Toxicogenomics: Using cDNA Microarrays to Detect

Effects of Environmental Exposures

Research Triangle Park, NCOctober 15, 2002

Mary Jane Cunningham, Ph.D.Director of DiscoveriesMolecular Mining Corporation55 Rideau St.Kingston, ON Canada K7K 2Z8613-547-9752E-mail: mjcunningham@molecularmining.com

Definition of Toxicogenomics

The use of genomic technologies for the measurement and analysis

of gene and protein expressionin assessing the risk of

new chemical entities (NCEs)

Three Different Approaches

To study sex differences: Indirect gene expression

-Gene sequencing-Electronic comparison of cDNA library abundances

Direct gene expression

-Gene expression microarrays

Protein expression

-2D gels annotated by mass spectroscopy

Gene Sequencing

cDNA Library Technologies Oligo dT: Standard method of library production

Normalized rare clone-biased: Modified Soares* protocol used to bias library towards low abundance transcripts (75-90+% rare), increases % unique singletons in rat by 2X

Subtracted: Modified Soares* protocol used to target differentially expressed transcripts between 2 tissues, increases % unique singletons in rat by 2X

Hybridized: Top 100 highest abundance genes & 1st strand cDNA probes used to screen out high abundance clones; preferential enrichment of middle abundance clones; increases %unique singletons but only 1.5X

*Soares et al, PNAS 91:9228 (1994);

Bonaldo et al, Genome Research 6:791 (1996)

ZooSeqTM Analysis

Subsetting analyses of hepatotoxin-treated cDNA libraries:-APAP timecourse [12h, 24h, d3, d7, d14, d28]

-B(a)P timecourse [12h, 24h, d3, d7, d14, d28]

-CLO timecourse [12h, 24h, d3, d7, d28]

-4-AAF, ANIT, CCl4, Fenofibrate, Hydrazine (6 h, 24h)

-Female vs. Male [B(a)P, CLO]

-Subtracted CLO library

-Subtracted CLO libraries [female vs. male]

ZooSeqTM Analysis (Continued)Known Genes (with abundant transcripts present):APAP: QAbunAP56=Acetaminophen binding protein 8Cytochrome P450 IIIA 7B(a)P:Cytochrome P450c (3-MCA induced) 16Cytochrome P450d (3-MCA induced) 6CLO:Cytochrome P450 4A (LA-omega) 34Cytochrome P450 4A2 6Carnitine octanoyltransferase 11Acyl-CoA oxidase 6Peroxisomal bifunctional enzyme (PBE) 21CCl4:Alpha-1 acid glycoprotein 9Fenofibrate:Peroxisomal bifunctional enzyme (PBE) 19

Possible Toxicity Markers252 Incyte Uniques

(QAbun>3)

APAP only11

B(a)P only35 BP female

3

4-AAFANITCCl4

FenofibrateHydrazine

60

CLO female1

CLO93CLO only93

Possible Sex Markers

CLO female20

Known Genes-female:Comp. Gene ID Description QAbun B(a)P fvsm g2224669 KIAA0364 79CLO fvsm g2224669 KIAA0364 10CLO fvsm g204091 testis-specific farnesyl pyrophosphate synthetase 7B(a)P fvsm g57670 ribonuclease inhibitor 6CLO fvsm g286245 oligomycin sensitivity conferring protein (OSCP) 6 B(a)P fvsm g5007031 transgelin 3B(a)P fvsm g203773 CYP2E1 3CLO fvsm g4894587 growth hormone receptor

binding protein 3

Unique Genes-female:B(a)P fvsm 1 Incyte Unique 13B(a)P fvsm 2 Incyte Unique 10B(a)P fvsm 3 Incyte Unique 7CLO fvsm 4 Incyte Unique 3

Summary RNA transcripts of genes known to be involved in the

response of B(a)P, APAP and CLO were observed with high abundance in rat liver cDNA libraries.

Unique genes whose functions are not yet known were also observed.

In comparisons of cDNA libraries made from both male and female rats, transcripts from both known and unknown genes were detected in higher abundance in female rats than in males.

cDNA Microarray Technology Array Design: cDNAs are deposited onto glass surface

Sample Prep: PolyA mRNA isolated from cells/tissues is reverse transcribed to cDNA from control (red label) and treated (green label) samples

Hybridization: Fluorescently-labeled cDNA hybridized to cDNAs on array

Red=down-regulatedGreen=up-regulatedYellow=control and treated probes co-hybridize at same

frequency-no change in expression

Rat GEMTM has approx. 7400 cDNAs deposited(predominantly rat liver and kidney genes and genes which are related to toxic response)

Original References: Science 270: 467-470 (1995) Genome Res. 6: 639-645(1996)

cDNA Microarray Technology

+ =

OUTPUT: Ratio of Expression

Rat Hepatocyte Cell Lines

Cell Line Sex Morphology

Strain Isolated From

Year Isolated

BRL3A Female Epithelial Buffalo 1968

Clone 9 Male EpithelialSprague-Dawley 1968

H4-II-EC-3 Male Epithelial A x C 1961

McA-RH7777 Female Epithelial Buffalo 1974

Experimental Plan

Cell LineState of Growth

InitialPassage

Passage at Harvest

Cell Number

BRL3A Preconfluent Unknown 15 3.8(10)7

BRL3A Confluent Unknown 12 5.4(10)7

Clone 9 Preconfluent 16 33 6.0(10)7

Clone 9 Confluent 16 33 2.9(10)8

H4-II-EC-3 Preconfluent Unknown 15 1.7(10)8

H4-II-EC-3 Confluent Unknown 15 7.4(10)8

McA-RH7777 Preconfluent 70 79 4.7(10)8

Photomicrographs

Preconfluent or Randomly-Proliferating Culture

Confluent or Contact-Inhibited Culture

Proliferating vs. ConfluentHierarchical Clustering:Distance Metric - Euclidean, Complete LinkageFilter - Balanced Differential Expression >4-Fold

Results from GeneLinker TM Platinum

Female Cell Line vs. Male Cell LinesHierarchical Clustering:Distance Metric - Euclidean, Complete LinkageFilter - Balanced Differential Expression >9-Fold

Results from GeneLinker TM Platinum

Summary Similar gene expression profiles from randomly-

proliferating and contact-inhibited cultures were observed for Clone 9 and H4-II-EC-3 cells.-BRL3A cells resulted in a slightly different profile.

In comparisons of expression profiles from Clone9, H4-II-EC-3, BRL3A and McA-RH7777 cells, differences were observed in the female rat-derived cell lines versus the male rat-derived cell lines. McA-RH7777 cells were observed to have the highest differences

followed by BRL3A.

In Vivo 30-Day Study in Rat

*Genotoxic: Benzo(a)pyrene [BP]*Nongenotoxic: Acetaminophen [APAP] Clofibrate [CLO]*Control vehicle (DMSO)

14 287310.5

Days of Dosing-BP

APAPCLO

Days Post Last Dose

Dosing: BP-3 doses/wk, 2wks APAP, CLO-single dose

Timepoints: 12h, 24h, d3, d7, d14 and d28Tissues harvested: liver, kidney, lung, brain, pancreas and spleen

Hepatotoxins• metabolized by P450

• detoxification products:-glucuronides-sulfates-glutathione conjugates

• active metabolite: NAPQI

HO- -NHCOCH3

Acetaminophen:

Benzo(a)pyrene: •metabolized by P450 and epoxide hydrolase to two active metabolites:

-Bay area diol epoxide [BPDE] -K-region diol epxide [BP-4,5-oxide]•activation/detoxification•persistance of DNA adducts in liver out to 56 days

Hepatotoxins (Continued):Clofibrate:

•Peroxisome Proliferator Activating Receptor (PPAR) Inducer

-primarily PPAR--stimulates:-oxidation of fatty acids

insulin sensitivityglucose metabolism

-hyperlipidemia treatment•Induces Hepatocellular Carcinoma in Rodents

-nongenotoxic transformationstimulates cell growthsuppresses apoptosis

Cl- -OC(CH3)2COOCH2CH

3

Animal Monitoring Physical Signs:

-mild tremors, decreased activity, rigid body tone

-mortality=17% CLO, 6% APAP

Gross pathology at necropsy: -Liver: APAP>CLO>BP

-Spleen: BP>CLO>APAP

Liver to body weight ratios:

-significant increase with BP over time course

Serum enzyme levels:

-AST and ALT: increases in APAP, BP and CLO treatments (mostly at 12h, 24h)

GEMTM Results12 hr. B(a)P-Treated Male Rat Liver

GEM Results for Time Courses• Total number of genes:

APAP: 269BP: 146CLO: 271

• Total number of Incyte uniques:APAP: 69 (26%)BP: 37 (25%)CLO: 58 (21%)

Reproducibility Examples

Day BP1 BP2 CLO1 CLO2 APAP1 APAP2 0.5 1.9 2.0 2.9 2.9 3.0 3.7

1 5.2 3.8 7.5 7.1 1.7 2.7 3 1.4 1.4 -1.1 -1.2 5.0 6.0 7 2.6 2.9 1.1 1.1 1.3 2.0

14 1.9 2.0 ---- ---- 1.8 2.6 28 3.7 4.6 -1.2 -1.2 1.7 2.8

28

First Pair of cDNAs:Day BP1 BP2 CLO1 CLO2 APAP1 APAP2 0.5 1.2 1.0 2.9 2.9 11.1 3.9

1 11.7 11.8 7.5 7.1 29.1 36.8 3 1.1 1.0 -1.1 -1.2 6.0 5.0 7 -1.1 -1.3 1.1 1.1 1.1 1.3

14 10.8 12.9 ---- ---- 2.2 2.4 28 1.7 1.8 -1.2 -1.2 1.3 1.4

Second Pair of cDNAs:

Top 44 Expressed Genes – 12 Hr.

Possible Toxicity Markers

Genes

Bal

ance

d D

iffe

ren

tial

Exp

ress

ion

Distribution of N-Fold Scoring Genes

APAP

89 (28%)

B(a)P

98 (31%)

CLO

36 (11%)

9(3 %)5

(2 %)10

(3 %)

69

(22%)172 (54%) 186 (59%)

60 (19%)

Expression Patterns 3 compounds have different known modes of action:

-Resulted in different gene expression profiles-Some common genes induced but at different times

Expression Patterns Isozymes were expressed but did not always

result in same pattern of expression

Individual Rats vs. Pooled SampleHierarchical Clustering:Distance Metric - Euclidean, Average LinkageFilter - Balanced Differential Expression >6-Fold

DMSO-1DMSO-2DMSO-3 DMSO-PoolCLO-1CLO-2CLO-3CLO-Pool

Results from GeneLinker TM Platinum

Sex DifferencesHierarchical Clustering:Distance Metric - Euclidean, Average LinkageFilter - Balanced Differential Expression >8-Fold

Results from GeneLinker TM Platinum

Sex Differences-Proteomics•Approximately 72,000 features curated•967 features differ from normal by > 2-fold

• 599 from clofibrate treatment- approximately 50% annotated to date

Peroxidases / peroxisome proteins - confirm catalase, glutathione peroxidase- confirm glutathione sulfotransferases- confirm enol-CoA hydratase

Metabolic enzymes - confirm cytochrome P450Adducts - none cataloged to dateSex differentials - confirm estrogen metabolism enzymes

Performed in collaboration with Oxford GlycoSciences

Summary Different gene expression profiles were observed from

liver tissues of rat treated with B(a)P, APAP and CLO. Several genes whose function is not yet known (unique

genes) were shown to be highly expressed in tissues from all 3 compounds.

A wide range of gene expression was seen in arrays from individual rat samples versus arrays using pooled samples Pooled samples tended to give an average expression

profile-an average of each individual rat’s value

Differences in gene expression profiles were also found between female and male rats. Similar differences were also detected in proteomic

profiles.

Acknowledgements cDNA Library Construction

Laura Stuve Laura KamigakiAnne Curtis Glenn Fu and group

• GEM Design, Array Manufacture and HybridizationGary Zweiger Jeff SeilhamerScott Panzer Microarray DivisionOlga Bandman

Data Analysis MethodsRoland Somogyi Stefanie FuhrmanShoudan Liang