Occurrence of ochratoxin A and citrinin in Czech cereals and comparison of two HPLC methods for ochratoxin A detection.

The aims of the study were to obtain information about the occurrence of ochratoxin A (OTA) and citrinin (CIT) in cereals harvested in the Czech Republic and to compare two analytical procedures for detecting OTA. A total of 34 cereal samples, including two matrix reference materials (R-Biopharm, Germany), were analysed. The results were compared with the limit for raw cereal grains used as a foodstuff according to Commission Regulation No. 1881/2006, which allows a maximum OTA level of 5 µg kg−1. Compared were two methods based on the high-performance liquid chromatography principle, one using the immunoaffinity columns OchraTest™ (VICAM) and the second based on solvent partition (PART), both followed by fluorescence detection. The highest OTA contents were found in two barley samples. According to the method employed, the results for the first sample (malting barley) were VICAM = 31.43 µg kg−1 and PART = 44.74 µg kg−1. For the second sample (feeding barley) they were VICAM = 48.63 µg kg−1 and PART = 34.40 µg kg−1. Two samples of bread wheat had an OTA content approaching the legal limit (VICAM = 4.71 µg kg−1 and PART = 6.03 µg kg−1; VICAM = 4.12 µg kg−1 and PART = 3.95 µg kg−1). CIT was analysed using the PART method only, and its highest content (93.64 µg kg−1) was found for the malting barley sample with high OTA content (44.74 µg kg−1 as analysed using PART)

Binding of Zearalenone, Aflatoxin B1, and Ochratoxin A by Yeast-Based Products: A Method for Quantification of Adsorption Performance

A methodology was developed to quantify the efficiency of yeast-based pro ducts for adsorption of three mycotoxins: zearalenone (ZEA), aflatoxin B 1 (AFB 1 ), and ochratoxin A (OTA). Eight products were tested (yeast cell wall or in activated yeast). The described experimental protocol based on in vitro tests provi ded reliable isotherms for each mycotoxin. The most suitable models were the Hill model for ZEA, the Langmuir model for AFB 1 , and the Freundlich model for OTA. From these models, original mathematical affinity criteria were defined to quantif y the product adsorption performances for each mycotoxin. The best yeast product, a yeast cell wall from baker’s yeast, can adsorb up t o68 % of ZEA, 29 % of AFB 1 , and 62 % of OTA, depending on the mycotoxin concentrations. The adsorption capacity larg ely depended both on yeast composition and mycotoxin, but no direct correlation between yeast composition and adsor ption capacity was found, confirming that adsorption of mycotoxin on yeast-based products involves complex phenomena. The resul ts of this study are useful for comparing the adsorption efficiency of various yeast products and understanding the me chanisms involved in adsorption

Development of an in vitro method for the prediction of mycotoxin binding on yeast-based products: case of aflatoxin B1, zearalenone and ochratoxin A

To date, no official method is available to accurately define the binding capacity of binders. The goal is to define general in vitro parameters (equilibrium time, pH, mycotoxin/binder ratio) for the determination of binding efficacy, which can be used to calculate the relevant equilibrium adsorption constants. For this purpose, aflatoxin B1 (AFB1), zearalenone (ZEA) or ochratoxin A (OTA) were incubated with one yeast cell wall in pH 3, pH 5 or pH 7 buffers. The percentage of adsorption was recorded by quantitation of remaining mycotoxins in the supernatant and amount of mycotoxin adsorbed on the residue. The incubation of yeast cell wall in the presence of mycotoxins solved in buffer, lead to unexpected high adsorption percentage when the analysis was based only on remaining mycotoxins in the supernatant. The decrease of mycotoxins in the supernatant was not correlated to the amount of mycotoxins found in the residue. For this reason we modified the conditions of incubation. Yeast cell wall (5 mg) was pre-incubated in buffer (990 μl) at 37 °C during 5 min and then 10 μl of an alcoholic solution of mycotoxin (concentration 100 times higher than the final concentration required in the test tube) were added. After incubation, the solution was centrifuged, and the amount of mycotoxins were analysed both in the supernatant and in the residue. A plateau of binding was reached after 15 min of incubation whatever the mycotoxins and the concentrations tested. The adsorption of ZEA was better at pH 5 (75 %), versus 60 % at pH 3 and 7. OTA was only significantly adsorbed at pH 3 (50 %). Depending on the pH, the adsorptions of OTA or ZEA were increased or decreased when they were together, indicative of a cooperative effect

Study of the effect of a multi mytoxin contamination on the reproductive system and on the developement of urany tract cancer

Tout au long de la chaîne alimentaire, des moisissures peuvent se développer et produire des mycotoxines. Ce sont des composés toxiques naturels issus du métabolisme secondaire des moisissures, susceptibles de contaminer l'alimentation animale et humaine, provoquant de nombreuses pathologies (hépatotoxicité, néphrotoxicité, neurotoxicité, mutagénicité, tératogénicité, cancérogénicité,…). La première étape de ce travail était d'évaluer la présence simultanée de l'ochratoxine A (OTA), de la citrinine (CIT), des aflatoxines (AFs), de la zéaralénone (ZEA), de la fumonisine (FB) et des trichothécènes dans des aliments destinés aux humaines (céréales, lait, café, jambon) et aux animaux (croquettes de chat et chien, foins). En général plusieurs mycotoxines coexistaient. Certains échantillons pour les humains dépassaient les limites autorisées en mycotoxines dans l'Union Européenne. Suite à l'étude de simulation d'apport en mycotoxines dans une ration quotidienne, nous avons constaté que les doses journalières admissibles (DJA) peuvent être dépassées. La deuxième phase consistait à étudier l'impact des mycotoxines seules ou en combinaison sur la viabilité cellulaire et la génotoxicité sur des modèles cellulaires (cellules rénales d'opossum (OK), cellules rénales humaines (HK2), cellules humaines de glandes mammaires (MCF7)) et chez des animaux (porc, rat). Nous avons montré que la CIT, la FB1 et la ZEA agissent en synergie sur la génotoxicité de l'OTA. Chez les animaux, nous avons montré qu'à des doses (5 ng d'OTA/kg poids corporel/ jour et de 200ng FB1/kg pc/j) correspondantes aux DJA, il y avait des effets génotoxiques (formation d'adduits à l'ADN). Nous avons mis en évidence l'implication des mycotoxines dans l'alimentation animale sur la baisse de fertilité et la tératogénicité chez les chats, ainsi que sur la mort des chevaux. Au cours de la troisième partie de cette étude, nous avons testé sur des cultures cellulaires (HK2 et MCF7) et in vivo (poulet) l'effet protecteur du glutathion (GSH) et de la sélénométhionine (SeMet) contre l'OTA responsable de cancers de voie urinaire et la ZEA responsable de baisse de fertilité. Le GSH est un puisant antioxydant et le sélénium est un oligoélément indispensable qui intervient comme co-facteur de nombreuses enzymes ayant des propriétés antioxydantes, comme les glutathion peroxydases. D'une manière générale, au niveau des cellules rénales, le GSH seul et la levure correspondante ont un effet bénéfique vis-à-vis de la génotoxicité de l'OTA ; par contre la sélénométhionine et la levure séléniée augmentent la génotoxicité de l'OTA et de la ZEA. Dans les cellules des glandes mammaires, il y a une nette amélioration vis-à-vis de la génotoxicité des deux mycotoxines lorsque les cellules sont exposées à une seule mycotoxine simultanément au GSH, à la sélénométhionine et aux levures enrichies. Chez les poulets, la diminution de la génotoxicité n'est pas exclusivement corrélée à la capacité des parois de levure ou des levures à adsorber l'OTA. Ces dérivés de levure ont gardé la propriété de partiellement métaboliser l'OTA dans l'intestin. Les parois de levures et les levures enrichies en GSH ont un meilleur pouvoir protecteur que celles enrichies en SeMetThroughout the food chain, mold can grow and produce mycotoxins. These are toxic compounds "natural" from the secondary metabolism of molds that may contaminate the feed and food, causing many diseases (hepatotoxicity, nephrotoxicity, neurotoxicity, mutagenicity, teratogenicity, carcinogenicity, ...). The first stage of this work was to assess the level of multi-contamination by mycotoxins (OTA, CIT, Afs, ZEA, FB, DON) in food (cereals, milk, coffee, ham) and feed (pet food). Some samples analyzed exceeded the limits of mycotoxins in the European Union. Through the simulation study of mycotoxin intake in a daily diet, we found that the acceptable daily intake (ADI) may be exceeded. The second phase was to study the impact of mycotoxins alone or in combination on cell proliferation, genotoxicity in cellular models (OK, HK2, and MCF7) and animal (pig, rat). We have demonstrated genotoxic effects (formation of DNA adducts) at doses (5 ng OTA / kg bw / day and 200 ng FB1/kg bw / day) considered safe (ADI). We have shown that the CIT, FB1 and ZEA act synergistically on the genotoxicity of OTA. We pointed to the involvement of mycotoxins in animal feed on declining fertility and teratogenicity in cats, as well as the death of horses. In the third part of this study, we tested in cell cultures (HK2 and MCF7) and in vivo (chicken) the protective effect of glutathione (GSH) and selenomethionine (SeMet) against OTA responsible for urinary tract cancers and ZEA reducing fertility. GSH is considered as a potent antioxidant and selenium is a trace essential element that acts as a cofactor of enzymes such glutathione peroxidase. In summary, in kidney cells, GSH and GSH enriched yeast decrease OTA genotoxicity whereas SeMet and SeMet enriched yeast increase genotoxicity of OTA and ZEA. In mammary cells, whatever the compounds gentoxicty of OTA and ZEA significantly decrease. Decrease of OTA genotoxicity in chicken kidney cannot be exclusively explained by adsorption of OTA on yeast by products. The yeast products retain their ability to metabolize the OTA. GSH enriched yeast and yeast cell wells are more efficient than SeMet enriched yeas

Structure-activity relationships imply different mechanisms of action for ochratoxin a-mediated cytotoxicity and genotoxicity.

Ochratoxin A (OTA) is a fungal toxin that is classified as a possible human carcinogen based on sufficient evidence for carcinogenicity in animal studies. The toxin is known to promote oxidative DNA damage through production of reactive oxygen species (ROS). The toxin also generates covalent DNA adducts, and it has been difficult to separate the biological effects caused by DNA adduction from that of ROS generation. In the current study, we have derived structure-activity relationships (SAR) for the role of the C5 substituent of OTA (C5-X = Cl) by first comparing the ability of OTA, OTBr (C5-X = Br), OTB (C5-X = H), and OTHQ (C5-X = OH) to photochemically react with GSH and 2'-deoxyguanosine (dG). OTA, OTBr, and OTHQ react covalently with GSH and dG following photoirradiation, while the nonchlorinated OTB does not react photochemically with GSH and dG. These findings correlate with their ability to generate covalent DNA adducts (direct genotoxicity) in human bronchial epithelial cells (WI26) and human kidney (HK2) cells, as evidenced by the ³²P-postlabeling technique. OTB lacks direct genotoxicity, while OTA, OTBr, and OTHQ act as direct genotoxins. In contrast, their cytotoxicity in opossum kidney epithelial cells (OK) and WI26 cells did not show a correlation with photoreactivity. In OK and WI26 cells, OTA, OTBr, and OTB are cytotoxic, while the hydroquinone OTHQ failed to exhibit cytotoxicity. Overall, our data show that the C5-Cl atom of OTA is critical for direct genotoxicity but plays a lesser role in OTA-mediated cytotoxicity. These SARs suggest different mechanisms of action (MOA) for OTA genotoxicity and cytotoxicity and are consistent with recent findings showing OTA mutagenicity to stem from direct genotoxicity, while cytotoxicity is derived from oxidative DNA damage