Effects of silver nanoparticles and ions and interactions with first line of defense

Summary Silver nanoparticles (Ag NPs) are among the most promising groups of NPs (particles with all dimensions below 100 nm) for application in numerous consumer products due to their broad spectrum antimicrobial activities. Examples are incorporation in textiles and plastics, personal care products, water filters, food supplements etc. The extensive application and use together with the not yet fully understood properties of Ag NPs as well as the toxicity of Ag itself has raised concerns on potential impact of Ag NPs on human and environmental health. The research conducted within this thesis aimed at the evaluation of potential hazards of Ag NPs and identification of some key factors that determine the toxicity of Ag NPs. A tiered approach was employed using a battery of standard bioassays with model aquatic organisms, followed by the determination of sub-lethal concentrations for mechanistic endpoints, the identification of target tissues and organisms for Ag NP exposure and uptake and the integration of a proteomic tool to identify subtle changes. One of the main uptake routes for Ag NPs is through ingestion making the gastrointestinal epithelium one of the first ports of potential NP uptake and cell-particle interactions. An in vitro co-culture model incorporating a mucus layer mimicking the gastrointestinal epithelium was established for a more realistic evaluation of Ag NP potential toxicity than using intestinal epithelial cells alone. Indeed, the absence of mucus resulted in an overestimation of Ag NP toxicity. To be able to elucidate subtle changes in cellular functions and identification of particle specific effects and NP modes of action, a proteomic approach was employed. Differences and commonalities were observed between the cellular responses induced Ag NPs of different sizes and AgNO3 as a source of free Ag ions. As the Ag NPs are expected to reach the aquatic environment, a combination of adapted standard ecotoxicity assays with organisms of different trophic levels were used to evaluate the toxic effects Ag NPs. Synthetically produced Ag NPs of different sizes (Ag 20 and 200 nm) as well as Ag NPs synthesized by a biological method (using plant leaf extracts of Ocinum sanctum and Azadirachta indica, Ag 23 and 27 nm, respectively) were used as model particles in order to elucidate the relation between size, synthesis method, NP surface properties, ion dissolution and toxicity. Based on earlier indications of interference of another type of NPs with the multi xenobiotic resistance mechanism (MXR), a first line of defense against xenobiotics, the effects of Ag NPs on the MXR mechanism were studied as well. The MXR mechanism is present in all animals, including humans and aquatic organisms. MXR can be compromised by chemical agents that are structurally and chemically unrelated, and interference with MXR could be the basis for enhanced toxicity by contaminant mixtures. A fast in vitro cellular efflux pump inhibition assay (CEPIA) was established evaluating first the effects of contaminants commonly found in the environment. Next, an in vivo CEPIA assay was established using the juvenile D. magna model aquatic organism and the potential of the MXR modulation by Ag NPs and ionic Ag was quantified in vitro and in vivo. This integrated approach revealed that the size and the synthesis method are the factors affecting most the uptake and toxicity in both cells in vitro as well as in vivo in freshwater crustaceans (daphnids) and dissolution in the different media with the biologically synthesized Ag NPs being more potent compared to the conventional Ag NPs. The gastrointestinal tract is expected to be a target site for Ag NPs exposure and a co-culture of Caco-2-TC7 and HT29-MTX cells optimized and employed in the current study represents a more realistic model compared to Caco-2 monocultures. The mucus layer provides an additional protective barrier and its absence can lead to overestimation of effects in in vitro studies. Ag was detected both in the cells in co-culture, in the gut of daphnids’ as well as specific areas, seemingly developing oocytes, indicating a potential translocation of Ag NPs that could have consequences for fecundity. MXR efflux transporters were found to be modulated by Ag at low concentrations (0.18 µg/L), that are slightly lower compared to the predicted environmental concentrations. The extent to which the Ag ions contribute to the effects of Ag NPs depends on the size and surface properties of the Ag NPs. For the conventional, uncoated Ag NPs, the Ag release is minimal and the size is the determining factor while for biologically synthesized particles the biomolecules present due to the synthesis method and Ag release affect most the uptake and effects. The simultaneous presence of Ag ions and NPs releasing ions can lead to an exacerbation of the effects. The proteomic approach was successfully applied and it proved to be a useful technique in discerning subtle cellular changes in response to Ag NP exposure that would otherwise be unnoticed. Ag NPs 20 nm regulated different sets of proteins with a distinct pattern of cellular responses compared to Ag 200 nm and AgNO3, suggesting a different mode of action with effects being particle- and size-dependent. These results obtained during this thesis are promising for future toxicity testing of new materials using invertebrate organisms and more realistic in vitro models leading to more meaningful results and more accurate assessment of Ag NP hazards.