ALCOHOL INDUCED P450S--EFFECTS ON TOXICITY & METABOLISM

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Long term goals of this proposal are: l) to understand interactions of ethanol, anesthetics and co-anesthetics at the P450 level in intact cells; 2) to understand the underlying mechanism by which human cytochrome P450 2El/3A4 and reactive oxygen intermediates (ROI) participate in the hepatotoxicity of ethanol, drugs and inhalational anesthetics; and 3) to design therapeutic interventions to prevent or ameliorate this toxicity by modulation of P450 activity or nutrition. Three specific objectives are derived from these goals and are addressed in this application which will utilize HepG2 cells that express CYP2EI (E-9 cells) and CYP3A4. Specific Aim (S. A.) I: To clone CYP3A4 into HepG2 and E-9 cells for the purpose of developing cell lines to serve as new models for the metabolism, interactions and toxicity of alcohol and drugs as mediated by CYP3A4 and/or CYP2EI in intact HepG2 cell lines. The working hypothesis is that this cloning produces stable cell lines that constitutively express 3A4 and that the expressed P450s exhibit known characteristics of this isoform. These cells will then be used to test the hypothesis that the membrane of an intact cell will modulate substrate metabolism, by acting as a selective barrier that can either impede or facilitate the availability of substrate (or inhibitors). S.A. ll: To characterize the metabolism and toxicity of halothane by the HepG2 cell lines. The working hypothesis is that the metabolism of inhalationaI anesthetics by these cell lines (E-9 cells, CYP3A4 cells and E-9/CYP3A4 cells) can be manipulated to reflect different in vivo conditions such that this model will enable one to ascertain which factors are important in toxicity. S. A. M: To characterize the generation of ROl by HepG2 cell lines. We will test the hypothesis that CYP2EI and/or CYP3A4 contribute to the generation of ROI by HepG2 cell lines or microsomes isolated from these cell lines, and that subsequent toxicity is modulated by the oxygen tension, the presence of xenobiotics and antioxidants in the environment.