Macrocyclic Trichothecenes Research Articles

Evaluation of the antiviral activity against Junin virus of macrocyclic trichothecenes produced by the hypocrealean epibiont of Baccharis coridifolia.

Four macrocyclic trichothecenes, roridin A, roridin E, verrucarin A and verrucarin J, produced by the hypocrealean epibiont of Baccharis coridifolia, were evaluated for their inhibitory activity against the arenavirus Junin (JUNV), the etiological agent of Argentine hemorrhagic fever. The trichothecenes achieved a dose-dependent inhibition of JUNV multiplication at concentrations not affecting cell viability. The 50 % inhibitory concentration (IC50) values determined by a virus yield inhibition assay were in the range 1.2 - 4.9 ng/ml. The most active compound was verrucarin J which reduced JUNV yield more than 2 log units and had a similar effect against the arenavirus Tacaribe. The trichothecenes lacked virucidal effects on JUNV virions. From time of addition and removal experiments, it can be concluded that verrucarin J inhibited a late stage in the replicative cycle of JUNV, after 5 h of adsorption.

Cytotoxic macrocyclic trichothecenes from the mycelia of calcarisporium arbuscula preuss

A new cytotoxic macrocyclic trichothecene calcarisporin B1 ( 1 ), and two known compounds roridin H ( 2 ) and roridin J ( 3 ) were isolated from the cultured mycelia of Calcarisporium arbuscula Preuss. The structure of 1 was determined to be 8 f -acetoxy roridin H on the basis of spectral data. The cytotoxic activities of 1 - 3 were evaluated in vitro .

Phytotoxicity and mammalian cytotoxicity of macrocyclic trichothecene mycotoxins from Myrothecium verrucaria

Macrocyclic trichothecene toxins produced by Myrothecium verrucaria (a phytopathogen of interest in biological weed control) and the non-trichothecene toxin atranone B from Stachybotiys atra were tested for phytotoxicity in duckweed (Lemna pausicostata L.) plantlet cultures and kudzu (Pueraria lobata L.) leaf disc assays, and for mammalian cytotoxicity in four cultured cell lines. Roridin E and H, epi-isororidin E, and verrucarin A and J were phytotoxic (half-maximal effect in the concentration range 0.1–9.7 μM on duckweed and 1.5–>80 μM on kudzu) and cytotoxic to mammalian cell lines (half-maximal inhibition of proliferation in the concentration range 1–35 nM). Trichoverrins A and B and atranone B were moderately phytotoxic (half-maximal effect in the concentration range 1 9–69 μM on duckweed and 13–>80 μM on kudzu) and weakly cytotoxic with mammalian cell lines (half-maximal inhibition of proliferation in the concentration range 0.3–>2 μM).

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 (⩽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.

Possible sources of sick building syndrome in a Tennessee middle school.

Sick Building Syndrome has been reported with increasing frequency during recent years. Buildings that have sustained water damage harbor various molds, some of which may be toxic. Students and staff at Central Middle School in Murfreesboro, Tennessee, reported symptoms similar to those associated with Sick Building Syndrome. Upon investigation, investigators noted that a black fungal growth occurred throughout the building on wet cellulose ceiling tiles. Fungal growth of this type is consistent with the genus Stachybotrys. Stachybotrys spores contain macrocyclic trichothecenes, which may cause harm when inhaled or ingested. Bulk sampling of the black mold was initiated, and the samples were cultured in a moist chamber. Testing of the samples confirmed the presence of Stachybotrys spp., a finding that implies that air sampling procedures should ensue. Professional remediation of this potentially hazardous fungal contaminant is therefore recommended.

Toxic principles of a poisonous mushroom Podostroma cornu-damae

From the culture broth of a poisonous mushroom Podostroma cornu-damae, roridin E, verrucarin J, and satratoxin H were isolated. From the fruit bodies of the same fungus, satratoxin H, satratoxin H 12′,13′-diacetate, satratoxin H 12′-acetate, and satratoxin H 13′-acetate were isolated. All these macrocyclic trichothecenes except for verrucarin J had a lethal effect on mice by at least 0.5mg per capita.

Stachybotrys-Toxine in einer Münchner Wohnung mit Wasserschaden

This paper presents a case report of indoor Stachybotrys toxins in Germany. In the summer of 2000, heavy rainfall and a leakage of the gutter system of an appartement house in Munich, Germany, caused a severe water damage in one of the appartments. The appartment was at that time occupied by two of the authors (ES, EU). A wall (covered with wallpaper) in one room in particular was most strongly affected, about 6–8 m2 of the wallpaper became very wet. Large parts of this wet wallpaper were within days infested by multiple circumscribed round, grey-black colonies (0.5–5 cm in diameter) of presumtiveStachybotrys spp.. At the same time, the air in this room became very damp and oppressive. Several persons reported burning sensations on the mucous membranes of the eye, nose, and larynx, followed by headache symptoms, as soon as a few minutes after they had entered this room. Inadvertent skin contact with one of the fungal colonies by one person resulted in burning sensations immediately, but symptoms disappeared after thorough rinsing of the skin with water. The infested room was evacuated, and samples were taken from the wet wallpaper at areas (1) in the center of the most heavy fungal colony growth, (2) several cm away from colony growth, and (3) from locations which were wet but about 1 m away from visible colony growth. The samples were extracted with methanol, the extract diluted with buffer solution and analysed by competitive enzyme immunoassay (EIA) for roridin A. Due to cross-reactivity, this EIA detects several macrocyclic trichothecenes produced byStachybotrys spp.. Heavily infected pieces of wallpaper were strongly contaminated with toxins, with maximum concentrations of 1.1 microgram per cm2 (Roridin A equivalents). Wet but visibly not infected samples of wallpaper were negative in the EIA. Considering a EIA cross-reactivity of satratoxin H (the major Stachybotrys-toxin) of 15%, the actual maximum toxin concentration would correspond to 6 microgram of satratoxin H per cm2. As far as we know, this is the first documented case of indoor Stachybotrys toxins in Germany. The toxin concentration by far exceeds the minimum toxin level required for skin toxicity, and was considered as unhealthy. Health authorities in Munich were informed but were unable to provide assistance, probably because awareness of the importance and the possible health risks caused by indoor mycotoxins is not yet widespread in Germany. Reporting and monitoring programs for mycotoxins in water-damaged buildings seem to be necessary to provide insight into the occurrence of similar cases as the one described here.

A new macrocyclic trichothecene, 12,13-deoxyroridin E, produced by the marine-derived fungus Myrothecium roridum collected in Palau.

A new macrocyclic trichothecene, 12,13-deoxyroridin E (1), and three known compounds, roridin E (2), verrucarin A (3), and verrucarin J (4), were obtained as cytotoxic components from the marine-derived fungus Myrothecium roridum, isolated in Palau. 12,13-Deoxyroridin E is the second example of a macrocyclic trichothecene possessing a double bond at C-12-C-13 and was about 80-fold less cytotoxic than roridin E, the epoxide variant.

Mouldy lucerne hay suspected to cause bovine abortion

In a farm at the province of Jujuy (Argentina), a cattle ate some mouldy bales of lucerne hay and some cows aborted immediatly after. Considering that there could be mycotoxicosis present, some samples of lucerne hay were collected and the following fungi were isolated: Myrothecium verrucaria (29,2%), Eurotium amstelodami (28,8%), Aspergillus versicolor (19,5%), Alternaria alternata (10,1%), Aspegillus ochraceus (5,28%), Penicilium aurantiogriseum (1,64%), Aspergillus flavus (0,70%), Aspergillus niger (0,47%), and others (4,33%). Ochratoxin A and aflatoxin B were detected in the fodder yet there was absence of macrocyclic trichothecenes. When the animals stopped eating the contaminated fodder, ochratoxin a was detected in the milk of one cow.

Trichothecenes Production by the Hypocrealean Epibiont of Baccharis coridifolia

: 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).

Fast methods for screening of trichothecenes in fungal cultures using GC-MS/MS

Fast methods for screening for production of simple and macrocyclic trichothecenes, produced on solid substrates were developed. Crude extracts fromFusarium cultures grown on yeast extract sucrose agar (approx. 0.3 cm(2) culture) were derivatised using pentafluoropropionic anhydride and analysed by GC - tandem mass spectrometry using a Finnigan GCQ(+) ion trap. The MS was operated in the EI(+) multiscan mode allowing simultaneous full scan and MS/MS of 3-4 parent ions. Production of acetyl T-2 toxin (AT-2), T-2 toxin, HT-2 toxin (HT-2), T-2 triol (T-2TR), T-2 tetraol (T-2TE), neosolaniol (NEO), iso-neosolaniol (I-NEO), scripentriol (SCR), 4,15-diacetoxyscirpenol (DAS), 15-acetoxyscripentriol (15MAS), 4-acetoxyscripentriol (4MAS), nivalenol (NIV), fusarenon-X (F-X), deoxynivalenol (DON), 15-acetoxy-DON (15DON) and 3-acetoxy-DON (3DON) was studied for severalFusarium species. In hydrolysed crude extracts ofStachybotrys albipes, Trichoderma harzianum, andMemnoniella echinata trichodermol was detected, in cultures ofS. chartarum both verrucarol and trichodermol were detected as the heptafluorobuturyl esters after derivatisation with a imidazole based reagent.

Macrocyclic Trichothecene Production by the Fungus Epibiont of Baccharis Coridifolia

Cultures of the fungus epibiont from the herbaceous shrub B. coridifolia yielded four macrocyclic trichothecenes. As these toxins are the same as those found in B. coridifolia, the relationship between the plant and the epibiont must be considered as mutualistic.

Mycotoxins from mould infested building materials

Only limited documentation of non-allergenic, especially toxic reactions after inhalation of microfungal spores in water damaged buildings exists. Recently attention has been drawn to the mycotoxins as causal compounds, as some the dominating genera found in buildings are well known mycotoxin producers.Penicillium chrysogenum and A. ustus do not seem to produce any known mycotoxins when growing on building materials, whereasP. brevicompactum produces mycophenolic acid, someP. polonicum produces verrucosidin and verrucofortine,A. versicolor produces sterigmatocystins,A. niger produces nigragillin, orlandin, naphtho-γ-pyrones and tetracyclic compounds, someA. ochraceus produces ochratoxin A,Alternaria spp. produce alternariol and alternariol monomethyl ether,Chaetomium globosum produce chaetoglobosins, and finally 30-40% ofStachybotrys chartarum isolates from buildings produce macrocyclic trichothecenes and a number of other biologically active compounds.

Stereochemistry of the roridins. Diastereomers of roridin E.

A careful investigation of cultures of Myrothecium verrucaria has shown that this fungus produces all four roridin E isomers (3a-d), diastereomeric at the C-6' and C-13' centers. The stereochemistries at these centers were established by a combination of X-ray crystallographic analysis, NMR spectroscopy, and chemical transformations. NMR data from these and other macrocyclic trichothecenes allows for the assignment of configurations at the C-6' and C-13' centers for most of these compounds, the exceptions being those congeners having a C-4' ketone group in the macrolide ring.

EFFECTS OF SATRATOXINS AND OTHER MACROCYCLIC TRICHOTHECENES ON IL-2 PRODUCTION AND VIABILITY OF EL-4 THYMOMA CELLS

The macrocyclic trichothecenes are a group of potent protein synthesis inhibitors that have been encountered in indoor air and food as a result of infestation by the fungus Stachybotrys. To evaluate the capacity of these mycotoxins to alter immune functions, the effects of satratoxin G, H, F, roridin A, and verrucarin A on interleukin 2 (IL-2) production and viability were evaluated in a murine T-cell model. EL-4 thymoma cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin and concurrently exposed to various concentrations of the trichothecenes. Enzyme-linked immunosorbent assay (ELISA) of supernatants revealed that IL-2 concentrations at 24 and 72 h were significantly increased in cultures that were incubated in the presence of 0.5 to 1 ng/ml of satratoxin H, 1 to 5 ng/ml of isosatratoxin F, 0.1 to 0.5 ng/ml of roridin A, and 0.25 to 0.5 ng/ml of verrucarin A. However, IL-2 levels at these time points were significantly depressed when incubated in the presence of higher concentrations of satratoxin G ( 2.5 ng/ml), satratoxin H and isosatratoxin F ( 5 ng/ml), and roridin A and verrucarin A ( 1 ng/ ml). Cell viability, as measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, was depressed by each of the trichothecenes in a concentration-dependent manner. MTT responses were significantly decreased by as little as 0.5 ng/ml satratoxin G, roridin A, and verrucarin A and by 2.5 ng/ml of isosatratoxin F and satratoxin H. When these data were compared to those found in EL-4 cells for the 8-ketotrichothecene vomitoxin ( deoxynivalenol) , a common food contaminant, the macrocyclic trichothecenes were at least 100 times more potent. The results indicate that, at low concentrations, macrocyclic trichothecenes as a group could superinduce IL-2 production even while partially decreasing cell viability, whereas higher concentrations suppressed cytokine production and were markedly cytotoxic. The capacity of these compounds to dysregulate cytokine production in a biphasic fashion may play an etiologic role in outbreaks of human illnesses associated with indoor Stachybotrys contamination.

Characterization of a Transcriptional Activator Controlling Trichothecene Toxin Biosynthesis

Trichothecene biosynthetic pathway genes are localized within a gene cluster in Fusarium sporotrichioides and require the zinc-finger containing protein, TRI6, for expression. We show here that TRI6 is able to bind within the promoter regions of nine different pathway genes and that TRI6 binding is involved in pathway gene activation. TRI6 binding occurs at three distinct sites in the TRI5 promoter, all of which contain the sequence TNAGGCCT. DNA fragments from the promoter regions of six other pathway genes containing this sequence are also substrates for TRI6 binding. Specific nucleotide changes in the TNAGGCCT sequence dramatically reduced TRI6 binding. Analysis of TRI6 binding within the TRI3 and TRI11 promoters and the TRI4–TRI6 intergenic region which do not contain the TNAGGCCT motif suggests that the minimum sequence required for TRI6 binding is YNAGGCC. Two potential TRI6 binding sites, T4A and T4B, were identified within the intergenic region for the divergently transcribed TRI4 and TRI6 genes. Alteration or deletion of the T4A site resulted in the loss of nearly all in vitro TRI6 binding and was correlated with the loss of promoter activity in vivo as measured by the expression of mutant TRI4p/GUS fusions. This establishes a physiological role for TRI6 binding and demonstrates that TRI6 is directly involved in the regulation of pathway gene expression. To determine if a predicted Cys2His2 zinc-finger motif at the C-terminus of TRI6 is involved in DNA binding, a C187A mutant was constructed in TRI6 using site-directed mutagenesis. The C187A mutant did not bind promoter DNA fragments, supporting the role of C187 in DNA binding. In addition, a TRI6 homologue in the distantly related macrocyclic trichothecene pathway of Myrothecium roridum (MRTRI6) was also shown to bind to the same TRI5 and TRI4 promoter fragments bound by TRI6. Together, these data confirm our previous proposal that TRI6 is an activator of trichothecene pathway gene expression and that DNA binding employs the C-terminal region of TRI6 containing three predicted Cys2His2 zinc fingers.

Antimalarial activity of macrocyclic trichothecenes isolated from the fungus Myrothecium verrucaria.

Bioassay-guided fractionation of an extract from the fungus Myrothecium verrucaria BCC 112 resulted in the first isolation of roridin E acetate (5) from nature together with four common macrocyclic trichothecene isomers (1-4). Trichothecenes 1-5, while known as mycotoxins, were evaluated for their high antimalarial activity.

Production of trichothecene mycotoxins on water damaged gypsum boards in Danish buildings

Water damaged gypsum boards heavily infested with Stachybotrys chartarum were found in a school and in a domestic residence in Copenhagen. Samples were extracted with dichloromethane and cleaned up on Sep-Pak ® C 18 modules. The trichothecenes were detected as the heptafluorobutyrlated derivatives by gas chromatography ion trap mass spectrometry with negative ion chemical ionisation. Extracts of samples from both locations yielded verrucarol after hydrolysis, strongly indicating the presence of toxic macrocyclic trichothecenes, probably satratoxin H and G. In addition the sample from the domestic residence yielded trichodermol.

Intoxicação experimental por Baccharis coridifolia (Compositae) em bovinos

Treze terneiros receberam Baccharis coridifolia recém-colhida ou dessecada, em doses únicas que variaram entre 0,5 e 5g/kg. Um terneiro recebeu quatro administrações diárias de 0,5g/kg da planta dessecada. Dois terneiros não receberam a planta e serviram como controles. A planta foi colhida mensalmente de julho de 1991 a maio de 1992 e em março de 1993, e foi analisada para seu conteúdo em tricotecenos macrocíclicos. Quando em floração, as amostras dos espécimes macho e fêmea da planta foram analisadas e administradas aos terneiros separadamente. Os níveis de tricotecenos macrocíclicos e seus glicosídeos foram muito maiores nas plantas fêmeas em floração do que os níveis observados nas plantas não em floração e nas plantas machos em floração. Dez bovinos morreram espontaneamente ou foram sacrificados devido à toxicose. Dois terneiros que receberam a planta fêmea em floração, adoeceram e morreram, enquanto que três terneiros que receberam a planta macho em floração não adoeceram. Os sinais clínicos nos terneiros intoxicados foram anorexia, desidratação, atonia ruminal, tensão e dores abdominais, diarréia líquida, focinho seco, instabilidade dos membros posteriores e decúbito esternal. Os achados patológicos incluiram lesões necróticas no tubo gastrintestinal, particularmente nos pré-estômagos, e nos tecidos linfóides com exceção do timo. Esse último achado sugere um efeito sobre as células B do tecido linfóide. A distribuição das lesões nos sistemas digestivo e linfóide foi sistematicamente determinada. Conclui-se que os tricotecenos macrocíclicos presentes na planta são responsáveis pelas lesões observadas na toxicose e que a planta fêmea em floração é substâncialmente mais tóxica que a planta macho em floração ou das plantas que não estão em floração. Essa diferença em toxicidade é devida à diferença no conteúdo de tricotecenos macrocíclicos.

Detection of polar and macrocyclic trichothecene mycotoxins from indoor environments.

A method is described for the qualitative and semi-quantitative simultaneous determination of both non-macrocyclic and macrocyclic trichothecene biotoxins from samples derived from indoor environments. The method includes extraction, sample pre-treatment and reversed-phase HPLC separation followed by tandem mass spectrometric identification and quantification using electrospray ionization on a quadrupole ion trap mass analyser. Aqueous methanol was used in the initial extraction and solvent partitioning and solid-phase extraction in the purification of samples. The HPLC separation was run on-line with electrospray ionization MS-MS detection. The detection limits and recoveries of the procedure varied from 1 to 1000 pg and from 31 to 92%, respectively. As the method includes few and not very labour intensive sample treatment steps, it should allow for a high throughput of samples with good prospects of automation.