The time and spatial effects of bystander response in mammalian cells induced by low dose radiation

Abstract

Bystander effects induced by low dose of ionizing radiation have been shown to widely exist in many cell types and may have a significant impact on radiation risk assessment. Though many studies have been reported on this phenomenological observation, the mechanisms underlying this process are not clear, especially on the questions of how soon after irradiation the bystander effects can be initiated and how far this bystander signal can be propagated once it is started. DNA double-strand breaks (DSBs) induced by ionizing radiation or carcinogenic chemicals can be visualized in situ using gamma-H2AX immunofluorescent staining. Our previous studies have shown that in situ visualization of DSBs could be used to assess irradiation-induced extranuclear/extracellular (bystander) effect at an early stage after irradiation. In the present studies, we used this method to investigate the time and spatial effects of damage signals on unirradiated bystander cells. The results showed that increased DSBs in irradiated and unirradiated bystander areas could be visualized 2 min after radiation and reached its maximum 30 min after radiation. The average levels of DSB formation at 30 min post-1cGy irradiation in the irradiated and unirradiated bystander areas were 3-fold and 2-fold higher than those of the sham-irradiated control cells, respectively. Afterwards, the formation of DSBs declined with incubation time and remained steady for at least 6 h at a level that was statistically higher than their controls. The results also showed that the bystander signal derived from irradiated cells could be transferred to anywhere in the dish and the percentage of DSBs in the unirradiated bystander cells was not dependent on the dose delivered. Moreover, the fraction of DSB positive cells in unirradiated bystander areas showed a time-dependent increase based on its distance to the irradiated area at very early stage post-irradiation. Both lindane and DMSO significantly suppressed the yield of DSBs in the cells of unirradiated bystander areas, which suggest that gap junctional intercellular communication and reactive oxygen species played important roles in the induction of the bystander effects, both in irradiated and unirradiated bystander areas.

DNA-PKcs and ATM influence generation of ionizing radiation-induced bystander signals

“…Frequency histograms (Figure 2b) illustrate that this is a general increase, rather than being limited to a subset of cells. It is also interesting to note that there was a similar increase in SCE frequency for both the 1:100 and 1:1000 dilutions, which is consistent with previous reports that the BSE appears to operate by an 'on/off' mechanism (Nagasawa and Little, 1992;Deshpande et al, 1996;Hu et al, 2006). Therefore, only the 1:100 dilution cell transfer method was utilized in subsequent experiments.…”

Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

“…Assuming that SCEs generated in bystander cells likewise result from HRR events, it seems unlikely this process involves directly induced ("frank") DNA DSBs as previously suggested [28,29]. The chromosome aberration and mutation spectra observed in bystander cells support our hypothesis that single-stranded oxidative lesions are the source of HRR-derived SCEs in S-phase cells.…”

“…The difference in the extent of this reduction between MMC and low dose α-particle exposures is likely related to the types and levels of DNA damage induced by these agents and specific repair systems employed to resolve this damage. It also must be noted that all cells are exposed in MMC-treated cultures whereas in the case of low dose α-particle irradiations, only a small percentage of cells are directly irradiated (≤2%) and a portion of non-irradiated cells may not be exposed to bystander molecules as a result of the limited diffusion potential of these signaling factors through gap-junction mediated intercellular communication (GJIC) or the cell culture medium [25,29,54].…”

Constitutive nitric oxide acting as a possible intercellular signaling molecule in the initiation of radiation-induced DNA double strand breaks in non-irradiated bystander cells

“…Many studies have demonstrated that ROS is very important for the induction of RIBE (reviewed by Azzam et al, 2004). Our previous studies (Hu et al, 2005(Hu et al, , 2006) also indicate that ROS is an important mediator for the induction of DSBs in non-irradiated cells. Furthermore, by treating medium donor cells with dimethyl sulfoxide, a quencher of ROS, before irradiation could effectively reduce the DIA of conditioned medium (Han et al, accepted).…”

“…In our previous studies, based on the immunochemistry of phosphorylated form of H2AX (g-H2AX), a reliable marker to reflect the induction of DNA double strand breaks (DSBs) in cellular nucleus (Rothkamm and Lobrich, 2003;Sedelnikova et al, 2002Sedelnikova et al, , 2004, we demonstrated that in situ visualization of DSBs could be used to assess the early events of radiation-induced extranuclear/extracellular (bystander) effects (Hu et al, 2005). In addition, the bystander DSBs were induced in a time-dependent manner and could be detected as early as 2 min post-irradiation (Hu et al, 2006). However, the nature of these intercellular signaling molecule(s) and how they participate in initiating radiation-induced bystander effects (RIBE) are not clear.…”