Organophosphate insecticides disturb neuronal network development and function via non-AChE mediated mechanisms

Exposure to organophosphate (OP) insecticides has been related to several adverse health effects, including neurotoxicity. The primary insecticidal mode of action of OP insecticides relies on (irreversible) binding to acetylcholine esterase (AChE), with -oxon metabolites having a much higher potency for AChE inhibition than the parent compounds. However, OP insecticides can also have non-AChE-mediated effects, including changes in gene expression, neuroendocrine effects, disruption of neurite outgrowth and disturbance of the intracellular calcium (Ca²⁺) homeostasis. Since Ca²⁺ is involved in neurotransmission and neuronal development, our research aimed to assess the effects of two widely used OP insecticides, chlorpyrifos (CPF) and diazinon (DZ) and their respective -oxon metabolites, on intracellular Ca²⁺ homeostasis in human SH-SY5Y cells and rat primary cortical cultures. Furthermore, we assessed the acute and chronic effects of exposure to these compounds on neuronal network maturation and function in rat primary cortical cultures using microelectrode array (MEA) recordings. While inhibition of AChE appears to be the primary mode of action of oxon-metabolites, our data indicate that both parent OP insecticides (CPF and DZ) inhibit depolarization-evoked Ca²⁺ influx and neuronal activity at concentrations far below their sensitivity for AChE inhibition, indicating that inhibition of voltage-gated calcium channels is a common mode of action of OP insecticides. Notably, parent compounds were more potent than their oxon metabolites, with exposure to diazinon-oxon (DZO) having no effect on both neuronal activity and Ca²⁺ influx. Human SH-SY5Y cells were more sensitive to OP-induced inhibition of depolarization-evoked Ca²⁺ influx than rat cortical cells. Acute exposure to OP insecticides had more potent effects on neuronal activity than on Ca²⁺ influx, suggesting that neuronal activity parameters are especially sensitive to OP exposure. Interestingly, the effects of DZ and chlorpyrifos-oxon (CPO) on neuronal activity lessened after 48 h of exposure, while the potency of CPF did not differ over time. This suggests that neurotoxicity after exposure to different OPs has different effects over time and occurs at levels that are close to human exposure levels. In line with these results, chronic exposure to CPF during 10 days impaired neuronal network development, illustrating the need to investigate possible links between early-life OP exposure and neurodevelopmental disorders in children and highlighting the importance of non-AChE mediated mechanisms of neurotoxicity after OP exposure.