Humans can be exposed to engineered and nonintentionally formed metal and metal oxide nanoparticles (Me NPs) in occupational settings, in public transportation areas, or by means of contact with different consumer products. A critical factor in the toxic potency of Me NPs is their ability to induce oxidative stress. It is thus essential to assess the potential reactive oxygen species (ROS) formation properties of Me NPs. A common way to assess the relative extent of ROS formation in vitro is to use fluorescence spectroscopy with the DCFH-DA (2 ',7'-dichlorofluorescein diacetate) probe, with and without HRP (horseradish peroxidase). However, this method does not provide any information about specific ROS species or reaction mechanisms. This study investigated the possibility of using complementary techniques to obtain more specific information about formed ROS species, both the type and reaction mechanisms. Cu NPs in PBS (phosphate buffered saline) were chosen as a test system to have the simplest (least interference from other components) aqueous solution with a physiologically relevant pH. ROS formation was assessed using fluorescence by means of the DCFH-DA method (information on relative amounts of oxygen radicals without selectivity), the Ghormley's triiodide method using UV-vis spectrophotometry (concentrations of H2O2), and electron paramagnetic resonance with DMPO as the spin-trap agent (information on specific oxygen radicals). This approach elucidates that Cu NPs undergo ROS-generating corrosion reactions, which previously have not been assessed in situ. In the presence of H2O2, and based on the type of oxygen radical formed, it was concluded that released copper participates in Haber-Weiss and/or Fenton reactions rather than in Fenton-like reactions. The new combination of techniques used to determine ROS induced by Me NPs provides a way forward to gain a mechanistic understanding of Me NP-induced ROS formation, which is important for gaining crucial insight into their ability to induce oxidative stress.

Diffusely dispersed metal and metal oxide nanoparticles (NPs) can adversely affect living organisms through various mechanisms and exposure routes. One mechanism behind their toxic potency is their ability to generate reactive oxygen species (ROS) directly or indirectly to an extent that depends on the dose, metal speciation, and exposure route. This review provides an overview of the mechanisms of ROS formation associated with metal and metal oxide NPs and proposes a possible way forward for their future categorization. Metal and metal oxide NPs can form ROS via processes related to corrosion, photochemistry, and surface defects, as well as via Fenton, Fenton-like, and Haber-Weiss reactions. Regular ligands such as biomolecules can interact with metallic NP surfaces and influence their properties and thus their capabilities of generating ROS by changing characteristics such as surface charge, surface composition, dissolution behavior, and colloidal stability. Interactions between metallic NPs and cells and their organelles can indirectly induce ROS formation via different biological responses. H2O2 can also be generated by a cell due to inflammation, induced by interactions with metallic NPs or released metal species that can initiate Fenton(-like) and Haber-Weiss reactions forming various radicals. This review discusses these different pathways and, in addition, nano-specific aspects such as shifts in the band gaps of metal oxides and how these shifts at biologically relevant energies (similar to activation energies of biological reactions) can be linked to ROS production and indicate which radical species forms. The influences of kinetic aspects, interactions with biomolecules, solution chemistry (e.g., Cl- and pH), and NP characteristics (e.g., size and surface defects) on ROS mechanisms and formation are discussed. Categorization via four tiers is suggested as a way forward to group metal and metal oxide NPs based on the ROS reaction pathways that they may undergo, an approach that does not include kinetics or environmental variations. The criteria for the four tiers are based on the ability of the metallic NPs to induce Fenton(-like) and Haber-Weiss reactions, corrode, and interact with biomolecules and their surface catalytic properties. The importance of considering kinetic data to improve the proposed categorization is highlighted.

Humans are exposed daily to metallic nanoparticles (Me NPs) from multiple sources which can have both natural and anthropogenic origins. Such exposures take place via different routes including inhalation and skin contact and may result in adverse health effects. The objectives of this thesis were to investigate surface interactions taking place on metallic nanoparticles upon simplified inhalation and how such interactions influence their toxic potency. 

Except for the particle and surface characteristics, the interface to the cell environment, the extent, speciation of the released metal fraction, and the ability of the particles to form reactive oxygen species (ROS) were investigated as these parameters are known to largely govern the cell toxicity. ROS naturally form in the cells as an essential part of our immune system, but can in excess, result in cell membrane damage and harmful effects. This has been studied using a multianalytical and interdisciplinary approach combining surface and material chemistry with toxicological investigations with the main focus on ROS formation. The study has for example elucidated which reactive oxygen species that are formed due to the presence of metallic NPs, and the underlying mechanisms. 

The reliability of using different ROS assays, and the possible artifacts induced by metallic NPs, have been investigated. Methods to assess ROS which previously have not been used in the field of nanotoxicology were introduced showing that copper NPs via corrosion reactions produce ROS and Haber Weiss or Fenton-reactions can decompose hydrogen peroxide into the reactive hydroxyl radical. A way to group NPs based on their surface reactions forming ROS was proposed. Other particle- and surface characteristics of metallic NPs of importance for the toxic potency and ability to induce oxidative stress, e.g. adsorption of biomolecules, particle agglomeration, surface composition, and release of metal ions including their speciation.

Människor utsätts dagligen för metalliska nanopartiklar från olika källor vilka kan ha både naturligt och antropogent ursprung. Sådan exponering sker via olika exponeringsvägar, till exempel genom inandning och hudkontakt, vilket kan leda till negativa hälsoeffekter. Syftet med denna avhandling var att undersöka vad som sker på ytan av metalliska nanopartiklar vid simulerad inandning och hur detta påverkar deras potentiellt toxiska egenskaper.

Partikelytans egenskaper undersöktes då denna yta är i direkt kontakt med den omgivande miljön. Egenskaperna av intresse var främst graden av metallfrisättning, dess kemiska form, samt partiklarnas förmåga att bilda syreradikaler och väteperoxid (ROS). ROS bildas naturligt i cellerna som en viktig del av vårt immunsystem, men kan i överskott resultera i negativa effekter. Studier genomfördes genom att tillämpa ett multianalytiskt och tvärvetenskapligt tillvägagångssätt där yt- och materialkemiska studier kombinerades med toxikologiska undersökningar, med huvudfokus på ROS-bildning. Studierna har bland annat belyst vilka typer av ROS som kan bildas på grund av metalliska NP samt de underliggande mekanismerna. 

Tillförlitligheten av att använda olika tekniker för ROS-analyser och deras möjliga artefakter inducerade av metalliska NP undersöktes. Metoder för att bedöma ROS som tidigare inte har använts inom nanotoxikologi introducerades vilka möjliggör detektion av specifika syreradikaler. Resultat för Cu NP visade på att både korrosionsreaktioner producerar ROS samt att Haber Weiss- eller Fenton(lika) reaktioner kan sönderdela väteperoxid och bilda den reaktiva hydroxylradikalen. Ett förslag för gruppering av metalliska NP baserat på deras ROS mekanismer föreslås. Studier av andra partikel- och ytegenskaper hos metalliska NP av betydelse för deras möjliga toxicitet omfattar även adsorption av ligander och biomolekyler, grad av partikelagglomeration, yt-sammansättning, metallfrisättning och deras speciering i lösning.

The fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) together with the enzyme horseradish peroxidase (HRP) is widely used in nanotoxicology to study acellular reactive oxygen species (ROS) production from nanoparticles (NPs). This study examined whether HRP adsorbs onto NPs of Mn, Ni, and Cu and if this surface process influences the extent of metal release and hence the ROS production measurements using the DCFH assay in phosphate buffered saline (PBS), saline, or Dulbecco’s modified Eagle’s medium (DMEM). Adsorption of HRP was evident onto all NPs and conditions, except for Mn NPs in PBS. The presence of HRP resulted in an increased release of copper from the Cu NPs in PBS and reduced levels of nickel from the Ni NPs in saline. Both metal ions in solution and the adsorption of HRP onto the NPs can change the activity of HRP and thus influence the ROS results. The effect of HRP on the NP reactivity was shown to be solution chemistry dependent. Most notable was the evident affinity/adsorption of phosphate toward the metal NPs, followed by a reduced adsorption of HRP, the concomitant reduction in released manganese from the Mn NPs, and increased levels of released metals from the Cu NPs in PBS. Minor effects were observed for the Ni NPs. The solution pH should be monitored since the release of metals can change the solution pH and the activity of HRP is known to be pH-dependent. It is furthermore essential that solution pH adjustments are made following the addition of NaOH during diacetyl removal of DCFH-DA. Even though not observed for the given exposure conditions of this study, released metal ions could possibly induce agglomeration or partial denaturation of HRP, which in turn could result in steric hindrance for H2O2 to reach the active site of HRP. This study further emphasizes the influence of HRP on the background kinetics, its solution dependence, and effects on measured ROS signals. Different ways of correcting for the background are highlighted, as this can result in different interpretations of generated results. The results show that adsorption of HRP onto the metal NPs influenced the extent of metal release and may, depending on the investigated system, result in either under- or overestimated ROS signals if used together with the DCFH assay. HRP should hence be used with caution when measuring ROS in the presence of reactive metallic NPs.

The increased use of nanoparticles (NPs) requires efficient testing of their potential toxic effects. A promising approach is to use reporter cell lines to quickly assess the activation of cellular stress response pathways. This study aimed to use the ToxTracker reporter cell lines to investigate (geno)toxicity of various metal-or metal oxide NPs and draw general conclusions on NP-induced effects, in combination with our previous findings. The NPs tested in this study (n = 18) also included quantum dots (QDs) in different sizes. The results showed a large variation in cytotoxicity of the NPs tested. Furthermore, whereas many induced oxidative stress only few activated reporters related to DNA damage. NPs of manganese (Mn and Mn3O4) induced the most remarkable ToxTracker response with activation of reporters for oxidative stress, DNA damage, protein unfolding and p53-related stress. The QDs (CdTe) were highly toxic showing clearly size-dependent effects and calculations suggest surface area as the most relevant dose metric. Of all NPs investigated in this and previous studies the following induce the DNA damage reporter; CuO, Co, CoO, CdTe QDs, Mn, Mn3O4, V2O5, and welding NPs. We suggest that these NPs are of particular concern when considering genotoxicity induced by metal-and metal oxide NPs. 

In this work we have experimentally studied the impact of bentonite clay on the process of radiation-induced copper corrosion in anoxic water. The motivation for this is to further develop our understanding of radiation-driven processes occurring in deep geological repositories for spent nuclear fuel where copper canisters containing the spent nuclear fuel will be embedded in compacted bentonite. Experiments on radiation-induced corrosion in the presence and absence of bentonite were performed along with experiments elucidating the impact irradiation on the Cu2+ adsorption capacity of bentonite. The experiments presented in this work show that the presence of bentonite clay has no or very little effect on the magnitude of radiation-induced corrosion of copper in anoxic aqueous systems. The absence of a protective effect similar to that observed for radiation-induced dissolution of UO2 is attributed to differences in the corrosion mechanism. This provides further support for the previously proposed mechanism where the hydroxyl radical is the key radiolytic oxidant responsible for the corrosion of copper. The radiation effect on the bentonite sorption capacity of Cu2+ (reduced capacity) is in line with what has previously been reported for other cations. The reduced cation sorption capacity is partly attributed to a loss of Al-OH sites upon irradiation.