Crosslinking Action of Chlorpyrifos Oxon as Mechanism of Chronic Illness

毒死蜱的交联作用作为慢性疾病的机制

• 批准号: 9893217
• 负责人: OKSANA LOCKRIDGE
• 金额: $ 19.06万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-06 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893217
• 关键词: Acetylcholinesterase; Active Sites; Adverse effects; Algorithms; Antibodies; Aspartic Acid; Axonal Transport; Biological Assay; Brain; Cell physiology; Cells; Chemicals; Chlorpyrifos; Chronic; Chronic Disease; Clinical; Cognitive; Cultured Cells; Cytoskeleton; Digestion; Dose; Exposure to; Future; Glutamic Acid; Goals; Half-Life; Human; Impairment; Isotopes; Lead; Liquid Chromatography; Literature; Lysine; Mass Spectrum Analysis; Measurement; Measures; Mental Depression; Metabolic Clearance Rate; Methods; Modification; Molecular Weight; Nerve Degeneration; Neurites; Neuroblastoma; Neurons; Outcome; Peptides; Pesticides; Proteins; Reaction; Reporting; Role; Serine; Side; Symptoms; System; Testing; Time; Toxic effect; Trypsin; Tubulin; Vertebral column; Work; acute toxicity; adduct; base; beta Tubulin; crosslink; density; innovation; link protein; loss of function; microtubule-associated protein 1B; misfolded protein; nervous system disorder; neuroblastoma cell; neuron loss; neurotoxic; neurotoxicity; polyacrylamide gels; protein aggregation; protein crosslink; tandem mass spectrometry; toxicant

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.

背景：接触有机磷农药毒死蜱可能会导致 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机制来解释神经毒性 长期低剂量接触有机磷农药。假设：本提案中提出的假设基于我们的质谱观察结果，该观察结果表明，用毒死蜱处理过的蛋白质会产生 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。交联的 蛋白质在聚丙烯酰胺凝胶上表现为高分子量聚集体。 科学文献将蛋白质聚集体与神经退行性病变联系起来，提出： 蛋白质聚集体抑制轴突运输。其后果是神经元缓慢丧失 脊柱密度、神经元之间连接的丧失和临床症状。 方法：该提案旨在确定引发神经毒性的步骤。计划是 使用液相色谱-串联质谱法表明交联 蛋白质与功能丧失相关。功能测试是神经突抑制 培养细胞中的生长。交联蛋白的形成和清除率 将被测量。结果：质谱分析有望识别之间的交联 毒死蜱处理人神经母细胞瘤细胞中的神经元细胞骨架蛋白 奥克森。交联蛋白的存在预计与抑制相关 神经突生长，从而显示交联和丧失之间的关系 功能。测量交联蛋白的形成和清除率 预计将支持错误折叠、交联的蛋白质积累的机制 在神经元中。积累的交联蛋白的后果是神经毒性。 创新：我们率先报道有机磷毒物正在交联 代理。意义：拟议的工作有望提供一种机制来解释 长期低剂量接触有机磷农药造成的神经毒性。未来 研究可能会以这项工作为基础，以了解接触某种物质后的神经毒性。 各种化学品。