| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
Natural trichothecene
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|---|---|
| ln Vitro |
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-Acetyl-deoxynivalenol (3-ADON) 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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| Enzyme Assay |
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]
|
| Cell Assay |
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. |
| Toxicity/Toxicokinetics |
The oral LD50 in mice was 34 mg/kg. Gastrointestinal effects included small intestinal ulceration or hemorrhage; kidney, ureter, and bladder effects included cystitis, necrosis, or scarring; blood effects included spleen changes. Food and Chemical Toxicology, 25(155), 1987 [PMID:3557238]
The intraperitoneal LD50 in mice was 113 mg/kg. Applied and Environmental Microbiology, 50(1304), 1985 [PMID:2936303] |
| References | |
| Additional Infomation |
15-Acetyldeoxynivalenol is a trichothecene toxin, the product of acetylation of deoxynivalenol at the C-15 oxygen atom. It is a skin and eye irritant and, along with its 3-acetyl reticulomer 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. 15-Acetyldeoxynivalenol has been reported and data are available for detection in Fusarium graminearum and Fusarium oxysporum. 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 types of metabolites obtained from cell culture media are numerous and require further investigation 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 investigations should be conducted to detect possible disturbances in biochemical processes in order to explain the differences in toxicity. [1]
|
| Molecular Formula |
C17H22O7
|
|---|---|
| Molecular Weight |
338.352385997772
|
| Exact Mass |
338.137
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| CAS # |
88337-96-6
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| Related CAS # |
15-Acetyl-deoxynivalenol-13C17;911392-39-7
|
| PubChem CID |
10382483
|
| Appearance |
White to off-white solid powder
|
| Density |
1.42g/cm3
|
| Boiling Point |
518.8ºC at 760 mmHg
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| Melting Point |
185°C-187°C
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| Flash Point |
2ºC
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| Index of Refraction |
1.595
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| LogP |
-0.7
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
7
|
| Rotatable Bond Count |
3
|
| Heavy Atom Count |
24
|
| Complexity |
657
|
| Defined Atom Stereocenter Count |
7
|
| SMILES |
CC1=C[C@@H]2[C@]([C@@H](C1=O)O)([C@]3(C[C@H]([C@H]([C@@]34CO4)O2)O)C)COC(=O)C
|
| InChi Key |
IDGRYIRJIFKTAN-HTJQZXIKSA-N
|
| InChi Code |
InChI=1S/C17H22O7/c1-8-4-11-16(6-22-9(2)18,13(21)12(8)20)15(3)5-10(19)14(24-11)17(15)7-23-17/h4,10-11,13-14,19,21H,5-7H2,1-3H3/t10-,11-,13-,14-,15-,16-,17+/m1/s1
|
| Chemical Name |
[(1R,2R,3S,7R,9R,10R,12S)-3,10-dihydroxy-1,5-dimethyl-4-oxospiro[8-oxatricyclo[7.2.1.02,7]dodec-5-ene-12,2'-oxirane]-2-yl]methyl acetate
|
| Synonyms |
5-Acetyldeoxynivalenol; 88337-96-6; 15-Acetyl Deoxynivalenol; 15-Acetyl-deoxynivalenol; Deoxynivalenol 15-Acetate; 15-Acetoxy-3alpha,7alpha-dihydroxy-12,13-epoxytrichothec-9-en-8-one; 15-Acetylvomitoxin; 15-Acetyl Deoxynivalenol (~90%);
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
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
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|---|---|
| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9555 mL | 14.7776 mL | 29.5552 mL | |
| 5 mM | 0.5911 mL | 2.9555 mL | 5.9110 mL | |
| 10 mM | 0.2956 mL | 1.4778 mL | 2.9555 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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