Mutagenic effects of a single and an exact number of alpha particles in mammalian cells

Abstract

One of the main uncertainties in risk estimation for environmental radon exposure using lung cancer data from underground miners is the extrapolation from high- to low-dose exposure where multiple traversal is extremely rare. The biological effects of a single alpha particle are currently unknown. Using the recently available microbeam source at the Radiological Research Accelerator Facility at Columbia University, we examined the frequencies and molecular spectrum of S1- mutants induced in human-hamster hybrid (A(L)) cells by either a single or an exact number of alpha particles. Exponentially growing cells were stained briefly with a nontoxic concentration of Hoechst dye for image analysis, and the location of individual cells was computer-monitored. The nucleus of each cell was irradiated with either 1,2,4, or 8 alpha particles at a linear energy transfer of 90 keV/microm consistent with the energy spectrum of domestic radon exposure. Although single-particle traversal was only slightly cytotoxic to A(L) cells (survival fraction approximately 0.82), it was highly mutagenic, and the induced mutant fraction averaged 110 mutants per 10(5) survivors. In addition, both toxicity and mutant induction were dose-dependent. Multiplex PCR analysis of mutant DNA showed that the proportion of mutants with multilocus deletions increased with the number of particle traversals. These data provide direct evidence that a single a particle traversing a nucleus will have a high probability of resulting in a mutation and highlight the need for radiation protection at low doses.

Figure 1

Fluorescent imaging of A_L cells stained with Hoechst dye viewed by the image analysis system under a 40× objective lens. The nucleus of each cell is outlined in white, and the circles indicate the area where the particle is delivered.

Figure 2

Survival of A_L cells irradiated in the nucleus with either a single or an exact number of α particles. Data were pooled from 3–4 independent experiments. Bar represents ± SEM.

Figure 3

Induced mutants per 10⁵ survivors at the S1 locus in A_L cells irradiated with an exact number of α-particle traversals at 90 keV/μm. Induced mutant yield = total mutant yield minus background incidence. The background mutant fraction in A_L cells used in these experiments averaged 45 per 10⁵ survivors. Data were pooled from three experiments, and the curves fitted using the least-square method. (Bars represent ± SEM.)

Figure 4

(Right) Gel electrophoresis of multiplex PCR products using DNA from S1⁻ mutants as templates and primers for parathyroid hormone (PTH), Wilms tumor (WT), catalase (CAT), apolipoprotein A1 (APO-A1), and RAS. HaeIII-digested φX174 DNA provided the size markers (lane M). Lane 1, wild-type A_L cells with all of the markers present. Lane 2, a positive control showing the loss of all the markers examined except RAS. Lanes 3 and 4, spontaneous mutants showing no marker loss. Lanes 5 and 6, mutants induced by a single α particle where none of these markers was lost. Lanes 7–12, mutants induced by eight α particles showing mostly deletions of various sizes. (Left) The relative location of the marker genes on human chromosome 11 used in the multiplex PCR and their relative distance from the M1C1 gene.

Figure 5

Cumulative deletion spectra of S1⁻ mutants either of spontaneous origin or from cells exposed to either a single or an exact number of α-particle traversals through the nucleus. Each line depicts the spectrum from a single, independent mutant. The absence or presence of marker genes among the mutants was determined by multiplex PCR. Blank spaces depict missing markers.