Endocrinedisruptingchemicals:

thenewbrain drain?

ASParent,JPBourguignon

Pediatricendocrinology

University ofLiège

Belgium

€4B

Male Reproductive

Disorders

€6B

Premature Death

€15B

Obesity & Diabetes

€132B

Neurological Impacts

(including ADHD)

HEALTH EFFECTS FROM ENDOCRINE DISRUPTING CHEMICALS* COST THE EU €157B EACH YEAR. This is the tip of the iceberg: Costs may be as high as €270B.

€157B Cost by Health Effect

SOME EDC-RELATED HEALTH OUTCOMES NOT INCLUDED: SOME EDCs NOT INCLUDED:

• Breast Cancer• Prostate Cancer• Immune Disorders• Female Reproductive Disorders• Liver Cancer

• Atrazine• 2, 4-D• Styrene• Triclosan• Nonylphenol

€157B Cost by EDC Type

NOTE: The economic estimates do not include all costs associated with these conditions.

See Trasande et al. The Journal of Clinical Endocrinology & Metabolismhttp://press.endocrine.org/edc

* Endocrine Disrupting Chemicals (EDCs) interfere with hormone action to cause adverse health effects in people.

• Parkinson’s Disease• Osteoperosis• Endometriosis• Thyroid Disorders

• Polycyclic Aromatic Hydrocarbons• Bisphenol S• Cadmium• Arsenic• Ethylene glycol

€120B

Pesticides

€9B

Flame Retardants

€2B

ChemicalMixtures

€26B

Plastic, Cans: Phthalates &

BPA

“ THE TIP OF THE ICEBERG”The data shown to the left are based on fewer than 5% of likely EDCs. Many EDC health conditions were not included in this study because key data are lacking. Other health outcomes will be the focus of future research.

M.Bellangeretal.,JCEMMarch2015

Scientific findings (Gore et al, 2015, …)

EDC effects are more likely to harm the fetus because² Crucial developmental processes (brain, sex differ.)² Crucial organizational processes (energy balance,

reproduction, behavior, cancer risk)² Period of very high sensitivity to hormones² Effects of low dose mixtures and different hazards

Early life exposureto hazards

Compounds/ toxicants

INDIVIDUALLY

D

E

F

…

EDCs

Air pollutants

Mutagens

…

Hazard categories

A

Trendsinchemicalindustryoutputworldwide

(GlobalChemicalsOutlook,UNEP2012)

http://www.unep.org/chemicalsandwaste/Mainstreaming/GlobalChemicalsOutlook/tabid/56356/Default.aspx

Out

put

(Bill

ions

US

D)

China

Africa + Middle East

Central + South Amer.

Other Asia

India

Centr + EastEurope

JapanKorea

Australia

WesternEurope

North America

1970 1980 1990 1998 2000 2010 2020estim.

YEAR

Only aminorityofchemicalsarestudiedforendocrinedisruptingproperties

ü≈130,000 chemicalslistedbyECHAinEU(1/3>1T/yr)

ü 10,517 consideredbyUSEPAforEDCscreening

ü 1,326 potentialEDCsstudiedinliterature(TEDX)

ü ≈30 EDCsidentifiedamong422pesticidesorbiocidesusingECcriteria(JRCstudy)

≈ 50 physiologicalhormonesinhumans

à 99%unstudied,unknownà 1% studied:causallyinvolved?

Early life exposureto hazards

Compounds/ toxicants

INDIVIDUALLY

Adverseeffects / systems

INDIVIDUALLY

D

E

F

…

X

Y

Z

…

EDCs

Air pollutants

Mutagens

…

Hazard categories

A

GenitalmalformationsUndescendedtestesDisordersofpuberty

MaleinfertilityLowtestosteroneTesticularcancer

Prostatecancer

IQLoss,intellectualdisabilityAttentiondeficit– hyperactivity

Autism

IQLoss,intellectualdisabilityAttentiondeficit– hyperactivity

Autism

ObesityType2diabetes

ObesityType2diabetes

UterinefibroidsEndometriosis

Disordersofpuberty

Breastcancer

Goreetal,2015;Acconcia etal,2015;Klenke etal,2016;Mattison etal,2014

AndrogenR

ProgesteroneR

Thyroid

HormoneR

Pregnane XR

Peroxysome proliferator-

activatedRγ

ArylhydrocarbonR

Na+,K+andCa2+ channel

δ subunit ofGABAA R

Nuclear

Estrogen R

ERα, ERβ

Membrane

ERα, ERβBisphenol A

ERRγ

GPR30

Ethinylestradiol

Early life exposureto hazards

Compounds/ toxicants

INDIVIDUALLY

Adverseeffects / systems

INDIVIDUALLY

One toxicantOne systemOne effect

One endpoint

Demonstrationof causality

INDIVIDUALLY

D

E

F

…

X

Y

Z

…

EDCs

Air pollutants

Mutagens

…

Hazard categories

A

Intheimmatureratmodel,brainisaffectedbyaBPAdose6,400,000timeslowerthanthedoseactiveonuterineweight

0.025(μg/kg.day)

0.25

2.5

25

250

2,500

25,000

160,000 brainGnRHYamasaki Ketal.,Env Health Perspect2000;108:1147

Franssen Detal,Endocrinology2016;157:1740

“Safe”doseEFSA

Averagehumanexposure

uterotrophic assay

From CLSisk andDLFoster,NatureNeuroscience2004

Internal cues: neurotransmitters

Glial growth factors

Peripheral cues:

leptine, IGF-1

Environnemental cues: endocrine

disrupters?

Endocrinedisrupters

andalterationofpubertalcontrol

Postnatal Age (days)10 20 30 40 50B 5 15 25

30

50

70

90

vaginal opening

estrus cycle

GnRH interpulseinterval (min)

GnRH secretion (pg/7.5 min)

Time (min)

0 60 120 180 240

5

10

15

5

10

15

5

10

15

5

10

15

5

10

15 fetal

5 days

15 days

25 days

50 days

Prepubertal accelerationofpulsatileGnRH secretionexvivo

BourguignonJPetal.,JClinInvest,1992,90:1736-1744

0

50

100

29 31 33 35 37 39 41

Vaginalopening

(cum

ulative%)

Age(days)

Controls(n=8) BPA25ng(n=14) BPA5mg(n=12) 25ng/kg.d (n=14) 5mg/kg.d(n=12)PND1-15

30

35

40

45

50

55

60

65

GnRHinterpulse

interval(m

in,n=4)

5

15

25

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255

GnR

Hsecretion

pg/7.5min

≤

≤5

15

25

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255

min

PND20***

**

Postnataldose-dependent andopposing BPAeffectsonmaturationofpulsatileGnRHsecretion andpubertal timing

*

Franssen Detal,Endocrinology 2016

Averageexp.50ng/kg.daySafe(EFSA)4000ng/kg.day

Log2 (fold change) Significant(qvalue)

0

1

2

3

4

-20 -10 0 10 20log2(fold change)

−log 10

(p val

ue)

significantno

yes

genes: b25ng/ctrl

4

3

2

1

0

-8-40+4+8 -8-40+4+8 -8-40+4+8

34genes

25ng/kg.dayvs

CONTROL

0

1

2

3

4

-20 -10 0 10 20log2(fold change)

−log 10

(p val

ue)

significantno

yes

genes: b5mg/ctrl

472genes

5mg/kg.dayvs

CONTROL

0

1

2

3

4

-20 -10 0 10 20log2(fold change)

−log 10

(p val

ue)significant

no

yes

genes: b5mg/b25ng

1407genes

5mg/kg.dayvs

25ng/kg.day

Significantly increased orreduced mRNA expressionofhypothalamic genesafter postnatalexposure tolow vshigh BPAdoses

PND20

Franssen Detal,Endocrinology 2016

-log10(p-value

)

GABAR

signal.

Fact.cardio-gen.vertebr.

Transcr.emb.stemcells

Basalcellcarc.signal.

Embr.cellspluripotency

Gaqsignaling

CatransportI

PKAsignaling

012345

GABAR

signal.

GProt.coupl.

Rsignaling

CRHsignaling

Α-adrenerg.signaling.

Glutam.dep.Acidresist

CXCR4signaling

PKAsignalingCatransportI

cAMP-mediat.signaling

012345678

GProt.coupl.

Rsignaling

GABAR

signal.

Axonguid.signaling

Bone/cart.Rh.Arthr.

GHsignal.

Gaqsignaling

Fact.cardio-gen.Vertebr

cAMP-mediat.signaling

012345678

-log10 (p-value) -log10 (p-value) -log10 (p-value)

INGENUITYanalysisofpathways

25ng/kg.dayvsCONTROL

5mg/kg.dayvsCONTROL

5mg/kg.dayvs25ng/kg.day

Alterations ofGABAergic tone after exposure toBPA

Franssen Detal,Endocrinology 2016

Nerveterminalofpresynapticneuron

Synapse

Cytoplasmofpostsynapticneuron

25ng/kg.day

vsCONTROL

Ingenuity

î GAD

RNA

ì GAT

RNA

î GABAA R

RNA(γ subunit)

Nerveterminalofpresynapticneuron

Synapse

Cytoplasmofpostsynapticneuron

5mg/kg.day

vsCONTROL

+ +++ Repressed

+ +++ Enhanced

+ +++ Repressed

+ +++ Enhanced

+ +++ Repressed

+ +++ Enhanced

ì GAD

RNA

î GAT

RNA

ì GABAA R

RNA(γ subunit)

+ +++ Repressed

+ +++ Enhanced

Control Muscimol 0.1mM

BourguignonJPetal.,JNeuroendocrinol 1997,9:193-199

Hypothesis:

BPA(verylow dose)

BPA(highdose)

Increased activity ofGABAergic inhibition?

Decreased activity ofGABAergic inhibition?

GnRH interpulse interval-IPI(min)

Control

GABAA Rantagonist

Bicuculline 100mM

15DAYS 60.0± 6.0 40.2± 8.0*

25DAYS 39.2± 3.0 40.3± 4.0

GABAA R agonist

Muscimol (agonist)abletoincreaseGnRHIPI?

Bicuculline (antagonist)abletoreduce GnRHIPI?

66.0± 4.060.0± 6.0

50.8± 3.0*39.2± 3.0

GABAergicinhibitionofGnRHreleaseisincreasedafterinfantile(2-weeks)exposuretolowBPAandreducedafterexposuretohighBPA

30

35

40

45

50

55

ControlMEM BPA25ngMEM BPA25ngBic BPA25ngMus BPA5mgMEM BPA5mgBic BPA5mgMusControl BPABPABPA BPABPABPA

25ng/kg25ng/kg25ng/kg 5mg/kg5mg/kg5mg/kg+ + ++

Bicuccul.Muscimol Bicuccul.Muscimol100mM 0.1mM 100mM 0.1mM

******

*** **PND20

GnR

Hinterpulse

interval(m

in,n=3-4)

Franssen Detal,Endocrinology 2016

Courtesy ofN.Cabaton andD.Zalko

BPAexposure GD8– PND16

0 25ng/kg.d 250ng/kg.d 25μg/kg.d *

BrainGABA(arbitrary

units)

(1Hnuclear

magne

ticresonancespectroscopy)

*EnvironHealthPerspect 121:586–593(2013)

PND21

Ametabolomicstudyofwholebrainshowsdose-dependenteffectsofBPAonGABA

HARM

FUL

REQUIRED

NONE

EFFECT

SONHEA

LTH

Testosterone

Estradiol

ThyroxineVitaminDSalt(NaCl)Iodine…

DOSE

Harmtohealth:adoseissue?

HARM

FUL

NONE

ED

DOSE

Early life exposureto hazards

Compounds/ toxicants

INDIVIDUALLY

Adverseeffects / systems

INDIVIDUALLY

One compound/toxicant

banned or restricted because identified through

mode of action

One toxicantOne systemOne effect

One endpoint

Demonstrationof causality

INDIVIDUALLY

Risk assessment/ Management

INDIVIDUALLY

D

E

F

…

X

Y

Z

…

EDCs

Air pollutants

Mutagens

…

Hazard categories

A

AnEDis “...anexogenous substanceormixturethat altersthefunction(s)oftheendocrinesystem andconsequentlycausesadverseeffects inanintactorganism,oritsprogeny,or(sub)populations”

(WHO/IPCS2002)

AnEDis “...anexogenous substanceormixturethat hasanendocrinemodeofactioni.e.altersthefunction(s)oftheendocrinesystem andshowsanadverseeffectrelevantforhumanhealthasaconsequence oftheendocrinemodeofaction”inanintactorganism,orits progeny,or(sub)populations ”

(ECproposal 2017)

Endocrinedisrupting:Alterationofthyroidhormone/sex

steroidsaction

Disruptionofintracellularsignaling(Calciumsignaling,Geneexpression,…)

Impairmentofneurotransmission(Dopaminergic,serotoninergicetcholinergic

system)

Psychomotordifficultiesandimpairedlearningandmemory

Altered neuronalplasticity anddevelopment

PCBs:mechanisms ofaction

PCB

Neurotoxicity

From Kodavanti 2005

Stewart,2008,Environmental Health Perspectives Roegge,2000,Toxicological Sciences

RodentHuman

Perinatal exposure toPCBs andcognitivefunction

Barnesetal,2010

Cerebral cortex development

From FPolleux,Neuron 2008

PCBlevelsafterprenatalexposuretoA1254

Mating Birth Weaning

A1254,6mg/kg

E6

ENaveau,Plos One2014

*

*

control

PCBeffect ontotalT4andfreeT4serum levels indams

*

*

ENaveau,Plos One2014

Aroclor

Control

PrenatalexposuretoAroclor1254doesnotaffectneuronalprogenitorsproliferation

Courtesy ofTZoellerENaveau,Plos One2014

Ki67+BrdU

Prenatal exposure toAroclor 1254delays neuronalmigration

NaveauEetal,unpublishedENaveau,Plos One2014

Aretroviral markerfornewly born cells

Overstreet etal,2014,JNeurosci

Atransgenic markerfornewly born granulecells

E6 P21 P56P7 P14

Study EARLY and/or DELAY, ACUTE or PERSISTENT effects of perinatal exposure to PCBs on hippocampal neurogensis

Oral exposure to 0.5 or 6 mg/kg/d of Aroclor1254 or corn oil (in wafer)

Levels of PCBs in brainLevels of thyroid hormones in serum

PCBaccumulationinthebrain andeffects ontotalT4andfreeT4serum levels inpups

PinsonAetal,Eur JNeurosci 2016

Perinatal exposure toAroclor 1254does notaffectearlyneurogenesis

PinsonAetal,Eur JNeurosci 2016

Delayed effect ofperinatal exposure toAroclor 1254onadultneurogenesis

PinsonAetal,Eur JNeurosci 2016

Aroclor 1254affectssynaptogenesis inthedentate gyrus

P21CTL

P21ARO

PinsonAetal,Eur JNeurosci 2016

Aroclor 1254excitatory synapsedevelopment

PinsonAetal,Eur JNeurosci 2016

A1254&(6&mg/kg/d)&or&corn&oil&exposure&

A&

0" 5" 10"

ATP"metabolic"process"ATP"genera6on"from"ADP"ATP"biosynthe6c"process"Electron"transport"chain"

Respiratory"electron"transport"chain"ATP"synthesis"coupled"proton"transport"

Oxida6ve"phosphoryla6on"ATP"synthesis"coupled"electron"transport"

Mitochondrial"ATP"synthesis"coupled"electron"transport"Mitochondrial"electron"transport,"ubiquinol"to"cytochrom"c"

Fold&enrichment&

0&

1000&

2000&

3000&

CTL& ARO&

Cox4i1&FPK

M&

RNA&sequ

encing&

POMCIEG

FP&posiLve&

POMCIEG

FP&negaL

ve&

Total&den

tate&gyrus&

Cox4i1&

0&

1&

2&

3&

4&

5&

CTL& ARO&

cox4i1&m

RNA&relaLv

e&expression

&&

0&

1&

2&

3&

4&

CTL& ARO&

cox4i1&m

RNA&relaLv

e&expression

&&

0&

1&

2&

3&

CTL&P21& ARO&P21& CTL&P56& ARO&P56&

cox4i1&m

RNA&relaLv

e&expression

&&&

*"

q"="0.002""

ns"

ns"(p"="0.07)"

ns"

Cycs&B&

0&

50&

100&

150&

200&

250&

CTL& ARO&

Cycs&FPK

M&

q"="0.01""

0&

0,5&

1&

1,5&

CTL&P21& ARO&P21& CTL&P56&& ARO&P56&

Cytcs&m

RNA&relaLv

e&expression

&&&&&

ns" ns"

0&

1&

2&

3&

4&

CTL& ARO&

Cycs&m

RNA&relaLv

e&expression

&

ns"

0&

0,5&

1&

1,5&

CTL& ARO&Cycs&RNA&relaLv

e&expression

& ns"

0"

20"

40"

60"

80"

CTL$ ARO$

Numbe

r$of$4

HNE$p

osi6ve$ce

lls$

GZ"POMC+"SGZ"POMC/"

ARO$CTL$

A

C

4HNE

$EG

FP$

Merge$

**"

*"

0"

100"

200"

300"

CTL$ ARO$

Intensity

$of$fl

uorescen

ce$in$

the$SG

Z$(%$of$C

TL)$

B

p"="0.08"

0"

20"

40"

60"

80"

CTL$ ARO$

Numbe

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$of$fl

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p"="0.08"

PinsonAetal,unpublished

Aroclor 1254induces oxydativestressinnewborn neurons

Wayman,JNeurosci 2014

Early life exposureto hazards

Compounds/ toxicants

INDIVIDUALLY

Adverseeffects / systems

INDIVIDUALLY

One toxicantOne systemOne effect

One endpoint

Demonstrationof causality

INDIVIDUALLY

Risk assessment/ Management

INDIVIDUALLY

D

E

F

…

X

Y

Z

…

Mixtures, differenthazards

Compounds/ toxicants

AS A WHOLE

All systems, immediate

and delayed

Adverseeffects

AS A WHOLE

Retroactiveevidence

through effectsof measures

Demonstrationof causality

AS A WHOLE

Environmentalhygiene for

global hazard reduction

Intervention(preventive)

AS A WHOLE

Management(regulatory)

AS A WHOLE

Equal opportunity access to

environmental health

EDCs

Air pollutants

Mutagens

…

Hazard categories

A

B

One compound/toxicant

banned or restricted because identified through

mode of action

Scientific findings (Gore et al, 2015, …)EDC effects are more likely to harm in the fetus because² Crucial developmental processes (brain, sex differ.)² Crucial organizational processes (energy balance,

reproduction, behavior, cancer risk)² Period of very high sensitivity to hormones² Effects of low dose mixtures and different hazards

Implicationsinapediatricendocrinologyperspective

ü Set up a global protective strategy during prenatal and early postnatal life: save time for next generations

ü Develop scientific studies showing impact of such a global strategy on exposure and health endpoints

ü Urge authorities to develop conditions of equal opportunity access to environmental health: consumer information and education of health care providers

University of LiègeGIGA Neurosciences

DevelopmentalNeuroendocrinology

AS. ParentE. NaveauD. FranssenA. PinsonA. GérardD. Lopez-RodriguezJ. FudvoyeG. RasierJP. Bourguignon

TranscriptomicsplatformB. Hennuy

Biostatistics UnitAF. Donneau

Acknowledgments

14 : 45 – 15 : 15 Dr Muriel Thomas Le traitement par hormone de croissance des enfants avec petite taille en Belgique : le rôle du registre « Belgrow « De groeihormoonbehandeling van kinderen met een kleine gestalte in België : de rol van het register « Belgrow 15 :15 – 15 :45 Dr Dominique Beckers Le traitement des enfants et adolescents diabétiques : le fonctionnement des conventions - les résultats de IQECAD De behandeling van kinderen en adolescenten met diabetes : de werking van conventies – de resultaten van IKEKAD 15 :45- 16 : 15 Pause café Koffiepause 16 : 15 – 16 :45 Dr Sylvie Tenoutasse La  communication  de  l’  information  sur  des  endocrinopathies : le rôle de « BELENDO» Het verschaffen van informatie over endocrinopathies : de rol van « BELENDO » 16 :45 – 17 : 15 Prof Dr Jean De Schepper La reconnaissance de la sous-spécialité pédiatre-endocrinologue : nécessité et opportunité De erkenning van de subdiscipline kinderarts- endocrinoloog : noodzaak en opportuniteit 17 : 15 – 18 : 30 Réception / Receptie

Inscription obligatoire / Inschrijving verplicht :

Mevrouw Inge Sienaert : inge.sienaert@uzbrussel.be

______________________________________

Accreditatie  “  ethiek  en  economie” 2.5 CP Accréditation  “  éthique  et  économie  “ 2.5 CP

Vollum Institute, OHSU

Pr G WestbrookA BensenC ChatziN Woods

Probabilityofcausalinvolvementofendocrinedisruptorsinneurodevelopmentaldisorders

Trasande etal.,Bellanger etal.,JCEM2015

LossofIQ

Organophosphate

pesticides

Polybrominated

flameretard.Attentiondeficit&

Hyperactivity

70-99%probability

PhthalatesAutism40-69%

probability

Brain PCBandserumthyroxinconcentrationsafterAroclor1254exposure(6mg/kg.d)fromE6toP21

E6# P21# P56#P7#

Serum&total&and&free&thyroxin&concentra3on&&

Aroclor#1254#(6mg/kg/d)#or#corn#oil#exposure#

PCBs&concentra3on&in&brain&

0#

1000#

2000#

3000#

4000#

5000#

6000#

P21# P56#

PCBs#con

centraCo

n#in#brain##

(ng/g#fresh#weight)#

CTL#

ARO#

A#

B# C#

**"

*"

0#

10#

20#

30#

40#

50#

60#

P7# P21# P56#To

tal#T4#concen

traC

on#in#

serum#(n

g/ml)#

***"

***"

0#

5#

10#

15#

20#

25#

30#

P7# P21# P56#

Free#T4#concen

traC

on#in#

serum#(p

g/ml)#

D#

*"

ParentASetal,Eur JNeurosc 2016

RVPND56ParentASetal,Eur JNeurosc 2016

Aroclor 1254reduces neuronalproliferation inadult mice (T4normalized)

*

Prenatal exposure toAroclor 1254increases cell cycleexitintheSVZ

n=6 ENaveau,Plos One2014