*2.1. The Effect of Deoxynivalenol on the Viability, Integrity, and Permeability of Jejunum Mucosa Explants*

The application of DON at the concentration of 30 µg/mL to the luminal compartment of the Ussing chamber, and incubation of mucosa explants in its presence for 90 min resulted in a significant drop of the transepithelial electrical resistance (TEER) value. It reached only 52.4 <sup>±</sup> 0.7 Ohm·cm<sup>2</sup> at the end of exposure, whereas the control incubation with no mycotoxin resulted in a TEER measurement of 77.1 <sup>±</sup> 1.2 Ohm·cm<sup>2</sup> (Figure 1).

DON caused a remarkable increase in paracellular permeability measured indirectly by the penetration rate of paracellular transport markers. Both Lucifer Yellow (LY) and mannitol (MAN), administered at concentrations of 100 µg/mL, underwent more intense transportation across mucosa explants in intestine specimens treated with DON than in the control chambers (Figures 2 and 3).

*Toxins* **2022**, *14*, x FOR PEER REVIEW 3 of 14

**Figure 2.** Lucifer Yellow transport through intestine explants during 90 min of incubation in buffer supplemented with: amoxicillin—AMX, doxycycline—DOX, deoxynivalenol—DON, combination AMX + DON or DOX + DON, or CTRL—control condition without antibiotics and DON. Bars

indicate a statistically significant difference at *p*-value < 0.05.

bars indicate a statistically significant difference at *p*-value < 0.05.

**Figure 3.** Mannitol transport through intestine explants during 90 minincubation in buffer supplemented with: amoxicillin—AMX, doxycycline—DOX, deoxynivalenol—DON, combination AMX + DON or DOX + DON, or CTRL—control condition without antibiotics and DON. Bars show the mean of the 6 replicates ± SEM (standard errors of the mean). Different letters above the bars indicate a statistically significant difference at *p*-value < 0.05. **Figure 3.** Mannitol transport through intestine explants during 90 minincubation in buffer supplemented with: amoxicillin—AMX, doxycycline—DOX, deoxynivalenol—DON, combination AMX + DON or DOX + DON, or CTRL—control condition without antibiotics and DON. Bars show the mean of the 6 replicates ± SEM (standard errors of the mean). Different letters above the bars indicate a statistically significant difference at *p*-value < 0.05.

show the mean of the 6 replicates ± SEM (standard errors of the mean). Different letters above the

The flux of LY and MAN amounted to 89.5 ± 3.2 and 306.0 ± 8.6 ng/min/cm2, respectively, in the presence of DON, and to 38.3 ± 1.7 and 217.3 ± 6.5 ng/min/cm2, respectively, in the absence of the mycotoxin. The flux of the transcellular transport marker (caffeine—CAF) did not change when mucosa explants were incubated in a DONcontaining buffer. The addition of mycotoxin caused CAF penetration through intestine explants at the level of 2.9 ± 0.2 µg/min/cm2, whereas in the control trial, the flux came to 2.6 ± 0.1 µg/min/cm2 (Figure 4). The flux of LY and MAN amounted to 89.5 <sup>±</sup> 3.2 and 306.0 <sup>±</sup> 8.6 ng/min/cm<sup>2</sup> , respectively, in the presence of DON, and to 38.3 <sup>±</sup> 1.7 and 217.3 <sup>±</sup> 6.5 ng/min/cm<sup>2</sup> , respectively, in the absence of the mycotoxin. The flux of the transcellular transport marker (caffeine—CAF) did not change when mucosa explants were incubated in a DONcontaining buffer. The addition of mycotoxin caused CAF penetration through intestine explants at the level of 2.9 <sup>±</sup> 0.2 <sup>µ</sup>g/min/cm<sup>2</sup> , whereas in the control trial, the flux came to 2.6 <sup>±</sup> 0.1 <sup>µ</sup>g/min/cm<sup>2</sup> (Figure 4). *Toxins* **2022**, *14*, x FOR PEER REVIEW 5 of 14

**Figure 4.** Caffeine transport through intestine explants during 90 min incubation in buffer supplemented with: amoxicillin—AMX, doxycycline—DOX, deoxynivalenol—DON, combination AMX + DON or DOX + DON, or CTRL—control condition without antibiotics and DON. Bars show the mean of the 6 replicates ± SEM (standard errors of the mean). Different letters above the bars indicate a statistically significant difference at *p*-value < 0.05. **Figure 4.** Caffeine transport through intestine explants during 90 min incubation in buffer supplemented with: amoxicillin—AMX, doxycycline—DOX, deoxynivalenol—DON, combination AMX + DON or DOX + DON, or CTRL—control condition without antibiotics and DON. Bars show the mean of the 6 replicates ± SEM (standard errors of the mean). Different letters above the bars indicate a statistically significant difference at *p*-value < 0.05.

Moreover, the use of DON did not provoke any cytotoxicity measured by LDH leakage. The activity of LDH detected in the buffer amounted to 4.9 ± 0.2% and 4.7 ± 0.2%

**Figure 5.** Relative LDH activity in the luminal compartment of intestine explants at 90 min of

deoxynivalenol—DON, combination AMX + DON or DOX + DON, or CTRL—control condition

incubation in buffer supplemented with: amoxicillin—AMX, doxycycline—DOX,

5).

Moreover, the use of DON did not provoke any cytotoxicity measured by LDH leakage. The activity of LDH detected in the buffer amounted to 4.9 ± 0.2% and 4.7 ± 0.2% of total LDH activity in the presence and absence of the mycotoxin, respectively (Figure 5). leakage. The activity of LDH detected in the buffer amounted to 4.9 ± 0.2% and 4.7 ± 0.2% of total LDH activity in the presence and absence of the mycotoxin, respectively (Figure 5).

Moreover, the use of DON did not provoke any cytotoxicity measured by LDH

**Figure 4.** Caffeine transport through intestine explants during 90 min incubation in buffer supplemented with: amoxicillin—AMX, doxycycline—DOX, deoxynivalenol—DON, combination AMX + DON or DOX + DON, or CTRL—control condition without antibiotics and DON. Bars show the mean of the 6 replicates ± SEM (standard errors of the mean). Different letters above the

bars indicate a statistically significant difference at *p*-value < 0.05.

*Toxins* **2022**, *14*, x FOR PEER REVIEW 5 of 14

**Figure 5.** Relative LDH activity in the luminal compartment of intestine explants at 90 min of incubation in buffer supplemented with: amoxicillin—AMX, doxycycline—DOX, deoxynivalenol—DON, combination AMX + DON or DOX + DON, or CTRL—control condition **Figure 5.** Relative LDH activity in the luminal compartment of intestine explants at 90 min of incubation in buffer supplemented with: amoxicillin—AMX, doxycycline—DOX, deoxynivalenol— DON, combination AMX + DON or DOX + DON, or CTRL—control condition without antibiotics and DON. The LDH activity measured after explant homogenisation was taken to be 100%. Bars show the mean of the 6 replicates ± SEM (standard errors of the mean).
