Comparison of Inflammatory and Cytotoxic Lung Responses in Mice after Intratracheal Exposure to Spores of Two Different Stachybotrys chartarum Strains

J. Flemming, B. Hudson and T. G. Rand1

Department of Biology, Saint Mary’s University, 923 Robie St., Halifax, Nova Scotia, Canada B3H 3C3

Received July 7, 2003; accepted December 11, 2003


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

Key Words: Stachybotrys chartarum; spores; inflammation; cytotoxicity; macrocyclic trichothecenes; atranones; intratracheal instillation; mouse.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well known that Stachybotrys chartarum (Ehrenb. ex Link) Hughes (= S. atra Corda) produces three major classes of secondary metabolites each with varying biological activities (Miller et al., in pressGo). However, it has only been recently recognized that S. chartarum can be separated into two chemotypes based on secondary metabolite production profiles (Andersen et al., 2002Go; Nielsen et al., 2002Go; Peltola et al., 2002Go). One chemotype produces macrocyclic trichothecenes; the other produces atranones (and sometimes simple trichothecenes, e.g., trichodermol and trichodermin).

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., 2002Go). 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., 1996Go, 1997Go). 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., 1996Go, 1997Go; Rand et al., 2002aGo) and rats (Yike et al., 2002aGo). 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., 1996Go, 1997Go; Rand et al., 2002aGo, bGo) 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)Go 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., 2002Go; Rand et al., 2002aGo, bGo; Sumarah et al., 1999Go), 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., 2002Go), 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)Go, which showed that the NOAEL for pure satratoxin-H in vitro to be about (0.005 µM), and those of Yike et al. (1999Go, 2002aGo) 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The trichothecene producing Stachybotrys chartarum isolate employed in this study was from Cleveland (Cleveland strain # JS 58-17 (= ATCC 201211) supplied by Dr. D. Dearborn, Rainbow Babies Children’s Hospital, Cleveland, OH). Protein translation inhibition assay revealed this isolate to contain 670fg satratoxin G (SG) equivalents/spore (Yike et al., 1999Go) while the ELISA revealed it to contain 980fg SG equivalents/spore (Chung et al., in pressGo). The atranone-producing S. chartarum (strain # JS 58-06) isolate employed was supplied by Dr. S. Vesper (U.S. Environmental Protection Agency, Cincinnati, OH). The Cladosporium cladosporioides isolate was used as a negative control and was recovered from an outdoor sampling site in Nova Scotia (T.G.R.). The two S. chartarum strains and Cladosporium cladosporioides were cultured on Stachybotrys specific medium© (LabCor, Seattle, WA) at 25°C for three weeks prior to instillation.

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., 2002Go; Rand et al., in press).

Animals.
Random-bred pathogen-free, Carworth Farms white (CFW), Swiss Webster, male mice, 21–28 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, 1993Go) and with approval from the Saint Mary’s 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)Go. 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, 1993Go).

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)Go. 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-{alpha}) analysis.
Concentrations of the proinflammatory and oxidative stress cytokines IL-6 and TNF-{alpha} 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. Tukey’s 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice exposed to spores of C. cladosporioides or S. chartarum strains did not show any clinical signs of illness or respiratory distress.

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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FIG. 1. Pooled data showing dose dependent vascular leakage through changes in both total protein and albumin concentrations in mice BALF. *,**Indicates where concentrations are significantly greater than C. clad controls p <= 0.01, p <= 0.001 respectively. (0 = UTC, 1 = saline, 2 = C. clad. 30 spores/g BW, 3 = C. clad. 300 spores/g BW, 4 = C. clad 3000 spores/g BW, 5 = S. chartarum 58-06 30 spores/g BW, 6 = S. chartarum 58-17 300 spores/g BW, 7 = S. chartarum 58-06 3000 spores/g BW, 8 = S. chartarum 58-17 30 spores/g BW, 9 = S. chartarum 58-17 300 spores/g BW, 10= S. chartarum 58-17 3000 spores/g BW).

 
Figure 2A shows that at the high JS 58-17 spore dose, total protein concentration increased precipitously within 3 h (p < 0.001) to highest levels at 24 h PI then declined to near saline and untreated control levels by 96 h PI. Total protein concentration increase in this treatment group was about 10x higher than in all of the other groups treated with highest spore loads for the first 25–30 h. For JS 58-06 and C. cladosporioides spore treated animals, total protein concentrations increased gradually from 3 h of exposure, to reach maximal levels at 48 h. They then remained elevated for the duration of the experiment. Total protein concentration at 48 h PI was significantly higher for JS 58-06 than for C. cladosporioides (p < 0.05) while at 96 h PI it was higher than in all of the other groups (p < 0.01). At the moderate dose (300 spores/g BW), protein concentration profiles for JS 58-06 and JS 58-17 were similar to those seen in the highest spore dose exposure, although the mean concentrations were lower (Fig. 2B). Protein concentration profiles in animals exposed to the lowest spore dose (30 spores/g BW) of the S. chartarum strains were similar (Fig. 2C) with total protein levels significantly higher throughout the 96 h exposure time (p < 0.05) than those in animals instilled with either the C. cladosporioides spores or saline.



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FIG. 2. Time profile for total protein concentration in BALF of mice. A = 3000 spores/g BW, B = 300 spores/g BW, C = 30 spores/g BW. a-S. chartarum 58-17 significantly greater than all treatments, b-S. chartarum 58-17 and 58-06 treatments, c-S. chartarum 58-06 significantly greater than all treatments. —— saline —–— C. cladosporioides –––––S. chartarum 58-06 -–-–-–- S. chartarum 58-17.

 
Proinflammatory Cytokines
Interleukin-1ß (IL-1ß), tumor necrosis factor-{alpha} (TNF-{alpha}), and interleukin-6 (IL-6) concentrations for all treatment groups were generally highest at 6 h PI and are shown in Table 1. The exceptions were for the IL-1ß and TNF-{alpha} concentration in mice treated with the two highest doses of S. chartarum (both strains), which showed highest levels at 24 h PI. The TNF-{alpha} data revealed a dose dependent-like response pattern for the C. cladosporioides treated animals, but not for the JS 58-06 or JS 58-17 spore treated animals. In these two last-mentioned treatment groups, highest TNF-{alpha} concentrations at 6 h PI were in the groups receiving 300 spores/ g BW. Interleukin-1 (IL-1 ß) concentrations at 6 h were significantly increased only in animals exposed to highest JS 58-06 spore load (p < 0.02). Interleukin-6 (IL-6) levels at 6 h PI also 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 (Table 1). Highest IL-6 production was in animals exposed to the 3000 JS 58-17 spores/ g BW (p < 0.001).


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TABLE 1 Mean Cytokine Concentrations in BALF for All Treatments at 6 h PI

 
TNF-{alpha} concentration in BALF from animals exposed to highest C. cladosporioides and S. chartarum spore loads increased rapidly (p <= 0.001) at 3 h compared to that in the saline treated animals (Fig. 3A). Concentration peaked at 6 h in C. cladosporioides spore exposed animals, and at 24 h in JS 58-06 and JS 58-17 treated animals. TNF-{alpha} levels associated with high dose JS 58-17 spore exposures were lower than those in animals exposed to high C. cladosporioides spore loads until 6 h PI (p <= 0.05) and in JS 58-06 exposed animals until 48 h PI (p < 0.01). At 96 h PI, TNF-{alpha} levels in both S. chartarum spore treated animals were similar and significantly higher than in C. cladosporioides and saline treated animal groups (p < 0.05). In moderate dose JS 58-17 treated animals, TNF-{alpha} production peaked (p < 0.05) at 24 h followed by decline to nonsignificant levels at 96 h PI. For 58-06 treated animals, TNF-{alpha} was highest at 6 h and remained significantly elevated (p < 0.05) until 96 h PI (Fig. 3B). TNF-{alpha} concentration profiles in animals exposed to the low spore dose of both S. chartarum strains were similar. Concentrations were highest at 3 h PI declining to levels similar to C. cladosporioides and saline treated animals (p < 0.05) by 48 h for JS 58-17, and by 96 h PI for JS 58-06 (Fig. 3C).



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FIG. 3. Time profile for TNF-{alpha} concentration in BALF of mice. A= 3000 spores/g BW, B = 300 spores/g BW, C = 30 spores/g BW. a-S. chartarum 58-17 significantly greater than all treatments, b-S. chartarum 58-17 and 58-06 significantly greater than all treatments, c-S. chartarum 58-06 significantly greater than all treatments, d-S. chartarum 58-17 and 58-06 and C. cladosporioides significantly greater than saline. —— saline —–— C. cladosporioides –––––S. chartarum 58-06 -–-–-–- S. chartarum 58-17.

 
IL-1ß concentrations in animals exposed to the highest doses of S. chartarum (both strains) increased rapidly to highest levels at 24 h PI (p < 0.01; Fig. 4A). Thereafter, concentrations in animals exposed to JS 58-06 spores remained significantly elevated (p < 0.05) compared to all other treatments for 96 h PI, while those in JS 58-17 exposed animals declined to nonsignificant levels by 48 h PI. IL-1ß concentration profiles in animals exposed to the moderate and low doses of S. chartarum (both strains) were similar (Figs. 4B and 4C). In these groups, IL-1ß concentrations increased rapidly and peaked within 6 to 24 h PI for JS 58-17, or 24 to 48 h for JS 58-06 (p < 0.05), followed by decline to nonsignificant levels within 96 h PI.



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FIG. 4. Time profile for Il-1ß concentration in BALF of mice. A = 3000 spores/g BW, B = 300 spores/g BW, C = 30 spores/g BW. a-S. chartarum 58-17 significantly greater than all treatments, b-S. chartarum 58-17 and 58-06 significantly greater than all treatments, c-S. chartarum 58-06 significantly greater than all treatments. —— saline —–— C. cladosporioides –––––S. chartarum 58-06 -–-–-–- S. chartarum 58-17.

 
IL-6 concentration profiles in animals receiving high, moderate, and low doses of fungal spores (Figs. 5A–5C) were similar. At all spore doses, IL-6 concentrations increased rapidly to highest levels within 6 h PI then declined and leveled off at about 24 h PI. However, IL-6 concentration was highest (p < 0.001) in animals exposed to the highest JS 58-17 spore dose, and about 10x higher than in the other treatment groups. It was also significantly elevated (p < 0.05) but only in animals instilled with low JS 58-17 spore dose at 6 h PI (Fig. 5C).



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FIG. 5. Time profile for Il-6 concentration in BALF of mice. A = 3000 spores/g BW, B = 300 spores/g BW, C = 30 spores/g BW. a-S. chartarum 58-17 significantly greater than all treatments. —— saline —–— C. cladosporioides –––––S. chartarum 58-06 -–-–-–- S. chartarum 58-17

 
Cytotoxicity
At highest spore loads, LDH concentration in JS 58-17 treated animals reached maximal levels (p < 0.05) at 24 h PI then declined to nonsignificant levels starting at 48 PI. In the JS 58-06 spore exposed animals LDH gradually increased from 3 h PI to significant levels at 48 h PI (p < 0.05) and maximal levels at 96 h (p < 0.01; Fig. 6A). LDH concentration pattern in C. cladosporioides exposed animals was similar to that of JS 58-17, although significant LDH changes were not detected. LDH concentrations in low dose S. chartarum (both strains) exposures were similar. Compared to the saline and C. cladosporioides spore treated animals, LDH in the S. chartarum treated animals increased to significantly elevated levels (p < 0.01) at 6 h PI and maximal levels (p < 0.001) at 96 h PI.



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FIG. 6. Time profile for LDH concentration in BALF of mice. A= 3000 spores/g BW, B = 30 spores/g BW. a-S. chartarum 58-17 significantly greater than all treatments, b- S. chartarum 58-17 and 58-06 significantly greater than all treatments, c- S. chartarum 58-06 significantly greater than all treatments, d- S. chartarum 58-17 and 58-06 and C. cladosporioides significantly greater than saline. —— saline —–— C. cladosporioides –––––S. chartarum 58-06 -–-–-–- S. chartarum 58-17.

 
Pearson correlation coefficient results for the pooled BALF data from high, moderate, and low spore dose treatment groups are summarized in Table 2. They reveal that the strongest positive associations were between protein and albumin (r = 0.757), protein and IL-6 (r = 0.682), and IL-6 and albumin (r = 0.510). The weakest association was between LDH and IL-6 (r = 0.012).


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TABLE 2 Pearson Correlation Matrix for All Inflammatory Parameters

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study has revealed that there are marked differences in vascular, inflammatory, and cytotoxic lung responses in mice intratracheally exposed to Cladosporium cladosporioides or Stachybotrys chartarum spores even at low spore doses. This result was not unexpected since previous studies have revealed significant differences in lung cyto- and histopathology (Rand et al., 2002, in press), associated with exposures to spores of these two species. Nevertherless, it was interesting that the NOAEL for the two species was so different. While the NOAEL for C. cladosporioides was between 300 and 3000 spores/g BW, it was less than 30 spores/g BW for both S. chartarum strains. This result, combined with those of the previous studies showing that the magnitude as well as temporal patterns of biochemical, anatomical, and gross pathological lung responses toward exposures to both of these spore types in mice is different (Gregory et al., 2002Go; Mason et al., 1998Go, 2001Go; McCrae et al., 2001Go; Rand et al., 2002, in press), further supports the position that exposure outcome is largely attributable to differences in spore toxicity. However, results showing that exposure of mice to the high C. cladosporioides but not to high S. chartarum 58-17 spore dose stimulated rapid TNF-{alpha} production also indicates that factors other than toxins may be involved. Jussila et al. (2002b)Go reported high TNF-{alpha} production in the BALF of mice exposed to Penicillium spinulosum, a nontoxigenic species, which they attributed to the high ß1, 3 glucan content in the spore walls of this species. Whether differences in ß1, 3 glucan content in the walls of spores of the two species accounts for at least some of the lung response differences is unknown and deserves further consideration.

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)Go 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, 1983Go). Additionally, it may also reflect differences in degree of sensitivity of the assays used in the respective studies. Rao et al. (2000b)Go 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., 1998Go; Jussila et al., 2001Go, 2002aGo, bGo, cGo; Ruotsalainen et al., 1998Go). Increased total protein and albumin concentrations in BALF are indicative of vascular leakage (Rhoades and Pflanzer, 1996Go) and have been reported in mice (Mason et al., 2001Go) and rats (Rao et al., 2000aGo, bGo) exposed to S. chartarum spores. Rand et al. (2002b)Go 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 12–24 h PI (Rand et al., 2002bGo) 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, 1997Go). 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-{alpha} 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-{alpha} 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, 1996Go). These toxins are also lost quickly (within minutes) from spores (Hinkley and Jarvis, 2000Go) and incorporated into lung cells including alveolar macrophages whereupon they bind to ribosomes (Rand et al., in pressGo). In vitro studies have clearly demonstrated that these toxins suppress cytokine synthesis in macrophages (Nielsen et al., 2002Go; Sorenson et al., 1987Go). Given that the activated macrophage is the predominant source of both IL-1ß and TNF-{alpha} in vivo, (Kuby, 1997Go) 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-{alpha}). 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., 2001Go).

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., 1987Go). 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)Go recently reported that S. chartarum spores contain high concentrations of chymotrypsin-like serine proteinases, which they identified as stachyrase A. Yike et al. (2002b)Go 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)Go and Yike et al. (2002b)Go have been shown to cleave a number of collagen types and other structural proteins found in the lung environment (Kordula et al., 2002Go). Rand et al. (2002b)Go 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, 1996Go), as well as invasive pulmonary mycosis caused by Aspergillus fumigatus (Washburn, 1996Go). 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.


    ACKNOWLEDGMENTS
 
This work was supported by an NSERC operating grant administered to T.G.R.


    NOTES
 

1 To whom correspondence should be addressed. Fax: (902) 475-1982. E-mail: thomas.rand{at}smu.ca


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