Environmental Toxicology and Chemistry, Vol. 35, No. 5, pp. 1239–1246, 2016
# 2015 SETAC
Printed in the USA
RELATIVE SENSITIVITIES AMONG AVIAN SPECIES TO INDIVIDUAL AND
MIXTURES OF ARYL HYDROCARBON RECEPTOR–ACTIVE COMPOUNDS
FENGHUA WEI,y JUANYING LI,y RUI ZHANG,y PU XIA,y YING PENG,y JOHN P. GIESY,yzxk and XIAOWEI ZHANG*y
yState Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, People’s Republic of China
zDepartment of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
xDepartment of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan, USA
kSchool of Biological Sciences, University of Hong Kong, Hong Kong Special Administrative Region, China
(Submitted 12 August 2015; Returned for Revision 6 September 2015; Accepted 3 October 2015)
Abstract: Dioxins and dioxin-like compounds (DLCs) are potent toxicants to most vertebrates. Sensitivities to DLCs vary among
species. In the present study, the sensitivities of avian species (chicken [Gallus gallus], ring-necked pheasant [Phasianus colchicus], and
Japanese quail [Coturnix japonica]) to some polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) were
determined by using species-specific, in vitro, transactivation assays based on a luciferase reporter gene under control of species-specific
aryl hydrocarbon receptors. In ring-necked pheasant and Japanese quail, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was not the most
potent inducer of toxic effects. Especially for Japanese quail, the relative potency values of most of 9 PCDD/Fs tested were greater than
for TCDD. The rank order of avian species sensitivities to DLCs was chicken > ring-necked pheasant > Japanese quail. Effects of binary
mixtures of TCDD, 2,3,7,8-tetrachlorodibenzofuran, and 2,3,4,7,8-pentachlorodibenzofuran were strictly additive. Moreover, we also
found that the primary DLCs that were responsible for most of the potency of the DLC mixtures can be deduced by using ordination in a
multidimensional space defined by the avian species sensitivities. Overall, the relative potency and the species sensitivities of these
chemicals could guide risk assessments to wild species when exposure to mixtures of DLCs in the environment. Environ Toxicol Chem
2016;35:1239–1246. # 2015 SETAC
Keywords: Dioxin-like compounds
Species sensitivity
Combined effect
Luciferase
In vitro
order of sensitivities to 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8TCDF) and TCDD in inducting CYP1A4/CYP1A5 messenger
RNA (mRNA) and EROD is chicken > ring-necked pheasant
> Japanese quail [6,10]. Among birds, chicken is the most
sensitive to DLCs [9]. For TCDD, the sensitivity of Japanese
quail is 100-fold less than that of chicken [11]. Although a
complete understanding of the reasons underling differences in
sensitivities among species is not yet available [12], a factor is
differential binding of DLCs to the ligand binding domain of the
AhR caused by differences among sequences of amino acids of
the ligand binding domain, especially at positions 324 and 380
of the AhR1–ligand binding domain of birds [9,13]. Thus, it
is necessary to assess the species sensitivities of individual
DLCs in addition to TCDD.
Because organisms usually are exposed to mixtures
simultaneously, there has been concern about combined effects.
Most evidence suggests that AhR-mediated potencies of
mixtures of DLCs are additive and the potential for synergisms
or supra-additive effects is small [5,14,15]. However, components of mixtures are also probably antagonistic if they are
less potent agonists of the AhR and their presence in environmental samples is relatively great. Some studies have found
that combined effects of 2,3,4,7,8-PeCDF and 1,2,3,4,7,8hexachlorodibenzofuran (1,2,3,4,7,8-HxCDF) or 2,3,30 ,4,40 ,5hexachlorobiphenyl (PCB 169) were additive [16,17]. A recent
study found that effects of co-exposure to PCBs and TCDD
varied as a function of dose and end points measured, with the
effects of mixtures being additive, synergistic, or antagonistic [18].
Information about species sensitivity and the relative
potency of DLCs was limited, especially for the AhR-luciferase
reporter gene (LRG) assays. The AhR-LRG assay is effective for monitoring AhR activity, which has been used to test
AhR-mediated activity and interspecies sensitivities of DLCs
INTRODUCTION
Dioxins and dioxin-like compounds (DLCs), including
polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated
dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs),
polychlorinated napthalenes (PCNs), and polycyclic aromatic
hydrocarbons (PAHs), cause toxicities in most vertebrates. In
vertebrates, the critical mode of toxic actions of DLCs are
mainly mediated by the aryl hydrocarbon receptor (AhR) [1].
A long evolutionary period of AhR gene replication and
diversification has led to a variety of forms of the AhR,
including AhR1, AhR2, and AhR3 [2,3].
To facilitate assessment of effects of DLCs, concepts of
toxic equivalents based on in vivo effects of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and toxicity equivalency
factors have been widely accepted [4,5]. However, recent
studies have found that TCDD is not the most potent
compound in some species [6,7]. For example, the potency
of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF) is
3-fold to 5-fold that of TCDD in ring-necked pheasant and
30-fold that of TCDD in Japanese quail [6]. The potency of
2,3,4,7,8-PeCDF is 21-fold, 9.8-fold, and 40-fold greater than
that of TCDD for the expression of RNA of cytochrome
P4501A (CYP1A) 4, CYP1A5, and the activity of the mixed
function monooxygenase enzyme as measured by ethoxyresorufin-O-deethylase (EROD) in primary embryonic herring
gull (Larus argentatus) hepatocytes, respectively [7]. Meanwhile, there are differences in sensitivity to DLCs both
within and among classes of vertebrates [6,8,9]. The rank
* Address correspondence to howard50003250@yahoo.com
Published online 7 October 2015 in Wiley Online Library
(wileyonlinelibrary.com).
DOI: 10.1002/etc.3269
1239
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Environ Toxicol Chem 35, 2016
F. Wei et al.
among avian species [12,19,20]. Moreover, the effects of
binary mixtures of DLCs needed to be verified. Therefore, 9
PCDDs and PCDFs (PCDD/Fs), which are known to be
primary contributors to toxic equivalents in environmental
media [21–26], were selected as test substances by use of in
vitro (cell-based), transactivation assays based on luciferase
as the reporter gene (LRG) under control of species-specific
AhR [13].
The specific objectives of the present study were to compare
relative potency of 9 PCDD/Fs in avian species (chicken, ringnecked pheasant, and Japanese quail) by use of avian AhR1LRG assay, to determine sensitivities of avian species to 9
PCDD/Fs, to determine the effects of binary mixtures of PCDD/
Fs, and to identify dominant substances based on their
contribution to toxic equivalents in mixtures of DLCs.
MATERIALS AND METHODS
Chemicals and solutions
A detailed description of the preparation of the PCDD/F
(Table 1; Sigma-Aldrich) solutions is provided elsewhere [27].
In brief, PCDD/Fs were diluted from stock solutions in dimethyl
sulfoxide (DMSO; Sigma-Aldrich) by use of a gradient.
Concentrations were determined by high-resolution gas
chromatography high–resolution mass spectrometry (HRGCHRMS) by use of isotope dilution following the US
Environmental Protection Agency method 1613 [28].
For the AhR1-LRG assays of chicken, ring-necked pheasant,
and Japanese quail, the concentration of the primary stock
solution of TCDD, prepared in DMSO, was 60 000 nM [12].
Stock solutions of each PCDD/F in DMSO were prepared
with different concentrations ranging from 60 000 nM to
600 000 nM. Test solutions of PCDD/Fs or TCDD were
prepared by dissolving serially diluted solutions with cell
culture medium. The final in-well concentration of DMSO
added to 96-well plates was 0.5%.
Culture, transfection of COS-7 cells, and avian AhR1-LRG assays
The COS-7 cells, provided by R. Haché (University of
Ottawa, Ottawa, ON, Canada), were cultured in Dulbecco’s
Modified Eagle medium with 10% fetal bovine serum in an
incubator (37 8C, 5% CO2, 99% humidity). Details for preparing
chicken, ring-necked pheasant, and Japanese quail AhR1
constructs are provided elsewhere [20]. A vector containing
sequence coding for the aromatic hydrocarbon transporter
(ARNT1) and promoter region of CYP1A5 of the common
cormorant (Phalacrocorax carbo) were provided by H. Iwata
(Ehime University, Ehime, Japan) [29,30]. Details of the
procedure for conducting the AhR1-LRG assay are provided
elsewhere [20]. Briefly, transfection was carried out 18 h after
plating of COS-7 cells at a concentration of 10 000 cells per well
in 96-well plates. Fifty nanograms of DNA and 0.2 mL Fugene 6
transfection reagent (Promega) were mixed in Opti-MEM
(Invitrogen) to make a 6-mL mixture which was transfected
into cells in each well. The 50 ng of DNA included 8 ng
of chicken, ring-necked pheasant, or Japanese quail AhR1
expression construct, 7.5 ng of CYP1A5 reporter construct,
1.55 ng of cormorant ARNT1, 0.75 ng of Renilla luciferase
vector, and 32.2 ng of salmon sperm DNA (Invitrogen). Then,
TCDD, PCDD/Fs, or DMSO (solvent control) was added to
the cells 5 h after transfection. The AhR activity was quantified
as luminescence by use of Dual-Glo luciferase assay kits
(Promega) in a Microplate reader (BioTek Instruments) 20 h
after dosing.
Potencies of DLCs singly or in combination
Potencies of 9 PCDD/Fs—including TCDD, 1,2,3,7,8pentachlorodibenzo-p-dioxin (1,2,3,7,8-PeCDD), 1,2,3,7,8,9hexachlorodibenzo-p-dioxin (1,2,3,7,8,9-HxCDD), 1,2,3,4,6,
7,8-heptachlorodibenzo-p-dioxin (1,2,3,4,6,7,8-HpCDD), 1,2,
3,4,6,7,8,9-octachlorodibenzo-p-dioxin (OCDD), 2,3,7,8TCDF, 1,2,3,7,8-pentachlorodibenzofuran (1,2,3,7,8-PeCDF),
2,3,4,7,8-PeCDF, and 1,2,3,4,6,7,8,9-octachlorodibenzofuran
(OCDF)—were determined by use of the AhR1-LRG assay.
Binary mixtures (B1–B7) among TCDD, 2,3,7,8-TCDF, or
2,3,4,7,8-PeCDF were made based on contribution rates to
toxic equivalents, respectively (Table 2). Based on preliminary
results, mixtures were diluted serially by 2-fold to make a
gradient of 8 concentrations, which were then used to determine
sensitivities of avian species by the AhR1-LRG assay procedure.
Avian AhR1-LRG assays data analysis
For the AhR1-LRG assays of chicken, ring-necked pheasant,
and Japanese quail, concentration–response curves of each
AhR1 construct and PCDD/F treatment were obtained from
3 independent assays. To minimize variability caused by
operation and transfection efficiency, a ratio of firefly luciferase
units to Renilla luciferase units was used to normalize luciferase
activity [31]. Concentration–response curves were calculated
with GraphPad (GraphPad Prism Ver 5.0) using a 4-parameter
logistic mode [32]. For each replicate concentration–response
curve, maximal response values were determined using a
Table 1. Relative sensitivities of aryl hydrocarbon receptor 1 (AhR1) constructs among chicken, ring-necked pheasant, and Japanese quail exposed to
polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans in the AhR1–luciferase reporter gene assaya
ReS
Compound
2,3,7,8-tetra-chlorodibenzo-p-dioxin
1,2,3,7,8-pentachlorodibenzo-p-dioxin
1,2,3,7,8,9-hexachlorodibenzo-p-dioxin
1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin
1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin
2,3,7,8-tetrachlorodibenzofuran
1,2,3,7,8-pentachlorodibenzofuran
2,3,4,7,8-pentachlorodibenzofuran
1,2,3,4,6,7,8,9-octachlorodibenzofuran
ReS10
ReS50
Abbreviation
CAS No.
Chicken
Pheasant
Quail
Pheasant
Quail
TCDD
1,2,3,7,8-PeCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
OCDF
1746-01-6
40321-76-4
19408-74-3
35822-46-9
3268-87-9
51207-31-9
57117-41-6
57117-31-4
39001-02-0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.20
0.41
0.22
0.62
0.060
0.24
0.47
0.20
0.51
0.020
0.27
0.11
0.43
NCb
0.080
0.36
0.13
0.21
0.10
0.50
0.34
0.52
NCb
0.16
1.0
0.57
0.71
0.010
0.47
0.25
0.57
NCb
0.051
1.2
0.28
0.36
a
Relative sensitivies (ReS) based on mean 10% and 50% effective concentration (EC10 and EC50) values (ReS10 and ReS50) calculated from the
AhR1–luciferase reporter gene assays are presented.
b
Not calculated because the EC10 or EC50 values are not available.
Relative sensitivities of DLCs and combined effect
Environ Toxicol Chem 35, 2016
Table 2. Binary mixtures (B1–B7) of 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD), 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF), and 2,3,4,7,8pentachlorodibenzofuran (2,3,4,7,8-PeCDF)a
%TEQs
Mixture
B1
B2
B3
B4
B5
B6
B7
TCDD
2,3,7,8-TCDF
2,3,4,7,8-PeCDF
4
1
1
4
/
/
/
/
/
/
1
1
1
4
1
1
4
/
4
1
1
Induction of luciferase activity in COS-7 cells transfected with
AhRs of birds
4-parameter logistic mode and were presented as the mean standard error (SE). If the concentration–response curve did not
reach a plateau, The 50% effective concentration (EC50) could
not be calculated. In this case, the greatest observed response
was reported instead of the maximal response.
Estimation of relative sensitivity and relative potency
The chicken is the most sensitive avian species to DLCs
tested to date. The relative sensitivity values (ReSx) based on
effective concentration (EC) values were calculated by dividing
effective concentration values of the chicken if available in
the AhR1-LRG assay. The relative sensitivity values were
calculated using Equation 1
ð1Þ
The relative potency (RePx) values were calculated using the
framework proposed by Villeneuve et al. [33], with some
modifications. The relative potency values based on effective
concentration derived from regression models were calculated.
When the concentration–response curve did not reach a plateau,
in addition to relative potency values based on the 10% effective
concentration (ReP10), relative potency values based on the
50% and 80% effective concentrations (ReP50 and ReP80,
respectively) were also calculated. The average of relative
potency and the range of relative potency were calculated from
ReP10, ReP50, and ReP80 values. The relative potency values
were calculated using Equation 2
RePx ¼ ECx;TCDD =ECx;dioxin
ð2Þ
Combined toxicity assessment
The concentration addition model is usually applied to
estimate effects of mixtures. Two components of binary mixtures
are expressed as toxic units, which were calculated using
Equation 3
TU ¼ Ci=EC50; i
TU is 1.0. Connecting TUs of 2 compounds and applying
95% confidence intervals generates an additivity belt. When the
TUs of mixtures overlap with the additivity belt, it shows that
2 compounds produce additive effects. Thus, a concentration
addition model can be applied [34].
RESULTS AND DISCUSSION
a
B1 to B7 were made up by TCDD, 2,3,7,8-TCDF, or 2,3,4,7,8-PeCDF
based on contribution rates (1:4, 1:1, 4:1) to toxic equivalents (TEQs),
respectively.
ReSx ¼ ECx;chicken =ECi;species
1241
ð3Þ
where TU is the toxic unit of component i, EC50,i is the EC50
of component i in a separate test, and Ci is the concentration of
component i equivalent to the EC50 in the mixture.
Based on TUs with 2 components in the mixture, an axis
coordinate system was established. When each compound
causes effects through an independent joint action, the value of
Concentration-dependent effects of TCDD and PCDD/Fs
on luciferase activity expressed by COS-7 cells transfected
with chicken, ring-necked pheasant, or Japanese quail AhR1
constructs are shown in Figure 1. Luciferase activity induced by
300 nM of TCDD (a positive control for normalization of the
luciferase activity data) reached a plateau in all 3 avian AhR1
constructs. All PCDD/Fs of interest, except OCDD, induced
significant luciferase activity and reached a plateau in cells
transfected with AhR1 of chicken, ring-necked pheasant, or
Japanese quail. The greatest responses in in cells containing the
3 kinds of AhR1 constructs were induced by 1,2,3,7,8-PeCDD,
1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, and 2,3,7,8-TCDF, whereas
OCDD and OCDF generally caused less responses. Although
OCDF in cells transfected with constructs containing AhR1 of
chicken, pheasant, or Japanese quail reached a plateau, maximal
responses in chicken and pheasant were less than 50% of the
positive control. This result made it more difficult to make
comparisons among species.
EC50 of AhR1 constructs to PCDD/Fs exposure
In the present study, EC50 was calculated by the systematic
framework proposed by Villeneuve et al. [33] with some
modifications to compare the potencies of DLCs and sensitivities among avian species (Figure 2 and Table 3). If DLCs did not
exhibit the same efficacies (maxima), an EC50 value could not
be calculated. The potencies of TCDD, 1,2,3,7,8-PeCDD, and
2,3,4,7,8-PeCDF were the greatest, whereas that of OCDF was
the least. The EC50 values of OCDF were 10- to 30-fold less
than that of 1,2,3,4,6,7,8-HpCDD, which was nearest to OCDF
in the same species. The rank order of EC50 values among
species was approximately Japanese quail ring-necked
pheasant > chicken. For TCDD, EC50 values differed by
almost an order of magnitude among the avian species.
It was postulated that, for responses of the avian species
exposed to mixtures from the environment for which the
constituents are unknown, types of DLCs causing observed
potencies could be inferred based on ordination of the sample.
This type of analysis would be useful when conducting a
potency balance study to determine if all of the DLCs in the
mixture had been identified. Although this type of bioassay is
not meant to replace instrumental analyses, it could be useful in
identifying unknowns or at lease suggesting the type of DLCs
for which to look, particularly in areas of the world where
access to HRGC-HRMS is limited or unavailable [35,36]. To
test this hypothesis, EC50 values of more compounds and
more species would be needed.
Relative sensitivity of AhR constructs to PCDD/Fs
The relative sensitivity values based on either 10% effective
concentration or EC50 values of some PCDD/Fs demonstrated
distinct rank orders of sensitivity among cells containing AhR1
constructs of birds that were different from that of TCDD
(Table 1). Generally, the rank order of sensitivities of species to
dioxins was chicken > ring-necked pheasant > Japanese quail,
1242
Environ Toxicol Chem 35, 2016
F. Wei et al.
Figure 1. Concentration-dependent effects of 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), polychlorinated dibenzo-p-dioxins (PCDDs), or polychlorinated
dibenzofurans (PCDFs) on luciferase activity based on hydrocarbon receptor 1 (AhR1) in COS-7 cells transfected with chicken (A), ring-necked pheasant (B), or
Japanese quail (C) AhR1 constructs. Data are presented as percent response values relative to that of a 300 nM TCDD positive control for each of the avian
constructs. Concentration–response curves for the PCDD/Fs are only presented for constructs that showed a significant (p < 0.05) concentration-dependent
increase in luciferase activity relative to the dimethyl sulfoxide (DMSO) response. Points represent mean, positive control-normalized luciferase ratios obtained
from 3 independent experiments, each with 4 technical replicates per concentration of PCDD/Fs or TCDD. Bars represent standard error. See Table 1 for
definitions of compound abbreviations.
which was consistent with results of previous studies (exposed
to TCDD, 2,3,7,8-TCDF, PCB 126, or PCB 77) [13,20,37].
Especially for TCDD, 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF, the
rank order of sensitivities was in agreement with the results of a
previous study [6]. However, 2,3,4,7,8-PeCDF had a different
rank order of relative sensitivities depending on endpoints
used [10]. For example, based on induction of EROD activity,
the rank order of sensitivity to 2,3,4,7,8-PeCDF was chicken
> ring-necked pheasant > Japanese quail. However, based on
expression of CYP1A4 mRNA, when exposed to PeCDF,
the pheasant was predicted to be equally sensitive as the
chicken [10]. Rank orders of ReS50 values for 1,2,3,7,8PeCDD, 1,2,3,4,6,7,8-HpCDD, and 1,2,3,7,8-PeCDF between
Figure 2. Median effective concentration (EC50) value of polychlorinated
dibenzo-p-dioxins or polychlorinated dibenzofurans in avian species, based
on a luciferase reporter gene assay with aryl hydrocarbon receptor 1. The
EC50 values represent the average of 3 replicates obtained from three
96-well plates for each compound. See Table 1 for definitions of compound
abbreviations.
the ring-necked pheasant and Japanese quail were generally
similar. Among the 3 avian species studied, the construct
containing chicken AhR1 was the most sensitive—that is, 1-fold
to 10-fold more sensitive than the pheasant. The ring-necked
pheasant construct was more sensitive (0.9 to 7.5 times) than
that of the Japanese quail construct. However, for 1,2,3,7,8PeCDF, sensitivities based on pheasant and Japanese quail
constructs were almost equal to the chicken construct. Rank
orders of sensitivities of birds among PCDD/Fs and other DLCs
might be attributable to the ligand-specific differences in AhR
conformation [9,13].
Relative potency of PCDD/Fs in different avian AhR1 constructs
The effective concentration values, maximal responses, and
relative potency values for each PCDD/F and AhR1 construct
are provided in Table 3. The PCDD/Fs demonstrated diverse
relative potencies in AhR-mediated activity among AhR1
constructs bioassays. Of the 9 DLCs tested, only 2,3,7,8-TCDF
and 2,3,4,7,8-PeCDF had been studied previously in the
LRG [20]. The relative potency values of 2,3,7,8-TCDF and
2,3,4,7,8-PeCDF were consistent with those reported previously
(Table 3). Based on relative potency values (Table 3), the
rank order of PCDD/Fs potency was TCDD > 2,3,4,7,8PeCDF > 2,3,7,8-TCDF 1,2,3,7,8-PeCDD > 1,2,3,7,8-PeCDF
1,2,3,7,8,9-HxCDD > 1,2,3,4,6,7,8-HpCDD > OCDF for the
chicken AhR1 construct; 2,3,4,7,8-PeCDF > 1,2,3,7,8-PeCDD
> 2,3,7,8-TCDF TCDD > 1,2,3,7,8-PeCDF > 1,2,3,7,8,9HxCDD > 1,2,3,4,6,7,8-HpCDD for the pheasant AhR1
construct; and 2,3,4,7,8-PeCDF > 1,2,3,7,8-PeCDD > 1,2,3,7,8PeCDF > 2,3,7,8-TCDF > 1,2,3,7,8,9-HxCDD > TCDD 1,2,
3,4,6,7,8-HpCDD > OCDF for the Japanese quail AhR1 construct. Rank orders of PCDD/Fs were generally similar among
AhR1 constructs of chicken and ring-necked pheasant with
the exception of TCDD (Table 3). Rank orders of PCDD/Fs
were generally similar among the ring-necked pheasant and
Japanese quail AhR1 constructs with the exceptions of TCDD,
Relative sensitivities of DLCs and combined effect
Environ Toxicol Chem 35, 2016
1243
Table 3. Relative potency, effective concentration, and maximal response values of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans in
aryl hydrocarbon receptor 1 (AhR) constructs of chicken, ring-necked pheasant, and Japanese quaila
AhR
Compound
Chicken
TCDD
1,2,3,7,8-PeCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
OCDF
Pheasant
TCDD
1,2,3,7,8-PeCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
OCDF
Quail
TCDD
1,2,3,7,8-PeCDD
1,2,3,7,8,9-HxCDD
1,2,3,4,6,7,8-HpCDD
OCDD
2,3,7,8-TCDF
1,2,3,7,8-PeCDF
2,3,4,7,8-PeCDF
OCDF
RePrangeb
Maximal
response SE
(% TCDD)
ReP50c
1.0
0.64
0.091
0.031
NA
0.56
0.099
1.2
1.5E-03
1.01.0
0.401.1
0.0800.14
0.0360.039
NA
0.240.90
0.0360.20
1.21.6
1.5E-032.3E-03
100 2.2
93 3.2
49 2.5
69 3.2
16 4.5
83 2.5
106 4.1
99 3.1
41 5.5
1.0
–
–
–
–
0.4
–
0.9
–
1.0
2.2
0.30
0.13
NA
0.89
0.90
1.8
0.013
1.0
2.4
0.23
0.12
NA
1.0
0.71
2.5
0.010
1.01.0
2.22.7
0.160.30
0.120.13
NA
0.891.1
0.480.90
1.64.3
5.7E-030.013
100 2.9
113 2.2
76 1.6
92 2.0
37 7.1
93 3.1
87 1.6
108 3.1
41 1.7
1.0
–
–
–
–
0.3
1.0
16
1.5
1.5
NA
4.7
5.4
20
0.049
1.0
18
1.2
1.2
NA
3.8
5.5
18
0.037
1.01.0
1619
0.971.5
0.951.5
NA
2.34.7
4.85.4
1320
0.0300.049
100 4.1
121 1.2
71 1.8
97 1.3
5.1 0.45
77 1.1
92 2.0
100 1.5
56 1.2
1.0
–
–
–
–
3
–
20
–
EC10 SE
(nM)
EC50 SE
(nM)
ReP10
ReP50
ReP80
RePavg
0.068 0.005
0.062 0.003
0.49 0.03
1.7 0.09
59 0.1
0.075 0.002
0.33 0.08
0.043 0.002
30 0.7
0.22 0.04
0.46 0.08
3.4 0.1
9.4 0.09
NC
0.43 0.07
3.7 0.07
0.25 0.08
175 0.1
1.0
1.1
0.14
0.039
1.2E-03
0.90
0.20
1.6
2.3E-03
1.0
0.43
0.053
0.018
NA
0.54
0.057
0.83
9.3 E-04
1.0
0.40
0.08
0.036
NA
0.24
0.036
1.2
1.4E-03
0.34 0.003
0.15 0.005
2.2 0.1
2.8 0.4
976 3
0.32 0.005
0.70 0.02
0.22 0.01
59 3.1
2.8 0.05
0.93 0.04
10 0.03
18 0.04
NC
2.8 0.06
3.5 0.03
0.44 0.04
246 0.05
1.0
2.2
0.16
0.12
3.5E-04
1.1
0.48
1.6
5.7E-03
1.0
2.7
0.23
0.12
NA
1.1
0.74
4.3
8.3E-03
4.4 0.01
0.23 0.06
4.6 0.06
4.1 0.08
NC
0.99 0.01
0.92 0.03
0.33 0.004
141 2.5
21 0.05
0.98 0.02
14 0.04
16 0.02
NC
9.4 0.02
3.3 0.03
0.92 0.02
484 0.02
1.0
19
0.97
1.1
NA
4.5
4.8
13
0.031
1.0
19
1.2
0.95
NA
2.3
6.3
21
0.03
b
4
–
a
Effect concentrations values and maximal response values represented the average of 3 replicates standard error (SE) obtained from three 96-well plates for
each compound. Maximal response observed for the polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans expressed as a percentage of the
mean of the maximal response observed for TCDD.
b
Mean relative potency (RePavg) values and ranges of relative potency values (RePrange) values were calculated from relative potency (ReP) values based on
the 10%, 50%, and 80% effective concentrations (EC10, EC50, and EC80, respectively). No RePrange values were presented when only the ReP10 could be
calculated.
c
ReP50 values were taken from a previous study [20].
SE ¼ standard error; ReP10, ReP50, ReP80 ¼ relative potency values based on the 10%, 50%, and 80% effective concentrations, respectively; TCDD ¼ 2,3,7,8tetra-chlorodibenzo-p-dioxin; 1,2,3,7,8-PeCDD ¼ 1,2,3,7,8-pentachlorodibenzo-p-dioxin; 1,2,3,7,8,9-HxCDD ¼ 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin;
1,2,3,4,6,7,8-HpCDD ¼ 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin; OCDD ¼ 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin; 2,3,7,8-TCDF ¼ 2,3,7,8-tetrachlorodibenzofuran; 1,2,3,7,8-PeCDF ¼ 1,2,3,7,8-pentachlorodibenzofuran; 2,3,4,7,8-PeCDF ¼ 2,3,4,7,8-pentachlorodibenzofuran; OCDF ¼ 1,2,3,4,6,7,8,9-octachlorodibenzofuran; NC ¼ not calculated because the maximal response was not reached; NA ¼ ReP estimates not available to calculate the value.
2,3,7,8-TCDF, and 1,2,3,7,8-PeCDF (Table 3). For the chicken
AhR1 construct, relative potency values of all PCDD/Fs were less
than that for TCDD. Except for OCDD and OCDF, relative
potency values of other PCDD/Fs in cells containing the Japanese
quail AhR1 construct were greater than that of TCDD, with
values 1-fold to 22-fold greater than that of TCDD. There were
differences between the 3 kinds of AhR1 constructs for PCDD/Fs
with an exception of 2,3,7,8-TCDF. Relative potency values
differed by an order of magnitude among the avian species.
Interspecies variation of relative potency values for PCDD/Fs
among birds might be attributable to differences in the AhR
amino acid sequence, leading to different sizes of the AhR1
binding cavity or differences in ligand-receptor conformation
among avian species [13].
In general, TCDD is considered to be the most potent inducer
of AhR-mediated responses in all taxa and has therefore been
used as the reference DLCs to which all other DLCs are
compared to derive relative potency and toxic equivalency
factors [5]. However, the present study found that 2,3,4,7,8PeCDF and 1,2,3,7,8-PeCDD have greater potency than TCDD
in pheasant. Furthermore, the potency of all dioxins selected
in the present study except OCDD and OCDF were greater than
that of TCDD for Japanese quail. In Japanese quail, relative
potency values for 2,3,4,7,8-PeCDF and 1,2,3,7,8-PeCDD were
almost 20-fold greater than that of TCDD. The greater potency
of 2,3,4,7,8-PeCDF compared with TCDD observed in the
present study is consistent with the results of previous studies
that demonstrated that 2,3,4,7,8-PeCDF was more potent than
TCDD in Japanese quail primary hepatocytes [6]. In addition
to Japanese quail, similar phenomena were observed for other
species, such as double-crested cormorant and Forster’s tern
(Sterna forsteri) [38], green frog (Rana esculenta) [39], and
herring gull [7].
To carry out assessments of hazard and risk to wild species
exposed to mixtures of DLCs, the findings included in the
present study and previous studies demonstrate the importance
of assessing relative potencies of DLCs and interspecies
sensitivities among species other than the chicken. Meanwhile,
the AhR1-LRG assay based on constructs in COS-7 cells makes
it possible to develop species and chemical-specific relative
potency for use in assessments of hazards or risks of DLCs to
birds. Because the chicken is thought to be among the most
sensitive birds to the effects of DLCs, it has been used as
a surrogate for birds when conducting hazard and risk
1244
Environ Toxicol Chem 35, 2016
assessments. Thus, an overestimate of the potential risks and
inappropriate decisions might result [40].
Combined effects of PCDD/Fs on avian species
The TU values for binary mixtures of TCDD, 2,3,7,8-TCDF,
and 2,3,4,7,8-PeCDF overlapped with the additivity belt,
which showed that mixture effects of PCDD/Fs were additive
(Figure 3). Results of previous studies have also found
the same combined effect of 2,3,4,7,8-PeCDF mixed with
1,2,3,4,7,8-HxCDF or 2,3,4,5,30 ,40 -hexachlorobiphenyl [16].
Although concentration of each congener of PCBs and PCDD/
Fs might vary among environmental mixtures, the overall
combined potency can be estimated by a simple additive
model [18].
In the present study, the effects of mixtures based on EC50
demonstrated that they were additive, which might be
attributable to the fact that all of the DLCs tested had effects
mediated solely via the AhR. Binary mixtures (B1–B7) among
TCDD, 2,3,7,8-TCDF, or 2,3,4,7,8-PeCDF were made up
based on contribution rates (1:4, 1:1, 4:1) to toxic equivalents
(Table 2). The binary mixtures samples (B1–B7) among TCDD,
F. Wei et al.
2,3,7,8-TCDF, and 2,3,4,7,8-PeCDF all resulted in similar
relative potency values (Figure 4). These mixtures, when
ordinated in the 2-dimensional spaces, demonstrated that the
mixtures ordinated near the dominant DLCs in the various
mixtures. For example, B1 was near TCDD, B1 and B5 were
near 2,3,4,7,8-PeCDF, and B4 and B7 were close to 2,3,7,8TCDF. Thus, if a substance is dominant in the mixtures of
DLCs substances, relative potency values of the mixtures will
be similar to that of the dominant substance. Therefore, our
results suggested that relative potency values of environmental
samples among different species could be used to predict the
dominant DLCs.
In summary, the results of the present study have contributed
to a growing number of relative potency values for DLCs
among avian species that can be used in the mass balance
analysis of environmental samples. Effects of binary mixtures
of TCDD, 2,3,7,8-TCDF, and 2,3,4,7,8-PeCDF were strictly
additive. Furthermore, TCDD was found to not be the most
potent inducer of toxic effects, especially for Japanese quail.
Most DLCs, including 1,2,3,7,8-PeCDD, 1,2,3,7,8,9-HxCDD,
2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, and 2,3,4,7,8-PeCDF, were
Figure 3. Evaluation of binary mixture toxicity of 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF), and
2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF). Axes are the toxic unit (TU) of TCDD, 2,3,7,8-TCDF, 2,3,4,7,8–PeCDF to which 3 avian species were
exposed separately. The solid line is the additive equivalent curve, whereas the area delineated by the 2 dashed lines is the belt of additive effects. Connecting the
TUs of 2 compounds and applying 95% confidence intervals generates an additivity belt.
Relative sensitivities of DLCs and combined effect
Environ Toxicol Chem 35, 2016
1245
Figure 4. Scatter diagram of relative potency based on 50% effective concentration (ReP50) values for polychlorinated dibenzo-p-dioxins or polychlorinated
dibenzofurans and binary mixtures of TCDD, 2,3,7,8-TCDF, and 2,3,4,7,8-PeCDF. The inset figure on the right is a partial enlargement of the figure on the left.
Binary mixtures (B1–B7) among TCDD, 2,3,7,8-TCDF, or 2,3,4,7,8-PeCDF were made up based on contribution rates (1:4, 1:1, 4:1) to toxic equivalents (TEQs)
respectively. See Table 1 for definitions of compound abbreviations.
more potent than TCDD. The rank order of sensitivities of birds
to DLCs was chicken > ring-necked pheasant > Japanese quail.
The relative potency values, together with the interspecies
variations based on the AhR-LRG assays, could provide
valuable information for the hazard and risk assessments of
complex environmental pollutants to which wild birds are
exposed.
Acknowledgement—The present project was supported by the National
Natural Science Foundation of China (Grants 21322704 and 21007025) and
the Jiangsu Province S & T Support Plan Social Development Funding
Project (BE2011776). J. Giesy was supported by the program of 2012
“High Level Foreign Experts” (GDT20143200016) funded by the State
Administration of Foreign Experts Affairs, the P. R. China to Nanjing
University, and the Einstein Professor Program of the Chinese Academy of
Sciences. J. Giesy also was supported by the Canada Research Chair
program.
Data availability—The data can be obtained from the corresponding author
by emailing howard50003250@yahoo.com.
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