Department of Biology, Saint Marys University, 923 Robie St., Halifax, Nova Scotia, Canada B3H 3C3
Received July 7, 2003; accepted December 11, 2003
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ABSTRACT |
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Key Words: Stachybotrys chartarum; spores; inflammation; cytotoxicity; macrocyclic trichothecenes; atranones; intratracheal instillation; mouse.
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INTRODUCTION |
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In vitro studies have revealed that the macrocyclic trichothecene producing isolates are highly cytotoxic while the atranone producers are associated with induction of strong inflammatory responses (Nielsen et al., 2002). In vivo studies have shown that histopathogical responses of mouse lung exposed to spores of a macrocyclic trichothecene-producing isolate are more severe than those associated with lungs exposed to spores of a nontrichothecene producing isolate (Nikulin et al., 1996
, 1997
). They have also revealed that exposure to Stachybotrys spores (both chemotypes) can result in massive lung damage with acute lethality in both mice (Nikulin et al., 1996
, 1997
; Rand et al., 2002a
) and rats (Yike et al., 2002a
). However, the in vivo experiments have been generally conducted beyond the maximum tolerated dose, using from about 3000 spores/g body weight (BW) in mice (Nikulin et al., 1996
, 1997
; Rand et al., 2002a
, b
) to some 100,000 spores/g BW in rats (Rao et al., 2000, Yike et al., 2002). Not only are these doses higher than those encountered by humans in most contaminated environments (Miller et al., 2003), use of high spore loads makes it unclear whether lung damage is a consequence of the cytotoxic and/or inflammatory toxic properties associated with the spores administered to the animals.
While there is an abundance of information concerning exposure outcome in animals at high spore doses, there is surprisingly little on the effects of low S. chartarum spore doses. Rao et al. (2000b) revealed that exposure doses of about 3000 spores/g BW represented the no adverse effect level (NOAEL) in Charles River-Dawley rats. However, whether these investigators were using a macrocyclic trichothecene- or an atranone-producing S. chartarum isolate is unknown. Interestingly, in mice, exposure to 3000 S. chartarum or Cladosporium cladosporioides (= nontoxigenic phylloplane fungus) spores/g BW results in a variety of pathological lung changes (Gregory et al., 2002
; Rand et al., 2002a
, b
; Sumarah et al., 1999
), indicating that the NOAEL for spores of these two species in mice is lower than it is for S. chartarum in Dawley rats. What constitutes the NOAEL for S. chartarum exposures in mice and whether it is the same for the two S. chartarum chemotypes and C. cladosporioides is unknown.
The purpose of this study was to use bronchoalveolar lavage fluid (BALF) to investigate dose-response and time-course relationships between exposure to macrocyclic trichothecene- (JS 58-17) and atranone-producing (JS 58-06) S. chartarum strains and Cladosporium cladosporioides spores, and vascular, inflammatory and/or cytotoxic lung responses in mice. Based on results of our previous studies, we hypothesized that the vascular, inflammatory, and cytotoxic responses in mice exposed to S. chartarum spores would be significantly more severe than those associated with C. cladosporioides (nontoxigenic) spore exposures. Based on in vitro assays (Nielsen et al., 2002), we also hypothesized that cytotoxicity would be significantly more pronounced in mouse lungs exposed to spores of the macrocyclic trichothecene producer compared to that associated with those of the atranone producer. Lastly, based on the in vitro studies of Sorenson et al. (1987)
, which showed that the NOAEL for pure satratoxin-H in vitro to be about (0.005 µM), and those of Yike et al. (1999
, 2002a
) that showed that spores of the macrocyclic trichothecene producing S. chartarum strain we used in our studies to contain 670 fg satratoxin G (SG) equivalents/spore, we hypothesized that the NOAEL for S. chartarum exposure in mice would be near the in vitro NOAEL for satratoxin H, which is equivalent to the satratoxin H content in about 30 spores/g BW in a 25 g wt. animal.
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MATERIALS AND METHODS |
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Spores of all these species were harvested by gently flooding the cultures with sterile physiological saline (0.9% NaCl), followed by gentle agitation to lift the spores but not mycelium from the medium surface. Spores were then collected with a glass pipette and suspended in sterile physiological saline. Spore concentrations were then determined using a hemacytometer and diluted to a working concentration of 1.5 x 106 spores/ml. To minimize toxin loss from spores, spore suspensions were used in the instillation experiments within 1 h of preparation. Previous work has shown that spores of both S. chartarum strains suspended in saline for this time will still retain toxins (Gregory et al., 2002; Rand et al., in press).
Animals.
Random-bred pathogen-free, Carworth Farms white (CFW), Swiss Webster, male mice, 2128 days old, (25.3 ± 1.2 g BW) were used in this experiment. The mice were housed according to the standards of the Canadian Council for Animal Care (CCAC, 1993) and with approval from the Saint Marys University and Dalhousie University Animal Care Committees. The mice were given food and water ad libitum and acclimatized for one week prior to use.
Intratracheal instillations.
A total of 260 mice were used in this experiment, including ten control (untreated) animals. The treatment animals (S. chartarum strains JS 58-17 and JS 58-06, C. cladosporioides and saline) were separated into groups of five mice. The mice were then lightly anesthetized with a 0.2 ml im injection of a mixture containing an average of 124.56 mg/kg of ketamine (Ketaleen) and 8.78 mg/kg of xylazine (Rompun) in physiological saline. Once anesthetized, each mouse was weighed to the nearest 0.1 g and placed upright dorsal side down, on an intratracheal instillation board seated 20° from the vertical as described in Mason et al. (1998). Each mouse with the exception of untreated control (UTC) mice was instilled with 50 µl of S. chartarum (strains 58-17 or 58-06) or C. cladosporioides spores or 0.9% NaCl. For the dose dependent study, groups of mice were given spore concentrations at 30, 300, or 3000 spores/g BW. Dilutions were made appropriately from the original stock solution (1.5 x 106 spores/ml). Mice were left in the upright position on the instillation board for approximately 2 min and were then put back into their cages on a warm pad and allowed to recover for 2 h. During recovery, mice were continuously monitored for signs of sickness or distress as outlined in CCAC guidelines (CCAC, 1993
).
Bronchoalveolar lavage fluid (BALF) recovery.
The treatment mice were killed after 3, 6, 24, 48, and 96 h post instillation (PI) using a 300 µl intraperitoneal injection of 65 mg/ml of sodium pentobarbital (Somnotol). They were immediately weighed to the nearest 0.1g. They were then exsanguinated by cutting the abdominal artery. The mouse lungs were then lavaged with 0.9% physiological NaCl in 4 x 0.8ml aliquots as previously described in Mason et al. (1998). BALF was then distributed into four 1.5 ml microcentrifuge tubes into which an anti-proteinase mixture was added as follows: 100 mM phenylmethylsulfonyl fluoride (PMSF) in iso-propanol was first added to each of the four tubes at a concentration of 10 µl/mlBALF, followed by 500 mM of ethylenediamine tetraacetic acid (EDTA) in ddH20 per 10 µl/mlBALF. The BALF sample tubes were then flash frozen in liquid N2 and stored at 36°C.
Total protein, albumin and lactate dehydrogenase (LDH) analysis.
Total protein concentration in BALF, an indicator of epithelial and cell membrane integrity, was quantified using the modified Lowry Method Protein Assay Kit (Sigma Chemical Co.). Briefly, this involved a modified trichloro-acetic acid protein precipitation reaction followed by a modified Lowry method for colorimetric concentration determination. Sample absorbance was recorded on a Novaspec Spectrophotometer© at a wavelength of 600 nm. For total protein, BALF samples were diluted 50:50 with dH20.
Albumin concentrations in BALF, an indicator of vascular permeability, were quantified using a quantitative enzyme linked immunosorbent assay (ELISA) purchased from Bethyl Laboratories, Inc. Samples were diluted 1:800 in diluent (Tris-buffered saline [TBS] with 1% bovine serum albumin [BSA], 0.05% Tween 20, pH 8). Final absorbencies were determined using a Bio-Tek Instruments© ELx800 automated microplate reader at a wavelength of 450 nm.
LDH levels were measured as an indicator of cytotoxicity. Concentrations were determined using the enzyme based Cytotoxicity Detection Kit (Roche Diagnostics). A standard curve was generated for quantification of LDH levels. This was prepared by serially diluting an L-LDH solution (Roche Diagnostics) in physiological 0.9% NaCl to concentrations of 10, 25, 50, 75, 100, 125, and 150 ng/ml. The final absorbance of the samples was determined at the 490 nm wavelength using the microplate reader.
Proinflammatory and oxidative stress cytokine (IL-1ß, IL-6, and TNF-) analysis.
Concentrations of the proinflammatory and oxidative stress cytokines IL-6 and TNF- in BALF were determined using sandwich ELISA procedures purchased from BD Biosciences. Assay diluent and tetramethyl benzoate (TMB) substrate reagent sets were also purchased from BD Biosciences. All standard curves for ELISA kits were generated according to respective kit instructions. IL-1ß concentrations were determined with a sandwich ELISA kit purchased from R&D Systems. The assay diluent used was a solution of TBS with 3% BSA and 0.05% Tween 20, pH 7.3). Samples were diluted appropriately in each assay and final absorbances were read at the 450 nm wavelength using the microplate reader.
Statistical analysis.
All data were tested for normality using a normal probability plot. Weight measurements followed normal distribution while all other data were transformed to improve normality and homoscedasticity using a log transformation. Significant differences between and amongst treatment groups were determined using one- or two-way ANOVA. The factor for one-way ANOVA was treatment, and for two-way ANOVA the factors were treatment and time. Tukeys multiple comparison test was used to find the location of any differences between means of treatment and control groups. The results are expressed as the mean ± SE. Correlation coefficients for all BALF parameters measured and pooled as a value for each treatment group were determined using Pearson correlation matrix to determine the nature and strength of any relationships. All tests were carried out using Systat version 5.1, and were considered significant at the 0.05 probability level.
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RESULTS |
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Vascular Leakage
Total protein and albumin concentrations in bronchoalveolar lavage fluid (BALF) from untreated control, saline, and low, moderate, and high spore dose treatment animals are shown in Figure 1. Total protein concentration was significantly increased in mouse lungs exposed to high dose S. chartarum strain 58-17 compared to all other treatments (p 0.001). Albumin concentrations were significantly increased in mouse lungs exposed to high dose S. chartarum strain 58-06 (p
0.001) and medium (p
0.01) and high (p
0.001) dose S. chartarum strain 58-17 compared to all other treatments (p
0.001). Total protein and albumin concentration profiles were similar and showed dose dependent-like profiles with lowest response levels in low C. cladosporioides and S. chartarum (both strains) spore dose animals and highest response in high spore dose animals. Figure 1 also indicates that the majority of the increased total protein in the BALF was albumin.
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DISCUSSION |
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The fact that we found the NOAEL response for S. chartarum exposures (both chemotypes) in mice to be less than 30 spores/g BW was surprising since Rao et al. (2000b) reported the NOAEL to be about 3000 S. chartarum spores/g BW in rats. This difference in result may reflect differences in animal species susceptibility (Ueno, 1983
). Additionally, it may also reflect differences in degree of sensitivity of the assays used in the respective studies. Rao et al. (2000b)
did not use ELISA assays but instead more insensitive biochemical and microscopic assays for inflammation such as hemogloblin and leukocyte differential counts, respectively.
Lung response patterns toward exposures to high and moderate spore doses of the two S. chartarum chemotypes also differed widely. While vascular, inflammatory, and cytotoxic responses in C. cladosporioides spore treated animals showed dose dependent-like responses in all parameters tested except IL-1, for both S. chartarum strains, responses showed dose dependency for only total protein, albumin, and IL-6. Dose dependent-like response patterns are not unusual and appear to reflect the magnitude of stimulation, especially in single exposure studies. They have been demonstrated in a variety of in vivo animal exposures to nonbiological particulates, bacteria, and fungi (Finch et al., 1998; Jussila et al., 2001
, 2002a
, b
, c
; Ruotsalainen et al., 1998
). Increased total protein and albumin concentrations in BALF are indicative of vascular leakage (Rhoades and Pflanzer, 1996
) and have been reported in mice (Mason et al., 2001
) and rats (Rao et al., 2000a
, b
) exposed to S. chartarum spores. Rand et al. (2002b)
proposed that increased total protein concentration in BALF might be due to granuloma formation resulting in increased blood perfusion pressure and vascular deficit in the affected lung. However, that significant protein exudation into BALF was within 3 h PI whereas granuloma formation is apparent only after 1224 h PI (Rand et al., 2002b
) suggests that protein exudation may be due to some endogenously derived substances produced in the lung. The strong correlation between total protein and albumin production and IL-6 concentration in BALF suggests that this cytokine may be involved. IL-6 is produced by activated alveolar macrophages, fibroblasts, and endothelial cells and results in increased vascular permeability when these cells are stimulated by foreign particulates (Kuby, 1997
). Whether other factors, such as endothelins, are associated with increased vascular permeability in lungs exposed to S. chartarum spores is unknown and deserves further attention.
While dose dependent-like response patterns were evident for total protein, albumin, and IL-6 levels, they were lacking for LDH in S. chartarum treated animals (both strains). LDH is a biomarker of cell damage and death. Detection of highest LDH concentrations in BALF from animals receiving lowest spore loads indicates that even low spore doses are potently cytotoxic. Moreover, that LDH production was a response in animals exposed to spores of both S. chartarum chemotypes does not support the position that the macrocyclic trichothecene producing strain (58-17) is more cytotoxic than the atranone producer (58-06).
Dose dependent-like responses were also lacking for IL-1ß and TNF- concentrations in the JS 58-17 spore treated animals and for IL-1ß in the JS 58-06 instilled animals. Differences between dose dependency profiles for the pooled TNF-
production in mice exposed to the two S. chartarum strains provides evidence that disease outcome towards both strains is different. These response differences may reflect differences in the potency and pharmacokinetics of the toxins found in the spores of each of these strains. For example, the macrocyclic trichothecene toxins sequestered in spores of JS 58-17 are amongst the most potent protein synthesis inhibitors known (Riley and Norred, 1996
). These toxins are also lost quickly (within minutes) from spores (Hinkley and Jarvis, 2000
) and incorporated into lung cells including alveolar macrophages whereupon they bind to ribosomes (Rand et al., in press
). In vitro studies have clearly demonstrated that these toxins suppress cytokine synthesis in macrophages (Nielsen et al., 2002
; Sorenson et al., 1987
). Given that the activated macrophage is the predominant source of both IL-1ß and TNF-
in vivo, (Kuby, 1997
) exposure to high enough concentrations of these toxins may result in decreased cytokine expression. Differences in disease outcome, especially inflammatory responses, toward both S. chartarum strains were also seen in the temporal concentration changes of cytokines. What is clear from these data is that at moderate and high spore doses, S. chartarum JS 58-17 spores evoked relatively fast lung responses, generally within 24 h PI followed by decline. Stachybotrys chartarum JS 58-06 spore exposure stimulated responses, the magnitude of which either increased throughout the 96 h period (total protein, albumin, LDH) or remained at significantly elevated levels for this time period (IL-1ß, TNF-
). Differences in temporal responses may have to do with the nature of the pharmacokinetics of each of the toxins. While trichothecenes are considered to act on cells rapidly leading to cell death or to depressed protein synthesis, at least one of the toxins sequestered in spores of JS 58-06, stachylysin, is known to act slowly causing cell membrane disruption after more long-lasting exposure (Vesper et al., 2001
).
An important result of the study was finding that exposure to low spore doses (30 spores/g BW) of the two S. chartarum strains still precipitated responses that were significantly higher than those associated with C. cladosporioides and saline exposures. However, differences in the inflammation response in mice towards the two S. chartarum strains at the low spore concentration are unapparent. This result is interesting because concentrations of macrocyclic trichothecenes in the 30 spore/g BW exposure of S. chartarum JS 58-17 are less than that associated with the NOAEL in in vitro exposures (Sorenson et al., 1987). The commonalities in the response profiles towards the lowest dose suggest that chemicals other than trichothecenes, atranones, and hemolysins sequestered in spores may be contributors of lung pathogenesis. Possibly, these substances include proteinases. Kordula et al. (2002)
recently reported that S. chartarum spores contain high concentrations of chymotrypsin-like serine proteinases, which they identified as stachyrase A. Yike et al. (2002b)
reported that the spores of both S. chartarum JS 58-17 and JS 58-06 contain high concentrations of proteinases although they did not fully characterize them. These proteinases reported by Kordula et al. (2002)
and Yike et al. (2002b)
have been shown to cleave a number of collagen types and other structural proteins found in the lung environment (Kordula et al., 2002
). Rand et al. (2002b)
also reported depressed collagen iv expression in lung granulomas surrounding S. chartarum spores, which they attributed to serine proteinase activity. Serine proteinases are associated with some entomopathogenic fungal diseases (Khachatourians, 1996
), as well as invasive pulmonary mycosis caused by Aspergillus fumigatus (Washburn, 1996
). These enzymes may also be contributing to some of the inflammatory responses in the animals exposed to low S. chartarum spore doses. This obviously deserves attention owing to its implication in understanding lung disease onset associated with S. chartarum spore exposures in humans and animals.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (902) 475-1982. E-mail: thomas.rand{at}smu.ca
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