Limits...
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.

No MeSH data available.


Related in: MedlinePlus

Effect of DON (+/− U0126) and DOM-1 on TEER and viability of differentiated IPEC-J2. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium, before addition of DON (1–20 µM). Alternatively, differentiated IPEC-J2 were treated only with DOM-1 (1–100 µM). (A) TEER was measured after 24, 48 and 72 h (p-values indicate significant differences between IPEC-J2 treated with DON alone or DON + U0126 at individual concentrations; significant differences (*) of DON-treated cells to untreated control; significant increases (▲) or decreases (▼) of DON+U0126-treated cells compared to untreated control; significant decreases (▽) of U0126-treated cells compared to U0126 control. (B) Following the final TEER measurement, viability was determined via NR uptake. Data represent mean ± SD, n = 4.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037490&req=5

toxins-08-00264-f003: Effect of DON (+/− U0126) and DOM-1 on TEER and viability of differentiated IPEC-J2. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium, before addition of DON (1–20 µM). Alternatively, differentiated IPEC-J2 were treated only with DOM-1 (1–100 µM). (A) TEER was measured after 24, 48 and 72 h (p-values indicate significant differences between IPEC-J2 treated with DON alone or DON + U0126 at individual concentrations; significant differences (*) of DON-treated cells to untreated control; significant increases (▲) or decreases (▼) of DON+U0126-treated cells compared to untreated control; significant decreases (▽) of U0126-treated cells compared to U0126 control. (B) Following the final TEER measurement, viability was determined via NR uptake. Data represent mean ± SD, n = 4.

Mentions: Effects of DON (1–20 µM) and DOM-1 (1–100 µM) were investigated on TEER of differentiated IPEC-J2. To explore the involvement of MAPK signaling in DON-induced effects on IPEC-J2 barrier function, cells were additionally pretreated with complete cultivation medium or the p44/42 inhibitor U0126 (10 µM), followed by addition of DON (1–20 µM) (Figure 3). Compared to the untreated control, DON significantly reduced TEER at 5–20 µM after 24 h (5 µM: p = 0.042; 10 µM: p = 0.006; 20 µM: p = 0.000), 48 h (5 µM: p = 0.022; 10 µM: p = 0.032; 20 µM: p = 0.000) and 72 h (5, 10, and 20 µM: p = 0.000) (*). After 72 h, TEER reached a minimum of 2.93 ± 1.11 kOhm × cm2 at 20 µM DON. However, IPEC-J2 treated with DON+U0126, displayed significant TEER reductions only at 20 µM DON (p = 0.002) after 72 h (▲), compared to the untreated control. Compared to the untreated control, TEER of U0126-pretreated cells exposed to DON + U0126, was significantly elevated at 1 µM (p = 0.000), 5 µM (p = 0.001) and 10 µM (p = 0.009) after 24 h and at 1 µM (p = 0.007) after 48 h (▼). Compared to the U0126 control at each time point, TEER of cells treated with a combination of DON and U0126 was significantly reduced at 20 µM after 24 h (p = 0.000), 48 h (p = 0.011) and 72 h (p = 0.000) (▽). These differences are due to the fact that the 24 h U0126 pre-treatment alone, already significantly raised TEER values compared to cells pretreated with complete cultivation medium. U0126 pretreatment alone increased TEER of differentiated IPEC-J2 by 1.68 kOhm × cm2 after 24 h (+17%; p = 0.004), 1.77 kOhm × cm2 after 48 h (+18%; p = 0.034) and 1.73 kOhm × cm2 after 72 h (+18%; p = 0.026)), compared to cells which were pretreated with complete cultivation medium. At all individual DON concentrations and time points, TEER of cells treated with a combination of DON and U0126 was significantly higher than that of cells treated with DON alone.


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 (+/− U0126) and DOM-1 on TEER and viability of differentiated IPEC-J2. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium, before addition of DON (1–20 µM). Alternatively, differentiated IPEC-J2 were treated only with DOM-1 (1–100 µM). (A) TEER was measured after 24, 48 and 72 h (p-values indicate significant differences between IPEC-J2 treated with DON alone or DON + U0126 at individual concentrations; significant differences (*) of DON-treated cells to untreated control; significant increases (▲) or decreases (▼) of DON+U0126-treated cells compared to untreated control; significant decreases (▽) of U0126-treated cells compared to U0126 control. (B) Following the final TEER measurement, viability was determined via NR uptake. Data represent mean ± SD, n = 4.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037490&req=5

toxins-08-00264-f003: Effect of DON (+/− U0126) and DOM-1 on TEER and viability of differentiated IPEC-J2. Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium, before addition of DON (1–20 µM). Alternatively, differentiated IPEC-J2 were treated only with DOM-1 (1–100 µM). (A) TEER was measured after 24, 48 and 72 h (p-values indicate significant differences between IPEC-J2 treated with DON alone or DON + U0126 at individual concentrations; significant differences (*) of DON-treated cells to untreated control; significant increases (▲) or decreases (▼) of DON+U0126-treated cells compared to untreated control; significant decreases (▽) of U0126-treated cells compared to U0126 control. (B) Following the final TEER measurement, viability was determined via NR uptake. Data represent mean ± SD, n = 4.
Mentions: Effects of DON (1–20 µM) and DOM-1 (1–100 µM) were investigated on TEER of differentiated IPEC-J2. To explore the involvement of MAPK signaling in DON-induced effects on IPEC-J2 barrier function, cells were additionally pretreated with complete cultivation medium or the p44/42 inhibitor U0126 (10 µM), followed by addition of DON (1–20 µM) (Figure 3). Compared to the untreated control, DON significantly reduced TEER at 5–20 µM after 24 h (5 µM: p = 0.042; 10 µM: p = 0.006; 20 µM: p = 0.000), 48 h (5 µM: p = 0.022; 10 µM: p = 0.032; 20 µM: p = 0.000) and 72 h (5, 10, and 20 µM: p = 0.000) (*). After 72 h, TEER reached a minimum of 2.93 ± 1.11 kOhm × cm2 at 20 µM DON. However, IPEC-J2 treated with DON+U0126, displayed significant TEER reductions only at 20 µM DON (p = 0.002) after 72 h (▲), compared to the untreated control. Compared to the untreated control, TEER of U0126-pretreated cells exposed to DON + U0126, was significantly elevated at 1 µM (p = 0.000), 5 µM (p = 0.001) and 10 µM (p = 0.009) after 24 h and at 1 µM (p = 0.007) after 48 h (▼). Compared to the U0126 control at each time point, TEER of cells treated with a combination of DON and U0126 was significantly reduced at 20 µM after 24 h (p = 0.000), 48 h (p = 0.011) and 72 h (p = 0.000) (▽). These differences are due to the fact that the 24 h U0126 pre-treatment alone, already significantly raised TEER values compared to cells pretreated with complete cultivation medium. U0126 pretreatment alone increased TEER of differentiated IPEC-J2 by 1.68 kOhm × cm2 after 24 h (+17%; p = 0.004), 1.77 kOhm × cm2 after 48 h (+18%; p = 0.034) and 1.73 kOhm × cm2 after 72 h (+18%; p = 0.026)), compared to cells which were pretreated with complete cultivation medium. At all individual DON concentrations and time points, TEER of cells treated with a combination of DON and U0126 was significantly higher than that of cells treated with DON alone.

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