Article pubs.acs.org/est
Chromosomal Aberrations in Large Japanese Field Mice (Apodemus speciosus) Captured near Fukushima Dai-ichi Nuclear Power Plant 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,○ Satoshi Yoshida,◆ and Yoshihisa Kubota*,† †
Fukushima Project Headquarters, National Institute of Radiological Sciences, National Institute for Quantum and Radiological Science and Technology, 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 § Center for Radiation Protection Knowledge, National Institute of Radiological Sciences, National Institute for Quantum and Radiological Science and Technology, 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 Campus, Miyazaki 889-2192, Japan ◆ Department of Management and Planning, National Institute for Quantum and Radiological Science and Technology, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan S Supporting Information *
ABSTRACT: Since the Fukushima Dai-ichi Nuclear Power Plant accident, radiation effects on nonhuman biota in the contaminated areas have been a major concern. Here, we analyzed the frequencies of chromosomal aberrations (translocations and dicentrics) in the splenic lymphocytes of large Japanese field mice (Apodemus speciosus) inhabiting Fukushima Prefecture. A. speciosus chromosomes 1, 2, and 5 were flowsorted in order to develop A. speciosus chromosome-specific painting probes, and FISH (fluorescence in situ hybridization) was performed using these painting probes to detect the translocations and dicentrics. The average frequency of the translocations and dicentrics per cell in the heavily contaminated area was significantly higher than the frequencies in the case of the noncontaminated control area and the slightly and moderately contaminated areas, and this aberration frequency in individual mice tended to roughly increase with the estimated dose rates and accumulated doses. In all four sampling areas, the proportion of aberrations occurring in chromosome 2 was approximately >3 times higher than that in chromosomes 1 and 5, which suggests that A. speciosus chromosome 2 harbors a fragile site that is highly sensitive to chromosome breaks induced by cellular stress such as DNA replication. The elevated frequency of chromosomal aberrations in A. speciosus potentially resulting from the presence of a fragile site in chromosome 2 might make it challenging to observe the mild effect of chronic low-dose-rate irradiation on the induction of chromosomal aberrations in A. speciosus inhabiting the contaminated areas of Fukushima.
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INTRODUCTION
Japan. Key areas of Fukushima Prefecture, in particular, were heavily contaminated, and the residents had to evacuate these areas after the accident. As a result of the evacuation, the
The Great East Japan Earthquake, which occurred in the Northwest Pacific Ocean approximately 130 km off the coast of northeastern Japan on March 11, 2011, and a subsequent devastating tsunami caused the Fukushima Dai-ichi Nuclear Power Plant (F1-NPP) accident. Massive amounts of radioactive nuclides were released into the atmosphere, which led to considerable radioactive contamination in large parts of eastern © 2017 American Chemical Society
Received: Revised: Accepted: Published: 4632
December March 30, March 31, March 31,
8, 2016 2017 2017 2017 DOI: 10.1021/acs.est.6b06210 Environ. Sci. Technol. 2017, 51, 4632−4641
Article
Environmental Science & Technology
moderately contaminated areas, only five and six mice, respectively, were captured in July 2012 and analyzed for chromosomal aberrations; thus, the mice captured in these areas in December 2013 and October 2014 were included here in order to increase the number of mice used to analyze chromosomal aberrations. In the noncontaminated control area, seven mice were captured in July 2013 and used to determine the radiosensitivity of A. speciosus. Detailed information on the sampling areas is presented in Supporting Information (SI) Table S1. The materials and methods used for the capture, rearing, and sacrifice of A. speciosus were the same as those described previously.15 For comparison with A. specious, 12−24-week-old female C3H/HeJ mice were purchased from a domestic breeder (Japan SLC, Shizuoka, Japan). 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. Culture of Spleen Cells and Preparation of Chromosome Slides. After mice were sacrificed, the spleen was aseptically dissected and spleen cells were isolated and cultured as reported by Tanaka et al.16 Briefly, single-cell suspensions of splenic lymphocytes were cultured for 24−26 h in RPMI 1640 medium containing 10 μg/mL of lipopolysaccharide (LPS), 3 μg/mL of concanavalin A (ConA), and 50 μM of 2mercaptoethanol (2-ME) in 95% air/5% CO2 with 95% humidity at 37 °C. Colcemid at a final concentration of 0.02 μg/mL was added for the last 6 or 24 h of culture to collect metaphase cells. The cultured spleen cells were used for preparing chromosome slides, which were then subjected to FISH as described below. Some of the cultured spleen cells obtained from the A. speciosus captured in the noncontaminated control area and from the purchased C3H/HeJ mice were irradiated with an X-ray machine (Shimazu Co. Ltd., Tokyo, Japan), at a dose rate of approximately 0.2 Gy min−1, in order to determine the in vitro radiosensitivity of splenic lymphocytes. The irradiated cells were incubated for 3−4 h in culture medium lacking mitogens and then cultured as described.16 Cell Cycle Analysis. Spleen cells obtained from A. speciosus captured in the noncontaminated control area and from C3H/ HeJ mice were cultured for 24−42 h in the presence of 1 μg mL−1 5-bromo 2′-deoxyuridine (BrdU) to differentiate metaphase cells into the first cycle (M1), second cycle (M2), and third or higher cycle (M3+) cells based on examining the differential staining pattern of sister chromatids.17,18 The remainder of the procedure used for analyzing the cell cycle was as described in detail previously.15 Approximately 1000 cells were analyzed for determining the mitotic indices. Radioactivity Measurement. After the spleen was removed from the sacrificed mice, two professional researchers analyzed tooth abrasion to estimate the age of the mice. The age of A. speciosus was comprehensively estimated based on the two breeding seasons, spring (February to May) and autumn (August to October), together with the age estimation according to tooth abrasion and body size; these details are shown in SI Table S2. Next, the entire body of the mice without the spleen was ashed using the method described by MEXT.19 Activity concentrations of named radionuclides in the ashed samples were measured through γ-spectrometry by 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,
residents’ radiation-exposure dose (rate) has been lowered to acceptable levels. By contrast, the nonhuman biota inhabiting heavily contaminated areas in Fukushima has been continuously exposed to radiation from the time of the accident until now. Given that the importance of radiological protection of the environment has been increasingly recognized over the past two decades,1,2 radiation effects on the nonhuman biota inhabiting Fukushima have been a major concern since the F1-NPP accident,3−5 and a few radioecological6−10 and biological11−14 studies have been conducted to date to address these effects. However, additional studies are necessary for reaching a consistent conclusion regarding whether the F1-NPP accident has had an impact on nonhuman biota. For analyzing the radiation effect on wildlife chronically exposed to low-dose (rate) radiation, such as the animals inhabiting Fukushima, chromosomal aberrations in the lymphocytes of wild animals serve as sensitive indicators; this is because lymphocytes in the peripheral blood or spleen live for long periods without cell division and thus retain any chromosomal damage after it has once occurred as a result of acute or chronic exposure to clastogenic agents such as radiation. Previously, we reported that the average frequencies of dicentric chromosomes, an unstable type of chromosomal aberration, was markedly increased in the splenic lymphocytes of small Japanese field mice (Apodemus argenteus) inhabiting the moderately and heavily contaminated areas around F1NPP.15 The small Japanese field mouse (A. argenteus) and the large Japanese field mouse (Apodemus speciosus) are species endemic to Japan and the main species of mice captured in Fukushima. We previously found that the dicentric assay performed with Cbanding to identify dicentric chromosomal aberrations could be applied to A. argenteus but not A. speciosus, because the Cbanding pattern used to distinguish the centromere was very subtle and faint in most of the A. speciosus chromosomes.15 Translocation, which is another representative chromosomal aberration, is regarded as a comparatively more suitable indicator in the case of long-term exposure because it is stable: the aberration is stably retained for extended periods after cell division. Thus, since the F1-NPP accident, an analysis of the translocation frequency in these two species of wild mice inhabiting Fukushima has been awaited. However, the nonavailability of FISH (fluorescence in situ hybridization) probes for readily detecting translocations in the genus Apodemus has precluded an examination of translocations in these mice until now. Here, we first sorted the chromosomes of A. speciosus and developed painting probes specific to individual chromosomes of A. speciosus by using the sorted chromosomes as templates. Next, we used these probes and investigated the frequency of chromosomal aberrations in the lymphocytes of A. speciosus inhabiting four areas around F1-NPP where the contamination level varies.
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MATERIAL AND METHODS Capture of A. speciosus. We sampled A. speciosus (large Japanese field mice) in the same areas as those described previously.15 According to the contamination level, the four sampling areas were referred to as the noncontaminated control (0.1 ± 0 μSv h−1), slightly contaminated (0.3 ± 0.1−0.4 ± 0.2 μSv h−1), moderately contaminated (7.5 ± 2.1−30.7 ± 7.4 μSv h−1), and heavily contaminated (80.0 ± 13.6 μSv h−1) areas. All mice from the heavily contaminated area that were used were captured in July 2012. However, in the case of the slightly and 4633
DOI: 10.1021/acs.est.6b06210 Environ. Sci. Technol. 2017, 51, 4632−4641
Article
Environmental Science & Technology Japan).20 The counting time was set at 600−100 000 s to reduce statistical error during measurement to
