Review

Are polyphenols antioxidants or pro-oxidants? What do we learn

from cell culture and in vivo studies?
Barry Halliwell
*
Department of Biochemistry, National University of Singapore, University Hall, Lee Kong Chian Wing,
UHL #05-02G, 21 Lower Kent Ridge Road, Singapore 119077, Singapore
Received 13 November 2007, and in revised form 26 December 2007
Available online 7 February 2008
Abstract
Diets rich in polyphenols are epidemiologically associated with lower risk of developing some age-related diseases in humans. This
apparent disease-protective eect of polyphenols is often attributed to their powerful antioxidant activities, as established in vitro. How-
ever, polyphenols can also exert pro-oxidant activities under certain experimental conditions. Neither pro-oxidant nor anti-oxidant activ-
ities have yet been clearly established to occur in vivo in humans, nor are they likely given the limited levels of polyphenols that are
achievable in vivo after consumption of foods and beverages rich in them. Other actions of polyphenols may be more important
in vivo. Many studies of the biological eects of polyphenols in cell culture have been aected by their ability to oxidise in culture media,
and awareness of this problem can avoid erroneous claims.
2008 Elsevier Inc. All rights reserved.
Keywords: Polyphenols; Cell culture; Antioxidant; Pro-oxidant; Epigallocatechin gallate; Hydrogen peroxide; Ascorbate; Dulbeccos modied Eagles
medium; Green tea; Red wine
Aerobic organisms produce a wide range of oxygen rad-
icals and other reactive oxygen species (ROS)
1
, both for
useful purposes (e.g. defence, redox signalling) and by
accidents of chemistry (reviewed in [1]). These ROS are
metabolised by a series of antioxidant defences, some syn-
thesised in vivo and other diet-derived [1]. The purpose of
the antioxidant defence network [2] is not to remove
all ROS, but to control their levels so as to allow useful
functions whilst minimising oxidative damage (Fig. 1) [1
4]. But how important are the diet-derived antioxidants
such as vitamins C and E to humans? In general, increased
intakes of these vitamins do not decrease levels of oxidative
damage very much (if at all) in well-nourished humans who
are already consuming the recommended dietary allow-
ances [1,57]. Indeed, it has been suggested that the main
biological function of a-tocopherol in humans is not as
an antioxidant [8].
Polyphenols as antioxidants
Foods and beverages rich in avonoids and other poly-
phenols have been associated with decreased risk of age-
related diseases in several (but not all) epidemiological
studies [915]. Flavonoids have powerful antioxidant activ-
ities in vitro, being able to scavenge [1623] a wide range of
reactive oxygen, nitrogen, and chlorine species, such as
superoxide O
2

, hydroxyl radical OH

, peroxyl radicals
RO
2

, hypochlorous acid (HOCl), and peroxynitrous acid

(ONOOH). Flavonoids can also chelate metal ions, often
decreasing the pro-oxidant activity of metal ions [20,22].
They can inhibit the ability of myeloperoxidase to oxidise
low-density lipoproteins (LDL), a potential anti-athero-
sclerotic eect [24]. Because considerable evidence indicates
that increased oxidative damage is associated with, and may
contribute to the development of, all major age-related
0003-9861/$ - see front matter 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2008.01.028
*
Corresponding author. Fax: +65 6775 2207.
E-mail address: bchbh@nus.edu.sg
1
Abbreviations used: ROS, reactive oxygen species; LDL, low-density
lipoproteins; DMEM, Dulbeccos Modied Eagles Medium.
www.elsevier.com/locate/yabbi
ABB
Available online at www.sciencedirect.com
Archives of Biochemistry and Biophysics 476 (2008) 107112
diseases [13], many have attributed the apparent disease-
protective eects of avonoids to their antioxidant ability
(e.g. reviewed in [20]).
Polyphenols as pro-oxidants
Polyphenols oxidise readily in beverages [2527] such as
green tea. They can also oxidise in cell culture media (see
below) and even in the oral cavity; holding or chewing
green tea in the mouth generates substantial levels of
H
2
O
2
[28]. Often, these pro-oxidant eects involve interac-
tions of polyphenols with transition metal ions [1,2935].
Oxidation of polyphenols produces O
2

, H
2
O
2
and a com-
plex mixture of semiquinones and quinones, all of which
are potentially cytotoxic [26,31,36,37]. It has been argued
that polyphenols may exert antioxidant and other cytopro-
tective eects in the gastrointestinal tract because of the
high levels that can be present [3840]. However, since
there are often unabsorbed transition metal ions (especially
iron [41,42]) in the gastrointestinal tract, pro-oxidant
eects could conceivably occur there as well. Indeed, such
eects have been demonstrated in the gastrointestinal tracts
of certain insects consuming high levels of phenols [43,44].
However, in practice pro-oxidant eects can also be ben-
ecial, since, by imposing a mild degree of oxidative stress,
the levels of antioxidant defences and xenobiotic-metabol-
ising enzymes might be raised, leading to overall cytopro-
tection [45], as illustrated in Fig. 1.
Are polyphenols pro-oxidants or antioxidants in vivo in
humans?
No data are available on whether polyphenols are anti-
oxidant or pro-oxidant in vivo in the human stomach, intes-
tines, and colon, where they can be present at signicant
levels [38,39,46,47]. As for eects after absorption into the
body, multiple well-designed human studies have been done
using reliable biomarkers of oxidative damage in plasma
(F
2
-isoprostanes) and urine (F
2
-isoprostanes, isoprostane
metabolites, 8-hydroxy-2
0
-deoxyguanosine [8OHdG]),
essentially testing for systemic antioxidant or pro-oxidant
activity. The results have been reviewed in detail elsewhere
[40] and are quite variable, but overall no evidence for sys-
temic pro-oxidant eects of polyphenols has emerged. A
few studies report that administration of high doses of epi-
gallocatechin gallate to animals leads to the formation of
cysteine conjugates detectable in the urine, indicative of
some degree of oxidation in vivo [36]. However, these eects
may not be important at lower doses and may not be rele-
vant to humans [36].
Similarly, only limited and variable evidence for antiox-
idant eects of avonoids in humans has been obtained
(reviewed in [40]). This is not, to the author, very surpris-
ing; although avonoids can be absorbed through the gas-
trointestinal tract, maximal plasma concentrations
achieved are low, usually not more that 1 lmol/L, in part
because of rapid metabolism by human tissues [4749].
Many of the products of metabolism, such as methylated
and glucuronidated forms, have decreased antioxidant (or
pro-oxidant) abilities because of the blocking of the pheno-
lic hydroxyl groups involved in such activities [23,48].
Therefore, plasma avonoid concentrations in vivo seem
insucient to exert systemic antioxidant eects.
Another point to consider in interpreting the published
human studies is that several groups have studied avo-
noid-rich foods (e.g. pomegranate [50] or chocolate/cocoa
[51,52]) or beverages (e.g. green tea) rather than pure avo-
noids, and such foods contain other constituents that might
be able to modulate oxidative damage. But are such foods
and beverages eective as antioxidants in vivo? Again, the
data are mixed. Some studies showed antioxidant eects
(e.g. [37,48,5153]), others no eects (e.g. [5457]) and yet
others some indication of mild pro-oxidant eects (e.g.
[58]). One must be careful in studies with foodstus, since
Fig. 1. Balance of antioxidants and reactive species in vivo.
108 B. Halliwell / Archives of Biochemistry and Biophysics 476 (2008) 107112
the mere act of eating in a fasted individual can alter
parameters of oxidative damage. For example, dark soy
sauce has powerful antioxidant abilities in vitro [59,60].
Recently, we attempted to see if dark soy sauce decreases
oxidative damage in vivo in human volunteers, and indeed
it was able to decrease levels of F
2
-isoprostanes [61]. We
administered the soy sauce with rice, using a control con-
sisting of a placebo colouring on the same amount of rice.
The rice meal (devoid of antioxidants) also had eects on
F
2
-isoprostanes and urinary 8OHdG excretion [61],
although the soy sauce did better than the placebo in low-
ering F
2
-isoprostane levels. Similarly, Richelle et al. [62]
and Lee et al. [63] suggested that fasting may raise plasma
F
2
-isoprostane levels. As another example [64], olive oil
administration to human volunteers decreased the propen-
sity of LDL subsequently isolated from their blood to
undergo oxidation in vitro, but feeding oil without antiox-
idants had the same eect.
So overall, in vivo we have no evidence of systemic pro-
oxidant eects of avonoids in humans, and little or no
clear evidence of antioxidant eects. Remember also that
avonoids are not only anti- and pro-oxidants. They have
many other biological eects including the ability to inhibit
cyclooxygenases, lipoxygenases, metalloproteinases and
NADPH oxidases (reviewed in [1,40,6569] and other
papers in the current volume). These other actions may
be more important in vivo than antioxidant eects,
although again many of them have been demonstrated
in vitro only at unphysiologically-high levels of
polyphenols.
Antioxidants in cell culture
Cell culture has often been used to study the cellular
eects of reactive species and of antioxidants, and many
useful data have resulted. However, one must be cautious,
for two reasons. First, normal culture conditions are a state
of hyperoxia [70,71]. Most cells in the human body are
exposed to O
2
concentrations in the range of 110 mm Hg
(obvious exceptions include corneocytes, corneal and respi-
ratory tract lining cells). Yet culture under 95% air/5% CO
2
is about 150 mm Hg of O
2
. Rates of production of ROS by
cellular enzymes (e.g. xanthine oxidase) or by leakage from
electron transport chains (especially in mitochondria)
appear to be O
2
-limited at 10 mm Hg and so production
of ROS will increase if O
2
levels are raised [7173]. In other
words, cells in culture are under an oxidative stress, which
can alter their properties in multiple ways [70], including
sometimes promoting proliferation [1,74].
A second problem is that cell culture media are fre-
quently decient in antioxidants, especially tocopherols
and ascorbate [75]. Vitamin E is rarely added because it
is insoluble in water, and vitamin C because it is unstable
(discussed below). Thus cells are deprived of these antiox-
idants, a situation which can lead to over-interpretations
of the benecial eects of added antioxidants. In other
words, antioxidants may appear to have benecial eects
when added to cultured cells, but this is because a de-
ciency is being corrected rather than being a real benecial
eect of extra antioxidants. Deciencies in selenium in
some cell culture media have been reported [76,77]. This
could decrease or prevent oxidative stress-triggered rises
in the activities of selenium-dependent antioxidant
enzymes, such as the glutathione peroxidase family and thi-
oredoxin reductase [77,78].
One factor that has bedevilled studies of the cellular
eects of avonoids and other polyphenols is their instabil-
ity in commonly-used culture media, especially Dulbeccos
Modied Eagles Medium (DMEM) [70,79]. Oxidation
products include H
2
O
2
and quinones/semiquinones, which
can often react with and deplete cellular GSH [37,70,84].
Fig. 2 shows a striking example; epigallocatechin gallate
(EGCG) added to DMEM begins oxidizing immediately
and rapidly generates cytotoxic levels of H
2
O
2
. Such eect
may have led to artefacts in interpretations of the cellular
eects of high concentrations of added polyphenols. Table
1 summarises some published examples. Not all the cellular
eects of polyphenols are due to such artefacts (e.g. some
of those observed when lower levels, e.g. the lM range,
are added to cells), but it is necessary to consider the poten-
tial for error when determining what the true cellular eects
really are. Oxidation artefacts can also lead to false-posi-
tive results in in vitro genotoxicity testing using cultured
cells, where the generated H
2
O
2
(or other oxidation prod-
ucts) rather than the compound under test is causing the
chromosomal aberrations detected [102,108110].
Why do these eects occur? As well as its normal iron
content (due to contamination and iron-containing pro-
teins in foetal calf serum), DMEM contains added ferric
nitrate, i.e. there is free pro-oxidant iron, which would
be expected to catalyse autoxidation reactions [1]. Surpris-
ingly however, iron ion chelating agents such as desferriox-
Fig. 2. Generation of H
2
O
2
on addition of epigallocatechin gallate
(EGCG) to Dulbeccos modied Eagles medium. The nal concentrations
of EGCG in the medium are shown. SD values are not shown to avoid
cluttering the gures. Note the rapid rate of H
2
O
2
production from EGCG
as soon as it is added to DMEM (see legend to Table 2). Adapted from
[79].
B. Halliwell / Archives of Biochemistry and Biophysics 476 (2008) 107112 109
Table 1
Examples of artefacts caused by oxidation of compounds added to cell culture media (Adapted with considerable updating from [70])
Observation Comment Reference
Induction of cell death by ascorbate in HL-60 or acute myeloid
leukaemia cells or human broblasts
Due to generation of H
2
O
2
by ascorbate oxidation in cell culture
media
[8082]
Induction of apoptosis by green tea in PC12 cells Due to generation of H
2
O
2
by oxidation of tea components in cell
culture media
[83]
Induction of cell death by L-DOPA and dopamine in PC12 and M14
cells
Due to H
2
O
2
, quinones, and semiquinones generated by oxidation
of L-DOPA and dopamine in the culture medium
[84]
Toxicity of apple phenolics to cancer cells Due to oxidation to produce H
2
O
2
in the culture medium [85]
Cytotoxicity of gallic acid Entirely or largely due to oxidation of gallic acid to produce H
2
O
2
in culture medium
[86,87]
Addition of grape seed extract to CaCo-2 cell culture medium
generates H
2
O
2
due to oxidation of phenolics in the medium
[88]
Eects of polyphenols on c-jun phosphorylation in bronchial
epithelial cell lines
Shown to involve H
2
O
2
, although H
2
O
2
was not specically
identied as coming from the culture medium
[89]
Epigallocatechin gallate induces apoptosis in human oral cell lines Due to production of H
2
O
2
in the culture medium [90]
Toxicity of myricetin to Chinese hamster lung broblast V79 cells Due to H
2
O
2
production, although H
2
O
2
was not specically
identied as coming from the culture medium
[91]
Cell culture media found to generate ROS as detected by spin traps
and uorescent dyes
[92]
Ascorbate observed to inhibit cell proliferation and bronectin
synthesis in human skin broblasts
Inhibition by catalase suggests may be due to H
2
O
2
generation in
the culture medium
[80,93]
Stimulation of SIRT1 activity by polyphenols in HT29 cells Results confounded by instability of polyphenols in the culture
medium
[94]
Cyanidin-3-rutoside toxic to HL60 cells. Shown to involve peroxide, although H
2
O
2
was not specically
identied as coming from the culture medium
[95]
EGCG and green tea extract cause oxidative stress responses in S.
cerevisiae
Involves H
2
O
2
production in the medium [96]
Cytotoxicity of EGCG to oral carcinoma cell lines Involves both H
2
O
2
and quinones, although these did not account
for all the eects
[97]
Activation of NF-jB in macrophages by coee Due to H
2
O
2
; coee contains substantial H
2
O
2
levels [98] [99]
Toxicity of catechols to PC12-AC cells Involves H
2
O
2
, mainly generated in the extracellular space [100]
Toxicity of EGCG to Jurkat T cells Involves H
2
O
2
generation in the culture medium [101]
Cytotoxicity and genotoxicity of green tea extract to H260 and
RAW264.7 cells
Involves H
2
O
2
generation, although H
2
O
2
was not specically
identied as coming from the culture medium
[102]
Toxicity of extracts of the oriental fungus Ganoderma lucidum to
human lymphocytes
Involves H
2
O
2
generation [103]
Toxicity of quercetin, catechin and ascorbate to pancreatic b-cells Involves H
2
O
2
generation in the cell culture medium [104]
Toxicity of 4-methylcatechol to murine tumor cells Involves oxidation to form H
2
O
2
and quinones/semiquinones in
the cell culture medium
[105]
Toxicity of EGCG to ovarian cancer calls in DMEM Due to H
2
O
2
formation, probably both intracellularly and in the
culture medium
[106]
Stimulatory eects of garcinol on growth of intestinal cells Involves ROS production in the culture medium; low levels of
H
2
O
2
often stimulate cell proliferation [1,74]
[107]
Table 2
Levels of hydrogen peroxide in culture media under 95% air/5% CO
2
containing 1 mM epigallocatechin gallate (EGCG)
Culture Media Mean H
2
O
2
(lM) SD at various times (h)
0 h 0.5 h 1.0 h 1.5 h 2.0 h
DMEM 75 22 183 8 275 21 317 35 334 34
F-10 21 10 34 4 35 3 37 2 43 3
F-12 65 11 89 4 85 3 92 4 121 14
RPMI with Hepes 33 3 99 12 159 9 212 13 259 17
RPMI without Hepes 82 3 184 32 257 16 299 23 332 22
Mc Coy 5A 24 1 79 14 136 10 192 16 251 19
Williams E 66 8 143 15 209 20 278 23 329 15
Data are means SD, n = 3. Data selected from [110].
Levels of H
2
O
2
were signicantly greater than in medium alone (P < 0.05) at all time points for all media. Note the rapid H
2
O
2
generation at t = 0,
indicating that EGCG has oxidized substantially when added to media in the few seconds before H
2
O
2
measurement can be made. Media alone generated
no signicant levels of H
2
O
2
(<2 lM) after 2 h incubation at 37 C.
110 B. Halliwell / Archives of Biochemistry and Biophysics 476 (2008) 107112
amine or ortho-phenantholine did not decrease the rate of
H
2
O
2
production when gallic acid was added to DMEM
[111]. Eects can be even more complex when mixtures of
antioxidants are used. Thus both ascorbate [80] and poly-
phenols [79] generate H
2
O
2
in DMEM, but if both are pres-
ent the amounts of H
2
O
2
produced are much less than the
sum of the amounts with each compound alone [88,111].
Thus one should always be alert when adding polyphe-
nols to cells in culture; one must check for reactions taking
place in the culture medium that could lead to artefacts and
carefully distinguish eects of oxidation products from
real eects of polyphenols. Addition of catalase can be
used to scavenge the H
2
O
2
, and GSH or N-acetylcysteine
to scavenge quinones and semiquinones [84]. Another
approach is to use a less pro-oxidant medium, since
other culture media seems less good at catalysing polyphe-
nol oxidation than is DMEM [109]. For example, Table 2
shows that F-10 and F-12 media seem far less pro-oxi-
dant than is DMEM.
Conclusion
Polyphenols are metabolized as typical xenobiotics by
the human body, and such metabolism decreases their anti-
oxidant and pro-oxidant abilities. It is now looking unli-
kely that polyphenols act as antioxidants in vivo, and
attention is turning to their other potential eects. Even
so, whether polyphenols contribute to human health by
any mechanism remains uncertain. Care is needed when
studying their eects in cell culture to use biologically-rele-
vant levels, to examine the eects of important metabolites,
and to allow for artefactual chemical processes in the cell
culture media.
Acknowledgment
I am grateful to the Biomedical Research Council of
Singapore for support (BMRC 01/1/21/18/213).
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