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Laser Microdissection-Targetted
Transcription Profiling and
Pathways Analysis for
Mechanism-Based Hazard
Assessment.
Simon Plummer
Genomics Meeting, Nice
1st October 2007
Time–dependent and Compartment-Specific Effects of In Utero Exposure
to Di(n-butyl) Phthalate on Gene/protein Expression in the Fetal
Rat Testis as Revealed by Transcription Profiling and Laser
Capture Microdissection
Plummer et al, Toxicological Sciences 97(2),
520-532, 2007
Phthalate Ester Toxicity
• Effects are most commonly observed in endocrine and reproductive organs and the liver.
• The functions of these tissues are tightly regulated by the nuclear hormone receptor super-family, which includes transcription factors such as PPARs, AR, ER, LXR, FXR, etc.
• In the fetal rat testes phthalates cause testicular mal development (hypospadias, cryptorchidism, infertility) accompanied by a reduction in testosterone production which is thought to underlythese effects.
Aims
• To gain insight into molecular mechanism(s) of phthalate(DBP)-induced testicular mal development in rats including assessment of the the possible role of nuclear hormone receptors.
• To develop mechanistically-based in vitromodels for cross-species comparison and hazard assessment .
In Vivo/In Vitro Extrapolation
Paradigm
Animal in vitro
Animal in vivo
Human in vitro
Human in vivo
?
Experimental Design
Molecular MarkersTestosterone
Fetus Weanling Puberty AdultBirth
DBP
GD 12 GD 21
Molecular MarkersCryporchidism
�Male Wistar rats exposed in utero to DBP (500mg/Kg)�Fetal testes taken at E15.5, E17.5, E19.5�Microarray analysis whole fetal testes/ sub-regions�Testicular testosterone�Incidence of cryptorchidism in adults (90 day, ~ 65%)�Immunohistochemistry
In Utero DBP-treatment Decreases Testicular Testosterone and Increases Cryptorchidism
Incidence
• DBP caused ~70%
decrease in whole fetal
testes testosterone level.
• High incidence (~90%) of
either uni- or bi-lateral
cryptorchidism in adult
male offspring in DBP-
treated litters.
Normalised Testosterone : whole e19.5 rat testes
0
0.2
0.4
0.6
0.8
1
1.2
Corn Oil DBP 500 mg/Kg
treatment
No
rmalised
Tests
ote
ron
e
Differential Gene Expression
• 1197, 2979 and 2171 differentially expressed (p<0.01) genes (‘signature lists’) were selected in the GD 15, 17 and 19 respectively.
• Filter lists to remove dye swaps artifacts.
• Remove genes of unknown function.
• Reduced size of lists to 100s of genes.
Pathways Affected by in utero DBP Exposure of Fetal Testes (GD19.5)
• Steroidogenesis e.g. Cyp11a, Cyp17a, NR5a1 (SF-1)
• Choleterol biosynthesis/transport (e.g. HMGCS, HMGCR, StAR …)
• Testes development (e.g. Insl3, INHA)
• Redox homeostasis (e.g. GSTA1, ALDH)
Hypothesis
Testosterone
SF-1
StAR Cyp 11A Cyp 17
Pregnenolone DHEACholesterol
SF-1 regulators
Insl3
HMG CoA synthase
Hypospadias
and infertility
Inhibin αααα
Impaired testis
development/tumours
Impaired
gubernacular
development/
cryptorchidism
DBP
HMG CoA reductase
Green = down regulation
RNA Expression –Real Time PCR, GD19.5
Expression of SF-1 RNA relative to b-actin for in utero DBP-treated foetal rat testes
normalised to control (untreated foetal rat testes)
0.00
0.50
1.00
1.50
2.00
GD19 control + Litter 26 + Litter 27 + Litter 28 + Litter 29 +
Rati
o o
f S
F-1
RN
A t
o b
-
acti
n e
xp
ress
ion
(%
of
co
ntr
ol)
* * *
Expression of StAR RNA relative to b-actin for in utero DBP-treated foetal rat testes
normalised to control (untreated foetal rat testes)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
GD19 control + Litter 26 + Litter 27 + Litter 28 + Litter 29 +Rati
o o
f stA
R R
NA
to
b-a
cti
n
ex
pre
ss
ion
(% o
f co
ntr
ol)
*** ********
SF-1
0.00
1.00
2.00
Control Litter 26 27mR
NA
no
rma
lise
d
* * *
0.00
0.40
0.80
1.20
*** *** *****
28 29
StAR
Control Litter 26 27 28 29
mR
NA
no
rma
lise
d
Expression of ISLF3 RNA relative to b-actin for in utero DBP-treated foetal rat testes normalised to
control (untreated foetal rat testes)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
GD19 control + Litter 26 + Litter 27 + Litter 28 + Litter 29 +Rati
o o
f IS
LF
3 R
NA
to
b-a
cti
n
exp
res
sio
n(%
of
co
ntr
ol)
***
*
*** *** ***
0.00
0.40
0.80
1.20
** ** **
0.00
0.40
0.80
1.20
***
*
*** *** ***
CYPscc
Control Litter 26 27 28 29
mR
NA
no
rma
lise
d
Insl3
Control Litter 26 27 28 29
mR
NA
no
rma
lise
d
Western blot and Immunohistology
analysis of SF1 protein in GD19.5
fetal testes
control DBP
Pathways Analysis Using
Ingenuity Pathways AnalysisTM (IPA)Software
The diagrams show upregulated (red)-and downregulated (green)- genes that were affected in whole fetal testes following in utero
exposure of rats dibutylphthalate (DBP) 500mg/Kg from GD 12 to GD 19. Orange lines show genes that are regulated by the transcription
factors peroxisome proliferator receptor alpha (PPARA) and steroidogenic factor 1 (SF-1). Grey lines represent associations (of genes) to
a particular functional category e.g steroidogenesis. Green oval shows genes involved in sterol biosynthesis, steroidogenesis and testes
development (mostly down-regulated).
synthesis/ transportof cholesterol
GD 19.5
Cytoplasm
Extracellular Space
Plasma Membrane
Nucleus
F2
APOA2
SLC27A2
ACADS
EBP
ACOX1
ADCYAP1
CYCS
APOA1
FABP1
HSD17B4
FADS1
-1.591
APOE
ACSL1
CYP17A1
-3.043
CYP11A1
-3.116
SERPINA1
CYP51A1
-1.956
STAR
-4.042
FDFT1
-1.535
SCP2
-1.331
FDPS
-1.434
SCARB1
-2.880
HMGCR
-1.918
APOA4
APOC1
PEBP1
-1.614
SLCO1B3ABCA4 (includes EG:24)
GOT2
ABCC3 ABCA3
SLCO2A1
PRDX2
-1.128
FAT
FABP3
-1.309
SREBF1PPARA NR5A1
HMGCS1
-2.061
INSL3
INHA
-1.641
NCOA1 CREB1
Fatty Acid Metabolismand Transport
Testes descent and
development
steroidogenesis
Network of genes in functional pathways postulated to be involved in phthalate-induced testicular mal-development (TMD) in Wistar rats at GD19.5
Network of genes in functional pathways postulated to be involved in phthalate-induced testicular mal-development (TMD) in Wistar rats at GD15.5
GD 15.5
Cytoplasm
Extracellular Space
Plasma Membrane
Nucleus
F2
24.985
APOA2
40.648
SLC27A2
3.935
ACADS
1.644
EBP
3.519
ACOX1
1.290
ADCYAP1
-2.966
CYCS
1.715
APOA1
38.886
FABP1
12.910
HSD17B4
1.329
FADS1
1.498
APOE
2.790
ACSL1
2.446
CYP17A1
-1.429
CYP11A1
-1.575
SERPINA1
CYP51A1
STAR
FDFT1
SCP2
FDPS
SCARB1
HMGCR
steroidogenesis
synthesis/ transportof cholesterol
Fatty Acid Metabolismand Transport
APOA4
-3.206
APOC1
3.646
PEBP1
SLCO1B3
-4.166
ABCA4 (includes EG:24)
-1.573
GOT2
1.403
ABCC3
-1.193
ABCA3
2.530
SLCO2A1
-1.619
PRDX2
1.298
FAT
-1.425
FABP3
SREBF1PPARA NR5A1
HMGCS1
INSL3
INHA
NCOA1
5.523
CREB1
Testes descent and
development
The diagrams show upregulated (red)-and downregulated (green)- genes that were affected in whole fetal testes following in utero
exposure of rats dibutylphthalate (DBP) 500mg/Kg from GD 12 to GD 19. Orange lines show genes that are regulated by the transcription
factors peroxisome proliferator receptor alpha (PPARA) and steroidogenic factor 1 (SF-1). Grey lines represent associations (of genes) to
a particular functional category e.g steroidogenesis.Red oval shows fatty acid metabolism/transport genes regulated by PPARA (mostly
up-regulated),
Time-course of the effect of DBP on SF-1-regulated genes
Cyp 11A Immunostaining in Foetal Rat Testes from Control and In Utero DBP-Exposed Rats –
GD 17.5
Cyp 11A is SF-1 regulated and is down-regulated in Leydig Cells
Corn oil DPBGD17.5 control GD17.5 DBP
Inhibin alpha Immunostaining in Foetal Rat Testes from Control and In Utero DBP-Exposed
Rats – GD 17.5
Inhibin alpha is SF-1 regulated and is down-regulated in Leydig Cells with no effect in Sertoli cells
Anti-Mulerian Hormone (AMH) Immunostaining in
Foetal Rat Testes from Control and In Utero DBP-
Exposed Rats – GD 19.5
AMH is SF-1 regulated, but is Sertoli Cell specific and is NOTdown-regulated by DBP
Corn oil DBP
Summary and conclusions
• Profiling identified battery of genes which facilitated the formulation of a biologically plausible hypothesis for testicular dysgenesiscentred on genes regulated by steroidogenic factor 1 (SF-1), a nuclear hormone receptor.
• Genes in this battery were co-ordinately down-regulated with SF-1. SF-1 protein not down-regulated.
• Effects of DBP on SF-1 most likely indirect.
• DBP effects on SF-1-regulated genes focussed primarily on Leydigcells.
• PPAR alpha-regulated genes induced at GD15.5
Cross-species comparison for
human hazard assessment requires in vitro models that
respond in a way that reflects the in vivo situation.
Gene Expression Analysis in
Specific Tissue Regions Using Laser Capture Microdissection
Laser Microdissection
(PALM)
Laser Microdissection of Fetal Testes Regions
Prior to RNA Extraction and Microarray Analysis
Region Specific Microarray
Data Analysis
Aims:
•To compare ‘signature’ genes: interstitial vs tubular using Venn diagram analysis.
•To identify cell-type (region) specific effects of DBP on gene expression.
Comparison of Interstitial and Cord ‘signature’gene lists
Cord ‘unique’
37
Interstitial ‘unique’
55
Common7
Interstitial
•Steroidogenic acute regulator (STAR)
•Inhibin alpha (INHA)
•Hmg coA sythase (HMGCS)
•Isopentyl diphosphate delta isomerase (IDI)
•Steroyl coA desaturase (SCD)
•Insulin-like factor 3 (INSL3)
•Cellular retinoic acid binding protein 2
•FAT tumour supressor homolog 1 (FAT)
•Farnesyl diphosphate synthase
DBP-induced Gene Expression changes that
were UNIQUE to the Interstitial (Leydig Cell)
region.
Function
Steroidogenesis (SF-1)
Steroidogenesis (SF-1)
Cholesterol syn (SF-1)
Cholesterol syn
Fatty acid met (PPARαααα)
Gubernacular dev (SF-1)
Testes morph (RARαααα)
Cellular organisation
Cholesterol syn (PPARγγγγ)
DBP-induced Gene Expression changes
that were Unique to the Cord (Sertoli
Cell) region.
Cord Function
•High-mobility group box 1 (HMGB1) Chromatin bending
•High-mobility group nucleosomal domain 2 (HMGN2) Chromatin bending
•Tumour protein translationally controlled (TPT1) Calcium signalling
•Myrystolate protein kinase C subatrate (MARKS) Phagocytosis
•Hypoxia inducible factor 1 alpha (HIF1A) Transcription factor
(response to hypoxia)
DBP-induced Gene Expression Changes
that were Common to both Interstitial
and Cord regions.Common Function
•Diazepam-binding inhibitor (DBI) Steroidogenesis
•Fatty acid binding protein 5 (FABP5) Steroidogenesis
•Scavenger receptor class B 1 (SCARB1) Steroidogenesis
•Cytochrome P450 17 A (Cyp 17A) Steroidogenesis
•Phosphoglycerate dehydrogenase (PHGDH) Cell/tissue assembly
•Actin related protein 2/3 complex 5 (ARPC5) Cell/tissue assembly
•Serine protease inhibitor member 1 (SERPING1) Cell/tissue assembly
•Transketolase (TKT) Multiple metabolic pathways
Role of NHR in effects of DBP gene expression
changes in fetal rat testes interstitium (Leydig cells)
SF-1-regulated genes down-regulated
• Steroidogenic genes (Cyp17A, STAR)
• Developmental genes (INSL3, INHA)
PPAR alpha-regulated genes up-regulated
•SCD, ACADS, ACOX
PPAR gamma-regulated genes down-regulated
•SCARB1, FDPS
Retinoic acid receptor-regulated genes down-regulated
•CRABP2
DBP and MBP are PPAR alpha
agonists.
Hurst and Waxman Tox Sci, 2003
Lapinskas et al, Toxicology, 2004
Hypothetical mechanism of PPAR alpha-
mediated repression of SF-1 /RARα genes
PPAR/ RXR SF-1
Coactivator Competition
CBP [Limiting]
RARα
DBP
Current hypothesis
Summary 2
•Regional microarray analysis showed that DBP altered expression of fetal rat testes genes that are regulated by several different NHR receptors (SF-1, PPARα, PPARγ, RARα).
•Almost all the NHR-regulated genes were uniquely altered in the Leydig cells and the majority, apart from SCD, were repressed
•Leydig cell-specificity of effects of DBP on NHR-regulated gene expression corroborated at protein level.
Overall conclusions
�Effects of DBP on gene expression in fetal testes were compartmentalised.
�Regional TP analysis confirmed that the anti-androgenic effects are focussed on the Leydig cells (INT region).
�The data suggest a role for PPARalpha-mediated effects as a possible mechanism underlying DBP-induced Leydig cell dysfunction (cofactor starvation?).
Future work• Analysis of genes (proteins) involved in TMD
pathways focussing on NHRs (SF-1, PPARs, others?)
• Immunohistological assessment of NHR and coactivator expression in rat fetaltestes
• In vitro model
• Effects of various phthalates/phthalate metabolites on gene expression in rat fetaltestes explants
• Cell culture (primary Leydig cells?)
• Extrapolation to Humans
• Human fetal testes explant cultures?
• Engineered cell lines
Acknowledgments
Richard Sharpe, Nina Hallmark, Kim Mahood
-MRC Human Reproductive Sciences Unit, Edinburgh
Ulrich Sauer – P.A.L.M.
Funded by European Council for Plasticisers and
Intermediates - (ECPI)