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Toxicon 93 (2015) 103e111

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Toxicon

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

Alternariol-induced cytotoxicity in Caco-2 cells. Protective effect of thephenolic fraction from virgin olive oil

C. Chiesi a, C. Fernandez-Blanco b, L. Cossignani a, G. Font b, M.J. Ruiz b, *

a Dipartimento di Scienze Economico-Estimative e degli Alimenti, Sezione di Chimica Bromatologica, Biochimica, Fisiologia e Nutrizione, Facolt�a diFarmacia, Universit�a degli Studi di Perugia, Perugia, Italyb Laboratory of Toxicology, Faculty of Pharmacy, University of Valencia, Valencia, Spain

a r t i c l e i n f o

Article history:Received 19 September 2014Received in revised form18 November 2014Accepted 20 November 2014Available online 21 November 2014

Keywords:AlternariolCytotoxic and cytoprotective effectROS generationExtra virgin olive oilPhenolic compoundsCaco-2 cells

* Corresponding author.E-mail address: M.Jose.Ruiz@uv.es (M.J. Ruiz).

http://dx.doi.org/10.1016/j.toxicon.2014.11.2300041-0101/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The extra virgin olive oil (EVOO) has been associated to antioxidant effects. The mycotoxin alternariol(AOH) can contaminate olives. The aims of this work were to determine the cytotoxic effects and reactiveoxygen species (ROS) produced by AOH, tyrosol and oleuropein (two polyphenols of olive oil) and a realEVOO extract in Caco-2 cells. The MTT assay and the ROS production by the H2-DCFDA probe were used.Results demonstrated that AOH reduces cellular proliferation depending on concentration, whereastyrosol and oleuropein did not (12.5e100 mM). The combination of AOH þ oleuropein (50 mM) increasedcell proliferation (24%) whereas, AOH þ tyrosol decreased (47%) it. Besides, AOH increased ROS gener-ation depending on time and concentration. Oleuropein þ AOH decreased ROS production. However,25 mM of tyrosol increased 1.2-fold the ROS production. Respect to the EVOO extract, cytoprotective effect(151%) was evidenced, even with the combination EVOO extract þ AOH (15%e55% respect to cellsexposed to AOH alone). ROS generation was significantly reduced compared to ROS generation producedby 25 mM of AOH alone. The phenolic antioxidant of EVOO decreases cytotoxicity and ROS production inCaco-2 cells exposed to AOH. Thus, polyphenols of EVOO could contribute to diminish the toxicologicalrisk that mycotoxins can produce to humans.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Mycotoxins are biologically active secondary fungal metabolitesfound as contaminants of food and feed. These compounds pose arisk for disease in human and animals (Ostry, 2008). Mycotoxins aremainly produced by genera Fusarium, Penicillium, Aspergillus,Alternaria and Claviceps. Alternariol (AOH), a diphenolic compoundproduced by Alternaria sp, is often found as contaminant in fruit(including olives) and cereals products (Logrieco et al., 2003). It hasbeen demonstrated that extracts of Alternaria alternata are geno-toxic and mutagenic in vitro (Solhaug et al., 2012). Therefore A.alternata has been implicated in an elevated incidence of oeso-phageal carcinogenesis (Liu et al., 1991). AOH at concentration of15e30 mM almost completely blocked cell proliferation (Solhauget al., 2012).

Olives are often affected by Alternaria sp. particulary if the fruitsremain for a long time on the soil after ripening (Logrieco et al.,

2003). Olive oil is the main fatty component of the Mediterra-nean diet. Extra virgin olive oil (EVOO) is a vegetable oil which canbe obtained directly from olive fruit using only mechanicalextraction and which can be consumedwithout further treatments.Its chemical composition has been regulated by CommissionRegulation EC No. 1989/2003. This oil exhibits numerous biologicalfunctions which are beneficial for the healthiness of humans (DiBenedetto et al., 2007; Driss and El-Benna., 2010). It is character-ized by high percentage of monounsaturated fatty acids as well asby its elevated content in antioxidant agents (Di Benedetto et al.,2007). On the other hand, under certain conditions, antioxidantscan act as pro-oxidants. The antioxidant or pro-oxidant activityintimately depends on antioxidant concentration. The conse-quences of pro-oxidant activity could be the possible damage to thebiomolecules such as DNA, proteins and lipids, and the consequentcellular death (Bouayed and Bohn, 2010).

Several important biological properties (antioxidant, anti-inflammatory, chemopreventive and anti-cancer) and the charac-teristic pungent and bitter tasty properties have been attributed toEVOO phenolic compounds (Servili et al., 2009). Olive polyphenolsare recognized as potential nutraceutical targets for food and

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pharmaceutical industries (Obied et al., 2007). Moreover, the an-tioxidants present in olive oil are able to scavenge free radicals andafford an adequate protection against peroxidation (de la Lastraet al., 2001). The main antioxidants of EVOO are polyphenols rep-resented by lipophilic and hydrophilic phenolic compounds. Thelipophilic phenolic compounds, among tocopherols and toco-trienols, can be found in other vegetable oils, but the hydrophilicphenolic compounds, including tyrosol or 2-(4-hydroxyphenyl)ethanol (p-HPEA) and oleuropein, two of the most representativeantioxidants present in the EVOO, are not generally present in otheroils and fats (Boskou, 1996). In vitro methods are widely used todetermine the implication of intracellular reactive oxygen species(ROS) in the cytotoxic effect produced by mycotoxins (Ferrer et al.,2009; Prosperini et al., 2013a,b). The cytoprotection effect ofpolyphenols in cells exposed to mycotoxins was also tested by cellculture methods (Manna et al., 1997, 2000; Lombardi et al., 2012).Among the in vitro systems to assess bioavailability, in recent years,the use of cell cultures have beenwidely employed (Sambruy et al.,2001). During the past few years, Caco-2 cells monolayer (CCM)have been widely accepted by pharmaceutical companies and byregulatory authorities as a standard permeability-screening assayfor prediction of drug intestinal permeability (Grajek and Olejnik,2004). Caco-2 model is considered as a model to study passivedrug or other toxic substances absorption across the intestinalepithelium due to the good correlation obtained between data onoral absorption in humans and the results in Caco-2 model(Artursson and Karlsson, 1991; Manyes et al., 2014; Meca et al.,2012; Prosperini et al., 2012).

In this study, the Caco-2 cells were selected to determine, a) thecytotoxic effects of AOH, b) cytotoxicity of tyrosol and oleuropein,in order to consider the concentrationwhich acts as an antioxidant,c) the cytotoxic effects of the binary combination of AOH and eachantioxidant selected; d) ROS generation of AOH, tyrosol andoleuropein; e) ROS generation of the binary combinations of AOHplus tyrosol and AOH plus oleuropein. And finally, the Caco-2 cellswere used to determine the antioxidant effect of a real extract of anEVOO sample obtained from a local producer in the Umbria region(Italy) after AOH exposure.

2. Materials and methods

2.1. Reagents

The reagent grade chemicals and cell culture components used,mainly Dulbecco's Modified Eagle's Medium (DMEM), penicillin,streptomycin, trypsin/EDTA solution, HEPES, thiazolyl blue tetra-zolium bromide (MTT), no essential aminoacids (NEAA), PhosphateBuffer Saline (PBS), Sorensen's glicine buffer, glucose, dimethylsulfoxide (DMSO), dichlorodihydrofluorescein diacetate (H2-DCFDA), tyrosol (138.16 g/mol; �98% purity), oleuropein (540.51 g/mol; �98% purity) and AOH (258.2 g/mol; �98% purity) were Sig-maeAldrich products (Sigma Co., St. Louis, Mo., USA). Foetal calfserum (FCS) was from Cambrex Company (Belgium). Deionizedwater (<18 MU cm resistivity) was obtained from a Milli-Q waterpurification system (Millipore, Bedford, MA, USA). All other re-agents were of standard laboratory grade. Stock solutions of AOH,tyrosol and oleuropeinwere prepared in DMSO. Final concentrationof AOH, tyrosol and oleuropein in the assay were achieved byadding the culture medium. The final DMSO concentration in themedium was �1% (v/v). Control cultures were exposed to theequivalent concentration of DMSO.

2.2. Cell culture of Caco-2 cells and treatment

The Caco-2 (ATCC HTB-37) cells were cultured in monolayer in9 cm2 polystyrene tissue culture dishes with DMEM supplementedwith 25 mM HEPES, 1% NEAA, 100 U/ml penicillin, 100 mg/mlstreptomycin, and 10% heat inactivated FCS. Incubation conditionswere pH 7.4, 37 �C and 5% CO2 in a 95% relative humidity atmo-sphere. The cells were subcultivated after trypsinization (trypsin-EDTA) once or twice per week and resuspended in complete me-dium in a 1:3 split ratio. Cell were subculture routinely with only asmall number of sub-passages (<70 subcultures) in order tomaintain the genetic homogeneity. Absence of mycoplasma waschecked routinely using the Mycoplasma Stain Kit (SigmaeAldrich,St. Louis, MO, USA).

2.3. Determination of cell viability by the MTT assay

Caco-2 cells were cultured into 96-well tissue-culture plates byadding 200 ml/well of a suspension of 3 � 104 cells/ml. After cellsreached 90% confluence, the culturemediumwas replaced and cellswere exposed to 200 ml of i) fresh medium containing differentconcentrations of AOH (12.5, 25, 37.5, 50, 75 and 100 mM) plus acontrol during 24, 48 and 72 h and ii) fresh medium containingdifferent concentrations of tyrosol or oleuropein (12.5, 25, 37.5, 50,75 and 100 mM) plus a control during 24 h. For the binary combi-nations (AOH þ oleuropein and AOH þ tyrosol), the cells wereexposed to oleuropein or tyrosol (concentrations: 25, 50 and100 mM) during 24 h. Afterwards, the medium containing oleur-opein or tyrosol was removed and cells were exposed to AOH (12.5,25, 50 and 100 mM) for 24 h of incubation. For the binary combi-nations procedure, from now on it will call pre-treatment assay.The plates were incubated for 24 h at 37 �C, and the cytotoxicitywas determined by the MTT assay.

The MTT assay determines the viability of cells by the reductionof yellow soluble tetrazolium salt (MTT), only in the metabolicallyactive cells, via a mitochondrial-dependent reaction to an insolublepurple formazan crystal. The MTT viability assay was performed asRuiz et al. (2006). Briefly: after exposure of AOH, tyrosol, oleuropeinor the binary combinations, the medium containing these com-pounds was removed and cells of each well received 200 ml freshmedium plus 50 ml of MTT. The plates were wrapped in foil andincubated for 4 h at 37 �C. After 4 h incubation, the medium con-tained the MTT was removed and the resulting formazan wassolubilised in DMSO. The absorbance was measured at 570 nmusing an ELISA plate reader Multiscan EX (Thermo Scientific, MA,USA). The results were expressed in relative form to cell cultureprotein content in order to avoid misinterpretation due to the in-fluence of the chemical tested on cell proliferation and detachment.

2.4. Determination of intracellular reactive oxygen species by H2-DCFDA

Intracellular ROS production was monitored in Caco-2 cells byadding the H2-DCFDA according to Ruiz-Leal and George (2004). Inbrief: 3 � 104 cells/well were seeded in a 96-well black culturemicroplate. Once cells exhibited 90% confluence, the culture me-diumwas replaced and cells were loaded with 20 mMH2-DCFDA for20 min and then, the medium with H2-DCFDA was removed andwashed with PBS before the addition of AOH (15, 30 and 60 mM),AOH þ tyrosol, AOH þ oleuropein or control (medium with 1%DMSO). For binary combinations, cells were simultaneously incu-bated with tyrosol or oleuropein (1, 2.5 and 25 mM) and AOH (15, 30and 60 mM). H2-DCFDA is non-fluorescent until it is hydrolysed byintracellular esterases and readily oxidized to the highly fluorescentDCF in the presence of ROS. Increases in fluorescence were

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measured at intervals up to 2 h at excitation and emission wave-lengths of 485 and 535 nm, respectively.

To determine oleuropein and tyrosol test concentrations, severalconsiderations were taken into account. Tyrosol and oleuropeinconcentrations reported were: 11.9 mg/kg olive oil (Servili et al.,2009) and 11 mg/kg olive oil (Perri et al., 1999), respectively. Tak-ing into consideration this value and considering that olive oilconsumption in Spain is 11.5 kg/year (EFSA, 2011), it could bepossible to calculate the daily intake of tyrosol and oleuropein,0.375 mg/day and 0.346 mg/day, respectively. Finally, the dailyintake of oleuropein and tyrosol corresponds to the concentrationtested of 2.5 and 25 mM, respectively. Therefore, to determine ROSgeneration, 1, 2.5 and 25 mM of AOH were tested to verify if theconcentration which corresponds to the daily intake could reallyprotect and for comparing them. Moreover, 1 mM oleuropein andtyrosol was selected considering worse situations, i. e. countrieswhere the consumption of olive oil is less.

2.5. Preparation of a real extract of extra virgin olive oil

An EVOO is a complex matrix which contains a great variety ofantioxidant components, so the analysis of the cytoprotective effectby the in vitro method was carried out. The application of in vitromethod with Caco-2 cells is very remarkable because of humanstudies conducted on patients fed with olive oil polyphenolsshowed that ingested olive oil phenols are absorbed, supporting theevidence that absorption occurs in the small intestine (Covas et al.,2010; Corona et al., 2014).

The EVOO sample was obtained from a local producer in theUmbria region (Italy) and stored at room temperature until anal-ysis. The EVOO extract was prepared according to Gutfinger, (1981),properly modified as follow: Two grams of oil were dissolved in1 ml hexane and the solution was extracted three times with 2 mlportions of 60% aqueous methanol. The mixtures were shaken for2 min and combined. The obtained extract was brought to drynessin a vacuum rotary evaporator at 65 �C. The residuewas dissolved in1 ml DMSO and stored at �20 �C until it was used.

To determine if EVOO extract alters Caco-2 proliferation cells,the MTT assay was used as previously described. For the MTT assay,the following dilution series of the extract with medium was per-formed: 1, 1/2, 1/4, 1/6 and 1/8. To investigate as the antioxidanteffect of EVOO extract can inhibit cytotoxic effect on AOH-inducedin Caco-2 cells, the EVOO extract and its different dilutions weresimultaneously incubated with AOH (25, 50 and 100 mM). Knowingthat AOH produce ROS in Caco-2 cells, EVOO extract was checked toknow if it could prevent ROS generation by AOH when they weresimultaneously exposed in Caco-2 cells. For this assay, only 25 mMof AOH was selected for the mixtures according to the maximumconcentration of AOH found in oilseeds in Europe (2300 ng/g olive;Logrieco et al., 2003) and according to the total daily per capitaconsumption of olive oil in Europe (FAO, 2009; EFSA, 2011).

2.6. Statistical analysis

Statistical analysis of data was carried out using SPSS version 19(SPSS, Chicago, IL, USA), statistical software package. Data wereexpressed as mean ± SD of three independent experiments. Thestatistical analysis of the results was performed by Student's t-testfor paired samples. Differences between groups were analysedstatistically with one-way ANOVA followed by the Tukey HDS post-hoc test for multiple comparison. The level of p � 0.05 wasconsidered statistically significant.

3. Results

3.1. Effect of AOH, oleuropein and tyrosol on Caco-2 cells viability

Fig. 1 shows the chemical structures of AOH, tyrosol and oleur-opein. The cytotoxic effect of AOH, tyrosol and oleuropein on Caco-2 cells is shown in Fig. 2. To determine the molar concentration ofAOH, oleuropein and tyrosol that reached 50% inhibition of cellularproliferation (IC50) under assay condition. The IC50 values weredetermined graphically from the dose-response curves. Fig. 2ashows that after 24 and 48 h of incubation the AOH significantlyreduced cell viability (about 40%) at the higher concentrationstested (75 and 100 mM). Similar cellular viability reduction wasobserved at 72 h of incubation from 50 mM to 100 mM of AOH.Moreover, AOH did not show IC50 value in the range of concen-trations tested and the time intervals assayed (Fig. 2a). However,morphological changes induced by AOH were reduction in the cellnumber, being these alterations more evident at 48 and 72 h afterthe exposure to AOH. However, no morphological studies wereperformed.

The cytotoxic effect of tyrosol and oleuropein on Caco-2 cells isshown in Fig. 2b. Not cytotoxic effect of oleuropein and tyrosol wasobserved. And no IC50 values were obtained for any of the twopolyphenols assayed. On other hand, they produce some stimula-tions of the mitochondrial function reaching 157% and 126%compared to control, for oleuropein and tyrosol, respectively.

3.2. Intracellular ROS production

The ability of AOH to produce ROS and consequent oxidativedamage was also evaluated. To determine the changes in the redoxstatus in response to AOH, Caco-2 cells were exposed to differentconcentrations of this mycotoxin from 0 to 120 min (Fig. 3). Theincrease of ROS production was time- and concentration depen-dent, when compared to the basal rate. These results indicated thathigh production of oxidizing species was produced immediatelyafter AOH exposure.

3.3. Cytoprotection and antioxidant activity of tyrosol andoleuropein

To investigate whether oleuropein and tyrosol can inhibitcytotoxic effect AOH-induced in Caco-2 cells, fresh medium con-taining simultaneously the phenolic compound (concentration 25,50 and 100 mM) and AOH (12.5, 25, 50 and 100 mM) plus a control(1% DMSO) were assayed. The effects of these binary combinationson the Caco-2 cells proliferation using the MTT assay is showed inFig. 4.

As shown in Fig. 4a after 24 h of incubation no significant dif-ferences were observed when AOH was simultaneously exposedwith 25 mM oleuropein respect to AOH tested alone. However,when Caco-2 cells were simultaneously incubated with AOH and50 mM oleuropein a slight cytoprotection (24%) was observed. FiftymM oleuropein prevented cell damage induced by 100 mM of AOHrespected AOH tested alone. However, 100 mM oleuropein incu-bated with 12.5 and 50 mM of AOH, decreased cell proliferationabout 20% and 10%, respectively respect to AOH tested alone(Fig. 4a). In relation to tyrosol, no differences were observed whenAOH and tyrosol were simultaneously exposed in Caco-2 cells overall concentration tested (Fig. 4b). Moreover, the lowest concentra-tion of tyrosol simultaneously exposedwith 100 mMAOH is not ableto protect Caco-2 cells producing a significantly increase in thecytotoxic effect. With this binary combination, cell proliferationdecrease about 47% respects to AOH tested alone (Fig. 4b). Even

Fig. 1. Chemical structure of a) alternariol, b) oleuropein and c) tyrosol.

oleuropeintyrosol

Fig. 2. Cytotoxic effects of a) AOH and b) oleuropein and tyrosol in Caco-2 cells after24 h of exposure by the MTT assay. Each point represents the mean value of at leastthree experiments. An asterisk indicates a significant difference from control value(p � 0.05).

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Fig. 3. Time dependence of ROS-induced fluorescence in Caco-2 cells exposed to AOH.Caco-2 cells were loaded with H2-DCFDA for 20 min in 96-well plates (30,000 cells perwell) and then exposed to AOH (15, 30 and 60 mM) or control (1% DMSO). Fluorescenceof oxidized DCF was followed by emission at 535 nm and the excitation of 485 nm.Mean ± SD, 24 replicates. (*p � 0.05) different significantly from the control.

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Fig. 4. Protective effect of (a) oleuropein and (b) tyrosol in Caco-2 cells exposed to AOH(12.5, 25, 50 and 100 mM concentration): Cells were pre-treated with oleuropein andtyrosol (25, 50 and 100 mM) during 24 h. Thereafter, AOH was exposed during 24 h andviability of these cells was measured using the MTT assay. An asterisk indicates asignificant difference from control value (p � 0.05).

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with this combination, an IC50 value of 70 mM was obtained(Fig. 4b).

On the other hand, to assay the capacity of oleuropein andtyrosol to protect Caco-2 cells from AOH-mediated oxidative injury,the cells were incubated in the presence of each phenolic com-pound (1, 2.5 and 25 mM concentration) simultaneously with AOH(Figs. 5 and 6). As can be observed in Fig. 5, after combinations ofAOH (15, 30 and 60 mM) and oleuropein (1, 2.5 and 25 mM) not ROSwere produced when compared to the basal rate. The oleuropeincould serve as ROS scavenger. Similarly, the treatment of Caco-2cells with AOH (15, 30 and 60 mM) and tyrosol (1 and 2.5 mM)prevented ROS production. However, when AOH (30 and 60 mM)was simultaneously exposed with 25 mM of tyrosol a slight pro-duction of ROS was produced at 5 min which is maintained up to120 min respect to 0 min of exposure. The highest relative intensityof fluorescence observed in Caco-2 cells was about 1.2-fold higherthan the basal rate (Fig. 6c).

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Fig. 5. Time dependence of ROS-induced fluorescence in Caco-2 cells exposed to AOH (15, 30 and 60 mM) simultaneously with oleuropein a)1 mM, b) 2.5 mM and c) 25 mM. Caco-2cells were loaded with H2-DCFDA for 20 min in 96-well plates (30,000 cells per well) and then exposed to the binary combination (AOH þ oleuropein) or control (1% DMSO). AOHwas tested alone at the same concentrations and time of exposure (see Fig. 3). Fluorescence of oxidized DCF was followed by emission at 535 nm and the excitation of 485 nm.Mean ± SD, 24 replicates. (*p � 0.05) different significantly from the control.

C. Chiesi et al. / Toxicon 93 (2015) 103e111 107

3.4. Effect of a real extract of EVOO in Caco-2 cells exposed to AOH

Fig. 7 shows the effect of a real extract of EVOO on Caco-2 cellsproliferation evaluated by the MTT assays over 24 h of exposure. Ascan be observed, cell viability (%) increased after all dilutions of theEVOO extract tested (Fig. 7); it means that cell proliferationincreased more than the number of cells in control showing aprotective effect of EVOO extract in Caco-2 cells. The highest pro-tective effect reached 151% (extract dilutions 1/2, 1/4 and 1/6)versus control (100%; Fig. 7). The effect of the simultaneous com-bination of the EVOO extract (and its dilutions, ½, ¼, 1/6, 1/8) andAOH (25, 50 and 100 mM) are shown in Fig. 7. As shown in Fig. 7,when AOH and the EVOO extracts were simultaneously exposed inCaco-2 cells cytoprotective effect was observed respect to AOHtested alone. Simultaneous exposition of the EVOO extract and 25

or 50 mM of AOH increased (from 24 to 55%) cell proliferationrespect AOH tested alone (Fig. 7). Smaller increase in cell prolifer-ation was obtained with the mixtures of 100 mM of AOH plus EVOOextracts (Fig. 7). The combinations of 100 mM AOH plus EVOO ex-tracts only increased significantly Caco-2 cell proliferation with ½dilution extract (15%) and the EVOO extract without dilution (36%)respect to AOH tested alone (Fig. 7).

As shown in Fig. 8, the increase of ROS generationwas producedin Caco-2 cells after 25 mM of AOH exposure. Twenty-five mM ofAOH increased ROS generation in time dependent manner, whencompared to the basal rate. Similar effect was observed when25 mM of AOH was simultaneously exposed with 1/8 dilution EVOOextract (Fig. 8) from 45 up to 120min of incubation. However, when25 mM of AOH was simultaneously exposed with EVOO extractwithout dilution, ROS generation was significantly reduced (from 5

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Fig. 6. Time dependence of ROS-induced fluorescence in Caco-2 cells exposed to AOH (15, 30 and 60 mM) simultaneously with tyrosol a) 1 mM, b) 2.5 mM and c) 25 mM. Caco-2 cellswere loaded with H2-DCFDA for 20 min in 96-well plates (30,000 cells per well) and then exposed to the binary combination (AOH þ tyrosol) or control (1% DMSO). AOH was testedalone at the same concentrations and time of exposure (see Fig. 3). Fluorescence of oxidized DCF was followed by emission at 535 nm and the excitation of 485 nm. Mean ± SD, 24replicates. (*p � 0.05) different significantly from the control.

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to 120 min) compared to ROS generation produced by 25 mM ofAOH tested alone (Fig. 8). So, EVOO extract could be considered as apotential protector against a condition of oxidative stress generatedby AOH.

4. Discussion

The resorcyclic lactone AOH represents one of the major sec-ondary metabolites produced by Alternaria alternate. This myco-toxin can be found in foods and feeds and represents a health riskfor humans and animals (EFSA, 2011). The present study

investigated the cytotoxic effects of the AOH in Caco-2 cells. Ourresults demonstrated that AOH decrease cell viability and prolif-eration in Caco-2 cells at higher concentrations (>50 mM). However,there is a wide variation in sensitivity to A. alternata mycotoxinsamong cell lines depending on the tissue of origin and possible thedegree of differentiation. Wollenhaupt et al. (2008) demonstratedthat AOH (from 3.12 to 12.5 mM) inhibits the metabolic activity ofporcine endometrial cells as determined by theMTTassay. Tiemannet al. (2009) shown that the cell viability measured as the activity ofmitochondrial dehydrogenases was more sensitive to toxic effectsof AOH (from 1.6 to 12.8 mM) in porcine granuloma cells than the

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Fig. 7. Protective effect of EVOO extract dilutions in Caco-2 cells exposed to AOH (25, 50 and 100 mM concentration). Cells were pre-treated with EVOO extract dilutions (1, ½, ¼, 1/6and 1/8) during 24 h. Thereafter, AOH was exposed during 24 h and viability of these cells was measured using the MTT assay. (*p � 0.05) different significantly from the control.(#p � 0.05) different significantly from each own EVOO extract without AOH treatment.

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number of cells (relative to untreated controls), suggesting that theAOH inhibits metabolic activity and proliferation rather than topromote cell death. Similar results were obtained by Tiessen et al.(2013). They found that AOH (from 0.1 to 50 mM) did not pro-duced IC50 in HT29 cells neither using the SRB assay nor the WST-1assay after 24 h of incubation. However, about 10e15% cell viabilityreduction was observed at concentration of 10 mM or higher.Brugger et al. (2006) determined the number of viable cells with anelectronic cell counter as a measure of cytotoxicity and prolifera-tion. They found a concentration-dependent reduction of viablecells after AOH treatment with up to 30 mM (V79 cells) or 20 mM(MLC) AOH, which reduced the number of cells to approximately35% and 69%, respectively. Bensassi et al. (2012) demonstrated thatthe human colon carcinoma (HCT116) cells are sensible to AOH.They demonstrated that AOH induced a decrease in HCT116 cellviability in a dose-dependent manner. Moreover, they found an IC50value of 65 mM after 24 h of exposure.

Although cytotoxicity of AOH is low compared to other promi-nent mycotoxins, its relevance in the context of fruit (includingolives) and cereal products has led to considerable interest in publichealth. On the other hand, virgin olive oil (VOO) is one of thecomponents of the Mediterranean diet, which is considered animportant source of phenolic compounds like hydroxytyrosol,tyrosol, oleuropein, ligstroside, lignans (Driss and El-Benna., 2010).The presence of potent antioxidant compounds in the VOO hascontributed to the increasing interest of scientists to investigate thepossible preventive effect of the Mediterranean countries, associ-ated high oil consumption, on chronic-degenerative diseases suchas cardiovascular diseases and cancer. Antioxidants have been oftenassociated with cytoprotective effects.

In this study, tyrosol and oleuropein do no decrease cell viabilityin small-intestinal epithelial Caco-2 cells. Bulotta et al. (2011)analysed the effect of oleuropein in two human breast cancercells (T-47D and MCF-7 cells) during 72 h of incubation. Theyproved that oleuropein caused a dose-dependent inhibition of cellsproliferation in T-47D cells, with a maximal reduction evidenced at100 mM (aprox. 40%). Whereas, no alteration in MCF-7 cell viabilitywas produced by oleuropein. Tyrosol is a simple phenol andoleuropein is a phenolic secoiridoid glycoside, and both of them

identified in VOOs. Moreover, secoiridoids comprised about50e70% of the total phenolic derivates (Gioffi et al., 2010; Bulottaet al., 2011). Once the absence of cytotoxic effect was demon-strated, the cytoprotection effect in cells treated with the AOH inconcentration within the daily intake range was assessed. From allconcentrations of tyrosol and oleuropein tested, only 50 mM oftyrosol prevented cell cytotoxicity (Fig. 4a) induced by AOH(increasing Caco-2 cells viability respect to AOH exposure) indi-cating that tyrosol suppresses the AOH-induced cytotoxicity. Thesefindings proved that tyrosol was higher effective in cell cytopro-tection than oleuropein. And, can be corroborated by Manna et al.(2000), whom demonstrated that the transport of tyrosol andother tyrosol derivates occurs via a passive diffusion mechanism.And, that uptake is linear in the 50e100 mM range at 37 �C.Furthermore, discoveries of Di Benedetto et al. (2007) support thisevidence because they evaluated the intracellular content of tyrosolwith time, demonstrating that tyrosol was detected early andshowed a time-dependent intracellular storage, doubling its con-centration from 5 min to 18 h. However, oleuropein can to beabsorbed in small intestine, albeit poorly. Therefore, to be highlyabsorbed oleuropein has to be degraded into hydroxytyrosol as themajor end product (Covas et al., 2010; Corona et al., 2014).

On the other hand, the AOH produces ROS at all concentrationstested leading to oxidative stress in Caco-2 cells. Oxidative stressproduced by AOH might be one mechanism of the cytotoxicity ofthis mycotoxin. Correspondingly, Tiessen et al. (2013) found thatAOH (10, 25 and 50 mM) enhances ROS production in HT29 cellsincubated for 1 h. Analogously, Schwarz et al. (2012) demonstratedthat the treatment of HT29 cells with AOH resulted in an increase ofthe DCF signal, up to 2.5-fold at 50 mM. This increase was significantfrom 10 mM of AOH. Bensassi et al. (2012) demonstrated that ROSproduced in response to AOH in HCT116 cells appears to be mainlymitochondrial superoxide anion. On the other hand, Schreck et al.(2012) no significant differences in ROS-levels observed betweenuntreated cells and cells treated with 20 and 40 mMof AOH and 1 or2 h after incubation with H2DCF-DA.

Tyrosol and oleuropein showed protective effect against theAOH-induced oxidative injury to the intestinal Caco-2 cells imme-diately after the addition of the two olive oil polyphenols (Figs. 5

Fig. 8. Time dependence of ROS-induced fluorescence in Caco-2 cells exposed simultaneously to 25 mM AOH and EVOO extract dilutions (1, ½, ¼, 1/6 and 1/8). Caco-2 cells wereloaded with H2-DCFDA for 20 min in 96-well plates (30,000 cells per well) and then exposed to 25 mM AOH, extract dilutionsþ25 mM of AOH or control (1% DMSO). Fluorescence ofoxidized DCF was followed by emission at 535 nm and the excitation of 485 nm. Mean ± SD, 24 replicates (*p � 0.05) different significantly from the control.

C. Chiesi et al. / Toxicon 93 (2015) 103e111110

and 6). Driss and El-Benna (2010) reported that hydroxytyrosol(structurally related phenol to tyrosol present in olive oil) can serveas scavenger of aqueous peroxyl radicals near the membrane sur-face, while oleuropein acts as a scavenger of chain-propagatinglipid hydroxyl radicals within membranes. Bulotta et al. (2011)observed that exposure of T-47D and MCF-7 cells to H2O2(250 mM) in presence of oleuropein (10 and 100 mM) reduced ROSgeneration respect to cells without oleuropein. Furthermore, Gioffiet al. (2010) demonstrated the moderate scavenging activity ofoleuropein in Caco-2 cells, against superoxide anion, one of themost aggressive ROS products. Equal results were obtained by DiBenedetto et al. (2007) in J774 A.1 cells, where tyrosol wasslightly effective at counteracting the early production of super-oxide anion radical (within 12 h of exposure) and hydrogenperoxide (after 24 h). However, pre-treatment of J774 A.1 cells withtyrosol for 2 h and then removed from the culture medium beforeoxidant compound addition, appeared highly effective, because ofcomplete inhibition of ROS rise (at 6 h of incubation). These resultssuggest that the effectiveness of tyrosol depend on its capability topenetrate the cells. Similarly to our results, they demonstrated thatwhen cells were exposed to oleuropein (250 mmol/l) an increase incell viability respect to the positive oxidant control (10 mmol/l ofH2O2) was observed. So, oleuropein suppresses the H2O2-inducedtoxicity.

The antioxidant activity of EVOO extract was determined withthe same procedure described previously. The EVOO extractshowed cytoprotection in Caco-2 cells (Fig. 7). Isik et al. (2012)determined the effects of olive extracts on Caco-2 cell viabilityand proliferation. They found that 1/20 dilution extract reducesignificantly the Caco-2 cells viability without cytotoxicity whilelow concentrations increased cell growth in a time-dependentmanner. And, when the extracts were simultaneously exposedwith AOH in Caco-2 cells, these cytoprotection was maintained atthe lower AOH concentrations tested (Fig. 7). However, 100 mM ofAOH blocks the cytoprotection function of the more diluted EVOOextract, showing no differences with the cytotoxic effect producedby AOH (Fig. 7). Similarly, EVOO extract (without dilution), which isthe highest polyphenol-rich extract tested, showed to be a potentscavenger of ROS produced by AOH in Caco-2 cells (Fig. 8). TheEVOO extract protected epithelial cell line Caco-2 cells from ROS-induced cytotoxicity. Thus, these findings demonstrated thatEVOO extract possess antioxidant/free-radical scavenging proper-ties, which are very likely due to the presence of high contents ofphenolic compounds. The results were in agreement with those ofother authors whose revealed that olive oil phenolic compoundsare effective scavengers of hydroxyl, superoxide and peroxyl

radicals (Gioffi et al., 2010; Driss and El-Benna, 2010; Sarria et al.,2012). Moreover, olive polyphenols are recognized as potentialantioxidant additives for food, dietary supplements, functionalfoods and natural cosmetics as well as for pharmaceutical in-dustries (Obied et al., 2007; Bulotta et al., 2011).

In conclusion, the protective effect of cells against cytotoxiccompounds depends on a balance between antioxidants and pro-oxidant components. Increased levels of antioxidants mayrespond to polyphenols in food commodities (as olives, olive oil,wine, etc.) and dietary supplements (oleuropein-rich diets, etc.)which have been studied extensively to improve antioxidantreserve and thereby help regulate oxidative damage. Our data alsoprovide experimental support for the hypothesis of the key roleplayed by phenolic antioxidant fraction of olive oil, thus contrib-uting to the positive effect of olive oil in lowering the risk of ROS inMediterranean diet.

The results obtained shown that olive oil phenols improveantioxidant defence system. Thus, food commodities containingpolyphenols (specially, EVOO) could contribute to diminish thetoxicological risk that natural contaminants in diet, as mycotoxins,can produce to humans.

Ethical statement

The authors have obeyed all the standards of ethical behaviourin relation to.

� Reporting standards� Data Access and Retention� Originality and Plagiarism� Multiple, Redundant or Concurrent Publication� Acknowledgement of Sources� Authorship of the Paper� Hazards and Human or Animal Subjects (not any used)� Disclosure and Conflicts of Interest� Fundamental errors in published works

Acknowledgements

This work was supported by the Economy and CompetitivenessSpanish Ministry (AGL2013-43194-P) and the fellowships grantedby Universit�a degli Studi di Perugia, Italy (C. Chiesi; LLP Erasmusgrant).

C. Chiesi et al. / Toxicon 93 (2015) 103e111 111

Transparency document

Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.toxicon.2014.11.230.

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