An integrated approach unveils novel mechanisms of nicotine-induced neurotoxicity in zebrafish larvae

Nicotine is a ubiquitous environmental pollutant which may exert neurotoxicity to aquatic organisms. However, the mechanisms at environmentally relevant concentrations remain inadequately understood. This study employed an integrated approach combining in silico predictions, in vivo zebrafish assays, transcriptomics and targeted metabolomics to decipher the mechanistic framework of nicotine-driven developmental neurotoxicity to zebrafish. Network toxicology and molecular docking identified six core targets (CXCR4, CHRNB1, CHRNA1, CHRNA3, STAT3, HIF1A) and predicted key pathways, including neuroactive ligand-receptor interaction and calcium signaling. Experimental validation in zebrafish larvae revealed that nicotine exposure, even at low environmental levels (0.03–3 μg/L), significantly induced developmental delays, heart rate imbalances, and neurobehavioral deficits. Targeted metabolomics demonstrated that nicotine caused a pronounced neurotransmitter imbalance, characterized by a sharp increase in acetylcholine and disrupted levels of dopamine, norepinephrine, and serotonin. Transcriptomics further confirmed the dysregulation of key pathways, including calcium signaling, MAPK inflammation cascade, and neuroactive ligand-receptor interaction. An integrated multi-omics analysis delineated a cohesive adverse outcome pathway: nicotine initially disrupts neuroactive ligand-receptor interactions, leading to intracellular calcium overload, which subsequently triggers MAPK-mediated inflammatory apoptosis and FoxO/p53-related oxidative stress, ultimately resulting in neuronal damage and behavioral dysfunction. Our findings provide novel and comprehensive insights into the mechanistic basis of nicotine-induced neurotoxicity, highlighting significant ecological risks at environmental concentrations and offering a robust framework for assessing the neurotoxic potential of environmental contaminants. • This study establishes a comprehensive research paradigm that spans in silico to in vivo and molecular to phenotypic levels. • Nicotine exposure compromises zebrafish larval development, behavior, and acetylcholine metabolism. • Network toxicology and transcriptomics reveal novel adverse outcome pathways of nicotine-induced neurotoxicity.