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Toxicon 84 (2014) 1–6

Contents lists ava

Toxicon

journal homepage: www.elsevier .com/locate/ toxicon

In vitro investigation of toxicological interactions between thefusariotoxins deoxynivalenol and zearalenone

Fatma Bensassi a,b,*, Cindy Gallerne c, Ossama Sharaf el dein c,Mohamed Rabeh Hajlaoui b, Christophe Lemaire c,d, Hassen Bacha a

a Laboratory for Research on Biologically Compatible Compounds, Faculty of Dentistry, Rue Avicenne, 5019 Monastir, Tunisiab Laboratory of Plant Protection, The National Institute for Agricultural Research, INRA Tunisia, Rue Hedi Karray, 2049 Ariana, Tunisiac INSERM U769, LabEx LERMIT, Signalisation et Physiopathologie Cardiaque, Faculté de Pharmacie, Châtenay-Malabry, FrancedUniversité de Versailles St. Quentin, Versailles 78035, France

a r t i c l e i n f o

Article history:Received 31 March 2013Received in revised form 18 September 2013Accepted 18 March 2014Available online 27 March 2014

Keywords:DeoxynivalenolZearalenoneCombinationApoptosisMitochondriaPTP

* Corresponding author. Laboratory for ReseaCompatible Compounds, Faculty of Dentistry, RMonastir, Tunisia. Tel./fax: þ216 73 42 55 50.

E-mail address: bensassifatma83@yahoo.fr (F. Be

http://dx.doi.org/10.1016/j.toxicon.2014.03.0050041-0101/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

It is expected that humans are exposed to combined mycotoxins, which occur simulta-neously in the food items, than to individual compounds and that can increase their po-tential toxicity. Considering this coincident production, deoxynivalenol (DON) andzearalenone (ZEN) as they are produced by several Fusarium species, can interfere at acellular level. Therefore, these two toxins were chosen to study their interactive effects onhuman colon carcinoma cells (HCT116), using the endpoints including cell viability, cellcycle analysis, mitochondrial transmembrane potential (DJm) determination andpermeability transition pore (PTP) opening. Our results showed that DON and ZEN causeda marked decrease of cell viability in a dose-dependent manner, mediated by an activationof the mitochondrial apoptotic process; characterized by PTP opening and the loss of DJm.Nevertheless, combined DON and ZEN reduced all the toxicities observed with the my-cotoxins separately. Therefore, the combination of the two mycotoxins appears as a sub-additive response.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction mycotoxin types A and B, and non-trichothecene com-

Mycotoxins are natural contaminants produced by fila-mentous fungi. The presence of these harmful compoundsin food and feed has long been recognized as a potentialhuman and animal health hazard (Bennett and Klich,2003). Numerous mycotoxins are produced during theharvest period, belonging mainly to those synthesized bythe genus Fusarium (Bottalico and Perrone, 2002). In theliterature, it is known that several Fusarium toxigenic spe-cies are capable of producing a number of trichothecene

rch on Biologicallyue Avicenne, 5019

nsassi).

pounds as zearalenone, beauvericin and moniliformin(Thrane, 2001; Desjardins, 2006). Several trichothecenesare of concern, including deoxynivalenol, nivalenol, diac-etoxyscirpenol and T-2 toxin.

Deoxynivalenol (DON), a representative mycotoxin ofthe trichothecene B group, is one of the most widespreadcereal contaminants worldwide (Larsen et al., 2004;Krysinska-Traczyk et al., 2007). DON consumption maycause feed refusal, emesis, vomiting and disrupt the im-mune system in different animal species (Pestka, 2007,2010). At a cellular level, the main toxic effect of DON isdue to the inhibition of protein and nucleic acid synthesis(Ueno et al., 1973; Shifrin and Anderson, 1999), via bindingto the ribosome and by activating cellular kinases involvedin signal transduction, which consequently results in thedecrease of the cell proliferation.

F. Bensassi et al. / Toxicon 84 (2014) 1–62

Zearalenone (ZEN) is also one of the most frequentfusariotoxin in small grain cereals (Muller et al., 1998;Scudamore and Patel, 2000). ZEN is a non-steroidal es-trogenic mycotoxin, which induces genotoxic and carci-nogenic disorders by producing DNA adducts, andmicronuclei, and by causing chromosome aberrations(Ouanes et al., 2005; Ayed-Boussema et al., 2007; Ayedet al., 2011). It has also been reported that ZEN inhibitsDNA and protein synthesis and induces oxidative stressmediated cell death (El Golli et al., 2009, Abid-Essefiet al., 2012).

The various biological effects of mycotoxins areattributed largely to the alteration of basic metabolicprocesses. Indeed, when cells exposed to these toxicmetabolites failed to restore their homeostasis, they cantrigger their own death. This programmed cell death iscalled apoptosis. It is a genetically directed process ofcell self-destruction that is marked by a series ofmorphological and biochemical changes, including cas-pase activation, chromatin condensation, and fragmen-tation of nuclear DNA (Zimmermann et al., 2001). Avariety of key events in apoptosis focus on mitochondria(Parsons and Green, 2010), such as the release of caspaseactivators, changes in electron transport, loss of mito-chondrial transmembrane potential (DJm), andinvolvement of pro and anti-apoptotic Bcl-2 familyproteins.

Most of the conducted investigations evaluate theeffect of mycotoxins taken individually. Nevertheless, it isvery likely that humans and animals are always exposedto the mixture rather than to individual compounds. Thisis particularly true for DON and ZEN, which can both beproduced by Fusarium culmorum (Hestbjerg et al., 2002;Glenn, 2007) and are quite common contaminants thatoccur jointly in several cereal grains. Mycological surveyscarried out on freshly harvested wheat grains from themain production regions at the Northern part of Tunisia,revealed that grains were heavily contaminated withmycotoxin-producing fungi, and the most recoveredgenus, in terms of frequency was Fusarium, predomi-nately F. culmorum and F. pseudograminearum (Kammounet al., 2010; Bensassi et al., 2011). Indeed, these FusariumHead Blight (FHB) associated pathogens are capable ofproducing a number of mycotoxins, including DON aswell as ZEN. Thus, chromatographic analyses conductedby Bensassi et al. (2010) and Zaied et al. (2012), showedthe natural occurrence of both DON and ZEN in wheatproduced under Tunisian agroclimatic conditions withlevels that exceeded in several samples the DON/ZENlimit fixed by the European Commission Reg. 1881/2006intended for human and animal consumption (EuropeanCommission, 2006). Thus, it would be interesting toassess the effects of the concomitant action of the twofusarial toxins DON and ZEN, additionally to their indi-vidual effects on intestinal cell line. We have performedour experiments on HCT116 cells, derived from a humancolon carcinoma, as alimentary intoxication is the mainroute of exposure to mycotoxins contamination. In thepresent study, we aimed to investigate whether thecombination of DON and ZEN would enhance theirrespective cytotoxic potential.

2. Materials and methods

2.1. Chemicals

DON and ZEN were purchased from Sigma Aldrich.Dulbecco’s modified eagle medium-F12 (DMEM-F12),foetal bovine serum (FBS), phosphate buffer saline (PBS),trypsin–EDTA, penicillin and streptomycin mixture, propi-dium iodide (PI), Fluorescein diacetate (FDA),chloromethyl-X-rosamine (CMXRos), Calcein-AM (C-3100),and Hanks’ balanced salt solution (HBSS) were supplied byInvitrogen. All others compounds were purchased fromSigma Aldrich and all the used chemicals were of analyticalgrade.

2.2. Cell culture and treatment

Human colorectal carcinoma cells HCT116 were main-tained as monolayer culture in DMEM-F12, supplementedwith 10% FBS, 1% L-glutamine (200 mM), 1% of mixturepenicillin (100 IU/ml) and streptomycin (100 mg/ml), in ahumidified incubator at 37 �C in an atmosphere of 5% CO2 inair.

DON and ZEN were dissolved in pure ethanol. To obtainthe studied concentrations in the cell culture media, themycotoxin treatment volume was negligible and repre-sented a maximum of 0.5% of the total medium volume. Inthese conditions, untreated cells and cells receiving thislow vehicle volume responded in the samemanner. For thisreason, we have chosen untreated cells as control.

2.3. Cell viability assay

The fluorescent probe fluorescein diacetate (FDA) hasbeen used to assess cell viability of HCT116 cells. FDA, a nonfluorescent compound, is able to cross the membrane ofliving cells. Once inside the cell, esterases will cut thediacetate and fluorescein is released resulting in ameasurable fluorescence signal that is directly related tothe viability of cells. At 50% confluence, cells were seeded in24-well multidishes and were treated with DON, ZEN orDONþ ZEN for 24 h. Then, theywere incubated for 5 min at37 �C with FDA at 0.2 mg/ml. Finally, cells were analyzed byflow cytometry. Living cells are FDA positive, whereas deadcells are FDA negative. IC50 value is defined as the con-centration inducing 50% loss of cell viability.

2.4. Cell death determination

During apoptosis, condensation and fragmentation ofnuclear DNA occurred. The hypoploid cells (Sub-G1 popu-lation) showing a loss of DNA content can be recorded byflow cytometry. HCT116 cells were exposed to DON(100 mM), ZEN (40 mM) or DON þ ZEN (100 þ 40 mM) for48 h. Negative control corresponds to untreated cells. Cellswere harvested and fixed with ice-cold 70% ethanol, storedat �20 �C overnight and stained with 50 mg/ml PI in thepresence of 250 mg/ml of RNAse A. The percentage of cellsin Sub-G1 was determined by flow cytometric analysis. Atotal of 10,000 cells were analyzed per sample.

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Fig. 1. Cytotoxic effects of individual and binary combination of DON andZEN in HCT116 cells using the FDA assay. Cells were treated with DON or ZENat indicated concentrations for 24 h (A, B). Cells were treated withDON þ ZEN at 100/40 mM for 24 h (C). Data are expressed as the mean � SDof three independent experiments. Values are significantly different(p < 0.05) from control.

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Fig. 2. Percentage of cells in Sub-G1 after individual and binary combinationof DON and ZEN in HCT116 cells. Data are expressed as the mean � SD ofthree independent experiments. (*) Values are significantly different(p < 0.05) from control.

F. Bensassi et al. / Toxicon 84 (2014) 1–6 3

2.5. Determination of mitochondrial transmembranepotential (DJm) and permeability transition pore (PTP)opening

To measure the mitochondrial membrane potential,CMXRos was used. This fluorescent probe specifically ac-cumulates in the mitochondria depending on the DJm.After toxins treatment, cells were stained with 100 nM ofCMXRos for 30 min at 37 �C and 10,000 cells were analyzedby flow cytometry. PTP opening was assessed as previouslydescribed (Deniaud et al., 2008). At 50% confluence, cellswere incubated for 15 min at 37 �C with 1 mM Calcein-AMand 1 mM CoCl2 in Hanks’ balanced salt solution (HBSS)supplemented with 1 mM HEPES, pH 7.3. Before toxinstreatment, Hanks’ solution was replaced by complete cul-ture medium.

2.6. Flow cytometric analysis

Cells were analyzed with Cell Lab Quanta MPL flow cy-tometer (Beckman–Coulter, Paris, France). Data wereanalyzed using Cell Lab Quanta Analysis software.

2.7. Statistical analysis

Each experiment was done independently three times.Values are presented as mean � SD. Statistical differencesbetween control and treated groups for all experimentswere determined by Student’s t-test. Differences wereconsidered significant at p < 0.05.

3. Results

3.1. Individual and combined toxicity of DON and ZEN onHCT116 cells

To evaluate the influence of mycotoxins on intestinalcells, the FDA assay was used. The data obtained herein,clearly indicated that the two fusarial mycotoxins induced amarked decrease of cell viability in a dose-dependentmanner (Fig. 1A,B). IC50 values determined after 24 h ofcell treatment from the viability curves were about 150 and60 mM for DON and ZEN, respectively. As shown by IC50values, ZEN inhibits the viability of HCT116 cells moststrongly than DON. The combination of two toxins at con-centrations which induce about 30% of mortality, 100 mMfor DON and 40 mM for ZEN, led to an increase of cell pro-liferation as compared to each toxin taken alone (Fig. 1C).The interaction between DON and ZEN seems to be of anantagonistic nature.

3.2. Cell cycle analysis

Flow cytometric experiments were performed in orderto analyze the condensation and fragmentation of thechromatin, considered as hallmarks of the apoptotic pro-cess (Ardestani et al., 2008). Indeed, the cells with frag-mented DNA appear as cells with a hypoploid DNA contentand are represented as a hypoploid Sub-G1 peak. HCT116cells were treated with DON at 100 mM, ZEN 40 mM orDON þ ZEN at 100/40 mM for 48 h, and then the percentage

of apoptotic cells was quantified (Fig. 2). The hypoploidpopulation significantly increased from 9.59 � 0.96% incontrol (untreated cells) to 32.6 � 2.3% and 24.4 � 1.3% inDON and ZEN-treated cells, respectively. Nevertheless,when we combined DON (100 mM) and ZEN (40 mM), thepercentage of cells at Sub-G1 decreased to 16.4 � 1.1%.

3.3. Mitochondrial apoptosis

In order to evaluate the role of mitochondria in myco-toxins dependent cell death, we performed flow cytometricexperiments to check the mitochondrial membrane

F. Bensassi et al. / Toxicon 84 (2014) 1–64

potential (DJm) after CMXRos staining (Fig. 3A). Whateverthe toxin used, DON or ZEN, both mycotoxins were able toinduce a significant loss of mitochondrial transmembranepotential (DJm). The percentage of cells CMXRos (�)reached about 3.3-fold and 2.7-fold to the control valuesafter 24 h of DON (100 mM) and ZEN (40 mM) exposure,respectively. However, the combination of DON at 100 mMand ZEN at 40 mM significantly decreased the loss of DJmto only 1.8 fold to the control, as compared to the effect ofDON and ZEN taken separately. These results indicate thatthe combination of these two mycotoxins induces sub-additive effects.

We used the calcein/cobalt method to examine openingof PTP and inner membrane (IM) permeabilization (Poncetet al., 2003). As observed in Fig. 3B, after 24 h of treatment,DON and ZEN induced PTP opening in about 31% and 25% ofcells, respectively. To further confirm the type of interactionbetween DON and ZEN on the PTP opening, we comparedthe percentage of cells Calcein (�) induced by the toxinstaken individually to the percentage of cells Calcein (�)when toxins are combined. The result recorded hereinshows that the combination of DON and ZEN induced theopening of PTP in only 17% of cells (Calcein �).

Taken together, we demonstrated that DON and ZENtrigger themitochondrial pathway of apoptosis by inducingPTP opening and mitochondrial membrane permeabiliza-tion (MMP), and most importantly, we proved that thecombined effects of the fusarial toxins are undoubtedly lesstoxic than their effects when considered individually.

4. Discussion

There is compelling evidence that mycotoxins areimplicated in many human health disorders. Nevertheless,

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Fig. 3. Effects of individual and binary combination of DON and ZEN onmitochondrial transmembrane potential (DJm) and PTP opening in HCT116cells. Loss of DJmwas assessed by flow cytometry after cells treatment withtoxins, and then stained with CMXRos (A). Opening of PTP was measured bythe calcein/cobalt assay in response to toxins treatment by flow cytometry(B). Data are expressed as the mean � SD of three independent experiments.(*) Values are significantly different (p < 0.05) from control.

it is broadly accepted that human diet can be contaminatedby a cocktail of mycotoxins which can lead to a possiblehigher risk of adverse effects than consumption of foodcontaminated with only one mycotoxin. The simultaneouspresence of mycotoxins may lead to additive, synergistic orantagonistic effects; however, data regarding the multi-exposure, relevant for hazard characterization, are scarce.

In the present study, we investigated the cytotoxic ef-fects of DON and ZEN combination. These fusarial toxinsindividually tested on HCT116 cells decrease cells viabilityas measured by the FDA assay in a dose-dependent mannerwith IC50 values of about 60 and 150 mM for ZEN and DON,respectively. This result shows that HCT116 cells weremoresensitive towards ZEN than towards DON. The combinationof the two toxins at their IC30 values led to a reducedcytotoxicity. This cell mortality was chiefly due to anapoptotic cell death. Indeed, both toxins trigger an increasein the hypoploid population (Sub-G1 cells) and a decreasein the mitochondrial transmembrane potential (DJm).Thus, we demonstrated that the two toxins, taken indi-vidually, induce apoptosis by involving the mitochondrialintrinsic pathway.

It is becoming increasingly clear that mitochondria playan important role in the regulation of cell death. Indeed,mitochondria enclose many pro-apoptotic soluble mito-chondrial intermembrane proteins (SIMPs) including cy-tochrome c, Apoptosis-Inducing Factor (AIF) andEndonuclease G (EndoG). These factors are released fromthe mitochondria following the mitochondrial membranepermeabilization (MMP) (Van Gurp et al., 2003; Kroemeret al., 2007). It is generally accepted that MMP is medi-ated by several mechanistically different models. Amongthe different models proposed, pro-apoptotic members ofBcl-2 family, such as Bax and/or Bak, or the mitochondrialpermeability transition pore (PTP) were demonstrated toform pores in mitochondrial membranes (Reed, 2006;Rasola and Bernardi, 2007; Ricchelli et al., 2011). Induc-tion of MMP results in the dissipation of the mitochondrialinner membrane potential (DJm) and leads to mitochon-drial swelling and subsequent release of apoptogenic pro-teins of the intermembrane space into the cytosol(Goldstein et al., 2005).

Furthermore, the combination of both mycotoxins; DONat 100 mMand ZEN at 40 mM; induced a significant decreasein the level of cell death, characterized by a reduction of theSub-G1 cell population and a decrease in the loss of DJm.These results strongly suggest a sub-additive mechanism ofinteraction between DON and ZEN as compared to the ef-fect of mycotoxins taken separately. In this work, we havemonitored the involvement of PTP in response to fusarialtoxins. Regarding binary combination, it leads to animportant decrease of PTP opening as compared to eachmycotoxin taken individually.

Our results clearly indicate that DON and ZEN combi-nation had no appreciable higher cytotoxic effect withrespect to each individual mycotoxin, although the mech-anisms of interaction remain unknown. These findingssuggest that the multi-exposure of mycotoxins in foodcommodities may be less cytotoxic than the presence ofmycotoxins alone. Our results are largely in agreement withthose observed for DON and ZEN at low concentrations

F. Bensassi et al. / Toxicon 84 (2014) 1–6 5

(Kouadio et al., 2007). However, Kouadio et al., 2007, re-ported synergism for high concentration for mixture ofDONþ ZEN. Moreover, Groten et al. (1998) and Tajima et al.(2002) showed that combined exposure to ZEN and DONresults in an additive effect. This is probably due to therelative complexity and intrinsic variability of cellularresponses.

At the best of our knowledge, although cytotoxic effectsof individual mycotoxins have been widely studied, dataexploring the in vitro cytotoxicity interaction of myco-toxins, particularly DON þ ZEN, are generally limited.Indeed, it has been proposed that DON exerts its toxicity tocells by a mechanism known as the ribotoxic stressresponse by binding to the peptidyl transferase region ofthe ribosome and interfering with initiation and elongation(Shifrin and Anderson, 1999; Li and Pestka, 2008). ZENconstitutes an important endocrine disrupters, whichcauses estrogenic effects and alter reproduction (Diekmanand Green, 1992). Oxidative damage is likely to be evokedas one of the main pathway of ZEN toxicity (Hassen et al.,2007). This oxidative damage may therefore be an initi-ating event and contribute, at least in part, to the mecha-nism of ZEN different genotoxic and cytotoxic effects. Thus,oxidative stress seems implicated in both DON and ZENtoxicities. Nevertheless, it is hard to predict the nature ofcombined toxic effects of these mycotoxins, because it de-pends on many aspects including chemistry and mecha-nism of action, toxicodynamics as well as toxicokinetics.This means that several mycotoxins can affect certain tar-gets and initiate more than one event in the cell machineryleading to extremely complicated cell response. The in-vestigations performed with binary mixture of ZEN withother mycotoxins highlight the idea that the observed sub-additive effect may be due to the estrogenic properties ofZEN which can interfere with other mycotoxins to raise thecell proliferation (Thuvander et al., 1999; Ruiz et al., 2011).

Taken together, our in vitro findings indicate that DONand ZEN, individually, can induce cell death through a PTP-dependent mechanism and their mixture significantly re-duces the level of mortality. This sub-additive effect is ofpractical importance since fusarial toxins often occur incombination. However, this finding should be validatedwith other cell lines.

Acknowledgments

This study was supported by “Le Ministère Tunisien del’Enseignement Supérieur, de la Recherche Scientifique etde la Technologie”. We thank DrMbarek Chetoui (Faculty ofSciences, Tunisia) for his help and Dr. Salwa Bacha (Uni-versity of La Manouba, Tunisia) for the correction of theEnglish manuscript.

Conflict of interest statement

There are no conflicts of interest.

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