The ToxCast TM Program – predicting hazard, characterizing toxicity pathways and prioritizing the toxicity testing of environmental chemicals This work was reviewed by EPA and approved for presentation but does not Office of Research and Development necessarily reflect official Agency policy. Mention of trade names or commercial National Center for Computational Toxicology products does not constitute endorsement or recommendation by EPA for use.
The Need For a New Approach Office of Research and Development 1 National Center for Computational Toxicolo
EPA’s Need for Chemical Prioritization Too Many Chemicals Too High a Cost 90,000 100000 11,000 Cancer 10000 IRIS DevTox TRI Pesticide Actives 1000 NeuroTox CCL 1&2 Pesticide Inerts ReproTox 100 HPV ImmunoTox MPV Current 10 MPV Historical PulmonaryTox Millions $ TSCA Inventory 1 Data Collection …and not enough data. Office of Research and Development January 23, 2009 2 National Center for Computational Toxicology
Ways to Prioritize: Animal studies • – cost, time, ethical considerations QSAR • – domain of applicability, availability of models Bioactivity Profiling • – biologically relevant chemical characterization – HTS methods – ToxCast Office of Research and Development January 23, 2009 3 National Center for Computational Toxicology
ToxCast TM : a computational toxicology approach based on high-throughput bioactivity profiling • 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 NIH: 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://actor.epa.gov/actor/ Office of Research and Development January 23, 2009 4 National Center for Computational Toxicology
Phased Development of ToxCast Target Number of Chemical Number of Cost per Phase Purpose Chemicals Criteria Assays Chemical Date Data Rich Signature I 320 552 $20k FY08 Development (pesticides) Ib 15 Nanomaterials Pilot 166 $10K FY09 Data Rich IIa >300 Validation >400 ~$20-25k FY09 Chemicals Known Human IIb >100 Extrapolation >400 ~$20-25k FY09 Toxicants Expanded IIc >300 Structure and Use Extension >400 ~$20-25k FY10 Diversity IId >12 Nanomaterials PMN >200 ~$15-20K FY09-10 Prediction and III Thousands Data poor >300 ~$15-20k FY11-12 Prioritization January 23, 2009 5
ToxCast_320 Phase I Chemicals 309 unique structures 3 triplicates, 5 duplicates for QC Acetylcholine esterase inhibitors 8 metabolites conazole fungicides Sodium channel modulators 291 total pesticide actives pyrethroid ester insecticides organothiophosphate acaricides 273 registered pesticide actives dinitroaniline herbicides 22 pesticide inerts MOA Classes with pyridine herbicides 33 antimicrobials thiocarbamate herbicides > 3 chemicals imidazolinone herbicides 56 of 73 proposed Tier 1 EDSP organophosphate insecticides phenyl organothiophosphate insecticides 23 IUR aliphatic organothiophosphate insecticides amide herbicides 13 HPV aromatic fungicides 11 HPV Challenge Misc chloroacetanilide herbicides chlorotriazine herbicides 73 OW PCCL growth inhibitors 11 CCL1 organophosphate acaricides 10 CCL2 oxime carbamate insecticides phenylurea herbicides 25 CCL3 pyrethroid ester acaricides strobilurin fungicides 122 IRIS chemicals unclassified acaricides unclassified herbicides Office of Research and Development January 23, 2009 6 National Center for Computational Toxicology Classification based on OPPIN
High-Throughput Screening Assays batch testing of chemicals for pharmacological/toxicological endpoints using automated liquid handling, detectors, and data acquisition LTS MTS HTS uHTS Gene-expression 1000s/day 10s-100s/yr 10,000s- 100,000s/day 10s-100s/day Human Relevance/ Cost/Complexity Throughput/ Simplicity Office of Research and Development National Center for Computational Toxicology
ToxCast Phase I Datasets 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) 20 Assay sources ToxCast 1.1 (January, 2008) • – Neurite outgrowth HCS (NHEERL) 554 Endpoints – Cell proliferation (NHEERL) – Zebrafish developmental toxicity (NHEERL) ToxCast 1.2 (June, 2008) • – XME Gene Regulation (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 Zebrafish vascular/cardiotoxicity (Zygogen) – microarray with metabolism (Solidus) – HTS stress response (NHEERL+NCGC ) Office of Research and Development January 23, 2009 8 National Center for Computational Toxicology
ToxCast Assays Biochemical Assays Cellular Assays Protein families Cell lines • • – HepG2 human hepatoblastoma – GPCR – A549 human lung carcinoma – NR – HEK 293 human embryonic kidney – Kinase – Phosphatase Primary cells • – Protease – Human endothelial cells – Other enzyme – Human monocytes – Human keratinocytes – Ion channel – Human fibroblasts – Transporter – Human proximal tubule kidney cells – Human small airway epithelial cells Assay formats • Biotransformation competent cells – Radioligand binding • – Primary rat hepatocytes – Enzyme activity – Primary human hepatocytes – Co-activator recruitment Assay formats • – Cytotoxicity – Reporter gene – Gene expression – Biomarker production Office of Research and Development – High-content imaging for cellular January 23, 2009 9 National Center for Computational Toxicology phenotype
228 Assays Biochemical Assay Results 320 Chemicals Log IC50 (M)
Examples of Chemical Activity in Biochemical Assays Prochloraz HPTE (IC50 log M) (IC50 log M) Boric Acid Bisphenol A (IC50 log M) (IC50 log M) Office of Research and Development January 23, 2009 11 National Center for Computational Toxicology
PCA Mapping of CYP Inhibition Conazoles OPs Office o Nation
CYP Inhibition Chemical Class Conazoles OPs • chemical class specificity • usually many cyps inhibited or none • may need to consider mechanism of inhibiton • may need to consider induction of cyps IC50 (log M) • may provide predictions of bioavailability
ToxCast and Biotransformation HPTE Methoxychlor Glucocorticoid receptor Opioid Dopamine, receptors Androgen dopamine Estrogen Progesterone receptor transporter Receptor receptor 14
Cellular Assays Types of Assays • – Known toxicity pathways and targets biomarker measurements • reporter gene assays • – General cytotoxicity – Toxicity cellular phenotypes Cell lines and primary cells • Generally screened at up to 100 M or used maximally • tolerated concentration defined by general cytotoxicity determination Concentration-response format used and EC 50 generated • Office of Research and Development January 23, 2009 15 National Center for Computational Toxicology
Primary Human Cell Systems (BioSeek, Inc.) 8 Assay systems • 87 endpoints • 4 concentrations • Office of Research and Development January 23, 2009 16 National Center for Computational Toxicology
Functional Similarity Map of ToxCast Library
Mitochondrial Dysfunction and Endoplasmic Reticulum Stress Classes myxothiazol pyraclostrobin trifloxystrobin benomyl fludioxonil paclitaxel Office of Research and Dev 23, 2009 18 National Center for Computati
Use of BioSeek Data in ToxCast Individual assay endpoints become part of larger ToxCast data set for • developing predictive models BioMAP signatures used to provide mechanistic understanding of • potential mechanism/mode of action May be able to validate signatures with other phenotypic assays • Office of Research and Development January 23, 2009 19 National Center for Computational Toxicology
High-Content Screening of Cellular Phenotypic Toxicity Parameters (Cellumen, Inc.) Technology: automated fluorescent microscopy • Objective: Determine effects of chemicals on toxicity • biomarkers in a cell culture of human liver hepatoma HepG2 Stress Pathway Activation Panel 1 design* : • Multiple mechanisms of toxicity Organelle • Acute, early & chronic exposure Functions • 384-well capacity • HepG2 Oxidative Stress • 1 o rat hepatocytes DNA Damage Cell Cycle CSK Integrity Office of Research and Development January 23, 2009 20 National Center for Computational Toxicology
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