Ozone toxicity in the rat. III. Effect of changes
in ambient temperature on pulmonary parameters
MILDRED J. WIESTER, WILLIAM P. WATKINSON, DANIEL L. COSTA, KAY M. CRISSMAN,
JUDY H. RICHARDS, DARRELL W. WINSETT, AND JERRY W. HIGHFILL
Pulmonary Toxicology Branch, Experimental Toxicology Division, National Health
and Environmental Effects Research Laboratory, United States Environmental
Protection Agency, Research Triangle Park, North Carolina 27711
bronchoalveolar lavage; lung volumes; adaptation; antioxidants; lung epithelial lining fluid
OZONE (O3 ) is a principal oxidant pollutant found in
ambient air in urban areas worldwide. Both epidemiologic and laboratory studies have reported that acute
exposure to this irritant gas causes concentrationrelated changes in pulmonary function, tissue injury,
and lung inflammation in both humans and animals
(28). Similar studies have shown that when O3 exposure is repeated daily, some of the pulmonary effects
attenuate as the exposures continue (6, 13, 17, 25). An
inhalation experiment in rats from our laboratory has
also demonstrated that the magnitudes of selected
extrapulmonary responses consequent to O3 exposure,
e.g., decreases in heart rate (HR) and core body temperature (Tco ), are concentration related and can be modified by changing ambient temperature (Ta ) during the
exposure (29). Presently, little is known about the
influence of Ta on O3 toxicity and adaptation in the
lung.
Human populations can be exposed to environmental
pollutant episodes that persist for several days, with
maximal O3 concentrations as high as 0.3–0.5 parts/
million (ppm) (34); these higher concentrations are
often associated with warmer Ta levels. Daily traffic
and weather patterns may combine to produce diurnal
exposure peaks. Environmental O3 concentrations and
Ta levels fluctuate over periods of pollutant exposure,
however, and are difficult to simulate experimentally.
Conversely, laboratory studies in humans and animals
permit more rigorous control and generally employ
stable exposure concentrations (frequently from 0.0 to
2.0 ppm) at Ta levels confined to a narrow range around
normal room temperatures (usually 18–26°C). When
realistic exposure scenarios are employed, data from
these studies provide insight into mechanisms and aid
in the interpretation of epidemiologic studies.
The exposure conditions used in the present study
were selected based on both environmental and experimental considerations. Rats were exposed to one of
three regimens of O3 at a Ta level of either 10, 22, or
34°C over 5 consecutive days. The particular combinations of exposure regimens and Ta levels were designed
to provide environmental relevance while facilitating
comparisons of the results among experimental studies. The concentration of O3 used (i.e., 0.5 ppm) was
selected to approximate the higher O3 levels seen
during severe air pollution episodes, to ensure the
induction of appropriate nonlethal pulmonary and extrapulmonary responses and to match the exposure
levels of previous laboratory studies (13, 25, 26, 29, 33).
The 6 h/day O3-exposure protocol was designed to
mimic peaks observed in urban exposures and induce
mild O3 effects, whereas the 23 h/day O3-exposure
protocol was designed to characterize steady-state exposure and produce decisive effects on all monitored
parameters (28, 29). The exposure temperatures used
in this study were chosen to span a normal ambient
range to examine the impact of moderate changes in Ta
on the observed responses.
Analyses and modeling of toxicity and adaptation for
the monitored extrapulmonary responses for this study
(i.e., HR, Tco, and activity) have been previously reported (9, 31). Briefly, these results showed significant
decreases in HR and Tco after exposure to O3, with the
magnitude of these decreases modulated by and inversely proportional to the exposure Ta. Mathematical
models were constructed to describe both normal circadian changes as well as O3-induced decreases in extrapulmonary parameters by using cosine and onecompartment functions, respectively. We now present
results for the Ta modulation of O3 toxicity on the lung.
O3 effects were assessed using changes in lung volumes
and in the cellular and biochemical composition of
bronchoalveolar lavage fluid (BALF) indexes that have
1691
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Wiester, Mildred J., William P. Watkinson, Daniel L.
Costa, Kay M. Crissman, Judy H. Richards, Darrell W.
Winsett, and Jerry W. Highfill. Ozone toxicity in the rat.
III. Effect of changes in ambient temperature on pulmonary
parameters. J. Appl. Physiol. 81(4): 1691–1700, 1996.—
Pulmonary toxicity of ozone (O3 ) was examined in adult male
Fischer 344 rats exposed to 0.5 parts/million O3 for either 6 or
23 h/day over 5 days while maintained at an ambient
temperature (Ta ) of either 10, 22, or 34°C. Toxicity was
evaluated by using changes in lung volumes and the concentrations of constituents of bronchoalveolar lavage fluid that
signal lung injury and/or inflammation. Results indicated
that toxicity increased as Ta decreased. Exposures conducted
at 10°C were associated with the greatest decreases in body
weight and total lung capacity and the greatest increases in
lavageable protein, lysozyme, alkaline phosphatase activity,
and percent neutrophils. O3 effects not modified by Ta included increases in residual volume and lavageable potassium, glucose, urea, and ascorbic acid. There was a progressive decrease in lavageable uric acid with exposure at 34°C.
Most effects were attenuated during the 5 exposure days
and/or returned to normal levels after 7 air recovery days,
regardless of prior O3 exposure or Ta. It is possible that
Ta-induced changes in metabolic rate may have altered
ventilation and, therefore, the O3 doses among rats exposed
at the three different Ta levels.
1692
OZONE AND TEMPERATURE EFFECTS ON RAT LUNG
been reported to characterize pulmonary injury, inflammation, and adaptation (11, 16, 20, 25, 33). A secondary
objective of this study was to estimate epithelial lining
fluid (ELF) concentrations for the various biochemical
components that were measured in BALF.
METHODS
Exposure Regimens
Ambient
Temperatures,
°C
0.0 ppm O3 3 24 h/day
0.5 ppm O3 3 6 h/day
0.5 ppm O3 3 23 h/day
10
22
34
Parameters Measured
Body weight
Total lung capacity
Residual volume
BALF total protein
BALF neutrophils
BALF lysozyme
BALF lactic acid dehydrogenase
BALF alkaline phosphatase
BALF urea
BALF creatinine
BALF glucose
BALF potassium
BALF uric acid
BALF ascorbic acid
BALF total glutathione
BALF, bronchealveolar lavage fluid; ppm, parts/million.
was allowed for chamber cleaning. The treatments and
measurements are listed in Table 1.
BALF and blood sampling. On a test day, 18 rats (n 5
6/O3-exposure group) were randomly selected and removed
from the exposure chambers. Rats were lavaged (33) in a
rotating fashion across exposure groups to normalize the time
of analysis. Continuous group rats were lavaged 1–5 h after
their last exposure, and Intermittent group rats were lavaged
19–24 h after their last exposure. Rats were anesthetized
with urethan (1.25 g/kg ip) and transorally intubated. The
lung was partially evacuated of air at 223 cmH2O, and then
passively filled to capacity with saline at 123 cmH2O. Immediately after saline instillation, BALF was evacuated at 223
cmH2O. One minute was allowed for saline instillation and
1.5 min for evacuation. By weighing the rat before and after
lavage and weighing the BALF, the lavage technique provided a quantitative sample of BALF from an anesthetized
rat. The sample represented one 37°C saline lung wash at a
volume equal to the total lung capacity (TLC) while lung
pressures were controlled between 223 and 123 cmH2O. The
method measured TLC and residual volume (RV) in rats and
permitted BALF components to be equated to units per
milliliter of TLC.
Blood samples were obtained from the abdominal aorta
after rats were anesthetized with urethan. Whole blood was
centrifuged (1,500 g, 30 min, 4°C), and the serum was
removed and stored at 280°C until analyzed.
Fig. 1. Sequence of events for an experiment lasting 25 days showing 1 ambient temperature (Ta ) phase of study.
Three separate Ta phases were conducted by using temperature levels of 10, 22, or 34°C. Acclimation to Ta started on
day 24, and O3 exposures started on day 0. Measurements were collected on days designated by hatched blocks
(e.g., lavage procedures were done on days 1-5 and 12). Surgery, recuperation, and telemetric monitoring refers to
rats that were examined and discussed in earlier reports from this study (9, 31).
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Animals. This study used 420 rats and examined both
extrapulmonary (n 5 45) and pulmonary (n 5 324) effects of
changes in Ta on O3 toxicity. Additional rats (n 5 51) were
used to examine biochemical relationships between blood
serum and ELF. Results regarding the effects on extrapulmonary parameters have been reported (9, 31) along with a
detailed discussion of the animal exposure conditions that
were used in this paper. These conditions are briefly summarized here. Ninety-day-old male Fischer 344 rats [CDF(F344)CrlBr, VAF1] were purchased from Charles River Breeding Laboratories, Raleigh, NC. At the time of the study, the
rats were 100–120 days old. Before each experiment, rats
were housed two to a cage with heat-treated pine shaving
bedding, and Ta was maintained at 22 6 1°C. Lights were
turned on at 0600 and off at 1800, and food (Purina rodent lab
chow, St. Louis, MO) and water were provided ad libitum.
During an exposure, rats were housed individually in 30 3
13 3 17-cm stainless steel cages without bedding, with food
and water provided ad libitum.
Study design. The study was conducted in three phases by
Ta with three O3-exposure groups per phase. Rats were
allowed 4 days to acclimate to the environmental conditions
and were then exposed to O3 or filtered air for 5 days. This
was followed by a 1-wk recovery period in filtered air while
the rats were still maintained at their respective Ta levels (Fig. 1).
Rats were lavaged on days 1-5 and day 12. Blood samples were
obtained from rats exposed to filtered air at 22°C on days 3 or 4.
O3 exposure. Cages were placed in one of three walk-in
inhalation exposure chambers (2.2 3 2.8 3 2.3 m) in which O3
concentration, humidity, temperature, and light cycle were
automatically controlled (4). Chamber temperatures were
maintained at 10, 22, and 34°C and will be referred to as Cold,
Room, and Warm, respectively (31). Rats at each Ta were
exposed to either filtered air (Air), 0.5 ppm O3 3 6 h/day
(Intermittent), or 0.5 ppm O3 3 23 h/day (Continuous). This
study employed only one O3 concentration to simplify comparisons among the Ta groups as well as comparisons with
previously published studies, and this concentration was
selected to ensure the induction of appropriate effects on both
pulmonary and extrapulmonary parameters without producing lethality. O3 exposures occurred between 0900–1500 or
1000–0900 for the Intermittent and Continuous animals,
respectively. The 1-h downtime for the Continuous exposure
Table 1. Exposures, treatments,
and physiological measurements
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OZONE AND TEMPERATURE EFFECTS ON RAT LUNG
RESULTS
Effects of Ta on air-exposed animals. A Ta effect was
seen in many of the monitored parameters, even though
the rats spent 1–5 h in the laboratory in cages with
bedding before they were lavaged. Analyses determined that there were differences due to Ta for some of
the parameters, but there were no differences due to
time or time 3 treatment for any of the parameters,
indicating that the Ta adjustments had occurred during
the acclimation period. Because the parameter values
were stable over the treatment period (days 1–12), they
were averaged over time, and the means are presented
in Table 2.
There was a strong Ta effect on body weight (P ,
0.001). Just before the acclimation period (day 24),
body weights were not different among the Ta groups of
Air rats, with a mean weight for all Air rats of 285 6 3 g
(n 5 102). By day 1, rats at the Warm Ta had lost ,25 g,
and those at the Cold and Room Ta showed no change.
Mean body weights were 283 6 2, 291 6 2, and 260 6 1
g for the Air rats at 10, 22, and 34 °C, respectively.
Total lung capacity was greatest for rats held at the
Cold Ta (P , 0.001), and RV was greatest (P , 0.02) for
rats held at the Warm Ta. The BALF composition was
also affected by Ta. At the Cold Ta, protein was reduced
(P , 0.01), and percent neutrophils was slightly but
significantly increased (P , 0.002). ALP was highest
(P , 0.0001) at Room Ta. Rats held at 34°C had
decreased levels of lysozyme (P , 0.0001) and AA (P ,
0.002), with increased levels of urea (P , 0.0001),
glucose (P , 0.0001), and K1 (P , 0.001). UA was
slightly higher in rats held at 34°C compared with that
of rats at 10°C (P , 0.05); however, it was not measured
at 22°C.
Effects of Ta on O3-exposed animals. In general, O3
was most toxic to rats when they were exposed at the
Cold Ta and least toxic when exposed at the Warm Ta.
The effects over time that O3-exposure regimens had on
body weight and pulmonary parameters at each Ta are
shown in Figs. 2–5 and are discussed below. These data
have been normalized to offset the effects of Ta and are
presented as the magnitude of the difference from data
obtained in air-exposed rats.
Continuous O3 exposure caused marked body weight
loss at both the Cold and Room Ta (Fig. 2A). After 3
Table 2. Body weight and pulmonary parameters
for air-exposed rats maintained at 3 different
ambient temperatures
Ambient Temperature, °C
Parameter
n
Body weight, g
Total lung capacity, ml
Residual volume, ml
Protein, mg/ml
Neutrophils, %
Lysozyme, µg/ml
Alkaline phosphatase,
U/l
Urea, mM/l
Creatinine, mg/dl
Glucose, mg/dl
Potassium, mM/ml
Uric acid, ng/ml
Ascorbic acid, ng/ml
10
22
36
30
283 6 2
291 6 2
12.5 6 0.1*
11.7 6 0.1
1.43 6 0.03
1.37 6 0.04
0.047 6 0.004* 0.155 6 0.01
0.8 6 0.2*
0.3 6 0.1
82.14 6 1.02
81.73 6 1.68
20.1 6 0.9
0.62 6 0.03
0.069 6 0.007
4.87 6 0.44
0.95 6 0.05
524 6 79
2,804 6 98
34
36
260 6 1*
12.0 6 0.1
1.54 6 0.05*
0.139 6 0.019
0.2 6 0.1
59.86 6 1.16*
32.5 6 0.9*
18.3 6 0.9
0.51 6 0.02
0.88 6 0.10*
0.049 6 0.009 0.084 6 0.005
4.62 6 0.29
7.98 6 0.72*
1.01 6 0.05
1.28 6 0.07*
1,004 6 208
2,715 6 92
2,267 6 135*
Values are means 6 SE; n 5 total no. of rats used in days 1–5 and
on day 12 for the means. Analyses determined that there were no
time effects on any of parameters, and therefore data represent
average over time. * Significant effect of ambient temperature for a
group when compared with other temperature groups, P , 0.05.
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BALF and blood analysis. An aliquot of BALF was used for
a differential cell count. Cells for counts were prepared by
using a Shandon Cytospin (Shandon, Pittsburgh, PA) and
stained with Diff-Quick (American Scientific Products, McGaw Park, IL). The remainder of BALF was centrifuged
(1,500 g, 15 min, 4°C), and acellular BALF was used for the
subsequent assays. K1 concentration was determined by
using an Astra-4 (Beckman Instrument, Brea, CA). Ascorbic
acid (AA), uric acid (UA), and total glutathione (GSH) were
assayed from perchloric acid filtrate. One milliliter of BALF
was mixed with 35 µl of 60% perchloric acid, and the mixture
was centrifuged (20,000 g, 20 min, 4°C). The supernatant was
stored at 280°C for subsequent analysis. Both AA and UA
were analyzed by liquid chromatography with electrochemical detection (18), and data were collected and analyzed by
using a Nelson Analytical 3000 series Chromatography Data
System (Cupertino, CA).
Assays of GSH, total protein (protein), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), urea, glucose,
creatinine, and lysozyme were modified for use on the Centrifichem System 500 centrifugal spectrophotometer (Baker
Instruments, Allentown, PA). GSH was determined by 5,58dithio-bis-(2-nitrobenzoic acid) (DTNB)-glutathione disulfide
reductase recycling assay, a modification of the method of
Anderson (1). The reagent contained 10 mg of NADPH, 6 mM
DTNB, and glutathione reductase in a 0.143 M sodium
phosphate, 6.3 mM EDTA, pH 7.4 buffer. Sample concentration of GSH was determined from a standard curve. Concentrations of creatinine, ALP, LDH, urea, and glucose were
determined by using commercially prepared kits and controls. Lysozyme activity was determined by measuring the
initial rate of lysis of a 1.38 mg/ml Micrococcus lysodeikticus
cell wall suspension in a 0.05 M sodium phosphate buffer at
pH 6.0. A standard curve using hen egg-white lysozyme was
employed. The method was modified from Konstan et al. (14).
All chemicals were obtained commercially (Sigma Chemical,
St. Louis, MO). Protein concentration was determined using
the Bio-Rad method (Bio-Rad Laboratories, Richmond, CA).
Sample protein concentration was determined from a standard curve by using bovine serum albumin standards. Assays
for blood serum were similar to those used for BALF.
Statistical analysis. Parameter measurements from nine
combinations of three O3 concentrations and three Ta levels
were recorded over time. Initially, the Air groups were
analyzed to examine the effects of Ta over time on the
response variables using a two-way analysis of variance
(ANOVA). Subsequent to finding significant effects due to Ta,
the O3-treatment group responses were normalized by subtracting the averaged daily Air group values to control for
these effects. The normalized measurements were analyzed
by using a three-way ANOVA to assess the nonzero significance of individual parameters in the model, with the three
variables being exposure (Intermittent and Continuous), Ta
(10, 22, and 34°C), and day (1, 2, 3, 4, 5, and 12). All data
values are reported as means 6 SE, and significance is
defined as P , 0.05.
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OZONE AND TEMPERATURE EFFECTS ON RAT LUNG
days, rats had lost 30 6 5 and 32 6 4 g at 10 and 22°C,
respectively, and the effect appeared to be attenuating
by day 5 in the Room Ta-exposed rats. There was no
detectable effect of O3 on body weight in rats exposed at
the Warm Ta. A small but significant weight loss O3
effect (i.e., 10 6 4 g) was seen with Intermittent
exposure on day 5 in rats exposed at 10°C (Fig. 2B).
Continuous O3 exposure caused significant decreases
in TLC at all three Ta levels, but rats were most affected
at the Cold Ta (Fig. 2C). Although significant effects
were seen with Intermittent exposure, the pattern of
changes was inconsistent among Ta levels (Fig. 2D).
Residual volumes increased after 1 day of Continuous
exposure at all Ta levels (Fig. 2E); however, exposure at
Warm Ta caused RV to diminish on days 3, 4, and 5. A
decrease in RV was seen with Intermittent exposure
and Warm Ta on days 3 and 4. Most of the O3 effects on
lung volumes were attenuated or had returned to
no-effect levels by the fifth day of exposure.
O3-exposure effects on BALF were differentially affected by Ta. Protein, percent neutrophils, lysozyme
levels, and ALP increased significantly with Continuous exposure, and all of these parameters showed the
greatest response when exposure was conducted at
10°C (Fig. 3, A, C, E, and G). These parameters all
appeared to adapt after 3 days of exposure, showing
diminished effects on days 4 and 5. Intermittent exposure caused minimal effects on protein, percent neutrophils, and level of lysozyme (Fig. 3, B, D, and F);
however, there were pronounced increases in ALP with
Cold Ta exposure on days 2 and 3 (Fig. 3H).
The Ta did not appear to affect the O3 responses for
urea, glucose, creatinine, or K1 in BALF. With Continuous exposure, urea, glucose, and K1 were significantly
increased, particularly on day 1 (Fig. 4, A, E, and G),
but there was no convincing O3 response seen in
creatinine levels (Fig. 4C). The creatinine response
with the Intermittent exposure regimen was variable
with respect to both O3 and Ta (Fig. 4, B, D, F, and H).
Although UA was significantly increased on day 1 for
Continuous exposure rats at 10°C, there was no further
effect in these animals. However, at Warm Ta, UA
decreased dramatically over the 5 days (Fig. 5A).
Similar effects on UA, in both direction and magnitude,
were found with the Intermittent exposure, except that
responses were delayed to days 4 and 5 with Cold Ta (Fig.
5B). The UA responses did not show attenuation but did
return to preexposure levels after 1 wk of recovery.
There was no effect of Ta on the AA response to O3. In
addition, and like UA, the AA response was similar for
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Fig. 2. Effect of Continuous (left) and
Intermittent (right) O3 exposure on body
weight (A, B), total lung capacity (C,
D), and residual volume (E, F) for rats
maintained at Ta levels of 10, 22, or
34°C. Data points were normalized for
effects of Ta, i.e., mean daily values
obtained from Air-exposed rat groups
(,6/group) were subtracted from those
obtained from respective O3-exposed
rats. Hatched vertical columns designate no. of hours per day that rats were
exposed to 0.5 ppm O3. See text for
further group description. Analyses
were performed on normalized data to
determine significance of an O3 effect
for each of 3 Ta levels. D, Change.
Significant data (P , 0.05) are designated as a, b, and c for rats exposed at
10, 22, and 34°C, respectively.
OZONE AND TEMPERATURE EFFECTS ON RAT LUNG
1695
both Continuous and Intermittent O3-exposure regimens (Fig. 5, C and D). AA levels increased over the 5
exposure days without any indication of attenuation.
AA levels were still elevated after 1 wk of recovery but
were only significant for rats exposed to the Continuous
regimen.
Lavageable GSH and LDH concentrations were below the level of detection in BALF samples with the
assay methods that were used in this study.
DISCUSSION
Previous work from this laboratory reported O3exposure concentration-related decreases in HR and Tco
in rats and that these responses were modified by
changes in Ta (29). The initial report from the present
study (31) supported this earlier work and demonstrated that HR and Tco were most depressed in rats
during the Cold O3 exposure and only slightly reduced
during the Warm exposure, suggesting that colder Ta
levels exacerbate toxicity. The O3 effects on HR and Tco
resolved with continued exposure and were not detected during the third, fourth, or fifth day of exposure
at any Ta, suggesting that the adaptation response was
not affected by Ta manipulations.
An association between effects on HR, Tco, and frequency of breathing (f ) has also been described (29).
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Fig. 3. Effect of Continuous (left) and
Intermittent (right) O3 exposure on lavageable protein (A, B), neutrophils (C,
D), lysozyme (E, F), and alkaline phosphatase (G, H) for rats exposed at 10,
22, or 34°C. Data were normalized and
analyzed as discussed for Fig. 2.
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OZONE AND TEMPERATURE EFFECTS ON RAT LUNG
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Fig. 4. Effect of Continuous (left) and
Intermittent (right) O3 exposure on lavageable urea (A, B), creatinine (C, D),
glucose (E, F), and potassium (G, H) for
rats exposed at 10, 22, or 34°C. Data were
normalized and analyzed as discussed
for Fig. 2.
Rats inhaling 1.0 ppm O3 for 2 h at a Ta of ,19°C
developed an increase in f in conjunction with decreases in HR and Tco. The HR and Tco responses were
not different for the exposures of 1.0 or 0.5 ppm O3.
Although measurements of spontaneous breathing were
not obtained for the rats exposed to 0.5 ppm, or in the
present study, other studies have shown that tachypnea (e.g., increased f and decreased tidal volume)
occurs with acute exposure to 0.5 ppm O3 in rats (25,
33) and that these breathing effects are attenuated
with repeated exposure after 2–3 days (25). Previous
studies from this laboratory have shown that O3 adaptation can develop during ‘‘environmental-type’’ daily
exposure and may be maintained for up to 18 mo of
exposure (32, 33). Thus it is reasonable to assume that both
the Continuous and Intermittent exposures in the present
study exerted an effect on spontaneous breathing (i.e.,
tachypnea) and that this effect adapted after 2–3 days,
much like that found for effects on HR and Tco (31).
Variations in Ta affected most of the parameters.
These Ta effects were seen on day 1, remained stable
over the 12 monitoring days, and were assumed to
reflect normal metabolic and physiological adjustments
to each of the three Ta levels. Because the O3 responses
were superimposed on Ta effects, the O3 response data
were normalized by using Air-exposure averaged val-
OZONE AND TEMPERATURE EFFECTS ON RAT LUNG
1697
Fig. 5. Effect of Continuous (left) and
Intermittent (right) O3 exposure on lavageable uric acid (A, B) and ascorbic
acid (C, D) for rats exposed at 10, 22, or
34°C. Data were normalized and analyzed as discussed for Fig. 2.
cient tissue injury or cell lysis to cause an increase (5)
and whether Ta would affect this parameter. Lavageable K1 was elevated in continuously exposed rats, but
there was no evidence that it was modulated by Ta. The
source of excess K1 is not clear. It likely did not come
from lysed red blood cells because the acellular BALF
samples were clear and colorless. There is also no
reason to believe that it was affected by extensive
O3-induced damage to the airways. Joris et al. (12)
found that K1 concentrations in airway surface fluid
were similar between normal subjects and patients
with sustained airway irritation, acute airway infection, cystic fibrosis, or asthma. The elevated levels of
K1 in our study may reflect O3-induced depletion of
airway and alveolar macrophages (8), cells that make
up ,6% of the total cell volume of the alveolar region of
the rat lung cell population (22). If some rat macrophages were destroyed by inhaled O3, then residual cell
debris may include excess lavageable K1.
Plasma-derived urea, glucose, and creatinine freely
cross semipermeable membranes and distribute rapidly throughout body fluids, including the transcellular
fluids (i.e., fluid separated from plasma by capillary
endothelium and a continuous layer of specialized
epithelium) such as ELF. Because the composition of
ELF is controlled by the pulmonary epithelium, it is
possible that a toxic insult to the epithelium would
affect the concentrations of these solutes in the BALF
samples. Both glucose and urea were increased after 1
day of Continuous exposure regardless of Ta, whereas
creatinine was not affected. Other biomarkers appear
to indicate that epithelial injury did occur after 23 h of
Continuous O3 exposure and that the injury lasted ,3
days. It is not clear what the nature of the injury was
that altered urea diffusion and glucose homeostasis
without affecting creatinine.
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ues before analyses to examine the specific O3 responses, as modified by Ta.
Although body weight was markedly decreased in
rats by the Warm Ta, O3 exposure caused no further
change in these animals. However, rats exposed to O3
continuously at Room or Cold Ta experienced profound
weight loss over the 5 exposure days, suggesting a
greater toxicity with the cooler Ta levels.
Lung function measurements such as TLC and RV
are useful in predicting the presence of obstructive or
restrictive pulmonary disease and are sensitive to
changes at the parenchymal level. It has been reported
that O3 exposure induces concentration-dependent decrements in TLC in humans (16) and in guinea pigs (20).
Rats exposed continuously in this study showed decreases in TLC that were more severe when exposures
were conducted at Cold Ta. The depression of TLC was
greatest on day 3 but still apparent on day 5. Residual
volume was increased after a single 23-h exposure,
similar to that reported by Yokoyama et al. (35) for rats
exposed to 1.0 ppm O3 for 24 h. At Cold and Room Ta
levels, RV returned to control levels by day 2 and was
no longer affected by O3. However, at Warm Ta, RV
dropped below baseline levels with either Continuous
or Intermittent exposure. Results indicate that the O3
effects on lung volumes are strongly modified by Ta,
with the cooler Ta levels being most effective in exacerbating toxicity.
This magnification of O3 toxicity with Cold Ta was
also reflected in BALF parameters. Continuously exposed rats had the greatest increases in lavageable
protein, percent neutrophils, lysozyme, and ALP activity, changes typically associated with increased respiratory epithelial permeability and inflammation (8, 10,
11, 27, 25). The K1 concentration in BALF was measured to determine whether O3 exposure induced suffi-
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OZONE AND TEMPERATURE EFFECTS ON RAT LUNG
relationship between Tco and toxicity. The seemingly
contrasting results of the present study may actually
reflect an inequality in dose (31). For an inhalation
exposure, dose is a function of an animal’s minute
ventilation (f 3 tidal volume). Rodents maintained at
cooler Ta levels exhibit higher metabolic and oxygen
consumption rates to maintain normothermia. Because
minute ventilation is directly correlated with these
parameters, it follows that rodents exposed at cooler Ta
levels would have higher ventilation rates and higher
initial doses of inhaled toxicants. Although all exposed
animals demonstrated a significant hypothermic response (presumably to limit their uptake of O3 ), this
limitation was likely neither complete nor sustained.
Thus, in the present study, it is possible that a higher
O3 dose at the Cold Ta may have overridden the more
subtle beneficial effects of hypothermia and assumed
the major role in determining the toxic response.
Previous work from this laboratory with CO2-induced
increases in ventilation during O3 exposure has demonstrated similar enhanced toxicity (26).
With the exception of AA, all of the O3 effects on
pulmonary and extrapulmonary physiological parameters returned to baseline within the 7 recovery days.
In fact, most of these parameters reached preexposure
levels by completion of the fifth exposure day. The
significance of differential adaptation of the various
parameters and the ultimate impact of this process on
protection of the lung remain unclear.
An ancillary objective of the present study was to
determine some of the normal biochemical relationships between serum and ELF, and no attempt was
made to examine the effects of Ta or O3 on this
relationship. Data were obtained from rats exposed to
Air at 22°C. The lavage technique provided a BALF
sample that consisted of ELF diluted to TLC plus any
additional substances that were secreted or leached
Table 3. Serum concentrations, estimated ELF
concentrations, and ELF-to-serum ratios for
substances found in lavage fluid from rats
exposed to air at 22°C
n
Alkaline phosphatase, U/l
Lysozyme, µg/ml
Potassium, mM/l
Ascorbic acid, ng/ml*
Uric acid, µg/ml*
Protein, g/dl
Glucose, mg/dl
Urea, mM/l
Creatinine, mg/dl
Serum
ELF
ELF-toSerum Ratio
28
153 6 5
30.96 6 1.28
5.5 6 0.1
5.06 6 0.41
6.69 6 0.91
5.86 6 0.05
252 6 11
8.96 6 0.29
0.86 6 0.04
30
3,790
9,550
118
317
50.2*
0.018
540
59.6
5.7
24.8
308
21.5
62.6
7.5
0.0031
2.14
6.65
6.63
Values are means 6 SE; n 5 no. of rats/group. Epithelial lining
fluid (ELF) concentrations were computed as [BALF] 3 TLC/0.1,
where TLC is total lung capacity. ELF-to-serum ratio was calculated
from data mean values shown; ELF values for ascorbic acid given in
µg/ml. * Serum and lavage fluid values for these parameters were not
available from rats examined in this study; therefore, data were
substituted from other control Fischer 344 rats (6/group) that were
exposed to air at room temperature (,22°C) and tested in our
laboratory.
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UA is an important respiratory system antioxidant
that contributes to airway defense mechanisms during
O3 exposure (19). Increased levels of UA in BALF have
been observed after O3 exposure in humans (15) and
guinea pigs (13). A recent study suggested that plasma
UA is taken up, concentrated, and secreted into the
nasal airway by mucosal cells in humans (21). The
present studies found no O3 effect on lavageable UA
with Continuous exposure at the Cold Ta; however, UA
was elevated on days 4 and 5 with Intermittent exposure. Interestingly, UA concentrations progressively
decreased in lavage fluids during the Continuous and
Intermittent exposures at the Warm Ta. Although the
UA response to O3 in rats appears to be strongly Ta
related, it is difficult to compare the findings of the
present study with those in humans or guinea pigs
because of the absence of Room Ta data.
AA, a widely distributed water-soluble antioxidant,
is sensitive to O3, and its exposure-related increase in
BALF has been associated with protection from oxidant
injury and O3 adaptation in rats and guinea pigs (13,
24, 25, 33). The relationship between O3 exposure and
lavageable AA is complex. In rats, AA concentration in
BALF is diminished immediately after an acute single
exposure to O3 (33). With no further exposure, the AA
level increases, resulting in overshoot. Full rebound
may require 1–2 days, and the higher AA level may be
sustained for a week. With repeated daily exposure, the
maximum increase appears to reflect a balance between the loss caused by each daily exposure and the
ability of the cellular mechanisms to provide more AA
to the ELF. There is some evidence to suggest that the
O3-induced increase in AA is more effective with exposure at lower O3 concentrations, compared with exposures that induce overt lung injury (24). Thus it may
help to explain the findings in the present study
showing that neither Ta nor exposure regimen affected
the AA-response patterns. They were similar for rats
exposed either continuously or intermittently over 5
days for all three Ta levels. The primary difference was
that the rebound level was still significantly elevated
after 7 days of recovery for the continuously exposed
rats.
Overall, the toxic effects of O3 on the lung were
greatest with longer exposures and cooler Ta levels.
Although longer exposures would predictably be associated with increased toxicity, the more severe effects at
the cooler Ta levels were not initially expected and may
be related to the route of exposure. A number of studies
from our laboratory and others have shown that chemical exposures in rodents often induce a mild hypothermia that is potentiated by cooler Ta levels. Studies of
toxic effects in groups of rats artificially maintained at
different Tco levels and exposed intraperitoneally to
equal doses of xenobiotic agents generated a U-shaped
toxicity curve, exhibiting an attenuation of the observed toxicity in moderately hypothermic rats, compared with animals in which the Tco response was
either blocked or potentiated (30). Although otherwise
similar in concept, the present studies employed inhalation exposures and demonstrated an inverse linear
OZONE AND TEMPERATURE EFFECTS ON RAT LUNG
We thank Donald L. Doerfler for statistical assistance; Dr. Urmila
P. Kodavanti, James A. Raub, and Brenda T. Culpepper for their
helpful reviews of the manuscript; and Joanne Cook for secretarial
services.
This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection
Agency, and has been approved for publication. Mention of trade
names or commercial products does not constitute endorsement or
recommendation for use.
Address for reprint requests: M. J. Wiester, Experimental Toxicology Division (MD-82), National Health and Environmental Effects
Research Laboratory, US Environmental Protection Agency, Research Triangle Park, NC 27711.
Received 25 January 1996; accepted in final form 10 June 1996.
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1699
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