Effects of Phenolic Acids on the Growth and Production of T-2 and HT-2 Toxins by Fusarium langsethiae and F. sporotrichioides.

The effect of natural phenolic acids was tested on the growth and production of T-2 and HT-2 toxins by Fusarium langsethiae and F. sporotrichioides, on Mycotoxin Synthetic medium. Plates treated with 0.5 mM of each phenolic acid (caffeic, chlorogenic, ferulic and p-coumaric) and controls without phenolic acid were incubated for 14 days at 25 °C. Fungal biomass of F. langsethiae and F. sporotrichioides was not reduced by the phenolic acids. However, biosynthesis of T-2 toxin by F. langsethiae was significantly reduced by chlorogenic (23.1%) and ferulic (26.5%) acids. Production of T-2 by F. sporotrichioides also decreased with ferulic acid by 23% (p < 0.05). In contrast, p-coumaric acid significantly stimulated the production of T-2 and HT-2 toxins for both strains. A kinetic study of F. langsethiae with 1 mM ferulic acid showed a significant decrease in fungal biomass, whereas T-2 production increased after 10 days of incubation. The study of gene expression in ferulic supplemented cultures of F. langsethiae revealed a significant inhibition for Tri5, Tri6 and Tri12 genes, while for Tri16 the decrease in gene expression was not statistically significant. Overall, results indicated that phenolic acids had a variable effect on fungal growth and mycotoxin production, depending on the strain and the concentration and type of phenolic acid assayed.

Reduction of T-2 and HT-2 mycotoxins by atmospheric cold plasma and its impact on quality changes and germination of wheat grains.

Wheat (Triticum aestivum) is susceptible to mycotoxin contamination, which can result in significant health risks and economic losses. This research examined the ability of air atmospheric cold plasma (air-ACP) treatment to reduce pure and spiked T-2 and HT-2 mycotoxins' concentration on wheat grains. This study also evaluated the effect of ACP treatment using different gases on wheat grain germination parameters. The T-2 and HT-2 mycotoxin solutions applied on round cover-glass were placed on microscopy slides and wheat grains (0.5 g) were individually spiked with T-2 and HT-2 on their surfaces. Samples were then dried at room temperature (∼24 °C) and treated by air-ACP for 1 to 10 min. Ten minutes of air-ACP treatment significantly reduced pure T-2 and HT-2 concentrations by 63.63% and 51.5%, respectively. For mycotoxin spiked on wheat grains, 10 min air-ACP treatment significantly decreased T-2 and HT-2 concentrations up to 79.8% and 70.4%, respectively. No significant change in the measured quality and color parameters was observed in the ACP-treated samples. Wheat grain germination parameters were not significantly different, when treated with ACP using different gases. Air-ACP treatment and ACP treatment using 80% nitrogen + 20% oxygen improved the germination of wheat grains by 10% and 6%, respectively. This study demonstrated that ACP is an innovative technology with the potential to improve the safety of wheat grains by reducing T-2/HT-2 mycotoxins with an additional advantage of improving their germination. PRACTICAL APPLICATION: Atmospheric cold plasma (ACP) technology has a huge potential to degrade mycotoxins in food grains. This study evaluated the efficacy of ACP to reduce two major mycotoxins (T-2 and HT-2 toxins) in wheat grains. The results of this study will help to develop and scale-up the ACP technology for mycotoxin degradation in grains.

T-2 toxin inhibits murine ES cells cardiac differentiation and mitochondrial biogenesis by ROS and p-38 MAPK-mediated pathway.

OBJECTIVE: To investigate the effect of T-2 toxin on murine embryonic stem cells (ESCs) cardiac differentiation and mitochondrial biogenesis in vitro. METHODS: Cardiac differentiation of the mouse ESCs was initiated by embryoid bodies (EBs) formation in hanging drops. EBs were exposed to 0.5ng/ml T-2 toxin for 24, 72 and 120h. Cultures were observed daily for the appearance of contracting clusters, and cardiac-specific protein (α-actiniin) were measured by Western blot and immunocytochemistry. Mitochondrial ultrastructure was observed by confocal laser scanning microscopy and transmission EM photography. Reactive oxygen species (ROS) was monitored by H2-dichlorofluorescein-diacetate (H2DCF-DA). The phosphorylation of the p38 (p-p38) and p38 mitogen-activated protein kinase (MAPK) and the expression of mitochondrial biogenesis proteins, including peroxisome proliferator activated receptor coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (mtTFA), and mitochondrial respiratory chain complex IV (COXIV) were analyzed using Western blot. In some experiments, mESCs were pre-treated with the antioxidant Trolox (200µM) for 30min, then exposed to Trolox (200µM) and T-2 toxin (0.5ng/ml) for 72h. RESULTS: Contracting clusters were observed under the microscope light and cardiac-specific protein (α-actinin) expressed positively indicated mESCs directly differentiated in cardiomyocytes. However, the cardiac differentiation was inhibited by T-2 toxin treatment 72 and 120h. ROS accumulated in murine ES cells in a time-dependent manner. The expression of p-p38 significantly increased in 24h group and decrease in 72 and 120h groups. The decrease of mitochondrial number and the mitochondrial biogenesis-related proteins expression, including PGC-1α, NRF-1, mtTFA, and COXIV decreased in a time-dependent manner with T-2 toxin treatment. However, the inhibition of mitochondrial biogenesis by T-2 toxin in differentiated mESCs was recovered significantly in the presence of the antioxidant Trolox. CONCLUSION: Taken together, T-2 toxin decreased the expression of PGC-1α, NRF-1, and mtTFA, inhibited mitochondrial biogenesis, and then inhibited the cardiac differentiation of murine ES cells, and the effect was partly responsible for the p38 MAPK mediated by ROS.

Assessment of potential therapies for acute T-2 toxicosis in the rat.

The efficacy of a variety of approaches for the treatment of animals with acute T-2 toxicosis was assessed utilizing young female rats. A single large dose of the water soluble salt of methylprednisolone significantly prolonged survival times in T-2 toxin treated animals. The use of diltiazem hydrochloride, dazemgrel, N-acetylcysteine, dimethyl sulfoxide, adenosine triphosphate (ATP), ATP combined with magnesium chloride, ascorbic acid, and aprotinin did not prolong survival times at the dosages administered. Trichodermin, a trichothecene similar in structure and biochemical activity to T-2 toxin but much less acutely toxic, had a detrimental effect on survival times whether given 1 hr prior to or after T-2 toxin.

Therapeutic efficacy of superactive charcoal in rats exposed to oral lethal doses of T-2 toxin.

Superactive charcoal, a compound known to complex with many toxins, was evaluated in this study for its effectiveness in preventing death in rats given an oral lethal dose of 8 mg/kg body weight of T-2 toxin. The median effective dose of oral superactive charcoal in preventing deaths in rats was 0.175 g/kg body weight. Concurrent use of cathartics, such as sorbitol, magnesium sulfate and sodium sulfate, to facilitate removal of the superactive charcoal:T-2 toxin complex formed in vivo did not enhance the survival rates of rats. One gram per kilogram body weight oral superactive charcoal enhanced survival times and survival rates in rats given 8 mg/kg of T-2 toxin as late as 3 hr after the T-2 toxin was administered. Some benefit in survival rate may be derived from giving the superactive charcoal as late as 5 hr after the T-2 toxin.

Effects of drugs and metabolic inhibitors on the acute toxicity of T-2 toxin in mice.

The antidotal effects of antiinflammatory agents, inhibitors of bioamine syntheses, an opioid antagonist and other pharmacological agents on lethal toxicity, leukocytosis and ear inflammation, were investigated in mice subcutaneously administered or topically exposed to T-2 toxin, a trichothecene mycotoxin of Fusarium species. The acute lethal toxicity of T-2 toxin was reduced by administration of the steroidal anti-inflammatory agents, prednisolone and dexamethasone, and prolongation of survival times was demonstrated with an antihistaminic agent, diphenhydramine, and an opioid antagonist, naloxone. Prednisolone also antagonized leukocytosis and the increment of ear weight caused by T-2 toxin. These findings suggest that the action site(s) of steroidal anti-inflammatory agents is involved in the development of the toxic actions of T-2 toxin, and the implications of the results with bioamines and opioids are also discussed.

In vitro inhibitory effects of antioxidants on cytotoxicity of T-2 toxin.

T-2 toxin is a secondary fungal metabolite produced by various species of Fusarium. It is capable of killing cells by causing extensive damage to the cellular membrane. In this study, cytotoxicity of T-2 toxin in combination with different antioxidant materials, including vitamin C (vit. C), vitamin E (vit. E) and selenium (sel) was investigated in vitro using the neutral red cytotoxicity assay. Eleven primary and transformed cell lines established from different tissues were used in pre-test experiments to identify the most sensitive and resistant lines by measuring the half lethal concentration (LC(50)) of the toxin. Three cell lines including human gingival fibroblast (HGF), the most sensitive (LC(50)=0.25 ng/ml), human colorectal adenocarcinoma (SW742), the most resistant (LC(50)=5.5 ng/ml) and human hepatoma (HepG2), with median susceptibility (LC(50)=2 ng/ml) were selected to investigate the inhibitory effects of the antioxidant agents, on cytotoxicity of T-2 toxin. Our results demonstrated that co-incubation of cell lines with different concentrations of T-2 toxin and antioxidants decreased significantly, but did not totally inhibit, the cytotoxicity of T-2 toxin (P<0.001). These findings suggest that in addition to lipid peroxidation, which is inhibited by antioxidants, other unidentified mechanism(s) seem to be involved in cytotoxicity of T-2 toxin.

Effects of testosterone on the prevention of T-2 toxin-induced adrenocortical necrosis in mice.

To evaluate the effect of exogenous testosterone on the development of T-2 toxin-induced necrosis of adrenal glands, mice were allotted to 3 treatment groups. Each treatment group contained castrated male, and castrated and sexually intact female mice. Each mouse in group 1 was given 0.16 mg testosterone propionate at 48-hour intervals for a total of 12 injections, group-2 mice were given similar injections of only the vehicle, and group-3 mice were given no treatment. Twenty-four hours after the last injection, the mice in all 3 groups were exposed for 10 minutes to an aerosol of T-2 toxin. All mice alive at 24 hours after exposure were euthanatized and the adrenal glands and thymuses were examined histologically. Necrosis of the adrenal cortex was not found in any of the mice given preexposure treatment with exogenous testosterone, whereas all mice given vehicle only or no treatment had T-2 toxin-induced necrosis of the inner portion of the adrenal cortex. Lymphocytolysis in the cortex of the thymus confirmed that each mouse of all 3 treatment groups had experienced systemic mycotoxicosis. The uniform severity of the lesion in all mice suggests that the thymus was not protected by exogenous testosterone administration or by the castration status of the mice. We propose that T-2 toxin-induced adrenal necrosis in mice is prevented by the presence of testosterone.

Efficacy of a hydrated sodium calcium aluminosilicate to reduce the toxicity of aflatoxin and T-2 toxin.

A hydrated sodium calcium aluminosilicate (HSCAS) was incorporated into diets (.5%) containing 3.5 mg of aflatoxin (AF) per kg and 8.0 mg of T-2 toxin (T-2) per kg, singly, and in combination. Male broiler chicks (n = 480) were provided with feed and water for ad libitum consumption from 1 to 21 days of age. Body weight gains were significantly depressed by AF and T-2, singly, and further decreased by the combination of the two toxins. Efficiency of feed utilization was not affected. The AF alone and the AF plus T-2 combination caused increases in relative liver, kidney, proventriculus, gizzard, spleen, and pancreas weights. Treatment-related changes in hematological and serum biochemical values and enzyme activities were observed. Oral lesions were observed only in chicks receiving the T-2 diets. The HSCAS fed singly did not alter any of the parameters measured but it did diminish the toxicity of AF for many parameters but did not appear to alter the toxicity of T-2. Addition of HSCAS to the AF plus T-2 combination diet diminished some of the effects of the toxin combination. These findings indicate that HSCAS can diminish many of the adverse effects of dietary AF in the chicken, but it has no effect on T-2 toxicity.

Effect of T-2 toxin on egg production and hatchability in laying hens.

The effect of diets containing different levels of T-2 toxin on egg production and hatchability was studied in a four-week experiment using 100 laying hens of the SSL hybrid line and 10 cocks divided into 10 groups. Another aim of the experiment was to investigate how effectively the increased dietary vitamin E content neutralized the adverse effects of T-2 toxin. The diet of the control group (C) contained no mycotoxin, while those of the experimental groups included the following levels of T-2 toxin: groups 1, 2 and 3: 1 mg/kg, groups 4, 5 and 6: 5 mg/kg; groups 7, 8 and 9: 10 mg/kg. Vitamin E was added to the diet of groups C, 1, 4 and 7 at a rate of 50 mg/kg while to that of groups 2, 5 and 8 at a rate of 100 mg/kg. To the diet of groups 3, 6 and 9 no vitamin E was added. Contamination of the diet with T-2 toxin markedly decreased egg production and impaired hatchability. The production decrease was proportional to the T-2 toxin concentration of the diet. Increased dietary vitamin E concentration exerted no influence on egg production. However, during the first week of the experiment it significantly (P < 0.01) decreased the number of infertile eggs and significantly (P < 0.01) improved the hatching percentage. Dietary vitamin E concentration was in positive correlation with the hatching percentage; this correlation was rather close (r = 0.74) in the first week of the experiment.

[Effects of bioflavonoids on the toxicity of T-toxin in rats. A morphological study]. / Vliianie bioflavonoidov na toksichnost' T-2-toksina dlia krys. Morfologicheskie issledovaniia.

Subacute toxicity of T-2 toxin in rats was characterized by a primary defeat of liver, thymus, spleen and intraorgan arteries. In 75% of animals found out increase of the size and adipose infiltration of a liver, in all animals--reduction of the size of thymus (sharp) and spleen (moderate) and pronounced hypoplasia of lymphoid tissue. In the majority of rats vacuolation of cytoplasma of smooth-muscular walls of coronary and intrarenal arteries was revealed. In animals received T-2 toxin against a background of a diet with addition a flour from seeds of milk thistle with high contents of flavonoids, described morphological changes were expressed to a lesser degree and were observed less often. Moderate periportal adipose infiltration of a liver was revealed in 30% of animals, occupancy by cells of lymphoid tissue increased, the quantity and sizes of vacuoles in walls of vessels decreased.

[Effects of bioflavonoids on the toxicity of T-toxin in rats. A biochemical study]. / Vliianie bioflavonoidov na toksichnost' T-2-toksina dlia krys. Biokhimicheskie issledovaniia.

The enrichment of a diet of rats by flavonoids of milk thistle, Silybum marianum, reduced toxicity of T-2 toxin and was accompanied by reduction of a degree of change of total and nonsedimentable activity of lysosomal enzymes and microsomal xenobiotic metabolizing enzymes.

Toxicity induced by F. poae-contaminated feed and the protective effect of Montmorillonite supplementation in broilers.

The T-2 and HT-2 toxins, the main metabolites of Fusarium poae, induce toxicity in broilers and accumulate in tissues. Consequently, during the breeding process of broilers, diets are frequently supplemented with physical adsorbents to protect birds against the toxicity induced by mycotoxins. In the present research, T-2 and HT-2 were produced in maize inoculated with F. poae. Mont, the strongest adsorbent based on in vitro adsorption ratios, was added to the contaminated diet. One-day-old chickens were randomly and equally divided into the following four groups: control diet group, Mont supplemented diet group, contaminated diet group and detoxification diet group. The experiment lasted for 42 days. Compared to the control group, the contaminated group showed significant decrease in body weight, feed intake and TP (P < 0.05), and marked increase in FCR, ALP, AST and ALT activity, T-2/HT-2 residues in the tissues and the relative expressions of apoptosis-related mRNAs (P < 0.05). Mont supplementation provided protection for the treated broilers in terms of performance, blood biochemistry, hepatic function, T-2/HT-2 residue of tissues and apoptosis. Therefore, Mont may be suitable as a detoxification agent for T-2/HT-2 in feed for broilers.

The role of mitochondria in T-2 toxin-induced human chondrocytes apoptosis.

T-2 toxin, a mycotoxin produced by Fusarium species, has been shown to cause diverse toxic effects in animals and is also a possible pathogenic factor of Kashin-Beck disease (KBD). The role of mitochondria in KBD is recognized in our recent research. The aim of this study was to evaluate the role of mitochondria in T-2 toxin-induced human chondrocytes apoptosis to understand the pathogenesis of KBD. T-2 toxin decreased chondrocytes viabilities in concentration- and time-dependent manners. Exposure to T-2 toxin can reduce activities of mitochondrial complexes III, IV and V, ΔΨm and the cellular ATP, while intracellular ROS increased following treatment with T-2 toxin. Furthermore, mitochondrial cytochrome c release, caspase-9 and 3 activation and chondrocytes apoptosis were also obviously observed. Interestingly, Selenium (Se) can partly block T-2 toxin -induced mitochondria dysfunction, oxidative damage and chondrocytes apoptosis. These results suggest that the effect of T-2 toxin on human chondrocytes apoptosis may be mediated by a mitochondrial pathway, which is highly consistent with the chondrocytes changes in KBD.

Toxic effects of T-2 toxin and deoxynivalenol on the mitochondrial electron transport system of cardiomyocytes in rats.

The in vitro effects of 2 representative mycotoxins, T-2 toxin and deoxynivalenol (DON), of trichothecene group on the electron transport system (ETS) of mitochondria in rat cardiomyocytes were investigated by measuring oxygen consumption rates (OCR). The ATP-linked OCR and the reserve capacity (RC) of the mitochondria ETS were quantified by a "mitochondria stress test" which was estimated by the OCR responses to oligomycin and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, with an extracellular flux analyzer. The basal OCR was significantly inhibited by the application of T-2 toxin at concentrations of 6 × 10⁻¹ to 6 × 10⁻5 µM and DON at concentrations of 0.78 to 100 µM for 24 hr. The threshold of cardiomyocyte toxicity was estimated to be between 6.0 × 10⁻6 and 6.0 × 10⁻5 µM for T-2 toxicity on both ATP-linked OCR and RC and between 0.39 and 0.78 µM on ATP-linked OCR or between 1.56 and 3.13 µM on RC for DON. The decrease in OCR of cardiomyocytes exposed to T-2 toxin with a concentration of 6.0 × 10⁻³ and 6.0 × 10⁻4 µM was significantly inhibited by antioxidants, catalase and vitamin C. In conclusion, the present study demonstrated, through the direct and real-time measurement of respiratory function in mitochondria, that a marked inhibition of mitochondrial ETS function in cardiomyocytes was induced by T-2 toxin and DON and that the mitochondrial dysfunction by T-2 toxin was largely associated with oxidative stress.

Effects of xanthotoxin treatment on trichothecene production in Fusarium sporotrichioides.

There are 4 P450 oxygenases involved in the biosynthesis of T-2 toxin in Fusarium sporotrichioides. Exactly how these enzymes react to antimicrobial plant defense compounds is unknown. Xanthotoxin (8-methoxypsoralen) is a phototoxic furanocoumarin that acts as a P450 oxygenase inhibitor. The current study shows that the addition of concentrations of 1.0 mmol/L or less of xanthotoxin to liquid cultures of F. sporotrichioides NRRL3299 can effectively block T-2 toxin production and cause an increase in accumulation of trichodiene, the hydrocarbon precursor of trichothecenes. The addition of xanthotoxin to liquid cultures of a trichodiene-accumulating F. sporotrichioides Tri4- mutant caused a 3- to 10-fold increase in trichodiene accumulation, suggesting that xanthotoxin not only blocks trichothecene oxygenation reactions, but may in some way also promote the synthesis of trichodiene. Feeding studies showed that 2 of the 4 P450 oxygenases, TRI4 and TRI1, were more sensitive to xanthotoxin, while oxygenases TRI11 and TRI13 were unaffected. Quantitative reverse-transcriptase PCR indicated that several of the genes in the toxin biosynthetic pathway were upregulated by xanthotoxin, with Tri4 showing the highest increase in expression. These results indicate that while xanthotoxin inhibits specific P450 oxygenase activity, it also has an effect on gene expression.

Monoclonal anti-idiotype induces protection against the cytotoxicity of the trichothecene mycotoxin T-2.

An IgG1 mAb, designated HD11, specific for the trichothecene mycotoxin T-2 and capable of neutralizing its cytotoxicity was used to generate a syngeneic monoclonal anti-Id antibody. The generated anti-Id mAb, designated DE8, specifically bound to HD11 anti-T-2 mAb, and not to IgG1 mAb of irrelevant specificity or to normal mouse Ig. DE8 inhibited the binding of HD11 anti-T-2 to T-2-BSA-coated plates, whereas a control anti-Id mAb did not, suggesting recognition of an Id determinant associated with the T-2 binding site of HD11. Moreover, the binding of HD11 to DE8 and that of DE8 to HD11 were specifically inhibited by free T-2 mycotoxin. DE8 mAb was efficient in abrogating the protective effect of HD11 in the cytotoxicity of T-2 on the human epidermoid carcinoma cell line Hep-2. In vivo immunization of BALB/c mice with DE8 conjugated to KLH induced an anti-T-2 antibody titer comparable to that obtained with T-2-OVA immunization, whereas immunization with unconjugated DE8 resulted in a lower titered anti-T-2 response. Immunization with DE8-keyhole limpet or with unconjugated DE8 induced anti-T-2 antibody responses characterized by expression of "HD11-like" Id and by protection against T-2 cytotoxicity. However, the T-2-OVA-induced anti-T-2 response lacked the HD11+ Id and was only partially protective against T-2 cytotoxicity. This represents the first demonstration of the use of an anti-Id based vaccine in the in vivo induction of a protective antibody response against the cytotoxicity of a nonproteinaceous, small m.w. biologic toxin, whose very toxic nature precludes its use as the immunogen.

Beneficial effect of dexamethasone in decreasing the lethality of acute T-2 toxicosis.

1. Steroidal and non-steroidal anti-inflammatory agents were evaluated for effectiveness for treatment of acute T-2 toxicosis in mice. 2. Non-steroidal agents, indomethacin, phenylbutazone, and acetylsalicylic acid, either were ineffective, or potentiated the lethality of T-2 toxin. 3. Of the anti-inflammatory steroids tested, dexamethasone was the most effective. 4. Dexamethasone was administered before, at the same time as, or after injection of T-2 toxin. 5. As the time between toxin exposure and treatment was increased, there was a corresponding increase in lethality. 6. In conclusion, steroidal, but not non-steroidal, anti-inflammatory agents were effective in decreasing T-2 toxin-induced lethality.

Cumulative effect of T-2 toxin and vitamin C on chromosomal abnormalities in the bone marrow cells of mice (Mus musculus).

A regular dose of 10 mg/kg body wt/day of vitamin C as a part of the daily diet markedly reduced T-2 toxin-induced abnormalities in the chromosomal cells in mice (Mus musculus). The preventive effect on individual types of change in chromosomes such as breakages, chromatid gaps, ring formations and widespread fragmentation, was increased.

In vitro neutralization of T-2 toxin toxicity by a monoclonal antibody.

A T-2 toxin specific monoclonal antibody, IgG1 K, with a low level of ELISA cross-reactivity to Acetyl T-2, HT-2, and iso T-2 toxins has been produced. The ability of this monoclonal antibody to neutralize the cytotoxicity of T-2 toxin in PHA stimulated cultures of human lymphocytes was determined by the MTT method. The complete neutralization of the toxic effect of 0.02 microM T-2 toxin was obtained with 0.03 microM of MoAb, whereas the 50% neutralizing dose (ND50) was observed at 0.009 microM of MoAb. Partial neutralization was observed with Acetyl T-2 toxin (ND50 = 0.038 microM) and HT-2 (ND50 = 0.94 microM). These results could represent a rational for clinical use of T-2 toxin specific monoclonal antibody in prophylaxis and therapy of T-2 toxemia.