Diagnosing Toxicant Specific Disruption of Sexual Development in Wild Fish using Metabolomics

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

Environmental monitoring is important for determining if man-made stressors, such as pollution and climate change, are damaging animals that live in our aquatic environment. Perhaps surprisingly, up to half of the water flowing in some UK rivers originates from the effluent of sewage treatment plants and this can affect the native wildlife. Several years ago we found that the reproductive systems in fish living in many UK rivers had been damaged by exposure to chemicals contained in the sewage treatment plant effluents that disrupt the body's sex hormone systems. Although scientists now have tools for determining if fish reproductive systems have been damaged, these generally involve killing the fish and examining dissected tissues, which is not ideal for extensive impact monitoring programmes. Also, the existing tools do not provide much information on the types of pollutants that are causing the damage.Such information would be very useful to environmental regulators,and could be used to help trace the source(s) of pollutant(s) and to help design strategies to remove these chemicals from the water before they damage fish and other animals. The aim of this research project is to develop and apply a new technology (called 'metabolomics') for assessing the health of fish by measuring large numbers of small molecules (called metabolites) in their blood and tissues. These measurements are so detailed that they can provide information on exactly which parts (or 'metabolic pathways') of the fish's reproductive system are damaged. This approach is now also being used successfully in hospitals to assess human diseases, and we will use it to look for unique patterns or 'signatures' in wild fish that would determine if they are damaged and if so by what types of pollutants in the river. To achieve this we will first need to expose fish (the roach, in which we have shown widespread sexual disruption) in the laboratory to single pollutants that are suspected to be involved with disruption of reproduction in wild fish, and measure the damage caused by each of these chemicals. These experiments will include short and long exposures of the fish, some of which will last for two years to accurately mimic real environmental scenarios. Then we will 'train' computers to recognize the specific patterns of pollutant-induced damage, and those computers will search for these patterns in the wild fish in much the same way they can search through genetic fingerprints to match those collected at a crime scene. Another advantage of 'metabolomics' is that unlike most existing environmental monitoring methods, it can be conducted rapidly using only a minute volume of blood taken from the fish, which can then be released back into the river. The final advantage is that the measurements of certain metabolites in the blood, particularly those called 'sex steroids', can tell us a lot about the fish's reproductive health, i.e., there is a close relationship between these molecular measurements and the overall fitness of the whole fish. The tools used in 'metabolomics' are more commonly associated with chemistry labs and include nuclear magnetic resonance spectroscopy and mass spectrometry. Also, because of the immense amount of data that will be produced, experts in mathematics will also be needed. Our multi-disciplinary team has expertise in chemistry, biology, toxicology and mathematics, and brings together scientists from the Universities of Birmingham and Exeter, as well as the Environment Agency, a Water company and a Mass Spectrometry company. Ultimately the results of this study will enable development of a more appropriate methodology and practical set of tools (called 'biomarkers') for end-users, which can contribute to environmental impact assessments and the regulation of discharges by the regulatory authorities. This will provide a more informed knowledge of the health of our rivers thus protecting our aquatic resources and biodiversity.

Publications

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Mark Viant (Author) (2008) Endocrine Disrupting Chemicals and their Environmental Impacts. in CRC Press

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Brown AR (2009) Genetic variation, inbreeding and chemical exposure--combined effects in wildlife and critical considerations for ecotoxicology. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

 
Description Environmental monitoring is important for determining if pollution is damaging the health of animals that live in our aquatic environment. Up to half of the water flowing in some UK rivers originates from the effluent of waste water treatment works (WWTW) and this has been shown to affect the native wildlife, including our own studies on fish. Previously we found that fish reproductive systems had been damaged by exposure to chemicals in WWTW effluent. Although scientists now have tools for determining if damage has occurred, they generally involve killing the fish and examining dissected tissues. Also, the existing tools provide little information on the classes of pollutants causing the damage, and this would be especially useful to environmental regulators. The aim of this project was to develop and apply a new technology ('metabolomics') for assessing the health of fish by measuring large numbers of 'metabolites' in their blood and tissues. These measurements can provide information on which 'metabolic pathways' in the fish are damaged. To do this, first we developed novel mass spectrometry methods to measure metabolites rapidly, and with high sensitivity and specificity. The postdoctoral researcher was also trained in targeted liquid chromatography-mass spectrometry (LC-MS) analysis of steroids, in collaboration with Dr Hill, University of Sussex.

We then used these metabolomics techniques (mass spectrometry, nuclear magnetic resonance spectroscopy and LC-MS) to study the metabolic responses of fish (roach) exposed in the laboratory for 28 days to pollutants. The selected pollutants were known or suspected to be involved in disrupting reproduction of wild fish. First we focused on the toxicity of a "Red Listed" (by the UK Environment Agency) pesticide called fenitrothion. Our studies revealed (1) the first evidence that this chemical affects steroid metabolism, (2) that it acts as a neurotoxin (its intended "mode of action", so this was expected), (3) and that many other metabolic changes occurred, in particular associated with energy metabolism and also a metabolic pathway involving phenylalanine metabolism. Arguably one of the most important discoveries was that the classic biomarker for fenitrothion toxicity, namely the increase in a metabolite called acetylcholine, was not observed, but instead we detected the opposite effect. This result questions the robustness of single molecular biomarker assays for studying the effects of real-world (chronic) pollution.

We also studied the biological responses of roach exposed to WWTW effluent. This resulted in considerable metabolic disruption in the fish, including to steroid metabolism (e.g. to 11-ketotestosterone, the most important male sex hormone in fish). This observation, and other more traditional measurements, confirmed that the WWTW effluent was 'oestrogenic'. Analyses of these large datasets are on going, with the aim to discover a metabolic signature that can be used as a diagnostic tool for exposure to oestrogen-like pollutants. We further conducted chronic exposures of roach to this WWTW effluent (and also to the model oestrogen, ethinyloestradiol) to show extensive disruption of the male fish, which in the worst cases after 3 years of exposure switched sex such that their reproductive systems appeared identical to that of females.

Overall our multi-disciplinary team, which comprised of experts in chemistry, biology, toxicology and statistics and in addition scientists from the Environment Agency and a Water company, has contributed towards developing and evaluating state-of-the-art methodologies for measuring metabolic responses in living organisms to environmental stress. Although the results from such experiments can appear complex, they have helped us to discover particular metabolic pathways that are disrupted in toxicant-exposure fish, and which could provide a practical tool for end-users concerned with monitoring the health of our environment.
Exploitation Route Regulatory toxicology
Sectors Environment

 
Description We have worked closely with UK Water Industry for some of the exposures undertaken and to inform them on the results obtained (notable Essex and Suffolk Water). Tyler has supported the so called Endocrine Disruption Demonstration Programme including the UK Water Industry, the Environment Agency and DEFRA to investigate which treatment technologies are the more effective at removing EDCs in WWTW before the discharge of effluents into UK Rivers. Extensive dialogue in our research on EDCs is on-going with government bodies, industry and international bodies including the OECD (as evidenced by the workshops attended/hosted and international meetings at which we have been invited to present our data. Our work on EDCs has appeared in the Times, Telegraph, Observer, and other national and international newspapers. It has been a feature article in US National geographic and Frontiers in Science. It has also appeared in NERC News, as NERC podcast, as an ACS webinar, as short videos on Youtube (e.g. http://www.youtube.com/universityofexeter#p/c/868210A42F3C27E1/6/rbD-8xnbezA) , featured on BBC televisions including Country File and features in various international news and televisions programmes.
 
Description Influencing the understanding and realisation of potential of OMICS technologies for environmental (in particular chemical and nanomaterial) risk assessments
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
 
Title Improved analytical and computational methods for direct infusion- and LC-mass spectrometry based metabolomics 
Description Improved analytical and computational methods for direct infusion- and LC-mass spectrometry based metabolomics 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Increased citations 
 
Description Collaboration / Partnership with OECD 
Organisation Organisation for Economic Co-operation and Development OECD
Country France 
Sector Public 
PI Contribution Expert advice in the field of environmental OMICS and the translation of OMICS technologies (in particular metabolomics) into regulatory science (in particular toxicology)
Collaborator Contribution International perspective
Impact See publications
Start Year 2006
 
Description Collaboration / Partnership with UK Government 
Organisation Environment Agency
Country United Kingdom 
Sector Public 
PI Contribution Expert advice in the field of environmental OMICS and the translation of OMICS technologies (in particular metabolomics) into regulatory science (in particular toxicology)
Collaborator Contribution Stake holder perspective, EU regulations
Impact See publications
Start Year 2006
 
Description Collaboration / Partnership with USEPA 
Organisation United States Environmental Protection Agency
Country United States 
Sector Public 
PI Contribution Expert advice in the field of environmental OMICS and the translation of OMICS technologies (in particular metabolomics) into regulatory science (in particular toxicology)
Collaborator Contribution Provide stake hold perspective
Impact See publications
Start Year 2006
 
Description 'Molecular fingerprinting holds clues to cleaner environment' - Original alumni magazine 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact 'Molecular fingerprinting holds clues to cleaner environment'. Article published in Original alumni magazine, University of Birmingham, Summer 2012.

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Year(s) Of Engagement Activity 2012
 
Description Member of the OECD Expert Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Member of the OECD Expert Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) (2014-present)

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Year(s) Of Engagement Activity 2014
 
Description Molecular study shows unexpected effects of toxin - NERC Planet Earth Online 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 'Molecular study shows unexpected effects of toxin'. Article published on NERC Planet Earth Online, August 2011. http://planetearth.nerc.ac.uk/news/story.aspx?id=1035

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Year(s) Of Engagement Activity 2011