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

Original Research COPD

Peroxynitrite Elevation in Exhaled Breath Condensate of COPO and Its Inhibition by Fudosteine* Grace O. Osoata, PhD; Toyoyuki Hanazawa, MD; Caterina Brindicci, MD; Misako Ito, BSc; Peter J. Barnes, DM, FCCP; Sergei Kharitonov, MD; and Kazuhiro Ito, PhD

Background: Peroxynitrite (PN) formed by the reaction of nitric oxide and superoxide is a powerful oxidantlnitrosant. Nitrative stress is implicated in COPD pathogenesis, but PN has not been detected due to a short half-life « I 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 nmollL; n = 10) were significantly higher than those of healthy volunteers (2.0 ± 1.1 nmollL; p < 0.0001; n 8) and smokers (2.8 ± 0.9 nmollL; p = 0.0017; n = 6). There was a good correlation between PN level and disease severity (FEV 1) 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 mmollL) reduced PN-enhanced interleukin (IL)-Ill-induced IL-8 release and restored corticosteroid sensitivity defected by PN more potently than those induced by H 2 0 2 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. (CHEST 2009; 135:1513-1520)

=

Abbreviations: BSA = bovine serum albumin; DCDHF = 2',T-dichIorofluorescein; EBC = exhaled breath condensate; EC so = median effective concentration; FDS = fudosteine; IC.5o = median inhibitory concentration; IL = interieukin; HRP = horseradish peroxidase; NAC = N-acetyI cysteine; NO = nitrite oxide; PBS = phosphate-buffered saline; PN = peroxynitrite, SIN-1 = 3-morpholinosydnonimine HCI; SOD = superoxide dismutase

Reactive nitrogen species (nitrosants) have been implicated in the pathogenesis of COPD.! An increased staining of the nitration marker 3nitrotyrosine 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 'From the Airway Disease Section, National Heart and Lung Institute, Imperial College London, London, 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. Manuscript received August 31, 2008; revision accepted November 29,2008. www.chesljournal.org

stress" may be exaggerated in the airways of patients. 2 ,3 One study:' 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,5-9 but we recently found that alveolar NO Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. orgtsite/misdreprints.xhtmI). Correspondence to: Kazuhiro Ito, PhD, Airway Disease Section, National Heart and Lung Institute. Dooehouse St, London SW3 6LY, UK; e-mail: [email protected] DOl: 1O.1378/chest.08-2105 CHEST 1135 1 61 JUNE, 2009

1513

was significantly elevated in COPD with disease severity," which is confirmed in another report.!" 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 (02-) to form peroxynitrite (ONOO-; PN).ll 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," metalloproteinase, p38MAPK,16 histone de acetylase 2,17 and transcriptional factors!" 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.l? Fudosteine ([-]-[R]-2amino-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 humanss" and

enhances ciliary beat impaired under cigarette smoke in vitro. 2 1 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 CO PO 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 AND METHODS 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); antinitrotyrosine antibody (Upstate; Charlottesville, VA); anti-BSA antibody (Serotec Ltd; Kidlington; Oxford, UK); anti-sheep! antimouse secondary antibodies (DakoCytomation; Clostrup, Denmark); 2',7'-dicWorofluorescein (DCDHF) [Invitrogen Ltd; Molecular Probe; Paisley, UK], and 3-morpholinosydnonimine HCI (SIN-I) [Qbiogene-Alexis Ltd; Nottingham, UK]. Subjects Eight healthy nonsmoking subjects (mean [:±: SD] age, 43.8 :±: 6.1 years; mean FEV!, 100 :±: 7.8% predicted), 6 smokers without COPD (mean age, 53.2:±: 10.1 years; mean FEV!, 102.7:±: 13.2% predicted), 10 subjects with moderate-to-severe COPD (mean age, 61.9 :±: 8.3 years; mean FEV!, 53.7 :±: 13.8% predicted), and 5 patients with mild asthma (mean age, 43 :±: 7.5 years; mean FEV!, 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 I-Characteristics of Subjects * Healthy Subjects Characteristics Gender, No. Male Female Age, yr FEV I' % predicted FEV /FVC ratio, % predicted Treatment

Smoking history, pack-yr

(n = 8)

6 2

Smokers (n = 6) 4 2 53.2 ::':: 10.1

43.8::':: 6.1 100 ::':: 7.8 83.9::':: 2.9

102.7::':: 13.2 79.9::':: 3.2

None

None

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Patients With Moderate-to-Severe COPD (n = 10)

Patients With Mild Asthma

6 4

4 1

61.9::':: 8.3 53.7::':: 13.8 46.6::':: 12.6

43.0::':: 7.5 92.6::':: 6.7 73.6::':: 3.4

Inhaled steroid (5 patients); combination therapy (4 patients); anticholinergic (8 patients); oral steroid (1 patient)

Albuterol only

35.8::':: 9.5

(n = 5)

o

*Values are given as the mean::':: SO, unless otherwise indicated. 1514

Original Research

Collection of EBC

Statistical Analysis

EBC was collected during 10 min of tidal breathing using a condenser (EcoScreen condenser; Jaeger; HOchberg, 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 umol/L). Cigarette smoking was stopped at least 1 h before EBC collection.

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, CAl. 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.

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.w 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 X 106 celis/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.w-" Different concentrations of PN (2 u.L) in 0.3 N NaOH (0, 1, 10, 100,200,300,400,500, and 1,000 nmollL) were

mixed with 2 floL of 0.3 N HCl, 7 floL of DCDHF stock solution, and 989 u.mol/L buffer (90 mmol/L sodium chloride, 50 mmollL 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 urnol/L diethylenetriaminepentaacetic acid added and readjusting the pH to 7.4 with 0,1 N HCl solution). Then, 100 u.L of EBC was added to the mixture of 7 u.L of DCDHF and 893 u.L 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 F'l ; Labtech International Ltd; Uckfield, UK). For the in vitro study, 1,000 nmollL PN (2 floL) was mixed with 2 floL of 0.3 N HCl, 7 floL of DCDHF stock solution, and 989 umol/L buffer after a 10-min pretreatment of different concentrations of FDS and NAC (0.01 to 100 u.mol/L). For sputum macrophaging, the wells were washed with the PN assay buffer, and cells were loaded with 100 u.mol/L DCDHF for 30 min. 28 The cells were washed and the fluorescence of the cells from each well was measured 30 min later.

RESULTS

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-I, 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 urnol/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 umol/L PN]. Stability of PN was found to be alkaline condition-dependent. As shown in Figure 1, top right, c, PN (1,000 nmollL) 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).

BSA Nitration by PN SIN-ll (500 umol/L) was added to BSA (1 flogt20 u.L phosphatebuffered saline [PBS) solution) and incubated at 37°C for 1 h in the presence of NaHC03 (200 mmoIlL). 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, CAl 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-l 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. www.chestjournal.org

PN Measurement in ERC in COPD Then 1,000 nmollL 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 nmollL, 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 nmollL at 10:00 AM and 6.4 ± 2.0 nmollL at 3:00 PM on the first day, and 6.4 ± 1.8 nmollL at 10:00 AM and 7.0 ± 3.2 nmollL at 3:00 PM on the second day (Fig 2, top left, a). NO and CHEST /135 / 6/ JUNE, 2009

1515

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FIGURE 1. PN measurement by DCDHF oxidation. PN oxidized DCDHF and induced fluorescent signal (top left, a). Effects of PN (open circle), SIN-I, a PN donor (closed reversed triangle), a mixture of H 2 0 2 and HRP (open diamond), and H 2 0 2 (closed triangle) on DCDHF oxidation were evaluated for a 3D-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, 50, and 180 min, and 24 h) in different conditions and mixed with DCDHF substrate (bottom right, d).

superoxide (0 2 - ) 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 urnol/L), PN level was decreased to almost half (8.7 ± 4.5 nmollL 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 nmollL and significantly higher than in healthy subjects (2.0 ± 1.1 nmollL; 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 FEVr (Spearman r = -0.88; P = 0.0016; n = 10) [Fig 2, bottom right, d] and FEV/FVC ratio (Spearman r = -0.93; P = 0.0003; n = 10).

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 1516

effective concentration (EC so) was 5.9 umol/L, which was similar to L-cysteine (6.3 urnol/L) but four times more potent than NAC (26.0 umol/L) [Fig 3, top right, b]. FDS also inhibited H 2 0 2-HRP-induced DCDHF oxidation with a lower efficacy (EC so to 43 u.mol/L) than that for PN-induced DCDHF oxidation and similar to NAC (38 umol/L). 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-l clearly nitrated BSA in vitro. Pretreatment of FDS inhibited PN-induced BSA nitration, and the potency (EC so to 3 urnol/L) was 13 times higher than NAC (EC so to 40 umol/L). PN was also produced from sputum macrophages. The PN level measured by DCDHF oxidation for 30 min was 190.8 ± 25.7 nmollL 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 mollL) significantly inhibited PN formation (42.3 ± 16.8 Original Research

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FIGURE 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 umol/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 (hottom left, c). Correlation between PN level in the presence of SOD and FEY" percent predicted (bottom right, d).

vs 190.8 ::t 25.7 nmol/L, respectively; p = 0.0047), but NAC (10- 4 mollL) had no significant effect (135.3 ::t 24.4 nmollL; p > 0.05).

Effect of FDS in PN-Induced Amplified IL-8 Production and Corticosteroid Insensitivity IL-1I3 significantly induced IL-8 production (nontreatment, 180 ::t 20 pglmL; IL-II3, 6,680 ::t 133 pgl mL) in A549 cells, which was enhanced by pretreatment of SIN-l (500 umol/L, mean, 11,050::t 484 pglmL). In addition, the ability of dexamethasone, a glucocorticoid, to inhibit IL-II3-induced IL-8 production was reduced 10-fold by pretreatment of SIN-l (dexamethasone-median inhibitory concentration [ICso] to 1.2 nmollL; dexamethasone-fCg., with SIN-l to 16.6 nmollL) [Fig 4, left]. Pretreatment of FDS inhibited SIN-I-enhanced IL-8 production by 80% (IL-II3, 6,500::t 133 pglmL; IL-1I3 + SIN-I, 11,050 ::t 484 pglmL; IL-II3 + SIN-I + FDS, 7,400 ::t 503 pglmL) and restored corticosteroid sensitivity (dexamethasone-ICg, with SIN1 + FDS, 3.4 nmol!L; dexamethasone-ICg., with SIN-I, 16.6 nmollL). However, NAC inhibited SIN-I-dependent IL-8 induction only by 36% (Fig 4, left) and did not restore www.chestjournal.org

steroid sensitivity. H 20 2 also enhanced IL-8 production (with H 20 2, 11,075::t 584 pglmL; without H 20 2 7,650::t 263 pglmL) and decreased steroid sensitivity (dexamethasone-K'g., in the presence of H 20 2, 3.5 vs 44.5 nmollL) [Fig 4, right], but FDS did not significantly inhibit them.

DISCUSSION

Elevation of oxidative/nitrative stress is one of the major characteristics of COPD.l 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.S" Although oxidation of rhodamine 123, which is oxidized by HCIO, PN, H 20 2 + HRP equally, has CHEST/135/6/ JUNE, 2009

1517

FIGURE 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 ofPN. EC so 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- I (500 umol/L). Representative image of western blotting for nitrated BSA (nBSA) and total BSA (upper panels). The band density was measured (lower panels). EC5Q 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/MannWhitney test, and comparisons of COPD with FDS/NAC treatment was made by one-way analysis of variance/Dunnett multiple comparison test.

been used for the detection of cellular oxidative/ nitrative stress,27 DCDHF was more sensitive to PN and SIN-I, 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.s" 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 0 2 - 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 1518

detected in EBC from all subjects. Because SOD does not affect DCDHF oxidation directly.?? 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." Furthermore, PN in EBC was more abundant in COPD than in smokers and healthy Original Research

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FIGURE 4. FDS inhibited PN-induced amplified IL-8 release and corticosteroid insensitivity. IL-113 (I ng/mLl-induced IL-8 release in the presence or absence of SIN-I (500 urnol/L) in A549 cells (top left). The value was shown as an increased IL-8 release by subtracting basal production without IL-113 in each case. A different concentration of dexamethasone was incubated for 30 min. SIN-I was added 30 min before dexamethasone treatment. FDS or NAC was pretreated 10 min before SIN-I addition. EC 5Il values were also calculated from the concentration-dependent curve (bottom left). IL-113 (I ng/mL)-induced IL-8 release in the presence or absence of H 20 2 (100 urnol/L) in A549 cells (top right). H 2 0 2 was added 30 min before dexamethasone treatment. EC so values were also calculated from the concentration-dependent curve (bottom right).

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 FEV 1 and the FEV 1 to FVC ratio (Fig 2, bottom right, d). Increasing evidence indicates that PN is involved in inflammation.v->' Our data also demonstrated that PN enhanced IL-ljj-induced IL-8 production. Nuclear factor-xb is known as a reactive oxygen species-sensitive transcriptional factor and causes IL-8 production." Iho and colleagues'< showed that nicotine produced PN and consequently induced IL-8 release via NF -KB activation. As we previously reported, nitrative stress caused enhancement of IL-8 production and reduced corticosteroid sensitivity via reduction of histone deacetylase.P-" 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. 2 1,38 ,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. www.chestioumal.orq

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.

REFERENCES

2

3

4

5

6

7

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