Chemosphere 144 (2016) 1754–1762
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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
c
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
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
Article history:
Received 9 July 2015
Received in revised form 29 September
2015
Accepted 29 September 2015
Available online 11 November 2015
Handling editor: David C. Volz
Keywords:
Toxicogenomics
Molecular initiating event
Ligand binding domain
RNA-seq
Cyp1A
Xenobiotic metabolism
a b s t r a c t
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, 2,2 ,3,3 ,4,5,6-hepta-CDPS, and 2,4,4 ,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.
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 their
∗
Corresponding author. School of the Environment, Nanjing University 163 Xianlin Avenue, Qixia Nanjing, 210000, China.
E-mail address: [email protected], [email protected] (X. Zhang).
http://dx.doi.org/10.1016/j.chemosphere.2015.09.107
0045-6535/© 2015 Elsevier Ltd. All rights reserved.
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 0.1–6.9 (ng/g, dry mass (dm)) in surface
sediment and 0.18–2.03 (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) 1754–1762
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 2,3,3 ,4,5,6-hexa-CDPS (Relative potency, 1.9 × 10−3 TCDD)
and 2,2 ,3,3 ,4,5,6-hepta-CDPS (Relative potency, 7.2 × 10−3 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.
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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
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 concentration–response 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. Materials and methods
2.4. H4IIE exposure and RNA sequencing
2.1. Chemicals and solutions
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; Sigma–Aldrich, 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.
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 μL 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 μL of DMSO (solvent control) or DMSO solutions of TCDD (1 × 10−4 –5 × 100 nM) or PCDPSs (3 × 100 –
5 × 104 nM). The final concentration of DMSO was 0.5%. No cytotoxic effect was previously observed in H4IIE-luc cells at 72 h after
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, 2,4,4 ,5-tetra-CDPS (S7), 2,2 ,3,3 ,4,5,6-heptaCDPS (S2) were chosen for the RNA-seq analysis because of their
relatively greater potencies compared with others. HII4E cells at
1 × 105 cells mL−1 were cultured in a six-well plate and treated
with 650 nM 2,2 ,3,3 ,4,5,6-hepta-CDPS, or 350 nM 2,4,4 ,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 = 3 for 2,2 ,3,3 ,4,5,6hepta-CDPS, n = 3 for 2,4,4 ,5-tetra-CDPS and n = 3 for DMSO control) were prepared from 8 μg total RNA using Dynabeads mRNA
DIRECT Micro Kit (Life technologies, AS, Oslo, Norway) and Ion Total RNA-Seq Kit v2 (Life technologies, Austin). Sequencing was performed on Ion Torrent Proton with Ion PI Template OT2 200 Kit v2
(Life technologies, Carlsbad, CA, USA) and Ion PI Sequencing 200
Kit v2 (Life technologies, Carlsbad, CA, USA).
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J. Zhang et al. / Chemosphere 144 (2016) 1754–1762
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 = 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 2,4,4 ,5-tetra-CDPS and 2,2 ,3,3 ,4,5,6-heptaCDPS 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 housekeeping gene β -actin (ACTB) using the Ct method (Schmittgen
and Livak, 2008). Fold change was calculated as 2−Ct .
3. Results
3.1. Induction of AhR mediated luciferase activity in H4IIE-luc cells
A concentration-dependent induction of luciferase activity in
H4IIE–luc 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 (2,2 ,3,3’-tetra-CDPS, 2,2 ,3-tri-CDPS, 2,4 ,5-tri-CDPS, 2,4 ,6tri-CDPS, 2,3,3 -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 (2,2 ,3,3 ,4,5,6-hepta-CDPS, 2,4,4 ,5-tetraCDPS, 2,3,3 ,4,4 ,5,6-hepta-CDPS; maximal response ≥ 40% of positive control response) and (2) lesser efficacy PCDPSs (the other
PCDPSs except PCDPSs, maximal response < 50% of positive control response) (Fig. 1).
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 2,2 ,3,3 ,4,5,6-hepta-CDPS, or 350 nM 2,4,4 ,5-tetra-CDPS,
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 2,2 ,3,3 ,4,5,6-hepta-CDPS and 2,4,4 ,5-tetra-CDPS altered
the transcriptome of untransfected H4IIE cells. Seventeen genes
were up-regulated and 18 genes down-regulated in 2,2 ,3,3 ,4,5,6hepta-CDPS treatment. Ten genes were up-regulated and 3 genes
down-regulated in 2,4,4 ,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 2,2 ,3,3 ,4,5,6-hepta-CDPS
or 2,4,4 ,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 = 0.9284) (Fig. 4). This gives further confidence in the results
obtained from RNA-seq.
4. Discussion
The majority of the tested PCDPSs significantly activated AhR
mediated effect in H4IIE–luc 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
dose–response 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 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 observation that lower-substituted PCDPSs decreased in mouse liver
J. Zhang et al. / Chemosphere 144 (2016) 1754–1762
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Table 1
Genes identified as differentially expressed (DEGs) in wild-type H4IIE cells exposed to 2,2 ,3,3 ,4,5,6-hepta-CDPS or 2,4,4 ,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.
(Zhang et al., 2012).
The effects of AhR activation effects by exposure to PCDPSs
could be explained by two possible mechanisms. Firstly PCDPSs
could bind to ligand binding domain (LBD) of AhR directly and
form the ligand:AhR:ARNT heterodimer which further stimulate
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
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
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J. Zhang et al. / Chemosphere 144 (2016) 1754–1762
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) 1754–1762
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
PC80 -based 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
2,3,3 ,4,5,6-hexa-CDPS
2,2 ,3,3 ,4,5,6-hepta-CDPS
2,2 ,3 ,4,5-penta-CDPS
2,4,4 ,5-tetra-CDPS
2,3,3 ,4,4 ,5,6-Hepta-CDPS
2,3,4,4 ,5,6-hepta-CDPS
2,2 ,4,5-tetra-CDPS
4,4’-di-CDPS
2,2 ,4,4 ,5-penta-CDPS
2,3,4,5,6-penta-CDPS
2,3 ,4,5-tetra-CDPS
3,4’-di-CDPS
2,2 ,3,3’-tetra-CDPS
2,3-di-CDPS
2,2 ,3-tri-CDPS
2,4 ,5-tri-CDPS
2,4 ,6-tri-CDPS
2,3,3 -tri-CDPS
1.0
NC
NC
4.2 × 10−5
NC
1.1 × 10−5
NC
NC
NC
NC
4.5 × 10−6
NC
NC
<1.1 × 10−6
NC
<5.3 × 10−8
<2.1 × 10−6
<2.1 × 10−6
<1.1 × 10−6
1.0
8.1 × 10−7
3.3 × 10−5
1.3 × 10−5
4.0 × 10−5
3.4 × 10−6
NE
8.9 × 10−7
NE
6.7 × 10−7
NE
1.6 × 10−6
NE
NE
NE
NE
NE
NE
NE
1.0
8.5 × 10−7
2.1 × 10−5
1.1 × 10−5
3.6 × 10−5
3.3 × 10−6
NE
8.7 × 10−7
NE
7.6 × 10−7
NE
1.4 × 10−6
NE
NE
NE
NE
NE
NE
NE
1.0
NE
1.6 × 10−5
NE
3.0 × 10−5
3.9 × 10−7
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
1.0
NE
NE
NE
2.8 × 10−5
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
1.0
8.3 × 10−7
2.3 × 10−5
1.2 × 10−5
3.3 × 10−5
2.4 × 10−6
NA
8.8 × 10−7
NA
7.2 × 10−7
NA
1.5 × 10−6
NA
NA
NA
NA
NA
NA
NA
1.0–1.0
8.1 × 10−7 ∼ 8.5 × 10−7
1.6 × 10−5 ∼ 3.3 × 10−5
1.1 × 10−5 ∼ 1.3 × 10−5
2.8 × 10−5 ∼ 4.0 × 10−5
3.9 × 10−7 ∼ 3.4 × 10−6
NA
8.7 × 10−7 ∼ 8.9 × 10−7
NA
6.7 × 10−7 ∼ 7.6 × 10−7
NA
1.4 × 10−6 ∼ 1.6 × 10−6
NA
NA
NA
NA
NA
NA
NA
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.
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 dioxinlike, 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 2,3,7,8tetrachlorodibenzo-p-dioxin (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 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
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 ).
(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 2,2 ,3,3 ,4,5,6-heptaCDPS, 2,2 ,3 ,4,5-penta-CDPS, 2,4,4 ,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 2–3 substituted Cl atoms
(n = 7) were all NA and PCDPSs with 4–7 substituted Cl atoms
(n = 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 2,2 ,3,3 ,4,5,6-heptaCDPS or 2,4,4 ,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
2,4,4 ,5-tetra-CDPS and 2,2 ,3,3 ,4,5,6-hepta-CDPS were, in all likelihood, primarily mediated through the AhR. However, these five
genes were not altered to the same extent by 2,4,4 ,5-tetra-CDPS
and 2,2 ,3,3 ,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 2,2 ,3,3 ,4,5,6-hepta-CDPS, and
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J. Zhang et al. / Chemosphere 144 (2016) 1754–1762
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 downregulated 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 2,2 ,3,3 ,4,5,6-hepta-CDPS and 2,4,4 ,5-tetra-CDPS treatment. Red means up-regulated and
green means down-regulated. 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) 1754–1762
1761
ments showed a similar result. The network affected by exposure
to 2,4,4 ,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 2,2 ,3,3 ,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).
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 2− Ct . Points stand for
gene fold-change in each treatment group.
350 nM 2,4,4 ,5-tetra-CDPS as indicated by GSEA analysis (FDR qvalue = 0.072 in 2,2 ,3,3 ,4,5,6-hepta-CDPS treatment and 0.034
in 2,4,4 ,5-tetra-CDPS treatment). The retinol metabolism pathway
(FDR q-value = 0.076) and steroid hormone biosynthesis pathway
(FDR q-value = 0.076) were also altered by 2,2 ,3,3 ,4,5,6-heptaCDPS at the tested concentration level. These results suggested
that 2,4,4 ,5-tetra-CDPS and 2,2 ,3,3 ,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 μM of 2,2 ,3,3 ,4,5,6-hepta-CDPS
and 100 uM of 2,4,4 ,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 2,2 ,3,3 ,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 2,2 ,3,3 ,4,5,6-hepta-CDPS first affected
genes in the xenobiotic metabolism pathway and then altered 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 treat-
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 2,2 ,3,3 ,4,5,6-hepta-CDPS, 2,2 ,3 ,4,5-penta-CDPS, 2,4,4 ,5-tetraCDPS 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 RNA-seq experiment showed that AhR-mediated
genes and pathways were the most significant molecular response
to PCDPSs at non-cytotoxic 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.
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.
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Activation of AhR-Mediated Toxicity Pathway by Emerging Pollutants
Polychlorinated Diphenyl Sulfides
Junjiang Zhang,† Xiaowei Zhang,*† Pu Xia†,† Rui Zhang, Yang Wu,† Jie Xia,†
Guanyong Su,† Jiamin Zhang,† John P. Giesy,†,‡,§,¶ Zunyao Wang,† Daniel L.
Villeneuve,║ Hongxia Yu†
†
State Key Laboratory of Pollution Control & Resource Reuse, School of the
Environment, Nanjing University, Nanjing, P. R. China, 210023
‡
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
¶
School of Biological Sciences, University of Hong Kong, Hong Kong, SAR, China
Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong,
SAR, China
║
United States Environmental Protection Agency, Mid-Continent Ecology Division,
Duluth, MN, USA.
List of Tables
Table S1.
Primer designed for qRT-PCR by NCBI/ Primer-BLAST software using
mRNA template for each gene in NCBI database.
Table S2.
Sequencing detail for each library exported from proton server. All the
data is calculated by the proton server. Bases mean total basepair for each library.
Map reads mean the total map reads.
Table S3.
Comparison of fold-changes in gene expression determined by RNA-seq
versus qRT-PCR. RNA-seq experiment fold-change were derived from EdgeR result.
qRT-PCR fold-change were calculated by 2-△△Ct
Table S1. Primer designed for qRT-PCR by NCBI/ Primer-BLAST software using
mRNA template for each gene in NCBI database.
Gene
Forward primer
Reverse primer
Cyp1a1
TCCTTCCTCACAGCCAAAGCAG
CCCAGAATCCAAGGCAGAATGT
Cyp1a2
TCGGTGGCTAATGTCATCGG
GCTCTTCACGAGGTTGAGCA
Cyp1b1
CGCAACTTCAGCAACTTCGT
CTTTTCGGCGGAGAGGATGA
Nqo1
ATTGTATTGGCCCACGCAGA
GATTCGACCACCTCCCATCC
Gstp1
GGGTCGCTCTTTAGGGCTTT
TTGCATCGAAGGTCCTCCAC
Ugt1a2
AAGGGCTTCGAACCACAACA
AGCTGCACAAGAATTTGCGT
Ugt2b7
CTCTGGGGTCGATGATCAGG
GGTGTCTGGTTTCTTGCCCT
Gsta2
TTGACATGTACACCGAAGGCA
ACCGGTTTTTGGTCCTGTCT
ACTB
CCCGCGAGTACAACCTTCTT
CGCAGCGATATCGTCATCCA
Table S2. Sequencing detail for each library exported from proton server. All the data
is calculated by the proton server. Bases mean total basepair for each library. Map
reads mean the total map reads.
Sample
Bases
Reads
Read Length
Mapped Reads
map ratio
2-1
1491305221
16440899
90 bp
10432766
63.46%
7-1
1398347533
15079074
92 bp
10364118
68.73%
d-1
1330985156
14439768
92 bp
9329427
64.61%
2-2
1324658424
17653093
75 bp
14326049
81.15%
7-2
1360379686
15399370
88 bp
12385183
80.43%
D-2
1454590485
16785143
86 bp
13754581
81.94%
2_3
1612824309
15641776
103 bp
10758046
68.78%
7_3
1816202281
18614618
97 bp
12609191
67.74%
D-3
1509333107
17595163
85 bp
11999985
68.20%
Table S3. Comparison of fold-changes in gene expression determined by RNA-seq
versus qRT-PCR. RNA-seq experiment fold-change were derived from EdgeR result.
qRT-PCR fold-change were calculated by 2-△△Ct
Gene
2,2´,3,3´,4,5,6-hepta-CDPS
RNA-seq
qRT-PCR
fold-change
fold-change
2,4,4´,5-tetra-CDPS
RNA-seq
qRT-PCR
fold-change
fold-change
Cyp1a1
183.3253
234.9198
24.34678
21.30416
Cyp1a2
34.03378
58.55449
7.392445
11.52668
Cyp1b1
6.610294
1.939669
2.270397
1.521777
Gsta2
2.296018
1.585404
1.732649
1.838271
Gstp1
1.357619
1.698134
1.358865
1.866885
Nqo1
2.216092
2.191308
1.543793
1.612305
Ugt1a2
1.402619
1.263627
1.085225
1.50004
Ugt2b7
1.747133
1.360305
1.497632
1.611084
Figure legend
Figure S1
Relationship between H4IIE-luc assays derived RePavg values of PCDPSs and the
number of substituted Cl atoms. The value of 10-7 was assigned when the RePavg value
could not be calculated according to our recent study.
Figure S2
Gene expression in metabolism of xenobiotics by cytochrome P450 pathway. Color in
each cell represent the expression value (log2 transformed normalized total exon
reads) for each sample minus the means expression value in DMSO group. Red means
up-regulated and green means down-regulated. 3 color bar on the left of each gene
cell stand for 2,2´,3,3´,4,5,6-hepta-CDPS and 3 on the right for 2,4,4´,5-tetra-CDPS.
Figure S3
Gene expression in retinol metabolism pathway. Color in each cell represent the
expression value (log2 transformed normalized total exon reads) for each sample
minus the means expression value in DMSO group. 3 color bar of each gene cell stand
for 2,2´,3,3´,4,5,6-hepta-CDPS treatment. Green means down-regulated and red
means up-regulated.
Figure S4.
Gene expression in steroid hormone biosynthesis pathway. Color in each cell
represent the expression value (log2 transformed normalized total exon reads) for
each sample minus the means expression value in DMSO group. 3 color bar of each
gene cell stand for 2,2´,3,3´,4,5,6-hepta-CDPS treatment. Green means
down-regulated and red means up-regulated.
Figure S1
Figure S2
Figure S3
Figure S4.