VOLUME 115 | NUMBER 1 | January 2007 • Environmental Health Perspectives
Epidemiologic studies and animal experimen-
tation have revealed that alterations in the
nutritional status of a developing fetus may
predispose individuals to hypertension and
coronary heart disease that become apparent
in adulthood (Sallout and Walker 2003).
Epidemiologic studies also suggest that the
intrauterine hormonal milieu may predispose
an individual to carcinogenesis. For example,
increased risk of breast cancer correlated with
twin dizygotic birth, a marker of high estrogen
exposure (Braun et al. 1995), and preeclampsia,
a marker of low estrogen exposure, was associ-
ated with lowered risk (Innes and Byers 1999).
Currently, the concern about effects of prenatal
estrogen exposure is focused on the exposure to
environmental estrogens, which may affect
mammary gland development and/or enhance
the risk of breast cancer later in life.
Over the last 60 years, a plethora of syn-
thetic hormonally active chemicals have been
released into the environment. Meanwhile, an
increase in endocrine-related diseases of the
male reproductive system (Sharpe and
Skakkebaek 1993) and testicular (Skakkebaek
et al. 1998) and breast cancers (Davis et al.
1993; Sasco et al. 2003) have been reported.
Among these endocrine disruptors,
bisphenol A (BPA) is receiving increased
attention because of its high potential for
human exposure. In fact, in a recent study,
Calafat et al. (2005) reported the presence of
BPA in 95% of urine samples. BPA has also
been measured in human sera (mean ± SE:
adult men, 1.49 ± 0.11 ng/mL; adult women,
0.64 ± 0.10 ng/mL) (Takeuchi and Tsutsumi
2002), and in human maternal and fetal
plasma and in placental tissue at birth (Ikezuki
et al. 2002; Schonfelder et al. 2002). BPA is
used in the preparation of epoxy resins and in
the manufacture of polycarbonate plastics and
other consumer products (Krishnan et al.
1993; Steiner et al. 1992). BPA has been
found in foods (4–23 μg/can), beverages
(7–58 μg/g), and saliva (90–913 μg/saliva col-
lected in a 1-hr period after application of
dental sealant) in concentrations that were
sufficient to induce the proliferation of estro-
gen target cells in culture (Biles et al. 1999;
Brotons et al. 1995; Olea et al. 1996).
Recognizing that it is not feasible to generate
accurate exposure levels from the existing
data, we have chosen to administer 25 μg
BPA/kg body weight(bw)/day, which falls
just 2.5-fold above the estimated daily intake
of 0.01 mg/kg/day set by the European
Commission (EC 2002).
In rodents, BPA has been shown to tra-
verse the placenta (Takahashi and Oishi
2000), and it is also present in follicular fluid,
amniotic fluid, and fetal serum during preg-
nancy (Zalko et al. 2003). The developing
embryo is particularly susceptible to chemicals
in general and hormones in particular (Bern
1992). As put succinctly by Gilbert (1997),
“the construction of an organ can be affected
by chemicals that have no deleterious effects
on the normal functioning of that organ.”
Previously, we demonstrated that perinatal
exposure to BPA has profound effects on
rodent hormone-dependent tissues long after
exposure elapsed (Markey et al. 2001; Muñoz-
de-Toro et al. 2005; Ramos et al. 2001,
2003). In the mammary gland, BPA altered
development at the biochemical, cellular, and
tissue levels of organization. Of particular
interest were the increase in the number of
terminal end buds and terminal ends (because
these are thought to be the sites where carci-
nomas originate) and the increase in ductal
density and sensitivity to estradiol, which also
suggests increased susceptibility to mammary
cancer (Markey et al. 2001; Muñoz-de-Toro
et al. 2005). Hence, we hypothesize that peri-
natal exposure to low doses of BPA increases
the risk of developing mammary cancer.
The rat mammary carcinogenesis model is
one of the most widely used surrogate models
because it closely mimics the human disease
allowing elucidation of the influence of host
factors, both on the initiation of the neoplastic
process and on the susceptibility according to
Address correspondence to M. Muñoz-de-Toro,
Laboratorio de Endocrinología y Tumores
Hormonodependientes, School of Biochemistry and
Biological Sciences, Casilla de Correo 242, (3000)
Santa Fe, Argentina. Telephone/Fax: 54-342-
4575207. E-mail: firstname.lastname@example.org
This work was supported by grants from the
Universidad Nacional del Litoral (CAI+D program),
the Argentine National Agency for the Promotion of
Science and Technology (ANPCyT), and the National
Institutes of Health (ES08314 and ES012301). M.D.
is a fellow, and L.K. and E.H.L. are career investigators
of the Argentine National Council for Science and
The authors declare they have no competing
Received 21 April 2006; accepted 29 August 2006.
Prenatal Bisphenol A Exposure Induces Preneoplastic Lesions in the
Mammary Gland in Wistar Rats
Milena Durando,1Laura Kass,1Julio Piva,1Carlos Sonnenschein,2Ana M. Soto,2Enrique H. Luque,1and
1Laboratorio de Endocrinología y Tumores Hormonodependientes, School of Biochemistry and Biological Sciences, Universidad
Nacional del Litoral, Santa Fe, Argentina; 2Department of Anatomy and Cellular Biology, Tufts University School of Medicine,
Boston, Massachusetts, USA
BACKGROUND: Humans are routinely exposed to bisphenol A (BPA), an estrogenic compound that
leaches from dental materials, food and beverage containers, and other consumer products. Prenatal
exposure to BPA has produced long-lasting and profound effects on rodent hormone-dependent tis-
sues that are manifested 1–6 months after the end of exposure.
OBJECTIVE: The aim of the present work was to examine whether in utero exposure to BPA alters
mammary gland development and increases its susceptibility to the carcinogen N-nitroso-N-
METHODS: Pregnant Wistar rats were exposed to BPA (25 µg/kg body weight per day) or to vehi-
cle. Female offspring were sacrificed on postnatal day (PND) 30, 50, 110, or 180. On PND50 a
group of rats received a single subcarcinogenic dose of NMU (25 mg/kg) and they were sacrificed
on either PND110 or PND180.
RESULTS: At puberty, animals exposed prenatally to BPA showed an increased proliferation/apopto-
sis ratio in both the epithelial and stromal compartments. During adulthood (PND110 and
PND180), BPA-exposed animals showed an increased number of hyperplastic ducts and aug-
mented stromal nuclear density. Moreover, the stroma associated with hyperplastic ducts showed
signs of desmoplasia and contained an increased number of mast cells, suggesting a heightened risk
of neoplastic transformation. Administration of a subcarcinogenic dose of NMU to animals exposed
prenatally to BPA increased the percentage of hyperplastic ducts and induced the development of
CONCLUSIONS: Our results demonstrate that the prenatal exposure to low doses of BPA perturbs
mammary gland histoarchitecture and increases the carcinogenic susceptibility to a chemical chal-
lenge administered 50 days after the end of BPA exposure.
KEY WORDS: bisphenol A (BPA), desmoplasia, endocrine disruptor, hyperplastic ducts, mammary
tumor, mast cells, N-nitroso-N-methylurea (NMU). Environ Health Perspect 115:80–86 (2007).
doi:10.1289/ehp.9282 available via http://dx.doi.org/ [Online 29 August 2006]
age and reproductive history. In rats, tumors
can be chemically induced by the administra-
tion of either dimethylbenz[a]anthracene
(Russo and Russo 1996) or N-nitroso-N-
methylurea (NMU; Thompson and Adlakha
1991). The sensitivity of the mammary gland
to neoplastic transformation depends on the
stage of mammary gland differentiation at the
time of carcinogenic stimuli (Russo and Russo
1996). NMU can also induce mammary carci-
nomas in parous rats (Yang et al. 1999),
although at a lower incidence than in sexually
immature (Thompson et al. 1995) and peri-
pubertal rats (Maffini et al. 2004; Thompson
and Adlakha 1991).
Therefore, the aim of the present work
was to extend our investigation on the suscep-
tibility of mammary tissue to NMU-induced
neoplasia following in utero exposure to BPA.
Materials and Methods
Animals. We used sexually mature female rats
of the Wistar-derived strain bred at the
Department of Human Physiology (School of
Biochemistry and Biological Sciences, Santa
Fe, Argentina). Animals were maintained in a
controlled environment (22 ± 2°C; 14 hr of
light from 0600 to 2000 hours) and had free
access to pellet laboratory chow (Cooperación,
Buenos Aires, Argentina). The concentration
of phytoestrogens in the diet was not evalu-
ated; however, because feed intake was equiva-
lent for control and BPA-treated rats
(unpublished observations) we assumed that
animals in the experimental and control
groups were exposed to the same levels of
phytoestrogens. To minimize other exposure
to endocrine-disrupting chemicals, rats were
housed in stainless steel cages and tap water
was supplied ad libitum in glass bottles with
rubber stoppers. Animals were treated
humanely and with regard for alleviation of
suffering in accordance with the principles and
procedures outlined in the Guide for the Care
and Use of Laboratory Animals (Institute of
Laboratory Animal Resources 1996).
Experimental procedures. Females in pro-
estrous were caged overnight with males of
proven fertility. The day that sperm was found
in the vagina was designated day 1 of preg-
nancy [gestation day 1 (GD1)]. Pregnant ani-
mals were assigned to one of two groups, with
11–14 mothers/group: dimethyl sulfoxide
(DMSO; vehicle-treated control) or 25 μg
BPA/kg bw/day (25 BPA). On GD8, corre-
sponding to the beginning of organogenesis in
the fetus, rats were weighed and implanted
subcutaneously with a miniature osmotic
pump (model 1002; Alza Corp., Palo Alto,
CA, USA), which delivered 25 BPA (Sigma-
Aldrich de Argentina S.A., Buenos Aires,
Argentina) or DMSO (99.9% molecular biol-
ogy grade; Sigma-Aldrich de Argentina S.A.).
Both BPA and DMSO were released continu-
ously for 14 days (from GD8 to GD23) at a
rate of 0.25 μL/hr. Offspring were delivered
on GD23 and weaned from their mothers at
postnatal day 21 (PND21). We evaluated the
effect of treatment in female offspring.
The first experiment was designed to study
whether in utero BPA exposure affects the
development of the rat mammary gland.
These animals were sacrificed at prepuberty
(PND30), after puberty (PND50), and adult-
hood (PND110 and PND180). Puberty onset
was determined by examining vaginal open-
ing, and body weight was recorded once each
week during the study period.
The second experiment was designed to
evaluate whether prenatal exposure to BPA
enhanced the responsiveness of the mammary
gland to chemically induced mammary pre-
neoplastic/neoplastic lesions. To select a sub-
carcinogenic dose of NMU, a pilot experiment
was performed in which we tested the carcino-
genic effect of 25 NMU (25 mg/kg bw; Sigma-
Aldrich de Argentina S.A.), taking as reference
the tumor incidence in rats receiving NMU
(50 mg/kg bw), a dose previously defined as
carcinogenic (Thompson et al. 1995). Fifty-
day-old virgin rats received a single intra-
peritoneal (ip) injection of either 25 NMU or
50 NMU (positive control) dissolved in 0.9%
NaCl, acidified to pH 4.0 with acetic acid
(Thompson and Adlakha 1991).
Once the subcarcinogenic NMU dose was
established, female offspring from the DMSO
group received either 25 NMU (control) or
50 NMU (positive control) in a single ip dose
at PND50, and female offspring from the
25 BPA group received 25 NMU. The groups
generated were as follows: DMSO + 25 NMU
(n = 16), DMSO + 50 NMU (n = 10), and
25 BPA + 25 NMU (n = 21). Whereas all rats
treated with 50 NMU were sacrificed at
PND180, rats receiving 25 NMU were sacri-
ficed either at PND110 or PND180. All ani-
mals were weighed and palpated biweekly for
mammary tumor detection beginning 1 week
after the NMU administration; however, to
detect early nonpalpable lesions, we sacrificed
a subset of 25 NMU rats and the correspond-
ing controls on PND110. To evaluate NMU
carcinogenic activity and rat strain susceptibil-
ity, we sacrificed animals injected with
50 NMU (positive control) on PND180.
Tissue samples. Two hours before sacrifice,
all rats were injected ip with bromodeoxy-
uridine (BrdU; 6 mg/100 g bw; Sigma-
Aldrich de Argentina S.A.) to determine the
proliferative index in the mammary gland
stroma and epithelium. Abdominal–inguinal
mammary gland chains were dissected out
bilaterally. One chain was processed for whole
mount (Thompson et al. 1995) and the other
was fixed in 10% buffered formalin for 6 hr at
room temperature and embedded in paraffin.
In animals that had received NMU and were
sacrificed on PND180 (DMSO + 25 NMU,
DMSO + 50 NMU, and 25 BPA + 25 NMU),
both mammary gland chains were whole
mounted to facilitate the visualization of
macroscopic and/or microscopic lesions. To
localize microscopic lesions, the whole mounts
were observed under a Leica stereomicroscope,
(Leica Inc., Buffalo, NY, USA) and micro-
scopic or macroscopic lesions were removed
and embedded in paraffin for histologic analysis
(Singh et al. 2000). Serial 5-μm paraffin sec-
tions were mounted on slides coated with
3-aminopropyl triethoxysilane (Sigma-Aldrich
de Argentina S.A.) and stained with hema-
toxylin and eosin (H&E) or used for immuno-
Immunohistochemistry. For immunohisto-
chemistry, sections were deparaffinized and
dehydrated in graded ethanols. We evaluated
BrdU incorporation by immunohistochemistry
(BrdU antiserum; Novocastra Laboratories
Ltd., Newcastle upon Tyne, UK) after acid
hydrolysis for DNA denaturation and a
microwave pretreatment for antigen retrieval
(Kass et al. 2000). Sections used for immuno-
staining cytokeratin 8 (CK8; The Binding
Site, Birmingham, UK) and p63 (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) were
also subjected to a microwave pretreatment for
antigen retrieval. Endogenous peroxidase
activity and nonspecific binding sites were
blocked. To identify mast cells, we used pro-
teinase I (specifically, the rat mast cell pro-
teinase I, RMCP-I; Moredun Scientific Ltd.,
Edinburgh, Scotland) immunostaining follow-
ing the immunoperoxidase technique after
periodic acid and sodium borohydrate treat-
ment to block endogenous peroxidase activity
(Varayoud et al. 2004). Primary antibodies
were incubated overnight at 4°C at dilutions
showed in Table 1. Reactions were developed
by the avidin-biotin peroxidase method using
diaminobenzidine (DAB) (Sigma-Aldrich de
Argentina S.A.) as a chromogen substrate.
Samples were counterstained with Harris’
hematoxylin (Biopur, Rosario, Argentina) for
BrdU labeling or with Mayer’s hematoxylin for
CK8, p63, and RMCP-I detection, and
mounted with permanent mounting medium
(PMyR, Buenos Aires, Argentina). Each
immunohistochemical assay included positive
and negative controls. Negative controls were
incubated with nonimmune serum (Sigma-
Aldrich de Argentina S.A.).
In situ detection of apoptosis. The apop-
totic cells were identified as previously
described (Ramos et al. 2002) using the
BPA exposure and cancer risk
Environmental Health Perspectives • VOLUME 115 | NUMBER 1 | January 2007
Table 1. Antibodies used for immunohistochemistry.
TUNEL technique (ApopTag, Serologicals
Corporation, Norcross, GA, USA). Briefly,
after incubation with proteinase K (5 μg/mL)
(Intergen Co., Purchase, NY, USA) for 10 min
at 37°C, sections were treated with hydrogen
peroxide in phosphate-buffered saline for
10 min at room temperature to quench
endogenous peroxidase activity. The incuba-
tion with a mixture containing digoxigenin
deoxynucleotide triphosphate, unlabeled
deoxynucleotide triphosphate, and terminal
transferase enzyme was developed in a humidi-
fied chamber at 37°C for 1 hr. Subsequently,
the reaction was visualized using anti-digoxi-
genin-peroxidase and DAB. Samples were
counterstained with Mayer’s hematoxylin, and
then dehydrated and mounted with PMyR.
The negative control slides were prepared simi-
larly, except that distilled water was added
instead of terminal transferase enzyme. For a
positive control, we processed an involuting
mouse mammary gland collected 4 days after
weaning in an identical manner.
Morphometry and image analysis. Per-
centage of hyperplastic ducts. The percentage
of hyperplastic ducts was quantified in H&E-
stained sections. The primary criterion used for
diagnosing hyperplasia was the presence of an
increased number of epithelial layers within the
ducts (Singh et al. 2000); only ducts with four
or more layers of epithelial cells were consid-
ered hyperplastic. To obtain the percentage of
hyperplastic ducts, we evaluated three mam-
mary gland sections, at least 30 μm apart, and
analyzed 50 ducts per section.
Stromal nuclei density. We recorded
images of the fat pad using a Sony ExwaveHAD
color video camera (Sony Electronics Inc., Park
Ridge, NJ, USA) attached to an Olympus
BH2 microscope (Olympus Optical Co., Ltd.,
Tokyo, Japan), which was prepared for Köhler
illumination. The resolution of the images was
set to 640 × 480 pixels and the final screen res-
olution was 0.103 μm/pixel. We evaluated two
sections for each specimen and 40 representa-
tive fields in each section, covering a total area
(at) of 0.1 mm2. To quantify the fat pad area
occupied by stromal nuclei (an), we created an
automated sequence operation using Auto-Pro
macro language (Image Pro-Plus 220.127.116.11 sys-
tem; Media Cybernetics, Silver Spring, MD,
USA). Infiltrating cells and blood vessels cells
were not included. The ratio between anand at
was designated as the stromal nuclei density.
Evaluation of immunostained tissues. We
evaluated immunostained tissue sections using
an Olympus microscope with a Dplan 100×
objective (Olympus). Percentages of BrdU
positives and apoptotic cells were determined
separately in each cellular compartment
(parenchyma and stroma) of the whole mam-
mary section (at least 2,000 epithelial cells and
1,000 stromal cells per section). The ratio
between BrdU positive cells and the apoptotic
index (AI) was also calculated.
To obtain data regarding the number of
mast cells in mammary tissue, we used a point
counting procedure (Luque et al. 1996). We
used a square grid inserted in a focusing eye-
piece and determined the volume densities of
mast cells. The volume density ratio was
defined as the number of incident points in
the studied cell (mast cell) divided by the total
number of incident points in the volume unit
(whole mammary gland).
We used immunostaining with CK8 (a
marker for epithelial cells) and p63 (a marker
for myoepithelial cells) to assess whether
epithelial cells invaded the surrounding stroma.
Statistics. Statistical analyses were per-
formed using the Mann-Whitney U test;
p < 0.05 was accepted as indicating a signifi-
cant difference between groups.
BPA exposure resulted in early puberty in
female offspring. Female offspring exposed
in utero to 25 BPA exhibited advanced
puberty, measured as early vaginal opening
compared to controls (Table 2). However,
early vaginal opening was not associated with
modifications in body weight. We found no
significant difference in body weight between
BPA-treated and control female offspring from
birth to adulthood (PND180) (Figure 1).
BPA-induced alterations of mammary
gland organization in female offspring were
evident after puberty. After puberty, at
PND50, both the mammary gland paren-
chyma and stroma of animals prenatally
exposed to BPA exhibited a higher BrdU/AI
ratio (Figure 2A, B). This alteration in cellular
turnover was mainly due to an inhibition of
apoptosis; significantly lower AIs were
observed (Figure 2E, F) while proliferative
indices were slightly increased (Figure 2C, D).
Changes in mammary tissue growth rate
(Figure 2A–F) and the morphologic features of
the stroma became apparent only after puberty.
At PND30, we found no differences between
the mammary glands of BPA- and vehicle-
treated rats. At PND110 and PND180, we
observed a significant increase in the percent-
age of hyperplastic ducts in BPA-treated ani-
mals relative to vehicle-treated controls
(Figure 3A). Even though structures resem-
bling hyperplastic ducts were identified in
control groups, they represented < 10% of
evaluated ducts. At PND110 and PND180,
an increase in both the stromal nuclear density
and the number of mast cells surrounding the
hyperplastic ducts was found (Figure 3B, C;
for a detailed view, see Figure 4E, F).
The mammary gland stroma of BPA-
treated animals also exhibited morphologic
changes in the extracellular matrix. A dense
stroma layer was formed around mammary
epithelial structures, and a fibroblastic stroma
replaced the normal adipose tissue of the
mammary gland exhibited by controls. The
presence of such fibroblastic-like stroma,
which also includes inflammatory cells, indi-
cates a desmoplastic reaction (Figure 4A–D).
Prenatal BPA exposure enhanced NMU
effects on rat mammary glands. Results from
the pilot experiment indicated that mammary
tumor incidence after NMU administration at
180 days of age (almost 19 weeks after chemi-
cal carcinogen injection) in Wistar rats were
0% (0/10) for the group receiving 25 NMU
and 83.3% (5/6) for 50 NMU. Thus,
25 NMU was considered a subcarcinogenic
dose and was used to test our hypothesis.
Females treated in utero with vehicle
(DMSO) and later with the subcarcinogenic
NMU dose (25 mg/kg) showed no change in
the number of hyperplastic ducts at PND110,
whereas at PND180 a significant increase was
found (Table 3). Moreover, in rats treated
in utero with 25 BPA, the subcarcinogenic
NMU dose (25 mg/kg) induced a significant
Durando et al.
VOLUME 115 | NUMBER 1 | January 2007 • Environmental Health Perspectives
Table 2. Pregnancy-, nursing-, and fertility-related variables in rats treated with BPA.
Percent successful pregnancies in pregnant dams
Mother’s weight gain during pregnancy [g (mean ± SD)]
Length of pregnancy (days)
No. of pups/litter (mean ± SD)
Percent females per litter (mean ± SD)
AGD of female offspring [mm (mean ± SD)]
Age at vaginal opening [days (mean ± SD)]
100 (n = 11)
112 ± 4
11 ± 3
49.0 ± 17.1
100 (n = 14)
116 ± 15
9 ± 3
42.6 ± 22.6
2.5 ± 0.5
3.3 ± 0.6
39 ± 3
1.8 ± 0.1
3.0 ± 0.2
34 ± 1*
AGD, anogenital distance.
*Significantly different from control at p < 0.05 (Mann-Whitney U test).
Figure 1. Body weight gain from birth to adulthood
in female rats treated in utero with BPA. The female
body weight was not modified by the BPA treatment
during the evaluated period.
Body weight (g)
increase in hyperplastic lesions at PND180
(Table 3). The differences between both treat-
ments were statistically significant at PND180,
indicating a positive interaction suggestive of
an additive effect (Table 3).
In addition to increasing the incidence of
preneoplastic lesions, in utero exposure to
25 BPA enhanced the response to the sub-
carcinogenic NMU dose: at PND180, 13.3%
(2/15) of animals developed mammary malig-
nancies. All tumors were encapsulated and of
solid consistency, and the stromal response
demonstrated by fibrosis and mononuclear
infiltration (mainly lymphocytes and eosino-
phils) was a common feature. CK8 immuno-
staining patterns ruled out stromal invasion by
epithelial cells. Tumors were classified as ductal
carcinoma in situ with cribriform (Figure 5 B),
papillary, or mixed pattern (cribriform and
papillary) (Figure 5C). Other than neoplastic
mammary lesions, we observed a salivary gland
neoplasia and a cytosteatonecrosis (a large
droplet of lipid surrounded by connective tis-
sue with abundant eosinophilic infiltration) in
the animals treated with a subcarcinogenic
dose of NMU. Mammary tumors in rats
treated with the carcinogenic dose of NMU
(positive control) reached an incidence of 70%
(7 of 10) and were classified as invasive adeno-
carcinoma of papillary, cribriform, or mixed
pattern. Two malignant thyroid gland tumors
of follicular origin were also diagnosed.
Results regarding incidence of tumors and/or
hyperplastic lesions and tumor multiplicity are
summarized in Table 3.
In the present study we examined the influ-
ence of prenatal BPA exposure on the post-
natal development of the female mammary
gland and on susceptibility to NMU-induced
mammary neoplasia. Prenatal exposure to
BPA resulted in an increased number of pre-
neoplastic lesions, namely, ductal hyperplasias
involving the epithelial compartment and stro-
mal alterations in the vicinity of the affected
ducts. Because these effects were not apparent
before puberty, it is plausible to infer that
mammary glands of BPA-exposed rats may be
more sensitive to estrogen than the mammary
glands of unexposed animals. In fact, increased
responses to estradiol were reported in the
mammary glands of mice exposed perinatally
to BPA (Muñoz-de-Toro et al. 2005).
Carcinogenesis is a complex process in
which interactions between stromal and
epithelial cells play an important role
(Barcellos-Hoff 2001; Maffini et al. 2004,
2005). Moreover, a recurrent concept in can-
cer biology is that neoplastic transformation
represents development gone awry. From this
perspective, it is reasonable to hypothesize
that extemporaneous exposure to estrogens or
xenoestrogens during fetal development may
alter the reciprocal interactions that induce
and maintain tissue organization, and that
these alterations in turn generate abnormal
tissue structures and altered control of cell
proliferation. Thus, a marked stromal reac-
tion and a deregulation of growth rate in both
the parenchyma and the stroma (Chrenek
et al. 2001; Noël and Foidart 1998) would be
observed even at early stages of neoplastic
transformation. At PND50 in the BPA-
exposed group (i.e., immediately after
puberty), we observed significant deregulation
of mammary gland growth as a consequence of
two trends: an increase in proliferation and a
decrease in apoptosis. Alterations in these two
processes modified cellular turnover, a phe-
nomenon observed during early stages of
mammary carcinogenesis (Shilkaitis et al.
2000). The present results support our previ-
ous finding that prenatal exposure to BPA
increases the sensitivity of the developing
mammary gland to endogenous estrogen,
thereby creating a permissive state that can lead
to malignancy (Muñoz-de-Toro et al. 2005).
BPA exposure and cancer risk
Environmental Health Perspectives • VOLUME 115 | NUMBER 1 | January 2007
Figure 3. Quantitative evaluation of the histo-
morphologic changes in the mammary gland of
female offspring treated in utero with BPA shown
as the percentage of hyperplastic ducts (A), stro-
mal nuclei density (B), and volume density (Vv) of
mast cells (C). Bars represent mean ± SE (at least
six animals per group).
*Statistically significant difference between BPA-treated
animals and controls (p < 0.05; Mann-Whitney U test).
Hyperplastic ducts (%)
Stromal nuclei density
Mast cells (Vv × 100)
Figure 2. Mammary gland growth rate (BrdU/AI ratio; A, B), BrdU incorporation (C, D), and AI (E, F) quantified
on PND30, PND50, and PND110 in mammary gland parenchyma and stroma of female offspring exposed
inuteroto BPA. Bars represent mean ±SE (at least six animals per group).
*Statistically significant difference between BPA-treated animals and their respective controls for each PND (p < 0.05;
Mann-Whitney U test).
BrdU (%)BrdU (%)
PND30PND50 PND110PND30PND50 PND110
AI (%) AI (%)
In this context, we suggest that the increased
incidence of hyperplastic ducts and increased
stromal nuclear density observed in adult
animals (PND180) may be a consequence of
the cellular turnover deregulation that
occurred earlier in life (i.e., around puberty).
Hyperplastic ducts are considered premalig-
nant structures and the precursors of neo-
plastic lesions (Singh et al. 2000).
The alterations observed in the mammary
gland stroma of females exposed to BPA
in utero may predispose to neoplastic develop-
ment. In this regard, recent observations sug-
gest that the desmoplastic reaction in breast
cancer is the result of altered epithelial–stroma
interactions and that accumulation of stromal
fibroblasts provides both structural and hor-
monal support for the tumor tissue (Deb et al.
2004; Meng et al. 2001). Other structural fea-
tures, such as an increase in matrix rigidity,
may perturb tissue architecture, enhancing cell
growth and tumor metastasis (Akiri et al.
2003; Paszek et al. 2005). Thus, the fibrotic
response observed in the mammary glands of
adult animals exposed prenatally to BPA may
play a permissive, if not cocausal, role regard-
ing NMU-induced carcinogenesis. In this
context, it is relevant to recall that Maffini
et al. (2004) observed that epithelial mam-
mary tumors could be induced after recombi-
nation of unexposed normal epithelial cells
with NMU-exposed stroma.
In BPA-exposed animals, we observed an
increased number of mast cells in the mam-
mary gland stroma that were spatially associ-
ated with hyperplastic ducts. Mast cells are
multifunctional effector cells of the immune
system that produce and release a wide variety
of mediators. Mast cells have been implicated
in promoting angiogenesis in reproductive tis-
sue (Varayoud et al. 2004) and within tumors
(Aoki et al. 2003), but their precise effects on
tumor growth remains unclear. Dabbous
et al. (1986, 1995) proposed that mast cells
increase proliferation of tumor cells and facili-
tate tumor invasion by promoting collageno-
lytic activities. Furthermore, using a mast
cell–stabilizing compound they observed inhi-
bition of tumor growth (Dabbous et al.
1991). Indeed, mast cells have been linked to
intraductal proliferations that could progress
to carcinoma in situ and to invasive carci-
noma (Russo and Russo 1996). The factors
that regulate the progression of normalcy to
preneoplasia and neoplasia are unknown;
however, mast cell degranulation could con-
tribute directly to this sequence by modifying
stroma–epithelium interactions either by
stimulating angiogenesis or through extra-
cellular matrix degradation (Aoki et al. 2003;
Dabbous et al. 1986, 1995; Folkman 1986)
The increase of the mast cell number in the
mammary gland of BPA-exposed animals also
buttresses the notion of the permissive effect
Durando et al.
VOLUME 115 | NUMBER 1 | January 2007 • Environmental Health Perspectives
Figure 4. Representative photomicrographs of mammary glands from adult females (PND110) exposed
in utero to vehicle (control; A, C, E) or 25 BPA (B, D, F, G, H). Tissue sections were either stained with H&E
(A–D) or immunostained to identify mast cells (E–F), myoepithelial cells (G), or epithelial phenotype (H).
Differences between normal ducts in control (A) and hyperplastic ducts (B) in BPA-treated animals are
shown. The adipose tissue of the control mammary gland (C) consists of mainly fat cells, with few fibrob-
lasts or blood vessels. Treatment with 25 BPA (D) promoted a significant increase of nuclear density in the
stromal compartment. After BPA treatment, we found an increase in the volume density of mast cells
(arrows) surrounding the hyperplastic duct (F) compared with few mast cells observed near the normal
duct (E). The insets in (E) and (F) show mast cells at higher magnification. (G) and (H) show a higher magni-
fication of a hyperplastic duct from a BPA-treated mammary gland; the epithelial phenotype of the cells
layers within the hyperplastic ducts was confirmed by the positive CK8 immunostaining (H), whereas
myoepithelial cells were labelled with p63 (G). Bars = 75 µm.
exerted by prenatal exposure to BPA on
chemically induced carcinogenesis.
Several factors contribute to the induction
of rat mammary tumors, among them, the age
of animals at the time of chemical carcinogen
exposure, the carcinogen itself, and the suscep-
tibility of the rat strain. We considered each of
these factors in our study. Although the highest
incidence of tumors induced by NMU has
been obtained by applying the carcinogen in
animals at 21 days of age (Thompson et al.
1995), we decided to inject our animals at
PND50 for two reasons: the 50- to 55-day
period is one where maximal density of highly
proliferating terminal end buds occurs (Russo
and Russo 1996), and our first experiment sig-
naled that this is the period in which BPA-
exposed animals showed the highest BrdU/AI
ratio in the mammary glands. Regarding the
carcinogen, we selected NMU because a) it has
a short half-life (< 30 min) and does not need
to be metabolized to become active; b) NMU
tumors are mainly estrogen dependent, like
human breast carcinoma (Rose et al. 1980);
and c) the induced carcinomas are usually
aggressive and locally invasive (Thompson and
Adlakha 1991). In addition, our model was
developed using Wistar rats, which are consid-
ered to have medium sensitivity to NMU
We observed a significantly increased num-
ber of ductal hyperplasias at PND110 and
PND180 in BPA-treated animals compared
with DMSO-treated animals; this suggests that
prenatal BPA exposure increased the sensitivity
of the gland to develop preneoplastic lesions.
The administration of subcarcinogenic doses of
NMU to animals exposed prenatally to vehicle
produced no observable effects until PND180.
At PND180, BPA-exposed animals that were
treated with NMU exhibited a significantly
higher number of ductal hyperplasias compared
to animals that were not exposed to BPA; this
suggests that prenatal BPA exposure increased
the susceptibility of the gland to develop pre-
neoplastic lesions as a response to NMU expo-
sure. In addition, treatment with the
subcarcinogenic dose of NMU (25 mg/kg/day)
only induced carcinomas in the mammary
glands of animals exposed prenatally to BPA.
In summary, we conclude that prenatal
exposure to low, environmentally relevant
doses of BPA may increase the risk of devel-
oping rat mammary tumors. The results
reported here indicate that in utero BPA expo-
sure a) induced alterations in the mammary
gland at cellular and tissue levels that could be
considered as preneoplastic lesions, and
b) increased the susceptibility to the chemical
carcinogen NMU, which resulted in the devel-
opment of carcinomas. It is relevant to ask
what is the significance of the results reported
herein using a widely accepted surrogate model
of breast carcinogenesis to that experienced in
BPA exposure and cancer risk
Environmental Health Perspectives • VOLUME 115 | NUMBER 1 | January 2007
Table 3. Effect of prenatal exposure to BPA and postnatal exposure to NMU on the incidence of premalignant
and malignant lesions in the rat mammary gland.
In utero NMU (mg/kg)a
Day of sacrifice Hyperplastic ducts (%)b Tumor incidence
25 BPA— PND110
25 BPA— PND180
25 BPA25 PND180
2.5 ± 2.1
1.5 ± 0.8
8.2 ± 1.5d
18.0 ± 3.2e
5.3 ± 1.3d
14.8 ± 2.8e
3.2 ± 1.3f
16.2 ± 2.3e
15.7 ± 1.2e
35.5 ± 3.7g
19.5 ± 2.2e
aAdministered at PND50. bMean ± SE. cNumber of mammary tumors per rat (mean ± SE). d–gDifferent letters denote statis-
tical differences between groups (p < 0.05; Mann-Whitney U test).
Figure 5. Whole mounts (A) and histologic sections of the abdominal inguinal mammary gland chains (B, C)
from female rats treated in utero with 25 BPA and exposed after puberty to the subcarcinogenic dose of
NMU. Abbreviations: LN, lymph nodes; M, muscle. Whole mounts (A) show gross lesions. H&E-stained
lesions are classified as ductal carcinoma in situ of the cribriform (B) and mixed (C; cribriform and papillar)
types. Bar = 70 µm (B, C).
Durando et al. Download full-text
VOLUME 115 | NUMBER 1 | January 2007 • Environmental Health Perspectives
the human condition. Our observations
strengthen arguments linking the recently
reported increased incidence of endocrine-
dependent human tumors, including those in
the breast, to in utero exposure to minimal
doses of xenoestrogens such as BPA, to which
pregnant women are exposed.
Akiri G, Sabo E, Dafni H, Vadasz Z, Kartvelishvily Y, Gan N, et al.
2003. Lysyl oxidase-related protein-1 promotes tumor fibro-
sis and tumor progression in vivo. Cancer Res 63:1657–1666.
Aoki M, Pawankar R, Niimi Y, Kawana S. 2003. Mast cells in
basal cell carcinoma expresses VEGF, IL-8 and RANTES.
Int Arch Allergy Immunol 130:216–223.
Barcellos-Hoff MH. 2001. It takes a tissue to make a tumor: epi-
genetics, cancer and the microenvironment. J Mammary
Gland Biol Neoplasia 6:213–221.
Bern HA. 1992. The fragile fetus. In: Chemically-induced
Alterations in Sexual and Functional Development: the
Wildlife/Human Connection (Colborn T, Clement C, eds).
Princeton, NJ:Princeton Scientific Publishing, 9–15.
Biles JE, White KD, McNeal TP, Begley TH. 1999. Determination
of the diglycidyl ether of bisphenol A and its derivatives in
canned foods. J Agric Food Chem 47:1965–1969.
Braun MM, Ahlbom A, Floderus B, Brinton LA, Hoover RN. 1995.
Effect of twinship on incidence of cancer of the testis,
breast, and other sites (Sweden). Cancer Causes Control
Brotons JA, Olea-Serrano MF, Villalobos M, Pedraza V, Olea N.
1995. Xenoestrogens released from lacquer coatings in
food cans. Environ Health Perspect 103:608–612.
Calafat AM, Kuklenyik Z, Reidy JA, Caudill SP, Ekong J,
Needham JL. 2005. Urinary concentrations of bisphenol A
and 4-nonylphenol in a human reference population. Environ
Health Perspect 113:391–395.
Chrenek MA, Wong P, Weaver VM. 2001. Tumour-stromal inter-
actions. Integrins and cells adhesions as modulators of
mammary cell survival and transformation. Breast Cancer
Dabbous MK, Haney L, Nicolson GL, Eckley D, Woolley DE. 1991.
Mast cell modulation of tumour cell proliferation in rat mam-
mary adenocarcinoma 13762NF. Br J Cancer 63:873–878.
Dabbous MK, North SM, Haney L, Tipton DA, Nicolson GL.
1995. Effects of mast cell-macrophage interactions on the
production of collagenolytic enzymes by metastatic tumor
cells and tumor-derived and stromal fibroblasts. Clin Exp
Dabbous MK, Walker R, Haney L, Carter LM, Nicolson GL,
Woolley DE. 1986. Mast cells and matrix degradation at
sites of tumour invasion in rat mammary adenocarcinoma.
Br J Cancer 54:459–465.
Davis DL, Bradlow HL, Wolff M, Woodruff T, Hoel DG, Anton-
Culver H. 1993. Medical hypothesis: xenoestrogens as pre-
ventable causes of breast cancer. Environ Health Perspect
Deb S, Amin S, Imir AG, Vilmaz MB, Suzuki T, Sasano H, et al.
2004. Estrogen regulates expression of tumor necrosis
factor receptors in breast adipose fibroblasts. J Clin
Endocrinol Metab 89:4018–4024.
EC. 2002. Opinion of the Scientific Committee on Food on
Bisphenol A. Brussels:European Commission. Available:
[accessed 10 January 2006]
Folkman J. 1986. How is blood vessel growth regulated in normal
and neoplastic tissue? G.H.A. Clowes Memorial Award
Lecture. Cancer Res 46:467–473.
Gilbert SF. 1997. Developmental Biology. 5th ed. Sunderland,
Ikezuki Y, Tsutsumi O, Takai Y, Kamei Y, Taketani Y. 2002.
Determination of bisphenol A concentrations in human bio-
logical fluids reveals significant early prenatal exposure.
Hum Reprod 17:2839–2841.
Innes KE, Byers TE. 1999. Preeclampsia and breast cancer risk.
Institute of Laboratory Animal Resources. 1996. Guide for the Care
and Use of Laboratory Animals. Washington DC:National
Isaacs JT. 1986. Genetic control of resistance to chemically
induced mammary adenocarcinogenesis in the rat. Cancer
Kass L, Varayoud J, Ortega H, Muñoz-de-Toro M, Luque EH. 2000.
Detection of bromodeoxyuridine in formalin-fixed tissue.
DNA denaturation following microwave or enzymatic diges-
tion pretreatment is required. Eur J Histochem 44:185–191.
Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D. 1993.
Bisphenol-A: an estrogenic substance is released from
polycarbonate flasks during autoclaving. Endocrinology
Luque EH, Ramos JG, Rodriguez HA, Muñoz-de-Toro MM. 1996.
Dissociation in the control of cervical eosinophilic infiltra-
tion and collagenolysis at the end of pregnancy or after
pseudopregnancy in ovariectomized steroid-treated rats.
Biol Reprod 55:1206–1212.
Maffini MV, Calabro JM, Soto AM, Sonnenschein C. 2005.
Stromal regulation of neoplastic development: age-depen-
dent normalization of neoplastic mammary cells by mam-
mary stroma. Am J Pathol 67:1405–1410.
Maffini MV, Soto AM, Calabro JM, Ucci AA, Sonnenschein C.
2004. The stroma as a crucial target in rat mammary gland
carcinogenesis. J Cell Sci 117:1495–1502.
Markey CM, Luque EH, Muñoz-de-Toro M, Sonnenschein C,
Soto AM. 2001. In utero exposure to bisphenol A alters the
development and tissue organization of the mouse mam-
mary gland. Biol Reprod 65:1215–1223.
Meng L, Zhou J, Sasano H, Suzuki T, Zeitoun KM, Bulun SE.
2001. Tumor necrosis factor α and interleukin 11 secreted
by malignant breast epithelial cells inhibit adipocyte differ-
entiation by selectively down-regulating CCAAT/enhancer
binding protein α and peroxisome proliferator-activated
receptor γ: mechanism of desmoplastic reaction. Cancer
Muñoz-de-Toro M, Markey C, Wadia PR, Luque EH, Rubin BS,
Sonnenschein C, et al. 2005. Perinatal exposure to bisphenol
A alters peripubertal mammary gland development in mice.
Noël A, Foidart J-M. 1998. The role of stroma in breast carcinoma
growth in vivo. J Mammary Gland Biol Neoplasia 3:215–225.
Olea N, Pulgar R, Perez P, Olea-Serrano F, Rivas A, Novillo-
Fertrell A, et al. 1996. Estrogenicity of resin-based com-
posites and sealants used in dentistry. Environ Health
Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI,
Gefen A, et al. 2005. Tensional homeostasis and the malig-
nant phenotype. Cancer Cell 8:241–254.
Ramos JG, Varayoud J, Bosquiazzo VL, Luque EH, Muñoz-de-
Toro M. 2002. Cellular turnover in the rat uterine cervix
and its relationship to estrogen and progesterone receptor
dynamics. Biol Reprod 67:735–742.
Ramos JG, Varayoud J, Kass L, Rodríguez H, Costabel L,
Muñoz-de-Toro M, et al. 2003. Bisphenol A induces both
transient and permanent histofunctional alterations of the
hypothalamic-pituitary-gonadal axis in prenatally exposed
male rats. Endocrinology 144:3206–3215.
Ramos JG, Varayoud J, Sonnenschein C, Soto AM, Muñoz-de-
Toro M, Luque EH. 2001. Prenatal exposure to low doses of
bisphenol A alters the periductal stroma and glandular cell
function in the rat ventral prostate. Biol Reprod 65:1271–1277.
Rose DP, Pruitt B, Stauber P, Erturk E, Bryan GT. 1980. Influence
of dosage schedule on the biological characteristics of
N-nitrosomethylurea-induced rat mammary tumors.
Cancer Res 40:235–239.
Russo J, Russo IH. 1996. Experimentally induced mammary
tumors in rats. Breast Cancer Res Treat 39:7–20.
Sallout B, Walker M. 2003. The fetal origin of adult diseases.
J Obstet Gynaecol 23:555–560.
Sasco AJ, Kaaks R, Little RE. 2003. Breast cancer: occurrence,
risk factors and hormone metabolism. Expert Rev Anticancer
Schonfelder G, Wittfoht W, Hopp H, Talsness CE, Paul M,
Chahoud I. 2002. Parent bisphenol A accumulation in the
human maternal–fetal–placental unit. Environ Health
Sharpe RM, Skakkebaek NE. 1993. Are oestrogens involved in
falling sperm count and disorders of the male reproductive
tract? Lancet 341:1392–1395.
Shilkaitis A, Green A, Steele V, Lubet R, Kelloff G, Christov K.
2000. Neoplastic transformation of mammary epithelial
cells in rats is associated with decreased apoptotic cell
death. Carcinogenesis 21:227–233.
Skakkebaek NE, Rajpert-De Meyts E, Jorgensen N, Carlsen E,
Petersen PM, Giwercman A, et al. 1998. Germ cell cancer
and disorders of spermatogenesis: an environmental con-
nection? APMIS 106:3–12.
Singh M, McGinley JN, Thompson HJ. 2000. A comparison of
the histopathology of premalignant and malignant mam-
mary gland lesions induced in sexually immature rats with
those occurring in the human. Lab Invest 80:221–231.
Steiner S, Honger G, Sagelsdorff P. 1992. Molecular dosimetry
of DNA adducts in C3H mice treated with bisphenol A
diglycidylether. Carcinogenesis 13:969–972.
Takahashi O, Oishi S. 2000. Disposition of orally administered
2,2-bis(4-hydroxyphenyl)propane (bisphenol A) in pregnant
rats and the placental transfer to fetuses. Environ Health
Takeuchi T, Tsutsumi O. 2002. Serum bisphenol A concentrations
showed gender differences, possibly linked to androgen
levels. Biochem Biophys Res Commun 291:76–78.
Thompson HJ, Adlakha H. 1991. Dose-responsive induction of
mammary gland carcinomas by the intraperitoneal injection
of 1-methyl-1-nitrosourea. Cancer Res 51:3411–3415.
Thompson HJ, McGinley JN, Rothhammer K, Singh M. 1995.
Rapid induction of mammary intraductal proliferations,
ductal carcinoma in situ and carcinomas by the injection of
sexually immature female rats with 1-methyl-1-nitrosourea.
Varayoud J, Ramos JG, Bosquiazzo VL, Muñoz-de-Toro M, Luque
EH. 2004. Mast cells degranulation affects angiogenesis in
the rat uterine cervix during pregnancy. Reproduction
Yang J, Yoshizawa K, Nandi S, Tsubura A. 1999. Protective
effects of pregnancy and lactation against N-methyl-N-
nitrosourea-induced mammary carcinomas in female
Lewis rats. Carcinogenesis 20:623–628.
Zalko D, Soto AM, Dolo L, Dorio C, Rathahao E, Debrauwer L,
et al. 2003. Biotransformations of bisphenol A in a mam-
malian model: answers and new questions raised by low-
dose metabolic fate studies in pregnant CD1 mice. Environ
Health Perspect 111:309–319.