Analysis of buffer samples using LC/MSMS technique

- Mode: ESI negative

- Acquisition Method: DON+de-DON_190508_delay.dam - HPLC-conditions: Agilent Technologie

- Column: BETASIL Phenyl-Hexyl, 3 µm, 100 x 2,1 mm (Thermo) - Guard column: Phenyl, 4x2 mm (Phenomenex, Nr. AJO-4350), with

guard column holder of Phenomenex - Eluent A: 0.13 mM NH₄ Acetat, pH 7,4

- Eluent B: Acetonitrile

- Gradient: 0 – 2.0 min: 4% B; 2.0 – 5.0 min: 4-95% B; 5.0 – 7.0 min: 95% B; 7.0 – 7.5 min: 95-4% B; 7.5 – 12.0 min: 4%

B

- Flow rate: 400 µl/min - Injection volume: 10 µl - Column oven temperature: 20°C

- tR DON/De-epoxy DON : ca. 4.4/4.6 min 3.4.4 Determination of plasma DON concentrations

DON was analyzed in porcine plasma by HPLC-DAD after clean-up with immunoaffinity columns (IAC) according to (VALENTA et al. 2003) with slight modifications.

- Material and reagents:

- Sodium acetate buffer (PH 5.5) - β-glucuronidase (Sigma, G 0876)

- ChemElut cartridge (Varian, Middelburg, Netherlands) - IAC (DONtest HPLC^®, VICAM, Watertown, MA, USA) - Method:

- One ml of sodium acetate buffer (PH 5.5) was added to the plasma sample (1.5 ml) - The previous mixture was incubated for 16 h with β-glucuronidase

- Then the sample was extracted with ethyl acetate on a ChemElut cartridge and cleaned-up by IAC.

- The samples were measured by HPLC-DAD.

- The mean recovery of DON was 91% ± 8% (n=12; 10-30 ng/ml). The results were not corrected for recovery.

3.4.5 Determination of plasma TNF-α concentration

TNF-α was determined using ELISA kit (R&D Systems, Minneapolis, USA) based on porcine specific matched pairs of antibodies in combination with recombinant standards.

All reagents and samples were prepared at room temperature according to manufacture instructions.

1- Preparation of the reagents

a- Control was dissolved in 1 ml of ionized H2O

b- Wash buffer (25 ml) was mixed with 600 ml of ionized water.

c- Substrate solution was prepared 15 min before use in dark place; color reagents A and B were mixed in equal parts.

2- Standard series

a- Standard was dissolved in 2 ml Calibrator diluent RD6-33 and was left for 5 min.

b- Six Polypropylene tubes were used.

c- 200 µl of Calibrator diluent were added in each tube to make a standard series d- A Blank tube was filled with Calibrator diluent.

3- 50 µl of Assay Diluent RD1-63 were added to the plate.

4- 50 µl Standards (in duplicate), control (in duplicate), Blank (in duplicate) and samples (single determination) + Blank

5- Shaking for 1 min.

6- The plate was sealed with foil and was incubated for 2h at room temperature.

7- The plate was washed 5 times with 400 µl wash buffer.

8- 100 µl Conjugate was added.

9- The plate was sealed with foil and was incubated for 2h at room temperature 10- The plate was washed 5 times with 400 µl wash buffer.

11- 100 µl Substrate Solution were added (15 min before start).

12- The plate was incubated for 30 min in dark place.

13- 100 µl stop solution were added.

14- The plate was measured (within 30 min) at 450 nm, 540 nm, 570 nm.

3.4.5 Calculations and statistics

The measured DON concentrations (ng/ml) were converted to amounts (ng) by multiplying 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 2-factorial ANOVA analysis (DON in feed and ip LPS) including their interactions. One-factorial ANOVA analysis was applied to evaluate the effect of the group on both mucosal and serosal amounts of DON. All results were presented as means ± standard deviation (SD) whereas n represents the number of pigs and p-values <

0.05 were considered to be significant.

4. Chapter 1

Effects of deoxynivalenol and lipopolysaccharide on electrophysiological parameters in growing pigs

4. Chapter 1

Effects of deoxynivalenol and lipopolysaccharide on electrophysiological parameters in growing pigs

Amal Halawaª, Sven Dänicke^b, Susanne Kersten^b, Gerhard Brevesª

a Physiological Institute, University of Veterinary Medicine, Hannover, Germany

b Institute of Animal Nutrition, Friedrich Loeffler Institute (FLI), Federal Research Institute for Animal Health, Braunschweig, Germany

Submitted to Mycotoxin Research, at 06.05.2012, undergoing review

Corresponding author: Sven Dänicke, Institute of Animal Nutrition, Friedrich-Loeffler-Institute (FLI), Federal Research Friedrich-Loeffler-Institute for Animal Health, Bundesallee 50, D-38116 Braunschweig, Fax: + 49 531-596 3199, E-mail: sven.daenicke@fli.bund.de

4.1 Abstract

Deoxynivalenol (DON) is a major B-trichothecene that gains importance from its natural occurrence in cereals worldwide. It has many effects on rapidly dividing cells relying on intensive protein synthesis for growth and proliferation. Lipopolysaccharide (LPS) is an endotoxin released from most Gram-negative bacteria which plays a major role in induction of inflammation and sepsis under certain conditions. From our experiments we aimed at studying the effects of different concentrations of DON on electrogenic transport of nutrients and on tissue conductances using the Ussing chamber technique. The effect of DON contaminated feed on the respective parameters as well as the interactions between DON and LPS were assessed using porcine jejunal tissues. In vitro DON inhibited the absorption of alanine and glucose across pig jejunum at concentrations of 4000 and 8000 ng/ml, suggesting that DON had an inhibitory effect on the electrogenic transport of nutrients across porcine small intestines. Electrogenic transport of alanine and glucose across porcine small intestine varied regionally among intestinal segments with higher response in ileal tissues. A synergistic effect was observed between DON in feed and injected LPS on tissue conductances in response to glucose with higher short circuit currents across porcine jejunal mucosa were observed in nutrient stimulated conditions.

Keywords: deoxynivalenol; lipopolysacchaide; Ussing chamber; electrogenic transport.

4.2 Introduction

Deoxynivalenol (DON) is a secondary metabolite produced by different Fusarium species, such as Fusarium graminearum and Fusarium culmorum (BAKAN et al. 2002; BOTTALICO

1998; VISCONTI and BOTTALICO 1983), which grow on cereals such as corn, wheat, barley and maize (ABBAS et al. 1985; EFSA 2004; FDA 2010; MEGALLA et al. 1986; RICHARD 2007).

DON is known to affect both, the gastrointestinal tract and immune system (ROTTER et al.

1996) in which cells are mainly rapidly dividing such as lymphocytes, fibroblasts and epithelial cells (DÖLL et al. 2009b; ERIKSEN 2003; ROCHA et al. 2005) and depending in their proliferation on protein synthesis. DON is one of the most potent trichothecenes that has emetic activity (COPPOCK and JACOBSEN 2009). Swine are the most susceptible species to DON (ROTTER et al. 1996). Acute toxicity of DON, especially in pigs, is characterised by abdominal distress, vomiting and diarrhea (EFSA 2004;FORSYTH et al. 1977; WILLIAMS et al.

1988; YOUNG et al. 1983). Chronic toxicity of DON is characterised by anorexia, reduced weight gain, feed refusal and modulated immune function (ROTTER et al. 1996). DON is rapidly absorbed from the gastrointestinal tract, through paracellular or transcellular routes (AWAD et al. 2007a; SERGENT et al. 2006; VIDEMANN et al. 2007). The major absorption sites are the proximal parts of the small intestine of pigs after oral exposure (DÄNICKE et al.

2004a). DON can be detected in pig plasma within 30 min after both, oral and intragastric administration (ERIKSEN 2003; ERIKSEN and PATTERSSON 2004; PRELUSKY et al. 1988). Most of ingested DON in pigs is eliminated with urine as DON, and to a lesser degree as de-epoxy DON both unconjugated and as glucuronidated conjugates (DÄNICKE et al. 2004b). It was found that DON had inhibitory effects on glucose and proline uptake across intestinal mucosa of chickens and laying hens (AWAD et al. 2009; AWAD et al. 2005b) and reduced the transepithelial electrical resistance (TEER) across human and porcine cell lines (SERGENT et al. 2006; PINTON et al. 2010). DON was able to affect on the integrity of enterocytes (KOLF -CLAUW et al. 2009; PINTON et al. 2009) evidenced by shortened and coalescent villi, lysis of enterocytes and oedema.

Lipopolysaccharide (LPS) is a component of the outer bacterial membrane of Gram-negative bacteria such as Escherichia coli and Salmonella enterica (ALEXANDER and RIETSCHEL 2001;

MAYEUX 1997) and is released during bacterial growth or lysis (RIETSCHEL et al. 1994;

ROSENFELD et al. 2006). Both, humans and animals are susceptible to LPS intake (ROTH et al.

1997) in different degrees, depending on the disease state, age, presence of a xenobiotic agent and other factors (GANEY and ROTH 2001). It acts as a stimulator of the innate immunity of the host (ALEXANDER and RIETSCHEL 2001) but it can induce septic shock in high doses. LPS stimulates the immune cells to produce mediators and effector molecules (SCHLETTER et al.

1995) such as reduced oxygen species and cytokines (CHUNG et al. 2003). The intra-bacterial quantity of LPS molecules is ~ 2x10⁶ molecules of LPS/bacterial cell (MAYEUX 1997; RAETZ

1986). LPS can be absorbed through transcellular or paracellular pathway in the intestinal tract (TOMLINSON and BLIKSLAGER 2004) and thus reach the general circulation. LPS was found to reduce the intestinal uptake of leucine, fructose and galactose (ABAD et al. 2001;

GARCIA-HERRERA et al. 2008; AMADOR et al. 2008). In several studies the effects of DON alone or LPS alone on the electrophysiological parameters of intestinal epithelium as indicators for decreases in electrogenic sugar and amino acid uptake have been studied. The

interactions between DON and LPS were investigated with regard to immunological effects such as induction of proinflammatory cytokines such as TNF-α, IL-6 and IFN-γ or to induce apoptosis (DÖLL et al. 2009a; ISLAM et al. 2002; ISLAM and PESTKA 2006; PESTKA and ZHOU

2006; ZHOU et al. 2000).

The present experiments firstly aimed at studying the effects of different concentrations of DON in vitro on short circuit currents and tissue conductances along different segments of the small intestine of growing pigs fed an uncontaminated control diet by using the Ussing chamber technique as a physiological approach for studying nutrient and electrolyte transport across epithelia (CLARKE 2009).

Secondly, the effects of oral DON administration and/or injected LPS on electrophysiological parameters of porcine intestinal epithelia including their interactions were measured by the same technique to closer approach the in vivo situation (ex vivo study).

4.3 Material and Methods

Animal studies and procedures were conducted according to the European Community regulations concerning the protection of experimental animals and the guidelines of the Regional Council of Braunschweig, Lower Saxony, Germany (File number 509.42502/09-01.03).

4.3.1 Experimental design

In order to study the effect of in vitro DON on the electrophysiological parameters across porcine small intestine, different in vitro DON concentrations were examined across different intestinal segments using Ussing chamber technique. The studies were done in 2 series; each had different in vitro DON concentrations (see table 6).

For assessment of the effect of oral DON on the respective parameters, pigs were fed on DON-contaminated wheat for about 5 weeks in the 2^nd series (for compositions of experimental diet see table 5). For evaluation of the interaction effect between oral DON and LPS on the short circuit currents and tissue conductances, LPS was injected ip 3h before slaughtering of the animals in the 2^nd experimental series (see table 6).

4.3.2 Animals

The animals used in both series are male castrated pigs. They were of the breed "Deutsches Edelschwein". The average body weight was approximately 24.3 kg in the 2^nd series, at the

time of tissue preparation for the electrophysiological investigations. For the duration of the feeding experiments (~ 5 weeks) they were kept at the experimental facilities of the Institute of Nutrition, Friedrich Loeffler Institute Braunschweig, Germany, while the in vitro electrophysiological investigations using the Ussing-chamber technique were performed at the Department of Physiology, University of Veterinary Medicine, Hannover, Germany.

4.3.3 Chemicals

DON was purchased from (Sigma-Aldrich D-0156, München, Germany) diluted in isotonic saline. LPS was used from E. coli serotype 0111:B4 (Sigma-Aldrich L-2630, 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):

After stunning, slaughtering and bleeding of the animals, the gastrointestinal tract was removed within 5 min after bleeding. In the first experimental series intestinal segments of about 80 cm length were immediately taken from the duodenum, mid jejunum and ileum. In the second series, segments from the mid jejunum were taken. The segments were immediately rinsed with ice-cold saline (0.9% NaCl) and kept in a modified glucose-containing Krebs-Henseleit buffer solution at 4°C, being continuously gassed with carbogen (95% O2, 5% CO2) until mounting in Ussing chambers.

4.3.5 Ussing Technique

After longitudinal incision along the mesenteric border, the intestinal segments were washed free of any remaining intestinal contents and the muscle and serosal layers were stripped off before mounting the mucosal layer into Ussing chambers to minimize the effect of the intrinsic neuromuscular system on the electrophysiological parameters. The chambers were

connected to glass circulation reservoirs through silicon tubes. The glass reservoirs were filled with 12.5 ml of modified Krebs-Henseleit buffer solution at both sides of the tissues and were adjusted at 38.4°C and gassed permanently with carbogen to maintain continuous circulation and the pH at 7.45. The electrophysiological experiments were carried out by computer controlled voltage / current clamps. The clamps were connected to the chambers through 3 M KCl-containing agar bridges and Ag/AgCl electrodes through which the short circuit currents were recorded. Fluid resistance and potentials were measured before mounting the tissue segments and corrected for during the experiments. For determination of electrophysiological parameters tissues were mounted in Ussing chambers with an exposed area of 1.13 cm² with silicon rings and nets to prevent tissue damage and bulging. The tissues were clamped to 0 mV in order to eliminate the electrical gradients. Identical buffer solutions were used on both sides in order to abolish the chemical gradient. Thus the tissues were incubated in the absence of the transepithelial electrochemical gradient.

After an equilibration period (30 min), the basal values for potential differences, short circuit currents and electrical tissue conductances were recorded. DON was added to the mucosal side of the Ussing chambers at the previously mentioned concentrations (see table 6) to reach final volume of 13 ml in the glass reservoirs. An equal volume of modified glucose-containing Krebs-Henseleit buffer was added to the serosal side.

In order to assess the effects of DON and LPS on alanine and glucose transport, alanine (10 mM) was added to the mucosal side of the tissues in each Ussing chamber. Ten min later glucose (10 mM) was added to the luminal side. Responses of Isc after addition of alanine and glucose were recorded as differences between the constant values before addition and the maximal response (∆Isc).

4.3.6 Calculations and statistics

In both experimental series the short circuit currents (µEq.cm^-2.h^-1) and tissue conductances (mS.cm^-2) were recorded via computer controlled voltage/current clamps. ∆Isc as well as ∆Gt were calculated for each animal for alanine and glucose as differences between the constant values before their addition and the maximal response after nutrients addition and the averages of ∆Isc and ∆Gt for each pig were calculated. The previous data were analysed using 2-factorial ANOVA (tissue and DON in vitro) and 3-factorial ANOVA (DON in vitro, ip LPS and DON in feed) including their interactions for first and second series, respectively and all

the results were presented as means, standard deviations and probabilities for the main effects and interactions.

4.4 Results

4.4.1 Basic electrophysiological parameters along the small intestines

The mean basal short circuit currents (Isc) and conductances in the first experiment ranged between -0.480 and 0.267 µEq.cm^-2.h^-1 and 24.6 and 38.8 mS.cm^-2 (Table 8A), respectively in duodenum, mid jejunum and ileum. When alanine and glucose were added to the mucosal side of the tissues up to 27 fold increase in Isc could be detected with highest increases for both nutrients in ileal tissues. No significant changes of tissue conductances were seen in response to both nutrients.

4.4.2 Acute in vitro effects of DON on nutrient stimulated electrophysiological parameters

In the first series tissues from the duodenum, mid jejunum and ileum were exposed to increasing DON concentrations in two experiments. The mean basal Isc varied between 0.043 and 0.685 µEq.cm^-2.h^-1 without effect of different DON concentrations. The respective data for Gt ranged between 25.0 and 46.7 mS.cm^-2. In response to both nutrients increases in Isc between 0.240 and 1.1 (alanine) and 0.295 and 3.2 (glucose) irrespective of the DON concentrations were detected (Table 8A). Relatively higher values were obtained in the second experiment (Table 8B). No significant effects on Gt were recorded.

In contrast to these findings DON induced significant decreases in alanine and glucose response when the mucosal concentrations were adjusted to 4000 or 8000 ng/ml in the mucosal buffer solution with no effects on tissue conductances (Table 9).

4.4.3 The main effect of DON in feed

In the 2^nd experimental series 2.9 mg DON/kg feed was fed to pigs. The mean basal short circuit currents were -0.119 and -0.005 µEq.cm^-2.h^-1 in control and DON groups, respectively, without significant effect of DON in feed. The respective data for Gt were 29.43 and 26.73 mS.cm^-2 in control and DON groups, respectively. In response to both nutrients (alanine and glucose) increases in Isc were observed in both groups without significant changes between them. Tissue conductances showed no significant changes (Table 9).

4.4.4 The main effect of intraperitoneal LPS

In the 2^nd series ip 5 µg/kg LPS was applied. The mean basal Isc were -0.124 and 0.001 µEq.cm^-2.h^-1 in control and LPS groups, respectively. The respective data for Gt were 28.63 and 27.52 mS.cm^-2 without any effect of LPS. The short circuit currents were highly increased in response to alanine and glucose in both groups with a trend increasing effect of LPS for both nutrients. No significant effects were recorded in tissue conductances after addition of the nutrients (Table 9).

4.4.5 Interactions between DON in feed, intraperitoneal LPS challenge and DON in vitro The mean basal short circuit currents and tissue conductances ranged between -0.151 and 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

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

Im Dokument Toxicological and immunological effects of DON and LPS at the intestinal barrier (Seite 50-0)