Population success and individual fitness are proportional to reproductive success in all sexually-reproducing species. Steroids and their respective receptors are highly conserved across taxa,^4,6 and therefore persistent contaminants, collectively known as endocrine disrupting chemicals (EDCs),^4,6 may adversely impact fitness.^4,6,7 This study particularly focuses on the potential disruption of the reproductive endocrinology of Tursiops truncatus (Atlantic bottlenose dolphins), by certain persistent EDCs in the Indian River Lagoon (IRL), Florida and Charleston Harbor (CHS), South Carolina as part of the Dolphin Health and Risk Assessment study (HERA). This study utilizes in vivo, in vitro and Quantitative Structure Activity Relationships (QSAR) studies for estrogenic and androgenic compounds that disrupt either steroidogenesis or the native steroid-receptor binding site,¹ providing a means of sorting through candidate EDCs to determine which chemicals are most likely to adversely impact the reproductive success of an individual or population. Based on serum and blubber contaminant concentrations found in HERA study animals, and prior studies indicating endocrine-disrupting effect in marine mammals and humans, a number of legacy and emerging contaminants have been selected for this study.^2,3,5 Selected legacy compounds are: 4,4'-dichlorodiphenyltrichloroethane (DDT) and dichlorodiphenyldichloroethylene (DDE), 2,4'-dichlorodiphenyldichloroethane (DDD), and polychlorinated biphenyls (PCBs)--118, 138, 153, 180, and 183, and these will be assessed as potential estrogen and androgen receptor (ER and AR)--agonists and antagonists. Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), and polybrominated biphenyl ethers (PBDEs)--47, 99 and 153 were selected for assessment as potential ER and AR-antagonists. During the course of this study, enzyme-linked immunoassays (ELISA) of serum and urine for estradiol (E₂), testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are being developed, tested and validated, followed by Western Blot and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) for the hormones and their gonadal receptors. These initial assays allow confirmation of the presence of native hormones in given samples, assessment of endocrine function within the donor animal, and estimates of the relative concentrations, mass, sequences and structures of the hormones and receptors of interest. High performance liquid chromatography/electron spray ionization/mass-mass spectrometry (HPLC/ESI/MS-MS) will be performed on serum and gonadal samples to allow comparison of the three-dimensional structures of the binding regions of the hormones, their receptors and selected EDCs, followed by binding affinity studies to compare relative binding affinities of native hormones and EDCs.¹ These data will be analyzed with Molecular Shape Analyses (MSA) and Comparative Molecular Similarity Indices Analyses (coMSIA) to determine which EDCs are most likely to disrupt gonadal receptors. The strongest candidate EDCs will then be selected for further study in tissue and live model animals.

Acknowledgements

Grateful acknowledgement is extended to Dr. Gregory Bossart and Dr. Patricia Fair for support and access to samples and data, and to Ms. Holly Muraco, and Dr. Maya Rodriguez for samples. Dr. Janet Roser and the Roser Lab at UC-Davis have provided invaluable material support and information. Financial support has been provided by the FAU National Alumni Association, the FAU Graduate Student Association, FAU Student Financial Aid scholarships, the Department of Biological Sciences, and the Capt. Nathan Award.

References

1.  Blair RM, Fang H, Branham WS, Hass BS, Dial SL, Moland CL, Tong W, Shi L, Perkins R, Sheehan DM 2000. The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. Toxicol Sci. 54:138-153.

2.  Fair PA, Mitchum G, Hulsey TC, Adams J, Zolman E, McFee W, Wirth E, Bossart GD 2007a. Polybrominated diphenyl ethers (PBDEs) in blubber of free-ranging bottlenose dolphins (Tursiops truncatus) from two Southeast Atlantic estuarine areas. Arch Environ Contam Toxicol 53:483-494.

3.  Fair PA, Muir DCG, Small J, Sturman S, Adams J, Houde M, Bossart GD 2007b. Tissue distribution of perfluoroalkyl compounds in bottlenose dolphins (Tursiops truncatus) from southeast coastal USA. Organohalogen Compounds 69:849-852.

4.  Gray Jr. LE, Kelce WR, Wiese T, Tyl R, Gaido K, Cook J, Klinefelter G, Desaulniers D, Wilson E, Zacharewski T, Waller C, Foster P, Laskey J, Reel J, Giesy J, Laws S, McLachlan J, Breslin W, Cooper R, DiGuilio R, Johnson R, Purdy R, Mihaich E, Safe S, Sonnenschein C, Welshons W, Miler R, McMaster S, Colborn T 1997. Endocrine screening methods workshop report: detection of estrogenic and androgenic hormonal and antihormonal activity for chemicals that act via receptor or steroidogenic enzyme mechanisms. Reprod Toxicol 11(5):719-750.

5.  Houde M, Pacepavicius G, Wells RS, Fair PA, Letcher RJ, Alaee M, Bossart GD, Hohn AA, Sweeney J, Solomon KR, Muir DCG 2006. Polychlorinated biphenyls and hydroxylated polychlorinated biphenyls in plasma of bottlenose dolphins (Tursiops truncatus) from the Western Atlantic and Gulf of Mexico. Environ Sci Technol 40:5860-5866.

6.  Kavlock RJ, Daston GP, DeRosa C, Fenner-Crisp P, Gray LE, Kaattari S, Lucifer G, Luster M, Mac MJ, Maczka C, Miller R, Moore J, Rolland R, Scott G, Sheehan DM, Sinks T, Tilson HA 1996. Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the U.S. EPA-sponsored workshop. Environ Health Perspectives 104(Suppl. 4):715-740.

7.  Wingfield JC, Kitaysky AS 2002. Endocrine responses to unpredictable environmental events: stress or anti-stress hormones? Integr Comp Biol 42:600-609.