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The present work investigates the multigenerational effects of paternal pre-conceptional exposure to continuous low-dose-rate gamma rays in C56BL/6J mice. Male C57BL/6J (F0 sires) mice were exposed to low dose rates of 20, 1, and 0.05 mGy/day for 400 days, to total accumulated doses of 8,000, 400, and 20 mGy, respectively. Upon completion of the radiation exposure, the F0 male mice were immediately bred to non-irradiated 8-week-old C57BL/6J females (F0 dams) to produce the first-generation (F1) mice. Randomly selected F1 males and females were then bred to produce the second-generation (F2) mice. All the mice, except the F0 dams, were subjected to pathological examination upon natural death. Reproductive parameters, lifespan, causes of death, neoplasm incidences and non-neoplastic disease incidences were used as parameters to evaluate the biological effects of continuous pre-conceptional exposure of the sires (F0) to continuous low-dose-rate radiation. There were no significant differences in the pregnancy and weaning rates among the parent (F0) generation. Average litter size and average number of weaned pups (F1) from dams bred to males (F0) exposed to 20 mGy/day were significantly decreased compared to the non-irradiated controls. Significant lifespan shortening in the sires (F0) was observed only in the 20 mGy/day group due to early death from malignant lymphomas. Life shortening was also observed in the F1 progeny of sires (F0) exposed to 20 and 1 mGy/day, but could not be attributed to a specific cause. No significant differences in the causes of death were found between dose groups in any generation. The number of primary tumors per mouse was significantly increased only in the F0 males exposed to 20 mGy/day. Except for the increased incidence rate for Harderian gland neoplasms in sires (F0) exposed to 20 mGy/day, there was no significant difference in neoplasm incidences and tumor spectra in all 3 generations in each sex regardless of radiation exposure. No multi- or transgenerational effects in the parameters examined were observed in the F1 and F2 progeny of sires exposed to 0.05 mGy/day for 400 days., (© 2024 by Radiation Research Society. All rights of reproduction in any form reserved.)

Institute for Radiological Science (NIRS), National Institutes for Quantum Science and Technology (QST), and Institute for Environmental Sciences (IES) have conducted large-scale animal experiments for radiation risk analyses in terms of life shortening and cancer prevalence. It is important to store data and biological samples from these large-scale experiments for sharing and future use since the economic and practical limitations, as well as the ethical considerations, make it difficult. QST/NIRS established an archive called the Japan Storehouse of Animal Radiobiology Experiments (J-SHARE) for the purpose of storing and sharing these historic collections. We plan to integrate the data and tissue specimen images obtained at the IES into J-SHARE by standardizing the archive format, with the aim of maximizing the results of radiation biology research. This integration is expected to contribute to the generation of new knowledge for radiation risk assessment and the provision of scientifically based information on radiation protection., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

ApcMin/+ mouse was a model mouse for human familial adenomatous polyposis, and irradiation at an early age increases tumors in the small and large intestine. To study the effects of antioxidant administration on tumor incidence after continuous whole-body exposure to gamma rays, ApcMin/+ mice were exposed to a medium-dose-rate, 200 mGy/d, from postnatal Day 0 to 21 of age or a high-dose-rate of 0.65 Gy/min (total dose 4.2 Gy) on postnatal Day 7. The dams and pups were supplied with the N-acetylcysteine (NAC) in drinking water (7 g/L), from gestation Day 15 until weaning (21 days-old). A significant increase in the number of intestinal tumors were observed in ApcMin/+ mice irradiated with high dose-rate gamma rays as compared with the non-irradiated controls, but there was no significant difference in tumor counts between the non-irradiated controls and the medium-dose rate irradiation groups. NAC administration did not have any significant effect at least at this dose. These results suggest that the supplementation of anti-oxidant at the early stage of tumorigenesis does not suppress the formation of irradiation-induced small intestinal tumors., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The present work investigates the long-term effects of continuous low dose-rate (20 mGy/day to total doses of 1-8 Gy) gamma-ray exposure on the hematopoietic cells of specific pathogen-free C3H/HeN mice. Peripheral white blood cell (WBC) counts decreased on days 206, 471, and 486, with no significant changes in red blood cell (RBC) and platelet (PLT) counts. The number of colony forming units (CFU-S and CFU-GM) in the bone marrow and spleen from irradiated mice decreased with increasing total dose on day-12 and day-7. The decrease in bone marrow CFU-S persisted throughout the 400-day irradiation period and did not show any recovery up to 210 days postirradiation. These findings suggest that the effects of low-dose-rate (LDR) radiation on the hematopoietic system remain long after the 400-day irradiation was completed. Further investigation showed no significant difference in life spans of non-irradiated W/Wv (c-kit-deficient) mice inoculated with hematopoietic stem cells from irradiated (20 mGy/day for 400 days) or nonirradiated wild-type mice. These results suggest that the effects of continuous low-dose-rate irradiation are more pronounced in hematopoietic stromal cells than in HSCs themselves., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (©2022 by Radiation Research Society. All rights of reproduction in any form reserved.)

Previously, we reported that while low-dose-rate (LDR) gamma-ray exposure to 20 mGy/day for the entire gestation period (gestation days 0-18) did not result in any significant effect in B6C3F1 pups up to 10 weeks of age when compared to the non-irradiated controls, exposure to medium-dose-rates (MDR, 200 and 400 mGy/day) resulted in growth retardation and gonadal hypoplasia, in addition to delayed ossification (only at 400 mGy/day). In the present work, we investigated the late effects of continuous in utero exposure to gamma rays at LDRs (0.05, 1.0 and 20 mGy/day) and at an MDR of 400 mGy/day, on life span, causes of death, neoplastic and non-neoplastic disease incidences in B6C3F1 mice. Reproductive parameters such as litter size and weaning rates was not significantly different among the LDR groups, but was significantly decreased in the MDR group, when compared to the non-irradiated controls. Mean life spans were not significantly different among the LDR exposed groups compared to the non-irradiated controls, whereas the life spans of those exposed to the MDR were significantly shorter than the non-irradiated controls. There was no significant difference in tumor spectra between the non-irradiated and LDR nor MDR irradiated groups. In mice exposed to MDR in utero, the over-all incidence rates shifted with increased incidences in the number of neoplasms of liver (both sexes) and endocrine (adrenals, pituitary and ovaries in females) origin with corresponding decreases in the incidence of malignant lymphomas (both sexes) and lung neoplasms (males). Multiple primary neoplasms were significantly increased only in females exposed to MDR. Results show that B6C3F1 mice exposed to gamma-rays in utero at LDRs of 0.05, 1 and 20 mGy/day for the entire gestation period (18 days) does not significantly alter lifespan, cause of death, neoplasm incidence rates and tumor spectra., (©2022 by Radiation Research Society. All rights of reproduction in any form reserved.)

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., (©2022 by Radiation Research Society.)

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., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Life span shortening and increased incidences of cancer and non-cancer diseases were observed in B6C3F1 mice irradiated with gamma-rays at a low dose-rate (LDR) of 20 mGy/d for 400 d. A genome-wide gene expression profiling of livers from mice irradiated at a LDR (20 mGy/d, 100-400 d) was performed. LDR radiation affected specific pathways such as those related to lipid metabolism, e.g. 'Cholesterol biosynthesis' and 'Adipogenesis' in females irradiated for 200 and 300 d at 20 mGy/d, with increased expression of genes encoding cholesterol biosynthesis enzymes (Cyp51, Sqle, Fdps) as age and radiation dose increased. No significant alterations in the expression of these genes were observed in male mice exposed similarly. However, the genes encoding adipogenesis regulators, Srebf1 and Pparg, increased with age and radiation dose in both sexes. Comparison between LDR-irradiated and medium dose-rate (400 mGy/d) male mice revealed quite different gene expression profiles. These results seem to be consistent with the increased incidence of fatty liver and obesity in female mice exposed to LDR radiation and suggest that metabolism is an important target of LDR radiation., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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.

The effects of radiation exposure on germ cells and the gonads have been well studied at acute high-dose exposures, but the effects of chronic low-dose-rate (LDR) irradiation, particularly relevant for radiation protection, on germ cells and the gonads are largely unknown. Our previous study revealed that chronic exposure of mice to medium-dose-rate (MDR, 200 or 400 mGy/day) gamma-rays in utero for the entire gestation period (18 days) induced only a mild degree of general growth retardation, but with very drastic effects on the gonads and germ cells. In the current study, we further investigated the histomorphological changes in the gonads and the number of germ cells from gestation day (GD) 18 fetuses irradiated with MDR throughout the entire gestation period. The germ cells in the testes and ovaries of the MDR-irradiated fetuses were almost obliterated. Gestation day 18 fetuses exposed to LDR (20 mGy/day) radiation for the entire gestation period showed decreases in the number of the germ cells, which were not statistically significant or only marginally significant at most. Further investigations on the effects of LDR irradiation in utero using more sensitive methods are necessary., (©2021 by Radiation Research Society. All rights of reproduction in any form reserved.)

Studies of environmental enrichment are progressing in the fields of nervous system, stress and exercise. Recently, housing in enriched environment have shown to influence to carcinogenesis and life span. However, the study for antitumor effect of environmental enrichment are difficult to reproduce due to the complexity of the experimental technique. Thus, a simpler experiment system is needed for antitumor study using environmental enrichment. In this research, we propose a minimum environmental enrichment, which is an experimental system by placing one mouse igloo which is normally used as a mouse shelter in the rearing environment. The experimental system of minimum environmental enrichment is not only easy to reproduce but also have enhanced activity to suppress the growth of transplanted tumor significantly. It was found that the activation of NK cells is involved also in the immune system related to tumor immunity of minimum environmental enrichment. Because minimum environmental enrichment is effective in activating antitumor immunity to transplanted tumor cells in mice, we believe this will be useful for promoting antitumor studies using environmental enrichment.

Calorie restriction is known to influence several physiological processes and to alleviate the late effects of radiation exposure such as neoplasm induction and life shortening. However, earlier related studies were limited to acute radiation exposure. Therefore, in this study we examined the influence of chronic low-dose-rate irradiation on lifespan. Young male B6C3F1/Jcl mice were divided randomly into two groups, which were fed either a low-calorie (65 kcal/ week) or high-calorie (95 kcal/week) diet. The latter is comparable to ad libitum feeding. The animals in the irradiated group were continuously exposed to gamma rays for 400 days at 20 mGy/day, resulting in a total dose of 8 Gy. Exposure and calorie restriction were initiated at 8 weeks of age and the diets were maintained for life. The life-shortening effects from chronic whole-body irradiation were compared between the groups. Body weights were reduced in calorie-restricted mice irrespective of radiation treatment. Radiation induced a shortened median lifespan in both groups, but to a greater extent in the calorie-restricted mice. These results suggest that calorie restriction may sensitize mice to chronic low-dose-rate radiation exposure to produce a life-shortening effect rather than alleviating the effects of radiation.

Purpose: Many studies are focusing on the biological effects of gamma irradiation at low-dose rates. Studies have shown that chronic exposure to gamma irradiation at low-dose rates shortened the lifespan of mice due to neoplasm formation. The aim of this study was to clarify the physiological effects of long-term exposure to gamma irradiation at low-dose rates in mice, measured with noninvasive parameters such as blood pressure., Materials and Methods: Specific-pathogen-free female B6C3F 1 mice were irradiated with gamma rays at a low dose of 20 mGy/day - a dose rate shown to shorten the life span in previous studies. The blood pressure parameters (systolic, diastolic, and mean blood pressure), heart rate, tail blood volume, and blood flow of the mice were measured every 7 weeks. Age-matched, non-irradiated mice were used as controls., Results and Conclusion: The blood pressure levels of the irradiated mice decreased at an earlier age compared to the non-irradiated control mice. The expression levels of the marker genes of aging that are also associated with regulation of blood pressure showed significant differences between non-irradiated and irradiated mice. These results indicated that long-term exposure to gamma irradiation at low-dose rates induce the expression levels of Rap1a and reduces Panx1 and Sirt3, which may have contributed to the accelerated blood pressure decline in female mice.

This review summarizes the results of experiments conducted in the Institute for Environmental Sciences for the past 21 years, focusing on the biological effects of long-term low dose-rate radiation exposure on mice. Mice were chronically exposed to gamma rays at dose-rates of 0.05, 1 or 20 mGy/day for 400 days to total doses of 20, 400 or 8000 mGy, respectively. The dose rate 0.05 mGy/day is comparable to the dose limit for radiation workers. The parameters examined were lifespan, neoplasm incidence, antineoplasm immunity, body weight, chromosome aberration(s), gene mutation(s), alterations in mRNA and protein levels and trans-generational effects. At 20 mGy/day, all biological endpoints were significantly altered except neoplasm incidence in the offspring of exposed males. Slight but statistically significant changes in lifespan, neoplasm incidences, chromosome abnormalities and gene expressions were observed at 1 mGy/day. Except for transient alterations in the mRNA levels of some genes and increased liver neoplasm incidence attributed to radiation exposure, the remaining biological endpoints were not influenced after exposure to 0.05 mGy/day. Results suggest that chronic low dose-rate exposure may induce small biological effects.

Purpose: Chronic low-dose-rate (20 mGy/day) γ-irradiation increases the incidence of hepatocellular adenomas (HCA) in female B6C3F1 mice. The purpose of this study is to identify potential serum biomarkers for these HCAs by a new approach., Material and Methods: Microarray analysis were performed to compare the gene expression profiles of HCAs from mice exposed to low-dose-rate γ-rays with those of normal livers from non-irradiated mice. From the differentially expressed genes, those for possibly secretory proteins were selected. Then, the levels of the proteins in sera were analysed by ELISA., Results: Microarray analysis identified 4181 genes differentially expressed in HCAs (>2.0-fold). From these genes, those for α-fetoprotein (Afp), α-1B-glycoprotein (A1bg) and serine peptidase inhibitor Kazal type-3 (Spink3) were selected as the genes for candidate proteins. ELISA revealed that the levels of Afp and A1bg proteins in sera significantly increased and decreased, respectively, in low-dose-rate irradiated mice with HCAs and also same tendency was observed in human patients with hepatocellular carcinomas., Conclusion: These results indicate that A1bg could be a new serum biomarker for liver tumor. This new approach of using microarray to select genes for secretory proteins is useful for prediction of novel tumor markers in sera.

Pre- and postnatal male mice were acutely (659-690 mGy/min) and continuously (0.303 mGy/min) exposed to 2 Gy γ-rays to evaluate spermatogenic potential and chromosome damage in their germ cells as adults. Acute irradiation on Days 15.5, 16.5, and 17.5 post-coitus affected testicular development, as a result of massive quiescent gonocyte loss; the majority of the seminiferous tubules in these testes were devoid of germ cells. Acute irradiation on Days 18.5 and 19.5 post-coitus had less effect on testicular development and spermatogenesis, even though germ cells were quiescent gonocytes on these days. Adverse effects on testicular development and spermatogenesis were observed following continuous irradiation between Days 14.5 and 19.5 post-coitus. Exposure to acute and continuous postnatal irradiation after the differentiation of spermatogonial stem cells and spermatogonia resulted in nearly all of the seminiferous tubules exhibiting spermatogenesis. Neither acute nor continuous irradiation was responsible for the increased number of multivalent chromosomes in primary-spermatocyte descendents of the exposed gonocytes. In contrast, a significant increase in cells with multivalent chromosomes was observed following acute irradiation on Days 4 and 11 post-partum. No significant increases in unstable structural chromosomal aberrations or aneuploidy in spermatozoa were observed, regardless of cell stage at irradiation or the radiation dose-rate. Thus, murine germ cells that survive prenatal and postnatal irradiation can restore spermatogenesis and produce viable spermatozoa without chromosome damage. These findings may provide a better understanding of reproductive potential following accidental, environmental, or therapeutic irradiation during the prenatal and postnatal periods in humans., (© 2017 Wiley Periodicals, Inc.)

The Xist (X inactive specific transcript) gene plays an essential role in X chromosome inactivation. To elucidate the mechanisms controlling Xist expression and X inactivation, we examined in vivo DNA-protein interactions in the Xist promoter region in a female mouse cell line (BMSL2), which has distinguishable Xist alleles. In vivo footprinting was accomplished by treatment of cells with dimethyl sulfate or ultraviolet light, followed by ligation-mediated polymerase chain reaction of purified DNA. The expressed allele on the inactive X chromosome and the silent allele on the active X chromosome were separated by the use of a restriction fragment length polymorphism prior to ligation-mediated polymerase chain reaction. The chromatin structure of the Xist promoter was found to be consistent with the activity state of the Xist gene. The silent allele (on the active X chromosome) showed no footprints, while the expressed allele (on the inactive X chromosome) showed footprints at a consensus sequence for a CCAAT box, two weak Sp1 sites, and a weak TATA box.

A major role of DNA-methyltransferase (MTase) is thought to be maintaining the DNA methylation profile through DNA replication. However, previous surveys of mRNA distribution in different tissues by Northern-blot analysis have shown unexpectedly high levels of expression of DNA-MTase mRNA in adult mouse brain, which consists mostly of slowly proliferating glial and nonproliferating neuronal cells. In order to identify cells expressing the gene in the brain, we performed an in situ hybridization analysis of mature brain as well as whole embryos of different ages. In addition to various embryonic tissues with active cell proliferation such as the ventricular neurogenic layer, hair follicle epithelia, thymus and epithelia of the base of intestinal villi, almost all mature neurons in brain of adult and even aged mice expressed DNA-MTase mRNA at substantial levels. No significant expression of the gene was detected in the white matter. These findings suggest some neuron-specific biological function of DNA methylation, unrelated to DNA replication.