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◆ Low dose-rate irradiation and threshold dose-rate for radiation risk.
◆ A comparison of the mutagenic effects of tritium rays and 137Cs rays on wild and p53-deficient mice.
Toshiyuki Umata
Radioisotope Research Center, University of Occupational and Environmental Health, Japan
EMRAS , Theme 3Ⅱ WG tritium
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◆ Low dose-rate irradiation and threshold dose-rate for radiation risk.
◆ A comparison of the mutagenic effects of tritium rays and 137Cs rays on wild and p53-deficient mice.
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OBJECTIVE OBJECTIVE
To investigate the biological effects of tritium on mouse at low dose-rate, mice were exposed to -rays by continuous administration of various concentration of tritiated drinking water throughout their lives at low dose-rates.
CONCLUSION CONCLUSION
These studies revealed that there exists two types of threshold dose-rates, not only in the frequency of thymic lymphomas but also in the life-shortening. In the life-shortening, it seems that the effect of tritium rays is greater than that of rays.
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blood
organ tissue(average of brain,Liver, muscle)
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Dose-rate, Gy/day 0.24 0.0 96 0.048 0.024 0.010 0Number of mice used 45 38 60 60 53 67MST, days 165±36 259±52 414±66 481±112 622±121 811±134
Thymic lymphoma 29(64)[162±28] 22(58)[273±51] 15(25)[415±53] 4(7)[508±202] 3(6)[589±32] 0(0)
Non-thymic lymphoma 5(11)[146±27] 4(11)[229±24] 12(20)[433±82] 9(15)[504±120] 11(21)[609±70] 12(18)[787±129]Reticular cell neoplasm 2(5)[179±15] 5(8)[390±67] 12(20)[485±144] 10(19)[570±150] 4(6)[760±161]Ovarian tumor 2(5)[201±18] 4(7)[431±60] 8(13)[511±98] 11(21)[641±114] 4(6)[868±149]Haemangiosarcoma 2(5)[331±21] Fibrosarcoma 2(3)[431±58] 4(7)[467±97] 6(11)[607±90] 4(6)[871±179]Harderian gland tumor 2(3)[423±81] 2(3)[537±75]Lung tumor 1(2)[464] 3(5)[460±30] 8(15)[736±84] 4(6)[812±24]Skin tumor 1(2)[401] Bladder tumor 1(2)[580]Rhabdomyosarcoma 1(2)[298] Mammary tumor 2(4)[582±58]Hepatic tumor 2(4)[685±23] 3(4)[696±41]Adenal gland tumor 1(2)[623]Splenic tumor 2(3)[827±19]Stomac tumor 1(2)[912]Double tumor-bearing 0(0) 0(0) 0(0) 2(3) 10(19) 5(8)Tumor-bearing mouse 34(76) 32(84) 42(70) 42(70) 4(83) 41(54)
( );% [ ];Mean latent period ±SD, dayMST: mean survival time or mean time of death after the initiation of the exposure.
Table1 Tumor developments in mice at different dose-rates of HTO
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Number of mice 69 51 120 55 58 120MST, days 810 795 804 790 758 804Thymic lymphoma 0(0) 0(0) 0(0) 0(0) 0(0) 3(5)[556±40]
Non-thymic lymphoma 16(23)[773±147] 14(27)[799±113] 30(25)[787±129] 14(25)[769±137] 17(29)[779±84] 11(20)[728±97] Fibrosarcoma 5(7)[860±161] 4(8)[81±126] 9(8)[838±145] 5(9)[836±36] 13(22)[767±116] 5(9)[835±18] Ovarian tumor 4(6)[868±149] 2(4)[885±80] 6(5)[875±121] 3(5)[829±187] 2(3)[811±197] 5(9)[819±111] Liver tumor 6(9)[800±151] 3(6)[729±202] 9(8)[776±168] 4(7)[939±62] 10(17)[844±120] 1(2)[729] Lung tumor 3(4)[811±24] 2(4)[967±22] 5(4)[873±23] 4(7)[847±115] 1(2)[852] Other tumors 3(4)[855±40] 3(6)[775±139] 6(5)[815±110] 4(7)[920±78] 3(5)[879±91] 2(4)[941±3]Multiple tumor-bearing 4(6) 2(4) 6(5) 3(5) 4(7) 2(4)Tumor-carrying mice 33(48) 26(51) 59(49) 27(49) 45(78) 26(46)
Dose-rate, mGy/day Control 1* Control 2 Total control 0.2 0.9 3.6(Cumulative dose, Gy) (0) (0) (0) (0.17±0.03) (0.71±0.13) (2.62±0.41)
( );% [ ];Mean latent period ±SD, dayMST: mean survival time or mean time of death after the initiation of the exposure. *Previously reported (Yamamoto et al. 1995)
Table2 Tumor developments in mice at different dose-rates of HTO
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50 mGy/day=18 Gy/y
25%
400 days
2×109 Bq/L
20 Gy
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SUMMARYSUMMARY
◆ There exists two types of threshold dose-rates, essential and practical, not only in the frequency of thymic lymphoma but also in the life-shortening. In the life-shortening, it seems that the effect of tritium rays is greater than that of rays.
◆ Threshold dose-rate in the frequency of thymic lymphomas was12 (0.9) mGy/day. And that in the life-shortening was 2 (0.2) mGy/day (1.8 Gy).
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◆ Low dose-rate irradiation and threshold dose-rate for radiation risk.
◆ A comparison of the mutagenic effects of tritium rays and 137Cs rays on wild and p53-deficient mice.
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OBJECTIVES OBJECTIVES
Although nuclear fusion facilities, such as ITER, are ex
pected to require about 2,000 PBq of tritium for their “fu
el”, only a small part of these tritium may be released fr
om the facilities. Therefore, an exposure condition of tri
tium radiation from nuclear fusion reactor could be a lo
ng-term exposure at a low dose-rate. So, analytical met
hods sensitive to radiation at low dose or low dose-rate
are needed. In the present study, the mutagenic effects
of tritiated water at a low dose-rate was investigated usi
ng wild type and p53-/- mice, then compared that of 137C
s rays.
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Features of p53-/- mice
・ p53 is a tumor suppressor gene.
・ p53 plays the role as guardian of genome.
・ p53-/- mice have no apoptotic activity, which remove damaged cells from tissue.
・ Life of p53-/- mice are short cause of occurrence of tumor. : ~ 8 months (wilt-type mice : ~28 months)
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Preparation of T lymphocytes for Mutation Analysis
spleens
gently dissociate
filtration
hemolysisof red cells
nylon wool
37℃ 1 h
FACS
cell suspension
5 x 105 cells
T-lymphocytes
・ PE conjugated anti-CD4 antibody・ FITC conjugated anti-CD3 antibody
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TCR mutation assay (principle)
If mutation is formed in T cell receptor, CD3 is not able to be transfered into cell membrane.
Extracellular
Cytoplasm
CD4CD3T cell receptor
irradiation
Extracellular
Cytoplasm
Mut Res 1997, Meth Mol Biol 2005, Radiat Res 2006
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PE
-ant
i CD
4
PE
-ant
i CD
4
FITC-anti CD3 FITC-anti CD3
0 Gy (control mouse) 3 Gy (irradiated mouse)
CD3-CD4+ CD3-CD4+CD3+CD4+ CD3+CD4+
50191
37= = 7.4×10-4
TCR VFCD3+CD4+
CD3-CD4+
=
50097
188= = 37.5×10-4
TCR VFCD3+CD4+
=CD3-CD4+
Estimation of TCR variant fraction (mutation frequency)
17Mice were irradiated by 137Cs -rays simulation-irradiation system in 2.3 days as an effective half-life.
Days after the start of irradiation
Intraperitoneal injection of HTO (266 MBq/mouse)and start of simulation-irradiation (0.7 mGy/min at start)
Analysis of apoptosis[ p53+/+, p53-/-]
Acute irradiation(3Gy)
Analysis of mutation[p53+/+, p53-/-]
absorbed dose
dose rate
0 1 2 3 4 5 6 7 80
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Dos
e ra
te (
mG
y/m
in)
0
1
2
3
4
Cum
ulat
ive
abso
rbed
dos
e (G
y)
0.8
9 10 11 12 13 14 15 16 17 18 19
Days after the start of irradiation
Irradiation methods Irradiation methods8 week old normal and p53-/- mice were used.
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137Cs -rays simulation- irradiation systemfor low dose-rate
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137Cs -rays irradiation equipment for high dose-rate
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0
5
10
15
20
25
30
*
**
Var
iant
fra
ctio
n (x
10-4)
P < 0.01*
p53+/+ mice p53-/- mice
35
40
: spontaneous: HTO: 137Cs - rays ( < 0.7 mGy/min): 137Cs - rays (0.86 Gy/min)
3 Gy
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Induced TCR variant fractions
p53+/+
p53-/-
exposure
HTO
simulation
acute -rays
HTO
simulation
acute -rays
induced variant fraction
7.6 x 10-4
4.5 x 10-4
20.1 x 10-4
miceRelative value
14.2 x 10–4
1.0
1.7
3.9 x 10-4
0
-
-
0.3
1.0
0.4
1.0-
-
-
-
A / B A / C
A
B
C
A
B
C
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A D
B E
C F
A
p53+/+ p53-/-
spontaneous
HTO
137Cs
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SUMMARYSUMMARY
◆ To evaluate the mutagenic effects of tritiated water, TCR variant fractions using wild type and p53-/- mice
were investigated.
◆ When compared on the basis of the induced TCR variant fractions in p53 deficient mice at 3 Gy, tritium rays appear to be 1.7 times more mutagenic han rays.
◆ p53-dependent apoptosis could suppress the increase of TCR variant fraction induced by tritium rays.
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Thank you for your attention
Radioisotope Research CenterRadioisotope Research Center
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27Mice were irradiated by 137Cs -rays simulation-irradiation system in 2.3 days as an effective half-life.
Days after the start of irradiation
Intraperitoneal injection of HTO (266 MBq/mouse)and start of simulation-irradiation (0.7 mGy/min at start)
Analysis of apoptosis[ p53+/+, p53-/-]
Acute irradiation(3Gy)
Analysis of mutation[p53+/+, p53-/-]
absorbed dose
dose rate
0 1 2 3 4 5 6 7 80
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Dos
e ra
te (
mG
y/m
in)
0
1
2
3
4
Cum
ulat
ive
abso
rbed
dos
e (G
y)
0.8
9 10 11 12 13 14 15 16 17 18 19
Days after the start of irradiation
Irradiation methods Irradiation methods8 week old normal and p53-/- mice were used.
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Induced TCR variant fractions
p53+/+
p53-/-
exposure
HTO
simulation
acute -rays
HTO
simulation
acute -rays
induced variant fraction
7.6 x 10-4
4.5 x 10-4
20.1 x 10-4
miceRelative value
14.2 x 10–4
1.0
1.7
3.9 x 10-4
0
-
-
0.3
1.0
0.4
1.0-
-
-
-
A / B A / C
A
B
C
A
B
C
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A D
B E
C F
A
p53+/+ p53-/-
spontaneous
HTO
137Cs
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End Point administration dose(Gy) control RBE reference
LD50/30 i.p 4~8 1.7 Furchner Radiat Res 1957
Contraction of spleen i.p 1 ~10 1.3 - 1.5 Storer et al. Radiat Res and Thymus 1957 genomic instability of 0.6 1 - 2 Kozkowski et al.
lymphocyte Int J Radiat Biol 2001
apoptosis of crypt cell 0.13~0.28 1.4 - 2.1 Ijiri Radiat Res 1989
survival of oocyte 0.055 1.6 - 3 Dobson et al. Radiat Res 1976
Researches for biological effect of HTO on mice
・対照放射線i.p. : intraperitoneally
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The T lymphocyte is one of the most radiosensitive cells in the body. Several genetic end points are used to measure mutant fractions in blood cells. The T-cell receptor (TCR) gene is utilized for in vivo and in vitro mutation assays in both human and animal cells. It is known that the TCR-α and TCR-β chains are only expressed on the cell surface following the formation of large molecular complexes with CD3 chains. If either the TCR-α or -β chain gene is not expressed, the TCRαβ/CD3 complex cannot be transported to the cellular membrane, resulting in the accumulation of defective complexes in the cytoplasm. Thus, inactivation mutations in either the TCR-α or TCR-β genes in CD4+ T cells manifest as CD3-CD4+ variant T cells, that can be detected via two-color flow cytometry using monoclonal antibodies against the CD3 and CD4 molecules. In the present study, the TCR assay was utilized to quantify the genotoxic effects of tritium exposure in mice.
principle principle
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Days Spleen Kidney Liver Thymus Ovary Muscle Blood Femur Tibia
1 0.062 0.0582 0.057 0.0567 0.0365 0.0435 0.0624 0.0959 0.0767
2 0.108 0.101 0.0885 0.0985 0.0644 0.0763 0.109 0.169 0.134
4 0.166 0.157 0.138 0.152 0.102 0.119 0.169 0.266 0.205
7 0.208 0.198 0.174 0.191 0.132 0.152 0.212 0.341 0.257
12 0.23 0.221 0.194 0.211 0.15 0.17 0.236 0.386 0.285
18 0.236 0.226 0.199 0.217 0.156 0.176 0.242 0.397 0.293
23 0.237 0.228 0.2 0.218 0.158 0.177 0.244 0.4 0.295
30 0.237 0.228 0.2 0.218 0.159 0.178 0.245 0.402 0.297
37 0.238 0.229 0.201 0.219 0.159 0.178 0.246 0.403 0.298
∞ 0.239 0.231 0.201 0.219 0.16 0.184 0.232 0.395 0.303∞
J UOEH 1988
Table Tissue cumulative absorbed dose by HTO (Gy)
・ 18.5MBq HTO were i.p. injected
7日間で3 Gy の吸収線量を達成するには3/ 0.208 X 18.5 = 266 MBq を接種する。
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低線量でのがん発生の線量ー効果モデル低線量でのがん発生の線量ー効果モデル
a0
0Dose
( low dose )
( high dose )
spontaneous
推定域
実測域
LNT hypothesis
Fre
quen
cy o
f T
umor
(%
)Dose-effect model
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35
p53 System against Genotoxic Stressp53 System against Genotoxic Stress
3 Gy3 Gy3 Gy3 Gy
p53p53 (–/–) (–/–)
Background level
p53p53 (+/+) (+/+)
HighDose-rate
LowDose-rate
HighDose-rate
LowDose-rate
Va
rian
t fr
act
ion
Background level
p53 (–/–)p53 (–/–)
repair repair errorrepair
apoptosis
abnormal cellremoval of abnormal cell
normal cell
p53 (+/+)p53 (+/+)
normal cell
repair error
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Dose response curves at different dose-rate of rays for the TCR variant fractions in the splenic T-lymphocytes of p53+/+ and p53–/– mice
Kato et al, J Radiat Res 2002
0
0 1 2 3
Dose (Gy)
Var
iant
fra
ctio
n (x
10
–4)
50
40
20
10
30
p53 –/–
p53 +/+
(@ 1,017 mGy/min)
1 2 3
Dose (Gy)
00
Var
iant
fra
ctio
n (x
10
–4)
50
40
20
10
30 p53 –/–
p53 +/+
@ 1.2 mGy/min
37
OBJECTIVE OBJECTIVE
There are a number of important variables that may alter the precise estimation of the radiation weighting factor (WR) for tritium rays. For instance, the interpretation of published experimental studies is complicated by the fact that the dose rates of reference radiations are frequently much higher than those normally received by tritium internal exposure. To resolve these uncertainties and determine the most appropriate WR for tritium rays, the mutagenic effects of tritium rays on spleen T lymphocytes of Trp53+/+ and Trp53-/- mice was compared to chronic or acute 137Cs irradiation.
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