Cytotoxicity of silver nanoparticles on tumoral and normal human cells

S202

Abstracts / Toxicology Letters 211S (2012) S43–S216

Purpose: The purpose with this project was to study the toxicity of different metal nanoparticles in vitro. Nanotechnology is developing fast and nanoparticles are used within many areas, e.g. clothing, cosmetics and medicine. Concerns of health effects have been raised and certain nanoparticles have been shown to increase levels of oxidative stress, inflammation and DNA damage. Understanding of cellular mechanisms behind nanotoxicity is important for assessment of human health risks. Methods: Human cell lines (lung and blood cells) have been used to evaluate toxic effects of a range of metal and metal oxide nanoparticles. Various methods have been used to assess the toxicity in terms of cytotoxicity using trypan blue staining, mitochondrial damage using the fluorescent probe tetramethylrhodamine methyl esther and apoptosis analysis using Annexin-V and PI staining. Results and conclusions: Studies of 38 nanoparticles show that the cytotoxicity varies greatly, some nanoparticles generated up to 100% cytotoxicity after 18 hours exposure of 80 ␮g/ml. Dose-response curves show that certain nanoparticles are highly cytotoxic with IC50-values varying from 15-90 ␮g/ml for the most potent nanoparticles. Differences in toxicity between cell types are also seen when comparing three leukemia cell lines and lymphocytes. Necrosis or secondary apoptosis is the main type of cell death. Results from analysis of mitochondrial damage show that the nanoparticles are diverse in their mitochondrial interaction, in terms of time and dose dependency. Conclusion from this study is that certain metal nanoparticles are highly toxic and that the mechanisms behind the toxicity depend on composition and size. doi:10.1016/j.toxlet.2012.03.724

P33-15 Detection of nanoparticle-induced DNA oxidation using the comet assay Johanna Kain, Hanna Karlsson, Lennart Möller Karolinska Institutet, Sweden Purpose: One increasingly used method for detection of DNA damage induced by nanoparticles is the comet assay, often used to measure DNA breaks 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 different 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. Methods: The ability of nanoparticles (Ag, CeO2 , Co3 O4 , Fe3 O4 , NiO and SiO2 ) to induce DNA breaks and oxidatively damaged DNA was assessed using the comet assay, intracellular production of reactive oxygen species (ROS) by 2 ,7 -dichlorofluorescin diacetate fluorescence and non-cellular oxidation of the DNA base guanine using HPLC-EC. Results and conclusion: DNA breaks were induced by all nanoparticles except from SiO2 , but no or low levels of FPG-sites could be detected. In contrast, several nanoparticles induced intracellular ROS, and especially Ag nanoparticles caused non-cellular oxidation of guanine. Incubation of FPG with the particles led to less FPG activity, particularly with nanoparticles of Ag due to released Ag ions, but also with CeO2 , Co3 O4 and SiO2 . 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. doi:10.1016/j.toxlet.2012.03.725

P33-16 Toxicity of zinc oxide nanoparticles towards a fish and mammalian cell line Tobias Lammel 1 , Maria-Luisa Fernández-Cruz 1 , Mona Connolly 1 , Barrado Ana Isabel Conde Estefania 2 , Sylvain Derick 3 , Yolanda Pérez 4 , Marta Fernández 2 , Christophe Furger 3 , José Maria Navas 1 INIA, Spain, 2 CIEMAT, Spain, 3 NOVALEADS, France, 4 Universidad Rey Juan Carlos, Spain

1

Purpose: The objective of this study was to assess the toxic effects that zinc oxide (ZnO) nanoparticles (NPs) exert at the (sub)cellular level. It was further aimed to extend toxicity assessment of ZnO NPs to non-mammalian in vitro models. In addition, the applicability of a recently developed cytotoxicity assay (DAP) to assess nanomaterial toxicity was tested. Methods: The toxicity of three different ZnO NPs and their bulk counterpart was studied in the fish and the human hepatoma cell lines PLHC-1 and HepG2. Morphology and size distribution of particles were characterized by TEM and DLS, respectively. Real concentrations were measured using ICP-MS. Cytotoxicity was determined with the MTT, NRR, LDH, and DAP assay. ROS generation was examined using DCFH-DA. Contribution of the dissolved ZnO fraction to toxicity was investigated through exposure to supernatants of centrifuged particle suspensions. Results and conclusion(s) of the study: All tested ZnO particles caused a dose-dependent decrease in cell viability, with HepG2 being more sensitive than PLHC-1. The DAP assay was succesfully applied to the fish cells. The dissolved ZnO fraction was found to contribute to the cytotoxicity observed in both cell lines. Induction of oxidative stress could not explain ZnO NP cytotoxicity. When taking into account measured concentrations, cytotoxicity appeared to depend only on concentration and not on other factors such as NP size. These results stress the importance of determining real NP concentrations to avoid erroneous conclusions on cytotoxic potential. doi:10.1016/j.toxlet.2012.03.726

P33-17 Cytotoxicity of silver nanoparticles on tumoral and normal human cells Alicia Ávalos 1 , Ana Isabel Haza 2 , Paloma Morales 2 1

Facultad de Veterinaria, UCM, Spain, 2 Facultad de Veterinaria, Spain An improved understanding of the potential risks of nanomaterials is necessary to check its toxicity or safety. Therefore, the aim of this study was to evaluate the cytotoxic effects of silver nanoparticles (AgNps) of different sizes on tumoral [leukemia (HL-60) and hepatoma (HepG2)] and normal [dermal (NHDF) and pulmonary (HPF) fibroblasts]. MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction and lactate dehydrogenase (LDH) leakage were used for cytotoxicity assessment. 4.7 nm silver nanoparticles showed a strong decrease in HepG2 from 3.36 to 13.45 ␮g/mL (14%, 5% and 4% of cell survival, respectively), in HL-60 at 13.45 ␮g/mL for 24–72 h (3%, 3.7% and 3.8% of cell survival, respectively) and increased dramatically the percentage of LDH leakage (80%, 98% and 99%, respectively) in HepG2 and to 100% in HL-60. NHDF cell viability decreased drastically at concentrations of 6.72 and 13.45 ␮g/mL (10% and 8% of survival,

Abstracts / Toxicology Letters 211S (2012) S43–S216

respectively), in HPF cells at concentration of 13.45 ␮g/mL (15%, 6% and 6% of cell viability, respectively) and increased dramatically the percentage of LDH leakage (93–97%) in both cell lines. However, silver nanoparticles of 42 nm showed a large decreased in cell viability at concentrations of 100 ␮g/mL (HepG2, 33% of survival), 50 ␮g/mL (HL-60, 39%) and 2000 ␮g/mL (NHDF and HPF, 62% and 36 to 5%, respectively). The release of LDH increased to 65% (HepG2), 69% (HL-60), 40% (NHDF) and 90% (HPF). Silver nanoparticles of size 4.7 nm showed the highest cytotoxicity in all cells. Tumoral cells were more sensitive than normal cells to the effects of silver nanoparticles. doi:10.1016/j.toxlet.2012.03.727

P33-18 Hemolysis of silica particles: Importance of surface properties and plasma corona Jingwen Shi 1 , Yolanda Hedberg 2 , Maria Lundin 2 , Inger Odnevall Wallinder 2 , Hanna Karlsson 1 , Lennart Möller 1 Karolinska Institutet, Sweden, 2 KTH Royal Institute of Technology, Sweden

1

Purpose: Novel silica materials incorporating nanotechnology are promising materials for biomedical applications, such as drug and gene delivery. Even though micro-sized silica is well-documented to induce hemolysis, little is known about the hemolysis of nanostructured silica materials. The purpose of this study was to investigate the hemolytic activities of synthetic nanostructured silica materials for their rational design in nanomedicine. Methods: Amorphous nano silica particles with primary sizes of 7–14 nm, 5–15 nm, 20 nm, and 80 nm, and porous silica particles with pore diameters of 40 nm and 170 nm were investigated. A crystalline silica sample (0.5–10 ␮m) was included for benchmarking purposes. Physicochemical characterization was performed by electron microscopy, X-ray diffraction, dynamic light scattering, zeta-potential, and X-ray photon electron spectroscopy. Silica particle-induced hemolysis of human red blood cells (RBC) was investigated, and compared with the trypan blue assay of human promyelocytic leukemia cells (HL-60). Results and conclusions: Hemolysis of nanostructured silica particles was dependent on the temperature and solution complexity (solvent, plasma), as well as the surface properties (silanol groups, gel-like structure) of these particles. Hydrophobic modified silica nanoparticles induced less hemolysis than their hydrophilic counterparts. Furthermore, a significant correlation was observed between the hemolytic of RBC and the cytotoxicity of HL-60 induced by silica particles, suggesting a potential universal mechanism of action. Importantly, the generated results suggest that the protective effect of plasma towards silica particle-induced hemolysis as well as cytotoxicity was primarily due to the protein/lipid layer shielding the silica particle surface. doi:10.1016/j.toxlet.2012.03.728

S203

P33-19 Carbon-based nanomaterials trigger interleukin-1␤ secretion in human macrophages Fernando Torres Andon 1 , Lisong Xiao 2 , Xuefeng Song 2 , Elena R. Kisin 3 , Ashley R. Murray 3 , Anna A. Shvedova 3 , Sanjay Mathur 2 , Bengt Fadeel 1 1 3

Karolinska Institutet, Sweden, 2 University of Cologne, Germany, NIOSH/CDC and West Virginia University, United States

Purpose: The aim of this work is to investigate the toxic effects of three carbon-based nanomaterials on primary human monocyte-derived macrophages (HMDM), these include: graphene oxide (GO), a 2-D nanomaterial composed of layers of carbon atoms; single-walled carbon nanotubes (SWCNT), a 1-D nanomaterial formed by the rolling of graphene sheets and 3-D hollow carbon spheres (HCS). Methods: Monocytes were differentiated into macrophages for 3–4 days with M-CSF. In addition, to study inflammasome activation, HMDM were primed for 3 h with lipopolysaccharide (LPS) prior to exposure to the nanomaterials. Trypan Blue assay was utilized for assessment of cell viability. Cellular uptake of nanomaterials was monitored by transmission electron microscopy. Production of pro-inflammatory cytokines was determined by ELISA. Results and conclusions: No significant toxicity is noted with SWCNT or GO up to 100 ␮g/ml during 24 and 48 h of treatment, while the cytotoxic effect induced by HCS is dose-dependent. HMDM are able to internalize all the materials after 2 h of co-incubation. Dose- and time-dependent activation of mediators of innate immunity is detected with HCS, while SWCNT and GO only induce the secretion of IL-1␤, but to a lesser degree, after 24 h of treatment using the highest dose (100 ␮g/ml). The inhibition of caspase-1 prevents the secretion of IL-1␤, indicative of inflammasome-dependent cytokine secretion. This work demostrates the immunotoxicity (cell death) and/or immunostimulation effect induced by three carbon-based nanomaterials with differences in terms of size, shape and surface properties. doi:10.1016/j.toxlet.2012.03.729

P33-20 Biocompatibility of polyester dendrimers in comparison to polyamidoamine dendrimers ˜ 2, Feliu Torres Neus 1 , Marie V. Walter 2 , Maria I. Montanez Andrea Kunzmann 1 , Anders Hult 2 , Andreas Nyström 1 , Michael Malkoch 2 , Bengt Fadeel 1 Karolinska Institutet, Sweden, 2 Royal Institute of Technology, Sweden

1

Dendrimers are a new class of polymer materials that can be designed for several biomedical applications, such as drug delivery, diagnosis etc., due to dendrimers present high degree of branching, globular architecture, multivalency and well-defined molecular weight. The present study focused on the in vitro biocompatibility evaluation of a library of aliphatic polyester dendrimers based on 2,2-bis(methylol)propionic acid (bis-MPA) with an overall diameter of 0.5–2 nm. In addition, dendrons corresponding to the structural fragments of the dendrimers were evaluated as well as commercial polyamidoamine dendrimers (PAMAM) with cationic (amine) or neutral (hydroxyl) end group that were included for comparison. Cell viability studies were conducted in human cervical cancer (HeLa) and acute monocytic leukemia cells (THP.1) differenti-