Differential expression of the p27Kip1 mRNA in IFN-sensitive and resistant cell lines

STUDY OBJECTIVES: In a previous study published by our group, six out of nine subjects with mild allergic asthma were shown to have an enhanced response to allergen challenge following a 1-h exposure in an 0.8-m3 exposure chamber (modified from a body plethysmograph) to an average of 120 parts per billion (ppb) ozone at rest. Other studies failed to confirm this effect. In the present study, using a similar design, we reexamined this effect using a larger group of asthmatics and a larger chamber allowing minimal fluctuations in ozone levels during exposures. DESIGN: Prospective, randomized single-blinded crossover study. SETTING: Pulmonary function laboratory equipped with an exposure chamber. SUBJECTS: Fifteen subjects had mild allergic asthma; 9 men and 6 women; the mean (SD) age was 32.5 (10) years; FEV1 was 3.4 (0.8) L; baseline methacholine provocation concentration causing a 20% fall in FEV1 was (PC20) 3.28 (4.1) mg/mL. INTERVENTIONS: Each participant was exposed, at rest, on 1 day to filtered air and on another day to ozone (mean level=120 ppb) in a larger exposure chamber than the one used in our first study with less variability in ozone level (110 to 130 vs 85 to 175 ppb) using a random, single-blinded design. After each exposure, the subject was challenged with allergen (nine with grass pollen extract and six with ragweed extract) and allergen PC15 was measured. RESULTS: Ozone preexposure did not affect allergen PC15 when compared with clean air preexposure (allergen PC15 dilution 1/114 vs 1/119, respectively). Ozone vs air preexposure resulted in an allergen PC15 that was lower in five subjects, higher in six, and unchanged (within one doubling dose) in four. CONCLUSIONS: At this low level with less variability and lower peaks than our previous study, ozone had no significant effect on airway allergen responsiveness.     

Airway responsiveness to hyperosmolar saline challenge in cystic fibrosis: a pilot study

Hyperosmolar aerosols are used to assess airway responsiveness in subjects with asthma. Using a 10% NaCl aerosol, we investigated airway responsiveness in 23 cystic fibrosis (CF) subjects (12 females, 11 males; 19.1 +/- 3.3 years) who had asthma-like symptoms. The pre-challenge predicted forced expiratory volume in 1 second (FEV1) was 74.7 +/- 21.5. The aerosol was generated by a MistO2gen 143A ultrasonic nebulizer and inhaled for 0.5, 1, 2, 4, 8, 8, and 8 minutes or part thereof. Spirometry was performed before and 1 minute after each inhalation period. The challenge was stopped when a > or = 20% fall from the baseline FEV1 was recorded, after the last inhalation period, or when requested by the subject. We recorded different responses to 10% NaCl among subjects. In 7, the FEV1 fell progressively throughout the challenge in a manner similar to asthmatics. By contrast, in 15 subjects the FEV1 was higher at the completion of challenge compared to during challenge, i.e., the fall in FEV1 was transient. In 7 of these subjects, the final FEV1 at the end of the challenge was higher than the pre-challenge FEV1. We conclude that inhaled 10% hyperosmolar saline causes either progressive and sustained or transient airway narrowing during challenge in the majority of CF subjects. The cause of the transient airway narrowing requires further investigation.     

Regional clearance of solute from peripheral airway epithelia: recovery after sublobar exposure to ozone

The influence of local exposure to ozone (O3) on respiratory epithelial permeability of sublobar lung segments was studied by using aerosolized 99mTc-diethylenetriamine pentaacetic acid (DTPA; mol wt, 492). Two bronchoscopes were inserted through an endotracheal tube in anesthetized, mechanically ventilated, mixed breed dogs and were wedged into sublobar bronchi located in the right and left lower lobes, respectively. Segments were ventilated via the bronchoscope with 5% CO2 in air delivered at 200 ml/min, and an aerosol of 99mTc-DTPA was generated and delivered through the scope and into the sublobar segment over a 30-s period. Clearance of 99mTc-DTPA was measured simultaneously from right and left lower lung segments at baseline and 1, 7, and 14 days after a 6-h sublobar exposure to filtered air or 400 parts per billion O3. O3 treatment significantly decreased the clearance halftime (t50) of 99mTc-DTPA by 50% from the baseline mean of 32.3 to 16.0 min at 1 day postexposure. After 7 days of recovery, t50 was still reduced by 28. 8%; however, by 14 days postexposure, clearance of 99mTc-DTPA had recovered, and the t50 had a mean value of 30.0 min. 99mTc-DTPA clearance was not altered by exposure to filtered air, and t50 values were comparable to baseline at 1, 7, and 14 days postexposure. These results reveal that a single local exposure to O3 increases transepithelial clearance, but only for epithelia directly exposed to O3, and that 7-14 days of recovery are required before permeability to small-molecular-weight solutes returns to normal.     

Effects of acute passive smoking on exercise-induced bronchoconstriction in asthmatic children

This study was designed to investigate the acute effects of environmental tobacco smoke (ETS) in children with mild asthma during rest and exercise. We studied 13 children [8 males, 5 females; mean age 10 (range 8-13) yr; mean forced expired volume in 1 s (FEV1) 93% (range 82-108%) of predicted] with exercise-induced bronchoconstriction [46 +/- 4% (SE) fall in FEV1 after exercise during cold air breathing]. Children were exposed to ETS (20 ppm carbon monoxide) or ambient air (AA) for 1 h. During the first 54 min of exposure, children were at rest, and during the last 6 min they exercised on a bicycle ergometer (2 W/kg body wt). Spirometry was performed before and during exposure and after exercise. Respiratory symptoms were recorded before and after exposures. In seven children the experiments with AA and ETS were done in duplicate. FEV1 between 5 and 54 min of exposure at rest decreased by 3.2 +/- 0.8% (SE) during AA and by 7.2 +/- 2.3% during ETS exposure compared with preexposure values; the difference between AA and ETS was statistically significant (P = 0.04). The drop in FEV1 was achieved within 5 min and did not change with ongoing exposure. Analysis of individual data revealed that the mean changes during ETS were mainly effected by three children with a significant fall and one child with a significant improvement in FEV1 (P < 0.05). Maximum postexercise fall of FEV1 was 25 +/- 4% after AA and 24 +/- 3% after ETS, which did not differ significantly. Upper and lower respiratory tract symptoms were not significantly different between exposures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ozone-induced inflammation is attenuated with multiday exposure

It is well known that ozone (O3) causes acute lung inflammation. What is not known is whether there is progression of the inflammatory response in humans with repeated short-term exposures. Our study was designed to test the hypothesis that repeated exposures to a high-ambient concentration of O3 (0.2 ppm) over several days would cause more inflammation than a single exposure. Fifteen healthy volunteers were exposed in random fashion to 0.2 ppm ozone for 4 h on a single day and to 0.2 ppm O3 for 4 h on 4 consecutive days while exercising moderately for 30 min of each hour. Pulmonary function tests were obtained immediately before and after each 4-h exposure. Bronchoscopy was performed 20 h after the completion of each exposure arm to obtain bronchoalveolar lavage (BAL) for measurement of markers of inflammation. Our results show initial progression followed by attenuation of the acute physiologic response to O3 with repeated daily exposures. We found a significant difference in percent change in FEV1, FVC, and specific airway resistance (SRaw) across the single-day exposure when compared with the change across Day 4 of the 4-d exposure. Bronchial fraction (the first 15 ml of BAL return) and BAL were analyzed for the following end points: total and differential cell counts, total protein, lactate dehydrogenase (LDH), fibronectin, interleukin-6 (IL-6), interleukin-8 (IL-8), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In the bronchial fraction the number of polymorphonuclear cells (PMN)s and fibronectin concentration were significantly decreased after 4-d exposure compared with single-day exposure. In BAL, significant decreases in the number of PMNs, fibronectin, and IL-6 were found after 4-d exposure versus single-day exposure. These results suggest that there is attenuation of the O3-induced inflammatory response in both proximal airways and distal lung with repeated daily exposures.     

Glycoprotein secretion by tracheal explants cultured from rats exposed to ozone

Tracheal explants from rats exposed to 0.8 ppm (1.9 mg per m3) of ozone 8 hours per day for 1 to 90 days were incubated in culture with glucosamine labeled with carbon-14 or hydrogen-3. Compared with tracheas from control rats exposed to filtered air, the explants demonstrated a decreased rate of glycoprotein secretion for exposure intervals of as long as one week, followed by a rebound to an increased rate of glycoprotein secretion for at least 12 weeks of continued exposure to ozone. Detailed study of the behavior of labeled glycoproteins from the culture medium on chromatography on columns of BioGel A-150m demonstrated that the ratio of the low to high molecular weight peaks increased when there was an increased rate of glycoprotein secretion. This is the first report of a direct biochemical effect induced by ozone on airway metabolism.     

Exposing guinea pigs to ozone for 1 wk enhances responsiveness of rapidly adapting receptors

Acute exposure to ozone causes changes in breathing pattern and lung function which may be caused in part by stimulation of rapidly adapting receptors (RARs). The consequences of repeated daily ozone exposure on RAR responsiveness are unknown, although ozone-induced changes in pulmonary function diminish with repeated exposure. Accordingly, we investigated whether repeated daily ozone exposure diminishes the general responsiveness of RARs. Guinea pigs (n = 30) were exposed to 0.5 parts/million ozone or filtered air (8 h/day for 7 days). The animals were then anesthetized, and RAR impulse activity, dynamic compliance (Cdyn), and lung resistance were recorded at baseline and in response to four stimuli: substance P, methacholine, hyperinflation, and removal of positive end-expiratory pressure. Repeated daily ozone exposure exaggerated RAR responses to substance P, methacholine, and hyperinflation without causing physiologically relevant effects on baseline or substance P- and methacholine-induced changes in Cdyn and lung resistance. Because agonist-evoked changes in RAR activity preceded Cdyn changes, the data suggest that repeated daily ozone exposure enhances RAR responsiveness via a mechanism other than changes in Cdyn.     

In vivo salicylate hydroxylation: a potential biomarker for assessing acute ozone exposure and effects in humans

Ozone is known to yield hydroxyl radical, which may contribute to ozone-mediated lung injury. In the presence of hydroxyl radical, salicylate is hydroxylated to form 2,3-dihydroxybenzoic acid (2,3-DHBA). There is no evidence of enzymatic formation of 2,3-DHBA. We hypothesized that salicylate hydroxylation might be used as a biomarker indicating human exposure to ozone. Healthy, nonsmoking volunteers, 18 to 34 yr of age, were given acetylsalicylic acid (975 mg) or placebo orally 0.5 h before an exposure. Subjects were exposed to ozone (0.12 or 0.4 ppm) or filtered air in an environmental chamber for 2 h, while performing intermittent exercise. Results indicate significant decrements in FVC, FEV1.0, forced expiratory flows at 50% and 75% of FVC, and peak expiratory flow rate, and an increase in airway resistance, after exposure to 0.4 ppm ozone in comparison with air control (p < 0.05). Exposure to 0.4 ppm ozone also resulted in increased symptom numbers and severity (p < 0.05). When subjects were exposed to 0.12 ppm ozone, changes of pulmonary function and symptoms reported were minimal. Plasma concentration of 2,3-DHBA was significantly increased after exposure to 0.12 and 0.4 ppm ozone in comparison with air control (p < 0.05). There was a significant correlation between ozone-induced changes of pulmonary function and normalized salicylate hydroxylation (p < 0.05). The results indicate that exposure to ozone can initiate in vivo production of hydroxyl radical, a potent reactive agent. Salicylate hydroxylation may then serve as a sensitive dosimetric biomarker for ozone exposure, even at subclinical ozone exposure levels.     

Cardiovascular effects of ozone exposure in human volunteers

We hypothesized that ozone (O3) exposure acutely affects cardiovascular hemodynamics in humans and, in particular, in subjects with essential hypertension. We studied 10 nonmedicated hypertensive and six healthy male adults. Each subject, after catheterization of the right heart and a radial artery, was exposed in an environmentally controlled chamber to filtered air (FA) on one day and to 0.3 ppm O3 on the following day for 3 h with intermittent exercise. Relative to FA exposure, O3 exposure induced no statistically significant changes in cardiac index, ventricular performance, pulmonary artery pressure, pulmonary and systemic vascular resistances, ECG, serum cardiac enzymes, plasma catecholamines and atrial natriuretic factor, and SaO2. The overall results did not indicate major acute cardiovascular effects of O3 in either the hypertensive or the control subjects. However, mean preexposure to postexposure changes were significantly (p < 0.02) larger with O3 than with FA for rate-pressure product (1,353 beats/min/mm Hg) and for heart rate (8 beats/min); these responses were not significantly different between the hypertensive and the control subjects. Significant O3 effects were also observed for mean FEV1 (-6%), and AaPO2 (> 10 mm Hg increase), which were not significantly different between the two groups. These results suggest that O3 exposure can increase myocardial work and impair pulmonary gas exchange to a degree that might be clinically important in persons with significant preexisting cardiovascular impairment, with or without concomitant lung disease.     

L-arginine uptake and metabolism by lung macrophages and neutrophils following intratracheal instillation of silica in vivo

Cold air inhalation and exercise-induced bronchoconstriction (EIB) have both been used as measures of bronchial responsiveness. Both stimuli are often combined in the Nordic climate. The main objective of the present study was to investigate the climatic influence of cold temperatures upon exercise-induced asthma. The secondary aims were: (a) to assess metacholine bronchial hyper-responsiveness and EIB in children with bronchial asthma (n = 32; mean age 10.8 years) compared to children with other chronic lung diseases (CLD) (n = 26, mean age 10.1 years); and (b) to assess the influence of cold air inhalation upon EIB in the two groups of children. Methods used were: (a) the metacholine concentration causing a reduction in FEV1 of 20% (PC20-M), (b) maximum FEV1 fall (delta FEV1) after submaximal treadmill run (EIB test); and (c) delta FEV1 after submaximal treadmill run while inhaling cold (-20 degrees C) dry air (CA-EIB test). Geometric mean PC20-M did not differ significantly between the asthma children (1.28 mg ml-1) and the CLD children (2.90 mg ml-1). In the asthma children, mean delta FEV1 after EIB test was 12.8% vs 21.8% after adding cold air (P < 0.0001), compared to 5.2 and 7.4%, respectively (P = 0.03), in the CLD group. Maximum sensitivity and specificity for the EIB test were 69.8% at a fall in FEV1 of 6.8%; for the CA-EIB test, 72% at a fall in FEV1 of 10.2%; and for metacholine provocation, 56% at a PC20-M of 1.5 mg ml-1. In conclusion, children with bronchial asthma are substantially more sensitive to cold air than children with CLD, and EIB is markedly increased by cold air inhalation in asthmatic children, maintaining the specificity of the EIB test and increasing the sensitivity. The low sensitivity of the EIB test is probably influenced by the use of inhaled steroids. Metacholine inhalation test has less specificity and sensitivity in discriminating asthma from other chronic lung diseases.     

Contractile responses and structure of small bronchi isolated from rats after 20 months' exposure to ozone

Short-term exposure to high concentrations of ozone has been shown to increase airway responsiveness in normal humans and in all laboratory animal species studied to date. While our knowledge concerning the pulmonary effects of single exposures to ozone has increased rapidly over recent years, the effects of repeated exposures are less understood. The goal of the present study was to determine whether airway responsiveness is increased after near-lifetime exposure to ozone. Airway segments representing approximately eighth generation airways were isolated from Fischer 344 rats of both genders that had been exposed for 6 hr per day, 5 days per week for 20 months to 0, 0.12, 0.5, or 1.0 parts per million (ppm) ozone. Circumferential tension development was measured in isolated airways in response to bethanechol, acetylcholine, and electrical field stimulation. Responsiveness of the airways to the contractile stimuli was described by the effective dose or frequency that elicited half-maximum contraction (ED50) and the maximum response. Since ozone exposure is associated with remodeling of peripheral airways, smooth muscle area was determined and tension responses were normalized to the area measurements. Before normalization of tension data to smooth muscle area, neither the ED50 nor maximum response of small bronchi to the contractile stimuli was altered after chronic ozone exposure. Smooth muscle area was greater in airways isolated from animals that had been exposed to 0.5 ppm ozone. After accounting for smooth muscle area, maximum responses of the small bronchi isolated from male rats were significantly reduced after 0.12 and 0.5 ppm ozone. Although not significant statistically, a similar trend was observed in airways isolated from female rats. These results suggest that the increase in airway responsiveness associated with acute ozone exposure does not persist during near-lifetime exposure. Although the mechanism responsible for the adaptation to the effects of O3 on airway responsiveness is unknown, the results indicate that smooth muscle cell function was compromised by the chronic exposure. The mechanism(s) responsible for mediating this effect and the relevance of these results to humans remains to be determined.     

Tolerance of volunteers to cyclosporine A-dilauroylphosphatidylcholine liposome aerosol

Cyclosporine A (CsA) in liposomes of dilauroylphosphatidylcholine (DLPC), containing 118 micrograms of CsA/L of aerosol with a particle size of 1.6 to 1.7 micron diameter, was inhaled by 10 nonsmoking, normal volunteers each for 45 min. Aerosol was administered through an Aerotech II nebulizer (CIS-US, Inc., Bedford, MA) mouthpiece. Eight of the 10 volunteers had tracheal irritation and intermittent coughing following exposure. FEV1 and FVC values were mildly reduced, but returned to normal in 1 h. Blood chemical and hematologic values were unchanged at any time point after as opposed to before inhalation. Nine of the 10 volunteers later inhaled DLPC only, administered through the nebulizer mouthpiece. There was no change in FEV1 or FVC values, and there was no coughing or tracheal irritation. Subsequently, five of the volunteers who had previously had respiratory reactions inhaled CsA-DLPC liposome aerosol for 45-min, but through a mouth-only face mask. There was no tracheal irritation, coughing, or changes in spirometric measures. Blood concentrations of CsA at 15 min after the 45-min inhalation with a face mask averaged 83 +/- 42 ng/ml (mean +/- SD). At 24 h after treatment, CsA was undetectable in blood of the initial 10 volunteers. These studies indicate that CsA-DLPC liposome aerosol can be safely explored as a treatment for patients with moderately severe asthma.     

Antioxidant supplementation and respiratory functions among workers exposed to high levels of ozone

Ozone exposure has been related to adverse respiratory effects, in particular to lung function decrements. Antioxidant vitamins are free-radical scavengers and could have a protective effect against photo-oxidant exposure. To evaluate whether acute effects of ozone on lung functions could be attenuated by antioxidant vitamin supplementation, we conducted a randomized trial using a double-blind crossover design. Street workers (n = 47) of Mexico City were randomly assigned to take daily a supplement (75 mg vitamin E, 650 mg vitamin C, 15 mg beta carotene) or a placebo and were followed from March to August 1996. Pulmonary function tests were done twice a week at the end of the workday. During the follow-up, the mean 1-h maximum ozone level was 123 ppb (SD = 40). During the first phase, ozone levels were inversely associated with FVC (beta = -1.60 ml/ppb), FEV1 (beta = -2.11 ml/ppb), and FEF25-75 (beta = -4.92 ml/ppb) (p < 0.05) in the placebo group but not in the supplement group. The difference between the two groups was significant for FVC, FEV1, and FEF25-75 (p < 0.01). During the second phase, similar results were observed, but the lung function decrements in the placebo group were smaller, suggesting that the supplementation may have had a residual protective effect on the lung. These results need to be confirmed in larger supplementation studies.     

Association between ozone and asthma emergency department visits in Saint John, New Brunswick, Canada

This study examines the relationship of asthma emergency department (ED) visits to daily concentrations of ozone and other air pollutants in Saint John, New Brunswick, Canada. Data on ED visits with a presenting complaint of asthma (n = 1987) were abstracted for the period 1984-1992 (May-September). Air pollution variables included ozone, sulfur dioxide, nitrogen dioxide, sulfate, and total suspended particulate (TSP); weather variables included temperature, humidex, dewpoint, and relative humidity. Daily ED visit frequencies were filtered to remove day of the week and long wave trends, and filtered values were regressed on air pollution and weather variables for the same day and the 3 previous days. The mean daily 1-hr maximum ozone concentration during the study period was 41.6 ppb. A positive, statistically significant (p < 0.05) association was observed between ozone and asthma ED visits 2 days later, and the strength of the association was greater in nonlinear models. The frequency of asthma ED visits was 33% higher (95% CI, 10-56%) when the daily 1-hr maximum ozone concentration exceeded 75 ppb (the 95th percentile). The ozone effect was not significantly influenced by the addition of weather or other pollutant variables into the model or by the exclusion of repeat ED visits. However, given the limited number of sampling days for sulfate and TSP, a particulate effect could not be ruled out. We detected a significant association between ozone and asthma ED visits, despite the vast majority of sampling days being below current U.S. and Canadian standards.     

The detection of cytochrome P450 2E1 and its catalytic activity in rat testis

The aim of this study was to compare the efficacy of 100 micrograms of salbutamol inhaled from a new metered-dose powder inhaler (MDPI, Leiras Taifun, Finland) with that of a same dose of salbutamol inhaled from a conventional pressurized metered-dose inhaler with a large volume spacer (pMDI + S) in protecting against methacholine (Mch) induced bronchoconstriction. This was a 3 day, randomized, cross-over, partly blinded, placebo-controlled multicentre study where the pMDI + S was used as an open control. Twenty-six asthmatic outpatients with a baseline FEV1 > or = 60% of predicted and with bronchial hyperreactivity (PD20 FEV1 < or = 890 micrograms of Mch) were studied. On each study day the patients underwent an Mch provocation 30 min after inhaling placebo from the MDPI or a dose of 100 micrograms of salbutamol from the MDPI and from the pMDI + S. PD20 FEV1 and dose-response slope [DRS; maximal change in FEV1 (%)/dose of Mch (mumol)] were used to evaluate efficacy. The median values of PD20 FEV1 were 250, 622 and 1737 micrograms after placebo MDPI, salbutamol pMDI + S and salbutamol MDPI, respectively. The corresponding DRS values were -11.0%, -4.5% and -2.0% mumol-1. With both parameters, all differences were statistically significant (P < 0.05). In conclusion, 100 micrograms of salbutamol inhaled from Leiras Taifun MDPI offers better protection against Mch-induced bronchoconstriction than 100 micrograms of salbutamol from a pMDI connected to a large volume spacer device.     

Longitudinal and cross sectional analyses of exposure to coal mine dust and pulmonary function in new miners

The association between exposure to dust and pulmonary function was studied by longitudinal and cross sectional analyses in a group of United States underground coal miners beginning work in or after 1970. Quantitative estimates of exposure to respirable coal mine dust were derived from air samples taken periodically over the entire study period. The cohort included 977 miners examined both in round 2 (R2) (1972-5) and round 4 (R4) (1985-8) of the National Study of Coal Workers' Pneumoconiosis. Multiple linear regression models were developed for both cross sectional (pulmonary function at R2 and R4) and longitudinal (change in pulmonary function between R2 and R4) analyses with exposure partitioned into pre-R2 and post-R2 periods and controlled for covariates including smoking history. The results indicate a rapid initial (at R2) loss of FVC and FEV1 in association with cumulative exposure of the order of 30 ml per mg/m3-years. Between R2 and R4 (about 13 years) no additional loss of function related to dust exposure was detected although the percentage of predicted FVC and FEV1 did decline over the period. After some 15 years since first exposure (at R4), a statistically significant association of cumulative exposure with FEV1 of about -5.9 ml per mg/m3-years was found. These results indicate a significant non-linear effect of exposure to dust on pulmonary function at dust concentrations present after regulations took effect. The initial responses in both the FVC and FEV1 are consistent with inflammation of the small airways in response to exposure to dust.     

Consequences of prolonged inhalation of ozone on F344/N rats: collaborative studies. Part XIII. A comparison of changes in the tracheobronchial epithelium and pulmonary acinus in male rats at 3 and 20 months

A limitation of the NTP/HEI Collaborative Ozone Project conducted with F344/N rats at the Battelle Pacific North-west Laboratories in Richland, WA (1991-1993) was that the study used only one time point (20 months) to examine the chronic effects of exposure to ozone. Issues the design of that study could not address were (1) the status of cellular differentiation at earlier time points during the course of ozone exposure; (2) whether changes that appeared to be compensatory after 20 months of exposure were due to ozone, or were aspects of the natural aging process in rats; (3) the inability to define adequately which effects were related specifically to the prolonged duration of exposure; and (4) how and what changes brought about by the natural aging process may have overridden or confounded a clear definition of the effects of exposure to ozone at ambient concentrations (e.g., 0.12 parts per million [ppm]), which are of most concern with long-term exposure to this pollutant. The present study examined the effects of a 3-month exposure to ozone under conditions identical to those of the 20-month NTP/HEI Collaborative Ozone Project. In our facilities at the University of California, Davis, we exposed 42 male F344/N rats to either filtered air or 0.12 or 1.0 ppm ozone. After 3 months of exposure to 1.0 ppm ozone, changes in the distribution of superoxide dismutase (SOD) in the copper-zinc (Cu-Zn) form were shown by a pattern of reduced staining in terminal bronchioles and the centriacinar region; and the manganese (Mn) form of SOD was elevated within the centriacinar region. Further analysis by transmission electron microscopy and immunogold labeling confirmed that Mn SOD was elevated within epithelial type II cells immediately distal to the bronchiole-alveolar duct, junction (BADJ). The trachea, three major bronchi, and a short-length and long-length airway path relative to the trachea were examined by morphometric techniques. The pulmonary acini arising from each of these two paths were also examined morphometrically as a function of distance into the alveolar duct. Cellular changes occurring in each of these anatomical regions after 3 months of exposure were analyzed and compared to the changes noted after the 20-month ozone exposures. We found significant increases in the volume density of nonciliated epithelial cells lining the trachea and caudal bronchi as well as in the proximal and terminal bronchioles of the cranial region at a concentration of 1.0 ppm ozone after both 3 and 20 months of exposure. Remodeling of the centriacinar region, particularly within the cranial region of the lungs after exposure to 1.0 ppm ozone, was statistically significant at both 3 and 20 months. No statistically significant effects were noted following exposure to 0.12 ppm ozone for either 3 or 20 months. An important finding was that age did not influence the effect of ozone on the lungs of rats. We conclude that long-term exposure to ozone, rather than the effects of aging, lead to significant alterations of epithelial cell populations lining the airways and centriacinar region of the lung. Marked cellular changes were noted after exposure to 1.0 ppm ozone, but not to 0.12 ppm.     

Bronchodilator response to salbutamol after spontaneous recovery from nonspecific bronchial provocation tests in asthma

Assessment of airway responsiveness by bronchoprovocation and bronchodilatation tests is important in the diagnostic work-up protocol of bronchial asthma and it would be convenient to undertake both tests on the same occasion. However, it is not known whether this can be done accurately. Therefore, this study evaluated the effect of a prior bronchial provocation test on the bronchodilator response to salbutamol after spontaneous recovery of the forced expiratory volume in one second (FEV1) in a group of asthmatic subjects. On two separate occasions at the same time of day, concentration-response studies with inhaled histamine or methacholine, or a sham challenge with normal saline were carried out in a blinded, randomized manner. Changes in airway calibre were followed as FEV1 and agonist responsiveness expressed as the provocative concentration causing a 20% fall in FEV1 (PC20). After either spontaneous recovery or a fixed-duration wait of 45 min (when appropriate), the subjects received 2x100 microg of salbutamol from a metered dose inhaler with a spacer. The bronchodilator response to salbutamol was expressed as a percentage of initial FEV1 (deltaFEV1% init). Bronchial challenge with both agonists failed to alter significantly the airway response to salbutamol, with the deltaFEV1% init mean value (range) being 16.9% (9.0-31.9) and 17.5% (11.6-31.2) on the sham and histamine/methacholine challenge day respectively. It was shown that the degree of bronchodilatation achieved after salbutamol 200 microg is not affected by prior bronchoprovocation testing when enough time is allowed for the airways to recover spontaneously to baseline forced expiratory volume in one second. Thus evaluation of airway responsiveness by both bronchial provocation tests and bronchodilator testing can be assessed reliably within a few hours in asthmatic patients.     

Expression of the Bcl-2 protein in nasal epithelia of F344/N rats during mucous cell metaplasia and remodeling

Exposure to ozone induces mucous cell metaplasia in rat airway epithelia. During the regeneration process, apoptotic mechanisms may be responsible for eliminating metaplastic cells. Therefore, the present study investigated expression of Bcl-2, a regulator of apoptosis, in ozone-induced mucous cell metaplasias. Adjacent metaplastic mucous cells in nasal airway epithelia that were exposed to ozone were heterogeneous in their expression of Bcl-2; some cells expressed high levels, whereas others expressed low levels or no Bcl-2. On Western blot analysis, Bcl-2 was detected in protein extracts from nasal epithelia of rats exposed to 0.5 ppm ozone for 1 mo but not in control rats exposed to filtered air. The number of metaplastic mucous cells in transitional epithelia of rat nasal airways was increased from 0 to about 200 after 3 and 6 mo of exposure to ozone; only 0 to 10 metaplastic mucous cells remained after a recovery period of 13 wk in rats exposed to ozone for 3 mo. The number of mucous cells of the respiratory epithelium lining the midseptum did not change after ozone exposure or recovery. The percentage of Bcl-2-positive cells lining the midseptum increased from 7 to 14% after a 3- and 6-mo ozone exposure, respectively. In transitional epithelia of the lateral wall and the nasoturbinates and maxilloturbinates, 35 to 55% of cells were Bcl-2-positive after a 1-mo exposure and 10 to 18% after both a 3- and a 6-mo exposure to ozone. Bcl-2 reactivity decreased to 0 to 8% after a recovery period of 13 wk. These observations suggest that Bcl-2 plays a role in the development and resolution of mucous cell metaplasias. This model may be useful in uncovering the role of Bcl-2 during the development and maintenance of metaplastic mucous cells. Disregulation of Bcl-2 expression may be responsible for the sustained mucous cell metaplasia in asthmatics or may allow cells to accumulate and become more susceptible to transformation leading to neoplasia.     

Role of cytokine-induced neutrophil chemoattractant (CINC) in ozone-induced airway inflammation and hyperresponsiveness

Cytokine-induced neutrophil chemoattractant (CINC) is a rat chemokine with potent chemoattractant effects on neutrophils. We determined the involvement of CINC in ozone-induced airway neutrophilia and bronchial hyperresponsiveness (BHR) in the rat. We found a marked increase in lung CINC messenger RNA (mRNA) within 2 h after cessation of ozone exposure (1 ppm for 3 h), as measured by Northern blot analysis, whereas rats exposed to room air had no detectable CINC mRNA. Ozone exposure induced a significant neutrophilia in bronchoalveolar lavage fluid (BALF) at 24 h after exposure (air-exposed rats: 4.2 +/- 2.0 x 10(4), versus ozone-exposed rats: 16.1 +/- 3.7 x 10(4)); prior treatment with a goat anti-CINC antibody (1 mg, intravenously) suppressed the neutrophilia (3.1 +/- 0.9 x 10(4)). When administered intratracheally, the antibody (230 micrograms) partially inhibited the influx of neutrophils. The increase in bronchial responsiveness to acetylcholine observed after ozone exposure was not inhibited by the anti-CINC antibody. The anti-CINC antibody (1 mg, intravenously) also inhibited BALF neutrophilia induced by exposure to a higher concentration of ozone (3 ppm, 3 h), without an effect on BHR. CINC is an important chemokine causing ozone-induced neutrophil chemoattraction, but is not involved in the induction of ozone-induced BHR. The neutrophil is unlikely to contribute to BHR in this model.