Metal/Metal Oxide Nanomaterials and Oxidative Stress- Are there Harmful Health Effects in Fish for Environmental Exposures?

Summary
The nanotechnology industry is worth $ billions to the global economy. Unique properties (e.g. surface reactivity) exhibited at the nanoscale (particles with at least one dimension less than 100nm) and exploited for use in these industries, however, can result in toxic properties and there is increasing experimental evidence for this. From a mechanistic perspective the ability to generate reactive oxygen species (ROS) and induce oxidative stress is increasingly being recognized as a paradigm to explain some of the toxic effects related to the particle for metal/metal oxide NMs. In this proposal we will undertake exposures of fish models to some of the most widely used and commercially important nanomaterials [silver (Ag) and cerium oxide (CeO2)] including for environmentally realistic scenarios to investigate for harmful health effects via oxidative stress. This work will be enhanced greatly by the ability to detect, quantify and characterise NMs in complex environmental media and organisms at ambient levels, through the use of labelling NMs with highly enriched stable isotopes, advanced imaging methods (e.g. Coherent Antistoke Raman Scattering) and the development of a novel biosensor zebrafish model.
A biosensor zebrafish will be developed with a convenient response system for detecting oxidative stress. In the zebrafish model ROS triggers an electrophile responsive element (EpRE) reporter - normally found in the 'starter' (promoter) sequences of genes involved in protecting the cell from oxidative damage - and this will induce a green fluorescent protein (GFP) that can be detected via imaging. The genetic approach to develop this new biosensor fish has been applied successfully at Exeter for other pollutants. The model will enable identification of the target tissues for oxidative responses in real time. Transcriptomics - measuring the responses of many thousands of genes - will be used to identify the effect mechanisms (molecular pathways) in the responsive (green glowing) tissues for selected NM exposures. Findings from the studies with transgenic zebrafish will inform a NM exposure in a UK natural water with a fish species native to UK rivers (carp) to assess for chronic health in a partnership with South West Water. Detailed characterisation of the particles in the water will be undertaken to inform on particle fate and behaviour and to quantify dose and help interpret biological effects analysis.
This project will build upon significant track records and publications of the interdisciplinary team, bringing together extensive infrastructure and technological capability, and industry partnerships, to advance understanding on the potential for commercially important NMs to induce harm under realistic environmental conditions. The work will support NM risk assessment for both protection of the freshwater aquatic environment and sustainable development of the nanotech industry. All data generated will be published in the peer reviewed literature, in leading journals, and disseminated via public fora and liaison groups to stakeholders (e.g. public, regulators, industry), which the partners have extensive existing links.
The project proposed is directly relevant to NERC's science themes for Biodiversity, Sustainable use of natural resources and Environment, Pollution and Human health, and also to the EC European Water framework Directive - to develop our understanding of the environmental risks and hazards of pollution and wastes. The project furthermore supports the objectives of the NERC's mission to promote innovative, interdisciplinary high quality research that relates to the understanding and exploitation of biological systems.

Impact Summary
Given the global economic importance of the nanotechnology industry - currently in $billions worldwide - our findings will be of very wide interest (inter)nationally to a very wide range of industries, government regulatory bodies, environment protection groups and the wider public. The major part of our impact activities will be centred on training, engagement with industry partners, and national and international government regulatory bodies and informing the wider public.
We expect the work to benefit industry and government regulatory bodies (inter)nationally, by supporting evidence-based decision-making, through advanced understanding on health impacts and risks associated with nanomaterials exposure, and academic and industry researchers working in similar or related fields through provision of advanced tools (e.g. a new transgenic fish model, isotopically enriched nanomaterials, microscopy techniques, including CARS,) and associated expertise for nanomaterials research. The transgenic zebrafish model for systems-wide effects analysis of oxidative stress will facilitate greatly mechanisms based effects analysis for pollutants generally with potentially very wide utility. The public will also benefit from better evidence of nanoparticle effects that may affect their own health. The project is directly relevant to NERC's science themes for Biodiversity, Sustainable use of natural resources, and Environment, Pollution and Human health.
The applicants have considerable experience of commercial engagement and research associations with large nanomaterial organizations (including with the Nanomaterial Industry Association, and various nanotechs through major international projects including, nanoBEE, the PROSPECT consortium and various large EU consortia), with other large multinationals (AstraZeneca, Syngenta), with UK water companies, and various SMEs. PIs on this proposal hold KTP and LINK awards with various industry partners, and CASE studentship awards. The project team also has a strong track record in supporting UK and international governments for screening and testing guidelines, including for nanomaterials, and policy development and provides evidence and input to policy influencers and policy decision makers via a number of routes (e.g. Defra commissioned reports OECD test guidelines, Min of Environ for Japan and Royal Comm. task and RCEP advisory groups). All of these links will provide conduits for the sharing of knowledge developed in this project and creating impact with industry and government bodies.
Results of this work are expected to make a significant scientific impact and major routes for dissemination will be via relevant and high impact ISI scientific journals, and national and international conferences and workshops. The applicants will also disseminate knowledge from this research work via the press (as and where appropriate and in agreement with NERC), as we have done on many occasions previously, into local schools, and into higher education programmes, and at public meetings, including at defra, to the Nanotech Industries. We will also organize and run a one-day workshop (in month 24 of the project) and invite a cross section of the key stakeholders (approx 30-40 people) to disseminate knowledge on our research findings, support its uptake by the end users, and develop further research opportunities.
The RCo-I and PDRAs will be provided with the opportunity to develop their awareness of, and skills in, knowledge transfer, designating major roles for PDRAs in the proposed workshop, and School visits. The participating laboratories have impressive records on employment for their students that include permanent positions in academia (both nationally and internationally), and within various industries and government agencies.