Original Contribution

PYOCYANIN INDUCES OXIDATIVE STRESS IN HUMAN ENDOTHELIALCELLS AND MODULATES THE GLUTATHIONE REDOX CYCLE

MICHAEL MULLER

Centre for Infectious Diseases, University of Sydney, Westmead Hospital, Westmead, NSW, Australia

(Received 15 February 2002;Revised 20 June 2002;Accepted 13 August 2002)

Abstract—Pyocyanin is a redox active virulence factor produced by the human pathogenPseudomonas aeruginosa.Treatment of endothelial cells with pyocyanin (1–50�M) resulted in the dose-dependent formation of hydrogenperoxide that was detected in the extracellular medium. Total intracellular glutathione levels decreased in response topyocyanin in a dose-dependent manner from a control value of 19.9� 2.7 nmol/mg protein to 10.0� 2.4 nmol/mgprotein. Prior treatment of cells with catalase afforded complete protection against loss of glutathione. Total intracellularsoluble thiols decreased from 95.0� 6.2 nmol/mg protein to 78.6� 2.3 nmol/mg protein at the highest test dose.Intracellular levels of NADPH increased up to 2.4-fold in response to pyocyanin exposure. It is concluded thatpyocyanin exposes endothelial cells to oxidative stress by the generation of hydrogen peroxide, which subsequentlydepletes intracellular glutathione and increases intracellular levels of mixed disulfides. © 2002 Elsevier Science Inc.

Keywords—Pseudomonas aeruginosa, Pyocyanin, Oxidative stress, Hydrogen peroxide, Glutathione, Redox status,Free radicals

INTRODUCTION

Pseudomonas aeruginosa is a common nosocomialpathogen of immunocompromised patients that cancause either acute or chronic disease. Acute conditionsinclude pneumonia [1,2] and vasculitis [3], while chronicinfection typically involves patients with cystic fibrosis[4,5]. A characteristic ofP. aeruginosa is the productionof substantial quantities of a blue redox active phenazinepigment, pyocyanin (1-hydroxy-5-methylphenaziniumhydroxide inner salt). Pyocyanin has been isolated fromthe sputum of infected patients at levels as high as 27.3�g/ml (�130�M) [6]. The pigment is reported to mod-ify several host cell responses, including inhibition ofneutrophil superoxide generation [7–9], inhibition oflymphocyte proliferation [10,11] and inhibition of theeicosanoid response, which is responsible for the pro-duction of several important proinflammatory mediators[12–15]. The toxicity of pyocyanin has been attributed toits ability to generate reactive oxygen species due tointracellular redox cycling [16]. Pyocyanin undergoes

nonenzymatic reduction by NAD(P)H and the reducedpigment rapidly reacts with molecular oxygen to producesuperoxide [17] and, by dismutation, hydrogen peroxide.The presence of these oxidants can induce oxidativestress in susceptible cells.

Oxidative stress has been implicated as an importantfactor in many diverse pathologies including cardiovas-cular and chronic lung disease [18]. Host cells possessseveral mechanisms by which oxidant species can beneutralized. Hydrogen peroxide is detoxified by two sep-arate cellular systems, the glutathione-peroxidase systemlocated in the cytosol and mitochondrial matrix and bycatalase located in peroxisomes [19]. The glutathioneperoxidase system is effective at removing both hydro-gen peroxide and lipid hydroperoxides, which are re-duced to water and the corresponding lipid alcohol, re-spectively. This occurs at the expense of reducedglutathione (GSH), which is oxidized to glutathione di-sulfide (GSSG). To ensure an adequate supply of GSH isavailable to the peroxidase system, GSSG is reduced toGSH by GSSG-reductase. Reducing equivalents for thisprocess are derived from intracellular NADPH. Underconditions of oxidative stress the level of NADPH can beaugmented by stimulation of the pentose phosphate path-way. In contrast to the glutathione peroxidase system,

Address correspondence to: Dr. M. Muller, Centre for Education andResearch on Ageing, ANZAC Research Institute, Concord RG Hospi-tal, Concord, NSW 2139 Australia; Fax:�61 2 9767 9160; E-Mail:mmuller@med.usyd.edu.au.

Free Radical Biology & Medicine, Vol. 33, No. 11, pp. 1527–1533, 2002Copyright © 2002 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/02/$–see front matter

PII S0891-5849(02)01087-0

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catalase is specific for hydrogen peroxide and is ineffec-tive at low micromolar concentrations [19,20].

The ability of pyocyanin to consume cellular NADPHand to generate reactive oxygen species suggested thatpyocyanin might adversely affect the intracellular redoxstatus and particularly the GSH content of host cells. AsP. aeruginosa frequently becomes localized at the post-capillary venules [1] this study was conducted to exam-ine the effect of pyocyanin on endothelial cell glutathi-one status.

MATERIALS AND METHODS

Reagents

All reagents were purchased from Sigma (St. Louis,MO, USA) unless otherwise stated. Reagents were pre-pared just prior to use in Hanks’ balanced salts solution(HBSS) containing calcium and magnesium but withoutphenol red (pH 7.4) and protected from light.

Synthesis of pyocyanin

Pyocyanin was synthesized by the photochemicalmethod of Knight et al. [21] and purified as previouslydescribed [13].

Cell culture

Human umbilical vein endothelial cells (HUVECs)were prepared essentially according to the method ofJaffe et al. [22] and cultured with the modificationsdescribed [23]. HUVECs were grown and maintained inMedium 199 (M199) containing 20% fetal bovine serum(CSL, Ryde, Australia), 100 U/ml penicillin, 100 �g/mlstreptomycin, 2.5 �g/ml amphotericin B, 75 �g/ml en-dothelial cell growth factor, and heparin 100 �g/ml. Allincubations were conducted at 37°C under an atmo-sphere of 5% CO2. To ensure consistent glutathionecontent between different batches of HUVECs fresh me-dium was added to each culture the day prior to conduct-ing experiments. The same production batch of fetalbovine serum was used for all experiments. Tightly con-fluent cultures of HUVECs (passages 1 to 4) on 6 wellplates were used for all experiments.

Assessment of cell viability

The viability of HUVECs after treatment with pyocy-anin for 1 h was assessed by trypan blue dye exclusionand by exclusion of the fluorescent nucleic acid stain,propidium iodide (Research Organics Inc., Cleveland,OH, USA). At the end of the incubation period theHUVECs were washed three times with PBS and freshPBS containing propidium iodide (50 �g/ml) was addedand allowed to incubate in the dark at room temperaturefor 15 min. Cells were washed three times with PBS

followed by fixation with glutaraldehyde (2%) in PBSfor 60 min. The cells were then examined by fluores-cence microscopy.

Hydrogen peroxide assay

The presence of hydrogen peroxide in the extracellu-lar medium was assayed for by the method of Thurmanet al. [24]. Confluent cultures of HUVECs in 6 welltissue culture plates were incubated in HBSS for 30 or 60min with pyocyanin (0 to 50 �M). After incubation, 500�l of medium was removed and centrifuged for 5 min at1000 � g to remove any cellular debris. To the super-natant was added trichloroacetic acid to give 2.5%, fer-rous ammonium sulfate to give 1 mM and ammoniumthiocyanate to give 0.25 M in a total volume of 1 ml. Thesamples were incubated for 10 min at room temperatureand the absorbance determined at 480 nm. The hydrogenperoxide content of samples was subsequently read from astandard curve. In order to confer greater specificity on theassay the catalase-inhibitable fraction was determined.

Glutathione assay

Intracellular levels of GSH and GSSG were assayedby the recycling method of Griffith [25]. After exposureto pyocyanin the cells were washed three times with PBSand lysed with 10 mM HCl followed by three freeze/thaw cycles. An aliquot was removed for protein deter-mination and the remainder of the lysate was treated with5-sulfosalicylic acid to give 5%. The samples were cen-trifuged at 10,000 � g for 5 min and the supernatantsassayed for GSH and GSSG. For the analysis of GSSG,GSH was first removed by the addition of 2 �l of2-vinylpyridine per 100 �l of sample.

Determination of soluble thiols

Soluble thiols (i.e., protein –SH and nonprotein –SH)were determined essentially according to the method ofEllman [26]. HUVECs were washed twice with phenolred-free HBSS and overlaid with HBSS containing pyo-cyanin (50 �M) and incubated for 30 min. The cells werethen washed three times with HBSS and lysed by theaddition of 200 �l of 10 mM HCl followed by threefreeze/thaw cycles. Soluble thiols were assayed by incu-bating 25 �l aliquots of cell lysate with 190 �l of 0.4 MTris-HCl (pH 8.9), 675 �l of methanol and 100 �l of5,5'-dithio-bis(2-nitrobenzoic acid) (3.96 mg/ml in meth-anol; DTNB) for 15 min. Following centrifugation at3000 � g for 15 min, the absorbance was measured at412 nm using GSH as the standard.

Treatment of HUVECs with pyocyanin and catalase

HUVECs were washed twice with phenol red-freeHBSS and cells overlaid with HBSS � 0.5% human

1528 M. MULLER

serum albumin (HBSS/A). Pyocyanin was dissolved inHBSS just prior to use and added to the HUVECs to givethe required concentrations and incubated for the appro-priate time at 37°C. Catalase (final concentration 1000U/ml in HBSS; preservative free) was added just prior tothe addition of pyocyanin.

NADPH assay

Intracellular levels of NADPH were assayed essen-tially according to the method described by Burch et al.[27].

Assay of GSSG-reductase activity

Confluent monolayers of HUVECs were lysed by theaddition of 400 �l of lysis buffer consisting of Tris-HCl(10 mM), ethylenediaminetetraacetic acid (EDTA; 1mM), Triton X-100 (0.2%) and phenylmethanesulphonylfluoride (50 �M) pH 7.5 at room temperature for 30 min.The lysate was centrifuged at 2500 � g for 10 min and50 �l aliquots assayed for GSSG-reductase activity inthe presence of NADPH (0.2 mM), GSSG (0.5 mM), andDTNB (0.6 mM) in assay buffer (KH2PO4, 125 mM;EDTA, 6.3 mM; pH 7.5). The absorbance was continu-ously monitored for 10 min at 412 nm.

Protein assay

Cellular protein was assayed by the method of Lowryet al. [28].

Statistical analysis and presentation of results

Statistical analysis was performed using the programGraphPad Prism (V2.01) (GraphPad Software, San Di-ego, CA, USA). Significance levels were determined byone-way analysis of variance with Dunnett’s multiplecomparison test or Student’s t-test. Data are presented asthe mean � standard deviation of at least three separateexperiments.

RESULTS

Effect of pyocyanin on cell viability

Incubation of HUVECs for 1 h with pyocyanin atconcentrations up to 50 �M did not result in the uptakeof trypan blue or propidium iodide, thus indicating thecells were viable after the incubation period.

Generation of hydrogen peroxide by endothelial cellsexposed to pyocyanin. Treatment of HUVECs with pyo-cyanin (0–50 �M) resulted in significant levels of hy-drogen peroxide being detected in the extracellular me-dium (p � .01) for all concentrations of the pigmentgreater than 1 �M (Fig. 1). Extending the incubationtime with pyocyanin from 30 to 60 min did not result in

a significant increase in formation of the oxidant. Pro-duction of hydrogen peroxide reached a plateau above apyocyanin concentration of 12.5 �M suggesting thatrate-limiting conditions had been reached. The maximumamount of hydrogen peroxide produced at 60 min was 24� 7.4 nmol/mg protein.

Effect of pyocyanin on endothelial cell GSH levels

Exposure of HUVECs to pyocyanin (0–50 �M) for30 min resulted in a decrease in total glutathione levels.Significant depletion occurred at a pyocyanin concentra-tion of 12.5 �M (p � .05; Fig. 2). Determination ofintracellular levels of GSSG showed that treatment withpyocyanin did not produce a significant increase beyond

Fig. 1. Hydrogen peroxide formation by endothelial cells exposed topyocyanin. Confluent cultures of HUVECs were incubated in HBSS for30 or 60 min with pyocyanin (0–50 �M) after which the medium wasassayed for the presence of hydrogen peroxide. Results represent themean � standard deviation of six separate experiments.

Fig. 2. Effect of pyocyanin on intracellular glutathione levels. Totalglutathione and GSSG levels were assayed after exposure to pyocyanin(0 to 50 �M) in HBSS/A for 30 min. Results represent the mean �standard deviation of four separate experiments. *p � .50, **p � .01.

1529Pyocyanin depletes cellular glutathione

control levels of 2.0 � 1.8 nmol/mg protein for anypyocyanin concentration.

No changes were observed in extracellular total GSHconcentrations after incubation with pyocyanin com-pared to control cells (data not shown).

Effect of pyocyanin on soluble thiol content

The content of soluble thiols (protein –SH and non-protein –SH) decreased in response to treatment withpyocyanin (50 �M) for 30 min (Fig. 3). Pyocyanintreatment produced 78.6 � 2.3 nmol –SH/mg proteincompared to 95.0 � 6.2 nmol –SH/mg protein for con-trols (p � .04).

Effect of pretreatment with catalase on intracellularglutathione

To determine if the presence of hydrogen peroxidewas directly responsible for the decrease in GSH levels,HUVECs were treated with catalase (1000 U/ml) prior tothe addition of pyocyanin (50 �M). In the absence ofcatalase, pyocyanin reduced the total glutathione contentby 34.3 � 3.5% (p � .01) with respect to control levelsafter a 1 h incubation while in the presence of pyocyaninand catalase the total glutathione content was not signif-icantly different from control levels but did differ signif-icantly from pyocyanin treatment alone (p � .01; Fig. 4).In preliminary experiments some commercial prepara-tions of catalase were found to reduce HUVEC GSHlevels in the absence of any other stimulus, possibly due

to the presence of preservatives; such catalase prepara-tions were not used in the experiments reported here.

Nonenzymatic conjugation and reduction of pyocyaninwith GSH

Reduced glutathione forms conjugates with manycompounds, by enzymatic and nonenzymatic means. Toexclude the possibility that either oxidized or reducedpyocyanin depleted GSH by nonenzymatic conjugation,GSH (50 �M) was incubated with pyocyanin (50 �M)for 30 min with or without NADPH (100 �M). At theend of the incubation DTNB (100 �M) was added andthe remaining GSH determined by absorbance at 412 nm.While oxidized pyocyanin was without effect, there wasa 56% reduction in GSH concentration when GSH wasincubated in the presence of reduced pyocyanin (p �.0001), however, no reduction occurred when superoxidedismutase (SOD; 250 U/ml) and catalase (1000 U/ml)were present demonstrating that pyocyanin-derived reac-tive oxygen species were responsible for the decrease inGSH (Fig. 5).

As pyocyanin can be directly reduced by several thiolcompounds (unpublished observations) the ability ofGSH to reduce pyocyanin was also investigated. At pH7.4 no significant reduction of pyocyanin (10 �M) oc-curred in the presence of GSH (1 mM) over 60 min inPBS when monitored between 200 and 400 nm (data notshown).

Effect of pyocyanin on intracellular NADPH

As increasing the incubation time of HUVECs withpyocyanin from 30 to 60 min did not result in greaterhydrogen peroxide formation, nor did increasing the doseof pyocyanin above 12.5 �M, the results suggested thata rate-limiting condition had been reached. As the reduc-

Fig. 3. Effect of pyocyanin on cellular soluble thiol content. Totalsoluble thiols (protein –SH and nonprotein –SH) were determined afterexposure of HUVECs to pyocyanin (50 �M) in HBSS for 60 min.Results represent the mean � standard deviation of five separateexperiments (*p � .04).

Fig. 4. Effect of catalase on glutathione depletion by pyocyanin. Cata-lase (Cat; 1000 U/ml) was added to confluent HUVECs immediatelyprior to the addition of pyocyanin (50 �M) or and equal volume ofHBSS/A and incubated for 60 min. Results represent the mean �standard deviation of four separate experiments. *p � .01 compared tocontrol.

1530 M. MULLER

tion of pyocyanin is dependent on the presence ofNADPH and depletion of this factor may cause a rate-limiting condition, intracellular levels of NADPH weredetermined. In the absence of pyocyanin, intracellularlevels of NADPH were found to be 0.60 � 0.06 nmol/mgprotein. For all pyocyanin concentrations tested, a sig-nificant increase (p � .01) in NADPH levels over thecontrol value was observed over 30 min. Pyocyanin at 5�M resulted in a 2.4-fold elevation in NADPH. How-ever, pyocyanin concentrations greater than 5 �M re-sulted in a steady decline in NADPH from the maximallevel of 1.44 � 0.12 nmol/mg protein (Fig. 6).

Effect of pyocyanin on GSSG reductase activity

To determine if pyocyanin affected GSSG reductaseactivity, cell-free preparations of the enzyme were pre-

pared and GSH formation in the absence and presence ofpyocyanin was determined. As pyocyanin is reduced in anonenzymatic manner by NADPH, which may lead tolimiting conditions with respect to NADPH in theGSSG-reductase assay, preliminary experiments wereperformed (not shown) to establish the nonlimiting con-centration of NADPH required, which proved to be 200�M.

Under cell-free conditions, concentrations of pyocya-nin of 5 �M or greater resulted in a significant increasein GSH formation (Fig. 7). When NADH was substitutedfor NADPH in the cell-free system pyocyanin failed toproduce an increase in GSH formation above controllevels (data not shown). In order to determine if a dif-ference in the rate of oxidation of NADH and NADPHby pyocyanin accounted for the difference, the aerobicreduction rates for pyocyanin were determined in PBS at25°C. The initial rates were determined to be 14.3 nmol/min for NADH and 23.8 nmol/min for NADPH in thepresence of pyocyanin (5 �M).

DISCUSSION

The maintenance of intracellular redox status is es-sential for the normal functioning of cellular systems.Inflammatory conditions, particularly those associatedwith infection, can alter cellular redox status as a resultof increased oxidative stress, either due to host defenses[29,30] or to microbial-derived oxidants [31]. Cellularredox status is maintained by antioxidant mechanismsand when these systems become impaired cells are ex-posed to the deleterious effects of oxidative stress. Hostcells possess a substantial armamentarium to defendagainst the effects of oxidants. The well-described func-tions of superoxide dismutase and catalase offer specificprotection against superoxide and hydrogen peroxide

Fig. 5. Depletion of glutathione by pyocyanin-derived reactive oxygenspecies. GSH (50 �M) was incubated with pyocyanin (50 �M) withand without NADPH (100 �M) for 30 min. The control consisted ofGSH (50 �M) and NADPH (100 �M). In the presence of superoxidedismutase (SOD; 250 U/ml) and catalase (Cat; 1000 U/ml) the pyocy-anin-dependent depletion of GSH did not occur. Results represent themean � standard deviation of three separate experiments. *p � .0001compared to control.

Fig. 6. Effect of pyocyanin on intracellular NADPH levels. ConfluentHUVECs were exposed to pyocyanin (0–50 �M) for 30 min. Resultsrepresent the mean � standard deviation of three separate experiments.

Fig. 7. Effect of pyocyanin on glutathione reductase activity. A cell-free preparation of HUVEC GSSG-reductase was incubated withNADPH (200 �M) and pyocyanin (0–50 �M) for 10 min. Resultsrepresent the mean � standard deviation of three separate experiments.

1531Pyocyanin depletes cellular glutathione

respectively. However, for the most part, general protec-tion against a wide range of oxidants is achieved by thepresence of high intracellular levels of GSH. Scavengingof oxidants at the intracellular level within the cytosolappears to rely on GSH and glutathione peroxidase foreliminating low micromolar levels of hydrogen peroxideand lipid hydroperoxides [20]. Additional protection canbe derived from dietary antioxidants such as ascorbicacid and �-tocopherol that protect the cellular aqueousand lipid phases, respectively.

The present study has shown that pyocyanin, a majorvirulence factor of P. aeruginosa, modulates the redoxstatus of endothelial cells in a complex manner (Fig. 8).By undergoing a redox cycle with intracellular NADPH,the cofactor becomes limiting, thus leading to an increasein NADPH production, which can be achieved by stim-ulation of the pentose phosphate pathway. Stimulation ofthe pentose phosphate pathway by pyocyanin has beenobserved in human neutrophils [9] and other cellularsystems with related phenazine derivatives [32,33]. Thereduced pyocyanin then exports reducing equivalents tothe extracellular space where molecular oxygen is re-duced to superoxide, which dismutates to hydrogen per-oxide. On entering the cell, hydrogen peroxide can causeformation of mixed disulfides by oxidation of protein andnonprotein thiol groups including GSH. In order to main-tain intracellular GSH levels, glutathione reductase mustthen compete with pyocyanin for NADPH in order toreduce GSSG to GSH. Although this study has shownthat pyocyanin can enhance the activity of glutathionereductase in a cell-free system, under cellular conditionswhere NADPH is limiting the contribution is likely to benegligible. The net effect is to lower intracellular levelsof free sulfhydryl groups, including GSH. While redoxactive compounds can deplete intracellular GSH by theformation of hydrogen peroxide, depletion can also occurby conjugation [34]. Under cell-free conditions no evi-

dence was found for the conjugation of GSH with eitherthe oxidized or reduced pigment. That catalase was ableto completely abrogate the effect of pyocyanin on GSHlevels further indicate that, under cellular conditions,conjugation of pyocyanin does not occur in these cells.

The depletion of intracellular glutathione by pyocya-nin is of particular interest in the context of chronicpseudomonal lung disease, and particularly cystic fibro-sis. As a result of their exocrine dysfunction, patientswith cystic fibrosis have impaired antioxidant defensesthat manifest as a systemic deficiency of GSH, includingthe epithelial lining fluid of the lung [35,36], and, forthose with pancreatic insufficiency, reduced plasma lev-els of �-tocopherol [37]. While dietary supplementationwith �-tocopherol adequately addresses the latter effect,raising and maintaining normal levels of GSH is moredifficult, a situation that may well be compounded by theeffect of pyocyanin-dependent depletion of GSH.

In conclusion, this study has demonstrated that pyo-cyanin at pathophysiological concentrations adverselyaffects endothelial cell redox status by the generation ofhydrogen peroxide with a consequent decrease in intra-cellular glutathione and the formation of mixed disul-fides.

Acknowledgements — This work was supported by grants from theWestmead Hospital Scientific Advisory Committee, the WestmeadInstitutes of Health, the Ageing and Alzheimer’s Research Foundationand a NSW Department of Health Infrastructure grant.

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Fig. 8. Mechanism of endothelial cell glutathione depletion by pyocyanin. GPx � glutathione peroxidase; GR � glutathione reductase;PPP � pentose phosphate pathway; Pox � oxidized pyocyanin; Pred � reduced pyocyanin.

1532 M. MULLER

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ABBREVIATIONS

DTNB—5,5'-dithio-bis(2-nitrobenzoic acid)GSH—reduced glutathioneGSSG—glutathione disulfideHBSS—Hanks’ balanced salts solutionHBSS/A—HBSS � human serum albuminHUVECs—human umbilical vein endothelial cellsPBS—phosphate buffered salinePyo—pyocyaninSOD—superoxide dismutase

1533Pyocyanin depletes cellular glutathione