Institute of Pharmacology and Toxicology, University of Wuerzburg, Versbacher Straße 9, 97078, Wuerzburg, Germany.
Department of General and Visceral, Vascular and Pediatric Surgery, University Hospital Wuerzburg, Wuerzburg, Germany.
Arch Toxicol. 2021 May;95(5):1831-1841. doi: 10.1007/s00204-021-03012-4. Epub 2021 Mar 5.
The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction.
彗星试验是一种常用于测定多种样本中 DNA 损伤和修复活性的方法。近年来，由于彗星试验有多种改良版本，可用于测定血液样本中的个体易感性，因此在人体生物监测中的应用极具吸引力。然而，在人体生物监测研究中，由于需要处理大量样本且检测时间跨度较长，因此需要考虑一些额外的因素。其中最重要的问题之一是样本的储存及其对彗星试验结果的影响。另一个重要的问题是不同血液制备物的适用性。在本研究中，我们分析了冷冻保存对人血样 DNA 损伤和修复活性的影响。此外，我们还研究了不同血液制备物的适用性。我们应用了碱性彗星试验、FPG 彗星试验以及两种不同类型的修复彗星试验和体外过氧化氢挑战。我们的研究结果证实，冷冻保存的血液制备物适用于全血、血涂片和 PBMCs 中碱性彗星试验和 FPG 彗星试验中 DNA 损伤的研究。在分离的 PBMCs 中，体外过氧化氢挑战可产生最佳效果。采用 UVC 或过氧化氢诱导的损伤和阿霉素阻滞的修复彗星试验在新鲜 PBMCs 中效果良好。冷冻保存的 PBMCs 不能在解冻后立即使用。然而，在有丝分裂刺激或无有丝分裂刺激的情况下进行 16 小时的恢复，可使修复彗星试验得以应用，尽管只能在存活细胞部分应用。
