4.1.4. Toxicological Analysis of the Apex of the Heart

#### Tissues Samples

Five prepubertal gilts from every group were euthanized on analytical dates 1 (D1 exposure day 7) and 2 (D2—exposure day 21) by intravenous administration of pentobarbital sodium (Fatro, Ozzano Emilia BO, Italy) and bleeding. Samples were collected from the myocardium (the apex of the heart) immediately after cardiac arrest and were rinsed with phosphate buffer. The collected samples were stored at a temperature of −20 ◦C.

#### Extraction Procedure

The presence of ZEN, α-ZEL and β-ZEL in the apex of the heart was determined with the use of immunoaffinity columns (Zearala-TestTM Zearalenone Testing System, G1012, VICAM, Watertown, MA, USA). All extraction procedures were carried out in accordance with the recommendations of column manufacturers. 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% acetonitrile (ACN) was added to dry residues to dissolve the mycotoxin. The process was monitored with the use of internal standards, and the results were validated by mass spectrometry.

#### Chromatographic Quantification of ZEN and Its Metabolites

Zearalenone and its metabolites were quantified at the Institute of Dairy Industry Innovation in Mr ˛agowo. The biological activity of ZEN, α-ZEL and β-ZEL in the myocardium was determined by combined separation methods, immunoaffinity chromatog-

raphy (Zearala-TestTM Zearalenone Testing System, G1012, VICAM, Watertown, MA, USA), liquid chromatography (LC) (Agilent 1260 LC system) and mass spectrometry (MS). 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. 984 730 109, Merck-Hitachi, Mannheim, Germany), 20% methanol (LiChrosolvTM, No. 1.06 007, Merck-Hitachi, Mannheim, Germany) and 10% deionized water (MiliporeWater Purification System, Millipore S.A. Molsheim-France) with the addition of 2 mL of acetic acid per 1 L of the mixture. The column was flushed with 99.8% methanol (LIChrosolvTM, No. 1.06 007, Merck-Hitachi, Mannheim, Germany) to remove the bound mycotoxin. The eluents were placed in a water bath with a temperature of 50 ◦C, and the solvent was evaporated in a stream of nitrogen. Mycotoxin concentrations were determined with an external standard and were expressed in ppb (ng/mL). Matrixmatched 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 and percentage content of ZEN, α-ZEL and β-ZEL were determined in each group and on both analytical dates (Table 1).

#### Carry-Over Factor

Carry-over toxicity takes place when organisms exposed to low doses of mycotoxins survive. Mycotoxins can compromise tissue or organ functions [59] and modify their biological activity [4,15]. The carry-over factor (CF) was determined in the myocardium 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 560–6255 µg ZEN/kg of the complete diet, depending on daily feed intake. Mycotoxin concentrations in tissues were expressed in terms of the dry matter content of the samples.

The CF was calculated as follows:

$$carry-over\ factor = \frac{\text{taxin concentration in tissue [ng/g]}}{\text{taxin concentration in diet [ng/g]}} \tag{1}$$

#### Statistical Analysis

Data were processed statistically at the Department of Discrete Mathematics and Theoretical Computer Science, Faculty of Mathematics and Computer Science of the University of Warmia and Mazury in Olsztyn. The bioavailability of ZEN and its metabolites in the apex of heart was analyzed in group C and three experimental groups on two analytical dates. The results were expressed as means (*x*) with standard deviation (SD). The following parameters were analyzed: (i) differences in the mean values for three ZEN doses (experimental groups) and the control group on both analytical dates, and (ii) differences in the mean values for specific ZEN doses (groups) on both analytical dates. In both cases, the differences between mean values were determined by one-way ANOVA. If significant differences were noted between groups, the differences between paired means were determined by Tukey's multiple comparison test. If all values were below LOD (mean and variance equal zero) in any group, the values in the remaining groups were analyzed by one-way ANOVA (if the number of the remaining groups was higher than two), and the means in these groups were compared against zero by Student's t-test. Differences between groups were determined by Student's *t*-test. The results were regarded as highly significant at *P* < 0.01 (\*\*) and as significant at 0.01 < *P* < 0.05 (\*). Data were processed statistically using Statistica v.13 software (TIBCO Software Inc., Silicon Valley, CA, USA, 2017).

#### *4.2. Laboratory Analyses*

#### 4.2.1. Sampling for In Vitro Analyses

Tissue samples for in vitro analyses were collected from the same animals as the tissues for the toxicological analysis. The animals were euthanized by intravenous administration of pentobarbital sodium (Fatro, Ozzano Emilia BO, Italy) and exsanguinated.

#### 4.2.2. In Vitro Analysis

In vitro analyses were performed at the Department of Pharmacology and Toxicology of the Faculty of Medicine at the University of Warmia and Mazury in Olsztyn, Poland.

#### 4.2.3. Drugs

Acetylcholine (ACh) chloride, sodium nitroprusside (SNP), bradykinin acetate salt, indomethacin, NG-nitro-L-arginine methyl ester (L-NAME) and potassium chloride (KCl) (Chempur, Poland) were obtained from Sigma-Aldrich unless stated otherwise. Stock solutions (10 mM) of indomethacin and L-NAME were prepared in DMSO. These solutions were stored at −20 ◦C, and appropriate dilutions were made in Krebs-Henseleit solution (KHS in mM: NaCl 120, KCl 4.76, NaHCO<sup>3</sup> 25, NaH2PO4·H2O 1.18, CaCl<sup>2</sup> 1.25, MgSO4·7H2O 1.18, glucose 5.5) on the day of the experiment. At these concentrations, DMSO did not alter the reactivity of coronary arteries.

#### 4.2.4. Vascular Reactivity Analyses

Porcine hearts were transported on ice and rinsed with cold aerated KHS. The left anterior descending branches of the coronary artery (MID LAD, D1-D2 sections) were excised at room temperature (*n* = 5). The surrounding connective tissues were removed, the arteries were cut into 4 mm rings and suspended between two stainless-steel rods positioned in 5 mL tissue baths (Graz Tissue Bath System, Harvard Apparatus) filled with aerated (95% O<sup>2</sup> + 5% CO2) KHS at 37 ◦C (pH 7.4). The resting tension of 1.5 g was applied and further readjusted every 15 min during a 60 min equilibration period before further analysis.

The viability of each porcine coronary artery was determined by contraction with 30 mM KCl before relaxation with 1 mM bradykinin. Rings that failed to produce an average contraction greater than or equal to 4.0 g when challenged with KCl and relaxation greater than or equal to 40% when treated with bradykinin were excluded from the study (approx. 10–20% of prepared rings). The rings were washed three times with fresh KHS, and baseline tension was readjusted before the examination.

After an initial equilibration period of 60 min, contractile responses elicited by either a cumulative concentration of KCl (2.5–30 mM) or a single maximum depolarizing concentration of KCl (30 mM) were assessed. The rings were also assessed for contractile responses generated by a cumulative concentration of acetylcholine (ACh: 0.1–10 µM). The cumulative concentrations of sodium nitroprusside (SNP: 0.1–100 µM) were assessed to determine the possible contribution of exogenous nitric oxide (NO) to relaxation responses on endothelium-intact rings that had been precontracted with submaximal concentrations of KCl (30 mM).

In experiments evaluating the influence of COX and e-NOS-inhibitors on contractile responses, indomethacin (4.4 <sup>×</sup> <sup>10</sup>−<sup>6</sup> M) and L-NAME (4.4 <sup>×</sup> <sup>10</sup>−<sup>5</sup> M) were added to the chambers 30 min before the arteries were constricted with acetylcholine. Each tissue was exposed to the contracting agent only once.

#### 4.2.5. Statistical Analysis

Data were expressed as means ± SD (Standard Deviation), where *n* denoted the number of porcine hearts from which arterial rings were obtained. The contraction elicited by KCl was expressed in g of tension. ACh-induced contraction and SNP-induced relaxation were expressed as a percentage of the initial contraction elicited by 30 mM KCl. Doseresponse curves were analyzed for the area under the curve, Emax and EC<sup>50</sup> in GraphPad

Prism 9.0.2. Data were processed statistically by comparing the curves obtained in each experimental group with the control curve in two-way ANOVA, followed by Tukey's multiple comparisons test. Differences were regarded as significant at *P* ≤ 0.05.

**Author Contributions:** The experiments were conceived and designed by M.G., M.S.M. and M.T.G. The experiments were performed by M.G., M.S.M., E.O. and Ł.Z. Data were analyzed and interpreted by M.G., M.S.M., W.G., J.J. and S.L.-Z. The manuscript was drafted by M.G. and M.S.M. and critically ˙ edited by A.B., Ł.Z. and M.T.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The study was supported by the "Healthy Animal—Safe Food" Scientific Consortium of the Leading National Research Centre (KNOW) pursuant to a decision of the Ministry of Science and Higher Education No. 05-1/KNOW2/2015. The project was financially co-supported by the Minister of Science and Higher Education under the program entitled "Regional Initiative of Excellence" for the years 2019-2022, 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.

#### **References**


*Study Protocol*
