## 5 RIBE and RIAE Studies at the AS and ESRF Synchrotron Facilities  
### Studies at the AS  
#### 5.1.1 In Vitro RIBE Studies at the AS  
Earlier reports of bystander endpoints following partial irradiation of animals with conventional X- or \(\gamma\)-rays have attracted criticism asserting that the observed effects may have been due to low doses of scattered radiation, rather than true bystander signaling. To address this possibility, RIBE experiments were conducted at the IMBL at the AS by [PERSON]'s team [97; 98]. The design of irradiations was such that a defined zone of a cell population grown in one well of a two-well chamber slide was irradiated with various doses of MRT or BB at the dose rate of 49.3 Gy/s. The chambers were divided by a plastic wall; therefore, the cells grown in the non-irradiated well were separated from the irradiated cells and not exposed to the factors that the irradiated cells secrete to the medium. Scoring of \(\gamma\)-H2 AX foci in different cell populations--directly irradiated, out-of-the irradiated zone bystander cells in the irradiated well, and separated cells in the non-irradiated well--allowed a quantitative analysis of the impact of scattered radiation on the cellular response.  
Doses of scattered radiation were determined by a sensitive radiochromic film XRQA2 dosimetry at various distances from the irradiated zone. As expected, the scattered dose at the given distance correlated with the absorbed dose in the irradiated zone measured by a radiochromic film, EB3. The sensitivity of the \(\gamma\)-H2 AX assay was sufficient to measure as low as 0.01 Gy of scattered radiation at 30 min post-exposure [97], the time point that corresponds to maximum foci formation. Therefore, while the \(\gamma\)-H2 AX assay has not been shown to be an appropriate biodosimeter for MRT valley doses [99], it seems to be good for estimating the effects of scattered radiation after exposure of biological targets to synchrotron radiation. This quantification provided a tool for answering an important question: whether scattered radiation contributes to the appearance of RIBE.  
[PERSON] et al. [97] observed RIBE (manifested as increased DNA damage measured by \(\gamma\)-H2 AX foci) in naive human keratinocytes sharing medium with the cells in the irradiated well. An overall trend for a larger number of \(\gamma\)-H2 AX foci was observed in bystander cells compared to the cells in a separated well, only affected by scattered radiation. In the following study by [PERSON] et al. [98], human colon cancer cells were subjected to the similar study design as described above for MRT and BB irradiations. Scattered radiation substantially contributed to overall DNA damage accumulation in naive cells sharing medium with irradiated cells. Moreover, the cells exposed to the low-dose scatter also generated unrepairable DNA damage, possibly due to an ongoing exchange of bystander signaling induced by low dose-irradiation. A more pronounced bystander response was observed in unirradiated cells co-cultured with cells exposed to BB compared to MRT. A question as to whether loss of p53, the major tumor suppressor gene [100], would modify DNA damage response in non-irradiated cell cultures was also addressed. For p53-null cells, an overall reduced response to low-dose scatter irradiation, but not for RIBE, was detected. As a clinical implication, slower DNA damage repair and maintenance of unrepairable DNA damage in out-of-field tumor and normal tissuesmay cause over-sensitivity to low-dose scattered radiation in cancer radiotherapy patients, especially those carrying p53 deficiencies. Since p53 plays a central role in DNA damage responses, such as cell cycle arrest, senescence, apoptosis, and DNA repair [101; 102], the implication may be valid for deficiencies in the related genes.