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T-2 toxin inhibits murine ES cells cardiac differentiation and mitochondrial biogenesis by ROS and p-38 MAPK-mediated pathway
Accepted Manuscript Title: T-2 toxin inhibits murine ES cells cardiac differentiation and mitochondrial biogenesis by ROS and p-38 MAPK-mediated pathway Author: Haiqin Fang Liangzi Cong Yuan Zhi Haibin Xu Xudong Jia Shuangqing Peng PII: DOI: Reference:
S0378-4274(16)32252-4 http://dx.doi.org/doi:10.1016/j.toxlet.2016.06.2103 TOXLET 9477
To appear in:
Toxicology Letters
Received date: Revised date: Accepted date:
19-4-2016 19-6-2016 26-6-2016
Please cite this article as: Fang, Haiqin, Cong, Liangzi, Zhi, Yuan, Xu, Haibin, Jia, Xudong, Peng, Shuangqing, T-2 toxin inhibits murine ES cells cardiac differentiation and mitochondrial biogenesis by ROS and p-38 MAPK-mediated pathway.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2016.06.2103 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
T-2 toxin inhibits murine ES cells cardiac differentiation and mitochondrial biogenesis by ROS and p-38 MAPK-mediated pathway
Haiqin Fanga,b†, Liangzi Congc†, Yuan zhi b, Haibin Xu b, Xudong Jiab,, Shuangqing Penga
a
Evaluation and Research Centre for Toxicology, Institute of Disease Control and
Prevention, Academy of Military Medical Sciences, Beijing 100071, China b
Key Laboratory of Food Safety Risk Assessment of Ministry of Health, China
National Center for Food Safety Risk Assessment, Beijing 100021, China c
Huaiyin District Center for Disease Control and Prevention, Jinan, Shandong
Corresponding author. Tel./fax: 8610-66948462, E-mail address: [email protected] (S Peng); Tel./fax: +8610 67770977, E-mail address: [email protected] (X Jia). † These authors contributed equally.
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Highlights T-2 toxin induced oxidative stress in mESCs in a time-dependent manner. T-2 toxin inhibited mESCs differentiated into cardiacmyocytes during spontaneous differentiation. T-2 toxin inhibited mitochondrial biogenesis of mESCs by ROS, and then inhibited the differentiation of mESCs. The effect of T-2 toxin on mESCs differentiation and mitochondrial biogenesis was partly responsible for the p38 MAPK mediated by ROS. Mitochondrial biogensis and mitochondrial pathway plays an important role in the mechnism of T-2 embryotoxicity.
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 0.5 ng/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 (200 μM) for 30 min, then exposed to Trolox (200 μM) and T-2 toxin (0.5 ng /mL) for 72h.
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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.
Key Words: T-2 toxin, mitochondrial biogenesis, cardiac differentiation, ROS, p38MAPK, PGC-1α
1. Introduction As a cytotoxic fungal metabolite that belongs to the trichothecene mycotoxin family, T-2 toxin is produced by various species of Fusarium and can infect corn, wheat, barley, and rice crops in the field or during storage, being characteristically stable under changing environmental conditions (Yagen et al., 1993; Desjardins et al., 1993). Ingestion by humans or livestock of cereals contaminated by T-2 toxin can cause adverse reactions, such as vomiting, diarrhea, and even death (Rotter et al., 1996; Nelson et al., 1994; Magnuson et al., 1987). In view of the great harm to the health of humans and livestock, the toxicological effects of T-2 toxin was reported in the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives (FAO/WHO 2002) .
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On experimental animals, livestock, and humans, T-2 toxin has been shown to produce a variety of toxic effects, including brain lesions, hematopoietic, lymphoid, gastrointestinal tissues, suppresses reproductive organ functions, and developmental Toxicity (Yagen et al., 1993; Desjardins et al., 1993; Escrivá et al., 2015). T-2 toxin readily passes through the placenta and is distributed to fetal tissues (Lafarge-Frayssinet et al., 2002), resulting in embryo/fetal death, fetal brain damage, fetal liver damage, and fetal bone malformation (Blakey et al., 1987; Sehata et al., 2003; Sehata et al., 2005; Doi et al, 2008). Embryonic stem cell test (EST), which has been approved by the European Centre for the Validation of Alternative Methods (ECVAM) (Genschow et al., 2002), is a novel model in the evaluation for chemical embryotoxicity and developmental risk assessment of environmental toxicants (Rezvanfar et al., 2016), and a good tool in investigation underlying mechanisms. EST consists of two sets of procedures: cytotoxicity tests conducted with the mESC line, D3, and a differentiation assay using D3 cells. In our previous study, the IC50D3 of T-2 toxin, which expresses the cytotoxicity of T-2 toxin to embryonic stem cell D3, was 1.5 ng/ml, and the ID50 of T-2 toxin, which expresses as the concentration of test chemical inhibiting the development of contracting cardiac muscle cells by 50% was 0.57ng/ml (Fang et al., 2012). Studies have provided evidence that proper mitochondrial biogenesis and function is essential to proliferation and differentiation of ES cells. It is suggested that mitochondrial biogenesis and metabolic switches might be hallmarks of the ES cell differentiation processes (Wanet et al., 2015). In self-renewal state, ES cells were supported by glycolysis metabolism and by mitochondrial properties including low mitochondrial DNA (mtDNA) copy number, immature organelle shape with under-developed cristae, and low levels of oxidative stress (Prigione et al., 2010; St John et al., 2006). However, during differentiation, mitochondria in mouse and human ES cells dramatically increased in number, cristae elongated, and an extensive reticular network of tubular structures generated (Prigione et al., 2010; St John et al., 2006; Chung et al., 2007; Facucho-Oliveira et al., 2007; Mandal et al., 2011). 4
Oxidative stress is one of the most important underlying toxic mechanisms for T-2 toxin. It was reported that free radicals, including reactive oxygen species, were generated by T-2 toxin both in vitro and in vivo, and then lipid peroxidation leading to changes in membrane integrity and cellular redox signaling (Chang et al., 1988; Fang et al., 2012; Schuster et al., 2004; Wu et al., 2011; Yang et al., 2016). It was reported that mitochondria was an derect target in human Kashin-Beck disease induced by T-2 toxin (Liu et al., 2014). Exposure to T-2 toxin can reduce activities of mitochondrial complexes III, IV and V, MMPand the cellular ATP, and increased intracellular ROS. In our previous study, we demonstrated that T-2 toxin was a potent embryonic toxin and mitochondria was an important target. T-2 toxin induced oxidative damage in differentiated mouse ES cells, including a significant decrease of glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity, and an increase in the ROS formation and malonaldehyde (MDA) contention. Moreover, T-2 toxin inhibited mESCs mitochondrial function, including mitochondrial membrane potential (MMP) loss, and induced apoptosis by mitochondrial pathway in differentiated mESCs (Fang et al., 2012). It is assumed that mitochondria biogenesis plays an important role in ES cells differentiation and mitochondria are an major target of T-2 toxin. The present study was undertaken to observe the effect of T-2 toxin on mitochondrial biogenesis and differentiation ability in murine differentiated ES cells, and to investigate the underlying mechanism of T-2 toxin embryonic toxicity by ROS-mediated mitochondrial pathway.
2. Materials and Methods 2.1 Reagents T-2 toxin was kindly provided by the Evaluation and Research Centre for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences. T-2 toxin was dissolved in DMSO to prepare a stock solution of 1 mg/ml and diluted to final concentrations with knockout DMEM. Dulbecco’s modified Eagle’s minimal essential medium (DMEM), knockout DMEM and non-essential amino acids were 5
purchased from Gibco (Gibco BRL, Germany). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonsaure (Trolox), β-mercaptoethanol, and 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma Aldrich (Munich, Germany). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, USA). Mouse leukemia inhibitory factor (mLIF) was purchased from Chemicon (Milipore, USA). Anti-PGC-1α, anti-NRF-1, anti-mtTFA, anti-COXIV, anti-p-p38MAPK, anti-p38MAPK, anti-α-actinin, and anti-GADPH antibodies were obtained from Abcam Biotechnology, Inc (Abcam, USA). Enhanced chemiluminescence (ECL) reagent were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, USA). All other reagents were of analytic grade. 2.2 Embryonic stem cell culture and T-2 toxin treatment The murine ES D3 cells (American Type Culture Collection, CRL-1934) were cultivated in an undifferentiated state on feeder of mouse embryonic fibroblasts (MEF) as described previously . Cultures of differentiating ES cells were established by the formation of EBs in hanging drop cultures with the differentiation medium as previously described (Fang et al., 2012). After cultivated in hanging drops for 3 days and suspended in Petri dishes for another 2 days, EBs were plated separately onto gelatin-coated 24-well culture plates in the differentiation medium on day 5. At the time of EBs plating 24, 72 and 120h, 0.5ng/ml T-2toxin were added in culture medium, and EBs were harvested at the time of plating 144h. In another word, EBs were exposed to T-2 toxin for 120, 72, 24, and 0h respectively. Cultures were observed daily with an inverted microscope for observing the appearance of contracting clusters and counting the numbers of beating EBs. In some experiments, ES cells were pre-treated with the antioxidant Trolox (200 μM) for 30 min, then exposed to Trolox (200 μM) and T-2 toxin (0.5 ng/ml) for 72h. 2.3 Western blot analysis Western blot analysis for PGC-1α, NRF-1, mtTFA, COXIV, p-p38MAPK, p38 MAPK, α-actinin, and GAPDH expression were performed as previously described (Fang et al., 2012). After incubation with 0.5 ng/ml T-2 toxin for 0, 24, 72, and 120 hours, differentiated ES cells were washed in PBS and lysed in lysis buffer (50 mM 6
Tris-HCl, 150 mM NaCl, 1% NP-40, pH 7.5) containing protease-inhibitor (1 mM PMSF). Homogenized tissues were then centrifuged at 4 °C and 14000 g for 15 min and supernatants were subjected to Western blot analysis. Protein (30 μg) was loaded for 1-dimensional 12% SDS-PAGE. Detection was measuring using the chemiluminescence of the ECL reagent. The photographs generated were quantitatively analyzed for PGC-1α, NRF-1, mtTFA, COXIV, p-p38MAPK, p38 MAPK, α-actinin, protein levels with a Quantity One image densitometer. The molecular weights of the protein bands were determined by reference to the standard molecular weight markers. Protein levels were standardized by comparison with GAPDH. 2.4 Confocal laser scanning microscopy Differentiated ES cells that had been grown on coverslips were fixed for 20 min in methanol at -20°C, followed by permeabilization in 0.1% Tween 20 in phosphate-buffered solution (PBS). After washing in PBS 3 times, the EB on coverslips were transferred to PBS containing 10% goat serum (Sigma, USA) for 30 min at room temperature. The EBs were then placed into PBS containing mouse monoclonal anti-α-actinin (Sigma, USA; dilution 1:100) and incubated overnight at 4 °C. The EB were washed in PBS 3 times, followed by incubation in PBS containing the FITC-conjugated antimouse IgG (Sigma, USA; dilution 1:500). Fluorescence recordings were performed by means of confocal laser scanning setup (Leica TCS SP2, Bensheim, Germany) connected to an inverted microscope. 2.5 Transmission electron microscope scanning Transmission electron microscopy imaging was performed at the Instrument Center, Academy of Military Medical Sciences, China. Differentiated murine ES cells fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, were washed in 0.1 M cacodylate buffer and treated with 0.1% Millipore-filtered buffered tannic acid, postfixed with 1% buffered osmium tetroxide for 30 min, and stained with 1% Millipore-filtered uranyl acetate. The samples were washed several times in water, then dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in LX-112 medium. ES cells 7
were polymerized in a 60 °C oven for about 2 days. Ultrathin sections were cut in a Leica Ultracut microtome (Leica), stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a JEM 1010 transmission electron microscope (JEOL) at an accelerating voltage of 80 kV. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques). 2.6 Statistical analysis Data were expressed as mean ± S.D. and analyzed by one-way ANOVA, followed by LSD’s post hoc test by SPSS 11.5 statistical software. The level of significance was accepted as p<0.05.
3. Results 3.1 T-2 toxin inhibited the murine ES cells differentiation According to the protocol recommended by ECVAM, a differentiated ESC model was established. On the gelatin-coated plate, colonies of ES-D3 cells (Fig. 1A: arrows refer to colonies of ES-D3 cells) resulted in the establishment of the EST model by the formation of EBs in hanging drop cultures, and caused the cells to spontaneously differentiate into synchronously-contracting cardiac clusters derived from EBs on day 5 +5 (Fig. 1B). Staining with α-actinin, a cardiac myoblasts specific protein, which appeared as red fluorescence, was observed in differentiated ES-D3 cells (Fig. 1C), and significant decrease in expression of α-actinin were observed in differentiated ES cells following 72 and 120 h exposure to T-2 toxin at 0.5ng/ ml( Fig. 1D). As shown in Fig. 1F, there was no beating EB observed under the microscope light in 72 and 120 h groups, which indicated that T-2 toxin inhibited the differentiation of ES-D3 cells. 3.2 T-2 toxin decreased the mitochondrial biogenesis murine ES cells Mitochondrial morphology was evaluated in differentiated mouse ESCs by staining with a mitochondrial-specific dye, MitoTracker Red. Consistent with previous reports, we found that ESCs mitochondria were distributed as perinuclear clusters that appeared punctate and globular (Fig. 2A–2D), and ESCs mitochondria in normal group generated an extensive reticular network of tubular structures. Although the 8
common punctate and rudimentary appearance of mitochondria were seen in all differentiated ESCs, the amount of mitochondrial mass varied between independent groups of ESCs, that is, the ES cells of normal (Fig. 2 A) or T-2 toxin treatment 24 h (Fig. 2 B) appeared to have more mitochondria than T-2 toxin treatment 72 (Fig. 2 C)and 120 h (Fig. 2D). As shown in Fig. 2E-2H, compared with the normal differentiated ESCs (Fig. 2 E) or T-2 toxin treatment 24 h (Fig. 2 F), significant decrease in the mitochondrial number, deformation of mitochondria, and lack of complete cristae were observed through TEM in the groups of T-2 toxin exposed for 72 (Fig. 2 G)and 120 h (Fig. 2 H). It was obvious that T-2 toxin inhibited the mitochondrial biogenesis in differentiated murine ES cells after a long term exposure (72 and 120h). 3.3 T-2 toxin-induced oxidative stress in differentiated ES cells Oxidative stress is involved in the toxicity of trichothecene mycotoxins, including T-2 toxin. In the present study, differentiated ES cells were exposed to 0.5 ng/ml T-2 toxin for 0, 24, 72 and 120 h, and oxidative stress was monitored in terms of production of ROS. Significant increase in the ROS formation was seen after exposure to 24, 72, and 120 h (Fig. 3A, B). 3.4 T-2 toxin effected the expression of mitochondrial biogenesis-related proteins in differentiated murine ES cells The protein expression of mitochondrial biogenesis modulation factors p38MAPK, phosphorylated p38MAPK (p-p38), NRF-1, mTFA, and COXIV were examined in differentiated ES cells treated with 0.5 ng/ml T-2 toxin for 0, 24, 72 and 120 h followed by Western blot analysis of these proteins. In the mouse ES cells exposed to T-2 toxin 24h, the protein expression of p-p38 increased significantly, however, there was no significant change in other indexes in this group. In the groups of T-2 toxin treatment 72 and 120 h, except p38 protein, the expression of PGC-1ɑ, NRF-1, mTFA, and COXIV decreased significantly. (Fig. 4). 3.5 Trolox recovered the inhibition of mitochondrial biogenesis and differentiation ability induced by T-2 toxin in differentiated ES cells
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To confirm the role of ROS in T-2 toxin-inhibited differentiation through the inhibition of mitochondrial biogenesis by p-38MAPK, differentiated ES cells were pre-treated with the antioxidant Trolox (200 μM) for 30 min, and exposure to Trolox was continued during subsequent T-2 toxin (0.5 ng/ml) treatment for 72 h. Trolox significantly inhibited T-2 toxin induced increase of ROS generation by 21% (Fig.5A,B). As Fig. 5 shown, compared with the control group(Fig.5C), ES cells treated with Trolox alone did not change in mitochondrial biogenesis (Fig.5D). However, compared with the ES cells exposed to T-2 toxin for 72 h (Fig.5E), the amount of mitochondrial mass in Trolox pretreated was greater (Fig.5F). As mitochondrial biogenesis-associated protein expression, compared with the ES cells exposed to T-2 toxin for 72 h, Trolox increased the expression of p-p38 by 22.9%, PGC-1α by 27.3%, NRF-1 by 27.4%, mTFA by 26.9% and COXIV by 26.7% (Fig.5G).
4. Discussion In our previous study, T-2 toxin’s was evaluated strong embryonic toxicity by EST (Fang et al., 2012). Following the IC50D3 for T-2 toxin, different concentration of T-2 toxin were selected to investigate the cytotoxicity mechanism of T-2 toxin to ESC D3. ROS-mediated mitochondrial pathway was found to be involved in T-2 toxin-induced apoptosis in murine differentiated ES cells in a dose-dependent manner(Fang et al., 2012). In the present study ( according to the ID50 of T-2 toxin is 0.57 ng/ml) a concentration of 0.5 ng/ml and different exposure time (24 h, 72 h, 120 h) were chosen to observe the effect of T-2 toxin on ESC differentiation, and try to investigate the modulation of T-2 toxin on ES mitochondrial biogenesis and ES differentiation. Differentiated ES cells can be used as a model to investigate the mechanism of chemical’s embryonic toxicity. During ES cells differentiation, ES cells spontaneously differentiate into different cell types of all three germ layers in vitro, tissue-specific genes and proteins are expressed in a developmentally, regulated manner, recapitulating the processes of early embryonic development (Guan et al., 1999; Rohwedel et al., 2001; Rolletschek et al., 2004). In the protocal of the embryonic stem 10
cell test (EST) published by ECVAM, the cardiac differentiation was defined as the marker of mESCs differentiation (Genschow et al., 2002). In the present study, the differentiation ability of ES cells were observed both as spontaneously contracting cardiomyocytes and as the expression of cardiac myoblasts -specific protein α-actinin. However, this ability could be inhibited when T-2 toxin exposure for 72 and 120 h. Metabolism and mitochondria regulate ES cell fate (Wanet et al., 2015; Folmes et al., 2016). Recent studies have demonstrated that differentiated ES cells show altered mitochondrial function and metabolic profiles and production of reactive oxygen species. This raises an emerging paradigm about the role of mitochondria in stem cell biology and urges the need to identify mitochondrial pathways involved in these processes (Lopes et al., 2016).Although anaerobic metabolism is sufficient to meet the energy demand of undifferentiated mouse and human ESCs, successful differentiation of ESCs requires activation of mitochondrial aerobic metabolism in order to promote the synthesis of greater levels of ATP and the maintenance of homeostasis in the differentiated cell. An increase in mitochondrial oxidative phosphorylation has to occur for differentiation to succeed (Lopes et al., 2016). Indeed, fully differentiated cells such as neurons and cardiomyocytes express high levels of nDNA- and mtDNA-encoded electron transport chain (ETC) subunits, produce ATP through oxidative phosphorylation (OXPHOS) (Cho et al., 2006; Sart et al., 2015) and contain enriched mtDNA content (Prigione et al., 2010; St John et al., 2006; Chung et al., 2007; Facucho-Oliveira et al., 2007). In addition, differentiation of ESCs results in changes in mitochondrial structure, morphology, tubular structure, numerous elongated cristae, dense matrices, and high membrane potential suggesting the initiation of metabolic activity through OXPHOS (Cho et al., 2006; Facucho-Oliveira et al., 2007; Sart et al., 2015). In the present study, numerous mitochondria with complete cristae, and the extensive reticular network of tubular structures were shown in differentiation ES cells of control groups. COXIV, a symbol complex of mtDNA-encoded ETC subunits was expressed
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strongly in differential mESCs. These results implied that the cardiomyocyte differentiation of ES cells was accompanied by an organization of mitochondrial biogenesis. Compared with the control group, we found the significant decrease of the mitochondrial number, deformation of mitochondrial structure, lack of complete mitochondrial cristae, and significant decrease in COXIV protein expression were observed in the groups of T-2 toxin exposed for 72 and 120 h. ROS play dual role in stem cell homeostasis, depending on its level of production. Once cells start to produce large amount of ATP through the mitochondrial ETC, ROS will be produced as by-products of oxidative phosphorylation (Sart et al., 2015). Nevertheless, the intracellular levels of ROS are higher in differentiating ESCs than in undifferentiated ESCs due to the increase in OXPHOS metabolism (Cho et al., 2006; Turrens et al., 2003). Although an increase in ROS levels might have a role in cell signaling and regulation of proliferation and differentiation, exposure to low levels of ROS has been reported to enhance ESC differentiation towards the cardiomyogenic and vascular lineages, whereas continuous exposure to high levels of ROS results in inhibition of differentiation (Schmelter et al., 2006). As Fig.6 shown, tight regulation of biochemical and biomechanical environment can control stem cell oxidative status and thus the stem cell fate decision (Sart et al., 2015). An increasing body of evidence show that T-2 toxin can induce oxidative damage in biological systems due to increased generation of hydroxyl radical(Chang et al., 1988; Schuster et al., 2004; Liu et al., 2014; Yang et al., 2016). In the present study, T-2 toxin stimulated ROS generation in a time-dependent manner was observed. Accompanied with cellular continuously increasing ROS, T-2 toxin inhibited the differentiation of murine ESCs exposed to T-2 toxin at 72 and 120 h. We also found T-2 toxin decreased mitochondrial biogenesis after T-2 toxin treatment at 72 and 120 h. After the inhibition of T-2 toxin on mitochondrial biogenesis and differentiation ability were found (0.5 ng/ml T-2 toxin for 72 and 120h), we focused our study on the modulation factors in the ROS-mediated mitochondrial pathway in differentiated mESCs.
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PGC-1α (peroxisome-proliferator-activated receptor γ coactivator-1α) is a co-transcriptional regulation factor that induces mitochondrial biogenesis by activating different transcription factors, including nuclear respiratory factor 1 and nuclear respiratory factor 2, which activate mitochondrial transcription factor A. The latter drives transcription and replication of mitochondrial DNA. Matthew reported that PGC-1α also played a key role in regulating hESC-derived cardiomyocyte mitochondrial respiration, contractile automaticity, and superoxide production (Matthew et al., 2013). PGC-1α itself is regulated by several different key factors involved in mitochondrial biogenesis. AMPK (AMP-activated protein kinase) is of major importance, and AMPK acts as an energy sensor of the cell and works as a key regulator of mitochondrial biogenesis. In the signaling events leading to activation of the transcription cascade, p38 MAPK has been suggested to play an important role in myogenic cell differentiation (Cho et al., 2006). Transgenic mice specifically overexpressing p38 MAPK in skeletal muscle show enhanced PGC-1α expression and increased mitochondrial proteins (Sart et al., 2015). Interestingly, accumulating evidence indicates that the p38 MAPK plays a critical role in cardiomyocyte differentiation of murine carcinoma stem cells and ES cells in vitro (Schmelter et al., 2006). Ding reported that intracellular ROS generation and p38MAPK could be triggered when murine ES cells are exposed to icariin which resulted in cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro (Ding et al., 2008). In the present study, p38MAPK was spontaneously activated in T-2 toxin exposure at 24h, and the mitochondrial biogenesis-related protein expression showed an increased trend in same group, however, there was no significant difference in statistics in mitochondrial biogenesis and differentiation. Moreover, the inhibition of p38MAPK phosphorylation was accompanied with ROS continuous accumulation, when T-2 toxin was present long term (72 and 120h). These results indicated that ROS at low level can be a signal factor to activate p38MAPK; however, the activation effect of ROS on
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p38MAPK gradually disappeared with an overwhelming accumulation after long-term T-2 toxin exposure. PGC-1α, as a direct downstream target of the p38 MAPK, also as the master regulator of mitogenesis, can directly or indirectly stimulate the transcription of OXPHOS genes and the mitochondrial transcription factor A (mtTFA) gene (Kelly et al., 2004; Lin et al., 2002; Akimoto et al., 2005). In the present study, the expression of PGC-1α, NRF-1, mTFA, and COXIV decreased significantly in T-2 toxin exposure at 72 and 120 h. It suggests that accumulation of ROS induced by T-2 toxin inhibited the phosphorylate p38MAPK and subsequently inhibited of PGC-1α-regulated mitochondrial biogenesis in the differentiation process of murine embryonic stem cells in a time-dependent manner in vitro. To confirm our hypothesis, the antioxidant Trolox, a water-soluble vitamin E derivative, was used in our study. It is reported that vitamin E supplementation can reduce the DNA damage or lipid peroxidation induced by T-2 toxin in vivo (Vila` et al., 2004). In our experiments, T-2 toxin induced ROS generation in murine differentiated ES cells, which was significantly inhibited by Trolox at a concentration of 200 μM. This result showed that the antioxidant participated in differentiated ES cells’ redox reactions and neutralized the oxidative stress induced by T-2 toxin. Meanwhile, T-2 toxin-inhibited the mitochondrial biogenesis in differentiated murine ES cells was significantly recovered after Trolox treatment. However, unlike the expression of COXIV, there was no significant recovery of the expression of α-actinin. This suggests that the differentiation of ES cells is complicated and sensitive, and the factors what regulate stem cell differentiation remains to be determined. Our data indicated that a p38MAPK-involved and ROS-mediated mitochondrial pathway played an important role in the T-2 toxin-inhibited mitochondrial biogenesis and cardiac differentiation of mESCs. However, the differences of relevant mitochondrial biogenesis and ES cell differentiation indicators between the control group and the group treated with T-2
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toxin plus Trolox are very significant, suggesting that other pathways might participate in the process in addition to the p38MAPK-involved and ROS-mediated pathway. Taken together, we demonstrated T-2 toxin inhibited mitochondrial biogenesis-triggered by the phosphorylation of the p38 MAPK, which was mediated by ROS, and then inhibited murine ES cells differentiation in a time-dependent manner. We speculate that this might be a possible underlying molecular mechanism of T-2 toxin induced embryonic toxicity. This study focuses on the metabolic profile of ESC and how mitochondrial biogensis can influence the differentiation processes. Indeed, mitochondria appear to have a crucial role in the maintenance of a pluripotent state and in differentiation. Therefore, in vitro differentiation of ESCs into cardiomyocytes can be compromised if those mechanisms are impaired, such as the influence of environmental toxicants. We also found ROS played dual role in ES cells differentiation into cardiomyocytes. Future research should shed light on how mitochondrial impairment occurring in differentiation stage may contribute for the etiopathogenesis of cardio developmental and how to prevent cardiac developmental disorders by mitochondrial pathway.
Acknowledgements This project was supported by the High-Level Talents and Experts Introducing Program for “Project 523” of the China National Center for Food Safety Risk Assessment (1311613106702), Natural Science Foundation of Beijing City (7142128), and National Natural Science Foundation of China (81302462, 81172699).
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References Akimoto, T., Pohnert, SC., Li, P., Zhang, M., Gumbs, C., Rosenberg, PB., Williams, RS., Yan, Z., 2005. Exercise stimulates Pgc-1α transcription in skeletal muscle through activation of the p38 MAPK pathway. J. Biol. Chem. 2005; 280(20):19587-19593. Birket, MJ., Casini, S., Kosmidis, G., Elliott, DA., Gerencser, AA., Baartscheer, A., Schumacher, C., Mastroberardino, PG., Elefanty, AG., Stanley, EG., Mummery, CL., 2013. PGC-1ɑ and reactive oxygen species regulate human embryonic stem cell-derived cardiomyocyte function. Stem Cell Reports. 1(6):560-574. Blakley, BR., Hancock, DS., Rousseaux, CG., 1987. Embryotoxic effects of prenatal T-2 toxin exposure in mice. Can J Vet Res. 51(3):399-403. Chang, IM., Mar, WC., 1988. Effect of T-2 toxin on lipid peroxidation in rats: elevation of conjugated diene formation. Toxicol Lett. 40(3):275-280. Cho, YM., Kwon, S., Pak, YK., Seol, HW., Choi, YM., Park, do J., Park, KS., Lee, HK., 2006. Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of human embryonic stem cells. Biochem Biophys Res Commu. 348 (2006) 1472-1478. Chung, S., Dzeja, PP., Faustino, RS., Perez-Terzic, C., Behfar, A., Terzic, A., 2007. Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells. Nat Clin Pract Cardiovasc Med. 4(suppl 1):S60-S67. Desjardins, AE., Hohn, TM., McCormick, SP., 1993. Trichothecene biosynthesis in Fusarium species: chemistry, genetics, and significance. Microbiol Rev. 57(3):595-604. Ding, L., Liang, XG., Hu, Y., Zhu, DY., Lou, YJ., 2008. Involvement of p38MAPK and Reactive Oxygen Species in Icariin-Induced Cardiomyocyte Differentiation of Murine Embryonic Stem Cells In Vitro. Stem Cell Dev. 17:751-760. Doi, K., Ishigami, N., Sehata, S., 2008. T-2 toxin-induced toxicity in pregnant mice and rats. Mol Sci. 9(11):2146-2148. Escrivá, L., Font, G., Manyes, L., 2015. In vivo toxicity studies of fusarium mycotoxins in the last decade: A review. Food Chem Toxicol. 78:185-206.
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Facucho-Oliveira, JM., Alderson, J., Spikings, EC., Egginton, S., St John, JC., 2007. Mitochondrial DNA replication during differentiation of murine embryonic stem cells. J Cell Sci 120(pt22):4025-4034. Fang, H.,Wu, Y., Guo, J., Rong, J., Ma, L., Zhao, Z., Zuo, D., Peng, S., 2012. T-2 toxin induces apoptosis in differentiated murine embryonic stem cells through reactive oxygen species-mediated mitochondrial pathway. Apoptosis. 17(8):895-907. FAO/WHO, 2002. Evaluation of certain mycotoxins in food. Fifty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives. World Health Organ Tech Rep Ser. 906:i-viii, 1-62. Folmes, CD., Ma, H., Mitalipov, S., Terzic, A., 2016. Mitochondria in pluripotent stem cells: stemness regulators and disease targets. Curr Opin Genet Dev. 38:1-7. [Epub ahead of print] Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Bradym M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European centre for the validation of alternative methods. Altern Lab Anim 30(2):151-176. Guan, K., Rohwedel, J., Wobus, AM., 1999. Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro. Cytotechnology. 30(1-3):211-226. Kelly, DP., Scarpulla, RC., 2004. Transcriptional regulatory circuits controlling mitochondrial biogenesis and function. Genes Dev. 18 (2004) 357-368. Lafarge-Frayssinet, C., Chakor, K., Lafont, P., Frayssinet, C., 1990. Transplacental transfer of T2 toxin: pathological effect. J Environ Pathol Toxicol Oncol. 10(1-2):64-68. Lin, J., Wu, H., Tarr, PT., Zhang, CY., Wu, Z., Boss, O., Michael, LF., Puigserver, P., Isotani, E., Olson, EN., Lowell, BB., Bassel-Duby, R., Spiegelman, BM., 2002. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres. Nature. 418:797-801. Liu, J., Wang, L., Guo, X., Pang, Q., Wu, S., Wu, C., Xu, P., Bai, Y., 2014. The role of mitochondria in T-2 toxin-induced human chondrocytes apoptosis. PLoS One. 9(9):e108394.
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Lopes, C., Rego, AC., 2016. Revisiting Mitochondrial Function and Metabolism in Pluripotent Stem Cells: Where Do We Stand in Neurological Diseases? Mol Neurobiol. Feb 18. [Epub ahead of print] Mandal, S., Lindgren, AG., Srivastava, AS., Clark, AT., Banerjee, U., 2011. Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells. Stem Cells. 29(3):486-495. Magnuson, BA., Schiefer, HB., Hancock, DS., Bhatti, AR., 1987. Cardiovascular effects of mycotoxin T-2 after topical application in rats. Can J Physiol Pharmacol. 65(5): 799-802. Nelson, PE., Dignani, MC., Anaissie, EJ., 1994. Taxonomy, biology, and clinical aspects of Fusarium species. Clin Microbiol Rev. 7(4): 479-504. Prigione, A., Fauler, B., Lurz, R., Lehrach, H., Adjaye, J., 2010. The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. Stem Cells. 28:721-733. Rezvanfar, MA., Hodjat, M., Abdollahi, M., 2016. Growing knowledge of using embryonic stem cells as a novel tool in developmental risk assessment of environmental toxicants.Life Sci. pii: S0024-3205(16)30308-3. [Epub ahead of print] Rohwedel, J., Guan, K., Hegert, C., Wobus, AM., 2001. Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects. Toxicol In Vitro. 15(6):741-753. Rolletschek, A., Blyszczuk, P., Wobus, AM., 2004. Embryonic stem cell-derived cardiac, neuronal and pancreatic cells as model systems to study toxicological effects. Toxicol Lett 149:361-369. Rotter, BA., Prelusky, DB., Pestka, JJ., 1996. Toxicology of deoxynivalenol (vomitoxin). J Toxicol Environ Health. 48(1):1-34. Sart, S., Song, L., Li, Y., 2015. Controlling Redox Status for Stem Cell Survival, Expansion, and Differentiation. Oxid Med Cell Longev. 2015:1-14. Schuster, A., Hunder, G., Fichtl, B., Forth, W., 1987. Role of lipid peroxidation in the toxicity of T-2 toxin. Toxicon. 25(12): 1321-1328. Schmelter, M., Ateghang, B., Helmig, S., Wartenberg, M., Sauer, H., 2006. Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation. FASEB J. 20(8):1182-1184. 18
Sehata, S., Teranishi, M., Atsumi, F., Uetsuka, K., Nakayama, H., Doi, K., 2003. T-2 Toxin-induced morphological changes in pregnant rats. J Toxicol Pathol. 16:59-65. Sehata, S., Kiyosawa, N., Atsumi, F., Ito, K., Yamoto, T., Teranishi, M., Uetsuka, K., Nakayama, H., Doi, K., 2005. Microarray analysis of T-2 toxin-induced liver, placenta and fetal liver lesions in pregnant rats. Exp Toxicol Pathol. 57(1):15-28. St John, JC., Amaral, A., Bowles, E., Oliveira, JF., Lloyd, R., Freitas, M., Gray, HL., Navara, CS., Oliveira, G., Schatten, GP., Spikings, E., Ramalho-Santos, J., 2006. The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells. Methods Mol Biol. 331:347-374. Turrens, JF., 2003. Mitochondrial formation of reactive oxygen species. J Physiol. 552(Pt 2):335-344. Vila`, B., Jaradat, ZW., Marquardt, RR., Frohlich, AA., 2002. Effect of T-2 toxin on in vivo lipid peroxidation and vitamin E status in mice. Food Chem Toxicol. 40(4):479-486. Wanet, A., Arnould, T., Najimi, M., Renard, P., 2015. Connecting Mitochondria, Metabolism, and Stem Cell Fate.Stem Cells Dev. 2015 24(17):1957-1971. Wu, J., Jing, L., Yuan, H., Peng, SQ., 2011. T-2 toxin induces apoptosis in ovarian granulosa cells of rats through reactive oxygen species-mediated mitochondrial pathway. Toxicol Lett. 2 02(3):168-177. Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A., Cinti, S., Lowell, B., Scarpulla, RC., Spiegelman, BM., 1999. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell. 98:115-124. Yagen, B., Bialer, M., 1993. Metabolism and pharmacokinetics of T-2 toxin and related trichothecenes. Drug Metab Rev. 25(3): 281-323. Yang, L., Yu, Z., Hou, J., Deng, Y., Zhou, Z., Zhao, Z., Cui, J., 2016. Toxicity and oxidative stress induced by T-2 toxin and HT-2 toxin in broilers and broiler hepatocytes.Food Chem Toxicol. 87:128-137.
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Figure Legends Fig. 1 Murine ES cells spontaneously differentiated into cardiac clusters (A, B,C) and T-2 toxin inhibited the differentiation ability of ES cells (D). Data were mean ± SD from three independent experiments, *P<0.05 versus control values Fig.2 T-2 toxin decreased the mitochondrial biogenesis murine ES cells. Fluorescence micrograph observation(A-D): A, control; B, T-2 toxin 24h; C, T-2 toxin 72h; D, T-2 toxin 120h. Ultrastructural observation by TEM photo (E-H): E, control; F, T-2 toxin 24h; G, T-2 toxin 72h; H, T-2 toxin 120h. Fig. 3 Effects of T-2 toxin treatment for different time on ROS generation. These results show that the redox balance was broken and oxidative stress was induced. Data were mean ± SD from three independent experiments, *P<0.05 versus control values Fig.4 T-2 toxin effect the expression of mitochondrial biogenesis-related proteins in differentiated murine ES cells. Identical results were obtained from three independent experiments. Data were mean ± SD. *P<0.05 versus control Fig.5 The effect of Trolox on the mitochondrial biogenesis and differentiation of murineES cells. Differentiated murine ES cells were pretreated with Trolox (200 μM) for 30 min, and T-2 toxin (0.5 ng/ml) treatment for 72 h. Trolox decreased the accumulation of ROS induced by -2 toxin (A,B). Trolox recoved mitochondrial biogenesis inhibited by -2 toxin (C-F): C, control; D, Trolox control; E, T-2 toxin 72h; F, Trolox pretreatment + T-2 toxin 72 h. Trolox increase the expression of mitochondrial biogenesis-related proteins decreased by -2 toxin (G). Data were mean ± SD from three different experiments, *P<0.05 versus control; #P<0.05, compared with the T-2 toxin treatment group
Fig. 6 Modulation of ROS level regulates the stem cell fate decision. (cited from Controlling Redox Status for Stem Cell Survival, Expansion, and Differentiation, 2015.Sébastien Sart, et al)
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S0378-4274(16)32252-4 http://dx.doi.org/doi:10.1016/j.toxlet.2016.06.2103 TOXLET 9477
To appear in:
Toxicology Letters
Received date: Revised date: Accepted date:
19-4-2016 19-6-2016 26-6-2016
Please cite this article as: Fang, Haiqin, Cong, Liangzi, Zhi, Yuan, Xu, Haibin, Jia, Xudong, Peng, Shuangqing, T-2 toxin inhibits murine ES cells cardiac differentiation and mitochondrial biogenesis by ROS and p-38 MAPK-mediated pathway.Toxicology Letters http://dx.doi.org/10.1016/j.toxlet.2016.06.2103 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
T-2 toxin inhibits murine ES cells cardiac differentiation and mitochondrial biogenesis by ROS and p-38 MAPK-mediated pathway
Haiqin Fanga,b†, Liangzi Congc†, Yuan zhi b, Haibin Xu b, Xudong Jiab,, Shuangqing Penga
a
Evaluation and Research Centre for Toxicology, Institute of Disease Control and
Prevention, Academy of Military Medical Sciences, Beijing 100071, China b
Key Laboratory of Food Safety Risk Assessment of Ministry of Health, China
National Center for Food Safety Risk Assessment, Beijing 100021, China c
Huaiyin District Center for Disease Control and Prevention, Jinan, Shandong
Corresponding author. Tel./fax: 8610-66948462, E-mail address: [email protected] (S Peng); Tel./fax: +8610 67770977, E-mail address: [email protected] (X Jia). † These authors contributed equally.
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Highlights T-2 toxin induced oxidative stress in mESCs in a time-dependent manner. T-2 toxin inhibited mESCs differentiated into cardiacmyocytes during spontaneous differentiation. T-2 toxin inhibited mitochondrial biogenesis of mESCs by ROS, and then inhibited the differentiation of mESCs. The effect of T-2 toxin on mESCs differentiation and mitochondrial biogenesis was partly responsible for the p38 MAPK mediated by ROS. Mitochondrial biogensis and mitochondrial pathway plays an important role in the mechnism of T-2 embryotoxicity.
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 0.5 ng/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 (200 μM) for 30 min, then exposed to Trolox (200 μM) and T-2 toxin (0.5 ng /mL) for 72h.
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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.
Key Words: T-2 toxin, mitochondrial biogenesis, cardiac differentiation, ROS, p38MAPK, PGC-1α
1. Introduction As a cytotoxic fungal metabolite that belongs to the trichothecene mycotoxin family, T-2 toxin is produced by various species of Fusarium and can infect corn, wheat, barley, and rice crops in the field or during storage, being characteristically stable under changing environmental conditions (Yagen et al., 1993; Desjardins et al., 1993). Ingestion by humans or livestock of cereals contaminated by T-2 toxin can cause adverse reactions, such as vomiting, diarrhea, and even death (Rotter et al., 1996; Nelson et al., 1994; Magnuson et al., 1987). In view of the great harm to the health of humans and livestock, the toxicological effects of T-2 toxin was reported in the Joint Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives (FAO/WHO 2002) .
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On experimental animals, livestock, and humans, T-2 toxin has been shown to produce a variety of toxic effects, including brain lesions, hematopoietic, lymphoid, gastrointestinal tissues, suppresses reproductive organ functions, and developmental Toxicity (Yagen et al., 1993; Desjardins et al., 1993; Escrivá et al., 2015). T-2 toxin readily passes through the placenta and is distributed to fetal tissues (Lafarge-Frayssinet et al., 2002), resulting in embryo/fetal death, fetal brain damage, fetal liver damage, and fetal bone malformation (Blakey et al., 1987; Sehata et al., 2003; Sehata et al., 2005; Doi et al, 2008). Embryonic stem cell test (EST), which has been approved by the European Centre for the Validation of Alternative Methods (ECVAM) (Genschow et al., 2002), is a novel model in the evaluation for chemical embryotoxicity and developmental risk assessment of environmental toxicants (Rezvanfar et al., 2016), and a good tool in investigation underlying mechanisms. EST consists of two sets of procedures: cytotoxicity tests conducted with the mESC line, D3, and a differentiation assay using D3 cells. In our previous study, the IC50D3 of T-2 toxin, which expresses the cytotoxicity of T-2 toxin to embryonic stem cell D3, was 1.5 ng/ml, and the ID50 of T-2 toxin, which expresses as the concentration of test chemical inhibiting the development of contracting cardiac muscle cells by 50% was 0.57ng/ml (Fang et al., 2012). Studies have provided evidence that proper mitochondrial biogenesis and function is essential to proliferation and differentiation of ES cells. It is suggested that mitochondrial biogenesis and metabolic switches might be hallmarks of the ES cell differentiation processes (Wanet et al., 2015). In self-renewal state, ES cells were supported by glycolysis metabolism and by mitochondrial properties including low mitochondrial DNA (mtDNA) copy number, immature organelle shape with under-developed cristae, and low levels of oxidative stress (Prigione et al., 2010; St John et al., 2006). However, during differentiation, mitochondria in mouse and human ES cells dramatically increased in number, cristae elongated, and an extensive reticular network of tubular structures generated (Prigione et al., 2010; St John et al., 2006; Chung et al., 2007; Facucho-Oliveira et al., 2007; Mandal et al., 2011). 4
Oxidative stress is one of the most important underlying toxic mechanisms for T-2 toxin. It was reported that free radicals, including reactive oxygen species, were generated by T-2 toxin both in vitro and in vivo, and then lipid peroxidation leading to changes in membrane integrity and cellular redox signaling (Chang et al., 1988; Fang et al., 2012; Schuster et al., 2004; Wu et al., 2011; Yang et al., 2016). It was reported that mitochondria was an derect target in human Kashin-Beck disease induced by T-2 toxin (Liu et al., 2014). Exposure to T-2 toxin can reduce activities of mitochondrial complexes III, IV and V, MMPand the cellular ATP, and increased intracellular ROS. In our previous study, we demonstrated that T-2 toxin was a potent embryonic toxin and mitochondria was an important target. T-2 toxin induced oxidative damage in differentiated mouse ES cells, including a significant decrease of glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity, and an increase in the ROS formation and malonaldehyde (MDA) contention. Moreover, T-2 toxin inhibited mESCs mitochondrial function, including mitochondrial membrane potential (MMP) loss, and induced apoptosis by mitochondrial pathway in differentiated mESCs (Fang et al., 2012). It is assumed that mitochondria biogenesis plays an important role in ES cells differentiation and mitochondria are an major target of T-2 toxin. The present study was undertaken to observe the effect of T-2 toxin on mitochondrial biogenesis and differentiation ability in murine differentiated ES cells, and to investigate the underlying mechanism of T-2 toxin embryonic toxicity by ROS-mediated mitochondrial pathway.
2. Materials and Methods 2.1 Reagents T-2 toxin was kindly provided by the Evaluation and Research Centre for Toxicology, Institute of Disease Control and Prevention, Academy of Military Medical Sciences. T-2 toxin was dissolved in DMSO to prepare a stock solution of 1 mg/ml and diluted to final concentrations with knockout DMEM. Dulbecco’s modified Eagle’s minimal essential medium (DMEM), knockout DMEM and non-essential amino acids were 5
purchased from Gibco (Gibco BRL, Germany). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carbonsaure (Trolox), β-mercaptoethanol, and 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma Aldrich (Munich, Germany). Fetal bovine serum (FBS) was obtained from Hyclone (Logan, USA). Mouse leukemia inhibitory factor (mLIF) was purchased from Chemicon (Milipore, USA). Anti-PGC-1α, anti-NRF-1, anti-mtTFA, anti-COXIV, anti-p-p38MAPK, anti-p38MAPK, anti-α-actinin, and anti-GADPH antibodies were obtained from Abcam Biotechnology, Inc (Abcam, USA). Enhanced chemiluminescence (ECL) reagent were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, USA). All other reagents were of analytic grade. 2.2 Embryonic stem cell culture and T-2 toxin treatment The murine ES D3 cells (American Type Culture Collection, CRL-1934) were cultivated in an undifferentiated state on feeder of mouse embryonic fibroblasts (MEF) as described previously . Cultures of differentiating ES cells were established by the formation of EBs in hanging drop cultures with the differentiation medium as previously described (Fang et al., 2012). After cultivated in hanging drops for 3 days and suspended in Petri dishes for another 2 days, EBs were plated separately onto gelatin-coated 24-well culture plates in the differentiation medium on day 5. At the time of EBs plating 24, 72 and 120h, 0.5ng/ml T-2toxin were added in culture medium, and EBs were harvested at the time of plating 144h. In another word, EBs were exposed to T-2 toxin for 120, 72, 24, and 0h respectively. Cultures were observed daily with an inverted microscope for observing the appearance of contracting clusters and counting the numbers of beating EBs. In some experiments, ES cells were pre-treated with the antioxidant Trolox (200 μM) for 30 min, then exposed to Trolox (200 μM) and T-2 toxin (0.5 ng/ml) for 72h. 2.3 Western blot analysis Western blot analysis for PGC-1α, NRF-1, mtTFA, COXIV, p-p38MAPK, p38 MAPK, α-actinin, and GAPDH expression were performed as previously described (Fang et al., 2012). After incubation with 0.5 ng/ml T-2 toxin for 0, 24, 72, and 120 hours, differentiated ES cells were washed in PBS and lysed in lysis buffer (50 mM 6
Tris-HCl, 150 mM NaCl, 1% NP-40, pH 7.5) containing protease-inhibitor (1 mM PMSF). Homogenized tissues were then centrifuged at 4 °C and 14000 g for 15 min and supernatants were subjected to Western blot analysis. Protein (30 μg) was loaded for 1-dimensional 12% SDS-PAGE. Detection was measuring using the chemiluminescence of the ECL reagent. The photographs generated were quantitatively analyzed for PGC-1α, NRF-1, mtTFA, COXIV, p-p38MAPK, p38 MAPK, α-actinin, protein levels with a Quantity One image densitometer. The molecular weights of the protein bands were determined by reference to the standard molecular weight markers. Protein levels were standardized by comparison with GAPDH. 2.4 Confocal laser scanning microscopy Differentiated ES cells that had been grown on coverslips were fixed for 20 min in methanol at -20°C, followed by permeabilization in 0.1% Tween 20 in phosphate-buffered solution (PBS). After washing in PBS 3 times, the EB on coverslips were transferred to PBS containing 10% goat serum (Sigma, USA) for 30 min at room temperature. The EBs were then placed into PBS containing mouse monoclonal anti-α-actinin (Sigma, USA; dilution 1:100) and incubated overnight at 4 °C. The EB were washed in PBS 3 times, followed by incubation in PBS containing the FITC-conjugated antimouse IgG (Sigma, USA; dilution 1:500). Fluorescence recordings were performed by means of confocal laser scanning setup (Leica TCS SP2, Bensheim, Germany) connected to an inverted microscope. 2.5 Transmission electron microscope scanning Transmission electron microscopy imaging was performed at the Instrument Center, Academy of Military Medical Sciences, China. Differentiated murine ES cells fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, were washed in 0.1 M cacodylate buffer and treated with 0.1% Millipore-filtered buffered tannic acid, postfixed with 1% buffered osmium tetroxide for 30 min, and stained with 1% Millipore-filtered uranyl acetate. The samples were washed several times in water, then dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in LX-112 medium. ES cells 7
were polymerized in a 60 °C oven for about 2 days. Ultrathin sections were cut in a Leica Ultracut microtome (Leica), stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a JEM 1010 transmission electron microscope (JEOL) at an accelerating voltage of 80 kV. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques). 2.6 Statistical analysis Data were expressed as mean ± S.D. and analyzed by one-way ANOVA, followed by LSD’s post hoc test by SPSS 11.5 statistical software. The level of significance was accepted as p<0.05.
3. Results 3.1 T-2 toxin inhibited the murine ES cells differentiation According to the protocol recommended by ECVAM, a differentiated ESC model was established. On the gelatin-coated plate, colonies of ES-D3 cells (Fig. 1A: arrows refer to colonies of ES-D3 cells) resulted in the establishment of the EST model by the formation of EBs in hanging drop cultures, and caused the cells to spontaneously differentiate into synchronously-contracting cardiac clusters derived from EBs on day 5 +5 (Fig. 1B). Staining with α-actinin, a cardiac myoblasts specific protein, which appeared as red fluorescence, was observed in differentiated ES-D3 cells (Fig. 1C), and significant decrease in expression of α-actinin were observed in differentiated ES cells following 72 and 120 h exposure to T-2 toxin at 0.5ng/ ml( Fig. 1D). As shown in Fig. 1F, there was no beating EB observed under the microscope light in 72 and 120 h groups, which indicated that T-2 toxin inhibited the differentiation of ES-D3 cells. 3.2 T-2 toxin decreased the mitochondrial biogenesis murine ES cells Mitochondrial morphology was evaluated in differentiated mouse ESCs by staining with a mitochondrial-specific dye, MitoTracker Red. Consistent with previous reports, we found that ESCs mitochondria were distributed as perinuclear clusters that appeared punctate and globular (Fig. 2A–2D), and ESCs mitochondria in normal group generated an extensive reticular network of tubular structures. Although the 8
common punctate and rudimentary appearance of mitochondria were seen in all differentiated ESCs, the amount of mitochondrial mass varied between independent groups of ESCs, that is, the ES cells of normal (Fig. 2 A) or T-2 toxin treatment 24 h (Fig. 2 B) appeared to have more mitochondria than T-2 toxin treatment 72 (Fig. 2 C)and 120 h (Fig. 2D). As shown in Fig. 2E-2H, compared with the normal differentiated ESCs (Fig. 2 E) or T-2 toxin treatment 24 h (Fig. 2 F), significant decrease in the mitochondrial number, deformation of mitochondria, and lack of complete cristae were observed through TEM in the groups of T-2 toxin exposed for 72 (Fig. 2 G)and 120 h (Fig. 2 H). It was obvious that T-2 toxin inhibited the mitochondrial biogenesis in differentiated murine ES cells after a long term exposure (72 and 120h). 3.3 T-2 toxin-induced oxidative stress in differentiated ES cells Oxidative stress is involved in the toxicity of trichothecene mycotoxins, including T-2 toxin. In the present study, differentiated ES cells were exposed to 0.5 ng/ml T-2 toxin for 0, 24, 72 and 120 h, and oxidative stress was monitored in terms of production of ROS. Significant increase in the ROS formation was seen after exposure to 24, 72, and 120 h (Fig. 3A, B). 3.4 T-2 toxin effected the expression of mitochondrial biogenesis-related proteins in differentiated murine ES cells The protein expression of mitochondrial biogenesis modulation factors p38MAPK, phosphorylated p38MAPK (p-p38), NRF-1, mTFA, and COXIV were examined in differentiated ES cells treated with 0.5 ng/ml T-2 toxin for 0, 24, 72 and 120 h followed by Western blot analysis of these proteins. In the mouse ES cells exposed to T-2 toxin 24h, the protein expression of p-p38 increased significantly, however, there was no significant change in other indexes in this group. In the groups of T-2 toxin treatment 72 and 120 h, except p38 protein, the expression of PGC-1ɑ, NRF-1, mTFA, and COXIV decreased significantly. (Fig. 4). 3.5 Trolox recovered the inhibition of mitochondrial biogenesis and differentiation ability induced by T-2 toxin in differentiated ES cells
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To confirm the role of ROS in T-2 toxin-inhibited differentiation through the inhibition of mitochondrial biogenesis by p-38MAPK, differentiated ES cells were pre-treated with the antioxidant Trolox (200 μM) for 30 min, and exposure to Trolox was continued during subsequent T-2 toxin (0.5 ng/ml) treatment for 72 h. Trolox significantly inhibited T-2 toxin induced increase of ROS generation by 21% (Fig.5A,B). As Fig. 5 shown, compared with the control group(Fig.5C), ES cells treated with Trolox alone did not change in mitochondrial biogenesis (Fig.5D). However, compared with the ES cells exposed to T-2 toxin for 72 h (Fig.5E), the amount of mitochondrial mass in Trolox pretreated was greater (Fig.5F). As mitochondrial biogenesis-associated protein expression, compared with the ES cells exposed to T-2 toxin for 72 h, Trolox increased the expression of p-p38 by 22.9%, PGC-1α by 27.3%, NRF-1 by 27.4%, mTFA by 26.9% and COXIV by 26.7% (Fig.5G).
4. Discussion In our previous study, T-2 toxin’s was evaluated strong embryonic toxicity by EST (Fang et al., 2012). Following the IC50D3 for T-2 toxin, different concentration of T-2 toxin were selected to investigate the cytotoxicity mechanism of T-2 toxin to ESC D3. ROS-mediated mitochondrial pathway was found to be involved in T-2 toxin-induced apoptosis in murine differentiated ES cells in a dose-dependent manner(Fang et al., 2012). In the present study ( according to the ID50 of T-2 toxin is 0.57 ng/ml) a concentration of 0.5 ng/ml and different exposure time (24 h, 72 h, 120 h) were chosen to observe the effect of T-2 toxin on ESC differentiation, and try to investigate the modulation of T-2 toxin on ES mitochondrial biogenesis and ES differentiation. Differentiated ES cells can be used as a model to investigate the mechanism of chemical’s embryonic toxicity. During ES cells differentiation, ES cells spontaneously differentiate into different cell types of all three germ layers in vitro, tissue-specific genes and proteins are expressed in a developmentally, regulated manner, recapitulating the processes of early embryonic development (Guan et al., 1999; Rohwedel et al., 2001; Rolletschek et al., 2004). In the protocal of the embryonic stem 10
cell test (EST) published by ECVAM, the cardiac differentiation was defined as the marker of mESCs differentiation (Genschow et al., 2002). In the present study, the differentiation ability of ES cells were observed both as spontaneously contracting cardiomyocytes and as the expression of cardiac myoblasts -specific protein α-actinin. However, this ability could be inhibited when T-2 toxin exposure for 72 and 120 h. Metabolism and mitochondria regulate ES cell fate (Wanet et al., 2015; Folmes et al., 2016). Recent studies have demonstrated that differentiated ES cells show altered mitochondrial function and metabolic profiles and production of reactive oxygen species. This raises an emerging paradigm about the role of mitochondria in stem cell biology and urges the need to identify mitochondrial pathways involved in these processes (Lopes et al., 2016).Although anaerobic metabolism is sufficient to meet the energy demand of undifferentiated mouse and human ESCs, successful differentiation of ESCs requires activation of mitochondrial aerobic metabolism in order to promote the synthesis of greater levels of ATP and the maintenance of homeostasis in the differentiated cell. An increase in mitochondrial oxidative phosphorylation has to occur for differentiation to succeed (Lopes et al., 2016). Indeed, fully differentiated cells such as neurons and cardiomyocytes express high levels of nDNA- and mtDNA-encoded electron transport chain (ETC) subunits, produce ATP through oxidative phosphorylation (OXPHOS) (Cho et al., 2006; Sart et al., 2015) and contain enriched mtDNA content (Prigione et al., 2010; St John et al., 2006; Chung et al., 2007; Facucho-Oliveira et al., 2007). In addition, differentiation of ESCs results in changes in mitochondrial structure, morphology, tubular structure, numerous elongated cristae, dense matrices, and high membrane potential suggesting the initiation of metabolic activity through OXPHOS (Cho et al., 2006; Facucho-Oliveira et al., 2007; Sart et al., 2015). In the present study, numerous mitochondria with complete cristae, and the extensive reticular network of tubular structures were shown in differentiation ES cells of control groups. COXIV, a symbol complex of mtDNA-encoded ETC subunits was expressed
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strongly in differential mESCs. These results implied that the cardiomyocyte differentiation of ES cells was accompanied by an organization of mitochondrial biogenesis. Compared with the control group, we found the significant decrease of the mitochondrial number, deformation of mitochondrial structure, lack of complete mitochondrial cristae, and significant decrease in COXIV protein expression were observed in the groups of T-2 toxin exposed for 72 and 120 h. ROS play dual role in stem cell homeostasis, depending on its level of production. Once cells start to produce large amount of ATP through the mitochondrial ETC, ROS will be produced as by-products of oxidative phosphorylation (Sart et al., 2015). Nevertheless, the intracellular levels of ROS are higher in differentiating ESCs than in undifferentiated ESCs due to the increase in OXPHOS metabolism (Cho et al., 2006; Turrens et al., 2003). Although an increase in ROS levels might have a role in cell signaling and regulation of proliferation and differentiation, exposure to low levels of ROS has been reported to enhance ESC differentiation towards the cardiomyogenic and vascular lineages, whereas continuous exposure to high levels of ROS results in inhibition of differentiation (Schmelter et al., 2006). As Fig.6 shown, tight regulation of biochemical and biomechanical environment can control stem cell oxidative status and thus the stem cell fate decision (Sart et al., 2015). An increasing body of evidence show that T-2 toxin can induce oxidative damage in biological systems due to increased generation of hydroxyl radical(Chang et al., 1988; Schuster et al., 2004; Liu et al., 2014; Yang et al., 2016). In the present study, T-2 toxin stimulated ROS generation in a time-dependent manner was observed. Accompanied with cellular continuously increasing ROS, T-2 toxin inhibited the differentiation of murine ESCs exposed to T-2 toxin at 72 and 120 h. We also found T-2 toxin decreased mitochondrial biogenesis after T-2 toxin treatment at 72 and 120 h. After the inhibition of T-2 toxin on mitochondrial biogenesis and differentiation ability were found (0.5 ng/ml T-2 toxin for 72 and 120h), we focused our study on the modulation factors in the ROS-mediated mitochondrial pathway in differentiated mESCs.
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PGC-1α (peroxisome-proliferator-activated receptor γ coactivator-1α) is a co-transcriptional regulation factor that induces mitochondrial biogenesis by activating different transcription factors, including nuclear respiratory factor 1 and nuclear respiratory factor 2, which activate mitochondrial transcription factor A. The latter drives transcription and replication of mitochondrial DNA. Matthew reported that PGC-1α also played a key role in regulating hESC-derived cardiomyocyte mitochondrial respiration, contractile automaticity, and superoxide production (Matthew et al., 2013). PGC-1α itself is regulated by several different key factors involved in mitochondrial biogenesis. AMPK (AMP-activated protein kinase) is of major importance, and AMPK acts as an energy sensor of the cell and works as a key regulator of mitochondrial biogenesis. In the signaling events leading to activation of the transcription cascade, p38 MAPK has been suggested to play an important role in myogenic cell differentiation (Cho et al., 2006). Transgenic mice specifically overexpressing p38 MAPK in skeletal muscle show enhanced PGC-1α expression and increased mitochondrial proteins (Sart et al., 2015). Interestingly, accumulating evidence indicates that the p38 MAPK plays a critical role in cardiomyocyte differentiation of murine carcinoma stem cells and ES cells in vitro (Schmelter et al., 2006). Ding reported that intracellular ROS generation and p38MAPK could be triggered when murine ES cells are exposed to icariin which resulted in cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro (Ding et al., 2008). In the present study, p38MAPK was spontaneously activated in T-2 toxin exposure at 24h, and the mitochondrial biogenesis-related protein expression showed an increased trend in same group, however, there was no significant difference in statistics in mitochondrial biogenesis and differentiation. Moreover, the inhibition of p38MAPK phosphorylation was accompanied with ROS continuous accumulation, when T-2 toxin was present long term (72 and 120h). These results indicated that ROS at low level can be a signal factor to activate p38MAPK; however, the activation effect of ROS on
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p38MAPK gradually disappeared with an overwhelming accumulation after long-term T-2 toxin exposure. PGC-1α, as a direct downstream target of the p38 MAPK, also as the master regulator of mitogenesis, can directly or indirectly stimulate the transcription of OXPHOS genes and the mitochondrial transcription factor A (mtTFA) gene (Kelly et al., 2004; Lin et al., 2002; Akimoto et al., 2005). In the present study, the expression of PGC-1α, NRF-1, mTFA, and COXIV decreased significantly in T-2 toxin exposure at 72 and 120 h. It suggests that accumulation of ROS induced by T-2 toxin inhibited the phosphorylate p38MAPK and subsequently inhibited of PGC-1α-regulated mitochondrial biogenesis in the differentiation process of murine embryonic stem cells in a time-dependent manner in vitro. To confirm our hypothesis, the antioxidant Trolox, a water-soluble vitamin E derivative, was used in our study. It is reported that vitamin E supplementation can reduce the DNA damage or lipid peroxidation induced by T-2 toxin in vivo (Vila` et al., 2004). In our experiments, T-2 toxin induced ROS generation in murine differentiated ES cells, which was significantly inhibited by Trolox at a concentration of 200 μM. This result showed that the antioxidant participated in differentiated ES cells’ redox reactions and neutralized the oxidative stress induced by T-2 toxin. Meanwhile, T-2 toxin-inhibited the mitochondrial biogenesis in differentiated murine ES cells was significantly recovered after Trolox treatment. However, unlike the expression of COXIV, there was no significant recovery of the expression of α-actinin. This suggests that the differentiation of ES cells is complicated and sensitive, and the factors what regulate stem cell differentiation remains to be determined. Our data indicated that a p38MAPK-involved and ROS-mediated mitochondrial pathway played an important role in the T-2 toxin-inhibited mitochondrial biogenesis and cardiac differentiation of mESCs. However, the differences of relevant mitochondrial biogenesis and ES cell differentiation indicators between the control group and the group treated with T-2
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toxin plus Trolox are very significant, suggesting that other pathways might participate in the process in addition to the p38MAPK-involved and ROS-mediated pathway. Taken together, we demonstrated T-2 toxin inhibited mitochondrial biogenesis-triggered by the phosphorylation of the p38 MAPK, which was mediated by ROS, and then inhibited murine ES cells differentiation in a time-dependent manner. We speculate that this might be a possible underlying molecular mechanism of T-2 toxin induced embryonic toxicity. This study focuses on the metabolic profile of ESC and how mitochondrial biogensis can influence the differentiation processes. Indeed, mitochondria appear to have a crucial role in the maintenance of a pluripotent state and in differentiation. Therefore, in vitro differentiation of ESCs into cardiomyocytes can be compromised if those mechanisms are impaired, such as the influence of environmental toxicants. We also found ROS played dual role in ES cells differentiation into cardiomyocytes. Future research should shed light on how mitochondrial impairment occurring in differentiation stage may contribute for the etiopathogenesis of cardio developmental and how to prevent cardiac developmental disorders by mitochondrial pathway.
Acknowledgements This project was supported by the High-Level Talents and Experts Introducing Program for “Project 523” of the China National Center for Food Safety Risk Assessment (1311613106702), Natural Science Foundation of Beijing City (7142128), and National Natural Science Foundation of China (81302462, 81172699).
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References Akimoto, T., Pohnert, SC., Li, P., Zhang, M., Gumbs, C., Rosenberg, PB., Williams, RS., Yan, Z., 2005. Exercise stimulates Pgc-1α transcription in skeletal muscle through activation of the p38 MAPK pathway. J. Biol. Chem. 2005; 280(20):19587-19593. Birket, MJ., Casini, S., Kosmidis, G., Elliott, DA., Gerencser, AA., Baartscheer, A., Schumacher, C., Mastroberardino, PG., Elefanty, AG., Stanley, EG., Mummery, CL., 2013. PGC-1ɑ and reactive oxygen species regulate human embryonic stem cell-derived cardiomyocyte function. Stem Cell Reports. 1(6):560-574. Blakley, BR., Hancock, DS., Rousseaux, CG., 1987. Embryotoxic effects of prenatal T-2 toxin exposure in mice. Can J Vet Res. 51(3):399-403. Chang, IM., Mar, WC., 1988. Effect of T-2 toxin on lipid peroxidation in rats: elevation of conjugated diene formation. Toxicol Lett. 40(3):275-280. Cho, YM., Kwon, S., Pak, YK., Seol, HW., Choi, YM., Park, do J., Park, KS., Lee, HK., 2006. Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of human embryonic stem cells. Biochem Biophys Res Commu. 348 (2006) 1472-1478. Chung, S., Dzeja, PP., Faustino, RS., Perez-Terzic, C., Behfar, A., Terzic, A., 2007. Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells. Nat Clin Pract Cardiovasc Med. 4(suppl 1):S60-S67. Desjardins, AE., Hohn, TM., McCormick, SP., 1993. Trichothecene biosynthesis in Fusarium species: chemistry, genetics, and significance. Microbiol Rev. 57(3):595-604. Ding, L., Liang, XG., Hu, Y., Zhu, DY., Lou, YJ., 2008. Involvement of p38MAPK and Reactive Oxygen Species in Icariin-Induced Cardiomyocyte Differentiation of Murine Embryonic Stem Cells In Vitro. Stem Cell Dev. 17:751-760. Doi, K., Ishigami, N., Sehata, S., 2008. T-2 toxin-induced toxicity in pregnant mice and rats. Mol Sci. 9(11):2146-2148. Escrivá, L., Font, G., Manyes, L., 2015. In vivo toxicity studies of fusarium mycotoxins in the last decade: A review. Food Chem Toxicol. 78:185-206.
16
Facucho-Oliveira, JM., Alderson, J., Spikings, EC., Egginton, S., St John, JC., 2007. Mitochondrial DNA replication during differentiation of murine embryonic stem cells. J Cell Sci 120(pt22):4025-4034. Fang, H.,Wu, Y., Guo, J., Rong, J., Ma, L., Zhao, Z., Zuo, D., Peng, S., 2012. T-2 toxin induces apoptosis in differentiated murine embryonic stem cells through reactive oxygen species-mediated mitochondrial pathway. Apoptosis. 17(8):895-907. FAO/WHO, 2002. Evaluation of certain mycotoxins in food. Fifty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives. World Health Organ Tech Rep Ser. 906:i-viii, 1-62. Folmes, CD., Ma, H., Mitalipov, S., Terzic, A., 2016. Mitochondria in pluripotent stem cells: stemness regulators and disease targets. Curr Opin Genet Dev. 38:1-7. [Epub ahead of print] Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Bradym M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European centre for the validation of alternative methods. Altern Lab Anim 30(2):151-176. Guan, K., Rohwedel, J., Wobus, AM., 1999. Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro. Cytotechnology. 30(1-3):211-226. Kelly, DP., Scarpulla, RC., 2004. Transcriptional regulatory circuits controlling mitochondrial biogenesis and function. Genes Dev. 18 (2004) 357-368. Lafarge-Frayssinet, C., Chakor, K., Lafont, P., Frayssinet, C., 1990. Transplacental transfer of T2 toxin: pathological effect. J Environ Pathol Toxicol Oncol. 10(1-2):64-68. Lin, J., Wu, H., Tarr, PT., Zhang, CY., Wu, Z., Boss, O., Michael, LF., Puigserver, P., Isotani, E., Olson, EN., Lowell, BB., Bassel-Duby, R., Spiegelman, BM., 2002. Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres. Nature. 418:797-801. Liu, J., Wang, L., Guo, X., Pang, Q., Wu, S., Wu, C., Xu, P., Bai, Y., 2014. The role of mitochondria in T-2 toxin-induced human chondrocytes apoptosis. PLoS One. 9(9):e108394.
17
Lopes, C., Rego, AC., 2016. Revisiting Mitochondrial Function and Metabolism in Pluripotent Stem Cells: Where Do We Stand in Neurological Diseases? Mol Neurobiol. Feb 18. [Epub ahead of print] Mandal, S., Lindgren, AG., Srivastava, AS., Clark, AT., Banerjee, U., 2011. Mitochondrial function controls proliferation and early differentiation potential of embryonic stem cells. Stem Cells. 29(3):486-495. Magnuson, BA., Schiefer, HB., Hancock, DS., Bhatti, AR., 1987. Cardiovascular effects of mycotoxin T-2 after topical application in rats. Can J Physiol Pharmacol. 65(5): 799-802. Nelson, PE., Dignani, MC., Anaissie, EJ., 1994. Taxonomy, biology, and clinical aspects of Fusarium species. Clin Microbiol Rev. 7(4): 479-504. Prigione, A., Fauler, B., Lurz, R., Lehrach, H., Adjaye, J., 2010. The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells. Stem Cells. 28:721-733. Rezvanfar, MA., Hodjat, M., Abdollahi, M., 2016. Growing knowledge of using embryonic stem cells as a novel tool in developmental risk assessment of environmental toxicants.Life Sci. pii: S0024-3205(16)30308-3. [Epub ahead of print] Rohwedel, J., Guan, K., Hegert, C., Wobus, AM., 2001. Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects. Toxicol In Vitro. 15(6):741-753. Rolletschek, A., Blyszczuk, P., Wobus, AM., 2004. Embryonic stem cell-derived cardiac, neuronal and pancreatic cells as model systems to study toxicological effects. Toxicol Lett 149:361-369. Rotter, BA., Prelusky, DB., Pestka, JJ., 1996. Toxicology of deoxynivalenol (vomitoxin). J Toxicol Environ Health. 48(1):1-34. Sart, S., Song, L., Li, Y., 2015. Controlling Redox Status for Stem Cell Survival, Expansion, and Differentiation. Oxid Med Cell Longev. 2015:1-14. Schuster, A., Hunder, G., Fichtl, B., Forth, W., 1987. Role of lipid peroxidation in the toxicity of T-2 toxin. Toxicon. 25(12): 1321-1328. Schmelter, M., Ateghang, B., Helmig, S., Wartenberg, M., Sauer, H., 2006. Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation. FASEB J. 20(8):1182-1184. 18
Sehata, S., Teranishi, M., Atsumi, F., Uetsuka, K., Nakayama, H., Doi, K., 2003. T-2 Toxin-induced morphological changes in pregnant rats. J Toxicol Pathol. 16:59-65. Sehata, S., Kiyosawa, N., Atsumi, F., Ito, K., Yamoto, T., Teranishi, M., Uetsuka, K., Nakayama, H., Doi, K., 2005. Microarray analysis of T-2 toxin-induced liver, placenta and fetal liver lesions in pregnant rats. Exp Toxicol Pathol. 57(1):15-28. St John, JC., Amaral, A., Bowles, E., Oliveira, JF., Lloyd, R., Freitas, M., Gray, HL., Navara, CS., Oliveira, G., Schatten, GP., Spikings, E., Ramalho-Santos, J., 2006. The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells. Methods Mol Biol. 331:347-374. Turrens, JF., 2003. Mitochondrial formation of reactive oxygen species. J Physiol. 552(Pt 2):335-344. Vila`, B., Jaradat, ZW., Marquardt, RR., Frohlich, AA., 2002. Effect of T-2 toxin on in vivo lipid peroxidation and vitamin E status in mice. Food Chem Toxicol. 40(4):479-486. Wanet, A., Arnould, T., Najimi, M., Renard, P., 2015. Connecting Mitochondria, Metabolism, and Stem Cell Fate.Stem Cells Dev. 2015 24(17):1957-1971. Wu, J., Jing, L., Yuan, H., Peng, SQ., 2011. T-2 toxin induces apoptosis in ovarian granulosa cells of rats through reactive oxygen species-mediated mitochondrial pathway. Toxicol Lett. 2 02(3):168-177. Wu, Z., Puigserver, P., Andersson, U., Zhang, C., Adelmant, G., Mootha, V., Troy, A., Cinti, S., Lowell, B., Scarpulla, RC., Spiegelman, BM., 1999. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell. 98:115-124. Yagen, B., Bialer, M., 1993. Metabolism and pharmacokinetics of T-2 toxin and related trichothecenes. Drug Metab Rev. 25(3): 281-323. Yang, L., Yu, Z., Hou, J., Deng, Y., Zhou, Z., Zhao, Z., Cui, J., 2016. Toxicity and oxidative stress induced by T-2 toxin and HT-2 toxin in broilers and broiler hepatocytes.Food Chem Toxicol. 87:128-137.
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Figure Legends Fig. 1 Murine ES cells spontaneously differentiated into cardiac clusters (A, B,C) and T-2 toxin inhibited the differentiation ability of ES cells (D). Data were mean ± SD from three independent experiments, *P<0.05 versus control values Fig.2 T-2 toxin decreased the mitochondrial biogenesis murine ES cells. Fluorescence micrograph observation(A-D): A, control; B, T-2 toxin 24h; C, T-2 toxin 72h; D, T-2 toxin 120h. Ultrastructural observation by TEM photo (E-H): E, control; F, T-2 toxin 24h; G, T-2 toxin 72h; H, T-2 toxin 120h. Fig. 3 Effects of T-2 toxin treatment for different time on ROS generation. These results show that the redox balance was broken and oxidative stress was induced. Data were mean ± SD from three independent experiments, *P<0.05 versus control values Fig.4 T-2 toxin effect the expression of mitochondrial biogenesis-related proteins in differentiated murine ES cells. Identical results were obtained from three independent experiments. Data were mean ± SD. *P<0.05 versus control Fig.5 The effect of Trolox on the mitochondrial biogenesis and differentiation of murineES cells. Differentiated murine ES cells were pretreated with Trolox (200 μM) for 30 min, and T-2 toxin (0.5 ng/ml) treatment for 72 h. Trolox decreased the accumulation of ROS induced by -2 toxin (A,B). Trolox recoved mitochondrial biogenesis inhibited by -2 toxin (C-F): C, control; D, Trolox control; E, T-2 toxin 72h; F, Trolox pretreatment + T-2 toxin 72 h. Trolox increase the expression of mitochondrial biogenesis-related proteins decreased by -2 toxin (G). Data were mean ± SD from three different experiments, *P<0.05 versus control; #P<0.05, compared with the T-2 toxin treatment group
Fig. 6 Modulation of ROS level regulates the stem cell fate decision. (cited from Controlling Redox Status for Stem Cell Survival, Expansion, and Differentiation, 2015.Sébastien Sart, et al)
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