Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells

Abstract

Ever since x-rays were shown to induce mutation in Drosophila more than 70 years ago, prevailing dogma considered the genotoxic effects of ionizing radiation, such as mutations and carcinogenesis, as being due mostly to direct damage to the nucleus. Although there was indication that alpha particle traversal through cellular cytoplasm was innocuous, the full impact remained unknown. The availability of the microbeam at the Radiological Research Accelerator Facility of Columbia University made it possible to target and irradiate the cytoplasm of individual cells in a highly localized spatial region. By using dual fluorochrome dyes (Hoechst and Nile Red) to locate nucleus and cellular cytoplasm, respectively, thereby avoiding inadvertent traversal of nuclei, we show here that cytoplasmic irradiation is mutagenic at the CD59 (S1) locus of human-hamster hybrid (AL) cells, while inflicting minimal cytotoxicity. The principal class of mutations induced are similar to those of spontaneous origin and are entirely different from those of nuclear irradiation. Furthermore, experiments with radical scavenger and inhibitor of intracellular glutathione indicated that the mutagenicity of cytoplasmic irradiation depends on generation of reactive oxygen species. These findings suggest that cytoplasm is an important target for genotoxic effects of ionizing radiation, particularly radon, the second leading cause of lung cancer in the United States. In addition, cytoplasmic traversal by alpha particles may be more dangerous than nuclear traversal, because the mutagenicity is accomplished by little or no killing of the target cells.

Figure 1

Dual fluorescent imaging of A_L cells stained with Hoechst 33342 (nucleus) and Nile Red (cytoplasm) by the image analysis system under a 40× objective lens. The nucleus of each cell is outlined in white. The image analysis system determines the length of the major axis of each nucleus to calculate the irradiation positions that are chosen to be 8 μm from each end of the nucleus, as shown by the small, numbered circles.

Figure 2

Survival of A_L cells irradiated with a single or an exact number of 90 keV/μm α-particles targeted to areas of the cytoplasm. Each data point was obtained from three to seven independent experiments. (Bars represent ± SEM.)

Figure 3

Induced CD59⁻ mutant fractions per 10⁵ survivors in A_L cells irradiated with exact numbers of α-particle traversals through the cytoplasm. Induced mutant frequency equals total mutant yield minus background incidence. Data are pooled from 11 experiments. Induced mutant yield at 32 particles was the same as at eight particles and showed no further increase (data not shown). (Bars represent ± SEM.)

Figure 4

Mutational spectra of CD59⁻ mutants either of spontaneous origin or from cells exposed to eight α-particles delivered either to the cytoplasm or the nucleus. Each line represents the spectrum for a single, independent mutant. Blank spaces depict missing markers. The absence or presence of marker genes in each mutant was determined by multiplex PCR. Nuclear irradiation with eight α-particles resulted in 384.6 ± 116 mutants per 10⁵ survivors at a surviving fraction of 12% (3).

Figure 5

Effects of the free radical scavenger DMSO and the thiol-depleting drug BSO on induced mutant yield in A_L cells irradiated with four α-particles through the cytoplasm. Cells were treated with 8% DMSO for 10 min before and 10 min after irradiation or with 10 μm BSO for at least 18 hr before irradiation. Data were pooled from three to six experiments. Neither DMSO nor BSO was mutagenic alone. (Bars represent ± SEM.)

Figure 6

Relative immunoperoxidase staining intensity for 8-OHdG in control and A_L cells irradiated with eight α-particles through the cytoplasm. Data are averaged from three independent experiments with 60–90 nuclei each. (Bars represent ± SEM.)