MOLECULAR MECHANISMS OF DIKETONE NEUROTOXICITY

二酮神经毒性的分子机制

• 批准号: 3420199
• 负责人: ANTHONY P DECAPRIO
• 金额: $ 4.36万
• 依托单位: NEW YORK STATE DEPARTMENT OF HEALTH
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-05-01 至 1987-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3420199
• 关键词: affinity chromatography; aminoacid analyzer; axon reaction; chemical structure function; colorimetry; covalent bond; cytoskeleton; environmental toxicology; gas chromatography; gel electrophoresis; gel filtration chromatography; high performance liquid chromatography; lysine; mass spectrometry; molecular biology; molecular site; neurofilament; neuronal transport; neurotoxins; occupational hazard; pyrroles; toxicant interaction

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.

各种工业和商业上可获得的化学品引起 一种以异常蓄积为特征的特异性中毒性神经病 脆弱轴突中的神经丝 广泛使用的溶剂正己烷 和甲基丁基酮，这是造成许多情况下， 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γ-二酮，据信可直接与 与轴突成分，以诱导观察到的效果。 这项建议是 旨在评估γ-二酮与赖氨酸反应的假设 神经丝（NF）和其他轴突细胞骨架的ε-氨基 蛋白质在体内形成疏水的2，5-二烷基吡咯加合物，这 加合物形成是诱导神经病变的关键事件， 所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化细胞骨架的破坏 轴突内的蛋白质运输机制和随后的 NF蛋白。 由于改变的蛋白质存在于轴突内， 无法到达神经末梢进行蛋白水解，营养物质转运进入 远端轴突中断，并发生轴突变性。 虽然 吡咯加合物的形成发生在非神经蛋白，清除 机制有效地去除改变的蛋白质，防止毒性。 旨在证明这一假设的具体目标包括定量 吡咯加合物的神经和非神经组织蛋白质从大鼠 长期经口暴露于2，5-HD，并表征理化性质 这些蛋白质内的变化和分子结合位点。 额外 目的是检查体内蛋白胺结合的非神经毒性 2，4-HD，评估该异构体影响时间的能力 2，5-HD神经病的病程和体外蛋白胺的检查 相关神经毒素二硫化碳、丙烯酰胺和 β，β ′-亚氨基二丙腈，其长期目标是阐明 这些化合物的共同作用机制。 分析技术 采用的方法包括聚丙烯酰胺凝胶电泳、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HPLC和沉降分析， 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表征，并且 2，4-和2，5-HD的组织水平将通过气体定量 层析 结果将提供实质性进展， 阐明这些重要的分子作用机制 神经毒性化学物质