International Workshopon

The Biological Effects of Low Dose Radiation

PROGRAM&
ABSTRACTS

21 October 2013

Sanjo Conference HallThe University of Tokyo

Institute for Environmental Sciences

International Workshop on
the Biological Effects of Low Dose Radiation
21 October 2013
Venue:

Sanjo Conference Hall, The University of Tokyo
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654 JAPAN

Organized by

Institute for Environmental Sciences
http://www.ies.or.jp/
1-7Ienomae, Obuchi, Rokkasho, Aomori 039-3212 JAPAN
Phone: +81-175-71-1200 Fax: +81-175-72-3690

WELCOME

Dear Colleagues,
On behalf of the organizing committee, I welcome you to the “IES International Workshop
on the Biological Effects of Low Dose Radiation.”
Although the estimation of the effects of low dose and low dose rate radiation is importantfor human welfare, it has been hampered by inevitable low signal to noise ratios. New
technologies in various fields of biological sciences however, enable us to overcome thisdifficulty.
In this workshop, recent prominent progress in our knowledge on the effects of low doseand low dose rate radiation will be presented. I hope that the future research on this subject will
be promoted by penetrating opinion in lively discussion.

Yours sincerely,Jun-ichiro Komura
Institute for Environmental Sciences

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21 October, 2013, Monday, Tokyo
IES International Workshop on the Biological Effects of Low Dose Radiation
Sanjo Conference Hall, The University of Tokyo
9:00-9:30

Registration

9:30-9:35

Opening

9:35-12:30

Session I

Presentations by Invited Speakers(Chair: Dr. Hiroshi MITANI, The University of Tokyo)

I-1 Dr. Mary Helen BARCELLOS-HOFF (New York University)
Radiation Carcinogenesis: Microenvironment Matters
I-2 Dr. Yoshiya SHIMADA (National Institute for Radiological Sciences)
Age Dependence of Radiation Carcinogenesis in Animal Models(Coffee Break, 10:40-10:55)
I-3 Dr. Christophe BADIE (Public Health England)
A Mouse Model of Radiation-Induced Leukemia, Past, Present and Future WorkI-4 Dr. Michael M. WEIL (Colorado State University)
Low Dose Rate Exposure Enhances Interindividual Differences in DNA DSB Repair
I-5 Dr. Keiji SUZUKI (Nagasaki University)
DNA Damage Accumulation in Tissues/Tissue Stem Cells Exposed to
Low-Dose-Rate Radiation
12:30-13:30

Lunch

13:30-16:25

Session II

Presentations by IES speakers(Chair: Dr. Toshiyasu IWASAKI, Central Research Institute of Electric Power Industry)
II-1 Dr. Tetsuya ONO
Outline of the IES Research Projects on Biological Effects of Low-Dose-Rate Radiation
II-2 Dr. Ignacia Braga TANAKA
Transgenerational effects in Mice Exposed to Continuous Low-Dose-Rate Gamma-rays,
Pathological Study
II-3 Dr. Keiji OGURA
Transgenerational effects in Mice Exposed to Continuous Low-Dose-Rate Gamma-rays,
Genome Mutation Study
II-4 Dr. Daisaku TAKAI
Response of B6C3F1 Mice Continuously Irradiated with Low-Dose-Rate Gamma-rays toTransplanted Tumor Cells(Coffee break, 14:55-15:10)(Chair: Dr. Keiji SUZUKI, Nagasaki University)
II-5 Dr. Shingo NAKAMURA
Relationship between Radiation-Induced Menopause and Body Weight Gain in Mice
Continuously Irradiated with Low-Dose-Rate Gamma-rays
II-6 Dr. Tokuhisa HIROUCHI
Mutations and Alterations of Expressions in Genes Associated with DNA Repair System
in Leukemia Induced by Continuous Exposure to Gamma-rays at Low-Dose-Rates
II-7 Dr. Atsushi KOHDA
Chromosomal Translocation Rates in Splenocytes of Mice Chronically Exposed to LowDose-Rate Gamma-rays
16:25-16:30

Closing

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I-1
Radiation Carcinogenesis: Microenvironment Matters
Mary Helen BARCELLOS-HOFF
Departments of Radiation Oncology and Cell Biology, New York University School of
Medicine, New York, NY 10016 USA.
Ionizing radiation can alter genomic sequence as a result of damage due to targeted effects from theinteraction of energy and DNA, and the response to damage can alter phenotype and multicellularinteractions, referred to herein as non-targeted effects (NTE). We have previously demonstrated thathigh and low LET radiation affects epithelial phenotype, e.g. epithelial to mesenchymal transition (EMT),
stromal remodeling and genomic instability (GIN), in human cells and mouse tissues, and have recently
shown radiation affects stem/progenitor self-renewal in vivo (reviewed in (Barcellos-Hoff and Nguyen,
2009; Nguyen et al., 2010).

We have demonstrated the relevance of NTE to radiation carcinogenesis

using a mouse model to show that radiation effects on the microenvironment significantly alter thefrequency and course of mammary cancer (Nguyen et al., 2011a). Our hypothesis is that radiationinduced signaling disrupt tissue-level interactions that suppress malignant progression of genomicallyunstable epithelial cells.
Tissue microenvironments can either suppress or promote tumorigenesis. Our published andpreliminary studies suggest that radiation exposure has very early and persistent effects on the tissue
microenvironment prior to the development of cancer. We have identified one critical signal, TGFβ, inprior studies. Our published low LET studies have identified three event modified by radiation in
irradiated mammary gland: Stem cell self-renewal, macrophage activation, and persistent stromal
activation, all of which occur well before cancer develops (Nguyen et al., 2011b). Recent analysis ofexpression profiles from HZE irradiated tissues confirms that high LET irradiation also affects key
macrophage and fibroblast pathways.
Our model is shown in Figure 1. We propose that 1) a pre-cancer niche is established between
activated stromal cells (fibroblasts and/or macrophages) and epithelial cells undergoing neoplastictransformation; and 2) that such pre-cancer niches mediate the efficiency of mammary cancer
development (Barcellos-Hoff et al., 2013). Our data lead shows that radiation actually primes the target
epithelium by increasing the frequency of stem cells by inducing Notch and TGFβ stimulated symmetric
division (Tang et al., 2013). This primes the host by recruitment of macrophages and activation offibroblasts to form pre-cancer niches, which in turn malignant progression. This component of radiation
carcinogenesis is particularly important to preventing cancer risk in susceptible populations such as thosetreated with radiation for childhood cancers.

References
Barcellos-Hoff, M. H., Lyden, D., and Wang, T. C. (2013). The evolution of the cancer niche
during multistage carcinogenesis. Nat Rev Cancer 13, 511-518.Barcellos-Hoff, M. H., and Nguyen,
D. H. (2009). Radiation carcinogenesis in context: How do irradiated tissues become tumors?
Health Physics 97, 446-457.
Nguyen, D. H., Bochaca, I. I., and Barcellos-Hoff, M. H. (2010). The biological impact of radiation
exposure on breast cancer development. In Breast Cancer and the Environment, J. Russo, ed. (NewYork: Springer ).
Nguyen, D. H., Oketch-Rabah, H. A., Illa-Bochaca, I., Geyer, F. C., Reis-Filho, J. S., Mao, J. H.,
Ravani, S. A., Zavadil, J., Borowsky, A. D., Jerry, D. J., et al. (2011a). Radiation Acts on theMicroenvironment to Affect Breast Carcinogenesis by Distinct Mechanisms that Decrease Cancer
Latency and Affect Tumor Type. Cancer Cell 19, 640-651.
Nguyen, N. H., Oketch, H. A., Geyer, F. C., Reis-Filho, J. S., Mao, J.-H., Ravani, S. A., Zavadil, J.,
Borowsky, A. D., Jerry, D. J., Dunphy, K. A., et al. (2011b). Radiation Acts on theMicroenvironment to Affect Breast Carcinogenesis by Distinct Mechanisms that Decrease Breast
Cancer Latency and Affect Tumor Type. Cancer cell In press.
Tang, J., Fernandez-Garcia, I., Vijayakumar, S., Martinez-Ruiz, H., Illa-Bochaca, I., Nguyen, D. H.,
Mao, J.-M., and Barcellos-Hoff, M. H. (2013). Irradiation of juvenile, but not adult, mammary
gland increases stem cell self-renewal and estrogen receptor negative tumors. Stem Cells
10.1002/stem.1533.





I-2
Age Dependence of Radiation Carcinogenesis in Animal Models
Yoshiya Shimada1, Mayumi Nishimura1, Kentarou Ariyoshi1, Shusuke Tani1, Yuka Ishida2,
Chizuru Tsuruoka1, Tatsuhiko Imaoka1, Yutaka Yamada1, Shino Takeda1, Yoshiko Amasaki1,
Yi Shang1, Tomoko Sawai1, Shinobu Hirano1, Ken-ichi Iwata1, Ayaka Hosoki1, Takamitsu
Morioka1 and Shizuko Kakinuma1
1Radiobiology for Children’s Health Research Program,2
Department of Technical Support and Development,
National Institute of Radiological Sciences

With the advance of radiological techniques in medicine, radiation dose of child patients has
been rapidly increased in these two decades. Since children are highly susceptible to radiationinduction of cancer, the cancer risk of radiation has been a great concern. Therefore, it isimportant to access the cancer risk after childhood exposure accurately.
Recently we have started the project to elucidate the age dependence of radiogenic cancer risk
and the underlying mechanism. We here introduce the scheme of our project, and show recent
results on the effect of early-life radiation exposure on lifespan shortening and cancer induction
in mouse and rat models.
Life span shortening: Life shortening was investigated in both genders of B6C3F1(C57BL/6 x C3H) mice exposed to gamma rays, carbon ions (290-MeV/u; 13 keV/um) andneutrons (1-2 MeV). Mice were irradiated in utero or at 1, 3, 7 and 15 weeks after birth. Linear
dose-response curves were obtained. For mice exposed to gamma rays at 7 weeks of age, theslope was steeper for female than male, indicating that female is more susceptible than male.
Irradiation at 1 week of age further increased the slope and no gender difference was observed.
In utero exposures had negligible effect for both genders. The experiments on carbon ions andneutrons are now in progress.
Cancer induction: It was reported that there is a susceptible age-window for radiation
carcinogenesis in a tissue dependent (Figures). We further extended the tissue types withadditional tumor models and demonstrated the susceptible age-window for each organ. These
include brain, kidney, intestine, lung and mammary glands.
Biological mechanisms of age dependence: High incidence of tumors after irradiation atjuvenile is ascribed to extensive cell division during growth, which results in the rapid fixationof DNA damage and gives chance for mutant cells to clonally expand. We introduce the unique
response of immature/progenitor cells and tissue microenvironment to ionizing radiation, which
are dependent on the age-at-exposure.





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I-3
A Mouse Model of Radiation-Induced Leukemia,
Past, Present and Future Work
Christophe BADIE
Cancer Genetics and Cytogenetics group, Biological Effects Department
Centre for Radiation, Chemical & Environmental Hazards Public Health EnglandAnimal models of human cancers are extremely valuable for a better understanding of themolecular mechanisms of tumour initiation and development, in particular for cancers induced
by environmental agents such as ionizing radiation (IR). Radiation-induced acute myeloid
leukaemia (rAML) is one IR induced tumour for which mouse models are available. The
CBA/H mouse model of rAML has been studied for decades to bring to light the molecularmechanisms associated with multistage carcinogenesis. A specific interstitial deletion of mouse
chromosome 2 found in a high proportion of rAML is recognised as the initiating event; thispartial hemizygous deletion is a common feature in CBA/H and several other susceptible strains.
The deletion is an early event detectable 24 hours after exposure in bone marrow cells. The
deletion leads to the loss of Sfpi, a gene essential for haematopoietic development. Its product,
the transcription factor PU.1 acts as a tumour suppressor in this model. Although the deletioncan be detected by cytogenetic techniques, precise characterization of the haematopoietic cells
carrying the deletion and the study of their fate in vivo couldn’t so far be achieved. It is assumed
that leukaemia originates in an early progenitor cell or haematopoietic stem cell but it isunknown whether the original chromosome damage occurs at a similar frequency in committedprogenitors and stem cells. In a recent study we monitored the frequency of chromosome 2deletions in immature bone marrow cells (Lin−) and haematopoietic stem cells/multipotent
progenitor cells (LSK) by several techniques, using a reporter gene model. We showed thatpartial chromosome 2 deletions are present in the LSK subpopulation (Table 1). Aftertransplantation, these cells didn’t show an advantage for growth and in vivo repopulation, atleast at early stages following occurrence.
Using the same genetically engineered CBA/H mouse model expressing the GFP fluorescentmolecule under the control of the Sfpi1 promoter, we demonstrated that GFP expression did notinterfere with X-ray induced leukaemia incidence and that GFP fluorescence in live leukaemic
cells is a surrogate marker of radiation-induced chromosome 2 deletions (Fig.1). This is the firstexperimental evidence for the detection of this leukaemia initiating event in live leukemic cells.
New mouse models are currently being developed for investigating individual pre-leukaemic
cell fate within its natural microenvironment in order to dissect the steps in tumour initiation,
promotion and malignant progression. This could lead to new insights into the nature of themolecular mechanisms and identification of the cell of origin in radiation-induced leukaemia.
This report is commissioned by the National Institute for Health Research. Financial support
was also provided by the European Union FP7 DoReMi network of excellence.
Table 1

Olme CH et al. Mutat Res. 2013 Aug 30;756(1-2):119-26

Figure 1

Correlation between geometric mean of GFP expression and GFP copy number in unirradiatedmouse bone marrow cells and rAML
Olme CH et al. Leuk Res. 2013 Oct;37(10): 13

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I-4
Low Dose Rate Exposure Enhances Interindividual Differencesin DNA DSB Repair
Michael M. WEIL, Takamitsu KATO, Peter DEMANT, Donasian OCHOLA,
and Joel S. BEDFORD
Department of Environmental and Radiological Health Sciences, Colorado State University
Individuals differ in their capacity to repair radiation-induced DNA double strand breaks(DSB) efficiently. Low dose rate radiation exposure followed by immediate quantification of
repair foci in exposed cells provides a much more sensitive assay for DNA DSB repair
efficiency than time-delayed measurements after acute high dose rate exposures. We have used
this technique to assess the repair proficiency of fibroblasts derived from retinoblastoma
patients, ataxia-telangiectasia (A-T) patients, their unaffected family members and clinicallynormal, control individuals. The assay can identify members of A-T families who carry a single
mutated copy of the ATM gene. Fibroblasts from these individuals, while not nearly as deficientin DNA DSB repair as fibroblasts from A-T patients with two defective ATM alleles, arenevertheless less adept at repair than fibroblasts from most normal controls. While the numberof families studied is small, it also appears that the parents of retinoblastoma patients with denovo RB1 mutations are less repair proficient than most normal controls. This observation,
along with the finding of interindividual differences in DNA DSB repair in clinicallyunremarkable individuals, may contribute to our understanding of the events underlying thetransmission of de novo germline mutations.
We are currently using fibroblasts from recombinant inbred mouse strains to examine the roleof specific genetic polymorphisms in DNA DSB repair proficiency. Also using these mousestrains we are examining the possible connection between the genetically based differences inrelative susceptibilities that have been observed for radiogenic cancers in the recombinantcongenic mouse strains and the corresponding sensitivities as measured in the low dose rate
based DNA DSB repair assay. For this, we are using the molecular focus assay directly in cellsof relevant tissues in low dose rate irradiated mice. This will further allow a direct comparisonof possible tissue dependent heterogeneity in cellular sensitivity using this assay.

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I-5
DNA damage accumulation in tissues/tissue stem cells exposed to
low-dose-rate radiation
Keiji SUZUKI
Department of Radiation Medical Sciences, Atomic Bomb Disease Institute, Nagasaki
University.
Absorption of radiation energy into DNA gives rise to DNA damage, among which DNA
double strand breaks (DSBs) are the most lethal and carcinogenic ones. The spatiotemporal
dynamics of DSBs is well examined after high-dose and high-dose-rate exposure. However, theinformation is still limited on the effects of low-dose and low-dose-rate exposure, particularly invivo. Therefore, we aim to investigate accumulation of DSBs in various tissues and organs
obtained from mice exposed to γ-rays at different dose-rates.
B6C3F1 mice were irradiated with 137Cs γ-rays at low-dose-rate (LDR) of 400 mGy/22 h/day(0.3 mGy/min) for 10 days (Figure 1). For high-dose-rate (HDR) exposure, mice received 4 Gy
at 850 mGy/min. After completion of radiation exposure, various tissues and organs, including
lung, were isolated. Formalin-fixed samples were embedded in paraffin, and then, tissue slices
were obtained. Antigen retrieval was performed at 95°C for 40 min. DSBs and proliferating
cells were quantified by immunofluorescence using anti-53BP1 and anti-Ki-67 antibodies,
respectively. The images were captured and analyzed by FW4000 software.
DSBs were detectable in the epithelial cells of the bronchioles obtained from unirradiatedcontrol mice (approx. 0.04 foci/cell), however, HDR exposure significantly increased thenumber of DSBs in all bronchiolar epithelial cells, whose yield was more than 15 foci/cell(Figure 2). While DNA repair pathway efficiently removed DSBs, approximately 8 foci/cellwere still remained in 50% of cells even 3 days after irradiation. LDR exposure also induced
DSBs but to a much lesser extent, and approximately 1 focus/cell was observed in about 60% of
cells. After 3 days, the DSB levels were dropped down to 0.11 foci/cell, and foci-positive cells
were about 10%. Intriguingly, the frequency of Ki-67 was significantly increased in the
bronchioles 3 days after HDR exposure.
These results demonstrated that the induction and repair of DSBs were detectable in vivo.
HDR exposure efficiently induced DSBs, whereas not the same effect was observed by LDR
exposure, even if the total dose was the same. Moreover, a half of exposed cells still harbored atleast one focus 3 days after HDR exposure, which was in contrast to LDR exposed cells that
repaired DSBs in approximately 90% of them. Thus, the effect of HDR exposure is far greaterthan that of LDR exposure, indicating that multiplicity and complexity of DSBs could be
different between HDR and LDR exposures. Since excess tissue proliferative response wasobserved only after HDR exposure, this should take into account to estimate cancer risk fromradiation exposure.
This work has been performed in collaboration with IES.

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Figure 1

Figure 2
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II-1
Outline of the IES Research Projects on Biological Effects
of Low-Dose-Rate Radiation
Tetsuya ONO
Institute for Environmental Sciences
The biological effects of low dose radiation are not understood well and the risk estimation isbased on a hypothesis called a linear non-threshold model. It was not a big issue when the lowdose exposure was limited mostly to a specific group of people like radiation workers, medical
practitioners, scientists, etc. However, the two nuclear power plant accidents in Chernobyl and
Fukushima have pulled out people’s strong attention to the risk of low dose exposure.
Elucidation of the biological responses to the low dose radiation is now an urgent theme forradiation biology.
The low dose of radiation can be exposed in either acute or chronic ways. IES founded in1990 has chosen a long-term low-dose-rate chronic irradiation as a target to challenge. The
dose-rate was set at two levels, 400 mGy/400 day and 20 mGy/400 day. These are comparable
to the exposure in space station and the legal dose limit for radiation workers, respectively. As apositive control, the effects of 8,000 mGy/400 day were also examined in parallel. Mice wereused as experimental animals. They were irradiated with gamma-rays from 137Cs for 22 hours
every day and it was continued for 400 days under SPF (specific pathogen-free) conditions. The
remaining 2 hours (10:00-12:00) were not used for irradiation but spent for health check of themice, supply of food and water, etc. The biological indices studied so far were life span, causeof death, cancer incidence, pathological change, body weight, chromosome abnormality,
somatic mutation frequency, alteration in gene expression, immunological competence against
cancer cells, and trans-generational effects examined on life span, cancer incidence and genomicchange. All of these indices revealed some influence if the total dose of radiation was 8,000
mGy. One exception was cancer incidence in the offspring of irradiated males. Slight but
significant effects were observed for limited indices in 400 mGy-irradiated mice; life span infemales, chromosomal abnormality and gene expression. The total dose of 20 mGy, on the other
hand, revealed little detectable effects (Table 1).
  Now, we are planning to extend our studies to the following three questions:(1) Dose-rate dependency for trans-generational effects on life shortening and cancer induction.
(2) Effects of low-dose-rate irradiation on embryo and fetus.
(3) Molecular and cellular mechanisms of low-dose-rate irradiation.
Our goal is to clarify the biological effects of low-dose-rate irradiation in experimental
animals. It will provide important information for understanding low dose radiation effects inhumans.
This study was performed under contract with the Aomori Prefectural Government, Japan.

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II-2
Transgenerational Effects in Mice Exposed to Continuous
Low-Dose-Rate Gamma-Rays– Pathological Study –Ignacia TANAKA and Satoshi TANAKA
Department of Radiobiology, Institute for Environmental Sciences.
To study the effects of continuous low-dose-rate gamma-ray irradiation on the progeny ofmice, males (sires) were irradiated for 400 days with 137Cs gamma-rays at low-dose-rates of 20
mGy/22 h/day, 1 mGy/22 h/day, and 0.05 mGy/22 h/day with accumulated doses equivalent to8000 mGy, 400 mGy, and 20 mGy, respectively. Immediately after completion of irradiation,
the male mice were bred with non-irradiated females to produce F1 mice. Randomly selected F1
males and females were bred to produce F2 mice. All mice, except the dams of F1 mice, were
subjected to pathological examination upon natural death. Lifespan, cancer incidence andnumber of offspring were used as parameters to evaluate the biological effects of low-dose-rateirradiation (Fig 1). There were no significant differences in the pregnancy rate and weaning ratein the parent generation. There were, however, significant decreases in the mean litter size
(p=0.029), as well as the mean number of weaned pups (p=0.023) per female bred to males
exposed to 20 mGy/22 h/day compared to the non-irradiated controls (Table 1). Partial results
show that significant decreases in the lifespan of male parent mice (F0, p=0.001) exposed to 20
mGy/22 h/day and their male progenies (F1, p=0.006) were observed. No significant differenceswere found in the cause of death and cancer incidence in F1 and F2 progeny mice.
This study was performed under contract with the Aomori Prefectural Government, Japan.

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II-3
Transgenerational effects in Mice Exposed to Continuous
Low-Dose-Rate Gamma-Rays, Genome Mutation Study
Keiji OGURA1, Hidekazu NEGISHI2, Chihiro HARADA2, Katuyoshi FUJIKAWA1, Satoshi
TANAKA1, Ignacia BRAGA-TANAKA III1, Junichiro KOMURA1
1

Department of Radiobiology, Institute for Environmental Sciences, 2Japan Animal CareIn the past, trans-generation effects of radiation have been examined using phenotypicmarkers like coat color. Recent advances in technology enabled us to examine a whole genome
at molecular level. Here we adopted one of these techniques called comparative genome
hybridization (CGH) and studied trans-generational effects on DNA copy number aberrations(CNAs). C57BL/6J mice were exposed to low-dose-rate (LDR) gamma rays (20 mGy/22 h/day;909 µGy/h) for 400 days (total dose: 8000 mGy) from 8 weeks of age. We analyzed, so far, a
total of 333 genomes from mice (111 progenies from 20 pairs of parents in 20 mGy/22 h/day
irradiated group, and 140 progenies from 21 pairs of parents in non-irradiated group) using
oligo-microarray CGH (Agilent Technologies). The results indicate that the progeny from the
20 mGy/22 h/day irradiated group had significantly higher frequencies of genomic aberrations
compared to non-irradiated mice (21.6% vs 11.4%, Table 1). Next we asked if the F1 miceharboring CNAs could have shorter life spans than the F1 that did not have aberration (Table 2).
Although some trends were suggested, the statistical analysis reveled no significant difference
between these two groups. The difference in life shortening between mice with or without DNA
aberrations after exposure to LDR radiation was not significant due to the small number ofsamples. In addition, we will discuss the possibility of genomic instability in somatic tissues
being induced in the 20 mGy/22 h/day irradiated group mice.
This study was performed under contract with the Aomori Prefectural Government, Japan.

ࠉTable 1. Results of the genomic aberrations analysis using the oligo-microarray CGH

No. ofanalyzed
F1 miceNo. of micewithaberrations

No. of lociwithaberrations

111

24(21.6 %)

175
(Ave. 1.58
/generation)

140

16(11.4 %)

21
(Ave. 0.15
/generation)

P =0.03

P<0.0120 mGy/ 22h/day
irradiated group

Non irradiated group

Table 2. Relationship between the presence of aberrations and life span in F1 mice♂
20 mGy/ 22h/day
irradiated group(111 mice)non irradiated

♀

♂

group
(140 mice)♀

Aberrations

Life
span

Yes 13(20.6%)

794.8±170.7No50(79.3%)

843.7±231.2

Yes 11(22.9%)

706.1±182.7

No

37(77.1%)

799.8±154.3Yes

7(10.4%)787.9±340.5No60(89.6%)

905.5±172.2Yes

9(12.3%)699.0±167.0No64(87.7%)

804.5±158.5

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Cause of deathCategory
Major
Neoplasms
8 (66.6%)
Inﬂammation 1 (8.3%)
Others3 (25.0%)
Neoplasms 23 (48.9%)
Inﬂammation13 (27.7%)
Others11 (23.4%)
Neoplasms
9 (81.8%)
Inﬂammation 1 (9.1%)
Others1 (9.1%)
Neoplasms 29 (78.4%)
Inﬂammation 4 (10.8%)
Others4 (10.8%)
Neoplasms
3 (42.9%)Inﬂammation 2 (28.6%)
Others2 (28.6%)
Neoplasms 34 (58.6%)
Inﬂammation14 (24.1%)
Others10 (17.2%)
Neoplasms
7 (77.8%)
Inﬂammation 0 (0.0%)
Others2 (22.2%)
Neoplasms 43 (69.4%)
Inﬂammation12 (19.4%)
Others7 (11.2%)

Lymphoma, Malignant

38%

Lymphoma, Malignant

14%

Lymphoma, Malignant

45%

Lymphoma, Malignant

43%

Lymphoma, Malignant

14%

Lymphoma, Malignant

22%

Lymphoma, Malignant

67%

Lymphoma, Malignant

41%

II-4
Response of B6C3F1 Mice Continuously Irradiated with Low-Dose-Rate
Gamma-Rays to Transplanted Tumor Cells
Daisaku TAKAI
Department of Radiobiology, Institute for Environmental Sciences
Previously, Tanaka et al. reported significant life-shortening due to early neoplastic death inspecific-pathogen-free B6C3F1 mice irradiated continuously with low dose-rate (20 mGy/22 h/day)
gamma-rays to a total dose of 8000 mGy (high dose). To understand the mechanisms responsible for the
early neoplastic death, we transplanted syngeneic ovarian granulosa tumor cells, OV3121, into agematched irradiated and non-irradiated control mice and found enhanced transplantability of the tumor
cells in irradiated mice. Then we examined the expression of chemokine receptors relevant to tumor
immunity in recipient mice and found that compared with non-irradiated control mice, expressions ofsome chemokine receptors were reduced in irradiated mice. Moreover, mice with reduced expression ofchemokine (C-C motif) receptor type 5 (Ccr5) showed enhanced tumor transplantability. We
hypothesized that immune cells with Ccr5 migrated to the transplantation site in recipient mice andcontributed to the eradication of tumor cells. Based on the assumption that the migration of the immune
cells to the transplantation site would be induced by a chemokine ligand from the transplanted tumor
cells, we have examined whether the transplantability of OV3121 cells in non-irradiated B6C3F1 mice isenhanced if the expression of a chemokine (C-C motif) ligand 8 (Ccl8), which is known to bind Ccr5, isknocked down in the inoculated cells. The result showed that the tumor transplantability of OV3121(Ccl8-) was enhanced compared with OV3121 (Ccl8+).
These suggest that low dose-rate gamma-irradiation disturbs the balance of chemokine network andreduces anti-tumor immunity. The understanding of the detailed role of chemokine network in tumortransplantability in irradiated mice requires further investigation.
This study was performed under contract with the Aomori Prefectural Government, Japan.

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II-5
Relationship between Radiation-Induced Menopause and Body Weight Gainin Mice Continuously Irradiated with Low-Dose-Rate Gamma-Rays
Shingo NAKAMURA, Satoshi TANAKA, Ignacia Braga-Tanaka III
Department of Radiobiology, Institute for Environmental Sciences.
We previously reported a significant increase in body weights of B6C3F1 female micecontinuously exposed to low dose-rate (20 mGy/22 h/day) γ-rays as compared to that of agematched non-irradiated control mice (Radiat Res. 2010, 173, 333-341). To clarify theunderlying mechanisms of body weight gain observed after exposure to continuous low doserate irradiation, we examined adipose tissue weights, liver and serum lipid contents, and factors
related to lipid metabolism (adipocytokines), as well as ovarian dysfunction in female B6C3F1
mice continuously irradiated with γ-rays at 20 mGy/22 h/day from 9 to 44 weeks of age.
Significant body weight gains, related to tissue adiposity, were observed from 30 to 44 weeks ofage (accumulated dose = 3 to 5 Gy) in the irradiated mice (Fig. 1). Histopathological analysesof ovaries and vaginal smears from irradiated mice showed gradual depletion of viable oocytes
as irregular estrus cycles become increasingly frequent leading menopause and that these wereobserved to occur within the same period the increased body weight gain (Fig.2 & Fig. 3). These
results suggested a possibility that the radiation-induced premature menopause could trigger
body weight gain due to adiposity in B6C3F1 female mice continuously irradiated with low
dose-rate γ-rays at 20 mGy/22 h/day.
This study was performed under contract with Aomori Prefectural Government, Japan.

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II-6
Mutations and Alterations of Expressions in Genes Associated with DNA
Repair System in Leukemia Induced by Continuous Exposure to GammaRays at Low-Dose-Rates
Tokuhisa HIROUCHI
Department of Radiobiology, Institute for Environmental Sciences.
It is important to understand why mice exposed to high-dose γ-rays at a low-dose rate (LDR)
have a significantly higher leukemia incidence than non-irradiated mice, although LDR γ-ray
irradiation induces very few DNA double strand breaks. This study focused on characterizing
the leukemic stem cells of LDR radiation-induced leukemias by comparing them with those ofhigh-dose-rate (HDR) or middle-dose-rate (MDR) γ-ray-induced leukemias. The LDR, MDR
and HDR γ-ray-induced leukemias were obtained from mice that were exposed to 8,000 mGy at
20 mGy/22h/day, 4,000 mGy at 400 mGy/22h/day and 3,000 mGy at 890 mGy/min,
respectively. Leukemic stem cells in leukemias induced by irradiation at the three dose rates
were identified by transplanting 100 cells of different hematopoietic differentiation stages tosyngeneic mice. The stages were hematopoietic stem cell, multipotent progenitor, common
myeloid progenitor, common lymphoid progenitor, macrophage, erythrocyte, and B/NK cell.
The leukemic stem cells from HDR and MDR γ-ray-induced leukemias had hemizygous
deletions of chromosome 2 at around PU.1 allele and showed identical CD antigen profiles.
Microarray analysis of the mRNA revealed similar gene expression profiles to those of normal
common myeloid progenitor (Table 1). In contrast, leukemic stem cells from LDR and some of
the MDR γ-ray-induced leukemias with intact PU.1 allele did not reveal similar profiles of CDantigen and mRNA gene expression profiles to those of normal common myeloid progenitor,
but frequently showed profiles resembling those of common lymphoid progenitor cells,
granulocytes or monocytes. Thus, the origin of leukemic stem cells of LDR γ-ray-induced
leukemias seems to be different from that induced by HDR γ-ray. Annotation analysis of gene
expression profiles showed that down-regulation of p53 in the LDR and MDR leukemic stem
cells influenced on several DNA damage response-associated biological pathway in database of
Ingenuity Systems Co. such as, “Role of BRCA1 in DNA damage response”. However, noalterations of DNA damage responses were found in HDR leukemic stem cells. These suggest apossibility that DNA damage repair systems contribute to leukemogenesis in different ways
when dose rate is changed.
This study was performed under contract with the Aomori Prefectural Government, Japan.Table 1. CD antigen profiles and prediction of origin of the stem cells of radiation-induced
acute myeloid leukemias (rAML) on the basis of gene expression profiles.
AML sample

CD antigen profilea

Population with highest similarity
with rAML stem cellb

Confidence Measurec

LSK-

0.9 Gy/min-2
0.9 Gy/min-3
0.9 Gy/min-9
0 mGy/day-2
0.9 Gy/min-10
0.9 Gy/min-1
0.9 Gy/min-2
0.9 Gy/min-3
0.9 Gy/min-6
0.9 Gy/min-9
0.9 Gy/min-10
0 mGy/day-7
20 mGy/day-1
20 mGy/day-3
400 mGy/day-4
0.9 Gy/min-9
0.9 Gy/min-10
20 mGy/day-1
20 mGy/day-3
400 mGy/day-3
0.9 Gy/min-10

Common myeloid progenitor
Common myeloid progenitor
Multipotent progenitor
Multipotent progenitor
Multipotent progenitor
Common myeloid progenitor
Common myeloid progenitor
Common myeloid progenitor
Common myeloid progenitor
Common myeloid progenitor
Common myeloid progenitor
Common lymphoid progenitor
Multipotent progenitor
Multipotent progenitor
Common lymphoid progenitor
Common myeloid progenitor
Common myeloid progenitor
Common myeloid progenitor
Common lymphoid progenitor
Common lymphoid progenitor
Hematopoietic stem cell

0.63
0.94
0.95
0.95
0.83
0.97
0.89
0.98
0.97
0.93
0.91
0.66
0.65
0.95
0.91
0.95
0.93
0.83
0.97
0.88
0.55

LSK+
CMP

CLP

Gr-1 pos

CD45R/B220pos



II-7
Chromosomal Translocation Rates in Splenocytes of Mice Chronically
Exposed to Low-Dose-Rate Gamma-Rays
Atsushi KOHDA, Jun-ichiro KOMURA, Kimio TANAKA
Department of Radiobiology, Institute for Environmental Sciences.
The rates of chromosomal translocations were estimated in splenic lymphocytes of specificpathogen-free (SPF) female C3H mice exposed to low-dose-rate (LDR; 20 mGy/22h/day, 1
mGy/22h/day and 0.05 mGy/22h/day) 137Cs-γ-rays continuously from 8 weeks of age to a
maximum of about 400 days (Fig 1) and compared with those after irradiation at a high dose
rate (HDR; 890 mGy/min) or a medium dose rate (MDR; 400 mGy/day). Splenic lymphocytes
from irradiated and non-irradiated control mice were cultured for 46 h in the presence of LPS,
Con A, and 2-ME to obtain metaphase spreads, and translocations were identified under afluorescent microscope using the multiplex-fluorescence in situ hybridization (M-FISH) method.
Translocations increased linearly with the total dose of LDR (20 mGy/22h/day and 1
mGy/22h/day) as well as MDR (400 mGy/day) γ-rays. This was in contrast with a linearquadratic increase induced by HDR (890 mGy/min) γ-rays (Fig 2). So far, significant increases
in translocations have not been detected in mice irradiated at the lowest dose rate (0.05
mGy/22h/day). The dose rate effect was quite obvious when comparing HDR (890 mGy/min)and MDR (400 mGy/day), but negligible between 400mGy/day and 1 mGy/22h/day. The doseand dose-rate effectiveness factor (DDREF) was calculated to be about 2 from the rates oftranslocation for HDR and LDR at a total dose of 100 mGy. These results will be useful for
estimating the risk of LDR radiation.
This study was performed under contract with the Aomori Prefectural Government, Japan.

Irradiation procedures
Animals; SPF C3H/HeN female mice
Irradiation from 8 weeks of age
20 mGy/22h/day (909 Gy/h) Low-dose-rate (LDR)
0 240 5001000
0 12 25 50

2000
100

4000
200

6000
300

8000 mGy
400 days

300
300

400 mGy
400 days

1 mGy/22h/day (45.5 Gy/h) Low-dose-rate (LDR)
0
0

125
125

200
200

0.05 mGy/22h/day (2.27 Gy/h) Low-dose-rate (LDR) (ongoing)
0
0

6.25
125

10
203

15
307

20 mGy400 daysAge-matched and non-irradiated control

Chromosomal translocation rates
in mouse splenic lymphocytes
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Secretariat:

Institute for Environmental Sciences

http://www.ies.or.jp/
2-121 Hacchazawa, Takahoko, Rokkasho, Aomori 039-3213 JAPAN
Phone: +81-175-71-1966 Fax: +81-175-71-1982