Bixafen-induced immunotoxicity in zebrafish: TLR4/NF-κB activation and DNA replication disruption as critical pathways

Comprehensive understanding the toxic effects of pesticide with relative long half-life properties are always hot research topics in the world. In this study, the immunotoxic effects of Bixafen (BIX), a prominent Succinate Dehydrogenase Inhibitor (SDHI) fungicide, were systematically assessed at concentrations of 0.05, 0.1, and 0.2 μM, using a zebrafish ( Danio rerio ) larval model. Neutral red staining and confocal microscopy quantification showed that BIX exposure reduced immune cell numbers in a concentration-dependent manner. qPCR and Western blot validation revealed upregulated pro-inflammatory factors ( IL-1β , IL-8 , TNF-α ) and TLR4 gene expression, while the lymphocyte marker gene rag1 was downregulated. Protein - level detection found significantly increased p-p65/p65 and TLR4 expression. Molecular docking experiments showed that BIX forms multiple interactions with the TLR4 protein. RNA-Seq analysis further confirmed that BIX interferes with DNA replication and metabolic pathways. Our results suggest that BIX may activate proinflammatory responses and disrupt immune cell homeostasis via the TLR4 signaling pathway, posing a potential immunotoxicity risk to non-target organisms. These findings provide insights into the molecular mechanisms of BIX-induced immunotoxicity and are critical for assessing the environmental safety and health risks associated with SDHI fungicides.