1. PRENATAL TCDD EXPOSURE ALTERS CELL PROLIFERATION, APOPTOSIS,
AND PROTEIN EXPRESSION IN THE SPRAGUE-DAWLEY RAT UTERUS
by
VIVEK KALIA
CORAL A. LAMARTINIERE, COMMITTEE CHAIR
ASIM K. BEJ
STEPHEN A. WATTS
A THESIS
Submitted to the graduate faculty of The University of Alabama at Birmingham,
in partial fulfillment of the requirements for the degree of
Master of Science
BIRMINGHAM, ALABAMA
2006
2. PRENATAL TCDD EXPOSURE ALTERS CELL PROLIFERATION, APOPTOSIS,
AND PROTEIN EXPRESSION IN THE SPRAGUE-DAWLEY RAT UTERUS
VIVEK KALIA
ABSTRACT
Commonly known as dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has
been recognized as a potent human carcinogen, yet it remains ubiquitous in the environ-
ment as a byproduct of incineration procedures and waste disposal. It is a well-studied
endocrine disrupting chemical that alters cellular organization on a number of levels, both
macroscopically and at the molecular level. In this study, the goal was to determine the
modulatory effects TCDD has on uterine protein expression, cell proliferation, and apop-
tosis in a rat model. Pregnant Sprague-Dawley CD rats were treated with 3 μg TCDD/kg
body weight by gavage on gestational day 15. Female offspring were transferred to sur-
rogate mothers to minimize post-birth exposure to TCDD via lactation. Female offspring
were then sacrificed at day 21 and day 50, postpartum, and uteri were dissected out for
processing and analysis. Western immunoblot analysis and cell proliferation and apop-
tosis analysis were used to assess the differences between the control and TCDD-treated
animals at both 21 and 50 days of age.
By Western blot analysis, we found no differences in the control and TCDD-
treated animals at 21 days of age in a very wide set of biomarkers including growth fac-
tors, signal transducers, proinflammatory cytokines, cell survival proteins, steroid recep-
tors, and steroid receptor coactivators. At 50 days of age, we found six proteins to be dif-
ferentially regulated by TCDD:SOD1 (↓), SRC-1 (↓), SRC-2/GRIP-1 (↓), SRC-3 (↓),
EGFR (↑), and IGF-BP3 (↓). In cell proliferation analysis, we found a significant down-
regulation in both the glandular (four-fold decrease) and luminal epithelium (two-fold
ii
3. decrease) at 21 days of age. In the 50 day animals, we found a significant up-regulation
only in the luminal epithelium (two-fold increase). In apoptosis analysis, we found no
difference between control and TCDD-treated animals at 21 days, but a very significant
down-regulation of apoptosis at 50-days in both the glandular (ten-fold decrease) and lu-
minal epithelium (four-fold decrease). TCDD can modulate uterine proteins that are
known to play a role in uterine growth and disease as well as alter epithelial cell prolif-
eration and apoptosis in a manner that may enhance cancer susceptibility.
iii
4. DEDICATION
I dedicate this work to my loving family and closest friends, without whom I
would not have been able to come this far. You have all been a blessing in my life, and
for that, I am very grateful.
iv
5. ACKNOWLEDGMENTS
To begin, I thank my mentor Dr. Coral A. Lamartiniere. He has always been very
supportive of my research efforts and without his guidance and expertise I would have
been led astray many times. In addition, I appreciate the funding that was provided by Dr.
Lamartiniere that enabled me to pursue this master’s degree. I hope that my work will be
of great benefit to his lab in the future.
Next, I thank my other committee members, Dr. Stephen A. Watts and Dr. Asim
K. Bej. They have both been extremely helpful and insightful throughout this process. I
appreciate the time and efforts spent on helping to further me in my academic careers.
Next, I thank Mr. Timothy Whitsett for his assistance in the experimental and
written phases of this project. Without his help, I would have had many more roadblocks
in my intellectual path that would have prevented me from completing this research.
I thank Dr. Jun Wang, whose technical assistance and knowledge in the area of
cancer research helped me on countless occasions during my work.
Last, I thank my other coworkers for being supportive of my efforts and receptive
to my results, always encouraging me along the way. It was truly a pleasure to work in
this laboratory.
v
6. TABLE OF CONTENTS
Page
ABSTRACT........................................................................................................................ ii
DEDICATION................................................................................................................... iv
ACKNOWLEDGMENTS ...................................................................................................v
LIST OF TABLES........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
LIST OF ABBREVIATIONS..............................................................................................x
INTRODUCTION ...............................................................................................................1
MATERIALS AND METHODS.........................................................................................7
2.1 Animals..................................................................................................................7
2.2 TCDD exposure.....................................................................................................7
2.3 Cell proliferation analysis......................................................................................8
2.4 Apoptosis analysis .................................................................................................9
2.5 Western immunoblot analysis..............................................................................10
2.6 Statistical analysis................................................................................................12
RESULTS ..........................................................................................................................13
3.1 Body weights, uterine weights, and serum hormone levels.................................13
3.2 Cell proliferation..................................................................................................14
3.3 Apoptosis.............................................................................................................14
3.4 Uterine protein biomarkers..................................................................................19
DISCUSSION...................................................................................................................22
4.1 Body weights, uterine weights, and serum hormone levels.................................22
4.2 Changes in cell proliferation................................................................................23
4.3 Changes in apoptosis ...........................................................................................26
4.4 Changes in protein expression levels...................................................................28
4.5 Future directions..................................................................................................33
vi
7. TABLE OF CONTENTS (Continued)
Page
LIST OF REFERENCES...................................................................................................35
APPENDIX
A INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE
APPROVAL FORM..............................................................................................40
B WESTERN BLOTS OF PROTEINS SIGNIFICANTLY REGULATED BY
PRENATAL TCDD...............................................................................................42
vii
8. LIST OF TABLES
Table Page
1 Body and uterine weights, and uterine to body weight ratios in 21- and
50-day-old female rats exposed prenatally to TCDD.................................................13
2 Cell proliferation index in 21- and 50-day-old female rats exposed prenatally to
TCDD .........................................................................................................................19
3 Apoptosis index in 21- and 50-day-old female rats exposed prenatally to TCDD ....19
viii
9. LIST OF FIGURES
Figure Page
1 Structure of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ..........................................2
2 Cell proliferation staining in uteri of 21-day-old rats exposed prenatally to TCDD
or sesame oil...............................................................................................................15
3 Cell proliferation staining in uteri of 50-day-old rats exposed prenatally to TCDD
or sesame oil...............................................................................................................16
4 Apoptosis staining in uteri of 21-day-old rats exposed prenatally to TCDD
or sesame oil...............................................................................................................17
5 Apoptosis staining in uteri of 50-day-old rats exposed prenatally to TCDD
or sesame oil...............................................................................................................18
6 Protein biomarkers in uteri of 50-day-old rats exposed prenatally to TCDD and
sesame oil ...................................................................................................................21
ix
10. LIST OF ABBREVIATIONS
AhR aryl hydrocarbon receptor
AIB1 amplified in breast cancer 1
AIN American Institute of Nutrition
AR androgen receptor
ARNT aryl hydrocarbon receptor nuclear translocator
BAX BCL-associated X protein
BCL2 B-cell leukemia/lymphoma 2
BW body weight
CYP1A1 cytochrome P450, family 1, subfamily A, polypeptide 1
DAB 3,3' diaminobenzidine
DES diethylstilbestrol
DMBA 7,12-dimethylbenz(a)anthracene
EGF1 epidermal growth factor 1
EGFR epidermal growth factor receptor
ERα estrogen receptor α
ERβ estrogen receptor β
ERK extracellular regulating kinase
GRIP-1 glucocorticoid receptor interacting protein 1
IARC International Agency for Research on Cancer
ICR Institute for Cancer Research
x
12. 1
INTRODUCTION
In the past few decades, toxic impurities and contaminants in the environment
have gained much attention in the scientific community. It is becoming increasingly ap-
parent that estrogen exposure or exposure to estrogen-mimicking compounds is a major
risk factor for the development of breast cancer (1). Commonly known as dioxin, 2,3,7,8-
tetrachlorodibenzo-p-dioxin (TCDD) is one particular compound of growing concern for
its potential for endocrine disruption.
The polycyclic aromatic hydrocarbon TCDD (Fig. 1) is an extremely toxic envi-
ronmental contaminant that is formed during industrial incineration of wastes containing
polychlorinated benzenes and chlorophenoxy ethers (2). It is often referred to as the pro-
totype of the dioxin family and as the most toxic man-made chemical (3). TCDD has
been shown to modulate multiple growth factor signaling pathways, such as the epider-
mal growth factor (EGF) and the tumor necrosis factor α and β (TGFα and TGFβ) path-
ways. It has also been shown to alter cytokine and protooncogene expression levels (3).
Malformations such as splenic and thymic atrophy and cleft palate are common results of
in utero TCDD exposure (4).
Gray and Ostby have demonstrated that the external genitalia of female offspring
of Long Evans hooded rats exposed prenatally to TCDD (via the dam receiving 1 μg
TCDD/kg BW) were malformed, with some animals having partially cleft phallus, some
having completely cleft phallus, and others with vaginal threads (5).
14. 3
The malformations seen after gestational TCDD exposure can also be observed by
late gestational exposure to potent estrogens such as diethylstilbestrol (DES) and estra-
diol, though a much higher dose of other estrogens is required to induce similar malfor-
mations. The same study showed that TCDD treatment altered estrous cyclicity when ani-
mals reached 1 year of age, but not at 4 months and 5 months (the two earlier timepoints
when estrous cyclicity was evaluated). At necropsy, it was reported that TCDD caused a
slight decrease (though not statistically significant) in the weight of the ovaries and the
weight of the female reproductive tract (vagina, cervix, and uterus). Interestingly, it has
been reported that TCDD does not induce urogenital abnormalities and malformations
(no hypospadias, undescended testes, cleft phallus, or agenesis of the ventral prostate) in
male offspring (exposed at the same developmental stages) as it does in the females in an
antiandrogenic manner; it has thus been hypothesized that the female fetus may be more
sensitive to TCDD than the male (6).
It was reported that a number of important changes were present in the female re-
productive tract because of prenatal exposure to TCDD (7). Gray reported that rats prena-
tally exposed to TCDD on gestation day 15 had ovarian neoplasms, Sertoli cell tumors in
the female reproductive tract, and an increased incidence and severity of ovarian intersti-
tial hyperplasia. Additionally, growth and viability were reduced at prenatal doses of 0.8
and 1.0 μg TCDD/kg BW. Mann also reported gross malformations of the external geni-
talia of female rat offspring exposed to TCDD in utero (8). For cross-species comparison
purposes, it was reported by Theobald and Peterson that perinatal TCDD exposure had no
significant effect on dam and offspring body weights in outbred ICR mice (9).
15. 4
In a recent review, it was reported that dioxins cause effects at all levels of bio-
logical organization, affecting metabolism, macroscopic organ and tissue function, cellu-
lar communication mechanisms, and enzyme functioning (10). Safe et al. showed that
TCDD can alter the metabolism of estrogen by way of the induction of biotransformation
enzymes, certain types of which (e.g. cytochromes P450 and flavin monooxyenases) pro-
tect our bodies from harmful chemicals in the environment (11).
Not only is TCDD an endocrine disruptor; it is a known human carcinogen, as
proclaimed by the International Agency for Research on Cancer (IARC) in 1997. This
classification of TCDD as a carcinogen has been reviewed and evaluated since that time
(12). It was also reported that prenatal TCDD treatment led to twice as many mammary
tumors per rat several months later in rats treated at sexual maturity with 7,12-
dimethylbenz(a)anthracene (DMBA) (13). In that study, an increased number of terminal
end bud structures was observed at the time of DMBA exposure in TCDD-exposed ani-
mals, suggesting that TCDD delayed gland maturation. Furthermore, Fenton et al. found
that females perinatally-exposed to TCDD weighed significantly less than their control
counterparts and that peripubertal animals had delayed vaginal opening and persistent
vaginal threads (14). These investigators also noted that mammary glands taken from 4-
day-old offspring exposed perinatally to TCDD had reduced primary branches, decreased
epithelial elongation, and significantly fewer alveolar buds and lateral branches. Though
control animals developed well-differentiated terminal structures by postnatal day 68,
TCDD-exposed animals retained undifferentiated terminal structures. These determina-
tions were made by whole mount analyses on postnatal days 4, 33, 37, 45, 68, and 110.
The developmental defects and delays in migration of ductal structures through the
16. 5
mammary fat pad were consistently detectable in the whole mounts. Female offspring
exposed to TCDD on gestation day 15 exhibited stunted progression of epithelium
through the fat pad, decreased numbers of lateral branches, and delayed lobule formation
(14).
Sex steroid and growth factor signaling pathways are vital in the processes of de-
velopment and differentiation of hormone-responsive tissues such as the rat uterus. In re-
cent years it has been proposed that the uterus is particularly susceptible to environmental
factors such as dioxin, leading to diseases such as endometriosis and uterine cancer. Rier
et al. reported an increased incidence of endometriosis in rhesus monkeys following
chronic dietary exposure to TCDD (15). The severity of the disease corresponded to the
dosage of TCDD given (0, 5, or 25 ppt/day for 4 years).
Because of the well-established detrimental effects prenatal exposure to TCDD
has on the mammary gland (including delayed maturation and increased susceptibility to
carcinogenesis), potential for carcinogenicity and disease in other reproductive organs
must be addressed. The goal of this research is to identify proteins that are differentially
regulated in the uterus of prepubertal and sexually-mature rats exposed prenatally to
TCDD. The uterus has been reported to accumulate TCDD at very high levels, equivalent
to levels in the liver but lower than the fat tissue (16). In this study, we analyzed uterine
sections histologically to identify patterns of cell proliferation and apoptosis in 21- and
50-day old animals in control and TCDD-exposed groups and focused on proteins that
play a role in regulating sex steroid signaling (ERα, ERβ, AR, PR, SRC-1, SRC-2/GRIP-
1, SRC-3), apoptosis (BCL2, BAX), cell proliferation (Ki67, AKT, P-AKT), growth fac-
17. 6
tor signaling (IGF1, IGF1-R, IGF-BP3, EGF1, EGFR, ERKs, P-ERKs), and detoxifica-
tion/activation of dioxin mechanisms (SOD1, CYP1A1, AhR, ARNT).
18. 7
MATERIALS AND METHODS
2.1 Animals
Animal studies were performed according to the guidelines and protocols ap-
proved by the UAB Institutional Animal Care and Use Committee (Appendix A). We
purchased female Sprague-Dawley CD rats from Charles River Breeding Laboratories
(Raleigh, NC). All animals were fed AIN-93G base diet (Harlan Teklad, Madison, WI).
AIN-93G is a purified diet containing no detectable estrogens. TCDD was obtained from
Cambridge Isotope Laboratories Inc. (Andover, MA).
Sixty female rats were bred and the date of conception of each female (when
sperm is present in the vagina) was determined by doing vaginal smears daily according
to the protocol outlined by Cooper et al. (17). Animals were maintained with food and
water available ad libitum. Polypropylene cages and water bottles were used for housing
the rats and for the drinking supply. Animals were maintained on a light:dark cycle
(12:12) with lights on at 0800 hr and off at 2000 hr. The animal room temperature was
kept at 22 °C.
2.2 TCDD exposure
Pregnant females were treated with TCDD by gavage at a concentration of 3
μg/kg BW. The TCDD treatment was administered on day 15 postconception (recogni-
tion of sperm in vagina designated as day 1). Controls received an equivalent volume of
sesame oil on the same schedule. Pregnant females were gavaged in an alternating
19. 8
fashion until we reached 30 animals per treatment group. After birth, offspring were
weighed at 21- and 50-days of age.
At day 21 and day 50 postpartum, female offspring were weighed and subse-
quently anesthetized using ketamine and xylazine. Following sedation of the rats, live
collections of the uteri were performed in order to minimize protein degradation. Then,
animals were killed by decapitation and trunk blood was immediately collected. The
blood was centrifuged at 2300 revolutions per minute for 15 min, and serum was col-
lected and frozen at –80 °C. Tissues collected were weighed and paraffin blocked or fro-
zen in liquid nitrogen until tissue processing. Estradiol-17β and progesterone concentra-
tions in the serum were determined via radioimmunoassay in both sesame oil- and
TCDD-exposed animals.
2.3 Cell proliferation analysis
IHC analysis of Ki67, a protein expressed in all phases of active cell cycle, was
used as an indicator of cell proliferation. Paraffin-embedded uteri were deparaffinized in
xylene and rehydrated in a series of graded alcohols (100, 95, 70, 50%). Samples were
then washed in dH2O followed by PBS. Antigen retrieval was performed using the Vector
Antigen Unmasking Solution from Vector Laboratories (Burlingame, CA). Specimens
were boiled in the Antigen Unmasking Solution for 20 min and then allowed to cool to
room temperature. Endogenous peroxidases activity was blocked by incubating speci-
mens in 3% hydrogen peroxide (H2O2) at room temperature for 10 min. Blocking was
done using ready-to-use 2.5% Normal Horse Serum from the ImmPRESSTM
Reagent Kit
(Anti-Mouse Ig) from Vector Laboratories. Next, a monoclonal mouse anti-rat Ki-67 an-
20. 9
tigen antibody from DakoCytomation (Carpinteria, CA) was applied to the specimens for
30 min followed by washes in phosphate-buffered saline (PBS). A ready to use
ImmPRESSTM
Reagent secondary antibody was then applied to the samples for 30 min
followed by washes in PBS. For staining Ki67 antigen, a Peroxidase Substrate Kit, 3,3'
diaminobenzidine (DAB), was applied to samples for 10 min followed by a wash in tap
water for 5 min. To counterstain, hematoxylin QS was applied to the specimens for 60
sec followed by a dip in tap H2O for 10 sec. Clearing was performed by immersing the
specimens in a series of graded alcohols and then xylene. Specimens were mounted and
coverslips were applied using Vector Mounting Media (Burlingame, CA). The glandular
and luminal epithelial cells stained for Ki67 were counted as well as the total number of
epithelial cells per uterine section (at least 1000 cells). Sections from six animals in both
the control and treatment groups were analyzed and counted. A proportion score (prolif-
erative index) was given by the number of stained cells / total number of cells counted x
100.
2.4 Apoptosis analysis
The ApopTag®
Plus Peroxidase In Situ Apoptosis Detection Kit (Chemicon Inter-
national, Temecula, CA) was used to measure apoptosis following the manufacturer’s in-
structions. Briefly, paraffin-embedded tissue sections were deparaffinized and rehydrated
in graded alcohols (100, 95, and 70%). Tissues were treated with freshly diluted Pro-
teinase K (20 μg/mL) from Qiagen (Valencia, CA) for 15 min at room temperature and
then washed in dH2O. Endogenous peroxidases were inactivated with 3% H2O2 in PBS
for 5 min and then washed in PBS. Equilibration buffer was then added to the sample for
21. 10
20 min, followed by a 1-hr application of Terminal deoxynucleotidyl Transferase (TDT)
enzyme in a humidified chamber at 37 °C. Next, stop/wash buffer was added followed by
a 30 min incubation with an anti-digoxignenin conjugate at room temperature. Tissues
were then washed four times in PBS. To develop color in the peroxidase substrate,
specimens were covered with DAB substrate diluted by DAB dilution buffer for 10 min
at room temperature. Specimens were washed and then counterstained with 0.3% methyl
green for 10 min. Next, sections were washed in 3 changes of dH2O followed by three
washes in 100% n-butanol. The specimens were cleared using graded alcohols and xylene
and then mounted using Permount mounting solution and coverslips. The apoptotic index
was defined as the number of epithelial cells stained positive for apoptosis divided by the
total number of epithelial cells counted x 100. Separate indices were determined for glan-
dular and luminal epithelia. Visualization was performed using a Nikon light microscope
(with a 40x objective lens magnification) and Nikon digital camera, and images were
analyzed using Image J software (National Institutes of Health, Bethesda, MD).
2.5 Western immunoblot analysis
The following biomarkers were measured using western immunoblot analysis and
enzyme-linked immunosorbent assay (ELISA): aryl hydrocarbon receptor (AhR), ampli-
fied in breast cancer 1/steroid receptor coactivator 1 (AIB1/SRC-3), androgen receptor
(AR), aryl hydrocarbon receptor nuclear translocator (ARNT), BCL-associated X protein
(BAX), B-cell leukemia/lymphoma 2 (BCL2), cytochrome P450 family 1 subfamily A
polypeptide 1 (CYP1A1), epidermal growth factor 1 (EGF1), epidermal growth factor re-
ceptor (EGFR), estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), extracellu-
22. 11
lar regulating kinases (ERKs), glucocorticoid receptor interacting protein 1/steroid recep-
tor coactivator 2 (GRIP-1/SRC-2), insulin-like growth factor 1 (IGF1), IGF1 receptor
(IGF1-R), insulin-like growth factor binding protein 3 (IGF-BP3), phosphorylated EGFR
(P-EGFR), phosphorylated ERKs (P-ERKs), phosphorylated protein kinase B/P-AKT (P-
PRB/P-AKT), progesterone receptors (PR-A and PR-B), protein kinase B/AKT
(PRB/AKT), superoxide dismutase 1 (SOD1), and steroid receptor coactivator 1 (SRC-1).
Uteri of animals were processed and western immunoblot analysis was performed
using the protocol detailed by Wang et al. (18). Samples were homogenized using a mix-
ture of 1x RIPA Lysis Buffer (Upstate®
Cell Signaling Solutions) and the following pro-
tease inhibitors: Leupeptin, Aprotinin, Vanadate, and Phenyl Methyl Sulfonyl Fluoride
(PMSF). The samples were ground using a Sample Grinding Kit (Amersham Biosci-
ences, Piscataway, NJ) following the manufacturer’s protocol.
The protocol used for western immunoblot analysis included a Bradford protein
assay (BioRad, Hercules, CA), which was performed in duplicate to determine protein
concentrations for each sample. Equal amounts of protein extract were electrophoresed
using Criterion SDS-PAGE from BioRad and then transferred onto nitrocellulose mem-
branes. The membranes were blocked with 5% skim milk in wash buffer (containing 1 x
BioRad Tris-Buffered Saline with Tween 20) and incubated overnight with appropriate
primary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA; BD Biosciences,
San Jose, CA ; Cell Signaling Technology, Beverly, MA). The following antibodies were
used: ERα (C-311/sc-787) (Santa Cruz); ERβ (H-150/sc-8974) (Santa Cruz); PR (C-19/
sc-538) (Santa Cruz); AR (N-20/sc-816) (Santa Cruz); SRC-1 (M-341/sc-8995) (Santa
Cruz); SRC-2/GRIP-1/TIF2 (Cat:610985) (BD BioSciences); SRC-3/AIB1 (Cat:611105)
23. 12
(BD BioSciences); BCL2 (ΔC 21/sc-783) (Santa Cruz); BAX (P-19/sc-526) (Santa Cruz);
cyclin D1 (C-20/sc-717) (Santa Cruz); p21 (M-19/sc-471) (Santa Cruz); AKT (C-20/sc-
1618) (Santa Cruz); P-AKT (Ser-473 – R/sc-7985-R) (Santa Cruz); IGF1-R (H-78/sc-
7952) (Santa Cruz); IGF-BP3 (H-98/sc-9028) (Santa Cruz); EGFR (1005/sc-03) (Santa
Cruz); phospho-EGFR (Tyr1068 / # 2234S) (Cell Signaling Technology); p44/42 MAP
Kinase (#9102) (Cell Signaling Technology); phospho-p44/42 MAP Kinase (#9101S)
(Cell Signaling Technology); SOD1 (C-17/sc-8637) (Santa Cruz); CYP1A1 (G-18/sc-
9828) (Santa Cruz); AhR (N-19/sc-8088) (Santa Cruz); and ARNT (H-172/sc-5580)
(Santa Cruz). Following washes, the membranes were incubated with the appropriate
horseradish peroxidase-conjugated secondary antibodies. Membranes were washed one
final time and subjected to chemiluminesence with SuperSignal West Dura Extended Du-
ration Substrate from Pierce Biotechnology (Woburn, MA). The relative intensity of the
protein bands was obtained by autoradiogram and scanned using a VersaDoc 4000 densi-
tometer (BioRad). Quantity One software (BioRad) was used to quantify band intensity.
Proteins were blotted and confirmed at least twice for verification.
2.6 Statistical analysis
Statistics were performed using Microsoft Excel 2003 software. Student’s t-test
was used to perform comparative analysis between control (sesame oil) and treatment
(TCDD) groups. Statistical significance was defined as a P value of < 0.05. Outliers were
not included in calculations and were determined using the Grubbs outlier test (19).
24. 13
RESULTS
3.1 Body weights, uterine weights, and serum hormone levels
Twenty-one day old rats exposed prenatally to TCDD as compared to sesame oil
(controls) had significantly decreased body weight (20%; Table 1). Uterine wet weights
were slightly but not significantly decreased in TCDD-exposed rats (11%), and the calcu-
lated uterine to body weight ratios were not significantly different (12% increase) in the
21-day-old animals. At 50 days postpartum, TCDD-exposed rats had slight but signifi-
cantly reduced body weights (8%; Table 1) compared to controls. However, uterine and
uterine to body weight ratios were not significantly different between the two groups
(11.5 and 3.5% decreases, respectively).
Table 1
Body and uterine weights, and uterine to body weight ratios in 21- and 50-day-old female
rats exposed prenatally to TCDD
Animal Group (n) Body Weight
(g)
Uterine
Weight (mg)
Uterine to Body
Weight Ratio
(mg/g)
Treatment
21 day control (10) 54.6 ± 2.01 32.2 ± 2.34 0.59 ± 0.04 Sesame oil
21 day TCDD (10) 43.9 ± 2.98b
28.5 ± 2.10 0.67 ± 0.06 3 µg TCDD/kg BW
50 day control (20) 198.9 ± 3.41 338.5 ± 15.20 1.71 ± 0.08 Sesame oil
50 day TCDD (20) 183.7 ± 3.50a
299.5 ± 13.80 1.65 ± 0.17 3 µg TCDD/kg BW
Timed pregnant Sprague-Dawley CD female rats were gavaged with 3 μg TCDD/kg
body weight or an equivalent volume of sesame oil (controls) on day 15 post-conception.
At birth, offspring were cross-fostered to untreated dams (surrogate mothers). Each
treatment group contained 30 dams. Only one female from each litter was used at 21 and
50 days of age. Values represent means ± SEM. a
P < 0.005 and b
P < 0.010 as compared
with age-matched controls.
25. 14
Estradiol-17β and progesterone concentrations were not found to be different be-
tween control and TCDD-exposed groups. Control animals had concentrations of 13.7 ±
2.4 pg estradiol-17β/mL serum and 15.0 ± 2.4 ng progesterone/mL serum, while TCDD-
exposed animals had concentrations of 16.2 ± 3.2 pg estradiol-17β/mL serum and 16.9 ±
3.1 ng progesterone/mL serum.
3.2 Cell proliferation
Cell proliferation was monitored via Ki67 antigen IHC in the uterus. The two
types of epithelial cells counted, glandular and epithelial, were counted separately and
analyzed individually to determine differences between control and TCDD-exposed rats
at both 21 and 50 days. In the glandular and luminal epithelium of 21-day-old rats ex-
posed prenatally to TCDD compared to controls, cell proliferation was significantly de-
creased by four- and two-fold, respectively (Table 2 and Fig. 2). On the other hand, in 50-
day-old rats exposed prenatally to TCDD cell proliferation was significantly increased
(two-fold) in the luminal epithelium but there was no significant change of cell prolifera-
tion in the glandular epithelium of these sexually mature animals (Table 2 and Fig. 3).
3.3 Apoptosis
In 21-day rats exposed prenatally to TCDD, the rate of apoptosis was not signifi-
cantly changed in the glandular and luminal epithelia (Table 3 and Fig. 4). On the other
hand, in uteri of 50-day-old rats apoptosis was significantly decreased by ten- and four-
fold in the glandular and luminal epithelia, respectively following prenatal TCDD expo-
sure (Table 3 and Fig. 5).
26. 15
CONTROL
Positive for Proliferation (LE)
Positive for Proliferation (GE)
TCDD
Positive for Proliferation (GE)
Positive for Proliferation (LE)
Fig. 2. Cell proliferation staining in uteri of 21-day-old rats exposed prenatally to TCDD
or sesame oil. Both glandular (GE) and luminal epithelium (LE) can be seen in each pic-
ture. DAB staining (brown) for Ki67 was counted as indicator of cell proliferation in the
glandular and luminal epithelia. There is a significant down-regulation of cell prolifera-
tion in both the glandular and luminal epithelium of TCDD-exposed animals. Pictures
were taken at 400x magnification.
27. 16
CONTROL
Positive for Proliferation (LE)
Positive for Proliferation (GE)
TCDD
Positive for Proliferation (LE)
Positive for Proliferation (GE)
Fig. 3. Cell proliferation staining in uteri of 50-day-old rats exposed prenatally to TCDD
or sesame oil. Both glandular (GE) and luminal epithelium (LE) can be seen in each pic-
ture. DAB staining (brown) for Ki67 was counted as indicator of cell proliferation in the
glandular and luminal epithelia. There is a significant up-regulation of cell proliferation
in the luminal epithelium at 50 days and no change in the glandular epithelium. Pictures
were taken at 400x magnification.
28. 17
Positive for Apoptosis (GE)
Positive for Apoptosis (LE)
CONTROL
Positive for Apoptosis (GE)
Positive for Apoptosis (LE)
TCDD
Fig. 4. Apoptosis staining in uteri of 21-day-old rats exposed prenatally to TCDD or ses-
ame oil. Both glandular (GE) and luminal epithelium (LE) can be seen in each picture.
DAB staining (brown) was counted as indicator of apoptosis in the glandular and luminal
epithelia. There is no significant difference between control and TCDD-exposed groups
in either the luminal or glandular epithelium. Pictures were taken at 400x magnification.
29. 18
CONTROL
Positive for Apoptosis (GE)
Positive for Apoptosis (LE)
TCDD
Positive for Apoptosis (GE)
Negative for Apoptosis (LE)
Fig. 5. Apoptosis staining in uteri of 50-day-old rats exposed prenatally to TCDD or ses-
ame oil. Both glandular (GE) and luminal epithelium (LE) can be seen in each picture.
DAB staining (brown) was counted as indicator of apoptosis in the glandular and luminal
epithelia. There is a very significant down-regulation of apoptosis as detected by DNA
strand breaks in both the glandular and luminal epithelium. Pictures were taken at 400x
magnification.
30. 19
Table 2
Cell proliferation index in 21- and 50-day-old female rats exposed prenatally to TCDD
Animal Group (n) Glandular Epithelium Luminal Epithelium
21 day control (6) 5.18 ± 1.27 6.10 ± 0.91
21 day TCDD (6) 1.21 ± 0.52a
3.31 ± 0.42a
50 day control (6) 5.40 ± 1.03 7.83 ± 1.38
50 day TCDD (6) 5.91 ± 1.91 14.47 ± 3.27b
Timed pregnant Sprague-Dawley CD female rats were gavaged with 3 μg TCDD/kg
body weight or an equivalent volume of sesame oil (controls) on day 15 post-conception.
At birth, offspring were cross-fostered to untreated dams (surrogate mothers). Each
treatment group contained 30 dams. Only one female from each litter was used at 21- and
50-days of age. Values represent means ± SEM. a
P < 0.01 and b
P < 0.05 as compared
with age-matched controls.
Table 3
Apoptosis index in 21- and 50-day-old female rats exposed prenatally to TCDD
Animal Group (n) Glandular Epithelium Luminal Epithelium
21 day control (6) 10.00 ± 1.91 3.20 ± 0.46
21 day TCDD (6) 8.70 ± 1.55 4.10 ± 0.67
50 day control (6) 40.30 ± 3.50 43.40 ± 2.50
50 day TCDD (6) 4.00 ± 2.00 a
10.80 ± 2.50a
Timed pregnant Sprague-Dawley CD female rats were gavaged with 3 μg TCDD/kg
body weight or an equivalent volume of sesame oil (controls) on day 15 post-conception.
At birth, offspring were cross-fostered to untreated dams (surrogate mothers). Each treat-
ment group contained 30 dams. Only one female from each litter was used at 21- and 50-
days of age. Values represent means ± SEM. a
P < 0.000001 as compared with age-
matched controls.
3.4 Uterine protein biomarkers
Using western blot analysis, we investigated several proteins involved in cell pro-
liferation, apoptosis, activation/detoxification reactions, and sex steroid and growth factor
signaling. In uteri of 21-day-old rats exposed prenatally to TCDD compared to the con-
trols, we did not observe any significant changes in protein expression of AhR, SOD1,
ERα, ERβ, PR, SRC-1, SRC-2/GRIP-1, SRC-3/AIB1, p21, EGFR, IGF1-R, AKT, P-
AKT, and cyclin D1 (results not shown).
31. 20
In the uteri of 50-day-old rats, however, we did observe changes in some protein
biomarkers. SOD1, SRC-1, SRC-2/GRIP-1, SRC-3 and IGF-BP3 were found to be sig-
nificantly down-regulated in uteri of TCDD exposed rats by 17, 67, 74, 89, and 53%, re-
spectively (Fig. 6). On the other hand, EGFR was significantly up-regulated by two-fold
in uteri of 50-day-old rats exposed prenatally to TCDD. The following proteins were
blotted and their levels were not significantly altered at 50 days due to prenatal TCDD
exposure: Bcl-2, Bax, IGF1-R, AhR, PR, ARNT, AR, ERα, ERβ, CYP1A1, pERK,
tERK, and p21. Blots for these proteins are not shown. Western blots of proteins signifi-
cantly regulated by prenatal TCDD exposure are contained in Appendix B.
32. 21
Protein Biomarkers in Uteri of 50-Day-Old Rats Exposed
Prenatally to TCDD
300
ontrol)
0
50
100
150
200
250
Control
TC
DD
Control
TC
DD
Control
TC
DD
Control
TC
DD
Control
TC
DD
Control
TC
DD
ProteinExpression(PercentofC
SRC1SOD1 EGFR IGF-BP3SRC3GRIP1
p=0.003 p<0.0001 p=0.0019p=0.023 p=0.003
p=0.033
Fig. 6. Protein biomarkers in uteri of 50-day-old rats exposed prenatally to TCDD and
sesame oil. Western blot analysis was used to measure protein levels of SOD1, SRC-1,
SRC-2/GRIP-1, SRC-3, EGFR, and IGF-BP3. Representative protein blots are provided
for each protein and treatment. Values are shown as percent of control for each of the six
biomarkers. Each group contained 10 samples. Values represent mean ± SEM. Level of
statistical significance is listed above each set of graphs.
33. 22
DISCUSSION
Our laboratory is interested in the potential of TCDD as an endocrine disruptor,
focusing our attention on the rat mammary and uterus. Previously, we demonstrated that
exposure of pregnant rats on day 15 of gestation to 1 μg TCDD/Kg body weight resulted
in the adult offspring being more susceptible for chemically-induced mammary cancer.
Subsequent research efforts with this concentration of TCDD has not yielded any bio-
chemical basis for its actions in the mammary and uterus. Hence, we have increased the
TCDD concentration to 3 μg/kg BW and have investigated if this concentration would al-
ter body and uterine weights, circulating estrogen and progesterone concentrations, cell
proliferation and apoptosis, and specific uterine proteins associated with apoptosis, acti-
vation/detoxification mechanisms and sex steroid and growth factor signaling.
4.1 Body weights, uterine weights, and serum hormone levels
Our finding that prenatal TCDD exposure resulted in decreased body weights at
day 21 and day 50 postpartum is consistent with other reports that TCDD treatment (pre-
natal, perinatal, or prepubertal) results in decreased body weights in rats at doses that
range from 1 μg TCDD/kg BW to 75 μg TCDD/kg BW (13, 20-22). Although we did not
measure food intake in the present study, decreased appetite has been implicated to be a
contributing factor for the commonly seen decrease in body weights in TCDD-exposed
rats (23-25). Interestingly, the effect on body weight was more pronounced at day 21
compared to day 50 in the present study, from 20 to 8% decrease, respectively. This sug-
gests that the animals are able to “catch up” on body weight gain as they age, perhaps a
result of diminution of residual TCDD as the animals age or change in physiology.
34. 23
In this study, uterine wet weights were lower in the TCDD-exposed group at both
day 21 and day 50 postpartum, but the difference was not statistically significant. A study
done by Gray et al. using 1 μg TCDD/kg BW treatment of pregnant female rats on day 15
of gestation showed results consistent with our study: a slight, but not significant de-
crease in uterine weights in rats (5). Also, they showed that ovarian weight and the
weight of the female reproductive tract (vagina, cervix, and uterus) were slightly, but not
significantly, reduced. The downward trend in uterine weights may indicate a growth
suppressive effect on the uterus; this is similar to what occurs in the mammary gland,
which has been documented by several studies (10,13,14). These reports showed that
animals treated with TCDD have delayed maturation and growth of the mammary gland,
and the present study may indicate a similar effect for the rat uterus.
In the present study, no difference in the circulating estrogen (estradiol-17β) and
progesterone concentrations was detected at 50 days of age, suggesting that TCDD may
be exerting its antiestrogenic affects in another manner. Although no decrease in uterine
wet weight or decrease in circulating estrogen and progesterone concentrations was ob-
served in the present study, TCDD caused a significant decrease in cell proliferation in
both glandular and luminal epithelium of the uterus at 21 days, which may be attributed
to its well-documented antiestrogenicity. The signal transduction pathways involved in
bringing about this effect are unknown at this time.
4.2 Changes in cell proliferation
Using IHC, we stained and evaluated uterine sections for Ki67 antigen as an indi-
cator of cell proliferation. At 21 days postpartum, cell proliferation was down-regulated
35. 24
in both the glandular and luminal epithelial cells of animals exposed prenatally to TCDD.
The difference represents approximately a four-fold decrease in cell proliferation in the
glandular epithelium and a two-fold decrease in cell proliferation in the luminal epithe-
lium of the 21-day-old offspring. In a study examining the mouse uterus, Buchanan et al.
found that the anti-proliferative effects of TCDD on uterine epithelia appeared to be me-
diated indirectly through AhR in the stroma. The authors suggest that TCDD inhibits
uterine epithelial responses to 17β-estradiol by acting through the stromal AhR (26). Al-
though cell proliferation was down-regulated in both the glandular and luminal epithe-
lium in the present study at 21 days, expression levels of key cell cycle and growth factor
signaling proteins including cyclin D1, AKT, ERα, ERβ, PR, SRC-1, SRC-2/GRIP-1,
SRC-3, EGFR, EGF-1, IGF1-R, IGF-1, p21, and the ERKs were not found to be differen-
tially regulated at 21 days.
Cell proliferation was shown, however, to be up-regulated in the luminal epithe-
lium of 50-day-old animals exposed prenatally to TCDD, but unaffected in the glandular
epithelium. In the luminal epithelium, there is approximately a two-fold increase in cell
proliferation. The increase in cell proliferation in the luminal epithelium which coincides
with up-regulated EGFR (an important growth factor receptor) in 50 day animals exposed
prenatally to TCDD is of significance. However, 95% of detected uterine cancers in hu-
mans are adenocarcinomas, which arise from the glandular lining (made up of glandular
epithelium and stromal cells, collectively called the endometrium). Endometriosis is a
disease in which a number of endometrial glandular and stromal cells are found growing
outside of the uterine cavity. The progression of the disease can be modulated by hormo-
nal factors (27). The results of the present study suggest that the increased cell prolifera-
36. 25
tion in the luminal epithelium at 50 days of age, but not in the glandular epithelium, may
predispose these animals to uterine diseases other than adenocarcinomas by dramatically
increasing levels of proliferation.
Interestingly, the pattern of cell proliferation observed in the present study (down-
regulation at 21 days and up-regulation at 50 days) is exactly the opposite of that ob-
served when the same species of rat is exposed prepubertally to genistein, a purported
chemopreventive isoflavone found in soy. In a study using dietary genistein exposure at a
dose of 250 mg genistein/kg diet, Lamartiniere et al. (28) found that decreased cell prolif-
eration at 50 days postpartum was associated with reduced susceptibility to chemical car-
cinogenesis in the mammary gland. The same study noted that EGF signaling was associ-
ated with cell proliferation and that down-regulation of the EGF receptor likely contrib-
utes to genistein chemoprevention in the mammary. In the present study, we found EGFR
and cell proliferation to be up-regulated in the rat uterus at 50 days in the rat uterus. In-
asmuch as the study by Lamartiniere et al. has shown a direct relationship between EGFR
levels and cell proliferation in the mammary, the findings in the present study are consis-
tent for the uterus. In another study, Brown and Lamartiniere (29) noted that increased
cell proliferation in the epithelial cells of the rat uterus at 21 days (following prepubertal
genistein treatment) was associated with a paracrine mechanism involving elevated levels
of EGFR in the luminal and glandular epithelium.
From these studies, it appears that genistein and TCDD produce opposite effects
in regard to cell proliferation in the mammary gland and uterus: genistein increases cell
proliferation at 21 days and decreases cell proliferation at 50 days, resulting in reduced
susceptibility for chemically-induced mammary cancer. On the other hand, prenatal
37. 26
TCDD exposure decreases cell proliferation at 21 days and increases cell proliferation at
50 days. These findings lend credence to the body of evidence suggesting that genistein is
an effective chemopreventive agent and TCDD may make tissues more susceptible to
carcinogenesis. Since highly proliferative cells are associated with susceptibility for dis-
ease and/or cancer, these results suggest that TCDD, by increasing cell proliferation in
the luminal epithelium of the uterus at 50 days of age, renders the rat more susceptible to
uterine disease. Interestingly, it has been shown in vivo that glandular epithelium is the
one main cell type (of three) of the uterus that is most susceptible to the chemopreventive
effects of genistein (30). In the present study, we found the glandular epithelium to be
less sensitive to TCDD-induced proliferation and thus less susceptible to disease.
4.3 Changes in apoptosis
Using DAB staining to track DNA strand breaks by the indirect terminal dUTP
nick-end labeling (TUNEL) method, we measured apoptotic indices for both the glandu-
lar and luminal epithelium of 21-day-old and 50-day-old animals treated prenatally with
TCDD. At 21 days, apoptosis was unaffected by the prenatal TCDD treatment in both the
glandular and luminal epithelium.
At 50 days, apoptosis was drastically down-regulated in both the glandular and
luminal epithelium of rats exposed prenatally to TCDD. The difference represents a ten-
fold decrease in apoptosis in the glandular epithelium and a four-fold decrease in apop-
tosis in the luminal epithelium. The fact that apoptosis was not altered at day 21 postpar-
tum, but was at day 50, argues against residual TCDD concentrations being responsible
for apoptotic events. Rather, imprinting mechanisms and the response to puberty may
38. 27
play a role in causing effects early in development which are not displayed until later in
life, and in the case of the present study, after puberty (13).
The sharp decrease in the level of apoptosis seen at 50 days in the present study
coincides with a study done by Gray et al. which shows that permanent vaginal threads,
an anomaly many TCDD-exposed females possess, result from a direct inhibition of en-
dogenous estrogen-induced apoptosis of the vaginal membrane during puberty (7). Al-
though apoptosis was shown to be down-regulated at 50 days in both the glandular and
luminal epithelium, certain key apoptotic biomarkers (BCL2, BAX) were not found to be
down-regulated in the TCDD-exposed group in our study, suggesting that other apoptotic
signals may be playing a role.
The findings at 50 days of age have remarkable implications: the luminal epithe-
lium was found to have a two-fold increase in cell proliferation and a four-fold decrease
in apoptosis. With these two factors combined, the luminal epithelium of animals treated
prenatally with TCDD is highly susceptible to uterine disease and cancer later in life. Our
study suggests that TCDD may promote carcinogenesis in the rat uterus by increasing
cell proliferation and decreasing apoptosis, leading to a high turnover rate combined with
low programmed cell death. These endpoints were analyzed in detail in the present study
by measuring key protein biomarkers involved in growth and apoptosis pathways at both
21- and 50-days of age.
39. 28
4.4 Changes in protein expression levels
4.4.1 Sex steroid receptors and co-regulators
Uterine growth and cancer is often associated with de-regulation of ER and PR
mRNA and protein levels. Estrogen and progesterone have the ability to promote cell
proliferation in the cells of the breast and uterus. It could be hypothesized that the in-
crease in cell proliferation in the uterus, as was found in the present study at 50 days
postpartum, could be caused by an increase in levels of ERα, ERβ, and/or PR. However,
no significant modulation of either of the estrogen receptors or progesterone receptors at
21 or 50 days in the TCDD-exposed animals was observed. We suspect that the prolifera-
tive effects noticed at 50 days are not tied to differing levels of the hormone receptors
themselves but rather to other vital growth factors, steroid receptor co-regulators, and cell
survival proteins.
The SRCs are transcriptional co-activators for steroid and nuclear hormone recep-
tors. The p160 SRC gene family contains three homologous members: SRC-1 (NCoA-1),
SRC-2 (GRIP-1, TIF2, or NCoA-2), and SRC-3 (p/CIP, RAC3, ACTR, AIB1, and
TRAM-1, or NCoA-3). It has been shown that the SRCs are involved in a variety of tran-
scription regulation pathways, including breast cancer cell proliferation and invasion
(31). In the present study, all three members of the p160/SRC family were found to be
significantly down-regulated in the TCDD-exposed group at 50 days of age, though none
of them were found to be altered at 21 days of age. Significant decreases in all three
members of the SRC family in the present study are postulated to decrease sex steroid re-
sponsiveness in the animals exposed prenatally to TCDD due to the critical role the SRCs
play in transcription initiation. A study by Liao et al. suggested that a loss of SRC-3 func-
40. 29
tion causes a decrease in sensitivity of estrogen-mediated inhibition of growth (32). It is
unlikely that estradiol levels in these mature females contributed to the decrease in levels
of SRC’s because it has been shown previously that treatment with ER ligands did not al-
ter co-activator mRNA expression levels for SRC-1, GRIP-1, or SRC-3 in the rat uterus
(33). Modulation of this family of co-activators may signal an altered response to uterine
hormones and thus help to account for the changes in proliferation and apoptosis that
were observed. Transcriptional co-activator proteins, such as the p160/SRC family help
transcription factors stimulate transcription initiation after they bind to enhancer ele-
ments. One study found that SRC-1 enhances estrogen receptor, glucocorticoid receptor,
and thyroid hormone receptor transcriptional activities through their DNA response ele-
ments in the presence of hormone (34). In another study, it was demonstrated that SRC-
2/GRIP-1 stimulates transcriptional activity in a hormone dependent fashion by facilitat-
ing the assembly of basal transcription factors into a stable preinitiation complex (35).
SRC-3 has been found to play important roles in cell proliferation, cell migration, cell
differentiation, somatic growth, sexual maturation, female reproductive function, vaso-
protection, and breast cancer (32). The authors suggest that SRC-3 plays an important
role in serving as a bridge for kinase-mediated growth factor signaling to nuclear receptor
pathways. It has been demonstrated that the C-terminal domains of both SRC-1 and SRC-
3 possess histone acetyltransferase activities, and thus may play a role in chromatin re-
modeling during transcription initiation (36,37).
It has been shown previously that TCDD treatment increases mRNA levels for
TNF-α in human uterine endometrial adenocarcinoma RL95-2 cells (38). TNF-α is a po-
tent pyrogen that can cause inflammation by stimulation of interleukin-1 secretion. In-
41. 30
flammation is associated in some tissues with diminished responsiveness to steroid hor-
mone action (39). In this study, Leite et al. showed that proinflammatory cytokines such
as TNF-α reduce sensitivity to steroid hormones in uterine smooth muscle cells by reduc-
ing levels of key nuclear receptor co-activators, such as SRC-1 and SRC-2/GRIP-1. In
the present study, we found decreased levels of SRC-1, SRC-2, and SRC-3 in the rat
uterus at 50 days following prenatal TCDD treatment. This is consistent with the Leite et
al. study and suggests that accompanying increased levels of TNF-α may decrease the
sensitivity of the animals used in our study to hormone action.
It is likely that other growth factors are compensating for the down-regulation of
the SRC/p160 family of nuclear receptor co-activators and the accompanying decrease in
hormone sensitivity. A host of other growth pathways were investigated in the present
study, including the IGF-1 signaling axis. Like the EGFR, IGF-R acts via a tyrosine
kinase. Alterations in these pathways would also account for the changes in proliferation
and cell death in the uterine epithelial cells that was observed.
4.4.2 Growth factors and free radical damage
In the present study, there was a significant up-regulation of EGFR at 50 days, a
known player associated with cell proliferation in the uterus. A study conducted by La-
martiniere et al. (28) showed that EGFR is associated with cell proliferation in a direct
manner. It has also been shown by Pai et al. (40) that inactivation of EGFR by selective
inhibitors significantly decreases levels of ERK2 (an important mediator of signal trans-
duction by EGFR) activation, c-fos (an important oncoprotein for signal transduction, cell
proliferation, and differentiation) RNA expression, and cell proliferation. Thus, an up-
42. 31
regulation of EGFR, as was found in the present study, would promote cell proliferation,
as was also found in the present study in the luminal epithelium of 50 day animals treated
prenatally with TCDD.
In addition to EGFR up-regulation, we found IGF-BP3 to be significantly down-
regulated by prenatal TCDD treatment in 50 day animals. IGF-BP3 modulates the
amount of bioavailable IGF-1 and inhibits its transfer from the circulation to tissue sites
of action (41). All six IGF-BPs have been shown to inhibit IGF-1 action and prevent its
binding to IGF1-R (42). The down-regulation of IGF-BP3 observed in the present study
is an expected result of prenatal TCDD exposure. Numerous studies have shown an in-
verse association between levels of IGF-BP3 and risk for disease (43-46). In a study done
by Yu et al., the authors found a higher level of plasma IGF-BP3 to be associated with
reduced risk for lung cancer. The lower level of IGF-BP3 found in the TCDD-exposed
animals in the present study suggests a higher risk for biochemical insult later in life. The
decreased IGF-BP3 available for IGF-1 binding increases the effective concentration of
IGF-1, which can then freely bind to IGF1-R and promote cell proliferation. IGF-1 is
known to be a strong promoter of cell proliferation in a variety of cancers (47,48), and an
increased effective concentration of IGF-1 may contribute to the increased cell prolifera-
tion seen at 50 days in the TCDD-exposed animals. It may also contribute to an increased
susceptibility for cancer.
In the present study, we found SOD1 protein levels in uteri of TCDD-exposed
animals at 50 days postpartum to be significantly down-regulated as compared to con-
trols. The TCDD-exposed animals had approximately 20% less SOD1 when compared to
controls. This finding is important because SOD1 neutralizes supercharged oxygen mole-
43. 32
cules called superoxide radicals. Superoxide radicals, which are byproducts of normal
cell processes, can damage cells if their levels are not tightly controlled by superoxide
dismutase. The significant down-regulation of SOD1 by TCDD-treatment may increase
the potential for free radical damage that accompanies a decrease in SOD1 levels. These
radicals can bind to DNA, proteins, and lipids and cause permanent loss of structure and
play a significant role in initiation and promotion of carcinogenesis. The reduction in the
levels of SOD1 indicate that TCDD-exposed animals may be more susceptible to cellular
structure damage brought on by reactive oxygen radicals that cannot be converted as
readily to a more benign species by SOD1 and subsequent enzymes such as catalase and
glutathione peroxidase.
In summary, it has been well established that prenatal exposure to TCDD can af-
fect later susceptibility to breast cancer and uterine disease although the mechanisms for
this effect are not well understood. In this report, we show that prenatal exposure to
TCDD can affect cell proliferation, apoptosis, and the expression of several key proteins,
known to play a role in uterine growth and uterine diseases, including cancer. With pre-
natal TCDD exposure, there were several alterations in the uterus that could create an en-
vironment more favorable for uterine disease and carcinogen insult, including (1) up-
regulating cell proliferation in the luminal epithelium of the uterus, possibly rendering ac-
tively dividing cells more susceptible to cancer; (2) severely down-regulating apoptosis
in both the glandular and luminal epithelium, allowing damaged and tumorigenic cells to
remain viable; (3) up-regulating the protein expression of key growth factors such as
EGFR, which allows more ligand to bind to the receptor and cause signal transduction in
growth factor signaling pathways; (4) down-regulation of biomarkers that serve to main-
44. 33
tain the integrity of cellular infrastructure, such as SOD1; and (5) down-regulation of
growth factor sequestering proteins such as IGF-BP3, which allows for more IGF-1
ligand to bind to its membrane-bound receptor and induce signal transduction in growth
pathways. Twenty-one days of age (pre-puberty) appears to be too early a timepoint in
the animal’s life for prenatal TCDD to exert effects on levels of vital growth regulatory
protein. This may be due to dependence on postpubtertal endocrinology that regulates sex
steroid and growth factor signaling. Post-puberty, at 50 days of age, the rats are at an in-
creased risk for uterine disease and/or cancer because of changes in sex steroid and
growth factor signaling. An interesting question is why specific proteins (SOD1, SRC-1,
SRC-2/GRIP-1, SRC-3, IGF-BP3 and EGFR) are differentially regulated in 50-day-old
rats, but not 21-day-old rats exposed prenatally to TCDD, indicating a substantial role for
puberty and the exposure to endogenous sex hormones. Because it is unlikely that high
concentrations of TCDD cross the placenta and remain in the 50 day offspring, we postu-
late that gestational TCDD causes developmental alterations to uterine proteins that are
manifested as altered protein signatures. We strongly believe, based on the results of this
study, that prenatal exposure to TCDD could increase cancer and uterine disease suscep-
tibility later in life.
4.5 Future directions
To further the insights brought about by the present study, future experiments
could be aimed to address what other growth factor signaling pathways could explain the
decrease in cell proliferation found at 21 days in both the glandular and luminal epithe-
lium (since no change in the presently-studied protein levels was detected). Possibilities
45. 34
for factors that may be involved are those upstream and downstream of the ubiquitous
AKT protein (involved in a multitude of growth and proliferation pathways), such as
mammalian target of rapamycin (mTOR) and phosphatase and tensin homolog (PTEN).
mTOR is an important regulator of cell growth and proliferation, and PTEN is involved
in cancer suppression through negative regulation of proliferation and cell growth.
Based on the results of the present study, the effect of puberty on the uterus seems
to be critically important. To find if the difference between 21-day-old and 50-day-old
animals is a result of the estrogen exposure encountered during puberty, one group of es-
tradiol-17β-exposed animals could be used to compare to TCDD-exposed animals. An-
other possibility for further investigation would be to ovariectomize the animals after
TCDD treatment to eliminate the hormone fluctuations brought on at puberty, and then
compare the ovariectomized animals’ protein levels at 50 days to those of intact animals.
Any differences detected could be caused in part to influences brought on by steroid pro-
duction in the ovaries during and surrounding puberty.
To determine if TCDD increases the susceptibility for uterine cancer and/or uter-
ine abnormalities later in life in the rat model, animals could be carried to later timepoints
than the present study addressed, such as 200 days. Histological identification of pre-
cancerous cells or cancerous cells, if present, could then be carried out by a board-
certified pathologist.
46. 35
LIST OF REFERENCES
1. Markey, C.M., Luque, E.H., Munoz de Toro, M., Sonnenschein, C., Soto,
A.M., 2001. In Utero Exposure to Bisphenol A Alters the Development and Tis-
sue Organization of the Mouse Mammary Gland. Biol. Reprod. 65, 1215-1223.
2. Rappe, C., Buser, H.R., Bosshardt, H.P., 1979. Dioxins, dibenzofurans and
other polyhalogenated aromatics: production, use, formation and destruction.
Ann. NY Acad. Sci. 320, 1-5.
3. Birnbaum, L.S., 1995. Developmental effects of dioxins and related endocrine
disrupting chemicals. Environ. Health Perspect. 103, 89-94.
4. Couture, L.A., Harris, M.W., Birnbaum, L.S., 1990. Characterization of the
peak period of sensitivity for the induction of hydronephrosis in C57BL/6N mice
following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam. Appl. Toxi-
col. 15,142-150.
5. Gray, Jr., L.E., Ostby, J.S., 1995. In Utero 2,3,7,8-Tetrachlorodibenzo-p-
dioxin (TCDD) Alters Reproductive Morphology and Function in Female Rat
Offspring. Toxicol. Appl. Pharmacol. 133, 285-294.
6. Gray, Jr., L.E., Ostby, J.S., Marshall, R., Miller, D., 1993. Gestational busul-
fan (B) alters CNS, testicular and ovarian development and fertility in LE hooded
rat offspring. Toxicologist 13, 75.
7. Gray, Jr., L.E., Wolf, C., Mann, P., Ostby, J.S., 1997. In Utero Exposure to
Low Doses of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Alters Reproductive Devel-
opment of Female Long Evans Hooded Rat Offspring. Toxicol. Appl. Pharmacol.
146, 237-244.
8. Mann, P., 1997. Selected lesions of TCDD in laboratory rodents. Toxicol.
Pathol. 25(1), 72-79.
9. Theobald, H.M., Peterson, R.E., 1997. In Utero and Lactational Exposure to
2,3,7,8-Tetrachlorodibenzo-p-dioxin: Effects on Development of the Male and
Female Reproductive System of the Mouse. Toxicol. Appl. Pharmacol. 145,124-
135.
10. Birnbaum, L.S., Fenton, S.E., 2003. Cancer and Developmental Exposure to
Endocrine Disruptors. Environ. Health Perspect. 111, 389-394.
11. Safe, S., Wang, F., Porter, W., Duan, R., McDougal, A., 1998. Ah receptor
agonists as endocrine disruptors: antiestrogenic activity and mechanisms. Toxicol.
Lett. 102,103, 343-347.
47. 36
12. Steenland, K., Bertazzi, P., Baccarelli, A., Kogevinas, M., 2004. Dioxin Re-
visited: Developments Since the 1997 IARC Classification of Dioxin as a Human
Carcinogen. Environ. Health Perspect. 112, 1265-1268.
13. Brown, N.M., Manzolillo, P.A., Zhang, J.X., Wang, J., Lamartiniere, C.A.,
1998. Prenatal TCDD and predisposition to mammary cancer in the rat. Carcino-
genesis 19, 1623-1629.
14. Fenton, S.E., Hamm, J.T., Birnbaum, L.S., Youngblood, G.L., 2002. Persis-
tent Abnormalities in the Rat Mammary Gland following Gestational and Lacta-
tional Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD). Toxicol. Sci. 67,
63-74.
15. Rier, S.E., Martin, D.C., Bowman, R.E., Dmowski, W.P., Becker, J.L.
(1993). Endometriosis in Rhesus Monkeys (Macaca mulatta) Following Chronic
Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin. Fund. Appl. Toxicol. 21, 433-
441.
16. Li, B., Liu, H.-Y., Dai, L.-J., Lu, J.-C., Yang, Z.-M., Huang, L., 2006. The
early embryo loss caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin may be related
to the accumulation of this compound in the uterus. Reprod. Toxicol. 21, 301-306.
17. Cooper, R.L., Goldman, J.M., Vandenbergh, J.G. 1993. Monitoring of the es-
trous cycle in the laboratory rodent by vaginal lavage. In Methods in Reproduc-
tive Toxicology: Female Reproductive Toxicology, vol. 3B, pp. 45-56. New
York: Academic Press, New York.
18. Wang, J., Eltoum, I.-E., Lamartiniere, C.A., 2004. Genistein alters growth
factor signaling in transgenic prostate model (TRAMP). Mol. Cell. Endocrinol.
219, 171-180.
19. Grubbs, F., 1969. Procedures for detecting outlying observations in samples.
Technometrics 11 (1), 1-21.
20. Seefeld, M.D., Corbett, S.W., Keesey, R.E., Peterson, R.E., 1984. Characteri-
zation of the wasting syndrome in rats treated with 2,3,7,8-tetrachlorodibenzo-p-
dioxin. Toxicol. Appl. Pharmacol. 73(2), 311-322.
21. Christian, B.J., Menahan, L.A., Peterson, R.E. 1986. Intermediary metabo-
lism of the mature rat following 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment.
Toxicol. Appl. Pharmacol. 83(2), 360-378.
22. Van Birgelen, A.P., Van der Kolk, J., Fase, K.M., Bol, I., Poiger, H., Brou-
wer, A., Van den Berg, M., 1995. Subchronic dose-response study of 2,3,7,8-
tetrachlorodibenzo-p-dioxin in female Sprague-Dawley rats. Toxicol. Appl.
Pharmacol. 132(1), 1-13.
48. 37
23. Stahl, B.U., Rozman, K., 1990. 2,3,7,8-Tetrachlorodibenzo-p-dioxin
(TCDD)-induced appetite suppression in the Sprague-Dawley rat is not a direct
effect on feed intake regulation in the brain. Toxicol. Appl. Pharmacol. 106(1),
158-162.
24. Rozman, K., Pfeifer, B., Kerecsen, L., Alper, R.H., 1991. Is a serotonergic
mechanism involved in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced ap-
petite suppression in the Sprague-Dawley rat? Arch. Toxicol. 65(2), 124-128.
25. Syracuse Research Corporation, 1989. Toxicity. In Toxicological Profile for
2,3,7,8-tetrachlorodibenzo-p-dioxin, pp. 48-68. U.S. Public Health Service
Agency for Toxic Substances and Disease Registry (ATSDR).
26. Buchanan, D.L., Sato, T., Peterson, R.E., Cooke, P.S., 2000. Antiestrogenic
effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse uterus: critical role of the
aryl hydrocarbon receptor in stromal tissue. Toxicol. Sci. 57(2), 302-311.
27. Oral, E., Arici, A., 1997. Pathogenesis of endometriosis. Obstet. Gynecol.
Clin. North Am. 24(2), 219-233.
28. Lamartiniere, C.A., Cotroneo, M.S., Fritz, W.A., Wang, J., Mentor-Marcel,
R., Elgavish, A., 2002. Genistein chemoprevention: timing and mechanisms of ac-
tion in murine mammary and prostate. J. Nutr. 132(3), 552S-558S.
29. Brown, N.M., Lamartiniere, C.A., 2000. Genistein regulation of transform-
ing growth factor-alpha, epidermal growth factor (EGF), and EGF receptor ex-
pression in the rat uterus and vagina. Cell Growth Differ. 11(5), 255-260.
30. Eason, R.R., Till, S.R., Velarde, M.C., Geng, Y, Chatman, L. Jr., Gu, L.,
Badger, T.M., Simmen, F.A., Simmen, R.C., 2005. Uterine phenotype of young
adult rats exposed to dietary soy or genistein during development. J. Nutr. Bio-
chem. 16(10), 625-632.
31. Kishimoto, H., Wang, Z., Bhat-Nakshatri, P., Chang, D., Clarke, R., and
Nakshatri, H., 2005. The p160 family coactivators regulate breast cancer cell pro-
liferation and invasion through autocrine/paracrine activity of SDF-
1alpha/CXCL12. Carcinogenesis 26(10), 1706-1715.
32. Liao, L., Kuang, S.Q., Yuan, Y., Gonzalez, S.M., O'Malley, B.W., Xu, J.,
2002. Molecular structure and biological function of the cancer-amplified nuclear
receptor coactivator SRC-3/AIB1. J. Steroid. Biochem. Mol. Biol. 83(1-5), 3-14.
33. Nephew, K.P., Ray, S., Hlaing, M., Ahluwalia, A., Wu, S.D., Long, X., Hy-
der, S.M., Bigsby, R.M., 2000. Expression of estrogen receptor coactivators in the
rat uterus. Biol. Reprod. 63(2), 361-367.
49. 38
34. Onate, S.A., Tsai, S.Y., Tsai, M.J., O'Malley, B.W., 1995. Sequence and
characterization of a coactivator for the steroid hormone receptor superfamily.
Science 270(5240), 1354-1357.
35. Hong, H., Kohli, K., Garabedian, M.J., Stallcup, M.R., 1997. GRIP-1, a tran-
scriptional coactivator for the AF-2 transactivation domain of steroid, thyroid,
retinoid, and vitamin D receptors. Mol. Cell Biol. 17(5), 2735-2744.
36. Chen, H., Lin, R.J., Schiltz, R.L., Chakravarti, D., Nash, A., Nagy, L.,
Privalsky, M.L., Nakatani, Y., Evans, R.M., 1997. Nuclear receptor coactivator
ACTR is a novel histone acetyltransferase and forms a multimeric activation
complex with P/CAF and CBP/p300. Cell 90(3):569-580.
37. Spencer, T.E., Jenster, G., Burcin, M.M., Allis, C.D., Zhou, J., Mizzen, C.A.,
McKenna, N.J., Onate, S.A., Tsai, S.Y., Tsai, M.J., O'Malley, B.W., 1997. Steroid
receptor coactivator-1 is a histone acetyltransferase. Nature 389(6647), 194-198.
38. Charles, G.D., Shiverick, K.T. 1997. 2,3,7,8-Tetrachlorodibenzo-p-dioxin
increases mRNA levels for interleukin-1beta, urokinase plasminogen activator,
and tumor necrosis factor-alpha in human uterine endometrial adenocarcinoma
RL95-2 cells. Biochem. Biophys. Res. Commun. 238(2), 338-342.
39. Leite, R.S., Brown, A.G., Strauss III, J.F., 2004. Tumor necrosis factor-alpha
suppresses the expression of steroid receptor coactivator-1 and -2: a possible
mechanism contributing to changes in steroid hormone responsiveness. FASEB J
18(12), 1418-1420.
40. Pai, R., Soreghan, B., Szabo, I.L., Pavelka, M., Baatar, D., Tarnawski, A.S.,
2002. Prostaglandin E2 transactivates EGF receptor: a novel mechanism for pro-
moting colon cancer growth and gastrointestinal hypertrophy. Nat. Med. 8(3),
289-293.
41. Wu, X., Tortolero-Luna, G., Zhao, H., Phatak, D., Spitz, M.R., Follen, M.,
2003. Serum levels of insulin-like growth factor I and risk of squamous intraepi-
thelial lesions of the cervix. Clin. Cancer Res. 9(9), 3356-3361.
42. Baxter, R.C., 2000. Insulin-like growth factor (IGF)-binding proteins: inter-
actions with IGFs and intrinsic bioactivities. Am. J. Physiol. Endocrinol. Metab.
278(6), E967-976.
43. Wolk, A., Mantzoros, C.S., Andersson, S.O., Bergstrom, R., Signorello,
L.B., Lagiou, P., Adami, H.O., Trichopoulos, D. 1998. Insulin-like growth factor
1 and prostate cancer risk: a population-based, case-control study. J. Natl. Cancer
Inst. 90(12), 911-915.
44. Ma, J., Pollak, M.N., Giovannucci, E., Chan, J.M., Tao, Y., Hennekens, C.H.,
Stampfer, M.J., 1999 Prospective study of colorectal cancer risk in men and
50. 39
plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3. J.
Natl. Cancer Inst. 91(7), 620-625.
45. Yu, H., Spitz, M.R., Mistry, J., Gu, J., Hong, W.K., Wu, X., 1999. Plasma
levels of insulin-like growth factor-I and lung cancer risk: a case-control analysis.
J. Natl. Cancer Inst. 91(2), 151-156.
46. Kaaks, R., Toniolo, P., Akhmedkhanov, A., Lukanova, A., Biessy, C.,
Dechaud, H., Rinaldi, S., Zeleniuch-Jacquotte, A., Shore, R.E., Riboli, E., 2000.
Serum C-peptide, insulin-like growth factor (IGF)-I, IGF-binding proteins, and
colorectal cancer risk in women. J. Natl. Cancer Inst. 92(19),1592-1600.
47. Macaulay, V.M., 1992. Insulin-like growth factors and cancer. Br. J. Cancer
65(3), 311-320.
48. Jones, J.I., Clemmons, D.R., 1995. Insulin-like growth factors and their bind-
ing proteins: biological actions. Endocr. Rev. 16(1), 3-34.
54. 43
SRC-1
↓ T T T T C T C T C T C T C T C
GRIP-1
↓
T T T T C T C T C T C T C T C
SRC-3
↓
T T T T C T C T C T C T C T C
IGF-BP3
↓ T T T T C T C T C T C T C T C
EGFR
↑ T T T T C T C T C T C T C T C
↓
SOD1
T T T T C T C T C T C T C T C
Western immunoblots of proteins significantly regulated by prenatal TCDD treatment in
50-day old rats. Shown are blots of SRC-1, GRIP-1/SRC-2, SRC-3, IGF-BP3, EGFR,
and SOD1. For each of the six proteins, p < 0.05 comparing TCDD-exposed animals to
controls. Direction of regulation - up (↑) or down (↓) is indicated below each protein
name. C = Control, T= Treatment.
