Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115
Received January 7, 2004; accepted February 26, 2004
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ABSTRACT |
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Key Words: reactive oxygen species; oxidative stress; particulate air pollution; inflammation; CAPs.
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INTRODUCTION |
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Proinflammatory and toxic effects of PM have been observed in the laboratory, in human subjects, in animal models, and in cells in culture. PM inhalation or intratracheal instillation was shown to invoke an inflammatory response in humans (Ghio and Devlin, 2001; Ghio et al., 2001
), dogs (Clarke et al., 2000b
; Godleski et al., 2000
), and rats (Clarke et al., 1999
, 2000a
; Hatch et al., 1985
; Kennedy et al., 1998
; Li et al., 1997
; Saldiva et al., 2002
). Critical components of the inflammatory response to PM are the release of macrophage- and pneumocyte-derived cytokines (Kennedy et al., 1998
; Li et al., 1997
) and the concomitant recruitment of neutrophils (Ferin et al., 1992
; Kennedy et al., 1998
; Li et al., 1996
, 1997
).
Increased production of reactive oxygen species (ROS) by PM is suggested by the finding that many of the proinflammatory genes induced upon in vivo exposure to PM (TNF-, and ß, TGF-ß,
-IF, IL-6, and IL-8 among others, Shukla et al., 2000
) are regulated by redox-sensitive transcription factors such as NF-
B, AP-1 and C/EBP. Activation of some of these transcription factors and increased transcription of downstream genes have been also demonstrated in vitro in alveolar and bronchial epithelial cell lines treated with PM (Jimenez et al., 2000
; Kennedy et al., 1998
; Shukla et al., 2000
). A role for ROS in these systems is further supported by the prevention of cytokine upregulation by enzymatic (SOD and catalase) and nonenzymatic (N-acetylcysteine, NAC) antioxidants (Jimenez et al., 2000
; Kennedy et al., 1998
; Shukla et al., 2000
).
We have previously shown that inhalation exposure to concentrated ambient particles (CAPs), but not inert particles, leads to time-dependent increases in the steady-state concentrations of oxidants in the lung and heart (Gurgueira et al., 2002). The oxidative stress imposed by CAPs was associated with the metal content of the particles in a tissue specific manner, and led to mild increases in lung and heart edema. In this study, we investigated the role of ROS in the development of CAPs-induced pulmonary inflammation and toxicity. Our data show that short-term inhalation exposure to CAPs leads to significant accumulation of oxidized lipids and proteins in the lung. Pulmonary oxidative stress was associated with increased polymorphonuclear neutrophil (PMN) count in bronchoalveolar lavage (BAL), PMN infiltration. These biological effects were prevented and pretreatment of the animals with NAC at a dose that prevented accumulation of oxidants.
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MATERIALS AND METHODS |
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Tissue preparation.
At the end of the exposure, animals were removed from the HAPC and kept in room air for 24 h. The lungs were then removed and frozen in a dry ice bath. Separate samples were taken for the determination of thiobarbituric acid-reactive substances (TBARS), carbonyl content, and water content. Samples to be processed for the determination of TBARS and carbonyl content were homogenized in 120 mM KCl, 30 mM phosphate buffer (pH 7.2), added with protein inhibitors (1 µg/ml leupeptin, 1 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 1 µg/ml pepstatin, and 0.5 mM PMSF) at 04°C. The suspensions were centrifuged at 600 x g for 10 min at 04°C to remove nuclei and cell debris. The pellets were discarded and the supernatants were used as homogenates.
Determination of TBARS.
For measurements of TBARS, homogenates were precipitated with 10% trichloroacetic acid (TCA), centrifuged, and incubated with thibarbituric acid (Sigma, Chem. Co.) for 1 h at 100°C. TBARS were extracted using butanol (1:1). After centrifugation, the fluorescence of the butanol layer was measured at 515nm excitation and 555 nm emission using a PTI spectrofluorometer (Photon Technology International, Lawrenceville, NJ). The amount of TBARS formed was expressed in picomoles per milligram of protein. Malondialdehyde standards were prepared from 1,1,3,3,-tetramethoxypropane (Esterbauer and Cheeseman, 1990). Protein concentration in homogenates was measured by Lowry method (Lowry et al., 1951
) using bovine serum albumin as standard. Measurements were carried out in a Perkin Elmer Lambda 40 spectrophotometer.
Determination of carbonyl content.
The content of carbonyl groups in oxidatively modified proteins was measured in lung homogenates by determining the amount of 2,4-dinitrophenylhydrazone formed upon reaction with 2,4-dinitrophenylhydrazine (Fields and Dixon, 1971). After precipitation of nucleic acids with 1% streptomycin sulfate, samples (>1mg of protein per ml) were treated with 2 mM 2,4-dinitrophenylhydrazine at room temperature, usually for 1 h. Proteins were precipitated with 10% TCA, washed with ethanol/ethyl acetate (1:1), and redissolved in 6 M guanidine hydrochloride20 mM potassium phosphate (pH 2.3) (Levine et al., 1994
). Carbonyl content was calculated from the absorbance maximum of 2,4-dinitrophenylhydrazone at 390 nm normalized to the absorbance at 350 nm, with an
390350 of 22 mM1 cm1 (Levine et al., 1994
). Results are expressed in nanomoles of carbonyl groups per milligram of protein.
Bronchoalveolar lavage.
Rats exposed to CAPs or filtered air were anesthetized with sodium pentobarbital (50mg/kg body weight) 24 h after exposure, and their lungs lavaged through the trachea using 5-ml aliquots of PBS (total volume, 50 ml). Each aliquot represent one in and out recovery of fluid. The recovered fluid was centrifuged (400g) at 4°C, and the supernatant from the first lavage was saved for measurement of protein level and lactate dehydrogenase (LDH) activity. Total cell counts were determined after trypan blue stain using a Newbauer chamber. Differential cell counts were performed using modified Wright-Giemsa stain in cytospin preparations (200 cells counted per sample). Total protein levels, as measure of vascular permeability, and LDH activity, as indicator of general toxicity, were measured in the supernatant of the first lavage using the method of Lowry et al. (Lowry et al., 1951) and standard kits (Sigma Chem. Co.), respectively. These measurements were carried out in a Perkin Elmer Lambda 40 spectrophotometer.
Lung edema.
Lung samples (100 mg), taken from the same animals used for the determinations of TBARS and carbonyl content samples were weighed and then dried in a convention oven (
80°C) and reweighed 24 h after to obtain the wet/dry ratios.
Histopathology.
The final pair of rats was euthanized using an overdose of sodium pentobarbital 24 h after exposure. The lungs were excised and fixed by intratracheal instillation of 2.5% glutaraldehyde in 0.1 M potassium phosphate buffer at constant pressure of 20 cm H2O. After fixation, all lung lobes (except for the cardiac lobe) were cut horizontally into uniform 2-mm sections with a guided razor blade. Each section was numbered, and one randomly selected section from each lobe was processed for histology. Tissue samples were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin for analysis by light microscopy. Histologic slides were coded for blinded observation, and the observer was unaware of the code until the analysis was completed.
Statistics.
The numbers in tables and the bars in figures indicate the mean value ± standard error of the mean (SEM) of 410 independent experiments. Data were analyzed statistically by factorial analysis of variance (ANOVA) followed by Fisher's test for comparison of the means. For elemental composition correlation analyses, separate linear regression models were fit using actual elemental concentration univariately as predictors. All statistical analyses were performed using Statview software for Macintosh.
Animal Care.
The Harvard School of Public Health is accredited by the American Association for the Accreditation of Laboratory Animal Care, meets National Institutes of Health standard as set forth in the "Guide for the Care and Use of Laboratory Animals," and accepts as mandatory the NIH "Principles for the Use of Animals." The principal investigator and laboratory personnel involved in this project have demonstrated competence in the care, use, and handling of laboratory animals. We give assurance of humane practice in animal maintenance and experimentation. All protocols of exposure and other procedures used in this study have been approved by the Harvard Animal Use Committee.
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RESULTS |
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DISCUSSION |
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Our data suggest that CAPs-dependent oxidative stress is associated with the development of lung inflammation. The ability of CAPs from different urban areas to promote lung inflammation has been previously reported. Inhalation exposure to CAPs from the New York area for 6 h was shown to increased the mRNA levels of proinflammatory cytokines (including IL-6, TNF-, and IFN-
) in the mice lung 24 h after exposure (mass concentration: 300 µg/m3) (Shukla et al., 2000
). Similarly, 3-day exposures to CAPs aerosols from the Boston area (5 h/day, mass concentration: 205733 µg/m3) were shown to decrease BAL macrophages and increase BAL lymphocytes, PMNs, and total protein in rat 24 h after exposure (Clarke et al., 1999
; Saldiva et al., 2002
). However, composition and timing seem to be essential variables in the development of the proinflammatory effects of CAPs, since similar protocols of exposure run in the Research Triangle Park area in North Carolina (3 days, 6 h/day, mass concentration: 2651200 µg/m3) did not elicit inflammatory responses when measured immediately after exposure (Kodavanti et al., 2000
).
In this study we found strong associations between the accumulation of oxidized lipids (TBARS) and the CAPs content of Al, Si, Fe, Cu, Pb, and K (Table 4). However, due to the limited size of the dataset and the less dramatic effect of CAPs inhalation on the lung carbonyl content and BAL PMN count, we could only see trends of association for these outcomes. Nonetheless, the suggested association between BAL PMN count and Zn is in agreement with previous data from the Godleski's group in a model of exposure to CAPs, for 6 h a day on three consecutive days (Clarke et al., 2000b; Saldiva et al., 2002
).
Also in agreement with previously reported data, we found that CAPs-exposed animals displayed bronchiolar inflammation (Saldiva et al., 2002) and thickening of blood vessels (Batalha et al., 2002
) (Fig. 3). These observations, combined with the increase in lung edema reported in a previous paper (Gurgueira et al., 2002
), indicate mild but significant toxicity by CAPs.
In vitro data show that, in some cases, the toxicity induced by PM emission components and surrogates can be ameliorated by antioxidants. Residual oil fly ash-induced mucin secretion and cytotoxicity in airway epithelial cells were attenuated by preadministration of dimethylthiourea (Jiang et al., 2002). NAC pretreatment was shown to significantly prevent TNF-
production in primary alveolar macrophages treated with ultrafine nickel particles (Dick et al., 2003
) and to protect a macrophage cell line (THP-1) against diesel exhaust particle chemicals (Li et al., 2002
). However, under similar experimental conditions, bronchial epithelial cells were not protected by NAC against diesel emission particles toxicity (Li et al., 2002
).
Due to its potential for clinical use, NAC has been extensively tested as a generic antioxidant in in vivo models of oxidant-mediated toxicity. NAC has been successfully used to prevent PMN influx and lung damage in models of exposure to cigarette smoke (Balansky et al., 1992), paraquat intoxication (Hoffer et al., 1993
), and carrageenan-induced pleurisy (Cuzzocrea et al., 2001
). The mechanism by which NAC prevents inflammation would include inhibition of ROS production in response to stimuli, with concomitant decreases in NF
B activation and expression of cytokine-induced neutrophil chemoattractant (Blackwell et al., 1996
). In models of preexisting inflammation, it has been shown that NAC can also modulate phagocytotic activity by suppressing PMN oxidative burst (Koch et al., 1996
; Stolarek et al., 2002
; Villagrasa et al., 1997
), and by potentiating host defense (Koch et al., 1996
; Villa et al., 2002
). On the other hand, treatment with NAC failed to prevent TNF-
-mediated MCP-1 up-regulation in vascular smooth muscle cells (De Keulenaer et al., 2000
), only reduced LPS-induced neutrophil infiltration at high doses (500 mg/kg) (Rockse et al., 2000
), and even transiently increased tissue damage and oxidative stress in humans subjected to acute muscle injury (Childs et al., 2001
).
Here, we applied an in vivo model of inhalation exposure to "real world" particles to demonstrate the central role of ROS in PM biological effects. Our data show that NAC, at a dose sufficient to prevent increase in ROS and accumulation of TBARS and to partially reduce protein oxidation, effectively prevented CAPs-induced inflammation. The observed preventive effect of NAC suggests that treatment with low doses of this antioxidant could be used to ameliorate the toxic effects of particulate air pollution.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Physiology Program, Department of Environmental Health, 665 Huntington Ave, Boston, MA 02115. Phone: (617) 432-1277. Fax: (617) 432-0014. E-mail: bgonzale{at}hsph.harvard.edu
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