Pieter S. Steyn

Tremorgenic neurotoxins from perennial ryegrass causing ryegrass staggers disorder of livestock: structure elucidation of lolitrem B

The lolitrems, tremorgenic neurotoxins from perennial ryegrass, are implicated in ryegrass staggers disorder in livestock; the assignment of structure (1) to the major neurotoxin, lolitrem B, is based on its spectroscopic properties, particularly a detailed structure of its high-field 1H and 13C n.m.r. spectra, as well as chemical evidence.

Characterization of magnesium and calcium tenuazonate from Phoma sorghina

Abstract Phoma sorghina Sacc., the fungus implicated in the aetiology of onyalai, a haematologic disorder, produces magnesium and calcium tenuazonate as toxic constituents.

Tremorgenic mycotoxins from Penicillium crustosum: isolation of penitrems A–F and the structure elucidation and absolute configuration of penitrem A

The isolation and characteristics of six tremorgenic mycotoxins, penitrems A–F from cultures of Penicillium crustosum are reported. The assignment of structure (2) to penitrem A is based on a detailed study of its high-field 1H and 13C n.m.r. spectra. The conformation and relative configuration of penitrem A was deduced from the observed proton–proton nuclear Overhauser effects (n.O.e.) and the magnitude of the proton–proton coupling constants. The chirality of C-25 was determined as S by the ‘partial resolution’ method of Horeau and penitrem A must therefore have the (12R, 14S, 15R, 18S, 19R, 22S, 23S, 24R, 25S, 26R, 28S, 31R, 32S) absolute configuration.

Structure elucidation of fusarin C, a mutagen produced by Fusarium moniliforme

The assignment of structure (1) to fusarin C, a mutagen isolated from cultures of Fusarium moniliforme is based on a detailed study of its high-field 1H and 13C n.m.r. spectra and X-ray crystallography of the 8Z isomer of (1) which defined the substitution pattern and relative configuration of the 2-pyrrolidone moiety; nuclear Overhauser enhancement experiments indicate that the 2E,4E,6E,8E,10E polyene chromophore of (1) exists in solution as an equilibrium between two conformers with s-cis and s-trans topology of the C-5–C-6 single bond.

Regional selectivity to ochratoxin A, distribution and cytotoxicity in rat brain.

Abstract Ochratoxin A (OTA) a chlorodihydro-isocoumarin linked through an amide bond to phenylalanine, is a mycotoxin found as a contaminant in foodstuffs and shown to be nephrotoxic, teratogenic, immunosuppressive, genotoxic, mutagenic and carcinogenic in rodents. Ochratoxin A is known to induce teratogenic effects in neonates (rats and mice) exposed in utero, characterised by microcephaly and modification of the brain levels of free amino acids. Since OTA has been found to accumulate in the brain according to the duration of exposure to doses in the range of natural contamination of feedstuffs, experiments were designed to determine more precisely the structural target of OTA in the brain. After intracerebral injection, OTA (403 ng/10 μl) was not found in the following parts of the brain : the frontal cortex (FC), striatum (ST), ventral mesencephalon (VM) and the cerebellum (CB) in contrast to the rest of the brain, probably due to the detection limit of 0.1 ng/g of tissue. However lactate dehydrogenase (LDH) was increased in extracellular space in the VM to a greater extent than in the rest of the brain, indicating that this structure could be one of the targets of OTA in the brain. Contents of free amino acids were morever similarly modified in the VM and in the rest of the brain. Male rats were given OTA (289 μg/kg per 24 h) by gastric intubation for 8 days and the main brain structures analysed for OTA content and cytotoxicity. OTA was found in the following structures in decreasing order: rest of the brain (50.3%), cerebellum (34.4%), VM (5.1%), striatum (3.3%) and hippocampus (2.9%) of the total OTA amount found in the brain, which represents 0.022% to 0.028% of the given dose. Interestingly cytotoxicity as measured by lactate dehydrogenase (LDH) release in the extracellular space was much more pronounced in the VM, hippocampus, and striatum than in the cerebellum, whereas no cytotoxicity was observed in the rest of the brain. Similarly deoxyribonuclease (DNase) activity in relation to possible necrotic cells was increased in the VM and cerebellum. Altogether these results designated the ventral mesencephalon, hippocampus, striatum and cerebellum as the main OTA-targets in the brain of adult rats and excluded the rest of the brain.

Prevention by vitamin E of DNA fragmentation and apoptosis induced by fumonisin B1 in C6 glioma cells

Abstract Fumonisin B1 (FB1), produced by the fungus Fusarium moniliforme, belongs to a class of sphingosine analogue mycotoxins that occur widely in the food chain. Epidemiological studies have associated consumption of Fusarium moniliforme-contaminated food with human oesophageal cancer in China and South Africa. FB1 also causes equine leucoencephalomalacia. Evidence for induction of apoptosis by FB1 was first obtained when C6 glioma cells were incubated with fumonisin B1 (3–27 μM) causing DNA fragmentation profiles showing DNA laddering in gel electrophoresis and apoptotic bodies revealed by chromatin staining with acridine orange and ethidium bromide. Further confirmation experiments and comet assays have been performed under similar conditions. The results of the comet test show that FB1 at 9 and 18 μM induces respectively 50 ± 2% and 40 ± 1% of cells with a comet with an increased tail length of 93 ± 9 μm and 102 ± 17 μm respectively. Under these concentrations, FB1 induced DNA fragmentation and laddering and many apoptotic bodies. Pre-incubation of the cells with vitamin E (25 μM) for 24 h before FB1 (18 μM) significantly reduced DNA fragmentation and apoptotic bodies induced by FB1.

Tremorgenic mycotoxins from Penicillium crustosum. Biosynthesis of penitrem A

The biosynthesis of penitrem A has been studied with both 13C- and 2H-labelled precursors, viz. [1-13C]-, [2-13C]-, [1,2-13C2]-, and [1-13C,2-2H3]-acetate, [2-13C]-, [2,3-13C2]-, [2-2H2]- and [5-2H2]-mevalonate. The results show that penitrem A is derived from tryptophan, which contributes the indole moiety of the metabolite, geranylgeranylpyrophosphate, and two isopentenylpyrophosphate units. A 1,2-bond migration, involving the 2,3-bond of a mevalonate unit, occurs in the course of the biosynthesis and results in the observation of a one-bond (C,C) coupling between two [1-13C]acetate-derived carbon atoms and between two [2-13C]acetate-derived carbon atoms. Analysis of the one-bond (C,C)coupling constants in [2-13C]acetate-derived penitrem A showed that [1,2-13C2]acetate was formed during the fermentation. Although loss of water from an hydroxyisopropyl group to form the isopropenyl function present in penitrem A should proceed with retention of the stereochemical integrity of the two methyl groups, isomerization of the double bond causes equal distribution of 13C label between C-36 and C-38 and precludes any stereochemical deductions.

Structure and biosynthesis of the penitrems A–F, six novel tremorgenic mycotoxins from Penicillium crustosum

The structures of six new mycotoxins, penitrems A–F, isolated from cultures of Penicillium crustosum are deduced from their 13C and 1H n.m.r. spectra and from biosynthetic results.

Selective toxicity of ochratoxin A in primary cultures from different brain regions

Abstract Ochratoxin A (OTA) is a mycotoxin produced by moulds from the Aspergillus and Penicillium genera. It is a natural contaminant of a wide variety of both human and animal foodstuffs. Via dietary intake, OTA passes into the blood of both humans and animals and accumulates in several organs, such as the kidney and the brain with selective toxicity in the ventral mesencephalon and in the cerebellum. In order to confirm the regional selectivity to OTA cytotoxicity in rat brain, investigations were designed to study the mechanism of the cytotoxicity of OTA in primary cultures of the above-mentioned structures (ventral mesencephalon and cerebellum), and to compare their sensitivity to the toxin. Protein and DNA synthases, lactate dehydrogenase (LDH) release and production of malondialdehyde (MDA) were assayed in astrocytes and neurones of the selected structures in the presence of OTA. After 48 h incubation, OTA (10–150 μM) induced an inhibition of protein and DNA syntheses in a concentration-dependent manner with a selective higher toxicity in the cells of the ventral mesencephalon [50% inhibitory concentrations (IC50) of protein and DNA syntheses were 14 ± 2 μM for neurones and 40 ± 5 μM for astrocytes] compared to the cerebellum values (24 ± 7 μM for neurones and 69 ± 9 μM for astrocytes). In parallel, a significant increase in levels of MDA and LDH release were noted. Altogether these results indicate that OTA is also a neurotoxic substance in addition to its well-documented nephrotoxicity and that the effects are likely to be restricted within particular structures of the brain.

The biosynthesis of the ochratoxins, metabolites of aspergillus ochraceus

Abstract The biosynthesis of ochratoxin A (Ia) was studied in Aspergillus ochraceus Wilh. by experiments in which dl -phenylalanine [U- 14 C], sodium acetate [1- 14 C] and methionine [ 14 CH 3 ] were supplied to a resting culture of the fungus. The carboxyl carbon of the amide group is shown to be derived from the C 1 -pool.