24th International Conference on Nuclear Tracks in Solids

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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Study of influence of catechins on bystander
responses in alpha-particle radiobiological
experiments using thin PADC films Y.L. Law and
K.N. Yu Department of Physics and Materials
Science, City University of Hong Kong
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Objectives

• To investigate the relationship between the
  radiation dose and induction of DNA damages
• in unirradiated cells
• 2. To investigate the scavenging effect of
  catechins
• on reactive oxygen species in unirradiated
  cells.

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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Introduction

• Radiobiological effects of ionizing radiation
• Alpha particle
• Interacts with DNA
• ? Double-strand breaks (DSBs)
• Free radicals ? DSBs
• DSBs
• Most lethal
• Cell death, chromosome aberration,
• gene mutation

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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Introduction

• Catechins
• Polyphenolic compounds in green tea
• EGCG (epigallocatechin gallate)
• most abundant
• Cancer protection
• Antioxidant effects
• Scavengers of reactive oxygen species (ROS)

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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Introduction

• Radiation-induced bystander effect
• ?Induction of biological effects in cells that
  are not directly
• traversed by an ionizing radiation
• Underlying mechanisms
• Gap junction-mediated intercellular communication
  
• Soluble factor such as ROS released by irradiated
  cells

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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Introduction

• Polyallyldiglycol carbonate (PADC) films
• -Most commonly used solid-state nuclear
• track detectors (SSNTDs)
• -Transparent
• -Relatively biocompatible
• -Does not dissolve in alcohol
• during sterilization

Commercially available 100 µm thick PADC film
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Methodology
Set-up PADC film cell dish
1000 µm PADC films

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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Methodology
Set-up Alpha-particle irradiation
Cell type Chinese hamster ovary (CHO) Cell
culture substrate 22 1000 µm PADC films Alpha
source 241Am (activity 5 µCi and main alpha
energy 5.49 MeV under vacuum )
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Methodology
2
1
?-particle irradiation
BY cells only
add EGCG (in DMSO)
3
4
Trypsinize the cells
Pelleted by centrifugation for TUNEL assay
single cell
BY cells only
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Methodology

• TUNEL assay
• Terminal deoxynucleotidyl transferase dUTP
• transferase-mediated nick end-labeling
• Common method for detecting
• DNA fragmentation results from
• apoptosis
• Identified by enzyme TdT
• dUTPs marker

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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Methodology TUNEL assay
Fixation
Permeabilisation
Labeling reaction with TUNELreaction mixture
Flow Cytometry
2 paraformaldehyde in PBS 1h in RT To
stabilize the cell reaction stopped
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Methodology TUNEL assay
Fixation
Permeabilisation
Labeling reaction with TUNELreaction mixture
Flow Cytometry

• 0.1 Triton X-100 and 0.1 sodium citrate
• 10 min on ice
• Dissolve the cell membranes allow
• larger dye molecules access into the cell

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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Methodology TUNEL assay
Fixation
Permeabilisation
Labeling reaction with TUNELreaction mixture
Flow Cytometry
90 Label solution and 10 TUNEL enzyme - 37? in
dark for 1 h Labels DNA strand breaks generated
during apoptosis
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Methodology TUNEL assay
Fixation
Permeabilisation
Labeling reaction with TUNELreaction mixture
Flow Cytometry
Detect and quantify fluorescein labels using
laser beam Counting of 10000 cells
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Methodology
2
1
Fixation
IR cells
3
4
Etching
37?
Capture image of tracks and cells
Capture image
14 N KOH solution
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Results and Discussions
Number of cell hits
Alpha-particle track
CHO cells
After fixation (CHO cells only)
After etching (CHO cells and tracks)
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Results and Discussions
Number of cell hits

• As low as 1 of the cell nuclei were actually
  traversed
• by alpha particles in all the samples
• Bystander damages occur even at very low doses
• DNA damages in bystander cells seems to be
  independent
• of the number of irradiated cells

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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Results and Discussions
Results of TUNEL assay


For example Bystander sample
33.75 Unirradiated control 17.22 Result
16.33 of TUNEL positive signal

P lt 0.05, P lt 0.005 compared to the
corresponding control (0 µM)
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Results and Discussions
Results of TUNEL assay
Signal transduction from irradiated cells to
bystander cells occurred within a short time
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Results and Discussions
Results of TUNEL assay



P lt 0.05, P lt 0.005 compared to the
corresponding control (0 µM)
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Results and Discussions
Results of TUNEL assay

• Scavenging effect of EGCG in 1 µM on bystander
  cells is the
• most significant
• Previous study showed EGCG significantly reduced
  the amount of DNA strand breaks in 1-2 µM
  concentrations (Anderson et al., 2001)
• ROS might be involved in the induction of DNA
  breaks

Anderson, R.F., Fisher, L.J., Hara, Y., Harris,
T., Mak, W.B., Melton, L.D., Packer, J.E., 2001.
Green tea catechins partially protect DNA from
OH radical-induced strand breaks and base damage
through fast chemical repair of DNA radicals.
Carcinogenesis. 22(8), 1189-93.
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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Conclusions

• Bystander damages occur at very low doses
• The signal transduction from irradiated cells to
  unirradiated cells
• occur within a short time after irradiation
• EGCG scavenges ROS at a significant level in the
  bystander cells
• produced by alpha-particles irradiation
• Further study is needed to find out which types
  of ROS are involved
• in the medium-mediated bystander effects.

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24th International Conference on Nuclear Tracks
in Solids Bologna, 1-5 September 2008
Study of influence of catechins on bystander
responses in alpha-particle radiobiological
experiments using thin PADC films Y.L. Law and
K.N. Yu Thank you!