INTERACTION OF ETHANOL WITH HEPATIC MICROSOMES

阐明细胞色素P450IIEL在微粒体中的作用 乙醇氧化系统(MEOS)和提纯活性物质 从人的肝脏制备，我们现在建议测定 该同工酶与其他生物底物的亲和力 兴趣，如维甲酸、致癌物质和肝毒性物质 在一定程度上，使用了这种人形。我们计划将人类的特征 细胞色素P450IIEL激活细胞色素P450II的能力 结构不同的化学致癌物二甲基和二乙基- 亚硝胺、亚硝基吡咯烷和氯乙烯。我们也会 确定是否在诱导 乙醇特异形式的P450及其对细胞外信号转导的增强作用 各种溶剂(如四氯化碳)的肝脏毒性和 常用药物(如对乙酰氨基酚)和滥用物质 (例如可卡因)。毒性将由泄漏的 肝酶进入血流和通过形态变化 由光学和电子显微镜测定。微体诱导 将通过测定总细胞色素P450作为 以及各种同工酶的SDS-PAGE和 其中一些形式的免疫定量使用特定的 抗体。其他因素(如酮)在反应中的作用 肝微粒体的诱导及其增强作用 肝脏毒性也将被评估；这些后一项研究可能 提供了对相互作用问题的新见解 酒精中毒和营养不良。ADH的相对重要性， 肝脏总乙醇代谢的MEOS和过氧化氢酶将 在体内和在分离的肝细胞中进行定量，重点是 长期饮酒引起的变化。三 将采用的方法：将氚作为命运的放射性示踪剂 从乙醇、重氢乙醇中提取的还原当量 确定途径选择性的同位素效应和代谢 对缺乏ADH的鹿鼠品系的研究。 我们的主要目标是定义生物化学的差异 酗酒者和非酗酒者，并确定在多大程度上 乙醇消费所产生的变化在本质上 微粒体P450系统改变酒精者对酒精的反应 生理和药物底物的方式可以 最终影响酒精相关的病理，其预防和治疗 治疗。我们的最终目标是用分子术语定义一些 慢性乙醇带来的最深远的变化是什么 身体内部环境中的消耗以及由此产生的 对环境的反应。

Having-elucidated the role of cytochrome P450IIEl in the microsomal ethanol oxidizing system (MEOS) and having purified an active preparation from human liver, we now propose to determine the affinity of this isozyme for other substrates of biological interest such as retinoids, carcinogens and hepatotoxic agents using, in part, this human form. We plan to characterize the human cytochrome P450IIEl with respect to its ability to activate the structurally diverse chemical carcinogens dimethyl- and diethyl- nitrosamines, nitrosopyrolidine and vinylchloride. We also will determine whether a parallelism exists between the induction of the ethanol specific form of P450 and the potentiation of the hepatotoxicity of various solvents (e.g. carbon tetrachloride) and commonly used drugs (such as acetaminophen) and substances of abuse (e.g. cocaine). Toxicity will be determined by the leakage of liver enzymes into the bloodstream and by morphologic changes determined by light and electron microscopy. Microsomal induction will be evaluated by the determination of total cytochrome P450 as well as the visualization of the various isozymes by SDS-PAGE and the immunoquantitation of some of these forms using specific antibodies. The role of other factors (such as ketones) in the induction of liver microsomes and the potentiation of hepatotoxicity will also be assessed; these latter studies may provide new insight into the question of interactions between ethanol toxicity and malnutrition. The relative importance of ADH, MEOS and catalase for total hepatic ethanol metabolism will be quantitated in vivo and in isolated hepatocytes, focusing on changes resulting from chronic alcohol consumption. Three approaches will be employed: tritium as a radiotracer for the fate of reducing equivalents derived from ethanol, deuterated ethanol to determine pathway-selective isotope effects and metabolic studies with a deermouse strain lacking ADH. Our broad aim is to define biochemical differences between alcoholics and non-alcoholics and to determine to what extent the changes produced by ethanol consumption in the nature of the microsomal P450 system alter the response of the alcoholic to physiologic and pharmacologic substrates in a way which may eventually affect alcohol related pathology, its prevention and treatment. Our ultimate goal is to define in molecular terms some of the most far-reaching changes brought about by chronic ethanol consumption in the internal milieu of the body and the resulting response to the environment.