Metabolomics analysis of the 3D L-02 cell cultures revealing the key role of metabolism of amino acids in ameliorating hepatotoxicity of perfluorooctanoic acid

To simulate the real cell status and morphology in the living systems is substantial for using cell models to address the detrimental effects of toxic contaminants. In this study, the comparative profiles of metabolites in three-dimensional (3D) human normal liver (L-02) cell spheroids with perfluorooctanoic acid (PFOA) treatment were analyzed using a metabolomic approach. The uniform 3D cell spheroids were well formed in 3 days (e.g., sphericity index >0.9) and stably maintained over the subsequent 11 days. The cytotoxicity of PFOA to the 3D L-02 cell spheroids was highly dependent on both exposure concentration and duration. Comparative analysis of metabolomes showed that the number of differential metabolites in the 3D cell spheroids treated with 300 μM PFOA for 10 days (n = 59) was greater than those with a 4-day exposure to 300 μM PFOA (n = 17). Six metabolic pathways related to amino acids metabolism were only found in the 3D cell spheroids with a 10-day treatment of 300 μM PFOA, which could not be found in the 2D monolayer cells and those 3D cell spheroids with a 4-day exposure. The suppression of PFOA on glutamine metabolism substantially decreased glutathione (GSH) production and accordingly increased the level of reactive oxygen species in the 3D cell spheroids. On the contrary, the supplementation of glutamine increased GSH production and the viability of cell spheroids, indicating that glutamine metabolism played a critical role in the chronic toxic effects of PFOA. Our study strongly suggested that comprehensive toxicological methodologies based on the 3D cell models could currently be robust and suitable for addressing the chronic adverse effects of toxic contaminants.