Generation of Reactive Oxygen Species Mediated by1‑Hydroxyphenazine, a Virulence Factor of Pseudomonas aeruginosaSarmistha Sinha,† Xiulong Shen,† Fabio Gallazzi,§ Qian Li,∥,∇ Jaroslaw W. Zmijewski,⊥

Jack R. Lancaster, Jr.,#,○,¶ and Kent S. Gates*,†,‡

†Departments of Chemistry and ‡Biochemistry and §Structural Biology Core, University of Missouri, 125 Chemistry Building,Columbia, Missouri 65211, United States∥Departments of Anesthesiology, ⊥Pulmonary, Allergy & Critical Care Medicine, #Cell, Developmental, and Integrative Biology,○Environmental Health Sciences, and ∇Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham,Alabama 35205, United States¶Departments of Pharmacology and Chemical Biology, Medicine, and Surgery, University of Pittsburgh School of Medicine,Pittsburgh, Pennsylvania 15261, United States

*S Supporting Information

ABSTRACT: 1-Hydroxyphenazine (1-HP) is a virulencefactor produced by Pseudomonas aeruginosa. In this study,supercoiled plasmid DNA was employed as an analytical toolfor the detection of ROS generation mediated by 1-HP. Theseassays provided evidence that 1-HP, in conjunction withNADPH alone or NADPH and the enzyme NADPH:cyto-chrome P450 reductase, mediated the production of super-oxide radical under physiological conditions. Experiments withmurine macrophage RAW264.7 cells and profluorescent ROS probes dichlorodihydrofluorescein or dihydroethidine providedpreliminary evidence that 1-HP mediates the generation of intracellular oxidants. Generation of reactive oxygen species maycontribute to the virulence properties of 1-HP in P. aeruginosa infections.

■ INTRODUCTION

Pseudomonas aeruginosa is an opportunistic pathogen thatinfects the lungs of immunocompromised patients andindividuals with cystic fibrosis.1 This organism produces anumber of virulence factors, including the phenazine derivativespyocyanin, 1-phenazine carboxylic acid, and 1-hydroxyphena-zine (1-HP) (Chart 1).2 The best characterized of these ispyocyanin.3,4 Pyocyanin contributes to P. aeruginosa pathoge-nicity and tissue damage in the host by mechanisms involvingthe production of reactive oxygen species (ROS): superoxideradical, hydrogen peroxide, and hydroxyl radical.3−5 Pyocyaningenerates superoxide radical via redox cycling. This process isinitiated via direct (nonenzymatic) reduction of pyocyanin byNADPH, NADH, and, possibly, the cellular thiol gluta-thione.6−10 Subsequent reactions of one- or two-electron-reduced pyocyanin intermediates with molecular oxygengenerate superoxide radical anion (O2

•−).6−9 Superoxideradical, in turn, gives rise to hydrogen peroxide and hydroxylradical by the well-known cascade of reactions shown in(unbalanced) eq 1, involving disproportionation of superoxideto yield hydrogen peroxide followed by metal-mediatedFenton-type reactions that produce hydroxyl radical.11,12

→ → + → +•− + • + +O O H O M HO Mn n2 2 2 2

( 1)(1)

Although the phenazine virulence factors of P. aeruginosaoften are discussed as a group, their molecular structures are

distinct and they should be expected to possess differentchemical and biological properties. For example, the presenceof a methylated phenazine nitrogen in pyocyanin drasticallyalters its redox potential relative to 1-HP (making pyocyanin

Received: July 2, 2014Published: January 15, 2015

Chart 1

Article

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© 2015 American Chemical Society 175 DOI: 10.1021/tx500259sChem. Res. Toxicol. 2015, 28, 175−181

easier to be reduced).13,14 In contrast to pyocyanin, thechemical and biochemical mechanisms of the virulence factor 1-HP are not well understood. During the course of recentstudies related to the phenazine natural product myxin,15 weobserved that molecules structurally related to 1-HP mediatedROS generation when combined with the reducing agentNADPH. This was intriguing because 1-HP does not possess aclassic redox-cycling structural motif such as a quinone,heterocyclic di-N-oxide, phenazine methosulfate, or polysulfideunit.7,15−21 These observations, along with the important roleof 1-HP in the biology of P. aeruginosa, inspired us to examinethe ability of this compound to mediate ROS generation underphysiologically relevant conditions.

■ MATERIALS AND METHODSMaterials. Dulbecco’s modified Eagle’s medium (DMEM) and

fetal bovine serum (FBS) were purchased from Fisher Scientific(Fairlawn, NJ). 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) wasfrom Oxis International, Inc. (Portland, OR). 2,7-Diamino-10-ethyl-9-phenyl-9,10-dihydrophenanthridine (dihydroethidium, DHE) and2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) were fromSigma (St. Louis, MO) and Invitrogen (Carlsbad, CA), respectively.Other reagents were of the highest purity available and were obtainedfrom the following suppliers and used without purification: sodiumphosphate, NADPH, acetonitrile, desferal, NADPH:cytochrome P450reductase (CYPOR), catalase, and superoxide dismutase (SOD) werefrom Sigma-Aldrich Chemical Co. (St. Louis, MO); agarose was fromSeakem; HPLC grade solvents methanol and ethanol were from FisherScientific (Pittsburgh, PA); and ethidium bromide was from RocheMolecular Biochemicals (Indianapolis, IN). Plasmid DNA pGL2BA-SIC was prepared using standard protocols.22 1-HP was preparedusing a published procedure.23

Cleavage of Plasmid DNA by 1-HP. In a typical DNA cleavageassay, supercoiled plasmid DNA (pGL2BASIC, 1 μg) was incubatedwith 1-HP (100 μM) and NADPH (200 μM) in sodium phosphatebuffer (50 mM, pH 7.0), in a final volume of 40 μL. Some assayscontained NADPH:cytochrome P450 reductase (0.05 U/mL, whereone unit is defined as the amount of enzyme required to cause thereduction of 1 μmol of cytochrome c by NADPH per min at pH 7.4 at37 °C), SOD (100 μg/mL), catalase (100 μg/mL), desferal (1 mM),and methanol (1 mM) or ethanol (1 mM). Heat-denatured catalaseand SOD for control reactions were prepared by heating aliquots ofthe enzyme stock solutions in a boiling water bath for 4 h. Reactionswere initiated by addition of NADPH or NADPH:cytochrome P450reductase, wrapped with aluminum foil to prevent exposure to light,and incubated under air at 24 °C for 12 h. Following incubation, thereactions were mixed with 50% glycerol loading buffer22 (6.6 μL), andthe resulting reaction mixture was loaded onto a 0.9% agarose gel. The

gel was electrophoresed for approximately 2 h at 90 V in TAE bufferand then stained in a solution of ethidium bromide (0.3 μg/mL) for 3h. DNA in the gel was visualized by UV-transillumination, and theamount of DNA in each band was quantified using an Alpha InnotechIS-1000 digital imaging system. Yields of DNA strand breaks in eachreaction (S) were calculated using the equation S = −ln f1, where f1 isthe fraction of plasmid remaining as supercoiled form I.

Nitro Blue Tetrazolium Assay. In a typical assay, 1-HP (100μM), NADPH (500 μM), nitro blue tetrazolium chloride (500 μM),and NADPH:cytochrome P450 reductase (0.13 U/mL) wereincubated in sodium phosphate buffer (50 mM, pH 7, 200 μL) at24 °C under aerobic conditions for 12 h. After incubation, solidsodium carbonate was added to the assay to adjust the pH to 10. Theassay was diluted with water to a final volume of 1 mL, and theabsorbance at 560 nm was measured. Two control experiments wereperformed using the same conditions except lacking 1-HP or NADPH,respectively.

Cell Culture and Imaging of DHE and DCF Fluorescence.Murine macrophage cell line RAW264.7 (American Type CultureCollection, Manassas, VA) was routinely cultured in DMEMsupplemented with 10% FBS, 100 units/mL of penicillin, and 100μg/mL streptomycin, as described previously.24 For the experiments,cells were seeded into 4-well Permanox slides (Nalge NuncInternational Corp., Naperville, IL) to reach 90% confluenceovernight. Cells were washed and incubated in DMEM containing0.5% FBS (0.5 mL/well) with 1-HP or DMNQ at the indicatedconcentrations for 30 min. Then, 20 μM DHE or H2DCFDA wasadded, and confocal fluorescence microscopy was performed.25,26

Images were acquired from three or more independent fields/well andwere processed using IPLab Spectrum and Adobe Photoshop (AdobeSystems). The levels of fluorescence were averaged using SimplePCIsoftware (Compix, Cranberry Township, PA).

■ RESULTS AND DISCUSSIONEvidence That 1-HP, in Conjunction with NADPH or

NADPH and NADPH:Cytochrome P450 Reductase,Mediates ROS Generation. In this study, we employedsupercoiled plasmid DNA as an analytical tool for the detectionof ROS. This assay capitalizes on the fact that oxidative DNAstrand cleavage causes conversion of the supercoiled plasmid tothe open circular form.27−30 These two forms of plasmid DNAare easily separated and quantitatively measured using agarosegel electrophoresis, staining the DNA with ethidium bromide,and digital image analysis of the stained gel. In general,intracellular generation of superoxide radical causes a host ofdeleterious effects including oxidative damage to DNA,proteins, and lipids and cytotoxicity.6,7,12,31−35 In the contextof the plasmid assay used here, it is important to note that

Figure 1. DNA strand cleavage by 1-HP driven by NADPH and NADPH plus NADPH:cytochrome P450 reductase (CYPOR). Supercoiled plasmidDNA (1 μg) was incubated with 1-HP (1-HP, 100 μM) and the indicated concentration of NADPH (200 μM, 400 μM, 750 μM, 1 mM) in sodiumphosphate buffer (50 mM, pH 7.0) at 24 °C for 12 h. Some assays contained CYPOR (0.05 U/mL) or catalase (Cat, 100 μg/mL) and desferal (Des,1 mM) as indicated. Yields of DNA strand breaks in each reaction (S) were measured by agarose gel electrophoresis and calculated using theequation S = −ln f1, where f1 is the fraction of plasmid remaining as supercoiled form I. Lane 3, S = 0.29 ± 0.16 above background; lane 4, S = 1.02 ±0.39 above background; lane 7, S = 0.63 ± 0.10 above background; lane 8, S = 1.52 ± 0.27 above background; lane 11, S = 0.82 ± 0.19 abovebackground; lane 12, S = 1.61 ± 0.58; lane 15, S = 0.91 ± 0.13 above background; lane 16, S = 1.83 ± 0.44 above background.

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hydroxyl radical, or a species of similar reactivity, is the actualagent responsible for DNA strand cleavage induced bysuperoxide radical.12,36 Nonetheless, the use of additives suchas superoxide dismutase, catalase, trace metal chelators, andhydroxyl radical scavenging agents can reveal whether the O2

•−

→ H2O2 → HO• reaction cascade is operative.12,16,37 There area number of examples in which enzymatic reduction drivesredox cycling of small organic molecules.16,38,39 Accordingly, weexamined the effect of NADPH:cytochrome P450 reductase(CYPOR) on the production of ROS by 1-HP. This enzymehas been implicated in the redox cycling of a wide variety ofcompounds.16,38−43

We found that 1-HP (100 μM) in conjunction with NADPH(100 μM) in sodium phosphate buffer (50 mM, pH 7)mediated the formation of direct strand breaks in thesupercoiled plasmid DNA substrate (Figure 1). The amountof strand cleavage increased with increasing concentrations ofNADPH. At lower concentrations of NADPH (e.g., 100 μM),the yields of strand cleavage induced by 1-HP (100 μM) wereenhanced 3.5-fold by the presence of CYPOR. Control assaysshowed that 1-HP alone or NADPH and CYPOR without 1-HP did not yield substantial amounts of strand cleavage.In general, the generation of direct strand breaks (as opposed

to base-labile lesions) caused by 1-HP in the plasmid assay wasconsistent with radical-mediated strand cleavage.36 Therefore,we set out to characterize the nature of the DNA-cleavingradicals generated by 1-HP. We found that addition of classicalhydroxyl radical scavengers12 methanol or ethanol (1 mM)inhibited the strand cleavage process (Figure 2). Addition of

the metal chelator desferal inhibits strand cleavage, consistentwith the involvement of adventitious transition metals in aFenton-type generation of hydroxyl radical from H2O2 (Figure2).12 Similarly, the hydrogen peroxide-destroying enzymecatalase inhibited the strand cleavage process. A controlreaction showed that heat-denatured catalase did notsignificantly inhibit the strand cleavage process. (In fact, heat-denatured catalase slightly increased strand cleavage yields,probably due to the release of redox-active transition metalsthat can participate in Fenton-type reactions; see Figure S1.)Addition of the enzyme superoxide dismutase (SOD) had asmall inhibitory effect on the strand cleavage process. SOD

merely accelerates the already fast disproportionation ofsuperoxide to H2O2 and O2 and therefore may not be expectedto have a significant effect on strand cleavage that arises fromthe production of superoxide radical.12 Heat-denatured SODexerted little effect on the reactions. In cases where SODinhibits ROS-mediated strand cleavage, it may reflect a role forsuperoxide in the reduction of transition metals required for theFenton reaction.12,44

The yields of strand cleavage increased as a function of both1-HP and NADPH concentrations (Figures 3 and 4). A side-

by-side comparison showed that 1-HP (0.28 ± 0.02 strandbreaks above background) generated approximately 0.28 timesas many direct strand breaks as did the archetypal45,46 redox-cycling agent 2,3-dimethoxy-1,4-naphthoquinone (DMNQ,0.97 ± 0.13 breaks above background) when each agent (100μM) was incubated in sodium phosphate buffer (50 mM, pH 7)with plasmid DNA in the presence of CYPOR (0.05 U/mL)and NADPH (200 μM) for 3 h at 24 °C. This indicated thatDMNQ was more effective than 1-HP at the generation ofstrand-cleaving ROS, under these reaction conditions. Con-sistent with a redox-cycling mechanism underlying thegeneration of superoxide radical by 1-HP, using LC-MS, wedid not observe any metabolites generated from this moleculeunder the reaction conditions employed in the plasmid assays.Further evidence consistent with the generation of super-

oxide in these assays was provided by a separate UV−vis

Figure 2. Evidence that DNA strand cleavage by 1-HP involves O2•−

→ H2O2 → HO•. Supercoiled plasmid DNA (1 μg) was incubatedwith 1-HP (100 μM), NADPH (200 μM), and CYPOR (0.05 U/mL)in sodium phosphate buffer (50 mM, pH 7.0) at 24 °C for 12 h.Additive concentrations were as follows: catalase and SOD (100 μg/mL), desferal (1 mM), methanol (1 mM), and ethanol (1 mM). Yieldsof DNA strand breaks in each reaction were measured by agarose gelelectrophoresis and calculated using the equation S = −ln f1, where f1 isthe fraction of plasmid remaining as supercoiled form I.

Figure 3. Dependence of strand cleavage yields on the concentrationof 1-HP. Supercoiled plasmid DNA (1 μg) was incubated with 1-HP(100 μM), NADPH (200 μM), and CYPOR (0.05 U/mL) in sodiumphosphate buffer (50 mM, pH 7.0) at 24 °C for 12 h. Yields of DNAstrand breaks in each reaction were measured by agarose gelelectrophoresis and calculated using the equation S = −ln f1, wheref1 is the fraction of plasmid remaining as supercoiled form I.

Figure 4. Dependence of strand cleavage yields on NADPHconcentration. Supercoiled plasmid DNA (1 μg) was incubated with1-HP (100 μM), varying concentrations of NADPH (200−1000 μM),and CYPOR (0.05 U/mL) in sodium phosphate buffer (50 mM, pH7.0) at 24 °C for 12 h. This plot is derived from the experimentsillustrated in Figure 1. Yields of DNA strand breaks in each reactionwere measured by agarose gel electrophoresis and calculated using theequation S = −ln f1, where f1 is the fraction of plasmid remaining assupercoiled form I.

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spectrophotometric assay that detected the conversion of nitroblue tetrazolium to monoformazan.47,48 In this experiment, 1-HP (200 μM) and nitro blue tetrazolium chloride (500 μM)were incubated with NADPH (500 μM) and CYPOR (0.13 U/mL) in sodium phosphate buffer (50 mM, pH 7) at 24 °C for12 h. In order to detect monoformazan generated in the assay,the pH of the mixture was adjusted to 10 prior to measuring the

absorbance at 560 nm. In an assay containing 1-HP, CYPOR,and NADPH, we observed a 3.4-fold increase in absorbance at560 nm versus a control sample containing only CYPOR andNADPH and a 9.7-fold increase versus a control containing theCYPOR enzyme without the NADPH substrate.We also employed cytochrome c as a probe for superoxide

radical production. Reduction of the heme unit in cytochrome c

Figure 5. Evidence that 1-HP generates ROS in cells. Murine macrophage RAW264.7 cells were incubated with 1-HP (5−30 μM) or DMNQ (30μM) for 30 min; then, profluorescent ROS probes H2DCFDA (20 μM) or DHE (20 μM) was added, and confocal microscopy was performed. (A,C) Confocal microscopic images of cells treated with 1-HP or DMNQ, respectively. (B, D) Bar graphs showing the increase in fluorescence fromimages acquired from three or more independent fields.

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by superoxide radical anion generates an absorbance increase at550 nm.49 We found that incubation of 1-HP (50 μM),NADPH (100 μM), CYPOR (0.05 U/mL), and cytochrome c(100 μM) for 12 h at 24 °C gave an absorbance increase at 550nm above background (cytochrome c in buffer) that, basedupon the known difference in molar absorptivity of reducedminus oxidized cytochrome c of 21 000 M−1 cm−1,corresponded to the generation of 1.5 equiv of superoxideradical based on 1-HP. The signal was substantially diminishedin control samples containing SOD, but it was not significantlyaffected by the presence of heat-denatured SOD. Controlexperiments showed that 1-HP alone, NADPH alone, orCYPOR alone did not generate significant levels of superoxideradical (Figure S2).As noted above, superoxide radical rapidly disproportionates

to generate hydrogen peroxide. We used ferrous ion oxidationin the presence of xylenol orange to detect hydrogen peroxidein our assays.50 The resulting ferric ion−xylenol complex has anapparent extinction coefficient of 2.24 × 105 M−1 cm−1 at 560nm. Incubation of 1-HP (100 μM), NADPH (200 μM), andCYPOR (0.05 U/mL) for 4 h at 24 °C followed by addition toa solution containing xylenol orange and ammonium ferroussulfate gave a strong absorbance at 560 nm. In contrast, nosignificant absorbance above background was observed in aparallel reaction spiked with the hydrogen peroxide-destroyingenzyme catalase. Overall, the evidence indicated that 1-HPmediates the generation of superoxide radical and hydrogenperoxide in conjunction with NADPH alone or the CYPORenzyme system.Evidence That 1-HP Mediates Oxidant Generation in

Cells. Finally, we examined whether 1-HP mediated thegeneration of oxidants in cells. In these experiments, RAW264.7cells were incubated with 1-HP (5−30 μM), and ROSgeneration was probed by either H2DCFDA (20 μM) orDHE (20 μM). H2DCFDA and DHE are well-knownprofluorescent probes for ROS.51,52 H2DCFDA is convertedto the fluorescent dye dichlorofluorescein (DCF) by variousoxidants including HO•, CO3

•−, NO2•, cytochrome c, low

molecular weight organic radicals, and H2O2 in the presence ofvarious catalysts such as heme iron.51−54 DHE displays distinctreactivity and is converted to the fluorescent products 2-hydroxyethidium (HE) and ethidium (E+) by O2

•− and HO•,ONOO−, or H2O2, respectively.

51,53 Fluorescent microscopyexperiments cannot distinguish HE and E+.51,54 Confocalmicroscopy revealed concentration-dependent increases in thefluorescence of DCF or HE in cells treated with 1-HP,consistent with intracellular generation of ROS by 1-HP(Figure 5). It is known that H2DCFDA alone can induce greenfluorescence via autoxidation and peroxidatic mechanisms.51,54

This type of chemistry may contribute to the backgroundfluorescence observed in the control samples containing no 1-HP. However, it is important to emphasize that we observedincreases in DCF fluorescence that are above background levelsand are dependent upon the presence and concentration of 1-HP (Figure 5). The fluorescence increases of both DCF andHE generated by 1-HP (30 μM) were comparable to thosegenerated by the redox-cycling quinone DMNQ (30 μM,Figure 5D). It is important to acknowledge that seriousproblems such as the potential for redox cycling by theseprobes preclude quantitative conclusions. Furthermore, thenonselective reactivity of these probes does not allowimplication of a specific oxidant in the fluorescence increasesinduced by 1-HP in cells. Nonetheless, the results provided

preliminary evidence that 1-HP mediates the generation ofintracellular oxidants. The results of these cellular studies forgea tentative link to our own in vitro studies described above andare in line with earlier studies that providing evidence that 1-HP mediates the generation of H2O2 in tracheal epithelialcells55 and ESR data showing that phenazine 1-carboxylic acidleads to production of O2

•− in A549 cells.58

■ CONCLUSIONS

We have presented evidence that 1-HP mediates the nettransfer of electrons from NADPH to molecular oxygen. Thisprocess can be facilitated by the enzyme CYPOR. The resultinggeneration of ROS may contribute to the properties of 1-HP asa virulence factor for P. aeruginosa. Our results are consistentwith previous studies that measured the ability of 1-HP toinduce ciliary dysfunction in mammalian tracheal epithelialcells.55 The authors of this earlier work concluded that 1-HPacted by a mechanism involving the production of H2O2,although this was ascribed to an ability of the compound toactivate a respiratory burst in neutrophils rather to direct redoxcycling.55 Our results suggesting that 1-HP is susceptible toenzymatic reduction by CYPOR are generally in line with arecent study showing that 1-HP, pyocyanin, and phenazine 1-carboxylic acid serve as electron shuttles that promoteanaerobic survival of P. aeruginosa.56 It was suggested thatphenazine 1-carboxylic acid serves in this capacity by acceptingreducing equivalents from some as yet unidentified enzyme inP. aeruginosa.56 Finally, it is interesting to note that generationof ROS by the virulence factor phenazine 1-carboxylic acid hasbeen detected in cellular assays,57 although it has been reportedthat this agent is incapable of NAD(P)H-driven redox cycling.58

Redox-driven generation of ROS by small organic moleculesis important in drug action, toxicology, and high-throughputscreening assays.59−61 There is a growing awareness thatchemical structures beyond classical motifs such as quinonesand paraquat can undergo redox cycling.21,60−62 The resultsreported here contribute to a broader understanding of thestructural motifs that are capable of redox-driven ROSgeneration under physiological conditions.

■ ASSOCIATED CONTENT

*S Supporting InformationFigure S1: Control reactions for DNA strand cleavage by 1-hydroxyphenazine. Figure S2: Cytochrome c assay for thedetection and quantitation of superoxide radical. Figure S3:Ferrous ion oxidation (FOX) assay for the detection andquantitation of hydrogen peroxide. Figure S4: 1H and 13CNMR spectra of 1-hydroxyphenazine. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Phone: (573) 882-6763. Fax: (573) 882-2754. E-mail:gatesk@missouri.edu.

FundingWe thank the National Institutes of Health for partial supportof this work (CA 100757).

NotesThe authors declare no competing financial interest.

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■ ABBREVIATIONSCYPOR, NADPH:cytochrome P450 reductase; NBT, nitroblue tetrazolium chloride; SOD, superoxide dismutase; CAT,catalase; Des, desferal; 1-HP, 1-hydroxyphenazine; DHE,dihydroethidium; HE, 2-hydroxyethidium; H2DCFDA, dichlor-odihydrofluorescein diacetate; DCF, dichlorofluorescein; HE,2-hydroxyethidium; ROS, reactive oxygen species

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