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1 International Journal of Radiation Biology ISSN: (Print) (Online) Journal homepage: Experimental studies on the biological effects of chronic low dose-rate radiation exposure in mice: overview of the studies at the Institute for Environmental Sciences Ignacia Braga-Tanaka III, Satoshi Tanaka, Atsushi Kohda, Daisaku Takai, Shingo Nakamura, Tetsuya Ono, Kimio Tanaka & Jun-ichiro Komura To cite this article: Ignacia Braga-Tanaka III, Satoshi Tanaka, Atsushi Kohda, Daisaku Takai, Shingo Nakamura, Tetsuya Ono, Kimio Tanaka & Jun-ichiro Komura (2018) Experimental studies on the biological effects of chronic low dose-rate radiation exposure in mice: overview of the studies at the Institute for Environmental Sciences, International Journal of Radiation Biology, 94:5, , DOI: / To link to this article: Accepted author version posted online: 13 Mar Published online: 03 Apr Submit your article to this journal Article views: 732 View Crossmark data Citing articles: 3 View citing articles Full Terms & Conditions of access and use can be found at

2 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY, 2018 VOL. 94, NO. 5, REVIEW Experimental studies on the biological effects of chronic low dose-rate radiation exposure in mice: overview of the studies at the Institute for Environmental Sciences Ignacia Braga-Tanaka III, Satoshi Tanaka, Atsushi Kohda, Daisaku Takai, Shingo Nakamura, Tetsuya Ono, Kimio Tanaka and Jun-ichiro Komura Department of Radiobiology, Institute for Environmental Sciences, Rokkasho-mura Kamikita-gun, Aomori-ken, Japan ABSTRACT 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. ARTICLE HISTORY Received 23 October 2017 Revised 29 January 2018 Accepted 31 January 2018 KEYWORDS Chronic exposure; low doserate radiation; mice; biological effects Introduction Humans are exposed to low-dose ionizing radiation daily through various routes such as medical diagnostic radiation (X-rays, CT scans) and occupational exposure (medical doctors, radiation technicians and workers, space station astronauts) in addition to background radiation (e.g. cosmic rays, food) (Table 1). The effects of these low dose and low doserate radiation exposures on health are not well understood and is currently estimated by extrapolating the effects of high-dose exposures based on the linear no-threshold (LNT) model (Brenner 2003; ICRP 2007, 2005; Dauer et al. 2010; Shah et al. 2012; R uhm et al. 2015). The two nuclear power plant accidents in Chernobyl in 1986 and Fukushima in 2011 attracted much attention and has increased the need for accurate assessment of health risks due to low dose radiation exposure rather than an estimation. Current knowledge on the effects of low-dose radiation exposure are based on epidemiological studies on human populations exposed to low doses and on experimental studies conducted on animals, primarily rodents. The results of these epidemiological studies however are of limited value particularly when applied or extrapolated to dose levels in relevant populations. Experiments at low dose-rates would require large numbers of animals to maintain statistical precision and power (Brenner et al. 2003), or, highly sensitive methods to detect slight changes. To study the effects of chronic low dose-rate exposures, a facility capable of long-term exposure is essential. Although there are several institutions outside of Japan that maintain low dose-rate exposure animal facilities (Figure 1), the lowest dose-rate available is 0.12 mgy/day (MICADO, National Institute for Radiological Protection and Nuclear Safety, France) and exposure levels equivalent to annual dose limit for radiation workers of 20 msv/yr are not available. In 1990, the Institute for Environmental Sciences (IES) was established for the purpose of investigating the effects of low dose-rate radiation exposure on health and the environment. Five years later, the animal facility was built for long-term low dose-rate radiation exposure of mice under specific pathogenfree (SPF) conditions. The dose rates were set at 0.05 mgy/day and 1 mgy/day using Cs-137 as a gamma-ray source and the duration of exposure was set at 400 consecutive days. The 0.05 mgy/day dose rate are comparable to the annual dose limit for radiation workers. An additional dose-rate of 20 mgy/ day was used as a positive control. The facility was described in detail previously by Tanaka et al. (2003). Figure 2 shows the setup in one of the irradiation rooms in the facility. Experiments Mice Depending on the biological endpoint examined, the different strains of mice used, such as B6C3F1, C3H, C57Bl/6 and CONTACT Ignacia Braga-Tanaka III tanakaib@ies.or.jp Institute for Environmental Sciences, 1-7, Ienomae, Obuchi, Rokkasho-mura, Kamikita-gun, Aomori , Japan Color versions of one or more of the figures in the article can be found online at ß 2018 Aomori Prefectural Government

3 424 I. BRAGA-TANAKA III ET AL. transgenic (gpt delta SWR) F1, and the number of animals used in each study is summarized in Table 2. All mice were kept under SPF conditions in a controlled environment, as described previously (Tanaka et al. 2003), for the entire duration of the experiment. Radiation exposure The mice were housed in plastic cages, 4 or 5 to a cage, placed on shelves arranged at a pre-determined distance around the radiation source in each irradiation room. The mice were exposed to gamma-rays for 22 hours every day from 12:00 h (noon) to 10:00 h the following day and the remaining 2 hours (10:00 12:00) were used for health monitoring of the mice and husbandry procedures (Tanaka et al. 2003). The dose rates ranged from 0.04 to 0.06 mgy/day, mgy/day and mgy/day depending on the time period the experiment was performed because of the decay of the radiation sources (Cs-137). The total irradiation periods were adjusted and ranged from 400 to 485 days resulting to total accumulated doses ranging from 20 to 21, and mgy. For the purpose of this review, the dose rates, duration of exposure and the total doses are uniformly expressed as 0.05, 1, 20 mgy/day; 400 days; and, 20, 400, 8000 mgy, respectively. Biological endpoints The biological effects of the chronic low dose-rate exposure were evaluated using 10 different endpoints lifespan, neoplasm incidence, body weight, tumor cell transplantability, changes in chromosome structure, gene mutations, changes in mrna and protein levels, and transgenerational effects on lifespan and neoplasm incidence. Statistical analyses were performed for each endpoint and p <.05 was set as significant level. For neoplasm incidence, p <.01 was applied. Table 1. Examples of low-dose radiation exposure in humans. Type of exposure Dose/Dose rate Reference Acute Medical diagnostics msv ICRP (2005) Chronic Dose limit for radiation workers 20 msv/yr ICRP (2007) Dose limit for public 1 msv/yr ICRP (2007) Background radiation 1 10 msv/yr ICRP (2007) Results Lifespan Lifespan shortening is one of the most sensitive endpoints attributable to radiation exposure. B6C3F1 mice (500 per sex/ group; total n ¼ 4000) (Table 2) were exposed to gamma-rays at dose-rates of 0.05, 1 and 20 mgy/day for 400 days from FIGARO, Norwegian University of Life Sciences, Norway Central Research Institute of Electric Power Industry (CRIEPI), Japan Belgian Nuclear Research Centre (SCK-CEN), Belgium Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan National Institute of Radiological Sciences (NIRS), Japan Canadian Nuclear Laboratories (CNL), Canada Radiation Biology Center, Kyoto University, Japan MICADO, National Institute for Radiological Protection and Nuclear Safety, France Institute for Environmental Sciences (IES), Japan 0.05 mgy/d 1 mgy/d 20 mgy/d 200, 400 mgy/d 890 mgy/min Evacuation order (Fukushima); Occupational exposure dose limit 20 msv/yr Natural background radiation (world average) 2.4 msv/yr General public dose limit 1 msv/yr , ,000 Dose Rate (mgy/h) Figure 1. Dose rates in irradiation facilities for animal experiments and comparison with human exposure scenarios. References: FIGARO, Norwegian University of Life Sciences, Norway ( Belgian Nuclear Research Centre (SCK-CEN) ( Central Research Institute of Electric Power Industry (CRIEPI)( Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan ( National Institute of Radiological Sciences (NIRS)(personal communication with Dr. Tatsuhiko Imaoka); Canadian Nuclear Laboratories (CNL) (personal communication with Dr. Dmitri Klokov); Radiation Biology Center, Kyoto University ( MICADO, National Institute for radiological Protection and Nuclear Safety, France ( Scientific-tools/experimental-facilities-means/MICADO-Lab/Pages/MICADO-Lab-experimental-platform.aspx).

4 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 425 Figure 2. One of the irradiation rooms at IES with the Cs-137 gamma-ray source located at the center of the room. Table 2. Summary of the biological effects observed in mice exposed chronically to low dose-rate radiation. 20 mgy 400 mgy 8000 mgy Mouse Strain (0.05 mgy/day) (1 mgy/day) (20 mgy/day) (n) Publication Somatic effect Life span Shortened in female Shortened B6C3F1 (500/sex/dose group) Tanaka et al. (2003) Neoplasm incidence Increased in male Increased Increased B6C3F1 (500/sex/dose group) Tanaka et al. (2007) Tumor latency NA NA Shortened B6C3F1, females (60-90/sacrifice Tanaka et al. (2017) point/dose group) Anti-tumor immunity Decreased B6C3F1, females (n ¼ 13 40/ Takai et al. (2011, 2015) dose group) Body weight Increased in male Increased in female B6C3F1 (500/sex/dose group) Tanaka et al. (2007) Body weight NA NA Increased in female B6C3F1, females (29/dose group) Nakamura et al. (2010) Chromosome aberration Increased Increased C3H/HeN, females (3 13/age group/ dose group) Tanaka et al. (2009, 2014) Gene mutation Increased (gpt delta x SWR) F1 (3 11/dose group) Ono et al. (2009), Okudaira et al. (2010) Change in mrna level Altered Altered Altered C57BL/6J, males (3/dose group) Taki et al. (2009), Uehara et al. (2010) Change in protein level Increased Altered C57BL/6J, males (3/dose group) Nakajima et al. (2008, 2017) Transgenerational effect Life span Shortened in F1 male C57BL/6J /sex/dose Neoplasm incidence (in progress) (in progress) (in progress) (unpublished) : no significant alteration. NA: not applicable. same study (data set). 8 weeks of age. After completion of radiation exposure, the mice were moved to the animal rooms and kept until they died a natural death. Nonirradiated control mice (from the same batches as the irradiated groups) were kept in the animal rooms. The average lifespan was reduced in male and female mice exposed to 20 mgy/day and in females exposed to 1 mgy/day. Gamma ray exposure to 0.05 mgy/day did not affect the lifespan in both sexes (Table 2). No adaptive response, such as hormesis (e.g. lengthening of lifespan), was observed in all the dose rates examined (Tanaka et al. 2003). The amount of life shortening observed in mice exposed to 20 mgy/day was approximately 100 days in males and 120 days in females, corresponding to an 11% and 14% decrease, respectively, compared to the nonirradiated control mice. Based on the total dose of 8 Gy, calculated life shortening per Gy is equal to 1.4% and 1.7%, in males and females, respectively.

5 426 I. BRAGA-TANAKA III ET AL. Table 3. Neoplasms in B6C3F1 mice with changes in incidence rates (%) after exposure to chronic low dose-rate gamma rays for 400 days (Tanaka et al. 2007). MALES FEMALES NEOPLASM Non-irrad 0.05 mgy/day 1 mgy/day 20 mgy/day (Fold Inc) Nonirrad 0.05 mgy/day 1 mgy/day 20 mgy/day (Fold Inc) Harderian gland Adenoma (2.9) (5.2) Adenocarcinoma (2.4) (2.6) Liver Adenoma, Hepatocellular (1.5) (2.2) Hepatoblastoma Hematopoietic System Leukemia, Myeloid (3.4) Respiratory System Adenoma, Bronchiolo-alveolar (2.1) Carcinoma, Bronchiolo-alveolar (1.1) (2.9) Circulatory System Hemangiosarcoma (1.4) (2.2) Ovary Adenoma, Tubulostromal (37.5) Adenocarcinoma, Tubulostromal Granulosa cell tumor, Benign (11.8) Granulosa cell tumor, Malignant (29.0) Endocrine System Adrenal, Adenoma, subcapsular cell (9.7) Pituitary, Adenoma, pars distalis (0.6) Thyroid, Adenoma, follicular cell (0.2) Fold Inc: the ratio of neoplasm incidence rate in mice exposed to 20 mgy/day over the non-irradiated controls. This was calculated only when the change in the incidence rate for the neoplasm is statistically significant in the 20 mgy/day group. p <.01 Fisher s Exact Test. p <.01 Peto s Trend Test. Neoplasm incidence Lifespan study All mice in the lifespan study underwent gross and microscopic pathological examination to determine cause of death and identify neoplastic and non-neoplastic lesions (Tanaka et al. 2003, 2007). Neoplasms were the main cause of death in both nonirradiated control and irradiated mice and were found in over 90% of the mice in the study (Tanaka et al. 2007) and there was no difference in the tumor spectra between the nonirradiated and irradiated mice. The most common neoplasms that caused death and contributed to life shortening in the 20 mgy/day group were malignant lymphoma (both sexes), lung neoplasms (males), hemangiosarcomas (males) and soft tissue neoplasms (females). Other neoplasms with significantly increased incidences at 20 mgy/ day were hepatocellular adenomas and Harderian gland neoplasms in both sexes, in addition to myeloid leukemia in males, and hemangiosarcoma, ovarian, lung, adrenal and renal tumors in females. Analysis of the temporal distribution of malignant lymphoma showed that exposure to 20 mgy/ day caused early death by approximately 100 days in both sexes suggesting that the radiation exposure at this dose rate for 400 days either causes a shortening of latent period or increased proliferation of neoplastic lymphoma cells. The decrease in the frequency of hepatocellular carcinomas as cause of death in males was attributed to competing risk. The cause of death could not be determined in up to 1.4% of mice. Non-neoplastic diseases such as interstitial pneumonia, hyaline glomerulopathy, hemorrhagic enteritis, renal amyloidosis and septicemia, caused 8 13% of the overall deaths with no significant difference in incidence rates among the nonirradiated control and irradiated groups. Table 3 shows the influence of chronic low dose-rate gamma ray exposure on the incidence rates of neoplasms, lethal and nonlethal, compared to the nonirradiated controls. Increased incidence rates for Harderian gland neoplasms [adenoma (2.9-fold increase in males; 5.2-fold increase in females) and adenocarcinoma (2.4-fold increase in males; 2.6- fold increase in females)] in mice exposed to 20 mgy/day were attributed to radiation exposure (p <.01, Peto s trend test). Hepatocellular adenoma incidence rates in male mice were significantly increased at all dose-rates (1.5-fold increase at 20 mgy/day) and is the only neoplasm that was significantly increased at 0.05 mgy/day. From the 20 mgy/day group, only females showed significantly increased hepatocellular adenoma incidence rate (2.2-fold increase) (p <.01). Although the incidence rates for hepatocellular adenoma does not appear to increase with dose in the males, significant Peto s trend tests (p <.01) in both sexes indicate that the increases are radiation dose-related. Despite very low incidence rates, significant increases in hepatoblastoma (1 mgy/day) and myeloid leukemia (20 mgy/day) in males were attributed to radiation exposure and were radiation dose-dependent (p <.01 Peto s trend test). Incidence rate for hemangiosarcoma was significantly increased (2.2-fold) only in females, although trend tests show that the increased incidence were radiation dose-dependent in both sexes (p <.01, Peto s trend test). Bronchiolo-alveolar neoplasms of the lungs, adenoma (2.1-fold) and carcinoma (2.9-fold), were significantly increased only in females and were radiation dose dependent for adenomas in females and carcinomas in both sexes. Other neoplasms with significantly increased incidence

6 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 427 Table 4. Latency periods and changes in incidence rates of non-neoplastic and neoplastic lesions in female B6C3F1 mice exposed to 20 mgy/day gamma rays (Tanaka et al. 2017). Latency (Sacrifice day) Lesion/Neoplasm Non-irradiated 20 mgy/day Period when Increased Incidence Rate (%) was observed in 20 mgy/day group Adrenal Hyperplasia, Subcapsular cell 100 d 100 d from 200 d to 700 d Adenoma, Subcapsular cell 700 d 400 d from 500 d to 700 d Harderian gland Adenoma 400 d 300 d from 400 d to 700 d Adenocarcinoma 600 d none Liver Degeneration, Fatty 300 d 200 d from 300 d to 600 d Adenoma, Hepatocellular 500 d 400 d from 500 d to 700 d Carcinoma, Hepatocellular 300 d from 500 d to 700 d Lung Adenoma, Bronchiolo-alveolar 100 d 100 d 600 d Carcinoma, Bronchiolo-alveolar 600 d 400 d 700 d Ovary Atrophy 500 d 100 d from 100 d to 600 d Hyperplasia, Tubulostromal 700 d 300 d from 300 d to 500 d Adenoma, Tubulostromal 600 d 400 d from 400 d to 700 d Benign, Granulosa cell tumor 200 d from 600 d to 700 d Malignant, Granulosa cell tumor 500 d 700 d : not observed. rates in irradiated females that were dose dependent were ovarian neoplasms ( fold) and subcapsular cell adenomas of the adrenal (9.7-fold), suggesting that female hormones play a role as well. The decreasing incidence rates for adenoma, pars distalis of the pituitary gland in irradiated females, were attributed to competing risks (not significant Peto s trend test). The decrease in the incidence of follicular cell adenomas of the thyroid (p <.01 Peto s trend test) in relation to dose was attributed to radiation exposure but could not be considered hormesis since there was no corresponding increase in the life span. The results of the lifespan study show that gamma ray exposure for 400 days causes lifespan shortening (both sexes at 20 mgy/day; females at 1 mgy/day) and increased incidence rates of some neoplasms but with no apparent change in the tumor spectrum. Further investigation is needed to clarify the increase in incidence of hepatocellular adenoma in males exposed to the dose rates studied particularly 0.05 mgy/day. Tumor latency Our next concern was the effect of chronic low dose-rate radiation exposure on tumor latency. Using the same exposure protocol as the lifespan study, female B6C3F1 mice exposed to 20 mgy/day of gamma rays for 400 days (Table 2) and were serially sacrificed every 100 days from the start of irradiation exposure, until day 700 (300 days after the completion of the 400-day radiation exposure) (Tanaka et al. 2017). Based on shortened latencies and their corresponding increases in incidence rates (summarized in Table 4), the study shows that the neoplastic and non-neoplastic effects of chronic low dose-rate radiation exposure vary depending on the organ(s) affected. Non-neoplastic lesions that exhibited shortened latencies in irradiated mice were fatty degeneration in the liver (200 d), and, atrophy (100 d) and tubulostromal hyperplasia (300 d) of the ovaries. Changes observed in the ovary indicate that it is sensitive to low dose-rate chronic radiation exposure and manifests as ovarian atrophy in 20% of the mice as early as 100 days (total dose ¼ 2 Gy) after the start of radiation, and as tubulostromal hyperplasia from 300 d (total dose ¼ 6 Gy). Neoplasms with shortened latencies after radiation exposure were hepatocellular adenoma, subcapsular cell adenoma of the adrenal, tubulostromal adenoma of the ovaries and adenomas of the Harderian gland. Although adrenal tumorigenesis postgonadectomy is a well-established phenomenon in certain in-bred strains of mice (Bernichtein et al. 2008), the relationship between ovarian dysfunction (atrophy and tubulostromal hyperplasia) and the development of subcapsular hyperplasia/adenomas in the adrenal gland in B6C3F1 females exposed to 20 mgy/day gamma rays was not clear since not all mice with ovarian dysfunction presented with subcapsular cell hyperplasia/adenoma and vice versa. The shortened latencies and increased incidence rates of bronchiolo-alveolar adenomas of the lungs and adenomas of the Harderian gland indicate sensitivity to dose rates of 20 mgy/day. Compared to the nonirradiated controls, incidence rates for both neoplastic and non-neoplastic lesions increased as the irradiated mice aged. Although increased incidence rates for malignant lymphoma in irradiated mice were observed at a certain sacrifice point (600 d), the total incidence rate was not significantly different as compared to the nonirradiated mice and there was no shortening of the latency. Anti-tumor immunity Anti-tumor immunity was examined in female B6C3F1 mice exposed to a low dose-rate of gamma-rays by subcutaneously injecting cell suspensions of an ovarian granulosa cell tumor line, OV3121 (Yanagihara et al. 1995) and determining its transplantability (Table 2) (Takai et al. 2011). Tumor

7 428 I. BRAGA-TANAKA III ET AL. transplantability did not significantly differ among the nonirradiated control and in mice exposed to 0.05 nor 1 mgy/day for 400 days but was increased in mice exposed to 20 mgy/ day for 400 days, suggesting a weakened anti-tumor immunity (Takai et al. 2015). Body weight During the entire conduct of the experiments, body weights were monitored every 4 weeks and was found to significantly increase with chronic low dose-rate gamma-ray exposure. In the life span study (Table 2), statistically significant increases (p <.05) in body weights were observed in males exposed to 1 mgy/day and in females exposed to 20 mgy/day (Tanaka et al. 2007). Further studies on female B6C3F1 mice (Table 2) exposed to 20 mgy/day showed that the increase in body weights were due to adiposity with increased serum leptin and lipid content in both the liver and serum (Nakamura et al. 2010). Female adiposity when analyzed together with the results of the tumor latency experiment where ovarian atrophy and tubulostromal hyperplasia were observed prematurely in mice exposed to 20 mgy/day at 100 days, suggests that the pathogenesis may be related to ovarian dysfunction due to changes in female sex hormones, as seen in menopause (Sorensen et al. 2001). The cause of body weight gain in male mice exposed chronically to low dose-rates of gamma-rays require further investigation. Chromosome aberration Chromosome aberrations have been used as a sensitive biomarker for monitoring radiation exposure and the two most common types found are translocations and dicentric or ring chromosomes. Most, if not all translocations are stable and remain for a long time, whereas dicentric or ring chromosomes are unstable and disappear with time (Spruill et al. 1996; Tucker 2008). We have analyzed these two types of aberrations in splenic cells of female C3H/HeN mice (Table 2) after exposure to three different dose rates by Giemsa staining, centromere FISH and mfish (multicolor Fluorescence In Situ Hybridization). Tanaka et al. (2009) found significant increases of both translocation and dicentric plus ring aberrations after exposures to total doses of 400 and 8,000 mgy. The number of translocations induced per unit dose of radiation (efficiency) appeared similar in both dose-rates at 1 (total dose ¼ 400 mgy) and 20 mgy/day (total dose ¼ 8000 mgy), suggesting a dose rate independency (Tanaka et al. 2014). Dose response at high dose-rate exposure (890 mgy/min) showed more efficient induction of both types of aberrations (Tanaka et al. 2009, 2013). So far, we have not detected any increase in chromosome aberrations after the chronic exposure to a total dose of 20 mgy (Kohda et al. unpublished results). Data suggest that it is possible to monitor chronic low dose-rate exposure using chromosomal abnormalities. Gene mutation High doses of x-rays have been reported to induce mainly deletion type mutations in lacz transgenic mice (Ono et al. 1999, Nakamura et al. 2000). In 2010, Okudaira et al. examined mutation induction in the spleen and liver of (gpt-delta (Nohmi et al. 1999)SWR (Shaver-Walker et al. 1995)) F1 mice exposed gamma rays to total doses of 4 and 8 Gy at three different dose-rates: 920 mgy/min, 1 mgy/min and 12.5 lgy/min (approximately 20 mgy/day). The gpt-delta mice harbor lambda EG10 genomic DNA in chromosome 17 containing both red and gam genes in tandem allowing measurement of deletion type mutations that inactivate the two genes at a time, and was thought appropriate for detecting radiation-induced mutations more efficiently. Although a linear dose-dependent increase in mutation frequency was observed in the spleen and liver of (gpt-delta SWR) F1 mice after acute high dose-rate exposure (920 mgy/min), higher frequencies were observed in the spleen (Okudaira et al. 2010). While decreasing the dose rate also reduced the induction rate of mutations in both organs, mutation frequencies in liver of (gpt-delta SWR) F1 mice exposed to 1 mgy/min (medium dose-rate) and 12.5 lgy/min (low doserate) were similar, an effect also observed by Russell and Kelly (1982a, b) in spermatogonia. In the spleen, the efficiency decreased when the dose rate dropped from 1 mgy/ min to 12.5 lgy/min. Analysis of the molecular nature of the mutations induced with high and low dose-rate radiation revealed that the major changes induced were base pair-long deletions in both tissues (Okudaira et al. 2010). Mutation induction was not detected in (gpt-delta SWR) F1 mice after continuous gamma-ray exposure to 1 mgy/day (total dose ¼ 400 mgy) or 0.05 mgy/day (total dose ¼ 20 mgy) (Ono et al. 2009). Change in mrna level Alterations in the levels of mrna after chronic low dose-rate exposure were examined in the liver, kidney and testis of male C57Bl/6 mice (Taki et al. 2009; Uehara et al. 2010) using microarray that can analyze 20,000 46,000 kinds of mrna at a time. In the liver, 20, 11 and 3 genes showed altered levels of mrna when examined immediately after the completion of 400 days of exposure to 20 mgy/day, 1 mgy/day and 0.05 mgy/day, respectively. Most of the altered genes overlapped among the three dose groups, suggesting similarities. Based on changes in mrna, altered cellular functions include non-esterified fatty acid levels, obesity, endoplasmic reticulum (ER) stress response, among others (Uehara et al. 2010). Similar analyses were performed on kidney and testes by Taki et al. (2009) wherein increased mitochondrial oxidative phosphorylation was observed in kidneys exposed to 20 mgy/day and 1 mgy/day. In the testes, suppression of DNA metabolism, response to DNA damage and DNA replication suggest dependence on radiation dose. It is of interest to note that most of the mrnas altered after radiation exposure did not overlap between the kidney and testes, nor with those of the liver, suggesting that the chronic low dose-rate exposure elicits organ/tissue specific mrna alterations.

8 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 429 Vares et al. (2011) searched for possible transcription factors that may be involved in mrna responses to chronic low dose-rate exposure in liver through computer analyses and found some potential factors such as sterol regulatory element-binding protein 1 (SREBP-1), arylhydrocarbon receptor (AhR/Ar), glucocorticoid receptor (GR), hepatocyte nuclear factor 1 (HNF-1), etc. Examination of the mrna level alterations in the liver show that 20 kinds of mrnas were altered when measured immediately after completion of the 400-day exposure to 20 mgy/day. All mrna levels return to the same levels found in non-irradiated control mice at 1 day and 1 week after the completion of irradiation exposure indicating that the alterations observed immediately after exposure were temporary and do not persist (unpublished data). Changes in protein levels Nakajima et al. (2008) examined the changes in protein levels in liver after chronic exposure using two-dimensional gel electrophoresis and found that rhodanese levels were elevated after 1 and 20 mgy/day exposures but not after 0.05 mgy/day. Rhodanese is located in mitochondria and assumed to be involved in sulfur metabolism and detoxification of cyanide (Cipollone et al. 2007; Nakajima 2015). The levels of anti-oxidative enzyme SOD1 and SOD2 remained unchanged after exposure to all three dose-rates (Nakajima et al. 2008). Recently, Nakajima et al. (2017) adopted an antibody microarray assay system to study altered protein levels in the livers of male C57BL/6J mice after 400 days of exposure to 20 mgy/day, and found 10 altered proteins. At 3 months post-exposure, the levels of 6 out of the 10 proteins, returned to the levels similar to the non-irradiated control mice (Table 5) whereas the other 4 proteins, all with apoptosis as their cellular function, remained altered. Nakajima et al. (2017) also detected 3 new altered proteins at 3 months post-exposure, namely, Caspase12, Apaf1 and Phosphor-beta-catenin (pser 45 ), that were not detected when examined immediately after completion of radiation exposure regimen. Caspase12 and Apaf1 function in apoptosis while Phosphor-beta-catenin (pser 45 ) functions in the cytoskeleton. These results suggest that chronic low dose-rate radiation exposure may have long-lasting effects on apoptotic processes. Examination of protein levels 3 months post-exposure to an acute high dose of 4 Gy (0.72 Gy/min) show 5 altered proteins (Nakajima et al. 2017) different from those found 3 months post-exposure to chronic low dose-rates suggesting that the effects of chronic exposure are different from that of acute exposure. Uehara et al. (2010) noted that the altered Rhodanese level in the liver as observed by Nakajima et al. (2008) were not correlated with alterations at mrna level. Transgenerational effects Transgenerational effects of chronic low dose-rate gammaray exposure was examined in F1 and F2 mice born from irradiated C57Bl/6J sires using lifespan and neoplasm Table 5. Changes in protein levels in the liver of male C57BL/6J mice exposed to 20 mgy/d for 400 days immediately after completion of exposure and 3 months post-exposure (Nakajima et al. 2017). Change after exposure Protein þ 0 day þ 3 months Cellular function MBLN1 Increased Gene expression and regulation Laminin 2a2 Increased Basement membrane craf (pser 621 ) Decreased MEK signaling SynCAM Decreased Cell-cell adhesion ILP2 Decreased Apoptosis PUMA/bbc3 Decreased Apoptosis DRAK1 Decreased Decreased Apoptosis BID Decreased Decreased Apoptosis Bim Decreased Decreased Apoptosis Bmf Decreased Decreased Apoptosis Caspase12 Increased Apoptosis Phosphor-b-catenin Increased Cytoskeleton (pser 45 ) Apaf1 Decreased Apoptosis : no change. incidence as parameters. As in the life span study, C57Bl/6J sires (n ¼ 180 per dose group) were similarly exposed to 0.05, 1 and 20 mgy/day gamma-rays for 400 days and were mated to 8-week-old virgin C57Bl/6J females immediately after completion of irradiation exposure (approximately 456 days of age). Randomly selected F1 offspring were mated to produce the F2 generation. All the irradiated sires (F0) and progeny (F1 and F2) were kept until they died a natural death and were all subjected to gross and microscopic pathological examination to determine the cause of death as well as detect neoplasms and other non-neoplastic disease. Significant life shortening was observed in the irradiated sires exposed to 20 mgy/day by approximately 60.5 days (p ¼.003) and in F1 males born from the sires exposed to 20 mgy/day by approximately 43 days (p ¼.045) (unpublished data). Pathology results for this experiment are currently underway. Results from other studies on transgenerational effects (Sato et al. 1981; Nomura 1982; Sasaki 1991; Cosgrove et al. 1993; Cattanach 1995, 1998) are inconsistent and difficult to compare with the results from our experiments due to various factors such as mouse strain used, conventional vs. specific pathogen free (SPF) environment, age at exposure, dose and dose rate as well as radiation quality, to name a few. Discussion The dose rates used in IES experiments have been selected in an attempt to recreate chronic exposure conditions for nuclear power plant workers who risk exposure to very low dose-rates of radiation for the entire duration of their career. The lowest dose rate of 0.05 mgy/day is approximately twenty times the world s average external (cosmic and terrestrial) background radiation exposure of 0.87 mgy/yr. Studies using dose rates lower than 0.05 mgy/day remain difficult to perform because of technical problems as evidenced by the available dose rates in radiation facilities worldwide (Figure 1).

9 430 I. BRAGA-TANAKA III ET AL. At very low dose-rates of exposure, obtaining the dose and dose rate effectiveness factor (DDREF) through phenomenological studies similar to our life span study is very difficult to perform logistically, since its calculation requires that the total dose for all test groups to be equal resulting in different lengths of exposure. To illustrate, using the lowest dose-rate of 0.05 mgy/day in our life span study, a lifetime irradiation of 850 days (mean life span of mice) will only accumulate a total dose of 43 mgy. In the same perspective, life span irradiation results in different accumulated doses between mice with shorter life spans versus mice with longer life spans. Another issue that was taken into consideration in the life span study was wasted radiation (Mole 1955), where there was a possibility that the total accumulated dose is greater than the dose required to produce the same effect (e.g. cancer). In our life span study, we corrected cancer incidences for competing risks, as they have been shown to influence the shape of the dose response curves, estimates of latent periods and of risk from ionizing radiation (Groer 1980). Based on the assumption that increasing the number of exposed animals would result in a proportionate increase in neoplasm incidence and using data from mice exposed to 0.05 mgy/day in our life span study (Tanaka et al. 2007), we calculated that it would require at least 5000 mice/sex to detect a statistically significant difference from the nonirradiated controls. Hence, the absence of statistically significant effect at exposures to a low dose-rate of 0.05 mgy/day in the life span study does not imply the absence of radiation effects as statistical significance depends on the magnitude of the effect and sample size (Tanaka et al. 2007). Statistical power is also the same reason as to why no clear statement could be made regarding adaptive response at low dose exposures (BEIR VII). One of the most important issues that continue to beleaguer animal experiments, radiation-related or not, is the extrapolation of the results from animals to man particularly at low dose levels (Carnes 2002). Besides the similarities common to mammals, interspecies extrapolation must recognize that the differences between humans and rodents such as different life spans, food consumption, metabolic rate, anatomical differences, stomach ph and DNA excision repairs (rats) (Monro and Mordenti, 1995). Although it is recognized that molecular and cellular similarities in carcinogenic processes among mammals, much of differences in carcinogenic responses of various species (and strains) remain unexplained (Fung et al. 1995). Neoplasms and non-neoplastic diseases in experimental animals with known pathogenetic mechanisms that are not applicable to radiation carcinogenesis in humans should not be considered (BEIR VII). Analyses of the causes of death and tumor incidence data indicate that reducing the dose rate results in decreased incidence of radiation-induced tumors as well as alteration of the neoplastic spectrum (BEIR VII). Our studies in mice, however, depending on the dose, showed shortened tumor latencies (Tanaka et al. 2017), increased tumor burden (Tanaka et al. 2007, 2017) and increased incidence rates of some spontaneous neoplasms without altering the tumor spectrum (Tanaka et al. 2007, 2017). As seen in atomic bomb survivors for solid cancers (Shimizu et al. 1990), our studies using B6C3F1 mice (Tanaka et al. 2007, 2017) show that cancer rates increase in direct proportion to normal increases with aging. The biological significance of altered gene expressions in a several organs from mice exposed to radiation, particularly at low doses and low dose-rates, remain uncertain due to substantial knowledge gaps of protein response to radiation exposure in terms of dose, time from exposure, organ and tissue differences as well as inter-individual variability (Marchetti et al. 2006). Nakajima et al. (2017) showed that altered proteins after chronic low-dose radiation exposures largely mediate apoptosis signaling, whereas acute radiation exposure mediates proteins involved in defense pathways and inflammatory reactions. Understanding the effects of chronic radiation exposure on signaling pathways would contribute toward radiation protection, reducing risk, evaluation of radiation damage and treatment of the effects of radiation exposure. With regards to DNA damage, so far, no comparisons have been made between the biological effects associated with the repair of spontaneous damage versus damage due to ionizing radiation has been done (BEIR VII). Future perspectives The problems of studying the effects at low dose and dose rates remain because of limitations discussed. Since carcinogenesis is also dependent on species and strain predilection, further in vivo experiments, with sufficient sample sizes, using different species such as rats or guinea pigs, different strains of mice or genetically engineered mice (GEM) and animal models of human diseases (including cancer), may be useful in evaluating the interaction(s) of low dose and low dose-rate radiation exposures at each level of the biological organization (molecular, organelle, cellular, tissue and whole animals). Despite the difference in radiation sensitivity between the human and mouse ovary, mechanistic studies involving the hypothalamic regulation of food intake and energy expenditure (Korner and Aronne 2003) resulting from radiation-induced disruption in the pituitary and ovarian hormone functions may provide a clearer understanding of the radiation induced obesity. Other aspects of low dose and low dose-rate radiobiology that require further investigation include non-neoplastic diseases, late effects (malignant/benign neoplastic and nonneoplastic diseases) of parental exposure (transgeneration), and the effect of age (in utero, perinatal, juvenile) at the time of exposure. With advancing technology, contributions from mechanistic studies or omics approaches such as screening for genetic mutations or alterations in gene expression, serum biomarkers, epigenetics, post-translational modification (phosphorylation, acetylation and/or methylation), signal transduction and metabolomics, will increase the understanding of the basic mechanisms of low dose and low dose-rate radiation exposures. Using genetically engineered mice, further studies on the cardiovascular disease (atherosclerosis and stroke), neurologic conditions (Parkinson s, Alzheimer s diseases) and cataracts as seen in epidemiologic studies on

10 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 431 occupational exposures and cancer survivors who have undergone radiation therapy. Although there is an abundance of research on prophylactic or postexposure measures, chemical or biologic, for alleviating the effects of acute radiation exposure, studies on reducing the late effects of low dose and low dose-rate exposure have been lacking. Calorie restriction has been shown to increase the life span in mice exposed to acute radiation by delaying cancer-related deaths (Shang et al. 2014). Studies also show that environmental enrichment (such as increased housing space, nesting materials and play equipment) increases the life span in mice by improving the function of immune cells (Arranz et al. 2010) or by reducing tumor growth (Cao et al. 2010; Nachat-Kappes et al. 2012). Further investigation on whether calorie restriction or calorie restriction mimetic drugs and environmental enrichment could reduce the cancer risks at low dose and low dose-rate radiation exposures. A comprehensive analysis of the relationships between non-neoplastic lesions toward the development of neoplasms, as well as the total disease burden, cause of death and its relationship to life span in the whole individual animal is necessary in order to fully understand the effects low dose and low dose-rate radiation exposure on over-all health (Tanaka et al. 2017). A multidisciplinary systems biology approach to data analyses will facilitate toward understanding the effects of low dose and low dose-rate radiation as a whole. Conclusions The results of our studies on biological effects of chronic low dose-rate exposure in mice at 0.05, 1 and 20 mgy/day for 400 days demonstrated the following: (1) the total dose of 8000 mgy altered many biological endpoints; (2) the total dose of 400 mgy affected lifespan, neoplasm incidence, chromosome aberration, body weight and gene expression at mrna and protein levels suggests that radiation exposure at this dose for a year or longer may induce some health effects although small; and, (3) the total dose of 20 mgy delivered over 400 days increased the incidence of hepatocellular adenoma and suppressed mrna levels of three genes in the liver with no detectable dose-dependency for hepatocellular adenoma and lacked temporal consistency at the mrna level. More studies, empirical and mechanistic, are required to clarify the effects of low dose-rate radiation at the level of 0.05 mgy/day. Acknowledgements We thank all fellow researchers and technicians in IES for helping us in the conduct of studies as well as all collaborators in the other institutions. We thank Drs. Tatsuhiko Imaoka (NIRS) and Dmitri Klokov (CNL) for providing the doses used in their respective institutions. Our special gratitude is expressed to Drs. A. Shima, T. Matsumoto and F. Sato. The studies were performed under contract with Aomori Prefectural Government, Japan. Disclosure statement The authors report no conflict of interest. The authors alone are response for the content and writing of the paper. Notes on contributors Ignacia Braga-Tanaka III, DVM, PhD is a veterinary pathologist and is an associate senior researcher in the Department of Radiobiology at the Institute for Environmental Sciences. She investigates the late effects (neoplastic and non-neoplastic disease) of chronic low dose-rate radiation exposure on mice. Satoshi Tanaka, DVM, PhD is a veterinary pathologist and is a senior researcher in the Department of Radiobiology at the Institute for Environmental Sciences. He investigates the late effects (neoplastic and non-neoplastic disease) of chronic low dose-rate radiation exposure on mice. Atsushi Kohda, PhD is a researcher in the Department of Radiobiology at the Institute for Environmental Sciences. He currently investigates the chromosomal aberrations in mice exposed to chronic low doserate radiation. Daisaku Takai, PhD is a researcher in the Department of Radiobiology at the Institute for Environmental Sciences. He currently investigates antitumor immunology in mice exposed to chronic low dose-rate radiation. Shingo Nakamura, PhD is a researcher in the Department of Radiobiology at the Institute for Environmental Sciences. He currently investigates endocrine damage in mice exposed to chronic low doserate radiation. Tetsuya Ono, PhD is currently the President of the Institute for Environmental Sciences. His research interests are in DNA expression and mutation at low dose radiation exposures. Kimio Tanaka, PhD is currently an adviser to the Department of Radiobiology at the Institute for Environmental Sciences. His research interests are in radiation-induced cell response, gene abnormalities and carcinogenesis. Jun-ichiro Komura, PhD is currently the head of the Department of Radiobiology at the Institute for Environmental Sciences. His research interests are in molecular and cellular biology of radiation effects. References Arranz L, De Castro NM, Baeza I, Mate I, Viveros MP, Dela Fuente M Environmental enrichment improves age-related immune system impairment: long-term exposure since adulthood increases life span in mice. Rejuv Res. 13: Biological Effects of Ionizing Radiation (BEIR) Health risks from exposure to low levels of ionizing radiation. 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