McKim Conference, May 19, 2010 Duluth MN

1. Cellular Stress Response: Systems-based Approach to Toxicant Identification and Characterization: relevance to genotoxicity testingRam Ramabhadran McKim Conference, May 19, 2010 Duluth MN 0

2. Outline • Problem statement- current limitations • Need for novel approaches • 3 Rs and Tox Testing in 21st 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

3. 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- • 3R’s – reduce, refine and replace

4. Challenge and Approach • 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 3

7. Toxicity Pathways & Adaptive Stress Responses: Canaries in the Intracellular Coalmine Exposure Tissue Dose Biologic Interaction Perturbation Adapted from: Toxicity Testing in the Twenty-first Century: A Vision and a Strategy, National Research Council. 2007. Normal Biologic Function Biologic Inputs Early Cellular Changes Cell Injury Adaptive Stress Response Cell death, Regeneration Cancer? Morbidity and Mortalilty 6 6 6

8. Prototypic Toxicity PathwaysNAS Report pp 63-64 • Years of work

9. Cellular Stress Responses: From Pathways to Prediction 8 8

10. Major Stress Response Pathways (Relatively well understood) Oxidative Stress Genotoxic Stress Heat Shock ER Stress Hypoxia Inflammation Metal Response 9 9

11. 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 10 10

12. Stress Pathway Architecture Perturbation Sensor TF Transducers StRE Target Genes 11 11

13. Integrated Response System Transducers MAPK Erk p38 PKC CamK2 CK2 Plk1 ATM Jnk Chk1 Chk2 IKK PI3K Akt TKs PKA Msk1 CK1 Sensors/TFs Keap1 MDM2 BiP IKB Nrf2 p53 XBP/ATF NFkB hsp90 VHL ??? HSF1 HIF1 MTF1 NFAT5 12 12

14. Integration of Multiple Upstream Inputs: Pathways to Assays T2 T3 T1 T4 Sensor TF StRE Target Genes Luciferase 13 13

15. Reporter Assay Construction 14

16. Multi-Stress Response Strategies (Criticality of testing dose) No genotox pathway 15 15

17. Compound-Specific Profiling Simmons, et al., Toxicological Sciences 111(2), 202–225 (2009) 16 16

18. 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 17 17

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21. Moving Beyond Prioritization: QBAR? cell type cell type rotenone pathways compounds assays ? time dose-response chemicals 20 20

22. α-naphth MMS Metam Iodo HQ Maneb tBHQ Nabam CdCl2 ZnCl2 Propineb CuCl2 OPD B-naphth Thiram MeHg 1C-24DNB pBQ BME EtBr EMS AP-1 NFkB ARE hsp70 MT2A CMV GADD153 Grp94 p21 p53 Grp78 SV40 50nM 50uM 500uM inactive QBAR ConceptSupersedes QSAR, includes metal ions & contaminants, etc. 21 21

23. 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

24. ? p53: Master Switch for Genotoxicity Pluquet and Hainaut (2001) Cancer Letters 174,1–15 .

25. Activators of p53 Pluquet and Hainaut (2001) Cancer Letters 174,1–15 .

26. 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

27. Cell Cycle Arrest vs. Apoptosis Schlereth, et al. Molecular Cell 38, 356–368, May 14, 2010

28. 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

29. Cellumen Gentronix Reporter Antibody InVitrogen Reporter p53 based Genotoxicity AssaysCommercial Assays Knight, et al. (2009) Regulatory Toxicology and Pharmacology 55:188–199

30. p53 Binding Sites in Responder Genes RRRCWWGYYY (R = A, G; W = A,T; Y = C, T) separated by 0–14 base pairs

31. Luciferase Cp (Cignal) p53RE Luciferase Gp GADD45A Luciferase Pp CDKN1A (p21) Luciferase GpGi GADD45A GADD45A Luciferase GpPi CDKN1A (p21) GADD45A Luciferase PpPi CDKN1A (p21) CDKN1A (p21) Luciferase PpGi CDKN1A (p21) GADD45A Promoter sequence Luciferase Open Reading Frame • Promoters cloned 5’ to luciferase ORF; introns cloned 3’ to ORF. Intronic sequence p53 Reporter Constructs

32. Luciferase MpCp MDM2 p53RE Luciferase GpCp GADD45A p53RE Luciferase CpMp p53RE MDM2 Luciferase CpGp p53RE GADD45A Luciferase GiMp GADD45A MDM2 Luciferase GiGp GADD45A GADD45A • Promoter fused 5’ to luciferase ORF; introns fused to promoters and cloned 5’ to ORF. Promoter sequence Luciferase Open Reading Frame Intronic sequence p53 Reporter Constructs (con’t)

33. MMS

34. MMS

35. Chloroquine

36. 5-Fluorouracil

37. Doxorubicin

38. Doxorubicin

39. Cell-type Dependent ResponseCignal Reporter-Doxorubicin

40. Diversity of p53 responses Staib, et al. (2005) Cancer Res., 65: 10255-64

41. 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

42. 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

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44. p53 Activation by γ-Radiation Hamstra et al. Cancer Research, 66, 7482 (2006)

45. Utilizing In vivo Stress Assays control 0.2uM 5uM Blechinger SR, Warren JT Jr, Kuwada JY, Krone PH. Developmental toxicology of cadmium in living embryos of a stable transgenic zebrafish line. Environ Health Perspect. 2002 Oct;110(10):1041-6. 125uM 45

46. Acknowledgements Steven Simmons US EPA NHEERL Chun-Yang Fan (Sygenta) Jeanene Olin Theresa Freudenrich NIH Chemical Genomic Center Menghang Xia, Sunita Shukla Ruili Huang, Chris Austin Jim Inglese US EPA, National Center for Computational Toxicology David Reif, Bob Kavlock Keith Houck, David Dix National Toxicology Program Ray Tice,Kristine Witt Open Biosystems (Thermo-Fisher) John Wakefield, Attila Seyhan (Wyeth) The Hamner Institutes Rusty Thomas Brookhaven National Laboratory Carl Anderson 46 46