1. 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.

Wheat germ contains several bioactive ingredients, such

as flavonoids, dietary fibres, as well as lignins, oligosaccharides, and vitamins [1]. Hidvegi et al. [2] demonstrated that

wheat germ is rich in the glycosylated form of 2,6-

dimethoxy-p-benzoquinone (DMBQ). The conversion of

DMBQ into its biologically more active forms requires β-glucosidase enzyme [3]. Wheat germ is fermented by Saccharomyces cerevisiae yeasts [4] or treated with Lactobacillus

plantarum dy-1 [5]. Fermented wheat germ extract (FWGE)

is available in both human (Avemar®) and veterinary (Immunovet®) medicine. These products are aqueous extractions,

which are fermented by Saccharomyces cerevisiae, and they

contain several biologically active molecules [4, 6]. FWGE

is applied as an adjuvant in human cancer therapy, because

benzoquinones have antimetastatic [2], antimetabolic [6],

antiangiogenic [7], and antiproliferative properties and are

able to induce apoptosis [5, 8]. Furthermore, FWGE can

enhance the cellular immune response [4, 9] and has an antioxidant effect [2].

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.

2.3. Experimental Design and Cell Treatments. To investigate

the cell viability, the seeding density for the cells was 1 × 104

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, H2O2 production, and intracellular ROS

levels, the cell-seeding density was 1:5 × 105 cells/well in a

6-well polyester membrane insert (4.67 cm2

) 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

H2O2 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

× 104 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% CO2. 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).

2.5. 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 × 106 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).

2.6. Detection of Changes in the Extracellular H2O2

Concentrations. Extracellular H2O2 production was monitored in IPEC-J2 cells by using the Amplex Red Hydrogen

Peroxide Assay Kit (Invitrogen, Thermo Fisher Scientific,

Waltham, MA, USA). The Amplex Red reagent reacts with

H2O2 (in 1 : 1 stoichiometry) to produce a red fluorescent

product called resorufin in the presence of horseradish peroxidase. After 24 and 48 h incubation time, 50 μL of the

cell-free supernatants was collected from the basolateral

compartments and was mixed with the Amplex Red working

solution according to the manufacturer’s instructions. The

fluorescence intensity was measured at 590 nm with a fluorometer using 530 nm excitation wavelength (Victor X2

2030, Perkin Elmer, Waltham, MA, USA).

2.7. Assessing the Changes in Intracellular ROS Levels. Measurement of disruptions in the intracellular redox state of

IPEC-J2 cells was carried out using DCFH-DA dye (SigmaAldrich, Budapest, Hungary). DCFH-DA is oxidized to the

highly fluorescent form of dichlorofluorescein (DCF) by the

intracellular ROS [20]. Following a centrifugation process

for 10 min at 10 000 rpm at 5°

C, 100 μL of cell-free supernatant was collected and pipetted into a 96-well plate. Samples

of supernatant were collected at 24 and 48 h after treatments.

The fluorescence intensities of the supernatants were measured at 530 nm with a fluorometer using 485 nm excitation

wavelength (Victor X2 2030, Perkin Elmer, Waltham, MA,

USA).

2.8. Statistical Analysis. The statistical analysis of the results

was performed by using R Core Team (version of 2016)

[21]. Differences between groups were analyzed by one-way

ANOVA coupled with the post hoc Tukey test for multiple comparisons. ∗p < 0:05, ∗∗p < 0:01, and ∗∗∗p < 0:001 were

considered to be statistically significant.