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

Transcription

1 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 *This study was performed under contract with Aomori Prefectural Government, Japan Training Meeting, Biodosimetry in The 21 st Century, HICARE / IAEA, 2013, 6.11, Hiroshima

2 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

3 Institute for Environmental Sciences (IES) Rokkasho, Aomori, Japan AMBIC LERF Research on biological effects of lowdose-rate and low dose radiation on the environment, including humans Medium-Dose-Rates 400 mgy/day 200 mgy/day 20 mgy/day Low-Dose-Rates(LDRs) 21 mgy/day 1 mgy/day 1-1 mgy/day mgy/day* mgy/day (BG) 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 Mice are chronically irradiated with gamma-rays from a 137 Cs source 22 h/day under SPF conditions. SPF: specific pathogen free

4 Research on Low Dose and Low-Dose-Rate (LDR) Radiation Biology msv mgy High Dose Atomic Bomb Survey Results Human LD50:4,000 Epilation or Hair loss:3,000 Nausea/ Vomiting:1,000 10,000 1,000 Doses used in IES Biological Studies 8,000 (20 mgy/22h/day x 400 days) Leukopenia:500 Small increase in Human Cancers: (1 mgy/22h/day x 400 days) Low Dose Dose Limit for Radiation Workers (average/year) :20 CT Scan of the chest:6.9 Natural Background (average/year): (0.05 mgy/22h/day x 400 days) Dose Limit for Public (per year) International Space Station (per day) }:1 1 Diagnostic X-ray of the chest:0.05 Estimated Radiation released from the Recycling Nuclear Facility (per year): 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.

5 Institute for Environmental Sciences (IES) 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

6 Life-span and tumorigenesis study in mice exposed to low-dose-rate (LDR) γ- rays Animals: Specific Pathogen Free(SPF) B6C3F1 (C57BL/6J C3H/He) mice Method: Non-irradiated control (male 500 and female 500) 137 Cs γ-ray Irradiated (male 500 and female 500 each) Kept until the animals die a natural death 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 Kept until the animals die a natural death

7 Institute for Environmental Sciences (IES) Life-span and Tumorigenesis in Mice Continuously Exposed to γ-rays at Low-Dose-rates (LDRs) 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. Tanaka, S. et al. Radiat. Res. 160: (2003); Tanaka, I.B. III et al. Radiat. Res. 167: (2007)

8 Life shortening in B6C3F1 mice continuously exposed to γ-rays Dose rate (mgy/day) Total Dose (mgy) Number of mice Mean life span (days ± SE) Life shortening (days ± SE) Male Female (2) ± (5) ± ± ± ± (1) ± ± ± (5) ± ± (3) ± ± ± ± 9.3 Number in parentheses are losses from accidental death * : p<0.05, ** : p< ** * ** Tanaka, S. et al., Radiat. Res. 160: (2003)

9 Results of the Life-span and Tumorigenesis Study Male Dose rate (Total dose) Life-span Major cause of death Increased neoplastic incidence Low-dose rate 0.05 mgy/day (Low dose 20 mgy) Low-dose rate 1.1 mgy/day (Middle dose 400 mgy) No change - - 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

10 Atrophy of ovary increased body weight and development of adiposity*. BW(g) Increase of BW than non-exposed mice 2.2 Gy 175 day Non- Irradiation Slight atrophy Irradiation LDR at 20 mgy/day Age BW(g) Increase of BW than non-exposed mice day Non- Irradiation atrophy Irradiation Age Accumulated dose 1 2 Gy *Lipid content in the liver, serum and adipocytes were significantly higher in irradiated mice than the non-irradiated control. BW: body weight Nakamura, S. et al. Radiat. Res 173; 333 (2010)

11 20 mgy/day Relative number of CFU-S 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<0.05, **P<0.01). Number of primitive bone marrow cells (day 7 CFU-S) of C3H mice were decreased by chronic irradiation at low-dose-rate (LDR) of 20 mgy/day (*p<0.05, **p<0.01). 図 1 Yanai, T. et al. Proc. IES Symposium. (2003)

12 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) Oogenesis, body weight gain and adiposity in female Primitive bone marrow cells (CFU-S, CFU-GM) ーー + 3 ー (body weight) ー (body weight) +(> 2000 mgy) 4 N.D. ー +(> 3000 mgy) 5 Chromosome aberrations ー? + (> 200 mgy) + 6,7 Gene mutations ー mrna expression ,10 Protein expression ー ,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)

13 more than 2-3 Gy were developed in effective for biological effects total dose seems to be more

14 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 ( 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.

15 Low- and medium-dose-rate ranges and dose rate dependent biological effects Natural background radiation level Inst. Environ. Sci. (IES) (LERF)* Russell W.L. (1959) (Mutation rates in mice germ cell) Lyon M.F. (1968,1972) Russell W.L. (1965) Russell W.L. (1982) HPRT mutation in mice lymphocyte; Lorenze et al. (1994) Chrom. ab. in mice blood lymphocyte; Sorensen et al. (2000) IES, AMBIC SPF Csγ* Inverse dose- rate effects; Vilenchik M. & Knudson A.G. (2006) IES, AMBIC CV Csγ Many experiments for X and γ-rays (1 mgy/h) Dose Rate(Gy/h) 20 mgy/day 1 mgy/day 0.05 mgy/day 200 mgy/day mgy/day 400 mgy/day Low-dose-rate(< =134 mgy/22h/day= 0.1 mgy/h) * under specific pathogen free(spf) conditions

16 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) mgy days 2 1 mgy/22h/day (0.045 mgy/h)- LDR , 700 mgy , 700 days mgy/22h/day ( mgy/h)-ldr mgy days 4 Non-irradiated control mice (Mice of same age at the time of each accumulated dose)

17 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. Dicentric chromosomes

18 Frequency of translocations in 20 mgy/day ( < = 1000 mgy) and 1 mgy/day groups ( < = 700 mgy) Accumulated dose (mgy) Translocations per 100 cells Observed data before age-adjustment

19 Translocations increased with both accumulated dose and aging at low-dose-rates (LDRs ) of 1 mgy/day and 0.05 mgy/day Translocations per 100 cells 100 細胞あたりの転座型染色体異常個数 照射開始後日数 Accumulated Observed data dose with (mgy) 1 mgy/22h/day(45.5 μgy/h) 0.05 mgy/22h/day(2.27 μgy/h) 線量 (mgy) 線量 (mgy) Age(days after start of IR) Control 0.05 mgy/22h/ 日 照射開始後日数 Age(days after start of IR) Observed data before age-adjustment 1 mgy/22h/ 日 IR: irradiation

20 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+θ02t 2 +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 t 2 +θ1(0.05*t) 1 mgy/day group: β2(t) = θ01+θ02 t 2 +θ2(1*t) Compare correlation coefficient values (θ1, θ2, θ3) 20 mgy/day group: β3(t) = θ01+θ02 t 2 +θ3 (20*t) Total dose(d in mgy)=dose rate*t, t= Age -56 Multiple correlation coefficient R 2 =

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

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

23 Increase of translocations in 20 mgy/day, 1 mgy/day and 0.05 mgy/day groups with accumulated dose (mgy)

24 (Translocations) Table1. Estimated corelation coefficients in weighted multiple regression analysis Irradiated groups Regression coefficient Estimated S.E. z- value p-value θ Control θ mgy/day θ *0.000 * mgy/day θ * mgy/day θ *0.000 Table2. Test of Effects among irradiated groups Null hypothesis Likelihood ratio test statistics d.f. p-value Θ 1 =θ mgy/day vs. 1 mgy/day 1.1 Θ 2 = θ 3 1 mgy/day vs. 20 mgy/day 17.4 Θ 3 = θ mgy/day vs. 20 mgy/day 1.5 Θ 1 = θ 2 = θ , 1 mgy/day VS. 20 mgy/day * *0.000

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

26 Current formula of dose-rate effects based on classical DNA repair theory is not correct. < Chromosome-breakage-reunion hypothesis> High-Dose-Rate irradiation Y = βd 2 +α 1 D+c Y: chromosome aberration rate (Number of aberrations/100 cells) D: Dose (mgy) Low-Dose-Rate irradiation Y = α 2 D+c β= 0 α coefficient: independent on dose rate β coefficient: dependent 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)= (βd 2 +α 1 D)/α 2 D=D (β/α)+1 on the assumption that α 1= α 2. In a cell B A Interaction between A and B 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)

27 Dose-rate effects in cell population, associated with bystander effects in low-dose rate (LDR) irradiation High-dose rate- irradiation Cell to cell communication More cells are damaged in neighboring cells Low-dose rate- irradiation

28 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 % of aberrations 3 years 10 years 50 years Chronic Low Dose Rate(LDR) exposure % of aberrations Dicentric chromosome(estimated) Translocations Dicentric chromosomes (observed)

29 Incidence of dicentric chromosomes in low-dose-rateradiation exposed peoples % of Dicentric chromosome in LDR irradiated people was always higher than control. Dicentric chromosomes per 1000 cells Japanese English High background area in China Japanese medical radiotechnologist Residents of Badgastein hot spring 0.85x10-3 (Japanese), 0.55x10-3 (English) Nuclear ship dockyard workers Semipalatinsk NETS residents (Tanaka et al. 2006, 2013) Childrens in Chernobyl contaminated areas

30 Method for chromosome analysis at each day after finishing irradiation SPF C3H/HeN mice were irradiated form 8 weeks of age. 137 Cs-γ rays 20 mgy/22 h/day (909 µgy/h) for 200 days ( total dose: 4000 mgy) 日 Kept for SPF condition up to 300 days after exposure 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.

31 Reduction of chromosome aberration rates in spleen lymphocytes of high-doserate 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: ) translocations Translocations Dicentric chromosomes

32 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 12.0 Translocations 4.0 Dicentric chromosomes 転座型染色体異常個数 /100 細胞 Translocations per 100 cells Non-exposed 非照射群 4000 mgy 照射群 照射後放置日数 Days after exposure 二動原体染色体個数 /100 細胞 Dicentrics per 100 cells Non-exposed 非照射群 4000 mgy 照射群 照射後放置日数 Days after exposure Each point shows mean value of three mice and 95% CI.

33 Which type of chromosome aberrations will be applicable for biodosimetry in long-term chronic exposure? Analyzed cells Dicentric chromosomes (Cenromere FISH) Many Easily scoring But need automatic analysis Translocations (Chromosome painting FISH, M-FISH) 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 ( 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.

34 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

35 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