Toxicology Centre
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
ATW Moncton
October 7, 2013
Toxicology Centre
Sturgeon
All species of sturgeon are likely threatened
Anthropogenic causes:
• habitat alteration
• overfishing
• introduced species
• pollution
ATW Moncton
October 7, 2013
Toxicology Centre
Dioxin-like Compounds
•
•
•
•
Include PCBs, PCDFs, PCDDs, and others
Resistant to degredation and biotransformation
Bioconcentrate and undergo trophic transfer
Variety of adverse biological effects
ATW Moncton
October 7, 2013
Sturgeon
Toxicology Centre
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)
ATW Moncton
October 7, 2013
Toxicology Centre
Great differences in species sensitivity to dioxin-like
compounds
Most Sensitive
Lake Trout
40-fold difference in
embryo-lethality
Brook Trout
Red Seabream
Rainbow Trout
Fathead Minnow
Channel Catfish
Lake Herring
Japanese Medakafish
White Sucker
Northern Pike
Least Sensitive
Zebrafish
0
ATW Moncton
500
1,000
1,500
LD50 (pg/g egg)
2,000
2,500
3,000
October 7, 2013
Toxicology Centre
Research Question
How sensitive are sturgeon to dioxinlike compound exposure compared
to other species of fishes?
ATW Moncton
October 7, 2013
Toxicology Centre
Characterization of AhRs in Sturgeon
GCTGGATGTTTACTTCCCCTTTG
TGACCGCTGTGTGAACTTGCTTT
TTTAATCTGCTTCAAGGACATCT
T
GTGTCAGACGCGGGATTCTGGC
TTTTTAAATATAAAAAGCGTCTTC
AhR1
AhR2
ATTTTTTTAAATAAGTGAATTGCG
CCTTTTATCGAGACGGGATGATG
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%
ATW Moncton
AACATTAGTAGTAGTGGTAATAC
Identified Multiple Aryl
Hydrocarbon Receptors in
Sturgeon
C
October 7, 2013
Toxicology Centre
AhRs in Sturgeon
2.0
Transcript Abundance (Folddifference from AhR2 in liver)
Transcript Abundance (Fold-difference from Liver)
4
3
2
1.5
A
A
1.0
0.5
0.0
AhR1
AhR2
1
0
Liver
ATW Moncton
Brain
Gill
Heart
Spleen
Stomach
* Statistical significance not shown
Intestine
Head Kidney
Muscle
October 7, 2013
Toxicology Centre
Transcript Abundance (Fold-change)
AhRs in Sturgeon
16
14
12
10
8
*
6
*
4
2
0
0 mg/kg
50 mg/kg
Liver
ATW Moncton
I
I
I
I
I
I
I
I
I
I
I
I
I
*
*
0 mg/kg
50 mg/kg
Gill
I
I
I
I
I
I
I
I
I
I
I
I
I
*
0 mg/kg
*
50 mg/kg
Intestine
* indicates significant difference over basal (Kruskal Wallis, p < 0.05)
October 7, 2013
Toxicology Centre
Evolution
Postlethwait, 2004
ATW Moncton
October 7, 2013
Toxicology Centre
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
ATW Moncton
2,3,7,8-TCDD
2,3,4,7,8-PCDF
October 7, 2013
Toxicology Centre
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
ATW Moncton
October 7, 2013
Toxicology Centre
Amino Acid Identity at the Ligand
Binding Domain of the Sturgeon
AhR1
ATW Moncton
% Similarity
Pheasant AhR1
Quail AhR1
Chicken AhR1
Shark AhR1
Red Seabream AhR1
Salmon AhR1
Goldfish AhR1
Zebrafish AhR1
88%
88%
88%
84%
82%
80%
75%
50%
October 7, 2013
Toxicology Centre
Comparison of Critical Amino Acids in
the LBD of the AhR1
ATW Moncton
Type 1 (sensitive)
Type 2 (moderate)
I
I
S
A
Type 3 (insensitive)
V
A
Sturgeon
I
A
October 7, 2013
Toxicology Centre
Comparison of Critical Amino Acids in
the LBD of the AhR1
ATW Moncton
Type 1 (sensitive)
Type 2 (moderate)
I
I
S
A
Type 3 (insensitive)
V
A
Sturgeon
I
A
October 7, 2013
Toxicology Centre
Comparison of Critical Amino Acids in
the LBD of the AhR1
ATW Moncton
Type 1 (sensitive)
Type 2 (moderate)
I
I
S
A
Type 3 (insensitive)
V
A
Sturgeon
I
A
October 7, 2013
Toxicology Centre
Ongoing Work
• In vitro Sturgeon
Hepatocyte Assays
ATW Moncton
• Ligand Binding
Assays
October 7, 2013
Toxicology Centre
Ligand Binding Studies
ATW Moncton
October 7, 2013
Toxicology Centre
Stay tuned
Preliminary Data: Relative Potencies (RPs) of Dioxin-Like Chemicals
Bird TEF
Fish TEF
2,3,7,8-TCDD
Mammal
TEF
1.0
1.0
White Sturgeon
RP (AhR2)
1.0
White Sturgeon
RP (AhR1)
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
ATW Moncton
Van den Berg et al, 1998; Doering et al., unpublished data
October 7, 2013
Toxicology Centre
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.
ATW Moncton
October 7, 2013
Toxicology Centre
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
ATW Moncton
October 7, 2013
Toxicology Centre
Acknowledgements
Jon Doering
Steve Wiseman
Shawn Beitel
John Giesy
Markus Hecker
ATW Moncton
Reza Farmahin
Sean Kennedy
October 7, 2013
Toxicology Centre
Acknowledgements
ATW Moncton
October 7, 2013
Toxicology Centre
ATW Moncton
Questions ??
October 7, 2013