65, 239 –245 (2002)
Copyright © 2002 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Microanatomical Changes in Alveolar Type II Cells in Juvenile Mice
Intratracheally Exposed to Stachybotrys chartarum Spores and Toxin
T. G. Rand,* ,1 M. Mahoney,* K. White,* and M. Oulton†
*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
Stachybotrys chartarum is an important environmental fungus.
We have shown recently that alveolar type II cells are sensitive to
exposure to Stachybotrys chartarum spores and to the trichothecene, isosatratoxin-F, both in vitro and in vivo, in a juvenile
mouse model. This sensitivity is manifest as significant changes in
the composition and normal metabolic processing of pulmonary
surfactant. This study evaluated the effects of a single intratracheal exposure of S. chartarum spores and toxin on ultrastructure
and dimensions of alveolar type II cells from juvenile mice. This
was to determine whether there are concurrent morphological and
dimensional changes in the alveolar type II cell that reflect the
metabolic alterations in pulmonary surfactant that we observed in
the treated mice. Marked ultrastructural changes were associated
with alveolar type II cells in both S. chartarum and isosatratoxin-F
treated animals compared to untreated, saline, and Cladosporium
cladosporioides spore treated animals. These ultrastructural
changes included condensed mitochondria with separated cristae,
scattered chromatin and poorly defined nucleolus, cytoplasmic
rarefaction, and distended lamellar bodies with irregularly arranged lamellae. Point count stereological analysis revealed a
significant increase (p < 0.05) in lamellar body volume density in
S. chartarum and isosatratoxin-treated animals after 48 h exposure. Mitochondria volume density was significantly lower in the
isosatratoxin-F (48 h exposure) and S. chartarum treated (24 and
48 h exposure) animals compared to those in the other treatment
groups. These results reveal that exposure to S. chartarum spores
and toxin elicit cellular responses in vivo differently from those
associated with exposure to spores of a nontoxigenic mold species.
They also indicate that accumulation of newly secreted pulmonary
surfactant in the alveolar space of S. chartarum and isosatratoxin-F treated animals might be a consequence of cellular trauma
resulting in lamellar body volume density changes leading to
increased release of pulmonary surfactant into the alveolar space.
Key Words: ultrastructure; alveolar type II cells; Stachybotrys
chartarum; trichothecenes; intratracheal instillation; fungal
conidia; morphometrics.
Stachybotrys chartarum is an important environmental fungus. Inhalation exposure of building occupants to mycelial
1
To whom correspondence should be addressed. Fax: (902) 475-1982.
E-mail: [email protected].
fragments, spores, and building dust, all containing toxins
produced by this species, has been linked to a number of health
problems. These problems include the onset of a variety of
respiratory and nonrespiratory symptoms (Croft et al., 1986;
Dearborn et al., 1999; Hodgson et al., 1998; Johanning 1995;
Johanning et al., 1996). Despite the large number of respiratory
symptoms associated with exposure to this species in humans,
there are only a few in vivo animal studies evaluating the
impact of spores and toxins of this species on lung tissues.
Moreover, little is known about the specific mechanisms that
may lead to these last mentioned symptoms. Several workers
have shown that exposure of animals to spores of this species
elicit biochemical changes in the bronchoalveolar lavage fluid
(BAL) that accord with acute inflammation responses (see
Mason et al., 1998, 2001; McCrae et al., 2001; Rao et al.,
2000a,b) but there are few studies employing histology to
determine whether these respiratory symptoms accord with
anatomical changes in lung tissue. Nikulin et al. (1996, 1997)
have described some of the histological changes associated
with acute S. chartarum spore exposure in mouse lungs. They
showed that intranasal exposure of mice to aerosolized spores
of 2 different S. chartarum strains resulted in alveolar and
interstitial inflammation with hemorrhagic exudate in the alveoli. Most importantly, they also showed that exposure to spores
containing trichothecene toxins produced the most severe inflammation in the lungs, and that severity was dependent on
spore toxicity and concentration. Histological work in our
laboratory has also revealed that instillation exposure of young
mouse lungs to spores of this species initiates an inflammatory
response marked by the formation of granulomatous lesions
containing a pleomorphic inflammatory cell infiltrate comprising fibroblasts, macrophages, and the occasional polymorphonucleocyte that enclosed sites of spore impaction on lung tissue
(Rand et al., manuscript in prepartion).
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 bio-
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RAND ET AL.
chemical 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.
MATERIALS AND METHODS
Conidia collection. S. chartarum conidia as the test fungus, isosatratoxin-F as a positive control toxin, and Cladosporium cladosporioides as the
negative-control fungus were employed in this study. The S. chartarum isolate
used in this study was recovered from a mold-contaminated Cleveland home
and produces trichothecene toxins (Cleveland strain # 58 –17). The C. cladosporioides isolate was recovered from an outdoor sampling site in Nova Scotia
by T.G.R. Dr. Bruce Jarvis, Department of Chemistry, University of Maryland,
College Park, Maryland, provided the purified isosatratoxin-F, isolated from a
S. chartarum strain for use in this project.
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 5– 6 mice. Preweighed 21–28-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 ␮l of 1 of the following treatments: 0.9% NaCl, 0.02 ␮g
isosatratoxin-F/ml (⫽ 0.7 ng toxin or 35 ng/kg BW/animal), S. chartarum or
C. cladosporioides spores (1.4 ⫻ 10 6 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 ␮l 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 H 2O 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 mm 3 sections. Pieces from each
sectioned mouse lung were randomly selected, washed in 0.1 M cacodylate
buffer, postfixed in 1% osmium tetroxide (OsO 4) 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 ⫻ 11-in. paper or scanned using a flat-bed scanner at a
final magnification ⫻16,500 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 (V PII), lamellar
bodies (V LB), mitochondria (V mito), and nuclei (V nuc) using the appropriate
formulae (see Bozzola and Russell, 1992; Davies, 1980; Weibel and CruzOrive, 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: V v (fraction of cytoplasm occupied by organelle) ⫽ (points
counted on organelle (P organelle) ⫼ (total points on alveolar type II cell profile
(P T) – points counted on nucleus (P nuc)). Data were analyzed using normality
of variance and 2-way ANOVA, and at 95% (p ⱕ 0.05) confidence level.
RESULTS
Mice exposed to either 50 ␮l 10 6 conidia/ml of S. chartarum
conidia, C. cladosporioides conidia, or to 50 ␮l isosatratoxin-F, did not show any apparent clinical signs of respiratory
distress or sickness.
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
S. CHARTARUM AND ALVEOLAR TYPE II CELL ULTRASTRUCTURE
241
FIG. 1. Electron micrographs of alveolar walls. These are (A) a wall from a
saline treated animal and (B) a wall from
a Cladosporium cladosporioides treated
animal. Each micrograph shows an alveolar type II cell with prominent nucleus
(N), lamellar bodies (L), mitochondria
(M), and microvilli (arrows). AS, alveolar space; original magnification ⫻9000.
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.
Alveolar type II cells from animals treated with either S.
chartarum spores or isosatratoxin-F showed remarkable ultrastructural changes compared to the controls. Alveolar type II
cells from these animals often featured mitochondria that were
condensed (Figs. 2A and 2B), electron dense, and irregularly
aligned cristae (Fig. 2A). Nuclei of some alveolar type II cells
from treatment animals had scattered chromatin (Fig. 2C) and
a poorly defined nucleolus. These cells were swollen and their
cytoplasm often, but not always, showed rarefaction and clus-
tered electron dense granules (Figs. 2C and 2D). The plasmalemma of some of the treatment animals showed marked distension and some lacked microvilli (Fig. 2D).Lamellar bodies
in alveolar type II cells from these animal treatment groups
were also often swollen and contained lamellae with irregular
profiles (Figs. 2B and 2C). Other ultrastructural changes were
the vesicles and membranal figures that could also be found in
the alveolar space of treatment animals.
Stereology
Point count stereological analyses revealed that P t volumedensity estimates for alveolar type II cells from all the animal
groups were not significantly different (p ⬎ 0.05; Table 1).
Lamellar body abundance was increased in animals exposed to
S. chartarum for 48 h but this was not significant (p ⬎ 0.05).
However, Pi LB and V vlam estimates for this group and in animals
exposed to isosatratoxin-F for 48 h were significantly elevated
242
RAND ET AL.
FIG. 2. Electron micrographs of alveolar type II cells. (A) Cell with condensed mitochondria from a Stachybotrys chartarum treated animal. AS, alveolar
space; N, ⫽ alveolar type II cell nucleus; microvilli (single arrows); lamellar bodies (L); original magnification ⫻8000. (B) Cell from a Stachybotrys chartarum
treated animal. Note condensed mitochondria (double arrows); marked distension of lamellar bodies with irregularly arranged lamellae (L); microvilli (single
arrows). AS, alveolar space; original magnification ⫻10,500. (C) Cell from an isosatratoxin-F treated animal. Note condensed mitochondria (M); dispersed
nuclear chromatin (N2); electron dense aggregates (single arrows); and cytoplasmic rarefaction; original magnification ⫻10,500. (D) Cell from a Stachybotrys
chartarum treated animal. Note condensed mitochondria (double arrows) and membranal figures (V) in alveolar space (AS); cytoplasmic rarefaction; electron
dense aggregates (single arrows). AS, alveolar space. Note the lack of microvilli along the margin of the alveolar type II cell; original magnification ⫻16,500.
(p ⬍ 0.05), compared to the other treatment groups (Table 1).
Mitochondria abundance was not significantly different among
treatment groups, but Pi and V vmito estimates for mitochondria
were significantly lower (p ⬍ 0.05) in the isosatratoxin-F (PIH
48 h) and S. chartarum treated animals (PIH 24 and 48 h)
compared to the other treatment groups (Table 1). Nucleus Pi
and V vnuc estimates were the same for all treatment groups.
Volume-density outcome was not influenced by exposure time
in any of the treatments (p ⬎ 0.05).
DISCUSSION
Alveolar type II cells comprise some 15– 60% of the cells of
the pulmonary epithelium (see Burkitt et al., 1993; Mason and
Shannon, 1997). These cells synthesize and store major components of pulmonary surfactant, a phospholipid-rich substance (Haagsman and van Golde, 1991; Hawgood and Shiffer,
1991; Oulton et al., 1993; Possmayer, 1984) that lines the
alveolar surface. Surfactant promotes lung stability by reduc-
243
S. CHARTARUM AND ALVEOLAR TYPE II CELL ULTRASTRUCTURE
TABLE 1
Volume Density of Lamellar Bodies and Mitochondria in Alveolar Type II Cells from Untreated and Treated Mouse Groups
Group
PIH
n
No. of LB
No. of Mito
P i LB
P i Mito
Pt
P nuc
V vlam
V vmito
Control
Saline
Saline
Isosatratoxin-F
0
24
48
5(58)
5(59)
5(53)
16.5 ⫾ 3.4
17.2 ⫾ 2.3
16.5 ⫾ 4.1
14.3 ⫾ 3.4
13.7 ⫾ 2.6
15.4 ⫾ 3.2
10.7 ⫾ 1.3
9.9 ⫾ 1.8
9.4 ⫾ 1.4
5.9 ⫾ 1.8
5.4 ⫾ 2.5
5.1 ⫾ 1.9
59.4 ⫾ 15.1
58.2 ⫾ 14.9
60.6 ⫾ 15.7
13.3 ⫾ 1.9
14.5 ⫾ 1.8
14.2 ⫾ 1.9
0.23 ⫾ 0.02
0.22 ⫾ 0.02
0.21 ⫾ 0.03
0.13 ⫾ 0.01
0.12 ⫾ 0.01
0.11 ⫾ 0.01
24
48
5(57)
5(58)
18.3 ⫾ 3.3
17 ⫾ 3.8
12.6 ⫾ 1.9
11.4 ⫾ 2.1
11.2 ⫾ 1.8
12.1 ⫾ 2.1
4.6 ⫾ 2.2
4.3 ⫾ 2.1
61.4 ⫾ 15.9
63.7 ⫾ 16.3
13.6 ⫾ 2.3
15.4 ⫾ 1.8
0.23 ⫾ 0.04
0.32 ⫾ 0.06*
0.09 ⫾ 0.02
0.09 ⫾ 0.01*
24
48
5(55)
5(64)
19 ⫾ 4.2
21 ⫾ 4.1
13.2 ⫾ 2.2
11.8 ⫾ 2.1
10.8 ⫾ 2.5
12.3 ⫾ 2.7
4.1 ⫾ 1.7
3.9 ⫾ 1.9
62.2 ⫾ 16.8
64.4 ⫾ 17.6
17.4 ⫾ 1.9
16.9 ⫾ 2.1
0.28 ⫾ 0.03
0.57 ⫾ 0.08*
0.08 ⫾ 0.01*
0.08 ⫾ 0.01*
24
48
5(56)
5(54)
17 ⫾ 3.5
18 ⫾ 3.2
14.4 ⫾ 3.0
15.6 ⫾ 2.8
11.3 ⫾ 1.9
10.5 ⫾ 1.8
5.5 ⫾ 2.1
5.7 ⫾ 2.3
62.9 ⫾ 15.4
59.5 ⫾ 15.8
16.2 ⫾ 1.8
15.5 ⫾ 1.7
0.24 ⫾ 0.02
0.26 ⫾ 0.01
0.12 ⫾ 0.02
0.13 ⫾ 0.01
S. chartarum
C. clad
Note. Data are expressed as mean ⫾ SD of all counts in cranial lung lobes. LB, lamellar bodies; Mito, mitochondria; n, number of animals (number of cells
observed); P i LB, points of interest (lamellar bodies); P i Mito, points of interest (mitochondria); P t, total points whole cell; P nuc, total points on nucleus; V v, volume
density (P i/P t-Pn).
*Significantly elevated compared to all other treatment and control groups (p ⬍ 0.05).
ing the surface tension of the air-alveolar interface (Guyton et
al., 1984; Notter, 1984), and it performs important functions in
alveolar defense (Curti and Genghini, 1989; Jarstrand, 1984;
Schurch et al., 1990). Ultrastructural features of alveolar type
II cells have been described for many animals and, in healthy
tissues, have been found to exhibit remarkable similarity
amongst species (Adamson, 1990; Mason and Shannon, 1997).
Results of the present study have revealed that alveolar type II
cells from untreated and saline treated animals generally exhibited ultrastructural features similar to those reported for
cells from normal, healthy alveolar tissue (see Burkitt et al.,
1993).
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 (133)-␤-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 explana-
244
RAND ET AL.
tion 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.
ACKNOWLEDGMENTS
We thank C. Leggiadro and D. O’Neil, NRC Institute of Marine Biosciences
for their assistance and excellent technical support. We would also like to
thank Drs. B. Jarvis for the gift of isosatratoxin-F and M. Wiles for use of his
stereological templates. This study was supported by a Natural Sciences and
Research Council operating grant to T.G.R.
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