Tox21: Transforming Environmental Health Protection

Raymond Tice, Ph.D.Chief, Biomolecular Screening Branch

(tice@niehs.nih.gov)

LINCS Consortium Kick-Off MeetingRockville, MD

October 23-24, 2011

2007200620052004 2008 2009 2010 2011 2012

NCGC

EPA NCCTToxCast II

(~1000 cmpds x 550 assays)

ToxCast I (~300 cmpds x ~550 assays)

qHTS I (~2800 cmpds)qHTS II (10K

cmpds)

Tox21 - a “Community Resource” Project 2

The Tox21 Screening Timeline

Area of Expertise NIEHS NCGC EPA FDA

Lab Animal Toxicology Human Toxicology/Exposure Assessment Ultra High Throughput Screening Low to Mid Throughput Assays Stem Cell Assay Development Epigenetic Assays Engineered Tissue Models ‘Omic Based Systems Lower Organism Systems Genetic Variability in Response Databases & Informatic Tools Validation Experience 3

Tox21 Partners

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

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Identify toxicity pathways & corresponding assays

Review nominated assays

Prioritize assays for qHTS

Characterize assay output and evaluate assay performance

Develop prioritization schemes and prediction models

Make all data publicly accessible via CEBS, PubChem, ACToR

Evaluate the relevance of prioritization schemes and prediction models

Prioritize substances for more complex testing

Extrapolate in vitro conc to in vivo dose

Establish a 10K DMSO soluble compound library for qHTS

Establish QC procedures

Establish libraries of mixtures and aqueous soluble compounds for qHTS

Informatics Working Group

Co-ChairsRuili Huang, Ph.D. (NCGC)

Richard Judson, Ph.D. (EPA)Jennifer Fostel, Ph.D. (NIEHS)

Weida Tong, Ph.D. (FDA)

Chemical Selection Working Group

Co-ChairsWilliam Leister, Ph.D. (NCGC)Donna Mendrick, Ph.D. (FDA)

Ann Richard, Ph.D. (EPA)Cynthia Smith, Ph.D. (NTP)

Targeted Testing Working Group

Co-ChairsKevin Crofton, Ph.D. (EPA)

Michael DeVito, Ph.D. (NTP)David Gerhold, Ph.D. (NCGC)James Weaver, Ph.D. (FDA)

Assays & Pathways Working Group

Co-ChairsKevin Gaido, Ph.D. (FDA)Keith Houck, Ph.D. (EPA)Kristine Witt, M.S. (NTP)

Menghang Xia, Ph.D. (NCGC)

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Tox21 Phase I – Proof of Principle

• NCGC screened 1408 compounds (1353 unique) from NTP and 1462 compounds (1384 unique) from EPA in >100 qHTS at 14 conc (5 nM to 92 M typical).

• EPA via ToxCast™ screened 320 compounds (309 unique, primarily pesticide actives and some endocrine active compounds) in ~550 assays.

• Data released to the scientific community via:

− EPA ACToR (Aggregated Computational Toxicology Resource; http://epa.gov/actor)

− NLM PubChem (http://pubchem.ncbi.nlm.nih.gov/)

− NTP CEBS (Chemical Effects in Biological Systems; http://www.niehs.nih.gov/research/resources/databases/cebs/index.cfm)

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ToxCast 1.0 (April, 2007)

• Enzyme inhibition/receptor binding HTS (Novascreen)

• NR/transcription factors (Attagene, NCGC)

• Cellular impedance (ACEA)

• Complex cell interactions (BioSeek)

• Hepatocelluar HCS (Cellumen)

• Hepatic, renal and airway cytotoxicity (IVAL)

• In vitro hepatogenomics (IVAL, Expression Analysis)

• Zebrafish developmental toxicity (Phylonix)

ToxCast 1.1 (January, 2008)

• Neurite outgrowth HCS (NHEERL)

• Cell proliferation (NHEERL)

• Zebrafish developmental toxicity (NHEERL)

ToxCast 1.2 (June, 2008)

• NR Activation and translocation

(CellzDirect)

• HTS Genotoxicity (Gentronix)

• Organ toxicity; dosimetry (Hamner

Institutes)

• Toxicity and signaling pathways

(Invitrogen)

• C. elegans WormTox (NIEHS)

• Gene markers from microscale cultured

hepatocytes (MIT)

• 3D Cellular microarray with metabolism

(Solidus)

• Zebrafish vascular/cardiotoxicity

(Zygogen)

• HTS stress response (NHEERL+NCGC) 7

ToxCastTM Phase I Testing

• Phenotypic readouts

−Cytotoxicity

−Apoptosis: caspase 3/7, 8, 9

−Membrane integrity: LDH, protease release

−Mitochondrial toxicity (membrane potential)

−Genetox: p53, ELG1, DNA damage gene deficient lines (DT40 lines and mouse)

• Cell Signaling

−Stress response: ARE, ESRE, HSP, Hypoxia, AP-1

−Immune response: IL-8, TNF, TTP

−Other: AP-1, CRE, ERK, HRE, JNK3, NFkB, LDR

• 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 variation: 87 HapMap CEPH Panel

Phase I NCGC qHTS Assays

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Differential Compound Toxicity Among 13 Cell Types

>50 µM10-50 µM1-10 µM<1 µM

Hierarchical Clustering

7.55E-2 1

1 446 8 34 102

Xia et al., EHP 116:284, 2008

IC50 Values

H HM HH H R M HH HH R

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Tox21 Phase II

• EPA’s ToxCast™ Phase II: ~1000 compounds in ~550 assays.

• NCGC qHTS Phase II: >10K compounds 3x at 14 conc for:

− nuclear receptor activation or inhibition (AR, AhR, ER, FXR, GR, LXR, PPAR, PXR, RXR, TR, VDR, ROR)

− induction of stress response pathways (e.g., DNA damage, heat shock, hypoxia, inflammation, oxidative)

• Assay selection based on

− Information from in vivo toxicological investigations

− Phase I experience, advice of basic researchers, and nominated assays

− Maps of disease-associated cellular pathways

• Future focus on disease-associated pathways (e.g., obesity/diabetes, autism) using stem cells/differentiated cells and high throughput gene array assays

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Tox21 Phase II qHTS 10K Library

NCGC

• Drugs

• Drug-like compounds

• Active pharmaceutical ingredients

EPA

•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

•NTP-studied compounds

•NTP nominations and related compounds

•NICEATM/ICCVAM validation reference compounds for regulatory tests

•External collaborators (e.g., Silent Spring Institute, U.S. Army Public Health Command)

•Formulated mixtures

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The Tox21 Genomes Project (with I. Rusyn, UNC)

•Assessment of variation within and between populations

•Mapping of genomic regions associated with variation of responses to individual chemicals or classes

•In a cell-based system, with carefully controlled growth and environmental conditions, the assay may serve as an endo-phenotype, with a greater proportion of variation explained by genomic variation than for a typical complex trait

http://en.wikipedia.org/wiki/1000_Genomes_Project

Status:

•Phase I – 87 CEPH panel x 240 cmpds x 12 conc x 2 assays (cytotox & caspase 3/7)

•Phase II – 1090 lines (9 racial groups) x 180 cmpds x 8 conc x 1 assay (cytotox)

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The NTP DrugMatrix Rat Toxicogenomics Database

• Integrated Collection of Data– 637 unique chemicals (mostly drugs)– 5600 drug-treatment transcript profiles in

rat organs– 127,000 histopathology measurements– 100,000 blood chemistry measurements– 60,000 literature facts

• Over 500 validated signatures– Mode of action and pathology

• Comprehensive data mining– Formulate 100,000’s questions (phenotypes)– Test for ability to classify using transcript data

only

• ~122,000 frozen tissues

• Automated genomics analysis

Steatotic

Non-steatotic

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• Drugmatrix website: https://ntp.niehs.nih.gov/drugmatrix

• ToxFx website: https://ntp.niehs.nih.gov/toxfx/

The NCGC Universe of Human Pathways

~1100 human pathways mapped to the pathway globe

Detailed view of a pathway

Gene information

Pathways

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Development of an Integrated Prediction System

• Collaboration between NIEHS, Leadscope Inc., Lhasa Limited, and MultiCASE Inc.

• System to support the prioritization of chemicals through human-relevant toxicity predictions

• Designed for use by scientists with different backgrounds

• Brings together toxicity data and predictions from multiple geographically distributed locations

−qHTS data from the Tox21 project

−Historical in vitro and in vivo data

−(Q)SAR models for human adverse event endpoints as well as in vitro and in vivo endpoints

NTP Workshop:Role of Environmental

Chemicals in the Development of Diabetes and

Obesity

January 11-13, 2011

Michael Gallo, Workshop ChairDept. of Environmental & Occupational Health, University of

Medicine & Dentistry of New Jersey

Kristina Thayer, Director NTP Office of Health Assessment and Translation http://cerhr.niehs.nih.gov/evals/

diabetesobesity/ 16

Identifying Disease Pathways

• Well-characterized chemical libraries (identity, purity, concentration, stability)

• Well-characterized assays in terms of reliability and relevance

• Ability to incorporate xenobiotic metabolism

• Informatic tools to integrate and mine robust data from multiple sources

• Understanding the relationships between pathways and disease in animal models and humans

• Making the data freely accessible as quickly as possible

• Scientific outreach and training the next generation

Success depends on:

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