Abstract 1690: Analysis of benzo[a]pyrene and phenanthrene tetraol enantiomers in human urine: Relevance to the bay region diol epoxide hypothesis of benzo[a]pyrene carcinogenesis and to biomarker studies

Abstract

Abstract One widely accepted metabolic activation pathway of the prototypic carcinogenic polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) proceeds through the bay region diol epoxide BaP-(7R,8S)-diol-(9S,10R)-epoxide. However, few studies have addressed the analysis of human urinary metabolites of BaP which result from this pathway. Phenanthrene (Phe) is structurally related to BaP, but human exposure to Phe is far greater and its metabolites can be readily detected in human urine. Thus, we have proposed that Phe metabolites can be biomarkers of PAH exposure and metabolic activation. Phe-tetraols in particular could be biomarkers of the diol epoxide pathway. While BaP-tetraols and Phe-tetraols have been previously quantified in human urine, no published studies have determined their enantiomeric composition. This is important because different enantiomers would result from the bay region diol epoxide and “reverse” diol epoxide pathways, the latter being associated with weak mutagenicity and carcinogenicity. We addressed this problem using chiral HPLC to separate the enantiomers of Phe-1,2,3,4-tetraol and BaP-7,8,9,10-tetraol. Urine samples from smokers were subjected to hydrolysis with β-glucuronidase and arylsulfatase followed by solid-phase extraction on a Strata-X polymeric sorbent cartridge and a Bond Elute phenylboronic acid cartridge, chiral HPLC on a Cyclobond II or Pirkle column, and quantitation by gas-chromatography-negative ion chemical ionization-tandem mass spectrometry-selected reaction monitoring analysis of the Phe-tetraols, as their tetra-trimethylsilyl ethers. The results demonstrated that >96% of Phe-tetraol in smokers’ urine was Phe-(1S,2R,3S,4R)-tetraol, resulting from the “reverse” diol epoxide pathway, whereas less than 4% resulted from the bay region diol epoxide pathway of Phe metabolism. The corresponding (R,R)-stereochemistry of their precursor diols Phe-1,2-diol and Phe-3,4-diol was also confirmed. Urine from creosote workers with very high exposure to PAH was similarly analyzed for BaP-tetraol enantiomers. In contrast to the results of the Phe-tetraol analyses in smokers’ urine, >75% of BaP-tetraol in these human urine samples was BaP-(7R,8S,9R,10S)-tetraol resulting from the bay region diol epoxide pathway of BaP metabolism. These results provide the first direct support based on human urinary metabolite analysis for the bay region diol epoxide pathway of BaP metabolism and demonstrate differences in BaP and Phe metabolism which may be important when considering Phe-tetraols as biomarkers of PAH metabolic activation. 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 1690.