John B. Little Center of Radiation Sciences, Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, United States.
Center for Nanotechnology and Nanotoxicology, Department of Environmental Health, Harvard T.H. Chan School of Public Health, 655 Huntington Avenue, Boston, Massachusetts 02115, United States.
ACS Nano. 2021 Mar 23;15(3):4728-4746. doi: 10.1021/acsnano.0c09254. Epub 2021 Mar 12.
The potential genotoxic effects of engineered nanomaterials (ENMs) may occur through the induction of DNA damage or the disruption of DNA repair processes. Inefficient DNA repair may lead to the accumulation of DNA lesions and has been linked to various diseases, including cancer. Most studies so far have focused on understanding the nanogenotoxicity of ENM-induced damages to DNA, whereas the effects on DNA repair have been widely overlooked. The recently developed fluorescence multiplex-host-cell reactivation (FM-HCR) assay allows for the direct quantification of multiple DNA repair pathways in living cells and offers a great opportunity to address this methodological gap. Herein an FM-HCR-based method is developed to screen the impact of ENMs on six major DNA repair pathways using suspended or adherent cells. The sensitivity and efficiency of this DNA repair screening method were demonstrated in case studies using primary human small airway epithelial cells and TK6 cells exposed to various model ENMs (CuO, ZnO, and GaO) at subcytotoxic doses. It was shown that ENMs may inhibit nucleotide-excision repair, base-excision repair, and the repair of oxidative damage by DNA glycosylases in TK6 cells, even in the absence of significant genomic DNA damage. It is of note that the DNA repair capacity was increased by some ENMs, whereas it was suppressed by others. Overall, this method can be part of a multitier, hazard assessment of ENMs as a functional, high-throughput platform that provides insights into the interplay of the properties of ENMs, the DNA repair efficiency, and the genomic stability.
工程纳米材料 (ENM) 的潜在遗传毒性效应可能通过诱导 DNA 损伤或破坏 DNA 修复过程发生。DNA 修复效率低下可能导致 DNA 损伤的积累，并与包括癌症在内的各种疾病有关。迄今为止，大多数研究都集中在了解 ENM 诱导的 DNA 损伤的纳米遗传毒性，而对 DNA 修复的影响则被广泛忽视。最近开发的荧光多重宿主细胞复活 (FM-HCR) 测定法可直接定量活细胞中多种 DNA 修复途径，为解决这一方法学空白提供了极好的机会。在此，我们开发了一种基于 FM-HCR 的方法，用于筛选悬浮或贴壁细胞中 ENM 对六种主要 DNA 修复途径的影响。使用暴露于各种模型 ENM（CuO、ZnO 和 GaO）的亚细胞毒性剂量的原代人小气道上皮细胞和 TK6 细胞进行的案例研究证明了这种 DNA 修复筛选方法的灵敏度和效率。结果表明，ENM 可抑制 TK6 细胞中的核苷酸切除修复、碱基切除修复和 DNA 糖苷酶修复氧化损伤，即使在没有明显基因组 DNA 损伤的情况下也是如此。值得注意的是，一些 ENM 增加了 DNA 修复能力，而另一些则抑制了 DNA 修复能力。总体而言，该方法可以作为 ENM 危害评估的多层次、高通量平台的一部分，为深入了解 ENM 的特性、DNA 修复效率和基因组稳定性之间的相互作用提供信息。
