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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