Cytotoxicity and apoptosis induced by mixed mycotoxins (T-2 and HT-2 toxin) on primary hepatocytes of broilers in vitro. (April 2017)

• Record Type: Journal Article
• Authors: Yang, Lingchen; Tu, Di; Zhao, Zhiyong; Cui, Jun
• Is Part Of: Toxicon. Volume 129(2017)
• Page Start: 1
• Page End: 10
• Publication Date: 2017-04
• Subjects: T-2/HT-2 toxin -- Cytotoxicity -- Apoptosis -- Liver toxicity -- Hepatocytes; T-2 T-2 toxin -- HT-2 HT-2 toxin -- Bcl-2 B-cell lymphoma 2 -- Bax Bcl-2 Associated X protein -- MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide -- TUNEL Terminal transferase nick-end labeling -- ALT alanine aminotransferase -- AST aspartate aminotransferase -- PCR polymerase chain reaction -- DMEM Dulbecco's modified Eagle's medium -- D-HBSS D-Hank's balanced salt solution -- DMSO dimethyl sulfoxide -- FITC Fluorescein Isothiocyanate -- PI Propidium Iodide -- T-TBS Tris Buffered Saline−Tween -- S.E. standard error of measurement -- PVDF polyvinylidene fluoride -- T-TBS Tris Buffered Saline−Tween; Toxins -- Periodicals; Venom -- Periodicals; 615.9
• DOI: 10.1016/j.toxicon.2017.01.001
• Languages: English
• ISSNs: 0041-0101

Abstract

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 < 0.05) and markedly increased intercellular spaces, dead cells and ALT/AST activities. T-2/HT-2 treatment also significantly increased the number of apoptotic cells and the apoptotic ratio ( P < 0.05). T-2/HT-2 induced early stage apoptosis of the hepatocytes and levels of apoptosis-related mRNAs and proteins changed in a manner implicating them in the apoptotic process. These changes occurred from 0 to 24 h of T-2/HT-2 exposure. Expression of bax and caspase-7 mRNAs was significantly upregulated, in a time-dependent manner, during this period ( P < 0.05). Levels of mRNAs for caspase-3 and caspase-9 were increased from 0 to 12 h ( P < 0.05) and then decreased after 12 h ( P < 0.05). There were no significant effects on expression of bcl-2 mRNA ( 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 < 0.05). Overall, T-2/HT-2 induced cytotoxicity and apoptosis in hepatocytes. The resulting changes in mRNA and protein expression were shown that several apoptosis-related proteins were involved in the liver toxicity of these agents. Highlights: T-2 and HT-2 toxin caused cytotoxicity on primary hepatocytes of broilers in a dose-dependent manner. Mixture of T-2 and HT-2 caused apoptosis mainly in early stage. P53, Caspases-3, Caspases-7, Caspases-9 and Bax were all play key roles during apoptosis induced by mycotoxins.