Gene chip applications in environmental toxicology

Gene chip applications in environmental toxicology.

Patrick Larkin Ph.D.Vice President-Research and Development

EcoArray Inc, Alachua, FL USA(386) 418-1400

[email protected]

1

1 1

Advantages of Gene chips

-Biomarkers of exposure

-Compound discrimination and quantification

-Bioavailability

2

2 2

The company- what we do.

EcoArray Inc. is a company that manufactures gene chips and provides support and services

related to these products.

Our products and services are specifically tailored to the toxicology field.

3

3 3

Existing technologies that can measure compounds

Technology Limitations • Fail to report on what is • Water/sediment happening in an animal.chemistry tests

• In vitro assays • Can not report on

(ie. YES assay). metabolites of compounds.

• Whole animal bioassays. • Insensitive endpoints.

4

4 4

Existing technologies that can measure compounds

Technology Limitations • Fail to report on what is • Water/sediment happening in an animal.chemistry tests

• In vitro assays • Can not report on

(ie. YES assay). metabolites of compounds.

• Whole animal bioassays. • Insensitive endpoints.

… gene chips overcome these limitations.

5

5 5

Small glass slides or membranes that contain

genetic material.

What are gene chips?

6

66

How gene chips work- an overview

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77

How gene chips work- an overview

EcoArray programs

8

88

How gene chips work -In more detail…

liver

cellDNA

mRNA (expressed genes)

9

9 9


OR

liver

cellDNA

mRNA (expressed genes)

Compound present in the environment

Very sensitive tests!!! 10

10 10

Measure mRNA using gene chips

•Thousands of genes can be spotted onto chips

• mRNA changes can be quantitated

• Changes in mRNA can be used for the early detection of compounds BEFORE adverse effects are observed in animals.

11

11 11

Constant Exposure to 100 ng/L E2

Days of treatment 0 10 20 30 40 50

Vtg

mR

NA

(ng/

µg to

tal R

NA

)

0.01

0.1

1

10

100

1000

10000

Pla

sma

Vtg

(mg/

ml)

0.01

0.1

1

10

100

RNA Protein

Early detection

Denslow et al. 12

1212

Perc

ent M

easu

red

Effe

ct

Concentration x Time of Exposure

Popula

tion

Molecu

lar

(gene

)

100

50

0

Physio

logica

l

Irreversible effects

Reversible effects

Monitor for contaminants before irreversible effects occurs

Early detection

13

1313

Robotics

How gene chips are made

14

1414


Chip surface

mRNA

mRNA (expressed gene)

15

1515

How gene chips are used

Extract tissue

Label mRNA

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16 16

17 17

How gene chips are used

fingerprint

Label mRNA

Extract tissue

17

Obtain genetic

Model species

Fathead minnow freshwater species that is commonly used for toxicology testing.

Example: Biomarkers for exposure

Sheepshead minnow estuarine species that is commonly used for toxicology testing.

Example: Compound discrimination and quantification

Largemouth bass important game fish found throughout much of the United States.

Example: Bioavailability

18

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Experimental strategy

Controlled laboratory exposures

Genetic fingerprint database

Pollutant A Pollutant B Pollutant C

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19 19

Experimental strategy

Controlled laboratory exposures

Field site

Genetic fingerprintdatabase

Pollutant A Pollutant B Pollutant C

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20 20

Fathead minnow

male

female

21

2121

FHMinnow chip

• 200 gene chip.

•Genes obtained from a variety of methods (cDNA libraries, directed cloning, and subtraction libraries).

• Genes are parts of multiple pathways.

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22 22

Un-exposed female

Un-exposed male

Male exposed to 400 ng/L of E2 for 48hrs

FHMChips®

Genetic biomarker for estrogens

Fathead minnow experiments

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2323

Fathead minnow experiments

We are developing a 2000+ oligonucleotide based gene chip in fathead minnows with the EPA.

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2424

Sheepshead minnow

male

female

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25 25

Compound discrimination Strong Weak

estrogens estrogens 17-β estradiol Endosulfan

-SHMs exposed to Control

several estrogenic compounds

Ethinylestradiol Methoxychlor

NonylphenolDiethylstilbestrol

(65 ng/L)

(100 ng/L)

100 ng/L

(590 ng/L)

(5.6 ug/L)

11.8 ug/L

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26 26

Compound discrimination

1000010000

10001000

100100

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Methoxychlor NonylphenolMethoxychlor NonylphenolMethoxychlor NonylphenolMethoxychlor NonylphenolN

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-EDES EndosulfanDES EndosulfanDES EndosulfanDES Endosulfan

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27 27

1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

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1e+3

1e+4

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1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

l uni

ts

Vtg 2 Vtg 1

Quantification

-SHMs exposed to several different doses of EE2

1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

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l uni

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1e+4

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1e+6

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1e+3

1e+4

1e+5

1e+6

1e+7

1e+3

1e+4

1e+5

1e+6

1e+7

Pixe

l uni

ts

Choriogenin 3Choriogenin 2

1 Concentration

(ng/L)

0 10 100 10001Concentration

(ng/L)

0 10 100 1000 1 Concentration

(ng/L)


(ng/L)

0 10 100 1000

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

ER-α Coagulation Factor XI

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

1 Concentration

(ng/L)


(ng/L)

0 10 100 10001 Concentration

(ng/L)


(ng/L)

0 10 100 1000

1 Concentration

(ng/L)


(ng/L)

0 10 100 10001 Concentration

(ng/L)


(ng/L)


(ng/L)


(ng/L)

0 10 100 1000

Larkin et al, EHP 2003 28

2828

Transferrin AMBP protein (α-1-microglobulin)

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

1 Concentration


0 10 100 1000 1 Concentration




0 10 100 1000

Quantification

Beta actin

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

2e+3

1e+4

1e+5

1e+6

Pixe

l uni

ts

(ng/L)(ng/L)

1 Concentration

(ng/L)


(ng/L)

0 10 100 1000

(ng/L)(ng/L)(ng/L)(ng/L)

Larkin et al, EHP 2003 29

2929

Largemouth bass model

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3030

Area 7 field site

Eustis Muck Farm

OCPs levels in the muscles of fish :

Chlordane Dieldrin

p’p’-DDD p’p’-DDE p’p’-DDT

toxaphene

31

3131

LMBass gene chip

• 500 gene chip.

•Genes obtained from a variety of methods (cDNA libraries, directed cloning, differential display, and subtraction libraries).

• Genes are parts of multiple pathways.

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Largemouth bass model

Laboratory exposure • 17-β estradiol exposure (2.5 mg/kg inject, 48 hrs).

• p’p-DDE (100 mg/kg inject, 48 hrs).

• 11-ketotestosterone (2 mg/kg inject, 48 hrs).

• Characterize reproductive cycle

Field analysis

• Site of investigation

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Estradiol Treated (2.5 mg/kg for 48 hrs)

Controls

Estradiol laboratory exposures

Larkin et al, CBP 2002 34

3434

Estradiol laboratory exposures

0.10

1.00

10.00

100.00

1000.00

EA-L

MB

-348

Hem

opex

in-li

ke p

rote

in

EA-L

MB

-122

EA-L

MB

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ha-2

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cept

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lloge

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2A

Vite

lloge

nin

1 Fold

cha

nge

* * * **** * *** * **

P<0.05 P<0.01 * **


3535

DDE laboratory exposures

0.1

1

10

ER a

lpha

Prot

ein

disu

lfide

is

omer

ase

Vtg.

3

Asp

artic

pr

otea

se

Cho

rioge

nin

3

Cho

rioge

nin

2 Vtg.

2A

Vtg.

1

Pixe

l uni

ts

(Fol

d ch

ange

)


3636

11KT laboratory exposures

0.10

1.00

10.00

EA-L

MB

-348

Hem

opex

in-li

ke p

rote

in

EA-L

MB

-122

EA-L

MB

-106

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Prot

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Estr

ogen

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ptor

sub

type

I

EA-L

MB

-423

Asp

artic

pro

teas

e

Vite

lloge

nin

3

Cho

rioge

nin

3

Cho

rioge

nin

2

Vite

lloge

nin

2A

Vite

lloge

nin

1

Fold

cha

nge *

* * * *

P<0.05 P<0.01 * **

37

3737

Bass reproductive cycle

Need to define “normal” fingerprint pattern in bass before one can identify atypical gene expression patterns in the field

January April August

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Bass reproductive cycle

Need to define “normal” fingerprint pattern in bass before one can identify atypical gene expression patterns in the field

Study site

January April August

39

39 39

4040

October February April

0

5

10

15

20

25


0

200

400

600

800

Months

Vtg 2.

Vtg 1.

Vtg 2A.

Vtg 3.

Aspartic protease

ZP3

ZP2

Months

Pixe

l uni

ts(F

old

chan

ge)

Bass reproductive cycle(females)

40

4141

Months

Pixe

l uni

ts(F

old

chan

ge)

Bass reproductive cycle(males)

No significant change in the vtgs and choriogenins.


0

5

10

15

20

25

41

Eustis (study site)

Deleon Springs (clean site)

Field analysis

No change in ZP’s No change in Vtg’s

42

4242

Field analysis

0.10

1.00

10.00

EA

-LM

B-0

28

EA

-LM

B-4

43

Ser

ine

prot

ease

inhi

bito

r 2b

EA

-LM

B-0

35

Per

leca

n (h

epar

an s

ulfa

te

prot

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2)

EA

-LM

B-1

90

EA

-LM

B-3

99

AM

BP

pro

tein

pre

curs

or

EA

-LM

B-1

14

Tryp

sin

II pr

ecur

sor

Pixe

l uni

ts

* *

* ******* Study site

Clean

P<0.05*

43

4343

Gene ontology database

44

4444

Field analysis

0.10

1.00

10.00

EA

-LM

B-0

28

EA

-LM

B-4

43

Ser

ine

prot

ease

inhi

bito

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-LM

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35

Per

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-LM

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90

EA

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99

AM

BP

pro

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pre

curs

or

EA

-LM

B-1

14

Tryp

sin

II pr

ecur

sor

Pixe

l uni

ts

* *

* ******* Study site

Clean

P<0.05*

45

4545

Field analysis

Digestive pathways may be affected in

these animals

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Summary

-Gene chips can be used for biomarkers for exposure.

-Gene chips can be used to discriminate between compounds and can provide quantifiable data.

-Gene chips can provide information on bioavailability of compounds.

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Acknowledgements

EcoArray Inc University of Florida Barbara Carter Nancy Denslow, Ph.D.

Kevin Kroll

Fish and Wildlife Tara Sabo, Ph.D.

Conservation Commision, Eustis FL

Jamie Kelso Arianna Poston

Jaleh Khorsandian-Falleh Bill Johnson

Bill Farmerie, Ph.D.Li Liu

Anuj SahniUS EPAMichael Hemmer

Ira Adelman, PhD. Funding: Leroy Folmar Ph.D. SBIR grant #1R43ESA011882-01

SBRP (P42 ES 07375) 48

48 48

Additional information

See www.ecoarray.com

Our website contains links to manuscripts using this technology and other information

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Biosensing with Zebrafish

Elwood Linney, Ph. D.

Molecular Genetics and Microbiology

Duke University Medical Center

Biosensing with Zebrafish

Elwood Linney, Ph. D.

Molecular Genetics and Microbiology

Duke University Medical Center

Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC 50

50 50

Laboratory of Molecular Development Department of Molecular Genetics and Microbiology DUMC

transgenesis imaging

gene discovery

51

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Assumptions we make:Assumptions we make:

1) toxicants are impacting upon normal, existing pathways

2) there can be a differential sensitivity to a toxicant depending upon whether the organism or target organ is developing or fully formed

3) there are common pathways in different organisms

4) differences between organisms should be represented by “differences” in their genomes


52 52

Our changing view of “biosensors”

Use results to design new biosensor transgenics


Our changing view of “biosensors”

1) transgenic indicator mice for snapshots of “retinoic acid activity”

2) transgenic, fluorescent zebrafish for live 4-D studies of activity

3) using zebrafish themselves as indicators or sensors for toxicant events

4) using discovery microarray analysis for identifying genes affected

Use results to design new biosensor transgenics

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Reporter transgenic miceusing constructed retinoic acidresponsive promoter

RARE -TKpr-β gal

RXR RAR

RA

Subset of retinoic acid receptor activity in reporter transgenic mouse:

Reporter transgenic mice using constructed retinoic acid responsive promoter


5454

Size relationships, developmental time, changingin size with development:


relative size of embryos

Time-lapse 2 cell to 17 hours

mouse 9.5d to neonate

R.Kalstron and D. KaneSize relationships, developmental time, changing in size with development:

55

5555

Mouse and zebrafish homeobox genes:


Mouse and zebrafish homeobox genes:

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Parallels in axial development between vertebrate species:

Black bars denote spinal nerves of brachial plexus

level of curved line represents the level of limb or fin

shaded somites represent level of Hoxc-6 expression


Parallels in axial development between vertebrate species:

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Syntenic relationships


Vol. 10, Issue 12, 1890-1902, December 2000

Zebrafish Comparative Genomics and the Origins of Vertebrate Chromosomes John H. Postlethwait,1,3 Ian G. Woods,2 Phuong Ngo-Hazelett,1 Yi-Lin Yan,1 Peter D. Kelly,2 Felicia Chu,2 Hui Huang,2 Alicia Hill-Force,1 and William S. Talbot2

Syntenic relationships between vertebrate genomes --genes inherited as linked clusters during speciation

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Retinoic acid indicator embryos

RARE βgal GFP/YFP

RA


retinoic acid responsive day 8.5 mouse embryo expressing lacZ from RARE TKpr sequences

24 hr live zebrafish embryo expressing YFP from RARE zGT2 basal promoter sequence

sizes approximately to scale

Retinoic acid indicator embryos

RARE-promoter-βgal/GFP/YFP RXR RAR

RA

59

5959

Vitamin A-Retinoid Relationships:

retinol dehydrogenase

retinal dehydrogenase(RALDH)

cytochrome p450RAI(cyp26)


vitamin A(all-trans retinol)

retinaldehyde

retinoic acid [ligand for transcription factors]

4-oxoretinoic acid

Vitamin A-Retinoid Relationships:

retinol dehydrogenase

retinal dehydrogenase(RALDH)

cytochrome p450RAI(cyp26)

RA synthesis

RA metabolism

60

6060

Dual in situ hybridization for RALDH2(purple) and cyp26a1(red-orange):


Dual in situ hybridization for RALDH2(purple) and cyp26a1(red-orange):

head

tail

RALDH2 in somites

[cyp26 in growing caudal end]

61

6161

Model of some of the retinoid events occurring in the trunk neural tube:Model of some of the retinoid events occurring in the trunk neural tube:

18hpf Embryo

Somites

-Raldh2

- RA

- Cyp26A1

Neural tube tail budRA

RA


62 62

RALDH inhibitor[our work]

Neckless mutant inRALDH2

RALDH inhibitor Neckless mutant in [our work] RALDH2

Begemann, Schilling, Rauch, Geisler and Ingham untreated

Either chemical inhibition of Raldh’s DEAB in zebrafish with DEAB or an isolated

Raldh2 zebrafish mutant produced phenotypes paralleling the mouse Raldh2 knockout phenotypes:

hindbrain changes forelimb or fin inhibition

normal DEAB treated


63 63

In situs of zebrafish Raldh2 at different developmental stages[Kari Yacisin]:

Point:

In situs of zebrafish Raldh2 at different developmental stages[Kari Yacisin]:

tailbud stage ~10h 10 somites~14h

14 somites~16h 18 somites~18h Point: apparent turn-off of RALDH2 as neural tube ceases to grow

20 somites~<20h 23h


64 64

Two mouse KO studies ofcyp26A1 revealed caudal truncationsand occasional exencephaly


cyp 26 knockout mice Sakai, Meno, Fujii, Nishino, Shiratori, Saijoh, Rossant, and Hamada

cyp26 knockout mice Abu-Abed, Dolle, Metzger Beckett, Chambon and Petkovich [somewhat similar phenotypes from these authors]

Two mouse KO studies of cyp26A1 revealed caudal truncations and occasional exencephaly

65

6565

Summary:Summary:

1)Raldh2 and cyp26a1(and cyp26b1) can be found adjacent to each other in the developing embryo creating functional "microgradients" of RA ligand for RAR activity

2)expression patterns and available mutants for these genes in mouse and zebrafish show consider homology

3)in zebrafish the Raldh2 promoter is directly repressed by RA and the cyp26a1 promoter is directly induced by RA

4)this system is being studied to determine whether there might be a genetic and/or environmental basis for neural tube defects


66 66

Developmental changes flanking and including neural tube growthwhich we are analysing with 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24hmicroarray analysis:


8h 10h 11.3h 13h 15.5h

17h 19h 21.5h 24h

Developmental changes flanking and including neural tube growth which we are analysing with 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h microarray analysis:

67

6767

Progression of microarray development: Progression of microarray development:

1) oligos from 500 selected zebrafish genes

2)16k oligomer library from Compugen arrayed

3)now examining 22k zebrafish oligomer array produced by Agilent, bioinformatics through Paradigm

Affymetrix has produced zebrafish arrays but we have yet to use them


68 68

Genes regulated during segmentation


Genes regulated during segmentation [work done in collaboration with R. Malek at TIGR]

expression log base 2

upregulated at 12 hpf

downregulated at 12hpf

69

6969

Some elements of our zebrafish toolbox:Some elements of our zebrafish toolbox:

1) live, fluorescent, transgenic embryos

2) anti-sense morpholino knockdown of gene expression


70 70

Some transgenic lines we made:


Some transgenic lines we made:

GFP off constitutive promoter

GFP off artificial construct--lights up cells migrating along pronephric ducts

YFP of zHuC promoter that lights up developing nervous system

71

7171

Four transgenic lines for examining nervous system:


Four transgenic lines for examining nervous system:

72

7272

Antisense morpholino approaches use byzebrafish researchers to “knockdown” genes


AUG 5'

AAAAAAAAAAUAC

m orpho lino

A. Morpholino t o inhibit t ranslat ion

B. Morpholino t o inhibit splicing

exon a exon b exon c exon d

exon a exon b exon d exon a exon b exon c exon d

m orpho lino against sp lice accept or

norm al spliced m RNA

abnorm al sp liced product due t o m orpho lino

Antisense morpholino approaches use by zebrafish researchers to “knockdown” genes

73

7373

no allele) morpholino we injected into a 1-cellzebrafish embryo--these are 4 day larvae afterhatching--the phenotype is what is seen with realmutants in no-tail


no--tailtail (T(T--allele) morpholino we injected into a 1-cell zebrafish embryo--these are 4 day larvae after hatching--the phenotype is what is seen with real mutants in no-tail

74

7474

Chlorpyrifos studiesChlorpyrifos studies


75 75

mice

zebrafish


Acetycholinesterase

from Soreq and Seidman, Nature Reviews Neuroscience(2001)

classical function of hydrolysing the neurotransmitter, acetylcholine

mice have AChE plus a butyrylcholinesterase so mouse KOs in AChE allow animals to at least be born and live ~21 days

zebrafish only has AChE, so AChE-/-embryos show defects in muscle fiber formation, innervation, and primary sensory neurons die prematurely--embryos are initially motile and then develop paralysis and die

76

7676

Chlorpyrifos treating zebrafish embryos--our work, high dose(500ng/ml):


Chlorpyrifos treating zebrafish embryos--our work, high dose(500ng/ml):

77

7777

Experimental Plan:Experimental Plan: 1) expose embryos for 5 days with

chlorpyrifos

2) adult learning studies in E. Levin’s lab

3) acetylcholine esterase assays during embryogenesis

4) AChE morpholino titration to CPF inhibition studies

5) adult learning studies and microarray analysis


78 78

Acetylcholine esterase activity/chlorpyrifos exposure:


Averaged AChE Activity (0-5day exposure, 3 sets)

0

5

10

15

20

25

30

35

40

45

1 2 3 4 5 6 7 8 9

Age at Extraction

AChE

Act

ivity

(a

rbitr

ary

units

) 0 ng/ml CPF 10 ng/ml CPF

100 ng/ml CPF

Acetylcholine esterase activity/chlorpyrifos exposure:

79

7979

Collaborative work with E. Levin and E. Chrysanthis:


40

45

50

55

60

65

70

0 10 100

Chlorpyrifos (ng/ml)

Developmental Chlorpyrifos Exposure Effects on Average Choice Accuracy

* **

vs. Control (0 ng.ml CPF)* p<0.05** p<0.01

Collaborative work with E. Levin and E. Chrysanthis:

As part of our Superfund program we chlorpyrifos treated embryos for 5 days, released them and grew them up and they tested for learning in maze designed by E. Levin in our Psychiatry department:

80

8080

Targeting acetylcholine esterase with a morpholino:Targeting acetylcholine esterase with a morpholino:

Contol, MO, 100ng/ml CPF f.5/19/03

0 5

10 15 20 25 30 35 40 45

1 2 3 4 5 6 7 8 9

age at extraction

Rel

ativ

e A

ChE

Act

ivity

Control 0-5d DMSO MO-ache inj 100 ng/ml CPF 0-5d


81 81


-20

-15

-10

-5

0

5

Percent Correct Difference from Controls

CPF 0 CPF 10 CPF 100 Morpholino Control Morpholino

Delayed Spatial Alternation Choice Accuracywith Developmental Chlorpyrifos or Morphoino Zebrafish

vs. CPF 0 * p<0.05** p<0.01

vs. Morpholino Control# p<0.025

#

*

**

[work done in collaboration with E. Levin laboratory]

82

8282

Preliminary comparison of expression of MO-AChE, 100ng/ml CPF10ng/ml CPF versus control using filter of 2x or above expression:


H56788

BM

095875

AI8

78728

BI8

66260

BG

304327

BE016746

AA606013

AW

058804

BE201806

AI7

93723

BG

308438

BG

985503

AI8

78080

AI6

41757

BI6

73358

BI8

80088

BM

070938

AI3

31953

MO-ache

10ng/ml

0

5

10

15

20

25

30

MO-ache

100ng/ml

10ng/ml

Preliminary comparison of expression of MO-AChE, 100ng/ml CPF 10ng/ml CPF versus control using filter of 2x or above expression:

expression of experimental divided by control

each bar individual gene (15 out of 37 genes overlap of MO vs. 100ng/ml CPF)

[3 day treated and untreated embryos]

83

8383

Future--Biosensors:Future--Biosensors: 1) the generation of a series of responsive transgenics to small molecules (in progress, estrogen inducibility)

2) the use of the Sanger Centre zebrafish DNA assembly to identify clones for genes which show distinct responsiveness to environmental toxicants so that transgenics can be derived from their regulatory sequences

3) the analysis of the 22k array data to formulate potential pathways that toxicants are impacting upon

our zCyp26A1pr transgenics with RA inducible promoter

line 1 with 1 uM RA (18h-22h)


84 84

Research support from:


Lab individuals involved in our CPF work: Neural tube work: Sue Donerly Lucia Upchurch Betsy Dobbs-McAuliffe Stephen Huang Margaret Lai

past members Kari Yacisin Keenan O’Leary Qingshun Zhao

Collaborators: Ed Levin Elizabeth Chrysanthis Renae Malek(TIGR) Brad Smith(MRM) G.A. Johnson(MRM)

Research support from: NIEHS Superfund and Toxicogenomics Consortium, NICHD

[email protected] http://glowfish.mc.duke.edu

85

8585

http://glowfish.mc.duke.edu

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Gene chip applications in environmental toxicology