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PubMed2002

Volatile metabolites produced by three strains of Stachybotrys chartarum cultivated on rice and gypsum board.

Stachybotrys chartarum (atra) is a toxigenic fungus frequently found in water-damaged buildings. Although microbial volatile organic compounds (MVOCs) produced by Aspergillus, Penicillium, and other fungi have been investigated extensively, little information exists on what MVOCs can be produced by S. chartarum. In this study, three strains of S. chartarum isolated from water-damaged residential homes in Cleveland, Ohio, were cultivated on rice and gypsum board. Air samples were collected after one, two, three, four, and six weeks of cultivation using Tenax TA tubes. Unique MVOCs were determined and other alcohols, ketones, and terpenes were also investigated using gas chromatography/mass spectrometry after thermal desorption from the sampling tube. Four unique MVOCs, 1-butanol, 3-methyl-1-butanol, 3-methyl-2-butanol, and thujopsene, were detected on rice cultures, and only one of them (1-butanol) was detected on gypsum board cultures. For a given strain, volatiles were considerably different with different cultivation media. Concentration profiles of the volatile compounds varied among compounds; however, each compound exhibited corresponding concentration trends between the strains. In comparison with our previous studies of five Aspergillus species on gypsum board under the same experimental conditions, fewer unique MVOCs were produced by S. chartarum, and they were quite different. It thus may be possible to use marker-unique MVOCs as a fingerprint to distinguish fungi in indoor environments once enough information becomes available. Our findings also indicate that volatiles produced by S. chartarum may represent a relatively small fraction of the total volatiles present in problem buildings where Aspergillus spp., Penicillium spp., and other fungi usually coexist.

Biomarkers↗
PubMed2003

Stachybotrys chartarum: cause of human disease or media darling?

This is a review of the literature of associations of the saprotrophic fungus Stachybotrys chartarum sensu lato with human and animal illnesses. This fungus grows on very wet cellulose-based building materials. S. chartarum has been the subject of considerable media attention because of temporal associations of exposure with unexpected and dramatic outcomes such as infant pulmonary hemosiderosis and neurocognitive damage. It is generally accepted that living or working in mouldy environments is associated with building related asthma, exacerbating asthma in mould-sensitive asthmatics and increased rates of upper respiratory disease. However, such relationships are with building-associated moulds, comprising many species that colonize wet or damp building materials, and are not specific to S. chartarum. There is limited evidence that severe lung damage can occur from building exposure to S. chartarum but possibly only under conditions of exposure that approach those associated with handling contaminated straw. There is no positive evidence in the literature to account for putative neurological damage resulting from exposure to this mould.

Allergens↗
PubMed2006

Comparison of inflammatory responses in mouse lungs exposed to atranones A and C from Stachybotrys chartarum.

Stachybotrys chartarum isolates can be separated into two distinct chemotypes based on the toxins they produce. One chemotype produces macrocyclic trichothecenes; the other produces atranones (and sometimes simple trichothecenes, e.g., trichodermol and trichodermin). Studies using in vivo models of lung disease revealed that exposure to spores of the atranone producing S. chartarum isolates led to a variety of immunotoxic, inflammatory, and other pathological changes. However, it is unclear from these studies what role the pure atranone toxins sequestered in spores of these isolates exert on lung disease onset. This study examined dose-response (0.2, 1.0, 2.0, 5.0, or 20 microg atranone/animal) and time-course (3, 6, 24, and 48 h postinstillation [PI]) relationships associated with inflammatory cell and proinflammatory chemokine/cytokine responses in mouse lungs intratracheally instilled with two pure atranones (either A or C) isolated from S. chartarum. High doses (2.0 to 20 microg toxin/animal) of atranone A and C induced significant inflammatory responses manifested as differentially elevated macrophage, neutrophil, macrophage inflammatory protein (MIP)-2, tumor necrosis factor (TNF) and interleukin (IL)-6 concentrations in the bronchioalveolar lavage fluid (BALF) of intratracheally exposed mice. Compared to controls, BALF macrophage and neutrophil numbers were increased to significant levels from 6 to 48 h (PI). Except for macrophage numbers in atranone A treatment animals, cells exhibited significant dose dependent-like responses. The chemokine/cytokine marker responses were significantly and dose-dependently increased from 3 to 24 h PI and declined to nonsignificant levels at 48 h PI. The results suggest not only that atranones are inflammatory but also that they exhibit different inflammatory potency with different toxicokinetics. Data also suggest that exposure to these toxins in spores of S. chartarum in contaminated building environments could contribute to inflammatory lung disease onset in susceptible individuals.

Animals↗
PubMed2005

Comparison of immunomodulator mRNA and protein expression in the lungs of Stachybotrys chartarum spore-exposed mice.

Stachybotrys chartarum is an important toxigenic fungus that has been associated with respiratory disease onset in animals and humans. It can be separated into macrocyclic trichothecene-producing and nonproducing chemotypes based on secondary metabolite production. However, effects of spores of the two chemotypes on lung inflammatory responses are poorly understood. In this study, real-time reverse-transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) were used to investigate time-course (1, 3, 6, 24, and 48 h post-instillation [PI]) relationships in mice intratracheally exposed to 300 spores/g body weight of a macrocyclic trichothecene-producing (JS 58-17) and a nonproducing (JS 58-06) S. chartarum isolate and of Cladosporium cladosporioides. There were marked differences in the magnitude and temporal patterns of mouse lung immune responses to intratracheal exposure to spores of these species at this spore dose. Both macrophage inflammatory protein 2 (MIP-2) and surfactant protein-D (SP-D) mRNA expression were significantly upregulated in lungs of JS 58-17-treated animals compared to that of all other treatment animals at 6 and 24 h PI. Heightened mRNA expression of these immunomodulators combined with comparatively depressed MIP-2 and tumor necrosis factor (TNF)-a protein expression suggests that the action of macrocyclic trichothecenes sequestered in 58-17 spores is involved. Interestingly, TNF-a protein expression in all spore treatment animal groups was also significantly increased over that in saline controls. Similarities in expression among all spore treatment animals suggest that chemicals other than toxic secondary metabolites, and possibly spore-sequestered 1,3-beta-D-glucan, may contribute to lung pathogenesis.

Animals↗
PubMed2002

An extract of Stachybotrys chartarum causes allergic asthma-like responses in a BALB/c mouse model.

Environmental exposure to Stachybotrys chartarum has been associated with multiple adverse health effects in humans. The goal of this study was to assess soluble components of this fungus for their ability to cause an asthma-like response in a BALB/c mouse model. Five isolates of S. chartarum were combined and extracted to form a crude antigen preparation (S. chartarum extract 1 [SCE-1]). Female BALB/c mice were sensitized by involuntary aspiration of SCE-1 and subsequently reexposed at 2, 3, and 4 weeks. To distinguish immune from nonspecific inflammatory effects, mice were exposed to 3 doses of Hanks' balanced salt solution (HBSS) and a final dose of SCE-1; or to 4 doses of bovine serum albumin (BSA) as a negative control protein. Serum and bronchoalveolar lavage fluid (BALF) were collected before the fourth aspiration (Day 0), and at Days 1, 3, and 7 following the final exposure, and lungs were fixed for histopathological examination. SCE-1-exposed mice displayed increased BALF total protein on Days 0, 1, and 3 and increased lactate dehydrogenase (LDH) at Days 1 and 3 only, compared to HBSS controls. BALF total cell numbers were elevated on each day, and differential counts of BALF cells showed neutrophilia on Day 1, marked eosinophilia on all days, and increased numbers of lymphocytes at Days 1, 3, and 7. Serum and BALF total IgE levels were elevated at all days, and BALF IL-5 levels were greatly increased (7-fold) on Day 1. Mice exposed to a single dose of SCE-1 exhibited inflammatory responses but not allergic responses, while BSA-treated mice showed neither inflammatory nor allergic responses. Histopathology confirmed the biochemical findings. Barometric whole-body plethysmography was performed 10 min prior to (baseline) and one h following each aspiration exposure in a second group of mice, to assess immediate respiratory responses. Airway hyperresponsiveness to increasing concentrations of nebulized methacholine (MCh) was assessed on Days 1 and 3 following the fourth aspiration exposure. Exposure to HBSS or BSA did not alter baseline enhanced pause (PenH) values or PenH following the aspiration exposures, nor did it cause an increase in airway responsiveness to MCh. Exposure to SCE-1 resulted in a 4.7-fold increase in PenH over baseline after the third exposure, increasing to 5.6-fold after the final exposure, and increased responsiveness to a 32 mg/ml MCh aerosol challenge. We conclude that multiple respiratory exposures to SCE-1 cause responses typical of allergic airway disease in this mouse model. However, BSA was nonallergenic and did not generate respiratory physiological responses when administered by aspiration.

Administration, Inhalation↗
PubMed2004

Comparison of inflammatory and cytotoxic lung responses in mice after intratracheal exposure to spores of two different Stachybotrys chartarum strains.

Stachybotrys chartarum is an important toxigenic fungus that has been associated with respiratory disease onset in animals and humans. While it can be separated into macrocyclic trichothecene- and atranone-producing chemotypes based on secondary metabolite production, effects of spores of the two chemotypes on lungs are poorly understood. In this study we used bronchoalveolar lavage fluid (BALF) to investigate dose-response (30, 300, 3000 spores/g body weight [BW]) and time-course (3, 6, 24, 48, 96 h post instillation [PI]) relationships in mice to exposure of macrocyclic trichothecene- (JS 58-17) and atranone-producing (JS 58-06) S. chartarum strains, as well as Cladosporium cladosporioides spores. BALF total protein, albumin, pro-inflammatory cytokine (IL-1beta, IL-6, and tumor necrosis factor-alpha [TNF-alpha]), and lactate dehydrogenase (LDH) concentrations showed significant (p < 0.05) fungal species (S. chartarum vs. C. cladosporioides) and strain (58-17 vs. 58-06), spore dose and time dependent changes. The no adverse effect level (NOAEL) due to exposure to spores of JS 58-17 and JS 58-06 was < 30 spores/g BW; for C. cladosporioides it was < 300 spores/g BW. At moderate and high S. chartarum doses, BALF composition reflects differences in strain toxicity while at the lowest dose, BALF composition of either S. chartarum strain were similar. This suggests that at low spore doses, it is spore sequestered factors common to both strains not strain dependent toxins that are contributing to lung disease onset.

Albumins↗
PubMed2004

Stachybotrys chartarum alters surfactant-related phospholipid synthesis and CTP:cholinephosphate cytidylyltransferase activity in isolated fetal rat type II cells.

Stachybotry chartarum, a fungal contaminant of water-damaged buildings commonly grows on damp cellulose-containing materials. It produces a complex array of mycotoxins. Their mechanisms of action on the pulmonary system are not entirely clear. Previous studies suggest spore products may depress formation of disaturated phosphatidylcholine (DSPC), the major surface-active component of pulmonary surfactant (PS). If S. chartarum can indeed affect formation of this phospholipid, then mold exposure may be a significant issue for pulmonary function in both mature lung and developing fetal lung. To address this possibility, fetal rat type II cells, the principal source of DSPC, were used to assess effects of S. chartarum extract on formation of DSPC. Isolated fetal rat lung type II cells prelabeled with 3H-choline and incubated with spore extract showed decreased incorporation of 3H-choline into DSPC. The activity of CTP:cholinephosphate cytidylyltransferase (CPCT), the rate-limiting enzyme in phosphatidylcholine synthesis was reduced by approximately 50% by a 1:10 dilution of spore extract. Two different S. chartarum extracts (isolates from S. chartarum (Cleveland) and S. chartarum (Hawaiian)) were used to compare activity of CPCT in the presence of phosphatidylglycerol (PG), a known activator. PG produced an approximate two-fold increase in CPCT activity. The spore isolate from Hawaii did not alter enzyme activity. S. chartarum (Cleveland) eliminated the PG-induced activation of CPCT. These results support previous observations that mold products alter PS metabolism and may pose a risk in developing lung, inhibiting surfactant synthesis. Different isolates of the same species of fungus are not equivalent in terms of potential exposure risks.

Animals↗
PubMed2005

Acute inflammatory responses to Stachybotrys chartarum in the lungs of infant rats: time course and possible mechanisms.

Stachybotrys chartarum has been linked to building-related respiratory problems including pulmonary hemorrhage in infants. The macrocyclic trichothecenes produced by S. chartarum have been the primary focus of many investigations. However, in addition to trichothecenes this fungus is capable of producing other secondary metabolites and a number of protein factors. This study examines the effects of intact, autoclaved, and ethanol-extracted spores on the lungs of infant rats as an approach to differentiate between secondary metabolites and protein factors. Seven-day-old infant rats were exposed intratracheally to 1 x 10(5) spores/g body weight (toxic strain JS58-17) and sacrificed at various times up to 72 h. The inflammatory response was measured by morphometric analysis of the lungs and determination of inflammatory cells and cytokine concentrations in bronchoalveolar lavage (BAL) fluid. Alveolar space was greatly reduced in animals exposed to fungal spores compared to phosphate buffered saline (PBS)-treated controls. The largest effects were observed in pups treated with intact spores where alveolar space 24 h after treatment was 42.1% compared to 56.8% for autoclaved spores, 51.1% for ethanol-extracted spores, and 60.6% for PBS-treated controls. The effects of different spore preparations on inflammatory cells, cytokine, and protein concentrations in the BAL fluid can be ranked as intact > autoclaved > extracted. Tumor necrosis factor alfa (TNF-alpha), interleukin 1-beta (IL-1beta), and neutrophils were the most sensitive indicators of inflammation. The difference between autoclaved (100% trichothecene toxicity, denatured/enzymatically inactive proteins) and intact (100% trichothecene activity, unaltered/released proteins) spores indicates the involvement of fungal proteins in the inflammatory response to S. chartarum and sheds new light on the clinical importance of "nontoxic" strains.

Animals↗
PubMed2003

Clinical implications of mycotoxins and Stachybotrys.

There has been considerable interest and concern in recent years regarding the potential health effects of mycotoxins in the indoor environment. Although the existence of mycotoxins has been known for several decades, relatively little is known about their effects in humans. What is known comes almost exclusively from studies of ingestion as the route of exposure. This review summarizes what is known regarding health effects of mycotoxins in general and specifically examines the evidence for the role of indoor exposure to the fungi of the genus Stachybotrys as a cause of disease in humans. Much work remains to be done in the area of mycotoxin research. The risk of health effects from ingestion seems much more widespread than from indoor airborne exposure, although the latter has received considerably more media attention. Rigorously controlled studies are needed to clarify these issues.

Aflatoxins↗
PubMed2004

Selective targeting of a laccase from Stachybotrys chartarum covalently linked to a carotenoid-binding peptide.

Atwo-step targeting strategy was used to identify improved laccases for bleaching carotenoid-containing stains on fabric. We first applied a modified phage display technique to identify peptide sequences capable of binding specifically to carotenoid stains and not to fabric. Prior deselection on the support on which the carotenoid was localized, increased stringency during the biopanning target selection process, and analysis of the phage peptides' binding to the target after acid elution and polymerase chain reaction (PCR) postacid elution, were used to isolate phage peptide libraries with increased binding selectivity and affinity. Peptide sequences were selected based on identified consensus motifs. We verified the enhanced carotenoid-binding properties of the peptide YGYLPSR and subsequently cloned and expressed C-terminal variants of laccase from Stachybotrys chartarum containing carotenoid-binding peptides YGYLPSR, IERSAPATAPPP, KASAPAL, CKASAPALC, and SLLNATK. These targeted peptide-laccase fusions demonstrate enhanced catalytic properties on stained fabrics.

Carotenoids↗
PubMed1979

Hydrolysis of aryl beta-D-glucopyranosides and beta-D-xylopyranosides by an induced beta-D-glucosidase from Stachybotrys atra.

The induced beta-D-glucosidase from Stachybotrys atra hydrolyzes aryl beta-D-glucopyranosides and aryl beta-D-xylopyranosides by the same basic two-step mechanism. In the first step the aglycon group is split of with simultaneous formation of an enzyme-glycosyl complex. In the second step this intermediate complex reacts with water yeilding beta-D-glucose or beta-D-xylose. For beta-D-xyloside hydrolysis each of the two steps is partially rate-controlling, whereas for beta-D-glucoside hydrolysis the second step is rate-limiting. The enzyme is inhibited by high concentrations of substrate and the exact rate-concentration equation is a second-order equation. 1-Thio-beta-D-glycopyranosides with an aromatic aglycon inhibit the reaction in both a competitive and non-competitive way. A tentative mechanism is proposed to explain all types of inhibition. In this mechanism substrates and inhibitors with an aromatic aglycon group bind through hydrophobic forces to the 'aglycon subsite' of the intermediate enzyme-glycosyl complex. Binding of the second substrate molecule or of the inhibitor to this complex does not prevent the reaction of the glycosyl moiety with water, it only decreases the rate of the second step.

Enzyme Induction↗
PubMed1998

Respiratory effects in mice exposed to airborne emissions from Stachybotrys chartarum and implications for risk assessment.

Stachybotrys chartarum, a mycotoxin producing mould found in some damp buildings, was grown in aluminum dishes in closed exposure chambers. The loading factor, 5.12 m2/m3, corresponded to 2.8 times the loading in a normal room with all surfaces covered by mould. Sensory irritation, bronchoconstriction and pulmonary irritation effects were investigated using a sensitive mouse bioassay in which the airway reactions were measured plethysmographically. Little effect was seen from the vapours in agreement with the predicted effects of the low concentrations of volatile organic compounds measured. Even under the influence of an airflow about four times that measured in normal buildings, the concentration of liberated spores and other particles was very low, corresponding to the biological effects observed, and probably reflecting the high water content of the substrate. These results demonstrate that many factors are important for the transport of biologically active mould metabolites from building material to occupants and that no direct relationship may exist between immediate biological effects and surface area covered with mould. Therefore, risk assessments should be based on estimated effects of emitted vapours, effects of liberated particles, e.g. sensitization potentials of the mould spores and effects of the generated metabolites (mycotoxins).

Air Microbiology↗
PubMed1981

Macrocyclic trichothecene toxins produced by a strain of Stachybotrys atra from Hungary.

A strain of Stachybotrys atra isolated from a field case of stachybotryotoxicosis in Hungary was cultured in Hungary. All of the compounds toxic to brine shrimp were separated from the culture extract by solvent partition, column chromatography, and preparative thin-layer chromatography. Two of the toxic compounds were identified as verrucarin J and satratoxin H by comparison with pure standards resolved by high-pressure liquid chromatography and characterized by mass spectrometry. Two other toxic components were identified as roriden E and satratoxin G on the basis of their mass spectra. The fifth toxic compound was identified as a macrocyclic trichothecene based on the following findings: a positive 4-(p-nitrobenzyl)pyridine color reaction, hydrolysis resulting in verrucarol verified by combined gas chromatography-mass spectrometry, and a characteristic trichothecene proton-nuclear magnetic resonance spectrum. This macrocyclic trichothecene has a molecular ion (528) identical to satratoxin H, and its mass spectrum is similar; however, its Rf value on Silica Gel G differs.

Chemical Phenomena↗
PubMed1986

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.

Chromatography, High Pressure Liquid↗
PubMed1987

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.

Aerosols↗
PubMed1998

Study of toxin production by isolates of Stachybotrys chartarum and Memnoniella echinata isolated during a study of pulmonary hemosiderosis in infants.

A cluster of cases of pulmonary hemosiderosis among infants was reported in Cleveland, Ohio, during 1993 and 1994. These unusual cases appeared only in infants ranging in age from 1 to 8 months and were characterized by pulmonary hemorrhage, which caused the babies to cough up blood. A case-control study identified major home water damage (from plumbing leaks, roof leaks, or flooding) as a risk factor for development of pulmonary hemorrhage in these infants. Because of an interest in the possibility that trichothecene mycotoxins might be involved in this illness, a number of isolates of Stachybotrys chartarum were grown in the laboratory on rice, and extracts were prepared and analyzed both for cytotoxicity and for specific toxins. Two isolates of Memnoniella echinata, a fungus closely related to S. chartarum, were also included in these studies. S. chartarum isolates collected from the homes were shown to produce a number of highly toxic compounds, and the profiles of toxic compounds from M. echinata were similar; the most notable difference was the fact that the principal metabolites produced by M. echinata were griseofulvins.

Animals↗
PubMed1999

Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains.

Stachybotrys chartarum is an indoor air, toxigenic fungus that has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio, area. In this study, 16 strains of S. chartarum isolated from case (n = 8) or control (n = 8) homes in Cleveland and 12 non-Cleveland strains from diverse geographic locations were analyzed for hemolytic activity, conidial toxicity, and randomly amplified polymorphic DNA banding patterns. In tests for hemolytic activity, strains were grown at 23 degrees C on wet wallboard pieces for an 8-week test period. Conidia from these wallboard pieces were subcultured on sheep's blood agar once a week over this period and examined for growth and clearing of the medium at 37 or 23 degrees C. Five of the Cleveland strains (all from case homes) showed hemolytic activity at 37 degrees C throughout the 8-week test compared to 3 of the non-Cleveland strains. Five of the Cleveland strains, compared to two of the non-Cleveland strains, produced highly toxic conidia (>90 microgram of T2 toxin equivalents per g [wet weight] of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of the 28 strains examined both were consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland where an infant had idiopathic pulmonary hemorrhage.

DNA, Bacterial↗