http://informahealthcare.com/imtISSN: 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 thehuman 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

1RIKILT-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

Deoxynivalenol (DON) is one of the most abundant mycotoxins worldwide and mostly detectedin cereals and grains. As such, DON poses a risk for many adverse health effects to human andanimals. In particular, immune cells are very sensitive to DON, with the initiating step leading totoxicity being a binding to the eukaryotic 60S ribosomal subunit and induction of ribotoxicstress. The present study aimed to: (1) extend insight into the mechanism of action (MOA) ofDON in immune cells; and (2) understand why immune cells are more sensitive to DON thanmost other cell types. Previously published microarray studies have described the effects ofDON on immune cells. To build upon these findings, here, immunocytological and biochemicalstudies were performed using human T-lymphocyte Jurkat cells that were exposed for 3 h to0.5mM DON. Induction of ER stress by DON was confirmed by immunocytology demonstratingincreased protein expression of two major ER stress markers ATF3 and DDIT3. T-cell activationwas confirmed by induction of phosphorylation of protein kinases JNK and AKT, activation ofNF-�B (p65), and increased expression of NFAT target gene NUR77; each of these are knowninducers of the T-cell activation response. Induction of an oxidative stress response was alsoconfirmed by monitoring the nuclear translocation of major oxidative stress markers NRF2 andKEAP1, as well as by changes (i.e. decreases) in cell levels of reduced glutathione. Lastly, thisstudy 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 DONin immune cells, i.e. binding to eukaryotic 60S ribosomal subunit! ribotoxic stress! ERstress! calcium release from the ER into cytoplasm! T-cell activation and oxidativestress! apoptosis. It is proposed that immune cells are more sensitive to DON thanother cell types due to the induction of a T-cell activation response by increased intracellularcalcium levels.

Keywords

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

History

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

Introduction

Deoxynivalenol (DON) is a mycotoxin belonging to the type Btrichothecenes group. DON is a biologically active secondarymetabolite 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 tohigh temperatures and milling processes; as a result, its stabilityallows it to readily enter the food chain (Sugita-Konishi et al.,2006). Intake of high levels of DON by humans has beenimplicated in a number of incidents of intoxication, primarily inAsia (Bhat et al., 1989; Ramakrishna et al., 1989; Yoshizawa1983). In one example, outbreaks of acute human illness in Chinaduring 1961–1991 were reported, with symptoms of nausea,

vomiting, diarrhea, abdominal pain, headache, dizziness, andfever (Luo, 1994).

As noted, ingestion of DON contaminated food products posesa 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 – includingbread and cereals, DON levels up to 700mg/kg have been detected(Rasmussen et al., 2003; Schollenberger et al., 2005; Soubraet al., 2009).

An important question that persists is to what extent dailyexposure to non-acute DON levels affects human health? Manymodels have been used to ascertain the potential immunotoxicrisks from exposures to DON. For example, in mice, the immunesystem has been seen to be quite sensitive to DON. Oral dailyexposure of BALB/c mice to DON at410 mg/kg induced thymusatrophy and decreased spleen weight (Robbanabarnat et al., 1987).DON exposure was also reported to exacerbate infections withparasites, bacteria, or viruses across a wide range of animal

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

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host species (including non-rodents) (Antonissen et al., 2014;Pestka 2010). Thus, it is logical to assume then that DONexposure(s) might negatively impact on the immune response ofhumans as well.

The latter view has been borne out. For example, ingestion ofDON-contaminated foods caused adverse effects on human andanimal health, particularly their immune responses (Pestka 2004,2010; Rotter et al., 1996). At the cellular level, DON has beenshown to modulate the function of T- and B-lymphocytes, NKcells, and macrophages in rodents (Moon & Pestka, 2002; Rotteret al., 1996). DON also has been seen to inhibit the proliferationof mouse and human lymphocytes in vitro (Meky et al., 2001).Further, low doses of DON were found to cause an increasedproduction of cytokines and chemokines (Moon & Pestka, 2002,2003), while high doses caused apoptosis among human bloodmonocytes, Jurkat T-cells, B-cells, and macrophages (Pestkaet al., 1994, 2005b; Zhou & Pestka, 2003).

Despite a large number of studies on the subject, themechanisms of action of DON are not yet completely understood.It is known that DON binds the eukaryotic 60S ribosomal subunitleading to ribotoxic stress and inhibited protein synthesis (Pestka,2008). DON also induces several MAP kinases (MAPK) known tostimulate 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 themouse thymus and affected biological processes includingfunction of ribosomes and mitochondria, T-lymphocyte activa-tion, and apoptosis (van Kol et al., 2011). Our own studies of theeffects of DON on whole genome mRNA expression in Jurkatcells and peripheral blood mononuclear cells (PBMC)—usingDNA microarrays (Katika et al., 2012)—confirmed many of theknown effects of DON on ribosomes, RNA/protein synthesis, andapoptosis. Previously unreported effects included the induction ofboth endoplasmic reticulum (ER) and oxidative stress, activationof calcium-mediated signaling, and both NFAT and NF-�Bpathways, as well as the induction of T-lymphocyte activation.The present study aimed to build upon/strengthen those findingsusing immunocytological and biochemical analyses of the effectsin Jurkat cells specifically.

Materials and methods

Chemical compounds

Deoxynivalenol (DON, 97% pure) was purchased from SigmaAldrich Company (Zwijndrecht, the Netherlands) and dissolved in96% 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 SantaCruz Biotech (Heerhugowaard, the Netherlands). Antibodyagainst phospho-SAPK/JNK (#9251), p-NF-kB p65 (3031s), andcleaved caspase-3 (#9664) were obtained from Cell SignalingTechnology (Leiden, the Netherlands). Secondary antibodies usedhere were fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse-IgG1 (sc-2078) (Santa Cruz Biotech) and anti rabbit-IgG(H+L)/28176-FITC-H488 from Anaspec (Heerhugowaard).

Cell culture

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

Immunocytology

Jurkat cells were exposed to a final concentration of 0.5 mM DONfor 3 h. This exposure condition was based on outcomes of ourprevious microarray study (Katika et al., 2012) wherein Jurkatcells were exposed to 0.25 or 0.5 mM DON for 3, 6, or 24 h. It wasseen there that exposure to 0.5 mM DON for 3 h clearly affectedexpression of genes involved in various bio-processes, includingER/oxidative stress, activation of NFAT and NF-�B pathways,T-cell activation, and apoptosis. Further, that study showed thatthis DON exposure time and dose did not result in cytotoxicity. Assuch, this condition was selected for use in the presentexperiments.

Upon exposure, Jurkat cells were immobilized on poly-L-lysine coated slides (Memel-Glaser, Braunschweig, Germany)using mild cytospin centrifugation (5 min, 44� g) followed byincubation in 4% freshly prepared paraformaldehyde with 0.025%glutaraldehyde (in phosphate-buffered saline [PBS, pH 7.4]) for30 min. After blocking with a solution of 1% bovine serumalbumin (BSA)/0.01% Triton X-100 in PBS for 45 min, the cellswere washed with 0.1% acetylated BSA (Aurion, Wageningen,NL) in PBS, prior to immunolabeling overnight at 4 �C. Primaryantibodies against ATF3, DDIT3, phospho-AKT 1/2/3, phospho-SAPK/JNK, NUR77, NRF2, KEAP1, phospho NF-�B (p65), andcleaved caspase-3 were diluted 1:100 in 0.1% acetylated BSA inPBS. After extensive washing in 0.1% acetylated BSA in PBS, thecells were incubated with 300-fold diluted goat anti-rabbit IgG(H+L)-FITC or anti mouse-IgG1-FITC secondary antibody for120 min at 37 �C. The slides were then washed in PBS, mountedin Vectashield containing 1.5mg/ml DAPI (Vectashield,Amsterdam, the Netherlands) and imaged with an LSM510confocal microscope (Carl Zeiss, Germany). Images wereobtained with 420–480 nm BP filter for DAPI and 505–530 nmBP filter for FITC with a 63� Plan Apochromat objective NA1.4to obtain high z-resolution (51.0 mm optical slice). LSM 5 imageexaminer and Image J software packages were used for imageprocessing. Picture panels were processed in Adobe Photoshop.To assess the proportion of cells that responded to DON, at least100 cells were examined per slide.

DCFH-DA assay

Intracellular reactive oxygen species (ROS) levels in Jurkat cellswere measured by using a fluorometric DCFH-DA (5, 6-carboxy-29,79-dichlorofluorescin diacetate) assay. DCFH-DA (Invitrogen,Breda, the Netherlands) is a fluorescent non-polar molecularprobe 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 aconcentration of 0.25� 106/ml) were exposed to 0.5mM DON orvehicle 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 thenincubated in the dark for 30 min in a CO2 incubator (37 �C, 5%CO2) before fluorescence was measured at 480 nm excitation and530 nm emission in a microplate reader (BioTek, Winooski, VT).

Glutathione oxidation assay

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

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the cell lysate and the mixture was incubated at 37 �C in the darkfor 1 h in a 5% CO2 incubator. Fluorescence was then measured at360 nm excitation and 520 nm emission using the BioTekmicroplate reader. Protein concentrations in the lysates weredetermined by the BCA assay (Bio-Rad) and used for correctionof all glutathione results. All assays were performed inquadruplicate.

Comparative data analysis

To check whether pathways affected by DON in the Jurkat cellline were also affected in vivo, we analysed previously publishedmicroarray data of thymuses of mice 3, 6, or 24 h after oralexposure with 5, 10, or 25 mg DON/kg BW (van Kol et al., 2011).The microarray data were converted into 2 log ratios of treatmentvs the average of the controls. For comparison, genes wereselected on the basis of their differential expression in DON-treated Jurkat cells and involvement in relevant pathways orprocesses. Five pathways/processes were found affected in Jurkatcells, i.e. ribosome function, RNA synthesis, translation, ERstress, and T-cell activation response (p-value50.01 in combin-ation with an FDR-value50.1 according to GSEA statistics). Thegenes of the gene sets related to ribosome function, RNAsynthesis and translation were taken from Gene Ontology (http://www.geneontology. org/). Genes involved in ER stress werecollected from KEGG and upon literature mining. The set ofgenes up-regulated during T-lymphocyte activation was takenfrom the lymphocyte database (Shaffer et al., 2001). Genes werefiltered on �1.5-fold up- or down-regulation in at least one of thetreatments. The genes were hierarchically clustered and visualizedin heat maps.

Results

DON induces ER stress

Our previous microarray results indicated that DON induces theER stress response in Jurkat cells. Here, the effect of DON on theinduction of ER stress was examined by immunocytology, usingantibodies directed against two major ER stress markers, i.e.ATF3 and DDIT3. The data indicate DON clearly increased ATF3and DDIT3 protein expression in Jurkat cells within 3 h(Figures 1a and b).

Treatment with DON leads to apoptosis

Previous microarray and qRT-PCR results revealed that DONinduced apoptosis in Jurkat cells. The effect of DON on apoptosiswas verified by immunocytology using an antibody againstcleaved caspase-3. Cleavage and activation of caspase-3 plays acentral role in the process of apoptosis. The data show DONinduced caspase-3 cleavage in Jurkat cells within 3 h of exposure(Figure 2).

Exposure to DON results in phosphorylation of AKT andMAP protein kinases

The effect of DON on activation of MAP and AKT kinases inJurkat cells was confirmed by immunocytology using antibodiesagainst phosphorylated AKT 1/2/3 and JNK1/2 (alias SAPK/JNK). Exposure of Jurkat cells to 0.5 mM DON for 1 h inducedphosphorylation 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 indica-tions that DON induced up-regulation of NF-�B target genes.Thus, activation of NF-�B was examined via immunohistochem-istry using an antibody against phosphorylated NF-�B (p65).

The results indicate that treatment with 0.5 mM DON increasedphosphorylation of NF-�B (p65) after 3 h of exposure (Figure 4).

DON exposure leads to activation of the NFAT signalingcascade

To confirm the findings of the microarray study, the effect ofDON on NFAT signaling in Jurkat cells was investigated byexamining the expression of major NFAT target gene NUR77using immunocytology. As seen in Figure 5, exposure of Jurkatcells to 0.5mM DON increased NUR77 expression within 3 h ofexposure.

DON triggers oxidative stress response

Analysis of the microarray data revealed that DON activatedoxidative stress and the anti-oxidative NRF2 pathway in Jurkatcells. In the present study, the effect of DON on induction ofoxidative stress was examined using antibodies against NRF2 andKEAP1, each of which are known to be involved in oxidativestress responses. A concentration of 0.5 mM DON inducedtranslocation of NRF2 and KEAP1 from the cytoplasm to thenucleus after 3 h (Figures 6a and b). In addition, the induction ofreactive oxygen species (ROS) by DON in Jurkat cells wasassessed using the DCFH-DA assay. Compared to the controls atthe same timepoints, 0.5 mM DON treatment led to increased ROSlevels after 6 h (Figure 6c). In addition, the effect of DON onlevels of reduced glutathione in Jurkat cells was assessed. Theresults indicate that DON significantly reduced the amount ofreduced glutathione after 3 and 6 h of exposure (Figure 6d).

Comparative microarray data analysis

To verify whether the pathways and biological processes affectedby DON in Jurkat cells were also affected in vivo, this study tookadvantage of the availability of microarray data of the thymuses ofDON-treated mice. Most of the genes related to ribosomefunction, RNA biosynthesis, translation, ER stress, andT-lymphocyte activation that were affected by DON in Jurkatcells were affected in the same direction in the mouse thymusin vivo as well (Supplemental Figures 1i–v). The highest overlapin 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 vivofor 3 h.

Discussion

The present study aimed to verify the findings of our previousmicroarray study on DON (Katika et al., 2012). The biologicalinterpretation 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 apop-tosis. These effects of DON were not specific for Jurkat cells, butalso found in in vitro exposed human PBMC (Katika et al., 2012).Moreover, comparative microarray data analyses in the presentstudy revealed that most of the biological processes were similarlyaffected in vivo in the thymuses of DON-treated mice.

Previously, we demonstrated by microarray analysis and qRT-PCR that DON affected the expression of many ER stressresponse genes including ATF3, DDIT3 (alias CHOP10), andPMAIP1 (alias NOXA) (Katika et al., 2012). In the present work,it was shown that DON clearly up-regulated ATF3 and DDIT3 atthe protein level as well. ATF3 and DDIT3 are highly expressedduring ER stress and involved in ER stress-mediated apoptosis(Mashima et al., 2001; Oyadomari & Mori, 2004). Induction of

DOI: 10.3109/1547691X.2014.925995 Modes of action of DON in Jurkat cells 3

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ATF3 and DDIT3 by DON has been demonstrated before inHepG2 and HCT-8 cells using immunoblotting (Nielsen et al.,2009; Park et al., 2010).

Analysis of the microarray data also revealed that DONinduces the MAP kinase pathway in Jurkat cells (Katika et al.,2012). In the present work this finding was confirmed bydemonstrating the rapid phosphorylation of the MAP kinases

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

Figure 1. DON induces ER stress.(a) Exposure of Jurkat cells for 3 h to0.5mM DON increases ATF3 expression.(A, B) untreated; (C, D) treated. (A, C) ATF3staining; (B, D) DNA staining with DAPI.Scale bars: 8 (treated) and 6 mm (untreated).(b) Exposure of Jurkat cells for 3 h to 0.5mMDON increases the expression of DDIT3. (A,B) untreated; (C, D) treated. (A, C) DDIT3staining; (B, D) DNA staining with DAPI.Scale bars: 21 (treated) and 18mm(untreated).

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(Shi & Pestka, 2009; Zhou et al., 2005a). Besides JNK, AKT isknown to be activated via phosphorylation by ER stress and bothproteins play a protective role against ER stress-induced apoptosis(Hu et al., 2004). In accordance with this, immunofluorescencedemonstrated that DON rapidly phosphorylated AKT inJurkat cells.

The effect of DON on NF-�B target genes was evident fromthe microarray studies. In the present study we demonstrated thatDON induces the phosphorylation of NF-�B (p65). It has beendemonstrated that phosphorylation of MAP kinases by DON inmouse in vivo spleen cells activates transcription factors such asAP-1, C/EBP, and NF-�B (Zhou et al., 2003a). NF-�B activationis involved in the regulation of T-lymphocyte activation, prolif-eration, and apoptosis. The induction of the NF-�B pathway byDON has also been reported for the human intestinal Caco-2and monocytic U937 cell lines (Gray & Pestka, 2007; van deWalle et al., 2008).

Previously, we had confirmed that DON activated calcium-binding proteins M-calpain, calcineurin, and NFATC1 (Katikaet al., 2012). NFAT activation is an essential step forT-lymphocyte activation and apoptosis (Jayanthi et al., 2005).Here, it was demonstrated by immunocytology that DON inducedprotein expression of NUR77 (alias NR4A1), a major NFATtarget gene. NUR77 is a member of the orphan nuclear receptorfamily involved in various physiological functions such asproliferation, differentiation, and apoptosis (Wansa et al., 2002).NUR77 mediates apoptosis induction in thymocytes in responseto T-lymphocyte activation. A previous gene expression studyreported that DON induced NUR77 expression in mouse spleencells as well (Kinser et al., 2004).

As mentioned above, immunocytological examination of theJurkat cells showed that DON exposure led to phosphorylation ofNF-�B (p65), a process known to be involved in the induction ofT-lymphocyte activation response (Fisher et al., 2006). Themicroarray analysis demonstrated that many genes induced duringT-lymphocyte activation were also induced by DON (Katika et al.,2012). This group of genes included interleukin (IL)-4, a majorT-lymphocyte activation-related gene (Zamorano et al., 2001).

Up-regulation of IL-4 by DON was confirmed previously by qRT-PCR as well (Katika et al., 2012).

Biological interpretation of the microarray data revealed thatDON affects genes involved in oxidative stress and the NRF2pathway (Katika et al., 2012). In the present study, we showed thatDON induced nuclear translocation of NRF2 and KEAP1. Thesetwo proteins are present in the cytoplasm as a complex thatdissociates upon oxidative stress. NRF2 is a transcription factorthat binds to the anti-oxidant response element (ARE) andregulates the expression of genes encoding anti-oxidativeenzymes (Lee & Johnson, 2004). KEAP1 is an oxidative stressresponse protein that regulates cytoplasmic-nuclear shuttling anddegradation of NRF2 (Hu et al., 2004). KEAP1 is also known totranslocate into the nucleus and it has been demonstrated thatnuclear Keap1 is required for termination of Nrf2-ARE signalingby escorting nuclear export of Nrf2 (Sun et al., 2007).

In relation to this, DON significantly increased the amount ofintracellular reactive oxygen species after 6 h and decreased thelevels of reduced glutathione after 3 and 6 h of exposure. Reducedglutathione (GSH) acts as an effective anti-oxidant and helps toprotect cells against reactive oxygen species. In this process,reduced glutathione is converted to its oxidized form glutathionedisulfide (GSSG). The ratio of reduced glutathione to oxidizedglutathione is a measure for the production of reactive oxygenspecies (Baek et al., 2000). Induction of reactive oxygen speciesand depletion of reduced glutathione levels by DON have alsobeen reported for the human colon carcinoma cell line HT-29(Krishnaswamy et al., 2010). DON also has been reportedto induce oxidative stress in HT-29 colon cancer cells andinsect cells (Kalaiselvi et al., 2013; Krishnaswamy et al., 2010;Li et al., 2013).

Our previous microarray and qRT-PCR data indicated thatDON induces apoptosis in Jurkat cells. This was substantiated inthe present work by immunofluorescence demonstrating thatDON induced cleavage of caspase-3. Induction of cleavage ofcaspase-3 by DON has also been demonstrated in HT29 coloncancer cells using immunoblotting (Kalaiselvi et al., 2013).Activation and cleavage of caspase-3 is known to play a central

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

DOI: 10.3109/1547691X.2014.925995 Modes of action of DON in Jurkat cells 5

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role in the execution of apoptosis (Jeruc et al., 2006). DON hasbeen shown to induce mitochondrial-mediated apoptosis byrelease of cytochrome c and activation of caspase-3 and -9 inhuman colon cancer cells (HCT116 and HT-29) (Bensassi et al.,2012; Ma et al., 2012). Induction of apoptosis by DON has alsobeen 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 mMDON. To be relevant, the DON concentration used in our studyneeds to reflect back upon levels found in consumed foods.Several studies have reported DON levels in different food prod-ucts in Europe. The maximum levels of DON were 25.4 mg/kg for

corn-based foods products in Spain, 134mg/kg for organic breadsin Germany, and 500 and 20–257 mg/kg for durum wheat and ryeflour, respectively, in Denmark (Castillo et al., 2008; Rasmussenet al., 2003; Schollenberger et al., 2005). A recent study reportedon the levels of DON in animal feeds used in South Korea(Kim et al., 2014). The maximum levels of DON ranged from0.131–1.000 mg/kg in cattle feed, 0.037–0.982 mg/kg in swinefeed, and 0.035–1.492 mg/kg in poultry feed. Assuming that 1 kgfood equals to 1 L, then the concentration of 0.5 mM DON appliedin the present study was 3.4- and 10.1-fold lower than the highestconcentrations of DON detected in the Danish rye flour (1.7 mM)and the Korean poultry feed (&5mM), respectively.

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

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Conclusions

The present study confirmed the outcome of a previous DNAmicroarray study. The combined findings are schematicallyillustrated in Figure 7. The ribosome is likely a primary targetof DON, although our results do not exclude a direct effect on ER.The results demonstrated that DON induced ER stress, calcium-mediated signaling (activation of calcineurin and calpain), and

activated protein kinases AKT and JNK. Induction of calcium-mediated signaling activated NFAT and NF-�B pathways, result-ing in a T-lymphocyte activation response that, in combinationwith ER and oxidative stress, finally induced apoptosis. Based onthese findings, we propose that immune cells are more sensitive toDON than other cell types due to the fact that calcium leakagefrom the ER leads to induction of T-lymphocyte activationresponses. The functional consequences of our findings confirmed

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

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

DOI: 10.3109/1547691X.2014.925995 Modes of action of DON in Jurkat cells 7

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Figure 6. Treatment of Jurkat cells with DON induces oxidative stress. (a) Exposure of cells for 3 h to 0.5mM DON increases NRF2 expression andinduces 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 bar¼ 18mm. (b) Exposure of Jurkat cells for 3 h to 0.5 mM DON induces KEAP1 expression. (A, B) untreated; (C, D) treated. (A, C) KEAP1staining; (B, D) DNA staining with DAPI. Scale bars: 21 (treated) and 15 mm (untreated). (c) DON induces ROS production. Jurkat cells were exposedfor 3 and 6 h to 0.5mM DON and intracellular ROS formation was measured by DCFH-DA assay. The amount of reactive oxygen species is presented asfluorescence units. Results shown are mean ± SD from triplicate exposures. *p50.05 vs control at same exposure time (Student’s t-test). (d) DONdecreases 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 ispresented 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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ROS

Ca2+

CRAC

Ca2+

IP3R

DON Ca2+

MitochondriaRibosomal stress

CalcineurinCalpain

Ca2+

ROS

Ca2+

ROS

NFATPPP

P PP P Oxidative stress

Caspase-4

AKT JNKNucleus

NFAT

ER STRESS

FASL, NUR77and cytokines

T cell activationNF-kB

Caspase-3

ATF3 and DDIT3

Apoptosis

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

Figure 6. Continued.

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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). SinceJurkat cells are human in origin, we expect that DON would likelymodulate functions of T-lymphocytes, leading to immunotoxicityin humans.

Acknowledgements

The authors thank Peter Schmeits and Norbert de Ruijter for theirtechnical support.

Declaration of interest

The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of the paper. The study wassupported by a grant (MFA 6809) that the University of Maastrichtreceived from the Dutch Technology Foundation STW.

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Supplementary material available online

Supplementary Figures S1–S4

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