Predictive value of in vitro assays depends on the mechanism of toxicity of metal oxide nanoparticles (original) (raw)
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PLOS ONE, 2015
Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO 2 NMs with different surface chemistryhydrophilic (NM-103) and hydrophobic (NM-104), two forms of and coated with triethoxycapryl silane (NM-111) and two SiO 2 NMs produced by two different manufacturing techniquesprecipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO 2 >TiO 2 ). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO 2 and SiO 2 , while the embryonic stem cell test (EST) classified the TiO 2 NMs as potentially 'weakembryotoxic' and ZnO and SiO 2 NMs as 'non-embryotoxic'. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO 2 NM-203 > SiO 2 NM-200 > TiO 2 NM-104 > TiO 2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO 2 displayed higher toxicity compared with ZnO and SiO 2 . Importantly, the in vitro methodology applied could identify cell-and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.
Toxicological Sciences, 2007
Previous studies have reported little correlation between the relative toxicity of particle types when comparing lung toxicity rankings following in vivo instillation versus in vitro cell culture exposures. This study was designed to assess the capacity of in vitro screening studies to predict in vivo pulmonary toxicity of several fine or nanoscale particle types in rats. In the in vivo component of the study, rats were exposed by intratracheal instillation to 1 or 5 mg/kg of the following particle types: (1) carbonyl iron (CI), (2) crystalline silica (CS) (Min-U-Sil 5, a-quartz), (3) precipitated amorphous silica (AS), (4) nano-sized zinc oxide (NZO), or (5) fine-sized zinc oxide (FZO). Depending on particle type and solution state, these particles range in size from 90 to 500 nm in size. Following exposures, the lungs of exposed rats were lavaged and inflammation (neutrophil recruitment) and cytotoxicity end points (bronchoalveolar lavage [BAL] fluid lactate dehydrogenase [LDH] values) were measured at 24 h, 1 week, 1 and 3 months postexposure. For the in vitro component of the study, three different culture conditions were utilized. Cultures of (1) rat L2 lung epithelial cells, (2) primary alveolar macrophages (AMs) (collected via BAL from unexposed rats), as well as (3) AM-L2 lung epithelial cell cocultures were incubated with the particle types listed above, and the culture fluids were evaluated for cytotoxicity end points (LDH, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan [MTT]) as well as inflammatory cytokines (macrophage inflammatory 2 protein [MIP-2], tumor necrosis factor alpha [TNF-a], and interleukin-6 [IL-6]) at one (i.e., cytokines) or several (cytotoxicity) time periods. Results of in vivo pulmonary toxicity studies demonstrated that instilled CI particles produced little toxicity. CS particles produced sustained inflammation and cytotoxicity. AS particles produced reversible and transient inflammatory responses. NZO or FZO particles produced potent but reversible inflammation which was resolved by 1 month postinstillation exposure. Results of in vitro pulmonary cytotoxicity studies demonstrated a variety of responses to the different particle types, primarily at high doses. With respect to the LDH results, L2 cells were the most sensitive and exposures to nano-or fine-sized ZnO for 4 or 24 h were more cytotoxic than exposures to CS or AS particles. Macrophages essentially were resistant and epithelial macrophage cocultures generally reflected the epithelial results at 4 and 24 h incubation, but not at 48 h incubation. MTT results were also interesting but, except for nano-and fine-sized ZnO, did not correlate well with LDH results. Results of in vitro pulmonary inflammation studies demonstrated that L2 cells did not produce MIP-2 cytokines, but CS-or AS-exposed AMs and, to a lesser degree, cocultures secreted these chemotactic factors into the culture media. Measurements of TNF-a in the culture media by particle-exposed cells demonstrated little activity. In addition, IL-6 secretion was measured in CS, AS, and nano-sized ZnO-exposed cocultures. When considering the range of toxicity end points to five different particle types, the comparisons of in vivo and in vitro measurements demonstrated little correlation, particularly when considering many of the variables assessed in this study-such as cell types to be utilized, culture conditions and time course of exposure, as well as measured end points. It seems clear that in vitro cellular systems will need to be further developed, standardized, and validated (relative to in vivo effects) in order to provide useful screening data on the relative toxicity of inhaled particle types.
Particle and Fibre Toxicology, 2014
Background: Although ZnO nanoparticles (NPs) are used in many commercial products and the potential for human exposure is increasing, few in vivo studies have addressed their possible toxic effects after inhalation. We sought to determine whether ZnO NPs induce pulmonary toxicity in mice following sub-acute or sub-chronic inhalation exposure to realistic exposure doses. Methods: Mice (C57Bl/6) were exposed to well-characterized ZnO NPs (3.5 mg/m 3 , 4 hr/day) for 2 (sub-acute) or 13 (sub-chronic) weeks and necropsied immediately (0 wk) or 3 weeks (3 wks) post exposure. Toxicity was assessed by enumeration of total and differential cells, determination of total protein, lactate dehydrogenase activity and inflammatory cytokines in bronchoalveolar lavage (BAL) fluid as well as measurements of pulmonary mechanics. Generation of reactive oxygen species was assessed in the lungs. Lungs were evaluated for histopathologic changes and Zn content. Zn concentration in blood, liver, kidney, spleen, heart, brain and BAL fluid was measured. Results: An elevated concentration of Zn 2+ was detected in BAL fluid immediately after exposures, but returned to baseline levels 3 wks post exposure. Dissolution studies showed that ZnO NPs readily dissolved in artificial lysosomal fluid (pH 4.5), but formed aggregates and precipitates in artificial interstitial fluid (pH 7.4). Sub-acute exposure to ZnO NPs caused an increase of macrophages in BAL fluid and a moderate increase in IL-12(p40) and MIP-1α, but no other inflammatory or toxic responses were observed. Following both sub-acute and sub-chronic exposures, pulmonary mechanics were no different than sham-exposed animals. Conclusions: Our ZnO NP inhalation studies showed minimal pulmonary inflammation, cytotoxicity or lung histopathologic changes. An elevated concentration of Zn in the lung and BAL fluid indicates dissolution of ZnO NPs in the respiratory system after inhalation. Exposure concentration, exposure mode and time post exposure played an important role in the toxicity of ZnO NPs. Exposure for 13 wks with a cumulative dose of 10.9 mg/kg yielded increased lung cellularity, but other markers of toxicity did not differ from sham-exposed animals, leading to the conclusion that ZnO NPs have low sub-chronic toxicity by the inhalation route.
A practical approach to assess inhalation toxicity of metal oxide nanoparticles in vitro
Journal of applied toxicology : JAT, 2018
Exposure of humans to metal oxide nanoparticles (NPs) occurs mainly via air, and inhaled metal oxide NPs may generate inflammation. The aim of this study was to investigate the proinflammatory potential of six metal oxide NPs (CeO , Mn O , CuO, ZnO, Co O and WO ; 27-108 μg ml ) using human primary 3-dimensional airway epithelium (MucilAir™) and dendritic cell (DC) models. Metal oxide NPs were mainly aggregated/agglomerated in the cell media, as determined by dynamic light scattering, scanning electron microscopy and differential centrifugal sedimentation. WO and ZnO were highly soluble, both with and without respiratory mucus. Proinflammatory signalling by the epithelium was evaluated after a 24 hour exposure by increased interleukin-6 and -8 and monocyte chemoattractant protein 1 cytokine release, which occurred only for CuO. Moreover, maturation of immature human DCs, which play a key role in the lung immune system, were evaluated by expression of surface markers HLA-DR, CD80, CD8...
A review of mammalian toxicity of ZnO nanoparticles
Nanotechnology, Science and Applications, 2012
This review summarizes the literature on mammalian toxicity of ZnO nanoparticles (NPs) published between 2009 and 2011. The toxic effects of ZnO NPs are due to the compound's solubility. Whether the increased intracellular [Zn(2+)] is due to the NPs being taken up by cells or to NP dissolution in medium is still unclear. In vivo airway exposure poses an important hazard. Inhalation or instillation of the NPs results in lung inflammation and systemic toxicity. Reactive oxygen species (ROS) generation likely plays an important role in the inflammatory response. The NPs do not, or only to a minimal extent, cross the skin; this also holds for sunburned skin. Intraperitoneal administration induces neurological effects. The NPs show systemic distribution; target organs are liver, spleen, lung, and kidney and, in some cases, the heart. In vitro exposure of BEAS-2B bronchial epithelial cells and A549 alveolar adenocarcinoma cells results in cytotoxicity, increased oxidative stress, increased intracellular [Ca(2+)], decreased mitochondrial membrane potential, and interleukin (IL)-8 production. Decreased contractility of airway smooth muscle cells poses an additional hazard. In contrast to the results for BEAS-2B and A549 cells, in RKO colon carcinoma cells ZnO NPs and not Zn(2+) induce cytotoxicity and mitochondrial dysfunction. Short-term exposure of skin cells results in apoptosis but not in an inflammatory response, while long-term exposure leads to increased ROS generation, decreased mitochondrial activity, and formation of tubular intercellular structures. Macrophages, monocytes, and dendritic cells are affected; exposure results in cytotoxicity, oxidative stress, intracellular Ca(2+) flux, decreased mitochondrial membrane potential, and production of IL-1β and chemokine CXCL9. The NPs are phagocytosed by macrophages and dissolved in lysosomes. In vitro the Comet assay and the cytokinesis-blocked micronucleus assay show genotoxicity, whereas the Ames test does not. This is, however, not confirmed by in vivo genotoxicity assays. Protein binding results in increased stability.
Particle and Fibre Toxicology
Background Acute phase response (APR) is characterized by a change in concentration of different proteins, including C-reactive protein and serum amyloid A (SAA) that can be linked to both exposure to metal oxide nanomaterials and risk of cardiovascular diseases. In this study, we intratracheally exposed mice to ZnO, CuO, Al2O3, SnO2 and TiO2 and carbon black (Printex 90) nanomaterials with a wide range in phagolysosomal solubility. We subsequently assessed neutrophil numbers, protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, Saa3 and Saa1 mRNA levels in lung and liver tissue, respectively, and SAA3 and SAA1/2 in plasma. Endpoints were analyzed 1 and 28 days after exposure, including histopathology of lung and liver tissues. Results All nanomaterials induced pulmonary inflammation after 1 day, and exposure to ZnO, CuO, SnO2, TiO2 and Printex 90 increased Saa3 mRNA levels in lungs and Saa1 mRNA levels in liver. Additionally, CuO, SnO2, TiO2 and Printex 90 in...
Toxicology and Applied Pharmacology, 2014
The applicability of rat precision-cut lung slices (PCLuS) in detecting nanomaterial (NM) toxicity to the respiratory tract was investigated evaluating sixteen OECD reference NMs (TiO 2 , ZnO, CeO 2 , SiO 2 , Ag, multi-walled carbon nanotubes (MWCNTMWCNTs)). Upon 24-hour test substance exposure, the PCLuS system was able to detect early events of NM toxicity: total protein, reduction in mitochondrial activity, caspase-3/-7 activation, glutathione depletion/increase, cytokine induction, and histopathological evaluation. Ion shedding NMS (ZnO and Ag) induced severe tissue destruction detected by the loss of total protein. Two anatase TiO 2 NMs, CeO 2 NMs, and two MWCNT caused significant (determined by trend analysis) cytotoxicity in the WST-1 assay. At non-cytotoxic concentrations, different TiO 2 NMs and one MWCNT increased GSH levels, presumably a elsevier_YTAAP_12991 defence defense response to reactive oxygen species, and these substances further induced a variety of cytokines. One of the SiO 2 NMs increased caspase-3/-7 activities at non-cytotoxic levels, and one rutile TiO 2 only induced cytokines. Investigating these effects is, however, not sufficient to predict apical effects found in vivo. Reproducibility of test substance measurements was not fully satisfactory, especially in the GSH and cytokine assays.
Nanotoxicology, 2013
There are a multitude of nanoparticles (NPs) which have shown great potentials for medical applications. A few of them are already used for lung therapeutic and diagnostic purposes. However, there are few toxicological studies which determine possible adverse pulmonary responses. It is thus important to propose in vitro screening strategies to evaluate the pulmonary toxicity of NPs used in nanomedicine. Our goal was to determine the cellular effects of several biomedical NPs with different physico-chemical characteristics (chemical nature, size and coating) to establish suitable tests and useful benchmark NPs. The effects of poly(lacticco-glycolic acid) (PLGA), silica, iron oxide and titanium dioxide NPs were studied using human bronchial (16HBE) and alveolar epithelial cells (A549). We evaluated cytotoxicity, reactive oxygen species (ROS) production and pro-inflammatory response in both cell lines. We demonstrated that PLGA NPs are good candidates for negative control NPs and SiO 2 NPs were revealed to be the best benchmark NPs. Coating of Fe 3 O 4 with sodium oleate, a known biocompatible compound, led to an unexpected increase in cytotoxicity. Moreover, 16HBE cells are more sensitive than A549 cells and propidium iodide uptake is a more sensitive cytotoxicity test than WST-1. The measurement of oxidative stress does not systematically allow us to predict cellular responses and different other cellular endpoints should also be addressed. We conclude that a battery of assays and cell lines are necessary to accurately evaluate the pulmonary effects of NPs and that PLGA and SiO 2 NPs are suitable candidates respectively for negative and positive controls.
Environmental Health Perspectives, 2008
Nanotechnologies have the potential to improve many aspects of our life. Nanomaterials are either being used or have the potential to be widely incorporated in a range of applications, including textiles, finishes, and electronics, as well as having a spectrum of uses in medical imaging, disease diagnoses, and drug delivery. This potential is a result of unique physicochemical characteristics apparent at the nanoscale, such as large surface area, altered electronic properties, surface reactiv