Laetitia Gonzalez

Size‐Dependent Cytotoxicity of Monodisperse Silica Nanoparticles in Human Endothelial Cells

The effect that monodisperse amorphous spherical silica particles of different sizes have on the viability of endothelial cells (EAHY926 cell line) is investigated. The results indicate that exposure to silica nanoparticles causes cytotoxic damage (as indicated by lactate dehydrogenase (LDH) release) and a decrease in cell survival (as determined by the tetrazolium reduction, MTT, assay) in the EAHY926 cell line in a dose-related manner. Concentrations leading to a 50% reduction in cell viability (TC(50)) for the smallest particles tested (14-, 15-, and 16-nm diameter) ranging from 33 to 47 microg cm(-2) of cell culture differ significantly from values assessed for the bigger nanoparticles: 89 and 254 microg cm(-2) (diameter of 19 and 60 nm, respectively). Two fine silica particles with diameters of 104 and 335 nm show very low cytotoxic response compared to nanometer-sized particles with TC(50) values of 1095 and 1087 microg cm(-2), respectively. The smaller particles also appear to affect the exposed cells faster with cell death (by necrosis) being observed within just a few hours. The surface area of the tested particles is an important parameter in determining the toxicity of monodisperse amorphous silica nanoparticles.

Genotoxicity of engineered nanomaterials: A critical review

In view of the fast-growing industrial applications of engineered nanomaterials (ENMs), the evaluation of their genotoxic potential and of their mode of action is a necessity to conduct adequate hazard/risk assessment and to produce safer and sustainable ENMs. This review aims at: (i) Providing an evaluation of in vitro and in vivo genotoxicity data available for ENM, and (ii) proposing minimal criteria for conducting nano-genotoxicity assays. The possible modes of action of ENM (i.e., generation of reactive oxygen species (ROS) and mechanical interference with cellular components) and the potential cellular targets are discussed. The available studies are evaluated on the basis of specific quality criteria after categorisation according to ENMs type/size investigated. No definitive conclusion can be drawn concerning the genotoxic activity of ENMs, essentially because of the limited number of data, incomplete physico-chemical characterization of ENMs examined and shortcomings in experimental approaches. This evaluation revealed gaps to be considered in future studies (e.g., one-sided approach focusing mainly on ROS as mode of action) and the need to develop adequate positive controls for genotoxicity assays when conducted with nanomaterials.

The in vitro MN assay in 2011: origin and fate, biological significance, protocols, high throughput methodologies and toxicological relevance

Micronuclei (MN) are small, extranuclear bodies that arise in dividing cells from acentric chromosome/chromatid fragments or whole chromosomes/chromatids lagging behind in anaphase and are not included in the daughter nuclei at telophase. The mechanisms of MN formation are well understood; their possible postmitotic fate is less evident. The MN assay allows detection of both aneugens and clastogens, shows simplicity of scoring, is widely applicable in different cell types, is internationally validated, has potential for automation and is predictive for cancer. The cytokinesis-block micronucleus assay (CBMN) allows assessment of nucleoplasmic bridges, nuclear buds, cell division inhibition, necrosis and apoptosis and in combination with FISH using centromeric probes, the mechanistic origin of the MN. Therefore, the CBMN test can be considered as a “cytome” assay covering chromosome instability, mitotic dysfunction, cell proliferation and cell death. The toxicological relevance of the MN test is strong: it covers several endpoints, its sensitivity is high, its predictivity for in vivo genotoxicity requires adequate selection of cell lines, its statistical power is increased by the recently available high throughput methodologies, it might become a possible candidate for replacing in vivo testing, it allows good extrapolation for potential limits of exposure or thresholds and it is traceable in experimental in vitro and in vivo systems. Implementation of in vitro MN assays in the test battery for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified.

Nominal and Effective Dosimetry of Silica Nanoparticles in Cytotoxicity Assays

Because of their small size and large specific surface area (SA), insoluble nanoparticles are almost not affected by the gravitational force and are generally formulated in stable suspensions or sols. This raises, however, a potential difficulty in in vitro assay systems in which cells adhering to the bottom of a culture vessel may not be exposed to the majority of nanoparticles in suspension. J. G. Teeguarden et al., 2007, Toxicol. Sci. 95, 300-312 have recently addressed this issue theoretically, emphasizing the need to characterize the effective dose (mass or number or SA dose of particles that affect the cells) which, according to their model based on sedimentation and gravitation forces, might only represent a very small fraction of the nominal dose. We hypothesized, in contrast, that because of convection forces that usually develop in sols, the majority of the particles may reach the target cells and exert their potential toxicity. To address this issue, we exposed three different cell lines (A549 epithelial cells, EAHY926 endothelial cells, and J774 monocyte-macrophages) to a monodisperse suspension of Stöber silica nanoparticles (SNP) in three different laboratories. Four different end points (lacticodehydrogenase [LDH] release, LDH cell content, tetrazolium salt (MTT), and crystal violet staining) were used to assess the cell response to nanoparticles. We found, in all cell lines and for all end points, that the cellular response was determined by the total mass/number/SA of particles as well as their concentration. Practically, for a given volume of dispersion, both parameters are of course intimately interdependent. We conclude that the nominal dose remains the most appropriate metric for in vitro toxicity testing of insoluble SNP dispersed in aqueous medium. This observation has important bearings on the experimental design and the interpretation of in vitro toxicological studies with nanoparticles.

Synthesis and Characterization of Stable Monodisperse Silica Nanoparticle Sols for in Vitro Cytotoxicity Testing.

For the investigation of the interaction of nanoparticles with biomolecules, cells, organs, and animal models there is a need for well-characterized nanoparticle suspensions. In this paper we report the preparation of monodisperse dense amorphous silica nanoparticles (SNP) suspended in physiological media that are sterile and sufficiently stable against aggregation. SNP sols with various particle sizes (2-335 nm) were prepared via base-catalyzed hydrolysis and polymerization of tetraethyl orthosilicate under sterile conditions using either ammonia (Stober process (1) ) or lysine catalyst (Lys-Sil process (2) ). The series was complemented with commercial silica sols (Ludox). Silica nanoparticle suspensions were purified by dialysis and dispersed without using any dispersing agent into cell culture media (Dulbeccos Modified Eagles medium) containing antibiotics. Particle sizes were determined by dynamic light scattering. SNP morphology, surface area, and porosity were characterized using electron microscopy and nitrogen adsorption. The SNP sols in cell culture medium were stable for several days. The catalytic activity of the SNP in the conversion of hydrogen peroxide into hydroxyl radicals was investigated using electron paramagnetic resonance. The catalytic activity per square meter of exposed silica surface area was found to be independent of particle size and preparation method. Using this unique series of nanoparticle suspensions, the relationship between cytotoxicity and particle size was investigated using human endothelial and mouse monocyte-macrophage cells. The cytotoxicity of the SNP was strongly dependent on particle size and cell type. This unique methodology and the collection of well-characterized SNP will be useful for further in vitro studies exploring the physicochemical determinants of nanoparticle toxicity.

Influence of size, surface area and microporosity on the in vitro cytotoxic activity of amorphous silica nanoparticles in different cell types

Abstract Identifying the physico-chemical characteristics of nanoparticles (NPs) that drive their toxic activity is the key to conducting hazard assessment and guiding the design of safer nanomaterials. Here we used a set of 17 stable suspensions of monodisperse amorphous silica nanoparticles (SNPs) with selected variations in size (diameter, 2–335 nm), surface area (BET, 16–422 m2/g) and microporosity (micropore volume, 0–71 μl/g) to assess with multiple regression analysis the physico-chemical determinants of the cytotoxic activity in four different cell types (J774 macrophages, EAHY926 endothelial cells, 3T3 fibroblasts and human erythrocytes). We found that the response to these SNPs is governed by different physico-chemical parameters which vary with cell type: In J774 macrophages, the cytotoxic activity (WST1 assay) increased with external surface area (αs method) and decreased with micropore volume (r2 of the model, 0.797); in EAHY926 and 3T3 cells, the cytotoxic activity of the SNPs (MTT and WST1 assay, respectively) increased with surface roughness and small diameter (r2, 0.740 and 0.872, respectively); in erythrocytes, the hemolytic activity increased with the diameter of the SNP (r2, 0.860). We conclude that it is possible to predict with good accuracy the in vitro cytotoxic potential of SNPs on the basis of their physico-chemical characteristics. These determinants are, however, complex and vary with cell type, reflecting the pleiotropic interactions of nanoparticles with biological systems.

Adaptations of the in vitro MN assay for the genotoxicity assessment of nanomaterials

The issue of appropriate testing strategies has been raised for the genotoxicity assessment of nanomaterials. Recently, efforts have been made to evaluate the adequacy of Organisation for Economic Co-operation and Development-standardised tests to assess the genotoxicity of nanomaterials. The aim of this review was to examine whether the current guideline for the in vitro micronucleus (MN) assay is applicable for testing nanomaterials. From a Pubmed literature search, 21 available studies were identified for analysis. We reviewed all protocols used for testing nanomaterials with the in vitro MN assay. All studies were categorised based on the particle type and size. Different aspects of the protocols were evaluated such as the exposure (duration and doses), the cytochalasin-B treatment, serum levels and cytotoxicity assessment. Sixteen of the 21 studies demonstrated increased frequencies of MN. Some recommendations regarding the protocol were formulated to maximise sensitivity and avoid false negatives. Determination of the cellular dose was advised for a better interpretation of MN frequency results. The level of serum can modulate the cellular response, therefore the serum percentage used should enable cell growth and proliferation and a maximal sensitivity of the assay. Furthermore, different types of cytochalasin-B treatment were used, co-treatment, post-treatment and delayed co-treatment. In order to avoid decreased cellular uptake as a consequence of actin inhibition, post-treatment or delayed co-treatment is suggested. Exposure during mitosis should be recommended to allow contact with the chromatin or mitotic apparatus for nanomaterials that are unable to cross the nuclear membrane. With these adaptations, the in vitro MN assay can be recommended for genotoxicity testing of nanomaterials.

Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models

Abstract We explored how to assess the genotoxic potential of nanosize particles with a well validated assay, the in vitro cytochalasin-B micronucleus assay, detecting both clastogens and aneugens. Monodisperse Stöber amorphous silica nanoparticles (SNPs) of three different sizes (16, 60 and 104 nm) and A549 lung carcinoma cells were selected as models. Cellular uptake of silica was monitored by ICP-MS. At non-cytotoxic doses the smallest particles showed a slightly higher fold induction of micronuclei (MNBN). When considering the three SNPs together, particle number and total surface area appeared to account for MNBN induction as they both correlated significantly with the amplitude of the effect. Using nominal or cellular dose did not show statistically significant differences. Likewise, alkaline comet assay and FISH-centromeric probing of MNBN indicated a weak and not statistically significant induction of oxidative DNA damage, chromosome breakage and chromosome loss. This line of investigation will contribute to adequately design and interpret nanogenotoxicity assays.

Causes of genome instability: the effect of low dose chemical exposures in modern society

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genomes integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Oxidative stress induced by pure and iron-doped amorphous silica nanoparticles in subtoxic conditions.

Amorphous silica nanoparticles (SiO₂-NPs) have found broad applications in industry and are currently intensively studied for potential uses in medical and biomedical fields. Several studies have reported cytotoxic and inflammatory responses induced by SiO₂-NPs in different cell types. The present study was designed to examine the association of oxidative stress markers with SiO₂-NP induced cytotoxicity in human endothelial cells. We used pure monodisperse amorphous silica nanoparticles of two sizes (16 and 60 nm; S16 and S60) and a positive control, iron-doped nanosilica (16 nm; SFe), to study the generation of hydroxyl radicals (HO·) in cellular-free conditions and oxidative stress in cellular systems. We investigated whether SiO₂-NPs could influence intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, increase lipid peroxidation (malondialdehyde (MDA) and 4-hydroxyalkenal (HAE) concentrations), and up-regulate heme oxygenase-1 (HO-1) mRNA expression in the studied cells. None of the particles, except SFe, produced ROS in cell-free systems. We found significant modifications for all parameters in cells treated with SFe nanoparticles. At cytotoxic doses of S16 (40-50 μg/mL), we detected weak alterations of intracellular glutathione (4 h) and a marked induction of HO-1 mRNA (6 h). Cytotoxic doses of S60 elicited similar responses. Preincubation of cells being exposed to SiO₂-NPs with an antioxidant (5 mM N-acetylcysteine, NAC) significantly reduced the cytotoxic activity of S16 and SFe (when exposed up to 25 and 50 μg/mL, respectively) but did not protect cells treated with S60. Preincubation with NAC significantly reduced HO-1 mRNA expression in cells treated with SFe but did not have any effect on HO-1 mRNA level in cell exposed to S16 and S60. Our study demonstrates that the chemical composition of the silica nanoparticles is a dominant factor in inducing oxidative stress.