Bisphenol A alters the development of the rhesus monkey mammary gland.
ABSTRACT The xenoestrogen bisphenol A (BPA) used in the manufacturing of various plastics and resins for food packaging and consumer products has been shown to produce numerous endocrine and developmental effects in rodents. Exposure to low doses of BPA during fetal mammary gland development resulted in significant alterations in the gland's morphology that varied from subtle ones observed during the exposure period to precancerous and cancerous lesions manifested in adulthood. This study assessed the effects of BPA on fetal mammary gland development in nonhuman primates. Pregnant rhesus monkeys were fed 400 μg of BPA per kg of body weight daily from gestational day 100 to term, which resulted in 0.68 ± 0.312 ng of unconjugated BPA per mL of maternal serum, a level comparable to that found in humans. At birth, the mammary glands of female offspring were removed for morphological analysis. Morphological parameters similar to those shown to be affected in rodents exposed prenatally to BPA were measured in whole-mounted glands; estrogen receptor (ER) α and β expression were assessed in paraffin sections. Student's t tests for equality of means were used to assess differences between exposed and unexposed groups. The density of mammary buds was significantly increased in BPA-exposed monkeys, and the overall development of their mammary gland was more advanced compared with unexposed monkeys. No significant differences were observed in ER expression. Altogether, gestational exposure to the estrogen-mimic BPA altered the developing mammary glands of female nonhuman primates in a comparable manner to that observed in rodents.
- [show abstract] [hide abstract]
ABSTRACT: We measured serum dBPA in non-pregnant and pregnant female rhesus monkeys, fetuses and amniotic fluid. dBPA was administered by a daily oral bolus or sc implantation of Silastic capsules; both resulted in daily average serum unconjugated dBPA concentrations of <1 ng/ml. We observed lower serum concentrations of unconjugated dBPA in pregnant females relative to pre-pregnancy values, and generally lower concentrations in fetal serum than in maternal serum. Differences in pharmacokinetics of dBPA were evident between pre-pregnancy, early and late pregnancy, likely reflecting changes in maternal, fetal and placental physiology. The serum ratio of conjugated to unconjugated dBPA after continuous sc release of dBPA was similar to values reported in human biomonitoring studies and markedly lower than with oral administration, suggesting oral bolus exposure is not an appropriate human exposure model. We report elsewhere that there were numerous adverse effects on fetuses exposed to very low serum dBPA in these studies.Reproductive Toxicology 01/2014; · 3.14 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Bisphenol A (BPA) exposure results in numerous developmental and functional abnormalities in reproductive organs in rodent models, but limited data are available regarding BPA effects in the primate uterus. To determine if maternal oral BPA exposure affects fetal uterine development in a non-human primate model, pregnant rhesus macaques carrying female fetuses were exposed orally to 400 µg/kg BPA or vehicle control daily from gestation day (GD) 50-100 or GD100-165. Fetal uteri were collected at the completion of treatment (GD100 or GD165); tissue histology, cell proliferation, and expression of estrogen receptor alpha (ERα) and progesterone receptor (PR) were compared to that of controls. Gene expression analysis was conducted using rhesus macaque microarrays. There were no significant differences in histology or in the percentage of cells expressing the proliferation marker Ki-67, ERα, or PR in BPA-exposed uteri compared to controls at GD100 or GD165. Minimal differences in gene expression were observed between BPA-exposed and control GD100 uteri. However, at GD165, BPA-exposed uteri had significant differences in gene expression compared to controls. Several of the altered genes, including HOXA13, WNT4, and WNT5A, are critical for reproductive organ development and/or adult function. We conclude that second or third trimester BPA exposure does not significantly affect fetal uterus development based on morphological, proliferation, and steroid hormone receptor assessments. However, differences in expression of key developmental genes after third trimester exposure suggest that BPA could alter transcriptional signals influencing uterine function later in life.PLoS ONE 01/2014; 9(1):e85894. · 3.73 Impact Factor
- Clinical Oral Investigations 03/2014; 18(2):347-9. · 2.20 Impact Factor
Bisphenol A alters the development of the rhesus
monkey mammary gland
Andrew P. Tharpa,1, Maricel V. Maffinia,1, Patricia A. Huntb, Catherine A. VandeVoortc, Carlos Sonnenscheina,
and Ana M. Sotoa,2
aDepartment of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, MA 02111;bSchool of Molecular Biosciences and Center for
Reproductive Biology, Washington State University, Pullman, WA 99164; andcDepartment of Obstetrics and Gynecology and California National Primate
Research Center, University of California, Davis, CA 95616
Edited* by Joan V. Ruderman, Harvard Medical School, Boston, MA, and approved April 2, 2012 (received for review January 3, 2012)
The xenoestrogen bisphenol A (BPA) used in the manufacturing of
various plastics and resins for food packaging and consumer
products has been shown to produce numerous endocrine and
developmental effects in rodents. Exposure to low doses of BPA
during fetal mammary gland development resulted in significant
alterations in the gland’s morphology that varied from subtle ones
observed during the exposure period to precancerous and cancer-
ous lesions manifested in adulthood. This study assessed the
effects of BPA on fetal mammary gland development in nonhu-
man primates. Pregnant rhesus monkeys were fed 400 μg of BPA
per kg of body weight daily from gestational day 100 to term,
which resulted in 0.68 ± 0.312 ng of unconjugated BPA per mL
of maternal serum, a level comparable to that found in humans.
At birth, the mammary glands of female offspring were removed
for morphological analysis. Morphological parameters similar to
those shown to be affected in rodents exposed prenatally to
BPA were measured in whole-mounted glands; estrogen receptor
(ER) α and β expression were assessed in paraffin sections. Stu-
dent’s t tests for equality of means were used to assess differences
between exposed and unexposed groups. The density of mam-
mary buds was significantly increased in BPA-exposed monkeys,
and the overall development of their mammary gland was more
advanced compared with unexposed monkeys. No significant dif-
ferences were observed in ER expression. Altogether, gestational
exposure to the estrogen-mimic BPA altered the developing mam-
mary glands of female nonhuman primates in a comparable man-
ner to that observed in rodents.
endocrine disruptor|perinatal exposure|morphogenesis|internal dose|
chemical for three consecutive days stopped cycling and
remained in an estrous phase, Dodds and Lawson described BPA
as a “synthetic estrogenic agent” (2). Two years later, these sci-
entists developed diethylstilbestrol (DES), a much more potent
synthetic estrogen and, thus, BPA ceased to be considered for
pharmacological use. Decades later, BPA became the building
block of polycarbonate plastic and main component of epoxy
resins used in many industries. Worldwide, ≈3 million metric
tons of BPA are produced per year (3).
According to the Environmental Protection Agency (EPA),
humans appear to be primarily exposed through food packaging
(Bisphenol-A Action Plan, EPA, 2010, http://www.epa.gov/
oppt/existingchemicals/pubs/actionplans/bpa.html); however, it
is worth noting that BPA is also used in some cash register
receipts, medical devices, and other consumer products. As a re-
sult, humans are routinely exposed and most likely throughout life
(4). A 2008 study by the Centers for Disease Control and Pre-
vention (CDC) detected BPA in the urine of >90% of Americans
sampled (5). To date, few epidemiological studies have examined
the effects of BPA on humans. For instance, toddlers exposed to
high maternal levels of BPA during pregnancy showed altered
isphenol A (BPA) was first synthesized in 1891 (1). In 1936,
after observing that ovariectomized rats injected with the
behavior (6). Also, urinary BPA levels have been shown to be
associated with increased cardiovascular disease and diabetes in
adults (7) and sexual dysfunction in men (8).
Pharmacokinetics studies in rodents and nonhuman primates
showed that, once ingested, BPA is conjugated in the liver into
BPA-glucuronide and BPA-sulfate. Although most BPA in se-
rum is conjugated, unconjugated BPA was measured in these two
species (9) and in humans (10). Unlike its conjugated version,
unconjugated BPA is able to bind to estrogen receptors and
trigger a response. Unconjugated BPA was also measured in
some human tissues (e.g., fat and placenta) and fluids including
blood, breast milk, and amniotic fluid. Human serum levels of
unconjugated BPA were reported at ≈1 ng/mL (10). Importantly,
studies have suggested that the pharmacokinetics appear to be
similar across these species (9). The doses that produce similar
levels in rodents and in nonhuman primates suggest that the
exposure levels in humans are higher than expected. Although
oral exposure is thought to be the main source of BPA, a recent
human study showed that urinary levels of conjugated BPA
do not decrease rapidly after fasting, suggesting that nonoral
exposures may be significant (11). In fact, evidence of trans-
dermal BPA exposure has recently been documented (12).
BPA binds to estrogen receptors α and β (13) and to membrane-
alllevels ofbiological complexity in estrogen target organs (15–21).
Animal studies showed that BPA exposure at or below the refer-
ence dose of 50 μg/kg per day (http://cfpub.epa.gov/ncea/iris/index.
displayed a variety of alterations in the prostate, brain, ovary,
thyroid, and uterus (reviewed in ref. 22) as well as diminished
reproductive capacity in aging females (23). More importantly,
BPA exposure during gestation and organogenesis produces
effects that alter the structure and function of target organs
(24). In particular, these low environmentally relevant develop-
mental exposures caused changes in the tissue organization of the
mouse mammary gland observed during fetal development (25),
and further changes manifested long after the end of exposure
at puberty (18, 26) and in adulthood (18, 26–30). Female mice
exposed to BPA during gestation also displayed a heightened
response to estrogen compared with unexposed controls (31) and
developed intraductal hyperplasias, i.e., a precancerous lesion
(28). Furthermore, rats exposed to BPA in utero developed pre-
cancerous and cancerous lesions (32). Also, perinatal exposure
increased rodents’ sensitivity to chemical carcinogens (33–36).
Although a small human study found no association between
Author contributions: P.A.H. and A.M.S. designed research; A.P.T., M.V.M., and C.A.V.
performed research; A.P.T., M.V.M., C.S., and A.M.S. analyzed data; and A.P.T., M.V.M.,
C.S., and A.M.S. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1A.P.T. and M.V.M. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: email@example.com.
| May 22, 2012
| vol. 109
| no. 21www.pnas.org/cgi/doi/10.1073/pnas.1120488109
BPA concentrations and breast cancer incidence (37), exposure
to the synthetic estrogen DES increased the risk of breast cancer
(38). Additionally, women under 14 years of age who were
exposed to the estrogenic pesticide DDT have five times greater
risk of developing breast cancer (39), suggesting that mammary
glands are organs sensitive to estrogenic chemicals across species.
Rodents are by far the most commonly used animals in toxi-
cological studies. However, because of the potential implications
to human health, there is a strong interest in performing BPA
exposure studies in nonhuman primates. For instance, rodents
are considered a “low-estrogen level” model because the highest
level of estradiol in a female rodent is ≈40 pg/mL during pro-
estrous, with very low levels detected after ovulation. In com-
parison, estradiol levels peak in humans at 400 pg/mL during
ovulation and at ≈270 pg/mL in monkeys (40). Additionally,
monkeys also resemble humans in that they have a true luteal
phase characterized by the secretion of estrogen and progester-
one by the corpus luteum (41). Monkeys thus provide a “high-
estrogen level model” that closely resembles the human condi-
tion. Despite the differences in endogenous estrogen levels,
similar effects of BPA were reported in the developing brains of
rodents and nonhuman primates (42, 43).
By taking advantage of an ongoing study designed to assess the
effect of BPA on ovarian morphogenesis and to determine the
circulating levels of unconjugated BPA in female rhesus mon-
keys exposed orally, we assessed whether gestational exposure to
BPA would affect the mammary gland of these animals. A
quantitative analysis that compared the morphology of exposed
and unexposed female neonatal mammary glands was con-
ducted, and here we show that gestational exposure to BPA af-
fected the development of the nonhuman primate mammary
gland in a similar fashion as was observed in mice.
The results presented here are part of a series of studies that aim
at assessing the effects of BPA on organogenesis in nonhuman
primates by using an oral dose that results in serum levels of
unconjugated BPA similar to those observed in humans. The
original experiments were designed to study ovarian develop-
ment. Because of the expenses and ethical considerations of
producing these animals, several independent laboratories from
across the country were invited to analyze various tissues
obtained from the Macaca mulatta female offspring.
Ultrasound screens were performed to confirm pregnancy and
normal development. Only those animals carrying a female
offspring were used (44). The duration of gestation in these
monkeys is ≈160–165 d. Mammary glands were collected from
A previous pharmacokinetics study in nonpregnant female
rhesus monkeys and CD-1 mice (9) showed that an oral dose
of 400 μg of BPA per kg of body weight per day was necessary to
reach circulating levels of unconjugated, biologically active, BPA
similar to levels measured in humans (≈1 ng/mL). The average
unconjugated BPA in serum was 0.5 ng/mL in monkeys and
0.7 ng/mL in mice. It is important to also measure the conjugate
levels because they unequivocally represent BPA that was me-
tabolized in the organism, ruling out the possibility of spurious
contamination. Additionally, the pharmacokinetics of BPA in
the rhesus monkey exposed orally is known (9) and, thus, these
measurements provide a comparison with previous studies.
Maternal Serum Concentrations of BPA. Pregnant monkeys were fed
a small piece of fruit containing 400 μg of deuterated BPA per kg
of body weight; the daily dose was administered from gestational
day 100–165, which roughly corresponds to the third trimester of
human gestation. Control animals received vehicle (ethanol)
only, also delivered in a small piece of fruit. Maternal serum
samples from exposed and control animals were taken near the
time of delivery and were analyzed for conjugated and un-
conjugated BPA. Maternal serum levels were not available for
one of the control and one of the BPA-treated animals.
In the BPA-treated mothers, the average level of un-
conjugated BPA was 0.68 ± 0.312 ng/mL (range: 0.22–1.88 ng/
mL), and the average level of conjugated BPA was 39.09 ± 15.71
ng/mL (range: 11.42–94.82 ng/mL) (Fig. 1). Conjugated and
unconjugated BPA were quantifiable in all of the exposed ani-
mals analyzed. In the control mothers, the levels of conjugated
and unconjugated BPA were below the level of quantification in
three of four animals and four of four animals, respectively. Only
one control mother had a quantifiable level of conjugated BPA
(0.814 ng/mL); however, this level represents a mere 2% of the
mean value found in exposed animals. The average ratio of
conjugated to unconjugated BPA was 90.70 (range: 17.37–244.56
ng/mL) in the BPA-treated mothers, which is comparable to that
found in orally exposed nonpregnant monkeys (9).
BPA Alters the Development of the Rhesus Monkey Mammary Gland.
Nonhuman primate mammary gland morphology. The nonhuman pri-
mate gland contains 5–7 lactiferous ducts converging to the
nipple (Fig. 2); each of these ducts drain a lobe (suggesting 5–7
lobes per mammary gland) formed by multiple ductal-lobular
units. This structure closely resembles the human breast, which
consists of 15–25 lobes, each drained by one lactiferous duct, and
both are known as multilobular glands. This arrangement con-
trasts with that of the mouse mammary gland that has a single
lactiferous duct that branches and develops as a ductal tree and
is considered a unilobular gland.
Mammary gland size and quantification of morphometric structures. The
mammary gland architecture and developmental patterns are best
visualized by using the technique known as whole-mounting, in
which the entire tissue is spread on a glass and stained while pre-
serving the 3D structures and allowing for quantitative measure-
ments. Using comparable morphometric parameters to those
applied to the analysis of rodent fetal mammary glands, we mea-
sured the total mammary gland area, ductal area, and number of
buds, terminal ends, bifurcating ends, and branching points (Fig. 2).
We also calculated the relative abundance of various epithelial
structures and the density of ductal structures within the mam-
mary gland and the relative abundance of these structures
per lobe. These measurements allowed us to directly compare
previously published data on the mouse’s unilobular with the
monkey’s multilobular mammary gland.
Table 1 summarizes the measurements for the control and
BPA-treated animals. The mammary glands of the BPA-exposed
animals were more developed for every parameter assessed, in-
cluding the number of buds, terminal ends, branching points,
bifurcating ends, as well as the total mammary gland area, the
ductal area, and the number of ductal units (namely, the number
of lactiferous ducts that define the number of lobes) compared
with controls. Some endpoints showed striking differences be-
tween these two groups. For instance, there was a 1.46-fold in-
crease in total area, a 2.02-fold increase in the number of buds,
and a 1.54-fold increase in the number of buds and terminal ends
combined in the BPA group compared with controls. These
differences, however, were not statistically significant, most likely
due to the low number of samples (five controls and four BPA).
Fig. 3 shows two representative images of whole-mounted neo-
natal mammary glands in which the BPA-exposed gland appears
clearly more complex overall and contains a higher number of
buds than the control mammary gland.
Density of epithelial structures. The mammary gland density was
defined as the number of buds, terminal ends, branching points,
and bifurcating ends, normalized for the number of ductal units
present. These results are shown in Fig. 4. The BPA group had
significantly more buds per ductal unit than the control group
(P = 0.027).
Tharp et al.PNAS
| May 22, 2012
| vol. 109
| no. 21
Expression of steroid hormone receptors and markers of epithelial
development. In rodents, estrogen receptors first appear in the
stroma surrounding the mammary gland; epithelial localization is
first observed at embryonic day 18 (25), whereas epithelial lo-
calization in the human mammary gland is first observed around
the 30th week in a few cells (45). We observed that both ER α
and β are expressed in epithelial cells of the rhesus monkey at
birth. As in our previous rodent study, no differences in both the
expression of ER α and β were observed between BPA-treated
animals and controls. In mice, the differentiation of the myoe-
pithelial cell layer occurs mostly postnatally (46), whereas in
humans, the differentiation occurs mostly prenatally (45).
Smooth muscle actin (SMA) starts to be expressed by week 22,
and differentiation of basal and luminal cells expressing keratin
18 (K18) was first detected at the same time (45). We observed
a strong basal expression of SMA and strong K18 expression in
luminal cells, whereas keratin 14 (K14), a marker for myoepi-
thelial cells, is expressed both basally and luminally, as reported
in mice of peripubertal age (47); complete cytodifferentiation is
achieved comparatively later in mice than in humans, and the
data here show that the timing of cell differentiation is compa-
rable in humans and macaques (Fig. 5).
The aim of this study was to determine whether maternal cir-
culating levels of unconjugated BPA similar to those found in
human serum affected the development of the mammary glands
of female rhesus monkey offspring. Because very little is known
BPA measured were below the level of quantification (LOQ) of this assay (indicated by the dashed line).
Mean conjugated and unconjugated BPA concentrations in maternal serum. The values of the controls are not given with SE bars because amounts of
of the ductal area; the epithelial ducts are outlined in blue, and their respective area is indicated by the abutting blue figures. The red polygon indicates area
subtended by the ductal structures (total mammary gland area). (C) The four main structural parameters measured. BE, bifurcating end; BP, branching point;
BUD, bud; TE, terminal end. (Scale bars: A, 1 cm; B, 1 mm.)
Morphometric parameters used for whole-mount analysis. (A) A newborn mammary gland dissected for whole mount analysis. (B) The quantification
| www.pnas.org/cgi/doi/10.1073/pnas.1120488109Tharp et al.
about the fetal and neonatal development of the mammary gland
in rhesus monkeys, we explored the presence of markers of ep-
ithelial differentiation and hormone receptors and found that
the neonate monkey mammary gland is comparable to the hu-
man in the last trimester and to the neonatal mouse. Our results
show that the mammary glands of nonhuman primates are sen-
sitive to BPA exposure during fetal development, and this sen-
sitivity is manifested as increased complexity of the ductal system
compared with unexposed animals. This study is particularly
valuable because it provides mammary gland data regarding fetal
exposure to BPA in nonhuman primates.
BPA exposure in mothers resulted in detectable serum levels of
unconjugated BPA of 0.68 ± 0.312 ng/mL. These data are im-
portant for the following reasons. First, these levels of un-
conjugated BPA are similar to those measured in humans, i.e., ≈1
ng/mL (10), making the results of this study very relevant to hu-
man exposure. Second, the oral dose given to the mother is 8
times higher than the reference dose of 50 μg/kg per day, sug-
gesting that humans and the monkeys in this study are routinely
exposed to levels above the “safe dose.” And third, similar levels
of unconjugated BPA have been shown to trigger biological
effects in vitro and in vivo (48), some of which are mediated by
the classical ERs as demonstrated in experiments comparing
wild-type and receptor null mice (21). Furthermore, these levels
were within the range observed in nonpregnant female rhesus
monkeys and nongestating CD-1 mice, both of which had an
average 24-h unconjugated BPA concentration of ≈0.5 ng/mL (9).
We previously showed that fetal exposure to BPA affected
mammary morphology in a mouse model; these alterations
manifested during the period of exposure and throughout post-
natal life (18, 25, 26, 28). Although the mammary glands of mice
exposed s.c. to 250 ng of BPA per kg of body weight per day
during fetal development showed increased epithelial area,
ductal extension, and branching points at gestational day 18 (25),
monkeys born to mothers orally exposed to 400 μg of BPA per kg
of body weight per day had a significant increase in the number
of buds per ductal unit. Because buds are incipient branches,
these data point to an increased epithelial area and branching in
the nonhuman primate gland similar to those observed in BPA-
exposed mice. Because the morphological alterations observed
at birth in the mammary glands of rodents and nonhuman pri-
mates are comparable, we conclude that BPA exposure during
gestation can be detrimental to mammary gland development
across species. To determine whether the mammary glands from
BPA-exposed nonhuman primates follow similar altered patterns
as those displayed by rodents at puberty and adulthood, such as
precancerous and cancerous lesions, further studies are required.
From what is known about the role of endogenous hormones
in the development of the mammary gland, and from previous
studies in rodents showing that the effects of fetal estrogens
typically manifest after puberty (49, 50), we also conclude that
BPA affects several developmental parameters of the mammary
gland of rhesus monkeys, including some that are relevant to
breast cancer risk in humans, such as epithelial density (51).
From the similarity of mammary gland alterations observed
perinatally in mice and monkeys as a result of BPA exposure, we
infer that BPA will have comparable effects throughout the
lifespan of nonhuman primates. Our current studies reinforce
the concept that the rodent mammary gland is a reliable model
to study developmental exposures to chemicals with estrogenic
Materials and Methods
the California National Primate Research Center. All procedures for
Table 1.Incidence of various epithelial structures
Control (n = 5) BPA (n = 4)
Mean ± SEM(Range)Mean ± SEM (Range)
Total area, mm2
Ductal area, mm2
No. of ductal units
No. of ductal units/total area
7.80 ± 1.73
2.04 ± 0.45
6.00 ± 1.00
0.92 ± 0.18
14.20 ± 5.50
20.20 ± 4.00
13.80 ± 2.95
5.60 ± 0.87
11.44 ± 5.27
2.80 ± 0.71
6.75 ± 0.75
0.94 ± 0.31
28.75 ± 6.01
24.50 ± 6.22
17.75 ± 5.58
6.00 ± 2.27
Data are expressed as mean ± SEM.
of control and BPA dosed animals. (A) Control. (B) BPA dosed. (Scale bars:
Comparison between whole mounts of neonatal mammary glands
ductal units. Data in figures presented as stem-and-leaf plots were created in
Microsoft Excel. In these graphs, the central line marks the 50th percentile
(median), the outer bounds of the box represent the 25th and 75th per-
centiles, and the stems represent 1.5 × (75th − 25th percentiles). Asterisk
denotes significance (P = 0.027).
Number of morphological features normalized by the number of
Tharp et al.PNAS
| May 22, 2012
| vol. 109
| no. 21
maintenance and handling of the animals were reviewed and approved in
advance by the Institutional Animal Use and Care Administrative Advisory
Committee at the University of California at Davis. Animals were housed in-
and at a temperature maintained at 25–27 °C. Animals were fed a diet of
Purina Monkey Chow and provided water ad libitum. Water was delivered to
each cage via rigid PVC pipes and a “lixit” device. Additionally, seasonal pro-
duce, seeds, and cereal were offered as supplements for environmental en-
richment. Gravid rhesus monkeys were sonographically screened early in
analysis targeting the Y chromosome was performed in maternal serum by 40
d of gestation (44); only female fetuses were used in this study.
Oral Administration of BPA. Deuterated BPA (CDN Isotopes) was prepared as
described (9). Briefly, a daily dose of 400 μg of BPA per kg of body weight or
vehicle (100 μL of ethanol) was administered per os from gestational day
100–165. All animals were trained to accept small pieces of fruit before
beginning the BPA treatment period. Fruit was cut small enough that ani-
mals would take the fruit in one bite and would not try to pull it into smaller
pieces before consuming. Preferences of each animal were noted. The BPA
dose for each animal was calculated based on body weight at weekly
intervals. The BPA solution (100–150 μL) was measured with a Hamilton
200-μL syringe and delivered into the center of fruit pieces, such as grapes,
banana slices, dates or dried apricots, so that the animal could grasp the fruit
and place it in her mouth without touching the BPA. The BPA doses were
prepared by laboratory personnel and kept in a secure place before dosing
and were then administered to each animal for immediate consumption,
which was confirmed. All offspring were delivered naturally via vaginal birth
within 1–2 d of full-term gestation (165 d). Tissues were collected at nec-
ropsy within 1–3 d after birth. Maternal serum samples were taken near the
time of spontaneous birth, ≈4 h after oral dosing. These samples were an-
alyzed for conjugated and unconjugated BPA on a fee-for-service basis at
the University of Missouri as described (9). The limit of quantification for this
method is reported as 0.2 ng/mL.
Sample Processing and Whole-Mount Staining. The neonatal mammary glands
were surgically removed; one mammary gland of each neonate was whole-
mounted onto a slide and the other was processed for histological analysis.
Both were fixed overnight in 10% phosphate-buffered formalin. The whole
mounts were then washed twice with PBS, dehydrated in an ethanol series,
cleared in toluene, and rehydrated before staining with carmine alum (52).
After staining, the whole mounts were dehydrated in an ethanol series,
cleared in xylene, and mounted with Permount (Fisher Scientific). The fixed
tissue was processed, paraffin-embedded, and sectioned by following pro-
cedures described (25).
Morphometric Analysis. Two glands were not analyzed because one had an
abnormal polycystic phenotype and the other was from a stillborn animal
with many dilated blood vessels that hindered the analysis. Whole-mounted
mammary glands were used to quantify area, budding, and branching. A
total of five control and four BPA samples were analyzed. First, images of
dissection microscope with a Zeiss AxioCam HRc digital camera (Carl Zeiss).
These images were analyzed by using the Zeiss AxioVision program version
4.4. Mammary glands were coded and analyzed in a treatment-blind manner.
Once analysis was complete but before the code was revealed, the results
were used to sort the glands into two distinct morphological categories. Once
the code was broken, it was evident that all glands had been sorted correctly
by treatment. For each sample, thetotal mammarygland area was quantified
and moving clockwise to the next furthest point of that structure, or a new
structure, thatcould bereachedwithout excludinganyoftheepithelium(Fig.
2). The ductal area was quantified by outlining the epithelial ducts of each
gland. The four main structural parameters measured in each gland were:
number of terminal ends, buds, bifurcating ends, and branching points (Fig.
2). Terminal ends included the end of a duct and newly developed ducts
branching from a main duct that were >200 μm in length; buds were de-
fined as branches that were <200 μm in length. If one point of a ductal
structure clearly split into two separate ducts, it was deemed to be
a branching point, whereas bifurcating ends, where an invagination and
thus a soon-to-be-branching point was present, were counted as a terminal
end. The number of buds, terminal ends, branching points, and bifurcating
ends were analyzed. In addition, the relative abundance of these structures
was calculated via a correction for the number of ductal units (each unit
corresponds to a lobe) present in the mammary gland. This measurement,
referred to as density of epithelial structures allowed a direct comparison
between the unilobular mouse mammary gland with the multilobar rhesus
monkey mammary gland.
Immunohistochemical Analysis. Immunohistochemistry was performed for
smooth muscle actin (SMA) (Abcam), keratin 14 (Thermo), keratin 18 (Sigma),
ERα (Novocastra), and ERβ (Thermo). Sections were treated with xylene to
remove paraffin and were rehydrated. An antigen-retrieval method using
microwave pretreatment and 0.01 M sodium citrate buffer (pH 6) was per-
formed. Nonspecific binding was blocked with normal goat serum before
overnight incubation in a humidity chamber with primary antibody. Sec-
ondary antibody incubation was followed by visualization using the
chloride (Sigma-Aldrich) as the chromogen. Counterstaining was performed
with Mayer’s hematoxylin. Images were captured by using a Zeiss Axioscope
2 plus microscope (Carl Zeiss MicroImaging). Primary and secondary antibody
concentrations are as follows: SMA, 1:100/1:200; keratin 14, 1:100/1:200;
keratin 18, 1:500/1:500; ERα, 1:50/1:100; ERβ, 1:50/1:100.
Statistics. SPSS software package 15.0 (SPSS) was used for all statistical
analyses. Significance between groups was determined by using Student’s t
tests for equality of means. For all statistical tests, results were considered
significant at P < 0.05. All results are presented as mean ± SEM.
ACKNOWLEDGMENTS. We thank David Damassa for assistance with statis-
tical analysis and Cheryl Schaeberle and Laura Vandenberg for their editorial
assistance. This work was supported by the Passport Foundation, National
Center for Research Resources Grant RR00169 and National Institute on
Environmental Health Sciences Grants ES08314 and ES016770.
Keratin 18 (luminal cell marker). (B) Keratin 14 (myoepithelial cell marker).
(C) SMA (myoepithelial cell marker). (D) ERβ. (E) ERα (epidermal epithelium).
(F) ERα (glandular epithelium). (Scale bars: 50 μm.)
Immunolocalization of markers of epithelial differentiation. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1120488109 Tharp et al.
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| no. 21