Gut-brain axis dysfunction mediates neurotoxicity of embryonic p-phenylenediamine exposure in zebrafish.

P-phenylenediamine antioxidants (PPDs) are widely used in the rubber industry and are increasingly recognized as environmental contaminants, raising concerns about their potential risks to wildlife and human health. Our present study investigated the neurotoxicity of embryonic exposure to a sublethal concentration (0.05 mg/L) of three PPDs (CPPD, IPPD and 77PD) in zebrafish. Our results showed that PPDs impaired the fish growth and induced distinct behavioral alterations. Specifically, CPPD exposure induced hyperactivity, aggression and social deficit. IPPD caused hyperactivity and loose shoaling behavior. And 77PD led to social deficits and reduced social cohesion. Examination of head tissues indicated elevated oxidative stress markers, altered antioxidant enzyme activities, increased neurotransmitter levels and abnormality of neural markers (Gfap/Olig2) using transgenic zebrafish. Histopathological analyses revealed marked intestinal damage including villus structural distortion and reduced mucus secretion. Integrated head-transcriptome and gut-microbiome analyses elucidated compound-specific molecular mechanisms. CPPD and IPPD significantly affected ocular lens development and visual perception pathways, whereas CPPD and 77PD notably disrupted olfactory sensory signaling and G protein-coupled receptor pathways. 16S rDNA sequence indicated PPDs-induced intestinal flora disorder by changing the composition and structure of intestinal flora, where the decreased Firmicutes to Bacteroidetes ratio may account for the fish growth suppression. Dual-omics integration revealed regulatory interactions between brain metabolic genes and gut microbiota, corroborating elevated gut lysozyme activity and increased inflammatory markers (TGFβ, IL6). These gut disturbances were linked to head gene dysregulations (muc5.3, or109 and apip). Additional antibiotic metronidazole (MTZ) treatment alleviated PPDs-induced zebrafish behavioral changes and suppressed the gut-brain stress response indicators. Our findings demonstrate that embryonic PPD exposure induces neurotoxicity mediated through gut-brain axis disruption, providing critical insights into the ecological risks of PPD pollutants.