DNA Damage Caused by Bisphenol A and Estradiol through Estrogenic Activity

Article (PDF Available)inBiological & Pharmaceutical Bulletin 29(2):206-10 · March 2006with210 Reads
DOI: 10.1248/bpb.29.206 · Source: PubMed
Abstract
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.

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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.
1—3)
Estrogen causes DNA damage by estrogen-derived oxi-
dants,
4,5)
DNA adducts formed by estrogen metabolites
5,6)
and formation of micronuclei.
7,8)
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.
4,9)
Detoxifying enzyme activity
markedly decreases by treatment with 17
b
-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.
4)
Bisphenol A (BPA) was first shown to be estrogenic in
1938 in ovariectomized rats
10)
and later in MCF-7 human
breast cancer cell culture assay.
11)
BPA is an endocrine-dis-
rupting chemical and has a weak affinity for ER, estimated at
about 1/1000 of E2,
12)
and its additional estrogenic effects on
the hormonal homeostatic system has recently received much
attention.
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.
13)
At the sites of stalled replica-
tion forks, H2AX is phosphorylated to
g
H2AX, which forms
foci
14)
that appear immediately after DNA damage and re-
cruit proteins responsible for repair of DNA damage, includ-
ing Bloom helicase (BLM),
14,15)
the product of BLM that is
the causative gene of Bloom syndrome, an autosomal reces-
sive genetic disorder.
16,17)
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.
18,19)
BLM responds to DNA damage and accumulates at the site
of DNA double-strand breaks and physically interacts with
g
H2AX.
15)
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
colocalization of
g
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,
MO, U.S.A.).
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)
and 50
m
g/ml gentamycin (both from Sigma-Aldrich, St.
Louis, MO, U.S.A.) in a humidified atmosphere under 5%
CO
2
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
Activity
Takako ISO,
a,b
Takahide WATANABE,
a,b
Ter uaki IWAMOTO,
b,c
Akira SHIMAMOTO,
a
and
Yasuhiro F
URUICHI
*
,a,b
a
GeneCare Research Institute Co., Ltd.; 200 Kajiwara, Kamakura, Kanagawa 247–0063, Japan:
b
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
c
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
of 17
bb
-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
gg
H2AX, an early marker of DNA breaks, increased after treatment with E2 or BPA in
MCF-7 cells.
gg
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
b
-estradiol; endocrine-disrupting chemical; DNA damage; Bloom helicase
© 2006 Pharmaceutical Society of JapanTo whom correspondence should be addressed. e-mail: furuichi@genecare.co.jp
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.
20)
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
m
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
Dunnett’s test.
Immunofluorescence Microscopy Subnuclear localiza-
tion of
g
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-
g
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.
21)
RESULTS
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
9
to 10
7
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
(10
10
to 10
8
M),
22)
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
23)
: the activity of BPA at
concentration 10
6
M is almost equivalent to the activity of
E2 at concentration 10
8
M.
11,24)
Thus, the observation of the
effective concentrations of BPA (10
6
to 10
4
M) is consistent
with the difference in ER affinity previously reported.
11,24)
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
10
7
M E2 or 10
4
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:
10
7
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
4
M slightly
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
6
M
February 2006 207
Table1.Effect of E2 and BPA on Comet Formation in MCF-7 Cells after
3h Treatment
Treatment
Concentration Cell Comet
(M) number (%)
a)
tail length (
m
m)
b)
Control (0.1% ethanol) 100 12.802.00
E2 10
11
105 15.153.76
10
9
103 22.619.89**
10
7
101 28.219.70**
Control (0.1% ethanol) 100 11.542.78
BPA 10
8
104 15.946.95
10
6
93 20.738.16**
10
4
96 29.4711.69**
a) Percentage of control as assessed by WST-8 assay. b) MeanS.D., 30 cells.
∗∗ p0.01.
ICI182780 for 1 h and then were treated with 10
7
M E2
or 10
6
and 10
4
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-
age.
14,15)
H2AX is phosphorylated to
g
H2AX, which then
forms foci in response to DNA double-strand breaks result-
ing in replication arrest in cells.
13)
g
H2AX foci are formed
rapidly in response to DNA damage.
14)
In this study, 10—20
g
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
7
M E2 or 10
4
M BPA for 3 h show typical tails.
Fig. 2. Localization of BLM with
g
H2AX after DNA Damage Induced by E2 and BPA
MCF-7 cells treated with 10
10
and 10
7
M
E2 or 10
6
M
BPA for 3 h, were simultaneously stained with anti-BLM and anti-
g
H2AX antibodies. BLM and
g
H2AX were stained
green and red, respectively. Colocalization of BLM and
g
H2AX were stained yellow.
g
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.
g
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
g
H2AX foci,
suggesting that part of BLM was associated with damaged
DNA sites. BPA treatment produced a similar result. Notably,
large foci colocalized with BLM and
g
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
g
H2AX foci and accumulate
BLM in the foci.
DISCUSSION
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
cells
5)
and physiological doses of E2 induce oxidative DNA
damage in MDA-MB-231 cells.
9)
However, E2 induces mi-
cronuclei formation in ER-positive tumor cells from breast
and ovary, but not in ER-negative cells.
8,25)
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
g
H2AX
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.
8,9,25)
Also, E2 downregulates
detoxifying enzyme activity ER dependently.
4)
And, Fischer
et al.
8)
and Stopper et al.
25)
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.
26)
observed
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
410
6
—410
4
M BPA were cytotoxic. In our study, BPA
at doses of 10
6
—10
4
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
mouse tissue,
27)
consistent with E2 markedly suppressing en-
zymes to metabolize oxidative products in MCF-7 cells.
4)
To
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.
28)
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
Treatment
Concentration
1h 3h 24h
(M)
Cell Comet tail Cell Comet tail Cell Comet tail
number (%)
a)
length (
m
m)
b)
number (%)
a)
length (
m
m)
b)
number (%)
a)
length (
m
m)
b)
Control (0.1% ethanol) 100 14.707.32 100 11.302.95 100 12.992.33
E2 10
7
100 17.696.60 101 41.7914.40** 106 37.3312.87**
BPA 10
4
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)
3h 24h
Cell number (%)
a)
Comet tail length (
m
m)
b)
Cell number (%)
a)
Comet tail length (
m
m)
b)
Control (0.1% ethanol) 100 7.032.49 100 9.152.15
E2 10
7
99 9.342.44* 92 9.992.26
BPA 10
4
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
Treatment
Concentration Cell Comet tail
(M) number (%)
a)
length (
m
m)
b)
Control (0.1% ethanol) 100 3.263.10
E2 10
7
101 25.7712.84**
ICI 10
6
100 6.212.39
ICI/E2 10
6
/10
7
90 6.522.51
Control (0.1% ethanol) 100 9.482.51
BPA 10
6
101 26.416.62**
ICI 10
6
96 10.332.57
ICI/BPA 10
6
/10
6
102 11.133.19
Control (0.1% ethanol) 100 11.123.31
BPA 10
4
96 27.426.74**
ICI 10
6
90 12.834.66
ICI/BPA 10
6
/10
4
92 11.465.58
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
of
14
C- BPA in rats.
29—31)
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
g
H2AX, which forms
foci at the sites of DNA double-strand breaks. BLM at
g
H2AX sites and is considered to interact with the DNA
damage response protein 53BP1 and to participate in DNA
repair processes.
15)
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-
est.
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
    • "It should be noted that the association may exist between DNA damage and disruption of hormone system activity. For example, it was found that BPA caused ER-dependent (estrogen receptordependent ) DNA damage by induction of strand-breaks in ER positive MCF-7 cells (Iso et al., 2006). In this study, using comet assay, we have observed that bisphenols studied caused an increase in DNA in the comet tail, the parameter that correlated with alkali-labile sites and DNA strandbreaks . "
    [Show abstract] [Hide abstract] ABSTRACT: In the present study, we have investigated DNA-damaging potential of BPA and its analogs, i.e. bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF) in human peripheral blood mononuclear cells (PBMCs) using the alkaline and neutral versions of the comet assay, which allowed to evaluate DNA single strand-breaks (SSBs) and double strandbreaks (DSBs). The use of the alkaline version of comet assay made also possible to analyze the kinetics of DNA repair in PBMCs after exposure of the cells to BPA or its analogs. We have observed an increase in DNA damage in PBMCs treated with BPA or its analogs in the concentrations ranging from 0.01 to 10 μg/ml after 1 and 4 h incubation. It was noted that bisphenols studied caused DNA damage mainly via SSBs, while DNA fragmentation via double DSBs was low. The strongest changes in DNA damage were provoked by BPA and particularly BPAF, which were capable of inducing single strand-breaks even at 0.01 μg/ml, while BPS caused the lowest changes only at 10 μg/ml. We have also observed that PBMCs significantly repaired bisphenols-induced DNA damage but they were unable (excluding cells treated with BPS) to repair totally DNA breaks.
    Article · Dec 2016
    • "In which, catalase may reflect inability of liver mitochondria and microsomes to eliminate hydrogen peroxide produced after exposure to BPA [6] and GST protects cells or tissue against oxidative stress and damage by detoxifying various toxic substrates derived from cellular oxidative processes [51]. Previous study strongly suggested that DNA damage induced by estrogen is dependent on estrogen receptors (ER) [22]. Regarding to the effect of both examined doses of BPA on the expression of estrogen receptor (ER), our study revealed that there were mild to strong immunoreactions of ER in ovary, uterus and liver of both dam (groups 2 & 3) and F1 female groups (groups 5 & 6) in which the expression was more clear in F1 groups comparing with dam rats (Figs. "
    [Show abstract] [Hide abstract] ABSTRACT: Environmental xenoestrogen contaminant bisphenol A (BPA), widely used as a monomer in the manufacture of epoxy, polycarbonate plastics and polystyrene resins. However, exposure to BPA has raised concerns, and the negative impacts of its exposure on reproduction have been controversial. The purpose of this work was directed to assess the potential adverse effects of BPA on dam rats and their first generation females in a comparative toxicological study. Fifteen pregnant female rats were classified into three equal groups; first group was orally administered corn oil and served as control (group1), second and third groups were orally administered BPA at dose levels of 50 and 200 mg/kg b.wt respectively (groups 2 & 3). The administration was carried out daily from zero day through the gestation period (21 days) until the last day of the lactation period (21days) and was extended after weaning for three months, in which female off springs of first generation (F1) of the three groups of dams were classified into; F1control group (group 4), F1 group treated with low dose of BPA (group 5) and F1 group treated with high dose of BPA (group 6) which continued in daily oral administration of BPA at the same previously mentioned doses for three months. The results elucidated a clear marked DNA fragmentation in the ovary of both dam and F1 female groups especially at higher examined concentration. Also, the data demonstrated a significant increase in the serum levels of GGT, ALP, glucose, total cholesterol, triglycerides, LDH and also in the serum level of estrogen hormone. Meanwhile, our study recorded a significant decrease in total protein, catalase, GST, HDL and FSH hormone in both treated dam and F1 female groups which was more significantly decreased in F1 female rats. Moreover, our experiment illustrated up-regulation in the immunoexpression of ERβ in ovary, uterus and liver of dam and F1 female groups. The histopathological investigation showed degeneration in the epithelial lining of ovarian follicles, submucosal leukocytic infiltration and increase in vaculation of hepatic cells with proliferation of kupffer cells. The lesions were more sever in groups 3 & 6 of both dam and their F1 females. Our data speculated that long- term exposure to BPA at 50 and 200 mg/kg.b.wt. depicted total genomic damage, significant alterations in liver enzymes, lipid profile, antioxidant enzymes and reproductive hormones with up-regulation in the expression of ERβ which were more significantly perturbed in group 3 and group 6 of both dam and F1 female rats.
    Full-text · Article · Aug 2016
    • "Together these exposures can increase the damage load of genomic DNA and have implications for genomic stability and the development and progression of disease. While, the estrogenic properties of BPA are one source of concern, BPA exposure has been shown to cause DNA damage independent of its estrogenic properties over a range of doses from environmentally relevant nanomolar to high micromolar, in both in vitro and in vivo models (Iso et al. 2006; Nishimura et al. 2014; Tiwari et al. 2012; Wu et al. 2013; Yang et al. 2009). Yet, how the DNA damage response and repair pathways address BPA exposure has not been extensively investigated. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Exposure to bisphenol A (BPA) has been reported to alter global gene expression, induce epigenetic modifications, and interfere with complex regulatory networks of cells. In addition to these reprogramming events, we have demonstrated that BPA exposure generates reactive oxygen species and promotes cellular survival when co-exposed with the oxidizing agent potassium bromate (KBrO3). Objectives: To determine the cellular microenvironment changes induced by co-exposure of BPA and KBrO3 versus either agent alone. Methods: Ku70-deficient cells were exposed to 150 µM BPA, 20 mM KBrO3, or co-exposed with both agents. 4 and 24 h post-damage initiation by KBrO3, with BPA only samples timed to coincide with these designated time points, we performed whole genome microarray analysis and evaluated chromatin structure, DNA lesion load, glutathione content, and intracellular pH. Results: We found that 4 h post-damage initiation BPA exposure and co-exposure transiently condensed chromatin compared to untreated and KBrO3 alone treated cells and reduced the transcription of DNA repair proteins. At this time point, BPA exposure and co-exposure also reduced the change intracellular pH observed after KBrO3 alone. 24 h post-damage initiation, BPA exposed cells showed less condensed chromatin than KBrO3 alone; the intracellular pH of the co-exposed treatment was reduced significantly compared to untreated and KBrO3 treated cells; and significant up-regulation of DNA repair proteins was also observed after co-exposure. Conclusion: These results support the induction of an adaptive response by BPA co-exposure that alters the microcellular environment and modulates DNA repair. Further work is required to determine whether BPA induces similar DNA lesions in vivo at environmentally relevant doses; however, in the Ku-deficient mouse embryonic fibroblasts, exposure to a high dose of BPA was associated with changes in the cellular microenvironment that may promote survival.
    Full-text · Article · Apr 2016
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