Understanding the impact of more realistic low-dose, prolonged engineered nanomaterial exposure on genotoxicity using 3D models of the human liver

Abstract

Background With the continued integration of engineered nanomaterials (ENMs) into everyday applications, it is important to understand their potential for inducing adverse human health effects. However, standard in vitro hazard characterisation approaches suffer limitations for evaluating ENM and so it is imperative to determine these potential hazards under more physiologically relevant and realistic exposure scenarios in target organ systems, to minimise the necessity for in vivo testing. The aim of this study was to determine if acute (24 h) and prolonged (120 h) exposures to five ENMs (TiO2, ZnO, Ag, BaSO4 and CeO2) would have a significantly different toxicological outcome (cytotoxicity, (pro-)inflammatory and genotoxic response) upon 3D human HepG2 liver spheroids. In addition, this study evaluated whether a more realistic, prolonged fractionated and repeated ENM dosing regime induces a significantly different toxicity outcome in liver spheroids as compared to a single, bolus prolonged exposure. Results Whilst it was found that the five ENMs did not impede liver functionality (e.g. albumin and urea production), induce cytotoxicity or an IL-8 (pro-)inflammatory response, all were found to cause significant genotoxicity following acute exposure. Most statistically significant genotoxic responses were not dose-dependent, with the exception of TiO2. Interestingly, the DNA damage effects observed following acute exposures, were not mirrored in the prolonged exposures, where only 0.2–5.0 µg/mL of ZnO ENMs were found to elicit significant (p ≤ 0.05) genotoxicity. When fractionated, repeated exposure regimes were performed with the test ENMs, no significant (p ≥ 0.05) difference was observed when compared to the single, bolus exposure regime. There was < 5.0% cytotoxicity observed across all exposures, and the mean difference in IL-8 cytokine release and genotoxicity between exposure regimes was 3.425 pg/mL and 0.181%, respectively. Conclusion In conclusion, whilst there was no difference between a single, bolus or fractionated, repeated ENM prolonged exposure regimes upon the toxicological output of 3D HepG2 liver spheroids, there was a difference between acute and prolonged exposures. This study highlights the importance of evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

Influence of Pre-Dispersion Media on the Batch Reactor Dissolution Behavior of Al2O3 Coated TiO2 (NM-104) and Two ZnO (NM-110 and NM-111) Nanomaterials in Biologically Relevant Test Media

“…b OECD—Dossier on Zink Oxide (2015) [ 24 ]. c Llewellyn et al (2021) [ 25 ]. d Da Silva et al (2019) [ 17 ].…”

Chronic effects of two rutile TiO2 nanomaterials in human intestinal and hepatic cell lines

“…The accumulation of TiO 2 NMs in the liver following oral exposure [ 18 ] has been shown to be associated with hepatic toxicity including induction of reactive oxygen species [ 15 , 17 , 61 ], liver inflammation [ 18 , 61 ] and liver edema [ 13 ]. While several in vitro studies reported low cytotoxicity in liver cell models following acute treatment with TiO 2 NMs [ 29 , 30 , 62 ], an increasing number of reports on the effects of repeated exposures on human hepatic cell models are available [ 28 , 33 , 35 ]. In a recent study investigating the toxicity of TiO 2 NMs in HepG2 spheroids, no cytotoxicity was observed following acute and prolonged treatment (120 h) with these NMs [ 35 ].…”

“…While several in vitro studies reported low cytotoxicity in liver cell models following acute treatment with TiO 2 NMs [ 29 , 30 , 62 ], an increasing number of reports on the effects of repeated exposures on human hepatic cell models are available [ 28 , 33 , 35 ]. In a recent study investigating the toxicity of TiO 2 NMs in HepG2 spheroids, no cytotoxicity was observed following acute and prolonged treatment (120 h) with these NMs [ 35 ]. However, another study in a 3D human liver microtissue model showed increased cytotoxicity of TiO 2 NMs in prolonged and repeated exposure scenarios up to 360 h [ 28 ].…”

“…In addition, as the liver has been reported to be a major site of accumulation of TiO 2 NMs, a number of studies have investigated the effects of these NMs on hepatocyte cell models [ 20 , 29 – 31 ]. In addition, an increasing number of nanotoxicological studies using more complex and multi-cellular in vitro models of the liver [ 32 ], more representative of the situation in vivo, have provided additional information about the toxicological effects of NMs following acute, prolonged and repeated exposure to TiO 2 NMs [ 28 , 33 – 35 ].…”

How Structured Metadata Acquisition Contributes to the Reproducibility of Nanosafety Studies: Evaluation by a Round-Robin Test

“…However, considering the number of research groups in the field of nanosafety assessment, this time- and labor-intense approach cannot be realized throughout the community. Furthermore, the development of new approach methodologies (NAMs) as non-animal alternative models to mimic more realistic exposure scenarios and physiological conditions poses additional requirements due to their complexity [ 13 , 14 ]. These could only be partially described by already established toxicological assays, which is challenging for interlaboratory comparisons.…”