ALCOHOL OXYGEN RADICAL INTERACTIONS AS DETECTED BY ESR

申请者摘要：目前人们对……的角色很感兴趣 植物体内活性自由基物种的氧化应激与乙醇生成 乙醇毒性的机理(S)。很难确定 CYP2EL、氧化应激和乙醇毒性之间的直接联系。我们的 实验室已经建立了一种结构性表达的HepG2细胞系 人类的细胞色素P4E1.乙醇或多不饱和脂肪酸(PUFA)有毒 作用于表达细胞色素P450的E9细胞，而不作用于对照细胞。毒性由 乙醇和多不饱和脂肪酸被抗氧化剂阻止。这些单元格似乎是一个 评价细胞色素P450依赖的反应性形成作用的有价值的模型 1-羟乙基(HER)等物种在肝脏毒性中的作用 乙醇。 具体目的I是为了研究她的作用，脂质和其他自由 乙醇和多不饱和脂肪酸对细胞毒性作用中的自由基表达 Cyp2EL。将使用的主要分析技术是电子自旋共振波谱。 计划进行研究，以研究CYP2EL形成HER的机制，以及 HER和脂质自由基在乙醇和乙醇毒性中的作用 多不饱和脂肪酸；她与包括加合物在内的细胞成分的相互作用 将评估与CYP2EL的形成；转录的激活 因子，核因子-kB，通过乙醇或细胞色素P450产生的HER和其他自由基物种 将会被确定。这些研究应该确定她的作品和 表达细胞色素P450_2的细胞中的脂质自由基及其作用 乙醇和多不饱和脂肪酸毒性中的自由基。 AIM II将表征依赖NADPH和NADH的HER，02-， 以及来自细胞系的微粒体的其他活性中间体 只表达一种人类P450亚型。乙醇或多不饱和脂肪酸对这些植物的毒性 将对细胞进行评估，以表明人类细胞色素P450 1是否具有独特的活性 激活乙醇到Her，形成自由基，并在促进 乙醇毒性是通过自由基、氧化应激型的机制实现的。 一氧化氮对细胞色素P450酶催化活性及HER和HER生成的影响 其他自由基将在AIM Ill中进行评估。如果NO抑制细胞色素P450，那就没有 可能被证明是一种有用的保护剂，可以抵御乙醇的毒性 由CYP2EL激活的其他毒素；这将被直接检测 和E9细胞。这些研究将提供有关 NO对细胞色素P450酶催化活性的调节能力 中间体、乙醇和多不饱和脂肪酸毒性。

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.