CARCINOGEN METABOLIZING P450S--REGULATION BY CYTOKINES

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DESCRIPTION: Cytokines may act as physiological modulators of P450 levels, influencing members of many P450 subfamilies. Many of these, including the polycyclic aromatic hydrocarbon-inducible P450s, P450 1A1 and P450 1A2, metabolize carcinogens, contributing to their detoxification and/or activation. IL-1 and H2O2 have been reported to inhibit the transcriptional activation of P450 1A1 and P450 1A2 by polycyclic compounds. This inhibition can be blocked by raising cellular glutathione levels. Cytokine levels can vary dramatically within different tissues and organs and also systemically between individuals, due to particular physiological states or genetic factors (acute or chronic inflammation or infection, cigarette smoking, diet, immunoreactivity, etc.). The working hypothesis is that differences in cytokine levels or differences in the responsiveness of P450 genes to cytokines may differentially affect the expression of carcinogen- metabolizing P450s and be an important factor contributing to genetically and/or environmentally determined differences among individuals in their susceptibility to carcinogens. Two DNA elements have been reported to be involved in mediating the effect of IL-1-and H2O2: (i) XREs, the elements that mediate responsiveness to aryl hydrocarbons, and (ii) a novel element termed IL-1 responsive element, ILRE. This element binds a factor, ILRP, that is rapidly down- regulated in isolated hepatocytes treated with either IL-1 or H2O2.. The specific hypothesis being tested by this research is that the IL-1 and H2O2-dependent suppression of P450 1A1 transcription is mediated by suppression of cooperativity of ILREs with XREs and other enhancers due to down regulation of ILRP binding activity, and by suppression of cooperativity between XREs. Specific objectives are: 1. Determine the mechanism by which ILREs and XREs mediate down- regulation of P450 1A1 transcription by IL-1 and H2O2.. DNA-mediated gene transfer of expression vectors containing various combinations of XREs, ILREs and other enhancers will be done to determine how these elements cooperate to mediate responsiveness to IL-1 and H2O2. in isolated rat hepatocytes. 2. Characterize sequence specificity and DNA-protein interactions important for IL-1 and H2O2. responsiveness using in vitro and in vivo footprinting in association with electrophoretic mobility shift analyses with wild type and mutant ILREs. The tissue distribution of the ILRP and the regulation of ILRP binding activity by oxidants, antioxidants, growth factors, insulin, vanadate, and other factors will be determined using electrophoretic mobility shift assays. 3. Clone ILRP cDNA using either expression cloning, plaque hybridization or antibody based procedures.