LC-MS untargeted metabolomics reveals metabolic disturbance and ferroptosis in MWCNTs-induced hepatotoxicity of Cyprinus carpio

In recent years, there is a great concern about the potential adverse effects of carbon nanotubes (CNTs) on the aquatic systems due to their increasingly extensive application. In this study, juvenile Cyprinus carpio were exposed to multi-walled CNTs (MWCNTs) at concentrations of 0, 0.25, and 2.5 mg L^-1 for 28 days. Then, oxidative stress indicators and metabolite profile of the livers were assessed. Results showed the significant increase of malondialdehyde (MDA) content and decrease of glutathione (GSH) activities in fish treated with 2.5 mg L^-1 MWCNTs. LC-MS untargeted metabolomics demonstrated that 406 and 274 metabolites in fish treated with 2.5 mg L^-1 MWCNTs were significantly up- and down-regulated, respectively. KEGG functional annotation analysis showed the disturbance of amino acid metabolism, lipid metabolism, and nucleotide metabolism. In addition, ferroptosis signaling pathway was detected. Therefore, iron content analysis and quantitative real-time RT-PCR assay were performed furtherly to validate the contribution of ferroptosis to MWCNTs-induced hepatotoxicity. The iron content increased significantly and the mRNA levels of ferroptosis-related genes including STEAP3, ACSL4, NCOA4, TFR1, NRF2, SLC3A2, SLC7A11, GPX4, and FPN1 were also obviously changed. Taken together, our study suggested that MWCNTs exposure-induced ferroptosis were associated with iron overload and lipid peroxidation via NRF2/SLC7A11/GSH/GPX4 axis. Our findings provide essential information to understand the mechanism of CNTs-induced hepatotoxicity in fish and explore potential biomarkers.