The molecular mechanism(s) by which chemically complex air pollution particles mediate their adverse health effects is not known. We have examined the ability of combustion and ambient air particles to induce pulmonary matrilysin expression due to the well-documented role of matrix metalloproteinases in tissue injury and repair responses. Rats were exposed to saline, residual oil fly ash (2.5 mg/rat), or ambient air particles (2.5 mg/rat) via intratracheal instillation and examined 3–72 h after exposure. Saline-exposed animals had low levels of matrilysin mRNA, whereas the animals exposed to either complex particle showed an early induction of pulmonary matrilysin gene expression as well as of the 19-kDa activated form of matrilysin. Immunocytochemistry and in situ hybridization analyses identified the alveolar macrophages and monocytes as primary sources of air pollution particle-induced matrilysin expression. Matrilysin gene induction and protein activation by combustion and ambient air particles correlated with the early histopathological changes produced by these particles. These results demonstrate the ability of combustion and ambient air particles to induce pulmonary matrilysin expression and suggest a role for this matrix metalloproteinase in the initiation of lung injury produced by these particles.
- matrix metalloproteinase
- particulate matter
ambient air particulate matter (PM) represents a dynamic and physicochemically complex mixture of constituents originating from natural and anthropogenic emissions as well as from secondary atmospheric transformations of gaseous air pollutants. Exposure to ambient air PM appears to represent a significant public health concern. Epidemiological studies (8, 33) have reported positive correlations between ambient air PM levels and mortality and morbidity in susceptible individuals. The biological mechanism(s) responsible for the adverse health effects associated with ambient air PM exposure is not known and represents a critical issue in the area of environmental health effects research (6).
Chemically complex combustion particles contribute to the overall fine (≤2.5-μm mass mean aerodynamic diameter) ambient air PM composition. Understanding the molecular mechanism(s) by which these particles induce lung injury may provide some insight into the adverse health effects associated with ambient air PM exposure. Exposure to a complex combustion particle, residual oil fly ash (ROFA), was found to induce pulmonary inflammation and fibrosis in rats (9,44) and to induce pulmonary proinflammatory cytokine expression (23). Bioavailable transition metals were found to mediate ROFA-induced acute lung injury and proinflammatory cytokine gene expression (9, 23). Ambient air PM exposure was found to induce acute lung injury in animals, and bioavailable transition metals were implicated as causal constituents (4, 34). Although the pathophysiological mechanisms responsible for ambient air PM-induced lung injury have yet to be determined, preliminary studies have shown that cytokine-induced neutrophil chemoattractant expression mediates part of the acute inflammatory response elicited by ambient air PM exposure (21).
Matrix metalloproteinases (MMPs) represent a family of zinc-containing endopeptidases capable of degrading various components of the extracellular matrix (31, 32). Although MMPs are important in normal processes such as tissue repair and remodeling, they have also been implicated in the pathogenesis of certain lung diseases. Several reports have suggested that MMPs are involved in acute respiratory distress syndrome (35), emphysema (40), cystic fibrosis (7), idiopathic pulmonary fibrosis (30), and asthma (26). Recent studies (13, 14) have demonstrated that MMPs play a significant pathogenic role in several animal models of acute lung injury. Matrilysin, designated as MMP-7, is a member of the metalloproteinase family and has a broad substrate specificity that includes type IV collagen, fibronectin, laminin, and entactin. These extracellular matrix components are integral structural proteins in lung basement membranes. Matrilysin can also activate latent MMPs as well as inactivate intrinsic protease inhibitors such as α1-antitrypsin, leading to an amplification of proteolytic activities within the lung (37, 42). Furthermore, matrilysin is only poorly inhibited by tissue inhibitors of metalloproteinases (TIMPs), with an association constant 0.1% that of other MMPs (41). Matrilysin has been shown to modulate the release of matrix-bound cytokines such as tumor necrosis factor-α (15) and transforming growth factor-β (19), which are important mediators in inflammation and cell proliferation, respectively.
The molecular mechanism(s) responsible for combustion and ambient air PM-induced lung injury is currently unknown. Pulmonary edema, focal hemorrhage, and cell infiltration are common histopathological responses that have been observed after exposure to ROFA and ambient air PM (44). We have hypothesized that MMPs may participate in combustion- and ambient air PM-induced lung injury because of the wide range of biological activities that have been attributed to these proteases. In the present study, we investigated the ability of air pollution particles derived from oil combustion and ambient air to affect pulmonary matrilysin expression. The determination of the association between PM-induced pulmonary histopathological alterations and matrilysin expression was of particular interest. Our results demonstrated a temporal correlation between matrilysin expression and lung injury induced by combustion and ambient air PM as well as identified alveolar macrophages/monocytes as the cellular origin of PM-induced matrilysin expression. These findings suggest a potential role for matrilysin in acute lung injury induced by combustion and ambient air PM.
MATERIALS AND METHODS
Male 70- to 75-day-old Sprague-Dawley rats were purchased from Charles River Laboratory (Raleigh, NC) and housed in an animal facility approved by the American Association for Accreditation of Laboratory Animal Care (72 ± 2°F, 50 ± 5% relative humidity, 12:12-h light-dark cycle) during quarantine and after intratracheal instillation. Animals were used in accordance with institutional animal care and use committee guidelines. All animals received standard rat chow and water ad libitum.
Particle collection and exposure.
ROFA was collected by Southern Research Institute (Birmingham, AL) on a Teflon-coated fiberglass filter located downstream from the cyclone of a Florida power plant that was burning low-sulfur no. 6 residual oil. The collection temperature was 204°C. Physical and chemical properties of ROFA have been previously reported (9,17). A massive air volume sampler was used to collect milligram quantities of size-fractionated ambient PM [PM < 1.7-μm mass mean aerodynamic diameter (PM<1.7)]. The massive air volume sampler was operated at a flow of 18.5 m3/min at Howard University (Washington, DC) from the middle of May 1995 to the middle of June 1995. The particles were separated aerodynamically and recovered from electrostatic precipitation plates. Animals were exposed to PM by intratracheal instillation of 0.3 ml/rat at a dose of 2.5 mg/rat. Intratracheal instillation was performed as previously described (5). Control animals were exposed to pyrogen-free saline.
Animals were killed 3–96 h after particle instillation. Tissues were processed for analyses of matrilysin gene expression by reverse transcription-polymerase chain reaction (RT-PCR), protein activation by Western blot, histopathology by hematoxylin and eosin stain, and matrilysin localization by in situ hybridization and immunocytochemistry.
RNA isolation and RT-PCR.
Total lung tissue RNA was extracted as previously described (24). Primer sequences for PCR were derived from published sequences in GenBank with the software package Oligo 5 (National Bioscience, Plymouth, MN). Primers were synthesized commercially by BioSynthesis (Lewisville, TX). Primer pairs used were forward primer 5′-cagtgggaacaggcgcagaat-3′ (positions 133–153) and reverse primer 5′-cgtgatctccccttgcgaagc-3′ (positions 512–532) (1) for rat matrilysin, and forward primer 5′-ctgatccacatctgctggaaggtgg-3′ and reverse primer 5′-accttcaacaccccagccatgtacg-3′ (25) for rat β-actin. A sequence homology blast search using the rat matrilysin 21-bp primers was performed against the nonredundant GenBank, European Molecular Biology Laboratory, DNA Data Bank of Japan, and Protein Data Bank databases and failed to detect sequence homology with any other MMPs except rat and mouse matrilysin. RT-PCR was carried out with the RNA-PCR Core Kit (PerkinElmer, Norwalk, CT) according to manufacturer's recommendations. Briefly, total lung RNA (1.0 μg) was reverse transcribed with murine leukemia virus RT and random hexamers in a total volume of 100 μl for 45 min. PCR amplifications of matrilysin and β-actin were performed in parallel with 10-μl aliquots of cDNA per sample taken from the same reverse transcription reaction and amplified with a PerkinElmer thermal cycler (model 480). PCR conditions were optimized for each transcript to ensure that amplification was performed during the exponential phase. Amplification conditions were as follows: initial denaturation at 94°C for 2 min followed by 32 cycles at 92°C for 45 s, 60°C for 1 min, and 72°C for 2 min. Final extension was done at 72°C for 10 min. The authenticity of the 400-bp matrilysin PCR product was verified by restriction enzyme site cleavage with EcoR I, which generated the predicted DNA fragments of 190 and 210 bp.
Amplified DNA bands were separated on a 2% agarose gel stained with SYBR Green 1 (Molecular Probes, Eugene, OR) and visualized by ultraviolet epifluorescent illumination. Illuminated DNA bands were recorded by photography with type 55 positive/negative Polaroid film (Polaroid, Cambridge, MA). Negatives were scanned with an Astra UMAX 1220 scanner equipped with a transparency adapter. Quantitative densitometry was performed on the scanned DNA band images with the UN-SCAN-IT, version 5.1, software (Silk Scientific, Orem, UT). Matrilysin DNA band densities were normalized to their corresponding β-actin DNA band density values, which were determined in parallel RT-PCRs. Statistical analysis of normalized matrilysin RT-PCR data was performed with a one-way ANOVA followed by Newman-Keuls multiple comparison test. Significance was set at P < 0.05.
Antibody to rat matrilysin.
Matrilysin from involuting rat uterus was purified by the methods of Woessner and Taplin (47) through the steps of blue Sepharose chromatography. The material was then applied to gel electrophoresis, and the active and latent matrilysin in the bands was eluted, concentrated, and injected with Freund's complete adjuvant into New Zealand White rabbits. After two booster shots, the serum was collected. The antibody was found to be specific for the purified and the recombinant rat matrilysin in Western blot analysis and showed no cross-reactivity to human MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 or murine metalloelastase (MMP-12) (Su and Dreher, unpublished results).
Western blot analysis.
Flash-frozen rat lung tissue was homogenized in a buffer containing 100 mM Tris (pH 8.0), 0.25% (wt/vol) Triton X-100, and the protease inhibitors phenylmethylsulfonyl fluoride (100 μg/ml), aprotinin (1 μg/ml), and leupeptin (10 μg/ml). After centrifugation at 12,000g for 15 min at 4° C, 80 μl of supernatant were precipitated by the addition of trichloroacetic acid (1% vol/vol) and centrifugated at 12,000 g for 15 min (22). The resulting pellet was resuspended in 20 μl of Laemmli buffer (27). Recombinant rat matrilysin was employed as a standard and was applied after partial activation by pretreatment with 10 mM aminophenylmercuric acetate for 30 min at 37°C. Rat lung tissue extracts and recombinant rat matrilysin control samples were separated by SDS-PAGE on a 4–20% gradient polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and transblotted onto an Immobilon-P membrane (Millipore, Bedford, MA). Membranes were treated with 5% nonfat dry milk in PBS to block nonspecific binding sites. Subsequently, membranes were washed in PBS and incubated for 2 h in PBS containing 1% BSA and a 1:1,000 dilution of rabbit anti-rat matrilysin antiserum. Membranes were washed and incubated with peroxidase-conjugated goat anti-rabbit antibody (1:2,000 in PBS with 1% BSA; Organon Teknika, West Chester, PA) for 1 h. After being washed in PBS, the targeted protein was visualized by chemiluminescence with the ECL Western blot kit (Amersham Life Science, Arlington Heights, IL).
Histopathology, immunocytochemistry, and in situ hybridization.
Lungs were infused in situ with 4% paraformaldehyde in PBS (pH 7.0) for fixation. They were then removed and placed in the fixative overnight at 4° C. The lung tissue was subsequently embedded in paraffin, and 4-μm serial sections were cut, mounted on Superfrost Plus slides (Fisher, Pittsburgh, PA), and stained with hematoxylin and eosin for histopathological analysis. Additional mounted sections were analyzed for rat matrilysin expression by immunocytochemistry and in situ hybridization as described below.
For immunocytochemical analysis, sections were deparaffinized in fresh xylene followed by treatment in a series of graded ethanol and water mixtures. Sections were first incubated in PBS containing 10% normal goat serum for 30 min at room temperature to block nonspecific binding. Sections were subsequently incubated for 1 h in rabbit anti-rat matrilysin antiserum diluted 1:200 in PBS containing 1% normal goat serum. Sections were washed in PBS three times for 5 min. Sections were then incubated with biotinylated goat anti-rabbit IgG (BioGenex, San Ramon, CA) diluted 1:50 in PBS for 20 min and were again washed in PBS three times for 5 min. Streptavidin conjugated with alkaline phosphatase diluted 1:60 in PBS was then applied to each section for 20 min. After the sections were washed in PBS, they were incubated with a substrate buffer containing nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt with levamisole. Sections were counterstained with 2% methyl green and mounted with ShandonMount (Shandon, Pittsburgh, PA).
In situ hybridization was performed with a probe derived from a rat matrilysin cDNA plasmid (1). Riboprobe synthesis, characterization, and in situ hybridization procedures have been previously reported (44) and were used in this study with minor modifications. Briefly, digoxigenin-labeled sense and antisense matrilysin riboprobes were synthesized with the use of digoxigenin-11-UTP by in vitro transcription with T7 and Sp6 RNA polymerases (Boehringer Mannheim, Indianapolis, IN), respectively. For optimal hybridization, synthesized riboprobes were washed twice with 75% ethanol and subjected to limited alkaline hydrolysis to obtain a probe size of ∼200 bp. Tissue sections were deparaffinized in xylene and rehydrated in graded ethanol solutions. After proteinase K digestion (1 μg/ml) at 37°C for 20 min, slides were treated with 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8.0, for 10 min, dehydrated through a graded ethanol series, and air-dried. Sections were then prehybridized and hybridized as previously described (44). Detection of the matrilysin riboprobe within tissue sections was accomplished with a colorimetric detection kit used according to manufacturer's recommendations (Boerhinger Mannheim). Sections were counterstained with methyl green and mounted with ShandonMount (Shandon).
PM-induced lung histopathology.
Exposure of rats to a combustion particle, ROFA, or ambient air PM<1.7 collected in Washington, DC, was found to induce a significant amount of acute lung injury (Fig.1). Normal alveolar structure was present within lung tissue recovered from saline-exposed animals (Fig.1 A). However, increased thickness of alveolar septa and sequestration of red blood cells into the alveolar space or focal hemorrhage were observed within the lungs of ROFA-treated animals as early as 6 h after exposure (Fig. 1 B). By 24 h after ROFA exposure, alveolar hemorrhage was still evident and was accompanied by infiltration of inflammatory cells within the lung (Fig.1 C). The accumulation of the dysmorphic, eosinophilic materials in the alveolar space and in the interstitium were characteristic of pulmonary edema. In addition, the continuity of the alveolar septum was interrupted in lung tissues recovered from ROFA-exposed animals. These histopathological changes were compatible with the kinetics of pulmonary edema and cytotoxicity obtained from bronchoalveolar lavage analysis of ROFA-exposed animals (9), and they demonstrated the ability of this combustion particle to cause severe damage to the structural integrity of the alveolar region within the lung.
The histopathological alterations present in lung tissues recovered from rats exposed to the Washington, DC, ambient air PM<1.7 were qualitatively similar to those observed in the ROFA-exposed animals. The lungs recovered from the ambient air PM-exposed animals displayed more sporadic focal hemorrhage (Fig.1 D) and less severe pulmonary edema and inflammation (Fig.1 E). However, the pattern of distribution of the pathological lesions (most prominent around terminal bronchioles), the time course, the focal hemorrhage at 3–6 h, and the inflammation at 12–24 h after exposure were very similar between ROFA and ambient PM exposures.
PM-induced matrilysin gene expression.
ROFA exposure produced a significant induction of pulmonary matrilysin gene expression as early as 6 h after exposure (Fig.2 A). The early induction of matrilysin gene correlated with the early initiation of pulmonary pathology after ROFA exposure. A maximal 38-fold induction over control levels of pulmonary matrilysin gene expression was observed by 12 h after ROFA exposure and approached control levels of matrilysin gene expression by 48–72 h.
The ability of Washington, DC, ambient air PM<1.7 to induce matrilysin gene expression was also examined (Fig.2 B). As previously observed for ROFA exposure, ambient PM was able to induce early pulmonary matrilysin gene expression by 3 h after exposure. Again, the early induction of matrilysin gene correlated well with the early initiation of pulmonary pathology after ambient PM exposure. However, the extent of matrilysin gene induction was much lower than that observed for ROFA exposure (6-fold in ambient PM vs. 38-fold in ROFA exposure). In contrast to that observed with ROFA exposure, elevated matrilysin gene expression was maintained for as long as 96 h after ambient air PM exposure (Fig.2 B).
PM-induced matrilysin protein expression and activation.
Matrilysin, like other MMPs, is synthesized in an inactive proenzyme form. Therefore, we examined whether the observed particle-induced matrilysin gene expression was also translated to the protein level. In addition, it was important to determine if ROFA and ambient PM could induce expression of the activated form of matrilysin. Saline (control) lung tissue was found to contain extremely low levels of the 29-kDa proenzyme and 19-kDa activated forms of matrilysin (Fig.3 A). ROFA exposure induced the expression of the 19-kDa activated form of matrilysin as early as 3 h after exposure. Maximal levels of the 19-kDa activated form of matrilysin were observed 24 h after exposure and remained elevated as late as 48 h after ROFA exposure.
As seen in Fig. 3 B, ambient PM exposure was also able to induce the expression of the 19-kDa activated form of matrilysin, although at a much lower level than that of ROFA. The 19-kDa activated form of matrilysin could be detected as early as 3 h after exposure to ambient air PM. Similar to responses seen in ROFA-exposed animals, maximal levels of the 19-kDa activated form of matrilysin were observed 24 h after ambient air PM exposure. Lower levels of the 19-kDa activated form of matrilysin could still be detected in ambient PM-treated animals 96 h after exposure.
Cellular origin of PM-induced matrilysin expression.
In situ hybridization and immunocytochemistry were employed to identify the cellular origin of pulmonary matrilysin expression after exposure to the ROFA and ambient air PM. Tissue sections hybridized to the matrilysin sense riboprobe did not show any labeling (Fig.4 A). Matrilysin mRNA was detected in alveolar macrophages and monocytes in lung tissue recovered 12 h after ROFA exposure when sections were hybridized to matrilysin antisense riboprobe (Fig. 4 B). Alveolar epithelial and interstitial cells did not express matrilysin after ROFA exposure. Immunocytochemistry was performed on lung tissue obtained 24 h after either ROFA or ambient air PM exposure to further determine the localization of pulmonary matrilysin protein expression and deposition. Little matrilysin staining was observed in saline-treated control animals (Fig. 4 C) or in sections incubated with control serum (Fig. 4 D). However, ROFA and ambient air PM exposure were found to induce pulmonary matrilysin protein expression (Fig. 4, E and F). In both cases, prominent matrilysin staining was associated with alveolar macrophages and monocytes within the alveolar space. These results are consistent with those obtained from in situ hybridization analysis (Fig. 4 B).
Epidemiological studies have provided evidence that exposure to ambient air PM at levels below current National Ambient Air Quality Standards represents a health risk to susceptible individuals (8, 33). However, the cellular and molecular pathophysiological mechanisms by which ambient air PM mediates its adverse health effects are unknown. Ambient air PM represents a dynamic and physicochemically complex mixture of particles derived from natural and anthropogenic emission sources. In the present study, we demonstrated the ability of chemically complex particles present within ambient air and those derived from anthropogenic processes to induce pulmonary matrilysin expression at the gene and protein levels. The ability of ROFA to induce pulmonary matrilysin suggests that these emission particles may be one of the particle types responsible for the increase in lung matrilysin expression after exposure to ambient air PM.
MMPs were first recognized for their extracellular matrix degrading or remodeling capabilities. MMPs participate in a variety of normal responses such as wound healing (36), organ morphogenesis, and embryonic development (38). Subsequent research has provided evidence that MMPs may play a role in the pathogenesis of several diseases such as arthritis (20), cancer (12), and heart disease (45) and may be involved in a variety of lung disorders such as acute respiratory distress syndrome (35), idiopathic pulmonary fibrosis (30), emphysema (11), and asthma (29). Recent studies (13, 14) have provided evidence for a role of MMPs in several animal models of acute lung injury. Matrilysin (MMP-7) is a highly active MMP and is thought to play a major role in the lung response to injury (10).
In the present study, matrilysin gene induction and protein activation were observed as early as 3–6 h after exposure to combustion or ambient air PM and occurred during a period when the structural integrity of the alveoli was severely compromised as evidenced by histopathological changes. This early induction of matrilysin gene expression and protein activation suggests that matrilysin may play a role in the early stages of air pollution particle-induced lung injury. Activated matrilysin can injure the lung by a variety of mechanisms. Matrilysin activation can lead to direct structural damage to basement membranes within the lung. In addition, matrilysin activation could increase the protease activity within the lung by activating latent procollagenases and serine proteases, even in the presence of TIMPs, and by inactivating intrinsic protease inhibitors such as α1-protease inhibitor (37, 42,46). Su et al. (43) have recently reported that combustion and ambient air PM can induce pulmonary gelatinase A and B genes as well as the expression of their proteins. Structural degradation of the lung extracellular matrix by matrilysin could initiate as well as contribute to pulmonary inflammation through the release of proinflammatory cytokines and growth factors present at cell surfaces or stored within the extracellular matrix (15, 19). In addition, MMP-generated extracellular matrix fragments have been shown to be chemotactic for inflammatory cells (39). At the present time, however, the precise manner and extent to which matrilysin contributes to ambient air PM-induced lung injury and adverse health effects remain speculative.
The alveolar macrophages and monocytes were found to be the primary cellular origin of ROFA- and ambient air PM-induced matrilysin expression. Although this is a novel finding, it is not unexpected because the alveolar macrophages/monocytes are one of the first pulmonary cell types to respond to inhaled particles. A previous study (13) has demonstrated the ability of cultured alveolar macrophages to express a number of MMPs. In addition, other studies (2, 16) have also reported the ability of human mononuclear phagocytes and tissue macrophages to express matrilysin.
The molecular mechanism(s) by which ROFA or ambient PM induces pulmonary matrilysin gene expression and protein activation is not known. Previous studies (9, 34) have demonstrated that bioavailable metals, particularly the first row transition metals capable of generating oxidative injury by redox cycling, were responsible for PM-induced acute lung injury. It is quite plausible that PM-associated oxidants such as redox active metals may induce matrilysin gene expression as well as participate in the autolytic activation of newly synthesized matrilysin proenzyme. This may explain why ROFA, which contains eight times more transition metals than ambient air PM on a weight basis, is able to induce a greater degree of lung matrilysin expression compared with ambient air PM exposure (4).
Workers who service oil combustion boilers develop “boilermakers' bronchitis” and are exposed to high levels of ROFA (25 mg/m3) such that high levels of vanadium, the main metal constituent of ROFA, can be found in their urine (280 μg/ml) (18, 28). In addition, truck drivers who haul ROFA from utility plants have been exposed to very high levels of ROFA and require immediate and prolonged intensive care hospitalization (41a). Therefore, the high dose of ROFA reported in our study may be fairly representative of high occupational exposures. Recently, we have shown induction of matrilysin gene expression in bronchoalveolar lavaged cells recovered from rats exposed to ROFA at a dose of 0.5 mg/rat (Dreher, unpublished data). There was an early threefold induction of matrilysin geme expression in lavaged cells recovered 1 h after exposure and a maximal sixfold induction 24 h postexposure. Although we have not confirmed the cellular origin of matrilysin in the recovered cells, our results shown in Fig.4 E would predict that they would be of macrophage/monocyte origin. The importance of this finding is that pulmonary matrilysin expression can be induced at a much lower ROFA dose, indicating that the ROFA results presented in this study are not simply a high-dose response.
ROFA emissions from residential and utility boilers also contribute to ambient air PM levels. Therefore, we wanted to determine whether ambient air PM could also activate pulmonary lung matrilysin expression. Although a relatively high dose of ambient air PM was employed in our studies, our results, at the very least, provide the first evidence that ambient air PM can activate pulmonary matrilysin expression and suggest a possible role for this MMP in air PM-induced lung injury. Verification of this possibility must await additional dose-response studies that can only be done when human exposure levels to ambient air PM have been measured. These have not been determined at the present time.
The present study has provided insight to the possible involvement of MMP in the mechanism by which chemically complex air pollution particles induce lung injury. We have provided evidence that exposure to combustion and ambient air particles can induce pulmonary matrilysin gene expression and protein activation, the kinetics of which correlate with the pathogenesis of the developing lung lesion after exposure to combustion and ambient air particles. The specific mechanism(s) responsible for combustion- and ambient air PM-induced pulmonary matrilysin gene induction and protein activation, as well as its role in the adverse health effects associated with exposure to these particles, is currently under investigation.
We thank Dr. Urmila Kodavanti and James Lehmann for expert technical assistance. We thank Dr. Daniel L. Costa of the US Environmental Protection Agency (Research Triangle Park, NC) and Dr. Ines Pagan of North Carolina State University (Raleigh, NC) for critically reviewing the manuscript.
This work was supported by a Cooperative Agreement (CT826514) between the US Environmental Protection Agency and Duke University (Durham, NC).
Results from this study were initially presented at the American Thoracic Society 1998 annual meeting in Chicago, IL, and published in abstract form (Am J Respir Crit Care Med 157: A152, 1998).
This report has been reviewed by the National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, and approved for publication. Approval does not signify that the contents reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
Address for reprint requests and other correspondence: K. L. Dreher, National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Mail Drop 82, Research Triangle Park, NC 27711 (E-mail:).
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