Toxicology 303 (2013) 3442

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Toxicology
journal homepage: www.elsevier.com/locate/toxicol

Inhalation of chlorine causes long-standing lung inammation and airway

hyperresponsiveness in a murine model of chemical-induced lung injury
Soa Jonasson a, , Bo Koch a , Anders Bucht a,b
a
Swedish Defence Research Agency, Division of CBRN Defence and Security, Ume, Sweden
b
Department of Public Health and Clinical Medicine, Unit of Respiratory Medicine, Ume University, Ume, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: Chlorine is highly irritating when inhaled, and is a common toxic industrial gas causing tissue damage
Received 3 October 2012 in the airways followed by an acute inammatory response. In this study, we investigated mechanisms
Received in revised form 23 October 2012 by which chlorine exposure may cause reactive airways dysfunction syndrome (RADS) and we examined
Accepted 25 October 2012
the dose-dependency of the development of symptoms. Mice were exposed to 50 or 200 ppm Cl2 during
Available online 9 November 2012
a single 15 min exposure in a nose-only container. The experiment terminated 2, 6, 12, 24, 48, 72 h and
7, 14, 28 and 90 days post exposure. Inammatory cell counts in bronchoalveolar lavage (BAL), secretion
Keywords:
of inammatory mediators in BAL, occurrence of lung edema and histopathological changes in lung
Chlorine
Chemical-induced lung injury
tissue was analyzed at each time-point. Airway hyperresponsiveness (AHR) was studied after 24 and
Airway hyperresponsiveness 48 h and 7, 14, 28 and 90 days. The results showed a marked acute response at 6 h (50 ppm) and 12 h
Respiratory mechanics (200 ppm) post exposure as indicated by induced lung edema, increased airway reactivity in both central
Mouse model and peripheral airways, and an airway inammation dominated by macrophages and neutrophils. The
inammatory response declined rapidly in airways, being normalized after 48 h, but inammatory cells
were sustained in lung tissue for at least seven days. In addition, a sustained AHR was observed for at least
28 days. In summary, this mouse model of chlorine exposure shows delayed symptoms of hyperreactive
airways similar to human RADS. We conclude that the model can be used for studies aimed at improved
understanding of adverse long-term responses following inhalation of chlorine.
2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction described in the clinical investigation after the train derailment of

Chlorine is one of the most commonly produced industrial
chemicals and is used for various purposes; for example in the
purication of drinking water and in the production of bleached
paper, plastics, solvents, pharmaceuticals and various other chem-
icals. Chlorine accidental release from industrial plants and during
transportation may cause a large number of casualties due to the
high toxicity (Ball and Dworak, 2005; Evans, 2005; MMWR, 2005).
Furthermore, chlorine has historically been used as a chemical
weapon and is still considered a terrorist threat (Szinicz, 2005). In
contact with water, chlorine produces hydrochloric and hypochlor-
ous acids, of which inhaled chlorine exposure can lead to a wide
variety of respiratory injuries both in upper and lower airways
(Evans, 2005; White and Martin, 2010; Williams, 1997). There
are both acute and chronic respiratory consequences of chlorine
inhalation of which the acute effects were recently extensively
Corresponding author at: Division of CBRN Defence and Security, Swedish
Defence Research Agency, SE-901 82 Ume, Sweden. Tel.: +46 90 10 67 45;
fax: +46 90 10 68 00.
E-mail address: soa.jonasson@foi.se (S. Jonasson).
a chlorine tanker in Graniteville in 2005 (Ball and Dworak, 2005;
Duncan et al., 2011; Williams, 1997). The onset of acute symptoms
ranges from minutes to hours and due to the high reactivity of chlo-
rine the occurrence of effects is generally limited to the exposed
tissues (Evans, 2005; Kales and Christiani, 2004). In the respiratory
tract, the acute symptoms include pulmonary edema, tracheobron-
chitis, temporary airow dysfunction and adult respiratory distress
syndrome while the long-term consequences are described as reac-
tive airways dysfunction syndrome (RADS), bronchiectasis, decline
in lung volumes and increased airway resistance (Demnati et al.,
1998; Koohsari et al., 2007; Martin et al., 2003; Morris et al.,
2005; Tuck et al., 2008; Williams, 1997). RADS is a result from
a non-immunologic prolonged hyperresponsiveness and airow
obstruction caused by a single inhalation exposure of a highly
irritating substance; for example chlorine, ammonia and sulfuric
acid. The RADS is characterized clinically by asthma-like symptoms
including cough, wheezing, chest tightness, and breathlessness. The
onset of RADS symptoms occurs within 24 h after exposure and per-
sists for at least three months (Brooks and Bernstein, 2011; Evans,
2005).
Previous murine studies are mostly based on the acute effects
on the airways caused by chlorine exposure (Chang et al., 2012;

0300-483X/$ see front matter 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tox.2012.10.022
 S. Jonasson et al. / Toxicology 303 (2013) 3442
Demnati et al., 1998; Koohsari et al., 2007; Martin et al., 2003;
Morris et al., 2005; Tuck et al., 2008; Williams, 1997). From these
studies, marked damage to the airways, epithelial sloughing, and
increased protein content in bronchoalveolar lavage (BAL) uid, an
inammatory response with neutrophil and macrophage recruit-
ment into the airways, and decreased respiratory function has been
reported. In the present study, we have focused on the poten-
tial for chlorine exposure to promote chronic lung injury in both
central and peripheral airways, in particular to investigate the
mechanisms by which chlorine exposure may cause delayed symp-
toms such as RADS. In addition to our primary aim, we wanted to
examine whether the development of RADS is dependent on the
chlorine concentration by comparing exposure to a low and a high
concentration. The high concentration of chlorine was considered
to be relevant for a scenario of a chemical disaster at an indus-
trial plant or during transportation, resulting in life-threatening
lung injuries. However, such scenario would also include humans
exposed to lower concentrations displaying no observable acute
symptoms during the rst 24 h although there is a potential risk to
develop delayed effects. To address these aims we used a murine
nose-only exposure system together with a small animal ventilator
(exiVentTM , SCIREQ ) in order to evaluate respiratory mechanics
and airway reactivity in response to a low and a high concentration
of chlorine up to 90 days after exposure. In addition to the mea-
surement of airway physiology, we performed extensive evaluation
of short-term and long-term inammatory responses in airways,
histopathological changes in lung tissue, formation of lung edema
and occurrence of lung brosis.
2. Methods
2.1. Animals
Female BALB/c mice (910 weeks old, Harlan Laboratories, Netherlands) were
used in this study. Animals were housed in plastic cages with absorbent bedding
material and were maintained on a 12 h daylight cycle, 22 C, with a 30% relative
humidity. Food (LAB FOR, R36, Lantmnnen, Sweden) and water were provided ad
libitum. All mice were weighed before subjected to chlorine and following exposure,
their health condition was monitored. Their care and the experimental protocols
were approved by the regional ethics committee on animal experiments in Ume,
Sweden
2.2. Chlorine exposure protocol
Animals were placed in individual nose-only containers (EMMS, UK) and cou-
pled to an exposure battelle tower providing equal and simultaneous exposure to Cl2
(AIR LIQUIDE Germany; compressed gas in gas cylinders: 1 mol% chlorine, 99 mol%
nitrogen). The compressed gas mixture was diluted with air to the nal concen-
tration of 50 and 200 ppm respectively. Mice were subjected to a single exposure
of Cl2 air mixture (50 (12.5 ppm h) or 200 ppm (50 ppm h)) during 15 min. Control
animals were breathing room air for 15 min.
2.3. Study design
The experiment terminated 2, 6, 12, 24, 48, 72 h and 7, 14, 28 and 90 days post
exposure (n = 68 mice per group). To limit the number of control animals, mice
were allocated into three age-matched groups: control 1 (C1: 248 h), control 2 (C2:
7, 14, 28 days) and control 3 (C3: 90 days) groups.
2.4. Respiratory mechanics
On the day of respiratory mechanics assessment, the animals were weighed and
anesthetized with pentobarbital sodium (90 mg/kg, intraperitoneal (i.p.) injection).
Mice were tracheostomized with an 18-gauge cannula and mechanically ventilated
in a quasi-sinusoidal fashion with a small animal ventilator (exiVentTM , SCIREQ )
at a frequency of 3 Hz and a tidal volume (VT ) of 12 ml/kg body weight. A positive
end-expiratory pressure of 3 cmH2 O was applied. A warming pad prevented cooling
of the animal. Mice were paralyzed with pancuronium (0.1 mg/kg, i.p.) before four
sigh maneuvers at three times VT were performed at the beginning of the exper-
iment to establish stable baseline respiratory mechanics and to ensure a similar
volume history before the experiments. The mice were then allowed a 2-min res-
ting period before the experiment began. To measure airway hyperresponsiveness
(AHR), incremental doses of inhaled methacholine (MCh, acetyl--methylcholine
chloride, SigmaAldrich) were given at 5-min intervals. The MCh, diluted in saline
35
in a volume of 20 l, was given during 10 s as an aerosol (AeronebTM , SCIREQ ).
Each dose was aerosolized without any interference with the ventilation pattern.
Between each dose of saline and MCh, respiratory resistance was allowed to return
to baseline level before the next MCh dose was administered. All respiratory param-
eters were measured continuously using a standardized script and the maximum
response to a given concentration of MCh is reported. The parameters obtained are
respiratory resistance (RRS ), respiratory elastance (ERS ) and respiratory compliance
(CRS ), tissue resistance (G) and tissue elastance (H). The more detailed description of
the method can be found in a previously published paper of Wigenstam et al. (2012).
2.5. Differential cell count in BAL uid
The lungs were lavaged four times via a tracheal tube with a total volume of
1 ml + 3 1 ml Ca2+ /Mg2+ free Hanks balanced salt solution (HBSS, SigmaAldrich,
Steinheim, Germany). The BAL uid was centrifuged (10 min, 4 C, 1500 rpm). After
removing the supernatant, the cell pellet was resuspended and then diluted in 1 ml
PBS. The rst 1 ml of supernatant was saved for further analysis. Leukocytes were
counted manually in a hemocytometer and 30,000 cells were loaded onto slides
using a Cytospin centrifuge (Shandon cytospin 3 cyto-centrifuge, cell preparation
system). Cytocentrifuged preparations were stained with MayGrnwaldGiemsa
reagents and differential cell counts of pulmonary inammatory cells (macrophages,
neutrophils, lymphocytes, and eosinophils) were made in duplicates using standard
morphological criteria and counting 300 cells per cytospin preparation. BAL sam-
ples were also used for ELISA and Bio-PlexTM (Pro Mouse Cytokine 23-plex panel)
analyses.
2.6. Inammatory mediators in BAL uid and serum
2.6.1. ELISA analyses
Inammatory mediators in BAL and serum were analyzed for the presence of
interleukin (IL)1B, IL6 and KC. All cytokine analyses were performed individually
with a specic assay kit (R&D Systems, Inc.) according to the manufacturers instruc-
tions and analyzed using an ELISA reader (Thermo Scientic Mutilskan FC, Thermo
Fischer Scientic Oy, Vantaa, Finland). ELISA data was analyzed using the software
program for the ELISA reader (SkanIt for Multiskan FC 3.1. Ink), referring to the
standards added to each plate.
2.6.2. Bio-PlexTM analyses
Inammatory mediators in BAL were analyzed for the presence of IL1A, IL1B,
IL2, IL3, IL4, IL5, IL6, IL9, IL10, IL-12A, IL12B, IL13, IL17, CSF3, CSF2, IFNG, CCL11,
CXCL1, CCL2, CCL3, CCL4, CCL5, and TNFA. All cytokine analyses were performed
simultaneously with a multiplex kit (Bio-PlexTM Pro Mouse Cytokine 23-plex panel)
according to the manufacturers instructions (Bio-Rad) and analyzed on a Bio-PlexTM
system (Luminex Bio-PlexTM 200 System, Bio-Rad, Hercules, CA).
2.7. Analysis of lung edema formation
Mice were sacriced by cervical dislocation 12 or 24 h after exposure. The whole
lung package was dissected and washed in HBSS following removal of heart and tra-
chea. Excessive uid was carefully removed by drying the lung with a piece of tissue
paper. The lung was placed on a piece of aluminum folio and weighed immediately
(wet weight). The lungs were dried overnight in an oven at 50 C and weighed again
(dry weight). The ratio between wet and dry weight is a marker for edema formation.
2.8. Analysis of collagen deposition in lung tissue
Lungs were removed, rinsed in PBS, and frozen in liquid nitrogen until extraction.
They were thawed on ice, cut into small pieces, and incubated in 10 ml of pepsin
dissolved in 0.5 M acetic acid (1:10 ratio of pepsin/tissue wet weight) for 24 h at 4 C
with vigorous stirring. The solution was then centrifuged at 2000 rpm for 15 min
and the clear solution was used for collagen measurement. Collagen content was
measured by a spectrophotometric method, SircolTM Collagen Assay kit, according to
the manufacturers description (Biocolor Ltd., Belfast, UK). Plates were read using an
ELISA reader (Thermo Scientic Multiskan FC, Thermo Fischer Scientic Oy, Vantaa,
Finland) at 550 nm. Analysis of data was performed with the software program for
the ELISA reader (SkanIt for Multiskan FC 3.1. Inc.). The calibration curve was set
up on the basis of the collagen standard provided by the manufacturer. The assay
was performed in duplicate and the mean of two data was determined for each
individual sample.
2.9. Histopathology
Following BAL, the right lung lobe was removed and xed in 4% paraformalde-
hyde until parafn embedding. After embedding in parafn, the tissue was cut into
3 m thick sections and mounted on positively charged slides. To assess inam-
matory cell inltration, the sections were deparafnized, dehydrated, and stained
with hematoxylin and eosin. Histopathological evaluations of stained sections were
performed in a blinded manner using light microscopy.
36 S. Jonasson et al. / Toxicology 303 (2013) 3442
Fig. 1. (A) The acute effects of chlorine (Cl2 ) on weight loss. (B) Mean weight in
mice subjected to 200 ppm Cl2 . C1C3 are age-matched controls exposed to room
air (control 1 (C1: 248 h) control 2 (C2: 7, 14 and 28 days) and control 3 (C3: 90
days) groups). Values indicate means SEM, *p < 0.05, ***p < 0.001. *Compared to
control group.
2.10. Statistical analysis
Results are presented as the mean standard error of mean (SEM). Statistical
signicance was assessed by parametric methods using a two-way analysis of vari-
ance (ANOVA) to determine differences between groups, followed by Bonferroni or
Dunnetts post hoc test. When appropriate, a one-way ANOVA or Students unpaired
t-test was used. A statistical result of p < 0.05 was considered signicant. The sta-
tistical analyses were carried out and graphs were prepared with GraphPad Prism
program (version 5.0 GraphPad Software Inc., San Diego, CA, USA).
3. Results
3.1. Animals
A 15-min exposure to Cl2 was non-lethal but all mice dis-
played signs of anorexia, lethargy and hypothermia up to 24 h post
exposure. When compared to the initial start weight, all chlorine
exposed mice showed signicant and dose-dependent weight-loss
12 h post exposure (Fig. 1A). Mice exposed to the high concentration
(200 ppm) were not completely recovered 28 days after exposure
while mice subjected to 50 ppm Cl2 , regained start weight after 48 h
(Fig. 1B).
3.2. Inammatory cells in BAL uid
The inltration of inammatory cells into the airways follow-
ing Cl2 exposure was dose-dependent. The higher concentration of
200 ppm resulted in a decline in total BAL leukocyte numbers at the
early time-points 2 and 6 h post exposure while animals exposed
to 50 ppm did not show any reduction in BAL cells between 2 and
6 h (Fig. 2A). At 12 h, there was a signicant increase of leukocytes,
predominantly macrophages and neutrophils in the airways when
compared to unexposed control animals (Fig. 2B). At 24 h, there was
also a small but signicant increase of eosinophils (Fig. 2C). The
time-course of the inux of lymphocytes into the airways differed
between low and high dose Cl2 . In animals subjected to 50 ppm,
the number of lymphocytes was signicantly increased at 6 h post
exposure whereas exposure to 200 ppm resulted in a delayed lym-
phocyte response reaching the highest number of cells 12 h after
exposure (Fig. 2D). A similar dose-dependent time-course of cell
responses was observed for neutrophils and eosinophils. Neither of
the two Cl2 concentrations caused any detectable long-term effects
on the inammatory response in airways as indicated by similar
leukocyte counts as in unexposed control mice after 48 h up to 90
days after exposure (data not shown).
3.3. Inammatory mediators
3.3.1. ELISA analysis of cytokines in BAL uid and serum
The time-course of the early inammatory cytokines IL6, CXCL1
and IL1B was analyzed in BAL uid from animals exposed to
50 ppm (Fig. 3A) and 200 ppm Cl2 (Fig. 3B). In the 50 ppm group,
both IL6 and CXCL1 were signicantly increased 2 h after expo-
sure compared to control group while exposure to 200 ppm caused
a delayed induction of IL6 and CXCL1, reaching maximum levels
6 h after exposure. IL1B was expressed in relatively low levels in
both groups, reaching maximum levels after 2 h in mice exposed to
200 ppm while being undetectable in BAL uid from mice exposed
to 50 ppm.
The time-course of IL6, CXCL1 and IL1B was also analyzed
in serum from animals exposed to 50 (Fig. 3C) and 200 ppm
Cl2 (Fig. 3D). Maximum levels of IL6 and CXCL1 were signi-
cantly reached at time-point 6 h both after 50 and 200 ppm Cl2 .
Increased expression of IL1B could not be detected in serum after
Cl2 exposure (data not shown). The maximum response of CXCL1
was not signicantly different between the two dose groups. The
release of early cytokines, in both BAL uid and serum, corre-
lated with the inltration of inammatory cells analyzed in BAL
uid.
3.3.2. Bio-plexTM analysis of inammatory mediators in BAL uid
Twenty-three cytokines were analyzed in BAL uid at differ-
ent time-points following 200 ppm Cl2 (Fig. 4AD). The maximum
levels of IL1B, IL6, IL12A, IL13, CSF2, CSF3, IFNG, CCL11, CXCL1
and TNFA were reached 6 h post exposure and the levels of all
cytokines declined to baseline levels at 24 h after exposure except
for CSF3, TNFA and CCL4 which were sustained 24 h post exposure
and thereafter declined. The levels of CCL5, IL12B, CCL2 and CCL3
were signicantly increased 24 h post exposure. Other cytokines
such as IL1A, IL2, IL3, IL4, IL5, IL9, IL10 and IL17 were not signi-
cantly expressed in BAL uid (data not shown).
3.4. Respiratory mechanics
Exposure to Cl2 induced a signicant AHR following MCh chal-
lenge in both dose groups 24 h post exposure (Figs. 5 and 6).
All respiratory parameters measured were signicantly altered,
including respiratory resistance and elastance, as well as periph-
eral tissue resistance and tissue elastance. The MCh-induced AHR
of the central airways in the 200 ppm group was increased more
than two-fold compared to the 50 ppm group at 24 h post expo-
sure (RRS 200 ppm: 24.1 4.2 cmH2 Os/ml compared to RRS 50 ppm:
10.2 2.0 cmH2 Os/ml, p = 0.01).
Exposure to 50 ppm Cl2 caused reversible effects on peripheral
airways which were restored to normal responses within 48 h but
the AHR of the central airways were still present at 48 h (Fig. 5). The
high concentration of Cl2 caused adverse effects on both central and
peripheral airways up to 14 days post exposure and signs of per-
sistent airway hyperreactivity was observed up to day 28 (Fig. 6).
 S. Jonasson et al. / Toxicology 303 (2013) 3442 37

Fig. 2. Total and differential cell counts in bronchoalveolar lavage (BAL) uid from chlorine exposed animals. (A) Total leukocytes, (B) neutrophils, (C) lymphocytes and (D)
eosinophils. Values indicate means SEM, *p < 0.05, ***p < 0.001 compared to control (C1, 248 h) group.

During the three month study that was conducted, baseline RRS in
mice was unaffected by inhalation of Cl2 but the baseline respira-
tory compliance CRS was signicantly decreased after exposure to
200 ppm Cl2 , a reduction that persisted up to four weeks (Fig. 7). At
day 90, the baseline CRS was normalized.
3.5. Histopathological evaluation of lung tissue
Histopathological evaluation of hematoxylin and eosin stained
lung tissue sections revealed that injuries following exposure to
the 200 ppm concentration of Cl2 were restricted to the larger air-
ways while peripheral parts of the lungs appeared to be unaffected
regarding tissue changes and inammatory inltrates. Two to six
hours after the chlorine exposure, large bronchi and bronchioles
exhibited extensive loss of epithelium affecting both ciliated, secre-
tory and Clara cells. After 12 h, some areas in the bronchiole and
adjacent alveoli exhibited degeneration, necrosis and exfoliation
of epithelium. The alveoli contained low numbers of neutrophils
and macrophages, cell debris and amorphous granular and bril-
lar materials (possibly brin). Occasional bronchioles displayed
degeneration and necrosis of the epithelium leaving denuded,
eroded mucosal areas 12 h post exposure. The peribronchial tis-
sues exhibited edema with inltrated leukocytes, predominantly
neutrophils and occasional monocytes and macrophages. This was
sustained at 48 h post exposure, and the mucosa of the major
bronchus displayed an area of degeneration and necrosis of the
epithelium. This area was inltrated with low numbers of neu-
trophils, which together with damaged epithelial cells sloughed

Fig. 3. Cytokine analysis in (A and B) bronchoalveolar lavage (BAL) uid and (C and D) serum from mice exposed to 50 or 200 ppm chlorine during the acute phase of the
inammation (224 h). Values indicate means SEM, *p < 0.05, **p < 0.01, ***p < 0.001 compared to control (C1, 248 h) group.
38 S. Jonasson et al. / Toxicology 303 (2013) 3442

Fig. 4. Cytokine analyses in bronchoalveolar lavage (BAL) uid from mice, 6 h up to 28 days after exposure to 200 ppm Cl2 . Cytokines are grouped after increasing concentration
in BAL uid; (A) showing cytokines up to 30 pg/ml, (B and C) up to 150 pg/ml and (D) up to 6000 pg/ml. Values indicate means SEM. Maximum levels at time-point 6 h;
IL1B, CCL4, CCL11, TNFA, IL12A, IL13, CSF2, IL6, CSF3 and CXCL1, all values p < 0.001 compared to control. Maximum levels at time-point 24 h; CCL5 (p < 0.05) and IL12B, CCL2
and CCL3 (all p < 0.001) compared to unexposed control mice, C (C1 and C2 were pooled together).

off in the bronchial lumen leaving an eroded mucosa. Adjacent
epithelial cells exhibited occasional mitoses consistent with regen-
eration. At 72 h post exposure, the bronchial mucosa was markedly
hyperplastic and the number of leukocytes was very sparse. Seven
days following exposure, mice exhibited the most severe lesions
(Fig. 8A). The large bronchi and adjacent alveoli displayed dam-
aged cells, but also hyperplastic cells repopulating the areas of
epithelial cell loss. The bronchial lamina propria and the peri-
bronchial tissues were partly disrupted by granulation tissues with
spindle cells consistent with angioblasts, broblasts, and colla-
gen bers. Tissues were also richly inltrated with leukocytes
(granulocytes, monocytes, macrophages, lymphocytes and also low
numbers of plasma cells). The airways showed hypertrophy of the
smooth muscle layer. Observed lesions were consistent with an
on-going regenerative and reparative process. The alveolar ducts
and more peripheral structures were not affected. On days 14
and 28 no signicant lesions were found and the lung tissue was
normalized.

Fig. 5. Respiratory mechanics in mice exposed to 50 ppm chlorine. Measurements of MCh-induced respiratory (A) resistance, RRS , and (B) elastance, ERS , were performed
using the single compartment model. Changes in (C) tissue resistance, G, and (D) tissue elastance, H, were measured with the forced oscillation technique (Zrs measurements,
Prime-2). The maximum response of inhaled MCh (100 mg/ml) is shown. C1-C3 are age-matched controls exposed to room air (control 1 (C1: 248 h) control 2 (C2: 7, 14 and
28 days) and control 3 (C3: 90 days) groups). Values indicate means SEM, ***p < 0.001 compared to C1.
 S. Jonasson et al. / Toxicology 303 (2013) 3442 39

Fig. 6. Respiratory mechanics in mice exposed to 200 ppm chlorine. Measurements of MCh-induced respiratory (A) resistance, RRS , and (B) elastance, ERS , were performed
using the single compartment model. Changes in tissue (C) resistance, G, and (D) elastance, H, were measured with the forced oscillation technique (Zrs measurements,
Prime-2). The maximum response of inhaled MCh (100 mg/ml) is shown. C1C3 are age-matched controls exposed to room air (control 1 (C1: 248 h) control 2 (C2: 7, 14
and 28 days) and control 3 (C3: 90 days) groups). Values indicate means SEM, *p < 0.05, **p < 0.01, ***p < 0.001.

3.6. Lung edema formation 4. Discussion

Inhalation of chlorine signicantly induced lung edema as indi-
cated by increased ratio of wet/dry lung weight both at 12 and 24 h
post exposure in animals exposed to 200 ppm Cl2 (Fig. 9). Animals
exposed to 50 ppm Cl2 showed signicant lung edema only after
24 h. There was no signicant difference in the edema formation
when the 50 ppm group was compared to the 200 ppm group.
3.7. Collagen formation
Exposure to 200 ppm Cl2 induced signs of lung brosis as indi-
cated by increased collagen deposition in lung tissue on days 14
and 28 post exposure when compared to age-matched control ani-
mals (Fig. 10). After three months (day 90) the collagen content
was normalized to levels similar to that of unexposed control mice.
There was no signicant difference in lung tissue collagen content
between days 14 and 28.
In the present study, we have investigated the adverse inam-
matory and respiratory responses following inhalation of highly
toxic chlorine. In our murine exposure model we observed both
early and late toxic effects depending on the exposure dose. In
accordance with previously published data in similar animal mod-
els, we demonstrated acute neutrophilic inammation in lung
tissue and airways, pulmonary edema, AHR and peribronchial
distribution of brotic depositions resulting from the effects of
chlorine (Gunnarsson et al., 1998; Honavar et al., 2011; Martin
et al., 2003; Menaouar et al., 1997; Tian et al., 2008). The inam-
matory response was further characterized by a detailed analysis
of cytokines mediating the inammatory response. From our data
it is evident that inhalation of chlorine can lead to a long-standing
AHR.
Exposure to Cl2 rapidly ruptured the epithelial layer affecting
adjacent cells maybe as a consequence of oxidative stress and

Fig. 7. Measurements of baseline respiratory compliance (CRS ) were performed with the single compartment model in animals exposed to 200 ppm chlorine. (A) Short-term
effects; groups are compared to C1 and (B) long-term effects; 7 days, 14 days and the 28 days groups are compared to C2 and the 90 days group are compared to C3. C1C3
are age-matched controls exposed to air (control 1 (C1: 248 h) control 2 (C2: 7, 14 and 28 days) and control 3 (C3: 90 days) groups). Values indicate means SEM, *p < 0.05,
**p < 0.01, ***p < 0.001.
40 S. Jonasson et al. / Toxicology 303 (2013) 3442
Fig. 8. Lung histological evaluation of hematoxylin and eosin stained sections by
light microscopy revealed that seven days following exposure (A), mice exhibited
the most severe lesions. The large bronchi and adjacent alveoli displayed damaged
cells, but also hyperplastic cells repopulating the areas of epithelial cell loss. Tissues
were also richly inltrated with leukocytes. (B) Age-matched control group exposed
to air (10 light micrographs).
cell toxicity (Jonasson et al., 2009; Martin et al., 2003). This is
one possible explanation for the decline in leukocytes in BAL uid
immediately after high levels of Cl2 exposure. The immediate toxic
reaction is followed by an early expression of pro-inammatory
cytokines resulting in recruitment of inammatory cells to the air-
ways, lung injury and increased AHR. The most intriguing result
of the present study is that mice still are hypersensitive to MCh
challenge up to 28 days post exposure, an observation consistent
with the reactive airways dysfunction syndrome (RADS) previ-
ously reported in humans exposed to lung irritants (Aslan et al.,
Fig. 9. Edema formation measured 12 h and 24 h after exposure to 50 or 200 ppm
chlorine. Values indicate means SEM, *p < 0.05 and **p < 0.01 compared to their
age-matched control group (C1: 248 h).
Fig. 10. Long-term collagen deposition in the lung after 200 ppm chlorine exposure.
Exposed groups were compared to age-matched control animals ((C2: 7 days, 14
days and 28 days) and (C3: 90 days)). Values indicate means SEM, **p < 0.01 and
***p < 0.001 compared to C2.
2006; Brooks and Bernstein, 2011; Brooks et al., 1985). RADS is
a closely related to asthma that is manifested within minutes or
hours after a single lung exposure to irritating or toxic chemi-
cals such as chlorine, ammonia and sulphuric acid (Brooks and
Bernstein, 2011; Brooks et al., 1985; Quirce and Barranco, 2010).
In the worst case scenario, the acute asthma-like symptoms can
develop into more severe respiratory symptoms such as pulmonary
edema and in some cases death (Brooks et al., 1985; Cullinan et al.,
1997). In humans, the acute histopathological features of RADS are
rapid denudation of the mucosa with a brinohemorrhagic exudate
in the submucosa followed by subepithelial edema and signs of
regeneration of the epithelial layer. Bronchoalveolar lavage reveals
neutrophilia in the acute stage of RADS (Lemiere et al., 1996, 1997).
The respiratory symptoms of RADS can improve over time but some
symptoms such as bronchial hyperresponsiveness may last for sev-
eral years (Currie and Ayres, 2004; Schonhofer et al., 1996).
In our mouse model, the acute inammatory response in air-
ways was clearly detectable within 12 h after exposure and was
resolved within 72 h, while inammatory inltrates remained
in lung tissue for at least seven days. Differential cell counts
and histopathological evaluation demonstrated a predominance
of neutrophils with occasional macrophages, monocytes and
eosinophils. A small number of lymphocytes was detected in
BAL uid and at later time-points also in lung tissue, an obser-
vation supporting our previous ndings that adaptive immune
responses might contribute to the sustained adverse responses
in chemical-induced lung injuries (Ekstrand-Hammarstrom et al.,
2011; Wigenstam et al., 2009). Our results showed that Cl2 expo-
sure produced a rapid and transient intravascular leakage into
the lung, edema, which correlated with changes in respiratory
physiology 24 h after exposure. Histopathological evaluation of the
lung injury conrmed a marked acute lung injury, extensive bron-
chopneumonia, in the more central airways, correlating with the
appearance of inammatory cells in BAL uid and with the increase
of pro-inammatory cytokines. The injured epithelial layer, acute
airway inammation with inamed airway smooth muscle and
lung edema, all together may contribute to narrowing or even
closure of a signicant number of small airways during the MCh
challenge explaining the large effect on the peripheral parameters
G and H. The effect of baseline compliance observed 24 h following
exposure was likely due to extensive inammatory response and
edema formation.
Most of the inammatory mediators were detected in BAL uid
and serum between 2 and 6 h post exposure, indicating activa-
tion of leukocytes, predominantly macrophages and monocytes,
in the early phase of the pathogenesis. By releasing chemotactic
 S. Jonasson et al. / Toxicology 303 (2013) 3442
factors, macrophages and monocytes stimulate the migration of
activated neutrophils from the microcirculation to the airspaces in
the lung. Among the mediators we detected in BAL uid, CXCL1
and TNFA promote recruitment of neutrophils while CCL11 and
CCL5 are described as a major chemotactic factor for eosinophils.
Neutrophils and eosinophils may further exaggerate the epithe-
lial damage by release of reactive oxygen species and proteolytic
enzymes (Jonasson et al., 2009).
Many of the mediators produced by macrophages and mono-
cytes may also activate immune competent cells such as T
helper lymphocytes (TH ). By using an extended cytokine array, an
increased secretion of IL12 in BAL uid were detected, indicating
activation of a TH type 1 response (TH 1) in airways involving cell
mediated immunity and cytotoxicity. In BAL uid, an induction
of IL13 implicating a cytokine environment in the lung promot-
ing a systemic TH type 2 (TH 2) responses was also detected. Such
immune response is one of the major mechanisms for the activation
of eosinophilic inammation characteristic for asthma. IL13 is also
reported to be involved in the pathogenesis of pulmonary brosis
(Hancock et al., 1998) and AHR (Kasaian and Miller, 2008), indicat-
ing that this cytokine might be directly involved in the AHR and
brotic tissue remodeling observed in our study. Other mediators
important in the regulation of brosis are the chemokines CCL2,
CCL3 and CCL5 of which all were shown to be induced in BAL uid
of mice exposed to Cl2 in our study (Wynn, 2008).
Seven days after Cl2 exposure, lung tissues were still inltrated
by leukocytes, mainly granulocytes, monocytes, macrophages and
lymphocytes together with lung lesions consistent with an on-
going regenerative and reparative process. However, after day
seven, the only remaining pathological nding was AHR and
reduced baseline respiratory compliance. From these observa-
tions, we hypothesized that the transient inammatory response
and lung injury induced by Cl2 trigger the AHR, but that the
long-term respiratory dysfunction is maintained by more subtle
changes in the lung epithelium possibly due to airway remodel-
ing. It is likely that activation of immune competent cells may
persist beyond day seven in lymph nodes draining the respira-
tory system, thereby being an additional hypothetic mechanism
for the long-term effects. However, from our data it is not
possible to conclude the presence of such persistent immune
response.
The sensitivity to chlorine varied considerably among individ-
ual animals and could indirectly be due to differences in magnitude
of epithelial injury and the capability of restoring the injured tis-
sues. However, it should also be noted that the magnitude of
injury may also differ considerably between different strains of
mice due to the genetic variability. Another study where male mice
were exposed to 800 ppm Cl2 during 5 min (66.7 ppm h) acute lung
injuries were detected but the damages were completely restored
within ten days (Tuck et al., 2008). The lack of long-term effects in
that study could possibly be explained by strain and gender differ-
ences compared to our experimental approach, but could not be
due to difference in exposed dose according to Habers law (Miller
et al., 2000) and the results from Hoyle et al. indicated that even
for a constant dose, different patterns of lung injury arose depend-
ing on how the dose was delivered (Hoyle et al., 2010). Long-term
studies in rats have found histopathological changes such as inter-
stitial brosis and thickening of the septa 45 days following acute
exposure of Cl2 (1330 ppm, 332.5 ppm h) (Yildirim et al., 2004).
Another rat study reported that 1500 ppm Cl2 (125 ppm h) dur-
ing 5 min induced histopathological abnormalities which resolved
within 3090 days (Demnati et al., 1998). Due to strain and species
differences along with the great recovery, reversibility and capabil-
ity to repair brotic lesions of rodents, it is challenging to establish
an in vivo model that mimics the progressive characters of human
disease (Gauldie and Kolb, 2008).
41
5. Conclusion
This murine model offers a further contribution to the under-
standing of the mechanisms by which exposure to chemical
compounds, such as chlorine, causes long-term effects. Many of our
ndings in the mouse model are similar to symptoms described
for RADS in humans; in particular the inammatory response in
lung tissue and regeneration of the lung epithelium during the rst
seven days after exposure and the persistence of hyperreactive air-
ways for at least 28 days. These results provide a foundation for
future studies aimed at identication of biomarkers for adverse
inammatory responses and respiratory dysfunction, as well as for
evaluation of new concepts for treatment of chemical-induced lung
injury.
Conict of interest statement
None.
Acknowledgements
Elisabeth Wigenstam, Lina Thors, Lina gren, Christine Akfur,
Barbro Ekstrand-Hammarstrm, Linda Elfsmark and Karin Wall-
gren are gratefully acknowledged for excellent technical assistance
and for invaluable help with the animal experiments. This work was
supported by grants from the National Board of Health and Welfare,
the Swedish Ministry of Defence and the Swedish Armed Forces.
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