Chromosomal Aberrations in Wild Mice Captured in Areas

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Chromosomal Aberrations in Wild Mice Captured in Areas Differentially Contaminated by the Fukushima Dai-Ichi Nuclear Power Plant Accident Yoshihisa Kubota,*,† Hideo Tsuji,‡ Taiki Kawagoshi,† Naoko Shiomi,† Hiroyuki Takahashi,§ Yoshito Watanabe,† Shoichi Fuma,† Kazutaka Doi,∥ Isao Kawaguchi,† Masanari Aoki,⊥ Masahide Kubota,⊥ Yoshiaki Furuhata,⊥ Yusaku Shigemura,# Masahiko Mizoguchi,# Fumio Yamada,× Morihiko Tomozawa,⊗ Shinsuke H. Sakamoto,○ and Satoshi Yoshida† †

Project for Environmental Dynamics and Radiation Effects, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan ‡ Administration and Planning Unit, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan § Tokyo Nuclear Services Co., Ltd. 1-3-5 Taito, Taito-ku, Tokyo 110-0016, Japan ∥ Project for Investigation of Human Health Effects, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan ⊥ Japan Wildlife Research Center, 3-3-7 Koutoubashi, Sumida-ku, Tokyo 130-8606, Japan # Japan NUS Co., Ltd., 7-5-25 Nishi-shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan × Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan ⊗ Department of Biology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8521, Japan ○ Faculty of Agriculture, University of Miyazaki, Kibana, Miyazaki 889-2192, Japan S Supporting Information *

ABSTRACT: Following the Fukushima Dai-ichi Nuclear Power Plant accident, radiation effects on nonhuman biota in the contaminated areas have been a great concern. The induction of chromosomal aberrations in splenic lymphocytes of small Japanese field mice (Apodemus argenteus) and house mice (Mus musculus) inhabiting Fukushima Prefecture was investigated. In mice inhabiting the slightly contaminated area, the average frequency of dicentric chromosomes was similar to that seen in mice inhabiting a noncontaminated control area. In contrast, mice inhabiting the moderately and heavily contaminated areas showed a significant increase in the average frequencies of dicentric chromosomes. Total absorbed dose rate was estimated to be approximately 1 mGy d−1 and 3 mGy d−1 in the moderately and heavily contaminated areas, respectively. Chromosomal aberrations tended to roughly increase with dose rate. Although theoretically, the frequency of chromosomal aberrations was considered proportional to the absorbed dose, chromosomal aberrations in old mice (estimated median age 300 days) did not increase with radiation dose at the same rate as that observed in young mice (estimated median age 105 days).

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nized.1,2 Therefore, radiation effects on nonhuman biota have been a concern since the accident.3−5 Some radioecological6−10 and biological11−14 studies have been performed so far to elucidate the influence of the F1-NPP accident on nonhuman biota. UNSCEAR released the report including the assessment of the F1-NPP accident regarding nonhuman species.15

catastrophic earthquake in the northwest Pacific about 130 km off northeastern Japan occurred on March 11, 2011 and induced a gigantic tsunami that severely damaged the Fukushima Dai-ichi Nuclear Power Plant (F1-NPP), resulting in hydrogen explosions within the reactors. These explosions caused serious radionuclides releases into the atmosphere and subsequent wet and dry depositions in large parts of eastern Japan from the Kanto to the Tohoku districts, mainly affecting the Fukushima Prefecture. In the past 2 decades, the importance of radiological protection of the environment has been increasingly recog© XXXX American Chemical Society

Received: March 27, 2015 Revised: July 25, 2015 Accepted: July 28, 2015

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areas and in July 2013 in the noncontaminated control area. The ambient dose rate (less than 0.1 μSv h−1) in the noncontaminated control area was similar to, or slightly higher than, that measured before the F1-NPP accident in many areas of the Tohoku region including Fukushima prefecture (www. kankyo-hoshano.go.jp/01/0101flash/01012050.html). Therefore, the sampling year in the control area (one year after the sampling in the contaminated areas) was not considered to influence the results. Every morning, the traps were observed and the captured mice were immediately placed in individual rearing cages, with wooden chips provided as floor bedding. Captured mice were given water and pelleted chow ad libitum. The wild mice were then transferred to a laboratory and reared in a controlled environment at a temperature of approximately 26 °C. In 5−20 days after capture, the mice were sacrificed with a lethal dose of the anesthetic isoflurane. The possible gradual decrease in chromosomal aberrations attributable to the cessation of irradiation in the period from the capture to sacrifice was not considered because of the longer expected half-life of chromosomal aberrations. The 12 to 24-week-old female C3H/HeJ mice were purchased from a domestic breeder (Japan SLC, Shizuoka, Japan) for comparison with wild mice. All animal experiments were conducted in accordance with the Law of Humane Treatment and Management of Animals. The capture of wild mice in the sampling areas was permitted by the Fukushima Prefectural Office. In the sampling areas, the litter and soil samples were also collected separately. All litter, including undecomposed organic matter and partly or well-decomposed organic matter in the Ohorizon, was collected in a 25 cm × 25 cm-area as a litter sample, while soil was collected in a 19.6 cm2 area (a circle with a 5 cm diameter), 0−50 mm in depth from the top of the Ahorizon. Culture of Spleen Cells, Preparation of Chromosome Slides, and C-Band Treatment. After sacrifice, the spleen was aseptically dissected from the mouse and the spleen cells were isolated and cultured in the same manner as reported by Tanaka et al.23 The cultured spleen cells were used to prepare chromosome slides which were then subjected to C-band treatment.24 A part of the preparation of spleen cells obtained from small Japanese field mice captured in the control area and purchased C3H/HeJ mice was irradiated using a X-ray machine (Shimazu Co. Ltd., Tokyo, Japan), with a dose rate of approximately 0.2 Gy min−1, in order to determine the in vitro radiosensitivity of splenic lymphocytes. Irradiated cells were incubated for 3−4 h in the culture medium without mitogen and then cultured as described above. Cell Cycle Analysis. Spleen cells obtained from small Japanese field mice captured in the control area and C3H/HeJ mice were cultured for 24−42 h in the presence of 1 μg mL−1 5-bromo 2′-deoxyuridine (BrdUrd) to differentiate metaphase cells into their first cycle (M1), second cycle (M2), and third cycle or more (M3+) cells by observing the differential staining pattern of sister chromatids.25,26 The chromosome slides, prepared as described above, were stained with 1 μg mL−1 Hoechst 33258 in Sørensen’s phosphate buffer (pH 6.8) for 5 min and mounted in 2× SSC on coverslips. They were exposed to black light for 5 min at a distance of 1 cm at 65 °C and then stained with 2% Giemsa for 8 min. In M1 and M2 cells, all chromosomes were homogeneously stained or differentially stained, with one chromatid stained and another lightly stained, respectively. In M3+ cells, half of the chromosomes were stained differentially, while the other half was composed of a

However, further studies are necessary to reach a consistent conclusion regarding whether the F1-NPP accident has had an impact on nonhuman biota and to eliminate the uncertainty of a potential long- term impact, which is possible in the future. Wildlife inhabiting areas contaminated with radionuclides released by the F1-NPP accident have been chronically exposed to radiation at a range of low dose rates, much less than 0.1 mGy min−1 that is an upper limit of the dose rate defined as low dose rate for human exposures.16 As the useful indicator to analyze the radiation effect on wildlife exposed to low absorbed radiological dose (rate) such as those inhabiting Fukushima, stochastic effects17,18 should be adopted because the effects theoretically occur in a dose-dependent manner even at a low absorbed dose(rate) exposure, whereas deterministic effects (tissue or organ reactions) are expected to be relatively difficult to detect because of the existing threshold dose. In the present study, chromosomal aberrations in splenic lymphocytes of wild mice were examined as a stochastic effect of radiation on wildlife in Fukushima since the chromosomal aberrations induced in mice by irradiation have been well studied experimentally, and the materials and methods used to examine these aberrations were established in laboratory mice.19 Because the consequence of chromosomal damage at the population level is not fully understood or is unknown,1 some physiological functions important for the maintenance of the population growth rate or structure, such as reproduction, should be studied when significant chromosomal damage is demonstrated in wild mice captured in the contaminated areas of Fukushima. In order to ensure that the observed changes are truly due to the effects of the radiation, it is important to evaluate radiation exposure together with an assessment of biological effects. However, only a few studies have reliably evaluated the radiation exposure in actual field studies following the F1-NPP accident. In the present study, the absorbed doses to the wild mice which were captured to study the chromosomal aberrations in the splenic lymphocytes were also estimated to elucidate the dose−effect relationship.

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MATERIALS AND METHODS Capture of Wild Mice. The sampling area for wild mice and environmental media in Fukushima Prefecture was selected based on radioactive contamination levels deduced from the airborne monitoring survey data.20 Specific forests in Suetsugu, Hisanohama Town, Iwaki City; Murohara, Namie Town, Futaba District; and Ottozawa, Okuma Town, Futaba District were selected as the slightly, moderately, and heavily contaminated sampling areas, respectively (detailed information on the sampling areas is shown in Table S1). In addition, a specific forest in Bandai, Bandai Town, Maya District was selected as the noncontaminated control area. Ottozawa is one of most heavily contaminated areas and is included in the former “Restricted Area”21 and the present “Area 3” where it is expected that residents will be unable to return for an extended period of time.22 Murohara is also included in the former “Restricted Area” and the present “Area 3.” Permission to enter the sampling point of “Area 3,” which is part of the exclusion zone, was obtained from the Nuclear Emergency Response Headquarters via Ministry of the Environment. In each sampling area, wild mice were captured within a rectangular area of approximately 100 m × 25 m, using 100 Sherman aluminum live traps (H.B. Sherman, Tallahassee, FL), each placed individually in areas of roughly 5 m × 5 m. The sampling was carried out in July 2012 in the three contaminated B

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analyzing the activity concentrations of radiocesium in litter and soil samples collected in individual sampling areas with ERICA Assessment Tool 1.0 methods.30 As the settings in the ERICA Assessment Tool for dose estimation, a radiation weighting factor of 1 for β- and γ radiation and an occupancy factor of 1.0 on soil were used. For the radionuclide distribution patterns in soil and litter, the infinite plane isotropic source at a depth of 0.5 g cm−2 was assumed. A body weight of 20 g and a body size of 0.025 m high, 0.025 m wide, 0.05 m long were used as a representative of wild mice. Under these conditions, the dose conversion coefficient (DCC) of external β and γ radiation on soil was 5.26 × 10−6 and 1.91 × 10−6 μGy h−1/Bq m−2 for 134Cs and 137Cs, respectively. Internal dose rates were also calculated using the ERICA Assessment Tool. DCC of internal β and γ radiation in the mice was 1.25 × 10−4 and 1.51 × 10−4 μGy h−1/Bq kg−1 for 134Cs and 137Cs, respectively, which was slightly lower than the default set for rat (1.70 × 10−4 and 1.70 × 10−4 μGy h−1/Bq kg−1 for 134Cs and 137Cs, respectively). In the present study, after the captured mice were reared for 5−20 days, they were sacrificed and activity concentrations of radionuclides retained in the body were measured. Feces and urine, which contained radionuclides excreted from the mice, were not collected. However, our recent study28 demonstrated an exponential decrease over time of the radiocesium retained in the bodies of wild mice at the time of capture, with a biological half-life of 3.31 ± 0.7 days and relatively small variations among individual mice. This suggested that the radiocesium retained in the bodies at the time of capture could be accurately calculated based on days between the capture and sacrifice of the wild mice and the radioactivity retained in the bodies at the time of sacrifice. As such, the activity concentrations of radiocesium at the time of capture were calculated from those at the time of sacrifice, while the internal dose rates were calculated using the calculated activity concentrations of radiocesium at the time of capture. Total dose rates were the sum of external and internal dose rates to individual mice. Finally, the accumulated doses were calculated by multiplying the total dose rate (mGy d−1) by the age in days of the mice. In the present study, 131I, the major radionuclide released from F1-NPP, was not included in absorbed dose (rate) calculation, because mice significantly exposed to 131I were only two old mice captured in the slightly contaminated area and the accumulated exposure dose was estimated to be less than 4 mGy, rather small compared to total absorbed doses of radiocesium to each mouse, 13.0 and 15.8 mGy. The detailed explanation why 131I was not considered is described in the Supporting Information. Statistical Analyses. To compare the probability of dicentric chromosomes in mice inhabiting the control and contaminated areas, repeated measures models for binary data were used. The probability of dicentric chromosomes in each cell was estimated for each area (the slightly, moderately, and heavily contaminated area) compared with the control area using the GEE approach with a sandwich variance estimator to account for intrasubject correlations (compound symmetry structure was assumed). The area was also included as a continuous variable in the model to test linear trends. A P-value of less than 0.05 was considered statistically significant. All analyses were performed using the SAS statistical software package (SAS 9.3, SAS Institute Inc., Cary, NC).

pair of lightly stained chromatids. To determine the proportion of M1, M2, and M3+ cells, more than 200 metaphase cells were observed. Approximately 1000 cells were analyzed for determination of mitotic indexes. Observation of Chromosomal Aberrations. The slides were coded to avoid any bias in scoring aberrations due to preconception. In addition to dicentric chromosome (dic) in Cbanded chromosomes, representative chromosomal aberrations, including chromatid break (ctb), chromatid exchange (cte), single minute (Smin), chromosome break (csb), ring chromosome (r), fragment (f), double minute (Dmin), and others, including deletions and translocations, were recorded. Gaps were excluded from the score owing to the ambiguity in the definition of chromosomal aberrations. Deletions were identified as chromosomes with an obvious shortened length due to an interstitial or terminal deletion, while translocations were identified as the appearance of a pair of obviously shortened and lengthened chromosomes or as an obvious change in the ratio between the lengths of the short and long arms. Dicentric chromosomes with fragments were distinguished from those without fragments. These aberrations were recorded and photographed. Dicentric chromosomes were observed and confirmed independently by two professional researchers. Over 2000 metaphase cells were observed per individual mouse, and the frequency of dicentric chromosomes and aberrations per cell were calculated. In calculating the frequency of aberrations per cell, a dicentric chromosome accompanying a fragment was counted as one chromosomal aberration. Radioactivity Measurement. After the spleen was removed, two professional researchers analyzed tooth abrasion to estimate the age of mice. The age of mice was comprehensively expected based on the two breeding seasons, spring (February to May) and autumn (August to October) together with the age estimation by tooth abrasion and the body size (the details were shown in Table S4). Next, the entire body of the mice without the spleen was ashed using the method described by MEXT.27 Activity concentrations of named radionuclides in the ashed samples were measured by gamma spectrometry using Ge semiconductor detectors with 40% relative efficiency (GC2018 or GC4018, Canberra Industries Inc., Oak Ridge, TN or GEM45-76-LB, Seiko EG & G Co., Ltd., Tokyo, Japan).28 The counting time was set at 600−100 000 s to reduce the statistical error during measurement to less than 5%. Radioactivity in litter and soil samples were also measured similarly.28 Calculation of Dose Rates and Accumulated Doses. Recently, we demonstrated in one of the sampling areas, i.e., the heavily contaminated area, that the external dose rate, measured with glass rod dosimeters embedded in the abdominal space of wild mice carcass left on the ground, could be approximated by multiplying the ambient dose rate, measured 1 m above ground level, with an NaI(Tl) scintillation survey meter (TCS-161, Hitachi Aloka Medical Tokyo, Japan), by a factor of 1.138.28 The factor was thought to be applicable to the estimation of the external dose rate to wild mice inhabiting overall forests in Fukushima, since radiocesium deposited in the forests of Fukushima revealed a similar spatial distribution, with an exclusive retention in the soil near the top of the A-horizon.29 Therefore, external dose rates to wild mice inhabiting individual sampling areas were estimated from averaging multiple ambient dose rate measurements within each area. The external dose rates were also estimated by C

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Figure 1. Analysis of the number of cell cycle passage of metaphase cells in splenic lymphocytes after starting the in vitro culture and the mitotic indexes. Panels A and C represent the fractions of metaphase cells in C3H/HeJ mice and small Japanese field mice, respectively, that resided in first (circles), second (squares), and third or more (triangles) cell cycles, which were classified based on a differential staining pattern of sister chromatids in BrdUrd-labeled chromosomes. Panels B and D represent mitotic index, expressed as percentages of total splenic cells in C3H mice and small Japanese field mice, respectively.

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RESULTS Captured Wild Mice. Wild mice were captured on 4 consecutive days. Two species of mice were captured in all four sampling areas: small Japanese field mice (Apodemus argenteus) and large Japanese field mice (Apodemus speciosus, also called wood mice) (Table S1). House mice (Mus musculus) were captured only in heavily contaminated area. Although large Japanese field mice and small Japanese field mice were considered the most suitable species to study the effect of radiation at first, unfortunately large Japanese field mice were not used, as the centromere, usually distinguished as densely stained spots in C-banded chromosomes, could not be identified properly in the mice. Therefore, to examine the frequency of dicentric chromosomes in C-banded chromosomes, small Japanese field mice and house mice were used. As shown in Figure S2, the in vitro radiosensitivity of splenic lymphocytes with respect to the induction of dicentric chromosomes or total chromosomal aberrations was similar between small Japanese field mice and house mice of C3H strain. Therefore, two house mice captured in the heavily contaminated area were presumed to have the same radiosensitivity as the other wild mice and were included in the experimental analysis. The capture of house mice only in the heavily contaminated area might be explained by some specific nature of the habitat (e.g., a few private houses were within 1 km radius of the heavily contaminated area). Representative photographs of dicentric chromosomes with fragments detected in C-banded chromosomes in small Japanese field mice are shown in Figure S1. Examination of Culture Conditions. To analyze the number of cell cycles after starting in vitro culturing, differential staining of sister chromatids in BrdUrd-labeled metaphase cells

was used. As shown in Figure 1, around 10−20% of M2 metaphase cells appeared at 24 h of culture, and the number of these metaphase cells increased over time. Although the mitotic index at 24 h was relatively lower than that observed at longer culturing times, culturing for 24 h was adopted as adequate and appropriate to analyze chromosomal aberration in M1 metaphase cells as much as possible. Chromosomal Aberrations Observed in Mice Inhabiting Areas Contaminated at Different Levels. The frequencies of dicentric chromosomes and other chromosomal aberrations observed in splenic lymphocytes of small Japanese field mice and house mice inhabiting the slightly, moderately, and heavily contaminated areas and the noncontaminated control area are shown in Figure 2 and Table S2. The statistical analysis is shown in Table S3. The average frequency of dicentric chromosomes observed in mice inhabiting the slightly contaminated area was similar to that observed in mice inhabiting the control area. In contrast, the average frequencies of dicentric chromosomes observed in mice inhabiting the moderately and heavily contaminated areas were significantly higher than that observed in mice inhabiting the control areas (P = 0.02 and P < 0.001, respectively). As shown Table S3, a statistically significant linear trend of increase in the frequencies of dicentric chromosomes was observed from the slightly to the heavily contaminated areas (P < 0.001). The dicentric chromosomes without fragment were observed approximately at the same levels as those with fragment regardless of the sampling areas (Table S2). The average frequencies of other chromosomal aberrations also increased with the contamination levels (Table S2). Estimation of the Absorbed Dose Rates and the Accumulated Doses. The external dose rates to the mice D

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captured only in the slightly contaminated area, there was no indication that mice in the slightly contaminated area lived longer than those in moderately and heavily contaminates areas, because no significant differences in the age of mice was observed among sampling areas by the statistical analysis using ANOVA. Because of the variations in the internal dose rate and the age among individual mice captured in the same areas, the accumulated doses also varied. The relationships between the dose rate and the frequency of dicentric chromosomes or total chromosomal aberrations are shown in Figure 3. No consistent relation was observed between internal dose rate and dicentric chromosomes (Figure 3A). The frequency of dicentric chromosomes increased with external dose rate (Figure 3B), although the rate of this increase was lower between the moderately and heavily contaminated areas than between the slightly and moderately contaminated areas. Figure 3C,D shows the relationship between total dose rate and the frequency of dicentric and total chromosomal aberrations, respectively. Although the values of the individual mice are so dispersed that a clear relation was not seen, chromosomal aberrations tended to roughly increase with total dose rate. In a moderately contaminated area, 300-day-old mice (open triangles) showed slightly higher frequencies of chromosomal aberrations than 105-day-old mice (closed triangles). The chromosomal aberrations were rather lower in 300-day-old mice captured in the heavily contaminated area (open diamonds) than in 105-day-old mice (closed diamond), although we used only one 105-day-old mouse.

Figure 2. Frequencies of dicentric chromosomes in small Japanese field mice and house mice captured in the sampling areas. Values observed in individual small Japanese field mice inhabiting the control (open circle), slightly contaminated (open square), moderately contaminated (open triangle), and heavily contaminated area (open diamond) are indicated. Two crosses depicted in the heavily contaminated area indicate values of two individual wild house mice. Closed symbols with vertical bars indicate the average frequency of dicentric chromosomes, with standard deviation in each sampling area. The average frequencies of dicentric chromosomes in the moderately and heavily contaminated area were significantly different from that in the control area (P = 0.02 and P < 0.001, respectively).

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DISCUSSION Comparison of Chromosomal Aberrations between Laboratory and Wild Mice. The use of chromosomal aberrations as indicators of radiation effects has been previously proven useful.31,32 They are regarded as important biological dosimeters in accidental (acute) human exposure, since a correlation between the exposure and the frequency of chromosomal aberrations has been established.33−35 The induction of chromosomal aberrations by irradiation has been also well studied experimentally in laboratory mice. Theoretically, the frequency of chromosomal aberrations increases with radiation dose even in exposure at a low dose (rate). Therefore, it was reasonable to expect that increases in chromosomal aberrations might be detected in chronically exposed wild mice at a low dose rate following the F1-NPP accident. However, even in laboratory mice the effect of chronic low dose rate exposure on the frequency of chromosomal aberrations has been analyzed only in a few studies.23,36,37 The studies performed at the Institute for Environmental Sciences (Rokkasho village, Aomori, Japan), focusing on the effect of chronic exposure at low dose rates on the chromosomal aberrations in C3H laboratory mice, provided important insights.23,38 In these studies, the mice were exposed to γ radiation at dose rates of 0, 0.05, 1.0, and 20 mGy d−1 for a maximum of 400 d. The frequency of dicentric chromosomes in mice exposed at a dose rate of 0.05 mGy d−1 was similar to that in nonexposed control mice. Exposure at a dose rate of 1 mGy d−1 had a very subtle effect, while exposure at a dose rate of 20 mGy d−1 had a noticeable dose dependent effect. Even in wild mice inhabiting the heavily contaminated area selected in the present study, known as the area most severely contaminated by the F1-NPP accident, except for F1-NPP itself, the dose rate was assessed to be around 3 mGy d−1 at the time of capture (Table 1). Therefore, it is not expected that

inhabiting the slightly, moderately, and heavily contaminated areas, calculated by multiplying the average ambient dose rates (Table S1) by a factor of 1.138 (the ratio of 47.8 ± 5.5 μGy h−1, external dose rate measured with glass rod dosimeters embedded in the abdominal space of wild mice carcass left on the ground and 42.0 ± 6.5 μSv h−1, ambient dose rate measured 1 m above ground level, with an NaI(Tl) scintillation survey meter, as reported previously28), was 0.01, 0.84, and 2.2 mGy d−1, respectively (Table 1). The external dose rates were also estimated from the activity concentrations of radiocesium in litter and soil samples, using the ERICA Assessment Tool30 (Table S5). Although there was a huge variation in the activity concentrations of radiocesium in litter and soil samples, even among the samples collected within the same sampling area, the external dose rates calculated from the average activity concentrations in litter and soil samples (Table S5) were roughly similar to those estimated from the ambient dose rates (Table 1). Table 1 shows calculated internal dose rates to individual mice. Large variations were observed in the internal dose rates between mice captured in the same sampling area, in particular a difference of 40-fold was observed between the minimum and the maximum values in the heavily contaminated area. The accumulated dose was obtained by multiplying the total dose rate (the sum of the external dose rate and the internal dose rate) by the age in days of mice. The mice were categorized into three estimated age span of 2−5 months (60−150 days), 9−11 months (270−330 days), 14−17 months (420−510 days) and were expediently referred as 105-, 300- 465-day-old mice, respectively (the detailed explanation was described in the footnote of Table S4). Although 465-day-old mice were E

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F

5395 13140 7484 28249 14036 2016 403

15 21 23 28 31 37 38

moderately contaminated area

heavily contaminated area

Cs

7672 19202 10613 42423 21086 3340 542

1074 2280 19871 5718 5677 2912 6594 3449 876

245 43 81 72 83 112 107 83 83

137

6 8 8 8 10 12 11

13 6 5 12 12 12 14 14 13

14 18 20 18 20 18 20 18 19

rearing period,d days

3.5 5.4 5.4 5.4 8.1 12.4 10

15.2 3.5 2.8 12.4 12.4 12.4 18.8 18.8 15.2

18.8 43.4 65.8 43.4 65.8 43.4 65.8 43.4 53.4

folde Cs

18883 70756 40414 152545 113692 24998 4080

12130 4932 36716 48075 44342 24602 77136 43484 9637

2482 998 2698 2083 3356 2734 4540 2561 2456

134

Cs

26852 103691 57310 229084 170797 41416 5420

16325 7980 55639 70903 70395 36109 123967 64841 13315

4606 1866 5330 3125 5461 4861 7041 3602 4432

137

activity concn in mouse at capture, Bq kg−1 wet

0.16 0.6 0.33 1.3 0.97 0.23 0.032

0.096 0.044 0.31 0.41 0.39 0.21 0.69 0.37 0.078

0.024 0.01 0.028 0.018 0.03 0.026 0.04 0.021 0.024

internal dose rate,f mGy d−1

2.2 2.2 2.2 2.2 2.2 2.2 2.2

0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84 0.84

0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

external dose rate,g mGy d−1

2.36 2.8 2.53 3.5 3.17 2.43 2.23

0.94 0.88 1.15 1.25 1.23 1.05 1.53 1.21 0.92

0.034 0.02 0.038 0.028 0.04 0.036 0.05 0.031 0.034

total dose rate,h mGy d−1

105 300 300 300 300 300 300

300 105 105 105 300 300 105 105 105

105 105 105 465 300 300 300 105 465

(60−150) (270−330) (270−330) (270−330) (270−330) (270−330) (270−330)

(270−330) (60−150) (60−150) (60−150) (270−330) (270−330) (60−150) (60−150) (60−150)

(60−150) (60−150) (60−150) (420−510) (270−330) (270−330) (270−330) (60−150) (420−510)

estimated age at the time of capture,i days

248 (142−354) 840 (756−924) 759 (683−835) 1050 (945−1155) 951 (856−1046) 729 (656−802) 669 (602−736)

282 (254−310) 92.4 (52.8−132) 121 (69.0−173) 131 (75.0−188) 369 (332−406) 315 (284−347) 161 (91.8−230) 127 (72.6−182) 96.6 (55.2−138)

3.57 (2.04−5.10) 2.1 (1.20−3.00) 3.99 (2.28−5.70) 13 (11.8−14.3) 12 (10.8−13.2) 10.8 (9.72−11.9) 15 (13.5−16.5) 3.25 (1.86−4.65) 15.8 (14.3−17.3)

accumulated dose,j mGy

a

Dose (rate) estimation was not done for individual mice captured in the control area because of the negligible low dose (rate). Actually the external and internal dose rates were approximately 2 μGy d−1 (0.07 μSv h−1 × 1.138 × 24 h) and 0.7 μGy d−1, respectively. The internal dose rate was calculated using the ERICA Tool from the mean activity concentrations of 134Cs and 137Cs (63 and 137 Bq kg−1, respectively) in 7 mice that were not used for examination of chromosomal aberrations. The accumulated dose was only