Tim Zacharewski

1
Toxicogenomic Assessment of Estrogenic Endocrine
Disruptors Effects of Ethynyl Estradiol on Gene
Expression
Tim Zacharewski Department of Biochemistry
Molecular Biology Institute For Environmental
Toxicology and The National Food Safety
Toxicology Center Michigan State
University tel [phone] fax (517)
353-9334 e-mail tzachare_at_pilot.msu.edu http//www
.bch.msu.edu/zacharet






Research Program Supported by National
Institutes of Health US Environmental Protection
Agency American Chemistry Council
2
Endocrine Disruptor Definition
An endocrine disruptor is an exogenous substance
that causes adverse health effects in an intact
organism, or its progeny, secondary to changes
in endocrine function.
3
Endocrine Disruption - Issues
Human Concerns - increased incidence of
hormone-dependent cancers - impaired cognitive
abilities - compromised fertility - increased
incidence of reproductive tract
abnormalities Wildlife Concerns - intersex - abno
rmal reproductive behavior - increased incidence
of developmental abnormalities - compromised
reproductive fitness
4
U.S. LEGISLATION
-
Food Quality Protection Act (FQPA)
Develop screening tests for chemicals that
mimic estrogen, androgen and thyroid by August
1998 and implement program by August 1999
Includes effects on humans and wildlife
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U.S. Environmental Protection Agency Endocrine
Disruptor Screening Program http//www.epa.gov/osc
pmont/oscpendo/index.htm
Initial Sorting
Priority Setting
Tier 1 Screening
Tier 2 Testing
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Endocrine Disruptor Screening Program
Initial Sorting - 87,000 chemicals - 900
pesticide active ingredients - 2,500 other
pesticide formulate ingredients - 75,500
industrial chemicals - 8,000 cosmetics, food
additives and nutritional supplements Priority
Setting - based on - production volume,
environmental persistence, exposure -
quantitative structure activity relationships
(QSARs) - high throughput prescreening
assays competitive ligand binding
reporter gene induction
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Structural Diversity of Estrogenic Endocrine
Disruptors
Pharmaceuticals
Industrial Chemicals
Ethynyl Estradiol
OH
C CH
Methoxychlor
H3C
CH3
H3C
HO
CH3
HO
Environmental Pollutants
Phytoestrogens/Natural Products
Zearalenone
HO
O
Genistein
Polychlorinated Biphenyl (PCB)
HO
O
OH
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Proposed Mechanism of Action of


Estrogen Receptors
L
L
ER
hsp90
EREs
hsp90
transcriptional effects
protein level changes
PLEIOTROPIC RESPONSE
PLEIOTROPIC RESPONSE
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Other Possible Actions of Estrogenic Endocrine
Disruptors
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The Emerging Paradigm
In order to fully assess the risk of chronic and
subchronic exposure to synthetic chemicals and
natural products, a more comprehensive
understanding of the physiological, cellular and
molecular effects is required within the context
of the whole organism, its genome, transcriptome,
proteome and metabonome.
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Data Integration Across Biological Levels

• Biological Networks
• depicts interactions between
• gene, protein and metabolite
• levels
• regulation distributed over all
• levels
• each level can influence the other
• network provides molecular
• basis for phenotype

From Brazhnik et al, Trends Biotechnol, 2002
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Translational Toxicogenomic Research

• Integration of global assessment technologies
  into
• environmental health and drug development.
• - Used to rank and prioritize drug
  candidates/chemicals for further
  development/testing
• Earlier incorporation of toxicology in drug
  development pipeline
• Identification of biomarkers for exposure and
  clinical
• trail monitoring

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Toxicology
Comprehensive Safety Assessment
Strategy correlate molecular changes to
observed effects in order to enhance predictive
accuracy
Tissue
Cell
Metabolome Proteome Transcriptome Genome
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Ethynyl Estradiol Induction of Global Gene
Expression
Study Design
harvest tissue
hrs
0
72
2
24
12
8
48
vehicle /- estrogen

• ovx immature C57BL/6 mice
• 0.1 mg/kg EE or with vehicle (sesame oil) by
  gavage
• uteri, liver, bone and mammary gland were
  harvested
• uteri - Affymetrix Mu11KSubA GeneChips
• liver, bone, mammary gland cDNA microarray

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Ethynyl Estradiol Induction of Uterine Global
Gene Expression

• Uterus
• Affymetrix Mu11KSubA GeneChip

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Data Analysis Approach
6523
Probe sets on Mu11KSubA GeneChip

• Screen 1 Nonparametric empirical Bayes

881
Significant time and/or treatment effect

• Screen 2 ANOVA

392
Significant treatment or treatmenttime effect

• k-means clustering Annotation UniGene

268
Genes (not unknown ESTs)

• Annotation Gene Ontology

263
Physiological/Toxicological interpretation

• Used gene ontology and RefSeq annotation to link
• transcriptional and physiologic changes

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Affymetrix GeneChip
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Affymetrix GeneChipsPhotolithographic Synthesis
of GeneChips
Lamp
GeneChip
Mask
http//www.affymetrix.com
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GeneChip Expression Tiling Array Design
5
3
Gene Sequence
Multiple oligo probes
Perfect Match
Mismatch
Perfect match
A-C-T-G-T-T-T-A-C-G-C-T-C-A-G-T-C-G-G-G-T-C-A-A-T
Mismatch
A-C-T-G-T-T-T-A-C-G-C-T-A-A-G-T-C-G-G-G-T-C-A-A-T
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GeneChip Expression AnalysisHybridization and
Staining
Array
Hybridized Array
cRNA Target
Streptravidin-phycoerythrin conjugate
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Microarray Data Management, Analysis, and Storage
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Modular Architecture of dbZach
(http//dbZach.fst.msu.edu)

• Under Development
• Promoter Subsystem
• Pathway Subsystem
• Toxicology Subsystem
• Real-Time PCR
• Subsystem
• Sample Annotation
• Subsystem
• Gene Annotation Tool
• Correlation Tool
• QA/QC monitoring
• Feature Inspection Tool

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Verification by QRT-PCR

• 26 known genes selected based on p1z value
• Pearson correlations were calculated
• profiles for 23/26 genes exhibited strong
  correlation
• profiles for 2/26 exhibited marginal
  correlation
• profile for 1/26 genes did not correlate

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K-Means Clustering
7 K-means clusters
General response
Confirm previously reported estrogen-regulated
gene responses
1.
2.
3.
4.
5.
Describe new estrogen-regulated gene responses
and hypothesize about specific mechanisms
6.
7.
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Summary Temporal Trends
0 4 8
12 16 20
24 . . . 3x24 hr
Water imbibition
Increased dry mass, hyperplasia
Energy
ATP
ATP transport
Solute/ water transport
Cl- transport
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polyamine depletion
Arginine/Ornithine Utilization
tissue proliferation
polyamines
Myc/Max
-neg
Oaz2 2-3x24 hr
Oazi N.D.
-neg
-neg
ornithine
-neg
proteins
-neg
arginine
citrulline
vasodilation, immune stimulation, inhibition of
smooth muscle cell growth
nitric oxide
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2-24 hr Following EE Exposure
polyamine depletion
tissue proliferation
polyamines
Myc/Max
-neg
Oaz2 2-3x24 hr
Oazi N.D.
-neg
-neg
ornithine
-neg
proteins
-neg
arginine
citrulline
vasodilation, immune stimulation, inhibition of
smooth muscle cell growth
nitric oxide
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3x24 hr Following EE Exposure
polyamine depletion
tissue proliferation
polyamines
Myc/Max
-neg
Oazi N.D.
-neg
-neg
ornithine
-neg
proteins
-neg
arginine
citrulline
vasodilation, immune stimulation, inhibition of
smooth muscle cell growth
nitric oxide
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Ethynyl Estradiol Induction of Hepatic Global
Gene Expression

• Liver
• cDNA/EST microarray

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Construction and Use of cDNA Arrays
cDNA clones in bacteria
plasmid DNA
PCR
tissue/cells
agarose gel analysis
purified PCR product
total RNA
array production
probe generation by RT labeling
informatics db
probe hybridization
signal detection
data analysis
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Gene Expression Analysis Using cDNA Microarrays
Control
Treated
RNA Isolation
Cy5
Cy3
Reverse Transcription

Mix cDNAs and Apply to µArray
Hybridize Under Coverslip
Scan
cDNA µArray

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Current and Future cDNA/EST Arrays Includes ESTs
with gt70 similarity
Mus musculus dbZach NIA 15K Affy subset Lion
Biosciences UniGene build 113
Homo sapiens VAI 40K dbZach UniGene build 154
3636
10656
6432
1162
2625
3472
Rattus norvegicus Lion Biosciences UniGene
build 106
5801
STRATEGY Orthologs represented on each array in
order to examine in vitro and in vivo
extrapolation between species
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Ethynyl Estradiol Induction of Hepatic Global
Gene Expression

• Summary
• growth and proliferation
• cytoskeleton and extracellular matrix
• monoxygenases, antioxidants
• glutathoine transferases
• lipid metabolism and transport

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Hepatic vs. Uterine Global Gene Expression
Uterus (Affymetric Mu11KSubA GeneChip)
Liver (cDNA/EST microarray)
2,258 unique genes
5,543 unique genes
5,543 genes with LocusLink
2,150 genes with LocusLink
1,318 genes with common LocusLink
979 genes exhibit significant change in
expression in at least one tissue

• 264 genes only
• expressed in liver
• 429 genes only
• expressed in uterus

693 genes exhibited significant change in only
one tissue
286 genes exhibited significant change in both
tissues
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Other Global Gene Expression Comparisons

• Uterus vs. Liver vs. Mammary Gland vs. Bone
• no common ethynyl estradiol elicited gene
  expression profile
• significant differences in gene expression
  profile kinetics that can not be explained by
  metabolism
• Mouse Hep1c1c7 cells vs. Mouse Liver
• - only 10 overlap in ethynyl estradiol elicited
  gene expression profiles

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Summary
Ethynyl estradiol gene expression profile overlap
between tissues and in vitro vs. in vivo models
is minimal Examination of pathways and
elucidation of mechanisms will identify
biomarkers with greater predictive value e.g.
Arginase 1 indicates attenuation of
proliferation Several anomalous and absent
responses were observed suggesting estrogen
receptor-independent mechanisms and
post-transcriptional activities Predictive
ability of in vitro screens to identify endocrine
disruptors with in vivo activity is questionable
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Future Directions
Complementary omic technologies e.g.
proteomics, metabonomics Phenotypic
anchoring e.g. in situ hybridization,
immunohistochemistry, histology,
clinical chemistry, toxicology Mechanisms/Pathway
Discovery e.g. ChIP on Chip, reverse
engineering, support vector machines Computationa
l Biology e.g. PBPK, network elucidation,
computational modeling Risk Assessment
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Systems Toxicology
The iterative development of computational models
that integrate disparate biological (DNA, RNA,
protein, protein interactions, biomodules, cells,
tissues, etc.), chemical, and toxicological data
which can be used to further elucidate the
mechanisms of toxicity of a substance as well as
support risk assessment.
39
Acknowledgements
Chris Gennings Virginia Commonwealth
University Jennette Eckel Mayo Clinic
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Molecular Genomic Toxicology Lab
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TOXICOGENOMICS Research Associate, Post Doctoral
Fellow and Graduate Student Positions Available

• Positions available to investigate
• Gene expression profiles for estrogenic and
  dioxin-like
• chemicals and mixtures using human, rat and
  mouse
• in vitro and in vivo models
• Develop bioinformatic and computational resources
  
• (e.g. relational database, analysis tools,
  modeling) in
• support of toxicology studies

Further information regarding research activities
in the laboratory is available at
www.bch.msu.edu/zacharet
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Toxicogenomic Positions contd
These are multifaceted position that will
require a highly motivated and well organized
individual with excellent writing and verbal
communication skills. Knowledge of molecular
biology and/or biochemistry is essential.
Experience with animal handling, statistical
analysis, genomics, bioinformatics, computer
programming and database management is highly
desirable. Competitive salary, including
benefits, will be based on training and
experience.
Interested individuals are requested to submit a
cover letter outlining their research
experience, career aspirations, a curriculum
vitae and copies of relevant reprints to
Tim Zacharewski, PhD, Michigan State
University Department of Biochemistry
Molecular Biology 223 Biochemistry Building,
Wilson Road East Lansing, Michigan
48824-1319 USA Tel [phone]
Fax [phone] E-mail
tzachare_at_pilot.msu.edu