Jing Wu1, Li Jing, Hui Yuan, Shuang-qing Peng. 1. Evaluation and Research Center for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences, 20 Dongdajie Street, Fengtai District, Beijing 100071, PR China.
Abstract
OBJECTIVE: To investigate the reproductive toxicity and cytotoxicity of T-2 toxin, which is a mycotoxin, and to explore its potential apoptotic induction mechanism. METHODS: Ovarian granulosa cells of rats were treated with T-2 toxin (1-100nM) for 24h. The cytotoxicity was assessed with MTT bioassay; apoptotic cells were identified microscopically by chromatin condensation and nuclear fragmentation with Hoechst 33258; mitochondrial membrane potential with hodamine 123 and reactive reactive oxygen species (ROS) with 2',7'-dichlorofluoresceinacetate (DCFH-DA) was analyzed by fluorometry; p53 and other apoptosis-related proteins such as Bax, Bcl-2, caspase-3, caspase-9 were determined by Western blot analysis, and related mRNA expressions were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The caspase activity was measured by cleavage of the caspase substrate (Ac-DEVD-pNA for caspase-3, Ac-LEHD- pNA for caspase-9). RESULTS: T-2 toxin inhibited the growth of granulosa cells in a concentration-dependent way. The result of Hoechst 33258 staining indicated that T-2 toxin induces granulosa cells apoptosis based on the typical apoptotic morphological changes. Subsequently, we found that T-2 toxin treatment induced ROS accumulation in granulosa cells, resulting in reduction of mitochondrial transmembrane potential. The induction of cell apoptosis was caused by the upregulation of p53, Bax, Bcl-2, Bax/Bcl-2 ration, and the activation of the caspases pathways. T-2 toxin-induced apoptotic granulosa cells significantly decreased through the use of antioxidant Trolox. CONCLUSION: These data suggest a possible underlying molecular mechanism for T-2 toxin that induces the apoptosis signaling pathway in rat granulosa cells by regulation of ROS-mediated mitochondrial pathway.
OBJECTIVE: To investigate the reproductive toxicity and cytotoxicity of T-2 toxin, which is a mycotoxin, and to explore its potential apoptotic induction mechanism. METHODS:Ovarian granulosa cells of rats were treated with T-2 toxin (1-100nM) for 24h. The cytotoxicity was assessed with MTT bioassay; apoptotic cells were identified microscopically by chromatin condensation and nuclear fragmentation with Hoechst 33258; mitochondrial membrane potential with hodamine 123 and reactive reactive oxygen species (ROS) with 2',7'-dichlorofluoresceinacetate (DCFH-DA) was analyzed by fluorometry; p53 and other apoptosis-related proteins such as Bax, Bcl-2, caspase-3, caspase-9 were determined by Western blot analysis, and related mRNA expressions were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The caspase activity was measured by cleavage of the caspase substrate (Ac-DEVD-pNA for caspase-3, Ac-LEHD- pNA for caspase-9). RESULTS:T-2 toxin inhibited the growth of granulosa cells in a concentration-dependent way. The result of Hoechst 33258 staining indicated that T-2 toxin induces granulosa cells apoptosis based on the typical apoptotic morphological changes. Subsequently, we found that T-2 toxin treatment induced ROS accumulation in granulosa cells, resulting in reduction of mitochondrial transmembrane potential. The induction of cell apoptosis was caused by the upregulation of p53, Bax, Bcl-2, Bax/Bcl-2 ration, and the activation of the caspases pathways. T-2 toxin-induced apoptotic granulosa cells significantly decreased through the use of antioxidant Trolox. CONCLUSION: These data suggest a possible underlying molecular mechanism for T-2 toxin that induces the apoptosis signaling pathway in rat granulosa cells by regulation of ROS-mediated mitochondrial pathway.
Authors: Liliana O Rocha; Matthew H Laurence; Robert H Proctor; Susan P McCormick; Brett A Summerell; Edward C Y Liew Journal: Toxins (Basel) Date: 2015-11-05 Impact factor: 4.546