Toxicogenomic Assessment of Estrogenic Endocrine Disruptors: Effects of Ethynyl Estradiol on Gene Ex...
This presentation is the property of its rightful owner.
Sponsored Links
1 / 42

Tim Zacharewski Department of Biochemistry & Molecular Biology PowerPoint PPT Presentation


  • 91 Views
  • Uploaded on
  • Presentation posted in: General

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

Download Presentation

Tim Zacharewski Department of Biochemistry & Molecular Biology

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Tim zacharewski department of biochemistry molecular biology

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: (517) 355-1607

fax: (517) 353-9334

e-mail: [email protected]

http://www.bch.msu.edu/~zacharet

Research Program Supported by:National Institutes of Health

US Environmental Protection Agency

American Chemistry Council


Tim zacharewski department of biochemistry molecular biology

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.


Tim zacharewski department of biochemistry molecular biology

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

-abnormal reproductive behavior

-increased incidence of developmental abnormalities

-compromised reproductive fitness


Tim zacharewski department of biochemistry molecular biology

-

Safe Drinking Water Act (SDWA)

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


Tim zacharewski department of biochemistry molecular biology

U.S. Environmental Protection Agency

Endocrine Disruptor Screening Program

http://www.epa.gov/oscpmont/oscpendo/index.htm

Initial Sorting

Priority Setting

Tier 1 Screening

Tier 2 Testing


Tim zacharewski department of biochemistry molecular biology

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


Tim zacharewski department of biochemistry molecular biology

OH

H3CO

OCH3

CCl3

HO

Diethylstilbestrol (DES)

4-

t

-Octylphenol

Cl

HO

CH3

O

Cl

O

CCl3

HO

O

2

3

3`

2`

Cly

Clx

1`

4`

4

1

5

6

6`

5`

o,p

' -DDT

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


Tim zacharewski department of biochemistry molecular biology

ER*

ER*

L

L

Proposed Mechanism of Action of

Estrogen Receptors

L

L

ER

hsp90

EREs

hsp90

transcriptional effects

protein level changes

PLEIOTROPIC RESPONSE

PLEIOTROPIC RESPONSE


Tim zacharewski department of biochemistry molecular biology

Estrogenic

Endocrine

Disrupting

Chemical

Binding

Globulin

Extracellular

Membrane Bound

Estrogen Receptor

Binding Globulin

Receptor

Intracellular

Oxidative

Metabolism

Kinase

New Ligands

Transcription

Factors

Receptors

Gene

Expression

Protein

Cellular Effects

Tissue Effects

Other Possible Actions of Estrogenic Endocrine Disruptors


Tim zacharewski department of biochemistry molecular biology

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.


Tim zacharewski department of biochemistry molecular biology

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


Tim zacharewski department of biochemistry molecular biology

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


Tim zacharewski department of biochemistry molecular biology

Toxicology

Comprehensive

Safety Assessment

Strategy:

correlate molecular

changes to observed

effects in order to

enhance predictive

accuracy

Tissue

Cell

Metabolome

Proteome

Transcriptome

Genome


Ethynyl estradiol induction of global gene expression

Ethynyl Estradiol Induction of Global Gene Expression

Study Design

harvest

tissue

hrs

0

2

8

12

24

48

72

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


Ethynyl estradiol induction of uterine global gene expression

Ethynyl Estradiol Induction of Uterine Global Gene Expression

  • Uterus

  • Affymetrix Mu11KSubA GeneChip


Data analysis approach

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 treatment*time 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


Tim zacharewski department of biochemistry molecular biology

Affymetrix GeneChip


Tim zacharewski department of biochemistry molecular biology

Affymetrix GeneChips:Photolithographic Synthesis of GeneChips

Lamp

GeneChip

Mask

http://www.affymetrix.com


Tim zacharewski department of biochemistry molecular biology

GeneChip Expression Tiling Array Design

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


Tim zacharewski department of biochemistry molecular biology

GeneChip Expression AnalysisHybridization and Staining

Array

Hybridized Array

cRNA Target

Streptravidin-phycoerythrin

conjugate


Tim zacharewski department of biochemistry molecular biology

Microarray Data Management, Analysis, and Storage


Modular architecture of dbzach http dbzach fst msu edu

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


Verification by qrt pcr

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


Tim zacharewski department of biochemistry molecular biology

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.


Tim zacharewski department of biochemistry molecular biology

Cell cycle

G1/S

S

G2/M

DNA synthesis

(d)NTPs

dNDPs

dNTP recycling

RNA/protein

synthesis

RNAPol; polyA-BP;

tRNA-synth.; eIFs

Immune

+/- migration and cytokine signaling suppressors

complement

components

- protectin (inhibits complement-mediated lysis)

Summary: Temporal Trends

0 4 8 12 16 20 24 . . . 3x24 hr

Increased dry mass, hyperplasia

Water imbibition

Energy

ATP

ATP transport

Solute/ water transport

Cl- transport


Tim zacharewski department of biochemistry molecular biology

Sat

3x24 hr

Abp1

3x24 hr

Mxi1

8-24hr

Odc

8-24 hr

Arg1

3x24 hr

Rars

8-12 hr

Pdi2

3x24 hr

Nos3

2-8 hr

Arginine/Ornithine Utilization

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


Tim zacharewski department of biochemistry molecular biology

Sat

3x24 hr

Abp1

3x24 hr

Mxi1

8-24hr

Odc

8-24 hr

Arg1

3x24 hr

Rars

8-12 hr

Pdi2

3x24 hr

Nos3

2-8 hr

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


Tim zacharewski department of biochemistry molecular biology

Sat

3x24 hr

Abp1

3x24 hr

Mxi1

8-24hr

Odc

8-24 hr

Oaz2

2-3x24 hr

Arg1

3x24 hr

Rars

8-12 hr

Pdi2

3x24 hr

Nos3

2-8 hr

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


Ethynyl estradiol induction of hepatic global gene expression

Ethynyl Estradiol Induction of Hepatic Global Gene Expression

  • Liver

  • cDNA/EST microarray


Tim zacharewski department of biochemistry molecular biology

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


Tim zacharewski department of biochemistry molecular biology

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


Tim zacharewski department of biochemistry molecular biology

Current and Future cDNA/EST Arrays

Includes ESTs with >70% 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


Ethynyl estradiol induction of hepatic global gene expression1

Ethynyl Estradiol Induction of Hepatic Global Gene Expression

Summary

  • growth and proliferation

  • cytoskeleton and extracellular matrix

  • monoxygenases, antioxidants

  • glutathoine transferases

  • lipid metabolism and transport


Hepatic vs uterine global gene expression

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


Other global gene expression comparisons

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


Tim zacharewski department of biochemistry molecular biology

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


Tim zacharewski department of biochemistry molecular biology

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

Computational Biology

e.g. PBPK, network elucidation, computational modeling

Risk Assessment


Tim zacharewski department of biochemistry molecular biology

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.


Tim zacharewski department of biochemistry molecular biology

Acknowledgements

Chris Gennings

Virginia Commonwealth University

Jennette Eckel

Mayo Clinic


Tim zacharewski department of biochemistry molecular biology

Molecular & Genomic Toxicology Lab


Tim zacharewski department of biochemistry molecular biology

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


Tim zacharewski department of biochemistry molecular biology

Toxicogenomic Positions cont’d

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: (517) 355-1607

Fax: (517) 353-9334

E-mail: [email protected]


  • Login