A Passion for Natural History. Understanding biological/ecological systems work..

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1 A Passion for Natural History. Understanding biological/ecological systems work.. Generating knowledge for environmental protection.. Charles R.Tyler

2 Ecotoxicology: Integrating molecular biology and physiology to understand chemical exposure effects on wildlife... Charles R.Tyler

3 The Feminisation of Freshwater Nature (Fish) An Unnatural History Charles R.Tyler Lou Guilette Charles R.Tyler Professor Charles R.Tyler 500 μm 1 Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, United Kingdom.. c.r.tyler@ex.ac.uk contact: c.r.tyler@ex.ac.uk

4 The Feminisation of Freshwater Nature Presentation Outline Chemicals in the Environment Endocrine Disruption and Impacts on Aquatic Vertebrate Wildlife (Fish) HO OH Impacts of Environmental Oestrogens on Sex in Fish and Fish Populations Wider Health Implications of Exposure to Environmental Oestrogens and Glowing Fish Some Concluding thoughts and Future Prospects

5 Chemicals in the Environment We live in a contaminated world... global dimensions Satellite images provide powerful insights into the extent of our chemical influence on the planet Nitrogen dioxide Ocean acidification Ammonia All chemicals we use and discharge ultimately find their way to the aquatic environment

6 Chemicals in the Environment Our Dependence on Chemicals We use tens of thousands of chemicals in products (a $3 trillion global enterprise)... plasticizers, fertilisers, pesticides, pharmaceuticals, etc. Chemicals enter human bodies via direct application (cosmetics), the food chain, drinking water or as aerosols. Chemicals can also enter the bodies of wildlife and most notably animals living in, or closely associated with, aquatic environments. Some chemicals build up in our bodies potentially increasing any biological effects they might have.

7 Chemicals of Concern Priority Substances have been identified for which we have concern because there is good evidence for adverse health effects. Other Chemicals for which there is increasing health concern include: Industrial chemicals: Chemicals in the Environment Perfluoro octanoic acid (PFOAs) - associated with elevated cholesterol and chronic kidney disease Polybrominated diphenyl ethers - Flame retardants associated with thyroid dysfunction Polycarbonate polymers (e.g. Bisphenol A) and epoxy resins. Associated with various health disorders (e.g. obesity, reproduction) Pharmaceutical substances: 17 alpha-ethinyloestradiol (EE2), 17 beta-oestradiol (E2), Diclofenac, SSRIs (antidepressants) Pharmaceutical substances - $800 billion industry. Endocrine Disrupters...

8 Endocrine Disruption Endocrine Disruption What is it? Communication Systems: The nervous system together with the endocrine system make up the body s major signalling pathways in all animals: Nervous- telephone network Immune system Endocrine- postal system A hormone is a chemical message that instructs a specific response Endocrine Disrupter: a chemical (man-made or natural) that mimics a hormone(s) and disrupts growth, development and/or reproduction in an individual or its progeny

9 Endocrine Disruption Evidence in Humans Falling sperm counts Increase in Hormone Dependent Cancers

10 Endocrine Disruption in Wildlife The Evidence in Nature Charles R.Tyler

11 Endocrine Disruption in Wildlife The Evidence in Nature Charles R.Tyler Charles R.Tyler

12 Endocrine Disruption in Wildlife The Evidence in Nature Charles R.Tyler

13 Endocrine Disruption in Wildlife The Evidence in Nature Charles R.Tyler

14 Endocrine Disruption in Wildlife The Evidence in Nature Charles R.Tyler

15 Endocrine Disruption in Wildlife FSBI 2017 The Evidence in Nature: Fish Charles R.Tyler Feminised Fish have been reported from sites across Europe, America, Canada ( Rainbow Darter - Etheostoma caeruleum, Percidae), Africa and Asia Androgenised Fish have been reported from sites across America

16 Vitellogenin mg/ml Oestrogenic Effects in Fish Feminisation of Roach in UK Rivers An Overview Wastewater Treatment Works Effluents are Oestrogenic Vitellogenin (egg yolk) *** *** *** *** Initial observations Control Willington Consett Carlisle Winsford Burwarton Location of Sewage Treatment Works

17 Oestrogenic Effects in Fish Sex in Fish is altered by exposure to Steroid Hormones Genetic Males Oogenesis (egg formation) High doses of oestrogen Genetic males or females Intermediate doses of steroids Intersex Genetic Females High doses of androgen Spermatogenesis (sperm formation)

18 Oestrogenic Effects in Fish Feminisation of Roach (Rutilus rutilus) in UK Rivers A Brief Overview Widespread sexual disruption Impact of sewage treatment works effluents on fish health (endocrine active chemicals) All feminised effects can be induced by exposure to WWTW effluents Intersex present at 44 (86%) of 51 sites Overall incidence of intersex in males of 23% Jobling, S., Nolan, M., Tyler, C.R., Brighty, G., and Sumpter, J.P. (1998).. Environmental Science and Technology 32: Jobling,S., Williams, R., Johnson, A., Taylor, A., Gross-Sorokin, M., Nolan, M., Tyler, C.R,van Aerle, R., Santos, E.M.,and Brighty, G. (2006).Environmental Health Perspectives 114: Liney, K.E., Jobling, S. Shears, J., Simpson.P.,Tyler, C.R*. (2005).Environmental Health Perspectives 113 (10): Rodgers-Gray, T. P., Jobling, S., Kelly,C.,Morris, S., Brighty, G., Waldock, M, Sumpter, J.P and Tyler C.R.* (2001). Environmental Science and Technology 35:(3)

19 Absorbance (ABS) Absorbance (ABS) Environmental oestrogens entering fish from WwTW effluents Mesocosms. Oestrogenic Effects in Fish Control E2 E1 1.5 Bile Time (minutes) The oestrogenic activity of the test effluents measured HO OH Exposed DHQ E2 EE2 E1 NP + NPnEO (n=1-4) 17β-dihydroequilenin + (Equilenin) Time (minutes) Fish take up a very wide range of oestrogenic chemicals into their bodies from the WWTW effluent Bile analysed to identify (anti-)oestrogenic compounds and their metabolites Enzymic hydrolysis RP- HPLC + yeast screens LC-NMR-MS/GC-MSMS Gibson, R., Smith, M.D., Spary, C., Tyler, C.R., Hill, E.M. (2005). ES&T 39(8) Tyler C.R., Spary, C, Gibson, R., Shears, J., Santos, E., Sumpter, J.P., Hill, E.M. (2005). ES&T 39 (8) Tyler, C.R, Filby, A.L., Hill, E.M., Liney, K.E., Katsu, Y., Bickley, L.K., Lange, A., Winter, M., Gibson, R., Shears, J.A., Iguchi, T.(2009). ES&T. 43:

20 Oestrogenic Effects in Fish Environmental steroidal oestrogens can induce all the feminised phenotypes seen in wild roach... Exposure to EE2 (0.3ng/L) can induce vitellogenin synthesis Ethinyloestradiol Exposure to EE2 (0.3ng/L) can induce ovarian cavity formation and intersex Life-long exposures to EE2 can complete sex reversal (4ng/L) Complete feminisation of fathead minnow population in a Canadian Lake dosed with ethinyloestradiol (4-6ng/L): Kidd et al., 2007 PNAS 104: Lange, A. Katsu,Y. R. Ichikawa, Greg Paull, Laura L. Chidgey, Tobias S. Coe Iguchi, T, and Tyler, C.R. (2009).. Toxicological Sciences 106(1), Lange, A., Katsu, Y., R. Ichikawa, Chidgey, L. Iguchi, T.and. Tyler, C.R. (2009). Environmental Science and Technology 43 (4),

21 Endocrine disrupting chemicals in fish Mixtures of hormone affecting chemicals that enter into testes of fish exposed to wastewater effluents (and their effects) NSAIDs (diclofenac, ibuprofen, naproxen, mefenamic acid) SSRIs (fluoxetine, norfluoxetine, paroxetine, sertraline, norsertraline, citalopram, venlafaxine) EDCs (bisphenol A, triclosan, chlorophene, propiconazole) Other xenobiotics (nordiazepam, clozapine, norclozapine, quetiapine, norquetiapine, warfarin, carbamazepine, propranolol, gemfibrozil, clopidogrel Collectively these chemical can affect steroid hormone, serotonin ( well being hormone) and prostaglandin biosynthesis David, A., Abdul-Sada, A; Al-Salhi,R.,Lange, A., Tyler, C.R., Hill E.M. (2014). Journal of Chromatography A. As many as 1000 chemicals have been identified with endocrine disrupting activity (more than 200 with oestrogenic activity). 8% of chemicals may be anti-androgenic (Kortenkamp and Faust, 2010 Int J. Andrology 33: )

22 Reproductive success Oestrogenic Effects in Fish Individual Breeding Consequences of Feminisation in Roach (Rutilus rutilus) DNA microsatellites - highly variable primers were developed and applied for parentage analysis (100% success) Breeding trials with wild roach LME model coefficient = , p < Intersex Index For roach that are moderately to severely intersex (index >2) there was an estimated 15% reduction for each incremental increase in the intersex index (75% reduction for the most feminised individuals included in this study). Genotyped all parents and offspring ( fry from each tank). Histopathology analysis of adult gonads. Harris, Hamilton, Runnalls, Jobling, Vinciotti, Henshaw, Hodgson. Coe, Tyler,. Sumpter (2010). EHP. 119:

23 Oestrogenic Effects in Fish Populations Do oestrogenic effluents affect size of the breeding population in wild roach? Population structure of roach in the region of the Thames catchment studied. Lee Thames MolMea Nene Blackwater (Thames) Blackwater (Anglian) Aire StoTed Stor t Trent Lee Relationship between predicted oestrogen exposure (E2Eq) and N e (ABC), an indicator of the breeding size in the parental population. No significant relationship was found, but a 5.6% decrease for each increase in exposure of 1 ng/l E2Eq could not be ruled out due to the wide 95% confidence intervals). Arun Kennet 0.02 Genetic Distance Some populations have been confined to some of the most oestrogen contaminated river stretches over multiple generations, and have not been sustained by immigration. Roach populations living in some effluent-contaminated river are self-sustaining Are Roach populations adapting to Oestrogens and what are the consequences if this is the case? Patrick B. Hamilton, Elizabeth Nicol, Eliane S. R. De-Bastos, Richard J. Williams, John P. Sumpter, Susan Jobling, Jamie, R. Stevens, Charles R. Tyler (2014). BMC Biology 12:1

24 Wider Health Implications of Oestrogens and Glowing Fish Roles of Oestrogens in the Body Oestrogens mediate: Female (and male) reproduction Growth Bone development Calcium Homeostasis Defence (Immune function) Oestrogens signalling: Via specific receptors ( 3 subtypes in fish- ESR1, ESR2a, ESR2b) (+ membrane receptors, oestrogen related receptors) Cross talk Every cell in the body probably has an oestrogen receptor! For exposures to chemical that mimic oestrogens there are much wider potential health effects that those on reproduction.

25 Wider Health Implications of Oestrogens and Glowing Fish ERE-GFP Zebrafish - Assessing Environmental Oestrogens (Xeno-)Estrogen Primary Response : Estrogen induces synthesis of Gal4ff protein Amplification of the signal Oestrogen Receptor 3ERE Gal4ffmRNA Control Ethinyloestradiol Secondary Response : Gal4ff protein induces synthesis of GFP GFP protein GFPmRNA UAS Sensitivities in responding body tissues (96h) Temporal dynamics in responding body tissues Okhyun Lee, Aya Takensano, Masa Tada, Charles R. Tyler and Tetsuhiro Kudoh (2012) Environ Health Perspect. 120,

26 Wider Health Implications of Oestrogens and Glowing Fish Different Oestrogenic Chemicals induce different response patterns (different target tissues) in ERE-Transgenic (Casper) Zebrafish Ethinyloestradiol Nonylphenol Genistein Ctrl BPA Ctrl 1 ng/l Ctrl 62.5 µg/l Ctrl 2.5 ng/l 30 µg/l 125 µg/l 62.5 µg/l 5 ng/l 62.5 µg/l 250 µg/l 125 µg/l 10 ng/l 125 µg/l 500 µg/l 250 µg/l 25 ng/l 50 ng/l 250 µg/l 1000 µg/l 500 µg/l 100 ng/l 500 µg/l 2000 µg/l 1000 µg/l 1000 µg/l HEART 2000 µg/l Green et al. ES&T 2016

27 Wider Health Implications of Oestrogens and Glowing Fish Bisphenol A What is is? Bisphenol A 17β-estradiol (E2) Bisphenol A is a found in plastics and resins used for food and beverage containers It is a weak oestrogen and its mode of action is complex and debated MBP (4-methyl-2,4- bis(4hydroxyphenyl)pe nt-1- ene) In mammals BPA is metabolised to a chemical known as MBP reported to be more potent Exposure to BPA is ubiquitous, with detectable levels found in most human urine and blood samples and there are associated human health concerns

28 Wider Health Implications of Oestrogens and Glowing Fish GFP Expression in the Heart in ERE-Transgenic Zebrafish exposed to Bisphenol A and its metabolic product 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1- ene [MBP] Solvent Control Solvent Control BPA * 50 ug/l 100 ug/l 250 ug/l 500 ug/l 1000 ug/l MBP * 0.10 ug/l 0.25ug/l 0.50 ug/l 1.0 ug/l 2.5 ug/l Fluorescence intensity values measured from hearts of * * * * * * Control 50 µg/ l 0.1µg/l MBP 0.25µg/l MBP 0.5µg/l MBP Moreman 1.0µg/l et al, MBP unpublished 2.5µg/l data MBP AV valve Outflow cushion Contol 50µg/l BPA 100ug/l BPA 250µg/l BPA 500ug/l BPA 1000ug/l BPA Atriu m Ventricle

29 Wider Health Implications of Oestrogens and Glowing Fish ERE-Kaede Casper Zebrafish Model. Green fluorescence UV Red fluorescence Enhanced Sensitivity to Oestrogens for repeat exposures Control (non exposed) larvae and larvae exposed initially to 10 ng EE2/L over the period of 48h (0-2 dpf) were imaged at 3 dpf (A) and the Kaede response was then converted fully from green to red fluorescence via UV exposure Kaede: Japanese maple C- EE2 EE2 EE2 500 μm Quantification of target tissue responses in ERE-Kaede-Casper transgenic zebrafish exposed to estrogen treatment, as determined by fluorescence induction. Fluorescence intensity was quantified in liver and heart at 5 dpf. Responses were quantified after EE2, genistein and BPA exposures for 3-5 dpf. Data are reported as mean fold induction ± SEM (n=18). EE2-10ng/L, Genistein- 500mg/L, BPA- 2000mg/L) Green et al., Scientific Reports In Press

30 Mean Peak height (fluorescent units) Median voxel fluorescence intensity (Fluorescence units) Wider Health Implications of Oestrogens and Glowing Fish Elav1 model Monitoring for chemicals effects on the brain elavl3:gcamp6s pre-exposed to drug via immersion Untreated control 4-AP treated Larvae immobilised in agarose in capillary 300 Example temporal activity profile from a 5mM PTZ-exposed larva (all registered brain regions) Time (z-stack cycles) Tegmentum (Peak height) ** LSM imaging of live larva through brain 0 CONTROL 1.25mM 2.5mM 5mM PTZ concentration (External) Multiple script outputs provides quantitative data on a global and anatomical regional scale Custom image processing script undertakes 4D anatomical registration using a standard brain template Winter et al., Scientific Reports DOI: /s

31 Concluding thoughts and Future Prospects Some Concluding thoughts and Future Prospects Endocrine disruption has changed the way we think about risk associated with chemicals. Environmental oestrogens induce harm in individual male fish affecting the ability to breed. Genetic analysis suggest the size of the roach breeding populations in UK rivers is however, not unduly affected (accepting limitations of the analyses), but we know little about any other population level effects. Can fish adapt to EDCs and what are the fitness consequences of such adaptations? Such an understanding should help provide insight into population resilience in their contaminated world. Molecular technologies are playing a major role in unravelling how chemicals (EDCs) affect the physiology in fish and the consequences in fish populations. Transgenic fish provide advanced models for understanding where and how EDCs work in the body for informing more integrative approaches in health effects assessments.

32 Acknowledgements, Sponsors and Collaborators of the work presented: The Contributing Team: Aya Takesono, Okhyun Lee, Matthew Winter, John Moreman, Jon Green, Ruth Cooper, Sulay Mourabit, Dylan Windell, Molly Payne, Ross Brown, Malcolm Hetheridge, Tetsuhiro Kudoh, Patrick Hamilton, Anke Lange Max-Planck-Institut für Neurobiologie Herwig Baier University of Rennes Olivier Kah University College London Masa Tada Steve Wilson INERIS Francois Brion Technische Universität Braunschweig Reinhard Köster

33 Thank you for listening Questions? Charles R. Tyler