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.     

Mucous cell metaplasia in rat nasal epithelium after a 20-month exposure to ozone: a morphometric study of epithelial differentiation

The present study was designed to examine the effects of long-term ozone exposure on nasal epithelia and intraepithelial mucosubstances (IM) throughout the nasal airways of F344/N rats. Animals were exposed to 0 (controls), 0.12, 0.5, or 1.0 ppm ozone, 6 h/day, 5 days/wk, for 20 mo. Rats were killed 1 wk after the end of the exposure, and nasal tissues were processed for light and electron microscopy. Standard morphometric techniques were used to determine epithelial cell densities and the amounts of IM in the surface epithelium lining the nasal airways. No mucous cells or IM were present in the epithelia lining the nasal lateral meatus and maxillary sinus of rats exposed to 0 or 0.12 ppm ozone. In contrast, rats exposed to 0.5 or 1.0 ppm ozone had marked mucous cell metaplasia (MCM) with numerous mucous cells and conspicuous amounts of IM in the surface epithelium lining these upper airways. Ozone-induced increases in total epithelial cells (i.e., epithelial hyperplasia) were present only in rats exposed to 1.0 ppm. The results of this study indicate that rats chronically exposed to 1.0 or 0.5 ppm, but not 0.12 ppm, ozone can develop marked MCM with significant increases in IM in both proximal and distal nasal airways. The epithelial changes observed throughout the nasal passages of ozone-exposed rats may be adaptive responses in an attempt to protect the upper and lower respiratory tract from further ozone-induced injury.     

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.     

Prenatal care utilization in New York City: comparison of measures and assessment of their significance for urban health

Short-term exposure to the air pollutant ozone results in severe injury to the nares, trachea, and centriacinus. Long-term exposure however, leads to a state of tolerance that is characterized by remodeling in the centriacinar airways and markedly reduced cell necrosis and inflammation. This remodeling consists of hypertrophy and hyperplasia of Clara cells with a 2- to 5-fold increase in the intracellular content of the major protein synthesized by the Clara cell, Clara cell secretory protein (CCSP). Previous in vitro studies suggest that CCSP may moderate inflammation and bind reactive cytotoxicants. This study tested whether acute and chronic exposure to ozone alters the normal level of expression of the CCSP gene. Rats were exposed to ozone, 1 ppm 8 h nightly, for up to 90 days. Immunohistochemistry demonstrated repopulation of the alveolar ducts with CCSP positive Clara cells and an increase in the intensity of immunoreactive CCSP within Clara cells. The results showed that (1) CCSP mRNA expression, GAPDH, and beta-actin do not change as a result of ozone injury, (2) mRNA levels are more variable as a result of ozone injury and (3) CCSP mRNA expression increases with age.     

Relationship of inhaled ozone concentration to acute tracheobronchial epithelial injury, site-specific ozone dose, and glutathione depletion in rhesus monkeys

Acute pulmonary epithelial injury produced by short-term exposure to ozone varies by site within the tracheobronchial tree. To test whether this variability is related to the local dose of ozone at the tissue site or to local concentrations of glutathione, we exposed adult male rhesus monkeys for 2 h to filtered air or to 0.4 or 1.0 ppm ozone generated from 18O2. Following exposure, lungs were split into lobes and specimens were selected by microdissection so that measurements could be made on airway tissue of similar branching history, including trachea, proximal (generation one or two) and distal (generation six or seven) intrapulmonary bronchi, and proximal respiratory bronchioles. One half of the lung was lavaged for analysis of extracellular components. In monkeys exposed to filtered air, the concentration of reduced glutathione (GSH) varied throughout the airway tree, with the proximal intrapulmonary bronchus having the lowest concentration and the parenchyma having the highest concentration. Exposure to 1.0 ppm ozone significantly reduced GSH only in the respiratory bronchiole, whereas exposure to 0.4 ppm increased GSH only in the proximal intrapulmonary bronchus. Local ozone dose (measured as excess 18O) varied by as much as a factor of three in different airways of monkeys exposed to 1.0 ppm, with respiratory bronchioles having the highest concentration and the parenchyma the lowest concentration. In monkeys exposed to 0.4 ppm, the ozone dose was 60% to 70% less than in the same site in monkeys exposed to 1.0 ppm. Epithelial disruption was present to some degree in all airway sites, but not in the parenchyma, in animals exposed to 1.0 ppm ozone. The mass of mucous and ciliated cells decreased in all airways, and necrotic and inflammatory cells increased. At 0.4 ppm, epithelial injury was minimal, except in the respiratory bronchiole, where cell loss and necrosis occurred, and was 50% that found in monkeys exposed to 1.0 ppm ozone. We conclude that there is a close association between site-specific O3 dose, the degree of epithelial injury, and glutathione depletion at local sites in the tracheobronchial tree.     

Short-term exposures of rats to airborne hydrogen fluoride

A series of acute inhalation exposures was performed with airborne hydrogen fluoride (HF) to establish the concentration response for nonlethal effects in the rat. Exposures were either 2 or 10 min long; concentrations ranged from 135 to 8621 ppm. Three additional exposures (20 to 48 ppm) were performed for 60 min. A mouth-breathing (MB) model with a tracheal cannula was used in most of the exposures to maximize delivery of the HF to the lower respiratory tract. Endpoints on the day after exposure included hematology, serum chemistry, bronchoalveolar lavage, pulmonary function, organ weights, and histopathology. Nasal resistance was measured in nose-breathing (NB) groups. Effects of exposure were generally limited to the respiratory tract and included alveolitis, bronchial lesions, altered parameters of pulmonary function and bronchoalveolar lavage, and mucosal necrosis, inflammation, and fibrinopurulent exudate in airways. Observed changes were concentration related and appeared more pronounced in major airways near the point of entry (trachea in MB animals and nose in NB animals). One group of MB animals exposed for 10 min to 1454 ppm was evaluated at 3 and 14 wk after exposure; the acute effects had resolved by those times. The effects of 2-min exposures were consistently more severe than those from 10-min exposures to the same product of concentration x time. Exposures of MB animals for 60 min to 20 or 48 ppm HF did not result in observable adverse effects, although quasistatic pressure-volume curves were shifted upward slightly after 48 ppm. These data provide an integrated picture of the concentration-related effects of short nonlethal exposures to HF.     

Neurochemical and behavioural effects of neurotensin vs [D-Tyr11]neurotensin on mesolimbic dopaminergic function

Changes in bronchoalveolar lavage fluid (BALF) and blood were examined to assess the toxic effects of diborane (B2H6, CAS: 19287-45-7) on the lung. Male Wistar rats were exposed to diborane at 20 ppm (intended concentration) for 4 h (phase I study) to evaluate time-course changes up to 14 days, and at 10 or 1 ppm (intended concentrations) to assess the dose-effect relationship after 3 days (phase II study). BALF parameters [leukocyte counts, alpha 1-antitrypsin (alpha 1-AT), superoxide dismutase (SOD), total protein, phospholipids etc.] were examined and biochemical and histopathological studies were also carried out. In the phase I study, neutrophils (%) in BALF increased on the day of exposure and then decreased gradually for 3 days. Rapid and marked increases in alpha 1-AT and SOD activity in BALF were detected on the day of exposure, and phospholipids had sharply increased on day 3. After 14 days, these parameters in the exposed rats had returned to their background level and alpha 1-AT decreased significantly. In the phase II study, total protein, alpha 1-AT activity and phospholipids in BALF showed dose-dependent increases, and serum alpha 1-AT activity increased significantly. Alveolar capillary and alveolar cell damage were confirmed in rats exposed to 20 ppm, 10 ppm or 1 ppm diborane for 4 h by evaluating the parameters examined. The protection system appeared to start operating immediately after exposure, and the recovery mechanism seemed to start operating 1 day after exposure and cease by day 14. The no-observed-effect level could not be observed.     

Centriacinar remodeling and sustained procollagen gene expression after exposure to ozone and nitrogen dioxide

Sprague-Dawley rats were exposed to 0.8 ppm ozone (O3), to 14.4 ppm nitrogen dioxide (NO2), or to both gases simultaneously for 6 h per day for up to 90 d. The extent of histopathologic changes within the central acinus of the lungs was compared after 7 or 78 to 90 d of exposure using morphometric analysis by placement of concentric arcs radiating outward from a single reference point at the level of the bronchiole- alveolar duct junction. Lesions in the lungs of rats exposed to the mixture of gases extended approximately twice as far into the acinus as in those exposed to each individual gas. The extent of tissue involvement was the same at 78 to 90 d as noted at 7 d in all exposure groups. At the end of exposure, in situ hybridization for procollagen types I and III demonstrated high levels of messenger RNA within central acini in the lungs of animals exposed to the combination of O3 and NO2. In contrast, animals exposed to each individual gas had a similar pattern of message expression compared with that seen in control animals, although centriacinar histologic changes were still significantly different from control animals. We conclude that the progressive pulmonary fibrosis that occurs in rats exposed to the combination of O3 and NO2 is due to sustained, elevated expression of the genes for procollagen types I and III. This effect at the gene level is correlated with the more severe histologic lesions seen in animals exposed to both O3 and NO2 compared with those exposed to each individual gas. In contrast, the sustained expression of the procollagen genes is not associated with a shift in the distribution of the lesions because the area of change in each group after 7 d of exposure was the same as after 78 to 90 d of exposure.     

Acute effects of inhaled urban particles and ozone: lung morphology, macrophage activity, and plasma endothelin-1

We studied acute responses of rat lungs to inhalation of urban particulate matter and ozone. Exposure to particles (40 mg/m3 for 4 hours; mass median aerodynamic diameter, 4 to 5 microm; Ottawa urban dust, EHC-93), followed by 20 hours in clean air, did not result in acute lung injury. Nevertheless, inhalation of particles resulted in decreased production of nitric oxide (nitrite) and elevated secretion of macrophage inflammatory protein-2 from lung lavage cells. Inhalation of ozone (0.8 parts per million for 4 hours) resulted in increased neutrophils and protein in lung lavage fluid. Ozone alone also decreased phagocytosis and nitric oxide production and stimulated endothelin-1 secretion by lung lavage cells but did not modify secretion of macrophage inflammatory protein-2. Co-exposure to particles potentiated the ozone-induced septal cellularity in the central acinus but without measurable exacerbation of the ozone-related alveolar neutrophilia and permeability to protein detected by lung lavage. The enhanced septal thickening was associated with elevated production of both macrophage inflammatory protein-2 and endothelin-1 by lung lavage cells. Interestingly, inhalation of urban particulate matter increased the plasma levels of endothelin-1, but this response was not influenced by the synergistic effects of ozone and particles on centriacinar septal tissue changes. This suggests an impact of the distally distributed particulate dose on capillary endothelial production or filtration of the vasoconstrictor. Overall, equivalent patterns of effects were observed after a single exposure or three consecutive daily exposures to the pollutants. The experimental data are consistent with epidemiological evidence for acute pulmonary effects of ozone and respirable particulate matter and suggest a possible mechanism whereby cardiovascular effects may be induced by particle exposure. In a broad sense, acute biological effects of respirable particulate matter from ambient air appear related to paracrine/endocrine disruption mechanisms.     

Respiratory responses to repeated prolonged exposure to 0.12 ppm ozone

Repeated exposure to high concentrations of ozone results first in augmentation (typically on the second day) and then attenuation of pulmonary response in humans. To determine the effects of repeated prolonged low-concentration ozone exposure, we exposed 17 healthy nonsmoking male subjects to 0.12 ppm ozone for 6.6 h on 5 consecutive days. Subjects were also exposed once to filtered air. Volunteers exercised at a ventilation of approximately 39 L/min for 50 min of each hour during the exposure. Spirometry, plethysmography, and symptom responses were obtained before, during, and after each exposure. Nasal lavage and aerosol bolus dispersion were obtained before and after exposure. Spirometry decreased and symptoms increased on the first day. Responses were less on the second day compared with those on the first day, and they were absent compared with control values on the subsequent 3 days of ozone exposure. Percent change in FEV1 after ozone exposure compared with that after air exposure averaged -12.79, -8.73, -2.54, -0.6, +0.18% for Days 1 to 5 of ozone exposure, respectively. FEV1 responses ranged from a zero to 34% decrease on Days 1 and 2. After each exposure, we determined the ratio of SRaw after inhaling a fixed dose of methacholine to SRaw after inhaling saline aerosol, as an index of airway responsiveness. Airway responsiveness was significantly increased after each ozone exposure. The mean ratios were 2.22, 3.67, 4.55, 3.99, 3.24, and 3.74 for filtered air and ozone Days 1 to 5, respectively. Symptoms of cough and pain on deep inspiration increased significantly on ozone Day 1 only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects in rats and guinea pigs of short-term exposures to sulfuric acid mist, ozone, and their combination

Ozone and the oxides of sulfur are common environmental pollutants. The acute pulmonary lesions caused by ozone and sulfuric acid mist in rats and guinea pigs have been characterized. Rats are not affected by sulfuric acid mist in concentrations up to 100 mg/m3 except for reduced body weight at the higher doses. A true alveolitis develops in guinea pigs exposed to more than 20 mg/m3 sulfuric acid mist. The ozone lesion is primarily confined to the terminal bronchioles and proximal alveoli. In combination studies with up to 2 ppm ozone and up to 10 mg/m3 sulfuric acid mist, the pulmonary lesion and lung/body weight data were essentially the same as in exposure to ozone alone, and the number of statistically significant synergistic effects in rats and guinea pigs was about what one would expect to observe by chance alone.     

Short-term ozone exposure affects the surface activity of pulmonary surfactant

The effects of short-term ozone exposure on the lung function and surface activity of surfactant subtypes isolated from rat lung lavage were studied. Rats were exposed to 0.8 ppm ozone for 2 or 12 hr. The surface activity of surfactant was affected by ozone exposure, whereas distinct morphological changes in bronchoalveolar lavage or in the surfactant subtypes were not observed. Adsorption experiments indicated that bronchoalveolar lavage from rats exposed for 12 hr to ozone remained at lower equilibrium surface pressures than lavage from control rats. These observations suggest interference of inflammatory proteins with the surface film. Extracted surfactant, containing only lipids and surfactant proteins B and C, had a decreased adsorption rate after ozone exposure. These results suggest that the activity of one or both of the hydrophobic surfactant proteins (SP-B and SP-C) was affected by ozone.     

Particle clearance and histopathology in lungs of F344/N rats and B6C3F1 mice inhaling nickel oxide or nickel sulfate

The goals of this study were to (1) determine the effects of repeated inhalation of relatively insoluble nickel oxide (NiO) and highly soluble nickel sulfate hexahydrate (NiSO4.6H2O) on lung particle clearance, (2) investigate the effects of repeated inhalation of NiO or NiSO4 on the pulmonary clearance of subsequently inhaled 85Sr-labeled microspheres, (3) correlate the observed effects on clearance with accumulated Ni lung burden and associated pathological changes in the lung, and (4) compare responses in F344 rats and B6C3F1 mice. Male F344/N rats and B6C3F1 mice were exposed whole-body to either NiO or NiSO4.6H2O 6 hr/day, 5 days/week for up to 6 months. NiO exposure concentrations were 0, 0.62, and 2.5 mg NiO/m3 for rats and 0, 1.25, and 5.0 mg NiO/m3 for mice. NiSO4.6H2O exposure concentrations were 0, 0.12, and 0.5 mg NiSO4.6H2O/m3 for rats and 0, 0.25, and 1.0 mg NiSO4.6H2O/m3 for mice. After 2 and 6 months of whole-body exposure, groups of rats and mice were acutely exposed nose-only to 63NiO (NiO-exposed animals only), 63NiSO4.6H2O (NiSO4.6H2O-exposed animals only), or to 85Sr-labeled polystyrene latex (PSL) microspheres (both NiO- and NiSO4.6H2O-exposed animals) to evaluate lung clearance. In addition, groups of rats and mice were euthanized after 2 and 6 months of exposure and at 2 and 4 months after the whole-body exposures were completed to evaluate histopathological changes in the left lung and to quantitate Ni in the right lung. Repeated inhalation of NiO results in accumulation of Ni in lungs of both rats and mice, but to a greater extent in lungs of rats. During the 4 months after the end of the whole-body exposures, some clearance of the accumulated Ni burden occurred from the lungs of rats and mice exposed to the lower, but not the higher NiO exposure concentrations. Clearance of acutely inhaled 63NiO was also impaired in both rats and mice, with the extent of impairment related to both exposure concentration and duration. However, the clearance of acutely inhaled 85Sr PSL microspheres was not impaired. The repeated inhalation of NiO resulted in alveolar macrophage (AM) hyperplasia with accumulation of NiO particles in both rats and mice, chronic alveolitis in rats, and interstitial pneumonia in mice. These lesions persisted throughout the 4-month recovery period after the NiO whole-body exposures were terminated. In contrast, repeated inhalation of NiSO4.6H2O did not result in accumulation of Ni in lungs of either rats or mice and did not affect the clearance of 63NiSO4.6H2O inhaled after either 2 or 6 months of NiSO4.6H2O exposure. Clearance of the 85Sr-labeled microspheres was significantly impaired only in rats exposed to the microspheres after 2 months of exposure to NiSO4.6H2O. Histopathological changes in rats were qualitatively similar to those seen in NiO-exposed rats. Only minimal histopathological changes were observed in NiSO4.6H2O-exposed mice. These results suggest that repeated inhalation of NiO at levels resulting in AM hyperplasia and alveolitis may impair clearance of subsequently inhaled NiO. The potential effects of repeated inhalation of soluble NiSO4.6H2O on the clearance of subsequently inhaled poorly soluble particles are less clear.     

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.     

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.     

Carbon disulfide neurotoxicity in rats: VI. Electrophysiological examination of caudal tail nerve compound action potentials and nerve conduction velocity

The effects of subchronic exposure to carbon disulfide (CS2) on ventral caudal tail nerve compound nerve action potential (CNAP) amplitudes and latencies, and nerve conduction velocity (NCV) in rats were examined. Male and female Fischer 344 rats were exposed to 0, 50, 500, or 800 ppm CS2 for 6 hrs/day, 5 days/week. Using separate groups, exposure duration was 2, 4, 8, or 13 weeks. Exposure to 500 or 800 ppm CS2 for 13 weeks decreased NCV compared to the 50 ppm CS2 group. CNAP amplitudes were increased, and peak P1P2 interpeak latency decreased, after exposure to 500 or 800 ppm CS2 for 13 weeks. Most of the changes in NCV and CNAPs were not attributable to differences in tail or colonic temperature. However, the increases in peak P1 amplitude may relate to the proximity of the electrodes to the tail nerves. Assessment of tail nerve morphology after 13 weeks exposure to 800 ppm CS2 revealed only minor changes compared to the extent of axonal swelling and degeneration observed in the muscular branch of the tibial nerve and axonal swelling in the spinal cord. As anticipated, in older animals the NCV increased, the CNAP amplitudes increased, and the CNAP latencies decreased. The biological basis for the changes in CNAPs produced by CS2 is under investigation. Future studies will focus on electrophysiological evaluation of spinal nerve function, to allow better correlation with pathological and behavioral endpoints.     

Time-dependent changes of inflammatory mediators in the lungs of humans exposed to 0.4 ppm ozone for 2 hr: a comparison of mediators found in bronchoalveolar lavage fluid 1 and 18 hr after exposure

Acute exposure of humans to ozone results in reversible respiratory function decrements and cellular and biochemical changes leading to the production of substances which can mediate inflammation and acute lung injury. While pulmonary function decrements occur almost immediately after ozone exposure, it is not known how quickly the cellular and biochemical changes indicative of inflammation occur in humans. Increased bronchoalveolar lavage (BAL) fluid levels of neutrophils (PMNs) and prostaglandins (PGE2) have been reported in humans as early as 3 hr and as late as 18 hr after exposure. The purpose of this study was to determine whether a broad range of inflammatory mediators are elevated in BAl fluid within 1 hr of exposure. We exposed eight healthy volunteers twice: once to 0.4 ppm ozone and once to filtered air. Each exposure lasted for 2 hr during which the subjects underwent intermittent heavy exercise (66 liters/min). BAL was performed 1 hr after the exposure. Ozone induced rapid increases in PMNs, total protein, LDH, alpha-1 antitrypsin, fibronectin, PGE2, thromboxane B2, C3a, tissue factor, and clotting factor VII. In addition, there was a decrease in the recovery of total cells and alveolar macrophages, and decreased ability of alveolar macrophages to phagocytize Candida albicans. A comparison of these changes with changes observed in an earlier study in which subjects underwent BAL 18 hr after an identical exposure regimen indicates that IL-6 and PGE2 levels were higher 1 hr after exposure than 18 hr after exposure, fibronectin and tissue-plasminogen activator levels were higher 18 hr after exposure, and that PMNs, protein, and C3a were present at essentially the same levels at both times. These results indicate that (i) several inflammatory mediators are already elevated 1 hr after exposure; (ii) some mediators achieve their maximal levels in BAL fluid at different times following exposure. These data suggest that the inflammatory response is complex, depending on a cascade of timed events, and that depending on the mediator of interest one must choose an appropriate sampling time.     

Time-course of functional and pathological changes after a single high acute inhalation of chlorine in rats

Reactive airways dysfunction syndrome (RADS) is an asthma-like condition that follows exposure to very high concentrations of an irritant material. We assessed the time-course of pathophysiological alterations in a model of RADS. Sprague-Dawley rats were exposed to 1,500 parts per million (ppm) of chlorine for 5 min. Lung resistance (RL), responsiveness to inhaled methacholine (MCh), the airway epithelium and bronchoalveolar lavage (BAL) were assessed over a 3 month period after exposure. RL increased significantly up to 3 days after exposure, reaching a maximal change of 110+/-16% from baseline. There was a significant decrease in the concentration of MCh required to increase RL by 0.20 cmH2O x mL(-1) x s from days 1-7 after exposure. In some rats, MCh hyperresponsiveness and RL changes persisted after exposure for as long as 1 and 3 months, respectively. Histological evaluation with morphometric evaluation revealed epithelial flattening, necrosis, increase in smooth muscle mass and evidence of epithelial regeneration. BAL showed an increased number of neutrophils. The timing of maximal abnormality in the appearance of the epithelium (days 1-3) corresponded to that of the maximal functional changes. Acute high chlorine exposure results in functional and pathological abnormalities that resolve in the majority of animals after a variable period; however, these changes can persist in some animals. Functional abnormalities in the initial stages may be related to airway epithelial damage.     

Role of tachykinins in ozone-induced acute lung injury in guinea pigs

To examine the hypothesis that the acute reversible changes caused by ozone (O3) exposure are mediated by tachykinin release, guinea pigs were depleted of tachykinins by use of repeated capsaicin (CAP) injections before O3 exposure in an attempt to prevent O3-induced functional changes. Unexpectedly, CAP pretreatment caused divergent results in the functional responses to O3. Ventilatory measurements obtained from CAP-pretreated O3-exposed (CAP-O3) animals were exacerbated rather than diminished compared with the effects of O3 alone. Similarly, lavage fluid protein accumulation was enhanced in the CAP-O3 group compared with the O3-exposed group. In better agreement with our initial hypothesis, the CAP-O3 group was less responsive than the O3-exposed animals to histamine aerosol challenge. Additionally, Evans blue dye accumulation, a hallmark of tachykinin release, was increased in O3-exposed animals and was partially blocked in the CAP-O3 group. These data suggest that tachykinin-containing sensory fibers are unlikely to mediate the acute effects of O3 exposure on tidal breathing and lavage fluid protein accumulation but may play a role in causing post-O3 airway hyperreactivity and protein extravasation into the trachea.     

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.