The Mycotoxin Fumonisin B1 Alters the Proliferation and the Barrier Function of Porcine Intestinal Epithelial Cells

Sandrine Bouhet*, Edith Hourcade*, Nicolas Loiseau*, Asmaa Fikry*, Stéphanie Martinez*, Marianna Roselli{dagger}, Pierre Galtier*, Elena Mengheri{dagger} and Isabelle P. Oswald*,1

* Laboratoire de Pharmacologie-Toxicologie, Institut National de Recherche Agronomique, 31931 Toulouse, France; and {dagger} Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, 00178 Roma, Italy

Received August 18, 2003; accepted September 26, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides (formerly F. moniliforme), a fungus that commonly contaminates maize. FB1 causes toxicological effects in laboratory and domestic animals including pigs. Because the gastrointestinal tract represents the first barrier met by exogenous food compounds, the purpose of this study was to investigate the effects of FB1 on IPEC-1, a porcine intestinal epithelial cell line. We first verified that low concentrations of FB1 did not exert any cytotoxic effect on IPEC-1. Indeed, significant LDH release was only observed for FB1 concentrations greater than 50 and 700 µM on proliferating and nonproliferating cells, respectively. We then demonstrated that FB1 inhibits proliferation of IPEC-1. Fluorescence-activated cell sorting (FACS) analysis of the cell cycle indicated that FB1 blocks the proliferation of intestinal cells in the G0/G1 phase. Similar results were obtained with LLC-PK1, a renal porcine epithelial cell line, which is considered to be a good model for studying FB1 in vitro effects. We have also assessed the effects of FB1 on the integrity of the barrier formed by the intestinal epithelium. We demonstrated that FB1 decreases the transepithelial electrical resistance (TEER) of IPEC-1 in a time- and dose-dependent manner. This effect was only noticed after a long exposure (8–12 days of treatment). FB1 induced the TEER decrease independently of the cell differentiation stage, and this effect was partially reversible. Taken together, our data indicate that FB1 alters the proliferation and the barrier function of intestinal cells. These results may have implications for humans and animals consuming FB1-contaminated food or feed.

Key Words: fumonisin B1; intestinal epithelial cells; IPEC-1; swine; barrier function; transepithelial electrical resistance.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycotoxins are secondary metabolites of fungi that may contaminate animal and human feeds at all stages of the food chain. Their global occurrence is considered to be a major risk factor affecting human and animal health, as it is estimated that 25% of the world crop production is contaminated with mycotoxins (Bullerman, 1996Go; Shephard et al., 1996Go).

Fumonisins are a family of cytotoxic and carcinogenic mycotoxins produced by Fusarium verticillioides (syn F. moniliforme), one of the most common molds found on maize and other agricultural products throughout the World. Maize contaminated with fumonisin B1 (FB1) is of concern because this mycotoxin causes various animal diseases. FB1 induces leukoencephalomalacia in horses, nephrotoxicity in rats, rabbits, and lambs, and hepatotoxicity in all species examined (reviewed in Bolger et al., 2001Go; WHO, 2000Go). In pigs, FB1 is responsible for pulmonary edema, liver failure, and cardiovascular toxicity (reviewed in Haschek et al., 2001Go). This toxin has also been reported to be a carcinogen in rodents (Gelderblom et al., 1991Go) and a contributing factor in human esophageal cancers (Rheeder et al., 1992Go). FB1 is also known to be cytotoxic on several cell lines including epithelial cells (Caloni et al., 2002Go; Mobio et al., 2000Go; Tolleson et al., 1996Go; Yoo et al., 1992Go).

The gastrointestinal tract represents the first barrier against ingested chemicals, food contaminants, and natural toxins. Following ingestion of mycotoxin-contaminated food or feed, intestinal epithelial cells could be exposed to a high concentration of toxin (Prelusky et al., 1996Go; Shephard et al., 1996Go). It is thus of interest to analyze the effects of fumonisins on the intestine (Enongene et al., 2002Go; Schmelz el al., 1998Go; Stevens and Tang, 1997Go). Considering that the gastrointestinal tract of the pig and its digestive physiology are very similar to that of humans, the pig can be regarded as a good model of extrapolation to humans (Miller and Ullrey, 1987Go; Swindle, 1992Go).

In this study, we used a porcine epithelial intestinal cell line to determine the effects of FB1 on the intestinal epithelium. We report herein that FB1 affects both the proliferation of epithelial cells and the epithelial integrity.


    MATERIAL AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and toxin.
LLC-PK1 cells are well characterized renal proximal tubule epithelial cells derived from the New Hampshire Mini-pig. This cell line was obtained from the American Type Culture Collection (Rockville, MD). The IPEC-1 cell line is a newborn swine intestinal epithelial cell line that was derived from the small intestine of a newborn unsuckled piglet (Gonzalez-Vallina et al., 1996Go). This cell line was a generous gift from Drs. H. M. Berschneider and D. D. Black.

The cells were maintained in serial passage in 75 cm2 flasks at 37°C, in a humidified incubator with a 5% CO2 atmosphere. LLC-PK1 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Eurobio, Les Ulis, France) containing 10% fetal bovine serum (FBS; Perbio Sciences, Bezons, France), penicillin (50 µg/ml), streptomycin (4 µg/ml) (Eurobio), and 2 mM L-glutamine (Eurobio). IPEC-1 were grown in complete DMEM/F-12 medium (Eurobio) supplemented with antibiotics, 5% FBS, 2 mM L-glutamine, 15 mM Hepes (Eurobio), epidermal growth factor (5 µg/l; Becton Dickinson Labware, Le Pont de Claix, France), and ITS (Premix, Sigma, St Quentin Fallavier, France). The composition of ITS was: insulin (5 µg/ml), transferrin (5 µg/ml), selenium (5 ng/ml).

Purified FB1 obtained from Promec (Tygerberg, South Africa) was diluted in water to make a stock solution and further diluted in the culture media at the indicated concentrations.

Cell morphological observation.
Cells were seeded at 104 cells/well in 1 ml of medium in 24-well plates (area: 2 cm2, Polylabo-Nunc, Strasbourg, France). After 2 days of treatment with 50 µM FB1, cells were photographed using an inverted microscope (Zeiss, Göttingen, Germany).

Cytotoxicity assay.
The cytotoxic effect of FB1 on the IPEC-1 cells was evaluated by measuring the release of lactate dehydrogenase (LDH) in the culture media using the CytoTox 96® Assay Kit (Promega, Charbonnières, France). Indeed, release of LDH strongly correlates with the number of lysed cells and is widely used in cytotoxicity studies (Tipton et al., 2003Go). Briefly, IPEC-1 were seeded in 96-well plates at concentrations of 1 x 104 and 2.5 x 104 cells/well to investigate the cytotoxicity of FB1 on dividing cells and on confluent cells, respectively. After an overnight culture at 37°C, the medium was replaced by complete medium containing various concentrations (2 to 700 µM) of FB1. LDH activity was measured 48 h later on 50 µl of supernatant according to the manufacturer’s instructions, and the absorbance was read at 492 nm on an ELISA plate reader (Tecan, Trappes, France). The maximal LDH release (total lysis) was determined after lysing the cells with 0.8% (w/v) Triton X-100. Results were expressed as percentage of total LDH release: [(experimental value – blank value)/(total lysis – blank value) x 100].

MTS bioassay.
The effects of FB1 on the proliferation and viability of LLC-PK1 and IPEC-1 cells were studied in a colorimetric assay using the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay Kit (Promega). LLC-PK1 and IPEC-1 were seeded at a concentration of 3 x 103 cells/well in 100 µl of adequate medium in flat-bottomed 96-well plates (Polylabo-Nunc). After 24 h of culture, various concentrations of FB1 ranging from 10 to 200 µM were added to the cells. After 48 h, 20 µl of a freshly prepared MTS/PMS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate] solution was added to the wells, and the cells were further incubated for 2–4 h. The amount of soluble formazan produced, by cellular reduction of the MTS, was measured by the absorbance at 492 nm on an ELISA plate reader (Tecan).

Analysis of the cell cycle by flow cytometry.
For analysis of the cellular cycle, 1.25 x 105 LLC-PK1 cells and 0.75 x 105 IPEC-1 cells were seeded in 6-well plates (area: 9 cm2, Polylabo-Nunc), in 3 ml of complete medium supplemented with 5% and 2% FBS, respectively. After 24 h of culture, the cells were treated with FB1 at concentrations ranging from 5 to 100 µM. Forty-eight hours later, the cells between 50 and 80% of confluence at harvest, depending on their treatment, were trypsinized. Single-cell suspensions were fixed for 45 min in 1 ml of 70% ice-cold ethanol. After 3 washes, cells were resuspended in 800 µl of PBS supplemented with 200 µl of phosphate citrate buffer (Merck, Fontenay-sous-Bois, France; pH 7.8). Cells were then stained for 30 min at room temperature with a solution containing 100 µg/ml of RNaseA (Sigma) and 40 µg/ml of propidium iodide (Sigma) diluted in PBS. Cells were analyzed on a Facscan (Becton Dickinson Labware) with laser excitation at 488 nm, using a 639 nm band pass filter to collect the red propidium iodide fluorescence. The percentage of cells in each stage of the cell cycle was estimated using the CELL-QUESTTM software (Becton Dickinson Labware).

Measurement of transepithelial electrical resistance (TEER).
IPEC-1 cells were seeded at 105 cells in 0.3 cm2 Transwell filters with 0.4 µm pores (Becton-Dickinson Labware). When the cells were confluent, apical and basal compartments were filled with serum-free complete DMEM/F-12 containing dexamethasone (50 µg/ml, Sigma) to allow the cells to differentiate. Treatment with FB1 (0, 50, 200, and 500 µM) started either at the beginning of the differentiation process (when cells were confluent, before the addition of dexamethasone) or at the end of the differentiation process (when cells were fully differentiated, 10 days after the addition of dexamethasone). The integrity of tight junctions was assessed during 28 days by measuring the TEER using a Millicell volt-ohm meter (Millipore, Saint-Quentin en Yvelines, France). Experimental TEER values were expressed as k{Omega} x cm2.

To assay the reversibility of the effect of FB1, differentiated cells were treated for 26 days with various concentrations of FB1 and then cultured for 16 additional days in FB1-free medium.

Statistical analysis.
A nonparametric test, Mann–Whitney U-test, was used to determine differences induced by FB1 on cytotoxicity, cell growth, cell cycle, and TEER; p values < 0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Morphological Observation of IPEC-1 Cells After FB1 Treatment
IPEC-1 cells were cultured for 2 days in the presence of 50 µM FB1, and their overall cellular morphology was analyzed by light microscopy. As we observed with LLC-PK1 (data not shown), we noticed that 50 µM FB1 inhibited the cell growth. Two days after seeding, the untreated IPEC-1 cells (Fig. 1Go, left panel) were confluent, in contrast to the treated ones (Fig. 1Go, right panel). We observed only a few detached cells in FB1 treated cultures compared to the untreated cultures. However, the attached treated cells were loosing their epithelial morphology and beginning to appear fibroblast-like.



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FIG. 1. Microphotographs of IPEC-1 cells after FB1 treatment. IPEC-1 cells were seeded at low density in 24-multiwell plates. After 24 h, subconfluent cells were treated with 50 µM FB1. They were then photographed after 48 h of incubation. The morphology of control untreated cells is presented for comparison.

 
Cytotoxicity of FB1 on Dividing and Nondividing IPEC-1 Cells
IPEC-1 cells were cultured for 2 days in the presence of various concentrations of FB1, and the cytotoxic effect of the mycotoxin was evaluated by measuring LDH release. As shown in Figure 1Go, left panel, FB1 did not exert any cytotoxic effects on dividing IPEC-1 cells when used at concentrations ranging from 2 to 20 µM. At higher concentrations (50 µM and above) the cytotoxic effect of FB1 was proportional to the dose of mycotoxin added to the cell culture. On confluent nondividing cells (Fig. 1Go, right panel), FB1 did not induce any LDH release except when the toxin was used at the highest concentration (700 µM).

FB1 Inhibits Cell Growth of Both IPEC-1 and LLC-PK1 Cell Lines
The effect of FB1 on cell proliferation was then investigated using the MTS assay. The conversion of tetrazolium salt to colored formazan by succinate dehydrogenase, the main mitochondrial enzyme, is proportional to the number of living cells. The results presented in Figure 2Go demonstrated that FB1 inhibited the proliferation of both IPEC-1 (panel A) and LLC-PK1 (panel B) cell lines in a dose-dependent manner. A significant decrease of the cell growth was noticed at FB1 concentrations determined to be noncytotoxic for IPEC-1 (10 and 20 µM, see above) and known to be noncytotoxic for LLC-PK1 (10 and 20 µM, see Yoo et al., 1996Go). The two cell lines were equally sensitive to the mycotoxin with an IC50 (concentration of FB1 responsible for 50% inhibition of formazan conversion) of 33 and 36 µM for IPEC-1 and LLC-PK1, respectively.



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FIG. 2. Cytotoxicity of FB1 on dividing and nondividing IPEC-1 cells as measured by the LDH release assay. IPEC-1 cells were seeded in 96-well plates either at low density (panel A, dividing subconfluent cells) or high density (panel B, nondividing confluent cells). They were allowed to adhere and were treated for 48 h with increasing concentrations of FB1. LDH activity was then measured in the supernatant. Values are expressed as mean ± SD of six independent wells. Values significantly different from the control are shown with ** (p < 0.005).

 
FB1 Blocks the Cell Cycle of IPEC-1 and LLC-PK1 Cell Lines in G0/G1 Phase
In order to better understand the effect of FB1 on epithelial cell proliferation, the percentage of IPEC-1 and LLC-PK1 cells within the different phases of the cell cycle was determined by fluorescence-activated cell sorting (FACS) analysis. For this purpose, both cell lines were treated for 48 hours with increasing concentrations of FB1 (ranging from 5 to 200 µM), and their DNA contents were analyzed after propidium iodide staining. As shown in Table 1Go, treatment with FB1 blocked the cell cycle progression of IPEC-1 and LLC-PK1 cells in the G0/G1 phase in a dose-dependent manner. Indeed, 64.3 and 69.3% of untreated IPEC-1 and LLC-PK1 cells, respectively, were in G0/G1 phase, whereas after a 48 h treatment with 200 µM FB1, these percentages increased to 80.4 and 85.3% for IPEC-1 and LLC-PK1 cells, respectively. IPEC-1 cells were found to be more sensitive than the LLC-PK1 cells, since the percentage of IPEC-1 cells in the G0/G1 phase changed significantly after 48 h of treatment with 5 µM FB1, whereas a FB1 concentration of at least 20 µM was necessary to induce a significant change in the cell cycle for LLC-PK1.


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TABLE 1 Percentage of Cells in the G0/G1 Phase After 48 h Exposure to FB1
 
FB1 Decreases the TEER of Intestinal Cells
The potential toxic effect of this mycotoxin was also assayed by measuring the TEER of IPEC-1 cells grown on Transwell filters. FB1 treatment started either when the cells were confluent but undifferentiated (Fig. 3Go, panel A) or when the cells were fully differentiated, i.e., 10 days after the beginning of the differentiation process (Fig. 3Go, panel B).



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FIG. 3. Inhibitory effects of FB1 on cell growth of IPEC-1 and LLC-PK1 cell lines as measured by the MTS assay. IPEC-1 (panel A) and LLC-PK1 (panel B) cells were seeded at low density in 96-multiwell plates, allowed to adhere, and then treated for 48 h with increasing concentrations of FB1. The amount of soluble formazan produced by cellular reduction of the MTS was then measured by absorbance at 492 nm. Values are expressed as the percentage of the maximal absorbance observed in untreated cultures, mean ± SD of three independent experiments. Statistical significance between treated culture and control is shown with * (p < 0.05).

 
Electrophysiological measurements were performed at regular intervals for one month. Our results showed a dose-dependent and time-dependent effect of FB1 on the disturbance of monolayer integrity of IPEC-1 cells, independently of the differentiation stage. Interestingly, the development and the maintenance of the TEER values were unaffected during the first 5 days, irrespectively of the FB1 tested doses. Fifteen to eighteen days were necessary to observe a complete abrogation of the TEER when high concentrations of toxin (200 and 500 µM) were used. Moreover, 13 days were needed to notice a significant TEER decrease with a lower concentration (50 µM) of FB1, as compared to the control cells, and after 26–28 days a small TEER value was still present.

The reversibility of the effects of FB1 on the epithelium integrity was then analyzed. Table 2Go shows that when cells were cultured for 16 additional days in a FB1-free medium after the end of FB1 treatment, a significant increase of the TEER was observed, although it was lower than the control values.


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TABLE 2 Recovery of Disturbance of IPEC-1 TEER
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The digestive epithelia of humans and animals consuming low quality maize-based foods and feeds can be exposed to high concentrations of fumonisins. Pigs, which mainly consume maize in certain areas, are naturally exposed to these toxins. FB1 is poorly absorbed (Shier et al., 2000Go), and in pigs its bioavailability after intragastric administration is only 4% (Prelusky et al., 1994Go). Thus, intestinal epithelial cells can be in contact with high amounts of FB1. Indeed, Shephard et al.(1995)Go demonstrated that 24 h after administration of radiolabeled FB1, intestinal epithelial cells from nonhuman primates contained 25% of the administrated dose. Norred et al.(1993)Go showed that 80% of the radiolabeled FB1 administered to rats was excreted in feces within 48 hours. The major route of elimination of FB1 is via the bile (Norred et al., 1993Go; Shephard et al., 1995Go), and enterohepatic circulation probably increases the exposure of intestinal cells to this mycotoxin.

In the present study we observed significant in vitro effects of FB1 on epithelial intestinal cells at concentrations of 5 to 50 µM, depending on the tested parameters. This would correspond to feed contaminated at 4 to 40 ppm, concentrations that are in the range observed in contaminated feed, throughout the world (Shephard et al., 1996Go). Indeed FB1 was found in 50% of maize samples collected between 1988 and 1991 from the Midwestern United States (Murphy et al., 1993Go). In this survey, 10% of the samples had toxin levels between 10 and 50 ppm (Murphy et al., 1993Go). Very high concentrations of FB1 (up to 76 ppm) have also been observed in moldy corn samples from Hungary (Fazekas and Tothe, 1998). In our study, some effects caused by FB1 (such as TEER decrease) were only observed after long exposure to the toxin (10–15 days). This long exposure is very likely to occur, as animals would eat the same batch of feed for an extended period of time.

In order to investigate the effects of FB1 on the pig intestine, we have used an epithelial cell line derived from the small intestine of a piglet. Using such cells, we demonstrated that concentrations up to 10 µM FB1 inhibit the proliferation of epithelial cells (Fig. 2Go). Comparable results were obtained with another porcine epithelial cell line (LLC-PK1, Fig. 2Go). Indeed, the IC50 for IPEC-1 and LLC-PK1 cells were 33 and 36 µM, respectively. These results are in agreement with those obtained by Yoo et al.(1992)Go, who showed an inhibition of LLC-PK1 proliferation with FB1 concentrations ranging from 10 to 35 µM. Other proliferating cell lines are also sensitive to FB1. Shier et al.(1991)Go showed that the cell proliferation of H4TG (rat hepatoma) and MDCK (dog kidney) cell lines was affected by FB1 and FB2. Similarly, Tolleson et al.(1996)Go observed apoptotic and antiproliferating effects of FB1 on human keratinocytes, fibroblasts, hepatoma cells, and esophageal epithelial cells.

The mechanisms of toxicity for fumonisins are complex and may involve several molecular sites. The primary biochemical effect of fumonisin is to inhibit ceramide synthase, leading to the accumulation of sphingoid bases and sphingoid base metabolites and to the depletion of more complex sphingolipids (Wang et al., 1991Go). Increases in free sphinganine concentration and in the sphinganine/sphingosine (Sa/So) ratio have been observed in several cell lines as well as in different tissues including mouse intestine (Enongene et al., 2002Go). In pigs, the Sa/So ratio has been studied in several organs (Gumprecht et al., 1998Go) but not in the intestine or in intestinal epithelial cells. However, preliminary results of our laboratory indicate that FB1 increases the Sa/So ratio in the IPEC-1 cell line (Loiseau, Bouhet, and Oswald, unpublished results). Alterations of sphingolipid metabolism contribute to the decreased cell death and cytolethality of FB1 observed in LLC-PK1, CHO, or HT-29 cells (Schmelz et al., 1998Go; Yoo et al., 1996Go; Yu et al., 2001Go). Using myriocin, a specific inhibitor of the enzyme serine palmitoyltransferase, the antiproliferative and cytotoxic effects of FB1 were found to be mainly due to the accumulation of free sphinganine (Riley et al., 1999Go; Schmelz et al., 1998Go) and linked to the induction of calmodulin, a molecule involved in apoptosis (Kim et al., 2001Go).

In this study we have found that the effect of FB1 on epithelial cell proliferation correlated with an arrest of cell cycle progression in the G0/G1 phase (Table 1Go). This effect was observed on porcine epithelial cell lines of both renal and intestinal origins (Table 1Go). An inhibition of the cell cycle progression in G0/G1 phase has also been reported in African green monkey kidney cells CV-1 (Ciacci-Zanella et al., 1998Go; Wang et al., 1996Go) and in rat hepatocytes (Ramljak et al., 2000Go) treated with FB1. In the latter case, the interference with the cell cycle has been proposed as the potential mechanism for FB1-mediated hepatocarcinogenesis (Ramljak et al., 2000Go). Other studies have indicated that FB1 may inhibit the cell cycle progression in different phases of the cell cycle. An arrest in the G2/M phase was observed in C6 glioma cells (Mobio et al., 2000Go) and in WHCO3 esophageal cancer cells (Seegers et al., 2000Go).

FB1 exerts a cytotoxic effect only when present at high concentrations (over 50 µM, Fig. 1Go). In addition, this effect is different on proliferating and nonproliferating cells (Fig. 1Go). A significant LDH release was observed for FB1 concentrations higher than 50 and 700 µM on proliferating and nonproliferating IPEC-1 cells, respectively. An increased susceptibility of dividing cells compared to the nondividing ones has already been described in cell lines and primary cells (Li et al., 2000Go; Schmelz et al., 1998Go; Yoo et al., 1996Go). It correlates with a higher rate of de novo sphingolipid biosynthesis in dividing cells that leads to a greater accumulation of free sphinganine upon FB1 treatment (Schmelz et al., 1998Go; Yoo et al., 1996Go). This selective sensitivity of dividing cultures to FB1 compared to nondividing ones may be related to differences in the kinetics of the enzymes involved in the sphingolipid biosynthesis pathway (ceramide synthase, serine palmitoyltransferase, sphinganine kinase, etc.), as suggested by the recent work of Enongene et al.(2002)Go or to the differential expression of Lag1p, a newly discovered protein encoding for a longevity assurance gene (Riebeling et al., 2003Go).

One of the main outcomes of the present study is that a prolonged treatment with FB1 disturbs the establishment of the transepithelial electrical resistance of an intestinal epithelial cell culture and alters the TEER of an already established one (Fig. 3Go). These results are in agreement with a previous study showing that FB1 treatment disrupts the endothelial cell monolayer integrity as indicated by the reduced rate of albumin transfer across the endothelial monolayer (Ramasamy et al., 1995Go). In our study, we have shown that treatment with 50 µM of FB1 for at least 13 days alters the TEER of both undifferentiated and differentiated cells (Fig. 3Go). The lag of time between the first exposure to the toxin and the effect on the TEER could be explained by the time necessary to significantly alter the intracellular concentration of sphingoid bases and/or complex sphingolipids (Enongene et al., 2002Go). The mechanism of FB1 disruption of the TEER has never been investigated; however several hypotheses can be proposed. First, the FB1 alteration of second messenger, such as sphinganine and ceramide, may result in the activation of signaling pathways leading to a loss of TEER. Alternatively, FB1 may alter the sealing function via the depletion in glycosphingolipids. Indeed, recent data suggest that glycosphingolipids could be structural components of the tight junction and/or contribute to the "raft-like" environments around the tight junction (Nusrat et al., 2000Go).

Several studies have shown the poor absorption of FB1 in the intestine. In laying hens and in cows, the systemic absorption of orally given FB1 is below 1% (Prelusky et al., 1995Go). In pigs, the bioavailability of FB1 following intragastric administration is estimated at 4% (Prelusky et al., 1994Go). However, all these studies were conducted with a single administration of FB1. Our in vitro results indicated that chronic exposure to FB1 diminishes the TEER of intestinal monolayers. We could hypothesize that an increase in membrane permeability may lead to an increased FB1 absorption. Thus, animals chronically exposed to FB1 may have a higher absorption of the mycotoxin than naive animals, and this might contribute to greater systemic susceptibility in chronically exposed animals.

The intestinal epithelium is a barrier protecting the organism from chemical products and also from microbial pathogens. One consequence of the decrease of intestinal barrier function by FB1 could be the increased translocation of bacterial pathogens across the intestine. Moreover, because sphingolipids and glycosphingolipids could act as membrane receptors for bacteria (Khan et al., 2000Go), the disruption of sphingolipid metabolism by FB1 may modify bacterial receptors on the surface of epithelial cells. Both effects could contribute to an increased colonization of the intestinal tract by pathogenic bacteria. Indeed, recent results from our laboratory indicate that, in piglets, the oral administration of FB1 for 7 days significantly increases colonization of the small and the large intestine by pathogenic E. coli strains and augments the bacterial translocation to extraintestinal organs (Oswald et al., 2003Go).

In conclusion, our results indicate that FB1 has a toxic effect both on undifferentiated and differentiated intestinal epithelial cells. FB1 decreases the growth of proliferating epithelial cells by blocking them in the G0/G1 phase of the cell cycle. FB1 also impairs the establishment of the epithelial barrier and disrupts that already established. Taken together, all these results strengthen the hypothesis that a chronic consumption of FB1 contaminated food or feed can induce intestinal damage and may have consequences for animal and human health.



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FIG. 4. Time-course of FB1-induced TEER decrease in IPEC-1 cells. IPEC-1 cells cultured on Transwell filters were incubated for various times in absence or in presence of FB1 added in the apical compartment. The integrity of the tight junctions was assessed by measuring the TEER. Treatment with FB1 began either on confluent cells, before the beginning of their differentiation (panel A), or on fully differentiated cells (panel B). White squares, untreated cultures; black squares, black traingles, and black diamonds are cultures treated with 50, 200, and 500 µM FB1, respectively. The values are expressed as the mean ± SD of three independent wells.

 

    ACKNOWLEDGMENTS
 
We are grateful to Drs. Helen M. Berschneider and Denis D. Black for their kind gift of the IPEC-1 cells and to Neil Ledger for helpful comments regarding the manuscript. S.B. was supported by a Fellowship from the Ministère de l’Éducation Nationale, de la Recherche et de la Technologie. This work was supported in part by the Région Midi-Pyrénées, France (DAER-Rech/99008345) and by the Transversalité INRA (Mycotoxines-P00263).


    NOTES
 
1 To whom correspondence should be addressed at Laboratoire de Pharmacologie Toxicologie, INRA, 180 Chemin de Tournefeuille, 31931 Toulouse Cedex 9, France. Fax: 33 (0) 561285310. E-mail: ioswald{at}toulouse.inra.fr. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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