Mycotoxin deoxynivalenol (DON) mediates biphasic cellular response in intestinal porcine epithelial cell lines IPEC-1 and IPEC-J2

Toxicology Letters 200 (2011) 8–18

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Mycotoxin deoxynivalenol (DON) mediates biphasic cellular response in intestinal porcine epithelial cell lines IPEC-1 and IPEC-J2 Anne-Kathrin Diesing a , Constanze Nossol a , Patricia Panther a , Nicole Walk a , Andreas Post a , Jeannette Kluess a , Peter Kreutzmann b , Sven Dänicke c , Hermann-Josef Rothkötter a , Stefan Kahlert a,∗ a b c

Institute of Anatomy, Medical Faculty, Otto-von-Guericke University Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany Institute of Biochemistry and Cell Biology, Medical Faculty, Otto-von-Guericke University Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany Institute of Animal Nutrition, Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, Bundesallee 50, 38116 Braunschweig, Germany

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Article history: Received 16 July 2010 Received in revised form 1 October 2010 Accepted 5 October 2010 Available online 19 October 2010 Keywords: Tight junction Cell cycle Cell proliferation Apoptosis Viability

a b s t r a c t The Fusarium derived mycotoxin deoxynivalenol (DON) is frequently found in cereals used for human and animal nutrition. We studied effects of DON in non-transformed, non-carcinoma, polarized epithelial cells of porcine small intestinal origin (IPEC-1 and IPEC-J2) in a low (200 ng/mL) and a high (2000 ng/mL) concentration. Application of high DON concentrations showed significant toxic effects as indicated by a reduction in cell number, in cellular reduction capacity measured by MTT assay, reduced uptake of neutral red (NR) and a decrease in cell proliferation. High dose toxicity was accompanied by disintegration of tight junction protein ZO-1 and increase of cell cycle phase G2/M. Activation of caspase 3 was found as an early event in the high DON concentration with an initial maximum after 6–8 h. In contrast, application of 200 ng/mL DON exhibited a response pattern distinct from the high dose DON toxicity. The cell cycle, ZO-1 expression and distribution as well as caspase 3 activation were not changed. BrdU incorporation was significantly increased after 72 h incubation with 200 ng/mL DON and NR uptake was only transiently reduced after 24 h. Low dose effects of DON on intestinal epithelial cells were triggered by mechanisms different from those responsible for the high dose toxicity. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Deoxynivalenol (DON) is a trichothecene primarily produced by the plant pathogen Fusarium graminearum and Fusarium culmorum and most prevalent in crops like wheat, oat or barley. DON contaminated products represent a serious problem in animal nutrition. It has been found that pigs are the most susceptible species and DON ingestion leads to reduced growth and thus to economical loss (Pestka et al., 2008). Typically, feed contamination of 20 ppm DON triggers acute toxic effects in pigs characterized by vomiting (Young et al., 1983). On the other hand, concentrations as low as

Abbreviations: Abs, absorbance; ANOVA, analysis of variance; BrdU, 5-bromo2-deoxyuridine; DAPI, 4 ,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified eagle medium; DON, deoxynivalenol; EC50 , half maximal effective concentration; EGF, epidermal growth factor; FCS, fetal calf serum; GAPDH, glyceraldehyde 3phosphate dehydrogenase; IPEC-1, intestinal porcine epithelial cell line 1; IPEC-J2, intestinal porcine epithelial cell line J2; ITS, insulin-transferrin-selenium; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NR, neutral red; PBS, phosphate buffered saline; PI, propidium iodide; SD, standard deviation; SEM, standard error of the mean; ZO-1, zonula occludens-1 protein. ∗ Corresponding author. Tel.: +49 0391 6713602. E-mail address: [email protected] (S. Kahlert). 0378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2010.10.006

1 ppm DON lead to reduced feed intake and weight gain (Rotter et al., 1994). Whereas the DON contamination in feed is a measurable quantity, an approximation of the effective DON concentration on the apical enterocyte border is difficult. In human intestine DON concentrations were approximated between 160 ng/mL and 2000 ng/mL (Sergent et al., 2006). The intestinal mucosa represents a pivotal border between the organism and its environment. The large contact surface allows efficient nutrient absorption, acts as an important barrier for pathogens and toxins and participates in the innate immune response (Mariani et al., 2009; Pitman and Blumberg, 2000). In the gastrointestinal tract DON comes into contact with the epithelial surface and is rapidly absorbed (Prelusky et al., 1988). The effects of high DON concentrations on the enterocyte border comprise cell death and loss of the epithelial barrier integrity. In jejunal explant cultures of pigs morphological changes were observed with DON concentrations of 1500 ng/mL including lysis of enterocytes (KolfClauw et al., 2009). DON concentrations within the same range decreased the transepithelial electrical resistance (TEER) of Caco2 and IPEC-1 cells cultured on permeable supports. Additionally, an altered claudin-3 and claudin-4 expression was observed after application of approximately 9000 ng/mL DON for 48 h (Pinton et al., 2009). However, lower concentrations of the mycotoxin were not tested in this investigation.

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The effects of DON on cellular physiology are complex and depend on the tested DON concentration and cell type. In early investigations DON binding to ribosomes and inhibition of translation has been reported (Ehrlich and Daigle, 1987). This ribotoxic effect of DON is not an inherent ribosome-degrading property of the toxin because ribosomal RNA is not cleaved in a cell free system (Li and Pestka, 2008). The ribotoxicity of DON is paralleled by reduced protein biosynthesis as shown with 3000 ng/mL DON in renal proximal tubular epithelial cells (RPTEC) (Königs et al., 2007). In comparison to epithelial cells, cellular components of the immune system seem to be more sensitive to DON mediated effects. In the murine macrophage cell line RAW264.7 low concentrations of DON (250 ng/mL) triggered phosphorylation of AKT and ERK, finally leading to an activation of caspase 3 and internucleosomal DNA fragmentation (Zhou et al., 2005). So far, only few effects of low DON concentrations on epithelial cells or the epithelium of pigs were described. A reduced villus length was observed in pig jejunal explant cultures in response to 300 ng/mL DON (Kolf-Clauw et al., 2009). DON induced arrest in G2/M cell cycle in human HCT-116 and Intestinal-407 epithelial cells in the concentration range of 250 ng/mL and 1000 ng/mL (Yang et al., 2008). Because swine is the most susceptible species, and acute toxicity as well as chronic effects triggered by long-term exposure of DON have been reported, we opted for the use of an in vitro cell culture that resembles the in vivo situation as closely as possible. As recently shown, intestinal porcine epithelial cells IPEC-1 and IPECJ2 are promising cell culture models which retained most of their original epithelial nature (Mariani et al., 2009). Both cell lines, isolated from the small intestine of neonatal piglets are not carcinoma derived or genetically modified. IPEC-J2 originate from jejunum solely whereas IPEC-1 cells were isolated from both jejunum and ileum, representing a more distal epithelial cell fraction of the gut (Gonzalez-Vallina et al., 1996; Schierack et al., 2006). We hypothesize that high and low DON concentrations trigger a different pattern of cellular responses in intestinal epithelium. In this context we have analyzed the effect of DON on IPEC-1 and IPEC-J2 including the estimation of cellular viability, growth capacity, induction of apoptosis, organization of tight junction and cell cycle analysis. The applied DON concentrations (typically between 200 and 2000 ng/mL) cover the expected concentrations on the intestinal border in vivo (Sergent et al., 2006). 2. Materials and methods 2.1. Cell culture conditions IPEC-1 and IPEC-J2 cell lines were used in this study (Gonzalez-Vallina et al., 1996; Rhoads et al., 1994). They represent non-transformed intestinal porcine epithelial cell lines continuously maintained in cell culture. Cells were cultured in Dulbecco’s modified eagle medium (DMEM/Ham’s F-12 [1:1]) supplemented with 5% fetal calf serum (FCS), 1% insulin-transferrin-selenium (ITS), 16 mmol/L HEPES (all PAN-Biotech, Germany) and 5 ng/mL epidermal growth factor (EGF; BD Biosciences, Germany), incubated at 39 ◦ C and 5% CO2 (Schierack et al., 2006). Cell cultures were regularly tested and found to be free of mycoplasma contamination (Venor® GeM Mycoplasma Detection Kit; Minerva Biolabs, Germany). The cells were routinely seeded at a density of 0.6 × 105 mL−1 (IPEC-1) and 0.5 × 105 mL−1 (IPEC-J2) with 7.5 mL medium in plastic tissue culture flasks (75 cm2 Nunc, Denmark) and passaged every 3–4 d for a maximum of 20 times (IPEC-1 passages 103–123; IPEC-J2 passages 78–98). Cells formed a confluent monolayer within 4 d and were then used in experiments. 2.2. Preparation of DON The obtained DON (D0156; Sigma–Aldrich, Germany) was diluted in absolute ethanol (99.6%; Roth, Germany) to a 0.2 mg/mL stock solution and working dilutions were prepared in cell culture medium. A final concentration of 1% ethanol corresponding to the ethanol concentration of 2000 ng/mL DON solution was tested in all assays and results were not significantly different from control.

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2.3. Assays in 96 well plate format Analysis of cellular viability (cell count, lactate dehydrogenase (LDH) assay, neutral red (NR) uptake, MTT assay), proliferation (BrdU assay) and apoptosis (luminescence caspase 3/7 assay) were performed in 96 well plate format. IPEC-1 and IPEC-J2 cells were seeded in 96 well plastic tissue culture plates (Nunc, Denmark) and grown for 4 d until confluence. Medium was removed and after washing once with PBS, fresh medium was added containing increasing final concentrations of DON (100–4000 ng/mL). Cells were incubated for 24 h, 48 h or 72 h. For long term experiments (14 d) treatment was performed in the same manner, but with lower DON concentrations (50–500 ng/mL) and a regular exchange of medium + DON after every 3–4 d. All assays were performed in triplicates and in at least three independent experiments using a multiplate reader (SunriseTM or Infinite M200, TECAN, Germany). 2.3.1. Cell count Cells were treated as described, trypsinized, pelleted and after resuspending in PBS counted in a Neubauer chamber. 2.3.2. Alkaline phosphatase (AP) activity The cellular enzymatic activity of alkaline phosphatase was detected by formation of blue-colored diformazan precipitate within 22 d of cultivation. A working solution was prepared from 240 ␮L stock solution A (25 mg/mL nitro blue tetrazolium chloride [NBT] in 70% dimethylformamide [DMF]) and 60 ␮L B (50 mg/mL 5-bromo-4-chloro-3-indolylphosphate toluidine salt [BCIP] in 100% DMF) in 16 mL 0.1 M Tris–HCl. Wells were incubated with 200 ␮L working solution overnight in the dark, subsequently the solution was removed and the diformazan was dissolved for 3 h in 200 ␮L DMF. Absorbency was measured at 560 nm and a calibration curve was used for the diformazan calculation. 2.3.3. LDH activity Cellular membrane integrity (necrosis) was assessed in normal and FCS-free medium by measurement of LDH activity in the supernatant at an optical density of 492 nm using the colorimetric Cytotoxicity Detection KitPLUS (Roche, Germany). 2.3.4. NR uptake Cellular viability was measured by NR uptake at an optical density of 546 nm. NR (Sigma, Germany) was added in a final concentration of 1:40 in cell media with or without FCS, incubated for 3 h, washed three times with PBS and destained with a solution of 1% acetic acid and 50% ethanol. 2.3.5. Metabolic activity Metabolic activity of cell culture was measured by MTT (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl-tetrazolium bromide; Sigma–Aldrich, Germany) assay. After the accordant time periods 10 ␮L of MTT (in 5 mg/mL PBS) were added and the cells were incubated for 3 h additionally. After dissolving the crystalline formazan product (100 ␮L of 0.01 mol/L HCl/SDS for 3 h at room temperature in the dark) the optical density was measured at 570 nm. Antimycin (50 ␮mol/L) was tested as a positive control and MTT reduction capacity was found below 35% of the untreated control. 2.3.6. Cell proliferation DNA-synthesis during proliferation was quantified by BrdU (5 -brome-2 deoxyuridine) incorporation during the last 6 h of incubation using the colorimetric BrdU ELISA (Roche, Germany) according to manufacturer’s protocol. The optical density was measured at 450 nm. Mitomycin (10 ␮mol/L) was tested as a positive control and BrdU incorporation was found below 17% of the untreated control. 2.3.7. Apoptosis Cellular caspase 3 activity was measured by the luminescence Caspase-Glo® 3/7 Assay (Promega, Germany) as described in manufacturer’s protocol. 2.4. Analysis of ZO-1 structure by immunofluorescence IPEC-1 and IPEC-J2 cells were seeded in a Lab-Tek® Chamber Slide® System (Nunc, Germany) and experiments were performed with confluent cell layers (4 d). DON exposure was performed as described above and cells were fixed for 30 min with absolute ethanol at 4 ◦ C followed by an incubation with acetone for 3 min. Cells were washed with PBS and blocked with 1% normal goat serum (NGS; Axxora, Germany). Primary rabbit anti-ZO-1 antibody (diluted 1:100; Invitrogen, Germany) and secondary Alexa fluor 488 labeled goat anti-rabbit antibody (1:200; Invitrogen, Germany) were used. Nuclei were stained with 4 ,6-diamidino-2-phenylindole (DAPI; Partec, Germany). Fluorescence microscopy was performed using an Axiovert 200 M (Zeiss, Germany) with corresponding Axiovision software. 2.5. Protein isolation and immunoblot analysis of caspase 3 and ZO-1 IPEC-1 and IPEC-J2 cells were seeded in plastic tissue culture 6 well plates (Nunc, Denmark) and experiments were performed with confluent cell layers (4 d). Cell

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homogenate protein was obtained by 10 min incubation on ice with SDS-gel loading buffer (1 M Tris base pH 6.8; 1% glycerol, 10% SDS, 0.1% bromophenol blue; shortly before addition of 0.05% ␤-mercaptoethanol and 1% protease inhibitors; Complete, Roche, Germany) and cells were harvested with a cell scraper. Samples were denatured at 95 ◦ C for 5 min and loaded together with the prestained protein ladder (SM1811; Fermentas, Germany) onto 10% SDS-polyacrylamide gels. After electrophoresis and semi-dry blotting onto 0.45 ␮m nitrocellulose membranes (Whatman, Germany) the primary rabbit anti-ZO-1 antibody (1:500; Invitrogen, Germany), rabbit anti-caspase 3 antibody and mouse anti-GAPDH antibody (both 1:1000, Cell Signaling, Germany) were used in blocking reagent. The secondary antibody was purchased with the BM Chemiluminescence Western Blotting Kit mouse/rabbit (Roche, Germany). After ZO-1 or caspase 3 development the blots were stripped at 50 ◦ C for 30 min with stripping buffer (7.58 g Tris base, 20 g SDS, 7 mL ␤-mercaptoethanol in 100 mL H2 O, pH 6.8), washed and reprobed with antiGAPDH antibody. Blots were analyzed on an Alpha-Ease® FC Imaging System (Alpha Innotech, Canada). 2.6. Cell cycle analysis by flow cytometry IPEC-1 and IPEC-J2 cells were seeded in plastic tissue culture 6 well plates (Nunc, Denmark) and experiments were performed with confluent cell layers (4 d). Cells were then 24 h synchronized in FCS free medium and DON exposure was performed as described above. Cells were trypsinized, pelleted and resuspended in PBS. Ethanol fixation and propidium iodide (PI; Sigma, Germany) staining procedure were performed as previously described (Chen and Donovan, 2004). Cells were analyzed with a FACSCalibur flow cytometer using CellQuest Pro® software (both BD Biosciences, Germany).

2.7. Statistical analysis Data were analyzed by ANOVA and p values were calculated using Dunnett’s post test (SPSS Statistics 17.0). Mean values represent triplicate measurement of at least 3 independent experiments. Significant differences between treated cells and control are indicated by asterisks (*p < 0.05; **p < 0.01).

3. Results 3.1. Differentiation of IPEC-1 and IPEC-J2 cells Both cell lines were cultured for up to 22 d and expression of tight junction protein ZO-1 and enzyme activity of alkaline phosphatase was monitored. On day 4 after seeding immunofluorescence staining of ZO-1 exhibited a continuous lining around adjacent cells in both cell lines. This pattern was not changed until day 21 (Fig. 1A). Detection of alkaline phosphatase enzyme activity as a marker for enterocyte differentiation (Hinnebusch et al., 2004) showed a sudden and remarkable increase between days 2 and 3 that was paralleled by the formation of a confluent cell layer (Fig. 1B). In membrane cultures TEER exhibited a similar time kinetic with TEER values above 1 k/cm2 after 4 d (results not shown).

Fig. 1. Time course of ZO-1 expression and alkaline phosphatase activity. (A) IPEC-1 and IPEC-J2 cells were seeded (day 0) and ZO-1 was stained by immunofluorescence after 4 d and 21 d. Bar = 10 ␮m. (B) IPEC-1 and IPEC-J2 cultures were further analyzed for alkaline phosphatase activity and parallel cultured cells were counted. Enzyme activity is given as generated dye/5000 cell ± SD.

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3.2. Cell death detected by decrease in cell count and LDH release

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an increase in LDH release. Significantly increased LDH signals in IPEC-1 after 48 h and 72 h incubation with 2000 ng/mL in serumfree media and in IPEC-J2 after 48 h incubation with 2000 ng/mL in complete media were detected. The experiments were performed in complete and serum-free media to exclude effects of potential DON attachment to albumin and effects of growth factors in FCS. Lysis of control cells resulted in a signal reaching the maximal technical detection limit of the test (Abs > 3.0).

The effect of increasing DON concentrations during 24 h, 48 h and 72 h on the total cell count of confluent IPEC-1 and IPEC-J2 cells is shown in Fig. 2. Due to the larger size of IPEC-J2 cells a lower cell density (2.1 ± 0.2 × 103 mm−2 ) in comparison to IPEC-1 (5.6 ± 0.6 × 103 mm−2 ) was found. The cell count of control cells was not significantly changed within 72 h of observation. Application of DON resulted in a time and dose dependent reduction of cell counts. Incubation with 2000–4000 ng/mL DON significantly reduced the cell count in both cell lines and all tested time points. After 48 h treatment with 1000 ng/mL DON significantly lower cell counts were obtained in IPEC-1, but not in IPEC-J2. DON concentrations between 100 and 500 ng/mL were without significant effect on the cell count in both cell lines with exception of 100 and 200 ng/mL in IPEC-1. IPEC-1 appeared more sensitive to DON mediated effects than IPEC-J2. The release of LDH due to permeabilization of the cell membrane was quantified by detection of LDH enzymatic activity in the supernatant (Fig. 3). Surprisingly, the significant decrease in cell count after treatment with DON was not completely reflected by

3.3. Cell viability detected by lysosomal uptake of NR Lysosomal uptake of NR as an indicator of cell viability was determined in IPEC-1 and IPEC-J2 cells after 24 h, 48 h and 72 h application of 200 and 2000 ng/mL DON and compared with control (Fig. 4). Similar to the LDH assay the experiment was performed in complete and serum-free media to exclude binding effects of albumin. Significant differences between complete and serum-free test conditions were found in IPEC-1 cells after 72 h only. Application of 2000 ng/mL DON reduced NR uptake in both cell lines and all tested culture conditions. It is worth noting that a 24 h application of 200 ng/mL DON decreased the NR uptake significantly compared

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DON (ng/mL) Fig. 2. Total cell count of IPEC-1 and IPEC-J2 cells after treatment with increasing DON concentrations for 24 h, 48 h and 72 h. Confluent IPEC-1 (A) and IPEC-J2 (B) cell layers were incubated with DON (0–4000 ng/mL) for 24 h, 48 h and 72 h in 96 well plates. Each bar represents at least 3 independent experiments performed in triplicates. Cell counts were calculated as total amount of cells per mL ± SEM. Measurements significantly different from corresponding control were indicated as *p < 0.05 and **p < 0.01.

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Fig. 3. Release of LDH in response to DON treatment. Confluent IPEC-1 (A) and IPEC-J2 (B) cell layers were treated with DON in complete and serum-free medium as indicated. LDH activity of the supernatant was measured by commercial LDH assay. Each bar represents at least 3 independent experiments performed in triplicate ± SEM. Significant differences in comparison to corresponding control were indicated as *p < 0.05 and **p < 0.01.

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Fig. 5. Viability of IPEC-1 and IPEC-J2 cells after 24 h, 48 h and 72 h of DON treatment measured by MTT assay. Confluent IPEC-1 (A) and IPEC-J2 (B) cell cultures were incubated with DON (0–4000 ng/mL) in complete medium for 24 h, 48 h and 72 h in 96 well plates. Reduction of MTT was measured after dissolving the formazan product. Each bar represents at least 4 independent experiments performed in triplicates ± SEM. Measurements significantly different from corresponding control were indicated as *p < 0.05 and **p < 0.01.

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3.4. Metabolic activity detected by MTT assay The MTT assay was applied to detect cell viability by their capacity to reduce MTT to blue formazan dye. Application of DON concentrations of 500 ng/mL or higher decreased viability of both cell types after 48 h and 72 h incubation in a dose dependent manner (Fig. 5). The highest DON concentration (4000 ng/mL) decreased the MTT signal in IPEC-1 to 60% and 40% of the control after 48 h and 72 h, respectively. In IPEC-J2 the corresponding measurements were 70% (48 h) and 58% (72 h). The MTT signal after 24 h showed a less distinct pattern. The maximal reduction of the MTT signal (versus control) was 78% as response to 4000 ng/mL DON in IPEC1 and 76% after 100 ng/mL DON application in IPEC-J2, indicating a moderate influence of DON on cellular reduction capacity after 24 h. 3.5. Proliferation detected by BrdU incorporation

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DON (ng/mL) Fig. 4. DON-dependent uptake of neutral red in IPEC-1 and IPEC-J2 cells. Confluent IPEC-1 (A) and IPEC-J2 (B) cell layers were treated with DON in complete and serumfree medium as indicated. Neutral red was applied at the end of the experiment and after washing the remaining cellular dye was measured. Each bar represents at least 3 independent experiments performed in triplicates ± SEM. Significant differences in comparison to corresponding control were in indicated as *p < 0.05 and **p < 0.01.

Putative effects of DON on DNA synthesis and cell proliferation were studied using BrdU as a thymidine analogue, which is integrated in DNA during the S-phase of cell cycle (Fig. 6). A significant reduction of BrdU incorporation in IPEC-1 was detected after application of 2000–4000 ng/mL DON at all incubation periods. A dosage of 1000 ng/mL DON significantly reduced BrdU incorporation after 72 h, but not after 24 h or 48 h. In IPEC-J2 significant reduction of BrdU incorporation was found after 72 h at concen-

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trations of 2000 ng/mL or higher only. The calculated EC50 values after 72 h are 656 ng/mL (2.2 ␮mol/L) and 2118 ng/mL (7.2 ␮mol/L) for IPEC-1 and IPEC-J2, respectively. Surprisingly, in contrast to the reduced BrdU incorporation observed for high DON concentrations an increase was found in the low concentration range. Incubation of IPEC-1 with 200 ng/mL DON for 48 h and 72 h significantly increased the BrdU incorporation. A similar effect was found in IPEC-J2 after 72 h incubation period.

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Fig. 7. Cell viability and DNA synthesis of IPEC-1 and IPEC-J2 cell cultures after long term (14 d) incubation with DON. Confluent IPEC-1 and IPEC-J2 cell cultures were incubated with DON (0–500 ng/mL) in complete medium for 14 d in 96 well plates. DON was applied initially and replenished every 3–4 d with medium exchange. Cellular viability (MTT assay; A) and DNA synthesis (BrdU incorporation; B) were measured as described before. Each bar represents at least 3 independent experiments performed in triplicate ± SEM. Measurements significantly different from corresponding control were indicated as *p < 0.05 and **p < 0.01.

In IPEC-1 cell cultures the ZO-1 pattern resembled a more cobblestone structure, whereas the IPEC-J2 pattern was of irregular shape. A 48 h exposure to 200 ng/mL DON did not change the pattern in comparison to control, whereas an exposure to 2000 ng/mL DON disintegrated the ZO-1 structure in both cell lines dramatically. The analysis of ZO-1 protein in Western blotting suggests a gentle reduction of ZO-1 protein level in 2000 ng/mL treated IPEC-1 and IPEC-J2 cells in comparison to GAPDH (Fig. 8B). In contrast to the dramatic effect of 2000 ng/mL DON detectable in immunofluorescence staining of ZO-1 the difference in ZO-1 protein expression was moderate. Application of 200 ng/mL DON did not reduce the signal in Western blotting.

3.6. Long term exposure Subsequently, we analyzed the effect of low DON concentrations during prolonged exposure times in confluent IPEC-1 and IPEC-J2 cultures using the MTT and BrdU assay (Fig. 7). Exposure of cells to 200–500 ng/mL DON for 14 d decreased MTT signal significantly in comparison to control (Fig. 7A). Concentrations below 200 ng/mL were without significant effect. The lowest MTT reduction capacity was reached at the highest tested concentration of 500 ng/mL and represented 10% of the control level for both IPEC-1 and IPEC-J2. The capacity of BrdU incorporation was significantly reduced by application of 300 ng/mL DON or higher (Fig. 7B). However, in IPEC1 concentrations of 50 ng/mL and 100 ng/mL triggered a numerical increase of BrdU incorporation in comparison to control. 3.7. Structure and amount of tight junction protein ZO-1 Integrity of the epithelial cell layer was analyzed by cellular ZO1 distribution. Both cell lines exhibited a characteristic continuous lining of ZO-1 protein pattern, which links individual cells (Fig. 8A).

3.8. Induction of apoptosis Caspase 3 is one marker for the induction of apoptosis. We analyzed DON mediated apoptotic effects on IPEC-1 and IPEC-J2 cell lines by detection of caspase 3 enzymatic activity (Fig. 9A and B) and using immunoblotting technique (Fig. 9C). Between 6 h and 72 h caspase 3 activity was significantly increased after 6 h and 8 h of incubation with 2000 ng/mL DON in comparison to control in both cell lines. After 24 h treatment with 2000 ng/mL DON caspase 3 activity was reduced to the control level. However, a slight but significant increase of caspase 3 activity was again detected after 48 h and 72 h. Application of 200 ng/mL DON did not trigger an increase in caspase 3 activity at any tested time period. The obtained data from immunoblotting confirmed the results of the caspase 3 activity assay. The cleaved caspase 3 fragment (19 kDa) was detectable after 6 h and 8 h incubation with 2000 ng/mL DON in IPEC-1. After longer incubation (12, 16 and 24 h), this signal was triggered only faintly. The band of the caspase 3 fragment in IPEC-J2 was generally weaker in comparison to IPEC-1. Similarly, a positive control

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Fig. 8. Analysis of ZO-1 in IPEC-1 and IPEC-J2 cells after 48 h of DON treatment. Confluent IPEC-1 and IPEC-J2 cell cultures were incubated with DON (0, 200 and 2000 ng/mL) in complete medium for 48 h. (A) Immunofluorescence staining of ZO-1 protein in fixed cells indicated tight junctions at the cell border. IPEC-1 and IPEC-J2 exhibited characteristic cobblestone (IPEC-1) or elongated (IPEC-J2) structures as visible in untreated control and 200 ng/mL DON. Application of 2000 ng/mL DON disturbed continuous lining of ZO-1 on the cell borders (scale bar: 10 ␮m). (B) Total amount of ZO-1 protein was detected by Western blotting. The blot was reprobed with anti-GAPDH. Expression of ZO-1 after incubation with 2000 ng/mL DON was moderately decreased in comparison to GAPDH.

stimulus (100 ␮mol/L staurosporine for 6 h) resulted in a decreased signal intensity in IPEC-J2 in comparison to IPEC-1. 3.9. Cell cycle analysis by flow cytometry We investigated the cell cycle by nuclear staining using PI and flow cytometry analysis. The relative changes of the cell cycle phases in response to 200 ng/mL and 2000 ng/mL were analyzed after 24 h, 48 h and 72 h (Fig. 10). Incubation for 24 h did not significantly change the ratio of cell cycle phases in both cell lines treated with low or high DON concentrations (200 ng/mL or 2000 ng/mL). After 48 h IPEC-1 exhibited significantly increased percentages of pre-G1, S and G2/M phases and a decrease of the G0/G1 phase in response to 2000 ng/mL. In contrast, only the G0/G1 phase was significantly decreased in the IPEC-J2 cell cycle ratios. Most striking differences in the cell cycle ratio pattern were found after 72 h. IPEC-1 showed a strong increase in the pre-G1 percentage reflect-

ing cell death with partially degraded DNA. This increase was not detected in IPEC-J2. Both cell lines showed a significant decrease in the G0/G1 phase in response to 2000 ng/mL and 72 h incubation. The G2/M phase was significantly increased in both cell lines, however, this enhancement was more pronounced in IPEC-J2. The ratio of cells in the S phase did not significantly rise in any condition tested except for 48 h incubation with 2000 ng/mL in IPEC-1. 4. Discussion 4.1. Cell culture model Analysis of the IPEC-1 and IPEC-J2 cell culture model showed a comparably fast appearance of various differentiation markers. Whereas Caco-2 cells only show a homogenous polarization and differentiation after 30 d of cultivation (Sambuy et al., 2005) both porcine cell lines exhibit an immediate and homogenous appear-

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Fig. 9. Time course of caspase 3 activation of IPEC-1 and IPEC-J2 cells in response to DON. Confluent IPEC-1 and IPEC-J2 cell cultures were incubated with DON (2000 ng/mL) in complete medium for 6–72 h. (A) Caspase 3 activity was quantified by luminescence assay. Significant increases of activity in comparison to corresponding negative control are indicated as *p < 0.05 and **p < 0.01. Bars = ±SEM. (B) Analysis of caspase 3 and cleaved caspase 3 fragment by Western blotting. Staurosporine (100 ␮mol/L) was applied for 6 h as positive control (PC). The blot was reprobed with anti-GAPDH.

ance of tight junction protein ZO-1 after cells reached confluence. This fast differentiation is further documented by the appearance of alkaline phosphatase and the formation of electrically tight cell layers. Both cell lines are derived from native intestinal enterocytes, consequently this fast differentiation could be an inherent enterocytic property as the life time of intestinal enterocytes in vivo is restricted to 3 d (Karam, 1999). 4.2. High concentration range The comparison of our results with data obtained from other experiments exhibit both similar and contradictory results. Effects of DON on cellular viability were measured by various methods in different cell types. A frequently used assay for the estimation of cell death is the measurement of LDH. In our hands, the LDH assay was rather insensitive to DON toxicity. Nevertheless we found a significant release of LDH in IPEC-1 with 2000 ng/mL after 48 h and 72 h and in IPEC-J2 with 2000 ng/mL after 48 h of incubation. However, in explant cultures of weaning piglets concentrations of

15

10 ␮mol/L (3000 ng/mL) and 30 ␮mol/L (9000 ng/mL) DON induced necrosis within 4 h of incubation indicating a higher susceptibility of intact epithelial layer and lamina propria to DON in comparison to enterocytes alone (Kolf-Clauw et al., 2009). Unfortunately results from other epithelial cells are scarce. In RPTECs 48 h treatment with 5 ␮mol/L (1480 ng/mL) DON was sufficient to induce necrotic cell death as measured by LDH release. Normal human lung fibroblasts (NHLF) were obviously resistant to DON-induced necrosis as concentrations up to 100 ␮mol/L (30 ␮g/mL) during 120 h incubation did not result in significant LDH release. However, viability of NHLF tested by MTT analogue WST 8 assay was reduced with 0.1 ␮mol/L (30 ng/mL) DON applied for 48 h (Königs et al., 2007). In contrast, LDH release in hepatocytes occurred only after long incubation times due to a secondary necrosis (Königs et al., 2008). In primary hepatocyte cultures, cell death was observed with 10 ␮g/mL and 100 ␮g/mL DON after 6 h and LDH release was reported after 24 h (Mikami et al., 2004). However, in 3T3 fibroblasts 2500 ng/mL (0.8 ␮mol/L) DON reduced LDH detected viability to 74% of the untreated control within 24 h. For comparison, 50 ng/mL (0.1 ␮mol/L) of T-2 toxin reduced the viability of 3T3 fibroblasts to 40% (Widestrand et al., 1999). Accordingly, DON impairs cellular viability in a cell type-specific manner. This heterogeneity in sensitivity between various cell types and detection methods suggests a differentiated toxic mechanism of DON beyond the well known inhibition of the protein biosynthesis (Kouadio et al., 2005). The MTT assay is a standard method for the detection of metabolic activity and cell viability in cell culture. In Caco-2 cells a 80% reduction of MTT signal was significant at 10 ␮mol/L (3000 ng/mL) DON after 24 h (Kouadio et al., 2005). This result is in accordance to our findings that after a time interval of 24 h and application of 3000 ng/mL toxin the viability of IPEC-1 and IPEC-J2 was reduced by 80% and in IPEC-J2 by 85%, respectively. Other cell types are reported less sensitive to DON application in vitro. Primary porcine hepatocytes were nearly completely resistant after treatment with up to 16 ␮mol/L DON for 72 h (Döll et al., 2009). Similar to our results 24 h application of 2500 ng/mL reduced the MTT measured viability of 3T3 fibroblasts to approximately 70% of the untreated control (Widestrand et al., 1999). The analysis of DNA synthesis by integration of BrdU into the DNA resulted in an EC50 value of 2.2 ␮mol/L for IPEC-1 and 7.15 ␮mol/L for IPEC-J2 (72 h). These values are higher than estimated IC50 values for DON in Caco-2 cells (IC50 = 1.7 ␮mol/L, Kouadio et al., 2005) or 3T3 fibroblasts (IC50 = 0.8 ␮mol/L, Widestrand et al., 1999). In Hep G2 carcinoma cell line and fibroblast-like fetal lung cell line MRC-5 IC50 values between 0.8 ␮mol/L and 3.6 ␮mol/L were found (Ivanova et al., 2006). It has been previously shown that a high concentration of DON affects clearly cell growth by arresting the cell cycle in the G2/M phase (Yang et al., 2008). The data point to a cell cycle arrest in G2/Mphase in IPEC-J2, whereas the increase in pre-G1phase in IPEC-1 favors the apoptotic pathway in this cell line. The molecular mechanism of this cell cycle arrest involves the activation of p38, p53 and p21 (Zhou et al., 2005). Recently, activation and stabilization of p21 independent of p53 has been described (Yang et al., 2008). ZO-1 is a protein found on the cytoplasmic part of the zonula occludens and it is essential for the structural organization of tight junctions (Stevenson et al., 1986). It is an important factor in forming a largely impermeable intestinal barrier. Breakdown of this connection leads to enhanced paracellular permeability not only for nutrients but also for toxins and microorganisms (Turner, 2006). Furthermore, ZO-1 interacts with transcription factors and cell cycle regulators (Paris et al., 2008). The effect of DON on ZO-1 in epithelial cell cultures IPEC-1 and Caco-2 was recently analyzed (Pinton et al., 2009). In contrast to our findings Pinton et al. showed

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IPEC-1 preG1

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Fig. 10. FACS analysis of IPEC-1 and IPEC-J2 cell cycle after 24 h, 48 h and 72 h of DON incubation. Confluent IPEC-1 and IPEC-J2 cell cultures were incubated with DON for 24, 48 and 72 h in complete medium as indicated. Cells were harvested, fixed and stained with propidium iodide (PI). The PI signal was used to separate cells in pre-G1, G0/G1, S or G2/M cell cycle phase. Percentage of gated cells in the respective cell cycle phase was calculated from at least 4 independent experiments ± SEM. Significant changes in ratio in comparison to corresponding control are indicated as *p < 0.05 and **p < 0.01.

that DON concentration of 30 ␮mol/L (9000 ng/mL) did not affect the ZO-1 structure in both cell lines. Our results clearly demonstrated that the ZO-1 structure in IPEC-1 as well as in IPEC-J2 cells was massively disturbed after 48 h of 2000 ng/mL DON treatment. Moreover, immunoblotting suggested a gentle reduction in the ZO1 protein amount which can be a consequence of the ribosomal toxicity of DON. In comparison, the impact of DON was stronger on IPEC-J2 than on IPEC-1 cells. The activation of caspase 3 is an early event (6–8 h) in toxicity of high (2000 ng/mL) DON concentrations in both investigated cell lines, but more pronounced in IPEC-1 than in IPEC-J2. The caspase 3 activity decreased during DON challenge within 24 h to basal level. A significant increase of caspase 3 activity was found again after 48 h and 72 h in the high concentration range of both cell lines. The initial increase of caspase 3 activity is coincident with findings in human colon carcinoma cells (HT-29) challenged with 10 ␮mol/L DON for up to 24 h (Bensassi et al., 2009). Primary porcine hepatocytes exhibited a similar caspase 3 activation pattern after application of 10 ␮mol/L and 100 ␮mol/L DON (Mikami et al., 2004). In a recent review Lewis and McKay (2009) focused on the effect of metabolic stress on the intactness and function of the epithelial barrier. The physiological function of the barrier is dependent on an adequate energy supply predominately by mitochondrial ATP gen-

eration. The consequences of mitochondrial dysfunction include enhanced permeability of the epithelial barrier for, amongst others, bacteria, finally leading to a pathological immune response causing gut injury and inflammation. The toxicological effect of high DON dosage may fit in this scheme as we found indeed degradation of ZO-1 structures which makes an enhanced permeability for bacteria plausible. However, in pig experiments there are no indications of an inflammatory response in the gut despite the presence of characteristic clinical symptoms like vomiting (Goyarts et al., 2005). It is furthermore unlikely that DON concentrations in the intestine are high enough to trigger such an enhanced permeability by mitochondrial dysfunction. So far there are no indications that DON acts on the mitochondrial membrane potential or hampers the ATP generation. This lack of detectable gross toxic effects focused our search on detectable effects of low DON concentrations like the increase in DNA synthesis. 4.3. Low concentration range The effects of DON concentrations above 1000 ng/mL are extensively analyzed and displayed classical toxicological pathways. In the low concentration range, represented in this investigation by 200 ng/mL DON, most of the toxicological relevant mechanisms were absent. Moreover, long term exposure to DON shifted the

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onset of a biphasic proliferative response to a lower concentration. In vivo chronic inoculation of low DON dose affects growth and performance in pigs (Goyarts et al., 2005). In addition, the BrdU incorporation assay indicated a proliferative effect of low DON dosage in our study. Whereas the inhibitory effects of DON on DNA synthesis is well documented in various cells, indications for an enhanced DNA synthesis activity are scarce. In vivo, a potential proliferative effect was found in young piglets. A thickening of the esophageal region of the stomach was observed in response to DON diet (Rotter et al., 1994). In PHA-induced lymphocyte proliferation, DON concentrations up to 0.5 ng/mL enhanced proliferation whereas concentrations between 50 ng/mL and 100 ng/mL decreased proliferation (Miller and Atkinson, 1986). It has been early suspected that in vivo effects of trichothecenes on the immune system may vary according to the level of exposure (Miller and Atkinson, 1986). Similarly, it was found that DON in the range of 10 ng/mL can enhance the protein biosynthesis in EL-4 thymoma T cells (Dong et al., 1994). However, the mechanism of this proliferative effect of DON in epithelial cells remains to be elucidated. Concentration dependent effects on intestinal cells have been reported for the soy bean component genistein (Chen and Donovan, 2004). It is notable that genistein absorption is virtually as fast as DON uptake (Prelusky et al., 1988). Similar to our observations using DON, two distinct concentration ranges were found which triggered opposing effects in cell proliferation. Genistein concentrations around 3.7 ␮mol/L increased DNA synthesis rate whereas concentrations of 26 ␮mol/L or higher decreased the rate measured by [3H]-thymidine incorporation. In conclusion, DON affected the IPEC-1 and IPEC-J2 cell culture models of porcine enterocytes in a dose- and time-dependent manner. Two distinct concentration ranges were identified: high concentrations (2000 ng/mL) induce toxic effects whereas low concentrations (200 ng/mL) have modulatory effects on cellular regulation. More in detail, high concentrations of DON exhibit features of typical toxicity finally compromising the intestinal barrier integrity as illustrated by disturbance of tight junction structure. Low concentrations of DON did not exhibit toxic properties, however, an unexpected proliferative effect was detected suggesting a distinct mechanism. To our knowledge, this is the first time that DON mediated proliferation in intestinal epithelial cells has been shown. Further investigations are necessary to analyze this intriguing modulatory function of low DON dosage in the intestine. Conflict of interest No conflicts of interest. Acknowledgments We thank Brigitte Ketzler, Anke Schmidt and Sandra Vorwerk, Institute of Anatomy, Otto-von-Guericke University, Magdeburg for technical assistance in cell culture, protein analyses and morphological techniques. We also thank Dr. Roland Hartig, Institute of Molecular und Clinical Immunology, Otto-von-Guericke University Magdeburg, for his help in FACS analysis. Financial support: Supported by DFG RO 743/3-2 and DA 558/1-3. Authors’ disclosure: A-K. Diesing, C. Nossol, P. Panther, N. Walk, A. Post, J. Kluess, P. Kreutzmann, S. Dänicke, H.-J. Rothkötter, and S. Kahlert no conflicts of interest. References Bensassi, F., Golli-Bennour, E., Abid-Essefi, S., Bouaziz, C., Hajlaoui, M.R., Bacha, H., 2009. Pathway of deoxynivalenol-induced apoptosis in human colon carcinoma cells. Toxicology 264, 104–109.

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