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).