Edinburgh Napier University

  The liver is recognised as a potential target site for nanoparticle (NP) toxicity, as NPs have been observed to accumulate within this organ subsequent to exposure via injection, inhalation or instillation. The liver’s unique structure has to be taken into consideration when evaluating NP toxicity, as a variety of cell types of distinct morphology and function are evident, and potentially affected by NP exposure. Of particular interest are hepatocytes, due to their abundance and importance to the maintenance of normal liver function, and macrophages due to their role in host defence.

The uptake and intracellular fate of fluorescent polystyrene particles (20nm and 200nm) by hepatocytes was evaluated (with exposure times of up to 60 minutes). Within the studies conducted comparisons of the response of primary rat hepatocytes, with C3A and HepG2 hepatocyte cell lines to NP exposure were made in order to investigate whether cell lines are a relevant model of hepatocyte behaviour. It was found that the uptake of particles by the primary hepatocytes, and both cell lines was size and time dependent. Specifically, it appeared that the internalisation of 200nm particles was limited, occurred at later time points (60 minutes), with the majority of particles evident at the cell surface. Polystyrene NPs (20nm) were internalised by cells after a 10 minute exposure time, after which NPs compartmentalised either within and/or between adjacent cells. The nature of the NP ‘compartments’, and therefore fate of internalised NPs was then investigated to determine if the compartments developed as a consequence of the mechanism of uptake, or due to the attempted elimination of NPs from cells. It was found that NPs were not contained within early endosomes or lysosomes. However it was apparent that polystyrene NPs were eliminated to a limited extent within the bile canaliculi of all cell types, and may accumulate within the mitochondria of cell lines after a 60 minute exposure, which warrants further investigation.

The impact of the PARTICLE_RISK particle panel [consisting of ultrafine carbon black (ufCB), CB, carbon nanotubes (CNTs), C60 (carbon fullerene) QD621 (positively charged quantum dots) and QD620 (negatively charged quantum dots)] on hepatocyte function was then determined. It was consistently observed that QD621 and QD620 were able to elicit the greatest extent of toxicity, evidenced within their ability to deplete cellular GSH, induce cytotoxicity, initiate a pro-inflammatory response (indicated by an increase in IL-8 production) and decrease bile secretion, in the hepatocyte couplet, in vitro model. It was observed that the pattern of response was similar within the cell lines and primary cells.

Differentiated monocytic THP-1 cells (to represent the resident liver macrophages, Kupffer cells) were exposed to the PARTICLE_RISK particle panel to obtain conditioned medium (CM) that was exposed to hepatocytes, in order to gain insight into the ability of macrophages to influence NP mediated toxicity to hepatocytes. Firstly, the response of macrophages to particle exposure was considered and it was apparent that the toxicity that was observed within hepatocytes was paralleled within the response of differentiated monocytic cells (THP-1). Accordingly, QD621 were again proven to have the greatest toxic potential, with QD620 able to induce toxicity to a more limited extent. The exposure of hepatocytes to CM potentiated the toxicity observed when cells were exposed to particles alone, so that the pattern of response was comparable, but the extent of toxicity greater, and evident at earlier time points. It was apparent that QDs were able to induce an inflammatory response (characterised by TNFα and IL-8 production) within the liver that was primarily mediated by macrophages.

When considering the results from all experiments it is evident that some of the particles contained within the PARTICLE_RISK panel were more capable of eliciting toxicity within the liver, and that their toxicity can be ranked in the following order: QD621>QD620>CNT=ufCB=C60>CB.