title: T-2 toxin induces apoptosis in ovarian granulosa cells of rats through reactive oxygen species-mediated mitochondrial pathway.
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.
  
  
  CONCLUSIONS
  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.affiliation: 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.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
authors.forename: Li
authors.initials: L
authors.lastname: Jing
authors.forename: Hui
authors.initials: H
authors.lastname: Yuan
authors.forename: Shuang-qing
authors.initials: SQ
authors.lastname: Peng
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005260:Female; D005562:Formazans / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006107:Granulosa Cells / Q000187:drug effects* / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013605:T-2 Toxin / Q000633:toxicity*; D013778:Tetrazolium Salts / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D005562:Formazans; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013778:Tetrazolium Salts; C079782:MTT formazan; D013605:T-2 Toxin