Langie Sabine A S, Knaapen Ad M, Brauers Karen J J, van Berlo Damien, van Schooten Frederik-Jan, Godschalk Roger W L
Nutrition and Toxicology Research Institute Maastricht, NUTRIM, Department of Health Risk Analysis and Toxicology, Maastricht University, 6200 MD, PO Box 616, Maastricht, The Netherlands.
Mutagenesis. 2006 Mar;21(2):153-8. doi: 10.1093/mutage/gel013. Epub 2006 Mar 23.
There is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacities. Therefore, a modification of the alkaline comet assay was developed to determine the ability of human lymphocyte extracts to perform the initial steps of the nucleotide excision repair (NER) process, i.e. damage recognition and incision. Gel-embedded nucleoids from A549 cells, pre-exposed to 1 microM benzo[a]pyrene-diol-epoxide, were incubated with cell extracts from frozen or freshly isolated lymphocytes. The rate at which incisions are introduced and the subsequent increase in tail moment is indicative for the repair capacity of the extracts. Freshly prepared extracts from lymphocytes of human volunteers (n = 8) showed significant inter-individual variations in their DNA repair capacity, which correlated with the removal of bulky DNA lesions over a period of 48 h determined by (32)P-post-labelling (R(2) = 0.76, P = 0.005). Repeated measurements revealed a low inter-assay variation (11%). Storage of cell extracts for more than 3 weeks significantly reduced (up to 80%) the capacity to incise the damaged DNA as compared to freshly isolated extracts. This reduction was completely restored by addition of ATP to the extracts before use, as it is required for the incision step of NER. In contrast, extracts freshly prepared from frozen lymphocyte pellets can be used without loss of repair activity. DNA repair deficient XPA-/- and XPC-/- fibroblasts were used to further validate the assay. Although some residual capacity to incise the DNA was observed in these cells, the repair activity was restored to normal wild-type levels when a complementary mixture of both extracts (thereby restoring XPA and XPC deficiency) was used. These results demonstrate that this repair assay can be applied in molecular epidemiological studies to assess inter-individual differences in NER.
对DNA修复能力进行表型评估的简单可靠方法的需求日益增加。因此，开发了一种碱性彗星试验的改良方法，以确定人淋巴细胞提取物执行核苷酸切除修复（NER）过程初始步骤的能力，即损伤识别和切割。将预先暴露于1 microM苯并[a]芘二醇环氧化物的A549细胞的凝胶包埋核仁与来自冷冻或新鲜分离淋巴细胞的细胞提取物一起孵育。引入切割的速率以及随后尾矩的增加表明提取物的修复能力。来自人类志愿者（n = 8）淋巴细胞的新鲜制备提取物在DNA修复能力上表现出显著的个体间差异，这与通过（32）P后标记在48小时内确定的大分子DNA损伤的去除相关（R（2）= 0.76，P = 0.005）。重复测量显示测定间变异较低（11%）。与新鲜分离的提取物相比，细胞提取物储存超过3周会显著降低（高达80%）切割受损DNA的能力。在使用前向提取物中添加ATP可完全恢复这种降低，因为这是NER切割步骤所必需的。相比之下，从冷冻淋巴细胞沉淀中新鲜制备的提取物可以使用而不会损失修复活性。DNA修复缺陷的XPA-/-和XPC-/-成纤维细胞用于进一步验证该试验。尽管在这些细胞中观察到了一些切割DNA的残余能力，但当使用两种提取物的互补混合物（从而恢复XPA和XPC缺陷）时，修复活性恢复到了正常野生型水平。这些结果表明，这种修复试验可应用于分子流行病学研究，以评估NER中的个体间差异。
