Department Environmental Toxicology

Interactions of nanoparticles with fish cells

green: nanoparticles
blue: cell nucleus
purple: cytosol/sell membrane

Do small things lead to big problems? Mechanisms of uptake and toxicity of metal nanoparticles in intestinal cells (NanoTraffic). Dr. Matteo Minghetti. PEOPLE MARIE CURIE ACTIONS Intra-European Fellowships.
Using models of human and fish intestinal and hepatic cells this project aims at investigating subtoxic intracellular effects of metal (Ag- and TiO2-) NPs. To mimic more closely the in vivo scenario and allow cell polarization, intestinal cells are grown on transwells. Moreover, to evaluate the effect of metal-NPs following the intestinal uptake and metabolism, hepatic cells are co-cultured in the sub located well. This system allows the measurement of NP transport across polarized intestinal cells and at the same time the measurement of cellular and molecular effects. Particular emphasis is placed on lysosome function and disturbance of the homeostasis of essential elements such as iron, zinc and copper. Tight regulation of the homeostasis of essential elements is essential for the life of all living organisms. Several metal specific transporter proteins are involved to allow this process. In this study the function of a copper ATPase, that are known to transport silver as well as copper, is investigated. The impact of how metal and nano-metals interact with other metals and metalloids is poorly understood at the molecular level. My research is providing new evidence that silver in its ionic and nano-form is affecting the homeostasis of some essential metals.

The EU FP7 Project NanoValid (contract: 263147, www.nanovalid.eu) aims to develop new reference methods and certified reference materials for hazard identification, risk assessment and life cycle assessment (LCA) of engineered nanomaterials (ENMs). Our project is focused on the ecotoxicological effects of ENMs especially considering the aquatic environment. Cell lines from rainbow trout (Oncorhynchus mykiss) will be used to assess the ecotoxicological effects of ENMs to fish. Currently, a novel two-compartment intestinal barrier model is developed using the rainbow trout intestinal cell line RTgutGC  to investigate the toxicity and translocation of ENMs in fish intestinal epithelium. Initial results show that nanoparticles can cause toxicity to the cells and can also be translocated across the intestinal barrier –  either as intact particles or as dissolved species released from the particles. Taken together, these studies should yield valuable information of the effects of ENMs to aquatic organisms especially on the molecular and cellular level (Dr. M. Geppert).

In the MeNanoqa project (SNSF, NRP 64) aims at examining the toxicity mechanisms and uptake of AgNP on rainbow trout fish cell line (RTgill-W1). Main results show that AgNP toxicity depends on the composition of exposure media. AgNP concentration-response curves as a function of dissolved silver ions, and the limited prevention of toxicity by silver ligands, indicate that AgNP elicit a particle-specific effect on the cells. Electronic microscopy and ICP-MS results indicate that AgNP can be internalized in RTgill-W1 cells where they accumulate in endocytosis related cell organelles. Isolation and analysis of the accumulated AgNP show particles to be coated with various proteins that were identified by mass spectrometry. Analysis of the biochemical function of these proteins allow to link observed cytotoxic effects of the AgNP to initial mechanisms of toxicity (PhD thesis Y. Yue).

Contact

Prof. Dr. Kristin Schirmer Head of department Tel. +41 58 765 5266 Send Mail

Publications

An interlaboratory comparison of nanosilver characterisation and hazard identification: harmonising techniques for high quality data

Within the FP7 EU project NanoValid a consortium of six partners jointly investigated the hazard of silver nanoparticles (AgNPs) paying special attention to methodical aspects that are important for providing high-quality ecotoxicity data. Laboratories were supplied with the same original stock dispersion of AgNPs. All partners applied a harmonised procedure for storage and preparation of toxicity test suspensions. Altogether ten different toxicity assays with a range of environmentally relevant test species from different trophic levels were conducted in parallel to AgNP characterisation in the respective test media. The paper presents a comprehensive dataset of toxicity values and AgNP characteristics like hydrodynamic sizes of AgNP agglomerates and the share (%) of Ag+-species (the concentration of Ag+-species in relation to the total measured concentration of Ag). The studied AgNP preparation (20.4 ± 6.8 nm primary size, mean total Ag concentration 41.14 mg/L, 46–68% of soluble Ag+-species in stock, 123.8 ± 12.2 nm mean z-average value in dH2O) showed extreme toxicity to crustaceans Daphnia magna, algae Pseudokirchneriella subcapitata and zebrafish Danio rerio embryos (EC50 < 0.01 mg total Ag/L), was very toxic in the in vitro assay with rainbow trout Oncorhynchus mykiss gut cells (EC50: 0.01–1 mg total Ag/L); toxic to bacteria Vibrio fischeri, protozoa Tetrahymena thermophila (EC50: 1–10 mg total Ag/L) and harmful to marine crustaceans Artemia franciscana (EC50: 10–100 mg total Ag/L). Along with AgNPs, also the toxicity of AgNO3 was analyzed. The toxicity data revealed the same hazard ranking for AgNPs and AgNO3 (i.e. the EC50 values were in the same order of magnitude) proving the importance of soluble Ag+-species analysis for predicting the hazard of AgNPs. The study clearly points to the need for harmonised procedures for the characterisation of NMs. Harmonised procedures should consider: (i) measuring the AgNP properties like hydrodynamic size and metal ions species in each toxicity test medium at a range of concentrations, and (ii) including soluble metal salt control both in toxicity testing as well as in Ag+-species measurements. The present study is among the first nanomaterial interlaboratory comparison studies with the aim to improve the hazard identification testing protocols.
Jemec, A.; Kahru, A.; Potthoff, A.; Drobne, D.; Heinlaan, M.; Böhme, S.; Geppert, M.; Novak, S.; Schirmer, K.; Rekulapally, R.; Singh, S.; Aruoja, V.; Sihtmäe, M.; Juganson, K.; Käkinen, A.; Kühnel, D. (2016) An interlaboratory comparison of nanosilver characterisation and hazard identification: harmonising techniques for high quality data, Environment International, 87, 20-32, doi:10.1016/j.envint.2015.10.014, Institutional Repository

Critical influence of chloride ions on silver ion-mediated acute toxicity of silver nanoparticles to zebrafish embryos

The toxicity of silver nanoparticles (AgNP) to aquatic organisms, including zebrafish (Danio rerio), has been demonstrated, but differing opinions exist on the contribution of the physical properties of the particles themselves and the free dissolved silver ions (Ag+) to the observed effects. High concentrations of chloride ions (Cl−) in the routinely used exposure media can cause precipitation of Ag+ as AgCl, as well as complexation of silver in diverse soluble chlorocomplexes, thus masking the contribution of dissolved silver to AgNP toxicity. In the present study, we formulated a zebrafish exposure medium with a low chloride content and exposed zebrafish embryos to AgNO3 or carbonate-coated AgNP. The severity of toxicity caused by both silver forms depended on the time of exposure start, with younger embryos being most sensitive. Toxicity caused by both AgNO3 and AgNP was of the same order of magnitude when compared based on the total dissolved silver concentration and could be prevented by addition of the Ag+ chelator cysteine. Further, we have analyzed the data from several previous studies to evaluate the influence of interactions between Ag+ and Cl− on silver toxicity to zebrafish embryos. Our analysis demonstrates that the acute toxicity of AgNP to zebrafish embryos is largely mediated by Ag+. The influence of particle size and coating can at least partially be explained by the differences in Ag+ dissolution. High Cl− levels in the exposure medium indeed have a pivotal influence on the resulting toxicity of AgNP, appearing to significantly attenuate toxicity in several studies. This consideration should influence the choice of exposure medium to be used when evaluating and comparing AgNP toxicity.
Groh, K. J.; Dalkvist, T.; Piccapietra, F.; Behra, R.; Suter, M. J. -F.; Schirmer, K. (2015) Critical influence of chloride ions on silver ion-mediated acute toxicity of silver nanoparticles to zebrafish embryos, Nanotoxicology, 9(1), 81-91, doi:10.3109/17435390.2014.893379, Institutional Repository

Toxicity of silver nanoparticles to a fish gill cell line: role of medium composition

In aqueous solutions, silver nanoparticle (AgNP) behavior is affected by a variety of factors which lead to altered AgNP size and toxicity. Our research aims to explore the effect of media composition on citrate-coated AgNP (cit-AgNP) behavior and toxicity to the cell line from rainbow trout (Oncorhynchus mykiss) gill, RTgill-W1. Three different exposure media (L15/ex, L15/ex w/o Cl and d-L15/ex) were used. These were characterized by varying ionic strength and chloride content, both of which had a dominant effect on the behaviour of cit-AgNP. Comparing the behaviour and toxicity of cit-AgNP in the different media, stronger agglomeration of cit-AgNP correlated with higher toxicity. Deposition of cit-AgNP on cells might explain the higher toxicity of agglomerated cit-AgNP compared to that of suspended cit-AgNP. The cit-AgNP concentration-response curves as a function of dissolved silver ions, and the limited prevention of toxicity by silver ligands, indicated that cit-AgNP elicited a particle-specific effect on the cells. Furthermore, the lysosomal membrane integrity was significantly more sensitive to cit-AgNP exposure than cellular metabolic activity or cell membrane integrity and showed the weakest protection by silver ligands. This revealed that cit-AgNP toxicity seems to particularly act on RTgill-W1 cell lysosomes. The newly developed low ionic strength medium, d-L15/ex, which can stabilize cit-AgNP and better mimic the freshwater environment, offers an excellent exposure solution to study cellular and molecular effects of NP to gill cells.
Yue, Y.; Behra, R.; Sigg, L.; Freire, P. F.; Pillai, S.; Schirmer, K. (2015) Toxicity of silver nanoparticles to a fish gill cell line: role of medium composition, Nanotoxicology, 9(1), 54-63, doi:10.3109/17435390.2014.889236, Institutional Repository

Mechanisms of nanotoxicity

Understanding mechanisms governing the interaction of biota and nanomaterials is key to proper risk assessment and safe nanomaterial design. This applies to both the development of nanomaterials that are intended to interact with biological entities, such as for targeted drug delivery or for antibacterial activity, as well as to the identification and evaluation of unwanted effects, such as the destruction of healthy cells in human tissue or the deterioration of microbial populations in biological water treatment. Thus, knowledge of mechanisms of biota nanomaterial interaction provides a basis to derive a causally linked chain of arguments to evaluate whether a particular nanomaterial is likely hazardous or benign. This knowledge must then be placed in the context of exposure and bioavailability (Chapter 5) to assess the risk of an ensuing toxicity. Here, the term "toxic" implies a detrimental impact (or adverse outcome) that cannot be repaired and leads to effects that threaten vital functions of organisms or tissues either immediately or over a prolonged time span. Such a toxic outcome ensues when cellular barriers and stress responses are overwhelmed. Figure 6.1 illustrates the arrangement of environment organism and organism internal barriers which, if overcome, lead to true internalization and distribution of nanomaterials in the organism. Any of the cells forming barriers, or cells comprising tissues or organs, can elicit stress responses to maintain cellular homeostasis that maybe disrupted by a nanomaterial, leading to toxicity. [...]
Schirmer, K. (2014) Mechanisms of nanotoxicity, In: Lead, J. R.; Valsami-Jones, E. (Eds.), Nanoscience and the environment, 195-221, doi:10.1016/B978-0-08-099408-6.00006-2, Institutional Repository

Ecotoxicological aspects of nanomaterials in the aquatic environment

Given the growing variety of NP, along with the diversity of aquatic species and environments, a key to promote sound risk assessment in nanoecotoxicology is to understand the mechanisms that govern the fate of NP in aquatic environments and their behavior at the NP-biota interface. The aim of this chapter is to provide an overview of those mechanisms known or anticipated to date. We focus on metalbased NP (MeNP) because, for the areas addressed by us, research is more advanced for these compared to other types of NP. However, the mechanistic view provided here can in principle be transferred to other types of NP as well.
Schirmer, K.; Behra, R.; Sigg, L.; Suter, M. J. -F. (2013) Ecotoxicological aspects of nanomaterials in the aquatic environment, In: Luther, W.; Zweck, A. (Eds.), Safety aspects of engineered nanomaterials, 135-156, doi:10.1201/b15261-6, Institutional Repository