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Early Activation of MAPK p44/42 Is Partially Involved in DON-Induced Disruption of the Intestinal Barrier Function and Tight Junction Network

View Article: PubMed Central - PubMed

ABSTRACT

Deoxynivalenol (DON), produced by the plant pathogens Fusariumgraminearum and Fusarium culmorum, is one of the most common mycotoxins, contaminating cereal and cereal-derived products. Although worldwide contamination of food and feed poses health threats to humans and animals, pigs are particularly susceptible to this mycotoxin. DON derivatives, such as deepoxy-deoxynivalenol (DOM-1), are produced by bacterial transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Intestinal epithelial cells are the initial barrier against these food- and feed-borne toxins. The present study confirms DON-induced activation of MAPK p44/42 and inhibition of p44/42 by MAPK-inhibitor U0126 monoethanolate. Influence of DON and DOM-1 on transepithelial electrical resistance (TEER), viability and expression of seven tight junction proteins (TJ), as well as the potential of U0126 to counteract DON-induced effects, was assessed. While DOM-1 showed no effect, DON significantly reduced TEER of differentiated IPEC-J2 and decreased expression of claudin-1 and -3, while leaving claudin-4; ZO-1, -2, and -3 and occludin unaffected. Inhibition of p44/42 counteracted DON-induced TEER decrease and restored claudin-3, but not claudin-1 expression. Therefore, effects of DON on TEER and claudin-3 are at least partially p44/42 mediated, while effects on viability and claudin-1 are likely mediated via alternative pathways.

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Effect of DON (20 µM) +/− U0126 (10 µM) on tight junction components of differentiated IPEC-J2. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium before addition of DON (20 µM) for 72 h. Alternatively, differentiated IPEC-J2 were treated with DOM-1 for 1 h. The expression of tight junction components claudin-1, -3 and -4; ZO-1, -2 and -3; and occludin was determined by (A) immunoblotting and (B) densitometry after normalization with ß-actin. Data were normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b,c).
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toxins-08-00264-f006: Effect of DON (20 µM) +/− U0126 (10 µM) on tight junction components of differentiated IPEC-J2. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium before addition of DON (20 µM) for 72 h. Alternatively, differentiated IPEC-J2 were treated with DOM-1 for 1 h. The expression of tight junction components claudin-1, -3 and -4; ZO-1, -2 and -3; and occludin was determined by (A) immunoblotting and (B) densitometry after normalization with ß-actin. Data were normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b,c).

Mentions: As 20 µM significantly reduced TEER without reducing viability, we examined the effect of this DON concentration on tight junction proteins, in the presence and absence of U0126 (10 µM). Additionally, we tested the effect of DOM-1 (100 µM) on the expression of tight junction proteins claudin-1, -3 and -4; ZO-1, -2 and -3; and occludin (Figure 6). DON did not affect ZO-1, -2 and -3; occludin; or claudin-4, but significantly reduced claudin-1 to 0.32 ± 0.11 (p = 0.12) and claudin-3 to 0.27 ± 0.15 (p = 0.000), compared to the control. This is equivalent to reductions of 68% and 73%, respectively. Although the significant reduction of claudin-3 expression could be counteracted by U0126, this was not possible for claudin-1. In contrast to DON, DOM-1—even if applied at a five-fold higher concentration—did not affect any of the tested tight junction proteins.


Early Activation of MAPK p44/42 Is Partially Involved in DON-Induced Disruption of the Intestinal Barrier Function and Tight Junction Network
Effect of DON (20 µM) +/− U0126 (10 µM) on tight junction components of differentiated IPEC-J2. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium before addition of DON (20 µM) for 72 h. Alternatively, differentiated IPEC-J2 were treated with DOM-1 for 1 h. The expression of tight junction components claudin-1, -3 and -4; ZO-1, -2 and -3; and occludin was determined by (A) immunoblotting and (B) densitometry after normalization with ß-actin. Data were normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b,c).
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Related In: Results  -  Collection

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toxins-08-00264-f006: Effect of DON (20 µM) +/− U0126 (10 µM) on tight junction components of differentiated IPEC-J2. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium before addition of DON (20 µM) for 72 h. Alternatively, differentiated IPEC-J2 were treated with DOM-1 for 1 h. The expression of tight junction components claudin-1, -3 and -4; ZO-1, -2 and -3; and occludin was determined by (A) immunoblotting and (B) densitometry after normalization with ß-actin. Data were normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b,c).
Mentions: As 20 µM significantly reduced TEER without reducing viability, we examined the effect of this DON concentration on tight junction proteins, in the presence and absence of U0126 (10 µM). Additionally, we tested the effect of DOM-1 (100 µM) on the expression of tight junction proteins claudin-1, -3 and -4; ZO-1, -2 and -3; and occludin (Figure 6). DON did not affect ZO-1, -2 and -3; occludin; or claudin-4, but significantly reduced claudin-1 to 0.32 ± 0.11 (p = 0.12) and claudin-3 to 0.27 ± 0.15 (p = 0.000), compared to the control. This is equivalent to reductions of 68% and 73%, respectively. Although the significant reduction of claudin-3 expression could be counteracted by U0126, this was not possible for claudin-1. In contrast to DON, DOM-1—even if applied at a five-fold higher concentration—did not affect any of the tested tight junction proteins.

View Article: PubMed Central - PubMed

ABSTRACT

Deoxynivalenol (DON), produced by the plant pathogens Fusariumgraminearum and Fusarium culmorum, is one of the most common mycotoxins, contaminating cereal and cereal-derived products. Although worldwide contamination of food and feed poses health threats to humans and animals, pigs are particularly susceptible to this mycotoxin. DON derivatives, such as deepoxy-deoxynivalenol (DOM-1), are produced by bacterial transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Intestinal epithelial cells are the initial barrier against these food- and feed-borne toxins. The present study confirms DON-induced activation of MAPK p44/42 and inhibition of p44/42 by MAPK-inhibitor U0126 monoethanolate. Influence of DON and DOM-1 on transepithelial electrical resistance (TEER), viability and expression of seven tight junction proteins (TJ), as well as the potential of U0126 to counteract DON-induced effects, was assessed. While DOM-1 showed no effect, DON significantly reduced TEER of differentiated IPEC-J2 and decreased expression of claudin-1 and -3, while leaving claudin-4; ZO-1, -2, and -3 and occludin unaffected. Inhibition of p44/42 counteracted DON-induced TEER decrease and restored claudin-3, but not claudin-1 expression. Therefore, effects of DON on TEER and claudin-3 are at least partially p44/42 mediated, while effects on viability and claudin-1 are likely mediated via alternative pathways.

No MeSH data available.


Related in: MedlinePlus