The Aryl Hydrocarbon Receptor Signaling Pathway of White Sturgeon: Implications for Sturgeon Sensitivity to Dioxin-like Compounds
Jon Doering1, Steve Wiseman1 , Shawn Beitel1, John P. Giesy1,2,3, Markus Hecker1,4
1.Toxicology Centre, University of Saskatchewan, Saskatoon, SK, Canada 2. Dept. Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, Canada
3. Dept. of Biology & Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China 4. School of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, Canada
Background
• Worldwide many species of sturgeons (Acipenseridae) have faced
massive population declines with some species nearing extinction.
• These declines are attributed to a variety of human activities including
habitat alteration, overfishing, and pollution.
• Sturgeon are uniquely susceptible to bioaccumulation of dioxin-like
compounds relative to other species of fish because they:
 are
extremely long lived
 have a higher lipid content
 mature later
spawn intermittently
 live in close association with sediments
 have a diet rich in benthic prey

Methods
Molecular endpoints were characterized in white sturgeon in order to better
understand how sturgeon respond to AhR agonists.
Exposure Experiment: White sturgeon (Acipenser transmontanus) were
injected intraperitoneally (I.P.) with one of two doses of β-naphthoflavone (βNF)
dissolved in corn oil at 0 or 50mg/kg-bw. Rainbow trout (Oncorhynchus mykiss)
were used as a previously characterized reference species. Tissue samples were
collected three days following injection.
Discussion
Results
Hypothesized AhR1
Hypothesized AhR2
• White sturgeon express at least 3 different AhRs which appear to be very
unique to sturgeons.
Hypothesized AhR3
(Based on 1722bp)
Dog AhR (Predicted)
84%
(Based on 1284bp)
Nile Tilapia AhR2 (Predicted)
79%
(Based on 951bp)
Tasmanian Devil AhR (Predicted)
84%
Panda AhR (Predicted)
84%
Fugu AhR2β
78%
Grey Opossum AhR (Predicted)
80%
Great Cormorant AhR1
83%
Zebra Finch AhR1 (Predicted)
78%
Platypus AhR (Predicted)
80%
Domestic Chicken AhR1
83%
Gilt-head Seabream AhR1
75%
Chinese Hamster AhR
80%
Japanese Quail AhR1
82%
Common Goldfish AhR2
75%
House Mouse AhR
79%
Table 1: Maximum nucleotide identity of AhRs of white sturgeon compared with
• Deformities and enzyme induction have been observed in sturgeon
collected from some contaminated sites however, little is known about the
sensitivity of sturgeon to dioxin-like compounds.
publicly available sequences of AhRs in other species. Additional sequencing data
from multiple RACE-PCRs is still being analyzed.
• White sturgeon have been found to be highly responsive to exposure to a
model dioxin-like compound (β-naphthoflavone) with regard to
inducibility of cytochrome P450 1A (Demonstrated in Figure 1 from
Doering et al, 2012).
90
80
70
60
50
40
30
20
10
0
Rainbow
Trout
*
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
l
White
Sturgeon
Figure 2: I.P. injection of rainbow trout with βNF.
Identification of AhRs: Because only a single, partial AhR gene had been
previously identified in sturgeons, Illumina paired-end transcriptome sequencing
of white sturgeon liver was used to identify novel AhR-like genes. Full
sequences were acquired by use of RACE-PCR (Clontech).
*
2
1
Brain
Gill
Heart
Liver
Gill
Intestine
Figure 1: EROD activity in liver, gill, and intestine from white sturgeon
and rainbow trout following 3-days of exposure to βNF. EROD activity
was non-detectable in gills and intestines from unexposed and
exposed rainbow trout. * indicates a significant (p ≤ 0.05) difference
compared to basal activity. Figure adapted from Doering et al, 2012.
• All known effects of exposure to dioxin-like compounds are mediated
through the aryl hydrocarbon receptor (AhR) signaling pathway.
• The AhR is an integral part of the AhR signaling pathway as in birds it has
been found that the molecular structure of the ligand binding domain of
the AhR determines the receptors affinity for dioxin-like compounds and
dictates the sensitivity of any species of bird (Karchner et al, 2006).
• However, nothing is known about the AhRs of sturgeons and little is
known about these receptors in other ancient fishes.
• Therefore the objectives of this study were to:
1) Identify all AhRs expressed in tissues of white sturgeon
2) Investigate tissue-specific expression of each AhR
3) Investigate tissue-specific expression of each AhR
following exposure to an AhR agonist
Spleen Stomach Intestine Head Muscle
Kidney
Figure 4.1: Comparison of basal transcript abundance of AhRs among tissues of
white sturgeon. Statistical significance not shown.
16
Statistical Test: Significance was determined using the Kruskal-Wallis test
followed by the Mann Whitney U test. Statistical tests were conducted by use of
SPSS 19 software (SPSS Inc., Chicago, IL, USA).
14
12
10
8
*
*
6
4
2
0
0 mg/kg
50 mg/kg
Liver
I
I
I
l
I
I
I
I
I
I
I
I
I
l
l
l
l
l
*
*
0 mg/kg
*
50 mg/kg
Gill
I
I
I
l
I
I
I
I
I
I
I
I
I
l
l
l
l
l
Acknowledgements
All experimental procedures were approved by the University Committee on Animal Care and Supply (UCACS) at the University of Saskatchewan .
*
0 mg/kg
50 mg/kg
Intestine
white sturgeon following exposure to βNF. * indicates a statistically significant
difference from 0 mg/kg-bw ( p ≤ 0.05).
References
Doering J, Giesy J, Wiseman S, Hecker M. (2012). Enviro. Sci. Pol. Res. In Press.
Doering J, Wiseman S, Beitel S, Tendler B, Giesy J, Hecker M. (2012). Aquat. Toxicol. 114-115, 125-133.
Travel support for J. Doering from the University of Saskatchewan and the Ross Norstrom Memorial Travel Award
• Salmonids are known to be among the most sensitive species of fishes to
dioxin-like compounds and this sensitivity is hypothesized to be partially
driven by these species expressing multiple, functional AhRs. The presence
of 3 AhRs in sturgeon might, therefore, indicate that sturgeon are sensitive to
dioxin-like compounds as well .
binding studies with the
novel AhRs found in white
sturgeon to determine if each
receptor is capable of binding
dioxins and interacting with
dioxin-responsive elements on
the DNA. This will determine
which AhRs are involved in
mediating dioxin-like effects in
sturgeons.
Figure 5: Hepatocyte cultures used
for development of TEFs in sturgeons.
 Development
of sturgeon specific
toxic equivalency factors (TEFs)
to determine if the unique AhR
signaling pathway of sturgeons
alters sturgeon sensitivity to
different dioxin-like compounds.
 Investigate
Thanks to the Kootenay Trout Hatchery for their donation of white sturgeon embryos.
Stipend support for J. Doering from NSERC CREATE HERA, UofS Dean’s Scholarship, Sask. Innovation and Opportunity Scholarship, and the Toxicology Centre
• This up-regulation following exposure to an AhR agonist implies that all 3 of
these AhRs might be involved in mediating dioxin-like effects.
 Ligand
This research was supported through the Canada Research Chair Program to M. Hecker.
Also thanks to the Aquatic Toxicology Research Facility (ATRF) and Toxicology Graduate Program for facilitating this research.
• All 3 AhRs were up-regulated following exposure to an AhR agonist.
Ongoing Research
Figure 4.2: Transcript abundance of AhRs among liver, gill, and intestine of
Figure 3: Juvenile White Sturgeon
• All 3 AhRs had greatest expression in the liver, gill, and heart. The liver and
heart are of interest as these are target organs for dioxin-like compounds.
• The unique AhR signaling pathway of sturgeon might alter sturgeon
sensitivity to dioxin-like compounds compared to other fishes. Birds and
mammals have greater sensitivity to PCDFs and mono-ortho PCBs,
respectively, than fishes, however sturgeon appear to have AhRs similar to
that of birds (AhR1) and mammals (AhR3). Therefore, sturgeon might have
greater sensitivity to PCDFs and mono-ortho PCBs than some other fishes.
Liver
Endpoint: Primers against white sturgeon AhR1 and AhR3 were designed from
partial sequences acquired from white sturgeon by use of Illumina sequencing.
Primers against white sturgeon AhR2 and β-actin were designed by use of
publically available sequences of white sturgeon. Transcript abundance was
quantified by normalizing to β-actin according to the methods of Simon (2003).
• The hypothesized white sturgeon AhR3 appears to share greatest nucleotide
identity with mammalian AhRs.
• Sturgeon appear to have a unique AhR signaling pathway involving at least 3
novel AhRs and might represent an important evolutionary link between
ancient fishes and higher vertebrates.
*
*
• The hypothesized white sturgeon AhR2 appears to share greatest nucleotide
identity with piscine AhR2s.
Conclusion
0
Transcript Abundance (Fold-change)
EROD Activity (Fold Change)
100
Transcript Abundance (Fold-difference
from Liver)
3
• The hypothesized white sturgeon AhR1 appears to share greatest nucleotide
identity with avian AhR1s.
Karchner S, Franks D, Kennedy S, Hahn, M. (2006). Proc. Natl. Acad. Sci. U.S.A. 103, 6252-6257.
Simon P. (2003). Bioinfo. 19, 1439-1440.
the responsiveness to
AhR agonists of other ancient
fishes, including species of
sturgeons that have not yet been
studied.
Figure 6: Juvenile Lake Sturgeon