Cytotoxicity and apoptosis induced by mixed mycotoxins (T-2 and HT-2 toxin) on primary hepatocytes of broilers in vitro

Abstract T-2 and HT-2 (T-2/HT-2) induced cytotoxicity and apoptosis in hepatocytes from broilers. In this study, hepatocytes treated with T-2/HT-2 were analyzed for cytotoxic effects and apoptosis and for the associated mechanisms. To assay cytotoxicity, we used the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) viability assay, hematoxylin-eosin staining and aspartase transaminase and alanine transaminase (ALT/AST) activities. We evaluated apoptosis by fluorescence microscopy using the Terminal transferase nick-end labeling (TUNEL) assay. The apoptotic ratio and the apoptotic stage of the hepatocytes were next assessed with fluorescently labeled (FITC) Annexin V and propidium iodide (PI) staining. Finally, expression levels of apoptosis-related mRNAs were assessed by real-time PCR and those of apoptosis-related proteins by western blotting. We found that cells treated with T-2/HT-2 showed, in a dose dependent manner, significantly lower cell viabilities ( P P P P P P  > 0.05). Expression of all apoptosis-related proteins examined, except for bcl-2, was significantly increased from 0 to 24 h in a time-dependent manner ( P