Exposure to trace levels of metals and fluoroquinolones increases inflammation and tumorigenesis risk of zebrafish embryos

Exposure to trace-level heavy metals and antibiotics may elicit metabolic disorder, alter protein expression, and then induce pathological changes in zebrafish embryos, despite negligible physiological and developmental toxicity. This study investigated the single and combined developmental toxicity of fluoroquinolones (enrofloxacin [ENR] and ciprofloxacin [CIP]) (≤0.5 μM) and heavy metals (Cu and Cd) (≤0.5 μM) to zebrafish embryos, and molecular responses of zebrafish larvae upon exposure to the single pollutant (0.2 μM) or a binary metal-fluoroquinolone mixture (0.2 μM). In all single and mixture exposure groups, no developmental toxicity was observed, but oxidative stress, inflammation, and lipid depletion were found in zebrafish embryos, which was more severe in the mixture exposure groups than in the single exposure groups, probably due to increased metal bioaccumulation in the presence of ENR or CIP. Metabolomics analysis revealed the up-regulation of amino acids and down-regulation of fatty acids, corresponding to an active response to oxidative stress and the occurrence of inflammation. The up-regulation of antioxidase and immune proteins revealed by proteomics analysis further confirmed the occurrence of oxidative stress and inflammation. Furthermore, the KEGG pathway enrichment analysis showed a significant disturbance of pathways related to immunity and tumor, indicating the potential risk of tumorigenesis in zebrafish larvae. The findings provide molecular-level insights into the adverse effects of heavy metals and antibiotics (especially in chemical mixtures) on zebrafish embryos, and highlight the potential ecotoxicological risks of trace-level heavy metals and antibiotics in the environment.