T-2 Toxin Research Articles

Apoptosis in the developing mouse embryos from T-2 toxin-inoculated dams.

T-2 toxin (3 mg/kg b.w.) was orally inoculated to pregnant mice at 11 days of gestation to examine the effect of T-2 toxin on the developing embryos. At 24 hours after T-2 toxin-inoculation, moderate pyknosis or karyorrhexis was generally observed in some layers of the central nervous system, caudal sclerotomic segment, caudal region of the tongue to pharyngeal- to laryngeal-mesenchyma, trachea and facial mesenchyma. These pyknotic or karyorrhectic nuclei were strongly stained by the modified TUNEL method widely used for the in situ detection of apoptotic nuclei and also showed ultrastructural changes characteristic for apoptosis. This is the first report of mycotoxin-induced apoptosis in embryos.

Determination of Trichothecene Toxin (T2 Mycotoxin) in Aqueous Sample with Solid Phase Microextraction Technique Followed by Gas Chromatography with Flame Ionization Detection

Determination of Trichothecene Toxin (T2 Mycotoxin) in Aqueous Sample with Solid Phase Microextraction Technique Followed by Gas Chromatography with Flame Ionization Detection

Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains.

Stachybotrys chartarum is an indoor air, toxigenic fungus that has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio, area. In this study, 16 strains of S. chartarum isolated from case (n = 8) or control (n = 8) homes in Cleveland and 12 non-Cleveland strains from diverse geographic locations were analyzed for hemolytic activity, conidial toxicity, and randomly amplified polymorphic DNA banding patterns. In tests for hemolytic activity, strains were grown at 23 degrees C on wet wallboard pieces for an 8-week test period. Conidia from these wallboard pieces were subcultured on sheep's blood agar once a week over this period and examined for growth and clearing of the medium at 37 or 23 degrees C. Five of the Cleveland strains (all from case homes) showed hemolytic activity at 37 degrees C throughout the 8-week test compared to 3 of the non-Cleveland strains. Five of the Cleveland strains, compared to two of the non-Cleveland strains, produced highly toxic conidia (>90 microgram of T2 toxin equivalents per g [wet weight] of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of the 28 strains examined both were consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland where an infant had idiopathic pulmonary hemorrhage.

Inhibited Cytotoxic Leukocyte Activity in Tilapia (Oreochromis niloticus) Following Exposure to Immunotoxic Chemicals

The measure of the ability of cytotoxic immune cells to target and lyse foreign cells has been widely used as a predictor of im-munosuppression in chemical-exposed rodents. However, the efficacy of this function for predicting immunosuppressive chemical exposure in nonrodent species remains unknown. In the present report, tilapia ( Oreochromis niloticus) were exposed to 9 chemical agents known to inhibit rodent cytotoxic T lymphocyte (CTL) activity in mice, benzo[a]pyrene (B[a]P), 7, 12-dimethylbenzanthracene (DMBA), 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), dimethyl-nitrosamine (DMN), cadmium chloride (CdCl2), azathioprine (AZA), T2 mycotoxin (T2 toxin), hexachlorocyclohexane (lindane), and diethylstilbesterol(DES); and five chemical agents which do not inhibit this response, oxymethalone, acetonitrile, tert-butylhydro-quinone (TBHQ), toluene, and formaldehyde. Eight of the nine agents which inhibit rodent CTL responses also caused decreased cytolytic responses in fish. All five of the compounds with negative CTL effects in rodents were also negative in fish. Thus, 13 of the 14 chemical agents tested gave similar results in fish as reported in rodents, indicating a comparable pattern of inhibited immune cell cytolytic responses in chemical-exposed tilapia to that seen in the laboratory rodent models.

A novel colorimetric yeast bioassay for detecting trichothecene mycotoxins

A novel colorimetric microbial bioassay for toxicity has been developed; it shows particular sensitivity to trichothecene mycotoxins. The assay uses inhibition of expression of β-galactosidase activity within the yeast Kluyveromyces marxianus as a sensitive toxicity indicator, cultures remaining yellow, rather than turning deep green-blue, in the presence of X-gal, a chromogenic substrate. The assay is conducted in standard microtitre plates, permitting small volumes (160 μl) and many replicates, and can be scored either automatically by a plate-reader, or by eye. Factors likely to affect the efficacy of the bioassay, including carbon source, solvents, inoculum cell density, and the use of membrane-modulating agents (MMAs), were assessed. Polymyxin B nonapeptide was the most effective toxicity-enhancing MMA tested, enabling the trichothecene mycotoxin, verrucarin A, to be detected at a concentration of about 1 ng/ml. The assay's reproducibility was examined using polymyxin B sulfate, a cheaper MMA, and another trichothecene mycotoxin, T2 toxin: reproducibility and sensitivity were better for the β-galactosidase X-gal endpoint than for an alternative chromogenic toxicity indicator, the respiratory substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

Apoptotic cellular damage in mice after T-2 toxin-induced acute toxicosis.

By histopathologic, electron microscopic, and immunochemical observation, the mechanism of cellular death was investigated in thymus, spleen, and liver of mice given intraperitoneally sublethal doses of T-2 toxin, a trichothecene mycotoxin. In the thymus and spleen of mice given 5.0 mg/kg body weight of T-2 toxin and killed 12 hours later, a massive cellular destruction characterized by chromatin condensation was evident, and electron microscopy analysis revealed the presence of apoptotic bodies. In the liver of mice given 2.5 mg/kg of T-2 toxin and killed 2 hours later, the induction of apoptotic cellular lesions was observed by electron microscopy, and Kupffer cells phagocytosed the apoptotic bodies. Such lesions were not observed in the mice killed 12 hours after receiving the toxin. In situ nick translation analysis (Tunel method) revealed DNA fragmentation in thymus, spleen, and liver shortly after administration of T-2 toxin. As previously observed in vitro, these findings indicated that T-2 toxin is a potent inducer of apoptotic cell death in thymus, spleen, and liver in vivo; especially in liver, apoptosis is induced rapidly as compared with the other tissues observed, and Kupffer cells play an important role for clearance of apoptosis.

Effects of multiple doses of T-2 toxin on the erythroid response capacity of mice following an extensive experimental bleeding.

Total nucleated cellularity and total erythroid cell populations were measured in spleen and bone marrow of mice at different times after treatment with 3 daily doses of T-2 toxin (2.0 mg/kg). It was found that the initial depletion of hematopoietic cells produced by the toxin was rapidly reverted in spleen, giving way after 48 hr to a significant hypercellularity which after 10 days was 2.5 times the normal levels, but this effect was not observed in bone marrow, which slowly recovered normal cellularity after 5 days. The cytological analysis revealed that there was a highly significant shift in the ratio of erythroid to non-erythroid cells, since erythroid cell populations increased by about 8-fold in spleen and nearly 2-fold in bone marrow between 10 and 35 days after intoxication. In order to test the integrity of the hematopoietic reserve capacity, a hemorrhagic stress was produced in intoxicated animals at 10-50 days after toxin exposure. It was found that the erythroid response capacity was significantly higher in the intoxicated animals compared to anemic controls. The results suggest that the initial cytotoxic damage produced by T-2 toxin in the hematopoietic system is followed by a significant erythroid hypercellularity, which can confer an increased capacity for response to a hemorrhagic emergency.

B Lymphocyte Precursor Cells Represent Sensitive Targets of T2 Mycotoxin Exposure

Exposure of experimental animals and humans to the Fusarium trichothecene metabolite, T2 toxin, has been associated with a variety of immunosuppressive effects, including altered parameters of humoral-mediated immunity. Although T2 toxin is cytotoxic in vitro to lymphocytic cells, limited information is presently available regarding the contribution of such a mechanism to immunosuppression in vivo, or to potential immune cell targets. In the present report, subchronic T2 toxin treatment of timed-pregnant B6C3F1 mice resulted in significant and selective depletion of fetal liver cells expressing low levels of surface CD44 and CD45 antigens, suggestive of possible lymphoid progenitor cell sensitivity to this agent. Evaluation of CD45R antigen expression in fetal liver supported such a hypothesis, demonstrating a significant reduction in fetal liver B lymphocytic cells in animals exposed to T2 toxin. Subsequent in vitro T2 toxin exposure of fetal liver cells enriched for prolymphocytes by differential density gradient centrifugation demonstrated the presence of a highly sensitive subpopulation of cells that was eliminated in a selective, and near-complete, manner by T2 toxin exposure. This sensitive cell population was observed to have light-scatter characteristics of CD45R+ B-lineage lymphocytes. Additional studies in adult mice demonstrated a reduction in CD44lo and CD45R+ bone marrow cells similar to that seen in fetal liver, indicating that T2 toxin may also target immature B lymphocytes in this hematopoietic compartment. Taken together, these data suggest that the precursors of B cells may represent, for unknown reasons, highly sensitive targets of T2 toxin exposure.

Fine Structure of the Peptidyl Transferase Centre on 23 S-like rRNAs Deduced from Chemical Probing of Antibiotic-Ribosome Complexes

Ribosomal binding sites were investigated for the diverse group of antibiotics: anisomycin, anthelmycin, blasticidin S, bruceantin, carbomycin, chloramphenicol, griseoviridin, narciclasine, T2 toxin, tylosin and virginiamycin M1all of which are considered to inhibit the peptidyl transferase reaction by different mechanisms. The drugs also exhibit differing degrees of specificity for bacterial, archaeal and eukaryotic ribosomes despite a high level of conservation of sequence and secondary structure at the peptidyltransferase centre of the 23 S-like rRNAs. The drug binding sites were characterized by incubating each antibiotic with ribosomes from a bacterium, an archaeon and a eukaryote and chemically probing the 23 S-like rRNA. The complexity of the changes in reactivity ranged from one or two nucleotides (anthelmycin, narciclasine) to eight or nine (virginiamycin M1) and it was inferred, at least for those drugs producing complex changes, that they induce, and stabilize, a particular functional conformer in the peptidyl transferase centre. The results were correlated with literature data on both ribosomal ligand binding and the putative inhibitory mechanisms of the drugs, and the following inferences are made concerning the fine structure of the peptidyl transferase centre. (1) An irregular secondary structural motif, which includes unpaired A2439 (Escherichia colinumbering), lies close to the catalytic centre; (2) nucleotides A2451 and C2452 contribute to a site for the binding of the side chains of aromatic amino acids; (3) the P-substrate site encompasses U2585, U2506 and, possibly, a site in domain IV (A1787), and (4) the sequence A2058 to A2062 and nucleotide U2609 contribute to, or could be directly attributed to an A-site and the possibility is raised that, if it exists, it consists mainly of ribosomal proteins. However, two drugs T2 toxin and virginiamycin M1protected the only nucleotide in the peptidyl transferase loop region (C2394) associated with the E-site. Finally, it is proposed that the putative sub-sites are physically separated, that some drugs bind to more than one of them, and that they are conformationally interdependent.

Effects of spice oil treatment of rice on moulding and mycotoxin contamination

Moulding and mycotoxin production have been studied during storage of rice at 85 and 90% relative humidity (r.h.) following treatment with cinnamon and clove oils. The clove oil-treated rice was artificially inoculated with Aspergillus flavus. Aflatoxin B 1 and ochratoxin A were determined by monoclonal antibody-based enzyme-linked immunosorbent assay. Both moulding and mycotoxin were inhibited by 9 μl cinnamon oil g −1 but, with smaller doses, fungi could be isolated during the early part of storage, but not subsequently. Thus, following treatment with 3 or 6 μl cinnamon oil g −1, fungi were isolated from direct plated grains after 30 days but not after 45 days. Fungi were also isolated from washings of grain up to 30 days after treatment with 3 or 6 μl g −1, although fewer than from untreated grain. Propagules could also be detected up to 15 days after treatment with 9 μl g −1. Clove oil was much less effective than cinnamon oil in preventing moulding. After treatment with 8 μl g −1, the proportion of seeds infected with fungi and numbers of propagules in washings decreased only after 60 days' storage and with 4 μ g −1 after 90 days at both relative humidities. However, aflatoxin contamination could not be detected 90, 60 and 60 days after treatment with, respectively, 2, 4 and 8 μl g −1 at 85% r.h., although ochratoxin A was present in all treatments. At 90% r.h. aflatoxin was absent only after 90 days from treatment with 4 and 8 μl g −1. No T2 toxin was detected.

Evidence that the CryIA crystal protein from Bacillus thuringiensis is associated with DNA.

Toxin generated by activation of the Bacillus thuringiensis CryIA(c) crystal protein (protoxin) with bovine trypsin was separated into two components by anion-exchange chromatography. One component (T2) was DNA-associated toxin, and the other was the DNA-free toxin (T1). Only one major protoxin component was observed, and it was found to be associated with DNA. The DNA from the T2 toxin varied in size from 100 to 300 base pairs, whereas the crystal and the solubilized protoxin contained 20-kilobase DNA as the major DNA component. DNase treatment converted the T2 toxin to the DNA-free T1 toxin. In contrast, the DNA in the crystal and the solubilized protoxin was resistant to DNase digestion and was not dissociated from the protein by 1.5 M NaCl. The protoxin and DNA appeared to elute as a complex with a molecular mass of > 2 x 10(6) Da on gel-filtration chromatography. No toxin was generated from the protoxin with trypsin after extensive digestion of the protoxin with DNase or dissociation of the DNA by succinylation of the lysine residues. It is proposed that DNA binds to the COOH-terminal half of the crystal protein and is essential for maintaining the conformational integrity required for crystal formation and generation of toxin.

Metabolism and Pharmacokinetics of T-2 Toxin and Related Trichothecenes

(1993). Metabolism and Pharmacokinetics of T-2 Toxin and Related Trichothecenes. Drug Metabolism Reviews: Vol. 25, No. 3, pp. 281-323.

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.

Hypovolemic shock in acute lethal T-2 mycotoxicosis

Experiments were performed on pentobarbital-anesthetized cats to test the hypothesis that hypovolemia rather than cardiac failure is responsible for the acute lethal toxicity of the trichothecene mycotoxin, T-2 toxin (T2T). Measurements were made on mean arterial blood pressure (MAP), arterial pulse pressure (PP), and heart rate (HR) in eight otherwise untreated cats given T2T (2 mg/kg iv) and in three cats similarly injected with T2T but then transfused with plasma and blood. The transfusions to their available extent significantly delayed or counteracted the development of mycotoxic shock (i.e., depressed MAP and PP) and prevented or reversed a rise in the hematocrit. HR remained stable under all conditions. Plasmapheresis followed by whole-blood removal was found best to simulate mechanistically the mycotoxic shock syndrome in six blood donor cats free of T2T. It is concluded that hypovolemia with polycythemia resulting from plasma leakage and internal bleeding accounts for acute lethal T-2 mycotoxicosis.

Incidence of molds and mycotoxins in commercial animal feed mills in seven midwestern states, 1988-1989.

A total of 82 feed manufacturers located within seven midwestern states (Iowa, Nebraska, Minnesota, Illinois, Indiana, Ohio, Michigan) participated in a survey of mold and mycotoxin contamination of corn. Samples were submitted from a composite of the grading samples taken from each incoming load of corn. The survey was initiated in July 1988. During the 12-mo period, moisture content of the corn samples upon receipt at the laboratory ranged from 10.5 to 13.3%. The greatest variation occurred in the springtime. Iowa's corn samples were driest (11.2%), and samples submitted from Ohio were wettest (12.8%). Mold counts averaged 2.63 x 10(4) per gram during the year. The predominant mold found was Fusarium sp. Samples were checked by black light and averaged 25.4% positive during the period. When assayed for mycotoxins, 19.5% of the samples were positive for at least one of the following: aflatoxin, zearalenone, T2 toxin and deoxynivalenol (vomitoxin). Aflatoxin and T2 toxin made up the majority of these samples containing toxin. The highest incidence of mycotoxin-contaminated corn (48%) occurred in samples submitted in July of 1988. Over the 12-mo period, the highest mycotoxin contamination occurred in Iowa, Illinois and Michigan. When samples were subjected to 90% relative humidity and 32 degrees C, an average of 3.9 d was required for mold growth to appear. After incubation, 24.7% of the samples contained one of the four toxins. The data indicate that mold and mycotoxin contamination of mixed samples of corn is widespread, even in the midwestern corn belt of the U.S.

The structure of T-2 toxin

C 24 H 34 O 9 cristallise dans P2 1 2 1 2 1 avec a=7,808, b=15,800, c=40,891 A, z=8; affinement jusqu'a R=0,055. La formule structurale etablie chimiquement a ete corroboree. Les deux molecules dans la maille asymetrique different seulement par leurs angles de torsion des chaines ester laterales

Anti-T2 Monoclonal Antibody Immobilization On Quartz Fibers: Stability and Recognition of T2 Mycotoxin

Abstract Several methods for immobilizing anti-T2 mycotoxin monoclonal antibodies on quartz fibers, for use in optical sensor development, have been evaluated with respect to the surface density and stability of the immobilized proteins. the first method activates matrix hydroxyl groups using p-toluenesulfonyl chloride (TSC). the second method activates these groups using p-nitrophenyl chloroformate (NPCF). the third method requires an initial silanization using 3-aminopropyltriethoxysilane (APTES) followed by carrier activation with glutaraldehyde. the activated carrier in all three methods is then reacted with the amino groups of the protein. the first two non-silanizing coupling methods are simple, inexpensive and non-hazardous compared to the third, more complex method in which an initial Correspondance: to PVS

Targeting of mycotoxins to reticuloendothelial system of mice with carrier erythrocytes.

The tricothecene mycotoxin, T-2 toxin, was encapsulated in bovine erythrocytes for in vivo delivery of T-2 toxin to macrophages. Intraperitoneal injection of bovine carrier erythrocytes (5 X 10(8) cells) containing T-2 toxin saturated mouse liver uptake of erythrocytes by 6 h postinjection. At 24 h postinjection, 20% of the injected carrier cells containing toxin were localized in the liver of mice. Saturation of the liver uptake of bovine carrier cells was independent of encapsulated or free T-2 toxin. A dose of T-2 toxin sufficient to inhibit 50% of the macrophage protein synthesis was targeted to the liver via the carrier erythrocytes. A methodology for delivery of highly toxic molecules to liver macrophages is described.

Skin effects of trichothecenes and their amelioration by decontamination

The ability of trichothecenes, in particular T2 toxin (T2), to cause irritant effects on the skin has been investigated in laboratory rodents and rabbits. Quantitatively, T2 was found to be highly potent in this respect, causing irritant reactions on rat skin at contamination densities of < 1 μ g · cm −2 . The first appearance of skin effects was delayed for approximately 6 h after application, irrespective of the dose applied. Similarly, variation in solvent or injection into or beneath the skin failed to accelerate the onset of the irritant reaction. An aqueous soaps solution was largely effective in removing low doses of T2 from the skin, but was ineffective in removing larger doses. However, washing the contaminated skin with polyethylene glycol 300 was very effective at removing even large doses of T2 from the skin. The macrocyclic trichothecene verrucarin A was of comparable irritancy to T2 on rat skin. Diacetoxyscirpenol and nivalenol were less potent.

Effect of T2 toxin on the spontaneous antibody-secreting cells and other non-lymphoid cells in the murine spleen

The Fusarium secondary metabolite, T2 toxin was investigated for its effects upon spontaneous antibody-producing cells in the murine spleen. Changes in the number of non-lymphoid cells in the spleen were also studied. A statistically significant increase in the number of spontaneous antibody-secreting cells, as detected by the protein A plaque assay, was observed. A dose-dependent increase was found after chronic exposure to T2 toxin for 3 wk. An increased number of erythroblast cells was also found in the spleens of these animals. At all the T2 toxin concentrations studied, the mice showed a dose-dependent reduction in blood levels of haemoglobin. Reduction in the suppression of B cell growth and stimulation of B cells caused by erythropoiesis or activated macrophages could be responsible for the increase in antibody production.