Effects of Continuous Prenatal Low Dose Rate Irradiation on Neurobehavior, Hippocampal Cellularity, Messenger RNA and MicroRNA Expression on B6C3F1 Mice.

Epidemiological, experimental, and ecological data have indicated the controversial effect of in utero chronic low dose rate (<6 mGy/h) with accumulative low (≤100 mGy) or high (>100 mGy) dose radiation exposure. Our main goal of this study was to examine if different low dose rates of chronic pre- and/or post-natal radiation exposure with accumulative high doses could induce hippocampal cellular, mRNA, and miRNA changes leading to neuropsychiatric disorders. The comprehensive mouse phenotypic traits, organ weight, pathological, and blood mRNA and miRNA changes were also studied. Using different approaches including SmithKline, Harwell, Imperial College, Royal Hospital, Phenotype Assessment (SHIRPA), neurobehavioral tests, pathological examination, immunohistochemistry, mRNA and miRNA sequencing, and real-time quantitative polymerase chain reaction (qRT-PCR) validation, we found that in prenatally irradiated (100 mGy/d for 18 days with an accumulative dose of 1.8 Gy) 1-year-old mice, no cellular changes, including immature neurons in the subgranular zone, mature neurons and glial cells in the hilus of the dentate gyrus and development of cognitive impairment, neuropsychiatric disorders, occurred. However, a significant reduction in body weight and mass index (BMI) was indicated by the SHIRPA test. A reduced exploratory behavior was shown by an open field test. Organ weights showed significant reductions in the testes, kidneys, heart, liver and epididymides with no abnormal pathology. mRNA and miRNA sequencing and qRT-PCR validation revealed the upregulation of Rubcnl and Abhd14b, and downregulation of Hspa1b, P4ha1, and Banp genes in both the hippocampus and blood of mice prenatally irradiated with 100 mGy/d. Meanwhile, downregulation of miR-448-3p and miR1298-5p in the hippocampus, miR-320-3p, miR-423-5p, miR-486b-5p, miR-486b-3p, miR-423-3p, miR-652-3p, miR-324-3p, miR-181b-5p, miR-let-7b, and miR-6904-5p in the blood was induced. The target scan revealed that Rubcnl is one of the miR-181b-5p targets in the blood. We, therefore, concluded that prenatal chronic irradiation with a low dose rate of 100 mGy/d and accumulative dose of 1.8 Gy or below might not induce significant adverse health effects on the offspring. Further study of different low dose rate radiation exposures with accumulative high doses may provide threshold doses for authorities or regulators to set new radiation safety guidelines to replace those extrapolated from acute high dose/dose rate irradiation to reduce unnecessary emergency evacuation or spending once a nuclear accident or leakage occurs.

An analysis of the effects of chronic low dose-rate radiation exposure on cancer focusing on the differences among cancer types.

PURPOSE: The effect of chronic low dose-rate radiation exposure on cancers was investigated by analyzing the data of mice experiments conducted at the Institute for Environmental Sciences (IES). This analysis focuses on the differences between malignant lymphomas and solid cancers. MATERIALS AND METHODS: The analysis is conducted based on the mathematical model introduced in our previous work. The model is expanded to analyze malignant lymphomas and solid cancers separately. Using the expanded model, the effect of chronic low dose-rate radiation on malignant lymphomas and solid cancers are discussed based on their occurrences, progressions, and mortalities. RESULTS: Non-irradiated control group and 20 mGy/day × 400 days irradiated groups are analyzed. The analysis showed that radiation exposure shortened mean life expectancy for both malignant lymphomas and solid cancers (shorter by 89.6 days for malignant lymphomas and 149.3 days for solid cancers). For malignant lymphomas, both the occurrence and the progression are affected by radiation exposure. The mean age at which malignant lymphoma developed in mice was shortened by 32.7 days and the mean progression period was shortened by 57.3 days. The occurrence of solid cancer is also affected by radiation exposure, wherein the mean age at which solid cancer develops was shortened by 147.9 days. However, no significant change in progression period of solid cancers was seen in the analysis. CONCLUSIONS: The analysis showed that the occurrence and mean lifespan are affected in both malignant lymphomas and solid cancers. The shortening of the progression period is only seen in malignant lymphoma, no significant change was observed in solid cancers.

A mathematical model for radiation-induced life-shortening attributed to cancer.

PURPOSE: In this paper, we described our mathematical model for radiation-induced life shortening in detail and applied the model to the experimental data on mice to investigate the effect of radiation on cancer-related life-shortening. MATERIALS AND METHODS: Our mathematical model incorporates the following components: (i) occurrence of cancer, (ii) progression of cancer over time, and (iii) death from cancer. We evaluated the progression of cancer over time by analyzing the cancer incidence data and cumulative mortalities data obtained from mice experiments conducted at the Institute for Environmental Sciences (IES). RESULTS: We analyzed non-irradiated control and 20 mGy/day × 400 days irradiated groups. In the analysis, all malignant neoplasms were lumped together and referred to as 'cancer'. Our analysis showed that the reduction in lifespan (104 days in median) was the result of the early onset of cancer (68 days in median) and the shortening of the cancer progression period (48 days in median). CONCLUSIONS: We described in detail our mathematical model for radiation-induced life-shortening attributed to cancer. We analyzed the mice data obtained from the experiment conducted at the IES using our model. We decomposed radiation-induced life-shortening into the early onset of cancer and the shortening of the cancer progression period.

Frequencies of Chromosome Aberrations are Lower in Splenic Lymphocytes from Mice Continuously Exposed to Very Low-Dose-Rate Gamma Rays Compared with Non-Irradiated Control Mice.

Chromosome aberrations have been one of the most sensitive and reliable biomarkers of exposure to ionizing radiation. Using the multiplex fluorescence in situ hybridization (M-FISH) technique, we compared the changes, over time, in the frequencies of translocations and of dicentric chromosomes in the splenic lymphocytes from specific pathogen-free (SPF) C3H/HeN female mice continuously exposed to 0.05 mGy/day (18.25 mGy/year) gamma rays for 125 to 700 days (total accumulated doses: 6.25-35 mGy) with age-matched non-irradiated controls. Results show that the frequencies of translocations and of dicentric chromosomes increased significantly over time in both irradiated and non-irradiated control mice, and that the frequencies were significantly lower, not higher, in the irradiated mice, which differs from our previous reports of increased chromosome aberration frequencies at higher radiation dose rates of 1 mGy/day and 20 mGy/day. These results will be useful when considering the radiation risk at very low-dose rates comparable to regulatory dose limits.

Frequencies of Chromosome Aberrations are Lower in Splenic Lymphocytes from Mice Continuously Exposed to Very Low-Dose-Rate Gamma Rays Compared with Non-Irradiated Control Mice.

Chromosome aberrations have been one of the most sensitive and reliable biomarkers of exposure to ionizing radiation. Using the multiplex fluorescence in situ hybridization (M-FISH) technique, we compared the changes, over time, in the frequencies of translocations and of dicentric chromosomes in the splenic lymphocytes from specific pathogen-free (SPF) C3H/HeN female mice continuously exposed to 0.05 mGy/day (18.25 mGy/year) gamma rays for 125 to 700 days (total accumulated doses: 6.25-35 mGy) compare with age-matched non-irradiated controls. Results show that the frequencies of translocations and of dicentric chromosomes increased significantly over time in both irradiated and non-irradiated control mice, and that the frequencies were significantly lower, not higher, in the irradiated mice, which differs from our previous reports of increased chromosome aberration frequencies at higher radiation dose rates of 1 mGy/day and 20 mGy/day. These results will be useful when considering the radiation risk at very low-dose rates comparable to regulatory dose limits.

EXPERIMENTAL STUDIES AT THE IES ON THE BIOLOGICAL EFFECTS OF CHRONIC LOW DOSE-RATE RADIATION EXPOSURE IN MICE.

Research in the Department of Radiobiology at the Institute for Environmental Sciences (IES) has focused mainly on the biological effects of long-term low dose-rate radiation exposure on mice since its establishment 30 y ago. The IES has exposed thousands of mice of various strains, to gamma-rays, mostly chronically, at low dose-rates of 0.05, 1, 20 or 100 mGy/d, at medium dose-rates of 200 or 400 mGy/d or at acute high dose-rates of 0.7-0.9 Gy/min. The dose-rate 0.05 mGy/d is comparable with the dose limit for radiation workers of 100 mSv/5 y. The results will be presented based on the parameters examined at various endpoints such as life span, neoplasm (cancer incidence), chromosome aberrations frequencies, alterations in mRNA levels, tumour transplantability and developmental abnormalities after in utero exposures. The results from research collaborations with universities and institutions both domestic (within Japan) and international will be presented. Lastly, an outline of experiments (e.g. juvenile exposure, low dose tritium exposures) and projects (e.g. radiobiology archives) currently in progress and future research perspectives will be described.

ADAPTIVE RESPONSE IN MICE CONTINUOUSLY IRRADIATED WITH LOW DOSE-RATE RADIATION.

Previous reports showed a reduction in hematopoietic death in mice exposed to a high (challenge) radiation dose if exposed two weeks prior with a relatively small (priming) radiation dose (0.3-0.5 Gy). This in vivo acquisition of radioresistance, known as "adaptive response" or the "Yonezawa effect," was shown in the experiments performed using high dose-rates (HDR) for priming. In the present study, we used low (LDR) and medium dose-rates (MDR) of radiation for priming in male C57BL mice. A total dose of 0.45-0.46 Gy (LDR, 20 mGy/day × 23 days or MDR, 18 mGy/hour × 25 hours) was used for priming, and was followed by challenge exposure 12 days later at an HDR (0.8 Gy/min) to a total dose of 6.75 Gy. Increased survival rates were observed in mice exposed to priming radiation delivered at LDR or MDR, suggesting that the adaptive responses induced are comparable with those induced at HDR.

Development of an adverse outcome pathway for radiation-induced microcephaly via expert consultation and machine learning.

BACKGROUND: Brain development during embryogenesis and in early postnatal life is particularly complex and involves the interplay of many cellular processes and molecular mechanisms, making it extremely vulnerable to exogenous insults, including ionizing radiation (IR). Microcephaly is one of the most frequent neurodevelopmental abnormalities that is characterized by small brain size, and is often associated with intellectual deficiency. Decades of research span from epidemiological data on in utero exposure of the A-bomb survivors, to studies on animal and cellular models that allowed deciphering the most prominent molecular mechanisms leading to microcephaly. The Adverse Outcome Pathway (AOP) framework is used to organize, evaluate and portray the scientific knowledge of toxicological effects spanning different biological levels of organizations, from the initial interaction with molecular targets to the occurrence of a disease or adversity. In the present study, the framework was used in an attempt to organize the current scientific knowledge on microcephaly progression in the context of ionizing radiation (IR) exposure. This work was performed by a group of experts formed during a recent workshop organized jointly by the Multidisciplinary European Low Dose Initiative (MELODI) and the European Radioecology Alliance (ALLIANCE) associations to present the AOP approach and tools. Here we report on the development of a putative AOP for congenital microcephaly resulting from IR exposure based on discussions of the working group and we emphasize the use of a novel machine-learning approach to assist in the screening of the available literature to develop AOPs. CONCLUSION: The expert consultation led to the identification of crucial biological events for the progression of microcephaly upon exposure to IR, and highlighted current knowledge gaps. The machine learning approach was successfully used to screen the existing knowledge and helped to rapidly screen the body of evidence and in particular the epidemiological data. This systematic review approach also ensured that the analysis was sufficiently comprehensive to identify the most relevant data and facilitate rapid and consistent AOP development. We anticipate that as machine learning approaches become more user-friendly through easy-to-use web interface, this would allow AOP development to become more efficient and less time consuming.

Increased Frequency of Copy Number Variations Revealed by Array Comparative Genomic Hybridization in the Offspring of Male Mice Exposed to Low Dose-Rate Ionizing Radiation.

There is very little information on the transgenerational or genetic effects of low dose-rate ionizing radiation. We report the detection of the transgenerational effects of chronic low dose-rate irradiation in mice, at the molecular level in the whole genome, using array comparative genomic hybridization technology. We observed that the number of the mice with de novo copy number variations (specifically, deletions) was significantly increased in the offspring of C57BL/6J male mice exposed to 20 mGy/day gamma-rays for 400 days (total dose: 8000 mGy), as compared to non-irradiated controls. We did not detect any difference in the size of the de novo deletions between the irradiated and the non-irradiated groups. An analysis of the life span of the offspring suggested a possibility that de novo copy-number variations may be associated with shorter life spans.