Induction of a bystander mutagenic effect of alpha particles in mammalian cells

Abstract

Ever since the discovery of X-rays was made by Röntgen more than a hundred years ago, it has always been accepted that the deleterious effects of ionizing radiation such as mutation and carcinogenesis are attributable mainly to direct damage to DNA. Although evidence based on microdosimetric estimation in support of a bystander effect appears to be consistent, direct proof of such extranuclear/extracellular effects are limited. Using a precision charged particle microbeam, we show here that irradiation of 20% of randomly selected A(L) cells with 20 alpha particles each results in a mutant fraction that is 3-fold higher than expected, assuming no bystander modulation effect. Furthermore, analysis by multiplex PCR shows that the types of mutants induced are significantly different from those of spontaneous origin. Pretreatment of cells with the radical scavenger DMSO had no effect on the mutagenic incidence. In contrast, cells pretreated with a 40 microM dose of lindane, which inhibits cell-cell communication, significantly decreased the mutant yield. The doses of DMSO and lindane used in these experiments are nontoxic and nonmutagenic. We further examined the mutagenic yield when 5-10% of randomly selected cells were irradiated with 20 alpha particles each. Results showed, likewise, a higher mutant yield than expected assuming no bystander effects. Our studies provide clear evidence that irradiated cells can induce a bystander mutagenic response in neighboring cells not directly traversed by alpha particles and that cell-cell communication process play a critical role in mediating the bystander phenomenon.

Figure 1

Survival of A_L cells irradiated with an exact number of alpha particles in the nucleus. Data were pooled from three to four independent experiments. Error bars represent ± SEM.

Figure 2

Mutant fraction obtained from populations of A_L cells in which 0, 5, 10, or 20% of whose nuclei were traversed by 20 alpha particles. Data were pooled from three to eight independent experiments. Error bars represent ± SEM.

Figure 3

Mutational spectra of CD59⁻ mutants isolated from unirradiated populations or from populations in which 20% of the cells had been irradiated with 20 alpha particles through their nuclei. Each line depicts a single mutant. Blank spaces depict missing markers on chromosome 11 as determined by multiplex PCR.

Figure 4

Effect of lindane treatment (40 μM, 2 hr before and 3 days after irradiation) on mutant yields in A_L cells 20% of which had been irradiated with 20 alpha particles through their nuclei. Data were pooled from three independent experiments. Error bar represents ± SEM.