In search of certainty beyond the cholinergic system: a systematic review of developmental chlorpyrifos exposure and neurotransmitter systems disruption in preclinical models.

Chlorpyrifos (CPF) is one of the most widely used pesticides globally, despite being strictly regulated and banned in several developed countries. It remains in use across many developing and underdeveloped nations. While its primary mechanism of action is acetylcholinesterase inhibition, multiple preclinical and clinical studies have reported developmental and cognitive alterations at lower doses that do not trigger this mechanism. These effects are particularly concerning during early development, when the central nervous system is immature and more vulnerable. Although various alternative molecular targets have been proposed, growing attention has been given to neurotransmitter systems beyond the cholinergic pathway. However, empirical data is inconsistent, and no qualitative or quantitative reviews have provided a clear understanding of these mechanisms. This systematic review aims to address this gap, focusing on preclinical rodent studies. Using a rigorous methodology, 41 studies were included in the qualitative analysis, covering 126 outcomes related to the dopaminergic, serotonergic, GABAergic, glutamatergic, and endocannabinoid systems. Overall, the included studies showed a low level of methodological quality and a high risk of bias. Only a few molecular targets were systematically investigated. We introduce two new evaluative metrics-Weight of Evidence and Percentage of Convergence-to highlight five key findings. Notably, preweaning CPF exposure consistently reduced the activity of endocannabinoid-degrading enzymes (FAAH and MAGL), resulting in elevated endocannabinoid tone, particularly increased levels of AEA. Additionally, there is consistent support for CPF-induced serotonergic alterations, particularly upregulation of 5HT2 and 5HT1A receptors following neonatal exposure. Due to insufficient data convergence across laboratories, a meta-analysis was deemed inappropriate. In conclusion, while numerous molecules have been linked to low-dose developmental CPF exposure, only a limited number show consistent empirical support, and only under postnatal exposure conditions. Future research should investigate prenatal exposure effects more systematically and replicate postnatal findings across independent laboratories to strengthen the reliability of these promising results through robust, quantitative analyses.