Low Doses of Gamma-Irradiation Induce an Early Bystander Effect in Zebrafish Cells Which Is Sufficient to Radioprotect Cells

Abstract

The term “bystander effect” is used to describe an effect in which cells that have not been exposed to radiation are affected by irradiated cells though various intracellular signaling mechanisms. In this study we analyzed the kinetics and mechanisms of bystander effect and radioadaptation in embryonic zebrafish cells (ZF4) exposed to chronic low dose of gamma rays. ZF4 cells were irradiated for 4 hours with total doses of gamma irradiation ranging from 0.01–0.1 Gy. In two experimental conditions, the transfer of irradiated cells or culture medium from irradiated cells results in the occurrence of DNA double strand breaks in non-irradiated cells (assessed by the number of γ-H2AX foci) that are repaired at 24 hours post-irradiation whatever the dose. At low total irradiation doses the bystander effect observed does not affect DNA repair mechanisms in targeted and bystander cells. An increase in global methylation of ZF4 cells was observed in irradiated cells and bystander cells compared to control cells. We observed that pre-irradiated cells which are then irradiated for a second time with the same doses contained significantly less γ-H2AX foci than in 24 h gamma-irradiated control cells. We also showed that bystander cells that have been in contact with the pre-irradiated cells and then irradiated alone present less γ-H2AX foci compared to the control cells. This radioadaptation effect is significantly more pronounced at the highest doses. To determine the factors involved in the early events of the bystander effect, we performed an extensive comparative proteomic study of the ZF4 secretomes upon irradiation. In the experimental conditions assayed here, we showed that the early events of bystander effect are probably not due to the secretion of specific proteins neither the oxidation of these secreted proteins. These results suggest that early bystander effect may be due probably to a combination of multiple factors.

Zebrafish as an In Vivo Model to Assess Epigenetic Effects of Ionizing Radiation

“…Recently, Pereira et al showed that γ irradiation increased the global methylation in both irradiated and bystander embryonic zebrafish fibroblast (ZF4) cells through mass spectrometry analyses [223]. High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) has been a common technique to evaluate the relative degree of global DNA methylation through determining the genome-wide level of cytosine and 5-mC in DNA samples [224].…”

Proteomic overview and perspectives of the radiation-induced bystander effects

“…An analysis of the secretome of zebrafish embryonic cells showed no specific bystander proteins following low-doses irradiation with chronic X-rays [57]. The factors secreted by a human skin model were analysed following irradiation using a multidimensional LC-MS strategy [58].…”

Key mechanisms involved in ionizing radiation-induced systemic effects. A current review

“…cytokines and other molecules) by the irradiated cells, contributing to the upregulation of oxidative metabolism in the bystander cells. [37][38][39][40] Interestingly, RIBE have been observed in a plethora of biological endpoints including DNA damage induction, [41][42][43][44] micronucleation, 45,46 genomic instability, 20,47,48 alteration in the microRNA (miRNA)…”

“…One approach is the transfer of medium from cultured irradiated cells to non-irradiated cells. 20,44,49,67 This technique allows simultaneously the detection and analysis of secreted RIBE factors in the conditioned medium and the study of their effect on non-irradiated (bystander) cells.…”

“…Another method is the in vitro co-culture, 20,44,56,66 often with some kind of compartmentalization to separate the groups of irradiated and non-irradiated cells. In this case the medium is freely diffusing between compartments.…”