Effect of<Emphasis Type="Italic">in vitro</Emphasis> digestion on fumonisin B<Subscript>1</Subscript> in corn flakes

Low levels of fumonisins have been found frequently in corn based breakfast cereals and can occur bound to protein and other matrix components.In vitro digestion of two samples of corn flakes was carried out under "fed conditions." Fumonisins were measured as o-phthaldialdehyde/mercaptoethanol derivatives by LC-fluorescence. One sample of corn flakes (FN12) had high concentrations of fumonisin B₁ (FB) (average 125 ng/g) and total bound FB₁, (TB FB₁) (average 92 ng/g) and the other (FN11) had a low level of free FB₁ (average 29 ng/g) and no detectable TB FB₁. After incubation of the samples with gastrointestinal tract solutions simulating saliva plus stomach and duodenal juices, chyme was analysed for FB₁, hydrolyzed FB₁ (HFB₁) and partially hydrolyzed fumonisin B₁ (PHFB₁). The bioaccessibility (percentage of FB₁ released from corn flakes into chyme) was 38-78% for incurred FB₁ in FN12, 8-54% for incurred plus spiked FB₁ in FN12, and 19-66% for incurred plus spiked FB₁ in FN11. HFB₁ and PHFB₁ were not detected. If free FB₁ was first extracted from sample FN12, no FB1 was detected in the chyme, indicating no contribution from TB FB₁. Concentrations were corrected for method recovery of FB₁ or, for bound FB₁, partial method recovery of HFB₁ Presented at the XIIth IUPAC International Symposium on Mycotoxins and Phycotoxins, Istanbul, Turkey, 21–25 May, 2007     

Fumonisin and Beauvericin Induce Apoptosis in Turkey Peripheral Blood Lymphocytes

Fumonisins, a family of mycotoxins produced by Fusarium verticillioides (synonym Fusarium moniliforme Sheldon) and F. proliferatum, have been associated with various deleterious effects in different animal species. Serological, hematological and pathological effects and mortality have previously been observed in broiler chicks fed F. proliferatum culture material containing known concentrations of fumonisin, moniliformin and beauvericin. Turkey peripheral blood lymphocytes were exposed in vitro for 72 hours to fumonisin B₁(FB₁), fumonisin B₂(FB₂), hydrolyzed fumonisin B₁ (HFB₁), moniliformin and tricarballylic acid (TCA) (0.01-25 g/ml). A decrease in cell proliferation, as determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] bioassay, occurred in the order: FB₂ > FB₁ > HFB₁, with IC₅₀ = 0.6 M, 1 M and 10 M, respectively. Internucleosomal DNA fragmentation and morphological features characteristic of apoptosis were observed following exposure to fumonisin B₁ and beauvericin; cytoplasmic condensation and membrane blebbing were seen by light microscopy. Tricarballylic acid and moniliformin did not interfere with cell proliferation. Results suggested that fumonisin B₁ and beauvericin may affect immune functions by suppressing proliferation and inducing apoptosis of lymphocytes.     

Bioaccessibility of total bound fumonisin from corn flakes

Fumonisin B₁ (FB₁) is often found as a natural contaminant of corn and corn-based food. Several publications have demonstrated the presence of fumonisin bound to proteins and to other compounds of the matrix. In spite of the low oral bioavailability of FB₁ in rats, pigs, chickens, cows, and monkeys, FB₁ can cause agriculturally significant disease and possibly human cancer. The aim of this work was to determine the bioaccessibility of total bound FB₁ (TB FB₁) (percentage of TB FB₁, released from corn flakes to the chyme) after in vitro digestion. Two samples of corn flakes washed with solvents were incubated with gastrointestinal tract solutions simulating saliva plus stomach and duodenal juices. After hydrolysis of the chyme with KOH, TB FB₁ was determined as hydrolyzed FB₁ (HFB₁). The bioaccessibility of TB FB₁ in chyme from corn flakes was 37–64%, indicating that these derivatives should be considered in evaluation of exposure to fumonisin.     

Enhancement of solubility in <Emphasis Type="Italic">Escherichia coli</Emphasis> and purification of an aminotransferase from <Emphasis Type="Italic">Sphingopyxis</Emphasis> sp. MTA144 for deamination of hydrolyzed fumonisin B<Subscript>1</Subscript>

Background  

Fumonisin B₁ is a cancerogenic mycotoxin produced by Fusarium verticillioides and other fungi. Sphingopyxis sp. MTA144 can degrade fumonisin B₁, and a key enzyme in the catabolic pathway is an aminotransferase which removes the C2-amino group from hydrolyzed fumonisin B₁. In order to study this aminotransferase with respect to a possible future application in enzymatic fumonisin detoxification, we attempted expression of the corresponding fumI gene in E. coli and purification of the enzyme. Since the aminotransferase initially accumulated in inclusion bodies, we compared the effects of induction level, host strain, expression temperature, solubility enhancers and a fusion partner on enzyme solubility and activity.     

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B₁

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B₁ and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B₁. We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B₁. Pyruvate was found to be the preferred co-substrate and amino group receptor (K _M = 490 μM at 10 μM hydrolyzed fumonisin B₁) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 μM. The enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K _M = 1.1 μM and k _cat = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.     

Developmental toxicity of fumonisin in Syrian hamsters

The effects of fumonisin on development of Syrian hamster fetuses were studied using fumonisin B₁ and B₂ extracted fromFusarium moniliforme corn-culture and purified fumonisin B₁. A significant increase in litters with fetal deaths occurred with the high doses of purified (18 mg FB₁/kg) and culture-extracted (18 mg FB₁ plus 4.5 mg FB₂) fumonisin. It is concluded that prenatal exposure to fumonisin on days 8 and 9 of gestation is detrimental to fetal hamster survivability but does not induce clinical maternal intoxication at these doses. Equivalent doses of fumonisin B₁, whether from culture-extract or pure solution produced similar results.     

Occurrence of Fumonisin B<Subscript>1</Subscript> in Corn from the Main Corn-Producing Areas of China

Fumonisin B₁ (FB₁) is the most abundant of the fumonisin mycotoxins, mainly produced in maize by F. verticillioides and F. proliferatum. A total of 282 corn samples harvested in 2005 from six provinces, the main corn-producing areas of China, were analyzed for FB₁ using high-performance liquid chromatography. All samples except one were (99.6%) positive for FB₁ at levels varying from 3 to 71,121 ng/g with mean and median levels for all samples of 6,662 and 1,569 ng/g, respectively. During an analysis of the distribution pattern for FB₁, it became apparent that 43.6% of tested samples had FB₁ concentrations below 1,000 ng/g, while 25.2% contained in excess of 5,000 ng/g. The average exposure to FB₁ (1.1 μg/kg body weight/day) is within the provisional maximum tolerable daily intake of 2 μg/kg body weight/day set by the Joint FAO/WHO Expert Committee on Food Additives.     

Natural occurrence of fumonisins and their correlation to Fusarium contamination in commercial corn hybrids growth in Argentina

Fifty commercial corn hybrids with different endosperm characteristics, vegetative cycle length and cross class grown in the same geographical area (Cordoba Province, Argentina) were analysed for fumonisin accumulation. All hybrids analysed showed fumonisin B₁ and B₂ contamination ranging from 185 to 27,050 ng/g for FB₁ and from 40 to 9950 ng/g for FB₂. Although most of the hybrids analysed had flint-type endosperm, two hybrids with dent-type endosperm (e.g. Prozea 10 and AX 746) showed the highest level of fumonisin (37,000 ng/g) and more FB₂ than FB₁ (FB₂/FB₁ ratio 2.42), respectively. There was no correlation between fumonisin concentration and length of the vegetative cycle. Among 18 hybrids examined for Fusarium species contamination there was also no correlation between fumonisin contamination and the level of infection with Fusarium species (Section Liseola). Eighteen hybrids showed fumonisin levels lower than 1000 ng/g. This result suggests that there is some possibility of selecting hybrids resistant or less susceptible to fumonisin and Fusarium contamination.     

Production of fumonisins byFusarium species of Taiwan

Twenty-nineFusarium species isolated from various sources in different districts of Taiwan were tested for their ability to produce fumonisins in corn cultures. OnlyFusarium moniliforme produced fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂). The finding that the other 28Fusarium species produced neither FB₁ nor FB₂ is preliminary because only one strain per species was studied. The detection of FB₁ and FB₂ in cultures ofF. moniliforme was demonstrated by TLC and HPLC, and FB₁ was further confirmed by mass spectrometry. In a separate experiment, in which 38 strains ofF. moniliforme were tested for fumonisins, approximately 66% (25/38) produced FB₁ and/or FB₂. Of the 25 strains, 14 produced only FB₁ and 11 produced both FB₁ and FB₂, and the amounts of FB₁ and FB₂ produced by different strains varied greatly. This is the first report that fumonisins are found in corn cultures experimentally infected withF. moniliforme strains from Taiwan. It is safe to assume that fumonisin producing strains ofF. moniliforme are widely distributed among the economic crops such as corn, rice, sugarcane, and sorghum throughout the Island.Abbreviations FB₁ Fumonisin B₁ - FB₂ Fumonisin B₂ - OPA o-phthalidialdehyde     

In vitro toxicology of fumonisins and the mechanistic implications

The effects of fumonisins B₁FB₁, B₂(FB{2}), and the backbone of fumonisin B₁ remaining after hydrolysis of the tricarballylic groups with base (HFB₁) on sphingolipid biosynthesis were studied in both primary rat hepatocytes and pig kidney epithelial cells (LLC-PK₁). Fumonisins were potent inhibitors of sphingolipid biosynthesis in hepatocytes (IC₅₀ of FB₁=0.1 M), but overt toxicity was not observed. In renal cells, fumonisins also inhibited sphingosine biosynthesis (IC₅₀ for FB₁=35 M), and caused decreased cell proliferation as well. Higher doses (70 M) killed renal cells after exposure for 3 days. The inhibition of de novo sphingolipid biosynthesis was specific, and appeared to be at the site of ceramide synthase, which catalyzes the formation of dihydroceramide or ceramide by the addition of the amide-linked fatty acid to sphinganine or sphingosine. These results may account for the ability of fumonisins to cause equine leucoencephalomalacia and to promote tumor formation.     

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.Published as paper No. 19,598 of the contribution series of the Minnesota Agricultural Experiment Station based on research conducted under Project 22–34H, supported by HATCH funds.     

Natural occurrence of Fusarium species and fumonisin-production by toxigenic strains isolated from poultry feeds in Argentina

Fusarium species and fumonisin production by toxigenic strains were investigated. During 1996–1998, 158 samples of poultry feeds were collected from a factory located in the department of Río Cuarto Córdoba province, Argentina. The most common species of Fusarium were F. moniliforme (60.7%) and F. nygamai (35.4%) followed by F. semitectum, F. subglutinans, F. proliferatum, F. dlamini, F. solani, F. oxysporum and F. napiforme. Fungal counts ranged from 1 × 10³ to 8 × 10⁵ CFU/g with mean values from 1.5 × 10³ to 2.3 × 10⁵ CFU/g. The highest counts were for F. dlamini, F. subglutinans, F. moniliforme and F. nygamai. Strains of F. moniliforme, F. nygamai, and F. proliferatum were screened for their potential to produce fumonisin B₁ (FB₁), fumonisin B₂ (FB₂) and fumonisin B₃ (FB₃) in corn grain. The samples were analysed using a modified high performance liquid chromatography method. The strains assayed, 43 strains, produced three fumonisins. There was a high degree of variability in the quantities of FB₁, FB₂, and FB₃ produced. The toxin produced in highest levels by the majority of the strains was FB₁. The range of concentration varied from 5.4 to 3,991, 1.01 to 189 and 0.4 to 765 ppm per gram of corn for FB₁, FB₂ and FB₃ respectively. The toxigenic pattern of strains was normal, although two strains of F. moniliforme produced exceptionally high concentrations of FB₃ and minor concentrations of FB₂ and FB₁. This is the first report from Argentina on Fusarium species in poultry feeds and fumonisin production by these strains.This revised version was published online in October 2005 with corrections to the Cover Date.     

An in vitro study of alkaline phosphatase sensitivity to mixture of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript> in the hepatopancreas of coastal lagoon wild and farmed shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

This study aimed to establish the combined effect of aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) on wild Litopenaeus vannamei hepatopancreas alkaline phosphatase (AP) activity compared with that of farmed shrimp. AP activity in hepatopancreas extract was confirmed by several specific inhibitor assays. AP activity of wild shrimp was higher than that of farmed shrimp (p?<?0.05). However, AP activity from both wild and farmed shrimp was inhibited when incubated with AFB₁ and FB₁. The greatest inhibition occurred when AP was incubated with a mixture of AFB₁ and FB₁. The IC₅₀ for AFB₁ on AP activity of wild and farmed shrimp hepatopancreases was 0.790 and 0.398 μg/mL, respectively. The IC₅₀ of FB₁ was 0.87 μg/mL for wild shrimp and 0.69 μg/mL for farmed shrimp. These results suggest that, at the mycotoxins concentrations used in the study, AP from farmed L. vannamei was sensitive to the presence of both mycotoxins; however, AP is more sensitive to the combination of AFB₁?+?FB₁ suggesting a possible synergistic or potentiating inhibitory effect.     

Comparison of the amount of bioaccessible fumonisin B<Subscript>1</Subscript> and B<Subscript>2</Subscript> in maize and rice inoculated with <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> (MRC 826) and determined by in vitro digestion—preliminary results

In this study the occurrence of hidden fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) was analysed, on two cereal substrates (maize and rice), inoculated with Fusarium verticillioides (MRC 826), in order to determine the ratio of hidden FB₁ and FB₂. Two parallel methods were applied: an in vitro human digestion sample pre-treatment and the routine extraction procedure, in both cases with subsequent LC-MS analysis. It was found that all samples showed higher concentration of total fumonisin B₁ after digestion, as compared to that of free fumonisin analysed only after extraction. The percentage of the hidden form by maize was 18.8 % (±2.4) for FB₁ and 36.8 % (±3.8) for FB₂, while for rice it was 32.3 % (±11.3) and 58.0 (±6.8), respectively, expressed as the proportion to total fumonisin B₁, for the total dataset. Significant differences were found in the FB₁ and FB₂ concentration measured after the different digestion phases (saliva, gastric and duodenal) in case of both matrixes. The results are useful for human risk assessment, since both humans and animals may be exposed to markedly higher toxin load, as determined merely by conventional analytical methods.     

Production of fumonisins B1, B2 and B3 byFusarium proliferatum Isolated from rye grains

Using the seed- plate technique, we have isolated a strain ofF. proliferatum from rye grains that produces 3 fumonisins, fumonisin B₁ (FB₁), FB₂ and FB₃ on inoculated rice and corn. Inoculated corn and rice were extracted with an aqueous methanol solution and fumonisin concentrations estimated using high performance liquid chromatography. Production of all 3 fumonisins (FB₁, FB₂ and FB₃) was much higher on rice than corn; ranging from 3816, 1068 and 985 ppm to 1643, 350 and 162 ppm respectively. We conclude that all natural substrates whereF. proliferatum is present as a component of the mycoflora should be monitored for the presence of fumonisins.     

Mycoflora and fumonisin-producing strains ofFusarium moniliforme in mixed poultry feeds and component raw material

In a survey of the mycoflora and mycotoxins in foods and feeds, 66 samples of mixed poultry feeds and some component raw materials were investigated. Fungal counts ranged from < 10² to 1.3 × 10⁶ CFU/g.Fusarium spp. counts ranged from 10² to 1.0 × 10⁶ CFU/g. TheFusarium spp. strains isolated were screened for their potential to produce fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) in maize cultures. Samples and maize cultures were analysed for FB₁ and FB₂ using TLC and fluorescamine-derivative HPLC. No fumonisins were detected in the samples (<6 ppm).Fusarium moniliforme was isolated in 59.1% of samples, and 97.4% of the strains produced FB₁ and 79.4% of strains produced FB₂ in maize cultures. Some isolates produced higher FB₁ and FB₂ levels than the reference strainF. moniliforme MRC 826.     

Occurrence of Fumonisin in Forage Grass in New Zealand

Fumonisin B₁ (FB₁) was isolated from samples of forage grass originating in paddocks associated with an idiopathic disease of Canadian wapiti and wapiti-red deer hybrids characterized by “ill thrift” and liver dysfunction. Four of 40 samples contained 1, 3, 6, and 9 ppm (micrograms per gram) of FB₁ and 4, 0.5, 2, and 0.5 ppm, respectively, of the methyl ester of FB₁. Analyses were done by ion spray mass spectrometry and confirmed by both fast atom bombardment (solids probe) and mass spectral analysis by electron impact ionization of the trifluoroacetate derivative of the base hydrolyzed product (pentolamine) of FB₁. This article contains the first report of the presence of fumonisin B₁ in grass.     

Mutagenic potentials of fumonisin contaminated corn following ammonia decontamination procedure

Naturally contaminated corn implicated in an outbreak of equine leukoencephalomalacia (ELEM) in southeastern Arizona was analyzed for mutagenic potential using the Salmonella/microsome mutagenicity assay before and after treatment with the ammonia procedure. Crude acetonitrile: water (1+1) extracts of high-pressure/ambient temperature (HP/AT) ammonia decontaminated, HP/AT plus low pressure/high temperature (LP/HT), and non-ammoniated fumonisin contaminated corn were tested for mutagenic potentials. Relatively pure (approx. 90%) fumonisin B₁ standard was also tested for comparison purposes. The results of this experiment indicate that there was no mutagenic potential for the fumonisin B₁ standard at the concentrations tested (100 g/plate). Also, neither the naturally-contaminated corn nor the ammonia decontaminated samples elicited a positive mutagenic response. Fumonisin B₁ levels, as determined by HPLC methods, were reduced by an average of 79% via the ammonia decontamination process. It is encouraging to note that, while further work is necessary to increase the efficacy of the ammonia process to reduce fumonisin levels, the ammonia process did reduce fumonisin levels and no mutagenic potentials were apparent in the treated corn.Abbreviations HP/AT high pressure/ambient temperature - LP/HT low pressure/high temperature - ELEM equine leukoencephalomalacia - FB₁ fumonisin B₁ - FB₂ fumonisin B₂     

Production of Fumonisins by Fusarium moniliforme Cultures Isolated from Jimsonweed in Mississippi

Eight Fusarium spp. were isolated from greenhouse-grown jimsonweed (Datura stramonium L.) in Mississippi in 1990. Four isolates of Fusarium moniliforme were obtained and when grown on autoclaved rice, produced 115 to 3,200 mg/kg fumonisin B₁, (FB₁). Other fumonisin-related compounds, such as FB₂, FB₃ and FB₄ were also produced at levels of 240, 210 and 160 mg/kg, respectively. F. semitectum (1 isolates) was negative for production of fumonisin and other phyto-toxins. F. oxysporutn (1 isolate) produced only 3.5 g/kg moniliformin. This is the first report of production of fumonisins by F. moniliforme isolated from weeds such as jimsonweed.     

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.