PR3

Project Research on the New Applicant Development using the Characteristics of
the Particles from the Neutron Capture Reaction

and biological effects (cell killing and induction ofDNA double strand breaks (DNA-dsb) using humanglioblastoma cells, A172 and T98G. A172 are wild
type of p53, and T98G cells are mutant type.
The results showed that difference of radiation sensitivity between A172 and T98G was decreased byBPA addition irradiation of neutron beam. Theseresults indicate that the difference between the radiation sensitivity was observed between the T98Gcells and A172 cells boron neutron capture reaction.
Interestingly, it was reduced with BPA where theirradiation mainly depends on the particle radiation
emitted by BNCR (Boron Neutron Capture Reaction). This result suggests that BNCT is an effective
treatment with glioblastoma that can reduce the difference of radio sensitivity by p53 functional status.
PRS-3 compared the different damage effects onplants between BNCR and 60Coγ-ray. To determine
the effectiveness of BNCR for plant mutagenesis atirradiation of dry seeds, two-row-barley was easily
used because of its compact size of the seeds andhigher mutation rate. The dry seeds of Hordeumvulgare cv.Hayadori were immersed in different
concentrations of 10B-enrichedp-boronophenylalanine (BPA) for 24hours, and all
the materials were irradiated with thermal neutronfor 120 minutes in the Kyoto University Research
Reactor (KUR).60Coγ-ray irradiations were also
carried out on the „Hayadori‟ dry seeds in the different doses using the Kyoto University‟s 60Coγ-ray
irradiation facility. They found out that thesemi-lethality dose (LD50) and RD50in the reduced
rate of 4-week-seedlingswas 74.2μM BPA. The
469μM value was transformed to the total physicaldoses as17.98 Gy, and the 74.2μM as 11.2Gy byusing the transformation equation.
PRS-4 developed boron tracedrugs with their “ondemand” traceability and their physical force forneutron dynamic therapy (NDT). The boron tracedrug, UTX-51 was studied that their dynamic,beyond chemical, effects when acquired by weak
thermal neutron irradiation of glycated BSA as amodel of advanced glycation end-products (AGEs),
which is linked to diabetes and aged diseases. They
found that all doses of the boron tracedrug UTX-51
caused destructive dynamic damage against
Gly-BSA during thermal neutron irradiation, suggesting boron tracedrugs could be used as dynamicdrugs for NDT targeted glycated proteins, such as
Gly-BSA, for serum protein-quality-control treatmet of AGEs-related diseases.

Y. Kinashi
Research Reactor Institute, Kyoto University
Objectives and Participating Research Subjects
In this project, we are intending to develop the new
application using the characteristics of the particles
from the neutron capture reaction.
PRS-1 Analysis of mutation in the mammalian cells
induced by BNCR (boron neutron capture reaction)( Y. Kinashi et al. )PRS-2 Analysis of double strand breaks in the
mammalian cells induced by BNCR
( S.Takahashi et al. )PRS-3 Application of BNCR to plant tissue culturefor mutation breeding( T.Morikawa et al.)PRS-4 Development of pharmacokinetic using boron
trace drugs( H. Hori et al. )Main Results and Contents
PRS-1 inspected whether ascorbic acid was effective in protection of the mutation induction of neutron radiation beam used for BNCT in Kyoto University Research Reactor (KUR). The mutagenicity
measured by the frequency of mutations induced byneutron irradiation with or without boron compound.
The HPRT locus was examined in Chinese hamster
ovary (CHO) cells irradiated with neutrons of KUR.
High dose rate neutron irradiation was 0.2Gy/min
with 5MW of KUR, and low dose rate neutron irradiation was 0.04Gy/min with 1MW of KUR. They
investigated that dose rate effect is exist neutron
irradiation in BNCT. Ascorbic acid treatment reduced mutation induction following neutron radiation. In neutron irradiation at high dose rate withBPA, the mutation induction was controlled mosteffectively. This result suggests that the ascorbate
may protect the mutagenic effects of BNCT on thenormal tissue cells that take up the low dose of boron compounds. They confirmed that the ascorbic
acid was effective in protection of the mutation induction of neutron radiation beam used for BNCT.
PRS-2 investigated the most important biologicaleffects, i.e., DNA damages, after the irradiation withheavy ion particles from BNCR and neutron beam.
They advanced research related to the status of p53

採択課題番号 26P3 硼素中性子捕獲反応(BNCR)誘発粒子線の特性利用の新展開（京大・原子炉）木梨 友子
㻝㻠プロジェクト

PR3-1 The effect of ascorbic acid on the HPRT mutation induction after the neutron irradiation
Y. Kinashi1, K. Ono1, S.Takahashi1
1Research Reactor Institute, Kyoto University

INTRODUCTION: We have previously reported
the increased mutagenicity of thermal neutrons and thedose rate effect of the neutron radiation beam [1].
Clinically, the mutagenic effects of BNCT on the normal tissue cells that do not take up the boron compounds may cause the genetic instability and second
cancer decade years after BNCT. In this study, we inspected whether ascorbic acid was effective in protection of the mutation induction of neutron radiation
beam used for BNCT in Kyoto University Research
Reactor (KUR).

No BPA

With BPANo BPA

With BPAHigh dose rate

Low dose rate

(20cG/min)( 4cG/min)

Neutron irradiation

Neutron irradiation

Fig.1 Induction of mutations in the HPRT locus in
CHO cells with or without BPA under the ascorbic
acid treatment. VC means Vitamin C which is anascorbate popular name.

MATERIALS & METHODS: The mutagenicity
measured by the frequency of mutations induced byneutron irradiation with or without boron compound.
The hypoxanthine-guanin-phosphoribosyl-transferase(HPRT) locus was examined in Chinese hamster ovary(CHO) cells irradiated with neutrons of KUR. A stock
solution of 10B-para-boronophenylanine (BPA) wasused for this experiment. Cell suspensions were incubated with BPA at 10 ppm concentration 1 hour before
neutron irradiation. High dose rate neutron irradiation
was 0.2Gy/min with 5MW of KUR, and low dose rate
neutron irradiation was 0.04Gy/min with 1MW ofKUR. Neutron fluencies were measured by radioactivation of gold foil and gamma-ray dose by TLD. After
neutron exposure, L-ascorbic acid was added to cells
at a final concentration of 5mmol/L and removed after
150 min of neutron irradiation. To determine mutation
frequencies, each treated culture was incubated with
non-selective medium for 7-9 days to allow phenotype
expression. Then, 2x105 cells were added to each dishcontaining 6-thioguanine and incubated for 10-14 days,
after which time the mutant colonies were counted.
The mutation frequency is expressed as the number ofresistant colonies divided by the total number of viable
cells as determined by cloning efficiency at the time ofselection.

0.2Gy/m in was 1.5-1.9 times and more increased thanat 0.04Gy/min in the function dose over the 1.8Gy.
These results suggested that dose rate effect is exist
neutron irradiation in BNCT. Ascorbic acid treatment
reduced mutation induction following neutron radiation. This protective effect of mutation induction was
more effective with BPA than without BPA. This resultshows that an ascorbate scavenged long-lived radicals
due to the nuclear capture reaction that of alpha particles or 7Li nuclei produced by 10B(n,) 7Li reaction,
not due to the reaction with normal tissue hydrogenand nitrogen. In neutron irradiation at high dose rate
(0.2Gy/min) with BPA, the mutation induction wascontrolled most effectively. This result suggests thatthe ascorbate may protect the mutagenic effects ofBNCT on the normal tissue cells that take up the lowdose of boron compounds, for example the peri-tumoral stroma cells after BNCT.
In this study, we found that the ascorbic acid waseffective in protection of the mutation induction ofneutron radiation beam used for BNCT dose rate effectof the neutron radiation beam used for BNCT.
REFERENCES:[1] K. Kinashi et al., Appl. Radiat. Isot. , 88 (2014)
153-156.

RESULTS and DISCUSSION: Figure 1 shows themutation frequency in the HPRT locus in CHO cells
after neutron irradiation at 0.2Gy/min or at
0.04Gy/min with or without BPA. The frequency ofmutations after neutron irradiation with 10ppm BPA at

採択課題番号 26P3-1
BNC 反応により誘発される突然変異の解析（京大・原子炉）木梨友子、小野公二、高橋千太郎

㻝㻡プロジェクト

PR3-2Analysis of DNA Double Strand Breaks in the Mammalian Cells Induced by BNCR

S. Takahashi, Y. Kinashi, K. Seki, N. Yokomizo1, T.
Ikawa1, K. Ono, Y. Kubota2, and R. Okayasu2that difference of radiation sensitivity between A172 andT98G was decreased by BPA addition irradiation of neutron mixed beam.
These results indicate that the difference between theradiation sensitivity was observed between the T98G
cells and A172 cells boron neutron capture reaction. Interestingly, it was reduced with BPA where the irradiation
mainly depends on the particle radiation emitted by
BNCR (Boron Neutron Capture Reaction). This result
suggests that BNCT is an effective treatment withglioblastoma that can reduce the difference ofradiosensitivity by p53 functional status.

Research Reactor Institute of Kyoto University,
Graduate School of Agriculture, Kyoto University,
2
National Institute of Radiological Sciences
1

INTRODUCTION: Boron neutron capture therapy(BNCT) is a unique and effective treatment for cancer,
and now becoming a clinical application stage. However,
little is known about the biological effects of particle radiation induced by BNC reaction (BNCR) nor thermal-epithermal neutron beams for BNCT. In the presentstudy, we have investigated the most important biologicaleffects, i.e., DNA damages, after the irradiation withheavy ion particles from BNCR and neutron beam. In theFY 2014, the relationship between the status of p53 andbiological effects (cell killing and induction of DNA
double strand breaks (DNA-dsb) was investigated.
EXPERIMENTS: Two types of human glioblastoma
cells, A172 and T98G were cultured. Both cells werepurchased from Riken BRC Cell Bank. A172 are wild
type of p53, and T98G cells are mutant type. The cells
were cultured in MEMα medium (Invitrogen) suppl
emented with 10% heat-inactivated FBS (Biowest) andmaintained at 37℃ in a humidified atmosphere with
5.0% CO2. The cells were irradiated at the KUR irradiation field for BNCT, with/without BPA. As a reference
radiation, Co-60 gamma-ray was used at the same dose
rate as the mixed irradiation. The cells were assayed forconventional colony formation, and DNA double strand
breaks (DSBs) were detected by immune-staining using
53BP1 antibodies.
RESULTS & DISCUSSION: The data on the cell survival are shown in Fig. 1 for the cell lines of A172 andT98G. In the case of not BPA added cells, A172 got thesteep slope graph of survival fraction. This shows T98G
cells have lower sensitivity to neutron mixed beam irradiation than A172. On the other hand, in the case of BPA
added cells, survival fraction curves of both cells weresimilar. This shows difference of radiation sensitivity
which confirmed the case of not added BPA cells declined by addition of BPA. When compared at 10% survival, in the case of not added BPA cells, A172 cells wererequired 1.663Gy radiation dose to cause 90% cell death,but T98G required 5.245Gy. This shows T98G were required the dose of 3 times more than the A172 to cause
90% cell death in the case of not added BPA, neutron
mixed beam irradiation alone cases. On the other hand, inthe case of added BPA cells, D10 of A172 indicated 0.821
Gy, and T98G indicated 1.076 Gy. This data indicatedFig. 1. Survival of glioblastoma cells (A172 andT98G) irradiated with thermal- to epithermal neutron
beam of KUR with/without BPA.
PUBLICATION:[1] K. Seki, Y. Kinashi, S. Takahashi:
The influence of p53 status in glioblastoma on the effects
of boron neutron capture therapy. Anticancer Research,
35: 169〜174, 2015.

課題採択番号 26P3-2 BNC 反応に伴う DNA 損傷、特に二重鎖切断とその修復の解析 プロジェクト（京大原子炉）高橋千太郎、木梨友子、関敬子、横溝夏弥、井川智之、小野公二（放医研）久保田善久、岡安隆一

㻝㻢