VIII-Ⅱ-1. Project Research
Project 6PR6

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

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 fromthe 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 mammaliancells induced by BNCR
( S.Takahashi et al. )PRS-3 Application of BNCR to plant tissue culture formutation breeding( M. Kirihata T.Morikawa K Mishiba et al.)PRS-4 Development of pharmacokinetic using boron trace
drugs( H. Hori et al. )Main Results and Contents
PRS-1 performed the mutation analysis induced by a
bystander effect following neutron irradiation. The radiation-induced bystander effects refer to the induction of
biological effects in cells that are not directly traversed
by a charged particle, but are instead located in close
proximity to irradiated cells. They investigated the mutagenic effects of neutron on cells that did not containboron, but were located near cells that contained 10B.
Due to the bystander effect, the frequency of mutations
increased in the cells located nearby the 10B-containing
cells compared with control cells.
PRS-2 determined the cell survival and focus formation of repair protein of DNA double strand breaks in
CHO/K1 and xrs-5 cells after the irradiation of neutron
mixed beam of KUR heavy water facility at an operating
power of 5 MW. The mixed beam irradiation seemed tobe more effective in CHO-K1 cells and slightly effectivein xrs5 cells than control Co-60 gamma-ray irradiation.
Although the doses were available below 2.6 Gy, extrapolation of the best fit line indicated that the relative biological effectiveness for 10% survival is 3.3 and 1.2 for
CHO-K1 and xrs5 cells, respectively.
The aim of PRS-3 is to establish a novel mutagenesis
system using boron neutron capture reaction (BNCR) in

higher plants. The progeny test is required for getting the homozygous mutants in diploid plant follow-

ing irradiation. To determine the effectiveness of BNCR
for plant mutagenesis at irradiation of dry seed stage,
two-row-barley (Hordeum vulgare; 2n=2X=14) plants
were used in the present study because of its early maturity and sensitivity to radiation. Maximum aberration
rate (23.3%) was observed at 800μM BPA and after thatthe rate at 1000μM went down to 21.5%. Linear regression analysis was carried out between chromosome aberration rate (Y %) and BPA concentration (X μM). As theresult of the analysis, there was a linear regression at 5 %significant level. The formula was Y=0.12X+11.24.
Death rates after 60 days were recorded at fifteen treatedgroups. The death rate gradually increased and was top at
800μM BPA of 1 and 1.5 hours irradiations. This is thesame situation with the chromosome aberration rate in
the primary root-tip cells. The growth rate estimatedfrom seedling height for 4 weeks, however, was negatively correlated with the BPA concentrations of 1.5
hours irradiation. The optimum BNCR condition (at800μM BPA of 1 and 1.5 hours irradiations) for expecting the maximum mutation rate was found out.
PRS-4 previously developed boron tracedrugs UTX-42,
UTX-43, and UTX-44, which possess antioxidant potency and UTX-42, UTX-44, UTX-47, UTX-50, and
UTX-51 as boron tracedrugs for NDT targeted a model
protein BSA. Among boron tracedrugs tested previously,
they choose here the boron tracedrug, UTX-51, for ourpresent PK and NDT study to explore their dynamic,
beyond chemical, effects when acquired by weak thermal
neutron irradiation of low-density lipoprotein (LDL),
which is associated with significantly increased strokemortality, treated with the boron tracedrug UTX-51. As aresult, the combination of 4.6 µg LDL with three different concentration of 1, 10, 100 mM of the boron
tracedrug UTX-51 showed a decrease in band intensity
after neutron irradiation (1.830 Gy for 1 µM; 1.835 Gy
for 10 µM; 1.880 Gy for 100µM). In conclusion, all doses of the boron tracedrug UTX-51 caused destructive
dynamic damage against LDL during thermal neutron
irradiation, suggesting boron tracedrugs could be used as
dynamic drugs for NDT targeted LDL as well as a
tracedrug for PK.

採択課題番号 23P6 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

プロジェクトPR6-1

Mutation Analysis Induced by a Bystander Effect following Neutron Irradiation

Y. Kinashi, K. Ono and S.Takahashi

INTRODUCTION: We have described the mutagenicity of thermal neutrons in CHO cells and presented evidence for the increased mutagenicity ofthermal neutrons in the presence of boron (1, 2). In
those previous experiments, boron was located both
inside and outside of the cells. Clinically, the problem
with BNCT is the potential mutagenic effects of thetherapy on the normal tissue cells that do not take up
the boron compounds, but are located near the boron-containing tumor cells. Evidence for the existence
radiation-induced bystander effects exposed to alphaparticles was first reported by Nagasawa and Little.
These radiation-induced bystander effects refer to the
induction of biological effects in cells that are not directly traversed by a charged particle, but are instead
located in close proximity to irradiated cells. In thisstudy, we investigated the mutagenic effects of neutronon cells that did not contain boron , but were locatednear cells that contained 10B. And more, we have investigated the mutagenic effects of no-boron cells under the neutron-irradiated medium those were locatedcells that contained 10B.
MATERIALS & METHODS: The lethality andmutagenicity measured by the frequency of mutations
induced in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus were examined in Chinese
hamster ovary (CHO) cells irradiated with neutrons(Kyoto University Research Reactor).
Experiment 1/ Neutron were irradiated to the 1:1 mixtures of cells with and without 10B. The cells that did
not contain 10B made up 99.4% of the resulting cell
population. The mutant frequency was estimated as afunction dose of the neutron.
Experiment 2/ The 10B containing cells and no-boron
cells were separately irradiated by KUR neutron.
The neutron-irradiated medium located cells that contained 10B was taken and poured to the cells that did
not contain 10B. The mutant frequency was estimatedof neutron irradiated cells without 10B.

Due to the bystander effect of the experiment 1, thefrequency of mutations increased in the cells locatednearby the 10B-containing cells compared with control
cells. However, the experiment 2 did not induce thefrequency of mutations increased in the cells with themedium located at the 10B-containing cells comparedwith control cells. These results suggested that in
BNCT, the mutations caused by the bystander effectand those caused by the original neutron irradiation are
induced by different mechanisms.
20
18
16

Mutant fraction per 105 cells

Research Reactor Institute, Kyoto University

14
12
10
8
6
4
2
0

0

1

2

3

4

5

6

Radiation Dose (Gy)Fig.1 Induction of mutations in the HPRT locus in
Fig.3ACHO cells after the neutron irradiation.
Plots of the mutant frequencies obtained with irradiated samples of no-boron cells (●), Exp1-bystander
cells (▲), and Exp2-bystander cells (■) are shown.
REFERENCES:
1) Kinashi Y et.al., Mutat Res., 337(1997) 211-215.
2) Kinashi Y et al., Radiat Res., 154(2000)313-318.

RESULTS and DISCUSSION: Figure 1 shows themutation frequency in the HPRT locus in CHO cells
after neutron irradiation of the experiment 1 and 2.

採択課題番号 23P6-1
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

プロジェクトPR6-2Analysis of Double Strand Breaks in the Mammalian Cells Induced by BNCR

S. Takahashi, K. Okumura, Y. Kinashi, K. Ono,
Y. Kubota1 and R. Okayasu2
Research Reactor Institute of Kyoto University,
Graduate School of Agriculture, Kyoto University,
1
Radiation Protection Center, National Institute ofRadiological Sciences,2International Research Laboratory, National Institute ofRadiological Sciences
INTRODUCTION: Boron neutron capture therapy(BNCT) is a useful modality for cancer therapy in somecases with relatively low side effects. But, as similar to theother radiotherapy, normal as well as tumor cells are
exposed to a mixed radiation field (thermal, epithermal
and fast neutrons, and gamma-rays). However, little is
known about the biological effects of such radiation
exposures as used for BNCT. It is generally known thathigh LET radiation may have other biological reaction
than ordinal low LET radiation such as X-rays[1].Here,therelative biological effectiveness (RBE), and the dose anddose rate effectiveness factor (DDREF) for the mixedirradiation used for BNCT in Kyoto University Research
Reactor (KUR), were investigated. In the present study, as
the endpoints for biological reaction, DNA double strand
breaks were used because of its significance for cancer
induction and tissue damages.
MATERIALS & METHODS: Commercial cultured
cells CHO/K1were used. Their mutant cell line, xrs-5,
were given from J.Peggo through Dr. Okayasu. The cells
were irradiated at the KUR irradiation field for BNCT.
The details of radiation field were shown in Table 1. The
thermal neutron was approximately one fourth of total
physical dose, and half of physical dose was attributable
to the gamma ray. The average physical dose rates ofthermal (<0.5eV), epithermal (0.5eV-10keV), fast(>10keV) neutrons, and gamma-rays were 10.0, 1.1, 7.4,
and 20.5 mGy/min, respectively, when the reactor was
operated at 1MW. When operated at 5MW, the dose rates
became approximately 5 times higher than those for 1
MW.
Table 1. Physical radiation doses used for the experiments.
Values are expressed as mGy/min.
Thermal(<0.5eV)

Epi-thermal(0.5eV-10keV)

Fast(>10keV)

Gamma

Total

9.0-11

1.1-1.2

6.8-7.6

19-21

36-40

As a reference radiation, Co-60 gamma-ray was used at
the same dose rate as the mixed irradiation. The cells wereassayed for conventional colony formation, and DNA
double strand breaks (DSBs) were detected byimmune-staining using gamma-H2AX and 53BP1
antibodies.
RESULTS & DISCUSSION: In this year, we mainly
focused to establish an irradiation field for theexperiments. As shown in Table 1, relatively high physicaldoses of thermal neutron could be used for the experiment,
but the contamination of gamma ray was significantly
large. Thus, we need to use the prolong gamma irradiation
as a reference irradiation (control).
At first, cell survival was determined by colony
formation in both CHO/K1 and xrs-5 cells, respectively.
The experiment was under progress at present, thesensitivity was higher in xrs-5 cells than CHO/K1 cells,
indicating the cell survival (cell killing) under these
irradiation conditions also attributable to the DNA double
strand breaks, because xrs-5 is a cell line that deficient the
enzyme to repair DNA double strand breaks. The
experiment are being continued to determine relative
biological effectiveness (RBE) for this BNCT irradiation
condition.
We also tried to detect the DNA double strand breaks
with using the immune-chemical staining method. The
cells were fixed at scheduled time after the irradiation, andstained with anti-gamma-H2AX and 53BP1 antibody.
Although the data are under analysis now, but the numberof gamma-H2AX and 53BP1 foci 1hr post-irradiation
were similar for the mixed radiation and the reference
gamma-ray, and the size of foci seemed to be different.
This may indicate that the neutrons may have induced adifferent type of DNA-DSBs.
REFERRENCES:[1] Okayasu, R., Okada, M., Okabe, A., Noguchi, M.,
Takakura, K. and Takahashi, S.: Repair of DNA
damage induced by accelerated heavy ions inmammalian cells proficient and deficient in the
Non-homologous End-joining pathway, Radiation
Research, 165, 59-67, 2006.

(10.0)*(1.1)(7.4)(20.5)(39)*Values in parenthesis are averages of 3-6 experiments.

採択課題番号 23P6-2 BNC反応に伴うDNA損傷、特に二重鎖切断とその修復の解析（京大・原子炉）高橋千太郎、奥村覚二、木梨友子、小野公二（放射線医学総合研究所）久保田善久、岡安隆一

プロジェクトPR6-3

Application of Boron Neutron Capture Reaction (BNCR) to Barley Seeds for
Mutation Breeding

T. Morikawa, S. Iwamoto, K. Mishiba and M. Kirihata
Graduate School of Life and Environmental Sciences,
Osaka Prefecture University

INTRODUCTION: The aim of the present study is toestablish a novel mutagenesis system using boron
neutron capture reaction (BNCR) in higher plants.
Radiation (e.g. x-ray, γ -ray and thermal neutron)
usually knocks out one pair of alleles at a target locus
andgenerates
loss-of-function
mutationsat[1]heterozygote . Therefore, the progeny test is requiredfor getting the homozygous mutants in diploid plant. To
determine the effectiveness of BNCR for plantmutagenesis at irradiation of dry seed stage,
two-row-barley (Hordeum vulgare; 2n= 2X= 14) plants
were used in the present study because of its earlymaturity and sensitivity to radiation.
EXPERIMENTS: The dry seeds of barley plants were
immersed in different concentrations (0, 400, 600, 800
and 1000 μM) of 10B-enriched p-boronophenylalanine(BPA) for 24hours, and the seeds were irradiated withthermal neutron for 0, 1 a nd 1.5 hours in the Kyoto
University Research Reactor (KUR). The irradiated seeds
were kept at 4 ℃ for overnight, and then were
germinated on a p lastic petri-dish containing moisturefilter paper at 25 ℃.

was carried out between chromosome aberration rate
(Y %) and BPA concentration (X μM). As the result of
the analysis, there was a linear regression at 5 %significant level. The formula was Y= 0.12X+11.24.
Death rates after 60 days were recorded at fifteen treatedgroups. The death rate gradually increased and was top at
800μM BPA of 1 and 1.5 hours irradiations. This is thesame situation with the chromosome aberration rate in
the primary root-tip cells. The growth rate estimatedfrom seedling height for 4 weeks, however, wasnegatively correlated with the BPA concentrations of 1.5
hours irradiation.
We have found out the optimum BNCR condition (at800μM BPA of 1 a nd 1.5 hours irradiations) for
expecting the maximum mutation rate. This result
indicates that the dry seeds of two-row-barley plant seemto absorb equal amounts of BPA into the cells dependingon the BPA concentrations treated.
REFERENCES:[1] Y. Ukai and A.Yamashita, Breeding Science60(2010)121.
[2] Y.Ukai, I.Yamaguchi and H.Takaki, Breeding Science
59(2009)129.

RESULTS: Germination rate of the irradiated seeds
were measured. The seeds were germinated normally at
all fifteen treated groups (five BPA concentrations ×
three irradiation times) at the range of from 60% to 100%.
There was no relationship between germination rate anddose effect[2]. Chromosome aberrations at primaryroot-tip cells of barley can easily examine and determine
the effect of BNCR to plant-cell divisions. The anaphasecells with chromosome aberrations at 1.5 hours irradiatedgroups were counted. The frequency of chromosome
aberrations was positively correlated with theconcentration of BPA. Maximum aberration rate (23.3%)
was observed at 800μM BPA and after that the rate at
1000μM went down to 21.5%. Linear regression analysis

採択課題番号 23P6-3

硼素中性子捕獲反応(BNCR)の植物育種への応用（大阪府立大）切畑光統、森川利信、大門弘幸、三柴啓一郎、

プロジェクト2

Design of Boron Tracedrug for PK and NDT N

Institute of Technology and Science, Graduate School
The University of Tokushima
1
Research Reactor Institute, Kyoto University
INTRODUCTION: The evaluation of pharmacokinetic(PK) properties and ADME-tox of drug candidates, being
under development, and drugs, available on the market,
has recently become an increasingly important factor of
drug discovery and development, because of increased
needs of targeted drugs with less adverse effects.
This needs accelerate medicinal chemists to develop
drugs with higher traceability, even in their whole lifetime. Radiolabeled compounds have been still available
with some inherent problems such as their half-life andthe specific regulation of experimental facilities.
For the purpose of overcoming these problems and creating traceable drugs without RI forever, we are developing
boron tracedrugs with their “on demand” traceability andtheir physical force for neutron dynamic therapy (NDT).
We previously developed boron tracedrugs UTX-42,
UTX-43, and UTX-44, which possess antioxidant potency [1] and UTX-42, UTX-44, UTX-47, UTX-50, and
UTX-51 as boron tracedrugs for NDT targeted a model
protein BSA [2].
Among boron tracedrugs tested previously, we choose
the boron tracedrug, UTX-51, for our present NDT study
to explore their dynamic, beyond chemical, effects when
acquired by weak thermal neutron irradiation of
low-density lipprotein (LDL), which is associated withsignificantly increased stroke mortality, treated with theboron tracedrug UTX-51.
EXPERIMENTS: The curcuminoid boron tracedrugs
UTX-51 (it contains boron isotopes, B-10 and B-11 with
their natural abundance ratio) was used for neutron dynamic therapy. Thermal neutron irradiation was performed using a reactor neutron beam with a cadmium(Cd) ratio of 9.4. The neutron fluence was measured fromthe radioactivation of gold foils at the front of the sample
tubes, and the average neutron fluence determined fromthe values measured was used. Contaminating γ-ray doses, including secondary γ-rays, were measured withthermoluminescence dosimeter powder at the front of thesample tubes. The absorbed dose was calculated using theflux-to-dose conversion factor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
was performed to detect the decomposition by thermal
neutron irradiation of human LDL and PARP treated withthese five boron tracedrugs.
RESULTS:	 The combination of 4.6 µg LDL with three

BF2

HO

UTX-47

N 10, 100 µM of the boron
different concentration of 1,
tracedrug UTX-51 showed a decrease in band intensity
after neutron irradiation(1.830 Gy for 1 µM; 1.835 Gy for10 µM; 1.880 Gy for 100µM).
In conclusion, all doses of the boron tracedrug UTX-51
F2
damage against LDL during
caused destructive dynamic
Bsuggesting boron tracedrugs
thermal neutron irradiation,OO
could be used as dynamic drugs for NDT targeted LDL.

UTX-50OH

HO

F2
B第 ２ 章  結 果 と 考 察OO

ボロントレースドラッグとしては、これまでに開発した UTX-51 を用いた。また、ヒトの血液O

O LDL を破壊実験に用いた。ヒト単離 LDL 濃度を 0.23 g/L と 0.023 g/L で固定し、から精製したUTX-51UTX-51 の濃度をそれぞれ 1 μM、10 μM、100 μM とふった。その後、京都大学原子炉にて中性子照射をした。その後、LDLの破壊の有無を SDS-PAGE で評価した。その結果、LDLの濃OH
HO
度が 0.023 g/L の場合、再現性が取れなかった。これは中性子照射時の LDL 濃度（全量が）少な
すぎて誤差が出来たためと考えられる。しかしながら、化合物存在下において、中性子照射して第２章 結果と考察いないバンドも薄くなっていることがわかる。一方で、LDL
濃度がを用いた。また、ヒトの血液0.23 g/L の場合、化合物濃度
ボロントレースドラッグとしては、これまでに開発した UTX-51が増加するにつれLDLを破壊実験に用いた。ヒト単離のバンドの濃さも増加する傾向が確認された。しかしながら、UTX-51
から精製した LDL
LDL 濃度を 0.23 g/L と 0.023 g/L で固定し、 のUTX-51の濃度をそれぞれ
1 μM、10 μM、100 μM とふった。その後、京都大学原子炉にて
濃度が
100μM
の場合は再現性がとれなかった。今後、濃度や評価方法を検討する必要があると中性子照射をした。その後、LDL の破壊の有無を SDS-PAGE で評価した。その結果、LDL の濃考えられる。
度が 0.023 g/L の場合、再現性が取れなかった。これは中性子照射時の LDL 濃度（全量が）少な
すぎて誤差が出来たためと考えられる。しかしながら、化合物存在下において、中性子照射して実験結果 ①Fig.
1. The curcuminoid boron tracedrug,
UTX-51,いないバンドも薄くなっていることがわかる。一方で、LDL
濃度が 0.23 g/L の場合、化合物濃度
1. LDLが増加するにつれLDL のバンドの濃さも増加する傾向が確認された。しかしながら、UTX-51 の
for
NDT.
2. LDL＋中性子照射
F2
B
O
O
O
O
濃度が 100μM の場合は再現性がとれなかった。今後、濃度や評価方法を検討する必要があると

3.

LDL＋UTX-51（100μM）

4.

LDL＋UTX-51 (1μM)＋中性子照射、考えられる。

HO

5.

LDL＋UTX-51（10μM）＋中性子照射
実験結果 ①LDL＋UTX-51（100μM）＋中性子照射
1. LDL2.

LDL＋中性子照射

3.

LDL＋UTX-51（100μM）

4.

LDL＋UTX-51 (1μM)＋中性子照射、

OH

UTX-51

6.

O

1

2

3

O
O

HO

OH

4

Control

F2
B

O

5

5.
LDL＋UTX-51（10μM）＋中性子照射
Neutron
中性子照射
＋
‐
＋
‐
irradiation
6.
LDL＋UTX-51（100μM）＋中性子照射＋

UTX-51

100

100
1

2

3

中性子照射
80

‐

＋

‐

60

Control

40 120100

20 100

4

LDL
5

＋

45

1

2

40

＋

58
44

23

0 80
60

6

＋
UTX-51
39

LDL

6

UTX-51＋

120

Band intensity

H. Hori, Y. Nazaumi, Y. Uto, S. Takahashi1, K. Ono11

Band intensity

PR6-4

N

58

345
4
Band No. 39

5

6

44

Fig.
2.破壊実験結果。SDS-PAGE
NDT
of LDLによる評価結果。LDL
using a boron
tracedrug
Fig.1 LDL
濃度 0.23 g/L(中性子照射した全量
20
4.6μg)。UTX-51 濃度：1μM（全量0.0832µg，25:
nmol,
濃度 0.00216
0 Gy）、
0
UTX-51(lane
4: 1 µM,
lane
10 BµM,
laneppm、1.83
6: 100345Gy)、1006 μM（全量 8.32 µg，200
10μM(全量 0.832 µg，201 nmol, 2B 濃度 0.0216
ppm、1.835µM) .
Band No.
23

10

10

68
Fig.1 LDL 破壊実験結果。SDS-PAGE による評価結果。LDL
濃度 0.23 g/L(中性子照射した全量
4.6μg)。UTX-51 濃度：1μM（全量 0.0832 µg，2 nmol, 10B 濃度 0.00216 ppm、1.83 0 Gy）、10μM(全量 0.832 µg，20 nmol, 10B 濃度 0.0216 ppm、1.835 Gy)、100 μM（全量 8.32 µg，200

REFERENCES:
nmol, B 濃度 0.216 ppm、1.88 Gy）。中性子照射時間
45 分。Control を 100 としてバンドの濃68[1]さを数値化。
E. Nakata et al., Adv. Exp. Med. Biol., 737 (2012)
301-306.
実験結果 ②[2]H.
Hori et al., Anticancer Res., 30 (2010) 3233-3242.
10Band No.
1.

LDL

2.

LDL＋中性子照射

3.

LDL＋UTX-51（100μM）

4.

LDL＋UTX-51（1μM）＋中性子照射

5.

LDL＋UTX-51（10μM）＋中性子照射

6.

LDL＋UTX-51（100μM）＋中性子照射

採択課題番号 23P6-4	
  Boron をトレーサとする薬物の動態解析法の開発‫ 	࠻ࠢࠚࠫࡠࡊ 	ޓޓޓޓޓ‬（徳島大学院・ソシオテクノサイエンス研究部）堀	  均、那住善治郎、宇都	  義浩、（京大・原子 LDL炉）高橋	
  千太郎、小野	
  公二	
 
1
2
3
4
5
6
	
 

中性子照射

‐

＋

‐

＋

＋

tensity

Control
120
100
80

100

＋

UTX-51

88

89
71

82
59