Characterization of the modes of action of deoxynivalenol (DON) in the human Jurkat T-cell line.

http://informahealthcare.com/imt ISSN: 1547-691X (print), 1547-6901 (electronic) J Immunotoxicol, Early Online: 1–11 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/1547691X.2014.925995

RESEARCH ARTICLE

Characterization of the modes of action of deoxynivalenol (DON) in the human Jurkat T-cell line Madhumohan R. Katika1,2,3, Peter J. M. Hendriksen1,3, Henk van Loveren2,3,4, and Ad A. C. M. Peijnenburg1,3

Journal of Immunotoxicology Downloaded from informahealthcare.com by Selcuk Universitesi on 01/27/15 For personal use only.

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RIKILT-Institute of Food Safety, Wageningen University and Research Centre, Wageningen, the Netherlands, 2Department of Toxicogenomics, Maastricht University, Maastricht, the Netherlands, 3Netherlands Toxico-genomics Centre, Maastricht, the Netherlands, and 4National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands Abstract

Keywords

Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detected in cereals and grains. As such, DON poses a risk for many adverse health effects to human and animals. In particular, immune cells are very sensitive to DON, with the initiating step leading to toxicity being a binding to the eukaryotic 60S ribosomal subunit and induction of ribotoxic stress. The present study aimed to: (1) extend insight into the mechanism of action (MOA) of DON in immune cells; and (2) understand why immune cells are more sensitive to DON than most other cell types. Previously published microarray studies have described the effects of DON on immune cells. To build upon these findings, here, immunocytological and biochemical studies were performed using human T-lymphocyte Jurkat cells that were exposed for 3 h to 0.5 mM DON. Induction of ER stress by DON was confirmed by immunocytology demonstrating increased protein expression of two major ER stress markers ATF3 and DDIT3. T-cell activation was confirmed by induction of phosphorylation of protein kinases JNK and AKT, activation of NF-B (p65), and increased expression of NFAT target gene NUR77; each of these are known inducers of the T-cell activation response. Induction of an oxidative stress response was also confirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 and KEAP1, as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, this study showed that DON induced cleavage of caspase-3, an event known to mediate apoptosis. Taken together, these results allowed us to formulate a potential mechanism of action of DON in immune cells, i.e. binding to eukaryotic 60S ribosomal subunit ! ribotoxic stress ! ER stress ! calcium release from the ER into cytoplasm ! T-cell activation and oxidative stress ! apoptosis. It is proposed that immune cells are more sensitive to DON than other cell types due to the induction of a T-cell activation response by increased intracellular calcium levels.

Apoptosis, deoxynivalenol, ER stress, immunotoxicity, Jurkat cells, NF-B, NFAT, oxidative stress

Introduction Deoxynivalenol (DON) is a mycotoxin belonging to the type B trichothecenes group. DON is a biologically active secondary metabolite produced by various Fusarium strains (e.g. F. culmorum and F. graminearum) and is often found in wheat, barley, and maize (Rotter et al., 1996). DON is highly resistant to high temperatures and milling processes; as a result, its stability allows it to readily enter the food chain (Sugita-Konishi et al., 2006). Intake of high levels of DON by humans has been implicated in a number of incidents of intoxication, primarily in Asia (Bhat et al., 1989; Ramakrishna et al., 1989; Yoshizawa 1983). In one example, outbreaks of acute human illness in China during 1961–1991 were reported, with symptoms of nausea,

Address for correspondence: Dr Madhumohan R. Katika, RIKILTInstitute of Food Safety, Wageningen University and Research Centre, Wageningen, the Netherlands. Tel: 31317480284. E-mail: maddycdfd@ gmail.com

History Received 17 February 2014 Revised 30 April 2014 Accepted 14 May 2014 Published online 2 July 2014

vomiting, diarrhea, abdominal pain, headache, dizziness, and fever (Luo, 1994). As noted, ingestion of DON contaminated food products poses a health risk to human and animals (Bhat et al. 1989; Luo, 1988; Pestka 2010; Ramakrishna et al., 1989; Rotter et al., 1996; Yoshizawa 1983). The European and Food Safety Authority (EFSA) reported that DON is present in 44.6%, 43.5%, and 75.2% of unprocessed grains, processed food products, and feed samples, respectively (EFSA J., 2013). In some food products – including bread and cereals, DON levels up to 700 mg/kg have been detected (Rasmussen et al., 2003; Schollenberger et al., 2005; Soubra et al., 2009). An important question that persists is to what extent daily exposure to non-acute DON levels affects human health? Many models have been used to ascertain the potential immunotoxic risks from exposures to DON. For example, in mice, the immune system has been seen to be quite sensitive to DON. Oral daily exposure of BALB/c mice to DON at 410 mg/kg induced thymus atrophy and decreased spleen weight (Robbanabarnat et al., 1987). DON exposure was also reported to exacerbate infections with parasites, bacteria, or viruses across a wide range of animal


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host species (including non-rodents) (Antonissen et al., 2014; Pestka 2010). Thus, it is logical to assume then that DON exposure(s) might negatively impact on the immune response of humans as well. The latter view has been borne out. For example, ingestion of DON-contaminated foods caused adverse effects on human and animal health, particularly their immune responses (Pestka 2004, 2010; Rotter et al., 1996). At the cellular level, DON has been shown to modulate the function of T- and B-lymphocytes, NK cells, and macrophages in rodents (Moon & Pestka, 2002; Rotter et al., 1996). DON also has been seen to inhibit the proliferation of mouse and human lymphocytes in vitro (Meky et al., 2001). Further, low doses of DON were found to cause an increased production of cytokines and chemokines (Moon & Pestka, 2002, 2003), while high doses caused apoptosis among human blood monocytes, Jurkat T-cells, B-cells, and macrophages (Pestka et al., 1994, 2005b; Zhou & Pestka, 2003). Despite a large number of studies on the subject, the mechanisms of action of DON are not yet completely understood. It is known that DON binds the eukaryotic 60S ribosomal subunit leading to ribotoxic stress and inhibited protein synthesis (Pestka, 2008). DON also induces several MAP kinases (MAPK) known to stimulate production of cytokines and induction of apoptosis (Moon & Pestka, 2002; Pestka et al., 2005a; Zhou et al., 2003b). A recent study indicated DON inhibited cell proliferation in the mouse thymus and affected biological processes including function of ribosomes and mitochondria, T-lymphocyte activation, and apoptosis (van Kol et al., 2011). Our own studies of the effects of DON on whole genome mRNA expression in Jurkat cells and peripheral blood mononuclear cells (PBMC)—using DNA microarrays (Katika et al., 2012)—confirmed many of the known effects of DON on ribosomes, RNA/protein synthesis, and apoptosis. Previously unreported effects included the induction of both endoplasmic reticulum (ER) and oxidative stress, activation of calcium-mediated signaling, and both NFAT and NF-B pathways, as well as the induction of T-lymphocyte activation. The present study aimed to build upon/strengthen those findings using immunocytological and biochemical analyses of the effects in Jurkat cells specifically.

Materials and methods

J Immunotoxicol, Early Online: 1–11

Immunocytology Jurkat cells were exposed to a final concentration of 0.5 mM DON for 3 h. This exposure condition was based on outcomes of our previous microarray study (Katika et al., 2012) wherein Jurkat cells were exposed to 0.25 or 0.5 mM DON for 3, 6, or 24 h. It was seen there that exposure to 0.5 mM DON for 3 h clearly affected expression of genes involved in various bio-processes, including ER/oxidative stress, activation of NFAT and NF-B pathways, T-cell activation, and apoptosis. Further, that study showed that this DON exposure time and dose did not result in cytotoxicity. As such, this condition was selected for use in the present experiments. Upon exposure, Jurkat cells were immobilized on polyL-lysine coated slides (Memel-Glaser, Braunschweig, Germany) using mild cytospin centrifugation (5 min, 44 g) followed by incubation in 4% freshly prepared paraformaldehyde with 0.025% glutaraldehyde (in phosphate-buffered saline [PBS, pH 7.4]) for 30 min. After blocking with a solution of 1% bovine serum albumin (BSA)/0.01% Triton X-100 in PBS for 45 min, the cells were washed with 0.1% acetylated BSA (Aurion, Wageningen, NL) in PBS, prior to immunolabeling overnight at 4 C. Primary antibodies against ATF3, DDIT3, phospho-AKT 1/2/3, phosphoSAPK/JNK, NUR77, NRF2, KEAP1, phospho NF-B (p65), and cleaved caspase-3 were diluted 1:100 in 0.1% acetylated BSA in PBS. After extensive washing in 0.1% acetylated BSA in PBS, the cells were incubated with 300-fold diluted goat anti-rabbit IgG (H+L)-FITC or anti mouse-IgG1-FITC secondary antibody for 120 min at 37 C. The slides were then washed in PBS, mounted in Vectashield containing 1.5 mg/ml DAPI (Vectashield, Amsterdam, the Netherlands) and imaged with an LSM510 confocal microscope (Carl Zeiss, Germany). Images were obtained with 420–480 nm BP filter for DAPI and 505–530 nm BP filter for FITC with a 63 Plan Apochromat objective NA1.4 to obtain high z-resolution (51.0 mm optical slice). LSM 5 image examiner and Image J software packages were used for image processing. Picture panels were processed in Adobe Photoshop. To assess the proportion of cells that responded to DON, at least 100 cells were examined per slide. DCFH-DA assay

Deoxynivalenol (DON, 97% pure) was purchased from Sigma Aldrich Company (Zwijndrecht, the Netherlands) and dissolved in 96% ethanol. The primary antibodies against NUR77 (sc-5569), ATF3 (sc-188), DDIT3 (sc-7351l), AKT 1/2/3 (sc-7985-R), NRF2 (sc-13032), and KEAP1 (sc-33569) were purchased from Santa Cruz Biotech (Heerhugowaard, the Netherlands). Antibody against phospho-SAPK/JNK (#9251), p-NF-kB p65 (3031s), and cleaved caspase-3 (#9664) were obtained from Cell Signaling Technology (Leiden, the Netherlands). Secondary antibodies used here were fluorescein isothiocyanate (FITC)-conjugated goat antimouse-IgG1 (sc-2078) (Santa Cruz Biotech) and anti rabbit-IgG (H+L)/28176-FITC-H488 from Anaspec (Heerhugowaard).

Intracellular reactive oxygen species (ROS) levels in Jurkat cells were measured by using a fluorometric DCFH-DA (5, 6-carboxy29,79-dichlorofluorescin diacetate) assay. DCFH-DA (Invitrogen, Breda, the Netherlands) is a fluorescent non-polar molecular probe that enters cells and is then cleaved by esterase to DCFH. Reactive oxygen species oxidize DCFH to highly fluorescent 20 70 -dichlorofluorescein (DCF) (Luukkonen et al., 2009). Cells (at a concentration of 0.25 106/ml) were exposed to 0.5 mM DON or vehicle control in 6-well plates for 3, 6, or 24 h. After exposure, the cells were washed with Hank’s buffered salt solution (HBSS) and then DCFH-DA reagent was added. The cells were then incubated in the dark for 30 min in a CO2 incubator (37 C, 5% CO2) before fluorescence was measured at 480 nm excitation and 530 nm emission in a microplate reader (BioTek, Winooski, VT).

Cell culture

Glutathione oxidation assay

The human T-lymphocyte cell line Jurkat was obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U penicillin/ml and 100 mg streptomycin/ml (all Sigma Aldrich). Cells were cultured at 37 C in a humidified 5% CO2 atmosphere. Medium was refreshed every 2–3 days.

Reduced glutathione (GSH) and total glutathione (GSH + GSSG) levels in cells were determined using a glutathione oxidation assay kit (Sigma, St. Louis, MO), following manufacturer protocols. Cells (at 107/ml) were treated with DON (0.5 mM) for either 3, 6, or 24 h, then washed with PBS (twice), and lysed by adding 150 ml lysis buffer and placing them on ice for 15 min. Subsequently, monochlorobimane reagent (thiol probe), assay buffer, and glutathione-S-transferase (GST) enzyme were added to

Chemical compounds

Modes of action of DON in Jurkat cells

DOI: 10.3109/1547691X.2014.925995

the cell lysate and the mixture was incubated at 37 C in the dark for 1 h in a 5% CO2 incubator. Fluorescence was then measured at 360 nm excitation and 520 nm emission using the BioTek microplate reader. Protein concentrations in the lysates were determined by the BCA assay (Bio-Rad) and used for correction of all glutathione results. All assays were performed in quadruplicate.


Comparative data analysis To check whether pathways affected by DON in the Jurkat cell line were also affected in vivo, we analysed previously published microarray data of thymuses of mice 3, 6, or 24 h after oral exposure with 5, 10, or 25 mg DON/kg BW (van Kol et al., 2011). The microarray data were converted into 2 log ratios of treatment vs the average of the controls. For comparison, genes were selected on the basis of their differential expression in DONtreated Jurkat cells and involvement in relevant pathways or processes. Five pathways/processes were found affected in Jurkat cells, i.e. ribosome function, RNA synthesis, translation, ER stress, and T-cell activation response (p-value 50.01 in combination with an FDR-value 50.1 according to GSEA statistics). The genes of the gene sets related to ribosome function, RNA synthesis and translation were taken from Gene Ontology (http:// www.geneontology. org/). Genes involved in ER stress were collected from KEGG and upon literature mining. The set of genes up-regulated during T-lymphocyte activation was taken from the lymphocyte database (Shaffer et al., 2001). Genes were filtered on 1.5-fold up- or down-regulation in at least one of the treatments. The genes were hierarchically clustered and visualized in heat maps.

Results DON induces ER stress Our previous microarray results indicated that DON induces the ER stress response in Jurkat cells. Here, the effect of DON on the induction of ER stress was examined by immunocytology, using antibodies directed against two major ER stress markers, i.e. ATF3 and DDIT3. The data indicate DON clearly increased ATF3 and DDIT3 protein expression in Jurkat cells within 3 h (Figures 1a and b).

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The results indicate that treatment with 0.5 mM DON increased phosphorylation of NF-B (p65) after 3 h of exposure (Figure 4). DON exposure leads to activation of the NFAT signaling cascade To confirm the findings of the microarray study, the effect of DON on NFAT signaling in Jurkat cells was investigated by examining the expression of major NFAT target gene NUR77 using immunocytology. As seen in Figure 5, exposure of Jurkat cells to 0.5 mM DON increased NUR77 expression within 3 h of exposure. DON triggers oxidative stress response Analysis of the microarray data revealed that DON activated oxidative stress and the anti-oxidative NRF2 pathway in Jurkat cells. In the present study, the effect of DON on induction of oxidative stress was examined using antibodies against NRF2 and KEAP1, each of which are known to be involved in oxidative stress responses. A concentration of 0.5 mM DON induced translocation of NRF2 and KEAP1 from the cytoplasm to the nucleus after 3 h (Figures 6a and b). In addition, the induction of reactive oxygen species (ROS) by DON in Jurkat cells was assessed using the DCFH-DA assay. Compared to the controls at the same timepoints, 0.5 mM DON treatment led to increased ROS levels after 6 h (Figure 6c). In addition, the effect of DON on levels of reduced glutathione in Jurkat cells was assessed. The results indicate that DON significantly reduced the amount of reduced glutathione after 3 and 6 h of exposure (Figure 6d). Comparative microarray data analysis To verify whether the pathways and biological processes affected by DON in Jurkat cells were also affected in vivo, this study took advantage of the availability of microarray data of the thymuses of DON-treated mice. Most of the genes related to ribosome function, RNA biosynthesis, translation, ER stress, and T-lymphocyte activation that were affected by DON in Jurkat cells were affected in the same direction in the mouse thymus in vivo as well (Supplemental Figures 1i–v). The highest overlap in expression was observed for Jurkat cells exposed to DON for 3, 6, or 24 h, and in the thymuses of mice treated with DON in vivo for 3 h.

Treatment with DON leads to apoptosis Previous microarray and qRT-PCR results revealed that DON induced apoptosis in Jurkat cells. The effect of DON on apoptosis was verified by immunocytology using an antibody against cleaved caspase-3. Cleavage and activation of caspase-3 plays a central role in the process of apoptosis. The data show DON induced caspase-3 cleavage in Jurkat cells within 3 h of exposure (Figure 2). Exposure to DON results in phosphorylation of AKT and MAP protein kinases The effect of DON on activation of MAP and AKT kinases in Jurkat cells was confirmed by immunocytology using antibodies against phosphorylated AKT 1/2/3 and JNK1/2 (alias SAPK/ JNK). Exposure of Jurkat cells to 0.5 mM DON for 1 h induced phosphorylation of AKT/1/2/3 and SAPK/JNK (Figures 3a and b). DON induces the NF-B signaling pathway Biological interpretation of the microarray data provided indications that DON induced up-regulation of NF-B target genes. Thus, activation of NF-B was examined via immunohistochemistry using an antibody against phosphorylated NF-B (p65).

Discussion The present study aimed to verify the findings of our previous microarray study on DON (Katika et al., 2012). The biological interpretation of the microarray data provided evidence that DON: (1) affected the entire pathway for protein synthesis in cells, including RNA synthesis, ribosome functioning, and translation; (2) induced ER stress and calcium-mediated signaling; and (3) activated MAP kinases and induces NF-B and NFAT pathways, as well as T-lymphocyte activation, oxidative stress, and apoptosis. These effects of DON were not specific for Jurkat cells, but also found in in vitro exposed human PBMC (Katika et al., 2012). Moreover, comparative microarray data analyses in the present study revealed that most of the biological processes were similarly affected in vivo in the thymuses of DON-treated mice. Previously, we demonstrated by microarray analysis and qRTPCR that DON affected the expression of many ER stress response genes including ATF3, DDIT3 (alias CHOP10), and PMAIP1 (alias NOXA) (Katika et al., 2012). In the present work, it was shown that DON clearly up-regulated ATF3 and DDIT3 at the protein level as well. ATF3 and DDIT3 are highly expressed during ER stress and involved in ER stress-mediated apoptosis (Mashima et al., 2001; Oyadomari & Mori, 2004). Induction of

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Figure 1. DON induces ER stress. (a) Exposure of Jurkat cells for 3 h to 0.5 mM DON increases ATF3 expression. (A, B) untreated; (C, D) treated. (A, C) ATF3 staining; (B, D) DNA staining with DAPI. Scale bars: 8 (treated) and 6 mm (untreated). (b) Exposure of Jurkat cells for 3 h to 0.5 mM DON increases the expression of DDIT3. (A, B) untreated; (C, D) treated. (A, C) DDIT3 staining; (B, D) DNA staining with DAPI. Scale bars: 21 (treated) and 18 mm (untreated).

ATF3 and DDIT3 by DON has been demonstrated before in HepG2 and HCT-8 cells using immunoblotting (Nielsen et al., 2009; Park et al., 2010). Analysis of the microarray data also revealed that DON induces the MAP kinase pathway in Jurkat cells (Katika et al., 2012). In the present work this finding was confirmed by demonstrating the rapid phosphorylation of the MAP kinases

JNK1/2. These MAP kinases are activated by a variety of cellular and environmental stresses including ER stress (Nishina et al., 2004). An increase of JNK1/2 protein by DON has also been reported before in Jurkat cells using immunoblotting (Pestka et al., 2005a) and in the macrophage RAW 264.7 cell line (Zhou et al., 2005b). Induction of AKT levels by DON has been demonstrated before using immunoblotting in macrophages

DOI: 10.3109/1547691X.2014.925995


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Figure 2. Exposure of Jurkat cells to DON induces apoptosis. Cells were treated for 3 h with 0.5 mM DON and cleavage of caspase-3 was then measured. (A, B) untreated; (C, D) treated. (A, C) cleaved caspase-3 staining; (B, D) DNA staining with DAPI. Scale bar: 10 mm.

(Shi & Pestka, 2009; Zhou et al., 2005a). Besides JNK, AKT is known to be activated via phosphorylation by ER stress and both proteins play a protective role against ER stress-induced apoptosis (Hu et al., 2004). In accordance with this, immunofluorescence demonstrated that DON rapidly phosphorylated AKT in Jurkat cells. The effect of DON on NF-B target genes was evident from the microarray studies. In the present study we demonstrated that DON induces the phosphorylation of NF-B (p65). It has been demonstrated that phosphorylation of MAP kinases by DON in mouse in vivo spleen cells activates transcription factors such as AP-1, C/EBP, and NF-B (Zhou et al., 2003a). NF-B activation is involved in the regulation of T-lymphocyte activation, proliferation, and apoptosis. The induction of the NF-B pathway by DON has also been reported for the human intestinal Caco-2 and monocytic U937 cell lines (Gray & Pestka, 2007; van de Walle et al., 2008). Previously, we had confirmed that DON activated calciumbinding proteins M-calpain, calcineurin, and NFATC1 (Katika et al., 2012). NFAT activation is an essential step for T-lymphocyte activation and apoptosis (Jayanthi et al., 2005). Here, it was demonstrated by immunocytology that DON induced protein expression of NUR77 (alias NR4A1), a major NFAT target gene. NUR77 is a member of the orphan nuclear receptor family involved in various physiological functions such as proliferation, differentiation, and apoptosis (Wansa et al., 2002). NUR77 mediates apoptosis induction in thymocytes in response to T-lymphocyte activation. A previous gene expression study reported that DON induced NUR77 expression in mouse spleen cells as well (Kinser et al., 2004). As mentioned above, immunocytological examination of the Jurkat cells showed that DON exposure led to phosphorylation of NF-B (p65), a process known to be involved in the induction of T-lymphocyte activation response (Fisher et al., 2006). The microarray analysis demonstrated that many genes induced during T-lymphocyte activation were also induced by DON (Katika et al., 2012). This group of genes included interleukin (IL)-4, a major T-lymphocyte activation-related gene (Zamorano et al., 2001).

Up-regulation of IL-4 by DON was confirmed previously by qRTPCR as well (Katika et al., 2012). Biological interpretation of the microarray data revealed that DON affects genes involved in oxidative stress and the NRF2 pathway (Katika et al., 2012). In the present study, we showed that DON induced nuclear translocation of NRF2 and KEAP1. These two proteins are present in the cytoplasm as a complex that dissociates upon oxidative stress. NRF2 is a transcription factor that binds to the anti-oxidant response element (ARE) and regulates the expression of genes encoding anti-oxidative enzymes (Lee & Johnson, 2004). KEAP1 is an oxidative stress response protein that regulates cytoplasmic-nuclear shuttling and degradation of NRF2 (Hu et al., 2004). KEAP1 is also known to translocate into the nucleus and it has been demonstrated that nuclear Keap1 is required for termination of Nrf2-ARE signaling by escorting nuclear export of Nrf2 (Sun et al., 2007). In relation to this, DON significantly increased the amount of intracellular reactive oxygen species after 6 h and decreased the levels of reduced glutathione after 3 and 6 h of exposure. Reduced glutathione (GSH) acts as an effective anti-oxidant and helps to protect cells against reactive oxygen species. In this process, reduced glutathione is converted to its oxidized form glutathione disulfide (GSSG). The ratio of reduced glutathione to oxidized glutathione is a measure for the production of reactive oxygen species (Baek et al., 2000). Induction of reactive oxygen species and depletion of reduced glutathione levels by DON have also been reported for the human colon carcinoma cell line HT-29 (Krishnaswamy et al., 2010). DON also has been reported to induce oxidative stress in HT-29 colon cancer cells and insect cells (Kalaiselvi et al., 2013; Krishnaswamy et al., 2010; Li et al., 2013). Our previous microarray and qRT-PCR data indicated that DON induces apoptosis in Jurkat cells. This was substantiated in the present work by immunofluorescence demonstrating that DON induced cleavage of caspase-3. Induction of cleavage of caspase-3 by DON has also been demonstrated in HT29 colon cancer cells using immunoblotting (Kalaiselvi et al., 2013). Activation and cleavage of caspase-3 is known to play a central

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Figure 3. DON treatment results in phosphorylation of JNK 1/2 and AKT. (a) Jurkat cells were treated with 0.5 mM DON for 3 h. (A, B) untreated; (C, D) treated. (A, C) PhosphoSAP/JNK staining; (B, D) DNA staining with DAPI. Scale bar: 7 mm. (b) Cells were treated with 0.5 mM DON for 3 h. (A, B) untreated; (C, D) treated. (A, C) Phospho-AKT1/2/3 staining; (B, D) DNA staining with DAPI. Scale bar: 11 mm (untreated) and 14 mm (DON-treated).

role in the execution of apoptosis (Jeruc et al., 2006). DON has been shown to induce mitochondrial-mediated apoptosis by release of cytochrome c and activation of caspase-3 and -9 in human colon cancer cells (HCT116 and HT-29) (Bensassi et al., 2012; Ma et al., 2012). Induction of apoptosis by DON has also been described to occur in macrophages, primary thymocytes, and Jurkat cells (Pestka et al., 1994, 2005a). In the present study, the Jurkat cells were exposed to 0.5 mM DON. To be relevant, the DON concentration used in our study needs to reflect back upon levels found in consumed foods. Several studies have reported DON levels in different food products in Europe. The maximum levels of DON were 25.4 mg/kg for

corn-based foods products in Spain, 134 mg/kg for organic breads in Germany, and 500 and 20–257 mg/kg for durum wheat and rye flour, respectively, in Denmark (Castillo et al., 2008; Rasmussen et al., 2003; Schollenberger et al., 2005). A recent study reported on the levels of DON in animal feeds used in South Korea (Kim et al., 2014). The maximum levels of DON ranged from 0.131–1.000 mg/kg in cattle feed, 0.037–0.982 mg/kg in swine feed, and 0.035–1.492 mg/kg in poultry feed. Assuming that 1 kg food equals to 1 L, then the concentration of 0.5 mM DON applied in the present study was 3.4- and 10.1-fold lower than the highest concentrations of DON detected in the Danish rye flour (1.7 mM) and the Korean poultry feed (&5 mM), respectively.

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Figure 4. DON activates NF-B (p65) in Jurkat cells. Cells were treated with 0.5 mM DON for 3 h. (A, B) untreated; (C, D) treated. (A, C) NF-B (p65) staining; (B, D) DNA staining with DAPI. Scale bar: 23 mm.

Figure 5. DON induces NUR77 expression. Jurkat cells were treated with 0.5 mM DON for 3 h. (A, B) untreated; (C, D) treated. (A, C) NUR77 staining; (B, D) DNA staining with DAPI. Scale bar: 7 mm.

Conclusions The present study confirmed the outcome of a previous DNA microarray study. The combined findings are schematically illustrated in Figure 7. The ribosome is likely a primary target of DON, although our results do not exclude a direct effect on ER. The results demonstrated that DON induced ER stress, calciummediated signaling (activation of calcineurin and calpain), and

activated protein kinases AKT and JNK. Induction of calciummediated signaling activated NFAT and NF-B pathways, resulting in a T-lymphocyte activation response that, in combination with ER and oxidative stress, finally induced apoptosis. Based on these findings, we propose that immune cells are more sensitive to DON than other cell types due to the fact that calcium leakage from the ER leads to induction of T-lymphocyte activation responses. The functional consequences of our findings confirmed

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Figure 6. Treatment of Jurkat cells with DON induces oxidative stress. (a) Exposure of cells for 3 h to 0.5 mM DON increases NRF2 expression and induces its translocation from the cytoplasm to the nucleus. (A, B) untreated; (C, D) treated. (A, C) NRF2 staining; (B, D) DNA staining with DAPI. Scale bar ¼ 18 mm. (b) Exposure of Jurkat cells for 3 h to 0.5 mM DON induces KEAP1 expression. (A, B) untreated; (C, D) treated. (A, C) KEAP1 staining; (B, D) DNA staining with DAPI. Scale bars: 21 (treated) and 15 mm (untreated). (c) DON induces ROS production. Jurkat cells were exposed for 3 and 6 h to 0.5 mM DON and intracellular ROS formation was measured by DCFH-DA assay. The amount of reactive oxygen species is presented as fluorescence units. Results shown are mean ± SD from triplicate exposures. *p50.05 vs control at same exposure time (Student’s t-test). (d) DON decreases reduced glutathione levels in Jurkat cells. Cells were treated with 0.5 mM DON for 3, 6 and 24 h. The amount of reduced glutathione is presented as fluorescence units. Results shown are mean ± SD from triplicate exposures. **p50.01 vs control at same exposure time (Student’s t-test).



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Figure 6. Continued.

Ca2+ CRAC

Ca2+

DON

Ca2+

Ribosomal stress

Mitochondria

IP3R

ROS

ROS

Ca2+ Calpain

Ca2+

Calcineurin ROS

Caspase-4 P

AKT

JNK

P P P NFAT P P P NFAT

Nucleus

Oxidative stress FASL, NUR77 and cytokines

ER STRESS

T cell activation

NF-kB ATF3 and DDIT3

Caspase-3

Apoptosis

Figure 7. Diagrammatic presentation of proposed molecular mechanism of action of DON in Jurkat T-cells. DON binds to active ribosomes and exerts ribosomal stress. This, and possibly also a direct interaction of DON with the ER, induces ER stress and activates AKT and MAP kinase JNK. ER stress induces Ca2+ release from the ER lumen into the cytoplasm via inositol-1,4,5-triphosphate (IP3) receptors. This small cytoplasmic Ca2+ peak triggers a large Ca2+ influx into the cell through CRAC channels within the plasma membrane. The increased intracellular Ca2+ level activates the calcium binding proteins calcineurin and M-calpain. Activated calcineurin dephosphorylates NFAT, leading to its translocation to the nucleus and induction of expression of NFAT target genes (FASL, NUR77 and cytokines), resulting in T-lymphocyte activation. Activation of M-calpain cleaves ER resident caspase-4. Both ER stress and increased Ca2+ levels activate the NF-B pathway. Furthermore, oxidative stress is induced by reactive oxygen species that are produced in the ER due to ER stress and in the mitochondria due to the elevated cytoplasmic Ca2+ levels. ER stress also induces activation of apoptosis-promoting proteins including DDIT3 and ATF3. Finally, the activated caspases induce apoptosis. Unbroken lines: mechanisms based on the outcomes of the present study. Dashed lines: based on information found in literature.

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that DON affected various biological processes, such as ER/ oxidative stress, calcium mediated signaling, MAP kinases, T-lymphocyte activation, and apoptosis (in Jurkat cells). Since Jurkat cells are human in origin, we expect that DON would likely modulate functions of T-lymphocytes, leading to immunotoxicity in humans.

Acknowledgements The authors thank Peter Schmeits and Norbert de Ruijter for their technical support.


Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. The study was supported by a grant (MFA 6809) that the University of Maastricht received from the Dutch Technology Foundation STW.

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Supplementary material available online Supplementary Figures S1–S4

Characterization of the human immunodeficiency virus type 1 enhancer-binding proteins from the human T-cell line Jurkat.

Lipopolysaccharides (LPS) modulate the metabolism of deoxynivalenol (DON) in the pig.

UV detection for the determination of DON in crushed wheat (bulgur).

Mechanism of phosphatidylserine-induced inhibition of interleukin 2 synthesis in the human T cell line Jurkat.

Studies on the bioavailability of deoxynivalenol (DON) and DON sulfonate (DONS) 1, 2, and 3 in pigs fed with sodium sulfite-treated DON-contaminated maize.

Influence of temperature and pH on the degradation of deoxynivalenol (DON) in aqueous medium: comparative cytotoxicity of DON and degraded product.

Effect of deoxynivalenol (DON) mycotoxin on in vivo and in vitro porcine circovirus type 2 infections.

Deoxynivalenol (DON) sulfonates as major DON metabolites in rats: from identification to biomarker method development, validation and application.

In vivo effects of deoxynivalenol (DON) on innate immune responses of carp (Cyprinus carpio L.).

Impact of deoxynivalenol (DON) contaminated feed on intestinal integrity and immune response in swine.

In vitro effect of deoxynivalenol (DON) mycotoxin on porcine reproductive and respiratory syndrome virus replication.

A protein kinase C inhibitory activity is present in the human T lymphoblast cell line Jurkat.

Growth performance, serum biochemical profile, jejunal morphology, and the expression of nutrients transporter genes in deoxynivalenol (DON)- challenged growing pigs.

The Metabolic Fate of Deoxynivalenol and Its Acetylated Derivatives in a Wheat Suspension Culture: Identification and Detection of DON-15-O-Glucoside, 15-Acetyl-DON-3-O-Glucoside and 15-Acetyl-DON-3-Sulfate.

Dose-Dependent Responses of I3C and DIM on T-Cell Activation in the Human T Lymphocyte Jurkat Cell Line.

Residues of deoxynivalenol (DON) and its metabolite de-epoxy-DON in eggs, plasma and bile of laying hens of different genetic backgrounds.

Physiological characterization of the human EndoC-βH1 β-cell line.

Cross-reactivity features of deoxynivalenol (DON)-targeted immunoaffinity columns aiming to achieve simultaneous analysis of DON and major conjugates in cereal samples.

The purification and characterization of recombinant human renin expressed in the human kidney cell line 293.

Hexokinase plays a critical role in deoxynivalenol (DON) production and fungal development in Fusarium graminearum.

Organ damage and hepatic lipid accumulation in carp (Cyprinus carpio L.) after feed-borne exposure to the mycotoxin, deoxynivalenol (DON).

Characterization of a human plasmacytoma line.

Using near-isogenic barley lines to validate deoxynivalenol (DON) QTL previously identified through association analysis.

A comprehensive characterization of the impact of mycophenolic acid on the metabolism of Jurkat T cells.