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

LLEWELLYN, SAMANTHA

doi:10.23889/SUthesis.58889

E-Thesis 283 views

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

Swansea University Author: SAMANTHA LLEWELLYN

Redacted version - open access under embargo until: 30th September 2024

DOI (Published version): 10.23889/SUthesis.58889

Abstract

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 expos...

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Published:	Swansea 2021
Institution:	Swansea University
Degree level:	Doctoral
Degree name:	Ph.D
Supervisor:	Doak, Shareen
URI:	https://cronfa.swan.ac.uk/Record/cronfa58889
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first_indexed	2021-12-03T18:20:30Z
last_indexed	2023-01-11T14:39:47Z
id	cronfa58889
recordtype	RisThesis
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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. 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spelling	2022-10-05T16:22:19.3412427 v2 58889 2021-12-03 Development and Application of In Vitro 3D Liver Models for more Physiologically Relevant Hazard Assessment of Engineered Nanomaterials b88581b00fbb25d465cfe17b1714f66a SAMANTHA LLEWELLYN SAMANTHA LLEWELLYN true false 2021-12-03 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. E-Thesis Swansea 3 12 2021 2021-12-03 10.23889/SUthesis.58889 A selection of third party content is redacted or is partially redacted from this thesis due to copyright restrictions.ORCiD identifier: https://orcid.org/0000-0003-1576-5471 COLLEGE NANME COLLEGE CODE Swansea University Doak, Shareen Doctoral Ph.D 2022-10-05T16:22:19.3412427 2021-12-03T18:16:50.6253057 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine SAMANTHA LLEWELLYN 1 Under embargo Under embargo 2021-12-03T18:37:13.8865456 Output 13102714 application/pdf Redacted version - open access true 2024-09-30T00:00:00.0000000 Copyright: The author, Samantha V. Llewellyn, 2021. true eng
title	Development and Application of In Vitro 3D Liver Models for more Physiologically Relevant Hazard Assessment of Engineered Nanomaterials
spellingShingle	Development and Application of In Vitro 3D Liver Models for more Physiologically Relevant Hazard Assessment of Engineered Nanomaterials SAMANTHA LLEWELLYN
title_short	Development and Application of In Vitro 3D Liver Models for more Physiologically Relevant Hazard Assessment of Engineered Nanomaterials
title_full	Development and Application of In Vitro 3D Liver Models for more Physiologically Relevant Hazard Assessment of Engineered Nanomaterials
title_fullStr	Development and Application of In Vitro 3D Liver Models for more Physiologically Relevant Hazard Assessment of Engineered Nanomaterials
title_full_unstemmed	Development and Application of In Vitro 3D Liver Models for more Physiologically Relevant Hazard Assessment of Engineered Nanomaterials
title_sort	Development and Application of In Vitro 3D Liver Models for more Physiologically Relevant Hazard Assessment of Engineered Nanomaterials
author_id_str_mv	b88581b00fbb25d465cfe17b1714f66a
author_id_fullname_str_mv	b88581b00fbb25d465cfe17b1714f66a_***_SAMANTHA LLEWELLYN
author	SAMANTHA LLEWELLYN
author2	SAMANTHA LLEWELLYN
format	E-Thesis
publishDate	2021
institution	Swansea University
doi_str_mv	10.23889/SUthesis.58889
college_str	Faculty of Medicine, Health and Life Sciences
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hierarchy_top_id	facultyofmedicinehealthandlifesciences
hierarchy_top_title	Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id	facultyofmedicinehealthandlifesciences
hierarchy_parent_title	Faculty of Medicine, Health and Life Sciences
department_str	Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
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description	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.
published_date	2021-12-03T04:15:46Z
_version_	1763754059241619456
score	11.013686

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

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