DNA Damage Caused by Bisphenol A and Estradiol through Estrogenic Activity
Evidence exists that raises concern about genotoxic effects induced by estrogen: oxidative stress caused by estrogen-derived oxidants, DNA adducts formed by estrogen metabolites and estrogen-induced chromosomal aberration. Estrogen receptors (ER) participate in some of these genotoxic effects by estrogen. In this study, we showed the effects of bisphenol A (BPA), an endocrine-disrupting chemical eliciting weak estrogenic activity, and of 17beta-estradiol (E2), on DNA damage in ER-positive MCF-7 cells by Comet assay. Higher concentrations of BPA, more than 1000 times of E2, were needed to induce the same levels of effects by E2. Immunofluorescence microscopy showed that gammaH2AX, an early marker of DNA breaks, increased after treatment with E2 or BPA in MCF-7 cells. gammaH2AX foci colocalized with Bloom helicase, which is considered to be responsible for the repair of DNA damage after treatment with E2 or BPA. Interestingly, DNA damage was not as severe in ER-negative MDA-MB-231 cells as in MCF-7 cells. The ER antagonist ICI182780 blocked E2 and BPA genotoxic effects on MCF-7 cells. These results together suggest that BPA causes genotoxicity ER dependently in the same way as E2.
Epidemiological studies and animal experiments have
shown carcinogenic properties of estrogen. Studies to clarify
the molecular mechanisms of carcinogenesis by estrogen
suggest that estrogen causes carcinogenic effects by com-
bined genotoxicity and stimulation of cell proliferation.
Estrogen causes DNA damage by estrogen-derived oxi-
DNA adducts formed by estrogen metabolites
and formation of micronuclei.
Recent studies strongly sug-
gest that DNA damage induced by estrogen is dependent on
estrogen receptors (ER): the ER antagonist tamoxifen in-
hibits E2 effects in ER-positive MCF-7 cells, but not in ER-
negative MDA-MB-231 cells.
Detoxifying enzyme activity
markedly decreases by treatment with 17
-estradiol (E2) in
MCF-7 cells, leading to increased susceptibility of cells to
DNA damage, but E2 has no effect on detoxifying enzyme
activity in MDA-MB-231 cells.
Bisphenol A (BPA) was ﬁrst shown to be estrogenic in
1938 in ovariectomized rats
and later in MCF-7 human
breast cancer cell culture assay.
BPA is an endocrine-dis-
rupting chemical and has a weak afﬁnity for ER, estimated at
about 1/1000 of E2,
and its additional estrogenic effects on
the hormonal homeostatic system has recently received much
We also studied proteins involved in the repair of DNA
damage induced by E2 and BPA. Histone H2AX (H2AFX) is
responsible for maintaining genomic stability by recognizing
DNA double-strand breaks.
At the sites of stalled replica-
tion forks, H2AX is phosphorylated to
H2AX, which forms
that appear immediately after DNA damage and re-
cruit proteins responsible for repair of DNA damage, includ-
ing Bloom helicase (BLM),
the product of BLM that is
the causative gene of Bloom syndrome, an autosomal reces-
sive genetic disorder.
Clinical features of patients having
Bloom syndrome include growth retardation, immunodeﬁ-
ciency, male infertility but not female infertility, and a high
incidence of cancers. Cells from Bloom syndrome patients
show a high frequency of sister chromatid exchange.
BLM responds to DNA damage and accumulates at the site
of DNA double-strand breaks and physically interacts with
In this study we investigated the effects of E2 and
BPA on DNA damage in ER-positive MCF-7 and ER-nega-
tive MDA-MB-231 cells, both of which are derived from
adenocarcinomas; these effects were assessed by alkaline sin-
gle cell electrophoresis (Comet assay). We also investigated
H2AX and BLM at sites of DNA damage.
MATERIALS AND METHODS
Chemicals E2 and BPA were purchased from Wako
Pure Chemicals Industries, Ltd. (Osaka). Estrogen receptor
antagonist ICI182780 was obtained from TOCRIS (Ellisville,
Cells, Cell Culture and Chemical Treatment MCF-7
cells and MDA-MB-231 cells were obtained from the
American Type Cell Culture (Bethesda, MA, U.S.A.). Cells
were maintained in Dulbecco’s Modiﬁed Eagle’s Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS)
g/ml gentamycin (both from Sigma-Aldrich, St.
Louis, MO, U.S.A.) in a humidiﬁed atmosphere under 5%
at 37 °C. For all experiments cells were transferred to
phenol red-free DMEM supplemented with 10% charcoal-
dextran-stripped FBS (Hyclone, Logan, UT, U.S.A.) for 48 h
before use to avoid hormonal effects, including by estrogen
in FBS. Chemical treatments continued for 1, 3 or 24 h at the
indicated concentrations. Pre-treatment with ICI182780 was
done for 1 h and then by E2 or BPA treatments. Chemicals
were solubilized in ethanol and the ﬁnal concentration of
ethanol in the culture was 0.1%. A control culture was ex-
posed to a culture medium containing 0.1% ethanol.
Colorimetric Assay of Cell Number by WST-8 Method
The WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-
5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt)
206 Vol. 29, No. 2Biol. Pharm. Bull. 29(2) 206—210 (2006)
DNA Damage Caused by Bisphenol A and Estradiol through Estrogenic
Ter uaki IWAMOTO,
GeneCare Research Institute Co., Ltd.; 200 Kajiwara, Kamakura, Kanagawa 247–0063, Japan:
Core Research for
Evolution Science and Technology, Japan Science and Technology Agency; Kawaguchi Center Building, 4–1–8 Honcho,
Kawaguchi, Saitama 332–0012, Japan: and
Department of Urology, St Marianna University School of Medicine; 2–16–1
Sugao, Miyamae, Kawasaki 216–8511, Japan. Received September 15, 2005; accepted November 15, 2005
Evidence exists that raises concern about genotoxic effects induced by estrogen: oxidative stress caused by
estrogen-derived oxidants, DNA adducts formed by estrogen metabolites and estrogen-induced chromosomal
aberration. Estrogen receptors (ER) participate in some of these genotoxic effects by estrogen. In this study, we
showed the effects of bisphenol A (BPA), an endocrine-disrupting chemical eliciting weak estrogenic activity, and
-estradiol (E2), on DNA damage in ER-positive MCF-7 cells by Comet assay. Higher concentrations of
BPA, more than 1000 times of E2, were needed to induce the same levels of effects by E2. Immunoﬂuorescence
microscopy showed that
H2AX, an early marker of DNA breaks, increased after treatment with E2 or BPA in
H2AX foci colocalized with Bloom helicase, which is considered to be responsible for the repair of
DNA damage after treatment with E2 or BPA. Interestingly, DNA damage was not as severe in ER-negative
MDA-MB-231 cells as in MCF-7 cells. The ER antagonist ICI182780 blocked E2 and BPA genotoxic effects on
MCF-7 cells. These results together suggest that BPA causes genotoxicity ER dependently in the same way as E2.
Key words bisphenol A; 17
-estradiol; endocrine-disrupting chemical; DNA damage; Bloom helicase
© 2006 Pharmaceutical Society of Japan∗ To whom correspondence should be addressed. e-mail: firstname.lastname@example.org
method was used for assessment of cell number. According
to the manufacturer’s protocol (Nacalai Tesque, Kyoto),
WST-8 solution was added to the culture, and then cells were
incubated for another 4 h. The absorbance was measured
at 450 nm by using a spectrophotometer (ARVO MX,
PerkinElmer, Boston, MA, U.S.A.) with a reference wave-
lengh of 620 nm.
Comet Assay To detect DNA double-strand breaks in a
single cell by using Comet assay, alkaline lysis and then alka-
line gel electrophoresis were used.
Brieﬂy, cells were incu-
bated with various concentrations of E2 or BPA up to 24 h.
The cells were treated with trypsin to detach cells from the
dish and from each other, and then they were suspended in
phosphate-buffered saline (PBS) and were mixed with a 10-
fold volume of 1% low-melting-point agarose (FMC Bio-
products, Rockland, ME, U.S.A.). Aliquots (75
l) of the cell
suspension were layered on a fully frosted glass slide (Mat-
sunami, Osaka) pre-coated with 1% agarose. The gel was
covered with a cover slip and was incubated at 4 °C for
20 min. The cover slips were removed and the slides were
immersed in a lysis solution containing 2.5 M NaCl, 100 mM
ethylenediamine-N,N,N,N-tetraacetic acid (EDTA) at pH
10, 10 mM Tris, 1% lauryl sarcosinate and 1% Triton X-100
for 1 h at 4 °C. The slides were transferred to an alkali solu-
tion (300 mM NaOH, 1 mM EDTA, pH 13) at 4 °C for an
additional 25 min, and then electrophoresis was done in the
fresh alkali electrophoresis solution (300 mM NaOH, 1 mM
EDTA, pH 13) at 20 V for 25 min at 4 °C. The cells were
neutralized with 400 mM Tris–HCl at pH 7.5 and were ﬁxed
in 70% ethanol for 10 min at room temperature. The gel was
dried, and the DNA was stained with SYBR Green (Trevigrn,
Gaitherburg, MD, U.S.A.). All processes were done under
dimmed light to avoid damage by UV. Comet formation of
cells was observed at 400 magniﬁcation by using a ﬂuores-
cence microscopy (Fluoview, Olympus, Tokyo) and the
Comet tail length (CTL) was measured for 30 cells (10 cells
from each of three slides). Statistical analysis of the CTL val-
ues between treated and control groups was done by using
Immunoﬂuorescence Microscopy Subnuclear localiza-
H2AX and BLM proteins was investigated by using
confocal immunoﬂuorescence microscopy (Fluoview, Olym-
pus, Tokyo). Cells were grown to subconﬂuence on a cham-
ber slide (BD Falcon, Bedford, MA, U.S.A.) in the presence
or absence of E2 or BPA. They were ﬁxed with 4% formalde-
hyde for 10 min at room temperature and were washed three
times with PBS containing 0.05% Tween 20. The cells were
permeabilized with 0.1% Triton X-100 in PBS for 5 min, and
then were blocked with 3% skim milk in PBS for 30 min at
room temperature. The following primary antibodies were
used: mouse anti-
H2AX monoclonal antibody (Upstate,
Charlottesville, VA, U.S.A.) and rabbit anti-BLM polyclonal
antibody (Abcam, Cambridge, U.K.). Anti-mouse IgG la-
beled with Alexa Fluor 594 and anti-rabbit IgG labeled with
Alexa Fluor 488 (both from Molecular Probes, Eugene, OR)
were used as secondary antibodies. Cells were mounted
under cover slips on glass slides in DAKO ﬂuorescent
mounting medium (DAKO, Glostrup, Denmark). The de-
tailed procedure was described previously.
Genotoxic Effect of E2 and BPA DNA damage in
MCF-7 cells was assessed by measuring the CTL. E2 was
added at concentrations from 10
M, which induced
Comet formation after 3 h, after which the CTL increased
dose dependently (Table 1). Because physiological concen-
trations of E2 in the blood are between pg/ml and ng/ml
the signiﬁcant effective dose of E2 that
induced Comet formation is assumed to be within physiolog-
ical concentrations. A similar effect to induce Comet forma-
tion was observed with BPA, but the concentrations needed
to induce similar levels of CTL were much higher (Table 1).
BPA is generally used in the manufacture of polycarbonate,
and elicits weak estrogenic activity
: the activity of BPA at
M is almost equivalent to the activity of
E2 at concentration 10
Thus, the observation of the
effective concentrations of BPA (10
M) is consistent
with the difference in ER afﬁnity previously reported.
Notably, effective concentrations of BPA that induce geno-
toxicity did not affect the viability of MCF-7 cells, indicating
that genotoxicity was not due to cytotoxicity of BPA. Figure
1 shows typical Comet formations elicited by E2 and BPA.
MCF-7 cells without treatment did not show Comet tail. The
Comet assay was done at 1, 3 and 24 h after treatment with
M E2 or 10
M BPA, and a signiﬁcant increase in CTL
was detectable at 3 h after treatment with E2 and at 1 h after
treatment with BPA (Table 2). The increased levels of CTL
by treatment with E2 or BPA were remained after the 24-h
treatment (Table 2). However, the ER-negative MDA-MB-
231 cells were less sensitive to DNA damage by E2 or BPA:
M E2 slightly increased CTL in MDA-MB-231 cells at
3 h, but did not affect CTL at 24 h after treatment (Table 3),
nor at ten times higher concentration of E2 at 24 h after treat-
ment (data not shown). BPA at concentration 10
increased CTL in MDA-MB-231 cells at 3 and 24 h after
treatment, but its effect was much weaker compared with
MCF-7 cells (Table 2). These results indicate that ER-posi-
tive MCF-7 cells have much higher susceptibility than ER-
negative MDA-MB-231 cells and support the idea that ER
participates in genotoxicity by E2 or BPA.
Further Evidence of ER Participation in Genotoxicity
by E2 or BPA To investigate further if ER participate in
DNA damage by E2 or BPA, the effect of ER antagonist
ICI182780 on the effect of E2 or BPA to induce genotoxicity
was studied. MCF-7 cells were pre-treated with 10
February 2006 207
Table1.Effect of E2 and BPA on Comet Formation in MCF-7 Cells after
Concentration Cell Comet
(M) number (%)
tail length (
Control (0.1% ethanol) 100 12.802.00
Control (0.1% ethanol) 100 11.542.78
a) Percentage of control as assessed by WST-8 assay. b) MeanS.D., 30 cells.
ICI182780 for 1 h and then were treated with 10
M BPA for 3 h. The pre-treatment with
ICI182780 antagonized the genotoxic effect by E2 or BPA,
and an increase in CTL by E2 or BPA was not observed in
the presence of ICI182780 (Table 4). These results also
strongly support the idea that ER participate in the genotoxic
effect by E2 or BPA.
Replication Stress after Treatment with E2 or BPA
Histone H2AX has been implicated in the maintenance of
genomic stability by participating in the repair of DNA dam-
H2AX is phosphorylated to
H2AX, which then
forms foci in response to DNA double-strand breaks result-
ing in replication arrest in cells.
H2AX foci are formed
rapidly in response to DNA damage.
In this study, 10—20
H2AX foci appeared in the MCF-7 nucleus at 3 h after
treatment with E2 or BPA (Fig. 2). Fluorescence signals of
208 Vol. 29, No. 2
Fig. 1. Comet Formation Induced by E2 and BPA
MCF-7 cells treated with 10
M E2 or 10
M BPA for 3 h show typical tails.
Fig. 2. Localization of BLM with
H2AX after DNA Damage Induced by E2 and BPA
MCF-7 cells treated with 10
E2 or 10
BPA for 3 h, were simultaneously stained with anti-BLM and anti-
H2AX antibodies. BLM and
H2AX were stained
green and red, respectively. Colocalization of BLM and
H2AX were stained yellow.
H2AX foci intensiﬁed as the concentration of E2 increased,
but the foci remained indistinct in untreated MCF-7 cells.
These results are consistent with the results by Comet assay
that E2 and BPA induced DNA double-strand breaks.
H2AX foci were indistinct in ER-negative MDA-MB-231
cells treated with E2 or BPA (data not shown). By using anti-
BLM antibody BLM partially colocalized with
suggesting that part of BLM was associated with damaged
DNA sites. BPA treatment produced a similar result. Notably,
large foci colocalized with BLM and
H2AX, which were
shown by yellow staining and were obvious in cells treated
with E2 or BPA but not in untreated control cells. These re-
sults suggest that DNA double-strand breaks caused by E2 or
BPA stimulate formation of
H2AX foci and accumulate
BLM in the foci.
Although genotoxic effects of E2 are ER-dependent, the
sensitivity of ER-negative cells to E2 effects is inconsistent:
E2 metabolites are genotoxic in ER-negative MDA-MB-231
and physiological doses of E2 induce oxidative DNA
damage in MDA-MB-231 cells.
However, E2 induces mi-
cronuclei formation in ER-positive tumor cells from breast
and ovary, but not in ER-negative cells.
In this study, we
showed: 1) E2 or BPA produced statistically signiﬁcant geno-
toxic effects in ER-positive MCF-7 cells, but much less
genotoxic effects, if any, in MDA-MB-231 cells, 2) ER an-
tagonist ICI182780 weakened E2 and BPA genotoxic effects
in MCF-7 cells and 3) E2 stimulated formation of
foci colocalized with BLM. These results strongly support
the idea that genotoxic effects of E2 are mediated by ER. Our
results and conclusion are supported by the evidence that es-
trogen-induced DNA damage is inhibited by the estrogen re-
ceptor antagonist tamoxifen.
Also, E2 downregulates
detoxifying enzyme activity ER dependently.
and Stopper et al.
suggest DNA damage may be due
to an overriding checkpoint under ER-dependent cell prolif-
eration induced by hormone.
We conclusively showed, we believe for the ﬁrst time,
that BPA had essentially similar effects as E2 to cause DNA
damage depending on ER in MCF-7 cells, although higher
concentrations of BPA were needed. Lee et al.
genotoxicity of BPA by using Comet assay in mouse lym-
phoma cells, but they concluded that the effect was false
positive due to cell death, because effective doses of
M BPA were cytotoxic. In our study, BPA
at doses of 10
M were genotoxic in MCF-7 cells but
were not cytotoxic, excluding the possibility that genotoxic-
ity was due to cytotoxicity. Interestingly, BPA administration
reduces the activity of detoxifying enzymes, including super-
oxide dismutase, glutathine peroxidase and catalase, in
consistent with E2 markedly suppressing en-
zymes to metabolize oxidative products in MCF-7 cells.
cause DNA damage, BPA, an endocrine-disrupting chemical,
needed higher concentrations than the levels of BPA detected
in various kinds of human biological ﬂuids contaminated
with this compound.
Studies of the biological fate of BPA
by using animal tests have shown that most radioactivity is
February 2006 209
Table2.Time-Course Analysis of DNA Damage Induced by E2 and BPA in MCF-7 Cells
1h 3h 24h
Cell Comet tail Cell Comet tail Cell Comet tail
Control (0.1% ethanol) 100 14.707.32 100 11.302.95 100 12.992.33
100 17.696.60 101 41.7914.40** 106 37.3312.87**
97 29.7734.46* 96 37.6711.31** 109 43.9119.16**
a) Percentage of control as assessed by WST-8 assay. b) MeanS.D., 30 cells. ∗ p0.05, ∗∗ p0.01.
Table3.Effect of E2 and BPA on Comet Formation in ER-Negative MDA-MB-231 Cells
Treatment Concentration (M)
Cell number (%)
Comet tail length (
Cell number (%)
Comet tail length (
Control (0.1% ethanol) 100 7.032.49 100 9.152.15
99 9.342.44* 92 9.992.26
96 8.973.69* 94 10.632.22*
a) Percentage of control as assessed by WST-8 assay. b) MeanS.D., 30 cells. ∗ p0.05.
Table4.DNA Damage Induced by E2 and BPA in the Presence of
ICI182780 in MCF-7 Cells
Concentration Cell Comet tail
(M) number (%)
Control (0.1% ethanol) 100 3.263.10
Control (0.1% ethanol) 100 9.482.51
Control (0.1% ethanol) 100 11.123.31
a) Percentage of control as assessed by WST-8 assay. b) MeanS.D., 30 cells.
ICI/E2: pre-treatment with ICI182780 followed by E2 treatment. ICI/BPA: pre-treat-
ment with ICI182780 followed by BPA treatment. ∗∗ p0.01.
recovered in urine and feces at about 7 d after administration
C- BPA in rats.
These observations suggest BPA is
not likely to accumulate in the body. Overall, genotoxicity of
BPA may not be serious.
H2AX is rapidly phosphorylated to
H2AX, which forms
foci at the sites of DNA double-strand breaks. BLM at
H2AX sites and is considered to interact with the DNA
damage response protein 53BP1 and to participate in DNA
Our preliminary study (Iso et al., unpub-
lished data) showed that damage induced by E2 and BPA was
restored reversibly in MCF-7 cells: when cells were cultured
for 24 h in the absence of E2 or BPA after 24 h-treatment of
cells with E2 or BPA, few Comet forming cells were ob-
served. Thus, the integrity of DNA structure may be recov-
ered, probably by a system to stabilize the genome, including
DNA repair enzymes such as BLM.
To sum up, our ﬁndings contribute to show genotoxicity of
estrogenic agents, including BPA, ER dependently, and
whether genomic instability induced by estrogenic agents can
be overcome in a DNA repair system will be of further inter-
Acknowledgements We thank Dr. Masanobu Sugimoto,
GeneCare Research Institute, for his discussion and help in
reviewing this manuscript. This study was supported by the
Japan Science and Technology Agency, Japan.
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210 Vol. 29, No. 2