Study On Toxic Mechanism Of T-2Toxin In Porcine Primary Hepatocytes

T-2toxin belongs to a large group of trichothecene mycotoxins produced by various Fusarium molds. It is widely scattered in cereal products and can cause several human and animal diseases upon exposure. Besides, human health is also in threat when animal derived food contains T-2toxin or its metabolites. Understanding the toxic mechanism of T-2toxin at a cellular level is essential for development of counter-measures. To determine the toxic mechanism of T-2toxin, hepatocytes isolated from3days old domestic Chinese piglets (crossbred from the Duroc, Large Yorkshire, and Landrace breeds) were exposed for48hours with0,0.02,0.05, or0.1μg/mL T-2toxin. After exposure, DNA microarray and2-dimensional difference gel electrophoresis (2-D DIGE) combined with MALDI-TOF MS/MS were applied to investigate the toxic mechanism of T-2toxin based on gene mRNA expression and protein expression levels. The main results are as follows:(1) Data from the Porcine Genome GeneChip showed that gene expression patterns of porcine primary hepatocytes exposed to0.05μg/mL T-2toxin was evidently changed. In which1193genes were significantly altered by T-2toxin, with562genes upregulated and631genes down-regulated. These T-2toxin reaction genes are involved in a variety of biological processes, including oxidation and reduction reaction, immune response, transport, proteolysis, cell adhesion, lipid metabolism process, and apoptosis etc. In addition, Real-time PCR was used to identify alteration of mRNA expression level of CYP450genes:CYP3A39, CYP3A46, CYP2C42and epoxide hydrolase EPHX1, carboxylesterase CES1C4. RT-PCR results showed that mRNA expression level of all these genes were increased, although in different degrees.(2) Two-dimensional difference gel electrophoresis (2-D DIGE) was performed to investigate the protein expression patterns of porcine primary hepatocytes exposed to0.05μg/mL T-2toxin.We found260significantly altered proteins. Using MALDI-TOF MS/MS analysis technique, we successfully identified84proteins, including34upregulated proteins and50downregulated proteins. Gene Ontology, KEGG database and Pathway StudioTM (v7.0) software analysis indicated that these T-2toxin reaction proteins are involved in multiple biological processes, including protein binding, electron transport, signal transduction, stress response, lipid metabolism and constitute cytoskeleton etc., and many of them are associated with hepatic injury and disease.