Cytotoxicity, Accumulation and Translocation of Silver and Silver Sulfide Nanoparticles in contact with Rainbow Trout Intestinal Cells

Silver nanoparticles (Ag NPs) are widely used in consumer products especially because of their antimicrobial properties. However, this wide usage of Ag NPs is accompanied by their release into the environment where they will be rapidly transformed to other silver species - especially silver sulfide (Ag2S). In the present study, we synthesized Ag NPs and sulfidized them to obtain a core-shell system Ag@Ag2S NPs. Both types of particles form stable dispersions with hydrodynamic diameters of less than 100 nm when diluted in water, but tend to form micrometer-sized agglomerates in biological exposure media. Application of Ag and Ag@Ag2S NPs to rainbow trout intestinal cells (RTgutGC) resulted in a concentration-dependent cytotoxicity for both types of particles, as assessed by a three-endpoint assay for metabolic activity, membrane integrity and lysosomal integrity. The Ag NPs were shown to be slightly more toxic than the Ag@Ag2S NPs. Adding Ag or Ag@Ag2S NPs to RTgutGC cells, grown on a permeable membrane to mimic the intestinal barrier, revealed considerable accumulation of silver for both types of particles. Indeed, the cells significantly attenuated the NP translocation, allowing only a fraction of the metal to translocate across the intestinal epithelium. These findings support the notion that the intestine constitutes an important sink for Ag NPs and that, despite the reduced cytotoxicity of a sulfidized NP form, the particles can enter fish where they may constitute a long-term source for silver ion release and cytotoxicity.

Effect of media composition on bioavailability and toxicity of silver and silver nanoparticles in fish intestinal cells (RTgutGC)

Kristin Schirmer

To understand conditions affecting bioavailability and toxicity of citrate-coated silver nanoparticles (cit-AgNP) and dissolved silver at the luminal enterocyte interface, we exposed rainbow trout (Oncorhynchus mykiss) gut cells (RTgutGC) in media of contrasting composition: two amino acid-containing media, one of which was supplemented with proteins, as can be expected during digestion; and two protein and amino acid-free media contrasting low and high chloride content, as can be expected in the lumen of fish adapting to freshwater or seawater, respectively. Dose-response curves were generated measuring cell metabolic activity, membrane and lysosome integrity over a period of 72hours. Then, nontoxic doses were applied and total silver accumulation, metallothionein and glutathione reductase mRNA levels were determined. The presence of proteins stabilized cit-AgNP keeping them in suspension. Conversely, in protein-free media, cit-AgNP agglomerated and settled, resulting in higher cellular accumulation of silver and toxicity. Chloride concentrations in exposure media modulated the toxicity of AgNO3 but not of cit-AgNP. Moreover, while amino acid-containing media are protective against AgNO3, likely due to the formation of thiolate complexes, they are only partially protective against cit-AgNP. Viability assays indicated that lysosomes are targets of cit-AgNP, supporting the hypothesis that cit-AgNP exert toxicity intracellularly. Metallothionein, a sensor of metal bioavailability, was induced by cit-AgNP in high chloride medium but not in low chloride medium, indicating that chloride might have a role in mobilizing silver from intercellular vesicles. Overall, this study shows that AgNP bioavailability and toxicity in the intestine is linked to its luminal content.

Taylor & Francis Ltd2016

A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC

Carolin Drieschner, Kristin Schirmer, Hannah Inka Schug

The intestine of fish is a multifunctional organ: lined by only a single layer of specialized epithelial cells, it has various physiological roles including nutrient absorption and ion regulation. It moreover comprises an important barrier for environmental toxicants, including metals. Thus far, knowledge of the fish intestine is limited largely to in vivo or ex vivo investigations. Recently, however, the first fish intestinal cell line, RTgutGC, was established, originating from a rainbow trout (Oncorhynchus mykiss). In order to exploit the opportunities arising from RTgutGC cells for exploring fish intestinal physiology and toxicology, we present here the establishment of cells on commercially available permeable membrane supports and evaluate its suitability as a model of polarized intestinal epithelia. Within 3 weeks of culture, RTgutGC cells show epithelial features by forming tight junctions and desmosomes between adjacent cells. Cells develop a transepithelial electrical resistance comparable to in vivo measured values, reflecting the leaky nature of the fish intestine. Immunocytochemistry reveals evidence of polarization, such as basolateral localization of Na+/K+-ATPase (NKA) and apical localization of the tight junction protein ZO-1. NKA mRNA abundance was induced as physiological response toward a saltwater buffer, mimicking the migration of rainbow trout from fresh to seawater. Permeation of fluorescent molecules proved the barrier function of the cells, with permeation coefficients being comparable to those reported in fish. Finally, we demonstrate that cells on permeable supports are more resistant to the toxicity elicited by silver ions than cells grown the conventional way, likely due to improved cellular silver excretion.

Springer2017

In vitro acute toxicity assessment of tire tread particles using rainbow trout cell lines of gill and intestine

Florian Frédéric Vincent Breider, Benoît Jean Dominique Ferrari, Thibault Béranger Masset, Kristin Schirmer

Tire and road wear particles (TRWP) are generated by the abrasion of tires while driving, and recent questions were raised in regards to their potential contribution to microplastics released into the aquatic environment and their potential toxicological impacts. Our study aimed to determine the toxicity of TRWP and associated chemicals to fish using two rainbow trout (Oncorhynchus mykiss) cell lines representing the gill (RTgill-W1) and the intestinal (RTgut-GC) epithelium. Acute exposure to cryogenically milled tire tread (CMTT) particles and CMTT digestate following in vitro digestion was investigated. To obtain the effective concentrations (EC50) causing a 50% cell viability loss, tests were performed in accordance with OECD TG249: cell viability was assessed after 24 hours exposure using a multiple-endpoint assay indicative of cell metabolic activity, membrane integrity and lysosome integrity. Finally, chemical composition of the exposure medium was analyzed to assess which chemicals could be responsible for the observed acute effects. Our results show that metabolic activity was the most sensitive indicator of CMTT and CMTT digestate toxicity. EC50 values after direct exposure to RTgill-W1 and RTgutGC cells were estimated at 2.01 g/L and 3.80 g/L, respectively. 2-Mercaptobenzothiazole was found at the highest concentration among organic chemicals leaching out from the particles. It could be a major contributor to the loss in metabolic activity based on its toxicity to rainbow trout in vivo, although additional particle or mixture effects cannot be ruled out at this point. For digestate toxicity, EC50 was determined at 12.15 g of tire particle/L of digestive fluid. The observed toxicity could be due to high amounts of Zn, N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), 1,3-Diphenylguanidine (DPG) or 2-Mercaptobenzothiazole leaching out during in vitro digestion. Traces of 6PPD-quinone were also detected in the exposure medium, but additional cytotoxicity assays using the RTgill-W1 cell line showed no significant toxicity of this compound up to 3 mg/L. Overall, our in vitro toxicity data agree with reports of acute fish (in vivo) toxicity and show that acutely toxic concentrations of CMTT are well above concentrations measured in river water (0.3 to 4 mg/L). However, we determined the highest no-observed-effect concentration at 10 mg/L, while up to 100 mg/L were measured in road runoffs.

2021

In vitro acute toxicity assessment of tire tread particles using rainbow trout cell lines of gill and intestine

Florian Frédéric Vincent Breider, Benoît Jean Dominique Ferrari, Thibault Béranger Masset, Kristin Schirmer

2021