fusarenon-X Research Articles

Trichothecene and zearalenone production, in culture, by isolates of Fusarium pseudograminearum from western Canada

One hundred and twenty-seven isolates of Fusarium pseudograminearum were obtained from seeds and vegetative parts of cereals and other gramineae grown in western Canada. Culture of the isolates on agar selective for Fusarium graminearum allowed a successful differentiation from F. graminearum. Sterilized rice (40% moisture content) was inoculated with a single germinated spore of isolates identified as F. pseudograminearum and was incubated at 23 °C. After 14 days of incubation, mycelium was taken from each culture, and the DNA was extracted to detect the tri5 gene by polymerase chain reaction analysis and confirm the species identity. After 21 days of incubation, mycotoxins were quantified in dried rice. All isolates contained the tri5 gene. Deoxynivalenol (DON) was produced in 125 of the 127 isolates, 3-acetyldeoxynivalenol (3-ADON) in 122 isolates, 15-acetyldeoxynivalenol (15-ADON) in 2 isolates, diacetoxyscirpenol (DAS) in 17 isolates, and zearalenone in 100 isolates; both nivalenol (NIV) and fusarenon X (FX) were detected in 1 isolate. There appears to be three chemotypes: DON–3-ADON, DON–15-ADON, and NIV. Neither of the two 15-ADON producers or the NIV producer formed DAS. This is the first time that the production of DAS and FX is associated with F. pseudograminearum. None of the isolates produced HT-2 toxin or T-2 toxin at detectable levels.

Natural Occurrence of 16 Fusarium Toxins in Grains and Feedstuffs of Plant Origin from Germany

A total of 220 samples comprising cereals, cereal byproducts, corn plants and corn silage as well as non-grain based feedstuffs was randomly collected during 2000 and 2001 from sources located in Germany and analysed for 16 Fusarium toxins. The trichothecenes scirpentriol (SCIRP), 15-monoacetoxyscirpenol (MAS), diacetoxyscirpenol (DAS), T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), neosolaniol (NEO), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivealenol (15-ADON), nivalenol (NIV) and fusarenon-X (FUS-X) were determined by gas chromatography/mass spectrometry. Zearalenone (ZEA) and alpha- and beta-zearalenol (alpha- and beta-ZOL) were analysed by high performance liquid chromatography with fluorescence and UV-detection. Detection limits ranged between 1 and 19 microg/kg. Out of 125 samples of a group consisting of wheat, oats, corn, corn byproducts, corn plants and corn silage only two wheat samples did not contain any of the toxins analysed. Based on 125 samples the incidences were at 2-11% for DAS, NEO, T-2 Triol, FUS-X, alpha- and beta-ZOL, at 20-22% for SCIRP, MAS, T-2 tetraol and 3-ADON, at 44-74% for HT-2, T-2, 15-ADON, NIV and ZEA, and at 94% for DON. Mean levels of positive samples were between 6 and 758 microg/kg. Out of 95 samples of a group consisting of hay, lupines, peas, soya meal, rapeseed meal and other oil-seed meals, 64 samples were toxin negative. DAS, T-2 triol, NEO and FUS-X were not detected in any sample. The incidences of DON and ZEA were at 14 and 23% respectively, those of the other toxins between 1-4%, mean levels of positive samples were between 5 and 95 microg/kg.

Intake estimates for trichothecene toxins of the population in Southwest Germany in 1998 and in 1999

The intake of theFusarium toxins deoxynivalenol (DON), nivalenol (NIV), HT-2 and T-2 toxin (HT-2, T-2), 3-, 15-acetyldeoxynivalenol (3-, 15- ADON), and fusarenon-X (FUS-X) was calculated for adults, children and babies, for an area of southwest Germany and two years (1998, 1999). Estimates were based on consumption data of bread and pasta by both adults and children and of infant food by babies, reported for the German population in a study on behalf of the European Union, and on toxin contents of a total of 208 samples of these commodities. No exceeding of the tolerable daily intake (TDI) of DON, NIV and the sum of HT-2 and T-2, as stated by the EU, was found for adults (70 kg body weight (BW)) and for babies (10 kg BW), independent of year and level of consumption. For children (20 kg BW) the intake of DON exceeded the TDI in 1998 for high, and in 1999 for both mean and high consumers. For both years the intake of the sum of HT-2 and T-2 was below the TDI following mean but above this value following high consumption. The intake of NIV was far below the TDI for both levels of ingestion. The daily intake of each of the three toxins 3-, 15- ADON and FUS-X was below 0.03, 0.11 and 0.05 μg/kg/BW for adults, children and babies, respectively.

Mycotoxins Produced by Fusarium spp. in Isogenic Bt vs. non‐Bt Maize Hybrids under European Corn Borer Pressure

Stalk and ear rots caused by Fusarium subspecies are often related to mycotoxin accumulation in maize (Zea mays L.) kernels. Various mycotoxicoses in livestock and humans are triggered by the consumption of these toxins. The European corn borer (Ostrinia nubilalis Hübner) reportedly promotes the infection by Fusarium spp. The objectives of our study were to (i) evaluate the concentration of deoxinivalenol (DON), 3‐acetyl‐deoxynivalenol (3‐A‐DON), 15‐acetyl‐deoxynivalenol (15‐A‐DON), fumonisin (FUM), fusarenon‐X (FUS‐X), moniliformin (MON), and nivalenol (NIV) in kernels; (ii) determine the level of European corn borer (ECB) resistance; and (iii) investigate the association between the concentration of mycotoxins and ECB resistance. The study used early maturing European Bt (Bacillus thuringiensis) cultivars, their isogenic counterparts, and commercial hybrids. The field experiments were conducted at three locations in Germany. The mycotoxins most prevalent were DON, FUM, and MON. Plots infested by and protected from ECB differed significantly for DON and FUM concentrations. In addition, significant differences were found for concentrations of FUM between isogenic Bt and non‐Bt hybrids. The two Bt events—Bt176 and Mon810—were also significantly different for FUM concentrations. Not all mycotoxins were related to ECB damage. Insect management and, therefore, the use of Bt cultivars may be a short‐term solution to minimize toxins in kernels.

Induction of apoptosis and cytokine production in the Jurkat human T cells by deoxynivalenol: role of mitogen-activated protein kinases and comparison to other 8-ketotrichothecenes

The Jurkat T-cell line was used to study potential impact of deoxynivalenol (DON) and related 8-ketotrichothecenes on human immune function. DON (250-1000 ng/ml) readily induced caspase-3 and apoptosis in Jurkat cells. DON (62.5-500 ng/ml) also significantly upregulated IL-2 and IL-8 production following prestimulation with phorbol myristate acetate and ionomycin. DON markedly induced phosphorylation of p38, JNK 1/2, and ERK2. SB203580, a specific inhibitor of p38, reduced DON-induced apoptosis. The MEK1 inhibitor PD98059 which blocks ERK activation had only a small inhibitory effect on DON-induced apoptosis while the JNK inhibitor SP600125 was without effect. Inhibition of p38 attenuated DON-induced upregulation of IL-2 while all three MAPK inhibitors suppressed IL-8 upregulation. When effects of DON were compared to other 8-ketotrichothecenes, the concentrations of DON, 3-acetyl deoxynivalenol (3-ADON), 15-acetyl deoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon X (FX) causing 50% apoptosis were 0.6, 0.5, 0.5, 0.5 and 7.5 microg/ml, respectively. Relative to IL-2 upregulation, FX was suppressive whereas 3-ADON, 15-ADON and NIV had no effect at concentrations of 62.5-500 ng/ml. In contrast, 15-ADON at 62.5-500 ng/ml and 3-ADON at 625-5000 ng/ml upregulated IL-8 production but FX and NIV had no effect. Taken together, these data suggest that DON's effects on apoptosis and cytokine production were differentially regulated by MAPKs. Although DON shared its capacity to induce apoptosis and potentiate IL-8 production with other 8-ketotrichothecenes, it appeared to be unique in its capacity to upregulate IL-2.

Report from SCOOP task 3.2.10 “collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU member states” : Subtask: trichothecenes

In 2001 the SCOOP (SCOOP: Scientific Co-operation on Questions relating to Food) task 3.2.10 “Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU Member States” was established. The task was divided in three subtasks (zearalenone, fumonisins and trichothecenes). Results of the subtask trichothecenes, which is co-ordinated by The Netherlands, will be presented. About 35,000 results were received about occurrence of 12 different trichothecenes (deoxynivalenol (DON), nivalenol (NIV), 3 and 15 acetyl-deoxynivalenol (3/15-AcDON), fusarenon-X (FUS-X), T-2 and HT-2 toxin, T2-triol, diacetoxyscirpenol (DAS), neosolaniol (NEOSOL, monoacetoxyscirpenol (MAS) and verrucarol (VOL)) in various food and food raw materials from 12 countries. Only the results of DON, NIV, T-2 and HT-2 toxin are included in this paper, because most of the data refer to these toxins and only for these trichothecenes the Scientific Committee for Food sets (temporary)-Tolerable Daily Intakes (t-TDIs). Occurrence data: By far most of the occurrence data were obtained for DON in wheat. Among cereals, corn showed the highest level of contamination with trichothecenes. Consumption data: There is a significant lack of consumption data in some countries. In particular information on baby’s and children’s food is generally not available. Intake data: Wheat and wheat containing products (like bread and pasta) represent the major source of intake for the four trichothecenes. The mean intakes for DON are below the TDI, however for the young children groups the mean intakes are sometimes (very) close to the TDI. By comparing the high intake levels for DON with the TDI, it is clear that especially for the young children groups most of the intakes are above the TDI. For NIV, the (mean and high level) intakes are far below the TDI. The summarised T-2 and HT-2 toxin intakes are in most cases (for the mean as well as the high level intake) above the t-TDI.

Survey of Fusarium toxins in foodstuffs of plant origin marketed in Germany

A total of 219 samples of foodstuffs of plant origin, consisting of grain-based food, pseudocereals and gluten-free food as well as vegetables, fruits, oilseeds and nuts, were randomly collected during 2000 and 2001 in food and health food stores. A spectra of 13 trichothecene toxins including diacetoxyscirpenol (DAS), 15-monoacetoxyscirpenol (MAS), scirpentriol (SCIRP), T-2 and HT-2 toxins (T-2, HT-2), T-2 triol, T-2 tetraol, neosolaniol (NEO) of the A-type as well as deoxynivalenol (DON), 3- and 15-acetyl-DON (3-, 15-ADON), nivalenol (NIV), and fusarenon-X (FUS-X) of the B-type were determined by gas chromatography/mass spectrometry. Analysis of zearalenone (ZEA), α- and β-zearalenol (α- and β-ZOL) was made by high-performance liquid chromatography with fluorescence and UV-detection. Detection limits ranged between 1 and 19 μg/kg. Out of 84 samples of cereal-based including gluten-free foods, 60 samples were positive for at least one of the toxins DON, 15-ADON, 3-ADON, NIV, T-2, HT-2, T-2 tetraol and ZEA, with incidences at 57%, 13%, 1%, 10%, 12%, 37%, 4% and 38%, respectively, whereas SCIRP and its derivatives MAS and DAS, T-2 triol, Fus-X as well as α- and β-ZOL were not detected in any sample of this subgroup. Contents of DON ranged between 8 and 389 μg/kg, for all other toxins determined concentrations were below 100 μg/kg. The pseudocereals amaranth, quinoa and buckwheat were free of the toxins investigated. Ten of 85 samples of vegetables and fruits were toxin positive. ZEA and the type A trichothecenes MAS, SCIRP, DAS, HT-2 were detected in 7, 3, 2, 1 and 1 samples, respectively. Out of 35 samples of oilseeds and nuts, 7 samples were toxin positive. HT-2, T-2 and ZEA were detected in 4, 3 and 4 samples, respectively. In vegetables and fruits as well as in oilseeds and nuts, toxin levels were below 50 μg/kg. None of the B-type trichothecenes analysed was found for both subgroups.

Placental and milk transmission of trichothecene mycotoxins, nivalenol and fusarenon-X, in mice

In order to investigate the transfer of nivalenol (NIV) and fusarenon-X (FX) from pregnant to fetal mice and from lactating to suckling mice, 3H-NIV or 3H-FX was given p.o. to pregnant or lactating mice. Radioactivity was detected in the whole fetal tissues as well as the fetal liver and kidney, the levels being comparable to those of the maternal tissues. Radioactivity was also detected in the milk, and liver and kidney tissues taken from suckling mice of both 3H-NIV or 3H-FX administered dams. HPLC analysis of fetal tissue homogenates from non-labeled FX- or NIV-administered pregnant mice revealed transmission of NIV to fetuses after administration of either toxin. In mice given the non-labeled FX, major and minor peaks of NIV and FX on HPLC were noted in suckling pup tissue homogenates. The results demonstrate that NIV transfers in unchanged form to fetal or suckling mice via placenta or milk, respectively, and that FX does so mainly after being metabolized to NIV in maternal body.

Relationship between European corn borer resistance and concentration of mycotoxins produced by Fusarium spp. in grains of transgenic Bt maize hybrids, their isogenic counterparts, and commercial varieties

AbstractThe European corn borer (ECB), Ostrinia nubilalis Hb., is a major pest of maize in central Europe and promotes the infection of maize with Fusarium spp. In this study, transgenic Bt maize hybrids were compared with their isogenic counterparts, and with commercial hybrids from the recommended list with regard to their level of ECB resistance and their concentration of deoxynivalenol (DON), its 15‐acetyl (15‐A‐DON) and 3‐acetyl (3‐A‐DON) derivatives, nivalenol (NIV), fusarenon‐X (FUS‐X), fumonisins (FUM), and zearalenon (ZEN) in harvested grains. The field experiments were performed in Germany at four locations in 1999 and at five locations in 2000. Transgenic Bt hybrids showed significantly lower means than their corresponding isogenic counterparts and than commercial hybrids for all resistance traits: damage rating of stalks, number of larvae per plant, number of larvae per ear, and percentage of damaged plants or ears under infestation. Among all mycotoxins analysed, DON consistently showed the highest concentration across all year × location combinations. Mycotoxin concentrations varied significantly between locations, years and genotypes, whereas mycotoxin concentrations were not significantly different between infested and protected plots. Associations between ECB resistance traits and mycotoxin concentrations were not consistent across years. It is concluded that under central European conditions, the use of Bt maize hybrids will only slightly reduce the contamination of maize kernels with mycotoxins produced by Fusarium spp.

Fusarenon-X induced apoptosis in HL-60 cells depends on caspase activation and cytochrome c release

Fusarenon-X (FX), a trichothecene mycotoxin, is well known to be cytotoxic to mammalian cells. Our previous study revealed that FX induced apoptosis in mouse thymocytes both in vivo and in vitro. We investigated the mode of apoptosis induced by FX using HL-60 cell culture. When FX at a final concentration of 0.5 μg/ml was added, cell degradation was observed 5 h after exposure, and most of the cells had fallen into apoptosis 24 h after exposure. DNA fragmentation into 180-bp multimers was observed 5 h after exposure, and its dose-dependency was clear in the cells treated with 0.1 μg/ml and higher doses. The percentage of apoptotic cells (sub-G 0 population) increased dose- and time-dependently after exposure, when analyzed using flow cytometry. The activities of caspase-3, -8, and -9 were elevated within 2 h by exposure to FX. DNA fragmentation and an increase in the apoptotic population were abrogated by pre-treating the cells with broad-spectrum caspase inhibitors Z-VAD-fmk or Z-Asp-CH 2-DCB. Cytochrome c release from mitochondria to cytoplasm was observed clearly, and this release occurred caspase-independently. These findings suggest that FX induces apoptosis in HL-60 cells by stimulating cytochrome c release followed by its downstream events including the activation of multiple caspases.

Fusarium toxins in wheat flour collected in an area in southwest Germany

A total of 60 samples of wheat flour were collected during the first 6 months of 1999 from mills and food stores in an area in southwest Germany. Samples included whole-grain and two types of white flour with these three groups characterized by a high, medium and low ash content. The contents of deoxynivalenol (DON), nivalenol (NIV), 3- and 15-acetyldeoxynivalenol, HT-2 toxin (HT-2), T-2 toxin (T-2) and fusarenon-X (FUS-X) were determined by gas chromatography/mass spectrometry, and those of zearalenone (ZEA), α- and β-zearalenol (α- and β-ZOL) by high performance liquid chromatography with fluorescence detection. FUS-X, α- and β-ZOL were not detected in any sample. Based on incidence and level, DON was the predominant toxin followed by NIV and ZEA for all three flour types. The overall degree of toxin contamination was lower with decreasing ash content. This suggests a localization of the toxins analyzed primarily in the outer parts of the original wheat kernels. The median DON content was significantly ( P<0.05) higher for wheat flour originating from wheat of conventional than of organic production.

Further survey of the occurrence of fusarium toxins in wheat grown in southwest Germany

A total of 53, 54, 57, 52 and 60 wheat samples for feed use were collected randomly after the 1989, 1990, 1991, 1992 and 1993 crops, respectively, from farms in an area of southwest Germany. Deoxynivalenol (DON), 3‐ and 15‐acetyldeoxynivalenol (3‐, 15‐ADON), nivalenol (NIV), HT‐2 toxin (HT‐2), T‐2 toxin (T‐2), diacetoxyscirpenol (DAS), and fusarenon‐X (FUS‐X) were determined by gas chromatography, combined with mass selective detection (GC‐MS), zearalenone (ZEA), α‐ and β‐zearalenol (α‐ß‐ZOL) were determined by HPLC. DON was the major toxin, with incidences at 77 to 93% and mean contents at 167 to 735 μg/kg. In contrast, incidences of ZEA, 3‐ADON, NIV, HT‐2, and T‐2 were at 13 to 37%, 10 to 44%, 15 to 67%, 0 to 11%, and 0 to 12%, respectively, with mean contents in positive samples between 2 and 73 μg/kg, except for 948 μg/kg 3‐ADON in samples from 1993. 15‐ADON and FUS‐X were assayed in samples from 1991, 1992 and 1993. 15‐ADON was found in 0 to 11% of samples at mean levels ≤ 17 μ/kg, DAS, α‐ and β‐ZOL, and FUS‐X were not detected in any sample. Over the years, incidences and levels of toxins remained constant, decreased or increased, with most differences between years being slight and insignificant. The risk for livestock due to DON, HT‐2 and ZEA was estimated based on maximum tolerated levels recommended for these toxins in some countries.

Fast methods for screening of trichothecenes in fungal cultures using GC-MS/MS

Fast methods for screening for production of simple and macrocyclic trichothecenes, produced on solid substrates were developed. Crude extracts fromFusarium cultures grown on yeast extract sucrose agar (approx. 0.3 cm(2) culture) were derivatised using pentafluoropropionic anhydride and analysed by GC - tandem mass spectrometry using a Finnigan GCQ(+) ion trap. The MS was operated in the EI(+) multiscan mode allowing simultaneous full scan and MS/MS of 3-4 parent ions. Production of acetyl T-2 toxin (AT-2), T-2 toxin, HT-2 toxin (HT-2), T-2 triol (T-2TR), T-2 tetraol (T-2TE), neosolaniol (NEO), iso-neosolaniol (I-NEO), scripentriol (SCR), 4,15-diacetoxyscirpenol (DAS), 15-acetoxyscripentriol (15MAS), 4-acetoxyscripentriol (4MAS), nivalenol (NIV), fusarenon-X (F-X), deoxynivalenol (DON), 15-acetoxy-DON (15DON) and 3-acetoxy-DON (3DON) was studied for severalFusarium species. In hydrolysed crude extracts ofStachybotrys albipes, Trichoderma harzianum, andMemnoniella echinata trichodermol was detected, in cultures ofS. chartarum both verrucarol and trichodermol were detected as the heptafluorobuturyl esters after derivatisation with a imidazole based reagent.

Determination of trichothecenes in cereals

An effective method for the determination of seven trichothecenes – deoxynivalenol (DON), nivalenol (NIV), T-2 tetraol (T-24), fusarenon-X (FUS-X), diacetoxyscirpenol (DAS), T-2 toxin (T-2), HT-2 toxin (HT-2) in cereals is presented. Gel permeation chromatography on Bio-Beads S-X3 was used for clean-up of acetonitrile–methanol extract. GC–ECD was used for identification and quantification of trifluoroacetylated trichothecenes. The limit of quantitation for the method was in the range 40–200 μg/kg. Recoveries at a spiking level of 2 mg/kg ranged from 76 to 100%.

A survey of Fusarium toxins in cereal-based foods marketed in an area of southwest Germany.

A total of 237 commercially available samples of cereal-based foods including bread and related products, noodles, breakfast cereals, baby and infant foods, rice and other foods were randomly collected in southwest Germany during the first six months of 1998. The trichothecenes deoxynivalenol (DON), 3- and 15-acetyl-deoxynivalenol (3-,15-ADON), nivalenol (NIV), fusarenon-X (FUS-X), T-2 toxin (T-2) and HT-2 toxin (HT-2) were determined by gas chromatography/mass spectrometry following clean-up by a two stage solid-phase extraction. Detection limits ranged between 2 and 12 micrograms/kg. Based on all samples, the incidence of DON, HT-2, T-2, 3-ADON, 15-ADON, and NIV was at 71, 18, 4, 4, 4 and 2%, respectively; the average contents in positive samples were at 103, 16, 14, 17, 24 and 109 micrograms/kg, respectively. Fus-X was not detected in any sample. A lower (P < 0.05) DON content was found in baby and infant foods as well as in cookies and cakes compared to bread. Overall, based on the incidence and level of all six toxins, the degree of contamination was lowest in baby and infant foods. Foods produced from either white or whole grain flour did not differ (P > 0.05) with regard to the incidence and level of DON. In foods produced from cereals of organic production both the incidence and median content of DON was lower compared to conventional production. Zearalenone, alpha- and beta-zearalenol were determined by high performance liquid chromatography in 20 selected samples, mostly baby and infant foods. These toxins were not present in excess of the detection limit in any sample.

Natural occurrence of Fusavium toxins in oats harvested during five years in an area of southwest Germany

A total of 56, 56, 54, 51, and 55 oats samples used for feed production were collected ramdomly after the 1987, 1989, 1990, 1991 and 1992 crops, respectively, from farms located in an area of southwest Germany. Deoxynivalenol (DON), 3‐ and 15‐acetyl‐deoxynivalenol (3‐,15‐ADON), nivalenol (NIV), fusarenon‐X (FUS‐X), T‐2 toxin (T‐2), HT‐2 toxin (HT‐2) and diacetoxyscirpenol (DAS) were determined by gas chromatography with mass selective detection (GCMS), zearalenone (ZEA), α and β‐zearalenol (α‐,β‐ZOL) by GC‐MS or by HPLC. DON was the major toxin with incidences at 49–85% and mean levels in positive samples of 52–302 fig/kg. Incidences of ZEA, 3‐ADON, NIV, HT‐2, and T‐2 were at 20–37, 0–30, 18–67, 0–29, and 27–61%, respectively, with mean levels in positive samples at 8–25, 5–63, 11–192, 205–296, and 20–244 μg/kg, respectively, α‐ and β‐ZOL and DAS were not detected in any sample. 15‐ADON and FUS‐X were assayed in samples from 1987, 1991 and 1992. 15‐ADON was detected in 9, 4 and 0% of samples, with an average of 9 and 18 μg/kg, respectively; FUS‐X was not detected. The incidence and levels of toxins varied from year to year. The correlation between the occurrence of toxins and precipitation is discussed.

Direct analysis of several Fusarium mycotoxins in cereals by capillary gas chromatography–mass spectrometry

A method for qualitative and quantitative analysis of Fusarium mycotoxins by gas chromatography–mass spectrometry (GC–MS) using cold on-column injection was improved. Eight typical mycotoxins, including deoxynivalenol (DON), 3-acetyldeoxynivalenol (3ADN), fusarenon-X (FX), diacetoxyscirpenol (DAS), 15-monoacetylscirpenol (15MAS), T-2 toxin (T-2), scirpentriol (SCT), and zearalenone (ZEA) were subjected to GC–MS without chemical derivatization by means of the on-column injection technique. Chromatographic separation of the toxins extracted from barley was achieved as a single peak, and the specific EI mass spectra of each toxin were obtained. The fatty acids in the extract that interfere with measurements of the toxins on the gas chromatogram were removed by precipitation as an insoluble metal soap with zinc acetate. Additional clean-up was accomplished using a Bond Elut Florisil cartridge. The quantitative detection limit in barley ranged from 0.1 to 0.5 μg/g. The average recoveries of 93.1% for DON, 3ADN, 15MAS, DAS, T-2 and ZEA, and 46.0% for FX and SCT added to barley at the level of 1 μg/g were obtained.

Course of apoptotic changes in the rat gastric mucosa caused by oral administration of fusarenon-X.

Male 5-week-old Wistar rats orally (po) administered with fusarenon-X (FX) 1.5 mg/kg and control rats po-treated with distilled water were sacrificed at 0-48 hr after gavage. FX-administered rats showed significant dilatation of the stomach with increased fluid contents at 1-24 hr postadministration (PA). Histopathologically, karyopyknosis of chief cells in the basal region of the gastric glands began to appear at 1 hr, and nuclear fragments were seen in the neck cell region at 1.5 hr PA. At 2-4 hr PA, apoptotic cells appeared diffusely in the neck region and focally in the basal region. Electron microscopy revealed that cells phagocytosing apoptotic bodies were the surface epithelia, undifferentiated neck cells, parietal cells and chief cells. No evidence was detected to show that parietal cells underwent apoptotic changes. The apoptotic lesions peaked at 4-6 hr PA, gradually subsided at 12 hr PA, and became minimal leaving apoptotic remnants in the basal region at 24 hr PA. At 48 hr PA, however, diffuse apoptotic lesions reappeared in the basal region at a level similar to that at 2-3 hr PA. This might be attributable to absorption of FX retaining in the stomach for 24 hr. In situ detection of DNA breaks by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction was consistent with the histopathologic findings. Agarose gel electrophoresis of DNA fragments isolated from the gastric mucosae of FX-administered rats showed a ladder pattern after 1.5 hr PA and the pattern became more distinct at 2-4 hr PA.

Rapid Apoptotic Changes in the Gastric Glandular Epithelium of Rats Administered Intraperitoneally with Fusarenon-X.

Fusarenon-X (FX) 1.5 mg/kg was administered intraperitoneally (i.p.) to 6-week-old male Wistar rats for examination of pathologic effects on the glandular stomach. Rats ip-treated with sterilized physiological saline were used as control. FX-administered rats showed dilatation of the stomach with increased fluid contents after 1-4 hr. Light microscopically, a few apoptotic karyopyknosis were seen in chief cells in the basal region at 1 hr postadministration (PA) and mitotic inhibition was evident after 2 hr PA. Marked apoptosis of nuclear pyknosis and cytoplasmic inclusions in both zymogenic and oxyntic cells developed from basal to middle regions of the gastric mucous membrane at 2-4 hr PA with a peak at 3 hr. Apoptotic changes of differentiating neck cells and surface epithelia were less evident. Electron microscopy revealed that the chief cells were the main target of FX-induced apoptotosis. The parietal cells were secondarily involved because they phagocytosed chief cell-derived apoptotic bodies. In situ detection of DNA breaks by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction revealed the positive nuclei after 1 hr PA, which increased with time and reached a peak at 3 hr PA, in accordance with apoptotic changes in histological study. Agarose gel electrophoresis of DNA isolated from the gastric mucosae of FX-administered rats showed ladder pattern of DNA fragments after 1.5 hr PA with the maximum distinctness at 3 hr PA.

Purification and quantification of nivalenol

A mean of 2.7 g fusarenon-X (FUS-X)/L was produced in cultures ofFusarium sp Fn-2B on SSA, a synthetic medium containing sucrose and asparagine. When culturing Fn-2B on several other media, or using otherFusarium strains, much lower concentrations of FUS-X or nivalenol (NIV) were obtained. The SSA-incubations with Fn-2B in an optimal volume of 100 mL were stopped just before reaching maximal FUS-X concentration and the start of conversion to NIV. FUS-X was extracted from the 22 days old cultures and partially purified on a silica gel column. It was then hydrolysed to NIV, which was rechromatographed on silica and crystallized.The purity and the chemical and physical properties of the NIV produced were investigated. Potential sources of error when quantifying NIV were also studied.