application of toxcasthigh throughput screening to green...
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Office of Research and DevelopmentNational Center for Computational Toxicology
David DixDeputy Director, National Center for Computational Toxicology
Application of ToxCast High Throughput Screening to Green Chemical Design
NAS Green ChemistyWashington, DCSeptember 20-21, 2011
This work was reviewed by EPA and approved for presentation but does not necessarily reflect official Agency policy.
Office of Research and DevelopmentNational Center for Computational Toxicology
Too Many Chemicals Too Little Data (%)
Why ToxCast?Why ToxCast?
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Acute Cancer GentoxDev Tox Repro Tox
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IRIS TRI PesticidesInerts CCL 1 & 2 HPVMPV
Office of Research and DevelopmentNational Center for Computational Toxicology2
High Throughput Screening 101 (HTS)
96-, 384-, 1536 Well Plates
Target Biology (e.g., Estrogen Receptor)
Robots
Pathway
Chemical Exposure
Cell Population
HTS: High Throughput Screening2
Office of Research and DevelopmentNational Center for Computational Toxicology
ToxCast / Tox21 HTS Chemical Space
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ToxCast
EDSP21
Tox21
1,000 2,000 10,000
500 ToxCast assays
ToxCast EATSassays
Tox21 Assays
Pesticides (active and inert), industrial chemicals, consumer products,marketed and failed pharmaceuticals, food additives, water contaminants, natural human metabolites
Office of Research and DevelopmentNational Center for Computational Toxicology
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The Home of TFomics TM
attageneattageneattageneattagene
Over 500 ToxCast HTS Assays fromContractors and Collaborators
Compound Focus, Inc.a subsidiary of
EPA/ORD/NHEERL
ToxRefDB ToxCast Human Disease
A. B. C.ToxicityEndpoints
ToxCastAssays
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mic
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Office of Research and DevelopmentNational Center for Computational Toxicology
Deepwater Horizon
Oil Exploration Platform Explodes April 20, 2010 • Estimated 4.9 million barrels of South Louisiana Crude released
1.8 million gallons of dispersant used• 1072K surface; 771K subsea• Corexit 9500A (9527 early in spill)
EPA Administrator call for less toxic alternative• Verification of toxicity information on NCP Product Schedule• ORD involvement in assessments of dispersant toxicity
Office of Research and DevelopmentNational Center for Computational Toxicology
Goals of the NCCT Oil Dispersants Project
• Test 8 candidate dispersants for endocrine (ER, AR, TR) activity– Driven by fact that some dispersants contain nonylphenol ethoxylates,
known ER agonists
• Evaluate relative cytotoxicity
• Look for other types of bioactivity using broad in vitro screen
• Return analysis in ~6 weeks
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Office of Research and DevelopmentNational Center for Computational Toxicology
The Dispersants
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Sample NameVolume Received Comments Date Received
Manufacturer/Source
Corexit 9500 1 L hazy yellow 11-May-10 Nalco
JD 2000 10 ml clear yellow 27-May-10Ethox Chemicals, LLC
DISPERSIT SPC 1000 10 ml clear amber 27-May-10 PolychemSea Brat #4 10 ml hazy yellow 27-May-10 Alabaster Corp
Nokomis 3-AA 10 mlclear light
color 27-May-10MAR-LEN Supply inc.
Nokomis 3-F4 10 mlclear light
color 27-May-10MAR-LEN Supply inc.
ZI-400 25 ml clear yellow 29-May-10 ZI Chemical
SAF-RON GOLD 500 ml silver iridescent 4-June-10
Sustainable Environmental Technologies, Inc.
Office of Research and DevelopmentNational Center for Computational Toxicology
Dispersant Cytotoxicity Results
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More potent Less potent
Significantly more cytotoxic (statistically but not biologically)
Bottom line: no significant difference across products
Attagene: HepG2NCGC: Bla ER/ARNHEERL: T47D, MDA, CV1
Office of Research and DevelopmentNational Center for Computational Toxicology
Concentration-Response Profiles for ER
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Estradiol andNonylphenol compounds
ERa.T=Attagene ERa TRANSERE.C=Attagene ERa CIS
CIS Efficacy less than half TRANS efficacy for reference compounds
Dispersant Positives
TRANS assay efficacy neardetection threshold for these dispersants
Office of Research and DevelopmentNational Center for Computational Toxicology
Biological Pathway Results
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Xenobioticmetabolism
EstrogenReceptorActivity
Bottom line: 2 products show weak estrogen activity“Not Significant Biologically”
More potent Less potent
Multi-assay pile-upIndication of generalized cell stressPrelude to cytotoxicity
Office of Research and DevelopmentNational Center for Computational Toxicology
Dispersant Conclusions
• Weak evidence of ER activity in 2 dispersants– Seen in single, perhaps over-sensitive assay (1 of 6)– Not of biological significance– Consistent with presence of NPE– Activity only at concentrations >> seen in Gulf after dilution
• No AR activity• No ER activity seen in Corexit 9500• Corexit is in the middle of the pack for cytotoxicity• No worrisome activity seen in other NR assays
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Analysis of Eight Oil Spill Dispersants Using In Vitro Tests for Endocrine and Other Biological Activity, June 30, 2010 (PDF 47pp) http://www.epa.gov/bpspill/reports/EPADispersantInVitroReport30june2010FINALx.pdfEnviron Sci Technol. 2010 Aug 1;44(15):5979-85.Analysis of eight oil spill dispersants using rapid, in vitro tests for endocrine and other biological activity.Judson RS, Martin MT, Reif DM, Houck KA, Knudsen TB, Rotroff DM, Xia M, Sakamuru S, Huang R, Shinn P, Austin CP, Kavlock RJ, Dix DJ.
Office of Research and DevelopmentNational Center for Computational Toxicology
ToxPi: Prioritizing Chemicals for EDSP21 and Potential Endocrine Activity
Prioritization Index = ToxPi = f(HTS assays + Chemical properties + Pathways)
Bisphenol A Tebuthiuron
Ingenuity pathways
ER
AR
TROther
XME/ADME
KEGG pathways
LogP_TPSA
Predicted CaCO-2
Disease classes
Other NR
Reif et al (2010) Endocrine Profiling and Prioritization of Environmental Chemicals Using ToxCast Data. Environ Health Perspect. Epub PMID: 20826373
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Office of Research and DevelopmentNational Center for Computational Toxicology
ToxPi Ranking of Chemicals
ToxScore
Reif et al, 2010
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HPTE
Methoxychlor
Pyrimethanil
Tebuthiuron
309
Che
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sorte
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Pi
highestlowest
Bisphenol A
Linuron
Office of Research and DevelopmentNational Center for Computational Toxicology
Bioactivity Signature Workflow
ToxCast HTS data (AC50s)
ToxRefDB
UNIVARIATE ASSOCIATIONS• t-test (continuous assay data)• classifier (2x2 contingency table)• Fisher’s exact test (dichotomous data)
MULTIVARIATE MODELS• machine learning• 5-fold cross-validation (80/20 split)• sensitivity analysis (ROC curves)
MULTICELLULAR MODELS• knowledge-base (VT-KB)• computer simulations (VT-SE)• biological inferences / mechanisms
(+)ve (-)ve
(+)ve TP FP
(-)ve FN TN
Office of Research and DevelopmentNational Center for Computational Toxicology
ReproTox Predictions
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Biol Reprod. 2011 Aug;85(2):327-39. Epub 2011 May 12.Predictive model of rat reproductive toxicity from ToxCast high throughput screening.Martin MT, Knudsen TB, Reif DM, Houck KA, Judson RS, Kavlock RJ, Dix DJ.
Office of Research and DevelopmentNational Center for Computational Toxicology
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Office of Research and DevelopmentNational Center for Computational Toxicology
Toxicol Sci. 2011 Aug 26. [Epub ahead of print]Predictive models of prenatal developmental toxicity from ToxCast high-throughput screening data.Sipes NS, Martin MT, Reif DM, Kleinstreuer NC, Judson RS, Singh AV, Chandler KJ, Dix DJ, Kavlock RJ, Knudsen TB.
Developmental Toxicity Predictions
Office of Research and DevelopmentNational Center for Computational Toxicology
Systems Modeling: Blood Vessel Development
v Endothelial proliferation,migration & sprouting (VEGF, Chemokines)
• Extracellular matrix degradation(uPAR, PAI-1, MMPs)
v Neovascular stabilization(Ang/Tie2)
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Environ Health Perspect. 2011 Jul 25. [Epub ahead of print]Environmental Impact on Vascular Development Predicted by High Throughput Screening.Kleinstreuer NC, Judson RS, Reif DM, Sipes NS, Singh AV, Chandler KJ, Dewoskin R, Dix DJ, Kavlock RJ, Knudsen TB.
Office of Research and DevelopmentNational Center for Computational Toxicology
Cell-agent-based models
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• stochastic cellular behaviors• specified cellular activities• PDE solvers for biochemical gradients• toolbox of morphogenetic processes• executes collective cell behavior• enables emergent properties
CompuCell3D.org
Office of Research and DevelopmentNational Center for Computational Toxicology
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Molecular Signals and Cellular Behaviors of Angiogenesis
Office of Research and DevelopmentNational Center for Computational Toxicology
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Angiogenetic field at time=0ECs
ECt
MCIC
SOURCE: Kleinstreuer et al. (in preparation)
Office of Research and DevelopmentNational Center for Computational Toxicology
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CCL2
CXCL10
Ang1/Tie2
VEGF121
sFlt1
VEGF165
MMPs
Virtual angiogenesis
Office of Research and DevelopmentNational Center for Computational Toxicology
5HPP-33 5HPP-33
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In vitroSOURCE: Noguchi et al. 2005, Bioorg Med ChemLett. 15 :5509-13.(experimental, 100 uM)
Thalidomide 5HPP-33control
In silicoSOURCE: Kleinstreuer et al. 2011, in preparation
(ToxCastDB, 40 uM)
quantitative predictionemergent from ToxCast HTS data input (AC50s)
TEST CASE: Thalidomide and 5HPP-33
Office of Research and DevelopmentNational Center for Computational Toxicology
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PFOS
PFOSPFOS
PFOS
PFOA
PFAA_Alt1
PFAA_Alt2PFAA_Alt3PFAA_Alt4
Office of Research and DevelopmentNational Center for Computational Toxicology
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HUVEC Proliferation: PFAA_Alt2HUVEC Proliferation: PFAA_Alt1
HUVEC Proliferation: PFOS HUVEC Proliferation: PFOA
Inhibition of Endothelial Cell Proliferation
Office of Research and DevelopmentNational Center for Computational Toxicology
PFOA, PFOS, and PFAA Alternatives:in silico vasculogenesis assay
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PFOA PFOS
PFAA_Alt1 PFAA_Alt2
Office of Research and DevelopmentNational Center for Computational Toxicology
Quantitative Effects:in silico chemical exposure
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Total Cell Number Total VEGF Concentration
Office of Research and DevelopmentNational Center for Computational Toxicology
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Green Chemistry and CompTox
• Less expensive, higher throughput approach to hazard prediction
• Prioritization of green alternative chemicals for further development and testing
• High throughput exposure predictions also needed
in vitro testing
ToxicityPathways
Prioritization, Predictiveand Systems Models
Office of Research and DevelopmentNational Center for Computational Toxicology
Virtual EmbryoTom Knudsen Nicole KleinstreuerNisha SipesAmar Singh (LHM)Michael Rountree (SSC)Richard Spencer (EMVL)Rob DeWoskinSid Hunter Stephanie PadillaKelly Chandler
US EPA CompTox
EPA Funded STAR GranteesUniversity of HoustonUniversity of TexasIndiana UniversityUniversity of North Carolina
Virtual LiverImran Shah John WambaughJohn JackWoody Setzer
The Hamner InstitutesRusty ThomasBarbara Wetmore
ToxCastKeith HouckAnn RichardBob KavlockDavid DixMatt MartinDavid ReifRichard JudsonSteve LittleAmy WangPatra VolarathSumit Gangwal