The adrenal steroid products, glucocorticoids, appear to play an important role in the expression of hepatic cytochrome P-450 dependent monooxygenases and other enzymes involved in metabolism of foreign compounds, such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase. We have observed either a potentiation or repression in the induction of several hepatic enzyme activities by polycyclic aromatic hydrocarbons (PAH) in the presence of glucocorticoids, suggesting a cooperative modulation of Ah receptor action by the glucocorticoid receptor. The effects of glucocorticoids on the expression of hepatic monooxygenases and other enzymes in hepatocyte cell culture will be measured by enzyme assay, by immunoblotting techniques for enzyme protein, and by measurements of the levels and rates of synthesis of mRNA specific for these enzymes with Northern analyses. A second specific aim will address the role of protein phosphorylation on the Ah and ARE receptor function in the absence and presence of glucocorticoids. A battery of genes under the control of either the Ah or Antioxidant Response receptors will be tested for their dependence on protein kinase action for induction. A third Specific Aim involves use of adrenalectomized animals to ascertain whether lack of adrenal steroids diminishes induction of these enzymes by PAH or t-butylhydroquinone in vivo. Alterations in extent of enzyme induction, DNA alkylation, and hemoglobin alkylation will be measured as a function of age (gestational and neonatal), since our second animal model provides periods of high and low levels of circulating glucocortiods. A fourth Specific Aim will establish whether chloramphenicol N-acetyltransferase reporter constructs containing the putative glucocorticoid responsive elements (GREs) found in many of these genes function as enhancer elements. Subsequently, a CAT construct containing 1.2 kb of 5'-flanking region of the CYP1A1 gene with multiple cloning sites will be used to make additional constructs containing the intronic GREs of this gene or the palindromic and halfsite GREs of the GST Yal 5'-flanking sequence. These CAT constructs will test whether the GREs of GST Yal or other genes function as a secondary enhancer to the XREs in Ah receptor function. A final Specific Aim would address whether there are protein-protein interactions between the two receptors when the glucocortoid receptor positively interacts with the Ah receptor. These studies would establish whether cooperative (positive and negative) modulation of multiple receptors occurs for these genes.
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