Boucher Didier, Hindo Joëlle, Averbeck Dietrich
Institut Curie-Section de Recherche, UMR2027 CNRS/I.C., LCR V28 CEA, Bâtiment 110, Centre Universitaire d'Orsay, F-91405 Orsay CEDEX, France.
Can J Physiol Pharmacol. 2004 Feb;82(2):125-32. doi: 10.1139/y04-006.
DNA double-strand breaks (DSBs) are highly cell damaging. We asked whether for a given dose a longer irradiation time would be advantageous for the repair of DSBs. Varying the gamma-irradiation dose and its delivery time (0.05 Gy/min low dose-rate (LDR) compared with 3.5 Gy/min high dose-rate), confluent Chinese hamster ovary cells (CHO-K1) and Ku80 mutant cells (xrs-6) deficient in nonhomologous end-joining (NHEJ) were irradiated in agarose plugs at room temperature using a cesium-137 gamma-ray source. We used pulsed-field gel electrophoresis (PFGE) to measure DSBs in terms of the fraction of activity released (FAR). At LDR, one third of DSBs were repaired in CHO-K1 but not in xrs-6 cells, indicating the involvement of NHEJ in the repair of gamma-induced DSBs at a prolonged irradiation incubation time. To improve DSB measurements, we introduced in our PFGE protocol an antioxidant at the cell lysis step, thus avoiding free-radical side reactions on DNA and spurious DSBs. Addition of the metal chelator deferoxamine (DFO) decreased more efficiently the basal DSB level than did reduced glutathione (GSH), showing that measuring DSBs in the absence of DFO reduces precision and underestimates the role of NHEJ in the dose-rate effect on DSB yield.
DNA双链断裂（DSB）对细胞具有高度损伤性。我们探讨了对于给定剂量，延长照射时间是否有利于DSB的修复。改变γ射线照射剂量及其照射时间（低剂量率（LDR）为0.05 Gy/分钟，高剂量率为3.5 Gy/分钟），将汇合的中国仓鼠卵巢细胞（CHO-K1）和缺乏非同源末端连接（NHEJ）的Ku80突变细胞（xrs-6）在室温下于琼脂糖凝胶块中使用铯-137γ射线源进行照射。我们使用脉冲场凝胶电泳（PFGE）根据释放活性分数（FAR）来测量DSB。在低剂量率下，CHO-K1细胞中有三分之一的DSB得到修复，而xrs-6细胞中则未修复，这表明在延长的照射孵育时间下，NHEJ参与了γ射线诱导的DSB的修复。为了改进DSB测量，我们在PFGE实验方案的细胞裂解步骤中引入了一种抗氧化剂，从而避免DNA上的自由基副反应和假性DSB。与还原型谷胱甘肽（GSH）相比，添加金属螯合剂去铁胺（DFO）能更有效地降低基础DSB水平，这表明在没有DFO的情况下测量DSB会降低精度，并低估NHEJ在剂量率对DSB产量影响中的作用。
