Transcript Slide 1

ETHYNYLESTRADIOL, TAMOXIFEN AND O,P’-DDT INDUCE CONSERVED AND DIVERGENT
GENE EXPRESSION PROFILES DURING UTEROTROPHY IN THE RAT UTERUS
1,3
Kwekel ,
2
Williams
1,3
Zacharewski
JC
KJ
TR
1Departments of Biochemistry & Molecular Biology, 2Pathobiology & Diagnostic Investigation,
3Center for Integrative Toxicology and National Food Safety & Toxicology Center,
Michigan State University, East Lansing, MI 48824
ABSTRACT
UTERINE WEIGHT: DOSE RESPONSE AND TIME COURSE
DDT is an environmentally persistent organochlorine widely used
until the early 1970’s as a common pesticide.
Its wide spread
production and use was in large part due to its role
in vector control for malaria
(mosquitoes). The ortho, para’
enantiomer of DDT has been shown to bind the estrogen receptor
and disrupt normal reproductive health and development.
17α-EthynylEstradiol
Tamoxifen
Despite this knowledge, the subsequent transcriptional activities of
the TAM- or DDT-ER complex and the full spectrum of target
genes has not been fully delineated. This project has set out to
characterize the gene expression changes associated with TAM
and DDT activity in the rodent uterus in conjunction with its
physiological and morphological effects.
o,p’-DDT
The rodent uterotrophic assay has been used for decades to assess the estrogenicity of compounds by their ability
to stimulate an increase in uterine wet weight. This assessment, while being a gold standard for screening
estrogenic compounds, provides limited insight into the pathways involved. We therefore have employed and
extended the enhanced uterotrophic assay adapted from Diel, et al. 2001 in which in vivo estrogenicity is evaluated
in a three tiered approach while incorporating a comprehensive time course study. This approach combines
assessments of the uterotrophic, morphological and transcriptional responses of the rodent uterus into an integrated
evaluation, wherein:
140
Weight (mg)
EE wet
EE blot
Tam wet
Tam blot
120
DDT wet
DDT blot
100
80
60
40
Veh
EE
TAM
DDT
2
4
8
12
18
24
72
Uterine Water Content
40
20
35
Veh
0.01
0.1
1
10
100
Dose (g/kg)
1k
10k
Weight (mg)
0
100k
Figure 3 Dose Response experiments were
performed which indicate comparable potency
of TAM but lower efficacy of TAM and DDT in
uterine wet weight induction and water
imbibition.
TAM
DDT
45
120
110
100
90
80
70
60
50
40
30
20
10
0
B.
40
EE
Uterine Wet Weight
30
25
20
15
10
5
0
2
4
8
12
18
24
Figure 4 Time Course
experiments monitored
uterine wet weight (A)
and water content (B),
which was calculated as
the difference between
wet and blotted weights.
TAM and DDT exhibited
early water imbibition
(4hr-12hr), but not at
72hr.
Error bars represent the
SEM for the average.
N=5, *p < 0.05, student’s
t-test.
*
Fos
40
35
(r = 0.814)
30
*
18
Fos
12
25
10
10
0
8
15
6
10
4
10
5
2
5
8
12
18
24
0
0
72
2
4
8
12
18
24
Figure 9 Quantitative real time PCR confirmed
the temporal expression patterns of several
known estrogen responsive genes including Fos
and C3. Bars and lines represent fold change
relative to time matched vehicle control for QRTPCR and microarray data, respectively. Error bars
represent the SEM for the average fold change in
QRT-PCR data. N=5, *p < 0.05, student’s t-test.
20
20
10
15
72
*
5
*
2
4
8
12
18
24
72
0
Time (hr)
800
700
600
500
20
C3
800
700
600
*
(r = 0.870)
400
300
40
15
20
C3
400
300
40
C3
*
15
500
30
200
400
10
30
300
40
10
100
50
*
*
30
20
5
20
40
30
20
5
10
20
10
10
0
0
2
4
8
12
18
24
10
0
72
0
2
4
8
12
18
24
72
0
2
4
*
*
8
12
18
24
72
0
Time
MICROARRAY DATA: COACTIVITY ANALYSIS
72
Rat Array
Time (h)
A.
B.
8,567 spots
(4,717 genes)
MORPHOLOGICAL ASSESSMENTS OF ESTROGENIC RESPONSE
Figure
5
Histological
sections at the uterotrophic
time point (72hr) exhibit
classic changes in uterine
cell morphology including
increased luminal epithelial
cell height (LEH), thickened
basal lamina and proliferation as evidenced by
increased luminal diameter.
TAM
treated
samples
elicited comparable induction in stromal area and
increased induction of LE
cell height compared to EE.
DDT
treated
samples
showed increased incidence of apoptosis thickened
myometrium and immune
cell accumulation in comparison
to
EE
treated
samples.
50
20
0
4
Fos
*
14
30
30
*
25
20
2
100
16
Differential Expression
P1t ≥ 0.999
|fold change| ≥ 1.5
TAM 72hr
TAM
(2,087)
69
10
CoActivity
Analysis
CAS
CAD
DAS
DAD
1565
18
362
39
117
204
1,984
EE
(2,409)
268
183
Luminal Epithelial Height
a,b
0.04
a
0.03
a,b
0.02
0.01
0.00
Veh
EE
TAM
DDT
(2,458)
Figure 6 Morphometric analysis of EE,
TAM, and DDT induced changes in
luminal
epithelial
cell
height.
Measurement of LEH was repeated with
a smaller cohort of animals to confirm
TAM’s increased induction in LEH
relative to EE. DDT exhibited a lower
induction in LE cell height at 72hr.
Mean+/-SEM, n=5, p<0.05, student’s ttest, Tukey’s HSD test, a compared to
vehicle, b compared to EE.
0.05
DDT
Correlation of EE-DDT
Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM); prescribed as an adjuvant therapy for ERpositive, breast cancer treatment or prophylactically for patients with increased breast cancer risk or family history.
It acts by binding the ER and inhibiting estradiol’s proliferative effects in tumor progression and growth. Tamoxifen’s
activity as a SERM has been well documented clinically, exhibiting antagonistic properties in the breast but partial
agonistic activity in the reproductive tract as evidenced by an increased incidence of uterine tumors in patients
taking the drug.
160
Height (mm)
INTRODUCTION AND OBJECTIVES
A.
Uterotrophy Dose Response Curves
Relative Weight (mg)
Changes in uterine physiology, cell morphology and gene expression were evaluated in a comprehensive time
course study after oral administration of 100 µg/kg bw ethynylestradiol (EE), 100 µg/kg tamoxifen (TAM) or 300
mg/kg o,p’-DDT (DDT) to immature, ovariectomized Sprague-Dawley rats. Animals were dosed once or once daily
for 3 consecutive days and uteri were harvested 2, 4, 8, 12, 18, 24, or 72 hrs after treatment. EE, TAM, and DDT
elicited uterotrophic EC50s of 16.2 µg/kg, 16.5 µg/kg and >64.6 mg/kg, respectively, in the dose response studies.
TAM and DDT only induced a 3.3-fold increase in wet weight with a marked decrease in water imbibition compared
to the 7-fold induction with EE. DDT treated histological samples were characterized by marked apoptosis in the
luminal epithelia and stroma at 72 hr compared to EE while TAM treatment exhibited significantly higher induction of
luminal epithelial cell height at 72hr. Temporal gene expression profiles were measured using custom rat cDNA
microarrays consisting of 8,567 features representing 4,342 genes and identified conserved and divergently
regulated genes. Of the 1984 genes commonly regulated by all three compounds, 1574 demonstrated highly
correlated temporal profiles between all three datasets indicating a large degree of similarity in the expression
profiles. However 10 and 183 genes exhibiting expression profiles unique from EE for TAM and DDT, respectively,
suggest additional non-estrogen receptor mediated pathways being regulated by DDT but not TAM.
QRT-PCR VERIFICATION OF MICROARRAY DATA
Figure 10 Genes meeting
activity criteria for at least one
ligand were compared in a Venn
analysis (A) to find overlapping active genes.
Genes active in all three data sets were then
further processed for coactivity whereby genes exhibiting similarities or differences
in time of expression and direction of change were subsequently designated as
coactive-similar, CAS; coactive-divergent, CAD; displaced active-similar, DAS; or
displaced active-divergent, DAD using CAIT (CoActivity Index Tool). The temporal
correlations of EE-TAM, TAM-DDT and DDT-EE were plotted (B) for each gene
along with coactivity designations to visualize conserved responses.
CONSERVED AND DIVERGENT GENES
Treatment at 72 hr
TAM and DDT treatments will be compared to the prototypical oral estrogen: ethynylestradiol in the rat uterus.
Classic estrogenic endpoints of uterotrophy and luminal epithelial cell height will be quantified and compared.
Temporal gene expression profiles of EE, TAM and DDT will be examined by cDNA microarray analysis and
comparative analysis of the expression profiles will determine conserved or divergent pathways.
MICROARRAY DATA: COMPARATIVE ANALYSIS
Active Genes per Time Point
1250
Target
Tissues
Uterus
Tamoxifen
O-p-DDT
Bisphenol A
Genistein
Liver
Mammary
Kidney
Bone
Poland Sucks!
Uterotrophic
Response
Morphological
Response
Rodent
Models
Rat
Transcriptional
Response
Mouse
DOSE RESPONSE
EE (ug/kg bw)
0.01, 0.1, 1, 10, 100, 300
0
24
48
EE 100 ug/kg bw
Sesame Oil Vehicle
24
72 h
2 4 8 12 18 24
Sacrifice
750
500
2
4
8
12
18
24
72
Time (hr)
48
DDT 300 mg/kg bw
Sesame Oil Vehicle
1000
0
Dosing Times (h)
0
Figure 7 Microarray data for all three ligands were
filtered by a statistical cutoff (P1t ≥ 0.999) and fold
change cutoff (± 1.5) relative to vehicle controls at any
time point and were thus designated as differentially
regulated or “active”. Number of active genes per time
point (A) were calculated for each compound. EE and
DDT exhibit similar kinetics in time of gene response
while TAM lagged by 10-12 hr due to TAM’s need for
bioactivation to the 4-OH form. Numbers of active genes
were comparable at 72 hr.
250
TAM 100 ug/kg bw
TAM (ug/kg bw)
0.1, 1, 10, 100, 300, 1000
DDT (mg/kg bw)
1, 3, 10, 30, 100, 300
Figure 1
The enhanced uterotrophic assay
consists of three daily doses of estrogen, followed
by physiological, morphological and biochemical
assessments 72 hrs after initial treatment
(adapted from Diel, et al. 2002, ). Uterine wet
weight and water content; histopathological and
morphometric assessments; and gene expression
studies through microarray analysis and QRTPCR verification of active genes were assessed.
The current study extended the Diel protocol by
incorporating a comprehensive time course
throughout the first 24 hrs at which the above
endpoints were assessed.
TIME COURSE
Dosing Times (h)
# of Active Genes
EXPERIMENTAL DESIGN
EEDC
Compounds
Ethynylestradiol
EE
TAM
DDT
72
Sacrifice Time (h)
Figure 2 The Dose Response study consisted of three daily oral doses, of EE, TAM and DDT (6 dose groups) or sesame oil vehicle to
immature, ovariectomized Sprague Dawley rats followed by sacrifice and tissue harvest at 72 hr. The Time Course study consisted of the
same dosing schedule for 100 ug/kg bw of EE, 100ug/kg TAM, or 300 mg/kg bw DDT followed by sacrifice at times indicated. Tissues for
gene expression were snap frozen in liquid nitrogen while histological samples were fixed for 24 hours in 10% neutral buffer formalin. Each
treatment group consisted of five animals each for both studies.
4.0
Figure 8 Genes shown to be
active in response to at least one
ligand were compiled into a
common list of genes and
hierarchically clustered agglomeratively (GeneSpring) by gene for
visual representation of comparable levels of gene induction and
repression between all three
ligands. This cluster plainly shows
the
highly
conserved
and
correlated nature of the expression
profiles of EE, TAM and DDT in
the rat uterus.
1.0
 TAM shows its partial agonist activity in less efficacious uterotrophy as well as overall lower
induction of gene expression relative to EE while activating few if any unique targets.
0.25
Supported by: US EPA RD 83184701, R21 GM075838, ES07255-16
www.bch.msu.edu/~zacharet/
 DDT is less potent and efficacious than EE, but also exhibits an expression pattern almost
identical to that of EE in the immature, OVX rodent uterotrophic response.
 Conserved and divergent gene expression profiles are being further analyzed for unique ligandER activity.
2
Email: [email protected]
CONCLUSIONS
4
8 12 18
EE
24 72
2
4
8
12 18 24 72
TAM
2
4
8
12 18 24 72
DDT
 Further analysis of cross tissue and cross species comparisons will lend valuable insight into
TAM and DDT’s conserved and divergent mechanisms.