The induction of proliferative vascular smooth muscle cell phenotypes by benzo[a]pyrene does not involve mutational activation of ras gene

Previous studies in this laboratory have suggested that benzo[a]pyrene (BaP) challenge in vivo and in vitro induces genomic changes that result in the acquisition of proliferative vascular smooth muscle cell (SMC) phenotypes. Because this phenotypic change correlates with alterations in ras gene expression, and ras genes are known mutational targets of BaP, the present studies were conducted to determine if BaP induces activating mutation(s) of ras genes in SMCs. For in vivo studies, male Sprague-Dawley rats were given weekly injections of an atherogenic dose of BaP (10 mg/kg) or vehicle for 8 weeks and SMCs were isolated from the thoracic aorta and established in culture. For in vitro studies, naive rat aortic SMCs were challenged with 3 μM BaP for 24 h and then subjected to nine serial passages for the induction of proliferative phenotypes. Measurements of DNA synthesis and cell numbers were conducted to define patterns of proliferative behavior in BaP and control cells. In parallel studies, exons 1 and 2 of ras genes were amplified by reverse-transcription polymerase chain reaction and sequenced to screen for activating ras mutations. BaP treatment in vivo was associated with the development of aortic wall lesions characterized by loss of endothelial integrity, fragmentation of the elastic laminae, expansion of the smooth muscle cell mass, and change in the orientation of medial SMCs. Although BaP enhanced the growth rate of vascular SMCs relative to controls, no mutations were detected in the activating regions (codons 12, 13, 60, or 61) of c-Ha-, c-Ki-, or N-ras genes. These data demonstrate that BaP-induced alterations in the proliferative potential of SMCs do not involve activating mutations in the frequently mutated regions of ras genes.