Aspects of endocrine disruption in the Upper Olifants River and Lake Loskop - PowerPoint PPT Presentation

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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