* Pulmonary Toxicology Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina;
North Carolina Central University, Durham, North Carolina; and
China Medical University, Shenyang, China
Received August 4, 1999; accepted December 14, 1999
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: bronchitis; bronchoalveolar lavage fluid (BALF); concentrated ambient particles (CAPs); Sprague-Dawley rats.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Individuals with preexistent chronic obstructive pulmonary disease, including bronchitis, constitute an important subgroup potentially at risk of PM-induced health effects based on the prevalence of the disease. Nearly 14 million cases of bronchitis are reported annually nationwide (Ball, 1995; Iribarren et al., 1999
). Bronchitis is often associated with chronic cigarette smoking and is characterized by chronic productive cough due to mucus hypersecretion and proliferation of mucus-secreting cells, recurrent or persistent airway inflammation, airflow obstruction, and often airway hyperresponsiveness (Jeffery, 1991
; O'Byrne and Postama, 1999). A number of animal models of bronchitis have been developed to study the pathogenesis of the disease and the impact of pharmacologic manipulation (Drazen et al., 1982
., 1995; Levrier et al., 1992
; Shore et al., 1987
). The most widely used and characterized animal model involves exposure of animals (dogs and rats) to high levels of sulfur dioxide (SO2) for a duration of 48 weeks (Chakrin and Saunders, 1975; Drazen et al., 1982
; Shore et al., 1995
). SO2-induced bronchitis in the rat is characterized by increased mucus production, goblet cell hyperplasia, and increased airway responsiveness to methacholine, as in the case of human bronchitis. However, the severity and persistency of neutrophilic inflammation and chronic thickening of airways seen in humans are relatively mild or absent in the rat SO2 model (Farone et al., 1995
; Shore et al., 1995
).
Ambient particles contain a variety of components such as sulfates, nitrates, metals, organics, and biologic materials (National Research Council, 1998). Each of these components theoretically has the potential to elicit airway injury via one or more mechanisms. Airway mucus has been considered a first line of defense against inhaled toxicants. However, in pathologic conditions of bronchitis, which are associated with increased mucus production and epithelial cell injury, inhaled PM may be more hazardous. Bronchitic rats have been shown to retain more particles in their lungs upon inhalation, and have a multifocal deposition pattern that is different from the more uniform pattern in healthy rats (Sweeney et al., 1995
). The purpose of this study was to evaluate the pulmonary health effects of real-time concentrated ambient particles (CAPs) from Research Triangle Park, North Carolina, in healthy and bronchitic rats (airways disease induced by SO2 exposure). Because it is likely that PM concentration and composition vary dependent on the atmospheric conditions and the season, and can influence the health outcome, we elected to conduct the study on four occasions at different times of the year to determine the consistency of observed health effects, and how that relates to water-leachable constituents such as sulfate and metals. Sulfate and minerals are considered primary causative constituents of combustion source fine PM (National Research Council, 1998
). The study shows that under some conditions, the bronchitic rat model exhibits increased pulmonary injury from CAPs exposure (a response is not readily repeatable at different times), and that leachable sulfate and metal content do not seem to correlate with the observed effects.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Concentrated ambient particle (CAPs) exposure.
One day following the last air (healthy) or SO2 (bronchitic) exposure, rats of each category were randomized into two groups by first sorting them from low weight to high weight. Then starting with the lowest weights, animals equal to the number of groups were selected. These animals were randomly (based on computer random number generator) placed in a group. This process was repeated with the next lowest body weight animals until all animals were used and all groups were filled. One group was exposed to clean air and the other was exposed to CAPs using the concentrator exposure unit (Table 1). The concentrator exposure unit utilizes virtual impacter technology in which massive airflow (~4000 l/min) through a series of narrow slits allows minor flow containing ambient particles of ~0.1 to 2.5 µm to be concentrated (Sioutas et al., 1995
). The virtual impacters were set up in a bleed flow manner where only 20% of the total flow exits the impacter at each stage, resulting in a concentration factor including slit losses of about 3x at each stage. A series of 4 virtual impacters produced ambient PM2.5 concentration enhancement of about 40 times in the chamber. Air containing real-time CAPs was directed through a 80-liter stainless steel and glass exposure chamber with 7.5 air changes/h. This chamber accommodated a maximum of nine rats in individual wire mesh cages (one layer) to be exposed to CAPs. Chamber temperature and relative humidity were 80 ± 2°F and 68 ± 2%, respectively. Each study included exposure of five healthy and four bronchitic rats to CAPS for 6 h/day. Three healthy and four bronchitic rats were simultaneously exposed to filtered nonconcentrated ambient air in a similar manner. All animals were weighed before and after the CAPs or filtered air exposure to determine health status. The ambient concentrator exposure unit could not be operated during unfavorable weather conditions (i.e., rain) and therefore, although the exposures were planned for 6/day for 3 consecutive days, in some cases, the exposures were conducted only for two consecutive days.
PM mass and elemental analysis.
PM samples were collected on preweighed Teflon filters (Gelman, Teflon 47mm diameter, 2 µm; Gelman Sciences, Ann Arbor, MI) for the duration of each exposure. At the end of exposure, filters were weighed using a microbalance (Cahn-C33; Ryan Research, Beverly, ME). Concentrations were determined by sample mass/sample flow volume (µg/m3). Aerosol size distribution in the CAPs system inlet was determined for each exposure by an eight-stage MOUDI (MSP, Minneapolis, MN) cascade impacter. Chamber temperature, relative humidity, airflow, and pressure were monitored continuously.
Each filter was individually extracted in 8.0 ml distilled water for 1 h by continuous agitation. The filtrates were centrifuged at 17000 x g for 30 min and filtered through a teflon syringe filter, then acidified to a pH of 2.0 using concentrated HCl to keep soluble metal salts in soluble form (0.1 µM). The acidified filtrates were analyzed for presence of sulfur, zinc, iron, vanadium, nickel, manganese, and copper using Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES), as described in Kodavanti et al. (1998b). For calibration of the instrument, metals calibration standard (100 ppm multielement standard; p/n QC-21) and sulfate calibration standard (1000 ppm standard; p/n AS-SO49-2X/2Y) from SPEX Certiprep (Metuchen, NJ) were used. Quality control (QC) standards used to check calibration standards were from VHG Labs, Manchester, VT. For metals, the calibration standard is 10 ppm multielement standard; p/n LCAL6020100, and for sulfate, calibration standard is 3000 ppm standard; p/n ASW-100. The minimum detection limits for filter extract solutions were sulfate = 0.7 µg/ml, zinc = 3 ng/ml, manganese = 1 ng/ml, iron = 10 ng/ml, and copper = 8 ng/ml. In some filter extracts, the levels or iron and copper were below the detection limit, whereas sulfate, zinc, and manganese were clearly detectible in all samples.
Nose-only residual oil fly ash (ROFA) inhalation exposure.
In one study (#E) healthy or bronchitic rats were exposed to a dry aerosol of ROFA by nose-only inhalation (6 h/day x 3 days) at a concentration of 1 mg/m3 (Ledbetter et al., 1997). Briefly, the generator, designed on the principle of a carpenter's chalk line, used a continuously moving string to carry adherent particles upward from a reservoir containing ROFA. Aerosolization was accomplished using compressed air to pulse-dislodge particles from the string into the dilution airstream. The ROFA-laden air was directed through a 2-mCi 85Kr charge neutralizer/mixing tube (Thermal Systems, Inc., St. Paul, MN), then into a four-row, 24-port, nose-only exposure chamber. Each exposed animal was restrained in a conical, plastic restrainer. All rats were acclimatized for 1 h to the nose-only inhalation tubes at the end of the last SO2 exposure. Chamber aerosol concentration was determined gravimetrically on samples collected hourly via an unoccupied exposure port onto 47.0-mm Teflon filters (Gelman Sciences, Ann Arbor, MI). Determination of aerosol-size distribution was performed at least once per exposure using a seven-stage cascade impacter (Intox Products, Albuquerque, NM). Chamber temperature, relative humidity, airflow, and pressure were monitored continuously and maintained at constant levels (Ledbetter et al., 1998).
Bronchoalveolar lavage fluid (BALF) analysis for determining lung injury.
Either within 3 h post CAPs (studies #A#D) or ROFA (study #E), or 18 h post CAPs (study #F), rats were anesthetized with sodium pentobarbital (Nembutal, Abbott Lab., Chicago; 50100 mg/kg body weight, ip) and exsanguinated via the abdominal aorta. The tracheas were cannulated, and the left lungs were ligated. The right lung was lavaged using phosphate-buffered saline (pH 7.4) at a volume of 28 ml/kg body weight (approximately 75% total lung capacity). Three in-and-out washes were performed using the same fluid. Following the lavage, the right lung was ligated and the left lung bronchus was opened. The left lung was then fixed through tracheal infusion of 4% buffered paraformaldehyde at a volume based on 28 ml/kg total lung capacity and the left lung being 40% of the total lung mass. The trachea was tied and the lung was submerged in a jar containing 4% paraformaldehyde for histologic evaluation. One aliquot of lavage fluid was used for determining total cells using a Coulter Counter (Coulter, Inc., Miami FL), and a second aliquot was centrifuged using a Shandon 3 Cytospin (Shandon) for preparing cell differential slides. The slides were dried at room temperature and stained with LeukoStat (Fisher Scientific Co., Pittsburgh, PA). Macrophages, neutrophils, eosinophils, and lymphocytes were quantitated using light microscopy (200 cells/slide).
The remaining BALF was centrifuged at 1500 x g to remove cells, and the supernatant fluid was analyzed for protein, albumin, N-acetyl glucosaminidase (NAG) activity, and lactate dehydrogenase (LDH) activity. Assays for protein, albumin, NAG, and LDH activity were modified and adapted for use on a Hoffmann-La Roche Cobas Fara II clinical analyzer (Roche Diagnostics, Branchburg, NJ). Total protein content was determined using a Coomassie Plus Protein Assay Kit (Pierce, Rockford, IL) with bovine serum albumin as a standard. BALF samples were analyzed for albumin content using a commercially available kit and controls from ICN Star Corporation (Stillwater, MN). LDH activity was determined using Kit 228 and standards from Sigma Chemical Co. (St. Louis, MO). NAG activity was determined using a kit and controls from Boehringer Mannheim Corporation Products (Indianapolis, IN).
Histopathology.
After fixation, the left lung tissues were embedded in paraffin and 4-µm thick transverse sections were mounted and stained with hematoxylin and eosin (Experimental Pathology Laboratory, Research Triangle Park, NC). Pathology evaluations were made in a nonblinded fashion by Dr. Peter Mann (Experimental Pathology Laboratory, Research Triangle Park, NC) in studies #A, #C, #D, and #F.
Statistics.
The data were analyzed using a two-way analysis of variance (ANOVA) model. The independent variables were model (healthy or bronchitic) and exposures (air or CAPs). Pairwise comparisons were performed as subtests of the overall model. Effects and comparisons were declared significant if the p-value was < 0.05. Adjustment in the significance level, for multiple comparisons, was made using a modified Bonferroni correction. The p-value of 0.05 was indicated by an asterisk (*) for comparisons between healthy:air and healthy:CAPs or bronchitic:air and bronchitic:CAPs groups; and as
for comparisons between healthy:air and bronchitic:air or healthy:CAPs and bronchitic:CAPs groups.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
BALF protein levels in bronchitic rats subsequently exposed to clean air were not higher than in healthy rats exposed to clean air in any of the studies. Exposure to CAPs or ROFA did not result in increased BALF protein in healthy rats. However, in one study (#A), exposure to CAPs was associated with a significant increase in BALF protein in bronchitic rats when compared to air-exposed bronchitic rats; and there was a modest increase in another study (#D) (Fig. 3). BALF albumin followed the same pattern of changes as protein in all studies (Fig. 4
). BALF LDH activity did not reveal any significant increase associated with either CAPs or ROFA exposure in any study (Fig. 5
), except that there appeared to be an increase (nonsignificant) in study #A (Fig. 5
) following CAPs exposure in both healthy and bronchitic rats. BALF NAG activity, measured as one of the indicators of pulmonary injury, was not increased in healthy rats exposed to CAPs but was increased significantly in three of the four CAPs exposures in bronchitic rats (CAPs exposed healthy rats vs CAPs exposed bronchitic rats; Fig. 6
). ROFA exposure did not increase NAG activity in healthy or bronchitic rats (Fig. 6
, study #E).
|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SO2-induced bronchitis in the rat has been considered a model of chronic human bronchitis because mucus hypersecretion and chronic mucus cell metaplasia are persistent and progressively increased during the course of SO2 exposure (Farone et al., 1995; Knauss et al., 1976
). The concentrations of SO2 used for developing bronchitis range from 200 to 600 ppm, depending on animal species and strain. Increased mucus production and goblet cell hyperplasia have been noted with this concentration range when the duration of exposure is 48 weeks (5 days/week). These characteristic features of bronchitis were readily apparent in the present study. However, the results of this study show that chronic airways inflammation and fibrosis, which are critical pathophysiologic features of human bronchitis (Jeffery, 1991
; Thurlbeck, 1990
), were not readily apparent or persistent in rats following 200 ppm (used in this study) or 250 ppm SO2 exposure (data not shown). It is likely that, as in human bronchitis (Fietta et al., 1988
; Jansen et al., 1995
), host defense mechanisms involved in the clearance of particles and pathogens are impaired in the SO2-induced model; however these responses are not well characterized. Thus, the rat model of SO2-induced bronchitis could be considered a model of acute airways mucus hypersecretion and goblet cell metaplasia. We chose to expose rats to 200 ppm SO2 for a longer duration because this may allow relatively milder but likely more consistent and persistent mucus production. A similar protocol and rat strain have previously been employed in PM deposition (Sweeney et al., 1995
) and PM susceptibility studies (Clarke et al., 1999
).
This report demonstrates that CAPs exposure can result in pulmonary injury in an animal model of preexistent disease. The congestion and cellularity appeared to be increased in histologic evaluation. BALF markers of lung injury (protein, albumin, NAG activity) and neutrophilic inflammation seemed to be slightly increased only in bronchitic rats following CAPs exposure. It is likely that airway epithelial cells, stimulated via SO2 exposure to produce excess amounts of inflammatory mediator cytokines, elicit greater inflammatory and pulmonary injury response upon exposure to ambient particles that likely contain sulfate, metals, organics, and biologic materials. It has been presumed that in vitro macrophage activation occurs more readily when metals of ambient particles interact with biologic materials in a synergistic manner (Imrich et al., 1999). Previous reports have shown that PM deposition patterns in rats with SO2-induced bronchitis differ from those of healthy rats such that focal areas of heavy deposition are evident, presumably due to airway mucus and altered deposition mechanics (Sweeney et al., 1995
). Recently Clarke et al. (1999) have shown that bronchitic rats are susceptible to pulmonary injury caused by ambient particles in a relatively more industrialized and populated area in Boston, Massachusetts. The presence of detectable pulmonary injury from CAPs exposure could have been due to increased focal deposition of CAPs within the lung. Alternatively, clearance of particles via ciliary transport may also have been impaired in the rats with bronchitis, as SO2 exposure has been shown to damage ciliary cells lining the airways (Asmundsson et al., 1973
; Reid, 1970
). How extra mucus would affect the dissolution and transport of ambient particles to pulmonary epithelium, leading to increased vascular leakage and inflammation, is not known.
Although the epidemiologic evidence of PM and associated human health effects is consistent regardless of location (suggesting varied composition of PM) and the time of the year, variability in responsiveness is often encountered in humans and animal models studies (Bertranpetit and Calafell, 1996; Kodavanti et al., 1999
). To determine variability of pulmonary response to inhaled CAPs in bronchitic rats, the study was repeated four times during the fall/winter and the spring of 1997. CAPs exposure was associated with significant pulmonary vascular leakage and inflammation in one of the four studies, with a trend (not significant) in the other two studies that was not consistent with the maximum levels of CAPs achieved during the exposure. A borderline effect was noted in some of the BALF parameters in the third study, indicating considerable variability in the response to CAPs at different times during the fall/winter and the spring. The following speculations can be made regarding variability of the pulmonary response: a) the composition of CAPs collected at different times was likely different; b) the bronchitic response in rats obtained at different times could quantitatively vary in such a way that the degree of mucus hypersecretion and lesions present at the beginning of CAPs exposure was sufficiently different at different seasons to cause variation in the host responsiveness to CAPs. It is noteworthy that healthy animals did not show detectible CAPs effects.
The presence of significant amounts of leachable sulfate and zinc (2050% of total mass) suggests that the fine fraction of ambient PM may originate from anthropogenic combustion sources. Burning of sulfur-containing fossil fuel has been shown to contribute to the fine particulate fraction of many locations in the eastern United States (Lippmann and Thurston, 1996). High sulfate concentrations in the ambient air, including the London fog episode, has been frequently associated with the incidence of bronchitis and also the exacerbation of bronchitic symptoms in humans (Abbey et al., 1998
; Schremk et al., 1949). This study utilized a rat model of bronchitis to support the epidemiologic association of increased morbidity with ambient PM containing sulfur. It is likely that sulfur exists largely as sulfate (Stevens, 1986
) associated in part with the bioavailable metals, because metals such as zinc and manganese were detectible in extracts of CAPs samples. Ammonium ion is the most common ligand for sulfates, but this was not measured. No apparent relationship could be established between pulmonary injury and the concentration of CAPs achieved during the exposure or its leachable metal or sulfate content. Because ambient particles contain many other nonleachable and leachable components (e.g., organics, biologicals), it is possible that those components play a role in biologic responsiveness. Many studies need to be done in order to determine specificity and interactions of causative constituents of real ambient PM from different origins in causing pulmonary response.
Our previous studies using ROFA combustion PM have shown that the metallic constituents of the material were responsible for lung injury in healthy rats (Kodavanti et al., 1997, 1998b
). ROFA contains significant quantities of leachable sulfur, nickel, vanadium, and iron; biologic contamination has been shown to be negligible (Dreher et al., 1997
). In order to determine similarities and differences in pulmonary response from CAPs and ROFA, in an additional study, healthy and bronchitic rats were exposed nose-only to ROFA at 1 mg/m3 6 h/day for 3 consecutive days. The lack of response in the bronchitic rats following ROFA exposure and a positive response with CAPs at a similar concentration suggest that there may be components present in CAPs that are more hazardous than anticipated based on bioavailable metal composition. It is also likely that biologic organic constituents synergistically interact with CAPs components to elicit pulmonary response (Imrich et al., 1999
). Additionally, host responsiveness of bronchitic rats, depending on the degree or state of the impairment, may modulate pulmonary injury or response to different PM constituents. Constituents such as organics and biologicals were not determined in the present study because of the lack of sufficient sample. Thus, what caused the pulmonary responses in bronchitic rats remains to be investigated.
In summary, real-time CAPs exposure is associated with detectible pulmonary injury in a rat model of relatively mild bronchitis. However, the response is variable if the study is conducted at different times of the year. There seems to be no apparent association between CAPs concentrations achieved, or its leachable sulfate or metal, and the extent of pulmonary injury.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
1 To whom correspondence should be addressed at Pulmonary Toxicology Branch, MD 82, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-0026. E-mail: kodavanti.urmila{at}epa.gov.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asmundsson, T., Kilburn, K. H., and McKenzie W. N. (1973). Injury and metaplasia of airway cells due to SO2. Lab. Invest. 29, 4153.[ISI][Medline]
Ball, P. (1995). Epidemiology and treatment of chronic bronchitis and its exacerbations. Chest 108 (Suppl. 2), 43S52S.
Bertranpetit, J., and Calafell, F. (1996). Genetic and geographical variability in cystic fibrosis: evolutionary considerations. Ciba Found. Symp. 197, 97114.[ISI][Medline]
Chakrin, L. W., and Saunders, L. Z. (1974). Experimental chronic bronchitis. Pathology in the dog. Lab Invest 30, 145154.[ISI]
Clarke, R. W., Catalano, P. J., Koutrakis, P., Krishna Murthy, G. G., Sioutas, C., Paulauskis, J., Coull, B., Ferguson, S., and Godleski, J. J. (1999). Urban air particulate inhalation alters pulmonary function and induces pulmonary inflammation in a rodent model of chronic bronchitis. Inhal. Toxicol. 11, 637656.[ISI][Medline]
Dockery, D. W., Pope , A. C., III, Xu, X., Spengler, J. D., Ware, J. H., Fay, M. E., Ferris, B. G., Jr., and Speizer, F. E. (1993). An association between air pollution and mortality in six U.S. cities. N. Engl. J. Med. 329, 17531759.
Drazen, J. M., O'Cain, C. F., and Ingram, R. H., Jr. (1982). Experimental induction of chronic bronchitis in dogs: effects on airway obstruction and responsiveness. Am. Rev. Respir. Dis. 126, 7579.[ISI][Medline]
Dreher, K. L., Jaskot, R. H., Lehmann, J. R., Richards, J. H., McGee, J. K., Ghio, A. J., and Costa, D. L. (1997). Soluble transition metals mediate residual oil fly ash induced acute lung injury. J. Toxicol. Environ. Health 50, 285305.[ISI][Medline]
Farone, A., Huang, S., Paulauskis, J., and Kobzik, L. (1995). Airway neutrophilia and chemokine mRNA expression in sulfur dioxide-induced bronchitis. Am. J. Respir. Cell Mol. Biol. 12, 345350.[Abstract]
Fietta, A., Bersani, C., De Rose, V., Grassi, F. A., Mangiarotti, P., Uccelli, M., and Grassi, C. (1988). Evaluation of systemic host defense mechanisms in chronic bronchitis. Respiration 53, 3743.[ISI][Medline]
Imrich, A., Ning, Y. Y., Koziel, H., Coull, B., and Kobzik, L. (1999). Lipopolysaccharides priming amplifies lung macrophage tumor necrosis factor production in response to air particles. Toxicol. Appl. Pharmacol. 159, 117124.[ISI][Medline]
Iribarren, C., Tekawa, I. S., Sidney, S., and Friedman, G. (1999). Effect of cigar smoking on the risk of cardiovascular disease, chronic obstructive pulmonary disease, and cancer in men. N. Engl. J. Med. 340, 17731780.
Jansen, H. M., Sachs, A. P., and van Alphen, L. (1995). Predisposing conditions to bacterial infections in chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 151, 20732080.[Abstract]
Jeffery, P. K. (1991). Morphology of the airway wall in asthma and in chronic obstructive pulmonary disease. Am. Rev. Respir. Dis. 143, 11521158.[ISI][Medline]
Knauss, H. J., Medici, T. C., Chodosh, S, and Robinson, W. E. (1976). Cell vs. noncell airway temporal response in rats exposed to sulfur dioxide. Arch. Environ. Health 31, 241247.[ISI][Medline]
Kodavanti, U. P., and Costa, D. L. (1999). Animal models to study for air pollutant effects. In Air Pollution and Health. (S. T. Holgate, J. M. Samet, H. S. Koren, and R. L. Maynard, Eds.), pp. 165197. Academic Press Ltd., London, 1999.
Kodavanti, U. P., Costa, D. L., and Bromberg, P. A. (1998a). Rodent models of cardiopulmonary disease: their potential applicability in studies of air pollutant susceptibility. Environ. Health Perspect. 106 (Suppl. 1), 111130.
Kodavanti, U. P., Hauser, R., Christiani, D. C., Meng, Z. H., McGee, J., Ledbetter, A., Richards, J., and Costa, D. L. (1998b). Pulmonary responses to oil fly ash particles in the rat differ by virtue of their specific soluble metals. Toxicol. Sci. 43, 204212.[Abstract]
Kodavanti, U. P., Jackson, M. C., Ledbetter, A. D., Richards, J. R., Gardner, S. Y., Watkinson W. P., Campen, M. J., and Costa, D.L. (1999). Lung injury from intratracheal and inhalation exposures to residual oil fly ash in a rat model of monocrotaline-induced pulmonary hypertension. J. Toxicol. Environ. Health 57, 543563.[ISI]
Kodavanti, U. P., Jaskot, R. H., Costa, D. L., and Dreher, K. L. (1997). Pulmonary proinflammatory gene induction following acute exposure to residual oil fly ash: roles of particle-associated metals. Inhal. Toxicol. 9, 679701.[ISI]
Ledbetter, A. D., Killough, P., and Hudson G. F. (1998). A low-sample-consumption dry-particulate aerosol generator for use in nose-only inhalation exposures. Inhal. Toxicol. 10, 239251.[ISI]
Levrier, L., Duval, D., and Lloyd, K. G. (1992). Study on the effect of oral administration of carbocysteine on ventilatory parameters in the SO2 inhalation model of bronchitis in the rat. Fundam. Clin. Pharmacol. 6, 231236.[ISI][Medline]
Lippmann, M., and Thurston, G. D. (1996). Sulfate concentrations as an indicator of ambient particulate matter air pollution for health risk evaluations. J. Expo. Anal. Environ. Epidemiol. 6, 123146.[ISI][Medline]
Martin, A. E. (1964). Mortality and morbidity statistics and air pollution. Proc. R. Soc. Med. 57, 969975.[ISI]
National Research Council. (1998). Research priorities for airborne particulate matter. I. Immediate and a long-range research portfolio. National Academy Press: Washington D.C.
O'Byrne, P. M., and Postma, D. S. (1999). The many faces of airway inflammation. Asthma and chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 159, S41S63.[ISI][Medline]
Pope, C. A., Dockery, D. W., and Schwartz, J. (1995). Review of epidemiological evidence of health effects of particulate air pollution. Inhal. Toxicol 7, 118.[ISI]
Reid, L. (1970). Evaluation of model systems for study of airway epithelium, cilia, and mucus. Arch. Intern. Med. 126, 428434.[ISI][Medline]
Schrenk, H. H., Heimann, H., Clayton, G. D., Gafafar, W. M., and Wexler, H. (1949). Air pollution in Donora, PA. Epidemiology of the unusual smog episode of October 1948: preliminary report. Washington, DC: Public Health Service; Public Health Service bulletin no. 306.
Schwartz, J. (1994). Air pollution and daily mortality: a review and meta analysis. Environ. Res. 64, 3652.[ISI][Medline]
Shore, S., Kariya, S. T., Anderson, K., Skornik, W., Feldman, H. A., Pennington, J., Godleski, J., Drazen, J. M. (1987). Sulfur-dioxide-induced bronchitis in dogs. Effects on airway responsiveness to inhaled and intravenously administered methacholine. Am. Rev. Respir. Dis. 135, 840847.[ISI][Medline]
Shore, S., Kobzik, L., Long, N. C., Skornik, W., van Staden, C. J., Boulet, L., Rodger, I. W., Pon, D. J. (1995). Increased airway responsiveness to inhaled methacholine in a rat model of chronic bronchitis. Am. J. Respir. Crit. Care Med. 151, 19311938.[Abstract]
Sioutas, C., Koutrakis, P., and Burton, R. M. (1995). A technique to expose animals to concentrated fine ambient aerosols. Environ. Health Perspect. 103, 172177.[ISI][Medline]
Stevens, R. K. (1986). Modern methods to measure air pollutants. In: Aerosols: Research Risk Assessment and Control Strategies. Proceedings of the Second U.S.-Dutch International Symposium, Williamsburg, VA (D. S. Lee, T. Schneider, L. D. Grant, and P. J. Verkerk, Eds.), p. 69. Lewis Publishers, Inc., Chelsea, MI.
Sweeney, T. D., Skornik, W. A., Brain, J. D., Hatch, V., and Godleski, J. J. (1995). Chronic bronchitis alters the pattern of aerosol deposition in the lung. Am. J. Respir. Crit. Care Med. 151, 482488.[Abstract]
Thurlbeck, W. M. (1990). Pathology of chronic airflow obstruction. Chest 97 (Suppl. 2), 6S10S.
Vedal, S. (1997). Ambient particles and health: lines that divide. J. Air Waste Manag. Assoc. 47, 551581.[ISI]