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In Vitro Lung Models to Assess the Mechanistic Genotoxicity of Characterised Few-Layer Graphene / Michael J. Burgum

DOI (Published version): 10.23889/Suthesis.51928

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Nanotechnology sits at the forefront of molecular and macromolecular medical science with tremendous applications worldwide. Paradoxically, as the number of applications for engineered nanomaterials (ENMs) increases the comparable knowledge of their toxicity may be limited. This could culminate in n...

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Published: 2019
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
URI: https://cronfa.swan.ac.uk/Record/cronfa51928
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spelling 2019-09-18T09:23:51.1002734 v2 51928 2019-09-17 In Vitro Lung Models to Assess the Mechanistic Genotoxicity of Characterised Few-Layer Graphene 2019-09-17 Nanotechnology sits at the forefront of molecular and macromolecular medical science with tremendous applications worldwide. Paradoxically, as the number of applications for engineered nanomaterials (ENMs) increases the comparable knowledge of their toxicity may be limited. This could culminate in nano-safety issues relating to occupational, environmental and human exposure regarding ENM use without a full comprehension of their toxicological impact. The aim of this thesis was firstly, to provide a comprehensive approach to characterising the physico-chemical features of, few-layer graphene (FLG) engineered with i) no functionality (Neutral-FLG), ii) amine groups (Amine-FLG) or iii) carboxyl groups (Carboxyl-FLG) with a carbon black (CB) positive control. Secondly, the toxicological impact of these ENMs was investigated utilising relevant respiratory cell lines. This was performed using; in vitro monoculture exposures, allowing the detection of primary-indirect genotoxicity, then applying these ENMs to a dual-cell co-culture lung model to permit the detection of secondary genotoxicity. By encompassing a comprehensive approach to ENM physico-chemical characterisation, a battery of complementary techniques was employed to gauge particle and agglomerate size, morphology, surface charge and surface area. Each ENM was then evaluated for their potential to promote cytotoxicity and genotoxicity, using relative population doubling and the micronucleus assay respectively in a monoculture of the human bronchial cell line, 16HBE14o-. A deeper investigation was then launched into the potential routes of oxidative and mitochondrial stress. Neutral-FLG, Amine-FLG and CB particles promoted a genotoxic (predominantly clastogenic) response, however all tested ENMs induced oxidative & mitochondrial stress with significant elevation of interleukin (IL)-8. A transformed alveolar epithelial type-I (TT1) cell line and differentiated THP-1 (dTHP-1) macrophages formed the alveolar co-culture. This advanced cell model, which could detect secondary genotoxic mechanisms, highlighted the potential for each ENM to promote genotoxicity via secondary mechanisms. This was observed with a potency ranking of CB> Amine-FLG> Neutral-FLG> Carboxyl-FLG with overall 2-fold increases in chromosomal damage (ascertained from micronuclei frequencies) at 50µg/ml following 24 hours of exposure. The potential mechanistic role of the oxidant: antioxidant balance as a contributor to the observed secondary genotoxicity was investigated by a 2-hour pre-incubation with 1.5mM N-acetyl-cysteine (NAC). This was found to reduce chromosomal damage to non-significant (p<0.05) levels suggesting oxidative stress and secondary mediators to be strongly linked to the increased genotoxic response. In conclusion, co-culture exposures revealed the capacity of each ENM to elevate the frequency of micronuclei in binucleated (BN/Mn) cells above monoculture levels, indicative of secondary mechanisms of genotoxicity. The co-culture system has therefore highlighted the importance of complex in vitro models as a means of improving upon 2D monocultures as predictors of potential in vivo toxicity, which would have otherwise been overlooked. E-Thesis 31 12 2019 2019-12-31 10.23889/Suthesis.51928 A selection of third party content is redacted or is partially redacted from this thesis. COLLEGE NANME COLLEGE CODE Swansea University Doctoral Ph.D 2019-09-18T09:23:51.1002734 2019-09-17T10:30:31.9341437 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Michael J. Burgum 1 0051928-17092019110406.pdf Burgum_Michael_J_PhD_Thesis_Final_Redacted.pdf 2019-09-17T11:04:06.5230000 Output 9860754 application/pdf Redacted version - open access true 2019-09-16T00:00:00.0000000 true
title In Vitro Lung Models to Assess the Mechanistic Genotoxicity of Characterised Few-Layer Graphene
spellingShingle In Vitro Lung Models to Assess the Mechanistic Genotoxicity of Characterised Few-Layer Graphene
,
title_short In Vitro Lung Models to Assess the Mechanistic Genotoxicity of Characterised Few-Layer Graphene
title_full In Vitro Lung Models to Assess the Mechanistic Genotoxicity of Characterised Few-Layer Graphene
title_fullStr In Vitro Lung Models to Assess the Mechanistic Genotoxicity of Characterised Few-Layer Graphene
title_full_unstemmed In Vitro Lung Models to Assess the Mechanistic Genotoxicity of Characterised Few-Layer Graphene
title_sort In Vitro Lung Models to Assess the Mechanistic Genotoxicity of Characterised Few-Layer Graphene
author ,
author2 Michael J. Burgum
format E-Thesis
publishDate 2019
institution Swansea University
doi_str_mv 10.23889/Suthesis.51928
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
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
document_store_str 1
active_str 0
description Nanotechnology sits at the forefront of molecular and macromolecular medical science with tremendous applications worldwide. Paradoxically, as the number of applications for engineered nanomaterials (ENMs) increases the comparable knowledge of their toxicity may be limited. This could culminate in nano-safety issues relating to occupational, environmental and human exposure regarding ENM use without a full comprehension of their toxicological impact. The aim of this thesis was firstly, to provide a comprehensive approach to characterising the physico-chemical features of, few-layer graphene (FLG) engineered with i) no functionality (Neutral-FLG), ii) amine groups (Amine-FLG) or iii) carboxyl groups (Carboxyl-FLG) with a carbon black (CB) positive control. Secondly, the toxicological impact of these ENMs was investigated utilising relevant respiratory cell lines. This was performed using; in vitro monoculture exposures, allowing the detection of primary-indirect genotoxicity, then applying these ENMs to a dual-cell co-culture lung model to permit the detection of secondary genotoxicity. By encompassing a comprehensive approach to ENM physico-chemical characterisation, a battery of complementary techniques was employed to gauge particle and agglomerate size, morphology, surface charge and surface area. Each ENM was then evaluated for their potential to promote cytotoxicity and genotoxicity, using relative population doubling and the micronucleus assay respectively in a monoculture of the human bronchial cell line, 16HBE14o-. A deeper investigation was then launched into the potential routes of oxidative and mitochondrial stress. Neutral-FLG, Amine-FLG and CB particles promoted a genotoxic (predominantly clastogenic) response, however all tested ENMs induced oxidative & mitochondrial stress with significant elevation of interleukin (IL)-8. A transformed alveolar epithelial type-I (TT1) cell line and differentiated THP-1 (dTHP-1) macrophages formed the alveolar co-culture. This advanced cell model, which could detect secondary genotoxic mechanisms, highlighted the potential for each ENM to promote genotoxicity via secondary mechanisms. This was observed with a potency ranking of CB> Amine-FLG> Neutral-FLG> Carboxyl-FLG with overall 2-fold increases in chromosomal damage (ascertained from micronuclei frequencies) at 50µg/ml following 24 hours of exposure. The potential mechanistic role of the oxidant: antioxidant balance as a contributor to the observed secondary genotoxicity was investigated by a 2-hour pre-incubation with 1.5mM N-acetyl-cysteine (NAC). This was found to reduce chromosomal damage to non-significant (p<0.05) levels suggesting oxidative stress and secondary mediators to be strongly linked to the increased genotoxic response. In conclusion, co-culture exposures revealed the capacity of each ENM to elevate the frequency of micronuclei in binucleated (BN/Mn) cells above monoculture levels, indicative of secondary mechanisms of genotoxicity. The co-culture system has therefore highlighted the importance of complex in vitro models as a means of improving upon 2D monocultures as predictors of potential in vivo toxicity, which would have otherwise been overlooked.
published_date 2019-12-31T04:04:00Z
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