Carcinogenesis vol.19 no.9 pp.

15831589, 1998

Quercetin inhibits hydrogen peroxide (H2O2)-induced NF-B DNA

binding activity and DNA damage in HepG2 cells

Clement A.Musonda and James K.Chipman1

School of Biochemistry, The University of Birmingham, Edgbaston,
Birmingham B15 2TT, UK
1To

whom correspondence should be addressed

Email: j.k.chipman@bham.ac.uk

We have investigated the effect of the plant-derived

flavonoid quercetin in relation to potential oxidant and
antioxidant activity on nuclear factor B (NF-B) binding
activity and DNA integrity in HepG2 cells. Gel mobility
shift assays using a -32P-labelled NF-B oligonucleotide
probe showed that treatment of HepG2 cells with quercetin
(up to 10 M, sub-cytotoxic) did not elevate NF-B binding
activity of nuclear extract protein but did inhibit binding
activity of an extract from cells treated with the oxidant
H2O2. A similar inhibition by quercetin of H2O2-induced
NF-B transcriptional activation was demonstrated using
a cat reporter gene assay. Considering oxidative DNA
damage, using single cell gel electrophoresis (comet) assay
we have demonstrated that quercetin (10 M and below)
did not induce DNA strand breaks. However, a marked
and statistically significant (P < 0.01 at 10 M) inhibition
of strand breakage produced by H2O2 was detected. The
specific formation of 8-oxo-29-deoxyguanosine (8-oxodG)
in calf thymus DNA exposed to either -irradiation or the
Fenton reaction system was also inhibited (P < 0.01 at 10
M) by quercetin in a dose-dependent manner. This was
not accompanied by formation of 8-oxodG by quercetin
itself. The inhibition of 8-oxodG formation by -irradiation
was more potent (IC50 J 0.05 M) than that by the Fenton
reaction (IC50 J 0.5 M), implying that the mechanism of
protection may be different between the two systems. The
inhibition of both NF-B binding activity and oxidative
DNA damage suggests that its antioxidant potential outweighs its oxidative potential in a cellular environment,
which may contribute to anticarcinogenic and antiinflammatory effects.
Introduction
Quercetin, a naturally occuring plant-derived flavonoid, has
been at the centre of several genetic toxicity and carcinogenicity
investigations. Quercetin possesses a wide range of both
potentially detrimental and protective biological characteristics.
For example, it has been reported to inhibit rat mammary
carcinogenesis induced by 7,12-dimethylbenz[a]anthracene
and N-nitrosomethylurea (1,2). In addition, Hertog and
Hollman recently reviewed the potential health effects of
Abbreviations: 8-oxodG, 8-oxo-29-deoxyguanosine; 8-oxoG, 8-oxoguanine;
AP-1, activator protein-1; CAT, chloramphenicol acetyltransferase; DMF, N,Ndimethylformamide; EMSA, electrophoretic mobility shift assay; NF-B,
nuclear factor B; PBS, phosphate buffered saline; ROS, reactive oxygen
species.
Oxford University Press

quercetin (3). However, other reports have shown quercetin to

be mutagenic in the Ames assay (4,5) and carcinogenic at high
doses (6). Moreover, the micronucleus test for genotoxicity
provided contradictory findings (7,8). In vitro quercetin has
the potential to produce reactive oxygen species (ROS) (9,10),
but has also been reported as an antioxidant (11). Consequently,
the balance between its reported harmful and potential protective effects on human health, particularly in relation to ROS,
remains to be elucidated.
ROS have been reported to play a key role in carcinogenesis
by inducing oxidative DNA damage (1214). The abundant
oxidative DNA product, 8-oxo-29-deoxyguanosine (8-oxodG),
has been reported to be highly mutagenic (14). In addition,
ROS are thought to contribute to carcinogenesis through
interference with signal cascade systems (15,16), including
nuclear factor B (NF-B). NF-B has been associated with
cellular proliferation as well as apoptosis and induction of its
activity has been directly linked with cellular transformation
(17,18).
NF-B has been implicated in the inducible expression of
a wide range of genes in response to oxidative stress (16).
Conversely, antioxidants have been reported to inhibit its
activation (19). NF-B transcription factor has also been
reported to activate the expression of a variety of genes
involved in inflammatory, immune and acute phase responses
(20). The inactive form of NF-B is localized in the cytoplasm
and consists of three subunits: DNA binding p50 and p65
subunits and an inhibitory subunit, called IB, which is bound
to p65 (21,22). IB masks the nuclear localization sequence
and its release initiates activation of NF-B and its subsequent
translocation to the nucleus, where it can bind to target sites
in DNA (23,24). The classical activated DNA-binding NF-B
is, therefore, a heterodimer consisting of the two subunits p50
and p65 (Rel A) (22). Both subunits are members of a larger
class of transcription factors called the Rel family (25).
ROS (as well as other extracellular signals, such as cytokines,
viruses and liposaccharides) may activate NF-B via phosphorylation of IB by specific kinases (15,26). Phosphorylation
of IB renders it susceptible to ubiquitination and subsequent
proteolytic degradation by the proteosome (27). There may
also be other effects of ROS on IB (28).
Hydrogen peroxide (H2O2) has been reported to both enhance
NF-B transcription factor activity in HeLa cells and to induce
DNA strand breaks after conversion to the hydroxyl radical
via the Fenton reaction (12,29,30). In the current study we
have used human-derived HepG2 cells to assess the effects of
quercetin on H2O2-mediated activation of NF-B transcription
factor activity as well as on DNA integrity and on 8-oxodG
formation in calf thymus DNA exposed to either -irradiation
or the Fenton reaction system.
Materials and methods
Chemicals
All chemicals, unless otherwise stated, were of the highest quality obtainable
from either Sigma-Aldrich Chemical Co. (Poole, UK), Promega (Southampton,

1583

C.A.Musonda and J.K.Chipman

Fig. 1. Effect of quercetin on H2O2-induced NF-B DNA binding activity. (A) Controls including vehicle (0.1% v/v dimethyl sulphoxide) and cold NF-B
and AP-1 probes. (B and D) Quantitative analyses of the gels by phosphorimaging analysis. (C) Gel shift mobility assays were with nuclear extracts from
HepG2 cells treated with quercetin and/or H2O2 for 90 min. C.P.M., counts per min of the band shifted by binding of NF-B to the probe. Data are the
average from two independent experiments. Dimethyl sulfoxide vehicle 0.1% (in the absence of quercetin) did not reduce the effect of H2O2.
UK) or Stratagene (Cambridge, UK). Plasmids pHIV-CAT and pHIV-CAT
KB, containing NF-B-responsive element consensus sites from the human
immunodeficiency virus long terminal repeat fused to the cat gene, were
kindly provided by Dr Gary J.Nabel (University of Michigan Medical Centre,
Ann Arbor, MI).
Cell culture
HepG2 cells (European Collection of Animal Cell Cultures, Salisbury, UK)
were routinely grown in Williams E medium supplemented with 10% (v/v)
foetal calf serum, 2 mM glutamine, 0.25 mM cysteine, 100 U/ml penicillin
and 100 g/ml streptomycin.
Cell extraction and electrophoretic mobility shift assays (EMSAs)
HepG2 cells were washed with phosphate-buffered saline (PBS) and fresh
serum-free medium containing quercetin (0.1, 1.0 or 10.0 M), and H2O2
(150 M) was added. The cells were then incubated at 37C for 90 min.
Nuclear extracts were prepared as described previously (31). We have shown
previously that quercetin is not cytotoxic at these concentrations but is
cytotoxic at 50 M (32). The concentration of H2O2 used has been used
previously for activation of NF-B (15,29).
DNA binding reaction mixtures (10 l) contained 10 mM TrisHCl, pH 7.5,
50 mM NaCl, 1 mM EDTA and 2 g poly(dIdC). Nuclear extracts (10 g) were

1584

added prior to [-32P]ATP-labelled NF-B oligonucleotide probe (9.0 pmol)

(Amersham, UK). The reaction mixtures were incubated with the probe
(20 min) followed by analysis of DNA-binding activities by mobility shift assay
(33). Complexes were analysed by non-denaturing 4% PAGE. Quantitative
evaluation of NF-BDNA complex formation was determined by Molecular
Dynamics PhosphorImager analysis.
HepG2 cells transfection and cat activity assay
HepG2 cells were transfected at 80% confluency by a liposome-mediated
method (34). Briefly, the HepG2 cells were plated in 100 mm culture plates
(Corning) at a density of 123105 cells/plate and incubated at 37C overnight.
The medium was then replaced with fresh medium (6 ml) containing Tf320
reagent (Promega, UK) and 10 g plasmid pHIV-CAT KB (control) or pHIVCAT containing the NF-B long terminal repeats (test), and the cells incubated
at 37C for 1 h. Fresh medium (12 ml) containing either quercetin (10 M)
and/or H2O2 (150 M) was gently added and the plates were incubated further
for 48 h at 37C.
The cells were scraped (rubber policeman) and assayed for CAT activity
as described previously (35). In brief, cell extract (100 g protein) obtained
from treated or untreated cells after three cycles of freeze (70C) and thaw
(37C) were mixed with 27 mM acetyl-CoA (Sigma, Poole, UK) in 1 M Tris

Quercetin inhibits NF-B activity and DNA damage

trypsin in 0.02% EDTA). The cells were gently spun at 200 g for 3 min at
4C and resuspended in low melting point agarose (0.5%) for comet analysis.
Single cell gel electrophoresis (comet) assay
DNA breaks were detected using an adaptation of the method described
previously (36,37). HepG2 cells treated with quercetin or H2O2 were suspended
in 100 l 0.5% (w/v) low melting point agarose in PBS, pH 7.4, at 37C and
immediately pipetted onto frosted glass microscope slides precoated with a
layer of 0.5% (w/v) normal melting point agarose similarly prepared in PBS.
The agarose was allowed to set for 10 min on ice and then the slides were
immersed in lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris,
pH 10.0, with NaOH, 10% v/v dimethyl sulfoxide and 1% v/v Triton X-100)
at 4C for 1 h to remove cellular proteins. Slides were then placed in a
double row in a 260 mm wide horizontal electrophoresis tank containing
electrophoresis buffer (75 mM NaOH and 1 mM Na2EDTA) for 20 min before
electrophoresis at 25 V for 20 min. The slides were then washed three times
for 5 min each with neutralizing solution (0.4 M TrisHCl, pH 7.5) at 4C
before staining with 50 l ethidium bromide (20 g/ml).
Quantification of the comet assay
Ethidium bromide stained slides were examined with a Zeiss Axiovert 10
fluorescence microscope (Zeiss, Germany). One hundred comets on each slide
were quantified with computerized image analysis using Komet 3.0 (Kinetic
Imaging Ltd, Liverpool, UK) according to the relative intensity of fluorescence
in the tail and results were grouped according to the criteria as described
previously (38).
Exposure of DNA to FeCl2/H2O2 (Fenton reaction system)
The protection against 8-oxodG formation in calf thymus DNA (reported to
be highly susceptible to treatment with H2O2) (39) was investigated in this
part of the study. Calf thymus DNA was solubilized in ultra high quality
water to a concentration of 500 g/ml. Reaction mixtures (made up to 1.0 ml
with PBS) containing DNA (125 g), FeCl2 (250 M), H2O2 (100 M) and
different concentrations of quercetin (0.1, 1.0, 10 or 100 M) in 0.01% (v/v)
dimethylformamide (DMF) were incubated for 15 min at 37C. DMF was used
as the vehicle solvent for quercetin in reaction mixtures. This concentration of
DMF was found in our laboratory not to affect H2O2-induced 8-oxodG
formation (A.Harper, unpublished data). The reaction was terminated by
adding 100 l 0.3 M sodium acetate and 4.0 ml ice-cold ethanol. The DNA
was precipitated at 20C overnight followed by centrifugation (1000 g;
15 min). The DNA pellets were dissolved in 5 mM sodium citrate, 20 mM
sodium chloride buffer, pH 6.5, with HCl and centrifuged (2000 g, 10 min at
4C). The supernatant was used for 8-oxodG analysis.
Exposure of DNA to -irradiation

Fig. 2. Inhibition of NF-B-responsive element-driven CAT activity by

quercetin in HepG2 cells. (A) Chromatogram of a representative CAT assay
with cell extracts from control pHIV-CAT KB (lane 5) and test pHIV-CAT
(lanes 25) transfectant cells with or without treatment. 3AcCm and 1AcCm
refer to the quantity of acetylated products from chloramphenicol (Cm).
Cells were exposed to either quercetin (10 M; lane 2), H2O2 (150 M;
lane 1) or both in combination (lane 3) for 48 h and analysed for CAT
activity as described in Materials and methods. (B) Histogram of quantified
CAT activities (average 6 SEM from two independent experiments). pHIVCATKB and pHIV-CAT represent plasmids containing the deleted and
wild-type NF-B-responsive element consensus sites respectively.
HCl, pH 7.5, followed by addition of 1-deoxy[dichloroacetyl-1-14C]chloramphenicol (2 l, 57 mCi/mmol; Amersham Life Science, Little Chalfont, UK).
The mixture was incubated at 37C for 90 min and 0.6 ml ethyl acetate was
added to extract the chloramphenicol. The organic layer was dried and taken
up in 30 l ethyl acetate, spotted on thin layer silica gel plates (BDH,
Lutterworth, UK) and run with chloroform/methanol (95:5 v/v). After autoradiography, spots were quantified by phosphorimaging. Activity of CAT was
measured by determining the amount of acetylated chloramphenicol produced
from 1-deoxy[dichloroacetyl-1-14C]chloramphenicol.
H2O2-induced DNA damage in HepG2 cells
To induce DNA damage, HepG2 cells (initially seeded at 0.813106 cells/
flask) grown in 25 cm2 flasks were washed twice in PBS, pH 7.4, and then
treated with Ara C for 30 min to stop DNA polymerase activity before
exposure to H2O2 (200 M) in the presence or absence of quercetin (0.1, 1.0
or 10 M) in serum-free medium for 30 min at 37C in 5% CO2/95% air.
The cells were washed twice with PBS, pH 7.4, and then trypsinized (0.05%

The solutions containing DNA (125 g in 1 ml) and different concentrations

of quercetin dissolved in DMF (0.1%) were irradiated at 4C at a dose rate
of 2.34 Gy/min for 9 min from a 60Co source. DNA was then precipitated as
described above.
Determination of 8-oxodG and deoxyguanosine (dG)
The amount of 8-oxodG and dG was measured using HPLC with electrochemical and UV detection, respectively (40). Briefly, DNA (50 g) in 100 l
5 mM sodium citrate, 20 mM sodium chloride, pH 6.5, was heated to 95C
for ~4 min. The solution was cooled on ice and incubated with 20 mM sodium
acetate buffer, pH 4.8, containing 0.1 mM ZnCl2 and 5 U nuclease P1 at
45C for 30 min, followed by incubation with 90 l 50 mM TrisHCl, pH 7.4,
and 3 U alkaline phosphatase at 37C for 1 h. The samples were centrifuged
as described above. The DNA hydrolysates (50 l) were injected onto a
reverse phase C18 HPLC column. The mobile phase contained 12.5 mM citric
acid, 25 mM sodium acetate, 30 mM NaOH and 10 mM acetic acid, pH 5.1,
with a flow rate of 1 ml/min. A Pye Unicam LC-UV detector was used for
quantification of dG and a LC-4B amperometric detector (Bioanalytical
System, Luton, UK) with temperature control was used for detection of
8-oxodG.
The amounts of 8-oxodG and dG were calculated from the peak area based
on their corresponding standards (40) and the results expressed as the ratio
of 8-oxodG compared with dG.
Statistical analysis
The per cent tail DNA has been suggested as the most appropriate parameter
for quantifying DNA strand breaks and comet data have been described as
not following a normal distribution (38,41), thus, the comet data were analysed
using a non-parametric test (MannWhitney) as well as a parametric test
(ANOVA) using Minitab software. The latter test was also used for assessment
of 8-oxodG. Results were expressed as means 6 SEM and significance was
accepted at P , 0.05.

1585

C.A.Musonda and J.K.Chipman

Fig. 3. Effect of quercetin on 8-oxodG formation by -irradiation (A) and the Fenton reaction (B). Values represent the means 6 SDs of three separate
experiments (analysed in duplicate). Results are expressed as per cent of the control which contained the vehicle (0.01% v/v DMF). The background level of
8-oxodG was 0.001% of dG. Positive controls gave 0.005% (-irradiation) and 0.015% (Fenton reaction). Solvent (0.01% v/v DMF) alone did not
significantly elevate the control value (.90% of control).

Results
Effect of quercetin on H2O2-induced NF-B binding activity
A gel shift assay showed inhibition of H2O2-induced NF-B
activation by quercetin in HepG2 cells. In accord with other
reports (15), a major specific shifted band of probe DNA was
observed on addition of control cell extracts (Figure 1A, lanes
1 and 2). Specificity was confirmed by loss of the band on
addition of excess cold NF-B but not by addition of an
equivalent amount of cold activator protein-1 (AP-1) probe
(Figure 1A, lanes 3 and 4, respectively). Quantification of the
intensity of the shifted band was achieved by phosphorimaging
(Figure 1B and D). H2O2 (150 M) elevated NF-B activity
(Figure 1C and D, lane 4) compared with controls and quercetin
inhibited H2O2-induced NF-B activity in HepG2 cells (lanes
14).
The effect of quercetin on NF-B-responsive element-driven
CAT activity in HepG2 cells was also investigated. H2O2
produced approximately a doubling of the response seen in
control cells transfected with plasmid pHIV-CAT KB (lacking
the NFB-responsive element) (Figure 2) but in the presence
of quercetin (10 M) the effect of H2O2 was reduced by ~50%.
An identical pattern of response was seen in two independent
experiments.
Effect of quercetin on calf thymus DNA exposed to the Fenton
reaction system and -irradiation
Conversion of dG to 8-oxodG in intact cells by H2O2 was
found not to be at a sufficient level to investigate protective
effects of quercetin. Quercetin inhibited both Fenton reactionand -irradiation-induced 8-oxodG formation in a dose-dependent manner. The inhibition of -irradiation-induced 8-oxodG
formation was more potent (IC50 5 0.05 M) than that by the
Fenton reaction (IC50 5 0.5 M) (Figure 3). Quercetin inhibited
.90% of -irradiation-induced 8-oxodG formation, while only
~60% of Fenton reaction-induced 8-oxodG formation was
inhibited under the conditions employed. Inhibition was statistically significant (P , at least 0.05) (ANOVA).
1586

Effect of quercetin on DNA strand breakage in HepG2 cells

We also investigated the inhibitory effect of quercetin in
HepG2 cells on H2O2-induced DNA damage using the single
cell electrophoresis (comet) assay. Quercetin (10 M) inhibited
H2O2-induced DNA damage (Figures 4 and 5) and did not itself
produce DNA damage. Statistical significance was indicated by
both parametric and non-parametric tests (P , 0.05, values
for the latter are given). However, at a cytotoxic concentration
of quercetin (100 M) DNA damage was apparent (P , 0.01)
(Figure 6).
Discussion
Although a number of studies have suggested the potential
genotoxicity of quercetin (4244), a few investigations have
also reported various potential beneficial effects of quercetin,
especially regarding oxidative damage. Sahu and Washington
(45) and Rahman et al. (10) indicate the potential for high
concentrations of quercetin to produce ROS in the presence
of metal ions and this might contribute to mutagenicity.
However, Sahu (46) also recognized the dual role of quercetin
as a pro-oxidant and an antioxidant depending on its environment (47). ROS are known to cause oxidative DNA damage
(12,13), which is considered to play an important role in
carcinogenesis and ageing (14,48). An abundant DNA oxidation product is 8-oxodG, which can cause misreading (49),
can activate certain oncogenes such as H-ras and K-ras (50)
and has been associated with cancer of several organs (for
example, see 5153). Quercetin did not elevate 8-oxodG in
calf thymus DNA nor did it cause the formation of DNA
strand breaks in HepG2 cells at sub-cytotoxic concentrations.
The latter technique can detect oxidative DNA damage in cells
(54), such as that produced by H2O2, as demonstrated here.
These data argue against an ability of quercetin to produce
ROS under the conditions employed. The results agree with
those of Duthie et al. (55), who found quercetin-induced DNA
strand breaks in various cell types occurred only at 100 M
quercetin or above (concentrations which were cytotoxic in

Quercetin inhibits NF-B activity and DNA damage

Fig. 4. Representative comet images of HepG2 cells following treatment with different concentrations of quercetin (1100 M) and/or H2O2 (200 M).
(A) Control; (B and C) treated with quercetin (1 and 10 M, respectively); (D) 100 M quercetin (cytotoxic); (E) quercetin and H2O2 (10 and 200 M,
respectively); (F) H2O2 alone (200 M). The cell population profiles are shown in Figure 5.

our study). The results are also in accord with the conclusions
of Nakayama (56). Puppo (57) noted the likelihood that
reductants in the cell would preclude the activity of quercetin
in redox cycling of iron, thus providing a plausible explanation
for the apparent lack of DNA oxidation in HepG2 cells.
In contrast with the lack of damage, an antioxidant influence
of quercetin was apparent. Quercetin inhibited both -irradiation- and Fenton reaction-induced 8-oxodG formation in calf
thymus DNA in a concentration-dependent manner. It also
suppressed H2O2-mediated DNA strand breakage in HepG2
cells. Inhibition of irradiation-induced 8-oxodG formation was
more potent than that induced by the Fenton reaction system
(IC50 values of 0.05 and 0.5 M, respectively), implying that
different mechanisms may be involved. These findings agree
with our recent report of an antioxidant influence on dichlorofluorescin oxidation in HepG2 cells (32). Supportive evidence
for quercetin as an antioxidant in cells comes from studies of
its ability to scavenge O2 and OH based on the action of
hydroxyl groups in the aromatic B ring (5861). However,
iron chelation (11,62) may contribute to the antioxidant effect
in the case of our studies using Fenton chemistry, but not with

damage induced by irradiation. These effects have recently

been discussed by Paganga et al. (63). A further possibility is
that quercetin binding to DNA (64) may mask oxidation target
sites. This protective effect against DNA oxidation may
contribute to the reported cancer chemopreventative effect of
quercetin (1).
The balance between oxidative and antioxidative influences
of quercetin was also assessed by studying the effect on NFB transcription factor. The involvement of ROS in activation
of NF-B is now well established and accepted (28,65,66) and
this has been strongly supported by the observations that
antioxidants inhibit NF-B activity (15,29). We found that
quercetin did not enhance the level of NF-B transcription
factor as assessed in nuclear extracts by gel shift mobility
assays and by the use of a transient transfection and reporter
gene assay for NF-B activation in HepG2 cells. Again, in
contrast, quercetin inhibited H2O2-mediated NF-B activity.
Enhanced NF-B DNA binding activity is linked to chronic
inflammatory diseases (20) and thus its inhibition by quercetin
may relate to the anti-inflammatory effect of the flavonoid
(67,68). However, it is important to note that prolonged
1587

C.A.Musonda and J.K.Chipman

assistance. We thank Dr Gary Nabel for providing the plasmids containing
NF-B response elements. This work is part of the doctoral thesis of
C.A.M. and was supported by an Association of Commonwealth Universities
Scholarship.

References

Fig. 5. Inhibition by quercetin of H2O2-mediated DNA damage (comet

assay) in HepG2 cells. Grade of damage (% DNA in tail) was categorized
as follows: none, ,5%; low, 520%; medium, 2040%; high, 4095%;
total, .95% with respect to DNA. Cells were treated with H2O2 (200 M)
or quercetin (10 M) (or both) and compared with controls (containing
0.1% v/v dimethyl sulfoxide vehicle). Two hundred cells were scored in
total (two separate experiments). Using mean per cent tail DNA values,
H2O2-treated cells were significantly different from control (P , 0.001) and
quercetin gave a significant inhibition (P , 0.01) of the H2O2-induced
effect.

Fig. 6. The effect of quercetin on DNA damage (comet assay) in HepG2

cells. One hundred cells were scored per concentration in each of two
separate experiments and mean values were used to categorize damage as
indicated in Figure 5. The level of damage to DNA (as in Figure 5) was
assessed by the per cent of DNA in the comet tail.

inhibition of its activity may be detrimental, since it is involved

in the regulation of immune and defence genes (20).
Acknowledgements
We are indebted to Drs Tanya Franklin and Aristides G.Eliopoulos for the
help with the CAT assay as well as Ms Jane Davies for her excellent technical

1588

1. Verma,A.K., Johnson,J.A., Gould,M.N. and Tanner,M.A. (1988) Inhibition

of 7,12-dimethylbenz(a)anthracene- and N-nitrosomethylurea-induced rat
mammary cancer by dietary flavonol quercetin. Cancer Res., 48, 5754
5758.
2. Shimoi,K., Masuda,S., Esaki,S. and Kinae,N. (1994) Radioprotective
effects of flavonoids in gamma-ray irradiated mice. Carcinogenesis, 15,
26692672.
3. Hertog,M.G.L. and Hollman,P.C.H. (1996) Potential health effects of the
dietary flavonol quercetin. Eur. J. Clin. Nutr., 50, 6371.
4. Bjeldanes,L.F. and Chang,G.W. (1977) Mutagenic activity of quercetin
and related compounds. Science, 197, 577578.
5. Rueff,J., Laires,A., Borda,H., Chaveca,T., Gomes,M.I. and Halpern,M.
(1986) Genetic toxicology of flavonoids: the role of metabolic conditions
in the induction of reverse mutation, SOS functions and sister-chromatid
exchanges. Mutagenesis, 1, 179183.
6. NTP (1991) Technical Report on the Toxicology and Carcinogenesis
Studies of Quercetin in F344/N Rats, NIH publication no. 91-3140. US
Department of Health and Human Services, Public Health Service,
Research Triangle Park, NC.
7. MacGregor,J.T., Wehr,C.M., Manners,G.D., Jurd,L., Minkler,J.L. and
Carrano,A.V. (1983) In vivo exposure to plant flavonoids: influence on
frequencies of micronuclei in mouse erythrocytes and sister-chromatid
exchange in rabbit lymphocytes. Mutat. Res., 124, 255270.
8. Laires,A., Rueff,J., Borba,H., Chaveca,T., Gomes,M. and Halpern,M.
(1985) Food mutagensthe role of metabolic fate in the response of
flavonoids in short-term assays for carcinogenicity. Anticancer Res., 5,
617619.
9. Gaspar,J., Rodrigues,A., Laires,A., Silva,F., Costa,S., Monteiro,J.M.,
Monteiro,C. and Rueff,J. (1991) On the mechanisms of genotoxicity and
metabolism of quercetin. Mutagenesis, 9, 445449.
10. Rahman,A., Fazal,F., Greensil,J., Ainley,K., Parish,J.H. and Hadi,S.M.
(1992) Strand scission in DNA induced by dietary flavonoids: role of
Cu(I) and oxygen free radicals and biological consequences of scission.
Mol. Cell. Biochem., 111, 39.
11. Morel,I., Lescoat,G., Cogrel,P., Sergent,O., Pasdeloup,N., Brissot,P.,
Cillard,P. and Cillard,J. (1993) Antioxidant and iron-chelating activities
of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat
hepatocyte cultures. Biochem. Pharmacol., 45, 1319.
12. Halliwell,B. and Aruoma,O.I. (1991) DNA damage by oxygen-derived
species: its mechanism and measurement in mammalian systems. FEBS
Lett., 281, 919.
13. Floyd,R.A. (1990) Role of oxygen free radicals in carcinogenesis and
brain ischemia. FASEB J., 4, 25872597.
14. Floyd,R.A. (1990) The role of 8-hydroxydeoxyguanosine in carcinogenesis.
Carcinogenesis, 11, 14471450.
15. Schreck,R., Rieber,P. and Baeuerle,P.A. (1991) Reactive oxygen
intermediates as apparently widely used messengers in the activation of
the NF-B transcription factor and HIV-1. EMBO J., 10, 22472258.
16. Pinkus,R., Weiner,M.L. and Daniel,V. (1996) Role of oxidants and
antioxidants in the induction of AP-1, NF-B and glutathione S-transferase
gene expression. J. Biol. Chem., 271, 1342213429.
17. Beg,A., Sha,W., Bronson,R. and Baltimore,D. (1995) Constitutive NF-B
activation, enhanced granulopoiesis and neonatal lethality in IB deficient
mice. Genes Dev., 9, 27362746.
18. Verma,I.M., Stevenson,J.K., Schwarz,E.M., VanAntwerp,D. and
Miyamoto,S. (1995) Rel/NF-B/IB family: intimate tales of association
and dissociation. Genes Dev., 9, 27232735.
19. Baeuerle,P. and Henkel,T. (1994) Function and activation of NF-B in the
immune system. Annu. Rev. Immunol., 12, 141179.
20. Barnes,P.J. and Karin,M. (1997) Nuclear factor-Ba pivotal transcription
factor in chronic inflammatory diseases. N. Engl. J. Med., 336, 10661071.
21. Ghosh,S., Gifford,A.M., Riviere,L.R., Tempst,P., Noland,G.P. and
Baltimore,D. (1990) Cloning of the p50 DNA binding subunit of NF-B:
homology to rel and dorsal. Cell, 62, 10191029.
22. Urban,M.B. and Baeuerle,P.A. (1991) The role of the p50 and p65 subunits
of NF-B in recognition of cognate sequences. New Biol., 3, 279288.
23. Beg,A.A., Ruben,S.M., Scheinman,R.I., Haskill,S., Rosen,C.A. and
Baldwin,A.S. (1992) IB interacts with the nuclear localization sequences
of the subunits of NF-B: a mechanism for cytoplasmic retention. Genes
Dev., 6, 18991913.

Quercetin inhibits NF-B activity and DNA damage

24. Gilmore,T.D. and Morin,P.J. (1993) The IB proteins: members of a
multifunctional family. Trends Genet., 9, 427433.
25. Liou,H.C. and Baltimore,D. (1993) Regulation of the NF-B/rel
transcription factor and IB inhibitor system. Curr. Opin. Cell Biol., 5,
477487.
26. Zhong,H., Su-Yang,H., Erdjument-Bromage,H., Tempst,P. and Ghosh,S.
(1997) The transcriptional activity of NF-B is regulated by IB-associated
PKAs subunit through a cyclic AMP-independent mechanism. Cell, 89,
413424.
27. Palombella,V., Rando,O., Goldberg,A. and Maniatis,T. (1994) The
ubiquitinproteasome pathway is required for processing the NF-B1
precursor protein and the activation of NF-B. Cell, 78, 773785.
28. Schreck,R. and Baeuerle,P.A. (1994) Assessing oxygen radicals as
mediators in activation of inducible eukaryotic transcription factor NFB. Methods Enzymol., 234, 151163.
29. Meyer,M., Schreck,R. and Baeuerle,A.P. (1993) H2O2 and antioxidants
have opposite effects on activation of NF-B and AP-1 in intact cells:
AP-1 as secondary antioxidant-responsive factor. EMBO J., 12, 20052015.
30. Meneghini,R. (1988) Genotoxicity of active oxygen species in mammalian
cells. Mutat. Res., 195, 215230.
31. Moore,C.N., Girdlestone,J., Anderson,G., Owen,T.J.J. and Jenkinson,J.E.
(1995) Stimulation of thymocytes before and after positive selection results
in induction of different NF-kB/Rel protein complexes. J. Immunol., 155,
46534660.
32. Musonda,A.C., Helsby,N. and Chipman,J.K. (1997) Effects of quercetin
on drug metabolizing enzymes and oxidation of 29,7-dichlorofluorescin in
HepG2 cells. Hum. Exp. Toxicol., 16, 700708.
33. Toledano,M.B. and Leonard,W.J. (1991) Modulation of transcription factor
NF-B binding activity by oxidationreduction in vitro. Proc. Natl Acad.
Sci. USA, 88, 43284332.
34. Felgner,P.L., Gadek,T.R., Holm,M., Roman,R., Chan,H.W., Wenz,M.,
Northrop,J.P., Ringold,G.M. and Danielsen,M. (1987) Lipofection: a highly
efficient, lipid-mediated DNA-transfection procedure. Proc. Natl Acad.
Sci. USA, 84, 74137417.
35. Gordman,C.M., Moffat,L.F. and Howard,B.H. (1982) Recombinant
genomes which express chloramphenicol acetyltransferase in mammalian
cells. Mol. Cell. Biol., 2, 10441051.
36. Singh,N.P., Mcoy,M.T., Tice,R.R. and Scheider,E.L. (1988) A simple
technique for quantitation of low levels of DNA damage in individual
cells. Exp. Cell Res., 175, 184191.
37. Gedik,C.M., Ewen,S.W.B. and Collins,A.R. (1992) Single cell gel
electrophoresis applied to the analysis of UV-C damage and its repair in
human cells. Int. J. Radiat. Biol., 62, 313320.
38. Anderson,D., Yu,T.-W., Phillips,B.J. and Schmezer,P. (1994) The effect of
various antioxidants and other modifying agents on oxygen-radicalgenerated DNA damage in human lymphocytes in the comet assay. Mutat.
Res., 307, 261271.
39. Wei,H., Cai,Q. and Rahn,O.R. (1996) Inhibition of UV light- and Fenton
reaction-induced oxidative DNA damage by the soybean isoflavone
genistein. Carcinogenesis, 17, 7377.
40. Winyard,P., Faux,S.P., Smith,A.J., Davies,J.E. and Chipman,J.K. (1992)
Bleomycin-dependent unscheduled DNA synthesis in non-permeabilised
hepatocytes is not paralleled by 8-oxo-7-hydrodeoxyguanosine formation.
Biochem. Pharmacol., 44, 12551260.
41. Anderson,D., Dhawan,A., Yu,T.-W. and Plewa,M.J. (1996) An investigation
of bone marrow and testicular cells in vivo using the comet assay. Mutat.
Res., 370, 159174.
42. Hermann,K. (1976) Flavonols and flavones in food plants: a review.
J. Food Technol., 11, 433448.
43. Gaspar,J., Laires,A., Monteiro,M., Laureano,O., Ramos,E. and Rueff,J.
(1993) Quercetin and the mutagenicity of wines. Mutagenesis, 8, 5155.
44. Dunnick,J.K. and Hailey,J.R. (1992) Toxicity and carcinogenicity studies
of quercetin, a natural component of foods. Fundam. Appl. Toxicol., 91,
423431.
45. Sahu,S.C. and Washington,M.C. (1991) Effects of antioxidants on
quercetin-induced nuclear DNA damage and lipid peroxidation. Cancer
Lett., 60, 259264.
46. Sahu,S.C. (1994) Dual role of flavonoids in mutagenesis and carcinogenesis.
Environ. Carcinogen. Ecotoxicol. Rev., C12, 121.
47. Laughton,M.J., Halliwell,B., Evans,P. and Hoult,J.R.S. (1989) Antioxidant
and pro-oxidant action of plant phenolics quercetin, gossypol and myricetin.
Biochem. Pharmacol., 38, 28592865.
48. Ames,B.N. and Gold,L.S. (1991) Endogenous mutagens and the cause of
aging and cancer. Mutat. Res., 250, 316.
49. Cheng,K.C., Cahill,D.S., Kasai,H., Nishimura,S. and Loeb,L.A. (1992) 8Hydroxyguanosine, an abundant form of oxidative DNA damage, causes
GT and AC substitutions. J. Biol. Chem., 267, 166172.

50. Kamiya,H., Miura,K., Ishikawa,H., Nishimura,S. and Ohtsuka,E. (1992)

c-Ha-ras containing 8-hydroxyguanosine at codon 12 induces point
mutations at the modified and adjacent positions. Cancer Res., 52,
34833485.
51. Wei,H. and Frenkel,K. (1991) In vivo formation of oxidized DNA bases
in tumour promoter-treated mouse skin. Cancer Res., 51, 44434449.
52. Hattori-Nakakuki,Y., Nishigori,C., Okamoto,K., Imamura,S., Hiai,H. and
Toyokuni,S. (1994) Formation of 8-hydroxy-29-deoxyguanosine in
epidermis of hairless mice exposed to near-UV. Biochem. Biophys. Res.
Commun., 201, 11321139.
53. Olinski,R., Zastawny,T., Budzbon,J., Skosowski,J., Zegarski,W. and
Dizdaroglu,M. (1992) DNA base modification in chromatin of human
cancerous tissues. FEBS Lett., 309, 193198.
54. Collins,A.R., Ma,A.-G. and Duthie,S.J. (1995) The kinetics of repair of
oxidative DNA damage (strand breaks and oxidised pyrimidines) in human
cells. Mutat. Res., 336, 6977.
55. Duthie,J.S., Johnson,W. and Dobson,V.L. (1997) The effect of dietary
flavonoids on DNA damage (strand breaks and oxidized pyrimidines) and
growth in human cells. Mutat. Res., 390, 141151.
56. Nakayama,T. (1994) Suppression of hydroperoxide-induced cytotoxicity
by polyphenols. Cancer Res., 54 (suppl.), 1991s1993s.
57. Puppo,A. (1992) Effect of flavonoids on hydroxyl radical formation by
Fenton-type reactions; influence of the iron chelator. Phytochemistry, 31,
8588.
58. Robak,J. and Gryglewski,R. (1988) Flavonoids are scavengers of
superoxide anions. Biochem. Pharmacol., 37, 837841.
59. Husain,R.S., Cillard,J. and Cillard,P. (1988) Hydroxyl radical scavenging
activity of flavonoids. Phytochemistry, 26, 24892491.
60. Cotelle,N., Bernier,J.-L., Catteau,J.-P., Pommery,J., Wallet,J.-C. and
Gaydou,E.M. (1996) Antioxidant properties of hydroxy-flavones. Free
Radical Biol. Med., 20, 3543.
61. Bors,W., Heller,W., Michel,C. and Saran,M. (1990) Flavonoids as
antioxidants: determination of radical-scavenging efficiencies. Methods
Enzymol., 186, 343355.
62. van Acker,S.A.B.E., van den Berg,D.-J., Tromp,M.N.J.L., Griffioen,D.H.,
van Bennekon,W.P., van der Vijgh,W.J.F. and Bast,A. (1996) Structural
aspects of antioxidant activity of flavonoids. Free Radical Biol. Med., 20,
331342.
63. Paganga,G., Al-Hashim,H., Khodr,H., Scott,B.C., Aruoma,O.I., Hider,R.C.,
Halliwell,B. and Rice-Evans,C,A. (1996) Mechanisms of antioxidant
activities of quercetin and catechin. Redox Rep., 2, 359364.
64. Ahmed,M.S., Ramesh,V., Nagaraja,V., Parish,J.H. and Hadi,S.M. (1994)
Mode of binding of quercetin to DNA. Mutagenesis, 9, 193197.
65. Ghosh,S. and Baltimore,D. (1990) Activation in vitro of NF-B by
phosphorylation of its inhibitor IB. Nature, 344, 678682.
66. Schreck,R., Meier,B., Mannel,D.N., Droge,W. and Baeuerle,P.A. (1992)
Dithiocarbamates as potent inhibitors of nuclear factor B activation in
intact cells. J. Exp. Med., 175, 11811194.
67. Alcaraz,M.J. and Jimenez,M.J. (1988) Flavonoids as anti-inflammatory
agents. Fitoterapia, 59, 2538.
68. Busse,W.W., Kopp,D.E. and Middleton,E. Jr (1984) Flavonoid modulation
of human neutrophil function. J. Allergy Clin. Immunol., 73, 801809.
Received on February 25, 1998; revised on May 7, 1998; accepted on
May 7, 1998

1589