CARCINOGEN METABOLIZING P450S--REGULATION BY CYTOKINES

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Cytokines may be one of the most comprehensive physiological modulators of P450 levels, influencing members of many P450 subfamilies. Many of these, including the polycyclic aromatic hydrocarbon-inducible P450s, P4501A1 and P4501A2, metabolize carcinogens, contributing to their detoxification and/or activation. We have found that interleukin-1 inhibits the transcriptional activation of P4501A1 and P4501A2 by polycyclic compounds. Furthermore, this inhibition can be blocked by raising cellular glutathione levels. By modulating P450 levels, cytokines could substantially influence carcinogen metabolism. Furthermore, cytokine levels can vary dramatically within different tissues and organs and also systemically between individuals, due to particular physiological states (acute or chronic inflammation or infection, cigarette smoking, diet, cytokine set point, immunoreactivity, etc.). There may also be genetic differences in some of these variables, such as cytokine set point and immunoreactivity. Our long term working hypothesis is that differences in cytokine levels or differences in the responsiveness of P450 genes to cytokines could differentially affect the expression of carcinogen-metabolizing P450s and, therefore, could be an important factor contributing to genetically and/or environmentally determined differences among individuals in their susceptibility to carcinogens. It is also possible that the suppression of P450 expression by cytokines represents an important adaptive response, that of reducing P450-mediating carcinogen metabolism at times of increased free radical generation. The specific hypotheses tested by this research are that cytokine-dependent suppression of P4501A1 and P4501A2 transcription is mediated by the interaction of specific proteins with cis-acting regulatory sequences and that cellular redox potential modulates responsiveness to cytokines. Specific objectives are as follows: 1. Determine the cis-acting sequences responsible for mediating cytokine suppression of P4501A1 and P4501A2 transcriptional activation. We will use DNA-mediated gene transfer of expression vectors into isolated rat hepatocytes to characterize these cis-acting regulatory sequences and the mobility shift DNA binding assay and DNA footprinting techniques to identify DNA binding proteins. 2. Determine the relationship between reactive oxygen intermediates/cellular redox potential and the cytokine-dependent suppression of P450 expression. Inducer-dependent P4501A1 and P4501A2 transcription and mRNA expression will be determined in hepatocytes treated with cytokines and/or reagents that modify levels of reactive oxygen intermediates or cellular redox potential. 3. Accomplishing the above research objectives will provide information crucial for the identification of physiological factors which participate in and/or modify this adaptive response. This is necessary for determining the nature and importance of this response and how it might contribute to inter-individual differences in susceptibility to chemically-induced cancer.