Genotoxicity assessment of the flavouring agent, perillaldehyde
Biomarker for genotoxicity 2013
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Transcript of Biomarker for genotoxicity 2013
Sample & Assay Technologies- 1 -For Internal Use Only Sample & Assay Technologies
Pathway-Powered PCR Arrays
Bill Wang, Ph.D.QIAGEN
Discovery & Validation of Biomarkers to Monitor Genotoxicity by Gene Expression Profiling
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Table of Contents
1. Introduction to Genotoxicity
2. Development of Gene-Based In Vitro Genotoxic Biomarkers Using PCR Arrays
• Introduction to RT2 Profiler PCR Array• Experimental Design• Results
3. Identifying miRNA-Based In Vivo Biomarkers to Monitor Genotoxicity in Mouse
• Introduction to miScript PCR Array• Experimental Design• Results
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Drug Development At A Glance
7 millions compounds screened
1000 hits
12 candidates
6 candidates 1 product
Early Discovery Phase Exploratory DevelopmentPhase I, II
Clinical Development Phase III
0 5 10 15yr
12 to 24 years
Cost: ~ $900 millionMarketed Drug
Idea
Efficacy and Safety
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The inability to accurately predict toxicity early in drugdevelopment cost the pharmaceutical industry billio ns and approximately one-third the cost of all drug failur es.
Drug Discovery Today 2007 12:289-91
Why Toxicity Is Concerned
Reasons For Failure During Drug Discovery & Develop ment Process
FAIL IT EARLIER, FAIL IT CHEAPER !
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RenalToxicity
GenoToxicity
CardioToxicity
ImmunoToxicity
PulmonaryToxicity
NeuroToxicity
HepatoToxicity
Current Hot Spots in Toxicity Risk Assessment
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Classification of Carcinogens
Genotoxic carcinogens:Cause irreversible genetic damage or mutation by binding to DNA.
Direct: DNA cross-linking. DNA adduct formation.Indirect : Interfere with the function of proteins that are involved in DNA replication or chromosome stability.
Non-genotoxic carcinogens:Don’t directly affect DNA but act in other ways to initiate, promote or aggravate tumor development: multiple and diverse mechanisms.
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Current Testing Battery For Genotoxicity
Bacterial mutagenesis (Ames Test)In vitro mammalian mutagenesis In vitro chromosome aberration assayIn vitro micronucleus assay In vivo chromosome stability assay2 year rodent carcinogenicity test.
Accuracy: 38%, with high false positive rate.
High toxic dose (50-80% growth inhibition) used --- low sensitivityDifficulty at prediction of carcinogenicity at low doses to which human subjects are usually exposed.
For non-genotoxic carcinogens: no suitable model av ailable.
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Gene Expression Regulates Biology
All require molecular signaling for action
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Gene expression profiles should be profoundly diffe rent between genotoxic and non-genotoxic carcinogens.
Gene expression profiles should be able to discrimi nate among compounds having different mechanisms.
The toxicity of unknown compounds can be predicted by comparison of their molecular fingerprints with tho se obtained with compounds of known toxicity.
Gene Expression And Genotoxicity
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Biological Markers Define Processes
Cell cycle Angiogenesis
P53MDM2CyclinsCDKs
VEGFbFGF
ANGPTPDGF
IL8TLRIFNγTNFβIn
divi
dual
pa
rtic
ipan
ts
Inflammation
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Pat
hway
Cell cycle Angiogenesis Inflammation
Complete Biological StoryBuilt on Pathway / Network Analysis
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Q: How to Assess the Expression of Different mRNAs in a Sample involved in a Pathway and Compare it across Multiple Conditions?
A:
Differential Gene Expression Analysis
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Table of Contents
1. Introduction to Genotoxicity
2. Development of Gene-Based In Vitro Genotoxic Biomarkers Using PCR Arrays
• Introduction to RT2 Profiler PCR Array• Experimental Design• Results
3. Identifying miRNA-Based In Vivo Biomarkers to Monitor Genotoxicity in Mouse
• Introduction to miScript PCR Array• Experimental Design• Results
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Real-Time PCR
• Amplify & simultaneously quantify target DNA
Reverse Transcription Real-Time PCR
• Amplify & simultaneously quantify messenger RNA (mRNA)
Ct Values
• Threshold Cycle
Principles of qRT-PCR in PCR Arrays
Sample & Assay Technologies
• 84 Pathway-Specific Genes of Interest
• 5 Housekeeping Genes– Normalization
• Genomic DNA Contamination Detection Assay
• Reverse Transcription Controls (RTC) n=3– Spike-in control
• Positive PCR Controls (PPC) n=3
Anatomy of a PCR Array
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Exquisite Specificity
Conclusion: Each well in a PCR Array detects a single gene-specific product.
• Assays on Human TGFβ & BMP Signaling Pathway PCR Array used to analyze BMP genes in Universal Reference RNA.
• Single peak dissociation curves
• Single gel bands of predicted size
• High specificity for genes difficult to design specific primers for.
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Uniform Amplification Efficiency
CONCLUSION: Accurate and reliable ∆∆Ct results are guaranteed.
Representative set of 500 out of > 4,000 assays used in PCR Arrays
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Reproducibility Among Different Instruments
Human Drug Metabolism PCR Array in Universal Reference RNA
Conclusion: Same raw threshold cycle data can be obtained for the same samples even from different end users at different times or using different instruments .
Reproducibility Among Different Users
High Reproducibility
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Definition of “PATHWAY-FOCUSED” Analysis
“Pathway-Focused” describes examination of:
• biological signaling events,• classes of genes, or • those genes related to diseases.
���� PCR Arrays allow for easy & simultaneous analysis o f a Pathway-Focused set of genes.
Sample & Assay Technologies
** Over 150 Pathway- Powered PCR Arrays Available**
Cancer and Apoptosis Cytokines & Inflammation Develop ment & Stem Cells
Apoptosis Inflammatory Cytokines Stem Cells
Cell Cycle Th17 for Inflammation WNT Signaling / Notch Signaling
Human miRNA Array Common Cytokines / Chemokines Terminal Differentiation Markers
Breast Cancer & Estrogen Receptor Inflammasomes TGFβ / BMP Signaling
Tumor Metastasis NF-kB Signaling Pathway Endothelial Cell Biology
Epithelial-to-Mesenchymal Transition Th1-Th2-Th3 Osteogenesis
Angiogenesis TNF Ligands Growth Factors
Cancer Drug Resistance Toll-like Receptors ECM & Adhesion
Signal Transduction Toxicology & Drug Metabolism Neur oscience
Signal Transduction PathwayFinder Drug Metabolism / Drug Transporters Neuroscience Ion Channels
NFkB Signaling Drug Phase I Enzymes Neurotransmitter Receptors
Jak / Stat Signaling Molecular Toxicology PathwayFinder 384HT Neurotrophins & Receptors
DNA Damage Signaling Oxidative Stress Neurogenesis and Neural Stem Cell
Insulin Signaling Stress & Toxicity Parkinson’s Disease
MAP Kinase Signaling Other Diseases Custom PCR Arrays (H/M/R/Q/D/F/P/B)
cAMP / Calcium Signaling Atherosclerosis 96-Well, 384-Well Plate
p53 Signaling Diabetes 100-Well Disc, 96x96 Chip
PCR Arrays for ALL Biomedical Researchers
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.Biologically relevant gene content• Not simply biochemical pathways or kinase
cascades• Published association with the biological or diseas e
pathway gathered from overlapping sources, including:
� Multiple Publicly Accessible Databases� Text Mining Relevant Literature
Genes in Signaling Pathways
Genes with High Relevance
Technically relevant gene content • Use genes that are regulated at the mRNA level• Specific feedback from thought leaders
How Genes on PCR Arrays Are Selected
Human DNA Damage Signaling Pathway RT 2 PCR Array• Genes involved in DNA damage, repair, cell cycle
control, and apoptosis. • Specific feedback from thought leaders
Custom PCR Array• Additional tentative signature genes derived from
various microarray studies (GEO) in the context of genotoxicity.
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Development of Genotoxic BiomarkerCompound Selection
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Development of Genotoxic BiomarkerStudy Design
.Cells:
.HepG2 cells: 2 X 105 cells/ml at seeding.
.96-well plate for cytotoxicity assays --- 3 to 5 biological replicates.
.6-well plate for RNA isolation --- 4 biological replicates.
.Dose:
.IC20 - low dose but substantial to trigger gene expression changes.
.Time:
.24 hr – to avoid generic stress responses often observed at shorter incubation time (e.g. 3 hr or 6 hr).
.RNA Isolation and PCR Array Analysis:
.RT2 PCR Array protocols.
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Non-Genotoxic Genotoxic
Upload & Analyze Data15 minutes
RNA Isolation (RNeasy)RNase-Free DNase Treatment
RNA IsolationAutomatedSolutions
QIAxcel TL IIQIAcube
How RT 2 Profiler PCR Arrays Work
Cells & Tissues RNA (25ng- 5ug)*
1st strand cDNA
~45 minutes
gDNA Elimination Step
First Strand cDNA SynthesisArtificial RNA Spike In (RTC)
*RT2 PreAMP for 1 ng
Real-time PCR Detection of 89 Gene-specific Amplification on
RT2 Profiler PCR Array2 hours
+ SYBR Green Master Mix RGQ
Experiment:
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• Gene Content for each Pathway is Pre-Loaded• Custom Arrays: Gene List is all you need
• From Raw C t Values to Fold Change Results in Multiple Analysis Formats
Volcano Plot Scatter Plot Clustergram 3-D Histogram
RT2 Profiler ™ PCR Array Data AnalysisFREE Complete & Easy Analysis with Web-/Excel-Based Software
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Expression Profiles of 11 Signature Genes
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Gene Signatures Define Compound Class
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Expression Profiles From Different Modes of Action
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Summary 1
.We described the concept of pathway focused gene ex pression analysis using RT 2 Profiler PCR Arrays.
.We identified 11 genes in DNA damage repair and p53 pathways as a classifier for genotoxic and non-genotoxic com pounds.
.Genotoxic compounds with different modes of action elicit distinct gene expression profiles.
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Table of Contents
1. Introduction to Genotoxicity
2. Development of Gene-Based In Vitro Genotoxic Biomarkers Using PCR Arrays
• Introduction to RT2 Profiler PCR Array• Experimental Design• Results
3. Identifying miRNA-Based In Vivo Biomarkers to Monitor Genotoxicity in Mouse
• Introduction to miScript PCR Array• Experimental Design• Results
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miRNA Biology
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miRNAs as Master Regulators
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miScript miRNA PCR Array SystemmiRNA Pathway & Whole miRNome Profiling
.PCR Arrays: Pathways and Genomes� Serum (Circulating Disease) – New!� Neurological Development & Disease� Brain Cancer� Cell Differentiation & Development� Immunopathology� Inflammation� miFinder� Whole Genome (miRNome): Human, Mouse, Rat, Dog
miRNA Isolation & cDNA Synthesis
Universal Primer to 40 cycle qPCR
Accurate Results Easy Data Analysis
.Complete Workflow Solution for miRNA Expression Ana lysis
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.Goal: explore miRNAs as potential biomarkers for ge notoxicity
.Subject: 6-month old female mice
.Compound: N-ethyl-N-nitrosourea (ENU) 120mg/kg body weight
.Time course:
.Sample source: miRNA from liver
.Analysis: miRNA PCR Array for 384 most expressed mi RNAs
Days
1 3 7 15 30 120
1 30
ENU
DMSO
Mice Days
1 3 7 15 30 120
1 30
Days
1 3 7 15 30 120
1 30
ENU
DMSO
Mice
miRNA Profiling After Exposure To Genotoxic Carcino genStudy Design
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Temporal Changes In Differentially Expressed miRNA
BMC Genomics 2010, 11:609
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Clustering Of Differentially Expressed miRNAs
BMC Genomics 2010, 11:609
mir-34 family
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Temporal Changes Of mir-34 Family
01 3 7 15 30 1201
2
4
8
16
** *
*
mmu-miR-34a
01 3 7 15 30 1201
2
4
8
16
*
**
**
mmu-miR-34a
Fol
d ch
ange
s
Days after ENU treatment
PCR array TaqMan individual
01 3 7 15 30 1201
2
4
8
16
*
** *
*
mmu-miR-34b-5p
01 3 7 15 30 1201
2
4
8
* mmu-miR-34b-5p
01 3 7 15 30 1201
2
4
8
16
* **
*
mmu-miR-34c
01 3 7 15 30 1201
2
4
8
*
mmu-miR-34c
01 3 7 15 30 1201
2
4
8
16
** *
*
mmu-miR-34a
01 3 7 15 30 1201
2
4
8
16
** *
*
mmu-miR-34a
01 3 7 15 30 1201
2
4
8
16
*
**
**
mmu-miR-34a
01 3 7 15 30 1201
2
4
8
16
*
**
**
mmu-miR-34a
Fol
d ch
ange
s
Days after ENU treatment
PCR array TaqMan individual
01 3 7 15 30 1201
2
4
8
16
*
** *
*
mmu-miR-34b-5p
01 3 7 15 30 1201
2
4
8
16
*
** *
*
mmu-miR-34b-5p
01 3 7 15 30 1201
2
4
8
* mmu-miR-34b-5p
01 3 7 15 30 1201
2
4
8
* mmu-miR-34b-5p
01 3 7 15 30 1201
2
4
8
16
* **
*
mmu-miR-34c
01 3 7 15 30 1201
2
4
8
16
* **
*
mmu-miR-34c
01 3 7 15 30 1201
2
4
8
*
mmu-miR-34c
01 3 7 15 30 1201
2
4
8
*
mmu-miR-34c
BMC Genomics 2010, 11:609
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Top 10 Biological Functions Implicated By Different ially Expressed miRNA
Differentially expressed miRNA at day 7 and 15
Top 5% of target genes
Target prediction algorithm
Ingenuity Pathway analysis
BMC Genomics 2010, 11:609
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Summary 2
.We introduced the concept of miRNA and miRNA profil ing using miScript PCR Arrays.
.Exposure to carcinogen (ENU) elicits different miRN A profiles in mouse liver.
.miRNA profiles have the potential to serve as bioma rkers for genotoxicity.
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Take Home Message
.Gene expression not only regulates biology, but als o serves as a surrogate to monitor biology.
.Pathway focused analysis using PCR Array is an easi ly accessible and accurate platform for biomarker iden tification and mechanistic studies.
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Pathway-Focused Expression AnalysisComplete Experimental Systems: Sample Prep through Data Analysis
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