Photon Factory Activity Report 2022 #40 (2023)

BL-27B/2021G539

Radio-adaptive response induced by the targeted cytoplasmic
irradiation in normal human fibroblasts with X-ray microbeams via
bystander effects by gap-junction mediated cell-to-cellcommunication1

Masao SUZUKI 1,* and Noriko USAMI 2
Institute for Quantum Medical Science, QST, 4-9-1 Anagawa, Chiba, 263-8555, Japan2
Photon Factory, KEK, 1-1 Oho, Tsukuba, 305-0801, Japan

1 Introduction
Cellular response, such as bystander effect, forlow-dose or low-fluence irradiations must be one ofthe major concerns for radiology or health physics. Itis very important for us to understand low-dose or
low-fluence radiations induced radio-biologicaleffects via bystander effects, such as genomic
instability and radio-adaptive response. Such radiobiological effects are also very essential to investigate
secondary carcinogenesis after tumor radio-therapy
and evaluate radiation risk such as the accident ofFukushima Daiichi Nuclear Power Plants caused by
the Great East Japan Earthquake at 2011.
A microbeam-irradiation system is powerful
method to examine low-dose or low-fluence
radiations induced radio-biological effects and so far
there are many studies available to examine cellularresponses such using microbeam radiations.
However, the most of them were carried out using
high-LET-particle radiations and limited studies wereavailable to use low-LET electromagnetic radiations.
In this study we have planned to make clear low-doseor low-fluence radiations induced radio-biologicaleffects by low-LET electromagnetic radiations using
the X-ray microbeams produced by PF at BL-27.
We so far reported low-LET X-ray induced
bystander effects in normal human fibroblasts asfollows:
1) The cellular bystander effect for chromosomal
damage via gap-junction mediated cell-to-cellcommunication was not induced in cells
immediately after irradiations with both cell
nucleus and cytoplasm [1].
2) The bystander cell-killing effect was induced whencells were irradiated with targeted cell nucleus
alone [2].
3) The higher frequency of gene mutation at the
hypoxanthine-guanine
phosphoribosyltransferase (HPRT) locus was observed in theprogeny of the following 20-cell generations fromthe X-ray-microbeam irradiated cells than those in
the progeny of non-irradiated control cells and it
was reduced to the non-irradiated control level

when treating with a specific inhibitor of gapjunction mediated cell-to-cell communication [3, 4].
In this project (2021G539) we have been studying
radio-adaptive responses induced in cells irradiatedwith targeted cytoplasm beforehand and thenirradiated with targeted cell nuclear by X-ray
microbeams. The results suggest that when the cells
were irradiated the cytoplasm beforehand, the cellsurvival in cells irradiated with the targeted cell nuclei
was returned to the control level, suggesting theradio-adaptive response [5-10]. This year we
examined intercellular radio-adaptive response using
the gap-junction inhibitor.
2 Experiment
Normal human skin fibroblasts distributed by theRIKEN BioResource Center Cell Bank (Cell No.:RCB0222, Cell name : NB1RGB,) were used for thetarget cells. 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. Cell nuclei werestained by Hoechst 33342 and captured by thecomputerized-cell-irradiation system.
X-ray-microbeam irradiations were carried out
using monochromatic X-ray (5.35keV) at BL-27B.
Targeted cytoplasmic irradiations with the X-ray
microbeams were performed using the microbeamcell-irradiation system [5]. Briefly, we made themicrobeam collimating the beam size of 30µm x30µm in which the center of the microbeam the goldmade mask that was 22 µm in diameter and 20 µm inheight on a thin SiN film was set in order to shield thenucleus from X-ray exposure. When targeted cell
nuclei irradiations were carried out, we used the Xray microbeams collimating the beam size of 10µm x10µm. The irradiation dose was selected to be 10R(0.092Gy). In order to examine the radio-adaptive
response induced in intercellular response, targeted
cytoplasm randomly selected 10% of all captured
cells were irradiated with X-ray microbeams
beforehand and then targeted cell nuclei of 100%
cells were irradiated at the 180min interval withinPhoton Factory Activity Report 2022 #40 (2023)

which the cells were kept in a CO2 incubator at 37oC
after the first cytoplasmic irradiation. A specificinhibitor of gap-junction mediated cell-to-cellcommunication(40µMofgammahexachlorocyclohexane) was added to the culture
medium 1hr before the first cytoplasmic irradiation to
the second cell nucleus irradiation in order toexamine the effects of bystander responses via gapjunction mediated cell-to-cell communication. Cellkilling effect was detected with a colony-forming
assay as a reproductive cell death. Immediately afterirradiation, cells were trypsinized and a defined
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 morethan 50 cells were scored as a surviving cell.
3 Results and Discussion
This year we carried out the repeat experiments
using independent beam times for 100% of targeted
nuclear irradiation (10R=0.092Gy) alone with thegap-junction inhibitor. The result was shown in Fig.1.
The cell survival irradiated with all cell nuclei alone
was 79% and no cell-killing effect was observed in thecytoplasmic irradiation alone. The cell survivals wererising to 95-98% when either 10% or 100% of
cytoplasm was irradiated beforehand. When treatingwith the gap-junction inhibitor, the cell survivals fortargeted nuclear irradiation were around 72% with thepresence or absence of the targeted cytoplasmic
irradiation beforehand. The results clearly showedthat the bystander effect via intercellular responsewith
gap-junction
mediatedcell-to-cellcommunication plays an essential role of reducing
cell-killing effect, suggesting radio-adaptive response.

Fig.1: Cellular lethal effect of normal humanfibroblasts irradiated with either targeted nuclear or
cytoplasmic irradiation of monochromatic X-ray
microbeams. (1) 100% of targeted nuclear irradiation(10R=0.092Gy) alone, (2) 100% of targeted
cytoplasmic irradiation (0.092Gy) alone, (3) 100% of
targeted cytoplasmic irradiation (0.092Gy) --–>
(180min interval) --–> 100% of targeted nuclear
irradiation (0.092Gy), (4) Randomly selected 10% of

targeted cytoplasmic irradiation (0.092Gy) --–>
(180min interval) --–> 100% of targeted nuclear
irradiation (0.092Gy), (5) 100% of targeted nuclear
irradiation (10R=0.092Gy) alone with the gapjunction inhibitor, (6) Randomly selected 10% of
targeted cytoplasmic irradiation (0.092Gy) --–>
(180min interval) --–> 100% of targeted nuclear
irradiation (0.092Gy) with gap-junction inhibitor. The
data showed the average and the standard error of atleast 8 independent experiments.
References[1] N. Autsavapromporn et al., Radiat. Res. 180,367 (2013).
[2] M. Suzuki et al., Photon Factory Activity Report
2010, #28 Part B (2011).
[3] M. Suzuki et al., Photon Factory Activity Report
2014, #32, 54 (2015).
[4] N. Autsavapromporn et al., Int. J. Radiat. Biol. 91,
62 (2015).
[5] M. Suzuki and N. Usami, Photon Factory Activity
Report 2015, #33 (2016).
[6] M. Suzuki and N. Usami, Photon Factory Activity
Report 2016, #34 (2017).
[7] M. Suzuki and N. Usami, Photon Factory Activity
Report 2017, #35 (2018).
[8] M. Suzuki and N. Usami, Photon Factory Activity
Report 2018, #36 (2019).
[9] M. Suzuki and N. Usami, Photon Factory Activity
Report 2020, #38 (2021).
[10] M. Suzuki and N. Usami, Photon Factory Activity
Report 2021, #39 (2022).
* suzuki.masao@qst.go.jp