Occurrence of deoxynivalenol (DON) and ochratoxin A (OTA) in dog foods

The commercially available dog food samples (29 dry foods and 11 wet foods) were analysed for deoxynivalenol (DON) and ochratoxin A (OTA) using ELISA. All (100%) dry foods were contaminated with DON with various amount of the toxin (22-1837 μg/kg). In wet food 3 samples were found to be positive for DON in the range of 95-170 μg/kg. There were a few samples contaminated with OTA: 3 samples in dry foods (7-40 μg/kg) and 2 samples in wet foods (45 and 115 μg/kg).     

Beeinflussung der Zytokinsekretion von Maus-Thymom-Zellen (EL-4) durch Ochratoxin A

The murine thymoma cell line EL-4 was used as an in vitro T-cell model to assess the immunomodulating effect of pure Ochratoxin A (OTA) and an Aspergillus ochraceus raw toxin preparation. Cytokine production (IL-2, IL-4, IL-5, IL-6) and cell viability of PMA-stimulated EL-4 cells were investigated in the presence of OTA. The cytotoxic effect of the raw toxin could be observed at lower concentrations than pure OTA. The IC50 values were 3 µg/ml and 11 µg/ml, respectively. Increasing concentrations of both OTA preparations caused an inhibition of cytokine production, but the inhibition effect of the raw toxin was stronger than of pure OTA. This is supposed to be the effect of further up to now not characterized substances in the raw toxin. Differences in the susceptibility of the mechanisms of production and regulation of each cytokine are indicated by the different concentrations for inhibition effects. Both toxin preparations showed also stimulating effects on some cytokines (IL-2 and IL-6) while others (IL-4 and IL-5) were depressed at these OTA concentrations. This indicates the immunomodulating properties of the toxin.     

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.     

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.     

A molecular and bioinformatic study on the ochratoxin A (OTA)-producing Aspergillus affinis (section Circumdati)

Aspergillus affinis (section Circumdati) is a novel ochratoxin A (OTA)-producing species found in submerged riparian decomposing leaves. However, very little is known about its role on the breakdown of plant debris and its ability to degrade carbohydrate polymers. Moreover, its OTA biosynthetic pathway has not yet been explored. In the present paper, we investigated the gene encoding the extracellular alpha-amylase (amyAa) of A. affinis within the evolution of the Aspergillus lineages in relation to the possible use of this enzyme in starch processing. The novel amyAa, despite being related to branches of the Aspergillus species of the sections Terrei and Flavi, formed a distinct phylogenetic branch, which may be of outstanding importance from a biotechnological point of view. Moreover, we identified the polyketide synthase gene (pks) putatively required for the first step of OTA biosynthesis in A. affinis. This otapks was examined in relation to a limited number of orthologous genes available from Aspergillus species of the sections Circumdati and Nigri. Our study highlights the importance of otapks as target genes in the treatment of ochratoxigenic Aspergillus species on a more comprehensive evolutionary basis.     

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: 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.     

Extraktion von Ochratoxin A aus geröstetem Kaffee Mittels Accelerated Solvent Extraction (ASE)

Accelerated solvent extraction (ASE) is an alternative sample extraction procedure for ochratoxin A in roasted coffee. ASE results are comparable to that of the modified Koch method, but required less sample preparation time. Furthermore, ASE gave higher quantitative values than other methods reported for extraction of ochratoxin A. In the end less harmful water could be used for extraction.     

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.     

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.     

Ochratoxin A: study of grain contamination

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 micrograms/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 micrograms/kg.     

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.     

Formation of (4R)- and (4S)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A from Ochratoxin A by rabbit liver microsomes.

Three metabolites were formed from ochratoxin A in the presence of rabbit liver microsomal fractions and NADPH. They were isolated by extraction, thin-layer chromatography, and high-pressure liquid chromatography. Two of them were identified as (4R)- and (4S)-4-hydroxyochratoxin A. It is suggested on the basis of mass and nuclear magnetic resonance spectroscopy that the third metabolite is 10-hydroxyochratoxin A. The formation of the metabolites was inhibited by carbon monoxide and metyrapone and was stimulated when microsomes from phenobarbital-treated animals were used. The results suggest that cytochrome P-450 catalyzes the formation of these metabolites.     

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.     

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.     

Investigation of Ochratoxin A biosynthetic genes in<Emphasis Type="Italic">Penicillium verrucosum</Emphasis> by DDRT-PCR experiments: differential expression of OTA genes

A defined minimal medium has been developed which is either restrictive or permissive for the production of ochratoxin A byPenicillium verrucosum simply by changing the nitrogen and carbon source. The combination of ammonium/glycerin promoted, whereas the combination nitrate/glucose repressed ochratoxin production.In parallel mutants ofP verrucosum have been constructed by restriction endonuclease mediated integration, which were not able to produce ochratoxin. Different types of transformants, which either produce no detectable amounts of ochratoxin A but ochratoxin α, strains which produce only ochratoxin B and strains which produces none of these secondary metabolites, have been observed.     

赭曲霉毒素A对体外培养人肾小管上皮细胞凋亡的作用

探讨赭曲霉毒素A(ochratoxin A,OA)对体外培养的人肾小管上皮细胞(HKC)凋亡的影响.体外培养HKC,给予不同浓度OA处理24 h后,分别采用流式细胞定量检测术(FCM)检测细胞的凋亡率,应用免疫细胞化学染色(SP法)和免疫蛋白印迹(Western 印迹)方法,观察凋亡相关蛋白caspase-3的表达以及JNK的表达水平和磷酸化水平(p-JNK).FCM检测结果表明,经OA处理24 h,HKC的凋亡率明显升高.SP法和Western印迹结果表明,OA处理组HKC caspase-3的表达以及JNK的磷酸化水平(p-JNK)均明显高于空白对照组和溶剂对照组,但各组间细胞JNK的表达水平无明显变化.OA处理可促进体外培养的人肾小管上皮细胞发生凋亡,JNK激活以及caspase-3可能参与OA诱导HKC凋亡的过程.     

The removability of pesticides during the production of dialysis water (2)

In many cases it can be demonstrated that the amount of plant protectives and plant treatments (pesticides) in drinking-water exceeds the permitted levels of the drinking-water decree which will be effective on October 1st, 1989. These components are in parts toxicologically important. Therefore, an examination was made on how far pesticides are removed during the conventional purification of dialysis water, but especially during the reverse osmosis. Retention rates of a reverse osmosis plant for 14 different pesticides were discovered which were used in different concentrations and compositions. In part 2 of this contribution the results of the investigation are presented. The figures demonstrate that almost all of the examined components were retained with an effectiveness of 92-98%. The elimination efficiency did not depend on the basic concentration of the pesticides. After an initial phase of 50 h duration, the permeat concentration reached a constant value which did not alter even after more than 700 h.