Cloning of Hynobius lichenatus (Tohoku hynobiid salamander) p53 and analysis of its expression in response to radiation.

BACKGROUND: Caudata species such as salamanders are easily affected by environmental changes, which can drastically reduce their population. The effects of acute X-rays and chronic γ-irradiation on Hynobius lichenatus, the Japanese Tohoku hynobiid salamander, are known. However, the expression of radiation-inducible genes, such as the DNA-damage checkpoint response gene p53, has not been analyzed in H. lichenatus. This has not occurred because there is no established method for mRNA quantification in H. lichenatus due to a lack of information on available nucleotide sequences corresponding to both radiation-inducible genes and endogenous control genes such as ACTB (ß-actin). RESULTS: In this study, we aimed to evaluate the effects of radiation on gene expression in H. lichenatus. Using RNA extracted from irradiated salamanders, we performed rapid amplification of cDNA ends (RACE) and cloned H. lichenatus ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and p53. We confirmed that the cloned cDNAs were able to synthesize salamander proteins by western blotting after transfection into cultured HEK293 cells. Proliferation assays using HEK293 cells stably expressing H. lichenatus p53 protein showed that this protein has antiproliferative effects, similar to that of mammalian p53. Furthermore, RT-qPCR analysis using gene-specific primers revealed that p53 mRNA expression in H. lichenatus was upregulated upon exposure to radiation. CONCLUSION: Our results suggest that H. lichenatus p53 protein take an important role in regulating the cellular responses to various stimuli as mammalian p53 does. Furthermore, our study provides novel data to select appropriate primers to analyze internal control mRNA expression in H. lichenatus and to evaluate p53 expression as a marker of radiation and environmental stimuli.

Estimation of absorbed radiation dose rates in wild rodents inhabiting a site severely contaminated by the Fukushima Dai-ichi nuclear power plant accident.

The dose rates of radiation absorbed by wild rodents inhabiting a site severely contaminated by the Fukushima Dai-ichi Nuclear Power Plant accident were estimated. The large Japanese field mouse (Apodemus speciosus), also called the wood mouse, was the major rodent species captured in the sampling area, although other species of rodents, such as small field mice (Apodemus argenteus) and Japanese grass voles (Microtus montebelli), were also collected. The external exposure of rodents calculated from the activity concentrations of radiocesium ((134)Cs and (137)Cs) in litter and soil samples using the ERICA (Environmental Risk from Ionizing Contaminants: Assessment and Management) tool under the assumption that radionuclides existed as the infinite plane isotropic source was almost the same as those measured directly with glass dosimeters embedded in rodent abdomens. Our findings suggest that the ERICA tool is useful for estimating external dose rates to small animals inhabiting forest floors; however, the estimated dose rates showed large standard deviations. This could be an indication of the inhomogeneous distribution of radionuclides in the sampled litter and soil. There was a 50-fold difference between minimum and maximum whole-body activity concentrations measured in rodents at the time of capture. The radionuclides retained in rodents after capture decreased exponentially over time. Regression equations indicated that the biological half-life of radiocesium after capture was 3.31 d. At the time of capture, the lowest activity concentration was measured in the lung and was approximately half of the highest concentration measured in the mixture of muscle and bone. The average internal absorbed dose rate was markedly smaller than the average external dose rate (<10% of the total absorbed dose rate). The average total absorbed dose rate to wild rodents inhabiting the sampling area was estimated to be approximately 52 µGy h(-1) (1.2 mGy d(-1)), even 3 years after the accident. This dose rate exceeds 0.1-1 mGy d(-1) derived consideration reference level for Reference rat proposed by the International Commission on Radiological Protection (ICRP).

Cytokinesis block micronucleus assay in field plants for monitoring radiation-induced genotoxicity of the environment.

Effective biomonitoring for detection of radiation-induced genotoxicity of contaminants in natural environments involves testing of field plants for cytogenetic changes. To increase the efficiency and precision of cytogenetic analyses of field plants that have naturally high individual variability, an improved micronucleus assay is proposed that employs a cytokinesis block technique similar to the lymphocyte test system used in mammals. In seed embryonic meristems of the Japanese cedar, application of a methylxanthine derivative, 3-isobutyl-1-methylxanthine (IBMX), was found to be effective in inhibiting cytokinesis to make once-divided cells easily recognizable by their binucleate appearance. In the meristem of IBMX-treated seminal roots from X-ray-irradiated seeds, variation in micronucleus frequency in the binucleate cell population was reduced compared to that in the total cell population. The highest efficiency of measurement of micronucleus frequencies was obtained in the root meristems where 0.2- to 1.5-mm-long seminal roots were incubated with IBMX for 24 h. This result indicated that this root elongation stage corresponded to the first divisions of the root meristematic cells, and was therefore suitable for obtaining reliable estimations of accumulated genetic damage in the seeds. This cytokinesis block assay applied specifically at the root elongation stage was then used to examine dose-response relationships in Japanese cedar seeds irradiated either acutely with X-rays or chronically with γ-rays. The resulting dose-response curve for the acute X-ray irradiation was fitted onto a linear-quadratic regression curve, whereas the dose-response curve for the chronic γ-irradiation matched a linear regression line better. Both dose-response curves were consistent with the target theory of classical radiation biology. The good agreement of the micronucleus data to a simple dose-response model indicates the proposed accuracy of the cytokinesis block micronucleus assay for plant monitoring.

Effects of chronic γ-irradiation on growth and survival of the Tohoku hynobiid salamander, Hynobius lichenatus.

The Tohoku hynobiid salamanders, Hynobius lichenatus, were chronically irradiated with γ-rays from embryonic to juvenile stages for 450 days. At 490 µGy h(-1) or lower dose rates, growth and survival were not significantly affected by irradiation, and any morphological aberrations and histological damages were not observed. At 4600 µGy h(-1), growth was severely inhibited, and all the individuals died mostly at the juvenile stage. Chronic LD50 was 42 Gy as a total dose. In the liver, the number of hematopoietic cells was significantly reduced in the living juveniles, and these cells disappeared in the dead juveniles. In the spleen, mature lymphocytes were depleted in the living larvae, and almost all the heamtopoietic cells disappeared in the dead juveniles. These results suggest that this salamander died due to acute radiation syndrome, i.e., hematopoietic damage and subsequent sepsis caused by immune depression. The death would be also attributed to skin damage inducing infection. At 18,000 µGy h(-1), morphological aberrations and severe growth inhibition were observed. All the individuals died at the larval stage due to a multiple organ failure. Chronic LD50 was 28 Gy as a total dose. Assuming that chronic LD50 was 42 Gy at lower dose rates than 4600 µGy h(-1), a chronic median lethal dose rate could be estimated to be <340 µGy h(-1) for the whole life (>14 years). These results suggest that, among guidance dose rates, i.e., 4-400 µGy h(-1), proposed by various organisations and research programmes for protection of amphibians and taxonomic groups or ecosystems including amphibians, most of them would protect this salamander but the highest value may not on the whole life scale.

Isotopic evidence of plutonium release into the environment from the Fukushima DNPP accident.

The Fukushima Daiichi nuclear power plant (DNPP) accident caused massive releases of radioactivity into the environment. The released highly volatile fission products, such as (129m)Te, (131)I, (134)Cs, (136)Cs and (137)Cs were found to be widely distributed in Fukushima and its adjacent prefectures in eastern Japan. However, the release of non-volatile actinides, in particular, Pu isotopes remains uncertain almost one year after the accident. Here we report the isotopic evidence for the release of Pu into the atmosphere and deposition on the ground in northwest and south of the Fukushima DNPP in the 20-30 km zones. The high activity ratio of (241)Pu/(239+240)Pu (> 100) from the Fukushima DNPP accident highlights the need for long-term (241)Pu dose assessment, and the ingrowth of (241)Am. The results are important for the estimation of reactor damage and have significant implication in the strategy of decontamination.

Derivation of hazardous doses for amphibians acutely exposed to ionising radiation.

Derivation of effect benchmark values for each taxonomic group, which has been difficult due to lack of experimental effects data, is required for more adequate protection of the environment from ionising radiation. Estimation of effects doses from nuclear DNA mass and subsequent species sensitivity distribution (SSD) analysis were proposed as a method for such a derivation in acute irradiation situations for assumed nuclear accident scenarios. As a case study, 5% hazardous doses (HD5s), at which only 5% of species are acutely affected at 50% or higher lethality, were estimated on a global scale. After nuclear DNA mass data were obtained from a database, 50% lethal doses (LD50s) for 4.8 and 36% of the global Anura and Caudata species, respectively, were estimated by correlative equations between nuclear DNA mass and LD50s. Differences between estimated and experimental LD50s were within a factor of three. The HD5s obtained by the SSD analysis of these estimated LD50s data were 5.0 and 3.1 Gy for Anura and Caudata, respectively. This approach was also applied to the derivation of regional HD5s. The respective HD5s were 6.5 and 3.2 Gy for Anura and Caudata inhabiting Japan. This HD5 value for the Japanese Anura was significantly higher than the global value, while Caudata had no significant difference in global and Japanese HD5s. These results suggest that this approach is also useful for derivation of regional benchmark values, some of which are likely different from the global values.

Environmental protection: researches in National Institute of Radiological Sciences.

Some studies for radiological protection of the environment have been made at the National Institute of Radiological Sciences (NIRS). Transfer of radionuclides and related elements has been investigated for dose estimation of non-human biota. A parameter database and radionuclide transfer models have been also developed for the Japanese environments. Dose (rate)-effect relationships for survival, growth and reproduction have been investigated in conifers, Arabidopsis, fungi, earthworms, springtails, algae, duckweeds, daphnia and medaka. Also genome-wide gene expression analysis has been carried out by high coverage expression profiling (HiCEP). Effects on aquatic microbial communities have been studied in experimental ecosystem models, i.e., microcosms. Some effects were detected at a dose rate of 1 Gy day(-1) and were likely to arise from interspecies interactions. The results obtained at NIRS have been used in development of frameworks for environmental protection by some international bodies, and will contribute to environmental protection in Japan and other Asian countries.

Responses of the bacterial community to chronic gamma radiation in a rice paddy ecosystem.

PURPOSE: Public concern about radiation protection of the environment and non-human species is increasing. The potential impact of chronic gamma radiation on the bacterial community in a rice paddy ecosystem was investigated. MATERIALS AND METHODS: Paddy field microcosm samples were continuously exposed at a dose rate of 1 Gy day(-1) for five days after which the bacterial community structures in them were compared with those of unirradiated control samples by denaturing gradient gel electrophoresis (DGGE) of the 16S ribosomal ribonucleic acid (rRNA) gene. Dissolved ions in ponding waters of the microcosm samples were determined by ion chromatography. RESULTS: The DGGE band profiles were clearly different between the control and exposure samples. Sequencing of selected DGGE bands revealed that a specific species of the genus Clostridium and of the genus Massilia were grown under radiation exposure. Additionally, the concentrations of sulphate ion in the ponding water of the microcosm samples increased under radiation exposure. Concentrations of Na(+), Ca(2+), and F(-) inversely decreased. CONCLUSIONS: The data presented suggest that chronic gamma radiation affects the bacterial community structure and material cycles in the rice paddy ecosystem.