Co-assessment of cell cycle and micronucleus frequencies demonstrates the influence of serum on thein vitrogenotoxic response to amorphous monodisperse silica nanoparticles of varying sizes

Abstract

Serum proteins have been shown to modulate the cytotoxic and genotoxic responses to nanomaterials. The aim was to investigate the influence of serum on the induction of micronuclei (MN) by nanoparticles (NPs) of different sizes. Therefore, A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles (SNPs) were used as models. Assessment of the cell viability, cell cycle changes and induction of MN by SNPs ranging from 12 to 174 nm was performed in presence or absence of serum, applying the in vitro flow cytometry-based MN assay. Here, it has been demonstrated that serum has an influence on these end points, with a lower cell viability in absence of serum compared with the presence of serum. Further, cell cycle changes, specifically, G1 and S-phase arrest, were observed in absence of serum for four out of six SNPs tested. A size-dependent MN induction was observed: larger SNPs being more active in absence of serum. In addition, the serum influence was characterised by a size-dependency for cytotoxic and genotoxic effects, with a higher influence of serum for smaller particles. The data indicate that the in vitro micronucleus assay in presence and absence of serum could be advised for hazard assessment because it demonstrates a higher sensitivity in serum-free conditions than in conditions with serum. However, this recommendation applies only if the cell line used is able to proliferate under serum-free conditions because cell division is a prerequisite for MN expression.

Cytome micronucleus assays with a metabolically competent human derived liver cell line (Huh6): A promising approach for routine testing of chemicals?

“…Another relevant parameter which we studied is the effect of different serum concentrations. It was reported that the serum levels have an impact on the genotoxic and acute toxic properties of chemicals and nanoparticles by modulation of their bioavailability and/or alterations of the activity of drug metabolizing enzymes (Wiencke et al, ; Wang and Lee ; Gonzalez et al, ; Lubberstedt et al, ; Pisani et al, ). The serum effect was compound specific; it was observed with certain direct acting chemicals such as CDDP and Etop but not with other substances (MMS and H 2 O 2 ).…”

Genotoxicity of TiO2nanoparticles assessed by mini-gel comet assay and micronucleus scoring with flow cytometry

“…Furthermore, by analysing the increase of the frequency of hypodiploid nuclei, the flow cytometer approach is sensitive not only to clastogens but also to aneuploidogens that interfere with the migration of chromosomes to spindle poles (20). This is important to analyse in a high-throughput manner since NM seem to exert their genotoxic potential also by mechanically interfering with the microtubules (21). Interestingly, recent studies on HepG2 cells exposed to silver nanoparticles (20 and 50nm) show not only an increase in MN frequency but also a dose-dependent increase in hypodiploid cells, thus suggesting involvement of aneugenicity (22,23).…”

Emerging metrology for high-throughput nanomaterial genotoxicology

“…Thus plausible interference and/or cross-talk between different dyes/ENMs could pose a challenge in the interpretation of MNi data sets. However, one way to correct for potential interferences is to simultaneously combine cell cycle data with MNi data to overrule inaccuracies in the evaluation of the genotoxic effects of ENMs (90). …”