Toxigenic strains of Fusarium moniliforme and Fusarium proliferatum isolated from dairy cattle feed produce fumonisins, moniliformin and a new C21H38N2O6 metabolite phytotoxic to Lemna minor L.

Corn samples suspected of causing refusal-to-eat syndrome in dairy cattle were examined mycologically. Fusarium moniliforme (14 isolates) and F. proliferatum (12 isolates) were the predominant fungi present. These isolates were tested for mycotoxin production on rice at 25 degrees C. Each strain of F. moniliforme produced fumonisin B1 (FB1: 378-15,600 ppm) and fumonisin B2 (FB2: 2-1050 ppm). Each strain of F. proliferatum produced moniliformin (45-16,000 ppm), FB1 (27-6140 ppm), and FB2 (5-1550 ppm). In addition, a new Fusarium metabolite of molecular composition C21H38N2O6 was produced by 10 of the F. moniliforme isolates and 7 of the F. proliferatum isolates. The metabolite's 1H- and 13C-NMR, HRFAB/MS and IR spectra indicate an alpha amino acid. It is toxic to Lemna minor L. duckweed (LD50 100 micrograms/mL).

Correlation of moniliformin, but not fumonisin B1 levels, in culture materials of Fusarium isolates to acute death in ducklings.

Five isolates of Fusarium moniliforme and two isolates Fusarium proliferatum of the Section Liseola were each fermented on rice for 21 d at 25 C. Each Fusarium-fermented rice, when dried and mixed into a poultry diet (10% by weight), caused a varied degree of acute mortality in baby Pekin ducklings. The acute (death in less than 48 h) mortality correlated significantly only to the amount of moniliformin in fermented rice, thus in the diet, but not to the amount of fumonisin B1 in fermented rice. This correlation of moniliformin concentration and noncorrelation of fumonisin B1 concentrations to acute toxicity were confirmed by duckling assay using diets containing these purified mycotoxins.

Inhibition of gluconeogenesis in cultured chicken embryo hepatocytes by Fusarium metabolites.

Four Fusarium metabolites, 2,5-anhydro-D-mannitol, 2,5-anhydro-D-sorbitol, moniliformin, and fumonisin B1, were tested for their ability to inhibit gluconeogenesis and cell viability in a primary chicken embryo hepatocyte culture system. The hepatocyte system was established from fertilized chicken eggs that were incubated for 14 days. The hepatocytes produced and secreted glucose into the supernatant of a Krebs incubation solution amended with 3 mM of either lactate or fructose as a precursor for glucose formation. 2,5-Anhydro-D-mannitol and 2,5-anhydro-D-sorbitol inhibited gluconeogenesis in these cells from both lactate and fructose. The former anhydro sugar was more inhibitory when lactate was the precursor (50% inhibition, IC50, 6 mM) and the latter anhydro sugar more inhibitory when fructose was the precursor (IC50, 12 mM). Moniliformin was more inhibitory to glucose formation from lactate (IC50, 100 microM) than from fructose in these cells. The degrees of inhibition of gluconeogenesis by the two anhydro sugars and moniliformin were greater than their effect on cell viability. Fumonisin B1 as high as 1 mM neither inhibited gluconeogenesis, nor affected cell viability.

(all trans)-2,4,6,8,10,12-tetradecahexene-1,14-dial, a new pigment from Conidiobolus paulus.

A study of the coloring matter produced by Conidiobolus paulus Drechsler NRRL 2648 on potato-dextrose medium led to the isolation of a new dialdehyde unsaturated metabolite, (all trans)-2,4,6,8,10,12-tetradeca-1,14-hexenedial. The structure was characterized by MS and by 1H- and 13C-NMR. The compound inhibited the growth of the Gram-positive bacterium, Bacillus cereus, and the Gram-negative bacterium, Escherichia coli (MIC values of 10 micrograms and 50 micrograms, respectively).

Inhibition of protein serine/threonine phosphatases by fumonisin B1, a mycotoxin.

Fumonisin B1 (FB1), a mycotoxin produced by the fungus Fusarium moniliforme, which is a common contaminant of corn, is suspected to be a cause of human esophageal cancer. FB1 is hepatotoxic and hepatocarcinogenic in rats, and although the mechanisms involved have not been clarified, the latter is associated with a weak initiating activity. The effects of FB1 on the activity of protein serine/threonine phosphatases (PPs) (PP1, PP2A, PP2B, PP2C and PP5/T/K/H) were investigated in the present study. Inhibition of dephosphorylation was noted for all five PPs with IC50 values of 80 microM-3000 microM. Among the five PPs examined, PP5 was most sensitive with an IC50 of 80 microM. This concentration is comparable to that estimated to be reached in the rat body by feeding FB1 to obtain hepatic tumors. Inhibition of PP5 could thus play important roles in the toxicity and carcinogenic action of FB1.

Intravenous fumonisin B1 induces cell proliferation and apoptosis in the rat.

In the rat, the target organs of fumonisin B1, a mycotoxin produced by Fusarium moniliforme, are the kidney and liver. Fumonisin B1 is also hepatocarcinogenic in the rat and is associated epidemiologically with esophageal cancer in humans. We investigated the effect of a single intravenous dose of fumonisin B1 on cell proliferation, lesion development, and glutathione status in the major target organs of the rat. Male Sprague-Dawley rats were injected intravenously with fumonisin B1 at 0 or 1.25 mg/kg and were euthanized at 12 hr or, 1,2,3, or 5 days. An intraperitoneal injection of 5-bromo-2'-deoxyuridine at 100 mg/kg was given 90 min prior to euthanasia. In fumonisin B1 treated rats, serum cholesterol and serum urea nitrogen were elevated; however, the activity of hepatic enzymes was unaffected. Hepatic and renal glutathione concentrations were depressed at 12 and 24 hr, respectively, with subsequent recovery. Histologic changes were most prominent in the outer medulla of the kidney, with cell proliferation and apoptosis followed by nephrosis. Cell proliferation also occurred in the liver and esophagus, but in the absence of tissue injury. The labeling index peaked on day 1 for the liver and on day 3 for the esophagus. These results confirm that the primary target organ of fumonisin B1 in the rat is the kidney and support the concept that fumonisin B1-induced mitogenesis may be the mechanism of carcinogenesis.

Acute cardiotoxicity of moniliformin in broiler chickens as measured by electrocardiography.

Electrocardiography was used to examine the acute cardiotoxic effects of moniliformin on 3-week-old broiler chickens. Each of the seven pairs of anesthetized birds (pentobarbital sodium, 40 mg/kg body weight, intramuscular) was injected intravenously with moniliformin (1 mg/kg body weight) or an equal volume of normal saline (1 ml/kg body weight), and changes in electrocardiogram were monitored for 50 minutes. Three of the seven birds injected with moniliformin died within 50 minutes post-injection. Moniliformin caused a bradycardia, which became highly significant (P < 0.05) within 15 minutes post-injection. The P-R, Q-T, and S-T intervals of moniliformin-injected birds were significantly lengthened throughout the 50-minute observation (P < 0.05). The results indicate that the moniliformin-induced mortality is due primarily to cardiac failure.

Comparative cytotoxicity of fumonisin B1 and moniliformin in chicken primary cell cultures.

Two water-soluble Fusarium metabolites, fumonisin B1 (FB1) and moniliformin (MN) were compared for their cytotoxicity in a variety of chicken primary cell cultures. Cardiac and skeletal myocytes and hepatocytes derived from embryos, and splenocytes, macrophages, and chondrocytes derived from 3- to 4-week old chickens were cultured in media containing either FB1 or MN (0 to 1 mM) for 48 hr. The colorimetric tetrazolium cleavage assay was then used for measuring cell survival. FB1 was not toxic to macrophages, hepatocytes, cardiac and skeletal myocytes but toxic to splenocytes and chondrocytes. MN was not toxic to chondrocytes and macrophages, but toxic to splenocytes, cardiac and skeletal myocytes. Median effective concentration (EC50) of MN in skeletal myocytes was 42 mu M (fiducial limits: 33 to 50 mu M) and in cardiac myocytes was 95 mu M (fiducial limits: 84 to 122 mu M). Estimated EC50 of FB1 in chondrocytes and splenocytes and EC50 of MN in splenocytes were all greater than 200 mu M.

Effects of intravenous fumonisin B1 in rabbits: nephrotoxicity and sphingolipid alterations.

Fumonisin B1 is hepatotoxic in all species, but nephrotoxicity has only been reported in rats. It is a specific inhibitor of sphinganine N-acyltransferase. Our objective was to determine the target organs for fumonisin toxicosis in the rabbit. We administered fumonisin B1 ( > 95% pure) intravenously to adult rabbits and examined selected clinical, biochemical, and histological parameters for up to 5 days. In a pilot study, rabbits were given fumonisin B1 at 1, 0.5, 0.3, 0.15, or 0 mg/kg daily for 4 or 5 days and then euthanized. Additional rabbits were given a single dose of fumonisin B1 at 1 mg/kg and euthanized on day 2 or 4. In the formal time-course study, rabbits were given a single dose of fumonisin B1 at 0 or 1.25 mg/kg and euthanized on days 1, 3, or 5. Rabbits given multiple doses of fumonisin B1 were lethargic and anorectic, and had decreased urine production. Liver- and renal-associated clinical chemistry parameters were elevated. Renal lesions consisted of severe proximal tubular necrosis. Liver lesions were variable and consisted of mild necrosis, hepatocyte vacuolation, and bile stasis. The sphinganine-to-sphingosine ratio, in both target and nontarget tissues, was markedly elevated in treated rabbits. A single dose of fumonisin B1 induced renal but not hepatic injury. Therefore, the target organs for fumonisin B1 toxicity in rabbits are kidney and liver, with the kidney being more sensitive.

Fusarium species associated with banana fruit rot and their potential toxigenicity.

Banana fruits exhibiting signs of decay were collected from markets in the United States and Italy. Fungi isolated from the lesions on the banana fruits wereFusarium moniliforme, F subglutinans, andF. semitectum var.majus. When the fungal strains were cultivated on maize kernels, the cultures did not produce zearalenone (ZON), zearalenols (á-, â-ZOH), and trichothecenes [deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), T-2 toxin (T-2), diacetoxyscirpenol (DAS)]. Fumonisins and fusarin C (FUS-C) were not detected naturally nor in bananas purchased in the U.S. and artificially infected withFusarium. Moniliformin (M) (up to 267 mg/kg) was detected in maize kernel cultures ofF. subglutinans from bananas. No mycotoxins were detected in naturally infected fruits. Although no mycotoxins were detected in the extracts from corn cultures ofF. semitectum var.majus, the extracts were toxic to brine shrimp and mice.

Toxicity of corn culture material of Fusarium proliferatum M-7176 and nutritional intervention in chicks.

The toxicity of Fusarium proliferatum M-7176 cultured on corn (FPC) and nutritional intervention were investigated in baby chicks (New Hampshire x Single Comb White Leghorn) in three 2-wk feeding experiments. In Experiment 1, 30% FPC decreased weight gain (P < .05) and increased relative heart weight (RHW) (P < .01). Experiment 2 included a 2 x 2 factorial arrangement of FPC (0 or 30%) and Se (0 or 5 mg/kg) and two detached treatments of Se (2.5 mg/kg) or thiamin (B1, 25 mg/kg) supplementations to 30% FPC. Only B1 was inhibitory to the toxic effects of FPC on weight gain, feed efficiency, and RHW (P < .05). Experiment 3 included 2 x 2 factorial arrangement between FPC (0 or 30%) and Se (0 or 4 mg/kg), or B1 (0 or 50 mg/kg), or vitamin E (0 or 50 IU/kg) and additional supplementations of Se (2 mg/kg), B1 (10 or 25 mg/kg), or E (10 IU/kg) to 30% FPC. A new batch of FPC was used and it caused 36% mortality. Vitamin E did not interact with FPC, but SE interacted with FPC only on RHW (P < .01). Thiamin interacted with FPC on all measured variables with significance ranging from P < .1 to P < .01. Supplementation of B1 as low as 10 mg/kg was inhibitory to some toxic effects of FPC. However, B1 as high as 50 mg/kg did not completely negate the cardiotoxicity. Water-extractable B1 in FPC diets was 13 to 27% of the control diets. Water extract of FPC reduced B1 recovery from a standard solution by 40%. The anti-thiamin factor was heat-sensitive. Both fumonisins and moniliformin were present in FPC. However, the results indicate that the anti-thiamin factor is also a major toxic factor of F. proliferatum M-7176.

Comparison of the cytotoxicities of Fusarium metabolites and Alternaria metabolite AAL-toxin to cultured mammalian cell lines.

Four water-soluble Fusarium metabolites (fumonisin B1, fusaric acid, butenolide and moniliformin), water-insoluble pigment (8-O-methylbostrycoidin), and an Alternaria metabolite (AAL-toxin) were tested for relative cytotoxicity to five established mammalian cell lines. Butenolide was the most cytotoxic to all five cell lines. LC50s were; 1 microgram/ml to rat hepatoma (RH) (tumors derived from parenchymal cells), 7 micrograms/ml to baby hamster kidney (BHK-21) fibroblast cells, and 15 micrograms/ml to McCoy mouse (MM) fibroblast cells: LC100s were 1 microgram/ml to Chinese hamster ovary (CHO) fibroblast cells, and 5 micrograms/ml to dog kidney (MDCK) fibroblast cells. Fusaric acid was cytotoxic to the MDCK, MM, RH, and CHO cell lines; moniliformin was cytotoxic to the RH, CHO, and MDCK, cell lines. The pigment, however, was cytotoxic only to RH and CHO cell lines. Fumonisin B1 and a related toxin, AAL-toxin, at a high dose level (100 micrograms/ml) were not cytotoxic to the RH, BHK, MM, CHO and MDCK cell lines. T-2 toxin was used as a positive control, and inhibited all cell lines at the nanogram level. The difference in response of these five cell lines to the toxic metabolites, that were noted in this study, was then used to evaluate nine HPLC fractions obtained from a methanol-water extract of an F. moniliforme culture. The results indicated that this type of cytotoxicity assay may be useful in following the isolation of metabolites from extracts of Fusarium culture, especially F. moniliforme.

2-Hydroxyhexadecanoic and 8,9,13-trihydroxydocosanoic acid accumulation by yeasts treated with fumonisin B1.

Fumonisin B1 is a sphingolipid-like compound that enhances the accumulation of yeast sphingolipids and 2-hydroxy fatty acids. These lipids occur both as freely extractable and cell bound components in yeast fermentations. Both free and bound 2-hydroxy fatty acids produced by Pichia sydowiorum NRRL Y-7130 were increased when fumonisin B1 (50 mg/L) was added to the usual growth medium containing yeast extract/malt extract/peptone/glucose. Fumonisin-treated cultures contained 38 mg/L more 2-hydroxyhexadecanoic and 15 mg/L more 2-hydroxyoctadecanoic acids than did untreated cultures. By contrast, fumonisin inhibited the accumulation of free 8,9,13-trihydroxydocosanoic acid in Rhodotorula sp. YB-2501 cultures, leading to 240 mg/L lower trihydroxy acid production than by untreated cultures.

Isolation and purification of AAL-toxin from Alternaria alternata grown on rice.

An isolation and purification procedure is described for AAL-toxin, a secondary metabolite produced by Alternaria alternata. The method involves growth of the fungus on rice media, extraction with chloroform followed by methanol: water from fungus-infested rice, and purification of the aqueous layer that contains AAL-toxin by using chromatography methods. The AAL-toxin was of type A, and white in color, and its purity was > or = 95% with mol. wt 522 (M + 1).

Reexamination ofFusarium graminearum NRRL-13820and NRRL-13852 reported as type A trichothecene producers.

NRRL-13820 and NRRL-13852 are reported to be two atypicalFusarium graminearum strains type A trichothecene producers [T-2 toxin (T-2) and diacetoxy-scirpenol (DAS)]. These two strains were reexamined by morphological, genetical (DNA / DNA relatedness) and toxicological techniques and compared with 28 wildF graminearum isolates obtained from corn in Italy and the USA. The isolate NRRL-13820 was morphologically confirmed as a typical isolate ofF graminearum, while the isolate NRRL-13852 showed some peculiar characteristics. Nuclear DNA comparison between NRRL-13820 and NRRL-13852 displayed 49% similarity and showed 94 % and 44 % relatedness, respectively, when compared withF graminearum NRRL-13833, which is a well assessed type B trichothecene producer [deoxynivalenol (DON) and 15-acetyldeoxynivalenol]. NRRL-13820, NRRL-13852, and NRRL-13833, as well as the 28 wild isolates, were not able to synthesize T-2, HT-2 nor DAS. Finally, NRRL-13820 and NRRL-13833, but not NRRL-13852, were able to produce DON (120 and 40/µg/g, respectively). The data support the concept that the production of examined type A trichothecenes is very rare inF graminearum.

Effects of selected secondary metabolites of Fusarium moniliforme on unscheduled synthesis of DNA by rat primary hepatocytes.

The Fusarium moniliforme mycotoxins--fusarin C, fumonisin B1, moniliformin and bikaverin--were evaluated for genotoxicity by their ability to induce unscheduled DNA synthesis (UDS) in primary rat hepatocytes. Isolated hepatocytes were exposed to several concentrations of moniliformin (5.0-500 microM), bikaverin (1.0-500 microM), fumonisin B1 (0.5-250 microM), or fusarin C (1.0-100 microM). Aflatoxin B1, a known inducer of UDS, was included as a positive control. UDS was determined by autoradiography of cells after their exposure to [3H]thymidine. The highest doses of fusarin C and bikaverin caused cell death, but no cytotoxicity was observed in cells exposed to moniliformin or fumonisin B1. Fumonisin B1, moniliformin and bikaverin were not genotoxic in the UDS assay. The results of the UDS assay with fusarin C were inconclusive since a marginal effect on UDS was obtained.

AAL toxins, fumonisins (biology and chemistry) and host-specificity concepts.

The AAL toxins and the fumonisins (FB1 and FB2) are structurally related and produced respectively by Alternaria alternata f.sp. lycopersici and Fusarium moniliforme. AAL toxin is characterized as a host-specific toxin, toxic to tomato, whereas fumonisin B1 causes equine leukoencephalomalacia. FB1 and FB2 were biologically active in susceptible tomato tissue (Earlypak-7) and animal tissue culture (rat hepatoma H4TG and dog kidney MDCK). Conversely, AAL toxin was also active in the rat and dog tissue culture cells. Both fungi produce toxin/s in culture that causes death in rats; these toxins are other than AAL and fumonisin. The peracetylated derivatives of AAL and FB1 are biologically inactive in both the tomato bioassay and the animal tissue culture systems. Acetylation of the amine renders AAL inactive. The hydrolysis product of AAL (phentolamine) is toxic to the susceptible tomato line whereas the phentolamine of fumonisin is not. AAL and FB1 can be analyzed by Continuous Flow Fast Atom Bombardment (CFFAB) and Ionspray Mass Spectrometry (ISM), both sensitive to the picomole range. The N-acetyl of the TFA hydrolysis product of AAL and FB1 is determined by comparing the fragment ions at m/z 86 and 140 for FB1 and 72 and 126 for AAL.

Characterization of fumonisin toxicity in orally and intravenously dosed swine.

Fumonisin B1 (FB1), a recently identified mycotoxin produced by Fusarium moniliforme in corn, has been shown to cause death in swine due to pulmonary edema, an apparently species specific effect, and to interfere with sphingolipid metabolism in vitro. Here we characterize the toxicity of fumonisins, using female cross-bred swine weighing 6 to 13 kg, and present a hypothesis regarding the mechanism of fumonisin-induced pulmonary edema in swine. FB1 was given daily intravenously (IV) to pig 1 for 9 days for a total of 72 mg (7.9 mg/kg) and to pig 2 for 4 days for a total of 67 mg (4.6 mg/kg). Pig 3 (control) was given saline IV for 9 days. Corn screenings naturally contaminated with FB1 (166 ppm) and FB2 (48 ppm) were fed to pigs 4, 5, and 6, and ground corn was fed to pigs 7 and 8 (controls). Pigs 4 and 7 were killed on day 5; pig 5 was found dead on day 6; and pigs 6 and 8 were killed on day 15. Pigs 4 and 5 had ingested 187 and 176 mg total fumonisins, respectively, while pig 6 had ingested 645 mg. Feed consumption had decreased in pigs fed corn screenings, with an additional sharp decrease prior to onset of clinical signs. Increases in serum liver enzymes, total bilirubin, and cholesterol were present, but electrocardiograms, heart rate, and body temperature were unaffected. Pigs dosed IV with FB1, developed mild intermittent respiratory abnormalities, while those fed screenings developed respiratory distress within 5 days. Mild interstitial pulmonary edema was observed in pig 1. Severe interstitial pulmonary edema, pleural effusion, and increased lung wet/dry weight ratio were observed in pigs 4 and 5. All pigs given fumonisin (either IV or orally) had hepatic changes characterized by hepatocyte disorganization and necrosis; pancreatic acinar cell degeneration was also observed. Ultrastructural changes in orally dosed swine included loss of sinusoidal hepatocyte microvilli; membranous material in hepatic sinusoids; and multilamellar bodies in hepatocytes, Kupffer cells, pancreatic acinar cells and pulmonary macrophages. Pulmonary intravascular macrophages (PIMs) contained large amounts of membranous material. Thus, the target organs of fumonisin in the pig are the lung, liver, and pancreas. At lower doses, slowly progressive hepatic disease is the most prominent feature, while at higher doses, acute pulmonary edema is superimposed on hepatic injury and may cause death. We hypothesize that altered sphingolipid metabolism causes hepatocellular damage resulting in release of membranous material into the circulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential cytotoxicity and mycotoxin content among isolates of Fusarium moniliforme.

Ten different isolates of the common corn fungus, Fusarium moniliforme, were cultured on corn, and the production by the isolates of two important mycotoxins, fusarin C and fumonisin B1, was compared. Additionally, both aqueous and organic extracts of the cultures were tested for cytotoxicity to rat primary hepatocytes by measuring the effects of three dose levels on the ability of the cells to take up valine and to cause the release of the cytoplasmic enzyme, lactate dehydrogenase. The fungal isolates differed drastically in their ability to produce the two mycotoxins and in their cytotoxicity. However the toxic effects could not be accounted for by the content of the two toxins measured. Therefore it appears that there are other toxins, both organic and aqueous soluble compounds, that are toxic to liver cells.

Mycotoxin formation by different geographic isolates of Fusarium crookwellense.

Eighteen Fusarium crookwellense isolates from the continents of Australia, Europe, and North America were compared for their ability to produce mycotoxins on corn at 25 degrees C after 2 weeks. Extracts from corn fermented with each Fusarium isolate were analyzed by thin-layer chromatography (TLC) and gas chromatography/mass spectroscopy (GS/MS) for mycotoxins. Toxins detected were zearalenone (13 isolates), fusarin C (11 isolates), nivalenol (4 isolates), and diacetoxyscirpenol (2 isolates). Zearalenone and fusarin C were produced by isolates from each continent, while nivalenol was detected in the Fusarium isolates originating from Australia and one isolate from the United States.