Applied Radiation and Isotopes 106 (2015) 185188

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Applied Radiation and Isotopes

journal homepage: www.elsevier.com/locate/apradiso

Detection of DNA double-strand breaks in boron neutron capture

reaction
Emiko Okamoto a,b, Tetsuya Yamamoto a, Kei Nakai a,n, Fumiyo Yoshida a, Akira Matsumura a
a

Department of Neurosurgery, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Doctoral Program in Clinical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 3058575, Japan
b

H I G H L I G H T S

The number of double-strand breaks increased with the neutron dose.

The single-strand breaks increased with the neutron dose and the 10B concentration.

Our model can quantify the number of DNA breaks regardless of the repair mechanism.

art ic l e i nf o

a b s t r a c t

Article history:
Received 19 January 2015
Received in revised form
16 July 2015
Accepted 14 August 2015
Available online 17 August 2015

We evaluated DNA double-strand breaks (DSBs) induced by boron neutron capture reaction (BNCR) using
plasmid DNA, boron solution, and gel electrophoresis. The amount of the linear form of DNA produced by
DSBs increased with the neutron-beam irradiation dose. The amount of the open-circular form of DNA
produced by single-strand breaks (SSBs) increased with the neutron-beam irradiation dose and the 10B
concentration. The model facilitated quantication of BNCR-induced DSBs and SSBs, irrespective of the
DNA repair mechanism.
& 2015 Elsevier Ltd. All rights reserved.

Keywords:
Neutron capture therapy
Double-strand break
DNA damage

1. Introduction
Boron Neutron Capture Therapy (BNCT) is a tumor cell-targeted
radiotherapy based on the 10B (n,) 7Li reaction, which results in
the release of high linear energy transfer (LET) (4He) and 7Li
particles. These high LET particles cause DNA double-strand breaks
(DSBs) and produce strong biological effects. Because the path
lengths of (4He) and 7Li particles are almost equal to the diameter
of a single tumor cell, 10B-containing tumor cells are selectively
destroyed in theory, minimizing radiation injury to normal tissue.
DSBs induced by boron neutron capture reaction (BNCR) have
been evaluated by immunochemical staining for detecting the
phosphorylation of core histone variant H2AX (gammma-H2AX)
foci (Kinashi et al., 2011; Masutani et al., 2014) and p53-binding
protein 1 (53BP1) foci (Kinashi et al., 2011, 2014; Okumura et al.,
2013). Gamma-H2AX molecules appear in discrete nuclear foci at
the sites of DSBs immediately after irradiation (Rogakou et al.,
n

Corresponding author. Fax: 81 298 53 3214.

E-mail address: knakai@md.tsukuba.ac.jp (K. Nakai).

http://dx.doi.org/10.1016/j.apradiso.2015.08.019
0969-8043/& 2015 Elsevier Ltd. All rights reserved.

1999) and colocalize with DNA repair proteins, such as 53BP1

(Rappold et al., 2001). Since gammma-H2AX and 53BP1 foci are
markers of DNA repair as well as markers of DSBs, these foci are
unlikely to detect DSBs selectively. Strand break assays with
plasmid DNA and gel electrophoresis are conventionally used for
analyzing radiation-induced DSBs regardless of the DNA repair
mechanism (Hempel and Mildenberger, 1987; van Touw et al.,
1985). The plasmid DNA is originally in the supercoiled form (SCDNA) and changes to the linear form (Lin-DNA) following introduction of DSB or the open-circular form (OC-DNA) by introduction of single-strand break (SSB) (Fig. 1A). These different
forms of plasmid DNA can be separated by gel electrophoresis
(Hempel and Mildenberger, 1987; Roots et al., 1985; van Touw
et al., 1985).
Quantication of DSBs induced by BNCR would help to elucidate the mechanism of tumor cell death and to identify boron
compounds that could effectively induce DSBs. The strand break
assay has been used in fast neutron radiotherapy for measuring
additional SSBs and DSBs generated by BNCR as a method for
boron neutron capture enhancement (Sche et al., 2002); however,

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E. Okamoto et al. / Applied Radiation and Isotopes 106 (2015) 185188

90 min. The total absorbed doses are shown in Table 2. The absorbed dose rates of thermal ( o0.5 eV), epithermal (0.5eV
10 keV), fast neutrons (4 10 keV), and gamma rays in the neutron
mixed beam were 1.3  10  2, 1.3  10  3, 9.56  10  3, and
1.44  10  2 Gy min  1, respectively. The dose rate of 10B (n,)7Li
was 7.1  10  3 Gy min  1mg10B L  1. As a control for the OC-DNA
produced by SSBs, a plasmid DNA solution (10 L) in a polypropylene tube was irradiated with 137Cs gamma rays (Gamma
Cell-40) at a dose rate of 7.4  10  1 Gy min  1 (Fig. 1B).
2.3. Post-irradiation analysis

Fig. 1. (A) Conformational changes in plasmid DNA caused by DSBs and SSBs. (B)
Gel electrophoresis of a control sample with plasmid pUC18.

this technique has not yet been applied to BNCT. Therefore, the
objective of our study was to evaluate DSB independent of the
DNA repair mechanism and to assess the relationship between the
number of DSBs and the radiation dose in BNCT.

The irradiated samples were mixed with 1 L of loading dye

(TOYOBO, Osaka, Japan) and then placed into the wells of a 1%
agarose gel (Reliant Gel System, Lonza Japan, Tokyo) containing
0.5 mg L  1 ethidium bromide. The samples were typically run at
100 V in TBE buffer (90 mM Tris, 90 mM boric acid, 2 mM
EDTA2 Na, pH 8.0) for 35 min at room temperature. After electrophoresis, the gel images were captured with an imaging system
(Tyhoon FLA 7000; GE Healthcare Japan Corporation, Tokyo, Japan). The separated band intensities were measured by gel analysis software (ImageQuant TL; GE Healthcare Japan Corporation).
The amounts of Lin-DNA, OC-DNA, and SC-DNA relative to total
plasmid DNA were calculated. The data were analyzed by Tukey's
honestly signicant difference test. Differences with p values of
less than 0.05 were considered signicant.

3. Results

2. Materials and methods

2.1. Materials
Plasmid DNA, pUC18 (2686 bp; Takara Bio Inc., Shiga, Japan),
was diluted with 1  TrisEDTA (TE) buffer (10 mM TrisHCl, 1 mM
EDTA  2Na, pH 8.0) to make a 0.25 g L  1 solution. We used standard boron solution (1000 mg B L  1), which contained 19.9% of 10B
(Wako Pure Chemicals, Tokyo, Japan). As a control for the Lin-DNA
produced by DSBs, nonirradiated pUC18 solution was treated with
EcoRI (Fig. 1B).
2.2. Irradiation experiments
Neutron irradiation was performed at the Heavy Water Facility
of the Kyoto University Research Reactor. To elucidate the relationship between the number of DSBs and the dose of neutron
beam irradiation, a plasmid DNA solution (5 L) with boron solution (5 L) containing 100 mg L  1 of 10B in a polypropylene tube
(0.2 mL; NIPPON Genetics Co., Ltd., Tokyo, Japan) was irradiated for
different times (90, 180, and 270 min). The total absorbed doses
are shown in Table 1. To assess the relationship between the
number of DSBs and the 10B concentration, plasmid DNA solution
(5 L) was mixed with boron solution (5 L) containing different
concentrations of 10B (0, 20, 50, and 100 mg L  1) and irradiated for

Gel analysis of the samples containing 100 mg L  1 of 10B with

irradiation doses of 0, 67.5, 135, or 202.4 Gy showed that the relative amount of Lin-DNA in pUC18 increased with the total physical dose (Fig. 2). The maximal value of Lin-DNA was approximately 2.3% with a dose of 202.4 Gy (Fig. 2B). In addition, the relative amount of OC-DNA increased with the total physical dose
(Fig. 2). Signicant increases in OC-DNA were observed at 135 and
202.4 Gy (Fig. 2B).
Analysis of the samples containing different concentrations of
10
B (0, 20, 50, and 100 mg L  1) after a 90-min irradiation revealed
that the relative amount of Lin-DNA did not differ signicantly
among the 10B concentration, although there was a tendency to
increase with the 10B concentration (Fig. 3). The relative amount of
OC-DNA was signicantly higher at a 10B concentration of
100 mg L  1 than at a 10B concentration of 0 mg L  1 (Fig. 3B).

4. Discussion
In the present study, the relative amount of Lin-DNA increased
with the dose of neutron beam irradiation. Sche et al. (2002)
reported that the number of DSBs per plasmid increases linearly
with the dose of fast neutron beam irradiation (with a range of 0
15 Gy) using plasmid DNA (pOC203, 4565 bp) in the presence of

Table 1
Total doses of neutron irradiation for different irradiation times.
Irradiation time
(min)

0
90
180
270

10

B Concentration
(mg L  1)

100
100
100
100

Thermal neutron ux
(cm  2 s  1)

0
5.8  1012
2.9  1012
1.9  1012

Dose (Gy)

Total dose
(Gy)

Thermal
neutrons

Epithermal
neutrons

Fast
neutrons

Gamma rays

10

B (n,)7Li

0
1.2
2.4
3.6

0
0.12
0.24
0.36

0
0.86
1.72
2.58

0
1.3
2.6
3.9

0
64
128
192

0
67.5
135.0
202.4

E. Okamoto et al. / Applied Radiation and Isotopes 106 (2015) 185188

Table 2
Total doses of neutron irradiation for difference concentrations of
Irradiation time
(min)

90
90
90
90

10

B Concentration
(mg L  1)

0
20
50
100

10

B.

Thermal neutron ux
(cm  2 s  1)

5.8  1012
5.8  1012
5.8  1012
5.8  1012

187

Dose (Gy)

Total dose
(Gy)

Thermal
neutrons

Epithermal
neutrons

Fast
neutrons

Gamma rays

10

1.2
1.2
1.2
1.2

0.12
0.12
0.12
0.12

0.86
0.86
0.86
0.86

1.3
1.3
1.3
1.3

0
12.8
32
64

Fig. 2. (A) Gel electrophoresis of pUC18 samples containing 100 mg L  1 of 10B with
different doses of neutron mixed beam irradiation (0, 67.5, 135, and 202.4 Gy).
(B) Relative amounts of Lin-DNA and OC-DNA with different doses of neutron
mixed beam irradiation (0, 67.5, 135, and 202.4 Gy) in plasmid pUC18. *p o 0.05,
**p o 0.01 compared with 0 Gy; p o 0.05, p o 0.01 compared with 67.5 Gy.

0.8 M 10B. The higher number of DSBs in the report by Sche and
colleagues (2002) may be attributable to fast neutrons that can
induce DSBs and SSBs independent of BNCR (Spotheim-Maurizot
et al., 1990). This difference may also be related to their use of
different types of plasmids.
The relative amount of Lin-DNA did not differ signicantly
among the 10B concentrations. Although we used boric acid, it is
unclear whether the induction of DSBs is enhanced by the presence of a DNA-binding boron compound (Tietze et al., 2002) or
clinically applied boron delivery agents, such as p-dihydroxyborylphenylalanine (BPA) and sulfhydryl borane Na2B2H2SH (BSH). The
relative amounts of OC-DNA increased with the dose of neutron
beam irradiation and the 10B concentration. This nding indicates
that the neutron capture reaction 10B (n,)7Li enhances SSBs as
well. Thus, our model successfully estimated the approximate
number of DSBs and SSBs induced by BNCR with neutron mixed
beam, irrespective of DNA repair.
The mechanism of tumor cell damage produced by BNCT has
been analyzed in terms of apoptosis, cell cycle arrest (Faio-Flores
et al., 2011; Fujita et al., 2009; Kamida et al., 2008; Sun et al., 2013;
Wang et al., 2010), extracellular matrix changes (Faio-Flores et al.,
2013a, 2013b), and DNA damage (Dagrosa et al., 2003, 2011;

B (n,)7Li

3.5
16.3
35.5
67.5

Fig. 3. (A) Gel electrophoresis of pUC18 samples containing different concentrations of 10B (0, 20, 50, and 100 mg L  1) after a 90-min irradiation. (B) Relative
amounts of Lin-DNA and OC-DNA at each concentration of 10B (0, 20, 50, and
100 mg L  1) in plasmid pUC18. *p o 0.05 compared with 0 mg L  1.

Kinashi et al., 2011; Masutani et al., 2014; Okumura et al., 2013;

Perona et al., 2011; Pller et al.,1996; Sche et al., 2002). DNA
damage is closely related to DNA repair, and the DNA repair capacity is signicantly lower after the induction of DNA damage by
neutron irradiation than after that by X-ray irradiation (Pller
et al., 1996). In Chinese hamster ovary cells, the number of
gammma-H2AX foci is decreased by DNA repair depending on the
time after BNCT (Kinashi et al., 2011). Further studies will be
needed to develop an experimental model in which DNA damage
and repair can coexist, but can be quantied separately.
One limitation of our study was that the different forms of DNA
and the number of water molecules binding with DNA in our
model were different from those in actual living cells because
plasmid DNA disperses in solution whereas DNA in the nucleus
exists in the form of euchromatin or heterochromatin. Moreover,
we plan to further investigate the possibility of 10B concentration
nonuniformity.

5. Conclusion
In this study, we determined the number of DSBs induced by
BNCR using plasmid DNA, boron solution, and gel electrophoresis.

188

E. Okamoto et al. / Applied Radiation and Isotopes 106 (2015) 185188

The number of DSBs increased with the dose of neutron mixed

beam irradiation. In addition, the number of SSBs increased with
the dose of neutron mixed beam irradiation and the 10B concentration. These ndings indicated that our model could be used
to quantify the approximate number of DSBs and SSBs, irrespective
of the DNA repair mechanism.

Acknowledgments
This work was supported by Grant- for Challenging Exploratory
Research (Nos. 24659643 and 26460718) from the Ministry of
Education, Culture, Sports, Science and Technology, Japan. This
study was performed using the facilities of the Kyoto University
Research Reactor Institute. We are grateful to Professor Masunaga
Shinichiro (Kyoto University Research Reactor Institute, Osaka,
Japan) for supporting the use of the Kyoto University Research
Reactor. We also thank Dr. Sakurai Yoshinori (Kyoto University
Research Reactor Institute) and Dr. Tanaka Hiroki (Kyoto University
Research Reactor Institute) for measuring the radiation dose.

References
Dagrosa, M.A., Viaggi, M., Longhino, J., Calzetta, O., Cabrini, R., Edreira, M., Juvenal,
G., Pisarev, M.A., 2003. Experimental application of boron neutron capture
therapy to undifferentiated thyroid carcinoma. Int. J. Radiat. Oncol. Biol. Phys.
57, 10841092.
Dagrosa, M.A., Crivello, M., Perona, M., Thorp, S., Santa Cruz, G.A., Pozzi, E., Casal, M.,
Thomasz, L., Cabrini, R., Kahl, S., Juvenal, G.J., Pisarev, M.A., 2011. First evaluation of the biologic effectiveness factors of boron neutron capture therapy
(BNCT) in a human colon carcinoma cell line. Int. J. Radiat. Oncol. Biol. Phys. 79,
262268.
Faio-Flores, F., Coelho, P.R., Arruda-Neto, J., Maria, D.A., 2011. Boron neutron capture therapy induces cell cycle arrest and DNA fragmentation in murine melanoma cells. Appl. Radiat. Isot 69, 17411744.
Faio-Flores, F., Coelho, P.R., Toledo Arruda-Neto, J.D., Maria-Engler, S.S., Tiago, M.,
Capelozzi, V.L., Giorgi, R.R., Maria, D.A., 2013a. Apoptosis through Bcl-2/Bax and
cleaved caspase up-regulation in melanoma treated by boron neutron capture
therapy. PLoS One 8, e59639.
Faio-Flores, F., Coelho, P.R., Arruda-Neto, J.D., Maria-Engler, S.S., Maria, D.A., 2013b.
Cell cycle arrest, extracellular matrix changes and intrinsic apoptosis in human
melanoma cells are induced by Boron Neutron Capture Therapy. Toxicol. In
Vitro 27, 11961204.
Fujita, Y., Kato, I., Iwai, S., Ono, K., Suzuki, M., Sakurai, Y., Ohnishi, K., Ohnishi, T.,
Yura, Y., 2009. Role of p53 mutation in the effect of boron neutron capture
therapy on oral squamous cell carcinoma. Radiat. Oncol. 4, 63.

Hempel, K., Mildenberger, E., 1987. Determination of G-values for single and double
strand break induction in plasmid DNA using agarose gel electrophoresis and a
curve-tting procedure. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 52,
125138.
Kamida, A., Fujita, Y., Kato, I., Iwai, S., Ono, K., Suzuki, M., Sakurai, Y., Yura, Y., 2008.
Effect of neutron capture therapy on the cell cycle of human squamous cell
carcinoma cells. Int. J. Radiat. Biol. 84, 191199.
Kinashi, Y., Takahashi, S., Kashino, G., Okayasu, R., Masunaga, S., Suzuki, M., Ono, K.,
2011. DNA double-strand break induction in Ku80-decient CHO cells following
boron neutron capture reaction. Radiat. Oncol. 6, 106.
Kinashi, Y., Okumura, K., Kubota, Y., Kitajima, E., Okayasu, R., Ono, K., Takahashi, S.,
2014. Dose-rate effect was observed in T98G glioma cells following BNCT. Appl.
Radiat. Isot. 88, 8185.
Masutani, M., Baiseitov, D., Itoh, T., Hirai, T., Berikkhanova, K., Murakami, Y., Zhumadilov, Z., Imahori, Y., Hoshi, M., Itami, J., 2014. Histological and biochemical
analysis of DNA damage after BNCT in rat model. App. Radiat. Isot 88, 104108.
Okumura, K., Kinashi, Y., Kubota, Y., Kitajima, E., Okayasu, R., Ono, K., Takahashi, S.,
2013. Relative biological effects 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. 54, 7075.
Perona, M., Pontiggia, O., Carpano, M., Thomasz, L., Thorp, S., Pozzi, E., Simian, M.,
Kahl, S., Juvenal, G., Pisarev, M., Dagrosa, A., 2011. In vitro studies of cellular
response to DNA damage induced by boron neutron capture therapy. Appl.
Radiat. Isot. 69, 17321736.
Pller, F., Bauch, T., Sauerwein, W., Bcker, W., Wittig, A., Streffer, C., 1996. Comet
assay study of DNA damage and repair of tumour cells following boron neutron
capture irradiation with fast d(14) Be neutrons. Int. J. Radiat. Biol. 70,
593602.
Rappold, I., Iwabuchi, K., Date, T., Chen, J., 2001. Tumor suppressor p53 binding
protein 1 (53BP1) is involved in DNA damage-signaling pathways. J. Cell. Biol.
153, 613620.
Rogakou, E.P., Boon, C., Redon, C., Bonner, W.M., 1999. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905916.
Roots, R., Kraft, G., Gosschalk, E., 1985. The formation of radiation-induced DNA
breaks: the ratio of double-strand breaks to single-strand breaks. Int. J. Radiat.
Oncol. Biol. Phys. 11, 259265.
Sche, E., Sabattier, R., Bajard, J.C., Blondiaux, G., Breteau, N., Spotheim-Maurizot,
M., Charlier, M., 2002. Direct effect in DNA radiolysis. Boron neutron capture
enhancement of radiolysis in a medical fast-neutron beam. Radiat. Res. 158,
292301.
Spotheim-Maurizot, M., Charlier, M., Sabattier, R., 1990. DNA radiolysis by fast
neutrons. Int. J. Radiat. Biol. 57, 301313.
Sun, T., Zhang, Z., Li, B., Chen, G., Xie, X., Wei, Y., Wu, J., Zhou, Y., Du, Z., 2013. Boron
neutron capture therapy induces cell cycle arrest and cell apoptosis of gliomastem/progenitor cells in vitro. Radiat. Oncol. 8, 195.
Tietze, L.F., Griesbach, U., Bothe, U., Nakamura, H., Yamamoto, Y., 2002. Novel carboranes with a DNA binding unit for the treatment of cancer by boron neutron
capture therapy. Chembiochem 3, 219225.
van Touw, J.H., Verberne, J.B., Retel, J., Loman, H., 1985. Radiation-induced strand
breaks in phi X174 replicative form DNA: an improved experimental and theoretical approach. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med 48, 567578.
Wang, P., Zhen, H., Jiang, X., Zhang, W., Cheng, X., Guo, G., Mao, X., Zhang, X., 2010.
Boron neutron capture therapy induces apoptosis of glioma cells through Bcl-2/
Bax. BMC Cancer 10, 661.