Transcriptional Responses of White Sturgeon (Acipenser transmontanus)
Following Exposure to a Model Dioxin-like Compound
Jon Doering1,2, Steve Wiseman2 , Song Tang3, Shawn Beitel1,2, Sarah Patterson1,2, Bryanna Eisner2,4, John P. Giesy2,5,6, Markus Hecker2,3
1.Toxicology Graduate Program, University of Saskatchewan, Saskatoon, SK , CA 2.Toxicology Centre, University of Saskatchewan, Saskatoon, SK, CA
3. School of the Environment and Sustainability, University of Saskatchewan, Saskatoon, SK, CA 4.Toxicology Undergraduate Program, University of Saskatchewan, Saskatoon, SK , CA
5. Dept. Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK, CA 6. Dept. of Biology & Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China
Background
Results & Discussion Continued
Results & Discussion
Down-regulated
• Worldwide many species of sturgeons (Acipenseridae) have faced
massive population declines with some species nearing extinction.
2-fold
Up-regulated
• These declines are attributed to a variety of human activities
including pollution.
• Due to their benthic nature and longevity, sturgeons are at risk to
bioaccumulative contaminants, such as dioxin-like compounds.
• However, nothing is known about the transcriptional responses of
AhR activation in sturgeons or the resulting toxic responses.
Table 1: Sensitivity of AhRs of diverse species in vitro following
exposure to 2,3,7,8-TCDD (Doering et al, submitted).
Species
White Sturgeon AhR1
White Sturgeon AhR2
Red Seabream AhR1
Red Seabream AhR2
Rainbow Trout AhR2a
Zebrafish AhR1b
Zebrafish AhR2
Atlantic Tomcod AhR2
Atlantic Salmon AhR2a
Chicken AhR1
EC50 (nM)
0.036
0.070
0.073
0.51
0.20
5.9
0.7
1.9
0.10
0.22
2-fold
Figure 4: Crosstalk between AhR and Hypoxia pathways with
transcript responses following exposure to beta-naphthoflavone
based on Illumina Sequencing and confirmed with RT-PCR.
Down-regulated
78%
(~2500 genes)
Figure 2: Proportion of Up- and Down- regulated
Genes of Differentially Expressed Genes Following
Exposure to Beta-Naphthoflavone.
Table 2: Xenobiotic Responses in Livers Following Exposure to
Beta-Naphthoflavone by use of Illumina Sequencing.
Transcript
Fold-change
Phase I
CYP1A
CYP2K1
CYP3A
Phase II
GST
UDP
Sulfotransferase
Phase III
Multi-drug Resistant Proteins
40x
88x
34x
Methods
Exposure Experiment: Juvenile white sturgeon (Acipenser
transmontanus) were injected intraperitoneally (I.P.) with one of two
doses of a model dioxin-like compound, β-naphthoflavone (βNF)
dissolved in corn oil at 0 or 50mg/kg-bw. Liver and gill samples were
collected three days following injection.
Figure 2
• The AhR shares the aryl hydrocarbon receptor nuclear
translocator (ARNT) with the hypoxia inducible factor alpha
(HIFa) (Figure 4). It has been speculated that exposure to dioxinlike compounds might interfere with the hypoxia response
through competition for or depletion of available ARNT. However,
this phenomena not been observed in laboratory model fish
species that have been tested to date (Pohjanvirta, 2012).
• It is currently unclear whether these responses will impact the
hypoxia response in sturgeons following co-exposure to dioxinlike compounds and hypoxia.
• Additionally, ARNT is important in numerous developmental
processes and an altered expression of ARNT might impact
proper development in embryos of sturgeons exposed to dioxinlike compounds.
up to 109x
Conclusions
• The expression of greater than 3,000 transcripts was altered by
beta-naphthoflavone by greater than or equal to 2-fold in livers of
white sturgeon.
• Greater than ¾ of the altered transcripts were down-regulated
(Figure 2).
• Transcripts known to be up-regulated by dioxin-like compounds in
other fishes, such as phase I, II, and II xenobiotic metabolism
enzymes, were also up-regulated in white sturgeon (Table 2).
• Consistent with previously published work on white sturgeon, the
classical biomarker of exposure to dioxin-like compounds, CYP1A,
was highly up-regulated (Table 2; Doering et al, 2012).
• White sturgeon livers are highly responsive to exposure to a
model dioxin-like compound, beta-naphthoflavone, which is
consistent with the great sensitivity of AhR1 and AhR2 of white
sturgeon in vitro (Doering et al, submitted).
• Numerous critical pathways were altered, which is consistent
with other studies with fish.
• Depletion of dissolved oxygen is a growing concern in areas of
anthropogenic activity, and exposure to dioxin-like compounds
might impair the ability of sturgeons to respond to hypoxia.
• Next-generation sequencing technologies, such as Illumina,
could prove useful in the discovery of novel biological responses
to contaminants in non-model species through the adverse
outcome pathway framework.
Figure 3
Figure 1: Illumina HiSeq instrument used for whole
transcriptome analysis of white sturgeon liver.
*
Figure 1: Juvenile White Sturgeon.
RNA Extraction: Total RNA was extracted from livers of 0 mg/kg-bw
and 50 mg/kg-bw exposed sturgeon. RNA from 3 individuals from
each treatment group were pooled before sequencing.
Illumina and RNA-Seq: Liver transciptomes of exposed and
unexposed fish were analyzed by use of Illumina RNA-Seq after de
novo assembly of a reference transcriptome. Samples were
sequenced on an Illumina HiSeq 2000 (100-bp paired end reads).
Sequencing was performed at the National Research Council Plant
Biotechnology Institution (Saskatoon, SK, CA). Contigs were de
novo assembled by use of the CLC Genomics Workbench 5.0.
Identities of contigs were determined by use of Blast2Go 2.5.0.
Reads were mapped to the reference transcriptome and abundance
determined as RPKM.
Pathway Analysis: For selected transcripts related to AhR
mediated effects, the Agilent Literature Search plugin was used to
construct a biological interaction network within the software
Cytoscape 3.1.0. Within the plugin, the available literature on
protein-protein / protein-DNA interactions was searched for
zebrafish. Resulting network nodes were organized using “yFiles
Organic” method and colored according to fold-changes in
expression.
Poster template by ResearchPosters.co.za
• However, inconsistent with other studies, a down-regulation in
ARNT and HIFa transcript with an up-regulation in AhR transcript
was observed in livers of white sturgeon following exposure to
beta-naphthoflavone (Figure 4) which might represent a novel
toxic response in sturgeons.
13x
12x
24x
The research questions of this study were to identify:
1) How responsive is the AhR pathway of white
sturgeon to dioxin-like compounds?
2) Which biological processes are affected?
3) What are possible toxic responses in sturgeons?
Down-regulated
5-fold
• The pleiotropic responses of exposure to dioxin-like compounds
are mediated through activation of the aryl hydrocarbon receptor
(AhR) signaling pathway.
• AhR1 and AhR2 of white sturgeon have been shown to be the
most sensitive AhRs studied to date to exposure to 2,3,7,8-TCDD
in vitro using transfected COS-7 cells (Table 1).
Up-regulated
22%
(~700 genes)
Acknowledgements
Figure 3: Interaction network of white sturgeon transcripts related to
AhR signaling. Nodes represent single genes, edges are proteinprotein interactions. * does not conform to RT-PCR confirmation data.
All experimental procedures were approved by the University
Committee on Animal Care and Supply (UCACS) at the University of
Saskatchewan.
Thanks to the Kootenay Trout Hatchery for their donation of white
sturgeon embryos.
• Numerous biological processes were affected in livers of white
sturgeon after exposure to beta-naphthoflavone (Figure 3) The
observed changes were consistent with findings previously
reported in rainbow trout (Aluru & Vijayan, 2008).
• Inconsistent with other studies, only one gene associated with
reproductive functions, estrogen receptor alpha, was altered in
white sturgeon livers (Figure 3). However, fish analyzed in this
study were sexually immature juveniles, and thus, these genes
might be differentially regulated than in mature animals.
• Few immune responsive genes were altered in white sturgeon
livers while studies in rainbow trout found the immune system to be
among the most affected systems (Aluru & Vijayan, 2008). This
might indicate the primitive immune system of ancient sturgeons.
References
Aluru N & Vijayan M. (2008). Aquat. Toxicol. 87. 1-12.
Doering J, Wiseman S, Beitel S, Tendler B, Giesy J, Hecker M.
(2012). Aquat. Toxicol. 114-115, 125-133.
Pohjanvirta R. (2012). John Wiley & Sons. New Jersey.