Individual information for large Japanese field mice.
The Fukushima Daiichi nuclear power plant (FDNPP) accident drew global attention to the health risks of radiation exposure. The large Japanese field mice (Apodemus speciosus) are rodents endemic to, and distributed throughout, Japan. This wild rodent live in and around the ex-evacuation zone on the ground surface and/or underground. In this study, we evaluated the effect of chronic radiation exposure associated with FDNPP accident on the testes of large Japanese field mice. Morphological analysis and electron-prove X-ray microanalysis (EPMA) was undertaken on the testes. Morphological analysis of testes based on H&E staining showed that the spermatogenesis was observed normally in the breeding season of wild mice in the heavily contaminated area. However, caesium (Cs) was not detected in all testes of wild mice from FDNPP ex-evacuation zone. In conclusion, even if the testes and the process of spermatogenesis are hypersensitive to radiation, we could not detect radiation effects on the spermatogenesis and Cs in the examined large Japanese field mice testes following chronic radiation exposure associated with the FDNPP accident.
- EPMA analysis
- Fukushima nuclear power plant accident
- wild mice
The Fukushima Daiichi nuclear power plant (FDNPP) accident drew global attention to the health risks of radiation exposure. We have established an archive system composed of livestock and wild animals within a 20 km radius from FDNPP, that is, the ex-evacuation zone of the FDNPP accident [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13]. This system provides critical information for the understanding of environmental pollution, biodistribution, radionuclide metabolism, dose evaluation, and the biological effects of internal and external exposure to radiation caused by nuclear disasters. In particular, experimental studies of low-dose rate (LDR) radiation exposure induced effects on spermatogenesis, along with indications from the nuclear disaster in Fukushima, will provide a more comprehensive radiobiological understanding of response mechanisms leading to improved accuracy in the estimation of human reproduction and health risk .
The large Japanese field mice (
Electron probe X-ray microanalysis (EPMA) is a powerful tool used to detect trace amounts of chemical elements in single cells and tissues . This method measures the characteristic X-ray spectra of specific elements in samples using an accelerated electron beam. We previously investigated the effect of chronic LDR exposure to 134Cs and 137Cs on the testis of euthanised bulls, boars, and inobutas from the evacuation zone [3, 7].
Discharge of 134Cs and 137Cs that emit γ- and β-rays is of primary concern, because they were released in a large amount and have a long half-life. In this study, we evaluated the heavy contamination levels of LDR effects of 134Cs and 137Cs (between 4848 and 70,200 Bq/kg) on the large Japanese field mice after the FDNPP accident.
2. Materials and methods
2.1 Collections of large Japanese field mice
The study protocol followed laboratory animal care guidelines, and all procedures were conducted in accordance with the guideline of the Ethics Committee for Care and Use of Laboratory Animals for Research of Niigata University, Japan (approval number: H2611). Large Japanese field mice were captured using Sherman traps (H.B. Sherman Traps, Inc., Tallahassee, FL, USA) at three sites, Akogi, Ide, and Omaru of Namie town in the ex-evacuation zone of the FDNPP accident in November 2012, April 2013, and April 2016 (Figure 1). Control large Japanese mice were captured using Sherman traps in May 2012, November 2015, and April 2016 in Aomori Prefecture, and April and May 2016 in Niigata Prefecture. The ambient dose rate was measured at the sampling sites using NaI (Tl) scintillation survey meter TCS-171B (Hitachi Aloka Medical, Ltd., Tokyo, Japan) at the height of 1 m. The measurements were expressed as micrograys per hour at 1 m above the ground.
2.2 Measurement of radioactivity
Radioactivities of the organ samples were determined via gamma-ray spectrometry using high-purity germanium (HPGe) detector (GEM40P4-83, Ortec Co., Oak Ridge, TN, USA) as described previously . The duration of the measurement varied from 110,600 to 663,400 s, depending on the radioactivity of the sample. Absolute efficiency of the detector was determined with the standard point sources of 137Cs (10 kBq, CS402) and 152Eu (10 kBq, EU402, Japan Isotope Association, Tokyo, Japan). The samples were placed in a small space (1 mm thick and 6 mm diameter) which is the same size as the standard point sources. A nuclide was identified when its characteristic photopeak 3σ above the baseline observed in the spectrum. The activities due to decay were corrected to the sampling dates.
2.3 Morphological assessment of testes cells
The testes were fixed in Bouin’s solution, embedded in paraffin, and stained using haematoxylin and eosin (H&E), according to standard protocols, as described by Takino et al. . Subsequently, the testes were briefly dehydrated in different concentrations of alcohol. The testes were made transparent by using toluene, and then, then they were embedded in paraffin and cut into 5 μm-thick sections before staining.
2.4 Electron probe X-ray microanalysis
Qualitative analysis: An analytical method was used to investigate the composition of the sample to be analysed. From 6B to 92U can be measured with a combination of analytical crystal to be used. The analysis conditions were as follows: voltage was set to 15 kV, beam current was 100 nA, beam size was minimum, sample current was 92.8 nA, and time 30 ms/point (Figure 2).
Elements analysis: Chemical trace analyses of caesium (Cs), sulphur (S), and nitrogen (N) in the testes were performed using a Shimadzu 1720HT electron probe micro-analyser (Shimadzu Corporation, Tokyo, Japan) equipped for X-ray spectrometry and specifically adapted for the examination of ultrathin sections. Accordingly, 3 μm of each testis section was placed on the carbon plate, and subsequently, each section was carbon coated for the electrification of samples (Biopathology Institute Co., Ltd., Oita, Japan). For the analysis, the voltage of the electron microscope was set to 15 kV, and the electron beam rate was set to 100 nA. Other parameters were beam size minimum × region (260 × 195 μm) and time (30 ms/point). The sections were viewed as secondary electron images, and chemical elemental mapping was performed. We performed EPMA analysis duplicate including test analysis.
3. Results and discussion
To date, low-dose radiation effects on physiological processes including spermatogenesis remain unclear. Further studies are required to confirm these low-dose radiation effects .
In the present study, we examined the effects of LDR exposure associated with the FDNPP accident on the testes of large Japanese field mice from different contaminated areas in the ex-evacuation zone, at Namie town in Fukushima. The ambient dose rate at Akogi was 26.9 μGy/h in November 2012, and 15.2 μGy/h in April 2013. The dose rate at Omaru was 12.3 μGy/h in April 2016. The dose rate at Ide was 16.4 μGy/h in April 2013, and 5.3 μGy/h in April 2016 (Table 1).
|Area||Large Japanese field mice||Sampling date||Ambient dose rate (μGy/h)||Body weight (g)||Radioactive concentration of 134Cs and 137Cs|
|No.||ID||Site||134Cs (Bq/kg)||137Cs (Bq/kg)||Total (Bq/kg)|
The 134Cs and 137Cs radioactivity concentrations (Bq/kg) in large Japanese field mice organ samples were detected via gamma-ray spectrometry by using an HPGe detector (Table 1). The total radioactivity concentrations of 134Cs and 137Cs in large Japanese field mice organ samples from Omaru were 2510, 2750, 3860, and 37,630 Bq/kg. Those from Ide were 10,820 and 16,550 Bq/kg, and this level is highly contaminated for the large Japanese field mice in the ex-evacuation zone.
Okano et al.  reported that, although the concentrations of 134Cs and 137Cs in wild mice from Fukushima exceeded 4000 Bq/kg, there were no significant differences in the frequencies of apoptotic cells or morphologically abnormal sperm when compared with wild mice from the non-contaminated control area. However, Kawagoshi et al.  reported that the average frequencies of chromosomal aberrations in splenic lymphocytes of animals living in the heavily contaminated (approximately 3 mGy/day) area of Fukushima were higher than those of animals from the non-contaminated, slightly contaminated (approximately 0.03 mGy/day), and moderately contaminated (approximately 1 mGy/day) areas. Moreover, the aberration frequency in individual wild mice tended to increase with the estimated dose rates and accumulated doses. Takino et al.  reported that enhanced spermatogenesis occurred in large Japanese field mice living in and around the ex-evacuation zone of FDNPP. It remains to be elucidated whether the phenomenon, which is attributable to chronic LDR exposure, has a beneficial or adverse effect on large Japanese field mice.
Morphological analysis of testes based on H&E staining showed that the stages of the seasonal reproductive cycle were classified into reproductive, non-reproductive, and transition periods (Figure 3A–D; 1). During the reproductive seasons of the large Japanese field mice from Ide, spermatogonia, primary spermatocyte, secondary spermatocyte, and sperm were observed (Figure 3B). Interestingly, spermatogenesis was also observed normally in the breeding season of wild mice in the heavily contaminated area of Omaru (Figure 3C). Moreover, it was confirmed that the regression of sperm and seminiferous tubules during the non-breeding season of the wild mice in the most heavily contaminated area of Akogi were normally observed (Figure 3D).
Figure 3A–D (images 4–6) presents the phase maps obtained using the EPMA indicating micro-constituent concentrations namely, Cs, S and N. Colour imaging rapidly and effectively facilitates the overall analysis of the composite structure; specifically, decreasing levels of metal distribution are indicated from red to blue. Cs was not detected in all testes of wild mice from Ide, Akogi, and Omaru (Figure 3A–D: images 4). In the breeding samples, sulphur was detected inside seminiferous tubules, especially in sperm and was detected around the seminiferous tubules in the non-breeding seasons (Figure 3A–D: images 5). Nitrogen was detected inside both the seminiferous tubules and membranes (Figure 3A–D: images 6).
In conclusion, even if the testes and the process of spermatogenesis are hypersensitive to radiation, there were no significant radiation effects on the spermatogenesis and Cs in the examined large Japanese field mice testes following chronic LDR radiation exposure associated with the FDNPP accident.
This work was supported by the Japan Society for the Promotion of Science, and entrusted to the Japan Atomic Energy Agency (JAEA) by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT).
Fukuda T, Kino Y, Abe Y, Yamashiro H, Kuwahara Y, Nihei H, et al. Distribution of artificial radionuclides in the abandoned cattle in the evacuation zone of the Fukushima Daiichi nuclear power plant. PLoS One. 2013; 8, e54312
Isogai E, Kino Y, Abe Y, Yamashiro H, Shinoda H, Fukuda T, et al. Distribution of radioactive cesium in ostrich ( Struthio Camelus) after the Fukushima Daiichi nuclear power plant accident. Radiation Emergency Medicine. 2013; 2:68-71
Yamashiro H, Abe Y, Fukuda T, Kino Y, Kawaguchi I, Kuwahara Y, et al. Effects of radioactive caesium on bull testes after the Fukushima nuclear plant accident. Scientific Reports. 2013; 3:2850
Hosoda M, Tokonami S, Tazoe H, Sorimachi A, Monzen S, Osanai M, et al. Activity concentrations of environmental samples collected in Fukushima Prefecture immediately after the Fukushima nuclear accident. Scientific Reports. 2013; 3:2283
Fukuda T, Kino Y, Abe Y, Yamashiro H, Kobayashi J, Shimizu Y, et al. Cesium radioactivity in peripheral blood is linearly correlated to that in skeletal muscle: Analyses of cattle within the evacuation zone of the Fukushima Daiichi nuclear power plant. Animal Science Journal. 2015; 86:120-124
Takahashi S, Inoue K, Urushihara Y, Hayashi G, Kino Y, Sekine T, et al. A comprehensive dose evaluation project concerning animals affected by the Fukushima Daiichi nuclear power plant accident: Its setup and progress. Journal of Radiation Research. 2015; 56(S1):i36-i41
Yamashiro H, Abe Y, Hayashi G, Urushihara Y, Kuwahara Y, Suzuki M, et al. Electron probe X-ray microanalysis of boar and inobuta testes after the Fukushima accident. Journal of Radiation Research. 2015; 56(S1):i42-i47
Fukuda T, Hiji M, Kino Y, Abe Y, Yamashiro H, Kobayashi J, et al. Software development for estimating the cesium radioactivity in skeletal muscle from that in blood of cattle. Animal Science Journal. 2016; 87:842-847
Koarai K, Kino Y, Takahashi A, Suzuki T, Shimizu Y, Chiba M, et al. 90Sr in teeth of cattle abandoned in evacuation zone: Record of pollution from the Fukushima-Daiichi nuclear power plant accident. Scientific Reports. 2016; 6:24077
Urushihara Y, Kawasumi K, Endo S, Tanaka K, Hirakawa Y, Hayashi G, et al. Analysis of plasma protein concentrations and enzyme activities in cattle within the ex-evacuation zone of the Fukushima Daiichi nuclear plant accident. PLoS One. 2016; 11:e0155069
Takino S, Yamashiro H, Sugano Y, Fujishima Y, Nakata A, Kasai K, et al. Analysis of the effect of chronic and low-dose radiation exposure on spermatogenic cells of male large Japanese field mice ( Apodemus speciosus) after the Fukushima Daiichi nuclear power plant accident. Radiation Research. 2017; 187:161-168
Koarai K, Kino Y, Takahashi A, Suzuki T, Shimizu Y, Chiba M, et al. 90Sr specific activity of teeth of abandoned cattle after the Fukushima accident—Teeth as an indicator of environmental pollution. Journal of Environmental Radioactivity. 2018; 183:1-6
Ariyoshi K, Miura T, Kasai K, Akifumi N, Fujishima Y, Yoshida MA. Radiation-induced bystander effect in large Japanese field mouse ( Apodemus speciosus) embryonic cells. Radiation and Environmental Biophysics. 2018; 57:223-231
Fukunaga H, Butterworth KT, Yokoya A, Ogawa T, Prise KM. Low-dose radiation-induced risk in spermatogenesis. International Journal of Radiation Biology. 2017; 93:1291-1298
Suzuki H, Yasuda SP, Sakaizumi M, Wakana S, Motokawa M, Tsuchiya K. Differential geographic patterns of mitochondrial DNA variation in two sympatric species of Japanese wood mice, Apodemus speciosusand A. argenteus. Genes & Genetic Systems. 2004; 79:165-176
Pogorelov AG, Budantsev AY, Pogorelova VN. Quantitative electron probe microanalysis of acetylcholinesterase activity in rat brain sections. Journal of Histochemistry and Cytochemistry. 1993; 41:1795-1800
Okano T, Ishiniwa H, Onuma M, Shindo J, Yokohata Y, Tamaoki M. Effects of environmental radiation on testes and spermatogenesis in wild large Japanese field mice ( Apodemus speciosus) from Fukushima. Scientific Reports. 2016; 6:23601
Kawagoshi T, Shiomi N, Takahashi H, Watanabe Y, Fuma S, Doi K, et al. Chromosomal aberrations in large Japanese field mice ( Apodemus speciosus) captured near Fukushima Dai-ichi nuclear power plant. Environmental Science & Technology. 2017; 51:4632-4641