Comparative mechanistic study of developmental neurotoxicity of organophosphorus pesticides

有机磷农药（OP）是一个化学性质多样的大类，是最常用的农药， 全球经济上重要的杀虫剂，约占最近使用的杀虫剂的40% 在美国，虽然法律的OP浓度对人类没有急性毒性，但研究表明，慢性产前 而婴儿接触可导致终生神经损伤和行为障碍。急性OP中毒 由于乙酰胆碱酯酶（AChE）的抑制，但是，尽管有几十年的OP研究， 仍然存在争议，是否以及如何亚急性OP暴露在监管水平，不显着抑制 乙酰胆碱酯酶损害发育中的人脑。这一争议的大部分是因为目标之间的联系 分子/途径和不利的健康结果在很大程度上是未知的，因为哺乳动物中的有机体筛选 产生这些连接的方法是非常缓慢和昂贵的。因此，迫切需要高- 通过非哺乳动物生物体筛选来填补这一知识空白。此外，人类经常 同时遇到几种OP，因为它们普遍存在且经常混合使用。因此， 了解不同OP单独和组合的毒性机制，以揭示可能的非- 累加毒性效应，并准确预测组合暴露对人类的毒性。总体 本研究的目的是揭示OP和OP混合物DNT的机制，为产前诊断提供信息， OP暴露指南。具体目的是在涡虫中使用比较OP HT筛查来测试 假设与OP DNT相关的表型差异是由于与不同的 分子靶点PI正在进行的R15在无性恋者中使用自动行为筛查的结果 淡水涡虫Dugesia japonica支持这一假说。亚慢性暴露于7种OP产生不同的 表型，不能解释的乙酰胆碱酯酶抑制水平。其中五个行动方案产生了强有力的 在亚致死浓度下再生的真涡虫的行为缺陷。拟议的工作建立在这些基础之上 结果，以确定这5个OP的作用模式。该假设将通过调查 使用分层筛选方法（目标1）和 使用全转录组RNA鉴定这些时期内哪些分子途径受到影响 测序（目标2）。该项目是创新的，因为它是第一个结合国家的研究， 艺术行为HT筛选，细胞/神经解剖学研究，和转录组学，以阐明机制 的DNT的多个OP和建立之间的连接中断的神经发育过程， 有机体不良后果。这种机械的理解将提供一个框架，以解开 多OP相互作用的机制在未来的研究。在真涡虫中发现的毒性途径可用于 指导哺乳动物系统中有针对性的机制研究，大大加快测试流程， 允许将这些结果转化为人类健康。

Organophosphorus pesticides (OPs), a large and chemically diverse class, are the most commonly used and economically important insecticides worldwide, accounting for approximately 40% of recently used insecticides in the U.S. While legal OP concentrations are not acutely toxic to humans, studies suggest that chronic prenatal and infant exposures can lead to life-long neurological damage and behavioral disorders. Acute OP poisoning due to inhibition of acetylcholinesterase (AChE) is well-understood. But, despite decades of OP research, it remains debated whether and how subacute OP exposure at regulated levels that do not significantly inhibit AChE damages the developing human brain. Much of this controversy is because connections between target molecules/pathways and adverse health outcomes are largely unknown since organismal screening in mammals that could yield these connections is extremely slow and expensive. Thus, there is a critical need for high- throughput (HT) non-mammalian organismal screening to fill this knowledge gap. Additionally, humans often encounter several OPs simultaneously, due to their ubiquity and frequent use in mixtures. Thus, it is imperative to understand the toxicity mechanisms of different OPs individually and in combinations to unravel possible non- additive toxic effects and to accurately predict toxicity of combinatorial exposure to humans. The overarching goal of this research is to reveal the mechanisms underlying DNT of OPs and OP mixtures, to inform prenatal OP exposure guidelines. The specific objective is to use comparative OP HT screening in planarians to test the hypothesis that phenotypic differences associated with OP DNT are due to interactions with different molecular targets. Findings from the PI’s ongoing R15 using automated behavioral screening in the asexual freshwater planarian Dugesia japonica support this hypothesis. Subchronic exposure to 7 OPs produced different phenotypes that could not be explained by levels of AChE inhibition. Five of these OPs produced robust behavioral defects in regenerating planarians at sublethal concentrations. The proposed work builds upon these results to determine the mode of action of these 5 OPs. The hypothesis will be tested by investigating the developmental periods most vulnerable to OP exposure using a tiered screening approach (Aim 1) and by identifying which molecular pathways within these periods are affected using whole transcriptome RNA sequencing (Aim 2). The proposed project is innovative, because it is the first study that combines state-of-the- art behavioral HT screening, cellular / neuroanatomical studies, and transcriptomics to unravel the mechanisms of DNT of multiple OPs and establish connections between disrupted neurodevelopmental processes and organismal adverse outcomes. This mechanistic understanding will provide a framework to unravel the mechanisms of multi-OP interactions in future studies. Toxicity pathways identified in planarians can be used to guide targeted mechanistic studies in mammalian systems, dramatically speeding up the testing pipeline and allowing for the translation of these results to human health.