Azithromycin induces neurotoxicity in zebrafish by interfering with the VEGF/Notch signaling pathway

Azithromycin (AZM) is a widely used antibiotic in both human and veterinary medicine, and its use has significantly increased during the COVID-19 pandemic. However, potential adverse effects of AZM on aquatic organisms have not been well studied. In this study, we explored the neurotoxicity of AZM in zebrafish and delved into its underlying mechanisms. Our results showed that AZM exposure resulted in a spectrum of detrimental effects in zebrafish, encompassing abnormal behaviors, damaged neuronal development, aberrant lateral line nervous system development, vascular malformations and perturbed expression of genes related to neural development. Moreover, we observed a concentration-dependent exacerbation of these neurotoxic manifestations with increasing AZM concentrations. Notably, AZM induced excessive cell apoptosis and oxidative stress damage. In addition, alterations in the expression levels of the genes involved in the VEGF/Notch signaling pathway were evident in AZM-exposed zebrafish. Consequently, we hypothesize that AZM may induce neurotoxicity by influencing the VEGF/Notch signaling pathway. To validate this hypothesis, we introduced a VEGF signaling inhibitor, axitinib, and a Notch signaling agonist, valproic acid, alongside AZM exposure. Remarkably, the administration of these rescue compounds significantly mitigated the neurotoxic effects induced by AZM. This dual verification provides compelling evidence that AZM indeed induces neurotoxicity during the early developmental stages of zebrafish, primarily through its interference with the VEGF/Notch pathway. Innovatively, our study reveals the molecular mechanism of AZM-induced neurotoxicity from the perspective of the close connection between blood vessels and nervous system. These findings provide new insights into the potential mechanisms underlying the neurotoxic effect of antibiotics and highlight the need for further investigation into the ecotoxicological effects of antibiotics on aquatic organisms and the potential risks to human health.