Ozone adaptation in mice and its association with ascorbic acid in the lung.

We have previously shown that ozone (O(3)) adaptation occurred in rats after daily exposure to an "urban-type" concentration. The adaptation was positively associated with an excess of ascorbic acid (AA) in bronchoalveolar lavage fluid (BALF), suggesting that AA may play a role in the adaptation mechanism. This relationship was not seen at higher and more toxic exposures. The present work exposed mice to low and high levels of O(3) to see if the adaptation-AA relationship is common among rodent species. Male CD-1 mice were studied during repeated 6-h/day exposures to 0.0 or 0.25 ppm O(3) for 10 days and 10 days of recovery in air (experiment 1) and to 0.0, 0.5, or 1.0 ppm O(3) for 5 days (experiment 2). Approximately 20 h after each daily exposure, groups of mice were randomly selected from each concentration type and examined for patterns of response. They were anesthetized (urethane, ip), intubated, and the lungs were lavaged with 37 degrees C saline. BALF was assayed for cells, cell differential, protein, albumin, lactate dehydrogenase, lysozymes, N-acetyl-beta-D-glucosaminidase, gamma-glutamyl transferase, uric acid, glutathione, and AA. Body weight and total lung capacity were also measured. Mice from experiment 1 (10/exposure) were tested for adaptation on day 12 by challenging them with 1.0 ppm O(3) for 6 h and collecting BALF 20 h later. In experiment 2, adaptation was assessed by evaluating the attenuation in response to continued exposure. There was only minimal response to the daily O(3) exposures in experiment 1 except for AA, which was significantly increased in BALF by day 3 and remained elevated well into the recovery period. The O(3)-preexposed mice demonstrated adaptation when compared to their O(3)-naive counterparts. Daily exposure to 1. 0 ppm O(3) in experiment 2 caused weight loss and changes in BALF consistent with toxicity, and neither adaptation nor an excess quantity of AA was seen. The findings in mice were in agreement with those seen in rats and suggest that there may be a common O(3) adaptation mechanism among rodents that involves the regulation of AA in lung lining fluid.

Impact of the hypothermic response in inhalation toxicology studies.

Previous studies from this laboratory showed that the decreases in Tco and associated functional parameters often observed in rodents following exposure to xenobiotic agents are capable of modulating the subsequent toxic response and that the magnitude of this induced hypothermic response may itself be modified by a number of experimental conditions. A moderate hypothermic response, characterized by a temperature drop of approximately 2 degrees C, appears to afford the optimal protection. Studies in which exposures occur through inhalation of harmful gases or particles present a special set of problems. In such studies, the dose of the toxic agent to which the animal is exposed is a function of the concentration of the agent in the atmosphere and the minute ventilation of the animal. Although ambient concentrations is generally held constant in laboratory studies, minute ventilation varies directly with metabolism, and both of these parameters may change significantly across experimental conditions. Thus, at low Tas, metabolism and minute ventilation are relatively high and uptake of inhalable toxic agents is increased. However, the development of the hypothermic response during the exposure entails a directly correlated reduction in these parameters and, presumably, in dose. For the most part, inhalation toxicological studies are conducted using resting animals or exercising humans. Animals are sometimes concurrently exposed to CO2 to simulate the increased ventilation of exercise and more closely mimic human studies. The experimental protocols employed in the above inhalation studies permitted examination of (1) the impact of species, size, handling stress, and changes in Ta on both the induced hypothermic response and the concomitant pulmonary toxicity; (2) the additive impact of exercise stress on O3 toxicity; and (3) the toxicity of ambient-derived particulate matter in normal rats and in rats with preexisting pulmonary inflammation. The results of these studies demonstrate that the magnitude of the induced hypothermic response is directly proportional to the uptake of the toxic agent by the lung and inversely proportional to the mass of the animal and the ambient temperature at which the exposure is conducted. The hypothermic response is sensitive to a number of experimental stresses including handling and changes in cage conditions. Exercise attenuates the hypothermic response, whereas CO2-stimulated increases in ventilation employed as an exercise surrogate may potentiate the response. Toxic exposures conducted in animals with lung disease or compromised pulmonary function may induce a severe hypothermic response while comparable exposures in normal animals produce only mild or moderate responses. In general, the development of the hypothermic response in the presence of ambient pollutants serves to decrease the minute ventilation of the animal and therefore limits the uptake and dose of the airborne toxicant. The results of these inhalation studies support our previous conclusions concerning the impact of the hypothermic response on toxicity and emphasize the need to monitor and incorporate these changes in functional parameters into analyses of toxicological data. Furthermore, because humans do not demonstrate a robust hypothermic response following exposure to toxic agents, extrapolation of the results obtained from animal studies and comparisons with data from human studies are considerably more complicated.

Ozone toxicity in the rat. III. Effect of changes in ambient temperature on pulmonary parameters.

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 degrees 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 degrees 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 with exposure at 34 degrees 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.

Adaptation to ozone in rats and its association with ascorbic acid in the lung.

Ozone (O3) adaptation is a well-known, but poorly understood phenomenon that has been demonstrated in humans and laboratory animals. This study examined pulmonary function and bronchoalveolar lavage fluid (BALF) parameters in O3-adapted F-344 rats to explore possible mechanisms of adaptation. Of particular interest was ascorbic acid (AA), an antioxidant reported to be protective against O3 injury and found to be increased in O3-adapted rats. Adaptation was induced by exposure to 0.25 ppm O3, 12 hr/day for 6 or 14 weeks and evaluated with a challenge test, one that reexposed rats to 1.0 ppm O3 and measured attenuation in the O3 effect on frequency of breathing. Pulmonary function was assessed 1 day postexposure and adaptation and BALF were evaluated 1, 3, and 7 days postexposure. Results showed that forced vital capacity increased over time but decreased due to exposure and that the 14-week, O3-exposed rats had an increase in forced expiratory flow rate. All of the O3-exposed rats that were tested demonstrated adaptation on Postexposure Days 1, 3, and 7, but it was diminished on Day 7. Adaptation was also more pronounced in rats exposed for 14 weeks. Except for AA, BALF levels of total protein, potassium, lysozyme, uric acid, and alpha-tocopherol were unaffected by O3 exposure. Lactic acid dehydrogenase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, and total glutathione were also assayed but were always below detectable limits. Ascorbic acid concentrations were elevated on Days 1, 3, and 7, showing postexposure patterns similar to those found for adaptation. Significant correlation was found between AA concentration and the magnitude of adaptation (r = 0.91, p < 0.002). We conclude that AA may play an important role in mechanisms associated with O3 adaptation in rats.

Ozone uptake in healthy adult males during quiet breathing.

Experimental measurements of ozone (O3) uptake are needed for validation of dosimetry model parameters and in predictions as well as for determining factors affecting uptake and for making comparisons between subpopulations or across species. In this study, 10 healthy adult male subjects were exposed to 0.3 ppm O3 while seated and breathing naturally through the nose or mouth. Total respiratory tract O3 uptake, spontaneous breathing parameters, and respiratory gas exchange were measured for 10 min under steady-state conditions. The exposure protocol was replicated in each subject approximately 2 weeks after the first visit. On each visit, health exams were performed and spirometric lung measurements were obtained. The experimental design provided comparisons of total O3 uptake during nasal and oral breathing, differences in uptake in an individual at two time points, and an examination of between-subject variability in O3 uptake. Exposure to O3 had no effect on the breathing parameters or gas exchange. Oral and nasal breathing frequency averaged 16.2 +/- 1.1 (SE) and 16.0 +/- 1.2 breaths per minute with tidal volumes averaging 651 +/- 46 and 669 +/- 67 ml, respectively. A significant correlation (p < 0.01) was found for the minute volume during resting breathing with the percentage of uptake. The percentage of O3 uptake was consistently higher (p = 0.02) during oral breathing (76.5% +/- 3.3) than during nasal breathing (73.1% +/- 3.0) although this difference may not be biologically significant. The variability in percentage of uptake between subjects was substantial with calculated uptakes ranging from 51 to 96%, a difference of about 45%. Variability in percentage of uptake for an individual was less with the maximal difference between the first and second visits being about 20%; the average difference, however, was only about 3%. We conclude that total percentage of O3 uptake is approximately 75% in adult males during resting breathing. It is slightly greater during oral than during nasal breathing, will vary considerably among subjects, and is moderately reproducible within a subject.

Ozone toxicity in the rat. I. Effect of changes in ambient temperature on extrapulmonary physiological parameters.

These studies examined the effects of exposure to near environmental levels of ozone (O3) on the unanesthetized unrestrained rat as well as the influence of changes in ambient temperature (Ta) on the observed responses. Male Fischer 344 rats were implanted with radiotelemetry transmitters that permitted continuous monitoring of electrocardiogram, heart rate, core body temperature (Tco), and activity. Telemetry animals (n = 4-5/group) were combined with nontelemetry animals to produce nine treatment groups (n = 44-50/group) composed of combinations of one of three O3 exposure regimens (0.0 ppm x 24 h/day, 0.5 ppm x 6 h/day, or 0.5 ppm x 23 h/day) paired with one of three Ta levels (10, 22, or 34 degrees C). The experimental protocol consisted of a Control Period (filtered air; 1 day), Treatment Period (O3; 5 days), and Recovery Period (filtered air; 7 days). At specific intervals during the experiment, subgroups (n = 6) of nontelemetry animals were randomly selected from each treatment group, anesthetized with urethan, and intubated, and their lungs were lavaged with warm saline. In general, results from the bronchoalveolar lavage procedure indicated that toxicity increased in magnitude and duration as the length of time of O3 exposure increased and the Ta decreased. Similarly, whereas minimal extrapulmonary effects were observed at an Ta of 34 degrees C, O3 exposures at Ta levels of 22 and 10 degrees C produced significant decreases in heart rate (160 and 210 beats/min, respectively), Tco (2.0 and 3.5 degrees C, respectively), and body weight (15 and 40 g, respectively). Decreases in these functional parameters reached their maxima over the first 2 exposure days and returned to control levels after the 3rd day of exposure. These data demonstrate the profound impact of Ta on Tco and other extrapulmonary parameters in the conscious unrestrained rat exposed to O3. Furthermore, these results suggest an integral role for both Tco and Ta in determination of the uptake of inhaled pollutants and modulation of the subsequent toxic effects and may have important implications with respect to the assessment of toxic risk.

Ozone adaptation in rats after chronic exposure to a simulated urban profile of ozone.

Studies in both humans and rats have indicated that certain pulmonary responses induced by exposure to an acute provocative concentration of ozone (O3) will eventually attenuate if the exposure is repeated on a daily basis. This phenomenon is commonly referred to as O3 adaptation. Whether or not a "state" of adaptation develops due to long-term low level O3 exposure is unknown. Two human studies have reported adaptation in subjects living in Los Angeles during periods when ambient O3 concentrations have been relatively high. At present, however, we are not aware of comparable information from rats. This study assessed O3 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 O3 for 13 hr/day, 7 days/week; Monday-Friday, peak to 0.25 ppm O3, over 9 hr), was used. To assess whether adaptation had occurred and/or persisted, awake rats were challenged with high provocative concentrations of O3 for up to 2 hr. During a challenge, rats were monitored for typical O3-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 O3 as compared to that in "control" rats (chronically exposed to air). Adaptation was found in the rats within 8 hr following the chronic O3 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 O3 after long-term exposure to an urban-type O3 profile and that the adaptation was not seen 4 months postexposure. These results suggest that exposure to environmental O3 in Los Angeles air may have been responsible for the adaptation found in residential subjects.

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

Male Fischer 344 rats were exposed to a simulated urban profile of ozone (O3) (9-hr ramped spike, integrated concentration = 0.19 ppm) for up to 78 weeks. Small, but statistically significant, changes in breathing patterns and mechanics in unanesthetized, restrained rats were observed at Weeks 1, 3, 13, 52, and 78 during postexposure challenge with 0, 4, and 8% carbon dioxide (CO2). The data indicate that O3 exposure caused an overall increase in expiratory resistance (Rc), but particularly at 78 weeks. This increase in Rc most likely accounts for the rats' reduced ability to increase ventilation during CO2 challenge compared to control rats. Reductions in CO2-induced tidal volume increases were observed in all O3-exposed animals during postexposure challenges to 4 and 8% CO2. Cumulatively, over all time points, spontaneous frequency of breathing and CO2-induced hyperventilation were also reduced. The decrease in frequency was dependent on a significant increase in the inspiratory time relative to control without a change in expiratory time. Light microscopic evaluation of the lung did not reveal any lesions associated with O3 exposure at any time point. Although statistically significant effects were detected, the etiology of the above-mentioned functional changes remains speculative. The potential relevance of these data to acute and chronic O3 exposure in humans is also discussed.

Histamine and methacholine aerosol bronchial challenge in awake guinea pigs.

Cardiopulmonary responses to histamine and methacholine aerosol challenge were examined and compared in unanesthetized spontaneously breathing guinea pigs. All animals had surgically implanted intrapleural catheters and some had arterial or right heart catheters as well. Animals were placed in plethysmographs and exposed to ascending doses of aerosolized agonists. The provocative dose, i.e., the dose that caused a clear bronchospastic response, was defined as the concentration that increased intrapleural pressure to at least 20 cm H2O. Results showed that bronchospasm was characterized further by decreases in dynamic lung compliance and arterial PO2 and an increase in airway resistance. The accumulation of trapped gas in the lung, after challenge, measured in the excised collapsed lung correlated with the increase in intrapleural pressure and with the fall in lung compliance. Male guinea pigs between 3- and 15-weeks of age did not vary in sensitivity to histamine. Methacholine and histamine were equipotent and produced similar responses. Tachyphylaxis was not demonstrated for either compound. In addition, the provocative dose for the drugs remained stable on a day-to-day basis. These results offer insight into the nature of the bronchospastic response in guinea pigs and provide new data on response to methacholine.

Measurements of cardiopulmonary response in awake rats during acute exposure to near-ambient concentrations of ozone.

Cardiopulmonary responses during acute exposure to near-ambient (less than or equal to 1.0 ppm) concentrations of ozone (O3) have not been reported for the unanesthetized rat. Such data on species sensitivity are crucial for the extrapolation of animal data to man. Therefore, this study was conducted to obtain functional measurements on awake rats using head-out plethysmographs and intrapleural or carotid artery catheters during a 135-min exposure to 0.0, 0.12, 0.25, 0.5 or 1.0 ppm O3. Carbon dioxide was added during alternate 15-min periods of the exposure to increase ventilation, much like the use of exercise in human O3 exposure studies. The results established that frequency of breathing was increased and tidal volume was decreased as a function of both the concentration and the duration of exposure. Breathing mechanics and cardiopulmonary measures were only marginally affected. Differences in the response of individual rats revealed that as O3 concentration increased, the proportion of rats responding and the magnitude of the response was increased. These data indicate that, for similar functional responses, the rat's sensitivity to O3 is comparable to that observed in man.

Comparative study of ozone (O3) uptake in three strains of rats and in the guinea pig.

Ozone uptake was assessed in awake, spontaneously breathing Fischer-344 Sprague-Dawley, and Long-Evans rats and Hartley guinea pigs to provide data on the dosimetry of O3 in small laboratory animals. This information is needed for extrapolation of O3 toxicity data from experimental animals to man. Breathing measurements and O3 exposure data were obtained using a head-out body plethysmograph connected to a nose-only exposure system. The fractional uptake of O3 was computed by measuring flow and O3 concentration both upstream and downstream from the nose. The quantity of O3 removed by the animal, O2 consumption, CO2 production, and tidal breathing measurements were automatically recorded once each minute. All animal types were exposed for 1 hr to 0.3 ppm O3. Other Fischer-344 rats were also exposed for 1 hr to 0.0 or to 0.6 ppm O3. Exposure concentrations of O3 had no significant effect on percentage O3 uptake in Fischer-344 rats. Results showed that percentage O3 uptake (47%) did not differ significantly among the three strains of rats nor between the rats and the guinea pigs. Similarly, percentage O3 uptake was independent of animal age, lung weight, and lung volume as well as normal variations encountered in the resting breathing measures. However, species-specific ventilation and O3 concentration were the primary determinants of the accumulated lung dose of O3 during the exposures.

Ozone uptake in awake Sprague-Dawley rats.

Knowledge of ozone (O3) uptake in rats in integral to efforts to quantitatively extrapolate animal data to man. We have measured percentage O3 uptake in 30 adult Sprague-Dawley rats exposed, nose only, for 1 hr to 0.3, 0.6, or 1.0 ppm O3. Tidal volume and breathing rate measurements, obtained by plethysmography, were time-matched with changes in airstream concentrations of O3, oxygen, and carbon dioxide, due to animal breathing, for computation of O3 uptake and respiratory gas exchange values. Ozone uptake was calculated by a fractional deposition technique, i.e. O3 in vs O3 out. Total respiratory system O3 uptake was found to be approximately 40% of that inspired and did not vary with O3 concentration nor during the 1-hr exposure. The quantity of O3 inhaled and retained by the rat increased proportionally with O3 concentration.

Cardiopulmonary effects in awake rats four and six months after exposure to methyl isocyanate.

Cardiopulmonary function was assessed four and six months after Fischer 344 rats were exposed to 2 hr to 0, 3, or 10 ppm methyl isocyanate (MIC). During assessment, the rats were challenged with 4 and 8% carbon dioxide (CO2) to stimulate ventilatory drive. Minute ventilation (VE) during CO2 challenge was increased in MIC-treated rats compared to controls when examined 4 months after exposure to 10 ppm MIC, suggesting a ventilation/perfusion inequality. An increase in maximum expiratory flow and a decrease in expiratory time indicated increased lung recoil in these rats. Evidence of pulmonary hypertension was observed in electrocardiograms (ECGs) and supported by postmortem analysis that showed a positive association between increased ECG abnormalities and increased right ventricular weights in the rats treated with 10 ppm MIC. At 6 months, forced expiratory flow-volume curves indicated persistent airway obstruction; however, no changes in inspiratory or expiratory resistance were evident. Decreased dynamic compliance and changes in two new measures of lung function (volume and time at zero expiratory intrapleural pressure) suggest that MIC-induced lung dysfunction also exhibited elements of a restrictive disease.

Inhalation studies of Mt. St. Helens volcanic ash in animals: respiratory mechanics, airway reactivity and deposition.

Effects of fine volcanic ash aerosol on pulmonary mechanical properties of awake guinea pigs were evaluated during exposure by inhalation. Ash penetration into the lung as well as tissue response to ash were determined by transmission electron microscopy. The reactivity of airway epithelial irritant receptors following ash exposure was assessed using a histamine bronchoprovocation test. Results indicated that breathing 9,4 mg/m3 of ash for 2 hr did not cause a measurable change in pulmonary function of guinea pigs. Electron micrographs showed that ash particles in the lung below the hilus did not seem to produce any acute tissue reaction and were almost all phagocytized by macrophages. Airways of guinea pigs exposed to ash were significantly less responsive to histamine than were the airways of animals exposed only to air. It appears that even though Mt. St. Helens ash was well tolerated by the guinea pig during the exposure, its presence in the inhaled air did change the "histamine sensitivity" of airway epithelial irritant receptors.

Inhalation studies of Mt. St. Helens volcanic ash in animals : Respiratory mechanics, airway reactivity and deposition

This study was conducted to improve speculation of changes in breathing which might be expected while inhaling a dust like Mt. St. Helens volcanic ash. Effects of fine volcanic ash aerosol on pulmonary mechanical properties of awake Guinea pigs were evaluated during exposure by in halation. Ash penetration into the lung as well as tissue response to ash were determinated by transmission electron mycroscopy. The reactivity of airway epithelial irritant receptors following ash exposure was assessed using a histamine bronchoprovocation test. Airways of Guinea pigs exposed to ash were significantly less responsible to histamine tan were the airways of animals exposed only to air (AU)

Diastolic and systolic blood pressure measurements in monkeys determined by a noninvasive tail-cuff technique.

A noninvasive method to determine systolic, diastolic, and mean blood pressure values in rhesus monkeys is described and evaluated. Simultaneous measurements of abdonimal aortic and tail-cuff pressures were compared for unanesthetized, anesthetized, hemorrhaged, and methoxamine-treated animals. Data indicated that correlation between direct and indirect methods were highly significant (r greater than or equal to 0.94); the mean differences found between the two measurements were less than or equal to 8 mm. Hg, with the exception of the methoxamine-treated groups. Reliability of the technique was considerably reduced following administration of this drug.

Cardiovascular actions of palladium compounds in the unanesthetized rat.

The natural occurrence of palladium (Pd) is rare. It is obtained as a by-product during the extraction of platinum and, until the present time, its uses were small in number--principally in the manufacture of jewelry, dental alloys, chemical catalysts, and electrical contacts. Interest in Pd toxicology has been limited to occupational exposures and even that has been sparse. Recently Pd has been chosen as one of the metals to be incorporated into the automotive catalytic converter material. Palladium chloride has been shown to be extremely toxic when given to rabbits intravenously. Animals rapidly injected quickly died with damage chiefly to the heart. This heart damage was not explained or further defined, and other references addressing this subject have not been found. The objective of our study was to investigate and attempt to characterize toxic actions of soluble Pd compounds on the cardiovascular system of the unanesthetized rat. Surgically prepared rats were monitored for ECG, aortic blood pressure, cardiac contractility (dp/dt), and respiration before, during, and 1 hr following intravenous injections of Pd2+ salt solutions. Our findings indicate that injected Pd2+ profoundly disturbs the electrical integrity of ventricular myocardium. Effective doses induce cardiac arrhythmias with the consequential fall in blood pressure; the extent of the response is dose related. An immediate cardiovascular effect is seen when doses of 0.4 mg/kg or more of Pd ion as PdSO4 is administered over a 40-sec period of time via the femoral vein. Pd(NO3)2 and PdCl2 appear to have similar activity. To achieve and equal response three times as much Pd is necessary as (NH4)2PdCl4 or K2PdCl4.