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
This study investigated the conversion of ochratoxin C to ochratoxin A in rats after oral and intravenous administration. Following oral administration of equal amounts of ochratoxin C or ochratoxin A, the blood concentration of ochratoxin A showed the same trend over time. The highest concentration of ochratoxin A was measured 60 minutes after administration. After intravenous administration of ochratoxin C, it was also converted to ochratoxin A, reaching its highest concentration after 90 minutes. Therefore, it is concluded that ochratoxin C is rapidly converted to ochratoxin A after both oral and intravenous administration. It is reasonable to believe that the comparable toxicity of these two toxins is based on this conversion process, and only interference with the biotransformation mechanism could lead to differences in their toxicity. …The metabolomic profile of ochratoxin A was studied in mice and cultures of Aspergillus ochraceus producing ochratoxin A (OA). Ochratoxin α (Oα), ochratoxin β (Oβ), 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 Aspergillus ochraceus cultures and their structures were characterized by 1H NMR spectroscopy, mass spectrometry, and high-performance liquid chromatography. 4-R-OH OA and Oα were continuously produced and were the main biotransformation metabolites in the fungal cultures 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 biomolecules were isolated from the fungal cultures by detergent extraction, followed by cold acetone precipitation and gel filtration. Acid hydrolysis of these fluorescent macromolecules released various ochratoxins, including Oα (80%), OA (2%), and OC (5%), as well as other unidentified fluorescent compounds, but not OB and Oβ. Cross-reactivity studies of natural OA macromolecule conjugates with anti-OA polyclonal antibodies indicated that these conjugates are covalently linked to the macromolecules via groups other than the carboxyl group. These studies suggest that fungi can produce the same OA metabolites as rats, and that Oα, OA, and OC may be covalently linked to fungal macromolecules.

Non-Human Toxicity Values

Chick (1-day-old) LD50: 216 μg/dose.

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Adverse Reactions

Occupational Hepatotoxicity - Secondary Hepatotoxicity: Potential toxic effects in occupational environments are based on cases of human ingestion or animal studies.
Nephrogenicity - This chemical may be toxic to the kidneys in occupational environments.

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Antidotes and Emergency Treatment
/SRP:/ Immediate First Aid Measures: Ensure adequate decontamination has been performed. If the patient stops breathing, begin artificial respiration immediately, preferably using a ventilator on demand, bag-valve-mask, or simple breathing mask, following training instructions. Perform cardiopulmonary resuscitation if necessary. Immediately flush contaminated eyes with running water. Do not induce vomiting. If vomiting occurs, position the patient forward or on their side (head down if possible) to maintain an open airway and prevent aspiration. Keep the patient calm and maintain normal body temperature. Seek medical attention. /Toxins A and B/Currance, PL Clements, B., Bronstein, AC (eds.); First Aid for Hazardous Substance Exposure. 3rd ed., Elsevier Mosby, St. Louis, Missouri, 2005, p. 160. Hazardous Substances Database (HSDB)/SRP:/ Basic Treatment: Establish a patent airway (using an oropharyngeal or nasopharyngeal airway if necessary). Suction if necessary. Observe for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen using a non-invasive breathing mask at a flow rate of 10 to 15 liters per minute. Monitor for pulmonary edema and treat as necessary… Monitor for shock and treat as necessary… Anticipate seizures and treat as needed… If eyes are contaminated, flush with water immediately. During transport, continuously flush each eye with 0.9% normal saline (NS)… Do not use emetics. If ingested, rinse mouth; if the patient is able to swallow, has a strong gag reflex, and does not drool, dilute with 5 mL/kg to 200 mL of water… After cleansing, cover burns with a dry, sterile dressing… /Class A and B Poisons/
/SRP:/ Advanced Treatment: For patients with impaired consciousness, severe pulmonary edema, or severe respiratory distress, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation using a bag-valve-mask may be effective. Consider medical treatment for pulmonary edema… Consider the use of a β-receptor agonist (such as salbutamol) to treat severe bronchospasm… Monitor heart rhythm and treat arrhythmias if necessary… Begin intravenous infusion of 5% glucose solution (D5W/SRP: maintain patency, minimum flow rate). If signs of hypovolemia appear, use 0.9% normal saline (NS) or lactated Ringer's solution. Use fluids with caution in cases of hypotension accompanied by signs of hypovolemia. Note signs of fluid overdose… Treat seizures with diazepam or lorazepam… Use promecaine hydrochloride to assist eye irrigation… /Toxins A and B/

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Human Toxicity Excerpt
/Alternatives and In Vitro Tests/ An in vitro model was established to study the effects of long-term exposure to low concentrations of ochratoxin A (OTA) or ochratoxin C (OTC) on the human mononuclear cell line (THP-1). Cells were cultured in 24-well cell culture plates for 15 days. During the culture period, ochratoxin A (OTA) and ochratoxin C (OTC) formulations at concentrations of 1 ng/mL were added to the cell culture medium, respectively. After exposure, cell viability and cell function parameters were measured. After 15 days of exposure to ochratoxins, the viability and function of the THP-1 cell line changed. All mycotoxins increased mitochondrial activity and IL-6 production. Cell membrane integrity was disrupted, and cell proliferation, as well as the production of TNF-α and IL-8, were inhibited. These parameters were most severely affected by mycotoxin preparations containing OTC. Our results indicate that long-term exposure to low concentrations of OTA, especially OTC, can lead to subtle alterations in cell viability and function, which may have significant implications for human and animal health. Therefore, further investigation into OTC contamination in food and feed is necessary. 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 and an ethyl ester of ochratoxin A. It is a metabolite of both Aspergillus and Penicillium, and is also a mycotoxin. It is an α-amino acid ester, belonging to the phenylalanine derivative class and is a member of the heterochromatic esters. Its function is related to ochratoxin A. Ochratoxin C has been reported to exist in Aspergillus, and relevant data are available. Based on fluorescence studies, OTB and OTC form highly stable complexes with human serum albumin (HSA). Fluorescence quenching and circular dichroism (CD) experiments indicate that PAT has a low affinity for HSA, while DON and T2 may not interact with the protein or only form less stable complexes. In ultrafiltration experiments, OTB and OTC significantly replaced the I-site marker warfarin, but other tested mycotoxins did not affect the binding of warfarin or naproxen to albumin. The presence of albumin mitigates or even eliminates the cytotoxic effects of OTB and OTC, indicating that the formation of OTB-HSA and OTC-HSA complexes plays an important role in toxicokinetics. Since the binding of mycotoxins to albumin significantly affects their tissue distribution and elimination half-life, these results contribute to a deeper understanding of the toxicokinetics of mycotoxins. Cell experiments are helpful in exploring the toxicological significance of mycotoxin-albumin interactions, as other factors, such as the diffusion of compounds and the involvement of active transport mechanisms, also affect the uptake of mycotoxins from circulation by cells. Given the complexity of the toxicokinetics of these compounds, it is reasonable to conduct animal experiments in the future to better characterize mycotoxin-albumin interactions. For example, displacing highly albumin-bound mycotoxins (e.g., ochratoxin) from proteins may significantly alter 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.)