1. Development of a novel
assay to investigate changes
to global DNA methylation
levels in response to
radiation exposure in vivo
Michelle Newman

2. Linear No-Threshold model for radiation risk
assessment predicts cancer risk due to radiation
exposure
Risk (excess cancers)
Dose above background
High dose epidemiological data
Extrapolated low dose data
200 mGy

3. DNA damage increases mutations
and cancer risk
• Increase in cancer risk could be due to increased DNA damage
and mutations following radiation exposure
strand breaks

4. Inappropriate DNA methylation is a form
of DNA damage
Or:
Adapted from: http://www.nature.com/nature/journal/v441/n7090/images/441143a‐i2.0.jpg
CH3
CH3
CH3
CH3
DNA methylation
• Is a chemical modification to DNA
– occurs at cytosine residues
• DNA methylation is involved in the
control of gene expression
– affects the accessibility of DNA
– changes structure of DNA
• Can be altered by diet and exposure to
carcinogens

5. • Aberrant DNA methylation is linked with cancer
• Incorrect DNA methylation can be one of the initiating events
in tumour progression
Gene
Gene
X
loss of methylation
increased methylation
expression of genes that
promote tumour growth
suppression of genes that
prevent tumour growth
DNA methylation is a chemical
modification to DNA

6. • High doses of radiation have been reported to cause a loss in
global DNA methylation
• Based on LNT, low dose radiation exposure should cause a
loss in DNA methylation
– but on a lower scale than high dose radiation exposure
• Lowest dose studied (500 mGy) has been shown to cause:
– an increase or a decrease in DNA methylation (Kovulchuk et al., 2004; Raiche
et al., 2004; Pogribny et al., 2005)
– this was dependent on tissue investigated and sex of mice used in the
study
– doses similar to medical exposures have not been studied
DNA methylation can be altered by radiation
exposure

7. • Low doses of radiation have been shown to:
– reduce cancer risk and DNA mutations
– increase tumour latency
– also prevent the damage induced by high dose radiation
• if given prior to high dose
• Aim:
– to investigate if DNA methylation is affected by low doses of radiation
in vivo
Is DNA methylation affected by low dose
radiation exposure?

8. Novel application of an assay to detect
global DNA methylation changes
• Needed an assay that:
– is sensitive
– high throughput
– robust
– reproducible
• Most methods detect changes in methylation of single genes
• We wanted to detect (global) methylation changes throughout the
genome resulting from radiation exposure
• Other global assays are either not high throughput, reproducible or
sensitive
• Utilising a chemical that allows the distinction between methylated and
unmethylated DNA and
– applying to sequences of DNA known as LINE1 elements, which are
found throughout the genome

9. ATCGTA
TAGCAT
CH3
=≠
Temperature →
Double Stranded
Single Stranded
CH3
ATUGTA
TAGUAT
ATCGTA
TAGCAT
Bisulphite Modification Bisulphite Modification
0% Methylation
100% Methylation
x % Methylation

10. LINE1 methylation assay can detect
chemical demethylation
100% methylated
Vehicle
0.125 µM 5‐aza
0.5 µM 5‐aza
0 % methylated
↑ methylated*p<0.05
n=5
↓methylated
Double stranded DNASingle stranded DNA

11. Preliminary data – the temporal effect of 1 Gy and
10 µGy on spleen tissue global DNA methylation levels
n=5
*p<0.05
*
*

12. • Is this a phenomenon that occurs only at LINE1 elements?
– other repeat elements (SINE, LTR)
• Investigate total methylated cytosine content using another
technique
• Tissues, genetic background, sample size
DNA methylation does not appear to be
altered by low doses of radiation

13. Future directions
If a single exposure (high or low) has long
term effects on DNA methylation
young mice old mice
if a low dose can prevent the changes in
methylation induced by high dose

14. Summary and Significance
• This is the first example where temperature differences have
been used to detect global DNA methylation levels
• Results indicate:
– high dose exposure alters DNA methylation levels and this change is
dynamic over time
– low doses may not alter methylation levels
• effects may be subtle

15. • Highlights the importance of conducting longitudinal studies
– to assess the long term impact of a single exposure
• Understanding changes to DNA methylation in response to
relevant radiation exposures is important
– given that aberrant DNA methylation changes is associated with
cancer
Summary and Significance

16. • DOE Group :
– Supervisors: Pam Sykes and Rebecca Ormsby
– Lab Members: Ben Blyth, Mark Lawrence, Alex Staudacher, Ami
Cochrane, Katrina Bexis
• RAH:
– Eva Bezak, Medical Physicist
• Funding:
– Low Dose Radiation Research Program, U.S. Department of Energy
– BHP Billiton/FMC Low Dose Radiation Research Scholarship
Acknowledgements