The overall goal of this proposal is to understand the molecular mechanisms by which the classical inducer, phenobarbital, increases gene expression of cytochromes P450. Cytochromes P450 form a superfamily of enzymes responsible for activation or inactivation by oxidative metabolism of a wide variety of endogenous and foreign compounds. The ultimate therapeutic or toxic activity of many agents is determined by the balance between activation and inactivation catalyzed by different cytochromes P450. This balance can be dramatically altered by induction of subsets of these enzymes. The specific objectives of this proposal are to identify the cis-and trans-acting factors that mediate phenobarbital induction in the rabbit CYP2C subfamily. This subfamily contains at least 8 genes which exhibit different tissue-specific expression and have strikingly different responses to phenobarbital. The lack of a suitable continuous cell culture model system has prevented analysis of the phenobarbital- responsive elements in the genes and their associated transcription factors. To circumvent this problem, the transcriptional activity of CYP2C genes will be assessed by the introduction of CYP2C promoter - luciferase hybrid genes into transgenic mice and rat hepatocytes in primary culture. Detection of luciferase activity is a simple, rapid, sensitive assay for promoter activity. Transgenic mice provide an in vivo model in which liver phenobarbital regulatory mechanisms are intact and in which the activities of regulatory elements are often more authentic than in cell culture assays. Once transgenic lines expressing the hybrid CYP2C-luciferase genes have been established, tissue-specific expression and the ontogeny of CYP2C gene expression and phenobarbital induction will be studied. Rat hepatocytes plated on matrigel, under conditions shown to maintain phenobarbital-inducible expression of endogenous CYP genes, will be transfected with the CYP2C hybrid genes. Regulatory elements will be defined by deletion and point mutations in the CYP2C gene sequences. Binding of proteins to phenobarbital- responsive elements will be studied by gel retardation and DNase I footprint analyses and these proteins will be isolated or their cDNAs will be cloned. In studies using similar techniques, basal and tissue- specific regulatory elements and proteins will be studied by transfection or the hybrid genes into HepG2 and kidney cells. These studied should result in a comprehensive understanding of the basal, tissue-specific, and phenobarbital-inducible expression of the CYP2C genes at the molecular level.
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