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: 10079484
• 负责人: OKSANA LOCKRIDGE
• 金额: $ 22.88万
• 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 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10079484
• 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：暴露于有机磷的农药，毒死rif虫可以有 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 慢性低剂量暴露于有机磷农药。假设：该提案中提出的假设是基于我们的质谱观察结果，该观察结果表明用毒死rif虫牛治疗的蛋白质 赖氨酸的共价加合物，其中一些加合物发生了交联反应 与谷氨酸或天冬氨酸侧链形成异肽键。交联 蛋白质可视化为聚丙烯酰胺凝胶上的高分子量聚集体。 科学文献将蛋白质聚集物与神经变性联系起来，提出 蛋白质聚集体抑制轴突运输。结果是神经元缓慢丧失 脊柱密度，神经元之间的连接丧失和临床症状。 方法：该提案旨在建立启动神经毒性的步骤。计划是 使用液相色谱串联质谱法以表明交联 蛋白质与功能丧失相关。功能测试是抑制神经突 培养细胞中的生长。交联蛋白的形成速率和清除率 将测量。结果：预计质谱分析将识别在之间的交联 用毒死rif虫治疗的人神经母细胞瘤细胞中的神经元细胞骨架蛋白 牛。预计的交联蛋白的存在将与抑制 神经突生长，因此显示了交联与丧失之间的关系 功能。测量形成速率和交联蛋白的清除率 预计将支持一种机制，该机制堆积错误，交联的蛋白质累积 在神经元中。累积的交联蛋白的结果是神经毒性。 创新：我们是第一个报告有机磷有毒物质正在交联的人 代理商。意义：预计拟议的工作将提供一种解释的机制 慢性低剂量暴露于有机磷农药的神经毒性。未来 研究可能以这项工作为基础，以了解暴露于 多种化学物质。