1. Environmental Endocrine Disruption Effects Assessment
In San Francisco Bay Fishes
Shiner Surfperch & Pacific Staghorn Sculpin
Kevin M. Kelley
Environmental Endocrinology Laboratory
Inst. for Integrated Research on Materials, Environment & Society (IIRMES)
Center for Education in Proteomics Analysis (CEPA)
Marine Biology Program, Dept of Biological Sciences, Calif. St. Univ. Long Beach

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

3. Because endocrine systems are . . .
. . . highly sensitive, responsive
. . . respond to presence of environmental stressors to a degree commensurate with the need to maintain homeostasis
. . . reflective in their response to the specific type(s) of stressor present

4. . . . measures of ‘endocrine disruption’ are increasingly being pursued as sensitive bioindicators for water/environmental quality and specific contaminant effects
Endocrine-disrupting chemicals (EDCs) are present in marine and estuarine environments, particularly along highly populated coastal regions:
SF Bay Estuary

5. STUDY OBJECTIVES
1- determine incidence and magnitude of endocrine disruption in wild fishes, emphasizing assessment of endocrine systems regulating stress responses, growth & repair, thyroid & reproduction
2- determine whether spatial differences can be established between different SF Bay locations, with respect to endocrine disrupted condition(s)
3- determine liver contaminant concentrations in individuals, to identify correlations between specific contaminants and type(s) of endocrine disruption in fish sampled from different SF Bay locations

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

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

8. Far Field Sites
Bodega Bay
Tomales Bay

9. STRESS RESPONSE AXIS

10. adaptational responses
Stress!
Hypothalamo-
Pituitary-
Interrenal
Axis
(HPI Axis)
BRAIN
HYPOTHALAMUS
CRH
PITUITARY
anterior posterior
ACTH
Interrenal
tissues
circulation
↑↑CORTISOL
adaptational responses

11. Plasma Cortisol Concentration (ng/ml)
Stress Response
Shiner Surfperch a a
1200 b
Plasma Cortisol Concentration (ng/ml) b
800
400 21 13 21 10
Oakland Inner Harbor
Berkeley Waterfront
San Pablo Bay
Redwood City

12. Plasma Cortisol Concentration (ng/ml)
Stress Response
Pacific Staghorn Sculpin a a
1200
Plasma Cortisol Concentration (ng/ml)
800 b
400 c 17 16 13 19
Oakland Inner Harbor
Berkeley Waterfront
San Pablo Bay
Redwood City

13. REDUCED cortisol response
STRESSOR
impaired HPI Axis
BRAIN
HYPOTHALAMUS
CRH
PITUITARY
anterior posterior
ACTH
Interrenal
tissues
circulation
EDCs ?
REDUCED cortisol response

14. (Quabius et al., 2005; Vijayan et al., 2006)
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
(Quabius et al., 2005; Vijayan et al., 2006)

15. THYROID ENDOCRINE SYSTEM

16. variety of physiological actions
Hypothalamo-
Pituitary-
Thyroid
Axis
(HPT Axis)
BRAIN
HYPOTHALAMUS
TRH
PITUITARY
anterior posterior
TSH
Thyroid
gland
circulation
T4 , T3
variety of physiological actions

17. Thyroid Hormones T4 T3 broad physiological actions
3,5,3’,5’-tetraiodothyronine (thyroxine)
3,5,3’-triiodothyronine
broad physiological actions
essential for brain/neural development
permissive to somatic growth
important regulators of metabolism

18. Thryoxine (T4) Levels in Shiner Surfperch80
Shiner
Surfperch
Plasma Thyroxine (T4)
Concentration, ng/ml 60 40 20
(11)
(19)
(19)
(9)
(14)
San Pablo
Bay
Berkeley
Waterfront
Redwood
City
Oakland
Inner Harbor
Bodega
Bay

19. Thryoxine (T4) Levels in Pacific Staghorn Sculpin80
Plasma Thyroxine (T4)
Concentration, ng/ml 60 40 20
(8)
(13)
(17)
(12)
(5)
San Pablo
Bay
Berkeley
Waterfront
Redwood
City
Oakland
Inner Harbor
Tomales
Bay

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

21. EDCs ? impaired HPT Axis altered physiology Altered T4 (T3) production
BRAIN
HYPOTHALAMUS
TRH
PITUITARY
anterior posterior
TSH
Thyroid
Gland
circulation
EDCs ?
Altered
T4 (T3) production
altered physiology

22. PBDEs and PCBs are implicated in thyroid endocrine disruption:
…in mammalian studies, PBDE exposure causes reduction of thyroid hormone level and production (e.g., Skarman et al., 2005; Boas et al., 2006)
…in fish, PBDE exposure causes reduction in T4, but not T3 (Tomy et al., 2004 –lake trout; Lema et al., 2006 –fathead minnow)
…in fish, PCB exposure alters T4 and T3 levels (LeRoy et al., 2006 –Atlantic croaker)

23. Reproductive Steroids

24. Plasma 17β-Estradiol Concentrations
Pacific Staghorn Sculpin
400
E2 (pg/ml)
300
200
N.D. for Testosterone (selected samples)
100
Female [winter]
Female [summer]
Male [winter]
Female
Male
[immature]
all SF Bay locations tested
English Sole [Orange County]
[n=13-29/bar]

25. Shiner Surfperch ?
August Oakland Inner Harbor

26. Pacific Staghorn Sculpin
17α-Ethinylestradiol (EE2) in Fish Plasma
200
150
Plasma EE2 Concentration (pg/ml)
100 50
N.D.
N.D.
Turbot
WWTP-affected site
Shiner Surfperch
Pacific Staghorn Sculpin
(Tomales Bay, Redwood City, Oakland Inner Harbor, Berkeley, San Pablo Bay (Loch Lohmand)

27. The Growth Endocrine Axis
PITUITARY
The Growth Endocrine Axis
Growth Hormone
LIVER
IGFBP-3
IGFBP-1
(IGFBPs 2,4-6)
IGF-I
IGFBP
IGFBP
“IGF Axis”
IGF
IGF
IGF REC
target cell
GROWTH

28. Stress Effects on Plasma IGFBP Levels
Jack Mackerel
Trachurus symmetricus
Controls
Stressed
Rat Plasma Control
45 kDa
30 kDa IGFBP-1
30 kDa
28 kDa
increased IGFBP-1 >> growth inhibition
cell culture work supports this

29. * * * ?! Endocrine Disruption—SF Bay Fish POTW, seasonal effects??
[Yellow=Year-1 fish analyses] *
Cortisol & Cortisone *
Thyroxine & Triidothyronine
POTW, seasonal effects??
17β-Estradiol & 17α-Ethinylestradiol
Testosterone & 11-ketotestosterone ?!
IGF-I & IGFBPs
(June 2007)
Hepatic contaminants (chlorinated pesticides, PCB congeners, PAHs) *
=spatial differences detected

30. Study Sites 2007 July-August
San Pablo Bay (Loch Lohmand Marina)
Richmond (Laurentian Channel, DDTs)
Berkeley Waterfront
Oakland Inner Harbor
San Leandro Bay (PCBs)
Redwood City
South of Dunbarton (POTW)
far-field reference: Tomales Bay
Bodega Bay

31. Stress (HPI) Axis –Continuing Analyses 2007
Field season July-Aug 2007
a) interrenal histology
b) hepatic contaminants correlation
c) ACTH-challenge*
d) cortisone measurement* (11β-HSD-2)
e) ectoparasitic infestation assessment 
f) growth endocrine system (IGF-I, IGFBPs)
g) steroidogenic enzyme expression* (StAR, 11β-hydroxylase, 11β-HSD-2)
With an emphasis on Oakland Inner Harbor (San Pablo Bay, San Leandro Bay??) vs. control sites
*b-d = potential bioassays for disruption (EDC screening?)

32. Thyroid (HPT) Axis –Continuing Analyses 2007
Field season July-Aug 2007
a) T3 measurement*
b) hepatic contaminants correlation
c) thyroid histology [?]
d) TSH-challenge* [?]
e) peripheral deiodinases (D1-3)* [?]
f) thyroid hormone receptors (TRα & TRβ)* [?]
g) growth endocrine system (IGF-I, IGFBPs)
*b-d = potential bioassays for disruption (EDC screening?)

33. ?? QUESTIONS

35. Liver Contaminant Analysis, GC/MS
Selected Contaminants*
Shiner Surfperch
Pacific Staghorn Sculpin
Chlordane-gamma
1 – 4
2 – 5
Chlordane-alpha
3.5 – 15
6 – 10
trans-Nonachlor
3.5 – 12
5 – 20
cis-Nonachlor
1.5 – 5
4 – 13
4,4’-DDE
20 – 47
48 – 54
*range of ng/g wet weight values shown
So-Calif would have 4, 4’-DDT
Nov. 2006

36. Liver Contaminant Analysis, GC/MS
Selected Contaminants*
Shiner Surfperch
Pacific Staghorn Sculpin
PCB 095
6 – 12
8 – 14
PCB 149
11 – 35
25 – 35
PCB 138
27 – 70
56 – 63
PCB 180
14 – 47
28 – 58
PCB 194
nd – 8
6 – 21
PCB 153
27 – 66
56 – 68
Other congeners measured:
28, 31, 44, 49, 52, 66, 70, 74, 87, 99,101,
105, 110, 118, , 141, 151, 156,
158, , 170, 177, 183, 187, 200
1 – 16
2 – 35
*range of ng/g wet weight values shown
Nov. 2006

37. Liver Contaminant Analysis, GC/MS
Selected Contaminants*
Shiner Surfperch
Pacific Staghorn Sculpin
Naphthalene
13 – 19.5
15 – 20
2-methylnaphthalene
9 – 15
6 – 17
1-methylnaphthalene
5 – 13
6 – 8
Biphenyl
1 – 5.5
2 – 5
Fluorine
2.5 – 11
3.5 – 5
Phenanthrene
6.5 – 11
6.5 – 8
*range of ng/g wet weight values shown
Nov. 2006

38. Russell Fairey et al. Moss Landing Marine Laboratories

39. Southern California Sea Grant Program San Francisco Estuary Institute

40. steroidogenic targets
cholesterol
StAR, PBR
mitochondria
P450scc
pregnenolone
17a-hydroxypregnenolone
dehydroepiandrosterone
progesterone
17a-hydroxyprogesterone
androstenedione
11-deoxycorticosterone
11-deoxycortisol
testosterone
P45011b
P450arom
corticosterone
cortisol
estradiol-17b
Cortisone
(inactive)
11b-HSD-2
aldosterone

41. Thyroxogenesis

42. Stress!! Stress!! expensive processes Catabolic Pathways
handling
crowding / captivity
water quality
Stimulate the HPI Axis
elevated CORTISOL levels
Catabolic Pathways
Anabolic Pathways
↑ Glucose, other fuels
↑ Hepatic glucose production
↑ AA release from muscle
↑ Lipid breakdown
Overall: increased fuel mobilization & availability
↓Growth & Repair
↓Reproduction
↓Immune function
Overall: reduced energy-
expensive processes

43. Elthusa californica
. . . was commonly found in opercular chambers of shiner surfperch (staghorn sculpin, to lesser degree)