Toxicology and Applied Pharmacology 212 (2006) 110 – 118

www.elsevier.com/locate/ytaap

Quercetin protects human hepatoma HepG2 against oxidative stress

induced by tert-butyl hydroperoxidei
Mario Alı́a, Sonia Ramos, Raquel Mateos, Ana Belén Granado-Serrano,
Laura Bravo, Luis Goya*
Departamento de Metabolismo y Nutrición, Instituto del Frı́o (CSIC), C/José Antonio Novais, 10, Ciudad Universitaria, 28040 Madrid, Spain

Received 28 April 2005; revised 14 July 2005; accepted 19 July 2005

Available online 29 August 2005

Abstract

Flavonols such as quercetin, have been reported to exhibit a wide range of biological activities related to their antioxidant capacity. The
objective of the present study was to investigate the protective effect of quercetin on cell viability and redox status of cultured HepG2 cells
submitted to oxidative stress induced by tert-butyl hydroperoxide. Concentrations of reduced glutathione and malondialdehyde, generation of
reactive oxygen species and activity and gene expression of antioxidant enzymes were used as markers of cellular oxidative status. Pretreatment
of HepG2 with 10 AM quercetin completely prevented lactate dehydrogenase leakage from the cells. Pretreatment for 2 or 20 h with all doses of
quercetin (0.1 – 10 AM) prevented the decrease of reduced glutathione and the increase of malondialdehyde evoked by tert-butyl hydroperoxide
in HepG2 cells. Reactive oxygen species generation induced by tert-butyl hydroperoxide was significantly reduced when cells were pretreated
for 2 or 20 h with 10 AM and for 20 h with 5 AM quercetin. Finally, some of the quercetin treatments prevented the significant increase of
glutathione peroxidase, superoxide dismutase, glutathione reductase and catalase activities induced by tert-butyl hydroperoxide. Gene
expression of antioxidant enzymes was also affected by the treatment with the polyphenol. The results of the biomarkers analyzed clearly show
that treatment of HepG2 cells in culture with the natural dietary antioxidant quercetin strongly protects the cells against an oxidative insult.
D 2005 Elsevier Inc. All rights reserved.

Keywords: Polyphenols; Glutathione; Malondialdehyde; Reactive oxygen species; Antioxidant enzymes

Introduction genic, anti-inflammatory and antiviral actions. In addition,

Dietary antioxidants, including polyphenolic compounds,
are considered beneficial because of their potential protective
role in the pathogenesis of multiple diseases associated to
oxidative stress such as cancer, coronary heart disease and
atheroschlerosis. Flavonoids comprise a large group of
naturally occurring low molecular weight polyphenolic
compounds that are present in all plants (Bravo, 1998).
Flavonoids, and specifically flavonols such as quercetin,
have been reported to exhibit a wide range of biological
activities (Hertog and Holland, 1996), including anticarcino-
i diet. The average daily Western diet contains between 100 mg
Part of this work was presented at the 1st International Conference on
Polyphenols and Health, Vichy, France, 18 – 21 November 2003.
* Corresponding author.
E-mail address: luisgoya@if.csic.es (L. Goya).
0041-008X/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2005.07.014
they inhibit lipid peroxidation, platelet agregation, capillary
permeability and the activity of enzyme systems including
lipoxygenase (Aherne and O’Brien, 1999). The flavonoids
exert these effects as antioxidants, chelators of divalent
cations and free radical scavengers and thus may be
involved in preventing free radical mediated cytotoxicity
and lipid peroxidation which are associated with cell aging
and chronic diseases (Middelton, 1996; Mora et al., 1990).
The flavonoid used in this study, quercetin, is one of the
most common dietary flavonols with a well characterized in
vitro antioxidant activity. It is found in fruits, vegetables, tea,
wine, nuts, seeds and represents an integral part of the human
and 1000 mg total flavonoids, of which approximately 23 mg
has been estimated as the average intake of flavonols and
flavones in the Dutch population (Hertog and Holland, 1996).
 M. Alı́a et al. / Toxicology and Applied Pharmacology 212 (2006) 110 – 118
Quercetin is the predominant flavonol found in foods and
intakes of between 6 and 31 mg per day have been reported
(Hertog et al., 1995). Absorption of these compounds through
the gastrointestinal tract renders measurable amounts of
blood polyphenols which may affect the biochemistry of cells
from different tissues, in particular the liver.
The study of the effect of dietary polyphenols on the
regulation of antioxidant defense mechanisms at the cellular
level may benefit from the use of an established cell culture
line. Human hepatoma HepG2, a well differentiated trans-
formed cell line, is a reliable model, easy to culture, well
characterized and widely used for biochemical and nutri-
tional studies where many antioxidants and conditions can
be assayed with minor interassay variations (Alı́a et al.,
2005, in press). In addition, steady-state functioning of the
antioxidant defenses in human hepatoma HepG2 is rela-
tively higher than that in hepatocytes and other non-
transformed cells, therefore, variations of responses to
different conditions are more easily detected (Alı́a et al.,
2005, in press; Murakami et al., 2002a,b; Chen et al., 2002).
The objective of the present study was to investigate the
potential protective effect of different quercetin concentra-
tions against oxidative stress induced by tert-butyl hydro-
peroxide (t-BOOH) in HepG2 cells in culture. Cell integrity
and several markers of oxidative damage were used to
estimate the effect of quercetin in cell survival and the
response of the antioxidant system of HepG2 to t-BOOH.
Concentration of reduced glutathione (GSH); determination
of malondialdehyde (MDA) as marker of lipid peroxidation;
generation of reactive oxygen species (ROS); evaluation of
the activity of antioxidant enzymes: catalase (CAT), gluta-
thione peroxidase (GPx), superoxide dismutase (SOD) and
glutathione reductase (GR) and the gene expression of CAT,
GPx and SOD were measured.
Methods
Cell culture and quercetin treatment. Human hepatoma
HepG2 cells initially isolated from a liver biopsy in a 15-year-
old Caucasian male (Aden et al., 1979) were a gift from Dr.
Paloma Martin-Sanz (Instituto de Bioquimica, CSIC, Mad-
rid, Spain). This cell line was grown in a humidified incubator
containing 5% CO2 and 95% air at 37 -C. They were grown in
DMEM F-12 medium from Biowhitaker (Innogenetics,
Madrid, Spain), supplemented with 2.5% Biowhitaker fetal
bovine serum (FBS) and 50 mg/L of each of the following
antibiotics: gentamicin, penicillin and streptomycin (all from
Sigma, Madrid, Spain). The culture medium was changed
every other day and the cells were usually split 1:3 when they
reached confluence. Plates were changed to FBS-free
medium before the beginning of the assay. The serum added
to the medium favors growth of most cell lines but might
interfere in the running of the assays and affect the results.
Moreover, a fairly good growth of HepG2 cells in FBS-free
DMEM-F12 has been observed (Alı́a et al., in press). For all
111
assays except ROS, the different concentrations of quercetin,
dissolved in dimethylsulfoxide, were added in FBS-free
DMEM-F12 to the cell plates for 2 (short term) or 20 (long
term) h. Then the cell plates were washed with PBS and a new
medium containing 200 AM t-BOOH was added to all
cultures except controls for 3 h. After that time, cell culture
was collected (LDH assay) or eliminated (rest of assays) and
the cells washed with PBS, collected by scraping and treated
as described below for each assay. In the ROS assay, cells
were treated with quercetin as above but then they were
treated with t-BOOH for 90 min (see below).
Evaluation of cytotoxicity (lactate dehydrogenase leakage
assay). Cells were plated in 60 mm diameter plates at a
concentration of 1.5  106 per plate and the assay was
carried out 2 days later. Cells were pretreated with the
different concentrations of quercetin in FBS-free medium
for 2 or 20 h. After this pretreatment with the antioxidant,
the plates were washed twice and the vehicle (control plates)
or 200 AM t-BOOH (all other plates) was added for 3 h.
Lactate dehydrogenase (LDH) leakage was estimated from
the ratio between the LDH activity in the culture medium
and that of the whole cell content (Alı́a et al., 2005, in press;
Vasault, 1987; Welder and Acosta, 1994).
Determination of reduced glutathione and evaluation of
malondialdehyde levels. The content of reduced gluta-
thione was quantitated by the fluorometric assay of Hissin
and Hilf (1976). The method takes advantage of the reaction
of reduced glutathione with o-phthalaldehyde (OPT) at pH
8.0. Fluorescence was measured at an emission wavelenghth
of 460 nm and an excitation wavelength of 340 nm. The
precise protocol has been described elsewhere (Alı́a et al.,
2005, in press). Cellular malondialdehyde (MDA) was
analyzed by high-performance liquid chromatography
(HPLC) as its 2,4-dinitrophenylhydrazone (DNPH) deriva-
tive (Mateos et al., 2004). Cells were treated as in the LDH
assay and then collected. For MDA, values are expressed as
nmol of MDA/mg protein; protein was measured by the
Bradford method (1976).
Determination of reactive oxygen species. Cellular reac-
tive oxygen species were quantified by the dichlorofluorescin
(DCFH) assay using a microplate reader (Wang and Joseph,
1999). After being oxidized by intracellular oxidants, DCFH
will become dichlorofluorescein (DCF) and emit fluores-
cence. By quantifying fluorescence over a period of 90 min, a
fair estimation of the overall oxygen species generated under
the different conditions was obtained. This parameter gives a
very good evaluation of the degree of cellular oxidative
stress. The assay has been described elsewhere (Alı́a et al.,
2005, in press).
Determination of antioxidant enzymes activity. For the
assay of the activity of antioxidant enzymes, cells previously
treated, as in LDH, reduced glutathione and MDA assays
112 M. Alı́a et al. / Toxicology and Applied Pharmacology 212 (2006) 110 – 118

were suspended in phosphate buffered saline (PBS), sonica- 3 h treatment with 200 AM t-BOOH evoked a great increase
ted and centrifuged. All the enzyme activities were measured in LDH activity in the cell culture medium indicating cell
in the supernatants. Catalase (CAT) activity was determined damage in HepG2 (Fig. 1a). Pretreatment for 2 h of HepG2
by following the decomposition of H2O2 measured as a cultures with 0.1 –5 AM quercetin greatly reduced cell
decrease in absorbance at 240 nm (Aebi, 1987). Glutathione damage. Concentrations of quercetin of 5 AM decreased cell
reductase (GR) activity was determined by following the toxicity almost to control values (t-BOOH-untreated cells).
decrease in absorbance due to the oxidation of NADPH A higher dose of quercetin (10 AM) further reduced cell
utilized in the reduction of oxidized glutathione (Goldberg damage to values similar to those of control cells (Fig. 1a).
and Spooner, 1987). The determination of glutathione When the pretreatment was extended to 20 h, the cell toxicity
peroxidase (GPx) activity is based on the oxidation of induced by t-BOOH was greatly reduced to values around
reduced glutathione by GPx, using tert-butyl hydroperoxide
as a substrate, coupled to the disappearance of NADPH by
GR (Gunzler et al., 1974). SOD activity was determined by
the Oxis commercial kit Bioxytech SOD-525. The enzyme
activities of the three most discussed types of superoxide
dismutase (SOD), Cu/Zn-SOD, Mn-SOD and Fe-SOD, are
measured together in this assay. All these methods have been
described in more detail in Alı́a et al. (2005, in press). Protein
was measured by the method of Bradford (1976).

Preparation of RNA and Northern blot analysis. Cultured

cells treated as above were separated from the plastic
substrate by trypsinization and total cell RNA was prepared
with Rneasy kit (IZASA, Barcelona, Spain). A 498 bp human
Cu/Zn SOD insert, a 424 bp human catalase insert and a 316
bp human GPx insert were used as probes. All probes were
cloned into HindIII – BamHI sites of the pGEM4z (Promega
Biotech, Madison, WI) and kindly provided by Dr. Jonathan
L. Tilly (Vincent Center for Reproductive Biology, Massa-
chusetts General Hospital, Boston, MS, USA) and Dr. M.
Cascales (Instituto de Bioquimica, CSIC, Madrid, Spain). A
h-actin probe (0.6 kb EcoRI/HindIII fragment isolated from
VC18 vector kindly provided by Dr. P. Martin-Sanz from
Instituto de Bioquimica, CSIC, Madrid, Spain) was used in
order to validate the ethidium bromide method for loading
normalization. 32P-labeled probes were generated using a
random prime labeling kit (Amersham Pharmacia Biotech,
Barcelona, Spain). The protocol has been previously
described (Alı́a et al., in press).

Statistics. Statistical analysis of data was as follows, prior

to analysis, the data were tested for homogeneity of variances
by the test of Levene; for multiple comparisons, one-way
ANOVA was followed by a Bonferroni test when variances
were homogeneous or by Tamhane test when variances were
not homogeneous (only ROS 2 h pretreatment 20 min and
ROS 20 h pretreatment 90 min). The level of significance was
P < 0.05. A SPSS version 12.0 program has been used.
Results
Cytotoxicity
Lactate dehydrogenase (LDH) leakage was used as an
indicator of cytotoxicity. In our experimental conditions, a
Fig. 1. Effect of quercetin on cell viability and intracellular concentration of
reduced glutathione and malondialdehyde. HepG2 were treated with the
noted concentrations of quercetin for 2 or 20 h, then the cultures were
washed twice and 200 AM t-BOOH was added to all the cultures except
controls for 3 h. (a) Lactate dehydrogenase (LDH) leakage was used as
index of cell viability. Results are expressed as percent of lactate
dehydrogenase activity in the culture medium of the total activity, culture
medium plus intracellular. (b) The results of the fluorescent analysis for
reduced glutathione (GSH) are expressed as nmol GSH per mg protein. (c)
Quantitative analysis of malondialdehyde (MDA) was made by HPLC in
cytoplasmatic contents of HepG2. Values are means T SD, n = 4 – 8 data.
Different letters indicate statistically significant differences ( P < 0.05)
among different groups.
 M. Alı́a et al. / Toxicology and Applied Pharmacology 212 (2006) 110 – 118 113

and below 10% of LDH activity in the culture medium.

Again, 10 AM quercetin was enough to convey HepG2 with
100% protection against a cell insult such as that evoked by
200 AM t-BOOH for 3 h (Fig. 1a).

Reduced glutathione concentration

As an index of the intracellular nonenzymatic antiox-

idant defenses, the concentration of reduced glutathione
was measured in cells treated with t-BOOH which had
been pretreated for 2 or 20 h with 0.1, 1, 5 and 10 AM
quercetin. 200 AM t-BOOH evoked a dramatic decrease of
cytoplasmic GSH which was overcome by a pretreatment
for 2 or 20 h with any of the above doses of quercetin
(Fig. 1b).

Malondialdehyde levels

As a biomarker for lipid peroxidation, the cytoplasmic

concentration of MDA was measured in cells treated 3 h
with t-BOOH which had been pretreated for 2 or 20 h with
0.1, 1, 5 and 10 AM quercetin. The 3 h treatment of HepG2
with 200 AM t-BOOH evoked a significant increase of about
50% in the cellular concentration of MDA and pretreatment
for 2 or 20 h with all four doses of quercetin prevented the
MDA increase, indicating a reduced level of lipid perox-
idation in response to t-BOOH in cells that had been
previously incubated in the presence of doses of quercetin as Fig. 2. Effect of quercetin on intracellular reactive oxygen species (ROS)
generation. HepG2 cultures were treated with the noted concentrations of
low as 0.1 AM for 2 or 20 h (Fig. 1c). Furthermore, after quercetin for 2 (a) or 20 (b) h, then the cultures were washed twice and
pretreatment of cells with doses of quercetin of 5 and 10 AM 200 AM t-BOOH was added to all cells except controls and intracellular
for 2 h or 1, 5 and 10 AM for 20 h, levels of MDA showed a ROS production was evaluated at 0, 20, 50 and 90 min and expressed as
trend to be below those of control untreated cells, even after fluorescence units. Letters upon symbols indicate statistical differences
3 h with 200 AM t-BOOH. ( P < 0.05) when that group or close groups of data are compared to
control (letter a) or to t-BOOH (letter b). Values are means of 8 different
samples per condition. SD values were not included in the plot due to
Reactive oxygen species (ROS) generation intense bar overlapping.

Cells were treated for 2 h or 20 h with the same doses of

quercetin and then the flavonoid was removed from the
culture medium and 200 AM t-BOOH was added for the
length of the ROS assay, 90 min. A significant increase in
ROS production was observed over time in the presence of
200 AM t-BOOH as compared to unstressed controls (Fig.
2). Pretreatment for 2 h of HepG2 cultures with 5 AM
quercetin greatly decreased ROS production and 10 AM
quercetin reduced ROS levels to those of untreated cells
(Fig. 2a). Pretreatment of cells for 2 h with 0.1 or 1 AM
quercetin evoked a smaller, although significant at 50 and
90 min, decrease in ROS generation than that observed in
cells treated with 5 or 10 AM quercetin. When HepG2 cells
were pretreated for 20 h with 5 or 10 AM quercetin, ROS
production in the presence of t-BOOH was reduced to that
of control untreated cells, whereas 0.1 or 1 AM quercetin
had no preventive effect (Fig. 2b). Leakage of probe was not
observed in cells throughout the assay, as determined in our
laboratory in previous tests during method set-up (Alı́a et
al., 2005, in press). Thus, any potential contribution of
extracellularly oxidized DCF to the final fluorescence can
be ruled out.
Antioxidant enzymes
The presence of 200 AM t-BOOH in the culture medium
for 3 h induced significant increases in the enzyme activities
of GPx, GR, CAT and SOD (Fig. 3). When cells were
pretreated for 2 or 20 h with the same range of doses of
quercetin previously used in the above experiments, the t-
BOOH-induced increase in enzyme activity of GPx was
suppressed. Pretreatment for 2 h with 1, 5 and 10 AM
quercetin and for 20 h with all four doses of the flavonol
completely prevented the t-BOOH-induced increase in SOD
activity of HepG2 cells. The t-BOOH-induced increase in
CAT was prevented when cells were pretreated with 5 and
10 AM for either 2 or 20 h. Finally, all four doses of
quercetin prevented the t-BOOH-induced rise in GR activity
when cells were pretreated for 2 h and none of the doses
114 M. Alı́a et al. / Toxicology and Applied Pharmacology 212 (2006) 110 – 118

Fig. 3. Effect of quercetin on the activity of antioxidant enzymes. Activity of GPx (a), SOD (b), CAT (c) and GR (d) was evaluated in cultures of HepG2.
HepG2 cells were treated with the noted concentrations of quercetin for 2 or 20 h, then the cultures were washed twice and 200 AM t-BOOH was added for 3 h
to all cultures except controls. Different letters indicate statistically significant differences ( P < 0.05) among different groups. Specific enzyme activity was
expressed as indicated in each panel and represents the means T SD of 3 – 4 different experiments per condition.

prevented such increase when cells were pretreated for 20 h
(Fig. 3).
Gene expression of antioxidant enzymes
Gene expression of the antioxidant enzymes Cu/Zn SOD,
CAT and GPx was tested in cultures of HepG2 treated for 2
or 20 h with a range of doses of quercetin prior to the
treatment with 200 AM of t-BOOH for 3 h. The results,
referred to a constitutive control for RNA expression such
as h-actin, showed no significant changes in RNA levels of
Cu/Zn SOD, CAT and GPx in cells treated with 200 AM t-
BOOH with respect to controls (Fig. 4). A small but
significant increase in RNA of Cu/Zn SOD and GPx was
obtained in cells submitted to t-BOOH and pretreated 2 h
with 0.1 or 10 AM quercetin, whereas a decrease of the RNA
concentration for CAT was observed in cells pretreated 2 h
with 0.1 and 1 AM quercetin prior to t-BOOH (Fig. 4). A 20
h pretreatment of cells with 0.1, 1 or 5 AM quercetin evoked
an increase of the RNA levels for Cu/Zn SOD and GPx after
exposure to 200 AM t-BOOH for 3 h. On the contrary, a
decreased gene expression for CAT was found in cells
pretreated for 20 h with 0.1, 1, 5 and 10 AM quercetin prior
to exposure to t-BOOH (Fig. 4).
Discussion
There is considerable current interest in the cytoprotec-
tive effects of natural antioxidants against oxidative stress
and the different defense mechanisms involved. This study
demonstrates that quercetin, a common dietary flavonol
with a high in vitro antioxidant activity, has the ability to
protect the cell against an oxidative insult by modulating
ROS generation, reduced glutathione concentration, MDA
production and the main antioxidant enzymes in the cell.
Through the well documented antioxidant and free
radical scavenging activity of dietary flavonols, these
compounds can inhibit oxidative damage to DNA and,
therefore, prevent or reduce cellular oxidative stress and cell
overgrowth (Duthie et al., 1997; Noroozi et al., 1998; Lipkin
et al., 1999). Quercetin has been reported to be a potent
inhibitor of lipoxygenase (LOX) and cycloxygenase (COX)
activities, Na – K-ATPase, protein kinase C (PKC) and
various tyrosine kinases (Lipkin et al., 1999). Through
one or more of the above biochemical mechanisms,
quercetin has been reported to protect against cellular
oxidative stress and both chemically induced and sponta-
neous formation of tumors in animals (Lipkin et al., 1999;
Duthie et al., 2000). When human hepatoma HepG2 cells
were pretreated with quercetin for 2 or 20 h prior to being
submitted to an oxidative stress induced by t-BOOH for 3 h,
cell toxicity evaluated by the leakage of the intracellular
enzyme lactate dehydrogenase into the culture medium was
greatly or completely prevented, indicating that the anti-
oxidant-treated cells were partly or totally protected against
the oxidative insult. The choice of t-BOOH over other
common prooxidants such as hydrogen peroxide is based on
previous research from our laboratory (Alı́a et al., 2005),
where it was shown that a condition of cellular stress was
evoked when HepG2 in culture were treated with t-BOOH
but not with H2O2..
 M. Alı́a et al. / Toxicology and Applied Pharmacology 212 (2006) 110 – 118 115

Fig. 4. Effect of a 2 and 20 h treatment with quercetin on gene expression of antioxidant enzymes. RNA expression of SOD, CAT and GPx was determined in
cultures of HepG2. Cells were treated with the noted concentrations of quercetin for (a) 2 h or (b) 20 h, then the cultures were washed twice and 200 AM t-
BOOH was added for 3 h to all cultures except controls. Specific RNA bands from representative Northern blot experiments are shown. Densitometric
quantitation of the bands from two to three different experiments (a total of four to six different samples per condition) is depicted below (c). A h-actin probe
was used for loading normalization, and the results are expressed as percent of optical density of the specific band referred to optical density of h-actin in the
same sample. This ratio is then referred to the ratio obtained for control samples which is assigned a value of 100%. Different letters on bars indicate
statistically significant differences ( P < 0.05) among different groups.

Reduced glutathione (GSH) is the main nonenzymatic
antioxidant defense within the cell, reducing different
peroxides, hydroperoxides and radicals (alkyl, alkoxyl,
peroxyl, etc.) (Viña, 1990). It is usually assumed that GSH
depletion reflects intracellular oxidation. On the contrary, an
increase in GSH concentration could be expected to prepare
the cell against a potential oxidative insult (Rodgers and
Grant, 1998; Myhrstad et al., 2002; Scharf et al., 2003; Alı́a
et al., 2005, in press). In our experimental conditions, a
treatment of HepG2 cells with 200 AM t-BOOH induced a
remarkable decrease in the concentration of reduced
glutathione which was completely prevented by a pretreat-
ment with all four doses of quercetin for 2 or 20 h. An
increase in intracellular GSH concentration induced by
quercetin has been previously found in MCF7 human breast
cancer cells (Rodgers and Grant, 1998), monkey kidney
derived COS1 cells (Myhrstad et al., 2002) and HepG2
(Scharf et al., 2003; Alı́a et al., in press). Those studies report
that the increase in GSH is preceded by stimulation by
quercetin of the g-glutamylcysteine synthetase, the enzyme
involved in glutathione synthesis. Perhaps the concentration
of GSH increases during the pretreatment of cells with
quercetin preventing its decrease below steady-state levels in
the presence of t-BOOH (Alı́a et al., in press). Other reduced
soluble thiols including cysteine, gamma-glutamyl cysteine
and homocysteine, are able to react with OPT and, although
116 M. Alı́a et al. / Toxicology and Applied Pharmacology 212 (2006) 110 – 118
their relevance as compared to the reducing effect of
glutathione might be relatively low, some interference with
the results should not be ruled out (Alı́a et al., 2005, in
press). At any rate, the results of the GSH analysis
demonstrate, as in the case of LDH, that HepG2 cells treated
with quercetin are better prepared to face an oxidative insult.
An important step in the degradation of cell membranes
is the reaction of ROS with the double bonds of poly-
unsaturated fatty acids (PUFAs) to yield lipid hydroper-
oxides. On breakdown of such hydroperoxides, a great
variety of aldehydes can be formed (Pilz et al., 2000). MDA,
a three-carbon compound formed by scission of peroxidized
PUFAs, mainly arachidonic acid, is one of the main
products of lipid peroxidation (Suttnar et al., 1997). Since
MDA has been found elevated in various diseases thought
to be related to free radical damage, it has been widely used
as an index of lipoperoxidation in biological and medical
sciences (Suttnar et al., 2001). However, determination of
MDA levels in cell culture conditions is extremely scant in
the literature (Courtois et al., 2002; Mateos et al., 2004; Alı́a
et al., 2005, in press). We have recently established a new
method of evaluation of MDA in cultures of human
hepatoma cells that is sensitive enough to detect a
significant increase in MDA concentration in response to
an oxidative stress induced by t-BOOH (Mateos et al., 2004;
Alı́a et al., 2005, in press). By using this method, we have
found that the t-BOOH-induced increase of MDA was
completely avoided when cells were pretreated for 2 or 20 h
with all tested doses of quercetin. This protection against
lipid peroxidation in a cell culture by quercetin, reported
here for the first time to our knowledge, is in concert with
previous studies that showed a similar protection by tea
catechins (Chen et al., 2002; Murakami et al., 2002a,b) and
beta carotene or lutein (Chen et al., 2002) in the same cell
line. Thus, our results on the one hand, extend the protective
effect reported for other dietary polyphenols to one of the
most common of them in the diet, quercetin, and on the
other hand, support the experimental model of HepG2 cells
in culture as a reliable model for this sort of studies.
Direct evaluation of the reactive oxygen species (ROS)
yields a very good indication of the oxidative damage to
living cells (Wang and Joseph, 1999). Based upon the fact
that nonfluorescent 2V,7V-dichlorofluorescin (DCFH) crosses
cell membranes and is oxidized by intracellular ROS to
highly fluorescent DCF (LeBel et al., 1992), the intracellular
DCF fluorescence can be used as an index to quantify the
overall oxidative stress in cells (Wang and Joseph, 1999;
Alı́a et al., 2005, in press). A prooxidant such as t-BOOH can
directly oxidize DCFH to fluorescent DCF, and it can also
decompose to peroxyl radicals and generate lipid peroxides
and ROS, thus increasing fluorescence. The antioxidant and
free radical activity of quercetin determined by other
methods has been shown in different experimental models
(Noroozi et al., 1998). In this study, increased ROS ge-
neration in cultured HepG2 submitted to an oxidative stress
by t-BOOH was completely inhibited by a pretreatment for
20 h with doses of quercetin in the upper physiological range
and an almost complete or partial inhibition was obtained
with other doses and periods of pretreatment. This result
clearly shows that the natural antioxidant quercetin strongly
inhibits the generation of ROS induced by t-BOOH in
cultured HepG2, thus preventing or delaying conditions
which favor oxidative stress in the cell.
In the defense against oxidative stress, the cellular
antioxidant enzyme system plays an important role. In fact,
changes in the activity of antioxidant enzymes can be
considered as a biomarker of the antioxidant response (Sies,
1993). There are three forms of SOD in mammals that
catalyze the dismutation of the superoxide radical anion,
i.e., MnSOD located in mitochondria, CuZnSOD found
mainly in cytosol and an extracellular SOD localized in the
extracellular fluid (Röhrdanz et al., 2002). Catalase and GPx
convert H2O2 to H2O (Aebi, 1987; Ursini et al., 1995) and
GR recycles oxidized glutathione back to reduced gluta-
thione (Goldberg and Spooner, 1987; Duthie et al., 2000).
Scientific literature dealing with the effect of quercetin on
the activity of antioxidant enzymes in cultured cells is
scarce, but other natural antioxidants have been tested and
significant changes in the enzyme activity or gene expres-
sion of the antioxidant enzymes have been observed only at
very high doses (Breinholt, 1999; Duthie et al., 2000;
Röhrdanz et al., 2002). In our experimental conditions,
treatment of HepG2 cells with a potent oxidative stressor
such as t-BOOH evokes a significant increase of the activity
of all four tested antioxidant enzymes. Besides, we show, for
the first time, that treatment of human hepatoma cells with
quercetin concentrations in the physiological range prevents
the increase in the activity of SOD, CAT, GR and GPx
induced by oxidative stress. Other authors have shown that
some polyphenolic antioxidants can prevent but not repair
(Aherne and O’Brien, 1999) the oxidative damage to DNA
induced by hydrogen peroxide in cultured hepatoma cells.
However, no previous cell culture study has shown a
specific effect of a polyphenolic compound, such as
quercetin, on the antioxidant enzyme response to an
oxidative insult. This could represent a major benefit for
the cell when dealing with a stressful situation.
Evaluation by Northern blot of SOD, CAT and GPx gene
expression suggests that the increased enzyme activity
observed in the same conditions cannot be the result of
increased gene expression. Two different characteristic
regulatory elements, the antioxidant responsive element
(ARE) and xenobiotic responsive element (XRE), have been
located in the promoter region of the Cu/Zn superoxide
dismutase in human liver cells (Park and Rho, 2002). In
addition, sequence analysis of the mouse GPx and CAT
genes revealed putative binding motifs for NF kappa B and
AP-1, transcriptional regulators that are activated in
response to oxidative stress in various tissues (Zhou et al.,
2001). These findings imply that the antioxidant enzymes
can be transcriptionally induced by the above factors in
combination with or independently of the two regulatory
 M. Alı́a et al. / Toxicology and Applied Pharmacology 212 (2006) 110 – 118
elements to effectively defend cells from oxidative stress.
By mechanisms yet unknown, the doses of quercetin tested
seem to be able to up- or down-regulate gene expression of
the main antioxidant enzymes. However, in our experimen-
tal conditions, the lack of relationship between the changes
in the antioxidant enzyme activity and those in gene
expression seems to indicate that the main antioxidant
enzymes are mostly post-transcriptionally regulated.
In summary, the results of the biomarkers analyzed
clearly show that treatment of HepG2 in culture with the
natural dietary antioxidant quercetin strongly protects the
cells against an oxidative insult. The presence of quercetin
in a range including physiological doses may prepare the
antioxidant defense system of the cell to face a condition of
oxidative stress. Therefore, quercetin may contribute to the
protection afforded by fruit- and vegetable-rich diets against
diseases for which excess production of ROS has been
implicated as a casual or contributory factor.
Acknowledgments
This work was supported by the grant AGL2000-1314
from the Spanish Ministry of Science and Technology
(CICYT). S. Ramos has a contract from the Ramon y Cajal
program from the Ministerio de Educación y Ciencia. R.
Mateos is a postdoctoral fellow from the Ministerio de
Educación y Ciencia.
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