key: cord-0013065-48qe1faw
authors: WIESTER, M. J.; TEPPER, J. S.; DOERFLER, D. L.; COSTA, D. L.
title: Ozone Adaptation in Rats after Chronic Exposure to a Simulated Urban Profile of Ozone(1)
date: 1995-01-03
journal: Fundam Appl Toxicol
DOI: 10.1093/toxsci/24.1.42
sha: 913d76a5bdf8073d74b1aee885a423d161e99248
doc_id: 13065
cord_uid: 48qe1faw

Studies in both humans and rats have indicated that certain pulmonary responses induced by exposure to an acute provocative concentration of ozone (O(3)) will eventually attenuate if the exposure is repeated on a daily basis. This phenomenon is commonly referred to as O(3) adaptation. Whether or not a “state” of adaptation develops due to long-term low level O(3) exposure is unknown. Two human studies have reported adaptation in subjects living in Los Angeles during periods when ambient O(3) concentrations have been relatively high. At present, however, we are not aware of comparable information from rats. This study assessed O(3) adaptation in rats following chronic (12 or 18 months) exposure and after a 4-month recovery period. A chronic exposure pattern, similar to that found in an urban area during the summer (0.06 ppm O(3) for 13 hr/day, 7 days/week; Monday–Friday, peak to 0.25 ppm O(3), over 9 hr), was used. To assess whether adaptation had occurred and/or persisted, awake rats were challenged with high provocative concentrations of O(3) for up to 2 hr. During a challenge, rats were monitored for typical O(3)-induced alterations in spontaneous breathing parameters (e.g., increase in breathing frequency and decrease in tidal volume). Adaptation was defined as attenuation of breathing response during the challenge in rats chronically exposed to O(3) as compared to that in “control” rats (chronically exposed to air). Adaptation was found in the rats within 8 hr following the chronic O(3) exposure but not after the 4-month recovery period. Spontaneous breathing parameters that were significantly attenuated in the chronically exposed rats were breathing frequency, tidal volume, inspiratory and expiratory times, and maximum expiratory flow. We conclude that rats demonstrated adaptation to O(3) after long-term exposure to an urban-type O(3) profile and that the adaptation was not seen 4 months postexpo-sure. These results suggest that exposure to environmental O(3) in Los Angeles air may have been responsible for the adaptation found in residential subjects.

Los Angeles air may have been responsible for the adaptation found in residential subjects, o IWS society of Toxicology.

Several studies report that, in humans, the magnitude of the spirometric decrements from an acute provocative exposure of ozone (O 3 ) will diminish or attenuate if the exposure is repeated on a daily basis (U.S. Environmental Protection Agency, 1986) . The phenomenon, an acquired resistance to the nonlethal effects of O 3 , has been referred to as adaptation (Mustafa and Tierney, 1978) . Besides spirometry, other parameters have shown adaptation, including cellular and biochemical markers in bronchoalveolar lavage (BAL) fluid (Devlin et ai, 1993) and airway responsiveness (Dimeo el ai, 1981) . These studies indicate that with repeated daily exposure, subjects tend to have an augmented pulmonary response on the second exposure day, but a decreasing responsiveness on successive days. However, a more recent study, using different exposure conditions (0.12 ppm O 3 , 6.6 hr/day for 5 days), found that the O 3 -induced increase in airway responsiveness did not attenuate with repeated exposure (Folinsbee et ai, 1990) . Subjects in the latter study exercised for 5 hr at a ventilation of 40 liter/min during each exposure.

Adaptation to O 3 has also been reported in rats receiving acute repeated daily exposures of O 3 . Tepper et ai (1989) exposed rats to 0.35, 0.5, or 1.0 ppm O 3 for 2.25 hr for 5 consecutive days with CO 2 administered periodically during the O 3 exposures to stimulate breathing, thereby increasing the O 3 dose rate. The study found that, by the fifth day, O 3 effects on the spontaneous breathing parameters, frequency of breathing (/) and tidal volume (V T ), were attenuated. In addition, a flow limitation in the smaller airways, noticed after the second day in the 0.5-ppm O 3 exposure group, had also disappeared by the fifth day. Despite apparent attenuation of these pulmonary physiologic parameters, the 0.5-ppm O 3 -exposed rats continued to show a progressive pattern of lung epithelial damage, inflammation in terminal airways, and a sustained increase in lavageable protein over the 5 days. Furthermore, rats exposed to 1.0 ppm did not show attenuation of any of their respiratory responses. This study suggests that the attenuation of respiratory responses and cellular and biochemical responses are not coincident and are likely to be dependent on exposure parameters. On the other hand, Van Bree el al. (1989) examined cellular and biochemical responses in lungs of rats exposed to 0.4 ppm O 3 for 12 hr for 3 consecutive nights. They reported that the rats developed acute pulmonary injury and inflammation following the first nocturnal exposure; however, the injury appeared to be reversible, despite continuous exposure.

Even though the human and rat studies are largely in agreement with respect to O 3 adaptation when using relatively acute repeated exposure protocols, one cannot reliably extrapolate the data to predict adaptation after chronic exposure to urban levels of O 3 in either humans or rats.

At present, we are aware of only two studies of humans associating O 3 adaptation with long-term exposure to O 3 as found in polluted urban areas. One study (Hackney et al., 1977a) reported that subjects living in Los Angeles, surmised to have been exposed to high urban concentrations of O 3 , were less responsive to an experimental O 3 challenge than were Canadian subjects who lived in an area of very low O 3 concentrations. These investigators suggested that environmental exposure to O 3 , and possibly other oxidants present in the Los Angeles air, may have provided these subjects with some degree of adaptation. The other study (Linn et al., 1988) reported that the response to O 3 is a persistent individual characteristic that shows seasonal variability. Subjects who responded to experimental O 3 exposures in the spring appeared to have lost their O 3 sensitivity in the autumn. The authors suggested that these subjects adapted to the high ambient O 3 exposures during the summer/fall and thus had decreased responsiveness in the fall.

The objective of our investigation was to determine whether a long-term exposure to environmentally relevant 0 3 concentrations would induce O 3 adaptation in laboratory rats and, if so, whether adaptation persisted. Rats were exposed to O 3 using an urban-like concentration profile for 12 and 18 months and were subsequently tested for adaptation. A portion of the rats, from the 18-month exposure group, were held in filtered air for recovery and were tested 4 months postexposure.

Male, 60-day-old Fischer 344 rats [CDF (F-344) CrIBr, VAF+, Charles River Breeding Laboratories, Inc., KJngston, NY] were used for both the 12-and 18-month exposures. In order to save time, rats scheduled to be exposed for 18 months and those scheduled for 12 months were purchased separately and delivered to two different facilities. All rats were otherwise treated identically, except where noted.

On arrival, rats were ear-tagged, weighed, and housed in individual wire cages with food (Purina Rodent Lab Chow, St. Louis, MO) and water ad libitum. They were quarantined in barrier-maintained exposure facilities for 10 days, and then randomly assigned to either filtered air or O 3 exposure groups (Table 1) .

The health of the rats was monitored over the duration of the chronic exposure. Serum samples, taken from 5% of the rats upon arrival and from sentinel rats during the chronic exposure, were free of antibodies to reovirus Type 3, pneumonia virus of mice, encephalomyelitis virus, Sendai virus, mouse adenovirus, sialodacryoadenitis, Toolan H-l, Kilham rat virus, lymphocytic choriomeningitis, and rat coronavirus. Lung washes from these animals were free of Micoplasma pulmonis. and bacterial cultures from the nasopharynx, trachea, and gut revealed no abnormal findings. Rats were also free of ecto-and endoparasites.

Exposures were carried out in automatic inhalation exposure facilities designed for chronic exposure of rodents to gaseous pollutants, a complete description of which has been reported by Davies et al (1987) .

The 12-month exposure groups were housed in Hazleton-2000 exposure chambers and the 18-month groups were placed in 14.2-m 3 walk-in environmental rooms. Ozone concentrations, temperature (74 ± 3°F, SD), humidity (60 ± 10%, SD), and lighting (0600-1800 hr) were automatically monitored and controlled. These parameters were maintained under similar conditions in both chamber types. Air changes in the Hazelton-2000 chambers (37.4/hr) and in the environmental rooms (8/hr) were adequate to maintain equivalent O 3 exposures.

Ozone was generated by passing cylinder-supplied oxygen (National Specialty Gas Co.) through a silent arc O 3 generator (Ozone Research and Equipment Corp., Model 05V-0) and was monitored using continuous chemiluminescent analyzers (Bendix Model 8002) (RFOA-0176-007). Analyzers were calibrated at 2-week intervals using a uv standard (Federal Register, 1976 .

The O 3 exposure consisted of a background concentration of 0.06 ppm with a concentration spike reaching 0.25 ppm on each of the 5 week days (Fig. 1) . Only the background concentration was maintained over Saturday and Sunday. The concentration spike increased from 0.06 to 0.25 ppm and back to 0.06 ppm over a 9-hr period. A down time of 2 hr was included in each day for servicing. Integration of the concentration spike showed that the exposure corresponded to a square wave that averaged 0.19 ppm.

The Oj concentration profile ( Fig. 1 ) continued through Friday of the final week for both the 12-and 18-month studies. On the last day (Sunday) of the 18-month study, some of the rats were placed in filtered air for recovery. All of the 12-month rats and the remaining 18-month rats were tested Monday through Friday during the week following their respective exposures. During these testing periods, the background exposure pattern (0.06 ppm O 3 , 23 hr/day) was maintained. Rats designated for recovery were held for 4 months and were tested during the week following this period. On each test day, rats were selected at random and removed from the exposure chambers at 8:30 AM and tested between 11:00 AM and 4:00 PM, approximately 2.5-7.5 hr after leaving the chambers. All rats were tested so that the investigators were blind to previous exposure (O 3 or air), and each exposed group was randomly distributed over time on each testing day.

Two different challenge testing procedures were used. In Procedure I, individual rats were exposed to 1.0 ppm O 3 for 1 or 2 hr. In Procedure II, four rats were simultaneously exposed to 0.5 ppm O 3 for 2.25 hr with CO 2 added to stimulate ventilation. Reasons for using two procedures were (1) Procedure I was used to test adaptation and to measure percentage O 3 uptake, and (2) Procedure II was used because it had been previously used in our laboratory to differentiate between O r adapted and nonadapted rats following different exposure scenarios (Tepper el al., 1989) . In Procedure I, 12-month rats were exposed to the challenge for 2 hr to accommodate ' Rats were challenged for 2 hr to accommodate another investigator because the number of chronically exposed rats was very limited.

another investigator and 18-month rats were challenged for 1 hr. Challenge protocols are presented in Table 1 . Some of the equipment and measurement techniques were similar for both procedures. In both procedures, awake rats were individually restrained in acrylic holders (Wiester el al., 1987a) , placed in stainless-steel head-out plethysmographs, and exposed to the challenge gas. Spontaneous breathing parameters, collected during the challenge exposures, were derived from thoracic wall excursions inside the plethysmograph, as sensed by an attached pressure transducer (Validyne Engineering Corp.). The resultant analog signal, calibrated in milliliters, was continuously recorded on a polygraph chart and also digitized by an analog-to-digital converter every 4 msec during a 4-sec sample period. Each period was automatically triggered at preselected times. During an epoch, four breaths were analyzed by a computer (PDP 11/03, Digital Equipment Corp.) 

FIG. 1. The simulated urban profile of O 3 concentrations, used during the 12-and 18-month O 3 exposures, are plotted. Rats were exposed to the profile for 5 days each week and to a background level (0.06 ppm) on the remaining 2 days. Integration of the spike portion of the curve determined that the exposure was equivalent to a square wave averaging 0.19 ppm O 3 . minute volume (^E), maximum inspiratory and expiratory flows (V^â nd ^e_). ar >d inspiratory and expiratory times (7", and T e ) were computed (Tepper el al, 1988) . Following an experiment, polygraph tracings were examined and sample periods showing animal movement artifacts were deleted from the data set.

Procedure I. Details of this procedure have previously been reported (Wiester el al. 1987b; . Briefly, after the rat was placed in the plethysmograph, a glass cone-shaped chamber, designed for nose-only exposure (the apex of the cone, where the nose was located, opened into the side of a vertical glass tube), was placed over the rat's head and sealed around the neck to the plethysmograph.

Ozone was produced by passing oxygen through a uv generator. The 1.0-ppm Oj concentration was achieved by dilution, using cylinder air (Zero-grade air. Union Carbide Corp., Linde Division, Danbury, CT). The air mixture flowed upward past the nose through the out-flow tube at a rate sufficient to prevent rebreathing of downstream air. Flows were measured using a Gilibrator primary flow calibrator (Gilian Instrument Corp., West Caldwell, NJ). While the rat breathed spontaneously into the system, the downstream concentrations of O 3 ,0 2 , and CO 2 (upstream concentrations were established prior to putting the rat in the system) were monitored once each minute using a photometric O 3 analyzer (Model 8810, Monitor Labs Inc., San Diego, CA), a TCM2 Tc oxygen monitor, and a TCM20 carbon dioxide monitor (Radiometer America Inc., Westlake, OH), respectively.

During each challenge, the rat first breathed zero-grade air for 30 min in order to acclimate it to the testing apparatus. Then, the challenge exposure gas (1.0 ppm O 3 or zero air) was administered for 1 or 2 hr. Along with the spontaneous breathing parameters, oxygen consumption (HO 2 ), carbon dioxide production (rcO 2 ), and O 3 uptake data were obtained once each minute. Each 1-min measurement of a spontaneous breathing parameter represented mean data from four breaths. Immediately following a challenge, the rat received a lethal intraperitoneal injection of pentobarbital sodium. Data collection continued for 5 min after the cessation of breathing. These nonbreathing gas measurements provided an estimate of O 3 lost to the surface of the face and confirmation that upstream concentrations of O 3 , O 2 , and CO 2 remained relatively stable over the entire challenge pe-riod. Although these changes were usually very small, they were entered into a reanalysis program to correct the data.

Procedure II. In this procedure, rats were challenged four at a time. Four plethysmographs were attached to the door of a 0.33-m 3 Rochester exposure chamber so that when rats were placed in plethysmographs and the door closed, the rats' heads were inside the chamber. The challenge regimen consisted of filtered air for 15 min, followed by 0.5 ppm O 3 for 2.25 hr, and then back to filtered air for another 15 min. At four times (15 min each time) during the O 3 challenge, 8% CO 2 was administered concurrently with the O 3 . Detailed descriptions of exposure and breathing measurement techniques are reported in Tepper el al. (1988 Tepper el al. ( , 1989 . Spontaneous breathing parameters were collected and analyzed during the last 3 min of each 15-min interval. Each of the 11 samples for one rat was the average of 4-16 breaths.

Baseline data (Table 2) were analyzed using a multivariate analysis of variance (MANOVA) model. Effects from the chronic O 3 exposure, the exposure plus 4 months of recovery, and those due to time alone were examined.

Challenge data. To normalize pulmonary measurements among rats, baseline values were determined for each rat and the successive measure-ments, collected during the challenge, were adjusted by these baseline values (the averaged baseline value was subtracted from each successive measurement). Baseline-adjusted measurements are identified with delta (A). For Procedure I, the 1 -min breathing parameter data were averaged within successive 5-min intervals over the challenge time. These mean values were paired with the midpoint times of their respective intervals. Data from Procedure II were averaged at each of the 11 (3 min) data collection periods. These mean data were paired with respective 15-min lime points. A repeated measures analysis of variance was used to determine differences in response between rat groups along a time axis (Figs. 2,6, and 7) . Testing for changes and absolute differences in response between groups was performed through subtesting within the overall MANOVA. The experimentwise error rate was maintained (p * 0.05) by adjusting the significance level of each comparison using a Bonferroni adjustment.

Chronic exposure groups were also compared by analysis of the cumulative dose-response curves. The following methods of calculations provided cumulative dose estimates for each rat as the challenge exposure progressed (Figs. 4, 5, and 8) .

Procedure I:

Dose was accumulated over each successive minute of the challenge period. The cumulative dose was then divided into incremental increases at Note. V T , tidal volume;/ breathing frequency; V& expiratory minute volume; 7j and T^ inspiratory and expiratory times; V lmtx and V^^, inspiratory and expiratory maximum flows; K) 2 , oxygen consumed; KX>2, carbon dioxide produced. Data represent means ± SE. Data were obtained at the end of the challenge procedure aclimation period. Significant if p < 0.05.

" Effect due to exposure. (Deleted in proof.) * Effect due to time. c Effect due to exposure X time.

Breathing parameter values associated with each dose interval were averaged and these mean values were paired with the midpoints of their respective cumulative dose intervals. Procedure II:

O 3 dose (jigji 5 min) = V E X 15 X Oj concentration (jigjml).

Dose was accumulated over each successive 15 min during the O 3 exposure using V z values obtained at the beginning of each measurement sequence (see Fig. 6 ). The resultant cumulative dose was divided into incremental increases at lO-/ig intervals. Mean breathing parameter values (as stated above) were paired with their respective dose intervals.

A repeated measures analysis of variance was used to determine differences between chronic O 3 -exposed and chronic air-exposed rats along the cumulative dose axis (Figs. 5 and 8) . Data treatment was similar to that performed on the time-response data.

Analysis of baseline measurements (prechallenge) indicates that, overall, there were no detectable effects from the chronic O 3 exposures on body weight, spontaneous breathing patterns, or exchange of respiratory gases, although V e in Procedure II rats showed a significant effect due to exposure X time (Table 2 ). Data forf, T K , T e , VO 2 , and FCO 2 were similar among the treatment groups. Time-related effects were detected in measurements of V T , V lmmx , and V CimjL in rats examined in Procedure I and in V T for those examined in Procedure II. Body weights among the various temporal groups of rats differed because of differences in housing between facilities and the fact that they derived from different breeding groups, given the number of animals needed to conduct the study. When checked against growth charts provided by the breeder, all groups were within 95% of their normal weight range.

Twelve-and 18-month air-exposed and air-challenged rats (12-A/air and 18-A/air, respectively) served as procedural controls. During the first hour of challenge, A/and AV T data were not statistically different between 12-A/air and 18-A/air rats. These air challenge data sets were plotted and are enclosed within the gray bands in Fig. 2 . The boundaries of these bands represent ± 1 SE around the mean value. Since only the 12-month rats were challenged for 2 hr, the gray bands represent 12-A/air data during the second hour. Although not included in the plots, the A/and AV T data (mean ± 1 SE) obtained from 12-OZ/air rats also fitted within the respective gray bands over the 2-hr challenge period. The procedural control data indicate that as rats were held in the challenge apparatus and breathing air, they experienced a slight increase in / and a more pronounced but gradual decrease in V T over the 2-hr period. The fact that the 12-OZ/air rat data also fitted within the band suggests that the 12-month O 3 exposure did not influence the procedural effects on A/or A V T . show the range of data (± 1 SE around the mean) obtained from procedural control rats (12-and 18-A/air). All groups are represented during the first hour, and data collected during this period were analyzed. The A/and AV T responses were significantly attenuated in 12-and 18-OZ/O 3 rats after 50 min (*). Significant differences (+) were also found between age groups in that 12-A/Oj rats were less responsive to O 3 than 18-A/O 3 rats. *p < 0.05; + p «: 0.05. Numbers in parentheses are the number of rats per group.

The A/and AV T responses to 1.0 ppm O 3 in Procedure I for 12-and 18-month rats (i.e., 12-A/O 3 , 12-OZ/O 3 , 18-A/ O 3 , 18-OZ/O 3 ) are also plotted in Fig. 2 . After 50 min of challenge, both the A/ (Fig. 2a) and AV T (Fig. 2b ) responses were significantly attenuated in 12-OZ/O 3 and 18-OZ/O 3 rats when compared to their respective 12-A/O 3 and 18-A/ O 3 groups. These results indicate that chronic O 3 exposure induced adaptation. Significant differences in response to the O 3 challenge were found in both the A/and AV T responses between 12-A/O 3 and I8-A/O3 rats, indicating that the 12-month rats were less sensitive to the O 3 challenge than 18-month rats.

In Fig. 2 the plots show that by the end of the second hour the A/and A V T responses were comparable in magnitude in both treatment groups (i.e., 12-A/O 3 and 12-OZ/O 3 ) that were represented during that period. These data indicate that the diminished O 3 sensitivity, seen during the first hour in the 12-OZ/O 3 group, could be overcome with continued high-level O 3 exposure.

To examine this apparent adaptation more closely, plots using the A/response were constructed for individual rats (Figs. 3a and 3b) . These plots show that, by the end of the first hour, ah 1 of the 12-A/O 3 and 18-A/O 3 rats (Fig. 3a) but none of the 12-OZ/O 3 and 18-OZ/O 3 rats (Fig. 3b) experienced an increase in A/ By the end of the second hour, while the 12-A/O 3 rats continued to exhibit a strong response, only two of the four 12-OZ/O 3 rats (animals D and C) exhibited a comparable response. Interestingly, rats A and B remained resistant to the challenge for up to 2 hr.

To see if any or all of these apparent differences in O 3 sensitivity could be attributed to the accumulation of the O 3 dose during the challenge, the A/data were plotted against the cumulative dose of O 3 for individual rats (Figs. 4a and  4b) . These plots indicate that, when the cumulative O 3 dose is taken into account, the 12-A/O 3 and 18-A/O 3 rats are similar in O 3 sensitivity (Fig. 4a ). In addition, the two rats that failed to show a response with 2 hr of challenge (rats A and B in Fig. 3b ) did not accumulate as much O 3 during the 2-hr time period as the responders (rats C and D).

Cumulative dose-response curve data, over the first hour of the challenge for rats in the 12-and 18-month exposure groups, were tested (12- show Af{f, breathing frequency) data for individual rats over time using a 1.0-ppm O 3 challenge, (a) By the end of the first hour, all eight 12-and 18-A/O 3 rats had responded and none of the 12-or 18-OZ/Oj rats had responded to the challenge, (b) Rats A and B did not respond even after 2 hr. The two rats that did respond during the second hour, C and D, responded with the same vigor (rate and magnitude) as rats in (b). Three out of the four I8-A/O 3 rats in (a) responded before the 12-A/O 3 rats. cumulative dose reached 20-23 ug. Response parameters AK E , AT,, Ar e , AK W and AK W AVO 2 , and AKCO 2 were also tested within the dose-response analysis (data not shown). Among these parameters, significant attenuation in response was found in AT,, AT C , and AK^ in rats preexposed to O 3 . In addition, attenuation in response was always seen at the same cumulative dose levels.

In Procedure II, CO 2 administered during the O 3 challenge to stimulate breathing was effective in all four groups (i.e., 18-A/O 3 + CO 2 , 18-OZ/O 3 + CO 2 , 18-OZ-R/O 3 + CO 2 , and I8-A-R/O3 + CO 2 ) (Fig. 6 ). Analysis determined that cumulative ventilation (F E X time) was equivalent among the groups.

The response to O 3 challenge was attenuated in the 18-OZ/O 3 + CO 2 rats as compared to 18-A/O 3 + CO 2 rats for both A/and AV r (Fig. 7) . Analogous plots of AT U AT e , and AKe^ (not shown) showed the same attenuation in the 18-OZ/O 3 + CO 2 rats. However, analysis determined that none of these differences reached statistical significance. After 4 months of recovery, there was no evidence of O 3 adaptation in 18-OZ-R/O 3 + CO 2 rats (Fig. 7) .

Acute O 3 responses in Procedure II rats were reexamined using a dose-response approach similar to that used for the Procedure I rats (as discussed above). These data, represented by A/and AK T , are shown in Fig. 8 . Both responses were significantly attenuated in the 18-OZ/O 3 + CO 2 group 

This work provides evidence indicating that long-term exposure of laboratory rats to O 3 at concentrations found in the urban environment can induce O 3 adaptation. Our findings complement results from two human studies that re-ported decreased responsiveness to O 3 , using spirometric measurements, in subjects living in Los Angeles (Hackney et al, 1977a; Linn et al, 1988) . These authors speculated that their subjects had probably acquired some degree of adaptation after exposure to the high ambient summer levels of O 3 that often occur in this city. How closely the rat and human responses parallel each other is not known. However, among humans, rats, and other laboratory animals, there are a number of common lung responses to provocative concentrations of O 3 . In our study, adaptation was detected using some of these common responses, and the testing for adaptation was done, as it is in human subjects, in unanesthetized spontaneously breathing rats. Thus, the adaptation found in our rats may reflect the adaptation seen in humans after long-term urban exposure. We assessed responsiveness to O 3 in rats by acutely challenging them to a provocative concentration of O 3 and measuring O 3 -induced changes in spontaneous breathing parameters, primarily the increase in/and the decrease in V T . These O 3 responses are common to both humans (Folinsbee et al, 1975 (Folinsbee et al, , 1977 and animals (Lee et al, 1979; Murphy et al., 1964; .

After the rats were chronically exposed for 12 or 18 months to an urban profile of O 3 concentrations, their O 3 responsiveness was significantly attenuated, indicating that they had adapted to O 3 . Attenuation was seen in measures off, V T ,T i ,T e , and F w but not in V E , K w VO 2 , or KCO 2 . Similar results were obtained from challenge procedures I and II using parallel groups of chronically exposed animals.

In this study, neither body weight nor spontaneous breathing parameters were affected by the chronic exposure to the urban-like profile of O 3 . A previous report concerning rats from the same chronic exposure groups indicated that pulmonary function was compromised by the exposure . In that report, a cohort of unanesthetized rats, each with an intrapleural catheter surgically implanted 1 day prior to testing, was evaluated after 1, 3, 13, 52, and 78 weeks of the same urban profile O 3 exposure. In the 18-OZ/O, + CO 2 group, both responses were attenuated following the chronic exposure, although differences between 18-OZ/O 3 + CO 2 and 18-A/O3 + CO 2 rats did not reach significance. Recovered rats (18-OZ-R/O3 + CO 2 ) showed no effect of the prior O 3 exposure. Results are expressed as means ± SE.

These rats were challenged with 4 and 8% CO 2 in the same apparatus used in the present study (Procedure II). Tepper et al. (1991) found an overall increase in expiratory resistance and a T^-dependent reduction in / In the present study, resistance was not measured (because pleural catheters were not implanted), but a ^-dependent reduction in/ was observed, although it was not statistically significant. This difference may have been due to the difference in data analysis which included the CO 2 -produced effects and/or the introduction of an intrapleural catheter for measurement of breathing mechanics in the study by Tepper et al. (1991) . A variety of testing methodologies and endpoints have been used to assess adaptation in both animals and humans. There is no clear evidence that adaptation in terms of the spontaneous breathing parameters correlates with, or is mechanistically similar to, spirometrically determined adaptation. There is, however, some evidence that during a provocative O 3 challenge in humans, an increase in/and a decrease in V r occur concurrently with the decrements in the spirometric measurements. In humans, Folinsbee et al. (1977) showed that O 3 exposure caused changes in spontaneous breathing parameters as well as changes in pulmonary function. Subjects exposed to 0.75 ppm O 3 for 2 hr (15-min light exercise alternated with 15-min rest) showed a marked increase in /and a decrease in K T along with significant reductions in forced vital capacity, forced expiratory volume in 1 sec, maximal expiratory flow at 50% of vital capacity, expiratory reserve volume, and inspiratory capacity. Adaptation in rats (Tepper et al., 1989 ) has been previously expressed as an attenuation in O 3 -induced changes in /and V T using daily repeated exposures to 0.5 ppm O 3 . Changes of forced expiratory data, similar to spirometric changes observed in humans, were also observed. The fact that acute exposure to high concentrations of O 3 causes similar changes in/and V T in both humans and animals suggests that measurements of spontaneous respiratory responses may be as useful as the spirometric or the airway responsiveness measurements in assessing O 3 adaptation. In humans, Folinsbee et al. (1980) exposed subjects to 0.5 ppm O 3 on a daily basis and observed reduced effects in spirometric parameters by the third day and no effects on the fourth day. However, they found no significant changes in the spontaneous respiratory pattern that could be attributed to O 3 .

Rats exposed to urban-type O 3 for 18 months and then recovered for 4 months breathing filtered air showed no evidence of O 3 adaptation. In humans, environmentally induced attenuation in O 3 effects may be more persistent. Linn et al. (1988) found that the attenuated O 3 responses seen in sensitive subjects in the autumn (late in the high O 3 season) were still attenuated in the winter. Yet Horvath et al. (1981) , testing subjects exposed to 0.5 ppm O 3 on 5 consecutive days and adapted by the fifth day, found that adaptation lasted, on average, less than 2 weeks and was shortest for the more sensitive subjects. A study in our laboratory showed that adaptation in rats lasted for at least 1 week .

At the present time, mechanisms providing O 3 adaptation in humans or in animals have not been wholly explained by either cellular or biochemical studies, although adaptative changes have been reported in these lung parameters. In humans, Devlin et al. (1993) showed that acute exposure (0.4 ppm O 3 , 2 hr/day)-induced inflammation, measured by the presence of neutrophils in BAL fluid, was completely attenuated by the fifth consecutive exposure day. Other endpoints (e.g., increases in lactic acid dehydrogenase and elastase content in BAL fluid and a decrease in macrophage phagocytosis) were attenuated but to a lesser degree. In mice, Canning et al. (1991) found that O 3 -induced reduction in the phagocytic activity of macrophages and increased levels of protein and prostaglandins, recoverable in BAL fluid, tend to resolve with continued O 3 exposure (0.5 ppm O 3 for 14 days). In rats exposed to 0.5 ppm for 5 days, Tepper et al. (1989) reported that even though the respiratory responses to O 3 adapted with repeated exposure over time, there was a progressive pattern of lung epithelial damage, inflammation in terminal airways, and a sustained increase in bronchoalveolar lavageable protein. Van Bree et al. (1989) , however, examined biochemical and cellular changes in the lungs of rats exposed to 0.4 ppm O 3 for 12 hr for 3 consecutive nights and found that the acute pulmonary injury and inflammation, seen after the first exposure night, were reversible despite continuous exposures.

Adaptation appears to be a series of physiological and biochemical adjustments, shared by a number of species. Although there has been much speculation about various mechanisms that may contribute to O 3 adaptation, data from some studies suggest that antioxidants may play a role. Tepper et al. (1989) found that lung ascorbate and glutathione were increased in the BAL fluid from O 3adapted rats. Jackson and Frank (1984) observed that preexposure of rats to 0.8 ppm O 3 for 7 days made the animals cross-tolerant to hyperoxia. This tolerance corresponded with an increase in lung tissue activity of antioxidants [i.e., superoxide dismutase (SOD), glutathione peroxidase, glucose 6-phosphate dehydrogenase, and catalase]. In the present study, rats chronically exposed for 12 months had significantly increased concentrations of glutathione in whole lung homogenate; however, SOD and nonprotein sulfhydryls were not affected (Grose et al., 1989) . In addition, Norwood et al. (1989) found that antioxidants in BAL fluid and in lung tissue were changed in rats from these same exposures. Specifically, a-tocopherol in surfactant was decreased after 12 and 18 months, but was increased in lavaged cells and in lung tissue homogenate after 18 months. The ascorbic acid/protein ratio was increased by 85% in lavaged cells after 12 months and by 151% after 18 months. This ratio was also increased in the BAL fluid itself by 133% after 18 months. Wiester et al. (1991) recently reported data suggesting a relationship between O 3 adaptation in rats and an increase in ascorbic acid levels in the BAL fluid.

In summary, the rats in this study developed statistically significant O 3 adaptation after long-term exposure to an urban-type O 3 profile. These changes were apparent without concurrent O 3 -associated changes in body weight or resting breathing parameters. This adaptation appeared to be reversible because it was not seen after rats had been allowed to recover for 4 months in filtered air. Adaptation was most clearly observed when the data were normalized for the cumulative O 3 dose, suggesting that some type of competitive antioxidant mechanism may be induced with chronic exposure that is overcome with a sufficient challenge exposure.

Ozone reduces murine alveolar and peritoneal macrophage phagocytosis: The role of prostanoids

Evaluating the toxicity of urban patterns of oxidant gases. 1. An automated chronic gaseous animal inhalation exposure facility

Attenuation of cellular and biochemical changes in the lungs of humans exposed to ozone for five consecutive days

Threshold concentration of ozone causing an increase in bronchial reactivity in humans and adaptation with repeated exposures

Adaptation in human subjects to the effects of inhaled ozone after repeated exposure

40CFR Part 50

Respiratory responses in humans repeatedly exposed to low concentrations of ozone

Pulmonary function and airway responsiveness after repeated prolonged exposure to 0.12 ppm ozone

Exercise responses following ozone exposure

Decrease of maximum work performance following ozone exposure

The impact of a 12-month exposure to a diurnal pattern of ozone on pulmonary function, antioxidant biochemistry and immunology

Effects of ozone exposure in Canadians and Southern Californians

Adaption to short term respiratory effects of ozone in men exposed repeatedly

Adaptation to ozone: Duration of effect

Ozone-induced tolerance to hyperoxia in rats

Mechanism of rapid, shallow breathing after ozone exposure in conscious dogs

Repeated laboratory ozone exposures of volunteer Los Angeles residents: An apparent seasonal variation in response

Altered function in animals inhaling low concentrations of ozone and nitrogen dioxide

Biochemical and metabolic changes in the lung with oxygen, ozone, and nitrogen dioxide toxicity

The effect of chronic ozone (O 3 ) exposure on bronchoalveolar lavage (BAL) supernatant, cell, and whole lung antioxidants

Unattenuated structural and biochemical alterations in the rat lung during functional adaptation to ozone

The use of carbon dioxide challenge to detect toxicant-induced changes in cardiopulmonary function of awake rats

Chronic exposure to a simulated urban profile of ozone alters ventilatory responses to carbon dioxide challenge in rats

Air Quality Criteria for Ozone and Other Photochemical Oxidants

Pathobiochemical effects in rat lung related to episodic ozone exposure

Ozone tolerance (O 3 T) in the F-344 rat: Duration and lavage fluid correlates

Comparative study of ozone (O 3 ) uptake in three strains of rats and in the guinea pig

A restraining system for plethysmography in small animals

Ozone uptake in awake Sprague-Dawley rats

The authors thank Dr. G. E. Hatch, Mr. J. A. Raub, and Dr. J. A. Dye for their critical review of the manuscript and J. A. Cook for her secretarial services. Presented in part at the 28th annual meeting of the Society of Toxicology, held in Atlanta, Georgia in 1989.