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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

Abstract

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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Abstract: 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.
Item Description: A selection of third party content is redacted or is partially redacted from this thesis.
College: Faculty of Medicine, Health and Life Sciences

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