Mycotoxins of fungal strains from stored herbal plants and mycotoxin contents of Nigerian crude herbal drugs

The ability of fungi isolated from stored herbal drug plants to produce mycotoxins in semisynthetic media was studied. The results obtained show that aflatoxins and ochratoxin A, were produced by Aspergillus flavus, A. parasiticus and A. ochraceus isolates. The time-production courses of aflatoxins B₁, B₂, ₁ and ochratoxin A in crude herbal drug preparations show that more of these toxins were produced with increase in time of storage of the drugs. The results indicate that the potential exists for the toxigenic strains to elaborate mycotoxins in a large quantity in herbal drug substrates than in semisynthetic media.This revised version was published online in October 2005 with corrections to the Cover Date.     

Penicillium verrucosum in feed of ochratoxin A positive swine herds

Ochratoxin A contamination of cereal feed grain was monitored during October 1989–September 1990 by analysis of blood samples from slaughter swine in Sweden. The detection of ochratoxin A in swine blood was used as a method to identify swine herds fed ochratoxin A contaminated feed. The contamination level of ochratoxin A in the blood of the positive herds was in the range 2–45 ng/ml with the mean concentration 5.2 ng/ml. Feed samples for mycological analysis were collected from both ochratoxin A positive herds (2 ng/ml blood) and ochratoxin A negative herds (<2 ng/ml blood). From the ochratoxin A positive herds and the ochratoxin A negative herds 22 and 21 feed samples were collected, respectively. No quantitative differences in mould content, as determined by colony forming units, were observed between the two groups. However, there were differences in the mycoflora. The incidence of storage fungi (Penicillium and Aspergillus spp.) was significantly higher (p < 0.05) in feed from ochratoxin A positive herds. Particularly, Penicillium verrucosum was found to be significantly more common (p < 0.001). Altogether 274 isolates were screened for their ability to produce ochratoxin A. Ochratoxin A producers were found only within P. verrucosum; 38% of the 63 isolates produced detectable amounts of ochratoxin A. Ochratoxin A producing isolates of P. verrucosum were found in 60% of the feed samples collected from ochratoxin A positive swine herds and in one sample (5% ) of the feed samples collected from the ochratoxin A negative herds.     

Effects of the botanical composition and weather conditions on mycotoxins in winter forage from grassland

The paper focuses on possible effects of the botanical composition and weather conditions on the extend of zearalenone and ochratoxin A concentrations in various grass swards in winter pasture systems. Zearalenone is only detectable in pure stands ofLolium perenne or inLolio-Cynosuretum plant communities, respectively. The occurrence of ochratoxin A is more frequent and less specific concerning the botanical composition. This mycotoxin was found in both,Lolium perenne andFestuca arundinacea in varying years. The incidence of ochratoxin A depends on year and is apparently related to the weather conditions in autumn and winter. There was no evidence that particular locatio8/22/2008 12:57PMns have a higher or a lower risk for high ochratoxin A or zearalenone concentrations than others. Peak values in individual swards are not intermittent over the years. Presented at the 25th Mykotoxin-Workshop in Giessen, Germany, May 19–21, 2003     

Assessment of ochratoxin A and tenuazonic acid in Canadian ice-wines

Twenty-six samples of commercial ice-wine made from late-harvested grapes were assayed for the mycotoxins ochratoxin A and tenuazonic acid. Canadian wines originated in British Columbia (18) and Ontario (8). For comparison two German wines from Hesse were also studied. Four additional samples of research ice-wine originating in were also studied. In all wine samples, assays using immuno-affinity chromatography and fluorescence liquid chromatography indicated ochratoxin A below 0.15 μg/L, the limit of determination of the method. Tenuazonic acid was determined by solidphase micro-extraction and liquid chromatography and was below the limit of determination (70 μg/L) in all samples. The European Union food tolerance limit for ochratoxin A in wine is 2 μg/L. A tolerance for tenuazonic acid has not yet been established.     

Activation markers and cell proliferation as indicators of toxicity: A flow cytometric approach

Cell proliferation is an attractive endpoint inin vitro toxicity assays, since nearly any kind of damage in a cell may result in altered cell proliferation. In toxicological applications, liquid scintillation counting, measuring radioactivity from tritiated thymidine, has been the traditional way to estimate cell proliferation. An alternative approach is the measurement of BrdU incorporation by flow cytometry. Before the actual DNA synthesis starts, several proteins are expressed on the cell surface, as well as intracellularly. Among the markers on the cell surface CD69, CD25, and CD71 are sequentially expressed on human lymphocytes after a mitogenic stimulation. The aim of this study was to evaluate information obtained by analysis of expression of activation markers on cell surfaces in lymphocyte subsets and to compare it with data from cell proliferation studies performed by liquid scintillation counting and BrdU flow cytometry. The experiments were performed with phytohemagglutinin-stimulated human lymphocytes exposed to ochratoxin A and cyclosporin A. While ochratoxin A-treated cultures showed a steep inhibition with increasing concentration, the cyclosporin A treatment gave an inhibition curve with a less steep slope. Activation marker studies showed that the effect of treatment with both of the toxins was more pronounced on the late markers CD25 and CD71, while CD69 had the advantage that significant effects could be detected as early as 6 h after ochratoxin A treatment. Cyclosporin A treatment induced only minor alterations in CD69 expression. Certain differences in expression of activation markers between CD4⁺ and CD8⁺ subsets were found both in ochratoxin A- and cyclosporin A-treated cultures. A stimulating effect was found in cell cultures exposed to the lowest concentration of ochratoxin A on CD69 and CD25 expression. Signs of an increase in frequencies of proliferating cells measured with the BrdU flow cytometry method were also seen. This increase could not be detected with liquid scintillation counting. No other differences between the liquid scintillation counting and BrdU flow cytometry measurements of proliferation were obtained. We conclude that studies of activation marker expression by the flow cytometric approach used in this report are useful complements to traditional measurements of cell proliferation as they yield subsetspecific information about cellular processes which precede proliferation of lymphocytes.Abbreviations A pulse area - BrdU bromodeoxyuridine - CD cluster of differentiation - FBS fetal bovine serum - FITC fluorescein isothiocyanate - FL fluorescence - FSC forward light scatter - H pulse height - PBS phosphate-buffered saline - PI propidium iodide - R-PE R-phycoerythrin - RPMI Roswell Park Memorial Institute - SEM standard error of the mean - SSC orthogonal light scatter - W pulse width     

Citrinin,ochratoxin A and iron. Possible implications for their biological function and induction of nephropathy

Experiments with Neisseria meningitidis have shown that Fe³⁺ to some extent can reverse the toxicity of ochratoxin A and citrinin, as measured by inhibition zones around impregnated paper discs. Similar phenomena were observed with the less toxic ochratoxin B. Zearalenone also inhibited growth, but its effect was not counteracted by iron. The mycotoxins aflatoxin B₁ and deoxynivalenol did not inhibit bacterial growth at all. Desferal (deferoxamine) also inhibited growth of meningococci, but iron totally abolished this inhibition. The results indicate that ochratoxin A and citrinin interfere with iron metabolism in this organism but that other additional toxic mechanisms are involved as well since a marked growth inhibition by both toxins was also observed in the presence of iron. One function of ochratoxin A and citrinin in nature could consequently be to affect the iron uptake of other competing microorgansms.Since both toxins interfere with iron and both cause nephropathy, a possible connection between these properties and lipid peroxidation is also briefly discussed.Abbreviations DON deoxynivalenol - OA ochratoxin A - OB ochratoxin B     

Detection and quantification of Aspergillus ochraceus in green coffee by PCR

AIMS: The aim of this study was to detect and quantify DNA of the ochratoxinogenic fungus Aspergillus ochraceus in green coffee and to compare the results with the ochratoxin A content of naturally contaminated samples. METHODS AND RESULTS: A DNA extraction protocol based on a combination of ultrasonification and a commercial kit was tested for the recovery of fungal DNA. PCR and real-time PCR protocols were established for the detection of A. ochraceus. Sensitivity of the PCR was checked by the addition of inoculated green coffee and pure fungal DNA to uncontaminated green coffee samples. The A. ochraceus DNA content of 30 naturally contaminated green coffee samples was determined and compared with the ochratoxin A concentrations. CONCLUSIONS: Aspergillus ochraceus can be rapidly and specifically detected in green coffee by PCR. A positive correlation between the ochratoxin A content and the DNA quantity was established. Significance and Impact of the Study: This work offers a quick alternative to the conventional mycological detection and quantification of A. ochraceus in green coffee.     

Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability

AIMS: To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A-producing species in European cereals, Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP). METHODS AND RESULTS: A total of 321 isolates of P. verrucosum were isolated from ochratoxin A-contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A-producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185 P. verrucosum isolates, thus 84% of the isolates may represent different genets of P. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP-identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual species P. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection of P. verrucosum showed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone. CONCLUSIONS: Among 184 ochratoxin A-producing Penicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the species P. verrucosum is phenotypically distinct and valid. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP fingerprinting of Penicillium verrucosum indicates that genetic recombination takes place in this fungus.     

Induction of micronuclei by ochratoxin A is a sensitive parameter of its genotoxicity in cultured cells

In order to better characterize the ochratoxin A (OTA)-induced DNA damage and to further investigate factors which may modulate dose-effect relationships in cells, the induction of micronuclei was studied in V79 Chinese hamster fibroblast cells and in primary cultures of porcine urothelial bladder epithelial cells (PUBEC). OTA was able to induce micronuclei in PUBEC and V79 cells at concentrations below those which were overtly cytotoxic. OTA concentrations between 0.03 and 1 μM caused a dose-dependent increase of micronuclei in V79 cells (up to 3-fold compared to controls); but the lowest tested concentration of 0.01 μM OTA did not induce a higher frequency of micronuclei than in the solvent control, indicative of an apparent threshold. Clear evidence for genotoxic effects was also found in PUBEC cultures treated with OTA concentrations of 1 μM and more, although the dose-effect relationship in PUBEC was more variable for several freshly isolated cell batches, pointing to differences in susceptibility to OTA between bladder cells from different donor animals. The chromosomal genotoxicity of OTA demonstrated in this study is in general accord with previous findings on the induction of clastogenic effects and oxidative DNA damage by OTA. In both cases, the shape of the dose-response curve at very low OTA concentrations supports the existence of a threshold for its genotoxicity. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Isolation and characterization of six polymorphic microsatellite loci in Aspergillus niger

Certain strains of Aspergillus niger produce ochratoxin A in food and in animal feeds. Six polymorphic microsatellite markers suitable for population analysis were developed for A. niger through screening published sequences for microsatellite repeats. Polymorphism was evaluated for 28 isolates of A. niger, including toxigenic strains. Loci displayed six to 13 alleles. Investigation of cross‐species amplifications with Aspergillus carbonarius and Aspergillus japonicus showed limited success.     

Bestimmung von Citrinin in Getreide

A simultaneous reversed-phase HPLC determination of citrinin and ochratoxin A in cereals is proposed. Both mycotoxins are eluted on a RP-amid C16 column using a gradient eluent acidified with phosphoric acid. The limits of detection, for a signal-to-background ratio of 3, are 1 μg/kg for citrinin and 0,4 μg/kg for ochratoxin A.

Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Vorkommen von ochratoxin A und B in gewürzen

A total of 681 samples of spices, which comprised more than 50 different spice commodities were analysed for the natural occurrence of the mycotoxins ochratoxin A (OTA) and ochratoxin B (OTB). The analytical method involved chloroform extraction, clean-up by immunoaffinity column and HPLC determination of both mycotoxins. OTA and OTB were detected in 143 (21%) and 68 (10%) of the samples, respectively. The highest frequency of occurrence of both mycotoxins detected were in chili (100% for OTA and 55% for OTB), paprika (41% and 15%, respectively) and pepper (23% and 44%, respectively). The toxin concentrations ranged between the detection limit (0.01 ng/g) and 41.8 ng OTA (2.7 ng OTB)/g of chili, 18.9 ng OTA (1.4 ng OTB)/g of paprika and 3.8 ng OTA (4.6 ng OTB)/g of pepper. One sample of a extract of vanilla was found to be positive for OTB at 15 ng/g. However, median values of most samples showed to be below the detection limit. Comparison of the geographical origin of the samples showed that the predominant number of contaminated spices was from Southeast-Asia and India. Highly contaminated paprika samples were found to come from Israel.     

Formation of ochratoxin A and aflatoxins following the use of propionic acid as a grain preservative for storing damp barley

The growth of storage moulds was studied in barley at 22% and approximately 28% moisture content treated with the recommended and reduced commercial doses of propionic acid over a 6 month storage period at 20°C. Experimental sample size was 5 kg barley per lot. Barley was fully protected against the growth ofA. flavus and aflatoxin formation when the recommended dose was applied. However, the treatment was less effective in controlling growth ofP. verrucosum and preventing ochratoxin A formation such that by 4 to 6 months of storage, the fungus had started to develop and toxin had formed even in some of the samples treated with propionic acid. The risk of the development of ochratoxin A during storage increased as the optimum dose was reduced, particularly for barley at 22% moisture content.     

Uptake and genotoxic effects of ochratoxin A in cultured porcine urinary bladder epithelial cells

Uptake of radiolabelled ochratoxin A (OTA) into porcine urinary bladder epithelial cells (PUBEC) was measured at neutral (pH 7.5) or acidic (pH 5.0) conditions. Genotoxicity of OTA was evaluated with the Comet assay and cytotoxicity with the neutral red uptake assay. At acidic pH-conditions, the bladder cells were able to take up more OTA than at neutral conditions. Cytotoxic effects were not increased at pH 5.0 compared to pH 7.5, but higher OTA uptake correlated with stronger genotoxic effects in the Comet assay at pH 5.0 compared to pH 7.5. These results demonstrate that uptake of OTA has to be regarded as an important factor for the toxicity of OTA as adverse effects depend on the amount of OTA taken up by the cells. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

赭曲毒素A的危害及其检测方法研究进展

从赭曲毒素的产生、毒性、危害,及其检测方法和防治等几个方面综述了赭曲毒素的研究进展,对防止赭曲毒素中毒及其检测方法提出了自己的观点。对赭曲毒素中毒的预防最重要,而预防前最重要的工作是做好对赭曲毒素的监督检测工作。ELISA方法是目前最有前景的检测方法,值得进一步研究。     

Effects of ochratoxin A metabolites on yeast phenylalanyl-tRNA synthetase and on the growth and in vivo protein synthesis of hepatoma cells

The ochratoxin A (OTA) metabolite (4R)-4-hydroxyochratoxin A [4R)-OTA) inhibits the aminoacylation of phenylalanine tRNA catalyzed by phenylalanyl-tRNA synthetase (PheRS) with a Ki-value of 0.9 mM as compared to 1.3 mM for OTA. It also inhibits protein synthesis and cell growth in the same manner as OTA. Ochratoxin alpha (OT alpha) does not affect either protein synthesis or cell growth.     

Effects of a long-term exposure with OTA or OTC on functions of a human monocytic cell line

An in-vitro model was developed to study the effects of a long-term exposure with a low concentration of ochratoxin A (OTA) or ochratoxin C (OTC) on a human monocytic cell line (THP-1). Cells were propagated in 24-well cell culture plates for 15 days. OTA and OTC preparations, respectively, at a concentration of 1 ng/ml were included in the cell culture medium during the whole cultivation period. At the end of the exposure time, parameters of cell viability and cell function were examined. After 15 days of exposure to ochratoxins, viability and function of the THP-1 cell line were modulated. Mitochondrial activity and the production of IL-6 were increased by all mycotoxin preparations. Cell membrane integrity was disturbed, proliferation and the production of TNF-α and IL-8 were inhibited. These parameters were most severely affected by mycotoxin preparations containing OTC. Our results show that long term exposure to OTA and especially OTC in low concentrations can cause subtle alterations of cell viability and function which may have remarkable consequences for human and animal health. In this context it seems to be necessary to study the contamination of food and feed stuffs with OTC more intensively. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

A molecular imprinted polymer with recognition properties towards the carcinogenic mycotoxin ochratoxin A

A molecularly imprinted polymer which recognises the mycotoxin ochratoxin A was prepared using the mimic N-(4-chloro-1-hydroxy-2-naphthoylamido)-(L) -phenylalanine as a template. The polymer was obtained by dissolving the template, methacrylic acid and ethylendimethacrylate in chloroform and polymerising the mixture by thermal treatment at 60°C. The monolith obtained was crushed, sieved to 30–90 m and extensively washed till the template could no longer be found in the washing solution. The binding properties towards the template, ochratoxin A and several related molecules were measured by eluting with acetonitrile and chloroform a HPLC column packed with the imprinted polymer. The experimental results show that the polymer recognises not only the template well, but also the ochratoxin A. The specific molecular recognition effect is due to hydrogen bond interactions but in order to assure the full recognition effect adjunctive steric factors are necessary. The magnitude of these interactions can be controlled by the use of limited amounts of acetic acid in the mobile phase.From the measurement of the relative selectivity it was found that only the simultaneous presence of the carboxyl, the phenolic hydroxyl and certain peculiar substructures such as the chlorine atom assures the whole recognition of the template.