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Page 1: Molecular imaging of hydrogen peroxide produced for cell signaling

Molecular imaging of hydrogen peroxide produced forcell signalingEvan W Miller1, Orapim Tulyathan2, Ehud Y Isacoff2 & Christopher J Chang1

Hydrogen peroxide (H2O2) is emerging as a newly recognizedmessenger in cellular signal transduction1,2. However, asubstantial challenge in elucidating its diverse roles in complexbiological environments is the lack of methods for probing thisreactive oxygen metabolite in living systems with molecularspecificity. Here we report the synthesis and application ofPeroxy Green 1 (PG1) and Peroxy Crimson 1 (PC1), two newfluorescent probes that show high selectivity for H2O2 and arecapable of visualizing endogenous H2O2 produced in livingcells by growth factor stimulation, including the first directimaging of peroxide produced for brain cell signaling. Thecombined features of reactive oxygen species selectivity,sensitivity to signaling levels of H2O2, and live-cellcompatibility presage many new opportunities for PG1, PC1and related synthetic reagents for exploring the physiologicalroles of H2O2 in living systems with molecular imaging.

Hydrogen peroxide is a small-molecule metabolite that exerts diversephysiological and pathological effects in living systems1–3. H2O2 wasonce viewed only as a marker for oxidative stress and damage eventsconnected to disease and aging or as a killing agent released byimmune cells in response to microbial invasion, but mounting newdata suggest that H2O2 serves as a messenger in normal cellular signaltransduction1,2,4. NADPH oxidase (Nox)complexes assembled in a wide spectrum ofnonphagocytic cell types can produce burstsof H2O2 upon stimulation with various pep-tide growth factors5,6, cytokines7, hormones8,and neurotransmitters9. Upon its generationthrough receptor-mediated Nox activation,H2O2 can activate a host of specific down-stream targets, including phosphatases10,11,kinases12, transcription factors13, and ionchannels14, through chemoselective oxidationof cysteine15–17, histidine18, or methionine19

residues that can then be re-reduced in thecell. The emerging concept of H2O2 as aredox signal that triggers reversible post-translational modifications of preciseprotein targets has generated interest in

understanding how cells produce, partition and funnel H2O2 intospecific signaling pathways.

The implication that H2O2 can serve as a beneficial messenger forphysiological redox signaling is provocative, but detailed investigationsof its oxidation biology are hampered in part owing to the difficulty intracking this small and reactive oxygen metabolite in real time,especially in live-cell settings. Molecular imaging with H2O2-responsive fluorophores offers a potentially powerful approach forstudying aspects of peroxide accumulation, trafficking, and functionin living systems with spatial and temporal fidelity. The key challengeis devising ways to detect H2O2 selectively over similar reactive oxygenspecies (ROS), particularly superoxide, nitric oxide and hydroxylradical. Fluorometric H2O2 assays are available20–24 but have limitedutility in live-cell studies because of (i) interfering fluorescence fromnonspecific reactions with other ROS or from prolonged light expo-sure, (ii) potential reactivity with thiols that exist in high cellularconcentrations, (iii) the need for an external activating enzyme orgenetic encoding, (iv) ultraviolet excitation and emission profiles thatcan trigger sample damage or autofluorescence, and/or (v) a lack ofmembrane permeability. We now present the synthesis and appli-cation of two new small-molecule fluorophores for detecting endo-genous H2O2 produced for signaling in living cells. PG1 and PC1are boronate-based H2O2 probes that have high selectivity and

O O

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Scheme 1 Syntheses of two new fluorescent probes for H2O2. (a) PG1. (b) PC1. Me, methyl;

Ph, phenyl; TfO, trifluoromethanesulfonate; Ac, acetate; dppf, 1,1¢-bis(diphenylphosphino)ferrocene.

Received 14 November 2006; accepted 8 March 2007; published online 1 April 2007; corrected after print 3 May 2007; doi:10.1038/nchembio871

1Department of Chemistry, University of California, 532A Latimer Hall, Berkeley, California 94720, USA. 2Department of Molecular and Cell Biology, Universityof California, Valley Life Sciences Addition, Berkeley, California 94720, USA. Correspondence should be addressed to C.J.C. ([email protected]).

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visible-wavelength excitation and emissionprofiles. These new chemical tools are capableof identifying and tracking H2O2 withinliving cells, as well as directly visualizingH2O2 generated by Nox-mediated growthfactor signaling. Finally, these reagents helpprovide strong evidence for H2O2 signaling inbrain systems via growth factor activation inprimary neurons.

We recently discovered that deprotectionof aryl boronates to phenols provides a reac-tion-based approach to specific fluorescencedetection of H2O2 over other ROS in situa-tions of oxidative stress25,26. However, initialattempts to use these diboronate reagents todetect H2O2 under oxidative signaling condi-tions were unsuccessful (data not shown).Seeking to develop new chemical tools thatare sensitive enough to report H2O2 produc-tion at physiological signaling levels whilemaintaining H2O2 specificity, we turned ourattention to dyes that can be activated by asingle boronate deprotection. Scheme 1 sum-marizes the syntheses of PG1 and PC1 basedon this design. Briefly, treatment of 2-methyl-4-O-methyl Tokyo Green (1) with N-phenylbis(trifluoromethanesulfonamide) affords tri-flate 2, and palladium-catalyzed borylationdelivers PG1 (3). An analogous sequencegoing from resorufin sodium salt (4) throughits corresponding triflate 5 produces PC1 (6).

We evaluated PG1 and PC1 under simu-lated physiological conditions (20 mMHEPES, pH 7) (Fig. 1). The dyes showedsimilar responses in various other buffersystems (Supplementary Fig. 1 online).PG1 features one prominent absorptionband in the visible region centered at460 nm (e ¼ 5,500 M–1 cm–1) and showsweak fluorescence with an emission maxi-mum at 510 nm (F ¼ 0.075). PC1 has onemajor absorption maximum at 480 nm (e ¼4,800 M–1 cm–1), with a correspondingweak emission band centered at 584 nm(F ¼ 0.006). Addition of H2O2 resulted in marked increases ingreen and red fluorescence for PG1 and PC1, respectively. Reactionof PG1 with H2O2 triggered a ten-fold fluorescence turn-on, whereasH2O2 elicited a 40-fold increase in fluorescence for PC1. Absorptionand emission spectra, along with mass spectrometry data, establishthat the H2O2-mediated boronate deprotections of PG1 and PC1generate 2-methyl-4-O-methyl Tokyo Green and resorufin, respec-tively, as fluorescent products. Figures 1a and 1b show representativeturn-on fluorescence responses for both reagents to H2O2. Kineticsmeasurements of the H2O2-mediated boronate deprotections wereperformed under pseudo-first-order conditions (1 mM dye, 1 mMH2O2), giving modest observed rate constants of kobs ¼ 1.1(1) � 10–3

s–1 and kobs ¼ 1.0(1) � 10–3 s–1 for PG1 and PC1, respectively.Maximum turn-on responses were observed within 2–3 h, but the highoptical brightness of the probes allows for detection of H2O2 withoutundergoing the full intensity change. Figure 1e compares the relativeH2O2 responses of monoboronate PG1 and PC1 to those of their

diboronate analogs Peroxyfluor 1 (PF1, 7) and Peroxyresorufin 1 (PR1,8), and to the H2O2 response of the nonspecific ROS probe dichloro-dihydrofluorescein (DCFH, 9). The data reveal that monoboronatePG1 undergoes the greatest absolute change in fluorescence turn-onupon reaction with H2O2 relative to other probes tested. Notably,DCFH is far less responsive to H2O2 relative to the boronate reagents,which is consistent with previous reports that establish that thisnonspecific oxidant indicator is more sensitive to other ROS27.

Owing to their chemoselective boronate switch, PG1 and PC1 retainhighly specific responses to H2O2. Figures 1c and 1d show relativeturn-on fluorescence increases observed for PG1 and PC1, respectively,in the presence of various biologically relevant ROS. PG1 is 4500-foldmore responsive toward H2O2 than it is toward superoxide (O2

–) ort-butoxy radical (�OtBu), and it is 425-fold more selective forH2O2 than for t-butyl hydroperoxide (TBHP, 10), hypochlorite ion(–OCl), hydroxyl radical (�OH) and ozone (O3). The xanthenoneprobe also shows a 415-fold higher response toward H2O2 than

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Figure 1 Spectroscopic responses and selectivities for H2O2 probes. All spectra were acquired in

20 mM HEPES, pH 7, at 25 1C. (a,b) Representative fluorescence responses to 100 mM H2O2 for5 mM PG1 (a) and 5 mM PC1 (b) after 60 min at 25 1C. The dotted and solid line spectra were

recorded before and after H2O2 addition, respectively (lexc ¼ 450 nm for PG1, 550 nm for PC1).

(c,d) Fluorescence responses to various ROS for 5 mM PG1 (c) and 5 mM PC1 (d). Data shown are for

10 mM for O2–, 2 mM for 1O2 and 100 mM for all other ROS. Concentrations given for highly reactive

ROS, including �OH, �OtBu and 1O2, are cumulative. (e) Relative responses to 100 mM H2O2 for

5 mM DCFH, PF1, PG1, PR1 and PC1. Data were acquired under similar conditions to obtain an

accurate reflection of the relative brightness of the dyes in response to H2O2. Collected emission was

integrated between 460 and 700 nm (lexc ¼ 450 nm) for PG1 and PF1, between 570 and 800 nm

(lexc ¼ 560 nm) for PC1 and PR1, and between 470 and 700 nm (lexc ¼ 460 nm) for DCFH. Bars

represent relative responses at 5 min (white), 15 min (light gray), 30 min (dark gray), 45 min (black)

and 60 min (red) after addition of H2O2. Error bars represent s.e.m.

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toward singlet oxygen (1O2) and a 44-fold greater turn-on for H2O2

relative to nitric oxide (NO). PC1 also has good H2O2 selectivity,showing a 4500-fold higher response toward H2O2 relative to O2

–,�OH, or �OtBu, and a 450-fold greater turn-on toward H2O2

relative to TBHP and O3. The red-fluorescent probe has a 415-foldhigher sensitivity to H2O2 over 1O2, and a 44-fold greater selectivityto H2O2 relative to NO and –OCl. Finally, the boronate probes areunreactive toward high-valent metal-oxo species derived from hemeproteins and H2O2, whereas the combination of heme protein andH2O2 triggers a much higher response for DCFH than does H2O2

alone (Supplementary Fig. 2 online).The boronate dyes were then tested for detecting H2O2 in living

biological samples using confocal microscopy (Fig. 2). Live humanembryonic kidney (HEK) 293 cells loaded with 5 mM PG1 for 15 minat 25 1C showed low levels of background fluorescence from the latentdye (Fig. 2a). Addition of 10 mM H2O2 to PG1-labeled cells (Fig. 2b)triggered clear increases in intracellular green fluorescence. Controlexperiments without dye showed no background fluorescence. Also,the deprotected 2-methyl-4-O-methyl Tokyo Green dye is notmembrane-permeable under these conditions, which confirms thatPG1 can enter cells and image changes in [H2O2]. Similar experimentswith 543-nm excitation showed that the red-fluorescent analog PC1can also detect micromolar changes in intracellular H2O2 concentra-tions (Fig. 2d,e). Brightfield transmission measurements after incuba-tion with either PG1 (Fig. 2c) or PC1 (Fig. 2f) and H2O2 along withthe nuclear stain Hoescht 33342 (data not shown) revealed that thecells are viable throughout the imaging experiments.

To test whether these new chemical tools can be used to visualizenatural cellular H2O2 production, we sought to probe the

relationships between physiological H2O2 generation and growthfactor signaling. Experiments were performed with the PG1 probeowing to its superior fluorescence brightness in response to H2O2.A431 cells were chosen for initial studies owing to their high expres-sion of epidermal growth factor receptors (EGFR)28. Live A431 cellsloaded with 5 mM PG1 showed modest fluorescence (Fig. 3a). Incontrast, the same PG1-labeled cells activated with a physiologicallyrelevant dose of growth factor (500 ng ml–1 EGF for 15 min at 37 1C)6

showed bright fluorescence relative to pre-stimulation levels (Fig. 3b).Control experiments with EGF minus dye gave no backgroundfluorescence, thereby establishing that PG1 is capable of visualizingintracellular H2O2 produced by growth factor activation. Building onthis result, we next used PG1 to probe the molecular pathway of EGF-mediated H2O2 production in conjunction with chemical inhibitors.We found that the EGF-stimulated generation of H2O2 is attenuatedby inhibitors of the EGFR kinase domain (PD153035, 100 mM,Fig. 3c), inhibitors of phosphatidylinositol-3-OH kinase (wortman-nin, 100 mM, Fig. 3d) and inhibitors of Nox (apocynin, 100 mM,Fig. 3e). Moreover, the H2O2 signal was not affected by an NO synthaseinhibitor (L-NAME, 100 mM, Fig. 3f). Figure 3g compares the relativefluorescence intensities of cells undergoing each of these treatments.Taken together, the results establish that PG1 is capable of detectingnatural signaling levels of H2O2 generated by living cells and can beused to map molecular pathways associated with H2O2 production.

The foregoing experiments led us to explore the possibility ofexpanding H2O2-mediated growth factor signaling to new biologicalsystems, and we were specifically interested in the brain because of itsunusually high oxidative capacity. Initial experiments verified thepresence of EGFR in postnatal rat hippocampal neurons (DIV 14 to

a b c d e f

Figure 2 Live-cell H2O2 imaging with PG1 and PC1. (a) Fluorescence image of live HEK 293 cells incubated with 5 mM PG1 for 5 min at 25 1C. (b) PG1-

stained HEK 293 cells treated with 10 mM H2O2 for 30 min at 25 1C. (c) Brightfield transmission image of cells shown in b. (d) Fluorescence image of HEK

293 cells incubated with 5 mM PC1 for 5 min at 25 1C. (e) PC1-stained HEK 293 cells treated with 10 mM H2O2 for 30 min at 25 1C. (f) Brightfield

transmission image of cells shown in e. Excitation was provided at 488 nm and 543 nm, for PG1 and PC1, respectively. Scale bar, 40 mm.

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gFigure 3 Molecular imaging of H2O2 produced by growth factor signaling. (a) Confocal fluorescence image of

live A431 cells incubated with 5 mM PG1 for 15 min at 37 1C. (b) PG1-loaded A431 cells from a stimulated

with 500 ng ml–1 EGF for 15 min at 37 1C. (c) PG1-labeled cells pretreated with PD153035 before EGF

stimulation. (d) PG1-labeled cells pretreated with wortmannin before EGF stimulation. (e) PG1-labeled cells

pretreated with apocynin before EGF stimulation. (f) PG1-labeled cells pretreated with L-NAME before EGF

stimulation. (g) Graph showing relative fluorescence intensities for treatments in a–f. Error bars represent s.e.m.

* indicates that P o 0.05 when compared against EGF-treated cells. For all inhibitor experiments, cells were

pretreated with 100 mM inhibitor for 25 min at 37 1C before EGF stimulation. Loading PG1 before or after

inhibitor treatment gave the same fluorescence staining. Excitation was provided at 488 nm. Scale bar, 25 mm.

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DIV 20) by immunostaining on fixed samples (Fig. 4a–c)29. We nexttreated live neurons from the same culture with PG1 (5 mM, 10 min,37 1C) and stimulated with EGF (1 mg ml–1, 10 min, 37 1C). In theabsence of inhibitors, compared with unstimulated neurons (Fig. 4d)the EGF-treated neurons (Fig. 4e) showed markedly brighter fluore-scence. The EGF-induced rises in intracellular [H2O2] were muted byinhibitors of EGFR, phosphatidylinositol-3-OH kinase, Rac1 and Nox(Fig. 4f–i). Figure 4j compares the relative fluorescence intensities ofneurons undergoing each of these treatments. These data suggest thatthe primary neurons have the required cellular machinery for gene-rating H2O2 through a pathway analogous to the EGFR/Nox pathwayof the A431 line, and we are currently elucidating similarities anddifferences in H2O2 signaling in various cell types.

In closing, we have described two new chemical tools for selectivemolecular imaging of H2O2 at concentrations generated for cellsignaling. The monoboronate reagents PG1 and PC1 complementdiboronate counterparts that are useful for detecting cellular H2O2 insituations of oxidative stress. PG1 and PC1 afford specific andsensitive detection of H2O2 in aqueous solution and in living cells,including endogenous intracellular H2O2 generated by EGF/Noxactivation. In addition, PG1 provides strong evidence for H2O2

signaling in brain systems through growth factor signaling in livehippocampal neurons. The ability to specifically monitor H2O2 inliving cells at signaling concentrations opens new avenues for therapidly expanding field of oxidation biology.

METHODSSpectroscopic materials and methods. Millipore water was used to prepare all

aqueous solutions. All spectroscopic measurements were performed in 20 mM

HEPES buffer, pH 7. Absorption spectra were recorded using a Varian Cary 50

spectrophotometer, and fluorescence spectra were acquired using a Photon

Technology International QuantaMaster 4 L-format scanning spectrofluorometer

equipped with an LPS-220B 75-W xenon lamp and power supply, an

A-1010B lamp housing with integrated igniter, a switchable 814 photon-

counting/analog photomultiplier detection unit, and an MD5020 motor driver.

Samples for absorption and emission measurements were contained in 1-cm �1-cm quartz cuvettes (1.4-ml or 3.5-ml volume, from Starna). For ROSexperiments, H2O2, TBHP and OCl– were delivered from 30%, 70% and 5%aqueous solutions, respectively. O2

– was added as solid KO2. �OH and �OtBu weregenerated by reaction of 1 mM Fe2+ with 100 mM H2O2 or 100 mM TBHP,respectively. NO was added using NO gas. O3 was generated by photolysis of O2.1O2 was generated by photolysis of Sensitox II (polymer-supported Rose Bengal).

Preparation and staining of cell cultures. HEK 293 cells were cultured in

DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS,

Invitrogen) and glutamine (2 mM). A431 cells were cultured in DMEM plus

Glutamax-1 (Invitrogen) supplemented with 10% FBS. 1 d before imaging, cells

were passed and plated on 18-mm glass coverslips. Dissociated postnatal rat

hippocampal neurons (P0–P5) were plated at 200,000 cells ml–1 on 12-mm

poly-L-lysine–coated glass coverslips in serum-containing medium. All animal

care and experimental protocols were approved by the Animal Care and Use

Committee at the University of California, Berkeley. For experiments with

HEK 293 cells, solutions of probe and/or H2O2 were added by bath appli-

cation to the medium while on the microscope stage. For experiments with

A431 cells and primary neurons, aliquots of probe, inhibitor, and/or EGF

were added to cells in fresh phosphate-buffered saline (PBS) and incubated at

37 1C for the indicated times and then imaged. Apocynin and wortmannin

were purchased from Sigma, and PD153035 and NSC23766 were purchased

from Calbiochem.

Immunostaining experiments. Postnatal hippocampal neurons were cultured

for 14–20 d in vitro after harvesting. The cells were then fixed with 4%

paraformaldehyde in PBS for 15 min, followed by washing in 0.3% Triton

X-100 in PBS (wash solution) and blocking with 2% goat serum and 0.02%

NaN3 in wash solution for 1 h. The cells were then treated with rabbit anti-

EGFR (1005) (Santa Cruz Biotechnology) at 1:100 dilution in wash buffer for

2 h at 25 1C in the dark. Control cells were treated with wash buffer only. Cells

were then washed five times with wash buffer and stained with goat anti-rabbit

Cy3 conjugate (Jackson ImmunoResearch) at 1:400 dilution for 30 min at

25 1C in the dark. Cells were then washed five times and mounted before

confocal imaging.

Fluorescence imaging experiments. Confocal fluorescence imaging was

performed with a Zeiss LSM510 NLO Axiovert 200 laser scanning microscope

and a �40 (0.8 numerical aperture) or �63 (0.9 numerical aperture)

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Figure 4 4 H2O2 and growth factor signaling in living neurons. (a) Confocal fluorescence image of fixed hippocampal neurons from postnatal rat (DIV 20)stained with rabbit anti-EGFR primary antibody (1:50) followed by goat anti-rabbit Cy3 conjugate secondary antibody (1:400). (b) Control neurons stained

only with secondary antibody (1:400). (c) Brightfield image of b. Scale bar for a–c, 10 mm. (d) Confocal fluorescence image of live hippocampal neurons

from postnatal rat (DIV 14) incubated with 5 mM PG1 for 10 min at 37 1C. (e) PG1-loaded neurons from d stimulated with 1 mg ml–1 EGF for 10 min

at 37 1C. (f) PG1-labeled neurons pretreated with PD153035 before EGF stimulation. (g) PG1-labeled neurons pretreated with wortmannin before EGF

stimulation. (h) PG1-labeled neurons pretreated with NSC23766 (Rac1 inhibitor) before EGF stimulation. (i) PG1-labeled cells pretreated with apocynin

before EGF stimulation. Scale bar for d–i, 20 mm. (j) Graph showing relative fluorescence intensities for treatments in d–i. Error bars represent s.e.m.

* indicates P o 0.05 when compared against EGF-treated cells. For all inhibitor experiments, cells were pretreated with 100 mM inhibitor for 25 min at

37 1C before EGF stimulation. Loading PG1 before or after inhibitor treatment gave the same fluorescence staining. Excitation was provided at 488 nm.

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water-immersion objective lens. Excitation of PG1-loaded cells at 488 nm was

carried out with an argon laser, and emission was collected using a META

detector (Zeiss) from 495 to 559 nm. Excitation of PC1-loaded cells at 543 nm

was carried out with a HeNe laser, and emission was collected using a META

detector from 548 to 644 nm.

Additional methods. See Supplementary Methods online.

Note: Supplementary information and chemical compound information is available onthe Nature Chemical Biology website.

ACKNOWLEDGMENTSWe thank the University of California, Berkeley, the Dreyfus Foundation, theBeckman Foundation, the American Federation for Aging Research, the PackardFoundation, and the US National Institute of General Medical Sciences (NIHGM 79465) for funding this work. E.W.M. thanks the Chemical Biology GraduateProgram sponsored by the US National Institutes of Health (T32 GM066698)and a Stauffer fellowship for support. We thank D. Fortin and S. Szobota forproviding neuronal cultures, and we thank K. McNeill (University of Minnesota)for the polymer-supported Rose Bengal. Confocal fluorescence images wereacquired at the Molecular Imaging Center at UC Berkeley. We alsothank A. Fischer at the UC Berkeley Tissue Culture Facility for experttechnical assistance.

AUTHOR CONTRIBUTIONSE.W.M. performed all of the synthetic and imaging experiments. O.T. preparedthe neuronal cultures and helped with the immunostaining experiments. C.J.C.and E.W.M. designed experimental strategies with help from E.Y.I. C.J.C. andE.W.M. wrote the paper.

COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.

Published online at http://www.nature.com/naturechemicalbiology

Reprints and permissions information is available online at http://npg.nature.com/

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Corrigendum: Molecular imaging of hydrogen peroxide produced for cell signalingEvan W Miller, Orapim Tulyathan, Ehud Y Isacoff & Christopher J ChangNature Chemical Biology 3, 263–267 (2007); published online 1 April 2007; corrected after print 3 May 2007

In the version of this article initially published, the second author’s last name is misspelled. The author’s name should read Orapim Tulyathan. The error has been corrected in the HTML and PDF versions of the article.

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