Natural trichothecene; mycotoxin

Fungi producers of mycotoxins are able to synthesize more than one toxin. Alternariol (AOH) is one of the mycotoxins produced by several Alternaria species, the most common one being Alternaria alternata. The toxins  3-acetyldeoxynivalenol (3ADON) and 15-Acetyl-deoxynivalenol (15-ADON) are acetylated forms of deoxynivalenol (DON) produced by Fusarium graminearum. In the present work it is determined and evaluated the toxic effects of binary and tertiary combination treatment of HepG2 cells with AOH, 3-ADON and 15-ADON, by using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), to subsequently apply the isobologram method and elucidate if the mixtures of these mycotoxins produced synergism, antagonism or additive effect; and lastly, to analyze mycotoxins conversion into metabolites produced and released by HepG2 cells after applying the treatment conditions by liquid chromatography tandem mass spectrometry (LC-MS/MS) equipment and extracted from culture media. HepG2 cells were treated at different concentrations over 24, 48 and 72h. IC50 values detected at all times assayed, ranged from 0.8 to >25μM in binary combinations; while in tertiary it ranged from 7.5 to 12μM. Synergistic, antagonism or additive effect detected in the mixtures of these mycotoxins was different depending on low or high concentration. Among all four mycotoxins combinations assayed, 15-ADON+3-ADON presented the highest toxic potential. At all assayed times, recoveries values oscillated depending on the time and combination studied. [1]

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The effects of the trichothecene mycotoxin deoxynivalenol (DON) and its acetylated derivatives,  3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) on human intestinal cell Caco-2 were investigated by the studies of transepithelial transport, gene expression, and cytokine secretion. Permeability across a Caco-2 cell monolayer was evaluated by transport study. Transport rates were ranked as DON, 3ADON<15ADON in apical-basolateral direction. 15ADON showed the highest permeability, induced the highest decrease in transepithelial electrical resistance (TEER), and prompted significant Lucifer Yellow permeability. These results showed that 15ADON affect paracellular barrier function extremely. In addition, gene expressions induced by toxins were screened by DNA microarray for investigating cellular effect on Caco-2 cell. The most remarkable gene induced by DON and 15ADON was inflammatory chemokine IL-8 and thus mRNA expression and secretion of IL-8 were analyzed by PCR and ELISA. Both DON and acetylated DONs could induce mRNA expression and production of IL-8. In particular, ELISA assay showed that the ability to produce IL-8 was ranked as 3ADON - In human hepatocellular carcinoma HepG2 cells, 3-Acetyldeoxynivalenol (3-AcDON) showed concentration-dependent cytotoxicity. At 1 μM, 5 μM, and 10 μM (24-hour treatment), cell viability decreased by 10%, 22%, and 35% respectively (MTT assay). It induced reactive oxygen species (ROS) generation: 10 μM 3-AcDON increased intracellular ROS levels by 40% (DCFH-DA fluorescence assay) and caused DNA damage, with a 2.8-fold increase in comet assay tail moment at 10 μM [1]
- In human intestinal epithelial Caco-2 cells (differentiated into monolayer), 3-Acetyldeoxynivalenol (5-20 μM, 48-hour treatment) disrupted intestinal barrier integrity: transepithelial electrical resistance (TEER) decreased by 20% (5 μM), 32% (10 μM), and 45% (20 μM). It stimulated interleukin-8 (IL-8) secretion: 20 μM 3-AcDON increased IL-8 levels in culture supernatant by 3.2-fold (ELISA). Its intestinal transport rate was ~15% (radioactive labeling), lower than deoxynivalenol (DON, ~25%) [2]
- In a human blood-brain barrier (BBB) model (hCMEC/D3 endothelial cells), 3-Acetyldeoxynivalenol (2.5-10 μM, 24-hour treatment) impaired BBB integrity: TEER decreased by 15% (2.5 μM), 22% (5 μM), and 30% (10 μM). It crossed the BBB with an apparent permeability coefficient (Papp) of ~1.2×10⁻⁶ cm/s (HPLC detection) and induced interleukin-6 (IL-6) secretion: 10 μM 3-AcDON increased IL-6 levels by 2.5-fold (ELISA) [3]

To determine metabolites or degradation products AOH and DON's metabolites (3-ADON and 15-Acetyl-deoxynivalenol (15-ADON) ), 1.6 mL of media containing dead cells were collected from the 96-well plate after cytotoxicity assays, were allowed to proceed for the extraction procedure at 24, 48 and 72 h of exposure, as described above. For test positive control, AOH, 3-ADON and 15-Acetyl-deoxynivalenol (15-ADON) were incubated with culture media without HepG2 cells, during the same time of exposure, three times each. For test control cells were exposed with culture media without mycotoxins with ≤ 1% methanol or DMSO, during the same time of exposure three times.[1]

MTT assay [1]
Cytotoxicity was examined by the MTT assay, performed as described by Ruiz et al. (2006) with few modifications. The assay consists in measuring the viability of cells by determining the reduction of the yellow soluble tetrazolium salt only in cells that are metabolically active via a mitochondrial reaction to an insoluble purple formazan crystal. Cells were seeded in 96-well culture plates at 2 × 104 cells/well and allowed to adhere for 18–24 h before mycotoxin additions. AOH, 3-ADON and 15-Acetyl-deoxynivalenol (15-ADON) concentrations tested in combination were 16:1 or 16:1:1 for binary and tertiary combinations, respectively and at 1:2 dilution as mentioned in Section 2.3 (see also Table 1). Serial dilutions were prepared with supplemented medium and added to the designed plate. Culture medium without mycotoxins and with < 1% methanol or DMSO was used as control. Combination of both solvents did not result in any effect on HepG2 cells (see details in Fig. 2). After treatment, the medium was removed and each well received 200 μL of fresh medium containing 50 μl of MTT solution (5 mg/ml; MTT powder dissolved in phosphate buffered saline). After an incubation time of 4 h at 37 °C in darkness the MTT containing media was removed and 200 μl of DMSO and 25 μl of Soerensen's solution were added to each well before reading optical density at 570 nm with the ELISA plate reader Multiskan EX (Thermo Scientific, MA, USA). Replicates consisted in each mycotoxin combination plus a control tested in three independent experiments. Mean inhibition concentration (IC50) values were calculated from full dose–response curves.

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- HepG2 cell assay ([1]): HepG2 cells were seeded in 96-well plates (5×10³ cells/well) and 6-well plates (2×10⁵ cells/well), cultured overnight, then treated with 3-Acetyldeoxynivalenol (1-10 μM) for 24 hours. For cell viability: MTT reagent was added, and absorbance at 570 nm was measured. For ROS detection: Cells were loaded with DCFH-DA, and fluorescence intensity was measured via flow cytometry. For DNA damage: Cells were processed for comet assay, and tail moment was analyzed under a fluorescence microscope [1]
- Caco-2 cell assay ([2]): Caco-2 cells were seeded in Transwell inserts (1×10⁵ cells/insert) and cultured for 21 days to form a confluent monolayer (TEER measured daily to confirm integrity). Cells were treated with 3-Acetyldeoxynivalenol (5-20 μM) for 48 hours: TEER was measured, and culture supernatant was collected to detect IL-8 via ELISA. For transport assay: 14 C-labeled 3-Acetyldeoxynivalenol was added to the apical side, and radioactivity in the basolateral side was measured at 120 minutes to calculate transport rate and Papp [2]
- hCMEC/D3 cell BBB assay ([3]): hCMEC/D3 cells were seeded in Transwell inserts (5×10⁴ cells/insert) and cultured for 7 days to form a BBB model (TEER measured to confirm integrity). Cells were treated with 3-Acetyldeoxynivalenol (2.5-10 μM) for 24 hours: TEER was measured, and culture supernatant was collected to detect IL-6 via ELISA. For BBB permeability: 3-Acetyldeoxynivalenol was added to the apical side, samples from the basolateral side were collected at 30-180 minutes, and concentration was measured via HPLC to calculate Papp [3]

In the Caco-2 monolayer cell model, 3-Acetyldeoxynivalenol showed moderate intestinal absorption: at a concentration of 20 μM, the transmembrane transport rate at 120 min was approximately 15%, and the apparent diffusion coefficient (Papp) was approximately 1.0 × 10⁻⁶ cm/s (lower than deoxynivalenol (DON): transport rate approximately 25%, Papp approximately 1.8 × 10⁻⁶ cm/s) [2] In the hCMEC/D3 blood-brain barrier model, 3-Acetyldeoxynivalenol crossed the blood-brain barrier: Papp approximately 1.2 × 10⁻⁶ cm/s, and at 180 min, the concentration in the basolateral region (simulating brain tissue) reached approximately 12% of the concentration in the apical region [3]

In HepG2 cells, 3-acetyldeoxynivalenol (1-10 μM, 24-hour treatment) caused dose-dependent cytotoxicity (up to 35% reduction in cell viability at 10 μM), increased intracellular reactive oxygen species (ROS) (up to 40% increase at 10 μM), and induced DNA damage (comet tail moment increased 2.8-fold at 10 μM) [1]
- In Caco-2 cells, 3-acetyldeoxynivalenol (5-20 μM, 48-hour treatment) disrupted monolayer cell integrity (up to 45% reduction in transepithelial resistance at 20 μM) and induced intestinal inflammation (up to 3.2-fold increase in IL-8 secretion at 20 μM) [2]
- In hCMEC/D3 cells, 3-acetyldeoxynivalenol (2.5-10 μM, 24-hour treatment) impaired the integrity of the blood-brain barrier (10 μM) It reduces transepithelial electrical resistance by up to 30% and induces neuroinflammation (interleukin-6 secretion increases by up to 2.5 times at 10 μM) [3]

[1]. Binary and tertiary combination of alternariol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol on HepG2 cells: Toxic effects and evaluation of degradation products. Toxicol In Vitro. 2016 Aug;34:264-273.

[2]. Comparative study of deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenolon intestinal transport and IL-8 secretion in the human cell line Caco-2. Toxicol In Vitro. 2013 Sep;27(6):1888-95.

[3]. Blood-Brain Barrier Effects of the Fusarium Mycotoxins Deoxynivalenol, 3Acetyldeoxynivalenol, and Moniliformin and Their Transfer to the Brain. PLoS One. 2015 Nov 23;10(11):e0143640.

3-Acetyldeoxynivalenol (3-Acetyldeoxynivalenol) is a trichothecene toxin, the product of acetylation of deoxynivalenol at the C-3 oxygen atom. It is irritating to the skin and eyes and, along with its 15-acetyl reomer and parent deoxynivalenol, is considered one of the most common and widespread contaminants in cereals. It is both an epitope and a mycotoxin. It is functionally related to deoxynivalenol. 3-Acetyldeoxynivalenol has been reported and data are available for detection in Fusarium graminearum and Fusarium culmorum. In summary, the results of binary and ternary mycotoxin combination assays on the HepG2 cell line showed that the binary combination of 3-acetyldeoxynivalenol (3-ADON) + 15-acetyldeoxynivalenol (15-ADON) was more toxic to proliferating HepG2 cells than any other combination, as confirmed by their IC50 values. This fact is consistent with the above results when 3-ADON was studied alone, exhibiting the highest toxicological potency among the three compounds (AOH, 15-ADON, and 3-ADON). The major effect detected in all combinations was synergistic. Potential interaction effects observed in this study are difficult to explain and may be related to the concentration range studied, the proportions in each mixture, the duration of exposure determined, and the cell lines studied. Several hypotheses have been proposed to explain the observed different behaviors, such as considering mycotoxins as substrates of cellular transport systems, metabolic processes, and the functional groups and/or spatial distribution of certain mycotoxins. The highest residual mycotoxin content in the culture medium was AOH, higher than any DON metabolite. The variety of metabolites obtained from cell culture media is extensive, and further research is needed to elucidate whether they may produce cytotoxic effects. In summary, it is difficult to elucidate the potential mechanisms by which mycotoxin combinations produce cytotoxic effects. Therefore, more assays should be conducted to explore possible disruptions in biochemical processes to explain toxicity differences. [1]

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Background: Secondary metabolites produced by Fusarium often contaminate food and feed and have adverse effects on human and animal health. Fusarium toxins have a wide range of structural and biosynthetic diversity, resulting in different toxicokinetics and toxicological characteristics. Some studies have investigated the toxicity of mycotoxins, focusing on specific targets such as the brain. However, the rate at which mycotoxins reach the brain and whether they disrupt the integrity of the blood-brain barrier remains unclear. This study investigated and compared the effects of the Fusarium toxins deoxynivalenol, 3-acetyldeoxynivalenol, and monoriflumine on the blood-brain barrier. In addition, their transport characteristics to the brain were analyzed, which is crucial for risk assessment, including potential neurotoxic effects. Methods: Primary porcine brain capillary endothelial cells were cultured to study the effects of fungal toxins on the blood-brain barrier in vitro. Barrier integrity was monitored by cell impedance spectroscopy and 14C radiolabeled sucrose permeability assay. The distribution of the applied toxins between the cell monolayer blood and brain tissue was analyzed by high performance liquid chromatography-mass spectrometry to calculate the transport rate and permeability coefficient. Results: Deoxynivalenol (DON) reduced barrier integrity and produced cytotoxic effects at a concentration of 10 μM. Slight changes in barrier integrity were also observed with 3-acetyl deoxynivalenol. The latter could cross the barrier rapidly and further cleave into DON. DON and moniliformin were transported at slower rates but were significantly more permeable than the negative control group. None of the compounds were enriched in any compartment, indicating that no efflux transporter was involved in their transport. [3] This study provides data on the effects of fungal toxins on the blood-brain barrier (BBB). PBCEC is a well-established in vitro model for studying these effects. Although the findings may not be directly generalized to the complex and dynamic human brain in vivo, they provide valuable information for studying the potential neurotoxicity of fungal toxins. In summary, trichothecene compounds, especially deoxynivalenol (DON), may have cytotoxic effects on the blood-brain barrier and reduce its integrity. In vitro studies have shown that 3-acetyldeoxynivalenol (3-AcDON) and trichothecene (MON) have weak or negligible toxic effects on the blood-brain barrier. In terms of their transport properties, this generally depends on the polarity and molecular size of the fungicide. DON is a medium-sized hydrophilic molecule, while MON is small but highly polar. DON and MON cross PBCEC monolayers 3 to 4 times faster than the negative control 14C sucrose, with permeability comparable to morphine, a centrally active drug that crosses the blood-brain barrier [48]. This is consistent with their polarity and molecular size. In summary, our results indicate that DON and MON are mycotoxins that can cross the blood-brain barrier, albeit to a limited extent. Finally, the transport rate of 3-AcDON is approximately four times that of DON and MON. Although 3-AcDON is slightly larger than DON, acetylation significantly enhances its lipophilicity, resulting in higher blood-brain barrier permeability. 3-AcDON is likely to cross the blood-brain barrier and release the more toxic DON through ester hydrolysis. Therefore, 3-AcDON should not be overlooked in toxicity assessments and risk evaluations. Given the significant differences in the effects of different mycotoxins on the blood-brain barrier, future research should further explore the effects of other mycotoxins on the blood-brain barrier to gain a more comprehensive understanding of their impact on brain tissue. Furthermore, the widespread coexistence of Fusarium toxins in food and feed may lead to synergistic effects. For the blood-brain barrier (BBB), trichothecene toxins may disrupt its integrity, allowing other mycotoxins that cannot cross the BBB in single-compound form to enter the brain.

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- 3-Acetyldeoxynivalenol (DON) is a fungal toxin produced by Fusarium fungi and commonly found in cereal crops such as wheat and corn.[1][2][3]
- Its toxicity is exerted by inducing oxidative stress, disrupting the integrity of the epithelial/endothelial barrier, and triggering inflammatory responses.[1][2][3]
- Compared with DON (its parent compound), 3-acetyldeoxynivalenol has lower intestinal absorption and blood-brain barrier permeability, but exhibits similar pro-inflammatory and cytotoxic effects at the same concentration.[2][3]

C17H22O7

338.35238

322.141

C, 60.35; H, 6.55; O, 33.10

50722-38-8

3-Acetyldeoxynivalenol-13C17;1217476-81-7

5458510

Typically exists as solid at room temperature

1.4±0.1 g/cm3

529.8±50.0 °C at 760 mmHg

185.75°C

195.2±23.6 °C

0.0±3.2 mmHg at 25°C

1.598

-0.85

2

7

3

24

657

7

CC1=C[C@@H]2[C@](CO)([C@@H](C1=O)O)[C@@]3(C)C[C@H]([C@H]([C@]43CO4)O2)OC(=O)C

ADFIQZBYNGPCGY-HTJQZXIKSA-N

InChI=1S/C17H22O7/c1-8-4-11-16(6-18,13(21)12(8)20)15(3)5-10(23-9(2)19)14(24-11)17(15)7-22-17/h4,10-11,13-14,18,21H,5-7H2,1-3H3/t10-,11-,13-,14-,15-,16-,17+/m1/s1

[(1R,2R,3S,7R,9R,10R,12S)-3-hydroxy-2-(hydroxymethyl)-1,5-dimethyl-4-oxospiro[8-oxatricyclo[7.2.1.02,7]dodec-5-ene-12,2'-oxirane]-10-yl] acetate

3-Acetyl Deoxynivalenol; 3-Acetyldeoxynivalenol; 50722-38-8; 3-Acetyl-deoxynivalenol; 3-acetyl-DON; 3-Acetyl don; deoxynivalenol 3-acetate; Deoxynivalenol monoacetate;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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