Applied Radiation and Isotopes 106 (2015) 185–188

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Applied Radiation and Isotopes

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Detection of DNA double-strand breaks in boron neutron capturereaction

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, Japanb Doctoral Program in Clinical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan

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.

a r t i c l e i n f o

Article history:Received 19 January 2015Received in revised form16 July 2015Accepted 14 August 2015Available online 17 August 2015

Keywords:Neutron capture therapyDouble-strand breakDNA damage

x.doi.org/10.1016/j.apradiso.2015.08.01943/& 2015 Elsevier Ltd. All rights reserved.

esponding author. Fax: þ81 298 53 3214.ail address: knakai@md.tsukuba.ac.jp (K. Naka

a b s t r a c t

We evaluated DNA double-strand breaks (DSBs) induced by boron neutron capture reaction (BNCR) usingplasmid DNA, boron solution, and gel electrophoresis. The amount of the linear form of DNA produced byDSBs increased with the neutron-beam irradiation dose. The amount of the open-circular form of DNAproduced by single-strand breaks (SSBs) increased with the neutron-beam irradiation dose and the 10Bconcentration. The model facilitated quantification of BNCR-induced DSBs and SSBs, irrespective of theDNA repair mechanism.

& 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Boron Neutron Capture Therapy (BNCT) is a tumor cell-targetedradiotherapy based on the 10B (n,α) 7Li reaction, which results inthe release of high linear energy transfer (LET) α(4He) and 7Liparticles. These high LET particles cause DNA double-strand breaks(DSBs) and produce strong biological effects. Because the pathlengths of α(4He) and 7Li particles are almost equal to the diameterof a single tumor cell, 10B-containing tumor cells are selectivelydestroyed in theory, minimizing radiation injury to normal tissue.

DSBs induced by boron neutron capture reaction (BNCR) havebeen evaluated by immunochemical staining for detecting thephosphorylation of core histone variant H2AX (gammma-H2AX)foci (Kinashi et al., 2011; Masutani et al., 2014) and p53-bindingprotein 1 (53BP1) foci (Kinashi et al., 2011, 2014; Okumura et al.,2013). Gamma-H2AX molecules appear in discrete nuclear foci atthe sites of DSBs immediately after irradiation (Rogakou et al.,

i).

1999) and colocalize with DNA repair proteins, such as 53BP1(Rappold et al., 2001). Since gammma-H2AX and 53BP1 foci aremarkers of DNA repair as well as markers of DSBs, these foci areunlikely to detect DSBs selectively. Strand break assays withplasmid DNA and gel electrophoresis are conventionally used foranalyzing radiation-induced DSBs regardless of the DNA repairmechanism (Hempel and Mildenberger, 1987; van Touw et al.,1985). The plasmid DNA is originally in the supercoiled form (SC-DNA) and changes to the linear form (Lin-DNA) following in-troduction of DSB or the open-circular form (OC-DNA) by in-troduction of single-strand break (SSB) (Fig. 1A). These differentforms of plasmid DNA can be separated by gel electrophoresis(Hempel and Mildenberger, 1987; Roots et al., 1985; van Touwet al., 1985).

Quantification of DSBs induced by BNCR would help to eluci-date the mechanism of tumor cell death and to identify boroncompounds that could effectively induce DSBs. The strand breakassay has been used in fast neutron radiotherapy for measuringadditional SSBs and DSBs generated by BNCR as a method forboron neutron capture enhancement (Sèche et al., 2002); however,

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

E. Okamoto et al. / Applied Radiation and Isotopes 106 (2015) 185–188186

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

2. Materials and methods

2.1. Materials

Plasmid DNA, pUC18 (2686 bp; Takara Bio Inc., Shiga, Japan),was diluted with 1� Tris–EDTA (TE) buffer (10 mM Tris–HCl, 1 mMEDTA �2Na, pH 8.0) to make a 0.25 g L�1 solution. We used stan-dard boron solution (1000 mg B L�1), which contained 19.9% of 10B(Wako Pure Chemicals, Tokyo, Japan). As a control for the Lin-DNAproduced by DSBs, nonirradiated pUC18 solution was treated withEcoRI (Fig. 1B).

2.2. Irradiation experiments

Neutron irradiation was performed at the Heavy Water Facilityof the Kyoto University Research Reactor. To elucidate the re-lationship between the number of DSBs and the dose of neutronbeam irradiation, a plasmid DNA solution (5 μL) with boron so-lution (5 μL) containing 100 mg L�1 of 10B in a polypropylene tube(0.2 mL; NIPPON Genetics Co., Ltd., Tokyo, Japan) was irradiated fordifferent times (90, 180, and 270 min). The total absorbed dosesare shown in Table 1. To assess the relationship between thenumber of DSBs and the 10B concentration, plasmid DNA solution(5 μL) was mixed with boron solution (5 μL) containing differentconcentrations of 10B (0, 20, 50, and 100 mg L�1) and irradiated for

Table 1Total doses of neutron irradiation for different irradiation times.

Irradiation time(min)

10B Concentration(mg L�1)

Thermal neutron flux(cm�2 s�1)

Dose (Gy)

Thermalneutrons

0 100 0 090 100 5.8�1012 1.2

180 100 2.9�1012 2.4270 100 1.9�1012 3.6

90 min. The total absorbed doses are shown in Table 2. The ab-sorbed dose rates of thermal (o0.5 eV), epithermal (0.5eV –

10 keV), fast neutrons (410 keV), and gamma rays in the neutronmixed beam were 1.3�10�2, 1.3�10�3, 9.56�10�3, and1.44�10�2 Gy min�1, respectively. The dose rate of 10B (n,α)7Liwas 7.1�10�3 Gy min�1mg10B L�1. As a control for the OC-DNAproduced by SSBs, a plasmid DNA solution (10 μL) in a poly-propylene tube was irradiated with 137Cs gamma rays (GammaCell-40) at a dose rate of 7.4�10�1 Gy min�1 (Fig. 1B).

2.3. Post-irradiation analysis

The irradiated samples were mixed with 1 μL of loading dye(TOYOBO, Osaka, Japan) and then placed into the wells of a 1%agarose gel (Reliant Gel System, Lonza Japan, Tokyo) containing0.5 mg L�1 ethidium bromide. The samples were typically run at100 V in TBE buffer (90 mM Tris, 90 mM boric acid, 2 mMEDTA2 Na, pH 8.0) for 35 min at room temperature. After elec-trophoresis, the gel images were captured with an imaging system(Tyhoon FLA 7000; GE Healthcare Japan Corporation, Tokyo, Ja-pan). The separated band intensities were measured by gel ana-lysis software (ImageQuant TL; GE Healthcare Japan Corporation).The amounts of Lin-DNA, OC-DNA, and SC-DNA relative to totalplasmid DNA were calculated. The data were analyzed by Tukey'shonestly significant difference test. Differences with p values ofless than 0.05 were considered significant.

3. Results

Gel analysis of the samples containing 100 mg L�1 of 10B withirradiation doses of 0, 67.5, 135, or 202.4 Gy showed that the re-lative amount of Lin-DNA in pUC18 increased with the total phy-sical dose (Fig. 2). The maximal value of Lin-DNA was approxi-mately 2.3% with a dose of 202.4 Gy (Fig. 2B). In addition, the re-lative amount of OC-DNA increased with the total physical dose(Fig. 2). Significant increases in OC-DNA were observed at 135 and202.4 Gy (Fig. 2B).

Analysis of the samples containing different concentrations of10B (0, 20, 50, and 100 mg L�1) after a 90-min irradiation revealedthat the relative amount of Lin-DNA did not differ significantlyamong the 10B concentration, although there was a tendency toincrease with the 10B concentration (Fig. 3). The relative amount ofOC-DNA was significantly higher at a 10B concentration of100 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 increasedwith the dose of neutron beam irradiation. Sèche et al. (2002)reported that the number of DSBs per plasmid increases linearlywith the dose of fast neutron beam irradiation (with a range of 0–15 Gy) using plasmid DNA (pOC203, 4565 bp) in the presence of

Total dose(Gy)

Epithermalneutrons

Fastneutrons

Gamma rays 10B (n,α)7Li

0 0 0 0 00.12 0.86 1.3 64 67.50.24 1.72 2.6 128 135.00.36 2.58 3.9 192 202.4

Table 2Total doses of neutron irradiation for difference concentrations of 10B.

Irradiation time(min)

10B Concentration(mg L�1)

Thermal neutron flux(cm�2 s�1)

Dose (Gy) Total dose(Gy)

Thermalneutrons

Epithermalneutrons

Fastneutrons

Gamma rays 10B (n,α)7Li

90 0 5.8�1012 1.2 0.12 0.86 1.3 0 3.590 20 5.8�1012 1.2 0.12 0.86 1.3 12.8 16.390 50 5.8�1012 1.2 0.12 0.86 1.3 32 35.590 100 5.8�1012 1.2 0.12 0.86 1.3 64 67.5

Fig. 2. (A) Gel electrophoresis of pUC18 samples containing 100 mg L�1 of 10B withdifferent 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 neutronmixed beam irradiation (0, 67.5, 135, and 202.4 Gy) in plasmid pUC18. *po0.05,**po0.01 compared with 0 Gy; †po0.05, ††po0.01 compared with 67.5 Gy.

Fig. 3. (A) Gel electrophoresis of pUC18 samples containing different concentra-tions of 10B (0, 20, 50, and 100 mg L�1) after a 90-min irradiation. (B) Relativeamounts of Lin-DNA and OC-DNA at each concentration of 10B (0, 20, 50, and100 mg L�1) in plasmid pUC18. *po0.05 compared with 0 mg L�1.

E. Okamoto et al. / Applied Radiation and Isotopes 106 (2015) 185–188 187

0.8 M 10B. The higher number of DSBs in the report by Sèche andcolleagues (2002) may be attributable to fast neutrons that caninduce DSBs and SSBs independent of BNCR (Spotheim-Maurizotet al., 1990). This difference may also be related to their use ofdifferent types of plasmids.

The relative amount of Lin-DNA did not differ significantlyamong the 10B concentrations. Although we used boric acid, it isunclear whether the induction of DSBs is enhanced by the pre-sence of a DNA-binding boron compound (Tietze et al., 2002) orclinically applied boron delivery agents, such as p-dihydroxyboryl-phenylalanine (BPA) and sulfhydryl borane Na2B2H2SH (BSH). Therelative amounts of OC-DNA increased with the dose of neutronbeam irradiation and the 10B concentration. This finding indicatesthat the neutron capture reaction 10B (n,α)7Li enhances SSBs aswell. Thus, our model successfully estimated the approximatenumber of DSBs and SSBs induced by BNCR with neutron mixedbeam, irrespective of DNA repair.

The mechanism of tumor cell damage produced by BNCT hasbeen analyzed in terms of apoptosis, cell cycle arrest (Faião-Floreset al., 2011; Fujita et al., 2009; Kamida et al., 2008; Sun et al., 2013;Wang et al., 2010), extracellular matrix changes (Faião-Flores et al.,2013a, 2013b), and DNA damage (Dagrosa et al., 2003, 2011;

Kinashi et al., 2011; Masutani et al., 2014; Okumura et al., 2013;Perona et al., 2011; Pöller et al.,1996; Sèche et al., 2002). DNAdamage is closely related to DNA repair, and the DNA repair ca-pacity is significantly lower after the induction of DNA damage byneutron irradiation than after that by X-ray irradiation (Pölleret al., 1996). In Chinese hamster ovary cells, the number ofgammma-H2AX foci is decreased by DNA repair depending on thetime after BNCT (Kinashi et al., 2011). Further studies will beneeded to develop an experimental model in which DNA damageand repair can coexist, but can be quantified separately.

One limitation of our study was that the different forms of DNAand the number of water molecules binding with DNA in ourmodel were different from those in actual living cells becauseplasmid DNA disperses in solution whereas DNA in the nucleusexists in the form of euchromatin or heterochromatin. Moreover,we plan to further investigate the possibility of 10B concentrationnonuniformity.

5. Conclusion

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

E. Okamoto et al. / Applied Radiation and Isotopes 106 (2015) 185–188188

The number of DSBs increased with the dose of neutron mixedbeam irradiation. In addition, the number of SSBs increased withthe dose of neutron mixed beam irradiation and the 10B con-centration. These findings indicated that our model could be usedto quantify the approximate number of DSBs and SSBs, irrespectiveof the DNA repair mechanism.

Acknowledgments

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

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