High-throughput neurotoxicity study of neonicotinoids in C. elegans: Oxidative stress and serotonergic neuronal damage as key mechanisms

Neonicotinoids (NEOs) have been widely used as effective pesticides for decades, yet their adverse effects on non-target organisms remain a growing concern. Although the neurotoxicity of certain NEOs has been established, the potential neurotoxicity of the remaining large fraction of NEOs, as well as their underlying mechanisms, are yet to be clarified. In the present study, we employed a high-throughput toxicity screening platform to evaluate the neurotoxic potential of eight commonly used neonicotinoid insecticides in Caenorhabditis elegans. Our results revealed that acetamiprid (ACE), dinotefuran (DIN), clothianidin (CLO) and thiacloprid (THI) significantly impaired locomotion behavior in worms. Upon examining the potential mechanism of action, we found that ACE, CLO and THI could induce serotonergic neuronal damage, while cholinergic, dopaminergic and GABAergic neurons remained unaffected. Additionally, these three NEOs reduced bend frequency by predominantly enhancing oxidative stress, which was partially mitigated by the antioxidant N-acetyl-L-cysteine. In the neurotransmitter system, ACE mainly upregulated the mRNA expression of synthesis- and transport-related genes of neurotransmitters, while THI increased transport-related gene expression and decreased GABAergic receptor gene expression. In contrast, DIN elevated total acetylcholine levels without significantly altering gene expression, suggesting a distinct mechanism. In summary, our findings highlight that ACE, CLO and THI exert neurotoxicity through oxidative stress and serotonergic neuronal damage, while DIN primarily disrupts the neurotransmitter system. Our study provides insights into the high-throughput screening of NEO-induced neurotoxicity, offering a critical foundation for future in-depth studies to fully elucidate the toxic mechanisms of NEOs and to assess their environmental and health risks.