Environmental Pollution 241 (2018) 674e683

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

New insights into the mechanism of phthalate-induced

developmental effects*
Xiyan Mu a, *, Ying Huang a, Jia Li a, Ke Yang a, Wenbo Yang a, Gongming Shen a,
Xuxing Li a, Yunlei Lei a, Sen Pang b, Chengju Wang b, Xuefeng Li b, Yingren Li a, **
a
Fishery Resource and Environment Research Center, Chinese Academy of Fishery Sciences, Beijing, PR China
b
College of Sciences, China Agricultural University, Beijing 100193, PR China

a r t i c l e i n f o a b s t r a c t

Article history: To investigate the biological pathways involved in phthalate-induced developmental effects, zebrafish
Received 6 February 2018 embryos were exposed to different concentrations of di-(2-ethylhexyl) (DEHP) and di-butyl phthalate
Received in revised form (DBP) for 96 h. Embryonic exposure to DEHP and DBP induced body length decrease, yolk sac
5 May 2018
abnormities, and immune responses (up-regulation of immune proteins and genes). The lipidomic re-
Accepted 31 May 2018
sults showed that at a concentration of 50 mg/L, DEHP and DBP significantly reduced the levels of fatty
acids, triglycerides, diacylglycerol, and cholesterol. These effects are partly explained by biological
pathway enrichment based on data from the transcriptional and proteomic profiles. Co-exposure to DBP
Keywords:
Di-(2-ethylhexyl) phthalate
and ER antagonist did not significantly relieve the toxic symptoms compared with exposure to DBP
Di-butyl phthalate alone. This indicates that phthalate-induced developmental abnormities in zebrafish might not be
Developmental effects mediated by the ER pathway. In conclusion, we identified the possible biological pathways that mediate
Immune response phthalate-induced developmental effects and found that these effects may not be driven by estrogenic
Lipid homeostasis activation.
Mechanism © 2018 Elsevier Ltd. All rights reserved.

1. Introduction contamination occur (more than 100 mg/L) in heavily industrialised

Phthalates are commonly used in a variety of industrial and
consumer products, including food packaging, children's toys, and
building materials. Di-(2-ethylhexyl) phthalate (DEHP) is the most
commonly used phthalate plasticizer in polyvinyl chloride (PVC)
formulations; DEHP accounts for 80% of phthalate production in
China and approximately 18% of all plasticizers used in Western
Europe (Zolfaghari et al., 2014; European Union, 2008). Di-butyl
phthalate (DBP) is typically used in personal care products (Lien
et al., 2015). Because phthalates are not chemically bound to the
plastics, they can leach into food, medicine, water, indoor dust, and
air, resulting in human and animal exposure (Zota et al., 2016; Jia
et al., 2017; Net et al., 2015; Kang et al., 2012; Gaspar et al., 2014).
The detected concentration of DEHP and DBP in drinking water and
surface water is generally below 1 and 10 mg/L respectively (Liu
et al., 2014; Zeng et al., 2008). However, hotspots of
*
This paper has been recommended for acceptance by Dr. Chen Da.
* Corresponding author.
** Corresponding author.
E-mail addresses: muxiyan@cafs.ac.cn (X. Mu), liyr@cafs.ac.cn (Y. Li).
areas (Fromme et al., 2002; Yuwatini et al., 2006; Khan and Jung,
2008). As a result of widespread contamination, these chemicals
can enter the human body through daily ingestion and inhalation.
Phthalates are ubiquitously detected in urine samples of all age
groups from all around the world (Guo et al., 2011; Gao et al., 2017;
Me
rida-Ortega et al., 2016; Lee et al., 2017).
Given the wide distribution of phthalates, their negative effects
on humans and animals have drawn considerable attention. Most
research efforts have focused on endocrine-disrupting effects, pri-
marily the alteration of hypothalamus-pituitary-thyroid axis and
hypothalamus-pituitary-gonad axis. Previous studies found that
the urinary phthalate concentrations in humans are negatively
associated with the levels of urinary thyroid hormones in children
and serum T4 in adults (Boas et al., 2010; Huang et al., 2017). In
addition, a variety of studies have shown that phthalates cause
reproductive toxicity in both male and female individuals. Serum
phthalates are negatively associated with serum testosterone, sex
hormone-binding globulin, semen volume, semen quality, and total
sperm count, whereas they are positively associated with DNA
damage in the sperm of adult men (Joensen et al., 2012; Pant et al.,
2008; Specht et al., 2014). In male fish, decreased plasma

https://doi.org/10.1016/j.envpol.2018.05.095
0269-7491/© 2018 Elsevier Ltd. All rights reserved.
 X. Mu et al. / Environmental Pollution 241 (2018) 674e683
testosterone (T) concentrations, reduced ability to fertilise oocytes
spawned by untreated females, decreased sperm motility and ve-
locity, and altered spermatogenesis were observed after DEHP
exposure (Uren-Webster et al., 2010; Crago and Klaper, 2012;
Golshan et al., 2015). In females, urinary concentrations of DEHP
metabolites are negatively associated with oocyte yield, clinical
pregnancy, and live birth (Hauser et al., 2016). Ye et al. (2014) re-
ported that female marine medaka (Oryzias melastigma) showed
decreased egg production, increased concentrations of T and E2 and
up-regulation of ldlr.
In addition to the effects on reproduction and hormone levels,
the developmental effects of phthalates have been increasingly
studied in recent years. Tsai et al. (2016) demonstrated that daily
DEHP intake is negatively associated with height percentile,
weight percentile, bone age/chronological age, and insulin-like
growth factor 1 (IGF-1) levels in children. The levels of urinary
phthalate metabolites are associated with delayed pubic hair
development in boys and earlier breast development in girls
(Zhang et al., 2015). Yamasaki et al. (2009) reported that maternal
exposure to dicyclohexyl phthalate can induce a series of devel-
opmental effects in male offspring, including prolonged preputial
separation, reduced ano-genital distance, increased areola/nipple
retention, and decreased ventral prostate and levator ani/bulbo-
cavernosus muscle weights. In addition, neurodevelopment has
been identified as another target of phthalates. Research on
humans and other mammals has shown that phthalate exposure
can result in attention problems along with aggressive or rule-
breaking behavior in children and induce cell apoptosis in hip-
pocampal neurons as well as impair spatial learning and memory
in rat pups (Kobrosly et al., 2014; Engel et al., 2010; Li et al., 2013;
Li et al., 2009). However, the mechanism of action and pathways
involved in phthalate-induced developmental effects remain
uncharacterized, although these symptoms have been considered
as the downstream health outcomes that resulted from the dis-
order of hormone, for instance lower production of estradiol or
progesterone (Kay et al., 2013).
Currently, zebrafish (Danio rerio) embryos are an important
model for toxicological investigations of hazardous chemicals
(Zhang et al., 2017; Brox et al., 2016; Huang et al., 2016). Zebrafish
embryos possess many advantages, including in vitro fertilization,
high fecundity, rapid embryonic development, and optical trans-
parency, which make it easy to detect morphological endpoints or
observe the development process in early life stages (Mu et al.,
2016). Previous studies have shown that phthalates have several
negative effects on zebrafish, including steroidogenic alteration,
cardiac defects, reproductive toxicity and oxidative stress (Sohn
et al., 2016; Sun and Liu, 2017; Zhang et al., 2014; Corradetti
et al., 2013). In this study, we applied several biological ap-
proaches with an experimental morphological investigation using
zebrafish embryo as a model to identify the biological pathways
that mediate phthalate-induced developmental effects.
2. Materials and methods
2.1. Zebrafish maintenance and embryo collection
Wild-type zebrafish (AB strain) were purchased from a local
provider. All adult zebrafish were maintained in flow-through
feeding equipment (made by Esen Corp.) at 26
C with a photope-
riod of 14/10 (light/dark). The zebrafish were fed daily with live
brine shrimp (Artemia salina). The preparation and collection of
zebrafish embryos followed the procedure described in our previ-
ous work (Mu et al., 2013).
675
2.2. Chemicals and reagents
Standard water was prepared in the lab according to the formula
of iso-7346-3: 2 mmol L1 Ca2þ, 0.5 mmol L1 Mg2þ, 0.77 mmol L1
Naþ, and 0.08 mmol L1 Kþ (ISO, 1996). Di-(2-ethylhexyl phthalate
(DEHP, 99.5%, CAS: 117-81-7) and dibutyl phthalate (DBP, 99%, CAS:
84-74-2) were purchased from Sigma-Aldrich (Darmstadt, Ger-
many). Stock solutions of DEHP and DBP for drug-exposure ex-
periments were prepared using acetone AR. ICI-182,780 (98%, CAS
129453-61-8) was purchased from Sigma-Aldrich (Darmstadt,
Germany).
2.3. Exposure and sample collection
Experiments were performed in accordance with current Chi-
nese legislation and were approved by the independent animal
ethics committee at Chinese Academy of Fishery Sciences.
2.3.1. Exposure for morphological endpoints
Test solutions of DEHP and DBP with concentrations of 0 (con-
trol), 50, and 250 mg/L (based on pre-experiment data) were made
using standard water. Embryos at approximately 2 h post-
fertilization (hpf) were randomly transferred into the test solu-
tions in 24-well plates. Exposure solutions for solvent control and
all treatment groups contained the same concentration of acetone
(0.05 mL/L). Each treatment was replicated three times. The num-
ber of dead individuals was determined, and the dead embryos
were removed daily. Embryo death was judged using the lethal
toxicological endpoints proposed by Nagel (2002). The hatching
and development status of embryos were checked daily. Terato-
genic effects were identified and recorded using a ZEISS Vert.A1
microscope (Jena, Germany). Body length along with yolk sac width
and height were measured linearly using the microscope software.
See the Supplemental Information for more details.
2.3.2. Exposure for transcriptomic and proteomic analysis
Embryos were randomly transferred into test solutions (50 mg/L)
in 1-L beakers at 2 hpf. Each beaker contained 500 mL of exposure
solution and approximately 100 embryos, and there were three
beakers in each treatment group. At 96 hpf, 75 hatched (or decor-
ticated) larvae from each replicate (beaker) were collected and
washed twice with standard water (25 for RNA extraction and 50
for protein extraction). The embryo samples were stored at 80
C
until analysis.
2.3.3. Exposure for lipidomic analysis
Embryos were randomly transferred into test solutions (50 mg/L)
in 200-mL beakers at 2 hpf. Each beaker contained 100 mL of
exposure solution and 40 embryos, and there were eight beakers in
each treatment group. At 96 hpf, 20 embryos were collected from
each beaker and washed with standard water. The embryo samples
were stored at 80
C until analysis.
2.3.4. Exposure for immune response and estrogenic activity test
Embryos were randomly transferred into test solutions (50 and
250 mg/L) in 1-L beakers at 2 hpf. 50 and 250 mg/L 17b-estradiol was
used as positive control in the estrogenic activity test. Each beaker
contained 500 mL of exposure solution and approximately 200
embryos, and there were three beakers in each treatment group. At
96 hpf, 120 hatched (or decorticated) larvae from each replicate
were collected and washed twice with standard water (30 for
mRNA extraction and 90 for the ELISA assay of hormones, ERa and
immune protein levels). The embryo samples were stored at 80
C
until analysis.
676 X. Mu et al. / Environmental Pollution 241 (2018) 674e683
2.4. Illumina sequencing and transcriptomic analysis
Total RNA was extracted from zebrafish embryos using a spin
column adsorption method following the manufacturer's protocol
(Tiangen Biotech). RNA quality was assessed using an Agilent 2100
BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and a
Qubit Fluorometer (Invitrogen). Total RNA samples that met the
following requirements were used in subsequent experiments:
RNA integrity number >7.0 and 28S:18S > 1.8. Libraries were con-
structed from three samples of embryos exposed to the solvent
control and three samples of embryos exposed to 50 mg/L phtha-
lates using a Next Ultra RNA Library Prep Kit for Illumina (NEB).
mRNA molecules with poly(A) tails were enriched from 1 mg total
RNA using a Next Poly(A) mRNA Magnetic Isolation Module (NEB)
kit. After RT-qPCR validation, the libraries were subjected to paired-
end sequencing with pair end 125-base pair reading length using
an Illumina HiSeq 2500 sequencer (Illumina). Please see the
Supplemental Information for more details on RNA-Seq
(Tables S1eS3 and Figures S5eS6).
2.5. Proteomics
For each sample, 50 embryos were ground in liquid nitrogen and
lysed using protein-extraction buffer on ice for 30 min and then
centrifuged at 16,000  g for 15 min at 4
C. The supernatant was
collected, and the total protein content was determined using a
bicinchoninic acid protein assay (Pierce, USA). The lysis was stored
at 80
C before further processing. Tandem mass tags (TMT6/10,
Pierce, USA) with different reporter ions (126e131 Da) were
applied as isobaric tags for relative quantification. TMT labeling was
performed according to the manufacturer's instructions. LC-MS/MS
analysis was carried out at Capitbalbio Technology using a Q
Exactive mass spectrometer (Thermo Scientific, CA). Proteome
Discoverer software (Version 1.4, Thermo Scientific, USA) was used
to perform database searching against the Oryctolagus cuniculus
database (46601 proteins) using the Sequest algorithms. More de-
tails regarding sample digestion, TMT labeling, LC-MS/MS param-
eters and data analysis are provided in the Supplemental
Information (Tables S4eS5).
2.6. Lipidomics
Lipid analysis was performed using a Q Exactive Orbitrap mass
spectrometer (Thermo, CA, USA). Mobile phase A was prepared by
dissolving ammonium acetate (0.77 g) in 400 mL of water followed
by adding 600 mL of acetonitrile. Mobile phase B was prepared by
mixing 100 mL of acetonitrile with 900 mL of isopropanol. An
XSelect CSH C18 column was used for lipid analysis at a column
temperature of 45
C. Lipids were identified and quantified using
Lipid Search 4.1.30 (Thermo, CA, USA). Mass tolerances of 5 and
10 ppm were applied for the precursor and product ions. A reten-
tion time shift of 0.25 min was performed in “alignment.” The M-
score and chromatographic areas were used to reduce false posi-
tives. Please see the Supplemental Information for more details.
2.7. ELISA
Protein and hormone levels were measured via ELISA. 90 em-
bryos were homogenized with saline (1:10, w/v) on ice in centri-
fuge tube using an electric homogenizer (Tiangen Biotech, Beijing,
China). After centrifugation for 10 min at 3000 rpm and 4
C, the
supernatant was collected for ELISA. ELISA was conducted accord-
ing to the ELISA Kit instructions (Dongge Biotechnology Co., Ltd,
Beijing, China). Please see the Supplemental Information for more
details.
2.8. Gene expression analysis
RNA extraction, cDNA synthesis and real-time PCR reactions
were performed as previously described (Mu et al., 2018).
Zebrafish-specific primers were designed for the genes of interest
using Primer 5.0 software (Table S6). The housekeeping gene b-
actin was used as an internal control. Transcript quantification was
performed using the 2DdCt method. Please see the Supplemental
Information for more details (Tables S7eS8).
2.9. ER inhibition test
Exposure was conducted in the same way as for morphological
endpoints. Embryos were exposed to 250 mg/L DBP or co-exposed
to 250 mg/L DBP and 5 mM (z3.04 mg/L) ICI 182,780 in a 24-well
plate from 2 to 96 hpf. The body lengths and yolk sac sizes of the
embryos were assayed.
2.10. Chemical confirmation of phthalate concentration
The actual concentrations of DEHP and DBP were measured at
0 hpf (at the beginning of exposure), 24 hpf (before the first
replacement) and 96 hpf (at the end of exposure). Based on the
monitoring results, the deviation between the nominal and actual
phthalate concentrations was generally less than 20%
(Tables S9eS11). Therefore, the nominal concentrations of DEHP
and DBP could be used to represent the actual concentrations in
exposure solution in this work.
2.11. Statistical analysis
All statistical analyses were performed using SPSS 16.0 software.
Differences were determined by one-way ANOVA complemented
by Dunnett post hoc comparison. P < 0.05 was considered signifi-
cant. Semantic similarity scatter plots were constructed using
REVIGO online software according to the instructions (Supek et al.,
2011).
3. Results
3.1. Solvent effect
No significant difference was found between the solvent and
blank control for all tested indicators (data not shown). The control
data shown in all figures and tables are the experimental data for
the solvent control.
3.2. Developmental effects caused by phthalates
Under the tested concentrations, DEHP and DBP exposure had
no significant effects on the survival and hatching of zebrafish
embryos (Figures S1b and 1c). However, the body length of the
embryos in the DBP-250 group was significantly reduced compared
with that of the control (Figure S1a). Furthermore, the yolk sac was
significantly affected by DEHP and DBP, which is reflected in two
aspects: (1) the proportion of the fish body made up by the yolk sac
was larger in phthalate-treated embryos than in control embryos
(Fig. 1a); and (2) the yolk sac size was larger in phthalate-treated
embryos than in control embryos (Fig. 1b and c).
3.3. Global changes in protein abundance
Global proteome profiling indicated the significant alteration of
the abundances of 257 proteins (217 up-regulated and 40 down-
regulated) and 235 proteins (217 up-regulated and 18 down-
 X. Mu et al. / Environmental Pollution 241 (2018) 674e683 677

Fig. 1. a Yolk sac area (red dotted line) of embryos in the control and DEHP-250 groups at 48 hpf. The phthalate-treated yolk sac accounted for a higher proportion of the larvae body
than the control yolk sac. b Widths and heights of embryo yolk sacs in the control and phthalate-treated groups at 48 hpf. c Yolk sac size (including width and height) of embryos in
the control, DBP-50 and DBP-250 groups at 48 hpf. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

regulated) in embryos exposed to 50 mg/L DBP and DEHP, respec-
tively (Fig. 2a). The enrichment analysis of proteome data revealed
that several processes, including response to xenobiotic stimulus,
lipoprotein metabolism, regulation of lipid catabolism, regulation
of fatty-acid metabolism, and regulation of triglyceride catabolism,
were significantly induced in DBP-treated embryos. In DEHP-
exposed embryos, lipase activity regulation, lipid absorption, lipid
catabolism and lipid metabolism were significantly up-regulated
(Figure S2).
To identify the main pathway involved in phthalate exposure,
we further conducted a REVIGO similarity analysis. According to the
similarity scatterplot of significantly enriched GO terms, lipid
metabolism, lipid transport, cell locomotion, digestive system,
lipase activity regulation, and cell adhesion are the main molecular
pathways altered by DBP exposure (Fig. 2b). Similar to the DBP
results, the main active pathways affected by DEHP exposure are
lipid digestion, lipid metabolism, lipid transport and lipase activity
regulation (Fig. 2c). Please see the Supplemental Information for
more details on the REVIGO analysis (Table S12 and S13).
3.4. Changes in transcriptional profile
The transcript profiles also indicated that the biological pro-
cesses involved in lipid metabolism, skeletal development, and the
immune system (e.g., steroid binding, lipid binding, fatty-acid
metabolism, lipid transport, skeletal muscle tissue development,
stress response, and chemokine activity) were significantly altered
by DEHP and DBP (Figure S3 a-c). However, the transcription levels
of genes involved in the synthesis of sex hormones, including es-
trogen receptor isomers, androgen receptors, and estradiol syn-
thetase, remained unchanged (Figure S3d).
3.5. Phthalates altered lipid profiles
Since both the proteomic and transcriptomic results showed
that the pathways involved in lipid metabolism were significantly
altered by DEHP and DBP, we further assessed the influence of
phthalates on the zebrafish lipid profile. After the analysis of pos-
itive and negative adducts, 207 lipids [4 acyl carnitines (Acca), 26
678 X. Mu et al. / Environmental Pollution 241 (2018) 674e683

Fig. 2. a Hierarchical clustering of differentially expressed proteins (fold change compared to control > 1.5) in zebrafish embryos exposed to 50 mg/L DEHP and DBP. b-c REViGO
semantic similarity scatter plot of significantly enriched (p < 0.01) biological processes. Ontology terms after DBP (b) and DEHP (c) exposure.

ceramides (Cer), 11 neutral glycosphingolipids (Cerg1 and Cerg2),
two cholesterol esters (Che), 21 diglycerides (DG), 98 triglycerides
(TG), six lysophosphatidylcholines (LPC), four lysophosphatidyle-
thanolamines (LPE), 34 sphingomyelins (SM), and one sphingosine
(So)] and 254 lipids [21 cardiolipins (CL), five ceramides (Cer), 30
fatty acids (FA), 17 phosphatidylserines (PS), 13 phosphatidylino-
sitols (PI), two phosphatidylglycerols (PG), 70 phosphatidyletha-
nolamines (PE), 85 phosphatidylcholines (PC), two LPEs, and two
LPCs], respectively, were identified. Most of the identified lipid
species remained unchanged after phthalate exposure. Compared
with the control, exposure to 50 mg/L DBP significantly reduced the
levels of Cer, Cerg2, Che, PC, DG, TG, and FA, whereas exposure to
50 mg/L DEHP significantly reduced the levels of Cerg1, Che, DG, TG,
and FA (Figure S4a). The significantly altered lipids are detailed in
Figures S4b and S4c. Among all the lipids, TG (16:0/16:0/18:1) and
TG (16:0/18:1/18:1) were the most significantly affected by
phthalate exposure. In addition, CerG2 (d18:1/22:0), ChE (22:6), PC
(17:1/16:0), DG (18:0/18:0), DG (18:0/16:0), and FA (18:1) levels
were significantly reduced by DBP exposure; the relative levels
compared with the control were 0.44, 0.58, 0.56, 0.49, 0.5, and 0.49,
respectively. DG (16:0/16:1), DG (16:0/16:0), ChE (22:6), and FA
(18:1) were significantly reduced by DEHP, with relative levels of
0.59, 0.64, 0.57, and 0.59, respectively.
3.6. Phthalates induced immune responses
To further investigate the immune stress induced by DEHP and
DBP, the embryonic levels of immune proteins and genes were
assayed. Both the protein and transcription levels of TNFa, il-1b,
and il-8 apparently increased after DEHP and DBP exposure (Fig. 3a,
b, d, and e). Phthalate concentrations of 50 and 250 mg/L signifi-
cantly induced the level of protein tp63 (Fig. 3c), while 250 mg/L
DEHP and DBP inhibited the transcription level of tp63. In addition,
the expression level of NF-kb was elevated after phthalate
exposure.
3.7. Phthalate-induced endocrine disruption
We further assessed the estrogenic activation and hormone
disruption in zebrafish embryos exposed to phthalates. ELISA test
showed that 250 mg/L E2 performed significant activation towards
both 17-estradiol and ERa protein level, however, had no obvious
impact on transcription level of ESR1 and AR (Fig. 4c, d and 4e).
DEHP and DBP could significantly induce the level of 17b-estradiol
and ERa protein, and the induction is lower than that of E2. Further,
DEHP and DBP induced sharp up-regulation of ESR1 expression.
Although exposure to 50 mg/L phthalates had no apparent effect
 X. Mu et al. / Environmental Pollution 241 (2018) 674e683
Fig. 3. a Relative protein levels of TNFa, il-1b (b), tp63 (c) and il-8 (d). eRelative embryonic transcription levels of TNFa, NF-kB, il-8, il-1b, tp63 in the control and phthalate-treated
groups. Asterisks denote significant differences between treatments and the control (determined by Dunnett post-hoc comparison; p < 0.05*, p < 0.01**).
on thyroid hormone levels, at the concentration of 250 mg/L, the
phthalates significantly induced triiodothyronine (T3) and tetraio-
dothyronine (T4) levels (Fig. 4a and b).
3.8. ER inhibition test
To further investigate the receptor mechanism of the DBP-
induced developmental effects, embryos were simultaneously
exposed to DBP and an ER antagonist (ICI-182,780). The morpho-
logical results showed that the yolk sac size at 48 hpf and the body
length at 96 hpf were significantly affected in both the DBP group
and DBP-ICI co-exposure group compared with the control (Fig. 5a,
b, and 5c). Co-exposure to ICI did not provide any apparent relief for
the developmental effects of DBP.
4. Discussion
Phthalates, which are commonly used as plasticizers in flexible
PVC formulations, are a class of ubiquitous environmental toxi-
cants. Thus, humans and animals can be exposed to phthalates
through oral, dermal, and inhalation routes. In addition, fetal and
newborn individuals can be maternally exposed to phthalates
during early life stages. The present study showed that embryonic
exposure of zebrafish to DEHP and DBP during the period from 2 to
96 hpf induced negative effects, including yolk sac abnormalities
and reductions in body length. The change in the transcript profile
also indicated significant alteration in the expression levels of
genes participating in skeletal development, which might be
associated with the reduced body length. In zebrafish embryos, the
679
yolk sac stores and provides the nutrients needed for embryonic
development (Wiegand, 1996). Thus, the reduction in body length
observed in this study might be a down-stream symptom of
nutritional deficiencies originating from the yolk sac abnormities.
Sant et al. (2016) demonstrated that monoethylhexyl phthalic acid
(MEHP) exposure led to nutritional deficiencies in developing
mouse embryos, including changes in uptake and availability of
nutrients and cofactor uptake; this suggests that embryonic expo-
sure to phthalates may reduce essential nutrient supplies, resulting
in stunted growth in mammals, consistent with the present study.
Many previous studies have suggested that phthalates can alter
human development, including changes in body size, body weight
and brain development (Boas et al., 2010; Engel et al., 2010;
Maresca et al., 2016; Cho et al., 2010). The majority of these
symptoms have been recognized as downstream health outcomes
resulting from phthalate-induced endocrine disruption (e.g., es-
trogenic effects or thyroid disruption) (Kay et al., 2013; Meeker and
Ferguson, 2011). However, in the present study, developmental
indicators seemed to be more sensitive to DEHP and DBP exposure
than endocrine indicators. The concentration of DEHP or DBP
required to affect body length, yolk sac morphology, and skeletal
development transcripts was 50 mg/L, lower than the concentration
required to induce symptoms related to endocrine disruption
(250 mg/L for the alteration of ER protein, ER transcription, estrogen,
and thyroxine levels). Therefore, we infer that the developmental
abnormities caused by phthalates in this work may not have been
driven by estrogen effects. To further confirm whether the devel-
opmental effects caused by DEHP and DBP arose from the estrogen
receptor-mediated pathway, zebrafish embryos were co-exposed to
680 X. Mu et al. / Environmental Pollution 241 (2018) 674e683

Fig. 4. a Relative T3 level of embryos in control and phthalate-treated groups. b Relative T4 level of embryos in control and phthalate-treated groups. c Relative 17b-estradiol level of
embryos in control, positive control (E2) and phthalate-treated groups. d Relative embryonic transcription level of ESR1 and AR in control, positive control (E2) and phthalate-
treated groups. e Relative ERa protein level of embryos in control, positive control (E2) and phthalate-treated groups. Asterisks denote significant differences between treat-
ments and control (determined by Dunnett post-hoc comparison, p < 0.05*; p < 0.01**).

Fig. 5. a Body lengths of zebrafish embryos in the control, ICI (3.04 mg/L), DBP (250 mg/L) and DBP-ICI (250 mg/Lþ3.04 mg/L) groups at 96 hpf. b Yolk sac heights of zebrafish
embryos in the control, ICI, DBP and DBP-ICI groups at 48 hpf. c Yolk sac widths of zebrafish embryos in the control, ICI, DBP and DBP-ICI groups at 48 hpf. Asterisks denote
significant differences between treatments and the control (determined by Dunnett post-hoc comparison; p < 0.05*, p < 0.01**).

phthalate and an ER antagonist. The results showed that co-
exposure to the ER antagonist did not relieve the developmental
effects induced by DBP. This failure to antagonize DBP effects
indicated that DBP might not act through common nuclear or
membrane-bound ERs since the same concentration (5 mM) of ICI
182,780 was sufficient to antagonize ER activity in zebrafish em-
bryos (Figure S7) (Kinch et al., 2015). Considering the above dis-
cussion, the phthalate-induced growth defects observed in this
work may not be mediated by ER.
Lipid metabolism disorder is a global public health problem
attributable to complex interactions between genetic, behavioral,
and environmental factors. In recent years, a number of studies
have shown that exposure to phthalates can cause abnormal lipid
metabolism in humans and other mammals (Lind et al., 2012;
Schmidt et al., 2012). Schmidt et al. (2012) reported that exposure
to environmental concentrations of DEHP enhanced the expression
levels of genes related to adipogenesis (PPAR isoforms and FABP4)
and led to increases in body weight and visceral fat deposits in C3H/
N mice. Chiang et al. (2016) showed that the MEHP-treated adi-
pocytes exhibited significant increases in lipolysis and the tran-
scription of genes involved in adipogenesis and lipid metabolism.
However, less is known about the phthalate-induced disruption of
lipid metabolism in aquatic organisms. In this work, proteomic data
showed that DEHP and DBP may alter lipid homeostasis in zebrafish
 X. Mu et al. / Environmental Pollution 241 (2018) 674e683
embryos by inducing the expression of proteins involved in certain
biological processes. The transcription profile also showed that
genes involved in fatty-acid metabolism, steroid binding, and lipid
transport were up-regulated by DEHP and DBP exposure, which is
consistent with previous studies. However, the lipidomic results
showed that most lipid species remained unchanged after phtha-
late exposure, while several species were reduced, including di-
glycerides, triglycerides, ceramides, phosphatidylcholines,
cholesterol esters, and fatty acids; this contradicts previous results
for mammals (Schmidt et al., 2012; Chiang et al., 2016). In fish eggs,
the endogenous lipid reserves, mainly phospholipids and tri-
acylglycerols, are in the form of yolk globules, and alterations in
lipid metabolism may affect the yolk sac (Wiegand, 1996). Previous
studies have reported yolk sac abnormities occurred along with
disruptions in lipid metabolism in zebrafish embryos after expo-
sure to triazole fungicides (Mu et al., 2013; Hermsen et al.,
2011).39,52 Thus, the disruption of lipid metabolism is a possible
pathway that could explain the yolk sac abnormities observed in
this study.
Both in vivo and in vitro assays have demonstrated that
phthalates can induce immune response (Kimber and Dearman,
2010). In human tests, in utero exposure to DEHP induced the
expression of immune response markers and increased the serum
TNF level in postnatal day 60 male offspring (Campioli et al., 2014).
Xu et al. (2013) reported that the transcription of innate immune-
related genes including IL-1b, TNFa, CC-chemokine, CXCL-clc, and
lysozyme were up-regulated in zebrafish upon exposure to DBP and
DEP. In the present study, an apparent immune response was
observed after phthalate exposure, including the up-regulation of
the expressions of immune proteins and their encoding genes.
Previous studies also reported that DEHP and DBP can significantly
affect fish immune systems through the inhibition of macrophage
formation and neutrophil bodies as well as the induction of il-1b,
IFNg, CCL20, and rag1 transcription (Huang et al., 2015; Xu et al.,
2015). It has been demonstrated that estrogens are involved in
immune processes, and estrogen-receptor proteins in fish immune
cells participate in the regulation of immune response (Milla et al.,
2011). Thus, exogenous estrogen may act on the fish immune sys-
tem by altering the level of ER protein (Casanova-Nakayama et al.,
2011). However, in this work, 50 mg/L DEHP and DBP induced sig-
nificant immune responses but had no effect on estrogen level, ERa
protein level, or ESR1 transcription. Based on the above results, the
immune system disruption caused by DEHP and DBP might not
originate from the alteration of estrogen receptors. Previous
research have found that immune response is related to the alter-
ation of lipid homeostasis (Im et al., 2011; Blanc et al., 2011).
Further, levels of multiple lipid species were altered by DEHP and
DBP in the present work. Thus we inferred that the disruption of
lipid levels is a possible pathway mediating the phthalates-induced
immune response.
The endocrine-disrupting effect has been the focus of consid-
erable ecotoxicity research on phthalate contaminants. Over the
past decade, a large number of studies have focused on the effects
of phthalates on fish hormone levels. Wang et al. (2013) examined
the effects of long-term DEHP exposure at environmentally rele-
vant concentrations on the endocrine systems of adult rare min-
nows (Gobiocypris rarusrare). They found that the plasma 17b-
estradiol level was significantly increased in male fish and signifi-
cantly decreased in female fish after exposure to DEHP at concen-
trations of 39.4 mg/L or higher. Guo et al. (2015) reported that after
six months of DEHP exposure (from larvae to adult), the fecundity,
plasma 17-b estradiol level, and plasma testosterone level of female
rare minnows were significantly decreased, and Cyp17 and cyp19b
transcription were down-regulated in the ovaries. After 81 days of
exposure to low MEHP concentrations (0.46, 4.0, and 37.5 mg/L), the
681
level of plasma 17b-estradiol was significantly increased in both
male and female zebrafish (Zhu et al., 2016). In the present study,
the trends in hormone levels after phthalate exposure were
consistent with the above studies; however, the phthalate con-
centration (250 mg/L) required for hormone alteration was much
higher than reported in previous studies. This may be attributed to
the shorter duration of exposure in this study or differences in
phthalate sensitivity between different species/different develop-
mental stages. In addition, DEHP and DBP treatment resulted in up-
regulation of ESR1, which is different with the results of E2 groups
which showed no regulation towards ESR1. This indicated that the
action mode of DEHP and DBP in zebrafish embryos is not exactly
the same with E2. Further, T3 and T4 levels were significantly
elevated after exposure to 250 mg/L phthalates, which is in consis-
tent with a previous study in which 400 mg/L DEHP enhanced the
levels of T3 and T4 in zebrafish larvae (Jia et al., 2016).
Our research indicated that phthalates can induce a variety of
effects during zebrafish embryonic development, including
decreased body length, yolk sac abnormities, immune response,
estrogenic effects, and reduced lipid levels. The effective concen-
tration required to produce immune response, lipid homeostasis
and yolk sac development was lower than that required for estro-
genic effects and hormone disruption, indicating that these pro-
cesses might be more sensitive to phthalate exposure than
endocrine system. Furthermore, an estrogen receptor inhibition
test revealed that DBP-induced morphological symptoms are not
mediated by ERs. Taken together, the results identified a series of
developmental abnormities caused by phthalates that may not
originate from estrogenic effects.
Acknowledgements
This work was supported by the Central Public-interest Scien-
tific Institution Basal Research Fund, CAFS (No. 2016C008) and the
Central Public-interest Scientific Institution Basal Research Fund,
CAFS (NO. 2017HYZD0201).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.envpol.2018.05.095.
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