T-2 toxin inhibits murine ES cells cardiac differentiation and mitochondrial biogenesis by ROS and p-38 MAPK-mediated pathway

Toxicol Lett. 2016 Sep 6;258:259-266. doi: 10.1016/j.toxlet.2016.06.2103. Epub 2016 Jun 27.

Abstract

Objective: To investigate the effect of T-2 toxin on murine embryonic stem cells (ESCs) cardiac differentiation and mitochondrial biogenesis in vitro.

Methods: Cardiac differentiation of the mouse ESCs was initiated by embryoid bodies (EBs) formation in hanging drops. EBs were exposed to 0.5ng/ml T-2 toxin for 24, 72 and 120h. Cultures were observed daily for the appearance of contracting clusters, and cardiac-specific protein (α-actiniin) were measured by Western blot and immunocytochemistry. Mitochondrial ultrastructure was observed by confocal laser scanning microscopy and transmission EM photography. Reactive oxygen species (ROS) was monitored by H2-dichlorofluorescein-diacetate (H2DCF-DA). The phosphorylation of the p38 (p-p38) and p38 mitogen-activated protein kinase (MAPK) and the expression of mitochondrial biogenesis proteins, including peroxisome proliferator activated receptor coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (mtTFA), and mitochondrial respiratory chain complex IV (COXIV) were analyzed using Western blot. In some experiments, mESCs were pre-treated with the antioxidant Trolox (200μM) for 30min, then exposed to Trolox (200μM) and T-2 toxin (0.5ng/ml) for 72h.

Results: Contracting clusters were observed under the microscope light and cardiac-specific protein (α-actinin) expressed positively indicated mESCs directly differentiated in cardiomyocytes. However, the cardiac differentiation was inhibited by T-2 toxin treatment 72 and 120h. ROS accumulated in murine ES cells in a time-dependent manner. The expression of p-p38 significantly increased in 24h group and decrease in 72 and 120h groups. The decrease of mitochondrial number and the mitochondrial biogenesis-related proteins expression, including PGC-1α, NRF-1, mtTFA, and COXIV decreased in a time-dependent manner with T-2 toxin treatment. However, the inhibition of mitochondrial biogenesis by T-2 toxin in differentiated mESCs was recovered significantly in the presence of the antioxidant Trolox.

Conclusion: Taken together, T-2 toxin decreased the expression of PGC-1α, NRF-1, and mtTFA, inhibited mitochondrial biogenesis, and then inhibited the cardiac differentiation of murine ES cells, and the effect was partly responsible for the p38 MAPK mediated by ROS.

Keywords: Cardiac differentiation; Mitochondrial biogenesis; PGC-1α; ROS; T-2 toxin; p38MAPK.

MeSH terms

  • Animals
  • Antioxidants / pharmacology
  • Biomarkers / metabolism
  • Cell Differentiation / drug effects
  • Cell Line
  • Coculture Techniques
  • MAP Kinase Signaling System / drug effects
  • Mice
  • Mitochondrial Dynamics / drug effects*
  • Mouse Embryonic Stem Cells / cytology
  • Mouse Embryonic Stem Cells / drug effects*
  • Mouse Embryonic Stem Cells / metabolism
  • Mouse Embryonic Stem Cells / ultrastructure
  • Myoblasts, Cardiac / cytology
  • Myoblasts, Cardiac / drug effects*
  • Myoblasts, Cardiac / metabolism
  • Myoblasts, Cardiac / ultrastructure
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / drug effects*
  • Myocytes, Cardiac / metabolism
  • Myocytes, Cardiac / ultrastructure
  • Organelle Biogenesis
  • Oxidative Stress / drug effects*
  • Phosphorylation / drug effects
  • Protein Processing, Post-Translational / drug effects
  • Reactive Oxygen Species / agonists
  • Reactive Oxygen Species / antagonists & inhibitors
  • Reactive Oxygen Species / metabolism
  • T-2 Toxin / antagonists & inhibitors
  • T-2 Toxin / toxicity*
  • Teratogens / chemistry
  • Teratogens / toxicity*
  • p38 Mitogen-Activated Protein Kinases / metabolism

Substances

  • Antioxidants
  • Biomarkers
  • Reactive Oxygen Species
  • Teratogens
  • p38 Mitogen-Activated Protein Kinases
  • T-2 Toxin