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 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.     

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.     

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.     

Detection of airborne Stachybotrys chartarum macrocyclic trichothecene mycotoxins on particulates smaller than conidia

Highly respirable particles (diameter, <1 microm) constitute the majority of particulate matter found in indoor air. It is hypothesized that these particles serve as carriers for toxic compounds, specifically the compounds produced by molds in water-damaged buildings. The presence of airborne Stachybotrys chartarum trichothecene mycotoxins on particles smaller than conidia (e.g., fungal fragments) was therefore investigated. Cellulose ceiling tiles with confluent Stachybotrys growth were placed in gas-drying containers through which filtered air was passed. Exiting particulates were collected by using a series of polycarbonate membrane filters with decreasing pore sizes. Scanning electron microscopy was employed to determine the presence of conidia on the filters. A competitive enzyme-linked immunosorbent assay (ELISA) specific for macrocyclic trichothecenes was used to analyze filter extracts. Cross-reactivity to various mycotoxins was examined to confirm the specificity. Statistically significant (P < 0.05) ELISA binding was observed primarily for macrocyclic trichothecenes at concentrations of 50 and 5 ng/ml and 500 pg/ml (58.4 to 83.5% inhibition). Of the remaining toxins tested, only verrucarol and diacetylverrucarol (nonmacrocyclic trichothecenes) demonstrated significant binding (18.2 and 51.7% inhibition, respectively) and then only at high concentrations. The results showed that extracts from conidium-free filters demonstrated statistically significant (P < 0.05) antibody binding that increased with sampling time (38.4 to 71.9% inhibition, representing a range of 0.5 to 4.0 ng/ml). High-performance liquid chromatography analysis suggested the presence of satratoxin H in conidium-free filter extracts. These data show that S. chartarum trichothecene mycotoxins can become airborne in association with intact conidia or smaller particles. These findings may have important implications for indoor air quality assessment.     

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.     

Production and characterization of a monoclonal antibody to the trichothecene mycotoxin diacetoxyscirpenol

A monoclonal antibody was obtained by the fusion of mouse myeloma cells with splenocytes isolated from Balb/c mice, which had been immunized with diacetoxyscirpenol-hemiglutarate (DAS-hemiglutarate) and verrucarol-hemiglutarates covalently bound to ethylenediamine-modified bovine serum albumin. The anti-DAS-antibody that could be induced was of the IgM type with kappa-chains. The titer of the monoclonal anti-DAS-antibody in ascites fluid obtained from mice injected the selected cell line was much higher than those of conventional antisera. An enzyme-linked immunosorbent assay based on the competitive binding principle in which the antibody was applied had a sensitivity of 1 ng DAS per assay. The relative cross-reactivity of the monoclonal antibody in the CI-ELISA with the related trichothecenes such as triacetoxyscirpenol, 15-monoacetoxyscirpenol, diacetylverrucarol, 4-monoacetoxyscirpenol and scirpentriol were found to be 1.8, 0.8, 0.15, 0.02 and less than 0.001, respectively. The trichothecenes verrucarol, T-2 toxin, T-2 tetraol, deoxynivalenol, 3-acetyldeoxynivalenol and trichothecin showed no cross-reactivity.     

Detection of Airborne Stachybotrys chartarum Macrocyclic Trichothecene Mycotoxins on Particulates Smaller than Conidia

Highly respirable particles (diameter, <1 μm) constitute the majority of particulate matter found in indoor air. It is hypothesized that these particles serve as carriers for toxic compounds, specifically the compounds produced by molds in water-damaged buildings. The presence of airborne Stachybotrys chartarum trichothecene mycotoxins on particles smaller than conidia (e.g., fungal fragments) was therefore investigated. Cellulose ceiling tiles with confluent Stachybotrys growth were placed in gas-drying containers through which filtered air was passed. Exiting particulates were collected by using a series of polycarbonate membrane filters with decreasing pore sizes. Scanning electron microscopy was employed to determine the presence of conidia on the filters. A competitive enzyme-linked immunosorbent assay (ELISA) specific for macrocyclic trichothecenes was used to analyze filter extracts. Cross-reactivity to various mycotoxins was examined to confirm the specificity. Statistically significant (P < 0.05) ELISA binding was observed primarily for macrocyclic trichothecenes at concentrations of 50 and 5 ng/ml and 500 pg/ml (58.4 to 83.5% inhibition). Of the remaining toxins tested, only verrucarol and diacetylverrucarol (nonmacrocyclic trichothecenes) demonstrated significant binding (18.2 and 51.7% inhibition, respectively) and then only at high concentrations. The results showed that extracts from conidium-free filters demonstrated statistically significant (P < 0.05) antibody binding that increased with sampling time (38.4 to 71.9% inhibition, representing a range of 0.5 to 4.0 ng/ml). High-performance liquid chromatography analysis suggested the presence of satratoxin H in conidium-free filter extracts. These data show that S. chartarum trichothecene mycotoxins can become airborne in association with intact conidia or smaller particles. These findings may have important implications for indoor air quality assessment.     

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 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.     

Preparation of antibodies to roridin A 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, 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 micrograms/kg.     

Macrocyclic trichothecene toxins produced by Stachybotrys atra strains isolated in Middle Europe.

A total of 17 strains of Stachybotrys atra isolated in Hungary and Czechoslovakia were cultured on Sabouraud agar, and the toxins produced by them were chemically analyzed by gas-liquid chromatography, high-pressure liquid chromatography, and mass spectroscopy. Furthermore, brine shrimp (Artemia salina) bioassay was used for the determination of toxicity of the compounds examined. Macrocyclic trichothecenes (satratoxins H and G, roridin E, and verrucarin J as well as two other unidentified macrocyclic trichothecenes) were found in all of the cultures tested. The identities of satratoxins H and G, roridin E, and verrucarin J were qualitatively determined by high-pressure liquid chromatography and gas-liquid chromatography. The ratio of satratoxins H and G and roridin E was found to be similar in each of the strains tested, but the amount of verrucarin J found was different in each of them. One of the unidentified macrocyclic trichothecenes was equivalent to the compound isolated by Harrach et al. (Harrach et al., Appl. Environ. Microbiol. 41:1428-1433, 1981). The other one proved to be a newly isolated macrocyclic trichothecene toxin. Stachybotryotoxicosis, one of the oldest mycotoxicoses known, and a serious problem in Middle Europe (Gy. Danko, Magy. Allatorv. Lapja 31:226-232, 1976), is believed to be caused by macrocyclic trichothecene toxins produced by Stachybotrys atra (R. M. Eppley, in Rodricks et al., ed., Mycotoxins in Human and Animal Health, p. 285-293, 1977). Forty years ago, the death of animals in the Soviet Union was associated with this fungus (C. U. Ruhliada, in Proceedings of the All-Union Sci. and Tech. Conf., p. 47-51, 1980).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra.

Stachybotrys atra is the etiologic agent of stachybotryotoxicosis, and this fungus and its trichothecene mycotoxins were recently implicated in an outbreak of unexplained illness in homes. S. atra was grown on sterile rice, autoclaved, dried, and then aerosolized by acoustic vibration. The distribution of particles (mass and number) was monitored on an aerodynamic particle sizer interfaced with a computer. Dust was collected on preweighed glass-fiber filters and extracted with 90% aqueous methanol. Extracts were tested for the ability to inhibit protein synthesis in rat alveolar macrophages, the ability to inhibit the proliferation of mouse thymocytes, and the presence of specific trichothecene mycotoxins. Virtually all of the particles were less than 15 micron in aerodynamic diameter, and the mass median diameter was 5 micron. Thus, most of the particles were respirable. Microscopic analysis of the generated dust revealed that ca. 85% of the dust particles were conidia of S. atra, another 6% were hyphal fragments, and the remainder of the particles were unidentifiable. Thus, greater than 90% of the particles were of fungal origin. The extracts strongly inhibited protein synthesis and thymocyte proliferation. Purified satratoxin H was also highly toxic in the same systems. Each of the individual filters contained satratoxin H (average, 9.5 ng/mg of dust). Satratoxin G and trichoverrols A and B were found in lesser amounts in some, but not all, of the filters. The limit of analysis is ca. 50 ng. These results establish that the conidia of S. atra contain trichothecene mycotoxins. In view of the potent toxicity of the trichothecenes, the inhalation of aerosols containing high concentrations of these conidia could be a potential hazard to health.     

Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra

Stachybotrys atra is the etiologic agent of stachybotryotoxicosis, and this fungus and its trichothecene mycotoxins were recently implicated in an outbreak of unexplained illness in homes. S. atra was grown on sterile rice, autoclaved, dried, and then aerosolized by acoustic vibration. The distribution of particles (mass and number) was monitored on an aerodynamic particle sizer interfaced with a computer. Dust was collected on preweighed glass-fiber filters and extracted with 90% aqueous methanol. Extracts were tested for the ability to inhibit protein synthesis in rat alveolar macrophages, the ability to inhibit the proliferation of mouse thymocytes, and the presence of specific trichothecene mycotoxins. Virtually all of the particles were less than 15 micron in aerodynamic diameter, and the mass median diameter was 5 micron. Thus, most of the particles were respirable. Microscopic analysis of the generated dust revealed that ca. 85% of the dust particles were conidia of S. atra, another 6% were hyphal fragments, and the remainder of the particles were unidentifiable. Thus, greater than 90% of the particles were of fungal origin. The extracts strongly inhibited protein synthesis and thymocyte proliferation. Purified satratoxin H was also highly toxic in the same systems. Each of the individual filters contained satratoxin H (average, 9.5 ng/mg of dust). Satratoxin G and trichoverrols A and B were found in lesser amounts in some, but not all, of the filters. The limit of analysis is ca. 50 ng. These results establish that the conidia of S. atra contain trichothecene mycotoxins. In view of the potent toxicity of the trichothecenes, the inhalation of aerosols containing high concentrations of these conidia could be a potential hazard to health.     

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.     

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.