A Comet Assay for DNA Damage and Repair After Exposure to Carbon-Ion Beams or X-rays in Saccharomyces Cerevisiae

Abstract

Ionizing radiation (IR) can result in serious genomic instability and genotoxicity by causing DNA damage. Carbon ion (CI) beams and X-rays are typical IRs and possess high-linear energy transfer (LET) and low-LET, respectively. In this article, a comet assay that was optimized by decreasing the electrophoresis time (8 minutes) and voltage (0.5 V/cm) was performed to elucidate and quantify the DNA damage induced by CI or X-rays radiation. Two quantitative methods for the comet assay, namely, comet score and olive tail moment, were compared, and the appropriate means and parameter values were selected for the present assay. The dose-effect relationship for CI or X-rays radiation and the DNA repair process were studied in yeast cells. These results showed that the quadratic function fitted the dose-effect relationship after CI or X-rays exposure, and the trend for the models fitted the dose-effect curves for various repair times was precisely described by the cubic function. A kinetics model was also creatively used to describe the process of DNA repair, and equations were calculated within repairable ranges that could be used to roughly evaluate the process and time necessary for DNA repair.

Keywords: DNA damage; DNA repair; Saccharomyces cerevisiae; comet assay; model fitting.

Figure 1.

Survival curves of yeast cells with 0, 25, 50, 75, 100, 125, 150, and 175 Gy CI (diamonds) or X-rays (squares) radiation. Each point represents the mean (standard deviation) of 3 estimates. The nonirradiated sample (0 Gy) was used as the negative control. CI indicates carbon ion.

Figure 2.

Classification of the comets. Comet categories are defined by the size and fluorescence intensity of the head and tail as well as tail length. The differences among stages reflect the extent of DNA damage. Stage 1, normal nucleus (very little DNA damage); stage 2, halo around the nucleus (slight DNA damage); stages 3 to 5, gradual increase in the length and intensity of the comet tail in parallel with a decrease in nuclear DNA content (varying degrees of DNA damage).

Figure 3.

Comet score (AU) indicating DNA damage in yeast cells after CI or X-rays irradiation. DNA damage was evaluated in an alkaline comet assay and quantified as described in the material and methods. Data are expressed as the mean AU of 3 independent experiments; bars indicate standard deviations. AU indicates arbitrary units; CI, Carbon ion.

Figure 4.

The quantification of DNA damage in yeast cells irradiated with various doses CI or X-rays using the Freeware Cometscore. Mean (SD) for TL (A) and OTM (B) of 3 independent experiments, with at least 300 comets per experiment for each dose (50 comets per slide). CI, Carbon ion; OTM, olive tail moment; SD, standard deviation; TL, tail length.

Figure 5.

Image samples obtained by the application of the yeast comet assay for untreated cells (0 Gy) and cells treated with CI (A) or X-rays (B) radiation of 100 and 175 Gy. The images were acquired with fluorescence microscopy at ×400 magnification and correspond to a representative assay from 3 independent experiments. Here, the blue arrow represents the cells that did not form comets (ie, did not form protoplasts); white bar = 20 μm. CI indicates carbon ion.

Figure 6.

Relationship between radiation dose and DNA damage for various repair times. DNA damage of yeast cells treated by CI or X-rays radiation was repaired in normal growth conditions (30°C and 200 rpm/min) for 3, 6, 12, and 24 hours, respectively, and then the residual DNA damage was assessed by OTM. A, For CI radiation, the dose–effect curves for various repair times was fitted by the cubic function and the extent of repair of DNA damage decreased for increasing doses. B, For X-rays radiation, the dose–effect curves for various repair times were also fitted to a cubic function, but the DNA damage was almost completely repaired in the range of 25 to 175 Gy. CI indicates carbon ion; OTM, olive tail moment.

Figure 7.

Repair of DNA damage induced by CI or X-rays radiation in yeast cells. A, Cells were irradiated with various doses of CI before embedding in agarose and lysing, and then incubated in normal growth conditions (30°C, 200 rpm/min) for 3, 6, 12, and 24 hours to repair. B, Cells were irradiated with various doses of X-rays following the methods in (A). The data represented are the means of at least 3 independent experiments. CI indicates carbon ion.