Photon Factory Activity Report 2016 #34 (2017)

BL-27B/2015G065

Cell-killing effect by the targeted cytoplasmic irradiation in normal
human fibroblasts with monochromatic X-ray microbeams (2)Masao Suzuki1,* and Noriko Usami2
1
NIRS, QST, 4-9-1 Anagawa, Chiba, 263-8555, Japan2
Photon Factory, IMSS, KEK, 1-1 Oho, Tsukuba, 305-0801, Japan

1 Introduction
The research using a microbeam can provide us very
important information in the research field of radiation
science and it should be essential for us to understandradio-biological effects, such as bystander effect,
genomic instability and radio-adaptive response, induced
by low-dose or low-fluence irradiations. The study ofsuch biological effects for low linear energy transfer(LET) radiation, such as X rays or gamma rays, cansurely provide the important implications for evaluatingrisk such a low-dose (rate) exposure as the accident ofFukushima Daiichi Nuclear Power Plants.
We have been studying cellular responses induced by
the low-density irradiations using the cell-irradiation
system of X-ray microbeams at BL27B [1] in order toclarify radiation risk concerning the issue of the accidentof Fukushima Daiichi Nuclear Power Plants and alreadyreported that the cellular bystander effect via gap-junction
mediated cell-cell communication was not induced incells immediately after irradiation of the X-ray
microbeams [2]. However, the higher frequency of genemutation at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus was induced in the progeny of
the following 20-cell generations from the X-raymicrobeam irradiated cells than that in the progeny of
non-irradiated control cells. Furthermore, the higher
mutation frequency at 20-cell generations after irradiation
was reduced to the non-irradiated control level whentreating with a specific inhibitor of gap-junction mediatedcell-cell communication [3]. We conclude that genomic
instability detected with gene mutation occurs in future
generations by the bystander effect via gap-junction
mediated cell-cell communication, although no bystander
cellular effects were observed in cells immediately after
irradiation of the X-ray microbeams. Our study suggeststhat it should be very important for us to investigate
biologicallate
effects
inducedby low-LET
electromagnetic radiations as the accident of Fukushima
Daiichi Nuclear Power Plants in order to evaluate the riskof cancer by low-dose exposure.
Most studies for radiation-induced bystander effects
using a microbeam, however, have been performed using
high-LET heavy ions and only limited studies areavailable to understand cellular responses using low-LET
electromagnetic radiations. In this study we have been
studying low-LET-radiation induced bystander cellulareffects irradiated with targeted cell nucleus or cytoplasm
using the X-ray microbeams. This year, we examinedcell-killing effect of either targeted cell-nuclear or
cytoplasmic irradiations using the method described inthe last year’s reported.
2 Experiment
Normal human skin fibroblasts distributed by theRIKEN BioResource Center Cell Bank (Cell name :NB1RGB, Cell No. : RCB0222) were used in thisresearch project. Approximately 1,000 exponentiallygrowing cells were inoculated into the center of eachmicrobeam dish, which was stretching a 2.5μm-thick
Mylar film over the bottom of the hole for X-ray window,
one day before irradiations. Each cell nucleus stained by
Hoechst 33342 was captured by the computerized cellirradiation system. Targeted cytoplasmic irradiations withthe monochromatic X-ray microbeams (5.35keV) to thenormal human fibroblasts were carried out using the cellirradiation system according to the last year’s report.
Briefly, we made the microbeam covering the areas of
30μm x 30μm in which the center of the microbeams thegold-made mask that was 22 micrometer in diameter and
20 micrometer in height on a thin SiN film was set inorder to shield the nucleus (Fig.1, Fig.2).

Fig.1: The method for the targeted cytoplasmic irradiation
using X-ray microbeams. This method regarding thetargeted cytoplasmic irradiations to the normal humanfibroblasts were explained in the last year’s report.

Photon Factory Activity Report 2016 #34 (2017)not by the pre-irradiation with the X-ray microbeams tocytoplasm.

1
0.9
0.8
Surviving fraction

The cytoplasm of all cells captured by thecomputerized irradiation system was irradiated witheither 10R or 40R. Also the targeted nuclear irradiation of
all cells was performed either 10R or 40R of the X-ray
microbeams collimating the beam size of 10μm x 10μm
using the same computerized cell-irradiation system.
Cell-killing effect was measured with a colony-forming
assay as a reproductive dell death. Immediately after
irradiation, cells were trypsinized and a defined numberof cells plated onto 100mm plastic dish to make 60-70
colonies per dish. The colonies fixed and stained with20% methanol and 0.2% crystal violet for 14 days afterincubation. The colonies consisting of more than 50 cells
were scored as a survivor.

0.7
0.6
0.5
0.4
0.3
0.2

Fig.2: The computerized targeted cytoplasmic irradiation
system of X-ray microbeams. The upper photo in the left
side shows 60-square-micrometer microbeam and thelower photo in the left side shows 30-square-micrometer
microbeam with the gold post at the center of themicrobeams. This figure regarding the targeted
cytoplasmic irradiations to the normal human fibroblasts
was taken from the last year’s report.

3 Results and Discussion
The obtained results were shown in Fig.3. Thesurviving fractions for the targeted cytoplasmic
irradiations were around 1.0 for both 10R and 40R. The
data indicated that no cell-killing effect was induced by
the cytoplasmic irradiations with the 5.35keV
monochromatic X-ray microbeam ranging from 10R to40R. On the other hand, the surviving fractions for thetargeted cell-nuclear irradiation were 0.52 for 40R and0.79 for 10R. Although we got the data with 2 different
exposure doses, the cell-killing effect in cells irradiatedwith the targeted cell-nuclear irradiation was a dosedependent manner. The results clearly showed that cellkilling effect occurred in just only cells deposited X-ray
energy in cell nuclear.
Now we just start to examine a radio-adaptive responsein the cell-killing effect by the targeted cell-nuclear
irradiation when the cells were treated with the preirradiation to the targeted cytoplasm of the X rays. We
will check the surviving fraction obtained by the targetedcell-nuclear irradiation whether it will be suppressed or(10R)

cytoplasm
ic IR

cytoplasm
ic IR(40R)
nuclear IRnuclear IR(10R)

0(40R)

0.1

Fig.3: Cell-killing effect of normal human fibroblasts
irradiated with either targeted nuclear or cytoplasmic
irradiation (IR) of monochromatic X-ray microbeams(5.35keV). The data show the average and the standard
error of 5 independent experiments.

References[1] Y. Kobayashi et al., J. Radiat. Res. 50, Suppl., A29(2009).
[2] N. Autsavapromporn et al., Radiat. Res. 180, 367(2013).
[3] N. Autsavapromporn et al., Int. J. Radiat. Biol. 91, 62(2015).

* suzuki.masao@qst.go.jp