1 National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Research Triangle Park 27711; and 2 Departments of Medicine and Cell Biology, Division of Pulmonary and Critical Care Medicine, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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The mechanism of tissue injury after exposure to air pollution particles is not known. The biological effect has been postulated to be mediated via an oxidative stress catalyzed by metals present in particulate matter (PM). We utilized a transgenic (Tg) mouse model that overexpresses extracellular superoxide dismutase (EC-SOD) to test the hypothesis that lung injury after exposure to PM results from an oxidative stress in the lower respiratory tract. Wild-type (Wt) and Tg mice were intratracheally instilled with either saline or 50 µg of residual oil fly ash (ROFA). Twenty-four hours later, specimens were obtained and included bronchoalveolar lavage (BAL) and lung for both homogenization and light histopathology. After ROFA exposure, EC-SOD Tg mice showed a significant reduction in BAL total cell counts (composed primarily of neutrophils) and BAL total protein compared with Wt. EC-SOD animals also demonstrated diminished concentrations of inflammatory mediators in BAL. There was no statistically significant difference in BAL lipid peroxidation; however, EC-SOD mice had lower concentrations of oxidized glutathione in the BAL. We conclude that enhanced EC-SOD expression decreased both lung inflammation and damage after exposure to ROFA. This supports a participation of oxidative stress in the inflammatory injury after PM exposure rather than reflecting a response to metals alone.
transgenic mice; particles; free radicals; vanadium
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INTRODUCTION |
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EPIDEMIOLOGICAL STUDIES
DEMONSTRATE that current levels of air pollution particles in
American cities increase human mortality and morbidity (5, 6, 22,
23). The mechanism of tissue damage after exposure to air
pollution particles is not known. Oxidative stress catalyzed by metals
present in ambient air pollution particles has been postulated to
result in damage through phosphorylation-dependent cell signaling. An
activation of specific transcription factors such as nuclear
factor-B results. An increased expression of inflammatory mediators,
including those genes that have binding sites in their promoter regions
for these transcription factors, can then be affected. The
potential consequence of the resulting enhanced synthesis of mediators
is an inflammatory injury.
Metals in residual oil fly ash (ROFA), an emission-source air pollution particle, have been demonstrated to be responsible for both in vitro and in vivo oxidative stress (8, 24). Vanadium, nickel, and iron in ROFA catalyzed a majority of the oxidative stress measured both in vitro as thiobarbituric acid-reactive products of deoxyribose (24) and in vivo with the use of electron spin resonance (17). The in vivo radical generation after exposure of an animal model to the oil fly ash could be reproduced using either a mixture of metal sulfates or individual metal sulfates (17). Rats instilled with this same particle had increased products of lipid peroxidation in their bronchoalveolar lavage (BAL) fluid measured as carbonyls (29) by high-performance liquid chromatography (HPLC) after derivatization with 2,4-dinitrophenylhydrazine (19). Instillation of solutions of metal compounds contained in ROFA similarly induced a specific increase in CH3CHO. These data corroborate the hypothesis that metals included in particulate matter (PM) can participate in the introduction of an oxidative stress into the lower respiratory tract.
With the use of this same particle, associations between exposure to metals included in PM with in vitro and in vivo cell signaling (27, 28), in vitro transcription factor activation (25), cellular release of inflammatory mediators (2), and lower respiratory injury in vivo (8) have been delineated. However, although this group of studies supports an association between metals included in air pollution particles and lung injury, the participation of a metal-catalyzed oxidative stress in in vivo damage after exposure to an air pollution particle has not been demonstrated. Using a transgenic (Tg) mouse model with increased lung expression of extracellular superoxide dismutase (EC-SOD), we tested the hypothesis that lung injury after exposure to an air pollution particle can be associated with an oxidative stress.
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MATERIALS AND METHODS |
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Materials. NADPH was purchased from ICN Biomedicals (Irvine, CA). Sodium and potassium phosphate, EDTA, phosphoric acid, formic acid, and HPLC-grade methanol were from Mallinckrodt (Chesterfield, MO). All other materials were obtained from Sigma (St. Louis, MO) unless otherwise specified.
The oil fly ash used has a mass median aerodynamic diameter of 1.95 ± 0.18 µm and was acquired from Southern Research Institute (Birmingham, AL). This particle is unique in its pronounced quantities of metals but contains little to no organics or biological components. These characteristics make it a useful model particle to use in studies that test the association among metals present in PM, oxidative stress, and various biological endpoints. As a result of cost and availability, we elected to use CD1 mice for the oil fly ash dose-response and time-response studies. B6C3 mice were used for all other studies.Response of mice to varying doses of oil fly ash. The dependence of the inflammatory response and injury in the lung on both mass of the particle instilled and time was delineated in a mouse model. Thirty-day-old (25-30 g) CD1 mice (Charles River Laboratory, Raleigh, NC) were anesthetized with Metafane (Pitman-Moore, Mundelein, IL) and then intratracheally instilled with either 0.05 ml of normal saline or oil fly ash, varying from 5 to 250 µg in 0.05 ml of saline (n = 6/dose). Twenty-four hours after exposure, the mice were anesthetized with Metafane, euthanized by exsanguination, and tracheally lavaged with 35 ml of normal saline/kg body wt.
The lavagate was stored on ice. A 20-µl aliquot of the total BAL fluid was mixed with an equal volume of 0.4% trypan blue, and the total cell count was obtained using a hemocytometer slide. Employing a modified Wright's stain (Diff-Quick stain; American Scientific Products, McGaw Park, IL), we enumerated neutrophils by counting 300 cells per animal at a magnification of ×400, and values were expressed as the percentage of total cells recovered. After centrifugation at 600 g for 10 min to remove cells, lavage protein was determined using the Pierce Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL). Bovine serum albumin served as the standard. Lactate dehydrogenase (LDH) concentration in the lavage fluid was also measured using a commercially prepared kit (Sigma) as modified for automated measurement (Cobas Fara II centrifugal analyzer; Roche Diagnostic, Montclair, NJ).Response of mice at varying times after exposure to oil fly ash. Thirty-day-old CD1 mice were anesthetized and then intratracheally instilled with either 0.05 ml of normal saline or 50 µg of oil fly ash in 0.05 ml of saline. At times varying between 2 and 96 h (n = 6/time point) after exposure, the mice were anesthetized, euthanized, and tracheally lavaged. Total cell numbers, differentials, and concentrations of both protein and LDH were then determined.
EC-SOD Tg mouse model. Mice overexpressing a human EC-SOD transgene under the control of a 3.7-kb human surfactant protein C promoter were generated and characterized as previously described (10). Heterozygous B6C3 Tg mice and wild-type (Wt) littermates were bred and maintained in a specific pathogen-free unit. The human EC-SOD genotype was confirmed by PCR (10). The EC-SOD transgene protein was detected only in the lungs of PCR-positive Tg mice and not in liver, heart, kidney, brain, skeletal muscle, small intestine, or blood, indicating a highly tissue-specific pattern of transgene expression. The EC-SOD activity in the BAL fluid and lung tissue of Tg mice was significantly higher than that of their Wt littermates. In BAL fluid, there was a 2.9-fold increase in EC-SOD enzyme activity, whereas in the lung tissue there was a threefold increase. The increased expression of the human transgene did not measurably affect the basal expression of endogenous mouse lung EC-SOD (10). Additionally, no effect was observed on the expression of other BAL fluid and/or lung tissue antioxidants, namely Mn-SOD, CuZn-SOD, glutathione peroxidase, catalase, and total glutathione. All experiments were performed according to institutional guidelines for animal care and use at the Duke University Medical Center.
Exposure of Wt and EC-SOD Tg mice to oil fly ash. Thirty-day-old Wt and EC-SOD Tg mice were anesthetized with Metafane and intratracheally instilled with either 0.05 ml of normal saline or 50 µg of oil fly ash in 0.05 ml of saline (n = 8/exposure/mouse type). Twenty-four hours after exposure, the mice were anesthetized with pentobarbital, euthanized by exsanguination, and tracheally lavaged with 1.0 ml of normal saline (0.9%). The lavage was repeated twice (total volume of lavagate was 3.0 ml) and then stored on ice. Cell numbers, differentials, and concentrations of protein, LDH, cytokines, and glutathione were then determined.
In addition to lavage fluid, lung tissue was obtained from mice (n = 2/exposure/mouse type) 24 h after exposure to either saline or 50 µg of oil fly ash. Lungs were inflation fixed with 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. In some animals, lungs were removed immediately, snap-frozen in liquid nitrogen, and stored atLavage concentrations of cytokines.
After centrifugation at 600 g for 10 min to remove cells,
concentrations of tumor necrosis factor (TNF)- and macrophage
inflammatory protein-2 (MIP-2) were measured using commercially
available ELISA kits (R&D Systems, Minneapolis, MN).
Malondialdehyde in the BAL fluid. Lipid peroxidation in the BAL fluid was measured (35). BAL fluid (100 µl) was treated with 150 µl of 0.44 M phosphoric acid and 50 µl of 0.6% thiobarbituric acid. The mixture was heated to 100°C for 1 h. Samples were protein precipitated and neutralized by adding 300 µl of a 1 M NaOH-methanol (1:11) solution. Fifty microliters of the supernatant liquid were injected into a Supelcosil LC-18 column [15 cm length, 4.0 mm internal diameter (ID), 5 µm] using an HPLC system (Hewlett-Packard, Palo Alto, CA). The mobile phase was 40% methanol-60% 50 mM potassium phosphate buffer (pH 6.8). The flow rate was 1 ml/min. Detection was accomplished by a fluorescence detector using an excitation wavelength of 525 nm and an absorbance of 550 nm (Hewlett-Packard). The average retention time of the malondialdehyde (MDA) derivative was 2.8 min. The sensitivity of assay was 0.5 pmol per injection.
Total and oxidized glutathione. Total glutathione levels were analyzed by HPLC (18). For total glutathione, 100 µl of either BAL fluid or lung homogenate were treated with 100 µl of 0.1 M Tris · HCl, pH 8.5. After 30 min on ice, 250 µl of 3.75% 5-sulfosalicylic acid were added to precipitate proteins. The samples were then centrifuged at 11,000 g for 2 min. Two hundred microliters of supernatant were removed and treated with 200 µl of OPA solution (50 mg of o-phthaldialdehyde was dissolved in 0.5 ml of methanol and then made up to 10 ml with 0.4 M potassium tetraborate, pH 9.9) for 2 min at room temperature, followed by neutralization with 200 µl of 250 mM sodium phosphate, pH 7.0. One hundred microliters of sample were injected into an HPLC.
Oxidized glutathione (GSSG) in BAL fluid was determined using duplicate samples treated with 20 mM of N-ethylmaleimide (NEM) and 200 µl of 3.75% of 5-sulfosalicylic acid. After centrifugation at 13,000 g for 2 min, 100 µl of supernatant were mixed with 50 µl of 1 M Tris and 100 µl of 25 mM dithiothreitol (DTT). The remainder of the procedure was as described for total glutathione. GSSG was determined in whole lung sample after being homogenized in 5% 5-sulfosalicylic acid (10 vol) containing 20 mM NEM followed by a 10-fold dilution in 0.1 M Tris containing 20 mM NEM. Then 100 µl of this solution were mixed with 1 M Tris and 25 mM DTT as above. HPLC was performed using a Supelcosil LC18 column (15 cm length × 4.0 mm ID, 5 µm) from Supelco (Bellefonte, PA) with a mobile phase of 0.15 M sodium acetate-methanol (92.5:7.5) delivered at 1.5 ml/min. The HPLC was performed on a Hewlett-Packard 1100 system (Hewlett-Packard, Wilmington, DE) equipped with a 1046A fluorescence detector set at 340 nm excitation and 420 nm emission.Statistical analysis. Statistical analysis was performed using SigmaStat, version 2.03 (San Rafael, CA). Two-way ANOVA was used to analyze the data using a Tukey's test for all pairwise multiple comparison procedures. Statistical significance was reached using a P value of 0.05 unless otherwise indicated.
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RESULTS |
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Twenty-four hours after the instillation of the oil fly ash, there
was a neutrophilic injury in the lungs of CD1 mice. The inflammatory
influx was dose dependent with elevations in the percentage of
neutrophils as the mass of the ash instilled increased (Fig.
1A). This incursion of cells
appeared to reach its greatest values at masses of 25 µg of instilled
particle and above (50, 100, and 250 µg). Comparable with the influx
of neutrophils, lung injury after exposure of CD1 mice to oil fly ash
was dependent on the mass instilled (Fig. 1A). Lavage
protein reached greatest values at 50, 100, and 250 µg of instilled
ash. To delineate the relationship of both inflammatory influx and lung
injury with time, we instilled animals with one single dose of particle
(50 µg) and then lavaged at intervals varying from 2 to 96 h.
The percentage of neutrophils increased to maximal values at 24 and 48 h after exposure of the animal (Fig. 1B). The
response of lavage protein was analogous and increased to its greatest
values at 8, 12, and 24 h after instillation (Fig. 1B).
At 96 h after exposure of the mice to the oil fly ash, both
percent polymorphonuclear neutrophils (PMNs) and BAL protein declined
in value relative to their peak values. On the basis of these results,
it was elected to expose the Wt and Tg animals to 50 µg of oil fly
ash and evaluate for evidence of lung inflammation and injury 24 h
later.
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Instillation of the oil fly ash was associated with a 4.3-fold increase
in total cells in only the Wt mice (Fig.
2A). There were no significant
differences in total cell counts between the saline-treated Wt and Tg
mice and the ROFA-treated Tg mice. More dramatically, the increase in
total cells was mostly due to an influx of neutrophils in the Wt mice,
which after ROFA exposure accounted for 75% of the total number of
cells (Fig. 2B). In absolute terms, the total number of PMNs
increased 17.9-fold in Wt animals and only 4.8-fold in the Tg mice.
When expressed as a percentage, the percent PMNs present in Wt saline-,
Tg saline-, and Tg ROFA-treated groups did not differ. Furthermore, the
percentage of lymphocytes did not change between the saline- and
ROFA-treated Wt and Tg mice.
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The influx of inflammatory cells into the lung can be associated with an injury-type response. After ROFA treatment, we found BAL total protein levels significantly increased in both Wt and Tg mice compared with saline-treated controls (Fig. 2B). Although there was no differences noted between saline-treated Wt and Tg mice, lung overexpression of human EC-SOD significantly attenuated the increase in BAL total protein seen after ROFA treatment.
Those mediators that have a capacity to direct such an incursion of
cells into the lung are numerous. Lavage concentrations of both MIP-2
and TNF- were quantified. There were no differences between lavage
TNF-
levels after saline treatment (Fig.
3A). After ROFA treatment,
TNF-
values significantly increased only in Wt mice, but not in Tg
mice. MIP-2 levels in lavage were similar in both Wt and Tg mice after
saline treatment but significantly increased in both controls and
transgenics after instillation of the oil fly ash (Fig. 3B).
Although the Tg mice did show an elevation in MIP-2, the absolute level
was significantly lower compared with Wt littermate control mice
exposed to ROFA.
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A diminished oxidative stress in the lower respiratory tract of the Tg
mice is one possible reason for a diminished injury among these mice
after exposure to the oil fly ash. To evaluate this, we measured MDA
and total glutathione/GSSG concentrations in the lavage samples.
Glutathione measurements of the BAL showed no differences between the
total glutathione and GSSG content in Wt and Tg mice after saline
instillation (Fig. 4). Instillation of
the ROFA particle was associated with a significant increase in total
glutathione in Wt mice but did not significantly increase in the Tg
animals compared with saline controls. Although the data suggest that
Tg ROFA-treated mice may have a lower BAL total glutathione content
than Wt ROFA mice, this did not reach statistical significance
(P = 0.06). Moreover, when the BAL was analyzed for GSSG content, Wt ROFA mice showed significantly higher levels of GSSG
than Tg ROFA mice (1.66 ± 0.18 µM vs. 1.08 ± 0.20 µM, P = 0.01). When the whole lung GSSG content was
analyzed, saline treatment showed similar levels in Wt and Tg mice
(1.02 ± 0.02 µM vs. 0.70 ± 0.09 µM, respectively).
After ROFA treatment, whole lung GSSG significantly increased in Wt and
Tg animals to comparable levels (2.07 ± 0.14 µM vs. 2.06 ± 0.34 µM, respectively). Twenty-four hours after exposure, there
were no significant differences in the concentrations of MDA after
saline or particle exposure in the Wt and Tg mice (30.1 ± 4.2, 34.6 ± 3.9, 42.1 ± 3.9, 38.7 ± 3.9 nM, respectively).
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In support of decreased lung injury among the Tg animals after exposure
to the oil fly ash, histopathology demonstrated greater damage among Wt
after particle exposure relative to Tg mice (Fig. 5, A and B).
Overall, there was less neutrophilic incursion in the lung tissues
acquired from the EC-SOD transgenics and less interstitial edema
formation. In addition, there was diminished distortion of the normal
architecture of the lung in Tg mice relative to Wt controls exposed to
the particle.
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DISCUSSION |
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Oxidative stress has been postulated as a mechanism for the biological effect of ambient air PM. Both in vitro and in vivo, oxidant generation by ROFA is catalyzed by metals included in this emission source PM (17, 24). Similarly, biological effects of this ash are associated with the metal content of this emission source PM. Pulmonary inflammatory injury induced by ROFA in a rat model has been reproduced by instillation of a mixture of soluble forms of vanadium, nickel, and iron in the proportions found in saline leachate (8). Although metal is the component of ROFA immediately associated with effects, this relationship can be mediated by pathways other than oxidative stress (e.g., metal response elements). However, this investigation demonstrates an unequivocal relationship between in vivo injury in an animal model after PM exposure and the catalysis of reactive oxygen intermediates by this particle. Specifically, the overexpression of EC-SOD in the mouse lung diminished tissue damage after ROFA exposure.
SODs are a group of antioxidant enzymes that catalyze the dismutation
of O-glutamylcysteine synthetase (
GCS), the lack of glutathione
induction in the Tg mice could reflect a lack of oxidant stress-induced
GCS mRNA induction, which has been shown to be associated with an
activator protein (AP)-1 or an AP-1-like responsive element in the
human alveolar epithelial cell line A549 (26). In
these studies, immediately after oxidative stress imparted by either
menadione or hydrogen peroxide, total glutathione levels decreased
after 1 h only to increase by 24 h concomitantly with an
increase in both
GCS mRNA and
GCS enzyme activity. Furthermore,
this disruption of normal glutathione metabolism after instillation of
ROFA is comparable with previous investigation in cultured cells
(9, 15). Additionally, we cannot exclude the possibility
that the enhanced inflammatory influx into the lung contributed, at
least in part, to the elevated BAL glutathione.
There was a significant incursion of lymphocytes and neutrophils in mice instilled with ROFA, and this was diminished in those animals with an elevated expression of EC-SOD. An influx of inflammatory cells into the lung after ROFA appears to be the culmination of a series of cellular reactions to metals included in the particle. ROFA has been shown to induce the production and release of proinflammatory mediators in airway cells (2). Pretreatment with antioxidants attenuated this inflammatory mediator production (2), suggesting the oxidants participate in the pathways mediating proinflammatory responses in the lung after exposure to this emission-source PM. Similarly, concentrations of mediators directing the influx of neutrophils into the lung were diminished in those mice with increased expression of EC-SOD.
The expression of inflammatory cytokines can be regulated through
signaling pathways that involve an activation of transcription factors
(4, 7, 30, 31). ROFA activates and causes the translocation of transcription factors (25). This effect
is mediated by metals included in oil fly ash and inhibitable by an
antioxidant. Overexpression of catalase in BEAS-2B cells decreased both
p38 activation and the consequent B-dependent transcription after
vanadium, a metal contributing to the biological effect of ROFA
(14). However, proinflammatory mediators can also be affected by activation of mitogen-activated protein kinases (MAPKs) (1, 16), which can be oxidant independent. ROFA activates the MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun
NH2-terminal kinase (JNK), and P38. This occurs
both in vitro in airway cells (27) and in vivo in an
animal model (28). Metals appear to be that component in
oil fly ash responsible for MAPK activation (27). One
pathway that might be responsible for this activation of MAPKs is an
inhibition of phosphatase activity. Vanadium ions are potent inhibitors
of protein tyrosine phosphatase activity (13) and have
previously been shown to activate MAPKs in a variety of cell types
(21, 36). The vanadate ion is a phosphate analog that is
believed to act as a competitive inhibitor of tyrosine phosphatases
(20). Similarly, arsenite is a structural analog of
phosphate, and arsenic-containing compounds activate JNK and P38 by
inhibiting a dual-specificity threonine/tyrosine phosphatase
(3). Zinc has also been shown to inhibit the receptor tyrosine phosphatase HPTP-
(32). The resultant MAPK
activation could potentially mediate metal-induced expression of
cytokines and the resulting inflammatory injury. A formation of
reactive oxygen species is an alternative mechanism for kinase
activation in cells exposed to either ROFA or Fenton-reactive metals.
Exposures to oxidants activate MAPK cascades, particularly JNK and p38
MAPK (12). Antioxidants did not inhibit kinase activation
after ROFA exposure, suggesting a pathway not involving oxidant
generation by the emission source PM (27). However, the
diminished influx of inflammatory cells after ROFA instillation in
those mice with increased expression of EC-SOD indirectly suggests a
participation of an oxidative stress in this response.
Those mice with an elevated expression of EC-SOD had diminished injury after instillation of ROFA. This may reflect damage mediated by the inflammatory influx, which is decreased in these same mice. Alternatively, some portion of the lung injury after ROFA is independent of the incursion of neutrophils and macrophages, and this may be affected by the increased expression of EC-SOD. The protection afforded by this enzyme against PM-associated lung damage was specific to EC-SOD, as we have previously failed to show any effect of varying CuZn-SOD on injury in a mouse model after instillation of this same particle (data not published). In addition to injury after ROFA, SOD prevented damage associated with hyperoxia. Transgenic mice that overexpress CuZn-SOD and Mn-SOD are protected against injury after exposures to hyperoxia (33, 34), with the CuZn-SOD-protective effects associated with young mice that have higher levels of glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase. Similarly, the increased expression of EC-SOD diminished lung injury after hyperoxia (10), without changes in other major antioxidant enzymes. Diminished tissue injury after ROFA and hyperoxia exposures is assumed to be the result of the enhanced antioxidant activity of the enzyme.
We conclude that lung inflammation and injury after exposure to an emission-source air pollution PM can be mediated by metal-catalyzed oxidative stress. Therapies directed at inhibiting this radical generation may be successful in diminishing human morbidity and mortality. However, metals may still be responsible for some portion of inflammatory damage after particle exposure through pathways not requiring a catalysis of oxidants.
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ACKNOWLEDGEMENTS |
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The authors are grateful for the assistance of the Duke University Comprehensive Cancer Center's Transgenic Mouse Facility in generating the transgenic mice described in this report. This work was supported, in part, by National Institutes of Health Grants ES-08698 and HL-64894.
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FOOTNOTES |
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This report has been reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily 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: R. J. Folz, Duke Univ. Medical Center, Box 2620, Rm. 331 MSRB, Durham, NC 27710 (E-mail: rodney.folz{at}duke.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 29, 2002;10.1152/ajplung.00409.2001
Received 19 October 2001; accepted in final form 15 February 2002.
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