Table 10: Mucosal uptake and serosal release of DON at in vitro DON concentrations of 4000 and 8000 ng/ml over one hour incubation period; means ± SD; n = 4 pigs/group
DON in vitro DON in feed ip LPSa ∆ Amount of DON (µ g/h) (ng/ml) (mg/kg feed) (µg/kg BW) Mucosal side Serosal side
4000 0 0 34.563 ± 3.78 -0.075 ± 0.06
4000 0 5 29.726 ± 2.67 -0.087 ± 0.04
4000 2.9 0 23.771 ± 3.76 -0.068 ± 0.15
4000 2.9 5 23.522 ± 3.53 -0.123 ± 0.06
8000 0 0 61.616 ± 8.42 -0.160 ± 0.16
8000 0 5 58.940 ± 7.03 -0.137 ± 0.12
8000 2.9 0 48.864 ± 5.39 -0.234 ± 0.09
8000 2.9 5 42.414 ± 9.14 -0.325 ± 0.14
ANOVA (p values)
DON in vitro < 0.0001 < 0.0001
DON in feed < 0.007 0.030
Ip LPS 0.369 0.265
DON in vitro*DON in feed 0.071 0.034
DON in vitro *ip LPS 0.725 0.946
DON in feed*ip LPS 0.890 0.170
DON in vitro*DON in feed*ip LPS 0.173 0.530
Figure 11: Plasma DON concentrations [ng/ml; dots: medians, boxes: 25%, 75%, whiskers: min, max] in different groups; n = 4 pigs/group
Figure 12: Plasma TNF-α concentrations [pg/ml; dots: medians, boxes: 25%, 75%, whiskers: min, max] in different groups; n = 4 pigs/group
6. General Discussion
Electrogenic transport of alanine and glucose was assessed across porcine small intestines using Ussing technique. Both nutrients are cotransported with sodium ions via different transport systems thus including changes in Isc (AWAD et al.2004;2009; HOPFER et al.1973;
SIGRIST-NELSON et al.1975). In our study, the mucosal addition of alanine as well as glucose increased the Isc across different segments of the small intestine of growing pigs demonstrating their stimulating effects on Na+ coupled cotransporters. Similar results were obtained after mucosal addition of 5 mmol/L of glucose across jejunal mucosa of laying hens and the electrical response peaked at about 1 min after glucose addition (AWAD et al. 2005a).
In the same way, alanine stimulated absorption of Na+ and induced the Isc across porcine small intestine with maximal ∆Isc at mucosal concentration of 20 mmol/L alanine (GRØNDAHL and SKADHAUGE 1997). Addition of an amino acid to the mucosal side of the tissues induced the Isc and further addition of an active sugar such as glucose resulted in further enhancement of the short circuit currents (SCHULTZ andZALUSKY 1964b), indicating that the active transport of glucose stimulated the active transport of Na+ (SCHULTZ and ZALUSKY 1963). This increase diminished with time due to a gradual decrease in the active sugar and Na+ transport because the active sugar transport across the small intestine was a saturated function of the mucosal sugar concentration (SCHULTZ andZALUSKY 1964b). In the present work, ileum showed highest transporting response to nutrients, while duodenum and mid jejunum were higher under basal conditions. In domestic fowl colon exhibited highest Isc, while in White Leghorn chickens, rectum showed significantly higher Isc than the rest of intestine (GRUBB et al. 1987; AMAT et al.1999). The behaviour of jejunum and ileum was relatively similar, while duodenum showed higher resistance in both studies owing to relative tightness of the duodenum with lower paracellular permeability compared with other intestinal segments (GRUBB et al. 1987; AMAT et al.1999). Mid and distal parts of porcine small intestines expressed higher Isc compared with the proximal parts under basal and stimulated conditions (GRØNDAHL and SKADHAUGE 1997). Our study and the previous studies proved that there was a regional variation in the electrogenic transport of nutrients. This difference could be attributed to the species involved and the variation in the amount of cotransporters at the mucosal surface of the intestinal tissues.
To estimate the effect of DON on the electrophysiological parameters of porcine small intestines, DON was added in vitro in concentrations up to 8000 ng/ml to the mucosal side of the tissues. At DON concentrations of 4000 and 8000 ng/ml reductions in short circuit currents of alanine and glucose were observed across jejunal epithelia of growing pigs. This result reflected the inhibiting effect of DON on the electrogenic transport of both nutrients mainly via inhibition of the cotransporter’s protein. At higher concentrations of DON (10 µg/ml) the uptake of glucose was reduced across jejunal epithelia of laying hens and young chickens (AWAD et al. 2007a; 2009). Reduction in currents in a dose-dependent manner were observed across jejunal mucosa of laying hens at in vitro DON concentrations of 1000, 5000 and 10000 ng/ml (AWAD et al. 2005a). The mechanism by which DON could exert its inhibiting effect on nutrient transport was similar to that of phlorizin, a specific SGLT-1-inhibitor (AWAD et al. 2007a). The difference in DON concentrations between our study and others reflected higher sensitivity of pigs to DON than poultry. An inhibiting effect was observed on L-serine uptake at concentration of 10 µmol DON /L in HT-29-D4 human intestinal epithelial cells (MARESCA et al. 2002). Addition of 10 µg DON/ml after addition of L-proline to the luminal side resulted in reduction in Isc across jejunal mucosa of laying hens (AWAD et al. 2005b). DON as an inhibitor of protein synthesis has an inhibiting effect on Na+ -dependent transporters especially SGLT-1 and Na+/alanine co-transporter, evidenced by reduction in Isc values across porcine intestinal segments.
On the other hand, DON reduced the transepithelial electrical resistance (TEER) in a dose-dependent manner under basal conditions, suggesting disruption of the intestinal barrier that guards the paracellular transport which in turn can increase the paracellular permeability.
Such inhibition was observed after 24h incubation of Caco-2 monolayer with different in vitro DON concentrations (SERGENT et al. 2006), suggesting that DON disrupt the integrity of the intestinal barrier. At 10 µmol DON/L (2963.2 ng/ml) TEER was reduced to about 57% in treated HT-29-D4 cells, while at 100 µmol DON/L (29632 ng/ml) TEER was abolished (MARESCA et al. 2002). DON reduced TEER across IPEC-1 cells in a dose- and time-dependent manner with an increase in the paracellular flux of FITC-dextran (PINTON et al.
2009) due to reduction in claudin expression that is important to the tight junction barrier function (PINTON et al. 2010).
Feeding DON at 2.9 mg/kg diet had neither an effect on the electrophysiological parameters nor on weight gain, although the values of Isc were numerically lower and TEER was reduced under basal and nutrient stimulated conditions but without significant effect, indicating that this concentration was relatively small to affect the animals. Feeding of DON at 5.7 mg/kg feed for 5 weeks also showed no effect on weight gain of pigs or on Isc of glucose across pig jejunum (ZERULL et al. 2005). In mice, DON at 10 ppm for 6 weeks did not affect leucine uptake (HUNDER et al. 1991). Broilers and young chickens fed on 10 mg DON/kg feed for 6 weeks showed no influence on weight gain of the birds but glucose uptake was lower than controls (AWAD et al. 2004; 2009), suggesting that DON in feed has an inhibitory effect on glucose transport. At concentrations somewhat similar to our, feeding of 2.8 mg DON/kg diet reduced weight gain of pigs in the first week of the experiment that continued for 4 weeks (WACHÉ et al. 2009), while feeding up to 3.0 mg DON/kg significantly inhibited weight gains of pigs in the initial 7 days of exposure (ROTTER et al. 1994). However, at the end of the experimental trial the overall gains did not differ from that of the control, suggesting adaptation of pigs on the naturally contaminated diet. The concentration of DON in feed, the exposure time, species of the animal and kind of feed, all considered as factors can interact with the effect of oral DON on the tested parameters.
Intraperitoneal injection of 5 µg LPS/kg BW recorded no direct effect on Isc or Gt, although Isc values were higher compared with other groups. By contrast, LPS inhibited Na+ -dependent D-galactose intestinal absorption and L-leucine active transport across jejunal mucosa of rabbits (AMADOR et al. 2008; ABAD et al. 2001). This was explained by the action of LPS on the activity of Na+/K+-ATPase at the basolateral membrane, which maintain the activity of Na+-dependent transporters of sugars and amino acids. We assume that LPS exerted this effect on Isc indirectly via induction of other mediators. Generally, systemic application of LPS induces proinflammatory cytokine secretion such as TNF-α, 1ß and IL-6 (WEBEL et al. 1997; MOYA et al. 2006). Several cytokines are expressed by the intestinal cells but markedly upregulated in response to microbial infection (MAYRHOFER 1995; JUNG et al. 1995). The action of the cytokines on the intestinal epithelial barrier was previously assessed (MCKAY and BAIRD 1999; AL-SADI et al. 2009) revealing that some cytokines like TNF-α increased the tight junction permeability through reduction in TEER (MA et al. 2004) via activation of the transcription factor NF-κB. TNF-α was found to initiate a reduction in
TEER at 5 ng/ml and reached maximum reduction at concentrations between 50 and 100 ng/ml at 48h incubation with Caco-2 BBE human cell sheet (MARANO et al. 1998). TNF-α reduced TEER from 376 ± 26 to 73 ± 8 Ω.cm2 after 8h incubation with HT-29/B6 colonic epithelial cell line, suggesting that TNF-α altered the structure and functions of the tight junction (SCHMITZ et al. 1999). In order to support our assumption, plasma samples from all pigs were analyzed for the proinflammatory cytokines. A significant rise in plasma TNF-α concentrations in DON/LPS group was found compared with control and DON groups and reach maximum levels in LPS group compared with other groups, suggesting that the effect of LPS could be mediated, in part, by the action of TNF-α. However, a direct effect of LPS was reported on SGLT-1-transfected Caco-2 cells and was represented by higher uptake of alpha methyl glucopyranoside (α-MG), a specific SGLT-1 sugar (YU et al. 2005) under high glucose media. After infusion of LPS, Na+-dependent glucose transport was reduced across ileum in Yorkshire pigs and was induced in Meishans pigs (ALBIN et al. 2007). Collectively, the response of the intestinal tissue to LPS may differ according to the type of nutrient examined, the method of LPS application and the breed of the animal.
During Ussing experiments, buffer samples were collected from both the mucosal and serosal sides at different time points to estimate the transport of DON across porcine jejunal mucosa in vitro. Samples that collected at 30 min and 90 min were used to achieve one-hour incubation period at concentrations of 4000 and 8000 ng/ml. We found that the transport of in vitro DON was proportional to its concentration, agreed with AWAD et al. (2007a) in which the transport of DON was assessed at 1000, 5000 and 10000 ng DON/ml across jejunal epithelia of laying hens. The mucosal uptakes of DON were higher than its serosal releases, suggesting either tissue accumulation of DON or substantial DON metabolism. A one hour incubation period is a short time period to evaluate the behaviour of DON inside tissues. In a previous study, DON after a single iv dose (1 mg/kg) was detected in porcine intestine in the first 19.8 min in few amounts (PRELUSKY and TRENHOLM 1991), but the concentrations of DON reduced gradually over 24h incubation period suggesting no accumulation of DON. On the other hand, metabolism of DON in pigs was assessed revealing that DON is poorly metabolised (GOYARTS and DÄNICKE 2006; PRELUSKY et al. 1988). It could be metabolised to less toxic compounds such as DOM-1 (RAZZAZI et al. 2002), DON metabolite and de-epoxy DON metabolite (DÄNICKE et al. 2004b) and glucuronide conjugate of DON (DÖLL et al.
2009b). Plasma analysis for the presence of potential DOM-1, the de-epoxydized product of DON, revealed that DON had not been transformed to DOM-1. However, other metabolisable compounds of DON could be found but were not detected.
Though our concentration of DON in feed was small, it affected on both, the mucosal uptake and serosal release of in vitro DON. Lower mucosal and serosal amounts in DON-fed groups compared with controls at the two in vitro DON concentrations. This result suggestd disruption in the integrity of the intestinal barrier and in turn increased the paracellular passage of in vitro DON. DON at 4000 ng/ml reduced TEER in IPEC-J2 after 24h incubation period (DIESING et al. 2011b) and in DON-treated IPEC-1 cells at 30 µmol/L DON (PINTON
et al. 2010). TEER was reduced in a dose-dependent manner across human Caco-2 cells and IPEC-1 cells (PINTON et al. 2009). The mechanism by which DON reduced TEER is via reduction in the expression of the tight junction proteins, claudins and ZO-1.
The interaction between DON and LPS was assessed for induction of apoptosis or proinflammatory cytokine secretion (ISLAM AND PESTKA 2006; DÖLL et al. 2009a; ZHOU et al.
1999; 2000). Both oral DON and ip LPS were able to induce secretion of TNF-α, IL-1ß and IL-6 in mice (ZHOU et al. 1999; ISLAM andPESTKA 2006). In porcine alveolar macrophages, DON and LPS synergistically induced TNF-α and IL-1ß secretion (DÖLL et al. 2009a). In the present study, we examined the interaction effect between DON in feed and ip LPS at the intestinal barrier on the electrophysiological parameters and on DON transport in vitro. In LPS group there were higher Isc values compared with other groups followed by DON/LPS group, but without significant interaction effect. Tissue conductances were increased in DON/LPS group compared with control group, reflecting lower TEER. This effect became significant after addition of glucose. Therefore, we could assume that these observations could be due to the systemic action of the toxins under investigations. Both, DON and LPS induced TNF-α secretion, which might play a partial role at the intestinal barrier. On the other side, in DON transport study, the mucosal uptake of DON in DON/LPS group was nearly similar to that in DON group, reflecting that LPS had no obvious effect on DON transport across porcine jejunal epithelia.
7. Conclusions
The present study proved glucose and alanine induced Isc across porcine small intestines especially across ileum. DON was able to inhibit the electrogenic transport of alanine and glucose across porcine jejunal mucosa. DON as well as LPS was able to induce plasma TNF-α secretion. The permeability of the intestinal barrier could be indirectly increased after ip LPS treatment with further studies needed to assess the effect of TNF-α on the tight junction barrier in pigs. The transport of DON was proportional to its initial concentration with a respective effect of DON in feed on DON transport. DON might be accumulated in porcine epithelia under short incubation period with further investigation of DON transport over longer incubation period was advised. A synergistic interaction effect was found between oral DON and ip LPS on Gt across porcine jejunal tissues, but more investigations are needed to evaluate such interaction at the intestinal barrier.
8 Summary
Amal Halawa (2012)
Toxicological and immunological effects of DON and LPS at the intestinal barrier
The objectives of this study were to examine the effect of DON either in feed or in vitro as well as intraperitoneal LPS on the short circuit currents and tissue conductances across the small intestines of growing pigs in vitro, evaluation of DON transport in vitro across jejunal mucosa of growing pigs and estimation of the immunological effect of DON as well as LPS to induce cytokine secretion.
The study was done in two series, in the first series the effect of different in vitro DON concentrations on Isc and Gt was investigated in six animals across duodenum, mid jejunum and ileum using Ussing technique. In the second series segments from mid jejunum were collected from 16 animals. The animals were divided into two groups, control and DON-fed groups. In DON-fed group, the animals were kept on diet containing 2.901 mg DON/kg feed for about 5 weeks. From each group four animals were injected intraperitoneally with 5 µg LPS/kg BW 3h before slaughtering. Blood samples were collected from all animals directly after slaughtering for plasma DON and cytokine analysis.
Buffer samples were collected at 30 and 90 min after addition of DON from both, mucosal and serosal sides of Ussing chambers for measuring of DON concentrations using LC-ESI-MS/MS technique.
Under basal conditions, duodenum and jejunum showed higher Isc compared with ileum which expressed higher Isc values under nutrient stimulated conditions. Among the examined concentrations, in vitro DON at 4000 and 8000 ng/ml reduced the electrogenic transport of both alanine and glucose across jejunal mucosa of growing pigs. Feeding DON at 2.901 mg/kg feed did not affect significantly the Isc or Gt across porcine intestinal segments. In LPS groups the Isc values were higher compared with non-injected groups, but without significant effect of LPS. Plasma analysis revealed higher TNF-α concentrations in LPS-injected groups compared with control and DON groups. As expected, higher plasma DON concentrations were found in DON-fed groups compared with controls.
In DON transport study, lower mucosal amount of DON was observed at 4000 ng/ml compared to that at 8000 ng/ml in all groups. The mucosal uptake of DON in DON-fed groups was significantly higher than that in control groups. Intraperitoneal LPS did not affect on DON transport.
Results of the first series showed that the response of porcine small intestines to the electrogenic nutrient transport varied with the type of the intestinal segments particularly ileum.
In the second series, DON inhibited the electrogenic transport of alanine and glucose across jejunal mucosa of growing pigs. Both DON and LPS could induce secretion of the proinflammatory cytokines especially TNF-α. Systemic LPS tend to increase the Isc propably via the action of TNF-α, the point which is recommended to be evaluated in pigs. DON transport was proportional to its initial concentrations, with a respective effect of DON in feed on DON transport.
9. Zusammenfassung
Amal Halawa (2012)
Toxikologische und immunologische Effekte von DON und LPS auf die intestinale Barriere
Ziel dieser Studie war es, die Effekte von in vivo verabreichten DON-haltigen Futtermitteln, direkt in vitro appliziertem DON und in vivo intraperitoneal verabreichtem LPS auf die Kurzschlussströme und Gewebeleitfähigkeiten im Dünndarm wachsender Schweine zu untersuchen. Darüber hinaus wurden in vitro Messungen zum Transport von DON über das Darmepithel durchgeführt und eine Einschätzung der immunologischen Wirkung von DON und LPS vorgenommen, in dem die Stimulation der Zytokinsekretion in vivo quantifiziert wurde.
Die Studie wurde in zwei Serien durchgeführt. In der ersten Serie wurden Abschnitte aus dem Duodenum, dem mittleren Jejunum und dem Ileum von sechs Schweinen verwendet, um die in vitro Effekte von DON in verschiedenen Konzentrationen auf die Kurzschlussströme und die Gewebeleitfähigkeiten des Darmepithels in An- und Abwesenheit von Alanin und Glucose mithilfe der Ussing-Kammer-Technik zu untersuchen. In der zweiten, mit 16 Schweinen durchgeführten Serie wurden ausschließlich Segmente aus dem mittleren Jejunum verwendet. Die Tiere wurden in zwei Fütterungsgruppen aufgeteilt. Die DON-Gruppe erhielt über einen Zeitraum von zwei Wochen eine Ration mit 2,901 mg DON / kg, während die anderen Tiere mit Kontrollfutter versorgt wurden. Drei Stunden vor dem Schlachten wurden je vier Tiere der beiden Fütterungsgruppen mit 5 µg LPS / kg intraperitoneal behandelt.
Unmittelbar nach dem Töten wurden Plasmaproben zur Bestimmung von DON- und Zytokinkonzentrationen sowie das Jejunum für die Using-Kammer-Experimente entnommen.
Um den Transport von DON über das Epithel zu quantifizieren, wurden die DON-Konzentrationen in den serosalen und mucosalen Kompartimenten der Ussing-Kammer nach 30 und 90 min Inkubationszeit mithilfe der LC-MS/MS Technik analysiert.
Unter basalen Bedingungen zeigten das Duodenum und das Jejunums höhere Kurzschlussströme als das Ileum. Nach Zugabe der Nährstoffe Alanin und Glucose konnten
die höchsten Werte im Ileum beobachtet werden. Eine in vitro Applikation von 4000 bzw.
8000 ng DON / ml reduzierte die Alanin- und Glucose-induzierten Kurschlussströme im Jejunum signifikant, während die Fütterung des DON- kontaminierten Futters keinen signifikanten Einfluss auf die elektrophysiologischen Parameter hatte. Die Behandlung mit LPS führte zu einer Erhöhung der TNF-α-Konzentrationen im Plasma. Die Nährstoff-induzierten Kurzschlussströme behandelter Tiere waren im Vergleich zu unbehandelten Schweinen jedoch nur tendenziell höher.
Generell korrelierte der Transport von DON über das Epithel mit der mucosalen Konzentration von DON, wobei die mukosale Aufnahme von DON bei zuvor zwei Wochen
Generell korrelierte der Transport von DON über das Epithel mit der mucosalen Konzentration von DON, wobei die mukosale Aufnahme von DON bei zuvor zwei Wochen