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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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Cellular distribution of the tight junction protein ZO-1 in IPEC-J2 monolayers treated with deoxynivalenol (DON).Cells were grown on inserts and incubated for 24, 48 or 74 hours either without DON (upper panel) or with 2000 ng/mL DON applied from apical (middle panel) or basolateral side (lower panel) in complete medium. Monolayers were stained for the tight junction associated protein ZO-1 and nuclei stained with DAPI, then detected by immunofluorescence microscopy. All micrographs were taken under identical exposure time and in the centre of each membrane. Micrographs are representative for 3 separate experiments with similar results. Scale bar  =  50 µm.
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pone-0017472-g007: Cellular distribution of the tight junction protein ZO-1 in IPEC-J2 monolayers treated with deoxynivalenol (DON).Cells were grown on inserts and incubated for 24, 48 or 74 hours either without DON (upper panel) or with 2000 ng/mL DON applied from apical (middle panel) or basolateral side (lower panel) in complete medium. Monolayers were stained for the tight junction associated protein ZO-1 and nuclei stained with DAPI, then detected by immunofluorescence microscopy. All micrographs were taken under identical exposure time and in the centre of each membrane. Micrographs are representative for 3 separate experiments with similar results. Scale bar  =  50 µm.

Mentions: The expression of ZO-1 and claudin-3, proteins of the tight junction complex between adjacent cells, was investigated using immunoblotting and immunofluorescence. We found a slight difference after 48 hours between 2000 ng/mL DON from apical or basolateral side, the latter resulting in a lower protein expression. This difference was markedly enhanced after 72 hours incubation, i.e. the ZO-1 expression in cells treated with 2000 ng/mL DON from basolateral disappeared completely from the blot (Fig. 6). In contrast to these findings, the immunofluorescence staining of ZO-1 did not change the distribution pattern between apical or basolateral treated cells with 2000 ng/mL DON at any investigated time point (Fig. 7). ZO-1 was detected as a continuous lining around each epithelial cell independent of application route. However, at 72 hours incubation of 2000 ng/mL DON from basolateral the confluent cell layer was disturbed and thus only cell islets were distributed on the membrane. Interestingly, DON showed a more pronounced impact on claudin-3 protein expressions (Fig. 6) and structure (Fig. 8). Immunoblotting clearly demonstrated the absence of a claudin-3 signal when DON was applied from basolateral at each time point. This effect was absent at apical application. This was confirmed by the immunofluorescence staining, where claudin-3 appeared as a continuous lining around each cell in controls and in cells treated with DON from apical. Basolateral application elicited a definite disturbance of the continuous lining, beginning at 24 h and resulting in a complete disappearance oat 48 and 72 h.


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)

Cellular distribution of the tight junction protein ZO-1 in IPEC-J2 monolayers treated with deoxynivalenol (DON).Cells were grown on inserts and incubated for 24, 48 or 74 hours either without DON (upper panel) or with 2000 ng/mL DON applied from apical (middle panel) or basolateral side (lower panel) in complete medium. Monolayers were stained for the tight junction associated protein ZO-1 and nuclei stained with DAPI, then detected by immunofluorescence microscopy. All micrographs were taken under identical exposure time and in the centre of each membrane. Micrographs are representative for 3 separate experiments with similar results. Scale bar  =  50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045462&req=5

pone-0017472-g007: Cellular distribution of the tight junction protein ZO-1 in IPEC-J2 monolayers treated with deoxynivalenol (DON).Cells were grown on inserts and incubated for 24, 48 or 74 hours either without DON (upper panel) or with 2000 ng/mL DON applied from apical (middle panel) or basolateral side (lower panel) in complete medium. Monolayers were stained for the tight junction associated protein ZO-1 and nuclei stained with DAPI, then detected by immunofluorescence microscopy. All micrographs were taken under identical exposure time and in the centre of each membrane. Micrographs are representative for 3 separate experiments with similar results. Scale bar  =  50 µm.
Mentions: The expression of ZO-1 and claudin-3, proteins of the tight junction complex between adjacent cells, was investigated using immunoblotting and immunofluorescence. We found a slight difference after 48 hours between 2000 ng/mL DON from apical or basolateral side, the latter resulting in a lower protein expression. This difference was markedly enhanced after 72 hours incubation, i.e. the ZO-1 expression in cells treated with 2000 ng/mL DON from basolateral disappeared completely from the blot (Fig. 6). In contrast to these findings, the immunofluorescence staining of ZO-1 did not change the distribution pattern between apical or basolateral treated cells with 2000 ng/mL DON at any investigated time point (Fig. 7). ZO-1 was detected as a continuous lining around each epithelial cell independent of application route. However, at 72 hours incubation of 2000 ng/mL DON from basolateral the confluent cell layer was disturbed and thus only cell islets were distributed on the membrane. Interestingly, DON showed a more pronounced impact on claudin-3 protein expressions (Fig. 6) and structure (Fig. 8). Immunoblotting clearly demonstrated the absence of a claudin-3 signal when DON was applied from basolateral at each time point. This effect was absent at apical application. This was confirmed by the immunofluorescence staining, where claudin-3 appeared as a continuous lining around each cell in controls and in cells treated with DON from apical. Basolateral application elicited a definite disturbance of the continuous lining, beginning at 24 h and resulting in a complete disappearance oat 48 and 72 h.

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