Pieter G. Thiel

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.

Hepato- and cardiotoxicity of Fusarium verticillioides (F. moniliforme) isolates from Southern African maize

Abstract From crops of South African and Transkeian maize were isolated 21 Fusarium verticillioides ( F. moniliforme ) strains that do not produce moniliformin, and their toxicity to ducklings and rats was determined. The pathological effects of one strain (MRC 602) were fully characterized and used as a basis for comparison with the other isolates. Acute mortality was common in ducklings fed the isolates, while in rats the mean time to death was at least 24 days even with the most toxic isolate. All the rats fed the MRC 602 isolate died, the mean times to death being 49 and 78 days, respectively, for continual intake of 32 and 16% levels of the fungal-culture material in the diet. Cirrhosis and nodular hyperplasia of the liver, and the occurrence of acute and proliferative endocardial lesions and concurrent intraventricular thrombosis were frequently encountered and were considered to be the most important lesions. Toxic nephrosis, endothelial proliferation in the pulmonary vessels, and thrombosis of the larger vessels in the heart, liver, pancreas, small intestine and lungs were considered to be less important or occurred less often. Lesions considered to be secondary to the above changes were also encountered.

A mutagen produced by Fusarium moniliforme

A mutagenic compound produced by Fusarium moniliforme on maize was isolated by CHCl3--iso-PrOH extraction, solvent partitioning and liquid chromatography on silica gel and Sephadex LH-20. HPLC studies showed that different mutagenic and non-mutagenic forms can be derived from the mutagen (P3) and that prolonged exposure to longwave u.v. light and to high temperatures causes a total loss of its u.v. absorption and mutagenic characteristics. Spectral data presented for P3 include u.v., i.r., mass spectra as well as 1H NMR and 13C NMR. Mass spectral data indicated a molecular formula of C23H29NO7.

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.

Biliary excretion of the mycotoxin fumonisin B1 in rats

The biliary excretion of the mycotoxin fumonisin B1 (FB1), produced by the fungus Fusarium moniliforme Sheldon, has been measured in male Wistar rats. After ip injection of a solution of FB1 (7.5 mg/kg body weight), 67% of the applied dose was recovered in bile over a 24-hr period, 88% of this recovery being excreted in the first 4 hr after dosing. In contrast to these results, a similar dose of FB1 given by gavage resulted in only 0.2% recovery of the toxin in bile over a 24-hr period. Hence, although these results show that biliary excretion is a major route of elimination of FB1 from the circulation, only small amounts of the toxin appeared to be absorbed from the gut in rats.

Toxicokinetics of the mycotoxin fumonisin B2 in rats

Fumonisin B2 (FB2), a secondary metabolite of the fungus Fusarium moniliforme, was administered at a dose of 7.5 mg/kg body weight to male BD IX rats by ip injection or by gavage. FB2 was rapidly absorbed from the peritoneum, its level in plasma reaching a maximum within 20 min after injection. It was rapidly eliminated from plasma with a half-life of 26 min. After 24 hr, FB2 could not be detected in plasma (< 20 ng/ml). Analysis of rat plasma for FB2 following a gavage dose failed to detect any toxin over a 6-hr period after dosing. The elimination of FB2 in the urine and faeces was determined over a 3-day period after dosing. After i.p. injection, the mean urinary excretion over this period was 1.2% and faecal elimination accounted for 84.1% of the dose. Similarly, after dosing by gavage, 0.2 and 82.0% of the dose was recovered in urine and faeces, respectively. FB2 appeared to be excreted unmetabolized.

Liquid chromatographic determination of the mycotoxin fumonisin B2 in physiological samples

The fungus Fusarium moniliforme produces a group of mycotoxins, the fumonisins, of which the most abundant are fumonisins B1 (FB1) and B2 (FB2). Previously developed analytical methods for the determination of FB1 in physiological samples have been modified for the determination of FB2 by the use of less polar extraction solvents. Plasma and urine extracts were purified on strong anion-exchange solid-phase extraction cartridges and fecal extracts on reversed-phase (C18) cartridges. FB2 in purified extracts was determined by reversed-phase HPLC with fluorescence detection using performed o-phthaldialdehyde derivatives. These methods were reproducible (R.S.D. of less than 6%) with recoveries greater than 85%. In a short preliminary study, they have been applied to the determination of the fate of FB2 dosed to rats by gavage. Of the dose given to the animals, over 90% was recovered unmetabolised in the feces within 48 h.

Ear-rot fungi and mycotoxins in South African corn of the 1989 crop exported to Taiwan

A shipment of South African corn (1989) exported to Taiwan, was analyzed for various ear-rot fungi andFusarium mycotoxins. Two sets of samples, one from the points of origin in South Africa prior to shipment, and the other from the end-point distributors in Taiwan, were studied. Surface-sterilized kernels were plated onto two different agar media and the fungal colonies identified. High Performance Liquid Chromatography was used to analyze mycotoxin levels. The predominant ear-rot fungi, in decreasing order of isolation frequency, wereFusarium subglutinans, F. moniliforme, Diplodia maydis andF. graminearum. Aspergillus flavus andA. parasiticus were not isolated from samples prior to export, but a small number ofA. flavus isolates were found after shipment. The predominant mycotoxins were fumonisins B1 (0–865 ng/g) and B2 (0–250 ng/g). Low levels of moniliformin (≤390 ng/g) were detected in some samples before shipment. Zearalenone (25 ng/g), and nivalenol (120 ng/g) were detected in two out of 32 samples taken in Taiwan. The samples contained no detectable levels of either aflatoxins (>0.5 ng/g) or deoxynivalenol (>100 ng/g) before or after shipment.

Polyclonal Antibody-Based ELISA and HPLC Methods for the Determination of Fumonisins in Corn: A Comparative Study

The performance of an experimental polyclonal antibody (PAb)-based competitive direct enzyme-linked immunosorbent assay (CD-ELISA) developed for the analysis of fumonisins in corn was assessed by comparison with an established high-performance liquid chromatography (HPLC) method. The comparative study was conducted using a series of 20 corn samples naturally contaminated with combined fumonisin levels ranging from <0.05 to >5 μg/g (ppm). Linear regression analysis between the results generated by HPLC and CD-ELISA provided correlation coefficients (r) and regression slopes (b) of r = 0.960, b = 1.493 (P < 0.001); r = 0.865, b = 3.903 (P < 0.001); and r = 0.832, b = 0.107 (P < 0.001) for the individual fumonisins B1 (FB1), B2 (FB2) and B3 (FB3), respectively, while corresponding values of r = 0.967, b = 1.059 (P < 0.001) were obtained for the combined FB1, FB2, and FB3 concentrations. In 3 of 18 fumonisin-positive corn samples, combined fumonisin levels determined by CD-ELISA were between 85 and 100% higher than those determined in the same extracts by HPLC, while in 13 other samples, CD-ELISA results were between 1.8 and 53% higher than those determined by HPLC. Conversely, in 2 of 18 samples, CD-ELISA results were lower than those determined by HPLC. The differences recorded between HPLC and the experimental PAb-based CD-ELISA were far less than those previously recorded for other mono- and polyclonal antibody-based CD-ELISA systems. The results indicate that the experimental PAb-based CD-ELISA may be effectively applied for the initial screening for fumonisins in corn.

Determination of fumonisins in corn: Evaluation of two purification procedures.

The performance of two solid phase extraction (SPE) purification procedures, used in the determination of fumonlsin B1 (FB1), B2 (FB2) and B3 (FB2) In corn, was evaluated using both thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Fewer interferences were observed In extracts prepared using the strong anion exchange (SAX) media, in contrast to those purified on C18 media, where on occasions, visual discernment of the TLC bands was hampered by the presence of interfering compounds. Precipitate formation, resulting In the blocking of SPE cartridges was also encountered when using the C18 procedure. HPLC analyses of extracts prepared by both media indicated that they gave comparable fumonlsin recoveries from naturally contaminated corn samples. The results suggest that the C18 procedure, originally developed for the TLC analyses of FB1 in mixed feeds, may also be applied to the determination of FB2 and FB2. However, where TLC is used quantitatively for fumonlsin levels <1 μg/g, purification of sample extracts on SAX media is recommended.