Metabolomic and transcriptomic analysis reveals endocrine disruption in Skeena River (British Columbia) Sockeye salmon during the 2008 spawning migration

John R. Cosgrove,1 Jonathan P. Benskin,1,2 Michael G. Ikonomou,2 Nik Veldhoen,3 Cory Dubetz,2 Caren C. Helbing3

1. AXYS Analytical Services Ltd. 2045 Mills Road West, Sidney BC, Canada, V8L 5X22. Institute of Ocean Sciences, Fisheries and Oceans Canada (DFO), 9860 West Saanich Road, Sidney BC, Canada, V8L 4B23. Department of Biochemistry & Microbiology, University of Victoria, P.O. Box 3055 Stn CSC, Victoria, B.C. Canada, V8W 3P6

•Sockeye are a major commercial and wildlife food source in British Columbia.•Over 80% of sockeye production from two systems, the Fraser and Skeena rivers.•Dramatic fluctuations in the number of salmon returning to spawn raised concern over the health of BC Sockeye Salmon

Background

Harrison River

Weaver Creek

Fulton Creek

Pinket Creek

• Hypothesis: Exposure to environmental contaminants during in-stream migration effects spawning success.

• Hypothesis: Exposure measurable via alterations in hepatic gene transcription.

• Results: Greater alterations in sex-specific hepatic gene transcripts in Fulton and Pinkut Creek salmon than in Weaver R. & Harrison Cr.

Background: Veldhoen et al. (2013) Comp. Biochem. Physio;. 157:150-161.

Objectives

1) To investigate whether fish displaying altered hepatic vitellogenin A expression can also be differentiated metabolomically.

2)To assess gender-specific changes in hepatic metabolome observed during in-migration up the Skeena River

Methods: Salmon sampling

• In 2008, livers (n=76) harvested from fish caught at the mouth of the Skeena (later confirmed as Fulton stock using DNA-based fingerprinting).

• Additional livers harvested from adult male and female sockeye salmon collected from the Fulton River (n=43) and Pinkut Creek (n=50 fish) spawning grounds.

• Genotypic sex: QPCR using genomic DNA from each animal.

• Gametic sex: individual salmon inspected visually for the presence of milt or roe.

• Transcriptomic sex: classified using hepatic vitellogenin A expression fold changes:

• Male: <100• Female: ≥100

Methods: Gender determination

Genotypic and phenotypic sex

% of total fish from each gender class (Genetic-Gametic-Transcriptomic)

Site MMM FFF MMF FFM Other

Skeena Mouth (n=75; Fulton

stock)53 41 3 1 1

Fulton (n=37) 43 32 0 16 8

Pinkut (n=50) 8 44 40 8 0

“Normal” “Abnormal”

Methods: Metabolite analysis

• Metabolites measured with kit-based approach (Biocrates Life Sciences) using FIA-MS/MS or LC-MS/MS instrumental analysis.

• Liver tissue was homogenized with liquid nitrogen; subsample extracted with 100% Methanol.

Targets (186)

R2

R1

O

O

C

CO

O

CH2

CHCH2

XO

O-

O

O

P

O

NH

XO

OH

CH3

CH3

R

NH2

CH3

O

CH3O

O

R

O

OCH3

CH3

CH3

N+

O

OH

HHH

OH

H

OH OH

H

OH

R

NH2

∑Hexose (H1)

Biogenic amines

Amino acids

Acylcarnitines

Sphingomyelins (SMs)

Glycerophospholipids (PCs)

QA/QC

• Method Validation– Extraction efficiency

– Spike/recovery

– Temperature

– Solvent choice

• Ongoing verification– Inter- and intra-plate QCs (reference plasma)

– Blanks

– Replicates

– Challenge to measure all targets in livers (146/186)

Results

• Associations between Metabolite Concentrations and:– Migration point– Spawning sites– Gender classification

Hepatic Metabolome Through Migration

FultonSkeena Mouth

Skeena Mouth

Fulton River

Lipids

Carnitine

Hepatic Metabolome Through Migration: Females

*p<0.05 **p<0.01

lysoPCa = ∑lysophosphatidylcholines PCaa = ∑phosphatidylcholines

PCaa = ∑acylalkylphosphatidylcholines SM(OH) = ∑hydroxysphingomyelins

SM = ∑sphingomyelins alpha-AAA= -aminoadipic acid

CO = carnitine

*

***

**

** **

**

*

*

Hepatic Metabolome Through Migration:Males

*p<0.05 **p<0.01

lysoPCa = ∑lysophosphatidylcholines PCaa = ∑phosphatidylcholines

PCaa = ∑acylalkylphosphatidylcholines SM(OH) = ∑hydroxysphingomyelins

SM = ∑sphingomyelins alpha-AAA= -aminoadipic acid

CO = carnitine

Gender: Pinkut Creek

FFF vs FFM MMM vs MMF

Significant metabolites: carnitine, Orn, Lys, DOPA, -aminoadipic acid

Significant metabolites: phosphatidyl cholines, Met, Arg, Thr, -aminoadipic

acid

Pinket

Gender: Fulton RiverFFF vs FFM

Significant metabolites: phosphatidyl cholines, -aminoadipic acid

Fulton

Mean -aminoadipic acid concentrations for Fulton River & Pinkut Creek Salmon:(Low vitellogenin A status with elevated -aminoadipic acid)

p<0.05 p<0.05

Altered -aminoadipic acid & vitellogenin A?

• Connection between changes in vitellogenin expression and alpha-aminoadipic acid unclear.

• Relevance of elevated alpha-aminoadipic acid?– Alpha-aminoadipic acid: intermediate in metabolism of

Lysine to Carnitine, integral to FA metabolism/ mitochondrial ß-oxidation.

• Chatzitos et al. (1996) – limited availability of carnitine increased hepatic long chain FA (indicative of reduced ß-oxidation in sea bream).

• Rathore et al (2010) – Lysine limitation alters storage patterns of protein, lipid and glycogen in Atlantic salmon.

Migratory Impacts on Metabolism: - More demanding migration in Skeena River may result in differential mobilization/metabolism of lipids/proteins.- This may increase lipophilic contaminant exposure, endocrine disruption & altered hepatic gene expression.

Skeena Fraser River

Fulton Pinkut Harrison Weaver

In-river distance (km)

500 550 113 120

Elevation gain (m)

711 711 10 26

Kelly et al. (2007)-Lipophilic contaminants are mobilized during upstream migration (Sockeye).•Zhang et al. (2011)• Drop in -AAA concentrations following exposure to PFDoA (rats).

Summary

•Measured gender- and location specific changes in the hepatic metabolome of migrating Sockeye Salmon.

•Elevated -aminoadipic acid and hepatic lipids (but decreased carnitine) at spawning sites versus river entry.

•Higher -aminoadipic acid concentrations associated with “male” hepatic vitellogenin A status.

•Causes of association unclear:• Disrupted ß-oxidation vs. protein-based glycolysis• Contaminant exposure (in-river or via lipid release)

Fisheries and Oceans

CanadaPêches et OcéansCanada