This grant will define the molecular controls regulating transcriptional activation of hepatic cytochrome P4502B1 /2 (CYP2B1 /2) by phenobarbital (PB) and phenobarbital-like agents, including organochlorine pesticides (Kepone and o,p-DDT) and certain polychlorinated biphenyls (PCBs) in rat liver. The regulatory nucleotide sequences involved in induction of the CYP2B1/2 genes will be defined by introduction of the chimeric gene constructs (2B1/2-TK-CAT) containing 5'-flanking sequences of the 2B1 or 2B2 gene fused to the reporter gene chloramphenicol acetyltransferase (CAT), into a functional assay system which is responsive to the PB signal, the system of primary rat hepatocytes cultured on matrigel. We will test the hypothesis that drug induced transcriptional activation of 2B1/2 involves the interaction of specific proteins with selective DNA sequences 5' to the transcriptional start site of these genes. We will determine the """"""""functionality"""""""" of putative 2B1/2 control sequences by transient transfection analysis. Concurrent with these studies DNA-protein interactions will be defined by electromobility gel shift analysis and southwestern blotting. In some cases, where antibodies to putative DNA binding factors are available, the gel shift will be followed by immunoblot analysis to define the role of candidate DNA binding proteins in transcriptional activation. Finally, the exact bases involved in DNA- protein interactions will be identified by methylation interference. The second specific aim is to test the novel hypothesis that the estrogen receptor participates in modulating drug induction of 2B1/2. This will be accomplished by transfecting hepatocytes with an expression vector for the estrogen receptor and determining whether it enhances drug-inducible expression of the native 2B1/2 mRNA, and determining if the expressed estrogen receptor is capable of transactivating co-transfected 2B1/2-TK- CAT chimeric genes. Using gel shift analysis followed by immunoblot analysis with antibody to the estrogen receptor we will determine the nature of the estrogen receptor-DNA interaction. The availability of the primary hepatocyte culture system, coupled with the successful method to introduce DNA into the cells, and chimeric 2B1/2-TK-CAT genes should allow the identification of regulatory elements governing transcriptional activation of 2B1/2 by xenobiotics and the role of the estrogen receptor in modulating this process.
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