Photon Factory Activity Report 2019 #37 (2020)

BL-27A/2009G679

Chromatid-type aberrations induced by monoenergetic soft X
rays with resonance energy of phosphorus K-shellabsorption edge
Masao SUZUKI 1,* Hiroshi MAEZAWA2 and Noriko USAMI 2
1NIRS, QST, 4-9-1 Anagawa, Chiba, 263-8555, Japan
2 Photon Factory, KEK, 1-1 Oho, Tsukuba, 305-0801, Japan
1 Introduction
We reported the effect for chromatin-breakinduction in Chinese hamster lung fibroblast-likecells (V79) irradiated with monochromatic soft X
rays near K-shell absorption edge of phosphorus,which is one of major elements formed DNAbackbone. Monochromatic soft X rays were
producedusinganInSb
double-crystal
monochrometer at beam-line 27A in Photon Factory,
KEK. Chromatin-break induction was measured bycounting the number of chromatin fragments at G1
phase detected by the premature chromosome
condensation (PCC) technique. The results
indicated that about 60% of initially measured
chromatin breaks induced by the X rays at 2146eVand 25% of breaks induced by the X rays at 2160eV
rejoined after 16 hours of post-irradiation incubation,
while only 3% of repairable breaks were observed at2153eV [1]. This repair value of 60% was the samelevel with high linear-energy-transfer (LET) carbonion beams with 100 to 150 keV/µm. We concluded
that there produces much non-rejoining chromatin
breaks by the inner shell absorption of phosphorus.
The data indicated the quantitative differences in
induced chromatin breaks near K-shell absorption
edge of phosphorus, however, it is still unclear whatkinds of qualitative differences in cellular damage
between K-shell absorption edge and out of edge
are. In this study we tried to identify qualitative
differences in induced chromatin aberrations near Kshell absorption edge of phosphorus using achemical induced G2-PCC technique.
2 Experiment
Normal human skin fibroblasts distributed by theRIKEN BioResource Center Cell Bank (Cell name,NB1RGB; Cell No., RCB0222) were used in thisstudy. Cells were cultured in Eagle’s minimum
essential medium (MEM) containing kanamycin (60mg/l), supplemented with 10% fetal bovine serum ina 5% CO2 incubator at 37 oC. The frozen stockedcells were thawed in a water bath at 37°C for
irradiation, then inoculated and subcultured in the75-cm2 plastic flask until they reached a confluent
state. The cells were trypsinized and inoculated onto
irradiation dishes. The following day, medium in thedishes was removed by aspiration and the dishes

were covered with 5-µm thick polyester PET film formonochromatic X-ray irradiation.
Thecells
wereirradiated
500R
with
monochromatic X-rays of 2146, 2153 and 2160eV,
which were monochromatized with an InSb doublecrystal monochrometer. The calibration of energy forresonance of K-absorption peak (2153eV) ofphosphorus was carried out using the absorption
spectrum of a DNA film [2]. The dosimetry of theexposure (R) was carried out using a free air
ionization chamber [3]. It was converted theexposure (R) into the absorbed dose (Gy) using themass energy-absorption coefficient of air and themass attenuation coefficient of cell [4]. We
calculated the conversion factors (Gy/R) to be
0.0085949 for 2146eV, 0.0088479 for 2153eV and
0.0086908 for 2160eV, respectively.
Immediately after irradiations, the irradiated cells
were treated with Calyculin A (Wako Chemicals,Tokyo, Japan) at a final concentration of 50nM for
30 min in a CO2 incubator at 37oC. The PCC
samples at a G2 phase were prepared according toa conventional cytogenetic procedure [5]. Briefly,cells were treated with a 75mM KCl solution for
20min at 37oC, and fixed in 3:1 methanol : acetic
acid. The cell suspension was dropped onto
ethanol-cleaned slides, air dried, and stained with a5% Giemsa solution. The PCC samples of 50 G2phases were scored under a light microscope. The
yield of induced chromatid-type aberrations wasdetermined as previously reported by Savage [6].
We scored chromatid breaks, gaps, isochromatid
deletions, and exchanges as chromatid-typeaberrations.
3 Results and Discussion
The ratios of DNA absorbance near K-shellabsorption edge of phosphorus based on the value
at 2146eV were to be 3.36 at 2153eV and 1.43 at
2160eV [7].
The results were summarized in Table 1. The
values of any categories of observed chromatid-type
aberrations were higher at 2153 and 2160eV, whichwere in the side of higher energies at K-shellabsorption edge of phosphorus, than at 2146eV.
Especially the ratios of chromatid-type breaks andexchanges were much higher than those of the DNA

Photon Factory Activity Report 2019 #37 (2020)

absorbance between lower energy and higher
energies of K-shell absorption edge. The datasuggests that chrotmatid-type aberrations areproduced other process(es) of the DNA absorbance,such as the effect of Auger electrons. Also,
isochromatid breaks did not produce at 2146eV.
They must be produced by 2-hit events close to both
single strand DNAs at the same time. The data,
which shows the values of isochromatid breaks at
2153 and 2160eV, indicates that dense energy
depositions occur near phosphorus in DNA strand at
the higher energies of K-shell absorption edge ofphosphorus, looking like the damage caused byhigh-LET radiations. The results in this study
suggest that energy depsition at K-shell absorption
edge of phosphorus enable to induce serious
damage in DNA/chromatin level, which are similar tohigh-LET radiations.
Table.1 Chromatid-type aberrations in normal
human fibroblasts immediately after irradiations
Energy(eV)Control2146
21532160

Gap
0.10
0.30
0.45
0.36

Chromatid-type aberrations per cellBreak
Exchange
Isochromatid break
0.10
00
1.2
0.05
0
5.9
0.64
1.7
3.2
0.21
0.78

The exposure of each energy was 500R. The
absorbed doses were calculated using theconversion factors (Gy/R) to be 4.297Gy for 2146eV,
4.424Gy for 2153eV and 4.345Gy for 2160eV.
References[1] H. Maezawa et al., Photon Factory Activity
Report, 420, #14 (1996).
[2] K.Kobayashi et al., .J. Radiat. Biol., 28, 243-253(1987).
[3] K.Kobayashi et al., Int. J. Radiat. Biol., 59, 643650 (1991).
[4] M.Tomita et al., Int. J. Radiat. Biol., 92, 724-732(2016).
[5] M.Suzuki et al., Radiat. Res., 155, 432-439(2001).
[6] J.R.K. Savage, J. Med. Genet., 13, 103-122(1976).
[7] H.Maezawa et al., Acta Oncologica, 35, 889-894(1996).
* suzuki.masao@qst.go.jp