Copper oxide (CuO) nanoparticles (NPs) are known to trigger cytotoxicity in a variety of cell models, but the mechanism of cell death remains unknown. Here we addressed the mechanism of cytotoxicity ...in macrophages exposed to CuO NPs versus copper chloride (CuCl
).
The mouse macrophage cell line RAW264.7 was used as an in vitro model. Particle uptake and the cellular dose of Cu were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. The deposition of Cu in lysosomes isolated from macrophages was also determined by ICP-MS. Cell viability (metabolic activity) was assessed using the Alamar Blue assay, and oxidative stress was monitored by a variety of methods including a luminescence-based assay for cellular glutathione (GSH), and flow cytometry-based detection of mitochondrial superoxide and mitochondrial membrane potential. Protein aggregation was determined by confocal microscopy using an aggresome-specific dye and protein misfolding was determined by circular dichroism (CD) spectroscopy. Lastly, proteasome activity was investigated using a fluorometric assay.
We observed rapid cellular uptake of CuO NPs in macrophages with deposition in lysosomes. CuO NP-elicited cell death was characterized by mitochondrial swelling with signs of oxidative stress including the production of mitochondrial superoxide and cellular depletion of GSH. We also observed a dose-dependent accumulation of polyubiquitinated proteins and loss of proteasomal function in CuO NP-exposed cells, and we could demonstrate misfolding and mitochondrial translocation of superoxide dismutase 1 (SOD1), a Cu/Zn-dependent enzyme that plays a pivotal role in the defense against oxidative stress. The chelation of copper ions using tetrathiomolybdate (TTM) prevented cell death whereas inhibition of the cellular SOD1 chaperone aggravated toxicity. Moreover, CuO NP-triggered cell death was insensitive to the pan-caspase inhibitor, zVAD-fmk, and to wortmannin, an inhibitor of autophagy, implying that this was a non-apoptotic cell death. ZnO NPs, on the other hand, triggered autophagic cell death.
CuO NPs undergo dissolution in lysosomes leading to copper-dependent macrophage cell death characterized by protein misfolding and proteasomal insufficiency. Specifically, we present novel evidence for Cu-induced SOD1 misfolding which accords with the pronounced oxidative stress observed in CuO NP-exposed macrophages. These results are relevant for our understanding of the consequences of inadvertent human exposure to CuO NPs.
The European Commission's Green Deal is a major policy initiative aiming to achieve a climate-neutral, zero-pollution, sustainable, circular and inclusive economy, driving both the New Industrial ...Strategy for Europe and the Chemicals Strategy for Sustainability. Innovative materials can help to reach these policy goals, but they need to be safe and sustainable themselves. Thus, one aim is to shift the development of chemicals to Safe- and Sustainable-by-Design, and define a new systems approach and criteria for sustainability to achieve this. An online workshop was organised in September 2020 by the Joint Research Centre and the Directorate-General Research and Innovation of the European Commission, with participants from academia, non-governmental organisations, industry and regulatory bodies. The aims were to introduce the concept of Safe- and Sustainable-by-Design, to identify industrial and regulatory challenges in achieving safer and more sustainable Smart Nanomaterials as an example of innovative materials, and to deliver recommendations for directions and actions necessary to meet these challenges. The following needs were identified: (i) an agreed terminology, (ii) a common understanding of the principles of Safe- and Sustainable-by-Design, iii) criteria, assessment tools and incentives to achieve a transition from Safe-by-Design to Safe- and Sustainable-by-Design, and (iv) preparedness of regulators and legislation for innovative chemicals/nanomaterials. This paper presents the authors' view on the state of the art as well as the needs for future activities, based on discussions at the workshop and further considerations. The case of Smart Nanomaterials is used to illustrate the Safe- and Sustainable-by-Design concept and challenges for its implementation. Most of the considerations can be extended to other advanced materials and to chemicals and products in general.
•Safe- and Sustainable-by-Design chemicals contribute to the transformation of policy ambitions into a circular economy.•Smart nanomaterials are a case study of the challenges of Safe- and Sustainable-by-Design.•The development of criteria to guide the design of chemicals facilitates achieving sustainability.•Broad SSbD expert consultation is needed for consensus on principles and implementation.•Agreed terminology and assessment tools for Smart Nanomaterials help to achieve the EU green policy goals.
Background: Advanced materials are most likely to bring future economic, environmental and social benefits. At the same time, they may pose challenges regarding their safety and sustainability along ...the entire lifecycle. This needs to be timely addressed by the stakeholders (industry, research, policy, funding and regulatory bodies). As part of a larger foresight project, this study aimed to identify areas of scientific research and technological development related to advanced materials, in particular advanced nanomaterials and the sub-group of smart nanomaterials. The study identified and collected data to build relevant research and innovation indicators and analyse trends, impact and other implications.
Methods: This study consisted of an iterative process including a documentation phase followed by the identification, description and development of a set of core research and innovation indicators regarding scientific publications, EU projects and patents. The data was extracted mainly from SCOPUS, CORDIS and PATSTAT databases using a predefined search string that included representative keywords. The trends, distributions and other aspects reflected in the final version of the indicators were analysed, e.g. the number of items in a period of time, geographical distribution, organisations involved, categories of journals, funding programmes, costs and technology areas.
Results: Generally, for smart nanomaterials the data used represent around 3.5% of the advanced nanomaterials data, while for each field analysed, they represent 4.4% for publications, 13% for projects and 1.1% for patents. The study shows current trends for advanced nanomaterials at a top-level information that can be further extended with sub-indicators. Generally, the results indicated a significant growth in research into advanced nanomaterials, including smart nanomaterials, in the last decade, leading to an increased availability of information.
Conclusion: These indicators identify trends regarding scientific and technological achievements and represent an important element when examining possible impacts on society and policy implications associated to these areas.
Metal and metal oxide nanoparticles are an important class of materials with numerous applications. Understanding how such nanoparticles interact with living systems is of considerable relevance both ...from a toxicological and biomedical perspective. The physicochemical features of nanoparticles are sometimes referred to as the synthetic identity, while the acquired properties of nanoparticles in a biological milieu resulting from the adsorption of biomolecules on the surface of the particles can be considered the biological identity. In this article, we explore the dynamic changes in the identity of nanoparticles resulting either from acquisition of a so-called bio-corona or through the process of biotransformation and how this impacts cellular recognition of nanoparticles and toxicological outcomes, with an emphasis on inflammation—an orchestrated host response against harmful stimuli, including pathogens as well as particles.
The European Green Deal policy ambitions set out in the Chemicals Strategy for Sustainability and the Zero Pollution Action Plan identify the transition to a Safe and Sustainable by Design (SSbD) ...approach to chemicals and materials. The H2020 SUNSHINE project has developed an approach to operationalize SSbD, specifically addressing multi-component nanomaterials (MCNMs), and applied it to two case studies. This approach enables assessment of safety and sustainability aspects at each stage of product development from a lifecycle perspective. This is achieved via a tiered approach that uses qualitative (Tier 1), semi-quantitative (Tier 2) and quantitative (Tier 3) assessment methods. The present work focuses on the Tier 1 (self-assessment) methodology designed to evaluate the safety, functionality and sustainability in the early R&D stages of the lifecycle of chemicals and materials. This approach was developed to be implementable by industries in a straightforward manner as often there is lack of time and/or expertise to engage in resource-intensive safety and sustainability evaluations. The approach was tested using two real industrial case studies, namely nano-enabled PFAS (Polyfluoroalkyl substances)-free anti-sticking coating for bakery molds, and nano-drops of essential oil anchored to the surface of nano clays and encapsulated in a polymeric film. The results indicate that these innovative materials have a high probability to have better safety, functionality and sustainability performance compared to conventional benchmark materials.
•A Safe and Sustainable by Design (SSbD) approach for advanced materials is proposed.•The qualitative SSbD self-assessment methodology is addressed to industry and SMEs.•The SSbD evaluates safety, functionality and sustainability in the early R&D stages.•The qualitative SSbD approach was applied to two industrial case studies.
Graphene-related two-dimensional nanomaterials possess very technically promising characteristics, but gaps exist regarding their potential adverse health effects. Based on their nano-thickness and ...lateral micron dimensions, nanoplates exhibit particular aerodynamic properties, including respirability. To develop a lung-focused, in vitro/in vivo screening approach for toxicological hazard assessment, various graphene-related nanoplates, i.e., single-layer graphene (SLG), graphene nanoplatelets (GNP), carboxyl graphene, graphene oxide, graphite oxide and Printex 90® (particle reference) were used. Material characterization preceded in vitro (geno)toxicity screening (membrane integrity, metabolic activity, proliferation, DNA damage) with primary rat alveolar macrophages (AM), MRC-5 lung fibroblasts, NR8383 and RAW 264.7 cells. Submerse cell exposure and material-adapted methods indicated material-, cell type-, concentration-, and time-specific effects. SLG and GNP were finally chosen as in vitro biologically active or more inert graphene showed eosinophils in lavage fluid for SLG but not GNP. The subsequent 28-day inhalation study (OECD 412) confirmed a toxic, genotoxic and pro-inflammatory potential for SLG at 3.2 mg/m3 with an in vivo-ranking of lung toxicity: SLG > GNP > Printex 90®. The in vivo ranking finally pointed to AM (lactate dehydrogenase release, DNA damage) as the most predictive in vitro model for the (geno)toxicity screening of graphene nanoplates.
Introduction:
Significant progress has been made in terms of best practice in research data management for nanosafety. Some of the underlying approaches to date are, however, overly focussed on the ...needs of specific research projects or aligned to a single data repository, and this “silo” approach is hampering their general adoption by the broader research community and individual labs.
Methods:
State-of-the-art data/knowledge collection, curation management FAIrification, and sharing solutions applied in the nanosafety field are reviewed focusing on unique features, which should be generalised and integrated into a functional FAIRification ecosystem that addresses the needs of both data generators and data (re)users.
Results:
The development of data capture templates has focussed on standardised single-endpoint Test Guidelines, which does not reflect the complexity of real laboratory processes, where multiple assays are interlinked into an overall study, and where non-standardised assays are developed to address novel research questions and probe mechanistic processes to generate the basis for read-across from one nanomaterial to another. By focussing on the needs of data providers and data users, we identify how existing tools and approaches can be re-framed to enable “on-the-fly” (meta) data definition, data capture, curation and FAIRification, that are sufficiently flexible to address the complexity in nanosafety research, yet harmonised enough to facilitate integration of datasets from different sources generated for different research purposes. By mapping the available tools for nanomaterials safety research (including nanomaterials characterisation, nonstandard (mechanistic-focussed) methods, measurement principles and experimental setup, environmental fate and requirements from new research foci such as safe and sustainable by design), a strategy for integration and bridging between silos is presented. The NanoCommons KnowledgeBase has shown how data from different sources can be integrated into a one-stop shop for searching, browsing and accessing data (without copying), and thus how to break the boundaries between data silos.
Discussion:
The next steps are to generalise the approach by defining a process to build consensus (meta)data standards, develop solutions to make (meta)data more machine actionable (on the fly ontology development) and establish a distributed FAIR data ecosystem maintained by the community beyond specific projects. Since other multidisciplinary domains might also struggle with data silofication, the learnings presented here may be transferrable to facilitate data sharing within other communities and support harmonization of approaches across disciplines to prepare the ground for cross-domain interoperability.
Engineered nanomaterials (ENMs) have tremendous potential to produce beneficial technological impact in numerous sectors in society. Safety assessment is, of course, of paramount importance. However, ...the myriad variations of ENM properties makes the identification of specific features driving toxicity challenging. At the same time, reducing animal tests by introducing alternative and/or predictive in vitro and in silico methods has become a priority. It is important to embrace these new advances in the safety assessment of ENMs. Indeed, remarkable progress has been made in recent years with respect to mechanism-based hazard assessment of ENMs, including systems biology approaches as well as high-throughput screening platforms, and new tools are also emerging in risk assessment and risk management for humans and the environment across the whole life-cycle of nano-enabled products. Here, we highlight some of the key advances in the hazard and risk assessment of ENMs.
The rapid dissolution of copper oxide (CuO) nanoparticles (NPs) with release of ions is thought to be one of the main factors modulating their toxicity. Here we assessed the cytotoxicity of a panel ...of CuO NPs (12 nm ± 4 nm) with different surface modifications, i.e., anionic sodium citrate (CIT) and sodium ascorbate (ASC), neutral polyvinylpyrrolidone (PVP), and cationic polyethylenimine (PEI), versus the pristine (uncoated) NPs, using a murine macrophage cell line (RAW264.7). Cytotoxicity, reactive oxygen species (ROS) production, and cellular uptake were assessed. The cytotoxicity results were analyzed by the benchmark dose (BMD) method and the NPs were ranked based on BMD20 values. The PEI-coated NPs were found to be the most cytotoxic. Despite the different properties of the coating agents, NP dissolution in cell medium was only marginally affected by surface modification. Furthermore, CuCl2 (used as an ion control) elicited significantly less cytotoxicity when compared to the CuO NPs. We also observed that the antioxidant, N-acetylcysteine, failed to protect against the cytotoxicity of the uncoated CuO NPs. Indeed, the toxicity of the surface-modified CuO NPs was not directly linked to particle dissolution and subsequent Cu burden in cells, nor to cellular ROS production, although CuO-ASC NPs, which were found to be the least cytotoxic, yielded lower levels of ROS in comparison to pristine NPs. Hierarchical cluster analysis suggested, instead, that the toxicity in the current in vitro model could be explained by synergistic interactions between the NPs, their dissolution, and the toxicity of the coating agents.
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•CuO nanoparticles (NPs) were cytotoxic for RAW264.7 macrophages.•Surface modification altered the cytotoxicity of the CuO NPs.•Particle dissolution or ROS production failed to explain the cytotoxicity.•The coating agents themselves also contributed to the overall cytotoxicity.
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 TiO2 NMs with different surface chemistry - hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO - uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques - precipitated (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>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially 'weak-embryotoxic' and ZnO and SiO2 NMs as 'non-embryotoxic'. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. 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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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