Defining the biologically effective dose for the pro-inflammatory effects of nanoparticles in lung target cells

Nanoparticles are extremely small particles, each individual one of which is less than one ten millionth of a metre in size. We are exposed to nanoparticles in busy streets as they are present in traffic exhaust produced from combustion of fuel and these nanoparticles are considered to be important in causing the harmful effects of air pollution. There are other situations where nanoparticles are found in the air because of the normal chemical reactions that occur in air, in plumes of volcanic ash and in some dusty workplaces. However, a new source of novel types of nanoparticles has arisen with the rise of nanotechnology. Part of nanotechnology aims to design and develop new nanoparticles, because they have properties which make them attractive to industry for a range of purposes. This has caused alarm bells to ring since perhaps some of these new types of nanoparticles could be harmful. For these reasons there is a need for testing of these new nanoparticles. The huge number of nanoparticle types and their variants in size and coatings mean that there are a huge number of particles to be tested and new ones are being produce all the time. However, there is huge ethical pressure against animal testing and so short-term tests that use cells instead of animals are needed. To carry out such testing it is necessary to know which tests to carry out and a thorough knowledge of how particles cause cell damage and disease would greatly help in choosing the tests. This project puts forward a study that aims to relate the physico-chemical characteristics of the particles, such as size and surface chemistry, with the activation of the process of inflammation. Inflammation has been chosen because it is key response seen when there is injury to the body and inflammation itself plays a role in leading to disease. This approach, trying to relate chemical structure to cellular response, represents an approach that could provide the essential information on how we can test the large number of nanoparticle types that need to be tested.

The present application specifically addresses research objectives set out in the DEFRA response to the Royal Society/Royal Academy of Engineering Report on the risks associated with nanotechnology, which form the basis of an MRC highlight on nanotoxicology. It focuses on practical nanotoxicology issues and is predicated on the importance of the inflammatory response in the lungs following airborne exposure to manufactured nanoparticles (MNP). It addresses the hypothesis that oxidative stress is the biologically effective dose (BED) for MNP, with special emphasis on the nature of the cellular oxidative stress response, the cellular pathways involved in pro-inflammatory gene expression and the best cell type and endpoints to use for predicting inflammogenicity of MNP. The study will contribute to predictive hazard assessment of nanoparticles that is sorely needed. The study utilises two panels of nanoparticles ? 1) a panel of fully characterised industrial in-use particles used by the applicants in a concurrent NERC-funded study; 2) a ?designer? panel of polystyrene latex nanoparticles that can be made to tight size ranges and specific surface characteristics and in fluorescent forms. Since the most appropriate lung target cell is not known, three different cell models will be used ? macrophage, epithelial cells and a combined model involving supernatants from macrophages treated with particles added to epithelial cells. MNP exposure in these models will be followed by assessment of a number of endpoints associated with particle free radical activity, cellular oxidative stress and pro-inflammatory gene expression. The size, surface chemistry and, in the case of particles available in fluorescent form, sub-cellular localisation will be related to these outcomes. All of the particles will then be assessed for their ability to cause inflammation in rat lung. The activity of the MNP in the endpoints as measured in the 3 cell models will be related to their ability to cause inflammation using correlation analysis. The study should therefore yield a number of important outcomes. 1) Comparison of macrophages and epithelial cells and the combined model, to allow validation of the best model for predictive testing strategies for MNP. 2) Knowledge of the most relevant pathways to inflammatory gene expression will allow rational choice of endpoints in short-term assays that allow prediction of inflammatory potential in untested nanoparticles. 3) Confirmation or otherwise of oxidative stress as the BED for MNP will enable approach towards a rational exposure metric for nanoparticles that can be linked to risk of lung inflammation.