Download - By JOSE ANIBAL CARDE-SERRANO
ENDOCRINE DISRUPTING CHEMICALS: ENDOCRINE DISRUPTING CHEMICALS: EFFECTS AND MECHANISM OF ACTION IN EFFECTS AND MECHANISM OF ACTION IN VTG GENE EXPRESSION IN THE TROPICAL VTG GENE EXPRESSION IN THE TROPICAL
LIZARDLIZARD Anolis pulchellus Anolis pulchellus
ByBy
JOSE ANIBAL CARDE-SERRANOJOSE 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 DilemmaNon-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.
University of Puerto Rico Intercampus Doctoral Program in Biology
Definitions:Endocrine Disrupting Chemicals (EDCs)
Xenoestrogens (XEs)
Endpoints
Estrogen Estriol Estrone
Diethylstilbestrol op DDT Methoxychlor
Bisphenol A
Diehtylhexyl Pthalate Tamoxifen
E2 E1 E3
DES DDT MTX
BPA DEHP TAM
Structures of EDCs – (QSAR)
University of Puerto Rico Intercampus Doctoral Program in Biology
COMPOUND EFFECTS
PesticidesDDT, 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
EstroneEstriol
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)
PlasticizersBPA
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).
Some EDCs and Their Effects
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
University of Puerto Rico Intercampus Doctoral Program in Biology
From Geneka Biotechnology
http://www.biolynx.ca/active.html
E2- Mechanism of Action
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
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
Tip Skin Body
3 [H]
cpm
University of Puerto Rico Intercampus Doctoral Program in Biology
★
★
3.0
1.0
0 Tip Skin Visc
Objective Ia - In vivo induction of VTG expression in Anolis pulchellus lizards transdermally treated with E217β.
Male Lizards - E217β, EtOH95%
Western Blot
Anti S1 (Vtg)
RT-PCR
- DDT, DDE, Mtx- DEHP, BPA- E1, E3, DES
University of Puerto Rico Intercampus Doctoral Program in Biology
Minimal Dose for Estradiol 17B: Transdermal (TD) vs Intramuscular (IM)
Vtg
169 153 116
Vtg
169 153 116
IM C- 0.0005 0.005 0.05 0.5 (μg)
TD C- 0.0005 0.005 0.05 0.5 (μg)
Anti S1 (Vtg)
Anti S1 (Vtg)
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
- DEHP, BPA
- E1, E3, DES
- 3Rxs every other day
Western Blot
Anti S1 (Vtg)
RT-PCR
Treatments
University of Puerto Rico Intercampus Doctoral Program in Biology
DDT (μg) DDE (μg) Mtx (μg)
60 125 125 250 375 125 250
Vtg
169
153
116
Effects of Pesticides on VTG synthesis
1 2 3 4 5 6 7 Anti S1 (Vtg)
University of Puerto Rico Intercampus Doctoral Program in Biology
DEHP (10 mg)
C+ C- 2 3 4
Vtg
169 153
116
Effects of Plasticizers on Vtg Synthtesis
1 2 3 4 5 Anti S1 (Vtg)
University of Puerto Rico Intercampus Doctoral Program in Biology
(μg) 250 375 500 1000 C+
Effects of Plasticizers on Vtg Synthtesis
Vtg
169 153
116
Alb
1 2 3 4 5 6
BPA
Anti S1 (Vtg)
University of Puerto Rico Intercampus Doctoral Program in Biology
Effects of Steroids and Non-steroids compound on VTG synthesis
Vtg
169 153
116
E217β E1 E3 DES 5 0.5 0.5 0.005 (μg)
1 2 3 4Anti S1 (Vtg)
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
- EtOH 95%
- DDT, DDE, Mtx
Western Blot
Anti S1 (Vtg)
RT-PCR
Captivity
Treatment
- 15 days
University of Puerto Rico Intercampus Doctoral Program in Biology
C15 Glf DDT DDE Mtx
- - - 1000 500 500 500 (μg)
Vtg
169 153
116
Vtg Synthesis Reverted by Pesticides in Females
1 2 3 4 5 6 7 8Anti 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
1 2 3 4 5 6 7 Vtg
mRNA RT-PCR
University of Puerto Rico Intercampus Doctoral Program in Biology
Effect of Methoxychlor on Vtg SynthesisEffect 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
Endocrine Disrupting Chemicals
Metoxychlor(Mtx)
Bisphenyl A(BPA)
Diethylstilbestrol(DES)
Dose (µg)
Vtg +
Vtg -
Dose (µg)
Vtg +
Vtg -
Dose (µg)
Vtg +
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
Dose Response Experiments
(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
SDS PageCompetitive
Binding Assays
Cytoplasmic Liver Protein Extraction (100xg)
or Blood plasma
University of Puerto Rico Intercampus Doctoral Program in Biology
33[H]-E[H]-E221717ββ Binding to Intracelullar Proteins:ER Binding to Intracelullar Proteins:ER
University of Puerto Rico Intercampus Doctoral Program in Biology
★★
★
★
8.0
6.0
4.0
2.0
0
C NRx Rx
EDCs binding to Intracelullar Proteins: EREDCs binding to Intracelullar Proteins: ER
University of Puerto Rico Intercampus Doctoral Program in Biology
Competingcompound
% 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
Relative Binding of EDCs to ER
HPTE
4-0HTam
University of Puerto Rico Intercampus Doctoral Program in Biology
EDCs binding to Plasma SSBPEDCs binding to Plasma SSBP
University of Puerto Rico Intercampus Doctoral Program in Biology
Competingcompound
% 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
Relative Binding of EDCs to SSBP
University of Puerto Rico Intercampus Doctoral Program in Biology
EDCs: Qualitative Ranking for Estrogenicity
University of Puerto Rico Intercampus Doctoral Program in Biology
CompounCompoundd
Vtg indVtg ind EERBEERB SSBPRBSSBPRB
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/+++
Tamoxifen -- 11/- 40/+E3>DDT>E1=DES=DDE=BPA>Mtx>DEHP
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
SDS Page EMSA
Cytoplasmic Liver Protein Extraction
University of Puerto Rico Intercampus Doctoral Program in Biology
Electrophoretic Mobility Shift Assay (EMSA)Electrophoretic Mobility Shift Assay (EMSA)
University of Puerto Rico Intercampus Doctoral Program in Biology
EMSA – ER -12hr - EMSA – ER -12hr - AnolisAnolisExtract : Old New
C- 25 50 100 25 50 100 (μg)
Free probe
ER ?
1 2 3 4 1 2 3
Pomega
ERE_KC
University of Puerto Rico Intercampus Doctoral Program in Biology
Bioinformatics Analysis: EREBioinformatics Analysis: ERE
5’ GTCCAAAGTCAGGTCACAGTGACCTGATCAAAGTT 3’
* - - - - * - - * - * * * * * * - * * * * * * * * * - - - * - - - - -
ERE_CS
ERE_KC
University of Puerto Rico Intercampus Doctoral Program in Biology
EMSA – ER*- EMSA – ER*- AnolisAnolis/HeLa/HeLaExtract : Anolis HeLa
C- 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 ComparisonComponentsComponents Promega (04)Promega (04) Ruiz-Ruiz-
Echevarria(00)Echevarria(00)Kumar- Kumar-
Chambom (88)Chambom (88)
GlicerolGlicerol 20%20% 5%5% 10%10%
MgCl2MgCl2 5mM5mM 3mM3mM --
EDTAEDTA 2.5mM2.5mM -- --
DTTDTT 2.5mM2.5mM 1mM1mM 1mM1mM
NaClNaCl 250mM250mM -- --
KClKCl -- 40mM40mM 20-100mM20-100mM
MercaptoEtOHMercaptoEtOH -- 1.5%1.5% --
Poly dI-dCPoly dI-dC 0.25mg/ml0.25mg/ml -- --
TrisHClTrisHCl 50mM50mM -- 10mM10mM
HepesHepes -- 10mM10mM --
University of Puerto Rico Intercampus Doctoral Program in Biology
TFII SP1
-UV +UV -UV +UV
P RE KC C- P RE KC P RE KC C- P RE KC
SP1
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 – General Transcription Factors - EMSA – General Transcription Factors - AnolisAnolis
TFII
EMSA – ER- 4hr - EMSA – ER- 4hr - AnolisAnolis♂ ♂ -UV +UV
TFII P RE KC C- P RE KC
Free probe
ER
1 2 3 4 5 6 7 8ERE_CS
University of Puerto Rico Intercampus Doctoral Program in Biology
EMSA – ER- 1hr- EMSA – ER- 1hr- AnolisAnolis
Extract : ♂ ♀
C+ SpC NSC C- C+ SpC NSC
Free probe
ER
1 2 3 4 5 6 7RE
ERE_CS
University of Puerto Rico Intercampus Doctoral Program in Biology
EMSA – ER- EMSA – ER- AnolisAnolis Specific Competitor (fmoles)
C- C+ 10 50 100 500
Free probe
ER
1 2 3 4 5 6RE
ERE_CS
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
AAAAA
ERENucleus
Cytoplasm
Detox Liver Cells
Enzymes
Vtg protein
Vtg mRNA
(2)
(3a) (4)
(3b)
Extracellular Milieu
(1)
University of Puerto Rico Intercampus Doctoral Program in Biology
Future Experiments
University of Puerto Rico Intercampus Doctoral Program in Biology
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.
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 sperm-counts due to estrogenic contamination
was mixed…
University of Puerto Rico Intercampus Doctoral Program in Biology