The Impact of Fermented Wheat Germ Extract on Porcine Epithelial Cell Line Exposed to Deoxynivalenol and T-2 Mycotoxins

The effect of fermented wheat germ extract (FWGE) (Immunovet®) was evaluated with cotreatments with deoxynivalenol (DON) and T-2 toxin (T-2). These mycotoxins are produced by Fusarium mold species. The effects of FWGE on IPEC-J2 with DON and T-2 have not been studied until now. The IPEC-J2 porcine, nontumorigenic cell line was selected to investigate the outcome of the individually and simultaneously added compounds, as it has in vivo-like properties. The cells were treated for 24 h with the selected solutions; then, the IPEC-J2 cells were allowed to regenerate in a culture medium for an additional 24 h. In our results, DON and T-2 significantly increased the adverse impacts on cell viability and integrity of the cell monolayer. To elucidate the extent of oxidative stress, extracellular H2O2 concentrations and intracellular reactive oxygen species (ROS) were measured. FWGE appeared to be beneficial to IPEC-J2 cells given the separately and significantly decreased ROS levels. 1% and 2% FWGE could significantly reduce mycotoxin-induced oxidative stress. In conclusion, the results demonstrate that FWGE exerted protective effects to counteract the oxidative stress-provoking properties of applied fusariotoxins in the nontumorigenic IPEC-J2 cell line.


Introduction
The number of studies involving natural products and dietary supplements has shown rapid growth recently. Natural products contain extensive chemical diversity, which makes it difficult to replace the collection of naturally occurring molecules with synthetized drugs.
It is of key importance worldwide to produce good quality feedstuff for livestock with the least amount of mycotoxin contaminants. Fusarium fungi are abundant in temperate climate zones and contaminate wheat and other cereals. This genus is capable of producing a wide variety of mycotoxins. One of these groups is the trichothecene mycotoxins, including deoxynivalenol (DON), nivalenol (NIV), T-2 (T-2) toxin, and HT-2 toxin [10,11]. In general, these fusariotoxins generate reactive oxygen species (ROS) and interfere with the normal functions of mitochondria, which can lead to apoptosis. They also inhibit protein synthesis in eukaryotic cells [12], especially in epithelial and immune cells, where the rate of cell replication is high [13,14]. Among farm animals, swine is the most sensitive species to fusariotoxin contamination; side effects are decreased feed intake, feed refusal, and vomiting [15].
The most frequently used cell line for oxidative stressrelated studies are intestinal porcine epithelial cell line-1 (IPEC-1) and intestinal porcine epithelial cell line 2 (IPEC-J2), which are suitable for in vitro modelling of nontumorigenic epithelium. There are only few publications regarding IPEC-J2 cells treated with both DON and T-2 toxin. In these studies, transepithelial electrical resistance (TEER) decreased while cellular permeability was enhanced in parallel [16] by these toxic substances. Both DON and T-2 increase the ROS level intracellularly [17,18].
The main objective of this study was to describe the beneficial effects of fermented wheat germ extract on the IPEC-J2 cell line induced with DON and T-2 mycotoxins. This study was focused on TEER and two oxidative stress markers: extracellular H 2 O 2 and intracellular ROS productions.

Materials and Methods
2.1. Reagents. DON and T-2 were purchased from Merck (Darmstadt, Germany). Acetonitrile was obtained from Thermo Fisher Scientific (Waltham, MA, USA). The final concentration of acetonitrile in the cell culture medium was <0.5% (v/v). FWGE was diluted from a commercial product in powder form (Immunovet Pets, Immunovet Ltd., Hungary).
Prior to the experiments, cell viability studies were performed to select the working concentrations to DON, T-2, and FWGE (data not shown). 8 μmol/L DON, 5 nmol/L T-2, and 1% and 2% FWGE concentrations were chosen from these results for further investigations ( Figure 1).

Cell and Culturing
Conditions. The porcine intestinal epithelial cell line IPEC-J2 (ACC 701) is nontumorigenic, intestinal columnar epithelial cells, which were isolated from neonatal piglet midjejunum. IPEC-J2 closely mimics in vivo pig and human physiology, which makes it a good model to study foodborne and plant-derived components.

Experimental Design and Cell Treatments.
To investigate the cell viability, the seeding density for the cells was 1 × 10 4 cells/well of a 96-well plate (Transwell, Sigma-Aldrich, Corning Costar, Merck, Darmstadt, Germany). The cells were treated the next day after reaching a confluent state. When studying TEER, H 2 O 2 production, and intracellular ROS levels, the cell-seeding density was 1:5 × 10 5 cells/well in a 6-well polyester membrane insert (4.67 cm 2 ) containing plates (Transwell, Sigma-Aldrich, Corning Costar, Merck, Darmstadt, Germany). These inserts were useful for the apically and basolaterally added treatments and for transepithelial electrical resistance measurements.
The stock solutions were freshly made with phenol redfree DMEM/F12 (Merck, Darmstadt, Germany). The DON and T-2 were diluted with acetonitrile (final concentration: <0.5% (v/v)); then, the following concentrations were made: 8 μmol/L DON, 5 nmol/L T-2, and 1 and 2% final concentrations of FWGE. Cell cultures were exposed to the treatments for an incubation time of 24 hours; then, the IPEC-J2 cells were treated only with phenol red-free DMEM:F12 culture medium for an additional 24 h as regeneration. After the treatment and the regeneration, the TEER was measured, the cell-free supernatants were collected for extracellular H 2 O 2 determination, and the DCFH-DA assay was added to the cells.
2.4. Evaluation of Cell Viability. Cytotoxicity was examined with an MTS reagent (CellTiter96 Aqueous One Solution, Promega, Bioscience, Budapest, Hungary) [19]. This test measures the rate of viable cells by determining the soluble tetrazolium salt conversion in the metabolically active cells to a coloured formazan product with the advantage over MTT that the solubilization step is not required for avoiding formazan precipitation in the aqueous medium.
IPEC-J2 cells were seeded in 96-well culture plates at 2 × 10 4 cells/well and allowed 24 hours to reach confluence. Mycotoxin and FWGE solutions were added to the cells using a multichannel pipette and were incubated for 24 h at 37°C, 5% CO 2 . After the incubation time, the treatments were removed, and each well received 100 μL of fresh phenol redfree medium containing 20 μL of MTS solution. After an incubation time of 1 h at 37°C, the absorbance values were measured at 490 nm using an ELISA Plate Reader (EZ Read Biochrom 400, Cambridge, UK).

Determination of Cell Membrane
Integrity. The integrity of the IPEC-J2 cell monolayer can be followed by measuring transepithelial electrical resistance (TEER) between the apical and basolateral compartments of the IPEC-J2 cells ( Figure 2). Cells were seeded to 6-well Transwell insert containing plates (polyester, 0.4 μm pore size, Corning, Merck, Darmstadt, Germany), and the seeding density was 3 × 10 6 cells/well. After the cells reached a confluent state, the barrier function was evaluated by measuring with an EVOM Epithelial Tissue Volt/Ohmmeter (World Precision Instruments, Berlin, Germany). 10 days after 2 Oxidative Medicine and Cellular Longevity seeding, the IPEC-J2 monolayer achieved the 600 Ω/well values. The results were calculated as kΩ × cm 2 by multiplying the values by the surface area of the monolayer (4.67 cm 2 ). The high TEER value of IPEC-J2 monolayers grown on Transwell polyester filters demonstrates the functional integrity of the continuous cell association, acting as a single-layer tight physical barrier.  [21]. Differences between groups were analyzed by one-way ANOVA coupled with the post hoc Tukey test for multiple  Oxidative Medicine and Cellular Longevity comparisons. * p < 0:05, * * p < 0:01, and * * * p < 0:001 were considered to be statistically significant.

Discussion
According to Hernández et al. [22], the wheat germ extract's main active components include DMBQ, hydroxybenzoic acids, hydroxycinnamic acids, and apigenin. These naturally occurring compounds are glycosylated and physiologically not active [23]. There were only few experiments treating livestock animals with FWGE (Immunovet®). Three-weekold chickens were infected with Mycoplasma gallisepticum and treated with FWGE, and poultry remained clinically healthy [24]. In another research, FWGE was beneficial for the maintenance of general health conditions including 4 Oxidative Medicine and Cellular Longevity biochemical and physiological parameters, increasing weight gain, and improved immune response to vaccination [25]. In growing pigs, FWGE enhanced weight gain and had a beneficial effect on cellular immunity. This effect is instrumental in promoting resistance against facultative pathogens [9]. As reported by Jerzsele et al. [26], 2% FWGE helped broiler chickens to gain greater body weight than the control group. It was also established that animals infected with Salmonella Typhimurium under controlled conditions and obtained FWGE treatments were not spreading the pathogens to other chickens. A prominent issue in feed production is mycotoxin contamination. As major contaminants of cereals, DON and T-2 have been implicated in various gastrointestinal problems in farm animals, such as vomiting, feed refusal, diarrhoea [27], and oesophageal perforation as well as malabsorption [28]. DON can also be present in glycosylated form (for example, as DON-3-β-d-glycoside) in plants, which increases the  5 Oxidative Medicine and Cellular Longevity toxicological effect of DON after consumption [29]. Before 2006, low-dose antibiotics were used to help the growth promotion of farm animals. These additional antibiotics were particularly not effective against mycotoxins but were beneficial to the general health status. Fortunately, numerous recent studies have been conducted in order to find a naturally occurring or plant-based solution to reduce the negative effects of mycotoxins and support the condition of farm animals. FWGE has several beneficial properties, such as its anti-oxidant effect [2], which can make this extract useful against oxidative stress generated by the two most common mycotoxins. Although mycotoxins negatively affect all farm animals, the swine is particularly sensitive to it.
The gastrointestinal epithelium is the first barrier for mycotoxin-contaminated feed. There are several in vitro studies regarding the impact of mycotoxins on epithelial cell metabolism, toxicity, and barrier integrity. Both DON and T-2 have demonstrated time-and concentration-dependent  Oxidative Medicine and Cellular Longevity cytotoxicity. Szakács et al. [30] established that FWGE boosted the immune responses compared to T-2-treated weaned pigs. The IC 50 for DON and T-2 was 23.52 μmol/L and 20.4 nmol/L for 72 hours of incubation time on IPEC-J2 [16]. In this study, DON was added at 8 μmol/L and T-2 at 5 nmol/L concentrations for 24 hours on IPEC-J2. Both DON and T-2 were shown to decrease the metabolic activity of the cells significantly. This result is in good correlation with that in the study by Sergent et al. [31]. The IC 50 of DON was determined on the Caco-2 cell line at 2.22 μmol/L, but a 0.67 μmol/L concentration of DON inhibited the proliferation of cells for 48 hours [31]. In our studies, both 1% and 2% of FWGE increased cell viability compared to control cells. In the literature, FWGE was mostly examined on tumor cell lines where FWGE induced apoptosis and cell death in several cases [32]. 1 or 2% of FWGE did not preserve cell viability when 8 μmol/L DON was added to the cells. In contrast, 1% and 2% FWGE enhanced the survival of IPEC-J2 cells treated simultaneously with T-2 compared to cells treated only with T-2.
IPEC-J2 cells can polarize and form a strong barrier through the development of tight junctions between cells [33]. The intercellular tight junction is the rate-limiting barrier in the paracellular pathway for permeation by ions and larger solutes. The TEER of cell monolayers can be considered a good indicator of the degree of organization of the tight junctions within the cell monolayer as well as that of epithelial integrity [34].
The authors did not find earlier results of studies using TEER to detect the effects of FWGE on the integrity of nontumorigenic intestinal cell monolayers exposed to fusariotoxins. On the other hand, TEER utilization in mycotoxin research has a more extensive representation in the literature. As reported by Goossens et al. [16] and Kang et al. [35], DON treatments significantly reduce the TEER values, depending on the dosage. Goossens et al. [16] also found that T-2 up to 210 nmol/L concentration for 72 h significantly lowered the integrity of the IPEC-J2 monolayer. Springler et al. [36] confirmed that DON reduced TEER significantly at 5-20 μmol/L after 24 h incubation. Our study found that 8 μmol/L DON and 5 nmol/L T-2 significantly reduced the TEER values during and after the treatments, while 1% and 2% FWGE alone significantly increased them. Based on our findings, 1% FWGE cotreatment with mycotoxins did not elevate the TEER, while 2% FWGE added for 24 h with 5 nmol/L T-2 helped the cells reach a higher TEER value.
Oxidative stress develops if concentrations of ROS exceed the antioxidant capacity of living entities. ROS are reactive species of radicals with a single unpaired electron, such as superoxide anion radical (O 2− ) and the hydroxyl radical (OH − ), along with nonradical ROS such as hydrogen peroxide (H 2 O 2 ). Sharply increasing intracellular ROS can cause oxidative stress with irreversible cell damage [37]. ROS can initiate the process of lipid peroxidation in the lipid membrane causing damage to the cell membrane's phospholipids and lipoproteins, and it can also damage DNA by propagating a chain reaction [38]. Moreover, oxidative stress mediated by ROS may increase cell apoptosis [39]. To counterbalance the prooxidant agents, the cells have intracellular nonenzymatic and enzymatic antioxidants, namely, tripeptide glutathione [40] and catalase, which can protect them from oxidative damage [41].
We examined oxidative stress by measuring extracellular H 2 O 2 production and intracellular ROS generation in IPEC-J2 cells. We found that both 8 μmol/L DON and 5 nmol/L T-2 significantly increased intracellular ROS levels during the 24 h treatment and after the 24 h regeneration. This is in agreement with the findings of Kang et al. [35] who published that DON at 6.7μmol/L in IPEC-J2 cells significantly elevated intracellular ROS levels after 24 h of mycotoxin exposure. Both 1% and 2% FWGE significantly decreased the ROS after a 24 h treatment. These findings are in good correlation with Karancsi et al. [42], who elucidated firstly beneficial effects of FWGE in case of LPS-evoked oxidative damage. FWGE could decrease excessive intracellular ROS levels after LPS administration and exerted protective effect on the integrity of the IPEC-J2 cell monolayer exposed to LPS treatment. Their study also showed that FWGE in different concentrations (1%, 2%, and 4%) did not affect cell death; moreover, FWGE in 2% concentration improved cell viability significantly after 24 h treatment. These results are seemingly contradictory to Otto et al. [6] who published that 24 μmol/L DMBQ from FWGE inhibits cell cycle progress, induce apoptosis, and increase intracellular DCF fluorescence after a 24 h treatment in nine human cancer cell lines. Hidvegi et al. [2] resolved this contraversion by clarifying firstly that the effects of FWGE are not solely attributable to benzoquinones and secondly that IPEC-J2 is a nontumorigenic cell line.
When treated simultaneously, both 1% and 2% FWGE significantly decreased the DON-and T-2-mediated ROS levels. An interesting phenomenon was detected in terms of 2% FWGE as the extracellular H 2 O 2 significantly decreased during the 24 h treatment; however, IPEC-J2 cells produced significantly higher H 2 O 2 at the end of the regeneration period when cells were previously exposed to DON or T-2 toxins for 24 h.

Conclusions
In conclusion, 1% and 2% FWGE has favourable properties on IPEC-J2 cell lines as FWGE helps the cells to proliferate. 2% FWGE is a beneficial agent against intracellular ROS when treated with DON and T-2 simultaneously. To our knowledge, this is the first published report of FWGE cotreated with DON or T-2 in which TEER was utilized to determine the impact of FWGE on the integrity of the cell monolayer.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.