Ronald F. Vesonder

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. This material is phagocytosed by the PIMs thus triggering the release of mediators which ultimately results in pulmonary edema.

AAL Toxins, funionisms (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 hostspecific 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 cause 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 (pentolamine) is toxic to the susceptible tomato line whereas the pentolamine 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.

Comparison of the cytotoxicities ofFusarium metabolites andAlternaria metabolite AAL-toxin to cultured mammalian cell lines

Four water-solubleFusarium metabolites (fumonisin B1, fusaric acid, butenolide and moniliformin), water-insoluble pigment (8-O-methylbostrycoidin), and anAlternaria 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 μg/ml to rat hepatoma (RH) (tumors derived from parenchymal cells), 7 μg/ml to baby hamster kidney (BHK-21) fibroblast cells, and 15 μg/ml to McCoy mouse (MM) fibroblast cells: LC100s were 1 μg/ml to Chinese hamster ovary (CHO) fibroblast cells, and 5 μg/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 μg/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 anF. moniliforme culture. The results indicated that this type of cytotoxicity assay may be useful in following the isolation of metabolites from extracts ofFusarium culture, especiallyF. moniliforme.

Phytotoxic activity of selected water-soluble metabolites of Fusarium against Lemna minor L. (Duckweed)

Phytotoxicity and inhibitory effects of the fusarial toxins fumonisin B1 (FB1) [m.p. 103–105 °C], fusaric acid [m.p. 106–107 °C], butenolide (4-acetamido-4-hydroxy-2-butenoic acid lactone) [116–117 °C], 9, 10-dihydroxyfusaric acid [m.p. 150–155 ° C], and moniliformin on chlorophyll synthesis in the aquatic macrophyte Lemna minor (duckweed) were examined. FB1 proved to be most active, reducing the growth of L. minor fronds and their ability to synthesize chlorophyll by 53% and 59%, respectively, at 0.7 μg/ml. The growth rate of L. minor was reduced 59% by 6.7 μg/ml fusaric acid, 62% by 66.7 μg/ml butenolide, and 22% by 66.7 μg/ml 9,10-dihydroxyfusaric acid. Moniliformin was the least phytotoxic to L. minor, with only a 16% suppression of growth rate and a 54% reduction in chlorophyll at 66.7 μg/ml.

Phytotoxic effects of fumonisin B1 on maize seedling growth

Fumonisin B1 toxin is produced by the fungusFusarium moniliforme Sheldon, which is systemic to maize (Zea mays L.) and maize seeds. The effects of zero to 100 parts per million fumonisin B1 on the germination process of maize seeds was determined. The presence of fumonisin had no effect on percent seed germination, but fumonisin inhibited radicle elongation by up to 75% after 48 hours of imbibition. An analysis of amylase secretion in the maize endosperm indicated that fumonisins inhibited amylase production in the germinating seed. Isoelectric focusing of endosperm extracts indicated that secretion of the low pI class of amylases was affected more that other amylase isozymes. The results suggested that the presence of high levels of fumonisin in maize seed may have deleterious effects on seedling emergence.

Comparative phytotoxicity of the fumonisins, AAL-toxin and yeast sphingolipids in Lemna minor L. (duckweed)

Fumonisin B1 and AAL-toxin, both of which contain sphingolipid-like substituents, are water-soluble metabolites of Fusarium moniliforme and Alternaria alternata, respectively. These two toxins were compared to each other and to tetraacetylphtosphingosine (TAPS) and triacetyldihydrosphingosine (TADS) for effects on chlorophyll production and growth in Lemna minor L. (duckweed). Fumonisin B1 (0.7 μg), TAPS, and TADS all produced parallel effects on growth rate and chlorophyll content; however, FB1 did so at a 33-fold lower concentration. The AAL-toxin at 0.7 μg affects chlorophyll content more than plant growth (36% versus 73%, respectively), whereas at 3.3 μg concentration, the growth rate was less than 50% and chlorophyll content was reduced by 80%. By contrast, the hydrolysis product of FB1 that does not contain the tricarballylic acid (TCA) substituent is 23 times less active, which suggests that this component somehow enhances activity. The yeast sphingolipids are completely acetylated and do not contain TCA groups but also affect chlorophyll content and growth rate of duckweed. However the effect was substantially less than with AAL-toxin and FB1, which contain one and two TCA groups, respectively.

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

Two water-solubleFusarium 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 µM (fiducial limits: 33 to 50 µM) and in cardiac myocytes was 95 µM (fiducial limits: 84 to 122 µM). Estimated EC50 of FB1 in chondrocytes and splenocytes and EC50 of MN in splenocytes were all greater than 200 µM.

Fumonisin-stimulatedN-acetyldihydrosphingosine,N-acetylphytosphingosine, and phytosphingosine products ofPichia (Hansenula) ciferri, NRRL Y-1031

Pichia (Hansenula) ciferri Y-1031 grown in the presence of 25–100 mg fumonisin B1/L for 4–5 days accumulated sphingolipids as evident in the centrifuged cells and extracellular particles (c/p fraction). The c/p fraction of fumonisin-treated (100 mg/L) cultures elicited a 15-fold increase ofN-acetyldihydrosphingosine and 31-fold increase of combinedN-acetylphytosphingosine and phytosphingosine over those from untreated cultures. During exponential growth of 1 day, fumonisin-treated cultures appeared to transfer sphingolipid bases into the medium (22 mg/L) rather than into the c/p (2 mg) fraction. Upon saponification, a residue from the c/p fraction contained 440 mg of additional, unknown polar lipids per liter that was not sphingolipid (14 mg/L).

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 byPichia 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 inRhodotorula sp. YB-2501 cultures, leading to 240 mg/L lower trihydroxy acid production than by untreated cultures.

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.