Fish serum and liver proteins responsive to environmental

Fish serum and liver proteins responsive to environmental pollutants: Bluefish as a model? Joe Kunkel University of Massachusetts Amherst

Bluefish, Pomatomus saltatrix

Estrogen and CYP 1 A • Xenobiotics are natural substances that are foreign to the body. (plant and human industry) • Cytochrome P 450 1 A (CYP 1 A) is a cellular protein induced by various xenobiotics and is being used as a biomarker for them. • Some xenobiotics are also estrogen-like and will induce vitellogenin, the major precursor of egg yolk proteins, produced in the liver. • The bluefish is a potential sensor species in which studying CYP 1 A and Vg induction may allow some discrimination between xenobiotic types.

Caudal Vein Bleed • Caudal vein nested ventral to vertebrae. • Allows 0. 5 -1 ml sample. • Heparinized syringe. Photo by Bruce Dockum

Serum Separation • Heparinized serum separates. • Red Cells are nucleated and are a legitimate source of DNA. • Add 95% Et. OH to the clot. • Serum contains accumulated Vitellogenin since no active ovary exists. Photo by Bruce Dockum

Serum Separation • Heparinized serum separates. • Red Cells are nucleated and are a legitimate source of DNA. • Add 95% Et. OH to the clot. • Serum contains accumulated Vitellogenin since no active ovary exists. Photo by Bruce Dockum

Process Fish • Live Sampling. • Return to recovery tank. - or- • Sample tissues. Photo by Bruce Dockum

Fast muscle Slow muscle

Stomach Liver Lobe GI tract Pancreas

From: • Fish 1 • Fish 2 Tissue: ü S- Serum ü G- Gill ü L- Liver ü M- Muscle Sample: o 1 o 2

Our NOAA/CMER Proposal • Use Commercial Antisera to CYP 1 A conserved peptide. • Develop an anti-Lipovitellin from purified bluefish Lv. • This depends on finding developing or ripe ovaries from bluefish. • Treat bluefish with natural estrogens and see how this affects their CYP 1 A levels.

Alternate CYP 1 A Assay • A non-lethal method to estimate CYP 1 A expression in laboratory and wild Atlantic salmon (Salmo salar) Rees, Mc. Cormick and Li (2005). Comp Phys Biochem C 141: 217 -24.

RT-PCR: Alternate CYP 1 A Assay • Compare Millers River Salmon to Control brook. • Compare the CYP 1 A c. DNA to an internal standard. Rees, Mc. Cormick and Li (2005). Comp Phys Biochem C 141: 217 -24.

Vitellogenin Structure and Evolution Finn & Kristoffersen (2007) PLo. S One 2: e 169.

Vitellogenin Structure and Evolution • Vg produced outside the oocyte. • Processing occurs before and after it enters the oocyte. • Vg -> Lv + Pv Vg Lv 1 Lipovitellin Pv Lv 1 Lv 2 + Phosvitin Lv 2 Pv

Structure of Lv • Large dimeric lipoprotein • Large subunit ca 115 k. D • Small subunit ca 31 k. D and highly phosphorylated • Twenty percent lipid http: //www. bioscience. org/2003/v 8/s/1071/fig 1. jpg

Gel Permeation Chromatography • Bio Gel A-1. 5 agarose beads. • Smaller molecules penetrate more. • Large molecules travel outside beads eluting 1 st. • Protein measured using A 280

Winter Flounder Lv • Compare native with heat stable • Subtract: n - hs = hl heat labile

Cod LV heat stability • Somewhat variable denaturation curve between species.

Cod, Hagfish vs Chicken Lv

Cod Lv heat stability Old Tech • • • Heat for 5 min Centrifuge Run SDS gel Densitometry Fit a surface New Tech • Microcalorimetry measures calories used.

DSC • Stages of denaturation using Differential Scanning Microcalorimeter http: //polymer. physik. uni-ulm. de/images/mcalor 2. gif

Cod LV heat stability • Lv heat stability discovered by Ruth Hartling in NOAA/CMER project. • Generalized to Cod, Shad, Tautog, Hagfish, but not Spiny Dog.

Δt Lucey model: Native Lv Heat Denatured Lv HLv ca 115 k. D Lipid LLv ca 31 k. D P- P- PP- P- P- 15°C 60°C 85°C The purified heat-stable Lv is used to make polyclonal antisera used to measure the titer of the Lv.

How have anti-fish-Lvs worked? • Case histories of heat stable anti-fish-Lvs: – Winter flounder (Ruth Hartling, Ph. D. ) • Boston harbor cleanup ended project. – Cod • Used to show Vg is not secreted in slime. • Vg in serum does not predict stages. – Shad • Used to test non-feeding hypothesis in fresh water. – Bluefish • Estrogen interaction with CYP 1 A during xenobiotic induction.

Use of anti-cod-Lv on Cod Sera Yes/No Vg! Truth? • • { A - Rabbit antiserum against heat-stable and gel filtration purified cod Lv I - should have no Vg. D - should have high Vg. R - should have stopped Vg synthesis. U - rare individuals during these cruises. S - might have Lv from involuting follicles which would react with Ab. T - should have zero Vg or Lv in their serum?

Cod Egg Staging 400 m Mid developing 100 m Early developing Late developing

[Vg] in 168 f & 210 m cod sera • Cod classified by cutter into: immature, developing, ripe, spent, resting, m/f. Cutter: JGK • Many developing cod with no serum Vg! • Vg in 4/210 males.

People Involved in Yolk Protein UMass • • • Francis Juanes Dave Leonard Jack Nordin Willy Bemis Joe Zydlewski Ruth Hartling Don Wojchowski John Storella Sean Lucey • NMFS Personnel – – – Jose Pereira Frank Almeida Jay Burnett Dean Perry Josef Idoine Ashok Deshpande

The End