The Ecotoxicology and Aquatic Biology Research Group – Our Science.

Endocrine Disruption

A major focus of our research work in EABRG is endocrine disruption, the study of chemicals that alter hormone function which can lead to adverse impacts on health. Our work with colleagues at Brunel University was some of the very first research to show that effluents from wastewater treatment works causes feminisation of male fish, inducing female characteristics including oocytes (developing eggs) in the testis of males. Through controlled, including long term (up to 4 year) exposures, we have subsequently proven that feminisation of wild roach living in UK rivers is caused by exposure to treated sewage effluents. Using a targeted biological screening of chemically fractionated bile from effluent exposed fish, with collaborators (University of Sussex), we have also identified the suite of feminising chemicals in effluent discharges, principally environmental oestrogens, and through laboratory exposures proven they contribute to the feminisation of wild fish in UK rivers. These chemicals include natural and synthetic steroid oestrogens, and alkylphenolic compounds. This original work has been very highly cited. Combining empirical studies with models including Concentration Addition/Joint Independent Action, we have further established that oestrogenic chemicals can be additive in their effects and these findings have had a widespread and international influence. In more recent work we have shown the widespread presence of anti-androgenic chemicals in wastewater effluent discharges and are now studying their possible contribution to the feminisation of fish living in UK rivers.

A major focus for our research work in the EABRG has been to establish whether wild fish (roach, Rutilus rutilus) populations are impacted adversely by exposure to oestrogenic effluent discharges. Undertaking controlled breeding studies in the laboratory and applying DNA microsatellites to assess parentage with we have proven that moderately to severely feminised males have a reduced capacity to compete with normal males to sire offspring. We have further shown that life long exposure to an oestrogenic effluent (at full strength) causes complete gonadal feminisation of males, some of which were able to breed as females. In some of our current work we are applying DNA microsatellites to establish whether roach populations living in rivers heavily polluted with wastewater effluents have a reduced effective population breeding size. If so, this will show that exposure to wastewater effluents at these sites is fundamentally affecting population genetics.

The mechanisms of action and pathways of effects have been established for very few endocrine active chemicals and this has been another key focus of our work conducted in EABRG. We have successfully employed both targeted single gene approaches, gene arrays and more recently sequencing technologies to unravel some of the molecular mechanisms of oestrogenic disruption (including for mixture effects) and other forms of chemical toxicity. We also involved with projects studying the metabolome and xenometabolome in fish in response to exposure to effluents and individual EDCs with Profs Hill (University of Susses) and Prof Viant (University of Birmingham). In our work, EABRG has cloned a very extensive range of hormone receptors, growth regulars and enzymes mediating steroidogenesis that span the hypothalamus-pituitary-gonadal-liver–axis, developed various complimentary PCR assays to quantify their expression, and helped developed gene arrays for zebrafish, fathead minnow, stickleback and roach. The EABRG has also worked on tumourigenesis and was the first to clone and sequence some of the p53 tumour suppresser genes in fish. Most recently, working with Dr Kudoh, also at Exeter, we have developed a ERE-GFP transgenic zebrafish that is exquisitely sensitive to oestrogens that allows us to identify – through the expression of a green fluorescent protein - where different oestrogens interact in the body and in real time. This is a very power tool that we are now using to investigate the potential for wider health impacts of exposure to environmental, oestrogens. We are currently attempting to develop a similar model for (anti-) androgens. Members of the EABRG also work on other chemical contaminants of environmental and human health concern including pesticides and pharmaceuticals.

Much of our work on endocrine disruption is focused on in vivo studies, spanning development of relatively short term bioassays to measure exposure (bioavailability) and effects on reproduction, to very long term exposures to assess life time exposure impacts. We do however also have projects focused on the development and application of in vitro systems for understanding mechanisms of action and reducing animal use. These systems include cell culture (especially hepatocytes), recombinant cell lines and receptor transactivation assays. Working with Taisen Iguchi at the National Institute of Basic Biology in Japan we have used receptor transactivation assays to compare responses to oestrogens in a very wide range of fish species.

Almost all of our work on endocrine disruption is focused on fish, but we have also worked on birds (Dipper, Cinclus cinclus), mammals (Otter, Lutra lutra) and various invertebrates.

Nanotoxicology

pictures of nanoparticles

Nanotechnology is an emerging field of science that deals with engineered molecules of an extremely small size (1-100 nanometres). Because of size – related changes in physico – chemical properties, substances at the nanoscale display optical, magnetic and chemical properties that can differ greatly from the standard material enabling diverse commercial applications. Nanoparticles are currently used in products such as suntan lotion, cosmetics, clothing, cleaning materials, medicines and food and assessing the potential health risks associated with exposure to nanoparticles is now recognised as being a major international research priority. The EARBG researches into the effects of metal and metal oxide nanoparticles, principally in fish, but also some invertebrates. Most of the research on nanomaterials in the EARBG has been focused on understanding their bioavailability, including for exposures in natural waters and in effluents. Our work is also investigating the absorption, distribution , biological effects, metabolism and elimination. This work is being complemented by in vitro studies with various fish cell types including hepatocytes, gut and gill cells to investigate how nanoparticles cross cell membranes and their fate within cells. Further methods applied in this work include whole mount in situ hybridisation to investigate specific gene responses in the bodies of whole embryos. Various analytical techniques and state of the art imaging techniques are being employed to monitor particle fate and behaviour in the water and fish, including RAMAN scattering. In our work to date we have found that many metal nanomaterials ( ceria, zinc, titania) show little toxicity to fish. In contrast silver nanoparticles are toxic, albeit that this toxicity may result from dissolution and free silver, rather than an innate function of the particles themselves. In our current work we are applying whole genome sequencing on exposed zebrafish embryos and the target tissues for specific nanomaterials in larger fish to further establish the effect mechanisms of metal/metal oxide nanoparticles in the body and thus help unravel some of the possible health concerns linked with these materials. This work is funded by very diverse sources including Research Councils, DEFRA, and various industries reflecting the multi-stakeholder interest.

Disease

EABRG has a growing interest in the effects of combined environmental stressors on wildlife, including features of climate change. A project that has recently been initiated with the Centre for Environment, Fisheries and Aquaculture Science is the application of sequencing technologies to understand the pathogenic process of white spot virus, a major disease organism for cultures and wild crustaceans.

Basic Biology

Underpinning all of our ecotoxicology work is our interest in the basic biology of fish and other wildlife species. Here are primary interests are in understanding the mechanisms controlling sexual development and reproduction. To be able to assess what is abnormal for exposure to an environmental stressor, it is important to understand the normal basic processes and variation. Major research activities and findings under this theme include:

Sexual differentiation: We have established the timing and mapped histologically the process of sexual differentiation in the roach, Rutilus rutilus, a sentinel species for studies on chemical effects in wild fish in UK freshwaters, and in the fathead minnow and zebrafish, both key laboratory species. We have further identified some of the molecular mechanisms controlling sexual differentiation in these fish.

Oogenesis (egg development) and Spermatogenesis: We have mapped histologically the process of oogenesis and spermatogenesis in roach, fathead minnow and zebrafish. In this work we have especially focused on the process of vitellogenesis (provision and uptake of yolk into oocytes) and established some of the endocrine and molecular mechanisms controlling yolk uptake and processing. Our work has included the cloning and sequencing of the first cDNA for a fish vitellogenin receptor, establishing the developmental expression and cellular localisation of the vitellogenin receptor mRNA in fish oocytes, and the cloning and sequencing of some of the first yolk processing enzymes (cathepsins) in oocytes. We also developed some of the first assays to quantify vitellogenins in fish, which have been vital for our work (and other laboratories worldwide) on endocrine disruption.

Endocrine control of fecundity: Very little is known about the determinants of fecundity in any animal. Fish, because they are highly fecund, provide excellent model systems for investigating the endocrine control of oocyte recruitment and growth - the associated titres of plasma hormones are considerably higher than for most other animals. We have applied the technique of unilateral ovariectomy (to manipulate oocyte recruitment) to identifiy some of the hormones control ling ovarian development. In this work, an assay we developed for follicle stimulation hormone (FSH) in trout has been used effectively to determine the effects of endocrine active chemicals on pituitary function.

Puberty: Fairly recently in mammals a novel pituitary receptor, KiSS I receptor and its associated ligands (KiSS peptins) have been cloned and shown to play key roles in initiating puberty. We have cloned the KiSS I receptor in various fish species, identified and cloned some of the KiSS peptin ligands (the first to do so in fish) and mapped them to their chromosomes. We have established the ontogeny and localization of expression of KiSS 1 receptor in the brain in both male and female fathead minnow during sexual development and shown how this expression aligns with the expression of an extensive suite if other genes involved in mediating sexual development (e.g. the GnRHs). The ability to control puberty in fish would be of great significance in the aquaculutre of many fish species.

Sexual behaviour: The zebrafish is one of the major models for studies in ecotoxicology, yet very little is known about its basic biology. We have contributed to filling this knowledge gap through extensive studies on its reproductive biology. In this work we have projects studying the behaviour of the zebrafish in breeding colonies. This work is principally focused on understanding the interactions and hierarchies that develop in zebrafish colonies to advance our understanding on their breeding dynamics in natural populations. In this work, detailed studies on male-male, male-female and female-female interactions are being established for different sized colonies and the reproductive success of individuals in those colonies are determined through genotyping of the offspring produced (through the use of DNA microsatellites). Our work on zebrafish is principally laboratory based, but we also have some ongoing studies on natural colonies in Bangladesh. This work also includes the development and application of population models.

New Models: We are always interested in exploring new models for understanding basic reproductive processes and/or exploiting them for better understanding possible health effects of environmental contaminants. A model we are currently working on is a Xenotoca spp, that gives birth to live young. Currently we are studying the basic reproductive biology, but our longer term plan is to use this model to investigate maternal tyransfer of EDCs, pharmaceuticals and nanomaterials and their health effects on developing embryos.