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[The detection of Alternaria mycotoxins in laboratory culture].

Analytical methods are described for detection of the Alternaria mycotoxins alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT) and tenuazonic acid (TeA) in natural and semisynthetic laboratory cultures. After extraction and purification of the crude extract by column chromatography on silica gel the qualitative and quantitative analyses were carried out by thin layer (TLC)- and high performance liquid chromatography (HPLC). HPLC separations were achieved using a Hypersil ODS column with methanol/water containing a complexing agent as eluent. Detection at 340 nm (AOH, AME, ALT) and 280 nm (TeA), respectively, has proved to be favourably. AME and TeA were produced in high purity and high yields as standard substances by two Alternaria strains. The identity of the toxins could be confirmed by EI-, CI- and FAB-mass spectrometry.

Alternaria↗

[The combined toxity of two kinds of mycotoxin in Sprague-Dawley rats].

OBJECTIVE: To study the combined toxic effects of fumonisin B(1) (FB(1)) and aflatoxin B(1) (AFB(1)) on Sprague-Dawley (SD) rats. METHOD: All 60 SD male rats were divided into five groups randomly according to the body weight (12 every group). They were given FB(1) (100 microg/kg bw), AFB(1) (100 microg/kg bw), FB(1) plus AFB(1) (100 microg/kg bw respectively), FB(1) plus AFB(1) (50 microg/kg bw respectively) and distilled water respectively by gavage. The experiment persisted 30 days to observe the changes of growth and development, the food used rate, the haematological indexes, the blood biochemical indexes and the viscera histopathology. RESULTS: At the end of the experiment, the mean body weight increased in the FB(1) plus AFB(1) (100 microg/kg bw respectively) group was (164.9 +/- 19.8) g and the mean body weight increased in the control group was (203.7 +/- 17.1) g. And the food used rate in the FB(1) plus AFB(1) (100 microg/kg bw respectively) group was (25.3 +/- 1.6)% and the food used rate in the control group was (28.1 +/- 1.2)%. There were significant differences in the mean body weight increased and the food used rate between the FB(1) plus AFB(1) (100 microg/kg bw respectively) group and the control group (P < 0.05). While there were no significant differences of body weights and food used rates between controls and AFB(1), FB(1), and low dose AFB(1) + FB(1) groups (P > 0.05). The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutaminetransferase (gamma-GT) in serum of all of the treatment groups were increased, but the increasing extent was severe in the AFB(1) + FB(1) high dose group. At the same time the liver weight and kidney weight were decreased and the liver occurred with the remarkable histopathological lesions in the AFB(1) + FB(1) high dose group. The activity of superoxide dismutase (SOD) in serum was decreased and the level of malondialdehyde (MDA) in serum was elevated in treatment groups. CONCLUSIONS: The combined toxic effects of AFB(1) and FB(1) existed in male SD rats. Our results provided the basic data for studying the combined effects on human exposed to these two mycotoxin at the same time.

Aflatoxin B1↗

The biosynthesis of polyketide-derived mycotoxins.

The biosynthesis of a few representative polyketide-derived mycotoxins is reviewed: patulin, citrinin, diplosporin, ochratoxin A, maltoryzine, xanthomegnin, cytochalasans, ergochromes, zearalenone, citreoviridin, and the aflatoxins. Particular attention is given to the biosynthetic sequence: acetate----averufin----versiconal acetate----versicolorin A----sterigmatocystin----aflatoxin B1.

Mycotoxins↗

[A review of the studies on fungi and mycotoxins in foodstuffs in Beijing during 1976-1986].

Fungi and mycotoxins contaminating foods in Beijing during 1976-1986 were studied. A total of more than 5,000 food samples classified into 104 kinds of 16 types were examined of analysed. The results showed that the natural occurrence of Aflatoxin B1 in foods in Beijing was not serious, but from the results of analysis 59 cases of food poisoning and food seriously contaminated with moulds, the hazards of fungal contamination of food should receive serious consideration. In order to improve food hygiene and facilitate supervision the author suggested that a mycological standard should be established for marketing foods in China, such as grain, and beverage which are highly susceptible to fungal contamination.

Aflatoxin B1↗

Changes in transmitter release patterns in vitro induced by tremorgenic mycotoxins.

The neurochemical effects of the tremorgenic mycotoxins Verruculogen and Penitrem A, which produce a neurotoxic syndrome characterized by sustained tremors, were studied using sheep and rat synaptosomes. The toxins were administered in vivo, either by chronic feeding (sheep) or ip injection 45 min prior to sacrifice (rat). Synaptosomes were subsequently prepared from cerebrocortical and spinal cord/medullary regions of rat, and corpus striatum of sheep. Penitrem A (400 mg mycelium/kg) increased the spontaneous release of endogenous glutamate, GABA, and aspartate by 213%, 455%, and 227%, respectively, from cerebrocortical synaptosomes. Verruculogen (400 mg mycelium/kg) increased the spontaneous release of glutamate and aspartate by 1,300% and 1,200% respectively, but not that of GABA, from cerebrocortical synaptosomes. The spontaneous release of the transmitter amino acids or other amino acids was not increased by the tremorgens in spinal cord/medullary synaptosomes. Penitrem A pretreatment reduced the Veratrine (75 microM) stimulated release of glutamate, aspartate and GABA from cerebrocortical synaptosomes by 33%, 46%, and 11% respectively, and the stimulated release of glycine and GABA from spinal cord/medulla synaptosomes by 67% and 32%, respectively. Verruculogen pretreatment did not alter the Veratrine-induced release of transmitter amino acids from cerebrocortex and spinal cord/medulla synaptosomes. Penitrem A pretreatment increased the spontaneous release of aspartate, glutamate and GABA by 68%, 62%, and 100%, respectively, from sheep corpus striatum synaptosomes but did not alter the synthesis and release of dopamine in this tissue. Verruculogen was shown to cause a substantial increase (300-400%) in the miniature-end-plate potential frequency at the locust neuromuscular junction. The response was detectable within 1 min, rose to a maximum within 5-7 min, and declined to the control rate over a similar period. No change in the amplitude of the m.e.p.p.s was observed. These effects of the tremorgens on transmitter release are interpreted in terms of their mode of action.

Amino Acids↗

Mycotoxin research in the Hungarian Central Veterinary Institute.

Mycotoxin research conducted in the Hungarian Central Veterinary Institute is reviewed briefly. The effect of zearalenone was studied by experiments in several animal species (cattle, pig, sheep, chicken, goose, duck, guinea fowl, fish) and in cell cultures in vitro. The chicken (Gallus domesticus) has proved to be resistant to the toxin. In the susceptible species zearalenone causes the most severe damage to the sexual organs. The metabolism of zearalenone, T-2 toxin and diacetoxyscirpenol was also studied. These toxins are resistant to physicochemical factors but are easily transformed in biologically active environment. The acute toxicity study of trichothecene toxins is described along with morphological changes, with particular respect to those of the lymphoid and myeloid organs. Trichothecene toxins impair the natural defence mechanism of the organism and result in the manifestation of different diseases (swine dysentery, caecal coccidiosis). Deoxynivalenol contamination of the feeds, first of all of wheat, is common in Hungary. Its effect on pig fattening was investigated in a field trial. The toxins of different storehouse moulds were also examined.

Animals↗

Decontamination and detoxification of mycotoxins.

Product decontamination and chemical detoxification are needed because preventive measures are not fully able to avoid contamination by mycotoxins. Criteria for safety evaluation studies of decontaminated products have to be established. Few chemical methods are available on an industrial scale; among them, ammoniation and the mixture monomethylamine-calcium hydroxide treatments show greatest promise of short-term application to oilseed cakes. Technical, economic, and public health aspects of these treatments are considered. Other decontamination techniques are briefly reviewed.

Decontamination↗

In vitro effects of cyclopiazonic acid mycotoxin on turkey papillary muscles.

An in vitro bioassay system was used to study the effects of cyclopiazonic acid (CPA) mycotoxin on cardiac muscle. Acute exposure to 6 micrograms of CPA/ml of modified Krebs-Henseleit solution significantly (P less than 0.05) decreased 5 in vitro turkey cardiac muscle performance criteria: maximal weight a muscle could lift; maximal contraction velocity; relaxation velocity; time to peak contraction; and total time for muscle contraction and relaxation. The effect on these 5 criteria appeared to result from intracellular changes partially associated with calcium availability and were irreversible, suggesting that physiologic changes had developed after acute exposure to CPA.

Animals↗

Mycotoxin bioassay, using Bacillus stearothermophilus.

Spores of Bacillus stearothermophilus in standardized spore strips are pretreated with solutions of the mycotoxins aflatoxin B1, patulin, rubratoxin B, and diacetoxyscirpenol and subsequently incubated in a nutrient solution containing bromocresol purple as pH indicator. After 16.5 hr of inclubation the color of the indicator medium inoculated with untreated spore strips of B. stearothermophilus changes from purple to yellow; no color change occurs in the indicator medium inoculated with spore strips treated 15 min with 0.01 mug of any of the 4 mycotoxins/ml during a 60 hr incubation.

Aflatoxins↗

[Conditions for the formation of mycotoxins in animal feed].

The development of moulds and the production of mycotoxins are closely related, but the optima of environmental factors for both do not necessarily coincide. Depending on the species, the percentage of toxinogenic strains differs considerably. Complex substrates, with a high content of carbohydrates, are favoured. Over a wide range of temperature toxin-production is possible, also at a very low partial pressure of oxygen. Too few is known about the factors, which promote toxin-production in the field. There seems to be a dependence on the area, where the grain is grown, and on the weather during the year of cropping, but the influence of methods of plant production, fertilization, plant protection and choice of cultivars have to be investigated.

Animal Feed↗

[A biological method for the determination of mycotoxins--Tetrahymena pyriformis].

The toxic action of selected mycotoxins on a biological subject was tested in the protozoan Tetrahymena pyriformis. The following toxins were tested: toxin T-2 at doses from 1.52 micrograms to 100.0 mg per litre of medium, ochratoxin A at doses from 12.21 micrograms to 800.0 mg per litre of medium, and rubratoxin B at doses from 12.21 micrograms to 800.0 mg per litre of medium. In toxin T-2 the LD100 was found to be about 390.63 micrograms and LD50 about 48.83 micrograms per litre of medium. In ochratoxin A the LD100 was about 25.0 mg and LD50 about 3.13 mg per litre of medium. In rubratoxin B the LD100 was about 200.0 mg and LD50 about 25.0 mg per litre of medium.

Biological Assay↗

Use of ten gram samples of corn for determination of mycotoxins.

Data were gathered, during a study on the development of an automated system for the extraction, cleanup, and quantitation of mycotoxins in corn, to determine if it was scientifically sound to reduce the analytical sample size. Five, 10, and 25 g test portions were analyzed and statistically compared with 50 g test portions of the same composites for aflatoxin concentration variance. Statistical tests used to determine whether the 10 and 50 g sample sizes differed significantly showed a satisfactory observed variance ratio (Fobs) of 2.03 for computations of pooled standard deviations; paired t-test values of 0.952, 1.43, and 0.224 were computed for each of the 3 study samples. The results meet acceptable limits, since each sample's t-test result is less than the published value of the /t/, which is 1.6909 for the test conditions. The null hypothesis is retained since the sample sizes do not give significantly different values for the mean analyte concentration. The percent coefficients of variation (CVs) for all samples tested were within the expected range. In addition, the variance due to sample mixing was evaluated using radioisotope-labeled materials, yielding an acceptable CV of 22.2%. The variance due to the assay procedure was also evaluated and showed an aflatoxin B, recovery of 78.9% and a CV of 11.4%. Results support the original premise that a sufficiently ground and blended sample would produce an analyte variance for a 10 g sample that was statistically comparable with that for a 50 g sample.

Aflatoxins↗

Mutagenicity and antibacterial activity of mycotoxins produced by Penicillium islandicum Sopp and Penicillium rugulosum.

Twelve mycotoxins produced by Penicillium islandicum Sopp and Penicillium rugulosum in solid-state fermentation on grains were purified and tested for mutagenicity and antibacterial activity in Salmonella/mammalian microsome assays. The mutations studied were reversions of histidine auxotrophs to prototrophy in strains TA98 and TA100 and forward mutations to 8-azaguanine resistance (8AGR) in strain TM677. Rubroskyrin, (+)rugulosin, lumiluteoskyrin [a photoproduct of (-)luteoskyrin], and simatoxin [a new water-soluble metabolite of unknown structure] induced 8AGR mutations in strain TM677 but not histidine reversions in strains TA98 and TA100. Mutagenic potency was reduced by rat-liver microsomes. The carcinogens (-)luteoskyrin and cyclochlorotine were antibacterial but not mutagenic. (+)Rugulosin, rubroskyrin, lumiluteoskyrin, and high concentrations of simotoxin were also antibacterial. Antibacterial activity but not mutagenicity was observed with pibasterol and skyrin. Chrysophanol, islandicin, iridoskyrin, and emodin were inactive as mutagens or as antibacterial agents.

Animals↗

Use of small charcoal/alumina cleanup columns in determination of trichothecene mycotoxins in foods and feeds.

Small charcoal/alumina cleanup columns have been effectively used to remove interfering materials from grain, feed, and food extracts prior to chromatographic determination of trichothecene mycotoxins. A thin layer chromatographic method has been developed that can simultaneously detect part per billion concentrations of deoxynivalenol, fusarenon X, nivalenol, T-2 toxin, HT-2 toxin, neosolaniol, and diacetoxyscirpenol in food and feed samples. Recoveries of 90-99% can be obtained. The use of charcoal/alumina cleanup columns in conjunction with liquid chromatography and gas chromatography of trichothecenes is also discussed.

Aluminum Oxide↗

Mycotoxins in animal feedstuffs: sensitive thin layer chromatographic detection of aflatoxin, ochratoxin A, sterigmatocystin, zearalenone, and T-2 toxin.

Improvements have been made to a previously described multi-mycotoxin method that involved a membrane cleanup step. Using 2-dimensional thin layer chromatography and appropriate solvent systems, aflatoxin B1 can be detected in mixed feedstuffs and various ingredients at levels ranging from 0.1 to 0.3 microgram/kg. Corresponding detection limits for ochratoxin A and sterigmatocystin are 5 to 20 microgram/kg and for T-2 toxin and zearalenone 20 to 200 microgram/kg.

Aflatoxins↗

Sample preparation of some shelled treenuts and peanuts in a vertical cutter-mixer for mycotoxin analysis.

A procedure has been devised for more rapid preparation of large samples of nutmeats (10-80 lb) for mycotoxin analysis without the use of grinding aids, by combining the grinding and mixing steps into one operation. Ease of cleaning equipment facilitates the preparation of several samples in a short time. The procedure was tested using shelled raw and roasted peanuts, and raw almonds, walnuts, and pecans. The reduction of particle size to pass a 20 mesh screen was attained using a 25 qt Hobart vertical cutter-mixer (VCM) equipped with standard serrated blades and a 40 qt VCM using either a standard serrated blade or a smooth-edge blade modified with sharp-edge notches to increase its cutting ability. Portions of ground composite were removed at various time intervals and particle sizes were measured. Original and check analyses of 12 naturally contaminated samples over a 3 year period indicate that this procedure is practical and reproducible.

Arachis↗

[Recent knowledge on the mechanism of action and metabolism of mycotoxins].

By investigations on domestic and laboratory animals great progress was achieved in the clarification of the mechanism of action and of the metabolism of mycotoxins. The aflatoxins which inhibit the synthesis of RNA in the liver and which lead to a liver necrosis are all over the world of great importance as harmful substances for man and animals. The chronic intake in subtoxic doses has a tumour-evoking effect. The amatoxins of the green Amanita phalloides are also hepatotoxic: they inhibit the synthesis of messenger RNA in the hepatocytes. The ergot alcaloids unite with alpha-adrenoceptors and evoke an inhibition of the effectiveness of beta-adrenoreceptors: they effect a vasoconstriction, a contraction of the uterus and an inhibition of the prolactin secretion. The ochratoxins have an influence on the transcription and have a nephrotoxic and teratogenic effect. The trichothezene toxins inhibit the protein synthesis and already in a low dose decrease the formation of coagulation factors and of immunoglobulins. The zearalenon has an estrogen effect: it is bound to estrogen receptors and influences the transcription in the cell nuclei. Patulin has a tumour-evoking effect. Nutrients infested by fungi are to be excluded from the consumption by man.

Aflatoxins↗