A single-molecule assessment of the protective effect of DMSO against DNA double-strand breaks induced by photo-and γ-ray-irradiation, and freezing

Abstract

Dimethyl sulfoxide (DMSO) is widely used as a cryoprotectant for organs, tissues, and cell suspension in storage. In addition, DMSO is known to be a useful free radical scavenger and a radio-protectant. To date, many in vitro assays using cultured cells have been performed for analysing the protective effect of DMSO against genomic DNA damage; however, currently it has been rather difficult to detect DNA double strand breaks (DSBs) in a quantitative manner. In the present study, we aimed to observe the extent of DNA damage by use of single molecular observation with a fluorescence microscope to evaluate DSBs induced by photo- and γ-ray-irradiation, or freeze/thawing in variable concentrations of DMSO. As a result, we found that 2% DMSO conferred the maximum protective effect against all of the injury sources tested, and these effects were maintained at higher concentrations. Further, DMSO showed a significantly higher protective effect against freezing-induced damage than against photo- and γ-ray-irradiation-induced damage. Our study provides significant data for the optimization of DNA cryopreservation with DMSO, as well as for the usage of DNA as the protective agent against the injuries caused by active oxygen and radiations.

Figure 1

Example of the real-time observation of DSB caused by photo-irradiation-induced ROS. Fluorescence microscopic images of a single T4 DNA molecule under photo-irradiation (upper), and the corresponding quasi-three-dimensional profiles of the fluorescence intensity distribution (bottom). (Fluorescence dye: 0.05 μM YOYO-1).

Figure 2

Photo-induced DSBs. (a) Time-dependence of the percentage of damaged DNA molecules at different DMSO concentrations. (b) The relationship between t ² and log₁₀ P, where P is the percentage of surviving DNA molecules, which was calculated as [100% − (percentage of damaged DNA)]. (The kinetic constants, K _v’s (s⁻²), are evaluated from the slopes of Fig. 2b. For DMSO’s concentration on 2%, 3% and 5%, the slopes are essentially the same within experimental errors, indicating the presence of saturation effect).

Figure 3

DSBs induced by γ-ray. (a) Fluorescence microscopic images of DNA molecules fixed on a glass surface after irradiation with different doses of γ-ray. (b) Average DNA lengths, 〈L〉, vs. the irradiation dose of γ-rays. (c) Number of DSBs per 10 kbp, 〈n〉, vs. the irradiation dose of γ-rays. (The kinetic constants, Kγ ‘s (Gy), are evaluated from the slopes of Fig. 3c.) The slopes are essentially the same for the DMSO concentrations above 2%, indicating saturation effect.

Figure 4

Freezing-induced DSBs. (a) Single DNA image after freeze/thawing to −25 °C (upper: slow frozen) and −80 °C (lower: quick frozen). (b) Average lengths of DNA, 〈L〉, vs. the concentration of DMSO. (c) The number of DSBs per 10 kbp, 〈n _f〉, vs. the concentration of DMSO.

Figure 5

Difference in the protective effect of DMSO. Vertical axis is the relative kinetic constant k = K/K ₀ for the generation of DSBs at different concentrations of DMSO, where K ₀ is the rate constant in the absence of DMSO. With respect to freezing, ‘quick freezing’ is to −80 °C, and ‘slow freezing’ is to −25 °C.