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]

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Deoxynivalenol (DON) is a trichothecene mycotoxin that adversely affects growth and immune function in experimental animals. Low doses can induce rapid, transient upregulation of proinflammatory cytokines (e.g., IL-1β, IL-6, TNF-α), leading to immune stimulation, while high doses cause apoptosis in lymphoid tissues, resulting in immunosuppression. [5]
In mice, acute oral exposure to DON (25 mg/kg bw) sequentially induces in the spleen: phosphorylation of JNK 1/2, ERK 1/2, and p38 within 15-30 minutes; transcription factor activation within 1-2 hours; and cytokine mRNA expression within 1-4 hours. [5]
DON also exhibits neuroendocrine effects, including anorexia and emesis, which may be mediated by the serotoninergic system. [5]

Transport studies [1]
\nFor 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.\n

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\n\nAssessment of MAPKs phosphorylation [1]
\nThe 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.\n

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\nAlkaline phosphatase (AP) activity [2]
\nThe cellular enzymatic activity of alkaline phosphatase was detected by formation of blue-colored diformazan precipitate within 22 d of cultivation.\nA 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.\n

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\n\nMeasurement of Deoxynivalenol (DON) by competitive direct ELISA [5]
\nDeoxynivalenol (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]
\nThe 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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\nFor 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]
\nFor 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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\n
\nPharmacokinetic analysis [5]
\nA 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]

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\nAcute Oral Exposure Study: Male B6C3F1 mice (7 weeks old) were fasted for 1 hour prior to administration. Deoxynivalenol was dissolved in endotoxin-free water and administered via oral gavage at a dose of 25 mg/kg body weight in a volume of 250 μL. Food and water were restored immediately after gavage. At time points of 5, 15, 30 minutes and 1, 2, 4, 8, 24 hours post-dosing, mice were anesthetized, blood was collected, and then mice were euthanized. Liver, kidney, heart, spleen, and brain were harvested. Plasma was separated and stored at -20°C. Organs were frozen at -80°C, pulverized, homogenized with phosphate-buffered saline (PBS) at a 1:5 ratio, centrifuged, and the supernatant was heated to 100°C for 5 minutes to inactivate enzymes and precipitate proteins. After another centrifugation, the supernatant was used for ELISA analysis. [5]
\nSubchronic Feeding Study: Purified DON was mixed into powdered high-fat AIN-93G Purified Rodent Diet at concentrations of 0, 2, 5, 10, and 20 mg/kg diet. Mice were fed these diets for 4 weeks. Blood was collected from the saphenous vein for plasma DON analysis. [5]

Absorption, Distribution and Excretion
Metabolic studies have been conducted in animals, primarily using T-2 toxin and less frequently deoxynivalenol (DON). These trichothecene toxins are rapidly absorbed from the digestive tract…the toxins are distributed fairly evenly and do not accumulate significantly in any particular organ or tissue. Trichothecene toxins are metabolized into less toxic metabolites through reactions such as hydrolysis, hydroxylation, deepoxidation, and glucuronidation. Trichothecene toxins, such as T-2 toxin and deoxynivalenol (DON), are rapidly excreted in feces and urine. In rats, 96 hours after administration, 25% of DON was excreted in urine and 65% in feces. Although deoxynivalenol is detectable in the blood within 30 minutes of ingestion in sheep, its systemic bioavailability is only 7.5%. Four one-year-old male sheep were administered a single oral gavage dose of 5 mg/kg body weight, with blood samples collected repeatedly over a 30-hour period. Deoxynivalenol and its decyclic metabolites were determined using gas chromatography-electron capture detector (GC-ECD). No deoxynivalenol or its decyclic metabolites were detected in plasma during the 30-hour observation period. Three other male sheep were administered a single intravenous injection of 0.5 mg/kg body weight, with blood samples collected and analyzed following the same procedure as after oral administration. Systemic bioavailability was calculated as the ratio of the area under the concentration-time curve to the dose, applicable to both oral and intravenous administration. The decyclic metabolite detected in plasma accounted for <0.3% of the oral dose and <2% of the intravenous dose. Free deoxynivalenol accounted for an average of 24.8% of the absorbed blood dose; the remainder consisted of decyclic metabolites or glucuronide conjugates of deoxynivalenol. Based on the recovery rates of urine and bile collected within 36 hours after oral administration of 5 mg/kg body weight of deoxynivalenol in two sheep, the oral absorption rate of deoxynivalenol in sheep was approximately 7%. Deoxynivalenol and its decyclic metabolites were analyzed using gas chromatography-electron capture detector (GC/ECD). The average recovered amount of deoxynivalenol in urine was 6.9% of the administered dose, and in bile, it was 0.11%. Only glucuronide-conjugated decyclic metabolites were detected in bile (limit of detection: 0.1 mg, equivalent to 0.04% of the administered dose). The average recovered amount of decyclic metabolites or their conjugates in urine was 1.3% of the administered dose, and the average recovered amount of deoxynivalenol or its conjugates was 5.7%. Compared to the low bioavailability in sheep and cattle, the bioavailability in pigs was relatively high. Blood, urine, bile, and feces were collected within 24 hours following administration of 0.6 mg/kg body weight of [(14)C]deoxynivalenol by gavage or 0.3 mg/kg body weight by intravenous injection. The proportion of radiolabeled material was assumed to represent the proportion of administered deoxynivalenol, and this assumption was confirmed by GC/MS, showing minimal metabolic or binding reactions. Based on measurements of the area under the concentration-time curves of three intravenously administered and three intragastrically administered animals, the mean 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 in rats has not been determined, 25% of the oral dose of 10 mg/kg body weight was recovered in urine after 96 hours, suggesting that absorption in rats may be higher than in sheep or cattle. High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC/MS) analysis showed that 25% of the radiolabeled substances in urine within 0–24 hours were associated with unmetabolized deoxynivalenols, and 10% were associated with decyclic metabolites. Similarly, within 96 hours, after oral administration of a dose of 6 mg/kg body weight to Wistar rats, 4.5% and 4.4% were recovered from urine as free deoxynivalenols and decyclic metabolites, respectively. More complete data on the absorption, distribution, and excretion of deoxynivalenols (13 in total) can be found on the HSDB record page.

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Metabolism/Metabolites
In three Yorkshire pigs weighing 10–15 kg, the lowest single emetic doses for deoxynivalenol and 15-acetyldeoxynivalenol administered via gavage or intraperitoneal injection were 0.050 and 0.075 mg/kg body weight, respectively. Following gavage administration, 3 out of 15 pigs received the 15-acetyl metabolite, and 4 out of 15 pigs received deoxynivalenol, with doses ranging from 20–200 μg/kg body weight. Following intraperitoneal injection, 9 out of 15 pigs vomited at all doses.
Following oral gavage or intraperitoneal injection, the no-observed-effect level (NOEL) for deoxynivalenol was 0.025 mg/kg body weight, and the NOEL for the 15-acetyl metabolite was 0.050 mg/kg body weight…
Deoxynivalenol (DON) can produce toxicity without activation, but individual differences in detoxification capacity may affect its toxicity sensitivity. This study reports the results of measurements of unmetabolized deoxynivalenol (DON) (free DON) and DOM-1 in urine samples from 34 British adults. These samples had previously been used to analyze the combined concentrations of free DON + DON-glucuronide (fD+DG), both >5 ng/mL. Urine samples were collected from 22 subjects over four consecutive days for analysis, while only a single urine sample was collected from 12 subjects. The mean (median) concentration of urinary fD+DG in this subset was 17.8 ng/mL (13.8 ng/mL), ranging from 5.0 to 78.2 ng/mL. Free DON was detected in 23 of the 34 subjects (68%), with a mean concentration of 2.4 ng/mL, ranging from 0.5 to 9.3 ng/mL. Urinary DOM-1 was detected in 1 of the 34 subjects (3%). The concentration of free DON and DG in this individual's urine was approximately 1%. The total concentration of fD+DG was significantly correlated with the concentration of free DON in urine (p<0.001, R²=0.65), but not significantly correlated with the percentage of free DON in fD+DG (p=0.615, R²=0.01), indicating that within the observed range, DON exposure levels did not affect its metabolism to DG. In this survey, DOM-1 was not detected in the urine of most individuals, and 68% of individuals failed to detoxify all ingested DON into DON-glucuronide…
Deoxynivalenol-3-β-D-glucoside (D3G) is a plant stage II metabolite of the fusarium toxin deoxynivalenol (DON), found in naturally contaminated wheat, corn, oats, barley, and their products. Although D3G is considered a plant detoxification product, its toxicity to mammals remains unclear. A major concern is that during mammalian digestion, D3G conjugates may be hydrolyzed back to their toxic precursor mycotoxin DON. The authors used an in vitro model system to simulate different stages of digestion, investigating the stability of D3G under acidic conditions, hydrolytic enzymes, and the action of gut bacteria. The study found that D3G could tolerate 0.2 M hydrochloric acid for at least 24 hours at 37°C, indicating that it is not hydrolyzed in the mammalian stomach. Human cytoplasmic β-glucosidase had no effect, while fungal cellulase and cellobiase preparations could cleave a considerable portion of D3G. More importantly, several lactic acid bacteria, such as Enterococcus durans, Enterococcus montelukast, and Lactobacillus plantarum, showed strong D3G hydrolytic ability. These combined data indicate that D3G has toxicological significance and should be considered a cryptic mycotoxin. A 20% (w/w) suspension of rat cecal contents was incubated with 35 μg/mL [(14)C]deoxynivalenol under anaerobic conditions for up to 24 hours. The proportions of radiolabeled substances associated with deoxynivalenol and its deepoxidized forms were quantitatively analyzed using standard co-elution by high-performance liquid chromatography (HPLC). The latter, immediately after the addition of deoxynivalenol, accounted for 1.3% of the added radiolabeled substance, 29% after 7 hours, and 90% after 24 hours; 60% was co-eluted with deoxynivalenol at 7 hours, and 2% at 24 hours. In another study, 1 mL of a 1 μg/mL deoxynivalenol solution was added to 2 g of porcine large intestine contents (including the cecum, but not specified) and cultured under anaerobic conditions for 96 hours. The results showed that deoxynivalenol was not converted to decyclic metabolites. This study reported that almost all intact deoxynivalenol was recovered. However, after culturing chicken intestinal contents under the same treatment for 96 hours, deoxynivalenol was almost completely converted to decyclic metabolites. After 24 hours of culture, the conversion rates were 56%, 69%, and 70% at concentrations of 0.014 μg/mL, 0.14 μg/mL, and 1.4 μg/mL, respectively. Similarly, after 96 hours of incubation in bovine rumen fluid, 35% of deoxynivalenol was metabolized to decyclic metabolites. More complete metabolite/metabolite data for deoxynivalenol (a total of 8 metabolites) can be found on the HSDB record page.
Biological half-life
/In pigs, peak plasma concentrations were reached within 15–30 minutes after oral administration, lasting approximately 9 hours, and then decreased over a half-life of 7.1 hours.
After a single intravenous injection of deoxynivalenol (1 mg/kg body weight) in pigs, the estimated elimination half-life was 3.9 hours.
Following oral administration of a 25 mg/kg body weight dose of deoxynivalenol (DON), it was rapidly absorbed into plasma. The highest concentrations were 12.1 and 11.5 μg/ml, detected at 5 minutes and 15 minutes, respectively (Figure 1). Its clearance was rapid and conformed to a two-compartment kinetic model, with a distribution half-life (t1/2α) and a clearance half-life (t1/2β) of 20.4 min and 11.8 h, respectively. [5] The distribution and clearance kinetics of deoxynivalenol (DON) in the liver (Fig. 2A) and kidney (Fig. 2B) also followed a two-compartment kinetic model. Similar to what was observed in plasma, the concentrations of DON in the liver (19.6 and 12.1 μg/g, respectively) (Fig. 2A) and kidney (7.6 and 9.0 μg/g, respectively) (Fig. 2B) peaked between 5 and 15 min. The half-lives (t1/2α) and (t1/2β) of deoxynivalenol (DON) in the liver were 22 min and 19.0 h, respectively, while those in the kidney were 47 min and 20.9 h, respectively. [5]

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Similar to the spleen and liver, in the above tissues, the highest DON concentrations in the spleen occurred at 5 minutes (7.3 μg/g) and 15 minutes (7.9 μg/g) (Fig. 3A). T1/2α and T1/2β were 29 minutes and 9.0 hours, respectively. In the heart, the highest DON concentrations occurred at 5 minutes (6.7 μg/g) and 15 minutes (6.8 μg/g), with T1/2α and T1/2β at 41 minutes and 12.3 hours, respectively (Fig. 3A 3B). [5]

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Compared with other tissues, deoxynivalenol (DON) enters the brain much more slowly and at much lower peak concentrations (0.7–1.0 μg/g), lasting from 5 minutes to 2 hours (Fig. 4). [5]

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A 4-week feeding study also evaluated the effect of subchronic dietary exposure to deoxynivalenol (DON) on plasma concentrations of this mycotoxin. When mice were fed diets of 2, 5, 10 and 20 mg/kg, plasma DON concentrations were observed to increase in a dose-dependent manner, ranging from 20 to 100 ng/ml (Figure 5). All four doses reduced weight gain (data not shown), consistent with previous reports in B6C3F1 mice (Forsell et al., 1986) [5]

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The results presented in this paper indicate that the absorption and distribution of deoxynivalenol (DON) in mice can be monitored by ELISA. DON is rapidly absorbed into tissues after exposure, with the highest concentrations in the order of liver > plasma > kidney > spleen > heart > brain. Except for brain tissue, DON was initially cleared rapidly in all tissues, followed by relatively slow clearance. The data in this paper are highly consistent with our previous [3H]-DON studies in mice (Azcona-Olivera et al., 1995). This study also reported the two-compartment kinetics of DON, namely, after exposure to DON at 5 mg/kg body weight (t1/2α = 21.6 min, t1/2β = 7.6 h) and 25 mg/kg body weight (t1/2α = 21.6 min, t1/2β = 7.6 h), the initial clearance rate of DON in plasma was faster, while the final clearance rate was slower (33.6 min and 88.9 h, respectively). When the absorption and clearance of DON in plasma, liver and kidney in the first 4 hours of this study were compared graphically with the results of the above study at the 25 mg/kg body weight dose, the two were found to be very similar (Figure 6). Therefore, the ELISA method and the radiolabeling method are comparable in monitoring the tissue concentration and toxicokinetics of DON in mice. The main advantages of this immunoassay method are: (1) high sensitivity and specificity; (2) simple sample preparation scheme without complicated purification steps; (3) no need for evaporation or concentration. [5]

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Deoxynivalenol (DON) was rapidly absorbed and distributed in mice after a single oral dose of 25 mg/kg body weight. The highest plasma DON concentration (12 μg/mL) was reached within 5 to 15 minutes. [5]
Plasma clearance subsequently followed a two-compartment pharmacokinetics model with a rapid distribution half-life (t₁/₂α) of 20.4 minutes and a slower terminal elimination half-life (t₁/₂β) of 11.8 hours. At 8 hours and 24 hours post-administration, the plasma drug concentrations were 5% and 2% of the maximum concentration, respectively. [5]
Deoxynivalenol (DON) was detected in plasma, liver, spleen, and brain tissue within 5 minutes to 24 hours post-administration; DON was detected in the heart and kidneys within 5 minutes to 8 hours post-administration. [5]
Five minutes after administration, the tissue concentrations (mean ± standard error) were as follows: liver 19.5 ± 1.9 μg/g, kidney 7.6 ± 0.5 μg/g, spleen 7.3 ± 0.8 μg/g, heart 6.8 ± 0.9 μg/g, and brain 0.8 ± 0.1 μg/g. Distribution and clearance kinetics: Tissue DON levels were similar to plasma DON levels. [5]
In subchronic feeding studies, plasma DON levels increased in a dose-dependent manner with increasing dietary DON concentration (0 to 20 mg/kg diet). [5]

Interactions
Deoxynivalenol (DON) and fumonisin (FB) are the most common mycotoxins produced by Fusarium spp. and are also the most common co-occurring substances in animal feed. This study investigated the toxicity of these mycotoxins to piglets and analyzed multiple parameters, including plasma biochemical indicators, organ histopathology, and immune responses. Twenty-four 5-week-old piglets were randomly divided into four groups and fed different diets for 5 weeks: a control group, a group supplemented with DON (3 mg/kg), a group supplemented with FB (6 mg/kg), or a group supplemented with a mixture of the two toxins. On days 4 and 16 of the experiment, piglets were subcutaneously immunized with ovalbumin to assess their specific immune response. Different diets had no effect on the growth performance of piglets and had little effect on hematological and biochemical indicators. However, both DON and FB induced histopathological damage in the liver, lungs, and kidneys of piglets. The liver was more significantly affected when both mycotoxins were present. Contaminated feed can also alter specific immune responses following vaccination, manifested as decreased plasma levels of anti-ovalbumin IgG and reduced lymphocyte proliferation after antigen stimulation. Since cytokines play a crucial role in immunity, we detected the expression levels of IL-8, IL-1β, IL-6, and macrophage inflammatory protein-1β by RT-PCR at the end of the experiment. The results showed that the expression of these four cytokines was significantly reduced in the spleens of piglets exposed to multiple contaminated feeds. In summary, our data indicate that ingestion of multiple contaminated feeds leads to more severe histopathological damage and greater immunosuppression compared to ingestion of a single contaminated feed.
Bovidia oleifera (BEA), deoxynivalenol (DON), and T-2 toxin (T-2) are important foodborne mycotoxins closely related to human health. This study used the MTT assay and the neutral red (NR) assay to detect the acute toxicity of single and combined mycotoxins (BEA, DON, and T-2) in immortalized hamster ovary cells (CHO-K1) after 24, 48, and 72 hours of exposure. The IC50 values for all mycotoxins, determined by the MTT and NR assays, ranged from 0.017 to 12.08 μM and 0.042 to 17.22 μM, respectively. Both single and combined mycotoxins exhibited significant dose-dependent cytotoxic effects on CHO-K1 cells. When mycotoxins were tested individually, T-2 showed the strongest IC50 values (0.017 to 0.052 μM) at both endpoints, followed by DON (0.53 to 2.30 μM), while BEA showed the weakest IC50 values (2.77 to 17.22 μM). Furthermore, cytotoxic interactions were assessed using an isotropic line plot method. In acute binary trials, DON+BEA (CI = 1.60–25.07) and DON+T-2 (CI = 1.74–7.71) showed antagonistic effects at 24, 48, and 72 hours after exposure. In contrast, the BEA+T-2 binary combination (CI = 0.35–0.78) showed synergistic effects at all tested exposure time points. The ternary compound BEA+DON+T-2 combination showed synergistic effects at 24 and 48 hours after exposure (CI = 0.47–0.86); however, it showed moderate antagonistic effects at lower doses after 72 hours of exposure (CI = 1.14–1.60). These results provide quantitative evidence that there may be important interactions among BEA, DON, and T-2, and that these interactions depend on exposure time. The combination index-equivalent line graph equation method can serve as an effective tool for food risk assessment. Due to the potent toxicity of BEA, DON, and T-2, combined exposure to these substances may be a significant contributing factor to various human diseases from a fungal toxicology perspective, especially after long-term exposure. This study investigated the ability of cyproheptadine (a 5-HT2 receptor antagonist and a known appetite stimulant) to mitigate the adverse effects of deoxynivalenol in 21-day-old male ICR mice weighing 15-18 grams across three trials. Groups of ten mice were fed a diet containing a combination of cyproheptadine and deoxynivalenol (99% purity), with deoxynivalenol at doses of 4-16 mg/kg (equivalent to 0.6-2.4 mg/kg body weight/day) and cyproheptadine at doses of 1.2-20 mg/kg (equivalent to 0.19-3 mg/kg body weight). Cyproheptadine was added to the diet two days prior to the addition of deoxynivalenol, followed by simultaneous addition of both substances for 12 days. Cyproheptadine effectively counteracts the reduced feed intake induced by deoxynivalenol (DON), but only at specific doses. When the DON concentration in the feed is 4 mg/kg, the optimal dose of cyproheptadine is 1.2–2.5 mg/kg of feed. When the DON concentration in the feed is 8 mg/kg, the required dose is 2.5 mg/kg; when the DON concentration is 12 mg/kg, the required dose is 2.5–5.0 mg/kg; and when the DON concentration is 16 mg/kg, the required dose is 5–10 mg/kg. At lower doses of cyproheptadine (5 mg/kg), whether used alone or in combination with the lowest tested dose of DON, a slight increase in body weight was observed, but this was not observed at higher DON concentrations. Therefore, it is concluded that a serotonergic mechanism may mediate the DON-induced reduction in feed intake. Studies have found that cyproheptadine can significantly attenuate the effects of deoxynivalenol, indicating that the 5-hydroxytryptamine 2 receptor is involved in this process. This study used 3-6 pigs weighing 60 kg to investigate the health effects of adding purified deoxynivalenol and ochratoxin A, alone or in combination, to their diets, as well as the residues 90 days after ingestion. Pigs were fed the following diets: diets containing 0.1 mg/kg ochratoxin A and 1 mg/kg deoxynivalenol (equivalent to 0.004 mg ochratoxin A and 0.04 mg deoxynivalenol per kg body weight, respectively); diets containing only 0.1 mg/kg ochratoxin A; or diets containing only 1 mg/kg deoxynivalenol. Two control groups were also included, fed diets without ochratoxin A and deoxynivalenol. Pigs fed with mycotoxins showed no clinical or hematological changes. Pigs fed with both mycotoxins simultaneously showed gastric mucosal congestion, and one pig in each treatment group showed renal tubular epithelial cell changes. Although pathological damage was minimal, the committee noted the small number of animals in this study. Observed pseudorabies (Ojerseysky disease or "rabies") antibody titers (as an indicator of immune system effects) suggest that nonspecific defense mechanisms were not affected. The mean concentration of ochratoxin A in the kidneys of animals treated with both toxins simultaneously was approximately 50% higher than in the group treated with ochratoxin A alone, suggesting a possible interaction. Ochratoxin A concentrations also appeared to be slightly elevated in the muscles of animals treated with both mycotoxins simultaneously.
For more complete data on interactions with deoxynivalenol (6 items in total), please visit the HSDB record page.

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Non-human toxicity values
Intraperitoneal LD50 of mice (male): 70 mg/kg
Intraperitoneal LD50 of mice (female): 76.7 mg/kg
Oral LD50 of mice: 46 mg/kg
Subcutaneous LD50 of mice: 45 mg/kg
For more complete (11) non-human toxicity values of deoxynivalenol, please visit the HSDB record page.

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The acute oral dose of 25 mg/kg body weight used in this study is equivalent to one-third of the previously reported LD₅₀ in female B6C3F1 mice. [5]
DON is known to cause anorexia, gastroenteritis, vomiting, and hematologic disorders. [5]

[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 (DON) is a trichothecene toxin produced by fungi of the genus Fusarium, which contaminates wheat, barley, corn, and their products. It is a fungal toxin. DON is a trichothecene compound belonging to the cyclic ketone, secondary α-hydroxy ketone, primary alcohol, enone, and triol classes. DON has been reported to be detected in Fusarium graminearum, Fusarium culmorum, and Euglena gracilis, and relevant data are available. Mechanism of Action: This study aimed to investigate whether proteomic analysis of thymoma cells treated with deoxynivalenol (DON) compared to untreated (control) cells could reveal differentially expressed proteins, thus contributing to a better understanding of its toxic mechanism. To this end, the mouse thymoma cell line EL4 was exposed to 0.5 μM DON for 6 hours. Thirty proteins were affected after EL4 cells were exposed to DON. Most of these proteins were upregulated, including key metabolic enzymes (such as fatty acid synthases, aldose reductases, carbamoyl phosphate synthases, and glucose-6-phosphate isomerases), molecular chaperones (such as HSP9AB1 and HSP70), enzymes involved in protein folding (PDI and ERO1-1α), and proteins involved in protein degradation (ubiquitin-binding enzyme (E1) and proteasome α-subunit 1). Furthermore, the expression of a 60 kDa IgE-binding protein and transcription factor My-binding protein 1a (MYBBP1A) was also upregulated by DON. The upregulation of MYBBP1A (a known inhibitor of multiple transcription factors, such as PGC-1α, C-myb, and p65 of the NF-κB family) suggests that this protein may play a role in the DON toxicity mechanism. Deoxynivalenol (DON) is one of the most common trichothecene toxins in cereals and has been associated with fungal poisoning in humans and livestock. Low-dose toxicity manifests as reduced weight gain, decreased nutrient utilization, and impaired immune function. The extent and pattern of DON contamination in human food suggest that DON intake constitutes a public health concern. The stability of DON during processing and cooking explains its high prevalence in human food. This study characterizes DON poisoning by demonstrating that the toxin affects feeding behavior, body temperature, and motor activity after both oral and central administration. Using c-Fos expression profiling, the authors identified DON-activated neuronal structures and observed that the pattern of toxin-activated neuronal populations was similar to that induced by inflammatory signaling. Evidence of DON-induced central nervous system inflammation was reported by real-time PCR, characterized by significant upregulation of interleukin-1β, interleukin-6, tumor necrosis factor-α, cyclooxygenase-2, and microsomal prostaglandin synthase-1 (mPGES-1) messenger RNA. However, silencing the prostaglandin E2 signaling pathway in mPGES-1 knockout mice resistant to cytokine-induced disease behavior did not alter the response to the toxin. These results indicate that, despite numerous similarities, the behavioral changes observed after DON poisoning differ from classic disease behaviors induced by inflammatory cytokines. Trichothecene compounds are toxic to actively dividing cells, such as intestinal crypt epithelial cells and hematopoietic cells. Their cytotoxicity is associated with impaired protein synthesis or cell membrane dysfunction due to the binding of compounds to eukaryotic ribosomes. Inhibition of protein synthesis is associated with the induction of unstable and regulatory proteins (such as IL-2) in immune cells. Even very low concentrations of trichothecene compounds can impair the transport of small molecules across the cell membrane. /Trichothecene Compounds/ Most trichothecene compounds inhibit protein synthesis; their potency depends on structural substituents and requires unsaturated bonds at the C9-C10 positions and the integrity of the 12,13-epoxy ring. Trichothecene toxins bind to the 60S subunit of eukaryotic ribosomes, interfering with peptidyl transferase activity. Deoxynivalenol (DON) lacks a substituent at the C-4 position and inhibits peptide chain elongation. Inhibition of protein synthesis is considered a major toxic effect of trichothecene toxins, including DON. DON has an ID50 of 2 μg/mL for inhibiting protein synthesis in rabbit reticulocytes. In vitro studies have shown that DON is approximately 100 times less toxic than T-2 toxin, which has been more extensively studied due to its effects on macromolecules. Due to its lipophilicity and other potential effects, the in vivo toxicity of deoxynivalenol is greater than the expected toxicity based on its in vitro effects on protein synthesis. For more complete data on the mechanisms of action of deoxynivalenols (10 in total), please visit the HSDB record page. Deoxynivalenol (DON, vomitoxin) is a trichothecene fungal toxin commonly produced by Fusarium graminearum and found in contaminated grains such as wheat, barley, and corn. [5]
The presence of DON in food is a significant human health concern worldwide. [5]
All animal species are sensitive to DON, with the sensitivity ranking as follows: pigs > mice > rats > poultry > ruminants. Differences in metabolism, absorption, distribution, and excretion may be the cause of these differences. [5]
The competitive direct ELISA method described in this study is a sensitive and specific tool for detecting DON in biological samples, with recoveries of 90% or higher in DON-added tissue samples. [5]

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.)