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Environ Health Perspect. 2006 July; 114(7): 1099–1107. Published online 2006 February 27. doi: 10.1289/ehp.8854. | PMCID: PMC1513335 |
Copyright This is an Open Access article: verbatim copying and redistribution of
this article are permitted in all media for any purpose, provided this
notice is preserved along with the article's original DOI Research Satratoxin G from the Black Mold Stachybotrys chartarum Evokes Olfactory Sensory Neuron Loss and Inflammation in the Murine Nose
and Brain Zahidul Islam,1,2,3 Jack R. Harkema,1,4 and James J. Pestka1,2,3 1 Center for Integrative Toxicology 2 Department of Microbiology and Molecular Genetics 3 Department of Food Science and Human Nutrition and 4 Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA Received November 14, 2005; Accepted February 27, 2006. Satratoxin G (SG) is a macrocyclic trichothecene mycotoxin produced by Stachybotrys chartarum, the “black mold” suggested to contribute etiologically
to illnesses associated with water-damaged buildings. Using an intranasal
instillation model in mice, we found that acute SG exposure specifically
induced apoptosis of olfactory sensory neurons (OSNs) in the
olfactory epithelium. Dose–response analysis revealed that the
no-effect and lowest-effect levels at 24 hr postinstillation (PI) were 5 and 25 μg/kg
body weight (bw) SG, respectively, with severity
increasing with dose. Apoptosis of OSNs was identified using immunohistochemistry
for caspase-3 expression, electron microscopy for ultrastructural
cellular morphology, and real-time polymerase chain reaction
for elevated expression of the proapoptotic genes Fas, FasL, p75NGFR, p53, Bax, caspase-3, and CAD. Time-course studies with a single instillation of SG (500 μg/kg
bw) indicated that maximum atrophy of the olfactory epithelium occurred
at 3 days PI. Exposure to lower doses (100 μg/kg bw) for 5 consecutive
days resulted in similar atrophy and apoptosis, suggesting
that in the short term, these effects are cumulative. SG also induced
an acute, neutrophilic rhinitis as early as 24 hr PI. Elevated mRNA
expression for the proinflammatory cytokines tumor necrosis factor-α, interleukin-6 (IL-6), and IL-1 and the chemokine macrophage-inflammatory
protein-2 (MIP-2) were detected at 24 hr PI in both the ethmoid
turbinates of the nasal airways and the adjacent olfactory bulb of
the brain. Marked atrophy of the olfactory nerve and glomerular layers
of the olfactory bulb was also detectable by 7 days PI along with mild
neutrophilic encephalitis. These findings suggest that neurotoxicity
and inflammation within the nose and brain are potential adverse health
effects of exposure to satratoxins and Stachybotrys in the indoor air of water-damaged buildings. Keywords: apoptosis, fungus, inflammation, inhalation, mycotoxin, neurotoxicity, olfactory sensory neuron, rhinitis, trichothecene Numerous adverse human health effects have been attributed to damp indoor
air environments generated by aberrant water exposure due to excessive
condensation and failure of water-use devices, as well as building
envelope breach during heavy rains or flooding, as occurred during Hurricanes
Katrina and Rita on the Gulf Coast of the United States. An Institute
of Medicine (IOM) expert panel concluded that an association
exists between damp buildings and upper respiratory tract symptoms, wheeze, cough, and
exacerbation of chronic lung diseases such as asthma, whereas
supportive data for other reported outcomes such as neurocognitive
dysfunction, mucous membrane irritation, fatigue, fever, and immune
disorders are lacking ( IOM 2004). Building-related illnesses are often linked to dampness-promoted growth
of fungi ( Fog Nielsen 2003) and, most notably, Stachybotrys chartarum, a saprophytic “black mold” that grows on cellulosic materials, including
wall-board, ceiling tiles, and cardboard ( Hossain et al. 2004). Incidences of indoor S. chartarum contamination often generate costly litigation and remediation, are extensively
reported by the media, and have evoked intense public and scientific
controversy ( Hardin et al. 2003). The IOM panel suggested that although in vitro and in vivo research on S. chartarum and its mycotoxins suggests that adverse effects in humans are indeed “biologically
plausible,” their association with building-related
illnesses requires rigorous validation from the perspectives
of mechanisms, dose response, and exposure assessment ( IOM 2004). The satratoxins, macrocyclic trichothecenes produced by S. chartarum, are potent inhibitors of protein translation that initiate both inflammatory
gene expression and apoptosis in vitro after upstream activation of mitogen-activated protein kinases (MAPKs) ( Chung et al. 2003; Yang et al. 2000). Satratoxin equivalent airborne concentrations ranging from 2 to 34 ng/m 3 ( Yike et al. 1999) and from 54 to 330 ng/m 3 ( Vesper et al. 2000) have been previously estimated, by a translational bioassay, to occur
in rooms of water-damaged homes heavily contaminated with Stachybotrys. These water-soluble mycotoxins occur in the outer plasmalemma surface
and the inner wall layers of conidiospores ( Gregory et al. 2004) as well as in nonviable airborne particulates ( Brasel et al. 2005), which could facilitate entry and release into respiratory airway tissue. Indeed, pulmonary
toxicity of the spores of S. chartarum and associated trichothecenes has been demonstrated in animal studies
using intranasally or intratracheally exposed laboratory rodents ( Yike and Dearborn 2004; Yike et al. 2005). Under normal resting, nonexercising conditions, the nose functions to filter, warm, and
humidify inhaled air before it enters more delicate airway
and alveolar tissues in the distal lung ( Cole 1993). Nasal passages serve as “scrubbing towers” for the respiratory
tract by efficiently a) absorbing water-soluble and reactive gases and vapors, b) trapping inhaled particles, and c) metabolizing airborne xenobiotics ( Harkema 1991). Given these air conditioning and defensive roles, we hypothesized that
the nasal airways are another critical site for interaction with S. chartarum mycotoxins. To test this hypothesis, we employed a murine intranasal instillation
model previously used by our laboratory and others to study
the adverse effects of harmful toxic agents ( Giannetti et al. 2004), allergens ( Farraj et al. 2004), and pathogens ( Wiley et al. 2001) to investigate potential nasal toxicity of satratoxin G (SG), one of
the most potent trichothecenes produced by S. chartarum ( Yang et al. 2000). Toxins SG and isosatratoxin F (ISF) were purified from S. chartarum cultures and kindly provided by B. Jarvis (University of Maryland, College
Park, MD). SG and ISF yielded a single peak at 254 nm by the HPLC
method of Hinkley and Jarvis (2001). SG and ISF identities were further confirmed by electrospray ionization/collision-induced
dissociation (ESI-CID) tandem mass spectroscopy at
the Michigan Sate University mass spectrometry facility by a modification
of a published method ( Tuomi et al. 1998) using a LCQ-DECA device (Finnigan, San Jose, CA) fitted with an ESI probe. Deoxynivalenol, T-2 toxin, and verrucarin A (Sigma Chemical Co., St. Louis, MO) had
reported purities of > 98%, 98%, and 95%, respectively. Laboratory animals and intranasal instillation Mice were maintained under humane conditions according to National Institutes
of Health guidelines ( Institute of Laboratory Animal Resources 1996) as overseen by the All University Committee on Animal Use and Care at
Michigan State University. Pathogen-free female C57Bl/6 mice (7–8 weeks
of age; Charles River, Portage, MI) were randomly assigned
to experimental groups ( n = 5–6) and housed in polycarbonate cages containing Cell-Sorb
Plus bedding (A & W Products, Cincinnati, OH) covered with
filter bonnets and provided free access to food and water. Room lights
were set on a 12-hr light/dark cycle, and temperature and relative humidity
were maintained between 21 and 24°C and 40 and 55%, respectively. For
each experiment, mice were anesthetized with 4% halothane
and 96% oxygen and then instilled intranasally
at 50 μL/mouse with SG or other trichothecenes dissolved
in a vehicle of pyrogen-free saline (Abbott Laboratories, Abbott Park, IL) or
with the vehicle alone. Animal necropsies and tissue processing for light microscopic examination For histopathology and morphometry, mice were deeply anesthetized via intraperitoneal (ip) injection of 0.1 mL of 12% sodium pentobarbital
in saline at designated times post-instillation (PI), from 6 hr
to 28 days, and killed via exsanguination by cutting the abdominal aorta
or renal arteries. Heads from each mouse were immediately removed, and 1 mL
of 10% neutral buffered formalin (Fisher Scientific Co., Fairlawn, NJ) was
flushed retrograde through the nasopharyngeal meatus. After
the lower jaw, skin, muscles, eyes, and dorsal cranium were
removed, the head with brain intact was immersed and stored in a large
volume of the fixative for at least 48 hr before further tissue processing. Lungs
were also removed and intratracheally perfused with formalin
fixative at a constant pressure of 30 cm of water for approximately 1 hr
and then similarly immersed and stored for a minimum 48 hr. After fixation, transverse tissue blocks from the head and left lung lobe
of these mice were selected for light microscopy as previously described ( Steiger et al. 1995). Before sectioning, the heads were decalcified in 13% formic
acid for 7 days and then rinsed in tap water for at least 4 hr. The nasal
cavity of each mouse was transversely sectioned at four specific anatomic
locations, designated T1–T4 ( Mery et al. 1994; Young 1981). The most proximal nasal section was taken immediately posterior to the
upper incisor teeth (proximal, T1); the middle section was taken at
the level of the incisive papilla of the hard palate (middle, T2); the
third nasal section was taken at the level of the second palatal ridge (T3); and
the most distal nasal section (T4) was taken at the level
of the intersection of the hard and soft palate and through the proximal
portion of the olfactory bulb (OB) of the brain (). In addition, two transverse tissue blocks from the left lung lobe were
also taken for microscopic examination at the level of airway generation 5 (proximal) and
generation 11 (distal) along the main axial airway. Tissue
blocks were embedded in paraffin, and the anterior face of
each block was sectioned at a thickness of 5 μm, and stained
with hematoxylin and eosin (H&E). Immunohistochemistry Unstained and hydrated paraffin sections from nasal blocks T3 and T4 were
incubated first with a nonspecific protein-blocking solution containing
normal sera (Vector Laboratories Inc., Burlingame, CA) and then with
specific dilutions of primary polyclonal antibodies directed against
activated caspase-3 (1:100, rabbit anti-caspase-3 antibody; Abcam, Inc., Cambridge, MA), olfactory marker protein (OMP; 1:4,000, goat anti-OMP
antibody, provided by F. Margolis, University of Maryland), or infiltrating
neutrophils (1:600, rabbit anti-rat neutrophil antibody, provided
by R. Roth, Michigan State University). Tissue sections used for
caspase-3 or OMP detection were pretreated before the blocking solution
with 3% hydrogen peroxide in methanol to destroy endogenous
peroxidase. With these tissue sections, the primary antibody was followed
by the secondary antibody, biotinylated anti-species IgG. Immunoreactivity
of caspase-3 and OMP was visualized with Vector R.T.U. Elite
ABC-Peroxidase Reagent followed by Nova Red (Vector Laboratories Inc.) as
the chromagen. Anti-neutrophil antibody treatment was followed
by biotinylated anti-rabbit IgG, and then streptavidin-phosphatase complex (KPL
laboratories, Gaithersburg, MD) and Vector red as the chromagen. After
immunohistochemistry, slides were lightly counterstained with
hematoxylin. Semiquantitative scoring of nasal histopathology Nasal sections from mice that received a single instillation of SG at various
doses and were sacrificed 24 hr PI were scored for the amount of
toxin-induced, light microscopic lesions in the olfactory epithelium (OE). A
veterinary pathologist, without previous knowledge of exposure
history of the individual mice, ranked severity of SG-induced OE apoptosis
with atrophy in the examined nasal tissue sections (T1–T4) using
the following histopathologic numeric scores: 0, no SG-induced
nasal lesions in OE; 1 (minimal), 25% of OE with lesions; 2 (mild), 25–50% of OE with lesions; 3 (moderate), 50–75% of
OE with lesions; or 4 (marked), ≥ 75% of
OE with lesions. Light microscopic morphometry Thickness of the OE lining the medial surface of the second ethmoid turbinates (2E) in
T3 () was morphometrically evaluated as previously described for airway epithelium ( Hyde et al. 1990, 1991; Plopper et al. 1994). Measurements were conducted at a final magnification of 3,540× using
a light microscope (Olympus BX40; Olympus America Inc., Melville, NY) coupled
to a 3.3-megapixel digital color camera (Q-Color 3 Camera; Quantitative
Imaging Corp., Burnaby, British Columbia, Canada), and
a personal computer (Dimension 8200; Dell, Austin, TX). The morphometric
analyses were performed using a cycloid grid overlay and software
for counting points and intercepts (Stereology Toolbox; Morphometrix, Davis, CA) ( Hyde et al. 1990, 1991). The percentage volume density, Vv, the proportion of the epithelium composed of cytoplasm, nuclei, or apoptotic
nuclear fragments, was determined by point counting and calculated
using the following formula: where Pp is the point fraction of Pn, the number of test points hitting the structure of interest, divided
by Pt, the total points hitting the reference space (OE). The volume of the
epithelial component of interest (e.g., apoptotic nuclei) per unit of
basement membrane (Sv) was determined by point and intercept counting and was calculated using
the following formula: where Io is the number of intercepts with the object (epithelial basal lamina) and Lr is the length of test line in the reference volume (epithelium). To determine
thickness of the OE, a volume per unit area of basal lamina (cubic
micrometers per square micrometer) was then calculated using the
following formula for arithmetic mean thickness (τ): Other standard morphometric and image analysis techniques were used to
determine the numeric cell density of mature olfactory sensory neurons (OSNs) in
OE. Morphometric estimates of the numeric cell density of OSNs
immunohistochemically reactive for OMP (protein indicator of mature
OSNs) were determined via light microscopy (790× final magnification) by
counting the number of nuclear profiles of these immunoreactive
neuroepithelial cells in the OE lining the medial surface of 2E
in T3 () and dividing by the length of the underlying basal lamina. The length
of the basal lamina was determined from the contour length of a computerized
digital image of the basal lamina using the Scion Image program (Scion
Corporation, Frederick, MD). All numeric cell density data were
expressed as the number of OSN nuclei per millimeter of basal lamina. Ultrastructural examination of the olfactory mucosa and OB via transmission
electron microscopy Mice designated for transmission electron microscopy (TEM) analysis were
anesthetized with an ip injection of 0.1 mL 12% pentobarbital
containing 1 IU heparin. Immediately after anesthesia, the whole body
received an intravascular perfusion via the left heart with a saline
solution containing 10 IU heparin for 2–3 min, followed by a 7–10 min
perfusion with 4% glutaraldehyde fixative solution (Ted
Pella, Inc., Redding, CA). The nasal cavity and brain were
then removed and stored in the fixative until TEM processing after the
nasal cavity was decalcified with 10% EDTA for 3–4 weeks; selected
tissues from ethmoid turbinates and OB were postfixed in 1% phosphate-buffered osmium tetroxide, dehydrated through a
graded series of ethanol and propylene oxide, and embedded in Poly/Bed-Araldite
resin (Polysciences, Inc., Warrington, PA). Sections (1 μm) were
cut and stained with toluidine blue for light microscopic
identification of tissue sites for TEM. Ultrathin tissue sections for
TEM were cut at approximately 75 nm with a diamond knife, mounted on
copper grids, and stained with lead citrate and uranyl acetate. Sectioning
was done with an LKB Ultratome III (LKB Instruments, Inc., Rockville, MD). Ultrastructural
tissue examination and photography were performed
with a JEOL JEM 100CXII electron microscope (JEOL Ltd., Tokyo, Japan). Real-time polymerase chain reaction Mice used for polymerase chain reaction (PCR) analyses of nasal and brain
tissues were anesthetized and killed at designated times after SG instillation
as described above. Immediately after death, the head of each
mouse was removed from the carcass; after the skin, muscles, eyes, and
lower jaw were removed from the head, the nasal airways were opened
by splitting the nose in a sagittal plane adjacent to the midline. The
nasal septum was removed, thereby exposing the nasal turbinates projecting
from the lateral wall of each nasal passage (). Using a dissecting microscope and ophthalmic surgical instruments, all
ethmoid turbinates and the OB were dissected from both nasal passages
and brain, respectively. These excised tissues were stored in RNAlater (Ambion
Inc., Austin, TX) within 5 min, and RNA was isolated using
RNeasy Protect Mini kit (Qiagen Inc., Valencia, CA) within 7 days. Absence
of RNA degradation was routinely verified by agarose electrophoresis; real-time
PCR for apoptosis-related genes [ Fas, FasL, p75-nerve growth factor receptor ( p75NGFR), p53, Bax, Bcl-2, Apaf-1, caspase-3, and caspase-activated DNase ( CAD)], cytokine genes [interleukin-1 ( IL-1), tumor necrosis factor-α ( TNF-α), IL-6], and the chemokine gene MIP-2 (macrophage-inflammatory protein-2) were performed on an ABI PRISM 7900HT
Sequence Detection System using Taqman One-Step RT-PCR (reverse-transcriptase-PCR) Master
Mix and Assays-on-Demand primer/probe gene expression
products according to the manufacturer’s protocols (Applied
Biosystems, Foster City, CA). Relative quantification of apoptotic
and cytokine gene expression was carried out using an 18S RNA as the
loading control and an arithmetic formula method ( Audige et al. 2003). Statistics All data were analyzed with SigmaStat (version 3.1; Jandel Scientific, San
Rafael, CA) with the criterion for significance set at p < 0.05. Morphometric and RT-PCR data were statistically analyzed using
one-way analysis of variance with Student-Newman-Keuls posttest. Data
from histopathologic severity scores of SG-induced lesions were analyzed
using the Mann Whitney rank sum test (nonparametric test) with
Bonferroni correction for multiple comparisons. OE targeted by nasal SG exposure Light microscopic evaluation of four specific anatomical sites (T1–T4) revealed
that mice exposed to SG [500 μg/kg body
weight (bw)] and sacrificed at 1, 3, or 7 days PI had conspicuous
nasal epithelial and inflammatory lesions in the dorsocaudal half
of the nasal passages that is normally lined by OE. These lesions were
not apparent in the nasal cavity of saline vehicle–treated
controls (). SG-related alterations were neither present in regions of the nasal
airways lined by other nasal epithelial types, including respiratory, transitional, or
squamous epithelium, nor found in the lungs of exposed
mice. In addition, mice that were sacrificed only 6 hr PI had no exposure-related
lesions in their nasal cavities. SG-induced OE lesions at 1 day
PI consisted of numerous individual epithelial cells with morphologic
features characteristic of apoptosis in the OE lining all the
ethmoid turbinates and the adjacent lateral walls that border the lateral
meatus in the distal regions of both nasal passages (T3 and T4), with
the most dorsolateral ethmoid turbinates (1E and 2E) being most severely
affected (). SG-induced apoptosis was also present in the OE of the mid and ventral
septum lining the middle medial meatus in the distal nasal passages (T3 and
T4). In the middle of the nasal passages (T2), before the distal
regions containing the ethmoid turbinates (T3 and T4), SG-induced
apoptotic lesions in the OE were detected only in a small mucosal region
of the lateral walls and septum lining the middle medial meatus where
the OE meets the respiratory epithelium. SG-induced nasal epithelial
lesions were undetectable in the most proximal regions of the nasal
passages (T1). Interestingly, the OE lining the dorsal medial meatus throughout
the nasal passages (T1–T4) had no microscopic evidence
of SG-induced apoptosis or any other epithelial alterations. Apoptosis induction in OE Dose–response analysis of SG-induced apoptotic lesions indicated
that the no-effect level was 5 μg/kg bw (80 ng/mouse) and the
lowest effect level was 25 μg/kg bw (400 ng/mouse; ). SG-induced apoptosis was defined by condensation and shrinkage of individual
epithelial cells; clumping, fragmentation, and margination of
nuclear chromatin; and numerous widely scattered cellular fragments (apoptotic
bodies) (–C). Apoptosis was restricted to OSNs whose cell bodies and nuclei
reside in the middle nuclear layers of the OE below the distinct apical
row of sustentacular (support) cell nuclei and above the basal cell
nuclei near the basal lamina. Shrunken OSNs and associated apoptotic
bodies expressed the inducible apoptotic marker protein caspase-3 at 1 day
PI (). Prominent anti-caspase-3 staining was also present in olfactory nerve
bundles (axons of OSNs) in the underlying lamina propria of the mice
instilled with SG. Real-time PCR analysis of microdissected OE-lined
ethmoid turbinates from SG-treated mice demonstrated a marked up-regulation
of the proapoptotic genes Fas, FasL, p75NGFR, p53, Bax, caspase-3, and CAD, whereas expression of Apaf-1 and the anti-apoptotic gene Bcl-2 was unchanged (). SG-mediated OE atrophy Concurrent with SG-induced OSN apoptosis, OE atrophy was detectable at 1 day
PI (). SG-induced OE lesions were similarly apparent at 3 and 7 days PI along
with increased atrophy. Morphometry of OE lining 2E () confirmed that greater atrophy occurred in mice sacrificed 3 and 7 days
PI than in mice sacrificed 1 day PI, with an approximately 50% reduction
in epithelial thickness compared with control mice (). Restoration of the normal thickness of the OE compared with that of
control was still not complete at 28 days PI (). Compared with SG-exposed OE at 1 day PI, volume density of apoptotic
nuclei within the OE was remarkably reduced at 3 days PI and absent at 7 and 28 days
PI (). Exposure to a lower daily dose of SG (100 μg/kg bw or 1.6 μg/mouse) for 5 consecutive days resulted in similar atrophy and
apoptosis of OE compared with those alterations caused by the single 500 μg/kg
bw SG (,D), suggesting that, in the short term, these effects were cumulative. Role of trichothecene structure in OE atrophy The nasal effects of trichothecenes not associated with Stachybotrys were also assessed. Mice intranasally exposed to deoxynivalenol, T-2, and
verrucarin A, which are type A, type B, and macrocyclic trichothecenes, respectively, using
doses of equivalent to one-third to one-fifth
of LD 50 values (doses lethal in 50% of test animals) ( Ueno 1984). These trichothecenes had no effect on OE compared with the Stachybotrys mycotoxins SG and ISF, which exhibited robust toxicity (). Thus, the trichothecene nucleus with characteristic 12–13 epoxide
found in trichothecenes was insufficient to induce OE atrophy in
mice; rather, the effect appeared to be dependent on satratoxin structure (). Selective apoptosis induction in OSNs OMP, a specific peptide found only in mature OSNs ( Kream and Margolis 1984), was markedly reduced in the OE of SG-instilled mice compared with saline-instilled
control mice (). Morphometric analysis revealed a 67, 94, and 81% loss in OSNs
per millimeter of OE in the nasal mucosa lining the dorsolateral meatus
at 1, 3, and 7 days PI, respectively, compared with the identical
region in vehicle-instilled mice (). Consistent with partial recovery of OE thickness at 28 days PI (), there was also incomplete restoration of the numbers of OMP-positive
OSNs () in the nasal mucosa lining the dorsolateral meatuses of OSNs and OE thickness. OSNs, unlike
neurons in other parts of the body, have the ability
to regenerate from OE basal cells and restore their synaptic connections
in the OB ( Graziadei and Graziadei 1978). Dosing with five consecutive daily SG instillations (100 μg/kg
bw) resulted in an 87% loss of OSNs, again suggesting that
neuronal effects were cumulative. At the ultrastructural level, SG-induced
atrophic OE had a conspicuous loss of nuclear and cytoplasmic profiles
of OSNs and their ciliated dendritic knobs that contain the animal’s
odorant receptors and normally project above the microvillar
apical surfaces of the sustentacular cells (). Consistent with OSN loss, both ultrastructural and immunohistochemical
examination demonstrated a marked reduction of the normally dense mat
of cilia projecting from the dendritic knobs and lining the surface
of the nasal airway lumen (). Concurrent with the initial loss of OMP-positive OSNs, there was also noticeable
atrophy of the OMP-positive olfactory nerve bundles located
in the lamina propria underlying the atrophic OE (). This corresponded to marked bilateral atrophy of the olfactory nerve
layer and adjacent glomerular layer comprising the outer two tissue layers
of the OB in SG-instilled mice (). Loss of OMP-stained olfactory nerves was most marked in the lateral
and medial aspects of each of the OBs (). Inflammatory gene up-regulation and neutrophil infiltration in OE and OB At 1 day PI, SG also induced conspicuous accumulations of exfoliated and
degenerating cellular debris from the dendritic portions of the apoptotic
OSNs in the nasal airways along the luminal surfaces of the atrophying
OE. With secondary degeneration of these exfoliated dendritic fragments, there
was accompanying infiltration of numerous phagocytic cells
consisting mainly of polymorphonuclear leukocytes (neutrophils) and
only occasional mononuclear cells (monocytes and macrophages). Many
of the luminal neutrophils had engulfed apoptotic cellular fragments (, ). Phagocytosis of apoptotic bodies by sustentacular cells within the OE
was also evident by ultrastructural examination. Lesser numbers of infiltrating
neutrophils were also widely scattered in lamina propria of
the SG-altered olfactory mucosa. Consistent with leukocyte influx was
a markedly increased expression of mRNAs for the cytokines TNF-α, IL-1α, IL-1β, and IL-6 as well as the chemokine MIP-2 associated
with acute inflammatory cell infiltration (). Elevated MIP-2, TNF-α, and IL-6 mRNA expression was also observed in the OB (). Slightly decreased severity of SG-induced neutrophilic rhinitis corresponded
with time-dependent disappearance of epithelial apoptosis and development
of epithelial atrophy. A mild-to-moderate influx of neutrophils
persisted in the lamina propria of the affected nasal mucosa underlying
the atrophic OE even at 7 days PI (). Remarkably, there were mild, widely scattered infiltrations of neutrophils
in the remaining olfactory nerve bundles penetrating the bony cribiform
plate that separates the nasal cavity and OB of the brain (). These infiltrations extended bilaterally into the atrophic olfactory
nerve and glomerular layers of the OB at 7 days PI (). Ultrastructural examination revealed that the infiltrating neutrophils
were closely associated with focal areas of degeneration and loss of
OSN axons in both of these outer layers of the OB (). A few isolated neutrophils were even detected in the deeper external
plexiform layer, although no neuronal damage was evident in this or other
areas of the OB of SG-exposed mice. The causes of damp-building syndrome are likely to be multifactorial and
involve toxic, inflammatory, and allergic responses to microbes and
their products; however, the underlying mechanisms, relative contributions
of individual organisms, and potential for interactions remain poorly
understood ( IOM 2004). Although exposure to either S. chartarum spores or associated satratoxins has been previously shown to initiate
acute inflammatory responses in the rodent lung ( Yike and Dearborn 2004), our observations that very low doses of SG are directly toxic to OSNs
and initiate an inflammatory response in the nose (rhinitis) that extends
into the brain (mild focal encephalitis) () are heretofore unreported. These findings raise significant new questions
about hazards associated with indoor exposure to this fungus in water-damaged
buildings. Several aerosol studies have demonstrated that
there is substantial deposition (> 50%) of either very large (> 5 μm
in particle diameter; e.g., a fungal spore) and
very small particles (< 10 nm in diameter; nanoparticles) in the nasal
airways of humans and laboratory animals when inhaled through the
nose ( Cheng et al. 1990, 1991, 1996; Yeh et al. 1997). Therefore, it is very likely that the nasal airways will filter out
the inhaled spores or extremely small fragments emitting from the mold, preventing
deposition in the lower respiratory tract, including the
lungs. Like other epithelial cells in the body, but unlike most neuronal cell
populations in the mammalian nervous system, OSNs undergo apoptosis and
genesis throughout the life of the animal as part of the normal turnover
of mature OE. OSNs are unique in that they have relatively short
life spans compared with other neurons and are continuously being replaced
through basal cell proliferation and differentiation (neuronal regeneration) ( Graziadei and Graziadei 1978). Most OSNs live for 30–40 days, but some cells have life spans
of 3 months or even longer. OSNs of laboratory animals may be induced
to die in vivo by experimentally manipulative methods that include olfactory bulbectomy, transection
of the olfactory nerve at the cribiform plate, and intranasal
exposure to chemicals known to be toxic to the OE, such as zinc
sulfate and methyl bromide ( Cowan and Roskams 2002). Exposures to most olfactory chemical toxins result in necrosis (oncosis) of
the OSNs along with other epithelial cells in the OE, unlike the
selective cell death of OSNs by apoptosis observed in the present study. Recently, however, exposure of mice to some chemotherapeutic agents, such
as vincristine, was found to induce marked apoptosis of OSNs
with subsequent OE atrophy that resembles SG-induced lesions described
herein but without obvious nasal inflammation ( Kai et al. 2004). In contrast to our study, mice in these previous studies were given
the chemical agents systemically and at much higher doses relative to
body weight (milligrams per kilogram vs. micrograms per kilogram). SG might drive both extrinsic (death receptor–mediated) and intrinsic (mitochondrial-mediated) apoptotic pathways in OSNs. The trichothecenes
induce gene expression and apoptosis via a ribotoxic stress response
that involves MAPKs ( Shifrin and Anderson 1999; Yang et al. 2000) and is mediated upstream by double-stranded RNA–activated protein
kinase ( Zhou et al. 2003) and Src-family kinases ( Zhou et al. 2005b). Notably, SG-induced genes that have previously been associated with
death receptor–mediated OSN apoptosis include TNF-α, Fas, FasL, and p75NGFR ( Cowan and Roskams 2002), as well as the downstream apoptotic genes p53 ( Huang et al. 1995), Bax ( Ge et al. 2002), and caspase-3 ( Cowan and Roskams 2004). Relative to the intrinsic pathway, trichothecene deoxynivalenol induces
p38-mediated mitochondrial-dependent caspase-3 activation and apoptosis
in cloned macrophages ( Zhou et al. 2005a). Furthermore, satratoxin H–induced caspase-3 activation and apoptosis
in the PC12 neural cell model have recently been reported to
be both p38 and JNK dependent ( Nusuetrong et al. 2005). It is unclear why SG specifically targeted OSNs when nasal respiratory
epithelium and other cell types in the OE were unaffected. OSN sensitivity
to SG might relate to longer regional exposure to epithelial cells
in OE compared with the exposure to cells in respiratory epithelium. This
is possibly due to a much slower rate of mucociliary clearance of
inhaled agents from OE-lined ethmoid turbinates, which are covered by
immotile cilia, compared with other parts of the nasal cavity that are
lined by respiratory epithelium containing motile cilia with high ciliary
beat frequencies. This latter movement generates rapid regional
flows of mucus out of the nasal cavity and through the nasopharynx into
the upper digestive tract ( Morgan et al. 1984). A slower rate of intranasal SG clearance from OE compared with respiratory
epithelium may also be due to differences in other factors known
to affect the clearance of chemicals from the nasal airway, such as
mucosal metabolism or blood flow. Alternatively, based on our observations that satratoxin-induced OE atrophy
is highly dependent on chemical structure () and that one region of the nasal cavity lined by OE (dorsomedial meatus) was
consistently spared from toxicant-induced injury (), it is tempting to speculate that these trichothecenes or as yet unidentified
metabolites bind to specific OSN receptors, thus facilitating
uptake and resultant toxicity. In support of this contention, populations
of distinct odorant receptors can be divided into four specific topographical
regions of the OE, one of which lines the dorsomedial meatus ( Ressler et al. 1993). Taken together, our observations that the OE and OB are targets of SG and
ISF should be a critical consideration in future studies of damp-building–related
illnesses and the potential etiologic role of S. chartarum. The profile of induced cytokines and MIP-2 is likely to contribute to OSN apoptosis as well as accompanying rhinitis
and mild focal encephalitis observed in the present study. In the
future, it will be necessary to ascertain the dose–response effects
and latency of recovery in nasal tissue after chronic exposure to
satratoxins alone, as well as the contributions of spore matrix, or
coexposures to other indoor air contaminants such as endotoxin. Particularly
intriguing will be understanding the basis for OSN specificity
and the role of toxin metabolism. Of further critical importance will
be the extent to which toxicant-induced inflammation and neuronal injury
occur in other parts of the brain along the olfactory pathway and whether
this contributes to neurocognitive dysfunction. Ultimately, all
such information must be framed against accurate quantitative assessments
of human exposure to satratoxins using both state-of-the-art sampling
and analytical methods and relevant biomarkers. - Audige A, Yu ZR, Frey BM, Uehlinger DE, Frey FJ, Vogt B. Epithelial sodium channel (ENaC) subunit mRNA and protein expression in
rats with puromycin aminonucleoside-induced nephrotic syndrome. Clin Sci (Lond). 2003;104:389–395. [PubMed]
- Brasel TL, Douglas DR, Wilson SC, Straus DC. Detection of airborne Stachybotrys chartarum macrocyclic trichothecene mycotoxins on particulates smaller
than conidia. Appl Environ Microbiol. 2005;71:114–122. [PubMed]
- Cheng YS, Hansen GK, Su YF, Yeh HC, Morgan KT. Deposition of ultrafine aerosols in rat nasal molds. Toxicol Appl Pharmacol. 1990;106:222–233. [PubMed]
- Cheng YS, Yeh HC, Guilmette RA, Simpson SQ, Cheng KH, Swift DL. Nasal deposition of ultrafine particles in human volunteers and its relationship
to airway geometry. Aerosol Sci Technol. 1996;25:274–291.
- Cheng YS, Yeh HC, Swift DL. Aerosol deposition in human nasal airway for particles 1 nm to 20 mm: A
model study. Radiat Prot Dosimetry. 1991;38:41–47.
- Chung YJ, Jarvis B, Pestka J. Modulation of lipopoly-saccharide-induced proinflammatory cytokine production
by satratoxins and other macrocyclic trichothecenes in the murine
macrophage. J Toxicol Environ Health A. 2003;66:379–391. [PubMed]
- Cole P. 1993. The Respiratory Role of the Upper Airways. A Selective Clinical
and Pathophysiological Review. St. Louis, MO:Mosby Year Book.
- Cowan CM, Roskams AJ. Apoptosis in the mature and developing olfactory neuroepithelium. Microsc Res Tech. 2002;58:204–215. [PubMed]
- Cowan CM, Roskams AJ. Caspase-3 and caspase-9 mediate developmental apoptosis in the mouse olfactory
system. J Comp Neurol. 2004;474:136–148. [PubMed]
- Farraj AK, Harkema JR, Kaminski NE. Allergic rhinitis induced by intranasal sensitization and challenge with
trimellitic anhydride but not with dinitrochlorobenzene or oxazolone
in A/J mice. Toxicol Sci. 2004;79:315–325. [PubMed]
- Fog Nielsen K. Mycotoxin production by indoor molds. Fungal Genet Biol. 2003;39:103–117. [PubMed]
- Ge Y, Tsukatani T, Nishimura T, Furukawa M, Miwa T. Cell death of olfactory receptor neurons in a rat with naso-sinusitis infected
artificially with Staphylococcus. Chem Senses. 2002;27:521–527. [PubMed]
- Giannetti N, Moyse E, Ducray A, Bondier JR, Jourdan F, Propper A, et al. Accumulation of Ym1/2 protein in the mouse olfactory epithelium during
regeneration and aging. Neuroscience. 2004;123:907–917. [PubMed]
- Graziadei P, Graziadei GA. 1978. The olfactory system: a model for the study of neurogenesis and
axon regeneration in mammals. In: Neuronal Plasticity (Cotman CW, ed). New
York:Raven Press, 131–153.
- Gregory L, Pestka JJ, Dearborn DG, Rand TG. Localization of satratoxin-G in Stachybotrys chartarum spores and spore-impacted mouse lung using immunocytochemistry. Toxicol Pathol. 2004;32:26–34. [PubMed]
- Hardin BD, Kelman BJ, Saxon A. Adverse human health effects associated with molds in the indoor environment. J Occup Environ Med. 2003;45:470–478. [PubMed]
- Harkema JR. Comparative aspects of nasal airway anatomy: relevance to inhalation toxicology. Toxicol Pathol. 1991;19:321–336. [PubMed]
- Hinkley SF, Jarvis BB. Chromatographic method for Stachybotrys toxins. Methods Mol Biol. 2001;157:173–204. [PubMed]
- Hossain M, Ahmed M, Ghannoum M. Attributes of Stachybotrys chartarum and its association with human disease. J Allergy Clin Immunol. 2004;113:200–208. [PubMed]
- Huang CC, Chen K, Huang TY. Immunohistochemical studies of sensory neurons in rat olfactory epithelium. Eur Arch Otorhinolaryngol. 1995;252:86–91. [PubMed]
- Hyde DM, Magliano DJ, Plopper CG. Morphometric assessment of pulmonary toxicity in the rodent lung. Toxicol Pathol. 1991;19:428–446. [PubMed]
- Hyde DM, Plopper CG, St George JA, Harkema JR. 1990. Morphometric cell biology of air space epithelium. In: Electron
Microscopy of the Lung (Schraufnagel DE, ed). New York:Marcel Dekker, 71–120.
- Institute of Laboratory Animal Resources 1996. Guide for the Care and Use of Laboratory Animals. 7th ed. Washington, DC:National
Academy Press.
- IOM (Institute of Medicine) 2004. Damp Indoor Spaces and Health. Washington, DC:National Academies
Press.
- Kai K, Satoh H, Kajimura T, Kato M, Uchida K, Yamaguchi R, et al. Olfactory epithelial lesions induced by various cancer chemotherapeutic
agents in mice. Toxicol Pathol. 2004;32:701–709. [PubMed]
- Kream RM, Margolis FL. Olfactory marker protein: turnover and transport in normal and regenerating
neurons. J Neurosci. 1984;4:868–879. [PubMed]
- Mery S, Gross EA, Joyner DR, Godo M, Morgan KT. Nasal diagrams: a tool for recording the distribution of nasal lesions
in rats and mice. Toxicol Pathol. 1994;22:353–372. [PubMed]
- Morgan KT, Jiang XZ, Patterson DL, Gross EA. The nasal mucociliary apparatus. Correlation of structure and function
in the rat. Am Rev Respir Dis. 1984;130:275–281. [PubMed]
- Nusuetrong P, Yoshida M, Tanitsu MA, Kikuchi H, Mizugaki M, Shimazu K, et al. Involvement of reactive oxygen species and stress-activated MAPKs in satratoxin
H-induced apoptosis. Eur J Pharmacol. 2005;507:239–246. [PubMed]
- Plopper CG, Duan X, Buckpitt AR, Pinkerton KE. Dose-dependent tolerance to ozone. IV. Site-specific elevation in antioxidant
enzymes in the lungs of rats exposed for 90 days or 20 months. Toxicol Appl Pharmacol. 1994;127:124–131. [PubMed]
- Ressler KJ, Sullivan SL, Buck LB. A zonal organization of odorant receptor gene expression in the olfactory
epithelium. Cell. 1993;73:597–609. [PubMed]
- Shifrin VI, Anderson P. Trichothecene mycotoxins trigger a ribotoxic stress response that activates
c-Jun N-terminal kinase and p38 mitogen-activated protein kinase and induces
apoptosis. J Biol Chem. 1999;274:13985–13992. [PubMed]
- Steiger D, Hotchkiss J, Bajaj L, Harkema J, Basbaum C. Concurrent increases in the storage and release of mucin-like molecules
by rat airway epithelial cells in response to bacterial endotoxin. Am J Respir Cell Mol Biol. 1995;12:307–314. [PubMed]
- Tuomi T, Saarinen L, Reijula K. Detection of polar and macrocyclic trichothecene mycotoxins from indoor
environments. Analyst. 1998;123:1835–1841. [PubMed]
- Ueno Y. 1984. General toxicology. In: Tricothecenes: Chemical, Biological and
Toxicological Aspects (Ueno Y, ed). Developments in Food Science, Vol 4. New
York:Elsevier, 135–145.
- Vesper S, Dearborn DG, Yike I, Allan T, Sobolewski J, Hinkley SF, et al. Evaluation of Stachybotrys chartarum in the house of an infant with pulmonary hemorrhage: quantitative assessment
before, during, and after remediation. J Urban Health. 2000;77:68–85. [PubMed]
- Wiley JA, Hogan RJ, Woodland DL, Harmsen AG. Antigen-specific CD8(+) T cells persist in the upper respiratory tract
following influenza virus infection. J Immunol. 2001;167:3293–3299. [PubMed]
- Yang GH, Jarvis BB, Chung YJ, Pestka JJ. Apoptosis induction by the satratoxins and other trichothecene mycotoxins: relationship
to ERK, p38 MAPK, and SAPK/JNK activation. Toxicol Appl Pharmacol. 2000;164:149–160. [PubMed]
- Yeh HC, Muggenburg BA, Harkema JR. In vivo deposition of inhaled ultrafine particles in the respiratory tract
of rhesus monkeys. Aerosol Sci Technol. 1997;27:465–470.
- Yike I, Allan T, Sorenson WG, Dearborn DG. Highly sensitive protein translation assay for trichothecene toxicity in
airborne particulates: comparison with cytotoxicity assays. Appl Environ Microbiol. 1999;65:88–94. [PubMed]
- Yike I, Dearborn DG. Pulmonary effects of Stachybotrys chartarum in animal studies. Adv Appl Microbiol. 2004;55:241–273. [PubMed]
- Yike I, Rand TG, Dearborn DG. Acute inflammatory responses to Stachybotrys chartarum in the lungs of infant rats: time course and possible mechanisms. Toxicol Sci. 2005;84:408–417. [PubMed]
- Young JT. Histopathologic examination of the rat nasal cavity. Fundam Appl Toxicol. 1981;1:309–312. [PubMed]
- Zhou HR, Islam Z, Pestka JJ. Induction of competing apoptotic and survival signaling pathways in the
macrophage by the ribotoxic trichothecene deoxynivalenol. Toxicol Sci. 2005a;87:113–122. [PubMed]
- Zhou HR, Jia Q, Pestka JJ. Ribotoxic stress response to the trichothecene deoxynivalenol in the macrophage
involves the SRC family kinase Hck. Toxicol Sci. 2005b;85:916–926. [PubMed]
- Zhou HR, Lau AS, Pestka JJ. Role of double-stranded RNA-activated protein kinase R (PKR) in deoxynivalenol-induced
ribotoxic stress response. Toxicol Sci. 2003;74:335–344. [PubMed]
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