Photon Factory Activity Report 2018 #36 (2019)

BL-27B/2017G695

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

1 Introduction
The study using a microbeam can provide us very
important scientific evidence in the research field ofradiation science and it should be essential to understandradio-biological responses induced by low-dose or lowfluence irradiations. 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. Furthermore, the study of suchbiological responses for low linear energy transfer (LET)
radiation can surely provide the critical 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 out
using high-LET-particle radiations and so far only limited
data is available to examine biological effects induced by
low-LET electromagnetic radiations, such as X or gamma
rays.
Radiation-induced bystander effects are described as
the ability of cells affected by irradiation to convey
manifestations of damage to neighbor cells that are notdirectly irradiated. We already reported that the cellular
bystander effect via gap-junction mediated cell-cellcommunication was not induced in cells immediately
after random irradiations with both cell nucleus andcytoplasm of the X-ray microbeams [1]. However, thebystander cell-killing effect was induced in cells targetedirradiations with cell nucleus alone. And also the higher
frequency of gene mutation at the hypoxanthine-guanine
phosphoribosyl transferase (HPRT) locus was induced in
the progeny of the following 20-cell generations from theX-ray-microbeam irradiated cells than that in the progenyof non-irradiated control cells and it was reduced to thenon-irradiated control level when treating with a specificinhibitor
ofgap-junction
mediatedcell-cellcommunication [2]. We also have been studying theradiation-quality dependent bystander cellular effects,
such as cell-killing effect and gene mutation induced byhigh-LET heavy-ion microbeams at the Takasaki Ion
Accelerator for Advanced Radiation Application,
Takasaki Advanced Radiation Research Institute.
Together with the data of X-ray microbeams in PF, so far
we understand the cellular responses as follows:(1) In the case of both irradiations with cell nucleus andcytoplasm at the same time, the bystander cellulareffects were observed in the cells irradiated withmedium-LET
carbon-ion
microbeams,
but
not higher-LET neon- or argon-ion microbeams
and lower-LET X-ray microbeams.
(2) Secondary radiations with low-LET components
were calculated to produce from higher-LET heavyion tracks in proportion to LET values by the Monte
Carlo simulation. They irradiated the cells located in
the neighborhood of the heavy-ion tracks.
(3) The bystander cellular effects were induced by thetargeted cell-nucleus irradiations with low-LET Xray microbeams.
(4) The biological effects of low-LET X rays werehigher in the cells with the targeted cell-nucleus
irradiations than those in the cells of both irradiations
with cell nucleus and cytoplasm at the same time.
Thus we can set up a hypothesis from the abovescientific evidence as follows:“When the cytoplasm of targeted cells are irradiated with
low-LET X rays, unknown cellular response(s) is inducedin the cell and in consequence the cell becomes protective
to radiation damage.”
It should be a powerful source for the microbeam oflow-LET electromagnetic radiations to use thesynchrotron radiations. And we have trying to verify thehypothesis using the X-ray microbeams in this study.
2 Experiment
Normal human skin fibroblasts distributed by theRIKEN BioResource Center Cell Bank (Cell No.:RCB0222, Cell name : NB1RGB),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 [3]. When cell

Photon Factory Activity Report 2018 #36 (2019)

nucleus was irradiated, we used the X-ray microbeams
collimating the beam size of 10µm x 10µm. The
irradiation doses were selected to be 10R (0.092Gy) and
40R (0.37Gy).
This year, we examined the radio-adaptive response
induced in the cells pre-irradiated cytoplasm with X-ray
microbeams. The cytoplasm of all cells captured by thecomputerized irradiation system was irradiated with 10R
first, and then the cell nucleus stained by Hoechst 33342
of all cells captured was irradiated with 10R at the180min interval, within which the cells were kept in a
CO2 incubator at 37oC after the first cytoplasmic
irradiation. 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.

3 Results and Discussion
The plating efficiencies (PE) of Hoechst + UV
scanning, which was used for capturing the cell nucleus
by the computerized cell irradiation system, targeted
nuclear irradiation alone and targeted cytoplasmic
irradiation alone were shown in Fig.1. Now the surviving
fraction (SF), which was one of the indicators for a cellkilling effect, was calculated as the following formula;

In the case of the experiment to examine the radioadaptive response, the cell samples were scanned under
the condition of Hoechst + UV twice times putting 180min incubation in a CO2 incubator at 37oC between twoirradiations. The PE of Hoechst + UV scanning twice andtargeted cytoplasmic and nuclear irradiations within 180min interval were shown in Fig.2. The data suggested thatthese were almost the same, indicating the SF of thetargeted cytoplasmic and nuclear irradiations within 180min interval was around 1.0.

Fig.2: Plating efficiencies of normal human fibroblasts
for each treatment; (1) Hoechst + UV scanning, (2)targeted cytoplasmic irradiation (10R) --–> (180mininterval) --–> targeted nuclear irradiation (10R). The data
showed the average and the standard error of 6
independent experiments.

SF = PE (irradiation) / PE (Hoechst + UV scanning)
The data clearly showed the cell-killing effect for thetargeted nuclear irradiation alone increased with dosedependent manner, but no cell-killing effect was observedin targeted cytoplasmic irradiation alone.

Fig.1: Plating efficiencies of normal human fibroblasts
for each treatment; (1) Hoechst + UV scanning, (2)targeted nuclear irradiation (10R) alone, (3) targeted
nuclear irradiation (40R) alone, (4) targeted cytoplasmic
irradiation (10R) alone, (5) targeted cytoplasmic
irradiation (40R) alone. The data showed the average andthe standard error of 6 independent experiments.

To make clear the radio-adaptive response induced in
the cells pre-irradiated cytoplasm, the SF data wereplotted for targeted nuclear irradiation (10R) alone,targeted cytoplasmic irradiation (10R) alone and targeted
cytoplasmic and nuclear irradiations (10R) within 180min interval (Fig.3). The SF for the targeted cell nucleus
irradiation was around 0.80 and no cell-killing effect wasinduced by the targeted cytoplasm irradiation (SF = 1.0).
On the other hand, the SF in the cells irradiated with thetargeted cell nuclei when the cells were irradiated thecytoplasm beforehand was increased at 0.97. The
obtained data showed that the SF was drastically
recovered by the pre-irradiated cytoplasm of the lowdose-X-ray irradiation, by which dose the SF was almost
1.0. There is clear evidence that the radio-adaptive
response should occur in the cells pre-irradiated tocytoplasm with the low-dose irradiation of the X-ray
microbeams. And we can conclude that our hypothesis isproven by this study.
In the first step, we can identify the radio-adaptive
response of the cell-killing effect as the intracellularresponse. In the next step, we must make clear what kindsof factor(s) are activated by a low-dose irradiation incytoplasm. In this study we chose 3hr as the interval
between the first cytoplasmic and the second nuclearirradiations. The result indicates that the cellular responseled to the radio-adaptive response will be completed

Photon Factory Activity Report 2018 #36 (2019)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.
Also, we will try to examine the intercellular response i.e.
participating bystander effect. We have a plan to examine
the radio-adaptive response induced by the intercellularresponse focused on gap-junction mediated bystander
effect in the next research project.

Fig.3: Cell-killing effect of normal human fibroblasts
irradiated with either targeted nuclear or cytoplasmic
irradiation of monochromatic X-ray microbeams. (1)targeted nuclear irradiation (10R) alone, (2) targeted
cytoplasmic irradiation (10R) alone, (3) targeted
cytoplasmic irradiation (10R) --–> (180min interval) --–>
targeted nuclear irradiation (10R). The surviving fractions
were calculated as PE (irradiation) / PE (Hoechst + UV
scanning) and the data showed the average and thestandard error of 6 independent experiments.

References[1] N. Autsavapromporn et al., Radiat. Res. 180, 367(2013).
[2] N. Autsavapromporn et al., Int. J. Radiat. Biol. 91, 62(2015).
[3] M. Suzuki and N.Usami, Photon Factory Activity
Report 2017, #35 (2018).

* suzuki.masao@qst.go.jp