Development and Application of In Vitro 3D Liver Models for more Physiologically Relevant Hazard Assessment of Engineered Nanomaterials

Due to the expanding use of nanotechnology in consumer applications, human and environmental exposure to engineered nanomaterials (ENM) is inevitable. Hepatic toxicology is important when considering ENM exposure, as the liver is the major site of ENM secondary deposition and accumulation post exposure, as well as being vital in metabolic homeostasis and detoxification. The vast range of ENMs available deems it untenable to rely on in vivo based methods to elucidate the immediate and lasting effects of ENM exposure. Therefore, this research project aimed to develop an advanced 3D in vitro liver model with enhanced physiological relevance to better understand the human health hazards, specifically genotoxicity, associated with ENM exposure. The in vitro model developed was a HepG2 3D liver spheroid model with 14-day viability and liver-like functionality, as well as proliferating capabilities required to support the evaluation of fixed DNA damage endpoints. Utilising this model, the next objective was to evaluate several toxicological endpoints (e.g. liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) for a variety of ENMs (TiO2, ZnO, Ag, BaSO4 and CeO2) under different exposure regimes designed to better mimic human exposure routes. To achieve this, the ENM were 1) pre-treated in a series of biological simulant fluids to mimic inhalation and ingestion exposure routes, and 2) applied to the 3D liver model for both short- (24hr) and prolonged (120hr) single-bolus, and repeated-fractionated daily ENM exposure regimes, prior to hazard characterisation. The effects of material biotransformation upon reactivity, cytotoxicity, (pro-)inflammatory and genotoxic potential of Ag and TiO2 was demonstrated, and illustrated that the necessity of ENM pre-treatment prior to in vitro hazard assessment should be reserved for ENM that exhibit high degrees of physico-chemical transformation and reactivity (i.e. a tiered testing strategy). When comparing dosing durations, no cytotoxicity or significant reduction in liver-like functionality was observed across either acute, prolonged or repeated exposure regimes. Acute exposure to all ENMs induced a significant increase (p≤0.05) in genotoxicity, albeit not dose-dependently. ZnO, which rapidly dissolves into ions, was the only material to exhibit genotoxicity at both an acute and prolonged exposure. For the materials selected in this study, there was no significant difference between prolonged, bolus or repeated exposure regimes, indicating that the added complexity of fractionated dosing was not necessary. In conclusion, 3D in vitro hepatic spheroid models have the capacity to be utilised for evaluating more realistic ENM exposures, thereby providing a future approach to better support in vitro ENM hazard assessment in a routine and easily accessible manner