Acute Copper Exposure Induces Immunotoxicity and Metabolic Reprogramming in Rainbow Trout (Oncorhynchus mykiss) Muscle via FoxO/mTOR/PPAR Pathways

Abstract

Copper sulfate (CuSO4) is widely used in aquaculture for disease prevention and control, which poses significant potential risks to fish health and welfare. This study investigated the response mechanisms of immune-related molecules in the muscle tissue of rainbow trout (Oncorhynchus mykiss) following acute sublethal copper concentrations and during the recovery period. Rainbow trout were exposed to nominal Cu2+ concentrations of 0, 75, 150, and 300 μg/L for 4 d, followed by a 2 day depuration period. Integrated biochemical assays, transcriptomics and metabolomics were applied to muscle tissue, focusing on immune relevant pathways. Copper exposure significantly inhibited acetylcholinesterase and Ca2+-ATPase activities, indicating neurotoxicity, and induced oxidative stress, as evidenced by altered antioxidant enzyme activities and increased lipid peroxidation. It is worth noting that, during the stress induction process, the pro-inflammatory cytokine TNF-α and genes associated with lipid metabolism (Srebp1, FAS) were continuously up-regulated, and remained slightly higher than in the control group even during the recovery period, leading to the accumulation of saturated fatty acids and inflammatory signaling; this indicates persistent dysregulation of transcription. Muscle copper content paradoxically decreased while total protein increased, suggesting active metal efflux and metabolic reprogramming. Integrated multi-omics identified MYH4 and SCD1 as key nodes, and KEGG and O2PLS analyses showed that copper-induced immunotoxicity was associated with coordinated dysregulation of genes enriched in FoxO, mTOR and PPAR signaling pathways—central regulators of inflammation, protein homeostasis and lipid metabolism. These pathway-level findings are correlative and require causal validation. These findings suggest that short-term exposure to acute sublethal copper concentrations is associated with persistent immunotoxic transcriptional alterations in rainbow trout muscle, characterized by sustained inflammatory signaling and metabolic disruption during the short recovery period. The identified pathways and targets (TNF α, MYH4, SCD1, FoxO/mTOR/PPAR) provide candidate biomarkers for assessing copper-induced immunotoxicity and contribute to understanding the immune risks of copper pollution in aquatic ecosystems.