Neurotoxin/mycotoxin from A. ochraceus; food contamination.

Ochratoxins, patulin, deoxynivalenol, and T-2 toxin are mycotoxins, and common contaminants in food and drinks. Human serum albumin (HSA) forms complexes with certain mycotoxins. Since HSA can affect the toxicokinetics of bound ligand molecules, the potential interactions of ochratoxin B (OTB), ochratoxin C (OTC), patulin, deoxynivalenol, and T-2 toxin with HSA were examined, employing spectroscopic (fluorescence, UV, and circular dichroism) and ultrafiltration techniques. Furthermore, the influence of albumin on the cytotoxicity of these xenobiotics was also evaluated in cell experiments. Fluorescence studies showed the formation of highly stable OTB–HSA and OTC–HSA complexes. Furthermore, fluorescence quenching and circular dichroism measurements suggest weak or no interaction of patulin, deoxynivalenol, and T-2 toxin with HSA. In ultrafiltration studies, OTB and OTC strongly displaced the Sudlow’s site I ligand warfarin, while other mycotoxins tested did not affect either the albumin binding of warfarin or naproxen. The presence of HSA significantly decreased or even abolished the OTB- and OTC-induced cytotoxicity in cell experiments; however, the toxic impacts of patulin, deoxynivalenol, and T-2 toxin were not affected by HSA. In summary, the complex formation of OTB and OTC with albumin is relevant, whereas the interactions of patulin, deoxynivalenol, and T-2 toxin with HSA may have low toxicological importance.[1]

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A broad-specific photoelectrochemical (PEC) immunosensor was developed for the simultaneous detection of ochratoxin A, ochratoxin B and ochratoxin C (OTA, OTB, OTC) by using the direct growth of CdS nanorods on FTO as the photoelectrode and Au nanoflowers-modified glass carbon electrode (GCE) as the bioelectrode. The bioelectrode was used to capture antigens and then associate corresponding antibodies, followed by using SiO2@Cu2+ nanocomposites to conjugate the secondary antibody (Ab2) and a DNA strand as the initiator. After the hybridization chain reaction (HCR) and the addition of hemin, numerous DNAzymes (G-quadruplex/hemin) were produced. Due to the similar enzymatic property with horseradish peroxidase (HRP), G-quadruplex/hemin can accelerate the oxidation of 4-chloro-1-naphthol (4-CN) with H2O2 to yield the biocatalytic precipitation (BCP) on the bioelectrode. Then, the bioelectrode was further treated with moderate acid and thus Cu2+ was released, which can decrease the photocurrent of the photoelectrode by the formation of CuS. Due to the advantages of surface effect of Au nanoflowers, DNA amplification and high photoelectrocatalytic activity, the proposed broad-specificity PEC immunosensor can detect OTA, OTB and OTC with a detection limit of 0.02, 0.04 and 0.03 pg/mL, respectively. In addition, the acceptable stability and selectivity suggest its possible application in the detection of OTA, OTB and OTC in water samples. [2]

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Ochratoxins A, B, and C (OTA, OTB, and OTC) can be found in cereals and feeds; the simultaneous detection of these ochratoxins holds a great need in food safety. In this study, four antibodies raised from two ochrotoxin haptens and two coating antigens were compared, and then a sensitive and broad-specificity enzyme-linked immunosorbent assay (ELISA) was established for the simultaneous determination of three ochratoxins, where the detection limits were 0.005, 0.001, and 0.001 ng/mL for OTA, OTB, and OTC, respectively, and recoveries of three ochratoxins were between 84.3% and 111.7%. This developed method had been successfully applied to detect ochratoxins in both millet and maize. Molecular modeling revealed that the broad-specificity was related with the chlorine electronegativity on OTA and OTC and the potential of the acetyl ester group on OTC. The proposed ELISA can be used for simultaneous detection of three ochratoxins[3].

Cell Culturing and Viability Assay [1]
Cell experiments were performed on HepG2 (human hepatocellular carcinoma) adherent cell line. The cells were cultured in DMEM with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin (5% CO2, 37 °C). Cells (104/well in 96-well plates) were treated for 48 h with OTB (5.0, 10.0 and 20.0 µM), Ochratoxin C (OTC) (0.05, 0.1, and 0.5 µM), PAT (1.0, 2.0, and 5.0 µM), DON (1.0, 2.0, and 5.0 µM), and T2 (0.02, 0.25, and 1.0 µM) in the absence and presence of 10% FBS or 40 g/L HSA. ATP levels were quantified applying the previously described method without modifications.

Metabolism / Metabolites
The conversion of ochratoxin C to ochratoxin A was studied in rats after oral and intravenous administration. The concentration of ochratoxin A in the blood as a function of time was the same after oral administration of equivalent amounts of either ochratoxin C or ochratoxin A. The maximum ochratoxin A concentrations were measured 60 min after administration. Given intravenously, ochratoxin C was also converted to ochratoxin A. Maximum concentrations were reached after 90 min. It is concluded that ochratoxin C is readily converted to ochratoxin A after both oral and intravenous administration. There is reason to believe that a comparable toxicity of the two toxins is based upon this conversion and that only interference with the biotransformation mechanisms may cause a difference in their toxicity.
...The metabolic profile of ochratoxin A (OA) /was studied/ in rats and in a culture of OA-producing Aspergillus ochraceus. Ochratoxin alpha (O alpha), ochratoxin beta (O beta), 4-R-hydroxyochratoxin A (4-R-OH OA), 4-R-hydroxyochratoxin B (4-R-OH OB), and 10-hydroxyochratoxin A (10-OH OA) were isolated from a culture of A. ochraceus and structurally characterized by 1H nuclear magnetic resonance spectroscopy, mass spectrometry and high-pressure liquid chromatography. 4-R-OH OA and O alpha were consistently produced and were the dominant biotransformed metabolites in the fungal culture and in rats treated with OA and ochratoxin C (OC), while the formation of 10-OH OA was conditional in the fungal system. Green fluorescent biomacromolecules were isolated by detergent extraction of the fungal culture followed by cold-acetone precipitation and gel filtration. Acid hydrolysis of the fluorescent macromolecules resulted in the release of several ochratoxins, including O alpha (80%), OA (2%), and OC (5%), and other unidentified fluorescent compounds but not OB and O beta. Cross-reactivity studies of the natural macromolecule conjugates of OA with anti-OA polyclonal antibodies indicated that they were covalently linked to the macromolecules via a group other than the carboxyl group. These studies demonstrated that a fungus can produce some of the same metabolites of OA as the rat and that O alpha, OA, and OC may be covalently linked to fungal macromolecules.

Non-Human Toxicity Values
LD50 chicks (day-old) 216 ug/assay.

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Adverse Effects
Occupational hepatotoxin - Secondary hepatotoxins: the potential for toxic effect in the occupational setting is based on cases of poisoning by human ingestion or animal experimentation.
Nephrotoxin - The chemical is potentially toxic to the kidneys in the occupational setting.

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Antidote and Emergency Treatment
/SRP:/ Immediate first aid: Ensure that adequate decontamination has been carried out. If patient is not breathing, start artificial respiration, preferably with a demand valve resuscitator, bag-valve-mask device, or pocket mask, as trained. Perform CPR if necessary. Immediately flush contaminated eyes with gently flowing water. Do not induce vomiting. If vomiting occurs, lean patient forward or place on the left side (head-down position, if possible) to maintain an open airway and prevent aspiration. Keep patient quiet and maintain normal body temperature. Obtain medical attention. /Poisons A and B/ Currance, P.L. Clements, B., Bronstein, A.C. (Eds).; Emergency Care For Hazardous Materials Exposure. 3Rd edition, Elsevier Mosby, St. Louis, MO 2005, p. 160 Hazardous Substances Data Bank (HSDB) /SRP:/ Basic treatment: Establish a patent airway (oropharyngeal or nasopharyngeal airway, if needed). Suction if necessary. Watch for signs of respiratory insufficiency and assist ventilations if needed. Administer oxygen by nonrebreather mask at 10 to 15 L/min. Monitor for pulmonary edema and treat if necessary ... . Monitor for shock and treat if necessary ... . Anticipate seizures and treat if necessary ... . For eye contamination, flush eyes immediately with water. Irrigate each eye continuously with 0.9% saline (NS) during transport ... . Do not use emetics. For ingestion, rinse mouth and administer 5 mL/kg up to 200 mL of water for dilution if the patient can swallow, has a strong gag reflex, and does not drool ... . Cover skin burns with dry sterile dressings after decontamination ... . /Poisons A and B/

/SRP:/ Advanced treatment: Consider orotracheal or nasotracheal intubation for airway control in the patient who is unconscious, has severe pulmonary edema, or is in severe respiratory distress. Positive-pressure ventilation techniques with a bag valve mask device may be beneficial. Consider drug therapy for pulmonary edema ... . Consider administering a beta agonist such as albuterol for severe bronchospasm ... . Monitor cardiac rhythm and treat arrhythmias as necessary ... . Start IV administration of D5W /SRP: "To keep open", minimal flow rate/. Use 0.9% saline (NS) or lactated Ringer's if signs of hypovolemia are present. For hypotension with signs of hypovolemia, administer fluid cautiously. Watch for signs of fluid overload ... . Treat seizures with diazepam or lorazepam ... . Use proparacaine hydrochloride to assist eye irrigation ... . /Poisons A and B/

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Human Toxicity Excerpts
/ALTERNATIVE and IN VITRO TESTS/ An in-vitro model was developed to study the effects of a long-term exposure with a low concentration of ochratoxin A (OTA) or ochratoxin C (OTC) on a human monocytic cell line (THP-1).Cells were propagated in 24-well cell culture plates for 15 days. OTA and OTC preparations, respectively, at a concentration of 1 ng/mL were included in the cell culture medium during the whole cultivation period. At the end of the exposure time, parameters of cell viability and cell function were examined.After 15 days of exposure to ochratoxins, viability and function of the THP-1 cell line were modulated. Mitochondrial activity and the production of IL-6 were increased by all mycotoxin preparations. Cell membrane integrity was disturbed, proliferation and the production of TNF-a and IL-8 were inhibited. These parameters were most severely affected by mycotoxin preparations containing OTC.Our results show that long term exposure to OTA and especially OTC in low concentrations can cause subtle alterations of cell viability and function which may have remarkable consequences for human and animal health. In this context it seems to be necessary to study the contamination of food and feed stuffs with OTC more intensively. PMID:23604760

[1]. Probing the Interactions of Ochratoxin B, Ochratoxin C, Patulin, Deoxynivalenol, and T-2 Toxin with Human Serum Albumin. Toxins (Basel). 2020 Jun 13;12(6):392.
[2]. Broad-specificity photoelectrochemical immunoassay for the simultaneous detection of ochratoxin A, ochratoxin B and ochratoxin C. Biosens Bioelectron. 2018 May 30:106:219-226.
[3]. Broad-Specificity Immunoassay for Simultaneous Detection of Ochratoxins A, B, and C in Millet and Maize. J Agric Food Chem. 2017 Jun 14;65(23):4830-4838.

Ochratoxin C is a phenylalanine derivative that is the ethyl ester of ochratoxin A. It has a role as an Aspergillus metabolite, a Penicillium metabolite and a mycotoxin. It is an alpha-amino acid ester, a phenylalanine derivative and a member of isochromanes. It is functionally related to an ochratoxin A.
Ochratoxin C has been reported in Aspergillus ochraceus with data available.

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Based on fluorescence studies, OTB and OTC form highly stable complexes with HSA. Fluorescence quenching and CD experiments suggest the low-affinity interaction of PAT with HSA, while DON and T2 likely do not interact with the protein or form poorly stable complexes. In ultrafiltration experiments, OTB and OTC significantly displaced the site I marker warfarin but other mycotoxins tested did not affect the albumin binding either of warfarin or naproxen. Cytotoxic effects of OTB and OTC were alleviated or even abolished in the presence of albumin, suggesting the high toxicokinetic importance of OTB–HSA and OTC–HSA complex formations. Since albumin binding of mycotoxins can strongly affect their tissue distribution and elimination half-life, these results may help deepen the understanding of the toxicokinetics of mycotoxins. Cell experiments help to explore the toxicological importance of mycotoxin–albumin interactions, because several other factors (e.g., diffusibility of the compound and the involvement of active transport mechanisms) can also influence the cellular uptake of mycotoxins from the circulation. Due to the complex toxicokinetics of these compounds, it is reasonable to perform animal studies in the future for the better characterization of mycotoxin–albumin interactions. For example, the displacement of highly albumin-bound mycotoxins (e.g., ochratoxins) from the protein may strongly modify their toxicokinetics and toxicity. [1]

C22H22CLNO6

431.86

431.114

C, 61.19; H, 5.13; Cl, 8.21; N, 3.24; O, 22.23

4865-85-4

20997

White to off-white solid powder

1.328g/cm3

612.6ºC at 760 mmHg

324.3ºC

1.33E-15mmHg at 25°C

1.592

3.626

2

6

7

30

637

2

CCOC(=O)[C@H](CC1=CC=CC=C1)NC(=O)C2=CC(=C3C[C@H](OC(=O)C3=C2O)C)Cl

BPZZWRPHVVDAPT-PXAZEXFGSA-N

InChI=1S/C22H22ClNO6/c1-3-29-21(27)17(10-13-7-5-4-6-8-13)24-20(26)15-11-16(23)14-9-12(2)30-22(28)18(14)19(15)25/h4-8,11-12,17,25H,3,9-10H2,1-2H3,(H,24,26)/t12-,17+/m1/s1

ethyl (2S)-2-[[(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihydroisochromene-7-carbonyl]amino]-3-phenylpropanoate

HSDB 3439; OCHRATOXIN C; 4865-85-4; Ochratoxin A ethyl ester; UNII-0DY21HW450; HSDB-3439; 0DY21HW450; OCHRATOXIN C [MI]; OCHRATOXIN C [HSDB]; Ochratoxin C

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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.)