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Institut Méditerranéen de Recherche en Nutrition, Unité Mixte de Recherche-Institut National de la Recherche Agronomique, Faculté des Sciences de Saint-Jérôme, Marseille, France
2To whom correspondence should be addressed. E-mail: jacques.fantini{at}univ.u-3mrs.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • deoxynivalenol • intestinal absorption • nutrient • HT-29 • mycotoxin • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). These mycotoxin are potent inhibitors of protein synthesis (1
–4
). Deoxynivalenol (DON)3
is the most prevalent trichothecene in crops used for food and feed production (5
,6
). For instance, levels of 0.9–7.6 µg/kg have been detected in wheat samples (7
), representing a potential risk for both farm animals and human health. Toxic effects of DON on animals have been well documented and concern mainly the immune system and the gastrointestinal tract (1
). In particular, DON causes vomiting, necrosis, diarrhea, emesis and malabsorption of nutrients (1
,8
–10
). In vitro, DON has been shown to interfere with the differentiation of the intestinal epithelial cell lines Caco-2 and T-84 (11
). However, the effect of DON on the intestinal absorption of nutrients has not been investigated. In this report, we used the human intestinal cell line HT-29-D4 as an in vitro model for the intestinal epithelium. These cells have been used in the past for deciphering the molecular and cellular mechanisms of enteropathy induced by viruses (12
) and xenobiotics (13
). In particular, these cells express high levels of intestinal nutrient transporters, such as the sodium-dependent glucose/galactose transporter SGLT1, facilitated sugar transporters GLUT2 and GLUT5, and amino-acid transporters (12
,13
). Using this well characterized clonal cell line, we studied the effect of DON on the intestinal absorption of nutrients. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell culture media and reagents were from BioWhittaker (Emerainville, France), except glucose-free Dulbecco’s modified minimum Eagle medium (DMEM), which was purchased from Sigma (St. Louis, MO). Deoxynivalenol (DON), deoxycholate and cycloheximide were from Sigma. Stock solutions were prepared in ethanol and stored at -20°C before use. Fetal calf serum was from Dutscher (Strasbourg, France). Nonradioactive substrates were from Sigma. Radioactive substrates were from Amersham Pharmacia Biotech Europe (Saclay, France): methyl-
-D-[U-14C]glucopyranoside (250 mCi/mmol) (AMG), D-[U-14C]fructose (250 mCi/mmol) (D-Frc), L-[U-14C]serine (155 mCi/mmol) (L-Ser), [1-14C]Palmitic acid (55 mCi/mmol), [7-3H]cholesterol (20 Ci/mmol) and 2-Deoxy-D-[1-3H] glucose (20 Ci/mmol) (DOG).
Cell culture.
HT-29-D4 cells (14
,15
) were routinely grown in 75-cm2 flasks (Costar, Strasbourg, France) in DMEM/F12 medium supplemented with 10% fetal calf serum. To induce differentiation, half-confluent HT-29-D4 cells were grown in glucose-free DMEM supplemented with 5 mmol/L galactose and 10% dialyzed fetal calf serum for 16 d, as previously reported (12
). Before toxin exposure, the cells were washed three times with serum-free medium and the incubations with DON, deoxycholate or cycloheximide were performed in serum-free culture medium. All culture wells received the same ethanol volume, i.e., 0.5% of final volume.
Nutrient uptake measurements.
Differentiated HT-29-D4 cells cultured in 12-well plates were either not incubated or incubated with increasing DON, cycloheximide or deoxycholate concentrations for various times. Then, the cells were analyzed for nutrient uptake capacity. Dishes were washed three times with Ringer-Hepes medium [137 mmol/L NaCl, 5.36 mmol/L KCl, 0.4 mmol/L Na2HPO4, 0.8 mmol/L MgCl2, 1.8 mmol/L CaCl2, 20 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)] adjusted to pH 7.4 with NaOH. The initial rate of nutrient uptake was measured with a substrate concentration of 0.1 mmol/L (0.15 mCi/L) and for various times corresponding to a linear increase in nutrient transport: 20 min for AMG, 10 min for DOG, D-Frc, L-serine, palmitic acid, and 5 min for cholesterol. For sugar transport experiments, three substrates were used: AMG, that is specific of SGLT1; D-Frc, a specific substrate of the passive glucose transporter GLUT5; and DOG, that is transported by all members of the GLUT family. For L-serine, the total cellular uptake is the sum of a passive, Na+-independent and an active, Na+-dependent transport. For this reason, the uptake of L-serine was measured in both the classical Ringer-Hepes medium and Na+-free Ringer-Hepes medium with choline chloride and potassium phosphate replacing, respectively, sodium chloride and sodium phosphate (adjusted to pH 7.4 with KOH). The active part of L-serine uptake was calculated by subtracting the passive L-serine uptake measured in Na+-free medium from total L-serine uptake, measured in classical medium. At the end of the incubation with each nutrient, the medium was removed and the cells were extensively washed with ice-cold medium. The cells were disrupted with 0.1 mol/L NaOH, 1 g/L of sodium dodecyl sulfate, and the radioactivity was measured in a Packard ß-counter. The results are expressed as picomoles of nutrient per milligram of protein per minute. The specificity of sugar transport was assessed by the inhibitory effect of phlorizin (100 µmol/L) for SGLT1 and cytochalasin B (100 µmol/L) for GLUT transporters.
Cellular content in proteins.
The protein content was evaluated according to the Folin procedure using bovine serum albumin as the standard (13
).
Cell proliferation assay.
The effects of DON on the proliferation and viability of undifferentiated HT-29-D4 cells were studied in a colorimetric assay using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) as previously reported (16
). Briefly, HT-29-D4 cells were seeded in 96-well plates at a density of 2.5/104 cells/well in DMEM/F12 medium supplemented with 10% fetal calf serum. After a lag time of 4 h to allow cell adhesion, DON was added to the medium at concentrations ranging from 0.1 to 100 µmol/L. At 0, 24 and 48 h after DON exposure, the cells were used in the MTT assay.
LDH release assay.
To study the membrane integrity of cells exposed to the toxin, we measured the release of the cytosolic enzyme, lactate deshydrogenase (LDH). Differentiated HT-29-D4 cells were incubated with increasing concentrations of DON for 48 h. At the end of the incubation, the LDH activity was measured in the culture medium with a Sigma kit based on lactate oxidation by LDH with simultaneous reduction of nicotinamide adenine dinucleotide NAD to NADH. The total intracellular LDH activity was estimated after lysis of the cells with Triton X-100.
Electrophysiological measurements.
Differentiated HT-29-D4 cells were cultured in two-compartment cell culture chambers (Transwell-clear, catalogue number 3450; Costar) and analyzed for electrical parameters in a modified Ussing chamber as reported (12
). The cells were incubated in serum-free culture medium for 48 h in the presence of increasing concentrations of DON. At the end of the incubation, the cells were washed three times with Ringer-Hepes medium. Apical and basal compartments of the Ussing chamber were filled with Ringer-Hepes medium at 37°C. Transepithelial potential difference was measured with electrodes and continuously recorded using a voltage clamp unit (Physiologic Instrument, San Diego, CA). Bipolar current pulses (20 µA for 2 s, every 30 s) were passed through electrodes to measure the transepithelial electrical resistance according to the Ohm’s law.
Protein synthesis measurement.
Differentiated HT-29-D4 cells were cultured on 12-well plates. The cells were washed three times with DMEM and then incubated with increasing concentrations of either DON or cycloheximide for 1 h at 37°C. L-14C-serine was then added to the cells for a further incubation of 1 h at 37°C (final concentration 1 mCi/L). At the end of the incubation, the cells were washed three times with Ringer-Hepes medium and then lysed with 500 µL of 200 g/L NaOH at 37°C for 30 min. Cellular proteins were precipitated by addition of 1.5 mL of 400 g/L TCA at 4°C for 30 min. TCA precipitates were filtered on glass microfiber filters (GF/C membrane; Whatman, Tewksbury, MA). Membranes were washed three times with 50 g/L TCA and dried with ethanol at 95°C before counting radioactivity in a Packard ß-counter. The IC50 was determined as the concentration of toxin needed to inhibit 50% of the incorporation of L-14C-serine in a single experiment.
Acridine orange staining.
Induction of apoptosis in HT-29-D4 cells by DON was investigated with the fluorescence dye acridine orange. Using this staining procedure, DNA in nuclei of fixed cells appears yellow-green, whereas RNA appears light red (17
). Apoptotic cells showed a particular morphology with chromatin condensation and nuclear fragmentation (18
). Differentiated HT-29-D4 cells were cultured on glass coverslips and incubated with various concentrations of DON or cycloheximide for 48 h or deoxycholate for 24 h. At the end of the incubation, the cells were washed with phosphate-buffered saline (PBS) and fixed with 40 g/L paraformaldehyde in PBS for 20 min at room temperature. After PBS washes, the cells were stained for 3 min with 0.5 g/L acridine orange in PBS. After washing, the cells were incubated for 30 s in 100 mmol/L CaCl2, followed by three washes with PBS. The staining was observed with a Leica fluorescence microscope (Marseille, France).
Statistical analysis.
All experiments were conducted in triplicate and the results expressed as means ± SD. ANOVA and the Fisher multiple-comparison posthoc test were conducted. Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The toxicity of DON for proliferating HT-29-D4 cells was studied with the MTT assay (Fig. 1
). DON had no significant effect for concentrations 
0.1 µmol/L. At 1 µmol/L, DON inhibited cell proliferation 48 ± 8% (P < 0.01) after 24 h of incubation. At 10 µmol/L of DON, cell proliferation was inhibited by 79 ± 5% (P < 0.001). At 100 µmol/L, cell proliferation was blocked and significant cell loss was observed because the optical density of DON-treated cells at 24 and 48 h was less than the initial value (P < 0.001).
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). At concentrations 10 µmol/L, DON slightly but significantly increased LDH release (130% of the control value, P < 0.05). At 100 µmol/L, DON increased LDH release to 642% of the control value (P < 0.0001). DON did not alter the cellular protein content at concentrations less than 10 µmol/L (Fig. 2)
, but treatment with 100 µmol/L decreased the amount of cellular protein (0.776 ± 0.008 vs. 0.46 ± 0.017 mg/well for control cells and DON-treated cells, respectively, P < 0.0001). Consistent with these data, DON did not affect the cellular morphology at concentrations lower than 100 µmol/L (Fig. 3
). At this concentration, the mycotoxin caused cell damage, including rounding of cells and, in some areas, cell loss from the monolayer. The transepithelial electrical resistance (TER) for mock-treated cells was 520 ± 20 
.cm2 and this value remained stable after 48 h of treatment with up to 1 µmol/L DON (Fig. 2)
. TER was decreased 57% in cells treated with 10 µmol/L DON (P < 0.001). However, at 100 µmol/L of DON, TEER was abolished, showing that the cells no longer formed an intact epithelial monolayer (P < 0.00001).
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Dose-dependent studies of DON effects at 48 h on sugar uptakes (Fig. 4
) showed that: DON inhibited the uptake of AMG with a 50% decrease at 10 µmol/L (P < 0.05) and a maximal effect at 100 µmol/L (76 ± 1.6% of inhibition, P < 0.01); and DON decreased D-Frc uptake, with a minimal effective concentration of 10 nmol/L (79.6 ± 2.2% of control D-Frc uptake, P < 0.01) and a maximal inhibitory concentration of 10 µmol/L (58 ± 0.1% of control D-Frc uptake, P < 0.001). At 100 µmol/L of DON, the uptake of D-Frc did not differ from control values. The uptake of DOG was only slightly affected by DON for concentrations 1 µmol/L (17 ± 0.7% of inhibition at 1 µmol/L, P < 0.01), but was increased at 10 and 100 µmol/L [i.e., 115 (P < 0.05) and 304% (P < 0.001) of control uptake, respectively].
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) showed that the effects of 100 µmol/L DON on AMG uptake occurred after 24 h of treatment. At this time, the mycotoxin significantly inhibited AMG uptake (63% of control uptake, P < 0.05). After 48 h of DON incubation, the level of AMG transport was further decreased to 21% of the control value (P < 0.05). After 72 h of incubation, this value was not different from the 48-h value. DON increased the uptake of DOG after 48 h of exposure (310% of control uptake, P < 0.01), but not before.
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). In mock-treated cells, the uptake of AMG was strongly inhibited (98%) by phlorizin, showing that ethanol itself did not affect the activity of SGLT1 (P < 0.01). For DON-treated cells, residual SGLT1 activity was detected and the AMG uptake in this case was definitely phlorizin-sensitive. Control and 100 µmol/L DON treated cells had a similar level of phlorizin-independent AMG uptake. Similar results were obtained with D-Frc, because both control and 100 µmol/L DON-treated cells had comparable levels of cytochalasin B-independent uptake of D-Frc.
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Effect of DON on amino-acid uptake.
For concentrations less than 1 µmol/L, DON did not significantly affect L-serine uptake (Fig. 6
). At 1 and 10 µmol/L, DON decreased total (Fig. 6
A), Na+-independent (Fig. 6
B), and Na+-dependent (Fig. 6
C) uptakes of L-serine to various degrees (respectively, 31 ± 2.8; 38 ± 6.9 and 30 ± 2.4% inhibition for 10 µmol/L of DON, P < 0.05). At 100 µmol/L, DON increased L-serine uptake to 263 ± 24; 169 ± 13; 275 ± 26% of control, respectively, for total; passive; and active transports compared with control L-serine uptake (P < 0.05).
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At concentrations less than 1 µmol/L, DON did not alter the uptake of palmitate (Fig. 7
). Yet, at 10 and 100 µmol/L, DON dose-dependently increased palmitate uptake (135 ± 0.5 and 191 ± 7.5% of control uptake, respectively, P < 0.01). DON did not affect cholesterol uptake at concentrations less than or equal to 10 µmol/L. However, at 100 µmol/L, the uptake of cholesterol was significantly increased (161 ± 7.5% of control uptake, P < 0.01).
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Next, we tested the possibility that 100 µmol/L of DON could increase nutrient uptake by rendering accessible basolateral membrane transporters of HT-29-D4 cells to apically added nutrients. Cells were cultured in two-compartment culture chambers and incubated with 100 µmol/L of DON in serum-free culture medium for 48 h. At the end of the incubation, uptake studies were conducted with nutrients added to both sides (i.e., apical and basolateral) of the cellular monolayer. The presence of nutrients in both compartments of the culture chambers did not modify the effects of DON (100 µmol/L) on nutrient uptake, i.e., the inhibition of SGLT1 activity (P < 0.01) and the significant increases in all other nutrient transport activities tested (P < 0.05) except for D-Frc (Table 2
).
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To study whether DON effects were due to an inhibition of protein synthesis, we tested the effects of cycloheximide on nutrient uptake. Cycloheximide dose-dependently decreased AMG uptake with a minimal effective concentration of 100 nmol/L (68.0 ± 1.8% of control AMG uptake, P < 0.01), a IC50 of 10 µmol/L (P < 0.001), and a maximal effect at 1 mmol/L (15.5 ± 0.9% of control AMG uptake, P < 0.001; Table 3
). Moreover, cycloheximide increased the uptake of the other nutrients at concentrations above 100 µmol/L (P < 0.01). The remarkable similarity between the activity of cycloheximide and DON on nutrient uptake is consistent with their common ability to inhibit protein synthesis. Indeed, the incorporation of 14C-serine in nascent proteins was dose-dependently inhibited by both toxins in HT-29-D4 cells with an IC50 of 2.3 and 30 µmol/L for DON and cycloheximide, respectively (data not shown).
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In control HT-29-D4 cells, apoptosis was not detected (Fig. 8A
). When cells were incubated with 100 µmol/L of the apoptosis inducer deoxycholate (24 h), some with typical apoptotic morphology were observed (Fig. 8
B). Lower concentrations of deoxycholate did not alter cell staining. Similarly, DON did not cause apoptotic morphology at concentrations less than 100 µmol/L (data not shown). However, 48 h of exposure of differentiated HT-29-D4 cells to 100 µmol/L of DON or to 1 mmol/L of cycloheximide clearly induced cellular apoptosis (Fig. 8
, C and D). Finally, we studied the effects of a 24-h exposure of differentiated HT-29-D4 cells to deoxycholate on AMG and DOG uptakes (Fig. 9
). Deoxycholate dose-dependently decreased AMG uptake with 31 and 71% of inhibition at 50 (P < 0.05) and 100 µmol/L (P < 0.01), respectively. At 100 µmol/L, deoxycholate increased DOG uptake (263% of control uptake, P < 0.001). Phlorizin and cytochalasin B inhibited AMG and DOG uptakes of 100 µmol/L deoxycholate-treated cells to the same extent as in control cells (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), but also by specifically reducing nutrient absorption. Therefore, it has a potent, broad-range anti-nutritional effect (10
). Although feed refusal, reduced weight gain, diarrhea and emesis were observed after human exposure to DON (19
), studies on the effects of DON on human intestinal nutrient uptake are few. The HT-29-D4 cell line is a valuable model of the human intestinal epithelium because it expresses high levels of intestinal transporters. Moreover, HT-29-D4 cells have been recently used to caracterize the enteropathogenic effect of another mycotoxin, ochratoxin A (13
). In this study, this cell line was used to evaluate the effects of DON on the absorptive capacity of human enterocytes. Classical toxicity assays, i.e., LDH release, protein content, and TER measurement, showed that DON is not toxic to differentiated HT-29-D4 cells at concentrations below 100 µmol/L (29 µg/L). At this concentration, DON induced local cell detachment from the epithelial monolayer, which was correlated with a decrease in protein content, an increase in LDH activity in culture supernatants, alterations of cellular morphology and a sharp decrease in TER. These toxic effects are consistent with in vivo observations that demonstrated necrosis of the intestinal epithelium, inflammatory diarrhea and intestinal bleeding in animals fed high amounts of trichothecene mycotoxins (20
–24
). For example, an acute oral intoxication of mice with DON (78 µg/kg) induced extensive necrosis of the gastrointestinal tract (24
). At DON concentrations below 100 µmol/L, HT-29-D4 cells did not exibit typical signs of toxicity. Interestingly, proliferating HT-29-D4 cells appeared to be more sensitive to DON than terminally differentiated cells. Indeed, 1 µmol/L (296 ng/mL) DON inhibited the proliferation of undifferentiated HT-29-D4 cells (48% inhibition, P < 0.01), in agreement with previous studies performed on fibroblasts and T cells (25
,26
). In differentiated HT-29-D4 cells, the absorptive functions were affected by DON concentrations greater than 10 µmol/L (2.96 µg/mL). The difference in sensitivity between undifferentiated and differentiated HT-29-D4 cells was explained by the fact that the proliferation of differentiated cells was arrested by contact inhibition (14
).
In differentiated HT-29-D4 cells, the uptake of nutrients was specifically affected, depending on both the nutrient studied and the mycotoxin concentration. At DON concentrations less than or equal to 10 µmol/L, the active D-glucose transporter (SGLT1) appeared to be the most DON-sensitive transporter, followed by the passive D-Frc transporter (GLUT5). Passive and active L-serine transporters exhibited a moderate sensitivity, whereas passive sugar transporters of the GLUT family were only slightly affected by the mycotoxin. In contrast, concentrations of DON less than or equal to 10 µmol/L did not affect lipid uptake as assessed by experiments with palmitic acid and cholesterol. Taken together, these data suggest that low concentrations of DON may selectively affect the intestinal absorption of D-glucose and D-galactose. In vivo experiments are fully consistent with our in vitro data. Indeed, treatment with DON (10 µg/kg) decreased D-glucose absorption in mice (10
). Other trichothecene mycotoxins such as fusarenon-X and T2-toxin also have been shown to inhibit D-glucose absorption in animals (27
–29
). In addition to this anti-nutritional effect, the inhibition of SGLT1 by DON could also cause a watery diarrhea syndrome since SGLT1 is responsible for the daily absorption of 5 L of water (30
).
The differential effect of DON on SGLT1 vs. all other nutrient transporters was especially important for 100 µmol/L of DON. At this concentration, SGLT1 activity was inhibited by 76%, whereas the activity of the other nutrient transporters was increased. Because 100 µmol/L of DON induced the release of LDH, it was logical to consider that the inhibition of SGLT1 and the stimulation of passive uptakes could be due to membrane permeabilization. However, this hypothesis was rejected on the basis of two observations. First, among the nutrients tested, Na+-dependent L-serine uptake was increased by 100 µmol/L of DON although it is an active transport process that takes place against a concentration gradient, just like SGLT1. Second, the uptake of sugars, i.e., both active (AMG) and passive (DOG/D-Frc), remained sensitive to specific inhibitors of the corresponding membrane transporters (i.e., phlorizin and cytochalasin B) upon treatment of cells with 100 µmol/L of DON. Therefore, we conclude that DON effects are due to a specific modulation of the activity of intestinal transporters rather than a consequence of nonspecific cell damage. As a matter of fact, the increase in LDH activity in culture medium supernatants could be attributed to local cell loss and not to a general permeabilization of the epithelial monolayer (Fig. 3)
. Moreover, because dead cells were washed out before uptake studies, their contribution to transport activity measurements is certainly minimal.
An alternative explanation for the stimulation of nutrient uptake activities could be an improvement of the accessibility of membrane transporters resulting from the decrease of TER observed at 100 µmol/L of DON. Namely, in absence of functional tight junctions, nutrients added apically could theoretically be transported by both apical and basolateral transporters, explaining the increase in transport activities observed with 100 µmol/L of DON. This hypothesis was also dismissed because we observed that uptake measurements when nutrients were incubated on both sides of the epithelium gave the same results as when nutrients were added only apically.
How can we explain the selective effects of DON on nutrient uptakes? Previous studies have shown that inhibition of protein synthesis in adipocytes by cycloheximide increased the activity of GLUT. This increase could be attributed to an increase of GLUT mRNA content leading to an increase of GLUT transporter proteins in the plasma membrane (31
) and/or to a direct stimulation of GLUT transporters already present in the membrane (32
). The basal activity of GLUT was constitutively inhibited by one or more regulatory protein(s) with a very short half-life. While inhibited by protein synthesis, the expression level of such regulatory protein(s) was decreased and the constitutive inhibition of GLUTs activity was removed. In this respect, it is important to note that both DON and cycloheximide had similar effects on HT-29-D4 transporters and on the incorporation of 14C-Serine into nascent proteins. These data strengthen our conclusion that DON acts by inhibiting protein biosynthesis in HT-29-D4 cells.
Acridine orange staining showed that DON could induce apoptosis of differentiated HT-29-D4 cells. This effect has been previously described for DON and others trichothecene mycotoxins (33
–36
). To study the potential involvement of apoptosis induction in DON effects on nutrient uptakes, we used the bile salt deoxycholate, a well characterized inducer of apoptosis in the HT-29 cell model (37
,38
). Deoxycholate caused the same effects as DON or cycloheximide on sugar transport, i.e., a decrease in AMG uptake and an increase in DOG uptake. These data suggest that the various effects of DON on nutrient uptake may be due to the ability of the tricothecene to induce apoptosis through protein synthesis inhibition, as previously shown for cycloheximide-treated cardiomyocytes (39
). To the best of our knowledge, such a selective modulation of nutrient absorption in intestinal cells undergoing apoptosis has never been reported. Whether this regulation of nutrient uptake capacity reflects a general adaptation of intestinal cells to stressful conditions remains to be established.
Overall, our data shed some light on the mechanism by which DON could induce diarrhea in animals and humans. Exposure to low amounts of DON (less than or equal to 10 µmol/L) may cause an aqueous diarrhea through inhibition of the intestinal SGLT1 transporter, resulting in a decrease of D-glucose-associated water absorption and so in an increase of water content in the intestinal lumen. Exposure to high amounts of DON (greater than or equal to 100 µmol/L) may lead to the concomitant inhibition of SGLT1 and to local destruction of the epithelial barrier, resulting in an inflammatory diarrhea. Interestingly, similar conclusions could be drawn from the study of another mycotoxin that inhibits protein synthesis, ochratoxin A (13
).
As for the potential nutritional risk of DON contamined-food for humans, Kuiper-Goodman (40
) estimated the tolerable daily intake for adults to be 3.0 µg of DON per kg of body. Assuming that 9 L of water are present daily in the small intestine (41
), this quantity represents a maximal concentration of 77 nmol/L (0.02 µg/L) of DON. Accordingly, the consumption of controlled food with respect of the tolerable daily intake of 3.0 µg/kg of body should not represent a risk for human health. Nevertheless, the detection of greater amounts of DON (up to 7.6 µg/kg) in wheat samples (7
) indicates that great caution is needed to avoid the contamination of feed by hazardous mycotoxins.
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FOOTNOTES |
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3 Abbreviations used: AMG, -methyl-glucose; DMEM, Dulbecco’s modified minimum Eagle’s medium; DOG, 2-deoxy-glucose; DON, deoxynivalenol; Frc, fructose; GLUT, D-glucose; LDH, lactate deshydrogenase; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PBS, phosphate-buffered saline; TCA, trichloroacetic acid; TER, transepithelial electrical resistance.
Manuscript received 18 March 2002. Initial review completed 2 April 2002. Revision accepted 20 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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