Kaur B S, Blazek E R
Biology Department, Illinois Institute of Technology, Chicago 60616-3793, USA.
Radiat Res. 1997 May;147(5):569-78.
The apparent biological significance of DNA double-strand breaks (DSBs) has stimulated considerable effort toward quantification of this lesion. The neutral (or nondenaturing) filter elution assay at pH 7.2 or 9.6 has long been a standard method for the measurement of double-strand breakage and rejoining in eukaryotic cells, with a threshold dose for detection of DSBs of 5-10 Gy. Agarose gel electrophoresis, either pulsed- or constant-field, can detect DSBs induced by as little as 1 Gy of ionizing radiation, but electrophoresis assays may have inherent problems in measurement of break rejoining, and may be more susceptible than elution to factors other than break frequency, such as cell cycle stage or bromodeoxyuridine substitution. We report here that filter elution performed at pH 11.1 can detect DSBs produced by only 1 Gy of ionizing radiation, but is insensitive to the single-strand breaks that are formed when cells are exposed to hydrogen peroxide. Double-strand breaks produced in permeabilized cells by the restriction endonuclease HaeIII were used to demonstrate that the increase in the pH of the eluting solution from 9.6 to 11.1, although increasing assay sensitivity by a factor of five, converts few additional alkali-labile sites to DSBs. Thus validated, the pH 11.1 filter elution assay was applied to a low-dose measurement of induction and rejoining of DSBs in 9L cells.
DNA双链断裂（DSB）明显的生物学意义激发了人们对这种损伤进行量化的大量努力。长期以来，在pH 7.2或9.6条件下进行的中性（或非变性）滤膜洗脱测定法一直是测量真核细胞中双链断裂和重新连接的标准方法，检测DSB的阈值剂量为5-10 Gy。脉冲场或恒场琼脂糖凝胶电泳能够检测低至1 Gy电离辐射诱导产生的DSB，但电泳测定法在测量断裂重接方面可能存在固有问题，并且可能比洗脱测定法更容易受到除断裂频率之外的其他因素影响，如细胞周期阶段或溴脱氧尿苷取代情况。我们在此报告，在pH 11.1条件下进行的滤膜洗脱测定法能够检测仅由1 Gy电离辐射产生的DSB，但对细胞暴露于过氧化氢时形成的单链断裂不敏感。用限制性内切酶HaeIII在通透细胞中产生的双链断裂来证明，洗脱溶液的pH从9.6增加到pH 11.1，虽然测定灵敏度提高了五倍，但仅将少量额外的碱不稳定位点转化为DSB。经过验证后，pH 11.1滤膜洗脱测定法被应用于对9L细胞中DSB诱导和重接的低剂量测量。
