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Characterization of Benzo(a)pyrene-trans-7,8-dihydrodiol Glucuronidation by Human Tissue Microsomes and Overexpressed UDP-glucuronosyltransferase Enzymes¹

Divisions of Cancer Control and Molecular Oncology, H. Lee Moffitt Cancer Center, Interdisciplinary Oncology Program and Departments of Biochemistry and Pharmacology and Therapeutics, University of South Florida, Tampa, Florida 33612 [J-L. F., D. W., P. L.]; Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079 [F. A. B., D. R. D.]; Oncology and Molecular Endocrinology Research Center, CHUL Research Center (CHUQ), School of Pharmacy, Laval University, Quebec, Canada G1V-4G2 [C. G.]; and Centro de Química Estrutural, Complexo I, Instituto Superior Técnico, 1049-001 Lisboa, Portugal [M. M. M.]

UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of benzo(a)pyrene-trans-7,8-dihydrodiol (BPD), precursor to the potent mutagen benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide, may be an important pathway in the detoxification of benzo(a)pyrene. To better characterize this pathway in humans, high-pressure liquid chromatography (HPLC) was used to detect glucuronide conjugates of BPD formed in vitro. Three peaks were detected by HPLC after incubation of racemic BPD with human liver microsomes; these were identified as monoglucuronides by liquid chromatography-mass spectrometry analysis. Proton nuclear magnetic resonance spectroscopy of isolated fractions, combined with HPLC analysis of the glucuronide products from human liver microsomal incubations with purified benzo(a)pyrene-trans-7S,8S-dihydrodiol [(+)-BPD] and benzo(a)pyrene-trans-7R,8R-dihydrodiol [(-)-BPD] forms of BPD, indicated that peak 1 contained the 7-glucuronide of 7S,8S-BPD (BPD-7S-Gluc), peak 2 was a mixture of the 7-glucuronide of 7R,8R-BPD (BPD-7R-Gluc) and the 8-glucuronide of 7S,8S-BPD (BPD-8S-Gluc), and peak 3 contained the 8-glucuronide of 7R, 8R-BPD (BPD-8R-Gluc). In liver microsomes, peak 1 (BPD-7S-Gluc) was the largest peak observed, whereas in microsomes from aerodigestive tract tissues, peak 2 (both BPD-7R-Gluc and BPD-8S-Gluc) was the largest HPLC peak observed. The liver enzymes UGT1A1 and UGT2B7 formed BPD-7S-Gluc as the major diastereomer, whereas UGT1A8 and UGT1A10, extrahepatic enzymes present in the aerodigestive tract, preferentially formed both BPD-7R-Gluc and BPD-8S-Gluc. In addition, both UGT1A9 and UGT1A7 preferentially formed BPD-7R-Gluc. No detectable glucuronidating activity against BPD was observed by UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B15, or UGT2B17. The affinity of individual UGT enzymes as determined by K_m analysis was UGT1A10 > UGT1A9 > UGT1A1 > UGT1A7 for (-)-BPD and UGT1A10 > UGT1A9 > UGT2B7 UGT1A1 > UGT1A7 for (+)-BPD. These results suggest that several UGTs may play an important role in the overall glucuronidation of BPD in humans, with UGT1A1, UGT1A7, UGT1A9, UGT1A10 and potentially UGT1A8 playing an important role in the glucuronidation of the procarcinogenic (-)-BPD enantiomer, and that the stereospecific activity exhibited by different UGTs against BPD is consistent with tissue-specific patterns of BPD glucuronide diastereomer formation and UGT expression.

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