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)