1. Environmental Endocrine Disruption In San Francisco Bay Fish Effects Assessment Kevin Kelley 1-3, Jesus Reyes 1-3, Rusty Fairey 4, Zed Mason 2,3, Cathy Propper 5, Julianne Kalman 1,2,3 1 EEL --Environmental Endocrinology Laboratory, Marine Biology Program 2 IIRMES --Institute for Integrated Research on Materials, Environment & Society 3 CEPA --Center for Education in Proteomics Analysis California State University, Long Beach 4 Marine Pollution Laboratory, Moss Landing Marine Laboratories 5 Northern Arizona University 6 Cabrillo Marine Aquarium, San Pedro, CA

2. ENDOCRINE SYSTEMS regulate essential physiological functions Reproduction - Somatic Growth - Metabolism Defense/Immune - Thyroid - Developmental Stress Responses - Neuroendocrine - others …Homeostasis, Adaptation, Survival…

3. Some points about endocrine systems:... highly sensitive & responsive... respond to presence of environmental factors (stressors) to a degree commensurate with the need to maintain homeostasis... endocrine disturbances observed are typically indicative of the type of environmental factor(s) acting

4. endocrine disruptionsensitive screening toolassess effects (impacts) Monitoring programs can take advantage of endocrine disruption as a sensitive screening tool to assess effects (impacts) of water/environmental quality & specific contaminant effects Endocrine-disrupting chemicals (EDCs) are present in marine and estuarine environments, particularly along highly populated coastal regions : SF Bay Estuary http://toxics.usgs.gov/photo_gallery/sanfran.html

5. 1- determine incidence and magnitude of endocrine disruption in wild fishes thyroid, stress responses, growth & repair, reproduction 2- 2- determine whether spatial differences can be established between different SF Bay locations 3- determine liver contaminant concentrations in individuals Correlate specific contaminant concentrations with types/magnitude of endocrine disruption STUDY OBJECTIVES

6. Shiner Surfperch Cymatogaster aggregata Pacific Staghorn Sculpin Leptocottus armatus pelagic benthic

7. San Pablo Bay Berkeley Waterfront Oakland Inner Harbor Redwood City far-field reference: Tomales Bay 2006 Field Sites [pilot year]

8. THYROID ENDOCRINE SYSTEM

9. Thyroid Hormones broad physiological actions broad physiological actions essential for brain/neural development essential for brain/neural development permissive to somatic growth permissive to somatic growth important regulators of metabolism important regulators of metabolism 3,5,3’,5’-tetraiodothyronine (thyroxine) 3,5,3’-triiodothyronine T4T3

10. 0 20 40 60 80 100 San Pablo Bay Berkeley Waterfront Redwood City Oakland Inner Harbor Tomales Bay (11) (19) (9)(14) Plasma Thyroxine (T4) Concentration, ng/ml ShinerSurfperch Thryoxine (T4) Levels in Shiner Surfperch

11. 0 20 40 60 80 100 San Pablo Bay Berkeley Waterfront Redwood City Oakland Inner Harbor Tomales Bay (8)(13)(17)(12)(5) Plasma Thyroxine (T4) Concentration, ng/ml Pacific Staghorn Sculpin Thryoxine (T4) Levels in Pacific Staghorn Sculpin

12. 0 20 40 60 80 100 San Pablo Bay Berkeley Waterfront Redwood City Oakland Inner Harbor Tomales Bay (8) (11) (13)(19)(17) (19) (12)(9)(5)(14) Plasma Thyroxine (T4) Concentration, ng/ml ShinerSurfperch Pacific Staghorn Sculpin Thryoxine (T4) Levels in SF Bay Fish

13. Environmental Contaminants are Implicated in Thyroid Endocrine Disruption: … e.g., Phenols, Hg, Cd impair thyroxogenesis in thyroid follicle cells in fish (Bhattacharya et al., ‘89) … e.g., endosulfan, Aroclor 1254 and selected PCBs decrease T4 levels in fish, via effects on iodotyrosine diodinases (Coimbra et al., ‘05; LeRoy et al., ‘06) … e.g., ioxynil, certain PCBs and PBDEs bind transthyretin and TRs with high affinities, altering T4 and T3 levels/ actions in fish (Tomy et al., ‘04; Lema et al., ‘06; Morgado et al., ‘07)

14. STRESS RESPONSE ENDOCRINE SYSTEM

15. 0 400 800 1200 1600 Redwood City Oakland Inner Harbor Berkeley Waterfront San Pablo Bay 21 13 2110 Shiner Surfperch Plasma Cortisol Concentration (ng/ml) a a b b Stress Response

16. 0 400 800 1200 Redwood City Oakland Inner Harbor Berkeley Waterfront San Pablo Bay 17 16 1319 Pacific Staghorn Sculpin Plasma Cortisol Concentration (ng/ml) a a b c Stress Response

17. Other wild fish species living in affected marine environments exhibit impairment of their neuroendocrine stress response system...experimental studies have shown that contaminants, --such as PCBs-- impair stress responses in fish...experimental studies have shown that contaminants, --such as PCBs-- impair stress responses in fish ( Quabius et al., 2005; Vijayan et al., 2006 )

18. Thyroxine/ thyroid IGF-I/ growth San Pablo Bay Berkeley Waterfront Redwood City Oakland Inner Harbor Tomales Bay Shiner Perch Pacific Staghorn Sculpin Shiner Perch Pacific Staghorn Sculpin ?? ?? Endocrine Disruption—SF Bay Fish S U M M A R Y –Year 1 Cortisol/ stress response Shiner Perch Pacific Staghorn Sculpin

19. Testosterone/ Reprod 17 α -Ethinylestradiol/ Pharm. Endocrine Disruption—SF Bay Fish San Pablo Bay Berkeley Waterfront Redwood City Oakland Inner Harbor Tomales Bay Pacific Staghorn Sculpin 17 β -Estradiol/ Reprod. Pacific Staghorn Sculpin S U M M A R Y –Year 1 immature

20. Shiner Perch ? August 2006 Oakland Inner Harbor

21. Endocrine Disruption—SF Bay Fish San Pablo Bay Berkeley Waterfront Redwood City Oakland Inner Harbor Tomales Bay Shiner Perch Pacific Staghorn Sculpin Shiner Perch Pacific Staghorn Sculpin Shiner Perch Pacific Staghorn Sculpin ? ? ??? ? ? ??? S U M M A R Y –Year 1 17 β -Estradiol/ Reprod. 17 α -Ethinylestradiol/ Pharm. Testosterone/ Reprod

22. Contaminant Analyses: Feasible to measure individuals liver contaminant levels (PCBs, PAHs, pesticides) Feasible to measure individuals liver contaminant levels (PCBs, PAHs, pesticides) Several contaminants measured at high concentrations in shiner perch and Pacific staghorn sculpin Several contaminants measured at high concentrations in shiner perch and Pacific staghorn sculpin Relatively higher levels of contaminants in livers of Pacific staghorn sculpin Relatively higher levels of contaminants in livers of Pacific staghorn sculpin Feasible to correlate individual liver contaminant concentrations with endocrine and physiological indices Feasible to correlate individual liver contaminant concentrations with endocrine and physiological indices

23. Year – 2 Studies under way

24. San Pablo Bay (Loch Lohmand Marina) Richmond/Berkeley Waterfront Oakland Inner Harbor Redwood City Tomales Bay Bodega Bay Study Sites [August 2007] Richmond (Lauritzen Channel, DDTs ) San Leandro Bay (PCBs) San Francisco Waterfront Hunter’s Point (POTW) Two Harbors –Santa Catalina Island (Pacific side) L.A. Harbor –Inner Cabrillo Beach (collab. w/CMA)

25. THYROID Thyroxine & Triidothyronine REPRODUCTION Testosterone & 11-ketotestosterone REPRODUCTION 17 β -Estradiol, 17 α -Ethinylestradiol, Vitellogenin STRESS Cortisol & Cortisone GROWTH IGF-I & IGFBPs Endocrine Parameters in 2007-8

26. Altered thyroxine levels. -- T4 measurement Altered thyroxine levels. -- T4 measurement Altered T3 levels, T4:T3 ratios? -- T3 measurement, calculate T4:T3 Altered T3 levels, T4:T3 ratios? -- T3 measurement, calculate T4:T3 Altered thyroid gland morphology? -- Thyroid histology Altered thyroid gland morphology? -- Thyroid histology Impaired thyroid responsiveness? -- TSH-challenge Impaired thyroid responsiveness? -- TSH-challenge Altered T4 metabolism or TH receptors? -- peripheral iodotyrosine deiodinases (ID1-3), TRα, TR β Altered T4 metabolism or TH receptors? -- peripheral iodotyrosine deiodinases (ID1-3), TRα, TR β Emphasis: Thyroid Endocrine (HPT) Axis Impaired growth? -- Growth & IGF system indices [IGF-I, IGFBPs, cartilage growth activity] Impaired growth? -- Growth & IGF system indices [IGF-I, IGFBPs, cartilage growth activity] Relationships with specific bioaccumulated chemicals? -- Hepatic contaminants measurement & correlation Relationships with specific bioaccumulated chemicals? -- Hepatic contaminants measurement & correlation Underlying Mechanisms: Environmental Contaminants: Physiological Impacts:

27. Emphasis: Stress Endocrine (HPI) Axis Altered cortisol levels. -- Cortisol measurement Altered cortisol levels. -- Cortisol measurement Altered interrenal gland morphology? -- Interrenal histology Altered interrenal gland morphology? -- Interrenal histology Altered metabolism of cortisol? -- cortisone measurement Altered metabolism of cortisol? -- cortisone measurement Impaired interrenal responsiveness? -- ACTH-challenge Impaired interrenal responsiveness? -- ACTH-challenge Impaired production of cortisol? -- steroidogenic enzyme expression (StAR, 11β-hydroxylase, 11β-HSD-2) Impaired production of cortisol? -- steroidogenic enzyme expression (StAR, 11β-hydroxylase, 11β-HSD-2) Relationships with specific bioaccumulated chemicals? Relationships with specific bioaccumulated chemicals? -- Hepatic contaminants measurement & correlation -- Hepatic contaminants measurement & correlation Impaired defense? -- Parasitic infestation assessment Impaired defense? -- Parasitic infestation assessment Impaired growth? -- Growth & IGF system indices [IGF-I, IGFBPs, cartilage growth activity] Impaired growth? -- Growth & IGF system indices [IGF-I, IGFBPs, cartilage growth activity] Underlying Mechanisms: Environmental Contaminants: Physiological Impacts:

28. I.Endocrine responses and/or endocrine disruption serve as sensitive bio-markers of environmental perturbations II.Endocrine systems are: --highly sensitive to environmental stressors --respond proportionally to the need to maintain homeostasis --show alterations reflective of the kind of stressor(s) present (different EDCs affect different endocrine axes via distinct underlying mechanisms) SUMMARY POINTS

29. III. Endocrine-disrupted conditions are observed in SF Bay fish (urban, industrial, boat marinas) IV. Endocrine disruption is evident in different fish species V. Endocrine disruption is occurring in different endocrine systems (stress, thyroid, reproduction, growth & repair, defense, metabolism) VI. There may be interactions among endocrine systems (means wider impacts on physiological performance) SUMMARY POINTS continued

30. A.Identification of specific chemicals associated with endocrine disruption B. Understand mechanisms and test candidate EDCs, to establish cause and effect C. Build models to estimate ecologically relevant performance alterations from measures of endocrine disruption (growth, reproduction, disease/parasitism) D. Determine contaminant thresholds of effects E. Use new technologies (proteomics) to build integrated phenotypic biomarker fingerprints (multifactorial screen) DIRECTION

31. Long Beach Env’l. Endo. Lab. Jesus Reyes, Kathy Sak, Andrew Hamilton, Becki Hagstrom, Maelanie Galima, Dawn Petschauer, Navdeep Brar, Tiffany Price, Tuan Truong, Dana Cianfrani, Shari Smolko, Tami Gonzalez, Claire Wingard, Kenna Koons, Julianne Kalman, Scott Adams, Wendy Hall ? http://www.csulb.edu/depts/endo

32. *PARTNERS *PARTNERS Orange County Sanitation District Env’l Assessment Division City of Los Angeles Bureau of Sanitation Env’l Monitoring Division Los Angeles County Sanitation Districts Southern California Coastal Water Research Project University of California, Riverside University of California, San Diego California State University, Long Beach –IIRMES & CEPA Moss Landing Marine Labs Marine Pollution Laboratory Northern Arizona Univ. Regional Monitoring Program –San Francisco Estuary Institute Cabrillo Marine Aquarium * * * * *

33. ?? QUESTIONS