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Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Premature Chromosome Condensation (PCC) analysis
(Dose estimation by PCC method)
Mitsuaki Yoshida
Department of Radiation Biology Institute of Radiation Emergency Medicine Hirosaki University (IREM/HU)
BIODOSIMETRY IN THE 21st CENTURY Training Meeting HICARE in collaboration with the International Atomic Energy Agency Hiroshima, Japan, June 10-14, 2013.
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Radiological Accident ? Nuclear Plants Nuclear terrorism Nuclear Bomb Factory Medical field Scientific Research
6
Fukushima Dai- Ichi NPS
Atomic Bomb
CT scan laboratory
terrorism JCO in Tokai- Mura
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
1 . Medical systems for the radiation emergency 2. The system for Radiation Measurement Biological dose estimation Bioassay Physical dose estimation 3. Education of the professional responder
Preparedness for Radiation Emergency Medicine
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Most important thing in radiological accident is “medicine” to save a life of exposed person. The second is “to estimate the radiation dose”.
Why? Clinical symptom such as acute radiation syndrome depends on radiation dose. The information of the estimated dose will be useful for both the medical treatment and the protection of damage in the irradiated persons by suspecting the degree of damage.
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Biological dose estimation 1. Clinical symptom 2. Number of lymphocyte 3. Chromosome analysis -dicentric -translocation -PCC-ring -micronucleus
10
➸Chromosome is the most reliable biological marker (direct evidence) for the measurement of radiation damage (Gold standard)
Hirosaki University Chromosome Research Group
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Dose estimation (Dosimetry)
if possible
➸ quickly and accurately
8 4
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Dicentric assay: acute exposure (external) Translocation assay : old or long term (chronic) exposure (external)
Atomic bomb survivor in Hiroshima and Nagasaki Residents in high- background radiation area in China Children in Namie- Town of Fukushima Prefecture
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Dose estimation by PCC-ring method
suspected high dose exposure
Hirosaki University Chromosome Research Group
5 10 15 20
A: <1 Sv B: 1-2 Sv C: 2-5 Sv D: >5 Sv Days after the exposure
Lymphocyte
Thrombocyte(blood platelet)
Granulocyte After the cells are irradiated with high dose, the cell cycle will be stopped in G2 stage (G2 arrest) and will not proceed at the metaphase. or such cells will be dead (apoptosis) Chromosomes will not be Identified and analyzed. Radiation dose cannot be estimated.
Hirosaki University Chromosome Research Group
G1 S G2 M DNA replication
P M P M P M P M P M P M P M P M
metaphase
de-condensation
DNA
centromere
T-lymphocyte (G0 stage)
+PHA(The cell cycle starts by the stimulation of PHA)
The dynamics of DNA in the cell cycle.
P :Paternal M :Maternal
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
5
underestimate
: Y=αD2+βD+c
: real data of dic/cell
There is the difference between standard curve obtained from the dic analysis in the dose range from 0 – 5Gy data and the real data of dic/cell at the dose of over 5Gy.
If there is a possibility that the exposed dose may over 5Gy, the radiation dose will be underestimated.
Also, when lymphocytes are exposed By high dose radiation, apoptosis and G2 arrest are induced and chromosome Figure cannot be obtained. Therefore, in such case of possible high dose exposure, other technique should be applied in order to estimate radiation Dose.
PCC
M. S. Sasaki, et al.
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
PCC= Premature Chromosome Condensation
PCC is induced by fusing human lymphocytes with Chinese hamster ovary (CHO) mitotic cells in the presence of polyethylene glycol (PEG). PCC is also chemically induced by the treatment with a phosphatase inhibitor such as Okadaic acid or Calyculin A.
Hirosaki University Chromosome Research Group
Fusion by virus or PEG
Chromosome in Metaphase
Prematurely condensed chromosome
MPF: Mitosis Promoting Factor
Interphase Metaphase
Chromosome condensation is induced by activated MPF in interphase cell fused with metaphase cell.
PCC by cell fusion
Hirosaki University Chromosome Research Group
Functions of MPF
Triggers the formation of mitotic spindle through microtubule instability. Promotes mitosis i.e. chromatin condensation through phosphorylation of condensins. Causes nuclear envelope breakdown by phosphorylating the lamins that form an intermediate filament-type network (nuclear lamina) underlying the inner nuclear membrane. The three lamins present in the nuclear lamina, lamin A,B & C, are phosphorylated by MPF at serine amino residues. This leads to depolymerisation of the nuclear lamina & breakdown of nuclear envelope into small vesicles. Causes phosphorylation of GM130, which leads to the fragmentation of the Golgi and the ER
Hirosaki University Chromosome Research Group
Development of PCC assay - fusion PCC assay -
Johnson RT. Nature, 1970 Pantelias GE. Somatic Cell Genet, 1983
Virus-mediated PEG-mediated
Fusion PCC method has several issues, such as complicated operation, low condensation index, and maintenance of cells for fusion.
Hirosaki University Chromosome Research Group
Development of PCC assay - chemical induced PCC assay -
Gotoh E. Biomed Res, 1995 Dyban AP. Mol Reprod Dev, 1993
Mouse embryo Human PBL
PCC induced by okadaic acid PCC induced by calyculin A
Hirosaki University Chromosome Research Group
cdc25 cdc25
P
Protein Phosphatase Type 2A (PP2A) PP2A
CA, OA
Inactive MPF (Cyclin B/cdc2 complex)
P Cyclin B cdc2
inactive active
Cyclin B cdc2
Active MPF (Cyclin B/cdc2 complex)
dephosphorylation
Phosphorylation
promotion
inhibition
Chemical induced PCC(Hypothesis)
condensation
Hirosaki University Chromosome Research Group
Okadaic Acid : 500nM for 1hr
Calyculin A : 50nM for 1hr
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Premature Chromosome Condensation induced by Protein Phosphatase inhibitors
Okadaic acid Calyculin A
treat for 5~60min
PCC induced in lymphocytes at various stages of the cell cycle. A: G2/Meta-PCC, B: Meta/Ana-PCC, C: Ana-PCC Kanda et al.
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Typical images of G1-, S-, G2/M-, M/Ana, and Ana/Telo-PCC cells.
G1 S G2 M
PCC cells are classified into 5 categories as follows; G1-PCC cells showed univalent chromosomes, S-PCC cells had a "pulverized" appearance, G2/M-PCC cells displayed bivalent chromosomes, M/Ana-PCC cell showed separated sister chromatids, and Ana/ Telo-PCC cells had more distant separated sister chromatids than those in M/Ana-PCC cells.
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Problems in ring analysis by PCC 1. Chromosomes are over-condensed and become fuzzy. solution : optimization of chemical treatment (concentration and time) 2. Identification of ring chromosome solution: determination of criteria to identify ring
decreasing of detection sensitivity of ring chromosome
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Ana/Tel PCC G2/M PCC
It may be difficult to identify a ring chromosome
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Optimization Of
chemical treatment
Hirosaki University Chromosome Research Group
Conditions of calyculin A treatment used for chromosome aberration study by PCC assay
Cells Conc. Period Parameters References Whole blood 50 nM 30 min Translocation George K. Int J Radiat Biol, 2001
PBL 50 nM 30 min Chr #, PCC index Gotoh E. Int J Radiat Biol, 2005
PBL 50 nM 30 min Chromosome length ratio
Gotoh E. Int J Radiat Bio, 2005
Cell line 50 nM 5 min Chromatid break Bryant P. Mutagenesis, 2007
Whole blood 500 nM 60 min Chr #, chromosome length ratio
Wang ZZ. Radiat Environ Biophys, 2007
Cell line 80nM 60 min Fragment, translocation
Bergs JW, J Radiat Res (Tokyo), 2008
Cell line 50 nM 5 min Chromatid break Bryant P. Mutat Res, 2008
Whole blood 100 nM 60 min Fragment Wang ZZ. Mutat Res, 2009
Biopsy 80nM 60 min Chr #, translocation Darroudi F. Cancer Lett, 2010
Condition of calyculin A treatment in PCC assay is not fixed yet.
Radiation Biology IREM/HU
Radiation Biology IREM/HU
G2/M-PCC images in cell spread prepared using the optimized calyculin A treatment
0 Gy
7 Gy 20 Gy
3 Gy
Treatment with 50 nM Calyculin A 30 min
before harvesting
T. Miura and W. F. Blakely, Cytometry 2011
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Optimized condition of chemical treatment
Okadaic Acid : 1000nM for 60 min Calyculin A : 25 or 50nM for 30 min
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Dose response of PCC-ring in human lymphocytes
I. Hayata et al., J. RADIAT. RES., 42:SUPPL., S149-S155(2001)
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
The criticality accident in a uranium conversion test plant in Tokai-mura On September 30, 1999, at around 10:35 a. m., the criticality accident occurred in a uranium conversion test plant of the JCO Ltd., in Tokai-mura, Ibaraki Prefecture, Japan. As two workers (A and B) were pouring uranyl nitrate into a precipitation tank, the solution emitted a flash of blue light and the alarms went off to warn against gamma rays. Another worker (C) who was in the corridor next to the room immediately recognized that a criticality accident had taken place and ordered the two workers to evacuate. 60
61
Two workers (A and B) were pouring uranyl nitrate into a precipitation tank, the solution emitted a flash of blue light and the alarms went off to warn against gamma rays.
Hirosaki University Chromosome Research Group
Hematological change in the 3 patients in Tokai-mura criticality accident
A B C
62
Hirosaki University Chromosome Research Group
Hirosaki University Chromosome Research Group
Hirosaki University Chromosome Research Group
Biodosimetry by PCC assay in the JCO accident
Hayata I. J Radiat Res (Tokyo), 2001
Pt. A
Pt. B
Pt. C
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Preparedness for
Cytogenetic biodosimetry in
Hirosaki University
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Culture after stimulation by PHA
Taking Blood (heparinized)
Harvest
Staining
CO2 incubator (48 h)
Whole blood or
Isolated lymphocytes
Hypotonic treatment and
Cell fixation
Slide preparation Observation/analysis
Colcemid treatment to stop the cell cycle
Flow of chromosome analysis
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Equipment for the cytogenetic biodosimetry laboratory
1. Culture
Centrifuge : for isolation of lymphocytes
Safety cabinet CO2 incubator
2. Harvest: hypotonic treatment and fixation
Centrifuge and water bath
Cell Harvester :HANABI-PIII ADStec. Inc., Japan
Chromosome spreader : HANABI-PIV ADStec. Inc. Japan
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Automated Chromosome Spreader (HANABI-PIV, ADStec. Inc. Japan)
Automated Cell Harvester (HANABI-PII, ADStec. Inc., Japan)
A total of 24 blood samples are processed by this machine at one time.
A total of 96 slides can be prepared by this machine (Max. 96 samples, 1slide/sample).
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Slide preparation Scanning and analysis
Microscope and PC
Freezer Refrigerator
Phase contrast microscope
Manual HANABI 5 slides
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
High yield chromosome preparation technique (isolated lymphocytes culture) 1.Isolation of lymphocytes from 3 ml PB using CPT tube 2.Culture for 48 hrs in 20%FCS/RPMI medium containing PHA and/or Colcemid (3.Add Calyculin A for 30 min ~1 hour before starting harvest) 4.Hypotonic treatment with 0. 075M KCl for 20 min 5.Fix the cell with methanol- acetic acid (3: 1 ) sol. 6.Slide preparation by chromosome spreader
15ml tube
Hirosaki University Chromosome Research Group
Isolation of the lymphocytes from whole blood
BD CPT Tube Remove PHA in tube Add 20%FCS culture medium (1 ml) Add the whole blood (3ml)
Centrifugation 3000rpm for 1 5min
Remove the lymphocytes Culture (6ml) + PHA with or without Colcemid
Washing and Centrifugation 1 500rpm for 5min
Hirosaki University Chromosome Research Group
Heparinized whole blood 3~5ml
Remove this solution
Add 1ml of 20%FCS/RPMI1640
1. taking whole blood using heparin
2. Preparation of CPT tube (Becton Dickinson)
Hirosaki University Chromosome Research Group
3. Add 3ml of whole blood into CPT tube
Remove plasma and add 5~6ml of 2%FCS/ RPMI1640(4℃) and Pipetting to take lymphocytes
Centrifuge this CPT tube at 3000rpm for 15min at room temperature
Red blood cells
Lymphocytes You cannot see the layer of lymphocytes.
plasma
Transfer solution including lymphocyte Into 15ml tube
Hirosaki University Chromosome Research Group
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Education and Training experiences of Hirosaki University Chromosome Research Group for Cytogenetic biodosimetry
Laboratory of Dept. of Radiation Biology 1. Is a member of the REMPAN of WHO. 2. Is member of the exchange program of researcher between Japan and Asian countries supported by the MEXT in Japan. October, 2012-March 2013: Ms. Benchawan Rungsimaphorn from Thailand 2013, Exchange program by the MEXT 3. Is supporting the establishment of cytogenetic biodosimetry in Asian countries. Thailand, Korea, Taiwan 4. Is accepting researchers and technologist from Asian countries. 2010, October- December, Dr. Wol-Soon, Jo, Korea 2013, Researcher from Thailand, supported by the IAEA program
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Preparedness of
Radiation Emergency Medicine in
Hirosaki University
National Institute of Radiological Sciences Hiroshima
Hirosaki City Hirosaki University
Tokyo
Location of Hirosaki City Aomori Prefecture
Nagasaki
Hirosaki University
Radiation Biology IREM/HU
Chromosome Research Group
Fukushima NPP
Nuclear power facilities in Aomori Prefecture
Hirosaki University
Radiation Biology IREM/HU
Chromosome Research Group
Hirosaki University Radiation Safety
Organization
Graduate School of Medicine
School of Medicine
Graduate School of Health Science
Advanced Emergency & Disaster Medical Center in University Hospital
Institute of Radiation Emergency Medicine (IREM/HU)
Administrative office Hirosaki University
Radiation Biology IREM/HU
Chromosome Research Group
Advanced Emergency and Disaster Medical Center
Hirosaki University
Radiation Biology IREM/HU
Chromosome Research Group
July 1st, 2010 Advanced Emergency and Disaster Medical Center
The victims in radiological accident are transferred by helicopter.
Decontamination Room
Patient treatment room
body surface contamination monitor Hand and foot cross monitor Refrigerator for specimens
Whole body counter
Total body Thyroid monitor
WBC-301B ALOKA
Radiation Measurement room (Bioassay)
Equipment for bioassay
βmeasuring instruments ( liquid scintillation counter)ALOKA LSC-LB5 α measuring instrument
Low backgroundα/β automated counter ALOKA LBC-4302
Gamma measuring instruments
Sterilized room and treatment room for the victims with a burn
Radiation Biology IREM/HU
Radiation Biology IREM/HU
Hirosaki University Chromosome Research Group
Co-medical Education Program in Radiation Emergency Medicine
The grant of the Ministry of Education, Culture, Sports, Science and Technology, JAPAN (2008 - 2012)
The Projects in Hirosaki University
文部科学省科学技術振興調整費 地域再生人材創出拠点の形成
被被ばばくく 医医療療ププ ロロ フフ ェェ ッッ シシ ョョ ナナルル育育成成計計画画 MEXT Special Coordination Funds for Promoting Science and Technology Re-establishment of Vital regions through Fostering Talents
Education Program for Professionals in Radiation Emergency Medicine
The grant of the Ministry of Education, Culture, Sports, Science and Technology, JAPAN (2010 - 2014)
Hirosaki University
Aomori Prefecture
Hirosaki University Chromosome Research Group
Education Programs for Radiation Emergency Medicine
Seminar in “Education program for Professional in REM”
Dr. Makoto Akashi, NIRS, Japan Dr. Joan Francesc Barquinero, Barcelona University, Spain
Dr. Reiko Kanda, NIRS, Japan
Exercise and training on REM
Hirosaki University Chromosome Research Group
Education Programs for Radiation Emergency Medicine
International Conference, March 1 9th, 201 0 Dr. Gordon Livingston, REAC/TS, USA Dr. William F. Blakely, AFRRI, USA Dr. Horst Romm, Bundesamt fuer Strahlenschutz, Germany Dr. Ruth Wilkins, Health Canada, Canada
4th Seminar for “Education Program for Professional in Radiation Emergency Medicine”, January 24th, 201 1 Dr. Zhanat Carr, WHO Dr. David Lloyd, HPA , England Dr. Firouz Darroudi, Leiden University the Netherlands
Thank you for your attention!! Mt. Iwaki in Hirosaki
Hirosaki University
Radiation Biology IREM/HU
Chromosome Research Group