Can Identities of key amino acids in the ligand binding
domain of the AhR be used to predict the sensitivity
of endangered sturgeons to dioxins?
By: Jon Doering
Sturgeons
Sturgeons have faced population declines due to a
variety of anthropogenic causes:
Historic Overfishing
Sturgeons
Sturgeons have faced population declines due to a
variety of anthropogenic causes:
Historic Overfishing
Habitat Alteration
Sturgeons
Sturgeons have faced population declines due to a
variety of anthropogenic causes:
Introduced Species
Habitat Alteration
Sturgeons
Sturgeons have faced population declines due to a
variety of anthropogenic causes:
Introduced Species
Pollution
Dioxin-like Compounds
•
•
•
•
•
Includes Dioxins, Furans, and PCBs
Extremely persistent in the environment
Accumulate in organisms
Variety of adverse biological effects
Aryl hydrocarbon receptor (AhR) agonists
PCBs
Dioxins
Furans
Species Sensitivity
Most Sensitive
Great differences in
species sensitivity:
Lake Trout
Brook Trout
Crucian Carp
Mummichog
Red Seabream
Rainbow Trout
Fathead Minnow
Channel Catfish
Lake Herring
Japanese Medakafish
White Sucker
Northern Pike
Zebrafish
Pallid Sturgeon
Shovelnose Sturgeon
Least Sensitive
200-fold difference
in embryo-lethality
0
5,000
10,000
LD50 (pg/g egg)
15,000
Sturgeons
Problem: there are large differences in sensitivity to
dioxin-like compounds among species – presents a
major challenge to risk assessment.
Sturgeons
Problem: there are large differences in sensitivity to
dioxin-like compounds among species – presents a
major challenge to risk assessment.
Solution: develop methods to predict species
sensitivity without use of live animals – with special
interest in predicting the relative sensitivity of
sturgeons or other endangered species.
Birds
Birds
Question
Lake Trout
Brook Trout
Crucian Carp
Mummichog
Red Seabream
Rainbow Trout
Fathead Minnow
Channel Catfish
Lake Herring
Japanese Medakafish
White Sucker
Northern Pike
Zebrafish
Pallid Sturgeon
Shovelnose Sturgeon
Does the same hold true for fishes?
0
5,000
10,000
LD50 (pg/g egg)
15,000
Question
Can relative sensitivity among species of fishes
be predicted based upon relative differences
in the molecular initiating event?
Lake Trout
Brook Trout
Crucian Carp
Mummichog
Red Seabream
Rainbow Trout
Fathead Minnow
Channel Catfish
Lake Herring
Japanese Medakafish
White Sucker
Northern Pike
Zebrafish
Pallid Sturgeon
Shovelnose Sturgeon
0
5,000
10,000
LD50 (pg/g egg)
15,000
Sturgeons
3 Main Objectives:
1) Identification of AhR1 and AhR2 amino acid
sequences
Sturgeons
3 Main Objectives:
1) Identification of AhR1 and AhR2 amino acid
sequences
2) Quantification of sensitivity to activation of AhRs
by dioxin-like compounds
Sturgeons
3 Main Objectives:
1) Identification of AhR1 and AhR2 amino acid
sequences
2) Quantification of sensitivity to activation of AhRs
by dioxin-like compounds
3) Predict sensitivity of sturgeon of unknown in
vivo sensitivity based on other species of fish of
known in vivo sensitivity
Study Species
White Sturgeon (A. transmontanus)
Lake Sturgeon (A. fulvescens)
Luciferase
Ratio
AhR1 Comparison - TCDD
Ligand Concentration (nM)
Luciferase
Ratio
AhR1 Comparison - TCDD
No difference
in sensitivity.
Ligand Concentration (nM)
Luciferase
Ratio
AhR2 Comparison - TCDD
Ligand Concentration (nM)
Luciferase
Ratio
AhR2 Comparison - TCDD
10-fold
difference in
sensitivity.
Ligand Concentration (nM)
AhR Structure
Do specific amino acid differences in the ligand
binding domain of the AhR between species
result in differences in affinity of the receptor
and differences in sensitivity between species?
AhR2
305
313
321
388
Ligand binding region of the AhR2 of White and
Lake Sturgeon have four amino acid differences.
AhR2
305
313
321
388
Ligand binding region of the AhR2 of White and
Lake Sturgeon have four amino acid differences.
AhR2 Structure
AhR2 Structure
AhR2 Structure
AhR2 Structure
AhR2 Structure
Relative Sensitivity?
Lake Trout
Brook Trout
Crucian Carp
White Sturgeon?
Mummichog
Red Seabream
Where do white sturgeon
and lake sturgeon fit?
10-fold?
Rainbow Trout
Fathead Minnow
Lake Sturgeon?
Channel Catfish
Lake Herring
Japanese Medakafish
White Sucker
Northern Pike
Zebrafish
Pallid Sturgeon
Shovelnose Sturgeon
0
2,000
4,000
6,000
8,000
LD50 (pg/g egg)
10,000
12,000
14,000
Relative Sensitivity?
Lake Trout
Brook Trout
Crucian Carp
Mummichog
Red Seabream
Rainbow Trout
Fathead Minnow
Channel Catfish
Lake Herring
Japanese Medakafish
White Sucker
Northern Pike
Zebrafish
Pallid Sturgeon
Shovelnose Sturgeon
0
2,000
4,000
6,000
8,000
LD50 (pg/g egg)
10,000
12,000
14,000
Diverse Fishes
Lake Trout
Brook Trout
Crucian Carp
Mummichog
Total of 11 species
spanning ~70-fold
difference in LD50 to
embryos
Red Seabream
Rainbow Trout
Fathead Minnow
Channel Catfish
Lake Herring
Japanese Medakafish
White Sucker
Northern Pike
Zebrafish
Pallid Sturgeon
Shovelnose Sturgeon
0
2,000
4,000
6,000
8,000
LD50 (pg/g egg)
10,000
12,000
14,000
Diverse Fishes
Lake Trout
Brook Trout
Crucian Carp
Mummichog
Total of
10 AhR1s and
15 AhR2s
Red Seabream
Rainbow Trout
Fathead Minnow
Channel Catfish
Lake Herring
Japanese Medakafish
White Sucker
Northern Pike
Zebrafish
Pallid Sturgeon
Shovelnose Sturgeon
0
2,000
4,000
6,000
8,000
LD50 (pg/g egg)
10,000
12,000
14,000
AhR2
Relatedness tree for AhR2s
AhR2
Total of 30 positions with amino acid
differences among 17 AhRs from 13
species
AhR2
Initial sequence alignment indicates
no trends in structure among
sensitive vs insensitive fishes
AhR2
Can Identities of key amino acids in the ligand
binding domain of the AhR be used to predict
the sensitivity of endangered sturgeons to
dioxins?
AhR2
Can Identities of key amino acids in the ligand
binding domain of the AhR be used to predict
the sensitivity of endangered sturgeons to
dioxins?
Activation assays
Basal expression studies
ongoing
ongoing
Acknowledgements
Co-authors:
Steve Wiseman
Shawn Beitel
John Giesy
Markus Hecker
Co-authors:
Reza Farmahin
Sean Kennedy
Additional Support:
Questions ??
Author Contact:
Jon Doering
jad929@mail.usask.ca