MOLECULAR MECHANISMS OF DIKETONE NEUROTOXICITY

各种工业和商业上可获得的化学品会导致 特殊的中毒性神经病，其特征是异常堆积 脆弱轴突内的神经丝。广泛使用的溶剂正己烷 和甲基丁基酮，它们造成了许多 人类职业性神经毒性疾病，在体内转化为 2，5-己二酮(2，5-HD)，一种被认为直接相互作用的伽马二酮 用轴突成分来诱导观察到的效应。这项建议是 旨在评估伽玛二酮与赖氨酸反应的假说 神经丝和其他轴突细胞骨架的Epsilon-氨基 蛋白质在体内形成疏水性的2，5-二烷基吡咯加合物，这 加合物的形成是诱发神经病变的关键事件，并且 由此导致的关键赖氨酸部分的损失和/或 蛋白质的疏水性导致特殊细胞骨架的破坏 轴突内蛋白质运输机制及随后的聚集 核因子蛋白。因为改变后的蛋白质持续存在于轴突内 无法到达神经末梢进行蛋白质分解，营养物质转移到 远端轴突中断，发生轴突变性。虽然 在非神经蛋白中形成吡咯加合物，清除 这种机制可以有效地去除改变的蛋白质，防止中毒。 为证明这一假设而设计的具体目标包括量化 大鼠神经和非神经组织蛋白中的吡咯加合物 长期经口接触2，5-HD及其理化特性 这些蛋白质内结合的变化和分子位置。其他内容 目的是检测体内蛋白质胺结合的非神经毒性 2，4-HD，该异构体对时间影响能力的评估 2，5-羟色胺神经病的病程及体外蛋白质胺检测 相关神经毒素二硫化碳、丙烯酰胺和 β，β‘-亚氨基二丙腈，长期目标是阐明一种 这些化合物的共同作用机制。分析技术 受聘人员将包括聚丙烯酰胺凝胶电泳法，定量 氨基酸分析和分析肽图谱用于鉴定 改变的蛋白质赖氨酸部分，以及高效液相色谱和沉淀物分析 用于评估溶解度变化和蛋白质聚集的蛋白质。 赖氨酸反应产物将通过质谱学进行表征，以及 组织中2，4-和2，5-HD的水平将通过GAS进行定量 层析法。结果将在以下方面取得实质性进展 阐明这些重要的分子作用机制 神经毒性化学物质。

A variety of industrial and commercially-available chemicals cause a specific toxic neuropathy characterized by abnormal accumulation of neurofilaments within vulnerable axons. The widely used solvents n-hexane and methyl butyl ketone, which have been responsible for many instances of human occupational neurotoxic disease, are converted in vivo to 2,5-hexanedione (2,5-HD), a Gamma-diketone believed to interact directly with axonal components to induce the observed effects. This proposal is designed to evaluate the hypothesis that Gamma-diketones react with lysine Epsilon-amino groups of neurofilament (NF) and other axonal cytoskeletal proteins in vivo to form hydrophobic 2,5-dialkylpyrrole adducts, that this adduct formation is the critical event for induction of neuropathy, and that the resultant loss of critical lysine moieties and/or changes in protein hydrophobicity causes disruption of the specialized cytoskeletal protein transport mechanism within the axon and subsequent aggregation of NF protein. Since the altered protein persists within the axon and is unable to reach the nerve terminal for proteolysis, nutrient transport into the distal axon is interrupted and axonal degeneration occurs. Although pyrrole adduct formation occurs in non-neural proteins, clearance mechanisms effectively remove altered protein, preventing toxicity. Specific aims designed to prove this hypothesis include quantitation of pyrrole adduct in neural and non-neural tissue protein from rats with prolonged oral exposure to 2,5-HD, and characterization of physico-chemical changes and molecular sites of binding within these proteins. Additional aims are examination of in vivo protein amine binding of non-neurotoxic 2,4-HD, assessment of the ability of this isomer to influence the time course of 2,5-HD neuropathy, and examination of the in vitro protein amine reactivity of the related neurotoxins carbon disulfide, acrylamide, and Beta,Beta'-iminodipropionitrile, with the long-range goal of elucidating a common mechanism of action for these compounds. Analytical techniques employed will include polyacrylamide gel electrophoresis, quantitative amino acid analysis, and analytical peptide mapping for identification of altered protein lysine moieties, and HPLC and sedimentation analysis of proteins for assessment of solubility changes and protein aggregation. Lysine reaction products will be characterized by mass spectroscopy, and tissue levels of 2,4- and 2,5-HD will be quantitated by gas chromatography. Results will provide substantial progress toward elucidation of the molecular mechanism of action of these important neurotoxic chemicals.