Considerations on silver nanoparticle toxicity testing with the use of an intestinal co-culture model

Georgantzopoulou, A.1, Cuny, L.1, Balachandran, YL.2, Guignard, C.1, Audinot, J-N.1, Hoffmann, L.1, Gutleb, A.C.1

1 Centre de Recherche Public-Gabriel Lippmann

2 School of Biotechnology and Genetic Engineering, Bharathiar University, Coimbatore 641046, Tamilnadu, India

 

Our research focuses on Ag nanoparticles and their effects on organisms of different trophic levels such as bacteria, algae, crustaceans. An intestinal co-culture model has been established in order to evaluate the effects of Ag NPs as the gastrointestinal epithelium could be an important uptake route for NPs while not that many studies are dealing with this. We aim to understand the different mechanisms leading to differences in effects between different-sized and –synthesized Ag NPs and potentially identify new biomarkers through a proteomic approach.

Abstract: The aim of the study was to elucidate the observed differences in effects of similarly sized Ag NPs (23, 24 and 27 nm) synthesized in a biological way on an intestinal co-culture model (Caco-2/TC7: HT29-MTX cells).

Effects of Ag NPs and AgNO3 on metabolic activity, viability and monolayer integrity were assessed (alamar blue, MTS, LDH and transepithelial electrical resistance (TEER) measurement). Ag uptake was evaluated with NanoSIMS50 and the ion dissolution was assessed with ultrafiltration and ICP-MS.

AgNO3 and all Ag NPs induced a dose-dependent decrease in metabolic activity (alamar blue assay) of the un-differentiated cells in co-culture with Ag 23 nm being the most potent followed by Ag 24 and 27 nm. This decrease in metabolic activity was attenuated by the addition of N-acetyl cysteine (NAC) to AgNO3 and Ag 23 nm suspensions. However, this was not observed in the case of Ag 24 and 27 nm. At a concentration of 1 mg/L Ag 23 nm and Ag 24 nm similar levels of Ag were released (2.5 µg/L after 6 hours of exposure) followed by Ag 27 nm.

A dose-dependent increase in LDH release was observed for AgNO3, Ag 23 nm and Ag 24 nm. However, at higher AgNO3 and Ag 24 nm concentrations the amount of released LDH decreased with LDH not being detected at all at the two highest concentrations of AgNO3 and Ag 23 nm. Ag at high concentrations was found to interfere with the assay by LDH inactivation. In the presence of NAC no decrease in the amount of LDH was detected.

Ag 23 nm and AgNO3 led to a decrease in monolayer integrity of differentiated cells in co-culture in a dose-dependent manner which corresponds to an increase in the amount of Ag found in the basolateral compartment, suggesting an effect on the barrier function. A decrease was already observed at concentrations that had no effect in the metabolic activity assays. Therefore, that could be an important additional endpoint. According to NanoSIMS50 analysis an uptake of Ag was observed for all NPs and AgNO3 that seems to be homogenously distributed.

Our study highlights the importance of considering suitable assays for Ag NP toxicity testing and that NP size or ion dissolution alone cannot predict and explain differences in effects observed for similar sized Ag NPs.

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