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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

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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 (50 µM) on viability differentiated IPEC-J2 in the absence or presence of MAPK inhibitor U0126 (10 µM) after 24 h, 48 h and 72 h Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium, before addition of DON (50 µM). Viability was assessed according to the (A) neutral red (NR); (B) sulforhodamine B (SRB) and (C) lactate dehydrogenase (LDH) assay after 24, 48 and 72 h. Data was normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b).
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toxins-08-00264-f005: Effect of DON (50 µM) on viability differentiated IPEC-J2 in the absence or presence of MAPK inhibitor U0126 (10 µM) after 24 h, 48 h and 72 h Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium, before addition of DON (50 µM). Viability was assessed according to the (A) neutral red (NR); (B) sulforhodamine B (SRB) and (C) lactate dehydrogenase (LDH) assay after 24, 48 and 72 h. Data was normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b).

Mentions: According to the NR assay performed after the final TEER measurement (Figure 3b), we observed that neither DON (1–20 µM), nor the combination of DON (1–20 µM) + U0126 (10 µM), significantly reduced viability after 72 h. However, to further investigate whether U0126 is able to protect against DON-induced cytotoxicity, a cytotoxic dose of DON (50 µM) was applied to differentiated IPEC-J2 in the absence or presence of U0126. Subsequently, viability was assessed via the NR (lysosomal activity), SRB (total protein content) and the LDH (membrane integrity) assay after 24, 48 and 72 h (Figure 5). According to the NR and SRB assay, DON induced significant viability reductions after 24 h (NR: −27%; SRB: −60%), 48 h (NR: −61%; SRB: −81%) and 72 h (NR: −81%; SRB: −87%). Addition of U0126 did not counteract the cytotoxic effect of DON, producing similar viability reductions after 24 h (NR: −31%; SRB: −56%), 48 h (NR: −56%; SRB: −81%) and 72 h (NR: −85%, SRB: −92%), as cells treated with DON alone. According to the LDH assay, DON (+/− U0126) was not cytotoxic after 24 h. After 48 h and 72 h, viability was significantly reduced in both DON−treated (48 h: −33%; 72 h: −50%) and DON + U0126-treated (48 h: −26%; 72 h: −46%) cells. Thus, U0126 did not counteract or reduce DON−induced toxicity over an incubation period of 72 h, showing a similar time-dependent cytotoxicity profile as 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 (50 µM) on viability differentiated IPEC-J2 in the absence or presence of MAPK inhibitor U0126 (10 µM) after 24 h, 48 h and 72 h Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium, before addition of DON (50 µM). Viability was assessed according to the (A) neutral red (NR); (B) sulforhodamine B (SRB) and (C) lactate dehydrogenase (LDH) assay after 24, 48 and 72 h. Data was normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b).
© Copyright Policy
Related In: Results  -  Collection

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toxins-08-00264-f005: Effect of DON (50 µM) on viability differentiated IPEC-J2 in the absence or presence of MAPK inhibitor U0126 (10 µM) after 24 h, 48 h and 72 h Differentiated IPEC-J2 were either pretreated with U0126 (10 µM) or cultivation medium, before addition of DON (50 µM). Viability was assessed according to the (A) neutral red (NR); (B) sulforhodamine B (SRB) and (C) lactate dehydrogenase (LDH) assay after 24, 48 and 72 h. Data was normalized to control and represent mean ± SD, n = 4. Statistically significant differences (p < 0.05) are indicated by different letters (a,b).
Mentions: According to the NR assay performed after the final TEER measurement (Figure 3b), we observed that neither DON (1–20 µM), nor the combination of DON (1–20 µM) + U0126 (10 µM), significantly reduced viability after 72 h. However, to further investigate whether U0126 is able to protect against DON-induced cytotoxicity, a cytotoxic dose of DON (50 µM) was applied to differentiated IPEC-J2 in the absence or presence of U0126. Subsequently, viability was assessed via the NR (lysosomal activity), SRB (total protein content) and the LDH (membrane integrity) assay after 24, 48 and 72 h (Figure 5). According to the NR and SRB assay, DON induced significant viability reductions after 24 h (NR: −27%; SRB: −60%), 48 h (NR: −61%; SRB: −81%) and 72 h (NR: −81%; SRB: −87%). Addition of U0126 did not counteract the cytotoxic effect of DON, producing similar viability reductions after 24 h (NR: −31%; SRB: −56%), 48 h (NR: −56%; SRB: −81%) and 72 h (NR: −85%, SRB: −92%), as cells treated with DON alone. According to the LDH assay, DON (+/− U0126) was not cytotoxic after 24 h. After 48 h and 72 h, viability was significantly reduced in both DON−treated (48 h: −33%; 72 h: −50%) and DON + U0126-treated (48 h: −26%; 72 h: −46%) cells. Thus, U0126 did not counteract or reduce DON−induced toxicity over an incubation period of 72 h, showing a similar time-dependent cytotoxicity profile as 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