Divisions of 1Pulmonary Medicine and 2Gastroenterology, Second Department of Internal Medicine, Hospital of Johann Wolfgang Goethe University, Frankfurt/Main, Germany
Submitted 16 September 2004 ; accepted in final form 30 December 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
airway secretions; oxidants; respiratory epithelium
Given this as a background, it is surprising that, in sharp contrast to chronic obstructive pulmonary disease patients, H2O2 is not elevated in breath condensate samples of CF patients (14, 24). This lack of exhaled H2O2 may be explained by an efficient scavenging in CF airways. Previously (6), we reported high levels of GSH and glutathione peroxidase in CF sputum samples. These data are well in line with the findings by Worlitzsch et al. (24), who reported high levels of catalase in CF sputum samples. Catalase, GSH, and glutathione peroxidase are the principal antioxidants detoxifying H2O2. We hypothesized that antioxidants in CF airway secretions effectively detoxify H2O2 and tested the H2O2-scavenging properties of CF airway secretions, obtained via sputum induction, in an in vitro H2O2 cytotoxicity model.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
H2O2 cytotoxicity. 16HBE14o- cells (a gift from Dieter Gruenert, Univ. of California at San Francisco) were used to study H2O2 cytotoxicity in the presence of CF sputum. 16HBE14o- cells were cultured in MEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. The cells were collected by trypsinization, counted (Trypan blue exclusion), seeded on multiwell plates at concentration of 1.0 x 106 viable cells/ml, and allowed to grow for 24 h in MEM (10% FBS). After 24 h of incubation, cells reached 100% confluence, cell culture medium was renewed, and cells were cultured for additional 24 h. Then, the cells were exposed to H2O2 diluted from the 30% stock solution (Sigma-Aldrich Chemie, Munich, Germany). Dilutions were made in serum-/antibiotic-free MEM or MEM (10% FBS, antibiotic-free) (both as controls) or MEM (10% CF sputum, antibiotic-free). The effects of H2O2 were examined as described below.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. Confluent cells grown on 96-well plates were incubated for 24 h with 01,000 µM H2O2 in either serum-/antibiotic-free MEM or MEM (10% FBS). After exposure, cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test (17). The viability was expressed as a percentage of the viability of control cells exposed to 0 µM H2O2. The H2O2 concentration that caused more than 50% decrease in cell viability was considered cytotoxic.
Detection of intracellular oxidants. Dihydrorhodamine 123 (DHR123; Mo Bi Tec, Göttingen, Germany) was used to detect intracellular oxidants. DHR123 is a nonfluorescent, noncharged dye that easily penetrates cell membrane. Once inside the cell, DHR123 reacts with intracellular oxidants to yield rhodamine, a highly fluorescent compound. The rhodamine fluorescence is directly proportional to the amount of DHR123 oxidized by intracellular oxidants and can be detected by, e.g., flow cytometry.
For the current studies, 16HBE14o- cells grown on 12-well plates were loaded for 30 min with 5 µM DHR123 in serum-/antibiotic-free MEM. Subsequently, the cells were exposed for 2 h to H2O2 in serum-/antibiotic-free MEM, MEM (10% FBS), or MEM (10% CF sputum). After incubation, cell culture supernatants were aspirated, cells were trypsinized, and cell fluorescence was analyzed using flow cytometer (FACSCalibur, BD Biosciences; excitation with a 488-nm argon laser, emission acquired in red channel). The instrument settings were adjusted to set the autofluorescence of unstained, unstimulated cells between 100 and 101 log on the x-axis. In each experiment, the fluorescence intensity of 10,000 cells was analyzed. Data are demonstrated as a histogram of fluorescence intensity (x-axis) vs. relative cell number (y-axis) using WinMDI version 2.8 software (http://facs.scripps.edu/software.html, developed by J. Trotter).
Cell images. 16HBE14o- cells grown on LabTec cell chambers (Nunc, Wiesbaden, Germany) were exposed to H2O2 for 12 h. Cell images were captured using a phase-contrast, inverted microscope equipped with a videocamera (Zeiss, Jena, Germany).
Viability assay with propidium iodide. 16HBE14o- cells grown on 12-well plates were exposed to H2O2 for 24 h. After incubation, cell supernatants were transferred into sterile 12 x 75-mm polystyrene test tubes to collect detached cells. Attached cells were trypsinized and combined with respective supernatants. Cells were stained for 5 min in the dark with a solution of propidium iodide (PI, 1 µg/ml) in phosphate-buffered saline (PBS, Invitrogen). After incubation, cells were briefly washed with PBS to remove unbound PI, and fluorescence intensity was analyzed on flow cytometer (red channel).
Viable and dead cells can be easily distinguished from their fluorescence intensity (viable cells exhibiting low vs. dead cells with high fluorescence intensity) (20). In each experiment, 10,000 cells were analyzed, and distribution of PI fluorescence was demonstrated as a histogram of fluorescence intensity (x-axis) vs. relative cell number (y-axis).
Reduced thiols and GSH in CF sputum extracts.
For this study, CF mucus plugs were diluted in PBS to obtain 10% solution. CF sputum extracts were obtained as described above. Reduced thiols in CF sputum samples were probed using 5,5'-dithio(bis)2-nitrobenzoic acid (DTNB) (10). In reaction 1:
![]() | (1) |
This reaction can be made specific for GSH by adding glutathione reductase (GR) and NADPH to reactions 2 and 3 (Ref. 1).
![]() | (2) |
![]() | (3) |
Inactivation of catalase and heme peroxidases by NaN3 and exposure of 16HBE14o- cells to H2O2. NaN3 is a broad inhibitor of catalase and heme peroxidases. To inactivate these enzymes, CF sputum extracts were incubated with 1 mM NaN3 (1 h, 4°C). In preliminary experiments, NaN3 completely inhibited 1,000 mU/ml of catalase, the activity comparable to the one reported by Worlitzsch et al. (24).
16HBE14o- cells were incubated with H2O2 in different tested media ± NaN3. Exposed cells were analyzed for cell viability (PI staining).
Data analysis. Each experiment was repeated with at least four different CF sputum samples. Where appropriate, results are expressed as means (± SE). Data were compared by Student's t-test for paired comparisons using the SPSS statistical package.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intracellular oxidant levels. It was assumed that exposure of cells to H2O2 will result in a significant accumulation of intracellular oxidants unless H2O2 is detoxified before reaching the cell layer. When the cells were exposed to 600 µM H2O2 in either serum-free MEM or MEM (10% FBS), there was a marked increase in fluorescence intensity due to oxidation of DHR123 by intracellular oxidants (Fig. 1, A and B). In contrast, this was not the case in cells overlaid with CF sputum and H2O2. DHR123 oxidation was completely abolished, and cells exposed to the oxidant exhibited the same fluorescent intensity as control cells (Fig. 1C).
|
|
|
Reduced thiols and GSH in CF sputum extracts. CF sputum extracts (10% in PBS) were probed for the presence of GSH and other reduced thiols. After DTNB was added, CF sputum extracts exhibited strong absorbance at 412 nm, indicating a high content of reduced thiols (Fig. 4). In the case of CF sputum extracts, the increase in OD412 nm after incubation with DTNB in CF far exceeded the changes in OD412 nm in positive controls (1 or 8 µM GSH) (Fig. 4).
|
Both low- (e.g., GSH, cysteine, etc.) and high-molecular-weight (i.e., SH-protein) reduced thiols can increase OD412 nm in a reaction with DTNB. The contribution of protein thiols to the reduction of DTNB was estimated in the following experiment. Sputum proteins were acid precipitated and removed by high-speed centrifugation. After the pH of the supernatant, now containing only acid-soluble, low-molecular-weight thiols, was restored to 7.4 and DTNB was added, the increase in OD412 nm was profoundly lower than in the presence of proteins (data not shown). This indicates that a significant portion of reduced thiols in CF sputum can be attributed to sputum proteins.
Inactivation of catalase and heme peroxidases by NaN3 and exposure of 16HBE14o- cells to H2O2. CF sputum extracts retained their H2O2-scavenging activity after catalase and heme peroxidases were inactivated (Table 1). 16HBE14o- cells exposed to CF sputum extracts and H2O2 demonstrated viability similar to control cells (Table 1), again, in a marked contrast to both control culture media (Table 1).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previously, high levels of antioxidants, such as GSH, glutathione peroxidase, and catalase, were found in CF sputum samples (6, 24). In addition, there are published data (21, 24) with respect to the in vitro oxidant-scavenging properties of CF sputum extracts. In the present study, we were able to confirm and extend the previous findings. The CF sputum extracts were obtained by a specific protocol to minimize cell damage and to maximally preserve antioxidants (5, 6). Techniques such as ultrafiltration and ultracentrifugation were not applied as they were found to damage sputum cells (22). The damage of sputum cells would result in a leakage of intracellular antioxidants and affect the evaluation of H2O2-neutralizing capacity of CF airway secretions. Furthermore, dithiothreitol (DTT), a synthetic thiol donor commonly used to liquefy sputum, for various reasons was not used during sputum processing in the present study. First, DTT was found to affect glutathione recycling assay (5). Next, DTT is a dithiol able to reduce DTNB. Therefore, sputum's content of reduced thiols would be hugely overestimated by DTNB assay in the presence of molar excess of DTT (0.1% DTT, a commonly used concentration for sputum homogenization, corresponds to 6.5 mM DTT).
Furthermore, the urge to study CF upper airway secretions as close as possible to their natural environment also forced the authors not to include patients receiving N-acetylcysteine, which is a thiol, antioxidant, and a glutathione prodrug. The obtained data indicate, however, that CF upper airway secretions, even in the absence of a thiol donor, demonstrate a substantially reduced thiol content and possess a significant H2O2-detoxifying capacity. H2O2 concentrations as high as 1 mM were efficiently neutralized by CF sputum extracts.
In a marked contrast, both control media (serum-free MEM and MEM containing 10% FBS) exhibited only a negligible antioxidant capacity in the present study. Interestingly, the FBS-containing culture medium was as inefficient in H2O2 detoxification as the serum-free MEM. Indeed, FBS is rich in albumin, and albumin contains one SH group that theoretically can interact with H2O2. However, there are reports in the literature demonstrating that the antioxidant capacity of albumin is, in fact, lower than that of reduced glutathione (4, 7). Therefore, it can be postulated that the antioxidant efficiency of CF airway secretions is due to the presence of specific antioxidants and not a simple reflection of protein abundance.
In normal human airway secretions, H2O2 can be consumed in several ways. First, there is an antioxidant enzymatic system (glutathione peroxidase and catalase) that neutralizes H2O2 to water. The second enzymatic system, based on heme peroxidases [myeloperoxidase (MPO), lactoperoxidase (LPO), and eosinophil peroxidase], uses H2O2 as a substrate to produce highly active antimicrobial compounds (hypochloride, thiocyanate, and hydrobromide, respectively). Furthermore, there are also nonenzymatic H2O2 scavengers, such as reduced glutathione and other reduced thiols, etc. All these substances have been identified in CF upper airways secretions. The presence of abundant H2O2-scavenging sputum factors is likely to be the reason why CF breath condensate does not exhibit elevated H2O2 levels.
In the present study, we did not attempt to compare the antioxidative potential of CF airway secretions with that of healthy individuals. Because healthy individuals do not produce induced sputum in sufficient quantities, it would have been difficult to perform experiments such as outlined above. Moreover, it is the authors' experience that samples of healthy individuals exhibit a gel-like consistency that is in marked contrast to mucopurulent CF samples. Due to the mucopurulent character of CF sputum, it is easier to obtain sputum supernatant by dilution with PBS and centrifugation. In contrast, a complete liquefaction of a healthy sputum requires use of DTT, which was avoided in the present study due to the aforementioned reasons.
It is, however, interesting to compare the data obtained in the present study with the data from literature. El Chemaly et al. (9) analyzed tracheal secretions from healthy individuals and estimated that H2O2 detoxification predominantly occurs via the enzymatic route (i.e., via catalase, glutathione peroxidase, and heme peroxidases). According to their estimations, in upper airway secretions these enzymes neutralize up to 80% of exogenous H2O2. The nonenzymatic mechanisms account for the remaining 20%. Interestingly, the present and previous (6, 24) studies document both enzymatic (glutathione peroxidase and catalase) and nonenzymatic H2O2 scavengers (reduced glutathione and protein thiols) in CF sputum. The data from the present study suggest, however, that the reduced thiol and/or glutathione peroxidase system is fully capable to detoxify H2O2, as the H2O2-scavenging potential was sustained after both catalase and heme peroxidases were inactivated by NaN3. It is not clear yet what is the relative contribution of reduced thiol/glutathione peroxidase/catalase scavengers vs. MPO/LPO to H2O2 consumption in CF airways. It is interesting, though, that in the present study CF sputum extracts, with or without added H2O2, were not toxic for cells. In the authors' opinion, the latter observation may be suggestive of H2O2's being detoxified to water in the presence of sputum factors, i.e., via reduced thiol and/or glutathione peroxidase/catalase system, rather than to hypochlorous acid or thiocyanate. Therefore, it can be speculated that the abundant and efficient antioxidant system in CF upper airway secretions may be helpful to combat oxidant stress, but this may happen at the expense of producing antimicrobial substances deriving from H2O2.
Data from the present study provide no exact answer as to which high-molecular reduced thiols may be involved in H2O2 detoxification. However, it can be speculated that sputum mucins may, at least partially, be responsible for this detoxification, since they are very abundant in sputum.
If the antioxidant system is so efficient in CF upper airway secretions, is oxidative stress a relevant feature of the CF lung disease? Recent studies appear to confirm this suggestion. There is abundant evidence of airway oxidative stress in CF (for review, see e.g., Ref. 3). It can be speculated that the mucus layer possesses sufficient antioxidant properties in CF whereas the sol layer may not. Due to the impaired absorption of fat soluble antioxidants, such as vitamin E, and affected secretion of GSH by the CF respiratory epithelium, the complex antioxidant network in the sol phase may be severely disturbed in CF. Therefore, H2O2 released in the vicinity of epithelial surface, i.e., in the sol phase of the airway surface liquid, may still reach the cells. In addition, proinflammatory cytokines, such as TNF-, and bacterial products, such as pyocyonin and pyochelin, are known to trigger intracellular oxidative stress by disturbing mitochondrial respiration. These factors, in contrast to H2O2, are unlikely to be scavenged by extracellular CF mucus and antioxidants present in it and may, therefore, elicit oxidative insults in CF airways.
In conclusion, in the present study the in vitro cytotoxicity of up to 1 mM H2O2 was effectively prevented by CF sputum extracts. The reduced thiol and/or glutathione peroxidase system appears to be very efficient in H2O2 neutralization, even when catalase and heme peroxidases are inactivated. Further studies are needed to understand the complex nature of oxidative stress and inflammation in CF to better design therapeutic interventions.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |