2.4. Models of Radiation-Induced Bystander Effects (RIBE)

For the in vivo micronucleus assay, 0.2 mL serum from irradiated animals was administered by intraperitoneal puncture to each animal to elicit RIBE effects. In the BysRA group, the animals were given drinking water containing 0.2% RA ad libitum for a period of two weeks. In the mouse bone marrow micronucleus assay, bone marrow was harvested from the medullary canal of the femur 24 h after exposure to X-ray irradiation. In the MN-RET assay by flow cytometry, blood samples were obtained by intracardiac puncture 48 h post-irradiation (Figure 2a).

Models of radiation-induced bystander (Bys) effects (RIBE): (a) In vivo micronucleus assay (in mouse bone marrow (Micronuclei in polychromatic erythrocytes (MNPCEs)) and reticulocytes (Micronuclei in reticulocytes (MNRET))), (b) micronucleus test in vitro (Cytokinesis-block micronucleus assay (CBMN)), (c) MTT cytotoxicity assay.

In the vitro micronucleus assay (CBMN), 0.1 mL serum from irradiated blood was added to the blood samples before commencement of the cytochalasin B block micronucleus test with human lymphocytes. In the BysRA group, 25 microliters (µL) of a 25 micromolar (µM) solution of RA dissolved in PBS had previously been added (Figure 2b).

In the MTT assays, we used the protocol of “medium transfer” from irradiated to non-irradiated cells. For this protocol, PNT2, TRAMPC1 and B16F10 cells were seeded to confluence. At the time of starting X-ray exposure, we changed the medium to fresh medium in all cells (non-irradiated cells, non-irradiated cells + RA, irradiated cells and irradiated cells + RA); 4 h after irradiation, this medium was collected, where the factors that can induce Bys effects are found. This medium was subsequently centrifuged (200 g) and transferred to 96-well plates containing transient PNT2, TRAMPC1 and B16F10 cells in confluence. In the BysRA group, 25 microliters (µL) of a 25 micromolar (µM) solution of RA dissolved in PBS had been previously added. Bys cells were kept in culture with this medium for 48 h and analyzed as described in the MTT assays for the study of the direct effect induced by X-rays (Figure 2c).