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Tox21: A U.S. Federal Collaboration to Improve the

n. Tox21: A U.S. Federal Collaboration to Improve the Human Hazard Characterization of Chemicals Raymond Tice, Ph.D. Chief, Biomolecular Screening Branch (tice@niehs.nih.gov) BSRC Regulatory Science Symposium Shinagawa, Japan February 21, 2014. Topics. Background

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Tox21: A U.S. Federal Collaboration to Improve the

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  1. n Tox21: A U.S. Federal Collaboration to Improve the Human Hazard Characterization of ChemicalsRaymond Tice, Ph.D. Chief, Biomolecular Screening Branch (tice@niehs.nih.gov)BSRC Regulatory Science Symposium Shinagawa, Japan February 21, 2014

  2. Topics • Background • Phase I – Proof of principle • Phase II - Expanded compound screening • Phase III - Improving on biological coverage and relevance • Chemical prioritization efforts 2

  3. Our Need for Prioritization Too Many Chemicals Too Little Data (%) 9912 3 Judson et al., EHP 117:685-695 (2009)

  4. Toxicity Testing in the 21st Century This 2007 National Academy of Science report envisions a not-so-distant future in which virtually all routine toxicity testing would be conducted in vitro in human cells or cell lines by evaluating perturbations of cellular responses in a suite of toxicity pathway assays using high throughput robotic assisted methodologies. 4

  5. Exposure Tissue Dose Biologic Interaction Perturbation Low Dose Normal Biologic Function Biologic Inputs Early Cellular Changes Morbidity and Mortality Adaptive Stress Responses Cell Injury Activation of a Toxicity Pathway Higher Dose Higher yet NAS Report, 2007 5

  6. 5-year Memorandum of Understanding (MoU) on “High-Throughput Screening, Toxicity Pathway Profiling, and Biological Interpretation of Findings” released on Feb 14, 2008 signed by NHGRI (F.S. Collins), NIEHS/NTP (S.H. Wilson), and EPA (G.M. Gray). Formation of the U.S. Tox21 Community • Revised 5-year MoU to add FDA signed on July 19, 2010 http://ntp.niehs.nih.gov/go/28213) by NHGRI (E.D. Green), NIEHS/NTP (L.S. Birnbaum), EPA (P.T. Anastas), and FDA (J. Woodcock). • Known informally as Tox21 for Toxicology in the 21st Century 6

  7. Tox21 Goals • Identify patterns of compound-induced biological response in order to: • characterize toxicity/disease pathways • facilitate cross-species extrapolation • model low-dose extrapolation • Prioritize compounds for more extensive toxicological evaluation • Develop predictive models for biological response in humans 7

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  9. Assays & Pathways Working Group Co-Chairs Kevin Gaido, Ph.D. (FDA) Keith Houck, Ph.D. (EPA) Kristine Witt, M.S. (NTP) Menghang Xia, Ph.D. (NCGC) Informatics Working Group Co-Chairs Ruili Huang, Ph.D. (NCGC) Richard Judson, Ph.D. (EPA) Jennifer Fostel, Ph.D. (NIEHS) Weida Tong, Ph.D. (FDA) Targeted Testing Working Group Co-Chairs Michael DeVito, Ph.D. (NTP) David Gerhold, Ph.D. (NCGC) Timothy Shafer, Ph.D. (EPA) James Weaver, Ph.D. (FDA) Chemical Selection Working Group Co-Chairs William Leister, Ph.D. (NCGC) Donna Mendrick, Ph.D. (FDA) Ann Richard, Ph.D. (EPA) Suramya Waidanatha, Ph.D. (NTP) • Identify toxicity pathways & corresponding assays • Review nominated assays and prioritize for use at the NCGC • Establish compound libraries for qHTS (10K, mixtures, water-soluble) • Establish QC procedures for compound identity, purity, concentration, and stability • Evaluate relevance of prioritization schemes & prediction models • Extrapolate in vitro concentration to in vivo dose • Evaluate assay performance • Develop prioritization schemes and prediction models • Make all data publicly accessible Agency Points of Contact FDA - Thomas Colatsky, Ph.D. NCGC/NCATS –Anton Simeonov, Ph.D. EPA/NCCT –Russell Thomas, Ph.D. NIEHS/NTP - Raymond Tice, Ph.D. 9

  10. Tox21 Phase I – Proof of Principle (2005 – 2010) • EPA via ToxCast™ screened 320 compounds (309 unique, primarily pesticide actives and some endocrine active compounds) in ~550 assays. • Data made public via ACToR (Aggregated Computational Toxicology Resource; http://epa.gov/actor) • NCGC screened 1408 compounds (1353 unique) from NTP and 1462 compounds (1384 unique) from EPA in 140 qHTS assays representing 77 predominantly cell-based reporter gene endpoints. • Data made public via PubChem (http://pubchem.ncbi.nlm.nih.gov/) and will be available in CEBS (Chemical Effects in Biological Systems; http://www.niehs.nih.gov/research/resources/databases/cebs/) 10

  11. Quantitative High Throughput Screening (qHTS) at NCATS • DMSO soluble compounds • homogeneous assays • 1536-well plate format • 15-point concentration-response curve • 5 nM to 92 M typical • ~5 L assay volume • ~1000-2000 cells/well 11

  12. Phenotypic readouts Cytotoxicity Apoptosis: caspase 3/7, 8, 9 Membrane integrity: LDH, protease release Mitochondrial toxicity (membrane potential) Genetox: p53, ATAD5, DNA damage repair deficient lines (chicken DT40 lines) Cell Signaling Stress response: ARE, ESRE, HSP, Hypoxia, AP-1 Immune response: IL-8, TNF, TTP Other: AP-1, CRE, ERK, HRE, JNK3, NFkB Epigenetics Locus DeRepression (LDR) Drug metabolism CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4 Target specific assays Nuclear receptors: AR, AhR, ER, FXR, GR, LXR, PPAR, PPARδ, PPARγ, PXR, RXR, TRβ, VDR, ROR, RORγ hERG channel Isolated molecular targets: 12hLO, 15hLO1, 15hLO2, ALDH1A1, HADH560, HPGD, HSD17b4, APE1, TDP1, DNA polymerase III, RECQ1 helicase, RGS4, BRCA, IMPase, O-Glc NAc Transferase, Caspase-1/7, CBFβ-RUNX1, PK, Tau, Cruzain, β-Lactamase, PRX, YjeE , NPS, Proteasome, SF1, SMN2, beta-globin splicing, Anthrax Lethal Factor, TSHR Genetic variability: 87 HapMap CEPH Panel Tox21 Phase I qHTS Phase I NCGC qHTS Assays 12

  13. Conclusions from Tox21 Phase I • qHTS can be used to screen libraries of environmental compounds against targets with known biological significance • Cell lines are more suitable for qHTS assays than primary cells • High quality data are essential for automated data analysis and interpretation • Signal to background ratio (S/B) • CV and Z’ factor • Reproducibility of duplicate compounds within and between runs • Biological relevance is critical: do the results make sense based on available standard toxicity test data and/or human data • Importance of reference compounds • Significant limitation remains the lack of hepatic metabolic activation 13 Tice et al. EHP 121:756–765 (2013)

  14. Tox21 Phase II – Expanded Compound Screening (2011 – 2014) • EPA’s ToxCast™ Phase II: ~700 compounds in ~700 assays, ~1000 compounds in endocrine activity assays • NCGC qHTS Phase II: • 10K compound library screened 3 times at 15 concentrations in each qHTS assay • qHTS assays focused on: • nuclear receptor activation or inhibition • induction of cellular stress response pathways • characterizing human variability in response • Partner-lead projects • cardiotoxicity (FDA) • endocrine disruptors (EPA) • genotoxicity (NIEHS/NTP) • mitochondrial toxicity (NCATS) 14

  15. Tox21 10K Compound Library EPA(3726) NTP(3194) (1328) 367 (623) (553) Library tested 3x in each assay NCGC (3526) 88 single-sourced cmpds in duplicate on each plate Compound identity and structures available at http://www.epa.gov/ncct/dsstox/sdf_tox21s.html 15

  16. Tox21 10K Compound Library • EPA • 3726 cmpds • ToxCast I and II compounds • Antimicrobial Registration Program • Endocrine Disruptor Screening Program • OECD Molecular Screening Working Group List • FDA Drug Induced Liver Injury Project • Failed Drugs • NTP • 3194 cmpds • NTP-studied compounds • NTP nominations and related compounds • NICEATM/ICCVAM reference compounds from in vivo regulatory tests • External collaborators (e.g., Silent Spring Institute, U.S. Army Public Health Command) • Formulated mixtures NCGC • 3524 cmpds • Drugs • Active pharmaceutical ingredients 16

  17. Phase II Stress Response qHTS Assays 17 *Bolded text indicates completed assays

  18. Phase II Nuclear Receptor and Related qHTS Assays* NR assays conducted in agonist and antagonist modes 18 *Bolded text indicates completed assays

  19. Tox21 10K Screen Performance *Score = 2x%active match + %inactive match - %inconclusive – 2x%mismatch 19

  20. Tox21 10K Screen Performance (Continued) *Score = 2x%active match + %inactive match - %inconclusive – 2x%mismatch 20

  21. NIEHS-NCATS-UNC TOXICOgenETICS project: qHTS for Cytotoxicity in a Population-Based in vitro Model Population-wide study design: Goal: use crowdsourcing to better predict the toxicity of chemicals • Use the biological data (SNPs, basal gene expression) to develop a model that accurately predicts individual responses to compound exposure • Use the intrinsic chemical properties to develop a model that accurately predicts how a particular population will respond to certain types of chemicals • for information, go to https://www.synapse.org/#!Synapse:syn1761567 1086cell lines • To understand how genetic variation affects individual response to common environmental and pharmaceutical chemicals • The largest ever population-based ex-vivo cytotoxicity study • 1086 cell lines • 179 common, pharmaceutical, or important environmental chemicals (9 duplicates) • 8 concentrations (0.33 nM – 92 M) • 1-3 plate replicates • ~2,400,000 data points + 2-5x106 SNPs 21

  22. Tox21 Phase II Advantages and Limitations • Extent of pathway coverage • Focus on the use of reporter gene assays using immortal cell lines • Focus on single compounds • Limited capability for xenobiotic metabolism • Focus on simple biological systems • Limited to acute exposure scenarios • Limited availability of “big” data analysis tools • Limited availability of high quality humantoxicological data 22

  23. Tox21 Phase III – Improving on Biological Coverage and Relevance (2013 - ?) HepaRG Cells • Goal to develop more physiologically-relevant /predictive in vitro and lower organism approaches (models/assays/data interpretation strategies) to assess chemical toxicity potential • Incorporation of xenobiotic metabolism & longer-term cumulative exposures into current and new approaches • Increased use of in silico models and quantitative extrapolation models • Integrated, data-rich assay approaches capturing various molecular pathways & cellular phenotypes • Mechanistic studies integrating to Adverse Outcome Pathways (AOPs) • Expand collaborations and interactions Spheroids/Organoids • Near-Term Targeted Assays • High Content screening • Hoechst: Cell loss & nuclear size • DHE: Oxidative stress/ROS • p53: DNA damage • pH2A.X: Genotoxicity • JC-10: Mitochondrial damage (MMP) • Caspase 3: Apoptosis • Lipitox: Steatosis & Phospholipidosis • Reactive metabolites/ROS: GSH depletion • Gene expression assays • ~1000 genes, multiple species 23

  24. Identify Practicable Approaches for 10k chemicals • Metabolite Structure Predictions (human enzymes) • Extent of Metabolism Predictions (metabolic clearance) • Assess Predictivity of Approaches • Assess accuracy via known substrates • Do we predict true metabolites • Assess extent of metabolism predictions • Analyze 8,307 Chemical Structures (10k Library) • P450 & UGT Substrate Predictions • CLINT Predictions (combined via liver expression levels) • Predict Metabolite Structures • Assess accuracy of metabolite structure predictions • Assess Predicted Structures via QSAR models • Toxicity Predictions • ADME/Permeabilities • Physicochemical Properties • Rank/Prioritize Chemicals for Assays in Metabolically Competent Model Systems • Predicted Toxicities via chemical structure alerts • Extent of metabolism predictions • Reactive Metabolites? In Silico Analysis of the 10k Library for Xenobiotic Metabolism • QSAR Predictions • Physicochemical • LogP, Lipinski Ruleof5, pH, solubility(water,salt,GI),blood/plasma ratio • Human Metabolism • Substrate, inhibitor, CLINT, SOM, Metabolite Structures, • ADME • Gut absorption & permeability, skin permeability, cornea permeability, blood-brain barrier permeability, uptake transport, efflux transport, • Toxicity • 22 QSAR model toxicity predictions • Human, Rat, Mouse, Aquatic • LD50, Mutagenicity, ALT, AST, Alk. Phos., LDH, phospholipidosis, reproductive, ER binding, AR binding 24

  25. Extent of Metabolism Predictions Substrate Calls • CLINT predicted for 5 individual P450s • Combined CLINT from 5 Enzymes in ToxPi • Weighted each enzyme CLINT contribution by relative P450 expression levels 10k Library Xenobiotic Metabolism Predictions #4 • 168,805 unique metabolites predicted • Evaluating ability to predict known metabolites in library • Assessing 10k library & 169k metabolites with various toxicity prediction models 25

  26. High Throughput Transcriptomics Workshop On Gene Prioritization Criteria September 16-17, 2013National Institute of Environmental Health Sciences • 29 July 2013 Federal Register Request for Information • The nomination and prioritization of ~1000 environmentally responsive genes per species for use in screening large numbers of substances in cells or tissues from human, rat, mouse, zebrafish, and C. elegans, using high throughput toxicogenomic technologies. • Recommendations on criteria to use for prioritizing the genes that potentially would be the most useful in a screening paradigm, with a focus on effects that reflect general cellular responses, independent of cell type, and gene expression changes that are specific by cell type.

  27. The NCATS BioPlanet: the universe of biological pathways for assay selection and prioritization • Hosts the universe of human pathways (~1600 unique) • All pathway annotations from manually curated, public sources (e.g., KEGG, WikiPathways, Reactome, Science Signaling) • Integrates pathways from >10 different data sources • Annotates pathways by source, biological function/process, disease/toxicity relevance, assay availability • Easy visualization, browsing, analysis of pathways • Facilitates pathway assay selection/prioritization for Tox21 production phase • Web version in process for public release 27 From Ruili Huang, NCATS

  28. C. elegans (NIEHS/NTP) • Screened ToxCast Phase II compounds in growth assay • Screening subsets of compounds in assays that measure • feeding • larval lethality • reproduction Alternative Organisms – C. elegans and Zebrafish • Zebrafish – R. Tanguay (Sinnhuber Aquatic Research Laboratory, Oregon State University, Corvallis, OR) • Screened ToxCast Phase II compounds • Screening 3455 NTP compounds at ~ 64 μM • Assays include • 1 day photo induced behavior • 1 day assessment of mortality/developmental progression • 5 day photo motor response • 5 day assessment of 20 morphological endpoints 5 days

  29. Human Stem Cell Related Projects • Collaboration with Cellular Dynamics and Molecular Devices to screen 80 compounds (focus on neurotoxicants, cardiotoxicants, mitochondrial toxicants) in: • Neurite outgrowth assay/mitochondrial membrane potential • Beating cardiomyocyte assay/mitochondrial membrane potential • Collaboration with QPS, PhoenixSongs Biologicals, & the Hamner Institutes to evaluate biological activity of the 80-compound library in various human and rat neuronal cell culture systems (e.g., primary, embryonic stem cell–derived, induced pluripotent stem cell-derived, transformed neural cell lines) • Collaboration with XCell to characterize response of iPSC-derived neural populations (e.g., dopaminergic) from Parkinson’s disease (familial & sporadic) using the 80-compound library • Collaboration with the Buck Institution to screen the 80-compound library in hTERT astrocytic cell lines to identify senescence-inducing agents and verify results from the screen in primary human astrocytic cultures. • Collaboration with the Univ. Konstanz to screen the 80-compound library in assays that evaluate migration of neural crest cells/neurite outgrowth in a human cell line.

  30. NTP-CDI-Molecular Devices Stem Cell Project

  31. In Vitro Neurite Outgrowth Assay Cell Body Processes (4) connected to cell body Branch 31

  32. Tox21 Phase III • Greatly increased pathway coverage • Focus on high content screening using normal human cells • Focus is still on single compounds but increased interest in mixtures • Expanded capability for xenobiotic metabolism • Focus on more complex biological systems • Potential for more extended exposure scenarios • Expanded availability of “big” data analysis tools 32

  33. BioActivity Network for Prioritization Data + = + 33

  34. NICEATM Uterotropic Network Paint Uterotropic Actives Uterotropic Inactives 34

  35. Filter for 1st Neighbors of Uterotropic+ Utero Actives Utero Inactives 1st Neighbors 35

  36. Tox21/ToxCast Data Release Activities • Tox21 10K library data - Phase II data released in PubChem(86 entries to date at https://www.ncbi.nlm.nih.gov/pcassay?term=tox21) • On Dec 17, EPA publicly released ToxCast Phase II high-throughput screening data at http://www.epa.gov/ncct/toxcast/data.html - Data on 1,800 chemicals, screened in 700+ assays • Accessible via Chemical Safety for Sustainability Dashboards (http://actor.epa.gov/dashboard/) and raw data files • Release will include announcement of data challenges through TopCoder and InnoCentive to figure out how to use new data to predict chemical no effect levels. Winners will receive monetary prizes (http://epa.gov/ncct/challenges.html • EPA is hosting stakeholder workshops (information at: http://epa.gov/ncct/workshops.html

  37. Success depends on • Robust scientific collaborations • Well-characterized chemical libraries • Well-characterized assays in terms of reliability and relevance, with broad biological coverage • Incorporating xenobiotic metabolism into in vitro assays • Informatic pipelines/tools that integrate and mine diverse data streams • Understanding the relationships between pathways and disease in humans and animal models • Making all data public • Outreach to the scientific community on the usefulness and limitations of Tox21 data 37

  38. What will success bring? • Test methods for toxicity testing that are scientifically sound and more economically efficient • An increased ability to evaluate the large numbers of chemicals that currently lack adequate toxicological evaluation • Models for risk assessment that are more mechanistically based • Reduction and/or replacement of animals in regulatory testing 38

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