Role of Cytochrome P4501B1 in Benzo[a]pyrene Bioactivation to DNA-Binding Metabolites in Mouse Vascular Smooth Muscle Cells: Evidence from³²P-Postlabeling for Formation of 3-Hydroxybenzo[a]pyrene and Benzo[a]pyrene-3,6-quinone as Major Proximate Genotoxic Intermediates

Abstract

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 32 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.

The Role of Aryl Hydrocarbon Receptor in the Pathogenesis of Cardiovascular Diseases

“…In particular, it has been hypothesized that AhR ligands disrupt the endothelial cell wall integrity and permeability, which allows more uptake of cholesterol-rich lipoprotein and influx of inflammatory cells, such as T lymphocyte, into the plaque (Curfs et al, 2005), resulting in atheroma formation and ischemic heart diseases (Lee et al, 2003). This hypothesis has been supported by several studies that showed that DNA and protein adduct formation in atherosclerotic lesions in rats and mice SMCs pretreated with 3MC or BaP, or exposed to cigarette smoke, were significantly inhibited by the antioxidant, N-acetylcysteine (NAC), suggesting a direct link between ROS production and atherogenesis (De Flora et al, 1996;Izzotti et al, 1994;Izzotti et al, 1995;Izzotti et al, 1998;Moorthy et al, 2002;Moorthy et al, 2003). Furthermore, the ability of TCDD to induce ROS production and increase DNA degradation in Medaka fish embryonic heart and veins of the embryonic vasculature was partially inhibited by NAC and completely abolished with PBO treatment (Cantrell et al, 1996).…”

“…Oxidative stress and DNA adduct formation have been implicated in the pathogenesis and progression of several CVDs, in which they represent an initiator key of gene mutations and thus organ dysfunction and carcinogenesis (Dhalla et al, 2000;Moorthy et al, 2003;Morganti et al, 2002;Ross et al, 2001). Although it is not clear how oxidative stress causes cardiac dysfunction, several mechanisms have been postulated (Dhalla et al, 2000).…”

“…Most recently, Thackaberry and coworkers showed that exposure of high doses (6 mg/kg) of TCDD to fetal murine upregulated cardiac Cyp1a1 and Cyp1b1 gene expression by 13-and 5-fold, respectively, but did not alter Cyp1a2 gene expression (Thackaberry et al, 2005a). In addition, cultured mouse aortic smooth muscle cells (SMCs) have been shown to constitutively express aryl hydrocarbon hydroxylase (AHH) activity and Cyp1b1 protein levels, which were significantly induced following 3MC or BaP treatment (Kerzee and Ramos, 2001;Moorthy et al, 2002;Moorthy et al, 2003).…”

“…The parent AhR ligands such as BaP and 3MC are not toxic by themselves; however, they are metabolized by cardiac CYPs, particularly the CYP1A enzyme family, to carcinogenic and atherogenic initiators. The availability of an enzymatic system in CVS that bioactivates AhR ligands into potent cardiotoxic and atherogenic metabolites was demonstrated by the presence of multiple DNA adducts in the aortic SMCs exposed to 3MC (Moorthy et al, 2003;Thirman et al, 1994) or BaP and its major genotoxic metabolites, 3-OH-BaP and BaP-3,6-quinone (Moorthy et al, 2003). A strong link between cigarette smoking and the CYP1A1 Msp1 polymorphism on the risk of CVDs has been reported in humans, where the C allele carrier had a higher frequency of ischemic heart diseases, stroke, and diabetes mellitus type II, a major risk factor for CVD (Lanca et al, 2004;Wang et al, 2002).…”

Evaluation of 3-hydroxy-benzo[a]pyrene levels in Nile tilapia (Oreochromis niloticus) after waterborne exposure to Benzo[a]pyrene

“…Some studies evaluated the in vitro 3-OH-BaP effects and showed that low concentrations of 3-OH-BaP (from 8 to 67 ng mL À1 ) may exert estrogenic activity (van Lipzig et al 2005) and form covalent bonds with macromolecules (Moorthy et al 2003;Sugihara and James 2003), leading to adverse consequences at the organism level. Although adverse effects were not assessed and the levels of free 3-OH-BaP measured in plasma (0.4 ng mL À1 ) were lower than concentrations capable of producing toxic effects, as it was quantified in one pooled sample, the levels obtained in our study may have been diluted.…”