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.
Highlights
Increasing evidence suggests that genomic DNA damages induced by various endogenous or environmental factors
To compare the protective effects of Dimethyl sulfoxide (DMSO) against double strand breaks (DSBs), caused due to damage by different sources, based on experimentally available kinetic constants, Kv, Kγ, and Kf: nf, we introduced a relative constant, k = K/K0, where K0 is the kinetic constant of the control group for each injury source in the absence of DMSO
Using this renormalized constant k, it is possible to compare the degree of the protective effect of DMSO, regardless of any difference in the mechanism of double-strand breakage, including whether the mechanism involves either one-step or two-step reactions
Summary
Increasing evidence suggests that genomic DNA damages induced by various endogenous or environmental factors. When the DSBs occur in the preserved cells, the damage is hazardous to the cell, and the viability after preservation will be significantly lower, because the cell will not survive during subsequent cell mitosis after thawing This protective effect on DNA has been argued to be related to the strong solvation effect of DMSO on water molecules[23, 31,32,33, 41]. DMSO interacts with water molecules through the two hydrogen bonds of water[40] Despite these useful practical applications of DMSO, the protective effect of DMSO against DSBs induced due to radiation or freezing/thawing has not yet been evaluated quantitatively. In order to quantitatively evaluate the protective effects of DMSO against DSBs on genomic DNA molecules by use of single DNA observation, we induced DSBs by several different injury sources; photo-induced reactive oxygen, γ-ray irradiation, and freeze/thawing. Because thermal disruption is one of the critical factors in cryopreservation, application of our method will provide a critical knowledge about freezing/thawing stress on preserved DNA
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