1

◆ 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

9

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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

20

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

22

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

25

26

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

34

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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