Biological effects of low dose and low-dose-rate radiation on development of neoplasm and chromosome aberration＊

Kimio TANAKA, Ph.D. Institute for Environmental Sciences(IES)

Rokkasho, Aomori, Japan kmtanaka@ies.or.jp, http://www.ies.or.jp

Training Meeting, Biodosimetry in The 21st Century , HICARE / IAEA, 2013, 6.11, Hiroshima

＊This study was performed under contract with Aomori Prefectural Government, Japan

mailto:kmtanaka@ies.or.jp

Today’s topics 1. Biological effects on development of neoplasm and non-neoplastic lesions by long-term γ- rays irradiation at a low–dose-rate (LDR), detected in IES research projects

2. Dose response and dose-rate effects of chromosome

aberration frequencies at a low-dose-rate range

3. Application of chromosome analysis for biodosimetry in long-term LDR chronic exposure

Institute for Environmental Sciences (IES) Rokkasho, Aomori, Japan

AMBIC : Advanced Molecular Bio-Sciences Research Center LERF : Low-Dose Radiation Effects Research Facility * Lowest possible dose rate exposure at the IES BG: back ground

Research on biological effects of low- dose-rate and low dose radiation on the environment, including humans

AMBIC LERF

21 mGy/day

400 mGy/day

1 mGy/day

0.05 mGy/day*

0.0025 mGy/day (BG)

200 mGy/day

20 mGy/day

1 mGy/day

Mice are chronically irradiated with gamma-rays from a 137Cs source 22 h/day under SPF conditions. SPF: specific pathogen free

Medium-Dose-Rates

Low-Dose-Rates(LDRs)

0.001 -

0.01 -

0.1 -

1 -

10 -

100 -

1000 -

Atomic Bomb Survey Results Human LD50：4,000

Epilation or Hair loss：3,000

Nausea/ Vomiting：1,000

Leukopenia：500

Small increase in Human Cancers：200

CT Scan of the chest：6.9

Natural Background (average/year)：2.4

Dose Limit for Radiation Workers (average/year) ：20

Dose Limit for Public (per year) International Space Station (per day)

Diagnostic X-ray of the chest：0.05 Estimated Radiation released from the Recycling Nuclear Facility (per year)： 0.022

mSv mGy Doses used in IES Biological Studies

8,000 (20 mGy/22h/day x 400 days)

400 (1 mGy/22h/day x 400 days)

20 (0.05 mGy/22h/day x 400 days)

Gray (Gy) is the unit used to express the absorbed dose. Sievert (Sv) is the unit used to express the equivalent dose reflecting

radiation dose to tissue adjusted to the type of ionizing radiation. NOTE: Use of the proper corresponding units to differentiate absorbed

dose (Gy) from equivalent dose (Sv). For gamma rays and X-rays, absorbed dose (Gy) and equivalent dose

(Sv) are almost the same.

10,000

1,000

100

10

1

0.1

0.01

Research on Low Dose and Low-Dose-Rate (LDR) Radiation Biology

}：1

Low Dose

High Dose

Current Research Projects on Biological Effects of Low-Dose-Rate (LDR) γ-Rays Irradiation

I. Life-span and Tumorigenesis Cause of death, Neoplasm and non-neoplastic lesions II. Transgenerational Effects Effects of long-term paternal exposure to low-dose-rate gamma-rays on progeny III. Bio-Defense System Changes in the immune system and metabolism leading to initiation, development and

progression of tumors, and, non-neoplastic lesions IV. Tumor-Related Genes Alterations in tumor-related genes and their expressions leading to initiation, development

and progression of tumors V. Biological Dosimetry Estimation of exposure dose, dose and dose-rate effect on chromosomal aberrations Archives Storage of raw data, specimen from animal experiments

Institute for Environmental Sciences (IES)

137Cs γ-ray

Animals: Specific Pathogen Free（SPF) B6C3F1 (C57BL/6J×C3H/He) mice Method: Non-irradiated control (male 500 and female 500) Irradiated (male 500 and female 500 each)

Kept until the animals die a natural death

Kept until the animals die a natural death

Life-span and tumorigenesis study in mice exposed to low-dose-rate (LDR) γ- rays

0.05 mGy/22hours/day x 400 days = 20 mGy 1.1 mGy/22hours/day x 400 days = 400 mGy 21 mGy/22hours/day x 400 days = 8000 mGy

Life-span and Tumorigenesis in Mice Continuously Exposed to γ-rays at Low-Dose-rates (LDRs)

Tanaka, S. et al. Radiat. Res. 160: 376-379 (2003); Tanaka, I.B. III et al. Radiat. Res. 167: 417-437 (2007)

◆ The life spans of the both sexes irradiated at 21 mGy/day and those of females irradiated at 1.1 mGy/day were significantly shorter than those of the non-irradiated groups.

◆ This life shortening was due to early neoplastic death and increased progression of neoplastic cells.

Institute for Environmental Sciences (IES)

Dose rate (mGy/day)

Total Dose (mGy) Number of mice †

Mean life span (days ± SE)

Life shortening (days ± SE)

Male

0 0 498 (2) 912.7 ± 8.2

0.05 20 495 (5) 905.8 ± 8.3 6.9 ± 11.7

1.1 400 500 895.2 ± 8.2 17.5 ±11.6

21 8000 499 (1) 812.0 ± 7.6 100.7 ± 11.2

Female 0 0 500 860.5 ± 6.3

0.05 20 495 (5) 851.8 ± 6.7 8.7 ± 9.2

1.1 400 497 (3) 839.8 ± 7.5 20.7 ± 9.8

21 8000 500 740.9 ± 6.8 119.6 ± 9.3

Life shortening in B6C3F1 mice continuously exposed to γ-rays

**

* **

Tanaka, S. et al., Radiat. Res. 160: 376 - 379. (2003)

† Number in parentheses are losses from accidental death * : p

Results of the Life-span and Tumorigenesis Study

Dose rate (Total dose) Life-span Major cause of death Increased neoplastic incidence

Male

Low-dose rate 0.05 mGy/day (Low dose 20 mGy) No change - -

Low-dose rate 1.1 mGy/day (Middle dose 400 mGy) No change - -

Low-dose rate 21 mGy/day High dose (8,000 mGy)

Significantly shortened

(approx. 100 days)

Malignant lymphoma Lung and vascular

neoplasia Myeloid leukemia

Vascular neoplasia

Female

Low-dose rate 0.05 mGy/day (Low dose 20 mGy) No change - -

Low-dose rate 1.1 mGy/day (Medium dose 400 mGy)

Significantly shortened

(approx. 20 days) Malignant lymphoma -

Low-dose rate 21 mGy/day (High dose 8,000 mGy)

Significantly shortened

(approx. 120 days)

Malignant lymphoma Soft tissue and

vascular neoplasia

Ovary, soft tissue and vascular

neoplasia

Non- Irradiation Irradiation

Non- Irradiation Irradiation

BW

（g）

Age

BW

（g）

Age

Atrophy of ovary increased body weight and development of adiposity*.

175 day

210 day atrophy

Increase of BW than non-exposed mice

LDR at 20 mGy/day

Nakamura, S. et al. Radiat. Res 173; 333 (2010)

2.2 Gy

2.9

Accumulated dose 1 2 Gy BW: body weight *Lipid content in the liver, serum and adipocytes were significantly higher in irradiated mice than the non-irradiated control.

Slight atrophy

Increase of BW than non-exposed mice

図１

Fig. 1 Effects of continuous irradiation with low dose rate on hematopoietic progenitors and stem cells. CFU-S colonies were counted on the 7th day after irradiation (*P

Biological effects of low dose and low-dose-rates radiation, detected in IES projects Index Total dose

20 mGy at 0.05 mGy/day

Total dose 400 mGy at 1 mGy/day

Total dose 8,000 mGy at 20 mGy/day

References

Life shortening ー ＋ (in female) ＋ 1

Tumor incidence(including adenoma) ー ― ＋(>≑2000 mGy) 2

Transgenerational effects (life shortening)

ー ー ＋ 3

Oogenesis , body weight gain and adiposity in female

ー(body weight) ー(body weight) ＋（>≑2000 mGy) 4

Primitive bone marrow cells (CFU-S, CFU-GM)

N.D. ー ＋（>≑3000 mGy) 5

Chromosome aberrations ー ? ＋ (>≑200 mGy) ＋ 6,7

Gene mutations ー ＋ ＋ 8

mRNA expression ＋ ＋ ＋ 9,10

Protein expression ー ＋ ＋ 11,12 1. S. Tanaka et al. Radiat Res 160: 367 (2003) 2. IB Tanaka et al. Radiat Res 167: 417 (2003) 3. Unpublished results 4. S. Nakamura et al. Radia Res 173, 333 (2010) 5. T. Yanai et al. Proc IES Sympo. (2003) 6. K Tanaka et al Radiat Res 171: 290 (2009) 7. K Tanaka et al. J. Radiol. Prot. 33, 61(2013)

8. N. Okudaira et al. Radiat Res 173: 138 (2010) 9. K. Taki et al. J Radiat Res 50: 241 (2009) 10. Y. Uehara et al. Radiat. Res. 174, 611 (2010) 11. T. Nakajima et al. J. Radiat Res. 49: 661 (2008) 12. T Sugihara et al., Radiat. Res. 179, 221 (2013)

were developed in more than 2-3 Gy

total dose seems to be more effective for biological effects

Dose Response and Dose-Rate-Effects of Chromosome Aberration Frequencies at Low-Dose Rate Range

Background There is no report to show dose response and dose-rate effects at

the low dose rate (LDR) range in human populations.

Objectives

To observe dose response and dose-rate effects at the LDR range between 0.05 mGy/22h/day (0.0023 mGy/h) and 20 mGy/22h/day

( 9.1 mGy/h) in translocation, after adjusting for age-related differences, in spleen lymphocytes of irradiated mice.

Inst. Environ. Sci. (IES) (LERF)*

Russell W.L. (1959) (Mutation rates in mice germ cell)

IES, AMBIC SPF Csγ* IES, AMBIC CV Csγ

Many experiments for X and γ-rays

10-7 10-6 10-5 10-4 10-3 10-2 10-1 1 10 102 103

Lyon M.F. (1968,1972)

Russell W.L. (1965)

Inverse dose- rate effects; Vilenchik M. & Knudson A.G. (2006) Natu

ral b

ackg

roun

d ra

diat

ion

level

0.0025 mGy/day

0.05 mGy/day

1 mGy/day

20 mGy/day

200 mGy/day 400 mGy/day

Dose Rate（Gy/h） （1 mGy/h）

Low- and medium-dose-rate ranges and dose rate dependent biological effects

＊ under specific pathogen free(SPF) conditions

Chrom. ab. in mice blood lymphocyte; Sorensen et al. (2000)

HPRT mutation in mice lymphocyte; Lorenze et al. (1994) Russell W.L. (1982)

Low-dose-rate(< =134 mGy/22h/day= 0.1 mGy/h)

Irradiation protocol of mice Dose rates and accumulated dose for chromosome analysis

Irradiation exposure began at 8 weeks of age of Ｃ３Ｈ ＳＰＦ female mice

１ 20 mGy/22h/day (0.91 mGy/h)- Low-dose-rate (LDR) 0 250 500 750 1000 1200 2000 4000 8000 mGy 12.5 25 33 50 60 100 200 400 days

２ 1 mGy/22h/day (0.045 mGy/h)- LDR 0 125 187.5 250 312.5 375 412.5 500 615 , 700 mGy 125 188 250 313 375 413 500 615 , 700 days

３ 0.05 mGy/22h/day (0.0023 mGy/h)-LDR

0 5 10 15 20 30 35 mGy 100 200 300 400 600 700 days

４ Non-irradiated control mice (Mice of same age at the time of each accumulated dose)

Translocations

Dicentric chromosomes

M-FISH

Translocations

Materials and Methods Chromosome analysis： ・Spleen cells were cultured for 46h with ConA, LPS and 2 ME ・Translocations were detected by M-FISH.

Frequency of translocations in 20 mGy/day ( < = 1000 mGy) and 1 mGy/day groups ( < = 700 mGy)

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Accumulated dose (mGy)

Observed data before age-adjustment

Translocations increased with both accumulated dose and aging at low-dose-rates (LDRs ) of 1 mGy/day and 0.05 mGy/day

Observed data with

0.0

0.5

1.0

1.5

2.0

2.5

0 200 400 600 800

照射開始後日数

10

0細

胞あ

たり

の転

座型

染色

体異

常個

数

Control0.05 mGy/22h/日1 mGy/22h/日

Control0.05 mGy/22h/日1 mGy/22h/日

0.0

0.5

1.0

1.5

2.0

2.5

0 200 400 600 800

照射開始後日数

線量(mGy)0 35302010 25156.250 35302010 25156.250 700600400200 5003001250 700600400200 500300125

線量(mGy)

1 mGy/22h/day(45.5 μGy/h) 0.05 mGy/22h/day(2.27 μGy/h)

Tran

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Accumulated dose (mGy)

Age(days after start of IR) Age(days after start of IR)

IR: irradiation

Observed data before age-adjustment

WEIGHTED MULTIPLE LINEAR REGRESSION ANALYSIS FOR ADJUSTMENT OF AGE-RELATED DIFFERENCES

Weighted multiple linear regression analysis was used on the linear or linear quadratic model, adjusting for age-related differences, for translocations. Ｙ = θ01+θ02t2 +θ1(0.05*t )X1+θ2(1.00*t )X2+θ3(20.0*t )X3 Ｙ： chromosome aberration rate per 100 cells at t =Age-56, d ：an accumulated dose,

β0(t) = θ01+θ02t² expresses an age effect for covariates X1, X2 and X3 take 1 if observation belongs to 0.05mGy/day, 1mGy/day and 20mGy/day, otherwise 0, respectively.

We assume that βj, j=0,1,2 and 3 are linear curves. Control group： β0(t) = θ01+θ02t² 0.05 mGy/day group： β1(t) = θ01+θ02 t2 +θ1(0.05*t) １ mGy/day group: β2(t) = θ01+θ02 t2 +θ2(1*t) Compare correlation coefficient values (θ1, θ2, θ3) 20 mGy/day group: β3(t) = θ01+θ02 t2 +θ3 (20*t) Total dose(D in mGy)＝Dose rate＊t, t= Age -56 Multiple correlation coefficient R2 = 0.9671

：

Increase of translocations in 20 mGy/day, 1 mGy/day, 0.05 mGy/day and non-exposed groups with age

Increase of translocations in 1 mGy/day, 0.05 mGy/day and non-exposed groups with age

(mGy)

Increase of translocations in 20 mGy/day, 1 mGy/day and 0.05 mGy/day groups with accumulated dose

(Translocations) Table1. Estimated corelation coefficients in weighted multiple regression analysis

Table2. Test of Effects among irradiated groups

Irradiated groups Regression coefficient Estimated S.E. z- value p-value

Control θ01 0.3165 0.0898

4.5 *0.000

θ02 2×10-6 4×10-7 4.8 *0.000

0.05 mGy/day θ1 -0.0055 0.0062 -0.9

0.378

1 mGy/day θ2 0.0007 0.0003 2.6

*0.011

20 mGy/day θ3 0.0019 0.00004 53.3

*0.000

Null hypothesis Likelihood ratio test statistics d.f. p-value

Θ1 =θ2 0.05 mGy/day vs. 1 mGy/day 1.1 1

0.294

Θ2 = θ3 1 mGy/day vs. 20 mGy/day 17.4 1

*0.000

Θ3 = θ1 0.05 mGY/day vs. 20 mGy/day 1.5 1

0.215

Θ1 = θ2= θ3 0.05, 1 mGy/day VS. 20 mGy/day 17.7 2

*0.000

Dose-rate effects: Biological effects reduce with decreasing of dose rate of irradiation by DNA repair

・ High-dose-rate almost no repair of DNA damage High chrom. aberration & mutation rates Mutation prone repair

・ Medium-dose-rate (400 mGy/day) Some DNA damages are repaired. Aberration rates are reduced.

・ Low-dose-rate Most DNA damages are repaired. Aberration rates are more reduced. (20 mGy/day）

（1 mGy/day）

Ｉｒｒａｄｉａｔｉｏｎ ｔｉｍｅ （0.05 mGy/day） Time interval of photon beams influence with aberration rate. Long interval induces decrease of aberration

rates, and then existing a threshold. Threshold or consistent decrease to background level? Seems to be no threshold

Current formula of dose-rate effects based on classical DNA repair theory is not correct.

< Chromosome-breakage-reunion hypothesis> High-Dose-Rate irradiation Y = βD2+α1D+c Y: chromosome aberration rate (Number of aberrations/100 cells) In a cell D: Dose (mGy) Low-Dose-Rate irradiation Y = α2D+c β= 0 α coefficient: independent on dose rate To obtain dose and dose rate effectiveness (DDREF) ICRP(1990), BEIR Ⅶ Report (2006) et al. recommend DDREF= ratio of （HDR/LDR)= (βD2+α1D)/α2D＝D (β/α)+1 on the assumption that α1=α2 . Because in our previous* and present studies, values of α coefficient significantly decreased

with reduction of dose rate. Then, this formula is not correct, ad not applicable for irradiation at low –dose-rate range.

*Tanaka, K. et al. Radiat. Res.171;290 (2009); Tanaka, K. et al. J. Radiol. Prot. 33; 61(2013)

β coefficient: dependent on dose rate Interaction between A and B

B

A

Dose-rate effects in cell population , associated with bystander effects in low-dose rate (LDR) irradiation

More cells are damaged

in neighboring

cells

Cell to cell communication

High-dose rate- irradiation

Low-dose rate- irradiation

Dose response of chromosome aberrations in low-dose-rate(LDR)-irradiation Both dicentric chromosome and translocation increased with accumulated dose in LDR irradiation.

Acute exposure : eg. Atomic bomb survivors、Radiation accident victims、Cancer therapy patients Dicentric chromosomes Translocations

3 years 10 years 50 years Chronic Low –Dose Rate(LDR) exposure

Translocations Dicentric chromosome（estimated）

Dicentric chromosomes (observed)

% o

f abe

rrat

ions

%

of a

berr

atio

ns

Incidence of dicentric chromosomes in low-dose-rate-radiation exposed peoples

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0 2 4 6 8 10 12

Dic

entr

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mosom

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er

1000 c

ells

Japanese English

High background area in China

Residents of Badgastein hot spring

Japanese medical radiotechnologist

Nuclear ship dockyard workers

Childrens in Chernobyl contaminated areas

Semipalatinsk NETS residents (Tanaka et al. 2006, 2013)

0.85x10-3(Japanese), 0.55x10-3(English)

% of Dicentric chromosome in LDR irradiated people was always higher than control.

SPF C3H/HeN ♀ mice were irradiated form 8 weeks of age.

Kept for SPF condition up to 300 days after exposure 0 50 100 200 250 400 日 300 350 150

137Cs-γ rays 20 mGy/22 h/day (909 µGy/h) for 200 days ( total dose: 4000 mGy)

Method for chromosome analysis at each day after finishing irradiation

Chromosome analysis was performed periodically every 50 days up to 300 days after completion of LDR exposure. Spleen cells were incubated with LPS (10 μg/ml)、ConA (3 μg/ml)、2-ME (50 μM) for 46 h to obtain chromosome metaphases.

Reduction of chromosome aberration rates in spleen lymphocytes of high-dose- rate X-ray(1.0 Gy/min ）irradiated Swiss albino female mice, at the 8weeks after irradiation (Hande, M. P. and Natarajan A. T. 、Int. J. Rad. Biol. 74: 441-448.1998)

• translocations •

Translocations Dicentric chromosomes

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 100 200 300 400 500

照射後放置日数

二動

原体

染色

体個

数/100細

胞

非照射群

照射群

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0 100 200 300 400 500

照射後放置日数

転座

型染

色体

異常

個数

/100細

胞 非照射群

照射群

Each point shows mean value of three mice and 95% CI.

Translocations Dicentric chromosomes

Reduction of chromosome aberration rates in spleen lymphocytes of C3H mice after completion of long term irradiation of γ-ray for 4000 mGy at 20 mGy/day

Non-exposed 4000 mGy

Non-exposed 4000 mGy

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Days after exposure Days after exposure

Which type of chromosome aberrations will be applicable for biodosimetry in long-term chronic exposure?

Dicentric chromosomes (Cenromere FISH)

Translocations (Chromosome painting FISH, M-FISH)

Analyzed cells ・Many ・Easily scoring ・But need automatic analysis

・Not many ・Complex and time consuming analyses ・Expensive ・Need automated cytogenetic analysis

Detection level High ・Much higher, because 3 times higher incidence than dicentrics. ・Higher when age-dependent translocation is excluded.

Age effects ・Weak ・Slightly linear or no increase

・Strong aging effects in curve linear fashion

Applicable for low dose rate range (20 -0.05 mGy/day =2.3 μGy/h）

・Possibly applicable. ・Need calculation of adjusting reduction rate after exposure. ・Combined with Qdr method using X1Cu cells (cells with Dic+frag) ?

・ Applicable for younger age people, but not for aged person. ・Need calculation of age-related adjusting.

Frequencies of translocations were increased linearly with dose

Dose-rate effects on the frequencies of translocations were observed in the dose rate range between 20 mGy/day to 1 mGy /day

translocation will be a suitable indicator to estimate total accumulated dose

Mt. Hakkoda viewing from Obuchi Lake in Rokkasho Village, Aomori Prefecture

Atushi KOHDA, Satoshi Tanaka, Braga Tanaka Department of Radiobiology Institute for Environmental Sciences Rokkasho, Aomori, Japan Kenichi SATOH Department of Environmetrics Institute for Radiation Biology and Medicine Hiroshima University, Hiroshima, Japan

Slide Number 1Today’s　topicsSlide Number 3Slide Number 4Slide Number 5Life-span and tumorigenesis study in mice exposed to �low-dose-rate (LDR) γ- raysLife-span and Tumorigenesis in Mice Continuously Exposed to γ-rays at Low-Dose-rates (LDRs)Slide Number 8Results of the Life-span and Tumorigenesis StudySlide Number 10図１　Biological effects of low dose and low-dose-rates radiation, detected in IES projects�Conclusion 1�Dose Response and Dose-Rate-Effects of Chromosome Aberration Frequencies at Low-Dose Rate Range��　　Slide Number 15Irradiation protocol of mice �Dose rates and accumulated dose for chromosome analysisSlide Number 17Frequency of translocations in 20 mGy/day ( < = 1000 mGy)�and 1 mGy/day groups ( < = 700 mGy)Translocations increased with both accumulated dose and aging at low-dose-rates (LDRs ) of 1 mGy/day and 0.05 mGy/day Weighted multiple linear regression analysis for adjustment of age-related differences：Slide Number 22Slide Number 23(Translocations)�Table1. Estimated corelation coefficients in weighted multiple regression analysisDose-rate effects: Biological effects reduce with decreasing of dose rate of irradiation by DNA repairCurrent formula of dose-rate effects based on classical DNA repair theory is not correct.�Dose-rate effects in cell population�, associated with bystander effects in low-dose rate (LDR) irradiation 　Dose response of chromosome aberrations in low-dose-rate(LDR)-irradiation�Both dicentric chromosome and translocation increased with accumulated dose in LDR irradiation. Slide Number 29Slide Number 30Reduction of chromosome aberration rates in spleen lymphocytes of high-dose- rate X-ray(1.0 Gy/min ）irradiated Swiss albino female mice, at the 8weeks　after irradiation�(Hande, M. P. and Natarajan A. T. 、Int. J. Rad. Biol. 74: 441-448.1998) 　�Slide Number 32����������Conclusion 2�