Speit G, Trenz K, Schütz P, Rothfuss A, Merk O
Universitätsklinikum Ulm, Abteilung Medizinische Genetik, Germany.
Toxicol Lett. 1999 Oct 29;110(1-2):73-8. doi: 10.1016/s0378-4274(99)00137-x.
The alkaline comet assay (single-cell gel electrophoresis) is becoming established as a genotoxicity test with many fold applications in vitro and in vivo. While the underlying principles are identical, various modifications of the method are in use which clearly affect the sensitivity and resolving power of the assay. One variable of potential importance that has been disregarded until now is temperature during alkaline treatment and electrophoresis. We therefore performed comet assay experiments with human blood and V79 Chinese hamster cells using two different temperatures (4 and 20 degrees C, i.e. room temperature) during alkaline treatment and electrophoresis. DNA damage was induced by the two standard mutagens gamma irradiation and methyl methanesulfonate (MMS). The results clearly indicate significant differences in the detection of background and mutagen-induced DNA damage at these two temperatures. Under otherwise identical test conditions (including the duration of alkaline treatment and electrophoresis), increased temperature during alkaline treatment and electrophoresis strongly enhances DNA migration. Our findings suggest that the comet assay should be performed under strictly controlled and reproducible temperature conditions. In any case the temperature during alkaline treatment and electrophoresis should be stated in a publication to allow for a critical evaluation of results obtained with the comet assay.
碱性彗星试验（单细胞凝胶电泳）正逐渐成为一种遗传毒性检测方法，在体外和体内有多种应用。虽然其基本原理相同，但该方法有多种改进形式，这显然会影响检测的灵敏度和分辨能力。一个到目前为止被忽视的潜在重要变量是碱性处理和电泳过程中的温度。因此，我们使用人血和V79中国仓鼠细胞进行彗星试验，在碱性处理和电泳过程中采用两种不同温度（4℃和20℃，即室温）。通过两种标准诱变剂γ射线照射和甲基磺酸甲酯（MMS）诱导DNA损伤。结果清楚地表明，在这两种温度下，背景和诱变剂诱导的DNA损伤检测存在显著差异。在其他相同的测试条件下（包括碱性处理和电泳的持续时间），碱性处理和电泳过程中温度升高会强烈增强DNA迁移。我们的研究结果表明，彗星试验应在严格控制且可重复的温度条件下进行。无论如何，碱性处理和电泳过程中的温度应在出版物中说明，以便对彗星试验获得的结果进行批判性评估。
