Ecotoxicology and Environmental Safety 229 (2022) 113069

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Comparison of the mechanisms of estrogen disrupting effects between

triphenyl phosphate (TPhP) and tris(1,3-dichloro-2-propyl)
phosphate (TDCIPP)
Xiaoya Ji a, b, Na Li a, *, Mei Ma a, c, *, Xinyan Li d, Kongrui Zhu a, c, Kaifeng Rao a, Zijian Wang c,
Jingfeng Wang e, Yanjun Fang e
a
Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
b
School of Public Health, Qingdao University, Qingdao 266000, China
c
College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100049, China
d
Institute of Environmental and Ecological Engineering, Guangdong University of Technology, Guangzhou 510006, China
e
Tianjin Institute of Environmental and Operational Medicine, Key Laboratory of Risk Assessment and Control for Environment & Food Safety, Tianjin 300050, China

A R T I C L E I N F O A B S T R A C T

Edited by Dr. G. Liu As the typical aryl-organophosphate flame retardants (OPFRs), triphenyl phosphate (TPhP) and tris(1,3-dichloro-
2-propyl) phosphate (TDCIPP) were reported to be estrogen disruptors. However, estrogen receptor α (ERα)
Keywords: binding experiments could not explain their biological effects. In this study, their action on ERα, G protein-
Triphenyl phosphate (TPhP) coupled estrogen receptor (GPER) and the synthesis of 17β-estradiol (E2) were investigated using in vitro as­
Tris(1,3-dichloro-2-propyl) phosphate
says and molecular docking. The results showed that TPhP acted as an ERα agonist and recruited steroid receptor
(TDCIPP)
co-activator 1 (SRC1) and 3 (SRC3), which was found for the first time. Unlike TPhP, TDCIPP acted as an ERα
Estrogen disrupting effects
Estrogen receptor α (ERα) antagonist. However, both TPhP and TDCIPP activated the estrogen pathway by GPER in SKBR3 cells which were
G protein-coupled estrogen receptor (GPER) lack of ERα. Although molecular docking results revealed that both TPhP and TDCIPP could dock into ERα and
Estrogen biosynthesis GPER, their substituent groups and combination mode might affect the receptor activation. In addition, by using
estrogen biosynthesis assay in H295R cells, both of TPhP and TDCIPP were found to promote E2 synthesis and
E2/T ratio involving their different alteration on levels of progesterone, testosterone and estrone, and expression
of various key genes. Our data proposed estrogen-disrupting mechanism frameworks of TPhP and TDCIPP.
Moreover, our results will contribute to future construction of adverse outcome pathway (AOP) framework of
endocrine disruptors.

1. Introduction the environment, leading to their frequent detection in varied environ­

In response to the gradual restriction of polybrominated diphenyl
ethers (PBDEs), there is growing need in the production and consump­
tion of organophosphate flame retardants (OPFRs) as one of the poten­
tial alternatives (van der Veen and de Boer, 2012). Triphenyl phosphate
(TPhP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) as shown in
Fig. 1 are typical aryl-OPFR and chlorinated-OPFR, respectively, and
widely used in baby products, residential furniture, electronic equip­
ment and building materials (Cooper et al., 2016; Stapleton et al., 2011;
Wei et al., 2015). Because TPhP and TDCIPP as additive flame retardants
do not form chemical bonds with materials, they are easily released into
ment media (water, house dust, soil), aquatic organism and even in
human samples (Brandsma et al., 2014; Kim et al., 2014; Reemtsma
et al., 2008; Wan et al., 2016; Ya et al., 2019). In the 150 samples
consisting of river water, lake water, tap water, seawater and rainwater
collected from New York State, the average levels of TPhP and TDCIPP
ranged from 0.53 to 11.0 and 4.75–21.1 ng/L, respectively (Kim and
Kannan, 2018). The highest concentrations of TPhP and TDCIPP in 60
floor dust samples located in Beijing, China reached 3514 and 3741
ng/g, respectively (Wu et al., 2016). TPhP and TDCIPP have also been
found in human breast milk samples (Zheng et al., 2021), children’s
urine (Y. Li et al., 2019; N. Li et al., 2019), human placenta (Ding et al.,

* Correspondence to: Key Laboratory of Drinking Water Science and Technology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,
18 Shuangqing Road, Haidian District, Beijing 100085, China.
E-mail addresses: nali@rcees.ac.cn (N. Li), mamei@rcees.ac.cn (M. Ma).

https://doi.org/10.1016/j.ecoenv.2021.113069
Received 9 October 2021; Received in revised form 30 November 2021; Accepted 5 December 2021
Available online 8 December 2021
0147-6513/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
X. Ji et al. Ecotoxicology and Environmental Safety 229 (2022) 113069

effects of EDCs on GPER. The H295R cells are sensitive to the EDCs that
disrupt the steroidogenic processes including the E2 biosynthesis. In the
present study, the action on ERα of TPhP and TDCIPP was determined by
the MVLN cell assay, molecular docking and the ERα-SRCs recruitment
assay was conducted to characterize the SRCs recruitment of the ERα
agonist. Their interaction with GPER was confirmed by molecular
docking and their activation on GPER-mediated signaling was further
investigated in SKBR3 cells. Moreover, their effects on steroid hormone
levels (progesterone, estrone, testosterone and E2) and expression of
genes involved were measured in H295R cells.

2. Materials and methods

2.1. Chemicals and cell culture

Fig. 1. The structures of TPhP and TDCIPP.

Triphenyl phosphate (TPhP, 99%) and Tris(1,3-dichloro-2-propyl)

2016) and human blood (Zhao et al., 2016), which has aroused great
phosphate (TDCPP, 99%) were purchased from J&K Chemical (Bei­
concern about their potential adverse effects.
jing, China). The chemicals were dissolved in dimethyl sulfoxide
Several studies have demonstrated that exposure to TPhP and
(DMSO) and stored at − 20 ◦ C. To avoid cytotoxicity from DMSO, its
TDCIPP are associated with developmental toxicity (Fu et al., 2013; Y. Li
final concentration in the culture medium was below 0.1% (V/V).
et al., 2019; N. Li et al., 2019), endocrine-disrupting effects (Bajard
SKBR3 cells and H295R cells were purchased from the cell bank of
et al., 2021; Rosenmai et al., 2021; Zhang et al., 2016), immunotoxicity
the Chinese Academy of Sciences (Beijing, China). MVLN cells were
(Farhat et al., 2014), cardiotoxicity (Mitchell et al., 2018), obesity
MCF-7 cells that contained estrogen responsive element (ERE)-
(Hoffman et al., 2015) and neurotoxicity (Hong et al., 2018; J. Wang
controlled luciferase reporter gene, and were cultured in DMEM
et al., 2015; Q.W. Wang et al., 2015; Qiangwei Wang et al., 2015).
(HyClone, USA) with 10% fetal bovine serum and 1% penicillin/strep­
Currently, there is increasing evidence that TPhP and TDCIPP also have
tomycin (Gibco, USA) in an incubator at 37 ◦ C with 5% CO2. SKBR3 cell
potential to disrupt estrogen function which is thought to act vital role in
were cultured in RPMI 1640 medium (HyClone, USA) with 10% fetal
reproduction, growth and development (Kojima et al., 2013; Liu et al.,
bovine serum and 1% penicillin/streptomycin in an incubator at 37 ◦ C
2016; J. Wang et al., 2015; Q.W. Wang et al., 2015; Qiangwei Wang
with 5% CO2. H295R cells were cultured in DMEM/F12 medium
et al., 2015). In in vivo, exposure to TPhP and TDCIPP affect
(HyClone, USA) with 1% Ultroser G (Pall Corporation, USA), 1% ITS
hypothalamic-pituitary-gonadal (HPG) axis of zebrafish along with
(Gibco, USA) and 1% penicillin/streptomycin in an incubator at 37 ◦ C
alteration of plasma 17β-estradiol (E2) and testosterone (T), and sig­
with 5% CO2.
nificant increases of vitellogenin (VTG) (Liu et al., 2013), which suggests
that they can alter hormone metabolism and biosynthesis to induce es­
2.2. Cell viability
trogenic activities. However, the underlying mechanisms of TPhP and
TDCIPP to exert their estrogen disrupting effects were still unclear.
MVLN cells (1.5 × 104 cells/well), SKBR3 cells (3 × 103 cells) and
Although TPhP and TDCIPP have been reported to interact with estrogen
H295R cells (3 × 103 cells/well) were seeded in 96-well plates with
receptor α (ERα) to exert estrogen-disrupting activities. However, con­
100 μL medium for 24 h. The MVLN cells, SKBR3 cells and H295R cells
troversy remains regarding their effects on ERα in different in vitro
were treated with serial dilution of test compounds for 72 h, 24 h and
systems (Liu et al., 2012; Zhang et al., 2014). Therefore, the
48 h exposure, respectively. After treatment, cells were stained with
estrogen-disrupting activities of TPhP and TDCIPP might result from
Hoechst 33,342 for overnight. The images were acquired by an Oper­
various pathways, and understanding mechanisms involved is un­
etta™ High Content Screening instrument (PerkinElmer, Waltham, MA,
doubtedly critical to assessing their toxicity.
USA), and the cell number was analyzed by Harmony™ software. The
It is well known that the molecular mechanisms of estrogen-
relative cell viability was calculated as the ratio the cell number of the
disrupting effects induced by endocrine disrupting chemicals (EDCs)
DMSO control. The following experiments used non-cytotoxic concen­
are complicated. One of the classical genomic pathways is EDCs interact
trations of compounds.
with ERα to recruit co-activators to promote the transcription of target
genes (Zhu et al., 2019). Moreover, some EDCs such as bisphenol A
2.3. MVLN cell assay
(BPA) (Vinas and Watson, 2013), cadmium (Liu et al., 2019), hydrox­
ylated polybrominated diphenyl ethers (OH-PBDEs) (Cao et al., 2018b)
MVLN cells (1.5 × 104 cells/well) were seeded in 96-well plates and
and thiodiphenol (Lei et al., 2017) have potential to active G
cultured in phenol red-free DMEM with 10% charcoal-stripped FBS.
protein-coupled estrogen receptor (GPER)-mediated rapid non-genomic
After 24 h, the cells were treated with 20 μM, 10 μM, 2 μM, 1 μM,
pathways to exert effects including proliferation and migration of breast
0.2 μM and 0.1 μM of TPhP, and 10 μM, 2 μM, 1 μM, 0.2 μM and 0.1 μM
cancer cells. In addition, EDCs also can disrupt the E2 biosynthesis to
of TDCIPP, respectively. After 72 h exposure, the luciferase activity was
cause estrogen-disrupting effects, involving alteration in the levels of
detected by the Steady-Glo-Luciferase Assay System (Promega Corpo­
various steroid hormones and the expression of multiple key enzymes
ration, USA) and determined using a microplate reader (Tecan GENios
(Feng et al., 2016; He et al., 2008; J. Wang et al., 2015; Q.W. Wang et al.,
A-5002, Salzburg, Austria). The relative luciferase activity for each
2015; Qiangwei Wang et al., 2015). However, the toxicity mechanisms
sample well was normalized as the ratio the luciferase activity of the
involved in estrogen-disrupting activities of TPhP and TDCIPP had not
DMSO control. The anti-estrogenic activity of the test compounds was
been well understood and their action on various pathways remains to
determined by coincubation of MVLN cells with E2.
be elucidated.
Various in vitro cell models were developed to determine the modes
2.4. ERα-SRCs recruitment assay
of action of EDCs. The ERα positive human breast MVLN cells that
contained ERE-controlled reporter genes are used to investigate the ef­
The LanthaScreen ERα SRCs kit and 10 fluorescein-tagged SRCs
fects of EDCs on ERα-ERE pathway (Pons et al., 1990). The human breast
peptides including SRC1–1, SRC1–2, SRC1–3, SRC1–4, SRC2–1, SRC2–2,
SKBR3 cells (ERα-, GPER+) were routinely conducted to study the
SRC2–3, SRC3–1, SRC3–2 and SRC3–3, were purchased from Life

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X. Ji et al.
Technology (CA, USA). According to the manufacturer’s instructions,
0.25 μM SRC peptide, 5 nM Tb anti-GST antibody, 3.5 nM ERα-LBD, and
serial concentrations including 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM
and 1000 μM of test compounds were gently mixed in a 384-well plate
(Corning, NY, USA). After 1 h incubation at room temperature from
light, the fluorescence was read on Spark 10 M (TECAN) with TR-FRET
systems (EX = 340 nm, EM = 495 and 520 nm).
2.5. Docking analysis
The GPER homology protein model was kindly provided by Men­
dez-Luna et al. (2016), and has been widely performed in reported
studies (Deng et al., 2017; Sarmiento et al., 2018). The Discovery Studio
4.0 software package was used to dock test compounds to ERα and GPER
(Accelrys Software Inc., San Diego, CA). The protocol was performed
according to the CDOCKER protocol, in which protocol 20 random
ligand conformations were generated and refined by grid-based simu­
lated annealing in the binding site of GPER, and the top-ranking poses
from the docking were selected.
2.6. cAMP
The LANCE Ultra cAMP kit (PerkinElmer, Norwalk, CT, USA) was
used to determine the cAMP levels. SKBR3 cells (3 × 103 cells) were
seeded in a 96-well plate (PerkinElmer, USA) with 100 μL phenol red-
free RPMI 1640 medium for 24 h. The cells were rinsed with HBSS
and treated with 20 μL mixture containing serial concentrations
including 0.1 μM, 1 μM and 10 μM of test compounds, 0.5 mM 3-isobu­
tyl-1-methylxanthine and Alexa-labeled antibodies for 30 min at room
temperature. Then, 20 μL detection solutions were added and incubated
for 60 min at room temperature. Finally, the signals at the module of
time-resolved fluorescence resonance energy transfer were measured by
Spark 10 M (TECAN, Switzerland), and the cAMP concentration was
normalized according to the manufacturer’s instructions. For the G15 (a
GPR30 inhibitor) inhibitory experiments, the cells were pretreated with
1 μM G15 for 30 min
2.7. Hormone measurement
H295R cells (3 × 105 cells/well) were seeded in 24-well plates with
1 mL complete medium for 24 h. The cells were treated with serial
concentrations including 0.1 μM, 1 μM, 2 μM and 10 μM of test com­
pounds. After 48 h exposure, the medium was centrifuged, collected and
stored at − 80 ◦ C. According to the manufacturer’s instructions of ELISA
kits (Cayman Chemical, USA), the concentrations of progesterone,
testosterone and estradiol were determined. The concentrations of
estrone were determined by an Estrone ELISA Kit (Abnova, Taiwan)
according to the manufacturer’s protocol.
2.8. RNA isolation and qPCR
H295R cells (3 × 105 cells/well) were seeded in 24-well plates with
1 mL complete medium for 24 h. The cells were treated with serial
concentrations including 0.1 μM, 1 μM, 2 μM and 10 μM of test com­
pounds. After 48 h exposure, the total RNA was extracted from the cells
using TRIzol reagent (Life Technologies, USA) and reverse transcribed in
a 20 μL reaction system by the FastQuant RT kit (TIANGEN, Beijing,
China). qPCR was performed using an ABI 7500 real-time quantitative
PCR system (Life Technologies, USA) according to the guidelines. The
primer pairs are shown in the Supporting Information (Table S1), and
β-actin was used as the housekeeping gene for the mRNA expression
analysis. The expression of steroidogenesis gene was calculated with the
2− ΔΔCt method.
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Ecotoxicology and Environmental Safety 229 (2022) 113069
2.9. Data analysis
All data was analyzed by OriginPro 2016 (Origin Lab, Northampton,
USA). All results were expressed as the mean ± SD (standard deviation)
of at least three independent experiments. Levene’s test was used to
check homogeneity of variances. Statistical significance was analyzed by
one-way analysis of variance (ANOVA) followed by Tukey’s test and the
level of significant difference was p < 0.05.
3. Results and discussion
3.1. The effects of TPhP and TDCIPP on ERα
The ERα activation is a significant target of EDCs to induce estrogenic
effects, and the molecular docking analysis and MVLN cells that contain
estrogen respective element-controlled luciferase gene were used to
determine the action of TPhP and TDCIPP on ERα. The results showed
that E2 formed hydrogen bonds with His524, Arg394 and Glu353
(Fig. 2A). TPhP formed a pi-sigma bond with Arg394 (Fig. 2B), and
TDCIPP formed a hydrogen bond with Arg394 (Fig. 2C), suggesting that
they had different interaction with ERα. In MVLN cells, the positive
compound E2 had concentration-dependent agonistic activities with
EC50 (the concentration that had 50% of maximal activity) of 2.06 × 10-
11
mol/L (Fig. S1). According to the results of MVLN cells viability
(Fig. S2), the concentrations (≤ 2 ×10-5 M for TPhP and ≤ 1 ×10-5 M for
TDCIPP) that had no cytotoxicity were used. TPhP showed agonistic
response in a dose-dependent manner with EC50 value and maximal
relative luciferase of 1.02 × 10-5 mol/L and 28.31%, respectively
(Fig. 2D). TDCIPP did not induce agonistic activity (Fig. S3), but it
showed strong antagonistic activity inhibiting the luciferase activity
induced by 1 × 10-8 mol/L E2 with a maximal inhibition of 24.82% and
IC50 (the concentration that had 50% of inhibitory activity) value of
1.63 × 10-6 mol/L (Fig. 2E), suggesting that TPhP and TDCIPP had
opposite effects on ERα. Specifically, TPhP acted as an ERα agonist to
activate ERE transcription, while TDCIPP acted as an ERα antagonist to
compete with E2 for binding to ERα. Our results were also similar to
publicly available data from ToxCast that TPhP had agonistic activity on
ERα with EC50 of 7.31 × 10-6 mol/L, while TDCIPP had antagonistic
activity on ERα with IC50 of 7.02 × 10-5 mol/L.
Generally, in the ERα-mediated ERE pathway, the ERα agonist binds
to ERα to form an agonist-ERα complex, and then various co-activators
are recruited to enhance target gene transcription. Moreover, the p160
steroid receptor co-activators (SRCs) family containing SRC1, SRC2 and
SRC3 are the first discovered co-activators, and a recent study has
demonstrated that differential recruitment of BPA and its analogs to
SRCs were implicated with their effects on ERα-mediated ERE tran­
scription. In order to investigate the SRCs involved in estrogenicity of
TPhP, the ERα-SRCs recruitment assay containing 10 SRCs peptides was
used. E2-ERα recruited 9 SRCs peptides except for SRC3–3 (Fig. S4).
TPhP showed the concentration-dependent ERα agonistic interaction
with SRC1–1 and SRC3–3 with EC50 of 2.93 × 10-4 and 1.30 × 10-5 mol/
L, respectively (Fig. 2F), and had no effects on other 8 SRCs peptides
(Fig. S5), suggesting that TPhP-ERα recruited SRC1–1 and SRC3–3. As
far as we know, this is the first study to investigate the interaction be­
tween ERα and SRCs induced by TPhP, and it was content with our
previous studies that TPhP did not induce the ERα interaction with SRC2
in the two-hybrid yeast system (Ji et al., 2020b).
3.2. The activation of TPhP and TDCIPP on GPER
The action of GPER has been demonstrated to be involved in
mammalian uterine proliferation, the progression of multiple
reproduction-related cells and endocrine-related cells, and GPER is also
implicated in the E2-activation of cancer-associated fibroblasts (Pross­
nitz and Barton, 2011). There is increasing studies that some EDCs have
potential to activate GPER and trigger various downstream pathways,
X. Ji et al. Ecotoxicology and Environmental Safety 229 (2022) 113069

Fig. 2. The effects of TPhP and TDCIPP on ERα action. (A-C) Molecular docking analysis of E2, TPhP and TDCIPP with ERα. (D) The agonistic activity of TPhP in the
MVLN cell assay. Data are presented as relative luciferase activity that was normalized to that of the DMSO control. (E) The antagonistic activity of TDCIPP in the
MVLN cell assay. Data are presented as the percent of activity that was inhibited relative to the maximum activity induced by 17β-estradiol (E2, 1 × 10-8 mol/L). (F)
TPhP induced ERα interaction with SRC1–1 and SRC3–3 peptides. Data are presented as the ratio of fluorescent units (520 nm/495 nm) emitted after excitation at
340 nm. Values represent the mean ± SD of three independent experiments.

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X. Ji et al. Ecotoxicology and Environmental Safety 229 (2022) 113069

such as cyclic adenosine monophosphate (cAMP), phosphoinositide
3-kinase/protein kinase B (PI3K/AKT) and intracellular calcium mobi­
lization, to exert estrogen-disrupting effects, resulting in proliferation
and migration of breast cancer cells including MCF-7 and SKBR3 (Cao
et al., 2017; Lei et al., 2017). In order to investigate the activation of
TPhP and TDCIPP on GPER, the molecular docking analysis was per­
formed to determine their interaction with GPER and their effects on
cAMP levels were measured in SKBR3 cells. In molecular docking
analysis, the results showed that E2 formed hydrogen bonds with
Tyr123 and Glu275, and Van der Waals forces with Cys205. G1, a GPER
agonist, had a hydrogen bond and a pi-pi bond with Gln138 and Phe208,
respectively (Fig. S6). TPhP formed a pi-sigma bond with Tyr142
(Fig. 3A), and the CDOCKER interaction energy was − 29.60 kcal/mol.
TDCIPP formed a hydrogen bond with Gln138 that was similar to G1
(Fig. 3B), and the CDOCKER interaction energy was − 59.33 kcal/mol,
suggesting that they had different interaction with GPER. The results
from cAMP measurement showed E2 evoked a dose-dependent cAMP
levels ranging from 1 × 10-10 to 1 × 10-7 mol/L (Fig. S7). According to
the results of SKBR3 cells viability (Fig. S8), the concentrations
(≤1 ×10-5 M for TPhP and TDCIPP) that had no cytotoxicity were used.
TPhP and TDCIPP showed significant agonistic effects on cAMP pro­
duction (Fig. 3C), and for the cells pretreated with 1 μM G15, their ef­
fects were significantly inhibited (Fig. 3D), indicating that they
activated the GPER-mediated cAMP pathway. Here, it was found for the
first time that both TPhP and TDCIPP have potential to activate
GPER-cAMP, but their ways of interaction with GPER were different.
3.3. The effects of TPhP and TDCIPP on E2 synthesis and mechanisms
involved
In addition to estrogen receptors-mediated pathways, EDCs also have
been reported to disrupt non-receptor-mediated action concerning E2
synthesis to exert estrogen-disrupting activities including alteration of
VTG levels and promoting human breast cancer cell (MCF-7) prolifera­
tion (Liu et al., 2007; Wrobel and Gregoraszczuk, 2013). In response, the
USA Environmental Protection Agency (EPA) has recommended the
H295R cell line to evaluate the effects of EDCs on steroidogenesis, and
several EDCs, such as 2,4-dichlorophenol (Ma et al., 2012), naphthenic

Fig. 3. The effects of TPhP and TDCIPP on GPER action. (A)-(B) Molecular docking analysis of TPhP and TDCIPP with GPER. (C) cAMP production induced by
different concentrations of TPhP and TDCIPP in SKBR3 cells. (D) cAMP production induced by E2 (100 nM), 10 μM TPhP and TDCIPP in the absence or presence of
1 μM G15. The result of the cAMP production was expressed by setting the cAMP production of the DMSO control group as 100%. Values represent the mean ± SD of
three independent experiments. *p < 0.05, compared with the control. # p < 0.05, compared with the groups treated with the compounds in absence of G15.

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X. Ji et al. Ecotoxicology and Environmental Safety 229 (2022) 113069

acids (J. Wang et al., 2015) and phthalates (Sohn et al., 2016), have been
demonstrated to promote E2 synthesis using H295R cell model, which
were in accordance with in vivo studies showing the increase of E2 levels
in zebrafish. Therefore, the effects of TPhP and TDCIPP on four hormone
concentrations in H295R cells were measured. The positive compound
forskolin increased concentrations of progesterone, estrone, testosterone
(T) and E2 (Fig. S9). According to the results of H295R cells viability
(Fig. S10), the concentrations (≤1 × 10-5 M for TPhP and TDCIPP) that
had no cytotoxicity were used. The E2 levels and E2/T ratio significantly
increased when H295R cells were exposed to TPhP and TDCIPP
(Fig. 4A–C), indicating that they could cause estrogenic response.
However, there was some difference in their effects on the levels of other
hormones involved in the process of E2 synthesis. TPhP increased the
levels of progesterone, but decreased the levels of estrone and testos­
terone (Fig. 4A), similarly, TPhP has been reported to promote proges­
terone synthesis in human placental choriocarcinoma JEC-3 cells (Hu
et al., 2017) and inhibit testosterone levels in Japanese medaka (Li et al.,
2018). TDCIPP increased the levels of estrone and testosterone, and did Fig. 5. The effects of TPhP and TDCIPP on the expression of genes related to
not cause any effects on the levels of progesterone (Fig. 4B). steroidogenesis in H295R cells. Values represent the mean ± SD of three in­
In order to investigate the mechanisms involved, the genes expres­ dependent experiments. Data are presented as the relative mRNA expression
sion of 9 key enzymes (HMGR, StAR, CYP11A1, 3β-HSD2, CYP17, 17β- levels in the exposed group to those in the DMSO control. β-actin was used for
HSD1, 17β-HSD4, CYP19 and SULT2A1) that are associated with the normalization.
synthesis of steroidogenesis were measured. As shown in Fig. 5, the
positive compound forskolin significantly increased expression of 8 and StAR mediates the transfer of cholesterol. The significant increase of
genes and inhibited SULT2A1 expression. Both TPhP and TDCIPP HMGR and StAR gene expression levels was induced by them, which
significantly promoted HMGR, StAR, CYP19 and 17β-HSD1 genes suggested that they promoted cholesterol synthesis and its transfer.
expression and significantly inhibited SULT2A1 gene expression CYP19 and 17β-HSD1 catalyze the conversion of androgen to estrogen
(Table S2). HMGR is a rate-limiting enzyme of cholesterol biosynthesis and the reduction of estrone into E2, respectively. It was found that

Fig. 4. The effects of TPhP and TDCIPP on steroidogenesis in H295R cells. (A) The effects of TPhP on the levels of progesterone, estrone, testosterone and 17β-
estradiol (E2). (B) The effects of TDCIPP on the levels of progesterone, estrone, testosterone and 17β-estradiol (E2). (C) The effects of TPhP and TDCIPP on the E2/T
ratio. Data are presented as relative levels that were normalized to that of the DMSO control. *p < 0.05, compared with the DMSO control.

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X. Ji et al.
TPhP and TDCIPP up-regulated CYP19 and 17β-HSD1 expression to
promote the conversion of testosterone and estrone to E2, which resul­
ted in an increase of E2 concentrations, similarly, up-regulation of
CYP19A gene and 17β-HSD1 gene have been reported to support an
increase of E2 induced by 5 phthalates (DEP, MEP, BBzP, MBzP and
DiBP) (Sohn et al., 2016) and trichloroethylene (Tachachartvanich et al.,
2018), respectively. Moreover, the fold of 17β-HSD1 expression
(10 μM = 3.6, 2 μM = 2.7) promoted by TPhP was significantly higher
than that of CYP19 (10 μM = 2.9, 2 μM = 2.0), implying that the reason
for the lower estrone concentrations is that more estrone is converted to
E2 than synthetic estrone. On the contrary, the fold of 17β-HSD1
expression (10 μM = 2.7, 2 μM = 2.1) promoted by TDCIPP was
significantly lower than that of CYP19 (10 μM = 3.7, 2 μM = 2.7),
implying that the reason for the higher estrone concentrations is that
more estrone is synthesized than that for further conversion to E2.
SULT2A1 catalyzes the sulfonation of dehydroepiandrosterone, and
their inhibition of SULT2A1 expression might lead to more dehydro­
epiandrosterone participating in the production of E2. However, they
induced different effects on 4 genes (CYP11A1, 3β-HSD2, CYP17 and
17β-HSD4), which might cause their different action on other 3 hor­
mones levels. CYP11A1 catalyzes the conversion of cholesterol to
pregnenolone, 3β-HSD2 acts a crucial role in the synthesis of various
steroids, and CYP17 catalyzes pregnenolone/progesterone to andro­
stenedione. TPhP increasing progesterone levels could result from the
up-regulation of CYP11A1 and 3β-HSD2 genes expression, and TPhP
promoting CYP17 gene expression might cause more androstenedione
synthesis. In contrast, exposure to TDCIPP, since the expression of
Fig. 6. The proposed toxicity mechanisms framework of estrogen disrupting effects for TPhP and TDCIPP. Hormones including progesterone, estrone, testosterone
and 17β-estradiol which are marked with boxes and all genes in the figure were tested in this study. Red indicated a significant increase of levels, black indicated no
significant changes of levels and blue indicated a significant decrease of levels.
7
Ecotoxicology and Environmental Safety 229 (2022) 113069
CYP11A1, 3β-HSD2 and CYP17 was not affected, and then the synthesis
and conversion of progesterone is normal, so that its levels remain un­
changed. 17β-HSD4 catalyzes the reduction of androstenedione to
testosterone. No changes of 17β-HSD4 and increased CYP19 expression
by TPhP caused that more testosterone is converted to E2 than synthetic
testosterone, leading to decreased testosterone levels. This result is
content with a previous study that perfluorooctanoic acid (PFOA) and
perfluorooctane sulfonate (PFOS) inhibited testosterone levels due to
their up-regulation of CYP19 expression and no effects on 17β-HSD4
expression (Kang et al., 2016), While TDCIPP promoting 17β-HSD4
expression resulted in increased testosterone concentrations.
TPhP and TDCIPP have been demonstrated to act estrogenic activ­
ities in in vivo, they up-regulated VTG levels in zebrafish (Liu et al.,
2013), which is thought to be one of biomarkers concerning
estrogen-like EDCs. According to the adverse outcome pathway (AOP)
framework for EDCs, an adverse outcome at the organism level might
result from various separate pathways, and understanding toxicity
pathways of EDCs is better for their risk assessment on human health.
Thereby, in present study, the action on ERα, the activation on GPER and
E2 synthesis of TPhP and TDCIPP was investigated to form a systematic
and comprehensive toxicity mechanism framework of their
estrogen-disrupting activities. As shown in Fig. 6, it was found that TPhP
acted as a pure estrogen-like substance, it showed agonistic potential on
ERα to activate ERE transcription in MVLN cells, and induced ERα
interaction with SRC1 and SCR3. It also formed a pi-sigma bond with
GPER and activated GPER-mediated cAMP pathways. Additionally,
TPhP disrupted steroidogenesis which involved increased progesterone
X. Ji et al.
levels, decreased testosterone and estrone levels, and alteration of
various key genes, eventually resulting in up-regulation of E2 concen­
trations. In contrast, TDCIPP acted as a partial estrogen-like substance
leading to estrogen-disrupting activities. It showed antagonistic effects
on ERα in MVLN cells, but it formed a hydrogen bond with GPER to
activate GPER-cAMP pathways. Additionally, TDCIPP disrupted ste­
roidogenesis which involved increased testosterone and estrone levels,
and alteration of various key genes including 17β-HSD4, 17β-HSD1 and
CYP19, eventually resulting in up-regulation of E2 concentrations
(Fig. 6). Obviously, TPhP induced the synergy of multiple pathways
related to estrogenic effects including ERα and GPER agonistic response,
and promoting E2 synthesis, while TDCIPP showed more complicated
mechanisms involving in its synergic effects between GPER activation
and promoting E2 synthesis, but antagonistic effects on ERα. The dif­
ference in their toxicity mechanisms might be associated with their
different substituent. In our previous studies, we found that in contrast
to TmCP and TpCP, ToCP with methyl group at the ortho position of the
aromatic ring showed higher activity in activating GPER (Ji et al.,
2020a). Moreover, due to chlorination, TDCIPP induced more serious
toxicity than TnBP on DNA damage and apoptosis in C. fluminea (Yan
et al., 2020). Of note, similar to BPA analogs, though they have similar
structure, some of them also exert different action in estrogen-disrupting
activities, specifically, BPA, BPF, BFS and TCBPA showed agonistic ef­
fects on ERα in MVLN cells (Molina-Molina et al., 2013), while 9,9-bis
(4-hydroxyphenyl)-fluorene (BHPF) acted as an ERα antagonist due to
its inhibition on the coactivator recruitment (Cao et al., 2019). More­
over, BFAF and BFB displayed higher estrogenic response than BPA via
GPER with different binding affinity (Cao et al., 2017). Furthermore,
BPA and BPS inhibited hormone production in H295R cells by
down-regulating steroidogenic genes, while BPF led to increased pro­
gesterone and E2 (Feng et al., 2016). In addition, our data provided for
the first time a more complete framework of toxicity mechanisms of
estrogen disrupting effects for TPhP and TDCIPP, and it still requires
further study on their synergic effects involving various pathways.
Currently, the molecular initiating event in the AOP framework for risk
assessment of EDCs mainly focused on their activation on ERα, and their
effects on GPER and E2 synthesis are needed to be considered in the
future.
TPhP and TDCIPP have high frequency detection in aquatic envi­
ronmental media, and their concentrations were in the levels of ng/L. Of
note, TPhP at the environmentally relevant concentrations of ng/L have
been demonstrated to cause increased plasma E2 and decreased testos­
terone, resulting in intersex in male medaka (Li et al., 2018), while
20 μg/L TDCIPP could increase plasma E2 in female zebrafish (J. Wang
et al., 2015; Q.W. Wang et al., 2015; Qiangwei Wang et al., 2015).
Moreover, TPhP and TDCIPP have also been found in various biota
samples, such as glaucous gull at levels of 1.2 and 22.52 ng/g wet weight
(ww), respectively, arctic fox at levels of 1.3 and 89 ng/g ww, respec­
tively, polar bear at levels of 6.5 and 54 ng/g ww, respectively, and
freshwater shrimp at levels of 5.4 and 2.0 ng/g ww, respectively (Hal­
langer et al., 2015; Strobel et al., 2018; Zhao et al., 2018), implying that
their potential to cause adverse effects on wildlife including aquatic and
terrestrial organisms. Additionally, TPhP and TDCIPP were also detec­
ted in human samples. In human milk samples, their maximal concen­
trations were 140 and 162 ng/g lipid wt, respectively (Kim et al., 2014),
which suggested that susceptible infants were at risk for exposure to
TPhP and TDCIPP, and might be easier to be affected by their
estrogen-disrupting potential. Note that maximal levels of 1.21 and
3.41 ng/mL for TPhP and TDCIPP, respectively, were found in 257
human blood samples from Shenzhen, China (Zhao et al., 2016), obvi­
ously, they are much lower than lowest-observed effect concentrations
(LOECs) for TPhP and TDCIPP in present study of 326.3 (1 × 10-6 M)
and 430.9 ng/mL (1 × 10-6 M), respectively. However, our data sug­
gested that TPhP and TDCIPP had potential to activate multiple path­
ways of estrogen-disrupting effects, and they also have been reported to
interfere with various steroid hormone receptors, such as antagonism to
8
Ecotoxicology and Environmental Safety 229 (2022) 113069
androgen receptor (AR) with LOECs of 5 × 10-5 and 5 × 10-6 M for TPhP
and TDCIPP, respectively, antagonism to thyroid hormone receptor (TR)
with LOECs of 1 × 10-5 M for TDCIPP, antagonism to glucocorticoid
receptor (GR) with both LOECs of 1 × 10-6 M, antagonism to mineral­
ocorticoid receptor (MR) with LOECs of 1 × 10-7 and 5 × 10-6 M for
TPhP and TDCIPP, respectively, and antagonism to estrogen-related
receptor γ (ERRγ) with LOECs of 1 × 10-5 M for TPhP (Cao et al.,
2018a; Kojima et al., 2013; Zhang et al., 2016, 2017), indicating that the
action of TPhP and TDCIPP toward various receptors might lead to
complex endocrine disrupting effects. Therefore, there is a need for
further studies to assess their potential ecological and health risks
effects.
In summary, we aimed to investigate the toxicity mechanisms con­
cerning estrogen disrupting effects of TPhP and TDCIPP. The results
showed that TPhP acted as a pure estrogenic substance and TDCIPP
acted as a partial estrogenic substance. Specifically, TPhP promoted
ERα-ERE transcription and recruited SRC1 and SRC3 which was found
for the first time, while TDCIPP showed antagonistic effects on ERα.
Both TPhP and TDCIPP activated GPER-mediated cAMP pathways.
Although TPhP and TDCIPP could dock into ERα and GPER, their sub­
stituent groups and combination mode might affect the receptor acti­
vation. Additionally, TPhP and TDCIPP up-regulated E2 concentrations
and E2/T ratio, but they involved different alteration of progesterone,
testosterone and estrone levels, and various key genes expression. The
difference in their mechanisms of their estrogen disrupting effects might
be due to their different substituents. Our data proposed a framework of
toxicity mechanisms of estrogen disrupting effects for TPhP and TDCIPP,
and provided valuable insight into future construction of AOP frame­
work of EDCs.
CRediT authorship contribution statement
Xiaoya Ji: Conceptualization, Methodology, Validation, Formal
analysis, Investigation, Writing – original draft. Na Li: Methodology,
Formal analysis, Writing – review & editing, Funding acquisition. Mei
Ma: Visualization, Writing – review & editing, Project administration,
Supervision, Funding acquisition. Xinyan Li: Investigation. Kongrui
Zhu: Validation. Kaifeng Rao: Visualization. Zijian Wang: Project
administration, Supervision. Jingfeng Wang: Investigation. Yanjun
Fang: Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We thank Dr. Méndez-Luna for kindly providing us the GPER ho­
mology modeling. This work was financially supported by the National
Key Research and Development Program of China (2019YFC1804604),
the National Natural Science Foundation of China (41977208,
52030003), Excellent Innovation Project of Research Center for Eco-
Environmental Sciences, CAS (RCEES-EEI-2019), and the special fund
from Key Laboratory of Drinking Water Science and Technology,
Research Center for Eco-Environmental Sciences, Chinese Academy of
Sciences (19204KLDWST), and Special Fund from key topics
(AWS18J004).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.ecoenv.2021.113069.
X. Ji et al. Ecotoxicology and Environmental Safety 229 (2022) 113069

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