Role of gut microbiota dysbiosis in podophyllotoxin-induced neurotoxicity in zebrafish: linking purine metabolism to synaptic dysfunction

Podophyllotoxin (PPT) is a naturally occurring plant-derived insecticide, yet its potent cytotoxicity raises concerns regarding potential neurobehavioral toxicity in non-target organisms. However, the underlying mechanisms remain largely unclear. This study aims to investigate the neurotoxic effects of PPT in zebrafish. Three-day post-fertilization wild-type AB strain zebrafish were exposed to six concentration gradients of PPT (0.997–31.2 µg/mL). Based on the determined LC10 and MNLC values, four concentrations including 0.140, 0.420, 1.26, and 1.57 µg/mL were selected for intestinal-brain toxicity assessments and color preference tests were conducted. Histopathological damage in brain and intestinal tissues were evaluated through hematoxylin-eosin and acridine orange staining techniques. Subsequently, based on the maximum non-lethal concentration of 1.26 µg/mL, the mechanisms were further investigated using 16S rRNA sequencing, HM700 targeted metabolomics, and bulk RNA-seq, with key genes being validated via quantitative PCR. Correlation analysis was employed to elucidate the relationships among gut microbiota, differential metabolites, and gene expression. Our results showed that zebrafish exposed to various concentrations of PPT for 2 days exhibited significant intestinal and neurotoxic effects, including alterations in color preference behavior and structural damage in both the brain and intestinal tissues. 16S rRNA sequencing revealed reduced gut microbial diversity and increased levels of Acinetobacter and Perlucidibaca. Metabolomic profiling revealed that PPT disrupts purine metabolism, significantly reducing neuroprotective metabolites like adenosine and inosine, while increasing neurotoxic compounds such as quinolinic and oxalic acids. Transcriptomic analysis showed that genes associated with neuronal damage and inflammation were affected. qPCR confirmed that PPT exposure suppresses genes crucial for synaptic function (gad1b, drd2b) and upregulates genes related to the complement pathway (c3a.1, c1qc) and pro-inflammatory cytokines (IL-6, IL-1β, TNF-α). Correlation analysis linked changes in gut bacteria, Acinetobacter and Perlucidibaca, to altered purine metabolism and genes involved in synaptic and inflammatory pathways. These findings suggest that PPT-induced neurotoxicity is associated with alterations in gut microbial composition, dysregulation of purine metabolism, and changes in neuroinflammatory and synaptic-related pathways. This study highlights the potential association between microbiota-gut-brain axis disturbances and PPT-induced neurotoxicity and provides new insights for evaluating its environmental health risks.