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.     

A homogeneous immunoassay for the mycotoxin T-2 utilizing liposomes, monoclonal antibodies, and complement

The trichothecene mycotoxin T-2 is a fungal metabolite known to contaminate agricultural products and cause intoxication of humans and animals. We have developed a homogeneous competition inhibition assay for T-2 mycotoxin based on complement-mediated lysis of liposomes. The T-2 mycotoxin was converted to an acid chloride derivative, subsequently coupled to the amino group of phosphatidylethanolamine, and incorporated with the phospholipid into unilamellar liposomes. Carboxyfluorescein, which is self-quenched at high concentrations, was entrapped in the liposomes as a release marker. We used a monoclonal IgG1 antibody specific for T-2 mycotoxin and a polyclonal anti-mouse Ig as a secondary antibody since the anti-T-2 IgG1 does not activate complement. In the absence of free T-2, the liposomes were lysed within 30 min after the addition of complement, releasing carboxyfluorescein into the surrounding buffer. In the presence of free T-2 toxin, the binding of antibodies to the liposomes was reduced, causing a corresponding decrease in lysis. This assay proved to be sensitive to T-2 toxin levels as low as 2 ng, which is 10-fold more sensitive than the present enzyme immunoassay using the same antibodies.     

Effect of trichothecenes on growth and intracellular pool size of Mycoplasma gallisepticum

The mycotoxin T-2 inhibited the growth of Mycoplasma gallisepticum. The growth inhibition was most pronounced with the hydrophobic derivatives T-2 acetate and very little with the hydrophilic T-2 tetraol. The toxin had no effect on the biosynthesis of either protein, DNA, RNA or complex lipids but markedly reduced the intracellular pool size of soluble low molecular mass precursors. It seems that T-2 acetate, by virtue of its hydrophobic nature, may accumulate within the lipid backbone affecting the permeability properties of the cell membrane.     

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.     

Metabolic effects of trichothecene T-2 toxin

Cereals and other agricultural products contaminated with trichothecene mycotoxins are unfit for consumption. Until recently, the metabolic effects of T-2 toxin (T-2) were thought to reside in its ability to inhibit protein synthesis. It is now clear that trichothecenes have multiple effects, including inhibition of DNA, RNA, and protein synthesis in several cellular systems, inhibition of in vitro protein synthesis, inhibition of mitochondrial functions, effects on cell division, normal cell shape, and hemolysis of erythrocytes. It is argued that these effects are pleiotropic responses of the cell's biosynthetic network to protein synthesis inhibition. However, in studies with erythrocytes, which lack nuclei and protein synthesis, changes in cell shape and lytic response towards T-2 are observed. Susceptibility to lysis is species dependent and correlates with the presence of phosphatidylcholine. Owing to their amphipathic nature, T-2 and other trichothecenes could exert their cytotoxicity by acting on cell membranes. As for cell energetics, T-2 inhibits the mitochondrial electron transport system, with succinic dehydrogenase as one site of action. Although initial investigations of the metabolic effects of T-2 mediated cytotoxicity suggested the inhibition of protein synthesis as the principal site of action, current thought suggests that the effects of trichothecenes are much more diverse.     

Microtubules, colchicine, and lymphocyte blastogenesis.

We have studied the time course of disassembly of microtubules of resting and stimulated mouse lymphocytes caused by the drug colchicine, as well as the effect of this compound on DNA and RNA synthesis of human and mouse lymphocytes. Fine-structure studies with the electron microscope showed a great increase in number of microtubules resulting from stimulation of mouse lymphocytes by the mitogenic lectin Con A. The presence of a network of microtubules was demonstrated in resting lymphocytes by use of the technique of immunofluorescence; this technique was not effective for the study of the microtubules of stimulated lymphocytes in the blast stage. The disappearance of microtubular networks in some cells (approximately 25%) was caused by the protocol of colchicine treatment used in many laboratories (30 min at 10(6) M); a 6- to 8-h treatment was required to cause all cells to lose their microtubules. It is indicated in these findings that there is need for extreme caution in implicating microtubule disruption as the cause of certain colchicine effects, such as that on the Con A-induced inhibition of receptor-ligand migration. The addition of colchicine to stimulated cells at varying times of culture caused marked inhibition of DNA synthesis provided that sufficient time (approximately 20 h for maximum inhibition) elapsed between addition of the drug to the stimulated culture and assay of DNA synthesis. Our data on the time course of inhibition of DNA synthesis by alpha-methyl mannoside (alpha MM) and by colchicine do not exclude the possibility that the latter compound may act partially by affecting the commitment of stimulated lymphocytes to DNA synthesis but they show that it can inhibit well after commitment is complete. The later the time of assay of thymidine incorporation, the more disparate were the curves relating the effects of alpha MM and colchicine to DNA synthesis of human cells. In the case of mouse splenic lymphocytes, there was no resemblance between the time course of the alpha MM and of the colchicine effects. Synthesis of RNA after 12 h of culture of stimulated human lymphocytes was also sensitive to colchicine.     

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.     

The effect of a Clostridium perfringens 8-6 enterotoxin on viability and macromolecular synthesis in Vero cells

Clostridium perfringens type A 8-6 enterotoxin causes gross morphological damage to Vero cells grown in tissue culture. Damage was observed to occur after only 30 minutes exposure at concentrations as low as 20 ng/ml, and within 60 minutes 95% of the cells had detached. Concentrations as low as 0.01 ng were able to cause detectable inhibition of plating efficiency. The enterotoxin inhibited DNA, RNA and protein synthesis and caused the reversal of glucose transport. Heat inactivated enterotoxin had no effect on cell function or morphology.     

Interaction of T-2 toxin and murine lymphocytes and the demonstration of a threshold effect on macromolecular synthesis

Although T-2 toxin intoxications have been described as radiomimetic, we find that T-2 toxin does not preferentially affect multiplying cells. Among the targets of T-2 toxin toxicity, DNA, RNA and protein synthesis inhibition are analysed. All three types of macromolecular syntheses are affected by a threshold dose of T-2 toxin which corresponds to the interaction of approx. 1 X 10(5) T-2 toxin molecules with the same number of T-2 toxin receptors (Gyongyossy-Issa, M.I.C. and Kachatourians, G.G. (1984) Biochim. Biophys. Acta 803, 197-202). Since toxic effects occur faster at higher toxin concentrations than at lower levels, the time-toxic effect relationship may be defined by a constant. Based on these observations, we hypothesize that complete receptor-occupation is the critical first step in the course of T-2 toxin toxicity events.     

DNA-damaging activity of patulin in Escherichia coli

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

Comparative study of the influence of renal endogenic factors on the proliferation activity of epithelial cells

The proliferation activity of monolayer culture of Madin Darby Canine Kidney (MDSK) cells is suppressed by a thermostable protein factor of renal tissue of white rats and of humans. Under the influence of renal factors (RF), a decrease in cell number, and suppression of DNA synthesis and mitotic activity in MDCK cells occur. The inhibition of proliferative activity of cultured cells under the influence of RF was substantiated also by MTT assay. It was established that the inhibitory influence of RF is stipulated by suppression of RNA synthesis. It follows that RF may inhibit division of MDCK cells via suppression of gene expression in G1-phase. Similar factors were obtained from renal cells of different systematic groups of organisms (snail, frog, fish, pigeon, guinea pig, swine).     

Biological active peptides in human urine: III. Inhibitors of the growth of human leukemia, osteosarcoma, and HeLa cells.

Medium-sized peptides isolated from normal humans urine were tested for their effect on DNA, RNA, and protein synthesis, and mitosis, in tissue culture of human myeloblastic leukemia, osteosarcoma, and HeLa cells. Two types of antineoplastic peptides were found. One type consists of strongly acidic peptides (probably sulfated glycopeptides) which act specifically on different kinds of neoplasma. The other type comprises slightly acidic and neutral peptides, and has broad specificity. The active peptides produce up to 97% inhibition of DNA synthesis and mitosis in the neoplastic cells in tissue culture. The peptide fraction which has broad specificity was tested in different concentrations and gave good dose-response relationship.     

DNA-damaging activity of patulin in Escherichia coli.

At a concentration of 10 micrograms/ml, patulin caused single-strand DNA breaks in living cells of Escherichia coli. At 50 micrograms/ml, double-strand breaks were observed also. Single-strand breaks were repaired in the presence of 10 micrograms of patulin per ml within 90 min when the cells were incubated at 37 degrees C in M9-salts solution without a carbon source. The same concentration also induced temperature-sensitive lambda prophage and a prophage of Bacillus megaterium. When an in vitro system with permeabilized Escherichia coli cells was used, patulin at 10 micrograms/ml induced DNA repair synthesis and inhibited DNA replication. The in vivo occurrence of DNA strand breaks and DNA repair correlated with the in vitro induction of repair synthesis. In vitro the RNA synthesis was less affected, and overall protein synthesis was not inhibited at 10 micrograms/ml. Only at higher concentrations (250 to 500 micrograms/ml) was inhibition of in vitro protein synthesis observed. Thus, patulin must be regarded as a mycotoxin with selective DNA-damaging activity.     

Effects of Fusarium moniliforme culture extracts and fumonisin B1 on DNA,RNA and protein synthesis by baby hamster kidney cells

Baby hamster kidney cells (BHK-21) were exposed to culture filtrates of 4 Fusarium moniliforme isolates containing varying levels of fumonisin B1 (FMB1) and the effects upon RNA, DNA and protein synthesis were monitored. Cells were also grown on medium amended with FMB1 only for comparison. After 24 h incubation FMB1 (100 μg/100 ml medium) reduced protein synthesis by 4% and by 18% after 48 h. Culture filtrates containing the highest levels of FMB1 also caused the greatest inhibition in protein synthesis after 24 h but after 48 h protein synthesis levels were the same as controls even though the FMB1 level was 360 μg/100 ml. Only FMB1 reduced DNA synthesis, by 8% after 24 h but after 48 h DNA levels had increased by 40 % over controls. The culture filtrates containing the highest levels of FMB1 (360 μg/100 ml) reduced DNA synthesis more than 50% after 24 h and 48 h. Culture filtrates containing lesser amounts of FMB1 in some instances stimulated DNA synthesis and inhibited it in others. There was also no correlation in the level of FMB1 with the inhibition of RNA synthesis by BHK cells. It appears that metabolites other than fumonisin produced by F. moniliforme in culture can affect and both stimulate and inhibit RNA, DNA and protein synthesis by BHK cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Solvent comparison in the isolation, solubilization, and toxicity of Stachybotrys chartarum spore trichothecene mycotoxins in an established in vitro luminescence protein translation inhibition assay

It is well known that non-viable mold contaminants such as macrocyclic trichothecene mycotoxins of Stachybotrys chartarum are highly toxinigenic to humans. However, the method of recovering native mycotoxin has been without consensus. Inconsistencies occur in the methods of isolation, suspension, preparation, and quantitation of the mycotoxin from the spores. The purpose of this study was to provide quantitatively comparative data on three concurrent preparations of 10(6)S. chartarum spores. The experiments were designed to specifically evaluate a novel method of mycotoxin extraction, solubilization, and the subsequent inhibitory effect in an established in vitro luminescence protein translation assay from 30 day-old spores. The mycotoxin-containing spores swabbed from wallboard cultures were milled with and without glass beads in 100% methanol, 95% ethanol, or water. Milled spore lysates were cleared of cell debris by filter centrifugation followed by a second centrifugation through a 5000 MWCO filter to remove interfering proteins and RNases. Cleared lysate was concentrated by centrivap and suspended in either alcohol or water as described. The suspensions were used immediately in the in vitro luminescence protein translation assay with the trichothecene, T-2 toxin, as a control. Although, mycotoxin is reported to be alcohol soluble, the level of translation inhibition was not reliably satisfactory for either the methanol or ethanol preparations. In fact, the methanol and ethanol control reactions were not significantly different than the alcohol prepared spore samples. In addition, we observed that increasing amounts of either alcohol inhibited the reaction in a dose dependent manner. This suggests that although alcohol isolation of mycotoxin is desirable in terms of time and labor, the presence of alcohol in the luminescence protein translation reaction was not acceptable. Conversely, water extraction of mycotoxin demonstrated a dose dependent response, and there was significant difference between the water controls and the water extracted mycotoxin reactions. In our hands, water was the best extraction agent for mycotoxin when using this specific luminescence protein translation assay kit.     

Molecular cloning and expression of cardiac-specific myosin heavy chain gene sequences in chick embryo

A recombinant DNA plasmid, pMHC8, that contains gene sequences for embryonic chick cardiac myosin heavy chain was constructed, identified and characterized. The identity of the clone was established by hybridization with labeled probes that afford screening of MHC²² with high specificity, by inhibition of MHC synthesis in the in vitro hybrid-arrested translation assay, and by tissue-specific hybridization of labeled pMHC8 DNA to MHC messenger RNA.The pMHC8 DNA probe is highly specific for chick heart muscle tissue, since it hybridized poorly to chick skeletal muscle RNA and did not detectably hybridize to adult rat heart RNA. Upon screening the embryonic chick heart cells in culture, no detectable level of MHC mRNA was observed in dividing myoblasts, but the mRNA appeared in differentiated cardiac myocytes paralleling morphogenetic changes in the embryonic cells.     

Actinomycin Analogues Containing Pipecolic Acid: Relationship of Structure to Biological Activity

Streptomyces antibioticus synthesizes a mixture of actinomycins which differ at the "imino acid" site of the peptide chains. In the presence of exogenous pipecolic acid, several new actinomycins were synthesized and 70% of the proline in the antibiotic mixture was replaced by the analogue. Three new antibiotics (designated Pip 1alpha, Pip 1beta, and Pip 2) were isolated from culture filtrates, purified, and crystallized. The molar ratio of pipecolic acid to proline was: Pip 1alpha, 1:0; Pip 1beta, 1:1; Pip 2, 2:0. These compounds inhibited the growth and cell division of gram-positive, but not gram-negative, bacteria. The relative inhibitory activity against bacteria, Escherichia coli deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase in vitro, and RNA synthesis in Bacillus subtilis and mouse L-929 cells was: actinomycin IV = Pip 1beta > Pip 2 > Pip 1alpha. Protein synthesis in B. subtilis was less affected, and DNA synthesis was inhibited only at higher concentrations of antibiotic tested. In L cells, DNA formation was reduced less than RNA synthesis, whereas protein synthesis was not blocked under the experimental conditions employed. Kinetic studies with B. subtilis revealed that RNA synthesis was inhibited rapidly followed by an inhibition of protein synthesis. All four antibiotics markedly inhibited the replication of vaccinia virus and reovirus in tissue culture cells, but the production of poliovirus was resistant to the antibiotics. These actinomycins bind to DNA, resulting in an elevation of its T(m) and a decrease in the peak extinction of the actinomycins. The mode of action, as well as the structure-activity relationships among the actinomycins, are discussed relative to a previously proposed model of binding.