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Aspects of endocrine disruption in the Upper Olifants River and Lake Loskop

Aspects of endocrine disruption in the Upper Olifants River and Lake Loskop. Christoff Truter Stellenbosch University Presentation to the Olifants River Forum 7 December 2012 . The endocrine system. Communication network Hormones = Signals Reproduction/ sexual characteristics

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Aspects of endocrine disruption in the Upper Olifants River and Lake Loskop

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  1. Aspects of endocrine disruption in the Upper Olifants River and Lake Loskop Christoff Truter Stellenbosch University Presentation to the Olifants River Forum 7 December 2012

  2. The endocrine system • Communication network Hormones = Signals • Reproduction/ sexual characteristics • Growth and development • Metabolism • Immunity • Behaviour

  3. The endocrine system: Basic mechanism of action Hormones (testosterone) Bloodstream Testicle Cell Hormone receptor Transcription + Translation Nucleus fitnessanddefense.com DNA Glands Targettissues Phenotypic change

  4. Cattle in feedlots are dosed with a mixture of hormones including estrogens and androgens to inhance growth (90% of USA beef that is produced) (Schiffer et al. 2001) Synthetic andorogen: Trenbolone-beta has a half-life in liquid manure of > 260 days, suggesting that it could have ecologic impacts if released into the environment as runoff from feedlots (Schiffer et al. 2001). Hormones in the aquatic environment Natural and synthetic • Oral contraceptives • Hormone replacement therapy • Growth stimulators (Agriculture) • Human and animal excretes toptenz.net • Activity at parts per trillion concentration • 1 teaspoon in 50 million litres Orlando et al. 2004, Environ. Health. Persp. 112(3):353-358 unifiedlifestyle.com sexmedsonline.com

  5. Impacts on fish • Impaired reproduction • Intersex fish (Jobling et al. 1998) • Reduced egg production (Brionet al. 2001) • Skewed sex ratios (Nimrod and Benson 1998) • Altered oogenesis (Woodling et al. 2006) Gakuranman.com

  6. Whole lake experiment Kid et al. 2007 Synthetic estrogen (Ethinylestradiol) • 3 years exposure (5 ng/L) • Fathead minnow population • Male fish feminised • Altered oogenesis in females • Population collapse Ovary Fathead minnow denr.sd.gov Normal female Exposed female Testis Normal male Exposed male

  7. Upper Olifants River Oberholster et al. 2010

  8. Upper Olifants River: Human impacts Sewage works effluents Informal settlements Agriculture Acid mine drainage thesanitationcrisis.wordpress.co.za Industrial effluents

  9. Aims • Determine female hormone concentrations in the Upper Olifants River • Assess seasonal and spatialvariation Ethinylestradiol 17-β-estradiol

  10. Water collection • 6 Localities (Sub-surface samples) • Autumn (2011) • Winter (2011) • Spring (2011) • Summer (2011) Study sites Oberholster et al. 2010

  11. Water extraction and analysis • C18 Solid Phase Extraction of organic compounds • 17-β-Estradiol • Ethinylestradiol • Immunoassays (Commercial ELISA kits)

  12. 17-β-Estradiol Mean ± Standard Deviation (technical repeats)

  13. 17-β-Estradiol • US EPA Maximum Acceptable Toxicant Concentration (MATC) • Effects: • Egg production • Male secondary characteristics • Gonad histology US EPA predicted no-effect concentration (Fish) Mean ± Standard Deviation (technical repeats)

  14. Ethinylestradiol Mean ± Standard Deviation (technical repeats)

  15. Ethinylestradiol • US EPA Maximum Acceptable Toxicant Concentration (MATC) • Effects: • Impaired fertilization success Complete feminization of fathead minnows (Lange et al. 2001) US EPA predicted no-effect concentration (Fish) Mean ± Standard Deviation (technical repeats)

  16. Spatial variation among localities a ab ab ab ab b Mean ± Standard Deviation Pairwise differences assessed with Kruskal Wallis ANOVA

  17. Seasonal variationacross the Upper Olifants System Mean ± Standard Deviation Pairwise differences assessed with Kruskal Wallis ANOVA

  18. Conclusions Future work • Potential disruption of the gonadal endocrine axis throughout the system • Potential reproductive impairment at five of the six sites In vivo: • Expose juvenile Mozambique tilapia to water collected during summer • Expression of a selection of genes associated with the reproductive system

  19. Lake Loskop Mozambique tilapia • Metabolic disorders • Obesity and lipid peroxidation (pansteatitis) (Dabrowski et al. unpublished) Lake Loskop Allan Stratton Oberholster et al. 2011

  20. Lake Loskop Mozambique tilapia Metabolic disordersThyroid disruption • Loskop Fish: Hiperthyroidism relative to Lake Flag Boshello(Dabrowski et al. unpublished) • Potential links to thyroid disruption by endocrine disrupting chemicals • Industrial chemicals? • Metals? • Cyanotoxins (microcystins)? • Other? Reviewed in Casals-Casas and Desvergne 2011 Further investigation needed: Pathway specific investigation of the Hypothalamus-pituitary-thyroid endocrinal axis

  21. The Thyroid Cascade Hypothalamus Thyrotrophin releasing hormones (TRH) Thyroid-stimulating hormones (TSH) Pituitary Thyroid hormones (T3 and T4) Thyroid gland Thyroid Receptors (TR alpha and TR beta) Wikipedia.org Target tissues Transcription (Thyroid activity) Growth Development Metabolic regulation Thyroid-hormone therapy and thyoid cancer: a reassessment Bernadette Biondi, SebastianoFiletti and Martin Schlumberger Nature Clinical Practice Endocrinology & Metabolism (2005) 1, 32-40doi:10.1038/ncpendmet0020

  22. Aims • Investigate thyroid disruption in fish caught from Lake Loskop using gene expression based biomarkers • mRNA expression • TR alpha • TR beta

  23. Fish collection (August 2012) • Mozambique tilapia • Lake Loskop (Gill nets) • Stellenbosch University Aquaculture Department

  24. RNA isolation Brains tissue • Lake Loskop • 5 males • 3 females • Stellenbosch • 3 males • 3 females

  25. Gene expression (RT-qPCR) • Thyroid Receptor-α • Thyroid Receptor-β • Reference gene: β-actin • Relative quantification: 2-(ΔΔCt)-method (Pfaffl2001)

  26. Mozambique tilapiaLake Loskop vs Stellenbosch University N 8 6 8 6 Mean ± Standard Deviation * P = 0.02; student’s t-test

  27. Gender effects on TR expression TR alpha TR beta N 5 3 3 3 N 5 3 3 3 Mean ± Standard Deviation Pairwise differences assessed with student’s t-test

  28. Cyanobacteria (cyanotoxins) as culprit? 96 hour Zebrafish larvae exposure (Yan et al. 2012) Lake Loskop: August 2012 (Oberholster and Cheng, unpublished data) • Water • Riverine zone - 1.299 mg/L • Transitional zone - 1.302 mg/L • Lacustrine zone -  1.401 mg/L • Mozambique tilapia • Livers: 0.075 μg/kg • Muscle: below detection limit Microcystin-LR (μg/L) Microcystin-LR (μg/L)

  29. Future work • Measure TSHβ expression in adult Mozambique tilapia brains • Expose juvenile Mozambique tilapia to water collected from Lake Loskop • mRNA expression of a selection of genes associated with the thyroid system Yan et al. 2012 Microcystin-LR (μg/L)

  30. Acknowledgements

  31. References • Brion, F., Palazzi, X., Triffault, G., Laillet, B., Porcher, J. M., Garric, J., Tyler, C. R. and Flammarion, P. 2002. Reproductive effects of exposure of various life stages of zebrafish. R&D TECHNICAL REPORT P2-T04/1 103 • Casals-Casas, C., and Desvergne, B. 2011. Endocrine Disruptors: From endocrine to metabolic disruption. Annual Review of Physiology 73:135-162. • Jobling, S., Nolan, M., Tyler, C. R., Brighty, G., and Sumpter, J. P. 1998. Widespread sexual disruption in wild fish. Environmental Science and Technology 32:2498-2506. • Kid, K. A., Blanchfield, P. J., Mills, K. H., Palace, V. P., Evans, R. E., Lazorchak, J. M., and Flick, R. W. 2007. Collapse of a fish population after exposure to a synthetic estrogen. Proceedings of the National Academy of Sciences of the United States of America 104:8897-8901. • Länge, R., Hutchinson, T.H., Croudace, C.P., et al. 2001.Effects of the synthetic estrogen 17α-ethinylestradiol on the life-cycle of the fathead minnow (Pimephalespromelas). Environmental Toxicology and Chemistry 20, 1216-1227. • Nimrod, A.C. and Benson, W.H. 1998. Reproduction and development of Japanese medaka following an early life stage exposure to xenoestrogens. Aquatic Toxicology44:141-156. • Oberholster, P. J., Myburgh, J. G. , Ashton, P. J., Coetzee J. J., Botha, A-M. 2011. Bioaccumulation of aluminium and iron in the food chain of Lake Loskop, South Africa Ecotoxicology and Environmental Safety. • Oberholster, P. J., Aneck-Hahn, N. J., Ashton, P. J., Botha, A-M., et al. 2010. Risk Assessment of Pollution in Surface Waters of the Upper Olifants River System: Implications for Aquatic Ecosystem Health and the Health of Human Users of Water. Report to the Olifants River Forum. • Orn, S., Holbech, H., Madsen, T.H., et al. 2003. Gonad development and vitellogenin production in zebrafish (Dania rerio) exposed to ethinylestradiol and methyltestosterone. Aquatic Toxicology 65: 397-411. • Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research 29:e45. • Woodling, J. D., Lopez, M. E., Maldonado, T. A., Norris, D. O., Vajda, A. M. 2006. Intersex and other reproductive disruption of fish in wastewater effluent dominated Colorado streams. Comparative Biochemistry and Physiology, Part C 144:10-15 • Yan, W., Zhou, Y., Yang, J., Li, S., Hu, D., Wang, J., Chen, J. , Li, G. 2012. Waterborne exposure to microcystin-LR alters thyroid hormone levels and gene transcription in the hypothalamic–pituitary–thyroid axis in zebrafish larvae. Chemosphere 87:1301-1307

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