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.
ASJC Scopus subject areas
- Molecular Medicine