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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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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
slide4

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

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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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Cellular Stress Responses:

From Pathways to Prediction

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slide10

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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slide12

Stress Pathway Architecture

Perturbation

Sensor

TF

Transducers

StRE

Target Genes

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slide13

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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Integration of Multiple Upstream Inputs: Pathways to Assays

T2

T3

T1

T4

Sensor

TF

StRE

Target Genes

Luciferase

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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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slide18

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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slide19

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slide20

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slide21

Moving Beyond Prioritization: QBAR?

cell type

cell type

rotenone

pathways

compounds

assays

?

time

dose-response

chemicals

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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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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

slide30

p53 Binding Sites in Responder Genes

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

separated by 0–14 base pairs

slide31

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

slide32

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)

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)

slide45

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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