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Toxic Mold Research Articles

Discussion in 'Addressing Biotoxin, Chemical & Food Sensitivities' started by slayadragon, May 30, 2010.

  1. slayadragon

    slayadragon Senior Member

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    Per Gerwyn's suggestion, I'm in the process of putting together a comprehensive literature review on the effects of trichothecenes (a type of mold toxin made by Stachybotrys and certain other toxic molds) as they might relate to Chronic Fatigue Syndrome.

    There is a lot of this research, so the project is going to take a bit more time to finish.

    In the meantime, I'm going to post abstracts here.

    The ones in this post are related to oxidative stress, which many CFS doctors (e.g. Dr. Cheney) now believe are a core part of this disease.

    Best, Lisa

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    Markkanen Penttinen P, Pelkonen J, Tapanainen M, Mki-Paakkanen J, Jalava PI, Hirvonen MR. Co-cultivated damp building related microbes Streptomyces californicus and Stachybotrys chartarum induce immunotoxic and genotoxic responses via oxidative stress. Inhal Toxicol. 2009 Aug;21(10):857-67. PMID: 19459771

    Oxidative stress has been proposed to be one mechanism behind the adverse health outcomes associated with living in a damp indoor environment. In the present study, the capability of damp building-related microbes Streptomyces californicus and Stachybotrys chartarum to induce oxidative stress was evaluated in vitro. In addition, the role of oxidative stress in provoking the detected cytotoxic, genotoxic, and inflammatory responses was studied by inhibiting the production of reactive oxygen species (ROS) using N-acetyl-l-cysteine (NAC). RAW264.7 macrophages were exposed in a dose- and time-dependent manner to the spores of co-cultivated S. californicus and S. chartarum, to their separately cultivated spore-mixture, or to the spores of these microbes alone. The intracellular peroxide production and cytotoxicity were measured by flow cytometric analysis, nitric oxide production was analyzed by the Griess method, DNA damage was determined by the comet assay, and cytokine production was measured by an immunochemical ELISA (enzyme-linked immunosorbent assay). All the studied microbial exposures triggered oxidative stress and subsequent cellular damage in RAW264.7 macrophages. The ROS scavenger, NAC, prevented growth arrest, apoptosis, DNA damage, and cytokine production induced by the co-culture since it reduced the intracellular level of ROS within macrophages. In contrast, the DNA damage and cell cycle arrest induced by the spores of S. californicus alone could not be prevented by NAC. Bioaerosol-induced oxidative stress in macrophages may be an important mechanism behind the frequent respiratory symptoms and diseases suffered by residents of moisture damaged buildings. Furthermore, microbial interactions during co-cultivation stimulate the production of highly toxic compound(s) which may significantly increase oxidative damage.

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    Wang H, Yadav JS. DNA damage, redox changes, and associated stress-inducible signaling events underlying the apoptosis and cytotoxicity in murine alveolar macrophage cell line MH-S by methanol-extracted Stachybotrys chartarum toxins. Toxicol Appl Pharmacol. 2006 Aug 1;214(3):297-308. PMID: 16476459

    Spore-extracted toxins of the indoor mold Stachybotrys chartarum (SC) caused cytotoxicity (release of lactate dehydrogenase), inhibition of cell proliferation, and cell death in murine alveolar macrophage cell line MH-S in a dose- and time-dependent manner.

    The apoptotic dose of SC toxins did not induce detectable nitric oxide and pro-inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) but showed exacerbated cytotoxicity in presence of a non-apoptotic dose of the known pro-inflammatory agent LPS (10 ng/ml).

    Intracellular reduced glutathione (GSH) level showed a significant decrease beginning at 9 h of the toxin treatment whereas oxidized glutathione (GSSG) showed a corresponding significant increase, indicating a delayed onset of oxidative stress in the apoptosis process.

    The toxin-treated macrophages accumulated p53, an indicator of DNA damage response, and showed activation of the stress-inducible MAP kinases, JNK, and p38

    Chemical blocking of either p38 or p53 inhibited in part the SC toxin-induced apoptosis whereas blocking of JNK did not show any such effect.

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    Wang H, Yadav JS. Global gene expression changes underlying Stachybotrys chartarum toxin-induced apoptosis in murine alveolar macrophages: evidence of multiple signal transduction pathways. Apoptosis. 2007 Mar;12(3):535-48. PMID: 17186382

    The overall mechanism(s) underlying macrophage apoptosis caused by the toxins of the indoor mold Stachybotrys chartarum (SC) are not yet understood.

    The toxin-regulated genes corresponded to multiple cellular processes, including cell growth, proliferation and death, inflammatory/immune response, genotoxic stress and oxidative stress, and to the underlying multiple signal transduction pathways involving MAPK-, NF-kB-, TNF-, and p53-mediated signaling.

    Transcription factor NF-kB showed dynamic temporal changes, characterized by an initial activation and a subsequent inhibition.

    Up-regulation of a battery of DNA damage-responsive and DNA repair genes in the early stage of the treatment suggested a possible role of genotoxic stress in the initiation of apoptosis.

    Simultaneous expression changes in both pro-survival genes and pro-apoptotic genes indicated the role of a critical balance between the two processes in SC toxin-induced apoptosis.

    Taken together, the results imply that multiple signaling pathways underlie the SC toxin-induced apoptosis in alveolar macrophages.

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    Rakkestad KE, Skaar I, Ansteinsson VE, Solhaug A, Holme JA, Pestka JJ, Samuelsen JT, Dahlman HJ, Hongslo JK, Becher R. DNA damage and DNA damage responses in THP-1 monocytes after exposure to spores of either Stachybotrys chartarum or Aspergillus versicolor or to T-2 toxin. Toxicol Sci. 2010 May;115(1):140-55. PMID: 20150440

    We have characterized cell death in THP-1 cells after exposure to heat-treated spores from satratoxin G-producing Stachybotrys chartarum isolate IBT 9631, atranone-producing S. chartarum isolate IBT 9634, and sterigmatocystin-producing Aspergillus versicolor isolate IBT 3781, as well as the trichothecenes T-2 and satratoxin G. Spores induced cell death within 3-6 h, with Stachybotrys appearing most potent.

    IBT 9631 induced both apoptosis and necrosis, while IBT 9634 and IBT 3781 induced mostly necrosis. T-2 toxin and satratoxin G caused mainly apoptosis.

    Comet assay +/- formamidopyrimidine DNA glycosylase showed that only the spore exposures induced early (3h) oxidative DNA damage. Likewise, only the spores increased the formation of reactive oxygen species (ROS), suggesting that spores as particles may induce ROS formation and oxidative DNA damage.

    In conclusion, activation of Chk2 and H2AX correlated with spore- and toxin-induced apoptosis. For IBT 9631 and satratoxin G, additional factors may be involved in triggering apoptosis, most notably p38 activation.

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    Chaudhari M, Jayaraj R, Santhosh SR, Rao PV. Oxidative damage and gene expression profile of antioxidant enzymes after T-2 toxin exposure in mice. J Biochem Mol Toxicol. 2009 May;23(3):212-21. PMID: 19526462.


    T-2 toxin showed significant alterations in hepatic alanine amino transferase, aspartate amino transferase, and lactate dehydrogenase.

    Significant changes in hepatic lipid peroxidation, depletion of glutathione (GSH), and expression of heat shock protein-70 indicated oxidative damage.

    We also evaluated the activity of antioxidant enzymes and compared the gene expression profile by quantitative real-time reverse transcriptase-polymerase chain reaction.

    Except for glutathione reductase (GR), there was a significant increase in activity of glutathione-S-transferase (GST), glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase at 1 LD50 dose.

    At 2 LD50 dose, SOD showed decrease in activity, whereas GST, GPx, and catalase showed significant increase.

    In contrast, gene expression profile showed downregulation in GR, GPx, GST, and catalase at 1 LD50 dose. At 2 LD50 dose except GSH synthetase, all other genes were downregulated.

    The results clearly show oxidative stress as one of the mechanisms of T-2 toxin-mediated toxicity.

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    Zhang X, Jiang L, Geng C, Cao J, Zhong L. The role of oxidative stress in deoxynivalenol-induced DNA damage in HepG2 cells. Toxicon. 2009 Sep 15;54(4):513-8. PMID: 19486909.

    The aim of this study was to assess the role of oxidative stress in deoxynivalenol-induced DNA damage, using human hepatoma HepG2 cells.

    In order to clarify the underlying mechanisms, we evaluated the level of reactive oxygen species (ROS) production with the 2,7-dichlorofluorescein diacetate (DCFH-DA) assay. Significant increase in the level of ROS was observed in HepG2 cells at a higher concentration (60 microM). The involvement of lipid peroxidation in the DNA damage of DON was confirmed by using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS), the doses being 7.5-60 microM and 3.75-15 microM, respectively. These results indicate that the DNA damage induced by DON in HepG2 cells is probably related to the oxidative stress.

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    Borutova R, Faix S, Placha I, Gresakova L, Cobanova K, Leng L. Effects of deoxynivalenol and zearalenone on oxidative stress and blood phagocytic activity in broilers. Arch Anim Nutr. 2008 Aug;62(4):303-12. PMID: 18763624


    Then chickens of Group 1 received a diet contaminated with DON and ZEA, both being 3.4 mg kg(-1), while Group 2 received DON and ZEA at 8.2 and 8.3 mg kg(-1), respectively.

    Intake of both contaminated diets resulted in a significantly decreased activity of glutathione peroxidase (GPx) and increased level of malondialdehyde (MDA) in liver tissue, while in kidneys the concentration of MDA was significantly increased only in Group 1. On the other hand, activities of blood GPx and plasma gamma-glutamyltransferase (GGT) were elevated in Group 2 only. Activities of thioredoxin reductase in liver and GPx in duodenal mucosa tissues, superoxide dismutase (SOD) in erythrocytes as well as levels of MDA in duodenal mucosa and alpha-tocopherol in plasma were not affected by dietary mycotoxins.

    Blood phagocytic activity was significantly depressed in Group 1 and 2.

    These results demonstrate that diets contaminated with DON and ZEA at medium levels are already able to induce oxidative stress and compromise the blood phagocytic activity in fattening chickens.


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    Kouadio JH, Mobio TA, Baudrimont I, Moukha S, Dano SD, Creppy EE. Comparative study of cytotoxicity and oxidative stress induced by deoxynivalenol, zearalenone or fumonisin B1 in human intestinal cell line Caco-2. Toxicology. 2005 Sep 15;213(1-2):56-65. PMID: 16019124

    Fusarium toxins such as, deoxynivalenol (DON), zearalenone (ZEN) and fumonisin B1 (FB1) have been shown to cause diverse toxic effects in animals and also suspected of disease causation in humans. From the literature and mechanistic point of view, DON binds to the ribosomal peptidyl-transferase and inhibits protein synthesis specifically and DNA synthesis consequently. ZEN known to be genotoxic, binds to 17-beta-estradiol receptors, induces lipid peroxidation, cell death and inhibits protein and DNA synthesis. FB1 disrupts sphingolipid metabolism, induces lipid peroxidation altering the cell membrane and causing cell death.

    We intended to compare DON, ZEN and FB1 (1-150 microM) cytotoxic effect and the pathways leading to cell death and related to oxidative stress and macromolecules syntheses in a human intestinal cell line in order to tentatively classify them according to their respective potential toxicity.

    The comparison reveals that all three mycotoxins bear, at variable degree, the capability of inducing lipid peroxidation (MDA production) and could be classified above 10 microM in decreasing potency order FB1>DON>ZEN.

    This effect seems to be related to their common target that is the mitochondria as revealed by MTT test and seemingly not related to sphingoids accumulation concerning FB1.

    DON and ZEN also adversely affect lysosomes in contrast to FB1.

    The three mycotoxins inhibit protein synthesis with respective IC50 of 5, 8.8 and 19 microM for DON, FB1 and ZEN confirming that protein synthesis is a specific target of DON.

    DNA synthesis is inhibited by DON, ZEN and FB1 with respective IC50 of 1.7, 10 and 20 microM. However at higher concentrations DNA synthesis seems to be restored for FB1 and DON suggesting a promoter activity.

    Altogether the potency of the three mycotoxins in macromolecules inhibition is DON>ZEN>FB1 in Caco-2 cells. It appears then that FB1 acts rather through lipid peroxidation while DON affects rather DNA and protein synthesis.


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    Nusuetrong P, Pengsuparp T, Meksuriyen D, Tanitsu M, Kikuchi H, Mizugaki M, Shimazu K, Oshima Y, Nakahata N, Yoshida M. Satratoxin H generates reactive oxygen species and lipid peroxides in PC12 cells. Biol Pharm Bull. 2008 Jun;31(6):1115-20. PMID: 18520041

    Satratoxin H, a mycotoxin, is thought to induce apoptosis of PC12 cells through the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in a glutathione (GSH)-sensitive manner.

    Satratoxin H increased reactive oxygen species (ROS) production and lipid peroxidation, as determined by malondialdehyde formation. These effects were attenuated by incubation of cells with GSH, suggesting that satratoxin H-induced increase in apoptosis of serum-deprived PC12 cells may be partially mediated through the generation of ROS.

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    Kovcikov Z, Ttrai E, Pieckov E, Tulinsk J, Pivovarov Z, Matausic-Pisl M, Kuricov M, Wsolov L. An in vitro study of the toxic effects of Stachybotrys chartarum metabolites on lung cells. Altern Lab Anim. 2007 Mar;35(1):47-52. PMID: 17411351

    During a study of indoor fungal colonisation, several isolates of Stachybotrys chartarum were recovered, and the effects of metabolites from four isolates on lung epithelial Type II cells and alveolar macrophages were studied in vitro. All the isolates showed toxic effects on both cell types,

    In Type II cells, the number of alkaline phosphatase positive cells was reduced, the pattern of Maclura pomifera agglutinin (MPA) binding was changed, and acid phosphatase activity in alveolar macrophages was diminished. In both cell types, the production of monocyte chemotactic protein-1 (MCP-1) and tumour necrosis factor-alpha (TNF-alpha) was changed, and parameters related to antioxidant status (superoxide dismutase, glutathione peroxidase, glutathione) were decreased.


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    Costa S, Schwaiger S, Cervellati R, Stuppner H, Speroni E, Guerra MC. In vitro evaluation of the chemoprotective action mechanisms of leontopodic acid against aflatoxin B1 and deoxynivalenol-induced cell damage. J Appl Toxicol. 2009 Jan;29(1):7-14. PMID: 18636399


    Several in vitro studies showed that free radical scavengers possess chemopreventive properties against mycotoxin-induced cell damage which are at least partially associated with the induction of phase II detoxifying enzymes and antioxidant enzymes like glutathione S-transferase (GST) and glutathione peroxidase (GPx).

    The aim of this project was to study the chemopreventive effects of leontopodic acid (LA), a potent natural occurring free radical scavenger isolated from the aerial parts of Leontopodium alpinum.

    Cell viability and reactive oxygen species concentration were determined, and the effects of pre-treatment with LA on these parameters were investigated together with the GST and GPx activity as well as the concentration of reduced glutathione.


    We hypothesize that the increase in detoxifying enzymes is probably the main mechanism of antioxidant mediated chemoprevention.


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    Rezar V, Frankic T, Narat M, Levart A, Salobir J. Dose-dependent effects of T-2 toxin on performance, lipid peroxidation, and genotoxicity in broiler chickens. Poult Sci. 2007 Jun;86(6):1155-60. PMID: 17495086

    The results of the present study indicate that impaired performance, DNA fragmentation in spleen leukocytes, and elevated serum IgA levels induced by T-2 toxin are dose-dependent.

    Based on our results, we could not confirm the hypothesis that oxidative stress is among the mechanisms by which T-2 toxin induces DNA fragmentation.


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    Dvorska JE, Pappas AC, Karadas F, Speake BK, Surai PF. Protective effect of modified glucomannans and organic selenium against antioxidant depletion in the chicken liver due to T-2 toxin-contaminated feed consumption. PMID: 17350343

    The aim of this work was to assess the effect of T-2 toxin on the antioxidant status of the chicken and to study possible protective effects of modified glucomannan (Mycosorb) and organic selenium (Sel-Plex).

    Inclusion of T-2 toxin in the chickens' diet (8.1 mg/kg for 21 days) was associated with significant decreases in the concentrations of selenium (Se)(by 32.2%), alpha-tocopherol (by 41.4%), total carotenoids (by 56.5%), ascorbic acid (by 43.5%) and reduced glutathione (by 56.3%) in the liver, as well as a decrease in the hepatic activity of Se-dependent glutathione peroxidase (Se-GSH-Px) (by 36.8%).

    However, inclusion of modified glucomannans into the T-2 toxin-contaminated diet provided a partial protection against the detrimental effects of the mycotoxin on the antioxidant defences in the chicken liver.

    *


    Nusuetrong P, Yoshida M, Tanitsu MA, Kikuchi H, Mizugaki M, Shimazu K, Pengsuparp T, Meksuriyen D, Oshima Y, Nakahata N. Involvement of reactive oxygen species and stress-activated MAPKs in satratoxin H-induced apoptosis. Eur J Pharmacol. 2005 Jan 10;507(1-3):239-46. PMID: 15659314

    Satratoxin H caused cytotoxicity, which was reflected from apoptosis determined by chromatin staining and flow cytometry. Satratoxin H stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Pre-incubation with SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor, but not PD98059, an ERK inhibitor, reduced satratoxin-induced cytotoxicity.

    Co-incubation of cells with glutathione, N-acetyl-L-cysteine or glutathione reductase inhibited cytotoxicity and the phosphorylation of p38 MAPK induced by satratoxin H.

    Our data suggest that satratoxin H-induced apoptosis in PC12 cells is dependent on the activation of p38 MAPK/JNK and the increase in reactive oxygen species.


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    Meissonnier GM, Laffitte J, Raymond I, Benoit E, Cossalter AM, Pinton P, Bertin G, Oswald IP, Galtier P. Subclinical doses of T-2 toxin impair acquired immune response and liver cytochrome P450 in pigs. Toxicology. 2008 May 2;247(1):46-54. PMID: 18355953

    This study was designed to investigate the effect of subclinical doses of T-2 toxin on liver drug-metabolizing enzymes and the immune response.

    Pigs fed 1324 or 2102microg T-2toxin/kg feed exhibited reduced anti-ovalbumin antibody production without significant alteration to specific lymphocyte proliferation.

    The livers of pigs exposed to T-2 toxin presented normal cytochrome P450 content, UGT 1A and P450 2B, 2C or 3A protein expression, and glutathione- and UDP glucuronosyl-transferase activities.

    However, P450 1A related activities (ethoxyresorufin O-deethylation and benzo-(a)-pyrene hydroxylation) were reduced for all pigs given T-2 toxin, with P450 1A protein expression decreased in pigs fed the highest dose. In addition T-2 toxin exposure reduced certain N-demethylase activities.

    The results of this study confirm the immunotoxic properties of T-2 toxin, in particular toward the humoral immune response.

    The reduction of monooxygenase activities, even though the liver presented no tissue lesion or lipid peroxidation, suggests possible deleterious interactions of T-2 toxin with these enzymes.
  2. slayadragon

    slayadragon Senior Member

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    Mold and Intestinal Cells

    Here are some articles detailing some effects of trichothecenes on intestinal cells.

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    Yang H, Park SH, Choi HJ, Moon Y. Epithelial cell survival by activating transcription factor 3 (ATF3) in response to chemical ribosome-inactivating stress. Biochem Pharmacol. 2009 Mar 15;77(6):1105-15. PMID: 19101521


    Ribotoxic stress responses lead to the expression of genes important for cellular homeostasis by modulating cell survival, proliferation and differentiation.

    ATF3 expression was up-regulated by chemical agents causing ribotoxic stress such as deoxynivalenol and anisomycin in different types of intestinal epithelial cells.

    Moreover, reduction of ATF3 gene expression promoted ribotoxic stress-triggered programmed cell death, implicating a protective role of ATF3 in epithelial cell survival.

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    Van De Walle J, Romier B, Larondelle Y, Schneider YJ. Influence of deoxynivalenol on NF-kappaB activation and IL-8 secretion in human intestinal Caco-2 cells. Toxicol Lett. 2008 Apr 1;177(3):205-14. PMID: 18343055


    We hypothesized a link between DON ingestion and intestinal inflammation, and used Caco-2 cells to assess the effects of DON, at plausible intestinal concentrations (250-10,000 ng/ml), on inflammatory mediators acting downstream the MAPKs cascade i.e. activation of nuclear factor-kappaB (NF-kappaB) and interleukin-8 (IL-8) secretion.

    Dose-dependent increases in NF-kappaB activity and IL-8 secretion were observed,

    Phosphorylation of inhibitor-kappaB (IkappaB) increased (1.6-fold) at DON levels <0.5 microg/ml.

    Exposure of Caco-2 cells to pro-inflammatory agents, i.e. 25 ng/ml interleukin-1beta, 100 ng/ml tumor necrosis factor-alpha or 10 microg/ml lipopolysaccharides, activated NF-kappaB and increased IL-8 secretion.

    Synergistic interactions between these stimuli and DON were observed.

    These data show that DON induces NF-kappaB activation and IL-8 secretion dose-dependently in Caco-2 cells, and this effect was accentuated upon pro-inflammatory stimulation, suggesting DON exposure could cause or exacerbate intestinal inflammation.

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    Kouadio JH, Dano SD, Moukha S, Mobio TA, Creppy EE. Effects of combinations of Fusarium mycotoxins on the inhibition of macromolecular synthesis, malondialdehyde levels, DNA methylation and fragmentation, and viability in Caco-2 cells. Toxicon. 2007 Mar 1;49(3):306-17. PMID: 17109910.

    We studied the interactive effects of either binary or tertiary mixtures of Fusarium mycotoxins, deoxynivalenol (DON), zearalenone (ZEA), and fumonisin B1 (FB1) on the human intestinal cell line, Caco-2,

    Because FB1 antagonizes the effects of estrogenic Zearalenone, FB1 was assayed against estradiol.

    In NR assay, mixture of FB1 and estradiol and/or ZEA improves Caco-2 cells viability in contrast to individual effects. Mixtures of ZEA or FB1 and DON, display synergistic effects in lipid peroxidation.

    Altogether, the data indicate that mixtures of Fusarium toxins are able to induce lipid peroxidation, DNA damage, DNA fragmentation, DNA methylation, and cytotoxicity in Caco-2 cells, and suggest a potential promoter effect in human intestinal cells.

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    Li M, Cuff CF, Pestka JJ. T-2 toxin impairment of enteric reovirus clearance in the mouse associated with suppressed immunoglobulin and IFN-gamma responses. Toxicol Appl Pharmacol. 2006 Aug 1;214(3):318-25. PMID: 16504231


    Trichothecenes are exquisitely toxic to the gastrointestinal (GI) tract and leukocytes and thus are likely to impair gut immunity.

    The purpose of this research was to test the hypothesis that the Type A trichothecene T-2 toxin interferes with the gut mucosal immune response to enteric reovirus infection.

    As compared to vehicle-treated control, T-2-treated mice had dramatically elevated intestinal plaque-forming viral titers after 5 days and failed to completely clear the virus from intestine by 10 days.

    T-2 suppressed IFN-gamma responses in PP to reovirus at 3 and 7 days as compared to infected controls whereas IL-2 mRNA concentrations were unaffected. PP IL-6 mRNA levels were increased 2-fold 2 h after T-2 treatment, but no differences between infected T-2-exposed and infected vehicle-treated mice were detectable over the next 7 days.

    Overall, the results suggest that T-2 toxin increased both the extent of GI tract reovirus infection and fecal shedding which corresponded to both suppressed immunoglobulin and IFN-gamma responses.

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    Sergent T, Parys M, Garsou S, Pussemier L, Schneider YJ, Larondelle Y. Deoxynivalenol transport across human intestinal Caco-2 cells and its effects on cellular metabolism at realistic intestinal concentrations. Toxicol Lett. 2006 Jul 1;164(2):167-76. PMID: 16442754.

    Epidemiological studies suggest a link between DON and gastrointestinal illness.

    We investigated the interaction of DON with Caco-2 cells, a widely used in vitro model of the human intestinal barrier.

    These data imply that a chronic exposure to DON contaminated foods may negatively affect human health by altering the intestinal mucosa integrity and by inducing the MAPKs implicated in inflammation.

    *

    Kouadio JH, Mobio TA, Baudrimont I, Moukha S, Dano SD, Creppy EE. Comparative study of cytotoxicity and oxidative stress induced by deoxynivalenol, zearalenone or fumonisin B1 in human intestinal cell line Caco-2. Toxicology. 2005 Sep 15;213(1-2):56-65. PMID: 16019124

    Fusarium toxins such as, deoxynivalenol (DON), zearalenone (ZEN) and fumonisin B1 (FB1) have been shown to cause diverse toxic effects in animals and also suspected of disease causation in humans. From the literature and mechanistic point of view, DON binds to the ribosomal peptidyl-transferase and inhibits protein synthesis specifically and DNA synthesis consequently. ZEN known to be genotoxic, binds to 17-beta-estradiol receptors, induces lipid peroxidation, cell death and inhibits protein and DNA synthesis. FB1 disrupts sphingolipid metabolism, induces lipid peroxidation altering the cell membrane and causing cell death.

    We intended to compare DON, ZEN and FB1 (1-150 microM) cytotoxic effect and the pathways leading to cell death and related to oxidative stress and macromolecules syntheses in a human intestinal cell line in order to tentatively classify them according to their respective potential toxicity.

    The comparison reveals that all three mycotoxins bear, at variable degree, the capability of inducing lipid peroxidation (MDA production) and could be classified above 10 microM in decreasing potency order FB1>DON>ZEN.

    This effect seems to be related to their common target that is the mitochondria as revealed by MTT test and seemingly not related to sphingoids accumulation concerning FB1.

    DON and ZEN also adversely affect lysosomes in contrast to FB1.

    The three mycotoxins inhibit protein synthesis with respective IC50 of 5, 8.8 and 19 microM for DON, FB1 and ZEN confirming that protein synthesis is a specific target of DON.

    DNA synthesis is inhibited by DON, ZEN and FB1 with respective IC50 of 1.7, 10 and 20 microM. However at higher concentrations DNA synthesis seems to be restored for FB1 and DON suggesting a promoter activity.

    Altogether the potency of the three mycotoxins in macromolecules inhibition is DON>ZEN>FB1 in Caco-2 cells. It appears then that FB1 acts rather through lipid peroxidation while DON affects rather DNA and protein synthesis.
  3. slayadragon

    slayadragon Senior Member

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    Effects of Bacteria in Sick Building Environments

    Following are some abstracts of articles showing some effects of bacteria that have been identified as growing in water-damaged buildings, either on their own or in combination with toxic molds such as Stachybotrys.

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    Hirvonen MR, Huttunen K, Roponen M. Bacterial strains from moldy buildings are highly potent inducers of inflammatory and cytotoxic effects. Indoor Air. 2005;15 Suppl 9:65-70. PMID: 15910531

    We aimed to identify inflammatory and cytotoxic potential of individual indoor air bacterial and fungal strains, as well as extracts of indoor air filter samples containing bacteria and fungi. Mouse RAW264.7 macrophages were exposed in vitro to four bacterial strains; Streptomyces californicus, Mycobacterium terrae, Bacillus cereus and Pseudomonas fluorescens, and three fungal strains; Penicillium spinulosum, Aspergillus versicolor and Stachybotrys chartarum.
    Furthermore, RAW264.7 macrophages were exposed to indoor air filter sample extracts representing 'low' (n = 21) and 'high' (n = 20) exposure to viable fungi or bacteria.
    The results show that the bacterial strains induce more profound production of NO, TNF-alpha and IL-6 than the studied fungal strains. They also decrease the viability of mouse macrophages.
    Similarly, the indoor air filter samples with high concentration of bacteria induced a statistically significant increase in TNF-alpha and IL-6 production as well as a decrease in cell viability. Altogether, these results suggest that indoor air bacterial strains are potent inducers of inflammatory responses and thus possibly related to adverse health effects of the inhabitants.


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    Huttunen K, Pelkonen J, Nielsen KF, Nuutinen U, Jussila J, Hirvonen MR. Synergistic interaction in simultaneous exposure to Streptomyces californicus and Stachybotrys chartarum. Environ Health Perspect. 2004 May;112(6):659-65. PMID: 15121507

    we investigated the inflammatory responses of mouse RAW264.7 macrophages after exposure to six indoor air microbes (Aspergillus versicolor, Penicillium spinulosum, Stachybotrys chartarum, Bacillus cereus, Mycobacterium terrae, and Pseudomonas fluorescens) alone and together with the actinomycete Streptomyces californicus.

    The production of nitric oxide, levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), and cytotoxicity were measured.

    The coexposure to Sta. chartarum and Str. californicus caused a synergistic increase in the production of IL-6 but not other cytokines.

    In further experiments, the metabolites from Sta. chartarum or from closely related fungi (atranones B and E, satratoxin G, trichodermin, 7-alpha-hydroxytrichodermol, staplabin, and SMTP-7) and the known fungal toxins sterigmatocystin, citrinin, and ochratoxin A were each tested with Str. californicus.

    The testing revealed a synergistic response in TNF-alpha and IL-6 production after coexposure to Str. californicus with both trichodermin and 7-alpha-hydroxytrichodermol.

    Finally, the synergistic inflammatory response caused by Str. californicus and trichodermin together was studied by analyzing for the presence of nuclear factor-kappa-B (NF-kappa-B) in nuclear extracts of the exposed cells.

    The exposure to Str. californicus induced the binding of NF-kappa-B proteins to the NF-kappa-B consensus sequence as well as to the natural NF-kappa-B site of the IL-6 promoter. Adding trichodermin to the exposure did not increase the DNA binding.


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    Penttinen P, Huttunen K, Pelkonen J, Hirvonen MR. The proportions of Streptomyces californicus and Stachybotrys chartarum in simultaneous exposure affect inflammatory responses in mouse RAW264.7 macrophages. Inhal Toxicol. 2005 Feb;17(2):79-85. PMID: 15764485


    we aimed to evaluate the effects of simultaneous exposure with modified proportions of actinobacteria Streptomyces californicus and fungi Stachybotrys chartarum on inflammatory responses (cytokines macrophage inflammatory protein 2 [MIP2], interleukin 6 [IL-6] and tumor necrosis factor a [TNFa]; nitric oxide) and cytotoxicity (MTT-test and DNA content analysis) in mouse RAW264.7 macrophage cell line.

    The present results revealed that mutual proportions of fungal and bacterial spores in simultaneous exposure affect the nature of their interactions leading to increased or suppressed production of inflammatory mediators in RAW264.7 macrophages.


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    Penttinen P, Pelkonen J, Huttunen K, Toivola M, Hirvonen MR. Interactions between Streptomyces californicus and Stachybotrys chartarum can induce apoptosis and cell cycle arrest in mouse RAW264.7 macrophages. Toxicol Appl Pharmacol. 2005 Feb 1;202(3):278-88. PMID: 15667833

    Differences in cytotoxic and inflammatory responses in mouse (RAW264.7) macrophages were studied after exposure to the spores of co-cultivated microbes, the mixture of separately cultivated spores, and the spores of either of these microbes cultivated alone.

    Co-cultivation increased the ability of the spores to cause apoptosis by more than 4-fold and the proportion of RAW264.7 cells at the G2/M stage increased nearly 2-fold when compared to the response induced by the mixture of spores. In contrast, co-cultivation decreased significantly the ability of the spores to trigger the production of NO and IL-6 in RAW264.7 cells.

    In conclusion, these data suggest that co-culture of S. californicus and S. chartarum can result in microbial interactions that significantly potentiate the ability of the spores to cause apoptosis and cell cycle arrest in mammalian cells.
  4. slayadragon

    slayadragon Senior Member

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    Trichothecenes and LPS (Bacteria Endotoxin)

    Following are some articles that discuss the interactions that can occur between Stachybotrys and other toxic molds with lipopolysaccharides (LPS or "endotoxins").

    Endotoxins tend to be associated with bacteria growing in the intestinal tracts and oral cavities of mammals. They also are produced by bacteria growing in the environment.

    The general intestinal problems in CFS make it seem that these bacteria will be especially present in this patient population. Certain researchers/doctors (I believe Dr. Cheney?) have suggested that the oral cavity tends to be a particular problem in these patients as well.

    To the extent that CFS patients indeed have LPS issues, these studies suggest that they may be especially affected by toxic mold exposures.

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    Hymery N, Sibiril Y, Parent-Massin D. In vitro effects of trichothecenes on human dendritic cells. Toxicol In Vitro. 2006 Sep;20(6):899-909. PMID: 16517116

    The aim of this work was to study the in vitro effects of trichothecenes on human dendritic cells.

    Two mycotoxins (T-2 and DON) known to be immunotoxic have been tested on a model of monocyte-derived dendritic cells culture. Cytotoxic effects of T-2 toxin and DON on immature dendritic cells showed that DON is less potent than T-2 toxin.

    The exposure to trichothecenes during dendritic cell maturation upon addition of LPS or TNF-alpha markedly inhibited the up-regulation of maturation markers such as CD-86, HLA-DR and CCR7.

    Features of LPS or TNF-alpha -mediated maturation of dendritic cells, such as IL-10 and IL-12 secretions and endocytosis, were also impaired in response to trichothecenes treatment.

    These results suggest trichothecenes have adverse effects on dendritic cells and dendritic cell maturation process.


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    Chung YJ, Jarvis B, Pestka J. Modulation of lipopolysaccharide-induced proinflammatory cytokine production by satratoxins and other macrocyclic trichothecenes in the murine macrophage. J Toxicol Environ Health A. 2003 Feb 28;66(4):379-91. PMID: 12554543

    To test the potential immunomodulatory effects of these mycotoxins, RAW 264.7 murine macrophage cells were treated with various concentrations of satratoxin G (SG), isosatratoxin F (iSF), satratoxin H (SH), roridin A (RA), and verrucarin A (VA) for 48 h in the presence or absence of suboptimal concentra-tion of lipopolysaccharide (LPS, 50 ng/ml), and tumor necrosis factor-alpha (TNF-alpha ) and interleukin-6 (IL-6) production were assayed by enzyme-linked immunosorbent assay (ELISA).

    In LPS-stimulated cultures, TNF-alpha supernatant concentrations were significantly increased in the presence of 2.5, 2.5, and 1 ng/ml of SG, SH, and RA, respectively, whereas IL-6 concentrations were not affected by the same concentrations these macrocyclic trichothecenes.

    When cells that were treated with LPS and SG (2.5 ng/ml) were evaluated by real-time polymerase chain reaction (PCR),TNF-alpha mRNA was found to increase at 24, 36, and 48 h compared to control cells.

    At higher concentrations, cytokine production and cell viability were markedly impaired in LPS-stimulated cells.

    Without LPS stimulation, neither TNF-alpha, nor IL-6 was induced.

    These results indicate that low concentrations of macrocyclic trichothecenes superinduce expression of TNF-alpha, whereas higher concentrations of these toxins are cytotoxic and concurrently reduce cytokine production.

    The capacity of satratoxins and other macrocyclic trichothecenes to alter cytokine production may play an etiologic role in outbreaks of Stachybotrys-associated human illnesses.


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    Pestka J, Zhou HR. Toll-like receptor priming sensitizes macrophages to proinflammatory cytokine gene induction by deoxynivalenol and other toxicants. Toxicol Sci. 2006 Aug;92(2):445-55. PMID: 16687389

    Activation of the innate immune system might predispose a host to toxicant-induced inflammation.

    In vitro macrophage models were employed to investigate the effects of preexposure to Toll-like receptor (TLR) agonists on induction of proinflammatory cytokine gene expression by the trichothecene mycotoxin deoxynivalenol (DON) and other toxicants.

    Priming of the murine RAW 264.7 macrophage line or peritoneal murine macrophages with the TLR4 agonist lipopolysaccharide (LPS) at 100 ng/ml for 4, 8, and 16 h significantly increased DON-induced IL-1beta, IL-6, and TNF-alpha mRNA expression as compared to LPS or DON alone.

    Prior TLR activation might render macrophages highly sensitive to subsequent induction of proinflammatory gene expression by xenobiotics with diverse mechanisms of action.


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    Chung YJ, Yang GH, Islam Z, Pestka JJ. Up-regulation of macrophage inflammatory protein-2 and complement 3A receptor by the trichothecenes deoxynivalenol and satratoxin G. Toxicology. 2003 Apr 15;186(1-2):51-65. PMID: 12604170

    The trichothecenes are a group of mycotoxins that target leukocytes and have a wide range of immunomodulatory effects.

    Both MIP-2 and C3aR mRNAs were up-regulated by DON while only MIP-2 mRNA was induced by SG.

    MIP-2 protein was also found to be induced by both DON and SG in RAW 264.7 cell cultures.

    When mice were treated with DON (12.5 mg/kg), splenic MIP-2 mRNA and serum MIP-2 levels were increased. MIP-2 mRNA and serum MIP-2 levels were synergistically increased when mice were co-treated with DON and LPS. Up-regulation of MIP-2 and C3aR are consistent with previous reports of trichothecene-induced inflammatory gene up-regulation and suggest that the specific genes affected may depend on trichothecene structures.


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    Thrasher JD, Crawley S, The biocontaminants and complexity of damp indoor spaces: more than what meets the eyes. Toxicol Ind Health. 2009 Oct-Nov;25(9-10):583-615. PMID: 19793773

    Nine types of biocontaminants in damp indoor environments from microbial growth are discussed: (1) indicator molds; (2) Gram negative and positive bacteria; (3) microbial particulates; (4) mycotoxins; (5) volatile organic compounds, both microbial (MVOCs) and non-microbial (VOCs); (6) proteins; (7) galactomannans; (8) 1-3-beta-D-glucans (glucans) and (9) lipopolysaccharides (LPS--endotoxins). When mold species exceed those outdoors contamination is deduced. Gram negative bacterial endotoxins, LPS in indoor environments, synergize with mycotoxins.

    The gram positive Bacillus species, Actinomycetes (Streptomyces, Nocardia and Mycobacterium), produce exotoxins. The Actinomycetes are associated with hypersensitivity pneumonitis, lung and invasive infections. Mycobacterial mycobacterium infections not from M. tuberculosis are increasing in immunocompetent individuals. In animal models, LPS enhance the toxicity of roridin A, satratoxins G and aflatoxin B1 to damage the olfactory epithelium, tract and bulbs (roridin A, satratoxin G) and liver (aflatoxin B1). Aflatoxin B1 and probably trichothecenes are transported along the olfactory tract to the temporal lobe. Co-cultured Streptomyces californicus and Stachybotrys chartarum produce a cytotoxin similar to doxorubicin and actinomycin D (chemotherapeutic agents). Trichothecenes, aflatoxins, gliotoxin and other mycotoxins are found in dust, bulk samples, air and ventilation systems of infested buildings. Macrocyclic trichothecenes are present in airborne particles <2 microm. Trichothecenes and stachylysin are present in the sera of individuals exposed to S. chartarum in contaminated indoor environments. Haemolysins are produced by S. chartarum, Memnoniella echinata and several species of Aspergillus and Penicillium. Galactomannans, glucans and LPS are upper and lower respiratory tract irritants. Gliotoxin, an immunosuppressive mycotoxin, was identified in the lung secretions and sera of cancer patients with aspergillosis produced by A. fumigatus, A. terreus, A. niger and A. flavus.

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    Hymery N, Sibiril Y, Parent-Massin D. In vitro effects of trichothecenes on human dendritic cells. Toxicol In Vitro. 2006 Sep;20(6):899-909. PMID: 16517116

    The aim of this work was to study the in vitro effects of trichothecenes on human dendritic cells.

    Two mycotoxins (T-2 and DON) known to be immunotoxic have been tested on a model of monocyte-derived dendritic cells culture. Cytotoxic effects of T-2 toxin and DON on immature dendritic cells showed that DON is less potent than T-2 toxin.

    The exposure to trichothecenes during dendritic cell maturation upon addition of LPS or TNF-alpha markedly inhibited the up-regulation of maturation markers such as CD-86, HLA-DR and CCR7.

    Features of LPS or TNF-alpha -mediated maturation of dendritic cells, such as IL-10 and IL-12 secretions and endocytosis, were also impaired in response to trichothecenes treatment.

    These results suggest trichothecenes have adverse effects on dendritic cells and dendritic cell maturation process.

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    Kankkunen P, Rintahaka J, Aalto A, Leino M, Majuri ML, Alenius H, Wolff H, Matikainen S. Trichothecene mycotoxins activate inflammatory response in human macrophages. J Immunol. 2009 May 15;182(10):6418-25. PMID: 19414795


    In this report, we have examined the effect of S. chartarum and trichothecene mycotoxins on the proinflammatory cytokine response in human macrophages.

    Satratoxin-positive S. chartarum activated inflammasome-associated caspase-1, which is needed for proteolytic processing of IL-1beta and IL-18. Furthermore, purified trichothecene mycotoxins, roridin A, verrucarin A, and T-2 toxin activated caspase-1, and these mycotoxins also strongly enhanced LPS-dependent secretion of IL-1beta and IL-18.

    The satratoxin-positive strain of S. chartarum and the trichothecenes also triggered the activation of caspase-3, which is an effector caspase of apoptosis. Satratoxin-negative S. chartarum was not able to activate either caspase-1 or caspase-3

    human macrophages sense trichothecene mycotoxins as a danger signal, which activates caspase-1, and further enables the secretion of IL-1beta and IL-18 from the LPS-primed cells.

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    Pugliese A, Savarino A, Vidotto V, Cantamessa C, Pescarmona GP. Effect of Aspergillus terreus mycotoxins on nitric oxide synthase activity in human erythroid K-562 cells. Cell Biochem Funct. 1999 Mar;17(1):35-45. PMID: 10191507

    Because several stimuli of microbial origin enhance the activity of nitric oxide synthase (NOS) in human cells of the myeloid lineage, we decided to investigate whether cellular damage induced by Aspergillus terreus mycotoxins could be associated with an increase in NOS activity.

    A pool of mycotoxins rather than individual toxins was tested so that the natural conditions could be mimicked.

    Canavanine, an inhibitor of NOS, significantly reduced cell death in the presence of the extract, suggesting that cellular damage, induced by the mycotoxins of A. terreus is at least in part mediated by NOS activity.

    Moreover, Escherichia coli lipopolysaccharide (LPS), known to be a potent NOS inducer, increased NOS activity in our experimental model as well.

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    Sugiyama K, Muroi M, Tanamoto K, Nishijima M, Sugita-Konishi Y. Deoxynivalenol and nivalenol inhibit lipopolysaccharide-induced nitric oxide production by mouse macrophage cells. Toxicol Lett. 2010 Feb 1;192(2):150-4. PMID: 19857559


    Trichothecene mycotoxins cause immune dysfunction, thus leading to diverse responses to infection. The present study evaluated the effect of DON and NIV on nitric oxide (NO) production by RAW264 cells stimulated with lipopolysaccharide (LPS). LPS-induced NO production was reduced in the presence of these toxins.

    The transcriptional activation and expression of inducible NO synthase (iNOS) by LPS were also repressed by these toxins.

    These results indicate that DON and NIV inhibit the LPS-induced NO and IFN-beta production, which both play an important role for host protection against invading pathogens, and suggests that the inhibition of these factors may be involved in the immunotoxic effects of these mycotoxins.

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    Luongo D, Severino L, Bergamo P, D'Arienzo R, Rossi M. Trichothecenes NIV and DON modulate the maturation of murine dendritic cells. Toxicon. 2010 Jan;55(1):73-80. PMID: 19635492


    Nivalenol (NIV) and Deoxynivalenol (DON), mycotoxins of the trichothecene family are considered very common food contaminants.

    In this work, we investigated whether the immunotoxic effects ascribed to these trichothecenes may be mediated by perturbations in the activity of dendritic cells (DCs).

    Murine bone marrow-derived DCs were used to evaluate the effects of NIV and DON on the LPS-induced maturation process.

    We found that the expression of the class II MHC and of the accessory CD11c molecules, but not of the costimulatory CD86 marker, was down-regulated by NIV and DON exposure in LPS-treated DCs, as well as nitric oxide (NO) production.

    Interestingly, NIV, but not DON, induced DC necrosis.

    Moreover, the analysis of the cytokine pattern showed that IL-12 and IL-10 expressions induced by LPS exposure were suppressed by both trichothecenes in a dose-dependent fashion.

    On the other hand, the secretion of the proinflammatory cytokine TNF-alpha was increased as a direct consequence of DON and NIV exposure.

    Taken together, our data indicated that the immunotoxicity of NIV and DON was related to the capacity of both trichothecenes to interfere with phenotypic and functional features of maturing DCs.

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    Dll S, Schrickx JA, Dnicke S, Fink-Gremmels J. Interactions of deoxynivalenol and lipopolysaccharides on cytokine excretion and mRNA expression in porcine hepatocytes and Kupffer cell enriched hepatocyte cultures. Toxicol Lett. 2009 Oct 8;190(1):96-105. PMID: 19607891


    The effects of deoxynivalenol (DON) on the mRNA expression of cytokines and inflammation-related genes, as well as the cytokine secretion of porcine hepatocytes and Kupffer cell enriched hepatocyte cultures (co-cultures), were investigated in the absence or presence of LPS.

    DON and LPS acted in a synergistic manner with regard to a significantly increased mRNA expression of TNF-alpha in hepatocytes exposed to 500 nM or 2000 nM DON, or non-significant increase in co-cultures after 3h of exposure.

    TNF-alpha supernatant concentrations were increased due to LPS but did not reflect the synergistic effects with DON as observed at mRNA level.

    IL-6 mRNA in hepatocyte cultures at 6h paralleled the TNF-alpha supernatant pattern at this time point. In co-cultures and hepatocytes, a DON dose dependent induction of IL-6 mRNA was detected in cells not exposed to LPS. Supernatant concentrations of LPS-induced IL-6 were significantly decreased by 2000 nM DON in both types of cell cultures. Also the mRNA expression of the anti-inflammatory IL-10 was increased by DON to various degrees depending on DON-dose, stimulation with LPS and time point of measurement. After 6h, expression of iNOS was only induced by 2000 nM DON, but not in LPS treated cells.

    Even if mRNA induction was not paralleled by related supernatant concentrations of TNF-alpha, IL-6 and IL-10 under the conditions of the present investigations, it was clearly demonstrated that DON has the potential to provoke and modulate immunological reactions of porcine liver cells.

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    Islam Z, Amuzie CJ, Harkema JR, Pestka JJ. Neurotoxicity and inflammation in the nasal airways of mice exposed to the macrocyclic trichothecene mycotoxin roridin a: kinetics and potentiation by bacterial lipopolysaccharide coexposure. Toxicol Sci. 2007 Aug;98(2):526-41. PMID: 17483119

    The purpose of this investigation was to determine (1) the kinetics of nasal inflammation and neurotoxicity after a single intranasal instillation of roridin A (RA), a representative macrocyclic trichothecene; and (2) the capacity of lipopolysaccharide (LPS) to modulate RA's effects.

    Taken together, the results suggest that RA markedly induced the proapoptotic gene FAS and proinflammatory cytokine expression prior to evoking olfactory sensory neurons (OSNs) apoptosis and olfactory epithelium (OE) atrophy and that RA's effects were augmented by LPS.


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    Islam Z, Pestka JJ. LPS priming potentiates and prolongs proinflammatory cytokine response to the trichothecene deoxynivalenol in the mouse. Toxicol Appl Pharmacol. 2006 Feb 15;211(1):53-63. PMID: 16009389

    Simultaneous exposure to lipopolysaccharide (LPS) markedly amplifies induction of proinflammatory cytokine expression as well as IL-1-driven lymphocyte apoptosis by trichothecene deoxynivalenol (DON) in the mouse.

    The purpose of this research was to test the hypothesis that LPS priming will sensitize a host to DON-induced proinflammatory cytokine induction and apoptosis.

    Taken together, exposure to LPS rendered mice highly susceptible to DON induction of cytokine expression and this correlated with increased apoptosis in the thymus.


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    Chung YJ, Zhou HR, Pestka JJ. Transcriptional and posttranscriptional roles for p38 mitogen-activated protein kinase in upregulation of TNF-alpha expression by deoxynivalenol (vomitoxin). Toxicol Appl Pharmacol. 2003 Dec 1;193(2):188-201. PMID: 14644621

    Deoxynivalenol (DON, vomitoxin) is a trichothecene mycotoxin that potentially mediates toxicity by upregulating proinflammatory cytokine gene expression in vitro and in vivo.

    The purpose of this study was to test the hypothesis that DON-induced activation of mitogen-activated protein kinases (MAPKs) mediates transcriptional and posttranscriptional upregulation of TNF-alpha gene expression.

    RNAse protection assay revealed that DON at 100 to 500 ng/ml induced mRNA expression of TNF-alpha as well as IL-6, IFN-gamma, TGFbeta-1, and TGFbeta-3 and that these effects were potentiated by 100 ng/ml lipopolysaccharide (LPS).

    Taken together, these results suggest that relative to DON-induced TNF-alpha mRNA expression, p38 and ERK activation contribute to DON-induced transcriptional upregulation whereas p38 plays a role in increasing mRNA stability.

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    Uzarski RL, Islam Z, Pestka JJ. Potentiation of trichothecene-induced leukocyte cytotoxicity and apoptosis by TNF-alpha and Fas activation. Chem Biol Interact. 2003 Oct 25;146(2):105-19. PMID: 14597125

    Trichothecene mycotoxins cause immunosuppression by inducing apoptosis in lymphoid tissue. Trichothecene-induced leukocyte apoptosis can be augmented by bacterial lipopolysaccharide (LPS) but the mechanisms involved in this potentiating effect are not completely understood.

    Primary leukocyte suspensions were prepared from murine thymus (TH), spleen (SP), bone marrow (BM) and Peyer's patches (PP) and then cultured with DON in the absence or presence of LPS,

    DON was found to inhibit LPS-induced proliferation and dexamethasone-induced apoptosis in SP cultures. In contrast, potentiation of DON-induced apoptosis and cytotoxicity was observed in BM cultures treated with anti-Fas and in TH cultures treated with TNF-alpha.

    When potentiation of DON-induced apoptosis by TNF-alpha was assessed using pharmacological inhibitors, generation of ROS, intracellular Ca2+, p38/SAPK, and caspase-3 activation were found to play roles.

    Taken together, these data demonstrate that LPS and its downstream mediators can interact with trichothecenes to modulate proliferative, cytotoxic and apoptotic outcomes in leukocytes in a tissue-specific manner.

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    Islam Z, King LE, Fraker PJ, Pestka JJ. Differential induction of glucocorticoid-dependent apoptosis in murine lymphoid subpopulations in vivo following coexposure to lipopolysaccharide and vomitoxin (deoxynivalenol). Toxicol Appl Pharmacol. 2003 Mar 1;187(2):69-79. PMID: 12649039


    Lipopolysaccharide (LPS) and vomitoxin (VT) synergistically induce glucocorticoid- mediated apoptotic cell death in lymphoid tissues of the mouse.

    Taken together, these data suggest that LPS can interact with VT in mice to induce the glucocorticoid-driven apoptotic loss of immature thymocytes and cytotoxic T lymphocytes in thymus, mature-B lymphocytes in Peyer's patch, and pro/pre-B lymphocytes and mature-B lymphocytes in bone marrow in mice.

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    Rosenblum Lichtenstein JH, Molina RM, Donaghey TC, Brain JD. Strain differences influence murine pulmonary responses to Stachybotrys chartarum. Am J Respir Cell Mol Biol. 2006 Oct;35(4):415-23. PMID: 16690987

    The severity of human responses to S. chartarum in both occupational and home settings varies widely.

    We intratracheally instilled C3H/HeJ, BALB/c, and C57BL/6J mice with S. chartarum spores suspended in saline. One day later, the mice were humanely killed, bronchoalveolar lavage (BAL) was performed, and biochemical and cellular indicators of lung injury and inflammation were measured.

    BALB/c mice showed the highest myeloperoxidase activity, albumin and hemoglobin levels, and neutrophil numbers in their BAL among the three strains. BALB/c was the only strain to show significant increases in keratinocyte-derived cytokine (KC), monocyte chemotactic protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, MIP-1gamma, MIP-2, RANTES, IL-1alpha, IL-1beta, IL-3, IL-6, IL-18, leukemia inhibitory factor, macrophage colony-stimulating factor, and TNF-alpha.

    A model of allergen-induced airway inflammation was examined to assess whether underlying allergic inflammation might contribute to increased susceptibility to S. chartarum-induced pulmonary inflammation and injury.

    Surprisingly, in BALB/c mice, ovalbumin-induced airway inflammation produced a protective effect against some S. chartarum-induced pulmonary responses.

    This is the first report of mammalian strain differences affecting responses to S. chartarum.

    These responses differ from those reported for LPS and other fungi. Analogous underlying genetic differences may contribute to the wide range of sensitivity to Stachybotrys among humans.

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  5. lono

    lono

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    This is a good summary article that discusses the current scientific understanding related to mold mycotoxins and mold illness (a.k.a. sick building syndrome). The author, David Straus of Texas Tech University, was able to identify mold mycotoxins in the blood (sera) of people who had been exposed to a water-damaged building.

    Molds, mycotoxins, and sick building syndrome
    Toxicology and Industrial Health 2009 vol: 25 iss: 9-10 pg: 617
    Author: Straus
  6. slayadragon

    slayadragon Senior Member

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    Here's a new article on the neurological effects of satratoxns (chemicals made by Stachybotrys).

    The paper is from this dissertation, available for free on the web.

    http://etd.lib.ttu.edu/theses/available/etd-05252005-163223/

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    Mycopathologia. 2010 Jun 13. [Epub ahead of print]
    Building-Associated Neurological Damage Modeled in Human Cells: A Mechanism of Neurotoxic Effects by Exposure to Mycotoxins in the Indoor Environment.
    Karunasena E, Larraaga MD, Simoni JS, Douglas DR, Straus DC.

    Department of Animal & Food Sciences, Texas Tech University, Lubbock, TX, 79409, USA, enusha.karunasena@ttuhsc.edu.
    Abstract
    Damage to human neurological system cells resulting from exposure to mycotoxins confirms a previously controversial public health threat for occupants of water-damaged buildings. Leading scientific organizations disagree about the ability of inhaled mycotoxins in the indoor environment to cause adverse human health effects. Damage to the neurological system can result from exposure to trichothecene mycotoxins in the indoor environment. This study demonstrates that neurological system cell damage can occur from satratoxin H exposure to neurological cells at exposure levels that can be found in water-damaged buildings contaminated with fungal growth. The constant activation of inflammatory and apoptotic pathways at low levels of exposure in human brain capillary endothelial cells, astrocytes, and neural progenitor cells may amplify devastation to neurological tissues and lead to neurological system cell damage from indirect events triggered by the presence of trichothecenes.

    PMID: 20549560 [PubMed - as supplied by publisher]
  7. slayadragon

    slayadragon Senior Member

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    Measuring Exposures to Mycotoxins

    Croft WA, Jastromski BM, Croft AL, Peters HA. Clinical confirmation of trichothecene mycotoxicosis in patient urine. J Environ Biol. 2002 Jul;23(3):301-20. PMID: 12597576

    The investigations of four Cases involving mold-contaminated buildings and human reaction to exposure, documents tests of extracted urine containing trichothecene mycotoxins confirming exposure and the diagnosis of mycotoxicosis in humans. In each of four Cases, the urine demonstrated antibiotic activity, sulfuric acid charring, and protein release. Urine was extracted using ethyl acetate 40V/60V[EA]. Extracted mycotoxin spotted on (TLC) displayed color and a range of (rf) between 0.2-0.6 using various solvents. Extract was re-suspended using 50% ethanol V/V to inject mycotoxins into weanling female Sprague-Dawley rats. Degeneration and necrosis of the rat's tissue followed. Koch's Postulates conditions were fulfilled by isolation of the causative agent, the trichothecene mycotoxins and the reproduction of disease. Examination of human tissue within the urine extraction group confirms Koch's Postulates and comparative pathology confirms inhalation Mycotoxicosis, with severe necrosis of the central nervous system and severe scarring within the lungs. Extraction of mycotoxins from human patient urine is a very useful confirmatory test to demonstrate exposure and identify mycotoxicosis. Low concentrations (6%) of sodium hypochlorite were ineffective against the activity of trichothecene mycotoxin. The severity or stages of disease directly correlates the level of exposure or poisoning (Patent Pending).

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    Van Emon JM, Reed AW, Yike I, Vesper SJ. ELISA measurement of stachylysin in serum to quantify human exposures to the indoor mold Stachybotrys chartarum. J Occup Environ Med. 2003 Jun;45(6):582-91. PMID: 12802211.

    The goal of this research was to develop a measurable indicator of human exposure to Stachyborys chartarum. Antibodies were produced against the hemolytic agent stachylysin obtained from the mold S. chartarum. These antibodies were used to develop two enzyme-linked immunosorbent assay methods for the analysis of stachylysin in human and rat sera and environmental samples. Stachylysin was measured in rat pups that received nasal instillations of S. chartarum conidia but not in control rat serum. Stachylysin in the serum of five human adults exposed to S. chartarum in water-damaged environments was 371 ng/mL but none was detected in the control serum. Stachylysin was also quantified in spore, wallboard, mycelial, and dust samples. The measurement of stachylysin may be a useful indicator in assessing human exposure to S. chartarum and in determining the presence of this indoor mold.

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    Brasel TL, Campbell AW, Demers RE, Ferguson BS, Fink J, Vojdani A, Wilson SC, Straus DC. Detection of trichothecene mycotoxins in sera from individuals exposed to Stachybotrys chartarum in indoor environments. Arch Environ Health. 2004 Jun;59(6):317-23. PMID: 16238166

    To date, no study has effectively demonstrated a direct human exposure to mycotoxins in mold-contaminated buildings. Therefore, the authors investigated the presence of trichothecene mycotoxins in sera from individuals exposed to indoor molds (specifically Stachybotrys chartarum). Sera from occupants of contaminated (test samples, n=44) and uncontaminated (control samples, n=26) buildings were analyzed using a competitive enzyme-linked immunosorbent assay (ELISA) highly specific for macrocyclic trichothecenes. Twenty-three samples were significantly different (p < 0.05) from normal human serum tested in the same manner, whereas only 1 of the control samples tested positive. Mass spectrometry analysis could not confirm the presence of intact S. chartarum macrocyclic trichothecenes. The authors hypothesize that this result was caused by uncharacterized ELISA-reactive metabolic breakdown products. Data from this study suggest that trichothecene mycotoxins can be demonstrated in the tissues of certain individuals exposed to S. chartarum in contaminated buildings.

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    Yike I, Distler AM, Ziady AG, Dearborn DG. Mycotoxin adducts on human serum albumin: biomarkers of exposure to Stachybotrys chartarum. Environ Health Perspect. 2006 Aug;114(8):1221-6. PMID: 16882529

    OBJECTIVE: Despite the growing body of evidence showing adverse health effects from inhalation exposure to the trichothecene-producing mold Stachybotrys chartarum, controversy remains. Currently, there are no reliable assays suitable for clinical diagnosis of exposure. We hypothesized that satratoxin G (SG) -albumin adducts may serve as biomarkers of exposure to this fungus. DESIGN: We studied the formation of adducts of SG with serum albumin in vitro using Western blots and mass spectrometry (MS) and searched for similar adducts formed in vivo using human and animal serum. RESULTS: Samples of purified human serum albumin that had been incubated with increasing concentrations of SG showed concentration-dependent albumin bands in Western blots developed with anti-SG antibodies. MS analysis found that as many as 10 toxin molecules can be bound in vitro to one albumin molecule. The sequencing of albumin-adduct tryptic peptides and the analysis of pronase/aminopeptidase digests demonstrated that lysyl, cysteinyl, and histidyl residues are involved in the formation of these adducts. Serum samples from three patients with documented exposure to S. chartarum similarly revealed lysine-, cysteine-, and histidine-SG adducts after exhaustive digestion, affinity column enrichment, and MS analysis. These adducts were also found in the sera from rats exposed to the spores of S. chartarum in contrast to control human subjects and control animals. CONCLUSIONS: These data document the occurrence of SG-albumin adducts in both in vitro experiments and in vivo human and animal exposures to S. chartarum. Relevance to clinical practice: SG-amino acid adducts may serve as reliable dosimeter biomarkers for detection of exposure to S. chartarum.

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    Kim EJ, Jeong SH, Cho JH, Ku HO, Pyo HM, Kang HG, Choi KH. Plasma haptoglobin and immunoglobulins as diagnostic indicators of deoxynivalenol intoxication. J Vet Sci. 2008 Sep;9(3):257-66. PMID: 18716445

    This study aimed to discover potential biomarkers for dioxynivalenol (DON) intoxication. B6C3F1 male mice were orally exposed to 0.83, 2.5 and 7.5 mg/kg body weight (bw) DON for 8 days and the differential protein expressions in their blood plasma were determined by SELDI - Time-of-Flight/Mass Spectrometry (TOF/MS) and the immunoglobulins (Igs) G, A, M and E in the serum were investigated. 11.7 kDa protein was significantly highly expressed according to DON administration and this protein was purified by employing a methyl ceramic HyperD F column with using optimization buffer for adsorption and desorption. The purified protein was identified as a haptoglobin precursor by peptide mapping with using LC/Q-TOF/MS and MALDI-TOF/MS and this was confirmed by western blotting and ELISA. IgG and IgM in serum were decreased in a dose-dependent manner and IgA was decreased at 7.5 mg/kg bw DON administration, but the IgE level was not changed. To compare the expressions of haptoglobin and the Igs patterns between aflatoxin B1 (AFB1), zearalenone (ZEA) and DON intoxications, rats were orally administered with AFB1 1.0, ZEA 240 and DON 7.5 mg/kg bw for 8 days. Haptoglobin was increased only at DON 7.5 mg/kg bw, while it was slightly decreased at ZEA 240 mg/kg bw and it was not detected at all at AFB1 1.0 mg/kg bw. IgG and IgA were decreased by DON, but IgG, IgA, IgM and IgE were all increased by AFB1. No changes were observed by ZEA administration. These results show that plasma haptoglobin could be a diagnostic biomarker for DON intoxication when this is combined with examining the serum Igs.

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    Layton RC, Purdy CW, Jumper CA, Straus DC. Detection of macrocyclic trichothecene mycotoxin in a caprine (goat) tracheal instillation model. Toxicol Ind Health. 2009 Oct-Nov;25(9-10):693-701. PMID: 19793770

    This study demonstrates the detection and dynamics of macrocyclic trichothecene mycotoxin (MTM) tissue loading using a commercially available assay in a goat model. The detection of MTMs has been difficult and complex due to the uncertainty of what tissues to examine and when to sample. Twelve goats (two groups of each) were instilled with Stachybotrys chartarum conidial suspension via the trachea. The first group was challenged repeatedly with fungal conidia containing 1 mg/kg of MTM per instillation whereas the second group was exposed once, to spores with a calculated concentration of 5 microg/kg of mycotoxin. These toxin estimates were generated by the QuantiTox(TM) Kit assay; a conidium of S. chartarum possessed 8.5 pg of MTM. After repeated exposure of 3 days, MTM was detected in one of six animals. This animal and two others from the same group had mycotoxin detected in their serum 24 hours after challenge at a comparable level (1.69 ng/mL) to the six animals challenged with a single dose (2.02 ng/mL) at the same time post-instillation. Results showed that MTMs are detectable in experimental animals soon after challenge and contribute to the understanding of the role of these mycotoxins in the disease process following mold exposure.

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    Hooper DG, Bolton VE, Guilford FT, Straus DC. Mycotoxin detection in human samples from patients exposed to environmental molds. Int J Mol Sci. 2009 Apr 1;10(4):1465-75. PMID: 19468319

    The goal of this study was to determine if selected mycotoxins (trichothecenes, aflatoxins, and ochratoxins) could be extracted and identified in human tissue and body fluids from patients exposed to toxin producing molds in their environment. Human urine and methanol extracted tissues and sputum were examined. Trichothecenes were tested using competitive ELISA techniques. Aflatoxins B1, B2, G1, and G2, and ochratoxin A were tested by using immunoaffinity columns and fluorometry. Test sensitivity and specificity were determined. Levels of detection for the various mycotoxins varied from 0.2 ppb for trichothecenes, 1.0 ppb for aflatoxins, and 2.0 ppb for ochratoxins. Trichothecene levels varied in urine, sputum, and tissue biopsies (lung, liver, brain) from undetectable (<0.2 ppb) to levels up to 18 ppb. Aflatoxin levels from the same types of tissues varied from 1.0 to 5.0 ppb. Ochratoxins isolated in the same type of tissues varied from 2.0 ppb to > 10.0 ppb. Negative control patients had no detectable mycotoxins in their tissues or fluids. These data show that mycotoxins can be detected in body fluids and human tissue from patients exposed to mycotoxin producing molds in the environment, and demonstrate which human tissues or fluids are the most likely to yield positive results.
  8. slayadragon

    slayadragon Senior Member

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    Stachybotrys & Macrophage Activity

    Sorenson WG, Frazer DG, Jarvis BB, Simpson J, Robinson VA. Trichothecene Mycotoxins in Aerosolized Conida of Stachybotrys Atra. Appl Environ Microbiol. 1987 June, 53(6): 1370-1375.

    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.

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    Yang GH, Jarvis BB, Chung YJ, Pestka JJ. Apoptosis induction by the satratoxins and other trichothecene mycotoxins: relationship to ERK, p38 MAPK, and SAPK/JNK activation. Toxicol Appl Pharmacol. 2000 Apr 15;164(2):149-60. PMID: 10764628

    The satratoxins are members of the trichothecene mycotoxin family that are produced by the fungus Stachybotrys and that have been etiologically associated with building-related health problems. The purpose of this study was to relate cytotoxic and apoptotic capacities of satratoxins and other trichothecenes to the activation of three groups of mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated protein kinase (ERK), p38 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK)). Two myeloid models, RAW 264.7 murine macrophage and U937 human leukemic cells were used. Upon evaluating representative trichothecenes in the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) cleavage assay, cytotoxicity was evident according to the following rank order: satratoxin G, roridin A, and verrucarin A > T-2 toxin, satratoxin F, H > nivalenol, and vomitoxin. Comparable results were found when measuring trichothecene-mediated apoptosis using DNA fragmentation and fluorescence microscopy assays, thus suggesting that cytotoxicity was mediated through an apoptotic process. Assessment of MAPK activation using Western blot analysis revealed that trichothecenes activated not only SAPK/JNK and p38 MAPK but also ERK. Activation of MAPKs by satratoxins and other trichothecenes correlated with and preceded apoptosis. The concentration of satratoxin G sufficient for protein synthesis inhibition correlated with that required for apoptosis and activation of all three MAPKs. Cycloheximide had similar effects to trichothecenes, suggesting that ribosome binding or protein synthesis inhibition may play roles in MAPK activation and apoptosis induction. Apoptosis induction by satratoxin G and vomitoxin was markedly enhanced when ERK activation was selectively inhibited by ERK-specific inhibitor PD98059, thus indicating a negative role for ERK. Inhibition of p38 MAPK activity with the p38-specific inhibitor SB203580 had no effect on apoptosis induction by the highly toxic satratoxin G. However, SB203580 moderately inhibited apoptosis induction by the less toxic trichothecene vomitoxin, thus implying a partial role of p38 MAPK in trichothecene-induced apoptosis. The results suggest that the satratoxins are among the most potent trichothecenes and that MAPKs may play integral roles in the diverse toxic manifestations of these mycotoxins. Copyright 2000 Academic Press.

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    Nielsen KF, Huttunen K, Hyvrinen A, Andersen B, Jarvis BB, Hirvonen MR. Metabolite profiles of Stachybotrys isolates from water-damaged buildings and their induction of inflammatory mediators and cytotoxicity in macrophages. Mycopathologia. 2002;154(4):201-5. PMID: 12206322

    The metabolite profiles of 20 Stachybotrys spp. isolates from Finnish water-damaged buildings were compared with their biological activities. Effects of purified compounds on cytotoxicity and production of inflammatory mediators such as nitric oxide, IL-6 and TNFalpha in murine RAW264.7 macrophage cells were studied. The 11 isolates belonging to the satratoxin-producing chemotype were highly cytotoxic to the macrophages. The isolates inducing inflammatory mediators all belonged to the atranone-producing chemotype, but pure atranones B, and D did not elicit a response in the bioassay. Altogether, cytotoxicity of Stachybotrys sp. isolates appear to be related to satratoxin production whereas the specific component inducing inflammatory responses in atranone-producing isolates remains obscure.

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    Mbandi E, Pestka JJ. Deoxynivalenol and satratoxin G potentiate proinflammatory cytokine and macrophage inhibitory protein 2 induction by Listeria and Salmonella in the macrophage. J Food Prot. 2006 Jun;69(6):1334-9. PMID: 16786854

    Health risks from microbial pathogens and toxins encountered in food and the environment continue to be of worldwide concern. The purpose of this research was to test the hypothesis that trichothecene mycotoxins amplify inflammatory responses to foodborne bacterial pathogens. We assessed the capacity of deoxynivalenol (DON) and satratoxin G (SG) to potentiate chemokine and proinflammatory cytokine production in RAW 264.7 murine macrophages induced by Listeria monocytogenes and Salmonella Typhimurium. When macrophage cultures were incubated with killed irradiated suspensions of the pathogens for 24 h, the minimum Listeria concentrations for induction of macrophage inhibitory protein 2 (MIP-2), interleukin-1beta (IL-beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) were 0.01, 0.01, 1.0, and 1.0 microg/ml (P < 0.05) and the minimum Salmonella concentrations were 0.01, 0.01, 0.1, and 0.1 microg/ml, respectively (P < 0.05). Induction of all four mediators by both pathogens was potentiated by DON (at 100 and 250 ng/ml); observed responses were significantly higher than predicted additive responses (P < 0.05). SG (at 2 and 5 ng/ml) also significantly amplified induction of IL-1beta and TNF-alpha (P < 0.05) by both Listeria and Salmonella. These results indicate that DON encountered in Fusarium-contaminated food and SG from Stachybotrys-contaminated indoor environments could magnify innate inflammatory responses to foodborne bacterial pathogens.

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    Islam Z, Harkema JR, Pestka JJ. Satratoxin G from the black mold Stachybotrys chartarum evokes olfactory sensory neuron loss and inflammation in the murine nose and brain. Environ Health Perspect. 2006 Jul;114(7):1099-107. PMID: 16835065

    Satratoxin G (SG) is a macrocyclic trichothecene mycotoxin produced by Stachybotrys chartarum, the "black mold" suggested to contribute etiologically to illnesses associated with water-damaged buildings. Using an intranasal instillation model in mice, we found that acute SG exposure specifically induced apoptosis of olfactory sensory neurons (OSNs) in the olfactory epithelium. Dose-response analysis revealed that the no-effect and lowest-effect levels at 24 hr postinstillation (PI) were 5 and 25 microg/kg body weight (bw) SG, respectively, with severity increasing with dose. Apoptosis of OSNs was identified using immunohistochemistry for caspase-3 expression, electron microscopy for ultrastructural cellular morphology, and real-time polymerase chain reaction for elevated expression of the proapoptotic genes Fas, FasL, p75NGFR, p53, Bax, caspase-3, and CAD. Time-course studies with a single instillation of SG (500 microg/kg bw) indicated that maximum atrophy of the olfactory epithelium occurred at 3 days PI. Exposure to lower doses (100 microg/kg bw) for 5 consecutive days resulted in similar atrophy and apoptosis, suggesting that in the short term, these effects are cumulative. SG also induced an acute, neutrophilic rhinitis as early as 24 hr PI. Elevated mRNA expression for the proinflammatory cytokines tumor necrosis factor-alpha, interleukin-6 (IL-6) , and IL-1 and the chemokine macrophage-inflammatory protein-2 (MIP-2) were detected at 24 hr PI in both the ethmoid turbinates of the nasal airways and the adjacent olfactory bulb of the brain. Marked atrophy of the olfactory nerve and glomerular layers of the olfactory bulb was also detectable by 7 days PI along with mild neutrophilic encephalitis. These findings suggest that neurotoxicity and inflammation within the nose and brain are potential adverse health effects of exposure to satratoxins and Stachybotrys in the indoor air of water-damaged buildings.

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    Bae HK, Shinozuka J, Islam Z, Pestka JJ. Satratoxin G interaction with 40S and 60S ribosomal subunits precedes apoptosis in the macrophage. Toxicol Appl Pharmacol. 2009 Jun 1;237(2):137-45. PMID: 19306889

    Satratoxin G (SG) and other macrocyclic trichothecene mycotoxins are potent inhibitors of eukaryotic translation that are potentially immunosuppressive. The purpose of this research was to test the hypothesis that SG-induced apoptosis in the macrophage correlates with binding of this toxin to the ribosome. Exposure of RAW 264.7 murine macrophages to SG at concentrations of 10 to 80 ng/ml induced DNA fragmentation within 4 h that was indicative of apoptosis. To relate these findings to ribosome binding of SG, RAW cells were exposed to different toxin concentrations for various time intervals, ribosomal fractions isolated by sucrose density gradient ultracentrifugation and resultant fractions analyzed for SG by competitive ELISA. SG was found to specifically interact with 40S and 60S ribosomal subunits as early as 5 min and that, at high concentrations or extended incubation times, the toxin induced polysome disaggregation. While co-incubation with the simple Type B trichothecene DON had no effect on SG uptake into cell cytoplasm, it inhibited SG binding to the ribosome, suggesting that the two toxins bound to identical sites and that SG binding was reversible. Although both SG and DON induced mobilization of p38 and JNK 1/2 to the ribosome, phosphorylation of ribosomal bound MAPKs occurred only after DON treatment. SG association with the 40S and 60S subunits was also observed in the PC-12 neuronal cell model which is similarly susceptible to apoptosis. To summarize, SG rapidly binds small and large ribosomal subunits in a concentration- and time-dependent manner that was consistent with induction of apoptosis.

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    Pieckov E, Hurbnkov M, Cern S, Liskov A, Kovcikov Z, Kollrikov Z, Wimmerov S. Inflammatory and haematotoxic potential of indoor Stachybotrys chartarum (Ehrenb.) Hughes metabolites. Arh Hig Rada Toksikol. 2009 Dec;60(4):401-9. PMID: 20061240

    Mould Stachybotrys chartarum (Ehrenb.) Hughes is known to pose a health risk in indoor environments. Most of its strains can produce several intra- and extracellular trichothecene mycotoxins. Complex secondary metabolites of stachybotrys isolates from mouldy dwellings/public buildings in Slovakia were intratracheally instilled in Wistar male rats (4 microg in 0.2 mL of 0.2 % dimethylsulphoxide; diacetoxyscirpenol as the positive control). After three days, haematological parameters were measured in peripheral blood and inflammatory response biomarkers in bronchoalveolar lavage fluid (BALF), and the results were statistically analysed. Exometabolites proved to suppress red blood cell (RBC), decreasing the total RBC count, haemoglobin, and haematocrit. The exposed rats showed significantly higher total BALF cell count, indicating inflammation, lower alveolar macrophage counts, and increased granulocyte count related to the BALF cells. Due to haematotoxic and inflammation-inducing properties, metabolites of S. chartarum can cause damage to the airways and haematological disorders in occupants of mouldy buildings.

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    Kankkunen P, Rintahaka J, Aalto A, Leino M, Majuri ML, Alenius H, Wolff H, Matikainen S. Trichothecene mycotoxins activate inflammatory response in human macrophages. J Immunol. 2009 May 15;182(10):6418-25. PMID: 19414795

    Damp building-related illnesses have caused concern for years in many countries. Although the problem is extensive, the knowledge of the immunological reactions behind damp building-related illnesses is still quite limited. Trichothecene mycotoxins form one major group of toxins, which possibly contribute to the illnesses. Stachybotrys chartarum is a well-known, but also controversial damp building mold and many strains of this mold are capable of producing trichothecenes. In this report, we have examined the effect of S. chartarum and trichothecene mycotoxins on the proinflammatory cytokine response in human macrophages. As a result, satratoxin-positive S. chartarum activated inflammasome-associated caspase-1, which is needed for proteolytic processing of IL-1beta and IL-18. Furthermore, purified trichothecene mycotoxins, roridin A, verrucarin A, and T-2 toxin activated caspase-1, and these mycotoxins also strongly enhanced LPS-dependent secretion of IL-1beta and IL-18. The satratoxin-positive strain of S. chartarum and the trichothecenes also triggered the activation of caspase-3, which is an effector caspase of apoptosis. Satratoxin-negative S. chartarum was not able to activate either caspase-1 or caspase-3. In conclusion, our results indicate that human macrophages sense trichothecene mycotoxins as a danger signal, which activates caspase-1, and further enables the secretion of IL-1beta and IL-18 from the LPS-primed cells.

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