THE JOURNAL OF BIOLOGICAL CHEMISTRY
217–221, 1997
Epidermal Growth Factor (EGF)-induced Generation of
Hydrogen Peroxide
ROLE IN EGF RECEPTOR-MEDIATED TYROSINE PHOSPHORYLATION*
(Received for publication, July 25, 1996, and in revised form, October 2, 1996)
Yun Soo Bae‡, Sang Won Kang‡, Min Seok Seo‡, Ivan C. Baines§¶, Ephrem Teklei,
P. Boon Chocki, and Sue Goo Rhee‡**
From the ‡Laboratory of Cell Signaling, the §Laboratory of Cell Biology, and the iLaboratory of Biochemistry, NHLBI,
National Institutes of Health, Bethesda, Maryland 20892
Recent evidence indicates that reactive oxygen spe-
cies (ROS) may function as intracellular messengers in
receptor signaling pathways. The possible role of ROS in
epidermal growth factor (EGF) signaling was therefore
investigated. Stimulation of A431 human epidermoid
carcinoma cells with EGF resulted in a transient in-
crease in the intracellular concentration of ROS, meas-
ured with the oxidation-sensitive fluorescent probe
2*,7*-dichlorofluorescin diacetate and laser-scanning
confocal microscopy. The predominant ROS produced
appeared to be H2O2, because the EGF-induced increase
in fluorescence was completely abolished by incorpora-
tion of catalase into the cells by electroporation. The
elimination of H2O2 by catalase also inhibited the EGF-
induced tyrosine phosphorylation of various cellular
proteins including the EGF receptor and phospholipase
C-g1. The dependence of H2O2 production on the intrin-
sic tyrosine kinase activity of the EGF receptor and the
autophosphorylation sites located in its COOH-terminal
tail was investigated. EGF failed to induce H2O2 gener-
ation in cells expressing a kinase-inactive EGF receptor.
However, normal H2O2 generation was observed in cells
expressing a mutant receptor from which the 126 COOH-
terminal amino acids had been deleted to remove four
(out of the total of five) autophosphorylation sites.
These results suggest that EGF-induced H2O2 formation
requires the kinase activity but probably not the auto-
phosphorylation sites of the EGF receptor and that in-
hibition of protein tyrosine phosphatase activity by
H2O2 may be required for EGF-induced protein tyrosine
phosphorylation to be manifested.
Reactive oxygen species (ROS)1 are generally considered cy-
totoxic, because of the oxidative damage they can cause to
cellular components. However, at low concentrations, ROS may
function as physiological mediators of cellular responses (1).
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
¶ Present Address: Dept. of Extramural Affairs, NHLBI, NIH Be-
thesda, MD 20892.
** To whom correspondence should be addressed: NIH, Bldg. 3, Rm.
122, 3 Center Dr. MSC 0320, Bethesda, MD 20892-0320. Tel.: 301-496-
9646; Fax: 301-496-0599.
1
The abbreviations used are: ROS, reactive oxygen species; PDGF,
platelet-derived growth factor; EGF, epidermal growth factor; EGFR,
EGF receptor; PTPase, protein tyrosine phosphatase; DMEM, Dulbec-
co’s modified Eagle’s medium; FBS, fetal bovine serum; DCFH-DA,
29,79-dichlorofluorescin diacetate; DCF, 29,79-dichlorofluorescein; SH,
Src homology; MAP, mitogen-activated protein; PLC-g1, phospholipase
C-g1.
This paper is available on line at http://www-jbc.stanford.edu/jbc/
This is an Open Access article under the CC BY license.
Vol. 272, No. 1, Issue of January 3, pp.
Printed in U.S.A.
For example, hydrogen peroxide (H2O2) mimics the stimulatory
effects of insulin on glucose transport and lipid synthesis in
adipocytes (2, 3). Recently, the production of ROS has been
detected in a variety of cells stimulated with cytokines such as
transforming growth factor-b1 (4, 5), interleukin-1 (6), and
tumor necrosis factor-a (6, 7), with peptide growth factors such
as platelet-derived growth factor (PDGF) (8, 9) and basic fibro-
blast growth factor (7, 9), with agonists of receptors with seven
transmembrane spans such as angiotensin II (10) and lyso-
phosphatidic acid (11) or with phorbol ester (12).
The term ROS encompasses many species including singlet
oxygen, the superoxide anion radical (O2. ), H2O2, lipid perox-
ides, nitric oxide, peroxynitrite (ONOO2), the thiyl peroxyl
radical (RSOOz), the ferryl radical (FeO21) and the hydroxyl
radical (OHz) (13–16). However, the chemical nature of ROS
generated in response to the activation of various receptors has
not been well characterized. H2O2 was shown to be a major
component of ROS in cells activated by transforming growth
factor-b1 or PDGF (4, 8). The generation of ROS in response to
various external stimuli has been related to the activation of
transcription factors such as NF-kB (17) and AP-1 (7, 18),
mitogen-activated protein (MAP) kinases (8, 11), and phospho-
lipase A2 (19) to the triggering of apoptosis (20), and to the
inhibition of protein tyrosine phosphatases (PTPases) (21, 22).
H2O2 is a small, diffusible, and ubiquitous molecule that can be
synthesized, as well as destroyed, rapidly in response to exter-
nal stimuli. As such it fulfills the important prerequisites for
an intracellular messengers. We have now investigated the role
of ROS in epidermal growth factor (EGF) signal transduction
by the EGF receptor (EGFR) protein.
EXPERIMENTAL PROCEDURES
Materials—Bovine catalase was obtained from Boehringer Mann-
heim; Dulbecco’s modified Eagle’s medium (DMEM), modified Eagle’s
medium without phenol red, fetal bovine serum (FBS), penicillin, and
streptomycin were from Life Technologies, Inc.; enhanced chemilumi-
nescence (ECL) reagents were from Amersham Corp.; antibodies to
phosphotyrosine and the EGFR were from Upstate Biotechnology; an-
tibodies to catalase and a-tubulin were from Calbiochem and Oncogene
Science, respectively; protein A-Sepharose beads were from Pharmacia
Biotech Inc.; and 29,79-dichlorofluorescin diacetate (DCFH-DA) was
from Molecular Probes. A mixture of monoclonal antibodies that was
used for immunoprecipitation of phospholipase C-g1 (PLC-g1) was pre-
pared as described (23).
Cell Culture, Electroporation, and Stimulation with EGF—Human
A431 epidermoid carcinoma cells were maintained at 37 °C under an
atmosphere of 5% CO2 in 150-mm dishes containing DMEM supple-
mented with 10% FBS. At 80 –90% confluency, cells were deprived of
serum for 24 h and then harvested by trypsin treatment. Harvested
cells were suspended in serum-free DMEM at a density of ;1 3 107
cells/ml, and 100-ml portions of the cell suspension were placed in an
electroporation chamber in the absence or the presence of catalase (8
mg/ml). Electroporation was performed by subjecting cells to six pulses,
217
218 EGF Receptor-mediated H2O2 Generation
at intervals of 1 or 2 s, at a field strength of 850 6 50 V/cm and a
single-pulse width of 250 ms. Cell viability, assessed by trypan blue
exclusion, was typically 70 – 80% after electroporation under these con-
ditions. The construction, operation, and efficiency of the electropora-
tion apparatus have been described previously (24). The electroporated
cells were transferred to DMEM supplemented with 1% FBS and the
same concentration of catalase as was present in the electroporation
chamber and were placed in an incubator for 18 h.
For analysis of EGF-induced tyrosine phosphorylation, cells were
stimulated with EGF (500 ng/ml) for 10 min and then exposed to lysis
buffer (20 mM Hepes-NaOH (pH 7.2), 1% Triton X-100, 10% glycerol, 50
mM NaF, 1 mM Na3VO4, leupeptin (5 mg/ml), aprotinin (5 mg/ml), and 1
mM phenylmethylsulfonyl fluoride). The lysates were incubated on ice
for 5 min and then centrifuged for 5 min at 10,000 3 g. Protein
concentration of the supernatant was measured with the Bio-Rad de-
tergent-compatible assay kit and bovine serum albumin as standard.
Immunoprecipitation and Immunoblot Analysis—The lysate super-
FIG. 1. Time course of EGF-induced ROS generation in A431
natants were incubated with monoclonal antibodies to PLC-g1 or to
cells as revealed by DCF fluorescence. A431 cells were cultured in
phosphotyrosine for 8 h, after which protein A-Sepharose beads were
DMEM supplemented with 10% FBS until 80 –90% confluency, after
added, and the incubation was continued for an additional hour. The which they were incubated overnight in DMEM containing 0.5% FBS.
beads were pelleted at 10,000 3 g for 5 min, washed three times with Cells were washed with modified Eagle’s medium without phenol red
ice-cold lysis buffer, and subjected to SDS-polyacrylamide gel electro- and treated with EGF (500 ng/ml) for the indicated times. ROS gener-
phoresis on an 8% gel. The separated proteins were transferred to a ation was measured by DCF fluorescence as described under “Experi-
nitrocellulose membrane and probed with antibodies to PLC-g1, to mental Procedures.” Data are representative of three similar experi-
phosphotyrosine, or to EGFR. Immune complexes were detected with ments, in which the relative fluorescence intensity per cell was
appropriate secondary antibodies and ECL reagents. calculated by averaging the values for five groups each containing
Assay of Intracellular ROS—Intracellular ROS production was 20 –30 cells.
measured by the method of Bass et al. (25) as modified for confocal
microscopy by Ohba et al. (4). Briefly, dishes of confluent cells at various
times after stimulation with EGF were washed with modified Eagle’s
medium without phenol red and incubated in the dark for 5 min in
Krebs-Ringer solution containing 5 mM DCFH-DA. DCFH-DA is a non-
polar compound that readily diffuses into cells, where it is hydrolyzed to
the nonfluorescent polar derivative DCFH and thereby trapped within
the cells (25). In the presence of a proper oxidant, DCFH is oxidized to
the highly fluorescent 29,79-dichlorofluorescein (DCF). Culture dishes
were transferred to a Zeiss Axiovert 135 inverted microscope, equipped
with a 320 Neofluor objective and Zeiss LSM 410 confocal attachment,
and ROS generation was detected as a result of the oxidation of DCFH
(excitation, 488 nm; emission, 515–540 nm). The effects of DCFH photo-
oxidation was minimized by collecting the fluorescent image with a
single rapid scan (line average, 4; total scan time, 4.33 s) and identical
parameters, such as contrast and brightness, for all samples. The cells
were then imaged by differential interference contrast microscopy. Five
groups of 20 –30 cells each were randomly selected from the image in
the digital interserence contrast (DIC) channel for each sample, the
fluorescence intensity was then measured for each group from the
fluorescence image, and the relative fluorescence intensity was taken as
FIG. 2. Effect of catalase incorporated into cells by electropo-
the average of the five values. Therefore, the relative fluorescence
ration on EGF-induced ROS generation. A431 cells that had been
intensity (given in arbitrary units) reflects measurements performed on
subjected to electroporation in the absence or the presence of catalase (8
a minimum of 100 cells for each sample. All experiments were repeated mg/ml) were incubated in the absence or the presence of EGF (500
at least three times. ng/ml) for 5 min, after which the generation of ROS was measured.
Data are representative of three similar experiments.
RESULTS
Intracellular generation of ROS in A431 cells was measured mass of ;160 kDa that was recognized by antibody to the
with DCFH-DA and laser-scanning confocal microscopy. Expo- EGFR was particularly prominent. However, in cells contain-
sure of quiescent A431 cells to EGF (500 ng/ml) resulted in a ing exogenous catalase, EGF had no apparent effect on tyrosine
rapid increase in DCF fluorescence, with the maximal, 2-fold phosphorylation of the EGFR or other proteins. Phosphoryla-
increase apparent 5 min after stimulation (Fig. 1); fluorescence tion of PLC-g1, a well characterized target of the EGFR kinase,
had returned to the baseline value after 20 min. was studied further. Immunoblot analysis, with antibodies to
Introduction of catalase, an enzyme that specifically cata- PLC-g1, of PLC-g1 immunoprecipitated from control cells
lyzes the dismutation of H2O2 to O2 and H2O into A431 cells by treated with EGF revealed a broad immunoreactive band (Fig.
electroporation, prevented EGF-induced DCFH oxidation (Fig. 3B); the increased breadth of the band relative to that apparent
2), suggesting that the latter is mainly mediated by H2O2. The with unstimulated cell is indicative of increased tyrosine phos-
amount of catalase incorporated into cells was about five times phorylation (26). The effect of catalase was investigated by
that of the endogenous enzyme (see below). The introduction of subjecting PLC-g1 immunoprecipitated with antibodies to
catalase also reduced DCFH oxidation in cells not exposed to PLC-g1 to immunoblot analysis with antibody to phosphoty-
EGF. rosine (Fig. 3C). Tyrosine phosphorylation of PLC-g1 was ap-
We next assessed the effect of incorporated catalase on EGF- parent from control EGF-treated cells but not with that from
induced tyrosine phosphorylation. EGF induced a rapid in- EGF-treated cells containing exogenous catalase. As demon-
crease in tyrosine phosphorylation of several proteins in control strated previously (26, 27), coprecipitation of autophosphoryl-
cells as revealed by immunoblot analysis of cell lysates with ated EGFR with tyrosine-phosphorylated PLC-g1 was ob-
antibodies to phosphotyrosine (Fig. 3A). Increased tyrosine served. The requirement for H2O2 of the tyrosine
phosphorylation of a broad band with an apparent molecular phosphorylation of PLC-g1 was also demonstrated by precipi-
 EGF Receptor-mediated H2O2 Generation
FIG. 3. Effect of exogenous catalase on EGF-induced protein
tyrosine phosphorylation. A431 cells that had been electroporated in
the absence or the presence of catalase were incubated for 10 min in the
absence or the presence of EGF (500 ng/ml), after which cell lysates
were prepared. A, lysates were subjected to immunoblot analysis with
antibody to phosphotyrosine. The positions of prestained molecular size
markers (in kilodaltons) are indicated on the right and that of the
EGFR on the left. B and C, lysates were subjected to immunoprecipi-
tation (IP) with monoclonal antibodies to PLC-g1 (aPLCg1), and the
immunoprecipitates were subjected to immunoblot analysis with the
same PLC-g1-specific antibodies (B) or with antibodies to phosphoty-
rosine (aPY) (C). D, lysates were subjected to immunoprecipitation with
antibodies to phosphotyrosine, and the immunoprecipitates were sub-
jected to immunoblot analysis with antibodies to PLC-g1. E, lysates
were subjected to immunoblot analysis with antibodies to catalase. F,
lysates were subjected to immunoblot analysis with antibodies to
a-tubulin.
tating proteins with antibody to phosphotyrosine and immuno-
blot analysis with antibodies to PLC-g1 (Fig. 3D).
Because the exogenous bovine catalase and endogenous hu-
man catalase can be separated on a SDS-polyacrylamide gel
(Fig. 3E), the amount of exogenous catalase incorporated to
cells was estimated from immunoblot analysis to be approxi-
mately five times the amount of endogenous enzyme. Equal
application of lysate protein among gel lanes for all immuno-
blot experiments was confirmed by immunoblot analysis with
antibodies to a-tubulin (Fig. 3F).
To test whether the number of EGFR on the cell surface is
affected by electroporation or by the addition of catalase con-
current with electroporation, we measured the number of
EGFR using 125I-EGF. The binding of 125I-EGF was saturable
and inhibited in a concentration-dependent manner by the
addition of unlabeled EGF (figure not shown). The calculated
binding site densities per cell were 4 3 106, 6 3 106, and 5 3
106, respectively, for control cells, electroporated cells assessed
after 18 h of recovery, and cells electroporated in the presence
of catalase and assessed after 18 h of recovery, suggesting that
receptor number is not affected by the treatments. We also
found that the autophosphorylation activity of immunoprecipi-
tated EGFR is not affected by the presence of catalase (8
mg/ml) or H2O2 (1 mM) (data not shown). These results suggest
219
that the inhibition of EGF-induced tyrosine phosphorylation in
cells electroporated in the presence of catalase is not attribut-
able to either the reduction in EGFR number, the inhibition of
EGFR kinase by catalase, or the requirement of H2O2 for the
activation of EGFR kinase.
To assess the role of the intrinsic kinase activity and auto-
phosphorylation sites of the EGFR in the EGF-induced gener-
ation of H2O2, we studied cell lines that express either the
wild-type EGFR, a tyrosine kinase-negative EGFR in which
Lys721 is replaced with Met, or a truncated EGFR (CD-126)
lacking the COOH-terminal 126 amino acids (and therefore the
four tyrosine phosphorylation sites at positions 1173, 1148,
1086, and 1068). These cell lines were generated previously by
Margolis et al. (28, 29) by expression of human EGFR cDNAs in
NIH 3T3 cells that lack endogenous EGFR (2.2 cells). Receptor
density was 3 3 105, 3 3 105, 1.5 3 105 receptors/cell, respec-
tively, for cells expressing wild-type, kinase-negative, and CD-
126 mutant EGFR (30, 31). Treatment with EGF increased the
concentration of H2O2 in cells expressing the wild-type or CD-
126 mutant EGFR but not in the cells expressing the catalyt-
ically inactive mutant (Fig. 4). These results suggest that the
intrinsic tyrosine kinase activity, but not the four autophos-
phorylation sites, is essential for the EGF-induced H2O2
generation.
DISCUSSION
Extracellular release of ROS is a well characterized response
of phagocytic cells to a variety of agonists. However, recent
observations indicate that nonphagocytic cells also generate
ROS (4 –12). To identify the ROS responsible for the intracel-
lular oxidation of DCFH, Ohba et al. (4) added catalase to the
culture medium of osteoblastic cells. The addition of catalase
completely inhibited the transforming growth factor-b1-in-
duced increase in DCF fluorescence, suggesting that H2O2 was
important in DCFH oxidation in response to transforming
growth factor-b1. Because the cells are not permeable to cata-
lase, the researchers proposed that H2O2 was released into the
medium from the plasma membrane, the site of its production,
and then diffused into the cells. Whether this diffusion was
promoted by the trapped DCFH is not clear. Catalase was also
used to characterize the ROS generated in PDGF-treated rat
vascular smooth muscle cells (8). Unlike most other cells, these
muscle cells incorporated, by an unknown mechanism, catalase
that was added to the culture medium. Catalase incorporation
completely blocked the PDGF-stimulated increase in H2O2 pro-
duction, suggesting that H2O2 is also the predominant ROS
induced by PDGF in these cells.
We have now shown that EGF induces ROS production in
A431 cells and that the increase in DCF fluorescence was
primarily attributable to H2O2 on the basis of its sensitivity to
catalase introduced into the cells by electroporation. With in-
corporation of catalase by electroporation, no ambiguity arises
as to whether H2O2 is released first into the medium and is
then taken up by the cells. Unlike rat vascular smooth muscle
cells, the simple addition of catalase to the culture medium did
not result in uptake of the enzyme and a consequent decrease
in DCF fluorescence (data not shown). The incorporation of
catalase also reduced DCFH oxidation in A431 cells not ex-
posed to EGF, indicating that substantial amounts of H2O2 are
generated in the absence of EGF, probably as a result of res-
piratory activity and the presence of various growth factors in
1% FBS. Overnight incubation of electroporated cells in me-
dium containing 1% FBS was necessary for cells to recover from
the electroporation procedure and to exhibit a tyrosine phos-
phorylation response to EGF. A high background of DCF fluo-
rescence was apparent even in cells containing exogenous cat-
alase. Whether this background DCFH oxidation is caused by
220 EGF Receptor-mediated H2O2 Generation
FIG. 4. Effects of EGF on H2O2 generation in NIH 3T3 2.2 cells
expressing wild-type, kinase-defective, or a COOH-terminal de-
letion mutant (CD-126) EGFR. A, DCF fluorescence was measured
with a confocal laser-scanning microscope after incubation of NIH 3T3
2.2 cells expressing wild-type, kinase-defective, or CD-126 mutant
EGFR in the presence of EGF (500 ng/ml) for 5 min; Control represents
cells expressing wild-type EGFR incubated in the absence of EGF. Cells
expressing mutant receptors yielded similar basal DCF fluorescence in
the absence of EGF. B, from the images shown in A, relative fluores-
cence intensity per cell was calculated by averaging the values for five
groups each containing 20 –30 cells. Data are representative of three
similar experiments.
residual H2O2 not degraded by catalase, by cellular oxidants
other than H2O2, or by oxidants introduced by the experimen-
tal procedure (for example, by photo-oxidation) is not clear.
Therefore, we took great care to manipulate the cells under
identical conditions, with the exception of the indicated
additions.
The EGF-induced tyrosine phosphorylation of various cellu-
lar proteins was completely blocked in A431 cells containing
exogenous catalase. Furthermore, detailed experiments with
PLC-g1 indicated that its tyrosine phosphorylation in response
to EGF requires the EGF-induced increase in H2O2 concentra-
tion. Because tyrosine phosphorylation is essential for the ac-
tivation of PLC-g1 in growth factor-treated cells (32), an in-
crease in H2O2 would appear to be required for growth factor-
induced hydrolysis of phosphatidylinositol 4,5-bisphosphate.
Treatment of smooth muscle cells with H2O2 was previously
shown to release Ca21 from intracellular stores that are sen-
sitive to inositol 1,4,5-trisphosphate (33). We also observed a
rapid increase in intracellular Ca21 following addition of 1 mM
H2O2 to A431 cells in a Ca21-free medium (figure not shown).
These observations are consistent with the notion that H2O2
inhibits PTPases and thereby causes activation (tyrosine phos-
phorylation) of PLC-g1 (see below).
Our study with EGF, together with previous studies with
basic fibroblast growth factor (7, 9) and PDGF (8, 9), suggests
that the generation of H2O2 is a common signaling event for
peptide growth factors. However, the role of H2O2 in growth
factor signaling is not clear. Exogenously added H2O2 was
previously shown to elicit tyrosine phosphorylation in several
cell types (34, 35), whereas inhibition of the PDGF-induced
increase in H2O2 blocked various steps in signaling by this
growth factor, including tyrosine phosphorylation of MAP ki-
nase (8). Furthermore, H2O2 directly inhibits PTPase activity
in vitro, and this inhibition is completely reversed by incuba-
tion with dithiothreitol (21). All PTPases contain one essential
sulfhydryl group at their active site that is susceptible to oxi-
dation because of its unusually low pKa (,5) (36). These obser-
vations suggest that PTPases may be targets of intracellularly
generated H2O2. Inactivation of PTPases would result in in-
creased tyrosine phosphorylation. Furthermore, the specific
activities of PTPases in vitro are 10 –1000 times those of pro-
tein tyrosine kinases (37). Therefore, in most cells, the activa-
tion of a receptor tyrosine kinase by the binding of a growth
factor may not be sufficient to increase the steady-state level
of protein tyrosine phosphorylation; concurrent inhibition of
PTPases might be necessary, and this inhibition may be
achieved through H2O2.
Binding of various peptide growth factors to their cognate
receptors activates multiple signaling pathways, including
those mediated by PLC-g1, phosphatidylinositol 3-kinase, sig-
nal transducer and activator of transcription protein (STAT),
and MAP kinase (38, 39). The ligand-bound receptors dimerize
and transphosphorylate each other at several tyrosine resi-
dues, thereby creating binding sites for cellular proteins that
contain Src homology 2 (SH2) domains, including PLC-g1,
GTPase-activating protein of RAS, the 85-kDa subunit of phos-
phatidylinositol 3-kinase (p85), and SH2-containing collagen
protein. Studies with autophosphorylation site mutants of the
receptors for PDGF (40), colony-stimulating factor (41), fibro-
blast growth factor (42), and nerve growth factor (43) have
shown that elimination of specific individual sites selectively
abrogates the association of one or two SH2-containing pro-
teins with the receptors, suggesting that individual autophos-
phorylation sites mediate the binding of specific SH2-contain-
ing protein. However, the association of PLC-g1, GTPase-
activating protein, p85, or SH2-containing collagen protein
with the EGFR, which contains five autophosphorylation sites
(residues 992, 1068, 1086, 1148, and 1173) in the COOH-ter-
minal region, does not appear to stringently require individual
autophosphorylation sites, but decreases gradually as the sites
are removed one by one by COOH-terminal truncation (44).
The intrinsic tyrosine kinase activity and autophosphoryla-
tion sites of the EGFR are not required for all signaling path-
ways activated by EGF. The activation of MAP kinase can
occur independently of EGFR kinase activity (45, 46), and the
activation of signal transducer and activator of transcription
proteins requires none of the autophosphorylation sites (47).
Our data with the kinase-inactive mutant indicate that EGFR-
dependent H2O2 generation requires the intrinsic kinase activ-
 EGF Receptor-mediated H2O2 Generation 221
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