Atha Donald H, Tona Alessandro, Reipa Vytas
National Institute of Standards and Technology, Biosystems and Biomaterials Division, Material Measurement Laboratory, Gaithersburg, MD 20899, USA.
J Nucleic Acids. 2022 Sep 17;2022:9188636. doi: 10.1155/2022/9188636. eCollection 2022.
Application of DNA damage diagnostic tests is rapidly growing, in particular for ovarian, prostate, and skin cancers; environmental monitoring; chronic and degenerative diseases; and male infertility. Such tests suffer from significant variability among different laboratories due the lack of standardization, experimental validation, and differences in data interpretation. Reference methods and materials for quantitative measurement of UVA-induced DNA damage in mammalian cells are frequently needed. In this study, we examined the use of the single-cell gel electrophoresis (comet) assay to assess the UVA-induced DNA damage in surface-attached Chinese hamster ovary (CHO) cells treated with a photosensitizer as a candidate cellular oxidative damage reference material. We found that the comet images became diffused and the viability of the cells decreased substantially (>20%) as the UVA dose and benzo [a] pyrene (BaP) concentration exceeded 6.3 J/cm and 10 mol/L BaP. Maintaining the conditions of exposure within this range can improve DNA damage measurement fidelity, particularly if used as a quantitative reference method and to produce materials considered as an standard for the comet assay.
DNA损伤诊断测试的应用正在迅速增长，特别是在卵巢癌、前列腺癌和皮肤癌的检测中；环境监测；慢性和退行性疾病；以及男性不育症的诊断方面。由于缺乏标准化、实验验证以及数据解释的差异，不同实验室之间的此类测试存在显著差异。因此，经常需要用于定量测量哺乳动物细胞中紫外线A（UVA）诱导的DNA损伤的参考方法和材料。在本研究中，我们研究了使用单细胞凝胶电泳（彗星）试验来评估在用光敏剂处理的表面附着的中国仓鼠卵巢（CHO）细胞中UVA诱导的DNA损伤，该光敏剂作为候选细胞氧化损伤参考材料。我们发现，当UVA剂量和苯并[a]芘（BaP）浓度超过6.3 J/cm²和10 μmol/L BaP时，彗星图像变得模糊，细胞活力大幅下降（>20%）。将暴露条件维持在该范围内可以提高DNA损伤测量的保真度，特别是当用作定量参考方法并生产被视为彗星试验标准的材料时。
