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The widespread use of synthetic nematicides has caused significant problems to the environment as well as human health. To address this issue, eco-friendly control measures, such as microbial nematicides, are being developed. During the screening of Myrothecium strains with nematicidal activity against the root-knot nematode (RKN) Meloidogyne incognita, we found that the acetone extract of Myrothecium sp. KACC 40321 was highly effective against hatched juveniles of M. incognita at 7 days after exposure. The fungus was identified as Meloidogyne verrucaria. Two macrocyclic trichothecenes verrucarin A and roridin A were isolated and identified as major active metabolites by bioassay-guided fractionation and instrumental analysis. When the second-stage juveniles were treated with the chemicals, no juvenile mortality was observed. However, they effectively killed juveniles from treated eggs. The hatched juvenile mortality was used to evaluate the in vitro nematicidal activity of the compounds against M. incognita. The median effective concentrations were 1.88 μg/mL for verrucarin A and 1.50 μg/mL for roridin A. Among various liquid media, commercial malt extract broth (cMEB) was found to be the best for the production of verrucarin A and roridin A, followed by potato dextrose broth. The cMEB culture filtrate effectively reduced the formation of galls and egg masses on tomato roots in a pot experiment. In addition, the culture filtrate reduced the formation of galls on the roots of melon plants and the number of RKNs in the soils under field conditions. These results suggest that M. verrucaria KACC 40321 can be used as a biocontrol agent against RKNs in various crops. To the best of our knowledge, this is the first study to report the effectiveness of verrucarin A and rorridin A against hatched juveniles of M. incognita.

Verrucarin A2 (VerA) is a toxic trichothecene mycotoxin that can be produced indoors at very low level by moulds contaminating dwellings and may be associated with several human health problems. In this study we describe a spectroscopic label-free biosensor for VerA. This sensor is based on the high sensitivity of Fourier transform infrared-attenuated reflection (FTIR-ATR) spectroscopic detection and the use of a new anti-VerA rat monoclonal antibody (mAb). This antibody was directly grafted at the surface of the infrared element. Competitive ELISA and FTIR-ATR techniques were compared for detection of VerA in buffer and in complex dust samples obtained from dwellings. After optimization, the competitive ELISA showed a sensitivity of 7.43 ng/ml of VerA in PBS and a dynamic range below one order of magnitude. The FTIR technique improved the detection of the VerA by three orders of magnitude (2 pg/ml in buffer and 6 pg/ml when spiked in dust samples). The dynamic range for its detection extended over four orders of magnitude. The percentage of recovery of VerA spiked (1000 ng to 0.1 ng) in a complex dust matrix ranged from 99 to 68%. Our results clearly show that this antibody-based spectroscopic biosensor allow a better detection of VerA as compared to classical immunoassays and can be very efficiently used in the field of indoor mycotoxin detection.

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).

Fumonisin B1 (FB1) is the predominant member of a family of toxic metabolites produced by several species of Fusarium and is commonly found on corn. FB1 is a potent competitive inhibitor of ceramide synthase, which catalyzes the conversion of sphinganine and sphingosine to ceramide. The resultant accumulation of free sphingoid bases and the disruption of sphingolipid metabolism is believed to be the mechanism of toxicity of the fumonisins. The objectives of this study were to determine the relative potency of analogs of FB1 to inhibit ceramide synthase and to determine whether the inhibition is specific to mycotoxins with fumonisin-like structures. Fumonisins B1, B2, B3, B4, C4, and TA toxin (a structurally similar mycotoxin produced by the tomato pathogen, Alternaria alternata f. sp. lycopersici) were approximately equipotent inhibitors. Hydrolyzed fumonisins B1, B2, and B3, which lack the tricarballylic side chains, were only 30-40% as potent as the parent toxins. N-acetylated FB1 (FA1) did not block ceramide synthase, suggesting that FA1 is nontoxic. Inhibition of ceramide synthase by fumonisin analogs did not appear to be related to the lipophilicity of the compounds, as determined by computer estimation of log P values. The ability of relatively high (10 and 100 microm) doses of other mycotoxins that bear no structural similarity to fumonisins, including aflatoxin B1, cyclopiazonic acid, beauvericin, T-2 toxin, sterigmatocystin, luteoskyrin, verrucarin A, scirpentriol, and zearalenone, to block ceramide synthase was also determined. All of the toxins tested were negative in the bioassay with the exception of fumonisins, indicating that disruption of sphingolipid metabolism is a specific cytotoxic response.

The effects of trichothecene structure on cytokine secretion and gene expression were assessed in primary CD4 + T-cells from murine spleen. CD4 + T-cells were stimulated with concanavalin A (Con A) for 2 or 7 days in the presence of various concentrations of the trichothecenes, vomitoxin (VT or deoxynivalenol), nivalenol (NIV), 15-acetyl deoxynivalenol (15-ADON), 3-acetyl deoxynivalenol (3-ADON), T-2 toxin (T-2) and verrucarin A (Ver A). Culture supernatants were subsequently analyzed for interleukin (IL)-2, IL-4 and IL-5 by ELISA. At day 2, all trichothecenes were found to have inhibited production of IL-2, IL-4, and IL-S. However, at day 7, supernatant IL-2 was significantly increased (2–5.5-fold) in cultures containing VT, NIV, 3-ADON, and 15-ADON at 250, 250, 2500, and 1000 ng/ml doses, respectively, when compared to control Con A-stimulated cultures; significant increases in IL-2 were not observed with T-2 and Ver-A. Similarly, at day 7, IL-4 and IL-5 were significantly increased in the presence of VT (100 ng/ml), NIV (100 ng/ml), 3-ADON (1000 ng/ml), 15-ADON (500 ng/ml), T-2 (1 ng/ml), and Ver A (50 pg/ml, only IL-5) when compared to control cultures. IL production was inhibited at trichothecene concentrations exceeding the aforementioned optima. When total RNA of 2-day cultures was assessed by reverse transcriptase polymerase chain reaction (RT-PCR) in conjunction with Southern analysis, IL-2 mRNA was also found to be superinduced by VT (50 and 100 ng/ml), NIV (50, 100 and 250 ng/ml), 3-ADON (1500 ng/ml), 15-ADON (100 ng/ml), T-2 (0.5 ng/ml) and Ver A (25, 50 and 100 pg/ml); IL-4 mRNA by VT (50 ng/ml), NIV (50 ng/ml), and Ver A (25, 50 and 100 pg/ml); IL-5 mRNA by VT (50 ng/ml); and IL-6 mRNA by 15-ADON (100 ng/ml) and Ver A (50 pg/ml). As the trichothecene concentration increased from these levels, inhibition of mRNA transcript levels was also observed for many of the interleukins. Taken together, the results suggest that trichothecenes as a group can either inhibit or superinduce both IL secretion and mRNA levels in CD4 + T-cells. Superinduction exhibited a rank order of macrocyclic > type A > type B trichothecenes and was dependent on acylation of the trichothecene nucleus.

The uptake of the naturally occurring 3-alpha-hydroxy isomer of T-2 toxin (alpha-T-2 toxin) was investigated in a murine B cell hybridoma as a model for trichothecene-lymphocyte interactions. alpha-[3H]T-2 toxin was prepared by oxidation of T-2 toxin and reduction with [3H]NaBH4 followed by normal phase and reverse phase high-performance liquid chromatography. Uptake of alpha-[3H]T-2 toxin by hybridoma cells was both time- and concentration-dependent. The antibiotic anisomycin inhibited uptake of alpha-[3H]T-2 toxin by hybridoma cells, which suggests ribosomal involvement in the uptake mechanism. Uptake of alpha-[3H]T-2 toxin was also inhibited by verrucarin A, roridin A and deoxynivalenol, and the inhibition followed a trichothecene structure-activity rank similar to that established for protein synthesis inhibition and in vivo toxicity. The characteristics of uptake of alpha-[3H]T-2 toxin by isolated splenocytes were qualitatively similar to those of the hybridoma but accumulation at equilibrium was less. Accumulation of alpha-[3H]T-2 toxin by erythrocytes, cells lacking ribosomes, did not increase with time and was not affected by the presence of unlabelled toxin. The results suggested that specific accumulation and uptake of alpha-[3H]T-2 toxin by the murine B cell hybridoma and spleen cells were highly consistent with a model based on intracellular binding of T-2 toxin to ribosomes.

A spontaneous mutant of Saccharomyces cerevisiae resistant to trichodermin has been isolated. It displays cross resistance both in vivo and in vitro to a number of sesquiterpene antibiotics (fusarenon X, trichothecin and verrucarin A) and to the chemically unrelated antibiotic anisomycin. The mutation conferring resistance to anisomycin and trichodermin is expressed in the 60-S subunit of the yeast 80-S ribosome. Mutant ribosomes bind [ 14 C] trichodermin much less efficiently than wild type ribosomes, suggesting that resistance may be due, at least in part, to this property. However, both types of ribosomes bind [ 3 H] anisomycin equally. These results suggest that anisomycin and trichodermin have different binding sites on the 60-S subunit of eukaryotic ribosomes, even though previous results have shown that both antibiotics bind to mutually exclusive sites.

The biological properties of the macrocyclic trichothecenes roridin A, isororidin E, baccharinol B4, verrucarin A, verrucarin J and trichoverrin B were assayed with etiolated wheat coleoptiles ( Triticum aestivum L.), bean ( Phaseolus vulgaris L.), corn ( Zea mays L.) and tobacco ( Nicotiana tabacum L.). Metabolites were tested from 10 −3 to 10 −8 M in the coleoptile assay and from 10 −2 to 10 −4 M in intact plants. All inhibited coleoptile growth; verrucarins A and J, and trichoverrin B inhibited to 10 −7 M. All induced effects in plants that ranged from chlorosis, necrosis, malformations to stunting.

A disk diffusion type bioassay was developed for T-2 toxin using the yeast Kluyveromyces fragilis . The lower limit of detection for this in 0.2 μg of T-2 toxin. The growth of this yeast was sensitive to other trichothecenes such as verrucarin A (0.01 μg). Aflatoxin B 1 (50 μg) and zearalanone (20 μg) did not inhibit the growth of this yeast.

Verrucarin A (Ver A) and roridin A (Ror A), macrocyclic trichothecene mycotoxins, were examined for their immunomodulatory effects. Both mycotoxins were administered intraperitoneally at 0.35 mg/kg (1/2 the LD50) in CD-1 mice. Lymphocyte proliferation was studied on days 2, 4 and 7 after animals were dosed with Ver A. After day 2, no significant differences in [3H]thymidine (3H-TdR) incorporation were observed using concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) or lipopolysaccharide (LPS). On day 4 DNA synthesis induced by Con A, PHA and PWM was increased significantly. On day 7, PHA stimulation increased (p less than 0.001) above controls while Con A, PWM and LPS responses were not significantly different. The data indicated that Con A, PHA and PWM responses were time-dependent. Antibody production was evaluated by the hemolytic plaque assay; sheep red blood cells (SRBC) and Ver A were administered simultaneously, and Ver A was given 2 days after SRBC challenge. While spleen weights increased when Ver A was administered with SRBC or when the toxin was given 2 days after antigen, the antibody responses were not altered. In contrast, roridin A decreased PHA stimulation only on day 7 (p less than 0.05). There were no significant effects associated with the other mitogens. Antibody production did not differ significantly following administration of Ror A. Although Ver A and Ror A are equitoxic and structurally similar, their immunomodulatory properties for the given dose differ considerably. These different immunologic responses may be independent of other systemic toxic effects.

Trichothecenes are sesquiterpen mycotoxins characterized by the tetracyclic 12,13-epoxytrichothec-9-ene skeleton. We determined the effect of these mycotoxins on the growth and differentiation of the human acute promyelocytic leukemia (HL-60) cell line. Sixteen natural and semisynthetic trichothecenes were tested at concentrations of 0.2–60,000 ng/ml. The cytotoxicity exerted by these compounds varied: e. g., roridin A was found to be toxic at 1 ng/ml, whereas T-2 palmitate was not toxic even at 1 μg/ml. These compounds varied also in their potential to induce differentiation: 9,10-epoxy T-2 toxin and T-2 toxin induced differentiation at concentrations of 2–5 ng/ml, while 9,10-dihydro T-2 toxin was effective only at 100 ng/ml. Other trichothecenes (e.g., verrucarin A and verrucarol) did not induce differentiation at either subtoxic or toxic concentrations. Cell differentiation was always associated with cytotoxicity; optimal concentrations for induction of differentiation were usually 30–60% of the toxic concentrations. The HL-60 cell population was found to be heterogenous with respect to the ability to differentiate in response to trichothecenes, while in some clones up to 70% of the cells underwent differentiation, and other clones were completely resistant. The latter clones could, however, be induced to differentiate by other agents such as retinoic acid, dimethyl sulfoxide and 12-O-tetradecanoylphorbol-13-acetate. Some of the inducible clones differentiated into neutrophilic granulocytes while others into mature macrophages. Thus, a single trichothecene could induce differentiation into either cell types, depending on the clone used. This study presents a new group of differentiation inducers. Further investigation is required to evaluate their possible therapeutic application.

Three small grain cereals—barley, oats and wheat—were inoculated with six isolates of Myrothecium spp. and the amounts of roridin A and verrucarin A quantified. The highest levels of roridin A and verrucarin A were detected in oats and wheat, respectively, whilst the lowest level was reported in barley.

The purpose of this research was to determine the rate of cutaneous penetration and metabolism of [3H]diacetoxyscirpenol (DAS) and [3H]verrucarin A (VCA) and compare these values to previously determined values for [3H]T-2 toxin (T-2), to compare the cutaneous penetration and metabolism of DAS in human and guinea-pig skin, and to compare the effects of dose and of two vehicles, methanol and dimethylsulphoxide (DMSO), on penetration rates. DAS or VCA was applied to the epidermal surface of excised skin, and the receptor fluid bathing the dermal surface was sampled periodically for 48 hr. Whether the applied dose (581 ng/cm2) was dissolved in methanol or DMSO, the rate of penetration through human skin was lower for VCA than for DAS or T-2, the rates for the two latter compounds being similar at this dose. Metabolism of DAS occurred during penetration through excised human skin and did not occur in the receptor fluid as a result of enzymes leaching out of the skin. VCA appeared to be metabolized by human skin, but this conclusion is tentative because of the relative instability of this compound. DAS penetrated significantly (P less than 0.05) faster through excised guinea-pig skin than through human skin. Metabolism of DAS was greater in human skin than in guinea-pig skin. When compared with methanol, DMSO increased the penetration of DAS and VCA by factors of between 7 and 52. At the low dose (79 ng/cm2) DAS penetrated human and guinea-pig skin significantly (P less than 0.05) faster than T-2 using either vehicle.

W. L. Thompson and R. W. Wannemacher , Jr . Structure - function relationships of 12,13-epoxytrichothecene mycotoxins in cell culture: comparison to whole animal lethality. Toxicon24, 985 – 994, 1986. — Nineteen 12, 13-epoxytrichothecene mycotoxins were tested for their relative capabilities to inhibit protein synthesis in Vero cells and rat spleen lymphocytes. Although the lymphocytes were generally more sensitive to the mycotoxins, good correlation existed between the relative potencies of the various trichothecenes in the two cell systems. The most potent mycotoxins (T-2, verrucarin A and roridin A) have acetyl side groups on, or a hydrocarbon chain between, carbons 4 and 15 of the basic ring structure. Loss of side groups from either of these positions or an isovaleryl group at carbon 8 resulted in reduced protein synthesis inhibition (T-2 to HT-2, neosolaniol or diacetoxyscirpenol). Any combination of loss from all three positions (T-2 triol, T-2 tetraol, 15-monoacetyl DAS, scirpentriol, fusarenon X and deoxynivalenol) further weakens their effect. Reduction of the hydroxyl groups to hydroxides, forming verrucarol and deoxyverrucarol, reduced their effectiveness by over a thousand-fold compared to the most potent mycotoxins. Addition of side groups resulted in reduced effectiveness only when an acetyl group was added to the carbon 3 position of T-2 (acetyl T-2) and deoxynivalenol (3-acetyl deoxynivalenol) or on substitution of an epoxide across the 9,10 carbons of diacetoxyscirpenol (β-epoxide DAS). Effects of combining these and other mycotoxins were additive and showed no synergism or competition for binding to the active site. When in vitro effects of the mycotoxins were compared with results from whole animal lethality tests, several of the trichothecenes were weak inhibitors of protein synthesis in vitro but had in vivo toxicities similar to that of T-2 toxin. Thus, the in vitro cell response of a given trichothecene is not always an accurate predictor of toxicity in whole animals.

195 yeast strains, 74 strains of moulds and 20 strains of bacteria were screened for sensitivity to 1 μg ml −1 of T-2 toxin. Growth inhibition was assessed by measuring changes in impedance, using a Bactometer 32 impedimeter. Twelve (60%) bacteria, 20 (27%) moulds and 38 (19%) yeasts were found to be sensitive to T-2 toxin. Eight bacterial strains, eight moulds and 16 yeast strains were tested against four other trichothecene mycotoxins also at 1 μg ml −1 diacetoxyscirpenol (DAS); deoxynivalenol (DON); roridin A and verrucarin A. Sensitivity to these varied. Little response was obtained to DON. The macrocyclic trichothecenes (roridin A and verrucarin A) were markedly more toxic than the others to the fungi, but not to the bacteria.

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.

The mass spectra of verrucarin J and the trimethylsilyl derivatives of verrucarin A, roridins A and E, satratoxins G and H, and baccharin B5 have, for the first time, been obtained after sample introduction by gas chromatography (GC). This has been accomplished by the use of a short-length fused-silica capillary column with on-column injection. The macrocyclic trichothecenes studied are separable by GC except for verrucarin J, derivatized verrucarin A and derivatized roridin E. However, these materials are distinguishable by selected ion monitoring. The fragmentation pathways for each of the compounds studied have been partially elucidated. Except for roridin E and satratoxin G, the macrocyclic trichothecenes give usably intense electron impact ions at high mass range for selected ion monitoring at 1–10 ng.

Syntheses of the acyclic chains of verrucarin A and J are described; the revised stereochemistry of verrucarin J is confirmed.

We have studied the effects of a number of sesquiterpene antibiotics of the trichodermin group on different steps of the elongation phase in human tonsils and yeast cell-free systems. We have observed that these antibiotics are more active in the human tonsils than in the yeast cell-free systems. When a similar system is considered the antibiotic trichodermin is more active than its derived alcohol trichodermol. Sesquiterpene antibiotics of the trichodermin group do not affect non-enzymatic binding of Ac-Phe-tRNA and either enzymatic or non-enzymatic binding of Phe-tRNA to human tonsil ribosomes. These antibiotics are also without effect on translocation to the P site of Ac-Phe-tRNA bound to the ribosomal A site. All the sesquiterpene antibiotics of the trichodermin group tested (trichodermin, trichodermol, verrucarin A, fusarenon X) are very active inhibitors in the peptide bond formation step. This inhibitory effect of antibiotics of the trichodermin group was observed in the “puromycin reaction” and in the “fragment reaction” assays. Binding of the substrates CACCA-Leu-Ac and UACCA-Leu, respectively, to the donor and the acceptor sites of the peptidyltransferase centre was also studied in this work. We have observed in this system an inhibitory effect in binding of substrates to both the donor and the acceptor site of the peptidyltransferase centre by all the antibiotics of the trichodermin group.

Summary Twenty-four strains of Myrothccium roridum and M. verrucaria were isolatedfrom leaves of pasture plants. Cultures of 18 strains produced either A- or B-type cytotoxic effects on mammalian cells in tissue culture, 15 at dilutions of 10-4–10-7. A-type cytotoxic cultures were confined to M. roridum and produced no toxic effects in vivo. B-type cytotoxic cultures caused inflammation of rabbit eyes, acute disease and death in sheep and calves given large oral doses, and weight loss without obvious clinical signs in sheep given small daily doses. In the acute disease, severe haemorrhagic gastro-entero-colitis was the dominant lesion. Sheep surviving for several days after dosing showed extensive caseous necrosis and ulceration of the epithelium in the fore-stomachs. Symptoms and lesions in sheep dosed B-type cultures were similar to those in lambs given verrucarin A and roridin A isolated from 3 cultures of M. verrucaria. A culture of M. roridum was the most toxic of the B-type group, and no verrucarin A, roridin A or similar metabolites were isolated from it. Assay in vitro gave good agreement ivtih in vivo activity. Large doses of non-cytotoxic cultures had no detectable effects on sheep.

Using the paper-disk method with Chlorella-seeded agar plates, 15-acetoxyscirpenol, HT-2 toxin, acetyl T-2 toxin and neosolaniol inhibited growth at a concentration of 1 mg/ml, whereas verrucarol, T-2 tetraol, nivalenol, fusarenon-X, deoxynivalenol and 3-acetyldeoxynivalenol were inactive. Taking into account that verrucarin A, roridin A, T-2 toxin and diacetoxyscirpenol had previously been found to strongly inhibit Chlorella growth, esterification at R15 appears to be important for growth inhibitory activity. The most active agents are also esterified at R4. Inhibition of protein synthesis appears to be involved in the toxicity.