Role of Reactive Oxygen and Nitrogen Species in
Olfactory Epithelial Injury by the Sulfur Mustard
Analogue 2-Chloroethyl Ethyl Sulfide
Heidi C. O’Neill1, David J. Orlicky2, Tara B. Hendry-Hofer3, Joan E. Loader3, Brian J. Day1,3, and Carl W. White3,4
1Department of Pharmaceutical Sciences, and2Department of Pathology, University of Colorado at Denver Health Sciences Center, Denver,
Colorado; and3Department of Medicine, and4Pulmonary Division, Department of Pediatrics, National Jewish Health, Denver, Colorado
The inhalation of sulfur mustard (SM) causes substantial deposition
in the nasal region. However, specific injury has not been character-
ized. 2-chloroethyl ethyl sulfide (CEES) is an SM analogue used to
model injury and screen potential therapeutics. After the inhalation
of CEES, damage to the olfactory epithelium (OE) was extensive.
Terminal deoxynucleotidyl transferase–mediated dUTP nick-end
labeling-positive cells were present by 4 hours, and maximal at 18–
72 hours. Cleaved caspase 3 immunohistochemistry (IHC) was
maximal at 18 hours after the inhalation of 5% CEES. Olfactory
marker protein (OMP)–positive olfactory neurons were markedly
decreased at 18 hours. IHC-positive cells for 3-nitrotyrosine (3-NT)
absent by 72 hours. AEOL 10150, a catalytic manganoporphyrin
antioxidant, administered both subcutaneously (5 mg/kg) and in-
tranasally (50 mM, ‘‘combined treatment’’), decreased OE injury.
CEES-induced increases in markers of cell death were decreased
by combined treatment involving AEOL 10150. CEES-induced
by combined treatment involving AEOL 10150. The selective in-
ducible nitric oxide synthase inhibitor 1400W (5 mg/kg, subcutane-
ous), administered 1 hour after inhalation and thereafter every
4 hours (five doses), also reduced OE damage with improved OMP
and 3-NT staining. Taken together, these data indicate that reactive
oxygen and nitrogen species are important mediators in CEES-
induced nasal injury.
Keywords: sulfur mustard; olfactory; cell death; oxidative stress;
Sulfur mustard (SM; bis 2-chloroethyl sulfide) is a bifunctional
alkylating and vesicating chemical warfare agent used in
numerous military conflicts during the last century. SM poses
a threat to both military and civilian populations because of its
ease of synthesis and potential for terrorist deployment. SM is
highly reactive, and forms stable adducts with cellular macro-
molecules (including DNA and proteins), resulting in an in-
crease or decrease in function of key cellular components. The
(CEES) lacks one of the terminal chlorines, but retains similar
alkylating and vesicating properties. CEES provides a useful
model for investigating the mechanisms of, and screening
therapeutic agents for, SM.
Exposure to SM in the battlefield or by civilian populations
typically occurs via inhalation. Although notable ocular and
skin injury occurs, those exposed will more likely succumb to
respiratory damage (2). Interestingly, early studies of SM ab-
sorption indicated that 80–90% of the compound was absorbed
through the nose (1). Even with percutaneous or intravenous
administration of SM using35S-labeled SM, the highest con-
centrations were found, in descending order, in the nasal region,
kidney, liver, and intestines (3). Numerous clinical studies also
indicate that the inhalation of SM results in anosmia (2, 4, 5).
Despite reports of high levels of SM absorption in the nose and
of secondary anosmia, nasal injury after the inhalation of SM or
CEES has not been characterized to date, to the best of our
Oxidative stress plays a role in the pathophysiology of SM/
CEES inhalation injury (6, 7). Oxidants are crucial for host
defense, but an overabundance of oxidants can overwhelm
repair and antioxidant adaptive responses, resulting in oxidative
stress and injury. After exposure to SM, the alkylation and
depletion of reduced glutathione contribute to an impaired
ability to respond to oxidative stress (8–10). Treatment with
exogenous superoxide dismutase (SOD), catalase, or antioxi-
dant mimetics has proven beneficial in CEES-induced lung
injury (6, 11–13). These data indicate a role for oxidative stress
after exposure to CEES. What, if any, role oxidative stress plays
in CEES-induced nasal injury remains unknown.
The nasal region serves a number of key functions, including
the conditioning of incoming air, filtration, and olfactory sensa-
tion. Inspired air is both heated and humidified in the nasal
inhaled particles and mucociliary clearance (15). In the nasopha-
ryngeal region, an impaction of 5–30 mm particles occurs, with
particles smaller than 5 mm depositing in a size-dependent
manner from the trachea to the alveolar spaces (16). After the
inhalation of toxicants, nasal injury tends to be site-specific, with
varied depositions because of airflow patterns or specific tissue
susceptibilities (17). Inhalation is highly likely to affect the
olfactory epithelium, given the reported anosmia. However,
Humans inhaling sulfur mustard (SM) have manifested
anosmia. Through the use of a rat nose-only inhalation
model and the sulfur mustard analogue 2-chloroethyl ethyl
sulfide (CEES), this study demonstrates that the inhalation
of CEES results in oxidative damage to the olfactory
neurons of the olfactory epithelium (OE). Treatment with
antioxidants or the use of a selective inducible nitric oxide
synthase inhibitor decreased OE damage. These data
indicate that oxidative damage may play a role in the
anosmia reported in humans after exposure to CEES, and
provides the foundation for future studies of SM.
(Received in original form May 26, 2010 and in final form November 1, 2011)
This research was supported by the CounterACT Program of the Office of the
Director at the National Institutes of Health and National Institute of Environ-
mental Health Sciences grant U54 ES015678.
Correspondence and requests for reprints should be addressed to Carl W. White,
M.D., Pulmonary Division, Department of Pediatrics, National Jewish Health,
1400 Jackson St., Denver, CO 80206. E-mail: firstname.lastname@example.org
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2010-0214OC on June 3, 2011
Internet address: www.atsjournals.org
Vol 45. pp 323–331, 2011
been reported. This study sought to characterize CEES inhala-
tion injury to the nasal region, and to investigate the role of oxi-
foundation for investigations of actual SM-induced injury.
MATERIALS AND METHODS
We used male Sprague-Dawley rats (Harlan, Indianapolis, IN) weigh-
ing 275–350 g. Animals were provided with food and water ad libitum.
These experiments were approved by the Animal Care and Use
Committee at National Jewish Health.
Rats were anesthetized with ketamine (75 mg/kg), xylazine (7.5 mg/kg),
and acepromazine (1.5 mg/kg). Animals were exposed to CEES for
15 minutes, as previously described (6).
Treatment with AEOL 10150. Rats received an injection of
AEOL 10150 (5 mg/kg, subcutaneous) or PBS vehicle (1 ml/kg) at 1
and 9 hours after exposure to CEES. For intranasal treatment, a 25-ml
volume at concentrations of 5 mM, 500 mm, 250 mm, or 50 mm was
delivered to each nare through insufflation during anesthesia with
isofluorane at both 1 and 9 hours after inhalation.
Treatment with 1400W. Rats received 1400W (5 mg/kg, subcuta-
neous) immediately after the exposure to inhalation and every 4 hours
thereafter, for a total of five doses during the 18-hour study period.
At 4, 8, or 18 hours after exposure, animals were killed with 130 mg
Sleepaway per animal (Fort Dodge Animal Health, Fort Dodge, IA).
Heads were removed by guillotine and skinned. The lower jaw and
musculature were removed, and heads were placed in 4% paraformal-
dehyde in PBS for 48 hours. After fixation, heads were decalcified
(Immunocal; Decal Chemicals, Tallman, NY) for 7 days. Transverse
sections of nasal passages were removed, based on the methods of
Young (18). The section representative of olfactory epithelium, T3, was
cut between the second palatal ridge and the middle of the first molars.
In Situ Cell Death Detection Using Terminal Deoxynucleotidyl
Transferase–Mediated dUTP Nick-End Labeling Assay
Cell death was detected using the DeadEnd Colorimetric TUNEL Kit
(Promega, Madison, WI). Briefly, antigen retrieval was performed
using proteinase K (30 mg/ml) for 10 minutes. Sections were incubated
with terminal deoxynucleotidyl transferase for 1 hour at 378C. An
application of streptavidin–horseradish peroxidase (HRP) for 30
minutes was followed by detection using 3,39-diaminobenzidine
(DAB). Four fields at the entrances to the ethmoturbinates were
counted. The apoptotic index was determined by counting apoptotic
cells 3 100 and dividing by total cells in a field.
Antigen retrieval was performed using a decloaking chamber (Biocare
Medical, Concord, CA) in 10 mM sodium citrate buffer, pH 6.0, except
for cleaved caspase 3 (CC3), which used 1 mM EDTA buffer, pH 8.0.
to CC3 (1:50; US Biologicals, Swampscott, MA), olfactory marker
protein (OMP; 1:200, Abcam ab62144; Abcam, Cambridge, MA), or
3-nitrotyrosine (3-NT, 1:300, Millipore AB5532; Millipore, Billerica,
MA). Secondary antibody (biotinylated anti-rabbit IgG; Vector Labo-
ratories, Burlingame, CA) was followed by the avidin–biotin complex
(Vector Laboratories) for OMP and 3-NT, or streptavidin–HRP for CC3.
IgG-negative controls were generated.
Data are presented as means 6 SEM. Comparisons between multiple
groups were performed using one-way ANOVA and the Tukey test for
post hoc analysis (GraphPad Prism version 4.0c; GraphPad, San Diego,
CA), with significance set at P < 0.05.
Nasal Injury and Cell Death in the Olfactory Epithelium
Attributable to the Inhalation of CEES
Investigation of the T3 section of the nasal cavity indicated that
damage was most prominent in the olfactory epithelium (OE) at
the medial meatus of the septum, and at the entrances to the
ethmoturbinates (ETs) (Figure 1, red). For consistency, all
sections were photographed at the entrance to the ETs. Figure
2A shows a normal, organized OE layer from an animal that
inhaled ethanol (EtOH) 18 hours after exposure. At 4 hours
after the inhalation of CEES, the organization of the OE layer
remained intact (Figure 2B), although pyknosis and nuclear
clearing are evident. By 18 hours after the inhalation of CEES,
marked disorganization of the full epithelial layer had occurred
Cell death in the OE was investigated using terminal
deoxynucleotidyl transferase–mediated dUTP nick-end labeling
(TUNEL). The OE of rats exposed to EtOH shows that a low
number of TUNEL-positive, DAB-stained cells was present
constitutively (see Figure E1A in the online supplement). By
4 hours after the inhalation of CEES, TUNEL-positive cells
were also present (Figure E1B). TUNEL-positive cells were
increased by the 18-hour time point (Figure E1C). Numbers of
TUNEL-positive and TUNEL-negative cells were counted in
four high-power fields at the edges of the ETs (3400 total
magnification), to calculate the percentages of TUNEL-positive
cells. As shown in Figure 2D, the percentage of TUNEL-
positive cells had significantly increased after the inhalation of
CEES in a time-dependent manner at the 4-hour and 18-hour
time points, compared with rats exposed to EtOH.
The cleavage of cysteine–aspartic acid proteases, or caspases,
commits a cell to apoptosis. CC3 is a terminal caspase. Its
cleavage is indicative of apoptosis, and complements TUNEL
dominately of olfactory epithelium (OE). The region in red shows
consistently high damage after the inhalation of sulfur mustard
analogue 2-chloroethyl ethyl sulfide (CEES). The septum along the
middle of the nasal cavity, and especially the medial meatus of the
septum (MS), exhibited marked injury. The entrances to the ethmo-
turbinates (ET) near E1 and E2, and the more medial E3, were regions
of most severe damage.
Representation of nasal cross section comprised pre-
324AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 45 2011
staining as a marker of cell death. A low level of CC3 staining
is present after the inhalation of EtOH (Figure E1D). Cells
staining positive for CC3 were also present by 4 hours after the
inhalation of CEES (Figure E1E). The number of CC3-positive
cells increased by 18 hours after the inhalation of CEES (Figure
E1F). The counting of CC3-positive cells indicated a significant
increase at the 18-hour time point after the inhalation of CEES,
compared with EtOH (Figure 2E).
Olfactory neuronal cell bodies at various levels of maturity
are present below the more apical sustentacular cell bodies, or
support cell layer. OMP is expressed specifically in the cytosol
of olfactory neurons (19). In Figure 2F, arrows indicate that
dark Nova Red–stained, OMP-positive olfactory neurons are
located along the central region after the inhalation of EtOH.
After exposure to CEES, the loss of this region is evident. No
changes were noted at the 4-hour time point (not shown). At
8 hours after the inhalation of CEES (Figure 2G), the OMP-
positive region was present but beginning to lose the structured
organization seen in rats exposed to EtOH. By 18 hours after
the inhalation of CEES, punctate OMP-positive regions were
evident but greatly reduced in number (Figure 2H).
Increased Oxidative Stress in the OE Is Attributable
to the Inhalation of CEES
3-NT is a stable end product of the formation of peroxynitrite,
and is commonly used as a marker for the formation of reactive
nitrogen species (RNS). No constitutive 3-NT staining was
evident in rats exposed to EtOH (Figure 2I). No changes were
evident at 4 hours after the inhalation of CEES (not shown).
Eight hours after exposure to CEES, an increased deposition
of 3-NT was evident in the OE (Figure 2J). The deposition of
3-NT was present 18 hours after the inhalation of CEES, but
appeared to be declining as of 8 hours (Figure 2K).
Neutrophils Were Minimally Evident at Sites of Injury after
the Inhalation of CEES
Polymorphonuclear leukocytes (PMNs) are commonly impli-
cated in inflammatory tissue damage, especially when present at
high levels. Esterase staining can be used to detect neutrophils.
Figure E2 demonstrates the presence or absence of these cells,
indicating minimal neutrophil involvement after the inhalation
of CEES. After the inhalation of EtOH, no PMNs were evident
(Figure E2A). No PMNs were noted near the injured OE ETs
by 8 hours after exposure to CEES (Figure E2B). PMNs were
observed to be extravasating by 18 hours after the inhalation of
CEES in small numbers (2–6 per 3400 field), as indicated by
arrows in Figure E2C.
AEOL 10150 Treatments and OE Damage
Previous studies indicated that treatment with AEOL 10150
resulted in improved outcomes in lung injury markers (6).
Because the maximal histopathological effect was evident at
18 hours after the inhalation of CEES, this time point was
chosen to investigate the therapeutic effects of AEOL 10150 in
the nose. Hematoxylin-and-eosin–stained sections indicated
severe apical damage to the OE after exposure to CEES only
cellular degeneration by 4 hours after inhalation (B), and marked apical disruption by 18 hours (C). CEES-induced cell death, as measured by TUNEL,
was significantly increased over rats inhaling EtOH at 4 and 18 hours (D; n 5 3, mean 6 SEM). (E) Cleaved caspase 3 (CC3) staining was significantly
increased over EtOH controls at 18 hours (n 5 3, mean 6 SEM). (F) Staining for olfactory marker protein (OMP), a specific marker for olfactory
neurons, was normally distributed in the mid-OE layer (arrows) after the inhalation of EtOH. (G) Because minimal change was evident at 4 hours, an
8-hour time point was investigated and showed decreased OMP-positive staining. (H) By 18 hours, some punctate OMP staining was evident, but
was in decline. 3-nitrotyrosine (3-NT) staining was absent in EtOH-exposed animals (I), but was prominent 8 hours after exposure to CEES (J). (K) By
18 hours, 3-NT staining was waning, but still present. Bars 5 50 mm.
Characterization of CEES-induced injury to the OE. Compared with rats inhaling ethanol (EtOH) (A), rats inhaling CEES show early
O’Neill, Orlicky, Hendry-Hofer, et al.: ROS and RNS in CEES-Induced Olfactory Epithelial Injury325
(Figure E3A) or CEES combined with PBS (diluent) treatment,
administered both subcutaneously (1 ml/kg) and intranasally
(25 ml per nare) at 1 and 9 hours after the inhalation of CEES
(Figure E3B). In contrast with previous studies in the lung,
AEOL 10150 delivered only by the subcutaneous route (5 mg/kg,
1 and 9 hours after the inhalation of CEES) did not improve nasal
injury (Figure E3C). Based on effective dosing in vitro, AEOL
10150 delivered intranasally (50 mM, 25 ml per nare) alone was
not effective against CEES-induced nasal injury (Figure E3D) (7).
A combination of subcutaneous and intranasal delivery of AEOL
10150 was then investigated, with the subcutaneous dose main-
tained at 5 mg/kg, and with intranasal dose concentrations of
5 mM, 500 mM, 250 mM, or 50 mM. Both subcutaneous and
intranasal treatments were administered at 1 and 9 hours after the
inhalation of CEES. The delivery of intranasal concentrations of
5 mM (Figure E3E), 500 mM (Figure E3F), or 250 mM (Figure
E3G) did not appear to result in improvement. Treatment with
50 mM AEOL 10150 did result in less apical damage, and was
investigated further (Figure E3H), and is hereafter referred to as
‘‘combined AEOL 10150 treatment.’’
Combined AEOL 10150 Treatment Decreased Histopathology
The double-blind scoring of apoptosis, apical damage, basement
membrane injury, the presence of edema, and overall severity of
injury was measured around four regions of the entrances to the
ETs. All injuries were graded as either absent (2) or present
(1). Animals exposed to EtOH showed no signs of histologic
injury in any of the five markers investigated. Rats exposed to
CEES alone showed increased apical membrane disruption
(‘‘ruffling’’), evidence of edema, areas of basement membrane
disruption, regions of pyknotic cells indicative of apoptosis, and
overall increased injury, evident in the four entrances to the
ETs. When animals received intranasal and subcutaneous
treatment with AEOL 10150, levels of apical membrane
disruption showed some improvement. Pyknotic cells, indicative
of apoptosis, had also decreased in some areas. No appreciable
change was evident in edema as a result of AEOL 10150
combined treatment, although areas of basement membrane
compromise were not evident, compared with rats only exposed
to CEES. The overall severity of injury was improved with
AEOL 10150 combined treatment, primarily because of the
appearance of decreased apical membrane disruption and de-
Impact of Combined AEOL 10150 Treatment on OE Cell
Death, Neuronal Loss, and Nitrotyrosine
Hematoxylin-and-eosin–stained sections were obtained after
treatment with CEES alone (Figure 3A), after treatment with
CEES combined with PBS (Figure 3B), or after treatment
with CEES combined with AEOL 10150 (Figure 3C).
TUNEL-positive cells had increased 18 hours after exposure
to CEES alone (Figure 3D) and after exposure to CEES
combined with PBS, 1 and 9 hours after exposure (Figure 3E),
and appeared to decrease with combined AEOL 10150
treatment (Figure 3F). The quantitation of DNA nick-end
labeling shows that AEOL 10150 combined treatment, ad-
ministered 1 and 9 hours after the inhalation of CEES,
resulted in decreased TUNEL-positive staining, compared
with CEES alone or combined treatment with CEES and
PBS (Figure 3G).
A similar concentration of DAB-stained, CC3-positive cells
was evident 18 hours after the inhalation of CEES with either
CEES alone (Figure 3H) or CEES with PBS, 1 and 9 hours after
exposure (Figure 3I). CEES with combined AEOL 10150
treatment, 1 and 9 hours after exposure, appeared to result in
decreased CC3 staining (Figure 3J). The quantitation of CC3-
positive cells showed a significant decrease with AEOL 10150
combined treatment administered 1 and 9 hours after the
inhalation of CEES, compared with CEES alone or combined
treatment with CEES and PBS (Figure 3K).
A decrease in OMP-positive staining cells was evident 18
hours after the inhalation of CEES with either CEES alone
(Figure 3L) or CEES combined with PBS, 1 and 9 hours after
exposure (Figure 3M). CEES with combined AEOL 10150
treatment (Figure 3N) preserved olfactory neurons, as evi-
denced by OMP staining in the OE.
3-NT is a marker of reactive nitric oxide metabolites,
including peroxynitrite. CEES alone or CEES combined with
PBS (Figures 3O and 3P, respectively) produced similar levels
of 3-NT staining, whereas CEES with AEOL 10150 combined
treatment produced decreased 3-NT staining in the OE
Treatment with the Selective Inducible Nitric Oxide Synthase
Inhibitor 1400W Improved OE Damage 18 Hours after the
Inhalation of CEES
N-(3-(aminomethyl) benzyl) acetamidine (1400W) is a novel
selective inhibitor of inducible nitric oxide synthase (iNOS)
(20). Figure 4 demonstrates the impact of iNOS inhibition with
1400W treatment 18 hours after the inhalation of CEES,
compared with rats receiving PBS after the inhalation of CEES.
When rats inhaled CEES and received PBS (1 ml/kg) immedi-
ately and then every 4 hours after exposure to CEES, the loss of
OE structural integrity was apparent (Figure 4A). In contrast,
when rats received 1400W (5 mg/kg) immediately after expo-
sure to CEES and then every 4 hours, the structural integrity of
the OE was maintained (Figure 4B).
Cell death was measured by both TUNEL staining and the
more apoptosis-specific CC3 immunohistochemistry staining.
The inhalation of CEES with subsequent PBS treatment
resulted in the presence of TUNEL-positive cells 18 hours after
the inhalation of CEES (Figure 4C). TUNEL positivity was
reduced when exposure to CEES was followed by 1400W
treatment (Figure 4D). The quantitation of DNA nick-end
labeling shows that treatment with 1400W resulted in decreased
TUNEL-positive staining, compared with CEES combined with
PBS (Figure 4E).
CC3 positive cells were evident after the inhalation of CEES
followed by treatment with PBS, as detected 18 hours after
exposure (Figure 4F). CC3-positive cells were evident with
1400W treatment, but were less prominent in the region near
the basement membrane (Figure 4G). As shown in Figure 4H,
treatment with 1400W after the inhalation of CEES resulted in
a significant decrease in CC3-positive cells, compared with
CEES combined with PBS.
Representative OMP staining after treatment with either
PBS or 1400W is also shown. The darker Vector Red–stained
regions of OMP staining were evident at the center of the OE
when rats inhaled EtOH (Figure 4I, arrow). When rats
inhaled CEES and then received PBS at 1 hour and then
every 4 hours, areas of decreased olfactory neurons, as
indicated by reduced OMP staining, were evident (Figure
4I). In contrast, when rats received 1400W in the same dosing
protocol, the olfactory neuronal layers were visible and were
more organized 18 hours after the inhalation of CEES
Representative 3-NT staining after the inhalation of CEES
with either PBS or 1400W is also shown. When rats inhaled
CEES and then received PBS at 1 hour after inhalation injury
326AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 452011
and then every 4 hours, diffuse areas of 3-NT staining were
evident in the epithelium and lamina propria (Figure 4K).
When rats received 1400W in the same dosing protocol as the
vehicle, areas of 3-NT staining were less evident in the OE 18
hours after the inhalation of CEES (Figure 4L).
Plasma Concentrations of Nitrate and Nitrate after Inhalation
of CEES Alone or in Conjunction with AEOL 10150
or 1400W Treatment
As illustrated in Figure E4, plasma concentrations of nitrate and
nitrite after the inhalation of CEES were significantly increased
over the concentrations in control rats exposed to EtOH.
Treatment with AEOL 10150, both intranasally and subcuta-
neously, did not significantly alter CEES-induced concentra-
tions of nitrate and nitrite. Treatment with 1400W significantly
decreased plasma concentrations of nitrate and nitrite, com-
pared with animals that only inhaled CEES.
Previous studies showed that the accumulation of SM is greatest
in the nasal region after exposure to inhalation (1).35S-labeled
SM, delivered percutaneously or intravenously, also resulted in
high levels of deposition in the nasal region (3). Despite this
knowledge, specific injury to the nasal region has never been
characterized, to the best of our knowledge. The present study
indicates that inhalation of the SM analogue CEES resulted in
marked damage to the OE, which may explain the SM-induced
Figure 3. Treatment with AEOL 10150 decreased CEES-induced injury to the OE. Rats receiving CEES alone (A) or CEES combined with PBS (both
subcutaneously and intranasally) (B) caused extensive epithelial apical disruption. (C) AEOL 10150 combined treatment (both subcutaneously and
intranasally) resulted in decreased apical disruption. (D and E) TUNEL staining was evident in both CEES and CEES combined with PBS. (F) A
reduction in TUNEL-positive cells was evident with AEOL 10150 combined treatment. (G) This decrease was significant (n 5 3, mean 6 SEM). (H and
I) CC3-positive cells were similarly evident after both CEES alone and CEES combined with PBS. (J) CC3-positive cells were evident after AEOL
10150 combined treatment. (K) CC3-positive cells were significantly decreased by AEOL 10150 versus CEES alone (n 5 3, mean 6 SEM). (L and M)
OMP staining was punctate and disorganized with either CEES alone or CEES combined with PBS. (N) OMP staining, indicating olfactory neurons,
was better preserved when treatment with AEOL 10150 followed the inhalation of CEES. 3-NT staining was also evident after CEES alone (O) and
CEES combined with PBS (P). (Q) 3-NT staining was reduced in the epithelium with AEOL 10150 combined treatment, although 3-NT staining
remained evident in the lamina propria. Bars 5 50 mm.
O’Neill, Orlicky, Hendry-Hofer, et al.: ROS and RNS in CEES-Induced Olfactory Epithelial Injury 327
anosmia after exposure. Treatment with AEOL 10150, de-
livered as a combination of intranasal and subcutaneous dosing,
improved nasal injury indices. The inhibition of iNOS, using the
selective inhibitor 1400W, also reduced cell damage in the OE,
indicating that peroxynitrite and/or other toxic metabolites of
?NO may play a role in nasal injury. Taken together, these data
confirm that oxidative stress plays a role in CEES-induced nasal
In recent years, investigations of the histopathology of nasal
injury have increased, with reports of both inhaled and system-
ically delivered compounds resulting in nasal injury. For exam-
ple, systemically delivered cancer chemotherapeutics such as
Figure 4. The selective inducible nitric oxide synthase (iNOS) inhibitor 1400W decreased CEES-induced injury to the OE. (A) After the inhalation of
CEES, apical epithelial disorganization was evident in the OE of rats receiving PBS. (B) In contrast, rats inhaling CEES and receiving treatment with
1400W maintained their OE structural organization. (C) TUNEL-positive cells were present after CEES inhalation with PBS treatment. TUNEL-positive
cells were also present when the inhalation of CEES was followed by treatment with 1400W (D), and TUNEL-positive were decreased with 1400W
treatment compared with PBS treatment (E; n 5 3, mean 6 SEM). (F) CC3-positive cells were evident throughout the OE with PBS treatment. CC3-
positive cells were still apparent when the inhalation of CEES was followed by treatment with 1400W (G), but CC3-positive cells were decreased with
1400W versus PBS treatment (H; n 5 3, mean 6 SEM). (I) OMP-stained OE was disorganized by the inhalation of CEES followed by treatment with
PBS. (J) OMP staining indicated that olfactory neurons were preserved by treatment with 1400W. 3-NT staining was evident with the inhalation of
CEES and treatment with PBS (K ), whereas the inhalation of CEES and treatment with 1400W resulted in reduced, but not absent, 3-NT staining in
OE (L). Bars 5 50 mm.
328 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 45 2011
vincristine sulfate were shown to induce the selective apoptosis
of olfactory neurons, with subsequent atrophy of the OE (21).
Systemic methimazole, an antithyroid drug, also results in
extensivedamagetothe OEsustentacular cellsandtoBowman’s
glands in the lamina propria, because of the high concentrations
of cytochrome P450 in these cell types and the P450-mediated
metabolism of methimazole (22). The inhalation of a mixture of
exposure ultimately results in the denudation of OE 24 hours
after exposure (23). The intranasal instillation of a single low
dose of the tricothecene mycotoxin satratoxin G (derived from
the black mold Stachybotrus chartarum) resulted in apoptosis of
the olfactory neurons (24). Non–cell type–specific damage to the
OE was also evident after the inhalation of chlorine or sulfur
of cell death to various cell types in the OE.
Because olfactory sensory neurons (OSNs) are in direct
contact with the environment, inhaled toxins such as CEES can
directly induce injury or cell death (27). Marked changes were
evident in the OE after the inhalation of CEES. Although
changes by 4 hours after inhalation were minimal according to
histology, by 18 hours after inhalation, marked changes in OE
structural integrity were noted. Cell death in the OE occurred
afterthe inhalation of CEES. This result was demonstrated using
both the histochemical assessment of the cleavage of caspase 3,
In the present study, fragmented DNA, as measured by TUNEL
staining, was significantly increased at 4 and 18 hours after the
inhalation of CEES, compared with the inhalation of EtOH. In
contrast, staining for CC3, an early-phase apoptosis marker, had
significantly increased only 18 hours after inhalation. Cytosolic
after the inhalation of CEES, indicating damage to the OSNs.
Cell death and the specific loss of OSNs after the inhalation of
CEES may provide an explanation for how anosmia occurs after
the inhalation of SM.
Reactive oxygen species (ROS), derived from high concen-
trations of inflammatory cells such as PMNs, are often contrib-
uting factors in oxidative stress. However, an investigation of
the nasal region at multiple time points indicated that few
PMNs were present in the injured area, and are thus not
primarily responsible for oxidative stress in nasal injury. Given
the extensive cell death in the OE, and the possibility that not
all cell death is attributable to controlled apoptosis, mitochon-
drial dysfunction attributable to dying cells may constitute
a major contributor to OE oxidative stress. To investigate
further the role of oxidative stress in CEES-induced damage
to the OE, studies with the catalytic antioxidant manganopor-
phyrin AEOL 10150 were undertaken.
AEOL 10150 is an antioxidant effective in scavenging
superoxide, hydrogen peroxide, peroxynitrite, and lipid perox-
ides (28–31). The formation of 3-NT after 18 hours was
decreased by AEOL 10150, suggesting that the scavenging of
superoxide and/or peroxynitrite may have played an important
role in the improved outcomes. Another possible mechanism by
which manganoporphyrins confer protection may involve the
inhibition of NO production (32). AEOL 10150 was shown to
redox-cycle with flavin-dependent oxidoreductases, including
nitric oxide synthases (33). Although redox cycling is generally
considered deleterious, AEOL 10150 and similar compounds
can scavenge superoxide produced at the site and thus may
reduce injury. In addition, AEOL 10150 may mitigate leak of
superoxide attributable to mitochondrial dysfunction. Previous
in vitro studies indicated that exposure to CEES results in
decreased mitochondrial potential (7). Insofar as measureable
cell death occurs in the OE, mitochondrial dysfunction and the
resultant production of ROS may also have occurred in vivo in
the present model. iNOS may constitute another source of
ROS, because that enzyme is known to produce superoxide
under basal conditions (34).
Treatment with AEOL 10150, when administered only sub-
cutaneously or intranasally, was not protective against CEES-
induced nasal injury. When subcutaneous treatment was bolstered
with intranasal treatment, a relatively low (50 mM) concentra-
tion (AEOL 10150 combination treatment) was effective in
reducing nasal injury. As the concentration of AEOL 10150
administered via the intranasal route increased, no visible
protection was afforded. This finding is consistent with those
in previous studies reporting a hormetic, bell-shaped curve, with
beneficial tissue-protective effects increasing and then waning
as exogenously administered concentrations of SOD increased
(35). Although we did not demonstrate that the effect of higher
intranasal concentrations of AEOL 10150 was toxic above and
beyond CEES-induced injury, we did see a clear lack of efficacy.
The 50-mM concentration was also most effective at preventing
CEES-induced epithelial cell injury in a previous in vitro model
(36). OE histologic markers were significantly decreased with
AEOL 10150 combination treatment, compared with either
CEES alone or CEES combined with PBS. In addition, AEOL
10150 combination treatment decreased cell death in the OE.
Olfactory neurons appeared to be protected 18 hours after the
inhalation of CEES when combined AEOL 10150 treatment
was administered, and 3-NT staining was reduced. These data
indicate that damage to the OE was decreased through the
combination of subcutaneous and intranasal treatments with
The generation of even a moderate level of ONOO2over
extended periods of time may result in cellular dysfunction, the
disruption of cell signaling pathways, and the induction of cell
death through both apoptosis and necrosis (37). Previous studies
demonstrated an increase in iNOS activity after the administra-
tion of SM in keratinocytes in a time-dependent and dose-
dependent manner (38). In an in vitro model of exposure to
SM in primary chick neurons, the nonspecific NOS inhibitor
L-NAME conferred significant protection, implying a role for
?NO (39). In the present study, the formation of 3-NT increased
in the OE after the inhalation of CEES, indicative of increased
production of RNS. Therefore, we investigated whether the
N-(3-(aminomethyl)benzyl) acetamidine (1400W) is a tightly
binding, extremely slowly reversible inhibitor of iNOS. 1400W
has a 1,000-fold greater selectivity for iNOS than for eNOS (20).
In contrast to bisthioureas (shown to be biologically inactive)
and arginine analogues (which are nonspecific for the various
NOS isoforms), 1400W is both biologically active in vivo and
selective for iNOS (20). 1400W significantly decreased TUNEL-
positive and CC3-positive cells. Olfactory neurons, as indicated
by OMP staining, were substantially better preserved by
treatment with 1400W compared with PBS after the inhalation
of CEES, suggesting that a decrease in iNOS activity is
beneficial to the survival of olfactory neurons (40). Although
3-NT staining was still evident after treatment with 1400W, it
appeared to have decreased in the epithelium. Overall, the
inhibition of iNOS by treatment with 1400W decreased appar-
ent nasal injury, implying a significant role for RNS in CEES-
Previous studiesindicated thatametalloporphyrincompound
similar to AEOL 10150 was effective at reducing the activity of
NOS (32). The present study indicated, at least systemically, that
treatment with AEOL 10150 was not effective at decreasing
CEES-induced concentrations of nitrate and nitrite. This finding
indicates that mechanistically, any improvement in nasal injury
O’Neill, Orlicky, Hendry-Hofer, et al.: ROS and RNS in CEES-Induced Olfactory Epithelial Injury329
derived from treatment with AEOL 10150 was likely related to
either superoxide or peroxynitrite scavenging. In contrast,
1400W did decrease systemic CEES-induced nitrate and nitrite
concentrations, indicating that a reduction of iNOS activity was
effective and caused beneficial effects in protecting nasal epithe-
lium. A schematic diagram of the potential modes of action of
AEOL 10150 and 1400W in the reduction of CEES-induced
nasal injury is presented in Figure 5.
The interpretation of these data in the context of human
inhalation must be performed with caution. In contrast to
humans, who have a poorly developed sense of smell (micro-
smatic), rodents have a highly developed sense of smell (macro-
smatic) (27). These differences are especially obvious when
considering the percentage of total nasal epithelium committed
to olfaction in humans (3%), compared with that in rats
(z 50%) (27, 41). Although differences exist between species,
the loss of sense of smell after the inhalation of SM in humans is
likely attributable to a loss of olfactory neurons. Our data
provide strong support for the concept that RNS and/or ROS
are critical in the pathogenesis of injury to the OE after
inhalation of the SM analogue CEES.
Author Disclosure: B.J.D. served as a consultant for and owns stock in Aeolus
Pharmaceuticals, has received reimbursement for lectures from Kyowa Hakko,
and has a patent with National Jewish Health for treatments that rescue injury
from alkylating species. None of the other authors have a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
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