T-2 toxin is a secondary metabolite produced by Fusarium species and commonly contaminates food and animal feed. T-2 toxin can induce hepatotoxicity through apoptosis and oxidative stress; however, the underlying mechanism is not clear. Recent studies indicated that RASSF4, a member of the RASSF family, participates in cell apoptosis and some cancers due to its inactivation via DNA hypermethylation. However, its role in T-2 toxin-induced liver toxicity is poorly understood. Therefore, in this study, female Wistar rats were given a single dose of T-2 toxin at 2 mg/kg b.w. and were sacrificed at 1, 3 and 7 days post-exposure. A normal rat liver cell line (BRL) was exposed to different concentrations of T-2 toxin (10, 20, 40 nM) for 4, 8, 12 h, respectively. Histopathological analysis revealed with apoptosis in some liver cells and clear proliferation under T-2 toxin exposure. Expression analysis by immunohistochemical assays, quantitative real-time PCR (qPCR) and western blot demonstrated that T-2 toxin activated PI3K-Akt/Caspase/NF-κB signaling pathways. Additionally, DNA methylation assays revealed that the expression of RASSF4 was silenced by promoter hypermethylation after exposure to T-2 toxin for 1 and 3 days as compared to the control group. Moreover, joint treatment of 5-Aza-2'-deoxycytidine (DAC) (5 μM) and T-2 toxin (40 nM) increased expression of RASSF4 and PI3K-Akt/caspase/NF-κB signaling pathways-related genes, inducing cell apoptosis. These findings for the first time demonstrated that DNA methylation regulated the RASSF4 expression under T-2 toxin, along with the activation of its downstream pathways, resulting in apoptosis.

College of Life Science Yangtze University Jingzhou 434025 China; Department of Chemistry Faculty of Science University of Hradec Kralove Hradec Kralove 50003 Czech Republic

Department of Biosciences COMSATS University Islamabad Sahiwal campus Pakistan

MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products Hubei 430070 China

National Reference Laboratory of Veterinary Drug Residues and MAO Key Laboratory for Detection of Veterinary Drug Residues Huazhong Agricultural University Wuhan Hubei 430070 China

National Reference Laboratory of Veterinary Drug Residues and MAO Key Laboratory for Detection of Veterinary Drug Residues Huazhong Agricultural University Wuhan Hubei 430070 China; MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products Hubei 430070 China

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Hypoxia, oxidative stress, and immune evasion: a trinity of the trichothecenes T-2 toxin and deoxynivalenol (DON)