Production of antibodies to roridin and their applications

Polyclonal rabbit antibodies against a conjugate synthesized through condensing BSA and disubstituted roridin A hemisuccinate allowed roridin A to be determined in solutions at a sensitivity of 0.2 ng/ml. The cross-reactivity of structural analogues—roridin A, verrucarin A, and verrucarol—amounted to 100, 2.5, and 0.03%, respectively. The data showed that these antibodies determine roridin A in an indirect heterogeneous enzyme immunoassay in cereal straw samples at a sensitivity of 20 μg/kg.     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

Enzyme immunoassay for the macrocyclic trichothecene roridin A: production, properties, and use of rabbit antibodies.

Antisera against roridin A were prepared by using a roridin A-hemisuccinate derivative coupled to human serum albumin as the immunogen. Antibodies could be detected in the sera of the immunized rabbits as early as 4 weeks after the initial exposure. After one booster injection at week 14, high antibody titers were measured over a period of 21 weeks. The specificity and sensitivity of the antibodies were tested by using roridin A-hemisuccinate coupled to horseradish peroxidase as an enzyme-linked toxin in a competitive assay with a double-antibody solid phase. The assay was most specific for the tested macrocyclic trichothecenes, and the relative cross-reactivities with roridin A, roridin J, verrucarin A, satratoxin H, and satratoxin G were 1, 0.41, 0.15, 0.15, and 0.07, respectively. When 16 nonmacrocyclic trichothecenes were tested, only diacetylverrucarol (0.0015) and verrucarol (0.0005) showed minor cross-reactivity. The sensitivity of the enzyme immunoassay for the detection of roridin A was in the range of 5 to 50 ng/ml (0.16 to 1.6 ng per assay).     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A.

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.     

Production and characterization of monoclonal antibodies to the macrocyclic trichothecene roridin A

Two murine monoclonal antibodies to the macrocyclic trichothecene roridin A are described. Screening for antibody production was performed on absorbed anti-mouse immunoglobulin serum as double-antibody solid phase, and further characterization was done on affinity-purified anti-mouse IgG serum. The antibodies, designated 5G11 and 4H10, had affinity constants for roridin A of 9.25 X 10(7) and 1.7 X 10(7) liters/mol, respectively. In monoclonal antibody-based direct enzyme immunoassays, these IgG1 antibodies had detection limits for roridin A of 0.4 ng/ml (0.02 ng per assay) and 1.8 ng/ml (0.09 ng per assay), respectively. Both antibodies were most specific for the tested macrocyclic trichothecenes. The relative cross-reactivities of antibody 5G11 with roridin A, roridin J, verrucarin A, satratoxin G, and satratoxin H were 100.0, 43.8, 16.7, 3.7, and 18.9%, respectively; for antibody 4H10 they were 100.0, 6.3, 64.0, 4.4, and 4.9%, respectively.     

Trichothecenes Production by the Hypocrealean Epibiont of Baccharis coridifolia

Abstract: The Hypocrealean epibiont of Baccharis coridifolia has been shown to synthesize de novo a series of macrocyclic trichothecene antibiotics, such as roridin A, roridin E, verrucarin A and verrucarin J. The structures of the compounds were determined by spectroscopic methods (¹H- and ¹³C-NMR and FABMS).     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

Growth hormone-like activities of macrocyclic trichothecenes in in vitro callus induction and growth of four Baccharis species

The ability of two plant-produced macrocyclic trichothecenes (baccharinoid B4 and roridin E) to induce callus growth of two trichothecene-producing Baccharis species (B. coridifolia and B. megapotamica) and two nontrichothecene-producing species (B. halimifolia and B. neglecta) was investigated. Roridin E had no effect in the induction of callus of B. coridifolia, a roridin-producing plant, but induced callus of nonroridin-producing plants (B. megapotamica, B. halimifolia, and B. neglecta). Baccharinoid B4 stimulated callus growth of B. megapotamica, a baccharinoid-producing plant, and inhibited growth of B. coridifolia, B. halimifolia, and B. neglecta callus tissues. The ability of roridin E to induce callus was most effective at concentrations of 10^–8 and 10^–6 M and when synergistically coupled with auxin, 2,4-dichlorophenoxyacetic acid (2,4-D). The ability of baccharinoid B4 to stimulate callus growth appeared to increase with increased concentration in the culture medium. Analysis of callus cultures grown in medium amended with roridin E showed that B4, roridin E, and 8-hydroxyroridin E and verrucarols were formed in the tissues but not in the medium. The results of this study indicated that while the callus-inducing ability of roridin E seemed to be nonspecies-specific in nature, the ability of B4 to stimulate callus was a highly species-specific phenomena. Callus-inducing activity of roridin E may depend on the capacity of plant species to transform exogenous roridin E into baccharinoids or other macrocyclic trichothecene derivatives.     

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.     

Verrucarin A and roridin E produced on spinach by <Emphasis Type="Italic">Myrothecium verrucaria</Emphasis> under different temperatures and CO<Subscript>2</Subscript> levels

The behavior of Myrothecium verrucaria, artificially inoculated on spinach, was studied under seven different temperature conditions (from 5 to 35 °C) and under eight different combinations of temperature and CO₂ concentration (14–30 °C and 775–870 or 1550–1650 mg/m³). The isolate used for this study was growing well on spinach, and the mycotoxins verrucarin A and roridin E were produced under all tested temperature and CO₂ conditions. The maximum levels of verrucarin A (18.59 ng/g) and roridin E (49.62 ng/g) were found at a temperature of 26–30 °C and a CO₂ level of 1550–1650 mg/m³. Rises in temperature as well as in temperature and CO₂ concentrations had a significant effect by increasing Myrothecium leaf spots on spinach. The biosynthesis of verrucarin A was significantly increased at the highest temperature (35 °C), while roridin E was influenced by the CO₂ concentration. These results show that a positive correlation between climate condition and macrocyclic trichothecene production is possible. However, because of the ability of M. verrucaria to produce mycotoxins, an increase in temperature could induce the spread of M. verrucaria in temperate regions; this pathogen may gain importance in the future.     

Spontaneous regression of pulmonary paracoccidioidomycosis

The Fusarium and Myrothecium mycotoxins roridin A, diacetoxyscirpenol, verrucarin A, T-2 toxin and zearalenone (10(-2) and 10(-3) mg/ml) inhibit the unspecific dehydrogenase activity of baker's yeast (Saccharomyces cerevisiae) in vivo. The action of these toxins is in the same order as that of aflatoxin B1. It is suggested that at least the trichothecenes decrease the dehydrogenase activity by an interaction with thiol groups of the active center of the enzymes.     

Macrocyclic trichothecenes are undetectable in kudzu (Pueraria montana) plants treated with a high-producing isolate of Myrothecium verrucaria.

Myrothecium verrucaria was found to be an effective pathogen against kudzu grown in the greenhouse and the field. M. verrucaria produced large amounts of macrocyclic trichothecenes when cultured on solid rice medium, including epiroridin E (16.8 mg/g crude extract), epiisororidin E (1 mg/g), roridin E (8.7 mg/g), roridin H (31.3 mg/g), trichoverrin A (0.6 mg/g), trichoverrin B (0.1 mg/g), verrucarin A (37.4 mg/g), and verrucarin J (2.2 mg/g). Most of these toxins were also isolated from M. verrucaria spores and mycelia grown on potato dextrose agar medium, including epiroridin E (32.3 mg/g), epiisororidin E (28.6 mg/g), roridin E (0 mg/g), roridin H (60 mg/g), trichoverrin A (1.3 mg/g), trichoverrin B (1.8 mg/g), verrucarin A (13.8 mg/g), and verrucarin J (131 mg/g). When M. verrucaria was cultured on liquid media, the numbers but not the amounts of toxins decreased. Only epiroridin E (28.3 mg/g), epiisororidin E (29.6 mg/g), verrucarin B (195 mg/g) and verrucarin J (52.6 mg/g) were measured when the fungus was cultured on cornsteep medium. On soyflour-cornmeal broth M. verrucaria produced several toxins, including epiroridin E (58.1 mg/g), epiisororidin E (5.8 mg/g), verrucarin B (29.9 mg/g) and verrucarin J (32 mg/g). In contrast, no macrocyclic trichothecenes were detected by HPLC analysis of plant tissues of kudzu, sicklepod, and soybean treated with aqueous suspensions of M. verrucaria spores formulated with a surfactant. Chloroform-methanol extracts of kudzu leaves and stems treated with M. verrucaria spores were less cytotoxic to four cultured mammalian cell lines than the corresponding extracts from control plants. Purified macrocyclic trichothecenes (verrucarin A and T-2 toxin) were very cytotoxic to the same cell lines (< or = 2 ng/ml). These results show that neither intact macrocyclic trichothecenes nor toxic metabolites could be detected in plant tissues after treatment with M. verrucaria spores. These results argue for both safety and efficacy for the use of M. verrucaria in biological control of kudzu and other noxious weeds, and support proceeding to animal feeding trials for further evaluation of safety.     

A comparison of the effect of three fungicides on growth and roridin E production by Myrothecium roridum

Three fungicides, chlorothalonil, dichloran and mancozeb were studied to determine the effects on growth and production of roridin E by Myrothecium roridum in vitro. With increasing concentrations of fungicides, both growth and production of roridin E were inhibited. Of the three fungicides, chlorothalonil was much more effective in the inhibition of growth and production of roridin E, than dichloran and mancozeb.     

A Colorimetric Technique for Detecting Trichothecenes and Assessing Relative Potencies

We tested a novel colorimetric toxicity test, based on inhibition of β-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B₁, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 μg/ml, nor could aflatoxins B₁ and M₁ be detected at concentrations up to 25 μg/ml. This test should be useful for trichothecene detection and for studies of relevant interactions—both between trichothecenes themselves and between trichothecenes and other food constituents.     

Trichothecenes produced by Stachybotrys atra from Eastern Europe

A total of 17 isolates of Stachybotrys atra isolated from various parts of Hungary and Czechoslovakia were grown on rice, and the toxin production of each isolate was analyzed by high-performance liquid chromatography. Of the 17 isolates, 14 produced macrocyclic trichothecenes (satratoxins F, G, and H, roridin E, and verrucarin J) as well as trichoverrols A and B. Most isolates produced satratoxins G and H in higher quantities than the other trichothecenes. The yield (in milligrams) of trichothecenes produced by one isolate grown on 800 g of rice was as follows: roridin E, 12; satratoxin F, 10; satratoxin G, 75; satratoxin H, 100; trichoverrol A, 15; and trichoverrol B, 30.     

Trichothecenes produced by Stachybotrys atra from Eastern Europe.

A total of 17 isolates of Stachybotrys atra isolated from various parts of Hungary and Czechoslovakia were grown on rice, and the toxin production of each isolate was analyzed by high-performance liquid chromatography. Of the 17 isolates, 14 produced macrocyclic trichothecenes (satratoxins F, G, and H, roridin E, and verrucarin J) as well as trichoverrols A and B. Most isolates produced satratoxins G and H in higher quantities than the other trichothecenes. The yield (in milligrams) of trichothecenes produced by one isolate grown on 800 g of rice was as follows: roridin E, 12; satratoxin F, 10; satratoxin G, 75; satratoxin H, 100; trichoverrol A, 15; and trichoverrol B, 30.     

Macrocyclic trichothecenes produced by Stachybotrys isolated from Egypt and Eastern Europe

Twenty seven isolates of Stachybotrys chartarum, S. albipes, S. kampalensis and S. microspora from Egypt and Eastern Europe were tested for production of macrocyclic trichothecenes. Twenty of the 27 isolates, grown on rice seeds, were toxic to brine shrimp larvae. Based on TLC and HPLC analyses, 5 macrocyclic trichothecenes (verrucarin J, roridin E, satratoxins F, G & H) as well as trichoverrols were identified. When grown in liquid culture on rice extract medium, only 3 isolates were toxic and produced verrucarin J, roridin E and satratoxins G & H. Extracts from mycelial mats were more toxic than culture filterates of two isolates grown on rice extract and both contained the same macrocyclic trichothecenes (285.5 mg/4 L), in addition to trichoverrols A & B (31 mg/4 L) found in mycelial mats only. When grown on 3% sucrose Czapek's medium supplemented with peptone and yeast extract (still cultures), all isolates were non-toxic to brine shrimp and no trichothecenes could be detected in the extracts.     

Candidusin A and B: New p-Terphenyls with Cytotoxic Effects on Sea Urchin Embryos

Three fungal trichothecenes, verrucarin A, roridin A and 8-β-hydroxyroridin E, were isolated as callus-initiating promoters from Myrothecium sp. 301. These trichothecenes promoted callus induction, synergistically coupled with a low concentration of 2,4-dichlorophenoxyacetic acid.     

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.     

Rhodotorula rubra bioassay for T-2 toxin: Increased sensitivity and wider application

Mutant strains of Rhodotorula rubra sensitive to 0.019–2.5 μg of T-2 toxin have been selected using ethylmethane sulphonate and ultraviolet light. When used as test organism in a bioassay for mycotoxins, these strains are also sensitive to trichothecin, diacetoxyscirphenol, verrucarin A and roridin A. A modification of the bioassay suitable for the rapid screening of large numbers of fungal strains without purification of the toxin is described.