This document discusses bystander effects from low-dose radiation exposure and an experimental model to study them in vivo. It summarizes that: 1) Bystander effects have been demonstrated in vitro but their relevance in vivo is still unclear, as most studies used much higher doses than typical environmental exposures. 2) The authors developed a mouse model using adoptive cell transfer to simulate rare radiation hits in vivo and study biological effects in surrounding bystander cells. 3) Initial experiments found no evidence of apoptosis or proliferation in bystander spleen cells after low-dose radiation exposure, suggesting bystander effects may not increase cancer risk from very low doses as has been hypothesized. 4) More research

1. The contribution of bystander effects to the The contribution of bystander effects to the
risks posed by low radiation dosesrisks posed by low radiation doses
Benjamin J. Blyth & Pamela J Sykes
Haematology & Genetic Pathology
School of Medicine
Flinders University and Medical Centre

2. Atomic Bomb Survivors - LSS
Preston 2007, Radiation Research
( 0 – 100 mSv )
Natural background, inc. radon
Radiography
Occupational exposures
Airline travel, inc. security screening

3. Atomic Bomb Survivor Data
Radiation Dose (mGy)
0 – 5 mGy Control Group
X and γ‐rays
3.7 MeV α‐particles

4. 100 mGy, 100 kVp X‐rays
or
5 Gy, 3.5 MeV α‐particles
0.1 mGy, 100 kVp X‐rays
or
50 mGy, 3.5 MeV α‐particles

5. Security Backscatter X‐Ray Screening
Effective dose 0.05 μSv / scan
Health Physics Society (2009)
50 – 125 kVp photons, dose concentrated near the
skin’s surface
1 μGy to skin surface / scan
0.1 – 1% cells traversed by an electron track
All exposed cells receive their dose within a 10 second period.

6. Domestic Radon Exposure
222Rn: 100 Bq m‐3
346 lung basal cell nuclei traversed by
a single alpha particle per day
(out of 1.5 × 109 cells) Harley et al. 2008
1 cell in the lung hit on average, every 4 minutes
Basal bronchial cells are not hit more than once
during their ‘lifetime’

7. Long‐Haul Airline Flights
Effective dose rate = 5 μSv h‐1
2.75 μSv h‐1 low‐LET + 2.25 μSv h‐1 high‐LET Bottollier‐Depois et al. 2000
0.5 – 2% of cells receiving energy deposition for a 10 h flight
LET range from 0.1 – 1000 keV μm‐1
Cellular dose per hit
<1 mGy to >100 mGy

9. Biophysical argument for linear extrapolation
Even the smallest radiation energy deposition to a cell will
carry a small, but non‐zero risk of causing a carcinogenic
mutation. The net risk will therefore decrease in proportion
with the number of cells receiving energy deposition.
10 x lower dose = 10 x fewer hit cells = 10 x lower risk

10. Radiation‐Induced Bystander Effects
Radiation‐induced effects in unirradiated cells remaining
within an irradiated tissue.
Cell death
DNA damage
Mutation
Proliferation
Genomic instability

11. In Vitro Bystander Effect Methods
Proof‐of‐Principle: Unirradiated cells can, under the right
conditions, respond to signals from irradiated cells.

12. Hypothetical Bystander Impact on Dose‐Risk Curve
What if all cells were at
increased cancer risk even if
only a small fraction actually
received energy deposition?

13. Bystander Effects*
• Many experimental systems have used
cellular doses and/or irradiated cell numbers
far above the relevant range
• Bystander effects are not universally observed
nor are they always reproducible
• Contradictory bystander responses
• Bystander effects remain to be demonstrated
under relevant conditions in vivo

14. A better understanding of Bystander mechanisms
and the extent to which they are active in vivo
‘are needed before they can be confirmed as factors to be included in the
estimation of potential risk’
International Commission for Radiological Protection
2006 Report

15. Our Aim
To create an in vivo experimental system that
could be used to re‐create realistic bystander
scenarios.
In vivo: Mouse
Tissue of interest: Spleen
Relevant radiation exposure: low‐LET
Single energy depositions: 3H, 1 d h‐1, 2.61 mGy hit‐1
Rare, known irradiated cells: < 1 in 1000

16. Adoptive Transfer Model
Stimulate
Cells to Divide
Incorporate 3H‐Thymidine
or Non‐Radiolabelled
Thymidine
Label with Fluorescent Dye
Recipient MouseDonor Mouse
Isolate Spleen
Isolate Lymphocytes
Culture cells
Inject
Radiolabelled or
Non‐Radiolabelled
Cells
via Tail Vein
Collect, Freeze
and Analyse
Recipient Mouse
Spleen

17. Apoptosis: TUNELProliferation: Ki-67
50 μm
3H +
-

18. Bystander Effects In Vivo?
No induction of apoptosis or
proliferation in unirradiated bystander
cells
– After 22 h or 72 h
– 1 × 3H disintegration h‐1
– 100 × 3H disintegrations h‐1
– 0.1 or 1 Gy X‐rays
– Irradiated cells lodged @ 2 – 20 × 10‐4
Blyth et al. 2010, Radiation Research
Local area around
donor cells
Whole spleen
P = 0.69 P = 0.58
More tissues, endpoints, times, genetic background, doses etc.
Lodged
Cells
Radioactive
Lodged
Cells
Non-
Radioactive
Lodged
Cells
Radioactive
Lodged
Cells
Non-
Radioactive

19. Bystander effects are a concern because they could theoretically increase the
carcinogenic risk of very low dose exposures.
However, bystander effects are yet to be demonstrated in vivo under the
relevant exposure conditions.
Bystander effects have been characterised mostly in terms of ‘negative’
endpoints, however, it is unclear whether the net effect of these biological
changes would be pro‐ or anti‐carcinogenic.
Bystander effects, if relevant in vivo, will likely be a dynamic and complex
response to radiation exposure, dependent on dose, dose‐rate, radiation‐
quality, stress, cell/tissue type, endogenous signalling and health of the
individual.
The adoptive transfer model can now be used to explore many of these
parameters in an in vivo experimental system.

20. US Department of Energy
Low Dose Radiation Research Program, Grant # DE‐FG02‐05ER64104
Rebecca Ormsby (Flinders University)
Alex Staudacher (Flinders University)
Edouard I Azzam (UMDNJ)
Roger W Howell (UMDNJ)
Flinders University and Medical Centre