endocrine disrupting chemicals: effects and mechanism of action in vtg gene expression in the...

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.


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


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.


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)


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


The Model System: Anolis pulchellus - ♂

Tropical lizards

Humid grassy areas

Night recollection

Easily maintained

Ubiquitous

Sentinel ?


The Model System: Anolis pulchellus - ♀

Vitellogenesis

Vitellogenins

Captivity Effects

mRNA/Vtg

Reverted by E2



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


Efficiency of Transdermal Treatment

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

Tip Skin Body

3 [H]

cpm


★

★

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


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)


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.


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


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)


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)


(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)


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)


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


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)


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


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)


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.


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.


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).


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


33[H]-E[H]-E221717ββ Binding to Intracelullar Proteins:ER Binding to Intracelullar Proteins:ER


★★

★

★

8.0

6.0

4.0

2.0

0

C NRx Rx

EDCs binding to Intracelullar Proteins: EREDCs binding to Intracelullar Proteins: ER


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


EDCs binding to Plasma SSBPEDCs binding to Plasma SSBP


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


EDCs: Qualitative Ranking for Estrogenicity


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.


Objective III

To determine if E2 activate the ER and if this ligand-receptor complex interact with an ERE from the VTG gene.


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


Electrophoretic Mobility Shift Assay (EMSA)Electrophoretic Mobility Shift Assay (EMSA)


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


Bioinformatics Analysis: EREBioinformatics Analysis: ERE

5’ GTCCAAAGTCAGGTCACAGTGACCTGATCAAAGTT 3’

* - - - - * - - * - * * * * * * - * * * * * * * * * - - - * - - - - -

ERE_CS

ERE_KC


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


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 --


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


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


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


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


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.


AAAAA

ERENucleus

Cytoplasm

Detox Liver Cells

Enzymes

Vtg protein

Vtg mRNA

(2)

(3a) (4)

(3b)

Extracellular Milieu

(1)


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.

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.


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.


Scientific reaction to the news of declining sperm-counts due to estrogenic contamination

was mixed…


endocrine disrupting chemicals: effects and mechanism of action in vtg gene expression in the...

Documents

anolis pulchellus lizards

estrogenic1994 ddt

induction of vtg expression

declining populations1972

mtx dehp

biologysome edcs

vtg gene expression

male lizards e217