Lethal toxicity of metformin on zebrafish during early embryonic development by multi-omics analysis

Metformin is an antidiabetic drug used in type 2 diabetes as well as indicators in polycystic ovary syndrome (PCOS) and cancer. Due to their increase in popularity, high amounts of metformin are being released into aquatic environments. However, the toxic effect of metformin on embryonic development in aquatic organisms remains limited. Therefore, this study aimed to elucidate the lethal embryotoxicity of metformin and determine the underlying molecular pathways influencing embryonic development using a zebrafish model through multi-omics analysis. Metformin was microinjected into zebrafish embryos at the 1-cell stage with varying concentrations (50 mM, 100 mM, 200 mM, 400 mM, and 800 mM). From the results, hatching rates decreased in a dose dependent manner. Fetal malformation and mortality (LC₅₀ = 339.8 mM) increased in a dose dependent manner. In situ hybridization of whole-embryo assays demonstrated that metformin exerts a significant impact on the initial stages of embryonic development, leading to aberrant differentiation of the germ layers, perturbed organogenesis, and delayed development. Furthermore, transcriptomics, metabolomics, and lipidomics were used to study the molecular mechanisms of embryonic toxicity. The results showed that the cell cycle, dorsoventral axis formation, and collecting duct acid secretion pathways were significantly altered in treated embryos. In brief, these results provide useful information on the lethal toxicity mechanism of metformin overdose and provide clues for further studies in humans.