Invitroeffects of EndocrineActive Substances (EASs)inhuman placenta - - PowerPoint PPT Presentation

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LuanaRicciPaulesu

UniversityofSiena(Italy)

“Invitroeffects of EndocrineActive Substances (EASs)inhuman placenta

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• Transferofnutrients,

gases andwaste products

• Immunologicalbarrier
• Secretionofhormones
• Metabolism

Human placenta

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Adverse factors ?

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Para&nonylphenol (p&NP):alkylphenol usedindetergents, emulsifiersandsolubilizers Bisphenol&A (BPA):additiveofplastics,mainly polycarbonates

Selected chemicals:

BPAandp&NP:two estrogen&like substances

Soto et al.,Environ HealthPerspect,1991 Welshons et al., Endocrinology,2006

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Primary cultures: PlacentaVillous explants Trophoblast&like cell lines: BeWo, HTR&8/Svneo.

Invitromodels for studies inhuman placenta

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Placentaestablishmentanddevelopmentintheuterus

HTR&8/Svneo BeWo

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48&96h

matrigel

24h

Themodelofplacentavillous explants

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1.Toxicologicalstudies (ontrophoblast cell lines) 2.Functional studies (ontrophoblast cell lines and

• nplacentavillous explants)

InvitrostudiesontheeffectsofEDCs in humanplacenta:

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Treatment: BeWo/HTR! 8/Svneo cells

Toxicological studies:

Cell viability 24h !NP /BPA

(0.01pM – 1mM)

(Control) Vehicle:

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BISPHENOL&A (BPA)

• %Cell viability

40 80 120 160

p&NONYLPHENOL (!NP)

1M 10M 1mM 0.01pM 0.1pM 1pM 0.001nM 0.01nM 1nM 10nM 100nM C 0.1nM 1M 10M 1mM 0.01pM 0.1pM 1pM 0.001nM 0.01nM 1nM 10nM 100nM C 0.1nM

1.Analysis of awiderange of concentrations

Toxicological studies:

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!7 !6 !5 !4 !3 !2 20 40 60 80 100 120

Log[BPA](M) Cellviability(%overCTRL)

3

• ?

M 1

• ?

M 1 4

• ?

M 1 8

• ?

M 2 2 5

• ?

M . 5

• m

M 1

• ?

M 1 5

• ?

M 6

• ?

M 1 2 5

• ?

M 1 6

• ?

M 2

• ?

M . 2 5

• m

M 1

• m

M

20 40 60 80 100 120

BPA

!7 !6 !5 !4 !3 !2 20 40 60 80 100 120

Log[p!NP](M) Cellviability(%overCTRL)

20 40 60 80 100 120 1E&06 1E&05 3E&05 6E&05 0,0001 0,0001 0,0002 0,0005 0,001

1 ? M 1 5

• ?

M 3

• ?

M 6

• ?

M 1

• ?

M 1 2 5

• ?

M . 2 5

• m

M . 5

• m

M 1

• m

M

p&NP Cell viability (%)

LC50 LC50

Toxicological studies

2.Analysis of therange of concentrations near toxicity

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Both chemicals tested (BPAandp&NP) causedtoxicityinhumantrophoblast cellsatconcentrations ranging from 10?Mto 1mM. Results from thetoxicological studies:

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10pM ! 0.1

• M

Theconcentrations selected were also easily found inthe environment andinhuman tissues

Non&toxic concentrations selected for functional studies inhuman trophoblast:

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Differentiation of trophoblast: β β β β&hCG secretion Caspase&3 ST

Functional studies:

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Treatment: BeWo / placenta explants !NP /BPA

β β β βhCG C BPA pNP

0.0 1.5 3.0 4.5 6.0 7.5

m I U /

• g o fp r o te in

β β β β!hCG

CNP BPA

Caspase!3 (Control) Vehicle:

Functional studies:

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β β β β&hCG Caspase 3 BPA p&NP Non&toxic,environmentally relevant concentrations of BPAandp&NP induce theβ β β β&hCG andcaspase&3activation in human trophoblast

10pM!0.1

• M

10pM!0.1

• M

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• Both chemicals tested (BPAandp&NP)exerted

toxicity onhuman trophoblast atconcentrationsnot achievedintheenvironment(from10?Mto 1mM); Both chemicals however increased thedifferentiative pathway of trophoblast atnon&toxic,environmentally relevant concentrations (from 10pM to 0.1

• M);

Theresultsobtainedraisegreatconcernaboutthe environmentalrisktohealth,andpointtotheneedfor protectingbothpre&natallifeanddevelopmentofthe foetus fromcontamination.

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UniversityofSiena Siena(Italy)

PartiallysupportedbytheEuropean Sixthframeworkprogramme (Project:ReProTect) FrancescaIetta Nicoletta Bechi RobertaRomagnoli Chiara Mannelli Antonella Spagnoletti