Effects of irradiated medium with or without cells on bystander cell responses

Abstract

Recent studies have indicated that extranuclear or extracellular targets are important in mediating the bystander genotoxic effects of alpha-particles. In the present study, human-hamster hybrid (A(L)) cells were plated on either one or both sides of double-mylar dishes 2-4 days before irradiation, depending on the density requirement of experiments. One side (with or without cells) was irradiated with alpha-particles (from 0.1 to 100 Gy) using the track segment mode of a 4 MeV Van de Graaff accelerator. After irradiation, cells were kept in the dishes for either 1 or 48 h. The non-irradiated cells were then collected and assayed for both survival and mutation. When one side with cells was irradiated by alpha-particles (1, 10 and 100 Gy), the surviving fraction among the non-irradiated cells was significantly lower than that of control after 48 h co-culture. However, such a change was not detected after 1h co-culture or when medium alone was irradiated. Furthermore, co-cultivation with irradiated cells had no significant effect on the spontaneous mutagenic yield of non-irradiated cells collected from the other half of the double-mylar dishes. These results suggested that irradiated cells released certain cytotoxic factor(s) into the culture medium that killed the non-irradiated cells. However, such factor(s) had little effect on mutation induction. Our results suggest that different bystander end points may involve different mechanisms with different cell types.

Fig. 1

Schematic diagram of a representative 35 mm i.d. double-mylar cell culture dish where only the upper layer contains a monolayer of attached cells (A). When α-particles traverse the bottom mylar layer, no cells are hit. In contrast, when a monolayer of cells are plated on both mylar layers which are separated by 9.5 mm (B), α-particles would traverse the bottom but not the top layer of cells. A hole on each side of the ring serves as a filling port for medium.

Fig. 2

SF of non-irradiated cells in double-mylar dishes where medium only was irradiated by α-particles ranging in doses from 0.1 to 100 Gy. Data were pooled from three independent experiments. Bar represents ±S.D.

Fig. 3

SF of non-irradiated cells in double-mylar dishes where the bottom layer of cells were irradiated with graded doses of α-particles from 0.1 to 100 Gy. Data were pooled from three independent experiments. Bar represents ±S.D.

Fig. 4

Mutation fraction of the upper, non-irradiated layer of A_L cells in double-mylar dishes where only media were irradiated by α-particles ranging in doses from 0.1 to 100 Gy. Data were pooled from three independent experiments. Bar represents ±S.D.

Fig. 5

Mutation fraction of the upper, non-irradiated layer of A_L cells in double-mylar dishes where the bottom layer of cells were irradiated with graded doses of α-particles from 0.1 to 100 Gy. Data were pooled from three independent experiments. Bar represents ± S.D.