| T-2toxin belongs to a large group of trichothecene mycotoxins produced by various fusarium molds. T-2toxin is the most toxic of trichothecene mycotoxin. T-2toxin is harmful toward animal health and livestock production. T-2toxin is a main mycotoxin to cause vomiting and diarrhea at high dietary concentrations, while lower doses and long exposure induce growth inhibition, severe damage in endocrine system and immune system. One of the main toxic targets of T-2toxin is pituitary system, previous laboratory research had shown that T-2toxin inhibited synthesis and secretion of growth hormone in rat pituitary GH3cells. However, T-2toxin induced growth inhibition was not deeply, the molecular mechanism and signal transduction of inhibition synthesis and secretion of growth hormone in rat pituitary GH3cells is not clear. Today, genomic and proteomic technologies have become the effective methods for the study of mechanism of drug actions and targets. Therefore, rat pituitary adenoma GH3cells were used as an vitro model, after exposing GH3cells to low (10nM) and moderate dose (40nM) T-2toxin for12h, gene expression profiles were analysed by microarray, protein expression profiles were analysed by2-dimensional difference gel electrophoresis (2-D DIGE) combined with MALDI-TOF MS/MS, to investigate the growth retardant mechanism of T-2toxin based on gene mRNA expression and protein expression levels. The study of mechanism of T-2toxin inhibited synthesis and secretion of growth hormone would provide more comprehensive information for toxicology research in line with international and national standards.1. Toxin selection and toxin treatment concentration and time Literature and laboratory studies have shown that T-2toxin is strongest toxicity in trichothecene mycotoxins, so we compared T-2toxin and metabolic toxicity. The effect of T-2toxin and metabolic (5-80nM) on the cell viability of GH3cells were tested by MTT method, treated with12h. It was found that T-2toxin was the most toxicity and decreased cell viability, and the significant dose-dependent cell injury was observed. So T-2toxin was more meaningful to explore the growth mechanism of trichothecenes.To find out the genes and proteins directly connected with the T-2toxin and the relationship between concentration of T-2toxin and the change of differentially expressed genes, to monitor synthesis and secretion of growth hormone in GH3cells treated with T-2toxin (5-80nM) by using ELISA method. The results showed that T-2toxin could dramatically inhibited synthesis and secretion of GH in dose-dependent. GH secretion were90.45%±4.32and88.67%±3.57, GH synthesis were185.23%±3.47and170.17%±2.1, statistical analysis showed significant difference. As this concentration effectively inhibited hormone secretion without affecting cell viability,10nM,40nM T-2toxin and12h were chosen in our analysis.2. The differentially expressed genes for GH3cells with treatment of T-2toxinTo better understand the mechanism of T-2toxin inhibits synthesis and secretion of growth hormone, GH3cells treated with T-2toxin (10nM and40nM) for12h were used as low-dose and high-dose treatment groups and a vehicle control group was used as the control group in accordance with the above results. The gene chip was adopted to identify differentially expressed genes in T-2toxin treated GH3cells, afterwards gene function classification was carried on.Data from the rat agilent4×44K genome genechip showed that gene expression patterns of GH3cells exposed to T-2toxin were evidently changed. The results showed that, compared with blank group,10nM of T-2toxins group,2452significantly altered genes, including the1408up-regulated genes, in which1261genes were named and their function have been studied, and1044genes down-regulated, in which877genes were named.40nM of T-2toxins group,3346significant altered genes, including1736up-regulated genes, in which1541genes were named and their function have been studied, and1610genes down-regulated, in which1194genes have been studied. These T-2toxin reaction genes were involved in energy metabolism, transcription regulation, cell stress, cell transport and apoptosis etc. Significant genes function analysis suggested that cysteinyl-tRNA synthetase, and ribosome stress might be the main molecular targets of T-2toxin on growth hormone synthesis and secretion toxicity. In addition, RT-PCR was used to identify alteration of mRNA expression level of Ergic2, Nqo1, Snap25, HCK, PKR, caspase3. RT-PCR results showed that the gene chip results were reliable. The tendency of significant genes expressed was similar. Sensitivity of RT-PCR was higher than gene chip.3. The differentially expressed proteins for GH3cells with treatment of T-2toxinIn order to complement and verify the results of the differentially expressed genes in translation level, two-dimensional difference gel electrophoresis was performed to investigate the protein expression patterns of GH3cells exposed to T-2toxin for12h and24h. Using MALDI-TOF MS/MS analysis technique, we successfully identified91proteins at treated with T-2toxins for12h group, including30upregulated proteins and61downregulated proteins. At treated with T-2toxins for24h group had63proteins, including25upregulated proteins and38downregulated proteins.Gene Ontology, KEGG software analysis indicated that these T-2toxin reaction proteins were involved in multiple biological processes, including energy metabolism, signal transduction, stress response and constitute cytoskeleton etc. Changes in expression levels of energy metabolism enzyme, such as pyruvate kinase isozymes M1/M2, isocitrate dehydrogenase [NAD] subunit alpha, malate dehydrogenase, showed that T-2toxin affected the normal metabolic processes of GH3cells, so affected growth hormone synthesis and secretion. Protein disulfide-isomerase has played a key role in the synthetic process of GH, T-2toxin downregulated PDI protein, directly affected the synthesis and secretion of GH. In addition, cytoskeleton protein (Coficin-1, TBA1C, Actin) and calmodulin and other protein had a important role to the inhibition of synthesis and secretion of GH induced by T-2toxin. Growth hormone protein as our purpose, GRP78protein helped GH fold correctly, changed significantly after T2toxin exposed, the expression of GH and GRP78protein were detected with western blot, confirmed that2DE results.In conclusion, based on overall analysis of genes and proteins, T-2toxin induced toxicity mechanism of secretion of growth hormone may be:the normal GH3cells have secretory function and protein synthesis, the GH3cells treated with T-2toxin inhibitory effect on eukaryotic protein, T-2toxin induced JNK signaling cascades via ribotoxic stress response. T-2toxin also affected ammonia acyl-tRNA synthase and protein disulfide isomerase (PDI). T-2toxin induced endoplasmic reticulum stress response via Creb311gene, endoplasmic reticulum chaperones were down regulation, which can lead to protein misfolding of GH cannot correct folding and degradation, so reduced growth hormone synthesis. T-2toxin interfered with vesicles transport process, reduced growth hormone secretion; T-2toxin reduced energy metabolism in GH3cells, interfered with the synthesis and secretion process of growth hormone, further influenced the synthesis and secretion of growth hormone. |
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