Environmental Endocrine Environmental Endocrine Disruption Effects AssessmentDisruption Effects Assessment

In San Francisco Bay FishesIn San Francisco Bay FishesShiner Surfperch & Pacific Staghorn SculpinShiner Surfperch & Pacific Staghorn Sculpin

Kevin M. KelleyKevin M. Kelley

Environmental Endocrinology LaboratoryEnvironmental Endocrinology Laboratory

Inst. for Integrated Research on Materials, Environment & Inst. for Integrated Research on Materials, Environment & Society (IIRMES)Society (IIRMES)

Center for Education in Proteomics Analysis (CEPA)Center for Education in Proteomics Analysis (CEPA)

Marine Biology Program, Dept of Biological Sciences, Calif. St. Marine Biology Program, Dept of Biological Sciences, Calif. St. Univ. Long BeachUniv. Long Beach

ENDOCRINE SYSTEMSENDOCRINE SYSTEMS

regulateregulate

essential physiological functionsessential physiological functions

Reproduction Reproduction - - Growth & RepairGrowth & Repair - - MetabolismMetabolism Defense/ImmuneDefense/Immune - - ThyroidThyroid

- - Developmental Developmental Stress Stress ResponsesResponses - - NeuroendocrineNeuroendocrine - - othersothers

Homeostasis, Adaptation, Homeostasis, Adaptation, SurvivalSurvival

Because endocrine systems Because endocrine systems areare . . . . . .

. . . highly sensitive, responsive. . . highly sensitive, responsive

. . . respond to presence of . . . respond to presence of environmental stressors to a degree environmental stressors to a degree commensurate with the need to commensurate with the need to maintain homeostasismaintain homeostasis

. . . reflective in their response . . . reflective in their response to the specific type(s) of stressor to the specific type(s) of stressor presentpresent

Endocrine-Endocrine-disrupting disrupting

chemicals (EDCs)chemicals (EDCs) are present in are present in marine marine and estuarine and estuarine environments, environments, particularly particularly along highly along highly

populated coastal populated coastal regions: regions:

SF Bay EstuarySF Bay Estuary

. . . measures of ‘endocrine . . . measures of ‘endocrine disruption’ are increasingly being disruption’ are increasingly being

pursued as sensitivepursued as sensitive bioindicators bioindicators for for

water/environmental quality and water/environmental quality and specific contaminant effectsspecific contaminant effects

http://toxics.usgs.gov/photo_gallery/sanfran.html

1- 1- determine incidence and magnitude of determine incidence and magnitude of endocrine disruption in wild fishesendocrine disruption in wild fishes, , emphasizing assessment of endocrine systems regulating stress responses, growth & repair, thyroid & reproduction2- 2- determine whether spatial differences determine whether spatial differences can be establishedcan be established between different SF Bay locations, with respect to endocrine disrupted condition(s)3- 3- determine liver contaminant determine liver contaminant concentrations in individualsconcentrations in individuals, , to identify correlations between specific contaminants and type(s) of endocrine disruption in fish sampled from different SF Bay locations

STUDY OBJECTIVESSTUDY OBJECTIVES

Shiner Surfperch

Cymatogaster aggregata

Pacific Staghorn Sculpin Leptocottus armatus

San Pablo BaySan Pablo Bay

Berkeley Berkeley WaterfrontWaterfront

Oakland Inner Oakland Inner HarborHarbor

Redwood CityRedwood City

far-field far-field referencereference:: Tomales BayTomales BayBodega BayBodega Bay

2006 Field 2006 Field SitesSites

Tomales Tomales BayBay

Bodega Bodega BayBay

Far Field Far Field SitesSites

STRESS STRESS RESPONSE RESPONSE

AXISAXIS

↑↑↑↑CORTISOLCORTISOL

adaptational responses

StressStress!! BRAIN

HYPOTHALAMUS

CRHCRH PITUITARYanterior posterior

ACTHACTHInterrenal

tissues

circulation

Hypothalamo-Hypothalamo-Pituitary-Pituitary-Interrenal Interrenal

AxisAxis(HPI Axis)(HPI Axis)

0

400

800

1200

Redwood City

Oakland Inner

Harbor

Berkeley Waterfro

nt

San Pablo Bay

21 13 21 10

Shiner SurfperchPla

sma C

ort

isol

Conce

ntr

ati

on (

ng/m

l)

aa aa

bb

bb

Stress ResponseStress Response

0

400

800

1200

Redwood City

Oakland Inner

Harbor

Berkeley Waterfro

nt

San Pablo Bay

17 16 13 19

Pacific Staghorn SculpinPacific Staghorn Sculpin

Pla

sma C

ort

isol

Conce

ntr

ati

on (

ng/m

l) aa aa

bb

cc

Stress ResponseStress Response

REDUCED REDUCED cortisol cortisol responseresponse

BRAIN

HYPOTHALAMUS

CRHCRH PITUITARYanterior posterior

ACTHACTHInterrenal

tissues

circulation

impairedimpaired HPI AxisHPI Axis

EDCs EDCs ??

STRESSORSTRESSOR

Other wild fish species living in affected Other wild fish species living in affected marine environments exhibit marine environments exhibit

impairment of their neuroendocrine impairment of their neuroendocrine stress response systemstress response system

...experimental studies have ...experimental studies have shown that contaminants, shown that contaminants,

such as such as PCBsPCBs, , impair stress responses in fishimpair stress responses in fish

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

THYROID THYROID ENDOCRINENDOCRINE SYSTEME SYSTEM

T4 , T3T4 , T3

variety of physiological actions

BRAIN

HYPOTHALAMUS

TRHTRH PITUITARYanterior posterior

TSHTSHThyroid

gland

circulation

Hypothalamo-Hypothalamo-Pituitary-Pituitary-Thyroid Thyroid

AxisAxis(HPT Axis)(HPT Axis)

Thyroid Hormones Thyroid Hormones

•broad physiological actionsbroad physiological actions•essential for brain/neural essential for brain/neural developmentdevelopment

•permissive to somatic growthpermissive to somatic growth•important regulators of important regulators of metabolismmetabolism

3,5,3’,5’-tetraiodothyronine 3,5,3’,5’-tetraiodothyronine (thyroxine)(thyroxine)

3,5,3’-triiodothyronine 3,5,3’-triiodothyronine

T4T4 T3T3

0

20

40

60

80

San PabloBay

Berkeley Waterfront

RedwoodCity

Oakland Inner Harbor

Bodega Bay

(11) (19) (19) (9) (14)

Plasma Thyroxine

Plasma Thyroxine

(T4)

(T4)

Concentration,

Concentration,

ng/ml

ng/ml

ShinerShiner

SurfpeSurfperchrch

Thryoxine (T4) Levels in Shiner Thryoxine (T4) Levels in Shiner SurfperchSurfperch

0

20

40

60

80

San PabloBay

Berkeley Waterfront

RedwoodCity

Oakland Inner Harbor

Tomales Bay

(8) (13) (17) (12) (5)

Plasma Thyroxine

Plasma Thyroxine

(T4)

(T4)

Concentration,

Concentration,

ng/ml

ng/ml

Pacific Pacific StaghoStaghorn rn SculpiSculpinn

Thryoxine (T4) Levels in Pacific Thryoxine (T4) Levels in Pacific Staghorn SculpinStaghorn Sculpin

0

20

40

60

80

100

San PabloBay

Berkeley Waterfront

RedwoodCity

Oakland Inner Harbor

Tomales/ Bodega

Bay

Plasma Thyroxine

Plasma Thyroxine

(T4)

(T4)

Concentration,

Concentration,

ng/ml

ng/ml

ShinerShiner

SurfpeSurfperchrch

Pacific Pacific StaghoStaghorn rn SculpiSculpinn

Thryoxine (T4) Levels in SF Bay Thryoxine (T4) Levels in SF Bay FishFish

(8) (13) (17) (12) (5)(11) (19) (19) (9) (14)

Altered Altered T4 (T3) productionT4 (T3) production

BRAIN

HYPOTHALAMUS

TRHTRH PITUITARYanterior posterior

TSHTSHThyroid

Gland

circulation

impairedimpaired HPT AxisHPT Axis

altered altered physiologyphysiology

EDCs EDCs ??

PBDEs and PCBs are implicated in PBDEs and PCBs are implicated in thyroid endocrine disruptionthyroid endocrine disruption::

……in mammalian studies, PBDE exposure causes in mammalian studies, PBDE exposure causes reduction of thyroid hormone level and reduction of thyroid hormone level and production production (e.g., Skarman et al., 2005; Boas et al., (e.g., Skarman et al., 2005; Boas et al., 2006)2006)

……in fish, PBDE exposure causes reduction in T4, in fish, PBDE exposure causes reduction in T4, but not T3but not T3 (Tomy et al., 2004 (Tomy et al., 2004 –lake trout–lake trout; Lema et al., ; Lema et al., 2006 2006 –fathead minnow–fathead minnow))

……in fish, PCB exposure alters T4 and T3 levelsin fish, PCB exposure alters T4 and T3 levels (LeRoy et al., 2006 (LeRoy et al., 2006 –Atlantic croaker–Atlantic croaker))

ReproductiReproductive Steroidsve Steroids

400

300

200

100

0

Pacific Staghorn Pacific Staghorn SculpinSculpin

Plasma 17Plasma 17ββ-Estradiol -Estradiol ConcentrationsConcentrations

FemalFemale e [winte[winter]r]

Female Female [summe[summe

r]r]

Male Male [winte[winte

r]r]

FemaleFemale MaleMale[immatu[immature]re]

English Sole English Sole [Orange County][Orange County]

all SF all SF Bay Bay

locations locations testedtested

[n=13-29/bar][n=13-29/bar]

E2 (

pg

/ml)

E2 (

pg

/ml)

N.D.N.D. for for Testosterone Testosterone

(selected (selected samples)samples)

Shiner Surfperch ?Shiner Surfperch ?A

ug

ust

2006

Oakla

nd

In

ner

Au

gu

st

2006

Oakla

nd

In

ner

Harb

or

Harb

or

0

50

100

TurbotWWTP-affected

site

Shiner Surfperch

Pla

sma E

E2

Conce

ntr

ati

on (

pg/m

l)1717αα-Ethinylestradiol -Ethinylestradiol ((EE2) in Fish PlasmaEE2) in Fish Plasma

150

200

Pacific Staghorn Sculpin

(Tomales Bay, Redwood City, Oakland Inner Harbor, Berkeley, San Pablo Bay (Loch Lohmand)

N.N.D.D.

N.N.D.D.

The The Growth Growth

EndocrinEndocrine Axise Axis

PITUITARYPITUITARY

LIVER

IGFIGF-I-I

IGFBP-3IGFBP-3IGFBP-1IGFBP-1(IGFBPs 2,4-(IGFBPs 2,4-

6)6)

IGFBPIGFBP

GROWTGROWTHH

target cell

Growth HormoneGrowth Hormone

IGF IGF RECREC

IGFIGFIGFBPIGFBP IGFIGF

““IGF IGF Axis”Axis”

45 kDa

30 kDa

28 kDa

ControlsControls

30 kDa 30 kDa IGFBP-1IGFBP-1

Rat Plasma Rat Plasma ControlControl

Stressed Stressed

Jack MackerelTrachurus symmetricus

increased IGFBP-1 >> growth increased IGFBP-1 >> growth inhibitioninhibition

Stress Effects on Stress Effects on Plasma IGFBP Plasma IGFBP

LevelsLevels

cell culture work supports cell culture work supports thisthis

ThyroxineThyroxine & & TriidothyronineTriidothyronine

TestosteroneTestosterone & 11- & 11-ketotestosterone ketotestosterone

1717ββ-Estradiol-Estradiol & & 1717αα--EthinylestradiolEthinylestradiol

IGF-I & IGF-I & IGFBPsIGFBPs

Endocrine Disruption—SF Endocrine Disruption—SF Bay FishBay Fish

Cortisol Cortisol & Cortisone & Cortisone

** **

POTW, POTW, seasonaseasona

l l effectseffects

??????!!

((June 2007June 2007))

[[YellowYellow=Year-1 fish =Year-1 fish analyses]analyses]

**=spatial differences detected=spatial differences detected

Hepatic contaminants Hepatic contaminants (chlorinated pesticides, PCB (chlorinated pesticides, PCB congeners, PAHs)congeners, PAHs)

San Pablo Bay San Pablo Bay (Loch Lohmand (Loch Lohmand Marina)Marina)

Berkeley WaterfrontBerkeley Waterfront

Oakland Inner HarborOakland Inner Harbor

Redwood CityRedwood City

far-field far-field referencereference:: Tomales BayTomales BayBodega Bodega BayBay

Study Sites Study Sites 20072007 July-AugustJuly-August

RichmondRichmond (Laurentian Channel, (Laurentian Channel, DDTs)DDTs)

San Leandro BaySan Leandro Bay (PCBs)(PCBs)

South of DunbartonSouth of Dunbarton (POTW)(POTW)

Stress (HPI) Axis –Stress (HPI) Axis –Continuing Continuing Analyses 2007Analyses 2007

Field season July-Aug 2007Field season July-Aug 2007

• a) interrenal histologya) interrenal histology• b) hepatic contaminants correlation b) hepatic contaminants correlation • c) ACTH-challengec) ACTH-challenge**• d) cortisone measurementd) cortisone measurement** (11(11ββ-HSD-2)-HSD-2)

• e) ectoparasitic infestation assessment e) ectoparasitic infestation assessment

• f) growth endocrine system (IGF-I, IGFBPs) f) growth endocrine system (IGF-I, IGFBPs) • g) steroidogenic enzyme expressiong) steroidogenic enzyme expression**

(StAR, 11(StAR, 11ββ-hydroxylase, 11-hydroxylase, 11ββ-HSD-2)-HSD-2)

*b-d = potential bioassays for disruption *b-d = potential bioassays for disruption (EDC (EDC screening?)screening?)

With an emphasis on With an emphasis on Oakland Inner HarborOakland Inner Harbor (San Pablo Bay, San (San Pablo Bay, San Leandro Bay??) Leandro Bay??) vsvs. . control sitescontrol sites

Thyroid (HPT) Axis –Thyroid (HPT) Axis –Continuing Continuing Analyses 2007Analyses 2007

Field season July-Aug 2007Field season July-Aug 2007

• a) T3 measurementa) T3 measurement**• b) hepatic contaminants correlation b) hepatic contaminants correlation • c) thyroid histology [?]c) thyroid histology [?]• d) TSH-challenged) TSH-challenge** [?] [?]• e) peripheral deiodinases (D1-3)e) peripheral deiodinases (D1-3)** [?] [?] • f) thyroid hormone receptors (TRf) thyroid hormone receptors (TRαα & TR & TRββ))** [?][?]

• g) growth endocrine system (IGF-I, IGFBPs)g) growth endocrine system (IGF-I, IGFBPs)

*b-d = potential bioassays for disruption *b-d = potential bioassays for disruption (EDC screening?)(EDC screening?)

?? ?? QUESTIONS QUESTIONS

Liver Contaminant Analysis, Liver Contaminant Analysis, GC/MSGC/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’-DDTSo-Calif would have 4, 4’-DDT Nov. 2006Nov. 2006

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, 128+167, 141, 151, 156, 158, 168+132, 170, 177, 183, 187, 200

1 – 16 2 – 35

*range of ng/g wet weight values shown

Nov. 2006Nov. 2006

Liver Contaminant Analysis, Liver Contaminant Analysis, GC/MSGC/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. 2006Nov. 2006

Liver Contaminant Analysis, Liver Contaminant Analysis, GC/MSGC/MS

Russell Fairey Russell Fairey et al.et al. Moss Moss

Landing Marine Landing Marine LaboratoriesLaboratories

Southern California Sea Grant Program Southern California Sea Grant Program

San Francisco Estuary InstituteSan Francisco Estuary Institute

cholesterol

pregnenolone

progesterone

11-deoxycorticosterone

corticosterone

aldosteronealdosterone

mitochondria

17-hydroxypregnenolone

17-hydroxyprogesterone

11-deoxycortisol

cortisol

dehydroepiandrosterone

androstenedione

testosterone

estradiol-17

Cortisone (inactive)

1111-HSD-2-HSD-2

steroidogenic targets

StAR, PBRStAR, PBR

P450scP450sccc

P450P4501111

P450P450aroaromm

Thyroxogenesis

handlinghandlinghandlinghandling crowding / captivitycrowding / captivitycrowding / captivitycrowding / captivity water qualitywater qualitywater qualitywater quality

Stimulate the HPI AxisStimulate the HPI Axis

elevatedelevated CORTISOLCORTISOL levelslevelselevatedelevated CORTISOLCORTISOL levelslevels

↑ Glucose, other fuels ↑ Hepatic glucose production ↑ AA release from muscle ↑ Lipid breakdown

Overall: increased increased fuel fuel mobilization & mobilization & availabilityavailability

↑ Glucose, other fuels ↑ Hepatic glucose production ↑ AA release from muscle ↑ Lipid breakdown

Overall: increased increased fuel fuel mobilization & mobilization & availabilityavailability

↓Growth & Repair ↓Reproduction ↓Immune function

Overall: reduced energy- reduced energy- expensive processesexpensive processes

↓Growth & Repair ↓Reproduction ↓Immune function

Overall: reduced energy- reduced energy- expensive processesexpensive processes

Anabolic PathwaysAnabolic PathwaysCatabolic PathwaysCatabolic Pathways

StresStress!!s!!

StresStress!!s!!

Elthusa Elthusa californicacalifornica

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