Project ResearchProject 13

PR13

Project Research on a Study on Biological Character and use of the Particle Induced by theBoron Neutron Capture Reaction

Y. Kinashi

has been stopping during this year.

Research Reactor Institute, Kyoto University

PRS-3 was not able to carry out its experiments
because KUR has been stopping during this year.

Objectives and Participating Research Subjects
In this project, we are intending to develop thenew application using the characteristics of the particles from the neutron capture reaction.

PRS-4 was not able to carry out its experiments
because KUR has been stopping during this year.

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 themammalian cells induced by BNCR
( S.Takahashi et al. )PRS-3 Development of the PARP repressor reinforced in its function by BNCR
( Y.Uto et al. )PRS-4 Development of the model animal shouing theblood vessel damage by BNCR
( R. Wate et al. )Main Results and Contents
PRS-1 studied gamma-H2AX focus data and investigated the DNA-dsb (double strand breaks)damage and association of the mutation induction
following the neutron radiation beam used for
BNCT in KUR. The mutagenic effects of BNCT on
the normal tissue cells that do not take up the boron
compounds may cause the genetic instability andsecond cancer decade years after BNCT.
The mutagenicity measured by the frequency ofmutations induced by neutron irradiation with orwithout boron compound.
The HPRT locus wasexamined in Chinese hamster ovary (CHO) cells
irradiated with neutrons of KUR.
The frequency of mutations after neutron irradiation with 10ppm BPA was about 5 times and moreincreased than 1Gy gamma-ray irradiation. thegamma-H2AX foci size at after 1Gy of neutron irradiation with 10ppm BPA. After 2 hours of neutronirradiation, about 20% of cells had big foci,The result suggestted that The mutation induction
might be increased due to the inferiority of the ability of DNA damage repair following BNCR.
PRS-2 was not able to carry out its experiments
because KUR (Kyoto University Research Reactor)
27P13- 58 -PR13-1

Association of the DNA-double Strand Breaks Damage andthe Mutation Induction Following the Neutron Radiation Beam used for BNCTY. Kinashi and S.Takahashi

RESULTS and DISCUSSION: Table 1 shows themutation frequency in the HPRT locus in CHO cells
after 1Gy neutron irradiation. The frequency of mutations after neutron irradiation with 10ppm BPA wasabout 5 times and more increased than 1Gy gamma-ray irradiation.

Research 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, weanalyzed gamma-H2AXfocus data and investigatedthe DNA-dsb (double strand breaks) damage and association of the mutation induction following the neutron radiation beam used for BNCT in Kyoto University Research Reactor (KUR).

Table 1 HPRT mutation frequencies of CHO cells exposed to 1Gy of gamma-rays, neutron and BNCR
GammaNeutron
BNCR(10ppmrayBPA)

HPRT mutationfrequency / 105cells

1.2±1.5

3.2±2.5

6.8±3.5

Figure 1 shows the gamma-H2AX foci size at after
1Gy of neutron irradiation with 10ppm BPA. After 2hours of neutron irradiation, about 20% of cells had
big foci (>5.0 m2), On the other hand, the cell withthe big focus was less than 1% at 2 hours after gamma
ray 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) wasused for this experiment. Cell suspensions were incubated with BPA at 10 ppm concentration 1 hour before
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 dish containing
6-thioguanine and incubated for 10-14 days, after
which time the mutant colonies were counted. Themutation frequency is expressed as the number of resistant colonies divided by the total number of viable
cells as determined by cloning efficiency at the time ofselection. DNA double-strand breaks (DSBs) in CHO
cells following neutron irradiation were detected using
the gamma H2AX foci assay. Immunofluorescencestaining was performed as described previously (2).
Briefly, irradiated cells were incubated 30 min.or 120min., and washed with cold PBS. After fixation with
3.6% formalin Immunofluorescence images were acquired using a fluorescence microscope (KEYENCE,BZ-9000), and then analyzed using the BZ-9000 optional software and Image-J (National Institutes ofHealth).

%Fig.1 gamma-H2AX foci size at 30min. (white bars) (m )and 120min. (black bars) after 1Gy of neutron irradiation with 10ppm BPA.
2

In this study, we found that the large foci which are aserious DNA damage increased by BNCR. The mutation induction might be increased due to the inferiorityof the ability of DNA damage repair following BNCR.
REFERENCES:[1] K. Kinashi et al., Appl. Radiat. Isot. , 88 (2014)
153-156.
[2] K. Kinashi et al., Radiation Oncology, 6:106 (2011)27P13-1- 59 -