ALCOHOL INDUCED P450S--EFFECTS ON TOXICITY & METABOLISM

这项建议的长期目标是：L)了解 乙醇、麻醉剂和复合麻醉剂在完整细胞中的P450水平； 2)了解人类 细胞色素P450 2EL/3A4与活性氧中间体 参与酒精、药物和吸入性药物的肝毒性 麻醉药；以及3)设计治疗干预措施，以防止或 通过调节P450的活性或营养来减轻这种毒性。 从这些目标派生出三个具体目标，分别是 在本申请中提出，该申请将利用 表达细胞色素P450 2EI(E-9细胞)和细胞色素P3A4。 特定目标(S.A.)I：将CYP3A4克隆到HepG2和E-9细胞中 为了开发细胞系作为新的细胞模型 酒精和药物的代谢、相互作用和毒性 通过CYP3A4和/或CYP2EI在完整的HepG2细胞系中表达。在工作中 假设是这种克隆产生了稳定的细胞系 结构性表达3A4，并且表达的P450展示已知 这种异构体的特征。然后，这些电池将被用于测试 认为完整细胞的膜会调节底物的假说 新陈代谢，通过充当选择性屏障，既可以阻碍或 促进底物(或抑制剂)的可用性。 研究氟烷的代谢和毒性。 HepG2细胞系。工作假说是新陈代谢 这些细胞系(E-9细胞、CYP3A4细胞)对吸入麻醉药的抑制作用 和E-9/CYP3A4细胞)可以被操纵以在体内反映不同的 该模型将使人们能够确定哪一个 影响毒性的因素很重要。 S.A.M：研究HepG2细胞系产生rol的特性。 我们将检验这一假设，即CYP2EI和/或CYP3A4对 HepG2细胞系或分离的微粒体产生感兴趣区 从这些细胞系中分离出来，随后的毒性是由 氧分压，外源物质和抗氧化剂的存在 环境。

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.