24th international conference on nuclear tracks in solids bologna, 1-5 september 2008 study of...

24th International Conference on Nuclear Tracks in Solids

Bologna, 1-5 September 2008

Study of influence of catechins Study of influence of catechins on bystander responses in on bystander responses in

alpha-particle radiobiological alpha-particle radiobiological experiments using thin PADC experiments using thin PADC

filmsfilms

Y.L. Law and K.N. YuDepartment of Physics and Materials Science,

City University of Hong Kong

24th International Conference on

Nuclear Tracks in Solids

Bologna, 1-5 September 2008ObjectivesObjectives

1. 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.



Bologna, 1-5 September 2008IntroductionIntroduction

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…




Catechins

•Polyphenolic compounds in green tea

•EGCG (epigallocatechin gallate) – most abundant

•Cancer protection

•Antioxidant effects

•Scavengers of reactive oxygen species (ROS)

http://hk.wrs.yahoo.com/_ylt=A3xsaCbO5rFI9bsAiQK.ygt./SIG=128h8g51l/EXP=1219704910/**http%3A//www.organicteatrust.com/images/green-tea2.jpg




Radiation-induced bystander effect

Induction of biological effects in cells that are not directly traversed by an ionizing radiation

Underlying mechanisms:

1. Gap junction-mediated intercellular communication 2. Soluble factor such as ROS released by irradiated cells




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



Bologna, 1-5 September 2008MethodologyMethodology

22 µm PADC film base

cell dish

+1000 µm PADC

films =

Set-up: PADC film cell dish




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 )

-particle beam

nutritive

medium

22 µm PADC film

1000 µm PADC film




21

3 4

-particle irradiation

add EGCG(in DMSO)

BY cells only

Trypsinize the

cells Pelleted by centrifugation

for TUNEL assay single

cell BY cells only




TUNEL assayTerminal deoxynucleotidyl transferase dUTP transferase-mediated nick end-labeling

•Common method for detecting DNA fragmentation results from apoptosis

•Identified by enzyme TdT

•dUTPs – marker



Bologna, 1-5 September 2008Methodology: TUNEL Methodology: TUNEL assayassay

FixationFixation PermeabilisationPermeabilisationLabeling reaction

with TUNELreaction mixture

Labeling reaction with TUNEL

reaction mixtureFlow CytometryFlow Cytometry

2% paraformaldehyde in PBS – 1h in RT To stabilize the cell – reaction stopped








Methodology: TUNEL Methodology: TUNEL assayassay

0.1% Triton X-100 and 0.1% sodium citrate-10 min on iceDissolve the cell membranes – allow larger dye molecules access into the cell









90% Label solution and 10% TUNEL enzyme - 37℃ in dark for 1 hLabels DNA strand breaks generated during apoptosis









Detect and quantify fluorescein labels using laser beamCounting of 10000 cells




21

3 4

Fixation

Capture image

Etching 37

℃

14 N KOH solution

Capture image of tracks and

cells

IR cells



Bologna, 1-5 September 2008Results and Results and DiscussionsDiscussionsNumber of cell hits

After fixation (CHO cells

only)

After etching(CHO cells and tracks)

Alpha-particle track

CHO cells



Bologna, 1-5 September 2008Results and Results and DiscussionsDiscussions

• 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

Number of cell hits




0 μM 1μM 10 μM 20 μM

* *

**

*P < 0.05, **P < 0.005 compared to the corresponding control (0 μM)

Results of TUNEL assay

For example:

Bystander sample= 33.75%

Unirradiated control

= 17.22%

Result: 16.33% of TUNEL positive

signal





* *

**


Without any treatment


Signal transduction from irradiated cells to bystander cells occurred within a short time





* *

**







• 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.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11470748&dopt=Abstract






Bologna, 1-5 September 2008Conclusions 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.

24th International Conference on Nuclear Tracks in Solids


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!Thank you!

24th international conference on nuclear tracks in solids bologna, 1-5 september 2008 study of...

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