| T-2toxin belongs to type A trichothecenes mainly produced by several fugal species, which commonly contaminates cereal-based foods worldwide. T-2toxin is most toxic trichothecene mycotoxin, and serious harm to human and anmials health. Toxic effect of T-2toxin is characterized by vomiting, leukopenia, hematopoietic system atrophy and immunosuppression reaction, inhibiting protein synthesis, and even may causing cancer. One of the main toxic target of T-2toxin is hematopoietic system, and it can cause lesions in animal bone marrow and spleens, depletion hematopoietic stem cells (CFUs), granulo-monocytic progenitors (CFU-GM), colony-forming unit erythroid (CFU-E), erythroblastic progenitors (BFU-E) in hematopoietic system, and decrease the counts of red blood cells and platelet. However, the research of hematopoietic system damage induced by trichothecenes not deeply, the molecular mechanism and signal transduction of T-2toxin inhibits hematopoietic stem cell differentiation is not clear. Therefore, K562cells, a human erythroleukemia cell line were chosed as the research object. The mechanism was researched by using microarray technology and2-dimensional electrophoresis (2-DE) technology. Discussed the molecular mechanism of T-2toxin on hematopoietic stem cells differentiate into erythroid cells, to clarify the potential toxicity mechanism of T-2toxin on hematopoiesis and provide an antitoxin for its detoxification.The effect of T-2toxin (5-50nM) on the proliferation of K562cells were tested by using MTT method and LDH release rate, incubated for24～72h. It was found that the activity of K562cells was suppressed, and a significant dose and time-dependent cell damage was observed.Flow cytometry was used to detect the numbers of erythroid differentiated cells of K562cells, which could specific bind to CD235-FITC antibody, to ensure the cytotoxicity of T-2toxin on erythroid differentiation of K562cells. It was found that the ratio of erythroid cells was significantly increased in the group dealed with sodium butyrate which was set as a negative control. And the differentiation ratio was5.36±0.59times of blank control at48h most significant. Low concentration (5nM) of T-2toxin can also induced differentiation of K562cell to erythroid cells. While high concentrations of T-2toxins (50nM) inhibited erythroid differentiation, and the differentiation ratio was0.23±0.03times of blank control in48h. T-2toxin (5～50nM) incubation with K562cells for48h,15nM T-2toxin could inhibit K562cell differentiation significantly. Therefore, in the subsequent experiment, the best incubated time was set at48h, and15nM T-2was chosed as the effect dose.The induce of oxygen free radical ROS release by T-2toxin (15nM) was detected by DCFH-DA fluorescent probe in K562cells for2,4,8,12h. Fluorescence probe marked cells were detected by flow cytometric, to ensure the ROS release induced by T-2toxin in K562cells. The results showed that T-2toxins could induce a significant time-dependent ROS released in K562cells. The ROS release of K562cells induced by T-2toxin for4h reach the highest point at2.25±0.10times of control. With time prolonged, ROS release reduced to normal level.Gene expression microarray detected K562cell mRNA expression change induced by T-2toxins (15nM), sodium butyrate (0.6mM) and hydrogen peroxide (1mM). As the results showed, compared with blank, T-2toxins group had1670significant changed genes, including653up regulated genes, in which583genes have been named and their function have been studied, and1017genes down regulated, in which398genes have been studied. Sodium butyrate treatment group had317significant genes, which included170genes have known names and function. The hydrogen peroxide treatment group had282significant genes, included116genes have known names and function. There were67significant genes were co-up regulated and56co-down regulated in T-2toxin and sodium butyrate. T-2toxin and hydrogen peroxide induced25significant genes up regulated and167down regulated. These significant genes were classified to9categories in terms of their function:nucleic acid transcription regulation, biosynthetic process, energy metabolism, cells process, cell transport, hematopoietic differentiation, cellular morphogenesis, cellular signal transduction and response to stress.Analysis of significant genes function, it was presumed that transferrin (TF), and hemoglobin synthesis might be the main molecular targets of T-2toxin on hematopoietic cell toxicity. Cell cycle related genes differentially expressed in a large number, such as cyclin B2, cyclin G1and cyclin G2, CDK2, ATM, and cyclin D1. T-2toxin induced K562cell cycle arrest detected by flow cytometry, the results showed that the high concentrations T-2toxin (15nM) could cause the cells cycle arrest in S phase. Cell proliferation was stopped, and the number of hematopoietic progenitor cells was reduced, which can affect the erythroid differentiation cells number, Cell proliferation was stopped, and the number of hematopoietic progenitor cells was reduced, which can affect the erythroid differentiation cells number, which may resulting in the decrease of the number of blood cells in peripheral blood system. T-2toxin also induced metabolism-related genes differentially expressed, interferesed normal biosynthetic and energy metabolism of K562cells, which may lead to the lack of energy and matters in cell proliferation and differentiation. Ultimately, it could reduced erythroid differentiation ability and lead to hematopoietic toxic. T-2toxin inhibited K.562cells erythroid differentiation through activation of MAPK and JAK-STAT signal transduction pathway. RT-PCR confirmed that the cDNA microarray results were reliable, the tendency of significant genes expressed the similar. Sensitivity of RT-PCR was higher than microarray.T-2toxin induced22differentially expressed proteins on K562cells identified by the two-dimensional electrophoresis (2-DE) and time of flight mass spectrometry (MALDI-TOF/TOF-MS) compared with control cells. These significant proteins includes7categories:genetic modification and repair proteins, cell transport protein, apoptosis-related proteins, molecular chaperones, signaling pathway kinase factors, intracellular oxidation reduction proteins and cells metabolism related proteins. Multiple energy metabolism enzyme expression levels changed, such as citrate synthase, NADPH dehydrogenase and glucosidase, suggested that T-2toxin affected the normal metabolic processes in K562cells. In addition, DNA-binding protein B, annexin A1, apoptosis related proteins, proteasome activator, the peroxiredoxins and other proteins played a important role in the inhibition of the erythroid differentiation induced by T-2toxin. Sodium butyrate and hydrogen peroxide groups had a lot of protein differential expressed on cell citric acid cycle and energy metabolism, such as acetyl Co A transferase,3-hydroxyacetyl-CoA dehydrogenase, malate dehydrogenase, NADPH, and pyruvate kinase. These differential expressed proteins indicated that the energy metabolism and substance metabolism involved in sodium butyrate induced erythroid differentiation and hydrogen peroxide inhibition to erythroid differentiation of hematopoietic cells. The results and their mechanisms were further discussed. The change at gene and protein level were consistent. HSP27protein expression detected by western blot, confirmed the results of2-DE.In summary, this research firstly found the transferrin and hemoglobin synthesis process were important molecular targets in the progress of hematopoietic cell differentiation when incubated with T-2toxin. JAK2-STAT5B played important role in T-2toxin toxicity on the hematopoietic system and K562cell cycle arrest. Cell metabolism related genes and protein levels changed were another important ways of T-2toxin-mediated toxicity on hematopoietic system. |
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