Tekneci Seda İpek, Üstündağ Aylin, Duydu Yalçın
Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, 06560, Ankara, Türkiye.
Ankara University, Graduate School of Health Sciences, 06110, Ankara, Türkiye.
Toxicol Res (Camb). 2024 Dec 4;13(6):tfae195. doi: 10.1093/toxres/tfae195. eCollection 2024 Dec.
Endogenous and exogenous factors cause DNA damage through chemical changes in the genomic DNA structure. The comet assay is a versatile, rapid, and sensitive method for evaluating DNA integrity at the individual cell level. It is used in human biomonitoring studies, the identification of DNA lesions, and the measurement of DNA repair capacity. Despite its widespread application, variations between studies remain problematic, often due to the lack of a common protocol and appropriate test controls. Using positive controls is essential to assess inter-experimental variability and ensure reliable results. Hydrogen peroxide (HO) is the most commonly used positive control, while potassium bromate (KBrO₃), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), -ethyl--nitrosourea (ENU), and etoposide are used less frequently. However, differences in concentrations and exposure durations prevent the confirmation of test method efficacy. This study investigates the dose-response relationship for HO, KBrO, MMS, EMS, ENU and etoposide in the comet assay for 30 and 60-minute exposure durations in 3T3 cell lines. Accordingly recommended concentrations and exposure durations were found to be 50 μM 30 minutes (HO); 500 μM 60 min. (MMS); 10 μM 30 min. (Etoposide); 0.2 mM 30 min. and 2 mM 60 min. (EMS); 2 mM 30 min. (ENU); 500 μM 30 min. and 50 μM 60 min. (KBrO). Our findings will contribute to reducing inter-laboratory variability by offering guidance on selecting doses and exposure durations for positive controls in the alkaline comet assay.
内源性和外源性因素通过基因组DNA结构的化学变化导致DNA损伤。彗星试验是一种在单个细胞水平上评估DNA完整性的通用、快速且灵敏的方法。它用于人体生物监测研究、DNA损伤的鉴定以及DNA修复能力的测量。尽管其应用广泛，但研究之间的差异仍然存在问题，这通常是由于缺乏通用方案和适当的测试对照。使用阳性对照对于评估实验间的可变性并确保获得可靠结果至关重要。过氧化氢（HO）是最常用的阳性对照，而溴酸钾（KBrO₃）、甲基磺酸甲酯（MMS）、乙基磺酸甲酯（EMS）、N-乙基-N-亚硝基脲（ENU）和依托泊苷的使用频率较低。然而，浓度和暴露持续时间的差异使得无法确认测试方法的有效性。本研究调查了在3T3细胞系中进行30分钟和60分钟暴露时长的彗星试验中，HO、KBrO、MMS、EMS、ENU和依托泊苷的剂量反应关系。据此，发现推荐的浓度和暴露持续时间分别为：50 μM 30分钟（HO）；500 μM 60分钟（MMS）；10 μM 30分钟（依托泊苷）；0.2 mM 30分钟和2 mM 60分钟（EMS）；2 mM 30分钟（ENU）；500 μM 30分钟和50 μM 60分钟（KBrO）。我们的研究结果将通过为碱性彗星试验中阳性对照的剂量和暴露持续时间选择提供指导，有助于减少实验室间的可变性。
