VIII-Ⅱ-1. Project Research
Project 8PR8Project Research on the New Applicant Development Using the Characteristics of
the Particles from the Neutron Capture Reaction

Y. Kinashi
Research Reactor Institute, Kyoto University
OBJECTIVES and PARTICIPATINGRESEARCH SUBJECTSIn 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 mammaliancells 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 culture
for mutation breeding( T.Morikawa et al.)PRS-4 Development of pharmacokinetic usingboron trace drugs( H. Hori et al. )tional colony formation, and DNA double strand
breaks (DSBs) were detected by immune-staining
using gamma-H2AX and 53BP1 antibodies. The
focus assay used for detecting the DNA double
strand breaks showed that the number of gamma-H2AX and 53BP1 foci 1hr post-irradiation
were similar for the mixed radiation and the reference gamma-ray. The disappearance of focus
delayed in the xrs5 cells, indicating the impaired
DNA damage repair in these cells. These results
agree well with the high sensitivity of xrs5 cells.
The mean of the foci sizes was not significantly
different between the cell irradiated with gamma-rays and neutron, but the size distribution
seemed to be larger in those irradiated with neutron.
PRS-3 tried to detect the BNCR condition for leaf
tissue culture and callus conditions. To determine
the effectiveness of BNCR for plant mutagenesis
at irradiation of dry seed, leaf disc and callus
stages, haploid and diploid tobacco (Nicotiana
tabacum L.cv. Red Russian) plants were usedbecause of its high callus induction, regenerationand totipotency. Tobacco callus is quite sensitive
to BNCR, and there is little protection of nuclear
proteins during active somatic cell division andhaving weak single strand of DNA. There was nodifference of BNCR sensitivity between haploidand diploid tobacco plants. They found out the
optimum BNCR conditions in tobacco leaf discs,
which were 400, 600 or 800μM BPA and 30 or 60
minutes irradiations for expecting the maximum
mutation rate. This result indicates that the leaf
disc of tobacco plants seem not to absorb enoughamounts of BPA into the cells for overnight
treatment.
PRS-4 developed boron tracedrugs with their “ondemand” traceability and investigated their physical force for neutron dynamic therapy (NDT).
The curcuminoid boron trace drugs UTX-51 wasused for neutron dynamic therapy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS -PAGE) was performed to detect the decomposition by thermal neutron irradiation ofhuman serum Gc protein treated with UTX-51.
They found that the all doses of UTX-51 caused
destructive dynamic damage against Gc protein
during thermal neutron irradiation. These results
suggested that borontrace drugs could be used as
dynamic drugs for NDT targeted Gc protein forserum protein-quality-control treatment.

MAIN RESULTS and CONTENTS
PRS-1 investigated dose rate effect of the neutron radiation beam used for BNCT in Kyoto
University Research Reactor (KUR) for the mutation induction following neutron irradiation.
Neutron irradiation for cells was operated at
0.2Gy/min with 10ppm BPA, at 0.2Gy/min or at
0.04Gy/min without BPA. The frequency of mutations after neutron irradiation at 0.2Gy/min with
10ppm BPA was two times and more increased
than that at 0.04 Gy/min without BPA in the function dose over the 1.8Gy. These results suggested
that dose rate effect is existence concerning mutation induction following the neutron irradiationin BNCT.
PRS-2 investigated the biological effects, i.e.,
DNA damages, after the irradiation with heavy
ion particles from BNC reaction. The induction of
DNA double strand breaks (DNA-dsb) was detected by using the focus assay of repair protein.
The Chinese hamster ovary cells (CHO/K1) andxrs-5, were irradiated at the KUR irradiation fieldfor BNCT. As a reference radiation, Co-60 gamma-ray was used at the same dose rate as themixed irradiation. The cells were assayed forconventional colony formation, and DNA double
strand breaks (DSBs) were detected by immune-staining using gamma-H2AX and 53BP1
antibodies.
cells were assayed for conven採択課題番号 24P8 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Evaluation of Dose Rate Effect for Mutation Induction following
Neutron Irradiation

Y. Kinashi, K. Ono and S.Takahashi
Research Reactor Institute, Kyoto University
INTRODUCTION: We have described the mutation
frequency of CHO cells after neutron irradiation andpresented evidence for the increased mutagenicity ofthermal neutrons [1,2]. High dose rate effect has been
generally known in gamma or x-ray irradiation. On theother hand, little is known about dose rate effect ofneutron irradiation. Clinically, the problem with
BNCT is the potential mutagenic effects of the therapy
on the normal tissue cells that do not take up the boron
compounds. We investigated dose rate effect of theneutron radiation beam used for BNCT in Kyoto University Research Reactor (KUR) for the mutation induction following neutron irradiation.
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) was used for this experiment. Cell suspensions
were incubated with BPA at 10 ppm concentration 1hour before neutron irradiation. High dose rate neutron
irradiation was 0.2Gy/min with 5MW of KUR, andlow dose rate neutron irradiation was 0.04Gy/min with
1MW of KUR. Neutron fluencies were measured byradioactivation of gold foil and gamma-ray dose by
TLD. After neutron exposure, 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 of
selection.
RESULTS and DISCUSSION: Figure 1 shows themutation frequency in the HPRT locus in CHO cells
after neutron irradiation with 10ppm BPA at
0.2Gy/min, without BPA at 0.2Gy/min or at

採択課題番号 24P8-1

0.04Gy/min. The frequency of mutations after neutron
irradiation with 10ppm BPA at 0.2Gy/min was twotimes and more increased than that without BPA at
0.04Gy/min in the function dose over the 1.8Gy.
These results suggested that dose rate effect is exist-

ence concerning mutation induction following
neutron irradiation in BNCT.

Mutation Fraction (per 105 cells)

PR8-1

Radiation Dose (Gy)Fig.1 Induction of mutations in the HPRT locus in
CHO cells after the neutron irradiation.
Plots of the mutant frequencies obtained with irradiated samples following KUR neutron irradiation with
10ppm BPA at 0.2 Gy/min (▲), without BPA at 0.2
Gy/min (■) and 0.04Gy/min (●) are shown. Plot of
the mutant fraction as a function of the radiation dose.
In this study, we found dose rate effect of the neutronradiation beam used for BNCT for the mutation induction of normal cells following neutron irradiation.
This dose rate effect of BNCT was mainly due to the
nuclear capture reaction that of alpha particles or 7Li
nuclei produced by 10B(n,α) 7Li reaction, not due tothe reaction with normal tissue hydrogen and nitrogen.
REFERENCES[1] Kinashi Y et al., Radiat Res., 154(2000)313-318.
[2] Kinashi Y et al., Int J Radiat Oncol Biol Physics,
68(2007)508-514.

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PR8-2Analysis of DNA Double Strand Breaks in Mammalian Cells Inducedby BNCR

S. Takahashi, K. Okumura1, Y. Kinashi, K. Ono,
Y. Kubota2 and R. Okayasu2
Research Reactor Institute of Kyoto University,
1
Graduate School of Agriculture, Kyoto University,
2
National Institute of Radiological Sciences,
nternational Research Laboratory, National Institute ofRadiological Sciences
INTRODUCTION: Boron neutron capture therapy(BNCT) is unique and effective treatment for cancer, andnow becoming a clinical application stage. However, little
is known about the biological effects of such radiation
exposures as used for BNCT. In the projective study, we
have investigated the most important biological effects,
i.e., DNA damages, after the irradiation with heavy ion
particles from BNC reaction. In the FY 2012, theinduction of DNA double strand breaks (DNA-dsb) was
detected by using the focus assay of repair protein.
MATERIALS & METHODS: The cells used were
Chinese hamster ovary cell line (CHO/K1) from Riken
Cell Bank. Their mutant cell line, xrs-5, were given from
P. Jeggo through Dr. Okayasu, NIRS. The cells were
irradiated at the KUR irradiation field for BNCT.
The radiation beam used was a mixed neutron beam
consisting of the thermal, epithermal and fast neutrons,
and gamma-rays. The detail of the irradiation condithons
were described the 2010 Progress Report. The average
physical dose rates of thermal (<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.
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: The cell survival was
determined by colony formation in both CHO/K1 andxrs-5 cells, respectively. As expected, xrs5 cells defective
in repair were more sensitive to both mixed beam andgamma-rays irradiation than CHO-K1 cells.
The focus assay used for detecting the DNA double strand
breaks showed that the number of gamma-H2AX and
53BP1 foci 1hr post-irradiation were similar for the mixed

radiation and the reference gamma-ray. The disappearanceof focus delayed in the xrs5 cells, indicating the impaired
DNA damage repair in these cells. These results agree
well with the high sensitivity of xrs5 cells for the mixed
neutron beam. At present, we did not find any differencein the characteristics and kinetics of focus between themixed neutron beam and the reference gamma-rays. .
The size of foci were also analyzed in some samples. As
shown in Fig. 1, the mean of the foci sizes was not
significantly different between the cell irradiated withgamma-rays and neutron, but the size distribution seemed
to be larger in those irradiated with neutron.

Fig. 1 Size distribution of foci in CHO (K1) and xrs cells
after irradiation with gamma-ray and neutron beam.
Larger foci are observed in the cells irradiated withneutron.
PUBLICATION:
K. Okumura, Y. Kinashi, Y. Kubota, E. Kitajima,
R.Okayasu, K. Ono, and S. Takahashi, Relative biologicaleffects of neutron mixed-beam irradiation for boron
neutron capture therapy on cell survival and DNA
double-strand breaks in cultured mammalian cells. J
Radiat Res (2013) 54 (1): 70-75.

課題採択番号 24P8-2 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、奥村覚二、木梨友子、小野公二（放医研）久保田善久、岡安隆一

PR8-3

Application of Boron Neutron Capture Reaction (BNCR) to
Tobacco Plants for Mutation Breeding

T. Morikawa, S. Iwamoto, Y. Hattori and
M. Kirihata
Graduate School of Life and Environmental Sciences, Osaka Prefecture University
INTRODUCTION: The present study was carriedout to establish a novel mutagenesis system usingboron neutron capture reaction (BNCR) in higher
plants. Radiation (e.g. x-ray, gamma-ray andthermal neutron) usually knocks out one pair ofalleles at a target locus and generates
loss-of-function mutations at heterozygote. Thistime, we try to detect the BNCR condition forleaf tissue culture and callus conditions. To determine the effectiveness of BNCR for plant mutagenesis at irradiation of dry seed, leaf disc andcallus stages, haploid and diploid tobacco (Nicotianatabacum L.cv. Red Russian) plants wereused because of its high callus induction, regeneration and totipotency.
EXPERIMENTS: The dry seed, leaf disc and cal-lus of haploid and diploid tobacco plants were
immersed in different concentrations (0, 400, 600,
10and
800μM)
ofB-enrichedp-boronophenylalanine (BPA) for 24hours, andall the materials were irradiated with thermal
neutron for 0, 30 and 60 minutes in the Kyoto
University Research Reactor (KUR). The irradiated seeds were kept at room temperature forovernight, and then were germinated on a plastic
petri-dish containing moisture filter paper at 25 .
The irradiated leaf disc and callus were instantly
transferred to new MS medium with containing
Auxin(2,4-D 2ppm) and Cytokinin(Kinetine
0.25ppm) for callus induction of leaf disc andcallus sub-cultures. After regeneration of them
the plantlets were transplanted to the pots filledwith mixtures of soil and compost. Survival, dedifferentiation and regeneration rates were examinedin the leaf discs and calluses.

times) at the range of from 60% to 100%. There
was no relationship between plant size of seedlings and dose effect of BPA and irradiation times.
Tobacco seeds seem to be highly resistant to
BNCR treatment. Survival, dedifferentiation andregeneration rates were also not affected with
BPA concentrations in the leaf discs. Some mutants appeared in the M 1 V 1 regenerated plants
obtained from leaf disc calluses. For example,
slender, dwarf, flower abnormal, male sterility,
multi-branching and variegation plants were obtained in M 2 V 1 generation. All irradiated calluses,
however, were suddenly dead showing chlorosisduring one month culture. This result indicatedthat tobacco callus is quite sensitive to BNCR
because there is little protection of nuclear proteins during active somatic cell division and having weak single strand of DNA. There was nodifference of BNCR sensitivity between haploidand diploid tobacco plants.
We have found out the optimum BNCR conditions in tobacco leaf discs,whichwere 400, 600 or
800μM BPA and 30 or 60minutes irradiations for
expecting the maximum mutation rate. This result
indicates that the leaf disc of tobacco plants seemnot to absorb enough amounts of BPA into thecells for overnight treatment.

RESULTS: The irradiated tobacco seeds were

germinated normally at all fifteen treated groups(five BPA concentrations × three irradiation

採択課題番号 24P8-3 硼素中性子捕獲反応(BNCR)反応の植物育種への応用
プロジェクト（大阪府立大学・生命環境科学研究科）森川利信・切畑光統・服部能英・岩本千里

PR8-4

Design of Boron Tracedrug for PK and NDT

H. Hori, R. Tada, Y. Uto, S. Takahashi1and K. Ono1
Institute of Technology and Science, Graduate School
The University of Tokushima
1
Research Reactor Institute, Kyoto University

RESULTS: The combination of 0.5 nmol Gc protein(54 KD, 27 µg) with three different B-10 concentration of
10, 25. and 50 nmol of the boron tracedrug UTX-51(MW 416.12 g/mol; 50 nmol or 20.8 µg, 125 nmol or
52.0 µg, and 250 nmol or 104.0 µg, for 10 nmol B-10, 25
nmol B-10, and 50 nmol B-10, respectively) showed adecrease in band intensity after neutron irradiation.
In conclusion, all doses of the boron tracedrug UTX-51
caused destructive dynamic damage against Gc protein
during thermal neutron irradiation, suggesting boron
tracedrugs could be used as dynamic drugs for NDT targeted Gc protein for serum protein-quality-control treatmet.

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
F2
drugs with higher traceability, even in their whole lifeB
O
O
OH
time. ORadiolabeled
compounds have been still available
Owith some inherent problems suchas theirBFhalf-life
O
O
3OEtand2
the specific regulation of experimental facilities.
Et 2O andr.t.
1h!"タンパク質を標的とした#$%&'(の分子破壊力評価䈊
For the purposeof overcoming OH
these problems
creatHOOH
F
ing traceable drugs without
RI forever, we are developingBOO
97％ andboron tracedrugs
with their “on demand”
traceability
OO'()*(+,-!"#$%&
their physical force for neutron dynamic therapy (NDT).
←!"タンパク質/#$%&'(HOOH
We previously developed boron tracedrugs UTX-42,////
2''/3456/
E>F>/B(A>(@UTX-43, and UTX-44, which possess antioxidant potenFig. 1. The curcuminoid boron tracedrug, UTX-51,
cy [1] and UTX-42, UTX-44, UTX-47, UTX-50, andfor NDT.
・クルクミンに!"$%ｔを反応させて'()*+,を合成した。
C'/ !"タンパク質を標的とした#$%&'(の分子破壊力評価䈊
'0/(0/# )*"+,-./&
UTX-51 as boron#$%&'(
tracedrugsfor
NDT targeted a model
(01
*<=+
*<=+ *<=+!"タンパク質を標的とした#$%&'(の分子破壊力評価䈊
!"タンパク質を標的とした#$%&'(の分子破壊力評価䈊
protein BSA [2] and UTX-51 for LDL [3].

15*-/)*7.*8)79
(>00 0>?@
0>?A 0>@? 0>BC
Among boron tracedrugs
tested previously,
we choose‐＋＋＋＋$:.;<5+/*.,7;=*/←!"タンパク質/!"タンパク質/#$%&'(the boron tracedrug,
UTX-51, for our present NDT study←
Gc protein#$%&'(← !"タンパク質/#$%&'(////2''/3456/////
2''/3456/
E>F>/B(A>(@////2''/3456/
D=*7;=+/
to explore their dynamic, beyond chemical,
effects when #$%&'( ＋；With
E>F>/B(A>(@
E>F>/B(A>(@irradiation‐；Without irradiation
acquired by weak thermal neutron irradiation of human
照射前の各サンプル中の!"タンパク質量 ： 0>'/*<=+
C'/ '0/'0/C'/ (0/'0/ C'/(0/
serum Gc protein, which is related with serum pro(01(01#$%&'()*"+,-./(0/*<=+*<=+#$%&'()*"+,-./*<=+*<=+*<=+*<=+ *<=+
(01
*<=+
#$%&'()*"+,-./
GcProtein treated with boron tracedrug on SDS-PAGE gel stained
with
*<=+
tein-qualityCoomassie
control, treatedwith (CBB)
the boron
tracedrug(>00 (>00
0>?@ 0>?A
0>BC 0>@? 0>BC
Brilliant Blueand Bandintensity. Irradiation:15*-/)*7.*8)79
thermalneutron15*-/)*7.*8)79
0>?@0>@?
0>?A
15*-/)*7.*8)79
0>?@
0>?A
‐ (>00＋＋＋＋ 0>@? 0>BC
UTX-51. (Absorbed dose 0.44 Gy at the present of 10 nmol 10B ; Time 45$:.;<5+/*.,7;=*/
min).
Band
‐
＋
＋
＋
＋
$:.;<5+/*.,7;=*/!"#$%&の合成

O

HO

2

O

O

F2O
B
O
O

F
F2 B 2
OB
O
O

O

O

O

O

HO
HO

HO

OH

OH
OH

‐
$:.;<5+/*.,7;=*/ D=*7;=+/intensity were measured with SWEDAY Just TLC soft (control = 1.00).

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 serum Gc protein treatedwith the boron tracedrug UTX-51.

＋

D=*7;=+/
D=*7;=+/＋#$%&'(＋＋＋；Withirradiation#$%&'(#$%&'(‐；Withoutirradiation
＋；With irradiation

照射前の各サンプル中の!"タンパク質量 ： 0>'/*<=+
＋；With irradiation‐；Without irradiation
照射前の各サンプル中の!"タンパク質量0>'/*<=+
GcProteintreated with boron tracedrug on SDS-PAGE
gel stained with ‐；Without irradiation
照射前の各サンプル中の!"タンパク質量：：0>'/*<=+
Coomassie
Brilliant
Blue
(CBB)
and
Band
intensity.
Irradiation:
thermalneutronGcProtein treated with boron tracedrug 10
on SDS-PAGE gel stained with
GcProtein
tracedrug
gelBandstained with; Time 45 min).
(Absorbed
dosetreated
0.44 Gywith
at theboronpresentof 10 nmolonBSDS-PAGE
Coomassie
Brilliantwith
Blue(CBB) Just
and TLC
Band intensity.
Irradiation:
thermal neutron
intensity
were measured
SWEDAY(control =1.00).
Coomassie
Brilliant Blue
(CBB) and
Bandsoft
intensity.
Irradiation:
thermal neutron
10(Absorbed dose 0.44 Gy at the present of 10 nmol B ; Time 45 min). Band
(Absorbed dose 0.44 Gy at the present of 10 nmol 10B ; Time 45 min). Bandintensity were measured with SWEDAY Just TLC soft (control = 1.00).
intensity were measured with SWEDAY Just TLC soft (control = 1.00).

Fig. 2. NDT of Gc protein using a boron tracedrug
UTX-51REFERENCES:[1] E. Nakata et al., Adv. Exp. Med. Biol., 737 (2012)
301-306.
[2] H. Hori et al., Anticancer Res., 30 (2010)3233-3242.
[3] H. Hori et al., Adv. Exp. Med. Biol., accepted, 2013.

採択課題番号 24P8-4	
 Boron をトレーサとする薬物の動態解析法の開発	
 （徳島大学院・ソシオテクノサイエンス研究部）堀 均、多田 竜、宇都義浩（京大・原子炉）高橋千太郎、小野公二	
 プロジェクト