Transcript Presentation Title

Tox21: Transforming Environmental
Health Protection
Raymond Tice, Ph.D.
Chief, Biomolecular Screening Branch
([email protected])
LINCS Consortium Kick-Off Meeting
Rockville, MD
October 23-24, 2011
The Tox21 Screening Timeline
NCGC
ToxCast I (~300
cmpds x ~550
assays)
EPA NCCT
2004
qHTS II (10K
cmpds)
qHTS I (~2800 cmpds)
2005
2006
2007
2008
Tox21 - a “Community
Resource” Project
2009
ToxCast II
(~1000 cmpds
x 550 assays)
2010
2011
2012
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Tox21 Partners
Area of Expertise
NIEHS
NCGC
EPA
FDA
Lab Animal Toxicology
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Human Toxicology/Exposure
Assessment
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Ultra High Throughput Screening
Low to Mid Throughput Assays
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Stem Cell Assay Development
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Epigenetic Assays
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Engineered Tissue Models
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‘Omic Based Systems
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Lower Organism Systems
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Genetic Variability in Response
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Databases & Informatic Tools
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Validation Experience
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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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Agency Points of Contact
Christopher Austin, M.D. (NCGC)
Thomas Colatsky, Ph.D. (FDA)
Robert Kavlock, Ph.D. (EPA)
Raymond Tice, Ph.D. (NTP)
Assays & Pathways
Working Group
Chemical Selection
Working Group
Informatics
Working Group
Targeted Testing
Working Group
Co-Chairs
Kevin Gaido, Ph.D. (FDA)
Keith Houck, Ph.D. (EPA)
Kristine Witt, M.S. (NTP)
Menghang Xia, Ph.D. (NCGC)
Co-Chairs
William Leister, Ph.D. (NCGC)
Donna Mendrick, Ph.D. (FDA)
Ann Richard, Ph.D. (EPA)
Cynthia Smith, Ph.D. (NTP)
Co-Chairs
Ruili Huang, Ph.D. (NCGC)
Richard Judson, Ph.D. (EPA)
Jennifer Fostel, Ph.D. (NIEHS)
Weida Tong, Ph.D. (FDA)
Co-Chairs
Kevin Crofton, Ph.D. (EPA)
Michael DeVito, Ph.D. (NTP)
David Gerhold, Ph.D. (NCGC)
James Weaver, Ph.D. (FDA)
 Identify toxicity
pathways &
corresponding assays
 Establish a 10K DMSO
 Review nominated
assays
 Establish QC
 Prioritize assays for
qHTS
soluble compound
library for qHTS
procedures
 Establish libraries of
mixtures and aqueous
soluble compounds 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 5
Tox21 Phase I – Proof of Principle
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•
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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:
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EPA ACToR (Aggregated Computational Toxicology Resource;
http://epa.gov/actor)
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NLM PubChem (http://pubchem.ncbi.nlm.nih.gov/)
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NTP CEBS (Chemical Effects in Biological Systems;
http://www.niehs.nih.gov/research/resources/databases/cebs/index.cfm)
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ToxCastTM Phase I Testing
ToxCast 1.0 (April, 2007)
ToxCast 1.2 (June, 2008)
•
Enzyme inhibition/receptor binding HTS
(Novascreen)
• NR Activation and translocation
(CellzDirect)
•
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•
NR/transcription factors (Attagene, NCGC)
• HTS Genotoxicity (Gentronix)
Cellular impedance (ACEA)
• Organ toxicity; dosimetry (Hamner
Institutes)
•
Zebrafish developmental toxicity (Phylonix)
Complex cell interactions (BioSeek)
Hepatocelluar HCS (Cellumen)
• Toxicity and signaling pathways
(Invitrogen)
Hepatic, renal and airway cytotoxicity (IVAL)
In vitro hepatogenomics (IVAL, Expression
Analysis)
ToxCast 1.1 (January, 2008)
•
•
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Neurite outgrowth HCS (NHEERL)
Cell proliferation (NHEERL)
Zebrafish developmental toxicity (NHEERL)
• 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)
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Phase I NCGC qHTS Assays
•
Phenotypic readouts
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Cytotoxicity
Apoptosis: caspase 3/7, 8, 9
Membrane integrity: LDH, protease
release
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Mitochondrial toxicity (membrane
potential)
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Genetox: p53, ELG1, DNA damage
gene deficient lines (DT40 lines and
mouse)
•
Cell Signaling
−
Stress response: ARE, ESRE, HSP,
Hypoxia, AP-1
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Immune response: IL-8, TNF, TTP
Other: AP-1, CRE, ERK, HRE, JNK3,
NFkB, LDR
Epigenetics
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Drug metabolism
•
CYP1A2, CYP2C19, CYP2C9, CYP2D6,
CYP3A4
Target specific assays
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Nuclear receptors: AR, AhR, ER, FXR,
GR, LXR, PPAR, PPARδ, PPARγ, PXR,
RXR, TRβ, VDR, ROR, RORγ
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hERG channel
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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
Locus DeRepression (LDR)
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Differential CompoundHierarchical
Toxicity Among
Clustering 13 Cell Types
1
8
34
102
7.55E-2
446
1
IC50 Values
>50 µM
10-50 µM
1-10 µM
<1 µM
H
M
H
H
H
H
R
M
H
H
H
Xia et al., EHP 116:284, 2008
H
R
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Tox21 Phase II
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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
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Drugs
Drug-like
compounds
Active
pharmaceutical
ingredients
EPA
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ToxCast I and II
compounds
Antimicrobial
Registration Program
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Endocrine Disruptor
Screening Program
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OECD Molecular
Screening Working
Group List
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FDA Drug Induced
Liver Injury Project
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Failed Drugs
NTP
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NTP-studied compounds
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NICEATM/ICCVAM
validation reference
compounds for regulatory
tests
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External collaborators
(e.g., Silent Spring
Institute, U.S. Army Public
Health Command)
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Formulated mixtures
NTP nominations and
related compounds
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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
Status:
http://en.wikipedia.org/wiki/1000_Geno
mes_Project
•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
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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
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Mode of action and pathology
• Comprehensive data mining
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Steatotic
Formulate 100,000’s questions (phenotypes)
Test for ability to classify using transcript data
only
• ~122,000 frozen tissues
• Automated genomics analysis
• Drugmatrix website: https://ntp.niehs.nih.gov/drugmatrix
• ToxFx website: https://ntp.niehs.nih.gov/toxfx/
Non-steatotic
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The NCGC Universe of Human Pathways
Detailed
view of a
pathway
Pathways
Gene
information
~1100 human pathways mapped to the pathway globe
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Development of an Integrated Prediction System
•
•
•
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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
Identifying Disease Pathways
NTP Workshop:
Role of Environmental Chemicals
in the Development of Diabetes
and Obesity
January 11-13, 2011
Michael Gallo, Workshop Chair
Dept. 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/diabe
tesobesity/
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Success depends on:
• 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
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