Are BPA substitutes safe ? René Habert Université Paris-Diderot - INSERM - CEA Equipe de Développement des Gonades- INSERM U 967. Centre CEA de Fontenay aux roses - 92-265 - France

Bisphenol A BPA is largely produced. Worldwide production is 3 millions tons per year. BPA is largely used 1) as monomer that is polymerized into : polycarbonate plastic

: bottles, food containers, optic lens, electrical & electronics, hard drive, stretch films, automobile, housewares & applicances, construction, medical use….

epoxy resins :

inner coating of food and beverage cans, adhesive, dental restorative material…).

2) as anti-oxidant or inhibitor of polymerization in some plasticizers and PVC …, 3) in thermal printer paper

Humans are constantly exposed to BPA, primarily through oral and dermal routes 2

BPA regulations and risk assessment ANSES (september 2011) 2012 : French Parliament has banned the use of BPA in all food containers, packaging, utensils intended to come into direct contact with food or beverage : - for children under 3 years since 2013 onwards - for the general population since 2015 onwards. ANSES (march 2013) Critical effects on

Key study

TDI

Route of exposure µg/kg/d

µg/kg/d

µg/kg/j

Brain  and   behaviour  

oral  

0,17

Female   reproduc3on  

oral  

0,33

Metabolism   Obesity  

oral     (drinking)  

0,28

Mammary   gland  

oral     (gavage)  

0,08

Maximum intake = 1 µg/kg/d

Necessity to find substutes to BPA 3

Numerous BPA substitutes

4

Some present uses of BPA substitutes BPS Used - as monomer for preparation of high temperature plastic resistant (polysulfone et poly ether sulfone) and epoxy resin, - thermal paper, - chemical additive in pesticides, dye-stuffs, dye dispersants....

BPF : Used as monomer for preparation of epoxyresin and polycarbonates, for use in the manufacture of lacquer, varnishes, coating, adhesive plastics..

BPAF : Used as monomer for polyamide, polyester, elastomer, 5

Internal exposure Urine concentration

10-9 M

10-8 M

Liao et al 2012

6

In vitro studies

7

Estrogen Receptor binding Radioligand receptor binding assay for ER

E2 >>> BPA > BPF > BPS hERα

x7000

x3

x5

hERβ

x2000

x4

x2

Molina-Molina (2013)

8

Estrogen Receptor activity MCF-7

ligand

BPA BPS ER

ERE

Kitamura et al. (2005)

luciferase

Luciferase reporter gene assay

BPA > BPS

E2 >>> BPAF > BPA > BPF = BPS x6000

x13

x2

x3

Kuroto-Niwa et al. (2005)

BPA > BPF > BPS x4

x2

E2 >> BPA > BPS MELN BG1

x2 x13

Grignard et al. (2012)

BPA > BPF = BPS x3

Molina-Molina et al. (2013)

1) Despite some difference as a function of cell lines, agreement for an estrogenic activity of BPS and BPF at least 2 times lower than BPA. 2) On the contrary, BPAF activity is 13 times stronger than BPA

9

How do bisphenols bind to ER ? Agonist-bound conformation R394

R394

R394

H524

B E353

Antagonist-bound conformation

H524

hERα/E2

T347

A

A350

E353

B T347

hERα/BPA    

A350

B H524

T347

E353

hERα/BPC   R394

R394

E : acide glutamique H : histidine R : arginine T : thréonine

(Delfosse et al., PNAS 2012) PNRPE

A

H524

A A

A350

hERα/BPAF    

H524

E353

B T347

A350

E353

hERα/BPAF  

10

Putative membrane receptors for bisphenols G15 : selective inhibitor for GPR30 / GPER

150

BPA

140

BPS

Lignée GH3 / B6 / F10 140

*

*

130

E2 10-9M

120 110 BPS + E2 100

Vehicule

pERK (% of vehicle)

pERK (% of vehicle)

Lignée GH3 / B6 / F10

130

*

120 110 100 90

90 -15 -14 -13 -12 -11 -10 -9

-8

-7

Bisphenols concentration (log M)

Vinas et Watson 2013

Similarly to BPA, very low doses of BPS are This effect involves GPR30/GPER able to trigger a membrane signaling pathway.

at least partly 11

General cell biology Lignée MCF-7 BPA BPF BPS

100 90

Molina-Molina et al. (2013)

Necrotic cells (%)

E2

Primary culture of human mononuclear leucocytes

BPA > BPF > BPS

70 60 50 40 30 20 10 0

BPAF BPA BPF BPS

0

10-4

2.10-4

3.10-4

4.10-4

Bisphenols concentration (M)

5.10-4

BPAF > BPA > BPF > BPS 60

BPF BPA BPS BPAF

Feng et al. (2016)

BPAF > BPA = BPS > BPF The relative bioactivity of one BPA substitute depends on the cell line and/or on the analysed parameter

Apoptotic cells (%)

Lignée H295R

BPA BPF BPS BPAF

50 40 30 20 10

0

10-4

2.10-4

3.10-4

4.10-4 5.10-4

Bisphenols concentration (M)

Mokra et al. (2015)

BPA > BPF = BPS > BPAF The relative bioactivity of one BPA substitute depends on the analysed parameter .

12

Hepatocyte cell line

Steroidogenic cell line

HepaRG cell line

H295R cell line

Cyp2B6 (WB) 1 0

1

Fatty Acid Synthase (RTPCR)

0

BPS = 0 Peyre et al. Food Chem. Tox. 2014 (PNRPE)

The bioactivity of one BPA substitute may be non-detectable

Progesterone (ng/ml) Testosterone (ng/ml)

Fold Change

Fold Change

Specific functions

BPA = BPS > BPAF

BPF =0

BPA ≠ BPS ≠ BPAF = BPF

0 0.1 1 10 50 70 BPF (µM)

0 0.1 1 10 50 70 BPA (µM)

0 0.1 1 10 50 70 BPS (µM)

0 0.1 1 10 50 BPAF (µM)

Feng et al 2016

The bioactivity of one BPA substitute depends qualitatively on the parameter analysed.

13

Ex vivo studies

14

Rat heart Isolated female heart Heart rate

Isolated female myocytes

Premature ventricular beats

Beats / min

Female myocytes contractibility

Control

10-9 M BPS

Control

-12 -11 -10 -9 -8 -7 -6

Control BPS Iso BPS BPA +Iso +Iso (10-9 M)

BPS

-9 (log M)

BPA

Non - monotonic

BPS = BPA Gao et al. 2015

In a model which is close to in vivo, BPS has the same effect as BPA

15

In vivo studies

16

OECD Test Enhanced OECD Test Guideline no. 407 Oral gavage with BPS of 8 week-old rats for 28 days

Control

Female body weight (g) 250 240 D====

230 220

D

210

* *

* *

200

1

3

8

12

* * *

17

D

** *

21

28

20 mg/kg/d * 600 * mg/kg/d * 100 mg/kg/d

Higashihara et al. 2007

Days of treatment

LOAEL for BPS = 20 mg / kg / j

17

Zebra fish Adult exposure for 21 days

Embryonic exposure from 0 to 5 dpf

BPS (7nM)-

AroB-MO BPS (7nM) -

Ji et al. 2012

As low as 0.5 µg/L (2.10-9 M) BPS is sufficient to alter reproductive functions.

AroB-MO BPA (7nM) -

+

+ -

+

+ -

+

+ +

+ +

Kinch et al. 2015

Identically to BPA, embryonic exposure to BPS induces hyperactivity via an increase of aromatase activity. 18

Our studies

19

200

Cancer testiculaire

France 74 M en 1989

150 100

50 M en 2005

50 0 1960

1970

1980 1990

2000 2005

Incidence pour 10 000

Nombre de spermatozoïdes (x106) par ml of sperm

Comptage spermatique 6 5 4 3,5

1973

1989

1997

Années

Années

Hypospadias

Cryptorchidie 4

4

3

2

1970

1975 1980 1985 1990

Années Toppari et al.Human Reprod Update (2001), Rolland et al Human Reprod (2012)

Fréquence pour 1 000 naissances

Fréquence pour 1 000 naissances

1981

3

2

1970

1975 1980 1985 1990

Années Sharpe & Irvine, Mol cell Endocrinol (2004),

Hypothesis 1 : these abnormalities are caused by environmental endocrine disruptors (Sharpe et Skakkebaek 1993)

Hypospermie, cancer testiculaire Spermatogonies souches Différenciation Prolifération Gonocytes C. Sertoli

Cordon séminifère

C. Leydig Testosterone, Insl 3

Fetal testis

Masculinization

Hypospadias, cryptorchidie Hypothesis 1 : these abnormalities are caused by environmental endocrine disruptors (Sharpe et Skakkebaek 1993)

Hypothesis 2 : these abnormalities are various symptoms of the same syndrome : the testicular dysgenesis syndrome which results from disturbances in the development of the (Skakkebaek et al 2001) fetal testis

Creation of an organotypic culture system which preserves the testicular architecture and the numerous intercellular intratesticular relationships

Factors

Seminiferous cord

SertoliGonocyte

-  IGF-I -  TGFß, activin, - Inhibin, AMH -  c Kit / Steel factor -  Testosterone -  Retinoic acid -  LIF, OSM, CNTF -  FGF2 - FGF9 -  IL3, IL4, IL11 -  TGFα, EGF -  PDGF -  TNFα

Leydig 23

Creation of an organotypic culture system -  Rat

Fetus

testis

control

filter Medium without any biological factor

(Habert et al. MCE 1991, Olaso et al. Endocrinology 1998, Livera et al. Biol Reprod 2000) -  Mouse (Livera et al. Cell Tissue research 2006) - Human (Lambrot et al. JCEM 2006, Lambrot et al. Biochimie 2006)

testis

Collaboration with the Gynaecology and Obstetric Department in Antoine Béclère Hospital (René Frydman / Alexandra Benachi)

+ ED to assay

Human fetal testis

maintenance in vitro of the development of each testicular cell types

Reprotoxicity assay : r / m / h FeTA : rat, mouse, human Fetal Testis Assay 24

Effect of BPA in human compared with rat and mouse Testosterone production on D3 (% of control)

FeTA

100

Fetus

*

80 60

*

40

control

BPA

*

* *

Internal BPA concentration in humans

20

10-12 M

10-8 M 10-7 M

*

Mouse (12.5 dpc) Rat (14.5 dpc) Human (7-11 GW)

*

10-6 M 10-5 M

BPA concentrations (M) N’Tumba Byn et al. 2012

A BPA concentration as low as 10-8 M (2.3 µg/L) is sufficient to reduce the endocrine activity of the human fetal testis.

For this parameter, rat and mouse are less sensitive to BPA than human 25

Screening of BPA substitutes! Bisphénol A BADGE Bisphenol A Diglycidyl Ether

Bisphénol S

Effets du BPA et des substituts à 10-5M Irgonox Premiers résultats

DCDPS

Bisphénol F

Dichlorodiphenyl Sulfone

Testosterone production (% of control)

300,00% 250

**

Mouse 10-5 M

250,00% 200

200,00% * *

150

150,00% 100

100,00%

** *

** *

* *

*

* * *

* ** * * *

*

* * ** * *

* * *

50 50,00% 0 0,00%

J1 1 Day

J2 2 Day

BPA BPS BPF BADGE IRGONOX DCDPS

Fetus

J3 3 Day 26

Effect of BPS and BPF in human compared with mouse Fetus

control

Fetus

BPA

control

Fetus

control

BPS

BPF

Testosterone production

Testosterone production

Testosterone production

(% of control)

(% of control)

(% of control)

Mouse

100 80

*

60

* *

* Human

40

Mouse

100

*

80

*

60

* *

40

* *

* Human *

20

20 10-12

10-8 10-7 10-6 10-5

[BPA] (M)

10-12

Mouse

100

10-8 10-7 10-6 10-5

80

*

60

*

40

Human*

*

20 10-12

[BPS] (M)

10-8 10-7 10-6 10-5

[BPF] (M) Eladak et al Fertil Steril 2015

Antiandrogenic effect of BPS on the fetal testis is similar to that of BPA

27

Conclusions Bisphenols used as BPA substitutes should be regulated as potential Endocrine Disruptors. Data on the exposure and on the toxicity of BPA-substitutes is scarce. Pub med : bisphenol A: 9663 papers bisphenol S : 89 papers However, most of the data demonstrate an ED activity of various bisphenols that may be equal or scarcely higher than that of BPA. Because there would be no value in trading one health hazard for another, health risk assessment of BPA substitutes is an urgent necessity. Health risk assessment of BPA-substitutes is a very huge difficult worksite : -  Multiplicity of the substitutes, -  Relative potential effect of one substitute depends on the model, the studied parameter, the environment. -  The responsiveness in human species may be different from that in rat or mouse, -  The mechanism of action of BPA is not yet clearly established.

28

Laboratoire de développement des gonades (CEA, INSERM, Université Paris-Diderot) Gabriel LIVERA (PU P7) Virginie ROUILLER-FABRE (PU P7) Marie-Justine GUERQUIN (MCU P7)

Service de Gynécologie Obstétrique Hôpital A Béclère Pr R FRYDMAN Pr A BENACHI

Sébastien MESSIAEN (IE P7) Delphine MOISON (AI P7) Sophie TOURPIN (IE P7) Tiphany GRISIN (IE CDD) Soria ELADAK (doctorante) Vincent MUCZYNSKI (doctorant) Thierry N TUMBA BYN (doctorant)

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