Epidemiological studies highlight a direct association between the decline in respiratory health of the human population and increased environmental ultrafine particulate (UFP) exposure. This evidence, coupled with research identifying shared characteristics and toxicity between UFP and engineered nanomaterial (ENM), suggests that increased levels of ENM associated with the nanotechnology revolution could have a detrimental effect on human health. Although the link between respiratory disease and air pollution is well-established, toxicological data for ENM is limited. Current methods for the assessment of particle toxicity utilise a combination of both in vitro assays and in vivo animal testing. In some cases, these conventional assays provide unreliable results on account of nanoparticle interference. In this thesis, assays were undertaken to more fully understand the impact of a panel of ENMs on alveolar epithelial cell function and survival, as well as to assess the potential value of an alternative method for nanotoxicological screening. Eicosanoid profiling was used to assess both toxicity and inflammatory markers associated with a panel of ENMs, this technique is novel for the use in testing of ENM and the results show it has potential to be introduced/applied as an effective tool to predict a broad spectrum of detrimental effects of ENM in lung function.
Submerged A549 cells, were used as a model of lung epithelial cells throughout. The secondary cell line is commonly used in in vitro research to examine the effect of toxins on respiratory health, specifically the alveolar region. A panel of ENM (SiO2, TiO2, NiO, ZnO and CuO) were selected to span from the benign to the highly toxic. ENM prepared in suspension were applied to the cells at 100cm2/mL for 24 h.
This doctoral thesis focused on addressing the following aims:
1. To assess whether metallic ENM of differing chemical composition damage the cell membrane and/or mitochondria.
2. To determine whether ENM induce mitochondrial dysfunction through delivery or over-production of harmful reactive oxygen species (ROS) and, if so, to determine whether mitochondrial dysfunction results in activation of apoptosis.
3. To ascertain whether ENM alter the release of lipid inflammatory mediators using eicosanoid profiling.
Mitochondrial function and membrane integrity assays revealed that CuO and ZnO induced mitochondrial dysfunction (~ 100% reduction in mitochondrial function), and promoted cell death (85 ± 7.5% cell lysis, ***P<0.001), respectively, when compared to control. In addition, superoxide production was increased by TiO2 alone (~ 100% increase, 0.0394 ± 0.0081 AU, **P<0.01), creating a discrepancy between assays. Analysis also revealed that metallic ENMs, specifically ZnO and CuO, significantly increased the production of prostaglandin E2 (~ 50%, 828 ± 119pg/sample, **P<0.01) and ~ 100%, 1439 ± 248pg/sample, ***P< 0.001), a pro-inflammatory eicosanoid, and elevated generation of a range of hydroxy-eicosatetraenoic acids (HETEs), suggesting induction of lipid peroxidation by these oxide derived ENMs.
In conclusion, through the use of in vitro assays and eicosanoid analysis it was determined that ZnO and CuO ENM induce cell damage and death. However, although traditional in vitro assays are able to identify highly toxic ENM from the rest, they lack the ability to identify more subtle changes and, in some cases, are unreliable. By contrast, eicosanoid profiling has the ability to provide more detailed information regarding generation of both pro- and anti-inflammatory mediators, as well as oxidative stress, whilst avoiding the issues that are encountered through the use of current in vitro tests.
|Date of Award||1 Nov 2016|
- The University of Edinburgh
|Supervisor||Ian Megson (Supervisor)|
Nanotoxicity of oxide-derived engineered nanomaterials: impact on cell viability and function, with conventional assays and evaluation of novel eicosanoid profiling
Garrison, E. (Author). 1 Nov 2016
Student thesis: Doctoral Thesis › Doctor of Philosophy (awarded by OU/Aberdeen)