Metabolism of benzo[a]pyrene in human bronchoalveolar H358 cells using liquid chromatography-mass spectrometry.
Benzo[ a]pyrene (B[ a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways: by peroxidases (e.g., cytochrome P450 peroxidase) to yield radical cations, by P4501A1/1B1 monooxygenation and epoxide hydrolase to yield diol epoxides, and by P4501A1/1B1 monooxygenation, epoxide hydrolase, and aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung; however, the profile of B[ a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[ a]P, and metabolites generated by peroxidase (B[ a]P-1,6- and B[ a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation [3-hydroxy-B[ a]P, B[ a]P-7,8- and 9,10- trans-dihydrodiols, and B[ a]P- r-7, t-8, t-9, c-10-tetrahydrotetrol (B[ a]P-tetraol-1)], and from AKRs (B[ a]P-7,8-dione) were detected and quantified by RP-HPLC, with in-line photo-diode array and radiometric detection, and identified by liquid chromatography-mass spectrometry (LC-MS). Progress curves showed a lag phase in the formation of 3-hydroxy-B[ a]P, B[ a]P-7,8- trans-dihydrodiol, B[ a]P-tetraol-1, and B[ a]P-7,8-dione over 24 h. Northern blot analysis showed that B[ a]P induced P4501B1 and AKR1C isoforms in H358 cells in a time-dependent manner, providing an explanation for the lag phase. Pretreatment of H358 cells with 10 nM 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) eliminated this lag phase but did not alter the levels of the individual metabolites observed, suggesting that both B[ a]P and TCDD induction ultimately yield the same B[ a]P metabolic profile. The one exception was B[ a]P-3,6-dione which was formed without a lag phase in the absence and presence of TCDD, suggesting that the peroxidase responsible for its formation was neither P4501A1 nor 1B1. Candidate peroxidases that remain include PGH synthases and uninduced P450 isoforms. This study shows that the P4501A1/1B1 and AKR pathways are inducible in human lung cells and that the peroxidase pathway was not. It also provides evidence that each of the pathways of PAH activation yields their distinctive metabolites in H358 human lung cells and that each pathway may contribute to the carcinogenic process.
Jiang
Hao
H
Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084, USA.
Gelhaus
Stacy L
SL
Mangal
Dipti
D
Harvey
Ronald G
RG
Blair
Ian A
IA
Penning
Trevor M
TM
D015089:20-Hydroxysteroid Dehydrogenases / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001564:Benzo(a)pyrene / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D065633:Cytochrome P-450 CYP1B1; D006801:Humans; D013058:Mass Spectrometry; D015394:Molecular Structure; D010544:Peroxidases / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000187:drug effects* / Q000378:metabolism*
D001564:Benzo(a)pyrene; D015089:20-Hydroxysteroid Dehydrogenases; C115140:3 alpha-beta, 20 beta-hydroxysteroid dehydrogenase; D010544:Peroxidases; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1
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