Reversal of neurodevelopmental toxicity induced by combined exposure to the neonicotinoid insecticides acetamiprid and imidacloprid in zebrafish through targeting DRP-1: insights into mitophagy and apoptosis mechanisms.

Neonicotinoid pesticides, including acetamiprid (ACE) and imidacloprid (IMI), are widely used due to their high efficacy and broad-spectrum activity. Despite IMI prohibition in certain countries by 2023, its residual effects, particularly in conjunction with ACE, which is frequently detected at elevated concentrations in rivers and surface waters, remain inadequately explored. In this study, the combined neurodevelopmental toxicity of ACE and IMI in zebrafish was investigated, with emphasis on mitochondrial dynamics and potential intervention strategies. We developed a 120-h acute exposure model using zebrafish embryos and a long-term chronic exposure model involving six months of environmentally relevant doses in adult zebrafish. Various parameters were systematically assessed using these models, including general toxicity, larval behavioral characteristics, outcomes of the novel tank diving test and light-dark test in adults, neurodevelopmental status, associated gene expression levels, and mitochondrial function. Furthermore, exposure experiments were conducted using transgenic zebrafish larvae Tg (huc:eGFP) and Tg (hb9:eGFP) to elucidate specific effects on the nervous system. Human neuroblastoma SK-N-SH cells were used to evaluate apoptosis, oxidative stress, ATP levels, mitochondrial membrane potential, and calcium ion concentrations. Protein markers associated with apoptosis (Bax, BCL-2, and Cleaved Caspase-3) and mitophagy (LC3A/B, P62, Parkin, and Pink-1) were analyzed. Additionally, DRP-1, Bax, and CytC levels were quantified in both mitochondrial and cytoplasmic fractions. The DRP-1 inhibitor Mdivi-1 was used to substantiate the role of mitochondrial dynamics. The results revealed a synergistic neurotoxic effect resulting from the combined exposure to ACE and IMI, which was characterized by impaired neural development, behavioral abnormalities, and mitochondrial dysfunction. Treatment with Mdivi-1 ameliorated these effects, reducing neurotoxicity in zebrafish. This study elucidates the synergistic neurodevelopmental toxicity of ACE and IMI, underscores the pivotal role of mitochondrial pathways, and provides insights into potential mitigation strategies for neonicotinoid-induced neurotoxicity.