Ultrastructural Analysis of Large Japanese Field Mouse (Apodemus speciosus) Testes Exposed to Low-Dose-Rate (LDR) Radiation after the Fukushima Nuclear Power Plant Accident.

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, great attention has been paid to the impact of chronic low-dose-rate (LDR) radiation exposure on biological systems. The reproductive system is sensitive to radiation, with implications connected to infertility. We investigated the testis ultrastructure of the wild large Japanese field mouse (Apodemus speciosus) from three areas contaminated after the FDNPP accident, with different levels of LDR radiation (0.29 µSv/h, 5.11 µSv/h, and 11.80 µSv/h). Results showed good preservation of the seminiferous tubules, comparable to the unexposed animals (controls), except for some ultrastructural modifications. Increases in the numerical density of lipid droplet clusters in spermatogenic cells were found at high levels of LDR radiation, indicating an antioxidant activity rising due to radiation recovery. In all groups, wide intercellular spaces were found between spermatogenic cells, and cytoplasmic vacuolization increased at intermediate and high levels and vacuolated mitochondria at the high-level. However, these findings were also related to the physiological dynamics of spermatogenesis. In conclusion, the testes of A. speciosus exposed to LDR radiation associated with the FDNPP accident showed a normal spermatogenesis, with some ultrastructural changes. These outcomes may add information on the reproductive potential of mammals chronically exposed to LDR radiation.

Estimation of external dose for wild Japanese macaques captured in Fukushima prefecture: decomposition of electron spin resonance spectrum.

The signal intensities of CO2- radicals in teeth can be utilised as an individual indicator of the cumulative external dose for animals. To accurately determine the external dose, it is desirable to analyse the CO2- radical intensity and improve its detection limit. We recently reported a dose-response in the range of 0-200 mGy and estimated the absorbed dose for seven wild Japanese macaques captured in/around the related areas to the Fukushima Daiichi Nuclear Power Plant accident. Herein, for further improvement of this method, we examined the electron spin resonance spectra of the teeth of these seven and an additional four macaques captured in Fukushima by applying two spectrum-decomposition algorithms.

Detection limit of electron spin resonance for Japanese deciduous tooth enamel and density separation method for enamel-dentine separation.

Electron spin resonance (ESR) dosimetry is one of the most powerful tools for radiation dose reconstruction. The detection limit of this technique using human teeth is reported to be 56 mGy or 67 mGy; however, the absorbed dose of Fukushima residents after the Fukushima Daiichi Nuclear Power Plant (FNPP) accident was estimated to be lower than this detection limit. Our aim is to assess the absorbed radiation dose of children in Fukushima Prefecture after the accident; therefore, it is important to estimate the detection limit for their teeth. The detection limit for enamel of deciduous teeth of Japanese children separated by the mechanical method is estimated to be 115.0 mGy. The density separation method can effectively separate enamel from third molars of Japanese people. As we have collected thousands of teeth from children in Fukushima, the present technique may be useful to examine their external absorbed dose after the FNPP accident.

Environmental radiation on large Japanese field mice in Fukushima reduced colony forming potential in hematopoietic progenitor cells without inducing genomic instability.

PURPOSE: To study the environmental radiation effects of wild animals after the Fukushima Dai-ichi nuclear power plant accident, we assessed effects on hematopoietic progenitor cells (HPCs) in large Japanese field mice (Apodemus speciosus). MATERIALS AND METHODS: A. speciosus were collected from three contaminated sites and control area. The air dose-rates at the control and contaminated areas were 0.96 ± 0.05 µGy/d (Hirosaki), 14.4 ± 2.4 µGy/d (Tanashio), 208.8 ± 31.2 µGy/d (Ide), 470.4 ± 93.6 µGy/d (Omaru), respectively. We investigated possible DNA damage and pro-inflammatory markers in the bone marrow (BM) cells. The colony-forming potential of BM cells was estimated by the number of HPC colony-forming cells. Radiation-induced genomic instability (RIGI) in HPCs was also analyzed by quantifying delayed DNA damage in CFU-GM clones. RESULTS: Although no significant differences in DNA damage and inflammation markers in BM cells from control and contaminated areas, the number of HPC colonies exhibited an inverse correlation with air dose-rate. With regard to RIGI, no significant differences in DNA damage of CFU-GM clones between the mice from the control and the three contaminated areas. CONCLUSIONS: Our study suggests that low dose-rate radiation of more than 200 Gy/d reduced HPCs, possibly eliminating genomically unstable HPCs.

Radioactivity and radionuclides in deciduous teeth formed before the Fukushima-Daiichi Nuclear Power Plant accident.

The Fukushima-Daiichi Nuclear Power Plant (FNPP) accident in March of 2011 released substantial amounts of radionuclides into the environment. We collected 4,957 deciduous teeth formed in children before the Fukushima accident to obtain precise control data for teeth formed after the accident. Radioactivity was measured using imaging plates (IP) and epidemiologically assessed using multivariate regression analysis. Additionally, we measured 90Sr, 137Cs, and natural radionuclides which might be present in teeth. Epidemiological studies of IP showed that the amount of radioactivity in teeth from Fukushima prefecture was similar to that from reference prefectures. We found that artificial radionuclides of 90Sr and 137Cs, which were believed to have originated from past nuclear disasters, and natural radionuclides including 40 K and daughter nuclides in the 238U and 232Th series contributed to the generation of radioactivity in teeth. We also found no evidence to suggest that radionuclides originating from the FNPP accident significantly contaminated pre-existing teeth. This is the first large-scale investigation of radioactivity and radionuclides in teeth. The present findings will be indispensable for future studies of teeth formed after the FNPP accident, which will fall out over the next several years and might be more contaminated with radionuclides.

Morphological reproductive characteristics of testes and fertilization capacity of cryopreserved sperm after the Fukushima accident in raccoon (Procyon lotor).

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex-evacuation zone. Wildlife within the alert zone have been exposed to low-dose-rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex-evacuation zone of the FDNPP accident. The radioactivity of caesium-137 (137 Cs) was measured by gamma-ray spectrometry, and the measured radioactivity concentration was 300-6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze-thawing sperm penetration ability was confirmed using the interspecific zona pellucida-free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons.

Structure-Dependent Effects of Bisphosphonates on Inflammatory Responses in Cultured Neonatal Mouse Calvaria.

Bisphosphonates (BPs) are classified into two groups, according to their side chain structures, as nitrogen-containing BPs (NBPs) and non-nitrogen-containing BPs (non-NBPs). In this study, we examined the effects of NBPs and non-NBPs on inflammatory responses, by quantifying the inflammatory mediators, prostaglandin E2 (PGE2) and nitric oxide (NO), in cultured neonatal mouse calvaria. All examined NBPs (pamidronate, alendronate, incadronate, risedronate, zoledronate) stimulated lipopolysaccharide (LPS)-induced PGE2 and NO production by upregulating COX-2 and iNOS mRNA expression, whereas non-NBPs (etidronate, clodronate, tiludronate) suppressed PGE2 and NO production, by downregulating gene expression. Additionally, [4-(methylthio) phenylthio] methane bisphosphonate (MPMBP), a novel non-NBP with an antioxidant methylthio phenylthio group in its side chain, exhibited the most potent anti-inflammatory activity among non-NBPs. Furthermore, results of immunohistochemistry showed that the nuclear translocation of NF-κB/p65 and tyrosine nitration of cytoplasmic protein were stimulated by zoledronate, while MPMBP inhibited these phenomena, by acting as a superoxide anion (O2-) scavenger. These findings indicate that MPMBP can act as an efficacious agent that causes fewer adverse effects in patients with inflammatory bone diseases, including periodontitis and rheumatoid arthritis.

Intestinal Bacteria as Powerful Trapping Lifeforms for the Elimination of Radioactive Cesium.

In March 2011, an accident at the Fukushima Daiichi Nuclear Power Plant led to major problems, including the release of radionuclides such as Cesium (Cs)-137 into the environment. Ever since this accident, Cs-137 in foods has become a serious problem. In this study, we determined the concentration of Cs-137 in the feces, urine, and ruminal contents of cattle and demonstrated the possibility of its elimination from the body by intestinal bacteria. The results revealed a high Cs-137 concentration in the feces; in fact, this concentration was higher than that in skeletal muscles and other samples from several animals. Furthermore, intestinal bacteria were able to trap Cs-137, showing an uptake ratio within the range of 38-81% in vitro. This uptake appeared to be mediated through the sodium-potassium (Na+-K+) ion pump in the bacterial cell membrane. This inference was drawn based on the fact that the uptake ratio of Cs-137 was decreased in media with high potassium concentration. In addition, it was demonstrated that intestinal bacteria hindered the trapping of Cs-137 by the animal. Cattle feces showed high concentration of Cs-137 and intestinal bacteria trapped Cs-137. This study is the first report showing that intestinal bacteria contribute to the elimination of Cs-137 from the body.

[MPMBP, a novel bisphosphonate with an antioxidant side chain, stimulates bone formation through inhibition of NF-κB nuclear translocation].

Bisphosphonates (BPs) are chemically characterized by a P-C-P bond with two lateral side chains on the carbon atom, and have been widely used as anti-resorptive agents in various metabolic bone diseases. 4-[(methylthio) phenylthio] methanebisphosphonate (MPMBP) is a novel non-nitrogen-containing BP with an antioxidant side chain that possesses anti-inflammatory properties. Since inflammation is known to be a cause of the pathological bone resorption, we investigated the effects of MPMBP on bone metabolism both in vitro and in vivo. The results showed that: i) MPMBP dose-dependently increased alkaline-phosphatase activity in a culture of osteoblastic MC3T3-E1 cells, ii) MPMBP increased the synthesis of collagen (type-I) in an organ culture of mouse calvaria, iii) local injection of MPMBP to alveolar bone induced prominent increases in both the bone mass and thickness of alveolar bone at the local site of injection in rabbits, iv) MPMBP increased the mRNA expression of alkaline-phosphatase, type-I collagen, osteocalcin, and bone sialoprotein in MC3T3-E1 cells, v) MPMBP inhibited the translocation of NF-κB/p65 to the nuclei in osteoblasts of cultured mouse calvaria. Taken together, these findings suggest that MPMBP is a promising agent to prevent bone loss, or even accelerate new bone formation, through inducing an uncoupling between bone resorption and bone formation, which is preferable to maintain bone mass and quality.

Haematological analysis of Japanese macaques (Macaca fuscata) in the area affected by the Fukushima Daiichi Nuclear Power Plant accident.

Several populations of wild Japanese macaques (Macaca fuscata) inhabit the area around Fukushima Daiichi Nuclear Power Plant (FNPP). To measure and control the size of these populations, macaques are captured annually. Between May 2013 and December 2014, we performed a haematological analysis of Japanese macaques captured within a 40-km radius of FNPP, the location of a nuclear disaster two years post-accident. The dose-rate of radiocaesium was estimated using the ERICA Tool. The median internal dose-rate was 7.6 µGy/day (ranging from 1.8 to 219 µGy/day) and the external dose-rate was 13.9 µGy/day (ranging from 6.7 to 35.1 µGy/day). We performed multiple regression analyses to estimate the dose-rate effects on haematological values in peripheral blood and bone marrow. The white blood cell and platelet counts showed an inverse correlation with the internal dose-rate in mature macaques. Furthermore, the myeloid cell, megakaryocyte, and haematopoietic cell counts were inversely correlated and the occupancy of adipose tissue was positively correlated with internal dose-rate in femoral bone marrow of mature macaques. These relationships suggest that persistent whole body exposure to low-dose-rate radiation affects haematopoiesis in Japanese macaques.

90Sr specific activity of teeth of abandoned cattle after the Fukushima accident - teeth as an indicator of environmental pollution.

90Sr specific activity in the teeth of young cattle that were abandoned in Kawauchi village and Okuma town located in the former evacuation areas of the Fukushima-Daiichi Nuclear Power Plant (FNPP) accident were measured. Additionally, specific activity in contaminated surface soils sampled from the same area was measured. (1) All cattle teeth examined were contaminated with 90Sr. The specific activity, however, varied depending on the developmental stage of the teeth during the FNPP accident; teeth that had started development before the accident exhibited comparatively lower values, while teeth developed mainly after the accident showed higher values. (2) Values of 90Sr-specific activity in teeth formed after the FNPP accident were higher than those of the bulk soil but similar to those in the exchangeable fraction (water and CH3COONH4 soluble fractions) of the soil. The findings suggest that 90Sr was incorporated into the teeth during the process of development, and that 90Sr in the soluble and/or leachable fractions of the soil might migrate into teeth and contribute to the amount of 90Sr in the teeth. Thus, the concentration of 90Sr in teeth formed after the FNPP accident might reflect the extent of 90Sr pollution in the environment.

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.

In this study we analyzed the effect of chronic and low-dose-rate (LDR) radiation on spermatogenic cells of large Japanese field mice ( Apodemus speciosus ) after the Fukushima Daiichi Nuclear Power Plant (FNPP) accident. In March 2014, large Japanese field mice were collected from two sites located in, and one site adjacent to, the FNPP ex-evacuation zone: Tanashio, Murohara and Akogi, respectively. Testes from these animals were analyzed histologically. External dose rate from radiocesium (combined 134Cs and 137Cs) in these animals at the sampling sites exhibited 21 µGy/day in Tanashio, 304-365 µGy/day in Murohara and 407-447 µGy/day in Akogi. In the Akogi group, the numbers of spermatogenic cells and proliferating cell nuclear antigen (PCNA)-positive cells per seminiferous tubule were significantly higher compared to the Tanashio and Murohara groups, respectively. TUNEL-positive apoptotic cells tended to be detected at a lower level in the Murohara and Akogi groups compared to the Tanashio group. These results suggest that enhanced spermatogenesis occurred in large Japanese field mice living in and around the FNPP ex-evacuation zone. It remains to be elucidated whether this phenomenon, attributed to chronic exposure to LDR radiation, will benefit or adversely affect large Japanese field mice.

Correction: Analysis of Plasma Protein Concentrations and Enzyme Activities in Cattle within the Ex-Evacuation Zone of the Fukushima Daiichi Nuclear Plant Accident.

[This corrects the article DOI: 10.1371/journal.pone.0155069.].

The novel bisphosphonate disodium dihydrogen-4-[(methylthio) phenylthio] methanebisphosphonate increases bone mass in post-ovariectomy rats.

The novel bisphosphonate (BP) disodium dihydrogen-4-[(methylthio) phenylthio] methanebisphosphonate (MPMBP) is a non-nitrogen-containing BP with an antioxidant side chain that possesses anti-inflammatory properties. We investigated the systemic effects of this compound on bone loss induced by ovariectomy (OVX) in adult rats. Micro-computed tomography revealed that MPMBP increased bone mass and density in both the metaphysis and diaphysis, and improved the structural properties important for mechanical strength of osteoporotic bone. Sequential bone labeling with tetracycline and calcein indicated that MPMBP decreased longitudinal growth of the primary spongiosa (PS), but stimulated cortical bone formation in the diaphysis. MPMBP increased type I collagen accumulation in the PS, and decreased the number and size of adipocytes in the bone marrow, suggesting inhibition of increased bone marrow adipogenesis induced by OVX. Furthermore, MPMBP reduced the number of bone resorbing cathepsin K-positive osteoclasts induced by OVX. These results suggest that MPMBP could improve bone loss induced by estrogen deficiency. Both stimulation of bone formation and inhibition of bone resorption might play a role in the increase in bone mass and bone density after MPMBP treatment.

Analysis of Plasma Protein Concentrations and Enzyme Activities in Cattle within the Ex-Evacuation Zone of the Fukushima Daiichi Nuclear Plant Accident.

The effect of the Fukushima Daiichi Nuclear Power Plant (FNPP) accident on humans and the environment is a global concern. We performed biochemical analyses of plasma from 49 Japanese Black cattle that were euthanized in the ex-evacuation zone set within a 20-km radius of FNPP. Among radionuclides attributable to the FNPP accident, germanium gamma-ray spectrometry detected photopeaks only from 134Cs and 137Cs (radiocesium) commonly in the organs and in soil examined. Radioactivity concentration of radiocesium was the highest in skeletal muscles. Assuming that the animal body was composed of only skeletal muscles, the median of internal dose rate from radiocesium was 12.5 µGy/day (ranging from 1.6 to 33.9 µGy/day). The median of external dose rate calculating from the place the cattle were caught was 18.8 µGy/day (6.0-133.4 µGy/day). The median of internal and external (total) dose rate of the individual cattle was 26.9 µGy/day (9.1-155.1 µGy/day). Plasma levels of malondialdehyde and superoxide dismutase activity were positively and glutathione peroxidase activity was negatively correlated with internal dose rate. Plasma alanine transaminase activity and percent activity of lactate dehydrogenase (LDH)-2, LDH-3 and LDH-4 were positively and LDH-1 was negatively correlated with both internal and total dose rate. These suggest that chronic exposure to low-dose rate of ionizing radiation induces slight stress resulting in modified plasma protein and enzyme levels.

(90)Sr in teeth of cattle abandoned in evacuation zone: Record of pollution from the Fukushima-Daiichi Nuclear Power Plant accident.

Here we determined the (90)Sr concentrations in the teeth of cattle abandoned in the evacuation area of the Fukushima-Daiichi Nuclear Power Plant (FNPP) accident. (90)Sr activity concentrations in the teeth varied from 6-831 mBq (g Ca)(-1) and exhibited a positive relationship with the degree of radioactive contamination that the cattle experienced. Even within an individual animal, the specific activity of (90)Sr (Bq (g Sr)(-1)) varied depending on the development stage of the teeth during the FNPP accident: teeth that were early in development exhibited high (90)Sr specific activities, while teeth that were late in development exhibited low specific activities. These findings demonstrate that (90)Sr is incorporated into the teeth during tooth development; thus, tooth (90)Sr activity concentrations reflect environmental (90)Sr levels during tooth formation. Assessment of (90)Sr in teeth could provide useful information about internal exposure to (90)Sr radiation and allow for the measurement of time-course changes in the degree of environmental (90)Sr pollution.

Software development for estimating the concentration of radioactive cesium in the skeletal muscles of cattle from blood samples.

The 2011 earthquake severely damaged the Fukushima Daiichi Nuclear Power Plant (FNPP), resulting in the release of large quantities of radioactive material into the environment. The deposition of these radionuclides in rice straw as livestock feed led to the circulation of contaminated beef in the market. Based on the safety concern of the consumers, a reliable method for estimating concentrations of radioactive cesium in muscle tissue is needed. In this study, we analyzed the concentrations of radioactive cesium in the blood and skeletal muscle of 88 cattle, and detected a linear correlation between them. We then developed software that can be used to estimate radioactive cesium concentrations in muscle tissue from blood samples. Distribution of this software to the livestock production field would allow us to easily identify high-risk cattle, which would be beyond the safety regulation, before shipping out to the market. This software is planned to be released as freeware. This software would contribute to food safety, and aid the recovery of the livestock industry from the damage creacted by the 2011 Tohoku earthquake and tsunami.

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.

The accident at the Fukushima Daiichi Nuclear Power Plant (FNPP) released a large amount of radioactive substances into the environment. Furthermore, beef contaminated with radioactive cesium above the 500 Bq/kg safety standard was circulated in the food chain in 2011. Japanese consumers remain concerned about the safety of radioactively contaminated food. In our previous study, we detected a linear correlation between radioactive cesium ((137) Cs) activity in blood and muscle around 500 to 2500 Bq/kg in cattle. However, it was unclear whether the correlation was maintained at a lower radioactivity close to the current safety standard of 100 Bq/kg. In this study, we evaluated 17 cattle in the FNPP evacuation zone that had a (137) Cs blood level less than 10 Bq/kg. The results showed a linear correlation between blood (137) Cs and muscle (137) Cs (Y = 28.0X, R(2) = 0.590) at low radioactivity concentration, indicating that cesium radioactivity in the muscle can be estimated from blood radioactivity. This technique would be useful in detecting high-risk cattle before they enter the market, and will contribute to food safety.

Effects of radioactive caesium on bull testes after the Fukushima nuclear plant accident.

We aimed to investigate the effect of chronic radiation exposure associated with the Fukushima Daiichi Nuclear Plant accident on the testis from 2 bulls. Estimated dose of internal exposure in one bull was 0.7-1.2 mGy (¹³4Cs) and 0.4-0.6 mGy (¹³7Cs) and external exposure was 2.0 mGy (¹³4Cs) and 0.8 mGy (¹³7Cs) (196 days). Internal dose in the other was 3.2-6.1 mGy (¹³4Cs) and 1.8-3.4 mGy (¹³7Cs) and external dose was 1.3 mGy (¹³4Cs) and 0.6 mGy (¹³7Cs) (315 days). Sperm morphology and spermatogenesis were within normal ranges. ¹³4,¹³7Cs radioactivity was detected but Cs was not detectable in the testis by electron probe microanalysis. Thus, adverse radiation-induced effects were not observed in bull testes following chronic exposure to the above levels of radiation for up to 10 months. Since we could analyse a limited number of testes, further investigation on the effects of ionizing radiation on spermatogenesis should be extended to more animals.

Distribution of artificial radionuclides in abandoned cattle in the evacuation zone of the Fukushima Daiichi nuclear power plant.

The Fukushima Daiichi Nuclear Power Plant (FNPP) accident released large amounts of radioactive substances into the environment. In order to provide basic information for biokinetics of radionuclides and for dose assessment of internal exposure brought by the FNPP accident, we determined the activity concentration of radionuclides in the organs of 79 cattle within a 20-km radius around the FNPP. In all the specimens examined, deposition of Cesium-134 ((134)Cs, half-life: 2.065 y) and (137)Cs (30.07 y) was observed. Furthermore, organ-specific deposition of radionuclides with relatively short half-lives was detected, such as silver-110m ((110m)Ag, 249.8 d) in the liver and tellurium-129m ((129m)Te, 33.6 d) in the kidney. Regression analysis showed a linear correlation between the radiocesium activity concentration in whole peripheral blood (PB) and that in each organ. The resulting slopes were organ dependent with the maximum value of 21.3 being obtained for skeletal muscles (R(2) = 0.83, standard error (SE) = 0.76). Thus, the activity concentration of (134) Cs and (137)Cs in an organ can be estimated from that in PB. The level of radioactive cesium in the organs of fetus and infants were 1.19-fold (R(2) = 0.62, SE = 0.12), and 1.51-fold (R(2) = 0.70, SE = 0.09) higher than that of the corresponding maternal organ, respectively. Furthermore, radiocesium activity concentration in organs was found to be dependent on the feeding conditions and the geographic location of the cattle. This study is the first to reveal the detailed systemic distribution of radionuclides in cattle attributed to the FNPP accident.