Toxicogenomic Investigation into False Positive Responses in the Local Lymph Node Assay (LLNA) Darrell Boverhof, Ph.D. Toxicology & Environmental Research and Consulting (TERC) The Dow Chemical Company Midland, MI Rboverhof@dow.com 1
Acknowledgements Study supported by: DOW CEFIC-LRI David Adenuga Michael Woolhiser Bhaskar Gollapudi Lindsay Sosinski Rachel Golden University of Manchester, UK Ian Kimber Rebecca Dearman The Hamner Institutes for Health Sciences Russell Thomas Michael Black 2
Background- Allergic Contact Dermatitis GPMT (Guinea pig maximization test) LLNA Proliferation/memory T-cells 3 http://sensitive-learning.eu/mod/resource/view.php?id=77
Background- Local Lymph Node Assay Assay which detects the contact sensitization potential of chemicals (sensitization phase) Dorsal 3 H-thymidine Incorporated 3 H-thymidine Incorporated surface of into DNA of dividing cells into DNA of dividing cells Sensitization / Sensitization / ears Lymph Node Lymph Node Proliferation Proliferation Rest Rest 5 hours 5 hours Days 4, 5 Days 4, 5 Treat Treat Inject 3 H-thymidine Inject 3 H-thymidine Remove Lymph Nodes Remove Lymph Nodes Days 1, 2, 3 Days 1, 2, 3 Day 6 Day 6 Day 6 Day 6 Auricular Nodes Auricular Nodes Reported as a stimulation index (SI) Prepare Single, Prepare Single, Ratio Treatment/Vehicle Cell Suspension Cell Suspension Cutoff for positive response SI >= 3 Measure 3 H-thymidine Incorporation Measure 3 H-thymidine Incorporation 4
The Problem of False Positive Chemistries in the LLNA
The Problem of False Positive Chemistries in the LLNA Can differential gene expression responses (toxicogenomics) in the lymph node be applied to distinguish true sensitizers from false positives in the LLNA? Functional insights into different mechanisms Development of molecular classifiers to distinguish between these two classes 6
Study Approach Critical Elements: Chemical Selection Selection of false positive chemistries # of Chemistries Dose All chemicals tested at equipotent doses in LLNA Time Examined multiple time points Comprehensive Anchor gene expression responses to traditional LLNA endpoint 2 Phases- Development and Confirmation 7
Study Approach- Comprehensive Phase 1 9 Sensitizers 7 False positives Functional evaluation of differential gene expression Whole genome array Molecular classifier development and assessment Phase 2 6 Sensitizers 6 False Positives Confirmation of functional gene expression responses Focused QRTPCR arrays Evaluation of classifier performance 8
Phase 1 Test Materials Sensitizer Class Test Material Vehicle Dose Dinitrochlorobenzene (DNCB) Acetone 0.10% Hexylcinnamic aldehyde (HCA) Acetone 25% Isoeugenol Acetone 10% para -phenylene diamine(PPD) Acetone 1% Sensitizers Hydroquinone (HQ) Acetone 0.25% Methyldibromo glutaronitrile (MDBGN) Acetone 20% Toluene diisocyanate (TDI) Acetone 0.04% Trimellitic anhydride (TMA) Acetone 0.65% Ammonium Hexachloroplatinate (AHCP) DMSO 0.70% Oleic acid DMSO 50% Maleic acid DMSO 11.50% Sodium lauryl sulphate (SLS) DMSO 25% Presumptive Tetraethylene glycol Monotetradecyl ether (TGME) Acetone 20% False Positives Polyaminofunctional siloxane Acetone 45% N-decylphenol polyethyleneglycol ether (DPP) Acetone 35% Hexadecan-1-ol Ethoxylated (EO2) C16 (HDE) Acetone 30% Equipotent doses (SI 6-9) calculated based on results from screening LLNA 9
Phase 1- Stimulation Index response 10
Phase 1- Toxicogenomic evaluation Test materials grouped into a 3 class coding scheme – Vehicles controls Isolate Auricular nodes Isolate Auricular nodes Sensitizers False positives Isolate RNA Isolate RNA Isolate RNA Cy3 Cy3 Cy3 Cy3 Reverse Reverse Reverse Data filtered on expression ( ± 1.5 Transcription Transcription Transcription FC) and 2 way ANOVA linear contrasts (FDR < 0.05) Hybridize on array Hybridize on array Commonly regulated genes Responses unique to Sens and FP Functional evaluation- Gene Data Extraction and Data Extraction and Analysis Analysis Ontology 11
Toxicogenomic evaluation Commonly Expressed Genes Genes similarly regulated by sensitizers and false positives relative to controls Functional Categories involved in Cell Proliferation Initiation of mitosis Cell cycle regulation The metaphase checkpoint Chromosome condensation in prometaphase Spindle assembly and chromosome separation o Indicates both Sensitizers and False positives induce a LN proliferative response o Offer no ability to discriminate between sensitizers and false positives 12
Toxicogenomic evaluation Sensitizer-Specific Genes 4 Genes Fxyd4 Thbs4 differentially Normalized Mean Intensity (Log 2 ) Cphx Lgals7 regulated by 2 Il21 Sens relative to both FPs and Controls 0 -2 Vehicles Sensitizers False Positives -4 Untreated Acetone DMSO AHCP TDI TMA DNCB HCA HQ Isoeugenol PPD MDBGN DPP HDE Siloxane TGME Maleic Oleic SLS Key functional categories – 1. Positive regulation of immune system process 2. Leukocyte activation and migration 13
Toxicogenomic evaluation False Positive-Specific Genes Genes differentially regulated by FPs relative to both Sens and Controls Key functional categories – 1. Acute inflammatory response 2. Innate defense response (Neut/Mac markers – Mpo, Lcn2) 14
Phase 2 Test Materials Sensitizer Class Test Material Conc. DNCB 0.05% Benzoquinone (BZQ) 0.10% 2-hydroxyethyl acrylate (2-HEA) 20% Sensitizers Phenyl acetaldehyde (PA) 20% Citral 30% Propyl Gallate (PG) 3% DPP 35% Benzalkonium Chloride (BZC) 1% Presumptive Squalene 50% False Positives Methyl Oleate (MO) 50% Hexaethylene glycol monododecyl ether (HGME) 25% Linolenic Acid (LA) 50% Equipotent doses (SI 6-9) calculated based on results from screening LLNA 15
Phase 2- Stimulation Index response SI=3 Stimulation Index 10 15 0 5 Acetone DNCB Sensitizers BZQ 2-HEA PA Citral PG DPP False Positives BZC Squalene MO HGME LA
Phase 2 Sensitizer-Specific Genes 15 Cphx Il21 Lgals7 12 Thbs4 Fold change to Veh 9 6 3 0 DNCB BQ 2-HEA PA Citral Propyl galate DPP Benzalk Cl Squalene Me oleate HGME Linoleic acid Sensitizers False Positives Key functional categories – 1. Positive regulation of immune system process 2. Leukocyte activation and migration 17
Phase 2 False Positive-Specific Genes 18 Cd5l 15 Dao Defa-rs2 Il12b Fold change to Veh Syn3 12 9 6 3 0 DNCB BQ 2-HEA PA Citral Propyl galate DPP Benzalk Cl Squalene Me oleate HGME Linoleic acid Sensitizers False Positives Key functional categories – 1. Acute inflammatory response 2. Innate defense response (Neut/Mac markers – Mpo, Lcn2) 18
Development of Statistical Classifiers- Phase 1 Total of 50 iterations Gene Expression Microarrays Select Features Averaged parameter values Build Partition Set 1 Set provide broad- Model Aside Data into 5 based evaluation Sets Predict of predictive Hold- performance Out Set Accuracy Sensitivity Specificity AUC Repeat 10X – Tested in a total of 84 optimized models 19
Development and Evaluation of Statistical Classifiers PHASE 1 30 genes PHASE 2
Summary/Conclusions Sensitizer- and False Positive-specific gene expression responses were identified Sens- antigen-mediated T-cell response. FPs- consistent with a pro-inflammatory response Class-specific gene expression responses were reproducible in an independent experiment Molecular classifiers were developed that had very good performance Genes that made up the classifier were consistent with those identified through the functional analysis Approach could be used as part of a WoE analysis for suspected false positives 21
Acknowledgements Study supported by: DOW CEFIC-LRI David Adenuga Michael Woolhiser Bhaskar Gollapudi Lindsay Sosinski Rachel Golden University of Manchester, UK Ian Kimber Rebecca Dearman The Hamner Institutes for Health Sciences Russell Thomas Michael Black 22
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