Photon Factory Activity Report 2017 #35 (2018)

BL-27B/2017G695

Cell-killing effect by the targeted cytoplasmic irradiation in normal
human fibroblasts with monochromatic X-ray microbeams (2)Masao SUZUKI1,* and Noriko USAMI2
1NIRS, QST, 4-9-1 Anagawa, Chiba, 263-8555, Japan2
Photon Factory, KEK, 1-1 Oho, Tsukuba, 305-0801, Japan

1 Introduction
The biological research using a microbeam can provide
us very important implications in the research field ofradiation science. Especially, a targeted irradiation toeither cell nucleus or cytoplasm using a microbeam
enable us to understand biological cellular responses,
such as bystander effects, genomic instability and radioadaptive response, induced by low-dose or low-fluence
irradiations more in detail. The study of such biologicalresponses for low linear energy transfer (LET) radiation
can surely provide the important information forevaluating risk such a low-dose (rate) exposure as theaccident of Fukushima Daiichi Nuclear Power Plants.
However, most studies for such biological effects inducedin cells irradiated with a microbeam have been carried outusing high-LET-particle radiations and so far only limited
data is available to understand biological effects induced
by low-LET electromagnetic radiations, such as X orgamma rays. It should be a powerful source for the
microbeam of low-LET electromagnetic radiations to use
the synchrotron radiations.
We have been studying cellular responses induced by
the targeted irradiations using the cell-irradiation system
of X-ray microbeams at BL27B [1]. Our final goal is toidentify the molecular mechanism(s) of radiation induced
cellular responses, and then to clarify radiation risk
concerning the issue of the accident of Fukushima Daiichi
Nuclear Power Plants.
We already reported that the cellular bystander effectvia gap-junction mediated cell-cell communication was
not induced in cells immediately after random irradiations
with both cell nucleus and cytoplasm of the X-ray
microbeams [2]. However, the bystander cell-killing
effect was induced in cells irradiated with just cell
nucleus when using the system of the targeted cell nuclear
irradiation [1]. And also 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 and it was reduced to the nonirradiated control level when treating with a specificinhibitorof
gap-junction
mediatedcell-cellcommunication [3]. Our study suggests that it should bevery important to investigate low-LET-radiation induced
bystander cellular effects irradiated with targeted cell
nucleus or cytoplasm using the X-ray microbeams as theaccident of Fukushima Daiichi Nuclear Power Plants inorder to evaluate the risk of cancer by low-dose exposure.
This year, we examined cell-killing effect of either
targeted cell-nuclear or cytoplasmic irradiations
expanding the last year’s experiment.
2 Experiment
Normal human skin fibroblasts distributed by theRIKEN BioResource Center Cell Bank (Cell name :NB1RGB, Cell No. : RCB0222) were used in this study.
Approximately 1,000 exponentially growing cells wereinoculated into the center of each microbeam dish, which
was stretching a 2.5µm-thick Mylar film over the bottomof the hole for X-ray window, one day before irradiations.
Each cell nucleus stained by Hoechst 33342 was capturedby the computerized cell irradiation system. Targeted
cytoplasmic irradiations with the monochromatic X-ray
microbeams (5.35keV) to NB1RGB cells were carried out
using the cell-irradiation system according to the lastyear’s report. Briefly, we made the microbeam covering
the areas of 30µm x 30µm in which the center of themicrobeams the gold-made mask that was 22 micrometerin diameter and 20 micrometer in height on a thin SiN
film was set in order 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 2017 #35 (2018)

This year, we tried to examine the radio-adaptive
response induced in the cells pre-irradiated cytoplasm
with low-LET electromagnetic radiations. The cytoplasmof all cells captured by the computerized irradiation
system was irradiated with 10R first, and then the cell
nucleus of all cells captured was irradiated with 10R at
the 3hr interval, within which the cells were kept in a CO2
incubator at 37oC after the first cytoplasmic irradiation,using the X-ray microbeams collimating the beam size of
10µm x 10µm. Cell-killing effect was measured with acolony-forming assay as a reproductive dell death.
Immediately after irradiation, cells were trypsinized and adefined number of cells plated onto 100mm plastic dish to
make 60-70 colonies per dish. The colonies fixed andstained with 20% methanol and 0.2% crystal violet for
16-day incubation. The colonies consisting of more than
50 cells were scored as a survivor.

conditions for inducing the radio-adaptive response, suchas interval times or irradiation doses of the firstirradiation.

Fig.3: Cell-killing effect of normal human fibroblasts
irradiated with either targeted nuclear or cytoplasmic
irradiation of monochromatic X-ray microbeams(5.35keV). (1); targeted nuclear irradiation (10R) alone,(2); targeted cytoplasmic irradiation (10R) alone, (3);targeted cytoplasmic irradiation (10R) --–> (3hr interval)--–> targeted nuclear irradiation (10R). The data show theaverage and the standard error of 3 independent
experiments.
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 preliminary result was shown in Fig.3. The
surviving fraction for the targeted cell nucleus irradiation
was around 0.79 and no cell-killing effect was induced by
the targeted cytoplasm irradiation. The surviving fractionin the cells irradiated with the targeted cell nuclei whenirradiated the cytoplasm beforehand was increased at 0.96.
The obtained results clearly suggest that the radioadaptive response should occur in the cells pre-irradiatedto cytoplasm with the X-ray microbeams.
In this experiment we chose 3hr as the interval between
the first cytoplasmic irradiation and the second nuclearirradiation. The result indicates that the cellular response
led to the radio-adaptive response will be completed
within 3hr after the first cytoplasmic irradiation, but it isstill unclear whether the “3h interval” is maximum
efficient for inducing the radio-adaptive response or not.
We will continue to study biological responses inducedby the targeted cytoplasmic irradiation to make clear theReferences[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