Developmental exposure to echimidine induces locomotor hyperactivity in zebrafish larvae via inhibiting acetylcholinesterase activity

Pyrrolizidine alkaloids (PAs) are natural toxins generated as secondary metabolites in plants, predominantly consisting of unsaturated PAs with diverse toxicities, such as hepatotoxicity. Echimidine, a prominent PA, is believed to exert various toxicological effects, including survival inhibition and induction of apoptosis of hepatocytes (Gleńsk et al., 2022). However, the effects of echimidine on development remain unclear. We selected three concentrations of 0.02, 0.2, and 2 mg/L to investigate the developmental toxicity of echimidine on zebrafish embryos. After a 7-day exposure, we observed hyperactivity and anxiety-like behavior in zebrafish larvae. Furthermore, we found that echimidine exposure significantly promoted embryonic motor neurodevelopment in genetically modified zebrafish. Next, we detected that echimidine exposure significantly increased the content of the excitatory neurotransmitter acetylcholine (ACh), accompanied by a significant decrease in acetylcholinesterase (AChE) activity. Conversely, echimidine led to a significant reduction in the content of the sedative neurotransmitter γ-aminobutyric acid (GABA), accompanied by abnormal gene expression of enzymes related to GABA synthesis. Moreover, we elucidated the strong direct binding of echimidine to zebrafish and human AChE protein through molecular docking. In summary, our study found that echimidine induced ACh accumulation possibly by inhibiting AChE activity, leading to motor neurodevelopmental abnormalities and hyperactivity in zebrafish larvae. This work provides important scientific knowledge on the effects and mechanisms of PAs on neural development, which is helpful for controlling the risk of PAs in food and protecting public health.