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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: [email protected]
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