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A consistent association between exposure to high concentrations of ambient particulate matter (PM) and excess cardiopulmonary-related morbidity and mortality has been observed in numerous epidemiological studies, across many different geographical locations. To elicit a similar response in a controlled laboratory setting, spontaneously hypertensive rats were exposed to an oil combustion-derived PM (HP-12) and monitored for changes in pulmonary function and indices of pulmonary injury. Rats were implanted with radiotelemeters to monitor electrocardiogram, heart rate, systemic arterial blood pressure, core temperature, and activity. Animals were divided into four groups and exposed via intratracheal instillation (IT) to suspensions of HP-12 (0.0, 0.83, 3.33, and 8.33 mg/kg; control, low, mid, and high dose, respectively) in saline vehicle. Telemetered rats were monitored continuously for 4-7 days post-IT and pulmonary function was examined using a whole-body plethysmograph system for 6 h/day on post-IT days 1-7. At 24, 96, and 192 h post-IT, bronchoalveolar lavage fluid (BALF) was obtained from sub- sets of nontelemetered animals in order to assess the impact of HP-12 on biochemical indices of pulmonary inflammation and injury. Immediate dose-related changes in pulmonary function were observed after HP-12 exposure, consisting of decreases in tidal volume (↑12-41%) and increases in breathing frequency (↑52-103%), minute ventilation (↑12-25%), and enhanced pause (↑113-187%). These functional effects were resolved by 7 days post-IT, although some average BALF constituents remained elevated through day 7 for mid- and high-dose groups when compared to those of the saline-treated control group. This study demonstrates significant deficits in pulmonary function, along with significant increases in BALF indices of pulmonary inflammation and injury in SH rats after IT exposure to HP-12. [ABSTRACT FROM AUTHOR]

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 α-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. [ABSTRACT FROM AUTHOR]

Epidemiological studies demonstrate a significant association between arrhythmias and air pollution exposure. Sensitivity to aconitine-induced arrhythmia has been employed to examine the factors that increase the risk of such dysfunction. We used aconitine to test whether a single exposure to diesel exhaust (DE) would increase the risk of arrhythmia being triggered in hypertensive rats. We hypothesised that DE exposure increases the risk of arrhythmia due to sensory irritation during and after inhalation. Spontaneously hypertensive rats surgically implanted with radiotelemeters were exposed to 150⇔…⇔1/4g/m³ of DE or filtered air for 4⇔…h. Arrhythmogenesis was assessed 24⇔…h later in urethane-anaesthetised animals by continuous intravenous infusion of aconitine while heart rate (HR) and electrocardiogram (ECG) were monitored. Rats exposed to DE had lower HR when compared to air-exposed animals. Exposure to DE resulted in significantly shorter PR intervals, and significantly prolonged corrected QT (QTc) and corrected JT (JTc) when compared to air exposure. Sensitivity to arrhythmia was measured as the threshold dose of aconitine required to produce ventricular premature beats (VPB), ventricular tachycardia (VT) and ventricular fibrillation (VF). Rats exposed to DE successively developed VPB's, VT, and VF at significantly lower doses of aconitine than air-exposed animals. Pre-exposure treatment of rats exposed to DE with a transient receptor potential A1 (TRPA1) antagonist prevented the heightened sensitivity to aconitine-induced arrhythmia. These findings suggest that a single exposure to DE increases arrhythmogenic sensitivity. This heightened sensitivity may be mediated by activation of TRPA1 on airway sensory nerves, which are particularly sensitive to inhaled irritants. (This abstract does not reflect EPA policy.) [ABSTRACT FROM AUTHOR]

We have recently demonstrated that pulmonary exposure to residual oil fly ash (ROFA) resulted in enhanced sensitization to house dust mite (HDM) and augmented the development of allergic lung disease after allergen challenge. This effect was associated with increased tumor necrosis factor alpha (TNF-alpha), a macrophage- and epithelial cell-derived cytokine that promotes granulocyte migration to the lung. The present study examined whether exogenous administration of TNF-alpha enhances sensitization to HDM. One day prior to pulmonary sensitization with 10 microg HDM (5 microg each on days 1 and 3), female Brown Norway rats were instilled via the trachea with either 2.0 microg recombinant rat TNF-alpha, 2.0 microg bovine serum albumin (BSA), or 1,000 microg ROFA, and were challenged with 10 microg HDM 14 days later. Antigen-induced immediate bronchoconstriction responses, antigen-specific immunoglobulin E (IgE) titers, lymphocyte proliferation, (cytokines (TNF-alpha and interleukin [IL]-13), and eosinophils were elevated in rats treated with ROFA or TNF-alpha compared with BSA-treated controls after HDM challenge. Intratracheal administration of anti-TNF-alpha monoclonal antibody during ROFA exposure did not reduce ROFA-enhanced lymphocyte proliferation or IgE titers, but had a trend for reduced pulmonary inflammation. This study demonstrates that TNF-alpha has similar adjuvant activity as ROFA, but other factors may fulfill this function when TNF-alpha activity is blocked.

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.

Cardiovascular disease is considered a probable risk factor of particulate matter (PM)-related mortality and morbidity. It was hypothesized that rats with hereditary systemic hypertension and underlying cardiac disease would be more susceptible than healthy normotensive rats to pulmonary injury from inhaled residual oil fly ash (ROFA) PM. Eight spontaneously hypertensive (SH) and eight normotensive Wistar-Kyoto (WKY) rats (12-13 weeks old) were implanted with radiotelemetry transmitters on Day -10 for measurement of electrocardiographic (ECG) waveforms. These and other nonimplanted rats were exposed to filtered air or ROFA (containing leachable toxic levels of metals) on Day 0 by nose-only inhalation (ROFA, 15 mg/m(3) x 6 h/day x 3 days). ECGs were monitored during both exposure and nonexposure periods. At 0 or 18 h post-ROFA exposure, rats were assessed for airway hyperreactivity, pulmonary and cardiac histological lesions, bronchoalveolar lavage fluid (BALF) markers of lung injury, oxidative stress, and cytokine gene expression. Comparisons were made in two areas: (1) underlying cardiopulmonary complications of control SH rats in comparison to control WKY rats; and (2) ROFA-induced cardiopulmonary injury/inflammation and oxidative burden. With respect to the first area, control air-exposed SH rats had higher lung and left ventricular weights when compared to age-matched WKY rats. SH rats had hyporeactive airways to acetylcholine challenge. Lung histology revealed the presence of activated macrophages, neutrophils, and hemorrhage in control SHrats. Consistently, levels of BALF protein, macrophages, neutrophils, and red blood cells were also higher in SH rats. Thiobarbituric acid-reactive material in the BALF of air-exposed SH rats was significantly higher than that of WKY rats. Lung inflammation and lesions were mirrored in the higher basal levels of pulmonary cytokine mRNA expression. Cardiomyopathy and monocytic cell infiltration were apparent in the left ventricle of SH rats, along with increased cytokine expression. ECG demonstrated a depressed ST segment area in SH rats. With regard to the second area of comparison (ROFA-exposed rats), pulmonary histology indicated a slightly exacerbated pulmonary lesions including inflammatory response to ROFA in SH rats compared to WKY rats and ROFA-induced increases in BALF protein and albumin were significantly higher in SH rats than in WKY rats. In addition, ROFA caused an increase in BALF red blood cells in SH rats, indicating increased hemorrhage in the alveolar parenchyma. The number of alveolar macrophages increased more dramatically in SH rats following ROFA exposure, whereas neutrophils increased similarly in both strains. Despite greater pulmonary injury in SH rats, ROFA-induced increases in BALF GSH, ascorbate, and uric acid were attenuated when compared to WKY rats. ROFA inhalation exposure was associated with similar increases in pulmonary mRNA expression of IL-6, cellular fibronectin, and glucose-6-phosphate dehydrogenase (relative to that of beta-actin) in both rat strains. The expression of MIP-2 was increased in WKY but attenuated in SH rats. Thus, SH rats have underlying cardiac and pulmonary complications. When exposed to ROFA, SH rats exhibited exacerbated pulmonary injury, an attenuated antioxidant response, and acute depression in ST segment area of ECG, which is consistent with a greater susceptibility to adverse health effects of fugitive combustion PM. This study shows that the SH rat is a potentially useful model of genetically determined susceptibility with pulmonary and cardiovascular complications.

Epidemiological studies have shown an association between elevated levels of particulate matter air pollution and increased morbidity and hospital visits in asthmatics. Residual oil fly ash (ROFA) is a primary combustion particle containing sulfate and metals such as vanadium, nickel, and iron. In this study the effect of ROFA on sensitization to house dust mite (HDM) was examined in a Brown Norway rat model of pulmonary allergy. Rats were instilled via the trachea with 200 or 1000 micrograms ROFA 3 days prior to local sensitization with 10 micrograms HDM and were challenged with 10 micrograms HDM 14 days later. Immunological endpoints were examined at 2, 7, and 14 days after sensitization and at 2 and 7 days after challenge (16 and 21 days post-sensitization, respectively). Antigen-specific immunoglobulin E and associated immediate bronchoconstriction responses to antigen challenge were increased in the ROFA-treated groups compared with the HDM control group. Lymphocyte proliferation to antigen was enhanced at Days 7 and 21 in the bronchial lymphocytes of ROFA-treated groups. Bronchoalveolar lavage fluid (BALF) eosinophil numbers and lactate dehydrogenase were significantly increased in the 1000 micrograms ROFA group at Days 2 and 16, BALF total proteins were elevated at Days 2 and 7 in both ROFA-treated groups, and BALF interleukin (IL)-10 was elevated in the 1000 micrograms ROFA group at Day 2. These results suggest that ROFA has an adjuvant effect on sensitization to HDM., (Copyright 1999 Academic Press.)

House dust mite (HDM) antigen is one of the most common allergens associated with extrinsic asthma. In a model of allergic lung disease, Brown Norway (BN) rats sensitized to HDM with alum and Bordetella pertussis adjuvants produce high levels of IgE antibody and experience bronchoconstriction, increased airway hyperresponsiveness (AHR) to acetylcholine (ACh), and pulmonary inflammation after antigen challenge. The purpose of this study was to determine whether these asthmatic symptoms could be transferred from sensitized animals to naive recipients via humoral or cellular factors. Syngeneic recipient rats were injected (intraperitoneally with 4 x 10(7) cells (precultured overnight with either HDM or bovine serum albumin [BSA]) from lymph nodes of sensitized or control rats, respectively. Other groups received a tail-vein injection of serum from either HDM-sensitized or control rats. Antigen challenge in rats injected with sensitized cells caused increases in pulmonary inflammation and in AHR, but no changes in immediate bronchoconstriction as compared with control recipients. Antigen challenge in serum recipients resulted in immediate bronchoconstriction but had no effect on AHR or on pulmonary inflammation. These data show that immune-mediated lung inflammation and AHR are promoted by antigen-specific lymphocytes, whereas immediate allergic responses are caused by serum factors.

The biological effects of particulate matter (PM) deposition in the airways may depend on aqueousleachable chemical constituents of the particles. The effects of two residual oil fly ash (ROFA) PM samples of equivalent diameters but different metal and sulfate contents on pulmonary responses in Sprague-Dawley rats were investigated. ROFA sample 1 (R1) had approximately twice as much saline-leachable sulfate, nickel, and vanadium, and 40 times as much iron as ROFA sample 2 (R2), while R2 had a 31-fold higher zinc content. Four groups of rats were intratracheally instilled with a suspension of 2.5 mg R2 in 0.3 ml saline (R2), the supernatant of R2 (R2s), the supernatant of 2.5 mg R1 (R1s), or saline only. By 4 days after instillation, 4 of 24 rats treated with R2s or R2 had died, compared with non treated with R1s or saline, and pathological indices were greater in both R2 groups compared with the R1s group. In surviving rats, baseline pulmonary function parameters and airway hyperreactivity to acetylcholine challenge were significantly worse in R2 and R2s groups than in the R1s group. Numbers of bronchoalveolar lavage neutrophils, but not other inflammatory cells or biochemical parameters of lung injury, were greater in both R2 groups compared with the R1s group. These results reinforce the hypothesis that the composition of soluble metals and sulfate leached from ROFA, an emission source particle, is critical in the development of airway hyperreactivity and lung injury.

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.

Lung exposures to complexes of coordinated iron can be associated with a neutrophilic alveolitis. We tested the hypothesis that lung inflammation after intratracheal instillation of mineral oxides in rats increases with surface-complexed [Fe3+]. The 10 mineral oxides employed had measurable [Fe3+] complexed to the dust surface. The metal was incompletely coordinated, as demonstrated by the ability of the particles to catalyze electron transfer and generate thiobarbituric acid (TBA) reactive products of deoxyribose. After exclusion of those silicates containing structural iron within the crystal lattice, there was a significant correlation between the concentration of chelatable metal and TBA-reactive products (r = 0.82; p = .04). Four days after intratracheal instillation of the 10 mineral oxide particles into rats, lavage neutrophils and protein were significantly increased for all dusts compared to injected saline. Among those dusts with no structural iron, the correlation between chelatable iron concentrations and percentage neutrophils did not reach significance (r = 0.73; p = .10), but that between metal and lavage protein did (r = 0.80; p = .05). We conclude that (1) mineral oxides complex iron cations at the surface, (2) in vitro measures of oxidant generation increase with the concentration of surface iron among those dusts with no structural iron, and (3) acute inflammation following introduction of these particles into the lower respiratory tract also increases with surface iron concentrations among those mineral oxides with no structural iron.

We have previously shown that rats dosed intratracheally with indium trichloride (InCl3) develop severe lung damage and fibrosis. However, it is not clear what pulmonary effects would result following accidental occupational exposure to low concentrations of indium by inhalation. The present study uses a model of acute lung injury based on single 1-hr nose-only exposures to 0.2, 2.0, or 20 mg InCl3/m3. Exposure to 0.2 mg InCl3/m3 was capable of initiating an inflammatory response. Seven days following inhalation of 20 mg InCl3/m3 the total cell number, fibronectin, and TNF alpha levels in the bronchial alveolar lavage fluid were 8, 40, and 5 times higher than the control, respectively. Commensurate with the level of lung injury 7 days after exposure, an acute restrictive lung lesion and increased airway responsiveness to acetylcholine were observed. Forty-two days after exposure a compensatory increase in lung volume and carbon monoxide diffusing capacity in the 20 mg InCl3/m3 group suggested recovery from the lung injury. Lung collagen levels were increased in a concentration-dependent manner 42 days postexposure. These data indicate that inhalation of InCl3/m3 causes acute inflammatory changes in the lung.

To investigate the potential for up to a near-lifetime exposure to high-ambient levels of nitrogen dioxide (NO2) to induce functional lung damage, groups of rats were exposed to air or a simulated urban profile of NO2 (0.5 ppm background, 1.5 ppm peak) for 1, 3, 13, 52, or 78 weeks. The dynamic, static, and diffusional characteristics of the lung were evaluated postexposure in anesthetized rats. Furthermore, for the 13-, 52-, and 78-week groups, additional animals were tested after a 6-, 26-, or 17-week period in filtered air, respectively. No significant NO2 differences between exposed and control animals were found for the nitrogen washout, compliance, lung volume, or diffusion capacity of carbon monoxide measurements. At 78 weeks, however, a reduction in delta FEF25%, an estimate of convexity in the later portion of the forced expiratory flow volume curve, was observed. Breathing patterns and mechanisms were also assessed postexposure in a parallel group of similarly exposed unanesthetized rats. These rats were examined during a filtered air, 4 and 8% carbon dioxide (CO2) challenge. In the unanesthetized rat, frequency of breathing was significantly decreased and tidal volume, expiratory resistance, and inspiratory and expiratory times tended to increase. For several of these variables, the largest response also occurred at 78 weeks and seemed to be exacerbated by CO2 challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

To examine the role of nicotine in the augmentation of elastase-induced emphysema by cigarette smoke, animals that had been pretreated with porcine pancreatic elastase (PPE) were exposed to cigarette smokes that had a five-fold difference in their nicotine concentrations. Young adult female Long-Evans rats were divided into seven groups: (1) untreated controls; (2) low nicotine cigarette smoke exposure (Kentucky 2A1 reference cigarettes; 35.0 mg total particulate matter, 0.42 mg nicotine, and 0.38 mg nitrogen oxides per cigarette); (3) high nicotine cigarette smoke exposure (Kentucky 2R1 reference cigarettes; 38.8 mg total particulate matter, 2.2 mg nicotine, and 0.34 mg nitrogen oxides per cigarette; (4) PPE alone; (5) PPE + sham smoke exposure; (6) PPE + 2A1 smoke exposure; and (7) PPE + 2R1 smoke exposure. Three days after intratracheal administration of PPE (400 IU/kg), animals in the smoke-treated groups were exposed to 10 puffs of cigarette smoke daily, 7 days/wk for 14 wk. Sham-treated animals received room air in place of cigarette smoke. After the exposures, pulmonary function tests were performed under general anesthesia. Whole lungs were examined for gross pathologic changes, and samples of lung tissue were harvested for quantitative morphometry. Cigarette smoke exposure alone did not produce significant changes in pulmonary function or structure. On the other hand, treatment with elastase alone produced a constellation of pulmonary functional and structural changes that were pathognomonic of emphysema. Exposure to 2R1 but not 2A1 cigarette smoke significantly augmented the emphysematous changes produced by PPE.(ABSTRACT TRUNCATED AT 250 WORDS)

Rats were treated with a single endotracheal dose of purified porcine pancreatic elastase (400 IU/kg), exposed to undiluted cigarette smoke from Kentucky 2RI reference cigarettes (one 35-ml puff/min for 10 min daily, 5 days per week, for 12 wk) or with a combination of elastase followed by smoke exposure. A number of significant functional abnormalities were observed in the lungs of rats receiving elastase; these included reduced spontaneous ventilation, enlarged subdivisions of lung volume, loss of elastic recoil, and diminished gas exchange capacity. Rats receiving both elastase and cigarette smoke had significantly more severe pulmonary dysfunction than did rats treated with elastase alone. Morphometric measurements of mean linear intercept demonstrated a loss of alveolar fine structure, with enlargement of distal air spaces in elastase-treated rats. These changes were significantly more severe in rats treated with both elastase and cigarette smoke. Pulmonary function tests and morphometric measurements in sham-treated rats and in rats exposed to cigarette smoke only were not significantly different from those in untreated control animals. It is concluded that elastase-induced emphysema in rats is enhanced by exposure to whole cigarette smoke.