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Cytogenetic biomarkers of ionising radiation exposure: a multiparametric approach

M. Prakash HANDE, Ph.D., M.P.H.

Genome Stability Laboratory Department of Physiology

Yong Loo Lin School of Medicine National University of Singapore

Singapore

Genome Stability Laboratory @ Department of Physiology

•  Telomeres and telomerase • DNA damage response and repair • Experimental Cancer Therapeutics • Radiation Biology/Biodosimetry •  Toxicogenomics/Environmental

Toxicology

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Frequencies of dicentrics and genomic translocations in mouse splenocytes following whole body, 2 Gy X-irradiation

Genomic Translocations Dicentrics

Days after exposure

Freq

uenc

y pe

r cel

l

Hande et al. (1996a) Int. J. Radiat. Biol. 69:437-446

D(t)=0.30 e-0.05t

T(t)=0.35 e-0.006t

0.50

0.00

0.10

0.20

0.30

0.40

-20 0 80 60 20 40 120 100

d t

f

Days after exposure

Freq

uenc

y pe

r 100

cel

ls

Translocations in mouse splenocytes after in vivo X-irradiation analysed by FISH

0 10

20

30

40

50

60

0 50 100 150 200 250

Hande and Natarajan (1998) Int. J. Radiat. Biol. 74, 441-448

Hande and Natarajan (2009) In: Radiation Risk Health Sciences, Springer Japan. Pp 143-149.

0.5 Gy 1.0 Gy 2.0 Gy 3.0 Gy

0

10

20

30

40

50

60

0 50 100 150 200 250

Reciprocal Non-reciprocal

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Frequencies of micronuclei and fragments in mouse splenocytes following 2 Gy X-irradiation

Freq

uenc

y pe

r 100

cel

ls

Days after exposure

Hande et al. (1996) Int. J. Radiat.Biol. 70, 375-383

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Detection of intra-chromosomal exchanges in human lymphocytes

Boei et al. (1999) Mutat Res 404: 45-53

April 2001 – Ozeorsk, Russia MAYAK PRODUCTION ASSOCIATION

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Past exposure to densely ionising radiation leaves a unique permanent signature in the genome

Intrachromosomal aberrations: pericentric, paracentric inversions and intra-arm inversions measured in chromosome 5; Interchromosomal aberrations: Apparently simple translocations between heterologous pair of chromosome

Hande et al (2003) Am J Hum Genet 72:1162-1170

Simple versus Complex Chromosomal exchanges in Mayak workers

0 2 4 6 8 2 4 6 8

Controls

Reactor Workers Zero Pu, High γ

Moderately exposed Pu workers

Highly exposed Pu workers

Aberration Frequency (%)

Simple Complex

82/1414

10/444

67/1424

4/582

41/1414

1/444

3/1424

0/582

88/1414*

3/444*

7/1424*

0/582*

*total aberrations from complex aberrations Hande et al. 2005 Genes Chromosomes Cancer. 2005 Sep;44(1):1-9

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Complex Chromosome Aberrations in Mayak Workers

Hande et al. 2005 Genes Chromosomes Cancer. 2005 Sep;44(1):1-9

Approach

•  Individual end points were studied independently by different laboratories

•  Few or no attempts were made to study different biomarkers on a single sample

•  Need for a multi-parametric approach on a single sample following irradiation

Initiated the work in 2006

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Ionising Radiation

Gamma radiation

Whole blood (A)

Peripheral blood derived

lymphocytes (B)

Heavy Ion Radiation**

Carbon (whole blood)

(C)

Iron (whole blood)

(D)

(n=10) γH2AX cronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression Cytokine profile

(n=10) γH2AX Micronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression miRNA expression Proteomic profile

(n=3) γH2AX Micronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression

(n=3) Micronucleus Chromosomal aberration (PNA FISH, mFISH) Gene expression

Doses : 0.1 Gy, 0.25 Gy, 0.5 Gy, 1 Gy (1.06 Gy/min, 137Cs source)

Time points post irradiation : 2 h and 24 h

**National Institute of Radiological Sciences, Chiba, Japan (R Okayasu)

Micronuclei detection in mouse/human cells

Hande et al. (1996) Int. J. Radiat.Biol. 70, 375-383 Poonepalli et al. (2005) Cancer Research 65: 10977-109883

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Single and Two colour FISH

Interphase nuclei: Single colour and two colour FISH

Minor satellite Lambda DNA and c-myc gene

Cop

yrig

ht ©

199

4 M

. Pra

kash

Han

de

Cop

yrig

ht ©

199

4 M

. Pra

kash

Han

de

Frequency of centromere-positive micronucleus in mouse splenocytes

Hande et al. (1996) Int. J. Radiat.Biol. 70, 375-383

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PNA FISH in the detection of telomeres

Blasco, Lee, Hande et al. 1997, Cell 91:25-34

Use of PNA FISH with telomere probe in mouse studies

Hande et al. 1999 J. Cell Biol.144: 589-601

Di fagagna, Hande et al. 1999 Nature Genetics

Di Fagagna, Hande et al., 2001 Current Biology

RLC

Dicentric

RLC

translocation

Translocation

Dicentric

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Use of centromere and telomere PNA probes to identify chromosome aberrations in human cells

Low et al. 2008 Int J Biochem Cell Biol 40:2583 – 2595

Gopalakrishnan et al. 2010 Genome Integr 1:16

Vinoth et al. 20008 Stem Cells Dev 17:599 – 607

Banerjee et al 2008 Breast Cancer Res Treat 107: 25 -31

Boei, Vermeulen, Natarajan 2000 Int J Radiat Biol 76:163-167

Differential gene

expression profile

following irradiation: two

way hierarchical

clustering

0 0.1 0.25 0.5 0 0.1 0.25 0.5

0.25 Gy

24 h

Radiation Dose (Gy)

2 h

3 ANOVA (p<0.05)

ROW : GENE COLUMN : SAMPLE

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Gene expression profile following gamma irradiation

PBMCs Principle component analysis

Whole Blood

High-throughput expression profiles of

transcriptome, miRNA, etc

Time/dose of known radiation exposure

Dose-response following exposure to

human peripheral Blood (ex vivo)

Dose-response clinical endpoints of all parameters

Biological data from public

domains and other literatures

Low, medium & high-throughput profiles of

radiation exposure

Risk estimation, elucidation of underlying

mechanism

Transcriptomic Knowledgebase

REFIRE Knowledgebase

Transcriptomics miRNA Cytogenetics

Risk Estimation Following (low-dose) Ionising Radiation Exposure (REFIRE)

Jayapal, Zeegers, Sethu, Banerjee, Hande 2011 Defence Science Journal, Vol. 61, 133-137

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REFIRE Knowledgebase - Our Tool Box for Radiation Biology

Dose, Time

Networks

Bioinformatics analysis

MN, CA DNA damage

Today

Optimised Dose, time catalogue

Complex pathways

Advance complex analysis

databases

Dose,/Time based catalogue

of MN and CA

Tomorrow

Biomarkers

Catalogue of Time / dose

specific markers

From a basic toolbox today to…

Integrated design, measurements & tools to enable predictable radiation exposure

Pilot population

Broad population spectrum

Summary

•  Donor specific effects and individual variation should be taken in to consideration for biomarker identification

•  Gene expression – changes may depend on time after exposure than on dose

•  More than one biological response marker may be necessary in biodosimetry

•  Influence of life-style factors

•  Validation of these biomarkers will be done in a larger population and radiotherapy patients

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Study design for CRP

•  To generate calibration curves for gamma radiation in human blood lymphocytes

•  Human PBL •  3 apparently healthy donors •  0, 0.25, 0.5, 1, 2, 3 and 4 Gy; 137Cs •  Dicentric and Micronucleus assay

Acknowledgements

Defence Innovative Research Programme, Defence Science and Technology Agency, Singapore; IAEA CRP E3. 50.08

• Swaminathan Sethu • Grace Low • Aik Kia Khaw • Birendranath Banerjee • Dimphy Zeegers • Manikandan Jayapal

• Susan Loong Li Er, National Cancer Centre, SGH, Singapore

• Rajamanickam Baskar (Radiation Oncology, National Cancer Centre, Singapore)

Ryuichi Okayasu and Takamitsu Kato, NIRS, Chiba