DNA damage induced by micro- and nanoparticles-interaction with FPG influences the detection of DNA oxidation in the comet assay
DNA damage induced by micro- and nanoparticles-interaction with FPG influences the detection of DNA oxidation in the comet assay
复制标题
DOI：
10.1093/mutage/ges010
复制
发表时间：
2012-07-01
期刊：
影响因子：
2.7
通讯作者：
Moller, L.
中科院分区：
文献类型：
--
作者：
Kain, J.;Karlsson, H. L.;Moller, L.
需要可靠的方法来评估颗粒（例如人造纳米颗粒）的毒性。一种有前景的工具是彗星测定，通常用于测量 DNA 断裂（链断裂和碱不稳定位点）以及氧化损伤的 DNA，后者通过添加特定的 DNA 修复酶（例如甲酰胺嘧啶 DNA 糖基化酶 (FPG)）来实现。本研究的目的是研究使用彗星测定来分析肺细胞系 A549 和 BEAS-2B 中一系列微米颗粒和纳米颗粒的 DNA 氧化，并检验在测定过程中存在于细胞中的纳米颗粒可能与 FPG 相互作用的假设。这是通过研究微米颗粒和纳米颗粒（不锈钢、地铁颗粒、MnO2、Ag、CeO2、Co3O4、Fe3O4、NiO 和 SiO2）诱导 DNA 断裂、DNA 氧化损伤（FPG 位点，主要是 8-氧代鸟嘌呤）、细胞内活性氧 (ROS) 产生和 DNA 碱基鸟嘌呤非细胞氧化的能力，以及研究颗粒及其释放的离子与 FPG 的相互作用来完成的。一些颗粒导致 DNA 断裂，但 FPG 位点水平较低。然而，FPG 具有检测光敏剂诱导的 DNA 氧化的能力。颗粒的氧化能力通过细胞内ROS水平的增加来表明，尤其是Ag和地铁颗粒引起鸟嘌呤的非细胞氧化。 FPG 与颗粒一起孵育导致 FPG 活性降低，尤其是 Ag 纳米颗粒，还有 CeO2、Co3O4 和 SiO2。对这些颗粒的进一步研究表明，对于 Ag，活性降低主要是由于释放的 Ag 离子，而对于 CeO2 和 Co3O4，FPG 相互作用是由于颗粒所致。我们得出的结论是，由于与 FPG 的相互作用，在彗星测定中，暴露于纳米粒子的细胞中氧化损伤 DNA 的测量结果可能被低估。
Reliable methods for evaluation of toxicity from particles, such as manufactured nanoparticles, are needed. One promising tool is the comet assay, often used to measure DNA breaks (strand breaks and alkali-labile sites) as well as oxidatively damaged DNA, the latter by addition of specific DNA repair enzymes such as formamidopyrimidine DNA glycosylase (FPG). The aim of this study was to investigate the use of the comet assay for analysis of DNA oxidation by a range of micro- and nanoparticles in the lung cell lines A549 and BEAS-2B and to test the hypothesis that nanoparticles present in the cells during the assay performance may interact with FPG. This was done by investigating the ability of micro- and nanoparticles (stainless steel, subway particles, MnO2, Ag, CeO2, Co3O4, Fe3O4, NiO and SiO2) to induce DNA breaks, oxidatively damaged DNA (FPG sites, dominantly 8-oxoguanine), intracellular production of reactive oxygen species (ROS) and non-cellular oxidation of the DNA base guanine, as well as by studying interactions of the particles and their released ions with FPG. Several particles caused DNA breaks, but low levels of FPG sites. The ability of FPG to detect DNA oxidation induced by a photosensitiser was however shown. An oxidative capacity of the particles was indicated by increased levels of intracellular ROS, and especially Ag and subway particles caused non-cellular oxidation of guanine. Incubation of FPG with the particles led to less FPG activity, particularly with nanoparticles of Ag but also with CeO2, Co3O4 and SiO2. Further investigations of these particles revealed that for Ag, the decreased activity was mainly due to released Ag ions, whereas for CeO2 and Co3O4, FPG interactions were due to the particles. We conclude that measurement of oxidatively damaged DNA in cells exposed to nanoparticles may be underestimated in the comet assay due to interactions with FPG.