DNA/RNA synthesis; Mycotoxin

Deoxynivalenol (DON) is a mycotoxin of the trichothecenes family to which human exposure levels can be high. Epidemiological studies suggest a link between DON and gastrointestinal illness. We investigated the interaction of DON with Caco-2 cells, a widely used in vitro model of the human intestinal barrier. The apical to basolateral (absorption) and basolateral to apical (excretion) transports of DON were found strictly proportional to both the initial concentration and the duration of the incubation. The absorption and excretion mean rates were similar to those of mannitol and were increased in the presence of EGTA, a calcium chelator. These data suggest that DON crosses the intestinal mucosa by a paracellular pathway through the tight junctions although some passive transcellular diffusion may not be ruled out. The DON transport was not affected by P-glycoprotein (PgP) or multidrug resistance-associated proteins (MRPs) inhibitors. A prolonged exposure to DON provokes the phosphorylation of the mitogen-activated protein kinases (MAPKs) Erk1/2, p38 and SAPK/JNK, as well as a decrease of the transepithelial resistance, suggesting that DON could trigger intestinal inflammation. These data imply that a chronic exposure to DON contaminated foods may negatively affect human health by altering the intestinal mucosa integrity and by inducing the MAPKs implicated in inflammation. [1]

Deoxynivalenol (DON, vomitoxin), is one of the most common contaminants of cereal grains world-wide. The effects of DON on fetal development were assessed in Charles River Sprague-Dawley rats. Pregnant female rats were gavaged once daily with DON at doses of 0, 0.5, 1, 2.5, or 5 mg/kg body weight on gestation days (GD) 6-19. At cesarean section on GD 20, reproductive and developmental parameters were measured. All females survived to cesarean section. DON caused a dose-related increase in excessive salivation by the pregnant females, a reaction probably linked to the lack of emetic reflex in rats. At 5 mg/kg, feed consumption and mean body weight gain were significantly decreased throughout gestation, mean weight gain (carcass weight), and gravid uterine weight were significantly reduced, 52% of litters (12/23) were totally resorbed, the average number of early and late deaths per litter was significantly increased, average fetal body weight and crown-rump length were significantly decreased, the incidence of runts was significantly increased, and the ossification of fetal sternebrae, centra, dorsal arches, vertebrae, metatarsals, and metacarpals was significantly decreased. At 2.5 mg/kg, DON significantly decreased average fetal body weight, crown-rump length, and vertebral ossification. These effects may be secondary to maternal toxicity and the reduced size of the fetuses. The incidence of misaligned and fused sternebrae was significantly increased at 5.0 mg/kg. No adverse developmental effects were observed at 0.5 and 1.0 mg/kg. Dose-related increases in maternal liver weight-to-body weight ratios were observed in all treated groups (significant at 1, 2.5, and 5 mg/kg). The weight changes were correlated with dose-related cytoplasmic alterations of hepatocytes. The NOEL for maternal toxicity for this study is 0.5 mg/kg based on the dose-related increase in liver-body weight ratio at 1 mg/kg. The NOEL for fetal toxicity is 1 mg/kg based on the general reduction in fetal development at 2.5 and 5 mg/kg. DON is considered a teratogen at 5 mg/kg day in Sprague-Dawley rats based on the anomalous development of the sternebrae. [3]

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Deoxynivalenol (DON or vomitoxin) is a trichothecene mycotoxin commonly found in cereal grains that adversely affects growth and immune function in experimental animals. A competitive enzyme-linked immunosorbent assay (ELISA) was used to monitor the kinetics of distribution and clearance of DON in tissues of young adult B6C3F1 male mice that were orally administered 25mg/kg bw of the toxin. DON was detectable from 5 min to 24h in plasma, liver, spleen and brain and from 5 min to 8h in heart and kidney. The highest DON plasma concentrations were observed within 5-15 min (12 microg/mL) after dosing. There was rapid clearance following two-compartment kinetics (t(1/2)alpha=20.4 min, t 1/2 beta=11.8h) with 5% and 2% maximum plasma DON concentrations remaining after 8 and 24h, respectively. DON distribution and clearance kinetics in other tissues were similar to that of plasma. At 5 min, DON concentrations in mug/g were 19.5+/-1.9 in liver, 7.6+/-0.5 in kidney, 7.3+/-0.8 in spleen, 6.8+/-0.9 in heart and 0.8+/-0.1 in the brain. DON recoveries in tissues by ELISA were comparable to a previous study that employed (3)H-DON and 25mg/kg bw DON dose. The ELISA was further applicable to the detection of DON in plasma of mice exposed to the toxin via diet. This approach provides a simple strategy that can be used to answer relevant questions in rodents of how dose, species, age, gender, genetic background and route/duration of exposure impact DON uptake and clearance.[5]

Transport studies [1]
For transport experiments, the culture medium was replaced by Hank's balanced salt solution (HBSS) containing 5 mM glucose and 10 mM Hepes (pH 7.4) for the lower compartment of the inserts or 10 mM Mes (pH 6.0) for the upper compartment. The lower compartment was further supplemented with 1% (w/v) bovine serum albumin. Deoxynivalenol (DON) or 4.25 μM [14C]mannitol were added to the upper or lower compartment containing, respectively, 1.8 ml or 2.8 ml of transport medium. Deoxynivalenol (DON) was introduced in the donor compartment at 2 μg/ml for the kinetic experiments or at various concentrations, from 0.16 to 7.5 μg/ml, in the dose–effect experiments. At each sampling time, an aliquot of transport medium was withdrawn from the acceptor compartment and replaced by fresh transport medium. Samples were analyzed for their Deoxynivalenol (DON) content by HPLC or for their [14C]mannitol content by liquid scintillation spectrometry fter dispersion in 2 ml of Aqualuma®. To detect the possible appearance of Deoxynivalenol (DON)-conjugated metabolites, some samples were treated with a mixture of β-glucuronidase and arylsulfatase, for 24 h at 37 °C in a 0.2 M acetate buffer at pH 5, before their analysis by HPLC.

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Assessment of MAPKs phosphorylation [1]
The cells were cultivated on a microporous membrane for 21–27 days, as described for transport studies. They were then incubated overnight in the same medium without EGF and insulin. Deoxynivalenol (DON) was then added, at 2 μg/ml in the upper compartment for specified durations or at various concentrations during 24 h.

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Alkaline phosphatase (AP) activity [2]
The cellular enzymatic activity of alkaline phosphatase was detected by formation of blue-colored diformazan precipitate within 22 d of cultivation. A working solution was prepared from 240 μL stock solution A (25 mg/mL nitro blue tetrazolium chloride [NBT] in 70% dimethylformamide [DMF]) and 60 μL B (50 mg/mL 5-bromo-4-chloro-3-indolylphosphate toluidine salt [BCIP] in 100% DMF) in 16 mL 0.1 M Tris–HCl. Wells were incubated with 200 μL working solution overnight in the dark, subsequently the solution was removed and the diformazan was dissolved for 3 h in 200 μL DMF. Absorbency was measured at 560 nm and a calibration curve was used for the diformazan calculation.

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Measurement of Deoxynivalenol (DON) by competitive direct ELISA [5]
Deoxynivalenol (DON) was analyzed using a commercial Deoxynivalenol (DON) ELISA which is based on a previously described direct competitive ELISA (Casale et al., 1988). The monoclonal antibody used in this assay binds to DON and 3-ADON but not to other trichothecenes. Since this assay was designed to analyze DON in grain and grain products, the manufacturer's protocol was adapted for use on plasma and organ tissue samples by preparing separate DON standards (4 to 1,000 ng/ml) in 10% (v/v) human plasma or 1% bovine serum albumin (w/v) in PBS which functioned to prevent non-specific binding. Samples or standards (100 μl each) were combined with 100 μl of DON horseradish peroxidase conjugate solution in mixing wells provided by the manufacturer. A 100 μl aliquot of this mixture was transferred to antibody-coated wells and incubated for 5 min at 25°C. Wells were washed, 100 μl of K-blue Max TMB substrate added and the wells incubated for an additional 5 min at 25°C. The reaction was stopped with 100 μl of 2N sulfuric acid and absorbance read on a Molecular Devices ELISA reader using 450 nm filter. DON concentrations were quantified from a standard curve using Molecular Devices Softmax software. DON recoveries of 90 percent or higher were observed in preliminary studies either in which heated liver extracts were spiked directly with the toxin at 12.5 to 250 ng/g tissue and analyzed or in which liver homogenates were spiked with the toxin at 10 to 250 ng/g tissue, heated and supernatants analyzed. Since this ELISA might detect DON as well as some of its metabolites, data were reported as DON equivalents per ml plasma or per g organ tissue.

Cytotoxicity assays [1]
The colorimetric 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to assess the effect of Deoxynivalenol (DON) on Caco-2 cells proliferation. Cells, seeded in 96-wells plates (Nunc, Roskilde, Denmark), at a density of 35,000 cells/cm2, were incubated, 24 h after seeding, with Deoxynivalenol (DON) at concentrations between 0 and 10 μg/ml for 48 h. The MTT assay was carried out as in Mosmann (1983), using 100 μl of MTT (0.5 mg/ml in PBS), 2 h incubation at 37 °C, crystals solubilization in 100 μl DMSO and reading at 500 nm. At the end of MTT assays or at the end of transport experiments, the cytotoxic effect of Deoxynivalenol (DON) was determined by the LDH assay, purchased as a kit. Maximal LDH release was obtained by exposing the cells to 1% (v/v) Triton X-100. The reduced formazan reaction product was measured at 500 nm.

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Apoptosis assays [1]
Cell death detection ELISA [1]
The apoptotic cell death was determined by DNA fragments detection using the Cell Death detection ELISAPLUS kit. Cells, seeded in 96-wells plates (Nunc) at a density of 35,000 cells/cm2, were incubated, 24 h after seeding, with Deoxynivalenol (DON) at 30, 90 and 300 ng/ml for 48 h. Cells incubated with sodium butyrate at 50 mM in the same conditions as Deoxynivalenol (DON) were used as a positive control. The cellular assay was performed according to the manufacturer's instructions. [1]

Fluorescence microscopy assay of apoptotic cells [1]
The nuclear morphology was assayed by fluorescence microscopy after acridine orange/propidium iodide staining. Caco-2 cells seeded in BD Falcon™ CultureSlides at a density of 35,000 cells/cm2 were incubated, 24 h after seeding, with Deoxynivalenol (DON) at 30, 90 and 300 ng/ml for 48 h. Cells incubated with sodium butyrate at 50 mM in the same conditions were used as a positive control. Cells were then treated briefly with a mixture of acridine orange (1 mg/ml)/propidium iodide (88 μg/ml). Apoptotic cells (containing highly condensed or fragmented nuclei) and non-apoptotic cells (containing intact nuclei) were counted under a fluorescence microscope.

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Assays in 96 well plate format [2]
Analysis of cellular viability (cell count, lactate dehydrogenase (LDH) assay, neutral red (NR) uptake, MTT assay), proliferation (BrdU assay) and apoptosis (luminescence caspase 3/7 assay) were performed in 96 well plate format. IPEC-1 and IPEC-J2 cells were seeded in 96 well plastic tissue culture plates and grown for 4 d until confluence. Medium was removed and after washing once with PBS, fresh medium was added containing increasing final concentrations of Deoxynivalenol (DON) (100–4000 ng/mL). Cells were incubated for 24 h, 48 h or 72 h. For long term experiments (14 d) treatment was performed in the same manner, but with lower Deoxynivalenol (DON) concentrations (50–500 ng/mL) and a regular exchange of medium + Deoxynivalenol (DON) after every 3–4 d. All assays were performed in triplicates and in at least three independent experiments using a multiplate reader.

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Cell cycle analysis by flow cytometry [2]
IPEC-1 and IPEC-J2 cells were seeded in plastic tissue culture 6 well plates and experiments were performed with confluent cell layers (4 d). Cells were then 24 h synchronized in FCS free medium and Deoxynivalenol (DON) exposure was performed as described above. Cells were trypsinized, pelleted and resuspended in PBS. Ethanol fixation and propidium iodide staining procedure were performed as previously described.

Following acclimation, the females were mated (two females per male). Cohabitation began at approximately 4:30 p.m. on each mating day. The next morning, each female was examined for the presence of sperm in the vaginal lavage. Any sperm-positive female was presumed pregnant (GD 0) and a stratified random procedure was used to assign each animal to the control group or one of the four treatment groups. Five groups, each containing 24 females, were dosed by gavage with 0, 0.5, 1.0, 2.5, or 5.0 mg Deoxynivalenol (DON)/kg body weight/day on GD 6–19. Feed and water consumption and body weight were measured daily during treatment and on GD 20. Females were weighed daily and the volume of water or Deoxynivalenol (DON) solution administered to each animal was based on its body weight. Deoxynivalenol (DON) was administered in a maximal volume of 1 ml/100 g body weight. [3]
The doses in this study were based on toxicity data obtained from a repeated-dose range-finding study in which pregnant female rats were gavaged with Deoxynivalenol (DON) from 0.25 to 7.5 mg/kg body weight on GD 6–19. The dose of 7.5 mg/kg body weight was fetotoxic and was therefore not used in this study. In the dose range-finding study, acclimation, housing conditions, mating, animal treatment, feed, water, compound administration, analysis of fetuses, etc. were identical to those of the study reported here.[3]

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For the acute exposure study, food and water were withdrawn from cages 1 h prior to Deoxynivalenol (DON) administration. Deoxynivalenol (DON) was administered at 25 mg/kg bw by oral gavage in 250 μl of endotoxin-free water. Food and water were restored immediately after gavaging. Ungavaged animals were used as controls. At 5, 15, 30 min and 1, 2, 4, 8, 24 h, mice were anesthetized and blood collected. Mice were then euthanized and liver, kidney, heart, spleen and brain removed. Plasma was isolated from blood and stored at -20°C. Organs were frozen at -80°C, pulverized using a mortar and pestle and then mixed with phosphate-buffered saline (PBS) at a 1:5 ratio. The tissue extract was centrifuged for 10 min at 14,000 × g. The resultant supernatant fraction was heated to 100°C for 5 min to inactivate enzymes and precipitate proteins. The heated extract was centrifuged for 10 min at 14,000 × g and the supernatant fraction subsequently used with appropriate dilution for ELISA. [5]
For the subchronic feeding study, purified Deoxynivalenol (DON) was added at 0, 2, 5, 10 and 20 mg/kg of powdered high fat AIN-93 G Purified Rodent Diet 101847 as detailed previously (Pestka et al., 1989). Experimental diets were placed in special containers to minimize spillage. Cages were kept in class II ventilated cabinets for the duration of the experiment. Mice were fed for 4 wk and blood collected from the saphenous vein as described previously (Hem et al., 1998).[5]

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Pharmacokinetic analysis [5]
A two-compartment open model (Shargel et al., 2004) was employed to calculate toxicokinetic parameters. Deoxynivalenol (DON) concentrations in plasma and tissue were fitted to biexponential expression to calculate clearance rates. [5]

Absorption, Distribution and Excretion
Metabolic studies have been carried out on animals, principally with T-2 toxin, but a few with Deoxynivalenol (DON) . These trichothecenes are rapidly absorbed from the alimentary tract... . The toxins are distributed fairly evenly without marked accumulation in any specific organ or tissue. Trichothecenes are metabolically transformed to less toxic metabolites by such reactions as hydrolysis, hydroxylation, de-epoxidation, and glucuronidation. Trichothecenes, such as T-2 toxin and  Deoxynivalenol (DON) , are rapidly eliminated in the feces and urine. ...In the rat, 25% of DON was eliminated in the urine and 65% in the feces, 96 hr after dosing.
Although deoxynivalenol appeared in the blood within 30 min after intake by sheep, the systemic bioavailability was only 7.5%. A single dose of 5 mg/kg bw of deoxynivalenol was administered by oral intubation to four 1-yr-old male sheep, and repeated blood samples were taken over 30 hr. Deoxynivalenol and the de-epoxy metabolite were determined by GC with electron capture detection (ECD). No deoxynivalenol or the de-epoxy metabolite could be detected in plasma within the 30-hr observation period. Three male sheep were given a single iv dose of deoxynivalenol at 0.5 mg/kg bw, with blood sampling and analysis as after oral dosing. Systemic bioavailability was calculated from the ratio of the integrated area under the concentration-time curve times the dose for both oral and iv administration. <0.3% of the oral dose and <2% of the iv dose was detected in plasma as the de-epoxy metabolite. Free deoxynivalenol accounted for an average of 24.8% of the absorbed dose measured in blood; the remainder was made up of the de-epoxy metabolite or the glucuronide conjugate of deoxynivalenol. The oral absorption rate of deoxynivalenol in sheep was approximately 7% on the basis of recovery rates from urine and bile collected over 36 hr from two sheep given 5 mg/kg bw of deoxynivalenol orally. Deoxynivalenol and the de-epoxy metabolite were analysed by GC/ECD. An average of 6.9% of the administered dose was recovered from urine and 0.11% from bile. Glucuronide-conjugated de-epoxy metabolite was the only form detected in bile (detection limit, 0.1 mg, corresponding to 0.04% of the administered dose). An average of 1.3% of the administered dose was recovered from urine as the de-epoxy metabolite or its conjugate, and 5.7% was recovered as deoxynivalenol or its conjugate.
In contrast to the low bioavailability seen in sheep and cows, relatively high bioavailability was observed in pigs. Blood, urine, bile, and feces were collected over 24 hr after an intragastric dose of 0.6 mg/kg bw of [(14)C]deoxynivalenol or an iv dose of 0.3 mg/kg bw. The proportions of radiolabel were assumed to represent those of administered deoxynivalenol, and the validity of this assumption was confirmed by GC/MS, which showed very little metabolism or conjugation. On the basis of measurements of the integrated area under the concentration-time curve for three animals treated intravenously and three treated intragastrically, the average systemic bioavailability of deoxynivalenol in pigs was estimated to be 55%. Approximately 95% of the administered dose was recovered as deoxynivalenol... .
Although the absolute bioavailability of deoxynivalenol has not been measured in rats, 25% of an oral dose of 10 mg/kg bw was recovered in urine at 96 hr, suggesting that the absorption rate in rats may be higher than in sheep or cows. HPLC and GC/MS analysis indicated that 25% of the radiolabel in 0-24 hr urine was associated with unchanged deoxynivalenol and 10% with the de-epoxy metabolite. Similarly, 4.5 and 4.4% of an orally administered dose of 6 mg/kg bw was recovered in the urine of Wistar rats as free deoxynivalenol and the de-epoxy metabolite, respectively, within 96 hr.
For more Absorption, Distribution and Excretion (Complete) data for DEOXYNIVALENOL (13 total), please visit the HSDB record page.

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Metabolism / Metabolites
The minimum single emetic doses of deoxynivalenol and 15-acetyldeoxynivalenol in groups of three Yorkshire pigs weighing 10-15 kg were 0.050 and 0.075 mg/kg bw, respectively, when given either by gavage or ip. After gavage, 3 of 15 pigs given the 15-acetyl metabolite and 4 of 15 given deoxynivalenol showed emesis at all doses from 20-200 ug/kg bw. After ip administration, 9 of 15 pigs showed emesis at all doses. The NOELs were 0.025 mg/kg bw for deoxynivalenol and 0.050 mg/kg bw for the 15-acetyl metabolite after either oral intubation or ip injection...
Deoxynivalenol (DON) requires no activation for toxicity, though susceptibility may reflect individual variations in detoxification. This study reports the measurement of un-metabolised urinary  Deoxynivalenol (DON) (free DON) and DOM-1 in samples previously analyzed for the combined measure of free DON+DON-glucuronide (fD+DG), with a concentration >5 ng/mL, for 34 UK adults. Four consecutive daily urine samples were analyzed from twenty-two individuals, whilst from 12 individuals only a single sample was analyzed. The mean (median) concentration of urinary fD+DG in this sub-set was 17.8 ng/mL (13.8 ng/mL), range 5.0-78.2 ng/mL. In 23/34 (68%) individuals, free DON was detected, mean 2.4 ng/mL; range 0.5-9.3 ng/mL. Urinary DOM-1 was detected in 1/34 (3%) of individuals; present at /about/ 1% of urinary fD+DG concentration for that individual. The concentration of fD+DG combined was significantly correlated with urinary free DON (p<0.001, R(2)=0.65), but not with the percentage of free DON to fD+DG (p=0.615, R(2)=0.01), suggesting that the level of DON exposure did not affect the metabolism to DG within the range observed. In this survey most individuals had no detectable urinary DOM-1 and 68% did not detoxify all of the ingested DON to DON-glucuronide...
Deoxynivalenol-3-beta-D-glucoside (D3G), a plant phase II metabolite of the Fusarium mycotoxin deoxynivalenol (DON), occurs in naturally contaminated wheat, maize, oat, barley and products thereof. Although considered as a detoxification product in plants, the toxicity of this substance in mammals is currently unknown. A major concern is the possible hydrolysis of the D3G conjugate back to its toxic precursor mycotoxin DON during mammalian digestion. /The authors/ used in vitro model systems to investigate the stability of D3G to acidic conditions, hydrolytic enzymes and intestinal bacteria, mimicking different stages of digestion. D3G was found resistant to 0.2 M hydrochloric acid for at least 24 hr at 37 °C, suggesting that it will not be hydrolyzed in the stomach of mammals. While human cytosolic beta-glucosidase also had no effect, fungal cellulase and cellobiase preparations could cleave a significant portion of D3G. Most importantly, several lactic acid bacteria such as Enterococcus durans, Enterococcus mundtii or Lactobacillus plantarum showed a high capability to hydrolyze D3G. Taken together these data indicate that D3G is of toxicological relevance and should be regarded as a masked mycotoxin.
Cultures of 20% w/w suspensions of rat cecal contents were incubated anaerobically with [(14)C]deoxynivalenol at a concentration of 35 ug/mL for up to 24 hr. A standard-co-elution method involving high-performance liquid chromatography (HPLC) was used to quantify the proportions of radiolabel associated with deoxynivalenol and with the de-epoxidated form. The latter represented 1.3% of the administered radiolabel immediately after addition of deoxynivalenol, 29% at 7 hr, and 90% at 24 hr; 60% co-eluted with deoxynivalenol at 7 hr and 2% at 24 hr. ...Deoxynivalenol was not converted to the de-epoxy metabolite in cultures of the contents of pig large intestine (including caecum, but not otherwise specified) in another study in which 1 mL of a 1-ug/mL solution of deoxynivalenol was added to 2 g of large intestinal contents and incubated anaerobically for 96 hr. Nearly complete recovery of intact deoxynivalenol was reported. The intestinal contents of chickens treated identically showed nearly complete conversion of deoxynivalenol to the de-epoxy metabolite after 96 hr. After 24 hr of incubation, the rate of conversion was 56% of an applied concentration of 0.014 ug/mL, 69% of 0.14 ug/mL, and 70% of 1.4 ug/mL. Similarly, 35% of the applied deoxynivalenol was metabolized to the de-epoxy metabolite in bovine rumenal fluid after 96 hr of incubation.
For more Metabolism/Metabolites (Complete) data for DEOXYNIVALENOL (8 total), please visit the HSDB record page.

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Biological Half-Life
/In pigs/ after oral intake, the peak concentration in plasma was reached within 15-30 min, remained elevated for about 9 hr and then declined with a half-time of 7.1 hr.
Following a single iv injection (1 mg/kg bw) of deoxynivalenol to swine, ... the elimination half-life was estimated at 3.9 hr.

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 Deoxynivalenol (DON) was rapidly absorbed into plasma following oral exposure to a 25 mg/kg bw dose. The highest concentrations, 12.1 and 11.5 μg/ml, were detectable at 5 and 15 min, respectively (Fig. 1). There was rapid clearance following two-compartment kinetics with half-lives of distribution (t1/2α) and clearance (t1/2β) being 20.4 min and 11.8 h, respectively.[5]

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 Deoxynivalenol (DON) distribution and clearance kinetics in liver (Fig. 2A) and kidney (Fig. 2B) similarly followed a two-compartment kinetic pattern. As observed for plasma, concentrations of DON peaked at 5 to 15 min in liver (19.6 and 12.1 μg/g, respectively) (Fig. 2A) and kidney (7.6 and 9.0 μg/g, respectively) (Fig. 2B). The t1/2α and t1/2β in liver were 22 min and 19.0 h, respectively, whereas the t1/2α and t1/2β for kidney were 47 min and 20.9 h, respectively.[5]

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As seen in spleen and liver, the above tissues, the highest  Deoxynivalenol (DON) concentrations in spleen were observed at 5 min (7.3 μg/g) and 15 min (7.9 μg/g (Fig. 3A). The t1/2α and t1/2β were 29 min and 9.0 h, respectively. The highest DON concentrations were observed in the heart at 5 min (6.7 μg/g) and 15 min (6.8 μg/g) with t1/2α and t1/2β being 41 min and 12.3 h, respectively (Fig. 3B).[5]

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Compared to other tissues,  Deoxynivalenol (DON) entered the brain much more slowly and peaked at much lower concentrations (0.7-1.0 μg/g), lasting from 5 min to 2 h (Fig. 4).[5]

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The effects of subchronic dietary exposure to  Deoxynivalenol (DON) on plasma concentrations of this mycotoxin were also assessed in a 4 wk feeding study. Dose-dependent increases in plasma DON were observed in mice fed the toxin at 2, 5, 10 and 20 mg/kg diet with concentrations ranging from 20 to 100 ng/ml (Fig.5). All four of these doses reduced weight gain (data not shown) as has been reported previously for the B6C3F1 mouse (Forsell et al., 1986)[5]

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The results presented here suggested that  Deoxynivalenol (DON) uptake and distribution in the mouse could be monitored by ELISA.  Deoxynivalenol (DON) was rapidly taken up into tissues after exposure and maximum concentrations followed the rank order liver> plasma> kidney> spleen> heart> brain. DON was cleared initially at a rapid rate in all tissues except for brain and this was followed by a relatively slower rate of clearance. The data presented here were highly consistent with a previous [3H]-DON study conducted in the mouse by our laboratory (Azcona-Olivera et al., 1995). That prior study also reported two compartment kinetics with an initial rapid clearance rate and slower terminal elimination in plasma following exposure to  Deoxynivalenol (DON) doses of 5 mg/kg bw (t1/2α = 21.6 min and t1/2β =7.6 h, respectively) and 25 mg/kg (t1/2α = 33.6 min and t1/2β = 88.9 h, respectively). When DON uptake and clearance in plasma, liver and kidney observed here during the first 4 h were graphically compared to the aforementioned study at the 25 mg/kg bw dose, the results were found to be remarkably similar (Fig. 6). The ELISA was thus comparable to the radiolabel method for monitoring DON tissue concentrations and toxicokinetics in the mouse. Key advantages of this immunoassay approach are that (1) it was very sensitive and specific, (2) it employed a facile sample preparation protocol that did not require extensive clean-up and (3) it did not require evaporation or concentration.[5]

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Interactions
Deoxynivalenol (DON) and fumonisins (FB) are the most frequently encountered mycotoxins produced by Fusarium species and most commonly co-occur in animal diets. These mycotoxins were studied for their toxicity in piglets on several parameters including plasma biochemistry, organ histopathology and immune response. Twenty-four 5-wk-old animals were randomly assigned to four different groups, receiving separate diets for 5 wk, a control diet, a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg) or both toxins. At days 4 and 16 of the trial, the animals were subcutaneously immunized with ovalbumin to assess their specific immune response. The different diets did not affect animal performance and had minimal effect on hematological and biochemical blood parameters. By contrast, DON and FB induced histopathological lesions in the liver, the lungs and the kidneys of exposed animals. The liver was significantly more affected when the two mycotoxins were present simultaneously. The contaminated diets also altered the specific immune response upon vaccination as measured by reduced anti-ovalbumin IgG level in the plasma and reduced lymphocyte proliferation upon antigenic stimulation. Because cytokines play a key role in immunity, the expression levels of IL-8, IL-1beta, IL-6 and macrophage inflammatory protein-1beta were measured by RT-PCR at the end of the experiment. The expression of these four cytokines was significantly decreased in the spleen of piglets exposed to multi-contaminated diet. Taken together, our data indicate that ingestion of multi-contaminated diet induces greater histopathological lesions and higher immune suppression than ingestion of mono-contaminated diets.
Beauvericin (BEA), deoxynivalenol (DON) and T-2 toxin (T-2) are important food-borne mycotoxins that have been implicated in human health. In this study, the acute toxicity of individual and combined mycotoxins (BEA, DON and T-2) were tested in immortalized hamster ovarian cells (CHO-K1) at 24, 48 and 72 hr of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC50 values obtained for all mycotoxins by the MTT and NR assays ranged from 0.017 to 12.08 uM and from 0.042 to 17.22 uM, respectively. Both, individual and combined mycotoxins demonstrated a significant cytotoxic effect in CHO-K1 cells in a dose-dependent manner. When mycotoxins were assayed individually, T-2 showed the strongest IC50 values (from 0.017 to 0.052 uM), by both endpoints tested, followed by DON (0.53-2.30 uM) and BEA, showing this last one, the weakest IC50 values (from 2.77 to 17.22 uM). On the other hand, cytotoxicity interactions were evaluated by the isobologram method. In acute binary tests, DON+BEA (CI=1.60-25.07) and DON+T-2 (CI=1.74-7.71) showed antagonism at 24, 48 and 72 hr of exposure. By contrast, the binary BEA+T-2 combination (CI=0.35-0.78) showed synergism at all time of exposure tested. The tertiary BEA+DON+T-2 combination demonstrated synergism effect (CI=0.47-0.86) after 24 and 48 hr of exposure; however moderate antagonistic effect (CI=1.14-1.60) was presented after 72 hr of exposure at the lower doses. These results provide quantitative evidence regarding potentially important interactions between BEA, DON and T-2 depending of the time of exposure. The combination index-isobologram equation method can serve as a useful tool in food risk assessment. Due to the potent toxic effects of BEA, DON and T-2, its combined exposure might be an important trigger for development of several diseases in humans, from the mycotoxicological point of view, especially after long period of exposure time.
The ability of cyproheptadine, a serotonin antagonist at the serotonin2 receptor and a known appetite stimulant, to attenuate the adverse effect of deoxynivalenol was investigated in 3 trials with 21-day-old male ICR mice weighing 15-18 g. Groups of 10 mice received diets containing combinations of cyproheptadine and deoxynivalenol (purity, 99%), providing doses of deoxynivalenol of 4-16 mg/kg (equivalent to 0.6-2.4 mg/kg bw/day) and of cyproheptadine of 1.2-20 mg/kg (equivalent to 0.19-3 mg/kg bw). Cyproheptadine was administered in the feed for 2 days before addition of deoxynivalenol, and the two agents were then administered concurrently for 12 days. Cyproheptadine effectively offset the reduction in feed intake caused by deoxynivalenol, but only at certain doses. At a dose of deoxynivalenol of 4 mg/kg of feed, the optimal dose of cyproheptadine was 1.2-2.5 mg/kg of feed; at 8 mg/kg of feed, cyproheptadine was required at 2.5 mg/kg of feed; at 12 mg/kg of feed, the required dose of cyproheptadine was 2.5-5.0 mg/kg feed; and at 16 mg/kg of feed, cyproheptadine at 5-10 mg/kg feed was required. At lower doses of cyproheptadine (5 mg/kg of feed), alone or in combination with the lowest dose of deoxynivalenol tested, a modest increase in weight gain was noted, but this was not seen at higher concentrations of deoxynivalenol. /It was/ concluded that serotininergic mechanisms probably mediate the deoxynivalenol-induced reduction in feed intake. The finding that cyproheptadine significantly attenuated the effect of deoxynivalenol indicates the involvement of the serotonin2 receptor in this process.
Groups of 3-6 pigs weighing 60 kg were used to study the health effects of purified deoxynivalenol and ochratoxin A in their feed, singly or in combination, and the presence of residues 90 days after intake. The pigs received diets containing ochratoxin A at 0.1 mg/kg with deoxynivalenol at 1 mg/kg, equivalent to 0.004 mg of ochratoxin A and 0.04 mg of deoxynivalenol/kg bw, respectively; ochratoxin A alone at 0.1 mg/kg; or deoxynivalenol alone at 1 mg/kg. Two controls received feed containing neither ochratoxin A nor deoxynivalenol. The pigs that received mycotoxins in their feed did not show clinical or hematological changes. The pigs that received both mycotoxins had hyperemia in the gastric mucosa, and changes in the tubular epithelium were observed in one animal in each treated group. Few pathological lesions were found, but the Committee noted that there were few animals in the study. The observed antibody titres against pseudorabies (Aujeszky disease or "mad itch"), as a measure of effects on the immune system, suggest that non-specific defense mechanisms were not affected. The mean concentration of ochratoxin A in the kidneys of animals treated with both toxins was about 50% higher than that in the group given ochratoxin A alone, indicating a possible interaction. The concentration of ochratoxin A also appeared to be slightly increased in muscle of animals receiving both mycotoxins.
For more Interactions (Complete) data for DEOXYNIVALENOL (6 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Mouse (male) ip 70 mg/kg
LD50 Mouse (female) ip 76.7 mg/kg
LD50 Mouse oral 46 mg/kg
LD50 Mouse sc 45 mg/kg
For more Non-Human Toxicity Values (Complete) data for DEOXYNIVALENOL (11 total), please visit the HSDB record page.

[1]. Deoxynivalenol transport across human intestinal Caco-2 cells and its effects on cellularmetabolism at realistic intestinal concentrations. Toxicol Lett. 2006 Jul 1;164(2):167-76.

[2]. Mycotoxin deoxynivalenol (DON) mediates biphasic cellular response in intestinal porcine epithelial cell lines IPEC-1 and IPEC-J2. Toxicol Lett. 2011 Jan 15;200(1-2):8-18.

[3]. Effects of deoxynivalenol (DON, vomitoxin) on in utero development in rats. Food Chem Toxicol. 2006 Jun;44(6):747-57.

[4]. Comparison of acute toxicities of deoxynivalenol (vomitoxin) and 15-acetyldeoxynivalenol in the B6C3F1 mouse. Food Chem Toxicol. 1987 Feb;25(2):155-62.

[5]. Immunochemical assessment of deoxynivalenol tissue distribution following oral exposure in the mouse. Toxicol Lett. 2008 May 5;178(2):83-7.

Deoxynivalenol is a trichothecene mycotoxin produced by Fusarium to which wheat, barley, maize (corn) and their products are susceptible to contamination. It has a role as a mycotoxin. It is a trichothecene, a cyclic ketone, a secondary alpha-hydroxy ketone, a primary alcohol, an enone and a triol.
Deoxynivalenol has been reported in Fusarium graminearum, Fusarium culmorum, and Euglena gracilis with data available.

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Mechanism of Action
The objective of this work was to investigate whether proteomic analysis of thymoma cells treated with the trichothecene deoxynivalenol (DON) as compared to non-treated (control) cells would reveal differential protein expression, and thus would contribute to a better understanding of the mechanisms of its toxicity. For that purpose the mouse thymoma cell line EL4 was exposed to 0.5 uM DON for 6 hr. A total of 30 proteins were affected after exposure of EL4 cells to DON. Most of these proteins were up-regulated and included key metabolic enzymes (e.g., fatty acid synthase, aldose reductase, carbamoyl phosphate synthetase, glucose-6-phosphate isomerase), chaperones (e.g., HSP9AB1 and HSP70), enzymes implicated in protein folding (PDI and ERO1-l alpha), and proteins involved in protein degradation (ubiquitin-conjugating enzyme (E1) and proteasome subunit alpha type-1). In addition, an IgE-binding protein with a molecular weight of 60 kDa and My-binding protein 1a (MYBBP1A), a transcription factor, were found to be up-regulated by DON. The observed up-regulation of MYBBP1A, a known repressor of a number of transcription factors such as PGC-1 alpha, C-myb, and p65 of the NF-kappaB family, suggests that this protein might play a role in the mechanism of DON toxicity.
Deoxynivalenol (DON), one of the most abundant trichothecenes found on cereals, has been implicated in mycotoxicoses in both humans and farm animals. Low-dose toxicity is characterized by reduced weight gain, diminished nutritional efficiency, and immunologic effects. The levels and patterns of human food commodity contamination justify that DON consumption constitutes a public health issue. DON stability during processing and cooking explains its large presence in human food. /The authors/ characterized here DON intoxication by showing that the toxin concomitantly affects feeding behavior, body temperature, and locomotor activity after both per os and central administration. Using c-Fos expression mapping, /the authors/ identified the neuronal structures activated in response to DON and observed that the pattern of neuronal populations activated by the toxin resembled those induced by inflammatory signals. By real-time PCR, /the authors/ report the ...evidences for a DON-induced central inflammation, attested by the strong upregulation of interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, cyclooxygenase-2, and microsomal prostaglandin synthase-1 (mPGES-1) messenger RNA. However, silencing prostaglandins E2 signaling pathways using mPGES-1 knockout mice, which are resistant to cytokine-induced sickness behavior, did not modify the responses to the toxin. These results reveal that, despite strong similarities, behavioral changes observed after DON intoxication differ from classical sickness behavior evoked by inflammatory cytokines.
Trichothecenes are toxic for actively dividing cells, such as the intestinal crypt epithelium and the hematopoietic cells. The cytotoxicity has been associated with either impairment of protein synthesis by the binding of the compounds to the ribosomes of eukaryotic cells, or the dysfunction of cellular membranes. Inhibition of protein synthesis has been associated with the induction of labile and regulatory proteins, such as IL-2 in immunocytes. Transport of small molecules is impaired in cell membranes by extremely low concentrations of trichothecenes. /Trichothecenes/
Most trichothecenes inhibit protein synthesis, their potency depending on structural substituents and requiring an unsaturated bond at the C9-C10 position and integrity of the 12,13-epoxy ring. Trichothecenes bind to the 60S subunit of eukaryotic ribosomes and interfere with the activity of peptidyltransferase. Deoxynivalenol, which lacks a substituent at C-4, inhibits chain elongation. Inhibition of protein synthesis is considered to be the primary toxic effect of trichothecenes, including deoxynivalenol. The ID50 for inhibition of protein synthesis in rabbit reticulocytes was 2 ug/mL... . In vitro, deoxynivalenol is about 100 times less toxic than T-2 toxin, which has been more widely studied for its macromolecular effects. Owing to differences in lipophilicity and other possible effects, the toxicity of deoxynivalenol in vivo is greater than would be expected from its effects on protein synthesis in vitro.
For more Mechanism of Action (Complete) data for DEOXYNIVALENOL (10 total), please visit the HSDB record page.

C15H20O6

296.3157

296.125

C, 60.80; H, 6.80; O, 32.40

51481-10-8

Deoxynivalenol-13C15;911392-36-4

40024

Fine needles from ethyl acetate + petroleum ether
Crystals from methanol (aqueous)

1.5±0.1 g/cm3

543.9±50.0 °C at 760 mmHg

151-153ºC

206.9±23.6 °C

0.0±3.3 mmHg at 25°C

1.632

Fusarium specie; Fusarium graminearum, Fusarium culmorum, and Euglena gracilis

-1.41

3

6

1

21

558

7

O1C([H])([H])[C@]21[C@@]1([H])[C@@]([H])(C([H])([H])[C@]2(C([H])([H])[H])[C@@]2(C([H])([H])O[H])[C@@]([H])(C(C(C([H])([H])[H])=C([H])[C@@]2([H])O1)=O)O[H])O[H]

LINOMUASTDIRTM-QGRHZQQGSA-N

InChI=1S/C15H20O6/c1-7-3-9-14(5-16,11(19)10(7)18)13(2)4-8(17)12(21-9)15(13)6-20-15/h3,8-9,11-12,16-17,19H,4-6H2,1-2H3/t8-,9-,11-,12-,13-,14-,15+/m1/s1

(3alpha,7alpha)-3,7,15-Trihydroxy-12,13-epoxytrichothec-9-en-8-one

Vomitoxin; 3-epi-DON; DEOXYNIVALENOL; 51481-10-8; 4-Deoxynivalenol; CRIS 7801; Dehydronivalenol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Ethanol : ~30 mg/mL (~101.24 mM)
DMSO : ~25 mg/mL (~84.37 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.44 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.44 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)