ToxicogenomicsToxicogenomics

Heather Handley

JP Student

ToxicogenomicsToxicogenomics

“… field of study that combines clinical, genomic, and proteomic data into a unified framework for understanding the biochemical and genetic basis for various diseases.” (Ballatori et al., 2003)

“… a new scientific field that elucidates how the entire genome is involved in biological responses of organisms exposed to environmental toxicants/stressors. It combines information from studies of genomic-scale mRNA profiling, cell-wide or tissue-wide protein profiling (proteomics), genetic susceptibility, and computational models to understand the roles of gene-environment interactions in disease.” (Selkirk and Tennant, 2002)

ToxicogenomicsToxicogenomics

PopulationGenomics

ComparativeGenomics

Transcriptomics(Gene Expression)

FunctionalGenomics

TOXICOGENOMICS&

PHARMACOGENOMICS

AgendaAgenda

MICROARRAY OVERVIEW

TOXICOGENOMICS EXAMPLES

Sequence Analysis

1. Pharmacogenomics and individualized medicine

2. Comparative and functional toxicogenomics

Gene Expression Profiling

3. Biomarkers of exposure and effect

4. Genomic approaches to study of toxic mechanisms

5. Toxicant “signature” profiling and predictive toxicology

• Monooxygenase enzymes responsible for metabolism of >80% of all clinical drugs and many organic pollutants

• Known roles in pollutant toxicity/carcinogenicity, drug-drug interactions, adverse drug effects, drug reactivity

• Large, complex gene superfamily … ~2,500 individual genes in bacteria, fungi, plants and animals

• Most animals have ~100 genes thought to be derived from a single common ancestor via extensive gene/genome duplication events

• Many inducible CYPs regulated by transcription factors in nuclear receptor or bHLH-PAS gene (super)families

Cytochromes P450Cytochromes P450CyCytochromes tochromes PP450450

Microarrays 101Definitions:• Platforms for massively parallel hybridization assays• High-density arrays of 100s to 1000s of probe-containing

features immobilized on a solid substrate

Terminology:Traditional hybridizations (e.g. Northern, Southern, Western):

target = immobilized sample (e.g.all RNAs/DNAs)probe = specific molecule of intrest in liquid phase

Microarrays:target = sample in liquid phaseprobes = molecules of interest immobilized on substrate

Microarrays 101• Probe type

– cDNA: gene expression profiling– Genomic DNA: CGH, ChIP-on-CHIP– Oligonucleotides

25-80mers

spotted or synthesized in situ (photolithography or inkjet)

– Proteins: enzyme activity, protein-protein interactions– Antibodies: protein expression

– Cells: biochemical functions, gene expression– Tissue sections (TMA): high-throughput ISH or IHC

Microarrays 101

• Substrate– Membranes (nylon, cellulose, etc.)– Coated glass slides

o Poly L-lysine -amino poly-silane (GAPS)o sugars

– Membrane-on-slide

• Probe density– Low-density macroarrays (10-100s of features)– Moderate-density microarrays (1,000s)– High-density microarrays (10,000s)

Microarrays 101

• Radioactive detection– Single sample per array

– Good sensitivity

• Fluorescent detection– CAN apply multiple samples per

array– Less sensitive but more

quantitative for changes

A BA + B

PharmacogenomicsPharmacogenomics

• Effect of polymorphisms on drug metabolism and toxic side effects of pharmaceutical agents

• Oligonucleotide arrays can be used to identify presence of specific alleles in individuals, or to quantify allele ratios in populationse.g. Affymetrix CYP chip (18 known mutations defining 10 alleles of CYP2D6 and 2 alleles of CYP2C19)CYP2D6 poor metabolizer genotypes protect against hepatitis C & cyrrhosis progression

• Microelectronic arrays can improve sensitivity/accuracy in detecting single nucleotide differences

The Thousand The Thousand Dollar GenomeDollar Genome

Genome Resequencing Technologies• Sequencing by hybridization

• Solid-phase multiplex PCR

• Solexa TotalGenotyping with Single Molecule Arrays

• Single molecule sequencing– Protein nanopore

– U.S. Genomics

Comparative Comparative ToxicogenomicsToxicogenomics

• Species differences may exist at the level of gene complement, enzyme function (coding sequence), or transcriptional regulation (flanking genomic or intronic sequences)

• Improved homology searching tools (combined with gene prediction) can be used to detect all members of a gene (super)family in a given genome

• Evolutionary analyses full gene complements facilitate distinction of orthologous and paralogous relationships in large gene superfamily

FunctionalFunctionalToxicogenomicsToxicogenomics

• Species comparisons can be used to identify regions of functional constraint or positive selection

• Algorithms for motif detection can be used to predict regulatory elements (and possibly transcription factors)

• cDNA microarrays can be used to assess gene expression

• Antibody arrays can be used to assess protein levels

• Protein arrays can be used to assess enzyme function

Biomarker Arrays

• Biomarker = biological response (ideally quantifiable) to an environmental chemical, which provides a metric of exposure and sometimes toxic effect(s); may be at the molecular, cellular or whole organism level.

• Multiple-gene biomarkers are likely to be more sensitive

and discriminating than single genes

e.g. Larkin et al. (2003) macroarrays for assessing exposure of fish to estrogenic compounds

– Cost-effective– Well implemented– Non-model organism

Genomic ApproachesGenomic Approachesto Toxic Mechanismsto Toxic Mechanisms

• Toxicity of many compounds due to altered transcriptional regulation of gene expression

• Gene expression profiling provides opportunities to identify affected molecular pathways and cellular functions

• Platform(s) cDNAs for oligonucleotides on glass slides

• Competitive hybridization with two-channel fluorescent detection used to compare gene-specific relative expression levels between two conditions(except Affymetrix)

AHR signaling and Dioxin Toxicity

? AHR Gene Battery• Cytochrome P450 1A (CYP1A)• Glutathione S Transferase

OncogenesCytokines

Estrogen-responsive genes

TOXICITY

AHR-ARNT*

Connective tissue

Smooth Muscle

Endothelium

Blood (>2 cell types)

Bulbus arteriosus Ventricle

Atrium

Sinusvenosus TISSUE

• 4+ cell-types• 10,000 genes?

cDNA LIBRARY • 76,800 clones

– Unsequenced– Redundant

““Adult Heart” LibraryAdult Heart” Library

4,896 AH clones

+ ~100 controls/genes

of interest

5,186 features5,186 features

(~2,500 genes?)(~2,500 genes?)

Adult Heart MicroarraysAdult Heart Microarrays

Gene (mRNA)Expression Profiling

Control Sample

Prepare total RNA

Generate amino-allylmodified cDNA

Label with fluorescentgreen dye (Cy3)

Experimental Sample

Prepare total RNA

Generate amino-allylmodified cDNA

Label with fluorescentred dye (Cy5)

MicroarrayAnalysis

More ExpressionIn Experimental

Equal Expression

More ExpressionIn Control

Control

Experimental

Data AnalysisData Analysis

2-fold

CYP1A

Dioxin-responsive genesDioxin-responsive genes

GENERAL TRENDS• 361 clones differentially

expressed (p-value 1*10-4)– 6 previously identified clones– 99 assembled into 17 contigs

(15 known genes, 2 ESTs)– ~75 low quality sequence

• Predominantly induction

• Low-dose responses prevalent

SUPPRESSION

INDUCTION

159 9441

0.5nM TCDD 5nM TCDD1

54 58

Chemical Profiling Chemical Profiling and Predictive Toxicologyand Predictive Toxicology

• Diagnostic features determined from training set of compounds with known mechanisms of action

• Methods for determining diagnostic subset include:– Self Organizing Maps (SOMs) and Neural networks– Bayesian statistics

• Caveats– Must be certain about mechanisms of training compounds

– Can only distinguish states represented in training set

Thomas et al., 2001

Thomas et al., 2001