2.3.2. Biotransformation of ZEN Tissue Samples

In each group, 5 animals will be euthanized on analytical dates 1 (D1—exposure day 7), 2 (D2—exposure day 21), and 3 (D3—exposure day 42) by the intravenous administration of pentobarbital sodium (Fatro, Ozzano Emilia BO, Italy) and bleeding. Immediately after cardiac arrest, tissue samples (approximately 1 × 1.5 cm) will be collected from entire intestinal cross-sections, from the following segments of the gastrointestinal tract: the duodenum—the first part (duodenal cap) and the horizontal or third part; the jejunum and ileum—middle parts; colon—middle parts of the ascending colon, transverse colon, and descending colon; the cecum—1 cm from the ileocecal valve. The samples will be rinsed with phosphate buffer and prepared for analyses [5,6].

#### Extraction and Purification

Zearalenone, α-ZEL, and β-ZEL will be extracted from tissues with the use of immunoaffinity columns (Zearala-TestTM Zearalenone Testing System, G1012, VICAM, Watertown, MA, USA) according to the manufacturer's recommendations. The obtained eluents will be placed in a water bath at 50 ◦C, and will be evaporated in a stream of nitrogen. The dry residue will be stored at –20 ◦C until chromatographic analysis. The procedure will be monitored with the use of internal standards, and the results will be validated by mass spectrometry.

Chromatographic Determination of the Concentrations of Zen and its Metabolites

The tissue concentrations of ZEN, α-ZEL, and β-ZEL will be determined with the Agilent 1260 liquid chromatograph (LC) and the Agilent 6470 mass spectrometer (MS). The prepared samples will be analyzed with the use of the Zorbax rapid resolution chromatographic column (2.1 × 50 mm; 1.8 micron Agilent Eclipse Plus C18) in gradient mode. The mobile phase will contain 0.1% (*v*/*v*) formic acid in water (solvent A) and 0.1% (*v*/*v*) formic acid in acetonitrile (solvent B). Gradient conditions will be as follows: initially, 20% B that increases to 100% B in 4.0 min and back to 20% B in 0.1 min.

Mycotoxin concentrations will be determined according to an external standard and will be expressed in ppb (ng/mL). The quantification process will involve matrix-matched calibration standards to eliminate matrix effects that can decrease sensitivity. Calibration standards will be dissolved in matrix samples based on the procedure described for the remaining samples. The material for preparing calibration standards will be free of mycotoxins. A signal-to-noise ratio of 3:1 will be used to estimate the limits of detection (LOD) for ZEN, α-ZEL, and β-ZEL. The LOQ will be estimated as the triple LOD value.

The specificity of the method will be determined by comparing the chromatograms of a blank sample with those corresponding to a spiked tissue sample.

Mass Spectrometric Conditions

The mass spectrometer was operate with ESI in the negative ion mode. The MS/MS parameters were opimized for each compoud. The linearity was tested by a calibration curve including six levels. Table 2 shows the optimized analysis conditions for the mycotoxins tested.


**Table 2.** Optimaized conditions for mycotoxins tested.

A chromatogram of standard mixtures of all analytes is presented in Figure 1.

**Analyte Precursor** 

**(m/z)** 

**Production (m/z)** 

**Table 2.** Optimaized conditions for mycotoxins tested.

*α*-ZEL 319.2 275.2 160.1 144 21 33 0.3 0.9 0.997 *β*-ZEL 319.2 275.2 160.1 144 21 33 0.3 1 0.993 A chromatogram of standard mixtures of all analytes is presented in Figure 1.

**Collision Energy (eV)** 

**LOD (ng mL-1)**

**LOQ (ng mL-1)**  **Linearity (%R2)**

**Fragmentor Voltage (V)** 

**Figure 1.** The chromatogram of standard solution. **Figure 1.** The chromatogram of standard solution.

#### 2.3.2.5. Statistical Analysis Statistical Analysis

The results of the study will be processed at the Department of Discrete Mathematics and Theoretical Computer Science at the Faculty of Mathematics and Computer Science of the University of Warmia and Mazury in Olsztyn. The bioavailability of ZEN and its metabolites in the intestinal tissues of pre-pubertal gilts will be analyzed in three experimental groups and the control group, on different sampling dates. The results will be expressed as mean values ( *x* ) and standard deviation (SD). The following tests will be carried out: (i) analyses of differences between the mean values in three experimental groups (receiving different doses of ZEN) and the control group on three analytical dates; (ii) analyses of differences between the mean values within groups (receiving the same ZEN dose) on each analytical date. In both tests, differences between mean values will be determined by one-way ANOVA. If the differences between groups are statistically significant, differences between pairs of means will be estimated by Tukey's multiple comparison test. If all values are below LOD (mean and variance are equal to zero) in any group, the values in the remaining groups will be processed by one-way ANOVA, and the differences between means in these groups will be compared with the population mean difference of zero in Student's t-test. Differences between groups will be estimated by Student's t-test. The results of each analysis will be considered to be highly significant at *p* < 0.01 (\*\*) and significant at 0.01 < *p* < 0.05 (\*). Data will be analyzed in the Statistica v.13 program (TIBCO Software Inc., Silicon Valley, CA, USA, 2017). The results of the study will be processed at the Department of Discrete Mathematics and Theoretical Computer Science at the Faculty of Mathematics and Computer Science of the University of Warmia and Mazury in Olsztyn. The bioavailability of ZEN and its metabolites in the intestinal tissues of pre-pubertal gilts will be analyzed in three experimental groups and the control group, on different sampling dates. The results will be expressed as mean values (*x*) and standard deviation (SD). The following tests will be carried out: (i) analyses of differences between the mean values in three experimental groups (receiving different doses of ZEN) and the control group on three analytical dates; (ii) analyses of differences between the mean values within groups (receiving the same ZEN dose) on each analytical date. In both tests, differences between mean values will be determined by one-way ANOVA. If the differences between groups are statistically significant, differences between pairs of means will be estimated by Tukey's multiple comparison test. If all values are below LOD (mean and variance are equal to zero) in any group, the values in the remaining groups will be processed by one-way ANOVA, and the differences between means in these groups will be compared with the population mean difference of zero in Student's t-test. Differences between groups will be estimated by Student's t-test. The results of each analysis will be considered to be highly significant at *p* < 0.01 (\*\*) and significant at 0.01 < *p* < 0.05 (\*). Data will be analyzed in the Statistica v.13 program (TIBCO Software Inc., Silicon Valley, CA, USA, 2017).

### *2.4. Expression of ERα, ERβ, CYP1A1, and GSTP1 Genes*

#### 2.4.1. Collection and storage of samples for RNA Extraction

Immediately after cardiac arrest, tissue samples will be collected from the duodenum the first part (duodenal cap) and the horizontal or third part; the jejunum and ileum middle parts, and the colon—middle parts of the ascending colon, transverse colon, and descending colon. The samples will be stored in RNA*later* solution (Sigma-Aldrich; Germany), in accordance with the manufacturer's instructions. Tissue samples will be collected on the same dates.

#### 2.4.2. Total RNA Extraction and CDNA Synthesis

Total RNA will be extracted from the tissues preserved in RNA*later* (approximately 20 mg per sample; *n* 1 4 3 in each experimental group) using the Total RNA Mini isolation kit (A&A Biotechnology; Poland) according to the manufacturer's protocol. RNA samples will be incubated with RNase-free DNase I (Roche Diagnostics; Germany) to prevent contamination of genomic DNA. Total RNA quality and the purity of all samples will be estimated with the BioPhotometer (Eppendorf; Germany), and the results will be used to synthesize cDNA with the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas; Canada). The cDNA synthesis reaction mixture for each sample will contain 1 µg of total RNA and 0.5 µg of oligo (dT)18 primers, and the reaction will be performed according to the manufacturer's protocol. The first synthesized cDNA strand will be stored at −20 ◦C for further analysis.

#### 2.4.3. qPCR

Real-time PCR primers for ER*α* and ER*β* mRNAs, and CYP1A1 and GSTP1 mRNAs will be designed using the Primer-BLAST tool based on the reference species (Table 3). *β*-actin will be used as the endogenous reference gene. The real-time PCR assay will be performed in the ABI 7500 real-time PCR system thermocycler (Applied Biosystems, Foster City, CA, USA) in singleplex mode. Further treatments will be applied as recommended by the manufacturer.


**Table 3.** Real-time PCR primers for the proposed study.

Quantitative cycle (Cq) values from qPCR will be converted to copy numbers using a standard curve plot (Cq versus log copy number) according to the methodology.

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

#### 2.4.4. Statistical Analysis

The expression of ERα and ERβ in the digestive tract of gilts, and the expression of *CYP1A1* and *GSTP1* genes in the ascending colon and the descending colon will be presented as mean values (±) SD for each sample. The results will be analyzed using Statistica software (StatSoft Inc., USA). The mean values in the control and experimental groups will be compared by repeated-measures one-way ANOVA based on the ZEN dose administered to pre-pubertal gilts. If differences between groups are found, Tukey's post hoc test will be performed to determine which pairs of group means are significantly different. In ANOVA, group samples will be drawn from normally distributed populations characterized by the same variance. If the above assumptions are not met in all cases, the equality of group means will be tested using the Kruskal–Wallis test of ranks and the multiple comparisons test in ANOVA. Different group pairs will be identified by post hoc multiple comparisons of mean ranks for all groups.

#### **3. Discussion**

Mycotoxins have always been and will always be present in foodstuffs and feedstuffs this is a banal truth. Higher mycotoxin doses can produce symptoms of mycotoxicosis (poisoning) in macroorganisms. However, little is known about the fate of mycotoxins and the responses of the host organism during exposure to low doses of mycotoxins in the range of NOAEL and MABEL doses [16]. Macroorganisms have developed various coping strategies that enable them to maintain homeostasis. These coping mechanisms can involve tolerance to very low mycotoxin doses [16]. Alternatively, mycotoxins can participate in vital life processes, as briefly noted in the Introduction. Mycotoxins are accumulated and absorbed not only in target tissues [66,67], which suggests that the intestinal mucosa containing ERs is the most exposed tissue and the first line of defense against undesirable substances [68]. Therefore, the relevant diagnostic tests and laboratory analyses will be performed in the proposed study.

Due to the general scarcity of published research into low-dose mycotoxicosis, additional in vivo data are needed to increase the safety of foodstuffs and feedstuffs, and minimize the risk of toxicity in the decision-making process [69]. The proposed study will attempt to determine whether low doses of ZEN can affect (i) the degree and site of β-ZEL accumulation in the porcine gastrointestinal tract at different exposure times; (ii) the expression of ERs in the porcine gastrointestinal tract, analyzed in vivo, in particular, the expression of Er*α,* which regulates intestinal function in the proximal segments of the gastrointestinal tract; (iii) the involvement of intestinal enzymes (expression effect) in the distal segment of the intestines in ZEN detoxification processes.

The results of the study will support the development of biomarkers of prolonged low-dose ZEN mycotoxicosis in pre-pubertal gilts within the framework of veterinary precision medicine.

**Author Contributions:** Conceptualization, M.G. and M.T.G.; methodology, P.B., E.O., Ł.Z. and K.L.-I.; software, Ł.Z.; validation, Ł.Z. and P.B.; formal analysis, A.B. and M.T.G.; investigation, P.B., Ł.Z., K.L.-I. and E.O.; data curation, M.G.; writing—original draft preparation, M.G., M.M., K.E.P. and M.T.G.; writing—review and editing, M.G., M.T.G., P.B. and A.B.; supervision, Ł.Z. and M.T.G.; project administration, Ł.Z.; funding acquisition, Ł.Z. and M.T.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The project was financially co-supported by the Minister of Science and Higher 613 Education under the program entitled "Regional Initiative of Excellence" for the years 2019–2022, 614 Project No. 010/RID/2018/19, amount of funding PLN 12,000,000.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
