* Department of Biology, Saint Mary's University, 923 Robie Street, Halifax, Nova Scotia, Canada B3H 3C3; and
Department of Physiology and Department of Obstetrics and Gynaecology, Dalhousie University, Halifax, Nova Scotia, Canada B3J 3G9
Received August 3, 2000; accepted November 6, 2001
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ABSTRACT |
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Key Words: ultrastructure; alveolar type II cells; Stachybotrys chartarum; trichothecenes; intratracheal instillation; fungal conidia; morphometrics.
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INTRODUCTION |
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We have also been studying a mouse model of lung injury induced by exposure to S. chartarum spores and have shown that pulmonary alveolar type II cells are sensitive to exposure to Stachybotrys spores and isosatratoxin-F (see Mason et al., 1998; McCrae et al., 2001
; Sumarah et al., 1999
). We have shown that alveolar type II sensitivity is manifest at the biochemical level as changes in alveolar surfactant phospholipid composition and concentration (McCrae et al., 2001
; Sumarah et al., 1999
), and in surfactant production and homeostasis (Hastings et al., 2001
; Mason et al., 1998
, 2001
). One of the interesting results of some of this work is that exposure of lungs to S. chartarum spores leads to increased release of newly secreted surfactant and accumulation of metabolically used surfactant fractions (Mason et al., 1998
). Reasons why increased surfactant secretion by alveolar type II cells is a response to exposure to S. chartarum are unclear. However, it may be that increased alveolar surfactant production in mice intratracheally exposed to S. chartarum spores is due to changes in organelle structure and function in alveolar type II cells. It is known that exposure of rats to bacterial lipopolysaccharide (LPS) results in ultrastructural changes in alveolar type II cells in rats, especially in cellular, lamellar body, and mitochondrial profiles (see Fehrenbach et al., 1998
; Lopez et al., 1987
; Young and Nicholls, 1996
). The objectives of this study were to determine whether a single intratracheal exposure of juvenile mice to S. chartarum spores and toxin results in ultrastructural and morphometric changes in alveolar type II cells.
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MATERIALS AND METHODS |
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For in vivo analysis, random-bred, pathogen-free Carworth Farms White (CFW) male mice were obtained. The animals were housed in accordance with the standards set forth by the Canadian Council for Animal Care (CCAC, 1993), and with the approval of the Saint Mary's University Animal Care Committee.
Intratracheal instillations.
Control and treatment mice were separated into groups of 56 mice. Preweighed 2128-day-old juvenile mice were anesthetized via an intramuscular injection containing ketamine (Ketaleen) and xylazine (Rompun) as previously described by Mason et al. (1998).
With the exception of untreated controls, each mouse was inoculated intratracheally with 50 µl of 1 of the following treatments: 0.9% NaCl, 0.02 µg isosatratoxin-F/ml (= 0.7 ng toxin or 35 ng/kg BW/animal), S. chartarum or C. cladosporioides spores (1.4 x 106 conidia/ml = 70,000 spores/animal), as described by Mason et al. (1998). Based on previous work by Sorenson et al. (1987) who determined that 1 mg S. chartarum spore dust can contain from 7.2 to 12.7 ng satratoxin/mg dust we estimated toxin concentration in the S. chartarum spore loads administered to mice to be about 0.5 ng toxin or 25 ng/kg BW satratoxin equivalents/animal.
The mice were placed back into their cages immediately after instillation. During recovery, mice were continuously monitored for signs of sickness or distress as outlined in the CCAC guidelines (CCAC, 1993). If the animals showed distress, they were immediately euthanized using a sodium pentobarbital (65 mg/ml) overdose and excluded from the study.
Fixation.
Mice were killed with a 300 µl ip injection of sodium pentobarbital (65 mg/ml), 24 and 48 h postinoculation. These times were chosen because results of our previous studies (see Mason et al., 1998; Sumarah et al., 1999
) indicated that the most significant changes in surfactant composition and homeostasis were manifest between 24 and 48 h postinoculation. Once euthanized, each mouse was immediately weighed then placed ventral side up. The body cavity was opened and the abdominal artery severed for exsanguation. The chest cavity was then exposed and the lungs degassed by puncturing the pleural sac. The trachea was cannulated using an 18-gauge butterfly needle. The needle was connected by plastic tubing to a 30 ml syringe filled with a fixative mixture of 1.5% glutaraldehyde and 1.5% freshly prepared paraformaldehyde in 0.1 M cacodylate buffer (pH 7.35; Bozzola and Russell, 1992
), for fixation by positive pressure instillation at 18 cm H2O as described by Davies (1980), for 30 min. After this time, the inflated lungs with instilled fixative were then carefully excised from the thoracic cavity in toto, disconnected from the catheter, and placed into a 250-ml beaker filled with the fixative solution for 12 h before processing for transmission electron microscopy (TEM). After primary fixation, the right cranial lung lobe from each mouse was excised and cut into roughly 1 mm3 sections. Pieces from each sectioned mouse lung were randomly selected, washed in 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide (OsO4) in buffer for 1 h, washed in buffer, dehydrated through an ascending acetone series, and flat-embedded in Epon-Araldite.
Thin sections (gray to silver interference colors) were cut from at least 3 blocks of embedded lung per animal using a Reichert UM 2 Ultramicrotome. Sections were mounted onto #300 mesh copper grids, stained using 5% uranyl acetate and Reynold's lead citrate (see Bozzola and Russell, 1992), and examined using a JOEL 100 STEM microscope at 6600 magnifications and operated at 80 kV.
For morphometrics, 10 to 12 alveolar type II cells per animal, for a total of between 50 and 60 cells per group, were identified and photographed using 70 mm Kodak plate film at 6600 magnifications. Care was taken to ensure that each cell photographed included a lumenal surface and nuclear profile. Electron micrographs of alveolar type II cells for analysis were either printed on Ilford Polycontrast 8 x 11-in. paper or scanned using a flat-bed scanner at a final magnification x16,500 and used to determine the volume density of alveolar type II cells, lamellar bodies, mitochondria, and nuclei.
Point counting with an unbiased double square lattice grid was used to determine relative volume density measurements. For this, a transparent, unbiased lattice grid with 112 lines, each 1-cm long, was superimposed over alveolar type II (PII) cell images. Electron micrograph images were identified by number only to ensure that evaluations were unbiased. Volume density (points per structure) was considered for alveolar type II cells (VPII), lamellar bodies (VLB), mitochondria (Vmito), and nuclei (Vnuc) using the appropriate formulae (see Bozzola and Russell, 1992; Davies, 1980
; Weibel and Cruz-Orive, 1997
). Points for cells or organelles were only considered when intersecting grid lines fell entirely within the structure observed. Fraction of the cell cytoplasm occupied by lamellar body or mitochondria was calculated using the following formula: Vv (fraction of cytoplasm occupied by organelle) = (points counted on organelle (Porganelle) ÷ (total points on alveolar type II cell profile (PT) points counted on nucleus (Pnuc)). Data were analyzed using normality of variance and 2-way ANOVA, and at 95% (p
0.05) confidence level.
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RESULTS |
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Ultrastructure
Alveolar type II cells from untreated controls, saline and C. cladosporioides treated animals were similar (Figs. 1A and 1B). Generally, the cytoplasm of cells from these animals was moderately electron dense. The cells supported a centrally to basally located nucleus with peripherally dispersed chromatin and usually a prominent, centrally located nucleolus. The cells contained moderate numbers of elongate mitochondria, and well-defined, separate, membrane bound lamellar bodies. The surface of cells exposed directly to the alveolar space supported numerous, small microvilli. Mild mitochondrial swelling was seen in a few alveolar type II cells of the C. cladosporioides treated animals.
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DISCUSSION |
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This study has also revealed that instillation exposure to mold spores and toxin will elicit ultrastructural changes in alveolar type II cells, although the nature and degree of the cellular response toward spores from different species appears to vary considerably. Exposure to Cladosporium cladosporioides spores resulted in some ultrastructural changes, which was evidenced as modest mitochondrial swelling in alveolar type II cells. Mitochondrial swelling is a commonly observed, early manifestation of cell injury and has been reported in cases of reversible lung damage resulting from acute oxygen toxicosis (Adamson, 1990), and exposure to bacterial endotoxin (Fehrenbach et al., 1998
; Lopez et al., 1987
; Young and Nicholls, 1996
). However, this response in the C. cladosporioides treated animals was mild compared to the response of alveolar type II cells from animals exposed to S. chartarum and isosatratoxin-F.
Alveolar type II cells from the S. chartarum and isosatratoxin-F treated animals clearly showed evidence of cytological damage, manifest at both the ultrastructural and morphometric levels. Differences in the degree of cellular response associated with exposure to C. cladosporioides spores and to S. chartarum spores and isosatratoxin-F supports the position that the nature and degree of the alveolar type II cell response toward pollutants can vary considerably. More importantly, it also supports the position that exposure to S. chartarum spores and toxin results in cellular responses in vivo different from those associated with exposure to spores of nontoxigenic mold species.
One of the most striking results of the study was the significant increase in lamellar body volume density estimates following exposure to S. chartarum spores and isosatratoxin-F. Because these changes were not observed in the C. cladosporioides treated animals, this result suggests that exposure to fungal spore-wall bound (13)-ß-D-glucans is not contributing to this response, but that metabolites, including toxins, produced by S. chartarum may be responsible. It is well understood that trichothecenes interact with cellular membranes and cause their disfunction (Riley and Norred, 1996
). Possibly, exposure to S. chartarum toxins sequestered in and liberated from spores results in lamellar body membrane deregulation resulting in lamellar body swelling and lamellae rearrangement. However, because similar changes in lamellar body volume density to those observed in this study have been reported for alveolar type II cells from rats exposed in vitro and ex vivo to Salmonella minnesota lipopolysaccharide (LPS; Fehrenbach et al., 1998
; Young and Nicholls, 1996
), this effect does not appear to be a specific sign of S. chartarum spore or toxin induced lung injury. Nevertheless, that exposure to S. chartarum spores and toxin induces lamellar body fusion helps to explain some of the results reported by Mason et al. (1998). They observed that exposure of mouse lungs to S. chartarum spores resulted in an increase in the production of newly secreted surfactant compared to the untreated, saline and C. cladosporioides controls. Mason et al. (1998) suggested that the increase in newly secreted pulmonary surfactant might reflect a defense mechanism to rid alveolar surfaces of S. chartarum spores. This study provides an alternative explanation that this accumulation could reflect a cellular trauma resulting in lamellar body membrane fusion and possibly, in changes in the amount of pulmonary surfactant exported via lamellar body exocytosis into the alveolar space. Obviously more work, employing pulse-chase radioactive tracer studies, is required to address this issue.
S. chartarum spore and isosatratoxin-F exposure also had a significant impact on alveolar cell mitochondria. It is well known that trichothecene exposure can result in mitochondrial function alterations in vitro (Riley and Norred, 1996). It is also recognized that these inhalation exposure to these toxins can cause cell damage and kill animals if exposure concentration is sufficiently high (Creasia et al., 1987
, 1990
). However, the effect of fungal spores and mycotoxins on mitochondrial structure is poorly documented. Pertola et al. (1999) reported mitochondrial swelling as a cytological lesion in boar spermatozoa exposed to S. chartarum spores and T-2 toxin. However, these investigators did not evaluate sperm mitochondria objectively using morphometrics. We also noted some swollen mitochondria in alveolar type II cells from S. chartarum and isosatratoxin-F treated animals examined during this study. However, mitochondrial condensation was the overall consequence of exposure. This result supports work of Okumura et al. (1999), who also found that exposure of a mouse cell line to T-2 toxin caused mitochondrial condensation. Okumura et al. (1999) suggested that this change in mitochondrial profile might be due to increased mitochondrial trans-membrane potential due to a rise in the intracellular concentration of calcium, which would result in organelle condensation, although unequivocal evidence for this is lacking. As far as we are aware, work has not been done to evaluate the impact of S. chartarum spores on mitochondrial function.
Okumura et al. (1999) also suggested that cellular changes they observed in mouse cells exposed to T-2 toxin may be linked to preprogrammed cell death, apoptosis. This may be a relatively common cellular response due to exposure to these toxins as Yang et al. (2000) have also found that other trichothecenes, derived from S. chartarum isolates, induce apoptosis in cells exposed to these toxins, in vitro. However, results of our study do not support the position that alveolar type II cells exposed to S. chartarum and isosatratoxin-F are undergoing apoptosis. Instead, we believe that the alveolar type II cells were exhibiting pre-lethal cytological changes more consistent with oncosis onset (see Trump and Berezesky, 1998). Oncosis is a form of accidental cell death (Majno and Joris, 1995
), which differs from the first mentioned remarkably in structure and function (Trump and Berezesky, 1998
). Ultrastructural features of apoptosis include rapid condensation of the cytoplasm and nuclear chromatin (Zakeri, 1998
), which we did not observe in this study. However, important diagnostic signs of end-stage oncosis are swollen cells with cytoplasmic paling (rarefaction), mitochondrial condensation, nucleolus fragmentation, and the redistribution of euchromatin to the periphery of the nuclear membrane (see Trump and Berezesky, 1998
). These features are similar to those we observed in some of the alveolar type II cells from S. chartarum and isosatratoxin-F treated animals. Oncosis often follows a variety of injuries brought about by exposure to toxins, among others, that interrupt ATP synthesis and alter membrane integrity (Trump and Berezesky, 1998
). Clearly, subsequent studies should be conducted to evaluate the impact of S. chartarum spores and toxin on cell membrane structure and functions.
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ACKNOWLEDGMENTS |
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NOTES |
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