Ochratoxin A

Magnetic microspheres-based cytometric bead array assay for highly sensitive detection of ochratoxin A.[Pubmed:28334625]

Biosens Bioelectron. 2017 Aug 15;94:420-428.

Accurate and sensitive quantification of a specific class of mycotoxins at trace levels in complex matrices with greener approaches is of significant importance. In this study, a green and economical protocol of magnetic microspheres-based cytometric bead array (CBA) assay on indirect competitive principle was developed for sensitive and rapid detection of Ochratoxin A (OTA) in malts with a small number of standard and sample solutions. The protocol included the competition of OTA in malt samples and that covalently coupled on the surface of microspheres with its monoclonal antibodies, the separation and aggregation of the magnetic microspheres, and the fluorescence detection of fluorescein isothiocyanate labeled goat anti-mouse immunoglobulin G probes. The magnetic microspheres-based CBA assay allowed for ultralow limit of detection (0.025mugkg(-1)) for OTA and showed higher sensitivity compared with the common polystyrene beads-based CBA method. This is the first report on the magnetic microspheres-based CBA assay by using a simple and easy-to-operate magnetic separator for highly sensitive and rapid detection of OTA in complex malt samples. By consuming less solvent, time and cost, as well as fewer standard and samples, the developed green protocol expressed high potential for one-site real-time detection of trace components in complex matrices.

Adsorption of aflatoxin B1, zearalenone and ochratoxin A by microorganisms isolated from Kefir grains.[Pubmed:28376398]

Int J Food Microbiol. 2017 Jun 19;251:1-7.

A strategy to reduce the deleterious effects of mycotoxins is to use dietary supplements that contain microorganisms that bind mycotoxins and decrease their gastrointestinal absorption. Novel strains were isolated from a Kefir culture and assessed for their mycotoxin adsorption and biotransformation ability. The most active strains were identified using DNA sequencing, and the stability of microorganism/mycotoxin complexes was evaluated using buffer solutions to simulate the pH conditions in the gastrointestinal tract. Our results showed that the microorganism consortium of Kefir grains adsorbed 82 to 100% of aflatoxin B1 (AFB1), zearalenone (ZEA) and Ochratoxin A (OTA) when cultivated in milk. The main strains that were capable of mycotoxin adsorption were identified as Lactobacillus kefiri, Kazachstania servazzii and Acetobacter syzygii. The strain L. kefiri KFLM3 was the most active, adsorbing 80 to 100% of the studied mycotoxins when cultivated in milk. Nonetheless, the strain K. servazzii KFGY7 retained more mycotoxin after the desorption experiments (65, 69 and 67% for AFB1, OTA and ZEA, respectively). These findings suggest that Kefir consumption may help to reduce gastrointestinal absorption of these mycotoxins and consequently reduce their toxic effects. The isolated strains may be of interest for the development of fermented dairy products for human consumption that have a new probiotic characteristic, the adsorption of mycotoxins.

A Review: Epigenetic Mechanism in Ochratoxin A Toxicity Studies.[Pubmed:28333080]

Toxins (Basel). 2017 Mar 23;9(4). pii: toxins9040113.

Ochratoxin A (OTA) is a natural contaminant that has displayed nephrotoxicity and hepatotoxicity in mammals. It contaminates a great variety of foodstuffs and threatens people's lives. The molecular mechanism of OTA-induced toxicity has been studied since 1965. Moreover, epigenetic mechanisms are also studied in OTA-induced toxicity. Additionally, the mode of OTA epigenetic research has been advanced in research hotspots. However, there is still no epigenetic study of OTA-induced toxicity. In this review, we discuss the relationship between these epigenetic mechanisms and OTA-induced toxicity. We found that studies on the epigenetic mechanisms of OTA-induced toxicity all chose the whole kidney or liver as the model, which cannot reveal the real change in DNA methylation or miRNAs or histone in the target sites of OTA. Our recommendations are as follows: (1) the specific target site of OTA should be detected by advanced technologies; and (2) competing endogenous RNAs (ceRNA) should be explored with OTA.

Determination of Ochratoxin A in wine by packed in-tube solid phase microextraction followed by high performance liquid chromatography coupled to tandem mass spectrometry.[Pubmed:28318567]

J Chromatogr A. 2017 Apr 14;1493:41-48.

Ochratoxin A (OTA), a widely studied mycotoxin, can be found in a variety of food matrices. As its concentration in food is generally low (in the order of mug kg(-1)), sample preparation techniques are necessary for the analyte purification and pre-concentration in order to achieve the required low detection limits. The separation and detection methods used for OTA analysis should also offer proper sensitivity in order to allow the adequate quantification of the analyte. This manuscript addresses the development of a methodology aiming the analysis of OTA in wine samples by packed in-tube SPME in flow through extraction mode coupled to HPLC-MS/MS. The in-tube SPME set up utilized a PEEK tube packed with C18 particles as the extraction column. The method was optimized by a central composite design 2(2)+3 extra central points, having as factors the percentage of ACN and time in the sample load step. The functionalities of the method were attested and its analytical conditions, enhanced by using 22% of ACN and 6min in the sample load step. Validation of the method was also accomplished prior to analyses of both dry red wine and dry white wine samples. The method demonstrated proper sensitivity, with detection and quantification limits equal to 0.02 and 0.05mugL(-1), respectively. Linearity and precision exhibited a 0.996 correlation coefficient and RSD under 6%, respectively. The method proved to be accurate at medium and higher concentration levels with a maximum recovery of 73% at higher concentration levels. OTA was not detected in either dry red and dry white wine samples evaluated in this work. If present, it would be at concentrations lower than the detection and quantification limits established for the proposed method, and considered not a potential danger to human health according to our present knowledge.

Ochratoxin A induces JNK activation and apoptosis in MDCK-C7 cells at nanomolar concentrations.[Pubmed:10869383]

J Pharmacol Exp Ther. 2000 Jun;293(3):837-44.

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.

Alterations in ATP-dependent calcium uptake by rat renal cortex microsomes following ochratoxin A administration in vivo or addition in vitro.[Pubmed:1417961]

Biochem Pharmacol. 1992 Oct 6;44(7):1401-9.

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.

Mechanism of action of ochratoxin A.[Pubmed:1820332]

IARC Sci Publ. 1991;(115):171-86.

Ochratoxin A has a number of toxic effects in mammals, the most notable of which is nephrotoxicity. It is also immunosuppressive, teratogenic and carcinogenic. The biochemical and molecular aspects of its action were first studied in bacteria. The appearance of 'magic spots' (ppGpp and pppGpp) pointed to inhibition of the charging of transfer ribonucleic acids (tRNA) with amino acids. This suggestion was confirmed by the demonstration that Ochratoxin A inhibits bacterial, yeast and liver phenylalanyl-tRNA synthetases. The inhibition is competitive to phenylalanine and is reversed by an excess of this amino acid. As a consequence, protein synthesis is inhibited, as shown with hepatoma cells in culture, with Madin Darby canine kidney cells (which are much more sensitive) and in vivo in mouse liver, kidney and spleen, the inhibition being more effective in the latter two organs. An excess of phenylalanine also prevents inhibition of protein synthesis in cell cultures and in vivo. Analogues of Ochratoxin A in which phenylalanine has been replaced by other amino acids have similar inhibitory effects on the respective amino acid-specific aminoacyl tRNA synthetases. 4R-HydroxyOchratoxin A, a metabolite of Ochratoxin A, has a similar action, whereas Ochratoxin Alpha (the dihydroisocoumarin moiety) and ochratoxin B (Ochratoxin A without chlorine) have no effect. Ochratoxin A might act on other enzymes that use phenylalanine as a substrate. We showed recently that it inhibits phenylalanine hydroxylase. In addition, the phenylalanine moiety of Ochratoxin A is partially hydroxylated to tyrosine by incubation with hepatocytes and in vivo. This competitive action with phenylalanine might explain why this amino acid prevents the immuno-suppressive effect of Ochratoxin A and partially prevents its teratogenic and nephrotoxic actions. The effect of Ochratoxin A on protein synthesis is followed by an inhibition of RNA synthesis, which might affect proteins with a high turnover. Ochratoxin A also lowers the level of phosphoenolpyruvate carboxykinase, a key enzyme in gluconeogenesis; this inhibition is reported to be due to a specific degradation of mRNA that codes for this enzyme. Recently, Ochratoxin A was also found to enhance lipid peroxidation both in vitro and in vivo. This inhibition might have an important effect on cell or mitochondrial membranes and be responsible for the effects on mitochondria that have been shown by several authors. Finally, the recent results of Pfohl-Leszkowicz et al. (this volume), who showed the formation of DNA adducts mainly in kidney but also in liver and spleen, explain the DNA single-strand breaks observed previously in mice and rats after acute and chronic treatment.