Functionality of Aryl Hydrocarbon Receptors (AHR1 and AHR2) of White Sturgeon and Implications for the Risk Assessment of Dioxin-like Compounds
Jon Doering1,2, Reza Farmahin3,4, Steve Wiseman2 , Sean Kennedy3,4, John P. Giesy2,5,6, Markus Hecker2,7
1.Toxicology Graduate Program, University of Saskatchewan 2.Toxicology Centre, University of Saskatchewan 3. Dept. Biology, University of Ottawa 4. Environment Canada, National Wildlife Research Centre 5. Dept. Veterinary
Biomedical Sciences, University of Saskatchewan 6. Dept. of Biology & Chemistry, City University of Hong Kong 7. School of the Environment and Sustainability, University of Saskatchewan
Background
• Most species of sturgeons (Acipenseridae) are endangered worldwide.
• There is particular concern about declines in white sturgeon (Acipenser
transmontanus) in northwestern USA and British Columbia, Canada.
• It is hypothesized that anthropogenic chemicals, including dioxin-like
compounds (DLCs), might be contributing to these declines.
• Currently, toxic equivalency factors (TEFs) developed for the World
Health Organization (WHO) and which are based on studies of embryolethality in salmonids are used in the risk assessment of DLCs.
• However, sturgeons are evolutionarily distinct from salmonids and it is
uncertain whether the current TEF approach is protective of sturgeons.
Results
Table 1: Relative potencies (RePs) of selected dioxin-like compounds (DLCs) to AHR1
and AHR2 of white sturgeon compared to in vivo derived RePs and current toxic
equivalency factors (TEFs).
White sturgeon AHR1
TCDD
1.0
PeCDF
1.0
TCDF
0.4
PCB 126
0.04
PCB 77
0.001
PCB 105
-
White sturgeon AHR2
1.0
1.3
1.0
0.04
0.002
-
Sturgeona
Embryos of Pallid
Embryos of Shovelnose Sturgeona
Embryos of Rainbow Troutb
1.0
NA
NA
0.08
NA
NA
1.0
1.0
NA
0.3
NA
0.03
0.07
0.005
NA
0.0002
NA
-
TEFWHO-Fishc
1.0
0.5
0.05
0.005
0.0001
< 0.000 005
a Buckler 2011
1) Develop in vitro relative potencies (RePs) for DLCs of concern to white
sturgeon by use of a luciferase reporter gene (LRG) assay with aryl
hydrocarbon receptors (AHRs) of white sturgeon.
2) Compare Toxic Equivalency Quotients (TEQs) by use of TEFs to TCDDEquivalents (TCDD-EQs) by use of calculated RePs in white sturgeon.
Methods
Identification of Aryl Hydrocarbon Receptors (AHRs): Illumina
paired-end transcriptome sequencing of white sturgeon livers was used
to identify nucleotide sequences of AHR1 and AHR2 genes.
b Zabel et al, 1995
A
Liver
Muscle
Measured TCDD-
TEQ
Measured
TCDD-
Conc.
EQ
Eggs
TEQ
Measured
TCDD-
Conc.
EQ
TEQ
Conc. a
EQ
TCDD
20.0
20.0
20.0
34.8
34.8
35.0
4.20
4.20
4.20
PeCDF
3.80
4.94
1.90
7.60
9.88
3.80
0.80
1.04
0.40
TCDF
390
390
19.5
520
520
26.0
42.6
42.6
2.10
PCB 126
7.80
0.312
0.390
10.7
0.428
0.054
1.80
0.072
0.009
PCB 77
59.0
0.118
0.006
62.9
0.126
0.006
7.0
0.014
0.001
PCB 105
9 795
0.098
0.049
21 337
0.213
0.110
2 707
0.027
0.014
415
41.8
565
65.0
48.0
6.72
B
• Concentrations of TCDD-EQs calculated by use of sturgeon specific
RePs were approximately 10-fold greater than WHO-based TEQs in
liver, muscle, and eggs from white sturgeon in the Fraser River and
Columbia River (Table 2).
• It is uncertain whether the concentrations of TEQs reported in liver,
muscle, or eggs of Fraser River or Columbia River white sturgeon
represent a risk.
Figure 3: Response curves for COS-7 cells transfected
with AHR1 (A) or AHR2 (B) of white sturgeon following
exposure to six DLCs. Curves are presented as a
percentage relative to maximal response of TCDD. Data
are presented as means +/- standard error of the mean
(SE).
Muscle
Measured TCDDConc. b
EQ
TCDD
0.146
0.146
PeCDF
0.276
TCDF
TEQ
Measured TCDD-
Measured
TCDD-
Conc.
EQ
EQ
0.150
0.172
0.172
0.170
2.37
2.37
2.40
0.359
0.140
0.166
0.216
0.083
0.550
0.719
0.280
20.6
20.6
1.0
13.3
13.3
0.670
67.2
67.2
3.40
PCB 126
16.1
0.644
0.081
5.15
0.206
0.026
15.9
0.636
0.080
PCB 77
33.5
0.067
0.003
24.5
0.049
0.003
27.7
0.055
0.003
PCB 105
4 680
0.047
0.023
3 740
0.037
0.019
3 750
0.038
0.019
21.9
1.40
14.0
0.971
Total
a MacDonald et al, 1997
b Kruse & Webb, 2006
71.0
6.18
Conclusions & Future Research
Figure 5: Embryo of
white sturgeon.
• Development of sturgeon specific RePs
could be essential for the objective risk
assessment of sturgeons worldwide.
TEQ
Conc.
• The sensitivity of sturgeons to DLCs warrants further investigation.
• Because sturgeons are endangered and can
have elevated exposure to mixtures of
DLCs, future research should investigate
whether in vitro RePs accurately represent
RePs derived by use of in vivo endpoints of
biological relevance, such as embryolethality.
Eggs
TEQ
• However, concentrations of TCDD-EQs for liver, muscle, and eggs of
Fraser River and Columbia River white sturgeon exceed effect
concentrations for several fishes, including some other species of
sturgeons.
• Based on RePs derived in vitro, it appears
that use of TEFWHO-Fish might underestimate
risk of DLCs to white sturgeon in the Fraser
and Columbia Rivers.
Columbia River White Sturgeon
Liver
Luciferase Reporter Gene (LRG) Assay: White sturgeon AHR1 or
AHR2 genes, white sturgeon ARNT gene, and rat CYP1A1 reporter gene
were transfected into AHR-deficient COS-7 cells. The LRG assay was
performed on three replicate assays in quadruplicate according to the
methods described in Farmahin et al (2012).
Data Analysis: Response curves were fit to a 4-parameter logistic model
by use of Graphpad Prizm 5.0 software. RePs were calculated by use of
the average of EC20, EC50, EC80 and standardizing to 2,3,7,8-TCDD.
• RePs developed for AHR1 and AHR2 of white sturgeon were distinctly
greater than TEFs developed by use of salmonids, but comparable to
those previously developed for other sturgeons (Table 1).
c Van den Berg et al, 1998
Fraser River White Sturgeon
Total
Figure
2:
Flow
chart
showing steps involved in
the LRG assay.
• AHR1 and AHR2 of white sturgeon were activated in a concentrationdependent manner by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD),
2,3,4,7,8-pentachloro-dibenzofuran
(PCDF),
2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3’,4,4’,5-pentachlorobiphenyl (PCB126), and
3,3’,4,4’-tetrachlorobiphenyl (PCB77). However, concentrations of
2,3,3’,4,4’-pentachlorobiphenyl (PCB105) did not activate either AHR
up to a maximum concentration of 9,000 nM.
Table 2:TEQs and TCDD-EQs of Fraser River (A) and Columbia River (B) white sturgeon
liver, muscle, and eggs for select DLCs based on maximum measured concentrations.
TCDD-EQs based on RePs for AHR2 of white sturgeon. Concentrations in pg/g-ww.
Figure 1: White sturgeon.
Objectives
Results & Discussion
Acknowledgements
Thanks to the Kootenay Trout Hatchery for donation of white sturgeon.
Figure 4: Map of British Columbia, Canada showing
locations of the Fraser River and Columbia River were
white sturgeon were collected for chemistry data.
These results have been published as Doering J, Farmahin, R, Wiseman S, Kennedy S, Giesy J, Hecker M (2014).Functionality of Aryl Hydrocarbon Receptors (AhR1 and
AhR2) of White Sturgeon (Acipenser transmontanus) and Implications for the Risk Assessment of Dioxin-like Compounds. Environ. Sci. Technol. 48: 8219-8226.