Primary and Secondary Genotoxicity of Nanoparticles: Establishing a Co-Culture Protocol for Assessing Micronucleus Using Flow Cytometry

Abstract

Genotoxicity is an important endpoint to assess for understanding the risks associated with nanoparticles (NPs). Most genotoxicity studies performed on NPs have focused on primary genotoxicity analyzed by comet- or micronuclei (MN) assay using microscopic scoring. Here, we established a protocol for a more efficient version of MN assessment using flow cytometry and, importantly, both primary and secondary (inflammation-driven) genotoxicity was assessed. Human bronchial epithelial cells (HBEC-3kt) were exposed to nickel oxide (NiO) NPs directly or indirectly. The indirect exposure was done to assess secondary genotoxicity, and in this case immune cells (THP-1 derived macrophages) were exposed on inserts and the HBEC were cultured in the lower compartment. The results in monocultures showed that no increased MN formation was observed in the HBEC cells but instead a clear MN induction was noted in THP-1 cells indicating higher sensitivity. No MN formation was either observed when the HBEC were indirectly exposed, but an increase in DNA strand breaks was detected using the comet assay. Taken together, the present study emphasizes the feasibility of assessing primary and secondary genotoxicity and, furthermore, shows a clear MN induction in THP-1 monoculture following NiO NPs exposure.

Characteristics and health risks of the inhalable fraction of metal additive manufacturing powders

“…The corrosion potential (E corr ) and corrosion current (I corr ) were determined by Tafel fitting of the polarization curve using the VersaStudio software. In addition, the anodic branch current (I ab ), [ 41 ] defined as the current at a potential +200 mV from E corr , was determined.…”

The pulmonary effects of nickel-containing nanoparticles: cytotoxicity, genotoxicity, carcinogenicity, and their underlying mechanisms

“…A dose-and time-dependent decrease in cell viability or decrease in colony-forming efficiency after NiO NP exposure has been confirmed in various human lung cells, including lung epithelial A549 19,[29][30][31][32][33][34][35][36][37] and H460, 20 airway epithelial HEp-2, 38 bronchial epithelial BEAS-2B 31,39 and NHBE, 20 alveolar epithelial HPAEpiC, 40 and pulmonary artery endothelial HPAEC cells. 41 Akerlund et al reported that exposure of HBECs to 5-50 μg Ni mL −1 of NiO NPs for 3 and 24 h did not cause significant cytotoxicity by the alamarBlue assay, 22 but significant cytotoxicity was observed when HBEC cells were exposed to 25 μg Ni mL −1 of NiO NPs for 48 h. 42 Increased LDH activity was also detected in the cell culture supernatant of A549 cells exposed to NiO NPs. 37,43 Increased ROS generation was observed in BEAS-2B, but not A549, cells exposed to 60 and 100 μg mL −1 NiO NPs for 45 min 31 or 25 and 50 μg Ni mL −1 of NiO NPs for 5 min to 2 h. 19,24 Others found increased ROS, perturbation of the mitochondrial membrane potential, lipid peroxidation, increased tHODE levels, or depletion of antioxidants, such as decreased GSH, SOD, or CAT, in A549 cells exposed to NiO NPs.…”

“…113 Direct exposure to, co-culture of, or conditioned media from THP-1* macrophages exposed to NiO NPs all caused DNA damage in HBEC cells by the comet assay, suggesting that NiO NPs appear to cause both primary and secondary (inflammationdriven) genotoxicity. 22,42 NiO NP exposure also caused chromosomal damage and rearrangements in BEAS-2B cells. 24 In addition, exposure of BEAS-2B or A549 cells to NiO NPs caused a significant nuclear translocation of DNA damage response proteins, p-ATM and p-ATR.…”

“…31 However, a lack of γH2AX focus induction or micronucleus formation was observed when HBEC cells were exposed to NiO NPs. 42,111 Capasso et al reported that exposure of BEAS-2B or A549 cells to NiO NPs caused cell cycle alterations, which were reflected by decreased cell population in the G1 phase, increased cell population in the G2/M phase, and no change in the S phase in A549 cells, and increased cell population in the G1 phase and decreased cell population in both G2/M and S phases in BEAS-2B cells. 31 Gliga et al showed that exposure of BEAS-2B cells to 0.5 μg Ni mL −1 NiO NPs for six weeks did not cause induction of micronuclei and hypodiploid nuclei, and no apparent changes in cell transformation or cell motility were observed.…”

Assessing the Genotoxicity of Cellulose Nanomaterials in a Co-Culture of Human Lung Epithelial Cells and Monocyte-Derived Macrophages

“…The combination of the comet and the micronucleus (MN) assays was intended to provide information on several endpoints related to cytotoxicity, genotoxicity and oxidative damage [ 44 ]. Indeed, the comet assay detects DNA damage/strand breaks and oxidant damage to DNA (FPG-comet assay) with high sensitivity, whereas the MN assay identifies chromosomal breaks or loss, thus characterizing biological events that are linked to cell transformation and cancer [ 44 , 45 , 46 ]. Due to the close association between genotoxicity and carcinogenicity, the assessment of the genotoxic effects of these CMNMs is pivotal to identify potential risks from human exposure to these materials before they enter the market.…”