Smith Catherine C, Adkins Deborah J, Martin Elizabeth A, O'Donovan Michael R
Genetic Toxicology, Macclesfield, Cheshire SK10 4TG, UK.
Mutagenesis. 2008 May;23(3):233-40. doi: 10.1093/mutage/gen008. Epub 2008 Mar 6.
Although the rodent comet assay is gaining acceptance as a standard technique for evaluating DNA damage in vivo, there is no internationally accepted guideline for its conduct and several aspects of its experimental design have not been optimized. For example, no standard positive control is used, there is no agreement on how tissue toxicity should be measured and sources of experimental variability have not been considered in relation to experimental design. This study showed that methylnitrosourea is a good alternative positive control inducing DNA damage in all tissues examined (stomach, liver, blood and bone marrow) over a dose range of 25-100 mg/kg at both 3 and 24 h after treatment. At the highest dose, significant toxicity was seen in all tissues using the neutral diffusion assay and also by histopathological/haematological analysis, except in the liver where no change was seen even 7 days after dosing. Analyses using control data pooled from several studies showed that, as expected, the greatest variability was seen between tissue preparations from different animals and that different numbers of animals were required to detect the same fold increases in different tissues. Power analyses showed that, preparing three gels for each tissue and scoring 50 nuclei per gel, a group of six animals allows 2-fold increases over control in the liver, bone marrow and stomach and a 3-fold increase in blood to be detected with 80% probability. It is recommended that similar investigations of experimental variability should be performed to determine optimal experimental design in any laboratory using the rodent comet assay.
尽管啮齿动物彗星试验作为评估体内DNA损伤的标准技术正逐渐被接受，但目前尚无国际认可的该试验实施指南，其实验设计的几个方面也尚未优化。例如，未使用标准阳性对照，对于如何测量组织毒性尚无共识，且在实验设计中未考虑实验变异性的来源。本研究表明，甲基亚硝基脲是一种良好的替代阳性对照，在治疗后3小时和24小时，在25 - 100 mg/kg的剂量范围内，能在所有检测组织（胃、肝、血液和骨髓）中诱导DNA损伤。在最高剂量下，使用中性扩散试验以及组织病理学/血液学分析，在所有组织中均观察到显著毒性，但肝脏除外，即使给药7天后肝脏也未见变化。对来自多项研究的对照数据进行分析表明，正如预期的那样，不同动物的组织样本间变异性最大，且在不同组织中检测相同倍数增加所需的动物数量不同。功效分析表明，每个组织制备3块凝胶，每块凝胶对50个细胞核进行评分，一组6只动物有80%的概率能检测到肝脏、骨髓和胃中比对照增加2倍以及血液中增加3倍的情况。建议在任何使用啮齿动物彗星试验的实验室进行类似的实验变异性研究，以确定最佳实验设计。
