4.6 Tables
5.3.3 Analyses
DON was purchased from (Sigma-Aldrich D-0156, München, Germany) diluted in isotonic saline. The buffer solutions (Modified Krebs-Henseleit solution) that bathed the mucosal and serosal surfaces of the epithelial tissues consisted of (mmol/L): mucosal side: 113.6 NaCl; 5.4 KCl; 0.25 HCl; 1.2 MgCl2.6 H2O; 1.2 CaCl2.2 H2O; 21.0 NaHCO3; 1.2 Na2HPO4.2 H2O; 0.3 NaH2PO4.H2O; and 31.96 mannitol. For the serosal side, the buffer consisted of: 113.6 NaCl;
5.4 KCl; 0.4 HCl; 1.2 MgCl2.6 H2O; 1.2 CaCl2.2 H2O; 21.0 NaHCO3; 1.2 Na2HPO4.2 H2O;
0.3 NaH2PO4.H2O; 23 mannitol and 10 glucose. All solutions were prepared with Indomethacin (10-5M) and were adjusted to an osmolality of 300 mosmol/kg with mannitol.
The buffers were continuously gassed with carbogen (95% O2, 5% CO2) at 38.4°C throughout the experiment.
2.3.2. Buffer samples
After de-freezing, the samples were mixed with vortex and an aliquot of 500 µl of each sample was diluted in equal volume of water. The total volume of the diluted sample was transferred to a ChemElut cartridge 1 ml (Varian, Nr. 12198002). After about 5 min during which the sample was absorbed and distributed within the column, the sample was extracted with 10 ml ethyl acetate and the collected extract was evaporated to dryness on a rotary evaporator. The extract was redissolved in 200 µl acetonitrile/water mixture (13/87, v/v) in an
ultrasonic bath for 5 min. The extract was filtered using PVDF-membrane filter (0.45 µm, 4 mm, Amchro) and kept frozen at -20°C until analysis.
Samples were analysed by LC-ESI-MS/MS (liquid chromatography-electrospray ionization tandem mass spectrometry, API 4000 QTrap, Applied Biosystems, Darmstadt, Germany, coupled with a 1200 series HPLC system, Aligent Technologies, Böblingen, Germany) as (HPLC) with diode-array detection (DAD) after clean-up with immunoaffinity columns (IAC) according to (VALENTA et al. 2003) with slight modifications. DON was analyzed as total DON (free plus conjugated) after incubation with b-glucuronidase. Briefly, after adding of one mL of sodium acetate buffer (pH 5.5) to 1.5 mL of plasma, the mixture was incubated for 16 hours with 6000 U of β-glucuronidase (Sigma, G 0876). The samples were extracted with ethyl acetate on a ChemElut cartridge (Varian, Middelburg, Netherlands), cleaned up by IAC (DONtest HPLC®, VICAM, Watertown, MA, USA) and measured by HPLC-DAD. The mean recovery of DON was 91% ± 8% (n=12; 10-30 ng/ml). The results for mycotoxin analyses the initial and final DON concentrations with the respective buffer volumes. The differences between the beginning and the end was calculated and presented as ∆ amount of DON [µg/h]
at both the mucosal and the serosal side, thus providing data on mucosal uptake and serosal release. Those data were analysed using 3-factorial ANOVA design (DON in feed, ip LPS and DON in vitro) including their interactions. Live weight gain was evaluated by simple t-test. The results were presented as means, standard deviations and probabilities for the main
effects and interactions. Plasma DON and TNF-α levels were evaluated by the Mann-Whitney-U test and presented as box whisker plots with medians, 25th and 75th percentile and minimum and maximum values.
5.4 Results
5.4.1 Effect of feeding DON on live weight gain
Live weight gain for the control group was 0.677 ± 0.117 g/d and that of DON-fed group was 0.649 ± 0.252 g/d without significant effect of DON in feed (P < 0.782).
5.4.2 Plasma DON and TNF-α concentrations
Plasma samples from all pigs were analysed for DON and TNF-α concentrations. The median concentrations of plasma DON in control and LPS groups were 3.03 ± 1.3 and 1.65 ± 1.2 ng/ml, respectively. A significant rise in plasma DON concentrations was observed in DON-fed group and DON/LPS group to 16.93 ± 6.7 and 16.43 ± 3.7 ng/ml, respectively (Figure 11). While plasma TNF-α concentrations ranged between 55.49 ± 14.45 and 774.71 ± 1126.9 pg/ml, with highest plasma TNF-α concentration in LPS group. Plasma TNF-α concentration in DON/LPS group was higher than that in control and DON groups (Figure 12).
5.4.3 DON transport study
5.4.3.1 The main effect of in vitro DON concentration
The mucosal uptake and serosal release of DON was assessed at two different in vitro DON concentrations (4000 and 8000 ng/ml) over one hour incubation period. The mean mucosal amounts of DON were 27.836 ± 5.7 and 52.265 ± 10.5 µg at 4000 and 8000 ng/ml, respectively, with a significant effect of in vitro DON concentration (Table 10). While the respective data for serosal amounts were -0.087 ± 0.085 and -0.213 ± 0.144 µg with a significant effect of DON in vitro.
5.4.3.2 The main effect of DON in feed
DON was fed to the animals at 2.901 mg/kg diet. The mean mucosal amount of DON was significantly reduced from 44.7 ± 15.3 in control animals to 34.4 ± 12.7 µg in DON-fed animals, respectively (Table 10). Serosal amounts also showed a significant reduction in DON-fed animals from -0.115 ± 0.106 to -0.188 ± 0.151 µg.
5.4.3.3 The main effect of ip LPS
The animals were injected intraperitonealy with 5 µg LPS/kg BW 3h before slaughtering. The mean mucosal amounts of DON were 40.9 ± 14.98 and 37.4 ± 14.65 µg in control and LPS-treated animals, respectively without significant effect of LPS. The respective data for serosal amounts were -0.131 ± 0.134 and -0.169 ± 0.133 µg without significant effect.
5.4.3.4 Interaction effects
The mucosal amounts of DON tend to decrease in DON-fed group at both in vitro DON concentrations. The serosal amounts of DON were declined in DON-fed animals with a significant interaction effect between DON in vitro and DON in feed (Table 10).
5.5 Discussion
5.5.1 Effect of oral DON on live weight gain
In the present study the amount of analysed DON in wheat was 2.901 mg/kg and the respective amount of DON in control wheat was 0.335 mg/kg. No difference in the live weight gain between control and DON-feeding groups was found. Feeding DON at concentrations ranging between 3 and 19.1 mg DON/kg feed resulted in lower feed intake as well as lower weight gains soon after starting of feeding (ROTTER et al. 1994; TRENHOLM et al. 1994; PRELUSKY et al. 1994; ROTTER et al. 1995) but the animals were able to adapt at the end of the feeding period. The reductions in both parameters remained over the experimental period in pigs fed on DON-naturally contaminated diet containing 1.7 and 3.47 mg DON/kg feed (BERGSJØ et al. 1993). While piglets receiving lower DON concentrations (≤ 1.2 mg/kg) showed no effect on feed intake and no great variation in weight gain (Drochner et al. 2004).
Although feed intake was depressed at 2 mg DON/kg (HOUSE et al. 2002) it did not affect average daily gain. From the previous studies, by increasing concentrations of DON in feed, the feed intake could be reduced and in turn the growth rate was also reduced (DERSJANT-LI et al. 2003). However, the effect of oral DON on feed intake or weight gain is transient and can be recovered afterward (FOSTER et al. 1986; ROTTER et al. 1994; FERRARI et al. 2009), especially with small concentrations of DON.
5.5.2 Plasma DON and TNF-α
Significant plasma DON concentrations were found in DON-fed groups compared to the groups fed the virtually DON-free control feed. DON was also detected in serum of pigs fed
on 3.7, 5 and 9.57 mg DON/kg diet (DÄNICKE et al. 2005; HE et al. 2009) DON appeared in plasma in a dose-dependent manner (DÄNICKE et al. 2004b), rapidly and efficiently absorbed and thus early detected in plasma (DÄNICKE et al. 2004a; VIDEMANN et al. 2007). Presence of modestly DON levels in plasma of the control groups reflects the natural DON background level measured in the control diet (Table 5).
In our study, intraperitoneal injection of 5 µ g LPS/kg BW significantly induced TNF-α 3h post injection compared with unchallenged control groups. Agreed with another study in which the same concentration was injected in pigs and plasma TNF-α level increased 2h post injection (WEBEL et al. 1997). Similar systemic stimulations of LPS to induce TNF-α levels were observed at 100 µ g LPS/kg BW at 2 and 4h and at concentrations up to 2000 µg/kg BW at 1h in pigs in a dose-dependent manner (WRIGHT et al. 2000; NORIMATSU et al. 1995). Also in vitro studies proved that 100 ng LPS/ml induced TNF-α mRNA at 2 and 7h in murine macrophage RAW 264.7 cell line (CHUNG et al. 2003). On the other hand, DON at 2.9 mg/kg feed stimulated TNF-α secretion which agrees with previous studies that found the ability of oral DON to induce TNF-α secretion either alone or with LPS co-treatment (ZHOU et al. 1998;
ZHOU et al. 1999; CHUNG at al. 2003; ISLAM and PESTKA 2006; PESTKA and ZHOU 2006;
AMUZIE et al. 2009; DÖLL et al. 2009a).
5.5.3 DON transport study
Many studies have been carried out to determine trichothecene contents including DON in grain extracts (RAZZAZI-FAZELI et al. 1999; BERGER et al. 1999; PLATTNER and MARAGOS
2003; ABRAMOVIĆ et al. 2005; HÄUBL et al. 2006) using different chromatographic techniques especially liquid chromatography (MATEO et al. 2001; NEUHOF et al. 2009; KADOTA et al.
2010). LC-MS/MS is a sensitive, rapid and specific technique used for identification of mycotoxins (PLATTNER and MARAGOS 2003; KRSKA and MOLINELLI 2007).
In the present study the amount of DON was determined both at mucosal and serosal sides of Ussing chambers at two in vitro DON concentrations by using LC-MS/MS. The results revealed higher mucosal uptake of DON compared with the serosal release. In order to explain this result, we could assume two possibilities; the first one is the metabolism of DON inside jejunal epithelia and the accumulation of DON in the tissues is the second one. It was proved that DON is rapidly and efficiently absorbed and poorly metabolised in pigs, the reasons make pigs very sensitive to DON toxicity (GOYARTS and DÄNICKE 2006; PRELUSKY
et al. 1988). This absorption mainly occurs from the proximal parts of the small intestine (DÄNICKE et al. 2004a) especially in jejunal tissues (AVANTAGGIATO et al. 2004). Most of orally administered DON in pigs is eliminated as DON and DON-glucuronide while de-epoxy DON (DOM-1), the de-epoxydized metabolite of DON, and its conjugates are of minor importance (DÄNICKE et al. 2004b, DÄNICKE et al. 2008). In the present experiment, the buffer samples were analysed for the presence of DOM-1 to prove whether the de-epoxydation of DON occurs in the jejunal mucosa. However, serosal buffers were virtually free from DOM-1 suggesting that this reaction obviously not takes place in jejunal mucosa. It was proved that the microflora of porcine lower gut were able to transform DON to a less toxic metabolite, while duodenum and jejunum showed no or less transforming activity (KOLLARCZIK et al.
1994; DÄNICKE et al. 2004b). Based on these literature findings and on the present results demonstrating no de-epoxydizing capacity of jejunal mucosa the presence of de-epoxy-DON and its conjugates in blood and urine of pigs originates probably solely from intestinal microbial activity. This view is further supported by the poor or absent capacity of the liver to contribute to DON de-epoxydation. Thus, the lack of de-epoxy-DON detection in serosal buffers can not explain the low serosal DON release.
However, other breakdown products of DON which are currently not known and thus non-detectable could also contribute to the low serosal DON release.
Regarding to the second possibility of accumulating DON in intestinal tissues, previous studies proved that DON was not accumulated in tissues to any appreciable extent either after oral or intravenous application (PRELUSKY and TRENHOLM 1991; 1992). Also no accumulation of DON was detected at the cellular level (KÖNIGS et al. 2007). After a single iv dose of 1 mg DON/kg, DON was detected in the tissues in the first 19.8 min and maximum concentrations were detected at 19.8-60 min with lower concentrations in porcine intestine (20 -165 ng/g) compared to tissues containing higher DON concentrations (1-2 µg/g) such as kidney and liver (PRELUSKY and TRENHOLM 1991). The concentrations of DON declined gradually over 24h post dosing. After feeding of DON-contaminated diet (6 - 7.6 mg/kg feed) for 3-7 weeks trace levels were detected in porcine tissues suggesting no retention or accumulation of DON residues in the tissues (PRELUSKY and TRENHOLM 1992).
An analysis of the jejunal epithelial samples after terminating the Ussing chamber experiment for DON was not possible due to the amounts of tissue too low to extract sufficient toxin
required for LC-MS/MS based quantification. Thus, the question of the low DON recovery with buffers cannot be answered at present.
In the present study the mucosal amount of DON at an in vitro concentration of 8000 ng/ml was higher than that at 4000 ng DON /ml proving that the transport of DON was proportional to its concentration. Similar observations were recorded at in vitro DON concentrations of 1000, 5000 and 10x103 ng/ml with mean flux rates were 109 ± 16, 221 ± 31 and 332 ± 34 ng/cm2.h, respectively, and the flux rate increased with increasing the flux periods with a linear increase in DON transport across jejunal epithelia of laying hens (AWAD et al. 2007a).
At the cellular level, the passage of in vitro DON at concentration of 2000 ng/ml was proportional to the duration of incubation with similar transporting rate in the apical to basolateral and basolateral to apical directions across differentiated Caco-2 cell monolayer (SERGENT et al. 2006). The mechanisms of absorption and secretion of DON at concentrations of 5, 10, 20 and 30 µM (1.4816, 2.9632, 5.9264 and 8.8896 mg) across human Caco-2 cells was investigated and it was found that the apical-basolateral and basolateral-apical directions was time- and dose-dependent (VIDEMANN et al. 2007).
A reduction in the mucosal amounts of DON at both in vitro DON concentrations was observed in DON and DON/LPS groups with a significant effect at 4000 ng/ml compared with controls. The question here is if this reduction could be attributed to the effect of oral DON and/or LPS challenge on the integrity of the intestinal barrier. It was suggested that DON transport occurred via paracellular pathway through simple diffusion (AWAD et al.
2007a) or via the transcellular route (VIDEMANN et al. 2007), and our previous work showed reduction in the transepithelial electrical resistance (TEER) in DON-fed group and in DON/LPS group, with significant interaction effect under glucose stimulation in the latter group (HALAWA et al. 2012). Previous studies showed that DON decreased TEER in treated HT-29-D4 cells and IPEC-1 cells (MARESCA et al. 2002; PINTON et al 2009; 2010) with an increase in the paracellular flux of FTIC-dextran, suggesting that reduction in TEER could increase the paracellular transport. Also in IPEC-J2 cells, basolateral exposure to 2000 and 4000 ng DON/ml resulted in reduction in TEER after 24h (DIESING et al. 2011b). LPS increased the uptake of α-methyl glucopyranoside in LPS-treated-Caco-2 cells under high glucose medium (YU et al. 2005) and impaired barrier function of tight junction in rat IEC-6 cell monolayer (KIMURA et al. 1997). On the other hand, the reduction in TEER might be
attributed, in part, to the systemic action of LPS via induction of TNF-α secretion, which was proved to reduce TEER (SCHMITZ et al. 1999; MA et al. 2004). This reduction in TEER could be a reason for increasing the paracellular passage of DON leaving comparatively lower mucosal amounts in DON-fed groups.
Conclusions
The present study proved that the transport of DON across porcine jejunal mucosa was proportional to DON concentration wihtout relation between the mucosal uptake and serosal release. Feeding of DON increased its intestinal transport. A small concentration of DON in feed was able to induce a proinflammatory response indicated by induction of TNF-α secretion. The effect as well as action of LPS at porcine intestinal barrier needs to be clarified by further assessement.
Acknowledgements
The assistance of the co-workers of the Institute of Physiology in Hannover in performing Ussing experiments for in vitro transport studies, and of the Institute of Animal Nutrition in Braunschweig in performing buffer and plasma analysis is gratefully acknowledged. The authors thank the "Deutsche Forschungsgemeinschaft" (DFG) for financial support (DA 558/1-3).
5.6 Figures and tables
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
Such inhibition was observed after 24h incubation of Caco-2 monolayer with different in vitro