5.3.1. Toxicological Analysis of Intestinal Tissues Tissues Samples

Five prepubertal gilts from every group were euthanized on analytical date 1 (D1—exposure day 7), date 2 (D2—exposure day 21), and date 3 (D3—exposure day 42) by intravenous administration of pentobarbital sodium (Fatro, Ozzano Emilia BO, Italy) and bleeding. Immediately after cardiac arrest, tissue samples (approximately 1 × 1.5 cm) were collected from entire intestinal cross-sections, from the following segments of the gastrointestinal tract: duodenum—third part; jejunum and ileum—middle part; cecum—1 cm from the ileocecal valve; colon—middle part of the centrifugal gyri of the ascending colon and centripetal gyri of the ascending colon (ascending colon), transverse colon, and descending colon. The samples were rinsed with phosphate buffer and prepared for analyses. The collected samples were stored at a temperature of −20 ◦C.

#### Extraction Procedure

The presence of ZEN, α-ZEL, and β-ZEL in tissue samples was determined with the use of immunoaffinity columns. Tissue samples were transferred to centrifuge tubes and homogenized with 7 mL of methanol (99.8%) for 4 min. The tubes were vortexed 4 times at 5 min intervals, after which they were centrifuged at 5000 rpm for 15 min. Samples of 5 mL were collected from the suspension and combined with 20 mL of deionized water, and 12.5 mL of the resulting solution was used to extract ZEN. The supernatant was carefully collected and passed through immunoaffinity columns (Zearala-TestTM Zearalenone Testing System, G1012, VICAM, Watertown, MA, USA) at a rate of 1–2 drops per second. The immunoaffinity bed in the column was subsequently washed with demineralized water (Millipore Water Purification System, Millipore S.A., Molsheim, France). Isocratic elution was performed with 99.8% methanol (LIChrosolvTM, No. 1.06 007, Merck-Hitachi, Germany) to remove the bound mycotoxin. After extraction, the eluents were placed in a water bath at a temperature of 50 ◦C and were evaporated in a stream of nitrogen. Dry residues were stored at −20 ◦C until chromatographic analysis. Next, 0.5 mL of 99.8% acetonitrile (ACN) was added to dry residues to dissolve the mycotoxin. The process was monitored with the use of internal standards (Cayman Chemical 1180 East Ellsworth Road Ann Arbor, Michigan 48108 USA, ZEN-catalog number 11353; Batch 0593470-1; *a*-ZEN-catalog number 16549; Batch 0585633-2; *β*-ZEN-catalog number 19460; Batch 0604066-7).

#### Chromatographic Quantification of ZEN and Its Metabolites

Zearalenone and its metabolites were quantified at the Institute of Dairy Industry Innovation in Mr ˛agowo, Poland. The biological activity of ZEN, α-ZEL, and β-ZEL in the bone marrow microenvironment was determined by combined separation methods involving immunoaffinity columns (Zearala-TestTM Zearalenone Testing System, G1012, VICAM, Watertown, MA, USA), Agilent 1260 liquid chromatography (LC) system, and a mass spectrometry system (MS, Agilent 6470). Samples were analyzed on a chromatographic column (Atlantis T3, 3 µm 3.0 × 150 mm, column No. 186003723, Waters, AN Etten-Leur, Ireland). The mobile phase was composed of 70% acetonitrile (LiChrosolvTM, No. 984730109, Merck-Hitachi, Mannheim, Germany), 20% methanol (LiChrosolvTM, No. 1.06 007, Merck-Hitachi, Mannheim, Germany), and 10% deionized water (Milipore-Water Purification System, Millipore S.A. Molsheim-France) with the addition of 2 mL of acetic acid per 1 L of the mixture. The flow rate was 0.4 mL/min., and the temperature of the oven column was 40 ◦C. The chromatographic analysis was completed in 4 min. The column was flushed with 99.8% methanol (LIChrosolvTM, No. 1.06 007, Merck-Hitachi, Mannheim, Germany) to remove the bound mycotoxin. The flow rate was 0.4 mL/min., and the temperature of the oven column was 40 ◦C. The chromatographic analysis was completed in 4 min.

Mycotoxin concentrations were determined with an external standard and expressed in ppb (ng/mL). Matrix-matched calibration standards were applied in the quantification process to eliminate matrix effects that can decrease sensitivity. Calibration standards were dissolved in matrix samples based on the procedure that was used to prepare the remaining samples. The material for calibration standards was free of mycotoxins. The limits of detection (LOD) for ZEN, α-ZEL, and β-ZEL were determined as the concentration at which the signal-to-noise ratio decreased to 3. The concentrations of ZEN, α-ZEL, and β-ZEL were determined in each group and on three analytical dates (see Table 1).

#### Mass Spectrometric Conditions

The mass spectrometer was operated with ESI in the negative ion mode. The MS/MS parameters were optimized for each compound. The linearity was tested by a calibration curve including six levels. Table 5 shows the optimized analysis conditions for the mycotoxins tested.


**Table 5.** Optimized conditions for mycotoxins tested [71].

#### Carry-Over Factor

Carry-over toxicity occurs when the body is able to survive under the influence of low doses of mycotoxins. Mycotoxins may impair the functions of tissues or organs [72] and modify their biological activity [5,7]. CF was determined in the intestinal tissues when the daily dose of ZEN (5 µg ZEN/kg BW, 10 µg ZEN/kg BW or 15 µg ZEN/kg BW) administered to each animal was equivalent to 560–32251.5 µg ZEN/kg complete diet, depending on the daily feed intake. The concentrations of mycotoxins in the tissues were expressed as the dry matter content of the samples.

The CF was calculated as follows:

CF = toxin concentration in tissue [ng/g]/toxin concentration in diet [ng/g]

#### Statistical Analysis

The data were statistically processed at the Department of Discrete Mathematics and Theoretical Computer Science, Faculty of Mathematics and Computer Science, University of Warmia and Mazury in Olsztyn. The bioavailability of ZEN and its metabolites in gut tissues was analyzed in group C and three experimental groups on three analytical dates. Results are expressed as means (±) with standard deviation (SD). The following parameters were analyzed: (i) the differences in the mean values for the various doses of ZEN (experimental groups) and the control group on the three analytical dates, and (ii) the differences in the mean values for the individual doses (groups) of ZEN at the three dates. Differences between mean values were determined using one-way ANOVA. If there were significant differences between groups, the differences between the pairs of means were determined using Tukey's multiple comparison test. If all values were below the LOD (mean and variance equal to zero) in either group, values in the remaining groups were analyzed by one-way ANOVA (if the number of remaining groups was greater than two), and the means of these groups were compared with zero on Student's t-test. The differences between the groups were determined by Student's t-test. The results were considered to be highly significant at *p* <0.01 (\*\*) and significant at 0.01 < *p* <0.05 (\*). The data were statistically processed using Statistica v.13 software (TIBCO Software Inc., Silicon Valley, CA, USA, 2017). Dose–response relationships were established using Pearson's correlation

analysis. Differences were considered significant at *p* ≤ 0.05. Results are presented as the means ± standard error of the mean (SEM).

#### *5.4. Expression of CYP1A1 and GSTπ1*

#### 5.4.1. Sampling and Storage for RNA Extraction

Immediately after cardiac arrest, tissue samples were collected from the mid-ascending and descending colon. The samples were stored in RNAlater (Sigma-Aldrich; Taufkirchen, Germany) according to the manufacturer's instructions. Tissue samples were collected at the same time.

#### 5.4.2. Complete RNA Extraction and cDNA Synthesis

Total RNA was extracted from RNAlater-preserved tissues (approx. 20 mg per sample; *n* = 5 in each treatment group) using the Total RNA Mini isolation kit (A&A Biotechnology; Gdansk, Poland) according to the manufacturer's protocol. RNA samples were incubated with RNase-free DNase I (Roche Diagnostics; Mannheim, Germany) to prevent contamination of genomic DNA. The overall RNA quality and purity of all samples were assessed with a BioPhotometer (Eppendorf; Hamburg, Germany), and the results were used for cDNA synthesis with the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas; Burlington, Canada). The cDNA synthesis reaction mixture for each sample contained 1 µg of total RNA and 0.5 µg of oligo (dT) primer, and the reaction was performed according to the manufacturer's protocol. The first synthesized cDNA strand was stored at −20 ◦C until further analysis.

#### 5.4.3. qPCR

Real-time PCR primers for the CYP1A1 and GSTπ1 mRNAs were designed using the Primer-BLAST tool based on the reference species (Table 6). The real-time PCR test was performed on an ABI 7500 Real-time PCR system thermalcycler (Applied Biosystems; USA) in singleplex mode. Subsequent treatments were applied in accordance with the producents' recommendations.


**Table 6.** Real-time PCR primers for the proposed study.

The quantitative cycle (Cq) values of qPCR were converted to copy number using a standard curve plot (Cq vs. log copy number) according to the methodology developed by [74] and described by Spachmo and Arukwe [75].

The rationale for the use of the standard curve is based on the assumption that the unknown samples have an equal amplification efficiency (usually above 90%), which is checked before extrapolating the unknown standards to the standard curve [75]. To generate standard curves, the purified PCR products of each mRNA were used to prepare a series of 6 10-fold dilutions with known copy number amounts that were used as templates in real-time PCR. The Cq values obtained for each dilution series were plotted against the log copy number and used to extrapolate the unknown samples to the copy number. The mRNA copy numbers of the samples collected from all experimental groups in each exposure date were divided by the averaged numbers from the C group, determined at the beginning of the experiment (control 0d), to obtain relative expression values, which were presented as the expression ratio (R).
