Pseudomonas aeruginosa pyocyanin directly oxidizes glutathione and decreases its levels in airway epithelial cells
Yunxia Q. O'Malley,1
Krzysztof J. Reszka,2,3
Douglas R. Spitz,3
Gerene M. Denning,1,2,4 and
Bradley E. Britigan1,2,3,4
1Department of Internal Medicine and 2Research Service, Veterans Affairs Medical Center-Iowa City; and 3Free Radical and Radiation Biology Program, Department of Radiation Oncology, and 4Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242
Submitted 29 January 2004
; accepted in final form 11 March 2004
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ABSTRACT
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Production of pyocyanin enhances Pseudomonas aeruginosa virulence. Many of pyocyanin's in vitro and in vivo cytotoxic effects on human cells appear to result from its ability to redox cycle. Pyocyanin directly accepts electrons from NADH or NADPH with subsequent electron transfer to oxygen, generating reactive oxygen species. Reduced glutathione (GSH) is an important cellular antioxidant, and it contributes to the regulation of redox-sensitive signaling systems. Using the human bronchial epithelial (HBE) and the A549 human type II alveolar epithelial cell lines, we tested the hypothesis that pyocyanin can deplete airway epithelial cells of GSH. Incubation of both cell types with pyocyanin led to a concentration-dependent loss of cellular GSH (up to 50%) and an increase in oxidized GSH (GSSG) in the HBE, but not A549 cells, at 24 h. An increase in total GSH, mostly as GSSG, was detected in the culture media, suggesting export of GSH or GSSG from the pyocyanin-exposed cells. Loss of GSH could be due to pyocyanin-induced H2O2 formation. However, overexpression of catalase only partially prevented the pyocyanin-mediated decline in cellular GSH. Cell-free electron paramagnetic resonance studies revealed that pyocyanin directly oxidizes GSH, forming pyocyanin free radical and O2·. Pyocyanin oxidized other thiol-containing compounds, cysteine and N-acetyl-cysteine, but not methionine. Thus GSH may enhance pyocyanin-induced cytotoxicity by functioning as an alternative source of reducing equivalents for pyocyanin redox cycling. Pyocyanin-mediated alterations in cellular GSH may alter epithelial cell functions by modulating redox sensitive signaling events.
human bronchial epithelial cells; type II alveolar epithelial cells; glutathione; lung
PSEUDOMONAS AERUGINOSA SECRETES a variety of factors that have been suggested to play key roles in lung injury that results from acute and chronic forms of P. aeruginosa lung infection (17). The low-molecular-weight product pyocyanin (N-methyl-1-hydroxyphenazine) (Fig. 1) appears to be such a factor based on its cytotoxicity in many in vitro and in vivo model systems (17, 25, 34, 54). In most cases, pyocyanin's cytotoxicity has been strongly linked to its potential to redox cycle. It will directly accept electrons from either NADH or NADPH. Under aerobic conditions it will pass those electrons to O2, leading to the generation of the reactive oxygen species (ROS) superoxide (O2·) and hydrogen peroxide (H2O2) (28). By the use of cellular reducing equivalents (e.g., NADH or NADPH), addition of pyocyanin to cellular systems places them under increased oxidative stress (28).

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Fig. 1. Structure of pyocyanin and its protic equilibrium. Shown is the structure of pyocyanin at 2 different protonation states (A). In near neutral and alkaline pH the pigment exists in the form of zwitter ion (blue color), whereas at acidic pH it is red in color. Also shown (B) for comparison is the structure of another P. aeruginosa secretory product 1-hydroxyphenazine. pKa, negative log of acidic dissociation constant.
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Most human cells possess several key mechanisms to limit their exposure to ROS. One of the major components of cellular antioxidant defenses is the thiol compound glutathione (15, 36). When cells are exposed to oxidant species such as H2O2, reduced glutathione (GSH) is oxidized to a dimer [oxidized glutathione, or glutathione disulfide (GSSG)] by the action of glutathione peroxidase (Fig. 2). GSSG is reduced back to GSH through the action of glutathione reductase. This cycling of GSH is felt to be an important means of limiting cellular exposure to and cytotoxicity from H2O2 (26, 36). Consumption and cellular loss of GSH are a consequence of cellular oxidant exposure and contribute to oxidant-mediated cell injury (12, 26, 36).

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Fig. 2. Formation of superoxide (O2·) and hydrogen peroxide (H2O2) following the reaction of NAD(P)H with pyocyanin and the subsequent interaction of pyocyanin-derived H2O2 with GSH. GPx, glutathione peroxidase.
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In recent years it has been found that cellular GSH concentration, by modulating the "redox tone" of the intracellular environment, also plays an important role in regulating cellular signaling events resulting from the action of redox-sensitive transcription factors such as NF-
B, activator protein (AP)-1, and hypoxia-inducible factor (HIF)-1
(2, 19, 22, 37, 50, 52). Thus agents that modify cellular GSH levels can in turn modulate cell function by altering the activity of these transcription factors. Consistent with this, we have previously shown that pyocyanin enhances epithelial cell release of interleukin (IL)-8 (14), a chemokine whose production is regulated by oxidative stress.
Given that pyocyanin exposure leads to the generation of intracellular ROS, we postulated that it would alter the levels of GSH within airway epithelial cells. Furthermore, since NADH and NADPH can directly reduce pyocyanin to the pyocyanin free radical (10, 24, 28, 29), we speculated that GSH might also directly reduce pyocyanin, thereby enhancing the formation of O2· and H2O2. This would create a unique circumstance in which GSH could enhance the formation of ROS, rather than serving an antioxidant function in the cell. Data reported in this communication provide solid support for each of these hypotheses. The implication of these findings to the pathophysiology of P. aeruginosa-associated lung injury is discussed.
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MATERIALS AND METHODS
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Cell culture.
The human alveolar type II cell line A549 [CL-185; American Type Culture Collection (ATCC), Rockville, MD] and the SV40 transformed normal human bronchial epithelial 16HBE14o cell line (27, 53) (HBE, provided by Dr. Michael Apicella, Dept. of Microbiology, University of Iowa) were cultured in Dulbecco's modified Eagle's medium (DMEM; Cellgro, Herndon, VA) that was supplemented with 5% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, penicillin (500 units/ml), and streptomycin (500 µg/ml). Each cell type was used between passages 73 and 100. All experiments were performed when the cell cultures were
95% confluent.
For human erythrocyte studies, human peripheral venous blood (10 ml) was collected into heparinized containers by venipuncture. The blood was centrifuged at 200 g for 5 min. The resulting plasma was discarded. The erythrocytes were washed twice with calcium- and magnesium-free PBS and then suspended in 10 ml of the same DMEM and supplements as for the epithelial cells. The erythrocytes were then placed in a T-75 flask and maintained at 37°C and 5% CO2. No attempt was made to remove the contaminating leukocytes, as they represented only
0.1% of the total cells present. We assessed viability of the erythrocytes in culture by monitoring cell-associated hemoglobin over time using the standard spectrophotometric assay for hemoglobin (55).
Pyocyanin purification.
Pyocyanin was extracted from the broth culture of P. aeruginosa by previously described methods (11). Briefly, P. aeruginosa strain PAO1 (ATCC, 15692) was grown in glycerol-alanine minimal medium, following which the bacteria were removed by centrifugation. Pyocyanin in the culture supernatant was extracted into chloroform. The blue pigment in chloroform phase was back extracted into acidified water (10 mM HCl). The water was then adjusted to pH 7 by the addition of NaOH. This process was repeated at least five times. The pyocyanin (in acidified form) was stored frozen or at 4°C protected from light. Pyocyanin purity was confirmed by HPLC with a Beckman System Gold apparatus and a reverse-phase C18 column (Microsorb-MV-C18, 250 x 4.6 mm; Varian, Walnut Creek, CA) and a solvent system comprising 0.05% trifluoracidic acid (TFA) in water and 0.05% TFA in acetonitrile using 25- to 30-min runs. 1-Hydroxyphenazine was purchased from TCI America (Portland, OR) and was used as received from the supplier.
Glutathione measurements.
Glutathione was determined by a modification of the method of Tietze (56). For HBE and A549 cells, the cells were cultured in six-well plates and were lysed by 0.2 ml of 10 mM HCl. The cell lysate (50 µl) was mixed with the same volume of 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) buffer (110 mM Na2HPO4, 40 mM NaH2PO4, 15 mM EDTA, and 0.3 mM DTNB), followed by 50 µl of glutathione reductase buffer (50 mM imidazole, 15 mM HCl, and 1 mM EDTA). The absorbance from reduced DTNB was measured at 405 nm by a plate reader (Ultramark, BIO-RAD microplate imaging system) at 0 and 5 min. We quantitated GSSG by blocking GSH with L-vinyl-pyridine. The cell lysate (100 µl) was mixed with L-vinyl-pyridine (10 µl) for 2 h at room temperature. GSSG was converted to GSH by the glutathione reductase buffer, and then the sample was analyzed for GSH as above.
For erythrocytes, 5 x 106 cells were collected, centrifuged (200 g, 5 min), washed twice with PBS, and then lysed with distilled water (1 ml), following which the lysate was frozen at 80°C. Samples were then thawed and vortexed vigorously, and then cell debris was removed by centrifugation (14,000 g, 1 min). Glutathione determinations were then made as above.
Spin trapping.
GSH, N-acetyl-cysteine (NAC), cysteine, methionine, NADPH, and SOD were obtained from Sigma (St. Louis, MO). Ultrapure 5,5-dimethylpiperidinyl N-oxide (DMPO) was generously provided by Labotec (Los Angeles, CA). The concentration of pyocyanin was determined by using its extinction coefficient at 520 nm, 2.46 x 103 M1·cm1, in 0.2 N HCl (33). To find out whether pyocyanin could react directly with GSH, we carried out electron paramagnetic resonance (EPR) experiments in buffered solutions of pyocyanin (
0.220.5 mM) to which GSH (10 mM) was added. The measurements were performed in acetate or phosphate buffers (100 mM) of pH ranging from
4 to
8, and the actual pH of the samples was checked after collection of the EPR spectra. Similar experiments were performed with cysteine, NAC, and methionine. The capacity of the pyocyanin-thiol system to generate O2· was examined by EPR/spin trapping with DMPO as the spin trapping agent.
We performed EPR measurements using a Brüker EMX EPR spectrometer operating at X-band with 100-kHz modulation equipped with TM110 cavity. Samples consisting of pyocyanin, a thiol, and DMPO (100 mM) in a buffer of desired pH were transferred to an EPR flat aqueous cell and positioned in the EPR cavity, and scans were executed promptly. We recorded spectra using the following instrumental settings: microwave power, 20 mW; modulation amplitude, 1 G; time constant, 81.92 ms, conversion time, 40.96 ms; and scan rate, 80 G/41.92 s. EPR spectra shown represent the average of 10 scans and are representative of the results obtained in at least three separate experiments.
Catalase overexpression.
Recombinant adenoviral vectors expressing catalase (Ad CMV Catalase), catalase with a mitochondrial targeting sequence (Ad CMV mCatalase) (3), or
-galactosidase (Ad CMV LacZ) were constructed by and purchased from the Vector Core Facility of The University of Iowa. Each adenoviral stock (46 x 1010 infectious viral particles/ml) was stored in 3% sucrose at 80°C. Multiplicities of infection (MOI) ranging from 10 to 200 were examined, and an MOI of 100 was routinely employed. Cells were exposed to adenovirus in media containing 5% FBS at 37°C for 24 h. We confirmed successful transfection by measuring expression of each antioxidant enzyme at both the protein and activity level by immunoblot and by native-gel activity assay staining, respectively (5, 9, 35).
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RESULTS
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Pyocyanin decreases airway epithelial cell GSH levels.
Pyocyanin induces the production of ROS in a variety of cell types (7), including the human HBE and A549 lung epithelial cell lines (13). Increased intracellular ROS formation leads to a loss of GSH in many cell systems (12, 26, 36). Therefore, we postulated that cellular GSH levels in airway epithelial cells would decrease as a consequence of pyocyanin exposure. We chose to examine this hypothesis over prolonged periods (hours to days) of pyocyanin incubation to better model the prolonged exposure of airway epithelial cells that would occur in the setting of cystic fibrosis. Furthermore, short-term observations may not allow sufficient time for the cells to adapt to pyocyanin exposure.
Consistent with our hypothesis, incubation of A549 cell monolayers with concentrations of pyocyanin previously detected in the sputum of patients with cystic fibrosis (up to 100 µM) (58) resulted in a time- and pyocyanin concentration-dependent decrease in A549 cell total GSH levels (Fig. 3). At 24 h, GSH was decreased by up to 40%, with a further decline with longer periods of incubation (Fig. 3). At pyocyanin concentrations of 10 µM and lower, the decrease in GSH did not progress over that seen at 24 h. This is likely due to the ability of the cells to metabolize the lower concentrations of pyocyanin to an inactive product(s) (40).
A549 cells are a lung cancer cell line, and antioxidant defense systems may be different in malignant, relative to normal, cells (41). To be certain that the above results were not unique to this cell line, we repeated the experiments using monolayers of HBE cells. At 24 h, total cellular GSH was significantly decreased as a function of the concentration of pyocyanin to which these cells were exposed (Fig. 4). Pyocyanin's ability to decrease cellular GSH appeared to require slightly more pyocyanin with HBE relative to A549 cells (Fig. 4). However, this remained well within the range of pyocyanin concentrations reported to occur in vivo (58).

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Fig. 4. Effect of pyocyanin on epithelial cell GSH and GSSG. 16HBE14o (HBE, A) or A549 (B) cells were incubated with the indicated concentrations of pyocyanin or vehicle for 24 h, following which cellular GSH and GSSG levels were determined. Results shown are means ± SD of cellular GSH (nmol/mg protein) and GSSG (nmol/mg protein) and the ratio of cellular GSH/GSSG as a function of the concentration of pyocyanin employed (n = 6). Pyocyanin concentrations of 30 µM significantly decreased total GSH in A549 and HBE cells (*P < 0.05, **P < 0.01, ***P < 0.001). No significant difference in GSSG or the GSH/GSSG ratio was observed in A549 cells (P > 0.05).
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Pyocyanin exposure also resulted in an increase in GSSG in HBE cells, as well as a decrease in the GSH/GSSG ratio (Fig. 4). This was not consistently seen with A549 cells (Fig. 4). With both cells lines, the magnitude of the decrease in cellular GSH was far greater than the increase in GSSG detected (Fig. 4).
To provide insight into whether pyocyanin's effect on cellular GSH levels involved nuclear regulatory processes, we examined the effect of pyocyanin on a cell type lacking a nucleus, the erythrocyte. Incubation of human peripheral blood erythrocytes with pyocyanin led to a depletion in total cellular GSH. Exposure to 50 µM pyocyanin for 24 h decreased erythrocyte GSH to 66% of untreated control cells, without altering cellular integrity, as reflected in a stable level of hemoglobin over time (n = 2, data not shown). These data suggest that the depletion of cellular GSH in response to pyocyanin occurs independently of nuclear regulatory events.
Pyocyanin induces efflux of GSH from airway epithelial cells.
We next pursued possible explanations whereby cellular total GSH was out of proportion to the apparent formation of GSSG. We hypothesized that the decrease in total cellular glutathione could occur if exposure to pyocyanin induced the cells to export cellular GSSG and/or GSH to the extracellular milieu. Secretion of GSSG/GSH has been reported to occur in a number of cell types, including A549 cells (43). To test this possibility, we exposed HBE and A549 cells to media alone or media plus 30100 µM pyocyanin for 24 h. The GSH and GSSG content of the media was then determined and compared. As shown in Fig. 5, the presence of pyocyanin led to an increase in the presence of total GSH (GSH plus GSSG) in the media. The vast majority of the GSH in the cell culture media was in the form of GSSG (Fig. 5). The magnitude of the increase in total GSH and GSSG in the media accounted for nearly all of the decrease in cellular GSH resulting from pyocyanin exposure (Fig. 5).

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Fig. 5. Pyocyanin causes efflux of GSH from epithelial cells. A: HBE or A549 cells were incubated with the designated concentrations of pyocyanin or vehicle for 24 h, following which GSH and GSSG levels in the media were determined and normalized to cellular protein content of each well. Results shown are means ± SD (n = 6). Cellular exposure to pyocyanin increased the level of total GSH and GSSG in the media at 24 h (***P < 0.001) for both cell types. Most of the increase in total GSH was in the form of GSSG. B: shown is the increase in media GSH and GSSG as a function of time of incubation of HBE cells with 50 µM pyocyanin (gray bars), relative to no pyocyanin exposure (black bars). Results shown are means ± SD of media GSH (nmol/mg protein) and GSSG (nmol/mg protein), n = 6. Statistically significant changes are designated as *P < 0.05, **P < 0.01, ***P < 0.001.
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Pyocyanin-mediated depletion of epithelial cell GSH may not solely involve enhanced H2O2 production.
Cellular glutathione peroxidase uses GSH to remove cytotoxic H2O2 (Fig. 2) (26, 36). Therefore, it seemed possible that the loss of cellular GSH could be the consequence of pyocyanin-induced formation of H2O2. To examine this possibility, we tested whether enhancing the H2O2-scavenging capability of the cells served to protect them from depletion of their GSH following pyocyanin exposure. HBE cells were transfected with adenovirus-expressing catalase or LacZ control. Catalase transfection resulted in a 150- and 180-fold increase in HBE cell catalase protein (Fig. 6) relative to untransfected or LacZ-transfected control cells, respectively. The cells were then incubated with pyocyanin for 24 h, following which cellular GSH and GSSG levels were measured. Overexpression of catalase only modestly prevented the pyocyanin-mediated decrease oxidation of GSH to GSSG and the export of GSH to the media (Fig. 6). Similar results were obtained when A549 cells were transfected with catalase (not shown).

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Fig. 6. Overexpression of catalase does not block the pyocyanin-mediated oxidation and export of cellular GSH. Top: representative immunoblot (n = 6) of HBE cell catalase protein expression following transfection with the designated MOI of catalase-expressing adenovirus [conventional and with mitochondrial targeting sequence (mCatalase)] and in the absence or presence of exposure to 50 µM pyocyanin. Middle, bottom: untreated HBE cells or those transfected with adenovirus 24 h previously at 100 multiplicities of infection (MOI) expressing LacZ or human catalase were incubated with 50 µM pyocyanin for 24 h (gray bars), following which cellular GSSG content was measured and compared with results observed in the absence of pyocyanin treatment (black bars). Shown are means ± SD of results for 6 separate experiments, expressed as GSSG. ***P < 0.001 relative to untreated control. Adenoviral transfection (LacZ) also led to an increase in GSSG levels above untransfected control cells (P < 0.05). WT, wild type.
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We previously obtained evidence that pyocyanin ROS formation occurs to a large extent in and/or near cellular mitochondria (40). Because catalase is a peroxisomal protein, it was possible that catalase overexpression did not occur at the site of pyocyanin-induced H2O2 production. Therefore, we also examined the effect of transfecting the cells with catalase linked to a mitochondrial targeting sequence, which leads to catalase localization in mitochondria. Mitochondrion-targeted catalase, however, also had no additional effect beyond that of conventional catalase on pyocyanin-induced oxidation of cellular GSH and export of GSH/GSSG (Fig. 6).
Together, these experiments show that catalase overexpression has only a modest ability to alter pyocyanin-mediated loss of epithelial cell GSH. This suggests that reaction of GSH with intracellular H2O2 generated by pyocyanin is not the only mechanism for the pyocyanin-dependent decrease in cellular GSH. Interestingly, for unknown reasons, adenoviral transfection appeared to slightly enhance the basal GSSG levels in these cells in the absence of pyocyanin (Fig. 6). This may relate to virus-mediated induction of cellular ROS production, as has been reported previously following infection of eukaryotic cells by various types of viruses (49).
Pyocyanin is directly reduced by GSH and other thiols.
The above data suggested that enhanced production of H2O2 may not be entirely responsible for the loss of GSH in A549 cells following pyocyanin exposure. Although O2· is also formed during pyocyanin redox cycling, that species exhibits a low rate constant for reaction with GSH (31, 59, 60). Therefore, we sought alternative explanations for our data. Because NADH and NADPH can directly reduce pyocyanin, we speculated that GSH might also directly reduce pyocyanin, thereby leading to oxidation of GSH to GSSG.
From the fact that thiols are one-electron donors, reduction of pyocyanin by thiols should be associated with formation of thiyl radicals (RS·). Furthermore, if the formation of pyocyanin radical leads to the formation of O2·, as occurs when pyocyanin is reduced by NAD(P)H (28), reduction of pyocyanin by thiols in aerobic solutions should also lead to formation of O2·. To ascertain whether interaction of thiols with pyocyanin generates RS
and O2· radical, a spin trapping technique was used. A representative EPR spectrum from a cell-free system consisting of pyocyanin and GSH in buffer containing DMPO is shown in Fig. 7, A (pH 5.8) and G (pH 7.1). The spectra contain contributions from DMPO/·OOH and DMPO/·OH, respectively (Fig. 7, B and C). The latter adduct was derived most likely from spontaneous degradation of DMPO/
OOH (20). At pH 7.0 or greater, the spectrum was dominated by DMPO/·OH (Fig. 7G), which is commonly seen under conditions of slower rates of O2· formation (8). In addition, because of the substantially higher rate of the addition of hydroperoxide to DMPO, the EPR spectrum of the DMPO/
OOH adduct is expected to be more pronounced at acidic pH. Based on simulation of the experimental spectrum at pH 7.1, DMPO/
SG was also present.

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Fig. 7. Pyocyanin reacts with GSH to generate O2·. Electron paramagnetic resonance (EPR) spectra generated by reacting pyocyanin ( 95 µM) with GSH (10 mM) in the presence of 5,5-dimethylpiperidinyl N-oxide (DMPO, 100 mM) in pH 5.8 buffer (50 mM phosphate) containing 1 mM diethylenetriaminepentaacetic acid (DTPA). A: experimental spectrum (solid line). Superimposed on the experimental spectrum is a simulated spectrum (dotted line) consisting of DMPO/ OOH and DMPO/ OH components. B: simulation of the DMPO/ OOH component using hyperfine splitting constants of aN = 14.16 G, a H = 11.26 G, a H = 1.23 G. C: simulation of the DMPO/ OH component with hyperfine splitting constants of aN = 15.2 G, a H = 14.78 G. D: same as A in the presence of SOD (38 µg/ml). E: same as A but with GSH omitted. F: same as A but with pyocyanin omitted. G: EPR spectrum at pH 7.1, [pyocyanin] = 0.35 mM and [GSH] = 11 mM. H: same as in G but in the presence of superoxide dismutase (SOD, 70 µg/ml). Instrument settings: microwave power, 20 mW; modulation amplitude, 1 G; time constant, 81.92 ms; scan rate, 80 G/41.92 s. Spectra represent the average of 10 scans.
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When SOD was added to the sample before pyocyanin, no signal was observed (Fig. 7, D and H). This indicates that formation of both DMPO/
OOH and DMPO/·OH was mediated by O2·. Further control experiments confirmed that the formation of O2· was fully dependent on the simultaneous presence of pyocyanin and the thiol, since when one of these components was omitted, the DMPO spin adducts were not detected (Fig. 7, E and F, respectively). These data indicate that the direct interaction of GSH with pyocyanin leads to the direct oxidation of GSH with resultant formation of O2·.
Cell free EPR spectrometry studies also confirmed that pyocyanin can directly oxidize GSH to form a pyocyanin free radical, the one-electron reduction product of pyocyanin (Fig. 8A). The spectrum from the pyocyanin/GSH system is identical to that from the pyocyanin/NAD(P)H system (Fig. 8E), suggesting that they both originate from one and the same species, that is, from the pyocyanin radical. Like the formation of O2·, the generation of pyocyanin radical in the presence of thiols was more efficient at acid pH compared with physiological pHs (Fig. 9B).

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Fig. 8. Pyocyanin is directly reduced by thiols. EPR spectra of a pyocyanin radical generated by reduction of pyocyanin ( 0.5 mM) with 10 mM GSH (A), cysteine (B), and N-acetyl-cysteine (NAC, C). Methionine is unable to reduce the pigment and generate pyocyanin radical (D). Spectrum of pyocyanin radical generated by reduction of pyocyanin (0.48 mM) with NADPH (0.8 mM) at pH 7.4 (50 mM phosphate) is shown for comparison (E). The concentration of thiols was 10 mM. pH values were determined to be 5.16 for GSH and cysteine and 5.0 for NAC and methionine after the EPR measurements were made. Instrument settings for spectra AD: microwave power, 40 mW; modulation amplitude, 1 G; time constant, 81.92 ms; scan rate, 80 G/41.92 s. Spectra AD are the average of 10 scans. Scan E is the average of 5 scans (microwave power, 20 mW; modulation amplitude, 1 G).
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Fig. 9. Effect of pH on reduction of pyocyanin by GSH. Shown are EPR tracings obtained in phosphate buffer at pH 7.1 (scan A) vs. pH 6.2 (scan C) following the addition of 0.55 mM pyocyanin to buffer containing 11 mM GSH. The solution had been bubbled with N2 before the addition of the pyocyanin to prevent subsequent reoxidation of the pyocyanin radical by dissolved O2. This was necessary to maintain sufficient stability of the radical at pH 7.1 so as to allow its detection by EPR at room temperature. If this was not done, the pyocyanin radical could not be detected at the higher pH (scan B). Instrument settings: microwave power, 40 mW; modulation amplitude, 1 G; time constant, 81.92 ms; scan rate, 80G/41.92 s; microwave power, 20 mW; and modulation amplitude, 1 G. The spectra are the average of 10 scans.
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We next assessed to what extent the reaction between pyocyanin and GSH extends to other thiols. Addition of pyocyanin to either NAC or cysteine also gave rise to O2· (Figs. 10 and 11, respectively) and pyocyanin radical (Fig. 8, B and C). When NAC was used in the DMPO spin-trapping experiments, an EPR spectrum containing a contribution from DMPO/
OOH and DMPO/
OH was detected (Fig. 10A). Both of these species were sensitive to SOD (Fig. 10E). With cysteine, DMPO/·OH was detected (Fig. 11A), which was not formed when SOD was present (Fig. 11E). Formation of RS
by pyocyanin/NAC and pyocyanin/cysteine was confirmed by detection of DMPO adducts with the corresponding RS
(Figs. 10 and 11). In contrast to the results with GSH, cysteine, and NAC, it was found that pyocyanin will not oxidize methionine (Fig. 8D), i.e., there is no formation of pyocyanin radicals. The inability of methionine to generate the pyocyanin radical indicates that the presence of the redox-active sulfhydryl group is necessary for the successful reduction of the pigment.

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Fig. 10. Pyocyanin reacts with NAC to generate O2·. EPR spectra generated by reacting pyocyanin ( 95 µM) with NAC (30 mM) in the presence of DMPO (100 mM) in pH 6.1 buffer (50 mM phosphate) containing 1 mM DTPA. A: experimental spectrum. Superimposed on the experimental spectrum is the simulated spectrum (dotted line) consisting of DMPO/ OOH, DMPO/ OH, and DMPO/ S-NAC. B: simulation of the DMPO/ OOH component (aN = 14.6 G, a H = 11.34 G, a H = 1.25 G). C: simulation of the DMPO/ OH component (aN = 15.02 G, a H = 14.78 G). D: simulation of DMPO/ S-NAC (aN = 15.12 G, a H = 16.96 G). E: same as A in the presence of SOD (38 µg/ml). Instrument settings: microwave power, 20 mW; modulation amplitude, 1 G; time constant, 81.92 ms; scan rate, 80 G/41.92 s. Spectra represent the average of 10 scans.
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In contrast to the results obtained with pyocyanin, 1-hydroxyphenazine, another cytotoxic phenazine compound secreted by P. aeruginosa (39, 44, 51, 58), failed to oxidize any of the thiols studied (not shown). Thus not all phenazines produced by P. aeruginosa are capable of directly oxidizing these thiols. These data suggest that in the presence of pyocyanin, GSH might promote the formation of intracellular ROS, a situation that clearly runs contrary to its usual role as an antioxidant.
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DISCUSSION
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Cellular GSH plays an important role in protecting the cell from oxidant-mediated injury and in modulating the action of redox-regulated transcription factors (2, 12, 36). Cells placed under increased oxidative stress often exhibit a decrease in GSH, as well as a decrease in their ratios of GSH/GSSG (48). As expected with a compound that results in the production of intracellular ROS, the addition of pyocyanin to HBE or A549 cells resulted in a decrease in total cellular GSH levels. Somewhat surprisingly, this occurred without a significant increase in the detection of GSSG in A549 cells, and in the case of HBE cells the magnitude of the increase in GSSG was less than the decrease in total GSH. Subsequent studies suggest that this is likely due in large part to the export of GSH and/or GSSG. Export of GSH is known to occur in many cell types, including A549 cells (43).
The airways of most patients with cystic fibrosis become chronically infected with P. aeruginosa, and pyocyanin (up to 100 µM) has been detected in the sputum of these patients (58). Both systemic and pulmonary GSH levels are reportedly decreased in cystic fibrosis patients (30, 32, 46, 57). Although this may be due to increased oxidative stress and alterations in glutathione transport resulting directly from the responsible mutation in CFTR itself (23, 30, 32, 46, 57), some of the loss of cellular GSH could be secondary to the effects of P. aeruginosa-derived pyocyanin.
Redox-active compounds are assumed to deplete cellular GSH levels by undergoing reduction via cellular systems with subsequent transfer of electrons to O2 forming ROS. It is this production of ROS, particularly H2O2, that leads to the oxidation of GSH to GSSG. We were able to only partially prevent the pyocyanin-mediated loss of GSH by increasing the intracellular H2O2-removing capacity of either HBE or A549 cells by overexpressing catalase within the cell, either in peroxisomes or mitochondria.
Recent work from Muller (38), found that a short-term (3060 min) exposure of human umbilical vein endothelial cells to pyocyanin in phosphate buffer results in a loss of cellular GSH, but with a coincident decrease in the GSH/GSSG ratio. This study did not address the possibility of GSH export. Although Muller also reported that extracellular catalase limited the loss of GSH in endothelial cells exposed short term (3090 min) to pyocyanin (38), we have been unable to observe any consistent alteration in the loss of epithelial cell GSH resulting from more prolonged exposure to pyocyanin by a similar approach to increase extracellular catalase (data not shown). Whether the disparity between our results and those of Muller reflects the differences in the cell type (endothelial vs. epithelial), duration of pyocyanin exposure (minutes vs. days), the medium in which the exposure occurred (simple buffer vs. complex medium), or other factors is unclear and requires further study. Regardless, our data are consistent with the possibility that H2O2 production may not be the sole means for the loss of GSH in airway epithelial cells exposed to pyocyanin for prolonged time periods.
The unique redox properties of pyocyanin allow it to accept electrons directly from NAD(P)H (10, 24, 28, 29). We now report that pyocyanin can directly and rapidly oxidize GSH without the need for cell-derived reducing equivalents. This leads to the formation of thiyl, pyocyanin, and O2· radicals. To our knowledge the ability of pyocyanin or other phenazine compounds to directly oxidize thiol-containing molecules has not been previously demonstrated. In fact, we find that another P. aeruginosa-derived phenazine compound, 1-hydroxyphenazine, does not have this capacity.
The direct oxidation of GSH by pyocyanin with resultant formation of O2· was readily detected at physiological pH but occurred more rapidly at lower pH. This dependence on pH may be due to protonation of pyocyanin at acid pH, negative log of acidic dissociation constant = 4.9 (21), resulting in the pyocyanin cation, which is a more powerful oxidant than the zwitter ion present in neutral and alkaline solutions (Fig. 1). In contrast, generation of pyocyanin radical by reaction of the pigment with NAD(P)H does not require acidic conditions (10, 24, 29). Although the exact values are not known, the rate of reaction between pyocyanin and NADPH appears to be greater at physiological pH than for the reaction of pyocyanin with GSH; the much higher concentrations of cellular GSH (low millimolar) compared with NAD(P)H (nanomolar) indicate that the reaction of pyocyanin with GSH would likely compete effectively with that of NAD(P)H in the cellular cytoplasm. In the mitochondria, where concentrations of NADPH may be closer to those of glutathione, NADPH may be the major reducing source for pyocyanin.
Our data suggest that intracellular GSH could serve as both a promoter of ROS formation and an antioxidant in pyocyanin-treated cells. By directly transferring electrons to pyocyanin, GSH would serve to enhance the rate of pyocyanin redox cycling, thereby promoting the intracellular formation of O2·, H2O2, and other ROS. These could in turn react with GSH to form GSSG in the antioxidant mode of the molecule. Both reactions could contribute to the cellular depletion of epithelial cell GSH over time.
Given earlier work indicating that GSH concentrations are abnormally low in patients with advanced cystic fibrosis (30, 32, 46, 57), it has been suggested that there may be a role for therapeutic replacement of GSH in such patients through the administration of GSH or NAC (1, 16, 46). NAC is a thiol compound that has direct antioxidant properties and also is converted to GSH by cells and thereby limits oxidant-mediated cell injury. Our data raise the possibility that such an approach could prove to be ineffective, or perhaps even deleterious, in the airways of patients infected with P. aeruginosa strains producing pyocyanin by "adding fuel to the fire." Further investigation of altering the cellular GSH levels on pyocyanin-mediated cytotoxicity should be assessed. Furthermore, the interaction of pyocyanin with other intracellular thiols such as thioredoxin may be of interest.
Acute and chronic pulmonary infection with P. aeruginosa is associated with an intense neutrophil inflammatory response that contributes to lung injury (4, 18). We have previously shown that pyocyanin is among the P. aeruginosa-derived secretory products that are capable of inducing production of the neutrophil chemokine IL-8 by airway epithelial cells via a process that involves MAP kinases (14). IL-8 expression is transcriptionally regulated in part by oxidant-sensitive transcription factors (6, 42, 45). Cellular GSH levels have been reported to influence the activity of a number of transcription factors, including NF-
B, AP-1, and HIF-1
(2, 47, 50). In preliminary studies, we found that exposure to pyocyanin alters the activity of each of these transcription factors in A549 cells. It enhances the levels of HIF-1
, but it has a bimodal effect on AP-1 and NF-
B, decreasing them at lower pyocyanin concentrations and enhancing them at higher ones. Work is currently ongoing to define the mechanism(s) whereby pyocyanin alters these transcription factors and to what extent the changes in cellular glutathione levels that we report in the present work may contribute to these events.
Nevertheless, our data suggest that pyocyanin may alter epithelial cell functions by directly or indirectly (via H2O2 production) oxidizing GSH and modulating redox-sensitive signaling events. The role of such events in the pathogenesis of P. aeruginosa-associated lung injury could prove to be of considerable interest and warrants further investigation.
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GRANTS
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This work was supported in part by grants from the Research Service of the Department of Veterans Affairs (to B. E. Britigan and G. M. Denning); by Public Health Service Grants RO1AI-43954, P30 DK-54759, P01-CA-66081; and by the Heartland Affiliate of the American Heart Association (G. M. Denning and K. J. Reszka).
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ACKNOWLEDGMENTS
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We thank Ling Li from the Antioxidant Core Facility of The University of Iowa for technical expertise with some of the measurements of glutathione efflux, funded through National Institutes of Health Grant P01-CA-66081 and DE-FG0202ER-63447 (Department of Energy).
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FOOTNOTES
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Address for reprint requests and other correspondence: B. E. Britigan, Univ. of Iowa Hospitals and Clinics, Dept. of Internal Medicine, SW54, GH, Iowa City, IA 52242.
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.
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