Robert E. Peterson

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.

A new fumonisin from solid cultures of Fusarium moniliforme

A new fumonisin has been isolated from Fusarium moniliforme isolate MRC826 grown on corn. It was shown by NMR and mass spectrometry to be an isomer of fumonisin B2 that has free hydroxyl groups at C-3 and C-10 instead of the normal C-3 and C-5. This new fumonisin was detected in cultures of most isolates of F. moniliforme that were examined and was usually present at concentrations similar to those of fumonisin B2. Two isolates of F. moniliforme that produce significantly higher levels of this new isomer were identified.

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.

Hydroxy fatty acids through hydroxylation of oleic acid with selenium dioxide/tert.-butylhydroperoxide

Oleic acid was hydroxylated in the allylic positions with the selenium dioxide/tert.-butylhydroperoxide system to give 8-hydroxy-9(E)-octadecenoic acid, 11-hydroxy-9(E)-octadecenoic acid and the novel 8,11-dihydroxy-9(E)-octadecenoic acid. This is a viable method for obtaining hydroxy fatty acids. The unsaturated hydroxy acids were hydrogenated with the hydrazine/air system to give the cor-responding saturated products. 8,11-Dihydroxyoctadecanoic acid thus obtained is also a novel compound. The saturated and unsaturated dihydroxy products were obtained aserythro/threo isomers as determined by nuclear magnetic resonance.

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.

Identification of NRRL strain B-18602 (PR3) as Pseudomonas aeruginosa and effect of phenazine 1-carboxylic acid formation on 7,10-dihydroxy-8(E)-octadecenoic acid accumulation

A new compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD), produced from oleic acid by a new bacterial isolate PR3, was discovered in 1991. We have now identified isolate PR3 as a strain of Pseudomonas aeruginosa by DNA reassociation studies. Strain PR3 also produced a crystalline yellowish compound the structure of which, as determined by GC/MS and NMR, is phenazine 1-carboxylic acid (PCA). In cultures of PR3, high PCA production was associated with low DOD accumulation.

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.

Viridicatumtoxin mycotoxicosis in mice and rats

LD50 studies of viridicatumtoxin were done in rats and mice using oral, intraperitoneal (i.p.), and subcutaneous (s.c.) routes. Mice were given oral doses of viridicatumtoxin up to 350 mg/kg body weight and rats were given doses of viridicatumtoxin up to 150 mg/kg. No deaths occurred in the animals dosed by the oral route. Hepatic alterations of hydropic change and necrosis of centrolobular hepatocytes were observed in mice given 250, 300, or 350 mg/kg viridicatum toxin and were more severe in the mice given the higher doses. No histopathologic alterations were present in the rats dosed orally. Mice were given doses of viridicatumtoxin up to 300 mg/kg, s.c., and rats up to 400 mg/kg. No deaths occurred in the animals dosed by this route. Microscopic alterations in both mice and rats were limited to the injection sites and consisted of coagulation necrosis. The single-dose, 72-h i.p. LD50 for the mouse and the rat was 70 mg/kg and 80 mg/kg respectively. Histopathologic alterations in mice given viridicatumtoxin i.p. included fibrinous peritonitis, large subcapsular areas of hepatic necrosis, single-cell hepatocytic necrosis, splenic lymphoid depletion, and vacuolar degeneration of the myocardium. Rats had splenic lymphoid depletion and fibrinous peritonitis.