Reproduictive Toxicology: of moca and men and molecules

Integrated testing
strategies for
reproductive toxicity
Aldert H. Piersma
Center for Health Protection
RIVM Bilthoven-NL
1
NVT Arbeidstoxicologie Den Bosch 150312
The Reproductive Cycle
postnatal
development
Sexual
fertility
maturation
Growth and
Gamete
development
production
Postnatal
Fertilisation
development
Zygote
Birth
transport
Foetogenesis
prenatal
development
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Implantation
Embryogenesis
OECD416
OECD414
OECD421
± 65% of experimental
animals is required for
reproductive toxicity testing
in REACH
3
20 November 2012
Van der Jagt et al., ECB 2004
Alternatives in
Developmental Toxicology
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type of test
test
end points
continuous cell lines
HEPM
MOT
V79
N115
EC/EST
proliferation
adhesion
metabolic cooperation
differentiation
differentiation
primary cell cultures
brain
limb bud
differentiation
differentiation
organ cultures
limb bud
development
embryo cultures
Hydra
frog
rodent
regeneration
development
development
Developmental Toxicity Alternative Methods
ZFET (photo Sanne Hermsen, RIVM)
WEC (photo Aart Verhoef, RIVM)
EST (photo Dorien van Dartel, RIVM)
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Pluripotent embryonic
stem cells
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A
B
C
Embryonic stem cell differentiation
related gene expression
D
Regulated genes can be divided into 7 clusters with
different gene expression profile in time
F
G
Gene expression level
E
Time dependent gene expression clusters
Predominant gene cluster function
A: Pluripotency
B: Cell adhesion
C: Proliferation
D: Unidentified
E: Unidentified
F: Early differentiation
G: Late differentiation
time
-3.5
7
±1
+3.5
Van Dartel et al., 2010
Prediction of compound embryotoxicity
Compound-exposed
experimental group
Differentiation and
compound modulation
(26 genes)
PenG
0.051
RA
5.10E-07
MAA
1.10E-16
VPA
2.60E-03
MBP
1.10E-03
5-FU
2.80E-13
Van Dartel et al., 2010
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Flusilazole-induced GO term enrichment
Van Dartel et al., 2011
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Zebrafish Embryotoxicity Test (ZET)
1 hpf
4 hpf
18 hpf
24 hpf
48 hpf
72 hpf
Hermsen et al., 2011
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Principal Component Analysis - Flusilazole
205 genes
FDR<0.05 and
concentration
response
Hermsen et al., 2011
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Selected gene set
regulation - Flusilazole
control
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high dose
20 November 2012
Hermsen et al., 2011
Rodent Postimplantation
Whole Embryo Culture
Rat embryos before
(gd10, 2 mm)
and after culture
(gd12, 4.5 mm)
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Neural Tube Effects
control
14
exposed
Retinoic Acid - Induced Morphology
Robinson et al., 2012
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RA Impacts in RA Metabolism Genes with Time
Robinson et al., 2012
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Adverse Outcome Pathway
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Retinoic acid and neural differentiation
rostro-caudal patterning of the spinal cord
paraxial
mesoderm
RALDH2
diffn
regressing
node
growth
FGF8
RA
spinal motor neurons
RA
FGF8
neural
differentiation
NeuroM
Delta1 Ngn1/2
pax6 Irx3
Wnt3a Cdx4
Brachyury(T)
RALDH2
cervical
brachial
thoracic
lumbar
Hoxc5/6
Hoxc6/8
Hoxc8/9
Hoxc10
FGF8
node
after Maden 2006 and Rhinn 2012
Aldert Piersma 121106
RA metabolism
disruption
RA biosynthesis
disruption
Retinoic acid and neural differentiation
retinoic acid exposure regulation and malformations
Rhinn 2012
An RA neural tube - axial patterning AOP Framework
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Sipes et al., Chem. Res. Toxicol. 2013, 26, 878-895
ChemScreen Ring Trial Battery Approach
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Battery approach - detailed results table
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AOP
test 1
test 1
AOP 2
test 2
test 6
AOP 3
test 3
test 7
3
AOP 4
test 4
AOP 5
test 5
AOP potencies
Adverse Outcome Pathway approach
test 8
•toxicity profile
•overall potency
20 November 2012
Dedicated to the Prevention of Birth Defects
methyl mercury
26
thalidomide
alcohol
Thank You
Dorien van Dartel
Sanne Hermsen
Esther de Jong
Ahmed Osman
Joshua Robinson
Sjors Schulpen
Peter Theunissen
Ilse Tonk
Aart Verhoef
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