Mechanism-based genotoxicity screening of metal oxide nanoparticles using the ToxTracker panel of reporter cell lines

Abstract

BackgroundThe rapid expansion of manufacturing and use of nano-sized materials fuels the demand for fast and reliable assays to identify their potential hazardous properties and underlying mechanisms. The ToxTracker assay is a recently developed mechanism-based reporter assay based on mouse embryonic stem (mES) cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress upon exposure. Here, we evaluated the ability of the ToxTracker assay to identify the hazardous properties and underlying mechanisms of a panel of metal oxide- and silver nanoparticles (NPs) as well as additional non-metallic materials (diesel, carbon nanotubes and quartz).MethodsThe metal oxide- and silver nanoparticles were characterized in terms of agglomeration and ion release in cell medium (using photon cross correlation spectroscopy and inductively coupled plasma with optical emission spectroscopy, respectively) as well as acellular ROS production (DCFH-DA assay). Cellular uptake was investigated by means of transmission electron microscopy. GFP reporter induction and cytotoxicity of the NPs was simultaneously determined using flow cytometry, and genotoxicity was further tested using conventional assays (comet assay, γ-H2AX and RAD51 foci formation).ResultsWe show that the reporter cells were able to take up nanoparticles and, furthermore, that exposure to CuO, NiO and ZnO nanoparticles as well as to quartz resulted in activation of the oxidative stress reporter, although only at high cytotoxicity for ZnO. NiO NPs activated additionally a p53-associated cellular stress response, indicating additional reactive properties. Conventional assays for genotoxicity assessment confirmed the response observed in the ToxTracker assay. We show for CuO NPs that the induction of oxidative stress is likely the consequence of released Cu ions whereas the effect by NiO was related to the particles per se. The DNA replication stress-induced reporter, which is most strongly associated with carcinogenicity, was not activated by any of the tested nanoparticles.ConclusionsWe conclude that the ToxTracker reporter system can be used as a rapid mechanism-based tool for the identification of hazardous properties of metal oxide NPs. Furthermore, genotoxicity of metal oxide NPs seems to occur mainly via oxidative stress rather than direct DNA binding with subsequent replication stress.

Development of HSPA1A promoter-driven luciferase reporter gene assays in human cells for assessing the oxidative damage induced by silver nanoparticles

“…Thus, the results from our study suggested that the oxidative stress responses to AgNPs could be explained by the released Ag + ions from the nanoparticles. Our results were in agreement with other studies in which the stress response indicated by gene expression profile following AgNPs exposure was induced by the released Ag + ions (Prasad et al, 2013;Karlsson et al, 2014). For equal mass of AgNPs with different sizes, the smaller particles will be greater in number and have higher total surface area, thus probably contributing to the more toxic potential observed with smaller AgNPs (Prasad et al, 2013).…”

“…The luciferase activity induced by AgNPs were generally in accordance with the oxidative damage indicated by MDA concentration, ROS level and GSH depletion, and the Ag + ions release in cell medium. Compared with the other three luciferase reporter cells in this study or other reporter system reported for detecting the oxidative stress (Prasad et al, 2013;Karlsson et al, 2014), the HSPA1A promoter in HepG2-luciferase cells is generally more sensitive to nanoparticles. The HepG2 cells, derived from a human liver hepatoma, are reported to retain many properties of primary cells such as containing many functional phase-Ι and-II enzymes which are lost in most cultured cell lines (Doostdar et al, 1993;Westerink and Schoonen, 2007).…”

Acute and subacute pulmonary toxicity and mortality in mice after intratracheal instillation of ZnO nanoparticles in three laboratories

“…In vitro studies have shown that ZnO is cytotoxic at relatively low concentrations as compared to other types of engineered nanomaterials such as titanium dioxide, carbon nanotubes [7][8][9][10] . Similar findings on cytotoxicity have been observed in broncho-alveolar lavage cells after pulmonary exposure to ZnO NP, which has been attributed to the dissolution of Zn ions [11;12] .…”

Emerging metrology for high-throughput nanomaterial genotoxicology

“…Recently, some reports showed that ENMs increase γ-H2AX levels in in vitro experiments (102–105). Metal-oxide NPs in particular, such as CuO, ZnO and NiO, etc., induced the formation of γ-H2AX foci (102,104).…”