Experimental and epidemiological studies have established a relationship between exposure to toxic environmental chemicals and the development of atherosclerosis. However, only limited information is available regarding the molecular mechanisms of chemical atherogenesis. In the case of benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), induction of atherosclerotic lesions is associated with deregulation of vascular smooth muscle cell (SMC) proliferation. The ability of BaP to influence SMC proliferation involves upregulation of c-Ha-ras transcription and binding of BaP/protein complexes to DNA. The induction of c-Ha-ras and SMC proliferation can be inhibited by alpha-naphthoflavone, an antagonist of the aryl hydrocarbon receptor (AhR) and an inhibitor of BaP metabolism. Studies are proposed in this application to test the hypothesis that enhancement of ras gene transcription by BaP is mediated by activation of a transacting factor(s) that interacts with a cis-regulatory element in the promoter region of the ras gene. As part of aim 1, studies will be conducted to determine if the AhR is the transacting factor involved in the upregulation oc c-Ha-ras gene expression. The effects of BaP on c-Ha-ras expression will be examined by northern analysis in aortic SMCs from congenic Ahbb and Ahdd C57BL/6J mice, animals of differential aryl hydrocarbon (Ah) responsiveness. Electrophoretic mobility shift assays (EMSA) and nuclear run-on experiments using ligands of varying affinity for the AhR will then be carried out to define the specificity of ligand/receptor interactions. Patterns of gene expression will then be correlated with proliferation profiles in response to BaP and related chemicals. Because the antioxidant responsive element (ARE), present at - 543 in the promoter region of ras may also participate in the transcriptional response, studies will be conducted as part of aim 2 to examine by Northern and nuclear run-on analysis the ability of chemicals which undergo redox cycling to modulate ras expression. These experiments will be followed by progressive 5',3', and internal deletions of the promoter coupled to transient CAT assays to define specific regions within the promoter which confer specificity for BaP interactions. Finally, EMSA using oligonucleotides that contain critical promoter sequence will be used to define protein/DNA interactions.
In aim 3, studies will be conducted to determine if ras up-regulation by BaP occurs in vivo and the role of ras in atherogenesis. The effects of repeated doses of BaP on aortic DNA synthesis will be examined in atherosclerosis prone apoE-knock out mice, in heterozygote controls, and in wild type mice. Conventional histopathologic analysis will be completed to monitor the cellular and acellular composition of lesions. Finally, in situ hybridization for ras will be correlated with the burst of proliferative activity in vivo. The data generated from these studies will be used to define the mechanisms of BaP atherogenicity and to better understand the role that toxic environmental chemicals play in the onset or progression of atherosclerotic vascular disease.
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