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TDK conference 2019

In vitro evaluation of DON-induced intestinal barrier damage
Gatt Katrina - year 6
University of Veterinary Medicine Budapest, Department of Pharmacology and Toxicology
Supervisor: Dr. Erzsébet Pásztiné Gere


Deoxynivalenol (DON) produced by Fusarium fungi can induce pathological lesions such as intestinal barrier damage. It affects the integrity of the intestinal epithelium through alterations in cell morphology and differentiation. Quercetin (Que) is a polyphenolic compound that shows good anti-inflammatory and antioxidative effects.

The aim of this study was to determine how the non-tumorigenic porcine intestinal epithelial (IPEC-J2) cells react to administration of DON, and whether Que has protective properties against DON-induced damage. Hence, we examined the effects of DON and Que on cell viability, membrane integrity and oxidative state.

Cell viability was measured using MTS assays. The research work was carried out on two sets of DON and Que exposure: 1) simultaneous treatment of cells with DON and Que for 1 hr (1 hr DON + Que), 2) and preincubation of cells with Que for 24 hr followed by 1 hr DON (24 hr Que + 1 hr DON). To evaluate the induced changes in cell membrane integrity, transepithelial electrical resistance (TER) and fluorescent labeled dextran-4 kDa (FD-4) transport was measured. For assessment of redox status, hydrogen peroxide concentration in the extracellular space was measured with Amplex Red Assay. Intracellular oxidative stress was investigated using 2’,7’-dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

Based on the MTS data, 2 µM DON and 20 µM Que were chosen for subsequent experiments. Cells treated with DON alone showed a significant decrease in TER, and cells treated with Que alone exhibited an increase in TER value. 1 hr DON + Que showed a lesser decline in TER than in case of only DON-treated cells. 24 hr Que + 1 hr DON caused significant decrease in cell integrity. 2 µM DON for 25 hours significantly increased FD-4 transport to the basolateral compartment, while no difference in FD-4 concentration was recorded following administration of 20 µM Que. Alteration in location of FD-4 was notable following 1 hr DON + Que. Que-treated cells did not show any changes in FD-4 basolateral concentration compared to the control.

H2O2 concentration was significantly increased following 25 hr treatment with 2 µM DON, and the 20 µM Que did not alter its production. 24 hr Que + 1 hr DON did not change the H2O2 concentration, whereas 1 hr DON + Que-treated cells showed significant increase. 20 µM Que-treated cells did not cause any increase in reactive oxygen species (ROS), but 2 µM DON considerably increased intracellular ROS, which could not be suppressed by Que application.

This research demonstrates that cytotoxic concentrations of DON disrupt barrier integrity and increases oxidative stress markers and Que helps the cell monolayer to strengthen the barrier function. However, both simultaneous treatment and pretreatment with Que was unable to fully counteract the harmful effects of DON toxicosis.

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