Mapping the molecular network of VX-induced neurotoxicity in male zebrafish through spatial metabolomics and...

VX is a persistent organophosphorus nerve agent with non-negligible environmental risks. Conventional homogenate-based omics approaches cannot resolve the spatial heterogeneity of toxin distribution or corresponding local molecular alterations. Spatially resolved mass spectrometry imaging overcomes this limitation by directly visualizing the spatial distribution of VX and its metabolites with dysregulated endogenous molecules within brain regions. In this study, male zebrafish were continuously exposed to three concentrations of VX (low = 19.36 μg/L, medium = 77.43 μg/L, high = 154.86 μg/L) for 96 h. Behavioral assays showed dose-dependent reductions in locomotor activity and exploratory behavior. Histopathological analysis revealed progressive brain lesions, and TUNEL staining demonstrated a clear dose-related increase in neuronal apoptosis. Correspondingly, brain acetylcholinesterase (AChE), superoxide-dismutase (SOD) and catalase (CAT) activities decreased markedly with increasing VX levels. DESI-MSI visualized dose-dependent accumulation and spatial distribution of VX within the brain. Integrated transcriptomic and spatial metabolomic analyses identified dysregulation of arachidonic-acid metabolism and ferroptosis as primary neurotoxic pathways. Importantly, this study establishes an integrative link from the spatial distribution of VX in the brain to local molecular disturbances and systemic toxicity, demonstrating that behavioral and histological deficits were accompanied by AChE inhibition, oxidative-stress-related changes, apoptosis, and alterations in lipid-metabolism and regulated-cell-death pathways. This work provides a novel multi-scale analytical framework for evaluating the ecological neurotoxicity and health risks of chemical warfare agents in aquatic ecosystems.