercapturate products of conjugation in the bay region of BPDE were detected in only one of ten replicate incubations (Upadhyaya et al, 2010). These studies indicate that detoxification of BPDE by GSH conjugation is a minor pathway in humans, and that the gene polymorphisms of GST are not an important risk factor in the diol epoxide pathway of benzo(a)pyrene carcinogenesis.…”