1. 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. Helbing 3 1. AXYS Analytical Services Ltd. 2045 Mills Road West, Sidney BC, Canada, V8L 5X2 2. Institute of Ocean Sciences, Fisheries and Oceans Canada (DFO), 9860 West Saanich Road, Sidney BC, Canada, V8L 4B2 3. Department of Biochemistry & Microbiology, University of Victoria, P.O. Box 3055 Stn CSC, Victoria, B.C. Canada, V8W 3P6

2. 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

3. 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.

4. 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

5. 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.

6. 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

7. Genotypic and phenotypic sex % of total fish from each gender class (Genetic-Gametic-Transcriptomic) SiteMMMFFFMMFFFMOther Skeena Mouth (n=75; Fulton stock) 5341311 Fulton (n=37)43320168 Pinkut (n=50)8444080 “Normal” “Abnormal”

8. 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.

9. Targets (186) ∑Hexose (H1) Biogenic amines Amino acids Acylcarnitines Sphingomyelins (SMs) Glycerophospholipids (PCs)

10. 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)

11. Considerations of a kit-based analytical approach Many (146 of 186 targets) were consistently quantifiable (but most acylcarnitines and several biogenic amines were <LOD in salmon liver). Pre-concentrating or increasing sample size resulted in numerous analytes being above ULOQs. Probably not possible to analyze all 186 tissue metabolites using a single kit.

12. Considerations of kit-based approach Spike/Recovery Experiment (salmon tissue) % recovery Several interferences noted in salmon liver which are not observed in human plasma. Not possible to confirm presence/absence of other interferences without acquiring additional standards.

13. Results Associations between Metabolite Concentrations and: –Migration point –Spawning sites –Gender classification

14. Hepatic Metabolome Through Migration Fulton Skeena Mouth Fulton River Lipids Carnitine

15. 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 * * **

16. * * 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

17. Gender: Pinkut Creek FFF vs FFMMMM vs MMF Significant metabolites: carnitine, Orn, Lys, DOPA,  -aminoadipic acid Significant metabolites: phosphatidyl cholines, Met, Arg, Thr,  -aminoadipic acid Pinket

18. Gender: Fulton River FFF vs FFM Significant metabolites: phosphatidyl cholines,  -aminoadipic acid Fulton

19. Mean  -aminoadipic acid concentrations for Fulton River & Pinkut Creek Salmon: (Low vitellogenin A status with elevated  -aminoadipic acid) p<0.05

20. 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.

21. 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. SkeenaFraser River FultonPinkutHarrisonWeaver In-river distance (km) 500550113120 Elevation gain (m) 711 1026 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).

22. 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)

23. Fisheries and Oceans Canada Pêches et Océans Canada

25. Results: Hepatic Metabolome & Migration Site Fulton Skeena Mouth Fulton River Carnitine Lipids

26. Sockeye are a major commercial and wildlife food source in British Columbia. Over 80% of sockeye production comes from two primary systems, the Fraser and Skeena rivers. Background Skeena River Fraser River

27. ** * * * * Hepatic Metabolome Through Migration *p<0.05**p<0.01 lysoPCa = ∑lysophosphatidylcholines; PCaa = ∑phosphatidylcholines

28. Hepatic Metabolome During Migration

29. Changes in Hepatic Metabolome During Migration

30. Mean  -aminoadipic acid concentrations for Fulton River and Pinket Creek Salmon