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. Pestka~~2,3 

'Center for Integrative Toxicology, ZDepartment of Microbiology and Molecular Genetics, 3Department of Food Science and Human 

Nutrition, and 4Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA 

Satratoxin G (SG) is a macrocyclic trichothecene mycotoxin produced by Stachybotrys chartarrrm, 

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 pg/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 pg/kg bw) indicated that maximum atrophy of the olfactory epithelium occurred at 3 days PI . Exposure to lower doses (100 pg/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-a, 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. Key words: 

apoptosis, fungus, inflammation, inhalation, mycotoxin, neurotoxicity, olfactory sensory neuron, 

rhinitis, trichothecene. Ertviran Health Perspect 114:1099-1107 (2006) . doi:10 .1289/ehp.8854 

available via http://dx.doi.org/ [Online 27 February 20061 

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 (lOM) 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 

cbartarum, a saprophytic "black mold" that 

grows on cellulosic materials, including wallboard, ceiling tiles, and cardboard (Hossain 

et al. 2004). Incidences of indoor S. chartaratrn 

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, macrocyelic 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/m3 (Yike et al . 1999) and from 54 to 

330 ng/m3 (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. cbartaracrn nrycotoxins . 'I o 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. chartarrsm (Yang 

et al . 2000). 

Materials and Methods 

Toxins . SG and isosatratoxin F (ISF) were 

purified from S. cbartarurrt 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 

Address correspondence to J .J . Pestka, 234 G.M . 

Trout Building, Michigan State University, East 

Lansing, MI 48824 USA. Telephone : (517) 353-

11-09 . Fax: (517) 353-8963 . E-mail : pestkaC msu .edu 

We thank L . Bramble, M . Perry, A. Porter, 

R. Rosebury, K. Campbell, R . Common. D. Craft, 

L . Chen, B . Chamberlin, and A. Thelen for their 

technical assistance . 

This research was funded by a Nlichigan State 

University Foundation Strategic Partnership Grant, 

the Michigan State Health and Biomedical Research 

Initiative I, and U.S . Public Health Service grant 

ES03358 (J .J .P .) from the National Institute of 

Environmental Health Sciences . 

The authors declare they have no competing 

financial interests . 

Received 14 November 2005 ; accepted 27 February 

2006. 

Environmental Health Perspectives " VOLUME 1141 NUMBER 7 1 July 2006 1099 
Islam et al . 

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 tinder 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 . Pathogenfree female C57B1/6 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 ItL/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 postinstillation (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 

111 

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, TI); 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 (Figure 1) . 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 ltm, 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 

0 Normal OE 

- Atrophic OE 

0 T1 

marker protein (OMP; 1 :4,000, goat antiOMP 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 SGinduced 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), a 75% of OE with 

lesions . 

Light microscopic morphometry. Thickness 

of the OE lining the medial surface of the second ethmoid turbinates (2E) in T3 (Figure 1) 

T3 

T2 

T4 

Figure 1 . Diagrammatic representation of the nasal passages of the murine nose . Abbreviations: DM, dorsal medial meatus (airway); E, ethmoid turbinate; HP, hard 

palate ; L, lateral meatus; MM, middle meatus ; MS, maxillary sinus; MT, maxilloturbinate ; N, nasoturbinate ; Na, naris; NP, nasopharynx; NPM, nasopharyngeal meatus ; 

S, septum; V, ventral meatus . (A) Right nasal passage with the nasal septum removed, exposing the luminal surfaces of nasal turbinates IN, MT, 1 E-6E) projecting 

from the lateral wall; vertical lines indicate the anterior surfaces of transverse tissue blocks (T1-T4) that were selected for microscopic examination. (8) Crosssectional views of Tl-T4. Colored areas in (A) and (B) represent OE that exhibited SG-induced apoptosis and atrophy 1-7 days PI or were free of toxin-induced injury. 

1100 VOLUME 114 1 NUMBER 7 1 July 2006 " Environmental Health Perspectives 
Satratoxin-induced olfactory epithelium atrophy 

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 3,540x 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, b,., the proportion of the 

epithelium composed of cytoplasm, nuclei, or 

apoptotic nuclear fragments, was determined 

by point counting and calculated using the 

following formula: 

vU = Pp = P,,/P,, III 

where PP is the point fraction of P , the number of test points hitting the structure of 

interest, divided by Pi, 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 (S,,) was determined by point and 

intercept counting and was calculated using 

the following formula: 

Sv= 21oLr, [21 

where Io is the number of intercepts with the 

object (epithelial basal lamina) and L, 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 (T) : 

t = V /S . [31 

Other standard morphometric and image 

analysis techniques were used to determine the 

numeric cell density of mature olfactory sensory neurons (OSNs) in OE. Morphometric 

`o u 

N 

. a 

m 

N 

V 

. 0 

0 

r 

r 

G 

N 

x 

4 

3 

2 

1 

0 

0 5 25 100 250 500 

SG (lug/kg) 

Figure 2. Effect of SG dose on OE toxicity shown as 

histologic severity score in nasal airways of treated 

mice (see "Materials and Methods" for details) . 

Bars represent group means + SEs (n= 5-6) . 

`Significantly different from saline vehicle control (p < 0 .05) . 

estimates of the numeric cell density of 

OSNs immunohistochemically reactive for 

OMP (protein indicator of mature OSNs) 

were determined via light microscopy (790x 

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 (Figure 1) 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 ml12% pentobarbital containing 1 IU heparin . 

Immediately after anesthesia, the whole bodv 

received an intravascular perfusion via the lefr 

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 pm) 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 uranvl 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 

IOOCXII electron microscope (JEOL Ltd ., 

Tokyo, Japan) . 

Real-time polymerase ehain 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 

Pro-/anti-apoptotic genes 

Figure 3. Apoptosis of OSNs in OE 24 hr after initial exposure to SG . Abbreviations: 2E, ethmoid turbinate 2; 

Be, basal cell ; LP, lamina propria; kn, dendritic knob of OSN ; mv, microvilli ; n, axons of OSNs ; Sc, sustentacular cell cytoplasm; Sn, sustentacular cell nucleus; TB, turbinate bone . (A and B) Immunohistochemical 

detection of activated caspase-3 (brownish red) in OE lining the medial aspect of 2E in T3 and olfactory 

nerve bundles in the underlying LP in mice treated with saline vehicle alone (A) or 500 pg/kg bw SG (B). The 

open arrow in (B) indicates the immunohistochemical staining of nerve bundles in LP ; solid arrows indicate 

stained OSNs in OE . (C) Electron photomicrograph of SG-exposed (500 pg/kg bw) OE with numerous apoptotic bodies or shrunken OSNs with condensed or fragmented nuclei or marginated nuclear chromatin 

(solid arrows); open arrows indicate neutraphils and arrowheads indicate subepithelial basal lamina . 

(D) Relative mRNA expression of apoptosis-related genes in excised ethmoid turbinates 1 day PI with saline 

or 500 pg/kg bw SG ; data are means ± SEs (n = 6) . 

*Significantly different from the saline vehicle control (p < 0 .05) . 

Environmental Health Perspectives " VOLUME 1141 NUMBER 7 1 July 2006 1 101 
Islam et al . 

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 

(Figure 1) . 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, TN) 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 caspaseactivated DNase (CAD)], cytokine genes 

[interleukin-1 (IL-1), tumor necrosis factor-" 

(TNF-a), IL-61, and the chemokine gene 

/L11P-2 (macrophage-inflammatory protein-2) 

were performed on an ABI PRISM 7900HT 

Sequence Detection System using Taqman 

One-Step RT-PCR (reverse-transcriptase-PCR) 

1 PI 

31`1 

7 PI 

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-NewmanKeuls 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 . 

Results 

OE targeted by nasal SG exposure. Light 

microscopic evaluation of four specific anatomical sites (T1-T4) revealed that mice exposed 

to SG [500 11g/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 vehicletreated controls (Figure lA) . 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 (Figure 1B). SG-induced apoptosis 

was also present in the OE of the mid and 

© 

T3 

O Saline 

SG 

FDJ 

-- 14 

E 

00'_ 

9 V 

E ° 

. 

c 0 

m 

12 

10 

B 

6 

4 

2 

0 

Single instillation 

Multiple 

instillation 

B 

1 3 7 28 6 

Days PI 

Figure 4. Time-dependent OE atrophy and OSN loss . Abbreviations: DM, dorsal medial meatus (airway) ; HP, hard palate ; LP, lamina propria; MM, middle meatus ; 

MS, maxillary sinus; NPM, nasopharyngeal meatus ; S, septum ; TB, turbinate bone . (A) OE at 1, 3, and 7 days PI in mice treated with saline vehicle alone or SG 1500 

pg/kg bwl. Tissues were stained with H&E; bars = 50 um . Arrows indicate apoptotic nuclei . (B) Intranasal site (2E, red asterisks) used for morphometry. 

Morphometric analysis of epithelial thickness (atrophy) (C) and volume density of apoptotic nuclei (D) in OE at 1, 3, 7, or 28 days after a single instillation with 

saline or SG 500 pg/kg bw or 6 days after the start of five daily instillations with saline or SG (100 Ug/kg bw). Bars represent group means ± SE In =61. Bars labeled 

with different letters are significantly different (p < 0.05). 

1 102 VOLUME 114 1 NUMBER 7 1 July 2006 " Environmental Health Perspectives 
Satratoxin-induced ol factory epithelium atrophy 

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 

(TI) . Interestingly, the OE lining the dorsal 

medial meatus throughout the nasal passages 

(TI-T4) had no microscopic evidence of SGinduced 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 lrg/kg 

bw (80 ng/mouse) and the lowest effect level 

was 25 Itg/kg bw (400 ng/mouse ; Figure 2) . 

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) (Figure 3A-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 III 

(Figure 3B). 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. Realtime 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, 

Baa, caspase-3, and CAD, whereas expression 

of Apaf-1 and the anti-apoptotic gene Bcl-2 

was unchanged (Figure 3D) . 

SG-mediated OE atrophy. Concurrent 

with SG-induced OSN apoptosis, OE atrophy 

was detectable at 1 day PI (Figure 4A) . SGinduced OE lesions were similarly apparent at 

3 and 7 days PI along with increased atrophy. 

Morphometry of OE lining 2E (Figure 413) 

confirmed that greater atrophy occurred in 

mice sacrificed 3 and 7 days PI than in mice 

sacrificed 1 day PI, with an approximately 5090 

reduction in epithelial thickness compared with 

control mice (Figure 4C) . Restoration of the 

normal thickness of the OE compared with 

that of control was still not complete at 28 days 

III (Figure 4C). 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 

(Figure 4D). Exposure to a lower daily dose of 

SG 

OH 

Deoxynivalenol 

ISF 

oH 

Verrucarin A 

Figure 5. Comparative effects of trichothecenes on the induction of atrophy in OE . Abbreviations: LP, lamina propria ; N, olfactory nerve; TB, turbinate bone . 

(A) Light photomicrographs of H&E-stained OE lining 2E in T3 from mice 3 days PI with saline vehicle alone, deoxynivalenol (10 mg/kg bw), verrucarin A (500 pg/kg 

bw), ISF (500 pg/kg bw), SG (500 pg/kg bw), or T-2 toxin (1 mg/kg bw); bars = 50 pm . (B) Chemical structures of tested trichothecenes . 

H,C-CH 

CH, 

0+ , 

,c 

SG (100 pg/kg bw or 1 .6 pg/mouse) for 5 consecutive days resulted in similar atrophy and 

apoptosis of OE compared with those alterations caused by the single 500 pg/kg bw SG 

(Figure 4C,D), suggesting that, in the short 

term, these effects were cumulative . 

Role of trichotbeeene structure in OE atrophy. The nasal effects of trichothecenes not 

associated with Stacbybotrys 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 onethird to one-fifth of LD50 values (doses lethal 

in 50% of test animals) (Ueno 1984) . These 

trichothecenes had no effect on OE compared 

with the Stacbybotrys mycotoxins SG and ISF, 

which exhibited robust toxicity (Figure 5A) . 

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 (Figure 5B) . 

Selective apoptosis induction in 0SNs. 

OMP, a specific peptide found only in 

mature OSNs (Kream and Margolis 1984), 

was markedly reduced in the OE of SGinstilled mice compared with saline-instilled 

control mice (Figure 6A-C) . Morphometric 

analysis revealed a 67, 94, and 81% loss in 

OSNs per millimeter of OE in the nasal 

mucosa lining the dorsolateral meatus at l, 3, 

CH, 

T-2 toxin 

Environmental Health Perspectives " VOLUME 1141 NUMBER 7 1 July 2006 1 103 
Islam et al . 

and 7 days PI, respectively, compared with 

the identical region in vehicle-instilled mice 

(Figure 6D). Consistent with partial recovery 

of OE thickness at 28 days PI (Figure 4C), 

there was also incomplete restoration of the 

numbers of OMP-positive OSNs (Figure 6D) 

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 Itg/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 

(Figure 6E-G). 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 

(Figure 6B,G). 

Concurrent with the initial loss of OMPpositive OSNs, there was also noticeable atrophy of the OMP-positive olfactory nerve 

bundles located in the lamina propria underlying the atrophic OE (Figure 6B). 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 (Figure 7A,B). 

Loss of OMP-stained olfactory nerves was 

most marked in the lateral and medial aspects 

of each of the OBs (Figure 7C). 

Inflamneatory gene up-regulation and 

neutrophil infiltration in OE and OB. At 

I 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 

(Figures 3C, 6F). 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-a, IL-lot, IL-1(3, and IL-6 as well as the 

chemokine MIP-2 associated with acute 

inflammatory cell infiltration (Figure 8A). 

Elevated MIP-2, TNF-a, and IL-6 mRNA 

expression was also observed in the OB 

(Figure 8B) . 

Slightly decreased severity of SG-induced 

neutrophilic rhinitis corresponded with timedependent disappearance of epithelial apoptosis and development of epithelial atrophy . 

© T3 

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 (Figure 8C). 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 (Figure 8D). These infiltrations 

extended bilaterally into the atrophic olfactory 

121 

E 

E 

H 

d 

d > 

.y 

Q. 

d 

0 

700 

600 

500 

400 

300 

200 

100 

0 

Single instillation 

A 

II I A A 

z 

F---l Saline 

SG - 

Multiple 

instillation 

A 

L 

3 7 28 6 

Days PI 

Figure 6. SG exposure depletes OSNs in OE . Abbreviations: AL, airway lumen; BC, basal cells; DM, dorsal 

medial meatus (airway) ; HP, hard palate ; LP, lamina propria; MM, middle meatus; MS, maxillary sinus; MV, 

microvillar apical surface; S, septum ; SC, sustentacular cells ; TB, turbinate bone . (A,8) Light photomicrographs of OE lining 3E in T3 nasal section (illustrated in C) from mice 1 day PI with (A) saline vehicle alone or 

(B) SG 1500 ug/kg bw). Sections were immunohistochemically stained with anti-OMP antibody and counterstained with hematoxylin; immunoreactivity for OMP (marker for mature OSNs) is identified by the brownish 

red chromagen in OE and olfactory neurons (n) in the LP . Arrows in (A) and (8) indicate OMP-stained cilia 

along the airway surface; (CD) Results of morphometric analyses of 3E (box) in T3 nasal section (C) are 

graphically illustrated in (D). Bars represent group means ± SEs. Bars labeled with different letters are significantly different (p < 0.05)_ (f-G) Three transmission electron photomicrographs of the apical third of the 

OE from a mouse i day after saline vehicle alone instillation (E) or 1 day after a single SG instillation (F), and 

the full thickness of remaining atrophic OE from a SG-exposed mouse 3 days after a single intranasal instillation (G); bars = 10 Nm. Dotted arrows in (E) indicate normal dendritic knobs of OSNs, but there is a loss of 

these dendritic portions of OSNs in (Fand G) . In (F), white arrows indicate apoptotic OSNs and black arrows 

indicate luminal cellular debris and phagocytizing neutrophils in the nasal AL . Arrowheads in (G) indicate 

subepithelial basal lamina ; only a few OSNs remain in the atrophic OE in (G). 

1 104 VOLUME 114 1 NUMBER 7 1 July 2006 " Environmental Health Perspectives 
Satratoxm-induced olfactory epithelium atrophy 

nerve and glomerular layers of the OB at 

7 days PI (Figure 8E). 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 (Figure 9A,B) . 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. 

Discussion 

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 

Figure 7. Depletion of OSNs in OBs. Abbreviations: CB, cranial bone ; G, glomerular layer; HP, hard palate ; ONL, olfactory nerve layer; NPM, nasopharyngeal meatus ; 

S, nasal septum . (A and B) Immunohistochemical detection of OMP (brownish red) in ONL and G layer in the OB of mice 7 days PI with (A) saline vehicle alone or 

(B) SG (500 ug/kg bw). (C) T4 nasal section containing OB and sites of ONL and G layer atrophy due to SG exposure (red asterisks) . Square in (C) represents the 

location in OB of (A) and IB). 1,000 

a .N 

N 

N 100 

G7 y 

Z C 

E? 

m ,~o 

. 

ad ¢ 

O Saline 

SG 

10 

0 

5 

.y e 

N 

n °~ 

x y 

md 

Q~ 

Z 

. e 

E? 0 

. a= 

s 

IL-I, IL-711 IL-6 TNf,, MIP-2 

Inflammatory cytokine gene 

B 

4 

3 

2 

0 

Inflammatory cytokine gene 

Vehicle SG 

D 

Figure 8. Acute inflammatory responses in mouse nasal passage (rhinitis) and OB (mild focal encephalitis). Abbreviations : CP, cribiform plate; EPL, external plexiform 

layer; ONL, olfactory nerve layer; and GL, glomerular layer; LP, lamina propria; ON, olfactory nerve bundles; TB, turbinate bone . (A, B) Real-time PCR measurements of 

proinflammatory gene mRNAs in ethmoid turbinates (A) and OB (B) of mice 1 day PI with saline vehicle or SG (500 Ug/kg bw); bars represent group means ± SE (n = 6). 

(C-E) Immunohistochemical detection of neutrophils (red stained cells and indicated by arrows) in OE (C, ON passing through the CP from nose to OB (D), and the ONL 

and GL in the OB (E) from mice 7 days PI with saline vehicle alone or SG (500 ug/kg bw); bars = 50 Um . 

"Significantly differentfrom control group (p < 0 .05) . 

IL-la IL-1{i IL-6 TNFci MIP-2 

Environmental Health Perspectives " VOLUME 114 1 NUMBER 7 1 July 2006 1 105 
Islam et al . 

underlying mechanisms, relative contributions 

of individual organisms, and potential for 

interactions remain poorly understood (IOM 

2004). Although exposure to either S. cbartarum spores or associated satratoxins has been 

previously shown to initiate acute inflammatory responses in the rodent lung (Yike and 

Uearborn 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) (Figure 10) 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 pm 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 Srcfamily kinases (Zhou et al . 20056) . Notably, 

SG-induced genes that have previously been 

associated with death receptor-mediated 

OSN apoptosis include TNF-a, 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 

Figure 9. Ultrastructural alterations in ONL and GL of OB 7 days PI with saline vehicle alone (A, C) or SG 

(500 pg/kg bw) (B, D) . Abbreviations: CL, capillary lumen; GL, glomerular layer; ML, meningeal layer; N, 

neutrophil; ON, olfactory nerves; ONL, olfactory nerve layer; PG, periglomerular cell . (A, 8) Focal areas of 

axonal degeneration (long arrows) present in the markedly atrophic ONL in the SG-treated mouse (8) but 

not the vehicle-treated control mouse (A). In IB), infiltrating Ns with segmented nuclei are closely associated with areas of degeneration (arrows) . (C, D) Dense staining axons of ON in the GL of the vehicle control mouse (C) are absent in the GL of the SG-exposed mouse (asterisk in D) . An N is also present in the 

SG-exposed mouse but not in the control mouse . 

OSN Depletion 

OE Atrophy 

Rhirp6s 

OSN Apoptosis Encephalitis 

SG 

Exposure 

Rhinitis 

24h 7days 

rime After Exposure 

s 

Figure 10. Diagrammatic representation of SGinduced pathology in OE and OB with time after 

exposure . Abbreviations: BC, basal cells; C, capillary in LP ; CP, cribiform plate; G, glomerular ; LP, 

lamina propria; MC, mitral cell ; N, neutrophil ; ON, 

olfactory nerve; ONL, olfactory nerve layer; SC, 

sustentacular cells . OE is shown in yellow, normal 

OSN in orange, and neutrophils phagocytosing 

apoptotic OSNs in blue . 

1 106 VOLUME 114 1 NUMBER 7 1 July 2006 " Environmental Health Perspectives 
Satratoxin-induced ol factory epithelium atrophy 

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 (Figure 5) and 

that one region of the nasal cavity lined by OE 

(dorsomedial meatus) was consistently spared 

from toxicant-induced injury (Figure 1), 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. chartarunt . 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 . 

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