Environmental Health: From Genomics to Analytical Chemistry Louis J. Guillette Jr., Ph.D. Professor of Obstetrics & Gynecology; Director of Marine Biomedicine; Endowed Chair of Marine Genomics Medical University of South Carolina & Hollings Marine Laboratory Genotype Genetic Determinism DNA RNA Protein Phenotype Genotype EpiGenetic Determinism DNA Environment expression RNA Protein Phenotype Stress Diet Behavioral & Nutrients & Environmental Contaminants (pesticides, metals, plasticizers, phytoestrogens) Body Burden Drugs & Stored Contaminants – Fat & Bone Supplements Legal & Illegal (smoking, alcohol, vitamins) Personal Care Products Genetics (phthalates, musk xylene) Parental Genetics & Epigenetics From Hamlin & Guillette, 2011, Embryo Today Sentinel Species 'Watch and Warn' • Powerful for studying environmental X gene perturbations • Weak for some mechanistic studies Environmental Factors Abiotic Xenobiotic Nutrition Factors Chemicals •Signaling Disruption •Endocrine •Nervous •Immune Environmental Factors Steroid Thyroid Obsogenic Signaling Signaling Signaling • Altered fertility & embryonic development • Altered metabolism & energy utilization • Altered fat storage and functioning OH H3C HO OH H3C HO OH H3C HO OH H3C OH OH H C H C RNA 3 3 HO Pol ER HO ER ER HO ER ERE TATA ER ER http://www.ks.uiuc.edu/Research/pro_DNA/ster_horm_rec/dbd/ Transactivation assays with steroid hormone receptors Kohno et al. (2008) Integ. Comp. Biology 48:527- 534 HRE-Luciferase System Steroid or Test Chemicals Expression Plasmid Ligand Receptor Receptor- Ligand HRE Complex Reporter Plasmid Luciferase cDNA Transcription Translation Luminescenc Reporter Protein e (Luciferase) estrogen response element–thymidine kinase–luciferase consensus palindromic ERE ERa Ligand Domain Sequence Similarity Bottlenose Dolphin vs other vertebrates Iguchi & Guillette, unpubl. data In vitro Transactivation Assay Iguchi & Guillette, unpubl. data ESR1 (ERa and Contaminants Lungfish Sturgeon Gar Zebrafish Medaka Stickleback Fold Induction Giant Salamander Tokyo Salamander Axolotl Katsu et al. Mol Cell Endo 2006, 2007; Endocrinology 2008a,b Using Liquid Chromatography Tandem Mass Spectrometry for Hormone Analyses Ashley Boggs et al. (NIST, NOAA & MUSC) • Uses retention times and fragmentation to detect hormones • Allows DIRECT detection of hormones • Applicable to all vertebrate wildlife species • Multiple hormones • single sample Cholesterol Pregnenolone Progesterone 17-OH-Pregnenolone 11-Deoxy-Corticosterone 17-OH Progesterone DHEA Corticosterone 11 Deoxy-Cortisol Androstenedione Cortisol Estrone Testosterone Adrenosterone Steroid Pathways Estradiol Estriol Steroid Hormone Profile17 from a Single Sample 4.4e5 4.2e5 Boggs et al. 4.0e5 1. Estriol 21 2. Estradiol-13C3, 3.8e5 3. Estradiol 4. 17-OH-Pregnenolone 3.6e5 5. DHEA 3.4e5 23 6. Cortisol 7. Cortisol-d4 3.2e5 8. Equilin 3.0e5 18 9. Estrone 10. 11-Deoxycortisol 2.8e5 11. Pregnenolone-13C2d2 2.6e5 12. Pregenolone 13. DHT 2.4e5 14. 17-OH-Progesterone 10 2.2e5 15. 17-OH-Progesterone-13C3 22 16. Corticosterone 2.0e5 14 17. Testosterone 1.8e5 13 18. Testosterone-13C3 15 19. Adrenosterone 1.6e5 3 20. 11-Deoxy-corticosterone Intensity, counts per second per counts Intensity, 24 21. Androstenedione, 1.4e5 22. Androstenedione-13C3 1.2e5 19 23. Progesterone 6 8,9 24. Progesterone-13C2 1.0e5 2 11 8.0e4 4 20 7 6.0e4 12 16 4.0e4 5 2.0e4 1 0.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 Time, min Female Pilot Whale Blubber • Definite Targets Defined peak & RSD < 15% ▫ Progesterone (P4) ▫ Cortisol (F) ▫ 11-Deoxy-Cortisol (S) • Probable Targets Above LOD & RSD < 20% ▫ Androstenedione (AE) ▫ Adrenosterone (AT) ▫ 11-Deoxy- Corticosterone (DOC) ▫ Pregnenolone (P5) Boggs et al. .