Baumgartner A, Schmid T E, Cemeli E, Anderson D
Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK, GSF Research Center, Institute of Molecular Radiobiology, 85764 Neuherberg, Germany.
Mutagenesis. 2004 Jul;19(4):313-8. doi: 10.1093/mutage/geh032.
In recent years, two techniques for detecting genetic damage in the whole genome have gained importance: the alkaline comet assay, to detect DNA damage such as strand breaks and alkali-labile sites, and a multicolour FISH method, spectral karyotyping (SKY), to identify chromosomal aberrations simultaneously in all metaphase chromosomes. In the present study, the induction of DNA damage in human sperm and lymphocytes in vitro has been studied employing an anticancer drug, doxorubicin (DX). An increase in DNA damage was observed with the comet assay as the median per cent head DNA of sperm significantly decreased from 82.07 and 85.14% in the untreated control groups to 63.48 and 72.52% at doses of 0.8 micro M DX. At 1.6 micro M the percentage declined to 60.96% (the corresponding tail moment increased from 4.42 to 12.19). In stimulated lymphocytes, a significant increase was observed in tail moment, from 0.72 and 0.53 in controls to 15.17 and 12.10 at 0.2 micro M DX, continuing at the same level to a final concentration of 1.6 micro M. Structural aberrations found in the parallel SKY study in stimulated lymphocytes at 0.2 micro M DX consisted of 14% chromatid-type and 2% chromosome-type aberrations; none were found in controls. The SKY results correlate very well with the findings of the comet assay in lymphocytes where DNA damage was observed at similar doses. This study is the first reporting use of the comet assay and SKY analysis in parallel after chemical treatment. The potential of the two techniques together is evident, as they represent a set of assays feasible for evaluating damage in human somatic and germ cells after chemical treatment (i) by direct observation of two different end-points, detecting general DNA damage and chromosomal aberrations and (ii) by extrapolation from lymphocytes to sperm, which provides a 'parallelogram' approach in human cells.
近年来，有两种检测全基因组遗传损伤的技术变得越发重要：碱性彗星试验，用于检测DNA损伤，如链断裂和碱不稳定位点；以及一种多色荧光原位杂交方法——光谱核型分析（SKY），用于在所有中期染色体中同时识别染色体畸变。在本研究中，使用一种抗癌药物阿霉素（DX），对体外培养的人类精子和淋巴细胞中的DNA损伤诱导情况进行了研究。通过彗星试验观察到DNA损伤增加，精子头部DNA的中位数百分比从未处理对照组的82.07%和85.14%显著下降至0.8微摩尔DX剂量时的63.48%和72.52%。在1.6微摩尔时，该百分比降至60.96%（相应的尾矩从4.42增加到12.19）。在受刺激的淋巴细胞中，尾矩显著增加，从对照组的0.72和0.53增加到0.2微摩尔DX时的15.17和12.10，并在相同水平持续到最终浓度1.6微摩尔。在平行的SKY研究中，在0.2微摩尔DX刺激的淋巴细胞中发现的结构畸变包括14%染色单体型和2%染色体型畸变；对照组未发现畸变。SKY结果与淋巴细胞彗星试验的结果非常吻合，在相似剂量下观察到了DNA损伤。本研究首次报道了化学处理后平行使用彗星试验和SKY分析。这两种技术结合的潜力显而易见，因为它们代表了一组可行的检测方法，可用于评估化学处理后人类体细胞和生殖细胞中的损伤：（i）通过直接观察两个不同的终点，检测一般DNA损伤和染色体畸变；（ii）通过从淋巴细胞推断到精子，这在人类细胞中提供了一种“平行四边形”方法。
