Toxicology Centre

October 7, 2013 ATW Moncton

The Aryl Hydrocarbon Receptor Pathway of Sturgeon: Evolutionary and Ecotoxicological Implications to

Dioxin Sensitivity in Fishes

Jon Doering, Steve Wiseman, Shawn Beitel, John Giesy & Markus Hecker

Toxicology Centre

October 7, 2013 ATW Moncton

Sturgeon All species of sturgeon are likely threatened Anthropogenic causes: • habitat alteration

• overfishing • introduced species • pollution

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October 7, 2013 ATW Moncton

Dioxin-like Compounds • Include PCBs, PCDFs, PCDDs, and others • Resistant to degredation and biotransformation • Bioconcentrate and undergo trophic transfer • Variety of adverse biological effects

Toxicology Centre

October 7, 2013 ATW Moncton

Sturgeon White Sturgeon & Dioxin-Like Chemicals • Maybe of particular risk due to life-style Benthic life-style Long-lived 15-20 years to sexual maturity Spawn intermittenly

• Have been show to be particularly sensitive to other contaminants such as metals (Vardy et al. 2011,2013)

Toxicology Centre

October 7, 2013 ATW Moncton

Great differences in species sensitivity to dioxin-like compounds

0 500 1,000 1,500 2,000 2,500 3,000

Zebrafish

Northern Pike

White Sucker

Japanese Medakafish

Lake Herring

Channel Catfish

Fathead Minnow

Rainbow Trout

Red Seabream

Brook Trout

Lake Trout

LD50 (pg/g egg)

40-fold difference in embryo-lethality

Most Sensitive

Least Sensitive

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October 7, 2013 ATW Moncton

Research Question How sensitive are sturgeon to dioxin-

like compound exposure compared to other species of fishes?

Toxicology Centre

October 7, 2013 ATW Moncton

Identified Multiple Aryl Hydrocarbon Receptors in Sturgeon

AhR1 AhR2

Peregrine Falcon AhR1 78% Zebrafish AhR2 89%

Chicken AhR1 77% Goldfish AhR2 88%

Albatross AhR1 77% Salmon AhR2 84%

Cormorant AhR1 77% Shark AhR2 83%

Pheasant AhR1 76% Turtle AhR2 82%

GCTGGATGTTTACTTCCCCTTTG

TGACCGCTGTGTGAACTTGCTTT

TTTAATCTGCTTCAAGGACATCT

T

GTGTCAGACGCGGGATTCTGGC

TTTTTAAATATAAAAAGCGTCTTC

ATTTTTTTAAATAAGTGAATTGCG

CCTTTTATCGAGACGGGATGATG

AACATTAGTAGTAGTGGTAATAC

C

Characterization of AhRs in Sturgeon

Toxicology Centre

October 7, 2013 ATW Moncton

AhRs in Sturgeon

* Statistical significance not shown

0

1

2

3

4

Liver Brain Gill Heart Spleen Stomach Intestine Head Kidney Muscle

Tran

scrip

t Abu

ndan

ce (F

old-

diffe

renc

e fr

om L

iver

)

0.0

0.5

1.0

1.5

2.0

AhR1 AhR2

Tran

scrip

t Abu

ndan

ce (F

old-

diffe

renc

e fr

om A

hR2

in li

ver)

A

A

Toxicology Centre

October 7, 2013 ATW Moncton

0

2

4

6

8

10

12

14

16

0 mg/kg 50 mg/kg 0 mg/kg 50 mg/kg 0 mg/kg 50 mg/kg

Tran

scrip

t Abu

ndan

ce (F

old-

chan

ge)

Liver Gill Intestine

*

AhRs in Sturgeon

* *

*

*

*

* indicates significant difference over basal (Kruskal Wallis, p < 0.05)

I I I I I I I I I I I I I

I I I I I I I I I I I I I

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October 7, 2013 ATW Moncton

Evolution

Postlethwait, 2004

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October 7, 2013 ATW Moncton

Sturgeon Are there toxicological implications of sturgeon expressing an avian-like AhR?

Could the unique AhR pathway of sturgeon alter sturgeon sensitivity to certain dioxin-like compounds?

PCB 105 2,3,7,8-TCDD 2,3,4,7,8-PCDF

Toxicology Centre

October 7, 2013 ATW Moncton

Predicting Sensitivity The amino acid sequence of the ligand binding

domain of the AhR can predict the sensitivity of any avian species to dioxins

Relatively little information available for fishes

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October 7, 2013 ATW Moncton

Amino Acid Identity at the Ligand Binding Domain of the Sturgeon AhR1

% Similarity Pheasant AhR1 88% Quail AhR1 88% Chicken AhR1 88% Shark AhR1 84% Red Seabream AhR1 82% Salmon AhR1 80% Goldfish AhR1 75% Zebrafish AhR1 50%

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October 7, 2013 ATW Moncton

Comparison of Critical Amino Acids in the LBD of the AhR1

Type 1 (sensitive) I S Type 2 (moderate) I A

Type 3 (insensitive) V A

Sturgeon I A

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October 7, 2013 ATW Moncton

Comparison of Critical Amino Acids in the LBD of the AhR1

Type 1 (sensitive) I S Type 2 (moderate) I A

Type 3 (insensitive) V A

Sturgeon I A

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October 7, 2013 ATW Moncton

Comparison of Critical Amino Acids in the LBD of the AhR1

Type 1 (sensitive) I S Type 2 (moderate) I A

Type 3 (insensitive) V A

Sturgeon I A

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October 7, 2013 ATW Moncton

Ongoing Work • In vitro Sturgeon

Hepatocyte Assays • Ligand Binding

Assays

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October 7, 2013 ATW Moncton

Ligand Binding Studies

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October 7, 2013 ATW Moncton

Mammal TEF

Bird TEF Fish TEF White Sturgeon RP (AhR2)

White Sturgeon RP (AhR1)

2,3,7,8-TCDD 1.0 1.0 1.0 1.0 1.0

2,3,7,8-TCDF 0.1 1.0 0.05 1.1 0.9

2,3,4,7,8-PCDF 0.5 1.0 0.5 2.2 0.6

PCB 126 0.1 0.1 0.005 0.04 0.04

PCB 77 0.0001 0.05 0.001 0.002 0.001 PCB 105 0.0001 0.0001 <0.000005 <0.00009 <0.00004

Van den Berg et al, 1998; Doering et al., unpublished data

Preliminary Data: Relative Potencies (RPs) of Dioxin-Like Chemicals

Stay tuned

Toxicology Centre

October 7, 2013 ATW Moncton

Ongoing Analysis 1) Correlate relative sensitivity of white sturgeon

AhR1 and AhR2 with in vitro hepatocyte responses to identify which receptor drives sensitivity to dioxin-like compounds in sturgeons.

2) Investigate diversity in sensitivity to dioxin-like compounds among different species of endangered sturgeons based on LRG assay and hepatocyte assays.

Toxicology Centre

October 7, 2013 ATW Moncton

Conclusions 1) The AhR pathway of sturgeon appears unique

compared to other fishes 2) The unique AhR pathway might alter sturgeon

sensitivity to some DLCs 3) This altered sensitivity has implications for the

risk assessment of endangered sturgeons to dioxin-like compounds

Toxicology Centre

October 7, 2013 ATW Moncton

Jon Doering Steve Wiseman Shawn Beitel John Giesy Markus Hecker

Reza Farmahin Sean Kennedy

Acknowledgements

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October 7, 2013 ATW Moncton

Acknowledgements

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October 7, 2013 ATW Moncton Questions ??