N-Acetylcysteine Prevents Lung Inflammation After Short-Term Inhalation Exposure to Concentrated Ambient Particles

Claudia Ramos Rhoden, Joy Lawrence, John J. Godleski and Beatriz González-Flecha1

Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115

Received January 7, 2004; accepted February 26, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lung inflammation is a key response to increased levels of particulate air pollution (PM); however, the cellular mechanisms leading to this response are poorly understood. To determine whether oxidants are implicated in PM-dependent lung inflammation, we tested the ability of N-acetylcysteine (NAC) to prevent lung inflammation in a rat model of short-term exposure to concentrated ambient particles (CAPs). Adult Sprague-Dawley rats were exposed to either CAPs aerosols (CAPs mass concentration 1060 ± 300 µg/m3) or filtered air (Sham controls) for 5 h. NAC-treated rats received 50 mg/kg (ip) NAC 1 h prior to exposure to CAPs. Oxidative stress and recruitment of inflammatory cells into bronchoalveolar lavage were evaluated 24 h after removal of the animals from the exposure chamber. Rats breathing CAPs aerosols showed significant oxidative stress, determined by the accumulation of thiobarbituric reactive substances (TBARS, 90 ± 15 pmol/mg protein; sham control: 50 ± 5 pmol/mg protein, p < 0.02) and oxidized proteins (1.6 ± 0.4 nmol/mg protein, sham: 0.70 ± 0.02 nmol/mg protein, p < 0.01) in their lungs. CAPs-induced oxidative stress was associated with increased numbers of polymorphonuclear leukocytes in bronchoalveolar lavage (BAL) (9 ± 2%; sham: 1.6 ± 0.5%, p < 0.001) and slight lung edema (wet/dry ratio: 4.77 ± 0.03, sham: 4.69 ± 0.02). No significant change was found in BAL protein concentration, total cell count, or lactate dehydrogenase (LDH) activity. NAC pretreatment effectively prevented CAPs-induced TBARS accumulation (30 ± 10 pmol/mg protein, p < 0.006), lung edema (4.64 ± 0.08, p < 0.05), and polymorphonuclear neutrophil (PMN) influx into the lungs (2.1 ± 0.5%, p < 0.001), but did not alter the protein carbonyl content. Histological evaluation of tissue samples confirmed the BAL findings. CAPs-exposed animals showed slight bronchiolar inflammation and thickened vessels at the bronchiole, whereas NAC treated animals showed no histological alterations. Regression analyses showed strong associations between increased TBARS accumulation and the CAPs content of Al, Si, and Fe, and trends of association between carbonyl content and Cr and Na concentrations, and between BAL PMN count and Cr, Zn, and Na. These data demonstrate that oxidants are critical mediators of the inflammatory response elicited by PM inhalation.

Key Words: reactive oxygen species; oxidative stress; particulate air pollution; inflammation; CAPs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ambient air particles are complex mixtures of organic and inorganic components that may vary in proportion and composition daily. Epidemiological studies show that transient high-level increases in ambient particulate matter (PM) of respirable size are strongly associated with increased exacerbation of respiratory disease, cardiopulmonary morbidity, and mortality (reviewed in Schwela, 2000Go). The respiratory effects include exacerbation of preexisting diseases, such as asthma and obstructive pulmonary disease, and increased hospital admissions for pneumonia.

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, 2001Go; Ghio et al., 2001Go), dogs (Clarke et al., 2000bGo; Godleski et al., 2000Go), and rats (Clarke et al., 1999Go, 2000aGo; Hatch et al., 1985Go; Kennedy et al., 1998Go; Li et al., 1997Go; Saldiva et al., 2002Go). Critical components of the inflammatory response to PM are the release of macrophage- and pneumocyte-derived cytokines (Kennedy et al., 1998Go; Li et al., 1997Go) and the concomitant recruitment of neutrophils (Ferin et al., 1992Go; Kennedy et al., 1998Go; Li et al., 1996Go, 1997Go).

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-{alpha}, and ß, TGF-ß, {gamma}-IF, IL-6, and IL-8 among others, Shukla et al., 2000Go) are regulated by redox-sensitive transcription factors such as NF-{kappa}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., 2000Go; Kennedy et al., 1998Go; Shukla et al., 2000Go). 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., 2000Go; Kennedy et al., 1998Go; Shukla et al., 2000Go).

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., 2002Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Concentrated ambient particles (CAPs).
The Harvard Ambient Particle Concentrator (HAPC) concentrates ambient air particles for subsequent aerosol exposure of animals (Sioutas et al., 1995Go). The principle of virtual impaction is used to concentrate ambient particles in the size range of 0.1–2.5 µm (fine particles, concentration factor: 26 ± 4, [Sioutas et al., 1995Go]). CAPs remain in suspension without physical or chemical alteration for inhalation exposures or for collection onto filters for mass and composition analysis. Mass concentrations and the size of the particles, determined gravimetrically and using a microorifice impactor, respectively, were monitored continuously during the operation of the HAPC. Trace metal concentrations were determined using x-ray fluorescence (Chester LabNet, Tigrad, OR). The CAPs total mass and elemental composition of the CAPs used in this study are presented in Table 1. The average CAPs composition was comparable to the values obtained previously (Gurgueira et al., 2002Go). Twelve out of the eighteen metals analyzed were present at equivalent concentrations. However, in average, these samples had three-fold higher concentrations of Al, Zn, and Br, five-fold higher concentrations of S and Mn, a four-fold higher total mass concentration, and almost no Cl.


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TABLE 1 Elemental analysis of CAPs

 
Exposure to CAPs.
Pathogen-free male Sprague Dawley rats (Taconic Farms, Germantown, NY) weighing 250–300 g were used. Animals were fed a conventional laboratory diet and water ad libitum. Rats were exposed to CAPs aerosols (CAPs group) or filtered air (control group) as described previously (Clarke et al., 2000aGo). The animals were awake and unrestricted during the exposures. The CAPs and control groups were exposed and tested simultaneously. The exposure was repeated ten times on ten different days from June 20 to August 16, 2002 (average CAPs mass concentration: 1228 µg/m3, Table 1). Each exposure was carried out for 5 h and included animals exposed to CAPs or filtered air (sham controls) with and without N-acetylcysteine pretreatment. Four animals (1 per group: CAPs, CAPs/NAC, sham, and sham/NAC) were assessed, 24 h after exposure, for oxidative stress and tissue damage, and eight animals (two per group) were assessed for markers of inflammation. On July 31, August 9, 14, and 16, additional animals (two per group each day) were added to the exposure protocol to be processed for histopathology. The temperature in the room and chamber was 25°C.

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 0–4°C. The suspensions were centrifuged at 600 x g for 10 min at 0–4°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, 1990Go). Protein concentration in homogenates was measured by Lowry method (Lowry et al., 1951Go) 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, 1971Go). 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 hydrochloride–20 mM potassium phosphate (pH 2.3) (Levine et al., 1994Go). Carbonyl content was calculated from the absorbance maximum of 2,4-dinitrophenylhydrazone at 390 nm normalized to the absorbance at 350 nm, with an {epsilon}390–350 of 22 mM–1 cm–1 (Levine et al., 1994Go). 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., 1951Go) 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 4–10 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative Stress and Oxidative Damage in Rats Exposed to CAPs Aerosols
In agreement with our previous findings, inhalation exposure to CAPs led to significant increases in the accumulation of oxidized lipids (Fig. 1A) and proteins (Fig. 1B). Rats breathing CAPs for 5 h showed greater than two-fold increases in their pulmonary levels of TBARS (Fig. 1A) and protein carbonyls (Fig. 1B). Pretreatment of the animals with 50 mg/kg NAC, 1 h prior to the initiation of the exposure effectively prevented the CAPs-dependent increases in oxidized lipids (Fig. 1A) and partially prevented the accumulation of oxidized proteins (Fig. 1B).



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FIG. 1. Oxidant damage in CAPs-exposed rats. Rats were exposed to CAPs (average mass concentration: 1060 ± 300 µg/m3) or filtered air (sham controls) for 5 h. At the end of the exposure the lungs were removed, processed, and assayed for TBARS (A) and protein carbonyl content (B) as described in Materials and Methods. The bars represent the mean of ten determinations ± SEM. *p < 0.0005 in (A) and p < 0.01 in (B) compared to the respective sham controls. **p < 0.0002 in (A) and p < 0.002 in (B) compared to CAPs.

 
Oxidant-Dependent Inflammation and Toxicity after CAPs Inhalation
To test the hypothesis that PM-induced pulmonary inflammation and toxicity are mediated by ROS, we evaluated the effect of NAC pretreatment on a series of markers of inflammation and damage in rats exposed to CAPs. Bronchoalveolar lavage fluid was collected from animals exposed to CAPs aerosols or filtered air for 5 h and evaluated for total and differential cell count, LDH activity, and total protein levels as described in the methods section. Inhalation exposure to CAPs led to significant accumulation of PMN leukocytes in BAL (Fig. 2 and Table 2). PMN influx was not accompanied by significant changes in LDH activity and total protein levels (Table 3). CAPs-dependent lung inflammation was effectively prevented by preadministration of NAC (Fig. 2 and Table 2). Histological evaluation of tissue samples from animals treated with CAPs or filtered air in the presence or absence of NAC confirmed the BAL findings (Fig. 3). CAPs-exposed animals showed slight bronchiolar inflammation and thickened vessels at the bronchiole (Fig. 3B) as previously reported (Batalha et al., 2002Go; Saldiva et al., 2002Go). However, CAPs-exposed animals pretreated with NAC showed no inflammation in the bronchiolar location and thin-walled vessels (Fig. 3C).



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FIG. 2. PMN influx in CAPs-exposed rats. BAL samples were collected 24 h after the end of the exposure to CAPs. Total and differential cell counts were performed as described in Materials and Methods. The bars represent the mean of ten determinations ± SEM. *p < 0.0004 compared to the respective sham controls; **p < 0.001 compared with CAPs exposure.

 

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TABLE 2 Differential Cell Count in BAL Fluid from Rats Exposed to CAPs

 

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TABLE 3 LDH Activity and Total Protein in BAL Fluid from Rats Exposed to CAPs

 


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FIG. 3. Morphological appearance of lung tissue in selected areas illustrating the responses of control and NAC-treated rats to CAPs exposure. (A) In sham rats, the bronchoalveolar area has normal histology. (B) In CAPs-exposed rats, thickened vessels walls (arrow in vessel) and slight inflammation in the area of the bronchiolar alveolar junction are visible (see arrows in lung). (C) In CAPs-exposed rats pretreated with NAC, lung histology was normal. Scale bar = 50 µm.

 
Finally, NAC treatment significantly decreased the wet/dry ratio, a measurement of global tissue damage, in CAPs exposed animals (Fig. 4).



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FIG. 4. Lung edema in CAPs-exposed rats. Animals were exposed to CAPs as described in Fig. 1. Lung samples were collected simultaneously for the determination of the wet/dry ratios. The bars represent the mean of four determinations ± SEM. *p < 0.05 compared to CAPs exposure.

 
Metal Content as a Determinant of CAPs Pulmonary Effects
One of the advantages of working with CAPs aerosols is that they reflect the day-to-day changes in composition typical of urban environments, and in that way they provide a set of samples with a wide range of metal concentrations suitable for statistical analyses (Table 1). Using univariate regression we identified several components with significant associations to increased TBARS accumulation in the lung (Table 4). Due to the strong effect of CAPs inhalation on this outcome, many elements show positive correlations to the TBARS levels. However, the stronger and more significant associations were found for Al, Si, and Fe (Table 4). We failed to see significant associations between metal concentrations and lung carbonyl content or PMN influx into the lung, probably due to the somewhat limited number of days analyzed. However, there was a trend of association between carbonyl content and Cr and Na concentrations, and between BAL PMN count and Cr, Zn, and Na (Table 4).


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TABLE 4 Statistical Parameters for Lung TBARS, Carbonyl Content, and PMN Count, and CAPs Elemental Components

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of PM to increase the intracellular production of ROS, although assumed to be essential for their biological effects, has not been extensively tested in vivo. In a recent report we showed that inhalation exposure to CAPs, following the same protocol used in this paper, increases by two-fold the steady-sate concentration of ROS in the rat lung and heart (Gurgueira et al., 2002Go). Our current data show that CAPs-dependent increases in ROS are sufficient to cause accumulation of oxidized lipids and proteins (Fig. 1). CAPs exposure led to about a two-fold increase in the levels of TBARS and protein carbonyls, the same magnitude of increase previously observed for ROS concentrations (Gurgueira et al., 2002Go). Pretreatment of the animals with 50 mg/kg NAC (ip) effectively prevented accumulation of oxidants as shown by TBARS (Fig. 1A). On the other hand, NAC treatment could only partially prevent accumulation of oxidized proteins in CAPs-exposed animals (Fig. 1B). This relative inefficiency could reflect constant accumulation of oxidized proteins over the 5 h of exposure and consumption of NAC.

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-{alpha}, and IFN-{gamma}) in the mice lung 24 h after exposure (mass concentration: 300 µg/m3) (Shukla et al., 2000Go). Similarly, 3-day exposures to CAPs aerosols from the Boston area (5 h/day, mass concentration: 205–733 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., 1999Go; Saldiva et al., 2002Go). 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: 265–1200 µg/m3) did not elicit inflammatory responses when measured immediately after exposure (Kodavanti et al., 2000Go).

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., 2000bGo; Saldiva et al., 2002Go).

Also in agreement with previously reported data, we found that CAPs-exposed animals displayed bronchiolar inflammation (Saldiva et al., 2002Go) and thickening of blood vessels (Batalha et al., 2002Go) (Fig. 3). These observations, combined with the increase in lung edema reported in a previous paper (Gurgueira et al., 2002Go), 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., 2002Go). NAC pretreatment was shown to significantly prevent TNF-{alpha} production in primary alveolar macrophages treated with ultrafine nickel particles (Dick et al., 2003Go) and to protect a macrophage cell line (THP-1) against diesel exhaust particle chemicals (Li et al., 2002Go). However, under similar experimental conditions, bronchial epithelial cells were not protected by NAC against diesel emission particles toxicity (Li et al., 2002Go).

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., 1992Go), paraquat intoxication (Hoffer et al., 1993Go), and carrageenan-induced pleurisy (Cuzzocrea et al., 2001Go). The mechanism by which NAC prevents inflammation would include inhibition of ROS production in response to stimuli, with concomitant decreases in NF{kappa}B activation and expression of cytokine-induced neutrophil chemoattractant (Blackwell et al., 1996Go). In models of preexisting inflammation, it has been shown that NAC can also modulate phagocytotic activity by suppressing PMN oxidative burst (Koch et al., 1996Go; Stolarek et al., 2002Go; Villagrasa et al., 1997Go), and by potentiating host defense (Koch et al., 1996Go; Villa et al., 2002Go). On the other hand, treatment with NAC failed to prevent TNF-{alpha}-mediated MCP-1 up-regulation in vascular smooth muscle cells (De Keulenaer et al., 2000Go), only reduced LPS-induced neutrophil infiltration at high doses (500 mg/kg) (Rockse et al., 2000Go), and even transiently increased tissue damage and oxidative stress in humans subjected to acute muscle injury (Childs et al., 2001Go).

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.


    ACKNOWLEDGMENTS
 
We thank Mr. Gonzalo Bedia-Diaz for technical assistance. C.R.R. acknowledges funding from the FFFCMPA (Brazil). This work was supported by grants from NIH (RO1 HL/ES68073 and P01 ES08129), and a Research Award from the U.S. EPA (R827353).


    NOTES
 
Data presented in part at the Annual Meeting of the American Thoracic Society 2003, Seattle, Washington.

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


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Balansky, R. B., D'Agostino, F., Zanacchi, P., and DeFlora, S. (1992). Protection by N-acetylcysteine of the histopathological and cytogenetical damage produced by exposure of rats to cigarette smoke. Cancer Lett. 64, 123–131.[ISI][Medline]

Batalha, J. R., Saldiva, P. H., Clarke, R. W., Coull, B. A., Stearns, R. C., Lawrence, J., Murthy, G. G., Koutrakis, P., and Godleski, J. J. (2002). Concentrated ambient air particles induce vasoconstriction of small pulmonary arteries in rats. Environ. Health Perspect. 110, 1191–1197.[ISI][Medline]

Blackwell, T. S., Blackwell, T. R., Holden, E. P., Christman, B. W., and Christman, J. W. (1996). In vivo antioxidant treatment suppresses nuclear factor-kappa B activation and neutrophilic lung inflammation. J. Immunol. 157, 1630–1637.[Abstract]

Childs, A., Jacobs, C., Kaminiski, T., Halliwell, B., and Leeuwenburgh, C. (2001). Supplementation with vitamin C and N-acetyl-cysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise. Free Radic. Biol. Med. 31, 745–753.[CrossRef][ISI][Medline]

Clarke, R. W., Catalano, P. J., Coull, B., Koutrakis, P., Krishna Murthy, G. G., Rice, T., and Godleski, J. J. (2000a). Age-related responses in rats to concentrated urban air particles (CAPs). Inhal. Toxicol. 12, 73–84.[CrossRef][ISI]

Clarke, R. W., Catalano, P. J., 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, 637–656.[CrossRef][ISI][Medline]

Clarke, R. W., Coull, B., Reinisch, U., Catalano, P., Killingsworth, C., Koutrakis, P., Kavouras, I., Krishna Murthy, G. G., Lawrence, J., Lovett, E., et al. (2000b). Inhaled concentrated ambient particles are associated with hematological and bronchoalveolar lavage changes in canines. Environ. Health Prespect. 108, 1179–1187.

Cuzzocrea, S., Mazzon, E., Dugo, L., Serraino, I., Centorrino, T., DeSarro, A., and Caputti, A. P. (2001). Protective effects of N-acetylcysteine on lung injury and red blood cell modification induced by carrageenan in the rat. FASEB J. 15, 1187–1200.[Abstract/Free Full Text]

De Keulenaer, G. W., Ushio-Fukai, M., Yin, O., Chung, A. B., Lyons, P. R., Ishizaka, N., Rengarajan, K., Taylor, W. R., Alexander, R. W., and Griendling, K. K. (2000). Convergence of redox sensitive and mitogen-activated protein kinase signaling pathways in tumor necrosis factor-alpha-mediated monocyte chemoattractant protein-1 in vascular smooth muscle cells. Arteroscler. Thromb. Vasc. Biol. 20, 385–391.[Abstract/Free Full Text]

Dick, C. A. J., Brown, D. M., Donaldson, K., and Stone, V. (2003). The role of free radicals in the toxic and inflammatory effects of four different ultrafine particle types. Inhal. Toxicol. 15, 39–52.[ISI][Medline]

Esterbauer, H., and Cheeseman, K. H. (1990). Determination of aldehydic lipid peroxidation products: Malonaldehyde and 4-hydroxynonenal. Meth. Enzymol. 186, 407–421.[Medline]

Ferin, J., Oberdorster, G., and Penney, D. P. (1992). Pulmonary retention of ultrafine and fine particles in rats. A. J. Respir. Cell. Mol. Biol. 6, 535–542.[ISI]

Fields, R., and Dixon, H. B. F. (1971). Micro method for the determination of reactive carbonyl groups in proteins and peptides using 2,4-dinitrophenyl-hydrazine. Biochem. J. 121, 587–593.[Medline]

Ghio, A. J., and Devlin, R. B. (2001). Inflammatory lung injury after bronchial instillation of air pollution particles. Am. J. Respir. Crit. Care Med. 164, 704–708.[Abstract/Free Full Text]

Ghio, A. J., Kim, C., and Devlin, R. B. (2001). Concentrated ambient air particles induce mild pulmonary inflammation in healthy human volunteers. Am. J. Respir. Crit. Care Med. 162, 981–988.[ISI]

Godleski, J. J., Verrier, R. L., Koutrakis, P., Catalano, P., Coull, B., Reinisch, U., Lovett, E. G., Lawrence, J., Murthy, G. G., Wolfson, et al. (2000). Mechanisms of morbidity and mortality from exposure to ambient air particles. Res. Rep. Health Eff. Inst. 91, 5–88.[Medline]

Gurgueira, S. A., Lawrence, J., Coull, B., Krishna Murthy, G. G., and González-Flecha, B. (2002). Rapid increases in the steady-state concentration of reactive oxygen species in the lungs and heart after particulate air pollution inhalation. Environ. Health Perspect. 110, 749–755.

Hatch, G. E., Boykin, E., Graham, J. A., Lewtas, J., Pott, F., Loud, K., and Mumford, J. L. (1985). Inhalable particles and pulmonary host defense: In vivo and in vitro effects of ambient air and combustion particles. Environ. Res. 36, 67–80.[ISI][Medline]

Hoffer, E., Avidor, J., Benjaminov, O., Shenker, L., Tabak, A., Tamir, A., Merzbach, D., and Taitelman, U. (1993). N-acetylcysteine delays the infiltration of inflammatory cells into the lungs of parqaut-intoxicated rats. Toxicol. Appl. Pharmacol. 120, 8–12.[CrossRef][ISI][Medline]

Jiang, N., Dreher, K. L., Dve, J. A., Li, Y., Richards, J. H., Martin, L. D., and Adler, K. B. (2002). Residual oil fly ash induces cytotoxicity and mucin secretion by guinea pig tracheal epithelial cells via an oxidant-dependent mechanism. Tox. Appl. Pharmacol. 163, 221–230.

Jimenez, L. A., Thompson, J., Brown, D. A., Rahman, I., Antonicelli, F., Duffin, R., Drost, E. M., Hay, R. T., Donaldson, K., and MacNee, W. (2000). Activation of NF-kappaB by PM(10) occurs via an iron-mediated mechanism in the absence of IkappaB degradation. Toxicol. Appl. Pharmacol. 166, 101–110.[CrossRef][ISI][Medline]

Kennedy, T., Ghio, A. J., Reed, W., Samet, J., Zagorski, J., Quay, J., Carter, J., Dailey, L., Hoidal, J. R., and Devlin, R. B. (1998). Copper-dependent inflammation and nuclear factor-kappaB activation by particulate air pollution. Am. J. Respir. Cell Mol. Biol. 19, 366–378.[Abstract/Free Full Text]

Koch, T., Heller, S., Heissler, S., Breil, J., Schiefer, H. G., van Ackern, K., and Neuhof, H. (1996). Effects of N-acetylcysteine on bacterial clearance. Eur. J. Clin. Invest. 26, 884–892.[ISI][Medline]

Kodavanti, U. P., Mebane, R., Ledbetter, A., Krantz, T., McGee, J., Jackson, M. C., Walsh, L., Hilliard, H., Chen, B. Y., Richards, J., et al. (2000). Variable pulmonary responses from exposure to concentrated ambient air particles in a rat model of bronchitis. Toxicol. Sci. 54, 441–451.[Abstract/Free Full Text]

Levine, R. L., Williams, J. A., Stadtman, E. R., and Shacter, E. (1994). Carbonyl assays for determination of oxidatively modified proteins. Meth. Enzymol.. 233, 346–357.[ISI][Medline]

Li, N., Wand, M., Oberley, T. D., Sempf, J. M., and Nel, A. E. (2002). Comparison of the pro-oxidant and pro-inflammatory effects of organic diesel exhaust particle chemicals in bronchial epithelial cells and macrophages. J. Immunol. 169, 4531–4541.[Abstract/Free Full Text]

Li, X. Y., Gilmour, P. S., Donaldson, K., and MacNee, W. (1996). Free radical activity and pro-inflammatory effects of particulate air pollution (PM10) in vivo and in vitro. Thorax 51, 1216–1222.[Abstract]

Li, X. Y., Gilmour, P. S., Donaldson, K., and MacNee, W. (1997). In vivo and in vitro proinflammatory effects of particulate air pollution (PM10). Environ. Health Perspect. 105 (Suppl. 5), 1279–1283.[ISI][Medline]

Lowry, O. H., Rosembrough, A. L., Farr, A. L., and Randall, R. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry. 193, 265–275.[Free Full Text]

Rockse, D., Lilliebook, B., Larsson, R., Jobansson, T., and Bucht, A. (2000). Differential anti-inflammatory and anti-oxidative effects of dexamethasone and N-acetylcysteine in endotoxin-induced lung inflammation. Clin. Exp. Immunol. 122, 249–256.[CrossRef][ISI][Medline]

Saldiva, P. I., Clarke, R. W., Coull, B., Stearns, R. C., Lawrence, J., Murthy, G. G., Diaz, E., Koutrakis, P., Suh, H., Tsuda, A., et al. (2002). Lung Inflammation induced by concentrated air particles is related to particle composition. Am. J. Resp. Crit. Care Med. 165, 1610–1617.[Abstract/Free Full Text]

Schwela, D. (2000). Air pollution and health in urban areas. Rev. Environ. Health 15, 13–42.[Medline]

Shukla, A., Timblin, C., BeruBe, K., Gordon, T., McKinney, W., Driscoll, K., Vacek, P., and Mossman, B. T. (2000). Inhaled particulate matter causes expression of nuclear factor (NF)-kappaB-related genes and oxidant-dependent NF-kappaB activation in vitro. Am. J. Resp. Cell Mol. Biol. 23, 182–187.[Abstract/Free Full Text]

Sioutas, C., Koutrakis, P., and Burton, R. M. (1995). A technique to expose animals to concentrated fine ambient aerosols. Envrion. Health Perspect. 103, 172–177.

Stolarek, R., Biaasiewicz, P., and Mowak, D. (2002). N-acetylcysteine effect on luminol-dependent chemiluminescence pattern of reactive oxygen species generation by human polymorphonuclear leukocytes. Pulmonary Pharmacology & Therapeutics 15, 385–392.[CrossRef][ISI][Medline]

Villa, P., Saccani, A., Sica, A., and Ghezzi, P. (2002). Glutathione protects mice from lethal sepsis by limiting inflammation and potentiating host defense. J. Infect. Diseases 185, 1115–1120.[CrossRef][ISI][Medline]

Villagrasa, V., Cortio, J., Marti-Calvera, M., Ortiz, J. L., Berto, L., Esteras, A., Bruseghinin, L., and Morcillo, E. J. (1997). Inhibitory effect of N-acetylcysteine on superoxide anion generation in human polymorphonuclear leukocytes. J. Pharm. Pharmacol. 49, 525–529.[ISI][Medline]