ALCOHOL OXYGEN RADICAL INTERACTIONS AS DETECTED BY ESR

申请人摘要：目前， 氧化应激和乙醇生成的反应性自由基物种在 乙醇有毒的机制。 很难确定 CYP 2 E1、氧化应激和乙醇毒性之间的直接联系。 我们 一个实验室已经建立了HepG 2细胞系，其组成型表达 人CYP 2 E1。 乙醇或多不饱和脂肪酸（PUFA）是有毒的 对表达CYP 2 E1的E9细胞，但对对照细胞不敏感。 毒性 乙醇和多不饱和脂肪酸被抗氧化剂阻止。 这些细胞似乎是 有价值的模型，以评估CYP 2 E1依赖的反应性形成的作用， 如1-羟乙基自由基（HER）的肝毒性作用 乙醇。 特定目的I旨在研究HER、脂质和其他游离 自由基在乙醇和多不饱和脂肪酸对表达 CYP2E1。 使用的主要分析技术是ESR光谱法。 计划研究CYP 2 E1形成HER的机制， HER和脂质自由基在乙醇毒性中的作用， HER与包括加合物在内的细胞成分的相互作用 将评价与CYP 2 E1的形成;转录的激活 因子，NF-κ B，通过乙醇或CYP 2 E1衍生的HER和其他自由基物质 将被确定。 这些研究应确定HER的产生， 脂质自由基在细胞表达CYP 2 E1和评估这些作用 自由基的毒性由乙醇和多不饱和脂肪酸。 目的II将表征HER，02-， OH和其他活性中间体， 仅表达一种人P450同种型。 乙醇或多不饱和脂肪酸对这些 将评估细胞，以指示人CYP 2 E1是否在细胞中具有独特的反应性。 激活乙醇到HER，形成自由基，并促进 乙醇毒性通过自由基，氧化应激类型的机制。 NO对CYP 2 E1催化活性和HER产生的影响， 其它自由基将在Aim III中评价。如果NO抑制CYP 2 E1，则NO 可以证明其可用作对抗乙醇毒性的保护剂， 由CYP 2 E1激活的其他毒素;这将直接测定 E9细胞。 这些研究将提供有关 NO调节CYP 2 E1催化活性的能力，产生反应性 中间体，以及乙醇和PUFA毒性。

APPLICANT'S ABSTRACT: There is much current interest in the role of oxidative stress and ethanol generation of reactive radical species in the mechanism(s) by which ethanol is toxic. It has been difficult to establish direct linkage between CYP2El, oxidative stress, and ethanol toxicity. Our laboratory has established a HepG2 cell line which constitutively expresses the human CYP2El. Ethanol or a polyunsaturated fatty acid (PUFA) was toxic to the E9 cells which express CYP2El, but not to control cells. Toxicity by ethanol and PUFA was prevented by antioxidants. These cells appear to be a valuable model to assess the role of CYP2El-dependent formation of reactive species such as the 1-hydroxyethyl radical (HER) in the hepatotoxic actions of ethanol. SPECIFIC Aim I is designed to study the role of HER, lipid, and other free radicals in the toxic actions of ethanol and PUFA to cells expressing CYP2El. The major analytical technique to be used is ESR spectroscopy. Studies are planned to study the mechanism of HER formation by CYP2El, and the role of HER and lipid radicals in the toxicity exerted by ethanol and PUFA; the interaction of HER with cellular constituents including adduct formation with CYP2El will be evaluated; activation of the transcription factor, NF-kB, by ethanol or CYP2El derived HER and other radical species will be determined. These studies should identify production of HER and lipid radicals in cells expressing CYP2El and assess the role of these radicals in the toxicity by ethanol and PUFA. Aim II will characterize NADPH- and NADH-dependent formation of HER, 02-, OH, and other reactive intermediates by microsomes from cell lines which express only one human P450 isoform. Toxicity by ethanol or PUFA to these cells will be evaluated, to indicate if human CYP2El is uniquely reactive in activating ethanol to HER, in formation of free radicals, and in promoting ethanol toxicity via a free radical, oxidative stress type of mechanism. The effect of NO on CYP2El catalytic activity and generation of HER and other free radicals will be evaluated in Aim Ill. If NO inhibits CYP2El, NO may prove to be useful as a protectant against the toxicity of ethanol and other toxins which are activated by CYP2El; this will be directly determined with the E9 cells. These studies will provide new information on the ability of NO to modulate CYP2El catalytic activity, generation of reactive intermediates, and ethanol and PUFA toxicity.