The gut-liver axis and TLR4/NF-κB pathway mediate Cu/Cd-induced hepatotoxicity in largemouth bass

Heavy metal pollution, particularly copper (Cu) and cadmium (Cd), poses a serious threat to aquatic ecosystems due to its toxicity and bioaccumulation potential. This study investigated the effects of individual and combined exposure to Cu and Cd on largemouth bass, focusing on the gut-liver axis and the TLR4/NF-κB pathway in hepatotoxicity. A total of 480 size-matched fish were randomly assigned to four groups (Control, Cu, Cd, and Cu + Cd) and exposed to sublethal concentrations of CuSO₄ (9.275 mg/L) and CdCl₂·2.5H₂O (1.15285 mg/L) for 14 days, followed by physiological, molecular, and omics analyses. Results showed that both single and combined exposures disrupted intestinal structure and barrier function, accompanied by downregulation of tight junction genes. Oxidative stress responses were tissue-specific, with increased reactive oxygen species (ROS) and MDA levels in the intestine but decreased levels in the liver, along with suppressed antioxidant enzyme activities. Inflammatory responses were activated, as indicated by elevated cytokine levels and upregulation of TLR4/MyD88/NF-κB signaling in the liver. Microbiota analysis revealed that Cd exposure increased gut microbial diversity, whereas Cu + Cd co-exposure reduced α-diversity and altered microbial composition, with increased Proteobacteria and decreased beneficial taxa such as Bacteroidota and Firmicutes. Untargeted metabolomics showed that hepatic metabolic profiles were altered, mainly affecting lipid, energy, and immune-related pathways. Overall, combined Cu + Cd exposure exerted stronger toxic effects than single exposures, inducing more severe intestinal damage, microbial dysbiosis, and metabolic disturbances. These findings highlight the critical role of the gut-liver axis in mediating heavy metal toxicity.