Toxicology Letters 172 (2007) 146–158

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces oxidative

stress, DNA strand breaks, and poly(ADP-ribose) polymerase-1
activation in human breast carcinoma cell lines
Po-Hsiung Lin a,∗ , Chia-Hua Lin a , Chuan-Chen Huang a ,
Ming-Chien Chuang a , Pinpin Lin b
a Department of Environmental Engineering, National Chung Hsing University, Taichung 402, Taiwan
b Division of Environmental Health and Occupational Medicine, National Health Research Institutes, Miaoli, Taiwan
Received 3 April 2007; received in revised form 6 June 2007; accepted 6 June 2007
Available online 16 June 2007

Abstract
The formation of reactive oxygen species (ROS) plays a critical role in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced
toxicities in mammalian cells since it promotes cell proliferation, growth arrest, and apoptosis. In this study, we investigated whether
TCDD induces oxidative stress and DNA damage in human ER␣(+)/MCF-7 and ER␣(−)/MDA-MB-231 breast cancer cells and
whether this is accompanied by the initiation of DNA repair events. Results indicated that viability of MCF-7 and MDA-MB-231
cells was concentration- and time-dependently reduced by TCDD. Further, we observed significant increases in ROS formation
and decreases in intracellular glutathione (GSH) in these two cell lines after TCDD treatment. Overall, the extent of cell death was
greater in MCF-7 cells than in MDA-MB-231 cells whereas the magnitude of ROS formation and GSH depletion was greater in
MDA-MB-231 cells than in MCF-7 cells. In addition, we observed that at non-cytotoxic concentration (1 nM for 5 h), TCDD induced
decreases in intracellular NAD(P)H and NAD+ in MCF-7 and MDA-MB-231 cells. These decreases were completely blocked by
three types of poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors. The catalytic activation of PARP-1 in cells treated with TCDD
was confirmed by detection of the presence of polymers of ADP-ribose-modified PARP-1 using Western blotting. Moreover, we
demonstrated increases in the number of DNA strand breaks in MCF-7 and MDA-MB-231 cells exposed to TCDD as measured
by the single-cell gel electrophoresis (Comet) assay. Overall, this evidence confirms that TCDD induces decreases in intracellular
NAD(P)H and NAD+ through PARP-1 activation mediated by formation of DNA strand breaks. In addition, we demonstrated that
the extent of oxidative stress and DNA damage was greater in MDA-MB-231 cells than in MCF-7 cells, with a strong correlation
to estrogen receptor (ER) status. In conclusions, our findings add further support to the theme that ROS formation is a significant
determinant factor in mediating the induction of oxidative DNA damage and repair in human breast cancer cells exposed to TCDD
and that the TCDD-induced oxidative stress and DNA damage may, in part, contribute to TCDD-induced carcinogenesis.
© 2007 Elsevier Ireland Ltd. All rights reserved.

Keywords: Oxidative stress; DNA damage; GSH; Dioxin; Poly(ADP-ribose) polymerase-1

Abbreviations: 3-AB, 3-aminobenzamide; 4-OHT, 4-hydroxyltamoxifen; BA, benzamide; DCFH-DA, 2 ,7 -dichlorofluorescin diacetate;
DMSO, dimethyl sulfoxide; ER␣, estrogen receptor ␣; ␣-NF, ␣-naphthflavone; PARP-1, poly(ADP-ribose) polymerase-1; ROS, reactive oxygen
species; SRB, sulforhodamine B; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin
∗ Corresponding author. Tel.: +886 4 22840441x515; fax: +886 4 22858970.

E-mail address: pohsiunglin@yahoo.com (P.-H. Lin).

0378-4274/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2007.06.003
 P.-H. Lin et al. / Toxicology Letters 172 (2007) 146–158
1. Introduction
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is
ubiquitous in the environment due to its resistance to
degradation, tending to accumulate in environment, in
animals, and even in human tissues (Bertazzi et al., 2001;
Baccarelli et al., 2005; Chen et al., 2003; Domingo et
al., 2002; Lee et al., 2006). Prolong exposure to TCDD
may result in a wide variety of adverse health effects
in experimental animals and in humans, including
immunotoxicity, neurotoxicity, hepatotoxicity, teroto-
genesis, and carcinogenesis (Alaluusua et al., 2004;
Baccarelli et al., 2005; Eskenazi et al., 2005; Warner et
al., 2002; Wyde et al., 2002). Numerous studies have
been conducted to uncover the mechanism by which
TCDD exerts its carcinogenic effects in mammals.
It is thought that the induction of the aryl hydrocar-
bon receptor (AhR) signal transduction pathway by
TCDD followed by altered gene expression, oxidative
stress, and tumor promotion may be responsible for
TCDD-induced carcinogenesis (Poland and Glover,
1979; Mimura and Fujii-Kuriyama, 2003; Wassom et
al., 1977). Over-expression of CYP subfamily may
generate oxygen radicals by futile cycling (Parke et
al., 1991). Interaction of TCDD with AhR and the
subsequent activation of the cytochrome P450 (CYP)
1A and 1B subfamily contribute significantly to the
induction of reactive oxygen species (ROS) formation
and liver tumor promotion (Chen et al., 2004; Lin et al.,
2004; Moennikes et al., 2004; Viluksela et al., 2000).
However, up to date, the mechanisms underlying the
carcinogenicity of TCDD still remain unclear.
Evidence for mutagenicity of TCDD in bacterial
mutagenicity assay failed to demonstrate mutagenic
activity of TCDD (Giri, 1986; Huff et al., 1991; Seiler,
1973). However, circumstantial evidence suggests that
TCDD induces DNA damage and chromosome abnor-
malities in mammalian cells (Park et al., 1996; Iannuzzi
et al., 2004). It is known that oxidative stress is capable
of inducing oxidative DNA lesions to generate oxidized
bases, abasic sites as well as DNA single-strand breaks
(Breen and Murphy, 1995). TCDD has been shown to
induce CYP1A1 as well as an elevation in the excretion
rate of 8-oxoguanine, the major repair product of 8-
oxo-2 -deoxyguanosine (8-oxo-dG) in mouse hepatoma
cells and in human HepG2 hepatoma cells (Park et al.,
1996; Knerr et al., 2006). In addition, increased 8-oxo-
dG adducts has also been reported in rats chronically
exposed to TCDD (Wyde et al., 2001). Exposure to
TCDD also resulted in the production of ROS, lipid
peroxidation, and DNA damage in brain tissue of mice
and rats ingested TCDD (Hassoun et al., 2004). This
147
evidence suggests that induction of CYP enzymes to
leak oxidants and the subsequent formation of oxidative
DNA damage may contribute to TCDD-induced carcino-
genesis (Knerr et al., 2006). It is known that oxidative
DNA damage is repaired mainly via base excision repair
process. DNA single-strand breaks are introduced as
intermediates of this repair pathway (Krokan et al.,
1997). PARP-1 is an enzyme mediating the cellular
response to DNA single- or double-strand breaks and
is involved in early DNA damage recognition and base
excision repair (Ishizuka et al., 2003). Accumulation of
DNA strand breaks activates PARP-1 that catalyzes the
formation of polymers of poly(ADP-ribose) and NAD+
depletion. In addition to NAD+ and ATP depletion,
repair-mediated indirect DNA strand break formation
leads to NAD(P)H depletion through activation of PARP-
1 in mammalian cells (Lin et al., 2005; Nakamura et
al., 2003). However, there is little information regarding
the link between TCDD-induced ROS generation and
the subsequent induction of DNA damage and repair in
human breast cancer cells.
Breast cancer continues to be a major cause of cancer
incidence and mortality in women worldwide (American
Cancer Society, 2006). With only a small proportion of
all breast cancer etiology being attributed to hereditary
origins, other causative factors, including environmental
exposure to persistent organic pollutants, are postulated
to account for the high incidence rate of breast cancer
in women. New epidemiological evidence suggests that
individual serum TCDD is highly associated with breast
cancer incidence among women in the Seveso Women’s
Health Study cohort (Warner et al., 2002). In this study,
we examined the action of TCDD on human breast can-
cer cells and investigate the roles of TCDD in mediating
the ROS formation, glutathione (GSH) depletion, and
DNA damage and repair in human MCF-7 and MDA-
MB-231 breast cancer cells. We demonstrated that at
physiologically relevant concentrations (0.001–10 nM)
TCDD induced ROS generation and the subsequent
PARP-1 activation through formation of DNA strand
breaks in both MCF-7 and MDA-MB-231 cells. In addi-
tion, we provided evidence of the roles of estrogen
receptor ␣ (ER␣) in modulating the TCDD-induced ROS
formation in human breast cancer cells.
2. Materials and Methods
2.1. Cell culture and Chemicals
The human breast carcinoma MCF-7 and MDA-MB-231
cells were purchased from Culture Collection and Research
Center (Hsinchu, Taiwan). MCF-7 cells were grown in a
148 P.-H. Lin et al. / Toxicology Letters 172 (2007) 146–158
humidified atmosphere containing 5% CO2 in Dulbecco’s
Modified Eagle’s Medium (DMEM) (Sigma) supplemented
with 5% fetal bovine serum (Biological Industries), 1%
antibiotics (Gibco), and 0.6 ␮g/ml insulin at 37 ◦ C. MDA-
MB-231 cells were cultured in Leibovitz’s L-15 medium
supplemented with 10% fetal bovine serum (Biological Indus-
tries), 1% antibiotic–antimycotic, and 0.06 mg/ml insulin at
37 ◦ C. TCDD were purchased from Chem. Service Inc. Com-
pany (Pennsylvania). CCK-8 solution was purchased from
Dojindo Molecular Technology. 2 ,7 -Dichlorofluorescin diac-
etate (DCFH-DA) was purchased from Molecular Probes,
Invitrogen Detection Technology. Dimethyl sulfoxide (DMSO)
and PARP inhibitors, 3-aminobenzamide (3-AB), benzamide
(BA) and coumarin were purchased from Sigma Chemical
Company (St. Louis, MO). All other chemicals were purchased
from Sigma, Aldrich, or Fisher unless stated otherwise, and
used without further purification.
2.2. Analysis of cytotoxicity by the sulforhodamine B
(SRB) assay
The SRB assay was used in this experiment to determine
survival of cells following the exposure to TCDD (dissolved in
0.1% DMSO). MCF-7 and MDA-MB-231 cells were seeded
in 96-well plates (8 × 103 cells/well). The total cell number
was measured by estimating the cellular proteins as previously
described (Skehan et al., 1990) with modifications (Lin et al.,
2005). The samples were determined by a spectrophotometer
and compares to the values of controls. Visible absorbance was
recorded in a 96-well plate reader at 492 nm.
2.3. Analysis of ROS formation by the
2 ,7 -dichlorofluorescin diacetate (DCFH-DA) assay
The DCFH-DA assay was used in this experiment to deter-
mine the formation of ROS in cells following the exposure
to TCDD as previously described by Sasaki et al. (1997).
MCF-7 and MDA-MB-231 cells were seeded in black 96-
well plates (8 × 103 cells/well). The media was discarded from
the plates and replaced with Earle’s Balanced Salt Solution
containing 10 ␮M DCFH-DA. TCDD was added to the wells
and ROS generation relative to controls were determined peri-
odically (30 min) by a fluorescent plate reader (λex = 485 nm,
λem = 535 nm). When necessary, specific AhR inhibitors, ␣-
naphthflavone (␣-NF) (0.15 ␮M), and an ER␣ inhibitor,
4-hydroxyltamoxifen (4-OHT) (100 nM), were applied 2 h
prior to the treatment and kept in the medium during TCDD
exposure until the cells were analyzed. H2 O2 (20 mM) was used
as a positive control in this assay (induces a 15- and 18-fold
increase in ROS formation in MCF-7 and MDA-MB-231 cells
over control after 2 h of exposure).
2.4. Analysis of GSH by the o-phthalaldehyde assay
Total GSH content was measured using the method as pre-
viously described by Siraki et al. (2004) with modifications.
Cells (seeded in the 96-well black plates) were treated with
TCDD for 0.5 and 2 h and 10 ␮l of 62.5% trichloroacetic
acid were then added to the medium. After 10 min, 40 ␮l
of 1 M sodium phosphate (pH 7) was added to each well
and the reaction was carried out for 20 min. One hundred
micro-liters of 0.16 M sodium phosphate solution contain-
ing 37.5 mM o-phthalaldehyde were added to each well.
The fluorescence was recorded (λex = 355 nm excitation and
λem = 460 nm) by a fluorometer after 30 min of reaction at
room temperature in the dark and was compared to the val-
ues of control. NEM (100 ␮M) was used as a positive control
in this assay (induces ∼50% decreases in GSH depletion in
MCF-7 and MDA-MB-231 cells over control after 30 min of
exposure).
2.5. Analysis of intracellular NAD(P)H by a
water-soluble tetrazolium salt
Intracellular NAD(P)H was assayed using a commercially
available assay as previously described by Nakamura et al.
(2003) with modifications (Lin et al., 2005). A water-soluble
tetrazolium salt was used to monitor the amount of NAD(P)H
through its reduction to a yellow colored formazan dye,
and determined periodically by a spectrophotometer. Cells
were seeded in 96-well plates (8 × 103 cells/well) and were
treated with TCDD for 2 and 5 h. After incubation, the reac-
tion medium was discarded and the wells were washed with
PBS. Then, 100 ␮l of fresh medium and 1/10 volume of
CCK-8 solution were added. Cells were further cultured for
up to 4 h and were examined periodically (30 min) by a
spectrophotometer. Visible absorbance was recorded in a 96-
well plate reader at 450 with 650 nm as a reference filter.
The decrease in the intracellular NAD(P)H was assessed by
comparing the absorbance of a well containing cells treated
with naphthalene quinonoids against the control, which was
treated with DMSO only. When necessary, specific PARP-1
inhibitors, including 3-AB (15 mM), BA (5 mM) and coumarin
(1.5 mM), were applied 2 h prior to the treatment and kept
in the medium during TCDD exposure until the cells were
analyzed.
2.6. Analysis of intracellular NAD+ by the enzyme
cycling assay
The cellular NAD+ level was determined by the enzyme
cycling assay (Nakamura et al., 2003) with modifications. In
brief, cells (7.5 × 105 cells/dish) were treated with TCDD for
5 h in 3.5-cm dishes. After washing with PBS, cells were
trypsinized, harvested, and transferred to microcentrifuge tubes
followed by centrifugation. The supernatants were removed,
and the pellets were resuspended in 100 mM potassium phos-
phate buffer containing 3% trichloroacetic acid. After 30 min
on ice, the samples were centrifuged at 12 000 × g. The
acid-soluble fractions were neutralized with 800 mM KOH
containing 200 mM Tris. Supernatants were mixed with a
reaction medium containing 0.09 mM WST-8, 3.75 ␮M 1-
 P.-H. Lin et al. / Toxicology Letters 172 (2007) 146–158 149

methoxy PMS, 15 U/ml alcohol dehydrogenase (Sigma) and 3. Results

120 mM ethanol in 100 mM potassium phosphate buffer. The
reactions were performed in 96-well plates and the plates 3.1. Induction of cytotoxicity by TCDD in MCF-7
were incubated at 37 ◦ C in the dark for 30 min. The decrease and MDA-MB-231 cells
in the intracellular NAD+ was assessed by comparing the
NAD+ content of well containing cells treated with TCDD
To determine if TCDD induces cytotoxic response
against that of a well with cells treated with DMSO (0.1%)
in human breast cancer cells, we incubated MCF-7 and
only.
MDA-MB-231 cells with TCDD for up to 72 h at con-
centrations ranging from 0.001 to 10 nM. Viable cell
2.7. Protein extraction and Western blot analysis number was determined by the SRB assay. After 2 and
5 h of exposure, no significant reduction in the num-
Levels of PARP and pADPr proteins were determined by
ber of viable cells was detected in both MCF-7 and
Western blot analysis (Vodenicharov et al., 2005). After the
MDA-MB-231 cells exposed to TCDD (Fig. 1). After
treatment, cells were washed three times with PBS, centrifuged
at 8000 × g for 5 min, and were soaked in liquid N2 . Cell 24 and 72 h of exposure, the total number of viable
pellets were thawed and lysed in 80 ␮l of protein extrac- cells in MCF-7 cells was significantly reduced by treat-
tion buffer (1 M Tris–HCl, pH 7.9, 3 M NaCl, 1% aprotinin, ment with TCDD in a concentration-dependent manner
2 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol) for (∼80% of control) (Fig. 1A). When MDA-MB-231 cells
30 min on ice and the extracts were centrifuged for 30 min at
10,000 × g. Protein concentration was measured with the Bio-
Rad protein assay kit (Bio-Rad, Hercules, CA). Protein were
loaded at 50 ␮g/lane and separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis, and blotted. Anti-PARP
and anti-pADPr monoclonal antibodies (Abcam, Cambridge,
UK) were used at a concentration of 1:1000. The secondary
antibodies, alkaline phosphatase-coupled anti-mouse antibody
were incubated at room temperature for 2 h at a concentra-
tion of 1:5000 dilutions, respectively. Immunoreactive bands
were visualized by incubating with chemiluminescence detec-
tion system (Amersham Life Science). The membranes were
also probed with anti-glyceraldehydes 3-phosphate dehydro-
genase (GAPDH) antibody to correct for differences in protein
loading.

2.8. Analysis of DNA strand breaks by single-cell gel

electrophoresis (Comet) assay

DNA strand breaks were quantified by the Comet assay

(Sing et al., 1988). After exposure, cells were mixed with
1.2% low-melting agarose and added to slides coated with
1% agarose. Cells were then denatured with lysis buffer (pH
13) overnight, subjected to electrophoresis at 25 V/300 mA for
25 min, and stained with YoYo-1. The tail moment of 50 ran-
domly selected cells was analyzed from each slide by using
Comet Scoring software.

2.9. Statistical analysis

All data are expressed as mean ± standard deviation. The

significance of differences in the results was evaluated with
ANOVA, followed by Dunnett’s multiple comparison tests.
All data are expressed as mean ± standard deviation. The
significance of differences in the results was evaluated with
one-way ANOVA, followed by Dunnett’s multiple comparison
tests.
Fig. 1. Induction of cytotoxicity in human (A) MCF-7 and (B) MDA-
MB-231 breast cancer cells treated with TCDD (0.001–10 nM) for up
to 72 h. Data represents the mean ± standard deviation of three deter-
minations. The horizontal dashed lines represent the standard deviation
for the control. (*), (**), (***) indicates statistically significant differ-
ence from control [DMSO] (p < 0.05), (p < 0.005), (p < 0.001).
150 P.-H. Lin et al. / Toxicology Letters 172 (2007) 146–158

were treated with TCDD for 24 h, a significant increase in

cytotoxic response was detected at concentrations 1 nM
or above (∼85% of control) (Fig. 1B). Similarly, signif-
icant increases in cytotoxic response were observed in
MDA-MB-231 cells after 72 h of exposure to TCDD at
concentrations ranging from 0.1 to 10 nM (∼85–90% of
control) (Fig. 1B). These results indicated that TCDD
induced concentration- and time-dependent increases in
cytotoxic response in human breast cancer cells and that
the degree of cytotoxic response induced by TCDD was
greater in MCF-7 cells than in MDA-MB-231 cells.

3.2. Induction of ROS by TCDD in MCF-7 and

MDA-MB-231 cells

To examine the differences in the induction of ROS

formation by TCDD, MCF-7 and MDA-MB-231 cells
were exposed to TCDD for up to 72 h at concentrations
ranging from 0.001 to 10 nM. The formation of ROS was
determined by the DCFH-DA assay. Results indicated
that TCDD induce ∼1.1–1.9-fold increases in ROS for-
mation in MCF-7 cells after 2 h of exposure (Fig. 2A)
whereas no significant increases in ROS formation was
observed after 5, 24, and 72 h of exposure (Fig. 2A). In
contrast, the level of ROS formation in MDA-MB-231
cells was significantly induced by treatment with TCDD
for 2–72 h (∼1.5–4.3-fold of control) (Fig. 2B). Over-
all, the extent of ROS formation induced by TCDD was
greater in MDA-MB-231 cells than in MCF-7 cells.
3.3. Effects of the AhR inhibitors on the formation
of ROS in MCF-7 and MDA-MB-231 cells exposed
to TCDD
To determine the effects of AhR status on the induc-
tion of ROS formation by TCDD in cells, we incubated
MCF-7 and MDA-MB-231 cells with TCDD in the
presence of an AhR inhibitor (␣-NF). After 2–72 h
of exposure, TCDD (10 nM) induce ∼1.1–4.3-fold
increases in ROS formation in MCF-7 and MDA-MB-
231 cells. Co-treatment of ␣-NF almost completely
blocked the TCDD-induced ROS formation in both
MCF-7 and MDA-MB-231 cells (Fig. 3A and B).
Overall, this evidence suggests that the status of AhR
modulates TCDD-induced ROS formation in human
breast cancer cells.
3.4. Induction of GSH depletion by TCDD in
MCF-7 and MDA-MB-231 cells
Induction of GSH depletion and inhibition of GSH
peroxidase activity by TCDD have been reported in
Fig. 2. Induction of ROS formation in human (A) MCF-7 and (B)
MDA-MB-231 breast cancer cells treated with TCDD (0.001–10 nM)
for up to 72 h. Data represents the mean ± standard deviation of
three determinations. The horizontal dashed lines represent the stan-
dard deviation for the control. (*), (**), (***) indicates statistically
significant difference from control [DMSO] (p < 0.05), (p < 0.005),
(p < 0.001).
mammalian system (Collier et al., 2006; Shen et al.,
2005; Hilscherova et al., 2003; Senft et al., 2002; Stohs,
1990; Wahba et al., 1989). To investigate the roles of
intracellular GSH in TCDD-induced toxicity in human
breast cancer cells, MCF-7 and MDA-MB-231 cells
were exposed to TCDD for up to 5 h at concentrations
ranging from 0.001 to 10 nM and the total GSH content
was determined by the o-phthalaldehyde assay. After 0.5
and 1 h of exposure, significant decreases in intracellular
GSH were detected in MCF-7 cells exposed to TCDD
at concentrations 0.001 nM or above (∼0.78–0.88-fold
of control) whereas no significant decreases in intracel-
lular GSH was observed after 1.5 and 2 h of exposure
(Fig. 4A). Similar pattern of the induction of GSH deple-
tion was observed in MDA-MB-231 cells treated with
TCDD (Fig. 4B). These results were in good agreement
 P.-H. Lin et al. / Toxicology Letters 172 (2007) 146–158
Fig. 3. Induction of ROS formation in human (A) MCF-7 and (B)
MDA-MB-231 breast cancer cells treated with TCDD (10 nM) in the
presence of AhR inhibitor (␣-NF) (0.15 ␮M) at 37 ◦ C for 2–72 h. Data
represents the mean ± standard deviation of three determinations. (*),
(**) indicates statistically significant difference from control [DMSO]
(p < 0.05), (p < 0.01).
with what has been observed in the induction of ROS
formation in TCDD-treated cells. Overall, we conclude
that TCDD is able to induce acute depletion of intra-
cellular GSH in MCF-7 and MDA-MB-231 cells and
that the extent of TCDD-induced GSH depletion is more
pronounced in MDA-MB-231 cells than in MCF-7 cells
after exposure.
3.5. Effects of PARP inhibitors on the decrease in
NAD(P)H induced by TCDD
In order to determine whether the reduction in
NAD(P)H is due to the depletion of NAD+ by PARP-1
activation, MCF-7 and MDA-MB-231 cells were treated
with non-cytotoxic concentrations of TCDD for 2 and
5 h, and were monitored for the decreases in NAD(P)H
151
Fig. 4. Induction of GSH depletion in human (A) MCF-7 and (B)
MDA-MB-231 breast cancer cells treated with TCDD (0.001–10 nM)
for up to 5 h. Data represents the mean ± standard deviation of
three determinations. The horizontal dashed lines represent the stan-
dard deviation for the control. (*), (**), (***) indicates statistically
significant difference from control [DMSO] (p < 0.05), (p < 0.005),
(p < 0.001).
levels. In some experiments, specific PARP inhibitors,
including 3-AB, BA, and coumarin, were included in
the medium (Nakamura et al., 2003; Shima et al., 1989;
Ying et al., 2001). Results indicated that significant
decreases in the amount of intracellular NAD(P)H in
MCF-7 (Fig. 5A) and MDA-MB-231 (Fig. 5B) cells
were detected in TCDD-treated cells when compared to
control (∼75–88% of control). The extent of TCDD-
induced decrease in intracellular NAD(P)H is more
obvious in MDA-MB-231 cells than in MCF-7 cells 2 h
after exposure. Further investigation indicated that all
PARP inhibitors blocked the TCDD-induced decrease
(∼75%) in the amount of intracellular NAD(P)H in
MCF-7 (Fig. 6A) and MDA-MB-231 cells (Fig. 6B).
This evidence suggests that decreases in intracellular
152 P.-H. Lin et al. / Toxicology Letters 172 (2007) 146–158
Fig. 5. Induction of intracellular NAD(P)H depletion in human (A)
MCF-7 and (B) MDA-MB-231 breast cancer cells treated with TCDD
(0.001–10 nM) for up to 5 h. Data represents the mean ± standard devi-
ation of three determinations. The horizontal dashed lines represent the
standard deviation for the control. (*), (**), (***) indicates statistically
significant difference from control [DMSO] (p < 0.05), (p < 0.005),
(p < 0.001).
NAD(P)H in TCDD-treated MCF-7 and MDA-MB-231
cells are mediated by PARP-1 activation.
3.6. Induction of intracellular NAD+ depletion by
TCDD in human MCF-7 and MDA-MB-231 cells
H2 O2 has been found to induce decreases in intracel-
lular NAD+ and increases in PARP activity in endothelial
cells (Junod et al., 1989; Skidmore et al., 1979). To
confirm the decrease in intracellular NAD(P)H in cells
induced by TCDD was due to the decrement in intra-
cellular NAD+ , MCF-7 and MDA-MB-231 cells were
exposed to TCDD for 5 h at concentration 1 nM. The
depletion of NAD+ in cells was determined by the
enzyme cycling assay after cell lysis. Results indicated
that after 5 h of exposure, TCDD induces decreases
Fig. 6. Induction of intracellular NAD(P)H depletion in human (A)
MCF-7 and (B) MDA-MB-231 breast cancer cells exposed to 1 nM
TCDD for 5 h in the absence or presence of PARP inhibitors, including
3-AB (15 mM), BA (5 mM), and coumarin (1.5 mM). Data represents
the mean ± standard deviation of three determinations. (**) and (#)
indicates statistically significant difference from control (p < 0.01) and
(p < 0.001).
in intracellular NAD+ in both MCF-7 (Fig. 7A) and
MDA-MB-231 (Fig. 7B) cells (∼80% of control).
This evidence suggests that decreases in intracellular
NAD(P)H induced by TCDD in MCF-7 and MDA-
MB-231 cells were primarily due to the decrement in
intracellular NAD+ pool mediated by PARP-1 activation.
3.7. Alteration of PARP and pADPr-modified PARP
protein expression induced by TCDD in MCF-7 and
MDA-MB-231 breast cancer cells
To confirm the TCDD-induced PARP-1 activation
in cells, we applied the polymer immunoblotting assay
using antibodies to pADPr and PARP to assess aver-
age PARP activation response in cells. The protein
 P.-H. Lin et al. / Toxicology Letters 172 (2007) 146–158
Fig. 7. Induction of intracellular NAD+ depletion in human (A) MCF-
7 and (B) MDA-MB-231 breast cancer cells treated with TCDD (1 nM)
for 5 h. Data represents the mean ± standard deviation of three deter-
minations. The horizontal dashed lines represent the standard deviation
for the control. (***) indicates statistically significant difference from
control [DMSO] (p < 0.001).
expression of PARP and its activated products, pADPr-
modified PARP were detected by immunoblotting with
anti-PARP and anti-pADPr monoclonal antibodies.
Results indicated that in TCDD-treated cells, strong sig-
nals for pADPr-modified proteins were seen as a smear
between 116 and 175 kDa in both MCF-7 (Fig. 8A)
and MDA-MB-231 cells (Fig. 8B). This evidence con-
firms that TCDD induces decreases in intracellular
NAD(P)H and NAD+ in MCF-7 and MDA-MB-231
cells are mediated by PARP-1 activation. In addition, we
noticed that increases in the protein expression of PARP
were observed in both MCF-7 and MDA-MB-231 cells
(Fig. 8B) exposed to TCDD (1 nM) for 5 h. These data
suggest that treatment of cells with TCDD may result in
a significant increase in PARP-1 in human breast cancer
cells.
153
Fig. 8. Western blot method to measure the pADPr, PARP-1 and
GADPH protein expression in human (A) MCF-7 and (B) MDA-MB-
231 breast cancer cells exposed to TCDD (1 nM) under physiological
condition at 37 ◦ C for 5 h.
3.8. Induction of DNA strand breaks by TCDD in
MCF-7 and MDA-MB-231 cells
To confirm the decreases in intracellular NAD(P)H
and NAD+ in TCDD-treated MCF-7 and MDA-MB-231
cells are mediated by PARP-1 activation through for-
mation of DNA strand breaks, we incubated MCF-7
and MDA-MB-231 cells with TCDD for 5 h at con-
centrations 1 and 10 nM. The DNA strand breaks were
determined by the Comet assay. After 5 h of exposure,
significant increases in DNA strand breaks were detected
in MCF-7 cells exposed to TCDD at concentrations 1 and
10 nM (∼150–250% of control) (Fig. 9A). Similar pat-
tern of the induction of DNA strand breaks was observed
in MDA-MB-231 cells treated with TCDD (Fig. 9B).
Overall, this evidence suggests that decreases in intracel-
lular NAD(P)H and NAD+ in TCDD-treated MCF-7 and
MDA-MB-231 cells are mediated by PARP-1 activation
through formation of DNA strand breaks.
3.9. Effects of the ERα inhibitor on the induction of
ROS formation and DNA strand breaks by TCDD in
MCF-7 and MDA-MB-231 breast cancer cells
To determine the effects of ER␣ status on the induc-
tion of ROS formation by TCDD, we incubated both
cells with TCDD (0.001–10 nM) for 48 h in the presence
of an ER␣ inhibitor, 4-OHT. The extent of ROS gener-
154 P.-H. Lin et al. / Toxicology Letters 172 (2007) 146–158
Fig. 9. Induction of DNA strand breaks in human MCF-7 and MDA-
MB-231 breast cancer cells treated with TCDD (1 and 10 nM) at 37 ◦ C
for 5 h. Data represents the mean ± standard deviation of three determi-
nations. (***) indicates statistically significant difference from control
[DMSO] (p < 0.001).
ation was determined by the DCFH-DA assay. Results
clearly showed that in the presence of an ER␣ antagonist
significant increases in ROS formation were detected in
MCF-7 cells treated with TCDD (0.001–10 nM) for 48 h
(Fig. 10A). This finding is comparable with that detected
in MDA-MB-231 cells exposed to TCDD.
In a separate experiment, we incubated both MCF-
7 and MDA-MB-231 cells with TCDD (1 nM) for 48 h
and the extent of DNA strand breaks were determined
by the Comet assay. In some experiments, acute loss
of ER␣ was evaluated using the anti-estrogen 4-OHT.
After 48 h of exposure, 4-OHT enhanced the extent of
DNA strand breaks in MCF-7 cells treated with TCDD
(Fig. 10B). This finding is comparable with that detected
in MDA-MB-231 cells treated with TCDD. In contrast
to the marked increases in DNA strand break in MCF-7
cells exposed to TCDD plus 4-OHT, we did not observe
any significant difference in the induction of DNA strand
breaks in MDA-MB-231 cells by TCDD with or without
co-treatment of 4-OHT. Overall, this evidence suggests
Fig. 10. Induction of (A) ROS formation and (B) DNA strand
breaks in human MCF-7 and MDA-MB-231 cells exposed to TCDD
(0.001–10 nM) for 48 h with or without the addition of ER␣ antagonist
(4-OHT) (100 nM). Data represents the mean ± standard deviation of
three determinations. (*), (**), (***) indicates statistically significant
difference from control [DMSO] (p < 0.05), (p < 0.005), (p < 0.001).
that the presence of ER␣ inhibits the TCDD-induced
ROS formation and DNA strand breaks in human breast
cancer cells.
4. Discussion
Much effort has been directed toward the characteri-
zation of the roles of oxidative stress in TCDD-induced
toxic responses. It has been shown that over-expression
of CYP 1A1 contributes to ROS generation and
the subsequent induction of oxidized bases in mam-
malian cells exposed to TCDD (Park et al., 1996;
Knerr et al., 2006). ROS-induced oxidized bases are
predominantly repaired by a base excision repair path-
way (Demple and Harrison, 1994). In this process,
8-hydroxy-2 -deoxyguanine-DNA glycosylase cleaves
the N-glycosylic bond between 8-hydroxyguanine and
deoxyribose, leaving 3 -nicked abasic sites (Rosenquist
 P.-H. Lin et al. / Toxicology Letters 172 (2007) 146–158
et al., 1997). In addition, ROS can induce sugar
lesions on DNA double-helix by hydrogen abstrac-
tion from deoxyribose resulting in DNA strand breaks
(Balasubramanian et al., 1998). Poly(ADP-ribose)
polymerase-1 (PARP-1) plays an important role in
BER by interacting with XRCC1, DNA polymerase
beta, and DNA ligase III at the site of DNA lesions
(Caldecott et al., 1994, 1995, 1996; Kubota et al.,
1996; Whitehouse et al., 2001). PARP-1 binds to DNA
strand breaks and uses NAD+ as a substrate to gener-
ate poly(ADP-ribose)polymer (Sugimura et al., 1967),
leading to dose-dependent NAD+ and NAD(P)H deple-
tion (Skidmore et al., 1979). In light of this, we attempted
to establish the links between TCDD-induced oxidative
events and the subsequent induction of DNA damage and
repair in human breast cancer cells. In the present study,
we examined the degree of TCDD-induced ROS forma-
tion, GSH depletion, DNA strand breaks, and PARP-1
activation in both human MCF-7 and MDA-MB-231
breast cancer cells. Results from the cell viability
assay indicated that TCDD induced concentration- and
time-dependent increases in cytotoxic response in both
MCF-7 and MDA-MB-231 cells (Fig. 1). In addition,
TCDD induced a concentration-dependent increase in
the formation of ROS in MCF-7 and MDA-MB-231
cells (Fig. 2). Overall, the extent of cytotoxic response
was greater in MCF-7 cells than in MDA-MB-231
cells whereas the magnitude of ROS formation was
greater in MDA-MB-231 cells than in MCF-7 cells.
Previous finding in ER␣-mediated regulation of oxida-
tive stress and DNA damage in human ER␣(+)/MCF-7
and ER␣(−)/MDA-MB-231 breast cancer cells by E2
indicated that antioxidant status is modulated through
the actions of ER␣. This evidence suggests that the
increased sensitivity to DNA damage induced by H2 O2
in MCF-7 cell is mediated through the ER␣ (Mobley
and Brueggemeier, 2004). We speculate that ER␣ status
may, in part, contribute to the variation in the induc-
tion of cell toxicity, ROS generation, and DNA damage
by TCDD between MCF-7 and MDA-MB-231 cells.
The data also demonstrate conclusive adverse effects
of TCDD toward human breast cancer cells at concen-
trations encountered with human exposures (Lee et al.,
2006). Further investigation indicated that addition of ␣-
NF blocked the TCDD-induced ROS formation in both
MCF-7 and MDA-MB-231 cells to levels comparable
to that of control (Fig. 3A and B). We speculate that
the increased CYP1A1 and CYP1B1 activities serve as
major sources of ROS formation in human breast cancer
cells exposed to TCDD. As glutathione acts as a free rad-
ical scavenger in mammalian cells (Shen et al., 2005),
depletion of GSH has been shown to result in increased
155
TCDD-induced oxidative damage in rats (Vuchetich et
al., 1996). In addition, TCDD produced changes in the
levels of mitochondrial GSH in C57BL/6J inbred mice
(Senft et al., 2002; Shen et al., 2005). In the work pre-
sented here, we have demonstrated GSH depletion in
MCF-7 and MDA-MB-231 cells after treatment with
TCDD for 0.5 and 1 h exposure (Fig. 4). We noticed that
the extent of GSH depletion was significantly greater in
MDA-MB-231 cells than in MCF-7 cells. This evidence
is in good agreement with what have been observed in
ROS formation where the extent of TCDD-induced ROS
generation was greater in MDA-MB-231 cells than in
MCF-7 cells (Fig. 2). The extent of GSH depletion in
TCDD-treated MCF-7 and MDA-MB-231 cells gradu-
ally dissipated after 2 and 5 h of exposure (Fig. 4). This
evidence points to the protective roles of intracellular
GSH in TCDD-induced ROS formation in human breast
cancer cells.
It is generally believed that the repair of DNA
single-strand breaks in mammalian cells is initiated
by PARP-1 (de Murcia and de Murcia, 1994). Repair-
mediated indirect DNA strand break formation leads to
NAD(P)H/NAD+ depletion through over-activation of
PARP-1 (Nakamura et al., 2003). Results from our obser-
vation confirmed that at non-cytotoxic concentrations
TCDD induced decreases in intracellular NAD(P)H in
MCF-7 and MDA-MB-231 cells (Fig. 5). Further, we
demonstrated that decreases in intracellular NAD(P)H
in both MCF-7 and MDA-MB-231 cells exposed to
TCDD were primarily due to reduction in intracellular
NAD+ pool (Fig. 7). In order to distinguish whether the
reduction in NAD(P)H is due to a reduction in mito-
chondria function or due to the depletion of NAD+
by PARP-1 activation, we co-exposed cells to TCDD
and three specific PARP-1 inhibitors, including 3-AB,
BA, and coumarin. After 5 h of exposure, all PARP-
1 inhibitors blocked the TCDD-induced decreases in
intracellular NAD(P)H in MCF-7 and MDA-MB-231
cells (Fig. 6A and B). These findings were confirmed
by polymer immunoblotting using monoclonal antibody
to pADPr (Fig. 8). We observed strong signals of pADPr-
modified PARP in TCDD-treated MCF-7 (Fig. 8A) and
MDA-MB-231 cells (Fig. 8B). These results suggest that
decreases in intracellular NAD(P)H induced by TCDD at
non-cytotoxic concentrations in both MCF-7 and MDA-
MB-231 cells are due to the depletion of NAD+ pool
by PARP-1 activation. In an effort to understand the ori-
gin of the TCDD-induced PARP-1 activation in human
breast cancer cells, we performed a regular Comet assay
to investigate the amount of tail moment in MCF-7 and
MDA-MB-231 cells exposed to TCDD at 1 and 10 nM
for 5 h. Results indicated that the amount of tail moment
156 P.-H. Lin et al. / Toxicology Letters 172 (2007) 146–158
was markedly increased in MCF-7 and MDA-MB-231
cells treated with TCDD when compared to controls
(Fig. 9). Overall, these findings suggest that TCDD-
induced ROS generation is the sources for the formation
of GSH depletion, DNA strand breaks, and PARP-1 acti-
vation in human breast cancer cells.
It has been shown that the human breast cancer cells
that contain estrogen receptor ␣ (ER␣) are more sensitive
to 4-hydroxyequilenin-induced oxidative DNA damage
as compared to ER␣ negative cells (Liu et al., 2002).
In addition, ER␣ is known to modulate the extent of
oxidative stress and DNA damage induced by estrogen
in human ER␣(+)/MCF-7 and ER␣(−)/MDA-MB-231
breast cancer cells (Mobley and Brueggemeier, 2004).
Our previous investigation indicates that the status of
ER␣ may play a role in modulating the PCB-induced
oxidative DNA damage and cell death in human breast
cancer cells (Lin and Lin, 2006). However, no significant
difference in the extent of estrogen-induced DNA strand
breaks between MCF-7 and MDA-MB-231 cells has also
been reported (Rajapakse et al., 2005). Thus, the status
of ER␣ in modulating the TCDD-induced DNA damage
in human breast cancer cells remains inconclusive. In
order to determine the effects of ER␣ status on the pro-
duction of ROS and DNA damage induced by TCDD
in MCF-7 and MDA-MB-231 cells, an ER␣ inhibitor,
4-OHT, was added to the medium used in MCF-7 cells.
Results clearly revealed that significant increases in the
extent of ROS generation and DND strand breaks were
detected in MCF-7 cells exposed to TCDD plus 4-OHT
(Fig. 10A and B). The fact that these responses can be
enhanced by 4-OHT reinforces the indication that these
effects are mediated by ER␣. This result is in good agree-
ment with that reported by Musarrat et al. (1996), where
the extent of oxidative DNA lesions was greater in breast
cancer tissues than in normal breast, with a strong cor-
relation to estrogen receptor (ER) status. This evidence
strongly suggests that functional ER␣ prevents the ROS
formation and the subsequent induction of DNA damage
induced by TCDD in human breast cancer cells.
In summary, our findings add further support to the
theme that ROS formation is a significant determinant
factor in mediating the induction of oxidative DNA dam-
age and repair in human breast cancer cells exposed to
TCDD and that the TCDD-induced oxidative stress and
DNA damage may, in part, contribute to TCDD-induced
carcinogenesis.
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