6PPD impairs liver growth through inflammatory pathways: Insights from zebrafish and human cell models

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), a widespread tire-derived contaminant, has drawn increasing concern for its environmental persistence and toxicity. However, its specific effects on early liver development remain poorly understood. In this study, we investigated the hepatotoxicity of 6PPD using zebrafish larvae and human L02 hepatocyte models. A novel exposure strategy was employed, initiating 6PPD treatment at 48 h post-fertilization, after liver budding, to minimize interference from early-stage developmental defects. 6PPD exposure led to a marked reduction in liver size without obvious morphological abnormalities, alongside downregulation of hepatocyte marker genes. Importantly, liver growth gradually recovered after 6PPD removal, suggesting acute and reversible toxicity. Mechanistically, 6PPD induced DNA damage and apoptosis in hepatocytes, as evidenced by elevated γ-H2AX, baxa, and casp3a expression, while hepatocyte proliferation remained unaffected. Transcriptomic and qPCR analyses revealed activation of inflammatory pathways and increased macrophage infiltration. Co-treatment with the anti-inflammatory agent dexamethasone rescued liver size and reduced DNA damage, indicating inflammation as a key mediator of 6PPD-induced toxicity. Similarly, 6PPD exposure in human hepatocytes reduced viability and increased apoptotic markers, which were alleviated by dexamethasone. These results demonstrate that 6PPD causes acute, inflammation-mediated liver toxicity during embryogenesis, with conserved mechanisms across species.