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Welcome to SWAMP Webinar

Title: Sublethal responses to environmental stressors in fish: an integrative multi-biomarker approach

Welcome to SWAMP Webinar

Title: Sublethal responses to environmental stressors in fish: an integrative multi-biomarker approach

Sublethal responses to environmental stressors in fish: an integrative multi-biomarker

approach

Sublethal responses to environmental stressors in fish: an integrative multi-biomarker

approach

Aquatic Toxicology LaboratorySchool of Veterinary Medicine - UC Davis

Richard Connon, Sebastian Beggel, Erika Fritsch, Richard Connon, Sebastian Beggel, Erika Fritsch, Leandro D’Abronzo, Becca Hudson-Davies, Leandro D’Abronzo, Becca Hudson-Davies,

Linda Deanovic, and Inge WernerLinda Deanovic, and Inge Werner2

Outline

• Molecular techniques background (5’-10’)• Links to relevant higher levels of

organization• Application of multiple biomarkers in delta

smelt• Ammonia/um in river water study• Current, future and proposed work• Other studies in model species (Inge

Werner)

3

www.mfi.ku.dk/ppaulev/chapter31/kap31.htm 4

wwwchem.leidenuniv.nl/metprot/armand/023.html

e.g. Metallothionein

5

Quantitative PCR

6

Time-dependent change in CYP1A mRNA transcription and protein activity (EROD) expression in striped bass exposed to 10mg.kg-1 -naphthoflavone.

0

50

100

150

200

250

300

0 1 2 3 4 5 6 7 8

Time after exposure (days)

Cyp

1a tr

ansc

riptio

n (r

elat

ive

to b

-Act

in)

0

10

20

30

40

50

60

70

80

ER

OD

Act

ivity

(pm

oles

/min

/mg

prot

)

mRNA

EROD

Contaminant insult (0h)

Peak mRNA response (6h)

Peak protein activity (12h)

Connon RE, Durieux EDH, D’Abronzo LS, Ostrach DJ, and Werner I. Cytochrome P450 induction in the striped bass (Morone saxatilis): signature biomarkers of past and present xenobiotic exposure (in prep).

7

-50

0

50

100

150

200

250

300

0 1 2 3 4 5 6 7 8 9


Cyp

1a

tra

nsc

rip

tion

(re

lativ

e t

o

-A

ctin

) Solvent control

BNF 10 mg/kg

T0

0

15

30

45

60

75

90

0 1 2 3 4 5 6 7 8 9


ER

OD

Act

ivity

(pm

oles

/mg

prot

/min

)

Solvent Control

BNF 10 mg.kg-1

T0

8

Time-dependent change in CYP1A mRNA transcription and protein activity (EROD) expression in striped bass exposed to 10mg.kg-1 -naphthoflavone.

Connon RE, Durieux EDH, D’Abronzo LS, Ostrach DJ, and Werner I. Cytochrome P450 induction in the striped bass (Morone saxatilis): signature biomarkers of past and present xenobiotic exposure (in prep).

Microarrayse.g. cDNA library, PCR amplified fragments, purified, and

printed onto epoxysilane coated glass slides.

9

What is a microarray and

how does it work?

Exposed Control

10

/ =

Exposed ControlRatio = ExposedControl

Smelt Clone ID Response Fold Gene most similar to Species Match Accession No E-ValueDS[96]_39_B_06 Up 2.36 1-acylglycerol-3-phosphate O-acyltransferase 3 Danio rerio NP_998590 4.00E-68DS[96]_77_C_03 Up 4.88 actin alpha 2, skeletal muscle Pagrus major BAF80060 1.00E-94DS[96]_03_E_12 Up 3.75 actin, alpha 2, smooth muscle, aorta Danio rerio AAH75896 e-107DS[96]_19_B_07 Up 2.92 actin, alpha, cardiac muscle 1 like Danio rerio NP_001001409 e-141DS[96]_34_A_06 Up 3.11 actin, alpha, cardiac muscle 1 like Danio rerio NP_001001409 e-148DS[96]_29_G_10 Up 3.28 actin, alpha, cardiac muscle 1 like Danio rerio NP_001001409 e-145DS[96]_12_G_03 Up 4.58 actin, alpha, cardiac muscle 1 like Danio rerio NP_001001409 e-125DS[96]_21_H_11 Up 6.10 actin, alpha, cardiac muscle 1 like Danio rerio NP_001001409 e-127DS[96]_38_C_06 Up 2.51 actin, beta Acanthopagrus schlegelii AAR84618 e-122DS[96]_05_A_08 Up 1.83 acyl-CoA synthetase long-chain family member 5 Tetraodon nigroviridis CAG06540 e-102DS[96]_47_G_04 Up 3.47 aldolase a, fructose-bisphosphate Danio rerio NP_919358 e-124DS[96]_63_E_08 Up 1.86 alpha tubulin, (protein LOC573122) Danio rerio NP_001098596 e-120DS[96]_05_B_06 Up 3.06 amylase-3 protein Pseudopleuronectes americanus AAF65827 e-144DS[96]_70_B_05 Up 3.36 amylase-3 protein Tetraodon nigroviridis CAC87127 3.00E-54DS[96]_34_D_11 Down 4.54 APEX nuclease (apurinic/apyrimidinic endonuclease) 2 Xenopus tropicalis NP_001006804 6.00E-25DS[96]_01_C_12 Up 1.80 apolipoprotein Tetraodon nigroviridis CAG03661 1.00E-38DS[96]_88_F_11 Up 2.28 apolipoprotein A-I Danio rerio NP_571203 1.00E-81DS[96]_74_G_02 Up 3.34 apolipoprotein A-I Danio rerio NP_571203 1.00E-81DS[96]_72_D_09 Up 7.67 apolipoprotein A-I Danio rerio NP_571203 6.00E-73DS[96]_11_H_09 Up 3.99 apolipoprotein A-I-1 precursor (Apo-AI-1) Oncorhynchus mykiss O57523 8.00E-76DS[96]_88_D_02 Up 4.81 apolipoprotein A-I-2 precursor Oncorhynchus mykiss O57524 4.00E-71DS[96]_02_A_11 Up 2.72 apolipoprotein A-IV Danio rerio AAH93239 1.00E-73DS[96]_41_F_02 Up 2.23 apolipoprotein B Salmo salar CAA57449 3.00E-24DS[96]_62_B_05 Up 2.02 apolipoprotein CII Oncorhynchus mykiss AAG11410 1.00E-19DS[96]_65_E_08 Up 2.17 apolipoprotein CII Oncorhynchus mykiss AAG11410 3.00E-19DS[96]_45_B_03 Up 4.16 apolipoprotein Eb Danio rerio NP_571173 2.00E-38DS[96]_79_H_01 Up 4.42 arachidonate 12-lipoxygenase Danio rerio NP_955912 4.00E-23DS[96]_77_B_08 Up 2.56 arachidonate 12-lipoxygenase Danio rerio NP_955912 5.00E-35DS[96]_86_F_12 Up 3.87 astacin like metallo-protease Oryzias latipes NP_001098207 2.00E-83DS[96]_17_B_07 Up 4.34 astacin like metallo-protease Oryzias latipes NP_001098207 7.00E-50DS[96]_07_A_09 Up 2.17 c1q-like protein Dissostichus mawsoni ABN45966 3.00E-38DS[96]_54_A_03 Down 1.69 calcitonin receptor-like receptor Oncorhynchus gorbuscha CAD48406 5.00E-56DS[96]_53_D_10 Down 1.58 calcium binding protein 39 Danio rerio NP_998666 1.00E-76DS[96]_71_F_06 Up 2.27 calpain 1 protein Danio rerio AAH91999 2.00E-68DS[96]_37_C_02 Up 1.82 carboxypeptidase H Paralichthys olivaceus AAO92752 1.00E-82DS[96]_27_A_09 Up 2.91 cardiac muscle ATP synthase,alpha 1, Danio rerio NP_001070823 7.00E-62DS[96]_05_C_08 Up 5.76 cell division cycle 14 homolog A Danio rerio CAP09233 3.00E-19DS[96]_30_G_11 Up 2.34 chitin binding Peritrophin-A domain Danio rerio AAH45331 4.00E-69DS[96]_33_H_06 Up 3.75 chitinase Oncorhynchus mykiss CAD59687 9.00E-68DS[96]_69_C_08 Up 4.25 chitinase1 Paralichthys olivaceus BAD15059 e-127DS[96]_71_C_06 Up 3.93 chymotrypsinogen 2-like protein Sparus aurata AAT45254 1.00E-20DS[96]_56_B_04 Down 2.01 cofilin 2 (muscle) Danio rerio NP_991263 5.00E-84DS[96]_59_A_01 Down 2.35 cofilin 2 (muscle) Danio rerio NP_991263 3.00E-83DS[96]_67_A_02 Up 5.20 corticotropin-lipotropin A precursor Oncorhynchus mykiss Q04617 7.00E-63DS[96]_26_G_05 Down 1.59 cytochrome P450, family 46, subfamily A, polypeptide 1 Danio rerio NP_001018358 2.00E-65DS[96]_11_H_05 Up 1.86 DAZAP2-like protein (deleted in azoospermia-associated) Takifugu rubripes NP_001072102 5.00E-59DS[96]_18_C_06 Down 1.78 dopachrome tautomerase Salmo salar ABD73808 1.00E-85DS[96]_20_B_11 Down 1.66 E3 ubiquitin-protein ligase MARCH2 Danio rerio Q1LVZ2 2.00E-87DS[96]_68_G_10 Up 5.08 elastase 2-like protein Sparus aurata AAT45251 2.00E-89

Microarray Microarray

differentially expressed differentially expressed

gene listgene list

Microarray Microarray

differentially expressed differentially expressed

gene listgene list

12

mindpowersystem.com/15/interrogation-methods/

• Provides information on the mode of action

• Allows test organisms to tell us what we should be looking at

• Permits the development and selection of biomarkers without preconceived ideas

• No prior knowledge of the system required

• Identifies specific effects on organism/condition in question

• Provides information on the mode of action

• Allows test organisms to tell us what we should be looking at

• Permits the development and selection of biomarkers without preconceived ideas

• No prior knowledge of the system required

• Identifies specific effects on organism/condition in question

Microarrays, biomarkers in monitoring

13

mRNAmRNA

PopulationPopulation

Linking Genomic Profiles with Relevant Higher Level

Effects:Adverse Outcome Pathways

Linking Genomic Profiles with Relevant Higher Level

Effects:Adverse Outcome Pathways

14

Molecules

Population and Community

Physiology

Immunology

Histopathology

Bioenergetics

withLinking

MoleculesHigher Levels of

Biological Organization

Cells

…

15

Development and Reproduction

Detoxification

Molecules

Population and Community

Physiology

Immunology

Histopathology

Bioenergetics

Detoxification

Development and Reproduction

with

Adverse Outcomes

Linking Molecules

Higher Levels of Biological Organization

Cells

…

16

Measure it

Swim it…

Bridge it

17

Swimming performance and aspartoacylase transcription in

larval delta smelt exposed to esfenvalerate

Connon RE, et al (2009). Linking mechanistic and behavioral responses to sublethal esfenvalerate exposure in the endangered delta smelt; Hypomesus transpacificus (Fam. Osmeridae). BMC Genomics.

0

10

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100

0.00 0.05 0.10 0.15 0.20 0.25 0.30Esfenvalerate Concentration (mg.L-1)

Sw

imm

ing

Per

form

ance

(re

spo

nse

%)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Asp

arto

asyl

ase

tran

scri

pti

on

(fo

ld-

chan

ge)

Swimming

Aspartoacylase

****** ***

18

Muscle Contraction

19

Genomic profiling: Responses in juvenile

delta smelt exposed for 7 days to ambient

samples from four sites in the Bay-Delta System,

along with respective high and low EC controls

20

Key:

Activity † Structure §

† † † † † † §§§§ §

Genomic profiling: Hood

21

Survival and swimming of Larval Delta Smelt after 7-d Exposure to

Ammonium Chloride(in Sacramento River water

from Garcia Bend)

NH4+Cl(mg.L-1

NH3/NH4+)

7-d LC50 7.5

0

20

40

60

80

100

0.00 2.00 4.00 6.00 8.00

SRGB

Hatchery Control

Concentration mg.L-1 NH3/NH4+

Per

cen

t S

urv

ival

(m

ean

, S

E)

0%

20%

40%

60%

80%

100%

2 38 71 91 116

Immobile Mobile Highly mobile


% o

f T

ime

Sw

imm

ing

D

uri

ng

90

Sec

on

ds

<20% 20-60% >60%

Ctr 2 4 6 8

* * *

22

Functional classification of genes responding to4 mg.L-1 NH3/NH4

+ from NH4Cl

Ammonium Transport, 1 Urea cycle, 3

Digestion, 2

Iron storage, 1

Immunoregulation, 1

Muscle, 3

Neurological, 5

Olfactory receptor, 1

Protein degradation, 5

Skin Integrity, 2

Structural (bone & vertebra), 2

Other Cellular processes, 18

Apoptosis, 2

Sacramento River at Garcia Bend(upstream from Wastewater Treatment Plant)

23

-10

-5

0

5

10

15

20

0 2 4 6

-6

-4

-2

0

2

4

6

8

0 2 4 6

Muscular responses Neurological responses

-6

-4

-2

0

2

4

6

0 2 4 6

Direct link to ammonia responses

NH4 transp

C/EBPD1

* * **

Molecular biomarker responses (n=10)

Genes were selected from top 10 statistically significant responses identified with microarray – and others from previous tests (e.g. Creatine Kinase, Aspartoacylase)

Gen

e tr

ansc

rip

tio

n (

rela

tive

to

-A

ctin

)

Ammonia/um Concentration (mg.L-1)


24

-10

-5

0

5

10

15

20

0 2 4 6

-6

-4

-2

0

2

4

6

8

0 2 4 6

Muscular responses Neurological responses

Gen

e tr

ansc

rip

tio

n (

rela

tive

to

-A

ctin

)


Decr. High Mobility

-6

-4

-2

0

2

4

6

0 2 4 6

Direct link to ammonia responses

NH4 transp

C/EBPD1

* * **

0%

20%

40%

60%

80%

100%

2 38 71 91 116

Immobile Mobile Highly mobile


% o

f T

ime

Sw

imm

ing

D

uri

ng

90

Sec

on

ds

<20% 20-60% >60%

Ctr 2 4 6 8

* * *

Decr. High Mobility

25

Hung CYC, et al (2007) Rhesus glycoprotein gene expression in the mangrove killifish Kryptolebias marmoraturs exposed to elevated environmental ammonia levels and air. J. Experimental Biology.

Quantitative PCR mRNA expression of RHbG, RhCG1 and RhCG2 in control and ammonia-exposed K. marmoratus.

26

Current, future and proposed work

• Generating contaminant-specific genomic profile database, using microarray and qPCR technology

• Assaying field based exposures

• Developing biomarkers linked to reproduction

Questions we’re seeking answers to:

• Can we use profiling system to aid TIE investigations based on chemical modes of action?

• Do all fish species respond comparatively to equitoxic contaminant concentrations?

27

Molecular Biomarkers as Tools in Toxicity Testing and Field

Monitoring ?

Major Challenges:

Laboratory toxicity testing with resident species;

Often difficult to culture and handle.

Require extensive method development.

Can generally not be used for in-depth studies evaluating ecologically important chronic endpoints, such as reproductive success, behavior and/or growth.

Interpretation of biomarker data collected in field monitoring studies.

Model Fish Species in Toxicity Testing

• Rainbow Trout (survival, embryo test, growth)

• Fathead Minnow (growth, survival, reproduction)

• Zebrafish (everything?)

Important Questions:

• Are expression patterns of selected biomarker genes reflective of species sensitivity and stressor type across species?

• Are molecular biomarkers mechanistically linked to contaminant type and ecologically relevant effects?

….. Connecting the Dots…..

Delta SmeltBiomarkers

Other Available BiomarkerInformation

Fathead Minnow

Establish Linkages (the “Bridge”) to Ecologically Relevant Adverse Effects: growth, reproduction, behavior, survival

Toxicant Identification Tools for Model Species

Mechanism-Based,

InterpretableBiomarkers

for Field Studies with

Resident Species

Neuromuscular Effects of Pesticides: Swimming Behavior of Larval Fathead Minnow after 24 h

Exposure to Talstar® (Bifenthrin)

Control 20% LC10 33% LC10 50% LC10

0

10

20

30

40

50

60

70

80

0 0.2 0.4

24 h (exposed)

48 h (24h recovery)

168 h (6-d recovery)Nr. Lines crossed/ min

Concentration bifenthrin pure a.i. [μg/L]

***

***

***

***

***

20% LC10=140 ng/L

Swimming Activity in Fathead Minnows Exposed to Bifenthrin for 24 h

Beggel S, Werner I, Connon RE, Geist JP. (2010) Science of The Total Environment. 408: 3169-3175.

**

*

*

*

Growth in Fathead Minnows

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Control SolvControl 10% LC10 20% LC10 33% LC10 50% LC10

We

igh

t p

er

fis

h (

mg

)

bifenthrin pure A.I. bifenthrin formulation

Gene Function GeneGeneral Stress Response HSP70, HSP90

Detoxification Metallothionein, Glutathione-S Transferase, CYP1A

Endocrine Disruption/Development

Vitellogenin, CYP3A, Zona Pellucida Protein, Glucocorticoid Receptor

Iron Storage Ferritin

Neuromuscular Function/Energy

Parvalbumin, Creatine Kinase, Epithelial Ca+ Channel, Aspartoacylase, Titin, Myozenin, G6PD

Growth Insuline-Like Growth Factor, Growth Hormone

Apoptosis Caspase 1

Immune System Microglobulin, IL8, Mx Protein, Nramp

Selected Molecular Biomarkers in Fathead Minnow

Fold

chan

gein

gen

eex

pres

sion

(EF1

a no

rmal

ized

)

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Vitellogenin

Zona Pellucida

glucocorticoidreceptor

Growthhormoneigf

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Cyp3a

Cyp1a

HSP90

GST

Metallothionein

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

parvalbumin

creatine kinase

Aspartoacylase

eCaCH

G6PD

Concentration bifenthrin [µg/L]

Fold

chan

gein

gen

eex

pres

sion

(EF1

a no

rmal

ized

)

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Vitellogenin

Zona Pellucida


Growthhormoneigf

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Cyp3a

Cyp1a

HSP90

GST

Metallothionein

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

parvalbumin

creatine kinase

Aspartoacylase

eCaCH

G6PD


-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Vitellogenin

Zona Pellucida


Growthhormoneigf

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

Cyp3a

Cyp1a

HSP90

GST

Metallothionein

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

parvalbumin

creatine kinase

Aspartoacylase

eCaCH

G6PD


-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 20 40 60 80 100 120 140 160 180

Cyp3A

Cyp1A

HSP90

GST

Metallothionein

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 20 40 60 80 100 120 140 160 180

Vitellogenin

Zona Pellucida

glucocorticoidreceptorGrowthhormoneigf

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 20 40 60 80 100 120 140 160 180

Parvalbumin

creatine kinase

Aspartoacylase

eCaCh

G6PD

Concentration fipronil [µg/L]

Fo

ldch

an

ge

in g

en

ee

xpre

ssio

n(E

F1

a n

orm

aliz

ed)

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 20 40 60 80 100 120 140 160 180

Cyp3A

Cyp1A

HSP90

GST

Metallothionein

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 20 40 60 80 100 120 140 160 180

Vitellogenin

Zona Pellucida

glucocorticoidreceptorGrowthhormoneigf

-6

-5

-4

-3

-2

-1

0

1

2

3

4

0 20 40 60 80 100 120 140 160 180

Parvalbumin

creatine kinase

Aspartoacylase

eCaCh

G6PD

Concentration fipronil [µg/L]

Fo

ldch

an

ge

in g

en

ee

xpre

ssio

n(E

F1

a n

orm

aliz

ed)Impaired Swimming Impaired Swimming

Homeostasisdisrupted

Homeostasisdisrupted

Genomic fingerprinting (Heatmaps). Molecular biomarkers distinguish between bifenthrin and fipronil exposed fathead minnow.

Beggel S, Werner I, Connon RE, Geist JP. (in preparation). Changes in gene expression in larval fathead minnow (Pimephales promelas) following short-term exposure to bifenthrin and fipronil.

Endocrine disruption in larval fathead minnows following exposure to Triclosan.

Erika Fritsch (Current work)

IGF1=Insulin Growth FactorVASA: sexual dimorphismDMRT: sexual dimorphism

Expression of selected biomarker genes in

Columbia River Steelhead Trout

*

Rela

tiv

e g

en

e e

xp

ress

ion

(-

Acti

n n

orm

ali

zed

)

-5

0

5

10 nRAMP MX MT CXC HSP70 HSP90

-30

-25

-20

-15

-10

-5

0

5

10

Excellent Good Fair Poor

Physical appearance classification

Gill

Kidney

***

***

***

******

***

***

**

*

*

Rela

tiv

e g

en

e e

xp

ress

ion

(-

Acti

n n

orm

ali

zed

)

-5

0

5


-30

-25

-20

-15

-10

-5

0

5

10



Gill

Kidney

***

***

***

******

***

***

**

*

*

-5

0

5


-30

-25

-20

-15

-10

-5

0

5

10



Gill

Kidney

***

***

***

******

***

***

**

*

*

D’Abronzo et al., in prep.

Conclusions

• Microarray technology allows us to identify new mechanism-based molecular biomarkers.

• The multi-biomarker approach (heatmaps) integrates old and new information.

• Shows great promise in distinguishing the impact of different stressors (TIE).

• Allows mechanism based interpretation of biomarker signals.

• Leads to a better understanding of linkages (“adverse outcome pathways”).

AcknowledgmentsAcknowledgments

The Interagency Ecological Program, Sacramento, CA, for funding the microarray and biomarker work.

The UC Davis Fish Conservation and Culture Laboratory, Byron, CA, for providing delta smelt.

The staff of the UCD Aquatic Toxicology Lab.

Sebastian Beggel & Erika Frisch, Graduate Students in I. Werner’s lab.

The Interagency Ecological Program, Sacramento, CA, for funding the microarray and biomarker work.

The UC Davis Fish Conservation and Culture Laboratory, Byron, CA, for providing delta smelt.

The staff of the UCD Aquatic Toxicology Lab.

Sebastian Beggel & Erika Frisch, Graduate Students in I. Werner’s lab.

Documents

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