| T-2 toxin is a cytotoxic fungal metabolite that belongs to the trichothecene mycotoxin family. It is produced by various species of Fusarium, which can infect corn, wheat, barley, and rice crops in the field or during storage, and are characteristically stable under changing environmental conditions. T-2 toxin has been shown to cause a variety of toxic effects in experimental animals, livestock, and humans. Oral, parenteral, and cutaneous exposure to T-2 toxin induces lesions in the brain, hematopoietic, lymphoid, and gastrointestinal tissues, and suppresses reproductive organ functions. T-2 toxin has been paid more attention in nowaday society, however, there isn’t an overall project to risk assessment of T-2 toxin for the deficiency of inspection standard and measurement. Moreover, the effect of T-2 toxin on embryonic development in vitro remains unclear, which in that T-2 toxin-induced immediate maternal toxicity or fetal toxicities caused by passing through the placenta. Therefore, it is important to evaluate T-2 toxin embryotoxicity and investigate the underlying mechanism.The murine ES cells are used in an in vitro embryo-toxicity test known as the embryonic stem cell test (EST) for developmental toxicity, which has been approved by the European Centre for the Validation of Alternative Methods (ECVAM). In the present study, EST model was established to evaluate T-2 toxin embryo-toxicity and to investigate the underlying mechanisms by the model. In the study, the mitochondrial toxicity of T-2 toxin was focused, including the immediate mitochondrial toxicity and the inhibition of mitochondrial biogenesis and function in differentiated ES cells by T-2 toxin.1. In the present study, two permanent cell lines, embryonic stem cells (ES cells D3) and fibroblast 3T3 cells, were used to establish the EST model, and two chemicals, Penicillin G and 5-Fluorouracil, were used to confirm the accuracy and availability of the model. Penicillin G was evaluated as a non-embryo-toxic chemical and 5-Fluorouracil was a strong embryo-toxic chemical, which are consistent with reports. It confirm the prediction of chemicals’embryotoxicity by the model established in the present stuady was accurate.2. Using the EST model, T-2 toxin was evaluated as a strong embryo-toxic chemical. The endpoints were detected, including 50% inhibition of cell growth in ES cells (IC50D3) was 1.48ng/ml and 50% inhibition of differentiation of ES cells into cardiac myoblasts (ID50D3) was 0.57 ng/ml.3. According the IC50D3 was 1.48ng/ml, differentiated mouse ES cells were cultivated as embryoid bodies along with T-2 toxin at different concentrations (0.5,1, and 2 ng/ml) for 24 hours. The balance of redox in ES cells was broken by dose-dependence. Increases in cellular reactive oxygen species (ROS), lipid oxidative damage were observed at 1 and 2 ng/ml concentrations by spectrophotometry. And DNA oxidative damage was also detected by comet assay.4. Furthermore, the mitochondrial permeability transition pores open was detected by spectrophotometry, the release of cytochrome c from mitochondria to cytoplasm was detected by Weatern blot, and loss of mitochondrial transmembrane potential were observed at 1 and 2 ng/ml concentrations.5. Flow cytometry showed that T-2 toxin induced cell cycle arrest and apoptosis at a dose dependent relation. Moreover, the the upregulation of gene and protein expression of p53, caspase-9, caspase-3 and the ratio of Bax/Bcl-2 were detected by RT-PCR and western blot. It suggested that T-2 toxin opened the mitochondrial permeability transition pore, caused the release of cytochrome c from mitochondria and induced the upregulation of p53, caspase-9, caspase-3 expression and increased the ratio of Bax/Bcl-2. However, T-2 toxin-induced oxidative damage and apoptosis in differentiated ES cells decreased significantly in the presence of the antioxidant Trolox. Taken together, these results demonstrate that T-2 toxin induces oxidative stress and apoptosis in differentiated murine ES cells, and ROS-mediated mitochondrial pathway plays an important role in T-2 toxin induced apoptosis.6. ES cells can be cultivated as cellular aggregates, also known as embryoid bodies (EBs), and differentiate spontaneously into different cell types of all three germ layers in vitro. As a significant marker of cardiac myoblast, the expression of MHC gene represented the ability of ES cells differentiation in cardiac myoblast. In the present study, ES cells shown the differention ability by the gene expression a-MHC, and the protein expression of a-actinin, the markers of cardiac myoblastsa-actinine. According 50%inhibition of differentiation of ES cells into cardiac myoblasts (ID50D3) was 0.57ng/ml, differentiated mouse ES cells were exposured to 0.5ng/ml T-2 toxin for 24 hours (1 day),72hours (3 days) and 120 hours (5 days). However, the ES cells differention ability was inhibited, which shown by the decreas of bio-marker proteins expression, such as PPAR-y and a-actinin in a long-term treatment of T-2 toxin.7. In a long-term exposure to T-2 toxin (3 and 5 days), with the excessive accumulation of ROS, the oxidative damage was detected by MDA, and the ability of mitochondrial oxidative phosphorylationrespiration was suppressed, such as mitochondrial respiratory rate and the ability of ATP-ase were inhibited. At the same time, the defect of mitochondria number and construction were observed by fluorescence images and transmission electron microscope photo.8. Futhermore, the decrease in the expression of mitochondria biogenesis signal factors,such as phosphorylation p38MAPK, PGC-la, NRF-1, mtTFA, and COXIV genes and proteins were detected by real-time PCR and western blot. However, the decrease of mitochondria biogenesis could been reversed partly by antioxidant Trolox. However, T-2 toxin caused ES cells oxidative damage, inhibited mitochondrial function and biogenesis recovered significantly in the presence of the antioxidant Trolox.In summary, T-2 toxin was evaluated as a strong embryo-toxicity by using the EST model established in the present study. Our study also demonstrate that mitochondria plays an important role in the embryotoxicity mechanism of T-2 toxin. The study can provide experimental data and scientific advice to prevente the harm of T-2 toxin and promote the usage EST in the drug developmental toxicity evaluation. |
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