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Pulmonary Toxicology Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
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.
bronchoalveolar lavage; lung volumes; adaptation; antioxidants; lung epithelial lining fluid
INTRODUCTION 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 concentration-related
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 one-compartment
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 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 Animals. This study used 420 rats and
examined both extrapulmonary (n = 45)
and pulmonary (n = 324) effects of
changes in Ta on
O3 toxicity. Additional rats
(n = 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(F-344)CrlBr, VAF+] 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 ± 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 × 13 × 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.
Table 1.
Exposures, treatments, and physiological measurements
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 
4, 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).
[View Larger Version of this Image (18K GIF file)]
Exposure Regimens
Ambient Temperatures, °C
Parameters Measured
0.0 ppm O3 × 24 h/day
10
Body weight
0.5 ppm O3 × 6 h/day
22
Total lung capacity
0.5 ppm O3 × 23 h/day
34
Residual volume
BALF total protein
BALF neutrophils
BALF lysozyme
BALF
lactic acid dehydroge nase
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.
23
cmH2O, and then passively filled
to capacity with saline at +23
cmH2O. Immediately after saline
instillation, BALF was evacuated at 23 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 23 and +23 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 80°C until analyzed.
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.
K+ 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 80°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 5,5
-dithio-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 ± SE, and significance is defined as
P < 0.05.
RESULTS
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4), body weights were not different among the Ta groups of
Air rats, with a mean weight for all Air rats of 285 ± 3 g
(n = 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 ± 2, 291 ± 2, and 260 ± 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 K+
(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, 3, 4, 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.
, Change.
Significant data (P < 0.05) are designated as a, b, and c for rats
exposed at 10, 22, and 34°C, respectively.
Continuous O3 exposure caused marked body weight loss at both the Cold and Room Ta (Fig. 2A). After 3 days, rats had lost 30 ± 5 and 32 ± 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 ± 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 K+ in BALF. With Continuous exposure, urea, glucose, and K+ 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 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 O3-exposure 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). 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 values 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 K+ concentration in BALF was measured to determine whether O3 exposure induced sufficient tissue injury or cell lysis to cause an increase (5) and whether Ta would affect this parameter. Lavageable K+ was elevated in continuously exposed rats, but there was no evidence that it was modulated by Ta. The source of excess K+ 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 K+ 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 K+ 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 K+.
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.
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 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 × 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 into it from epithelial lining cells during its dwell time in the lung. Using volumes for ELF (estimated for rats as ~0.1 ml; Refs. 3, 7) and TLC (determined by lavage), ELF concentrations were computed and are shown in Table 3 along with serum concentrations and ELF-to-serum ratios.
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The high ELF-to-serum ratios found for ALP, lysozyme, K+, and AA (24.8, 30.8, 21.5, and 62.2, respectively) support previous studies that suggest that these substances are secreted into ELF from luminal epithelial cells (2, 14, 23, 24), and the very low ratio for protein (i.e., 0.0031) indicates that it is primarily derived from serum (10, 11), especially in ELF from normal control rats. UA, also thought to be a secretory product of airway mucosa in humans (21), had a ratio of only 7.5. In humans, UA appears to be a major pulmonary antioxidant compared with AA, whereas in rats AA is more prominent (7, 15, 24). The membrane-permeable crystaloids in serum (e.g., glucose, urea, and creatinine) also had low ELF-to-serum ratios (2.14, 6.65, and 6.63, respectively). The actual ratios for these free-moving solutes in vivo are probably closer to 1.0; however, the BALF values were higher, possibly due to rapid diffusion into the saline during the lavage procedure (3). Because there is no reason to assume that the ELF composition is homogenous throughout the luminal spaces, the regional concentrations for these substances, except for the crystaloids, probably exceed the estimates shown in Table 3.
In conclusion, the pulmonary toxicity of O3 is enhanced in rats exposed for longer periods of time and at lower Ta levels. It is possible that the modulation of O3 toxicity by Ta is primarily a dose effect that is related to the special ventilatory requirements associated with Ta-altered metabolic states.
ACKNOWLEDGEMENTS
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.
FOOTNOTES
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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