Phenobarbital is a prototype chemical widely known for its propensity to induce expression of a number of biotransformation systems, including microsomal epoxide hydrolase, UDP-glucuronyltransferase, certain glutathione transferases, and some members of the cyotchrome P450 monooxygenase gene superfamily. Together these enyzmes attack a broad array of endogenous and xenobiotic substances. The balance between activation versus detoxication pathways, potentially perturbed by inducers such as PB, will dictate individual susceptibility to mutagenic, carcinogenic, teratogenic, and otherwise toxic effects associated with exposures to certain environmental chemicals. This research program seeks to examine expression mechanisms for PB-inducible members of the CYP2B subfamily of P450s, for which little molecular data is available. The CYP2Bl and CYP2B2 genes are the primary systems under analysis. Their transcription is largely repressed in most tissues, but markedly activated in rat liver upon PB exposure. Through the use of molecular approaches, our primary aims are to: 1) identify critical sequence elements within these genes that confer PB responsiveness; 2) identify repressor/silencer sequence elements responsible for the low levels of constitutive expression observed normally in most tissues; and, 3) characterize novel regulatory proteins, identified as key positive or negative transcriptional modulators. Isolated P450 genes will be analyzed with a battery of techniques, including DNase I hypersensitivity determinations, DNA footprinting and gel retardation assays, in vitro expression of P450/chloramphenicol acetyl transferase gene fusion constructs, direct expression of P450 genes in cultured cells, and in vitro reverse transcription procedures. The results of these experiments should enable a detailed level of understanding of the molecular events responsible for PB-inducible gene regulation. These investigations will provide a framework for determining the genetic basis underlying differential susceptibilities of individuals to the toxic effects associated with chemical exposures.
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