Ochratoxin A: Contamination of grain

Ochratoxin A was quantitatively monitored in grain extracts by indirect solid-phase enzyme immunoassay with the use of an immobilized conjugate of the toxin with gelatin and polyclonal rabbit antibodies raised against the ochratoxin A-BSA conjugate. This monitoring found that 1.7 to 18.5% of the samples were contaminated with the toxin at a concentration of 25.9–291.7 μg/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 μg/kg.     

Biosynthesis of Ochratoxin A

Biosynthesis of ochratoxin A by Aspergillus ochraceus Wilh. was investigated by radiolabeling experiments in which phenylalanine-1-(14)C and sodium acetate-2-(14)C were supplied to the fungus in sucrose-yeast extract medium. Results showed that phenylalanine was incorporated unaltered into the phenylalanine moiety of ochratoxin A, whereas the isocoumarin moiety of ochratoxin A was mostly derived via acetate condensation.     

Biosynthesis of Ochratoxin A

Biosynthesis of ochratoxin A by Aspergillus ochraceus Wilh. was investigated by radiolabeling experiments in which phenylalanine-1-¹⁴C and sodium acetate-2-¹⁴C were supplied to the fungus in sucrose-yeast extract medium. Results showed that phenylalanine was incorporated unaltered into the phenylalanine moiety of ochratoxin A, whereas the isocoumarin moiety of ochratoxin A was mostly derived via acetate condensation.     

Ochratoxin A in getrockneten Weinbeeren

In the Lebensmittelinstitut Braunschweig 89 samples of sultanas were analysed for Ochratoxin A in 2003. The samples were slurried with water to get a homogeneous test-material. After the extraction with acetonitrile the sample-filtrates were cleaned-up by immuno affinity columns. The measurement was performed by HPLC with fluorescence-detection. As the result we asserted a high contamination of sultanas with OTA. The median concentration amounted 2,7 μg/kg at a contamination rate of 95%. 8% of all samples above the limit value of 10 μg/kg with a maximum value of 28,5 μg/kg.     

Ochratoxin A: an important western Canadian storage mycotoxin

Ochratoxin A (OA) is a mycotoxin produced by certain species of storage fungi of the Penicillium and Aspergillus genera. Toxin production by these fungi is influenced by species and even strain of fungi, time and temperature of incubation, moisture content of substrate, and type of substrate. OA has been shown to occur in various grains, cereals and other plant products, animal feeds, meats, and human tissues in countries throughout the world. Of interest is the discovery of OA in a high percentage of blood from humans in Germany. OA is acutely toxic to many different animals and in addition to being a nephrotoxin, it is a hepatotoxin, a teratogen, a very potent carcinogen, possibly a mutagen, and an immunosuppressive agent. OA 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. OA is hydrolyzed by the intestinal microflora into nontoxic compounds (7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin (O alpha) and phenylalanine). It is excreted as either OA, hydroxylated OA, or O alpha in both the urine and feces. OA 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 OA. It would appear that this compound presents a true potential hazard for humans as its occurrence is wide spread and it is highly carcinogenic.     

赭曲霉毒素 A 的高灵敏时间分辨荧光免疫分析

采用时间分辨荧光免疫分析 (TRFIA) 技术建立快速的高灵敏度的赭曲霉毒素 A (OTA) 全自动检测方法 . 将 OTA-BSA 作为免疫分子免疫 Balb/c 小鼠, OTA-KLH 为筛选用抗原包被板,用间接酶联免疫分析 (ELISA) 法筛选出专一针对 OTA 的高效价的阳性克隆 3G9 ,用杂交瘤细胞制备抗 OTA 单克隆抗体 . 用 OTA-BSA 包被 96 孔板为固相抗原,与游离 OTA 共同竞争有限的抗 OTA 单克隆抗体,以稀土离子 Eu3+ 标记的羊抗鼠抗体进行示踪,采用间接竞争免疫分析方法在解离增强荧光免疫分析体系中建立 OTA-TRFIA. 该方法的灵敏度为 0.03 μg /L ,测量范围为 0.03 ~1 000 μg /L ,批内和批间变异分别为 3.7% 和 5.3% ,平均回收率为 94.2% ,与赭曲霉毒素 B 的平均交叉反应为 3.7% ,与黄曲霉毒素 B1 、 牛血清白蛋白和苯基丙氨酸无交叉反应,说明抗体的特异性很好 . 8 条不同时间进行的间接竞争 OTA-TRFIA 的效应点均值 ED80 、 ED50 、 ED20 分别为 (0.33±0.02) μg /L、 (1.44 ±0.08) μg /L 和 (5.22 ± 0.12) μg /L ,说明方法的稳定性好,样品经 TRFIA 和 ELISA 试剂盒同时检测 OTA ,两者的相关系数为 0.925 ,结果相符 . 研究表明, OTA-TRFIA 是目前报道的 OTA 检测中最灵敏的方法,该分析方法稳定性好,可测范围宽,具有很好的应用前景 .     

Ochratoxin A adsorption phenotype: An inheritable yeast trait

This study aimed to evaluate the inheritance of the trait ochratoxin A adsorption in two wine strains of Saccharomyces cerevisiae and their 46 descendants. Each strain was inoculated in triplicate in test tubes containing 10 ml of must obtained from the Calabrian Zibibbo white grape variety, artificially contaminated with ochratoxin A to reach a total content of 4.10 ng/ml. The microvinification trials were performed at 25°C. After 30 days, ochratoxin A values ranged from 0.74 to 3.18 ng/ml, from 0.01 to 2.69 ng/ml, and from 0.60 to 2.95 ng/ml respectively in wines, in lees after washing, and in the saline solution used to wash the lees. The analysis of OTA in wines was performed to find the residual toxin content after yeast activity, thus obtaining technological evidence of yeast influence on wine detoxification. The analysis of OTA in lees after washing was performed to distinguish the OTA linked to cells. The analysis of OTA in the saline solution used to wash the lees was performed to distinguish the OTA adsorbed on yeast cell walls and removed by washing, thus focusing on the adsorption activity of wine yeast through electrostatic and ionic interactions between parietal mannoproteins and OTA. Ploidy of the two parental strains was controlled by flow cytometry. Results demonstrated that the ochratoxin A adsorption is genetically controlled and is a polygenic inheritable trait of wine yeasts. The majority of the descendants are characterized by a great and significant diversity compared to their parents. Both the parental strains had genome sizes consistent with their being diploid, so validating the observed results. These findings constitute an initial step to demonstrate the mechanisms of inheritance and establish breeding strategies to improve the ochratoxin A adsorption trait in wine yeasts. This will allow a decrease in the ochratoxin A content of contaminated musts during winemaking, by using genetically improved wine yeasts.     

Assessment on pollution of Ochratoxin A in grain in China and its apoptosis effect on vitro-cultured human tubular kidney cells

Ochratoxin A (OTA) is nephrotoxic, immunosuppressive, and teratogenic in many species and is a possible human carcinogen. In this study, we investigated the OTA pollution situations of grains in northern China and the signaling pathway that mediated OTA-induced apoptosis in human tubular kidney cells (HKCs). Samples of grains collected from three representative areas were determined by using high-performance liquid chromatography fluorescence method. The effects of OTA on cell apoptosis, caspase-3, Bax, and Bcl-2 expression, and phosphorylation of c-Jun NH(2) terminal kinase (JNK) were detected in cultured HKCs via flow cytometry (FCM), Hoechst 33258 staining, and Western blot. It showed that OTA pollution of edible grains was very common in north China. OTA could affect caspase-3, Bax, and Bcl-2 expression and increased cell apoptosis in cultured HKCs. The JNK signalling pathway might play an important role during these cellular events.     

Aptamer-DNAzyme hairpins for biosensing of Ochratoxin A

We report an aptasensor for biosensing of Ochratoxin A (OTA) using aptamer-DNAzyme hairpin as biorecognition element. The structure of this engineered nucleic acid includes the horseradish peroxidase (HRP)-mimicking DNAzyme and the OTA specific aptamer sequences. A blocking tail captures a part of these sequences in the stem region of the hairpin. In the presence of OTA, the hairpin is opened due to the formation of the aptamer-analyte complex. As a result, self-assembly of the active HRP-mimicking DNAzyme occurs. The activity of this DNAzyme is linearly correlated with OTA concentration up to 10 nM, showing a limit of detection of 2.5 nM.     

Solid-Substrate Fermentor for Ochratoxin A Production

A laboratory-scale fermentor designed for solid-substrate fermentation was constructed and tested. Its capacity to produce ochratoxin under varied conditions was determined with wheat as substrate. Ochratoxin yields of 2,000 to 2,500 μg/g of wheat were regularly obtained, and occasionally yields as high as 4,000 μg/g were obtained. The most critical factor in the fermentation was initial substrate moisture content; wheat tempered at 30 to 31% moisture produced the highest yields. Other variables tested were agitation and aeration rates, initial static culture time, and inoculum types and volumes.     

Degradation of Ochratoxin A by a Ruminant

The fate of ochratoxin A during incubation with contents from the four stomachs of the cow was studied. It was concluded that ochratoxin A was cleaved into the nontoxic ochratoxin α and phenylalanine by the contents from all but the abomasum.     

Ochratoxin A: Occurrence as Natural Contaminant of a Corn Sample

Ochratoxin A was detected as a natural contaminant for the first time. It was present in a corn sample.     

Incorporation of Chlorine-36 into Ochratoxin A

Incubation of Aspergillus ochraceus NRRL 3174 in a medium containing Na(36)Cl incorporated (36)Cl into ochratoxin A. The highest incorporation (0.75 and 0.70%, respectively) was obtained when the radioactive chloride was added to the medium by the 2nd or 3rd day of incubation.     

Toxikologische Bewertung von Ochratoxin A: Offene Fragen und Forschungsbedarf

Ochratoxin A (OTA) is an important food and feed contaminant with potential adverse effects in humans and animals. In view of present discussions on limit values for OTA in foods, essential elements of a toxicological risk assessment are outlined. The exposure situation in Europe is now well documented. The data base, with respect to a characterization of hazard and dose-response relationships, allowed to calculate a provisional tolerable daily intake for OTA suited to protect the consumer against undesirable toxic effects. Nonetheless, further research on OTA is indicated in view of unresolved issues regarding the following points:

1. 1.
   mechanisms of action (mode of genotoxicity, role of bioactivation/metabolism, identification of DNA-adducts and dose-dependency);
    
2. 2.
   combinations of OTA and other mycotoxins (studies of relevant mixtures/conditions);
    
3. 3.
   individual susceptibility and/or situation-based vulnerability.
    

Better information on mechanistic aspects of mycotoxin-induced toxicities will further improve our knowledge on the “margin of safety” between a given exposure and a potential impairment of human health.     

Ochratoxin A induces apoptosis in neuronal cells

The mycotoxin ochratoxin A (OTA), which is produced by Aspergillus and Penicillium subspecies, is a frequently present contaminant of food and feedstuffs. OTA exhibits a wide range of toxic activities including nephro- and hepatotoxicity. However, little is known regarding potential neurotoxic effects of OTA. In the present study primary neurons as well as SH-SY5Y neuronal cells were incubated with increasing concentrations of OTA (0.1–2.5 μmol/L). OTA treatment resulted in a dose-dependent increase in cytotoxicity in both neuronal cell types. Caspase-9 and caspase-3 were activated in response to OTA treatment. Furthermore, caspase inhibitors were effective in partly counteracting OTA induced neurocytotoxicity. OTA induced apoptosis was accompanied by a loss of mitochondria membrane potential. Overall, present data indicated that OTA is neurotoxic at relatively low concentrations. OTA induced neurotoxicity seems to be, at least party, mediated by apoptosis. OTA may contribute to the pathogenesis of neurodegenerative diseases (e.g. Alzheimer’s and Parkinson’s disease) in which apoptotic processes are centrally involved.     

Ochratoxin A: Developmental and Reproductive Toxicity—An Overview

Ochratoxin A (OTA) is nephrotoxic, hepatotoxic, reprotoxic, embryotoxic, teratogenic, neurotoxic, immunotoxic, and carcinogenic for laboratory and farm animals. Male and female reproductive health has deteriorated in many countries during the last few decades. A number of toxins in environment are suspected to affect reproductive system in male and female. OTA is one of them. OTA has been found to be teratogenic in several animal models including rat, mouse, hamster, quail, and chick, with reduced birth weight and craniofacial abnormalities being the most common signs. The presence of OTA also results in congenital defects in the fetus. Neither the potential of OTA to cause malformations in human nor its teratogenic mode of action is known. Exposure to OTA leads to increased embryo lethality manifested as resorptions or dead fetuses. The mechanism of OTA transfer across human placenta (e.g., which transporters are involved in the transfer mechanism) is not fully understood. Some of the toxic effects of OTA are potentiated by other mycotoxins or other contaminants. Therefore, OTA exposure of pregnant women should be minimized. OTA has been shown to be an endocrine disruptor and a reproductive toxicant, with abilities of altering sperm quality. Other studies have shown that OTA is a testicular toxin in animals. Thus, OTA is a biologically plausible cause of testicular cancer in man.     

A new index for assessing heavy metals contamination in sediments: A case study

In this study, heavy metals contamination in surface sediments of northwest Persian Gulf was investigated and a new index was formulated for assessing severity of heavy metal pollution in aquatic environments. The surface sediment samples were collected from 45 stations. The concentrations of 8 metals (Fe, Cu, Zn, Cr, Ni, Pb, Cd and V) were analyzed. According to the results of potential ecological risk index (RI), pollution load index (PLI) and modified degree of contamination (mCD), based on the average shale, considerable and intense pollution of heavy metals in sediments of study area was not observed. Based on the sediments quality guidelines the concentration of Ni at whole study area has frequently adverse biological effect on aquatic organisms. The mean effects range-median quotient indicated that surface sediments in the vicinity of petrochemical zone and Bahrekan pier with 49% probability of toxicity can be risky for the biota. The Consequence of the cluster analysis and principal component analysis/factor analysis indicated that Fe, Cu, Zn and Cr are mainly originated from natural sources and Ni, Pb, Cd and V are mostly derived from anthropogenic sources. Results of introduced index in this research (Contamination Severity Index) demonstrated that pollution of heavy metals in sediments at several sites is very intense. Assessment of results illustrated that proposed index is more reliable and logical compared with other investigated indices for evaluating heavy metals pollution in sediments of aquatic environments.     

Enzyme-Linked Immunosorbent Assay for Detection of Ochratoxin A

A simple microtest plate enzyme-linked immunosorbent assay was developed for the detection of ochratoxin A at levels as low as 25 pg per assay. The relative cross-reactivities of the antibody in this system with ochratoxin A (OA), OB, OC, and Oα were found to be 1.0, 0.14, 0.44, and 0.01, respectively.     

Comparison of detection methods for Acarus siro (Acari: Acaridida: Acarididae) contamination in grain

Acarus siro L. 1758 (Acari: Acaridida: Acarididae) is an important pest of stored grain because it contaminates the grain by allergens and transfers pathogenous microorganisms. Rapid detection of contamination enables to intercept an early grain infestation by the pest. In this study, we compared the usability and efficiency of various detection approaches. Under laboratory conditions, grain samples of various sizes were infested by different levels of the following contaminants: eggs, adults, and feces of A. siro. The samples were analyzed by enzyme-linked immunosorbent assay (ELISA) by using anti-A. siro polyclonal antibody (immunochemical method), extracted in Berlese-Tullgren funnels, sieved, and processed by filth-flotation (conventional methods). The adults or juveniles of A. siro could be detected by all the three tested conventional methods and ELISA with detection limits in the range from 221 to 1,157 mites/kg grain. Eggs were detected by filth-flotation only; the detection limit was 1,950 eggs/kg grain. The feces of A. siro were detectable by ELISA test, only. ELISA enabled the detection of the feces with the minimal threshold level of 1.04 microg feces/g grain; it means the assay allowed to trace less than one metabolically active mite per gram of grain. The study thus demonstrated that reliable A. siro detection in grain can only be achieved by combining different detection methods. European Union and U.S. administratives dictate zero or near-zero tolerance level for mite infestation in stored products. This demand is difficult to fulfill, because every detection method is limited by its detection limit; thus, it is hard to reliably detect infestation levels lower than obtained detection limits. This methodical limitation is discussed in context with the determined detection limits of the tested methods.     

Ochratoxin A and zearalenone: a comparative study on genotoxic effects and cell death induced in bovine lymphocytes

Ochratoxin A (OTA) and zearalenone (ZEA), two naturally occurring contaminants of animal feed, have been implicated in several mycotoxicoses in farm livestock but there is little information on their genotoxicity and toxicity in these species. Therefore, we investigated on the cytogenetic and cytotoxic effects of both OTA and ZEA in in vitro cultures of bovine lymphocytes. We determined chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) as well as the mitotic index (MI) and cell viability following OTA and ZEA treatment. This report is the first to provide evidence of a statistically significant increase of structural CAs and of SCEs/cell associated with a reduction of the MI in all OTA- and ZEA-treated bovine lymphocyte cultures and a clear reproducible reducing effect of OTA on cell viability mediated by enhanced apoptosis. OTA-induced programmed cell death was not limited to bovine lymphocytes, as comparable data were demonstrated in the human leukemic T-cell line Jurkat.