Slow Releasing Long-lived radicals are induced by medium mediated bystander effect

Abstract

Because effects of bystander factors on the biology of cultured cells are very limited to compare with tremendous factors for those in directly irradiated cells, bystander systems are better for elucidating the mechanisms even these are still too complex. We have succeed in the direct measurement of slow releasing long-lived radicals (SRLLRs) by ESR in cultured cells. Levels of SRLLRs in irradiated cells have increased to +20% for five hours after irradiation. Suppression of SRLLRs by post-treatment of ascorbate has reduced point mutation frequency in control level. In this study we have measured the SRLLRs in bystander cells induced by medium mediated bystander effect with point mutation frequency. D-MEM(10% fcs) was gamma-ray irradiated in 1 Gy with donor CHO cells in cultur flasks and kept for 24 h. The medium was transferred to other flasks with non-irradiated bystander CHO cells and kept for 24h, then the bystander cells were harvested and put into ESR tube. The ESR spectra of bystander cells were recorded at 77 K. Levels of SRLLRs in bystander cells were increased to +20% in average, and frequency of HPRT- point mutation also increased four times larger than that of control. No increases detected when the medium was irradiated without donor cells. These results indicate that SRLLRs are induced by bystander factor produced by irradiated donor cells. Treatment of ascorbate (1 mM) to bystander cells for 24h from the medium transfer, both levels of SRLLRs and the mutation frequency was reduced with significant difference. The medium with myxothiazol, as an inhibitor of electron transportation in mitochondria, treated to donor cells before irradiation did not increase the levels of SRLLRs. These results strongly indicate that bystander factors are produced with dysfunction of mitochondria in donor cells and affect to produce SRLLRs in bystander cells. SRLLRs might be related to point mutation induction.