Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction

TIV-03861; No of Pages 9 Toxicology in Vitro xxx (2016) xxx–xxx

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Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction Seong-Hwan Park a,b,d, Juil Kim a,d, Dongwook Kim c, Yuseok Moon a,b,d,⁎ a

Laboratory of Mucosal Exposome and Biomodulation, Department of Biomedical Sciences, Pusan National University School of Medicine, Yangsan, South Korea Research Institute for Basic Sciences and Medical Research Institute, Pusan National University, Busan, South Korea c National Institute of Animal Science, RDA, Suwon, South Korea d Immunoregulatory Therapeutics Group, Brain Busan 21 Project, Busan, South Korea b

a r t i c l e

i n f o

Article history: Received 6 January 2016 Received in revised form 23 August 2016 Accepted 9 October 2016 Available online xxxx Keywords: Mycotoxin detoxifiers Porcine enterocytes Deoxynivalenol

a b s t r a c t Deoxynivalenol (DON), the most prevalent mycotoxin worldwide, leads to economic losses for animal food production. Swine is a most sensitive domestic animal to DON due to rapid absorption and low detoxification by gut microbiota. Specifically, DON can severely damage pig intestinal tissue by disrupting the intestinal barrier and inducing inflammatory responses. We evaluated the effects of several mycotoxin detoxifiers including bentonites, yeast cell wall components, and mixture-typed detoxifier composed of mineral, microorganisms, and phytogenic substances on DON-insulted intestinal barrier and pro-inflammatory responses using in vitro porcine enterocyte culture model. DON-induced disruption of the in vitro gut barrier was attenuated by all three mycotoxin detoxifiers in dose-dependent manners. These mycotoxin detoxifiers also suppressed DON-induced pro-inflammatory chemokine expression to different degrees, which was mediated by downregulation of mitogen-activated kinases and early growth response-1. Of note, the mixture-typed detoxifier was the most prominent mitigating agent at the cellular levels whereas the high dose of bentonite clay also had suppressive action against DON-induced pro-inflammatory insult. The in vitro porcine enterocyte-based assessment of intestinal barrier integrity and inflammatory signals provides sensitive and simplified alternative bioassay of feed additives such as detoxifiers against enteropathogenic mycotoxins with comprehensive mechanistic confirmation. © 2016 Published by Elsevier Ltd.

1. Introduction Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin secreted from Fusarium species in the world. This compound can infect monocotyledon-based crops such as maize, wheat and barley (Rasmussen et al., 2003; Yazar and Omurtag, 2008). Among the livestock, pigs are more sensitive to DON than poultry and typically show greater tolerance to DON exposure (Ghareeb et al., 2015). In terms of toxicokinetics, rapidly absorbed DON in pigs can be detected in the circulatory system even at 30 min after DON exposure, reaching its peak in the serum after 4 h (Sundstøl Eriksen, 2003). The proximal parts of the small intestine are the major site of DON absorption (Danicke et al., 2004). While early biotransformation of DON by gut microbiota plays crucial roles in its detoxification to DOM-1 (the non-toxic deepoxyDON) in many animals, the metabolic inactivation of DON by porcine microbiota is rarely observed, indicating that swine are highly sensitive ⁎ Corresponding author at: Department of Biomedical Sciences, Pusan National University School of Medicine, Yangsan 50612, South Korea. E-mail address: [email protected] (Y. Moon).

to DON exposure (Danicke et al., 2004; Frobose et al., 2015). Therefore, increased local levels of DON in the gut pose a greater risk of harmful injuries to the porcine enterocytes. In addition, DON adversely affects porcine growth, immune function, and reproductive performance in pigs (Goyarts and Danicke, 2005; Pinton et al., 2008; Tiemann and Danicke, 2007). In vivo and in vitro experiments show that the upper gastrointestinal tract (GIT) from the stomach to the proximal jejunum rapidly absorbs most of DON in the diet, leading to inhibition of protein synthesis and suppression of various target genes, including amino acid transporters (Danicke et al., 2006; Goyarts and Danicke, 2006). Intestinal epithelial cells (IECs) can also maintain immune homeostasis via contact with commensal bacteria, and the most crucial key to coexistence of commensal bacteria and IECs is the ability to segregate host cells from microorganisms (Lotz et al., 2006). However, if the intestinal epithelial barrier is disrupted, microbial colonization poses a risk of infection and inflammatory responses (Yan et al., 2013). DON is a well-known xenobiotic that increases intestinal permeability and induces inflammatory responses in human IECs as well as systemic inflammation (Moon et al., 2007; Park et al., 2010; Pestka, 2007). However, DON rarely reaches

http://dx.doi.org/10.1016/j.tiv.2016.10.003 0887-2333/© 2016 Published by Elsevier Ltd.

Please cite this article as: Park, S.-H., et al., Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction..., Toxicology in Vitro (2016), http://dx.doi.org/10.1016/j.tiv.2016.10.003

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the distal small intestine and colon at the luminal region. The upper GI tract (stomach until proximal jejunum) of swine is the most prominent site of DON absorption. Most of DON absorbed from the gut lumen enters the hepatic portal vein, circulates and then re-enters the intestinal tissues, after which it passes through the more distal parts of intestine via the basolateral side of the enterocytes (Diesing et al., 2011a). Therefore, IECs are exposed to DON at both the apical region facing the luminal portion of the intestine and the basolateral region via blood circulation. In terms of the absorption mechanism, DON is mostly absorbed via passive transcellular or paracellular diffusion through enterocytes (Sergent et al., 2006) whereas there is an evidence for the active transporting of DON via P-glycoprotein or multidrug resistanceassociated protein 2 (Videmann et al., 2007). In human and murine cells, DON-insulted ribosomes are scaffold organelles for signaling kinases that transduce stress signals to the downstream kinase cascades, including phosphorylation of mitogen-activated protein kinases (MAPKs) and subsequent gene regulation via activated transcription factors such as nuclear factor kappa B (NF-κB), AP-1, and early growth response protein 1 (EGR1-) (He et al., 2012; Liu et al., 2014; Moon et al., 2007; Oh et al., 2016; Pan et al., 2013; Wang et al., 2014). In terms of functionality, MAPK-linked signaling pathway has recently been shown to play key roles in both local immune stimulation (Pestka, 2007) and impairment of barrier integrity of the intestinal cells, leading to increased permeability and translocation of pathogens harboring the intestinal lumen, as well as reduction of the absorptive surface area and gene expression of transporters crucial for the absorption of key nutrients (Pinton et al., 2012). The porcine intestinal barrier is also susceptible to DON-induced inflammatory responses, which trigger the expression of various pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), as well as chemokines such as IL-8 (Cano et al., 2013). Several mycotoxin-detoxifying agents are currently added to feed to act in the digestive tract of the animals. Based on their mode of action, these feed additives can be classified into sequestering (absorbing) agents or biotransforming agents. While sequestering agents are able to bind mycotoxins and reduce their gastrointestinal absorption and bioavailability, biotransforming agents degrade mycotoxins to less or nontoxic metabolites via action of metabolic enzymes from microbes. Among the toxin detoxifying agents, bentonite clay, yeast cell wall constituents, and mixture-typed products composed of minerals (bentonite, inactivated yeast, and diatomaceous earth) and biological components (microorganisms and phytogenic substances) can be useful to reducing the toxic effects of mycotoxins on animals and are therefore manufactured as commercial mycotoxin-regulating products for feed additives (Huwig et al., 2001; Kong et al., 2014; Pasha et al., 2007). Yeast cell wall constituents such as mannan oligosaccharides (MOS) and β-glucans or mixture of bentonite and modified yeast cell wall reduce the effects of DON on the immune system of pigs, but do not affect the detoxification of DON in pigs (Shehata et al., 2004). Another toxin binder, bentonite, often called montmorillonite, is also an efficient adsorbent of aflatoxins. However, bentonite shows a very poor affinity for ochratoxin A (OTA) and thus low removing activity against serum and tissue OTA in intoxicated animals (Castellari et al., 2001; Kurtbay et al., 2008; Plank et al., 1990). Moreover, bentonite has the potential to bind aflatoxin B1 rather than DON in the condition which simulates the pH condition of the porcine GIT (Kong et al., 2014). In contrast to the effects of bentonites on DON, the inclusion of activated carbon in TNO (Netherlands Organization for Applied Scientific Research) dynamic laboratory model simulating the porcine GIT produced significant reductions of the jejunal absorption of DON (Avantaggiato et al., 2004). However, it is not clear how mycotoxin detoxifiers can attenuate mycotoxin-induced damage to GIT and DON-activated signals to mediate barrier integrity or pro-inflammatory responses in porcine IECs. In vivo feeding trials are close to the situation in practice but they are cost and labor intensive. Therefore, in vitro analysis is a powerful tool with economical efficacy as screening method to select the most potent

mycotoxin detoxifying agents (Devreese et al., 2013). In this study, we evaluated the action of mycotoxin detoxifiers on DON-induced disruption of the porcine intestinal epithelial barrier and pro-inflammatory responses using porcine IECs. Based on observation of the epithelial barrier integrity and production of pro-inflammatory chemokines in porcine enterocytes, the underlying mechanisms were investigated in terms of the stress signals reported in the murine and human models. Despite various analytical methods for detection of DON, conventional physicochemical monitoring technologies are often insufficient to assess their specific toxic actions in biological systems. In the present study, porcine enterocyte-based bioassay of DON-induced toxicity was developed to assess the actions of mycotoxin detoxifiers at the cellular levels. This in vitro assessment using porcine epithelial monolayer cells will provide an alternative sensitive method to screening and evaluation of mycotoxin detoxifiers, which also gives crucial insight into molecular evidence of action of toxin detoxifiers as feed additives against DON-induced disruption of the porcine intestinal barrier and pro-inflammatory responses in swine.

2. Materials and methods 2.1. Cell culture conditions and reagents The porcine normal intestinal epithelial cell line, IPEC-1, was purchased from Leibniz-Institute DSMZ (DSMZ, Braunschweig, Germany). IPEC-1 cells were maintained in DMEM/F-12 (Welgene, Daegu, South Korea) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Welgene), 50 units/ml penicillin and 50 μg/ml streptomycin (Welgene) in a 5% CO2 humidified incubator at 37 °C. Cell number was assessed by exclusion of trypan blue dye (Sigma-Aldrich Chemical Company, St. Louis, MO, USA) using a hemocytometer. Deoxynivalenol (DON) (97.6 ± 2.4% pure) isolated from Fusarium graminearum was obtained from Sigma-Aldrich. Two mycotoxin-binding agents (high purified calcium bentonite clay and the yeast cell wall components), and a mixture-typed product containing the biotransforming agent were kindly provided by Dr. Beob Gyun Kim (Konkuk University). High purified calcium bentonite clay belongs to the phyllosilicate group with a layered crystalline microstructure, consisting mostly of montmorillonite. Due to their montmorillonite content, the bentonite clay swells and forms thixotropic gels (Fowler et al., 2015). Cell walls derived from Saccharomyces cerevisiae yeast are also used as a dietary mycotoxin-adsorbing agent, consisting almost entirely of glucans and mannanslinked cell wall proteins (Fruhauf et al., 2012). The mixture-typed product is composed of minerals (including 38% bentonite, 30% inactivated yeast, 25% diatomaceous earth) and biological components (including microorganism BBSH797 and phytogenic substances). U0126 and SB203580 were purchased from Enzo Life Science (Farmingdale, NY, USA) and Calbiochem (Merck Millipore, Billerica, MA, USA), respectively. All other chemicals were purchased from Sigma-Aldrich. DON, U0126, and SB203580 were dissolved in DMSO while the mycotoxin detoxifiers were dissolved in complete DMEM/F-12 media.

2.2. Experimental design Each dose of DON was mixed with 0–10% mycotoxin detoxifiers in DMEM/F12 media, and then the reactants were incubated for 2 h at the room temperature. The mixed solution was centrifuged at 840 ×g for 3 min and then the supernatants were filtered through 0.45 μMpore-sized sterile filter disc (Sartorius Stedim Biotech, Gottingen, Germany). Free untransformed DON in the filtered supernatant was quantified by ELISA and HPLC/MS methods and used for the next porcine cell-based assays including measurement of TEER, paracellular tracer flux assay, RT-PCT, and Western blot analysis as described in the following methods.

Please cite this article as: Park, S.-H., et al., Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction..., Toxicology in Vitro (2016), http://dx.doi.org/10.1016/j.tiv.2016.10.003

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2.3. Quantitation of DON after reaction with mycotoxin detoxifiers For the quantitation of DON, enzyme-linked immunosorbent assay (ELISA) and HPLC/MS analysis were performed as the primary methods for quantifying levels of DON after reaction with mycotoxin detoxifiers. ELISA was performed using commercial ELISA kits (COKAQ4000, Romer Labs, Singapore). HPLC/MS analysis was used to confirm the ELISA analysis of DON. Sample clean-up steps were performed using DONtest WB (VICAM, Milford, MA, USA). Purification was performed by pushing the flange end of the cleanup column into the culture tube. The cleaned-up extract was then evaporated using Modul 4080C (Hanil Science Industrial, Kimpo, South Korea) and the residue was dissolved in mobile phase (water–acetonitrile–methanol, 90:5:5, v/v/v) to be injected into the HPLC column heated to 30 °C. A Shimadzu prominence UFLC Fast LC model (Shimadzu Corporation, Kyoto, Japan) liquid chromatography equipped with a quaternary pump, and an UV detector was used with a stainless steel reverse phase 250 × 4.6 mm (3 mm particle size) C18 Supelco HPLC column (Supelco, Bellefonte, USA). The flow rate used was 0.6 ml/min. DON levels were determined at a wavelength of 218 nm using an UV detector. Samples were confirmed using a LC/ mass spectrometry-selected ion monitoring system (QSTAR XL Pro System, Life Technologies Korea, Seoul, South Korea). Limit of detection (LOD) and limit of quantitation (LOQ) were 2.131 μg/kg and 6.458 μg/ kg, respectively. Recovery experiments were performed in triplicate by spiking blank with DON at levels of 300, 500 and 1000 μg/kg. The mean recovery was 94.68%. 2.4. Measurement of transepithelial electrical resistance (TEER) IPEC-1 cells were seeded at a density of 4 × 105 cells per well in 24well transwell filters with 0.4 μm pores (Becton-Dickinson Labware, Franklin Lakes, NJ, USA). Cells reached confluence within 2 d. For the cell differentiation, complete DMEM/F-12 media containing 100 nM dexamethasone was used and changed every other day until complete differentiation (Bouhet et al., 2004). At the end of the differentiation process (on 10th day after the addition of dexamethasone), cells were treated with DON. The transepithelial electrical resistance (TEER) was measured every 12 h with an EVOM2 epithelial voltohmmeter (World Precision Instruments, Sarasota, FL, USA). Experimental TEER values were expressed as Ω × cm2. All measures were made using three replicates from each experiment. 2.5. Paracellular tracer flux assay Differentiated IPEC-1 cells in 0.4 μm pore inserts as described above were treated with DON for 48 h. Next, 4 kDa fluorescein isothiocyanatedextran (FITC-dextran, Sigma-Aldrich Chemical Company) was dissolved in cell culture medium and added to the apical compartment of enterocytes at the final concentration of 2.2 mg/ml. After 1 h of incubation, the amount of fluorescence in the basolateral compartment was measured using the Victor3 fluorometer (Perkin Elmer, Waltham, MA, USA). The excitation and emission wavelengths were 490 and 535 nm, respectively. The presented data are representative of three independent experiments (Pinton et al., 2010). 2.6. Conventional and real-time RT-PCR The procedure was performed based on the methods reported in our previous study (Park et al., 2014). Briefly, total RNA was extracted with RiboEx (GeneAll Biotech, Seoul, South Korea) according to the manufacturer's instructions. RNA (300 ng) from each sample was transcribed to cDNA using TOPscript™ RT Dry MIX (Enzynomics, Deajeon, South Korea). For real-time PCR, FAM was used as a fluorescent reporter dye and conjugated to the 5′ ends of the probes used to detect the amplified cDNA. Real-time PCR was performed with a thermal cycler RoboGene Q (Qiagen, Hilden, Germany) using the following parameters:

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denaturation at 94 °C for 2 min followed by 50 cycles of denaturation at 98 °C for 10 s, annealing at 59 °C for 30 s, and elongation at 72 °C for 30 s. Each sample was tested in triplicate to ensure statistical significance. Relative quantification of gene expression was performed using the comparative Ct method. For this, the Ct value was defined as the point at which a statistically significant increase in fluorescence was observed. The number of PCR cycles (Ct) required for the FAM intensities to exceed a threshold value just above the background level was calculated for the test and reference reactions. In all experiments, GAPDH was used as the internal control. The results were analyzed in a relative quantitation study based on vehicle treated samples. The 5′ forward and 3′ reverse-complement PCR primers for amplification of each gene were as follows: Porcine IL-8 (5′-ACT TCC AAA CTG GCT GTT GC-3′ and 5′-TGC TGT TGT TGT TGC TTC TCA-3′), Porcine MCP-1 (5′-CTT CTG CAC CCA GGT CCT T-3′ and 5′-AGG CTT CGG AGT TTG GTT TT-3′) and Porcine GAPDH (5′-CAC GAC CAT GGA GAA GGC-3′ and 5′-GAA GCA GGG ATG ATG TTC TGG-3′). 2.7. Western immunoblot analysis All procedures of Western immunoblot analysis were performed based on the methods reported in our previous study (Park et al., 2014). Briefly, treated cells were washed with ice-cold phosphate buffer, lysed in boiling lysis buffer [1% (w/v) SDS, 1.0 mM sodium ortho-vanadate, and 10 mM Tris, pH 7.4], and sonicated for 5 s. Lysates containing proteins were quantified using a BCA protein assay kit (Welgene). Fifty micrograms of protein were separated using a BioRad mini gel electrophoresis system. Proteins were transferred onto PVDF membrane (Pall Corporation, NY, USA), after which the blots were blocked for 1 h with 5% skimmed milk in Tris-buffered saline plus Tween 0.1% (TBST) and probed with each primary antibody for an additional 2 h at room temperature or overnight at 4 °C. After washing three times with TBST, blots were incubated with horseradish-conjugated secondary antibody for 1 h and washed with TBST three times. Protein was detected by pico EPD (ELPIS Biotech. Inc., Taejon, South Korea). The primary antibodies used in the present study were rabbit polyclonal anti-Actin antibody, rabbit polyclonal anti-p65, rabbit polyclonal anti-EGR-1, mouse monoclonal anti-p-ERK1/2 (Dilution at 1/1000, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal p-NF-κB p65, rabbit-polyclonal pp38, and rabbit-polyclonal p-JNK (Dilution at 1/1000, Cell Signaling Technology, Beverly, MA, USA). 2.8. Construction of plasmid and transfection CMV-driven shRNA was constructed by inserting shRNA into a pSilencer 4.1-CMV-neo vector (Thermo Fisher Scientific, Waltham, MA, USA). The empty vector or shRNA of porcine EGR-1 insert-containing vector was denoted as the control or shEGR-1, respectively. Insert EGR-1 shRNA targeted the following sequence: 5′-GAT GAA CGC AAG AGG CAT A-3′. IPEC-1 cells were transiently transfected with control vector or shEGR-1 using jetPRIME™ (Polyplus Transfection SA, Illkirch, France) according to the manufacturer's protocols. All transfection efficiencies were maintained at ~50–60% and confirmed by expression of a pMX-enhanced GFP vector. After 4 h of incubation with the transfection mixture, the cell culture media was replaced and incubated for an additional 48 h. 2.9. Statistical analysis Data were analyzed using SigmaPlot for Windows (Jandel Scientific, San Rafael, CA, USA). A Student's t-test was used for comparative analysis of the two groups of data. For unpaired matched comparative analysis of multiple groups, analysis of variance (ANOVA) was performed. Data that did not meet normality assumptions were subjected to Kruskal–Wallis ranked ANOVA, after which pairwise comparisons were made using the Student–Newman–Keuls (SNK) method.

Please cite this article as: Park, S.-H., et al., Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction..., Toxicology in Vitro (2016), http://dx.doi.org/10.1016/j.tiv.2016.10.003

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3. Results 3.1. Mycotoxin detoxifiers rescue integrity of porcine intestinal epithelial function from DON-induced barrier disruption As mentioned in the introduction, DON absorbed from the lumen of the proximal intestine can affect the basolateral parts of enterocytes in the distal intestine. Therefore, we investigated which side of well-polarized porcine IECs is susceptible to DON exposure to disrupt the porcine intestinal epithelial barrier. Since transepithelial electrical resistance (TEER) is one of the standard measurements used to evaluate intestinal barrier integrity, we quantified real-time measurement of TEER in the porcine epithelial monolayer using polarized IPEC-1 cells exposed to DON (Fig. 1A–B). IPEC-1 cells were used since the porcine cells exhibit an immediate differentiation patterns such as the appearance of alkaline phosphatase and homogenous appearance of tight junction proteins after cells reached confluence when compared with Caco-2 cells (Diesing et al., 2011b). DON treatment at the basolateral side of well-polarized porcine IECs led to more rapid disruption of the intestinal barrier than treatment at the apical side (Fig. 1A). We also found that the exposure of the basolateral region of well-polarized porcine IECs to DON disrupted the intestinal epithelial barrier in a dose-dependent manner (Fig. 1B). Moreover, polarized IPEC-1 cell monolayers increased DONinduced paracellular permeability as shown by the passage of 4 kDa FITC-dextran (Fig. 1C). Taken together, these data show that the porcine intestinal epithelial barrier is more susceptible to disruption by DON at the basolateral region than the apical region, and that this occurs in a dose-dependent manner. Based on the results using the measurement of TEER and the paracellular tracer flux assay to assess the antagonistic action of mycotoxin detoxifiers against DON, the basolateral exposure model was used in the following studies because of its higher sensitivity than apical exposure. The mixture-typed detoxifier, which consisted of minerals, microorganisms, and phytogenic substances (MMP), was originally developed as a feed additive to metabolize trichothecenes such as DON into inactive de-epoxy forms. Each dose (%) of MMP was incubated

with DON, which retarded disruption of the porcine intestinal barrier significantly in a dose-dependent manner (Fig. 2A). Moreover, other mycotoxin detoxifiers such as yeast cell wall and bentonite clay were assessed for their ability to attenuate DON-mediated disruption of the porcine intestinal barrier. First, another readout of barrier integrity taken by measuring the paracellular passage of FITC-dextran demonstrated that all of three kinds of mycotoxin detoxifiers prevented DON-induced disruption of the intestinal epithelial barrier (Fig. 2B). Consistent with these data, measurement of TEER also showed that the yeast cell wall and bentonite clay prevented DON-induced disruption of the porcine intestinal barrier; however, each dose of mycotoxin detoxifier itself did not affect intestinal barrier disruption (Fig. 2C–E). In conclusion, DON-induced disruption of the porcine intestinal epithelial barrier was retarded by three mycotoxin detoxifiers (yeast cell wall, bentonite clay, and the mixture-typed detoxifier). Of note, the mixturetyped detoxifier was the most efficacious attenuator of DON-induced barrier disruption. In agreement with the comparative effects of mycotoxin detoxifiers on DON-induced barrier disruption, the actions of the mycotoxin detoxifiers to reduce the bioavailability of DON were confirmed in the present model. After each dose of mycotoxin detoxifiers was incubated with DON in the culture media, the chemical quantitation of free DON was performed using the immunological and chemical analysis methods. Based on the half inhibitory dose of each mycotoxin detoxifiers (ID50) against bioavailable DON (Lower panels in Fig. 2C– E), the mixture-typed detoxifier was confirmed as the most efficacious agent in mitigating the levels of bioactive DON in vitro, compared to the yeast cell wall and bentonite clay. 3.2. Mycotoxin detoxifiers inhibit DON-induced pro-inflammatory chemokines in porcine IECs DON can induce production of pro-inflammatory cytokines and chemokines via various mechanisms in human and murine intestinal epithelia and leukocytes (Choi et al., 2009; Park et al., 2010). DON also triggers inflammatory responses by inducing pro-inflammatory cytokines in swine (Cano et al., 2013), and we assessed the effects of DON

Fig. 1. Integrity of porcine intestinal epithelial monolayer is disrupted by DON. (A) Fully differentiated IPEC-1 cells were exposed to the vehicle control (DMSO) or 500 ng/ml DON from the apical or basolateral side of the cell layers for the indicated time. The symbol (#) indicates significant differences from each time control (p b 0.05). (B) Fully differentiated IPEC-1 cells were exposed to the vehicle control, 500 ng/ml or 1000 ng/ml DON from the basolateral side of the cell layers for the indicated time. The symbol (*) indicates significant differences from each time control group (p b 0.05). (C) Fully differentiated IPEC-1 cells were exposed to each dose of DON from the basolateral side of the cell layers for 48 h. The translocation of 2.2 g/l FITC dextran to the basolateral compartment was measured after one hour of DON exposure. The symbol (*) indicates significant differences from 0 ng/ml DON treatment (**; p b 0.01, ***; p b 0.001).

Please cite this article as: Park, S.-H., et al., Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction..., Toxicology in Vitro (2016), http://dx.doi.org/10.1016/j.tiv.2016.10.003

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Fig. 2. Mycotoxin detoxifiers inhibit DON-triggered disruption of porcine intestinal epithelial monolayer. (A, C, D, and E) Fully differentiated IPEC-1 were treated with detoxifier-incubated DON (1000 ng/ml) at the basolateral region for the indicated time. The symbol (*) indicates a significant difference from the group treated with 1000 ng/ml DON and 0% mixture-typed detoxifier at each time (p b 0.05). (B) Fully differentiated IPEC-1 were treated with the detoxifier-incubated DON (1000 ng/ml) at the basolateral region for 48 h. The translocation of FITCdextran to the basolateral compartment was measured at one hour after exposure. (**) indicates significant differences from the vehicle control group (p b 0.01). (##) indicates a significant difference from 1000 ng/ml DON incubated with 0% mixture-typed detoxifier (p b 0.01). (Lower panels in Fig. 2C, D, and E) Each dose of mycotoxin detoxifier was incubated with 1000 ng/ml DON in the DMEM/F12 culture media for 2 h. The remaining levels of active free DON was quantified. The symbol (*) indicates a significant difference from the group treated with DON and 0% mixture-typed detoxifier (*; p b 0.05, **; p b 0.01, ***; p b 0.001).

Please cite this article as: Park, S.-H., et al., Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction..., Toxicology in Vitro (2016), http://dx.doi.org/10.1016/j.tiv.2016.10.003

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exposure on the induction of porcine intestinal inflammatory chemokines including interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1), which are crucial to neutrophil recruitment in the early stages of inflammation. We also investigated whether mycotoxin detoxifiers can alter expression of DON-induced pro-inflammatory chemokines in porcine IECs. The results showed that DON triggers the expression of epithelial IL-8 and MCP-1, both of which were suppressed to different degrees by treatment with the mycotoxin detoxifiers (Fig. 3A–B). Of note, high doses (5%) of detoxifiers (the mixture-type N bentonite clay N yeast cell wall) efficiently suppressed both pro-inflammatory chemokines although effects of low doses (0.5%) are partial (Fig. 3A–B). In the following experiments, the high dose of the mixturetyped detoxifier and bentonite clay are more addressed for their molecular effects on the cellular signals.

expression in the porcine IECs (Fig. 5A). Among the MAP kinases, ERK1/2 is a well-known kinase that induces EGR-1 expression, which is a mediator of DON-induced pro-inflammatory chemokines in human IECs (Moon et al., 2007). ERK1/2 and p38 inhibition also attenuated EGR-1 induction by DON (Fig. 5B), demonstrating the positive modulation of EGR-1 expression by ERK1/2 or p38 MAPK activation. ERK1/2- or p38-activated EGR-1 was also positively involved in DON-induced chemokine expression in the porcine IECs since ERK1/2 and p38 inhibition also attenuated chemokine induction (Fig. 5C and D). Taken together, mixture-typed detoxifier and bentonite clay-mediated inhibition of ERK1/2 or p38 MAPK signals contributed to reduction of transcription factor EGR-1 and subsequent pro-inflammatory chemokine expression in porcine IECs. 4. Discussion

3.3. Mycotoxin detoxifiers suppress DON-induced EGR-1 expression via suppression of ERK1/2 and p38 signals As commented in the introduction, MAPKs and subsequent gene regulation via activated transcription factors such as NF-κB and EGR-1 are key signaling modulators in human and murine models in response to DON. In DON-exposed porcine epithelial cells, DON also activated various MAPKs such as ERK1/2, p38 and c-Jun N-terminal kinase (JNK) (Fig. 4A). Moreover, DON enhanced EGR-1 expression while suppressing expression and phosphorylation of p65, a crucial subunit of pro-inflammatory NF-κB (Fig. 4A). In terms of detoxifying agents, we tested whether the mixture-typed detoxifier or bentonite clay can modulate DON-activated stress kinases and their downstream transcription factors. The mixture-typed detoxifier and bentonite clay inhibited DON-activated ERK1/2 and p38 signals; however, JNK was enhanced in porcine IECs (Fig. 4B). In addition, ERK1/2 and p38 inhibition downregulated DON-induced chemokine expression (Fig. 4C–D), indicating the positive regulation of chemokine induction by MAPK signals. Moreover, the mixture-typed detoxifier or bentonite clay decreased DON-induced EGR-1

Fig. 3. Mycotoxin detoxifiers suppress DON-induced chemokine induction in porcine intestinal epithelial cells. DON (1000 ng/ml) was incubated in 0.5% or 5% of each mycotoxin detoxifier for 2 h at RT. IPEC-1 cells were exposed to the vehicle or detoxifier-incubated DON for 1 h. The IL-8 (A) and MCP-1 (B) mRNA were measured using RT real-time PCR. The symbol (*) indicates a significant difference from the group of DON incubated with 0% mycotoxin detoxifier (p b 0.05).

In the present study, DON was a potent trigger of pro-inflammatory responses that disrupted the intestinal epithelial barrier in porcine IECs. To mitigate the adverse actions of DON, three types of detoxifying agents were tested and found to reduce DON-induced disruption of the porcine intestinal epithelial barrier. Of note, the mixture-typed detoxifier was most efficient in attenuating DON-induced barrier disruption in in vitro model, which was in agreement with the degree of the bioavailability of free DON via reaction with the mycotoxin detoxifiers (Fig. 2C, lower panels). In addition, DON induced pro-inflammatory chemokine expression in porcine IECs, which was markedly suppressed by the mixture-typed detoxifier or bentonite clay (Fig. 3). Mechanistically, the mixture-typed detoxifier and bentonite clay reduced pro-inflammatory chemokine expression via suppressed MAPKs and EGR-1 expression, a key transcription factor of chemokine induction by DON in porcine IECs (Figs 4 and 5). Although the bentonite clay was less efficient in reducing bioavailable free DON than the mixture-typed detoxifier and the yeast cell wall, high dose (5%) of bentonite clay significantly interfered with the inflammatory cellular responses including chemokine production and activation of pro-inflammatory transcription factors. Although DON is not a good ionizable molecule with a more bulky epoxy group and therefore not easily bound by bentonite clay, it is possible that some degree of amounts of DON can incorporate into the planar gaps during the incubation time, which can be confirmed in the present assessment (Fig. 2C, lower panels). Therefore, these actions of bentonite clays against the pro-inflammatory signals can account for a potent usefulness of high doses of the agent to mitigate DON-induced toxicity in swine at the farm practice whereas the mixture-typed agent was the most efficient in reducing bioavailable DON among three tested detoxifiers. According to the recent in vivo study, dietary exposure to 2.3 mg DON/kg feed for 35 days induced significant histopathological changes and MAPK activation in the pig intestine whereas only the exposure to 2963.2 ng/ml (10 μM) DON for 1 h induces a significant increase in MAPK phosphorylation in ex vivo porcine gut explant model (Lucioli et al., 2013). Although it is hard to extrapolate from the one hour explant model to the sub-chronic effects of DON in the whole body, the ex vivo treatment dose (10 μM DON) is close to in vivo level (2.3 mg DON/kg feed) which corresponds to 7.7 μM DON (Pinton et al., 2009; Sergent et al., 2006). However, in terms of contamination levels, the previous feeding exposure level (2.3 mg DON/kg feed) is not frequent in cereals used for animal feed. According to the recent survey in 7049 feed samples sourced in North and South Americas, Europe and Asia during 2009–2011, 59% of raw feedstuffs and compound feed samples were contaminated with DON, and an average contamination level was 1 mg DON/kg feed (Rodrigues and Naehrer, 2012). Therefore, in our model, we used 100–1000 ng/ml (0.34–3.37 μM) DON which corresponds to in vivo exposure levels (0.1–1.0 mg DON/kg feed), which is supposed to be realistic within the average contamination level in feed. Moreover, the treatment doses in the present study are much lower than the levels of DON (1482–8799 ng/ml (5–30 μM)) in the

Please cite this article as: Park, S.-H., et al., Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction..., Toxicology in Vitro (2016), http://dx.doi.org/10.1016/j.tiv.2016.10.003

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Fig. 4. Mycotoxin detoxifiers inhibit DON-activated ERK1/2 and p38 signals, which mediates induction of pro-inflammatory chemokines. (A) IPEC-1 cells were exposed to vehicle or 1000 ng/ml DON for the indicated time. Cell lysates were subjected to Western blot analysis. (B) DON (1000 ng/ml) was incubated in 5% of each mycotoxin detoxifier for 2 h at RT. IPEC-1 cells were exposed to the vehicle or detoxifier-incubated DON (1000 ng/ml) for 30 min. Cell lysates were subjected to Western blot analysis. (C–D) DON (1000 ng/ml) was incubated in 5% of each mycotoxin detoxifier for 2 h at RT. IPEC-1 cells were pre-treated with control, 10 μM U0126 or SB203580 for 2 h, then exposed to vehicle or detoxifierincubated DON (1000 ng/ml) for 1 h. IL-8 (C) and MCP-1 (D) mRNA were measured using RT real-time PCR. Different letters over each bar represent significant differences between groups (p b 0.05).

previous studies using the porcine enterocyte or ex vivo gut explant model (Lucioli et al., 2013; Pinton et al., 2010). The dose regime (0.5–5%) for the commercially available mycotoxin detoxifiers in the present study were based on the in vitro quantitative assessment of bioavailable free DON after chemical reaction with the detoxifiers. Although no typical tolerance studies with mycotoxin detoxifiers were found, the varying levels of mycotoxin detoxifiers including bentonites (0.2–10%) are generally used in various safety

assessment for animal uses (Avantaggiato et al., 2005; FEEDAP, 2011; Sun et al., 2015). Twenty eight-week rat toxicity study shows dietary levels up to 2% calcium montmorillonite without toxicity and the FEEDAP Panel considers, as a conservative estimate, 0.5% bentonite to be safe for all target animal species although higher dose of bentonites can reduce the availability of the essential trace element manganese and can interact with other phytochemicals, but apparently not with vitamins in some animal models (FEEDAP, 2011). The lower levels (0.5%)

Fig. 5. Mycotoxin detoxifiers regulate EGR-1 expression via ERK1/2 and p38 signals in DON-exposed porcine intestinal epithelial cells. (A) DON (1000 ng/ml) was incubated in 5% of each mycotoxin detoxifier for 2 h at RT. IPEC-1 cells were exposed to vehicle or detoxifier-incubated DON (1000 ng/ml) for 2 h. Cell lysates were subjected to Western blot analysis. (B) IPEC-1 cells were pre-treated with control, 10 μM U0126 or SB203580 for 2 h and exposed to vehicle or 1000 ng/ml DON for 2 h. Cell lysates were subjected to Western blot analysis. (C–D) IPEC-1 cells transfected with control or porcine EGR1 shRNA (shEGR-1) were treated with vehicle or 1000 ng/ml DON for 1 h. IL-8 (C) and MCP-1 (D) mRNA expression was measured using RT real-time PCR. Different letters over each bar represent significant differences between groups (p b 0.05).

Please cite this article as: Park, S.-H., et al., Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction..., Toxicology in Vitro (2016), http://dx.doi.org/10.1016/j.tiv.2016.10.003

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of yeast cell wall-based detoxifier are conservatively recommended in mitigating DON exposure in the pig feeding models (Shehata et al., 2004). In case of the mixture-typed detoxifier, there is little information on the mixture toxicity and the working levels recommended by the producer are about 0.1–0.15%. However, treatment with higher levels (2–5%) of the mixture-typed detoxifier would not produce obvious toxicity since its major components are safety-confirmed bentonite, yeast cell wall, and the probiotic bacteria. In the present model, low levels (0.5%) of mycotoxin detoxifiers were also partly efficient in reducing DON-induced barrier disruption and pro-inflammatory responses. However, considering the short incubation time (2 h) of DON with mycotoxin detoxifiers in the present study, extended incubation would enhance the mitigating effects of the lower levels of detoxifiers against DON-induced toxicity and inflammatory stimulation in the digestive tract at the farm situation. DON is one of the mycotoxins that disrupts the intestinal epithelial barrier and increases intestinal permeability. Moreover, as mentioned in the introduction, DON absorbed from the lumen of the proximal intestine can affect the basolateral parts of enterocytes in the distal intestine after the portal and systemic circulation (Diesing et al., 2011a). Our results showed that the barrier disruption in the basolateral exposure was more severe than that in the apical exposure to DON. Therefore, the basolateral exposure model using the porcine enterocyte monolayer was useful in assessing the efficacy of DON detoxifiers in vitro to mimic the realistic in vivo toxicological performance in swine, which was also tested in the previous report using IPEC-J2, another porcine cell line (Devreese et al., 2013). In the present study, three mycotoxin detoxifiers retarded DON-induced disruption of the intestinal epithelial barrier and intestinal permeability, although the mycotoxin detoxifiers themselves did not affect the physical barrier integrity. Mechanistically, the detoxification was due to decreased bioavailability or metabolic inactivation of DON, which contributed to reduced barrier toxicity of the mycotoxin. Enterocytes are exposure-prone to the high local concentration of DON in the gut lumen and the porcine IECs are particularly susceptible to DON-induced disruption of the gut barrier via decreased expression of tight-junction proteins such as claudins, which leads to loss of barrier integrity (Pinton et al., 2010; Pinton et al., 2009). Mechanistically, this disruption is an orchestrated process that occurs in association with increases in phosphorylated MAPKs and subsequent decreases in the expression of tight junction proteins such as claudins (Pinton et al., 2012). According to previous studies, DON-induced disruption of the porcine intestinal barrier was mediated by inhibition of the tight junction protein, claudin-4, in humans and pigs (De Walle et al., 2010; Pinton et al., 2010). In addition to the reduction of claudin-4, expression of another tight junction protein, E-cadherin, is also suppressed by DON, leading to barrier disruption in the porcine intestine (Basso et al., 2013). Moreover, DON-impaired expression of tight junction molecules maintaining barrier integrity is linked to activation of MAPKs including ERK1/2 (Pinton et al., 2010). In our study, DON activated MAPKs such as ERK1/2, p38 and JNK. Among these three MAPKs, the mixture-typed detoxifier and bentonite clay inhibited ERK1/2 signals, which may account for the detoxifier-induced attenuation of barrier disruption of the porcine intestinal epithelial monolayer. In addition, p38 activation is associated with barrier disruption via redistribution of tight junction and adherens junction proteins or reorganization of F-actin in the human intestine (Elamin et al., 2014). In this study, the mixture-typed detoxifier and bentonite clay suppressed DON-activated p38 in the barrier in the porcine intestine, which can be associated with regulation of p38-mediated barrier disruption in the porcine intestinal barrier. In contrast to the effects of mycotoxin detoxifiers on ERK1/2 and p38 MAPK, JNK was more activated in the porcine intestinal epithelial cells. Activated JNK in the intestinal epithelial cells leads to increased cell number and villus length by regulating proliferation and migration of progenitor cells (Sancho et al., 2009). In particular, augmentation of the JNK signal enhances cd44 and Wnt target gene mRNA expression in the epithelial progenitor cells (Sancho et al., 2009). DON is well

known to regulate enterocyte growth and retard epithelial cell migration and restitution after ulcerative insults (Bianco et al., 2012). Therefore, DON-suppressed epithelial growth and migration can be antagonized by super-activated JNK in the presence of mycotoxin detoxifiers in swine enterocyte cells. However, the beneficial functions of JNK super-activated by mycotoxin detoxifiers cannot be a good news in the chronically DON-insulted enterocytes since this compensative growth and migration would be a bad prognosis biomarker of the progressive intestinal carcinogenesis (Bianco et al., 2012). In human IECs, many transcriptional or post-transcriptional regulators are associated with DON-induced pro-inflammatory chemokine expression (Choi et al., 2009; Moon et al., 2007). In particular, EGR-1 is a mediator of DON-induced pro-inflammatory chemokines via ERK1/2 activation in human IECs (Moon et al., 2007). However, NF-κB phosphorylation and expression are suppressed by DON in human IECs. In contrast, mediators or mechanisms of cytokine induction by DON in porcine IECs are poorly understood. This is the first study to report that ERK1/2- or p38 MAPK-induced EGR-1 is a positive regulator of DON-induced pro-inflammatory IL-8 and MCP-1 mRNA expression in porcine enterocytes. All of these events were counteracted by treatment with mixture-typed detoxifier and bentonite clay. In addition, NF-κB expression and phosphorylation were suppressed in DON-exposed porcine IECs, indicating NF-κB-independent induction of chemokines in DON-exposed porcine enterocytes as reported in the human cell exposure model (Moon et al., 2007; Park et al., 2010). Taken together, mycotoxin detoxifiers retarded DON-induced disruption of the intestinal epithelial barrier and suppressed DON-triggered pro-inflammatory chemokine expression via inhibition of ERK1/2- or p38 MAPK-activation and expression of EGR-1, a crucial pro-inflammatory transcription factor in response to DON in porcine IECs. Moreover, the pharmacological actions of dietary detoxifiers provide critical insights into preventive or therapeutic interventions with potent mycotoxin-derived intestinal disorders in swine via selective feed additives. Conflict of interest None of the authors have any conflict of interest. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements This work was supported by the 2012 Specialization Project Research Grant funded by Pusan National University. References Avantaggiato, G., Havenaar, R., Visconti, A., 2004. Evaluation of the intestinal absorption of deoxynivalenol and nivalenol by an in vitro gastrointestinal model, and the binding efficacy of activated carbon and other adsorbent materials. Food Chem. Toxicol. 42, 817–824. Avantaggiato, G., Solfrizzo, M., Visconti, A., 2005. Recent advances on the use of adsorbent materials for detoxification of Fusarium mycotoxins. Food Addit. Contam. 22, 379–388. Basso, K., Gomes, F., Bracarense, A.P., 2013. Deoxynivanelol and fumonisin, alone or in combination, induce changes on intestinal junction complexes and in E-cadherin expression. Toxins 5, 2341–2352. 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. Bouhet, S., Hourcade, E., Loiseau, N., Fikry, A., Martinez, S., Roselli, M., Galtier, P., Mengheri, E., Oswald, I.P., 2004. The mycotoxin fumonisin B1 alters the proliferation and the barrier function of porcine intestinal epithelial cells. Toxicol. Sci. 77, 165–171. Cano, P.M., Seeboth, J., Meurens, F., Cognie, J., Abrami, R., Oswald, I.P., Guzylack-Piriou, L., 2013. Deoxynivalenol as a new factor in the persistence of intestinal inflammatory diseases: an emerging hypothesis through possible modulation of Th17-mediated response. PLoS One 8, e53647.

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Please cite this article as: Park, S.-H., et al., Mycotoxin detoxifiers attenuate deoxynivalenol-induced pro-inflammatory barrier insult in porcine enterocytes as an in vitro evaluation model of feed mycotoxin reduction..., Toxicology in Vitro (2016), http://dx.doi.org/10.1016/j.tiv.2016.10.003