Acute effects of ozone exposure on lung function in mice sensitized to ovalbumin.
ABSTRACT Pulmonary responses to ozone exposure (1.0 ppm) were investigated in mice sensitized to ovalbumin compared with control mice receiving saline. Pulmonary function parameters were measured by pneumotachography. Arterial blood gases and the concentrations of soluble intercellular adhesion molecule-1 (sICAM-1) and tumor necrosis factor-alpha (TNF-alpha) in bronchoalveolar lavage fluid were analyzed. Ozone exposure, when compared with filtered air exposure, caused significantly larger decreases in dynamic compliance (P<0.05) and minute ventilation (P<0.05) in ovalbumin-sensitized mice but not in control mice. Moreover, the decrease in minute ventilation in response to ozone exposure was significantly greater (P<0.01) in ovalbumin-sensitized mice than in control mice. Ozone exposure caused a significant decrease in PaO2 in ovalbumin-sensitized mice but not in control mice. PaO2 after ozone exposure tended to be smaller in ovalbumin-sensitized mice than in control mice. The concentration of sICAM-1 in bronchoalveolar lavage fluid increased in ovalbumin-sensitized mice, but effects of ozone exposure were not observed. These results indicated that sensitization of the immune system to ovalbumin might be a risk factor which aggravates the effects of ozone exposure on the respiratory system.
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ABSTRACT: Environmental pollutants such as ozone may interact with airway responses to allergen in sensitised individuals. We examined the effects of a single exposure to ozone (1 ppm for 1 h) on bronchial responsiveness to acetylcholine (ACh) aerosol 3 and 24 h after single ovalbumin (OA) challenge in OA-sensitised anaesthetised guinea pigs. Broncho-alveolar lavage (BAL) was performed and protein content and differential cell counts were determined. Ozone increased bronchial responsiveness at 3 h but not at 24 h, while OA alone had no effect. At 3 h after ozone, OA induced further, but non-significant increases in bronchial responsiveness to ACh, but at 24 h after ozone, there was enhanced responsiveness. Neutrophil counts in BAL fluid increased at 3 and 24 h after exposure to ozone alone, but there were no further increases with ozone followed by OA exposure. Protein concentration was significantly increased in the ozone and OA groups at 3 and 24 h (163.4 +/- 25.6 and 128.7 +/- 7.4 mg/ml, respectively) compared to 54.0 +/- 18.1 mg/ml in the control group (p < 0.02 and p < 0.01, respectively). Our data demonstrate an interaction of OA with ozone exposure on bronchial responsiveness; one underlying mechanism could be through damage at the endothelial-epithelial barrier.International Archives of Allergy and Immunology 02/1997; 112(2):191-5. · 2.25 Impact Factor
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ABSTRACT: Homozygous mutant klotho (KL(-/-)) mice exhibit multiple phenotypes resembling human aging. In the present study, we focused on examining the pathology of the lungs of klotho mice and found that it closely resembled pulmonary emphysema in humans both histologically and functionally. Histology of the lung of KL(-/-) mice was indistinguishable from those of wild-type littermates up to 2 wk of age. The first histologic changes appeared at 4 wk of age, showing enlargement of the air spaces accompanied by destruction of the alveolar walls, and progressed gradually with age. In addition to these changes, we observed calcium deposits in type I collagen fibers in alveolar septa and degeneration of type II pneumocytes in 8- to 10-wk-old KL(-/-) mice. Pulmonary function tests revealed prolonged expiration time in KL(-/-) mice, which is comparable with the pathophysiology of pulmonary emphysema. The expression level of messenger RNA for type IV collagen, surfactant protein-A and mitochondrial beta-adenosine triphosphatase was significantly increased in KL(-/-) mice, which may represent a compensatory response to alveolar destruction. Additionally, the heterozygous mutant klotho mice also developed pulmonary emphysema late in life, around 120 wk of age. These findings indicate that klotho gene expression is essential to maintaining pulmonary integrity during postnatal life. The klotho mutant mouse is a useful laboratory animal model for examining the relationship between aging and pulmonary emphysema.American Journal of Respiratory Cell and Molecular Biology 01/2000; 22(1):26-33. · 4.15 Impact Factor
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ABSTRACT: To characterize the pulmonary response of asthmatic and healthy nonsmoking adult men to 0.20 ppm ozone by controlled chamber exposure. A prospective, crossover study of five atopic asthmatic and five normal subjects randomly exposed to ozone and filtered purified air (FPA) for 6 h, consisting of 30-min alternating periods of rest and moderate exercise. The two exposures were separated by at least 30 days. A controlled exposure in a stainless steel chamber. Five atopic asthmatic and five normal subjects between 18 and 45 years of age. Treatment with medications was withheld from asthmatics prior to the exposures. All subjects were nonsmokers. Symptoms were assessed throughout the exposures. Pulmonary function was measured at baseline, hourly throughout an exposure, and after an exposure. Bronchoalveolar lavage (BAL) was performed 18 h after the completion of an exposure. The BAL fluid (BALF) was analyzed for cell count and differential; the cell-free supernatant was analyzed for albumin, tumor necrosis factor (TNF), interleukin 1 (IL-1), interleukin 6 (IL-6), and interleukin 8 (IL-8). There were statistically significant increases in IL-8 levels, as well as percent polymorphonuclear neutrophils (PMNs) and PMNs per milliliter of lavage in asthmatics exposed to ozone as compared with the same asthmatics exposed to FPA and the same normal subjects exposed to ozone and FPA. Interleukin 6 was also significantly increased in asthmatics exposed to ozone. The BALF albumin, TNF, and IL-1 levels were not significantly different among the four groups. There were no differences between asthmatics and healthy controls exposed to ozone or FPA in baseline to postexposure FEV1, FVC, FEV1/FVC, and sRaw. We conclude that asthmatics exposed to ozone develop a significant BALF neutrophilia and increased levels of the cytokines, IL-8 and IL-6. These BALF findings occur even though the level of ozone exposure was not significant enough to reduce pulmonary function.Chest 01/1995; 106(6):1757-65. · 5.85 Impact Factor
Toxicology 172 (2002) 69–78
Acute effects of ozone exposure on lung function in mice
sensitized to ovalbumin
Tsuneo Yamauchia,*, Masayuki Shimaa, Tomoyuki Kuwakib,
Michiko Andoa, Masayoshi Ohmichic, Yasuichiro Fukudab,
aDepartment of Public Health, Chiba Uni?ersity School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
bDepartment of Physiology, Chiba Uni?ersity School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
cChiba City Institute of Health and En?ironment, 1-3-9 Saiwai-cho, Mihama-ku, Chiba 261-0001, Japan
Accepted 1 December 2001
Pulmonary responses to ozone exposure (1.0 ppm) were investigated in mice sensitized to ovalbumin compared with
control mice receiving saline. Pulmonary function parameters were measured by pneumotachography. Arterial blood
gases and the concentrations of soluble intercellular adhesion molecule-1 (sICAM-1) and tumor necrosis factor-?
(TNF-?) in bronchoalveolar lavage fluid were analyzed. Ozone exposure, when compared with filtered air exposure,
caused significantly larger decreases in dynamic compliance (P?0.05) and minute ventilation (P?0.05) in ovalbu-
min-sensitized mice but not in control mice. Moreover, the decrease in minute ventilation in response to ozone
exposure was significantly greater (P?0.01) in ovalbumin-sensitized mice than in control mice. Ozone exposure
caused a significant decrease in PaO2in ovalbumin-sensitized mice but not in control mice. PaO2after ozone exposure
tended to be smaller in ovalbumin-sensitized mice than in control mice. The concentration of sICAM-1 in
bronchoalveolar lavage fluid increased in ovalbumin-sensitized mice, but effects of ozone exposure were not observed.
These results indicated that sensitization of the immune system to ovalbumin might be a risk factor which aggravates
the effects of ozone exposure on the respiratory system. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Ozone; Ovalbumin; Pulmonary function test; Blood gas analysis
Ozone is recognized as a potent oxidant that
participates in photochemical air pollution and
affects human health by irritating the mucosa and
harming the respiratory system. In the lungs,
ozone can cause: (1) irritative cough and subster-
nal chest pain, (2) decrements in forced vital
capacity (FVC) and one second forced expiratory
volume (FEV1), (3) neutrophilic inflammation of
the airway submucosa (A committee of the envi-
ronmental and occupational health assembly of
* Corresponding author. Tel.: +81-43-226-2069; fax: +81-
E-mail address: email@example.com (T. Ya-
0300-483X/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.
T. Yamauchi et al. / Toxicology 172 (2002) 69–7870
the American Thoracic Society, 1996). Ozone ex-
posure results in greater FEV1decrements (Kreit
et al., 1989; Scannell et al., 1996), increased air-
way resistance (Scannell et al., 1996) and inflam-
matory responses in the lungs of asthmatics
(Basha et al., 1994; Scannell et al., 1996). Asth-
matic subjects are considered to be more suscepti-
ble to the harmful effects of ozone exposure, than
non-asthmatics. However, several previous con-
trolled clinical studies had not indicated hyper-re-
sponsiveness of asthmatics to ozone exposure
compared with healthy subjects (Linn et al., 1978;
Koenig et al., 1985, 1987).
In animal studies, it has been reported that
ozone exposure decreases dynamic compliance,
increases respiratory rate and causes neutrophilic
inflammation in the lungs (Watanabe et al., 1973;
Coleridge et al., 1993; Arito et al., 1997). Continu-
ous exposure to ozone during sensitization to
allergens resulted in elevated levels of specific
antibody and increased bronchial responsiveness
(Matthew, 1995; Sun et al., 1997). However, the
effects of ozone on already sensitized animals are
In this study, we have investigated the acute
effects of ozone exposure on pulmonary function
tests and blood gases in mice already sensitized to
ovalbumin. Moreover, we examined the effects of
ozone exposure on cell adhesion molecules, which
participate in neutrophilic inflammation.
Specific pathogen-free male C57BL/6 mice
weighing 18?22 g (6 week old, n=102) were
used in this study. They were purchased from
Japan SLC, Inc (Hamamatsu, Japan).
2.2. Aeroallergen treatment of mice
The sensitization scheme is outlined in Fig. 1.
Mice were sensitized by i.p. injection with 50 ?g
ovalbumin (Sigma Chemical Co, St. Louis, MO)/1
mg Al(OH)3 (Wako Pure Chemical Industries,
Ltd, Osaka, Japan), in 0.9% sterile saline on days
0 and 7. Non-sensitized mice received 1 mg
Al(OH)3in 0.9% saline. On days 14, 21, 28 and
35, mice were exposed to an aerosol of ovalbumin
(10 mg/ml) in 0.9% saline (non-sensitized mice
received saline only) for 30 min three times (at 30
min intervals) (Foster et al., 1996). The aerosol
was generated by an ultrasonic nebulizer (NE-12;
Omron, Tokyo, Japan). Mice were divided into an
OA-group and a saline-group.
2.3. Pulmonary function analysis
Physiologic parameters were measured in ure-
breathing mice, according to a slightly modified
Fig. 1. Aeroallergen treatment of mice.
T. Yamauchi et al. / Toxicology 172 (2002) 69–7871
method originally published for rats (Palecek,
1969). In brief, a short polyethylene tube was
inserted into the trachea and another was inserted
into the lower third of the esophagus. Respiratory
flow signals were measured through a Lilley-type
pneumotachograph connected to the intratracheal
tube. Respiratory volume was obtained by electric
integration of the flow signal. Intraesophageal
pressure was taken as intrathoracic pressure.
Body temperature was maintained at 36 °C
throughout the experiment. These data were fed
into a computer through an A/D converter
When breathing became stable for more than 5
min, respiratory resistance, dynamic compliance,
respiratory rate, and minute ventilation were mea-
sured. These data were used as baselines for the
pulmonary function tests (Onodera et al., 1997;
Suga et al., 2000).
2.3.1. Experiment 1: measurement of airway
reacti?ity to methacholine
Animals were studied 24 h after the last aerosol
inhalation. In vivo airway responsiveness was as-
sessed as an average of 10 breaths at peak re-
sponses after intravenous saline or methacholine
challenges in both the OA- and saline-groups. The
effects of the administration of five graded doses
of methacholine (0.005–0.1 ?g per gram body
weight, Sigma Chemical Co, St. Louis, MO) were
2.3.2. Experiment 2: acute effects of exposure to
ozone on the respiratory system
Animals were studied for 3 h after the last
aerosol inhalation. A previous study indicated
that the airway hyper-reactivity to acetylcholine
was observed between 3 and 24 h after the last
ovalbumin inhalation and hyper-reactivity was
slightly greater at 3 h than 24 h (Yamaguchi et al.,
1994). Therefore, we evaluated acute effects of
ozone exposure in OA-sensitized mice at 3 h after
the last inhalation, when the animals may be more
susceptible to ozone exposure. Pulmonary func-
tion parameters at baseline were measured and
changes during exposure to 1.0 ppm ozone, or
filtered air without ozone, were assessed.
Ozone was produced by ultraviolet lamps lo-
cated in the air intake duct of a Hinners-
type stainless steel chamber. The ozone was ex-
tracted from the chamber and led through a tube,
which was connected, to a flowmeter via a com-
pressor. Air containing ozone was fed through the
flowmeter into the polyethylene tube inserted
into the trachea. Filtered air without ozone
was provided in the same way. The ozone concen-
Japan), and controlled with a feedback circuit.
The ozone concentration in the chamber was
maintained at about 1.00 ppm (0.90–1.02 ppm)
Each parameter of pulmonary function was
compared with the 1 min average during exposure
at 10-min intervals from 0 to 60 min. In experi-
ments to evaluate the time course effects of ozone
exposure, results were expressed as percent of
baseline for comparison among groups.
2.4. Arterial blood gas analyses
After exposure to ozone or filtered air for 1 h,
the abdominal aorta was exposed, and arterial
blood was drawn into a heparinized syringe.
Blood gas analysis was immediately performed on
the sample with the model-ABL500 Blood Gas
System analyzer (Copenhagen, Denmark).
2.5. Bronchoal?eolar la?age (BAL) and
quantification of sICAM-1 and TNF-? in
bronchoal?eolar la?age fluid (BALF)
The lungs were lavaged three times with
1.0 ml of sterile saline, instilled by syringe
through a polyethylene tube inserted into the
trachea. The lavaged fluid was harvested by gentle
aspiration. The average volume retrieved was
greater than 80% of the 3.0 ml that was in-
stilled. The lavage fluid was centrifuged at 200×g
for 10 min and the supernatant was stored at
The concentrations of sICAM-1 and TNF-? in
BALF were determined with commercially avail-
able soluble ICAM-1 and TNF-? ELISA kits
(Endogen, Inc, Woburn, MA).
T. Yamauchi et al. / Toxicology 172 (2002) 69–7872
Fig. 2. Effect of intravenous methacholine on respiratory resistance. The significance of the differences between the experimental
groups was evaluated using the Mann–Whitney U-test. § P?0.05 compared with the saline-group.
2.6. Statistical analysis
Data are expressed as mean?S.E.M. The con-
centrations of sICAM-1 and TNF-? in BALF
They were compared as geometric means with the
95% confidence interval (CI). Statistical signifi-
cance between two groups in Experiment 1 was
determined by the Mann–Whitney U-test. In Ex-
periment 2, pulmonary parameters at baseline
were assessed by ANOVA. To compare with re-
peated measurements, a split-plot design was used
and P values were adjusted using the Huynh–
Feldt method. If the difference between the
groups was significant, contrast’s test was used to
distinguish between pairs of groups. The differ-
ences among four groups in both arterial blood
gas analyses and quantification of sICAM-1 and
TNF-? in BALF were assessed by ANOVA. If the
difference among groups was significant, Games–
Howell’s method was used to distinguish between
pairs of groups. We used a statistic package pro-
gram (Super ANOVA, Abacus Concepts, Berke-
ley, CA, USA).
3.1. Acute effects on pulmonary function tests
3.1.1. Experiment 1: measurement of airway
reacti?ity to methacholine
Mice in both the OA-group and the saline-
group demonstrated little response to intravenous
saline administration. Administration of more
than 0.025 ?g of methacholine per gram body
weight resulted in a significant increase in respira-
tory resistance in the OA-group compared with
the saline-group. The degree of increase in the
OA-group depended upon the concentration of
methacholine employed (P?0.05) (Fig. 2). This
shows that the OA-group was hyper-reactive to
3.2. Experiment 2: acute effects of exposure to
ozone or filtered air on lung function tests
As shown in Table 1, there were no significant
differences in pulmonary function values at the
baseline among the four groups. These values
T. Yamauchi et al. / Toxicology 172 (2002) 69–7873
generally agreed with previous reports in the liter-
ature (Martin et al., 1988; Henderson et al., 1996;
Suga et al., 2000).
Respiratory resistance increased immediately on
exposure to ozone in the OA-group (OA–ozone
group) but not in the other three groups (Fig. 3a).
After 60 min exposure, the respiratory resistance
stood at 171% of the baseline level in the OA–
ozone group. In contrast, the respiratory resistance
in the saline–ozone group remained essentially
unchanged compared with baseline over this pe-
riod. Filtered air exposure did not change the
respiratory resistance in either the saline–filtered
air group or the OA–filtered air group. Overall, the
OA–ozone group showed an increase compared
with the other three groups but this did not reach
Dynamic compliance decreased in the OA-group
immediately after exposure to ozone and was only
74% of baseline after 60 min (Fig. 3b). The decrease
was significantly greater (P?0.05) than the OA–
filtered air group. The saline–ozone group showed
a small and longer onset decrease which was 87%
of baseline after 60 min. However, the change in
not significantly different from that in the saline–
filtered air group. Thus, ozone exposure could
decrease the dynamic compliance compared with
filtered air exposure, but only in the OA-group and
not the saline group. Decrease of dynamic compli-
ance in the OA–ozone group tended to be greater
than in the saline–ozone group but this tendency
did not achieve statistical significance.
The respiratory rate in the OA–ozone group
decreased over time down to 81% of baseline after
60 min ozone exposure (Fig. 3c). In the saline–
ozone group, the respiratory rate increased on
ozone exposure, except at 50 min, and stood at
101% of baseline after 60 min. The OA–ozone
group showed the greatest decrease compared with
the other three groups, but this was also not
Minute ventilation per gram body weight in the
OA–ozone group decreased over exposure time
compared with the baseline value (Fig. 3d) and
sunk to 76% of baseline after 60 min. The other
three groups did not clearly show consistent re-
sponses. The OA–ozone group showed a signifi-
cant decrease compared with the saline–ozone
group (P?0.01). The OA–ozone group showed a
significant decrease compared with the OA–filtered
air group (P?0.05).
3.3. Acute effect on arterial blood gases
The OA–ozone group had a mean PaO2of
90.3 mmHg in arterial blood (Table 2). This
represented a significant decrease compared with
either the saline–filtered air group (P?0.01)
or the OA–filtered air group (P?0.05). On
the other hand, ozone exposure failed to decrease
PaO2in the saline pre-treated group. Although the
OA–ozone group showed a lower PaO2compared
with the saline ozone group, the difference did not
reach significance. There were no significant dif-
Pulmonary function at baseline
(cmH2O/ml per s)
Minute ventilation/body weight
(ml/min per g)
Values are means, with S.E.M. shown in parentheses.
T. Yamauchi et al. / Toxicology 172 (2002) 69–78 74
Fig. 3. Time course of pulmonary responses to 1.0 ppm ozone or filtered air. (a) Respiratory resistance; (b) dynamic compliance;
(c) respiratory rate; (d) minute ventilation per gram body weight. Values=mean?S.E.M. Each parameter expressed as per cent of
baseline. Number of animals in the four groups is as follows: the saline–filtered air group (n=10), the saline–ozone group (n=19),
OA–filtered air group (n=29) and OA–ozone group (n=31). To compare values in repeated measurements, we used the split-plot
method. The significance of differences between experimental groups was evaluated using contrast’s test.
ferences in mean PaCO2, HCO3−or pH among
the four groups.
3.4. Effects of ozone exposure on sICAM-1 and
TNF-? concentrations in BALF
As shown in Table 3, soluble ICAM-1 concen-
trations in BALF increased significantly (P?
0.001) inthe OA-groups
group=3390 ng/ml, OA–ozone group=3460 ng/
ml) compared with the saline-groups (saline–
filtered air group=2230 ng/ml, saline–ozone
group=2370 ng/ml). However, ozone exposure
did not induce significant differences in sICAM-1
concentrations either in the saline-group or the
OA-group. The TNF-? concentrations in BALF
were also not affected by OA sensitization or
ozone exposure (Table 3).
Recent studies have focussed on the effects of
ozone on susceptible groups, such as patients with
bronchial asthma. Ozone exposure has been re-
ported to increase bronchial responsiveness to
allergens (ragweed, grass) in atopic asthmatic sub-
jects (Molfino et al., 1991). From animal experi-
ments, it is known that ozone exposure can cause
an increase in airway resistance in non-sensitized
animals (Watanabe et al., 1973), a decrease in
dynamic compliance (Gordon and Amdur, 1980;
Coleridge et al., 1993) and an increase in the
respiratory rate (Arito et al., 1997). It is also clear
that inhalation of allergen can cause increases in
respiratory resistance, decreases in dynamic com-
pliance, increases in the respiratory rate and de-
creases of PaO2(Kessler et al., 1973; Chand et al.,
T. Yamauchi et al. / Toxicology 172 (2002) 69–7875
Effect of ozone on arterial blood gases
PaO2(mmHg)PaCO2(mmHg) Group HCO3−(mmol/l)pH
aP?0.01, versus the saline–filtered air group.
bP?0.05, versus the OA–filtered air group.
Values are means, with S.E.M. shown in parentheses.
1993). However, there have been few studies on
the acute effects of ozone exposure after sensitiza-
tion. Here, we have examined the acute effects of
ozone exposure on lung function in animals sensi-
tized to ovalbumin. We confirmed that our sensi-
tization protocol was clearly effective because
bronchial hyper-reactivity to methacholine com-
pared with control animals (Fig. 2).
In this study with 1.0 ppm ozone, mice sensi-
tized to ovalbumin showed an increase in respira-
methacholine but this was not significantly differ-
ent from the saline–ozone group. Airway resis-
tance changes are rarely seen in animals exposed
to ozone concentrations of less than 1.0 ppm
(Murphy et al., 1964; Tepper et al., 1990). There-
fore, we suggest that sensitization to ovalbumin
may increase susceptibility to ozone exposure, but
sensitization to ovalbumin is not definitely re-
quired to increase airway resistance during ozone
exposure compared with control mice.
In the present study, both the saline- and OA-
groups showed decreased dynamic compliance be-
low the baseline on ozone exposure, although
only the latter was statistically significant as com-
pared with filtered air exposure. It is known that
dynamic compliance is liable to decrease after
ozone exposure in guinea pigs (Chand et al., 1993)
and that the small airways are affected by acute
exposure to ozone in man (Foster et al., 1993).
This study indicated that the influence of ozone
exposure on the small airways in sensitized mice is
greater than in non-sensitized mice even if they
are from identical strains. However, this interpre-
tation should be regarded as tentative since the
difference in dynamic compliance between the
OA–ozone group and the saline–ozone group did
not reach statistical significance.
Here, the saline–ozone group did show a slight
increase in respiratory rate. This finding agrees
with previous reports that ozone exposure evokes
increases in the respiratory rate (Tepper et al.,
1990; McDonnell et al., 1983), by mechanisms
related to the stimulation of airway C-fiber
Effect of ozone on sICAM-1 and TNF-? concentrations mea-
sured in bronchoalveolar fluid
aP?0.01, versus the saline–filtered air group.
bP?0.01, versus the saline–ozone group.
Values are means, with 95%CI shown in parentheses.
T. Yamauchi et al. / Toxicology 172 (2002) 69–7876
(Hazucha et al., 1989; Coleridge et al., 1993).
Although there was no significant difference be-
tween the OA–ozone group and the saline–ozone
group, the OA–ozone group tended to show a
decrease. It has been reported that decreases in
the respiratory rate occur in non-sensitized mice,
24 h after ozone exposure (Currie et al., 1998).
Hence, further studies are required to clarify the
mechanism by which ozone exposure affects the
In the present study, the saline–ozone group
generally had increased minute ventilation, but
the OA–ozone group experienced a decrease in
showed a significant difference between the OA–
ozone group and the saline–ozone group. This
suggests that the effect of ozone exposure on
minute ventilation depends on the sensitization
conditions. The reasons for the decrease in minute
ventilation are thought to be related to defense
against the inhalation of ozone gases (Arito et al.,
1997) or alternatively, simply to be a result of
ozone-triggered airway inflammation. It has been
reported that minute ventilation in both animals
and humans during exercise is maintained at con-
trol levels during ozone exposure (Tepper et al.,
1990; McDonnell et al., 1983). However, another
study indicated that exposure to 3 ppm of ozone
for 8 h evoked decreases in minute ventilation in
rats (Paterson et al., 1992). These differences
might be associated with the experimental condi-
tions, such as ozone concentration, exposure time
or whether the studies included exercise or not.
The above considerations suggest that acute
effects of ozone exposure on pulmonary function
tests differ with sensitization conditions. One
mechanism may be related to asthmatic subjects
having epithelial destruction in the airways, so
that epithelial nerves are susceptible to specific or
non-specific stimulation (Laitinen et al., 1985).
Therefore, in the present study, OA-sensitization
might cause epithelial destruction in the airways
and acute effects of ozone exposure might be
exacerbated in sensitized mice.
Dynamic compliance is an index of small air-
way function, and it is thought that airway resis-
tance is an index of large airway function. For
this reason, in the present study ozone exposure
affected small airways more in sensitized mice.
The decreased dynamic compliance has been
shown to increase the impulse activity of rapidly
adapting receptors (Coleridge
Rapidly adapting receptors evoke augmented
breathing, which might lead to the decreased res-
Thus so far, no clear consensus has been
reached on how ozone exposure affects arterial
blood gases. Previous results suggest a depen-
dence on ozone concentration, exposure time, and
whether or not there was any underlying disease.
Exposure to 1, 2, 3 or 4 ppm of ozone did not
influence arterial blood gases after 3 h exposure in
sheep (Schelegle et al., 1990). However, in rats
exposed to 3 ppm of ozone, 4 and 8 h exposures
significantly decreased PaO2 and 4 h exposure
significantly increased PaCO2 (Paterson et al.,
The OA–ozone group in the present study
demonstrated a decrease in PaO2compared with
the other three groups. Rapidly adapting recep-
tors evoke reflex increases in bronchial secretion
and blood flow (Coleridge and Coleridge, 1994).
Bronchial secretion might cause the increase in
respiratory resistance and blood flow might be
associated with ventilation-perfusion ratio mis-
match. Ozone exposure may cause aggravation in
ventilation-perfusion ratio mismatch, leakage of
inflammatory proteins, inactivation of surfactants
and alveolar edema in sensitized mice. Ozone
exposure affected arterial blood gases only in the
mice with bronchial hyper-reactivity (OA-group)
not normal (saline-group). Thus, ozone exposure
is one of the factors that may aggravate respira-
tory conditions in sensitized mice. A decreased
oxygen supply to heart muscle cells after hypox-
emia may contribute to the mechanism by which
ozone can also affect the cardiovascular system.
Although ozone exposure can upregulate ex-
pression of ICAM-1 and P-selectin on the vessel
wall (Krishna et al., 1997; Blomberg et al., 1999),
it is not clear whether ozone exposure can affect
sICAM-1 in BALF. In our study, the concentra-
tion of sICAM-1 in the OA-group was found to
be higher than in the saline-group. However, there
was no effect of ozone exposure on the concentra-
tion of sICAM-1, which, in BALF, should be an
T. Yamauchi et al. / Toxicology 172 (2002) 69–78 77
indicator of inflammation caused by sensitization
rather than inflammation triggered by ozone, at
least under these experimental conditions. It has
been suggested that soluble ICAM-1 in lungs may
function as an anti-inflammatory mediator, espe-
cially by limiting leukocyte adhesion to endothe-
lial cells and diminishing leukocyte extravasation
(Rothlein et al., 1991). However, it may also
function as a pro-inflammatory mediator (Schmal
et al., 1998). Taken together, the data suggests
that the role of ICAM-1 in the lung may differ
depending on the causes of inflammation (OA or
ozone), or the organ on which it acts (vessel or
BALF) and on the timing during the inflamma-
TNF-? has been reported to upregulate IL-1,
IL-2, and PGE2 production, as well as, ICAM-1
and VCAM-1 expression. Increased TNF-? ex-
pression appears to be co-localized to the same
areas as increased ICAM-1 expression in lungs
(Nario and Hubbard, 1997). It has also been
reported that TNF-? was significantly elevated in
the BALF of atopic asthmatic patients 18 h after
segmental allergen challenges (Virchow et al.,
1995). Contrary to our expectations, however, the
concentrations of TNF-? in BALF were not af-
fected either by sensitization or ozone exposure in
the present study. The experimental conditions,
such as the period of time between the last aller-
gen challenge and starting the first experimental
measurements, might possibly contribute to dif-
ferences in the results. Alternatively, TNF-? may
not be such a sensitive indicator of inflammation
as the respiratory function parameters.
In summary, we have found that ozone expo-
sure causes an increase in respiratory resistance
and a decrease in dynamic compliance, respira-
tory rate, minute ventilation and PaO2in OA-sen-
sitized mice but does not have any impact on the
concentrations of sICAM-1 or TNF-? in BALF.
Decreases in dynamic compliance and minute
ventilation in response to ozone exposure were
significant in OA-sensitized mice but not in saline-
treated mice. However, in comparisons between
the OA–ozone and saline–ozone groups, only the
difference in minute ventilation reached statistical
significance. We conclude that sensitized mice
might be more susceptible to ozone exposure.
A part of the study was supported by grants
from the Naito Foundation, the Takeda Science
A committee of the environmental and occupational health
assembly of the American Thoracic Society, 1996. Health
effects of outdoor air pollution. Am. J. Respir. Crit. Care
Med. 153, 3–50.
Arito, H., Takahashi, M., Iwasaki, T., Uchiyama, I., 1997.
Age-related changes in ventilatory and heart rate responses
to acute ozone exposure in the conscious rat. Ind. Health
Basha, M.A., Gross, K.B., Gwizdala, C.J., Haidar, A.H.,
Popovich, J. Jr, 1994. Bronchoalveolar lavage neutrophilia
in asthmatic and healthy volunteers after controlled expo-
sure to ozone and filtered purified air. Chest 106, 1757–
Blomberg, A., Mudway, I.S., Nordenhall, C., Hedenstrom, H.,
Kelly, F.J., Frew, A.J., Holgate, S.T., Sandstrom, T., 1999.
Ozone-induced lung function decrements do not correlate
with early airway inflammatory or antioxidant responses.
Eur. Respir. J. 13, 1418–1428.
Chand, N., Nolan, K., Pillar, J., Lomask, M., Diamantis, W.,
Sofia, R.D., 1993. Aeroallergen-induced dyspnea in freely
moving guinea pigs: quantitative measurement by bias flow
ventilated whole body plethysmograpy. Allergy 48, 230–
Coleridge, H.M., Coleridge, J.C.G., 1994. Pulmonary reflexes:
neural mechanisms of pulmonary defense. Ann. Rev. Phys-
iol. 56, 69–91.
Coleridge, J.C.G., Coleridge, H.M., Schelegle, E.S., Green,
J.F., 1993. Acute inhalation of ozone stimulates bronchial
C-fibers and rapidly adapting receptors in dogs. J. Appl.
Physiol. 74, 2345–2352.
Currie, W.D., van Schaik, S.M., Vargas, I., Enhorning, G.,
1998. Ozone affects and pulmonary surfactant function in
mice. Toxicology 125, 21–30.
Foster, W.M., Silver, J.A., Groth, M.L., 1993. Exposure to
ozone alters regional function and particle dosimetry in the
human lung. J. Appl. Physiol. 75, 1938–1945.
Foster, P.S., Hogan, S.P., Ramsay, A.J., Matthaei, K.I.,
Young, I.G., 1996. Interleukin 5 deficiency abolishes
eosinophilia, airways hyperreactivity, and lung damage in a
mouse asthma model. J. Exp. Med. 183, 195–201.
Gordon, T., Amdur, M.O., 1980. Effect of ozone on respira-
tory response of guinea pigs to histamine. J. Toxicol.
Environ. Health 6, 185–195.
Hazucha, M.J., Bates, D.V., Bromberg, P.A., 1989. Mecha-
nism of action of ozone on the human lung. J. Appl.
Physiol. 67, 1535–1541.
T. Yamauchi et al. / Toxicology 172 (2002) 69–7878
Henderson, W.R. Jr, Lewis, D.B., Albert, R.K., Zhang, Y.,
Lamm, W.J., Chiang, G.K., Jones, F., Eriksen, P., Tien,
Y.T., Jonas, M., Chi, E.Y., 1996. The importance of
leukotrienes in airway inflammation in a mouse model of
asthma. J. Exp. Med. 184, 1483–1494.
Kessler, G.-F., Austin, J.H.M., Graf, P.D., Gamsu, G., Gold,
W.M., 1973. Airway constriction in experimental asthma
in dogs: tantalum bronchographic studies. J. Appl. Physiol.
Koenig, J.Q., Covert, D.S., Morgan, M.S., Horike, M.,
Horike, N., Marshall, S.G., Pierson, W.E., 1985. Acute
effects of 0.12 ppm ozone or 0.12 ppm nitrogen dioxide on
pulmonary function in healthy and asthmatic adolescents.
Am. Rev. Respir. Dis. 132, 648–651.
Koenig, J.Q., Covert, D.S., Marshall, S.G., Van Belle, G.,
Pierson, W.E., 1987. The effects of ozone and nitrogen
dioxide on pulmonary function in healthy and asthmatic
adolescents. Am. Rev. Respir. Dis. 136, 1152–1157.
Kreit, J.W., Gross, K.B., Moore, T.B., Lorenzen, T.J.,
D’Arcy, J., Eschenbacher, W.L., 1989. Ozone-induced
changes in pulmonary function and bronchial responsive-
ness in asthmatics. J. Appl. Physiol. 66, 217–222.
Krishna, M.T., Bromberg, A., Biscione, G.L., Kelly, F., Sand-
strom, T., Frew, A., Holgate, S., 1997. Short-term ozone
exposure upregulates P-selectin in normal human airways.
Am. J. Respir. Crit. Care Med. 155, 1798–1803.
Laitinen, L.A., Heino, M., Laitinen, A., Kava, T., Haahtela,
T., 1985. Damage of airway epithelium and bronchial
reactivity in patients with asthma. Am. Rev. Respir. Dis.
Linn, W.S., Buckley, R.D., Spier, C.E., Blessey, R.L., Jones,
M.P., Fischer, D.A., Hackney, J.D., 1978. Health effects of
ozone exposure in asthmatics. Am. Rev. Respir. Dis. 117,
Martin, T.R., Gerard, N.P., Gail, S.J., Drazen, J.M., 1988.
Pulmonary responses to bronchoconstrictor agonists in the
mouse. J. Appl. Physiol. 64, 2318–2323.
Matthew, I.G., 1995. Interaction of air pollutants and pul-
monary allergic responses in experimental animals. Toxi-
cology 105, 335–342.
McDonnell, W.F., Horstman, D.H., Hazucha, M.J., Seal, E.
Jr, Haak, E.D., Salaam, S.A., House, D.E., 1983. Pul-
monary effects of ozone exposure during exercise: does-re-
sponse characteristics. J. Appl. Physiol. 54, 1345–1352.
Molfino, N.A., Wright, S.C., Katz, I., Tarlo, S., Silverman, F.,
McClean, P.A., Szalai, J.P., Raizenne, M., Slutsky, A.S.,
Zamel, N., 1991. Effect of low concentrations of inhaled
allergen responses in asthmatic subjects. Lancet 338, 199–
Murphy, S.D., Ulrich, C.E., Frankowitz, S.H., Xintaras, C.,
1964. Altered function in animals inhaling low concentra-
tions of ozone and nitrogen dioxide. Am. Ind. Hyg. Assoc.
J. 25, 246–253.
Nario, R.C., Hubbard, A.K., 1997. Localization of intercellu-
lar adhesion molecule-1 (ICAM-1) in the lungs of silica-ex-
posed mice. Environ. Health Perspect. 105 (suppl 5),
Onodera, M., Kuwaki, T., Kumada, M., Masuda, Y., 1997.
Determination of ventilatory volume in mice by whole
body plethysmography. Jpn. J. Physiol. 47, 317–326.
Palecek, F., 1969. Measurement of ventilatory mechanics in
the rat. J. Appl. Physiol. 27, 149–156.
Paterson, J.F., Hammond, M.D., Montgomery, M.R., Sharp,
J.T., Farrier, S.E., Balis, J.U., 1992. Acute ozone-induced
lung injury in rats: structural-functional relationship of
developing alveolar edema. Toxicol. Appl. Pharmacol. 117,
Rothlein, R., Mainolfi, E.A., Czajkowskii, M., Marlin, S.D.,
1991. A form of circulating ICAM-1 in human serum. J.
Immunol. 147, 3788–3793.
Scannell, C., Chen, L., Aris, R.M., Tager, I., Christian, D.,
Ferrando, R., Welch, B., Kelly, T., Balmes, J.R., 1996.
Greater ozone-induced inflammatory responses in subjects
with asthma. Am. J. Respir. Crit. Care Med. 154, 24–29.
Schelegle, E.S., Gunther, R.A., Parsons, G.H., Colbert, S.R.,
Yousef, M.A., Cross, C.E., 1990. Acute ozone exposure
increases bronchial blood flow in conscious sheep. Respir.
Physiol. 82, 325–335.
Schmal, H., Czermak, B.J., Lentsch, A.B., Bless, N.M., Beck-
Schimmer, B., Friedl, H.P., Ward, P.A., 1998. Soluble
ICAM-1 activates lung macrophages and enhances lung
injury. J. Immunol. 161, 3685–3693.
Suga, T., Kurabayashi, M., Sando, Y., Ohyama, Y., Maeno,
T., Maeno, Y., Aizawa, H., Matsumura, Y., Kuwaki, T.,
Kuro-o, M., Nabeshima, Y., Nagai, R., 2000. Disruption
of the klotho gene causes pulmonary emphysema in mice:
defect in maintenance of pulmonary integrity during post-
natal life. Am. J. Respir. Cell Mol. Biol. 22, 26–33.
Sun, J., Koto, H., Chung, K.F., 1997. Interaction of ozone
and allergen challenges on bronchial responsiveness and
inflammation in sensitised guinea pigs. Int. Arch. Allergy
Immunol. 112, 191–195.
Tepper, J.S., Wiester, M.J., Weber, M.F., Manache, M.G.,
1990. Measurements of cardiopulmonary response in
awake rats during acute exposure to near-ambient concen-
trations of ozone. J. Appl. Toxicol. 10, 7–15.
Virchow, J.C.J., Walker, C., Hafner, D., Kortsik, C., Werner,
P., Matthys, H., Kroegel, C., 1995. T cell and cytokines in
bronchoalveolar lavage fluid after segmental allergen
provocation in atopic asthma. Am. J. Respir. Crit. Care
Med. 151, 960–968.
Watanabe, S., Frank, P., Yokoyama, E., 1973. Acute effects of
ozone on lungs of cats. Am. Rev. Respir. Dis. 108, 1141–
Yamaguchi, S., Nagai, H., Tanaka, H., Tsujimoto, M., Tsu-
ruoka, N., 1994. Time course study for antigen-induced
airway hyperreactivity and the effect of soluble IL-5 recep-
tor. Life Sci. 54, 471–475.