ALCOHOL-OXYGEN RADICAL INTERACTIONS AS DETECTED BY ESR

越来越多的证据表明，乙醇中毒可能与 提高了活性氧中间体的产量。在这些人中 乙醇产生氧化应激的机制 微粒体混合功能氧化酶系统和细胞色素的诱导 P450 2E1和乙醇衍生的NADH是特别感兴趣的。具体的 这项应用的目的是使用电子自旋共振(ESR) 光谱学来确定自由基的产生，如超氧化物， 羟基和碳中心自由基，如羟乙基 肝细胞细胞器，特别是微生物体，并评估其影响 乙醇处理对活性自由基产生的影响 中间体。ESR的主要优点是它提供了 明确、直接地测定自由基，是高度敏感的，并且 是检测HER等活性中间体的唯一方法。 将进行实验，比较NADH的有效性 与NADPH在促进自由基生成和相互作用中的作用 与多种铁络合物催化肝脏产生自由基 微生物体、线粒体、细胞核和胞浆等细胞器 膜。P450 2E1的作用将使用底物进行评估， 抑制剂和抗体。抗氧化剂和氧化还原的作用 环化剂，以及电子自旋共振结果与化学结果的比较 将会被检测到。慢性乙醇处理对血沉的影响 氧和羟基自由基及其产物的可检测率将 被评估。由于酒精中毒起源于静脉周围地带， 微生物体和其他细胞器将从门静脉周围分离出来 从对照组和乙醇处理的大鼠制备肝细胞，以及 由ESR确定的活性中间体的生产。要确定 微粒体酶在NADH和NADPH依赖中的作用 反应性自由基中间体的生成，与纯化的实验 NADH-b5还原酶、NADPH-P450还原酶、b5和P450(尤其是P450 2e1)，将进行选定的人体肝脏实验 微粒体与人肝P450 2E1.最终目标将是使用ESR来 评估甘油和其他多羟基醇的生产 激进分子。预计直接和具体的ESR研究将 提供有关反应性的产生和作用的新信息 酒精毒性中的自由基中间体。

There is increasing evidence that ethanol toxicity may be associated with elevated production of reactive oxygen intermediates. Among the mechanisms suggested by which ethanol produces oxidative stress, ethanol induction of the microsomal mixed-function oxidase system and cytochrome P450 2E1, and ethanol-derived NADH are of special interest. The specific objective of this application is to employ electron spin resonance (ESR) spectroscopy to determine production of radicals such as superoxide, hydroxyl and carbon center radicals such as the hydroxyethyl radical by liver cell organelles, particularly microsomes, and assess the influence of ethanol treatment on this production of reactive radical intermediates. The major advantages of ESR are that it provides unambiguous, direct determination of radicals, is highly sensitive, and is the only method to detect reactive intermediates such as HER. Experiments will be carried out which compare the effectiveness of NADH with that of NADPH in promoting radical generation and in interacting with a variety of iron complexes to catalyze radical generation by liver cell organelles such as microsomes, mitochondria, nuclei and plasma membranes. The role of P450 2E1 will be assessed using substrates, inhibitors and antibodies. The effects of anti-oxidants and redox cycling agents, and comparisons of results obtained by ESR and chemical detection, will be made. The effect of chronic ethanol treatment on ESR- detectable rates of oxygen and hydroxy radicals and HER production will be evaluated. Since ethanol toxicity originates in the perivenous zone, microsomes and other organelles will be isolated from periportal hepatocytes prepared from control and ethanol-treated rats, and production of reactive intermediates determined by ESR. To identify microsomal enzymes which play a role in the NADH-and NADPH-dependent generation of reactive radical intermediates, experiments with purified NADH-b5 reductase, NADPH-P450 reductase, b5 and P450 (especially P450 2E1) will be carried out, as will selected experiments with human liver microsomes and human liver P450 2E1. A final aim will be to use ESR to evaluate production of glycerol and other polyhydroxylated alcohol radicals. It is anticipated that direct and specific ESR studies will provide new information on the generation of, and the role of, reactive radical intermediates in alcohol toxicity.