Crosslinking Action of Chlorpyrifos Oxon as Mechanism of Chronic Illness

背景：接触有机磷农药毒死蜱， 不能用抑制乙酰胆碱酯酶来解释的慢性副作用。 目标：长期目标是建立一种机制来解释神经毒性， 长期低剂量接触有机磷农药。假设：在这个提议中提出的假设是基于我们的质谱观察，该观察表明，用毒死蜱处理过的蛋白质使 赖氨酸上的共价加合物，其中一些加合物发生交联反应 与谷氨酸或天冬氨酸侧链形成异肽键。交联 蛋白质在聚丙烯酰胺凝胶上显示为高分子量聚集体。 科学文献将蛋白质聚集体与神经变性联系起来，提出 蛋白质聚集体抑制轴突运输。结果是神经元的缓慢丧失 脊柱密度，神经元之间连接的丢失和临床症状。 方法：本提案旨在确定引发神经毒性的步骤。该计划是 使用液相色谱-串联质谱法显示交联的 与功能丧失相关的蛋白质。功能测试是神经突抑制 在培养的细胞中生长。交联蛋白的形成和清除速率 将被衡量。结果：预计质谱分析可识别 毒死蜱处理的人神经母细胞瘤细胞神经细胞骨架蛋白 Oxon。预期交联蛋白的存在与抑制细胞凋亡相关。 神经突生长，从而显示交联和损失之间的关系， 功能交联蛋白的形成和清除速率的测量 有望支持错误折叠的交联蛋白质积累的机制 在神经元中。累积的交联蛋白质的后果是神经毒性。 创新：我们是第一个报告有机磷毒物交联 剂.意义：拟议的工作预计将提供一种机制， 慢性低剂量接触有机磷农药的神经毒性。未来 研究可以建立在这项工作的基础上，以了解暴露于 各种化学品。

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.