Sestili Piero, Calcabrini Cinzia, Diaz Anna Rita, Fimognari Carmela, Stocchi Vilberto
Dipartimento di Scienze Biomolecolari, Università degli Studi di Urbino "Carlo Bo", Via Saffi, 2, 61029, Urbino, Italy.
Dipartimento di Scienze per la Qualità della Vita, Alma Mater Studiorum - Università di Bologna, Corso D'Augusto 237, 47921, Rimini, Italy.
Methods Mol Biol. 2017;1644:75-93. doi: 10.1007/978-1-4939-7187-9_6.
The need for express screening of the DNA damaging potential of chemicals has progressively increased over the past 20 years due to the wide number of new synthetic molecules to be evaluated, as well as the adoption of more stringent chemical regulations such as the EU REACH and risk reduction politics. In this regard, DNA diffusion assays such as the microelectrophoretic comet assay paved the way for rapid genotoxicity testing. A more significant simplification and speeding up of the experimental processes was achieved with the fast halo assay (FHA) described in the present chapter. FHA operates at the single cell level and relies on radial dispersion of the fragments of damaged DNA from intact nuclear DNA. The fragmented DNA is separated by diffusion in an alkaline solvent and is stained, visualized, and finally quantified using computer-assisted image analysis programs. This permits the rapid assessment of the extent of DNA breakage caused by different types of DNA lesions. FHA has proven to be sensitive, reliable, and flexible. This is currently one of the simplest, cheapest, and quickest assays for studying DNA damage and repair in living cells. It does not need expensive reagents or electrophoretic equipment and requires only 40 min to prepare samples for computer-based quantification. This technique can be particularly useful in rapid genotoxicity assessments and in high-throughput genotoxicity screenings.
在过去20年里，由于需要评估的新合成分子数量众多，以及采用了更严格的化学法规（如欧盟化学品注册、评估、授权和限制法规），对化学品的DNA损伤潜力进行快速筛选的需求日益增加。在这方面，诸如微电泳彗星试验等DNA扩散测定法为快速遗传毒性测试铺平了道路。本章所述的快速晕圈试验（FHA）实现了实验过程更显著的简化和加速。FHA在单细胞水平上运行，依赖于受损DNA片段从完整核DNA中的径向扩散。破碎的DNA在碱性溶剂中通过扩散分离，然后染色、可视化，最后使用计算机辅助图像分析程序进行定量。这使得能够快速评估由不同类型的DNA损伤引起的DNA断裂程度。FHA已被证明是灵敏、可靠且灵活的。它是目前研究活细胞中DNA损伤和修复最简单、最便宜且最快的测定方法之一。它不需要昂贵的试剂或电泳设备，仅需40分钟即可制备用于基于计算机定量的样品。该技术在快速遗传毒性评估和高通量遗传毒性筛选中可能特别有用。
