Developmental Toxicology II
Sid Hunter
6 November 2007
Hunter.Sid@EPA.GOV
Techniques and Models in Teratology
Issue?
Developmental Toxicity of Ethylene Glycol
MOUSE (mg/kg)
0
100
200
400
Fetal Body Weight
0.97 ± 0.01
0.88 ± 0.02
0.78 ± 0.02
0.71 ± 0.02
-9.2%
-19.6%
-26.8%
% Malformed/litter
0.25
± 0.25
10.0
± 1.9
37.8
± 6.3
56.5
± 6.8
Exencepha ly
Cleft Lip/Palate
Facial Cleft
0
0
0
3
1
0
4
4
0
6
13
4
RABBIT ( mg/kg)
0
100
500
1000
5000
Maternal Letha lity
0
0
0
0
42.1%
Fetal body weights
49.8
± 1.5
48.8
± 2.4
46.5
± 1.8
48.4
± 1.5
51.2
± 2.0
% malformed/litter
6.8 ± 3.1
5.5 ± 2.1
5.4 ± 2.0
6.7 ± 1.9
1.1 ± 1.1
G
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.
HO-CH 2-CH2-0H
Ethylene Glycol
Alcohol Dehydrogenase
RATE LIMITING STEP I
HO-CH -CH
2
||
O
Glycoaldehyde
Aldehyde deHase
HO-CH 2-C-OH
||
O
Glycolic acid
Lactate deHase or
Glycolate Oxidase
(RATE LIMITING II)
Formate
CO2 + H-C-OH
||
O
(60%)
Rabbit
Rat
(15%)
HC-C-OH
|| ||
O O
Glycoxylate
OTHER
PRODUCTS
Rabbit
Oxalate
Rat
HO-C-C-OH
|| ||
O O
Whole Embryo Culture System
Trophoblast
Parietal Yolk Sac
3-5 Somite Embryo
Amnionic Cavity
Allantois
Reichert's Membrane
Visceral Yolk Sac
Visceral Yolk Sac
Extraembryonic
Cavity
Ectoplacental Cone
Ectoplacental Cone
Fig. B
Fig. A
30 rpm
30 ml Flask
Fig. C
Fig. D
Schematic representation of the technique of embryo culture.
The conceptus is removed from the uterus (Fig.A), dissected free of maternal tissues,
trophoblasts, parietal yolk sac and Reichert's membrane (Fig.B), placed into a culture
flask of medium (Fig.C), and rotated in an incubator at 30 rpm at 38C (Fig.D).
Day 8 Mouse Embryo
Embryos after 24 hours in Culture
Developmental Effects of Ethylene Glycol Metabolites in
Mouse Whole Embryo Culture
B
GA
J
GOA
J
100
Percentage Malformations (Any Effect)
OA
H
F
J
J
B
H
Formate
B
B
90
H
80
70
H
B
60
50
B J
40
B
F
30
JH
20
J
10
B
0
100
H
F
1000
µM Xenobiotic
10000
20000
Physiological changes induced by Ethylene glycol administration
pH
Osmolality
Bicarbonate
Administration of sodium bicarbonate decreased the incidence of ethylene glycolinduced skeletal effects
Ethylene Glycol Example:
•Administration of ethylene glycol produced species dependent induction of
malformations
•Ethylene Glycol did not alter rat embryonic development when conceptuses were
directly exposed in vitro
•Ethylene glycol metabolites do induce malformations in rat/mouse embryos grown in
vitro
•Metabolites have different potencies
•Administration of Ethylene glycol produces physiological changes in the mother
•Amelioration of maternal physiological changes decreases (BUT DOES NOT
ELIMINATE) the teratogenic effects of Ethylene glycol administration
HYPOTHESIS
Physiological changes induced by ethylene glycol administration
are an important contributor to developmental effects
AND
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HO-CH 2-CH2-0H
Ethylene Glycol
Alcohol Dehydrogenase
RATE LIMITING STEP I
HO-CH 2-CH
||
O
Glycoaldehyde
Aldehyde deHase
HO-CH 2-C-OH
||
O
Glycolic acid
Lactate deHase or
Glycolate Oxidase
(RATE LIMITING II)
Formate
CO2 + H-C-OH
||
O
(60%)
Rabbit
Rat
(15%)
HC-C-OH
|| ||
O O
Glycoxylate
OTHER
PRODUCTS
Likely Proximate
Teratogen?
Rabbit
Oxalate
Rat
HO-C-C-OH
|| ||
O O
Does administration of glycolic acid produce malformation and how do
they compare to those produced by ethylene glycol
Ethylene Glycol Developmental Toxicity
The developmental effects produced by
ethylene glycol administration are may be due
to glycolic acid and maternal acidosis
Administration of glycolic acid recapitulates
most but not all of the effects of ethylene
glycol. (Follow-up study to evaluate
relationship between AUC and defects /
resorptions)
Toxicity enhanced by or produced by maternal
acidosis?
PBPK Model for Ethylene Glycol and
Glycolic Acid
Effects of a 6-hour exposure to the HAA-metabolites
100
2
80
60
1
40
20
J
J
J
0
0
Control
Glyoxylic Acid
Glycolic Acid
Oxalic Acid
Embryonic Dysmorphology Score
% Abnormal Development
J
Administration of drug (D) results in distribution of the drug
and its metabolites (M)
G
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dd™
e tc o oa smn e dp e ra et hs i ss
o pr i c
t u
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.
D
MOTHERp
MOTHERc
PLACENTA
AMNIOTIC FLUID CAVITY
FETUS
D
D
D
D
D
M
M
M
M
M
M
The placenta is NOT a barrier to D or M distribution
You can not use disposition information from male or non-pregnant
females to determine the distribution during pregnancy!!!!!
PHYSIOLOGICAL CHANGES IN PREGNANCY
System
Parameter
Body water
Body fat
Gastrointestinal
Total
Total
Motilit y
Gastric
Acid secretion
Mucou s secretion
Respir atory mi nut e vo lume
Alveo lar ventilation
Tidal vo lume
Residua l vo lume
Pulm onary blood fl ow
Cardiac output
Rate
Stroke
Peripher al b lood fl ow
Peripher al resist ance
Plasma flow
Glomerular fi ltration rate
Total vo lume
Plasma volume
Red cell m ass
Hemoglobin
Phospho li pids
Cho lesterol
Free fatty acids
Protein conc
Album in conc
Globul in conc
Creatinine conc
Urea conc
Oxygen consu mption
Body temperature
Hepatic metaboli sm
Placental and fetal
Respir atory
Cardiova scular
Renal
Blood
Metaboli sm
Change


































(% of Nonpregnan t
adult value
~7-8 lit ers
~3-4 kg
(30-50)
(40)
(20)
(30-40)
(0-20)
(10)
(85)
(70)
(35-40)
(50)
(18)
(30)
(55)
(40)
(15)
0.5°C
Present
How do chemicals cross the Placenta??
Carrier-mediated transport
facilitated diffusion: not energy dependent
high substrate specificity
follows [ ] gradient
D-glucose
active transport:
receptor-mediated
endocytosis:
simple diffusion
energy dependent
high substrate specificity
can function against [ ] gradient
amino acids; creatine; calcium
Immunogloblins (IgG)
Iron-transferrin
Not energy dependent
Follows [ ] gradient
lipophilic > hydrophilic
size, structure, pKa
depends on placental structure
sheep = multilayer = no lactate transfer
human = 3 layers = lactate transfer
Control of Rates of Xenobiotic Transfer across the Placenta
Xenobiotics - primarily Passive Diffusion
Rate Limiting
LIPOPHILIC Cmpd
Embryo
HYDROPHILIC Cmpd
Rate Limiting
Embryo
Some xenobiotics limit embryonic exposure by decreasing uterine
blood flow
Control of Rates of Xenobiotic Transfer across the Placenta
Xenobiotics - primarily Passive Diffusion
MOM
Embryo/Fetus
D
D
D
D
Protein Bound
Protein Bound
D-COO-
D-COO-
D-COOH
D-COOH
pHi
7.64 (day 9) Mouse
7.61 (day 11) Rat
7.05 (day 14) Rat
Plasma pH
7.26 Mouse
7.44 Rat
7.47 Rat
Sheep, Cow, Horse, Pig
Epitheliochorial
Dog, Cat, ferret
Endotheliochorial
Mouse, Rat, Rabbit, Human
Haemochorial
Gestational Age
8
9
9.5
10
12
17
22-term
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
-
+
-
+
+
+
+
+
+
-
-
-
-
Fetal
Endothelium
Mesenchyme
Cellular
Trophoblast
Maternal
Epithelium
Connective Tissue
Endothelium
Ratio of Chemicals in Fetus to Maternal Serum Concentrations
The rate of elimination may be as important as the rate of product formation for
accumulation of a toxic intermediate
sulfate
glucuronide
phenolic
tetrol
glucuronide
Glutathione
conjugate
Polycylic
aromatic
hydrocarbons
Arene oxide
Diol
diol-epoxide
glucuronide
sulfate
Macromolecular
conjugate
glutathione
conjugate
catechol
macromolecular
conjugate
methoxy
phenol
Techniques and Models in
Developmental Toxicology
in vivo veritas !!
Techniques and Models in
Developmental Toxicology
in vitro veritas !!
In vitro Developmental Toxicity Test Systems
Cultured Mammalian Embryos / Primorida
Non-Eutherian Embryos
Primary Cell Culture
Established Cell Lines
In vitro Developmental Toxicity Test Systems
Established Cell Lines
Human Embryonic Palate Mesenchyme cells
Mouse ovarian tumour cells
Neuroblastoma Cells
Mouse/Human Embryonic Stem Cells
In vitro Developmental Toxicity Test Systems
Non-Eutherian Embryos
Medaka
Zebrafish
Xenopus laevis
Frog Embryo Teratogenesis Assay Xenopus
(FETAX)
Drosophilia
Chick
Chick Embryotoxicity Screening Test (CHEST)
Crickets
Hydra
In vitro Developmental Toxicity Test Systems
Cultured Mammalian Embryos / Primorida
Whole Embryos - postimplantation
Mouse
Rat
Rabbit
Opossum
Palatal Shelves / Head
Limbs
Mandibles
It has been VERY difficult to successfully culture
preimplantation-staged embryos to post-implantation
stage.
Chen LT, Hsu YC. Development of mouse embryos in vitro: preimplantation to the limb bud
stage.Science. 1982 Oct 1;218(4567):66-8. PMID: 7123220
Whole Embryo Culture System
Trophoblast
Parietal Yolk Sac
3-5 Somite Embryo
Amnionic Cavity
Allantois
Reichert's Membrane
Visceral Yolk Sac
Visceral Yolk Sac
Extraembryonic
Cavity
Ectoplacental Cone
Ectoplacental Cone
Fig. B
Fig. A
30 rpm
30 ml Flask
Fig. C
Fig. D
Schematic representation of the technique of embryo culture.
The conceptus is removed from the uterus (Fig.A), dissected free of maternal tissues,
trophoblasts, parietal yolk sac and Reichert's membrane (Fig.B), placed into a culture
flask of medium (Fig.C), and rotated in an incubator at 30 rpm at 38C (Fig.D).
Mouse Embryos
PreCulture
24H Culture
Effects of LY294002 on embryonic development
10µM
75µM
50µM
G
are
Qraphics
uickTim
needed
e™
decom
toand
see
pressor
athis picture.
Be
nchmar kCon
centr at io
ns for H
aloacet icAcid
10000
NTD
AnyMalformation
Benchmark Concentration (µM)
1000
100
10
1
0.1
MIA
MBA
BCA
MCA
DBA
BDCA
DBCA
TCA
TBA
Log (1/BD) = a(Pka) - b(Elumo) + c;
r2 = 0.94
DCA
Morphological effects of HAAs and 3 metabolites
in Mouse WEC
DCA
ACETATE
TBA
TCA
Oxalic Acid
Glyoxalate
DBCA
Glycolate
BDCA
DBA
MCA
BCA
MBA
1
10
100
Benchmark Concentration
1000
3000
Control Medium
Morphological Evaluation
Begin Culture Period
Short Term Exposure to xenobiotics
Exposure to toxicant
Toxicity depends upon both concentration
and length of exposure
Time Dependent induction of dysmorphogenesis by DCA, DBA
and BCA in Whole Embryo Culture
100
90
Incidence of Dysmorphology
80
70
B
B
DCA
J
BCA
H
DBA
F
Glycolate
Ñ
Glyoxylate
É
Oxalate
Ñ
F
B
É
J
H
B
60
B
50
J
40
Ñ
J
30
20
H
10
B
F
B
J
É
0
100
1000
10000
AUC (µM * Hr)
100000
300000
Whole Embryo Culture
Advantages
No Maternal Metabolism
Disadvantages
Know
No Maternal
what embryo
Metabolism
is exposed to
No Maternal Disposition
Know
No Maternal
how long
Disposition
embryo is exposed
Select Embryos at
beginning of culture
All
Select
embryos
Embryos
haveatsame
beginning
# somites
of
All
culture
embryos have same sensitivity
Ability to manipulate
No ability to assess effects of
Embryo
compound on the mom
Overexpress genes
Knock-down expression
Compare sensitivity across strains and species
Mechanistic Studies
Embryonic Stem Cell Test (EST)
Embryonic stem cells are a permanent cell line
established from the ICM of embryos
The basis of the EST is to determine 3
endpoints
IC50 for inhibition of ESC differentiation to
beating heart cells
IC50 for induction of cell death in ESC
IC50 for induction of cell death in 3T3 cells
Current EST
(http://ecvam-sis.jrc.it/IP1685/published/indexed_113.html)
Cytotoxicity Assay: Grow D3 mouse embryonic stem cells and 3T3 embryonic fibroblasts in
presence of test chemical to determine IC50 for each cell line. Endpoint: cell viability after 10 days
(MTT assay).
D3 p14, cytotox (2/6/06)
3T3
norm, non-lin fit
% max response
110%
D3
90%
70%
50%
30%
5-FU
BCA
DCA
10%
-10%
-10
-9
-8
-7
-6
-5
-4
-3
-2
log [drug], M
Differentiation Assay: Allow D3 cells to differentiate for 10 days in presence of chemical to
determine ID50. Endpoint: microscopic inspection of beating cardiomyocyte development.
•
•
•
•
•
•
•
•
Day 0: Start “Hanging Drops” in medium
–LIF +chemical, 750 cells/ 20ul drop.
Incubate 3 days @ 37°C
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Day 10: Inspect each well for
presence of contracting
cardiomyocytes. Score % of
positive wells per plate.
Day 5: Deposit embryoid bodies
in multiwell plate for adherent
outgrowth, 5 days @ 37°C
• • • • • • •• • • •• •• • •••
Day 3: Grow embryoid bodies in
suspension culture, 2 days @
37°C
Apply Prediction Model to determine class of toxicity.
Linear discriminant functions I, II and III:
I: 5.916 Ig(IC50 3T3) + 3.500 Ig (IC50 D3) - 5.307 [(IC503T3-ID50) / IC503T3] -15.27
II: 3.651 Ig (IC503T3) + 2.394 Ig (IC50D3) - 2.033 [(IC503T3-ID50) / IC503T3] - 6.85
III: -0.125 Ig (IC503T3) - 1.917 Ig (IC50D3) +1.500 [(IC503T3-ID50) / IC503T3] - 2.67
Dose Response Curves for EST
EST Improved
Multiple Endpoint
Molecular Markers
Modified EST
• Differentiation Assay: modified to include a more quantitative assessment of multiple cell types
via Q-PCR analysis of selected gene expression markers.
Evaluation of Q-PCR probe sets for mouse Stem Cell Panel
Stem Cell Gene Expression Panel -- ABI probes
Results
+
+
+/+/-++
+
+
+
++
++
+/+/+
++
++
++
++
+
+/+
-++
++
+
+
+
++
++
+/+/+/+/+
+
++
+
+/-
Gene
PolR2a
Oct-4
Lox
Stat3
Dppa3
Ankrd1
Rex1/ ZFP42
Tdgf1/ Cripto
Akp2
LeftB
Utf1
Nanog
Tcl1
LIN28
Cldn7
5T4
GATA-5
GATA-4
AFP
HNF4a
Nkx2.2
Pdx1
Brachyury
MHC2a
PECAM
Flk-1/ VegfR2
MHCa
Nkx2.5
Col2a1
Cbfa1
Sox9
CD45
Tbx6
Lamin A
Sox1
Pax6
Fgf5
Prominin1
Nestin
NCAM
NeuroD1
Kcnma1
GFAP
Serpini1
Rab33a
marker for
endogenous control
stem cells
stem cells
stem cells
stem cells
stem cells
stem cells
stem cells
stem cells
stem cells
stem cells
stem cells
stem cells
stem cells
early differentiation
early diff., trophobl.
endoderm
endoderm/ mesoderm
endoderm, liver - visceral
endoderm, liver
endoderm, pancreas
endoderm, early pancreas
mesoderm, early - transient
mesoderm, skeletal muscle
mesoderm, vascular
mesoderm, vascular
mesoderm, cardiac muscle
mesoderm, cardiac
mesoderm, bone & cartilage
mesoderm, bone
mesoderm, early cartilage
mesoderm, hematopoietic
mesoderm
early ecto/meso, neuron, cardiac
ectoderm, early (decreased)
ectoderm, early
ectoderm, early (transient)
ectoderm
ectoderm
ectoderm, neural
ecto- brain, and endo- pancreas
ectoderm, neuron
ectoderm, glial cells, astrocytes
ectoderm, neural
ectoderm, neural
Results Key:
++ probe set amplified and matched expected pattern in all samples
+ probe set amplified and matched expected pattern in most samples
+/- probe set amplified and matched expected pattern in some samples
- probe set amplified, but did not match expected pattern
-- probe set failed to amplify
LocusLink Gene Nam e
LocusLink
Sym bol
Public RefSeq
Polr2a
NM_009089
Pou5f1
NM_013633
lysyl oxidase
Lox
NM_010728
signal transducer & act. of transcr. 3
Stat3
NM_213659
developmental pluripotency-assocd 3
Dppa3
NM_139218
ankyrin repeat domain 1 (cardiac muscle)
Ankrd1
NM_013468
zinc finger protein 42
Zfp42
NM_009556.2
teratocarcinoma-derived grow th factor
Tdgf1
NM_011562
alkaline phosphatase 2, liver
Akp2
NM_007431
left right determination factor 1
Lefty1
NM_010094
undifferentiated embryonic cell transcription factor
Utf11
NM_009482
Nanog homeobox,Gene mCG132219 Celera Annotation
Nanog
XM_132755
T-cell lymphoma breakpoint 1
Tcl1
NM_009337.2
lin-28 homolog (C. elegans)
Lin28
NM_145833.1
claudin 7
Cldn7
NM_016887.2
trophoblast glycoprotein
Tpbg
NM_011627
GATA binding protein 5
Gata5
NM_008093
GATA binding protein 4
Gata4
NM_008092
alpha fetoprotein
Afp
NM_007423
hepatic nuclear factor 4, alpha
Hnf4a
NM_008261
NK2 transcription factor related
Nkx2-2
NM_010919.1
insulin promoter factor 1, homeodomain transcription
Ipf1 factor NM_008814.2
brachyury
T
NM_009309
myosin, heavy chain 2, skeletal muscle
Myh2
NM_144961
platelet/endothelial cell adhesion molec. 1
Pecam1 NM_008816
kinase insert domain protein receptor
Kdr
NM_010612
myosin, heavy chain 6a, cardiac muscle
Myh6
NM_010856
NK2 transcription factor related
Nkx2-5
NM_008700
procollagen, type II, alpha 1
Col2a1
NM_031163
runt related transcription factor 2
Runx2
NM_009820
SRY-box containing gene 9
Sox9
NM_011448.2
protein tyrosine phosphatase, receptor type, C Ptprc
NM_011210.1
T-box 6
Tbx6
NM_011538
lamin A
Lmna
NM_001002011
SRY-box containing gene 1
Sox1
NM_009233
paired box gene 6
Pax-6
NM_013627.2
fibroblast grow th factor 5
Fgf5
NM_010203
prominin 1
Prom1
NM_008935
nestin
Nes
NM_016701
neural cell adhesion molecule 1
Ncam1
NM_010875
neurogenic differentiation 1
NeuroD1 NM_010894.1
potassium large conductance calc. channel
Kcnma1 NM_010610
glial fibrillary acidic protein,Gene mCG7451 Celera
Gfap
AnnotationNM_010277.2
serine (or cysteine) peptidase inhibitor, clade I, member
Serpini11 NM_009250.1
Function
polymerase (RNA) II polypeptide A
DNA-directed RNA polymerase, Nucleotidyltransferase
POU domain class 5, transcrip factor 1
Homeobox transcription factor, Nucleic acid binding
RAB33A, member of RAS oncogene family
Extracellular matrix, Oxidoreductase, Oxidase
Transcription factor, Nucleic acid binding
Molecular function unclassified
Transcription factor, Nucleic acid binding
Transcription factor, Nucleic acid binding
Signaling molecule, Grow th factor
Phosphatase, Other phosphatase
Signaling molecule, Cytokine, TGF-beta superfamily
Zinc finger transcription factor, Nucleic acid binding
Cell adhesion molecule, vesicle trafficking
Molecular function unclassified
Zinc finger transcription factor, Nucleic acid binding
Zinc finger transcription factor, Nucleic acid binding
Transfer/ carrier protein
Transcription factor, Nuclear hormone receptor
Nkx2.2,tinman,Nkx-2.2,MGI:97347
Transcription factor, Nucleic acid binding
Actin family cytoskel protein, Actin binding motor prot
Cell adhesion, Defense/immunity prot, Ig receptor
Tyrosine protein kinase receptor
Actin family cytoskel protein, Actin binding motor prot
Homeobox transcription factor, Nucleic acid binding
Extracellular matrix structural protein
Transcription factor, Nucleic acid binding
Transcription factor, Nucleic acid binding
Cytoskeletal protein, Intermediate filament
HMG box transcription factor, Nucleic acid binding
Homeobox transcription factor, Nucleic acid binding
Signaling molecule, Grow th factor
Membrane traffic protein
Molecular function unclassified
Cell adhesion molecule, CAM family adhesion molecule
Helix-loop-helix domain, transcription factor
Voltage-gated ion channel, potassium channel
NM_011228.1
Applied Biosystems 7900HT Real-time
PCR System
Q-PCR: Endoderm markers
Q-PCR: Mesoderm markers
Q-PCR: Ectoderm markers
mESC = hESC
Predictions of Developmental Toxicants
•
What do you want to Predict?
Is the compounds a human developmental
toxicant
What is the NOAEL/LOAEL in a rodent DT study
Should it predict all aspects of DT (weight,
function, structure)
What is the relative potency of the compound in
this model system
•
ECVAM Predict classification of compounds
• High, medium/low, or NO
•
Prediction of toxic potential is no better than the
best analysis in any system
In Vitro Developmental Toxicity Models
• For cytotoxic, cytostatic compounds good
predicitivity
• Need different prediction model to separate
compounds with moderate and low potential
• Need to integrate ADME information with in vitro test
results
• Good model for mechanistic research
• Comparing potency of parent and metabolite