Hanna L. Karlsson

Size-dependent toxicity of metal oxide particles—A comparison between nano- and micrometer size

Toxicological studies have shown increased toxicity of nanoparticles (<100 nm) compared to micrometer particles of the same composition, which has raised concern about the impact on human health from nanoparticles. However, if this is true for a wide range of particles with different chemical composition is not clear. The aim of this study was to compare the toxicity of nano- and micrometer particles of some metal oxides (Fe(2)O(3), Fe(3)O(4), TiO(2) and CuO). The ability of the particles to cause cell death, mitochondrial damage, DNA damage and oxidative DNA lesions were evaluated after exposure of the human cell line A549. This study showed that nanoparticles of CuO were much more toxic compared to CuO micrometer particles. One key mechanism may be the ability of CuO to damage the mitochondria. In contrast, the micrometer particles of TiO(2) caused more DNA damage compared to the nanoparticles, which is likely explained by the crystal structures. The iron oxides showed low toxicity and no clear difference between the different particle sizes. In conclusion, nanoparticles are not always more toxic than micrometer particles, but the high toxicity of CuO nanoparticles shows that the nanolevel gives rise to specific concern.

Size-dependent cytotoxicity of silver nanoparticles in human lung cells: the role of cellular uptake, agglomeration and Ag release

BackgroundSilver nanoparticles (AgNPs) are currently one of the most manufactured nanomaterials. A wide range of toxicity studies have been performed on various AgNPs, but these studies report a high variation in toxicity and often lack proper particle characterization. The aim of this study was to investigate size- and coating-dependent toxicity of thoroughly characterized AgNPs following exposure of human lung cells and to explore the mechanisms of toxicity.MethodsBEAS-2B cells were exposed to citrate coated AgNPs of different primary particle sizes (10, 40 and 75 nm) as well as to 10 nm PVP coated and 50 nm uncoated AgNPs. The particle agglomeration in cell medium was investigated by photon cross correlation spectroscopy (PCCS); cell viability by LDH and Alamar Blue assay; ROS induction by DCFH-DA assay; genotoxicity by alkaline comet assay and γH2AX foci formation; uptake and intracellular localization by transmission electron microscopy (TEM); and cellular dose as well as Ag release by atomic absorption spectroscopy (AAS).ResultsThe results showed cytotoxicity only of the 10 nm particles independent of surface coating. In contrast, all AgNPs tested caused an increase in overall DNA damage after 24 h assessed by the comet assay, suggesting independent mechanisms for cytotoxicity and DNA damage. However, there was no γH2AX foci formation and no increased production of intracellular reactive oxygen species (ROS). The reasons for the higher toxicity of the 10 nm particles were explored by investigating particle agglomeration in cell medium, cellular uptake, intracellular localization and Ag release. Despite different agglomeration patterns, there was no evident difference in the uptake or intracellular localization of the citrate and PVP coated AgNPs. However, the 10 nm particles released significantly more Ag compared with all other AgNPs (approx. 24 wt% vs. 4–7 wt%) following 24 h in cell medium. The released fraction in cell medium did not induce any cytotoxicity, thus implying that intracellular Ag release was responsible for the toxicity.ConclusionsThis study shows that small AgNPs (10 nm) are cytotoxic for human lung cells and that the toxicity observed is associated with the rate of intracellular Ag release, a ‘Trojan horse’ effect.

Surface characteristics, copper release, and toxicity of nano- and micrometer-sized copper and copper(II) oxide particles: a cross-disciplinary study.

An interdisciplinary and multianalytical research effort is undertaken to assess the toxic aspects of thoroughly characterized nano- and micrometer-sized particles of oxidized metallic copper and copper(II) oxide in contact with cultivated lung cells, as well as copper release in relevant media. All particles, except micrometer-sized Cu, release more copper in serum-containing cell medium (supplemented Dulbeccos minimal essential medium) compared to identical exposures in phosphate-buffered saline. Sonication of particles for dispersion prior to exposure has a large effect on the initial copper release from Cu nanoparticles. A clear size-dependent effect is observed from both a copper release and a toxicity perspective. In agreement with greater released amounts of copper per quantity of particles from the nanometer-sized particles compared to the micrometer-sized particles, the nanometer particles cause a higher degree of DNA damage (single-strand breaks) and cause a significantly higher percentage of cell death compared to cytotoxicity induced by micrometer-sized particles. Cytotoxic effects related to the released copper fraction are found to be significantly lower than the effects related to particles. No DNA damage is induced by the released copper fraction.

Intracellular Uptake and Toxicity of Ag and CuO Nanoparticles : A Comparison Between Nanoparticles and their Corresponding Metal Ions

UNLABELLED An increased understanding of nanoparticle toxicity and its impact on human health is essential to enable a safe use of nanoparticles in our society. The aim of this study is to investigate the role of a Trojan horse type mechanism for the toxicity of Ag-nano and CuO-nano particles and their corresponding metal ionic species (using CuCl2 and AgNO3 ), i.e., the importance of the solid particle to mediate cellular uptake and subsequent release of toxic species inside the cell. The human lung cell lines A549 and BEAS-2B are used and cell death/membrane integrity and DNA damage are investigated by means of trypan blue staining and the comet assay, respectively. Chemical analysis of the cellular dose of copper and silver is performed using atomic absorption spectroscopy. Furthermore, transmission electron microscopy, laser scanning confocal microscopy, and confocal Raman microscopy are employed to study cellular uptake and particle-cell interactions. The results confirm a high uptake of CuO-nano and Ag-nano compared to no, or low, uptake of the soluble salts. CuO-nano induces both cell death and DNA damage whereas CuCl2 induces no toxicity. The opposite is observed for silver, where Ag-nano does not cause any toxicity, whereas AgNO3 induces a high level of cell death. IN CONCLUSION CuO-nano toxicity is predominantly mediated by intracellular uptake and subsequent release of copper ions, whereas no toxicity is observed for Ag-nano due to low release of silver ions within short time periods.

The comet assay in nanotoxicology research

Nanoscale particles can have impressive and useful characteristics, but the same properties may be problematic for human health. From this perspective it is critical to assess the ability of nanoparticles to cause DNA damage. This review focuses on the use of the comet assay in nanotoxicology research. In the alkaline version of the assay, DNA strand breaks and alkali-labile sites are detected and oxidatively damaged DNA can be analyzed using the enzyme formamidopyrimidine glycosylase. The article reviews studies that have used the comet assay to investigate the toxicity of manufactured nanoparticles. It is shown that at least 46 cellular in vitro studies and several in vivo studies have used the comet assay and that the majority of the nanoparticles tested cause DNA strand breaks or oxidative DNA lesions. This is not surprising considering the sensitivity of the method and the reactivity of many nanomaterials. Interactions between the particles and the assay cannot be totally excluded and need further consideration. It is concluded that studies including several particle types, to enable the assessment of their relative potency, are valuable as are studies focusing both on comet assay end points and mutagenicity. Finally, the article discusses the potential future use of the comet assay in human biomonitoring studies, which could provide valuable information for hazard identification of nanoparticles.

Cell membrane damage and protein interaction induced by copper containing nanoparticles—Importance of the metal release process

Cu-containing nanoparticles are used in various applications in order to e.g. achieve antimicrobial activities and to increase the conductivity of fluids and polymers. Several studies have reported on toxic effects of such particles but the mechanisms are not completely clear. The aim of this study was to investigate the interactions between cell membranes and well-characterized nanoparticles of CuO, Cu metal, a binary Cu-Zn alloy and micron-sized Cu metal particles. This was conducted via in vitro investigations of the effects of the nanoparticles on (i) cell membrane damage on lung epithelial cells (A549), (ii) membrane rupture of red blood cells (hemolysis), complemented by (iii) nanoparticle interaction studies with a model lipid membrane using quartz crystal microbalance with dissipation monitoring (QCM-D). The results revealed that nanoparticles of the Cu metal and the Cu-Zn alloy were both highly membrane damaging and caused a rapid (within 1h) increase in membrane damage at a particle mass dose of 20 μg/mL, whereas the CuO nanoparticles and the micron-sized Cu metal particles showed no such effect. At similar nanoparticle surface area doses, the nano and micron-sized Cu particles showed more similar effects. The commonly used LDH (lactate dehydrogenase) assay for analysis of membrane damage was found impossible to use due to nanoparticle-assay interactions. None of the particles induced any hemolytic effects on red blood cells when investigated up to high particle concentrations (1mg/mL). However, both Cu and Cu-Zn nanoparticles caused hemoglobin aggregation/precipitation, a process that would conceal a possible hemolytic effect. Studies on interactions between the nanoparticles and a model membrane using QCM-D indicated a small difference between the investigated particles. Results of this study suggest that the observed membrane damage is caused by the metal release process at the cell membrane surface and highlight differences in reactivity between metallic nanoparticles of Cu and Cu-Zn and nanoparticles of CuO.

Mechanisms related to the genotoxicity of particles in the subway and from other sources.

Negative health effects of airborne particles have clearly been shown in epidemiological studies. People get exposed to particles from various sources such as the combustion of, for example, diesel and wood and also from particles arising from tire-road wear. Another source of importance for certain populations is exposure to particles in subway systems. We recently reported that these particles were more genotoxic when compared to that of several other particle types. The aim of this study was to further investigate and compare the toxicity of subway particles and particles from other sources as well as investigate some mechanisms behind the genotoxicity of subway particles. This was done by comparing the ability of subway particles and particles from a street, pure tire-road wear particles, and particles from wood and diesel combustion to cause mitochondrial depolarization and to form intracellular reactive oxygen species (ROS). Furthermore, the genotoxicity and ability to cause oxidative stress was compared to magnetite particles since this is a main component in subway particles. It was concluded that the subway particles and also street particles and particles from wood and diesel combustion caused mitochondrial depolarization. The ability to damage the mitochondria is thus not the only explanation for the high genotoxicity of subway particles. Subway particles also formed intracellular ROS. This effect may be part of the explanation as to why subway particles show such high genotoxicity when compared to that of other particles. Genotoxicity can, however, not be explained by the main component, magnetite, by water-soluble metals, or by intracellular mobilized iron. The genotoxicity is most likely caused by highly reactive surfaces giving rise to oxidative stress.

Microsomal Glutathione Transferase 1 Protects Against Toxicity Induced by Silica Nanoparticles but Not by Zinc Oxide Nanoparticles

Microsomal glutathione transferase 1 (MGST1) is an antioxidant enzyme located predominantly in the mitochondrial outer membrane and endoplasmic reticulum and has been shown to protect cells from lipid peroxidation induced by a variety of cytostatic drugs and pro-oxidant stimuli. We hypothesized that MGST1 may also protect against nanomaterial-induced cytotoxicity through a specific effect on lipid peroxidation. We evaluated the induction of cytotoxicity and oxidative stress by TiO2, CeO2, SiO2, and ZnO in the human MCF-7 cell line with or without overexpression of MGST1. SiO2 and ZnO nanoparticles caused dose- and time-dependent toxicity, whereas no obvious cytotoxic effects were induced by nanoparticles of TiO2 and CeO2. We also noted pronounced cytotoxicity for three out of four additional SiO2 nanoparticles tested. Overexpression of MGST1 reversed the cytotoxicity of the main SiO2 nanoparticles tested and for one of the supplementary SiO2 nanoparticles but did not protect cells against ZnO-induced cytotoxic effects. The data point toward a role of lipid peroxidation in SiO2 nanoparticle-induced cell death. For ZnO nanoparticles, rapid dissolution was observed, and the subsequent interaction of Zn2+ with cellular targets is likely to contribute to the cytotoxic effects. A direct inhibition of MGST1 by Zn2+ could provide a possible explanation for the lack of protection against ZnO nanoparticles in this model. Our data also showed that SiO2 nanoparticle-induced cytotoxicity is mitigated in the presence of serum, potentially through masking of reactive surface groups by serum proteins, whereas ZnO nanoparticles were cytotoxic both in the presence and in the absence of serum.

Epigenetic effects of nano-sized materials.

The term epigenetics includes several phenomena such as DNA methylation, histone tail modifications, and microRNA mediated mechanisms, which are able to mold the chromatin structure and/or gene expression levels, without altering the primary DNA sequence. Environmental agents can exert epigenetic properties and there is increasing evidence of epigenetic deregulation of gene expression in several human diseases, including cancer, cardiovascular diseases, autism spectrum disorders, autoimmune diseases, and neurodegeneration, among others. Given the widespread use and dispersion in the environment of nano-sized materials, this article summarizes the studies performed so far to evaluate their potential epigenetic properties. Those studies highlight the ability of certain nano-sized compounds to induce an impaired expression of genes involved in DNA methylation reactions leading to global DNA methylation changes, as well as changes of gene specific methylation of tumor suppressor genes, inflammatory genes, and DNA repair genes, all potentially involved in cancer development. Moreover, some nano-sized compounds are able to induce changes in the acetylation and methylation of histone tails, as well as microRNA deregulated expression. We also provided a detailed description of currently available methodologies to evaluate epigenetic modifications. Standard protocols are currently available to evaluate cytotoxic and genotoxic effects of nano-sized materials. By contrast, there are at present no available standard protocols to evaluate the epigenetic potential of any given compound. The currently available methodologies offer different, but often complementary information to characterize potential epigenetic changes induced by exposure to nano-sized compounds. Given the widespread use and dispersion in the environment of nano-sized materials, at present and foreseeable in the near future, and in light of the indication of potential epigenetic properties here reviewed, more attention should be paid to unravel the consequences of such effects in future studies.

DNA damage induced by micro- and nanoparticles—interaction with FPG influences the detection of DNA oxidation in the comet assay

Reliable methods for evaluation of toxicity from particles, such as manufactured nanoparticles, are needed. One promising tool is the comet assay, often used to measure DNA breaks (strand breaks and alkali-labile sites) as well as oxidatively damaged DNA, the latter by addition of specific DNA repair enzymes such as formamidopyrimidine DNA glycosylase (FPG). The aim of this study was to investigate the use of the comet assay for analysis of DNA oxidation by a range of micro- and nanoparticles in the lung cell lines A549 and BEAS-2B and to test the hypothesis that nanoparticles present in the cells during the assay performance may interact with FPG. This was done by investigating the ability of micro- and nanoparticles (stainless steel, subway particles, MnO(2), Ag, CeO(2), Co(3)O(4), Fe(3)O(4), NiO and SiO(2)) to induce DNA breaks, oxidatively damaged DNA (FPG sites, dominantly 8-oxoguanine), intracellular production of reactive oxygen species (ROS) and non-cellular oxidation of the DNA base guanine, as well as by studying interactions of the particles and their released ions with FPG. Several particles caused DNA breaks, but low levels of FPG sites. The ability of FPG to detect DNA oxidation induced by a photosensitiser was however shown. An oxidative capacity of the particles was indicated by increased levels of intracellular ROS, and especially Ag and subway particles caused non-cellular oxidation of guanine. Incubation of FPG with the particles led to less FPG activity, particularly with nanoparticles of Ag but also with CeO(2), Co(3)O(4) and SiO(2). Further investigations of these particles revealed that for Ag, the decreased activity was mainly due to released Ag ions, whereas for CeO(2) and Co(3)O(4), FPG interactions were due to the particles. We conclude that measurement of oxidatively damaged DNA in cells exposed to nanoparticles may be underestimated in the comet assay due to interactions with FPG.