Bystander cellular effects induced by low-fluence charged particles

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We have investigated bystander cellular responses in normal human fibroblasts irradiated with low-fluence ion-beams of low-energy carbon ions. Irradiation dishes were constructed by drilling 6.5mm holes in the center of 35mm-diameter tissue culture dishes and attaching a 2.5μm-thick Mylar film over the bottom of the hole to provide a window for low-energy carbon ion beams. Cells were inoculated onto the Mylar film in the dishes and then irradiated with 6MeV/n carbon ions at a fluence of 2 x 10^5 particles/cm^2. The beam was generated by the Medium Energy Beam Course at the Heavy Ion Medical Accelerator in Chiba (HIMAC). Cells were exposed using 4 different protocols ; (1)Cells were irradiated with all of the cells on the Mylar film ;. (2)Cells were irradiated with half of the cell population on the Mylar film ; (3)Irradiated cells and un-irradiated control cells were pooled in a 1:1 ratio and plated as a single culture ; and (4)Half of the cells were irradiated and treated with a specific inhibitor of gap junction mediated cell-cell communication (40μM lindane). Cell killing, which was assayed with a colony formation assay, showed that the surviving fraction of the irradiated group (2)was almost the same as for group (1).In addition, a similar level of survival was seen in the group (4)and in the mixed population group (3).Mutation induction, which was scored as the number of 6-thioguanine resistant clones arising at the HPRT locus, showed the same trend as the cell killing effect. Thus there is evidence that the bystander effect for both cell killing and mutation induction occurred in the un-irradiated half of the cells in group (2). Since lindane apparently inhibited these effects, this suggests that gap junction mediated cell-cell communication may play an important role in the induction of the bystander effect.