Activation of c-Ha-ras by benzo(a)pyrene in vascular smooth muscle cells involves redox stress and aryl hydrocarbon receptor

Repeated cycles of vascular injury by benzo(a)pyrene (BaP) increase the onset and progression of atherosclerotic lesions in laboratory animals. This atherogenic response is partly mediated by activation of cis-acting antioxidant/electrophile response elements that enhance c-Ha-ras transcription in vascular smooth muscle cells (vSMCs). Activation of antioxidant/electrophile responsive cis-acting elements may depend on metabolism of BaP by cytochrome P450s to intermediates that induce oxidative stress and modulate gene expression. To test this hypothesis, we evaluated mitogen-activated c-Ha-ras expression in vSMCs treated with BaP or its metabolic intermediates alone, and in combination with agents that modulate cellular redox status. BaP (0.3 and 3μM), BaP-3,6-quinone (0.3 μM), or hydrogen peroxide (50μM) enhanced serum-activated c-Ha-ras. Ellipticine (0.01 nM), a known inhibitor of cytochrome P450 metabolism and aryl hydrocarbon receptor (AhR) antagonist, inhibited c-Ha-ras induction by BaP (3μM). Serum challenge of G₀ synchronized cultures of vSMCs with DL- buthionine-(S,R)-sulfoximine (0.1 mM), a depletor of cellular glutathione, increased c-Ha-ras mRNA levels during the early phase of the mitogenic response. Combined BaP/DL-buthionine-(S,R)-sulfoximine challenge was cytotoxic to the cells and inhibited c-Ha-ras expression, whereas up- regulation of antioxidant capacity by N-acetylcysteine (0.5 mM) precluded BaP-induced ras expression. BaP increased formation of reactive oxygen species and depleted cellular glutathione, but these changes did not correlate with the kinetics of c-Ha-ras induction. BaP did not enhance c-Ha- ras expression in vSMCs from AhR knockout mice, although aryl hydrocarbon hydroxylase activity was constitutively expressed in these cells. These results suggest that c-Ha-ras activation in vSMCs by BaP involves a redox- sensitive mechanism that is coupled to AhR receptor-dependent functions.