Crosslinking Action of Chlorpyrifos Oxon as Mechanism of Chronic Illness

背景：接触有机磷杀虫剂毒死蜱，会有 不能通过抑制乙酰胆碱酯酶来解释的慢性不良反应。 目标：长期目标是建立一种解释神经毒性的机制 长期低剂量接触有机磷农药。假设：这项建议中的假设是基于我们的质谱学观察结果，这些观察表明，毒死蜱处理过的蛋白质会产生 赖氨酸上的共价加合物和其中一些加合物发生交联化反应 与谷氨酸或天冬氨酸侧链形成异肽键。交联体 蛋白质在聚丙烯酰胺凝胶上以高分子量聚集体的形式出现。 科学文献将蛋白质聚集体与神经退行性变联系起来，提出 蛋白质聚集体抑制轴突运输。其结果是神经元缓慢丧失。 脊椎密度，神经元之间的连接丢失，以及临床症状。 方法：本提案旨在建立启动神经毒性的步骤。我们的计划是 用液相色谱-串联质谱仪显示交联物 蛋白质与功能丧失有关。功能测试是抑制神经突起 在培养的细胞中生长。交联蛋白的形成和清除速度 将会被测量。结果：预计质谱学分析将确定 毒死蜱对人神经母细胞瘤细胞神经细胞骨架蛋白的影响 奥克森。交联蛋白的存在被认为与抑制 轴突的生长，因此表明交联剂和损失之间的关系 功能。交联蛋白形成速率和清除速率的测定 预计将支持一种错误折叠的、交联的蛋白质积累的机制 在神经元中。累积的交联蛋白的结果是神经毒性。 创新：我们是第一个报道有机磷毒物具有交联性的公司 探员们。意义：拟议的工作预计将提供一种机制来解释 长期低剂量接触有机磷农药的神经毒性。未来 这项研究可能会建立在这项工作的基础上，以了解暴露于 各种化学物质。

Background: Exposure to the organophosphorus pesticide, chlorpyrifos, can have chronic adverse effects that are not explained by inhibition of acetylcholinesterase. Goal: The long-term objective is to establish a mechanism to explain neurotoxicity from chronic low dose exposure to organophosphorus pesticides. Hypothesis: The hypothesis developed in this proposal is based on our mass spectrometry observations which show that proteins treated with chlorpyrifos oxon make covalent adducts on lysine and some of these adducts undergo a crosslinking reaction with glutamic or aspartic acid side chains to form isopeptide bonds. The crosslinked proteins are visualized as high molecular weight aggregates on polyacrylamide gels. The scientific literature links protein aggregates to neurodegeneration by proposing that protein aggregates inhibit axonal transport. The consequence is slow loss of neuronal spine density, loss of connections between neurons, and clinical symptoms. Method: This proposal aims to establish the steps that initiate neurotoxicity. The plan is to use liquid chromatography-tandem mass spectrometry to show that crosslinked proteins correlate with loss of function. The functional test is inhibition of neurite outgrowth in cultured cells. The rate of formation and clearance of crosslinked proteins will be measured. Outcome: Mass spectrometry analysis is expected to identify crosslinks between neuronal cytoskeleton proteins in human neuroblastoma cells treated with chlorpyrifos oxon. The presence of crosslinked proteins is expected to correlate with inhibition of neurite outgrowth, thus showing a relationship between crosslinking and loss of function. Measurement of the rates of formation and clearance of crosslinked proteins is expected to support a mechanism where misfolded, crosslinked proteins accumulate in neurons. The consequence of accumulated crosslinked proteins is neurotoxicity. Innovation: We are the first to report that organophosphorus toxicants are crosslinking agents. Significance: The proposed work is expected to provide a mechanism to explain neurotoxicity from chronic low dose exposure to organophosphorus pesticides. Future studies may build on this work to understand neurotoxicity following exposure to a variety of chemicals.