Transcript ENDOCRINE DISRUPTING CHEMICALS: EFFECTS AND …

University of Puerto Rico
Intercampus Doctoral Program in Biology
ENDOCRINE DISRUPTING CHEMICALS:
EFFECTS AND MECHANISM OF ACTION
IN VTG GENE EXPRESSION IN THE
TROPICAL LIZARD Anolis pulchellus
By
JOSE ANIBAL CARDE-SERRANO
University of Puerto Rico
Intercampus Doctoral Program in Biology
Historical Background
1950- DDT: estrogenic in birds.
1962- “Silent Spring”- Rachel Carson
1968- DDT: estrogenic in mammals
1969- DDE: declining populations
1972- DDT: banned in USA
1971-80 DES: reproductive/developmental
disorders in offspring of treated mothers.
University of Puerto Rico
Intercampus Doctoral Program in Biology
Historical Background …
1977- PCBs: restricted by EPA
1991- Plastic compounds: estrogenic
1993- E E2: - Sperm counts declination
1993- Sewage effluent: estrogenic
1994- DDT: strikes back, Lake Apopka, Fl
1996- “Our Stolen Future” Colborn et al.
1999- eHormone- Tulane University
University of Puerto Rico
Intercampus Doctoral Program in Biology
Today’s Dilemma
Non-human species as environmental sentinels?
Appropriate species to assess EDCs in lab and field?
Which are important endpoints to monitor the effects?
In vitro vs In vivo approaches.
Definitions:
Endocrine Disrupting Chemicals (EDCs)
Xenoestrogens (XEs)
Endpoints
University of Puerto Rico
Intercampus Doctoral Program in Biology
Structures of EDCs – (QSAR)
E2
Estrogen
Diethylstilbestrol
DES
BPA
Bisphenol A
Estriol
op DDT
E1
DDT
DEHP
Estrone
E3
Methoxychlor
Diehtylhexyl Pthalate
MTX
Tamoxifen
TAM
University of Puerto Rico
Intercampus Doctoral Program in Biology
Some EDCs and Their Effects
COMPOUND
EFFECTS
Pesticides
DDT, DDE, Mtx
Avian and reptile reproduction impaired, (Fry, 1981, Guillete
et al, 1994,1995)
Alterations in early development in amphibians (Bevan et al,
2002).
Estrogens
DES
Estrone
Estriol
Developmental defects on offspring reproductive system,
(vom Saal et al., 1995, Newbold 1995; McLachlan et al.,
1980).
Induction of female specific proteins in male fishes exposed
to water sewages. Sumpter and Jobling, 1993, Soto et al,
2004)
Plasticizers
BPA
Phthalates
(BBP, DEHP,
DBP)
Decrease in testicular size and sperm production (Sharpe et
al., 1995) and sterility in second generation males on rats
(Wine et al., 1997). Suspected for PSD (Colón et al., 2000).
Developmental abnormalities in amphibians (Sone et al.,
2004).
University of Puerto Rico
Intercampus Doctoral Program in Biology
The Model System: Anolis pulchellus - ♂
Tropical lizards
Humid grassy areas
Night recollection
Easily maintained
Ubiquitous
Sentinel ?
University of Puerto Rico
Intercampus Doctoral Program in Biology
The Model System: Anolis pulchellus - ♀
Vitellogenesis
Vitellogenins
Captivity Effects
mRNA/Vtg
Reverted by E2
University of Puerto Rico
Intercampus Doctoral Program in Biology
E2- Mechanism of Action
From Geneka Biotechnology
http://www.biolynx.ca/active.html
University of Puerto Rico
Intercampus Doctoral Program in Biology
Objective I
In vivo induction of VTG expression in
Anolis pulchellus lizards as indicator of
estrogenic activity of suspected EDCs.
A - Transdermal treatment, E217β
B - Males
C- Females
University of Puerto Rico
Intercampus Doctoral Program in Biology
Efficiency of Transdermal Treatment
★
4000000
H3 E217B-CPM (x106)
3500000
3.0
3
[H] cpm
3000000
2500000
★
2000000
1500000
1.0
1000000
500000
00
Tip
Tip
Skin
Skin
Visc
Body
University of Puerto Rico
Intercampus Doctoral Program in Biology
Objective Ia - In vivo induction of VTG expression in Anolis
pulchellus lizards transdermally treated with E217β.
Male Lizards
- E217β, EtOH95%
- DDT, DDE, Mtx
- DEHP, BPA
- E1, E3, DES
Western Blot
Anti S1 (Vtg)
RT-PCR
University of Puerto Rico
Intercampus Doctoral Program in Biology
Minimal Dose for Estradiol 17B:
Transdermal (TD) vs Intramuscular (IM)
TD
C- 0.0005 0.005 0.05 0.5 (μg)
Anti S1
(Vtg)
IM
Anti S1
(Vtg)
Vtg
169
153
116
C- 0.0005 0.005 0.05 0.5 (μg)
Vtg
169
153
116
University of Puerto Rico
Intercampus Doctoral Program in Biology
Problem
The need of model systems to be used as
sentinel species and for the assessment of
estrogenic activity in suspected EDCs.
Hypothesis
If Vtg is induced in lizard males by E217β, then
the EDCs with XEs activity will also induce the
Vtg gene expression. Similar mechanisms of
action can be suspected.
University of Puerto Rico
Intercampus Doctoral Program in Biology
Objective Ib - In vivo induction of VTG expression in Anolis
pulchellus lizards as indicative of estrogenic activity of
suspected EDCs.
Males
- E217β, EtOH95%
- DDT, DDE, Mtx
Treatments
- DEHP, BPA
- E1, E3, DES
- 3Rxs every other day
Western Blot
Anti S1 (Vtg)
RT-PCR
University of Puerto Rico
Intercampus Doctoral Program in Biology
Effects of Pesticides on VTG synthesis
DDT (μg)
DDE (μg)
60
125
125
1
2
3
Mtx (μg)
250 375 125 250
Vtg
169
153
116
Anti S1
(Vtg)
4
5
6
7
University of Puerto Rico
Intercampus Doctoral Program in Biology
Effects of Plasticizers on Vtg Synthtesis
DEHP (10 mg)
C+
C-
2
3
4
Vtg
169
153
116
1
2
3
4
5
Anti S1
(Vtg)
University of Puerto Rico
Intercampus Doctoral Program in Biology
Effects of Plasticizers on Vtg Synthtesis
BPA
(μg)
250
375 500 1000
C+
Vtg
169
153
116
Alb
1
2
3
4
5
6
Anti S1
(Vtg)
University of Puerto Rico
Intercampus Doctoral Program in Biology
Effects of Steroids and Non-steroids compound
on VTG synthesis
Vtg
E217β
5
E1
0.5
E3
0.5
DES
0.005
1
2
3
4
169
153
116
Anti S1
(Vtg)
(μg)
University of Puerto Rico
Intercampus Doctoral Program in Biology
Objective Ic - In vivo induction of VTG expression in Anolis
pulchellus lizards as indicative of estrogenic activity of
suspected EDCs.
Females
Captivity
- 15 days
Treatment
- EtOH 95%
- DDT, DDE, Mtx
Western Blot
Anti S1 (Vtg)
RT-PCR
University of Puerto Rico
Intercampus Doctoral Program in Biology
Vtg Synthesis Reverted by Pesticides in Females
C15
-
-
Glf DDT DDE Mtx
-
1000 500 500 500 (μg)
Vtg
169
153
116
1
2
3
4
5
6
7
8
Anti S1
(Vtg)
University of Puerto Rico
Intercampus Doctoral Program in Biology
Vtg Synthesis Reverted by Pesticides in Females
C15
Glf DDT DDE Mtx
1000 500 500 500 (μg)
500
300bp
Vtg
mRNA
RT-PCR
1
2
3
4
5
6
7
University of Puerto Rico
Intercampus Doctoral Program in Biology
Effect of Methoxychlor on Vtg Synthesis
Mtx 70 μg
C-
C+
Vtg
169
153
116
Alb
1
2
3
4
5
6
7
8
9
Anti S1
(Vtg)
University of Puerto Rico
Intercampus Doctoral Program in Biology
Dose Response Experiments
Endocrine Disrupting Chemicals
Metoxychlor
(Mtx)
Dose Vtg
(µg)
+
Bisphenyl A
(BPA)
Vtg
-
Dose
(µg)
Diethylstilbestrol
(DES)
Vtg Vtg Dose Vtg
+
(µg)
+
Vtg
-
70
4
3
375
2
5
0.3a
6
0
140
5
2
500
1
6
3.0
7
0
280
5
2
1000a
1
3
30a
5
1
(a) Dead animals.
University of Puerto Rico
Intercampus Doctoral Program in Biology
Conclusions
 A transdermal mode of administration for E217β and
suspected EDCs was developed and tested.
 The expression of the Vtg gene de novo in males can be
used as a confident endpoint of XEs activity in this totally
terrestrial anoline lizard.
 Pesticides as well as hormone-like compounds more likely to
be effectively and accurately tested in this model system.
University of Puerto Rico
Intercampus Doctoral Program in Biology
Objective II:
A) To demonstrate that the effect on VTG
expression that results from the E217β
and EDCs treatment is mediated via the
ER.
B) To demonstrate the interaction of
E217β and the EDCs with a plasma Sex
Steroid Binding Protein (SSBP).
University of Puerto Rico
Intercampus Doctoral Program in Biology
Objective IIa,b -To demonstrate the interaction of E217β with a
liver ER and plasma SSBP.
Males
- Rxs E217β
Cytoplasmic Liver Protein Extraction (100xg)
or Blood plasma
SDS Page
Competitive
Binding Assays
University of Puerto Rico
Intercampus Doctoral Program in Biology
H3 E217B-CPM (x106)
3[H]-E 17β
2
Binding to Intracelullar Proteins:ER
★
8.0
6.0
★
4.0
2.0
★
★
0
C
NRx
Rx
University of Puerto Rico
Intercampus Doctoral Program in Biology
EDCs binding to Intracelullar Proteins: ER
University of Puerto Rico
Intercampus Doctoral Program in Biology
Relative Binding of EDCs to ER
Competing
compound
% inhibition
% Relative
Binding(a)
E2-17β
60
100
BPA
47
78
DEHP
9
15
Mtx
13
22
DDT
40
67
DDE
27
45
DES
33
55
Estriol
44
73
Estrone
46
77
Tamoxifen
7
11
HPTE
4-0HTam
University of Puerto Rico
Intercampus Doctoral Program in Biology
EDCs binding to Plasma SSBP
University of Puerto Rico
Intercampus Doctoral Program in Biology
Relative Binding of EDCs to SSBP
Competing
%
compound inhibition
% Relative
Binding(a)
E2-17β
49
100
BPA
33
67
DEHP
0
0
Mtx
1
2
DDT
40
80
DDE
42
84
DES
16
33
Estriol
21
42
Estrone
38
76
Tamoxifen
20
40
University of Puerto Rico
Intercampus Doctoral Program in Biology
EDCs: Qualitative Ranking for Estrogenicity
Compound
Vtg ind
EERB
SSBPRB
E2-17β12
++++
1000/++++
100/++++
BPA
++
78/+++
67/++
DEHP
++
15/-
0/-
Mtx
+++
22/-
2/-
DDT
+++
67++
80/+++
DDE
+++
45/+
84/+++
DES
++++
55/++
33/+
Estriol
++++
73++
42+
Estrone
++++
77/+++
76/+++
-
11/-
40/+
Tamoxifen
E3>DDT>E1=DES=DDE=BPA>Mtx>DEHP
University of Puerto Rico
Intercampus Doctoral Program in Biology
Conclusions
 The presence of an E217β binding activity (ER) was
demonstrated in a liver cytosolic protein extract from Anolis
pulchellus for the first time. The interaction of 5 of 9
EDCs/XEs with the putative ER was also shown by this
binding assay.
 The presence of an E217β binding activity (SSBP) was
demonstrated in the blood plasma of the Anolis pulchellus
for the first time. Interaction with some of the EDCs/XEs with
the SSBP was also demonstrated.
 EDCs that require metabolic activation (MTX/HPTE) are
apparently appropriate for assessment in Anolis.
University of Puerto Rico
Intercampus Doctoral Program in Biology
Objective III
To determine if E2 activate the ER and
if this ligand-receptor complex interact
with an ERE from the VTG gene.
University of Puerto Rico
Intercampus Doctoral Program in Biology
Objective III- To determine if E2 activate the ER and if this
ligand-receptor complex interact with an ERE from the VTG
gene.
Males
- E217β
Cytoplasmic Liver Protein Extraction
SDS Page
EMSA
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Intercampus Doctoral Program in Biology
Electrophoretic Mobility Shift Assay (EMSA)
University of Puerto Rico
Intercampus Doctoral Program in Biology
EMSA – ER -12hr - Anolis
Extract :
Old
C-
New
25 50 100
25 50 100 (μg)
ER ?
Free
probe
Pomega
ERE_KC
1
2
3
4
1
2
3
University of Puerto Rico
Intercampus Doctoral Program in Biology
Bioinformatics Analysis: ERE
ERE_KC
5’ GTCCAAAGTCAGGTCACAGTGACCTGATCAAAGTT 3’
*---- *--*- *** ***- ********* --- *- ----
ERE_CS
University of Puerto Rico
Intercampus Doctoral Program in Biology
EMSA – ER*- Anolis/HeLa
Extract :
C-
Anolis
HeLa
C+ SpC NspC C-
C+
SpC NspC
ER
Free
probe
Promega
*ERE_CS
1
2
3
4
5
1
2
3
University of Puerto Rico
Intercampus Doctoral Program in Biology
Binding Buffers Comparison
Components
Promega (04)
RuizKumarEchevarria(00) Chambom (88)
Glicerol
20%
5%
10%
MgCl2
5mM
3mM
-
EDTA
2.5mM
-
-
DTT
2.5mM
1mM
1mM
NaCl
250mM
-
-
KCl
-
40mM
20-100mM
MercaptoEtOH
-
1.5%
-
Poly dI-dC
0.25mg/ml
-
-
TrisHCl
50mM
-
10mM
Hepes
-
10mM
-
University of Puerto Rico
Intercampus Doctoral Program in Biology
EMSA – General Transcription Factors - Anolis
TFII
SP1
-UV
+UV
-UV
P RE KC C- P RE KC
+UV
P RE KC C- P RE KC
SP1
TFII
Free
probe
1
2
3
4
5
6
7
8 9 10 11 12 13 14
University of Puerto Rico
Intercampus Doctoral Program in Biology
EMSA – ER- 4hr - Anolis♂
-UV
+UV
TFII P RE KC C- P RE KC
ER
Free
probe
ERE_CS
1
2
3
4
5
6
7
8
University of Puerto Rico
Intercampus Doctoral Program in Biology
EMSA – ER- 1hr- Anolis
♂
Extract :
♀
C+ SpC NSC
C-
C+ SpC NSC
ER
Free
probe
RE
ERE_CS
1
2
3
4
5
6
7
University of Puerto Rico
Intercampus Doctoral Program in Biology
EMSA – ER- Anolis
Specific Competitor (fmoles)
C-
C+
10
50
100 500
ER
Free
probe
RE
ERE_CS
1
2
3
4
5
6
University of Puerto Rico
Intercampus Doctoral Program in Biology
Conclusions
 The formation of an E217β-ER complex using the liver
cytosolic protein extract, capable of the recognition of a
consensus ERE for estrogen-regulated genes was
demonstrated. Confirming the presence of an E217β
binding activity (ER) in a liver cytosolic protein extract.
University of Puerto Rico
Intercampus Doctoral Program in Biology
(1)
Extracellular
Milieu
Detox Liver
Cells
Enzymes
(2)
(3a)
(3b)
Cytoplasm
(4)
ERE
Vtg mRNA
Nucleus
AAAAA
Vtg
protein
University of Puerto Rico
Intercampus Doctoral Program in Biology
Future Experiments
 Binding assays with active metabolites of
putative EDCs.
EMSAs with EDCs as activators or
promoters of XE-ER-ERE complex formation.
Super shifts to identify and characterize
transcription factors recruited in the ER-ERE
complex in the presence or absence of XEs.
University of Puerto Rico
Intercampus Doctoral Program in Biology
Agradecimientos
 Dra. Magda H. Morales, por la oportunidad de trabajar
con ella y aprender de ella.
 Los doctores Dra. Sandra Peña de Ortiz, Dr. José Ramón
Rodríguez Medina, Dr. Osvaldo Rosario y Dr. José Lasalde.
 Dr. Carlos I. González, por su cooperación y dirección
durante el último grupo de experimentos y por su
hospitalidad en su laboratorio, asi como a su grupo trabajo.
 Lorena Saavedra, Jhyoti Metha, Raúl Vázquez, Angel
Pérez, Ivelisse Castresana, Abimael de León.
 Rose Díaz, Juan Carlos Hernández, Nayda Ortiz, John
Santiago, y José E. Vidal.
University of Puerto Rico
Intercampus Doctoral Program in Biology
Agradecimientos
 La Universidad Adventista de las Antillas, el Departamento
de Ciencias y su Directora la Profesora Alicia Moradillos, asi
como a mis colegas y estudiantes por su apoyo y cooperación
durante este proyecto.
Mi esposa Lourdes, quién ha estado ahi al lado, luchando
conmigo para lograr esta meta. Al lado de cada hombre de
éxito … hay una mujer.
La Universidad de Puerto Rico, Recinto de Río Piedras, sus
programas Faculty Development, SUBE, FIPI y DEGI.
A Dios, por que a los que a Dios aman, todas las cosas les
ayudan a bien.
University of Puerto Rico
Intercampus Doctoral Program in Biology
University of Puerto Rico
Intercampus Doctoral Program in Biology
Scientific reaction to the news of declining spermcounts due to estrogenic contamination was mixed…