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Nanoparticle-induced genotoxicity can arise through different mechanisms, and generally, primary and secondary genotoxicity can be distinguished where the secondary is driven by an inflammatory response. It is, however, yet unclear how a secondary genotoxicity can be detected using in vitro methods. The aim of this study was to investigate inflammation and genotoxicity caused by agglomerated nickel (Ni) and nickel oxide (NiO) nanoparticles and, furthermore, to explore the possibility to test secondary (inflammation-driven) genotoxicity in vitro . As a benchmark particle to compare with, we used crystalline silica (quartz). A proteome profiler antibody array was used to screen for changes in release of 105 different cytokines and the results showed an increased secretion of various cytokines including vascular endothelial growth factor (VEGF) following exposure of macrophages (differentiated THP-1 cells). Both Ni and NiO caused DNA damage (comet assay) following exposure of human bronchial epithelial cells (HBEC) and interestingly conditioned media (CM) from exposed macrophages also resulted in DNA damage (2- and 3-fold increase for Ni and NiO, respectively). Similar results were also found when using a co-culture system of macrophages and epithelial cells. In conclusion, this study shows that it is possible to detect a secondary genotoxicity in lung epithelial cells by using in vitro methods based on conditioned media or co-cultures. Further investigation is needed in order to find out what factors that are causing this secondary genotoxicity and whether such effects are caused by numerous nanoparticles.

Background: Genotoxicity is an important toxicological endpoint due to the link to diseases such as cancer. Therefore, an increased understanding regarding genotoxicity and underlying mechanisms is needed for assessing the risk with exposure to nanoparticles (NPs). The aim of this study was to perform an in-depth investigation regarding the genotoxicity of well-characterized Ni and NiO NPs in human bronchial epithelial BEAS-2B cells and to discern possible mechanisms. Comparisons were made with NiCl 2 in order to elucidate effects of ionic Ni., Methods: BEAS-2B cells were exposed to Ni and NiO NPs, as well as NiCl 2 , and uptake and cellular dose were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS). The NPs were characterized in terms of surface composition (X-ray photoelectron spectroscopy), agglomeration (photon cross correlation spectroscopy) and nickel release in cell medium (ICP-MS). Cell death (necrosis/apoptosis) was investigated by Annexin V-FITC/PI staining and genotoxicity by cytokinesis-block micronucleus (cytome) assay (OECD 487), chromosomal aberration (OECD 473) and comet assay. The involvement of intracellular reactive oxygen species (ROS) and calcium was explored using the fluorescent probes, DCFH-DA and Fluo-4., Results: NPs were efficiently taken up by the BEAS-2B cells. In contrast, no or minor uptake was observed for ionic Ni from NiCl 2 . Despite differences in uptake, all exposures (NiO, Ni NPs and NiCl 2 ) caused chromosomal damage. Furthermore, NiO NPs were most potent in causing DNA strand breaks and generating intracellular ROS. An increase in intracellular calcium was observed and modulation of intracellular calcium by using inhibitors and chelators clearly prevented the chromosomal damage. Chelation of iron also protected against induced damage, particularly for NiO and NiCl 2 ., Conclusions: This study has revealed chromosomal damage by Ni and NiO NPs as well as Ni ionic species and provides novel evidence for a calcium-dependent mechanism of cyto- and genotoxicity.

Nickel (Ni) compounds are classified as carcinogenic to humans but the underlying mechanisms are still poorly understood. Furthermore, effects related to nanoparticles (NPs) of Ni have not been fully elucidated. The aim of this study was to investigate genotoxicity and mutagenicity of Ni and NiO NPs and compare the effect to soluble Ni from NiCl 2 . We employed different models; i.e., exposure of (1) human bronchial epithelial cells (HBEC) followed by DNA strand break analysis (comet assay and γ-H2AX staining); (2) six different mouse embryonic stem (mES) reporter cell lines (ToxTracker) that are constructed to exhibit fluorescence upon the induction of various pathways of relevance for (geno)toxicity and cancer; and (3) mES cells followed by mutagenicity testing (Hprt assay). The results showed increased DNA strand breaks (comet assay) for the NiO NPs and at higher doses also for the Ni NPs whereas no effects were observed for Ni ions/complexes from NiCl 2 . By employing the reporter cell lines, oxidative stress was observed as the main toxic mechanism and protein unfolding occurred at cytotoxic doses for all three Ni-containing materials. Oxidative stress was also detected in the HBEC cells following NP-exposure. None of these materials induced the reporter related to direct DNA damage and stalled replication forks. A small but statistically significant increase in Hprt mutations was observed for NiO but only at one dose. We conclude that Ni and NiO NPs show more pronounced (geno)toxic effects compared to Ni ions/complexes, indicating more serious health concerns. Environ. Mol. Mutagen. 59:211-222, 2018. © 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society., (© 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.)

Nickel (Ni) compounds are classified as carcinogenic to humans but the underlying mechanisms are still poorly understood. Furthermore, effects related to nanoparticles (NPs) of Ni have not been fully elucidated. The aim of this study was to investigate genotoxicity and mutagenicity of Ni and NiO NPs and compare the effect to soluble Ni from NiCl2. We employed different models; i.e., exposure of (1) human bronchial epithelial cells (HBEC) followed by DNA strand break analysis (comet assay and γ‐H2AX staining); (2) six different mouse embryonic stem (mES) reporter cell lines (ToxTracker) that are constructed to exhibit fluorescence upon the induction of various pathways of relevance for (geno)toxicity and cancer; and (3) mES cells followed by mutagenicity testing (Hprt assay). The results showed increased DNA strand breaks (comet assay) for the NiO NPs and at higher doses also for the Ni NPs whereas no effects were observed for Ni ions/complexes from NiCl2. By employing the reporter cell lines, oxidative stress was observed as the main toxic mechanism and protein unfolding occurred at cytotoxic doses for all three Ni‐containing materials. Oxidative stress was also detected in the HBEC cells following NP‐exposure. None of these materials induced the reporter related to direct DNA damage and stalled replication forks. A small but statistically significant increase in Hprt mutations was observed for NiO but only at one dose. We conclude that Ni and NiO NPs show more pronounced (geno)toxic effects compared to Ni ions/complexes, indicating more serious health concerns. Environ. Mol. Mutagen. 59:211–222, 2018. © 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society [ABSTRACT FROM AUTHOR]