ESD links tire-derived 6PPD-quinone exposure to NASH-related hepatic injury: an integrative multi-omics and in vivo study

As concern over tire-derived environmental contaminants continues to grow, the antioxidant N-(1,3-dimethylbutyl)- N ′-phenyl- p -phenylenediamine (6PPD) and its oxidative transformation product, 6PPD-quinone (6PPDQ), have attracted increasing attention for their hepatotoxic potential. Despite accumulating evidence of 6PPDQ-induced liver injury, the molecular mechanisms linking this contaminant to non-alcoholic steatohepatitis (NASH) remain undefined. In this study, an integrated framework combining network toxicology, machine learning, and multi-omics analysis was employed to investigate the association between 6PPDQ exposure and NASH. Intersection of 1451 predicted 6PPDQ targets with NASH-related transcriptomic signatures yielded 52 overlapping candidate mediators enriched in oxidative stress, metabolic regulation, and inflammatory signaling pathways including ErbB, Notch, Hedgehog, Hippo, and TNF. Machine learning analysis across six algorithms identified five hub genes, EPHX2, ESD, FAS, SSB, and ZNF436, with individual AUC values ranging from 0.711 to 0.825. ESD was prioritized for focused analysis, and functional network analysis, eQTL-GWAS colocalization, immune infiltration profiling, single-cell RNA sequencing, and molecular docking together placed ESD within the lipid metabolic, apoptotic, and immunoregulatory circuits central to NASH pathogenesis. In vivo, male BALB/c mice ( n  = 6 per group) exposed intraperitoneally to 0.4 or 4 mg/kg 6PPDQ for 28 days developed histological liver injury, hepatic lipid accumulation, and α-SMA-positive profibrotic activation, accompanied by significantly increased hepatic inflammatory and profibrotic gene expression. Serum ALT and AST rose dose-dependently, and hepatic Esd was upregulated at both the mRNA and protein levels. These findings support a proposed ESD-centered adverse outcome pathway framework linking 6PPDQ exposure to NASH-related hepatic injury.