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Cellular Stress Response: Systems-based Approach to Toxicant Identification and Characterization: relevance to genotoxicity testing Ram Ramabhadran. McKim Conference, May 19, 2010 Duluth MN. 0. Outline. Problem statement- current limitations Need for novel approaches

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Outline 1277445

Cellular Stress Response: Systems-based Approach to Toxicant Identification and Characterization: relevance to genotoxicity testingRam Ramabhadran

McKim Conference, May 19, 2010

Duluth MN

0


Outline
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


Regulatory challenges
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


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    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

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    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

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    6

    6


    Prototypic toxicity pathways nas report pp 63 64
    Prototypic Toxicity PathwaysNAS Report pp 63-64

    • Years of work


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    Cellular Stress Responses:

    From Pathways to Prediction

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    Major Stress Response Pathways

    (Relatively well understood)

    Oxidative Stress

    Genotoxic Stress

    Heat Shock

    ER Stress

    Hypoxia

    Inflammation

    Metal Response

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    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

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    10


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    Stress Pathway Architecture

    Perturbation

    Sensor

    TF

    Transducers

    StRE

    Target Genes

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    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

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    12


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    Integration of Multiple Upstream Inputs: Pathways to Assays

    T2

    T3

    T1

    T4

    Sensor

    TF

    StRE

    Target Genes

    Luciferase

    13

    13



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    Multi-Stress Response Strategies

    (Criticality of testing dose)

    No genotox pathway

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    Compound-Specific Profiling

    Simmons, et al., Toxicological Sciences 111(2), 202–225 (2009)

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    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

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    Moving Beyond Prioritization: QBAR?

    cell type

    cell type

    rotenone

    pathways

    compounds

    assays

    ?

    time

    dose-response

    chemicals

    20

    20


    Qbar concept supersedes qsar includes metal ions contaminants etc

    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.

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    21


    P53 master switch for genotoxicity
    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


    P53 master switch for genotoxicity1

    ?

    p53: Master Switch for Genotoxicity

    Pluquet and Hainaut (2001) Cancer Letters 174,1–15

    .


    Activators of p53
    Activators of p53

    Pluquet and Hainaut (2001) Cancer Letters 174,1–15

    .


    P53 signaling pathways
    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


    Cell cycle arrest vs apoptosis
    Cell Cycle Arrest vs. Apoptosis

    Schlereth, et al. Molecular Cell 38, 356–368, May 14, 2010


    P53 post translational modifications
    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


    P53 based genotoxicity assays commercial assays

    Cellumen

    Gentronix

    Reporter

    Antibody

    InVitrogen

    Reporter

    p53 based Genotoxicity AssaysCommercial Assays

    Knight, et al. (2009) Regulatory Toxicology and Pharmacology 55:188–199


    Outline 1277445

    p53 Binding Sites in Responder Genes

    RRRCWWGYYY (R = A, G; W = A,T; Y = C, T)

    separated by 0–14 base pairs


    Outline 1277445

    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


    Outline 1277445

    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)








    Cell type dependent response cignal reporter doxorubicin
    Cell-type Dependent ResponseCignal Reporter-Doxorubicin


    Diversity of p53 responses
    Diversity of p53 responses

    Staib, et al. (2005) Cancer Res., 65: 10255-64


    Causes for variable responses
    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


    Current efforts
    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



    P53 activation by radiation
    p53 Activation by γ-Radiation

    Hamstra et al. Cancer Research, 66, 7482 (2006)


    Outline 1277445

    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

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    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

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