U'S' Department of Energy Low Dose Radiation Research Program Developing a Scientific Basis for Radi

1
U.S. Department of Energy Low Dose Radiation
Research ProgramDeveloping a Scientific Basis
for Radiation Risk Estimates The Goal of the
DOE Low Dose Research Program
Antone L. Brooks Washington State University- Tri
Cities Richland, Washington 99352
Health Physics July
2004
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Problems with Low Dose Epidemiology

• Background radiation
• Background cancer
• High signal to noise ratio
• Radiation is a poor mutagen/carcinogen, but a
  very good cell killer

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Background Radiation
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Radiation is everywhere
Cosmic
Inhaled Radon
Bodies
Plants
Radioactive Elements
We live in a sea of radiation
Rocks
5

Normal annual exposure from man-made radiation

• About 70 mrem/yr
• Medical procedures
  53 mrems
• Consumer products
  10 mrems
• One coast to coast airplane flight
  2 mrems
• Watching color TV
  1 mrem
• Sleeping with another person
  1 mrem
• Weapons test fallout
  less that 1 mrem
• Nuclear industry
  less than 1 mrem

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Medical Radiation Exposures

• 200 million medical x-rays/year
• X-ray 1 mGy
• 100 million dental x-rays/year
• Dental 0.6 mGy
• 10 million doses of radiopharmaceuticals/yr
• 67 million CT scans/year
• Head scan 40-60 mGy/scan
• Body scan 10-40 mGy/scan
• Large doses from radiation therapy

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Dose Ranges
(mSievert)
Experimental Radiobiology
Cancer Epidemiology
DOE Low Dose Program
Medical Diagnostics
Regulatory Standards
Negligible Doses
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Background Cancer
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(No Transcript)
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Effects of Atomic Bomb

• Killed outright by the bomb or acute radiation
  effects.
• Survived for lifespan study

• 100,000 people
• 86,572 people

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Atomic Bomb Survivor Excess Cancer
Population of Survivors Studied 86,572
Total Cancers observed after the Bomb 8,180
Total Cancers Expected without Bomb 7,743
Total Cancer Excess 437
Excess Leukemia 104
Excess Tumor 334
437


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Why now?

• Standards set from high dose effects, but low
  dose effects have not been measurable until now
• New technological developments and biological
  discoveries have made it possible for mechanistic
  studies of low dose effects

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LNTH Assumption with Dose
Low dose x large number of subjects
High dose x small number of subjects
Energy to system
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Needed Shift in Research Emphasis
Low Dose Alteration of Homeostasis
High Dose Damage and Health Effects
Mechanisms of Action Science-based Standards

Common Pathways for Environmental Interaction
Repair and Protective Mechanisms
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DOE Low-Dose Radiation Research Program

• A 10 year program
• Focused on biological mechanisms of low-dose (lt
  0.1 Gy) and low dose-rate (lt 0.1 Gy / Yr)
  radiation
• International in scope (currently 70 projects)
• To develop a scientific basis for radiation
  standards
• http//lowdose.tricity.wsu.edu

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Key Research Areas

• Technological Advances
• Biological Advances

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Mechanisms for Cancer Induction

• High Doses
• Cancer
• Changes in gene expression
• Mutations
• Chromosome aberrations
• Genomic instability
• Cell killing
• Stimulate cell proliferation
• Tissue and matrix disruption
• Inflammation

• Low Doses
• Cancer?
• Changes in gene expression
• Mutations
• Chromosome aberrations
• Adaptive response

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Self sufficiency in growth signals
Insensitivity to anti-growth signals
Evading apoptosis
Sustained angiogenesis
Tissue invasion and metastasis
Limitless replicative potential
Hanahan and Weinberg 2000
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Gene Mutation and Expression in Cancer
Mutation Theory
Tissue Theory
Single cell origin of cancer
ROS status Matrix interactions Gene activation
Normal
Normal
Gene Activation
Down Regulation
Initiation
Promotion
Progression
Progression
Gene Mutation- a rare event
Tissue response- a frequent event
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Cell Transformation
Proliferation
X-Irradiation
300 Cells
13 cell divisions
Confluence
1/10
1/100
1/1000
Proliferation
1/10,000
Anne Kennedy 1985
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Cellular Changes

• Adaptive Response
• Small dose alters response to large dose
• Small dose decreases spontaneous damage
• Bystander Effects
• Cells respond without energy deposition
• Cell-cell communication
• Materials into the media
• Genomic Instability
• Loss of genetic control many cell generations
  after the radiation exposure

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Relationship between biological responses to
radiation
Adaptive Response
Genomic Instability
Bystander Effects
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Focused X-ray Microbeam
Spatial Resolution of the
Microfocus
Source
Cell
OSA
9mm
Apodized
Spot
Zone-plate
200µm
Michael et al.
Gray Laboratory
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Spatially-Resolved Electron Gun
Pacific Northwest National Laboratory
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Alpha-Particle Radiation System
Video Camera
Microscope Objective Lens
Newport Positioning Stage
Mylar Bottom Petri Dish
Texas AM
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Microbeam Hit Accuracy
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Bystander Effects in vitro
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Microbeam
10 of cells hit with 1 alpha particles
Each cell hit by one particle
Sawant et al. 2000
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Cell Transformation
Sawant et al.2000
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Radiation induced Bystander Effects From Tritium
1.0
1.0
Surviving Fraction
0.1
Surviving Fraction
0.01
0.1
0 10 20 30 40 50 60
0 50 100 150 200 250
Cluster Activity (kBq)
Lindane (?M)
Bishayee et al. 1999
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Radiation in Cytoplasm Produces Mutations
Zhou et al. 2000
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Mutation Frequency
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Mutation Frequency
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Bystander EffectAll-or-none dose response
Fraction of cells damaged
One cell targeted per dish Four cells
targeted per dish
Numbers of particles per targeted cell
Belyakov et al. 2001
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Bystander Effects in vivo

• Bystander effects after single, high-dose, acute
  radiation exposure.
• Tissue exposed
• Non-exposed organs or tissues
• Bystander effects following internally deposited
  radioactive materials.
• Tissues exposed
• Non-exposed organs or tissues

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Bystander Effects in vivo following Acute
radiation exposure

• Clastogenic factors
• Abscopal effects

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Clastogenic Factors

• A-bomb survivors
• Chernobyl clean-up
• Radiation therapy
• Experimental animals

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Induction of Clastogenic Factors in Experimental
Animals
Blood Sample
Blood Sample
Plasma
Blood Lymphocytes
Culture for chromosome analysis
Morgan 2003
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The Influence of Communication on
Radiation-induced Micronuclei in Lung
Exposed Cells
Khan et al 1998
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Bystander effects in vivo following low dose-rate
exposures

• Exposure to non-uniform radiation fields
• Exposure to internally deposited radioactive
  materials that target selected organs
• Exposure to internally deposited radioactive
  particles

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Induction of p53 in Rat Tracheal Epithelium by
Radon
All Cells Up-regulated
Few Cells Hit
Ford et al 1997
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Hot Particle Hypothesis

• Cellular dose and response are linked
• Non-uniform dose distribution results in
  increased risks
• Large doses to a few cells result in large risks
• Risk from inhaled plutonium particles too low by
  a factor of 100,000-200,000.
• Studies undertaken to test this hypothesis

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Non-Uniform Dose Distribution from Plutonium
Inhalation
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The Influence of 239PuO2 Particle Size on the
Dose-Distribution in the Liver of Chinese
Hamsters
Citrate
0.44µm
0.84µm
0.17µm
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The Influence of 239Pu Dose-Distribution on
Chromosome Aberration Frequency
Aberrations/Cell
Brooks et al
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Cumulative Liver Tumor Incidence After 239PuO2 or
239Pu Citrate Exposure
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Low Dose Rate exposures No Bystander Effects in
unexposed Tissues or Organs

• Cancer from internal emitters are at the site of
  radionuclide deposition
• Secondary cancers from radio-therapy located at
  the exposure site
• At low dose rates there is little evidence for
  cancer in non-exposed tissues

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Impact of Bystander Effects on Dose and Risk

• Risk of transformation
• Reponse is independent of the number of hit
  sites.
• Non-uniform distribution of dose

Sawant et al 2001
Prise et al. 2002
Brooks et al. 1978
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Relationship between biological responses to
radiation
Adaptive Response
Genomic Instability
Bystander Effects
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What Genes are Responsible for the Adaptive
Response ?
90
80
70
60
Aberrations
50
Observed
40
Expected
30
20
10
0
0
0.5
150
0.5 150
Dose cGy
Shadley and Wolff 1987
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Adaptive ResponseSub-linear dose response
Transformation Frequency
0
10
20
30
40
50
60
70
80
90
100
Dose (cGy)
Redpath et al. 2001
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Lymphomas inTrp53/- Mice
50 40 30 20 10 0
0 mGy 10 mGy 100 mGy
Number of Tumors
100 80 60 40
20 0
Survival ()
Mitchel et al. 2003
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Osteosarcomas inTrp53/- Mice
25 20 15 10 5 0
0 mGy 10 mGy 100 mGy
Number of Tumors
100 80 60 40
20 0
Survival ()
Mitchel et al. 2003
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Taiwan 60Co Accident

• Source Contaminated Steel
• Population exposed 10,000
• Residence time 9-20 yrs
• Average Dose 74 mSv/yr
• Average Cumulative Dose 600 mSv
• Collective Dose 6000 per-Sv

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Human Low Dose Response to 60Co exposure
Cancer
Birth Defects
Chen et al. 2004
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Relative risk as a function of dose for cancer
related end-points
5 4 3 2 1 0
Neoplastic transformation in vitro Human in vivo
Leukemia Human in vivo Breast cancer Human in
vivo All solid tumors
Relative Risk
0.1 1
10 100
Dose (cSv)
Redpath et al.2001
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Protective Response

• It was found that low-dose IR exposures
  modulated genes involved in stress response,
  synaptic signaling, cell-cycle control and DNA
  synthesis/repair, suggesting that low-dose IR may
  activate protective and reparative mechanisms as
  well as depressing signaling activity.

Yin 2003
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Instability Model NEOTRANS 2
Transient Problematic Instability
Normal Minor Instability
Stable Genome
Error-free repair
Mutant Cells
Error-free repair
Scott, et al. 2003
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Relationship between biological responses to
radiation
Adaptive Response
Genomic Instability
Bystander Effects
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Radiation-induced Genetic Damage
Old Paradigm
After a cell is mutated by radiation, all of its
prodigy are mutated Mutation is a rare event
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Genomic Instability
New Paradigm
After a cell is exposed to radiation, different
things can happen sometimes after many cell
divisions. This is a frequent event.
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Possible Outcomes of Radiation Damage
Cells may completely repair themselves
NO PROBLEM
NO PROBLEM
Aneuploidy
Chromosome Aberrations
Mutations
Micronuclei
Cells may cause cancer
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Detrimental early effects seen in cell
Chromosomal rearrangements Micronuclei Death
inducing factors Gene mutations Increased
Reactive Oxygen Species (ROS) Inflammatory
responses
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Detrimental late effects seen in progeny
Chromosomal re-arrangements Micronuclei Cell
Transformation Gene amplification Death inducing
effect (DIE) Gene mutations Cell death
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Genomic Instability in vitro and in vivo

• Morgan- in vitro
• Cellular and cytogenetic instability induced at
  high and low dose rates
• Ullrich- in vivoLinking cellular and molecular
  instability to whole animals and populations

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Radiation-induced hprt mutations
Genomic Instability
Total Mutations
Spontaneous Mutations
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Radiation-induced Genomic Instability
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Dose Rate
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Radiation-related Gene Induction (for the CDKN1A
gene)
70
60
50
40
Percent of genes induced
The percent of cells with this gene induced or
turned on increases each time the cell
reproduces.
30
20
10
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Generations from initial radiation exposure
Amundson et al. 1999
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Genomic Instability can be demonstrated in some
strains of mice
Hybrid Mouse Models
Some strains of mice such as BALBc are very
sensitive to radiation-induced breast cancer.
Others, such as C57BL/6 mice, are particularly
resistance to this radiation-induced effect.
After only a few generations of apparently
normal cell division, the sensitive mice show
increased chromosome aberrations per cell, while
the radiation resistant mice remain stable.
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Genomic Instability can be demonstrated in some
strains of mice
0.35
0.3
0.25
0.2
Aberrations/Cell
0.15
0.1
0.05
0
4
8
12
16
20
24
28
Population Doublings
B. Ponnaiya R.L. Ullrich, 1998
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Radiation-induced Genomic Instability

• High frequency event
• 3/Sv low LET
• 4/Sv high LET
• Independent of dose rate at high total dose
• Related to inflammation and the Redox status of
  the cell
• Produced both in vitro and in vivo

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Genomic Instability Impact on Standards

• Provides a mechanism to explain how radiation can
  produce the multiple steps needed to transform a
  normal cell to a malignant cell
• Supports the LNTH if genomic instability is
  produced by low doses
• Induction of genomic instability by low dose
  rates may question dose-rate reduction factors

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Relationship between biological responses to
radiation
Adaptive Response
Genomic Instability
Bystander Effects
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Mechanisms involved in new phenomena

• Altered gene expression
• Impact of oxidative status of the cell
• Radiation-induced changes in differentiation
  pathways
• Cell/cell, cell/matrix interactions
• Nutrition and radioprotectants

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Radiation-related Gene Induction
It has been shown that certain genes are
inappropriately induced, or turned on or
turned off by radiation. The effect of the
gene induction sometimes shows up more
frequently several generations after the
initial radiation exposure. Experiments on the
CDKN1A gene provide a good example.
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Radiation-induced Changes in Gene Expression
Low Dose Rate
High Dose
Low Dose
Identified genes Dose-response Time after
exposure Tissue type
Amundson
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DIFFERENCES IN TRANSCRIPTION PROFILES
BETWEEN LOW AND HIGH DOSE IRRADIATION IN
MURINE BRAIN CELLS







0.1 Gy
2Gy



191
299
213







Total gene set contains nearly 10,000 genes
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Radiation-induced changes in gene expression
Low Dose Genes
High Dose Genes
0 10 100
1000
Dose (cGy)
Wyrobek
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Radiation and the redox status of cells

• Radiation produces free radical and reactive
  oxygen species (ROS)
• Radiation triggers signals that alter the level
  of ROS and RNS for long periods of time (frequent
  event)
• Redox status regulates cell cycle,
  differentiation, and apoptosis
• Physiological factors can be used to modify redox
  signaling pathways induced by radiation
• These represent potential methods for
  intervention to modify radiation-induced damage
  and risk

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Inhibition of gamma ray -induced (50 cGy)
oxidative stress by ascorbic acid
Radiation only
No radiation, no ascorbic acid
Wan, et al 2004
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Radiation-induced changes in cellular switches
?
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Dynamic Interactions with Microenvironment
Organized epithelial cells
Integrin-mediated signaling
Fibroblasts
Microenvironment
Immune cells
Vasculature
Park et al. 2000
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Signaling from Microenvironment
Differentiation
Apoptosis
Proliferation
Microenvironment
Park et al. 2000
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Cell/Matrix Interactions

• Cell/Matrix communication and signaling
• Cell/matrix tension
• Signaling molecules and pathways
• Exposed matrix can produce transformation in
  non-exposed cells
• Change phenotype with matrix
• Frequent non-mutagenic event

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Efficiency of Colony Formation in Soft Agar
Kennedy et al 2004
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Dietary InterventionSupplements reduce Oxidative
Stress Levels after Radiation
Guan, et al 2004
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Observations suggesting major paradigm shifts

• Adaptive response vs additive or synergistic
  effects
• Hit theory vs. bystander effects
• Mutation vs. gene induction
• Single cell vs tissue responses
• Intervention vs non-intervention

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Alternative paradigm for dose-response
relationships
Adaptive Response Bystander Direct
Effects Bystander and direct effects
High-LET exposure
Chromosome Aberrations
Acute Low-LET exposure
Background
0 20 40
60 80 Dose (cGy)
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Frequent vs Rare Early Events in Radiation Biology

• Old Paradigm
• Rare early event one hit, one mutation, one
  chromosome aberration one cancer
• Support the LNTH
• New Paradigm
• Frequent early event changes in gene
  expression, changes in cell transformation,
  changes in redox status, alterations in adaptive
  responses, bystander effects, genomic instability
• Doesnt Support the LNTH

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Summary of New Paradigms

• Hit theory shift to bystander paradigm
• A cell does not have to be hit in order to be
  biologically altered
• Mutation theory shifts to gene expression
  paradigm
• Radiation induces changes in gene expression
  that may alter subsequent responses in a large
  fraction of the cell population
• Single mutation cancer theory shifts to tissue
  paradigm
• Tissues respond as whole and not as individual
  cell
• LNTH challenged by adaptive response genomic
  instability
• Adaptive response may result in protective,
  nonlinearity , but genomic instability could
  result in superlinear does responses

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Observations Suggesting Major Paradigm Shifts

• Adaptive response vs additive or synergistic
  effects
• Hit theory vs. bystander effects
• Mutation vs. gene induction
• Single cell vs tissue responses

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Mutation vs. Gene Induction
Gene Induction
Gene Mutation
Transient Change
Permanent Change
Gene Expression
Protein Production
Change in Phenotype Apoptosis Cell
Proliferation Cancer
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How will this Research Impact Standards?
SUB-LINEAR
Adaptive Response
Genomic Instability
Genetic Susceptibility Bystander Effects
LINEAR
SUPER-LINEAR
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Potential Impact of Research on Public Perception

• Provide scientific outreach
• Level of understanding increased
• Risk and fear put into perspective
• Scientific basis for standards

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Summary

• Mechanisms for radiation action are dose
  dependent.
• Bystander effects, adaptive responses, and
  genomic instability are interrelated.
• The observations suggest the need for paradigm
  shifts in radiation biology.
• New biology provides additional potential for
  intervention in radiation-induced disease
• Mechanism of action at low doses help define the
  relationships among bystander, adaptive response
  and genomic instability.