Chemosphere 144 (2016) 1754e1762
Contents lists available at ScienceDirect
Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
Activation of AhR-mediated toxicity pathway by emerging pollutants
polychlorinated diphenyl sulfides
Junjiang Zhang a, Xiaowei Zhang a, *, Pu Xia a, Rui Zhang a, Yang Wu a, Jie Xia a,
Guanyong Su a, Jiamin Zhang a, John P. Giesy a, b, c, d, e, Zunyao Wang a,
Daniel L. Villeneuve f, Hongxia Yu a
a
State Key Laboratory of Pollution Control & Resource Reuse, School of the Environment, Nanjing University, Nanjing, 210023, PR China
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA
d
School of Biological Sciences, University of Hong Kong, Hong Kong, China
e
Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China
f
United States Environmental Protection Agency, Mid-Continent Ecology Division, Duluth, MN, USA
b
c
h i g h l i g h t s
Thirteen PCDPSs caused AhR activation.
Xenobiotic metabolism pathway was the primary transcriptomic response.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2015
Received in revised form
29 September 2015
Accepted 29 September 2015
Available online xxx
Polychlorinated diphenyl sulfides (PCDPSs) are a group of environmental pollutants for which limited
toxicological information is available. This study tested the hypothesis that PCDPSs could activate the
mammalian aryl hydrocarbon receptor (AhR) mediated toxicity pathways. Eighteen PCDPSs were tested
in the H4IIE-luc transactivation assay, with 13/18 causing concentration-dependent AhR activation. Potencies of several congeners were similar to those of mono-ortho substituted polychlorinated biphenyls.
A RNA sequencing (RNA-seq)-based transcriptomic analysis was performed on H4IIE cells treated with
two PCDPS congeners, 2,20 ,3,30 ,4,5,6-hepta-CDPS, and 2,4,40 ,5-tetra-CDPS. Results of RNA-seq revealed a
remarkable modulation on a relatively short gene list by exposure to the tested concentrations of PCDPSs,
among which, Cyp1 responded with the greatest fold up-regulation. Both the identities of the modulated
transcripts and the associated pathways were consistent with targets and pathways known to be
modulated by other types of AhR agonists and there was little evidence for significant off-target effects
within the cellular context of the H4IIE bioassay. The results suggest AhR activation as a toxicologically
relevant mode of action for PCDPSs suggests the utility of AhR-related toxicity pathways for predicting
potential hazards associated with PCDPS exposure in mammals and potentially other vertebrates.
© 2015 Elsevier Ltd. All rights reserved.
Keywords:
Toxicogenomics
Molecular initiating event
Ligand binding domain
RNA-seq
Cyp1A
Xenobiotic metabolism
1. Introduction
Polychlorinated diphenyl sulfides (PCDPSs) have recently been
reported as priority pollutants because of their persistence and
environmental mobility properties (Mostrag et al., 2010). Due to
* Corresponding author. School of the Environment, Nanjing University 163
Xianlin Avenue, Qixia Nanjing, 210000, China.
E-mail addresses: howard50003250@yahoo.com, Zhangxw@nju.edu.cn (X. Zhang).
http://dx.doi.org/10.1016/j.chemosphere.2015.09.107
0045-6535/© 2015 Elsevier Ltd. All rights reserved.
their uses as lubricants and fire retardants (Naito et al., 1995;
Nakanishi and Umemoto, 2002), PCDPSs have been detected in a
wide range of environmental media, including dust from metal
recycling plants (Sinkkonen et al., 1994), and water and sediments
of the Elbe River (Schwarzbauer et al., 2000). Recently, congeners of
PCDPS were detected at concentrations of 0.1e6.9 (ng/g, dry mass
(dm)) in surface sediment and 0.18e2.03 (ng/L) in surface water,
respectively, from the Yangtze River (Zhang et al., 2014a). However,
information on mechanisms and thresholds for toxicological effects
of PCDPSs was limited.
J. Zhang et al. / Chemosphere 144 (2016) 1754e1762
It has been reported that PCDPSs could cause acute individual
mortality cross various taxa. In vitro studies have demonstrated that
several congeners of PCDPSs have antimicrobial and pesticidal activity (Ambrus et al., 2005; Logoglu et al., 2006). In vertebrates,
acute mortality and hepatic oxidative stress were observed in fish
and mice following exposure to PCDPS (Li et al., 2012a, 2012b;
Zhang et al., 2012). Our recent study demonstrated that some
PCDPS congeners can activate aryl hydrocarbon receptor 1 (AhR1)
in engineered luciferase reporter gene (LRG) assays based on avian
species (Zhang et al., 2014b). Further more, some PCDPSs like
2,3,30 ,4,5,6-hexa-CDPS (Relative potency, 1.9 103 TCDD) and
2,20 ,3,30 ,4,5,6-hepta-CDPS (Relative potency, 7.2 103 TCDD)
demonstrated higher relative potencies than that of OctaCDD,
OctaCDF, and most of the coplanar PCBs based on avian WHO-TEFs.
However, it was unknown if the activation of AhR by PCDPS was
conserved in mammalian systems and what role the AhR-mediated
pathway might play in toxic effects of PCDPSs.
The primary goal of this study was to test the hypothesis that
PCDPSs could activate the mammalian aryl hydrocarbon receptor
(AhR) mediated toxicity pathway. The capacity and relative potency
of PCDPSs to activate this molecular event could in turn suggest
relevant toxicological effects that may be of concern in mammals or
other vertebrates following exposure to PCDPSs. The specific objectives were three-fold: 1) to evaluate relative potencies of 18
PCDPSs to up-regulate the AhR-mediated pathways by use of the
mammalian cell-based transactivation reporter gene assay; 2) to
verify whether transcriptional pathways activated in wild type
H4IIE cells were consistent with AhR activation as a primary mode
of action in a mammalian hepatic cell context using of RNA-seq and
qRT-PCR; 3) to test the hypothesis that transcripts altered by PCDPS
are the same as those previously identified to be mediated by AHR
agonists.
1755
previously observed in H4IIE-luc cells at 72 h after dosing by MTS
cytotoxicity assay at tested concentration range of PCDPSs, which
was consistent with the previous report in COS-7 cells with the
same exposure method above (Zhang et al., 2014b). Three replicates
were conducted per treatment in the same plate and three independent experiments were conducted on three different plates. The
cells were lysed and luciferase activity was measured at the end of
72 h incubation using a LucLite kit (Promega, Madison, WI, USA) in
a Synergy H4 Hybrid Multi-Mode Microplate reader (BioTek Instruments, Winooski, VT).
2.3. H4IIE-luc data analysis
2. Materials and methods
Background-corrected luciferase activity elicited by PCDPSs was
normalized to percent response value relative to the maximal
luciferase activity induced by TCDD. The normalized luciferase activity data were imported into GraphPad (GraphPad Prism 5.0
software, San Diego, CA, USA) and fitted to a four parameters logistic model. Concentrations of PCDPSs that elicited a response
equal to x % of the positive control (PC) response, were referred to
as PCx, while ECx denotes the concentrations that elicited a
response equal to x% of the maximum response caused by tested
chemicals. EC50, PC10, PC20, PC50, PC80 and maximal response values
were determined for each replicate concentrationeresponse curve.
ReP values were calculated according to the systematic framework
previously proposed with some modifications (Villeneuve et al.,
2000). If no significant induction activity was observed, a RePEC50
value was estimated by dividing the EC50 value of TCDD by the
maximum concentration of PCDPSs tested. The relative potency of
PCDPSs compared to TCDD was defined as: EC50, PC10, PC20, PC50 or
PC80 of TCDD ÷ EC50, PC10, PC20, PC50 or PC80 of the PCDPSs. As
described previously, the RePEC50 was excluded from RePavg calculation because it can overestimate potency (Zhang et al., 2014a,
2014b).
2.1. Chemicals and solutions
2.4. H4IIE exposure and RNA sequencing
PCDPSs were synthesized and tested for the absence of
contamination with PCDDs/PCDFs as previously described (Zhang
et al., 2014b). Nominal concentrations of PCDPSs stock solution
ranging from 3 103 to 1 107 nM were prepared in dimethyl
sulfoxide (DMSO; SigmaeAldrich, St. Louis, MO, USA). Serial dilutions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were also
prepared from a stock solution with a nominal concentration of
2.48 105 nM in DMSO and used as a reference chemical in the
bioassay. For in vitro reporter gene assays, test solutions of each
individual PCDPS were prepared by dissolving the serially diluted
solutions with the cell culture medium before dosing.
HII4E rat, hepatoma cells that had not been transfected with a
stable construct containing the luciferase gene under control of the
DRE, were purchased from the Institute of Basic Medical Sciences
Chinese Academy of Medical Sciences (Beijing, China). Two of the
PCDPSs, 2,4,40 ,5-tetra-CDPS (S7), 2,20 ,3,30 ,4,5,6-hepta-CDPS (S2)
were chosen for the RNA-seq analysis because of their relatively
greater potencies compared with others. HII4E cells at
1 105 cells mL1 were cultured in a six-well plate and treated
with 650 nM 2,20 ,3,30 ,4,5,6-hepta-CDPS, or 350 nM 2,4,40 ,5-tetraCDPS, the PC50 concentration for each chemical, freshly dissolved in
dimethyl sulfoxide (DMSO), as well as with solvent control for 72 h.
Three independent experiments were conducted with three
different batches of cell culture and two replicate wells of each
treatment were tested in each experiment. HII4E cells were harvested after 72 h exposure and for each replicate, total RNA was
extracted using RNeasy mini kit (QIANGEN, GmbH, Hilden) and
stored at 80 C. Concentrations of RNA were measured using
Synergy H4 Hybrid Take3 reader and quality of RNA was determined by using Agilent 2100 bioanalyzer (Agilent technologies,
Santa Clara, CA, US). RNA integrity number (RIN) values for all
samples were 9.
For each batch of cells, two samples of RNA from each treatment
were pooled. Nine RNA libraries (n ¼ 3 for 2,20 ,3,30 ,4,5,6-heptaCDPS, n ¼ 3 for 2,4,40 ,5-tetra-CDPS and n ¼ 3 for DMSO control)
were prepared from 8 mg total RNA using Dynabeads mRNA DIRECT
Micro Kit (Life technologies, AS, Oslo, Norway) and Ion Total RNASeq Kit v2 (Life technologies, Austin). Sequencing was performed
on Ion Torrent Proton with Ion PI Template OT2 200 Kit v2 (Life
2.2. H4IIE-luc assay
The H4IIE-luc transactivation cell-based assay was used to
assess AhR-mediated activity and potency of chemicals as previously described (Eichbaum et al., 2014; Lee et al., 2013; Su et al.,
2012). The H4IIE-luc assay is based on rat hepatoma cells that
have been stably transfected with a luciferase reporter gene under
the control of the dioxin response enhancer (DRE) (Hilscherova
et al., 2000). The H4IIE-luc cells were plated at a concentration of
~6000 cells per well in 384-well plates at 75 mL per well
(~80,000 cells/ml). The cells were then maintained in Dulbecco's
Modified Eagle Medium at 37 C with 5% CO2 and 99% humidity.
Twenty-four hours later, the cells were dosed by multi-channel
pipette with 0.8 mL of DMSO (solvent control) or DMSO solutions
of TCDD (1 104e5 10 0 nM) or PCDPSs (3 100e5 104 nM).
The final concentration of DMSO was 0.5%. No cytotoxic effect was
1756
J. Zhang et al. / Chemosphere 144 (2016) 1754e1762
technologies, Carlsbad, CA, USA) and Ion PI Sequencing 200 Kit v2
(Life technologies, Carlsbad, CA, USA).
response) and (2) lesser efficacy PCDPSs (the other PCDPSs except
PCDPSs, maximal response < 50% of positive control response)
(Fig. 1).
2.5. RNA-seq data analysis
Raw sequence data were first trimmed of the adaptor with
barcode and filtered with the default parameter by the torrent
server. Filtered fastq files (n ¼ 9) were downloaded from the torrent
server and imported into the CLC Genomic Workbench 7.0.3 software (QIAGEN, Boston, MA, USA). Each file was mapped to the
Ensembl Rattus norvegicus 5.0.75 genome sequence annotated with
the R. norvegicus 5.0.75 gene transfer format (GTF) file. Mapped
total exon reads for 26312 genes were exported and EdgeR (Bioconductor R package, version 3.6.8) (Robinson et al., 2010) was used
to estimate differential expression. A significant change was
defined as False Discovery Rate (FDR) q-value < 0.1 and fold-change
1.5 or 0.667. Gene network analysis for differentially expressed
genes (DEGs) was performed on the GeneMania server (http://
genemania.org/) (Warde-Farley et al., 2010) using default parameter. Network data and expression data were imported into Cytoscape software 3.2.0 for visualization and further analysis (Doerks
et al., 2002). Gage (Bioconductor R package, version 2.14.4) (Luo
et al., 2009) and pathview (Bioconductor R package, version 1.4.2)
(Luo and Brouwer, 2013) package were used for gene set enrichment analysis (GSEA). Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathways affected by 2,4,40 ,5-tetra-CDPS and 2,20 ,3,30 ,4,5,6hepta-CDPS were determined as FDR q-value < 0.1.
2.6. qRT-PCR analysis
Eight genes (Cyp1a1, Cyp1a2, Cyp1b1, Gsta2, Gstp1, Nqo1, Utg1a2,
and Utg2b7, full gene description see Table 1) with a reference gene
(ACTB) were selected for qRT-PCR. The reverse transcription for
each pooled sample was performed using a QuantiTect Reverse
Transcription Kit (QIAGEN, GmbH, Hilden). Primers of target genes
were designed by NCBI/Primer-BLAST software using gene mRNA
template reported in the NCBI database (Table S1). qRT-PCR was
performed in 96-well plates using QuantiTect SYBR Green PCR
Master Mix (QIANGEN, GmbH, Hilden). The amplification was
performed on StepOne Plus (Life technologies, Singapore) with an
initial denaturation at 95 C for 5 min followed by 40 cycles of 95 C
for 10s, 60 C for 30s. 3 replicate for each gene were performed. The
Ct values of the target genes were normalized by a house-keeping
gene b-actin (ACTB) using the DDCt method (Schmittgen and Livak,
2008). Fold change was calculated as 2DDCt.
3.2. Differential transcriptomic expression profiles modulated by
PCDPSs demonstrated by RNA pyrosequencing
Alterations to the transcriptome of wild-type H4IIE cells
following exposure to two of the more potent PCDPS congeners
(Fig. 2), 650 nM 2,20 ,3,30 ,4,5,6-hepta-CDPS, or 350 nM 2,4,40 ,5-tetraCDPS, was used to evaluate whether AhR activation was the
dominant toxicological activity of these compounds and to test the
hypothesis that transcripts altered by PCDPS are the same as those
previously identified to be mediated by AHR activators. A total of
147,648,904 reads with an average length of 92 bp were obtained
from the torrent server. The average sequencing depth for each library was 16,405,434 ± 1,379,350 reads. In total, 105,959,346 reads
were mapped to the reference genome (Table S2). The mean
mapping ratio was 71.76%. 22,017 genes had been detected in total,
which showed sequencing depth was adequate for data analysis.
Both 2,20 ,3,30 ,4,5,6-hepta-CDPS and 2,4,40 ,5-tetra-CDPS altered
the transcriptome of untransfected H4IIE cells. Seventeen genes
were up-regulated and 18 genes down-regulated in 2,20 ,3,30 ,4,5,6hepta-CDPS treatment. Ten genes were up-regulated and 3 genes
down-regulated in 2,4,40 ,5-tetra-CDPS treatment (Table 1, Fig. 3a).
Five up-regulated genes (Cyp1a1, Cyp1a2, NAD(P)H dehydrogenase
quinone 1 (Nqo1), glutathione S-transferase alpha 2 (Gsta2), and
ENSRNOG00000047433) and two down-regulated genes (ENSRNOG00000033625 and ENSRNOG00000048373) were observed in
both treatments. Blastp against UniProt database were performed
for the uncharacterized genes (Table 1). Most of the DEGs, including
Cyp1a1, Cyp1a2, ENSRNOG00000047433 (similar to Cyp1b1), Nqo1,
and Gsta2, shared by two chemicals, are known to be regulated by
AhR. These five genes also ranked as the top significant DEGs (lesser
FDR q-value) with greater fold-changes.
Results of qRT-PCR further validated the conclusions based on
results of RNA sequencing. Most qRT-PCR results of the selected
genes in following exposure to 2,20 ,3,30 ,4,5,6-hepta-CDPS or
2,4,40 ,5-tetra-CDPS, were consistent with the RNA-seq results
(Table S3). Linear regression between log10 transformed RNA-seq
fold-change and log10 transformed qRT-PCR fold-change was statistically significant (least-squares linear regression, p < 0.0001;
R2 ¼ 0.9284) (Fig. 4). This gives further confidence in the results
obtained from RNA-seq.
4. Discussion
3. Results
3.1. Induction of AhR mediated luciferase activity in H4IIE-luc cells
A concentration-dependent induction of luciferase activity in
H4IIEeluc cells was observed after exposure to the majority of the
tested PCDPSs (Fig. 1, Table 2). Luciferase activity induced by TCDD
reached a plateau at higher concentrations, while the concentration
response curve of most of tested PCDPSs were failed to reach an
obvious plateau. 5 nM TCDD was used as positive control for the
normalization of luciferase activity data in H4IIE-luc assay since the
response induced was in the plateau phase. No significant luciferase activity was induced by several PCDPSs (2,20 ,3,3'-tetra-CDPS,
2,20 ,3-tri-CDPS, 2,40 ,5-tri-CDPS, 2,40 ,6-tri-CDPS, 2,3,30 -tri-CDPS) in
the tested concentration ranges. Overall PCDPSs can be grouped
into two general categories according to the maximal responses
induced in H4IIE-luc cells: (1) greater efficacy PCDPSs
(2,20 ,3,30 ,4,5,6-hepta-CDPS,
2,4,40 ,5-tetra-CDPS,
2,3,30 ,4,40 ,5,6hepta-CDPS; maximal response 40% of positive control
The majority of the tested PCDPSs significantly activated AhR
mediated effect in H4IIEeluc cells. Since contaminant-related artifacts have been reported in previous studies, the test PCDPSs were
checked for the presence of trace contaminants that could be AhR
agonists. The various PCDPS congeners were concentrated so that
even trace amounts of congeners of PCDF or polychlorinated
dibenzo dioxins (PCDD) or polychlorinated naphthalenes (PCNs)
sufficient to be detected in the bioassay would have been detected
by the high resolution gas chromatography high resolution mass
spectrometry (HRGC/HRMS), however none of these known AhR
agonists were observed (Zhang et al., 2014b). The confirmed lack of
contaminants in the present study, along with the fact that the
doseeresponse relationships observed among species for PCDPSs
were consistent, allows us to conclude that the AhR-mediated potencies observed for the PCDP congeners were not artifacts. The
activation of AhR mediated toxicity pathway in mammalian hepatic
cells could help to explain the previous observation that PCDPSs
caused acute lethality in mice (Zhang et al., 2012). Since activation
J. Zhang et al. / Chemosphere 144 (2016) 1754e1762
1757
Table 1
Genes identified as differentially expressed (DEGs) in wild-type H4IIE cells exposed to 2,20 ,3,30 ,4,5,6-hepta-CDPS or 2,4,40 ,5-tetra-CDPS, compared to DMSO-treated controls.
Differential expression was defined as FDR q-value 0.1 with fold-change 0.667 or fold-change 1.5 as determined using the EdgeR package. Red cell means up-regulated,
gray cell means down-regulated.
Gene Symbol
2,2´,3,3´,4,5,6-hepta-C
DPS treatment
Gene Description
Cyp1a1
Cyp1a2
cytochrome P450, family 1, subfamily a, polypeptide 1
cytochrome P450, family 1, subfamily a, polypeptide 2
cytochrome P450, family 1, subfamily b, polypeptide 1 (e-value=2e-103,
ENSRNOG00000047433*
score=816, identity=100%)
Cyp1b1
cytochrome P450, family 1, subfamily b, polypeptide 1
Mt1a
metallothionein 1a
Gsta2
glutathione S-transferase alpha 2
Nqo1
NAD(P)H dehydrogenase, quinone 1
Mt2A
metallothionein 2A
Ugt2b7
UDP glucuronosyltransferase 2 family, polypeptide B7
Gldc
glycine dehydrogenase
Areg
amphiregulin
Sod3
superoxide dismutase 3
PPIA
peptidylprolyl isomerase A
Aldh1a7
aldehyde dehydrogenase family 1, subfamily A7
Selenbp1
selenium binding protein 1
Tmem86b
transmembrane protein 86B
RGD1562259
similar to 40S ribosomal protein S20
Impad1
inositol monophosphatase domain containing 1
Actg1
actin, gamma 1
Heat shock cognate 71 kDa
ENSRNOG00000034066*
protein(e-value=0,score=2296,identity=99%)
ENSRNOG00000007930* Ribosomal protein S2(e-value=6e-80,score=619,identity=99%)
ENSRNOG00000033625* 60S ribosomal protein L35a (e-value=1e-74, score=581, identity=99.0%)
ECHS1
enoyl CoA hydratase, short chain, 1, mitochondrial
RNA polymerase I-specific transcription initiation factor RRN3 gene
ENSRNOG00000048373*
(e-value=2e-58, score=511, identity=83.0%)
ENSRNOG00000015559* 60S ribosomal protein L7a(e-value=2e-92,score=766,identity=91%)
Peptidyl-prolyl cis-trans isomerase A
ENSRNOG00000027864*
(e-value=2e-61,score=430,identity=72%)
RPS28
ribosomal protein S28
ENSRNOG00000028666* 60S ribosomal protein L21(e-value=8e-66,score=568,identity=75%)
Rps27l3
ribosomal protein S27-like 3
Rps19l1
ribosomal protein S19-like 1
RGD1562755
similar to 60S ribosomal protein L23a
ENSRNOG00000048958* 60S ribosomal protein L37 (e-value=3e-64, score=511, identity=97%)
Gns
glucosamine (N-acetyl)-6-sulfatase
RT1-DMb_1
major histocompatibility complex, class II, DM beta
AKR1B10
aldo-keto reductase family 1, member B10
SLC25A51
solute carrier family 25, member 51
Heat shock cognate 71 kDa
ENSRNOG00000034093*
protein(e-value=0,score=3235,identity=99%)
MRPL30_1
mitochondrial ribosomal protein L30
Itga7
integrin, alpha 7
Rps18-ps3
ribosomal protein S18, pseudogene 3
*, Uncharacterized genes were shown the blastp results against UniProt database.
of AhR could increase Superoxide dismutase 2 (SOD2) acetylation
and thereby decrease SOD2 activity via the mechanism of mitochondrial sirtuin deacetylase 3 (Sirt3), the PCDPSs activated AhR
activity found in the present study supported the previous
FDR
2,4,4´,5-tetra-CDPS
treatment
Fold-Change
1.83E+02
3.40E+01
5.64E-247
4.88E-95
Fold-Change
2.43E+01
7.39E+00
1.25E-68
3.79E-16
FDR
5.34E+00
4.50E-39
1.95E+00
1.08E-02
6.61E+00
2.54E+00
2.30E+00
2.22E+00
2.52E+00
1.75E+00
2.10E+00
1.98E+00
1.71E+00
3.26E+00
1.57E+00
1.50E+00
2.52E+00
3.01E-01
2.62E-01
2.81E-01
3.68E-23
5.49E-20
2.78E-16
6.04E-15
6.80E-10
1.95E-05
2.00E-04
1.14E-03
1.59E-03
4.20E-03
4.20E-03
3.62E-02
5.62E-02
8.47E-19
4.30E-14
5.48E-14
2.27E+00
1.40E+00
1.73E+00
1.54E+00
1.05E+00
1.50E+00
1.38E+00
1.66E+00
1.42E+00
1.77E+00
1.47E+00
1.43E+00
2.41E+00
6.98E-01
4.45E-01
4.83E-01
1.30E-01
4.35E-01
8.65E-06
1.08E-02
1.00E+00
3.17E-01
1.00E+00
1.27E-01
7.56E-01
1.00E+00
1.30E-01
3.73E-01
7.01E-01
1.00E+00
1.32E-01
4.63E-01
5.59E-01
2.31E-06
6.64E-01
1.30E-01
4.01E-01
2.51E-01
3.82E-01
3.37E-05
7.49E-05
2.00E-04
1.06E+00
3.57E-01
8.47E-01
1.00E+00
1.00E-02
1.00E+00
2.12E-01
3.31E-04
3.14E-01
4.05E-02
4.10E-01
5.16E-04
8.29E-01
1.00E+00
5.37E-01
2.92E-03
7.93E-01
1.00E+00
5.30E-01
3.62E-01
5.86E-01
5.01E-01
1.73E-01
4.36E-01
3.80E-01
5.67E-01
1.68E+00
2.08E+00
4.80E-03
5.16E-03
7.07E-03
3.38E-02
3.98E-02
4.95E-02
8.85E-02
9.78E-02
8.94E-01
2.56E-01
6.23E-01
4.74E-01
9.20E-01
5.72E-01
5.59E-01
6.51E-01
7.99E-01
1.02E+00
2.09E+00
2.78E+00
9.16E-01
1.30E-01
1.00E+00
1.00E+00
1.00E+00
1.00E+00
1.00E+00
1.00E+00
6.78E-04
1.08E-02
1.18E+00
1.00E+00
1.58E+00
6.19E-02
3.09E+00
1.84E+00
5.38E-01
4.74E-01
8.86E-01
1.97E-01
4.02E+00
2.45E+00
4.15E-01
6.94E-02
7.40E-02
2.00E-02
observation that lower-substituted PCDPSs decreased in mouse
liver (Zhang et al., 2012).
The effects of AhR activation effects by exposure to PCDPSs
could be explained by two possible mechanisms. Firstly PCDPSs
Fig. 1. PCDPS induced AhR mediated activity. a, Structural formulas of 18 PCDPSs tested in bioassays; b. Concentration-dependent effects of TCDD and PCDPSs on luciferase activity
in H4IIE-luc cells. Data are presented as percent response values relative to that of a 5 nM TCDD positive control. Concentration-response curves are only presented for the PCDPSs
that induced a significant (p < 0.05), concentration-dependent increase in luciferase activity relative to the DMSO response. Points represent mean, positive control-normalized
luciferase activities obtained from 3 independent experiments, each with 3 technical replicates per concentration of PCDPS or TCDD. Bars represent standard error.
J. Zhang et al. / Chemosphere 144 (2016) 1754e1762
1759
Table 2
Relative potency (ReP) values for PCDPSs in the H4IIE-luc assay. The average relative potency (RePavg) values and ReP ranges were calculated from PC10-, PC20-, PC50- and PC80based ReP values. If no induction of luciferase reporter gene activity was observed, RePEC50 values were estimated by dividing the TCDD EC50 value by the maximum concentration tested of PCDPS.
Compound
RePEC50
RePPC10
RePPC20
RePPC50
RePPC80
RePavg
ReP range
TCDD
2,3,30 ,4,5,6-hexa-CDPS
2,20 ,3,30 ,4,5,6-hepta-CDPS
2,20 ,30 ,4,5-penta-CDPS
2,4,40 ,5-tetra-CDPS
2,3,30 ,4,40 ,5,6-Hepta-CDPS
2,3,4,40 ,5,6-hepta-CDPS
2,20 ,4,5-tetra-CDPS
4,4'-di-CDPS
2,20 ,4,40 ,5-penta-CDPS
2,3,4,5,6-penta-CDPS
2,30 ,4,5-tetra-CDPS
3,4'-di-CDPS
2,20 ,3,3'-tetra-CDPS
2,3-di-CDPS
2,20 ,3-tri-CDPS
2,40 ,5-tri-CDPS
2,40 ,6-tri-CDPS
2,3,30 -tri-CDPS
1.0
NC
NC
4.2 105
NC
1.1 105
NC
NC
NC
NC
4.5 106
NC
NC
<1.1 106
NC
<5.3 108
<2.1 106
<2.1 106
<1.1 106
1.0
8.1
3.3
1.3
4.0
3.4
NE
8.9
NE
6.7
NE
1.6
NE
NE
NE
NE
NE
NE
NE
1.0
8.5
2.1
1.1
3.6
3.3
NE
8.7
NE
7.6
NE
1.4
NE
NE
NE
NE
NE
NE
NE
1.0
NE
1.6 105
NE
3.0 105
3.9 107
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
1.0
NE
NE
NE
2.8 105
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
1.0
8.3
2.3
1.2
3.3
2.4
NA
8.8
NA
7.2
NA
1.5
NA
NA
NA
NA
NA
NA
NA
1.0e1.0
8.1 107
1.6 105
1.1 105
2.8 105
3.9 107
NA
8.7 107
NA
6.7 107
NA
1.4 106
NA
NA
NA
NA
NA
NA
NA
107
105
105
105
106
107
107
106
107
105
105
105
106
107
107
106
107
105
105
105
106
107
107
106
~
~
~
~
~
8.5
3.3
1.3
4.0
3.4
107
105
105
105
106
~ 8.9 107
~ 7.6 107
~ 1.6 106
NC: Not calculated because the maximal response was not reached.
NE: Not estimated because the maximum observed response was below 10%, 20%, 50% or 80% of positive control response.
NA: ReP estimates not available to calculate the value.
could bind to ligand binding domain (LBD) of AhR directly and form
the ligand:AhR:ARNT heterodimer which further stimulate the
transcription of downstream genes. However, this “agonism”
mechanism still need further resting to validate. In general, PCDPSs
like polybrominated diphenyl ethers (PBDEs) are larger than PCDDs
and PCDFs. Because their ether and sulfide linkages are not planar,
they do not meet the structural criteria of more classic AhR agonists
(Villeneuve et al., 2002). While PCDPSs, like PBDEs do not seem to
conform to the size and shape of the AhR, there are generally a few
congeners in PBDEs that are able to elicit dioxin-like, AhR-mediated
responses (Koistinen et al., 1996; Villeneuve et al., 2002; Zhang
et al., 2014b). In most cases, the effects are comparatively weak,
up to 100,000-fold less potent than 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) in certain species. One possibility is that one end of
the molecule can interact with the LBD with sufficient affinity to
activate the receptor. However, for this to occur, the AhR would still
need to undergo the transformation of losing several heat shock
proteins and bind to the aromatic nuclear transport protein (ARNT)
and still bind to the DRE (Hilscherova et al., 2000). Given the difficulty readily predicting these atypical interactions with the AhR
LBD from structure alone, the rat-based bioassay used in the
Fig. 2. Relative potency (ReP) of PCDPSs in H4IIE-luc assay. Broader represented the
range of ReP values calculated for each compound (RePrange) and the bar in the middle
represented the average ReP value (RePavg).
present study provides an effective tool to rapidly screen and
identify PCDPS congeners that can activate AhR receptor and rank
their relative potencies.
Alternatively, AhR could be activated by the exposure to PCDPSs
via indirect mechanism. Indeed, recent studies have suggested that
the inhibition of CYP1 activities by compounds compatible with the
active sites can inhibit the degradation of endogenous AHR agonists
in the cell culture media (Wincent et al., 2012; Henry et al., 2006).
In this case AHR would be activated, but the actual agonist would
not be the exposure compound. Therefore, further study should
look for direct evidence of PCDPS binding to AHR.
REPavg values of 3 PCDPSs including 2,20 ,3,30 ,4,5,6-hepta-CDPS,
2,20 ,30 ,4,5-penta-CDPS, 2,4,40 ,5-tetra-CDPS were similar to or
greater than WHO-TEF of most mono-ortho substituted PCBs
(PCB118, PCB156, PCB189 et al. at 0.00003) (Van den Berg et al.,
2006). This highlights the potential toxicity that might be caused
by PCDPSs. Comparing RePavg values with the number of
substituted Cl atoms, RePavg of PCDPS with 2e3 substituted Cl
atoms (n ¼ 7) were all NA and PCDPSs with 4e7 substituted Cl
atoms (n ¼ 11) showed greater RePavg (Fig. S1). This was consistent
with the previous avian results that the relative potency (ReP) of
the PCDPSs increased with the increasing number of substituted Cl
atoms in avian AhR1 LRG assays (Zhang et al., 2014a, 2014b).
Remarkable modulation on a relatively short gene list was
observed in H4IIE cells exposed to the concentration that could
cause 50% AhR-mediated luciferase activity by either 2,20 ,3,30 ,4,5,6hepta-CDPS or 2,4,40 ,5-tetra-CDPS. Cyp1a1 and Cyp1a2 were the
two genes up-regulated with the greatest fold change in both
PCDPSs treatments. Both are known to be AhR-regulated genes and
are frequently used as molecular markers of exposure to dioxin-like
compounds (Kim et al., 2009). In other studies of the transcriptome
of cells or organisms exposed to TCDD (Boverhof et al., 2006;
Ovando et al., 2010) and other dioxin-like compounds like PCB
(Carlson et al., 2009; Ovando et al., 2010), TCDF, or 4-PeCDF
(Rowlands et al., 2007). CYP1 has been consistently identified as
the most significant gene with the greatest fold-change. This
indicated that transcriptomic responses in H4IIE cells exposed to
2,4,40 ,5-tetra-CDPS and 2,20 ,3,30 ,4,5,6-hepta-CDPS were, in all
likelihood, primarily mediated through the AhR. However, these
Fig. 3. Transcriptional response profile by PCDPS. a. Expression of DEGs in 3 treatments. Color in each cell stand for the log2 transformed fold change. Green means down-regulated
and red means up-regulated. Hierarchical clustering was performed using Manhattan distance with ward linkage. Uncharacterized genes were marked with * and shown with
blastp results against UniProt database. b. Gene network for DEGs in 2,20 ,3,30 ,4,5,6-hepta-CDPS and 2,4,40 ,5-tetra-CDPS treatment. Red means up-regulated and green means downregulated. Node size means the proportional to the node connectivity. The edge color is proportional to the connection weight between the two nodes. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
J. Zhang et al. / Chemosphere 144 (2016) 1754e1762
Fig. 4. Linear regression of log10 transformed RNA-seq fold-change and log10 transformed qRT-PCR fold-change. RNA-seq experiment fold-change were derived from
EdgeR result. qRT-PCR fold-change were calculated by 2DDCt. Points stand for gene
fold-change in each treatment group.
five genes were not altered to the same extent by 2,4,40 ,5-tetraCDPS and 2,20 ,3,30 ,4,5,6-hepta-CDPS, although the cells were
exposed to concentrations equivalent to the PC50 in the LRG assay.
This result might be due to variation in addition of chemicals to cell
culture or due to inherent differences between H4IIE and H4IIE-luc
cells or random errors in calculation of the PC50.
Key genes from xenobiotic metabolism pathways were significantly altered by 650 nM 2,20 ,3,30 ,4,5,6-hepta-CDPS, and 350 nM
2,4,40 ,5-tetra-CDPS as indicated by GSEA analysis (FDR qvalue ¼ 0.072 in 2,20 ,3,30 ,4,5,6-hepta-CDPS treatment and 0.034 in
2,4,40 ,5-tetra-CDPS treatment). The retinol metabolism pathway
(FDR q-value ¼ 0.076) and steroid hormone biosynthesis pathway
(FDR q-value ¼ 0.076) were also altered by 2,20 ,3,30 ,4,5,6-heptaCDPS at the tested concentration level. These results suggested that
2,4,40 ,5-tetra-CDPS and 2,20 ,3,30 ,4,5,6-hepta-CDPS have a similar
toxicological mechanism.
The activated AhR mediated pathway dominated global transcriptomic responses in the cells exposed to PCDPSs at the PC50
concentration, a concentration much less than that causes cytotoxicity (not observed in both the 10 mM of 2,20 ,3,30 ,4,5,6-heptaCDPS and 100 uM of 2,4,40 ,5-tetra-CDPS treatment) (Fig. S2).
Metabolism of xenobiotics was primarily mediated by CYP1A1, 1A2,
and 1B1 that is well known to be the AhR-mediated pathway
(Schmidt and Bradfield, 1996). Metabolism of xenobiotics by the
cytochrome P450 enzymes was one of only two pathways that were
significantly altered primary hepatocytes of both human and rat by
exposure of TCDD and PCB126 (Carlson et al., 2009). This suggests
that metabolism of xenobiotics via cytochrome P450 is the pathway
in mammalian liver cells most sensitive to alteration by AhR
agonists.
Beside the pathway of xenobiotic metabolism by cytochrome
P450-dependent related to metabolism of retinol (Fig. S3) and
synthesis of steroid hormones (Fig. S4) were the two affected by
2,20 ,3,30 ,4,5,6-hepta-CDPS. The DEGs associated with these two
pathways were Cyp1a1, Cyp1a2, Cyp1b1, Ugt2b7 (UDP glucuronosyl
transferase) in the steroid hormone biosynthesis pathway and
Cyp1a1, Cyp1a2, Aldh1a7 (aldehyde dehydrogenase), Ugt2b7 in the
retinol metabolism pathway. Most of these genes not only have
AhR-regulated expression, but also overlap with genes included in
xenobiotic metabolism pathways. These overlapping genes suggest
that activation of AhR by 2,20 ,3,30 ,4,5,6-hepta-CDPS first affected
genes in the xenobiotic metabolism pathway and then altered
1761
expression of genes in the steroid hormone biosynthesis and retinol
metabolism pathways through several overlapped genes (such as
Cyp1). These results further suggest that, effects on these two
pathways were likely the result of interactions with the AhR, rather
than other potential factors, such RXR, g-protein coupled receptors,
that are involved in modulating expression of genes associated
with those pathways.
The gene network associated with the DEGs in both treatments
showed a similar result. The network affected by exposure to
2,4,40 ,5-tetra-CDPS included up-regulation of genes activated by
the AhR, which then lead to up-regulation of the integrin alpha 7,
primary laminin receptor (Itga7) (Fig. 3). Expression of Itga7 on
skeletal myoblasts and adult myofibers and influences myogenic
differentiation of mice is down-regulated in mice exposed to TCDD
(Thornley et al., 2011). Changes in expression of genes after exposure to 2,20 ,3,30 ,4,5,6-hepta-CDPS showed that two metallothionein
genes, Mt1a and Mt2A linked with most DEGs (Fig. 3). Consistent
with the results observed here, expression of these metallothionein
genes have been reported to be up-regulated by exposure to AhR
agonists (Peijnenburg et al., 2010; Sato et al., 2013). Expression of
ribosomal proteins was also reported to be down-regulated when
exposed to TCDD (Hanlon et al., 2005). Most differential expression
was observed in mice exposed to TCDD except RT1-DMb_1 (major
histocompatibility complex, class II, DM beta), which was a molecular response that was specific to exposures to PCDPSs (Thornley
et al., 2011).
5. Conclusion
In summary, the H4IIE-luc assay showed 13 of the tested PCDPSs
could activate AhR-mediated molecular toxicological pathways in
mammals. The ReP values of three PCDPSs including 2,20 ,3,30 ,4,5,6hepta-CDPS, 2,20 ,30 ,4,5-penta-CDPS, 2,4,40 ,5-tetra-CDPS were
similar to or greater than WHO-TEF of some mono-ortho
substituted PCBs (for example PCB105,118). However, whether the
activation of AhR by PCDPSs was due to direct “agonism” or indirect
mechanism still need further investigation. The results of the RNAseq experiment showed that AhR-mediated genes and pathways
were the most significant molecular response to PCDPSs at noncytotoxic concentrations. This supported the hypothesis that the
activation of AhR is potentially the sensitive and relevant molecular
initiating response with regard to the toxicities caused by PCDPSs,
at least in hepatic cells. The activation of AhR mediated toxicity
pathway by PCDPSs could be connected by linking AhR mediated
transcriptional activation reported here, to the decreased SOD activity and oxidative stress in liver, and acute lethality in PCDPSs
exposed animals (Zhang et al., 2012). These results suggest that AhR
mediated toxicity pathway could be used for predicting hazards
associated with exposure to PCDPSs, particularly the more potent
congeners.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Grant No. 21322704 and 21007025), National
High-tech R&D Program of China (863 Program, Grant No.
2013AA06A309). The research is also supported by the Collaborative Innovation Center for Regional Environmental Quality. Dr.
Daniel L Villeneuve and Dr. John Giesy were supported by the
program of 2014 “High Level Foreign Experts” (#GDT20143200016)
of the State Administration of Foreign Experts Affairs, the P.R. China.
Dr. John Giesy was also supported by the Einstein Professor Program of the Chinese Academy of Sciences and by the Canada
Research Chair program.
1762
J. Zhang et al. / Chemosphere 144 (2016) 1754e1762
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.chemosphere.2015.09.107.
References
Ambrus, A., Füzesi, I., Susan, M., Dobi, D., Lantos, J., Zakar, F., Korsos, I., Olah, J.,
Beke, B., Katavics, L., 2005. A cost-effective screening method for pesticide
residue analysis in fruits, vegetables, and cereal grains. J. Environ. Sci. Health 40
(2), 297e339.
Boverhof, D.R., Burgoon, L.D., Tashiro, C., Sharratt, B., Chittim, B., Harkema, J.R.,
Mendrick, D.L., Zacharewski, T.R., 2006. Comparative toxicogenomic analysis of
the hepatotoxic effects of TCDD in Sprague Dawley rats and C57BL/6 mice.
Toxicol. Sci. 94 (2), 398e416.
Carlson, E.A., McCulloch, C., Koganti, A., Goodwin, S.B., Sutter, T.R., Silkworth, J.B.,
2009. Divergent transcriptomic responses to aryl hydrocarbon receptor agonists
between rat and human primary hepatocytes. Toxicol. Sci. 112 (1), 257e272.
Doerks, T., Copley, R.R., Schultz, J., Ponting, C.P., Bork, P., 2002. Systematic identification of novel protein domain families associated with nuclear functions.
Genome Res. 12 (1), 47e56.
Eichbaum, K., Brinkmann, M., Buchinger, S., Reifferscheid, G., Hecker, M., Giesy, J.P.,
Engwall, M., van Bavel, B., Hollert, H., 2014. In vitro bioassays for detecting
dioxin-like activityeapplication potentials and limits of detection, a review. Sci.
total Environ. 487, 37e48.
Hanlon, P.R., Zheng, W., Ko, A.Y., Jefcoate, C.R., 2005. Identification of novel TCDDregulated genes by microarray analysis. Toxicol. Appl. Pharmacol. 202 (3),
215e228.
Henry, E.C., Bemis, J.C., Henry, O., Kende, A.S., Gasiewicz, T.A., 2006. A potential
endogenous ligand for the aryl hydrocarbon receptor has potent agonist activity
in vitro and in vivo. Arch. Biochem. Biophys. 450 (1), 67e77.
Hilscherova, K., Machala, M., Kannan, K., Blankenship, A.L., Giesy, J.P., 2000. Cell
bioassays for detection of aryl hydrocarbon (AhR) and estrogen receptor (ER)
mediated activity in environmental samples. Environ. Sci. Pollut. Res. 7 (3),
159e171.
Kim, S., Dere, E., Burgoon, L.D., Chang, C.-C., Zacharewski, T.R., 2009. Comparative
analysis of AhR-mediated TCDD elicited gene expression in human liver adult
stem cells. Toxicol. Sci. 112 (1), 229e244.
Koistinen, J., Sanderson, J.T., Giesy, J.P., Nevalainen, T., Paasivirta, J., 1996. Ethoxyresorufin-O-deethylase induction potency of polychlorinated diphenyl ethers in
H4IIE rat hepatoma cells. Environ. Toxicol. Chem. 15 (11), 2028e2034.
, K., Giesy, J.P., Khim, J.S., 2013.
Lee, K.T., Hong, S., Lee, J.S., Chung, K.H., Hilscherova
Revised relative potency values for PCDDs, PCDFs, and non-ortho-substituted
PCBs for the optimized H4IIE-luc in vitro bioassay. Environ. Sci. Pollut. Res. 20
(12), 8590e8599.
Li, X., Ye, L., Wang, X., Wang, X., Liu, H., Qian, X., Zhu, Y., Yu, H., 2012a. Molecular
docking, molecular dynamics simulation, and structure-based 3D-QSAR studies
on estrogenic activity of hydroxylated polychlorinated biphenyls. Sci. total Environ. 441, 230e238.
Li, Y., Li, M., Shi, J., Yang, X., Wang, Z., 2012b. Hepatic antioxidative responses to
PCDPSs and estimated short-term biotoxicity in freshwater fish. Aquat. Toxicol.
120e121, 90e98.
€
Logoglu, E., Arslan, S., Oktemer,
A., 2006. In vitro antimicrobial activity studies of
thioethoxy-and thiophyenoxyhalobenzene derivatives. Heterocycl. Commun. 12
(3e4), 219e224.
Luo, W., Friedman, M.S., Shedden, K., Hankenson, K.D., Woolf, P.J., 2009. GAGE:
generally applicable gene set enrichment for pathway analysis. BMC Bioinform.
10 (1), 161.
Luo, W., Brouwer, C., 2013. Pathview: an R/Bioconductor package for pathway-based
data integration and visualization. Bioinformatics 29 (14), 1830e1831.
Mostrag, A., Puzyn, T., Haranczyk, M., 2010. Modeling the overall persistence and
environmental mobility of sulfur-containing polychlorinated organic compounds. Environ. Sci. Pollut. Res. Int. 17 (2), 470e477.
Naito Y., Akaboshi F., Goto T., Sugyama N., Ono S., Fukaya T., Kuwabara E., Kajii M.,
Nishimura H., and Sugiura M. (1995). Diphenyl Sulfides, Sulfoxides, and Sulfones for Prevention and Treatment of Eosinophil-related Diseases. In: Patent.
Nakanishi H., and Umemoto N. (2002). Thermal-resistant Lubricating Oil Composition for Gas Turbines or Jet Engines. 13. In: JP Patent 2,002,003,878 A.
Ovando, B.J., Ellison, C.A., Vezina, C.M., Olson, J.R., 2010. Toxicogenomic analysis of
exposure to TCDD, PCB126 and PCB153: identification of genomic biomarkers of
exposure to AhR ligands. BMC Genom. 11, 583.
Peijnenburg, A., Riethof-Poortman, J., Baykus, H., Portier, L., Bovee, T.,
Hoogenboom, R., 2010. AhR-agonistic, anti-androgenic, and anti-estrogenic
potencies of 2-isopropylthioxanthone (ITX) as determined by in vitro bioassays and gene expression profiling. Toxicol. In Vitro Int. J. Publ. Assoc. BIBRA
24 (6), 1619e1628.
Robinson, M.D., McCarthy, D.J., Smyth, G.K., 2010. edgeR: a bioconductor package for
differential expression analysis of digital gene expression data. Bioinformatics
26 (1), 139e140.
Rowlands, J., Budinsky, R., Gollapudi, B., Boverhof, D., Ferguson, S., Novak, R.,
Cukovic, D., Salagrama, S., Dombkowski, A., 2007. Comparative gene expression
analysis of TCDD-, 4-PeCDF-and TCDF-treated primary rat and human hepatocytes. Organohalogen Compd. 69, 1862e1865.
Schmidt, J.V., Bradfield, C.A., 1996. AH receptor signaling pathways. Annu. Rev. Cell
Dev. Biol. 12 (1), 55e89.
Schmittgen, T.D., Livak, K.J., 2008. Analyzing real-time PCR data by the comparative
CT method. Nat. Protoc. 3 (6), 1101e1108.
Schwarzbauer, J., Littke, R., Weigelt, V., 2000. Identification of specific organic
contaminants for estimating the contribution of the Elbe river to the pollution
of the German Bight. Org. Geochem. 31 (12), 1713e1731.
Sinkkonen, S., Vattulainen, A., Aittola, J.-P., Paasivirta, J., Tarhanen, J., Lahtiper€
a, M.,
1994. Metal reclamation produces sulphur analogues of toxic dioxins and furans. Chemosphere 28 (7), 1279e1288.
Su, G., Xia, J., Liu, H., Lam, M.H., Yu, H., Giesy, J.P., Zhang, X., 2012. Dioxin-like potency of HO- and MeO- analogues of PBDEs' the potential risk through consumption of fish from eastern China. Environ. Sci. Technol. 46 (19),
10781e10788.
Sato, S., Shirakawa, H., Tomita, S., Tohkin, M., Gonzalez, F.J., Komai, M., 2013. The aryl
hydrocarbon receptor and glucocorticoid receptor interact to activate human
metallothionein 2A. Toxicol. Appl. Pharmacol. 273 (1), 90e99.
Thornley, J.A., Trask, H.W., Ridley, C.J., Korc, M., Gui, J., Ringelberg, C.S., Wang, S.,
Tomlinson, C.R., 2011. Differential regulation of polysome mRNA levels in mouse
Hepa-1C1C7 cells exposed to dioxin. Toxicol. In Vitro Int. J. Publ. Assoc. BIBRA
25 (7), 1457e1467.
Villeneuve, D.L., Blankenship, A.L., Giesy, J.P., 2000. Derivation and application of
relative potency estimates based on in vitro bioassay results. Environ. Toxicol.
Chem. 19 (11), 2835e2843.
Villeneuve, D.L., Kannan, K., Priest, B.T., Giesy, J.P., 2002. In vitro assessment of
potential mechanism-specific effects of polybrominated diphenyl ethers. Environ. Toxicol. Chem. 21 (11), 2431e2433.
Van den Berg, M., Birnbaum, L.S., Denison, M., De Vito, M., Farland, W., Feeley, M.,
Fiedler, H., Hakansson, H., Hanberg, A., Haws, L., Rose, M., Safe, S., Schrenk, D.,
Tohyama, C., Tritscher, A., Tuomisto, J., Tysklind, M., Walker, N., Peterson, R.E.,
2006. The 2005 World Health Organization reevaluation of human and
Mammalian toxic equivalency factors for dioxins and dioxin-like compounds.
Toxicol. Sci. 93 (2), 223e241.
Warde-Farley, D., Donaldson, S.L., Comes, O., Zuberi, K., Badrawi, R., Chao, P.,
Franz, M., Grouios, C., Kazi, F., Lopes, C.T., Maitland, A., Mostafavi, S., Montojo, J.,
Shao, Q., Wright, G., Bader, G.D., Morris, Q., 2010. The GeneMANIA prediction
server: biological network integration for gene prioritization and predicting
gene function. Nucleic Acids Res. 38 (Web Server issue), W214eW220.
Wincent, E., Bengtsson, J., Mohammadi Bardbori, A., Alsberg, T., Luecke, S.,
Rannug, U., Rannug, A., 2012. Inhibition of cytochrome P4501-dependent
clearance of the endogenous agonist FICZ as a mechanism for activation of
the aryl hydrocarbon receptor. Proc. Natl. Acad. Sci. U. S. A. 109 (12),
4479e4484.
Zhang, X., Qin, L., Qu, R., Feng, M., Wei, Z., Wang, L., Wang, Z., 2014a. Occurrence of
polychlorinated diphenyl sulfides (PCDPSs) in surface sediments and surface
water from the Nanjing section of the yangtze river. Environ. Sci. Technol. 48
(19), 11429e11436.
Zhang, X., Liu, F., Chen, B., Li, Y., Wang, Z., 2012. Acute and subacute oral toxicity of
polychlorinated diphenyl sulfides in mice: determining LD50 and assessing the
status of hepatic oxidative stress. Environ. Toxicol. Chem. 31 (7), 1485e1493.
Zhang, R., Zhang, X., Zhang, J., Qu, R., Zhang, J., Liu, X., Chen, J., Wang, Z., Yu, H.,
2014b. Activation of avian aryl hydrocarbon receptor and inter-species sensitivity variations by polychlorinated diphenylsulfides. Environ. Sci. Technol. 48
(18), 10948e10956.