Radiation-induced bystander effects refer to the induction of biological changes in cells not directly hit by radiation implying that the number of cells affected by radiation is larger than the actual number of irradiated cells. Recent in vitro studies suggest the role of extracellular vesicles (EVs) in mediating radiation-induced bystander signals, but in vivo investigations are still lacking. Here, we report an in vivo study investigating the role of EVs in mediating radiation effects. C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2 Gy), and 24 h later, EVs were isolated from the bone marrow (BM) and were intravenously injected into unirradiated (so-called bystander) animals. EV-induced systemic effects were compared to radiation effects in the directly irradiated animals. Similar to direct radiation, EVs from irradiated mice induced complex DNA damage in EV-recipient animals, manifested in an increased level of chromosomal aberrations and the activation of the DNA damage response. However, while DNA damage after direct irradiation increased with the dose, EV-induced effects peaked at lower doses. A significantly reduced hematopoietic stem cell pool in the BM as well as CD4+ and CD8+ lymphocyte pool in the spleen was detected in mice injected with EVs isolated from animals irradiated with 2 Gy. These EV-induced alterations were comparable to changes present in the directly irradiated mice. The pool of TLR4-expressing dendritic cells was different in the directly irradiated mice, where it increased after 2 Gy and in the EV-recipient animals, where it strongly decreased in a dose-independent manner. A panel of eight differentially expressed microRNAs (miRNA) was identified in the EVs originating from both low- and high-dose-irradiated mice, with a predicted involvement in pathways related to DNA damage repair, hematopoietic, and immune system regulation, suggesting a direct involvement of these pathways in mediating radiation-induced systemic effects. In conclusion, we proved the role of EVs in transmitting certain radiation effects, identified miRNAs carried by EVs potentially responsible for these effects, and showed that the pattern of changes was often different in the directly irradiated and EV-recipient bystander mice, suggesting different mechanisms.

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Purpose: This work investigates the hypothesis that genetic background plays a significant role in the signalling mechanisms underlying induction and perpetuation of genomic instability following radiation exposure. Materials and methods:Bone marrow from two strains of mice (CBA and C57), were exposed to a range of X-ray doses (0, 0.01, 0.1, 1 and 3 Gy). Different cellular signalling endpoints: apoptosis, cytokine levels and calcium flux, were evaluated at 2h, 24h and 7d post-irradiation to evaluate immediate and delayed effects.Results: In CBA (radio-sensitive) elevated apoptosis levels were observed at 24h post X-irradiation, transforming growth factor-β (TGF-β) levels were additionally shown to increase with time and dose. C57 showed a higher background level of apoptosis compared to CBA, which was sustained 7 days after radiation exposure. Levels of tumor necrosis factor-α (TNF-α) were also increased at day 7 for higher X-ray doses. TGF-β levels were higher in CBA, whilst C57 exhibited a greater TNF-α response.Calcium flux was induced in reporter cells on exposure to conditioned media from both strains. Conclusions:These results show genetic and dose specific differences in radiation-induced signalling in the initiation and perpetuation of the instability process, which have potential implications on evaluation of non-targeted effects in radiation risk assessment.3

No effect of serotonin on the induction of either bystander effects of genomic instability was observed using this experimental system.