Cytotoxic and immunotoxic effects of Fusarium mycotoxins using a rapid colorimetric bioassay

A colorimetric MTT (tetrazolium salt) cleavage test was used to evaluate cytotoxicity of twenty-three Fusarium mycotoxins on two cultured human cell lines (K-562 and MIN-GL1) as well as their inhibitory effect on proliferation of phytohemagglutinin-stimulated human peripheral blood lymphocytes. The values of 50% inhibition of lymphocyte blastogenesis were very close to the 50% cytotoxic doses observed with the more sensitive cell line (MIN-GL1). T-2 toxin was the most cytotoxic with CD₅₀ and ID₅₀ values less than 1 ng/ml. Type A trichothecenes were the most cytotoxic followed by the type B trichothecenes; the non-trichothecenes were the least cytotoxic. The MTT cleavage test, in conjunction with cell culture, is a simple and rapid bioassay to evaluate cytotoxicity and immunotoxicity of Fusarium mycotoxins.Abbreviations Ac acetyl - ACU acuminatin - DAS diacetoxyscirpenol - DON deoxynivalenol - FUS fusarenon-X - HT-2 HT-2 toxin - MC mononuclear cell - MTT 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide - NEO neosolaniol - NIV nivalenol - NT-1 4,8-diacetoxy T-2 tetraol - PBS phosphate buffered saline - TAT-2T tetraacetoxy T-2 tetraol - T-2 T-2 toxin     

Trichothecene mycotoxins trigger a ribotoxic stress response that activates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase and induces apoptosis.

The trichothecene family of mycotoxins inhibit protein synthesis by binding to the ribosomal peptidyltransferase site. Inhibitors of the peptidyltransferase reaction (e.g. anisomycin) can trigger a ribotoxic stress response that activates c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinases, components of a signaling cascade that regulates cell survival in response to stress. We have found that selected trichothecenes strongly activate JNK/p38 kinases and induce rapid apoptosis in Jurkat T cells. Although the ability of individual trichothecenes to inhibit protein synthesis and activate JNK/p38 kinases are dissociable, both effects contribute to the induction of apoptosis. Among trichothecenes that strongly activate JNK/p38 kinases, induction of apoptosis increases linearly with inhibition of protein synthesis. Among trichothecenes that strongly inhibit protein synthesis, induction of apoptosis increases linearly with activation of JNK/p38 kinases. Trichothecenes that inhibit protein synthesis without activating JNK/p38 kinases inhibit the function (i.e. activation of JNK/p38 kinases and induction of apoptosis) of apoptotic trichothecenes and anisomycin. Harringtonine, a structurally unrelated protein synthesis inhibitor that competes with trichothecenes (and anisomycin) for ribosome binding, also inhibits the activation of JNK/p38 kinases and induction of apoptosis by trichothecenes and anisomycin. Taken together, these results implicate the peptidyltransferase site as a regulator of both JNK/p38 kinase activation and apoptosis.     

Protein synthesis by mammalian cells treated with C-3-modified analogs of the 12,13-epoxytrichothecenes T-2 and T-2 tetraol.

Modification at the C-3 position of the trichothecenes T-2 and T-2 tetraol affected their ability to inhibit protein synthesis in African green monkey kidney (Vero) and mouse erythroleukemia cells. Replacement of the 3-hydroxyl of T-2 with hydrogen caused a 24-fold decrease in activity, whereas acetylation resulted in a 500-to 1,000-fold decrease. Protection of the 3-hydroxyl with a tetrahydropyranyl moiety gave an analog that was 37-fold more inhibitory to Vero than to mouse erythroleukemia cells; with the other analogs a similar effect on protein synthesis was found for both types of cells. The analogs obtained after alkaline hydrolysis were much less potent than the parent trichothecenes. The 3-tetrahydropyranyl-modified analog was equivalent in potency to T-2 tetraol, while the deoxygenated species was at least threefold less potent. All T-2 analogs caused some degree of polysome "runoff," thereby demonstrating that these species inhibit protein synthesis at the chain initiation stage when added at their 50% infective dose concentrations or lower. From these results, we suggest that the 3-hydroxyl moiety is essential for T-2 to exhibit such high activity on eucaryotic cell protein synthesis and that modification at the C-3 position decreases but does not eliminate this activity.     

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.     

Fusarium phytotoxin trichothecenes have an elicitor-like activity in Arabidopsis thaliana, but the activity differed significantly among their molecular species

Phytopathogenic fungi such as Fusarium spp. synthesize trichothecene family phytotoxins. Although the type B trichothecene, deoxynivalenol (DON), is thought to be a virulence factor allowing infection of plants by their trichothecene-producing Fusarium spp., little is known about effects of trichothecenes on the defense response in host plants. Therefore, in this article, we investigated these effects of various trichothecenes in Fusarium-susceptible Arabidopsis thaliana. Necrotic lesions were observed in Arabidopsis leaves infiltrated by 1 microM type A trichothecenes such as T-2 toxin. Trichothecene-induced lesions exhibited dead cells, callose deposition, generation of hydrogen peroxide, and accumulation of salicylic acids. Moreover, infiltration by trichothecenes caused rapid and prolonged activation of two mitogen-activated protein kinases and induced expression of both PR-1 and PDF1.2 genes. Thus, type A trichothecenes trigger the cell death by activation of an elicitor-like signaling pathway in Arabidopsis. Although DON did not have such an activity even at 10 microM, translational inhibition by DON was observed at concentrations above 5 microM. These results suggested that DON is capable of inhibiting translation in Arabidopsis cells without induction of the elicitor-like signaling pathway.     

Protein synthesis by mammalian cells treated with C-3-modified analogs of the 12,13-epoxytrichothecenes T-2 and T-2 tetraol

Modification at the C-3 position of the trichothecenes T-2 and T-2 tetraol affected their ability to inhibit protein synthesis in African green monkey kidney (Vero) and mouse erythroleukemia cells. Replacement of the 3-hydroxyl of T-2 with hydrogen caused a 24-fold decrease in activity, whereas acetylation resulted in a 500-to 1,000-fold decrease. Protection of the 3-hydroxyl with a tetrahydropyranyl moiety gave an analog that was 37-fold more inhibitory to Vero than to mouse erythroleukemia cells; with the other analogs a similar effect on protein synthesis was found for both types of cells. The analogs obtained after alkaline hydrolysis were much less potent than the parent trichothecenes. The 3-tetrahydropyranyl-modified analog was equivalent in potency to T-2 tetraol, while the deoxygenated species was at least threefold less potent. All T-2 analogs caused some degree of polysome "runoff," thereby demonstrating that these species inhibit protein synthesis at the chain initiation stage when added at their 50% infective dose concentrations or lower. From these results, we suggest that the 3-hydroxyl moiety is essential for T-2 to exhibit such high activity on eucaryotic cell protein synthesis and that modification at the C-3 position decreases but does not eliminate this activity.     

Phytotoxic effects of trichothecenes on the growth and morphology of Arabidopsis thaliana

Non-volatile sesquiterpenoids, a trichothecene family of phytotoxins such as deoxynivalenol (DON) and T-2 toxin, contain numerous molecular species and are synthesized by phytopathogenic Fusarium species. Although trichothecene chemotypes might play a role in the virulence of individual Fusarium strains, the phytotoxic action of individual trichothecenes has not been systematically studied. To perform a comparative analysis of the phytotoxic action of representative trichothecenes, the growth and morphology of Arabidopsis thaliana growing on media containing these compounds was investigated. Both DON and diacetoxyscirpenol (DAS) preferentially inhibited root elongation. DON-treated roots were less organized compared with control roots. Moreover, preferential inhibition of root growth by DON was also observed in wheat plants. In addition, T-2 toxin-treated seedlings exhibited dwarfism with aberrant morphological changes (e.g. petiole shortening, curled dark-green leaves, and reduced cell size). These results imply that the phytotoxic action of trichothecenes differed among their molecular species. Cycloheximide (CHX)-treated seedlings displayed neither feature, although it is known that trichothecenes inhibit translation in eukaryotic ribosomes. Microarray analyses suggested that T-2 toxin caused a defence response, the inactivation of brassinosteroid (BR), and the generation of reactive oxygen species in Arabidopsis. This observation is in agreement with our previous reports in which trichothecenes such as T-2 toxin have an elicitor-like activity when infiltrated into the leaves of Arabidopsis. Since it has been reported that BR plays an important role in a broad range of disease resistance in tobacco and rice, inactivation of BR might affect pathogenicity during the infection of host plants by trichothecene-producing fungi.     

Morphological changes in CHO and VERO cells treated with T-2 mycotoxin. Correlation with inhibition of protein synthesis

Exposure of Chinese hamster ovary and African green monkey kidney cells to T-2 mycotoxin resulted in several morphological changes which were related to inhibition of protein synthesis, the basic in vitro mechanism of action of the toxin. These changes, which occurred in both cell types, included disassociation of polysomes and mitochondrial cristae alterations. In addition, CHO cells displayed membrane bleb formations similar to those found in CHO cells after exposure to established inhibitors of protein synthesis, puromycin and anisomycin. Blebs could be either a result of protein synthesis inhibition or a non-specific early pathological response. Bleb formations were not observed in VERO cells under any experimental condition.     

Preparation and characterization of monoclonal antibodies to the trichothecene mycotoxin T-2.

Two mouse immunoglobulin G1 monoclonal antibodies that bind to the trichothecene mycotoxin T-2 were prepared. These antibodies, designated 12C12 and 15H6, had affinities for T-2 of 3.5 X 10(6) and 5.8 X 10(7) liters/mol, respectively. A competitive inhibition enzyme immunoassay that employed these antibodies had a sensitivity for T-2 of 50 ng per assay. Both antibodies bound to the metabolite HT-2 but not to the related trichothecenes monoacetoxyscirpenol, diacetoxyscirpenol, deoxynivalenol, and deoxyverrucarol. Evidence is presented that T-2-protein conjugates inhibit protein synthesis in lymphoid cells and that this apparent immunotoxicity may be due to the release of T-2 from the protein carrier.     

Preparation and characterization of monoclonal antibodies to the trichothecene mycotoxin T-2

Two mouse immunoglobulin G1 monoclonal antibodies that bind to the trichothecene mycotoxin T-2 were prepared. These antibodies, designated 12C12 and 15H6, had affinities for T-2 of 3.5 X 10(6) and 5.8 X 10(7) liters/mol, respectively. A competitive inhibition enzyme immunoassay that employed these antibodies had a sensitivity for T-2 of 50 ng per assay. Both antibodies bound to the metabolite HT-2 but not to the related trichothecenes monoacetoxyscirpenol, diacetoxyscirpenol, deoxynivalenol, and deoxyverrucarol. Evidence is presented that T-2-protein conjugates inhibit protein synthesis in lymphoid cells and that this apparent immunotoxicity may be due to the release of T-2 from the protein carrier.     

The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria

The oxazolidinones are one of the newest classes of antibiotics. They inhibit bacterial growth by interfering with protein synthesis. The mechanism of oxazolidinone action and the precise location of the drug binding site in the ribosome are unknown. We used a panel of photoreactive derivatives to identify the site of action of oxazolidinones in the ribosomes of bacterial and human cells. The in vivo crosslinking data were used to model the position of the oxazolidinone molecule within its binding site in the peptidyl transferase center (PTC). Oxazolidinones interact with the A site of the bacterial ribosome where they should interfere with the placement of the aminoacyl-tRNA. In human cells, oxazolidinones were crosslinked to rRNA in the PTC of mitochondrial, but not cytoplasmic, ribosomes. Interaction of oxazolidinones with the mitochondrial ribosomes provides a structural basis for the inhibition of mitochondrial protein synthesis, which is linked to clinical side effects associated with oxazolidinone therapy.     

Crosstalk of JNK1-STAT3 is critical for RAW264.7 cell survival

T-2 toxin, a major compound of trichothecenes, inhibits protein synthesis and induces inflammation and cell apoptosis through the activation of MAPK pathway. The JAK/STAT pathway has recently been shown to be downstream targets of trichothecenes. However, whether there is any crosstalk between JNK and JAK/STAT pathways in trichothecene toxicity has not been studied. In the present study, we explored this potential in RAW264.7 cells treated with T-2 toxin. Our results revealed a crosstalk between JNK1 and STAT3 after T-2 toxin treatment, which was mediated by K-Ras. T-2 toxin treatment resulted in rapid phosphorylation, and more importantly, JNK1-STAT3 signaling pathway was shown to maintain the normal function of the mitochondria and to inhibit T-2 toxin-induced apoptosis. Therefore, this pathway was considered to be a potential cell survival pathway. Breakdown and degranulation of ribosomes in the rough endoplasmic reticulum and swelling of mitochondria were clearly visible after the cells had been incubated with T-2 toxin for 12 h. Our data suggest that T-2 toxin had a Janus face: it induced both apoptotic and cell survival pathways. These results suggest that the crosstalk and the balance between MAPK and JAK/STAT pathway might be involved in T-2 toxin-induced apoptosis in RAW264.7 cells.     

Trichothecene-induced cytotoxicity on human cell lines

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2, HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent cell proliferation assay assessing mitochondrial metabolic activity. Toxicity was expressed as the toxin concentration inhibiting 50% of cell viability (IC₅₀). Depending on the chemotype of the tested trichothecenes, relative cytotoxic activity differed by a factor of 100–1,000, and the corresponding IC₅₀ values were in the range from 2.2 nmol/l (satratoxin H on Jurkat and U937 cells) to 4,900 nmol/l (deoxynivalenol on HEp-2 cells). In contrast, the specific toxicity of each individual mycotoxin towards different cell lines was within remarkable close limits, and between-cell line differences were much smaller than previously reported. For the cell lines tested, IC₅₀ values were 4.4–10.8 nmol/l for T-2 toxin, 7.5–55.8 mol/l for HT-2 toxin, 600–4,900 nmol/l for DON, 300–2,600 nmol/l for NIV, and 2.2–18.3 nmol/l for satratoxins G/H. In addition, for the first time, the toxic activity of trichothecenes on primary cell culture of human endothelial cells (HUVEC) was tested. The susceptibility of this cell line was comparable to the other cell lines tested, with IC₅₀ values ranging from 16.5 nmol/l (T-2 toxin) to 4,500 nmol/l (DON). The results suggest that the current focus of cytotoxicological studies on trichothecenes on lymphoid cell lines may lead to an underestimate of their potential on other target cell systems.     

Effects of T-2 toxin on ethanol production by Saccharomyces cerevisiae.

A trichothecene mycotoxin, T-2 toxin, inhibits several aspects of cellular physiology in Saccharomyces cerevisiae, including protein synthesis and mitochondrial functions. We have studied growth of, glucose utilization by, and ethanol production by S. cerevisiae and show that they are inhibited by T-2 toxin between 20 and 200 micrograms/ml in a dose-dependent manner. At 200 micrograms/ml, T-2 toxin causes cell death. This apparent inhibition of ethanol production was found to be the result of growth inhibition. On the basis of biomass or glucose consumption, T-2 toxin increased the amount of ethanol present in the culture. This suggests that T-2 inhibits oxidative but not fermentative energy metabolism by inhibiting mitochondrial function and shifting glucose catabolism toward ethanol formation. As T-2 toxin does not directly inhibit ethanol production by S. cerevisiae, this system could be used for ethanol production from trichothecene-contaminated grain products.     

Metabolism of T-2 toxin in Curtobacterium sp. strain 114-2.

The metabolic pathway of T-2 toxin in Curtobacterium sp. strain 114, one of the T-2 toxin-assimilating soil bacteria, was investigated by thin-layer and gas-liquid chromatographic analyses. T-2 toxin added to the basal medium as a single carbon and energy source was biotransformed into HT-2 toxin and an unknown metabolite. Infrared, mass spectrum, proton magnetic resonance, and other physico-chemical analyses identified this new metabolite as T-2 triol. T-2 toxin was first deacetylated by the bacterium into HT-2 toxin, and this metabolite was then biotransformed into T-2 triol without formation of neosolaniol and T-2 tetraol. No trichothecenes remained in the culture medium after prolonged culture. Some properties of T-2 toxin-hydrolyzing enzymes were observed with whole cells, the cell-free soluble fraction, and the culture filtrate. Besides T-2 toxin, trichothecenes such as diacetoxyscirpenol, neosolaniol, nivalenol, and fusarenon-X were also assimilated by this bacterium.     

T-2 toxin inhibits mitochondrial function in yeast

T-2 toxin inhibits oxygen consumption of whole cells and purified mitochondria of Saccharomyces cerevisiae. Inhibition of mitochondrial respiration is not relieved by 2, 4-dinitrophenol, indicating that T-2 toxin inhibits mitochondrial function at the level of the electron transport chain. T-2 toxin inhibition of state 3 respiration (with succinate) is overcome by N, N, N', N'-tetramethyl-p-phenylenediamine, indicating inhibition of site II of the electron transport chain. T-2 toxin inhibits mitochondrial succinate dehydrogenase activity and increases mitochondrial NADH dehydrogenase activity.     

Detection and quantitation of T-2 mycotoxin with a simplified protein synthesis inhibition assay.

We describe a simple, rapid, and sensitive bioassay for the detection and quantitation of T-2 mycotoxin by using a protein synthesis assay in cultured cells. Increased sensitivity of the cells to the mycotoxin occurred with time up to ca. 60-min. Time and dose response curves show that an average of 10 to 20 ng of T-2 per ml was sufficient to cause 50% inhibition of protein synthesis in tissue culture cells. A wide range of tissue culture cells with varied type, tissue, and species sources and growth characteristics were tested by this system. All showed approximately the same sensitivity to the mycotoxin. A slight modification of the procedure was used for suspended cultures of mitogen-stimulated lymphocytes, which also showed an equal degree of sensitivity to the mycotoxin. By simply changing the labeled precursor, the inhibition of RNA, DNA, and protein synthesis by T-2 mycotoxin can be compared. Although T-2 mycotoxin had little effect on RNA synthesis, DNA and protein synthesis were equally inhibited. Because of its sensitivity and its capacity to quickly assay a large number of samples, this technique has been a valuable tool in screening samples for the presence of active toxin and has been used to help establish laboratory safety standards for the inactivation of T-2 mycotoxin by chemical agents. It is presently being used in studies of mycotoxin mechanism of action and approaches toward in vivo neutralization of the toxic effects of mycotoxins.     

Pyridalyl inhibits cellular protein synthesis in insect, but not mammalian, cell lines

To gain insight into the mechanism of action and selectivity of the insecticidal activity of pyridalyl, the cytotoxicity of pyridalyl against various insect and mammalian cell lines was characterized by measuring the inhibition of cellular protein synthesis. When the effect of pyridalyl on the cellular protein synthesis in Sf9 cells was evaluated by measuring the incorporation of [(3)H]leucine, rapid and significant inhibition of protein synthesis was observed. However, pyridalyl did not inhibit protein synthesis in a cell-free protein synthesis system, indicating that pyridalyl does not directly inhibit protein synthesis. No obvious cytotoxicity was observed against any of the mammalian cell lines tested. In the case of insect cell lines, remarkable differences in the cytotoxicity of pyridalyl were observed: the highest cytotoxicity (IC50 mM) was found against Sf9 cells derived from Spodoptera frugiperda, whereas no obvious cytotoxicity was observed against BmN4 cells derived from Bombyx mori. Measurements of the insecticidal activity of pyridalyl against Spodoptera litura and B. mori revealed a correlation between the cytotoxicity against cultured cell lines and the insecticidal activity. From these observations, it was concluded that the selective inhibition of cellular protein synthesis by pyridalyl might contribute significantly to the insecticidal activity and the selectivity of this compound.     

Trichothecene synergism, additivity, and antagonism: the significance of the maximally quiescent ratio.

The interactive effect of the combinations of trichothecene mycotoxins often found in fungus infected plants, contaminated grain, and other biological systems is poorly understood. Growth inhibition of the yeast Kluyveromyces marxianus was used to measure the effects of HT-2 toxin, roridin A, and T-2 toxin as individual toxins or as binary mixtures. A value, the combination index, was derived which indicates the interactive effects of a binary mixture of toxins. The interaction is affected by the ratio of the individual toxins, and the percent inhibition of yeast growth. Generally the interaction of T-2 toxin and roridin A or T-2 toxin and HT-2 toxin changes from antagonistic when they cause a low percent inhibition of yeast growth to synergistic when they cause a high percent inhibition of yeast growth. Additionally, any two trichothecenes have a unique ratio, which we name the maximally quiescent ratio (or MQR), where there is the least change in the type and intensity of their interaction. The maximally quiescent ratio in this case has helped to define the nature of toxin interactions and could be used to provide insights into hormone, immune system, developmental, enzyme, and gene regulation, combined drug therapy, and the action of mixtures of natural or synthetic toxins, carcinogens, pesticides, and environmental pollutants.