The superfamily of mammalian cytochrome P-450 (P450) genes codes for a diverse group of hemoenzymes known to play pivotal roles in the biotransformation of many endogenous and xenobiotic substances. In the adult rat liver, two major isozymes of highly homologous P450, P450b and P450e, are induced markedly by a number of compounds, including phenobarbital (PB). The goal of this research program is to delineate, for the P450b and P450e genes, the molecular mechanisms central to PB induction. Our hypothesis is that interactions between cis-sequence elements and specific trans- acting nuclear factors, coupled with alterations in gene architec- ture, contribute to the control of P450b/e gene transcription. We will define specific sequences conferring PB responsiveness by constructing expression vectors that combine various 5'-flanking regions of the P450b and P450e genes with the structural gene for chloramphenicol acetyl transferase (CAT). These vectors will be employed for transient CAT gene expression assays in a rat hepatocyte culture system that models the PB induction response observed in vivo. Upon identification of key regions that confer PB-responsiveness, site-specific mutagenesis will he employed to examine individual residues critical for induction. Interactions of trans-acting factors with these sequences will be assessed with cotransfection-competition assays, cycloheximide treatment, and DNA-footprinting experiments. Nuclear extracts of adult and fetal hepatocytes derived from control and PB treated Sprague-Dawley rats, and from Marshall 520/N rats, which are defective in P450e expression, will be utilized for trans-factor characterizations. Structural changes accompanying the developmental and PB-induced activation of rat hepatic P450b and P450e genes will be analyzed for differences in gene methylation and sites of nuclease sensitivity. The information gained from this research program will enhance our understanding of the molecular mechanisms responsible for PB induction and developmental regulation of P450 gene expression. The data will aid our ability to predict consequences of exposure to environmental inducing substances and help clarify differences in individual and developmental susceptibility to toxic chemicals that undergo biotransformation.
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