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 there are pathomorphological similarities between porcine mycotoxic nephropathy caused by ochratoxin A and Balkan endemic nephropathy (BEN), it has been suggested that the same aetiological agent has a role in BEN. Based on the results from several field and experimental studies carried out on pigs, an appropriate analytical method of monitoring possible human exposure to ochratoxin A was developed. The toxicokinetic properties of the toxin were species specific, although in all the animal species studied (with the exception of fish), as well as in humans, two binding proteins were found in the plasma. The monkey had the longest elimination half-life of the toxin, 510 hr, in contrast to the fish whose elimination half-life was only 0.68 hr. The fish kidney displayed a specific pattern of distribution. In the laying quail the most prominent observation was the accumulation of labelled ochratoxin A in egg yolk. Generally, (14C)ochratoxin A was eliminated rapidly from the quail body, but had a long retention time in the circulating blood in the mouse. Although the elimination of ochratoxin A from the body depending on its binding to plasma constituents, the existence of enterohepatic circulation might have been partially responsible for its prolonged retention and elimination from the body of mammals. The toxicokinetic profile of ochratoxin A did not contradict the mycotoxic hypothesis in the aetiology of BEN. PMID:1618443 Fuchs R, Hult K; Food Chem Toxicol 30 (3): 201-4 (1992)

... Ochratoxin A is rapidly absorbed throughout the entire gastrointestinal tract and distributes itself in the body as a two compartment open model and has a particular high affinity for serum albumin. Ochratoxin A is hydrolyzed by the intestinal microflora into nontoxic compounds (7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin (Ochratoxin alpha) and phenylalanine). It is excreted as either ochratoxin A, hydroxylated ochratoxin A or Ochratoxin alpha in both the urine and feces. Ochratoxin A appears to exert its toxic effect by promoting an increased level of lipid peroxidation by inhibition of an amino acylation reaction and possibly by conversion into metabolites that are capable of binding DNA. These in turn cause other secondary effects associated with ochratoxin A. It would appear that this compound presents a true potential hazard for humans as its occurrence is wide spread and it is highly carcinogenic. PMID:2200593 Marquardt RR et al; Can J Physiol Pharmacol 68 (7): 991-9 (1990)

Rats intubated daily with 500 ug ochratoxin A or fed 250 ug daily in barley. There was little accumulation of cmpd in liver or kidneys. Avg total amount excreted daily in urine & feces was just over 10% of administered dose. Small amount of hydrolysis product also excreted. VAN WALBEEK W ET AL; TOXICOL APPL PHARMACOL 20 (3): 439 (1971)

Rats given single ip injection of 1 mg ochratoxin A labelled with (14)C. Reached highest levels in serum (90%), liver (4.5%), & kidney (4.4%) 30 min later. Ochratoxin A was excreted primarily in urine as unchanged toxin or metabolites. Excretion in feces less significant. PMID:892675 CHANG FC, CHU FS; FOOD COSMET TOXICOL 15 (3): 199 (1977)

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Metabolism / Metabolites ... Ochratoxin A is hydrolyzed by the intestinal microflora into nontoxic compounds (7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin (Ochratoxin alpha) and phenylalanine). It is excreted as either ochratoxin A, hydroxylated ochratoxin A or Ochratoxin alpha in both the urine and feces. ... PMID:2200593 Marquardt RR et al; Can J Physiol Pharmacol 68 (7): 991-9 (1990)

Hydroxyochratoxin A was isolated & identified from urine of rats after injection with ochratoxin A. By incubating ochratoxin A with rat liver microsomes & NADPH, 1 major (90%) & 2 minor metabolites, more polar than ochratoxin A, were formed. PMID:7396488 Full text: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC291461 STORMER FC, PEDERSEN JI; APPL ENVIRON MICROBIOL 39 (5): 971 (1980)

Single oral or iv dose (2.5 mg/kg) ochratoxin A admin to healthy adult rats. Ochratoxin alpha only metabolite recovered from cecum & large intestine. Ochratoxin A excreted via urine & feces, both as free drug & ochratoxin alpha. Unidentified metabolites in urine. PMID:43233 GALTIER P ET AL; DRUG METAB DISPOS 7 (6): 429 (1979)

Ochratoxin A is cleaved into phenylalanine and a less toxic iso-coumarin derivative (ochratoxin alpha) by the microbial flora of the colon ... and by carboxypeptidase A and alpha-chymotrypsin ... . IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at:

Ochratoxin A is cleaved into phenylalanine and a less toxic iso-coumarin derivative (ochratoxin alpha) by the microbial flora of the colon, and by carboxypeptidase A and alpha-chymotrypsim. This is is the major metabolic pathway. 4-Hydroxyochratoxin A is the main hepatic metabolite and its formation appears to be via a polymorphic-like debrisoquine 4-hydroxylation. Some cytochrome P-450 enzymes, such as CYP2C9, and known to metabolize ochratoxin A into more cytotoxic compounds. (T35, A2870, A3099)

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Biological Half-Life Pregnant ICR mice were administered a single ip injection of 5 mg/kg ochratoxin A (OA) on day 11 or 13 of gestation. The half-life of OA in serum was calculated to be 28.7 hr on day 11 and 23.6 hr on day 13 of gestation. Fukui Y et al; Fd Chem Toxic 25: 17-24 (1987)

In rats ... the plasma half-life of ochratoxin A is about 60 hr ... . IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V31 198 (1983)

The apparent plasma elimination half-time of ochratoxin A after oral administration at 50 ug/kg bw varied from 0.68 hr in fish to 120 hr in rats and 510 hr in monkeys ... . IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. V56 499 (1993)

The fate of ochratoxin A has been studied in laboratory rodents and in breeding animals. In rats, orally administered ochratoxin A is readily absorbed, and considerable amounts of the toxin are detected in plasma, where maximal concentrations occur 2-4 hr after administration. Pharmacokinetic analysis of curves of plasma level versus time suggests its distribution in two distinct body compartments. The half-time of the toxin depends on both the dose and the animal species, varying from 0.7 hr in fish to 840 h in monkeys. In plasma, the toxin is bound to albumin, like many acidic compounds. This interaction is competitively inhibited by phenylbutazone, ethylbiscoumacetate and sulfamethoxy-pyridazine and is decreased in albumin-deficient rats. Galtier P; IARC Sci Publ (115): 187-200 (1991)

The toxicokinetic properties of the toxin were species specific, although in all the animal species studied (with the exception of fish), as well as in humans, two binding proteins were found in the plasma. The monkey had the longest elimination half-life of the toxin, 510 hr, in contrast to the fish whose elimination half-life was only 0.68 hr. The fish kidney displayed a specific pattern of distribution. In the laying quail, the most prominent observation was the accumulation of labelled ochratoxin A in egg yolk. Generally, (14)C-ochratoxin A was eliminated rapidly from the quail body, but had a long retention time in the circulating blood in the mouse. Although the elimination of ochratoxin A from the body depending on its binding to plasma constituents, the existence of enterohepatic circulation might have been partially responsible for its prolonged retention and elimination from the body of mammals. The toxicokinetic profile of ochratoxin A did not contradict the mycotoxic 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 has known human metabolites that include 2-[(5-Chloro-4,8-dihydroxy-3-methyl-1-oxo-3,4-dihydroisochromene-7-carbonyl)amino]-3-phenylpropanoic acid.

Toxicity Summary
Ochratoxin A has been shown to be weakly mutagenic, possibly by induction of oxidative DNA damage. The nephrotoxin ochratoxin A (OTA) causes a reduction of glomerular filtration rate (GFR) and of para-aminohippuric acid (PAH) clearance. It is a nephrotoxin which blocks plasma membrane anion conductance in Madin-Darby canine kidney (MDCK) cells. Some cytochrome P-450 enzymes, such as CYP2C9, are known to metabolize ochratoxin A into more cytotoxic compounds capable of forming DNA adducts. (A2869, A3099)

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Evidence for Carcinogenicity
Evaluation: There is inadequate evidence in humans for the carcinogenicity of ochratoxin A. There is sufficient evidence in experimental animals for the carcinogenicity of ochratoxin A. Overall evaluation: Ochratoxin A is possibly carcinogenic to humans (Group 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
Care is symptomatic and supportive. (A704)

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Interactions
... Ochratoxin A (OA) toxicity and the effect of supplemental ascorbic acid (AA) /was examined/ in laying hens housed under two environmental temperatures. /Two groups of/ ... 24 hens were randomly assigned to four dietary treatments in six replications. Treatments consisted of a control and three diets containing either 300 ppm AA, 3 ppm OA, or 300 ppm AA plus 3 ppm OA. Treatment diets were fed for 14 days following the feeding of the basal diet for 14 days. The test period temperature was 25 °C ... /for the first group/ and 33 °C in ... /the other group/. ... When laying hens were fed 3 ppm OA compared with those fed the control diet/, there were significant reductions in feed intake, body weight change, and egg production, and increased shell elasticity/. An analysis of plasma constituents showed that OA also increased Cl- concn and aspartate transaminase activity and decreased plasma calcium concentrations. Exposing hens to 33 °C (compared with 25 °C) appeared to aggravate the negative effects of OA. All the negative effects of OA, apart from body-weight changes, reductions in feed intake, and increases in egg shell elasticity at 33 °C were either moderated or significantly ... reversed by dietary AA supplementation. ... The results /indicate/ that the detrimental effects of OA in the diet of the laying hen can be counteracted by dietary /administration/ of AA. Haazele FM et al; Can J Anim Sci 73 (1): 149-57 (1993)

Aldrin concentration increased in liver of neonatal rats during 1st 6 hr after oral administration then decreased over 72 hr to less than 0.1% dose. Aldrin and ochratoxin given together; aldrin increased 1st 6 hr then decreased to 0.4% dose over 18 hr. FARB RM ET AL; PESTIC ENVIRON: CONTINUING CONTROVERSY, PAP INTER-AM CONF TOXICOL OCCUP MED, 8TH; 179 (1973)

Dieldrin, detected 2 hr after administration of aldrin to neonatal rats, increased to max 30% of initial aldrin by 18 hr. Aldrin & ochratoxin given together; dieldrin increased from 10% of aldrin dose at 2 hr to max 50% at 24 hr. FARB RM ET AL; PESTIC ENVIRON: CONTINUING CONTROVERSY, PAP INTER-AM CONF TOXICOL OCCUP MED, 8TH; 179 (1973)

Rainbow trout fed a diet containing 20 ug ochratoxin A/kg of diet; together with sterculic acid, developed hepatomas (number unspecified). IARC. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Geneva: World Health Organization, International Agency for Research on Cancer, 1972-PRESENT. (Multivolume work). Available at: https://monographs.iarc.fr/ENG/Classification/index.php, p. 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.

(R)N-(5-Chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)phenylalanine has been reported in Aspergillus ochraceus.

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Due to its toxicity and presence in numerous food products, Ochratoxin A (OTA) has drawn attention for decades. This article summarizes the first synthesis of a labeled analogue of Ochratoxin α (OTα), one of the main products generated by the metabolization of OTA by microorganisms. This synthesis also led to a new labeled analogue of OTA with the deuteration located on the dihydroisocoumarin moiety allowing thus both the accurate quantification of OTA and OTα and the establishing of a reliable detoxification rate.[2]

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The mycotoxin ochratoxin A is a potent inhibitor of the protein biosynthesis and known to be cytotoxic in nanomolar concentrations. In order to investigate the relationship between stereochemistry and cytotoxicity of this compound, all four ochratoxin A stereoisomers have been synthesized. Using the liver cell line Hep G2, the compounds were tested for cytotoxic and apoptotic potential. It could be shown, that the l-configuration of the phenylalanine moiety of the molecule is mostly responsible for the high cytotoxicity of ochratoxin A while the stereocenter at the dihydroisocoumarine structure is of less importance.[5]

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This study highlights the potential of bioactive compounds like P and FW in modulating the excretion of mycotoxins, specifically AFB1 and OTA, in Wistar rats. The observed effects on toxin bioavailability may result from altered absorption dynamics or enhanced excretion processes, potentially driven by mechanisms like physical adsorption or biochemical interactions. While the exact pathways remain to be clarified, these findings open new avenues for using FW and P as dietary interventions to mitigate mycotoxin exposure. Further research is needed to deepen our understanding of these mechanisms and to assess the practical applications of these compounds in mycotoxin detoxification strategies. Moving forward, research should explore the impact of these dietary interventions on gut microbiota, as shifts in the microbial community may influence the body’s ability to metabolize and eliminate mycotoxins, thereby reducing their toxic effects. Understanding these interactions may provide innovative strategies for improving animal health and public safety. In conclusion, the results suggest that dietary modifications, including the use of bioactive ingredients, could offer promising solutions for managing mycotoxin contamination in agricultural systems and may serve as a crucial strategy to safeguard both 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.)