Aptamer Molecular Beacon Sensor for Rapid and Sensitive Detection of Ochratoxin A.

Ochratoxin A (OTA) is a carcinogenic fungal secondary metabolite which causes wide contamination in a variety of food stuffs and environments and has a high risk to human health. Developing a rapid and sensitive method for OTA detection is highly demanded in food safety, environment monitoring, and quality control. Here, we report a simple molecular aptamer beacon (MAB) sensor for rapid OTA detection. The anti-OTA aptamer has a fluorescein (FAM) labeled at the 5' end and a black hole quencher (BHQ1) labeled at the 3' end. The specific binding of OTA induced a conformational transition of the aptamer from a random coil to a duplex-quadruplex structure, which brought FAM and BHQ1 into spatial proximity causing fluorescence quenching. Under the optimized conditions, this aptamer sensor enabled OTA detection in a wide dynamic concentration range from 3.9 nM to 500 nM, and the detection limit was about 3.9 nM OTA. This method was selective for OTA detection and allowed to detect OTA spiked in diluted liquor and corn flour extraction samples, showing the capability for OTA analysis in practical applications.

Ochratoxin A in Slaughtered Pigs and Pork Products.

Ochratoxin A (OTA) is a mycotoxin that is produced after the growth of several Aspergillus and Penicillium spp. in feeds or foods. OTA has been proved to possess nephrotoxic, hepatotoxic, teratogenic, neurotoxic, genotoxic, carcinogenic and immunotoxic effects in animals and humans. OTA has been classified as possibly carcinogenic to humans (Group 2B) by the IARC in 2016. OTA can be mainly found in animals as a result of indirect transmission from naturally contaminated feed. OTA found in feed can also contaminate pigs and produced pork products. Additionally, the presence of OTA in pork meat products could be derived from the direct growth of OTA-producing fungi or the addition of contaminated materials such as contaminated spices. Studies accomplished in various countries have revealed that pork meat and pork meat products are important sources of chronic dietary exposure to OTA in humans. Various levels of OTA have been found in pork meat from slaughtered pigs in many countries, while OTA levels were particularly high in the blood serum and kidneys of pigs. Pork products made from pig blood or organs such as the kidney or liver have been often found to becontaminated with OTA. The European Union (EU) has established maximum levels (ML) for OTA in a variety of foods since 2006, but not for meat or pork products. However, the establishement of an ML for OTA in pork meat and meat by-products is necessary to protect human health.

Spent Coffee Grounds Valorization as Bioactive Phenolic Source Acquired Antifungal, Anti-Mycotoxigenic, and Anti-Cytotoxic Activities.

Spent coffee grounds (SCGs), which constitute 75% of original coffee beans, represent an integral part of sustainability. Contamination by toxigenic fungi and their mycotoxins is a hazard that threatens food production. This investigation aimed to examine SCGs extract as antimycotic and anti-ochratoxigenic material. The SCGs were extracted in an eco-friendly way using isopropanol. Bioactive molecules of the extract were determined using the UPLC apparatus. The cytotoxicity on liver cancer cells (Hep-G2) showed moderate activity with selectivity compared with human healthy oral epithelial (OEC) cell lines but still lower than the positive control (Cisplatin). The antibacterial properties were examined against pathogenic strains, and the antifungal was examined against toxigenic fungi using two diffusion assays. Extract potency was investigated by two simulated models, a liquid medium and a food model. The results of the extract showed 15 phenolic acids and 8 flavonoids. Rosmarinic and syringic acids were the most abundant phenolic acids, while apigenin-7-glucoside, naringin, epicatechin, and catechin were the predominant flavonoids in the SCGs extract. The results reflected the degradation efficiency of the extract against the growth of Aspergillus strains. The SCGs recorded detoxification in liquid media for aflatoxins (AFs) and ochratoxin A (OCA). The incubation time of the extract within dough spiked with OCA was affected up to 2 h, where cooking was not affected. Therefore, SCGs in food products could be applied to reduce the mycotoxin contamination of raw materials to the acceptable regulated limits.

Ochratoxin a levels in fermented specialty coffees from Caparaó, Brazil: Is it a cause of concern for coffee drinkers?

Although postharvest coffee fruit fermentation can improve coffee flavour and quality, the mycotoxin ochratoxin A (OTA) can also be a result of microbiological activity, albeit in the later drying step of coffee processing. To evaluate the possible occurrence of OTA contamination in postharvest fruit fermentation, fourteen coffees that entailed two different postharvest fruit fermentation times were evaluated. These coffees originated in the surroundings of the village of Pedra Menina in the qualified Denomination of Origin and coffee producer region of Caparaó on the border between Minas Gerais and Espírito Santo states in Brazil. All coffees were classified according to the Specialty Coffee Association (SCA) protocol and 12 achieved specialty level. OTA was determined in all 14 coffees using immunoaffinity for sample clean-up and high-performance liquid chromatography with fluorescence detection for quantification. One sample presented an OTA concentration of 0.75 µg kg-1 and two samples showed OTA concentrations of 0.87 µg kg-1. The other samples had concentrations of OTA below the limit of quantification obtained in this work (0.64 µg kg-1). Thus, all samples showed OTA concentrations far below the most stringent maximum residue limit (MRL) of 5 µg kg-1 established for roasted coffees by European legislation. These low levels were similar to most of the previous results for Brazilian coffees listed and tabled in this work. This comparison showed that OTA contamination due to this kind of postharvest process - fruit fermentation - should not be a concern for producers and consumers of these fermented coffees.

Ochratoxin A and 2'R-Ochratoxin A in Selected Foodstuffs and Dietary Risk Assessment.

The aim of this study was to estimate the contamination of grain coffee, roasted coffee, instant coffee, and cocoa purchased in local markets with ochratoxin A (OTA) and its isomerization product 2'R-ochratoxin A (2'R-OTA), and to assess risk of dietary exposure to the mycotoxins. OTA and 2'R-OTA content was determined using the HPLC chromatography with immunoaffinity columns dedicated to OTA. OTA levels found in all the tested samples were below the maximum limits specified in the European Commission Regulation EC 1881/2006. Average OTA concentrations calculated for positive samples of grain coffee/roasted coffee/instant coffee/cocoa were 0.94/0.79/3.00/0.95 µg/kg, with the concentration ranges: 0.57-1.97/0.44-2.29/0.40-5.15/0.48-1.97 µg/kg, respectively. Average 2'R-OTA concentrations calculated for positive samples of roasted coffee/instant coffee were 0.90/1.48 µg/kg, with concentration ranges: 0.40-1.26/1.00-2.12 µg/kg, respectively. In turn, diastereomer was not found in any of the tested cocoa samples. Daily intake of both mycotoxins with coffee/cocoa would be below the TDI value even if the consumed coffee/cocoa were contaminated with OTA/2'R-OTA at the highest levels found in this study. Up to now only a few papers on both OTA and 2'R-OTA in roasted food products are available in the literature, and this is the first study in Poland.

Dechlorination and demethylation of ochratoxin A enhance blocking activity of PXR activation, suppress PXR expression and reduce cytotoxicity.

The pregnane X receptor (PXR) has been established to induce chemoresistance and metabolic diseases. Ochratoxin A (OTA), a mycotoxin, decreases the expression of PXR protein in human primary hepatocytes. OTA is chlorinated and has a methylated lactone ring. Both structures are associated with OTA toxicity. The study was to test the hypothesis that structural modifications differentially impact PXR blocking activity over cytotoxicity. To test this hypothesis, OTA-M and OTA-Cl/M were synthesized. OTA-M lacked the methyl group of the lactone-ring, whereas OTA-Cl/M had neither the methyl group nor the chlorine atom. The blocking activity of PXR activation was determined in a stable cell line, harboring both PXR (coding sequence) and its luciferase element reporter. OTA-Cl/M showed the highest blocking activity, followed by OTA-M and OTA. OTA-Cl/M was 60 times as potent as the common PXR blocker ketoconazole based on calculated IC50 values. OTA-Cl/M decreased by 90 % the expression of PXR protein and was the least cytotoxic among the tested compounds. Molecular docking identified that OTA and its derivatives interacted with different sets of residues in PXR, providing a molecular basis for selectivity. Excessive activation of PXR has been implicated in chemoresistance and metabolic diseases. Downregulation of PXR protein expression likely delivers an effective mechanism against structurally diverse PXR agonists.

Trial for reduction of Ochratoxin A residues in fish feed by using nano particles of hydrated sodium aluminum silicates (NPsHSCAS) and copper oxide.

This paper was designed to analyze the effect of ochratoxin A (OTA) contaminated feed on the growth outcomes, certain serum biochemical, histopathology, and OTA residue in the dorsal muscle, liver, and kidney in Nile tilapia. Also, to improve the drastic effect of OTA through dietary supplementation of hydrated sodium aluminum silicates nanoparticles or nano copper. For performing the present study, 270 fish were randomly allotted into 6 equal groups according to ochratoxin and nanoparticles of hydrated sodium aluminum silicates or copper oxide. The results indicated that supplementation of two levels of both nanoparticles (aluminum silicate or copper) as a mycotoxin adsorbent could prevent ochratoxicosis in Nile tilapia fish. In addition, they maintained optimal growth performance, feed efficiency without bad effect on serum profiles and vital organs function of fish in a dose-dependent manner. Histopathologically, the most interesting finding was the precipitation of calcium salts known as nephrocalcinosis, within the tubules, upon the degenerative tubules and tunica intima and media of the blood vessels in the control positive group. These pathological lesions were mitigated by nanoparticle supplementation. Thus increase the safety of fish products.

An Exonuclease I-Assisted Silver-Metallized Electrochemical Aptasensor for Ochratoxin A Detection.

Ochratoxin A (OTA)-a mycotoxin produced by Aspergillus and Penicillium fungi-is a carcinogen and common trace contaminant in agricultural and processed food products. As consumption is detrimental to human and animal health, regular product monitoring is vital, and highly sensitive and portable OTA sensors are necessary in many circumstances. Herein, we report an ultrasensitive, electroanalytical aptasensor for precise determination of OTA at trace levels. The sensor leverages a DNA aptamer to capture OTA and silver metallization as a signal enhancer. Exonuclease I is used to digest unbound aptamers, engendering excellent background signal suppression and sensitivity enhancements. Efficient optimization of assay conditions is achieved using central composite design (CCD), allowing rapid evaluation of both the electrode and square wave voltammetry parameter space. The sensor exhibits excellent analytical performance, with a concentration limit of detection of 0.7 pg mL-1, a limit of quantitation of 2.48 pg mL-1, and a linear dynamic range ( R2 = 0.968) of over 6 orders of magnitude (between 1 pg mL-1 and 0.1 µg mL-1). Direct comparison with ultraperformance liquid chromatography (UPLC) indicates excellent analytical performance for standard solutions ( R2 = 0.995) and spiked beer samples ( R2 = 0.993), with almost quantitative recovery and less than 5% relative standard deviation (RSD).

Aptamer-Structure Switch Coupled with Horseradish Peroxidase Labeling on a Microplate for the Sensitive Detection of Small Molecules.

Detection of small molecules with good sensitivity, high throughput, simplicity, and generality using aptamers is desired but still remains challenging. We described an aptamer-structure-switch assay coupled with horseradish peroxidase (HRP) labeling on microplates for sensitive absorbance and chemiluminescence detection of small molecules. This assay relies on competition for affinity binding to a limited HRP-labeled aptamer between small-molecule targets and immobilized short DNA strands complementary to the aptamer (cDNA) on a microplate. In the absence of targets, the HRP-labeled aptamer hybridizes with the cDNA on the microplate, and HRP catalyzes substrate into product, generating absorbance or chemiluminescence signals. The binding of small-molecule targets to aptamers causes displacement of HRP-labeled aptamers from the cDNA and signal decrease. In chemiluminescence-analysis mode, the assay achieved detection of aflatoxin B1 (AFB1), ochratoxin A (OTA), and adenosine triphosphate (ATP) with detection limits of 10 pM, 20 pM, and 20 nM, respectively. This assay does not require enzyme-labeled small molecules or the conjugation of small molecules on solid phase. HRP, as an enzyme label, here allows for easily obtainable and highly active signal amplification. This microplate assay is rapid and promising for high-throughput analysis. It shows potential for wide applications in the detection of small molecules.

Effects of curcumin on performance, antioxidation, intestinal barrier and mitochondrial function in ducks fed corn contaminated with ochratoxin A.

Curcumin has been attributed with antioxidant, anti-inflammatory, antibacterial activities, and has shown highly protective effects against enteropathogenic bacteria and mycotoxins. Ochratoxin A (OTA) is one of the major intestinal pathogenic mycotoxins. The possible effect of curcumin on the alleviation of enterotoxicity induced by OTA is unknown. The effects of dietary curcumin supplementation on OTA-induced oxidative stress, intestinal barrier and mitochondrial dysfunctions were examined in young ducks. A total of 540 mixed-sex 1-day-old White Pekin ducklings with initial BW (43.4±0.1 g) were randomly assigned into controls (fed only the basal diet), a group fed an OTA-contaminated diet (2 mg/kg feed), and a group fed the same OTA-contaminated feed plus 400 mg/kg of curcumin. Each treatment consisted of six replicates, each containing 30 ducklings and treatment lasted for 21 days. There was a significant decrease in average daily gain (ADG) and increased feed : gain caused by OTA (P<0.05); curcumin co-treatment prevented the decrease in BW and ADG compared with the OTA group (P<0.05). Histopathological and ultrastructural examination showed clear signs of enterotoxicity caused by OTA, but these changes were largely prevented by curcumin supplementation. Curcumin decreased the concentrations of interleukin-1ß, tumor necrosis factor-α and malondialdehyde, and increased the activity of glutathione peroxidase induced by OTA in the jejunal mucosa of ducks (P<0.05). Additionally, curcumin increased jejunal mucosa occludin and tight junction protein 1 mRNA and protein levels, and decreased those of ρ-associated protein kinase 1 (P<0.05). Notably, curcumin inhibited the increased expression of apoptosis-related genes, and downregulated mitochondrial transcription factors A, B1 and B2 caused by OTA without any effects on RNA polymerase mitochondrial (P<0.05). These results indicated that curcumin could protect ducks from OTA-induced impairment of intestinal barrier function and mitochondrial integrity.

Complexes of the Mycotoxins Citrinin and Ochratoxin A with Aluminum Ions and their Spectroscopic Properties.

The sensitive detection of the mycotoxin citrinin (CIT) utilizing its fluorescence requires approaches to enhance the emission. In this respect, we studied the complexation of CIT and ochratoxin A (OTA) with Al3+ in methanol using absorption and fluorescence spectroscopy. In this context, an isocratic high performance liquid chromatography (HPLC) method using a polymer column and a fluorescence detector was also developed that enables the separation of the metal ion complexes from the free ligands and non-complexed Al3+. CIT and OTA showed distinct changes in their absorption and fluorescence properties upon Al3+-coordination, and the fluorescence of CIT was considerably enhanced. Analysis of the photometrically assessed titration of CIT and OTA with Al3+ using the Job plot method revealed 1:2 and 1:1 stoichiometries for the Al3+ complexes of CIT (Al:CIT) and OTA (Al:OTA), respectively. In the case of CIT, only one ß-diketone moiety participates in Al3+ coordination. These findings can be elegantly exploited for signal amplification and provide the base to reduce the limit of detection for CIT quantification by about an order of magnitude, as revealed by HPLC measurements using a fluorescence detector.

Performance of purified grape pomace as a fining agent to reduce the levels of some contaminants from wine.

The quality of red wine depends on the absence of compounds which may affect its safety and/or stability such as ochratoxin A, biogenic amines and some metals and trace compounds. The presence of ochratoxin A in musts and wines is due to fungal contamination of the grapes and has been classified as a possible human carcinogen. Biogenic amines are formed by the microbiological decarboxylation of the corresponding amino acid precursors during the fermentation or ageing and storage, and, at high concentrations, they may induce adverse reactions in sensitive people. Trace elements may have both a nutritional and a toxic effect on health, but also can cause turbidity and stability problems. Their presence is affected mainly by natural factors such as soil mineral content and direct contact with tank surfaces and metallic tubing during winemaking. One of the best options to remove these compounds when present in excess in wine is fining. However, some fining agents commonly used may themselves present problems related with their allergenic properties or with their propensity to increase the protein content, which can cause turbidity problems. In an attempt to avoid such these problems, purified grape pomace was tested as a fining alternative since it has been seen to have a high capacity to reduce the astringency, turbidity and also the ochratoxin A content. The main aim of this work, therefore, was to study if this material can limit the presence of ochratoxin A, biogenic amines and metals and some trace elements in a Monastrell red wine, thus increasing the value and safety of this product.

Ochratoxin A: Toxicity, oxidative stress and metabolism.

Ochratoxin A (OTA) is a widespread mycotoxin commonly found as a corn contaminant. It has been shown to be nephrotoxic, hepatotoxic, teratogenic and immunotoxic to several species of animals and to cause kidney and liver tumors in mice and rats. The focus of this article is primarily intended to summarize the progress in research associated with oxidative stress as a plausible mechanism for OTA-induced toxicity as well as its metabolism. The present review shows that studies have been carried out for decades to elucidate the production of reactive oxygen species (ROS) and oxidative stress as a result of OTA treatment and have correlated them with various types of OTA toxicity, indicating that oxidative stress plays critical roles in the toxicity of OTA. The major metabolic pathways of OTA are hydrolysis and a small percentage of absorbed OTA is hydroxylated. CYP450, carboxypeptidase A, trypsin, α-chymotrypsin and cathepsin have been shown to be able to degrade OTA. Most metabolites of OTA are less toxic than OTA except OP-OTA. Further understanding of the role of oxidative stress in OTA-induced toxicity will throw new light on the use of antioxidants, scavengers of ROS, as well as on the blind spots of the metabolism and metabolic enzymes of OTA. The present review might contribute to reveal the oxidative stress-induced toxicity of OTA and help to protect against its oxidative damage.

Trienic α-pyrone and ochratoxin derivatives from a sponge-derived fungus Aspergillus ochraceopetaliformis.

A new trienic α-pyrone derivative asteltoxin G (1) bearing a tetrahydrofuran ring and a new ochratoxin derivative named ochratoxin A1 (5), along with seven known compounds, were isolated from a sponge-derived fungus Aspergillus ochraceopetaliformis. The compounds (1-9) were evaluated on the basis of spectroscopic analyses and comparison with those of the reported data. The new compound ochratoxin A1 (5) exhibited anti-inflammatory activity against IL-6 and TNF-α expression of the LPS-induced THP-1 cells with inhibitory rates of 74.4 and 67.7% at concentration of 10 µM, respectively.

Mutagenicity of Ochratoxin A: Role for a Carbon-Linked C8-Deoxyguanosine Adduct?

Ochratoxin A (OTA) is a fungal toxin that is considered to be a potent kidney carcinogen in rodent models. The toxin produces double strand breaks and has a propensity for deletions, single-base substitutions, and insertions. The toxin reacts covalently with DNA to afford a C8-2'-deoxyguanosine carbon-linked adduct (OT-dG) as the major lesion in animal tissues. Incorporation of model C-linked C8-aryl-dG adducts into the G3 site of the NarI sequence demonstrates a tendency to induce base substitutions and deletion mutations in primer extension assays using model polymerases. The degree of misincorporation induced by the C-linked C8-dG adducts correlates with an ability to adopt the promutagenic syn conformation within the NarI duplex as predicted by molecular dynamics (MD) simulations. MD simulations of the OT-dG adduct within the NarI duplex predict an even greater degree of conformational flexibility, suggesting enhanced in vitro mutagenicity compared to the simpler model C-linked C8-dG adducts. Together these findings support the role of OT-dG in promoting OTA-mediated mutagenicity and carcinogenicity in animal studies.

Ultrasensitive detection of ochratoxin A using aptasensors.

Regarding teratogenic, carcinogenic, and immunotoxic nature of ochratoxin A (OTA), selective and sensitive monitoring of this molecule in food samples is of great importance. In recent years, various methods have been introduced for detection of OTA. However, they are usually time-consuming, labor-intensive and expensive. Therefore, these parameters limited their usage. The emerging method of detection, aptasensor, has attracted more attention for OTA detection, due to distinctive advantages including high sensitivity, selectivity and simplicity. In this review, the new developed aptasensors for detection of OTA have been investigated. We also highlighted advantages and disadvantages of different types of OTA aptasensors. This review also takes into consideration the goal to find out which designs are the most rational ones for highly sensitive detection of OTA.

Molecular Modeling of the Major DNA Adduct Formed from Food Mutagen Ochratoxin A in NarI Two-Base Deletion Duplexes: Impact of Sequence Context and Adduct Ionization on Conformational Preference and Mutagenicity.

Exposure to ochratoxin A (OTA), a possible human carcinogen, leads to many different DNA mutations. As a first step toward understanding the structural basis of OTA-induced mutagenicity, the present work uses a robust computational approach and a slipped mutagenic intermediate model previously studied for C8-dG aromatic amine adducts to analyze the conformational features of postreplication two-base deletion DNA duplexes containing OT-dG, the major OTA lesion at the C8 position of guanine. Specifically, a total of 960 ns of molecular dynamics simulations (excluding trial simulations) were carried out on four OT-dG ionization states in three sequence contexts within oligomers containing the NarI recognition sequence, a known hotspot for deletion mutations induced by related adducts formed from known carcinogens. Our results indicate that the structural properties and relative stability of the competing "major groove" and "stacked" conformations of OTA adducted two-base deletion duplexes depend on both the OTA ionization state and the sequence context, mainly due to conformation-dependent deviations in discrete local (hydrogen-bonding and stacking) interactions at the lesion site, as well as DNA bending. When the structural characteristics of the OT-dG adducted two-base deletion duplexes are compared to those associated with previously studied C8-dG adducts, a greater understanding of the effects of the nucleobase-carcinogen linkage, and size of the carcinogenic moiety on the conformational preferences of damaged DNA is obtained. Most importantly, our work predicts key structural features for OT-dG-adducted deletion DNA duplexes, which in turn allow us to develop hypotheses regarding OT-dG replication outcomes. Thus, our computational results are valuable for the design and interpretation of future biochemical studies on the potentially carcinogenic OT-dG lesion.

Evaluation of Ochratoxin Recognition by Peptides Using Explicit Solvent Molecular Dynamics.

Biosensing platforms based on peptide recognition provide a cost-effective and stable alternative to antibody-based capture and discrimination of ochratoxin-A (OTA) vs. ochratoxin-B (OTB) in monitoring bioassays. Attempts to engineer peptides with improved recognition efficacy require thorough structural and thermodynamic characterization of the binding-competent conformations. Classical molecular dynamics (MD) approaches alone do not provide a thorough assessment of a peptide's recognition efficacy. In this study, in-solution binding properties of four different peptides, a hexamer (SNLHPK), an octamer (CSIVEDGK), NFO4 (VYMNRKYYKCCK), and a 13-mer (GPAGIDGPAGIRC), which were previously generated for OTA-specific recognition, were evaluated using an advanced MD simulation approach involving accelerated configurational search and predictive modeling. Peptide configurations relevant to ochratoxin binding were initially generated using biased exchange metadynamics and the dynamic properties associated with the in-solution peptide-ochratoxin binding were derived from Markov State Models. Among the various peptides, NFO4 shows superior in-solution OTA sensing and also shows superior selectivity for OTA vs. OTB due to the lower penalty associated with solvating its bound complex. Advanced MD approaches provide structural and energetic insights critical to the hapten-specific recognition to aid the engineering of peptides with better sensing efficacies.

Investigation of the Importance of Multidrug Resistance-Associated Protein 4 (Mrp4/Abcc4) in the Active Efflux of Anionic Drugs Across the Blood-Brain Barrier.

The importance of multidrug resistance-associated protein 4 (Mrp4/Abcc4) in limiting the penetration of Mrp4 substrate compounds into the central nervous system across the blood-brain barrier was investigated using Mrp4-/- mice. Significant adenosine triphosphate-dependent uptake by MRP4 was observed for ochratoxin A, pitavastatin, raltitrexed (Km = 43.7 µM), pravastatin, cyclic guanosine monophosphate, 2,4-dichlorophenoxyacetate, and urate. The defect in the Mrp4 gene did not affect the brain-to-plasma ratio (Kp,brain) of quinidine and dantrolene. Following intravenous infusion in wild-type and Mrp4-/- mice, the plasma concentrations of the tested compounds (cefazolin, cefmetazole, ciprofloxacin, cyclophosphamide, furosemide, hydrochlorothiazide, methotrexate, pitavastatin, pravastatin, and raltitrexed) were identical; however, Mrp4-/- mice showed a significantly higher (1.9- to 2.5-fold) Kp,brain than wild-type mice for methotrexate, raltitrexed, and cyclophosphamide. GF120918, a dual inhibitor of P-gp and Bcrp, significantly decreased Kp,cortex and Kp,cerebellum only in Mrp4-/- mice. Methotrexate and raltitrexed are also substrates of multispecific organic anion transporters such as Oatp1a4 and Oat3. GF120918 showed an inhibition potency against Oatp1a4, but not against Oat3. These results suggest that Mrp4 limits the penetration of methotrexate and raltitrexed into the brain across the blood-brain barrier, which is likely to be facilitated by some uptake transporters.

Assessing interactions of binary mixtures of Penicillium mycotoxins (PMs) by using a bovine macrophage cell line (BoMacs).

Penicillium mycotoxins (PMs) are toxic contaminants commonly found as mixtures in animal feed. Therefore, it is important to investigate potential joint toxicity of PM mixtures. In the present study, we assessed the joint effect of binary combinations of the following PMs: citrinin (CIT), ochratoxin A (OTA), patulin (PAT), mycophenolic acid (MPA) and penicillic acid (PA) using independent action (IA) and concentration addition (CA) concepts. Previously published toxicity data (i.e. IC25; PM concentration that inhibited bovine macrophage (BoMacs) proliferation by 25%) were initially analyzed, and both concepts agreed that OTA+PA demonstrated synergism (p<0.05), while PAT+PA showed antagonism (p<0.05). When a follow-up dilution study was carried out using binary combinations of PMs at three different dilution levels (i.e. IC25, 0.5∗IC25, 0.25∗IC25), only the mixture of CIT+OTA at 0.5∗IC25 was determined to have synergism by both IA and CA concepts with Model Deviation Ratios (MDRs; the ratio of predicted versus observed effect concentrations) of 1.4 and 1.7, respectively. The joint effect of OTA+MPA, OTA+PA and CIT+PAT complied with the IA concept, while CIT+PA, PAT+MPA and PAT+PA were better predicted with the CA over the IA concept. The present study suggests to test both IA and CA concepts using multiple doses when assessing risk of mycotoxin mixtures if the mode of action is unknown. In addition, the study showed that the tested PMs could be predicted by IA or CA within an approximate two-fold certainty, raising the possibility for a joint risk assessment of mycotoxins in food and feed.