Toxic Trichothecene Research Articles

免疫担当細胞に対するトリコテセンの毒性

免疫担当細胞に対するトリコテセンの毒性

Stachybotrys atra Growth and Toxin Production in Some Building Materials and Fodder under Different Relative Humidities

Growth of Stachybotrys atra and its toxin production on some building materials and in animal fodder were studied at relative humidities ranging from 78 to 100%. Toxins were detected by biological assays and chemical methods. Strong growth of the fungus and presence of macrocyclic trichothecenes, mainly satratoxins G and H, were detected on wallpaper and gypsum boards and in hay and straw at saturation conditions. On pine panels, S. atra grew well, but neither biological toxicity nor production of macrocyclic trichothecenes was observed.

Pigs fed Fusarium-infected maize containing zearalenone and nivalenol with sweeteners and bentonite

A 29-day growth assay involving 48 pigs assessed the value of sweeteners (raw sugar or dehydrated molasses) or bentonite (a natural alumino-silicate clay) at dietary concentrations of either 20 or 50 g/kg in overcoming the adverse effects of a basal diet containing 715 g/kg of mouldy maize. The maize was naturally infected with Fusarium graminearum and contained the mycotoxins zearalenone (ZEA, 3.0 mg/kg) and nivalenol (NIV, 11.5 mg/kg). A positive control diet, identical to the basal diet other than for the substitution of mouldy maize with sound maize, was also fed. Pigs given diets containing the mouldy maize showed marked feed rejection typical of trichothecene toxicity and some scouring and vomiting were also observed. Females exhibited signs of precocious oestrus typical of ZEA toxicity. The performance of pigs fed the control diet was better ( P < 0.01) than those fed the mouldy diets. None of the additives was effective in overcoming either the oestrogenic or depressed performance effects of the mouldy maize. Although sweeteners and bentonite may be useful in situations where moulds cause non-specific unpalatability, they appear ineffective where feed refusal is due to direct effects on appetite as appears to be the case with trichothecenes such as NIV.

Natural occurrence of Fusarium toxins in barley grown in a southwestern area of Germany.

The occurrence of toxins produced by Fusarium species in cereals and other agricultural commodities has increasingly raised interest over the last two decades (Gareis et al. 1989; M~ller and Schwadorf 1993). Efforts were mainly directed to the occurrence of zearalenone, an estrogenic Fusarium metabolite and to toxic trichothecenes (deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin, diacetoxyscirpenol). The occurrence of Fusarium toxins in barley grown in southwest Germany has been monitored by thin layer chromatography (TLC) and gas chromatography (GC) with FID or ECD detection (Thalmann et al. 1985). However, TLC is not sensitive enough to detect low levels of Fusarium toxins and confirmation is difficult, whereas GC method with FID and/or ECD can yield false positive results in extracts obtained from agricultural commodities for trichothecenes (Schwadorf and MUller 1991). In the present study, we used a gas chromatograph equipped with a mass selective detector, which is a sensitive and confirmatory method to determine Fusarium toxins in cereals (Schwadorf and M~ller 1991, 1992). Barley samples were collected soon after harvest because preharvest contamination is of main interest for agricultural practice. The kernels were microbiologically examined for invasion by Fusarium, as well as by Penicillium and Asperqillus species, since these latter two have been described as storage fungi (Christensen and Sauer 1982).

The toxicity of macrocyclic trichothecenes administered directly into the rat brain

The tested macrocyclic trichothecenes are produced by Myrothecium fungi and by the plant Baccharis megapotamica. The toxicity of five macrocyclic trichothecenes has been measured by intracerebral and subcutaneous injection into rats. It is assumed that the toxic effects are based on inhibition of protein synthesis. Intoxication of rats by these compounds finds expression in slowly progressing respiratory depression and paralysis of skeletal muscles. The macrocyclics are derived from verrucarol, which lacks ring D and exhibits only low toxicity. The high toxicity of the macrocyclics, established by intracerebral and subcutaneous applications, may thus be attributed to the presence of the large ring D.

Effects of growth temperature on toxicity of T-2 toxin and roridin A to yeast.

In yeasts, growth temperature is known to affect the membrane phospholipid content. The effect of temperature on the growth inhibition of Kluyveromyces marxianus and Saccharomyces cerevisiae by the trichothecene mycotoxins, T-2 toxin and roridin A, was investigated. Examination of EC50 values for T-2 toxin and roridin A showed that these toxins were least inhibitory to both yeasts at 30 and 25 degrees C, respectively. Increasing or decreasing growth temperature from these temperatures gradually increased the inhibitory effect of the trichothecene mycotoxins. Temperature may affect the toxicity of the trichothecenes to the yeasts by regulating the composition of yeast cell membranes.

Contents ofFusariumtoxins in Finnish and imported grains and feeds

The aim of the present study was to investigate the levels of trichothecenes in representative samples of Finnish and imported cereals and feeds from the 1987 and 1988 crops. Trichothecene concentrations were determined in samples as trimethylsilylether derivatives by a gas chromatograph equipped with a mass selective detector (GC-MSD) using selected ion monitoring (SIM) which proved to be a reliable and sufficiently sensitive technique. Representative samples of Finnish and imported oats, wheat, barley, rye, maize gluten, soy granules, rapeseed, turnip rapeseed, fish meal, poultry feed and pig feed were analysed employing 19-nortestosterone as an internal standard. Almost all grain and feed samples contained deoxynivalenol (DON) from 7 to 300 micrograms/kg and smaller amounts (13-120 micrograms/kg) of 3-acetyldeoxynivalenol (3-AcDON). The most toxic trichothecenes, T-2, HT-2 toxins and nivalenol (NIV) and also zeralenone (ZEN) were found at low concentrations in some samples. Six lots of oats containing 1.3-2.6 mg/kg of DON and 0.2-0.6 mg/kg of 3-AcDON were found in the Finnish grain samples. Silo samples of almost all imported grains contained DON but at levels below 50 micrograms/kg. Imported grains contained no other trichothecenes. DON, 3-AcDON and ZEN were found in imported feedstuffs at low concentrations.

The use of trichothecene-contaminated grains in feeds

A review is presented describing the relative efficiencies of the various technologies that have been proposed to permit incorporation of mycotoxin-contaminated grains into animal diets without adversely influencing growth rate or resulting in hazardous residues in edible animal tissues. When the degree of contamination is modest, it may be possible to dilute the contaminated materials with uncontaminated grain to lower the concentration of trichothecenes below the threshold of significant biological activity. A less useful alternative to dilution is the other mechanical approach of milling to remove the most heavily contaminated fractions of the grain. Chemical destruction of triochothecenes is also a possibility. An example is the use of sodium bisulfite treatment to destroy deoxynivalenol in contaminated corn. Such treatments may, however, reduce palatability and nutritional value. When the biochemical mechanism of trichothecene toxicity is known, in vivo therapeutic treatments may be possible. It has been shown, for example, that T-2 toxin-induced changes in brain prostaglandin production can be overcome by treatment with dexamethasone resulting in increased survival. A similar effect was seen using the selective platelet activating factor antagonist BN 52021. Another approach is the use of dietary treatments to either promote in vivo detoxification of mycotoxins or to reduce absorption from the digestive tract with the aid of nonnutritive binding agents.

Comparative Toxicity of Scirpentriol and Its Acetylated Derivatives ,

Scirpentriol (STO) and its seven acetylated derivatives, 3-, 4- and 15-monoacetoxyscirpenol (MAS), 3,4-, 3,15-, and 4,15-diacetoxyscirpenol (DAS), and 3,4,15-triacetoxyscirpenol (TAS) were compared for their acute oral lethality in broiler chicks, lethality in brine shrimp, and dermal toxicity in guinea pigs. Of the eight toxins, 4,15-DAS was the most toxic in the three assays, 3-MAS was die least toxic in brine shrimp and dermal assays, and 3,4-DAS was the least toxic in the chick assay. There was a difference of about a 100-fold and 20-fold, respectively, between 4,15-DAS and 3-MAS in dermal toxicity and brine-shrimp toxicity, as well as a difference of more than 16-fold between 4,15-DAS and 3,4-DAS in chick toxicity. In general, a free hydroxy group at the 3-position was a primary determinant of toxicity. Toxicity in the scirpenol family did not follow precisely the pattern reported earlier for the T-2 toxin family of trichothecene toxins, in which a decrease in the number of acyl groups was accompanied by a decrease in toxicity. At necropsy, the predominant sign in chicks was petechial hemorrhaging, primarily in the gastrointestinal tract and in the vascular beds of the beaks and the toe nails. The 4,15-DAS and 15-MAS were about 3 times more toxic in chicks than aflatoxin. All members of the scirpenol family of trichothecene mycotoxins appeared sufficiently toxic to warrant attention whenever field outbreaks occur. Apparently, brine shrimp and dermal assays are successful predictors of chick lethality by the more toxic trichothecenes and are less suitable for predicting the activity of the less toxic trichothecenes.

Preparation of the Fusarium toxin, nivalenol, by oxidation of the putative biosynthetic precursor, 7-deoxynivalenol.

Nivalenol is a toxic trichothecene metabolite which is produced by a number of different Fusarium species. However, the nature of its immediate biosynthetic precursor is not known. Oxidation of 7-deoxynivalenol(3 alpha,4 beta,15-trihydroxy-12,13- epoxytrichothec-9-ene-8-one) to nivalenol occurred with reagents known to react by a free radical pathway, such as hydrogen peroxide-ferrous ion-ascorbic acid or lead tetracetate, but not with electrophilic reagents requiring prior formation of the enol. These results suggest that 7-deoxynivalenol or an acetylated derivative could be the biosynthetic precursor of nivalenol.

Effects of radioprotective compounds and anti-inflammatory agents on the acute toxicity of trichothecenes

Antidotic effects of radioprotective and anti-inflammatory drugs against the acute toxicity of T-2 toxin and fusarenon-X, the trichothecene mycotoxins of Fusarium species, were examined in mice and rats with two toxicological parameters, the lethal toxicity and the increment of intestinal fluid volume. The s.c. and i.v. LD 50 values of T-2 in male 6-week-old mice were 2.1 and 3.8 mg kg , respectively, and the s.c. LD 50 in 4-week-old mice was 1.6 mg kg . Pretreatment of mice with steroidal anti-inflammatory agents such as prednisolone and hydrocortisone resulted in a marked reduction in the lethal toxicity of T-2 toxin. However, pretreatment of mice with several radioprotective agents exhibited neither a prolongation of survival time nor a decrease in lethal toxicity in the mice. Enteropooling assay revealed that the steroidal anti-inflammatory drugs also depressed the trichothecene-induccd increment of intestinal fluid volume in rats, while no significant effects were observed with non-steroidal inhibitors of the arachidonate cascade. In rats given fusarenon-X, the content of cyclic AMP in the mucosa of ileum was dose- and time-dependently increased, but its maximal increment was only 1.5-fold of the control value. These findings indicate an involvement of the action site of steroidal anti-inflammatory drugs in the development of acute toxicity of trichothecenes in rodents.

Impaired murine resistance to Salmonella typhimurium following oral exposure to the trichothecene T-2 toxin

On orally exposing Salmonella-resistant C3H/HeN mice to the trichothecene T-2 toxin (1 mg/kg body weight), challenging with Salmonella typhimurium, and continuing to dose with T-2 toxin on alternate days for 3 wk, the LD 50 for the organism decreased by five orders of magnitude, in comparison with control mice not treated with T-2 toxin. In the absence of S. typhimurium, T-2 toxin did not cause lethal effects when administered at this level. Increased mortality in response to S. typhimurium challenge was dependent on T-2 toxin dose in the range 0 to 1 mg/kg for this regimen. The toxin did not significantly affect intestinal infection but did increase splenic counts in mice challenged with a range of S. typhimurium doses and also accelerated body-weight loss in infected animals. Mice challenged with the organism exhibited similar mortality when T-2 toxin treatment was begun 1 day prior to infection or at 5 or 9 days after infection. A time-related decrease in mortality, relative to that found for the standardized co-challenge described above, was observed when T-2 toxin administration was begun at 9, 13 or 23 days after infection. The results indicated that, depending on the challenge dose of the organism, both early and late phase acquired immune response to S. typhimurium could be impaired by T-2 toxin. Markedly enhanced susceptibility to gram-negative bacterial infection is another manifestation of trichothecene toxicity and may be an important aetiological factor in animal health problems that are associated with these mycotoxins.

Short-term effects of two fusarium toxins, diacetoxyscirpenol and neosolaniol monoacetate, in male wistar rats

This study compared the pathological and haematological effects of neosolaniol monoacetate in male Wistar rats with those of diacetoxyscirpenol, a highly toxic trichothecene mycotoxin. Both toxins caused statistically significant decreases in erythrocyte counts and increased the proportion of larger platelets compared to controls when dosed at 1 mg/kg body weight three times/wk for up to 5 wk. Lesions caused by treatment with either toxin at this dosage level were mild and were seen—mainly after 2–4 wk—in the actively dividing cells of the thymus, spleen, lymph nodes and bone marrow. Reduced severity or disappearance of the lesions was noted towards the end of the treatment period. The type and severity of the toxic effects of neosolaniol monoacetate appeared to be similar to those of diacetoxyscirpenol.

Toxicity of epoxy trichothecenes in cultured mammalian cells

With four established cell lines, cytotoxicity of 23 epoxy trichothecenes was tested. Three cell lines from human origin have been found to have a similar degree of sensitivity to trichothecenes. A pig kidney cell line had a higher sensitivity to NT-1 toxin, neosolaniol, tetraacetyl T-2, and iso-T-2 toxin. The potential cause for discrepancies in literature were discussed. Activity of esterases in calf serum used for cell cultures should be recorded whenever conducting toxicity tests in cell cultures with trichothecenes.Relations of chemical structures of toxins to their biological activities were investigated. With verrucarol (scirpendiol), scirpentriol, and T-2 tetraol analogues the esters generally showed significantly higher cytotoxicity. But, loss of acetyl groups at single positions did not necessarily mean decline in toxicity. With macrocyclic trichothecenes minor differences in the side chain of verrucarol esters were found to contribute to differences in biological activity.

Mass Spectral Investigations on Trichothecene Mycotoxins. II. Detection and Quantitation of Macrocyclic Trichothecenes by Gas Chromatography/Negative Ion Chemical Ionization Mass Spectrometry

A general, sensitive gas chromatographic/negative ion chemical ionization mass spectrometric (GC/NICIMS) method of analysis was developed for the detection and quantitation of several polar, thermally labile, toxic macrocyclic trichothecenes. The procedure involves the conversion of the molecules to their corresponding alcohols (verrucarols) by alkaline hydrolysis, followed by derivatization of the hydrolysate with heptafluorobutyrylimidazole and analysis by GC/MS technique under negative ion chemical ionization conditions. Nanogram (250 ng) quantities of several macrocyclic trichothecenes with different verrucarol and ester moieties were analyzed successfully with good precision by this procedure. The method was applicable for the accurate determination of at least low ppb levels of these macrocyclic trichothecenes in environmental samples, such as fungal products, fermentation broths, and plant samples. This is the first reported, well developed, sensitive, and applicable method for the detection and quantitation of these compounds in naturally occurring samples.

Fast and Sensitive Screening Method for Detection of Trichothecenes in Maize by Using Protein Synthesis Inhibition in Cultured Fibroblasts

A fast and sensitive bioassay with hamster (BHK-21 C13) fibroblasts for the detection of toxic trichothecenes in maize is described. Cells are exposed to pure toxins or crude maize extracts for 30 min. The mixture is then incubated with [1-14C]-leucine for an additional 60 min and the radioactivity incorporated into the protein of the washed cells is determined. The sensitivity of the assay was in the range 1-10 ng/mL (or 50 ppb in maize) for T-2, HT-2, and diacetoxyscirpenol. At least 1000-fold higher concentrations of non-trichothecene mycotoxins and plant toxins were necessary to cause an inhibition of protein synthesis in the cells. Of 24 maize samples tested, 14 gave a positive response in this assay and the presence of trichothecenes could be confirmed chemically in 11 samples. Therefore, the described bioassay is proposed as a useful screening method for cytotoxic trichothecenes in maize.

Natural occurrence of Fusarium toxins in peanut, sorghum and maize from Mysore (India)

AbstractPeanut, sorghum and maize samples were collected from the wholesale market in Mysore, India, over a period of one year (October 1984 to September 1985). The samples were analysed for the natural occurrence of T‐2 toxin (T‐2), diacetoxyscirpenol (DAS) and zearalenone by thin‐layer chromatography, dermal toxicity test and gas chromatography. Of the total number of peanut samples analysed, 6.9% were positive for the toxic trichothecene(s) (T‐2, up to 38.89 mg kg−1; DAS, up to 2.03 mg kg−1); 4.8% of total sorghum samples analysed contained T‐2 up to 15 mg kg−1. Zearalenone was not found in any of the samples tested, and no toxins were detected in any of the maize samples. Samples marketed during winter and summer periods were contaminated with the toxins. All the toxin‐positive samples except one peanut sample were found in produce stored for more than a week.

Trichothecene Production in Liquid Stationary Cultures of Fusarium tricinctum NRRL 3299 (Synonym: F. sporotrichioides ): Comparison of Quantitative Brine Shrimp Assay with Physicochemical Analysis

Stationary liquid cultures of Fusarium tricinctum NRRL 3299 (synonym: F. sporotrichioides) produce T-2 toxin, neosolaniol, diacetoxyscirpenol, and HT-2 toxin when cultured on peptone-enriched Czapek Dox medium. At 15 and 27 degrees C, maximum T-2 toxin yield (265 and 50 mug/ml) was found after 10 to 14 and 7 days, respectively. The T-2 toxin in the culture medium was metabolized rapidly at 27 degrees C and slowly at 15 degrees C. Addition of 0.025% (wt/vol) sorbic acid to the medium resulted in an increased production of trichothecenes at 15 degrees C (400 mug of T-2 per ml after 14 days). Trichothecenes in the culture liquid were determined by the brine shrimp bioassay and physicochemical analysis. The brine shrimp assay was improved by using modern bioassay equipment, including tissue culture trays and multipipettes, and by a standardized approach with positive and negative controls. The physicochemical analysis was based on adsorption of the trichothecenes onto Amberlite XAD-2 columns, derivatization with trifluoroacetic anhydride followed by capillary gas chromatography, and identification by mass spectrometry (as many as 17 trichothecenes were detected in the culture medium). The brine shrimp assay offers an interesting monitoring system for the quantitation of T-2 toxin and should be useful for studies on production of this toxin in culture. Specific information on less toxic trichothecenes, however, requires a more time-consuming chemical analysis.

Optical motility test for the detection of trichothecenes using brine shrimps

A new optical bio-assay using brine shrimp larvae (Anemia salina L.) has been successfully applied for monitoring the presence of highly toxic trichothecenes. The motility of the swimming animals was determined instead of their lethality. This permits more objective and precise results. The inhibition of the motility can be seen earlier, than the death of animals in toxicity tests. The relationship between time and dose is indicated for two different concentrations. This test can also be applied in the control of other harmful compounds.

Chronic, including teratogenic and carcinogenic effects of trichothecenes: a short review.

Trichothecenes are toxic sesquiterpenes, produced as secondary metabolites by certain soil microfungi in agricultural products harvested during wet and cold weather, and/or stored inappropriately. When present in the ingested food they can cause haemorrhagic diathesis, acute and chronic disorders in many animal species, including man. Animals experimentally treated with T-2 toxin, one of the common, very toxic and immunosuppressive trichothecenes, develop gastrointestinal, skin, and neurological disorders. T-2 toxin given to pregnant animals can lead to abortion and fetal abnormalities. Rats surviving many months after several sublethal doses of T-2 toxin can develop cardiovascular lesions, high blood pressure and tumours, benign and malignant, of the brain, the pancreas, stomach, and certain other organs. The possibility has to be considered that trichothecenes may be involved in some similar “idiopathic” disorders and tumours in livestock and in man.