Multi-omics insights into 6PPD- and 6PPDQ-induced gut-liver axis disruption and non-alcoholic fatty liver disease progression in zebrafish (Danio rerio)

The pervasive environmental presence of N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) and its transformation product, 6PPD-quinone (6PPDQ), has raised concerns about their potential toxicity, yet their interactions with the gut microbiota at environmentally relevant concentrations remain poorly understood. Here, we investigated the effects of 6PPD and 6PPDQ on the gut-liver axis in zebrafish ( Danio rerio ). Zebrafish larvae exposed to 0.01, 1, and 100 μg/L of 6PPD or 6PPDQ for five days exhibited intestinal and hepatic developmental toxicity, including hepatic lipid accumulation and hepatomegaly. Adult zebrafish exposed for 21 days displayed compromised intestinal barrier integrity, gut dysbiosis, and lipidomic disturbances in the liver. Statistical analysis using the multi-response permutation procedure confirmed significant shifts in gut microbial community structure. Dysbiosis was characterized by reduced beneficial bacteria and an increase in pathogenic taxa, accompanied by elevated circulating lipopolysaccharide (LPS) and upregulated hepatic expression of lbp (LPS-binding receptor). Hepatic lipid accumulation resulted from increased triglyceride (TG) and total cholesterol synthesis, with lipidomics revealing distinct disruptions: 6PPD impaired phosphatidylinositol phosphate synthesis, while 6PPDQ affected TG homeostasis. Correlation analysis linked gut microbial shifts to hepatic lipid dysregulation. These findings suggest that 6PPD and 6PPDQ exposure disrupts gut-liver axis homeostasis, potentially driving non-alcoholic fatty liver disease development. This study underscores the need to integrate gut-liver-microbiota endpoints into environmental risk assessments for aquatic organisms.