Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 49 (2016)

Low-dose high-LET heavy ion-induced bystander signaling (II)M. Tomita,∗1,∗2 T. Tsukada,∗2 and M. Izumi∗2Radiation-induced bystander response (RIBR) is acellular response induced in nonirradiated cells thatreceive bystander signals from directly irradiated cells
within an irradiated cell population.1) RIBR induced
by low doses of high-LET radiations is an important
issue concerning the health of astronauts and in heavyion radiation cancer therapy. Here, we investigated theunderlying molecular mechanisms and biological implications of RIBR induced by such low doses of highLET radiations.
The clonogenic cell survival of normal human ﬁbroblast WI-38 cells irradiated with Ar ions (310 keV/µm)
is shown in Fig. 1. At a higher dose region (0.5 Gyand above), the surviving fractions of cells harvested
16–24 h after irradiation was similar to those of cells
harvested immediately (0 h) after irradiation [Fig. 1A].
On the other hand, a strong cell-killing eﬀect at doses
below 0.08 Gy was observed in the cells harvested 16–
24 h after irradiation [Fig. 1B]. Such an eﬀect was notobserved in the cells harvested immediately after irradiation. Previously, we reported that cells irradiatedwith high-LET Fe ions (1000 keV/µm) showed similar results.2) These results suggest that an adequate
incubation period is necessary for bystander signal induction and transfer.
Previously, we reported that gap-junction intercellular communication (GJIC), cyclooxygenase-2 (COX-2)
protein, and nitric oxide (NO) were involved in highLET Fe-ion-induced bystander signal transfer.2) Figure 2 shows the progress of results reﬂecting new data.
Lindane and NS-398 (an inhibitor of GJIC and COX-2,respectively) were dissolved in DMSO (a scavenger ofreactive oxygen species). c-PTIO is a scavenger of NO.
DMSO (0.1%), lindane (Lin, 50 µM), c-PTIO (20 µM),
or NS-398 (50 µM) was added to the medium 2 h before irradiation3) with 0.1 Gy of Fe ions (1000 keV/µm)[Fig. 2A] or 0.05 Gy of Ar ions (310 keV/µm) [Fig.
2B]. The obtained results for the cells irradiated withFe and Ar ions were almost similar. DMSO did not signiﬁcantly suppress the bystander cell killing. In contrast, lindane, NS-398, and c-PTIO signiﬁcantly (P <0.05) suppressed cell death to similar levels. Cells pretreated with both c-PTIO and lindane did not exhibit
a signiﬁcantly higher surviving fraction than those pretreated with lindane or c-PITO alone. These results
suggest that bystander signaling through GJIC and thecell culture medium induces the bystander cell killingeﬀect in a coordinated manner.

∗1∗2Radiation Safety Research Center, Central Research Institute of Electric Power Industry
RIKEN Nishina Center- 252 -Fig. 1. Survival curves of WI-38 cells. Conﬂuent monolayers of WI-38 cells were irradiated with 95 MeV/u Ar
ions and the cells were harvested immediately (0 h) or
16–24 h after irradiation. The surviving fraction wasdetermined by using a colony forming assay. Panel A
shows all data obtained in this study. Panel B shows thesurviving fractions at doses of 0.2 Gy and below. Theerror bars represent the standard errors of the mean
(SEM) (n = 3–6).

Fig. 2. Eﬀect of inhibitors or scavengers. Panels A and B
show the surviving fractions in the cells irradiated with0.1 Gy of Fe ions and 0.05 Gy of Ar ions, respectively.
*P < 0.05 and **P < 0.01, for comparison with control
and drug-treated cultures.

References
1) M. Tomita and M. Maeda: J. Radiat. Res. 56, 205(2015).
2) M. Tomita et al.: RIKEN Accel. Prog. Rep. 48, 302(2015).
3) M. Tomita et al.: Radiat. Res. 179, 200 (2013).