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Toxicogenomic Comparison of Rat Strains and Gender in an Acute Model of Cisplatin Induced Renal Injury

Joe Milano, MS, DABTAstraZeneca Pharmaceuticals

Global Safety AssessmentWilmington, Delaware

Outline

• Microarray analysis (transcriptomics)• What do microarrays really measure?

• Assumptions

• Rat Strain Study• Experimental design

• Data analysis work flow

• Pathway analysis

• Similarities in response

• Differences

• Individual gene responses in the Xenobiotic and Transporter ontologies.

What do Expression Arrays Really Measure?

• Gene Expression• mRNA levels in a cell• mRNA levels averaged over a population of cells in a sample• relative mRNA levels averaged over populations of cells in

multiple samples• relative mRNA hybridization readings averaged over

populations of cells in multiple samples

Ontario Genomics Innovation Centre

Central Assumption of Gene Expression Microarrays

• The level of a given mRNA is positively correlated with the expression of the associated protein.• Higher mRNA levels mean higher protein expression,

lower mRNA means lower protein expression

• Other factors:• Protein degradation, mRNA degradation, polyadenylation,

codon preference, translation rates, alternative splicing, translation lag, miRNA control

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Rat Strain Comparisons

Background

• Strain and gender differences in preclinical species may effect the synthesis of ADME and tox studies.• Safety Assessment at AstraZeneca traditionally uses the Han Wistar rat

for tox while DMPK employ the Sprague Dawley rat for ADME.

• This study attempts to address the differences among three rat strains using an acute dose model with well characterized toxicants and analyzing the transcript profiles of the primary organs of toxicity.• Doxorubicin• Heart

• Isonaizid• Liver

• Cisplatin• Kidney

Charles River Rat Strains Background

• Han Wistar - outbred strain• Origin traces back to the Wistar Institute in Philadelphia and refers to

animals whose origins can be traced back to Wistar animals maintained at the Hanover Institute in Germay.

• Charles River’s Han Wistar colony originated in1996.

• Sprague Dawley – outbred strain• Developed by Robert W. Dawley in about 1925 at the University of

Wisconsin.

• Charles River obtained a foundation stock from the Sprague-Dawley Company in 1950

• Fischer 344 – inbred strain• Developed by Curtis and Bullock at Columbia University in 1921

• 1960 transferred to Charles River and rederived and maintained as a continuously inbred strain since that time.

Lange, PL and Williams, WJ (1994) Pathobiology of the Ageing Rat. Volume 2

Cisplatin

• Cisplatin is a platinum-based chemotherapeutic used to treat various types of cancers.• Platinum complexes react in vivo binding to and causing DNA damage

that elicits DNA repair mechanisms, which in turn activate apoptosis when repair proves impossible.

• Approved for clinical use by the FDA in 1978

• Nephrotoxicity is manifested by renal tubular damage resulting in an elevation of the BUN, serum creatinine, decrease in creatinine clearance and glomerular filtration rate. Dose limiting.

Study Design

• This acute dose study consisted of male and female Han Wistar, Sprague Dawley and Fischer rats dosed IP with 5mg/kg or 10mg/kg cisplatin.

• Kidneys were harvested 24 hours after a single administration of drug.• Pathology and clinical chemistry measures were assessed

• The whole organ was homogenized for total RNA isolation then processed for Affymetrix analysis using the Rat 230 2.0 microarray.

• Expression data was normalized using PLIER and filtered base on minimum raw intensity of 50 units.

Analysis Workflow

QC performed on data collected for 54 kidney samples. Profiles normalized to the median for each gene.

All experimental groups compared using GeneSpring GX10 1-Way ANOVA p= 0.01 with Bonferoni adjustment N = 3

Analysis yielded 6564 genes.

List was used for hierarchical clustering of individual samples.

Enrichment analysis on lists grouped by gender to investigate similarity of response between females and males.

Gene lists were analyzed using GeneGo’s MetaCore platform

Transcript profiles of responding animals were identified and compared within experimental groups. i.e. Male Han Wistar, Female Fischer, etc. 1-Way ANOVA

Enrichment analysis on lists of individual experimental groups to investigate differences in response.

Pearson Correlation of all samples

• Raw gene expression data was filtered base on minimum raw intensity of 50 units then 18 experimental groups were analyzed using 1-Way ANOVA p= 0.01 with Bonferoni FDR.• Analysis yielded 6564 differentially

expressed transcripts.

• Many genes but remember there are 3 variables driving this – gender, dosing and strain

• This gene list was then used in a Pearson Correlation of individual kidney transcript profiles.• Individual profiles separate by

gender, cisplatin treatment and strain.

• Note the dosed samples that cluster with vehicle controls

Analysis by treatment groups – strain and Gender

• Responding animals and vehicle controls from each treatment group - male Han Wistar, Female Sprague Dawley were compare by 1-way ANOVA N= 2-3.• Enrichment analyses were performed in MetaCore to understand shared

pathways and toxicity networks by gender.

• Gene list differences for strains were separated by Venn diagram and analyzed.

• These tools suggest interacting networks and pathways that may be related to mechanism by transcript abundance.• Network – an interactive map that is drawn based on interactions that

have been curated from the literature

• Pathway – well established biochemical or signal transduction map. Eg. Insulin pathway, apoptosis pathway

• MetaCore has several analysis options.• Enrichment analysis with a 1.5 fold cut off.

MetaCore Toxicity Networks Enrichments

• In the absence of pathology, toxicity endpoint analyses show similar enrichment profiles for each experimental group related to the mechanism of cisplatin pharmacology.

MetaCore Pathway Maps Enrichments

• Female and male gene lists share 1 pathway• Signal transduction AKT signaling

Male Female

AKT Signaling is Common to Males and Females

Signal transduction AKT signaling

• p21- interacts with and inhibits the essential DNA replication factor, proliferating-cell nuclear antigen PCNA.

• MDM2- part of an autoregulatory negative feedback loop that is a transcriptional target of, and binds to and inhibits transcription factor p53.

• BAX – causes the opening of mVDAC which leads to the loss in membrane potential and the release of cytochrome c – apoptosis.

• PCNA - helps increase the processivity of DNA replication. In response to DNA damage, this protein is ubiquitinated and is involved DNA repair.

GeneGo Pathway Maps Enrichments

• Male specific pathway – DNA damage ATM/ATR regulation

• Female specific pathway – FAS signaling

Male Female

DNA Damage ATM/ATR Regulation of G1/S Checkpoint

• MDM2- part of an autoregulatory negative feedback loop that is a transcriptional target of, and binds to and inhibits transcription factor p53.

• p21- cyclin-dependent kinase inhibitor. Binds to and inhibits the activity of cyclin-CDK2 or -CDK4 and regulates cell cycle progression at G1

• C-Myc – protooncogene involved in cell cycle progression.

• PCNA - increases the processivity of DNA replication. In response to DNA damage, this protein is ubiquitinated and is involved DNA repair.

• GADD45 – growth arrest and DNA damage inducible protein.

Males

Apoptosis Signaling Cascade Fas Signaling

• The extrinsic apoptotic pathway, induced by FasR can crosstalk to the intrinsic pathway through the caspase-8-mediated cleavage of BID.

• BRE – may act as a death receptor-associated anti- apoptotic protein, which inhibits the mitochondrial apoptotic pathway.

• RAIDD - death domain containing protein and has been shown to induce cell apoptosis.

• BAX – causes the opening of mVDAC which leads to the loss in membrane potential and the release of cytochrome c – apoptosis

Venn Diagrams for males and females

• 72 genes were common for all male treated groups.

• 40 genes were common for all female treated groups

• Enrichment analysis of unique genes

Male Unique Genes

Male Fischer324 Genes

Distributions are driven by PKC, IP3R and PLC beta

Male Sprague768 Genes

Male Han Wistar206 Genes

Female Unique Genes show unique pathways

Female Han Wistar207 Genes

Female Sprague166 Genes

Female Fischer352 Genes

Effects on Transcripts in Transporters and Xenobiotic Metabolism Ontologies

• In general transcript data for males show greater induction or repression of several genes in the Transporter and Xenobiotic ontologies.

KidneyToxicity Panel

• Analytes from the rat renal tox panel show differences in transcript and urine protein for GSTYb1, alpha GST and Kim1.

• Kim1 transcript induction was mirrored by >2-fold increase in urine protein in Han Wistar males and females and Fischer males.

Summary• Pearson correlation shows greatest influence on sample separation by

gender followed by strain and cisplatin dose.

• In the absence of pathology, enrichment profiles show induction of DNA damage and apoptosis pathways related to the mechanism of cisplatin pharmacology.• Overlap of p21, MDM2, Bax and PCNA strongly suggest cell cycle arrest and

apoptosis

• Induction of GADD45 alpha and beta suggest cellular response to DNA damage.

• Strains responded similarly but showed different degrees of enrichment for common genes.• Strain to strain variability

• ADME

• Timing – this is a transcriptional snapshot in an acute dose model.

• Unique gene list analysis suggests difference in response of metabolic processes.

Conclusions

• Similarities in response to cisplatin are based on the enrichments for pathways involved in DNA damage and apoptosis directly related to known pharmacology.

• Differences in response to cisplatin are based on the magnitude of gene induction or repression of transcripts for both stain and gender.

Caveats when working with pathway analysis tools

• Redundancy

• Redundancy

• Redundancy• Specific sets of genes show multiple pathway and network enrichments

• IP3R, PKC and PLC beta and GnRH signaling in the kidney?

• Known Knowns• Information within the database is gleaned from the literature

• Known Unknowns• Novel interactions that are not in the literature will not be rendered

• Unknown Unknowns• Unknown ?

• Eg miRNA

Acknowledgements

• Yvonne Dragan• Omics enabler

• Brandon Jeffy• Experimental Design

• David Brott• Clinical Chemistry

• Trish Bentley• Urine protein biomarkers

• Martin Dyroff• $upport

Posters Wednesday Afternoon

Influence of Rat Strain and Gender on Toxicological Response to Doxorubicin

B. Jeffy, J. Milano, D. Brott, and Y. Dragan

Abstract 1642

Poster Board 430

Toxicogenomic Comparison of Rat Strains and Gender in and Acute Model of Cisplatin Induced Renal Injury

J. Milano, B. Jeffy, D. Brott, and Y. Dragan

Abstract 1641

Poster Board 429