Office of Research and Development NHEERL, Integrated System Toxicology Division 0 McKim Conference, May 19, 2010 Duluth MN Cellular Stress Response: Systems-based Approach to Toxicant Identification and Characterization: relevance to genotoxicity testing Ram Ramabhadran Office of Research and Development NHEERL, Integrated System Toxicology Division Outline Problem statement- current limitations Need for novel approaches 3 Rs and Tox Testing in 21 st Century report Cellular stress response as an early indicator of biological response Stress response biology and current approach to predict adverse outcomes Specific application and problems in predicting genotoxic responses to compounds Office of Research and Development NHEERL, Integrated System Toxicology Division Regulatory Challenges Large number of environmental compounds with limited toxicity information HPVs, etc. 90,000 chemicals on the EPA TSCA inventory and ~9,000 chemicals used in quantities >10,000 lbs. 1,468 chemicals have been tested in a rodent cancer bioassay (CPD, 2005). Inerts, Mixtures Extrapolation from model systems to human exposure effects Imperative to reduce the number of animal used in testing- 3Rs reduce, refine and replace Office of Research and Development NHEERL, Integrated System Toxicology Division 3 Need to develop cost-effective high-throughput screening approaches to facilitate prioritization of data-poor chemicals Need to reduce and refine current level of animals required for regulatory testing Need to collect data on human cell and tissues Incorporation of toxicity pathways Exploitation of screen-able pathway nodes Utility beyond prioritization? Data needs for QSAR Challenge and Approach Office of Research and Development NHEERL, Integrated System Toxicology Division Office of Research and Development NHEERL, Integrated System Toxicology Division the 21st Century: A Vision and a Strategy Office of Research and Development NHEERL, Integrated System Toxicology Division 66 6 Adapted from: Toxicity Testing in the Twenty-first Century: A Vision and a Strategy, National Research Council Normal Biologic Function Cell Injury Morbidity and Mortalilty Biologic Inputs Exposure Tissue Dose Biologic Interaction Perturbation Early Cellular Changes Adaptive Stress Response Toxicity Pathways & Adaptive Stress Responses: Canaries in the Intracellular Coalmine Cell death, Regeneration Cancer? Office of Research and Development NHEERL, Integrated System Toxicology Division Prototypic Toxicity Pathways NAS Report pp Years of work Office of Research and Development NHEERL, Integrated System Toxicology Division 88 Cellular Stress Responses: From Pathways to Prediction Office of Research and Development NHEERL, Integrated System Toxicology Division 99 Oxidative Stress Genotoxic Stress Heat Shock ER Stress Metal Response Inflammation Hypoxia Major Stress Response Pathways (Relatively well understood) Office of Research and Development NHEERL, Integrated System Toxicology Division 10 Adaptive Stress-Response Pathways Protective signaling pathways activated in response to environmental insults such as chemical toxicity Present in all metazoan cells and highly conserved Broad indicators of early cellular toxicity (perturbation) Triggered at low doses before more apical effects such as cell death or apoptosis Manageable number of key cellular stress pathways identified Pathways mechanistically well-characterized Share common architecture Office of Research and Development NHEERL, Integrated System Toxicology Division 11 StRE Target Genes TF Stress Pathway Architecture Perturbation Sensor Transducers Office of Research and Development NHEERL, Integrated System Toxicology Division 12 Integrated Response System MAPKErkp38PKC CamK2 CK2Plk1ATMJnkChk1 Chk2IKKPI3K Akt TKsPKAMsk1CK1 Transducers Sensors/TFs Keap1 Nrf2 hsp90 HSF1 MDM2 p53 VHL HIF1 BiP XBP/ATF IKB NFkB MTF1NFAT5 ??? Office of Research and Development NHEERL, Integrated System Toxicology Division 13 StRE Target Genes TF Integration of Multiple Upstream Inputs: Pathways to Assays Sensor T1 Luciferase T2 T3 T4 Office of Research and Development NHEERL, Integrated System Toxicology Division 14 Reporter Assay Construction PathwayTFReporter/responder Genes Oxidative StressNrf2AREx7, HMOX1, NQO1 Heat ShockHSF1HSPA6, DNAJB1, DNAJB11 ER StressXBP1ERSEx5, GRP78 HypoxiaHIF1HREx5, VEGF DNA damagep53p53REx4, CDKN1A, MDM2, GADD45A InflammationNFkBNFkBREx5, IL8, TNFA, IL2 Metal StressMTF1MT2A, MT1E Office of Research and Development NHEERL, Integrated System Toxicology Division 15 Multi-Stress Response Strategies (Criticality of testing dose) No genotox pathway Office of Research and Development NHEERL, Integrated System Toxicology Division 16 Compound-Specific Profiling Simmons, et al., Toxicological Sciences 111(2), 202225 (2009) Office of Research and Development NHEERL, Integrated System Toxicology Division 17 Recap Adaptive stress response pathways share a common exploitable architecture The transducer layer of the pathways is heavily cross-wired and plays a role in non-stress biology The transcription factor/sensor complex integrates multiple signaling inputs Activated TF can be measured using reporter genes that come in two basic flavors Low basal activity high dynamic range Because the patterns of activation vary by compound, a battery of such assays can be used to build compound- specific stress response profiles Office of Research and Development NHEERL, Integrated System Toxicology Division 18 Office of Research and Development NHEERL, Integrated System Toxicology Division 19 Office of Research and Development NHEERL, Integrated System Toxicology Division 20 Moving Beyond Prioritization: QBAR? pathways time cell type 20 chemicals assays cell type rotenone ? compounds dose-response Office of Research and Development NHEERL, Integrated System Toxicology Division 21 -naphth MMS Metam Iodo HQ Maneb tBHQ Nabam CdCl 2 ZnCl 2 Propineb CuCl 2 OPD B-naphth Thiram MeHg 1C-24DNB pBQ BME EtBr EMS AP-1NFkBAREhsp70MT2ACMVGADD153Grp94p21p53Grp78SV40 50n M 50u M 500u M inactive QBAR Concept Supersedes QSAR, includes metal ions & contaminants, etc. Office of Research and Development NHEERL, Integrated System Toxicology Division 22 Summary Adaptive stress response pathways are ideally suited for adaptation to qHTS assays These assays have been used successfully to screen large compound libraries at NCGC Low cost and automation of stress response assays facilitates multidimensional data collection Unsupervised hierarchical clustering should permit grouping of compounds based on similarities in biological response profiles instead of structural similarity With sufficient biological resolution of chemical space, compounds that cluster together should operate through similar modes/mechanisms of action Prior mode/mechanism knowledge is not a requisite Validation by whole animal study will be required Office of Research and Development NHEERL, Integrated System Toxicology Division p53: Master Switch for Genotoxicity One of the most studied proteins ($Bs) Mutations or loss found in 50 % of cancers- tumor suppressor Responds by stabilization to gentoxic stresses (both direct and indirect) Causes cell cycle arrest and apoptosis Office of Research and Development NHEERL, Integrated System Toxicology Division p53: Master Switch for Genotoxicity ? Pluquet and Hainaut (2001) Cancer Letters 174,115. Office of Research and Development NHEERL, Integrated System Toxicology Division Activators of p53 Pluquet and Hainaut (2001) Cancer Letters 174,115. Office of Research and Development NHEERL, Integrated System Toxicology Division P53 Signaling Pathways Anderson and Appella (2009): In: Handbook of Cell Signaling, 2nd edition. R. A. Bradshaw and E. A. Dennis, (Eds), Oxford: Academic Press, 2009 Office of Research and Development NHEERL, Integrated System Toxicology Division Cell Cycle Arrest vs. Apoptosis Schlereth, et al. Molecular Cell 38, 356368, May 14, 2010 Office of Research and Development NHEERL, Integrated System Toxicology Division p53:Post-translational Modifications Anderson and Appella (2009): In: Handbook of Cell Signaling, 2nd edition. R. A. Bradshaw and E. A. Dennis, (Eds), Oxford: Academic Press, 2009 Office of Research and Development NHEERL, Integrated System Toxicology Division p53 based Genotoxicity Assays Commercial Assays Reporter Antibody Gentronix InVitrogen Cellumen Knight, et al. (2009) Regulatory Toxicology and Pharmacology 55:188199 Office of Research and Development NHEERL, Integrated System Toxicology Division GenePromoterIntron p53RE (Cignal) 3N/A GADD45A01(3) CDKN1A (p21) 24 (1) MDM21N/A p53 Binding Sites in Responder Genes RRRCWWGYYY (R = A, G; W = A,T; Y = C, T) separated by 014 base pairs Office of Research and Development NHEERL, Integrated System Toxicology Division p53 Reporter Constructs GADD45A Luciferase p53RE Luciferase GADD45A Luciferase CDKN1A (p21) Luciferase GADD45A CDKN1A (p21) Luciferase CDKN1A (p21) GADD45A Luciferase CDKN1A (p21) Luciferase Cp Gp Pp GpGi GpPi PpPi PpGi Promoter sequence Luciferase Open Reading Frame Intronic sequence Promoters cloned 5 to luciferase ORF; introns cloned 3 to ORF. (Cignal) Office of Research and Development NHEERL, Integrated System Toxicology Division p53 Reporter Constructs (cont) p53RE Luciferase MpCp Promoter sequence Luciferase Open Reading Frame Intronic sequence MDM2 p53RE Luciferase GpCp GADD45A MDM2 Luciferase CpMp p53RE GADD45A Luciferase CpGp p53RE MDM2 Luciferase GiMp GADD45A Luciferase GiGp GADD45A Promoter fused 5 to luciferase ORF; introns fused to promoters and cloned 5 to ORF. Office of Research and Development NHEERL, Integrated System Toxicology Division MMS Office of Research and Development NHEERL, Integrated System Toxicology Division MMS Office of Research and Development NHEERL, Integrated System Toxicology Division Chloroquine Office of Research and Development NHEERL, Integrated System Toxicology Division 5-Fluorouracil Office of Research and Development NHEERL, Integrated System Toxicology Division Doxorubicin Office of Research and Development NHEERL, Integrated System Toxicology Division Doxorubicin Office of Research and Development NHEERL, Integrated System Toxicology Division Cell-type Dependent Response Cignal Reporter-Doxorubicin Office of Research and Development NHEERL, Integrated System Toxicology Division Diversity of p53 responses Staib, et al. (2005) Cancer Res., 65: Office of Research and Development NHEERL, Integrated System Toxicology Division Causes for Variable Responses Mode of action of compound- p53 modification Direct vs. indirect DNA damage Cell type level of p53 and other components Dose - growth arrest vs. apoptosis Need for dose response and cytotox Exposure duration- temporality of activation Need for time course Office of Research and Development NHEERL, Integrated System Toxicology Division Current Efforts Identify a gene that respond to multiple stimuli (single reporter assay) Use a set of responder genes that improve coverage- possibly multiplex Choose appropriate cell type that give the best response Lentiviral vectors Improve signal/noise by genetic manipulations Incorporate metabolism Office of Research and Development NHEERL, Integrated System Toxicology Division 43 Office of Research and Development NHEERL, Integrated System Toxicology Division p53 Activation by -Radiation Hamstra et al. Cancer Research, 66, 7482 (2006) Office of Research and Development NHEERL, Integrated System Toxicology Division 45 control 0.2uM 5uM 125uM Developmental toxicology of cadmium in living embryos of a stable transgenic zebrafish line. Environ Health Perspect Oct;110(10): Blechinger SR, Warren JT Jr, Kuwada JY, Krone PH. Utilizing In vivo Stress Assays Office of Research and Development NHEERL, Integrated System Toxicology Division 46 Acknowledgements US EPA NHEERL Chun-Yang Fan (Sygenta) Jeanene Olin Theresa Freudenrich US EPA, National Center for Computational Toxicology David Reif, Bob Kavlock Keith Houck, David Dix NIH Chemical Genomic Center Menghang Xia, Sunita Shukla Ruili Huang, Chris Austin Jim Inglese National Toxicology Program Ray Tice,Kristine Witt Open Biosystems (Thermo-Fisher) John Wakefield, Attila Seyhan (Wyeth) The Hamner Institutes Rusty Thomas Steven Simmons Brookhaven National Laboratory Carl Anderson