Glutathione-dependent generation of reactive oxygen species by the peroxidase-catalyzed redox cycling of flavonoids

Article abstract of DOI:10.1021/tx980271b

Catalytic concentrations of apigenin (a flavone containing a phenol B ring) and naringin or naringenin (flavanones containing a phenol B ring) caused extensive GSH oxidation at a physiological pH in the presence of peroxidase. Only catalytic H₂O₂ concentrations were required, indicating a redox cycling mechanism that generated H₂O₂ was involved. Extensive oxygen uptake ensued, the extent of which was proportional to the extent of GSH oxidation to GSSG and was markedly increased by superoxide dismutase. These results suggest that prooxidant phenoxyl radicals formed by these flavonoids co-oxidized GSH to form thiyl radicals which activated oxygen. GSH also prevented the peroxidase-catalyzed oxidative destruction of these flavonoids which suggests that phenoxyl radicals initiated the oxidative destruction. This is the first time that a group of flavonoids have been identified as prooxidants independent of autoxidation reactions catalyzed by the transition metal ions Fe³⁺, Fe²⁺, Mn²⁺, and Cu²⁺.

Full text of DOI:10.1021/tx980271b

Chem. Res. Toxicol. 1999, 12, 521-525
521
Glu ta th ion e-Dep en d en t Gen er a tion of Rea ctive Oxygen
Sp ecies by th e P er oxid a se-Ca ta lyzed Red ox Cyclin g of
F la von oid s
Giuseppe Galati, Tom Chan, Bin Wu, and Peter J . O’Brien*
Department of Pharmacology and Faculty of Pharmacy, University of Toronto,
Toronto, Ontario, Canada M5S 2S2
Received December 21, 1998
Catalytic concentrations of apigenin (a flavone containing a phenol B ring) and naringin or
naringenin (flavanones containing a phenol B ring) caused extensive GSH oxidation at a
physiological pH in the presence of peroxidase. Only catalytic H2O2 concentrations were
required, indicating a redox cycling mechanism that generated H2O2 was involved. Extensive
oxygen uptake ensued, the extent of which was proportional to the extent of GSH oxidation to
GSSG and was markedly increased by superoxide dismutase. These results suggest that
prooxidant phenoxyl radicals formed by these flavonoids co-oxidized GSH to form thiyl radicals
which activated oxygen. GSH also prevented the peroxidase-catalyzed oxidative destruction
of these flavonoids which suggests that phenoxyl radicals initiated the oxidative destruction.
This is the first time that a group of flavonoids have been identified as prooxidants independent
of autoxidation reactions catalyzed by the transition metal ions Fe3+, Fe2+, Mn2+, and Cu2+
.
flavonoids have been identified as prooxidants without
involving a transition metal-catalyzed autoxidation.
In tr od u ction
Flavonoids, which are widely distributed in green
vegetables and fresh fruits, have recently been identified
as a major cancer-preventive component of our diet
because of their antioxidative, oxygen radical scavenging,
and anti-inflammatory activities (1). They may also be
anti-atherosclerotic as they prevent Cu2+-catalyzed low-
density lipoprotein (LDL)1 oxidative modification by
scavenging reactive oxygen species (ROS) and eliminat-
ing LDL peroxyl and alkoxyl radicals (2, 3). Many
flavonoid preparations are also marketed as herbal
medicines or dietary supplements for a variety of alleged
nontoxic therapeutic effects which have yet to pass
controlled clinical trials. However, despite all these
apparently useful therapeutic properties, flavonols that
contain catechol or pyrogallol B rings can autoxidize in
the presence of transition metals to produce ROS which
accelerate LDL oxidation during the propagation phase
(4). The DNA strand scission (5) and the mutagenicity
induced by flavonols such as quercetin also likely result
from autoxidation that occurs during a lengthy in vitro
aerobic incubation (6). The carcinogenicity data are
conflicting (7), and it is possible that this prooxidant
activity as a result of autoxidation may not be important
in vivo, where transition metals are largely sequestered.
It is well-established that some phenolic compounds
are oxidized by peroxidases to phenoxyl radicals which
co-oxidize NAD(H) or GSH and cause oxygen activation
(8). In the following, extensive oxygen activation was
found when some flavones and flavanones containing
phenol B rings were metabolized by peroxidases in the
presence of GSH. This is the first time that a group of
Ma ter ia ls a n d Meth od s
Flavonoids, glutathione (GSH), horseradish peroxidase (HRP)
type VI, superoxide dismutase (SOD) type 1, cytochrome c, and
2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium 5-car-
boxanilide (XTT) were obtained from Sigma Chemical Co.
Phenol was obtained from Baker Chemical Co.
Mea su r em en t of th e Exten t of GSH Oxygen Con su m p -
tion . The reaction mixtures contained 2 mL of 0.1 M Tris-HCl/
1.0 mM EDTA buffer (pH 7.4), flavonoid or phenol (25 µM), H2O2
(25 µM), and GSH (400 µM). Reactions were started by the
addition of HRP (0.1 µM), and the extent of oxygen consumption
in this reaction mixture was measured with a Clarke type
electrode at 20 °C.
Mea su r em en t of th e Exten t of GSH Oxid a tion . The
reaction mixtures contained 2 mL of 0.1 M Tris-HCl/1.0 mM
EDTA buffer (pH 7.4), flavonoid or phenol (25 µM), H2O2 (1 µM),
and GSH (400 µM). Reactions were started by the addition of
HRP (0.1 mM). Aliquots were removed at different times for
the HPLC analysis of GSH and GSSG levels after the GSH
oxidation reaction was stopped with iodoacetic acid (15 mM) (9).
Mea su r em en t of th e Kin etics of GSH Dep letion . The
reaction mixtures contained 2.5 mL of 0.1 M Tris-HCl/1.0 mM
EDTA buffer (pH 7.4), flavonoid or phenol (25 µM), GSH (400
µM), HRP (0.1 µM), and H2O2 (25 µM). Aliquots of the reaction
mixture were removed every 30 s for a colorimetric analysis of
GSH with Ellman’s reagent (2 mM dithionitrobenzoic acid in
0.5% NaHCO3) after the reaction was stopped with catalase (400
units). The absorbance was followed at 412 nm using a Shi-
madzu UV-240 spectrophotometer (10).
Mea su r em en t of t h e E xt en t of Su p er oxid e R a d ica l
F or m a t ion . The extent of superoxide radical formation was
measured by determining the rate of SOD sensitive reduction
of the tetrazolium salt XTT. The assay mixture contained 2.5
mL of 0.1 M Tris-HCl/1.0 mM EDTA buffer (pH 7.4), flavonoid
or phenol (25 µM), H2O2 (25 µM), GSH (400 µM), HRP (0.1 µM),
and XTT (200 µM). The absorbance of the reduced form of XTT
* Corresponding author. Phone: (416) 978-2716. Fax: (416) 978-
8511. E-mail: peter.obrien@utoronto.ca.
1 Abbreviations: LDL, low-density lipoprotein; ROS, reactive oxygen
species.
10.1021/tx980271b CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/19/1999

522 Chem. Res. Toxicol., Vol. 12, No. 6, 1999
Galati et al.
that was produced (a water-soluble formazan) was monitored
at 470 nm using a Shimadzu UV-240 spectrophotometer as
described previously (11).
Assessm en t of GSH Con ju ga t es. The reaction mixtures
contained 5 mL of 0.1 M Tris-HCl/1.0 mM EDTA buffer (pH
7.4), GSH (400 µM), quercetin or luteolin (100 µM), HRP (0.1
µM), and H2O2 (100 µM). The extent of GSH conjugate formation
was determined by monitoring significant differences in the UV
absorbance of the reaction mixture versus the control (absence
of HRP/H2O2) using a Shimadzu UV-240 spectrophotometer
following the extraction of flavonoid or its oxidation products
from the reaction mixture with ethyl acetate.
Resu lts
The data in Figure 1A show that catalytic amounts of
apigenin, a flavone with a phenolic B ring, or naringenin,
a flavanone with a phenolic B ring (Table 1), promoted
GSH oxidation by peroxidase and H2O2 with a very high
efficiency. The rate of GSH oxidation was dependent on
apigenin or naringenin concentrations, indicating that
the rate of peroxidase/H2O2-catalyzed oxidation of api-
genin or naringenin was rate-limiting. GSH was also
rapidly oxidized by peroxidase and H2O2 in the presence
of catalytic amounts of naringin, a flavanone glycoside
of naringenin with a phenolic B ring (Table 1). Little GSH
oxidation by peroxidase and H2O2 occurred in the absence
of flavonoid which indicates that GSH at 0.4 mM is a
poor peroxidase substrate at pH 7.4, although others have
reported that GSH oxidation to thiyl radicals occurred
at pH 8.0 with 10 mM GSH (12). GSH oxidation occurred
at a pseudo-first-order rate with respect to peroxidase
when fixed concentrations of the other reactants were
used (Figure 1B) and pseudo-first-order with respect to
GSH concentration (results not shown). As shown in
Figure 1C, apigenin in the absence of GSH was rapidly
degraded by peroxidase and H2O2 but little degradation
occurred in the presence of GSH. GSH also prevented
naringenin oxidation by peroxidase and H2O2 (results not
shown).
As shown in Table 2, 25 µM apigenin, naringenin, or
naringin readily catalyzed the oxidation of 400 µM GSH
by 1 µM H2O2 and 0.1 µM HRP. With apigenin or
naringenin, a total of 143 or 155 mol of GSH, respectively,
was oxidized per mole of added H2O2. The order of
effectiveness of these flavonoids with respect to phenol
was as follows: naringin, naringenin, and apigenin >
phenol. The GSH oxidation catalyzed by naringenin was
inhibited by cytochrome c, a superoxide radical oxidant,
but was markedly stimulated by superoxide dismutase
(Table 2). This suggests that superoxide dismutase
generates H2O2 by dismutation of the superoxide anion
radicals generated by the GSH oxidation. Catechol or
flavanones or flavonols containing catechol B rings, i.e.,
luteolin, catechin, rutin, quercetin, and fisetin, did not
catalyze GSH oxidation, although some GSH depletion
did occur. This is likely due to GSH conjugate formation
as quercetin or luteolin products formed in the presence
of GSH could not be extracted by ethyl acetate (results
not shown).
F igu r e 1. (A) Dependence of the rate of GSH oxidation on
flavonoid concentration. The reaction vessel contained 2.5 mL
of 0.1 M Tris-HCl/1.0 mM EDTA buffer (pH 7.4), GSH (400 µM),
HRP (0.1 µM), H2O2 (25 µM), and varying concentrations of
apigenin (9) and naringenin ([) (5, 10, and 25 µM). A 25 µL
aliquot was removed at various times, and the absorbance was
followed at 412 nm using a Shimadzu UV-240 spectrophotom-
eter after the addition of catalase (400 units) and dithionitro-
benzoic acid (2 mM). Values represent the mean ( SE of three
separate experiments. (B) Dependence of the rate of GSH
oxidation on peroxidase concentration. The reaction vessel
contained 2.5 mL of 0.1 M Tris-HCl/0.1 mM EDTA buffer (pH
7.4), GSH (400 µM), apigenin (25 µM), H2O2 (25 µM), and
varying concentrations of HRP (0.01, 0.05, 0.1, and 0.5 µM). A
25 µL aliquot was removed at various times, and the absorbance
was followed at 412 nm using a Shimadzu UV-240 spectropho-
tometer after the addition of catalase (400 units) and dithionitro-
benzoic acid (2 mM). Values represent the mean ( SE of three
separate experiments. (C) Protective effect of GSH on apigenin
oxidative degradation. The reaction mixture contained 2.5 mL
of 0.1 M Tris-HCl/1.0 mM EDTA buffer (pH 7.4), apigenin (25
µM), and HRP (0.1 µM), and the reaction was started with H2O2
(25 µM). The effect of 1 mM GSH (9) at preventing apigenin
oxidation vs the absence of GSH ([) was followed at 363 nm
for 5 min using a Shimadzu UV-240 spectrophotometer. Values
represent the mean ( SE of three separate experiments.
Extensive oxygen uptake accompanied the stoichio-
metric oxidation of GSH to GSSG in the peroxidase/H2O2
reaction system with flavones or flavanones containing
phenol B rings (Table 2). No oxygen uptake occurred in
the absence of flavonoids, indicating that GSH is a poor
peroxidase substrate. The order of effectiveness of these
flavonoids was as follows: apigenin, naringenin, and

Glutathione Generation of ROS by Prooxidant Flavonoids
Chem. Res. Toxicol., Vol. 12, No. 6, 1999 523
Ta ble 1. Str u ctu r a l F or m u la s of th e F la von oid s Used in
Th is Stu d y
Ta ble 2. P er oxid a se-Ca ta lyzed Oxygen Activa tion by
F la von oid s a n d Glu ta th ion ea
substituent
one-electron redox
potential E° (mV)
at pH 7
GSSG
formed (µM consumed
GSH equiv)
total oxygen
3
5
7
3′
4′
flavones
apigenin
luteolin
diosmin
flavanones
naringenin
naringin
hesperetin
hesperidin
flavanonol
taxifolin
flavan-3-ol
catechin
flavonols
rutin
quercetin
galangin
kaempferol
fisetin
flavonoid
none
phenol
catechol
flavones
apigenin
luteolin
(PhO•/PhO-)
(µM)b
H
H
H
OH
OH
OH
OH
H
OH
OH
OH
OH
OMe
4 ( 2
96 ( 21
4 ( 1
2 ( 1
20 ( 3
2 ( 1
OH
860d
ORua
H
H
H
H
OH
OH
OH
OH
OH
H
H
OH
OH
OH
OH
OMe
OMe
>1000e
143 ( 15
4 ( 2
6 ( 3
52 ( 3
2 ( 1
3 ( 1
ORhb
OH
180,e 180,f 299h
diosmin
ORua
flavanones
naringenin
naringenin/SODc
naringenin/cyt cc
naringin
hesperetin
hesperidin
flavanonol
taxifolin
flavan-3-ol
catechin
flavonols
rutin
quercetin
galangin
600e
155 ( 16
245 ( 22
89 ( 9
173 ( 15
5 ( 2
6 ( 3
51 ( 3
98 ( 4
23 ( 2
31 ( 3
3 ( 2
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
440e
ORua
OH
OH
OH
OH
OH
OH
OH
OH
H
OH
OH
OH
OH
OH
OH
OH
H
H
OH
OH
OH
H
OH
OH
440,e 720g
3 ( 1
83h
4 ( 2
3 ( 1
3 ( 2
2 ( 1
160,e 130,f 570g
a
Ru is rutinoside [6-O-(6-deoxy-R-L-mannopyranosyl)-â-D-glu-
copyranosyl]. b Rh is rhamnoglucoside [2-O-(6-deoxy-R-L-mannopy-
ranosyl)-â-D-glucopyranosyl].
180,e 600,g 275h
30,e 0.06,f 398h
320,e 340f
3 ( 2
5 ( 2
4 ( 2
4 ( 1
5 ( 1
3 ( 1
3 ( 2
2 ( 1
2 ( 1
3 ( 1
kaempferol
fisetin
120,e 170,f 209h
120,e 140,f 214h
a
The reaction mixture contained in 2 mL of 0.1 M Tris-HCl/
1.0 mM EDTA buffer (pH 7.4), substrate (25 µM), HRP (0.1 µM),
H2O2 (1 µM), and GSH (400 µM) for 30 min. Reactions were started
by the addition of HRP. Means ( SEM for three separate
b
experiments are given. The extent of oxygen consumption was
determined as indicated in Materials and Methods. c SOD (1 µM)
d
and cyt c (20 µM) were added where indicated. E7 (mV) (32).e Ep/2
naringin > phenol. No oxygen uptake occurred with
catechol or flavanones or flavonols containing catechol
B rings, i.e., luteolin, catechin, rutin, quercetin, and
fisetin. Oxygen uptake was also stimulated by super-
oxide dismutase and inhibited by cytochrome c. A stoi-
chiometry of approximately 0.4 mol of O2 consumed per
mole of oxidized GSH was realized. No oxygen uptake
occurred in the absence of GSH under these conditions,
although some oxygen uptake occurred with naringenin
or apigenin at a much higher concentration (100 µM) in
the absence of GSH (results not shown), indicating that
some of the naringenin or apigenin breakdown products
autoxidize.
The effectiveness of flavonoids at catalyzing superoxide
radical formation during GSH oxidation was next inves-
tigated. As shown in Table 3, apigenin, naringenin, and
naringin on metabolic oxidation by peroxidase/H2O2 in
the presence of GSH caused XTT reduction which was
inhibited by superoxide dismutase. No XTT reduction
occurred in the presence of GSH alone or in the absence
of HRP or H2O2 or GSH. Flavonoids containing catechol
B rings were ineffective. Kaempferol, a flavonol, and
hesperetin, a flavanone, were also ineffective, even
though these flavonoids contain phenol B rings.
f
g
h
(mV) (33). E1/2 (V vs SCE) (34). E7 (V vs NHE) (35). E1° (mV)
(36).
Ta ble 3. F la von oid -Ca ta lyzed Su p er oxid e Ra d ica l
F or m a tion by th e H2O2/HRP /GSH System a
rate of XTT reduction
(nmol min-1 mL-1
total XTT reduction
(nmol/mL)
)
-SOD
+SOD
-SOD
+SOD
none
phenol
flavone
<1
16 ( 2
<1
<1
<1
55 ( 4
<1
<1
apigenin
flavanones
naringenin
naringin
hesperetin
hesperidin
flavanonol
taxifolin
45 ( 4
4 ( 1
143 ( 11
31 ( 3
27 ( 2
3 ( 1
<1
<1
<1
<1
<1
74 ( 6
21 ( 2
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
flavan-3-ol
catechin
flavonol
rutin
kaempferol
<1
<1
<1
<1
<1
<1
<1
<1
a
The complete system (2.5 mL) contained 400 µM GSH, 0.1 µM
HRP, 25 µM H2O2, 200 µM XTT, and 25 µM flavonoid or phenol
in 0.1 M Tris-HCl/1.0 mM EDTA buffer (pH 7.4). SOD (1 µM) was
added where indicated. The extent of XTT reduction was deter-
mined by following the increase in absorbance at 470 nm. Means
( SEM for three separate experiments are given.
Discu ssion
These results demonstrate that some flavones or
flavanones containing phenol B rings catalyze GSH
oxidation in the peroxidase/H2O2 system and that exten-
sive oxygen uptake accompanied the GSH oxidation. As
only catalytic amounts of these flavonoids and H2O2 were
required to catalyze the GSH oxidation, the flavonoid was
oxidized by peroxidase/H2O2 to a product which oxidized
GSH and underwent redox cycling.
Phenol also catalyzed GSH oxidation in the peroxidase/
H2O2 system, and previously, this was attributed to redox
cycling by phenoxyl radicals (8) in which GSH was
oxidized by phenoxyl radicals to form thiyl radicals which
generated reactive oxygen species (13). Phenol is a major
benzene metabolite, and a major mechanism proposed

524 Chem. Res. Toxicol., Vol. 12, No. 6, 1999
Galati et al.
for benzene-induced myelotoxicity involves the oxidation
of phenol by bone marrow myeloperoxidase in forming
phenoxyl radicals (14). Incubation of bone marrow cells
or HL-60 human promyelocytic leukemic cells with
phenolic metabolites of benzene resulted in GSH oxida-
tion, oxygen activation, H2O2 formation, oxidative DNA
damage, and cytotoxicity (15-17). Phenoxyl radicals have
also been shown to cause oxidative DNA damage directly
or via GSH-mediated oxygen activation (9).
The mechanism proposed for the apigenin- or narin-
genin-mediated GSH oxidation is as follows. The phen-
oxyl radical formed by the peroxidase-catalyzed oxidation
of the phenol B ring of these flavonoids oxidized GSH to
a thiyl radical (reaction 1) which reacted with GSH to
form a disulfide radical anion (reaction 2). The latter
radical anion rapidly reduced O2 to form GSSG and the
superoxide radical anion (reaction 3). In confirmation of
this mechanism, the addition of superoxide dismutase
markedly increased the amount of GSH that was oxidized
by apigenin presumably as a result of the formation of
H2O2 from superoxide generated by GSH oxidation.
cal formation) with myeloperoxidase and phenolic com-
pounds or methimazole (23). Others have also demon-
strated a GSH-dependent oxygen activation (and gluta-
thione radical formation) with phenolic compounds and
lactoperoxidase or thyroid peroxidase, other mammalian
peroxidases (24).
Phenoxyl radicals have also been implicated in the
initiation stage of atherosclerosis as myeloperoxidase
readily catalyzes the oxidation of tyrosine, a plasma
phenol (25), to a tyrosyl radical which can co-oxidize the
lipids and proteins of low-density lipoproteins (26, 27).
Myeloperoxidase was also found in human atherosclerotic
tissue (26), and the level of protein-bound o,o´-dityrosine
(a major tyrosyl radical product) was also markedly
increased in LDL isolated from human atherosclerotic
lesions (28). Interestingly, this peroxidase-catalyzed LDL
oxidation mechanism, unlike previously reported LDL
oxidation systems, does not require free transition metal
ions (copper or iron). In the plasma, these transition
metal ions are present almost exclusively in tightly bound
proteins (e.g., ceruloplasmin and transferrin) which do
not catalyze the oxidative modification of LDL.
The three most prooxidant flavonoids identified were
naringenin, naringin, and apigenin. These particular
flavonoids actually induce lipid peroxidation under the
same conditions under which other flavonoids prevented
lipid peroxidation (29). Presumably, the prooxidant phen-
oxyl radicals of naringenin, naringin, and apigenin
catalyze lipid peroxidation as do other prooxidant phen-
oxyl radicals (8). Naringin constitutes up to 10% of the
dry weight of grapefruit concentrations. Naringin con-
centrations in grapefruit juice are reported to range from
100 to 800 mg/L, and recently, there are concerns that
naringenin, the major naringin metabolite, may contrib-
ute to intestinal cytochrome P450 3A4 inhibition that
results in the reported clinical increase in the bioavail-
ability of dihydropyridine calcium channel blockers when
taken with grapefruit juice (30). Apigenin concentrations
in celery are reported to be 108 mg/kg of fresh weight
(1). Apigenin is also being considered as a skin cancer
preventive agent in sunscreens (31). Further research is
required to determine the consequences, if any, for
activated oxygen species formation by naringenin, nar-
ingin, and apigenin in peroxidase-containing tissues such
as bone marrow or thyroid or for plasma LDL oxidation.
PhO• + GSH f GS• + PhOH
GS• + GS- h GSSG•-
(1)
(2)
(3)
•-
GSSG•- + O2 h GSSG + O2
The disulfide radical, acetaminophen phenoxyl radi-
cals, and thiyl radicals have been detected and character-
ized by fast flow ESR spectroscopy in a similar peroxidase
system using acetaminophen as the phenol (18). Protein
thiol oxidation by acetaminophen phenoxyl radicals has
been implicated as a cause of liver necrosis induced by
high doses of acetaminophen (19).
Only flavones and flavanones containing phenol B
rings co-oxidize GSH, with resulting oxygen activation
when oxidized by peroxidase. Flavonoids containing a
catechol or benzene B ring did not cooxidize GSH when
oxidized by peroxidase. This is surprising as most of the
flavonoids in Table 1 contain a resorcinol A ring and
resorcinol when oxidized by peroxidase co-oxidizes GSH
with resulting oxygen activation (8). Furthermore, the
inactivation of thyroid peroxidase and lactoperoxidase by
flavonoids has been attributed to covalent binding of A
ring resorcinol radicals to the amino acid radical(s) of
peroxidase compound II (20).
This GSH prooxidant activity of flavones and fla-
vanones seemed to partly correlate with the one-electron
redox potential of their phenoxyl radicals (Table 1) as
only apigenin and naringenin had redox potentials that
were higher than the redox potential (E° ) 850 mV) for
the couple GS-/GS• (21). Hesperetin and kaempferol were
not active presumably because the one-electron redox
potential of their phenoxyl radicals was not high enough.
Flavonoids containing catechol B rings also have lower
one-electron redox potentials and depleted some GSH
without oxygen uptake or GSSG formation, suggesting
that GSH formed GSH conjugates with a B ring o-
quinone product.
Although the flavonoid substrate specificity of HRP
toward flavonoids cannot be directly transferred to myelo-
peroxidase as the entrance and the cleft leading to the
active site is tighter and more highly constrained than
HRP (22), we have previously demonstrated a similar
GSH-dependent oxygen activation (and glutathione radi-
Ack n ow led gm en t. This work was supported by a
grant from the National Sciences and Engineering Re-
search Council of Canada.
Refer en ces
(1) Manach, C., Regerat, F., Texier, O., Agullo, G., Demigne, C., and
Remesy, C. (1996) Bioavailability, metabolism and physiological
impact of flavonoids. Nutr. Res. 16, 517-544.
(2) Salah, N., Miller, N. J ., Paganga, G., Tyburg, L., Bolwell, G. P.,
and Rice-Evans, C. (1995) Polyphenolic flavonols as scavengers
of aqueous phase radicals and chain-breaking antioxidants. Arch.
Biochem. Biophys. 322, 339-346.
(3) Yamanaka, N., Oda, O., and Nagao, S. (1997) Prooxidant activity
of caffeic acid, dietary non-flavonoid phenolic acid, on Cu2+
-
induced low-density lipoprotein oxidation. FEBS Lett. 405, 186-
190.
(4) Yamanaka, A., Oda, O., and Nagao, S. (1997) Green tea catechins
such as epicatechin and epigallocatechin accelerate Cu2+-induced
low density lipoprotein oxidation in propagation phase. FEBS
Lett. 401, 230-234.
(5) Ahmad, M. S., Fazal, F., Rahman, A., Hodi, S. M., and Parish, J .
H. (1992) Activities of flavonoids for the cleavage of DNA in the

Glutathione Generation of ROS by Prooxidant Flavonoids
Chem. Res. Toxicol., Vol. 12, No. 6, 1999 525
presence of Cu(II): correlation with generation of active oxygen
species. Carcinogenesis 13, 605-608.
(6) Rueff, J ., Laires, A., Gaspair, J ., Booba, H., and Rodrigues, A.
(1992) Oxygen species and the genotoxicity of quercetin. Mutat.
Res. 265, 75-81.
(7) Ito, N. (1992) Is quercetin carcinogenic? J pn. J . Cancer Res. 83,
312-314.
(21) Sudhar, P. S., and Armstrong, D. A. (1990) Redox potential of
some sulfur containing radicals. J . Phys. Chem. 94, 5915-5917.
(22) Day, B. W., Tyurin, V. A., Tyurina, Y. Y., Liu, M., Facey, J . A.,
Carta, G., Kisin, E. R., Dubey, R. K., and Kagan, V. E. (1999)
Peroxidase-catalysed pro-versus antioxidant effects of 4-hydroxy-
tamoxifen: enzyme specificity and biochemical sequelae. Chem.
Res. Toxicol. 12, 28-37.
(8) O’Brien, P. J . (1988) Radical formation during the peroxidase-
catalysed metabolism of carcinogens and xenobiotics: the reactiv-
ity of these radicals with GSH, DNA and unsaturated lipid. Free
Radical Biol. Med. 4, 169-183.
(9) Reed, D. J ., Babson, J . R., Beatty, P. W., Boodie, A. E., Ellis, W.
W., and Potter, D. W. (1980) HPLC analysis of nanomole levels
of GSH, GSSG and related thiols and disulfides. Anal. Biochem.
106, 55-62.
(23) McGirr, L. G., J atoe, S. D., and O’Brien, P. J . (1990) Myeloper-
oxidase catalysed cooxidative metabolism of methimazole: oxida-
tion of glutathione and NADH by free radical intermediates.
Chem.-Biol. Interact. 73, 279-295.
(24) Nakamura, M., Yamazaki, I., Ohtaki, S., and Nakamura, S. (1986)
Characterisation of one- and two-electron oxidations of gluta-
thione coupled with lactoperoxidase and thyroid peroxidase
reactions. J . Biol. Chem. 261, 13923-13927.
(25) Savenkova, M., Mueller, D. M., and Heinecke, J . W. (1994) Tyrosyl
radical generated by myeloperoxidase is a physiological catalyst
for the initiation of lipid peroxidation in low-density lipoprotein.
J . Biol. Chem. 269, 20394-20400.
(26) Daugherty, A., Dunn, J . L., Rateri, D. L., and Reinecke, J . W.
(1993) Myeloperoxidase, a catalyst for lipoprotein oxidation, is
expressed with human atherosclerotic lesions. J . Clin. Invest. 91,
437-444.
(27) Heinecke, J . W., Li, W., Francis, G. A., and Goldstein, J . A. (1993)
Tyrosyl radical generated by myeloperoxidase catalyses the
oxidative cross-linking of proteins. J . Clin. Invest. 91, 2866-2872.
(28) Leeuwenburgh, C., Rasmussen, J . E., Hsu, F. F., Mueller, D. M.,
and Heinecke, J . W. (1997) Mass spectrometric quantification of
markers for protein oxidation by tyrosyl radical in low-density
lipoprotein isolated from human atherosclerotic intima. J . Biol.
Chem. 272, 3520-3526.
(29) Ratty, A. K., and Das, N. P. (1988) Effects of flavonoids on
nonenzymatic lipid peroxidation: structure-activity relationships.
Biochem. Med. Metab. Biol. 39, 69-79.
(30) Fuhr, U. (1998) Drug interactions with grapefruit juice. Drug
Safety 18, 251-272.
(31) Li, B., Robinson, D. H., and Birt, D. F. (1997) Evaluations of
properties of apigenin and analytic method of development. J .
Pharm. Sci. 86, 721-725.
(32) Wardman, P. (1989) Reduction potentials of one-electron couples
involving free radicals in aqueous solution. J . Phys. Chem. Ref.
Data 18, 1637-1755.
(33) Van Acker, S. A. B. E. (1996) Antioxidant activity of flavonoids.
Free Radical Biol. Med. 20, 331-342.
(34) Hodnick, W. F., Milosavljevic, E. B., Nelson, J . H., and Pardini,
R. S. (1988) Electrochemistry of flavonoids. Biochem. Pharmacol.
37, 2607.
(35) J ovanovic, S. V., Steenken, S., Tosic, M., and Simic, M. G. (1994)
Flavonoids as antioxidants. J . Am. Chem. Soc. 116, 4846-4851.
(36) Bors, W., Michel, C., and Schikora, S. (1995) Interaction of
flavonoids with ascorbate and determination of their univalent
redox potentials: A pulse radiolysis study. Free Radical Biol. Med.
19, 45-52.
(10) Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem.
Biophys. 82, 70-77.
(11) Ukeda, H., Maeda, S., Ishii, T., and Sawamura, M. (1997)
Spectrophotometric assay for superoxide dismutase based on
tetrazolium salt 3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-
bis(4-methoxy-6-nitro)benzensulfonic acid hydrate reduction by
xanthine-xanthine oxidase. Anal. Biochem. 251, 206-209.
(12) Mason, R. P., and Ramakrishna, R. (1990) Thiyl free radical
metabolites of thiol drugs, glutathione, and proteins. Methods
Enzymol. 186, 318-329.
(13) Stoyanovsky, D. A., Goldman, R., Claycamp, H. G., and Kagan,
V. E. (1995) Phenoxyl radical-induced thiol-dependent generation
of reactive oxygen species: implications for benzene toxicity. Arch.
Biochem. Biophys. 317, 315-323.
(14) Subrahmanyan, V. V., Ross, D., Eastmond, D. A., and Smith, M.
T. (1991) Potential role of free radicals in benzene-induced
myelotoxicity and leukemia. Free Radical Biol. Med. 11, 495-
515.
(15) Kolachana, P., Subrahmanyan, V. V., Meyer, K. B., Zhang, L.,
and Smith, M. T. (1993) Benzene and its phenolic metabolites
produce oxidative DNA damage in HL-60 cells in vitro and the
bone marrow in vivo. Cancer Res. 53, 1023-1026.
(16) Stoyanovsky, D. A., Goldman, R., J onnalagadda, S. S., and Kagan,
V. E. (1996) Detection and characterization of the EPR-silent
glutathionyl-5,5-dimethyl-1-pyrroline N-oxide adduct derived from
redox cycling of phenoxyl radicals in model systems and HL-60
cells. Arch. Biochem. Biophys. 330, 3-11.
(17) Rao, N. R., and Snyder, R. (1995) Oxidative modifications
produced in HL-60 cells on exposure to benzene metabolites. J .
Appl. Toxicol. 15, 403-409.
(18) Rao, R., Fischer, V., and Mason, R. P. (1990) Glutathione and
ascorbate reduction of the acetaminophen radical formed by
peroxidase. J . Biol. Chem. 265, 844-847.
(19) Weis, M., Rundgren, M., Nelson, S., and Moldeus, P. (1996)
Peroxidase-catalysed oxidation of 3,5-dimethylacetaminophen
causes cell death by selective protein thiol modification in isolated
hepatocytes. Chem.-Biol. Interact. 100, 255-265.
(20) Divi, R. L., and Doerge, D. R. (1994) Mechanism-based inactiva-
tion of lactoperoxidase and thyroid peroxidase by resorcinol
derivatives. Biochemistry 33, 9668-9674.
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