Aquatic Toxicology 169 (2015) 196–203
Contents lists available at ScienceDirect
Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox
Differential modulation of expression of nuclear receptor mediated
genes by tris(2-butoxyethyl) phosphate (TBOEP) on early life stages of
zebrafish (Danio rerio)
Zhiyuan Ma a,1 , Yijun Yu a,1 , Song Tang b , Hongling Liu a,∗ , Guanyong Su a , Yuwei Xie a ,
John P. Giesy a,c,d,e , Markus Hecker b,c , Hongxia Yu a
a
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023, China
School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
c
Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
d
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK S7N 5B3, Canada
e
Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Region
b
a r t i c l e
i n f o
Article history:
Received 24 August 2015
Received in revised form 23 October 2015
Accepted 25 October 2015
Available online 30 October 2015
Keywords:
Organophosphate
Flame retardant
Nuclear receptor
Estrogen receptor
Mineralocorticoid receptor
Receptor-mediated network
Endocrine disruption
Toxicity
a b s t r a c t
As one substitute for phased-out brominated flame retardants (BFRs), tris(2-butoxyethyl) phosphate
(TBOEP) is frequently detected in aquatic organisms. However, knowledge about endocrine disrupting
mechanisms associated with nuclear receptors caused by TBOEP remained restricted to results from
in vitro studies with mammalian cells. In the study, results of which are presented here, embryos/larvae of
zebrafish (Danio rerio) were exposed to 0.02, 0.1 or 0.5 ␮M TBOEP to investigate expression of genes under
control of several nuclear hormone receptors (estrogen receptors (ERs), androgen receptor (AR), thyroid hormone receptor alpha (TR␣), mineralocorticoid receptor (MR), glucocorticoid receptor (GR), aryl
hydrocarbon (AhR), peroxisome proliferator-activated receptor alpha (PPAR␣), and pregnane × receptor
(P × R)) pathways at 120 hpf. Exposure to 0.5 ␮M TBOEP significantly (p < 0.05, one-way analysis of variance) up-regulated expression of estrogen receptors (ERs, er1, er2a, and er2b) genes and ER-associated
genes (vtg4, vtg5, pgr, ncor, and ncoa3), indicating TBOEP modulates the ER pathway. In contrast, expression of most genes (mr, 11ˇhsd, ube2i,and adrb2b) associated with the mineralocorticoid receptor (MR)
pathway were significantly down-regulated. Furthermore, in vitro mammalian cell-based (MDA-kb2 and
H4IIE-luc) receptor transactivation assays, were also conducted to investigate possible agonistic or antagonistic effects on AR- and AhR-mediated pathways. In mammalian cells, none of these pathways were
affected by TBOEP at the concentrations studied. Receptor-mediated responses (in vivo) and mammalian
cell lines receptor binding assay (in vitro) combined with published information suggest that TBOEP can
modulate receptor-mediated, endocrine process (in vivo/in vitro), particularly ER and MR.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Commercial flame retardants (FRs) are used in textiles, floor polish, varnish, foams, plastics, furniture and electronic equipment
(Alaee et al., 2003). Due to their properties of persistence, bioaccumulation as well as potential to cause adverse effects pentaand octa-brominated diphenylethers (BDEs) have been phased out
globally (Stapleton et al., 2012). The organophosphate ester (OPE),
tris(2-butoxyethyl) phosphate (TBOEP), is being used as an alter-
∗ Corresponding author.
E-mail addresses: zhiyuan nju@163.com (Z. Ma), yjun.yu@gmail.com (Y. Yu),
hlliu@nju.edu.cn (H. Liu).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.aquatox.2015.10.017
0166-445X/© 2015 Elsevier B.V. All rights reserved.
native flame retardant in applications and products (McGee et al.,
2012). TBOEP is used not only as a flame retardant or plasticizer
but also as a leveling agent (Marklund et al., 2003). TBOEP is associated with a number of industries and products (Cao et al., 2012). The
reported global production of TBOEP ranges from 5000 to 6000 tons
per year (World Health Organization, 2000), which has resulted in
detectable concentrations in various matrices of the environment.
TBOEP was the most abundant FR in indoor air, dust, wastewater, effluent water, surface water, ground water and drinking water
(Andresen, 2006; Cequier et al., 2014; Fries and Puttmann, 2003;
Marklund et al., 2003, 2005; McGee et al., 2012; Reemtsma et al.,
2008; Rodriguez et al., 2006; Stapleton et al., 2009; Sundkvist et al.,
2010). TBOEP, which has a relatively large log Kow (3.75) (Reemtsma
et al., 2008), has potential to accumulate in sediment or bioaccumulate in aquatic organisms and wildlife. In China, concentrations
Z. Ma et al. / Aquatic Toxicology 169 (2015) 196–203
of TBOEP in sediment ranged from 1.0 to 5.0 mg/kg dm (dry mass)
and was the most abundant chemical in Tai Lake (Ch: Taihu) (Cao
et al., 2012).
Nuclear receptors (NRs) are a superfamily of ligand-activated
transcription factors that regulate a broad range of biological
processes including embryonic development, homeostasis, and
metabolic diseases (Bertrand et al., 2007; Castrillo and Tontonoz,
2004; Chinenov et al., 2013). NRs mediate transcriptional responses
and signaling such as sex steroids, adrenal steroids, thyroid, vitamin D3 and retinoid hormones in target cells (Knoedler and Denver,
2014; Omiecinski et al., 2011), and trigger a complex array of cellular responses (Kojima et al., 2013). The superfamily of NRs includes
6 subfamilies (ERs, AR, TR␣, MR, GR, PPAR␣) (Zhao et al., 2015)
and an orphan receptor subfamily that comprise the metazoan
transcription factors (Kojima et al., 2013). Recently, several OPEs
have been found to induce developmental toxicity and endocrine
disruption both in vivo and in vitro (Fu et al., 2013; Han et al.,
2014; Kojima et al., 2013; Liu et al., 2013, 2012b; McGee et al.,
2012; Porter et al., 2014; Sassaki et al., 2011; Wang et al., 2013a).
We had previously demonstrated that TBOEP could interfere
with endocrine axes, including hypothalamus–pituitary–thyroidal
(HPT), hypothalamus–pituitary–adrenal (HPA) and hypothalamuspituitary-gonadal (HPG) axes in early-stage zebrafish. TBOEP
caused irreversible changes in expression of genes along these
axes (Exposure period: 3–120 hpf) (Accepted, DOI information: 10.
1016/j.chemosphere.2015.10.049), which supports the hypothesis
that through effects on NRs TBOEP could modulate concentrations
of hormones. TBOEP has been reported to interfere with certain
endocrine functions by altering production or functions of hormones. In particular it has been shown to interact with the ER
as shown by results of studies H295R and MVLN cells (Liu et al.,
2012b). TBOEP has been shown to be an agonist of the human pregnane × receptor (P × R) transactivation reporter gene assays with
COS-7 cells (Kojima et al., 2013). Most previous studies have been
conducted in vitro with mammal cells. There was thus a lack of
sufficient information on in vivo toxic potency and critical mechanisms of toxic action of TBOEP, especially in aquatic vertebrates.
TBOEP has been reported to cause acute and chronic toxicity to the
water flea (Daphnia magna) (Giraudo et al., 2015) and adversely
affected viability of embryos, morphometry and concentrations of
thyroid hormone in embryos of the chicken (Gallus domesticus)
(Egloff et al., 2014). However, little in vivo information was available for aquatic vertebrates, especially for fish. In general, bony
fishes were highly sensitive to OPEs exposure especially during
early developmental stages (McGee et al., 2013). The notable signs
of OPEs toxicity in early life stages such as mortality, yolk sac
edema, pericardial edema, craniofacial malformation, hemorrhage
and retarded growth (McGee et al., 2012). Therefore, thresholds
and mechanisms of toxic action of TBOEP on expression of critical
genes along key biological pathways associated with eight typical NRs including the aryl hydrocarbon receptor (AhR), peroxisome
proliferator-activated receptor alpha (PPAR␣), estrogen receptor
(ER), thyroid hormone receptor alpha (TR␣), pregnane x receptor
(PxR), androgen receptor (AR), glucocorticoid receptor (GR), and
mineralocorticoid receptor (MR) were studied in zebrafish (Danio
rerio). In addition to the in vivo studies with embryos of zebrafish,
in vitro studies with mammalian cell cells were used to supplement
information on AhR and AR.
2. Materials and methods
2.1. Chemicals and reagents
TBOEP (95.8%, Augsburg, Germany) was purchased from
Dr. Ehrenstorfer GmbH (Germ: Gesellschaft Mit Beschraenkter
197
Haftung), tetrachlorodibenzo-p-dioxin (TCDD) and dihydrotestosterone (DHT) were purchased from Sigma–Aldrich (St. Louis, MO,
USA). The stock solution of TBOEP was prepared in dimethyl sulfoxide (DMSO, Nanjing Chemical Reagent Co., Ltd., Nanjing, China),
diluted with embryo rearing water (60 mg/L instant ocean salt
within aerated distilled water), and stored at −20 ◦ C before being
diluted to final concentrations immediately before use. The final
concentration of DMSO in test solutions did not exceed 0.1%.
RNAlater, RNA Stabilization Reagents, RNeasy Mini Kit, and Omniscript RT Kit were purchased from Qiagen (Hilden, Germany) and
SYBR Green Real time PCR Master Mix Plus Kit was obtained from
Toyobo (Tokyo, Japan).
2.2. Animal’s culture and chemical exposure protocol
Adult AB wild-type zebrafish (4-months old) were obtained
from the Institute of Hydrobiology, Chinese Academy of Sciences
(Wuhan, China) and cultured in a semiautomatic system (Zhongkehai Recycling Water Aquaculture System Co., Ltd., Qingdao, China)
with treated tap water (no residual ammonia, chlorine, chloramines, and disinfected with UV light) under a 14/10 h light/dark
photoperiod. All of the animal procedures were approved by the
Institutional Animal Care and Use Committee (IACUC) of Nanjing
University for laboratory animal use. Culture and breeding of fish
was performed according to OECD Guidelines for the Testing of
Chemicals (OECD, 1992). Briefly, fish were fed fairy shrimp three
times a day. Nylon nets were placed at the bottom of tanks in
order to isolate eggs from adult zebrafish. All fertilized embryos
were examined under a stereo microscope (Nikon, Tokyo, Japan),
embryos which showed signs of abnormal development were
excluded. Normal embryos were kept for 3 h post fertilization (hpf)
(during blastula period) and used for subsequent experiments.
Based on environmentally relevant concentrations (surface
water 127 ng/L, waste water up to 35 ␮g/L and sediment ranged
from 1.0 to 5.0 mg/kg dm (dry mass), respectively) (Cao et al., 2012;
Fries and Puttmann, 2003; Marklund et al., 2003) and log Kow (3.75)
of TBOEP, a gradient of nominal concentrations of TBOEP were
chosen (0.02, 0.1, and 0.5 ␮M, which are equivalent to 8, 40 and
200 ␮g/L, respectively). The water used in test had been aerated
for two or three days, followed by subsequent experiments. During
exposure experiment, to avoid disturbance on embryos/larvae, did
not aerated any more. The testing solutions were used immediately
after being made and did not change during the exposure period.
An illuminated incubator was used to maintain a stable environment over the course of the experiment (photoperiod: 14/10 h
light/dark; static; temperature: 25 ± 2 ◦ C). Twenty embryos were
distributed randomly to 25 mL glass beakers each containing a different concentration of TBOEP in 20 mL of culture medium. Each
vehicle control group and test group was triplicated. Duration of
exposure was from 3.5 to 120 h post-fertilization (hpf). To minimize
evaporation of test solution, beakers were covered with breathable
plastic wrap. Concentrations of TBOEP in exposure media were confirmed using LC–MS/MS according to previously described methods
(Han et al., 2014; Li et al., 2014). During the 120 h exposure, nominal concentrations were achieved and maintained and no extensive
depletion of TBOEP (<20%) was observed.
2.3. RNA isolation and qRT-PCR
Differential expressions of 66 genes, involved in eight receptormediated pathways, were examined in zebrafish exposed to 0.02,
0.1 or 0.5 ␮M TBOEP. Larvae were randomly sampled at termination of the experiment and stored in RNAlater solution (QIAGEN
Co., Ltd., Germany) at −20 ◦ C until isolation of RNA. Total RNA
was isolated by use of the RNeasy Mini Kit (QIAGEN Co., Ltd.,
Germany). Isolation of RNA with subsequent transcription of genes
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Z. Ma et al. / Aquatic Toxicology 169 (2015) 196–203
Fig. 1. Heatmap of expressions of genes, based on average expressions of genes three replicates per tests. Significant fold changes in expressions of genes are given in each
cell and gene involved in different receptor pathways are show as different colors. Significant difference (p < 0.05; ANOVA) from control group.
were quantified by qRT-PCR according to a previously reported protocol (Liu et al., 2012a). Briefly, the Omniscript RT Kit (QIAGEN Co.,
Ltd., Germany) was used to synthesize cDNA following the manufacturers’ instructions. qRT-PCR was performed using the Applied
Biosystems Stepone Plus Real-time PCR System (Applied Biosystems Inc., Foster City, CA, USA). The online Primer 3 program (http://
frodo.wi.mit.edu) was used to design primers for selected genes.
Primer sequences for qRT-PCR are listed in Supporting information
(Table S1). Conditions for qRT-PCR reactions were as follows: initial
denaturation step at 95 ◦ C for 2 min, followed by 40 cycles at 95 ◦ C
for 15 s and 60 ◦ C for 1 min. Melting curves were used to confirm
that a single product was amplified. Expression of mRNA for each
target was standardized to the housekeeping gene 18s RNA, and
changes of in expression of mRNA of related genes were analyzed
by the 2−Ct method.
2.6. Reporter gene assay for AR
MDA-kb2 cells were seeded at a density of 1 × 105 cells/mL, in
384-well culture plates (Corning Inc., NY, USA), with 75 ␮L medium
per well. After 12 h incubation, 4 ␮L of a given TBOEP (final concentration of 0.1% DMSO per well), DHT (positive controls/standards),
and L-15 (blank) were added to each well. After 72 h exposure, culture meidum was removed and 10 ␮L 1 × Cell Culture Lysis Reagent
(CCLR, Promega Corp., Madison, WI, USA) was added to each well
to lyse cells. 25 ␮L Luciferase Assay Reagent (Luciferase Assay System, Promega Corp.) was then added and luciference activities were
quantified by measuring luminescence in a microplate reader (Synergy H4 hybrid, BioTek Co., Ltd., Germany). The maximum response
of DHT was set to 100% and the relative light unit (RLU) for each
sample well was calculated as a percentage of the maximum induction of luciferase activity (% DHT).
2.4. NR pathway analysis
2.7. Reporter gene assay for AhR
For genes relating to the AhR and ER pathways, the Agilent
Literature Search application was used to construct a biological interaction network within the Cytoscape software v3.1.1
(Cytoscape Consortium, San Diego, CA, USA) (Cline et al., 2007;
Liu et al., 2015; Saito et al., 2012; Shannon et al., 2003). Within
the application, literature available on protein–protein/proteinDNA interactions was searched for zebrafish. Gene networks
of the other six NR pathways were retrieved by use of either
WikiPathways (http://www.wikipathways.org) (Pico et al., 2008)
or SABioscience Gene Network Central (http://www.sabiosciences.
com/genenetwork/genenetworkcentral.php), and integrated with
AhR and ER pathways as “associations” and visualized as one network by Cytoscape. Only genes of interest were shown in this
pathway network. The resulting network genes (nodes) were colored according to the significant fold changes of gene expressions
in respective treatments.
H4IIE-luc cells were seeded at a density of 4 × 104 cells/mL, in
384-well culture plates (Corning Inc., NY, USA), with 75 ␮L medium
per well. After 12 h incubation, 4 ␮L of a given TBOEP (final concentration of 0.1% DMSO per well), TCDD (positive controls/standards),
and DMEM (blank) were added to each well. After 72 h exposure, culture medium was removed and 10 ␮L 1 × Cell Culture Lysis
Reagent (CCLR, Promega Corp., Madison, WI, USA) were added to
each well to lyse cells. 25 ␮L Luciferase Assay Reagent (Luciferase
Assay System, Promega Corp.) was then added and luciferin activities were measured in a microplate reader (Synergy H4 hybrid,
BioTek Co., Ltd., Germany). The maximum response of TCDD was set
to 100% and the relative light unit (RLU) for each sample well was
calculated as a percentage of the maximum induction of luciferase
activity (% TCDD).
2.5. MDA-kb2 and H4IIE-luc cell culture
MDA-kb2 and H4IIE-luc cells, which are used to detect the chemicals agonistic or antagonistic potencies against AR and AhR in
reporter gene assay were cultured in our laboratory as described
previously (Liu et al., 2011; Su et al., 2012; Wang et al., 2013b).
In brief, the MDA-kb2 cells or H4IIE-luc cells were cultured in
medium (MDA-kb2 cells were in Leibowitz-15 medium (L-15,
Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% fetal
bovine serum (FBS, Gibco, Invitrogen Corporation, Carlsbad, CA,
USA) and H4IIE-luc cells were in Dulbecco’s modified Eagle’s
medium (DMEM, Sigma–Aldrich, St. Louis, MO, USA), respectively)
in the humidified incubator at 37 ◦ C (MDA-kb2 cells were without
additional CO2 and H4IIE-luc cells were with 5% CO2 , respectively).
The cells were then maintained in 0.25% Trypsin-EDTA (Gibco,
Invitrogen Corporation, Carlsbad, CA, USA) and medium at least
24 h and prepared for seeding.
2.8. Statistical analyses
IBM SPSS statistics 19 (SPSS Inc., Chicago, IL, USA) was used
for statistical analyses. The Kolmogorov–Smirnov test was used
to evaluate data for normality and Levene’s test was used to analyze homogeneity of variance. Statistically significant differences
among groups were determined by one-way analysis of variance
(ANOVA) followed by Tukey’s multiple range test. A value of P < 0.05
was considered statistically significant.
3. Results
3.1. Expression of genes in NR pathways
Exposure of zebrafish larvae to 0.02, 0.1 or 0.5 ␮M TBOEP
resulted in changes in expression of genes associated with each
of eight NR-mediated pathways. Percentages of genes expressing
significant fold changes relative to controls were 22.73%, 13.64%
and 34.85% of the 66 genes, respectively. A total of 31.8% genes
were altered more than 1.5-fold when exposed to 0.5 ␮M TBOEP
Z. Ma et al. / Aquatic Toxicology 169 (2015) 196–203
(Fig. 1 and Table S2). Expressions of genes associated with pathways mediated by the AhR were rarely affected in larvae exposed
to any of the three concentrations of TBOEP. Some downstream
genes among the AhR or GR pathways, such as arnt1la, arnt1lb and
rela, were significantly up-regulated by 1.72-, 1.54- and 2.07-fold,
respectively when exposed to 0.5 ␮M TBOEP. For the ThR pathway, expression of ppargc1a and thra were up-regulated by 1.89and 1.71-fold when exposed to 0.02 ␮M TBOEP, respectively. ncor
was significant up-regulated by 1.48-fold after exposure to 0.5 ␮M
TBOEP. TBOEP also significantly up-regulated a number of genes in
the PPAR␣, ER and AR pathways. For example, after exposure to
the least concentration of TBOEP (0.02 ␮M), dut, ppargc1a, ccnd1,
ctnnb1, ncoa4, pa2g4a and pa2g4b were significantly up-regulated
by 2.50-, 1.89-, 2.30-, 1.60-, 2.25-, 1.64- and 1.82-fold, respectively.
When exposed to 0.1 ␮M, dut, ccnd1and ctnnb1were up-regulated
by 1.81-, 1.65- and 1.49-fold and ncoa1was down-regulated by
2.27-fold, respectively. Exposure to 0.5 ␮M, TBOEP caused significant fold changes of related genes, including il8, vtg4, vtg5, pgr,
ncoa3, er2a, er2b, er1, ncoa4 up-regulated by 1.90-, 1.73-, 2.03-,
2.48-, 1.43-, 2.18-, 1.95-, 2.33-, 1.84-fold, and il6 down-regulated
by 2.22-fold, respectively. For PxR and MR, TBOEP caused significant down-regulation of genes when exposed to 0.02 ␮M TBOEP,
except ugt1a1which was up-regulated by 2.56-fold. The genes
ncoa1, ncoa2and ube2i were down-regulated by 2.70-, 1.54- and
1.49-fold, respectively. Exposure to 0.1 ␮M TBOEP, resulted in significant down-regulations of cyp3a65, ncoa1, 11ˇhsd and adrb2b,
by 2.22-, 2.27-, 2.33- and 4.17-fold, respectively. When exposure
to 0.5 ␮M TBOEP, only poulf1and hpse were up-regulated by 1.99and 1.53-fold, respectively, while cyp3a65, cyp24a1, 11ˇhsd, ube2i,
adrb2b and mr were all down-regulated by 2.56-, 2.17-, 3.23-, 1.72-,
3.03- and 2.38-fold, respectively.
3.2. Potency of TBOEP on expression of AR- and AhR-mediated
responses of MDA-kb2 and H4IIE-luc cells
Agonistic or antagonistic effects of TBOEP were determined by
use of responses of luciferase under control of the AR or AhR in
transactivation assays with MDA-kb2 and H4IIE-luc cells, respectively. Dose–response curves of TBOEP and the positive control
dihydrotestosterone (DHT) from the MDA-kb2 assay were developed (Fig. 2A). Exposure to TBOEP, were neither agonists nor
antagonists of the AR receptor. In H4IIE-luc cells (Fig. 2B), no
luciferase activity was detected after exposure to TBOEP compared
the positive control tetrachlorodibenzo-p-dioxin (TCDD) (Initial
data are shown in Supporting information Tables S3–S6).
4. Discussion
Exposure to 0.02, 0.1 or 0.5 ␮M TBOEP primarily altered expression of genes along two NR-mediated pathways, the ER and MR
which had good concentration-dependent effects in profiles of
transcription throughout NRs pathway network. In a previous study
zebrafish embryos/larvae have been employed to assess effects of
two OPEs, tri (2,3-dichloropropyl) phosphate (TDCPP) and triphenyl phosphate (TPP) on expression of genes in six NR-mediated
gene networks (AhR, PPAR␣, ER, TR␣, GR, and MR) (Liu et al., 2013).
Bioaccumulation/bioconcentration factors (BCF) for organophosphate flame retardants (OPFRs) are ranked as follows: TBOEP
(1080) > TPP (113) > TDCPP (13.5) (van der Veen and de Boer, 2012).
Concentrations of TBOEP in the environment were several-fold
greater than concentrations of TDCPP in Lake Trout and/or Walleye
collected from Canadian freshwaters (0.26 vs 0.11 ng/g wm), and in
egg yolk (1.89 vs 0.93 ng/g wm), egg albumen (8.09 vs 0.47 ng/g wm)
and fat tissue (13.4 vs 4.43 ng/g wm) of herring gulls from the North
American Great Lakes (Greaves and Letcher, 2014; McGoldrick
199
Fig. 2. Dose–response curves for positive controls and TBOEP to determine agonistic potency of TBOEP to AR (A) or AhR (B) by use of transactivation assays. Values
represent means ± SEM of two independent experiments and are presented as the
percentage of response. Maximum response of positive control was set to 100%
and the relative light unit (RLU) for each sample well was calculated as a percentage of the maximum induction of luciferase activity (% DHT) (A) and (% TCDD) (B),
respectively.
et al., 2014). Although TDCPP and TPP are structurally similar to
TBOEP, the unique structural properties of TBOEP appear to account
for the specific NR-transcription pattern and toxicity in zebrafish
larvae. The pathway network (Fig. 3) gives insight into molecular
functions of each receptor and its combinatorial regulatory network for NRs (Zhao et al., 2015). ER and MR were the primary
pathways altered by TBOEP.
Expression of genes under control of the ER receptors (er1, er2a
and er2b) were all significantly up-regulated in larvae exposed
to 0.5 ␮M TBOEP, which indicates that TBOEP is an ER agonist,
which could disrupt steroidogenesis in fish (Fig. 4 A). Activation
of the ER pathway by TBOEP is consistent with results of a previous report in which exposure of H295R cells to 1 or 10 mg/L
TBOEP for 48 h, significantly increased synthesis of the steroid hormones, 17␤-estradiol (E2) and testosterone (T) (Liu et al., 2012b).
Most of the downstream genes that are associated with the ER
pathway such as vtg4, vtg5, pgr and ncoa3were significantly upregulated relative to controls, when exposed to 0.5 ␮M TBOEP.
Expression of mRNA for these four genes were correlated with
concentrations of their respective proteins for which they code as
determined by proteomics technology for zebrafish larvae exposed
to 800 ␮g TBOEP/L (Han et al., 2014). Vitellogenins are the major
precursor of egg-yolk proteins and can bind to lipids, phosphates,
sugars, and metal ions to provide essential nutrients and energy
for embryonic development in oviparous organisms (Byrne et al.,
1989; Gundel et al., 2007). Induction of expression of the vtgs gene
is used as a molecular biomarker of exposure of oviparous vertebrates and invertebrates to estrogenic chemicals (Matozzo et al.,
2008; Sumpter and Jobling, 1995). Exposure to 0.02 or 0.1 ␮M
TBOEP resulted in significant up-regulation of ccnd1. It was previously reported that expression of ccnd1 (cyclin D1) along with
200
Z. Ma et al. / Aquatic Toxicology 169 (2015) 196–203
Fig. 3. Interaction network of selected genes in nuclear receptor pathways of zebrafish. Each node represents a single gene, edges either protein–protein or protein–DNA
interactions. Statistically significant (p < 0.05, ANOVA) differences in expression og genes relative to control group following different concentrations of treatment of TBOEP
(0.02 ␮M, 0.1 ␮M or 0.5 ␮M) at 120 hpf are given in respective boxes (see legend).
greater accumulation of TBOEP in egg yolk and albumen as well as
liver and brain of herring gull, suggest potential effects of TBOEP on
reproduction of vertebrates (Han et al., 2014). ERs regulate multiple
genes involved in growth, reproduction, development, metabolism,
and homeostasis of vertebrates (Shibata et al., 1997). Thus, any
effects of TBOEP mediated through the ER could result in changes
in more apical measurement endpoints for reproduction, such as
fecundity.
The mr gene and associated downstream genes, such as 11ˇhsd,
ube2i, adrb2b, were significantly down-regulated after exposure to
TBOEP (Fig. 4B). Reports of effects of TBOEP on the mineralocorticoid receptor (MR) are limited. But the MR is involved in transport
of sodium ions and water (Takahashi and Sakamoto, 2013). Previous studies have shown that MR-deficient (−/−) mice developed
symptoms, including dehydration, hyperkalemia, hyponatremia
and increase in renin and aldosterone plasma concentrations that
eventually resulted in death after 10 days (Berger et al., 1998).
MR is a member of the family of corticosteroid receptors (CR),
which function as ligand-inducible transcription factors. Mineralocorticoids (MC) are synthesized in interregnal tissue of the
head kidney in teleost fish, and the active MC, is the adrenocortical homologue, cortisol (Bury and Sturm, 2007; Nelson, 2003).
MCs are involved in maintenance of mineral homeostasis at systemic and cellular levels (Charmandari et al., 2005). Circulating
hormones, including cortisol and corticosteroid, have been found
to act as MC in fish (Bury et al., 2003; Colombe et al., 2000;
Greenwood et al., 2003; Sturm et al., 2005). In fish, MR is activated
by 11-deoxycorticosterone (DOC), a precursor of corticosterone
and 11-deoxycortisol as its natural endogenous ligand (Pascual-
Le Tallec and Lombes, 2005; Sturm et al., 2005). However, cortisol
binds with greater affinity, so under normal physiological conditions, the MR is occupied by cortisol instead of DOC (Prunet
et al., 2006). The enzyme 11␤-hydroxysteroid dehydrogenase
(11␤-HSD) converts corticosterone and cortisol to their respective inactive metabolites 11-dehydrocorticosterone and cortisone
(Bury and Sturm, 2007; Farman, 1999) (Fig. S1). TBOEP has been
reported to restrain the entire pathway for synthesis of steroids in
zebrafish, which could significantly suppress transport, uptake, and
metabolism of lipids, sugars, and minerals, which, in turn, could
decrease heart rate and delay development of larvae (Han et al.,
2014; Kojima et al., 2013; Liu et al., 2012b).
In vitro, receptor transactivation assays have an advantage in
determining potencies of agonists and/or antagonists for multiple
NRs. Previous studies have characterized the potency of TBOEP for
the human ER␣/␤ and PxR. Although TBOEP displayed PxR agonist
potency with 20% relative effective concentration (REC20 ) of 3.1 ␮M
in CHO-K1 cells, there was no displacement from human ER␣ and
ER␤ (Kojima et al., 2013). These results are inconsistent with the
results of the transcription studies reported herein, which demonstrated activation of the ER pathway. The contradiction might be
due to interspecies variability, differences in responses in vitro and
in vivo, or different metabolites of TBOEP. In this study, to expand
the knowledge regarding agonistic activity against human NRs of
OPFRs, MDA-kb2 and H4IIE-luc based cell report assays were carried out to test the binding capacity of TBOEP against human AR and
AhR. No activation on these two receptors were observed (Fig. 2),
which are consisted with transcription data of core receptor genes
in zebrafish larvae.
Z. Ma et al. / Aquatic Toxicology 169 (2015) 196–203
201
Fig. 4. Fold changes of genes related to ER (A) or MR (B) after exposure to TBOEP (0.02 ␮M, 0.1 ␮M or 0.5 ␮M) for 120 hpf. Results are shown as means ± SEM of three repicates.
The greatest concentration of TBOEP affected expression of genes related to ER or MR.
*Significant difference (p < 0.05, ANOVA) from control group. **Extremely significant difference (p < 0.01, ANOVA) from control group.
5. Conclusions
This present study demonstrated that TBOEP affected ERand MR-mediated endocrine pathways at the early-life stage of
zebrafish larvae. The findings revealed the possible molecular
mechanism for adverse outcomes induced by TBOEP, which might
be helpful to in understanding effects of TBOEP on endocrine
function and receptor mediated endocrine pathway. In addition,
the least TBOEP concentrations (0.02 ␮M; 8 ␮g/L) studied were at
least three times greater than those observed previously in some
river ecosystems, such as in Iberian rivers where concentrations
ranged from 5.3 to 659 ng/L. However, it should be noted that a
multitude of other OPFRs contaminants are frequently detected
in the aquatic ecosystems (e.g., TDCPP, TPP, tris(2-carboxyethyl)
phosphine (TCEP)), which could have the possibility of producing
additive or synergistic effects with TBOEP. These types of cocktails might be possible, albeit they have not been characterized to
date. Hence, the relevance of our findings to teleost exposure scenarios within aquatic environment is currently uncertain, but this
study is of great importance and raises concerns about the potential environmental risks of increasingly used, frequently detected,
and highly bioaccumulated OPFRs to early development of wildlife
and human.
Acknowledgements
This work has been co-financially supported by National Natural Science Foundation (No. 21377053 and 20977047) and Major
National Science and Technology Projects (No. 2012ZX07506-001
and 2012ZX07501-003-02) of China. Professor John P. Giesy was
supported by the Program of 2012 “Great Level Foreign Experts”
(#GDW20123200120) funded by the State Administration of Foreign Experts Affairs, PR China to Nanjing University, and the
Einstein Professor Program of the Chinese Academy of Sciences.
He was also supported by the Canada Research Chair Program and
a Visiting Distinguished Professorship in the Department of Biology
and Chemistry and State Key Laboratory in Marine Pollution at City
University of Hong Kong. Professor Markus Hecker was supported
by the Canada Research Chair Program.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.aquatox.2015.10.
017.
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