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

(2021) 19:193 Llewellyn et al. J Nanobiotechnol https://doi.org/10.1186/s12951-021-00938-w Journal of Nanobiotechnology Open Access RESEARCH Understanding the impact of more realistic low‑dose, prolonged engineered nanomaterial exposure on genotoxicity using 3D models of the human liver Samantha V. Llewellyn1 , Gillian E. Conway1, Ilaria Zanoni4, Amalie Kofoed Jørgensen5, Ume‑Kulsoom Shah1, Didem Ag Seleci2,3, Johannes G. Keller2,3, Jeong Won Kim6, Wendel Wohlleben2,3, Keld Alstrup Jensen5, Anna Costa4, Gareth J. S. Jenkins1, Martin J. D. Clift1 and Shareen H. Doak1* Abstract Background: With the continued integration of engineered nanomaterials (ENMs) into everyday applications, it is impor‑ tant to understand their potential for inducing adverse human health effects. However, standard in vitro hazard characteri‑ sation 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 frac‑ tionated 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. Interest‑ ingly, 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 expo‑ sure 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 pro‑ longed 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 provid‑ ing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner. Keywords: In vitro liver models, Engineered nanomaterials, Physiologically relevant exposure, Nanotoxicology, Genotoxicity *Correspondence: 1 In Vitro Toxicology Group, Institute of Life Science, Swansea University Medical School, Swansea University, Singleton Park, Swansea SA2 8PP, UK Full list of author information is available at the end of the article © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Llewellyn et al. J Nanobiotechnol (2021) 19:193 Background Nanotechnology is considered an important Key Enabling Technology (KET), underpinning a variety of novel applications across wide ranging sectors. As a global market, nanotechnology reached $75.8 billion in 2020 and is predicted to exceed $125 billion in the next three years, with engineered nanomaterials (ENMs) defined as having the greatest share of the global nanotechnology market [1]. ENMs are manufactured materials with advanced size specific physico-chemical properties derived from an unbound, monodispersed state, or as an aggregate/agglomerate where 50% or more of the particles possess one or more external dimensions in the size range 1–100 nm [2]. This greater surface area to volume ratio enables ENMs to harbour advantageous properties that improve the functionality of a plethora of applications (e.g. cosmetics, medicine, electronics, construction and energy industries) providing great opportunities for economic growth and life improving technologies. Consequently, with increasing human and environmental exposure comes the need to understand any potential associated safety risks. Human ENM exposure occurs via four primary routes; inhalation, ingestion, injection and dermal penetration. With the exception of certain medical treatments, the prospect of injecting ENMs into the body is relatively low for the majority of individuals. While for most ENM, the likelihood of occupational inhalation exposure is predominant, such as the use of barium sulfate (BaSO4) and cerium dioxide ( CeO2) in the automotive industry; other routes of potential relevant exposure could arise from the use of consumer products, with some examples being the ingestion of food grade titanium dioxide ( TiO2) or dermal penetration of sunscreen enhancing zinc oxide (ZnO) [3–6]. Silver (Ag) ENMs, with its popular anti-microbial properties, are deemed the most readily applied ENM in consumer products included in the top three applications found in medicine, textiles and cosmetic products [7–9]. Consequently, understanding the impact of repeated ENM exposure to human health over prolonged periods of time is imperative. Once ENMs have entered the body, if they have the ability to traverse biological barriers and enter circulation, the materials can translocate to secondary sites of deposition, including the spleen, liver and kidneys [10, 11]. Of these sites, the liver is of particular toxicological importance due to its high susceptibility to ENM deposit (...truncated)