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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
5
• 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)
6
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 (♂)
7
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
9
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
10
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
12
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
13
14
• 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
15
• 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
16
• 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
17
• 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
18
Hybridization plots with vs. # of samples
8 samples 7 samples 6 samples
• Refinement of analysis – I Excluded – Samples 2 & 3; Criteria: Outliers
19
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
20
• Refinement of analysis– I Quality of genes
21
Molecular function (28.74%)
Biological process (34.48%)
Cellular component
(36.78%)
• Refinement of analysis – I Gene cluster analysis
22
• 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
26
• Refinement of analysis – III Excluded – Samples 4, 5, 7 & 8; Criteria: Outliers in two dose rates
27
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
28
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
30
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
31
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
33
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
34
• 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
35
• 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
38
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
39
40
Thank you for your kind attention
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