Assessing the transferability and reproducibility of 3D in vitro liver models from primary human multi-cellular microtissues to cell-line based HepG2 spheroids.

To reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based methods for hazard assessment. When developing new approach methodologies, it is important to standardize the protocols and demonstrate the methods can be reproduced by multiple laboratories. The aim of this study was to assess the transferability and reproducibility of two advanced in vitro liver models, the Primary Human multicellular microtissue liver model (PHH) and the 3D HepG2 Spheroid Model, for nanomaterial (NM) and chemical hazard assessment purposes. The PHH model inter-laboratory trial showed strong consistency across the testing sites. All laboratories evaluated cytokine release and cytotoxicity following exposure to titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles. No significant difference was observed in cytotoxicity or IL-8 release for the test materials. The data were reproducible with all three laboratories with control readouts within a similar range. The PHH model ZnO induced the greatest cytotoxicity response at 50.0 µg/mL and a dose-dependent increase in IL-8 release. For the 3D HepG2 spheroid model, all test sites were able to construct the model and demonstrated good concordance in IL-8 cytokine release and genotoxicity data. This trial demonstrates the successful transfer of new approach methodologies across multiple laboratories, with good reproducibility for several hazard endpoints.

Deducing the cellular mechanisms associated with the potential genotoxic impact of gold and silver engineered nanoparticles upon different lung epithelial cell lines in vitro.

Human ENP exposure is inevitable and the novel, size-dependent physicochemical properties that enable ENPs to be beneficial in innovative technologies are concomitantly causing heightened public concerns as to their potential adverse effects upon human health. This study aims to deduce the mechanisms associated with potential ENP mediated (geno)toxicity and impact upon telomere integrity, if any, of varying concentrations of both ∼16 nm (4.34 × 10-3 to 17.36 × 10-3 mg/mL) Gold (Au) and ∼14 nm (0.85 × 10-5 to 3.32 × 10-5 mg/mL) Silver (Ag) ENPs upon two commonly used lung epithelial cell lines, 16HBE14o- and A549. Following cytotoxicity analysis (via Trypan Blue and Lactate Dehydrogenase assay), two sub-lethal concentrations were selected for genotoxicity analysis using the cytokinesis-blocked micronucleus assay. Whilst both ENP types induced significant oxidative stress, Ag ENPs (1.66 × 10-5 mg/mL) did not display a significant genotoxic response in either epithelial cell lines, but Au ENPs (8.68 × 10-3 mg/mL) showed a highly significant 2.63-fold and 2.4-fold increase in micronucleus frequency in A549 and 16HBE14o- cells respectively. It is hypothesized that the DNA damage induced by acute 24-h Au ENP exposure resulted in a cell cycle stall indicated by the increased mononuclear cell fraction (>6.0-fold) and cytostasis level. Albeit insignificant, a small reduction in telomere length was observed following acute exposure to both ENPs which could indicate the potential for ENP mediated telomere attrition. Finally, from the data shown, both in vitro lung cell cultures (16HBE14o- and A549) are equally as suitable and reliable for the in vitro ENP hazard identification approach adopted in this study.

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

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.

Simulating Nanomaterial Transformation in Cascaded Biological Compartments to Enhance the Physiological Relevance of In Vitro Dosing Regimes: Optional or Required?

Would an engineered nanomaterial (ENM) still have the same identity once it reaches a secondary target tissue after a journey through several physiological compartments? Probably not. Does it matter? ENM pre-treatments may enhance the physiological relevance of in vitro testing via controlled transformation of the ENM identity. The implications of material transformation upon reactivity, cytotoxicity, inflammatory, and genotoxic potential of Ag and SiO2 ENM on advanced gastro-intestinal tract cell cultures and 3D liver spheroids are demonstrated. Pre-treatments are recommended for certain ENM only.

In Vitro Three-Dimensional Liver Models for Nanomaterial DNA Damage Assessment.

Whilst the liver possesses the ability to repair and restore sections of damaged tissue following acute injury, prolonged exposure to engineered nanomaterials (ENM) may induce repetitive injury leading to chronic liver disease. Screening ENM cytotoxicity using 3D liver models has recently been performed, but a significant challenge has been the application of such in vitro models for evaluating ENM associated genotoxicity; a vital component of regulatory human health risk assessment. This review considers the benefits, limitations, and adaptations of specific in vitro approaches to assess DNA damage in the liver, whilst identifying critical advancements required to support a multitude of biochemical endpoints, focusing on nano(geno)toxicology (e.g., secondary genotoxicity, DNA damage, and repair following prolonged or repeated exposures).

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (≤14 days). These spheroid models (≥500 µm in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the 'gold standard' micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for 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.