Role of Cytochrome P4501B1 in Benzo[a]pyrene Bioactivation to DNA-Binding Metabolites in Mouse Vascular Smooth Muscle Cells: Evidence from P-Postlabeling for Formation of 3-Hydroxybenzo[a]pyrene and Benzo[a]pyrene-3,6-quinone as Major Proximate Genotoxic Intermediates

Benzo[a]pyrene (BP), a polycylic aromatic hydrocarbon (PAH), is a potent atherogen and carcinogen in laboratory animals. Since genotoxic mechanisms may contribute to the development of atherosclerosis by PAHs, we have tested the hypotheses that: 1) BP induces DNA adducts in mouse aortic smooth muscle cells (SMCs); 2) 3-hydroxybenzo[a]pyrene (3-OH-BP) and benzo[a]pyrene-3,6-quinone (BPQ) are proximate genotoxic metabolites; and 3) cytochrome P4501B1 (CYP1B1) mediates the activation of BP and its metabolites to ultimate genotoxic intermediates. Cultured mouse aortic SMCs were treated with BP, 3-OH-BP, or BPQ for 24 h, and DNA adduct formation was analyzed by P-postlabeling. In some experiments, cells were pretreated with the CYP1B1 inhibitor 1-ethynylpyrene (EP) prior to exposure to BP or its metabolites. BP, 3-OH-BP, and BPQ induced formation of several DNA adducts that were not observed in dimethylsulfoxide-treated cells. Reand cochromatography experiments indicated that 3-OH-BP and BPQ were proximate genotoxic metabolites of BP. DNA adduct formation was strongly inhibited by EP, a specific inhibitor of CYP1B1. BP treatment of SMCs resulted in induction of aryl hydrocarbon hydroxylase (AHH) activity and CYP1B1, but not CYP1A1, apoprotein. EP also blocked AHH induction by BP. In conclusion, the results of this study support the hypothesis that in SMCs, which are target sites for the development of atherosclerosis, the major bioactivation pathway of BP entails CYP1B1-mediated formation of the 3-OH-BP and BPQ, which are proximate genotoxic metabolites that may in turn get transformed to ultimate DNA-binding metabolites, which may contribute to atherogenesis by PAHs. Atherosclerosis is one of the major sequelae of cigarette smoking in humans (Ross et al., 2001; Salama et al., 2002). Atherosclerosis is a complex disease process involving elastic and muscular arteries that, like cancer, involves uncontrolled proliferation and dedifferentiation of vascular smooth muscle cells (SMCs) (Ramos and Parrish, 1995). Polycyclic aromatic hydrocarbons (PAHs) are important constituents of cigarette smoke, and animal and human studies have suggested that PAHs may be involved in the etiology of carcinogenesis and atherosclerosis associated with exposure to cigarette smoking (Majesky et al., 1983; Paigen et al., 1986; Izzotti et al., 1995; Ross et al., 2001; Salama et al., 2002). The molecular mechanisms of vascular damage by PAHs are not fully understood. Benzo[a]pyrene (BP), a potent PAH carcinogen that induces tumors in a variety of organs in experimental animals, is also a potent atherogen in laboratory animals (Bond et al., 1981). Several studies have suggested that there are similarities between carcinogenesis and atherogenesis in that both processes involve initiation and promotion stages (Majesky et al., 1983; Ramos and Parrish, 1995; Ross et al., 2001). The presence of PAH-DNA adducts in atherosclerotic lesions of humans suggests that DNA damThis work was supported in part by a grant-in-aid award from the American Heart Association (Texas Affiliate) and National Institute of Environmental Health Sciences (NIEHS) Grants R01 ES09132 (to B.M.) and R01 04849 (to K.S.R.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.044271. ABBREVIATIONS: SMC, smooth muscle cell; PAH, polycyclic aromatic hydrocarbon; BP, benzo[a]pyrene; BPDE, benzo[a]pyrene-7,8-dihydrodiol-9,10 epoxide; P450, cytochrome P450; AHH, aryl hydrocarbon hydroxylase; 3-OH-BP, 3-hydroxybenzo[a]pyrene; BPQ, benzo[a]pyrene-3,6quinone; EP, 1-ethynylpyrene; -NF, -naphthoflavone; DMSO, dimethyl sulfoxide; EH, epoxide hydrolase; MC, 3-methylcholanthrene; PEI-TLC; polyethyleneimine cellulose thin-layer chromatography; RAL, relative adduct labeling. 0022-3565/03/3051-394–401$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 305, No. 1 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 44271/1051630 JPET 305:394–401, 2003 Printed in U.S.A. 394 at A PE T Jornals on N ovem er 9, 2017 jpet.asjournals.org D ow nladed from age contributes to the development of atherosclerosis (Bond et al., 1981; Izzotti et al., 1995). Although parent PAHs by themselves are nontoxic, metabolic activation of PAHs by cytochrome P450 (P450) leads to the production of reactive metabolites capable of covalently binding to DNA (Gelboin, 1990). Benzo[a]pyrene-7,8-dihydrodiol-9,10 epoxide (BPDE) is the major ultimate carcinogen responsible for the carcinogenicity of BP (Gelboin, 1990). Recent studies have shown that BPDE-induced mutational hot spots are produced in lung tumors of smokers, providing direct evidence for a link between DNA adduction and cancer (Denissenko et al., 1996). In addition to BPDE, BP can also undergo one-electron oxidation, and subsequent radical reactions to form BP quinones, which have also been implicated in carcinogenesis (Cavalieri and Rogan, 1995) and atherogenesis (Bond et al., 1981; Kerzee and Ramos, 2000; Miller et al., 2000). Oxidative stress induced by BP has been implicated in atherogenic responses in animal models and cell culture studies (Kerzee and Ramos, 2000; Miller et al., 2000), with electrophile response elements playing an important role in atherogenic mechanisms. Since BP metabolism to quinones and semiquinones can lead to redox cycling and reactive oxygen species formation, it is plausible that direct DNA binding of BP semiquinones (Joseph and Jaiswal, 1994) and oxidative DNA damage may contribute to the atherogenicity of BP. Hepatic and extrahepatic CYP1 enzymes play major roles in the bioactivation of PAHs to genotoxic metabolites (Guengerich, 1988). In fact, PAH-DNA adduct formation has been demonstrated in several human cancer cell lines and organ explant cultures of extrahepatic origin (Melendez-Colon et al., 2000). However, BP activation leading to DNA adduct formation in cultured SMCs of aorta, a target site for the development of atherosclerosis, has not been demonstrated. Little progress has been made in the understanding of the relationship between P450 enzymes and DNA adducts in vascular tissues. Evidence to date suggests that BP induces CYP1A1 in rat aortic tissues (Thirman et al., 1994). Although CYP1A1 is localized in endothelial cells of porcine aorta (Stegeman et al., 1995) and in vascular SMCs from newborn rats (Giachelli et al., 1991), the enzyme is under negative regulation in adult quail and rodent aortic SMCs (Stegeman et al., 1995). Thus, other P450 isoforms probably also contribute to PAH metabolism in SMCs. A number of P450 isoforms are present within the vascular wall, including CYP1B1, which is preferentially expressed in SMCs, as opposed to vascular endothelium, and is coexpressed with CYP1A1 in several extrahepatic tissues (Kerzee and Ramos, 2001). CYP1A1 and 1B1 activities are frequently determined by measuring the activities of aryl hydrocarbon hydroxylase (AHH), which catalyzes the conversion of BP to 3-hydroxybenzo[a]pyrene (3-OH-BP) (Nebert and Gelboin, 1968). PAHs are potent inducers of CYP1B1 (Shimada et al., 1996), and metabolic activation of PAHs by CYP1B1 to oxidative intermediates and carcinogenic precursors (Bowes et al., 1996; Shimada et al., 1996; Moorthy et al., 2002) may have implications for PAH-induced atherogenesis. Taken together, it appears that CYP1B1-catalyzed BP metabolism to oxidative intermediates plays an important role in BP genotoxicity, which may in turn contribute to atherogenesis. To this end, we tested the hypotheses that 1) BP induces DNA adducts in mouse aortic SMCs, 2) 3-OH-BP and benzo[a]pyrene-3,6-quinone (BPQ) are proximate genotoxic metabolites, and 3) CYP1B1 plays an important role in the activation of BP and its metabolites to ultimate genotoxic intermediates. Materials and Methods Chemicals. BP (98% purity) was obtained from Sigma-Aldrich (St. Louis, MO), and 3-OH-BP and BPQ ( 99% purity by highpressure liquid chromatography) were obtained from the National Cancer Institute Chemical Carcinogen Reference Standard Repositories c/o Midwest Research Institute (Kansas City, MO). 1-Ethinylpyrene (EP) was a kind gift from Dr. William Alworth, Department of Chemistry, Tulane University (New Orleans, LA). All other chemicals were purchased from Sigma-Aldrich unless otherwise noted. BP, 3-OH-BP, BPQ, EP, ellipticine, and -naphthoflavone ( -NF) stock solutions were prepared in dimethyl sulfoxide (DMSO). Monoclonal antibody against CYP1A1 was a gift from Dr. Paul E. Thomas, Rutgers University (New Brunswick, NJ). Polyclonal antibodies to rat CYP1B1 that cross-react with mouse CYP1B1 were purchased from BD Gentest (Woburn, MA). Rat CYP1B1 protein standards were also obtained from BD Gentest. Cell Culture. Primary cultures of vascular SMCs were isolated from female C57BL/6 mouse aorta and maintained under standard conditions as described by Ramos and Cox (1993). Cells were grown in Medium 199 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA), 2 mM glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 g/ml amphotericin B (Invitrogen). Subcultures were prepared by trypsinization (Invitrogen) of subconfluent primary cultures. Cells were seeded at 100 cells/mm onto 100-mm plates for AHH assays, and at 75 cells/mm onto 150-mm plates for postlabeling experiments. The cells were allowed to acclimate for 48 h and then challenged with BP, 3-OH-BP, BPQ, or EP for the time periods specified in the figure legends. Concentrations of DMSO in all experiments