Deferoxamine

The present study tested the hypothesis that free radicals were involved in the pathogenesis of lung injury caused by diesel exhaust particles (DEP) and bacterial lipopolysaccharides (LPS). Intratracheal coinstillation of DEP and LPS in rat lungs resulted in synergistic enhancement of free radical generation in the lungs. The radical metabolites were characterized as lipid-derived by electron spin resonance (ESR). The free radical generation was paralleled by a synergistic increase in total protein and by infiltration of neutrophils in the bronchoalveolar lavage (BAL) fluid of the lungs. Experiments with NADP-reduced (NADPH) oxidase and iNOS knockout mice showed that NADPH oxidase and iNOS did not contribute to free radical generation. However, pretreatment with the macrophage toxicant GdCI^sub 3^, the xanthine oxidase (XO) inhibitor allopurinol, and the Fe^sup 111^ chelator Desferal resulted in a marked decrease in free radical generation, lung inflammation, and lung injury. These effects were concomitant with the inhibition of XO activity in BAL, suggesting that the activated macrophages and the activity of XO contributed to the generation of free radicals caused by DEP and LPS. This is the first demonstration that DEP and LPS work synergistically to enhance free radical generation in lungs, mediated by the activation of local XO.

Keywords: diesel exhaust particles; ESR spin trapping; free radicals; lipopolysaccharide; xanthine oxidase

The adverse health effects of particulate air pollution are of serious international concern. Fine-particle air pollutants less than 2.5 µm in aerodynamic diameter (PM^sub 2.5^) pose a particularly great risk to health, because fine particles (0.1-2.5 µm) reach the alveoli and are deposited there (1), whereas particles up to 10 µm reach only the proximal airways and are eliminated by mucociliary clearance. A number of cross-sectional studies have reported the association between daily mortality and fine particles (2,3). Fine particles from industrial sources and coal combustion are more significant contributors to human mortality than soil-derived particles.

Diesel exhaust particles (DEP) derived from diesel enginepowered automobiles are a major source of atmospheric PM^sub 2.5^. DEP contain an inert carbon core and a variety of organic compounds such as polyaromatic hydrocarbons (PAH), nitroaromatic hydrocarbons, heterocyclics, aldehydes, quinones, pyrenes (4-6), and traces of heavy metals (7). Experimental studies have shown that DEP cause lung injury (8-10). Human challenge studies with DEP reveal an increase in airway neutrophilic inflammation (11, 12).

It has been proposed that oxygen-centered free radicals such as Superoxide and hydroxyl radicals play an important role in the pathogenesis of DEP-induced lung injury because the formation of lung edema after intratracheal instillation of DEP into mice is markedly suppressed by pretreatment with polyethylene glycol-modified Superoxide dismutase (13). Metabolic activation of organic compounds such as quinones in DEP is also implicated in the mechanism of in vivo free radical generation (14). Although reactive oxygen species generated by DEP either directly or indirectly are believed to be associated with the adverse effects of DEP, no electron spin resonance (ESR) spin-trapping evidence of in vivo free radical generation by DEP has been reported.

A common component of paniculate matter, endotoxin, is implicated as an environmental stimulus that exacerbates lung diseases (15-19). Epidemiologic studies have reported that airborne endotoxin and respiratory symptoms (19, 20) are related to changes in lung function (19, 21). In animal studies, inhalation of endotoxin in a purified form known as lipopolysaccharide (LPS) has a strong potential to induce inflammation in the lung (22-24) through activation of transcriptional factors (25). In fact, bacterial endotoxin is present in bronchoalveolar lavage (BAL) fluid of patients with pneumonia (26), cystic fibrosis (27), and acute respiratory distress syndrome (28). Moreover, a recent experimental study has shown that coadministration of DEP and LPS exacerbated the development of acute lung injury, increasing neutrophil influx and cytokine release (29). Enhancement of acute lung injury related to bacterial endotoxin by the components of DEP has also been reported (30).

In this study, we tested the hypothesis that free radicals are generated after rats are treated simultaneously with DEP and LPS. After combined treatment with DEP and LPS, we applied the ESR spin-trapping technique to the detection of free radicals in the lung. Here we provide direct evidence that lipid-derived free radicals are a critical contributor to lung injury induced by DEP and LPS by mechanisms involving upregulation of the xanthine-xanthine oxidase (XO) system.

METHODS

Materials

Standard reference material SRM 2975 DEP was a gift from the National Institute for Standards and Technology (Gaithersburg, MD). α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) was obtained from Alexis (San Diego, CA). LPS (Escherichia coli O26:B6, contains not less than 500 EU/mg) from Sigma (St. Louis, MO) was used throughout this study. The endotoxin in saline was suspended by vortex. Allopurinol and deferoxamine mesylate (Desferal) also were obtained from Sigma. Gadolinium chloride hexahydrate (GdCl^sub 3^) was obtained from Aldrich (Milwaukee, WI). Pentobarbital (Abbott Laboratories, North Chicago, IL) and modified Wright's stain kit (Fisher Chemicals, Pittsburgh, PA) were used as received.

The endotoxin level in DEP, which was semiquantilicd by the Sigma E-TOXATE (Limulus Amoebocyte Lysate) test, was lower than the detection limit of 0.05 EU/ml in the vehicle solutions and DEP solutions. The endotoxin in LPS solutions and DEP + LPS solutions was 60 × 10^sup 4^ and 52 × 10^sup 4^ EU/ml, respectively. The value is the average of two determinations.

Animals and Treatment

Adult male Sprague-Dawley rats (250-300g; Charles River, Milmington, MA) were used and mice containing the disrupted gp91^sup phox^ gene (gp91^sup phox-/-^) and iNOS gene (iNOS^sup phox-/-^) obtained from The Jackson Laboratory (Bar Harbor, ME) were used. Age-matched mice of the C57BL\6 strain that possessed normal NADPH oxidase activity served as control animals for NADPH knockout experiments. The background of the iNOS-deficient mice is a mixture of two strains (C57BL/6 and SV129 mouse) (31); the control mice for iNOS knockout experiments were hybrids of these two strains. All mice were males 8 to 9 weeks of age. The animals were group-housed in a temperature-controlled room at 23 to 24°C with a 12/12-hour light/dark cycle and allowed free access to food and water. The studies adhered to the National Institutes of Health guidelines for the care and handling of experimental animals. All animal studies were approved by the Institutional Review Board.

The vehicle group received phosphate-buffered saline at pH 7.4 containing 0.05% Tween 80 (Sigma). The LPS group received 200 µg of LPS dissolved in the identical vehicle. The DEP group received suspended DEP (20 mg/kg) in the same vehicle. The DEP suspension was sonicated for 3 minutes under cooling conditions. The LPS + DEP group received the combined treatment of LPS and DEP. Each group (vehicle, LPS, DEP, or DEP + LPS) was dissolved in 0.3-ml aliquots and injected by the intratracheal route under anesthesia with 4% isoflurane (Baxter, Deerfield, IL). Another group of rats was intravenously pretreated with GdCl^sub 3^ (7 mg/kg) 18 hours before intratracheal instillation of DEP + LPS. Allopurinol (3 mg/kg orally) and Desferal (100 mg/kg, intraperitoncally) were given three times or twice a day, respectively, starting 24 hours before the intratracheal instillation with DEP + LPS. In the knockout mouse studies, the experimental procedure of the treatment with LPS, DEP, DEP + LPS, or the vehicle is essentially the same as in rats, but the dose of LPS was 50 µl/mouse.

ESR Studies

After the intratracheal instillation of LPS, DEP, DEP + LPS, or the vehicle, POBN was injected intraperitoneally (6 mmol/kg) under anesthesia with pentobarbital (30 mg/kg). Animals were killed 1 hour after administration, and the lipid phase of the lungs was extracted to quantify radical adduct content. Extraction was performed as previously described by Sato and colleagues (32) with some modification. Briefly, the lungs were homogenized in a mixture containing 4 ml 2:1 chloroform: methanol, 2 ml of 30 mM 2,2'-dipyridyl, 4 ml of 1.2 mM phenol, and 4 ml of deionized water using a homogenizer (Fisher Scientific PowerGen 125) under cooling conditions. The 2,2'-dipyridyl, a ferrous chelator, and the antioxidant phenol were used to inhibit ex vivo ferrousdependent and independent free radical generation. To the extraction, 36 ml 2:1 chloroform: methanol were added, shaken, and then centrifugea at 2,000 rpm for 20 minutes (Beckman Coulter, Inc., Fullerton, CA) (33, 34). The chloroform layer was then isolated and dried by passing through a sodium sulfate column.

After evaporating the sample by bubbling with N^sub 2^, ESR spectra were immediately recorded at room temperature using a quartz flat cell in a Bruker EMX EPR spectrometer equipped with a super-high Q cavity. Spectra were recorded on the IBM-compatible computer interfaced with the spectrometer using instrument settings of 9.79 GHz, 20.2 mW microwave power, 100 kHz modulation frequency, 1,300 millisecond conversion time, and 655 millisecond time constant. Simulations of ESR spectra were performed using the computer program WINSIM developed in this laboratory (35). The relative intensity of the signal was quantified by the WinEPR program provided by Bruker (Bruker Bio Spin Corporation, Billerica, MA).

BAL Fluid and Cell Counts

The trachea was cannulated after exsanguination. The lungs were lavaged with 10 ml of sterile saline containing 1 mM EDTA, instilled by syringe. The lavaged fluid was harvested by gentle injections and aspirations three times, and then centrifuged at 300 × g for 10 minutes. The total cell count was determined on a fresh fluid specimen using a hematocytometer. Differential cell counts were assessed on cytologie preparations. Slides were prepared using Cytospin (Shandon, Inc., Pittsburgh, PA) and stained with a modified Wright's stain. A total of 500 cells were counted under microscopy. Aliquots of the BAL supernatants were stored without further treatment at -80°C, and the total concentralions of protein in the supernatants were determined by the Bradford method (Bio-Rad) using bovine serum albumin as a standard.

Histologic Evaluation of Acute Lung Injury

After exsanguination, the lungs were fixed by intratracheal instillation with 10% neutral phosphate-buffered formalin at a pressure of 20 cm H2O for 72 hours. Slices of each pulmonary lobe 2 to 3 mm thick were embedded in paraffin. Sections 3 µm thick were stained with hematoxylin and eosin.

Quantitation of XO Activity in BAL Supernatant

XO activity was spectrophotometrically measured in BAL fluid of the lungs by monitoring the formation of uric acid from hypoxanthine as a substrate at 290 nm (36), employing a Beckman DU-640. Briefly, the BAL fluid of the lung was centrifuged at 400 × g for 10 minutes. The supernatant was filtered through an anisotropic membrane to remove the low molecular weight compounds (

Statistics

Data are expressed as mean ± SEM. Statistical significance of the difference was determined by Dunnett's multiple analysis. Differences between groups were considered statistically significant at the level of p

RESULTS

Synergistic Lung Free Radical Generation by DEP + LPS in Rats

To determine the combined effect of DEP and LPS on free radical generation, we evaluated the ESR signal of POBN radical adducts in lipid extracts of rat lungs 24 hours after the intratracheal instillations of DEP alone and in combination with 200 µg/rat LPS. In a previous study (32), we detected lipidderived free radical metabolites by intratracheal LPS dose of 500 µg/rat. We also found that using an LPS dose lower than 300 µg/rat would not yield free radical generation detectable by ESR spin-trapping technique. Therefore, to provide convincing evidence for the synergistic effect of DEP and LPS in this study, we chose a low LPS dose of 200 µg/rat, which alone would not generate free radicals at an ESR-detectable level.

As shown in Figure 1A, neither LPS (200 µg/rat) nor DEP given alone (20 mg/kg) gave an ESR spectra significantly higher than the control instillation. However, the combined instillation of DEP and LPS caused a marked increase in the six-line ESR spectrum of a radical adduct (Figure 1A, p

Synergistic Lung Injury and Neutrophilic Inflammation by DEP + LPS in Rats

For the animal model of lung injury caused by DEP, we chose an intratracheal dose of 20 mg/kg. Although there are reports that lower doses cause lung damage accompanied by free radical generation (29, 37), we were not able to detect free radicals using spin trapping at doses lower than 20 mg/kg (data not shown).

To determine the effect of DEP and LPS on lung injury, we evaluated the total concentration of protein in BAL fluid and lung specimens stained with hematoxylin and eosin 24 hours after intratracheal instillation (Figure 2). The instillation of LPS or DEP alone did not significantly change the protein concentrations in BAL fluid of the lungs as compared with vehicle administration alone. However, the combined administration of DEP and LPS resulted in a marked increase in the protein concentration in BAL fluid as compared with the LPS or DEP groups (p

To quantitate the magnitude of neutrophilic lung inflammation, we investigated the cellular profile of BAL fluid of the lungs 24 hours after the intratracheal instillations (Figure 2B). The instillation of LPS alone significantly increased the number of neutrophils in BAL fluid compared with vehicle administration alone (p

The histopathologic changes are shown in Figure 2C. In the LPS group, the infiltration of neutrophils was slight. A moderate infiltration of neutrophils was seen in the DEP group, especially around DEP accumulation sites. The combined instillation of DEP and LPS led to diffuse alveolar damage, including interstitial edema, infiltrating neutrophils, alveolar hemorrhage, and collapse of air spaces. Vehicle administration alone caused no histologie changes.

Computer Simulation of POBN Spectrum and Identification of Radical Species

After the combined instillation of DEP and LPS, we simulated the ESR spectra of POBN radical adducts (six-line radical adducts) in the lipid extracts of lungs using a computer program developed in this laboratory (35). The hyperfine coupling constants for the POBN adducts obtained in rats were a^sup N^ = 14.89 ± 0.04 G and a^sup H^β = 2.42 ± 0.02 G. These values were compared with the published hyperfine coupling constants to identify the radical species (32-34,38-39). The POBN radical adducts in the lung extracts instilled with LPS + DEP were very similar to other radical adducts identified as polyunsaturated fatty acid-derived. This result indicates that the POBN adducts induced by DEP + LPS reflect lipid-derived, carbon-centered radicals as a result of enhanced lipid peroxidation, a conclusion that is supported by previous in vivo (32-34) and in vitro studies (38-39).

GdCl^sub 3^ Decreased Lung Free Radical Generation, Neutrophilic Inflammation, and Protein Concentration in BAL Fluid Induced by DEP + LPS in Rats

To examine the role of phagocytes in free radical production in the lungs, we administered GdCl^sub 3^, an inhibitor of macrophage activation, 18 hours before the intratracheal instillation of DEP and LPS in combination. Intravenous administration of GdCl^sub 3^ (7 mg/kg) significantly decreased the ESR spectrum of POBN radical adducts in the lungs treated with DEP + LPS by ~ 50% (Figures 3A and 3B, p

The Effect of NADPH Oxidase Deficiency on Lung Free Radical Generation by DEP + LPS in Mice

To test the hypothesis that NADPH oxidase is the major source of free radical generation, we used gp91^sup phox^-deficient mice, which lack a critical membrane-bound subunit of this major source of reactive oxygen species in activated phagocytes. Combined instillation of DEP (20 mg/kg) and LPS (50 µg) into the lung led to a marked increase in the ESR spectrum of POBN radical adducts in the lungs of wild-type mice 24 hours after the instillation (p

The Effect of iNOS Deficiency on Lung Free Radical Generation by DEP + LPS in Mice

POBN adduct formation in the lungs of iNOS deficient mice was also examined after the combined instillation of DEP + LPS. LPS (50 µg) or DEP (20 mg/kg) alone gave a negligible six-line ESR signal 24 hours after instillation. In contrast, the typical POBN six-line radical adducts were observed in the lungs of iNOS wild-type mice treated with DEP 4 LPS in combination (p

Allopurinol and Desferal Inhibited Lung Free Radical Generation, Neutrophilic Inflammation, and Protein Concentration in BAL Fluid Induced by DEP + LPS in Rats

To evaluate whether xanthine/XO is involved in the mechanism of lung free radical generation, we examined the effects of allopurinol, a noncompetitive inhibitor of XO, and the ferric chelator Desferal on POBN adduct formation induced by DEP + LPS. We found that pretreatment with allopurinol three times per day (3 mg/kg by gavage) or Desferal twice per day (100 mg/kg, intraperitoneally) resulted in a marked reduction of the ESR spectrum of radical adduct in the lungs (p

Upregulation of XO Activity in BAL Fluid of the Lung by DEP + LPS and the Effects of GdCl^sub 3^, Allopurinol, and Desferal

To examine XO activity in the lungs, we measured uric acid, the end product of XO, in BAL fluid 24 hours after the instillation of vehicle, LPS, DEP, or the combination of DEP and LPS. XO activity in the BAL supernatant was markedly elevated after the instillation of DEP + LPS (p

GdCl^sub 3^, Allopurinol, and Desferal Ameliorated Histologic Changes in the Lungs Induced by DEP + LPS in Rats

Histopathologically, pretreatment with GdCl^sub 3^, allopurinol, or Desferal remarkably ameliorated alveolar damage induced by DEP + LPS such as interstitial edema, infiltrating neutrophils, alveolar hemorrhage, alveolar wall thickening, and collapse of airspace (Figure 8).

DISCUSSION

This study has provided the first demonstration that DEP and LPS work in synergy to form free radicals in the lung. This effect was paralleled by synergistic increases in total protein, infiltration of neutrophils in BAL fluid, and lung injury. GdCl^sub 3^ significantly inhibited free radical generation and lung inflammation. Furthermore, combined DEP- and LPS-stimulated free radical generation was unaffected by genetic inactivation of NADPH oxidase or iNOS, whereas pretreatment with the XO inhibitor allopurinol or the iron chelator Desferal markedly inhibited lung free radical generation, neutrophilic inflammation, and histologic changes by DEP and LPS. These effects are concomitant with inhibition of XO activity in B AL fluid of the lungs. These results suggest that activated macrophages and the local XO level, both potential sources of oxygen radicals, are important in the pathogenesis of lung injury by DEP and LPS.

The enzyme xanthine dehydrogenase (XDH) is the rate-limiting enzyme in purine metabolism. Under certain conditions, conversion of XDH into the oxidase form XO can lead to the generation of Superoxide and hydrogen peroxide. This generation of reactive oxygen species is thought to be the basis of XDWXO involvement in various pathologic conditions such as ischemia-reperfusion injury (40, 41), influenza virus infection (42), and neutrophil-mediated lung injury (43). In the present study, we found that the intratracheal coadministration of DEP and LPS synergistically enhanced free radicals in the lung, primarily in the form of lipid-derived free radicals detected as POBN radical adducts.

Our experiment with Desferal pretreatment may indicate that free iron plays a role in the formation of lipid-derived free radicals in the lung instilled with DEP and LPS. Desferal pretreatment decreased the amplitude of the signal generated by DEP + LPS by approximately 60%, and also ameliorated both total protein leakage in BAL fluid and histologie changes including interstitial edema and alveolar hemorrhage. These results may indicate that reactive oxygen species such as hydroxyl radical produced via a Fenton-like reaction are responsible for the formation of lipid-derived free radicals and are implicated in DEP- and LPS-induced lung injury.

Interestingly, Desferal pretreatment significantly reduced not only free radical generation but also neutrophil cell count in the BAL fluid, showing a decrease in neutrophil recruitment. It has been reported that alterations in leukocyte rigidity, proinflammatory cytokines, adhesion molecules, and chemotactic gradient via the local generation of chemotactic factors play an important role in the accumulation of leukocytes in lung inflammation (44). In a rat model of hepatic ischemia-reperfusion, treatment with Desferal attenuated tumor necrosis factor-α release, a major mediator of inflammation (45). Desferal has been shown to interfere with the adhesive functions of activated neutrophils (46). These findings suggest that in addition to the inhibition of iron's catalytic production of oxygen-derived radicals, Desferal-induced, antiinflammatory activities may be important in its protective effects. In addition, a previous report showed that Desferal decreased the activity of both XO and XDH in LPS-induced endothelial injury (47).

The major finding of this study is that DEP and LPS act synergistically to produce oxygen free radicals in the lung. Mechanistically, we first considered the possibility that free radicals would be produced by phagocytes because they express a multicomponent NADPH oxidase, the primary enzyme for the production of Superoxide in immune cells. Several studies regarding the effect of NADPH oxidase deficiency have been reported in complement-induced lung injury (48) and alcohol-induced liver disease (49). Free radical production in alcohol-induced liver disease, as detected by ESR, was inhibited in NADPH oxidase-deficient mice and is implicated in liver injury (49).

Surprisingly, in the present study, gp91^sup phox^-deficient mice that lack a critical membrane-bound subunit required for active NADPH oxidase were not resistant to the free radical generation in the lung induced by DEP and LPS, even though the neutrophil cell count drastically increased in BAL fluid. These results indicate that NADPH oxidase is not a main contributor to lung free radical generation or lung injury developed after exposure to DEP and LPS.

The effect of iNOS deficiency on combined DEP- and LPS-induced POBN adduct formation was also tested because either DEP or LPS alone induces iNOS expression in alveolar cells (50), and presumably produces peroxynitrite (51), a strong oxidant formed by the nearly diffusion-limited reaction of nitric oxide with Superoxide (52). However, the ESR spectrum of the radical adduct induced by DEP and LPS was unchanged in iNOS-deficient mice, indicating that NO derived from iNOS is not involved in the production of lipid-derived free radicals in the lung by DEP and LPS.

It has been reported that LPS induces increases in plasma XO activity (53). DEP- and LPS-stimulated production of free radicals was mainly mediated through the activation of XO because pretreatment with the XO inhibitor allopurinol resulted in a marked decrease in the formation of lipid-derived free radicals in the lung. The underlying mechanisms by which XO is regulated remain unclear. Our previous report demonstrated that LPS-stimulated production of free radicals was mediated through NADPH oxidase and not through xanthine oxidase (32). We attribute these differences to the different LPS doses used and different time points at which the enzyme activity was measured. Studies have shown that XDH/XO is upregulated in response to cytokines or hypoxia in endothelial cells and in an animal model of acute lung injury at the translational and posttranscriptional levels (54-56). For example, IFN-γ was found to be a potent inducer of XDH/XO gene expression and enzyme activity in rat pulmonary endothelial cells (57). XO enzyme activity is also enhanced in endothelial cells by tumor necrosis factor-α and chemotactic peptide (58). In addition, XDH/XO expression was enhanced under conditions associated with the inflammatory response in epithelial cells (59). These findings indicate that a variety of proinflammatory mediators play an important role in the regulation of XDH/XO activity during inflammation.

In the present study, we showed that DEP and LPS synergistically induced free radical generation and neutrophilic inflammation in the lung. DEP has been shown to release proinflammatory mediators from alveolar macrophages (60), airway epithelial cells, and endothelial cells that play a role in the pulmonary response (60). Other studies have shown that DEP suppress in vivo IFN-γ production by inhibiting cytokine effects on NK and NKT cells (61). It should be noted that other mechanisms of DEP toxicity may also operate, but their importance cannot be evaluated using the methodology reported here.

Because alveolar macrophages are key defenders of the lung and play an essential role in mediating the inflammatory response, we examined the effect of GdCl^sub 3^, a macrophage toxicant (32, 62), on free radical generation by DEP and LPS. The experiment with GdCl^sub 3^ pretreatment indicates that the participation of alveolar macrophages is indispensable for free radical generation, neutrophilic inflammation, and lung injury. The inhibition of neutrophilic infiltration by GdCl^sub 3^, which is probably the result of enzyme blockage and factors related to neutrophil migration and adhesion that are released from macrophages, has been reported in LPS- (32) and ozone-induced lung injury (63). Although NADPH oxidase was not responsible for the production of lipid-derived free radicals by DEP and LPS, proinflam matory mediators, including cytokines, chemokines, eicosanoids, and adhesion molecules released from phagocytes may play an important role in the free radical generation and lung injury induced by DEP and LPS.

Although the exact cause of DEP-induced pulmonary injury remains unknown, the interactions among a variety of environmental factors may set in motion a cascade of events leading to changes in lung function (64). One of those suspected factors is bacterial endotoxins because they are present in oral and nasal cavities and air pollutants. However, the interaction of DEP and bacterial endotoxin is little understood in the pulmonary reaction. The latter could be an important factor in the impairment of pulmonary function caused by particulate air pollution and the molecular mechanisms that regulate phagocyte cells in inflammation.

In conclusion, this is the first demonstration that DEP and LPS act synergistically to produce free radicals in the lung. Combined DEP- and LPS-induced free radical generation was not mediated through either NADPH oxidase or iNOS. The XO inhibitor allopurinol markedly inhibited free radical generation, neutrophilic inflammation, and lung injury by DEP and LPS. The synergistic production of free radicals by DEP and LPS is thus mediated, at least in part, through the increased local activity of XO, which appears to be important in the pathogenesis of combined DEP- and LPS-induced lung injury.

Conflict of Interest Statement: TA does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.B.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.P.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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Toyoko Arimoto, Maria B. Kadiiska, Keizo Sato, Jean Corbett, and Ronald P. Mason

Free Radical Metabolite Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina

(Received in original form February 26, 2004; accepted in final form October 4, 2004)

Correspondence and requests for reprints should be addressed to Ronald P. Mason, Ph.D., Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health (NIEHS/NIH), Research Triangle Park, NC 27709. E-mail: mason4@niehs.nih.gov

Am J Respir Crit Care Med Vol 171. pp 379-387, 2005

Originally Published in Press as DOI: 10.1164/rccm.200402-248OC on October 11, 2004

Internet address: www.atsjournals.org

Copyright American Thoracic Society Feb 15, 2005
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