Interactions between DON in feed, intraperitoneal LPS challenge and DON in vitro61

0.114 µEq.cm^-2.h^-1 and 24.33 and 28.84 mS.cm^-2,respectively with lowest Isc at in vitro DON concentration of 8000 ng/ml. After nutrients stimulation the Isc increased for alanine and glucose with higher increase in the control group (0 ng DON /ml) and lower increase at 8000 ng/ml in vitro DON. In the presence of DON in feed the respective data decreased in DON-fed groups at all in vitro DON concentrations especially at the later in vitro DON concentration without significant interaction effect between DON in feed and in vitro. On the other hand, intraperitoneal LPS resulted in an increase in Isc values at all in vitro DON concentrations compared to non-treated groups with lowest Isc values at 8000 ng DON/ml and non significant interaction effect. For evaluation of the interaction effect between DON in feed and ip LPS, the mean basal short circuit currents and conductances ranged between -0.243 and 0.006 µEq.cm^-2.h^-1 and 25.79 and 29.60 mS.cm^-2, respectively with lowest Isc in DON-fed group without significant effect. After mucosal addition of alanine and glucose up to 300-fold increases in Isc were observed with higher increases in LPS group compared with DON-fed groups without significant effects. A significant increase in Gt was observed in response to glucose compared with other groups. DON-feeding reduced LPS-induced Isc at all in vitro DON concentrations with lowest Isc at in vitro DON concentration of 8000 ng/ml without significant interaction effect between DON in feed, ip LPS and in vitro DON (Table 9).

4.5 Discussion

The present work proved that both alanine and glucose are able to stimulate Isc across intestinal mucosa especially across ileal tissues of pigs. Such findings are in agreement with

previous studies in which alanine and/or glucose were added to the mucosal side of porcine small intestines or the proximal jejuna of laying hens suggesting their stimulating effects in transepithelial transport of Na⁺ (AWAD et al. 2005a; GRØNDAHL and SKADHAUGE 1997). Mid and distal parts of porcine intestine were found to have significantly higher basal Isc and after addition of amino acids compared with the proximal parts (GRØNDAHL and SKADHAUGE

1997). While jejunum and ileum expressed relatively similar short circuit currents in domestic fowl (GRUBB et al. 1987). Higher transporting activity of porcine ileum to nutrient transport could be due to variation in the amount of the mucosal transporter proteins along the intestine.

In our study we focused on the effect of DON on electrogenic transport of nutrients, alanine and glucose, across porcine small intestines with different application methods of DON.

By increasing doses of in vitro DON in both series, only concentrations at 4000 and 8000 ng/ml were able to inhibit the transport of both nutrients with much reduction at the latter concentration suggesting an ability of DON to inhibit the electrogenic transport of both glucose and alanine. This ability could be attributed to the action of DON, as an inhibitor of protein synthesis, on glucose and alanine co-transporters proteins at the mucosal surface.

These findings agreed with other studies in which glucose uptake was inhibited from 3.28 ± 0.53 to 1.81 ± 0.24 nmol.cm^-2.min at 10 mg DON/L (10x10³ ng/ml) in laying hens and from 33 ± 7.75 to 26 ± 7.49 µA/cm² at 10 µg/ml (10x10³ ng/ml) across jejunal tissues of young chickens (AWAD et al. 2007a; 2009). In the present study, the inhibiting effect of DON was increased with increasing its in vitro concentration, reflecting that the action of DON depends on its concentration. DON at concentrations of 1, 5 and 10 µg/ml (1000, 5000 and 10x10³ ng/ml) reduced the short circuit currents in a dose dependent manner compared with basal values and values after addition of glucose across jejunal mucosa of laying hens (AWAD et al.

2005a). This effect of DON could be attributed to the inhibitory effect of DON on the transporter protein, SGLT-1, in a mechanism similar to that of phlorizin (AWAD et al. 2007a).

DON at concentration of 10 µg/ml (10x10³ ng/ml) inhibited the stimulated Isc after addition of 1 mmol/L of L-proline and returned to basal values (AWAD et al. 2005b). It was concluded that DON inhibited Na⁺/L-proline co-transport. Although our concentrations were lower compared to that used in the previous studies, but pigs are more sensitive to DON than other animal species due to the rapid and efficient absorption of DON (ROTTER et al. 1996). At the cellular level DON 10 µmol/L (2963.2 ng/ml) exhibited an inhibitory effect on L-serine

uptake across human intestinal epithelial cells (HT-29-D4) (MARESCA et al. 2002). While at higher concentration 100 µmol/L L-serine uptake was increased and SGLT-1 activity were inhibited compared to control cells, suggesting the selective action of DON. DON reduced TEER in a dose-dependent manner under basal conditions at 4000 and 8000 ng/ml, suggesting that DON affect on the integrity of the intestinal barrier. In human Caco-2 cells and porcine IPEC-1 cells non-cytotoxic doses of DON reduced TEER in a dose dependent manner with an increase in the paracellular flux of FTIC-dextran and higher sensitivity of porcine cell line than human intestinal cell line (PINTON et al. 2009; 2010). Such reduction was attributed to reduction in the expression of the tight junction proteins.

Preceding the in vitro assessment of DON on the electrophysiological properties of porcine small intestines, we applied a feeding trial to evaluate the effect of oral DON on Isc and Gt across porcine jejunal mucosa, in which pigs fed on DON-contaminated wheat for ~5 weeks.

Although the values of Isc in DON-fed group were lower than control and TEER was lowered, but the effect of oral DON was not significant. This could suggest that oral DON did not affect directly on the electrophysiological parameters under investigation. However, in a previous study in which a higher DON concentration (5.7 mg DON/kg feed) was applied, glucose uptake across pig jejunum was not affected (ZERULL et al. 2005). On the contrary, in case of broilers and young chickens fed on 10 mg DON/kg, oral DON induced an inhibitory effect on glucose uptake (AWAD et al. 2004; 2009). Although pigs are more sensitive to DON than chickens but the amount of DON used in the present study was much lower than that in the previous studies, proving that presence of 2.9 mg DON/kg feed has no direct impact on electrogenic nutrients transport in pigs especially with development of a kind of adaptation to the contaminated feed with prolonged exposure period (ROTTER et al. 1995).

It was proved that LPS was able to reduce the intestinal absorption of different nutrients such as galactose, fructose and leucine either after intravenous injection or by direct contact with the tissues (ABAD et al. 2001; AMADOR et al. 2007; 2008; GARCIA-HERRERA et al. 2003;

2008). This was attributed to the inhibitory effect of LPS on the mucosal transport systems and reduction of Na⁺/K⁺-ATPase at the basolateral membrane which maintain the activity of Na⁺-dependent transporters of sugars and amino acids. In contrast, in the present work ip LPS did not show significant effects but instead it increased the Isc across jejunal tissues in the 2^nd series compared with the control group and highest increases were observed in LPS group.

This augment in short circuit currents reflected higher transporting activity of the jejunal preparations. The increase in the transporting activity could be attributed to a reduction in TEER that resulted in disruption in tight junction barriers guarded the paracellular transport and in turn increased Isc. We assuming that, LPS affected indirectly on TEER via other mediators that secreted in response to the systemic action of LPS. Our assumption depending on previous studies which proved that LPS was able to induce cytokine secretion such as SGLT-1 sugar, was increased in the cells exposed to LPS under high glucose media (YU et al.

2005).

In our study, DON/LPS group has higher Isc responses compared to control and DON groups, without significant interaction effect. Only one exception, a significant interaction effect between oral DON and ip LPS on tissue conductances was found after addition of glucose, reflecting a synergistic action to decrease TEER and suggesting that LPS magnify the action of DON on TEER especially oral DON alone had no direct effect on TEER. This could assume that the presence of another toxin, like LPS, could increase the effect of small concentrations of oral DON on electrogenic transport or tissue conductances. To our Knowledge, our study is the first study that assessed the interaction effect between oral DON and ip LPS on electrophysiological parameters in growing pigs. Other studies estimated the synergistic effect between oral DON and ip LPS in induction of the proinflammatory cytokines TNF-α and interleukin 1β (IL-1β) in mice (ZHOU et al. 1999; ISLAM and PESTKA

2006) compared with administration of either of them. Suggesting that LPS reduced the minimum dose of DON needed to induce cytokine response, increased the toxic effects of DON and increased the sensitivity to DON and supporting our suggestion in magnification of the effect of small concentrations of DON.

This study proved the ability of in vitro DON to inhibit the electrogenic transport of nutrients across jejunal epithelia of growing pigs. A synergistic effect between DON and LPS was observed evidenced by reducing TEER in response to glucose. The response of porcine small

intestine to nutrient transport varied with the intestinal region. More investigations are needed to evaluate the interaction between DON and LPS in pigs. Further assessment of the systemic action of LPS in pigs is needed to declare its mechanism of action to increase nutrient uptake across porcine intestines.

Acknowledgements

The assistance of the co-workers of the Institute of Physiology in Hannover in performing Ussing experiments, and of the Institute of Animal Nutrition in Braunschweig in performing the analysis is gratefully acknowledged. The authors thank the "Deutsche Forschungsgemeinschaft" (DFG) for financial support (DA 558/1-3).