Jeannette Kluess

Vulnerability of polarised intestinal porcine epithelial cells to mycotoxin deoxynivalenol depends on the route of application.

Background and Aims Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated. Methods A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity. Results Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL. Conclusions Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity.

Mycotoxin deoxynivalenol (DON) mediates biphasic cellular response in intestinal porcine epithelial cell lines IPEC-1 and IPEC-J2

The Fusarium derived mycotoxin deoxynivalenol (DON) is frequently found in cereals used for human and animal nutrition. We studied effects of DON in non-transformed, non-carcinoma, polarized epithelial cells of porcine small intestinal origin (IPEC-1 and IPEC-J2) in a low (200 ng/mL) and a high (2000 ng/mL) concentration. Application of high DON concentrations showed significant toxic effects as indicated by a reduction in cell number, in cellular reduction capacity measured by MTT assay, reduced uptake of neutral red (NR) and a decrease in cell proliferation. High dose toxicity was accompanied by disintegration of tight junction protein ZO-1 and increase of cell cycle phase G2/M. Activation of caspase 3 was found as an early event in the high DON concentration with an initial maximum after 6-8 h. In contrast, application of 200 ng/mL DON exhibited a response pattern distinct from the high dose DON toxicity. The cell cycle, ZO-1 expression and distribution as well as caspase 3 activation were not changed. BrdU incorporation was significantly increased after 72 h incubation with 200 ng/mL DON and NR uptake was only transiently reduced after 24 h. Low dose effects of DON on intestinal epithelial cells were triggered by mechanisms different from those responsible for the high dose toxicity.

Studies on the toxicity of deoxynivalenol (DON), sodium metabisulfite, DON-sulfonate (DONS) and de-epoxy-DON for porcine peripheral blood mononuclear cells and the Intestinal Porcine Epithelial Cell lines IPEC-1 and IPEC-J2, and on effects of DON and DONS on piglets.

The in vitro effects of deoxynivalenol (DON), de-epoxy-DON, DON-sulfonate (DONS) and sodium metabisulfite (Na(2)S(2)O(5), SBS) on porcine peripheral blood mononuclear cells (PBMC), and on the Intestinal Porcine Epithelial Cell lines IPEC-1 and IPEC-J2 were examined by using the MTT assay. In addition, an uncontaminated and a DON contaminated triticale were included in diets either untreated (CON, FUS) or SBS treated (CON-SBS, FUS-SBS) and fed to piglets for 28 d starting from weaning. The diet concentrations of DON and DONS amounted to 0.156, 2.312, 0.084 and 0.275 mg and to<0.05, <0.05, <0.05 and 1.841 mg/kg, respectively. PBMC of the so-exposed piglets were also subjected to the MTT assay. Neither DONS and SBS nor de-epoxy-DON affected the viability of PBMC, IPEC-1 and IPEC-J2 significantly up to concentrations of 17, 8 and 23 microM, respectively. For DON, IC(50) values were estimated at 1.2+/-0.1, 1.3+/-0.5 and 3.0+/-0.8 microM for PBMC, IPEC-1 and IPEC-J2, respectively. PBMC from piglets fed the SBS treated diets were characterized by a significantly decreased stimulation index and an increased IgA supernatant concentration with the SBS effect being significantly more pronounced after feeding the FUS-SBS diet. Further studies should clarify the possible impact of SBS on the porcine immune system.

Air–liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC)

The specific function of the epithelium as critical barrier between the intestinal lumen and the organism’s internal microenvironment is reflected by permanent maintenance of intercellular junctions and cellular polarity. The intestinal epithelial cells are responsible for absorption of nutritional components, facing mechanical stress and a changing oxygen supplementation via blood stream. Oxygen itself can regulate the barrier and the absorptive function of the epithelium. Therefore, we compared the dish cell culture, the transwell-like membrane culture and the oxygen enriched air–liquid interface (ALI) culture. We demonstrated strong influence of the different culture conditions on morphology and function of intestinal porcine epithelial cell lines in vitro. ALI culture resulted in a significant increase in cell number, epithelial cell layer thickness and expression as well as apical localisation of the microvilli-associated protein villin. Remarkable similarities regarding the morphological parameters were observed between ALI cultures and intestinal epithelial cells in vivo. Furthermore, the functional analysis of protein uptake and degradation by the epithelial cells demonstrated the necessity of sufficient oxygen supply as achieved in ALI cultures. Our study is the first report providing marked evidence that optimised oxygen supply using ALI cultures directly affects the morphological differentiation and functional properties of intestinal epithelial cells in vitro.

Gene regulation of intestinal porcine epithelial cells IPEC-J2 is dependent on the site of deoxynivalenol toxicological action.

The intestinal epithelial cell layer represents the border between the luminal and systemic side of the gut. The decision between absorption and exclusion of substances is the quintessential function of the gut and varies along the gut axis. Consequently, potentially toxic substances may reach the basolateral domain of the epithelial cell layer via blood stream. The mycotoxin deoxynivalenol (DON) is a Fusarium derived secondary metabolite known to enter the blood stream and displaying a striking toxicity on the basolateral side of polarised epithelial cell layers in vitro. Here we analysed potential mechanisms of apical and basolateral DON toxicity reflected in the gene expression. We used the jejunum-derived, polarised intestinal porcine epithelial cell line IPEC-J2 as an in vitro cell culture model. Luminal and systemic DON challenge of the epithelial cell layer was mimicked by a DON application from the apical or basolateral compartment of membrane inserts for 72 h. We compared the genome-wide gene expression of untreated and DON-treated IPEC-J2 cells with the GeneChip® Porcine Genome Array of Affymetrix. Low basolateral DON (200 ng/mL) application triggered 10 times more gene transcripts in comparison to the corresponding apical application (2539 versus 267) despite the intactness of the challenged cell layer as measured by transepithelial electrical resistance. Analysis of the regulated genes by bioinformatic resource DAVID identified several groups of biochemical pathways modulated by concentration and orientation of DON application. Selected genes representing pathways of the cellular metabolism, information processing and structural design were analysed in detail by quantitative PCR. Our findings clearly show that apical and basolateral challenge of epithelial cell layers trigger different gene response profiles paralleled with a higher susceptibility towards basolateral challenge. The evaluation of toxicological potentials of mycotoxins should take this difference in gene regulation dependent on route of application into account.

The Fusarium toxin deoxynivalenol (DON) modulates the LPS induced acute phase reaction in pigs

The systemic effects of the Fusarium toxin deoxynivalenol (DON) and of bacterial lipopolysaccharides (LPS) were studied in male castrated pigs (40.4 ± 3.7 kg) infused intravenously with either DON or LPS alone (100 μg DON/kg/h, 7.5 μg/LPS/kg/h), or together (100 μg DON plus 7.5 μg/LPS/kg/h). The Control group received a saline infusion (n=6/treatment, 24h observation period). An additional DON infusion did not exacerbate the clinical signs observed in LPS-infused pigs. For example, rectal temperature climaxed after 4h (40.4 ± 0.2°C) and 5h (40.1 ± 0.3°C), in the LPS and LPS+DON group, respectively. Saline and DON alone did not induce an acute phase reaction as indicated by unaltered plasma levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) while LPS caused a significant rise of both cytokines. TNF-alpha plasma peak concentrations were significantly higher in the LPS compared to the DON+LPS group (94.3 ± 17.2 ng/mL vs. 79.2 ± 15.7 ng/mL) while IL-6 climaxed earlier in the latter group (3h p.i. vs. 2h p.i.). From the tested clinical-chemical plasma characteristics the total bilirubin concentration and the ASAT activity were strongly elevated by the LPS infusion and additionally increased and decreased by DON, respectively. In conclusion, the LPS-induced effects were only marginally modified by DON.

A chronic oral exposure of pigs with deoxynivalenol partially prevents the acute effects of lipopolysaccharides on hepatic histopathology and blood clinical chemistry

Lipopolysaccharides (LPS), a cell wall component of gram-negative bacteria, and deoxynivalenol (DON), a prevalent Fusarium-derived contaminant of cereal grains, are each reported to have detrimental effects on the liver. A potentiating toxic effect of the combined exposure was reported previously in a mouse model and hepatocytes in vitro, but not in swine as the most DON-susceptible species. Thus, pigs were fed either a control diet (CON) or a Fusarium contaminated diet (DON, 3.1mg DON/kg diet) for 37 days. At day 37 control pigs were infused for 1h either with physiological saline (CON_CON), 100μg/kg BW DON (CON_DON), 7.5μg/kg BW LPS (CON_LPS), or both toxins (CON_DON/LPS) and Fusarium-pigs with saline (DON_CON) or 7.5μg/kg BW LPS (DON_LPS). Blood samples were taken before and after infusion (-30, +30, +60, +120, and +180min) for clinical blood chemistry. Pigs were sacrificed at +195min and liver histopathology was performed. LPS resulted in higher relative liver weight (p<0.05), portal, periportal and acinar inflammation (p<0.05), haemorrhage (p<0.01) and pathological bilirubin levels (CON_CON 1.0μmol/L vs. CON_LPS 5.4μmol/L, CON_DON/LPS 8.3μmol/L; p<0.001). DON feeding alleviated effects of LPS infusion on histopathology and blood chemistry to control levels, whereas DON infusion alone had no impact.

Systemic and local effects of the Fusarium toxin deoxynivalenol (DON) are not alleviated by dietary supplementation of humic substances (HS)

The aim of this study was to examine the effects of a control diet (CON) or a Fusarium toxin contaminated diet (FUS) with and without HS (CON-HS and FUS-HS, respectively) on pigs during a 10-week growth trial starting at 35.1±3.2 kg live weight (n=12/group). Moreover, 2 additional choice feeding groups were included to test the ability of the pigs to differentiate between the CON and FUS diet. Feeding the FUS diets (∼3 mg DON/kg) did not depress feed intake irrespective of HS addition. However, the pigs of the choice feeding groups recognised the FUS diets and acquired an ability to avoid these diets. DON residues were detected exclusively in the blood of pigs exposed to the FUS diets (7-21 ng/mL) but their levels were not affected by HS, suggesting their inefficiency in preventing DON absorption. While zonula occludens-1 protein expression and villus height in jejunum and ileum were not compromised by FUS feeding, the jejunal crypts were significantly deepened at 31% compared to the CON group. These changes had no consequences for nutrient digestibility or LPS levels in systemic blood (0.02-0.08 EU/mL). As portal LPS levels were not measured, FUS effects on intestinal LPS translocation cannot be excluded.

Does Dietary Deoxynivalenol Modulate the Acute Phase Reaction in Endotoxaemic Pigs?--Lessons from Clinical Signs, White Blood Cell Counts, and TNF-Alpha.

We studied the interaction between deoxynivalenol (DON)-feeding and a subsequent pre- and post-hepatic immune stimulus with the hypothesis that the liver differently mediates the acute phase reaction (APR) in pigs. Barrows (n = 44) were divided into a DON-(4.59 mg DON/kg feed) and a control-diet group, surgically equipped with permanent catheters pre- (V. portae hepatis) and post-hepatic (V. jugularis interna) and infused either with 0.9% NaCl or LPS (7.5 µg/kg BW). Thus, combination of diet (CON vs. DON) and infusion (CON vs. LPS, jugular vs. portal) created six groups: CON_CONjug.-CONpor., CON_CONjug.-LPSpor., CON_LPSjug.-CONpor., DON_CONjug.-CONpor., DON_CONjug.-LPSpor., DON_LPSjug.-CONpor.. Blood samples were taken at −30, 15, 30, 45, 60, 75, 90, 120, 150, 180 min relative to infusion and analyzed for leukocytes and TNF-alpha. Concurrently, clinical signs were scored and body temperature measured during the same period. LPS as such induced a dramatic rise in TNF-alpha (p < 0.001), hyperthermia (p < 0.01), and severe leukopenia (p < 0.001). In CON-fed pigs, an earlier return to physiological base levels was observed for the clinical complex, starting at 120 min post infusionem (p < 0.05) and persisting until 180 min. DON_LPSjug.-CONpor. resulted in a lower temperature rise (p = 0.08) compared to CON_LPSjug.-CONpor.. In conclusion, APR resulting from a post-hepatic immune stimulus was altered by chronic DON-feeding.

Interactions between the Fusarium toxin deoxynivalenol and lipopolysaccharides on the in vivo protein synthesis of acute phase proteins, cytokines and metabolic activity of peripheral blood mononuclear cells in pigs

The in vivo effects of the Fusarium toxin deoxynivalenol (DON) on albumin and fibrinogen synthesis in pigs and metabolic activity of porcine peripheral blood mononuclear cells (PBMCs) were studied alone or in combination with lipopolysaccharides (LPSs) in order to examine proposed synergistic effects of both substances. A total of 36 male castrated pigs (initial weight of 26 kg) were used. Uncontaminated (Control) and naturally DON-contaminated (chronic oral DON, 3.1mg/kg diet) wheat was fed for 37 days. On the day of protein synthesis measurement, pigs recruited from the Control group were treated once intravenously with (iv) DON (100 μg/kg live weight (LW)/h), iv LPS (7.5 μg/kgLW/h) or a combination of both substances, and six pigs from the chronic oral group were treated once with iv LPS. A treatment with DON alone exhibited no alterations of acute phase protein synthesis and metabolic activity of PBMC. There was no evidence that the chosen dosing regimen of DON had influences on the induced sub-acute stage of sepsis, as the LPS challenge, irrespective of DON co-exposure, mediated an acute phase reaction with a typical decrease of albumin synthesis, as well as changes in cytokine concentration and a loss of metabolic activity in PBMC.