Predicting the Sensitivity of Endangered
Sturgeons to Dioxin-like Compounds: Molecular
Investigation into the AhR Pathway
By: Jon Doering
Sturgeons
24 species of sturgeon
White Sturgeon
(A. transmontanus)
Shovelnose Sturgeon
(S. platorynchus)
Russian Sturgeon
(A. gueldenstaedtii)
Sturgeons
• Historically supported lucrative fisheries
• Important cultural relevance
• Unique part of our biodiversity
Sturgeons
All species of sturgeon are likely threatened
Anthropogenic causes: • habitat alteration
•
•
•
•
•
overfishing
genetic bottlenecks
introduced species
pathogen exposure
pollution
Sturgeons
All species of sturgeon are likely threatened
Anthropogenic causes: • habitat alteration
•
•
•
•
•
overfishing
genetic bottlenecks
introduced species
pathogen exposure
pollution
Sturgeons
Pollution has been identified as a probable
cause of sturgeon population declines in:
•
•
•
•
•
•
Rainy River, Canada (1987)
Danube River, Central Europe (1997)
Volga River, Russia (1997)
St. Lawrence River, Canada (1998)
Delaware Estuary and Chesapeake Bay, USA (2006)
Yangtze River, China (2009)
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
Great differences in species sensitivity
Most Sensitive
Lake Trout
Brook Trout
Crucian Carp
200-fold difference
in embryo-lethality
Mummichog
Red Seabream
Rainbow Trout
Fathead Minnow
Channel Catfish
Lake Herring
Japanese Medakafish
White Sucker
Northern Pike
Zebrafish
Pallid Sturgeon
Shovelnose Sturgeon
Least Sensitive
0
2,000
4,000
6,000
8,000
LD50 (pg/g egg)
10,000
12,000
14,000
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…
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
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
Does sensitivity to activation of the AhR
differ between sensitive and
insensitive fishes?
Red Seabream
Rainbow Trout
Fathead Minnow
Channel Catfish
Lake Herring
Japanese…
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
Study Species
White Sturgeon (A. transmontanus)
Lake Sturgeon (A. fluvescens)
In Vivo Sensitivity
16
*
Fold-change in CYP1A transcript
14
White Sturgeon
12
10
8
7-fold difference in
up-regulation of
CYP1A transcript
Lake Sturgeon
6
4
**
2
0
0 mg/kg-bw
50 mg/kg-bw
Significant difference from 0 mg/kg-bw (T-test) denoted with * (p ≤ 0.05) or ** (p ≤ 0.1).
In Vitro Sensitivity
Luciferase Reporter Gene Assay (LRG)
In fishes:
AhR1
AhR2
AhR1 Comparison - TCDD
2,3,7,8-TCDD
AhR1 Comparison - Suite
AhR2 Comparison - TCDD
2,3,7,8-TCDD
AhR2 Comparison - Suite
Comparison
Sensitivity to TCDD In Vitro (nM)
Species
White Sturgeon AhR1
Lake Sturgeon AhR1
LOEC
0.01
0.01
EC20
0.008
0.009
EC50
0.04
0.04
EC80
0.2
0.2
White Sturgeon AhR2
Lake Sturgeon AhR2
0.03
0.1
0.02
0.3
0.07
0.8
0.3
2
Comparison
Sensitivity to TCDD In Vitro (nM)
Species
White Sturgeon AhR1
Lake Sturgeon AhR1
LOEC
0.01
0.01
EC20
0.008
0.009
EC50
0.04
0.04
EC80
0.2
0.2
White Sturgeon AhR2
Lake Sturgeon AhR2
0.03
0.1
0.02
0.3
0.07
0.8
0.3
2
Relative Sensitivity
3-fold
15-fold 11-fold 7-fold
In Vivo relative sensitivity:
7-fold
AhR1
351
• Ligand binding region of the AhR1 of White and
Lake Sturgeon have one amino acid difference.
AhR2
305
321
388
• Ligand binding region of the AhR2 of White and
Lake Sturgeon have four amino acid differences.
404
AhR2
388
• Ligand binding region of the AhR2 of White and
Lake Sturgeon have four amino acid differences.
AhR2
388
White Sturgeon
Lake Sturgeon
• Critical difference at equivalent of Position 386 in
zebrafish AhR1a and AhR2, 380 in avian AhR1, and
375 in mouse AhR.
Ongoing Research
AhR2
20
15
10
5
0
CYP1A
Ongoing Research
AhR2
20
15
CYP1A
10
5
0
Embryo-lethality studies will be used to
link differences in sensitivity to
ecologically relevant endpoints.
Ongoing Research
AhR2
20
15
CYP1A
10
5
0
Whole transcriptome sequencing
will be used to investigate
whether differences in the
molecular initiating event
result in whole transcriptional
differences.
(See poster MO214)
Conclusions
1) White sturgeon are more sensitive to
DLCs relative to lake sturgeon with
regard to up-regulation of CYP1A
transcript in vivo.
Conclusions
2) Relative differences between white and
lake sturgeon in the in vitro sensitivity of
AhR2, and not AhR1, is similar to the
relative differences in sensitivity in vivo.
Conclusions
3) The differences in sensitivity between
white and lake sturgeon in vivo and in
vitro appear to be a result of key amino
acid subsitutions in the LBD of the AhR2.
Acknowledgements
Co-authors:
Steve Wiseman
Shawn Beitel
John Giesy
Markus Hecker
Reza Farmahin
Sean Kennedy
Acknowledgements
Support:
Questions ??
Author Contact:
Jon Doering
jad929@mail.usask.ca