Mycotoxin

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with nephritogenic, carcinogenic, and teratogenic action. Epidemiological studies indicate that OTA may be involved in the pathogenesis of different forms of human nephropathies. Previously we have shown that OTA activates extracellular signal-regulated kinases 1 and 2, members of the mitogen-activated protein kinases (MAPK) family, in the C7-clone but not in the C11-clone of renal epithelial Madin-Darby canine kidney (MDCK) cells. Here we show that nanomolar concentrations of OTA lead to activation of a second member of the MAPK family, namely, c-jun amino-terminal-kinase (JNK) in MDCK-C7 cells but virtually not in MDCK-C11 cells, as determined by kinase assay and Western blot. Furthermore, OTA potentiated the effect of tumor necrosis factor-alpha on JNK activation. In parallel to its effects on JNK, nanomolar OTA induced apoptosis in MDCK-C7 cells but not in MDCK-C11 cells, as determined by DNA fragmentation, DNA ladder formation, and caspase activation. In addition, OTA potentiated the proapoptotic action of tumor necrosis factor-alpha. Our data provide additional evidence that OTA interacts in a cell type-specific way with distinct members of the MAPK family at concentrations where no acute toxic effect can be observed. Induction of apoptosis via the JNK pathway can explain some of the OTA-induced changes in renal function as well as part of its teratogenic action[3].

A disruption of calcium homeostasis, leading to a sustained increase in cytosolic calcium levels, has been associated with cytotoxicity in response to a variety of agents in different cell types. We have observed that administration of a single high dose or multiple lower doses of the carcinogenic nephrotoxin ochratoxin A (OTA) to rats resulted in an increase of the renal cortex endoplasmic reticulum ATP-dependent calcium pump activity. The increase was very rapid, being evident within 10 min of OTA administration and remained elevated for at least 6 hr thereafter. The increase in calcium pump activity was inconsistent with previous observations that OTA enhances lipid peroxidation (ethane exhalation) in vivo, a condition known to inhibit the calcium pump. However, no evidence of enhanced lipid peroxidation was observed in the renal cortex since levels of malondialdehyde and a variety of antioxidant enzymes including catalase, DT-diaphorase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione S-transferase were either unaltered or reduced. In in vitro studies, addition of OTA to cortex microsomes during calcium uptake inhibited the uptake process although the effect was reversible. Preincubation of microsomes with NADPH had a profound inhibitory effect on calcium uptake but inclusion of OTA was able to reverse the inhibition. Changes in the rates of microsomal calcium uptake correlated with changes in the steady-state levels of the phosphorylated Mg2+/Ca(2+)-ATPase intermediate, suggesting that in vivo/in vitro conditions were affecting the rate of enzyme phosphorylation[4].

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This study evaluates the effects of fermented whey (FW) and pumpkin (P) on the excretion of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in rats using immunoaffinity column cleanup and high-performance liquid chromatography-fluorescence detection (IAC-LC-FLD). The method achieved detection limits of 0.1 µg/kg for AFB1 and 0.3 µg/kg for OTA, with recovery rates ranging from 72-92% for AFB1 and 88-98% for OTA. A fecal analysis of 100 rats showed peak AFB1 concentrations of 418 µg/kg and OTA of 1729 µg/kg. In the toxin-exposed groups, OTA levels were higher than AFB1, with males in the OTA-only group showing significantly higher OTA (1729 ± 712 µg/kg) than females (933 ± 512 µg/kg). In the AFB1-only group, the fecal levels were 52 ± 61 µg/kg in males and 91 ± 77 µg/kg in females. The AFB1 + FW group showed notable AFB1 concentrations (211 ± 51 µg/kg in males, 230 ± 36 µg/kg in females). The FW + P combination further influenced excretion, with higher AFB1 and OTA levels. These findings suggest that FW and P modulate mycotoxin excretion and may play a role in mycotoxin detoxification, providing insight into dietary strategies to reduce mycotoxin exposure and its harmful effects [6].

Caspase-3 activity assay [5]
The assay was carried out according to the literature.17, 16 Cells were seeded in 24-well plates (2 × 104 cells/well). After toxin incubation, cells were washed with cold PBS buffer and incubated with 100 μL of cell lysis buffer (10 mM TRIS, 100 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, pH 7.5) for 15 min on ice. The cell lysates were centrifuged at 7000g for 10 min at 4 °C. Fifty microliters of the supernatant were incubated with 50 μL reaction buffer (50 mM PIPES, 10 mM EDTA, 0.5% CHAPS, 10 mM DTT) containing 8 μM fluorogenic caspase-3 substrate (Ac-Asp-Glu-Val-Asp-7-Amino-4-trifluoromethylcoumarin, DEVD-AFC) at 37 °C for 1 h. The fluorescence of 7-amino-4-trifluoromethylcoumarin (AFC), released by proteolytic cleavage, was measured with a microplate reader (excitation: λ = 400 nm; emission: λ = 505 nm). Released AFC concentrations were quantified using an AFC standard for the calibration and were normalized to the cellular protein content in each sample. Protein concentrations were determined with the bicinchoninic acid assay using bovine serum albumin (BSA) as standard for the calibration.

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Lactate dehydrogenase release assay [5]
The assay was carried out according to the literature.18 Cells were seeded in 24-well plates (2 × 104 cells/well). After toxin incubation, the cells were washed with cold PBS buffer and incubated with 100 μL of cell lysis buffer (10 mM TRIS, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, pH 7.5) for 15 min on ice. The cell lysates were centrifuged at 7000g for 10 min at 4 °C. Concordant samples of cell lysates and cell media were incubated with reaction buffer (2 μM NADH, 10 mM pyruvate, 100 mM HEPES, pH 7.0). The decrease of NADH related absorption at λ = 340 nm was measured every 2 min to monitor the kinetic parameters of the enzymatic reaction for 60 min at 37 °C using a microplate reader with temperature control. Thereof the amounts of total cellular lactate dehydrogenase (LDH) enzyme and released LDH were calculated for every sample and were normalized to positive and negative controls.

Cytotoxicity assay [5]
The cytotoxicity of ochratoxin A derivatives was evaluated colorimetrically using the Cell Counting Kit-8 (CCK-8) according to the literature and the manufacturer’s instructions. Briefly, cells were seeded on 96-well microplates (4 × 103 cells/well). After toxin exposure the dye solution (WST-8) was added to the cells, followed by the incubation for 1 h at 37 °C. The reduction of WST-8 dye by cellular dehydrogenases of viable cells increases the absorbance at λ = 450 nm and was measured with a microplate reader. The results for toxin-treated cells were normalized to the values of the untreated negative control.

In Vivo Study Design [6]
The current study builds upon the experimental framework established by Vila-Donat et al, investigating the effects of bioactive dietary ingredients on the urinary excretion of AFB1 and ochratoxin A/OTA in Wistar rats. The animals were housed under standard laboratory conditions (regulated temperature, humidity, and a 12 h light/dark cycle) and were provided ad libitum access to water and the assigned diets. Ethical guidelines for animal welfare were strictly followed throughout the experiment.

The study involved 120 rats (60 males and 60 females), divided into 12 groups, with each assigned a tailored diet. The diets included control feeds and feeds contaminated with AFB1 and ochratoxin A/OTA, which were prepared using grains inoculated with mycotoxin-producing fungi (A. flavus for AFB1 and A steynii for OTA). In addition, bioactive dietary ingredients, such as 1% P and 1% FW, were supplemented in specific diets to evaluate their impact on the fecal excretion of mycotoxins.

Fecal samples were periodically collected over the 28-day study period to measure AFB1 and ochratoxin A/OTA levels, facilitating the assessment of the effects of dietary interventions on mycotoxin elimination. Mycotoxin quantification in the feeds was conducted using LSE and LC-FLD methods. The detailed experimental procedures, including feed preparation recipes and the specific concentrations of mycotoxins in the feeds, have been previously detailed in Vila-Donat et al.

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Extraction of AFB1 and ochratoxin A/OTA from Feces [6]
The extraction of mycotoxins from feces was carried out using a clean-up process with the AflaOchra IAC, as described by Rodrigues et al. (2019) with slight modifications. The feces samples were first thawed and homogenized by grinding, and then, 1 g was mixed with 20 mL of 80% MeOH. The mixture was stirred for 45 min on a digital magnetic plate, followed by centrifugation at 4000 rpm for 10 min. Afterward, 10 mL of PBS were added to 2 mL of the supernatant, and the resulting buffered solution was purified using the AflaOchra IAC. For extraction, a vacuum-based SPE system was used, designed to concentrate, purify, or isolate analytes from complex matrices. Key components include a glass chamber and waste container, a position cover with luer adapters and a seal, luer connectors, stopcocks, guide needles, posts, shelves, a pressure gauge, and a mounting valve. The system accommodates 12 or 24 samples, ensuring efficient and precise sample preparation.

The column setup included a 10 mL syringe on top, an adapter, and a stopcock at the bottom to regulate the flow at 1 drop per second. Buffered samples were loaded into these columns. Then, the columns were washed, and finally, the elution of the compounds of interest was performed. AFB1 and ochratoxin A/OTA were eluted using a 1.5 mL mixture of MeOH and H2O (1:1, v/v) and collected in a glass vial. After the elution, air is passed through the system using the glass vacuum collector to ensure that all residual eluate is completely removed from the IAC columns. The extracted samples were then transferred to vials and directly injected into the LC-FLD system, as described in the following sections (Figure 4).

Absorption, Distribution, and Excretion
Since ochratoxin A-induced porcine mycotoxin nephropathy shares pathomorphic similarities with Balkan endemic nephropathy (BEN), it has been proposed that the pathogens of BEN may be the same. Based on the results of multiple field and experimental studies in pigs, we developed a suitable analytical method for monitoring potential human exposure to ochratoxin A. The toxicokinetics of this toxin are species-specific, although two binding proteins were found in plasma in all animal species studied (except fish) and in humans. The longest elimination half-life was observed in monkeys at 510 hours, compared to only 0.68 hours in fish. Fish kidneys exhibited a specific distribution pattern. In laying quail, the most significant observation was the accumulation of labeled ochratoxin A in the yolk. (14C)-ochratoxin A is typically rapidly cleared from quail, but remains in the circulating blood of mice for a longer period. Although clearance of ochratoxin A depends on its binding to plasma components, the presence of enterohepatic circulation may partially contribute to its long-term retention and clearance in mammals. The toxicokinetics of ochratoxin A do not contradict the fungal toxin hypothesis in the etiology of BEN. PMID:1618443 Fuchs R, Hult K; Food Chem Toxicol 30 (3): 201-4 (1992)
Ochratoxin A is rapidly absorbed throughout the gastrointestinal tract and distributed in vivo in a two-compartment open model, exhibiting a particularly high affinity for serum albumin. Ochratoxin A is hydrolyzed by intestinal flora into non-toxic compounds (7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin (ochratoxin α) and phenylalanine). It is excreted in urine and feces as ochratoxin A, hydroxylated ochratoxin A, or ochratoxin α. The toxic mechanism of ochratoxin A appears to be through the inhibition of aminoacylation, promoting elevated levels of lipid peroxidation, and possibly through conversion into metabolites capable of binding to DNA. These metabolites, in turn, induce other secondary effects associated with ochratoxin A. Due to its widespread presence and high carcinogenicity, this compound appears to pose a real potential hazard to humans. PMID:2200593 Marquardt RR et al; Canadian Journal of Physiological Pharmacology 68 (7): 991-9 (1990)
Rats were administered 500 μg of ochratoxin A daily via endotracheal intubation or fed 250 μg of ochratoxin A daily in barley. Little accumulation of the compound in the liver or kidneys was observed. The total amount excreted daily in urine and feces was on average slightly more than 10% of the administered dose. Small amounts of hydrolysates were also excreted. VAN WALBEEK W et al.; Toxicology and Applied Pharmacology 20 (3): 439 (1971)
A single intraperitoneal injection of 1 mg of ochratoxin A labeled with 14C in rats resulted in peak concentrations in serum (90%), liver (4.5%), and kidneys (4.4%) after 30 minutes. Ochratoxin A is primarily excreted in the urine as the unchanged toxin or as metabolites. Fecal excretion is minimal. PMID: 892675 CHANG FC, CHU FS; FOOD COSMET TOXICOL 15 (3): 199 (1977)

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Metabolism/Metabolites...Ochratoxin A is hydrolyzed by intestinal flora into non-toxic compounds (7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin (ochratoxin α) and phenylalanine). It is excreted in urine and feces as ochratoxin A, hydroxylated ochratoxin A, or ochratoxin α. ...PMID: 2200593 Marquardt RR et al.; Canadian Journal of Physiological Pharmacology 68 (7): 991-9 (1990)
Hydroxyochratoxin A was isolated and identified from the urine of rats injected with ochratoxin A. Ochratoxin A was incubated with rat liver microsomes and NADPH to produce one major metabolite (90%) and two minor metabolites, which were more polar than ochratoxin A. PMID:7396488 Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC291461 STORMER FC, PEDERSEN JI; Applied Environmental Microbiology 39 (5): 971 (1980)
Ochratoxin A was administered orally or intravenously (2.5 mg/kg) to healthy adult rats. Ochratoxin α metabolites were recovered only from the cecum and large intestine. Ochratoxin A was excreted in urine and feces as free drug and ochratoxin α. Unidentified metabolites were present in the urine. PMID: 43233 GALTIER P et al.; DRUG METAB DISPOS 7 (6): 429 (1979)
Ochratoxin A is cleaved by the colonic microbiota, carboxypeptidase A and α-chymotrypsin to phenylalanine and a less toxic isocoumarin derivative (ochratoxin α). International Agency for Research on Cancer (IARC). Monographs on Risk Assessment of Human Carcinogenic Chemicals. Geneva: World Health Organization, International Agency for Research on Cancer, 1972 to present. (Multi-volume). Available:
Ochratoxin A is cleaved by the colonic microbiota, carboxypeptidase A and α-chymotrypsin to phenylalanine and a less toxic isocoumarin derivative (α-ochratoxin). This is its main metabolic pathway. 4-Hydroxyochratoxin A is the main hepatic metabolite, and its formation appears to be through polymorphic 4-hydroxylation similar to norisopentenylquinoline. Some cytochrome P-450 enzymes, such as CYP2C9, are known to metabolize ochratoxin A into more toxic compounds. (T35, A2870, A3099)

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Biological half-life: Pregnant ICR mice were intraperitoneally injected with a single dose of 5 mg/kg ochratoxin A (OA) on day 11 or 13 of gestation. The half-life of ochratoxin A in serum on day 11 of gestation was calculated to be 28.7 hours, and on day 13 of gestation it was 23.6 hours. Fukui Y et al.; Food Chemistry and Toxicology 25: 17-24 (1987)
In rats…the plasma half-life of ochratoxin A is approximately 60 hours…. International Agency for Research on Cancer. Monographs on Risk Assessment of Human Carcinogenic Chemicals. Geneva: World Health Organization, International Agency for Research on Cancer, 1972 to present. (Multi-volume). Accessible at: https://monographs.iarc.fr/ENG/Classification/index.php, pp. 17-24. V31 198 (1983)
After oral administration of ochratoxin A at 50 μg/kg body weight, its apparent plasma elimination half-life is 0.68 hours in fish, 120 hours in rats, and 510 hours in monkeys… International Agency for Research on Cancer (IARC). Monographs on Risk Assessment of Carcinogenic Chemicals in Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972–present. (Multi-volume). Accessible at: https://monographs.iarc.fr/ENG/Classification/index.php, V56 499 (1993)
The metabolism of ochratoxin A in laboratory rodents and breeding animals has been studied. In rats, oral administration of ochratoxin A was readily absorbed, and a considerable amount of the toxin was detected in plasma, with peak concentrations occurring 2–4 hours post-administration. Pharmacokinetic analysis of plasma concentration-time curves indicated that the toxin was distributed in two distinct fluid compartments. The half-life of the toxin varied depending on the dose and animal species, ranging from 0.7 hours in fish to 840 hours in monkeys. In plasma, the toxin bound to albumin, as with many acidic compounds. This binding was competitively inhibited by phenylbutazone, ethyl dicumarol acetate, and sulfamethoxypyridazine, and was attenuated in albumin-deficient rats. Galtier P; IARC Sci Publ (115): 187–200 (1991)
The toxicokinetic properties of the toxin are species-specific, although both binding proteins were found in plasma in all studied animal species (except fish) and in humans. The longest elimination half-life was observed in monkeys at 510 hours, compared to only 0.68 hours in fish. The distribution of ochratoxin A in fish kidneys exhibits a specific pattern. In laying quail, the most significant observation is the accumulation of labeled ochratoxin A in the yolk. Typically, 14C-ochratoxin A is rapidly cleared from quail, but it remains in the circulating blood of mice for a longer period. Although the clearance of ochratoxin A depends on its binding to plasma components, the presence of enterohepatic circulation may be partly responsible for its prolonged retention and clearance in mammals. The toxicokinetic characteristics of ochratoxin A do not contradict the mycotoxin hypothesis in the etiology of BEN. PMID:1618443 Fuchs R, Hult K; Food Chem Toxicol 30 (3): 201-4 (1992)
Metabolism/Metabolites
3s14s-Ochratoxin A Known human metabolites include 2-[(5-chloro-4,8-dihydroxy-3-methyl-1-oxo-3,4-dihydroisocyanene-7-carbonyl)amino]-3-phenylpropionic acid.

Toxicity Overview
Ochratoxin A has been shown to be weakly mutagenic, likely acting by inducing oxidative DNA damage. The nephrotoxin ochratoxin A (OTA) causes a decrease in glomerular filtration rate (GFR) and para-aminohippuric acid (PAH) clearance. It is a nephrotoxin that blocks plasma membrane anion transport in Madin-Darby canine kidney (MDCK) cells. Several cytochrome P-450 enzymes, such as CYP2C9, are known to metabolize ochratoxin A into more cytotoxic compounds that can form DNA adducts. (A2869, A3099)

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Carcinogenicity Evidence
Assessment: There is insufficient evidence that ochratoxin A is carcinogenic to humans. There is sufficient evidence that ochratoxin A is carcinogenic to laboratory animals. Overall Assessment: Ochratoxin A is likely carcinogenic to humans (Class 2B).

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Toxicity Data
LD50: 20 mg/kg (oral, rat) (A716) LD50: 12,600 ug/kg (intraperitoneal, rat) (A716) LD50: 12,750 ug/kg (intravenous, rat) (A716) LD50: 46 mg/kg (oral, mouse) (A716)

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Treatment
Treatment was mainly symptomatic and supportive. (A704)

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Interactions
…This study investigated the toxicity of ochratoxin A (OA) and the effects of ascorbic acid (AA) supplementation on laying hens under two ambient temperatures. Twenty-four laying hens were randomly divided into two groups of 24 each, with four dietary treatment groups, each repeated six times. The treatment groups included a control group and three diet groups supplemented with 300 ppm AA, 3 ppm OA, or 300 ppm AA plus 3 ppm OA, respectively. After feeding the basal diet for 14 days, the treated diet was fed for another 14 days. The temperatures during the experiment were: 25°C for the first group and 33°C for the second group. …Compared to the control group, the hens fed 3 ppm OA showed significantly reduced feed intake, weight change, and egg production, and increased eggshell elasticity. Plasma component analysis indicated that OA also increased chloride ion concentration and aspartate transaminase activity, and decreased plasma calcium concentration. Exposure of hens to 33°C (compared to 25°C) appeared to exacerbate the negative effects of OA. Except for weight change, reduced feed intake, and increased eggshell elasticity at 33°C, all the negative effects of OA could be mitigated or significantly reversed by dietary supplementation with amino acids (AA). The results indicate that dietary supplementation with amino acids can counteract the harmful effects of OA in the diet of laying hens. Haazele FM et al.; Canadian Journal of Animal Science 73 (1): 149-57 (1993)
In neonatal rats, after oral administration of aldrin, the concentration of aldrin in the liver increased within the first 6 hours and then decreased to below 0.1% of the dose within 72 hours. When aldrin and ochratoxin were administered simultaneously, the concentration of aldrin increased within the first 6 hours and then decreased to 0.4% of the initial dose within 18 hours. FARB RM et al.; Pesticides and Environment: Ongoing Controversy, PAP International Conference on Occupational Medical Toxicology, 8th; 179 (1973)
Dieldrin was detected in neonatal rats 2 hours after aldrin administration, and the concentration of dieldrin could reach up to 30% of the initial dose after 18 hours. When aldrin and ochratoxin were administered simultaneously, the concentration of dieldrin increased from 10% of the aldrin dose at 2 hours to up to 50% at 24 hours. FARB RM et al.; Pesticides and the Environment: Ongoing Controversy, PAP International Congress on Occupational Medical Toxicology, 8th Session; 179 (1973)
Rainbow trout fed with ochratoxin A at 20 μg/kg of feed, along with cycloglutaric acid, developed liver cancer (quantity not specified). International Agency for Research on Cancer. Monographs on Risk Assessment of Carcinogenic Chemicals in Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972 to present. (Multi-volume). Accessible at: https://monographs.iarc.fr/ENG/Classification/index.php, page V10. 193 (1976)

[1]. Role of dopamine and selective dopamine receptor agonists on mouse ductus arteriosus tone and responsiveness. Pediatr Res. 2019 Dec 9.

[2]. First synthesis of a stable isotope of Ochratoxin A metabolite for a reliable detoxification monitoring. Org Lett. 2013 Aug 2;15(15):3888-90.

[3]. Ochratoxin A induces JNK activation and apoptosis in MDCK-C7 cells at nanomolar concentrations. J Pharmacol Exp Ther . 2000 Jun;293(3):837-44.

[4]. Alterations in ATP-dependent calcium uptake by rat renal cortex microsomes following ochratoxin A administration in vivo or addition in vitro. Biochem Pharmacol. 1992 Oct 6;44(7):1401-9.

[5]. Total synthesis and cytotoxicity evaluation of all ochratoxin A stereoisomers. Bioorg Med Chem. 2010 Jan 1;18(1):343-7.

[6]. Impact of Bioactive Ingredients on the Fecal Excretion of Aflatoxin B1 and Ochratoxin A in Wistar Rats. Molecules. 2025 Feb 1;30(3):647.

It has been reported that (R)N-(5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)phenylalanine is present in Aspergillus ochraceus. Ochratoxin A (OTA) has been a subject of concern for decades due to its toxicity and presence in a variety of foods. This paper summarizes the first synthesis of a labeled ochratoxin α (OTα) analog, one of the major products of microbial metabolism of OTA. The synthesis also yielded a novel labeled OTA analog with a deuterated group located in the dihydroisocoumarin moiety, which allows for precise quantification of OTA and OTα and the establishment of reliable detoxification rates. [2] Ochratoxin A is a potent inhibitor of protein biosynthesis and is known to be cytotoxic at nanomolar concentrations. To investigate the relationship between the stereochemistry and cytotoxicity of this compound, we synthesized all four stereoisomers of ochratoxin A. Using the Hep G2 hepatocyte cell line, we tested the cytotoxicity and apoptosis-inducing ability of these compounds. The results showed that the L-configuration of the phenylalanine moiety in the molecule was the main reason for the high cytotoxicity of ochratoxin A, while the stereocenter on the dihydroisocoumarin structure was relatively less important. [5] This study highlights the potential of bioactive compounds such as P and FW in regulating the excretion of fungitoxins, particularly aflatoxin B1 and ochratoxin A, in Wistar rats. The observed changes in toxin bioavailability may be due to alterations in absorption kinetics or enhanced excretion processes, which may be driven by mechanisms such as physical adsorption or biochemical interactions. Although the exact pathways remain to be elucidated, these findings open new avenues for using FW and P as dietary interventions to reduce fungal exposure. Further research is needed to deepen our understanding of these mechanisms and to evaluate the practical application of these compounds in mycotoxin detoxification strategies. Looking ahead, research should explore the effects of these dietary interventions on the gut microbiota, as changes in the microbial community may affect the body's metabolism and ability to clear mycotoxins, thereby reducing their toxicity. Understanding these interactions may provide innovative strategies for improving animal health and public safety. In summary, the findings suggest that dietary modifications, including the use of bioactive ingredients, may offer promising solutions for controlling mycotoxin contamination in agricultural systems and could become a key strategy for protecting food production and public health. [6]

C20H18NO6CL

403.81302

407.107

C, 59.49 H, 4.49 Cl, 8.78 N, 3.47 O, 23.77

303-47-9

12313657

White to off-white solid powder

1.4±0.1 g/cm3

632.4±55.0 °C at 760 mmHg

336.3±31.5 °C

0.0±2.0 mmHg at 25°C

1.628

Aspergillus ochraceus

4.31

3

6

5

28

608

2

C[C@@H]1CC2=C(C(O1)=O)C(O)=C(C(N[C@H](C(O)=O)CC3=CC=CC=C3)=O)C=C2Cl

RWQKHEORZBHNRI-GENIYJEYSA-N

InChI=1S/C20H18ClNO6/c1-10-7-12-14(21)9-13(17(23)16(12)20(27)28-10)18(24)22-15(19(25)26)8-11-5-3-2-4-6-11/h2-6,9-10,15,23H,7-8H2,1H3,(H,22,24)(H,25,26)/t10?,15-/m1/s1 SMILES Code

(R)-N-((5-Chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-benzo(c)pyran-7-yl)carbonyl)-3-phenylalanine

Ochratoxin-A; Antibiotic 9663; Alanine, (-)-; 1448049-50-0; ((R)-5-chloro-8-hydroxy-3-(methyl-d3)-1-oxoisochromane-7-carbonyl-3-d)-L-phenylalanine; (R)N-(5-Chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)phenylalanine; WLN: T66 BVOT & J D1 GG IVMYVQ1R & JQ; NSC-201422; L-Phenylalanine,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)carbonyl]-, (R)-; EX-A1468; EX-A-1468; EX-A 1468; EXA1468; EXA-1468; EXA 1468

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.)