TOXICOLOGICAL SCIENCES 111(1), 151–162 (2009)
Advance Access publication June 5, 2009
Activation of the Aryl Hydrocarbon Receptor during Different Critical
Windows in Pregnancy Alters Mammary Epithelial Cell Proliferation and
Betina J. Lew,* Loretta L. Collins,* Michael A. O’Reilly,† and B. Paige Lawrence*,1
*Department of Environmental Medicine; and †Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York
Received March 23, 2009; accepted May 27, 2009
Exposure to the aryl hydrocarbon receptor (AhR) agonist
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during pregnancy
causes severe defects in mammary gland development and
function; however, the underlying mechanism remains unclear.
Alterations in epithelial cell proliferation, differentiation, and
apoptosis during pregnancy-related mammary development can
lead to failed lactogenesis. To determine which of these processes
are affected and at what time periods, we examined proliferation,
differentiation and apoptosis in mammary glands following
exposure to TCDD during early, mid or throughout pregnancy.
Although AhR activation throughout pregnancy did not cause
early involution, there was a 50% decrease in cell proliferation,
which was observed as early as the sixth day of pregnancy (DP).
TCDD treatment on the day of impregnation only reduced
development and proliferation in early and mid-pregnancy,
followed by partial recovery by DP17. However, when AhR
activation was delayed to DP7, developmental impairment was
not observed in mid-pregnancy, but became evident by DP17,
whereas proliferation was reduced at all times. Thus, early
exposure to TCDD was neither necessary nor sufficient to cause
persistent defects in lactogenesis. These varying outcomes in
mammary development due to exposure at different times in
pregnancy suggest there are critical windows during which AhR
activation impairs mammary epithelial cell proliferation and
Key Words: TCDD; mammary gland development; lactogenesis;
ductal branching; alveologenesis.
The majority of the development of the mammary gland
occurs during postnatal life with the most dramatic morpho-
logical and physiological changes occurring with the beginning
of pregnancy, when the functional state of the gland is realized
upon synthesis of milk following the onset of lactogenesis.
With impregnation, the gland undergoes a proliferative phase,
which consists of epithelial cell proliferation, ductal branching,
and elongation (Hovey et al., 1999). This is followed by the
initiation of secretory differentiation, which involves formation
of lobulo-alveolar structures (Brisken, 2002). Secretory
activation begins later in pregnancy, or upon parturition, and
is marked by milk synthesis and secretion. All of these changes
take place under the influence of a wide range soluble
regulators that are produced both locally in the mammary
gland and by many other tissues and glands (Hennighausen and
Robinson, 2001). Some of the signals that regulate the early
events of development, such as ductal branching morphogen-
esis, also control processes that occur later in pregnancy,
such as lobule formation and their differentiation into alveoli
(alveologenesis). However, many factors participate in only
stage specific processes (Brisken and Rajaram, 2006). De-
regulation of the well-orchestrated process of mammary
development during pregnancy by extrinsic variables such as
nutrition and environmental pollutants can impair ductal
branching, alveologenesis or both events and cause decreased
ability to produce milk.
Our laboratory made a novel discovery that exposure to the
pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impairs
the normal development of mammary glands during pregnancy
to such an extent that dams failed to nutritionally support their
offspring (Vorderstrasse et al., 2004). This discovery is of
interest because it demonstrates very clearly that a known and
abundant pollutant has a profound and adverse effect on
TCDD is a ligand for the aryl hydrocarbon receptor (AhR),
a member of the per-arnt-sim (PAS) family of basic-helix-
loop-helix transcription factors. The AhR directs the expression
of many detoxification genes and functions as a modulator of
cellular signaling pathways critical for cell proliferation,
differentiation and apoptosis. In vertebrates, the AhR is found
in the cytosol in association with HSP90 chaperones, several
HSP90 accessory proteins and immunophilin-like proteins
(XAP2/ARA9/AIP). Upon ligand binding, the AhR trans-
locates to the nucleus where it associates with AhR nuclear
translocator/hypoxia-inducible transcription factor-1b (ARNT/
HIF-1b). The AhR/ARNT complex stimulates transcription of
several genes, such as those in the cytochrome P450 CYP1
1To whom correspondence should be addressed at Box 850, University of
Rochester Medical Center, Rochester, NY 14642. Fax: (585) 276-0239. E-mail:
? The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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family (Gonzalez and Fernandez-Salguero, 1998; Gu et al.,
2000; Puga et al., 2005). Mammary glands express AhR and
ARNT protein (Safe et al., 2000); however, the precise gene
targets of AhR in mammary tissue are not known.
Some of the regulatory pathways that are involved in the
development of the mammary gland are affected by TCDD
(and other AhR ligands) in other model systems. For example,
exposure to AhR ligands altered levels of circulating endocrine
hormones in rodents (De Krey et al., 1994; Jones et al., 1987),
and other reports suggest that TCDD can act as a modulator of
reproductive hormones (Sarkar et al., 2000; Tanaka et al.,
2007). Yet, the severe impairment in mammary differentiation
caused by AhR activation during pregnancy did not correlate
with diminished levels of circulating estradiol, progesterone, or
prolactin (Vorderstrasse et al., 2004). However, numerous
other factors that are locally and systemically produced drive
mammary development during pregnancy (Brisken, 2002).
Therefore, there are many other pathways that are potential
targets of AhR, and may mediate the disruptive effects of
TCDD on mammary gland development. In addition, in-
creasing evidence indicates that there are multiple mechanisms
through which AhR regulates cell proliferation and apoptosis in
different cell types (Camacho et al., 2001; Jin et al., 2004; Lei
et al., 1998; Miller et al., 1996; Mitchell et al., 2006; Singh
et al., 2008). Although the molecular mechanisms by which
AhR ligands impact these processes are not known, they show
that regulation of apoptosis and cell cycle arrest mechanisms
are targets of AhR.
Given that mammary gland development during pregnancy
involves proliferation and differentiation, and that TCDD
affects these processes in other system, the failed lactogenesis
caused by exposure to TCDD could be due to a decrease in
epithelial cell proliferation, impaired cell differentiation into
alveolar structures, premature induction of apoptosis, or
a combination of these events. The objectives of the present
study were to determine whether: (1) AhR activation by TCDD
affects mammary development during early pregnancy and is
associated with reduced induction of milk protein gene
expression, (2) exposure to TCDD during early pregnancy
impairs lactogenesis by suppressing mammary epithelial cell
proliferation or by increasing apoptosis, and (3) AhR activation
during very early pregnancy (i.e., the proliferative phase) is
necessary and sufficient to cause the defects in mammary gland
development observed at parturition.
MATERIAL AND METHODS
Animals and treatments. C57BL/6J mice (age 6 weeks) were obtained
from the Jackson Laboratory (Bar Harbor, ME) or NCI (Frederick, MD).
C57Bl/129J outbred p21 (?/?) mice (B6;129S2-Cdkn1atm1Tyj/J; ?p21KO)
were backcrossed 10 generations to C57BL/6J mice (O’Reilly et al., 2001)
obtained from Jackson laboratories (Bar Harbor, ME).
Female mice were housed with males, and checked daily for presence of
vaginal plugs. The day the vaginal plug was found was designated day 0 of
pregnancy (DP0). Pregnant mice were then individually housed in micro-
isolator units for the remainder of the study. Age-matched virgin mice included
as controls were not housed with males. Animals were given food and water
ad libitum, and were maintained on a 12:12-h light cycle. All animal treatments
were conducted with approval of the Institutional Animal Care and Use
Female mice were treated with 5 lg TCDD/kg body weight or vehicle
control by gavage. Stock TCDD—20 lg/ml (Cambridge Isotopes Laboratory,
Andover, MA) was dissolved in anisole (2%) and diluted in peanut oil, to
a final concentration of 0.5 lg/ml, 0.1% anisole. Vehicle control consisted of
peanut oil containing an equivalent concentration of anisole (0.1%). A single
dose of 50 mg/kg of 5-bromo-2#-deoxyuridine (BrdU—Sigma-Aldrich,
St Louis, MO) was injected (i.p.) 3 h before sacrifice for cell proliferation
studies. Mice were sacrificed by injection (i.p.) of avertin: (2% 2,2,2-
tribromoethanol, 2% tert-amyl alcohol), and blood was collected immediately
by cardiac puncture. Thoracic mammary glands were removed, immediately
frozen in liquid nitrogen and stored at -80?C; these glands were then used for
protein, enzyme (right side) and RNA analysis (left side). Abdominal glands
were removed and used for whole mounts (left side) and immunohistochemical
assays (right side).
Experimental groups. Treatment of animals with TCDD was conducted in
the context of several different exposure paradigms, which are described here.
Paradigm A: impregnated females were exposed to TCDD or vehicle on DP0
and 7 and sacrificed on DP6, 9 and 12 (Fig. 1A, n ¼ 6). This was used to
examine effects of sustained AhR activation on epithelial cell proliferation,
apoptosis, milk gene expression and early differentiation of the mammary
glands (Figs. 2–5). Paradigm B: Impregnated mice were exposed to TCDD or
peanut oil vehicle (Veh) in one of three different windows of time during
mental design. The cartoon represents the development of the mammary gland
during pregnancy. From DP0 until DP7 the mammary gland undergoes a highly
proliferative phase, followed by differentiation of the epithelial cells into
alveolar structures, which ultimately will produce milk. (A) To test the effects
of exposure to TCDD on early epithelial cell proliferation, apoptosis and early
mammary differentiation, impregnated females were exposed to TCDD (5 lg/
kg) or vehicle (veh.) on DP0 and 7 and sacrificed on DP6, 9, and 12. (B) The
effects of administering TCDD during three different windows of time were
evaluated using three exposure paradigms. TCDD early (Te): TCDD (5 lg/kg)
was administered one time, on DP0; TCDD delayed (Td): The first dose of
TCDD was delayed to the end of the highly proliferative phase (DP7). Animals
sacrificed after DP12 received a second dose of TCDD on DP14; TCDD
throughout (Tt): animals were treated throughout pregnancy (DP0, DP7, and
DP14). A fourth group of animals received peanut oil vehicle control. Animals
were sacrificed on DP6, DP9, DP12, and DP17. The number of animals in each
time in point varied from 6 to 9 for each treatment group.
Schematic representation of mammary development and experi-
LEW ET AL.
pregnancy (Fig. 1B, and Figs. 6–8). One group was treated with TCDD on
DP0, DP7, and DP14, and is referred as ‘‘TCDD throughout’’ (this is the same
exposure paradigm used in our previous report (Vorderstrasse et al., 2004) and
in our experiment with p21KO mice (Supplementary Data) Another group was
treated with a single dose of TCDD on DP0 and is referred to as ‘‘TCDD
early.’’ A third group of animals was treated with TCDD on DP7 and DP14,
and is referred to as ‘‘TCDD delayed.’’ As a control, age-matched pregnant
mice received the vehicle control on equivalent days.
Morphological development analyses. Mammary gland whole mounts
were prepared as described previously (Vorderstrasse et al., 2004). Briefly,
glands were removed, fixed in Carnoy’s fixative, stained with carmine alum,
gradually dehydrated, cleared in xylenes, and mounted with Permount (Fisher
Scientific, Pittsburgh, PA). Whole mounts were evaluated without knowledge
of treatment by at least two different scientists, using a Zeiss Stemi 2000C
microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY). Glands were
given a developmental score based on a four-point scale (1 ¼ poor development/
differentiation to 4 ¼ excellent growth and development). The subjective
scoring scales were specific to the stage of development at each time point
examined, and considered ductal branching, development of lobulo-alveolar
units, and the size of the structures. For quantitative morphometric analysis on
DP6, four sections randomly selected from different regions of mammary gland
whole mounts were photographed. Digital micrographs were taken using a Zeiss
Stemi 2000C Microscope with a Nikon KoolPix 995 digital camera. Images
were evaluated in a blinded fashion. The number of lobuloalveolar units was
determined using printed images representing 16 mm2of tissue. The number of
branches in a total length of about 35 mm of duct was evaluated using
a PlanWheel SA2 (Scalex Corporation, Carlsbad, CA). Mean scores for each
group were computed and analyzed for differences due to treatment.
Immunohistochemistry. Glands were removed, fixed overnight in 10%
buffered formalin, transferred to 70% ethanol, embedded in paraffin and
sectioned (4 lm). Deparaffinized sections were subjected to antigen retrieval,
which involved boiling in 10mM citrate buffer for 20 min. For BrdU
incorporation analysis, nonspecific staining was blocked using normal rabbit
serum prior to incubation for 1 h at room temperature with a mouse anti-BrdU
monoclonal antibody (1:100 dilution) (DakoCytomation, Denmark A/S). Next,
sections were incubated for 30 min at room temperature with biotinylated anti-
mouse IgG (1:500 dilution) (Vector Laboratories, CA) and StrepABComplex/
HRP (DakoCytomation). Antibody complexes were visualized using DAB
(Vector Laboratories, CA). Apoptotic cells were identified in separate sections
by terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end
labeling (TUNEL, Apoptag In Situ Apoptosis Detection Kit, Chemicon
International, Temecula, CA). Mammary glands obtained during involution and
lactation were used as positive and negative controls for apoptosis, respectively.
from animals treated with vehicle (Veh) or 5 lg/kg TCDD on DP0, 7, and 14. Whole mounts were fixed in Carnoy’s fixative and stained with carmine alum, as
detailed in the ‘‘Material and Methods’’ section. Representative images were obtained using a Zeiss dissecting microscope (1.253 magnification) with a Nikon
Coolpix E995 camera. (B) Impregnated mice were treated with vehicle or TCDD (5 lg/kg body weight) on DP0 and DP7 and sacrificed on DP9. Age-matched virgin
(AMV) mice were treated with two doses of vehicle or TCDD administered 9 and 2 days prior to sacrifice. The bar graph shows the fold change in expression of milk
protein genes in pregnant animals compared with AMV. Statistical analyses were performed by comparing mean gene expression levels between pregnant vehicle- and
TCDD-treated animals. Asterisks denote statistically significant differences between the vehicle and TCDD treatments (p < 0.05, n ¼ 3).
AhR activation during pregnancy impairs mammary gland development and lactogenesis. (A) Mammary glands were collected on the day of parturition
EXPOSURE TO TCDD AND MAMMARY DEVELOPMENT
using an Olympus BX51 microscope with a RT-Color diagnostic digital camera.
To determine the number of BrdU-positive cells and TUNEL positive cells, 10
different areas of each gland were randomly photographed (203 magnification)
and the average number of BrdU or TUNEL positive cells was determined.
Caspase-3 assay. Caspase-3 activity of nuclear extracts prepared from
frozen thoracic left mammary glands was measured using a fluorometric assay
(CaspACE Assay System, Fluorimetric, Promega, Madison, WI). The assay
was performed following the manufacturer’s information. Briefly, nuclear
extracts (100 lg protein) were mixed with caspase buffer, dimethyl sulfoxide,
1M dithiothreitol, and a synthetic 7-amino-4-methylcoumarin, N-acetyl-L-
aspartyl-Lglutamyl-L-valyl-l-aspartic acid amide caspase-3 substrate. The
emitted fluorescence was kinetically measured at 30?C for 50 min with
a Spectramax M5 Gemini Fluorometer (Molecular Devices, Sunnyvale, CA).
The relative cleavage was determined by calculating the slope of the
accumulation of 7-amino-4-methylcoumarin fluorochrome during the assay.
For statistical analyses, caspase activity in nuclear extracts from involuting
glands was set to 100% and the other values were calculated relative to this
measurement (Marti et al., 2001).
Milk protein gene expression. Mammary glands were removed on DP9
and from age matched virgin animals, and RNA was isolated using Trizol
reagent. Purity, integrity, and concentration of the RNA in each sample was
determined using a spectrophotometer and gel electrophoresis. Nondegraded
RNA (10 lg/sample) was amplified, biotinylated and fragmented for
hybridization at the Washington State University Genomics Core Facility.
Samples from each mouse were hybridized separately to Affymetrix Murine
Genome chips (Set 430A) using an Affymetrix instrument system (scanner,
fluidics station, and hybridization oven). Raw data were examined using
Microarray Suite software (Affymetrix, Santa Clara, CA) and the overall
quality of each chip was visually examined and found to be within normal
parameters. Quality control using spiked genes and housekeeping genes was
conducted to evaluate each chip. Four-way comparisons of the data were
analyzed using Genesifter.net (VizXlabs, Seattle, WA).
Western blots. Thoracic mammary glands were homogenized in the
following buffer: 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,
1mM ethylenediaminetetraacetic acid, 150mM NaCl, 0.6% NP-40, with 0.1M
phenylmethylsulfonyl fluoride, 10 lg/ml aprotinin, 10 lg/ml leupeptin. Protein
concentration was measured by Pierce BCA assay (Pierce, Rockford, IL) and
25 lg of protein from each sample was subjected to sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (Laemmli, 1970), and transferred to
polyclonal anti-mouse b-casein antibody, diluted 1:1000 and incubated overnight
at 4?C (sc-17971, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by
horseradish-peroxidase–conjugated donkey-anti-goat antibody, diluted 1:10,000
and incubated for 2 h at room temperature (Santa Cruz Biotechnology Inc).
Antibody complexes were visualized using chemiluminescent enhanced chem-
iluminescence reagents (Amersham Pharmacia, Piscataway, NJ).
Statistical analyses. Data were analyzed using StatView software (SAS
Software, Cary, NC). Using a two-way ANOVA followed by post hoc tests
(Bonferroni/Dunn test), differences between independent variables were
compared over time and between treatment groups. Differences between two
groups at a single point in time were evaluated using a Students t-test.
Differences were considered significant when p values were < 0.05.
Exposure to TCDD Impairs Mammary Development, with
Noticeable Effects as Early as DP6
We had previously discovered that TCDD treatment disrupts
mammary gland development, with morphological changes
vehicle (V) were collected on DP6 or from age matched virgin animals (n ¼ 6–9). Whole mounts were fixed in Carnoy’s fixative and stained in carmine alum. A.
Representative images were obtained using a Zeiss dissecting microscope (53 magnification) with Nikon Coolpix E995 camera. Branches are denoted by a solid
head arrow, and double-headed arrows point to lobulo-alveolar structures. The circle indicates a TEB. (B) The graph shows the average developmental scores on
DP6, determined as described in the Material and Methods section. (C) The average number of branches per linear mm was determined in mammary glands from
virgin and pregnant mice that were treated with vehicle or TCDD. Two areas of each gland (3.23 magnification) were photographed. The number of branches in an
average of 35 linear mm of duct was counted. (D) The average number of lobules was determined on DP6 in mammary glands from TCDD and vehicle treated
animals. Lobules were counted in four different areas of 4 mm2in each gland. Asterisks indicate statistically significant differences between TCDD and vehicle
treated animals (p < 0.05). Data are representative of two separate experiments.
Activation of AhR impairs mammary gland development as early as the DP6. (A) Mammary glands from mice treated with TCDD (T, 5 lg/kg) or
LEW ET AL.
observed between the DP9 and DP17 (Vorderstrasse et al.,
2004). Here, we extend these observation to include earlier and
later points in time. On the day of parturition, mammary glands
collected from vehicle-treated
populated with alveoli, to such extent that they covered the
adipose tissue. As a result of the dense alveoli present, ductal
structures of the parenchymal tissue are difficult to appreciate
at this stage (Fig. 2A). Moreover, during excision, the presence
of milk could be noticed in the tissue. In contrast, severe
defects were visible in glands collected from animals treated
with TCDD. For instance, the adipose tissue was apparent, with
parenchymal tissue containing fewer numbers of alveoli, and
when present they appeared unfilled and underdeveloped (Fig.
2A). In addition to the morphological changes observed,
TCDD altered the coordinated induction of milk protein genes,
which occurs around the DP9. The alpha-, beta-, and gamma-
casein genes encode common milk proteins and are markers of
mammary gland differentiation. Examination of their expres-
sion on DP9 revealed a severe decrease in the induction of
these genes in mammary glands from dams exposed to TCDD
when compared with glands collected from control animals
(Fig. 2B). These results suggest that the effects of exposure to
TCDD on mammary gland development are noticeable at the
beginning of the secretory differentiation phase of the
We next determined whether defects in pregnancy-induced
differentiation occur prior to DP9; during the proliferative
phase of early pregnancy. In addition to extensive mammary
epithelial cell proliferation, ductal branching morphogenesis
begins in early pregnancy. During this time, secondary and
tertiary branches fill the stromal tissue, forming a complex web
of branches. In addition, mature lobules start populating the
tissue and these structures eventually become alveoli, the milk
producing units of the gland. The initial stage of glandular
differentiation occurs during the first third of pregnancy, and
alterations during this phase could lead to impairment in milk
animals were completely
mammary gland. Mammary glands from pregnant mice treated with TCDD (T)
or vehicle control (V) were collected on DP6, DP9, and DP12 (n ¼ 6/group/
day). (A) Representative micrographs show TUNEL staining in mammary
glands during involution (þ), lactation (?), and pregnancy. The positive
control (þ) depicts apoptotic cells (arrow) in an involuting mammary gland.
Lactating glands served as the negative control (?) and had no TUNEL
staining. (B) The average caspase-3 levels (±SEM) in mammary tissue was
determined by colorimetric and fluorimetric caspase-3 activity assays.
Exposure to TCDD does not induce early involution in the
incorporation by mammary epithelial cells. Mice were treated as in Figure 1,
except that 5-bromo-2#-deoxyuridine (BrdU) was administered (i.p.) 3 h prior
to sacrifice. Details about the experimental groups and immunohistochemistry
can be found in the ‘‘Material and Methods’’ section. The graph represents the
average number of BrdUþcells from 10 different photographed areas of glands
on DP6, DP9, and DP12. The average number of BrdUþcells in 100 lm2tissue
was calculated. Asterisks represent statistically significant differences between
the vehicle and TCDD treatment groups (p < 0.01, n ¼ 6–9).
Exposure to TCDD in early pregnancy decreases BrdU
EXPOSURE TO TCDD AND MAMMARY DEVELOPMENT
production later in pregnancy. Therefore, we examined the
effects of exposure to TCDD on branching and lobule
formation during early pregnancy. As shown in Figure 3,
a single dose of TCDD on DP0 reduced glandular differenti-
ation on DP6. At this stage the best-developed glands collected
from control animals were scored as 4. Those glands showed
visible secondary branches throughout, with most areas being
populated with tertiary branches ending in mature lobules. In
contrast, the least developed glands, which were collected from
TCDD-treated animals scored as 1, had very few complex
structures, and retained some terminal end buds (TEBs; Fig.
3A, circle). On average, mammary glands collected from
vehicle control animals were scored 50% higher than glands
collected from TCDD-treated animals (Fig. 3B).
Impairment in mammary development observed on DP6
could be due to decreased ductal branching, reduced formation
of lobules, or a combination of both. Therefore, we in-
vestigated whether TCDD administration decreases ductal
(DP0 only), delayed (DP7 and DP14), or throughout pregnancy (DP0, DP7, and DP14). Control mice received vehicle treatment on equivalent days. Information
about the experimental groups can be found in Figure 5. Left abdominal mammary glands were collected, fixed in Carnoy’s fixative and stained with carmine alum,
as detailed in the ‘‘Material and Methods.’’ Representative images were obtained using a Zeiss dissecting microscope (53 magnification), with a Nikon Coolpix
E995 camera. Three different researchers scored the glands without knowledge of treatment group. A single asterisk represents a statistically significant difference
between vehicle TCDD treated groups (p < 0.05, n ¼ 6–9). Double asterisks indicate a significant difference (p < 0.05) from the group that received TCDD just
one time, on DP0.
TCDD exposure during different windows of time alters mammary development. Separate groups of nulliparous mice were treated with TCDD early
(i.p.) BrdU 3 h prior to sacrifice. Representative images were obtained using a Spot Pursuit camera (203 magnification). The number of BrdUþcells was
determined by quantitative morphometry, as described in the Materials and Methods. The average number of BrdUþcells in 100 lm2was calculated. Asterisks
represent statistically significant differences between the vehicle and indicated TCDD exposure groups (p < 0.05, n ¼ 6–9).
Exposure to TCDD during different windows of time decreases mammary epithelial cell proliferation. Mice were treated as Figure 5 and administered
LEW ET AL.
branching and/or alveolarization. Ductal morphogenesis starts
at puberty and continues during pregnancy (Hovey et al.,
2002); thus we measured ductal branching in glands collected
on DP6 and compared it with glands from vehicle- and TCDD-
treated virgin animals. Both vehicle- and TCDD-treated
pregnant animals had enhanced secondary and tertiary branch-
ing when compared with virgin animals; however, in vehicle-
treated animals the increase was much more robust (70%
higher than virgin) than in TCDD treated animals (about 40%
higher than virgin; Fig. 3C). In contrast to ductal branching,
alveolarization starts during pregnancy. We compared glands
from TCDD- and vehicle-treated dams at DP6 and found
a 30% decrease in number of lobules following exposure to
TCDD (Fig. 3D). There was no difference in the total surface
area of whole-mounted glands from different treatment groups
(data not shown). The mammary gland achieves its full surface
area prior to pregnancy; therefore an effect in total surface area
was not expected because TCDD exposure in virgin animals
did not alter mammary development (Vorderstrasse et al.,
2004). In summary, exposure to TCDD decreased number of
branches and mature lobules on DP6.
Exposure to TCDD during Pregnancy Decreases
Proliferation but Does Not Increase Apoptosis of
Mammary Epithelial Cells
The observed impairment in mammary development during
pregnancy followed by TCDD exposure could be a result of an
increase in apoptosis or decrease in proliferation of epithelial
cells. In contrast to involuting glands, which have a high index
of apoptotic cells, there was little to no TUNEL staining in
glands collected from pregnant mice regardless of TCDD
exposure (Fig. 4A). Apoptosis was also examined by caspase-3
patterns of BrdU incorporation by mammary epithelial cell and developmental scoring following exposure to TCDD early (A, B), delayed (D, E), or throughout
pregnancy (G, H). The line graphs represent the average number of BrdUþcells (A, D, G) and developmental scoring (C, F, I) on the indicated days of pregnancy.
Asterisks indicate statistically significant differences between the vehicle and TCDD treatment group (p < 0.05, n ¼ 6–9). Representative immunoblots show
b-casein protein levels in mammary glands collected on DP17 from animals exposed to vehicle control and TCDD early, (C), delayed (F), or throughout (I)
pregnancy (n ¼ 6–9). b-Actin was used as loading control.
Exposure to TCDD during different windows of time in pregnancy has distinct outcomes in the development of mammary gland. The different
EXPOSURE TO TCDD AND MAMMARY DEVELOPMENT
activity assay. Involuting glands were used as positive control
and had at about 70% higher caspase-3 activity than mammary
glands collected from pregnant animals. Importantly, there
were no statistically significant differences between vehicle-
and TCDD-treated pregnant animals (Fig. 4B). These results
suggest TCDD-induced impairment of mammary gland de-
velopment during pregnancy is not caused by premature
is dependant on epithelial cell proliferation, the defects in
mammary development observed following exposure to TCDD
could be due to a decrease in epithelial cell proliferation. In
contrast to apoptosis, which occurs primarily in involuting
glands, mammary glands have a high rate of proliferation,
especially during early and mid-pregnancy. Therefore, we next
examined the effects of exposure to TCDD on epithelial cell
proliferation. We analyzed proliferation through quantification
of BrdU incorporation by mammary epithelial cells in tissue
collected on DP6, DP9, and DP12. As early as DP6, there was
glands collected from TCDD-treated animals. Moreover, this
decrease persisted on DP9 and DP12 indicating that exposure to
TCDD decreases mammary epithelial cell proliferation during
early and mid-pregnancy (Fig. 5).
Exposure to TCDD in Early Pregnancy is Neither Necessary
Nor Sufficient to Impair Mammary Development and
On DP17 glands from all TCDD-treated groups had lower
developmental scores than glands from mice in the vehicle
group (Fig. 6). However, animals treated with TCDD only
early in pregnancy had significantly higher scores than animals
in which exposure to TCDD was delayed or maintained
throughout pregnancy. In contrast, developmental scores did
not differ between the TCDD delayed and TCDD throughout
treatment groups. When glands were analyzed on DP12, the
delayed TCDD treatment group did not show impaired
development when compared with vehicle treated animals.
However exposure to TCDD early and throughout pregnancy
reduced mammary gland developmental scores when compared
with vehicle-treated animals (Fig. 6).
In addition to examining development, we determined
whether TCDD treatment in early pregnancy is necessary and
sufficient to reduce cell proliferation. Compared with control
animals, delayed exposure to TCDD or throughout pregnancy
caused a 36 and 49% decrease in the number of BrdU-positive
cells in glands collected on DP17, respectively. Interestingly,
when animals were treated with TCDD on DP0 only, the
number of BrdU-positive cells on DP17 did not differ from
control. In contrast to differential effects observed on DP17,
glands collected on DP12 a showed significantly BrdU-positive
cells in all TCDD treated groups when compared with vehicle
control glands (Fig. 7).
When we integrate these findings over time, some interesting
observations standout. First, when animals were treated with
TCDD only early in pregnancy there was a decrease in the
number of BrdU-positive cells on DP6 and DP12 when
compared with vehicle treated mice. However, exposure to
TCDD did not prevent an increase in the number of BrdU-
positive cells between DP6 and 12. In other words, even
though there was a decrease in number of BrdU-positive cells
compared with the vehicle group, the number of epithelial cells
increased with progression of pregnancy. These findings
suggest that there is a retardation of the developmental process,
rather than a permanent injury to this process. In fact, on DP17
the number of BrdU-positive cells in the TCDD early group did
not differ from control animals, suggesting a partial recovery of
the glands (Fig. 8A). However, this partial recovery did not
translate into normal development, as evidenced by the
difference in developmental scoring between tissue from
TCDD early and vehicle treated animals (Fig. 8B). Yet despite
poorer development, the levels of b-casein protein in these
glands were similar to that of the vehicle group, suggesting that
these glands are potentially able to produce milk (Fig. 8C).
Another interesting observation stems from the group in
which TCDD treatment was delayed. In this group, there was
a statistically significant reduction in the number of BrdU-
positive cells at every time point examined. When compared
with glands from control animals, glands collected from
animals sacrificed on DP9 had a 40% decrease in the number
of BrdU-positive cells, and this decrease in proliferation
persisted on DP12 and DP17 (Fig. 8D). In contrast, de-
velopmental impairment was only evident in tissues collected
on DP17, but not at early points in time (Fig. 8E). Moreover, in
this paradigm, developmental impairment correlated with
decreased levels of b-casein on DP17, suggesting that glands
collected from animals treated with TCDD later in pregnancy
would not have the ability to produce milk (Fig. 8F). In
contrast to differing outcomes between the TCDD early and
TCDD delayed treatment groups, sustained activation of AhR
throughout pregnancy significantly reduced the number of
BrdU-positive cells (Fig. 8G) and the developmental score
(Fig. 8H) of mammary glands at all points in time, and these
effects correlated with a profound suppression in levels of
b-casein protein on DP17 (Fig. 8I).
Sustained AhR activation during pregnancy impairs mam-
mary development and suppresses lactation (Vorderstrasse
et al., 2004). The present data demonstrate that AhR activation
during different windows of time in pregnancy impairs
mammary gland development, but that the timing of TCDD
exposure influences the precise nature of the defect. Our
findings suggest that AhR activation by TCDD adversely
impacts both cell proliferation and glandular differentiation, but
LEW ET AL.
the mechanisms underlying these defects may be distinct and
independent. Indeed, exposure to TCDD has been shown to
decrease proliferation and impair differentiation in other model
systems (Elferink, 2003; Huang and Elferink, 2005; Puga et al.,
2005) however the precise mechanism by which AhR ligands
derail these processes is not fully understood.
When considering how AhR ligands could impact preg-
nancy-induced proliferation of mammary epithelial cells,
several mechanisms are possible. Reduced epithelial cell
number may be due to cell cycle arrest that results in fewer
cells in the S-phase of the cell cycle. TCDD has been shown to
cause fewer cells to progress from the G1 to S phase in an
hepatocyte-derived cell line, and the cell cycle inhibitor p21cip1
has been implicated the in this process (Barnes-Ellerbe et al.,
2004; Kolluri et al., 1999). However, Mitchell et al. (2006),
using liver regeneration as a model, found TCDD-mediated cell
cycle arrest to be independent of p21cip1. To determine the role
of p21Cip1in the cell cycle effects observed in our in vivo
model, we examined the effects of TCDD on the development
of mammary glands in pregnant p21Cip1knock out (p21KO)
mice and wild-type controls. Exposure to TCDD during
pregnancy impaired mammary gland development in p21KO
mice to the same extent as in wild type mice (Supplementary
Data). Thus, similar to the previous study (Mitchell et al.,
2006), our data suggest that the effects of TCDD on mammary
gland development during pregnancy are due to cell cycle
arrest, but that this arrest is not dependent on the presence of
the cell cycle inhibitor p21Cip1.
In addition to impacting cell cycle progression, the
impairment in mammary development caused by exposure to
TCDD could be due to premature involution of the gland.
Mammary gland involution is a process of apoptosis and
remodeling that occurs naturally in the mammary tissue in
response to weaning (Quarrie et al., 1996; Richert et al., 2000).
TCDD and other AhR ligands have been reported to increase
apoptosis in some experimental systems (Bock and Kohle,
2005; Puga et al., 2009; Ray and Swanson, 2009). However, in
our study, exposure to TCDD did not increase mammary
epithelial cell apoptosis during pregnancy. This is consistent
with several reports that TCDD treatment had either no effect
or reduced apoptosis in some systems (Davis et al., 2003;
Mitchell and Lawrence, 2003; Mitchell et al., 2006; Stinch-
combe et al., 1995; Teske et al., 2005). AhR ligands impact
apoptosis in a highly tissue and context-specific manner. In
systems where the development rate is high, such as in
differentiating mammary glands or regenerating liver, AhR
activation seems to have no effect on apoptosis rate; whereas in
tissues with an inherently high rate of apoptosis, such as the
thymus, AhR ligands may further enhance this process.
Regardless of differences in the impact of AhR activation on
apoptosis among these different systems, our data indicate that
exposure to TCDD did not alter apoptosis of mammary
epithelial cells during pregnancy. Thus rather than induction of
early involution, the impairment in pregnancy-associated
mammary development after exposure to TCDD is due, at
least in part, to a decrease in mammary epithelial cell
An intriguing observation is the presence of TEBs in
mammary glands collected from TCDD-treated animals on
DP6. TEBs are sites at which cells rapidly divide to advance
the elongation of ducts into the fat pad, and are typically seen
in pubescent animals (Humphreys et al., 1996). They disappear
once the entire fat pad has been filled with ducts, which
normally occurs in rodents at 12 weeks of age (Hennighausen
and Robinson, 2005). The mice in this study were 6 weeks old
when paired for breeding, and at this age TEBs are still present
in virgin animals. However, TEBs should no longer be present
by the DP6 (Brisken, 2002). The presence of these structures in
glands collected from pregnant animals exposed to TCDD is of
interest because it clearly denotes retardation in the process of
mammary development. Moreover, TEBs have been shown to
play a key role in mammary cancer development as they
represent structures that are most immature, proliferative and
thus susceptible to carcinogenesis (Jenkins et al., 2007; Russo
and Russo, 1978). In fact, the number of TEBs following
prenatal exposure to TCDD was decreased in pre-pubertal
young rats (Fenton et al., 2002) and increased in more mature
virgin animals (Brown et al., 1998; Lewis et al., 2001).
Overall, the previous reports concluded that in utero exposure
to TCDD results in mammary glands that are less differentiated
and more susceptible to carcinogenic exposure (Brown et al.,
1998; Fenton et al., 2002; Jenkins et al., 2007; Lewis et al.,
2001). Likewise, in our study the mammary glands from mice
exposed to TCDD early in pregnancy and collected on DP6
were immature and lacked substantial lobule development
when compared with control animals. These data suggest that
AhR activation during early pregnancy impairs mammary
development by a decrease in epithelial cell proliferation with
consequent abnormalities in ductal branching morphogenesis
and formation of mature lobules.
With progression of pregnancy, these mature lobules
populate the stromal tissue with alveolar-like units that
eventually become the milk-producing units of the mammary
gland (Hennighausen and Robinson, 2001). Thus we in-
vestigated if exposure to TCDD in early pregnancy is necessary
and sufficient to impair functional development of the
mammary gland. Using b-casein expression as a marker of
differentiation (Desprez et al., 1995), our data suggest that
glands collected from animals exposed to TCDD only on DP0
were potentially able to produce milk, which would suggest
that when AhR activation is not sustained throughout
pregnancy the gland partially recovers. The partial impairment
is likely due to decreased or abnormal ductal branching
morphogenesis, rather than diminished lobule formation and
alveolarization. The basis for this is that branching morpho-
genesis takes place in the beginning of pregnancy, which is
when TCDD was administered. In contrast, alveolarization
starts around DP7. In mice after DP14, alveoli are found all
EXPOSURE TO TCDD AND MAMMARY DEVELOPMENT
over the mammary gland, proliferation basically ceases and
functional differentiation leads to lactogenesis (Brisken, 2002).
Therefore, because the half-life of TCDD in rodents is about 8–
10 days (Gasiewicz et al., 1983) it is possible that animals only
treated on DP0 did not maintain a sufficient level of TCDD in
the system to impair later events of mammary development and
lactogenesis. In contrast, in glands collected from animals that
were exposed to TCDD starting later in pregnancy, the effects
are likely due to a decrease in lobule formation and
differentiation into alveoli. At the time of first exposure to
TCDD (DP7) these glands were already populated in terms of
branching, as this processes starts early in pregnancy
(Hennighausen and Robinson, 1998). However, proliferation
was still sensitive to TCDD treatment, resulting in fewer BrdU-
positive cells and a reduction in the formation of lobulo-
alevolar units. Taken together, our data suggest that an early
exposure to TCDD is neither necessary nor sufficient to cause
sustained and complete impairment in pregnancy-associated
mammary development. Furthermore, AhR activation after
DP7 appears to affect alvelolarization—by the time of first
exposure to TCDD, the mammary gland was already populated
with side branching.
The new findings reported herein suggest that exposure to
TCDD during pregnancy influences proliferation in early
pregnancy and differentiation in mid-pregnancy, and that these
effects may occur through independent mechanisms. For
example, the mechanisms that result in decreased proliferation
appear to be independent of defects in secretory differentiation
and lactogenesis. These findings explain, in part, the complex
biological puzzle involving activation of AhR following
exposure to TCDD during organogenesis and development.
Our data suggest there are critical windows during pregnancy
in which exposure to TCDD disrupts mammary gland
development; depending on time of exposure, there are
different outcomes in mammary gland development. Given
that the mammary gland provides an excellent model for
studying developmental processes because it develops mostly
postpartum, our findings may be useful when considering
mechanisms that underlie AhR-mediated alterations in pro-
liferation and differentiation in other developing tissues.
In addition to providing further insight into how AhR
regulates proliferation and differentiation, our findings address
an area that is clinically relevant but receives very little
attention. The American Association of Pediatrics recommends
that all infants receive breast milk for at least the first six
months after birth, and the consumption of breast-milk has
been found to be highly beneficial, with demonstrated effects
on brain development and metabolic status (Morley et al.,
2004; Mortaz et al., 2001; Singhal et al., 2004). Although the
data are limited, it is estimated that that each year 3–6 million
mothers of live infants worldwide are either unable to or have
significant difficulty initiating breast-feeding. The cause of this
problem is not clear, and numbers may be larger than this
estimate because lactation success receives insufficient atten-
tion. However, it has been suggested that exposure to
environmental contaminants, phytochemicals, and drugs may
adversely impact pregnancy-associated mammary differentia-
tion and milk production (Neville and Walsh, 1995). We show
definitively that exposure to the pollutant dioxin has a profound
and detrimental impact on mammary development during
pregnancy, resulting in reduced induction of milk proteins and
failure to nutritionally support offspring. Although it is
unknown whether dioxin-like compounds have similar effects
in humans, the regulatory processes in mice and humans are
sufficiently similar to speculate that environmental contami-
nants negatively impact human lactation.
Supplementary data are available online at http://toxsci.
National Institutes of Health (R01-ES013958 and K02-
ES012409) to B.P.L., (HL67392) to M.A.R., and (P30-
ES01247) EHSC Center Grant, and a ‘‘Seed the Scientist’’
award from the Art BeCAUSE Foundation to B.J.L.
We gratefully acknowledge Dr Suzanne Fenton (Environ-
mental Protection Agency) for sharing her knowledge of
scoring mammary glands and her helpful feedback with regard
to our data, Dr Beth A. Vorderstrasse (Washington State
University) for helpful discussion and thoughtful comments,
and Dr Ravikumar Manickam (University of Rochester) for his
help scoring the mammary glands.
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