Di-(2-ethylhexyl) phthalate exposure impairs cortical development in hESC-derived cerebral organoids

Abstract

Di-(2-ethylhexyl) phthalate (DEHP), a ubiquitous environmental endocrine disruptor, is widely used in consumer products. Increasing evidence implies that DEHP influences the early development of the human brain. However, it lacks a suitable model to evaluate the neurotoxicity of DEHP. Using an established human cerebral organoid model, which reproduces the morphogenesis of the human cerebral cortex at the early stage, we demonstrated that DEHP exposure markedly suppressed cell proliferation and increased apoptosis, thus impairing the morphogenesis of the human cerebral cortex. It showed that DEHP exposure disrupted neurogenesis and neural progenitor migration, confirmed by scratch assay and cell migration assay in vitro. These effects might result from DEHP-induced dysplasia of the radial glia cells (RGs), the fibers of which provide the scaffolds for cell migration. RNA sequencing (RNA-seq) analysis of human cerebral organoids showed that DEHP-induced disorder in cell-extracellular matrix (ECM) interactions might play a pivotal role in the neurogenesis of human cerebral organoids. The present study provides direct evidence of neurodevelopmental toxicity of DEHP after prenatal exposure.

Full text

Di-(2-ethylhexyl) phthalate exposure impairs cortical development in
hESC-derived cerebral organoids
Ling Yang
a,b,c,d
,JiaoZou
a
, Zhenle Zang
a
, Liuyongwei Wang
a
,ZhulinDu
a
,DandanZhang
a
, Yun Cai
a
,
Minghui Li
c,d
,QiyouLi
c,d
, Junwei Gao
a,
⁎, Haiwei Xu
c,d,
⁎⁎, Xiaotang Fan
a,
⁎
a
Department of Military Cognitive Psychology, School of Psychology, Third Military Medical University (Army Medical University), Chongqing 40038,China
b
Department of Physiology, College of Basic Medical Sciences, Third Military Medical University (Army Medical University), Chongqing 400038, China
c
Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, China
d
Key Lab of Visual Damage and Regeneration &Restoration of Chongqing, Chongqing 400038, China
HIGHLIGHTS GRAPHICAL ABSTRACT
•DEHP has negative impacton the develop-
ment of human cortical organoids.
•DEHP exposure inhibits cell proliferation,
induces apoptosis,and disrupts cell migra-
tion in cortical organoids.
•A series of components of cell-ECM inter-
actions are significantly changed after
DEHP exposure.
ABSTRACTARTICLE INFO
Editor: Lidia Minguez Alarcon
Keywords:
DEHP
Organoids
Cortical development
Migration
Cell-ECM interactions
Di-(2-ethylhexyl) phthalate (DEHP), a ubiquitous environmental endocrine disruptor, is widely used in consumer
products. Increasing evidence implies that DEHP influences the early development of the human brain. However, it
lacks a suitable model to evaluate the neurotoxicity of DEHP. Using an established human cerebral organoid model,
which reproduces the morphogenesis of the human cerebral cortex at the early stage, we demonstrated that DEHP ex-
posure markedly suppressed cell proliferation and increased apoptosis, thus impairing the morphogenesis of the
human cerebral cortex. It showed that DEHP exposure disrupted neurogenesis and neural progenitor migration, con-
firmed by scratch assay and cell migration assay in vitro. These effects might result from DEHP-induced dysplasia of
the radial glia cells (RGs), the fibersof which provide the scaffolds for cell migration. RNA sequencing (RNA-seq) anal-
ysis of human cerebral organoids showed that DEHP-induced disorder in cell-extracellular matrix (ECM) interactions
might play a pivotal role in the neurogenesis of human cerebral organoids. The present study provides direct evidence
of the neurodevelopmental toxicity of DEHP after prenatal exposure.
Science of the Total Environment 865 (2023) 161251
⁎Corresponding authors at: Department of Military Cognitive Psychology, School of Psychology, Third Military Medical University (Army Medical University), Chongqing, China.
⁎⁎ Correspondence to: H. Xu, Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing 400038, China.
E-mail addresses: nutdgjw@163.com (J. Gao), haiweixu2001@163.com (H. Xu), fanxiaotang2005@163.com (X. Fan).
http://dx.doi.org/10.1016/j.scitotenv.2022.161251
Received 19 October 2022; Received in revised form 24 December 2022; Accepted 24 December 2022
Available online 30 December 2022
0048-9697/© 2022 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv

1. Introduction
Di-(2-ethylhexyl) phthalate (DEHP) is a common plasticizer widely
used in many consumer products, including toys, food wrapping, and med-
ical devices, to render them softer and moremalleable (Wang et al., 2019b).
Since DEHP is a lipophilic compound, it is not chemically bound to the
product matrix, resulting in DEHP migrating with the material and gradu-
ally being released into the air, dust, and water (Ahmadpour et al., 2022;
Safarpour et al., 2022). People of all age groups may be exposed to DEHP
in various ways, such as dermal contact, ingestion, inhalation, and medical
injection, thereby resulting in serious adverse effects in different organs and
tissues (Liu et al., 2022;Liu et al., 2023). Consistently, a large number of an-
imal experiments have provedthat DEHP exposure could cause the disorder
in the spleen (Dai et al., 2022), kidney (Li et al., 2021), heart (Cui et al.,
2022), duodenum (Yang et al., 2022), and testicular (Zhao et al., 2022b).
Using household cleaning products in pregnant women was reported to
be correlated with higher phthalate metabolites transferred through the
maternal-fetal-infant pathway (Arbuckle et al., 2016;Valvi et al., 2015). Re-
cently, more attention has been focused on the neurotoxicity of DEHP in
brain development. Clinical evidence revealed that phthalate metabolite
concentrations in the urine were negatively correlated with the cortical
thickness of children (Park et al., 2015). Animal studies have reported
that prenatal or early development exposure to DEHP could cause changes
in cerebral cortex structure and function, resulting in abnormal behaviors
(Nadeem et al., 2021;Zhang et al., 2021). However, the exact mechanism
of DEHP-induced malformations of cortical development is still unclear.
The human cerebral cortex is a brain structure with rapid progression
during evolution. A series of cellular events, including progenitor cell pro-
liferation, neurogenesis, and radial glia-guided neural migration, are essen-
tial for human cortical expansion, lamination, and forming a highly
organized and interconnected network of neurons and glia (Jabali et al.,
2022;Krefft et al., 2021). Disruption of these events may result in malfor-
mations of human cortical development. In animal models, an increasing
body of studies has suggested that endocrine-disrupting chemicals
(EDCs), such as bisphenol A (BPA), phthalates, and pesticides, exert toxic
influences on neurogenesis and lamination construction in the cerebral cor-
tex (Komada et al., 2016;Lee et al., 2021a). While these classical animal
models are criticalin teasing apart aspects of cortical development, itis piv-
otal to appreciate that these observations cannot simply be transferred to
humans. Certain aspects of the complexity observed in the human cortex,
such as gyrus formation and outer radial glial cells (oRGs), can only be ex-
plored in the human system (Molnár et al., 2019).
Compared with the two-dimensional (2D) model in vitro, the three-
dimensional (3D) cell culture system has emerged as a novel platform to
mimic human cortical development with a sufficient level of spatial-
temporal complexity in a microenvironment resembling native tissue
(Fagerlund et al., 2021;Kaluthantrige Don and Kalebic, 2022). Cortical
organoids are suitable for simulating the processes of neuroepithelium ex-
pansion, generation of RGs, and ventricular zone (VZ) organization
(Lancaster et al., 2013). Cortical organoids generated from human embry-
onic stem cells (hESCs) have previously been employed to evaluate the ef-
fect of environmental factors on human cortical development. In 3D
human forebrain cortical spheroids, size- and concentration-dependent
microplastic exposure adversely affected embryonic brain-like tissue devel-
opment (Hua et al., 2022). Acrylamide exposure was reported to repress
neural differentiation and induce cell apoptosis in cerebral organoids (Bu
et al., 2020). Cadmium exposure induced neuroinflammation and impaired
ciliogenesis in cerebral organoids (Huang et al., 2021). Our previous studies
suggested that neural organoids, such as retinal organoids, can be success-
fully applied to investigate and clarify the neurotoxic mechanisms of envi-
ronmental contaminants, including tiny particulate matter (PM 2.5),
polybrominated diphenyl ethers (PBDEs), and bisphenols (Li et al., 2022c;
Li et al., 2022d;Zeng et al., 2021). However, the developmental neurotox-
icity of DEHP has not been assessed in cortical organoids. Therefore, hESC-
derived cortical organoids should be suitable for elucidating the mecha-
nisms of DEHP-induced malformations during cortical development.
In the current study, we used hESC-derived cortical organoids estab-
lished in our laboratory to investigate the neurotoxicity and molecular
mechanisms of DEHP exposure involved in the developing cerebral cortex.
Cortical organoids were exposed to different concentrations of DEHP from
Days 20–34, when the characteristics of cortical organoids were similar
to those in the first trimester of humans gestation (Kelava and Lancaster,
2016;Qian et al., 2016;Zang et al., 2022). The central nervous system
(CNS) is very sensitive to harmful environmental factors during early
pregnancy (Hu et al., 2018). DEHP exposure-induced morphological
changes, cell proliferation, and apoptosis were well evaluated in
hESC-derived cortical organoids. Transcriptomics approaches were em-
ployed to clarify the underlying mechanisms of DEHP-induced neuro-
toxicity.Notably,disruptedcellmigrationwasvalidatedinboth3D
and 2D models after DEHP exposure.Finally,thedevelopmentofRGs
was assessed to explain the obstructed cell migration in cortical
organoids, thus providing more insights into the adverse effects of
DEHP exposure in developing brains.
2. Materials and methods
2.1. Chemicals
DEHP (No. 36735; Sigma-Aldrich, MO, USA) was prepared at a stock
concentration of 400 mM in dimethyl sulfoxide (DMSO, No. D2650;
Sigma-Aldrich, MO, USA). Five final working concentrations (10, 50, 100,
200, and 400 μM) were selected in our experiments.
2.2. hESC culture and cortical organoids differentiation
The H9 hESC line was kindly provided by Stem Cell Bank from the Chi-
nese Academy of Sciences. hESCswere maintained in Essential 8™medium
(E8, #A1517001, Gibco) without a feeder and cultured on recombinant
human vitronectin-N (rhVTN-N, #A14700, Gibco) -coated plates. When
confluency reached 80–90 %, the cells were rinsed with Dulbecco's phos-
phate buffer (DPBS) and detached with versene solution (#15040066,
Gibco) at 37 °C for 3–4 min. After the versene solution was removed, the
cells were rinsed with E8 medium and reseeded at a ratio of 1:6 in E8 me-
dium containing 10 μM rock inhibitor Y27632 (#1293823, Peprotech)
for the first 24 h. The cells were maintained with daily medium changes
and incubated in a 5 % CO
2
incubator at 37 °C.
The cortical organoids were generated according to previous studies
(Giandomenico et al., 2021;Lancaster and Knob lich, 2014) with some mod-
ifications. Briefly, hESCs were dissociated into single cells using TrypLE
(#12605010, Gibco) at 37 °C f or 3–4min when confluency reached approx-
imately 80 %. On Day 0, about 9000 cells/well were seeded into ultralow-
attachment 96-well U-bottom plates (#CLS7007, Corning) with hESC me-
dium containing 80 % DME/F12 (#11330032, Gibco), 20 % KnockOut
Serum Replacement (KOSR) (#3181502, Gibco), 3 % ESC-quality FBS
(#30044333, Gibco), 1 % Glutamax (#35050061, Gibco), 1 % MEM-
NEAA (#11140050, Gibco), and 0.0007 % 2-mercaptoethanol
(#8057400250, Merck) for embryonic body (EB) formation. Y27632 (50
μM) and basic fibroblast growth factor (bFGF) (#100-18b, Peprotech) (4
ng/mL) were added to the medium for the first four days. The medium
was changed every two days. On Day 6, the EBs were transferred to low-
attachment 24-well plates (#CLS3473, Corning) with neural induction me-
dium supplemented with 97 % DME/F12, 1 % Glutamax, 1 % MEM-NEAA,
1 % N-2 supplement (#17502048, Gibco), 1 μg/mL heparin (#3149,
Sigma), and 5 μM smoothened receptor inhibitor cyclopamine (CycA,
#239803-5 mg, Millipore). The medium was changed every other day
and radiated neuroectoderm was formed during this period. On Day 12,
EBs were embedded into Matrigel droplets (#354234, Corning) and trans-
ferred to low-attachment 6-well plates (#CLS3471, Corning) with a differ-
entiation medium. The differentiation medium consisted of 50 % DME/
F12, 50 % Neurobasal medium, 1 % penicillin–streptomycin, 1 %
Glutamax, 0.5 % N-2 supplement, 0.5 % MEM-NEAA, 0.025 % insulin,
0.00035 % 2-mercaptoethanol, and 1 % B27 (from Days 12 to 16, B27
L. Yang et al. Science of the Total Environment 865 (2023) 161251
2

without vitamin A (#12587010, Gibco) was added, and from Day 16 on,
B27 with vitamin A (#17504044, Gibco) used). On Day 16, the organoids
were transferred to an orbital shaker (IKA, Germany) at a speed of 85
rpm. On Day 20, embedded organoids were mechanically dissociated
from Matrigel by syringe needles. The differentiation medium was changed
twice a week (Fig. 1A).
Fig. 1. Generation and identification of hESC-derived cortical organoids.
A: Schematic of induction andDEHP exposure protocols of cortical organoids and the sequential morphological changes of cortical organoids at differenttime points acquired
by phase-contrast microscope (Scalebars = 200 μm); B: Immunofluorescence staining of the H9 hESCline for OCT4, SOX2, Ki67, P-Vimentin,and DAPI (Scale bar = 20 μm);
C: Immunofluorescence staining of cortical organoids at Day 34 for SOX2, DCX, PAX6, MAP2B, P-Vimentin, Ki67, TUNEL, TBR2, CTIP2, NeuN, and DAPI (Scale bar = 50
μm).
L. Yang et al. Science of the Total Environment 865 (2023) 161251
3

2.3. DEHP exposure experiments
Studies have shown that the maximum dose of DEHP exposure could
reach 1000 μM through medical exposure (Inoue et al., 2005;Loff et al.,
2000). Therefore, on Day 20 of differentiation, the cortical organoids
were exposed to DEHP at concentrations of 0 (DMSO, negative control),
10, 50, 100, 200, and 400 μM. On Day 34, cortical spheroids were collected
for further analysis. The morphology and growth of cortical organoids were
determined by photography at each observation time using an Axio Ob-
server A2 (Zeiss, Germany). The 2D surface area and neuroepithelial bud
perimeter were assessed on Days 20, 27, and 34 by ZEN 2.6 software
(Zeiss, Germany).
2.4. Immunofluorescence staining
The cortical organoids were collected and fixed in 4 % PFA overnight at
4 °C. Then theywere washedtwice with PBSand dehydrated with 30 % su-
crose at 4 °C for 48 h. Subsequently, the organoids were embedded in opti-
mal cutting temperature (OCT) com pound (#4583, Sakura), snap-frozen on
dry ice, and stored at −80 °C. For immunofluorescence staining, 15 μm-
thick sections were cut with a cryostat (#819, Leica) and mounted onto
slides. Thecryosections were washed with PBS to remove excess OCT com-
pound and incubated in a blocking solution (0.3 % Triton with 5 % BSA) at
room temperature for 30 mins. The sections were then incubated in pri-
mary antibodies diluted with antibody diluent (#ZLI-9029, ZSGB-BIO) at
4 °C overnight. After washing three times with PBS, the corresponding
fluorophore-conjugated secondary antibodies diluted in PBS were added
and incubated at 37 °C for 2 h. The nuclei were counterstained with DAPI
(#D9564, Sigma). Following washing three times with PBS, the glass slides
were mounted using mounting medium (#SC-24941, Santa Cruz), ob-
served, and analyzed with an inverted fluorescence microscope equipped
with a Zeiss AxioCam digital color camera connected to the AxioVision
SE64 Rel. 4.9 software (Zeiss, Germany). The information of antibodies
used here is presented in Supplementary Table 1.
2.5. TUNEL assays
For labeling apoptotic cells, terminal deoxynucleotidyl transferase
(TdT)-mediated dUTP-X nick-end labeling (TUNEL) staining was per-
formed using the One-step in situ Cell Death Detection Kit (#KGA7073,
Keygen Biotech) according to the manufacturer's instructions. In brief,
after permeabilization with 0.3 % Triton, the cryosections were incubated
with a mixture of 45 μL equilibration buffer, 1 μL biotin-11-dUTP, and 4
μL TdT Enzyme at 37 °C for 30 min. After washing three times with PBS
(5 min each), a mixture of 5 μL streptavidin-fluorescein and 45 μL labeling
buffer was added and incubated at 37 °C for 30 min. The nuclei were coun-
terstained with DAPI. Following three-time washing, the sections were
mounted with a mounting medium and photographed with a fluorescence
microscope system (Zeiss, Germany).
2.6. Flow cytometry assays
The flow cytometry was conducted to assess neuronal viability on a BD
FACSCanto™II (Becton Dickinson). Briefly,after DEHP exposure, the spher-
oids were collected, cut into small pieces, and dissociated into single cells
with Accutase (#00–4555-56, Thermo fisher) at 37 °C for 5–10 min. For
Ki67 (a cellular marker for proliferation) staining, cells were fixed and
permeabilized with FOXP3 fixation buffer (#00–5523-00, eBioscience,
San Diego, CA, USA) according to the manufacturer's instructions. Fixed
cells were stained with 2.5 μL of Ki67-BV650 antibody (#151215,
Biolegend) in the dark for 20 min at room temperature and then washed
with perm/wash buffer (#554723, BD Bioscience). For apoptosis staining,
cells were stained with 200 μL of Annexin V Binding Buffer containing
2.5 μL of Annexin V-APC and 5 μL of 7-AAD antibodies (#640930,
BioLegend). After incubation in the dark for 60 min at 4 °C, the cells were
analyzed directly by flow cytometry without washing. Data were analyzed
using FlowJo software.
2.7. Cell migration assay
Cell migration assay was performed as previously described (Huo et al.,
2018). Briefly, cortical organoids from Days 27 to 34 were triturated into
small tissues by a Pasteur pipette. Neurospheres of similar sizes were plated
on Matrigel-coated coverslips in a neural differentiation medium. When the
neurospheres were attached, neural differentiation medium with or with-
out DEHP was used. After 72 h, neurons that migrated out from
neurospheres were assessed using bright-field microscopy with an Axio Ob-
server A2 (Zeiss, Germany) and immunofluorescence staining. The migra-
tion distance of neurons was evaluated by manual measurement of four
radii of the migration area and was defined as each neurosphere at perpen-
dicular angles from the edge of the neurosphere to the furthest migrated
MAP2B
+
neurons (a marker of the neuronal axon). The migration number
was the absolute cell count in the migration areas (Baumann et al., 2015).
2.8. Scratch assay
For the scratch assay (Huo et al., 2018), cortical spheres were dissoci-
ated into single cells by Accutase at 37 °C for 5 min, and then the cells
were collected and plated onto Matrigel-coated coverslips at a density of
5×10
5
/slip. When the cells adhered, the cultures were scratched using a
200 μL pipette tip. After two times wash, the neural differentiation medium
with or without DEHP was added. After 72 h, the neurons were fixed and
stained for migration analysis. The TUJ1
+
cells (a marker of neuronal den-
drite) in the scratched area were counted.
2.9. RNA sequencing
Cortical organoids were microdissected for RNA-sequencing (RNA-seq)
to evaluate the toxic effects of DEHP exposure on cortical development.
RNA-seq was conducted by Suzhou Transcriptome Biotechnology (Suzhou,
China). The purity, concentration, and integrity of RNA extractedfrom cor-
tical organoids in different groups were assessed. Sequencing libraries were
generated based on the extracted RNA. The expression of each gene was
measuredby Fragments Per Kilobase of exon per Million fragmentsmapped
(FPKM). Differentially expressed genes (DEGs) were identified according to
genes with an absolute fold-change (FC) >2 and false discovery rate (FDR)
<0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Ge-
nomes (KEGG) pathways were enriched to explore the functional annota-
tion of DEGs using the OmicShare platform (www.omicshare.com/tools).
A Protein-protein interaction (PPI) network was generated according to
the list of DEGs mapped to the STRING online database (confidence score
cutoff >700) using NetworkAnalyst web. Cytoscape (v3.8.2) was used for
network visualization.
Gene set enrichment analysis (GSEA) was conducted to fully utilize the
transcriptome expression data to filter and enrich biological processes sig-
nificantly affected by DEHP. A series of gene sets with concordant differ-
ences from the expression matrix of all genes were identified according to
the input gene sets in GSEA and used for further analysis (Subramanian
et al., 2005). The input data for the expression matrix in GSEA consist of
all genes that could be annotated to the KEGG gene database. The signifi-
cant pathways with P<0.05 and false discovery rate (FDR) <0.25 were
screened based on the KEGG database.
All human cerebral organoid RNA-seq dataare available in the NCBI Se-
quence Read Archive (SRA) through BioProject ID PRJNA885882.
2.10. Real-time polymerase chain reaction (RT–PCR)
RT–PCR was conducted according to our previously described method
(Zou et al., 2022). In brief, total RNA was extracted from cortical organoids
using TRIzol reagent (#15596026, Thermo Fisher), and reverse transcrip-
tion PCR was carried out using the PrimeScript RT Reagent Kit with
L. Yang et al. Science of the Total Environment 865 (2023) 161251
4

gDNA eraser according to the manufacturer's protocols (#RR047A,
Takara). The RT–PCR was performed with specific primers in the CFX con-
nect real-time PCR detection system (Bio-Rad, US) using SYBR® Premix Ex
Taq II (#RR820A, Takara, Japan). The program follows predegeneration at
95 °C for 2 min, (denaturation at 95 °C for 5 s, followed by annealing/exten-
sion step at 60 °C for 30 s) for 40 cycles. All primers used are listed in Sup-
plementary Table 2, and GAPDH was used as an internal control. The data
were analyzed using the 2
-△△Ct
method.
2.11. Statistical analysis
All data are presented as the mean ± standard error of the mean (SEM).
All results were obtained from at least three independent experiments in
this study. All data were analyzed using the SPSS 24.0 software (IBM Cor-
poration, Armonk, NY, USA). One-way analysis of variance (ANOVA)
followed by Tukey's multiple comparisons was employed to evaluate the
differences among different groups. A Pvalue <0.05 was considered statis-
tically different in the present study.
3. Results
3.1. Generation and identification of hESC-derived cortical organoids
hESC-derived cortical organoids were generated according to a previous
protocol (Giandomenico et al., 2021;Lancaster and Knoblich, 2014), of
which morphological alterations were photographed at different time
points (Days 0, 6, 12, 16, 20, and 34) (Fig. 1A). Neural rosettesappeared ob-
viously at approximately Day 20. To further verify whether this 3D model is
feasible, a series of markers were stained in the cortical organoid at differ-
ent stages. The stem cell markers, octamer-binding transcription factor 4
(OCT4) and SRY-box transcription factor 2 (SOX2), were stained in the
H9 hESC line. The proliferation of hESCs was analyzed using Ki67 and P-
Vimentin staining (Fig. 1B). At Day 34, different neural markers (DCX,
MAP2B, and PAX6), proliferation markers (Ki67 and P-Vimentin), apopto-
sis marker (TUNEL), and several cortical layer markers (TBR2, CTIP2, and
NeuN) were detected in cortical organoids (Fig. 1C), which validated the
feasibility and rationality of our 3D model.
3.2. Effects of DEHP exposure on the morphogenesis of hESC-derived cortical
organoids
Compared with the control group, the 2D surface area of cortical
organoids was decreased after various dosages of DEHP exposure (50 μM
VS. control, P= 0.073; 100 μMVS.control,P<0.01; 200 μMVS.control,
P<0.001; and 400 μM VS. control, P<0.001). However, the 2D surface
area of cortical organoids was not affected after 10 μM DEHP exposure. No-
tably, the 2D surface area of cortical organoids was significantly decreased
in the 200 μM(P<0.05) and 400 μM(P<0.05) DEHP exposure groups, in-
stead of the 100 μM group, when compared with the 50 μM group (Fig. 2A
and B). Thus, three DEHP doses (0, 50, and 200 μM) were selected for fur-
ther experiments. The 2D surface area was also evaluated at different time
points after DEHP exposure. At Day 20, there was no significant difference
in the size of cortical organoids before DEHP exposure. After DEHP expo-
sure for one week, there was no significant difference in the size of cortical
organoids among the different groups. However, the size of the organoids
was significantly decreased in the 200 μMDEHPgroup(P<0.01), instead
of the 50 μMgroup(P= 0.071), compared with the control after DEHP ex-
posure for two weeks (Fig. 2C). Similarly, the visible perimeter of individ-
ual neuroepithelial buds was statistically decreased in the 50 μM(P<
0.01) and 200 μM(P<0.001) DEHP exposure groups compared with the
control at Day 34, instead of Day 20 or Day 27 (Fig. 2D and E). These results
verified the feasibility of the DEHP exposure protocol in hESC-derived cor-
tical organoids. On closer examination, the thickness of the VZ/
subventricular zone (SVZ) of neural rosettes was significantly decreased
in the 200 μM DEHP exposure group (P<0.01), instead of the 50 μM
group (P= 0.087), compared with the control (Fig. 2FandG).
3.3. Effects of DEHP exposure on cell proliferation andapoptosis in hESC-derived
cortical organoids
Subsequently, to determine the causes affecting organoid size, we fur-
ther analyzed cell proliferation and apoptosis at the levels of whole cortical
organoids and neural rosettes, respectively. The ratio of Ki67
+
cells of the
whole organoid was significantly decreased in cortical organoids in the
50 μM(P<0.05) and 200 μM(P<0.01) groups compared with the control
group as determined by flow cytometry analysis (Fig. 3A and B). Similarly,
the ratio of Ki67
+
proliferating cells in the VZ/SVZ areas was markedly re-
duced in the 50 μM(P<0.001) and 200 μM(P<0.001) groups compared
with the control group (Fig. 3C and D). In addition, the viable cells were de-
creased in the whole organoids exposed to 200 μMDEHP(P<0.05), indi-
cating an increase in dead cells (Fig. 3E and F). At the substructural level,
TUNEL staining revealed that there were more apoptotic cells in the VZ/
SVZ areas of organoids in the 200 μM DEHP group (P<0.001), no signifi-
cant difference was found in the 50 μMgroup(P= 0.082), w hen compared
with the control group (Fig. 3G and H). These data demonstrated that DEHP
inhibited neural progenitor cells (NPCs) proliferation and induced apopto-
sis in cortical organoids.
3.4. Effects of DEHP exposure on transcriptome profiling in cerebral organoids
RNA-seq analysis was applied to detect transcriptome-wide alterations
and uncover the hazardous effects and molecular mechanisms of DEHP ex-
posure in cortical organoids. DEGs were identified with an absolute fold-
change (FC) >2 and a false discovery rate (FDR) <0.05. The volcano
plots showed that there were 590 (558 upregulated genes and 32 downreg-
ulated genes) and 762 (700 upregulated genes and 62 downregulated
genes) DEGsidentified in the 50 μM and 200 μM DEHP exposure groups, re-
spectively (Fig. 4A and B, Supplementary Fig. 1). The Venn plot showed
that 335 DEGs coexisted in both sets (approximately 40–60 % of DEGs
per group) (Fig. 4C). A heatmap plot was generated based on 335 DEGs
(Fig. 4D), and a detailed description of DEGs was in Supplementary
Table 3. Next, KEGG and GO enrichment analyses were carried out based
on 335 DEGs to obtain further insights into the mechanism of DEHP expo-
sure in the development of cortical organoids. GO analysis suggested signif-
icant enrichment of biological processes related to development and
morphogenesis, such as tube development and tube morphogenesis
(Fig. 4F). In the cellular component category, significant gene enrichment
was associated with ECM components, such as ECM, cell–cell junction,
and extracellular region (Fig. 4G). Following GO enrichment results, some
significant enrichments in the top 5 KEGG pathways were also correlated
with cell-ECM interactions, such as ECM-receptor interaction and focal ad-
hesion (Fig. 4E). These results indicated that the alteration of cell-ECM in-
teractions may play crucial roles in mediating the neurotoxicity of DEHP
in hESC-derived cortical organoids.
To further verify the reliability of the results based on DEGs enrichment,
we also performed GSEA using the transcriptome expression data. We
screened out 211 and 155 significant KEGG pathways in the 50 μM and
200 μM DEHP exposure groups compared with the control based on
GSEA, respectively. One hundred and thirty-five KEGG pathways coexisted
in these two sets (Fig. 5A and Supplementary Table 4). Of the 135 path-
ways, twenty-two were present in the previous KEGG enrichment based
on DEGs and categorized into four terms: cellular processes, environmental
information processing, organismal systems, and human diseases (Fig. 5B
and C). The normalized enrichment score (NES) of all gene sets that met
the condition was >0 in all DEHP exposure groups compared with the con-
trol group, suggesting that DEHP exposure may facilitate the expression of
genes related to these biological processes of hESC-derived cortical
organoids. Compared with the control group, the expression of genes asso-
ciated with ECM-receptor interaction was significantly upregulated in the
50 μM and 200 μM DEHP exposure groups, with NES values of 2.2133
and 2.1474, respectively (Fig. 5D and F). The expression of genes related
to focal adhesion was significantly upregulated in the 50 μM and 200 μM
DEHP exposure groups, with NES values of 1.8309 and 1.9113, respectively
L. Yang et al. Science of the Total Environment 865 (2023) 161251
5

Fig. 2. Effects of DEHP exposure on morphogenesis in hESC-derived cortical organoids.
A and D: Representative image of cortical organoids after DEHP exposure (Scale bar = 500 μm); B: Quantitative analysis of the 2D surface area of cortical organoids after
various doses of DEHP exposure (n=17forcontrol,n=17for10μM DEHP exposure group, n=17for50μM DEHP exposure group, n= 16 for 100 μM DEHP
exposure group, n= 15 for 200 μM DEHP exposure group, n= 15 for 400 μM DEHP exposure group); C: Quant itative analysis of the 2D surface area of cortical
organoids at Days 20, 27, and 34 (n= 16 organoids for each group); E: Quantitative analysis of the visible perimeter of individual neuroepithelial buds at Days 20, 27,
and 34 (n= 16 organoids for each group); F: Representative immunostaining image of VZ/SVZ of cortical organoids identified by immunofluorescence staining for SOX2
and DCX (Scale bar = 50 μm); G: Quantitative analysis of VZ/SVZ thickness of cortical organoids (n= 8 organoids for each group with 2–3 neural rosettes analyzed per
organoid). Data are presented as mean ± SEM. *P<0.05, **P<0.01, and ***P<0.001.
Fig. 3. Evaluation of cell proliferation and apoptosis in hESC-derived cerebral organoids after DEHP exposure.
A: Representative pseudocolor graph of proliferating cells in cortical organoids; B: Quantitative analysis of proliferating cells in cortical organoids determined by flow
cytometry (Ki67)(n= 4 organoids for each group); C: Representative immunostaining image of Ki67
+
proliferating cells in VZ/SVZ of cortical organoids after DEHP
exposure (Scale bar = 50 μm); D: Quantitative analysis of the ratio of Ki67
+
proliferating cells in VZ/SVZ of cortical organoids (n= 8 organoids for each group with 2–3
neural rosettes analyzed per organoid); E: Representative pseudocolor graph of viable cells in cortical organoids; F: Quantitative analysis of viable cells (Q4) in cortical
organoids determined by flow cytometry (7-AAD/Annexin V) (n= 4 organoids for each group); G: Representative immunostaining image of TUNEL apoptotic cells in
cortical organoids (Scale bar = 50 μm); H: Quantitative analysis of the ratio of TUNEL apoptotic cells in cortical organoids (n= 8 organoids for each group with 2–3
neural rosettes analyzed per organoid). Data are presented as mean ± SEM. *P<0.05, **P<0.01, and *** P<0.001.
L. Yang et al. Science of the Total Environment 865 (2023) 161251
6

L. Yang et al. Science of the Total Environment 865 (2023) 161251
7

(Fig. 5E and G). Simultaneously, the heatmaps were generated according to
the expression of genes related to ECM-receptor interaction and focal adhe-
sion in different groups (Fig. 5D-G). These results, in another sense, con-
firmed that the ECM-receptor interaction and focal adhesion pathways
played critical roles in the DEHP-induced neurotoxicity of hESC-derived
cortical organoids.
To explore how altered cell-ECM interactions affected the development
of cortical organoids after DEHP exposure, we then analyzed the hub genes
based on DEGs. The 335 DEGs modified by DEHP exposure were clustered
by the ClueGO plug-in of Cytoscape software into functional groups related
to KEGG pathways, resulting in16 terms, and ECM-receptor interaction and
focal adhesion pathways served crucial roles within the network (Supple-
mentary Fig. 2). Some common genes, such as fibronectin 1(FN1), alpha8
integrin (ITGA8), and ITGA11, are included in these two pathways. A
heatmap was drawn based on the expression of some critical genes, such
as ECM-receptor interaction and focal adhesion (Fig. 6A). To confirm the
Fig. 4. Transcriptome profile of hESC-derived cerebral organoids after DEHP exposure.
A: Volcano plot of altered gene expression patterns in hESC-derived cortical organoids after 50 μM DEHP exposure; B: Volcano plot of altered gene expression patterns in
hESC-derived cortical organoids after 200 μM DEHP exposure; C: Venn diagram of co-regulated DEGs in both 50 μM DEHP group and 200 μM DEHP group compared
with control; D: Hierarchical clustering heatmap for DEGs from 50 μM, 200 μM DEHP exposure, and control groups; E: Enriched significant 28 KEGG pathways. The
ECM-receptor interaction and focal adhesion pathways were significantly changed and highlighted in red fonts; F: Enriched top 10 GO terms in the biological processes
category; G: Enriched top 10 GO terms in the cellular component category.
L. Yang et al. Science of the Total Environment 865 (2023) 161251
8

RNA-seq reliability, the expression of key DEGs was determined by RT–
PCR. The levels of FN1,ITGA8,ITGA11, kinase insert domain receptor
(KDR), fraser syndrome 1 (FRAS1), fras1-related extracellular matrix 1
(FREM1), and FREM2 were significantly upregulated after DEHP exposure
(Fig. 6B-E and G-I).
Next, the 335 DEGs modified by DEHP exposure were analyzed using
the STRING online database to create an interaction network. The top 5
hub genes identified by the edge percolated component (EPC) method in
the cytoHubba plug-in of Cytoscape software were FN1,KDR, endothelial-
specificreceptortyrosinekinaseTEK, proto-oncogene tyrosine-protein ki-
nase MET,andfibroblast growth factor 19 (FGF19) (Supplementary
Table 5). The network and first expanded PPI subnetwork were presented
in Fig. 6J. One gene part was related to tube development, and others
were linked to ECM-receptor interactions and focal adhesion, such as
FN1,KDR,MET,ITGA8,andITGA11, mediating cell-ECM interactions.
We noted that the genes involved in cell migration were found in the two
parts, suggesting that cell migration may bedisturbed in the developmental
cortical organoids after DEHP exposure.
Fig. 5. Gene set enrichment analysis of hESC-derived cerebral organoids after DEHP exposure.
A: Venn diagram of co-regulated KEGG pathways with significance in the 50 μMand200μM DEHP groups compared with control; B: Venn diagram of co-regulated KEGG
pathways with significance both in GSEA and DEGs enrichments; C: The 22 co-regulated enrichment pathways of hESC-derived cerebral organoids after DEHP exposure; D
and F: Representative gene sets using GSEA (the genes on the left side of the zero cross shown in the plots were up-regulated genes), and hierarchical clustering heatmap for
ECM-receptor interaction pathway after different doses DEHPexposure; E and G: Representative genesets using GSEA (the geneson the left side of the zero cro ss shown in the
plots were up-regulated genes), and hierarchical clustering heatmap for focal adhesion pathway after different doses DEHP exposure.
L. Yang et al. Science of the Total Environment 865 (2023) 161251
9

3.5. Effects of DEHP exposure on cell migration in hESC-derived cortical
organoids
To examine the effects of DEHP exposure on cell migration in cortical
organoids, SOX2 for NPCs, TBR2 for intermediate progenitor cells (IPCs),
and CTIP2 for deep-layer cortical neurons were stained and quantitated
using bin analysis according to a previous study (Koizumi et al., 2006).
The neural tube was divided into five equally spaced divisions. The ratios
of SOX2
+
cells, TBR2
+
cells, and CTIP2
+
cells in each bin were analyzed.
There was nosignificant difference in the ratioof SOX2
+
cells in each binof
cortical organoids after DEHP exposure (Fig. 7A and B). In contrast, the per-
centage of TBR2
+
cells was significantly increased in bin 1 (P<0.01) and
bin 2 (P<0.05) of cortical organoids in the 200 μMDEHPexposuregroup
compared with the control group, and the ratio of TBR2
+
cells was
decreased in bin 5 of cortical organoids in the 200 μM DEHP exposure
group (P= 0.054) (Fig. 7A and C). In addition, the ratio of CTIP2
+
cells
was significantly increased in bin 1, bin 2, and bin 3 of cortical organoids
in the 200 μM DEHP exposure group (P<0.05, P<0.01, and P<0.05, re-
spectively) compared with the control group. In bin 5 of cortical organoids,
the ratio of CTIP2
+
cells was markedly decreased in the 50 μM(P<0.05)
and 200 μMDEHPexposure(P<0.01) groups compared with the control
group (Fig. 7D and E). These results suggested that DEHP exposure may af-
fect the distribution of IPCs and deep-layer cortical neurons in hESC-
derived cortical organoids.
To further validate the effects of DEHP exposure on cell migration, ad-
herent neurospheres from cortical organoids were treated with DEHP for
three days and analyzed with MAP2B staining. Fewer neurons migrated
from the 200 μM and 50 μM DEHP exposure neurospheres than the control
Fig. 6. Analysis of key genes of hESC-derived cerebral organoids exposed to DEHP.
A: Heatmap for DEGs correlated with ECM-receptor interaction and focal adhesion; B-I: Quantitative analysis of RT-PCR of correlated DEGs in cortical organoids (n=4
organoids for each group); J: The network and first expanded network of top 5 hub genes based on the STRING (11.5) database and the Cytohubba plug-in of Cytoscape
software (Green indicates that DEGs are associated with cell migration, yellow indicates that DEGs are associated with tube development, red indicates that DEGs take
part in ECM-receptor interaction, and blue indicates that DEGs take part in focal adhesion). Data are presented as mean ± SEM. *P<0.05 and **P<0.01.
L. Yang et al. Science of the Total Environment 865 (2023) 161251
10

neurospheres (Fig. 8A). Quantitatively, the distance of cells migrated from
neurospheres was markedly reduced in the 50 μM(P<0.05) and 200 μM(P
<0.01) DEHP exposure groups (Fig. 8B). To confirm the altered migration,
we analyzed cell migration by MAP2B staining and counting. Consistently,
the migrated MAP2B
+
cells were statistically reduced in the 50 μMDEHP
(P<0.01) and 200 μM DEHP exposure (P<0.001) groups compared
with the control group (Fig. 8C and D). In addition, we conducted a scratch
assay to evaluate cell migration abnormalities after DEHP exposure. Neu-
rons from cortical organoids were treated with DEHP for three days and
counted by TUJ1 staining. We found that numerous TUJ1
+
neurons were
distributed in the scratched area in the control group, but few appeared
in the 50 μM(P<0.05) or 200 μM DEHP exposure (P<0.01) groups
(Fig. 8E and F). These findings suggested that DEHP exposureinhibited cor-
tical neuron migration in 2D cell culture models.
3.6. Effects of DEHP exposure on RGs in hESC-derived cortical organoids
RGs play crucial roles in guiding cell migration in the development of
the brain. To verify whether retarded neural migration resulted from ven-
tricular RGs (vRGs), we conducted immunofluorescence staining for Nestin
and analyzed the results according to a previous study (Klaus et al., 2019).
The tortuosity of vRG fibers was significantly increased in the 50 μMand
200 μM DEHP exposure groups (both P<0.001) compared with the control
group (Fig. 9A and B). In addition, the fiber length of Nestin
+
vRGs was
markedly decreased in the 200 μM DEHP exposure group (P<0.001) com-
pared with the control group (Fig. 9A and C). DEHP exposure disrupted the
processes and caused the fibers with a twisted morphology, whereas the
aligned and straight fibers appeared in the controls. These results suggested
that DEHP could destroy the development of vRGs and cause the abnormal
morphology of vRGs, which may contribute to the inhibition of cell migra-
tion.
4. Discussion
DEHP, the most widely used phthalate ester, is reported to account for
50 % of global phthalate plasticizers (Li et al., 2022a). The toxic effects of
DEHP have been assessed in the digestive system, reproductive system, en-
docrine system, motor system, and immune system (Adam et al., 2022;Li
et al., 2022b;Li et al., 2020a;Mínguez-Alarcón et al., 2021;Mínguez-
Alarcón et al., 2022a;Mínguez-Alarcón et al., 2022b;Yang et al., 2022;
Fig. 7. Evaluation of neural distribution in hESC-derived cerebral organoids after DEHP exposure.
A: Representative immunostaining image of TBR2
+
intermediate progenitors in cortical organoids after DEHP exposure (Scale bars = 50 μm); B: Quantitative analysis of
SOX2
+
NPCs in cortical organoids using bin analysis (n= 8 organoids for each group with 2–3 neural rosettes analyzed per organoid); C: Quantitative analysis of TBR2
+
intermediate progenitors in cortical organoids using bin analysis (n= 8 organoids for each group with 2–3 neural rosettes analyzed per organoid); D: Representative
immunostaining image of CTIP2
+
deep-layer cortical neurons in cortical organoids after DEHP exposure (Scale bars = 50 μm); E: Quantitative analysis of CTIP2
+
intermediate progenitors in cortical organoids using bin analysis (n= 8 organoids for each group with 2–3 neural rosettes analyzed per organoid). Data are presented as
mean ± SEM. *P<0.05 and **P<0.01.
L. Yang et al. Science of the Total Environment 865 (2023) 161251
11

Zhao et al., 2022b). Recently, an increasing number of animal experiments
have revealed that perinatal exposure to DEHP in the early developmental
stage could impair neural development and result in abnormal behaviors
(Lv et al., 2022;Yirun et al., 2021). Moreover, DEHP could lead to neuro-
toxicity in different types of nerve cells (Huang et al., 2022;Zhao et al.,
2022c). The results of in vivo and in vitro studies showed that DEHP
could induce apoptosis, oxidative damage, proliferation inhibition, mitoph-
agy, and mitochondrial damage (Lee et al., 2019;Sims et al., 2014;Zhang
et al., 2022;Zhao et al., 2022a). However, these models face difficulty in
simulating human brain development due to the species barrier or lack of
spatiotemporal complexity. Therefore, 3D cell cultures have emerged to
overcome the shortcomings of these models in recent years. In the present
study, DEHP exposure inhibited cell proliferation and enhanced cell apo-
ptosis in cortical organoids. In addition, the altered transcriptome profile
suggested that DEHP exposure resulted in abnormal cell-ECM interactions,
which could delay cell migration on gene expression and signaling path-
ways at the molecular level. Therefore, the ability of cell migration was
assessed using migration and scratch assay. We found that DEHP exposure
interfered with the development of RGs, and disturbed cell migration,
which may be mediated by the alteration of cell-ECM interaction-
associated molecules. To our knowledge, this is the first study investigating
the toxic effects and molecular mechanisms of DEHP in cortical organoids.
The H9 hESC line was identified using the staining of the stem cell
markers, OCT4 and SOX2. Immunofluorescence staining identified the
characteristics of cerebral organoids. Various subtypes of nerve cells, such
as neural stem cells, NPCs, and cortical neurons were observed in the differ-
ent layers on Day 34. Critical cellular events such as cell proliferation and
apoptosis appeared in the developing cortical organoids. These results sug-
gested that the cortical organoid model was successfully established and
could be used to investigate the effects of DEHPexposure on cortical devel-
opment.
The U.S. Agency for Toxic Substances and Disease Registry (ATSDR) es-
timates that the maximum daily exposure to DEHP for the general popula-
tion is about 2 mg/day. Aseries of studies have indicated that occupational
and medical exposures can reach much higher levels. Exposure to DEHP
from blood transfusions can be as high as 250–300 mg, equivalent to a
dose of 3.5–4.3 mg/kg for an adult weighing 70 kg (Lovekamp-Swan and
Davis, 2003). During pregnancy and delivery, both mother and fetus may
Fig. 8. Effects of DEHP exposure on cell migration from neurospheres.
A: Representative images of cell migration from neurospheres afterDEHP exposure for three days (Scale bar = 100 μm); B: Quantitative analysis of cellmigration distance in
control, 50 μM, and 200 μM DEHP exposure group s (n= 8 neurospheres analyzed for each group); C: Representative immunostaining image of MAP2B
+
neurons migration
from neurospheres after DEHP exposure (Scale bar = 100 μm); D: Quantitative analysis of the number of MAP2B
+
neurons migrated from neurospheres in control, 50 μM,
and 200 μM DEHP exposure groups (n= 8 neurospheres analyzed for each group); E: Representative images of the scratch assay in control, 50 μM, and 200 μM DEHP
exposure groups after scratching and DEHP exposure (Scale bar = 200 μm); F: Quantitative analysis of the TUJ1
+
neurons presented in the scratched area in control, 50
μM, and 200 μM DEHP exposure groups after scratching and DEHP exposure (n= 9 scratched area analyzed for each group). Data are presented as mean ± SEM. *P<
0.05, **P<0.01, and ***P<0.001.
L. Yang et al. Science of the Total Environment 865 (2023) 161251
12

be exposed to DEHP through medical devices (Marsee et al., 2006). The
phthalate levels from blood bags showed that the levels of DEHP and
MEHP ranged from 4.6 to 213 μM and from 0.36 to 34.8 μM, respectively
(Inoue et al., 2005). Polyvinylchloride (PVC) infusion lines also resulted
in DEHP diffusion, the maximum doses of DEHP exposure in packed red
blood cells, platelet-rich plasma, and fresh frozen plasma were about 77.8
μM, 118.8 μM, and 1038 μM, respectively (Loff et al., 2000). According to
these data, the dose of DEHP exposed to people fluctuates on a large
scale. The DEHP doses used in our experiments were within the concentra-
tion range of DEHP exposure.
Morphological change is one of the visual indices used toassess the tox-
icity of harmful substances on cerebral development. A large body of evi-
dence has revealed that environmental pollutant exposure caused size
changes in organoids (Kim et al., 2022b;Li et al., 2022d). In our study, no
significant difference was found in the 2Dsurface area of cortical organoids
between the 10 μM exposure and control groups. Compared with the 50 μM
exposure group, the 2D surface area of cortical organoids was markedly re-
duced in the 200 μM and 400 μM DEHP exposure groups. In addition, we
found that the 2D surface area of cortical organoids was not altered signif-
icantly after different doses of DEHP exposure for one week. These results
suggested the neurotoxicity of DEHP was in a concentration- and time-
dependent manner, which was consistent with some previous studies (Lee
et al., 2019;Qiu et al., 2020). The cortical organoid development was seri-
ously delayed by DEHP exposure for two weeks. Consistently, the VZ/SVZ
thickness of neural rosettes became thinner in the DEHP exposure groups.
These abnormal morphologies of cortical organoids are usually related to
the cellular biological processes in terms of neurogenesis, cell apoptosis,
and neural migration. Neural proliferation inhibition and cell apoptosis
are common side effects of EDCs exposure, including DEHP, PBDEs, and
BPA, both in vitro and in vivo (Komada et al., 2016;Komada et al., 2020;
Li et al., 2022d;Sims et al., 2014;Yin et al., 2020). Neural proliferation
was significantly suppressed in the VZ/SVZ, and more apoptotic nerve
cells were found in the VZ/SVZ of cortical organoids after DEHP exposure,
which may contribute to the abnormal morphologies of cortical organoids.
Therefore, DEHP exposure may inhibit cell proliferation and apoptosis in
cortical organoids.
For exploring the hazardous effects and molecular mechanisms of DEHP
exposure in corticalorganoids,RNA-seq analysis was employed to examine
transcriptome-wide alterations. GO enrichment analysis showed that DEGs
were primarily related to tube development and morphogenesis in the bio-
logical process category, which provided some explanations for the mor-
phological changes in cortical organoids after DEHP exposure. In the
cellular component category, DEGs were significantly different in the clus-
ters in terms of extracellular components. Based on KEGG analysis of the
RNA-seq data, we noted that DEGs also gathered together in cell-ECM inter-
actions, such as ECM-receptor interactions and focal adhesion, which
ranked first and third, respectively. To avoid the interference of enrichment
only containing DEGs and preserve gene-gene correlations, GSEA was also
conducted based on the RNA-seq data (Subramanian et al., 2005). GSEA re-
sults suggested that two cell-ECM interaction correlated pathways, ECM-
receptor interaction (environmental information processing) and focal ad-
hesion (cellular processes), were upregulated in the cortical organoids
after DEHP exposure. These two pathways were also significantly enriched
according to the expression of DEGs in DEHP-induced hypospadias in rats
(Han et al., 2019). Similarly, ECM-receptor interaction and focal adhesion
were identified as significant pathways in our previous study evaluating
the neurotoxicity of PBDEs in retinal organoids (Li et al., 2022d). To further
clarify how altered ECM components affect cortical development, we con-
structed the hub genes and their first expanded network using the
STRING online database and the cytoHubba plug-in of Cytoscape software.
Fig. 9. Effects of DEHP exposure on the development of radial glia cells in hESC-derived cortical organoids.
A: Representative immunostaining image of Nestin
+
RG fibers in cortical organoids after DEHP exposure; B: Quantitative analysis of tortuosity of vRG fibers in cortical
organoids (n= 8 organoids for each group with 8 vRGs analyzed per organoid); C: Quantitative analysis of length of vRG fibers in cortical organoids (n= 8 organoids
for each group with 8 vRGs analyzed per organoid); Scale bars = 50 μm. Data are presented as mean ± SEM. ***P<0.001.
L. Yang et al. Science of the Total Environment 865 (2023) 161251
13

Some hub genes, such as FN1 and KDR, joined ECM-receptor interaction
and tube development sections together. Notably, most of the genes in
these two sections were correlated with cell migration, which implied
that DEHP exposure might disturb cell migration mediated by cell-ECM in-
teractions and affect cortical development.
Next, we assessed whether DEHP exposure disrupted cell migration
in cortical organoids. Notably, there was a significant difference in the
distribution of IPCs and deep-layer cortical neurons instead of hNPCs
after DEHP exposure. Similarly, we confirmed abnormal neuron migra-
tion caused by DEHP exposure in the 2D cytological experiments. In an
ICR mouse model, it has been reported that prenatal DEHP exposure
could result in an abnormal neuronal distribution and a reduction in
neurons in the neocortex of newborn mice, which was consistent with
our present results (Komada et al., 2016;Luo et al., 2018). In pigs,
early postnatal DEHP exposure impaired germ cell migration and signif-
icantly increased germ cell death (Lee et al., 2021b). However, DEHP
was reported to trigger cell migration in human endometrial and
endometriotic epithelial cells (Kim et al., 2022a). These inconsistent ef-
fects of DEHP on cell migration may be due to different cell types and
animal models.
Neural migration occurs at 10–20 weeks of gestation and persists
throughout the embryonic stage (Francis and Cappello, 2021). Neurons
originate in the VZ or SVZ and migrate to the developing cortical plate
(CP). These processes are guided by RGs attaching to other cells and
interacting with the ECM (Taverna et al., 2014). The radial processes of
RGs play critical roles in leading neurons via direct contactwith pivotal sig-
naling between them. This interaction is essential for neuron migration and
maintenance of the RG process (Stouffer et al., 2016). It has been reported
that abnormal morphology of RG blocks the migration of cortical neurons,
resulting in severe laminary defects of laminated structures, such as cere-
bellar cortices (Del Toro et al., 2020;Hatanaka et al., 2019;Muralidharan
et al., 2022;Xu et al., 2014). A growing number of studies have suggested
that harmful environmental factor exposure during early life contributes
to neuron migration disorders in the developing brain (Miyazaki et al.,
2005;Wang et al., 2019a;Wang et al., 2021). Consistently, our results re-
vealed the discontinuity and non-radial alignment of RG fibers in cortical
organoids after DEHP exposure. Therefore, DEHP exposure causes neural
migration disturbances correlated with RG fiber dysplasia in developing
cortical organoids. From the above findings, we infer that DEHP exposure
could lead to the disordered arrangement of different types of cells in corti-
cal organoids mediated by the disruption of RGs.
ECM proteins, cell–cell adhesion, interaction, and the interactions be-
tween ECM proteins and receptors influence neural migration during corti-
cal development (Zamecnik et al., 2012). The ECM is a complex meshwork
formed by highly cross-linked proteins, providing a critical cell-supporting
scaffold and physiological cues for neural migration, proliferation, and
function ofneural cells during cortical development.In addition, RG devel-
opment requires suitable ECM components (Amin and Borrell, 2020;
Cavalcante et al., 1996;Hynes, 2009). The altered ECM organization was
reportedto affect cellproliferation, disturb the development of RGs, impair
the migration potential and change the adhesive properties of cells (Franco
and Müller, 2011;Jen et al., 2009;Lathia et al., 2007;Ramírez-Rodríguez
et al., 2017). The interactions between ECM and RGs had influences on
the development of RGs and neuron migration in the developing brain
(Chou et al., 2018;de Agustín-Durán et al., 2021). A series of EDCs have
been identified to restrain cell proliferation, disrupt the ECM, and alter
cell motility in different tissues in vitro and in vivo (Chen et al., 2020;Li
et al., 2020b;Liu et al., 2021;Pu et al., 2022). It is tempting to speculate
that the abnormal ECM remodeling may cause abnormal development of
RGs and disturb cell migration during the development of cortical
organoids.
Based on these results, we hypothesized that DEHP exposure disturbed
cell migration in developing cortical organoids mediated by the abnormal
development of RGs. Although the altered components of the ECM may
be correlated with RGs development, the exact mechanism for DEHP expo-
sure directly linked to RGs development is still unclear.
Many common genes have been identified in ECM-receptor interac-
tions and focal adhesion pathways. These genes include FN1,IGTA8,
and IGTA11.FN1, a ubiquitous component of the ECM, contributes to
changes in the ECM via integr in transmembrane receptors, which are in-
volved in cellular adhesion and migration (Zollinger and Smith, 2017).
FN1 wasover-expressedinlipopolysaccharide-treatedweanedpiglets
(Xie et al., 2020). In addition, it has been demonstrat ed that DEHP expo-
sure causes upregulated expression of fibronectin (Rafael-Vázquez
et al., 2018).DysfunctionofFN1wasreportedtoinfluence neural
death and migration (Gao et al., 2014;Kwak et al., 2021;Tate et al.,
2007). Integrins are multifunctional receptors mainly responsible for
binding to ECM proteins, such as fibronectin and tenascin, mediating
cell-ECM interactions to initiate intracellular signaling cascades and
participating in various biological functions, including cell proliferation
and migration (Ikeshima-Kataoka et al., 2022). Allosteric conforma-
tional changes and single nucleotide polymorphisms (SNPs) of integrins
mediate mechanical bidirectional signals between the extracellular and
intracellular environment of nerve cells in the brain, which serves criti-
cal roles in the development of psychiatric disorders (Jaudon et al.,
2021). From these results, we hypothesized that DEHP exposure might
cause changes in correlated gene expression contributing to ECM re-
modeling. However, the exact molecular mechanism of DEHP-induced
ECMremodelingincorticalorganoidsremainstobeelucidatedinthe
future.
5. Conclusions
The developmental neurotoxicity of DEHP exposure was assessed in this
study using hESC-derived cerebral organoid models. The findings con-
firmed the multiple-biological defects implicated in cell proliferation and
apoptosis of DEHP exposure during the early development of cerebral
organoids. Notable, DEHP inhibited cell migration by disrupting the guid-
ance of RGs, which may be associated with cell-ECM interaction alteration.
In addition, our study provided an available cerebral organoid model to
evaluate the neurotoxicity of environmental endocrine-disrupting
chemicals.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2022.161251.
CRediT authorship contribution statement
Ling Yang conducted the experiments, collected and analyzed the data,
and wrote the original manuscript; Jiao Zou, Zhenle Zang, Liuyongwei
Wang, Zhulin Du, Dandan Zhang, Yun Cai, Minghui Li, and Qiyou Li ana-
lyzed the data and revised the manuscript; Junwei Gao, Xiaotang Fan,
and Haiwei Xu reviewed the experimental plan, provided technical and fi-
nancial support, and revised the manuscript. All authors contributed to the
article and approved the submitted version.
Funding
This study was supported by the National Key Research and
Development Program of China (2021YFA1101203); the National Natural
Science Foundation of China grants (31930068); the pre-research funding
from the Key Laboratory of Extreme Environment Medicine, Ministry of
Education, China (PF-KL2020-005); the Natural Science Foundation of
Chongqing (CSTB2022NSCQ-MSX1090).
Data availability
The data in this study are available from the corresponding author
based on the reasonable request.
Declaration of competing interest
The authors confirm that there are no conflicts of interest.
L. Yang et al. Science of the Total Environment 865 (2023) 161251
14

References
Adam, N., Lachayze, M.A., Parmentier, C., Hardin-Pouzet, H., Mhaouty-Kodja, S., 2022. Expo-
sure to environmentally relevant doses of plasticizers alters maternal behavior and re-
lated neuroendocrine processes in primiparous and multiparous female mice. Environ.
Pollut. 315, 120487.
de Agustín-Durán, D., Mateos-White, I., Fabra-Beser, J., Gil-Sanz, C., 2021. Stick around: cell-
cell adhesion molecules during neocortical development. Cells. 10, 118.
Ahmadpour, D., Mhaouty-Kodja, S., Grange-Messent, V.,2022. Effects and underlying cellular
pathway involved in the impairment of the neurovascular unit following exposure of
adult male mice to low doses of di(2-ethylhexyl) phthalate alone or in an environmental
phthalate mixture. Environ. Res. 207, 112235.
Amin, S., Borrell, V., 2020. The extracellular matrix in the evolution of cortical development
and folding. Front. Cell. Dev. Biol. 8, 604448.
Arbuckle, T.E., Fisher, M., MacPherson, S., Lang, C., Provencher, G., LeBlanc, A., et al., 2016.
Maternaland early life exposure to phthalates: the plasticsand personal-careproducts use
in pregnancy (P4) study. Sci. Total Environ. 551–552, 344–356.
Baumann, J., Dach, K., Barenys, M., Giersiefer,S., Goniwiecha, J., Lein, P., et al., 2015. Appli-
cation of the NeurosphereAssay for DNT Hazard Assessment: Challenges and Limitations.
Bu, Q., Huang, Y., Li, M., Dai, Y., Fang, X., Chen, K., et al., 2020. Acrylamide exposure re-
presses neuronal differentiati on, induces cell apoptosis and promotes t au
hyperphosphor ylation in hESC-d erived 3D cerebral organoids. Foo d Chem. Toxicol.
144, 111643.
Cavalcante, L.A., Garcia-Abreu, J., Moura Neto, V., Silva, L.C., Barradas,P.C., 1996. Heteroge-
neity of medianand lateral midbrain radial glia and astrocytes. Rev. Bras. Biol. 56 Su 1 Pt
1, 33–52.
Chen, H., Chen, K., Qiu, X., Xu, H., Mao, G., Zhao, T., et al., 2020. The reproductive toxicity
and potential mechanisms of combined exposure to dibu tyl phthalate and diisobutyl
phthalate in male zebrafish (Danio rerio). Chemosphere 258, 127238.
Chou, F.S., Li, R., Wang, P.S., 2018. Molecular components and polarity of radial glial cells
during cerebral cortex development. Cell. Mol. Life Sci. 75, 1027–1041.
Cui, J.G., Zhao, Y., Zhang, H., Li, X.N., Li, J.L., 2022. Lycopene regulates the mitochondrial
unfolded protein response to prevent DEHP-induced cardiac mitochondrial damage in
mice. Food Funct. 13, 4527–4536.
Dai, X.Y., Zhu, S.Y., Chen, J., Li, M.Z.,Zhao, Y., Talukder, M., et al., 2022.Lycopene alleviates
di(2-ethylhexyl) phthalate-induced splenic injury by activating P62-Keap1-NRF2 signal-
ing. Food Chem. Toxicol. 168, 113324.
Del Toro, D., Carrasquero-Ordaz, M.A., Chu, A., Ruff, T., Shahin, M., Jackson, V.A., et al.,
2020. Structural basis of teneurin-latrophilin interaction in repulsive guidance of migrat-
ing neuro ns. Cell 1 80, 323–339.e19.
Fagerlund, I., Dougalis, A., Shakirzyanova, A., Gómez-Budia, M., Pelkonen, A., Konttinen, H.,
et al., 2021. Microglia-like cells promote neuronal functions in cerebral organoids. Cells
11, 124.
Francis, F., Cappello, S., 2021. Neuronal migration and disorders - an update. Curr. Opin.
Neurobiol. 66, 57–68.
Franco, S.J., Müller, U., 2011. Extracellular matrix functions during neuronal migration and
lamination in the mammalian central nervous system. Dev. Neurobiol. 71, 889–900.
Gao, J., Wang, F., Wang, W., Su, Z., Guo, C.,Cao, S., 2014. Emodin suppresses hyperglycemia-
induced proliferation and fibronectin expression in mesangial cells via inhibiting cFLIP.
PLoS One 9, e93588.
Giandomenico, S.L., Sutcliffe, M., Lancaster, M.A., 2021. Generation and long-term culture of
advancedcerebral organoidsfor studying later stages of neuraldevelopment. Nat.Protoc.
16, 579–602.
Han, X., Shao, W., Yue, Z.,Xing, L., Shen, L., Long, C., et al.,2019. Di (2-ethylhexyl) phthalate-
inducedhypospadias in SD ratsis related with mafb expression:a transcriptome profiling-
based study. Nan Fang Yi Ke Da Xue Xue Bao 39, 456–463.
Hatanaka, Y., Kawasaki, T., Abe, T., Shioi, G.,Kohno, T., Hattori, M., et al., 2019;. Semaphorin
6A-Plexin A2/A4 interactions with radial glia regulate migration termination of superfi-
cial layer cortical neurons. iScience 21, 359–374.
Hu, J., Peng, Y., Zheng, T., Zhang, B., Liu, W., Wu,C., et al., 2018. Effects of trimester-specific
exposure to vanadium on ultrasound measures of fetal growth and birth size: a longitudi-
nal prospective prenatal cohort study. Lancet Planet Health 2, e427–e437.
Hua, T., Kiran, S., Li, Y., Sang, Q.A., 2022. Microplastics exposure affects neural development
of human pluripo tent stem cell- derived cortic al spheroids. J. Ha zard. Mater. 43 5,
128884.
Huang, Y., Dai, Y., Li, M., Guo, L., Cao, C., Huang, Y., et al., 2021. Exposure to cadmium in-
duces neuroinflam mation and impair s ciliogenesis in h ESC-derived 3D cer ebral
organoids. Sci. Total Environ. 797, 149043.
Huang, M.L., Yen, P.L., Chang, C.H., Liao, V.H., 2022. Chronic di(2-ethylhexyl) phthalate ex-
posure leads to dopaminergic neuron degeneration throughmitochondrial dysfunction in
C. Elegans. Environ. Pollut. 307, 119574.
Huo, H.Q., Qu, Z.Y., Yuan, F., Ma, L., Yao, L., Xu, M., et al., 2018. Modeling down syndrome
with patient iPSCs reveals cellular and migration deficits of GABAergic neurons. Stem
Cell Rep. 10, 1251–1266.
Hynes, R.O., 2009. The extracellular matrix: not just pretty fibrils. Science 326, 1216–1219.
Ikeshima-Kataoka, H., Sugimoto, C., Tsubokawa, T., 2022. Integrin signaling in the central
nervous system in animals and human brain diseases. Int. J. Mol. Sci. 23, 1435.
Inoue, K., Kawaguchi,M., Yamanaka, R., Higuchi, T., Ito, R., Saito, K., et al.,2005. Evaluation
and analysisof exposure levels of di(2-ethylhexyl) phthalatefrom blood bags. Clin. Chim.
Acta 358, 159–166.
Jabali, A., Hoffrichter, A., Uzquiano, A., Marsoner, F., Wilkens, R., Siekmann, M., et al., 2022.
Human cerebral organoids reveal progenitor pathology in EML1-linked cortical malfor-
mation. EMBO Rep. 23, e54027.
Jaudon, F., Thalhammer, A., Cingolani, L.A., 2021. Integrin adhesion in brain assembly: from
molecular structure to neuropsychiatric disorders. Eur. J. Neurosci. 53, 3831–3850.
Jen, Y.H., Musacchio, M., Lander, A.D., 2009. Glypican-1 controls brain size through regula-
tion of fibroblast growth factor signaling in early neurogenesis. Neural Dev. 4, 33.
Kaluthantrige Don, F., Kalebic, N., 2022. Forebrain organoids to model the cell biology of
basal radial glia in neurodevelopmental disorders and brain evolution. Front. Cell. Dev.
Biol 10, 917166.
Kelava, I., Lancaster, M.A., 2016. Dishing out mini-brains: current progress and future pros-
pects in brain organoid research. Dev. Biol. 420, 199–209.
Kim, H.G., Lim, Y. S., Hwang, S., Kim, H. Y., Moon, Y., Song, Y.J., et al., 20 22a. Di-(2-
ethylhexyl) ph thalate triggers proliferati on, migration, stemness, and epit helial-
mesenchymal transition in human endometrial and endom etriotic epithelial cells via
the tra nsform ing gro wth fac tor-β/Smad signaling pathway. Int. J. Mol. Sci. 23, 3938.
Kim, J., Lee, S., Lee, J., Park, J.C., Kim, K.H., Ko, J.M., et al., 2022b. Neurotoxicity of phenyl-
alanine on human iPSC-derived cerebral organoids. Mol. Genet. Metab. 136, 132–144.
Klaus, J., Kanton, S., Kyrousi, C., Ayo-Martin, A.C., Di Giaimo, R., Riesenberg, S., et al., 2019.
Altered neuronal migratory trajectories in human cerebral organoids derived from indi-
viduals with neuronal heterotopia. Nat. Med. 25, 561–568.
Koizumi, H., Tan aka, T., Gleeson, J.G., 2006. Doublec ortin-like kinase functions w ith
doublecortin to mediate fiber tract decussation and neur onal migration. Neuron. 49,
55–66.
Komada, M., Gendai, Y., Kagawa, N., Nagao, T., 2016. Prenatal exposure to di(2-ethylhexyl)
phthalate impairs development of the mouse neocortex. Toxicol. Lett. 259, 69–79.
Komada, M., Nagao, T., Kagawa, N., 2020. Postnatal di-2-ethylhexyl phthalate exposure af-
fects hippocampal dentate gyrus morphogenesis. J. Appl. Toxicol. 40, 1673–1682.
Krefft, O., Koch, P., Ladewig, J., 2021. Cerebral organoids to unravel the mechanisms under-
lying malformations of human cortical development. Semin. Cell Dev. Biol. 111, 15–22.
Kwak, J.H., Kim, S., Yu, N.K., Seo, H., Choi, J.E., Kim, J.I., et al., 2021. Loss of the neuronal
genome organizer and transcription factor CTCF induces neuronal death and reactive
gliosis in the anterior cingulate cortex. Genes Brain Behav. 20, e12701.
Lancaster, M.A., Knoblich, J.A., 2014. Generation of cerebral organoids from human pluripo-
tent stem cells. Nat. Protoc. 9, 2329.
Lancaster, M.A., Renner, M., Martin, C.A., Wenzel, D., Bicknell, L.S., Hurles,M.E., et al., 2013.
Cerebral organoids model human brain deve lopment and microcephaly. Nature 501,
373–379.
Lathia, J.D., Patton, B., Eckley, D.M., Magnus, T., Mughal, M.R., Sasaki, T., et al., 2007. Pat-
terns of laminins and integrins in the embryonic ventricular zone of the CNS. J. Comp.
Neurol. 505, 630–643.
Lee, D.G., Kim, K.M., Lee, H.S., Bae, Y.C., Huh, J.W., Lee, S.R., et al., 2019. Peroxiredoxin 5
prevents diethylhexyl phthalate-induced neuronal cell death by inhibiting mitochondrial
fission in mouse hippocampal HT-22 cells. Neurotoxicology 74, 242–251.
Lee, C.Y.,Hyun, S.A., Ko, M.Y., Kim, H.R., Rho,J., Kim, K.K., et al., 2021a. Maternal bisphenol
a (BPA) exposure alters cerebral cortical morphogenesis and synaptic function in mice.
Cereb. Cortex 31, 5598–5612.
Lee, Y., Rattan, S., Barakat, R., Inman, Z., De La Torre, K.M., Meling, D.D., et al., 2021b. Early
postnatalexposure to di(2-ethylhexyl) phthalate causes sex-specific disruption of gonadal
development in pigs. Reprod. Toxicol. 105, 53–61.
Li, X.N., Li, H.X., Yang, T.N., Li, X.W., Huang, Y.Q., Zhu, S.Y., et al., 2020a. Di-(2-ethylhexyl)
phthalate induced developmental abnormalities of the ovary in quail (Coturnix japonica)
via disruption of the hypothal amic-pituita ry-ovarian axi s. Sci. Total Env iron. 741,
140293.
Li, F., Luo, T., Rong, H., Lu,L., Zhang, L., Zheng, C.,et al., 2022a. Maternalrodent exposure to
di-(2-ethylhexyl) phthalate decreases muscle mass in the offspring by increasing myo-
statin. J. Cachexia. Sarcopenia Muscle 13 (6), 2740–2751.
Li, Y., Shen, Y., Li, J., Cai, M., Qin, Z., 2020b. Transcriptomic analysis identifies early cellular
and molecular events by which estrogen disruptstestis differentiation and causes femini-
zation in Xenopus laevis. Aquat. Toxicol. 226, 105557.
Li, M.Z., Zhao, Y., Wang, H. R., Talukder, M., Li, J.L., 2021. Lycopene preventing DEHP-
induced renal cell damage is targeted by aryl hydrocarbon rec eptor. J. Agric. Food
Chem. 69, 12853–12861.
Li, M., Gong, J., Ge, L., Gao, H., Yang, J., Yang, C., et al., 2022b. Development of human retinal
organoid models for bisphenol toxicity assessment. Ecotoxicol. Environ. Saf. 245, 114094.
Li, M., Zeng,Y., Ge, L., Gong, J., Weng,C., Yang, C., et al., 2022c. Evaluation of the influences
of low dose polybrominated diphenyl ethers exposure on human early retinal develop-
ment. Environ. Int. 163, 107187.
Li, Q., Zhu,Q., Tian, F., Li, J., Shi, L.,Yu, Y., et al., 2022d. Inutero di-(2-ethylhexyl) phthalate-
induced testicular dysgenesis syndrome in male newborn rats is rescued by taxifolin
through reducing oxidative stress. Toxicol. Appl. Pharmacol. 456, 116262.
Liu, Y., Wu, Y., Qin, G., Chen, Y., Wang, X., Lin, Q., 2021. Bioaccumulation and reproductive
toxicity of bisphenol a in male-pregnant seahorse (Hippocampus erectus) at environmen-
tally relevant concentrations. Sci. Total Environ. 753, 141805.
Liu, W., Sun, Y., Liu, N., Hou, J., Huo, X., Zhao, Y., et al., 2022. Indoor exposure to phthalates
and its burden of disease in China. Indoor Air 32, e13030.
Liu, Y., Guo, Z., Zhu, R., Gou, D., Jia, P.P., Pei, D.S., 2023. An insight into sex-specific neuro-
toxicity and molecular mechanisms of DEHP: a critical review. Environ . Pollut. 316,
120673.
Loff, S., Kabs, F., Witt, K., Sartoris, J., Mandl, B., Niessen, K.H., etal., 2000. Polyvinylchloride
infusion lines expose infants to large amounts of toxic plasticizers. J. Pediatr. Surg. 35,
1775–1781.
Lovekamp-Swan, T., Davis, B.J., 2003. Mechanisms of phthalate ester toxicity in the female
reproductive system. Environ. Health Perspect. 111, 139–145.
Luo, B.B., Feng, Q.W., Wu, D.J., Wang, Q.N., 2018. Experimental study on DEHP affect the
neurodevelopm ent through in terfering wit h placental thy roid hormones tr ansport.
Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi 36, 179–183.
Lv, J., Li, Y., Chen , J., Li, R., Bao, C. , Ding, Z., et al., 2 022. Maternal ex posure to bis(2 -
ethylhexyl) phthalate during the thyroid hormone-dependent stage induces persistent
emotional and cognitive impairment in middle-aged offspring mice. Food Chem. Toxicol.
163, 112967.
L. Yang et al. Science of the Total Environment 865 (2023) 161251
15

Marsee, K., Woodruff, T.J., Axelrad, D.A., Calafat, A.M., Swan, S.H., 2006. Estimated daily
phthalate expo sures in a populat ion of mothers of male infants exhibiting reduced
anogenital distance. Environ. Health Perspect. 114, 805–809.
Mínguez-Alarcón, L., Bellavia, A., Gaskins, A.J., Chavarro, J.E., Ford, J.B., Souter, I., et al.,
2021. Paternal mixtures of urinary concentrations of phthalate metabolites, bisphenol a
and parabens in relation to pregnancy outcomes among couples attending a fertility cen-
ter. Environ. Int. 146, 106171.
Mínguez-Alarcón, L., Burns, J., Williams, P.L., Korrick, S.A., Lee, M.M., Bather, J.R., et al.,
2022a. Urinary phthalate metabolite concentrations during four windows spanning pu-
berty (prepuberty through sexual maturity) and association with semen quality among
young russian men. Int. J. Hyg. Environ. Health 243, 113977.
Mínguez-Alarcón, L., Williams, P.L., James-Todd, T., Souter, I., Ford, J.B., Rexrode, K.M., et
al., 2022b. Association of Urinary Phthalate and Phthalate Replacement Metabolite Con-
centrations with serum lipid biomarker levels among pregnant women attending a fertil-
ity center. Toxics 10, 292.
Miyazaki, K., Narita, N., Narita, M., 2005. Maternal administration of thalidomide or valproic
acid causes abnormal serotonergic neurons in the offspring: implication for pathogenesis
of autism. Int. J. Dev. Neurosci. 23, 287–297.
Molnár, Z., Clowry, G.J., Šestan, N., Alzu'bi, A., Bakken, T., Hevner, R.F., et al., 2019. New in-
sights into the development of the human cerebral cortex. J. Anat. 235, 432–451.
Muralidharan, H., Guha, S., Madugula, K., Patil, A., Bennison, S.A., Sun, X., etal., 2022. KIFC1
regulates the trajectory of neuronal migration. J. Neurosci. 42, 2149–2165.
Nadeem, A., Al-Harbi, N.O., Ahmad, S.F., Alhazzani, K., Attia, S.M., Alsanea, S., et al., 2021.
Exposure to the plasticizer, Di-(2-ethylhexyl) phthalate during juvenile period exacer-
bates autism-like behavior in adult BTBR T + tf/J mice due to DNA hypomethylation
and enhanced in flammation in brain an d systemic immune c ells. Prog. Neuro -
Psychopharmacol. Biol. Psychiatry 109, 110249.
Park, S., Lee, J.M., Kim, J.W., Cheong,J.H., Yun, H.J., Hong,Y.C., et al., 2015. Association be-
tween phthalates and externalizing behaviors and cortical thickness in children with at-
tention deficit hyperactivity disorder. Psychol. Med. 45, 1601–1612.
Pu, Y., Ticiani, E., Waye, A.A., Dong, K., Zhang, H., Veiga-Lopez, A., 2022. Sex-specific extra-
cellular matrix re modeling during ea rly adipogenic d ifferentiatio n by gestational
bisphenol a exposure. Chemosphere 302, 134806.
Qian, X., Nguyen, H.N., Song, M.M., Hadiono, C., Ogden, S.C., Hammack, C., et al., 2016.
Brain-region-specific organoids us ing mini-bioreact ors for modeling ZIKV exposure.
Cell 165, 1238–1254.
Qiu, F., Zhou, Y., Deng, Y., Yi, J., Gong, M., Liu, N., et al., 2020. Knockdown of TNFAIP1 pre-
vents di-(2-ethylhexyl) phthalate-induced neurotoxicity by activating CREB pathway.
Chemosphere 241, 125114.
Rafael-Vázquez, L., García-Trejo, S., Aztatzi-Aguilar, O.G., B azán-Perkins, B., Quintanilla-
Vega, B., 2018. Exposure to diethylhexyl phthalate (DEHP) and monoethylhexyl phthal-
ate (MEHP) promotes the loss of alveolar epithelial phenotype of A549 cells. Toxicol.
Lett. 294, 135–144.
Ramírez-Rodríguez, G.B., Perera-Murcia, G.R., Ortiz-López, L., Vega-Rivera, N.M., Babu, H.,
García-Anaya, M., et al., 2017. Vascular endothelial growth factor influences migration
and focal adhesions, but not proliferation or viability, of human neural stem/progenitor
cells derived from olfactory epithelium. Neurochem. Int. 108, 417–425.
Safarpour, S., Ghasemi-Kasm an, M., Safarpo ur, S., Darban, Y.M ., 2022. Effect s of Di-2-
ethylhexyl pht halate on central nervous system functions: a narrative review. Curr.
Neuropharmacol. 20, 766–776.
Sims, J.N., Graham, B., Pacurari, M., Leggett, S.S., Tchounwou, P.B., Ndebele, K., 2014. Di-
ethylhexylphthalate (DEHP) modulates cell invasion, migration and anchorage indepen-
dent growth through targeting S100P in LN-229 glioblastoma cells. Int. J. Environ. Res.
Public Health 11, 5006–5019.
Stouffer, M.A., Golden, J.A., Francis, F., 2016. Neuronal migration disorders: focus on the cy-
toskeleton and epilepsy. Neurobiol. Dis. 92, 18–45.
Subramanian, A., Tamayo, P., Mootha, V.K., Mukherjee, S., Ebert, B.L., Gillette, M.A., et al.,
2005. Gene set enrichment analysis : a knowledge-bas ed approach for inte rpreting
genome-wide expression profiles. Proc. Natl. Acad. Sci. U. S. A. 102, 15545–15550.
Tate, C.C., García, A.J., LaPlaca, M.C., 2007. Plasma fibronectin is neuroprotective following
traumatic brain injury. Exp. Neurol. 207, 13–22.
Taverna,E., Götz, M., Huttner,W.B., 2014. The cell biology of neurogenesis: towardan under-
standing of the development and evolution of the neocortex. Annu. Rev. Cell Dev. Biol.
30, 465–502.
Valvi, D., Monfort, N., Ventura, R., Casas, M., Casas, L., Sunyer, J., et al., 2015. Variability and
predictors of urinary phthalatemetabolites in spanish pregnant women. Int. J. Hyg.Envi-
ron. Health 218, 220–231.
Wang, H., Zhou, L., Meng, Z., Su, M., Zhang, S., Huang, P., et al., 2019a. Clethodim exposure
induced development toxicity and behaviour alteration in early stages of zebrafish life.
Environ. Pollut. 255, 113218.
Wang, Y., Lafon, P.A., Salvador-Prince, L., Gines, A.R., Trousse, F., Torrent, J., et al., 2021.
Prenatal exposure to low doses of fungicides corrupts neurogenesis in neonates. Environ.
Res. 195, 110829.
Wang, Y., Zhu, H., Kannan, K., 2019b. A review of biomonitoring of phthalate exposures.
Toxics 7, 21.
Xie, Q., Feng, L., Zhan g, C., Gan, L., 2020. MicroRNA-183 regul ates lipopolysacc haride-
induced oxidative stress of hippocampal neurons by targeting the fibronectin 1 gene.
Neurosci. Lett. 72 5, 134902.
Xu, H.T., Han, Z., Gao, P., He, S., Li, Z., Shi, W., et al., 2014. Distinct lineage-dependent struc-
tural and functional organization of the hippocampus. Cell 157, 1552–1564.
Yang, T.N., Li, X .N., Wang, Y.X., Ma , X.Y., Li, J.L., 2022. Disrupted microbiota-bar rier-
immune interaction in phthalates -mediated barri er defect in the duode num.
Chemosph ere 308, 1 36275.
Yin, Z., Hua, L., Chen, L., Hu, D., Li, J., An, Z., etal., 2020. Bisphenol-a exposure induced neu-
rotoxicity and associated with synapse and cytoskeleton in neuro-2a cells. Toxicol. in
Vitro 67, 104911.
Yirun, A., Ozkemahli, G., Balci, A., Erkekoglu, P., Zeybek, N.D., Yersal, N., et al., 2021. Neu-
roendocrine disruption by bisphenol a and/or di(2-ethylhexyl) phthalate after prenatal,
early postnatal and lactational exposure. Environ. Sci. Pollut. Res. Int. 28, 26961–26974.
Zamecnik, J., Homola, A., Cicanic, M., Kuncova, K., Marusic, P., Krsek, P., et al., 2012. The
extracellular matrix and diffusion barriers in focal cortical dysplasias. Eur. J. Neurosci.
36, 2017–2024.
Zang, Z., Yin, H., Du, Z., Xie, R., Yang, L., Cai, Y., et al., 2022. Valproic acid exposure de-
creases neurogenic potential of outer radial glia in human brain organoids. Front. Mol.
Neurosci. 15, 1023765.
Zeng, Y., Li, M., Zou, T., Chen, X., Li, Q., Li, Y., et al., 2021. The impact of particulate matter
(PM2.5) on human retinal development in hESC-derived retinal organoids. Front cell.
Dev. Biol. 9, 607341.
Zhang, X., Huang, J., Zheng, G., Liang,J., Hu, B., Lou, Z., et al., 2021. Prenatal exposure to di
(2-ethylhexyl) phthalate causes autism-like behavior through inducing nischarin expres-
sion in the mouse offspring. Biochem. Biophys. Res. Commun. 585, 29–35.
Zhang, H., Zhao,Y., Cui, J.G., Li, X.N., Li, J.L., 2022. DEHP-induced mitophagy and mitochon-
drial damage in the heart are associated with dysregulated mitochondrial biogenesis.
Food Chem. Toxicol. 161, 112818.
Zhao, Y., Li, H.X., Luo, Y., Cui, J.G., Talukder, M., Li, J.L., 2022a. Lycopene mitigates DEHP-
induced hepatic mitochondrial quality control disorder via regulating SIRT1/PINK1/mi-
tophagy axis and mitochondrial unfolded protein response. Environ.Pollut. 292, 118390.
Zhao, Y., Li, X.N., Zhan g, H., Cui, J.G., Wang, J.X ., Chen, M.S., et al., 2022 b. Phthalate-
induced testosterone/androgen receptor pathway disorder on spermatogenesis and an-
tagonism of lycopene. J. Hazard. Mater. 439, 129689.
Zhao, Y.X., Tang, Y.X., Sun, X.H., Zhu, S.Y., Dai,X.Y., Li, X.N., et al., 2022c. Gap junction pro-
tein connexin 43 as a target is internalized in astrocyte neurotoxicity caused by Di-(2-
ethylhexyl) phthalate. J. Agric. Food Chem. 70, 5921–5931.
Zollinger, A.J., Smith, M.L., 2017. Fibronectin, the extracellular glue. Matrix Biol. 60–61,
27–37.
Zou, J., Shang, W., Yang, L., Liu, T., Wang, L., Li, X., et al., 2022. Microglia activation in the
mPFC mediates anxiety-like behaviors caused by Staphylococcus aureus strain USA300.
Brain Behav. 12, e2715.
L. Yang et al. Science of the Total Environment 865 (2023) 161251
16