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Vulnerability of polarised intestinal porcine epithelial cells to mycotoxin deoxynivalenol depends on the route of application.

Diesing AK, Nossol C, Dänicke S, Walk N, Post A, Kahlert S, Rothkötter HJ, Kluess J - PLoS ONE (2011)

Bottom Line: Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3.Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL.The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.

View Article: PubMed Central - PubMed

Affiliation: Medical Faculty, Institute of Anatomy, Otto-von-Guericke University, Magdeburg, Germany.

ABSTRACT

Background and aims: Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated.

Methods: A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity.

Results: Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL.

Conclusions: Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.

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Impact of deoxynivalenol (DON) on transepithelial electrical resistance (TEER) in polarised IPEC-J2 layers.Cells were grown on inserts and incubated for 24, 48 or 72 hours with DON (0–4000 ng/mL) applied from apical or basolateral side in complete medium. TEER values are expressed in kOhms per insert (0.3 cm2) with 1 kOhm being the level of confluence. Data are given as means (±SEM) from at least 14 separate experiments. ***p≤0.001 vs. DON0.
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pone-0017472-g005: Impact of deoxynivalenol (DON) on transepithelial electrical resistance (TEER) in polarised IPEC-J2 layers.Cells were grown on inserts and incubated for 24, 48 or 72 hours with DON (0–4000 ng/mL) applied from apical or basolateral side in complete medium. TEER values are expressed in kOhms per insert (0.3 cm2) with 1 kOhm being the level of confluence. Data are given as means (±SEM) from at least 14 separate experiments. ***p≤0.001 vs. DON0.

Mentions: TEER is an indicator for the tight junction integrity of a confluent epithelial cell layer. In control cells TEER was essentially constant throughout the experimental period. In our trials with apical DON exposure we did not see a significant change in TEER even with highest concentrations (4000 ng/mL) applied. It is noteworthy that DON added to the basolateral side showed a significant decrease in TEER at 2000 ng/mL and 4000 ng/mL DON already after 24 hours. TEER did not recover throughout prolonged incubation times (Fig. 5).


Vulnerability of polarised intestinal porcine epithelial cells to mycotoxin deoxynivalenol depends on the route of application.

Diesing AK, Nossol C, Dänicke S, Walk N, Post A, Kahlert S, Rothkötter HJ, Kluess J - PLoS ONE (2011)

Impact of deoxynivalenol (DON) on transepithelial electrical resistance (TEER) in polarised IPEC-J2 layers.Cells were grown on inserts and incubated for 24, 48 or 72 hours with DON (0–4000 ng/mL) applied from apical or basolateral side in complete medium. TEER values are expressed in kOhms per insert (0.3 cm2) with 1 kOhm being the level of confluence. Data are given as means (±SEM) from at least 14 separate experiments. ***p≤0.001 vs. DON0.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045462&req=5

pone-0017472-g005: Impact of deoxynivalenol (DON) on transepithelial electrical resistance (TEER) in polarised IPEC-J2 layers.Cells were grown on inserts and incubated for 24, 48 or 72 hours with DON (0–4000 ng/mL) applied from apical or basolateral side in complete medium. TEER values are expressed in kOhms per insert (0.3 cm2) with 1 kOhm being the level of confluence. Data are given as means (±SEM) from at least 14 separate experiments. ***p≤0.001 vs. DON0.
Mentions: TEER is an indicator for the tight junction integrity of a confluent epithelial cell layer. In control cells TEER was essentially constant throughout the experimental period. In our trials with apical DON exposure we did not see a significant change in TEER even with highest concentrations (4000 ng/mL) applied. It is noteworthy that DON added to the basolateral side showed a significant decrease in TEER at 2000 ng/mL and 4000 ng/mL DON already after 24 hours. TEER did not recover throughout prolonged incubation times (Fig. 5).

Bottom Line: Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3.Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL.The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.

View Article: PubMed Central - PubMed

Affiliation: Medical Faculty, Institute of Anatomy, Otto-von-Guericke University, Magdeburg, Germany.

ABSTRACT

Background and aims: Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated.

Methods: A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity.

Results: Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL.

Conclusions: Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.

Show MeSH
Related in: MedlinePlus