Yang Lingchen, Tu Di, Zhao Zhiyong, Cui Jun
College of Veterinary Medicine, Hunan Agricultural University, No.1 Nongda Road, Furong District, Changsha, 410128, People's Republic of China.
College of Veterinary Medicine, Hunan Agricultural University, No.1 Nongda Road, Furong District, Changsha, 410128, People's Republic of China.
Toxicon. 2017 Apr;129:1-10. doi: 10.1016/j.toxicon.2017.01.001. Epub 2017 Jan 16.
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.
T-2毒素和HT-2毒素（T-2/HT-2）可诱导肉鸡肝细胞产生细胞毒性并引发凋亡。在本研究中，我们对用T-2/HT-2处理的肝细胞进行了细胞毒性作用、凋亡情况及其相关机制的分析。为检测细胞毒性，我们采用了3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）活力检测法、苏木精-伊红染色以及天冬氨酸转氨酶和丙氨酸转氨酶（ALT/AST）活性检测。我们通过使用末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）检测法的荧光显微镜观察来评估凋亡情况。接下来，用荧光标记的（异硫氰酸荧光素）FITC-Annexin V和碘化丙啶（PI）染色来评估肝细胞的凋亡率和凋亡阶段。最后，通过实时聚合酶链反应评估凋亡相关mRNA的表达水平，并用蛋白质免疫印迹法评估凋亡相关蛋白的表达水平。我们发现，用T-2/HT-2处理的细胞呈现出剂量依赖性的显著较低细胞活力（P<0.05），且细胞间隙、死细胞数量以及ALT/AST活性显著增加。T-2/HT-2处理还显著增加了凋亡细胞的数量和凋亡率（P<0.05）。T-2/HT-2诱导了肝细胞的早期凋亡，并使凋亡相关mRNA和蛋白水平发生变化，表明它们参与了凋亡过程。这些变化发生在T-2/HT-2暴露后的0至24小时内。在此期间，bax和caspase-7 mRNA的表达呈时间依赖性显著上调（P<0.05）。caspase-3和caspase-9的mRNA水平在0至12小时升高（P<0.05），然后在12小时后下降（P<0.05）。对bcl-2 mRNA的表达没有显著影响（P>0.05）。除bcl-2外，所有检测的凋亡相关蛋白的表达在0至24小时内呈时间依赖性显著增加（P<0.05）。总体而言，T-2/HT-2诱导了肝细胞的细胞毒性和凋亡。mRNA和蛋白表达的变化表明，几种凋亡相关蛋白参与了这些药物对肝脏的毒性作用。
