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

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.

[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 is a trichothecene mycotoxin that is deoxynivalenol acetylated on the oxygen at C-3. A skin and eye irritant, along with its 15-acetyl regioisomer and its parent deoxynivalenol it is considered among the most commonly and widely distributed cereal contaminants. It has a role as an epitope and a mycotoxin. It is functionally related to a deoxynivalenol.
3-Acetyldeoxynivalenol has been reported in Fusarium graminearum and Fusarium culmorum with data available.

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In conclusion, the results of binary and tertiary mycotoxin combinations assayed on the HepG2 cell line showed that binary combination of 3-ADON + 15-ADON was more toxic for proliferating HepG2 cells than any other combination as demonstrated by their IC50. This fact concords when single treatment of 3-ADON was studied, which presented the highest toxicological potency out of three (AOH, 15-ADOn and 3-ADON). The major effect detected in all combination was synergism. The potential interaction effects obtained in this study are difficult to explain and could be related to the concentration range studied, ratio in each mixture, exposure time assayed and cell line studied. Some hypothesis have been postulated to explain the different behaviour obtained as: considering mycotoxins as substrates to cellular transport systems, metabolic processes, and functional groups of some mycotoxins and/or their spatial distribution. The highest amount of mycotoxin remaining in the media was for AOH than for any of the DON's metabolites. Metabolites obtained in the cell culture media are diverse and further studies would be necessary to clarify if they might contribute to produce cytotoxicity effects. In conclusion, it is difficult to clarify the mechanisms underlying the cytotoxic effects of the mycotoxin combinations; so that, more assays should be done regarding the possible disturbances in biochemical processes to explain the differences in toxicity.[1]

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Background: Secondary metabolites produced by Fusarium fungi frequently contaminate food and feed and have adverse effects on human and animal health. Fusarium mycotoxins exhibit a wide structural and biosynthetic diversity leading to different toxicokinetics and toxicodynamics. Several studies investigated the toxicity of mycotoxins, focusing on very specific targets, like the brain. However, it still remains unclear how fast mycotoxins reach the brain and if they impair the integrity of the blood-brain barrier. This study investigated and compared the effects of the Fusarium mycotoxins deoxynivalenol, 3-acetyldeoxynivalenol and moniliformin on the blood-brain barrier. Furthermore, the transfer properties to the brain were analyzed, which are required for risk assessment, including potential neurotoxic effects. Methods: Primary porcine brain capillary endothelial cells were cultivated to study the effects of the examined mycotoxins on the blood-brain barrier in vitro. The barrier integrity was monitored by cellular impedance spectroscopy and 14C radiolabeled sucrose permeability measurements. The distribution of the applied toxins between blood and brain compartments of the cell monolayer was analyzed by high performance liquid chromatography-mass spectrometry to calculate transfer rates and permeability coefficients. Results: Deoxynivalenol reduced the barrier integrity and caused cytotoxic effects at 10 μM concentrations. Slight alterations of the barrier integrity were also detected for 3-acetyldeoxynivalenol. The latter was transferred very quickly across the barrier and additionally cleaved to deoxynivalenol. The transfer of deoxynivalenol and moniliformin was slower, but clearly exceeded the permeability of the negative control. None of the compounds was enriched in one of the compartments, indicating that no efflux transport protein is involved in their transport.[3]

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In this study data on the effects of mycotoxins on the BBB is presented. PBCEC are a well-established model to study these effects in vitro. Although the results may not be transferred directly to the effects on the complex and dynamic human brain in vivo, they provide valuable information for research focusing on the potential neurotoxicity of mycotoxins. Taken together, it can be summarized that trichothecenes (especially DON) could cause cytotoxic effects at the BBB and reduce its integrity. 3-AcDON and MON were shown to exhibit much weaker or almost no adverse effects at the BBB in vitro. Referring to their transport properties, these are generally driven by polarity and molecular size of the mycotoxin. DON is a hydrophilic molecule of medium molecular size, whereas MON is very small, but also very polar. DON and MON were transferred three to four times faster across the PBCEC monolayer than the negative control 14C sucrose and reached permeabilities comparable to morphine, which is a CNS-active and BBB permeable drug [48]. This is in agreement with their polarity and molecular size. In conclusion the results of this study suggest that DON and MON are BBB permeable mycotoxins, although to a limited extent. Finally, 3-AcDON was transferred approximately four times faster than DON and MON. Although it is slightly larger than DON, the acetylation considerably increases the lipophilicity of 3-AcDON leading to a high BBB permeability. 3-AcDON is very likely to cross the BBB, and release the more toxic DON by ester hydrolysis. For this reason, it should not be neglected in toxicity assessment and risk evaluation. Considering the presented strong differences in their effects at the BBB, future research should investigate the effects of further mycotoxins on the BBB, to obtain a better overview of their effects on the cerebral tissues. In addition, the common co-occurrence of Fusarium mycotoxins in food and feed might lead to combination effects. In case of the BBB, trichothecenes could impair the integrity of the BBB and allow other mycotoxins, which would not pass the BBB as single compound, to reach the brain.

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.)