fusarenon-X Research Articles

Survey of natural occurrence of trichothecene mycotoxins and zearalenone in Korean cereals harvested in 1992 using gas chromatography/mass spectrometry

For the survey of the natural occurrence of trichothecene mycotoxins, produced by species of fungi imperfecti such as Fusarium and Trichothecium, a sensitive analytical method was developed for the simultaneous detection and quantitation of the major trichothecene mycotoxins, viz. T-2 toxin (T-2), HT-2 toxin (HT-2), nivalenol (NIV), fusarenon-X (F-X), deoxynivalenol (DON), 3-acetyl deoxynivalenol (3-Ac DON), and zearalenone (ZEN), using gas chromatography/mass spectrometry-selected ion monitoring (GC/MS-SIM) mode after trimethyl silyl derivatization. The incidence of NIV and DON in 30 barley samples were 93% and 67%, respectively; the average contents of NIV and DON in positive samples were 390 ng/g (range 40-2038) and 106 ng/g (range 5-361) respectively. In 15 maize samples, the incidences of NIV and DON were 53% and 93% respectively and the average contents were 168 ng/g and 145 ng/g, respectively. These results suggest that NIV and DON were the major contaminating trichothecene mycotoxins in Korean barley and maize samples harvested in 1992.

Mycotoxin producing potential of Fusarium graminearum isolates from Uruguayan barley.

Twelve isolates of Fusarium graminearum were obtained from barely grains collected from different Uruguayan regions (harvest 1993-94). This was the predominant fungal species contaminating the crop due to a particular humid and warm season with cold nights conductive to toxin production The isolates were grown on moist, sterile rice, extracted with aqueous methanol, and examined for mycotoxin production. Zearalenone (ZEA) and the trichothecenes deoxynivalenol (DON), 3- and 15-acetyl-DON (AcDON), nivalenol (NIV), nivalenol (NIV), fusarenon-X (FX) and T-2 toxin (T-2) were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) and confirmed by gas chromatography-mass spectrometry (GC-MS). Eleven of the 12 strains were DON and/or ZEA producers and 9 were AcDON positive. No NIV, or FX were detected. One strain produced T-2. The predominant acetyl-DON isomer was 15-AcDON. Mass-spectral analysis yielded detectable levels of other mycotoxins, 13-OH-apotrichothecenes, 11-epiapotrichothecenes, culmorin, sambucinol, and isotrichodermol being the most numerous. From the metabolic profiles it is suggested that Uruguayan F. graminearum strains belong to the chemotype IB (DON/15-AcDON). The predominance of this chemotype is in accordance with data from Canada, United States, Mexico and Argentina which have similar climatic conditions that would favor F. graminearum growth and mycotoxin production.

Antiviral activity of trichothecene mycotoxins (deoxynivalenol, fusarenon-X, and nivalenol) against herpes simplex virus types 1 and 2.

The effect of trichothecene mycotoxins, deoxynivalenol (DON), fusarenon-X (FX) and nivalenol (NIV), on plaque formation of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in HEp-2 cells was examined. The 50% effective concentrations (EC50) of DON, FX, and NIV for HSV-1 plaque formation were 160, 56, and 120 ng/ml, respectively. Those for HSV-2 plaque formation were 94, 26, and 50 ng/ml, respectively. These three mycotoxins showed about 2-fold higher selectivity to HSV-2 than to HSV-1. Plaque formation of HSV-1 was not inhibited with trichothecenes at concentrations completely inhibiting plaque formation when cells were treated during virus adsorption period or 15 hr before infection. These results indicate that trichothecenes affect replication of HSV-1 after virus adsorption, but not before or during virus adsorption to the host cells.

Optimization of Yeast Bioassay for Trichothecene Mycotoxins

An improved disc diffusion type bioassay was developed for T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), deoxynivalenol (DON), nivalenol (NIV), neosolaniol (NSL), fusarenon-X (FUS-X), trichothecin (TIN), roridin A (RDA) and verrucarin A (VCA) using the yeast, Kluyveromyces marxianus. Factors such as type of medium, agar volume per plate, preincubation time and temperature, incubation temperature, inoculum size and pH had variable, and in some cases a dramatic effect on the sensitivity of the assay. The effect of pH of the assay medium was particularly pronounced. The highest sensitivity was obtained when 6 ml per plate of a tryptic-soy-agar (TSA) medium (pH 7.5) containing 105 CFU inoculum per ml was incubated at 38°C for 18 h. All of the trichothecenes (TNN) were able to inhibit the growth of the yeast with the detection limit being 0.005, 0.01, 0.02, 0.02, 0.1, 0.5, 10, 10, 10 and 50 μg/disc for VCA, RDA, T-2, TTN, DAS, HT-2, DON, NSL, FUS-X and NIV, respectively. The detection limits for corn and wheat spiked with T-2 were 0.1 and 0.2 μg/g, respectively. Non-TNN mycotoxins that did not inhibit yeast at a concentration of 200 μg/disc were aflatoxin B1 (AFB1), ochratoxin A (OA), citrinin (CT), penicillic acid (PA), cyclopiazonic acid (CPA), penitrem-A (PTA) and zearalenone (ZEE). These results indicate that disc diffusion assay using K. marxianus under optimized conditions provides a sensitive method for the detection of low concentrations of several TNN.

Production of mycotoxins by selectedFusarium graminearumandF. crookwellenseisolates

Corn cultures (five isolates each of Fusarium graminearum Group 1 from wheat crowns, Group 2 from scabby wheat grains and from ear rot of corn and five isolates of F. crookwellense) were screened for their ability to produce deoxynivalenol (DON), nivalenol (NIV), fusarenon-x (FUS-X) and zearalenone (ZEA). Nine of the ten F. graminearum isolates from wheat produced DON (5-165 micrograms g-1) but none produced either NIV or FUS-X. Conversely, 3/5 and 2/5 of the F. graminearum isolates from corn produced NIV (5-40 micrograms g-1) and FUS-X (5-7 micrograms g-1), respectively, while none produced DON. All but one of the F. graminearum isolates produced ZEA (2-1160 micrograms g-1). None of the F. crookwellense isolates produced DON, but 5/5 and 4/5 produced NIV (6-170 micrograms g-1) and FUS-X (3-90 micrograms g-1), respectively, and all produced ZEA (605-1030 micrograms g-1). The results confirmed previous findings on the presence of two distinct F. graminearum chemotypes.

Bacterial overgrowth in the jejunum of ICR mice and Wistar rats orally administered with a single lethal dose of fusarenon‐X, a trichothecene mycotoxin

A single oral dose of fusarenon-X (F-X), a trichothecene mycotoxin, resulted in abnormal microflora in the jejunum in ICR mice and Wistar rats with some differences in dose response between the species. In the acute phase, enterobacteria, streptococci, Clostridium perfringens and bacteroides showed remarkably increased counts in the jejunum of mice and rats dosed with F-X while lactobacilli showed a decrease in count. F-X brought an invasion of Pseudomonas aeruginosa into the livers, lungs, kidneys and spleens of ICR mice. Changes in the jejunal microflora appeared after 7 h in ICR mice and after 24 h in Wistar rats after a single oral dose of F-X of 7.5 and 4.0 mg/kg b.w., respectively, and the microflora returned to its normal state at 72 h in mice and 96 h in rats. The changes of intestinal microflora were followed by alterations in the growth curves of both animal species. The pH in the glandular stomach was also greatly enhanced before changes in the jejunal microflora. Acute F-X intoxication may be an involved manifestation of essential cytotoxicity of F-X mycotoxin alone and secondary bacterial overgrowth in the bowel.

High-performance liquid chromatographic screening method for mycotoxins using new retention indexes and diode array detection.

A reversed-phase high-performance liquid chromatographic (RP-HPLC) screening method for the determination of mycotoxins has been developed. The toxins were characterized by retention indexes and on-line UV spectra produced with a diode array detector (DAD). Retention indexes of mycotoxins exhibiting a wide range of polarities and chemical structures were determined during linear gradient elution with an acetonitrile/water solvent system. The retention index scale was based on the use of a new homologous series of 1-[4-(2,3-dihydroxypropoxy)phenyl]-1-alkanones (D-compounds) as internal standards. The mycotoxins studied were: five trichothecenes of group A: T-2 toxin (T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), neosolaniol (NEO), and an isomer of neosolaniol (NEO isomer); three trichothecenes of group B: nivalenol (NIV), deoxynivalenol (DON), and fusarenon-X (FUS-X); four aflatoxins: B1, B2, G1, and G2; and five other mycotoxins: sterigmatocystin (STE), zearalenone (ZEA), ochratoxin A (OCH A), citrinin (CIT), and patulin (PAT).

Effects of fusarenon-X and T-2 toxin on intestinal absorption of monosaccharide in rats.

In order to delineate the effects of Fusarenon-X (FX) and T-2 toxin (T-2) on intestinal absorption of monosaccharide, jejunal absorption in vivo and uptake in vitro of 3-0-methyl glucose (m-glucose) and L-glucose were studied in rats. Jejunal absorption in vivo was assessed by determining the rate of appearance of the sugars in plasma of the mesenteric vein draining the jejunal segment, which was perfused with the medium containing radiolabelled m-glucose or L-glucose. Jejunal uptake in vitro was assessed by determining the m-glucose or L-glucose uptake by the everted jejunum taken from toxin-treated rats. m-Glucose absorption was reduced 1 or 3 h after either toxin was injected into the jejunal lumen. A reduction of m-glucose absorption was also noted after intravenous injection of the toxins, although the timing and magnitude of the reduction were slightly different from those seen after the luminal injection. These results suggest that both toxins impair the jejunal function relating to monosaccharide absorption in the early stages of intoxication. The reduction in m-glucose absorption was associated with a reduction in L-glucose absorption and unchanged or increased uptake of L-glucose. The active transport component, which was indicated by the difference between absorption or uptake of the two sugars, was also reduced in association with the reduction of m-glucose absorption. These results suggest that the toxins cause specific damage in the active transport system for monosaccharides on the one hand, and impairment of their diffusional movement from the epithelial layer to the mesenteric vein on the other.

Dermal toxicity of Fusarium toxins in combinations.

T 2 toxin (T 2), diacetoxyscirpenol (DAS), fusarenon X (FX) and butenolide (Bd) at concentrations of 0.2, 0.3, 5 and 10 micrograms/site, respectively, were applied individually and in combinations on shaved skin of guinea pigs. Erythema and induration were observed on skin patches treated with the toxins. Increase in the thickness of stratum malpighii was the major histological change observed. Mild to moderate degeneration of fibrocytes and cellular infiltration were found in the corium of skin treated with FX, Bd, DAS and T 2. The order of toxicity of individual toxins was T 2 greater than DAS greater than FX greater than Bd. Combinations of T 2 + FX and T 2 + Bd resulted in antagonism, while DAS + FX and DAS + Bd caused synergism.

Characteristics of inflammation induced by fusarenon-X, a trichothecene mycotoxin from Fusarium species

Fusarenon-X (F-X) is one of the trichothecene mycotoxins, which have been suggested to induce Fusarium mycotoxicoses. Administration of F-X to experimental animals causes diarrhea, skin irritation, and foot edema. In the present work, the properties of F-X-induced inflammation were examined. F-X increased capillary permeability of the abdominal blood vessels in a dose-dependent manner in mice. The F-X-induced increase of the abdominal capillary permeability was not inhibited by 200 mg/kg po of phenylbutazone, 200 mg/kg po of aspirin, 40 mg/kg po of phentolamine, 50 mg/kg po of diphenhydramine, 100 mg/kg po of dibenzyline, 200 mg/kg of hydrocortisone, and 4 mg/kg sc of reserpine. These results suggest that the permeability increment by F-X may not be mediated by serotonin, histamine, norepinephrine, prostaglandins, leukotrienes, and throm☐anes. The increment by F-X also appears to result from histological changes in the capillary blood vessels which are possibly induced by F-X-induced inhibition of protein synthesis, and/or from unidentified inflammatory chemical mediators.

Studies on mechanisms of diarrhea induced by fusarenon-X, a trichothecene mycotoxin from Fusarium species: Fusarenon-X-induced diarrhea is not mediated by cyclic nucleotides

Cardinal signs of red mold toxicosis in man and farm animals are vomiting, nausea, diarrhea, and food refusal. The red mold toxicosis has been suggested to be induced by trichothecenes, which are produced by Fusarium fungi. Fusarenon-X (F-X) is one of the trichothecene mycotoxins. The ip injection of F-X to rats causes an expansion of the small intestine and watery diarrhea. In this study, we measured the concentrations of protein, sodium, potassium, and calcium in the serum of rat treated with F-X for the sake of demonstrating the loss of serum protein and the decreases of serum sodium and calcium by F-X. Since it is well known that some diarrheal diseases are due to the increase of cyclic nucleotide level in the intestinal mucosa, we also measured cyclic AMP and cyclic GMP levels in the intestinal mucosa. It was demonstrated that F-X did not increase the cyclic AMP and cyclic GMP levels in the jejunal and the ileal mucosa at 8 and 24 hr after F-X treatment. The results obtained in this work suggest that F-X-induced diarrhea is not mediated by the cyclic nucleotide system.

Occurrence of Trichothecenes in feed and cereals in Finland

In 1985 82 samples of feed and food grain were analyzed for trichothecenes deoxynivalenol (DON), nivalenol (NV), diacetoxyscirpenol (DAS), T-2 toxin, HT-2 toxin and fusarenon-X (F-X). Trichothecenes were found in 77 of these samples. The highest amounts were DON 6300 ug/kg and DAS 1680 ug/kg.In 1986, in a corresponding study of 113 samples, trichothecenes were found in 110 samples. A new trichothecene in Finland, 3-acetyldeoxynivalenol (3-AcDON), was identified in 35 of these samples in concentration of 2-211 ug/kg.Analyzing methods were gas chromatography and GC-mass spectrometry. It is characteristic of the feed samples suspected of causing outbreaks in animals in Finland that several trichothecenes are often found in the same sample. As an example of this is a poultry feed with following results: DON 33 ug/kg, 3-AcDON 21 ug/kg, DAS 45 ug/kg, T-2 toxin 40 ug/kg, HT-2 toxin 12 ug/kg.

Efficient Method for Determination of Deoxynivalenol and Nivalenol in Wheat and Barley by Gas Chromatography

We have developed an analytical method for rapidly determining deoxynivalenol (DON) and nivalenol (NIV), typical mycotoxins in cereals infected by Fusarium spp., in wheat and barley. Since we used a solvent of high polarity and water to elute columns of mixed alumina-charcoal-celite and Amberlite for extraction and clean-up, we were able to omit partition and dehydration processes. The separated mycotoxin was determined by ECD-gas chromatography after trimethylsilylation. The limits of determination of DON and NIV are 12ng/g, and that of fusarenon-X (F-X) is 24ng/g. These limits are adequate for economic screening analysis. When DON and NIV were added to wheat and barley at the 120ng/g level and F-X at the 240ng/g level, the recoveries were 101 and 95%, 93 and 92%, and 96 and 96%, and the coefficients of variation were 2.9 and 3.4%, 3.7 and 2.4%, and 3.5 and 3.4%, respectively. When F-X was detected by this method, its identity was confirmed by mass chromatography. So far we have not detected F-X in cereals. Further studies are in progress. This method was designed for rapid determination of wheat and barley, but should also be applicable to the analysis of mixed feed. It is easy to change the limits of determination by changing the quantities of sample and solvent. One analyst can analyze 8 samples in a day, and two analysts, 20 samples.

Fusarenon X and nivalenol production by Gibberella zeae in liquid and rice cultures

Toxigenesis of Gibberella zeae strains NHL-F-1372 and NHL-F-1373 was compared in liquid and rice culture. Growth of both strains in glucose-yeast extract-peptone medium (GYEP) for 25 days resulted in peak levels of fusarenon X (FX) ranging from 40–200 mg/L with lower levels of nivalenol (NIV) (10 mg/L). Growth of these strains in modified Fries medium amended with 4% corn steep liquor (CSL) resulted in a much lower total 8-ketotrichothecene yield than in GYEP, with NIV being the primary compound produced. Although FX appeared initially in this latter medium, the toxin disappeared concurrently as the pH exceeded 8.0. Growth rates and total mycelial accumulation were lower in GYEP cultures than in the modified Fries with CSL cultures. Appearance of FX and NIV in modified Fries medium with CSL paralleled the order of appearance of these compounds in rice, but the total trichothecene yield in rice was much higher. In general, growth and toxigenesis by the nivalenol-producing fusaria in liquid and rice cultures was qualitatively similar to that previously found for deoxynivalenol-producing isolates.

Further survey on the Fusarium mycotoxins in Korean cereals.

Fifty-one samples of cereals from the 1984 harvest from Korea were analyzed for nivalenol (NIV), fusarenon-X (FX), deoxynivalenol (DON) and 3-acetyl-DON by gas chromatography (GC) utilizing a 63Ni electron capture detector (ECD), and were quantitated for zearalenone (ZEN) by high-performance liquid chromatography (HPLC) with a fluorescence detector (FD). Trichothecenes and ZEN in the positive samples were confirmed by GC-mass spectrometry (MS). Out of 51 samples, 51, 46 and 42 were positive for NIV, DON and ZEN, respectively, and one malt sample was heavily contaminated with NIV (2675 ng/g) and DON (246 ng/g), and one wheat sample was heavily contaminated with NIV (3169 ng/g). Neither FX nor 3-acetyl-DON was detected in any of the samples. The data reported here indicates that Korean cereals harvested in 1984 are simultaneously contaminated with NIV, DON and ZEN, and the incidences and levels are similar to those observed in the cereals harvested in 1983.

Studies of mechanisms of diarrhea induced by fusarenon-X, a trichothecene mycotoxin from Fusarium species; the effects of fusarenon-X and various cathartics on the digestion and the absorption of the mouse intestine

The red mold toxicosis, the major symptoms of which are diarrhea, vomiting and nausea, has been suggested to be induced by trichothecenes produced by Fusarium fungi. Fusarenon-X (F-X) is one of the trichothecene mycotoxins. In this study, we measured the blood glucose level, intestinal glucose contents and intestinal starch contents after the mouse small intestine was loaded with starch, in order to observe the effect of F-X and various cathartics on the digestive and absorptive abilities of the mouse small intestine. Mannitol, castor oil and magnesium sulfate tended to inhibit the dietary increase of blood glucose, the increase of intestinal glucose contents and the decrease of intestinal starch contents after the administration of starch. These results suggest that these cathartics may slightly inhibit the digestion or the absorption in the intestine, but their inhibitory effects seem to be too weak to induce a significant increase of osmotic pressure in the intestinal lumen. Dioctyl sodium sulfosuccinate (DSS) significantly inhibited the increase of blood glucose level, the increase of intestinal glucose contents and the decrease of intestinal starch contents after the starch administration, suggesting that DSS may induce malabsorption and maldigestion in the intestine which may be one of the factors of induction of DSS-induced diarrhea. In contrast, F-X induced no change on blood glucose level, intestinal glucose and starch contents. Therefore, F-X-induced diarrhea can not be related to the increase of osmotic pressure of the intestinal lumen which is induced by malabsorption and maldigestion. Present results support the mechanism of F-X-induced diarrhea proposed previously by us that F-X causes diarrhea through increasing the permeability of intestinal epithelium cells and resulting in the exudation of plasma contents and an eventual increase in the intestinal fluids.

Studies on mechanisms of diarrhea induced by fusarenon-X, a trichothecene mycotoxin from Fusarium species; characteristics of increased intestinal absorption rate induced by fusarenon-X.

Fusarenon-X (F-X) is one of the trichothecene mycotoxins. F-X enhanced D-xylose absorption rate in rat everted intestinal sac. In the present work, the mechanism of the F-X-induced enhancement of intestinal D-xylose absorption rate was studied. F-X had no detectable effect on D-glucose absorption of everted rat intestine. When 10 mM of D-xylose was employed, a concomitant application of oxygen-free condition and low temperature failed to affect the D-xylose absorption of normal rat, but inhibited that of F-X-treated rat. When D-glucose concentration was 5 mM, the concomitant application of oxygen-free condition and low temperature depressed the D-glucose absorption of normal rat, while it did not reduce that in F-X-treated rat. These results suggested that the sugar translocation mechanism of F-X-treated rat intestine was different from that of normal one. F-X-induced enhancement of D-xylose absorption in everted intestinal sac may depend on the increase in permeability of the intestinal membrane which is derived from the histological and functional damage of the intestinal mucosa induced by F-X.

Studies on mechanisms of diarrhea induced by fusarenon-X, a trichothecene mycotoxin from Fusarium species

Cardinal signs of red mold toxicosis in man and farm animals are vomiting, nausea, diarrhea, and food refusal. The red mold toxicosis has been suggested to be induced by trichothecenes which are produced by Fusarium fungi. Fusarenon-X (F-X) is one of the trichothecene mycotoxins. The ip injection of F-X to rats caused watery diarrhea 36 to 60 hr after injection. Autopsy done 24 hr after an injection of F-X revealed an expansion of the small intestine but no macroscopic bleeding into the lumen of the intestine. F-X increased the absorption rate of d-xylose from the intestine in vitro, caused leakage of intravenously injected Evan's blue dye into the intestinal lumen, and decreased the serum sodium level. However, F-X did not increase leakage of erythrocytes into the intestinal lumen. In histological observations, F-X shortened intestinal villi and caused an infiltration of erythrocytes into the intestinal lamina propria. These findings suggest that F-X may increase the permeability of the blood vessel wall and intestinal mucosal epithelium and thereby cause leakage of plasma contents into the intestinal lumen which eventually leads to diarrhea.

General pharmacological studies of fusarenon-X, a trichothecene mycotoxin from Fusarium species

Fusarenon-X (F-X) is one of the trichothecene mycotoxins. In the present work, pharmacological properties of F-X were examined. (1) F-X induced hypothermia but did not produce appreciable behavioral changes of mice. (2) In anesthetized rats, F-X caused a rise in the blood pressure and a decrease in the respiratory rate but induced no significant change in the heart rate. (3) In isolated tissues such as fundus muscle, vas deferens, tracheal muscle, ileum, and atrium, F-X had no detectable effects on spontaneous activity and the responsiveness to agonists. (4) Subplantar administration of F-X into rat hindpaw induced edema. F-X had little influence on the histamine release from mast cells and the membrane stability of erythrocytes. (5) F-X decreased the spontaneous peristalsis of intestine but increased the intestinal propulsion of charcoal meal. (6) F-X induced vomiting in dogs which was suppressed by preliminary administration of chlorpromazine and metoclopramide. F-X may induce vomiting by stimulating the chemoreceptor trigger zone in dogs.

Hypoglycemia in mice administered with fusarenon-X.

Histological observation combined with determination of the serum glucose level and histochemical detection of liver glycogen was undertaken to examine the acute toxicity of fusarenon-X (FX) in mice. Mice intraperitoneally injected with a sublethal dose of the toxin showed rapidly developed hypoglycemia followed by depletion of liver glycogen. Mitotic inhibition was observed in many visceral organs and most markedly in the intestinal crypt cells, where the mitotic figures completely disappeared prior to the increase in number of the degenerated and nucrotic cells. No glycosuria was found. The disturbing effect of FX on the oral glucose tolerance test suggested the involvement of accelerated glycolysis and, more likely, of intestinal malabsorption.