1. If bystander effects for apoptosis occur in spleen
after low dose irradiation in vivo then the magnitude
of the effect falls within the range of normal
homeostatic apoptosis
Pamela J Sykes
SA Pathology
Flinders University and Medical Centre
Can we see radiation bystander
effects in vivo at low doses?

2. Current radiation risk regulation
uses the
Linear No-threshold Model
LNT

3. LNT model of risk assessment
• Risk is directly proportional to dose
• Even the tiniest dose of radiation is
harmful
– ↑cancer
• Everyone must be protected from tiny
doses
– Billions of $’s

4. Cancer

DNA
damage
Radiation dose
LNT states that cancer risk is directly
proportional to dose
high exposure
Death
CancerData
Data???
LNT = Linear no-threshold
<100 mSv

5. Low dose radiation phenomena
which do not follow LNT
• Adaptive Response
• Bystander effects

6. Adaptive Response
• According to LNT, doses must be
directly additive

7. Adaptive response
Low dose protects from a high dose
Low dose protects from endogenous damage
Mutationfrequency
conditioning
dose
Challenge
dose
Expected
Observed
Low High
Radiation Dose

8. Adaptive responses
↓ Risk
in vitro and in vivo
Adaptive responses do not necessarily overly concern
regulators because they may be over-protecting the public
Identification of a safe threshold could save billions of
dollars in unnecessary radiation exposure regulation

9. Bystander Effect
• According to the LNT model “unhit” cells
cannot respond to radiation

10. BYSTANDER EFFECTS – IN VITRO
1 cell hit → many cells respond

11. Bystander effects at low doses?
• These worry regulators because they may
be under-protecting the population
• ↑ Risk or ↓ Risk ?
• Do bystander effects occur
– in vivo?
– at relevant doses?

12. Bystander effect at low doses in vivo?
• Can’t tell which cells have received direct hit
• At low doses, expected increase in any
biological end-point by direct effects would be
very small
– 1 mGy (most cells receive 1 electron track)
– 0.01 mGy (1/100 cells receive an electron track)
• However perhaps a bystander effect could be
observed?

13. Our approach to study bystander effects at
very low doses in vivo
• Whole animal – mouse
• Whole body X-irradiation
• Low doses relevant to OH&S and population exposure
– 0.01 mGy, 1 mGy
• Biological End-point
– Cell fate endpoint of apoptosis (programmed cell death)
– Often used in In vitro studies
• Spleen
• In situ analysis
• Temporal studies

14. 0, 0.01, 1 or 1000 mGy
(n= 10‐16/group)
Detection of apoptosis in mouse spleen after
single whole body X-irradiation
7 h, 1, 3 or 7 days
Remove spleen
Stain for Apoptosis
Alexander Staudacher
PhD student

15. Development of sensitive method of apoptosis in
spleen in vivo
• in situ
• Minimum of 110,000 cells studied in spleen/mouse
• Precision of 0.001%
• 14 experiments
– mean sham-treated apoptosis = 0.27±0.005%
– maximal difference of 1.2 apoptotic cells /1000 total cells
between sham-treated groups

16. Temporal study of apoptosis frequency in spleen
after low dose X-radiation
7 hours 1 day
3 days 7 days
N ≥ 10, Experiments performed twice
Total of 14 experiments 274 mice

17. You need a very large number of mice to detect apoptotic
changes based on LNT at these low doses

18. Even a substantial bystander effect will fall
within the homeostatic apoptosis range
25-fold25-fold
10-fold
Staudacher et al, radiation Research, in press

19. Summary
• Bystander effects have been observed in vitro which
could indicate a greater risk than predicted by LNT
• Do the in vitro results translate to in vivo?
• Chose apoptosis as a bystander end-point based on in
vitro studies
• Chose spleen as organ of study as used in other in vivo
low dose studies
• Used a precise in situ method of analysis

20. Summary (cont)
• No significant changes in apoptosis detected at low
doses in temporal studies
• Expected apoptosis changes due to direct effects based
on LNT at these low doses
– = small fraction of the natural variation in apoptosis
– Prohibitive numbers of mice required
• If bystander apoptotic effects exist in spleen
– fall within the homeostatic range of apoptosis
– may be a very specific sub-sets of cells affected?
• Need for methods for studying rare irradiated cells in
organs in vivo

21. Flinders University
and Medical Centre
Present members:
Pamela Sykes
Ben Blyth
Alex Staudacher
Rebecca Ormsby
Mark Lawrence
Monica Dreimanis
Katrina Bexis
Ami-Louise Cochrane
Past members:
Tony Hooker
Tanya Day
Guoxin Zeng
Royal Adelaide
Hospital
Eva Bezac
Kar Aun Giam
Research Funded by the Low Dose Radiation Research program,
Biological and Environmental Research, U.S. Department of Energy,
grant #DE-FG02-01ER63227 and DE-FG02-05ER64104.
Flinders Medical Centre Foundation
Lovelace Respiratory
Institute New Mexico
Bobby Scott
New Jersey Medical Centre
Edouard Azzam
Roger Howell
University of Chicago
David Grdina
University of Freiburg
Georg Bauer

22. P. Sykes