Comparative mechanistic study of developmental neurotoxicity of organophosphorus pesticides

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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.