Introduction

Pulmonary hemosiderosis in infants has been associated with exposure to Stachybotrys chartarum (atra) in 30 infants in Cleveland, Ohio.^1-4 The clinical profile of the infants was as follows: Most presented with severe pulmonary symptoms requiring intensive support; a few infants had less severe lung hemorrhage; three quarters of the infants required ventilator support and blood transfusions; eleven had transitory hemoglobinuria; five patients died and the remainder survived; several patients had continued low-grade alveolar hemorrhage for months after the initial bleeding, even after removal from their original homes.⁵ Moreover, pulmonary hemorrhage in an infant following a two weeks exposure to indoor molds (Penicillium and Trichoderma species) and tobacco smoke was reported in an infant in Greenville, N.C_.6 Finally, Stachybotrys atra (chartarum) was isolated from the home and the lungs of an infant with pulmonary hemorrhage in Houston, Tx.⁷ Several research papers have been published on toxic products from S. atra (chartarum) isolated from homes in Cleveland, Houston, Los Angeles and other countries. The published data demonstrate that in addition to trichothecenes, the S. atra isolates produce a hemolysin and a siderophore.

Hemorrhaging is not a typical response to purified trichothecenes.⁸ Agriculture workers exposed to S. chartarum reported nasal and tracheal bleeding.^9,10 In Eastern Europe and Russia in the 1930s thousand of horses died after consuming Stachybotrys contaminated fodder. The horses also had hemorrhaging.¹¹ Moreover, Stachybotrys was isolated from the spleen, liver, kidney, and lymphatic system of infected horses, indicating the organism is a potential pathogen of animals.¹¹ Finally, as mentioned above, Stachybotrys has been isolated from the lungs of an infant with pulmonary hemosiderosis, implicating its pathogenicity to humans.¹²

This communication will briefly review the current research in the scientific and medial literature on the isolation of and adverse effects of toxins produced by S. chartarum (atra) and their role in lung disease.

Definitions Pertinent to this Review

 

Pulmonary hemosiderosis (PH): The infants in Cleveland and the one in Houston developed pulmonary hemorrhaging and hemosiderosis, characterized by a chronic bleeding (alveolar hemorrhage) with coughing of blood. PH can occur in infants, children and young adults. Hemosiderin-laden macrohages can be present and the disease is often fatal. If the PH is recurrent and untreated it can lead to interstitial fibrosis.

Siderophores: Siderophores are small molecular weight (MW 500-1000) ligands that have a high-affinity for iron assimilation that is specific for ferric iron, and thus, supply iron to the bacterium and/or mold. They are of two basic types: catechols (phenolates) and hydroxamates. Enterobactin (produce by Enterobacteriaceae (e.g. E. coli) is an example of catechols. Ferrichrome is an example of hydroxamates. Hydroxamates are commonly found in Fungii. Siderophores are used to capture iron, which is then internalized in the cell and the iron can then be used for metabolic purposes.

Hemolysins: Hemolysins are proteins produced by many bacteria and some fungi. They are cytolytic, i.e. dissolve red blood cells, releasing organic iron compounds. Examples of bacterial hemolysins are: Streptolysin O and S, and beta hemolysin; Clostridia hemolysins; Staphyloccoccus aureus hemolysins. Fungal hemolysins have isolated from Aspergillus fumigatus and most recently, S. chartarum (atra).

BAL: Bronchoalveolar lavage (BAL) has been used to detect adverse pulmonary effects resulting from exposure to toxic compounds as well as molds. Some of the indicators used in this test are: pulmonary edema (albumin), cytoxicity (lactic acid dehydrogenase [LDH]), polymorpholeukocyte (PMN) activation (myeloperoxidase [MPO], and pulmonary hemorrhaging (hemoglobin). The technique has also been used to study the cellular immune mechanisms of lung diseases: e.g. asthma, reactive airway disease, hypersensitivity pneumonitis.

Pulmonary toxicity of S. chartarum spores

Several studies have been published using mice and rats as models to investigate the effects of toxic vs non-toxic spores from different strains of S. chartarum (atra) on pulmonary tissues. Essentially, these studies show that toxic spores cause greater lung damage when compared to non-toxic spores.

A toxin-producing strain isolated from a Southern California residence was used to investigate the pulmonary toxicity of S. chartarum spores in 10 week old rats. Spores were isolated into four different concentrations ranging from 2 x 10⁶ to 2 x 10⁷ spores per ml of saline.¹³ The spores before saline suspension were extracted with 100% methanol to remove mycotoxins (controls) and compared to non-extracted spores (experimentals) and saline treated animals (controls). The spores were instilled into the trachea under anesthesia. The animals were killed at 24 hours and BAL was performed on controls and experimental animals. Dose response effects were looked for. The results of the study clearly showed that instillation of spores caused the following lung damage: (1) Red tinged BAL fluid at the highest spore concentration occurred, indicating RBC lyses and the release of hemoglobin; and 2) Pulmonary injury and inflammation was demonstrated by a highly significant increased concentration in the BAL fluid of LDH, hemoglobin, albumin, and PMN counts. No trends were observed for MPO and total leukocyte count. Of interest, the hemoglobin concentration was not significantly different between the two spore groups (extracted and non-extracted), while it was different from the saline controls. This seems to indicate that the lyses of red blood cells and or hemorrhaging was not affected by the methanol extraction. Thus the hemolysin activity most likely is not associated with the mycotoxins, since mycotoxins are readily soluble in methanol.

An infant rat (4 days old) model was used to investigate the effects of toxic spores isolated from S. chartarum on developing lung tissues. Spores were either extracted with ethanol (non-toxic spores) or not extracted (toxic spores).¹⁴ The extracted spores and PBS (balanced salt solution) were used as controls. Conidia were instilled intra-tracheally at concentrations of 1.0 to 8.0 x 10⁵ per gram body weight. The lethal does response (LD50) was determined to be 2.7 spores x 10⁵/gram body weight). The results of the study showed minimal effects by ethanol extracted spore and PBS on all parameters studied. Toxic spores at 1.1 x 10⁵ spore/gram body weight caused increased respiratory resistance indicative of inflammation. Histological examination of lung tissues revealed fresh hemorrhage, hemosiderin-laden macrophages, and inflammation with thickened alveolar septa and infiltration of lymphocytes and intra-alveolar macrophages. BAL fluids showed a marked increase in lymphocytes, neutrophils, and hemoglobin. Proinflammatory mediators where increased: IL-1 beta (6-fold) and TNF-alpha (30-fold. Statistical evaluation of the observations showed that the observed changes were highly significant (p values ranged from 0.001 to 0.004). It should be pointed out that ethanol would extract the trichothecenes, but would not extract the hemolysin. The hemolytic activity of S. chartarum is due to a high molecular weight protein (see below).

Mice have also been used as models to investigate the effects of S. chartarum on lung tissues.^15,16 The experimental design (toxic vs non toxic spores) was essentially the same as described for the rat studies discussed above, except the spores were introduced intranasally. The toxic spores were found to contain Satratoxin G & H and stachybotrylactones and -lactams. No tichothecenes were found in the nontoxic spores, but they did contain minor amounts of stachbotrylactones and -lactams. In brief, the toxic spores caused considerable more tissue damage than did the non-toxic spores. Severe inflammatory changes occurred in the bronchioles and alveoli. Hemorrhaging occurred in the alveoli at 1 x 10⁵ spores/mouse. Spores at 1 x 10³/mouse showed less severe inflammatory changes. No inflammatory changes were observed in mice receiving 1 x 10³ non-toxic spores.

Extracts from S. charatrum have been demonstrated to cause an asthma-like response in a BALB/c mouse model. In brief, mice immunized intranasally with an extract from the fungus developed histopathological changes along with BAL changes. The BAL fluid contained increases in total protein, lymphocytes, neutrophils and eosinophils along with a total increase in serum IgE antibodies. Respiratory function studies revealed increased airway resistance and hyperesponsiveness to methacholine challenge.^17-18

These studies provide evidence in an animal model that acute and subchronic challenge exposures to Stachybotrys spores containing toxins cause a dose-related pulmonary response indicative of toxicity, asthma-like condition, and possible pathogenesis. However, no attempt was made to delineate the difference between the effects of hemolysins vs trichothecenes. Most likely the hemorrhaging results from the hemolysins (probably Stachylysin) since trichothecenes do not cause hemorrhaging.⁸

Alveolar Type II cells and lung surfactant

A series of studies, in vitro and in vivo, have shown that S. chartarum spores and isosatratoxin-F interfere with lung surfactant synthesis in juvenile mice. In addition, alveolar Type II cells develop ultrastructural changes consistent with the interruption of surfactant synthesis.^19-22 Type II alveolar cells develop condensed mitochondria, increased cytoplasmic rarefaction, and distended lamellar bodies with irregularly shaped lamellae following exposure. The incorporation of tritium labeled choline into dipalmitoylphoshatidylcholine is reduced. In addition, protein and phosolipid concentrations are increased in BAL fluid. These studies show that S. chartarum spores and isosatratoxin-F significantly affect alveolar Type II cells and homeostasis of lung surfactant. Lung surfactants are particularly important in the new born. They are synthesized shortly after parturition in full-term infants. Surfactants prevent alveoli from collapsing. Thus, decreased surfactants can give rise to respiratory distress syndrome (PDS). PDS is observed in many pre-term newborns and may also occur full term infants.

Stachybotrys: Siderophore and Hemolysin (Stachylysin)

Several scientific papers have been published on a hemolysin isolated from toxic strains of Stachybotrys chartarum. The hemolysin is termed Stachylysin. In addition, one of the studies has demonstrated that S. chartarum also produces a hydoxamate siderophore. The hemolysin cause pores to develop in the RBCs, in cell membranes of nucleated cells (neutrophils, monocytes, endothelial cells), and can effect the aggregation of platelets. Strains of S. chartarum were cultured on either 3/8 inch thick wall board or rice medium at 23 ^oC in these studies.

Hemolytic and toxic activity of several strains S. chartarum has been examined. The strains were obtained from the following: 16 strains came from Cleveland, Ohio, while 12 strains came from other sources (California, Canada, Finland, Egypt, United Kingdom).²³ The Cleveland strains consisted of 5 from homes that had infants (case homes) with pulmonary hemosiderosis (PH) and 7 from homes not reporting PH (control homes). Seven of the strains (five from Cleveland and two non-Cleveland) were consistently highly toxic as determined by an in vitro toxicity test (firefly luciferase). One Cleveland strain and two-non-Cleveland strains were moderately toxic, while the remaining 17 had consistently lower toxicities. In contrast, all 28 strains demonstrated hemolytic activity at 37 ^oC, and were inconsistent at 23 ^oC. The strains from case homes were hemolytic 89% of the time, while control houses were hemolytic 58% of the time at 37 ^oC. At 23 ^oC, the hemolysis was 53% (case homes) and 30% (control homes). The hemolytic activity of strains from case homes vs control homes was significantly different at both 37 ^oC and 23 ^oC (p <0.001). Of interest only three of the 28 strains tested were both highly toxic and consistently hemolytic. All three were from case homes where infants became sick. The data from this study suggests: (1) a combination of toxins and hemolysis may be required to induce PH and (2) after DNA analysis of all strains the three highly toxic and hemolytic strains differed from the remaining strains. Furthermore, Jarvis, et al²⁴ have shown that different strains of S. chartarum produce different quantities and types of various highly toxic trichothecenes.

More recently, both a siderophore and hemolysin have been isolated and characterized from strains of S. chartarum isolated from case homes as compared to control homes. The siderophore is of the hydroxamate-type. The five Cleveland strains and the Houston strain produced significantly more of this siderophore than did strains from control homes.²⁵ In addition, the Houston strain isolated from an infant lung with PH produced a hemolysin.²⁵ Of interest, DNA analysis demonstrated that Houston strain was related to the three highly toxic Cleveland strains discussed above. The hemolysin isolated from S. chartarum has been termed Stachylysin. Stachylysin is a high molecular weight protein containing 114 amino acids and is a pore-forming hemolysin.²⁶ Furthermore, in an earthworm model Stachylysin increased the permeability of blood vessels, causing leakage through blood vessel endothelium and walls.²⁷

Conclusions

S. chartarum has been isolated from the lungs of child with pulmonary hemosiderosis (PH) in Houston, Tx. This strain and others from Cleveland, Ohio where children had PH produce several toxic trichothecenes as well as a hemolysin, Stachylsin, and a hydroxamate siderophore. Toxic spores of S. chartarum cause pulmonary inflammation and hemorrhaging in infant mice and rat models. In addition, toxic spores also cause ultrastructrual changes in pulmonary Type II cells and disrupt surfactant homeostasis in the lungs of infant mice. Extracts from the fungus cause an asthma-like condition and results in the increase of serum IgE in mice. Furthermore, Stachybotrys is pathogenic to horses and causes hemorrhaging in these animals. It is evident from these observations that S. chartarum is pathogenic to animals and probably human infants. Research papers reviewed herein demonstrate that there are highly toxic and hemolytic strains of S. chartarum in case homes in the U.S. and probably other countries as well. Finally, some strains of S. chartarum produce principally macrocylic trichothecenes while others produce altrones. The altrone producing strains are less toxic than those that produced macrocyclic trichothecenes.²⁸

 

1. Etzel RA, et al (1996) Pulmonary hemosiderosis associated with exposure to Stachybotrys atra. Epidemiology 7:S38.

2. Dearborn D et al (1999) An overview of the investigations into pulmonary hemorrhage among infants in Cleveland, Ohio. Environ Health Perspect 107(Suppl):495-499

3. Montana E, et al (1997) Environmental risk factors associated with pediatric idiopathic pulmonary hemorrhage and hemosiderosis in a Cleveland community. Pediatrics 99:E5.

4. Montana E, et al (1995) Acute pulmonary hemorrhage in infancy associated with Stachybotrys atra Cleveland, Ohio. Am J Epidemiol 141:S83.

5. Dearborn DG, et al (2002) Clinical profile of 30 infants with acute pulmonary hemorrhage in Cleveland. Pediatrics 110:627-637.

6. Novotny WE, Dixit A (2000) Pulmonary hemorrhage in an infant following 2-weeks of fungal exposure. Arch Pediatr Adolesc Med 154:271-275.

7. Elidemer O, et al (1999) Isolation of Stachybotrys from the lung of a child with pulmonary hemosiderosis. Pediatrics 104:964-966.

8. Ueno Y (1983) Toxicology, p. 135-177. In: Y. Ueno (ed.) Development in Food Science, Col 4. Trichothecenes: chemical biological and toxicological aspects. Elsevier Scientific Publishing. Amserdam:The Netherlands.

9. Hintikka, E.-L (1978) Human stachybotrycosis, p. 87-89. In: TD Willie, LG Morehouse (eds.) Mycotoxic Fungi, Mycotoxins, Mycotoxicoses, an Encyclopedic Handbook, Vol 3. Marcel Dekker:New York, NY.

10. Sorenos WG, Lewis DM (1996) Organic dust toxic syndrome, p. 159-172. In: K Esser, PA Lempke (eds.) The Mycota, Volume III. Animal and Human Relationships. Pringer Verlag:Berlin, Germany.

11. Forgacs J (1972) Stachybotrycosis, p. 95-128. In: S Kadis, A Ciegler, SHJ Ajl (eds.) Microbial Toxins, Vol III. Academic Press:New York, NY.

12. Sarkisov AV, Orshanskaiya FW (1944) Laboratory diagnosis of Stachybotrys alternans. Veterinariya 21:38-40.

13. Rao CY, Brain JD, Burge HA (2000) Reduction of pulmonary toxicity of Stachybortys charartum by methanol extraction of mycotoxins. Appl Environ Microbiol 66:2817-2721.

14. Yike I, et al (2002) Infant animal model of pulmonary mycotoxicosis by Stachybotrys chartarum. Mycophathologia 154:138-152.

15. Nikulin M, et al (1996) Experimental lung mycotoxicosis in mice induced by Stachybotrys atra . Int J Exper Pathol 77:213-218.

16. Nikulin M, et al (1997) Effects of intranasal exposure to spores of Stachybotrys atra in mice. Fund Appl Toxicol 35:182-188.

17. Korpit A, et al (2002) Effects of erosols from nontoxic Stachybotrys chartarum on murine airways. Inhal Toxicol 14:521-540.

18. Viana ME, et al (2002) An extract of Stachybotrys charatum causes allergic asthma-like responses in a BALB/c mouse model. Toxicol Sci 70:98-109.

19. Rand TG, et al (2002) Microanatomical changes in alveolar type II cells in juvenile mice intrathecally exposed to Stachybotrys chartarum spores and toxin. Toxicol Sci 65:239-245.

20. Mason CD, et al (1998) Effects of Stachybotrys chartaum (atra) oncidia and isolated toxin on lung surfactant production and homeostasis. Nat. Toxins 6:27-33.

21. McCrae JC, et al (2001) Analysis of pulmonary surfactants by Fourier-transform infrared spectroscopy following exposure to Stachybotrys chartraum (atra) spores. Chem Phys Lipids 110:1-10.

22. Masson CD, et al (2001) Effects of Stachybotrys chartarum on surfactant convertase activity in juvenile mice. Toxicol Appl Pharm 172:21-28.

23. Vesper SJ, et al (1999) Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains. Appl Environ Microbiol 65:3175-3181.

24. Jarvis BB, et al (1998) Study of toxin production by isolates of Stachybotrys chartarum and Memnoniella echinata isolated during a study of pulmonary hemosiderosis in infants. App Environ Microbiol 64:3620-3625.

25. Vesper SJ, et al (2000) Quantification of siderophore and hemolysin from Stachybotrys chartarum strains, including a strain isolated from the lung of a child with pulmonary heorrhage and hemosiderosis. Appl Environ Microbiol 66:2678-2681.

26. Vesper SJ, et al (2001) Initial characterization of the hemolysin Stachylysin from Stachybotrys chartarum. Infection and Immunity 69:912-916.

27. Vesper SJ, Vesper MJ (2002) Stachylysin may be a cause of hemorrhaging in humans exposed to Stachybotrys chartarum. Infection and Immunity 70:2065-2069.

28. Jarvis BB (2002) Chemistry and toxicology of molds isolated from water-damaged buildings. In: Devries JW, Tucksess MW, Jackson LS (eds.) Kluwer Academic/Plenum Publishers:New York, pp. 43-52.

This is a brief summary on the poison of the month. For more information either e-mail Dr. Thrasher or call him at (505) 336-8317, New Mexico

 

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