van Loon A A, Sonneveld E, Hoogerbrugge J, van der Schans G P, Grootegoed J A, Lohman P H, Baan R A
TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
Mutat Res. 1993 Aug;294(2):139-48. doi: 10.1016/0921-8777(93)90022-9.
Alkaline elution has been used for quantitative detection of DNA damage caused by ionizing radiation in unlabeled somatic and germ cells. Both the induction and subsequent repair have been studied for two classes of DNA damage, viz. single-strand breaks (SSB), and base damage (BD) recognized by the gamma-endonuclease activity in a cell-free extract of Micrococcus luteus bacteria. The high sensitivity of the assay permitted the measurement of induction and repair of SSB and BD after in vitro exposure of hamster germ cells in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids), and of bone-marrow cells, to biologically relevant doses (0-8 Gy) of 60Co gamma-rays. A dose-dependent increase was observed for both types of lesions, which was similar for most cell types. The elongated spermatids, however, showed a lower induction frequency of SSB (and perhaps BD). Spermatocytes, round spermatids and bone-marrow cells had normal, fast repair of the SSB when compared with the repair reported for cultured rodent cells and human lymphocytes. In contrast, the elongated spermatids showed hardly any SSB repair. The initial rate of repair of BD in spermatocytes and bone-marrow cells was in the same range as that for SSB, but only 60-70% of the initial BD was repaired within 1 h, whereas after that period no SSB were detectable. The round spermatids hardly repaired any BD within the first hour after irradiation, but after 7 h only a few BD could be detected. In elongated spermatids repair of BD could not be measured due to a high background level of this type of damage.
碱性洗脱法已用于定量检测未标记的体细胞和生殖细胞中由电离辐射引起的DNA损伤。对于两类DNA损伤，即单链断裂（SSB）和碱基损伤（BD），人们已经研究了其诱导和后续修复过程。这两类损伤可通过藤黄微球菌无细胞提取物中的γ-核酸内切酶活性来识别。该检测方法的高灵敏度使得在体外将仓鼠生殖细胞（处于精子发生不同细胞阶段的精母细胞、圆形和伸长的精子细胞）以及骨髓细胞暴露于生物学相关剂量（0 - 8 Gy）的60Co γ射线后，能够测量SSB和BD的诱导和修复情况。观察到这两种损伤类型均呈剂量依赖性增加，大多数细胞类型的情况相似。然而，伸长的精子细胞显示出较低的SSB诱导频率（可能还有BD诱导频率）。与培养的啮齿动物细胞和人类淋巴细胞的修复情况相比，精母细胞、圆形精子细胞和骨髓细胞对SSB具有正常、快速的修复能力。相比之下，伸长的精子细胞几乎没有任何SSB修复。精母细胞和骨髓细胞中BD的初始修复速率与SSB的初始修复速率处于同一范围，但在1小时内仅修复了初始BD的60 - 70%，而在此之后就检测不到SSB了。圆形精子细胞在照射后的第一小时内几乎没有修复任何BD，但在7小时后只能检测到少量BD。由于这种类型损伤的背景水平较高，无法测量伸长精子细胞中BD的修复情况。
