Toxicology and Applied Pharmacology 285 (2015) 118–127

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Nivalenol induces oxidative stress and increases deoxynivalenol pro-oxidant effect in intestinal epithelial cells Marisanta Del Regno a, Simona Adesso a, Ada Popolo a, Andrea Quaroni b, Giuseppina Autore a, Lorella Severino c,1, Stefania Marzocco a,1,⁎ a b c

Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno, Italy Department of Biomedical Sciences, Cornell University, Veterinary Research Tower, Cornell University, Ithaca, NY 14853–6401, USA Department of Pathology and Animal Health, Division of Toxicology, School of Veterinary Medicine, University of Naples “Federico II”, Via Delpino 1, 80137 Naples, Italy

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Article history: Received 9 January 2015 Revised 2 April 2015 Accepted 3 April 2015 Available online 14 April 2015 Keywords: Nivalenol Deoxynivalenol Intestinal epithelial cells Oxidative stress Mycotoxins combination

a b s t r a c t Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6, the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol prooxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern. © 2015 Elsevier Inc. All rights reserved.

Introduction Mycotoxins are biologically active secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health (Wild and Gong, 2010). More than 400 different mycotoxins have been isolated and chemically characterized; those of major medical and agricultural concern are aflatoxins, ochratoxins, trichothecenes,

Abbreviations: (DON), deoxynivalenol; (NIV), nivalenol; (IECs), intestinal epithelial cells; (ROS), reactive oxygen species; (GI), gastrointestinal; (H2DCF-DA), 2′,7′-dichlorofluorescindiacetate; (DCF), 2′,7′-dichlorofluorescein; (DPI), diphenyleneiodonium chloride; (NAC), Nacetylcysteine; (PDTC), pyrolidine dithiocarbamate; (o-aminophenoxy), 1,2-bis; (BAPTA), ethane-N,N,N′,N′-tetraacetic acid; (PBS), phosphate buffer saline; (Fura 2-AM), Fura-2acetoxymethyl ester; (HBSS), Hank's balanced salt solution; (EGTA), ethylene glycol tetraacetic acid; (FCCP), carbonylcyanide p-trifluoromethoxy-phenylhydrazone; (NF-kB), nuclear factor-kB; (Erythroid-derived 2)-like 2 (Nrf2), nuclear factor; (NQO1), NAD(P)H: quinone oxidoreductase 1; (DAPI), 4′,6-diamidin-2-phenylindol; (HO-1), heme-oxygenase 1; (iNOS), inducible nitric oxide synthase. ⁎ Corresponding author at: Dpt. of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084, Fisciano, Salerno, Italy. E-mail address: [email protected] (S. Marzocco). 1 These authors equally contributed as last author.

http://dx.doi.org/10.1016/j.taap.2015.04.002 0041-008X/© 2015 Elsevier Inc. All rights reserved.

zearalenones and fumonisins. Data from the FAO (Food and Agriculture Organization of the United Nations) showed that about 25% of world food production is contaminated by at least one mycotoxin (Heussener et al., 2006). Trichothecenes mycotoxins are chemically related compounds produced by different fungal genera (Ueno, 1985). A data collection on the occurrence of Fusarium toxins in food in the European Union showed a 57% incidence of positive samples for the trichothecenes mycotoxin deoxynivalenol (DON) and 16% for trichothecenes mycotoxin nivalenol (NIV), out of several thousands of samples analyzed (Schothorst and van Egmond, 2004). Due to their toxic properties and their high stability to heat treatment, the presence of these mycotoxins in the food chain is potentially hazardous for human health. The clinical toxicological syndromes caused by ingestion of moderate to high amounts of mycotoxins have been well characterized: they range from acute mortality to slowed growth, and may include reduced reproductive efficiency, gastrointestinal disorders, and altered nutritional efficiency (Pestka and Smolinski, 2005). The intestinal epithelial layer represents the first barrier preventing the entry of foreign antigens, including natural toxins, into the underlying tissues. Intestinal epithelial cells (IECs) form a monolayer that constitutes a dynamic and selective barrier which mediates the transport of a variety of molecules. Following ingestion of mycotoxins-

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contaminated food or feed, IECs can be exposed to high concentrations of toxins (Maresca et al., 2008). Thus IECs are especially sensitive to trichothecenes and their exposure to these toxins may induce toxicity (Pinton et al., 2010; De Walle et al., 2010a, 2010b; Bianco et al., 2012). Humans and animals are often simultaneously exposed to several trichothecene mycotoxins because (a) most Fusarium can produce a number of mycotoxins simultaneously and (b) because food commodities may be contaminated by different fungi simultaneously or in quick succession and (c) because a common diet is made up of different food and feed components (Alassane-Kpembi et al., 2013). Humans may also be exposed to multiple trichothecenes via products from animals that have eaten contaminated feed (Streit et al., 2012). Rodrigues and Naehrer (2012) screened 7049 feed and feedstuff samples in a threeyear survey on the worldwide occurrence of mycotoxins, and reported 48% of samples contaminated by two or more mycotoxins. In particular, from 29 wheat samples, collected in three EU countries, they reported that 75% of the contaminated samples were positive for more than one type of mycotoxin (Monbaliu et al., 2010). Because of their natural co-occurrence, there is increasing concern about the hazard of exposure to mycotoxin mixtures also because their toxicity, when present together, cannot always be predicted based upon their individual toxicities (CAST, 2003; Grenier et al., 2011). Reactive oxygen species (ROS) are normal products of cellular metabolism. ROS have beneficial effects on several physiological processes including killing of invading pathogens, wound healing, and tissue repair processes. However, disproportionate ROS generation poses a serious problem to bodily homeostasis and causes oxidative tissue damage. The gastrointestinal (GI) tract is a key source of ROS. Despite the protective barrier provided by the epithelial layer, ingested materials and pathogens can cause inflammation by activating the epithelium, polymorphonuclear neutrophils and macrophages to produce inflammatory cytokines and other mediators that contribute further to oxidative stress. The pathogenesis of various GI diseases including peptic ulcers, gastrointestinal inflammatory bowel disease and GI cancers are in part due to excessive oxidative stress (Bhattacharyya et al., 2014). It has been previously demonstrated that DON can induce oxidative stress in various experimental models (Wu et al., 2014; Mishra et al., 2014), but there are no data regarding the effects of NIV in altering redox homeostasis both alone and in presence of DON, both of them at low concentrations. Thus, the aim of our study was to examine the pro-oxidative effects of NIV and DON, alone and in combination in a range of low concentrations, on the non-tumorigenic intestinal epithelial cells, IEC-6. Materials and methods Reagents. Unless stated otherwise, all reagents and compounds were purchased from Sigma Chemicals Company (Sigma, Milan, Italy). Cell culture. The IEC-6 cell line (CRL-1592) was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). IEC-6 cell originated from normal rat intestinal crypt cells (Quaroni and Hochman, 1996). This non-tumorigenic cell line was cultured using Dulbecco's modified Eagle's medium (4 g/L glucose) supplemented with 10% (v/v) heatinactivated fetal bovine serum, 2 mm L-glutamine, 1.5 g/L NaHCO3, and 0.1 unit/ml bovine insulin. Cells were used at the 17th–21st passage. Measurement of intracellular ROS and mithocondrial superoxide evaluation with MitoSOX red. ROS formation was evaluated by means of the probe 2′,7′-dichlorofluorescin-diacetate (H2DCF-DA) as previously reported (Adesso et al., 2013). H2DCF-DA is a non-fluorescent permeant molecule that passively diffuses into cells, where the acetates are cleaved by intracellular esterases to form H2 DCF and thereby traps it within the cell. In the presence of intracellular ROS, H2DCF is rapidly oxidized to the highly fluorescent 2′,7′-dichlorofluorescein (DCF). Briefly, IEC-6 cells were plated at a seeding density of 8 × 103 cells/well into 24-well plates. Cells

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were allowed to adhere for 24 h; thereafter, the medium was replaced with fresh medium and cells were incubated with NIV, DON and NIV and DON (5–0,5 μM) at the indicated times in Figures. In some experiments either diphenyleneiodonium chloride (DPI; 15–5 μM) or Nacetylcysteine (NAC; 15–5 mM) was added 1 h before incubation with mycotoxins. In another set of experiments, cells were pre-treated with pyrollidine dithiocarbamate (PDTC, 200 μM, 30 min before) or 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA, 10 μM, 2 h before) and then with mycotoxins (5–0,5 μM). Cells were then collected, washed twice with phosphate buffer saline (PBS) buffer and then incubated in PBS containing H2DCF-DA (10 mM) at 37 °C. After 45 min, cells fluorescence was evaluated using a fluorescence-activated cell sorter (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. In order to detect superoxide release from mitochondria in some experiments after cell treatment, as previously described, MitoSOX Red was used. MitoSOX Red (2.5 μM), was added for 10 min before fluorescence evaluation by means of flow cytometry. This indicator is a fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells and, once targeted to the mitochondria it is oxidized by superoxide and exhibits red fluorescence. MitoSOX is readily oxidized by superoxide but not by other ROS-generating systems. Measurement of intracellular Ca2 + signaling. Intracellular Ca2+ concentrations [Ca2 +]i were measured using the fluorescent indicator dye Fura-2-acetoxymethyl ester (Fura 2-AM), the membrane-permeant acetoxymethyl ester form of Fura 2, as previously described with minor revisions (Adesso et al., 2013). Briefly, IEC-6 cells (8 × 103/24 well plates) were incubated at 37 °C. Thereafter, the medium was replaced with fresh medium and NIV, DON or NIV and DON (5–0,5 μM) were added. After a proper incubation period (1 h), cells were washed in phosphate buffered saline (PBS), re-suspended in 1 ml of Hank's balanced salt solution (HBSS) containing 5 μM Fura 2-AM for 45 min. Thereafter, cells were washed with the same buffer to remove excess Fura 2-AM and incubated in Ca2+-free HBSS/0.5 mM ethylene glycol tetraacetic acid (EGTA) buffer for 15 min to allow hydrolysis of Fura 2AM into its active-dye form, Fura 2. IEC-6 cells then were transferred to the spectrofluorimeter (Perkin-Elmer LS-55). Treatment with ionomycin (1 mM final concentration) or with carbonylcyanide ptrifluoromethoxy-phenylhydrazone (FCCP, 0.5 μmol/L final concentration) was carried out by adding the appropriate concentrations of each substance into the cuvette in Ca2+-free HBSS/0.5 mM EGTA buffer. In the experiments involving the use of FCCP, a mitochondrial uncoupler used for mitochondrial calcium depletion, this compound was added to the medium for 10 min before the beginning of the recordings, as previously described. The excitation wavelength was alternated between 340 and 380 nm, and emission fluorescence was recorded at 515 nm. The ratio of fluorescence intensity of 340/380 nm (F340/F380) was used to estimate intracellular free calcium. Results are indicated as delta increase of fluorescence ratio (F340/F380 nm) induced by FCCP or ionomycin-basal fluorescence ratio (F340/F380 nm). Immunofluorescence analysis with confocal microscopy. For immunofluorescence assay, IEC-6 cells (2 × 104 for well) were seeded on coverslips in 12 well plate and treated with NIV and DON alone or in combination (5 μM) for 1 h. Then cells were fixed with 4% paraformaldehyde in PBS for 15 min and permeabilized with 0.1% saponin in PBS for 15 min. After blocking with BSA and PBS for 1 h, cells were incubated with nuclear factor-kB (NF-kB) p65 (sc-372 Santa Cruz Biotechnology), nuclear factor (erythroid-derived 2)-like 2 (Nrf2) antibody (sc-722 Santa Cruz Biotechnology), NAD(P)H dehydrogenase, quinone 1 (NQO1) antibody (sc-376023 Santa Cruz Biotechnology) or nytrotyrosine (06– 284 millipore) for 2 h at room temperature. The slides were then washed with PBS three times and fluorescein-conjugated secondary antibody (FITC) was added for 1 h. 4′,6-diamidin-2-phenylindol

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(DAPI) was used for counterstaining of nuclei. Coverslips were finally mounted in mounting medium and fluorescent images were taken under the Laser Confocal Microscope (Leica TCS SP5).

Measurement of heme-oxygenase 1 (HO-1) and inducible nitric oxide synthase (iNOS) by cytofluorimetry. IEC-6 cells were plated into 96well plates (1 × 104 cells/well) and were allowed to grow for 24 h.

Fig. 1. Effect of NIV DON alone or in combination (5–0,5 μM) on ROS formation (A), evaluated by means of the probe 2′,7′ dichlorofluorescein-diacetate (H2DCF-DA), in IEC-6. In some experiments a NADPH oxidase inhibitor, DPI (10 μM) and N-acetylcysteine, NAC (10 mM), were added to NIV (B) or to DON (C). In another set of experiments DPI (15–5 μM) and NAC (15–5 mM) were added to NIV and DON (5 μM; D). Values, mean ± s.e.m., are expressed as mean fluorescence intensity at least 3 independent experiments with three replicates each. Comparisons were performed using one-way analysis of variance and multiple comparisons were made by Bonferroni's test. ***, ** and * denote P b 0.001, P b 0.01 and P b 0.05 vs control. °°°, °° and ° denote P b 0.001, P b 0.01 and P b 0.05 vs NIV or DON alone.

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Thereafter the medium was replaced with fresh medium and cells were treated with NIV and DON alone or in combination (5 μM) for 1 h for further 24 h. IEC-6 were then collected, washed twice with PBS and then incubated in Fixing Solution for 20 min at 4 °C and then incubated in Fix Perm Solution for 30 min at 4 °C. Anti-HO-1 (SC-10789 Santa Cruz

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Biotechnology) or anti-iNOS (610431 BD Transduction Laboratories) were then added for further 30 min. The secondary antibody was added in Fix Solution and cells fluorescence was evaluated using a fluorescence-activated cell sorter (FACSscan; Becton Dickinson) and elaborated with Cell Quest software.

Fig. 2. Effect of NIV and DON alone and in combination on intracellular Ca2+ concentrations. Cells were treated with NIV, DON alone and in combination (5–0.5 μM) for 1 h. Intracellular calcium concentration was evaluated on IEC-6 cells in Ca2+-free medium. Effect of NIV and DON on mitochondrial Ca2+ pool (A) was evaluated on IEC-6 cells in Ca2+-free medium in the presence of FCCP (0.05 μM) after treatment. In some experiments in presence of BAPTA, a calcium chelator, ROS formation induced by NIV (B) and by DON (C) were evaluated. In the same experimental conditions we also evaluated superoxide production by mitochondria adding MitoSOX Red (D). Values, mean ± s.e.m., are expressed as mean ± s.e.m. of delta (δ) increase of Fura 2 ratio fluorescence (340/380 nm), as mean fluorescence intensity and as mitochondrial superoxide production at least 3 independent experiments with three replicates each. Data were analyzed by analysis of variance test and multiple comparisons were made by Bonferroni's test. ***, ** and * denote P b 0.001, P b 0.01 and P b 0.05 vs control. °°°, °° and ° denote P b 0.001, P b 0.01 and P b 0.05 vs NIV or DON alone.

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Data analysis. Data are reported as mean ± standard error mean (s.e.m.) values of at least three independent experiments. Statistical analysis was performed by analysis of variance test, and multiple comparisons were made by Bonferroni's test. A P-value less than 0.05 was considered significant.

Under the same experimental conditions DON-induced ROS release was inhibited both by DPI at (5–1 μM, P b 0.001 vs DON alone, Fig. 1D) and by NAC at the two highest concentrations (5–2.5 μM, P b 0.001 vs DON alone, Fig. 1D). These results indicated that also DON-induced ROS release resulted mainly from an influence on NADPH oxidase activity.

Results

NIV and DON affect [Ca2+]i concentrations

Effect of NIV and DON on ROS released from IEC-6

To analyze the involvement of mitochondrial Ca2 + content in mycotoxins-evoked responses, cells were incubated with NIV and DON alone or in combination (5–0,5 μM), in the absence of extracellular Ca2+, and then the mitochondrial calcium depletory, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP; 0.5 μmol/L), was added. As reported in Fig. 2A, in IEC-6 cells treated with mycotoxins for 2 h, delta increase in [Ca2 +]i induced by FCCP was significantly lower (P b 0.05) than control cells, indicating an impairment of mitochondrial calcium storage. In NIV and DON co-treated cells, delta increase in [Ca2+]i induced by FCCP was further reduced (P b 0.05).

To investigate whether NIV and DON alone or in combination could interfere in oxidative stress in IEC-6, we evaluated their effect on intracellular ROS by flow cytometry. As shown in Fig. 1, after 20 h treatment, NIV (A) induced a significant release of ROS in IEC-6 at all tested concentrations (P b 0.05 vs control). DON also induced a significant increase in ROS release but only to a concentration of 1 μM (Fig. 1B; P b 0.05). Interestingly when DON was added simultaneously with NIV in IEC-6 ROS release resulted significantly increased when compared to DON alone (Fig. 1B; P b 0.05).

Effect of NIV and DON on ROS production in presence of BAPTA NIV and DON induced-ROS release was inhibited both by DPI and NAC In order to investigate the mechanisms of NIV and DON-induced ROS in IEC-6, we examined the potential role of the pro-oxidative enzymeNAD(P)H oxidase. ROS production in IEC-6 was measured after treatment with NIV or DON (5–0.5 μM), in the presence of DPI, a NAD(P)H oxidase inhibitor. DPI significantly inhibited ROS release induced by all NIV concentrations (P b 0.05 vs NIV alone; Fig. 1C). We further analyzed whether the addition of the antioxidant-NAC could decrease oxidative stress induced by NIV. As shown in Fig. 1C, NIV-induced ROS production was significantly (P b 0.001) inhibited by NAC at the two highest concentrations (5–2.5 μM, P b 0.001 vs NIV alone), thus indicating a major involvement of NADPH oxidase in NIV-induced ROS release.

In order to confirm the involvement of calcium in mycotoxinsinduced oxidative stress, ROS formation was evaluated in presence of BAPTA (10 μM), a cell-permeant chelator, which is highly selective for Ca2 + and has been used to control the level of intracellular Ca2 +. BAPTA significantly (P b 0.05) reduced ROS production induced by NIV (Fig. 2B) and DON (Fig. 2C). Effect of NIV and DON on MitoSOX treated cells Mitochondrial ROS production was evaluated by means of MitoSOX red, a fluorogenic dye for highly selective detection of superoxide in the mitochondria. As shown in Fig. 2D, NIV and DON significantly increase

Fig. 3. Effect of NIV and DON alone and in combination (5 μM) on NF-kB p65 nuclear translocation in IEC-6. Nuclear translocation of NF-kB p65 subunit was detected using immunofluorescence assay at confocal microscopy (A). Green fluorescences indicate NF-kB p65 localization and blue fluorescences nucleus (DAPI) localization. Scale bar, 10 μm. ROS formation induced by NIV (B) and DON (C) were evaluated both alone and in presence of an NF-kB inhibitor, PDTC. *** and * denote P b 0.001 and P b 0.05 vs control. ###, ## and # denote P b 0.001, P b 0.01 and P b 0.05 vs NIV or DON alone. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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mitochondrial ROS production in IEC-6 at the highest three concentrations (P b 0.001 vs control). In NIV and DON co-treated cells mitochondrial ROS production was further increased (P b 0.001, at all tested concentrations, vs control and vs DON). NIV and DON induced p65 NF-kB nuclear translocation in IEC-6 Following p65 phosphorylation, the free NF-κB dimers translocate into the nucleus and bind to specific sequences to regulate downstream genes expression (Tak and Firestein, 2001). Thus we labeled p65 with a green fluorescence to track the influence of NIV and DON (5 μM), on NFκB p65 subunit nuclear translocation. As shown in Fig. 3A, nuclear p65 translocation was increased after 15 min by DON but in even greater extent by NIV. When both mycotoxins were present simultaneously in IEC-6 we observed that p65 NF-kB translocation was further enhanced respect to NIV and DON alone. NF-kB activation is involved in NIV and DON-induced ROS release To evaluate the possible involvement of NF-kB in NIV and DONinduced ROS release, we analyzed ROS production in the presence of the NF-kB inhibitor PDTC (200 μM). As showed in Fig. 3, PDTC significantly reduced ROS production induced both by NIV (P b 0,005 vs NIV alone; B) and DON (P b 0,001 vs DON alone; C).

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NIV and DON induced iNOS expression and nytrotyrosine formation in IEC-6 iNOS expression was induced in IEC-6 by NIV (P b 0,001 vs control; Fig. 4A) and by DON at the concentrations of 5.5 and 2.5 μM (P b 0.05 vs control; Fig. 4B); this effect was further enhanced when NIV and DON were contemporaneously added to IEC-6 at all concentrations tested (P b 0.001 vs control Fig. 4C). iNOS expression, leading to NO production that reacting with ROS induces peroxynitrite formation and 3-nitrotyrosine formation is a hallmark product. NIV and DON (5 μM) induced an increase of nitrotyrosine formation in IEC-6. This effect was greater for NIV when compared to DON and when IEC-6 was pretreated with both mycotoxins contemporary (Fig. 4D). NIV and DON induce Nrf2 activation and NQO1 and HO-1 expression in IEC-6 Nrf2 is an oxidative stress sensor and translocates into the nucleus where it binds to specific DNA sequences to regulate downstream gene expression. Thus we labeled Nrf2 with a green fluorescence to track the influence NIV and DON on its activation. As shown in Fig. 5, nuclear Nrf2 was increased after 1 h by NIV and DON. Also Nrf2 was increased mainly by NIV when compared to DON and mostly by both mycotoxins together in IEC-6. NQO1 induced by Nrf2, resulted increased by NIV and DON both alone and mostly in combination (Fig. 6A). The same effects were also

Fig. 4. Effect of NIV and DON alone and in combination (5 μM) on iNOS expression (A–C) and on nitrotyrosine formation (D) in IEC-6. iNOS expression was detected in IEC-6 treated with NIV (B) and DON alone and in combination (D) by cytofluorimetry and expressed as mean fluorescence intensity. ***, ** and * denote P b 0.001, P b 0.01 and P b 0.05 vs control. Nitrotyrosine formation was detected using immunofluorescence assay at confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and nitrotyrosine respectively. Analysis was performed by confocal laser scanning microscopy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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observed on HO-1 expression, another product of Nrf2 activation (Fig. 6B), Discussion The importance of studying the trichothecenes mycotoxins NIV and DON is related to food and feed contamination and to their capability in inducing severe consequences on both human and animal health. The gut mucosa is the first and principal site of mycotoxins exposure and toxic effects and constitutes the first barrier after their ingestion. Human microflora seems to be unable to produce de-epoxidated metabolites of NIV and a close relationship between the dietary exposure and DON recovery in human and animal urine samples has been reported (Meky et al., 2003). Therefore, IECs could contribute to NIV and DON toxicity by allowing their entry into the systemic circulation and thus their transport to the whole body. The amount of NIV in cereal products varies considerably among world regions (Tanaka et al., 1988; Hsia et al., 2004), depending on weather and culture conditions (Edwards, 2004). Estimated NIV intake of the French population is 88 ng/kg per day in adults, 163 ng/kg per day in children, and reaches 210 ng/kg per day in vegetarians (Leblanc et al., 2005). In European countries, the daily intake remains below the provisional maximum tolerable daily intake (PMTDI) value of 0.7 μg/kg per day, but in Asia, particularly in Japan and China, the daily intake may exceed the PMTDI. The lower DON concentration (0.16 μg/ml corresponding to 0.5 μM used in this study) corresponds to the mean estimated daily intake of French adult consumers eating food contaminated by mean DON levels, on a chronic basis. The DON concentration of

2 μg/ml corresponds to the highest level that can be reached after consumption of heavily contaminated food that can be encountered occasionally (e.g. unfavorable weather conditions) or among children and vegans/macrobiotics (Leblanc et al., 2005). The concentration range for NIV and DON used in this study are those plausibly encountered in the gastrointestinal tract after consumption of food contaminated by these mycotoxins. These values have been derived from estimated daily intakes of the SCOOP program (EC, 2003; EFSA, 2014) and other studies applied the same approach in order to use realistic NIV and DON levels to evaluate their effects in intestinal cells (Sergent et al., 2006; De Walle et al., 2010a, 2010b; Bianco et al., 2012; Alassane-Kpembi et al., 2013). Moreover recent attention has been focused on mycotoxins co-contamination (Alassane-Kpembi et al., 2013). Food and feed commodities are often contaminated by more than one mycotoxin; among the several combinations that frequently occur NIV and DON are often mentioned (Eskola et al., 1998). The main finding of this study is the pro-oxidant NIV effect in IECs, the stronger pro-oxidant effect of NIV versus DON and, interestingly, that NIV potentiates DON pro-oxidative effects. The results of our study provide evidences that the pathways underlying the observed oxidative stress induced by NIV and/or DON in IEC-6 involves: (i) NADPH oxidase and glutathione levels, (ii) alterations in calcium homeostasis, (iii) activation of NF-kB and (iv) activation of antioxidant response mediated by Nrf2. Moreover NIV and/or DON induces iNOS expression and nitrotyrosine formation in IEC-6 giving another insight into the toxic effects of these mycotoxins. In IEC-6 NIV induces a significant increase in ROS levels when compared to those observed with DON. Our study firstly reports the prooxidative effect of NIV and results regarding DON are well in accordance

Fig. 5. Effect of NIV and DON alone and in combination (5 μM) on Nrf2 nuclear translocation in IEC-6. Nuclear translocation of Nrf2 was detected using immunofluorescence assay at confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and Nrf2 respectively. Analysis was performed by confocal laser scanning microscopy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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with other studies reporting DON pro-oxidant activity (Kouadio et al., 2005; Mishra et al., 2014). The stronger NIV effect respect to DON is also in accordance with other studies, also from our laboratory, reporting the stronger NIV effect respect to DON in measuring other parameters of cell damage (Bianco et al., 2012; Marzocco et al., 2009). Mechanistic studies revealed that in NIV and DON-induced ROS release are involved both NADPH oxidase, as assessed by the presence of DPI, and glutathione homeostasis, as assessed by the presence of NAC in IEC-6 treated with mycotoxins. It has been previously reported that DON induces an alteration of calcium homeostasis (Sergeev et al., 1990) and since calcium is also important in regulating oxidative status in cells we evaluated its possible changes in IEC-6 treated with NIV and DON. Our results indicated that mitochondria are the main intracellular compartment involved in calcium variations induced by NIV and DON. In the presence of an inhibitor of calcium release, BAPTA, NIV and DON-induced ROS release was reduced indicating calcium involvement in NIV and DON-induced oxidative stress. The effects of NIV and DON on mitochondria were also indicated by means of MitoSOX red a selective indicator of mitochondrial ROS production in living cells and of mitochondrial damage (Mukhopadhyay et al., 2007). Owing to its well-established proinflammatory functions, NF-κB is regarded primarily as a potentially pathogenic factor that is harmful to the host when excessively or improperly activated. High ROS levels have been reported to promote activation and translocation of NF-κB into the nucleus, and in the inflamed gastrointestinal tract ROS production also increases (Gloire et al., 2006). This ROS/NF-κB self-sustaining regulatory loop may contribute to the perpetuation and to a progressive

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and uncontrolled inflammatory response (Dignass et al., 2004) and it has been recently reported that ROS production and NF-kB activation are critical events in colorectal cancer initiation (Myant et al., 2013). Our data indicate an increase in p65 NF-kB nuclear translocation in IEC-6 treated with NIV and DON and most of all with both of them together. In order to confirm the involvement of NF-kB activation on IEC-6 mycotoxin induced ROS release PDTC, an NF-kB inhibitor, was used. In the presence of PDTC, NIV and DON-induced ROS release resulted lower thus indicating an NF-kB influence in regulating oxidative stress induced by NIV and DON in IEC-6. Our data are in accordance with previous studies reporting DON ability to induce NF-kB activation (van De Walle et al., 2008) while, before this study, very little was known regarding NIV capability to activate this nuclear factor (Nagashima et al., 2011). NF-kB is also involved in iNOS expression, the main NOS isoform expressed during inflammation, and NO released by this isoform rapidly reacts with superoxide anion causing nitrosative stress and also generating the toxic metabolite peroxynitrite. Peroxynitrite can modify proteins, changing their structure through the reaction with various amino acids in the peptide chain. All of these reactions affect protein structure and function and thus have the potential to cause changes in the catalytic activity of enzymes, altered cytoskeletal organization and impaired cell signal transduction. Peroxynitrites are able to nitrate tyrosine residues in proteins resulting in nitrotyrosine formation. Because nitration of tyrosine is an alternative to phosphorylation at key residues, it can affect a protein's enzymatic activity (Schopfer et al., 2003). Nitrotyrosine is identified as an indicator or marker of cell damage, inflammation as well as NO production (Ischiropoulous, 1998). Furthermore, iNOS upregulation and nitrotyrosine immunoreactivity have also

Fig. 6. Effect of NIV and DON alone and in combination (5 μM) on NQO1 formation. NQO1 was detected using immunofluorescence assay at confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and Nrf2 respectively. Analysis was performed by confocal laser scanning microscopy (A). HO-1 expression was detected in IEC6 treated with NIV (B) and DON (C) alone and in combination (D) by cytofluorimetry, and expressed as mean fluorescence intensity. *** denotes P b 0.001 vs control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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been demonstrated in conditions such as endotoxemia, inflammatory bowel disease (IBD), Helicobacter pylori gastritis, as well as in animal models of colitis and ileitis suggesting that peroxynitrite may be a key mediator of mucosal injury and gut barrier failure (Potoka et al., 2003). In this study we reported the effect of NIV and DON in inducing iNOS expression and nitrotyrosine formation showing an additional toxic effect associated with both of these mycotoxins which lead to the expression of cytoprotective enzymes. A recurrent theme in oxidant signaling and antioxidant defense is reactive cysteine thiol-based redox signaling. Nrf2 is an emerging regulator of cellular resistance to oxidants. Nrf2 controls the basal and induced expression of an array of antioxidant response element-dependent genes that regulate the physiological and physiopathological outcomes of oxidant exposure such as (NQO1 and HO-1) (Kensler et al., 2007; Singh et al., 2010). NQO1 is a flavoprotein that catalyzes the metabolic detoxification of quinones and their derivatives to hydroquinones, using either NADH or NADPH as the electron donor. This protects cells against quinoneinduced oxidative stress, cytotoxicity, and mutagenicity (Chi et al., 2015). Another product of Nrf2 activation is HO-1. HO-1 is the rate limiting enzyme in the conversion of haem into biliverdin/bilirubin, iron and carbon monoxide (CO); all of them can potentially function as antioxidants (Choi and Otterbein, 2002). It is strongly suggested that HO-1 provides a potent cytoprotective effect, as shown in various in vitro and in vivo models of cellular and tissue injury (Otterbein et al., 1999; Soares et al., 1998). HO-1 is inducible by a variety of oxidative stresses and is thought to play an important role in the protection of tissues from oxidative injuries. Our results indicate an increase in NQO1 and HO-1 expression in IEC-6 treated with NIV and DON. In this study, to the best of our knowledge, we report for the first time that NIV and DON can induce Nrf2 activation in IEC-6, mostly when present simultaneously. Thus IEC-6 seem to react to the pro-oxidant mycotoxins injury by enhancing also defense systems such as Nrf2 activation. Conclusions Our results indicate that NIV and DON induce oxidative stress, in an intestinal epithelial non-tumorigenic cell line, and that they interact thus highlighting the importance of the toxic effect of these mycotoxins, mostly in combination, that are very important for their risk assessment process that are of growing concern. Transparency document The Transparency document associated with this article can be found, in the version. Acknowledgments This study was supported by FARB 2012 (ORSA127975), University of Salerno. The authors are grateful to Mr. Paolo Giannelli for editing the manuscript. References Adesso, S., Popolo, A., Bianco, G., Sorrentino, R., Pinto, A., Autore, G., Marzocco, S., 2013. The uremic toxin indoxyl sulphate enhances macrophage response to LPS. PLoS One 8 e76778. Alassane-Kpembi, I., Kolf-Clauw, M., Gauthier, T., Abrami, R., Abiola, F.A., Oswald, I.P., Puel, O., 2013. New insights into mycotoxin mixtures: the toxicity of low doses of type B trichothecenes on intestinal epithelial cells is synergistic. Toxicol. Appl. Pharmacol. 272, 191–198. Bhattacharyya, A., Chattopadhyay, R., Mitra, S., Crowe, S.E., 2014. Oxidative stress: an essential factor in the pathogenesis of gastrointestinal mucosal diseases. Physiol. Rev. 94, 329–354. Bianco, G., Fontanella, B., Severino, L., Quaroni, A., Autore, G., Marzocco, S., 2012. Nivalenol and deoxynivalenol affect rat intestinal epithelial cells: a concentration related study. PLoS One 7 e52051. CAST, 2003. Mycotoxins, Risks in Plant, Animal, and Human System, Task Force Report 139. Council for Agricultural Science and Technology, Ames Iowa (p10).

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