Integrated Cytotoxicity and Metabolomics Analysis Reveals Cell-Type-Specific Responses to Co-Exposure of T-2 and HT-2 Toxins.

T-2 toxin and HT-2 toxin are commonly found in agricultural products and animal feed, posing serious effects to both humans and animals. This study employed combination index (CI) modeling and metabolomics to assess the combined cytotoxic effects of T-2 and HT-2 on four porcine cell types: intestinal porcine epithelial cells (IPEC-J2), porcine Leydig cells (PLCs), porcine ear fibroblasts (PEFs), and porcine hepatocytes (PHs). Cell viability assays revealed a dose-dependent reduction in viability across all cell lines, with relative sensitivities in the order: IPEC-J2 > PLCs > PEFs > PHs. Synergistic cytotoxicity was observed at low concentrations, while antagonistic interactions emerged at higher doses. Untargeted metabolomic profiling identified consistent and significant metabolic perturbations in four different porcine cell lines under co-exposure conditions. Notably, combined treatment with T-2 and HT-2 resulted in a uniform downregulation of LysoPC (22:6), LysoPC (20:5), and LysoPC (20:4), implicating disruption of membrane phospholipid integrity. Additionally, glycerophospholipid metabolism was the most significantly affected pathway across all cell lines. Ether lipid metabolism was markedly altered in PLCs and PEFs, whereas PHs displayed a unique metabolic response characterized by dysregulation of tryptophan metabolism. This study identified markers of synergistic toxicity and common alterations in metabolic pathways across four homologous porcine cell types under the combined exposure to T-2 and HT-2 toxins. These findings enhance the current understanding of the molecular mechanisms underlying mycotoxin-induced the synergistic toxicity.