Hypothermia and radiation. Effects on DNA, chromatin and cellular survival

Abstract: The effect of ionising radiation on cells can be modulated by several physico-chemical factors. The aim of this study was to investigate if hypothermic (sub-physiological) temperatures (2°C-28°C) may influence the effect of X-rays on cellular damage in human normal fibroblasts and in a human breast adenocarcinoma cell line, MCF-7.The induction of double-strand breaks (dsb) was measured by constant field electrophoresis. Dsb induction was found to decrease with decreasing temperature when DNA from the malignant cell line MCF-7 was irradiated under different temperature conditions. This temperature effect was most prominent in a non-scavenging environment (PBS) with a dose modifying factor 3.4 found between 37°C and 2°C. This difference was abolished by a high concentration of scavenger (2.0 M DMSO), which indicates that the temperature effect is mediated by the indirect effects of ionising radiation. No difference between the two temperatures was found when intact MCF-7 cells were irradiated. However, the rejoining of dsb during the fast phase was slower in MCF-7 cells irradiated with 40 Gy at 37°C compared to cells irradiated at 2°C. A possible explanation may be that the damage is more complex and requires longer time to restore.The induction of chromatin damage was quantified in normal fibroblasts and MCF-7 cells by measuring the supercoiling ability of DNA in individual cells using the fluorescent nucleoid assay. Radiation-induced chromatin damage was decreased by low temperature in both normal fibroblasts and MCF-7 cells. The temperature during irradiation (2°C or 37°C) was more important than the temperature during a pre-incubation for 1h. No difference was found in the protective effect of low temperature between normal and transformed cells.MCF-7 cells irradiated at 2°C with 2, 3 and 4 Gy had a higher clonogenic survival than cells irradiated at 37°C using a colony forming assay in which the ability of cells to divide more than 6 times is quantified. The dose modifying factor was 1.23. No difference in cellular growth using the crystal violet assay was found in MCF-7 cells or normal fibroblasts during 19 days following irradiation at 2°C or 37°C.In conclusion, hypothermic temperatures during irradiation seemed to influence induction and rejoining of dsb as well as the induction of chromatin damage. The higher clonogenic survival found in cells irradiated at low temperature may reflect the modulation of these types of damage by the temperature.