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Original Research: COPD |

Peroxynitrite Elevation in Exhaled Breath Condensate of COPD and Its Inhibition by Fudosteine FREE TO VIEW

Grace O. Osoata, PhD; Toyoyuki Hanazawa, MD; Caterina Brindicci, MD; Misako Ito, BSc; Peter J. Barnes, DM, FCCP; Sergei Kharitonov, MD; Kazuhiro Ito, PhD
Author and Funding Information

*From the Airway Disease Section, National Heart and Lung Institute, Imperial College London, London, UK.

Correspondence to: Kazuhiro Ito, PhD, Airway Disease Section, National Heart and Lung Institute, Dovehouse St, London SW3 6LY, UK; e-mail: k.ito@imperial.ac.uk


This research was funded by Mitsubishi Pharma (Japan).

The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal.org/site/misc/reprints.xhtml).


© 2009 American College of Chest Physicians


Chest. 2009;135(6):1513-1520. doi:10.1378/chest.08-2105
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Background:  Peroxynitrite (PN) formed by the reaction of nitric oxide and superoxide is a powerful oxidant/nitrosant. Nitrative stress is implicated in COPD pathogenesis, but PN has not been detected due to a short half-life (< 1 s) at physiologic condition. Instead, 3-nitrotyrosine has been measured as a footprint of PN release.

Method:  PN was measured using oxidation of 2′,7′-dichlorofluorescein (DCDHF) in exhaled breath condensate (EBC) collected in high pH and sputum cells. The PN scavenging effect was also evaluated by the same system as PN-induced bovine serum albumin (BSA) nitration.

Results:  The mean (± SD) PN levels in EBC of COPD patients (7.9 ± 3.0 nmol/L; n = 10) were significantly higher than those of healthy volunteers (2.0 ± 1.1 nmol/L; p < 0.0001; n = 8) and smokers (2.8 ± 0.9 nmol/L; p = 0.0017; n = 6). There was a good correlation between PN level and disease severity (FEV1) in COPD (p = 0.0016). Fudosteine (FDS), a unique mucolytic antioxidant, showed a stronger scavenging effect of PN than N-acetyl-cysteine on DCDHF oxidation in vitro and in sputum macrophages, and also on PN-induced BSA nitration. FDS (0.1 mmol/L) reduced PN-enhanced interleukin (IL)-1β-induced IL-8 release and restored corticosteroid sensitivity defected by PN more potently than those induced by H2O2 in A549 airway epithelial cells.

Conclusion:  This noninvasive PN measurement in EBC may be useful for monitoring airway nitrative stress in COPD. Furthermore, FDS has the potential to inhibit PN-induced events in lung by its scavenging effect.

Figures in this Article

Reactive nitrogen species (nitrosants) have been implicated in the pathogenesis of COPD.1 An increased staining of the nitration marker 3- nitrotyrosine and of inducible nitric oxide (NO) synthase has been observed in induced sputum or cells from moderately stable COPD patients compared to nonsmokers, indicating that “nitrosative stress” may be exaggerated in the airways of patients.2,3 One study 4 showed that a higher number of 3-nitro-tyrosine positive cells was found in the submucosa of severe COPD patients compared to patients with mild/moderate COPD, smokers with normal lung function, and nonsmokers. Elevation of exhaled bronchial NO levels in COPD are controversial,59 but we recently found that alveolar NO was significantly elevated in COPD with disease severity,8 which is confirmed in another report.10 Thus, a lot of indirect evidence indicates that nitrative stress is abundant in COPD. However, there is no in vivo evidence bridging NO elevation and an increase in n-tyrosine deposition.

NO is a relatively unreactive radical, but it can form a potently reactive intermediate that affects protein function. NO reacts rapidly with superoxide (O2-) to form peroxynitrite (ONOO; PN).11 PN is an extremely powerful and cytotoxic oxidant in biologic systems, released predominantly by inflammatory cells at the site of injury in inflammatory disease and involved in tissue damage and airway inflammation.12,13 This can cause lipid peroxidation, DNA damage, and alterations in protein function in vitro. It also reacts with organic compounds or amino acids such as tyrosine, tryptophan, cysteine, and methionine residues. In general, nitrative stress is detected by measuring NO, nitrite/nitrate, and nitrotyrosine as a footprint of PN formation in clinical samples. It is extremely difficult to detect PN directly due to its short half-life (< 1 s at pH 7.4).

PN alters the function of certain proteins such as superoxide dismutase (SOD),14 glutathione s-transferase,15 metalloproteinase, p38MAPK,16 histone deacetylase 2,17 and transcriptional factors18 via nitration of tyrosine residues. These result in amplified inflammation and corticosteroid insensitivity, which are seen in COPD patients. The elimination of PN might be effective therapy for nitrative/oxidative stress-dominant pathogenesis, such as COPD. Antioxidants are under development, but the clinical effects of N-acetyl cysteine (NAC) were disappointing because of poor activity.19 Fudosteine ([-]-[R]-2-amino-3-[3-hydroxypropylthio] propionic acid; FDS) is a unique mucoactive agent launched in Japan in 1998.20 FDS inhibits lipopolysaccharide-induced goblet cell hyperplasia in rat lungs and humans20 and enhances ciliary beat impaired under cigarette smoke in vitro.21 FDS also showed 65% moderate improvement in the final global improvement rating of chronic respiratory diseases such as chronic bronchitis and pulmonary emphysema compared with placebo (24%) in a phase III study.22 The aim of this study was to detect PN in real time and noninvasively in exhaled breath condensate (EBC) obtained from COPD patients, and to evaluate a potent PN scavenging effect of FDS, with the potential outcome of improving inflammation and corticosteroid insensitivity induced under nitrative stress.

Materials

NAC, glycerol, SOD, and nitrated bovine serum albumin (BSA) were used (Sigma Ltd; Poole, UK). FDS was provided by Mitsubishi Pharma (Osaka, Japan). Other materials used included a complete protease inhibitor cocktail (Roche Diagnostics; Lewes, UK); PN (Calbiochem; Nottingham, UK); Bradford assay kit (Bio-Rad Laboratories; Hemel Hempstead, UK); anti-nitrotyrosine antibody (Upstate; Charlottesville, VA); anti-BSA antibody (Serotec Ltd; Kidlington; Oxford, UK); anti-sheep/antimouse secondary antibodies (DakoCytomation; Glostrup, Denmark); 2′,7′-dichlorofluorescein (DCDHF) [Invitrogen Ltd; Molecular Probe; Paisley, UK], and 3-morpholinosydnonimine HCl (SIN-1) [Qbiogene-Alexis Ltd; Nottingham, UK].

Subjects

Eight healthy nonsmoking subjects (mean [± SD] age, 43.8 ± 6.1 years; mean FEV1, 100 ± 7.8% predicted), 6 smokers without COPD (mean age, 53.2 ± 10.1 years; mean FEV1, 102.7 ± 13.2% predicted), 10 subjects with moderate-to-severe COPD (mean age, 61.9 ± 8.3 years; mean FEV1, 53.7 ± 13.8% predicted), and 5 patients with mild asthma (mean age, 43 ± 7.5 years; mean FEV1, 92.6 ± 6.7% predicted) were recruited (Table 1). This study was approved by the ethics committee of the Royal Brompton & Harefield Hospitals National Health Service Trust, and all subjects gave written informed consent.

Table Graphic Jump Location
Table 1 Characteristics of Subjects*

*Values are given as the mean ± SD, unless otherwise indicated.

Collection of EBC

EBC was collected during 10 min of tidal breathing using a condenser (EcoScreen condenser; Jaeger; Höchberg, Germany) as previously reported.23,24 Before collecting EBC, the collection tube was rinsed with 0.03 N NaOH in the presence or absence of SOD (10 μmol/L). Cigarette smoking was stopped at least 1 h before EBC collection.

Collection of Sputum Macrophages

Sputum was induced by nebulized hypertonic (3.5%) saline, and sputum cells were collected by < 1% dithiothreitol homogenization as previously described.25 After centrifugation at 3,000 revolutions/min for 5 min at 4°C, cells were resuspended in serum-free medium (Macrophage Medium; Invitrogen Ltd; Paisley, UK) [1 × 106 cells/mL] and sputum macrophage was collected by culture plate adhesion.

DCDHF Oxidation

A 14.5-mM stock solution of DCDHF was prepared as shown previously.26,27 Different concentrations of PN (2 μL) in 0.3 N NaOH (0, 1, 10, 100, 200, 300, 400, 500, and 1,000 nmol/L) were mixed with 2 μL of 0.3 N HCl, 7 μL of DCDHF stock solution, and 989 μmol/L buffer (90 mmol/L sodium chloride, 50 mmol/L sodium phosphate, 5 mM potassium chloride, pH 7.4, prepared with high-quality deionized water and passed over a Chelex-100 column to residual iron, with 100 μmol/L diethylenetriaminepentaacetic acid added and readjusting the pH to 7.4 with 0.1 N HCl solution). Then, 100 μL of EBC was added to the mixture of 7 μL of DCDHF and 893 μL of buffer and kept in dry ice. The mixture was incubated at 37°C for 30 min. The fluorescent signals were captured at 485-nm excitation and at 530-nm emission using a fluorometric plate reader (Biolite F1; Labtech International Ltd; Uckfield, UK).

For the in vitro study, 1,000 nmol/L PN (2 μL) was mixed with 2 μL of 0.3 N HCl, 7 μL of DCDHF stock solution, and 989 μmol/L buffer after a 10-min pretreatment of different concentrations of FDS and NAC (0.01 to 100 μmol/L). For sputum macrophaging, the wells were washed with the PN assay buffer, and cells were loaded with 100 μmol/L DCDHF for 30 min.28 The cells were washed and the fluorescence of the cells from each well was measured 30 min later.

BSA Nitration by PN

SIN-11 (500 μmol/L) was added to BSA (1 μg/20 μL phosphate- buffered saline [PBS] solution) and incubated at 37°C for 1 h in the presence of NaHCO3 (200 mmol/L). Nitration of BSA was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using immunoblot apparatus (Xcell SuperLock Mini-Cell and Blot module; Invitrogen). The band density was calculated by densitometry (UVP Bioimaging Systems; Upland, CA) using appropriate software (Labworks; Ultra-Violet Products; Cambridge, UK) and normalized to BSA expression, which was detected using anti-BSA antibody. FDS and NAC were incubated with BSA for 10 min before SIN-1 addition.

Enzyme-Linked Immunosorbent Assay

Interleukin (IL)-8 level in supernatant was determined by sandwich enzyme-linked immunosorbent assay (Duoset ELISA for human IL-8; R&D Systems Europe; Abingdon, UK) according to the manufacturer's instructions.

Statistical Analysis

Results are expressed as the mean ± SD. Analysis of variance was performed by the nonparametric Kruskal-Wallis test. When significant, a Mann-Whitney U test was performed for comparisons between groups using a statistical software package (GraphPad Prism; GraphPad Software Inc; San Diego, CA). The difference between treatment groups in the in vitro data was analyzed by one-way analysis of variance and the Dunnett multiple comparison test. Correlation coefficients were calculated using the Spearman rank method. Exact probability values were shown, and a p value < 0.05 was considered statistically significant. All p values are two-sided.

Optimization of DCDHF Oxidation by PN

PN was detected as fluorescence of dichlorofluorescein produced by oxidation of DCDHF (Fig 1, top left, a). As shown in Figure 1, bottom left, b, PN and SIN-1, a PN donor, concentration-dependently induced DCDHF oxidation, and the effects were more potent than hydrogen peroxide alone or a mixture of hydrogen peroxide and horseradish peroxidase (HRP). The sensitivity was > 0.008 μmol/L PN. In contrast, rhodamine 123, another molecular probe, could detect PN and hydrogen peroxide plus HRP equally (data not shown) [the sensitivity was > 0.045 μmol/L PN]. Stability of PN was found to be alkaline condition-dependent. As shown in Figure 1, top right, c, PN (1,000 nmol/L) was degraded completely in 30 min in < 0.003 N NaOH at 37°C, and the recovery rate was > 80% in > 0.03 N NaOH. The PN stability was compared between PBS solution (pH 7.4) at room temperature, PBS solution (pH 7.4) in dry ice, 0.03 N NaOH (pH approximately 12.2) at room temperature, or 0.03 N NaOH (pH approximately 12.2) in dry ice. In the condition of 0.03 N NaOH in dry ice, PN was stable up to 180 min (Fig 1, bottom left, b).

Figure Jump LinkFigure 1 PN measurement by DCDHF oxidation. PN oxidized DCDHF and induced fluorescent signal (top left, a). Effects of PN (open circle), SIN-1, a PN donor (closed reversed triangle), a mixture of H2O2 and HRP (open diamond), and H2O2 (closed triangle) on DCDHF oxidation were evaluated for a 30-min incubation (bottom left, b). Then 1,000 nmol/L PN was introduced into the DCDHF mixture in the presence of a different concentration of NaOH (different alkalized condition) [top right, c]. The PN stability was compared among normal PBS solution (pH 7.4) at room temperature (closed circle), normal PBS solution (pH 7.4) in dry ice (open circle), 0.03 N NaOH (pH approximately 12) at room temperature (closed triangle), and 0.03 N NaOH in dry ice (open triangle). The 1,000 nmol/L PN was kept for different periods (5, 30, 60, and 180 min, and 24 h) in different conditions and mixed with DCDHF substrate (bottom right, d).Grahic Jump Location
PN Measurement in EBC in COPD

Then 1,000 nmol/L PN was added to the collection tube prerinsed with 0.03 N NaOH and kept for 10 min at −20°C in a condenser (EcoScreen condenser; Jaeger). The sample was defrosted and added to the substrate mixture. The concentration of PN was 898 ± 121 nmol/L, and there was a sufficient recovery of introduced PN into the system (89.8%).

In the pilot study, EBC was collected in three patients with moderate COPD twice a day (10:00 am and 3:00 pm) for 2 consecutive days. The mean PN level was 6.6 ± 1.6 nmol/L at 10:00 am and 6.4 ± 2.0 nmol/L at 3:00 pm on the first day, and 6.4 ± 1.8 nmol/L at 10:00 am and 7.0 ± 3.2 nmol/L at 3:00 pm on the second day (Fig 2, top left, a). NO and superoxide (O2-) are expected to be exhaled and to produce PN outside the airway (or mouth) by their reaction to each other. When secondary PN formation was inhibited by SOD (10 μmol/L), PN level was decreased to almost half (8.7 ± 4.5 nmol/L without SOD; 4.5 ± 3.2 with SOD) [Fig 2, top right, b]. Therefore, all measurements were performed in the presence of SOD. As shown in Figure 2, bottom left, c, PN levels in patients with moderate-to-severe COPD were 7.9 ± 3.0 nmol/L and significantly higher than in healthy subjects (2.0 ± 1.1 nmol/L; p < 0.0001) and healthy smokers (2.8 ± 0.8 nmol/L; p = 0.0017). PN levels in patients with mild asthma (3.8 ± 1.7 nmol/L; p = 0.019) was significantly lower than that in patients with COPD. The PN level in COPD was inversely correlated with FEV1 (Spearman r = −0.88; p = 0.0016; n = 10) [Fig 2, bottom right, d] and FEV1/FVC ratio (Spearman r = −0.93; p = 0.0003; n = 10).

Figure Jump LinkFigure 2 PN measurement in COPD. PN was measured in EBC collected at 10:00 am and 3:00 pm on the first day and 10:00 am and 3:00 pm on the second day (top left, a). The total level of PN was compared in the presence and absence of SOD (10 μmol/L) [top right, b]. EBCs were collected from healthy subjects (Normal), smokers with normal lung function (Smoker), patients with moderate-to-severe COPD, and patients with mild asthma, and PN was measured in the presence of SOD (bottom left, c). Correlation between PN level in the presence of SOD and FEV1, percent predicted (bottom right, d).Grahic Jump Location
PN Scavenging Effect of FDS

The chemical structure of FDS is shown in Figure 3, top left, a. FDS concentration dependently inhibited PN-induced DCDHF oxidation and the median effective concentration (EC50) was 5.9 μmol/L, which was similar to L-cysteine (6.3 μmol/L) but four times more potent than NAC (26.0 μmol/L) [Fig 3, top right, b]. FDS also inhibited H2O2-HRP-induced DCDHF oxidation with a lower efficacy (EC50 to 43 μmol/L) than that for PN-induced DCDHF oxidation and similar to NAC (38 μmol/L).

Figure Jump LinkFigure 3 FDS has PN scavenging effect. Chemical structure of FDS, NAC, and L-cysteine (top left, a). Effects of FDS, NAC, and L-cysteine on PN-induced DCDHF oxidation (top right, b). These compounds were incubated for 10 min before addition of PN. EC50 was calculated and shown. Effects of FDS and NAC on PN-induced tyrosine nitration of BSA (bottom left, c). These compounds were incubated for 10 min before addition of SIN-1 (500 μmol/L). Representative image of western blotting for nitrated BSA (nBSA) and total BSA (upper panels). The band density was measured (lower panels). EC50 was also calculated and shown. DCDHF oxidation was measured in seeded sputum macrophages for 30 min (bottom right, d). PN level was calculated using PN standard curve. Comparisons between groups (healthy subjects, smokers, and COPD patients) were done using the Kruskal-Wallis/Mann-Whitney test, and comparisons of COPD with FDS/NAC treatment was made by one-way analysis of variance/Dunnett multiple comparison test.Grahic Jump Location

The effect of FDS on PN-induced nitration of tyrosine residue on BSA was also evaluated. As shown in Figure 3, bottom left, c, SIN-1 clearly nitrated BSA in vitro. Pretreatment of FDS inhibited PN-induced BSA nitration, and the potency (EC50 to 3 μmol/L) was 13 times higher than NAC (EC50 to 40 μmol/L).

PN was also produced from sputum macrophages. The PN level measured by DCDHF oxidation for 30 min was 190.8 ± 25.7 nmol/L in COPD patients (n = 4) and significantly higher than that of nonsmoking controls (24.0 ± 7.6 nmol/L; p = 0.029; n = 4). It was not significant but tended to be higher than in healthy smokers (111.5 ± 25.8 nmol/L; p = 0.057; n = 4). Pretreatment of an effective dose of FDS to eliminate 90% of PN formation (10−4 mol/L) significantly inhibited PN formation (42.3 ± 16.8 vs 190.8 ± 25.7 nmol/L, respectively; p = 0.0047), but NAC (10−4 mol/L) had no significant effect (135.3 ± 24.4 nmol/L; p > 0.05).

Effect of FDS in PN-Induced Amplified IL-8 Production and Corticosteroid Insensitivity

IL-1β significantly induced IL-8 production (nontreatment, 180 ± 20 pg/mL; IL-1β, 6,680 ± 133 pg/mL) in A549 cells, which was enhanced by pretreatment of SIN-1 (500 μmol/L; mean, 11,050 ± 484 pg/mL). In addition, the ability of dexamethasone, a glucocorticoid, to inhibit IL-1β-induced IL-8 production was reduced 10-fold by pretreatment of SIN-1 (dexamethasone-median inhibitory concentration [IC50] to 1.2 nmol/L; dexamethasone-IC50 with SIN-1 to 16.6 nmol/L) [Fig 4, left]. Pretreatment of FDS inhibited SIN-1-enhanced IL-8 production by 80% (IL-1β, 6,500 ± 133 pg/mL; IL-1β + SIN-1, 11,050 ± 484 pg/mL; IL-1β + SIN-1 + FDS, 7,400 ±503 pg/mL) and restored corticosteroid sensitivity (dexamethasone-IC50 with SIN1 + FDS, 3.4 nmol/L; dexamethasone-IC50 with SIN-1, 16.6 nmol/L). However, NAC inhibited SIN-1-dependent IL-8 induction only by 36% (Fig 4, left) and did not restore steroid sensitivity. H2O2 also enhanced IL-8 production (with H2O2, 11,075 ± 584 pg/mL; without H2O2 7,650 ± 263 pg/mL) and decreased steroid sensitivity (dexamethasone-IC50 in the presence of H2O2, 3.5 vs 44.5 nmol/L) [Fig 4, right], but FDS did not significantly inhibit them.

Figure Jump LinkFigure 4 FDS inhibited PN-induced amplified IL-8 release and corticosteroid insensitivity. IL-1β (1 ng/mL)-induced IL-8 release in the presence or absence of SIN-1 (500 μmol/L) in A549 cells (top left). The value was shown as an increased IL-8 release by subtracting basal production without IL-1β in each case. A different concentration of dexamethasone was incubated for 30 min. SIN-1 was added 30 min before dexamethasone treatment. FDS or NAC was pretreated 10 min before SIN-1 addition. EC50 values were also calculated from the concentration-dependent curve (bottom left). IL-1β (1 ng/mL)-induced IL-8 release in the presence or absence of H2O2 (100 μmol/L) in A549 cells (top right). H2O2 was added 30 min before dexamethasone treatment. EC50 values were also calculated from the concentration-dependent curve (bottom right).Grahic Jump Location

Elevation of oxidative/nitrative stress is one of the major characteristics of COPD.1 PN, produced by the reaction of superoxide and NO, is a powerful and cytotoxic oxidant,12,29 but it has not been detected in clinical samples due to its short half-life. Here we developed the quick measurement system of PN using DCDHF oxidation in samples collected noninvasively. Significant PN elevation was found in EBC obtained from COPD patients, which was confirmed in sputum macrophages. This is the first report to find elevation of PN in COPD, although a lot of reports have demonstrated elevation of nitrotyrosine deposition as a footprint of PN production.2,4

Although oxidation of rhodamine 123, which is oxidized by HCIO, PN, H2O2 + HRP equally, has been used for the detection of cellular oxidative/nitrative stress,27 DCDHF was more sensitive to PN and SIN-1, a PN donor, in our data and previous reports.27,28 In addition, the classic luminol method was less sensitive and not selective to PN (data not shown). Thus, the system we set up here with DCDHF under an alkaline condition will be more selective than another method. This oxidation was dependent on pH condition.26 DCDHF is oxidized well at pH 6.8 to 8.5. However, PN is not stable at this neutral condition (Fig 1, bottom right, d) and is more stable in a higher alkalized solution (Fig 1, top right, c). Therefore, we chose submaximal condition (0.03 N NaOH) for prerinsing the EBC collection tube, so as to adjust easily to a pH of approximately 7 to 8 following DCDHF oxidation reaction. In fact, prerinse of EBC collection tube with 0.03 N NaOH kept a sufficient recovery rate of PN at 89% in 1 h. Another factor influencing PN measurement is the contamination of secondarily synthesized PN by the reaction of O2- and NO after exhalation. As shown in Figure 2, top right, b, introduction of SOD into the EBC collecting tube inhibited half of the PN level detected in EBC from all subjects. Because SOD does not affect DCDHF oxidation directly,27 this suggests that almost half of PN detected in EBC is secondarily synthesized PN. Therefore, we measured PN levels in the presence of SOD in clinical samples. Thus, we overcame the problems of specificity, instability, and secondary formed PN.

The assessment of airway inflammation/cellular stress by biomarkers using noninvasive methods, especially EBC analysis, could be useful to recognize a signal to start anti-inflammatory treatment before the onset of symptoms and the impairment of lung function or to follow up the condition of patients with lung disease.30,31 A biomarker generally requires reproducibility, technical repeatability, and specificity. As shown in Figure 2, top left, a, the measurements were reasonably reproducible even in a limited number of samples. In addition, collection of EBC is a well-established noninvasive method and easy to do, suggesting it is very feasible and has good repeatability,32 although several limitations have been reported.31 Furthermore, PN in EBC was more abundant in COPD than in smokers and healthy subjects. Regarding disease specificity, we will need to collect from patients with several other diseases, such as severe asthma, pneumonia, and cystic fibrosis, in the future. At least we showed here that PN increases with severity of disease because the level was strongly correlated with FEV1 and the FEV1 to FVC ratio (Fig 2, bottom right, d).

Increasing evidence indicates that PN is involved in inflammation.33,34 Our data also demonstrated that PN enhanced IL-1β-induced IL-8 production. Nuclear factor-κB is known as a reactive oxygen species–sensitive transcriptional factor and causes IL-8 production.35 Iho and colleagues33 showed that nicotine produced PN and consequently induced IL-8 release via NF-κB activation. As we previously reported, nitrative stress caused enhancement of IL-8 production and reduced corticosteroid sensitivity via reduction of histone deacetylase.17,36 In addition, histone deacetylase activity and expression is reduced with the disease severity of COPD.37 Thus, elevation of PN might cause amplified inflammation and corticosteroid insensitivity in COPD.

FDS is a unique mucolytic agent with an antioxidant effect and inhibition of goblet cell hyperplasia in vitro.21,38,39 We have demonstrated here that FDS is a more potent antinitrosant than NAC. Thus, FDS might be useful in disease conditions exhibiting high levels of nitrative stress, such as COPD.

Taken together, the system for PN-dominant measurement will have benefits in monitoring nitrative stress and disease severity, and it is easily performed in a clinical laboratory. In addition, FDS could contribute to clarifying the pathogenesis of COPD and deserves further clinical investigation as a new class of PN scavenger.

BSA

bovine serum albumin

DCDHF

2′,7′-dichlorofluorescein

EBC

exhaled breath condensate

EC50

median effective concentration

FDS

fudosteine

IC50

median inhibitory concentration

IL

interleukin

HRP

horseradish peroxidase

NAC

N-acetyl cysteine

NO

nitrite oxide

PBS

phosphate-buffered saline

PN

peroxynitrite

SIN-1

3-morpholinosydnonimine HCl

SOD

superoxide dismutase

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Montuschi P, Collins JV, Ciabattoni G, et al. Exhaled 8- isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am J Respir Crit Care Med. 2000;162:1175-1177. [PubMed]
 
Keatings VM, Jatakanon A, Worsdell YM, et al. Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med. 1997;155:542-548. [PubMed]
 
Kooy NW, Royall JA, Ischiropoulos H. Oxidation of 2′,7′-dichlorofluorescin by peroxynitrite. Free Radic Res. 1997;27:245-254. [PubMed]
 
Crow JP. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitritein vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide. 1997;1:145-157. [PubMed]
 
Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med. 1999;27:612-616. [PubMed]
 
Beckman JS. Oxidative damage and tyrosine nitration from peroxynitrite. Chem Res Toxicol. 1996;9:836-844. [PubMed]
 
Montuschi P. Indirect monitoring of lung inflammation. Nat Rev Drug Discov. 2002;1:238-242. [PubMed]
 
Montuschi P. Review: analysis of exhaled breath condensate in respiratory medicine: methodological aspects and potential clinical applications. Ther Adv Respir Dis. 2007;1:5-23. [PubMed]
 
Zacharasiewicz A, Wilson N, Lex C, et al. Repeatability of sodium and chloride in exhaled breath condensates. Pediatr Pulmonol. 2004;37:273-275. [PubMed]
 
Iho S, Tanaka Y, Takauji R, et al. Nicotine induces human neutrophils to produce IL-8 through the generation of peroxynitrite and subsequent activation of NF-κB. J Leukoc Biol. 2003;74:942-951. [PubMed]
 
Zouki C, Jozsef L, Ouellet S, et al. Peroxynitrite mediates cytokine-induced IL-8 gene expression and production by human leukocytes. J Leukoc Biol. 2001;69:815-824. [PubMed]
 
Rahman I, Adcock IM. Oxidative stress and redox regulation of lung inflammation in COPD. Eur Respir J. 2006;28:219-242. [PubMed]
 
Ito K, Yamamura S, Essilfie-Quaye S, et al. Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-κB suppression. J Exp Med. 2006;203:7-13. [PubMed]
 
Ito K, Ito M, Elliott WM, et al. Decreased histone deacetylase activity in chronic obstructive pulmonary disease. N Engl J Med. 2005;352:1967-1976. [PubMed]
 
Komatsu H, Yamaguchi S, Komorita N, et al. Inhibition of endotoxin- and antigen-induced airway inflammation by fudosteine, a mucoactive agent. Pulm Pharmacol Ther. 2005;18:121-127. [PubMed]
 
Takahashi K, Kai H, Mizuno H, et al. Effect of fudosteine, a new cysteine derivative, on mucociliary transport. J Pharm Pharmacol. 2001;53:911-914. [PubMed]
 

Figures

Figure Jump LinkFigure 1 PN measurement by DCDHF oxidation. PN oxidized DCDHF and induced fluorescent signal (top left, a). Effects of PN (open circle), SIN-1, a PN donor (closed reversed triangle), a mixture of H2O2 and HRP (open diamond), and H2O2 (closed triangle) on DCDHF oxidation were evaluated for a 30-min incubation (bottom left, b). Then 1,000 nmol/L PN was introduced into the DCDHF mixture in the presence of a different concentration of NaOH (different alkalized condition) [top right, c]. The PN stability was compared among normal PBS solution (pH 7.4) at room temperature (closed circle), normal PBS solution (pH 7.4) in dry ice (open circle), 0.03 N NaOH (pH approximately 12) at room temperature (closed triangle), and 0.03 N NaOH in dry ice (open triangle). The 1,000 nmol/L PN was kept for different periods (5, 30, 60, and 180 min, and 24 h) in different conditions and mixed with DCDHF substrate (bottom right, d).Grahic Jump Location
Figure Jump LinkFigure 2 PN measurement in COPD. PN was measured in EBC collected at 10:00 am and 3:00 pm on the first day and 10:00 am and 3:00 pm on the second day (top left, a). The total level of PN was compared in the presence and absence of SOD (10 μmol/L) [top right, b]. EBCs were collected from healthy subjects (Normal), smokers with normal lung function (Smoker), patients with moderate-to-severe COPD, and patients with mild asthma, and PN was measured in the presence of SOD (bottom left, c). Correlation between PN level in the presence of SOD and FEV1, percent predicted (bottom right, d).Grahic Jump Location
Figure Jump LinkFigure 3 FDS has PN scavenging effect. Chemical structure of FDS, NAC, and L-cysteine (top left, a). Effects of FDS, NAC, and L-cysteine on PN-induced DCDHF oxidation (top right, b). These compounds were incubated for 10 min before addition of PN. EC50 was calculated and shown. Effects of FDS and NAC on PN-induced tyrosine nitration of BSA (bottom left, c). These compounds were incubated for 10 min before addition of SIN-1 (500 μmol/L). Representative image of western blotting for nitrated BSA (nBSA) and total BSA (upper panels). The band density was measured (lower panels). EC50 was also calculated and shown. DCDHF oxidation was measured in seeded sputum macrophages for 30 min (bottom right, d). PN level was calculated using PN standard curve. Comparisons between groups (healthy subjects, smokers, and COPD patients) were done using the Kruskal-Wallis/Mann-Whitney test, and comparisons of COPD with FDS/NAC treatment was made by one-way analysis of variance/Dunnett multiple comparison test.Grahic Jump Location
Figure Jump LinkFigure 4 FDS inhibited PN-induced amplified IL-8 release and corticosteroid insensitivity. IL-1β (1 ng/mL)-induced IL-8 release in the presence or absence of SIN-1 (500 μmol/L) in A549 cells (top left). The value was shown as an increased IL-8 release by subtracting basal production without IL-1β in each case. A different concentration of dexamethasone was incubated for 30 min. SIN-1 was added 30 min before dexamethasone treatment. FDS or NAC was pretreated 10 min before SIN-1 addition. EC50 values were also calculated from the concentration-dependent curve (bottom left). IL-1β (1 ng/mL)-induced IL-8 release in the presence or absence of H2O2 (100 μmol/L) in A549 cells (top right). H2O2 was added 30 min before dexamethasone treatment. EC50 values were also calculated from the concentration-dependent curve (bottom right).Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 Characteristics of Subjects*

*Values are given as the mean ± SD, unless otherwise indicated.

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Hanazawa T, Kharitonov SA, Barnes PJ. Increased nitrotyrosine in exhaled breath condensate of patients with asthma. Am J Respir Crit Care Med. 2000;162:1273-1276. [PubMed]
 
Montuschi P, Collins JV, Ciabattoni G, et al. Exhaled 8- isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am J Respir Crit Care Med. 2000;162:1175-1177. [PubMed]
 
Keatings VM, Jatakanon A, Worsdell YM, et al. Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med. 1997;155:542-548. [PubMed]
 
Kooy NW, Royall JA, Ischiropoulos H. Oxidation of 2′,7′-dichlorofluorescin by peroxynitrite. Free Radic Res. 1997;27:245-254. [PubMed]
 
Crow JP. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indicators of peroxynitritein vitro: implications for intracellular measurement of reactive nitrogen and oxygen species. Nitric Oxide. 1997;1:145-157. [PubMed]
 
Wang H, Joseph JA. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic Biol Med. 1999;27:612-616. [PubMed]
 
Beckman JS. Oxidative damage and tyrosine nitration from peroxynitrite. Chem Res Toxicol. 1996;9:836-844. [PubMed]
 
Montuschi P. Indirect monitoring of lung inflammation. Nat Rev Drug Discov. 2002;1:238-242. [PubMed]
 
Montuschi P. Review: analysis of exhaled breath condensate in respiratory medicine: methodological aspects and potential clinical applications. Ther Adv Respir Dis. 2007;1:5-23. [PubMed]
 
Zacharasiewicz A, Wilson N, Lex C, et al. Repeatability of sodium and chloride in exhaled breath condensates. Pediatr Pulmonol. 2004;37:273-275. [PubMed]
 
Iho S, Tanaka Y, Takauji R, et al. Nicotine induces human neutrophils to produce IL-8 through the generation of peroxynitrite and subsequent activation of NF-κB. J Leukoc Biol. 2003;74:942-951. [PubMed]
 
Zouki C, Jozsef L, Ouellet S, et al. Peroxynitrite mediates cytokine-induced IL-8 gene expression and production by human leukocytes. J Leukoc Biol. 2001;69:815-824. [PubMed]
 
Rahman I, Adcock IM. Oxidative stress and redox regulation of lung inflammation in COPD. Eur Respir J. 2006;28:219-242. [PubMed]
 
Ito K, Yamamura S, Essilfie-Quaye S, et al. Histone deacetylase 2-mediated deacetylation of the glucocorticoid receptor enables NF-κB suppression. J Exp Med. 2006;203:7-13. [PubMed]
 
Ito K, Ito M, Elliott WM, et al. Decreased histone deacetylase activity in chronic obstructive pulmonary disease. N Engl J Med. 2005;352:1967-1976. [PubMed]
 
Komatsu H, Yamaguchi S, Komorita N, et al. Inhibition of endotoxin- and antigen-induced airway inflammation by fudosteine, a mucoactive agent. Pulm Pharmacol Ther. 2005;18:121-127. [PubMed]
 
Takahashi K, Kai H, Mizuno H, et al. Effect of fudosteine, a new cysteine derivative, on mucociliary transport. J Pharm Pharmacol. 2001;53:911-914. [PubMed]
 
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