Abstract

Copper oxide nanoparticles (CuO NPs), with their superior catalytic activity, antimicrobial performance and photoelectric properties, are widely used in production and daily life, thereby increasing their environmental release risk. Compared with the well-studied effects of ionic copper, the chronic toxicity of CuO NPs remains largely overlooked. In this study, zebrafish were exposed to environmentally relevant concentrations (5 mg/L Cu) of CuO NPs or copper sulfate (CuSO 4 ) to systematically evaluate intestinal toxicity induced by long-term exposure to different forms of copper. An integrated strategy incorporating ultrastructural analysis, biochemical assays, gut microbiota profiling, and metabolomic analysis was employed. Both copper forms induced mitochondrial damage, metabolic perturbations, and gut microbial dysbiosis in intestinal epithelial cells. However, CuO NPs showed markedly stronger intestinal toxicity than ionic copper. Specifically, CuO NPs can directly penetrate mitochondria in particulate form, impair mitochondrial structure, and potently activate the FDX1-LIAS-DLAT cuproptosis pathway, thereby triggering significant disturbances in amino acid, lipid, and nucleotide metabolism. Furthermore, CuO NPs exerted a more robust impact on the bidirectional gut microbiota-host metabolite interactions. In conclusion, this study clarifies form-dependent differences in copper-induced intestinal toxicity and highlights the critical role of microbiota-metabolite crosstalk in CuO NP-mediated adverse effects. These findings yield critical mechanistic insights and provide a robust scientific basis for evaluating the potential health risks posed by chronic exposure to CuO NPs.