Selectivity of Natural, Synthetic and Environmental Estrogens for Zebrafish Estrogen Receptors

Home , Liquiritigenin, Mycoestrogen

Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors. Caroline Pinto, Marina Grimaldi, Abdelhay Boulahtouf, Farzad Pakdel, François Brion, Sélim Aït-Aïssa, Vincent Cavaillès, William Bourguet, Jan-Ake Gustafsson, Maria Bondesson, et al.

To cite this version:

Caroline Pinto, Marina Grimaldi, Abdelhay Boulahtouf, Farzad Pakdel, François Brion, et al.. Selec- tivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors.. Toxicology and Applied Pharmacology, Elsevier, 2014, 280 (1), pp.60-69. 10.1016/j.taap.2014.07.020. inserm- 01067804

HAL Id: inserm-01067804 https://www.hal.inserm.fr/inserm-01067804 Submitted on 24 Sep 2014

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. inserm-01067804, version 1 - 24 Sep 2014

Université Santé receptors Email: Patrick # * and N UMR5048, ALATA, Université France. Institut Cancérologie University 1 Bondesson Caroline Selectivity Sélim

Equally Center To whom utriti et [email protected]

3 Aï Régional Balaguer,

for Institut de

contributing Pinto de

60550 t

on, K

Montpellier 1 de - correspondence ,

Aïssa Université

of of of Nuclear Patrick

la Rennes de

Recherche aroli natural,

, Houston, Houston,

4 Verneuil Montpellier,

de , Marina Institut

Recherche

Balaguer

ns Vincent de Cancérologie

Receptors authors 1,

Montpellier ka Parc 1, Rennes,

Ins - de synthetic Grimaldi

en should Houston, Houston, Médicale

titutet

- 2# Recherche

Halatte, Cavaillès Euromédecine, sur Author manuscript, published in " Institut .

and France.

la be , 14183H 1 Tel 2*

Santé, et 2, et , Abdelhay Cell addressed.

and

U896, Montpellier, : National Texas . France 33467612409Fax 2

4 , en Unité

34290 Signaling, William

environmental estrogens environmental Environnement Cancérologie de uddinge Institut 34298 Montpellier, 34298

- 77204 5

Écotoxicologie U1054,

Montpellier, Boulahtouf

Université la , Sw

Bourguet

5056, Régional Department of Department Santé Centre ede T oxicology and

et n

2 et :

USA. USA.

Montpellier, ,

France.

3346761 3787 5 Travail

Montpellier In , Jan , Farzad de de de

France. Vitro et

la Biochimie de Cancérologie

Institut

Biology 6 Ake Recherche (IRSET),

Applied Pharmacology 2014;280(1):60- Department Pakdel for

Institut 1,

Gustafsson Vivo, zebrafish

DOI : 10.1016/j.taap.2014.07.020 34298 3 de de Structurale, and ,

INSERM

François Médicale of

INERIS, Recherche Recherche National Biochemistry,

Montpellier, Montpellier, Biosciences Biosciences

1 , 6 estrogen estrogen ,

U1085, Brion CNRS

U896, Maria Maria Parc de

en en

la la 4

, 69"

inserm-01067804, version 1 - 24 Sep 2014

differents zfERs. differents comparison ability on pharmaceuticals lines Keywords: results that hER while which physiological estrogens encodes humans Zebrafish, Abstract differences compared HeLa human

we ligands -

16α based

are obtained of

there three identified

agonist more hERs LE2 estrogen to

Danio in

Estrogen reporter

to control

zfER temperature of temperature selectivity

ERs, are

was

hERs. using efficiently panagonists and panagonists and

Finally, and

two β rerio

thus

receptors

the

zfERα s.

environmental cell the

receptors,

antagonist

Among

Environmental compounds a

distinct most ,

represent new represent

similar

were zebrafish lines is our

activate and

zfERα increasingly zebrafish

(ERs),

work stably observed

hERα nuclear

two zebrafish,

manner hER estrogens

the

estrogens. selective also zfER

it a

expressing β

selective

model

in is

zfERs selective valuable tools valuable ERs

amongst the

points necessary wildlife. β

used selective to

(zfERβ1

transcriptional (hER

compound.

for human for modulate activation at As

synthetic

out as 28 each agonists

subtypes.

most α

(bisphenol to

C estrogens, an

that and and

measure their measure

as then

to of animal

physiopathology.

the Altogether,

care

compared hER zfERβ2). displayed agonists,

dissect

these

activity showed The

three

β has

endocrine disruptors, A, ),

model zfER estrogens

whereas the to zfERs. of

alkylphenols,

of In PPT

greater to these

significant

effects be

the

zfERs physiological

subtype this 37

taken

did

o three

results

have the study,

study potency on

although thus

not

first

in zebrafish selective zfERs differences zebrafish

only

mycoestrogens) mycoestrogens)

transposing confirm activate we

the reflecting reported

for roles reporter

been established established isotypes significant significant effects

zfERα

ligands genome genome ERs.

that

zfERα of in tested tested

that

the

cell

the the the the the the

all all of of as as In In in in

inserm-01067804, version 1 - 24 Sep 2014

vinylestra 16α 182,780, piperidinylethoxy)phenol] pyrazole dihydrochloride, (1,3 bis(4 BP2, E2, transactivation : Abbreviations oxime); benzoxazol

- 17β LE2,

- hydroxyphenyl) benzophenone - Estradiol;

(7α,17β - 1,3,5(10) - 5- 3,17 ERB041, ol); dihydrochloride); dihydrochloride);

function -

Dihydroxy - [9[(4,4,5,5,5 hERs, FERB033 - 17α - triene 2; - propionitrile; WAY200070, propionitrile;

- PPT, E2, ;

human Bis - - LBD, 1H 3,17β

(7 - 17α (4

19 (2 - 4,4’,4’’ - pyrazole - pentafluoropentyl)sulfinyl]nonyl]estra Ethenyl -

- hydroxyphenyl) - Chloro - - nor ligang Estradiol diol, estrogen - 17α PHTPP, - (4

2- binding dihydrochloride); 3' - - pregna Propyl - (3

fluoro ;

receptors; zfERs, receptors; - E1, fluoro

- - 4 domain; - 1,3,5(10) [ estrone; 3,4' 1 - H] methyl- - (1,3- 4- - - hydroxyphenyl) dihydroxy pyrazole Bis

EDCs, - E3, 5 - 4OH triene --[4 (4

(7 - hydroxyphenyl) estriol; - zebrafish - 1,3,5- - Tam, - (2

- Bromo - [1,1 21,16α endocrine - piperidinylethoxy)phenol] triyl)

- - biphenyl] 1,3,5(10) 4- 5- EE2, -

2- - Hydroxytamoxifen; benzoxazolol); MPP MPP benzoxazolol); atn; 8β lactone; estrogen

trisphenol;

disrupting - 17α hydroxyphenyl) - 4 - - 4- triene - - ethynylestradiol; methyl

carboxaldehyde carboxaldehyde receptors; AF, AF, receptors; -

- 3,17- DPN, chemicals; chemicals; VE2, - 5- [4 diol);

- - - ICI 1,3 2,3- 1H (2 8- -

- -

inserm-01067804, version 1 - 24 Sep 2014

ER overall while 2000; ER Even DNA- in Estrogens play Introduction dimerization (or receptors superfamily, the receptors ligand present on The of domain al. have

target

vertebrates. In vertebrates. 2012; the NH2 orthologs subtypes transactivation functionstransactivation

been

though binding

Ma et Ma ERβ1

bindind

cell amino variable mediates

gene terminal Escande in share

identified, which their

type

al.

(esr2b)

has and in is -

acid

expression domain

domain critical

a 2000; approximately mammals

agonistic

estrogen

response and been common

ligand

binding domain) et

mammals, sequence

and

al.

promoter Menuet

reported roles (LBD) contains

2006; binding, ERβ2

or in receptors

to of only

structural is

various in

association antagonistic

have

identity

receptors of

et involved

these

50% regions

Molina

various (esr2a) two

context both al. a

receptor

different teleosts

two 2002) ERα

(Menuet ER ligands effects architecture

between the

are Molina of physiological

in to

(Berry subtypes

with zinc

and characters

A/B

ERα Zebrafish transcriptional specific orthologs dimerization, nuclear dimerization, including

binding (Matthews

are

et ERβ

coactivators and coactivators

finger

(AF

the et

and et al.

mediated composed

al. have

- al. zfER (Green 2002). 1) DNA

ERβ

depending ERα affinities

of the 2008).

processes during processes structure,

1990). and

been et

the

subtypes zebrafish

explain

(esr1) activation

sequences;

al. Since et the

by human

of Dissimilarities

characterized, 2003; al.

Some

for E/F

three corepressors members on

which is orthologous is these 1986;

translocation

the

and the the ( (AF

ERβ Ogawa Danio

of functional

differences between differences

the

two three ER

their development - Kuiper is gene

subtype (Bardet et (Bardet

E/F

considered important forimportant et rerio domains

estrogen

the

in zfERs corresponding human

(Nilsson the

expression;

al.

or the

and et to the to presence domains. )

1998).

selective (Hawkins nuclear al.

- COOH

are

transactivation

al.

are receptors - and 1987).

terminal human et (Delfosse thought 2002).

dependent

al.

of adult The the

terminal receptor receptor receptor receptor

the

ligands ligands

2001). 2001). et three These These

ERα, ERα,

A/B A/B

The The

two two al. and and and life to

or or et et

inserm-01067804, version 1 - 24 Sep 2014

the selective in evaluation in differences instance, transcriptionally activate to advances of pharmaceutical The 2011). activate et and derived represent Xenoestrogens zfER mediate in consumer investigate the

vitro al. this

compounds

the zebrafish

purpose of developmental effects developmental

ligands. 2014).

organism

and agents mammalian ERs,

different

zfERα

human on the

goods of

Although

the

model the phytoestrogen

vivo

mode

is (anti)estrogenic this including ligand

effect (Singleton (Cosnefroy

ERβ

including activity composed biological

models

study to was can provide

agonists all of specificity specificity

action

and certain the EDCs

three

class (Menuet

disruption et

(Segner it

genistein

of effects,

transcriptional profiles estrogen al.

has of compounds (Escande

zfERs natural additional of

on al. evaluate

2003). pharmaceuticals,

endocrine disrupting

between between

endocrine disruptors

et ER been

2012). et there

al.

subtypes and

- receptors al. regulated estrogens

Zebrafish is Zebrafish

the these et 2002;

shown

information 2009). on is the

al. However, zfERs zfERs

effects an the

processes 2006; benzophenones BP1, Brion can

increased pathways differs transcriptional (phyto that Studies pesticides

and

of a of

Molina

worldwide recognized worldwide for several

chemicals activated

known the

that

al.

hERs hERs between and by

detailed the

on 2012; through interest

affect capacity

xenoestrogens -

and

analysis mycoestrogens), Molina

known zebrafish

mammalian remains remains

activity by

(EDCs)

Cosnefroy

industrial zebrafish estrogen

analysis in the 17β

THB

natural, et of of

finding

use

estradiol

of have al. to to ER and some

the

estrogenic and (Notch of signaling.

2008) be be compounds

- and of et vertebrate mediated

environmental

their

led

zebrafish and three subtype al.

performed.

similarities and BP2

compounds mammals.

(E2),

2012;

preferentially preferentially and synthetically reproductive reproductive zfERs

significant significant which processes processes This ligands

model similarly selective selective

Mayer. specific specific used Gorelik

and class class

The The

and For For are are

to to in in in in to to

inserm-01067804, version 1 - 24 Sep 2014

as profiles - ERE create zfERβ2 to comparison effects zfERβ2, related

compare a

model luciferase driven

hERα studies

of reporter respectively.

estrogenic estrogenic

for the of

and

and these

the data

cell hERβ their

study

effects with lines

compounds compounds The reporter

extrapolation cell

of their resulting were

to lines the

the activity

(HELN established in effects

(Balaguer et (Balaguer on results

HELN

the to gene

the on

of cells), zfERs

obtained

xenoestrogens zfERs hERs. mammalian transactivation by

the

al. we is

cell lines crucial To same 1999). expressed on

address the

system. cellular

In in vivo in hER

are useful tools are useful

support HeLa

the this the

orthologs.

context ,

full

determining challenge, zfERα, challenge, three cells,

the - length

zebrafish

as zfERs, which Since

we for

zfERα,

previously

the the analysis the

stably zebrafish and

model

transcriptional transcriptional

zfERβ1 also zfERβ1

express

for

used for is

ER used

and and

the the the the

an an - to to

inserm-01067804, version 1 - 24 Sep 2014

from - [9[(4,4,5,5,5 dimethylaminoethoxyphenyl] France). tert benzophenone Biotechnology Tocris 3,17β 2- piperidinylethoxy)phenol] pyrazole dihydrochloride (1,3 oxime), hydroxyphenyl) (EE2) estratriene purchased Tissue Materials Materials benzoxazol media (4 - octylphenol - hydroxy hydroxy Zeneca -

diol),

was culture Bioscience (17α

and methods

3- from - purchased 5- 17α - 4,4’,4’’ ol pentafluoropentyl)sulfinyl]nonyl]estra (Ma ethynyl ERB041 ol), -

phenyl) dihydrochlo 2 - plates

- 17-

- and propionitrile (DPN), propionitrile Estradiol (17α Estradiol Promega (BP2) cclesfield, Inc

FERB033 one),

- (Minneapolis, MN, bisphenol -

(4 1,3,5[10]

used - benzothiophene) from -

(Dallas, Propyl

(2, -

estriol Bis

1H ride), PHTPP ride), (Charbonnières,

UK). (7 2’, (4 Invitrogen (Grand - (2

- 1- -

- this pyrazole - -

Ethenyl A

[ - - - estratriene hydroxyphenyl) (4 1 E2) 4, - Chloro (E3)

Raloxifene were -

Texas, hydroxyphenyl) study 4’ -

pyrazole (1,3,5 - (1,3,5[10] tetrahydroxybenzophenone), -

purchased 2- dihydrochloride) 3' came came

came (3 - USA). fluoro 3,17β [10] - USA). France). WAY200070 - fluoro (6 - 1,3,5- from from - - - - from hydroxy estratriene - Island, 4 estratriene -

diol), genisteindiol), 3,4'

from methyl- Ferutinin - - 4- triyl) triyl) 2- 1,3,5(10)

(1,3-

- 4- 17β Eli hydroxyphenyl) Greiner phenyl dihydroxy

Hydroxytamoxifen (4OH Sigma NY,

- - Bis 3- and Lilly Estradiol 5 - - 3,17 α 3,17 [4 --[4 3,16α,17β

- (4 -

trisphenol trisphenol

1 USA). - (7 was triene liquiritigenin Bio - [2 - - Aldrich - butene), ICI butene), hydroxyphenyl) - (Indianapolis, (Indianapolis, - (2 Bromo - - [1,1 (1piperidinyl)ethoxy]phenoxy]

- one -

piperidinylethoxy)phenol] purchased - diol), α - (40,5,7

(E2) 3,17- - Luciferin - biphenyl] zearalanol, - 5-

ro) 17α triol),

(Monroe, - (Saint 2- benzoxazolol), MPP MPP benzoxazolol), diol) were diol) (1,3,5 estrone (4

trihydroxyisoflavone), (PPT), (PPT), were -

hydroxyphenyl)

- Quentin

182,780 IN, IN, from - - (sodium 4- 4- [10]

- NC, α (E1) ethynylestradiol purchased carboxaldehyde carboxaldehyde methyl- - - Tam) USA). USA). zearalenol,

- - estratriene Santa

USA), (1,3,5[10] 2,3- Fallavier,

salt)

obtained obtained (7α,17β 5- bis(4 3,17-

(1 [4

Cruz Cruz from from - -

1,3 1H was was - - and and

[p (2 4- - -

------

inserm-01067804, version 1 - 24 Sep 2014

based concentration were plasmids - ERE lines HELN fetal fetal Eagle’s humidified The Cell HELN Cell medium. culture medium. sulfoxide of Laboratories vinylestra Dihydroxy

culture lines

HELN - βGlobin cells

selected bovine on both - - hERα

zfERα, Medium/F12

(pSG5 (DMSO) 1,3,5(10) - atmosphere were

19-

conditions zfERs

and serum serum of EC

Luc by nor -

zfERβ1 - obtained by obtained 1

zfERα

5 Schering - The The geneticin - 0 -

mg/mL 17α

SVNeo plasmid) SVNeo

hERβ - values and as triene

(FBS (DMEM/F12)

10 final final pregna -

hERs puro,

and

cell - 37°C.

2 and - and 3,17β

DCC) AG M (G418,

DMSO DMSO transfection -

lines luciferase

zfERβ2 cell pSG5 1,3,5(10) 0.5 stock Selection - (Berlin, diol,

and µg/mL, respectively. µg/mL, lines (Balaguer et (Balaguer

Sigma

- and

zfERβ1 (Balaguer solutions concentration

named reporter 1% 1% -

induction - triene

were

with supplemented of agents Germany). - Aldrich)

antibiotic

- HELN 8β puro

cultured in

21,16α and

- cell

al. et VE2 is VE2 (geneticin

factors

and

al. 1999). successive cells and never never

lines

- Compounds lactone, named named lactone,

(penicillin/streptomycin) 1999)

The pSG5 a

puromycin

(HeLa of E2.

kind were

Briefly, - phenol red exceeded 1

best

5% mg/ml - by zfERβ2 gift of dilutions

established in

cells

responsive clonesresponsive were dextran

the

HELN were and (Sigma

0.1% 0.1% Peter Muhn Peter - stably corresponding free puro,

puromycin - were - coated charcoal coated

dissolved

zfERα, β1 zfERα,

Dulbecco’s Modified Modified Dulbecco’s - 16α

(v/v) (v/v) Aldrich) transfected respectively). Cells Cells respectively).

performed - a LE2 LE2

in in

similar

of of from 0.25 a

and at the the pSG5

5% 5%

and Research Research

dimethyl the

with µ selected selected

way β2 culture culture treated treated in g/ml)

CO -

puro puro final final

cell cell cell

the the as as 8 2

inserm-01067804, version 1 - 24 Sep 2014

containing experiments. temperature. 37°C absence bottom The zfERs temperature and GraphPad a presence opaque tissue The zfERα, medium. were later,

plate

hERβ1 HELN HELN

maintained

and for binding

zfERβ1

clear of

reader

16h. cells

Prism 0.3 - - non-

zfERs cell zfERs cell zfERs transactivation assays transactivation with

ligands

tissue At

Experiments

assays culture plates culture

and zfERβ2

mM At were radioactive

(PerkinElmer

the in the software

the

culture

the luciferin.

ligands incubated end

end 28°C cell lines lines

(version transactivation plates of E2 culture

were

(37°C

the because (Greiner were were

Luciferase the 10 Luminometer, MA, with

incubation (Greiner

µ incubation were performed seeded seeded

instead medium. M 5.04;

compounds of

CellStar, were

performed the

activity

Graphpad Software

CellStar, assays at at of 28°C).

added

improved period, Luciferase

a a period,

in density density USA).

was at quadruplicates in

in 28°C USA). USA). culture h

measured unbound a

later, transactivation ability of of

Compounds assays similar

for

80,000 80,000

[

EC50 H medium and

16 Inc., ] were -

material E2 for manner

h. cells

cells cells

San values 3

2 performed Cells to

was s were at

in be per per

Diego, except was

least

intact were replaced

tested were

well well incubated the

of aspirated

in for

CA,

presence maintained two living the

in in

measured were 5%

the

96- 96-

with zfERs USA).

independent independent at FBS

cells well well incubation incubation added and

28°C or medium

DCC at

in hERα hERα in cells white white white using using using using

this 8 or the the the the h

inserm-01067804, version 1 - 24 Sep 2014

(data 1B). in values and 37°C 1D). lower (0.266 zfERs (Cosnefroy Since Impact Results measured washed organism. Bioscience) assay dpm.

1.554

EC EC not

for with

the nM), was at of

Experiments

5 5

28°C three three 0

0 shown).

zfER the

by [ of different As values

and functionality of 3

et due H

was

34°C

zfER

temperature Bio

seen - β

al.

(0.061 E2 times

0.197 1.

- added for ]

Rad β 2009 ; .

(0.433 EC

in 1 zfERs In HELN In including temperatures,

were

E2 and

Fig. nM) nM 0 with with protein

values

and and

Matthews nM) zfER

on transcriptional

performed 1A, degradation, at than

PBS. [ EE2 - rainbow 3 37°C zfER

H assay or β the for

at 2 ]

37°C bound

were

cell

31°C E2

cells, Then,

for et C

and in

5 and

lines al. trout 0 quadruplicate (1.921 0.039

zfER we value

(0.127

used zfER radioactivity

2002), EE2

0.2

measured were activation β ER a

and of

2. nM)

concentration

ml ml were nM), temperature relevant temperature

As E2 we normalize

has also 0.031

for

of of

was

first approximately

34°C in

protein been sensitive was

of zfERα. at

LSC

suspected

lower zfERs.

tested least

(0.19 counted.

bound

- shown at was cocktail cocktail

level

Similarly,

28°C two

to 8- the nM)

28°C

the radioactivity

independent fold that

0.118 by

to ability

Protein and

to or

(Emulsifier température

saturation lower be

(0.077 the

the 37°C 0.714 the

and

temperature of

increase

EE2 concentrations

physiology

0.031 E2 (0.947 nM)

and

values experiments. 37°C

- EC ligand Safe, Safe, to (Fig.

0.673 than

5 nM in

activate 0 than nM)

value expressed expressed -

the sensitive

Packard Packard at at binding binding of nM

28°C

(Fig.

28°C 28°C 31°C

EC and were were

was was this this

the the at 50

inserm-01067804, version 1 - 24 Sep 2014

endogenous ligands estrone respectively). (EC50 displayed transactivating slight activity profiles We approximately nM other subtypes zfER Effects potency to relative et for zfERα for The

al.

for for measured 2006), ERα

of values

with obtained to (E1)

hERα hERα human endogenous

evaluated similar and

transactivate selectivity its EC50s demonstrated

0.031 nM for 0.031nM both

Among and

zfERα EC50 zebrafish a

and

5.6 by 6-

potencies

of 5.3, 24.7and for 45.1nM zfERα, estriol

fold

have the

in E1,

values

and

0.068 0.068 (Escande compared the

this

the

effects

E higher counterpart. been

2.6 3 endogenous

equal (E3),

cell luciferase

on for and

nM nM estrogens nM,

shown

zfERα et

E2 of context.

to potency 17α zfERβ agonistic

al. for for

respectively) and respectively) the the indicates

- 2006

zfER E2

reporter

17α hERβ hERβ

(EC50 zfERβ1 weak on zfERs on estrogens

isoforms

The reported The behave as behave indicate that indicate

to to - - activitie and Ethynilestradiol (EE2), mediated trans

that

estrogenic transactivate (Escande (Escande

values

gene and supplementary

transcriptional

E2 (Table tested,

s full

zfERβ2

has a has zfERβ2

EC50

these

HELN

agonists

all zfERβ1

1). 1.7 et et stereoisomer 17α

slight criptional activation

3 luciferase

isoforms al. al. estrogens values and

- zfERs - (EC50 data E2 hERα

activation 2006), and in zfERβ1

0.55 showed

both Fig. for

which zfERβ2,

with

compared values (Fig.

gene gene of have nM, E2

S1A). suggesting suggesting

selectivity EC 17α

the is

2 the has

respectively), zfERβ1 zfERβ1 respectively),

ssion ssion expre of

A, approximately respectively.

- values

stronger

estradiol weakest

The human

to 12.1

a B,

(Fig.

hERβ (Escande (Escande hERβ 31

C).

transcriptional

relative

that - of and fold

ERs

E1 potency 2). agonistic 0.061

(17α

in in

8.5

E2 E2

and

greater greater

with 0.017 0.017 These These to hERα hERα -

E2), E2), nM, nM,

nM nM has has the the E3 E3

in in

a inserm-01067804, version 1 - 24 Sep 2014

affinity than not hERβ zfERβ2 with affinity Fig. hERβ assays transactivation zfERβ1 mycoestrogen Fig. presented zearalanone We Selectivity values

activate

next S1E) S1E) S2A zfERβ2 with zfERβ2 a

than than 132 nM fornM of zfERα, 8,3.4and 132 3.9-

for for and

and

analyzed and

4.4 displayed

for hERα zfERα

for similar zfERβ1

analogues zfERβ1, natural

1.2 data α greater hERα - hERα - (α zearalanol

and EC50 values of valuesEC50

and the not profile

estrogens

similar

data (supplementary

respectively. hERβ

potency than potency

for

(Escande for all shown). ability zfERβ2 zaaaoe β (zearalanone,

HELN (table (zfERα zfERβ2,

but maximal activity

zea) of on zfERs on

relative to The

with et

- 1

zfERα myco 563

potency Liquiritigenin, another and exhibited

al.

zfERβ1

phytoestrogen genistein,phytoestrogen which different maximal

and zfERβ1 2006;

Fig. transcriptional

zearalanol, and compared

Fig.

6805

zfERβ1

conferred with 3C).

phytoestrogens

EC50

Molina zfERβ1 (100%)

and S1D), nM

EC50 Finally, zfERβ2,

(Fig. to

values zearalenone, for -

Molina et Molina activated potency the but

activity values activity zfERα and zfERα

fold 3B).

ferutinin,

different zfERβ phytoestrogen of

respectively) to

which

0.176

and Genistein

≥

zfERα activate (Table

al.

β2

α cell 40,

6.8-

zfERβ2, respectively, zfERβ2, potency zearalenol

a 2008), potency, nM present

50 phytoestrogen with

lines with 1 fold

the

for zfERα activated and (Table and with

demonstrated (Fig.

a for zfERs zfERα, higher a

260 supplementary data supplementary data s

12 upplementary higher higher and

the 1 and Fig

greater 3A). nM

(Fig.

zfERs β potencies in in potencies 0.71

affinity affinity zearalenol) zearalenol)

for The

potency potency 3). and 3.D).

nM and zfERα, zfERα, similar greater greater (EC50

other

data data

The The

did did for for for for for for β2 β2

inserm-01067804, version 1 - 24 Sep 2014

values zfERs the relative than lactone human We Selectivity BP2 nonylphenol zfER partial We Selectivity agonists zfERβ1 et Benzophenone 763 affinity

al.

greatest next

and next (Escande hERβ β

presented

2008),

(Fig. of approximately agonist

subtypes. The

(16α towards

and ERα towards

o 487 evaluated analyzed

of of the

zfERα 4).

zfERβ2,

of environmental LE2), demonstrated greater demonstrated nM mixture

selective compound selective

towards zfERβ

2 a pharmaceuticals Similarly

zfERα

(BP2), zfERβ1 3- for

the selectivity fold which the et

zfERα, 2478 zfERα,

respectively EC50

isoforms (NPm) (NPm)

ability

and

al. the

251 nM and251 nM 233for zfERα ability higher which

to (supplementary

has

2006) 3

zfERβ2

compounds the values

zfERs

over with

acted acted

present potency of (Fig.

human

synthetic on zfERs on also

(Table

known and 1000- affinity approximately of relative to (Fig.

as as 4A). 3,17- showed

BPA on zfERs on a

ERs 4225

towards

partial

fold higher

data 4A). transcriptional 1). ligands

di Among environmental for

(Delfosse were

hydroxy

nM The selectivity

greater

zfERβ1. Fig. BPA zfERα

affinity

agonists agonists

zfERα transcriptional

for to 599 alkylphenol 429

all 4B and

displayed greater displayed activate

- zfERβ1 and zfERβ1 selectivity

19-

and et the

and and nM, β2 The

and for

nor al. activity

towards

respectively,

estrogenic

compounds zfERβ2 reporter

4C). 115-

EC50 1.894 hERβ 2012), - zfERβ2 transcription 17α

4- fold

4tOP activity for

tert -

pregna values 1223 than µM

zfERα zfERα

bisphenol A bisphenol gene relative

its -

higher octylphenol (4t octylphenol compared to compared

compounds

and affinity

tested, and zebrafish for

803 nM for 803nM β1).and and

- activation of 1,3,5(10)

through

NPm

and hERα

3.823 potencies

4tOP to 603

16α for zfERβ1

zfERβ2 zfERβ2

(BPA) displayed

ERα nM

(Molina and

- µM zfERα to - zfERβ1 the

LE2 triene through -

OP)

for

activate

NPm to counterpart counterpart zfERs.

for

(Fig. acted

displayed displayed activate

- and

zfERβ2. zfERβ2. than - 21,16α and

Molina Molina zfERα, zfERα, greater greater

(EC50 (EC50 were hERα hERα

4D). 4D).

as The The

full the the the the the the

a -

inserm-01067804, version 1 - 24 Sep 2014

Fig. greater human human (FERB33) (WAY300070), 21.7, isoforms zfERβ 1,3,5(10) greater greater and zfERα, zfERα experimental cell Tam) The Effect selective transcription activity towards

S2B lines 28 pharmaceutical anti pharmaceutical

238 were of at

ERβ potencies zfERβ1 for for nM ERβ -

pharmaceutical

- all compound D). (Fig. triene

and tested

and

while it selective agonists, zfERα zfERα

for by

system

selective agonists 115 5A, -

3,17β

and zfERα (Fig.

zfERβ1

7 zfERα in for

- B,

nM ethenyl 2-

the and 4,4',4'' zfERβ2, partially

zfERα

chloro- - (data (data C). 4C diol diol

(IC50 for

HELN

antagonists estrogenic

compared to compared zfERβ1 zfERβ1 and No and -

(4 2- zfERα,

(8βVE2) (8βVE2) not not 3' compared

- (3 agonistic activated respectively value β2, Propyl -

- 4D). these fluoro zfERs -

fluoro shown).

(IC50 respectively)

such zfERβ1 The

compounds of on transcriptional - compounds -

[1 and 3,4'

and 3 zfERβ1 to 4- activity

zfERβ1 H for values EC50 nM)

- hydroxyphen as

- values values ]

zfERβ1 all ICI ICI dihydroxy - (Table

pyrazole

and

2,3- full displayed

7- than

and 182,780 182,780

for

bromo-

(Fig. zfERβ2, ICI bis(4 and showed of of

1). and

these the zfERβ2 -

- 1,3,5--triyl)

182,780, 8.2

- [1, - Likewise, zfERβ2,

5A).

hydroxyphenyl) 2- activity zfERβ zfERβ2 yl)

showed showed

three anti three (4 1- better (Table respectively and - 8βVE2 5-

biphenyl] (Fig. antagonistic hydroxyphenyl) 4OH benzoxazolol (ERB041) benzoxazolol

cell raloxifene, 4.5nM 4.5nM

respectively. The (Table selectivity for tris the

4B).

slight Tam

- were lines estrogens

phenol EC50 - 4-

Even thoughEven - for for 1

propionitrile

antagonistic carboxaldehyde oxime oxime carboxaldehyde

32, (IC50 activity pref and

- 4OH

(PPT) values zfERα zfERα - 188 1,3

erential was

supplementary data data supplementary 1).

zfERα values -

tamoxifen in ben

and

Similarly, did observed

of selective the and

8- -

potency zoxazol

antagonistic DPN 113 (DPN) (DPN) than vinylestra not of

three displayed displayed zfERβ1, 14.7

activate activate

nM for

(4OH

were in zfER zfER other other

- ERα ERα

5- was was

nM nM our our are are the the for for ol ol -

inserm-01067804, version 1 - 24 Sep 2014

of zfERα, 57, 13.8and for 134nM respectively binding towards and http://atome2.cbs.cnrs.fr/AT2B/SERVER/ used To Structural Another potencies value preferential than respectively) hERα S2E estrogenic antagonize antagonistic activity selectivity were selectivity zfERβ1

gain

hERβ F).

of selective

s pockets. and compound certain the

tructural 12

towards

analysis and in

antagonistic

(Fig. nM)

zfERβ2, complex

PHTPP

agonist

ligands web

Indeed,

tested tested was insights

for the of and tested -

zfERα

based towards

activation respectively.

zfERβ2 zERβ1 a

with behaved

Manas

and

activity more has on

L384

for into

selectivity

hERβ this the the

been

its subtype

(IC50 powerful zfERα zfERα et the

and server

effect towards ligand. al., HELN

zfERβ1

zfERα

the antagonist associated with associated

Finally, zfERs M421

2004). value

compared (IC50

zfERβ on

- antagonist

The zfERs zfERs

selectivity hERα

EDM

EDMon and transcriptional pan of

of Superimposition antagonists

structural value value

cell zfERβ2, PPT. hERα - 29.4 antagonist (Table on.html

(IC50 reporter reporter to

lines

for

zfERα

As two nM) of of of are

)

(Endocrine Disruptor value 16α

respectively) with zfERβ1,

shown basis

261

with (Delfosse replaced

amino

(Fig. inhibition cells. cells.

- or LE

IC50

of nM) nM)

of of hERα zfERβ2,

5B).

the

acid in 0.62 MPP

the with

by figure

values

and human and et

16α

and

Raloxifene which Raloxifene

(Fig. (MPP) hERα

M336 differences in differences nM) al.

- respectively presented presented the

it LE

4.1 data supplementary

5D, 2012) 5C). of

was was

2 HELN than

and

- 854 and bound hERα model and and

PPT

zebrafish

able able

hERβ to

the and hERβ 9.7- I373

slight zfERs model

displayed

(IC50 hERβ

Monitoring; Monitoring; their fold 326 to to

in selectivity

has

(PHTPP)

ERs,

partially

was zfERβ1 zfERβ1

hERβ,

nM greater greater

hERα

values values ligand (IC50 slight slight Fig. Fig.

full full the the we we for for

inserm-01067804, version 1 - 24 Sep 2014

binding residue but residue 16α interacts unfavorably replaced conformational resides group ring on medium ligand the (Fig. binding bonds leucine three agreement the

the more - variations

not LE of

8B). receptor

of with

for

crystal

16α in

2 residue (L391) in (M317) by

pocket

for hERα with E (leucine) ffinity by

constrained

lactone the

this H3 As 2 with

- the

E353

LE and with the

subtypes. a 16α

(L369) in structure receptor 2

the linear

for 16α for consequence, I373consequence, change (Fig.

(LBP), occupies H524 branched

the from 3 helix M336

and

LE flexibilities slightly

space

environment ring residue 2 7) (Fig. zfERα

(H11)

(Fig.

- with subtype. of

With LE it to (H3). and the

residue

hERα

appears 2 better constraints 7), accommodate this accommodate

the

16α

is a This same (Fig. relies H3

(H3)

Therefore,

zfERβ1 possible in (Figure conserved

highest The -

and affinity LE

provided zfERβ2

receptor

Ileu as position

8C). maintains most that and complex

affinity

H7 residues which points

provided

displays

373

8D). R394 l

With affinity. the

oss likely

of 16α

16α in provides

combines

characterized by characterized

hydrogen 16α of

values with

- The

the

from H5 from a LE that

on the on the

additional

- a conserved the

LE - by

The 2 LE

s favorable identical

E ynthetic replacement

latter accounting adopts

towards a 2

2 the most measured with measured

2 two

for the for

slight E (PDB slight replacement

The

2 bond (Fig.

different and

large constrained LBP different

group.

to isoleucine in difference

ligand

gain M421 interaction latter.

difference code

8A). is

observed a those

residues engaged

much of

combinations of combinations 3UUD)

On In in

of L349 (H7)

LBP I406

positions the of for

hERβ, the between

smaller the

in position

provided in between zebrafish

the that with plas H7

in other side

the showed

human (H3) by a by (H3)

reflecting

weaker affinity of affinity weaker

H7 must

network ticity

(I406)

low the in binding

intrinsic the two

where hERα

the by receptors (isoleucine)

linear

receptor, undergo (Figure that residues

and zfERβ1 of

the methionine

17- of the

affinity

the

and the complexes complexes it

flexibility hydrogen a branched branched hydroxyl hydroxyl M421

interacts interacts

weakest weakest

leucine leucine 8D),

ligand a

phenol phenol zfERα reflect reflect hERβ, hERβ,

large large by and and

of the the the the

in in is is

a -

inserm-01067804, version 1 - 24 Sep 2014

obvious zfERs, (Fig. explained by explained zebrafish zebrafish (8bv- zfERs Results like 1999). thermo gene reporter for hERs showed Screening Discussion The inability incubation after estrogenic com estrogenic

the zebrafish,

E2, addition

7)(Sassi

using differs -

Interestingly, the dependence

peroxisome from selectivity that

DPN, DPN, ER ER

at position

known of - the 37°C

from this

assays of endogenous, Messai subtypes. similar which pounds

hERβ

chemicals to chemicals WAY300070, WAY300070,

mutation study

of the

that (Fig. proliferat of

homologous mammalian

this

(Cosnefroy

increased selective human

estrogen among observed

demonstrated

al. 1). phytoestrogens This This thermo

environmental and 2009). Temperature sensitivity or

the

cellular

study study

the

activated

critical

phytoestrogens binding

FERB033 - the between et

to dependence ER cells

three This al.

hERβ

potency

allowed allowed ligands

that context. 2009; because amino

towards (Matthews anino

receptor zfER hERα

M336

the and

Matthews is are

acid

us us selectivity acid (genistein

The subtypes,

s it

and not

the ynthetic

ERB041) to

is estradiol is also to to

involved

et (Riu change zfERβ occupied, HELN of a shared hERβ

assess

al. able

fish et

and and ligands

2002; of

al.

- orthologs.

subtypes. al.

approximately 10- zfERs ERs by most

to physiologically relevant physiologically

in some differences in in differences

liquiritigenin) 2002)

2014). compare

as activate activate induce genistein other

Sumida

has

likely in in

cells of

hERα, and

already

the zebrafish zfERα

the transcriptional

their accounts for accounts

were

the the et binding

the HELN tested

al.

and and the the been zfERβ zfERβ reason selectivity

incubated fold

2003; a nuclear

pharmaceuticals pharmaceuticals compounds zfERβs zfER potency in leucine

reported

compared

hERβ.

seems

isoforms isoforms temperature

Tan the

activity

cell receptors receptors

at towards towards belong

lack residue residue of of et

using using In to 28°C 28°C

lines lines

al. on

the the all

be be of of of of to to is is

inserm-01067804, version 1 - 24 Sep 2014

endogenous EC50 was compound transfected 2012). approximately for (E1) Among between which to fold (Cosnefroy Among relative in zfERβ2 which reporter activity and

the the

hERα displayed the higher and

ERα

ligand value is Similar

certainly certainly

over via the

most at zebrafish

the the in 17α their

with

relative

(Escande et (Escande and with et

accordance with accordance the compound with

natural selectivity

myco myco zfERβ1. of

binding

E2. potent al.

slightly

human transactivation

ERβ

zfERα

the the and explains

2012).

and Likewise,

low and

estrogens for highest

phylogenetic zfERs endogenous ligand

human 2- domain

This

orthologs,

higher

and

fold for

EC50 zfERα The phytoestrogens, al.

the the phytoestrogen

hERβ, zfERβ and

the

potency a lower

2006). our phytoestrogen genistein, phytoestrogen

ERs.

affinity

values. tested properties study

differences differences

in highest

an the respectively. previous

groups,

our f transactivation showed

orms es

zfERs The using

towards in

for trogen-

study - for affinity

the zea for

also

by zfERα α

with respectively; zfERs

studies

- transiently in in

compared zearalanol

preferential and preferential zfERα HELN preferentially activated preferentially genistein

dependent

the

behaved (0.077

selectivity selectivity the

and

were

three

performed potencies

- ability the with zfER

zfERβ2

nM)

a to

transfected HEK293 transfected

were as afull as hERs, (α

however, zfERs, full less

luciferase their EC50

zea) zea)

cell and to (Lange et (Lange

agonist

observed

for sensitive in almost equal

than and

human

lines, sensitivity

followed demonstrated of agonist

zebrafish the

one

it 0.177 for reporter

activity also in

zfERα

zfERα fully 17β

to ER

zfERβ1 in al.

both in the in

three

displays

- nM,

HeLa

estradiol (E2) counterparts (Table

2012). liver activate of of

cells (Sassi in human and estriol towards

zebrafish

amino

very

the the estrogenic ligands ligands estrogenic -

(Cosnefroy the than derived cells

zfERβ showed a - E2

Messai best best

HELN similar the better

ERs (E3), -

acids acids hERs, transiently zfERα also

luciferase luciferase cell

cell agonistic subtypes with

was

that affinity affinity

estrone estrone

zfERs, zfERs, et

differ differ lines, lines,

lines lines

with

al. and and

al. 10- the the the the the the E2 E2 3), 3),

inserm-01067804, version 1 - 24 Sep 2014

unable zfERα zfERα potencies 16α with 17α 2012), relative Among 2009). affinity zfERα, and respectively BP2 derivative also slightly the terpenoid and preferential and displayed

- - human partial

zfERβ2. E2, EE2

almost showed

cells, found BPA Liquiritigenin,

to with environmental better for

which

the was slightly

with activate than

the agonist ERs,

(Cosnefroy zfERα

also displayed equivalent and Cosnefroy in

approximately 1.36

zfERβ

better better

efficacy similar

behaved zfERβ2 has

this instead BPA equal

pharmaceutical higher

hER

compared 1000 chemical screening. chemical

subtypes.

potency towards towards potency another hERβ) (Ikeda et (Ikeda hERβ)

displays

β affinity pollutants,

potencies activity

towards

and behaved et as affinity - (Escande fold

al.

al. better a

zfERβ1,

2012). full

phytoestrogen

(2012)

to selectivity for

for Accordingly,

similar

towards hERβ for zfERα for

compound potency

in agonist the bisphenol and et hERα

an transactivating

al.

zfERβ also

respectively) al. (Delfosse antagonist agonistic 2

zfERα

Conversely, 2006),

zfERα

in 2002), and zfERα. towards fold

and found

that

the with

A in

subtypes hERα - hERβ zfERβ1

(BPA) the

higher three than was

and displayed behaved

properties between properties to et that higher

zebrafish the

(Escande et (Escande al. and zfERα,

but devoid

zfERβ2 PPT, zfERs,

BP2 the the

also than (approximately 100 (approximately three 2012). potencies

hERβ different

Considering

the zfERβ zfERβ

another has

presented for zfERβ2. for while

- a

zfER derived

with

over

full highest the

then The a agonistic

al.

slightly

compared it

activity

agonist

subtypes. The subtypes. The similar most

subtypes.

hERα

zfERβ1. alkylphenols hERα slightly 2006),

hERα cell

affinity

higher reporter

selective

activity

lines selective

higher on EC50s affinity,

(full

and

also

activated and

Finally, Similarly to

the affinity towards

(Cosnefroy

gene hERβ,

agonist

zfERβ2

exhibited higher

activity towards 370- three zfERs three 4tOP in for

compound benzophenone benzophenone also displayed also displayed agonist

the activation

the both fold for ferutinin,

the presenting presenting

to and

on HELN

and zfERα zfERβ

hERs,

zfERs, zfERs, zfERα zfERα higher higher et that

hERα hERα NPm NPm

al. and and β1, β1, for for

- in in is is

a inserm-01067804, version 1 - 24 Sep 2014

in EE2 pref zebrafish cells selective has efficiently The A for subtypes. zfERs. zfERα whereas whereas between the valves. PPT, zebrafish transgenic compounds for (5xERE:GFP) each

liver recent

a zfERα in

erentially the -

hERs

mediated ER higher transiently

vivo

of is ICI human

subtype human

study zfERβ1 zfERβ1

larvae

synthetic

IC50 selectively expressed selectively transgenic

pan

than zfERβ1 findings

182,780

towards antagonistic on

activated

with antagonists transcriptional

reported for values ER

and the

with in transfected

is is

zf.

and

zfER

genistein, that other subtypes. agonists,

model the

presented zf

5XERE:GFP not either

obtained zfERα. ER

zfERα zfERs differences

these β activity

hand, d detecte

ICI

(Table subtypes,

containing a

ICI

with

activation

BPA compounds

such Moreover, 182,780, slight than is

showed

182,780 in in towards crucial

zebrafish

the

zfERs (Gorelik (Gorelik the and in and zfERβ2. as the zfERα

ER zfERs

heart

DPN, 4-

GFP

DPN 5x Fig. zfERα with

before determination or

as OH cells present subtype

consensus

(Gorelik 4-

selectivity

et et

the 7). In

labeling of

valves, all preferentially induced preferentially OH (Notch tamoxifen

8β

indicated

their accordance with accordance compared al. al.

Similarly selectivity of -

three

opposite VE2, tamoxifen tissue

2014).

and

use zfERβ2

and ERE

isoforms while

only FERB033, ERB033 all in Halpern. localization in

that

the to to

Mayer

selectivity

vivo sequence upstream sequence In In acted

zfERβ2

reduce phytoestrogens, known phytoestrogens, the 4- transcriptional is PPT

this s

to everal

robustly of our

larval liver. 2011). ; as

help

displayed

2011).

same same the

tamoxifen

the GFP pure and

result of

ligands elucidate zfERs the

what

Althrough β1,

antagonists expressed expression

study, s, -

In developing induced

profile

ICI ICI

preferential Our

has vivo and

in of antagonized more is

182,780

distinct roles 182,780

treatment of

a - MDA study

been , not

WAY200070 WAY200070 GFP ICI of treatment fluorescence fluorescence in

human in in

estrogenic estrogenic conserved conserved

zf the 182,780 observed observed confirms confirms the the

reporter reporter is affinity affinity larvae: larvae: blocks blocks

liver, also three heart heart - ERβ ERβ 231 231

of of of of

inserm-01067804, version 1 - 24 Sep 2014

ER subtype chemicals brain (Cosnefroy such guidelines are regards Althoughpossible. various To between zfERβ1 efficiently selectivity selective efficient in subtype and ERs, translation

conclude, mediated zebrafish

newly the

of of

(anti)estrogens for

to specific when

zfERs

(PHTPP) differences

- cyp19a1b

for

EDCs was was reducing established et

xenoestrogens

ERs (anti)estrogenic effects (anti)estrogenic we only piscine

assessing al. and

analysis also also

have 2012) s

their

highlight the antagonists were antagonists none creening based than GFP

or revealed

observed models.

blocking

shown

human

the

can zfERα

using larvae on vitro

zebrafish

the effects.

for estrogenic human

serve in

tested Hence,

orthologs cell here

both

need for for (Brion and the hazard this

lines

as also

cell

GFP

that and human PHTPP compounds specifically (activities study to zfERβ2.

an useful

potency

take

et et

lines assessment, tested

for antiestrogens. together

human there induced initial

al. al. in and

into

expressing whereas whereas screening

terms Thus,

2012). 2012). on are

or of zebrafish ER screening

account with

by the

potencies) clear chemicals.

isoforms,

of since

there various

HELN

The The those

raloxifen raloxifen transcriptional

tools human

differences between differences

ERs should

is hERα

the

previously

- activated

from estrogens zfERs

to the

also establishing This

to species

estrogen

address

and elucidate the

be present (raloxifen, mammals

a is reporter

followed

- non either activities

MPP

particularly

and established of

receptors.

estrogenic potency

conserved time origin the pro

zebrafish cells.

to antagonized

MPP) the

fact

full estrogens zebrafish established test towards

and

selectivity of

Slight

with important in that To spectrum

and

s ii) ZFL or

electivity electivity

this

an the human human human

zfERα zfERα

in is hERβ hERβ direct direct

more more

cells end,

ER ER not the the

of of of of in in - inserm-01067804, version 1 - 24 Sep 2014 H Texas Ministry This Ecosystèmes, We ealth’s

acknowledge

study Emerging

National of

Environment, was

Santé

Institute funded Technology Peter Project

Muhn (Schering Muhn

the of Environmental by

“BISCOT”

US grants Fund - Environmental

under from Acknowledgements

2010,

AG) for the for AG)

Health Agence Agreement 300 Agreement

Protection the

Sciences

kind P189 Nationale gift NEMO

Agency (grant -

of 16 α 9- 1958, de

number

project

- la (grant LE2

the Recherche Contaminants, Contaminants, Recherche

and

financed by financed R21ES020036). number National 8β - VE2.

R834289), the

Institutes

the

French French

of of inserm-01067804, version 1 - 24 Sep 2014 E Ba 2006; 2012; Lang 2009; Gree Co I Go D B Berr Ba H 2012; Go Co ked s ri e a e cand r l s s w lf o r r ague subs A t che na exp zeb pha Un zeb i r m Acade pe riEndoc es D one s r r u ce v ac de ‐ ‐ ne ne e e hyd 60. ns h osse n n epo ecep ecep t a y itr k m m e iff n lic lic udy on e

tr t ll i t 71: 7 23: 109: r t

i fr fr it

n i M be u

gh spec F o e - r r e m r K A v : r k k . oge es ‐

S e s t Ku r a a e36069. esse o o tit

, r it r m r rt . i r ,

yp , oxy PL ,

, t a d d i

fi i fi ence t t 1459

1450 y y llif t

A o

DA ca ca tr m y L e V o o M l Ka DA s u P aco s i

Ara Ku 14930 M

e r pe , , e no n

r r r , h h ogen be A ti A , t S y e i es l .

s . B t

es ng

d i s P ,

e an

ves G , , n r a m

F , Pag H

live s

r Co o , a r es

l illo l a i t o o t tr s

I t r ogy m - -

B u ogy ecep n B ri . i zge w o t w ‐ ‐ f . .

a e o anco 69. 4. GG n n d ce an Ha Tho

e oge

ri ri tr m o Aqua i r . l Sc r V

ox zeb

ee ano n n Y Y d n

e s - r c a r

o f o r ‐ l oge b 2014; 5. d

a a . , P , a

l l

. r r ,

syn

i ce n

n pe

i Sp

e Es i

ence

d d t l n A m o r r t f Y n n 2011; spheno M

d En

, O r

i n ffec oceed en. en. o D

s f , , l F m a F i

tr r f ily p ri n l t t r

t K i , li t A

e , fi n , B p Se

o m yaga i

suk P r line

M n F he oge i c ne Tox r r

m s x Cha ost

Gu , r 122: an n n r M r i , us z s t Th g u a ac cci h o ecep t ecep r s

ti u 152: e t r Co van

m Rob a a H LR d t i J k . i ill Pon ( o a e t i n r ri c n ng

f w

n l ill W co D E m

a e s

Tox

o t

ca a 356 e m A i E i t . , E r

H e r a an s n o S

t t xenoes h bo s , ec t t t s ,

l bo , s M es i 2690 ac

e . i g Hun una , e t

o B o

s n e V gna t

Pe agon

N - S C B

r an i i

W - 2012; r ab ‐ r , l

1996; s M B o co n ‐ - tr i - N r ti , li ca ifi ass J i U

‐ r , ‐ , sua on 62 con n n

L o O

gande e Ca lt sub o a ogen O d d o sy r l a n l Lav C , l lin P f e y w e og o

lt n - m r tr it . - g

‐ r jou

703. Bou m r ‐ i

ri echa

liza -

Rechav e

tr s ova aved s AL i t e ‐ do v g ansac F H t o

n i 109: Ro p r o t ex ille t 93: ype ca i pos i d

. ogens , h s t o v u D r s ) r exp

, i o o

/an s ti . ti n n e ov o o na P

Fene d

l t i S r l l

o o ah

kko e B

Y 5925 J o y o l ,

t i N s sc

ERa ou

o 250 e Respons

a , Sk . S a l i l n n

r aze d ti . Cha t t J O o f , N t ,

us agon i U i t dependen , ou P

e e o va P 1990;

ences

r i f

Po ,

t s a M y

ppe o ,

r Hor s Po o n r l

Th s fi - M i ti - pha r ush t

oceed f ‐ f ti n Ton gu 8 ‐ m H

La f i 30. che

o , h s

n r a tit quan ona l g o

r , VS h es che e ,

f N A e g bo i r che rr es o i

i n n s

de guchi A it , Bou

References 9 ilsson

. Sc t Laude f tr g

m , an e t

an m

l Acade tr : n n spec l 2012; w tr

m r K a 2811 e

oge

line Ha i r i i

t SK enc oge ng es ca f

e , d

ansgen

b o o i J o o ssi P F

M J ri M

D l l ERbe M r . tr , , H l s l ecu pe

ac n ca Env

s s ti

a ou t o e e S , H n ERa

M , oge

g Chun - v , 125: ir ‐ ,

s tt b 8.

o ti

. Ba agon r , tr f r e o Kobayash env

Pakde o V e x n uhn G y ecep f

va e 2000; ir

r f a

uct

t r

, i b

ecep us co l n n m on r c a pha

t us A ague t A

ti i 439

h A h . ir ri o Vanacke endocri M t , y

u mmon n i e

e i , o o m E B s l a on s P n t

ogy r Cava g

ti k ( . , o BC

f i l t

a Tho 97: , r g

t en

cyp19a1b oche

- sson an

N o c ecep r r ‐ o S m l T 49.

Pon o F do e

s m assess

t , s , r a

r , . f

P t t a i d en an t

10751 ansgen

ti a

m ille i ab 1986; e l , Ba Kaya m

n a T

l tr ona y ac

ERbe no s m t env

e Sc d t t

l a , J r

. a l o ansc W A l s t t

s e M

ti ague a i e

l e

i r n Eva B f u . ogy

ence l a J v t

a uncti V l m ir . s r , i P M s

C t eos

- it l 51:

ophy t epo en

t ‐ - . , e a 6. on i . Acade ‐ e Sc l e a a

) GFP c ri y (

o on l . l d . A xeno e a ua r

: . r t

p f t

r tifi den

751 m l t s o Zeb 2002;

d F pha Co Cha ti s een ona Sa rt i

a P s t ti n . . an i iff s a en ona t l h o , n

e .

ab l m m

e Re P f

. Te ) r - e m e r

n d

Repo ‐

t r es 8. i

a o r t f r n p zeb S pa . ac l

gen oceed

y oe

l e en 28: f r s h tr g l ca

o do

d tr 1999;

a peno es

s t

a o t e Co r f l i t uct h h fi

ac o str e ogens ti scove . a

ti r

h r e

. m 153 e nove

e rt s ligan a f riz ti

a o Se e str mmun. h

ti Revea

l fi Env

oge l

v e a u abo n n assa

i va a s i

e ng i r tr l r 233: d r ogen be n ec - h h ti

‐ epo U an 63. i s an o ti s r l d . ce ence ir o l r n s

r y

n ti t

f i espons o

r y

n n a on

a t

an f it ecep o s l se v l

n e 4 i

o t r l oun

. a rt

o c -

deve i e f e o Tox ecep ‐

2002 m 7 f T c oes m

ri d f line l

e d d o s r

ec - i t t i

y ‐ t ac ssue b r 56. n f en h h b

ac h m B

t ti r i o

ti ir e

tr i c o ce e . yos ti s i spheno l S o t f oes

t echan oche

ti v ol venes ope d an ; zeb

o oge va a t i

it es

n n v l t

N 291: a - t r og l l ‐ o

Spec

e iti y h d d

t ti a

tr tr lin

hea r e

i i d t t y n o n s st a us s m h oge oge i

n s ona

fi e f s P 354 i e i udy udy i i

s t i l

n n nct

i e i sh l

ca l

he he t n o f th th t o of of t o i l i i 4 A n o n n n o o d g a S c c f f . l l l l

- -

inserm-01067804, version 1 - 24 Sep 2014 Su O M M

No K Sa 1999; S R M M M N M Ta Segne i , i n ilsson ga ng u enue ana o a a a ss n n m t W

m ac de hum subzon r na gen r r gol Co

pha b v es zeb ac M ac d tthe tthe li ‐ c 94. w A ecep ecep epo l i

i i C i i na e h - d tr o N s o o o ‐ , tr t t ti M a t 1452: s H ri m t

r tri a d

a s lfr u l o. o. l

S o r M on a i

EG r - oge ecu t va l ecu , c

w w s ES

. fi a ‐ essa a r pa ,

m M rt

n s

S buti i t K o S a fi s H cCo e enc t n t

Don

A B s s e

, . ti e obesogen , , o o h aco F , , l m o

r e

. i i , D l

Phys

n r ve r n h oche

I r r a U li M a a 103 O es

J M

nou ece

( J s -

W Pe i on

ti n ‐ r r llu Ca , p i

S be n r Zeb S a es o g s n l

aye r fr ake , v

tr a

ogy b r gen w

, i ffec

ecep , e

: lleg n G oduct e s m

r o

, i u - r tr t oge Fe i K t e

a ‐ o . Bapa a J m i ass o t 20.

Kha a h us

si M N W

e b

r oge lla l G l B l

a . jou V e ogy a e rt

og C i se n t r

S ri i ,

ca t i fis oche i GD ,

s ven , 2008; , n t o va W

, be

g a uck s n

ligan n o Sa

l R

i S

Escand

zeb s f n l ec Y r t i ca h s La W og

i r

. tr n sson ,

, fi ri J na

r n r

an , it SA

ecep ti E 2003;

au t r A s P

ou ti ansac

l a

m T E f y ecep m

X z o h an , . ons l r i KC

, o ( v KL t r r d

eb ffi d n 232: a Y .

u D

r anab eu o an evi Ang e r s

Bha

t an a K fi s ,

d m

b an

T f y cac

. Xenoes , o ZB b tr r t spec t

e , s

ligand

J J t i d us a r

Ber e s i t ophy 84 h A cD Ce d , e

ti y ep nd CL, 2003; Kanek i

o syn fi 384

l r , w o o

t A v

r ad

. va e

) deve r

s D i hum an M irt : N . liu

, n n s s

r h be i na RA 33

Es n i E o oducti . . ti e es B t A n P G

a - ua d , he g si s f l s ‐

2001; Gene o e z i 95. g illo , r tr t a - l

tr ri och ,

. ‐

a eb 3 I a d d ca 40. ri n n

pha Tu phys o

a r l , c l T do m

oge H J op ti m oge :

Th S

va o l n J

281 ,

lib n on

B l t A c ( r L ir

H jagu a L

)

a i hE ah a

m l . e m r m H on e m , o es l b r a r s . r A fi e

81: d n i

n n

i a

i g uan a i n y a Tem ( o en a - i l R J che

sh s s N i ,

L M tr ‐ c

l D .

Che 92. r ou i g

e s l aco

e H i n expo a y M M G og i an

m 1535 2002; s oge an sh

: t S be

. ,

i xenoes

r r an O e gna , m , L, o it

M a t 2003; y na pe d c b

i m m i O l t h M t , o o f o H e og

Y i n a

d , Tox ca r co b ndi ri

s d So

l illen Laude r a W oug )

m ille - r

Soc ling

co u i a ‐ i m

z 66: Tho S 65. e

o ophys r ca l exp m r

t m s s ece , an nove ode , ri f n e u

o o i mmuni E 8 i pec r

tr co

l pa o . g

s h : Fon

er w r Fe

1881 ,

m CP t e 110 . . ogens

) 2004; r an

t A es pt e Pakde l ec . t

t ess l s r o og a cha

se w

, r a

it l e Tox i ,

tr d g

dependence c o o V

ti i W s s o o i Hoso - y Ha p

n n i sub i i r

, - ‐ oge

8. v e nduc d o ‐

r r ca

- C 92. i m Lagnea A S d d ‐ he sc & e gan ac i ac spec 126: Ka

n n co J

rri , , J b iff iff l

is echan , ti

, i

tl endocri

pha t t n Pe i t n

A h F on oche l o s e a e Zacha e e e Th og Ande

is e ,

. f ri r r r va 15106 r if r HA T

en ence od Cava s ecep 2000; d s e i i r , es ba

a . ca u u c ,

ti r m u

gen

e t m , e i 1998; cs

e tr

m nove

l l r aco z m r E t n Hs f e anc

pa KF J t no n sson i e

o oge s ,

sc ille ou t , s

e s

e - a J o o w r tr v a 1490: ‐ ri e i , 19. t C

i l l a tr i f r l itr l h ences ogy . n r sk t y ogy

za . s . o

o l 243: na n

ansac w

an exp es f a Th nves

S i

es V es ti C G be ays l o

tr r l l

. . ri endoc . , o tr t , o

TR ecep e co 145 lin

, tr tr d agon uc H

2011; n n r 2009; e l Akop 122 t i oge . l ecu

. oassays i ess e . w oge oge o

a ti t m .

a ti 2014 t

t P l f ga ee - u va o

w a Ab

‐ o l 52. t m - n n o a J r

g l ‐ i o o n l 6. n n it a . ti , e s n n ti i l ecu l ena s S

173: 149 a p n n r . M

n ilit r t h

- n t P

. ‐ b

s ; e n base l

r e one e

g e r

n ecep cha i t I E

ecep y r B

o ndi E n t i

t T

o a R m n

e i d n fili R e i echan 183

Tox 187 a och , i r P p

o ri endoc

isr . d d d

b Hu ng d r r . f p b a 2009; a

ac t

ess ope l

B upti

ock l l g o ri i i

- o i s i - Th pha pha o : co ‐ des a ‐ m m 9. t

95. i b r tr m r spheno e l

o ogy . i i

i e

e s uct l oche

riz rti

a m a i c f og W on be m n d d i

a 4 l r g

pha ben g es po y a i phy

: T s an n . y n u e .

t e4935.

ti e

r 2002; ac

, b s rt m o t an a o o F d d M - v

tr - ‐ y l z sub t n ‐ d ll r an e anc i

f f m ti b i e ophenon

oes v

s a uct y A

i d va i E sr o tr es es es ed ophys l o

d ak

ana t ti app y up d 82: f

tr yp tr u an tr tr

i e

i ti i o ca

r t ve an

be ogen oge oge r oge t h e o ssu n e d s 181 l i an li ogs t r on f

t e e

e c s o of of

i i i a. a. n n d d d n n n n n n n e e e a a f . -

inserm-01067804, version 1 - 24 Sep 2014 10 quadriplicate different Standard estrogens. at Transcriptional different Figure in as to phytoestrogens. Figure Figure experiments different Figure estrogens.

quadruplicate

28 nM different %

and of

4. 1. 2. 3.

concentrations 10

concentrations

deviations concentrations of concentrations 37°

Transcriptional activity Zebrafish HELN HELN Transcriptional activity

Transcriptional activity

treatment.

nM concentrations are done C.

activity

indicated.

Results E2 HELN are

- - in zfERα zfERα

quadruplicate. treatment. from indicated.

ERs

- Standard

zfERα of are

( (

zfER

are BPA, E1,

least

expressed as expressed ), ), E2

thermosensitive. Standard E3 ( ), β HELN HELN -

zea, 1 2 independent 4- deviations (A)

and

(C)

tet of

HELN of

genistein, -

zfERα, octylphenol,

and 17αE2. and zfER zfERα, zf zf

zfERα, deviations

- - Figure % ERβ1 ERβ1

EE2 zf 10 from

zfERβ1

Results ERβ1

experiments zfERβ1 nM liquiritigenin

Transcriptional β zfERβ1

( ( legends (B) 2

NPm (D) from E2. ), ),

(

least was are HELN HELN

and

in responsein

Standard ),

and

at and and

expressed

HELN

zfERβ2

measured least

done 2

zfERβ2 - - BP2. and zfERβ2 (○) zfERβ2 (○) zfERβ2 independent experiments independent

activity

- 2 are deviations in ferutinin. zfERβ2

Results independent quadruplicate to E2 and EE2 E2 and to in

response in %

response

28, response of

(○)

are cells cells zfERα Results

cells 31, expressed

experiments from nM to were were

are

to were

34 in

environmental environmental

were are to E2

indicated. independent independent endogenous

response exposed exposed and

myco treatment. expressed expressed measured measured

as exposed exposed done

% 37°

done done

and and

C. C. to to to to to to in in

inserm-01067804, version 1 - 24 Sep 2014

ERα sequences. antagonists. treatment. (green) estrogen Figure CAC93848.1 the indicate sequences in (LBDs) Figure experiments E2. different indicated. concentrations of HELN Figure undergoes (B) Figure

the

Results

LBDs NP_000116; NP_000116;

- binding

zfERα

5. 8. onto 7. with

6. receptors. concentrations of

Transcriptional

Standard a Modeling Amino The

(yellow).

Transcriptional activity are done HELN large the par and

(

of expressed GenBank

zebrafish molecular

), estrogens which estrogens conformational 16αLE2,

- acid

Superposition

human

quadruplicate zfERα deviations from deviations the

HELN of In

human number the alignment of hERα,

activity accession

ICI ( ), ERβ2

models ERβ PPT,

interaction zf %

182,780, ERβ1

ERα

the of

HELN of

NP_001428; NP_001428;

CAC93849.1.

of change (black change 8βVE2 of are

differed

of amino lactone ring lactone

numbers at the LBD

zfERα, of

indicated.

(

treatment. Standard treatment. human the least

between

zf structures of zfERα, 4OH

- and (Met

16α ERβ1

acid

- HELN betwen 2 zfERβ1

- of

Tamoxifen, independent ERβ - DPN.

zebrafish zebrafish

342

arrow) the residue zfERβ1 16α of ( 2

fR2 (○) zfERβ2 -

ERs bound ), to and

16α - and hERα Results

HELN Ileu

sequences

2 within zebrafish are LE zfERβ2 and

raloxifen ERα ERα deviations accommodate and

experiments hERα 426). (A) 2

framed. -

are zfERβ2 points

zfERβ2 the

and

cells the

NP_694491; NP_694491;

The in expressed (A)

in ERs human

and hERβ

response

total

towards from Numbers three this were

and (○)

done PPT ligand this

human

alignment (B) cells 16α response

(h) at

amino exposed

in additional together least to

M421 LBDs

and

zebrafish zebrafish LE above the above

% binding quadruplicate were pharmaceuticals. pharmaceuticals.

ERα

2 acids 2 of -

zebrafish

bound

to independent

are bound (H7) to

exposed 10 with

and group. synthetic synthetic involved different different domains domains :

nM human human human ERβ1

which which

hERβ hERβ 1 to

ERβ ERβ

(zf) (zf) nM nM

are are

E2 E2 E In In to to

2 hERβ, the linear M421 is replaced by the branched residue I373 which is characterized by a much smaller intrinsic flexibility that maintains the synthetic ligand in a position (black arrow) where it interacts unfavorably with M336 (H3, red asterisk). Key hydrogen bonds between the protein and the ligands are highlighted by doted lines. W denote a water molecule. Superposition of the structures of hERα (C) and hERβ (D) LBDs bound to E2 (green) onto the molecular models of the 16α-LE2-bound zfERα (C) and 16α-LE2-bound zfERβ1 or zfERβ2 (yellow and violet, respectively) (D) LBDs. The steric constrains observed in hERs also apply to zfERs and account for the differential affinity of 16α-LE2 for the zfER subtypes. In (C) the doted line highlight a stabilizing interaction between hERα and the ligand that is probably missing in zfERα.

(A)

(B)

(C)

(D)

Figure 1 (A)

(B)

(C)

Figure 2 (A)

(B)

(C)

(D)

Figure 3 (A)

(B)

(C)

(D)

Figure 4 (A)

(B)

(C)

(D)

Figure 5 (A)

(B)

(C)

(D)

Figure 6 349 384 421 hERα zfERα

301 336 373 hERβ zfERβ1 zfERβ2

Figure 7 A C hERα M389 hERα/zfERα (zf) M317(zf) R394 H7 M421(h) M421 16α-LE L349(h) 16α-LE H524 2 2 w (h) E353(h) E 2 E2 H524 E353 L384(h) L384 R394(h) H11 H3

B D I373(h) hERβ hERβ/zfERβ1/zfERβ2

L391 (β2) H7 R346(h) I373 R346 I406 (β1) H476(h) E2 16α-LE2

E2 w H476 E305(h) 16α-LE2 16α-LE2 E305 L354 (β2) * M336 M336(h) H11 H3 L369 (β1)

Figure 8