ALCOHOL-OXYGEN RADICAL INTERACTIONS AS DETECTED BY ESR

越来越多的证据表明，乙醇毒性可能与 活性氧中间体的产生增加。 中 乙醇产生氧化应激的机制， 诱导微粒体混合功能氧化酶系统和细胞色素 P450 2E1和乙醇衍生的NADH是特别感兴趣的。 具体 本申请的目的是采用电子自旋共振（ESR）， 光谱学以确定自由基如超氧化物的产生， 羟基和碳中心基团如羟乙基， 肝细胞器，特别是微粒体，并评估影响 乙醇处理对这种反应性自由基的产生 中间体的 ESR的主要优点是它提供了 明确，直接测定自由基，是高度敏感的， 是检测HER等活性中间体的唯一方法。 将进行比较NADH的有效性的实验 与NADPH在促进自由基生成和相互作用中的作用 与各种铁络合物一起催化肝脏产生自由基 细胞器如微粒体、线粒体、细胞核和血浆 膜。 将使用底物评估P450 2E1的作用， 抑制剂和抗体。 抗氧化剂和氧化还原的影响 循环剂，并比较ESR和化学方法获得的结果 将进行检测。 慢性酒精治疗对ESR的影响- 可检测到的氧和羟基自由基和HER产生率将 被评价。 由于乙醇毒性起源于静脉周围区， 将从门静脉周围分离微粒体和其他细胞器 从对照和乙醇处理大鼠制备的肝细胞，和 通过ESR测定的活性中间体的产生。 以识别 在NADH和NADPH依赖性 反应性自由基中间体的产生，用纯化的 NADH-b5还原酶、NADPH-P450还原酶、b5和P450（尤其是P450 2E1）将进行，如将选择人类肝脏的实验 微粒体和人肝P450 2E1。 最后一个目标将是使用ESR， 评价甘油和其他多羟基化醇的生产 根的 预计直接和具体的ESR研究将 提供关于反应性物质的产生和作用的新信息， 酒精毒性的自由基中间体。

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.