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Radiation‐induced bystander effects – What? Why? And How?
Carmel Mothersill
Colin Seymour
McMaster University
Dose-Response 2010
Outline• Bystander effects and genomic instability
• In vivo relevance and evidence
• The fish model
• Mechanisms
• Multiple stressor issues
• Why do these effects happen?
Dose-Response 2010
Take home messages
• Target theory, LNT and DNA centric ideas of low dose effects are dead
• Chronic (background) exposure to radiation is in no way related, even by elaborate “fudge factors” to acute exposure
• Environmental effects are not simple and multiple stressor exposure HAS to be part of the discussion
• Evolution is all about adaptation (or not?) to changed conditions
Dose-Response 2010
1o and 2o
response
The bystander effectIonizing radiation, UVA, UVB, ELF‐EMF and heavy metals induce affected cell to signal to others.
Responses to the signals include apoptosis, micronucleus formation, transformation, mutation, induction of stress and adaptive pathways. Serotonin (5HT) and Calcium ions known to be involved in signal production.
bystander factormolecules
response
response
GJICconnexins
ROS/Nitric oxide/cytokinesBiogenic amines,TGFb, p53
????
Amplification/Cascade effects?
Ca2+
Ca2+
Ca2+
response
Ca2+
5HT
Dose-Response 2010
0
20
40
60
80
100
120
140
0.01 0.1 1 10 100 1000 10000
Dose (m
ICCMIrradiat
Bystander and direct dose survival curvesover six orders of magnitude 60 Co with calcium data
No Ca 2+ Ca 2+
Dose-Response 2010
Hit
Hit?
The link between bystander effects and genomic instability – twin pillars of the new paradigm
Old view‐ clonal outcome
New view‐non‐clonal, population‐determined outcome
Progeny cells are non‐clonal and may give rise to a variety of mutations or die
Cells continue to be produced with non‐clonal changes
Progeny are all i.e. identical and mutation is passed to all progeny
Dose-Response 2010
In vivo relevanceDose-Response 2010
In vivo evidence
• Clastogenic factors in blood of irradiated people and experimental animals
• Abscopal effects in distant organs
• Bone marrow ablated mice receiving opposite sex marrow transplant show instability in the regenerating marrow
• Soluble factors from explanted tissues after in vivo exposure
Dose-Response 2010
The fish model for studying bystander effectsDose-Response 2010
Dissect fresh tissue from animal
Chop tissue to provide explant pieces
Culture of explants
Measuring radiation induced bystander response in vivo
Harvest culture medium containing stress signal molecules/metabolomics
Add to unirradiated clonogenic cell line anddetermine survivalor other stress endpoints
Examine explant outgrowth/immunocytochemistry
±Metals in water
Irradiate fish
Do proteomics
Dose-Response 2010
X‐rayed fish
INDUCTION OF THE BYSTANDER EFFECT IN A DIFFERENT FISH
Waterborne bystander effect Partner bystander effect
Dose-Response 2010
Dose-Response 2010
BYSTANDER EFFECT INDUCED IN VIVO IN RAINBOW TROUT, MEDAKA & ZEBRAFISH
0.5Gy X‐ray dose Non X‐rayed fish
Non X‐rayed fish0.5Gy X‐ray doseWater soluble bystander signals
0.5Gy X‐ray dose Non X‐rayed fish
Rainbow trout (Mothersill et al 2006)
Zebrafish (Mothersill et al 2007)
Medaka (Mothersill et al 2009)
Dose-Response 2010
76.0
66.2
43.0
36.0
31.0
21.5
17.5
4.5 5.1 5.4 5.6 6.0 7.0 8.5
Molecular size (kDa)
Isoelectric point (pH units)
Hemopexin‐like protein
Pyruvate dehydrogenase (PDH)
RhoGDP dissociation inhibitor (RhoGDI)
Chromosome 1 SCAF protein
Annexin II
X‐RAY AND BYSTANDER EFFECT INDUCED CHANGES TO THE TROUT GILL PROTEOME
SCAF = SR‐like CTD‐associated factor (SR = Serine – argenine Rich, CTD = C Terminal Domain)Dose-Response 2010
Complement component C3
Warm temperature acclimation related 65 kDa protein (Wap65)Chromosome 5 SCAF protein
Creatine kinase
Lactate dehydrogenaseAnnexin A1
Annexin A4
ABC transporter
Medaka gill proteins affected
76.0
66.2
43.0
36.0
31.0
21.5
17.5
Molecular size (kDa)
4.5 5.1 5.4 5.6 6.0 7.0 8.5pI (pH units)
Dose-Response 2010
Bystander effect proteomics
• Protective response against reactive oxygen species
• Bystander effect proteomic changes are transcriptionally regulated (SCAF proteins)
• Virtually identical proteomic response in rainbow trout and medaka
• Evidence from trout and medaka suggests the bystander effect is immediately protective
• Additionally the induction of an adaptive response may be species specific and apply particularly in radiosensitive biological systems
Is the bystander effect…
… an immediate protective response (Smith et al 2007) or an adaptation to possible future radiation damage (Kadhim et al 2004)?
Dose-Response 2010
Legacy effect of single acute 0.5Gy X‐ray exposure to eggsDose-Response 2010
Transgenerational study(in progress)Dose-Response 2010
Sample #1
Time (s)
0 100 200 300 400 500 600
Rat
io o
f Flu
ores
cenc
e
0.95
1.00
1.05
1.10
1.15
1.20
1.25
1.30
1.35
Sample # 28
Time (s)
0 200 400 600 800 1000
Rat
io o
f Flo
ures
cenc
e
0.98
0.99
1.00
1.01
1.02
1.03
1.04
1.05
Sample # 10
Time (s)
0 100 200 300 400 500 600
Rat
io o
f Flo
ures
cenc
e
0.98
1.00
1.02
1.04
1.06
1.08
1.10
Sample # 11
Time (s)
0 100 200 300 400 500
Rat
io o
f Flo
ures
cenc
e
0.8
1.0
1.2
1.4
1.6
Transgenerational memory of irradiation calcium signal
sham Irradiated YSL stage parents
Sham Bystander to above parents
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Some mechanistic questions
• Role of DNA repair
• Role of serotonin
• Role of p53
Dose-Response 2010
DNA repair is important in vivo and in vitro
DNA repair deficient cell linesand transgenic medaka bothproduce highly toxic bystandersignals after low dose irradiation
Increased apoptosis in vivo
0
20
40
60
80
100
120
140
HPV-G SW48 GS3 1Br3 ATBr1 180Br CHO-K1 XRS 5 XR-1 Burkitts control Raji 9 Raji 10Cell Lin
0Gy0.5Gy5Gy
Reduced reproductive survival in vitro
Dose-Response 2010
Serotonin important in vivo and in vitro
Serotonin bound by irradiated cellsIn vitro, leading to Calcium pulse.
Reserpine inhibits serotonin binding and prevents the bystander effect in vitro and in vivo
Dose-Response 2010
Role of p53 in response to signal but not in generation of signal
0
20
40
60
80
100
120
1
PE
Treatments
HCT ‐/‐ Reporters
Direct 0Gy
"Direct 0.1Gy"
"Direct 0.5Gy"
got ‐/‐ 0Gy
got ‐/‐ 0.1Gy
got ‐/‐ 0.5 Gy
got +/+ 0Gy
got +/+ 0.1Gy
got +/+ 0.5Gy
0
20
40
60
80
100
120
1
PE
Treatments
HCT +/+ Reporters
Direct 0Gy
Direct 0.1Gy
Direct 0.5Gy
got ‐/‐ 0Gy
got ‐/‐ 0.1Gy
got ‐/‐ 0.5Gy
got +/+ 0Gy
got +/+ 0.1Gy
got +/+ 0.5Gy
Dose-Response 2010
Multiple stressors
• Radiation seldom acts alone
• Isotopes are by definition a radioactive chemical
• Little information on synergistic effects
• Little low dose information
• Little in vivo information
Dose-Response 2010
What the multiple stressor problem does to radiation protection
• Multiple inducers of stress effects therefore dose and effect are not simply linked
• Response based approach needed• How to link biological effect with adverse outcome at the organism, population and ecosystem level
• Mechanistic uncertainty at low doses• Non‐targeted effect predominate at low doses
Dose-Response 2010
Examples of complex senarios
• Radiation induces a cell to undergo apoptosis, removing it from the potentially carcinogenic pool. Substance 2 (eg Cd) interferes with the signaling cascade and the cell lives – survival assay suggests protective effect of interaction?
• Radiation induces an adaptive response in population A, a further stressor has little effect but pristine population B has no adaption and is devastated by the same stressor.
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Fish irradiation in Norway: (lets get some low dose data!)
• Exposure of fish in aerated tanks to mGy doses over 5‐48 hours
Metals in the water
Dose-Response 2010
Comparison of in vivo mGyradiation exposure±metalson production of bystander signals
Co60 Al+Cd
Dose-Response 2010
Eff
ect
Dose
Zone of “linearity”
Natural background
Proposed dose response relationship for radiation in the context of non-targeted effects
Purple arrows indicate mechanistic break points wherenew, more appropriate, responsepathways emerge
tolerance
saturation
New “coping” mechanisms induced
saturation
Blue line representsold LNT model
Zone of uncertainty
Hormesis area
Dose-Response 2010
Unifying Theory: transduction of bystander effect
Serotonin (nM range) binds -Activation of calcium channel
Ca2+
Activated system
Low dose stimulationof system
response In system
NOTES: “good” response at one level in system may be “bad” at another levelLinks stress even mental stress, physiological response and outcome
Dose-Response 2010
So why do these effects happen?Dose-Response 2010
WHY ARE THESE MECHANISMS SO WIDESPREAD AND PERSISTENT?
•In terms of evolution there is conservation of the mechanism and bystander pathway across species and this suggests a very primitive origin in the vertebrates since teleost fish split from the main vertebrate line early in vertebrate evolution.
Dose-Response 2010
Population based response?
• Are non‐targeted effects a reflection of population level regulation to optimise population fitness (tissue or individual level)?
• Is the function of radiation‐induced bystander signaling to co‐ordinate behaviour at higher hierarchical levels of organisation?
• Quorum sensing in bacteria is an example of this at the population level as are hormones at the organism level
Dose-Response 2010
SUMMARY
Non targeted effects existThey manifest at high frequency in many waysThey cause “stress‐like” symptomsWe know a lot about the mechanisms but little about the reasons why they are tolerated The underlying debate about purpose or chance is as old as Plato and Aristotle
Dose-Response 2010
Acknowledgements• Thanks to Colin Seymour, husband and long time collaborator
• Richard Smith and all the Salbu Lab
• NOTE EU IP collaborators
• Funded by The Canada Research Chairs and NSERC IRC Chairs programmes
Dose-Response 2010
Dose-Response 2010
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