Organophosphorus pesticide bioactivation and detoxification in an invertebrate rapid screening model to study mechanisms of neurotoxicity - Objectives. Organophosphorus pesticides (OPs) have been implicated in the dramatic surge of neurodevelopmental problems among U.S. children. Acute OP poisoning is due to inhibition of acetylcholinesterase (AChE). Chronic exposure to environmentally relevant OP levels has been suggested to cause developmental neurotoxicity (DNT) via alterations to cholinergic and non-cholinergic targets necessary for nervous system development and function. We have developed a cost-effective behavioral high-throughput screening (HTS) method for DNT studies using the asexual freshwater planarian Dugesia japonica, and shown it to be well-suited for assessing OP neurotoxicity/DNT. We have also shown that these planarians have two genes that are evolutionary ancestors of mammalian cholinesterases, with similar OP inhibition kinetics. These planarians can metabolize OPs at all developmental stages and exhibit carboxylesterase (CE) and calcium- dependent phosphoric triester hydrolase (PTEH) activity. To extrapolate planarian neurotoxicity data to human neurotoxicity further characterization of the OP bioactivation and detoxification pathways are needed. The overarching goal of this research is to establish planarians as a model for OP DNT. The specific objective of this R21 Exploratory/Developmental grant is to determine the key proteins involved in OP metabolism to contextualize planarian OP screening data, especially in the context of OP mixtures. We will test the hypothesis that key enzymes for OP bioactivation and detoxification are conserved between humans and planarians through 2 aims. In aim 1, we will identify and characterize OP detoxification enzymes, CE and paraoxonase (PON). We have shown that CE and PTEH activity exists in planarians but have not yet identified the responsible genes. Using standard molecular biology tools, we will identify gene homologs, characterize expression profiles, and use RNA interference to verify functional significance. In aim 2, we will identify and characterize the key cytochrome (CYP) P450s responsible for OP bioactivation and detoxification in planarians. We have shown that OP bioactivation exists in planarians but do not know which CYPs are involved. Because there are many putative CYPs in the D. japonica transcriptome and we don’t know a priori which CYPs will be relevant for OP bioactivation, we will first identify relevant CYPs using a CYP inhibitor screen. We will expose planarians to each OP +/- inhibitor for 30 minutes and quantify cholinesterase activity using an Ellman assay. Once we have identified the CYP subfamilies involved in OP bioactivation, we will use the same strategy as in Aim 1 to identify planarian CYP homologs and test for their functional significance. Impact: The data generated in this project will provide a foundation for future mechanistic studies of OP DNT, especially in the context of OP mixtures, which have been vastly understudied. By characterizing the key machinery involved in planarian OP metabolism, we can contextualize planarian OP screening data. This research will create unique opportunities for undergraduates in toxicology research at Swarthmore College.
