Krimpsiekte in a sheep following a single dose of Tylecodon ventricosus (Burm. f.) Toelken and the isolation of tyledoside D from this plant species.

Tylecodon ventricosus induced severe respiratory distress in two penned sheep without any electrocardiographic abnormalities being recorded. Based on the results it appears as if T. ventricosus predominantly induces the neuromuscular syndrome referred to as krimpsiekte. A single, relatively large intraruminal dose of 10.0 g/kg induced krimpsiekte in one sheep. Treatment with 5.0 g/kg activated charcoal on two consecutive days did not prevent the development of krimpsiekte. A bufadienolide, tyledoside D, was isolated from semi-dried plant material.

Krimpsiekte, associated with thalamic lesions, induced by the neurotoxic cardiac glycoside, cotyledoside, isolated from Tylecodon wallichii (Harv.) Toelken subsp. wallichii.

The specific neurotoxic principle of Tylecodon wallichii (Harv.) Toelken subsp. wallichii, the cause of krimpsiekte in small stock, was isolated and identified as the previously described cumulative bufadienolide, cotyledoside. Krimpsiekte was experimentally induced in two sheep by the repeated intravenous administration of cotyledoside at the rate of 0.01-0.015 mg/kg body mass. On day 9, both animals developed clinical signs typical of krimpsiekte, which is characterized by tremors, paresis and recumbency. Both sheep had difficulty in controlling their hindquarters when attempting to lie down. No significant electrocardiograph abnormalities were detected during the experiment which confirms that cotyledoside at low doses does not overtly affect the electrical activity of the heart. No gross lesions were observed in the sheep. The most significant microscopic lesions comprised mild brain oedema and pronounced vacuolation of the white matter of thalamic nuclei. These lesions might explain some of the motor function deficiencies clinically observed in this syndrome. The previously held contention that these neurotoxic cardiac glycosides are indeed the cause of krimpsiekte is, therefore, confirmed.

Physicochemical data for some selected Fusarium toxins.

Fusarium toxins are a major group of secondary metabolites, produced by several species, that may contaminate food cereals and animal feeds. We describe results of a study in which a number of physicochemical constants for 12 Important Fusarium mycotoxins (zearalenone, diacetoxyscirpenol, T-2 toxin, neosolaniol monoacetate, deoxynivalenol, nivalenol, fumonisin B1, fumonisin B2, moniliformin, fusarenon-X, HT-2 toxin, and beta-zearalenol) were determined. Nuclear magnetic resonance, mass spectrometric, UV spectral, molar absorption coefficients, fluorescence spectra, melting points, and specific rotation data are presented.

Determination of the mycotoxin fumonisin B1 and identification of its partially hydrolysed metabolites in the faeces of non-human primates.

A method has been developed for the determination of fumonisin B1 (FB1) in the faeces of non-human primates (vervet monkeys). The animals were dosed with 14C-labelled FB1, and the radioactive compounds in faeces were recovered by repeated extractions with 0.1 M ethylenediaminetetraacetic acid. The extracts were cleaned-up on a reversed-phase (C18) solid-phase extraction cartridge, and FB1 was determined by o-phthaldialdehyde derivatization and reversed-phase HPLC. The analytical method for the determination of FB1 in the faecal extracts was reproducible [2.6% relative standard deviation (RSD)] and accurate (recovery from spiked blank extracts of 93 +/- 2.9% RSD). Confirmation of the identification of FB1 in faeces was achieved using HPLC and thin-layer chromatography, which showed that the radioactivity extracted corresponded mainly to FB1 and a new metabolite with chromatographic properties similar to those of the mycotoxin. The new metabolite was identified by mass spectrometry and nuclear magnetic resonance spectroscopy to be an equilibrium mixture of the two structural isomers of partially hydrolysed FB1, which are formed by hydrolysis of one of the ester groups of the mycotoxin.

Production of [14C]fumonisin B1 by Fusarium moniliforme MRC 826 in corn cultures.

Kinetics of growth and fumonisin production by Fusarium moniliforme MRC 826 in corn "patty" cultures were investigated, and a technique was developed for the production of [14C]fumonisin B1 ([14C]FB1) by using L-[methyl-14C]methionine as the precursor. A significant (P < 0.01) correlation exists between fungal growth and FB1 (r = 0.89) and FB2 (r = 0.87) production in corn patties, beginning after 2 days and reaching the stationary phase after 14 days of incubation. [14C]FB1 was produced by adding L-[methyl-14C]methionine daily to cultures during the logarithmic phase of production. Incorporation of the isotope occurred at C-21 and C-22 of the fumonism molecule and was enhanced in the presence of unlabeled L-methionine. Although the concentration of exogenous unlabeled methionine is critical for incorporation of the 14C label, optimum incorporation was achieved by adding 50 mg of unlabeled L-methionine and 200 mu Ci of L-[methyl-14C]methionine to a corn patty (30 g) over a period of 9 days, yielding [14C]FB1 with a specific activity of 36 mu Ci/mmol.

Structure-activity relationships of fumonisins in short-term carcinogenesis and cytotoxicity assays.

A short-term rat liver cancer initiation/promotion model was used to monitor the cancer-initiating activity of the mycotoxins fumonisin B1 (FB1), fumonisin B2 (FB2) and fumonisin B3 (FB3) as well as the N-acetyl derivatives of FB1 and FB2, and their respective hydrolysis products the aminopolyols. The induction of resistant hepatocytes, which develop into hepatocyte nodules on selection by the 2-acetylaminofluorene-partial hepatectomy promoting treatment, was taken as the endpoint for cancer initiation. When fed at a level of 1000 mg/kg diet for 21 days, only the fumonisins B were found to initiate cancer. In addition, these mycotoxins caused a marked reduction in the rat body weight during the initiating treatment. Comparative cytotoxicity studies in primary rat hepatocytes indicated that FB2 exhibited the highest cytotoxic effect followed by FB3 and FB1. In general, the fumonisin B mycotoxins exhibited a low cytotoxic effect in hepatocyte cultures, and the concentrations of FB1 and FB2 that caused a 50% (CD50) release of the total lactate dehydrogenase (LDH) were in the order of 2000 and 1000 microM, respectively. The N-acetyl derivatives also exhibited a cytotoxic effect, but were not as cytotoxic as the parent molecules at high concentrations. The respective aminopolyols exhibited a higher cytotoxicity than did the parent compounds, while tricarballylic acid (TCA) exhibited no dose-response effect despite the fact that it had a higher background cytotoxicity compared with the control. The apparent inability of the aminopolyols to act as cancer initiators could be related to a lack in absorption from the gut.(ABSTRACT TRUNCATED AT 250 WORDS)

Fumonisins: isolation, chemical characterization and biological effects.

The fumonisin B mycotoxins (FB1 and FB2) have been purified and characterized from corn cultures of Fusarium moniliforme strain MRC 826. Fumonisin B1 (FB1), the major fumonisin produced in culture, has been shown to be responsible for the major toxicological effects of the fungus in rats, horses and pigs. Recent investigations on the purification of compounds with chromatographic characteristics similar to FB1 have led to the identification of two new fumonisins, FB3 and FB4. Fumonisins A1 and A2, the N-acetyl derivatives of FB1 and FB2 respectively, were also purified and shown to be secondary metabolites of the fungus. Short-term carcinogenesis studies in a rat liver bioassay indicated that over a period of 15 to 20 days, at dietary levels of 0.05-0.1%, FB2 and FB3 closely mimic the toxicological and cancer initiating activity of FB1 and thus could contribute to the toxicological effects of the fungus in animals. In contrast, no biological activity could be detected for FA1 under identical experimental conditions. These studies and others have indicated that the fumonisin B mycotoxins, although lacking mutagenicity in the Salmonella test or genotoxicity in the DNA repair assays in primary hepatocytes, appear to induce resistant hepatocytes similar to many known hepatocarcinogens.

Reversed-phase high-performance liquid chromatography of tenuazonic acid and related tetramic acids.

A reversed-phase high-performance liquid chromatographic system for the determination of the fungal toxin, tenuazonic acid, (5S,8S)-3-acetyl-5-sec.-butyltetramic acid, is described. The system utilizes a column packed with deactivated end-capped C18 silica with a high carbon load to overcome the problem of poor chromatographic performance of this beta-diketone on reversed-phase liquid chromatography which previously necessitated the use of anion-exchange, ligand-exchange or ion-pairing methods. The reversed-phase system allows the separation of tenuazonic acid from its (5R,8S)-diastereomer, allo-tenuazonic acid and was applied to the detection of tenuazonic acid in cultures of Alternaria alternata and Phoma sorghina. By means of diode-array ultraviolet detection, (5S)-3-acetyl-5-isopropyltetramic acid was observed in extracts of culture material. This metabolite was purified using the analytical reversed-phase system and was identified by 1H and 13C nuclear magnetic resonance spectroscopy.

Large-scale purification of the mycotoxins aflatoxin B1, B2 and G1.

The isolation and purification of gram quantities of the important mycotoxins aflatoxin B1, B2 and G1 are described. The method involves final purification on a Waters Prep LC-500 instrument, loaded with silica cartridges, and elution with chloroform.

Fumonisins--novel mycotoxins with cancer-promoting activity produced by Fusarium moniliforme.

Cultures on corn of Fusarium moniliforme MRC 826 are known to cause leukoencephalomalacia in horses and to be toxic and hepatocarcinogenic in rats. Culture material of this F. moniliforme isolate has also been shown to exhibit cancer-promoting activity in a short-term cancer initiation-promotion bioassay with diethylnitrosamine-initiated rats and the induction of gamma-glutamyl-transpeptidase-positive (GGT+) foci as an endpoint after 4 weeks of promotion. This bioassay was used as a monitoring system to isolate cancer-promoting compounds from cultures of F. moniliforme MRC 826. Culture material was successively extracted with ethyl acetate and CH3OH-H2O (3:1). Most of the cancer-promoting activity was recovered in the CH3OH-H2O extract and remained in the aqueous phase following partitioning of this extract between CH3OH-H2O (1:3) and CHCl3. The CH3OH-H2O fraction was chromatographed on an Amberlite XAD-2 column, and the active fraction was eluted with CH3OH. This fraction was chromatographed on a silica gel column with CHCl3-CH3OH-CH3COOH (6:3:1) as eluent and further purified on a C18 reverse-phase column. Two pure compounds were isolated, and these have been chemically characterized and given the trivial names fumonisin B1 and B2. At least 2 g of the major compound fumonisin B1 was purified from 1 kg of culture material. Fumonisin B1 in the diet (0.1%) significantly (P less than 0.001) induced the formation of GGT+ foci in the livers of initiated as well as noninitiated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of the effect of ochratoxin A analogues on yeast aminoacyl-tRNA synthetases and on the growth and protein synthesis of hepatoma cells.

Ochratoxin A (OTA), a naturally occurring mycotoxin of Aspergillus and Penicillium species, consists of a 5' chlorinated dihydromethyl isocoumarin linked to L,beta-phenylalanine by an alpha-amide bond. 8 analogues of OTA were prepared in which the phenylalanine was always substituted by another amino acid. The effects of these analogues on yeast tRNA amino acylation reaction and on growth and protein synthesis of hepatoma culture cells were compared with those of OTA. In addition, Ochratoxin B (OTB) and ochratoxin alpha (OT alpha) were examined. All the analogues of OTA had inhibitory effects in the 3 test systems, although to a lesser degree than OTA. The degree of inhibition depended on the kind of substituted amino acid, the tyrosine, valine, serine and alanine analogues being most effective, in contrast to the proline analogue. OTB and OT alpha were ineffective.

Moniliformin production and toxicity of different Fusarium species from Southern Africa.

Four new moniliformin-producing species of Fusarium were found, viz., F. acuminatum, F. concolor, F. equiseti, and F. semitectum. Isolates of F. acuminatum and F. concolor produced large amounts of moniliformin (3.4 and 9.5 g/kg, respectively), whereas isolates of the other three species yielded less than 30 mg/kg. The production of moniliformin by isolates of F. oxysporum and F. avenaceum from southern Africa is described. All 14 toxic isolates of F. oxysporum produced moniliformin. Most isolates of F. fusarioides and all six isolates of Fusarium moniliforme va. subglutinans tested produced moniliformin, as did 28 of 36 toxic isolates of F. moniliforme. A number of F. moniliforme isolates produced greater than 10 g/kg, and one isolate yielded 33.7 g/kg in corn after incubation for 5 weeks at 25 degrees C. Moniliformin production in the field in corn ears was shown by inoculating plants with known moniliformin-producing isolates of three Fusarium species. Yields of up to 645 mg/kg were recorded. Isolates of F. acuminatum, F. equiseti, F. fusarioides, and F. moniliforme were found that were highly toxic to ducklings but which did not produce moniliformin.

Mutagenicity of 5,6-dimethoxysterigmatocystin, a metabolite from Aspergillus multicolor, in the Salmonella/microsome system.

The natural sterigmatocystin derivative, 5,6-dimethoxysterigmatocystin, was found to be a mutagen for Salmonella typhimurium strains TA98 and TA100 after metabolic activation in a mammalian microsome system.