Rapid behavioral screening in the planarian Dugesia japonica is a biologically relevant system to study neurotoxicity of organophosphorus pesticides mixtures

Introduction Organophosphorus pesticides (OPs) are an economically important and chemically diverse class of compounds that share acetylcholinesterase (AChE) inhibition as their mode of action for acute toxicity. Chronic OP exposure at doses below significant AChE inhibition has been shown to cause neurotoxicity and developmental neurotoxicity in mammals, implying a role for other molecular targets. Because the large number of OPs are often used in mixtures and many require bioactivation, high throughput screening (HTS) methods with human-relevant xenobiotic metabolism are necessary to study how OPs interact and exert their toxic effects. Methods We screened 21 binary mixtures of 7 OPs (acephate, chlorpyrifos (CPF), diazinon, dichlorvos (DDVP), malathion, parathion, and profenofos) in equimolar ratios ranging from 0.1 to 10 µM in adult and regenerating Dugesia japonica planarians, which have metabolic competence for OPs. Using an automated screening platform with 22 readouts, we quantified effects on lethality, stickiness, and behavior on days 7 and 12 post-exposure. We compared experimental outcomes to predictions from a Loewe concentration addition model to evaluate whether the adverse outcomes of binary mixtures of OPs can be predicted based on their shared mode of action. To contextualize the behavioral results, we also tested four OP mixtures for AChE inhibition at day 12 post-exposure in regenerating planarians. Results The tested OP binary mixtures showed additive and non-additive interactions, including potentiation, synergism and antagonism, that differed across mixtures, readouts, and developmental stage (adult vs. regenerating planarians). Regarding AChE inhibition, CPF-parathion and CPF-malathion mixtures acted additively, while diazinon-DDVP showed antagonism, and diazinon-profenofos showed synergism. Some behavioral outcomes, such as stickiness, correlated with effects on AChE inhibition while others did not. Adult and regenerating planarians differed in the extent of non-additive OP interactions. Discussion The direct comparison of effects of OP mixtures on the adult vs. the developing nervous system allowed us to identify greater synergistic effects in regenerating planarians compared to adults, emphasizing the need to better understand the effects of OPs on the developing brain. Due to the conservation of key enzymes in OP toxicokinetics/toxicodynamics between planarians and humans, lessons learned from the planarian system are likely to be relevant to human health.