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What Can ToxCast Tell Us About Endocrine Disruption? Keith Houck, PhD ISRPT 2009 Endocrine Workshop 9-10 Sept 2009 This work was reviewed by EPA and approved for presentation but does not necessarily reflect official Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendation by EPA for use. Office of Research and Development National Center for Computational Toxicology What Can ToxCast Tell Us About Endocrine Disruption? • Probably not much but our focus is chemical prioritization for targeted testing Office of Research and Development National Center for Computational Toxicology 1 Our Challenge: Too Many Chemicals Too High a Cost 90,000 11,000 Cancer DevTox NeuroTox ReproTox ImmunoTox Millions $ PulmonaryTox …and not enough data. Office of Research and Development National Center for Computational Toxicology 2 Judson, et al EHP 117(5):685-95 (2009) ToxCastTM Background Research program of EPA’s National Center for Computational Toxicology Addresses chemical screening and prioritization needs for pesticidal inerts, anti-microbials, CCLs, HPVs and MPVs Comprehensive use of HTS technologies to generate biological fingerprints and predictive signatures Coordinated with NTP and NHGRI/NCGC via Tox21 Committed to stakeholder involvement and public release of data Communities of Practice- Chemical Prioritization; Exposure NCCT website- http://www.epa.gov/ncct/toxcast ACToR- Aggregated Computational Toxicology Resource http://www.epa.gov/actor/ Office of Research and Development National Center for Computational Toxicology 3 ToxCast_320 Phase I Chemicals 309 unique structures Replicates for QC 8 metabolites 291 total pesticide actives 273 registered pesticide actives 22 pesticide inerts MOA Classes with 33 antimicrobials > 3 chemicals 54 Tier 1 EDSP 23 IUR 13 HPV 73 OW PCCL 11 CCL1 10 CCL2 25 CCL3 Office of Research and Development National Center for Computational Toxicology Misc Acetylcholine esterase inhibitors conazole fungicides Sodium channel modulators pyrethroid ester insecticides organothiophosphate acaricides dinitroaniline herbicides pyridine herbicides thiocarbamate herbicides imidazolinone herbicides organophosphate insecticides phenyl organothiophosphate insecticides aliphatic organothiophosphate insecticides amide herbicides aromatic fungicides chloroacetanilide herbicides chlorotriazine herbicides growth inhibitors organophosphate acaricides oxime carbamate insecticides phenylurea herbicides pyrethroid ester acaricides strobilurin fungicides unclassified acaricides unclassified herbicides 4 EDSP Chemicals in ToxCast 320 CHEM_NAME 2,4-Dichlorophenoxyacetic acid (2,4-D) 2-Phenylphenol Abamectin Acephate Aldicarb Atrazine Azinphos-methyl Benfluralin Bifenthrin Bisphenol A Captan Carbaryl Chlorothalonil Cyfluthrin Cypermethrin d-cis,trans-Allethrin Diazinon Dibutyl phthalate Dibutyl phthalate Dichlobenil Dichlorvos Dicofol Diethylhexyl phthalate Dimethoate Dimethyl phthalate Disulfoton Endosulfan EPTC EPTC Esfenvalerate Ethoprop Flutolanil Folpet Imidacloprid Iprodione Linuron Malathion Metalaxyl Methamidophos Methidathion Methomyl Metolachlor Metribuzin MGK Myclobutanil Norflurazon Oxamyl Parathion-methyl Permethrin Piperonyl butoxide Propargite Propiconazole Propyzamide Pyriproxyfen Quintozene Resmethrin Simazine Triadimefon Trifluralin Vinclozolin Select Other Chemicals in ToxCast 320 Office of Research and Development National Center for Computational Toxicology Methoxychlor HPTE Perfluorooctane sulfonic acid Perfluorooctanoic acid 5 Types of Endocrine Assays Tested In ToxCast – Radioligand Receptor Binding (NRs) • Single conc at 25 mM followed by 7-point concentration response for actives – Multiplexed Reporter Gene Assay (Attagene) • Trans: GAL4-Ligand Binding Domain NR Assays • Cis: NR Response Element Assays • 5-point concentration-response up to 100 mM (unless limited by cytotoxicity) – Nuclear Receptor Coactivator Recruitment (NRs) • Single conc at 25 mM followed by 7-point concentration response for actives – CYP Enzyme Activity Assays • Single conc at 10 mM followed by 7-point concentration response for actives – NR Target Gene Assays (in primary human hepatocytes) • 5-point concentration-response up to 40 mM Office of Research and Development National Center for Computational Toxicology 6 Endocrine Related Assays ToxCast Assay (ToxMiner Name) ATG_AR_TRANS NCGC_AR_Agonist NVS_NR_rAR NVS_NR_hAR NVS_NR_hER NVS_NR_bER NCGC_ERalpha_Agonist ATG_ERa_TRANS ATG_ERE_CIS NCGC_TRbeta_Agonist ATG_TRa_TRANS NVS_NR_hTRa CLZD_UGT1A1_48 NVS_ADME_hCYP19A1 Office of Research and Development National Center for Computational Toxicology Assay Category AR AR AR AR ER ER ER ER ER TR TR TR TR ARO Assay Type Multiplexed Reporter Gene GAL4-Reporter Gene Radioligand Binding Radioligand Binding Radioligand Binding Radioligand Binding GAL4-Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene GAL4-Reporter Gene Multiplexed Reporter Gene Coactivator Recruitment Target gene expression Enzyme activity 7 Other Endocrine Related Assays ToxCast Assay (ToxMiner Name) NVS_NR_hCAR_Agonist NVS_NR_hFXR NVS_NR_hRAR NVS_ADME_hCYP1A1 NVS_ADME_hCYP1A2 NVS_ADME_hCYP1B1 NVS_ADME_hCYP2A6 NVS_ADME_hCYP2B6 NVS_ADME_hCYP2C18 NVS_ADME_hCYP2C19 NVS_ADME_hCYP2C19_Activator NVS_ADME_hCYP2C8 NVS_ADME_hCYP2C9 NVS_ADME_hCYP2D6 NVS_ADME_hCYP2E1 NVS_ADME_hCYP2J2 NVS_ADME_hCYP3A4 NVS_ADME_hCYP3A5 NVS_ADME_hCYP4F12 NVS_ADME_hCYP4F12_Activator NVS_ADME_rCYP1A1 NVS_ADME_rCYP1A2 NVS_ADME_rCYP2A1 NVS_ADME_rCYP2A2 NVS_ADME_rCYP2B1 NVS_ADME_rCYP2C11 NVS_ADME_rCYP2C12 NVS_ADME_rCYP2C13 NVS_ADME_rCYP2C6 NVS_ADME_rCYP2D1 NVS_ADME_rCYP2D2 Office of Research and Development NVS_ADME_rCYP2E1 National Center for Computational Toxicology NVS_ADME_rCYP3A1 NVS_ADME_rCYP3A2 Assay Category Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Assay Type Coactivator Recruitment Coactivator Recruitment Coactivator Recruitment Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity Enzyme activity 8 Other Endocrine Related Assays NVS_NR_hPPARg NVS_NR_hPPARa NVS_NR_hPXR NCGC_LXR_Agonist NCGC_PPARg_Agonist NCGC_PXR_Agonist_human NCGC_PXR_Agonist_rat NCGC_RXRa_Agonist ATG_Ahr_CIS ATG_CAR_TRANS ATG_ERRa_TRANS ATG_ERRg_TRANS ATG_FXR_TRANS ATG_GR_TRANS ATG_GRE_CIS ATG_LXRa_TRANS ATG_LXRb_TRANS ATG_PPARa_TRANS ATG_PPARd_TRANS ATG_PPARg_TRANS ATG_PPRE_CIS ATG_PXR_TRANS ATG_PXRE_CIS ATG_RARa_TRANS ATG_RARb_TRANS ATG_RARg_TRANS ATG_RXRa_TRANS ATG_RXRb_TRANS NVS_NR_bPR NVS_NR_hCAR NVS_NR_hGR NVS_NR_hPR CLZD_CYP1A1_48 CLZD_CYP1A2_48 CLZD_CYP2B6_48 Office of Research and Development National Center for Computational Toxicology CLZD_CYP3A4_48 CLZD_HMGCS2_48 CLZD_SULT2A1_48 Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Other Endocrine Related Fluorescent Ligand Binding (FP) Fluorescent Ligand Binding (FRET) Fluorescent Ligand Binding (FRET) GAL4-Reporter Gene GAL4-Reporter Gene GAL4-Reporter Gene GAL4-Reporter Gene GAL4-Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Multiplexed Reporter Gene Radioligand Binding Radioligand Binding Radioligand Binding Radioligand Binding Target gene expression Target gene expression Target gene expression Target gene expression Target gene expression Target gene expression 9 Example NR Binding Assay Actives hERa Office of Research and Development National Center for Computational Toxicology hAR hTRa 10 Overview of Assay Results • Generally found expected actives for ERa and AR binding assays • Little activity for Thyroid Receptor • Some species and/or assay sensitivity differences seen, e.g. low sensitivity of rat AR binding assay • Not always agreement between binding and cellular reporter assays • Many CYP inhibitors including those from expected chemical classes, i.e. conazoles and organophosphates • Potency mostly in the micromolar to tens of micromolar range; few nanomolar actives Office of Research and Development National Center for Computational Toxicology 11 Tox21 • Tripartite Collaboration: EPA/NTP/NCGC • Quantitative HTS Assays at NCGC • Accepting Nominations for Assays from All Sources • Chemical Libraries Being Screened: – EPA Library of 1462 Substances • Contains Toxcast 309 • Contains many NR reference compounds, e.g. pharmacological tools and drugs • Many different environmental chemicals, e.g. bisphenols, phthalates, PFAAs – 1408 substances in NTP library also screened – Expanding to 10,000 this year including all human pharmaceuticals Office of Research and Development National Center for Computational Toxicology 12 Tox21 Assays Nuclear Receptor Cellular Reporter Gene Assays – GAL4-Ligand Binding Domain System--principally ligand detection – Receptors screened in agonist and antagonist modes – 11-15 concentrations (highest 92 mM) • antagonist mode data interpretation confounded by cytotoxicity – Receptors screened: • Human – ERa, AR, TRa, VDR, MR, RXRa, PPARa, PPARb, PPARg, PXR, (AhR) • Rat – PXR Office of Research and Development National Center for Computational Toxicology 13 Office of Research and Development National Center for Computational Toxicology PPARalpha PPARdelta_Ref PPARdelta PPARgamma_Ref PPARgamma RXRa_Ref RXRa PPARdelta_Ref PPARdelta PPARgamma_Ref PPARgamma RXRa_Ref RXRa hPXR hPXR_Ref VDR VDR_Ref TRbeta TRbeta_Ref FXR FXR_Ref GR GR_Ref AR AR_Ref ERa ERa_Ref PPARalpha 0 PPARalpha_Ref 50 PPARalpha_Ref 100 rPXR 150 rPXR 200 rPXR_Ref % Efficacy rPXR_Ref hPXR hPXR_Ref VDR VDR_Ref TRbeta TRbeta_Ref FXR FXR_Ref GR GR_Ref AR AR_Ref ERa ERa_Ref Max Efficacy (% of Positive Control) Log AC50 (M) Tox21 Results EPA Library Actives: Agonists Potency -2 -4 -6 -8 -10 14 Tox21 EDSP Chemicals Agonist (All) Potency -2 -3 Log AC50 (M) -4 -5 -6 -7 -8 -9 hPXR VDR TRbeta RXRa PPARgamma PPARdelta PPARalpha GR FXR AhR AR ERa -10 Max Efficacy (% of Positive Control) Efficacy 400 300 200 100 80 60 40 20 hPXR VDR TRbeta RXRa PPARgamma PPARdelta PPARalpha GR FXR Office of Research and Development National Center for Computational Toxicology AhR AR ERa 0 15 FXR_antagonist FXR_antagonist_Ref RXRa_antagonist RXRa_antagonist_Ref PPARgamma_antagonist PPARgamma_antagonist_Ref PPAR-delta_antagonist PPAR-delta_antagonist_Ref PPARalpha_antagonist PPARalpha_antagonist_Ref VDR_antagonist VDR_antagonist_Ref TRbeta_antagonist TRbeta_antagonist_Ref Office of Research and Development National Center for Computational Toxicology GR_antagonist GR_antagonist_Ref AR_antagonist AR_antagonist_Ref ERa_antagonist ERa_antagonist_Ref Log AC50 (M) Tox21 EPA Library Antagonist Actives Antagonist -3 -4 -5 -6 -7 -8 -9 16 Partial Agonist Examples: ERa Bisphenol B 140 140 120 120 100 100 % of Control % of Control Phenolphthalein 80 60 40 80 60 40 20 20 0 0 -20 -4 -3 -2 -1 0 1 -2 0 2 Conc (log mM) 2 Conc (log mM) Phenolphthalin 140 140 120 120 100 100 % of Control % of Control Butyl benzyl phthalate 80 60 40 20 0 -20 80 60 40 20 0 -3 -2 -1 0 Conc (log mM) Office of Research and Development National Center for Computational Toxicology 1 2 -20 -3 -2 -1 0 1 2 Conc (log mM) 17 Importance of Biotransformation Methoxychlor (human ERa) Methoxychlor (bovine ERa) HPTE (human ERa) Parent/Metabolite ERa radioligand binding assay % of Inhibition 120 100 HPTE (bovine ERa) 80 Bottom Top LogIC50 HillSlope IC50 60 40 20 HPTE (hERa ) 2.448 = 100.0 -1.349 -0.9197 0.04476 HPTE (bERa ) 1.725 = 100.0 -1.697 -0.9877 0.02009 0 -20 -3 -2 -1 0 1 2 Conc (log mM) Parent/Metabolite 100 Methoxychlor HPTE ERa cellular (HEK293) transactivation assay % of Control 80 60 Bottom Top LogEC50 HillSlope EC50 40 20 Methoxy chlor 0.3017 41.86 0.5714 5.466 3.727 HPTE -3.912 35.70 -0.7131 2.258 0.1936 0 -3 -20 Office of Research and Development National Center for Computational Toxicology -2 -1 0 1 2 Conc (log mM) 18 In vivo/In vitro Correlation Physical chemical properties Cellular Assays Data Normalization In silico Predictions Biochemical Assays Office of Research and Development National Center for Computational Toxicology 19 Genomic Signatures Toxicology Endpoints Office of Research and Development National Center for Computational Toxicology ToxRefDB website: http://www.epa.gov/ncct/toxrefdb/ 20 Reproductive and Endocrine Organ Toxicity Endpoints from ToxRefDB Office of Research and Development National Center for Computational Toxicology 21 Using the Data for Prioritization In vitro assays (ToxCast) Chemical properties (descriptors) In vivo endpoints (ToxRefDB) Pathways (endocrine) Office of Research and Development National Center for Computational Toxicology 22 ide Thiodicarb Thiram Triadimef on Using the Data for Prioritization TR.assay .hit.percentage ER.assay .hit.percentage Other.assay .hit.percentage AR.assay .hit.percentage Lipinski_LS.normalized endpoint.hit.count.percentage Ingenuity .pathway .hit.percentage Bisphenol A Perfluorooctane sulfonic acid Office of Research and Development National Center for Computational Toxicology KEGG.pathway .hit.percentage Fenitrothion Symclosene Linuron 23 Summary of Preliminary Findings • Suite of assays useful in prioritization – endocrine-active chemicals score higher across assays – Individual assays may not be sufficient on their own • Moderate concordance across different assay types for same target – Differences in biotransformation between assays – Assay-specific false positives/false negatives – Error – Differences in biology we don’t understand • Sensitivity range of different assay types presents a challenge to interpret biological significance Office of Research and Development National Center for Computational Toxicology 24 ACKNOWLEDGEMENTS: NCCT/ORD/USEPA Matt Martin David Dix Richard Judson David Reif Ann Richards Robert Kavlock NCGC/NIH Menghang Xia Ruili Huang Chris Austin
