EL Bisphenol A incrementa los riesgos de Cáncer de mama

1. BIOLOGY OF REPRODUCTION 85, 490–497 (2011)
Published online before print 2 June 2011.
DOI 10.1095/biolreprod.110.090431
Bisphenol A Increases Mammary Cancer Risk in Two Distinct Mouse Models
of Breast Cancer1
Kristen Weber Lozada2,3 and Ruth A. Keri3,4,5
Departments of Pharmacology3 and Genetics4 and Division of General Medical Sciences-Oncology,5 Case Western
Reserve University, Cleveland, Ohio
Bisphenol A (BPA) is an industrial plasticizer that leaches from
food containers during normal usage, leading to human
exposure. Early and chronic exposure to endocrine-disrupting
environmental contaminants such as BPA elevates the potential
for long-term health consequences. We examined the impact of
BPA exposure on fetal programming of mammary tumor
susceptibility as well as its growth promoting effects on
transformed breast cancer cells in vivo. Fetal mice were exposed
to 0, 25, or 250 lg/kg BPA by oral gavage of pregnant dams.
Offspring were subsequently treated with the known mammary
carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). While no
significant differences in postnatal mammary development were
observed, both low- and high-dose BPA cohorts had a
statistically significant increase in susceptibility to DMBAinduced tumors compared to vehicle-treated controls. To
determine if BPA also promotes established tumor growth,
MCF-7 human breast cancer cells were subcutaneously injected
into flanks of ovariectomized NCR nu/nu female mice treated
with BPA, 17beta-estradiol, or placebo alone or combined with
tamoxifen. Both estradiol- and BPA-treated cohorts formed
tumors by 7 wk post-transplantation, while no tumors were
detected in the placebo cohort. Tamoxifen reversed the effects
of estradiol and BPA. We conclude that BPA may increase
mammary tumorigenesis through at least two mechanisms:
molecular alteration of fetal glands without associated morphological changes and direct promotion of estrogen-dependent
tumor cell growth. Both results indicate that exposure to BPA
during various biological states increases the risk of developing
mammary cancer in mice.
bisphenol A, BPA, breast cancer, development, DMBA, endocrine
disruptors, estrogen, mammary cancer, mammary glands, mouse,
puberty, rodents (rats, mice, guinea pigs, voles), tamoxifen
INTRODUCTION
Early and chronic exposure to pervasive environmental
contaminants raises the possibility of long-term health
consequences. In recent years, the increased exposure to
environmental synthetic estrogens, such a bisphenol A (BPA),
has been speculated to be involved in the increasing incidence
of breast cancer [1, 2]. BPA is an industrial plasticizer that is
1
Supported by grant RO1ES015768 from the National Institutes of
Health (NIH).
2
Correspondence: Ruth A. Keri, Department of Pharmacology, Case Western
Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH
44106-4965. FAX: 216 368 1300; e-mail: keri@case.edu
Received: 20 December 2010.
First decision: 11 February 2011.
Accepted: 11 May 2011.
Ó 2011 by the Society for the Study of Reproduction, Inc.
eISSN: 1529-7268 http://www.biolreprod.org
ISSN: 0006-3363
490
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utilized in the production of many products, including baby
bottles, food and water containers, medical supplies, and dental
fillings [3]. As a component of polycarbonate plastic, over 6
billion pounds of BPA are produced each year [4]. BPA
leaches from plastic containers during normal usage, and
detectable amounts can be found in many commercial food
products [5, 6]. It is chronically ingested by humans, and
multiple surveys have found that ;95% of adults and children
tested have detectable concentrations of total urinary BPA in
industrialized nations, indicating that exposure is both
ubiquitous and continuous [7]. BPA has also been measured
in maternal serum and ovarian follicular fluid, as well as in
fetal plasma and amniotic fluid, indicating passage across the
placenta [8, 9]. Recent studies have also demonstrated
placental transport of BPA-glucuronide and reactivation of
this apparently inactive metabolite in rat fetuses [10]. Hence,
there is significant risk of BPA exposure during critical
developmental periods that are particularly sensitive to changes
in the estrogenic environment. This may have lasting effects on
hormone responsiveness and homeostatic control of various
tissues much later in life [11].
Reports on the long-term reproductive effects of in vivo
BPA exposure have been diverse because of the disparity in the
use of animal models, strain, dose, timing, and route of
exposure [12–14]. The current Environmental Protection
Agency (EPA) reference dose for BPA is 50 lg kgÀ1 dayÀ1.
This level was derived by identifying the lowest observable
adverse effect level (LOAEL; 50 mg kgÀ1 dayÀ1) in Fischer
344 rats and reducing that dose by three orders of magnitude.
However, both transient and permanent effects have been
observed in various reproductive tissues of animal models with
doses that are lower than the EPA reference dose. Doses below
this reference are considered ‘‘low dose’’ because they are
within the range of human exposure and less than that typically
used in toxicology studies [15–17].
Exposure to endocrine disruptors during prenatal development may result in developmental effects and long-term
modification of organ systems in adults [18–20], and BPA
has been speculated to exert several estrogenic effects on the
rodent mammary gland [21]. This may be an indirect response
to the modulation of the timing of puberty because early
commencement of puberty can affect the development of the
mammary gland through premature exposure to ovarian
hormones, such as estrogen and progesterone, both of which
affect growth and development [22]. Indeed, exposure to BPA
during the perinatal period causes precocious puberty in female
CD-1 mice [3], and this has been associated with masculinization of the anteroventral periventricular preoptic area in the
brain [23]. This may occur through an estrogen-dependent
mechanism, as prenatal exposure to estrogen also induces
precocious puberty in ICR/Jcl mice [24].
In the mammary gland, fetal exposure of female CD1 mice
to BPA causes differences in ductal invasion as well the
number of ducts and terminal end and alveolar buds in adults
ABSTRACT

2. BISPHENOL A AND MOUSE MAMMARY CANCER RISK
100 ll corn oil; Sigma) or corn oil by oral gavage, one dose each at 5 and 6 wk
of age. At least 10 mice were included in each group. Mice were then palpated
weekly for the detection of mammary tumors. Tumor latency was measured
using Kaplan-Meier survival analysis. On the detection of tumors, mice were
killed and tumors collected. Any mice that were obviously sick or that died
during the experiment from causes other than mammary cancer were censored
from the study.
Xenograft Experiments
NCR nu/nu females were obtained from the Case Comprehensive Cancer
Center, Athymic Animal Facility. Recipient females were ovariectomized at 8
wk of age and implanted with a placebo (37.5 mg/60-day release), 17bestradiol (1.7 mg/60-day release), or low-dose BPA pellet (37.5 mg pellet/60day release; Innovative Research of America, Sarasota, FL). After recovery
from surgery, 1 3 106 MCF-7 cells in 150 ll of Matrigel (R&D Systems,
Minneapolis, MN) were subcutaneously injected into the flanks of the mice.
Tumor latency was then assessed by weekly palpation; tumor growth was
monitored by weekly measurement with calipers. Each treatment group
contains a minimum of five mice. After 60 days, mice were implanted with a
second 60-day release implant to ensure sustained exposure to 17b-estadiol or
BPA. Tumor volume was calculated with the equation (l 3 w2)/2, where w is
the smallest diameter measured.
Tamoxifen Treatment
Following tumor formation in NCR nu/nu mice xenografted with MCF-7
cells and treated with either BPA or estradiol, a small cohort was also treated
with the selective estrogen receptor modulator tamoxifen (1 mg mouseÀ1 dayÀ1
[Sigma] or 100 ll vehicle control, 50% PEG400/50% water [Sigma]) by oral
gavage for 5 continuous days per week (n ¼ 3 per group). Tumor growth or
regression was monitored by weekly measurement with calipers.
Mammary Gland Whole Mounts
MATERIALS AND METHODS
Animals
Animal care and use was conducted according to established guidelines
approved by the Institutional Animal Care and Use Committee at Case Western
Reserve University. All animals were treated humanely and with regard to the
alleviation of suffering. Animals were housed in a temperature- and humiditycontrolled animal facility with a 12-h light:dark cycle. All experimental mice
were housed in polypropylene cages with wood bedding and glass water bottles
with rubber stoppers. Weaned mice and lactating females were fed a
phytoestrogen-free diet, LabDiet 5001 and 5008, respectively (Purina LabDiet,
St. Louis, MO).
Vaginal Opening and Mammary Development Experiments
FVB/N mice (The Jackson Laboratory, Bar Harbor, ME) were bred and
observed for a copulatory plug. On evidence of mating, female mice were
placed in a separate cage. On Postcoital Day 8, female mice were treated with
vehicle (100 ll mineral oil), 25 lg/kg bisphenol A (Sigma, St. Louis, MO) or
250 lg/kg bisphenol A by oral gavage daily until parturition. BPA was
dissolved in 50% PBS/50% DMSO and the appropriate concentration added to
mineral oil. At parturition, offspring were culled to a maximum of six mice per
litter. Female offspring were then randomly divided into experimental groups.
Each experimental group contained a minimum of five mice from at least three
litters. Female mice were observed daily for vaginal opening beginning on
Postnatal Day 16. For analysis of development, mammary glands were
collected at 3, 5, and 8 wk of age. Ductal length was measured at 3 and 5 wk of
age as the distance from the midpoint of the lymph node to the terminal end
bud. The distance to the three longest ducts was measured, and these
measurements were averaged for each gland. For forced involution studies,
female mice (exposed prenatally to BPA) that were 8 wk of age were mated and
nursed a litter for 4 days, after which the pups were removed. Mammary glands
were collected 5 days after pup removal.
Tumor Susceptibility Experiments
We utilized the FVB/N strain, which is the most frequently used mouse
model for examining mammary cancer susceptibility because of its intrinsic
propensity to develop mammary tumors with various genetic manipulations
[34–36]. Female mice exposed prenatally to BPA or vehicle control as
described above were given 7,12-dimethyl benz[a]anthracene (DMBA) (1 mg/
When animals were 3, 5, and 8 wk of age or Day 5 postinvolution,
mammary gland number 9 was collected, fixed in Kahle’s solution for 2–4 h,
and stained by immersion in Carmine Alum stain (2% carmine, 5% aluminum
potassium sulfate in water) at 48C. Stained glands were dehydrated in repeated
ethanol washes, cleared with xylene, and mounted on glass slides with
Permount. At least five mice from two independent litters were measured for
each treatment group.
MKI67 Immunostaining
Tumors were fixed in 4% paraformaldehyde, sectioned, and mounted on
slides. Sections were cleared with xylene and dehydrated in ethanol washes.
Fifty milliliters of citrate buffer were used for antigen retrieval at 1208C for 10
min using a Biocare Medical pressure cooker. Sections were incubated with a
rabbit anti-mouse MKI67 antibody (Abcam, ab66155) at 4 lg/ml, overnight at
48C, followed by incubation with an HRP-conjugated secondary antibody
(Vectastain ABC kit; Vector Labs, Burlingame, CA). Positive cells were
identified with 3,3 0 -diaminobenzidine (Liquid DAB þ Substrate Chromogen
System; DAKO, Carpinteria, CA), and the sections were counterstained with
hematoxylin. Tissue sections were dehydrated through decreasing-concentration ethanol washes, cleared with xylene, and mounted under coverslips. Three
sections per mouse (n ¼ 3) and three fields per section were counted for positive
cells, with a minimum of 500 cells counted per field.
Statistical Analysis
Results are expressed as a mean 6 SEM. We performed unpaired Student ttests to assess significance of differences and considered a P-value of ,0.05 to
be statistically significant.
RESULTS
Effect of Prenatal Exposure to Bisphenol A
To begin evaluating the impact of BPA on mammary
tumorigenesis, we first examined whether fetal exposure of
FVB/N mice to BPA would induce precocious puberty or alter
the development of the mammary gland as described for
perinatal BPA exposure in CD1 mice [25–27]. Beginning on
Prenatal Day 8, prior to the onset of mammary morphogenesis
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compared to vehicle-exposed controls [25, 26]. Fetal exposure
also increased ductal area and ductal extension in embryonic
Day 18 female mammary glands in the same strain of mice
[27]. Terminal end buds are particularly sensitive to carcinogenic events, [28, 29]; hence, their increase following BPA
exposure suggests that these mice may be at increased risk for
mammary tumorigenesis. Supporting this postulate, intraductal
mammary epithelial hyperplasias have been observed in CD1
mice exposed to BPA during fetal life or perinatally [30].
While these lesions are suggestive of a potential procarcinogenic effect of BPA, it is unknown whether such changes result
in overt mammary tumors or increased mammary tumor
susceptibility [3, 31]. In contrast to the mouse studies, fetal
exposure of Sprague-Dawley and Wistar rats to BPA leads to
an increase in susceptibility to carcinogen-induced mammary
tumors, although no spontaneous tumors were observed [32,
33]. These data suggest that exposure to BPA during the
prenatal period could alter mammary susceptibility to additional insults; however, the specific carcinogenesis studies have
been limited to rat models.
We questioned whether the tumor-promoting effects of BPA
could be observed in other species and whether the impact of
this compound was restricted to prenatal exposures or whether
BPA could promote the growth of established breast tumors.
Herein, we report that fetal exposure to BPA also increases
mammary tumor susceptibility in mice and that BPA has
growth-promoting effects on human breast cancer xenografts.
Together, these studies indicate that BPA can act at multiple
stages to increase the acquisition of breast cancer.
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3. 492
LOZADA AND KERI
FIG. 1. Fetal exposure to BPA leads to early vaginal opening in FVB/N
female mice. Pregnant FVB/N females were treated with 25 lg kgÀ1 dayÀ1
bisphenol A or vehicle control by oral gavage beginning on Postcoital Day
8. Female offspring were observed for vaginal opening beginning on
Postnatal Day 16. Bars are means 6 SD (*P , 6 E-05). Each experimental
group contained a minimum of five mice from at least three litters.
Susceptibility to 7,12-Dimethylbenz[a]anthracene-Induced
Cancer
We next investigated whether fetal BPA exposure may exert
nonstructural effects on the mammary gland that could alter its
susceptibility to carcinogenic insults. We employed a model of
7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary
cancer [42, 43]. Fetal BPA exposure was repeated with a new
cohort of pregnant female mice with the same dosing schedule
(vehicle control or BPA [25 lg kgÀ1 dayÀ1 or 250 lg kgÀ1
dayÀ1]) as described above, and female offspring were
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that occurs on Embryonic Days 10–11 [37–39], pregnant dams
were treated with vehicle control or 25 lg kgÀ1 dayÀ1 BPA by
oral gavage [40]. At parturition, exposure to BPA was stopped,
and litters were culled to a similar size. Female offspring were
observed daily for vaginal opening, an external physical
indicator of puberty, beginning on Postnatal Day 16 [41].
Female FVB/N mice exposed prenatally to vehicle control
exhibited vaginal opening on Days 22–24, while mice exposed
to BPA exhibited accelerated vaginal opening on Days 21–22
(Fig. 1). The difference in age of vaginal opening was
statistically significant between vehicle- and BPA-treated
groups (P , 6 E-05). These results demonstrate that prenatal
exposure to BPA is manifesting an effect similar to the
previously reported effects of prenatal exposure to estrogens
and can influence changes in the hormonal milieu that establish
the onset of puberty.
To test the effect of prenatal BPA exposure on mammary
gland development, pregnant female mice were treated as
described above. The offspring were randomly separated into
various experimental end points, and the mammary glands
were harvested and whole mounted. Glands were collected at
various ages, including prepuberty, puberty, postpuberty,
pregnancy, lactation, and involution. At no time point
examined were there any notable morphological differences
in mammary gland development observed between the BPAand vehicle-treated cohorts of offspring (Fig. 2A; data not
shown). There was no difference in average duct length at 3
and 5 wk of age between the BPA and vehicle cohorts (Fig.
2B). By 8 wk of age, the ductal tree had filled the fat pad in
both cohorts. These results indicate that prenatal exposure to
BPA has no overt morphological effect on the development of
the mammary gland in FVB/N mice at the various time points
investigated.
FIG. 2. Fetal exposure to bisphenol A (25 lg kgÀ1 dayÀ1) by oral gavage
has no detectable effect on morphological development of the mammary
gland in FVB/N female mice. Pregnant FVB/N females were treated with
25 lg kgÀ1 dayÀ1 bisphenol A or vehicle control by oral gavage beginning
on Postcoital Day 8. Mammary glands were collected from female
offspring at 3, 5, and 8 wk of age as well as involution on Day 5 following
delivery and nursing of one litter. A) Histology of Carmine Alum-stained
whole mounts. B) The distance from mid-lymph node to terminal end bud
was quantified. At least five mice from two independent litters were
measured for each treatment group. Negative values occur in the 3-wk
cohorts because the ducts have not yet reached the midpoint of the lymph
node.
subsequently exposed to one dose of DMBA (1 mg mouseÀ1
dayÀ1) at 5 wk of age and an additional single dose of DMBA
at 6 wk of age. Tumor latency was assessed by weekly
palpation. Both the low-dose (25 lg kgÀ1 dayÀ1) and the highdose (250 lg kgÀ1 dayÀ1) BPA groups revealed a statistically
significant increase in susceptibility to tumor formation
compared to vehicle controls (Fig. 3). The high-dose BPA

4. BISPHENOL A AND MOUSE MAMMARY CANCER RISK
493
FIG. 3. Fetal exposure to bisphenol A or vehicle control results in
increased susceptibility to mammary carcinogenesis after exposure to
7,12-dimethylbenz[a]anthracene (DMBA). Pregnant FVB/N females were
treated with 25 lg kgÀ1 dayÀ1 or 250 lg kgÀ1 dayÀ1 bisphenol A or vehicle
control by oral gavage beginning on Postcoital Day 8. Prenatally exposed
female offspring were then treated with DMBA (1 mg/mouse) by oral
gavage one dose each at 5 wk and again at 6 wk of age. Female mice were
palpated weekly to monitor the formation of mammary tumors. KaplanMeier survival analysis is shown. At least 10 mice from each treatment
group represent at least three independent litters.
MCF-7 Cell Xenografts in NCR nu/nu Mice
The studies above indicate that BPA exposure during early
development can increase susceptibility to carcinogenic
events later in life. We next assessed whether BPA also
promotes the growth of established tumor cells to form an
overt tumor through an estrogenic mechanism. In this case,
we utilized a xenograft mouse model involving MCF-7
human breast cancer cells and immunocompromised NCR nu/
nu female mice. Recipients were ovariectomized at 8 wk of
age to remove the effects of endogenous ovarian hormones on
the xenografted mammary cells. MCF-7 cells require
estrogenic input from either the intact ovary or estrogen
pellet implant in ovariectomized mice to form tumors [45]. At
the time of ovariectomy, the mice were implanted with a
placebo (37.5 mg kgÀ1 60-day releaseÀ1), 17b-estradiol (1.7
mg/60-day release), or BPA pellet (37.5 mg pellet/60-day
release). After recovery from surgery, 1 3 106 MCF-7 cells
suspended in Matrigel were subcutaneously injected into the
flanks of the ovariectomized mice. The mice were palpated
weekly and tumors measured by calipers. The 17b-estradioltreated mice formed measurable tumors by 6 wk postinjection.
Similarly, BPA-treated females formed tumors by 7 wk
postinjection (Fig. 5). In contrast, no tumors formed in the
placebo cohort. By the conclusion of the experiment, five of
seven mice in the 17b-estradiol-treated cohort, five of six
mice in the BPA-treated cohort, and zero of seven mice in the
FIG. 4. Histology of 7,12-dimethylbenz[a]anthracene (DMBA)-induced
tumors from FVB/N female mice. Tumors were collected from FVB/N
females 1 wk after detection of a palpable tumor, fixed in 4%
paraformaldehyde, and stained with hematoxylin and eosin. The only
tumor that developed in the vehicle treated mice is shown with
representative images of tumors from BPA exposed animals. Magnification
320.
placebo-treated cohort formed tumors. On average, the tumors
formed in the 17b-estradiol-treated cohort were 3.0 times
larger in volume than the BPA-treated cohort at 9 wk post
tumor cell implantation (P , 0.001). These results indicate
that BPA alone can promote the growth of estrogendependent breast cancer cells in vivo.
FIG. 5. 17b-estradiol (solid line) or bisphenol A (dashed line) promote
growth of MCF-7 tumors in NCR nu/nu mice. Eight-week-old NCR nu/nu
females were ovariectomized and implanted with a 17b-estradiol (1.7 mg/
60-day release), bisphenol A (37.5 mg pellet/60-day release), or placebo
pellet (37.5 mg pellet/60-day release). One week later, 1 3 106 MCF-7
cells were injected subcutaneously into the flanks of each female (placebo
and 17b-estradiol, n ¼ 7; BPA, n ¼ 6). Mice were palpated for tumor
formation, and mammary tumor size was measured weekly. Average
tumor volume is statistically different between 17b-estradiol and bisphenol A treated cohorts, as indicated: *P , 0.001.
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cohort exhibited a mean tumor latency of 50.8 wk, and the lowdose BPA cohort exhibited a mean tumor latency of 69.3 wk.
Only one vehicle-treated mouse developed a DMBA-induced
tumor at 111 wk. There was a statistically significant difference
in tumor latency in both the low- and the high-dose BPAtreated cohorts compared to controls (P , 0.05). Histologically, all tumors were chemically induced pure squamous cell
carcinomas of the mammary gland, including the single tumor
that arose in the control group (Fig. 4) [44]. Hence, fetal BPA
exposure did not alter the type of mammary tumor that forms in
response to DMBA. These results indicate that BPA may
increase mammary tumorigenesis but does not exert a
dominant effect on the histopathology of resulting tumors.

5. 494
LOZADA AND KERI
FIG. 7. Tamoxifen treatment abrogates estrogen (solid line) or bisphenol
A (dashed line) induced growth of MCF-7 tumors. Eight-week-old NCR
nu/nu females were ovariectomized and implanted with either 17bestradiol (1.7 mg/60-day release), bisphenol A (37.5 mg pellet/60-day
release), or placebo pellets (37.5 mg pellet/60-day release). One week
later, 1 3 106 MCF-7 cells were injected subcutaneously into the flanks of
each female. Seven weeks after tumor formation was first detected,
females were treated daily with tamoxifen (1 mg mouseÀ1 dayÀ1) by oral
gavage. At 60 days post tumor cell injection, the mice were given a
second 17b-estradiol, BPA, or placebo pellet to maintain levels
throughout the remainder of the experiment. Mice were palpated and
mammary tumors measured weekly (n ¼ 3 for estradiol and BPA).
DISCUSSION
FIG. 6. A) MCF-7 tumors from mice treated with 17b-estradiol (1.7 mg/
60-day release) or BPA (37.5 mg/60-day release) are histologically
identical. Xenograft tumor sections from each cohort were stained with
hematoxylin and eosin. Representative sections are shown. B) MCF-7 cell
xenograft tumors from the 17b-estradiol cohort have a higher percentage
of proliferating cells as revealed with MKI67 immunostaining. Xenograft
tumor sections were immunostained with an anti-MKI67 antibody.
Representative sections are shown. Magnification 320. C) The percentage
of MKI67 positively stained cells was quantified in three mice from each
treatment group. Bars are means 6 SD (*P , 1.2 E-6).
Examination of hematoxylin and eosin-stained tumor
sections by light microscopy revealed similar histopathology
and tumor cell size between BPA and 17b-estradiol-treated
groups. Analysis of MKI67 immunohistochemistry, which
identifies proliferating cells, revealed that tumors from the 17bestradiol cohort had an average of 2-fold more proliferation
than the tumors of the BPA cohort (Fig. 6; P , 1.2 E-6). The
difference in the percentage of proliferating cells likely
underlies the difference in growth rate and tumor size between
the 17b-estradiol- and BPA-treated cohorts.
To determine if the effects of BPA were mediated by the
estrogen receptor, another cohort of mice was treated with the
selective estrogen receptor modulator tamoxifen (1 mg
mouseÀ1 dayÀ1). Following formation of measurable MCF-7
cell tumors in NCR nu/nu mice, the animals were treated with
tamoxifen for 3 wk while maintaining exposure to BPA or 17bestradiol. Tumors were measured weekly by calipers. Tumor
regression was observed in all mice treated with tamoxifen
regardless of whether the mice were treated with 17b-estradiol
or BPA (Fig. 7). Hence, although there is a difference in the
percent of proliferating cells between the 17b-estradiol- and the
BPA-treated cohorts, the estrogenic action of BPA is the
Here we report that prenatal exposure to BPA increased
tumor susceptibility in a dose-dependent manner in a DMBAinduced model of mouse mammary gland carcinogenesis. We
also found not only that BPA exposure during early
developmental stages increases susceptibility to mammary
gland cancer but also that adult exposure directly promotes
growth of estrogen dependent tumors in vivo.
In our first model of mammary gland cancer susceptibility,
we utilized FVB/N mice. FVB/N, an inbred strain, demonstrates sensitivity to the formation of mammary gland cancer,
unlike other models, such as C57BL/6, which are resistant to
such tumors [34, 35]. For this reason, the FVB/N strain has
been used extensively for analysis of mammary gland
morphogenesis and tumor susceptibility [35]. Characterization
of BPA-induced tumor susceptibility in an inbred strain such as
FVB/N should greatly facilitate identifying molecular mechanisms involved because genetic contributors to this process can
be readily examined. This contrasts with previous studies
utilizing outbred mice because genetic variability across these
strains adds a further level of complexity that can hamper
precise analyses of molecular mechanisms. Most important, we
have found that prenatal exposure of this strain to BPA results
in a substantial increase in carcinogen-mediated tumor
susceptibility. Determining whether BPA also increases
susceptibility to oncogene-induced tumorigenesis will be
essential for examining whether the increase in susceptibility
is restricted to a DNA damaging agent or whether other
tumorigenic events are also facilitated by the BPA-induced
molecular changes that occur during early mammary gland
development.
Potential routes of human exposure to BPA are being
discovered continually, such as the recent study demonstrating
the potential for BPA leaching from printed receipts [46]. As
the primary route of human exposure is ingestion, we exposed
mice to BPA by oral gavage. When administered in this
manner, BPA is subject to first-pass metabolism in the liver,
and remaining BPA is deposited throughout the body. The
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primary mechanism underlying its promotion of MCF-7 tumor
cell growth in vivo.

6. BISPHENOL A AND MOUSE MAMMARY CANCER RISK
mammary gland cancer are supported by previous studies using
rats. We report a dose-dependent increase in susceptibility to
mammary gland cancer in DMBA-induced model of mammary
gland cancer in mice that were prenatally exposed to BPA.
Prenatal exposure to BPA of Wistar rats, coupled with the
subsequent exposure to subcarcinogenic doses of N-nitroso-Nmethylurea in adulthood, results in increased susceptibility to
cancer, with the development of hyperplastic lesions and low
penetrance of focal tumors [33]. Increased susceptibility to
mammary cancer also occurs following neonatal and prepubertal exposure to BPA and DMBA in Sprague-Dawley rats
[32, 55]. In mice, it has been reported that prenatal exposure to
BPA caused the formation of hyperplastic lesions and ductal
carcinoma in situ, without any additional exposure to
carcinogens as an adult [31]. However, it is unclear whether
these lesions ultimately progress to cancers. Hence, to our
knowledge, our studies are the first to demonstrate that
mammary cancer susceptibility is indeed increased in mice
following prenatal BPA exposure, indicating that prenatal
exposure to BPA increases mammary tumor susceptibility in
multiple rodent models.
The terminal end bud of the mammary gland develops with
the onset of puberty, contains a large number of stem cells, and
is highly susceptible to carcinogenic events [60]. Thus, an
increase in terminal end bud number is frequently associated
with increased cancer susceptibility. Fetal exposure to BPA has
previously been reported to increase terminal end bud number
in CD1 mice, leading to speculation that this may increase
mammary tumor susceptibility. However, our studies demonstrated that prenatal exposure to BPA can increase tumor
susceptibility in the absence of morphological changes in
FVB/N animals. These data suggest that while BPA can alter
mammary morphology in some models, unassociated molecular changes must also contribute to the establishment of breast
cancer risk. Such changes may involve epigenetic alterations of
gene expression as has been observed following in vitro BPA
treatment of mammary-derived epithelial cells from adult
women [61]. Identifying the specific mechanism(s) by which
BPA establishes cancer risk during this period will require
extensive studies that examine BPA alteration of the specific
genetic programs that are established during anlagen morphogenesis that are coupled to tumor susceptibility later in life.
In addition to examining the impact of BPA on establishing
tumor susceptibility during development, we assessed whether
BPA could affect another stage of oncogenesis—specifically,
promoting the growth of transformed mammary epithelial cells
into overt tumors. We used a standard model of estrogendependent breast cancer involving MCF-7 cell xenografts.
Surprisingly, while BPA has been shown to promote growth of
these cells in vitro, no in vivo analyses have assessed whether
BPA can potentiate the growth of hormone-dependent breast
cancers. We found that, like 17b-estradiol, BPA can induce the
formation of tumors from these cells, albeit with reduced
growth rates. These results suggest that high circulating levels
of BPA may contribute to estrogen independent growth of
breast cancers in postmenopausal women; however, this will
require further study to determine if there is a correlation
between serum levels of BPA and apparent hormone
independence.
BPA has been reported to exert both estrogenic and
nonestrogenic effects. To determine if BPA was acting
primarily through its estrogenic properties to induce MCF-7
tumor growth, we treated mice with overt tumors with
tamoxifen, an established inhibitor of estrogen receptor a in
the mammary gland, in the sustained presence of BPA or 17bestradiol [62]. As evidenced by the regression of tumors in both
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timing, as well as route of exposure, is critical; hence, we chose
the prenatal time period during which development of the
mammary gland is initiated. In mice, mammary gland
development begins at E10, when the anlage of the mammary
gland begins to become visible as a small placode, with a small
epithelial bud forming around Day 12.5 [37, 38]. We began
treatment at E8, well before this critical time point, and
continued daily exposure until birth. BPA crosses the placental
barrier because it can be measured in the amniotic fluid and
fetal serum [47–49], and conjugated forms from the mother can
also be reactivated in the fetus [29]. Exposure of the fetus to
BPA could potentially be more detrimental than adult exposure
because of critical windows of fetal development that are
particularly susceptible to endocrine disruption [50]. The
period of anlagen morphogenesis has been described as one
such period of susceptibility for the mammary gland [51].
In addition to the potential to cross the maternal/placental
barrier and accumulate in the fetus, BPA has been shown to
have pathological effects at very low doses. It follows a
nonmonotonic dose-response curve. The dose-response curve
forms an inverted-U shape, with very low and high doses of
BPA eliciting profound responses in vivo [52–54]. To this end,
we chose a low dose (25 lg kgÀ1 dayÀ1), which is half of the
EPA recommended daily safe dose, and a high dose (250 lg
kgÀ1 dayÀ1), which is 10 times higher than that of our low
dose. Both of these doses have previously been utilized in other
mouse models of BPA exposure where perturbation of the
reproductive track has been demonstrated [13, 21, 31, 33, 55].
At both the low and the high doses of BPA delivered by oral
gavage, we observed early vaginal opening, an indicator of the
onset of puberty in mice. These data confirm previous studies
indicating that fetal BPA exposure alters the timing of female
puberty and confirm that the exposure paradigm was consistent
with previous reports. However, the impact of BPA exposure
on the puberty did not translate to morphological differences in
the mammary gland during the ages we investigated. By 8 wk
of age, the mammary tree had filled in the fat pad in both
cohorts, and there was no significant difference in ductal length
from the lymph node in 3- and 5-wk-old mice. These results
differ from similar rodent experiments that have previously
been reported. In several studies, prenatal and perinatal
exposure to BPA by osmotic pump results in marked
differences in mammary gland development, including differences in ductal invasion, number of ducts, and terminal end and
alveolar buds in adult female CD1 mice [25, 26]. Prenatal
exposure by osmotic pump also increases ductal area and
ductal extension in D18 embryonic mammary glands in CD-1
mice [27]. Morphological changes have also been reported for
female Sprague-Dawley rats that were prenatally exposed to
BPA [20]. Similar to the mammary gland, our study revealed
that no overt effects of prenatal low- or high-dose BPA were
observed on the morphology or histology of the ovaries. Other
groups have previously demonstrated an effect of BPA
exposure on the ovary, specifically reporting a disruption of
oogenesis and an impact on meiosis, resulting in an increase in
aneuploid oocytes and embryos [18, 56–59]. While we did not
collect information on the number and types follicles, the mice
that were exposed in utero to BPA were mated and were able to
become pregnant, carry a litter to term, and nurse the litter to
weaning age (data not shown). This indicates that the mice
were fully fertile at a young age; however, we have not
assessed whether these animals may undergo premature
ovarian failure due to an exhaustion of normal oocytes.
Although we observed differing results of prenatal exposure
to BPA on mammary gland development in FVB/N mice
compared to previous reports, our findings on susceptibility to
495

7. 496
LOZADA AND KERI
cohorts, BPA clearly fueled cell growth and tumor formation
via the estrogen receptor.
In conclusion, the results reported here indicate that BPA
may increase mammary tumorigenesis through at least two
mechanisms. One involves alterations of the developing fetal
mammary gland in the absence of morphological changes that
increases susceptibility to carcinogenic insults. The other
demonstrates promotion of tumor cell growth through
estrogenic signaling. Both results indicate that exposure to
BPA at various time points throughout the life span increases
the risk of developing mammary cancer in mice. If these
mechanisms extend to humans, BPA has the potential to
increase susceptibility to breast cancer at low doses if exposure
occurs at various important developmental time points.
17.
18.
19.
20.
21.
ACKNOWLEDGMENT
We thank Dr. Fadi Abdul-Karim, Department of Pathology, University
Hospitals-Case Medical Center, Cleveland, Ohio, for assistance with
tumor pathology analysis.
22.
23.
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