| T-2 toxin is a monoterminal mycoalkene compound produced by Fusarium sporotrichioides and F.poae under specific conditions.The natural occurrence of T-2 toxin is widely,and long-term intake of grains and feedstuffs contaminated by T-2 toxin will damage the health of animals and human beings,leading to anorexia,stunted growth and production performance of livestock and poultry,etc and causing human bone marrow and blood toxicity.However,there is no enough evidence about the main toxicity characteristics of T-2 toxin to animal acute injury,target organs and the molecular mechanism of T-2 toxin's toxic effect.Therefore,this paper which was financially supported by the Special Key Project of Biosafety Technologies for the National Major Research and Development Program of China(2017YFC1200901)mainly focuses on the toxicity of T-2 toxin from the following three aspects:one is to develop an acute toxicity evaluation model of T-2 toxin on animal injury,the second is to develop a cell evaluation model of T-2 toxin on kidney injury and its preliminary mechanism,and the third is to develop a cell evaluation model of T-2 toxin-induced immunotoxicity and its preliminary mechanism.The specific research contents and results are as follows:1.Female Wistar rats aged 7-8 weeks were selected according to the acute oral toxicity:up and down procedure(UDP)recommended by the Organization for Economic Cooperation and Development to develop an acute toxicity evaluation model of T-2 toxin on animal.According to the method of UDP,17.5,5.5 and 1.75 mg/kg·bw were selected as the toxic doses.The results showed that the LD50 of T-2 toxin to female Wistar rats was 2.957 mg/kg·bw with the 95%confidence interval of 1.75-5.5 mg/kg·bw.A total of 8 rats were used in the experiment.All rats except the control group showed toxic characteristic after exposure T-2 toxin for 3 hours,which accompanied by not eating or drinking water,vertical hair,closed eye.Rats showed hematuria,amorphous feces,and died after exposure 11 hours at the dose of 17.5 mg/kg·bw.Rats showed limp weakness,staggering gait,and finally paralysed,and died after exposure of 30 hours with the characteristic of cyan color in mouth,nose and tail at the dose of 5.5 mg/kg·bw dose.The symptoms of rats in the 1.75 mg/kg·bw dose group were relieved and improved after 24 hours of exposure,and they could eat and drink normally.The body weight of the experimental rats all decreased compared with control.Histopathological examination showed that heart,liver,spleen,lung,kidney,stomach,thymus,ovary and uterus were all damaged to varying degrees,among which kidney,spleen and thymus were the main target organs for the acute toxicity of T-2 toxin.2.Based on the acute toxicity evaluation model of T-2 toxin,human tubular epithelial cell HK-2 was used to develop a cell evaluation model of T-2 toxin on kidney injury.HK-2 cells cultured in vitro were treated with different concentrations of T-2 toxin for 72 h,CCK-8,Hoechst 33258 fluorescence staining,agarose gel electrophoresis,flow cytometry and spectrophotometry were used to detect the effects of T-2 toxin on the proliferation inhibition rate,apoptosis morphology,DNA fragmentation,apoptosis rate,cell cycle distribution,reactive oxygen species(ROS),mitochondrial membrane potential(MMP)and caspase-3 activity of HK-2 cells,respectively.The results showed that T-2 toxin had proliferative toxicity on HK-2 cells,and could obviously inhibit the proliferation of HK-2 cells in the concentration range of 20-250 nmol/L,and the inhibition rate increased as the concentration increase.The IC50 of T-2 toxin on HK-2 cells was 49.34 nmol/L.Hoechst fluorescence staining showed that the morphology of apoptotic cells such as chromatin condensation appeared in HK-2 cells after 20 nmol/L T-2 toxin treating.Flow cytometry showed that T-2 toxin induced the early apoptosis of HK-2 cells in a positive dose-dependent manner in the range of 10-40 nmol/L and affected the cell cycle distribution by causing G2/M phase arrest.In addition,T-2 toxin stimulated the increase of caspase-3 activity on HK-2 cells.Compared with the control group,caspase-3 activity increased to 1.93 times at 40 nmol/L(p<0.05).T-2 toxin caused the increase of ROS production.Compared with the control group,the ROS content increased significantly after 20 and 30 nmol/L T-2 toxin stimulating(P<0.05).T-2 toxin also led to a decrease in MMP.Compared with the control group,40 nmol/L T-2 toxin led to a significant decrease in MMP(P<0.05).In summary,T-2 toxin may activate caspase-3 through mitochondrial pathway to induce apoptosis.3.Based on the acute toxicity evaluation model of T-2 toxin,the human tissue cell lymphoma cell U937 was used to develop a cell evaluation model for T-2-toxin-induced immunotoxicity.U937 cells cultured in vitro were treated with different concentrations of T-2 toxin for 72 h,CCK-8,agarose gel electrophoresis,flow cytometry and spectrophotometry were used to detect the effects of T-2 toxin on the proliferation inhibition rate,DNA fragmentation,apoptosis rate,cell cycle distribution,ROS,MMP and caspase-3 activity of U937 cells,respectively.The results showed that T-2 toxin had proliferative toxicity on U937 cells,and could obviously inhibit the proliferation of U937 cells in the concentration range of 2.0-20 nmol/L,and the inhibition rate increased as the concentration increase.The IC50 of T-2 toxin on U937 cells was 4.31 nmol/L.Flow cytometry showed that T-2 toxin hardly caused cell apoptosis in the range of 1-4 nmol/L,but the proportion of total apoptotic cells increased significantly at 8.0 nmol/L,and the proportion of apoptotic cells reached 75.04%(P<0.05).T-2 toxin affected the cell cycle distribution by causing G0/G1 phase arrest in the range of 4-8 nmol/L.In addition,T-2 toxin stimulated the increase of caspase-3 activity on U937 cells,which increased to 2.57 times at 4 nmol/L compared with control(P<0.05).T-2 toxin had an effect on the production of ROS in U937 cells.Compared with the control group,the ROS was not significantly increased in the range of 1-4 nmol/L,while the ROS at 8.0 nmnol/L significantly decreased(p<0.05).T-2 toxin also led to a decrease in MMP.Compared with the control group,T-2 toxin could significantly reduce MMP in U937 cells at 8 nmol/L(p<0.05).In summary,T-2 toxin may activate caspase-3 through mitochondrial pathway to induce apoptosis. |
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