Bio-dosimetry in the 21st Century: Case of Molecular Bio-marker

Rajesh N. Sharan Radiation & Molecular Biology Unit North-Eastern Hill University Shillong 793022 (India) rrnsharan@nehu.ac.in.......

Title:   Preparedness to respond to possible acts of nuclear terrorism: Some strategies and recommendations

Author:   R. N. Sharan Publication:   INTERNATIONAL JOURNAL OF RADIATION

BIOLOGY

Jan 2003, Vol. 79, No. 3, Pages 217-219

Publisher:   Informa Healthcare Date:   Jan 1, 2003

2

Title:   Proteomic and genomic modulations induced by γ-irradiation of human blood lymphocytes

Author:   Andrei Turtoi1, Rajeshwar N. Sharan2, Alok Srivastava3, Frank H. A. Schneeweiss1

Publication:   INTERNATIONAL JOURNAL OF RADIATION BIOLOGY

October 2010, Vol. 86, No. 10 , Pages 888-904  

Publisher:   Informa Healthcare Date:   October, 2010 3

Dicentric Assay (DCA)

•  Cytogenetic bio-dosimetry

4

• PART A: Present

• R&D: Global protocol

Human blood under physiological condition

Ex vivo γ-irradiation of whole blood

Peripheral blood lymphocyte (PBL)

Cytogenetic analysis – DCA ( + ‘Cell culture’)

Global gene expression profiling – DNA Microarray analysis

Identification of “candidate” RRGs – Data mining/analysis

Validation by qRT-PCR to indentify “potential” RRP

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•  Ethnic population group of the North-East

Human blood under physiological condition

Ex vivo γ-irradiation (~ 8.39 Gy min-1) of heparinized blood (0.5,1 and 2 Gy)

Post-irradiation repair incubation at 37 0C for 1 h

Whole blood culture (RPMI-1640, 10% FCS; 0.01 mg ml-1 PHA)

0.05 mg ml-1culture Colcemid added after 24 h incubation

 Cultures harvested 52 h post-PHA stimulation

Cells treated with hypotonic KCl solution (60 mM) solution for 20 min at 37 0C

Cells fixed in 3:1 (methanol: acetic acid) fixative

Slides stained with 6 % Giemsa stain

Chromosomal aberration

• Experimental protocol (IAEA, 2011)

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Source (dose rate)

Dose in Gy

No. of meta-phases

Dicentrics

1 2 Yield

γ-rays (8.39 Gy/min;

60Co )

0.5 1100 40 0 0.036 + 0.005132

1 1343 62 1 0.047 + 0.002309

2 1393 153 7 0.120 + 0.021595

Radiation dose (Gy)

Fre

que

ncy

of d

ice

ntric

s/ c

ell γ-rays (8.39 Gy/min; 60Co )

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

control 0.5 1 2

male

• Dose-response curve: γ-rays (♂)

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Source (dose rate)

Dose in Gy

No. of meta-phases

Dicentrics

1 2 Yield

γ-rays (8.39 Gy/min;

60Co )

0.5 1145 38 0 0.033 + 0.003786

1 1178 68 1 0.059 + 0.016921

2 1333 131 6 0.107 + 0.022502

Radiation dose (Gy)

Fre

que

ncy

of d

ice

ntric

s/ c

ell γ-rays (8.39 Gy/min; 60Co )

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

control 0.5 1 2

female

• Dose-response curve: γ-rays (♀)

8

Radiation dose (Gy)

Fre

que

ncy

of d

ice

ntric

s/ c

ell

γ-rays (8.39 Gy/min; 60Co )

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

control 0.5 1 2

mean of all samples male

female

• Dose-response curve: γ-rays

Source (dose rate)

Dose in Gy

No. of meta-phases

Dicentrics

1 2 Yield + SE

γ-rays (8.39 Gy/

min; 60Co)

0.5 2245 78 0 0.0345 + 0.002121

1 2521 130 2 0.053 + 0.008485

2 2726 284 13 0.1136 + 0.009192

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Source (dose rate)

Dose in Gy

No. of meta-phases

Dicentrics

1 2 Yield

X-rays (1.5 Gy/min; 110

KVp )

0.5 500 0 0 0

1 500 15 0 0.042

2 500 31 3 0.074

Distribution of dicentrics in human (♂) lymphocytes exposed ex vivo X-rays (1.5 Gy/min; 110 kVp )

Radiation dose (Gy)

Fre

que

ncy

of d

ice

ntric

s/ c

ell

X-rays (1.5 Gy/min;110 KVp

Dose response calibration curve for dicentrics induced by X-ray

0

0.02

0.04

0.06

0.08

0.1

0.12

control 0.5 1 2

• DCA: X-rays

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Genomics to Proteomics approach

Radiation response gene (RRG)

Radiation response protein (RRP)

•  Molecular bio-dosimetry

3rd ACRR, Beijing, 2013 11

• PART B:

•  Molecular bio-marker identification •  Radiation Response Protein (RRP)

•  Specific to radiation

•  Showing linear dose response curve

•  Should be present in human blood (lymphocytes)

•  Raise antibody against the “identified” RRP •  Quantitative detection of RRP

•  Direct/indirect enzyme linked immuno-sorbant assay (ELISA)

•  Sensitive, specific and robust methodology

•  Easy to perform in short time

•  Compare to a similar calibration curve •  Easy automation for large sample size

•  Outlines of molecular bio-dosimetry

• Strategy for future

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Disclosure A patent (provisional)

application filed on the concept, RRG and RRP in

India under PTC vide # 486/KOL/2013 of

29 April 2013

• Global protocol

Human blood under physiological condition

Ex vivo γ-irradiation of whole blood

Peripheral blood lymphocyte (PBL)

Cytogenetic analysis – DCA (+ ‘Cell culture’)

Global gene expression profiling – DNA Microarray analysis

Identification of “candidate” RRGs – Data mining/analysis

Validation by qRT-PCR to indentify “potential” RRP

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•  60Co γ-rays •  Doses

•  0 (sham-control), 0.5, 1 & 2 Gy •  Dose rates

•  2.165 Gy min-1 (high) & 0.871 Gy min-1 (low)

• Human volunteers

•  Eight in number •  Five male (24 - 55 years) •  Three female (30 - 32 years)

•  Largest study - globally

• DNA Microarray

• Array •  Affimatrix GeneChip PrimeView - human

platform •  630 k probes; 38 k transcripts; 20 k genes

• Analysis & Data mining

•  GeneSpring GXT software

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• DNA Microarray RRGs in individuals at each dose point vs. respective controls

60Co γ-radiation Response # of RRG genes with ≥ 2 fold change

1 2 3 4 5 6 7 8

0.5 Gy

Vs

Control

Up regulated 20 161 682 31 25 25 170 260

Down regulated 52 8 50 93 52 32 239 186

Total 72 169 732 124 77 57 409 446

1 Gy

Vs

Control

Up regulated 43 55 5622 26 24 14 788 89

Down regulated 11 6 6004 68 38 34 688 204

Total 54 61 11626 94 62 48 1476 293

2 Gy

Vs

Control

Up regulated 22 728 404 16 17 15 157 325

Down regulated 47 430 978 26 8 2 121 85

Total 69 1158 1382 42 25 17 278 410

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• DNA Microarray RRGs in individuals at all 3 dose points vs. respective controls

Parameter   Response  

60  Co  γ-‐radia3on  

(0.5,  1  &  2  Gy)  Vs  Control  

1   2   3   4   5   6   7   8  

#  of  RRG  genes  with  ≥  2  fold  

change    Total     72   1240   11032   147   112   113   1408   631  

#  of  genes  showing  similar  expression  in  all  

doses    

Up  regulated   21   657   1407   28   25   22   337   195  

Down  regulated     23   216   1204     81   63   34   388   181  

Total     44   873   2611   109   88   56   725   376  

#  of  genes  showing  increasing  expression    

Up  regulated   3   115   30   4   0   1   16   31  

Down  regulated     3   122   173     1   0   1   18   12  

Total     6   237          203   5   0   2   34   43  

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• DNA Microarray RRGs in individuals at all 3 dose points vs. respective controls

Parameter   Response  

60  Co  γ-‐radia3on  (0.5,  1  &  2  Gy)  Vs  Control  

2.165  Gy  min-‐1   0.871  Gy  min-‐1  

P  =  0.05  FC  ≥  2    on    

P  =  0.05  

FC  ≥  1.5    on    

P  =  0.05  P  =  0.05  

FC  ≥  2    on    

P  =  0.05  

FC  ≥1.5    on    

P  =  0.05  

#  of  RRG  genes   Total     532   2   31   344   129   294  

#  of  genes  showing  similar  expression  in  all  

doses    

Up  regulated   82   0   6   38   28   36  

Down  regulated     135   0   16   50   34   47  

Total     217   0   22   88   62   83  

#  of  genes  showing  increasing  expression    

Up  regulated   9   0   1   1   0   1  

Down  regulated     20   0   1   5   1   4  

Total     29   0   2   6   1   5  

• Detailed analysis: Comparative Principal component analysis (PCA) vs. # of samples

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Hybridization plots with vs. # of samples

8 samples 7 samples 6 samples

• Refinement of analysis – I Excluded – Samples 2 & 3; Criteria: Outliers

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Sample    #  Age  of  donor    (years)  

Gender  of  donor  Dose  Rate  (Gy  

min-‐1)  

1   30   Female   2.165  

4   30   Female   0.865  

5   24   Male   0.865  

6   55   Male   2.165  

7   32   Female   1.9  

8   55   Male   0.810  

COMMON UP-REGULATED GENES = 6 COMMON DOWN-REGULATED GENES =

32

• Refinement of analysis - I One-way ANOVA with cut-off P = 0.05: Number of genes = 2043 Differentially expressed genes at fold change cut-off >= 1.5: Number of genes = 226

Venn diagram

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• Refinement of analysis– I Quality of genes

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Molecular function (28.74%)

Biological process (34.48%)

Cellular component

(36.78%)

• Refinement of analysis – I Gene cluster analysis

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• Refinement of analysis – II Excluded – Samples 7 & 8; Criteria: Outliers in two genders

23

Sample    #   Age  of  donor    (years)  

Gender  of  donor  

Dose  Rate  (Gy  min-‐1)  

1   30   Female   2.165  

4   30   Female   0.865  

5   24   Male   0.865  

6   55   Male   2.165  

COMMON UP-REGULATED GENES = 11 COMMON DOWN-REGULATED GENES =

16

• Refinement of analysis - II One-way ANOVA with cut-off P = 0.05: Number of genes = 3896 Differentially expressed genes at fold change cut-off >= 1.5: Number of genes = 113

Venn diagram

24

• Refinement of analysis – II Quality of genes

25

Biological process (35.14%)

Cellular component

(37.84%)

Molecular function (27.03%)

• Refinement of analysis – II Gene cluster analysis

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• Refinement of analysis – III Excluded – Samples 4, 5, 7 & 8; Criteria: Outliers in two dose rates

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Parameter      

Samples  1,  6  @  2.165  Gy  min-‐1  and    Samples  4,  5  at    0.871  Gy  min-‐1)  

Response  

60Co  γ-‐radia3on  (0.5,  1  &  2  Gy)  Vs  Control  

2.165  Gy  min-‐1   0.871  Gy  min-‐1  

P  =  0.05   FC     P  =  0.05   FC  

#  of  RRG  genes  with  ≥  1.5  fold  change         1942   24   3419   193  

#  of  genes  showing  similar  expression  

in  all  doses    

Up  regulated   458   0   718   4  

Down  regulated     157   4   991   16  

COMMON UP-REGULATED GENES = 4 COMMON DOWN-REGULATED GENES =

16

• Refinement of analysis - III One-way ANOVA with cut-off P = 0.05: Number of genes = 1866 Differentially expressed genes at fold change cut-off >= 1.5: Number of genes = 54

Venn diagram

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29

Present in samples exposed to both dose rates High ( ~2 Gy min-1) and low ( ~1 Gy min-1)

• Radiation Response Genes (RRG) ‘Candidates’

Present in all individuals ♂ as well as ♀

Present in samples exposed to all three doses of radiation 0.5, 1 and 2 Gy besides the control

Exhibit linear dose response curves Up- and down-regulated genes

To validate in a larger population-based study by RT-PCR

To select out “potential” RRP from the “candidate” RRG

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Present in samples exposed to both dose rates High ( ~2 Gy min-1) and low ( ~1 Gy min-1)

• Radiation Response Genes (RRG) ‘Candidates’

Present in all individuals ♂ as well as ♀

Present in samples exposed to all three doses of radiation 0.5, 1 and 2 Gy besides the control

Exhibit linear dose response curves Up- and down-regulated genes

To validate in a larger population-based study by RT-PCR

To select out “potential” RRP from the “candidate” RRG

• Approach: RT-PCR

Human blood under physiological condition

Ex vivo γ-irradiation of whole blood

Isolate total RNA (TRIzol)

cDNA (High–Capacity cDNA Reverse Transcription kit)

Taqman Gene Expression assay: 95ºC - 20 s ► 40 x 95°C - 03 s; annealing at 60ºC - 30 s

Average CT & ΔΔCT calculated - Fold Difference of each gene relative to the untreated and endogenous (18S) controls

SDS v1.4 software used to analyze

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Sample Gender Age (Years)

1 Female 33

2 Male 55

3 Female 27

4 Male 36

5 Male 24

6 Female 27

• Radiation Response Protein (RRP)

32

• RT-PCR: Primers

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Assay ID Gene

Amplicon size (bp)

Transcript

Exon boundary

Hs00402499_g1 ZNF440

149

NM_152357.2

3-4

Hs01675503_s1

ORC64

82

NM_001005494.1

1-1

Hs01631566_g1

GCSH

138

NM_004483.4

5 - 5

Hs00172459_m1

MCM3

66

NM_002388.3

12-13

Hs02596862_g1

MT-ATP6

150 NC_012920

Hs00232157_m1

NFIC

82 NM_205843

6-7

4319413E Eukaryotic 18S r RNA 187 X03205.1

NA

• RT-PCR: Analysis

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•  Comparative quantification algorithms - ΔΔCt

The ΔΔCt method compares results from experimental samples with both a calibrator (e.g., untreated or wild-type sample) and a normalizer (e.g., housekeeping gene expression). Cts for the gene of interest (GOI) in both the test sample(s) and calibrator sample are adjusted in relation to a normalizer (norm) gene Ct . The resulting ΔΔCt value is incorporated to determine the fold difference in expression.

Fold difference = 2-ΔΔCt ΔCt sample - ΔCt calibrator = ΔΔCt Ct GOIs - Ct norms = ΔCt sample Ct GOIc - Ct normc = ΔCt calibrator

FEMALE MALE

• RT-PCR: Results

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•  Gender sensitive RRP

FEMALE MALE

• RT-PCR: Results

36

•  Gender sensitive RRPs

• RT-PCR: Results

37

•  Gender neutral ‘potential’ RRP?

RRG/RRP

•  Twenty ‘candidate’ RRG identified

•  60Co γ-radiation •  Immediate response

genes •  Limited validation by RT-PCR

•  Screened 6 RRG •  Screening underway for

others •  Preliminary ‘potential’ RRP in

sight

•  Larger number of sample •  Different post-irradiation time

interval •  Identification of RRP as

molecular bio-marker •  Antibody •  Establishment of direct/

indirect ELISA based assay •  International cooperation

WAY FORWARD

• SUMMARY

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COWORKERS

•  Ms. Saibadaiahun Nongrum •  Dr. S. T. Vaiphei •  Mr. Joshua •  Dr. L. Kma •  Dr. R. C. Chaubey

•  IAEA, Vienna: CRP – II on bio-dosimetry

•  # 17047 •  BRNS, DAE, Govt. of India

•  # 2009/36/04-BRNS/263 •  DST, Govt. of India NE

Infrastructure Fund

GRANTS

• Acknowledgements

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Thank you for your kind attention