Benzophenone-3 Impairs Autophagy, Alters Epigenetic Status, and Disrupts Retinoid X Receptor Signaling in Apoptotic Neuronal Cells

Abstract

Benzophenone-3 (BP-3) is the most widely used compound among UV filters for the prevention of photodegradation. Population studies have demonstrated that it penetrates through the skin and crosses the blood-brain barrier. However, little is known about the impact of BP-3 on the nervous system and its possible adverse effects on the developing brain. We demonstrated that the neurotoxic effects of BP-3 were accompanied by the induction of apoptosis, as evidenced by apoptosis-related caspase-3 activation and apoptotic body formation as well as the inhibition of autophagy, as determined by the downregulation of autophagy-related genes, decreased autophagosome formation, and reduced LC3B-to-LC3A ratio. In this study, we showed for the first time that the BP-3-induced apoptosis of neuronal cells is mediated via the stimulation of RXRα signaling and the attenuation of RXRβ/RXRγ signaling, as demonstrated using selective antagonist and specific siRNAs as well as by measuring the mRNA and protein expression levels of the receptors. This study also demonstrated that environmentally relevant concentrations of BP-3 were able to inhibit autophagy and disrupt the epigenetic status of neuronal cells, as evidenced by the inhibition of global DNA methylation as well as the reduction of histone deacetylases and histone acetyl transferases activity, which may increase the risks of neurodevelopmental abnormalities and/or neural degenerations.

Full text

Benzophenone-3 Impairs Autophagy, Alters Epigenetic Status,
and Disrupts Retinoid X Receptor Signaling in Apoptotic
Neuronal Cells
Agnieszka Wnuk
1
&Joanna Rzemieniec
1
&Władysław Lasoń
1
&Wojciech Krzeptowski
2
&
Małgorzata Kajta
1
Received: 12 May 2017 /Accepted: 1 August 2017 /Published online: 16 August 2017
#The Author(s) 2017. This article is an open access publication
Abstract Benzophenone-3 (BP-3) is the most widely used
compound among UV filters for the prevention of
photodegradation. Population studies have demonstrated that
it penetrates through the skin and crosses the blood-brain bar-
rier. However, little is known about the impact of BP-3 on the
nervous system and its possible adverse effects on the devel-
oping brain. We demonstrated that the neurotoxic effects of
BP-3 were accompanied by the induction of apoptosis, as
evidenced by apoptosis-related caspase-3 activation and apo-
ptotic body formation as well as the inhibition of autophagy,
as determined by the downregulation of autophagy-related
genes, decreased autophagosome formation, and reduced
LC3B-to-LC3A ratio. In this study, we showed for the first
time that the BP-3-induced apoptosis of neuronal cells is me-
diated via the stimulation of RXRαsignaling and the attenu-
ation of RXRβ/RXRγsignaling, as demonstrated using selec-
tive antagonist and specific siRNAs as well as by measuring
the mRNA and protein expression levels of the receptors. This
study also demonstrated that environmentally relevant con-
centrations of BP-3 were able to inhibit autophagy and disrupt
the epigenetic status of neuronal cells, as evidenced by the
inhibition of global DNA methylation as well as the reduction
of histone deacetylases and histone acetyl transferases activity,
which may increase the risks of neurodevelopmental abnor-
malities and/or neural degenerations.
Keywords Benzophenone-3 .BP-3 .Retinoid X receptors .
RXR .Primary neuronal cell cultures .Autophagy
Introduction
Because of public anxiety about skin cancer caused by ultra-
violet light (UV), production and consumption of sunscreen
products are increasing. Nowadays, over 10,000 t of UV filters
are produced annually for the global market [1]. Chemical UV
filters are generally used as a mixture since none of the com-
pounds used individually get sufficient protection against UV.
Among the filters, benzophenones (BPs) are the primary
ingredients in the organic UV filter family. Benzophenone-3
(2-hydroxy-4-methoxybenzophenone, oxybenzone, 2OH-4
MeO-BP or BP-3) is the most widely used compound among
BPs for the skin prevention against photodegradation [2].
Human studies have demonstrated that after topical applica-
tion, BP-3 is absorbed through the skin, partially metabolized,
and is excreted in the urine. BP-3 was detected in almost all
(80–96%) urine samples collected from the general population
in the USA [3]; pregnant women in France [4,5]; and Danish
mothers and their children, adolescents, young men, and preg-
nant women [6,7]. BP-3 can be detected in the serum and
urine of adult volunteers shortly after dermal application,
proving that it can pass through the skin into the body [8]. It
has been estimated that 10% of the applied dermal BP-3 pen-
etrates skin into systemic circulation [9].
It is extremely disturbing that BP-3 has been detected in a
large proportion of milk samples indicating that breastfed
babies are exposed to BP-3 [10]. A recent study showed that
maternal exposure to BP-3 is strongly associated with the onset
of Hirschsprung’s disease in offspring [11]. However, data on
the effects of BP-3 on the nervous system are scarce. Espe-
cially little is known about the impact of BP-3 on individual
*Małgorzata Kajta
kajta@if-pan.krakow.pl
1
Department of Experimental Neuroendocrinology, Institute of
Pharmacology, Polish Academy of Sciences, Smetna Street 12,
31-343 Krakow, Poland
2
Department of Cell Biology and Imaging, Institute of Zoology,
Jagiellonian University, Gronostajowa Street 9,
30-387 Krakow, Poland
Mol Neurobiol (2018) 55:5059–5074
DOI 10.1007/s12035-017-0704-2
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

receptors that are strongly associated with brain development
such as retinoid X receptors. The only data on the apoptotic and
neurotoxic effects of BP-3 on the neural cells come from SH-
SY5Y neuroblastoma cells and our recently published original
paper [12,13]. BP-3 has been reported to act as endocrine
disrupting chemicals (EDCs). At present, it is only known that
BP-3 can weakly agonize estrogen signaling and strongly an-
tagonize androgen-related pathways [14–16]. An association
between BP-3 exposure and the estrogen-related disease endo-
metriosis has been found [17]. Recently, retinoid X receptors
have been postulated to be a target for EDCs.
The retinoid X receptor (RXR) is a type of nuclear receptor
family that is encoded by three genes: RXRα,RXRβ,and
RXRγ[18]. RXRs heterodimerize with one-third of the 48
human nuclear receptor superfamily members [19]. For most
of them, RXR is an obligatory partner for DNA binding and
transcriptional regulation. In addition, RXRαis able to form
homodimers and homotetramers, which is suggestive of the
self-regulation of specific RXRαsignaling pathways [20].
The diversity of RXRs suggests that they play critical roles
in a wide range of cellular pathways. Recent studies have
shown the prominence of RXR signaling in developing inner-
vation and myelination in health and disease of the central
nervous system [21]. Current studies in our research group
have shown the involvement of RXRs in the effects of EDCs
(specifically the pesticide dichlorodiphenyldichloroethylene
(DDE) and nonylphenol) [22–24].
One of the most important ways of regulating gene expres-
sion is the remodeling of chromatin, including post-
translational modifications of histones and DNA methylation.
It has been postulated that low doses of EDCs may cause
epigenetic changes, such as the incomplete methylation of
specific gene regions in the young brain [25,26]. Histone
post-translational modifications include the most studied
modifications—the acetylation of histones by histone acetyl
transferases (HATs) and the removal of acetyl groups from
histones by histone deacetylases (HDACs). These processes
play important roles in cognition as well as psychiatric and
neurologic diseases such as Alzheimer's disease, Huntington’s
disease, traumatic brain injury, post-traumatic stress disorder,
stress, depression, and addiction [27].
Autophagy is a process that is mainly responsible for elim-
inating the cells or keeping them alive, even in conditions
deprived of trophic factors. Autophagy is postulated to play a
housekeeping role in removing abnormal proteins or clearing
damaged organelles. The formation of autophagosomes de-
pends on several core Atg proteins, such as the following:
ULK1 complex, Beclin1:Vps34/Atg14L complex, and LC3
conjugation systems. During the process of autophagy, LC3
protein is cleaved by Atg4 to LC3A which next is modified
by ubiquitin-like systems to produce LC3B. Thus, LC3A and
LC3B are present in autophagosomes; both the ratio of LC3B
to LC3A and the amount of LC3B only can be used to estimate
the level of autophagy. Recent studies have proposed that gen-
erally autophagy is a survival mechanism, although its dysreg-
ulation may lead to non-apoptotic cell death [28].
The present study aimed to investigate the neurotoxic and
apoptotic effects of BP-3 and the impact of this chemical on the
expression and function of RXRs, including RXRα,RXRβ,
and RXRγ. Neurotoxicity was estimated by measuring lactate
dehydrogenase (LDH) release, which was complemented by
an assessment of caspase-3 activity. These data were supported
by Hoechst 33342/calcein acetoxymethyl (AM) staining,
which allowed for the visualization of apoptotic nuclei and cell
survival. The involvement of RXRs in the actions of BP-3 was
verified using selective antagonist and agonist as well as spe-
cific siRNAs. The levels of receptor mRNAs and proteins were
measured with qPCR, western blot, and enzyme-linked immu-
nosorbent assay (ELISA), and the cellular distributions of the
receptors were demonstrated using a confocal microscope. The
process of autophagy was assessed by measuring the expres-
sion of autophagy-specific genes using microarray analysis
and autophagosome detection, and the concentrations of
autophagy-selective proteins were measured by ELISAs.
Results regarding epigenetic modifications such as histone
post-translational modifications and DNA methylation were
complemented by an assessment of HAT and HDAC activity
and the measurement of global DNA methylation.
Materials and Methods
Materials
B27 and neurobasal media were obtained from Gibco (Grand
Island, NY, USA). L-glutamine, fetal bovine serum (FBS), N-
acetyl-Asp-Glu-Val-Asp-p-nitro-anilide (Ac-DEVD-pNA), di-
methyl sulfoxide (DMSO), HEPES, CHAPS, mouse monoclo-
nal anti-MAP2 antibody, ammonium persulfate, TEMED,
TRIZMA base, Tween 20, DL-dithiothreitol, Nonidet NP-40,
sodium deoxycholate, protease inhibitor (EDTA-free),
bromophenol blue, 2′,7′-dichlorofluorescein diacetate, RIPA
buffer, the Imprint Methylated DNA Quantification Kit, the
Histone Deacetylase Assay Kit, the Histone Acetyltransferase
Activity Assay Kit, the Autophagy Assay Kit, protease inhibitor
cocktail for mammalian tissues, and poly-ornithine were obtain-
ed from Sigma-Aldrich (St. Louis, MO, USA). Bradford re-
agent, SDS, 30% acrylamide, 0.5 M Tris-HCl buffer, 1.5 M
Tris-HCl gel buffer, and Laemmli sample buffer were from
Bio-Rad Laboratories (Munchen, Germany). DHA and HX
531 were from Tocris Bioscience (Minneapolis, MN, USA).
2-Mercaptoethanol was from Carl Roth GmbH + Co. KG
(Karlsruhe, Germany). Immobilon-P membranes were pur-
chased from Millipore (Bedford, MA, USA). Alexa 488-
conjugated anti-goat IgG, calcein AM, and Hoechst 33342 were
purchased from Molecular Probes (Eugene, OR, USA). Cy3-
5060 Mol Neurobiol (2018) 55:5059–5074
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conjugated anti-rabbit IgG and Cy5-conjugated anti-mouse
were obtained from Jackson ImmunoResearch, Inc. (West
Grove, PA, USA). The cytotoxicity detection kit and BM
chemiluminescence western blotting substrate (POD) were pur-
chased from Roche Diagnostics GmbH (Mannheim, Germany).
ELISA kits for RXRα,RXRβ,RXRγ, LC3A, and LC3B were
purchased from Shanghai Sunred Biological Technology Co.
(Sunred, China). The culture dishes were obtained from TPP
Techno Plastic Products AG (Trasadingen, Switzerland). The
rabbit polyclonal anti-RXRαantibody (sc-774), mouse mono-
clonal anti-RXRβantibody (sc-56869), mouse monoclonal
anti-RXRγantibody (sc-514134), mouse monoclonal anti-β-
actin antibody (sc-47778), as well as RXRαsiRNA (sc-
36448), RXRβsiRNA (sc-36446) and RXRγsiRNA (sc-
38879) were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA, USA). AllStars Negative Control siRNA AF
488, the RNeasy Mini Kit, RT
2
First Strand Kit, and RT
2
Profiler PCR Autophagy Array were obtained from Qiagen
(Valencia, CA, USA). INTERFERin was obtained from
PolyPlus Transfection (Illkirch, France), and the High
Capacity cDNA-Reverse Transcription Kit, the TaqMan Gene
Expression Master Mix, and TaqMan probes for specific genes
encoding hypoxanthine phosphoribosyltransferase coding gene
(Hprt), Rxrα,Rxrβ,andRxrγwere obtained from Life
Technologies Applied Biosystems (Foster City, CA, USA).
Quick-gDNA™MicroPrep was obtained from Zymo
Research (Irvine, CA, USA).
Primary Neocortical Cell Cultures
Neocortical tissue for primary cultures was prepared from
Swiss mouse embryos (Charles River, Germany) at 15–
17 days of gestation and cultured as previously described
[22,29]. All procedures were performed in accordance with
the National Institutes of Health Guidelines for the Care and
Use of Laboratory Animals and were approved by the
Bioethics Commission in compliance with Polish Law (21
August 1997). Animal care followed official governmental
guidelines, and all efforts were made to minimize suffering
as well as the number of animals used. The cells were
suspended in estrogen-free neurobasal medium supplemented
with B27 on poly-ornithine (0.01 mg/ml)-coated multi-well
plates at a density of 2.0 × 10
5
cells per cm
2
. The cultures
were maintained at 37 °C in a humidified atmosphere contain-
ing 5% CO
2
for 7 days in vitro (DIV) prior to experimentation.
The number of astrocytes, as determined by the content of
intermediate filament glial fibrillary acidic protein (GFAP),
did not exceed 10% for all cultures [22,30].
Treatment
Primary neuronal cell cultures were exposed to BP-3 (10–
100 μM) for 6 or 24 h. The involvement of RXR signaling
in BP-3-induced effects was verified with the high-
affinity RXR antagonist HX 531 (0.1 μM) and the
RXR agonist DHA (1 μM) as previously described
[22]. Specific ligands were added to the culture media
45–60 min before BP-3. To avoid nonspecific effects in
our study, agonist and antagonist of RXRs were used at
concentrations that did not affect the levels of caspase-3
activity or LDH release. All the compounds were orig-
inally dissolved in DMSO and were then further diluted
in culture medium to maintain DMSO concentrations
below 0.1%. The control cultures were supplemented
with DMSO in concentrations that were equal to those
used in the experimental groups.
Identification of Apoptotic Cells
Apoptotic cells were detected via Hoechst 33342 stain-
ing at 24 h after the initial treatment as previously de-
scribed [22,29]. Neocortical cells that were cultured on
glass coverslips were washed with 10 mM phosphate-
buffered saline (PBS) and stained with Hoechst 33342
(0.6 mg/ml) at room temperature (RT) for 5 min. The
cells containing bright blue fragmented nuclei, which
was indicative of condensed chromatin, were identified
as apoptotic cells. Qualitative analysis was performed
using a fluorescence microscope (NIKON Eclipse 80i,
NIKON Instruments Inc., Melville, New York, USA)
equipped with a camera with the BCAM Viewer©
Basler AG software. The level of cellular fluorescence
from fluorescence microscopy images was determined
using ImageJ software. To calculate the corrected total
cell fluorescence (CTCF), the following equation was
used: CTCF = Integrated density −(Area of selected
cell × Mean fluorescence of background).
Staining with Calcein AM
Intracellular esterase activity in the neocortical cultures
was measured by calcein AM staining at 24 h after the
initial treatment with BP-3 as previously described [22,
29]. To avoid the esterase activity present in the growth
media, the cells were washed with PBS and incubated in
2μM calcein AM in PBS at RT for 10 min. The cells
displaying bright green cytoplasm were identified as live
cells. Fluorescence intensity was monitored at Ex/Em 494/
520 nm using a fluorescence microscope (NIKON Eclipse
80i, NIKON Instruments Inc., Melville, New York, USA)
equipped with a camera with the BCAM Viewer© Basler
AG software. The level of cellular fluorescence from fluo-
rescence microscopy images was determined using ImageJ
software. To calculate the CTCF, the following equation
was used: CTCF = Integrated density −(Area of selected
cell × Mean fluorescence of background).
Mol Neurobiol (2018) 55:5059–5074 5061
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Assessment of Caspase-3 Activity
Caspase-3 activity was determined according to the protocol de-
scribed by Nicholson (1995) using samples treated for 6 or 24 h
with BP-3 alone or in combination with the test compounds. The
assessment of caspase-3 activity was performed as previously
described [22,30,31]. Cell lysates from neocortical cultures were
incubatedat37°CusingAc-DEVD-pNA, a colorimetric sub-
strate that is preferentially cleaved by caspase-3. The levels of p-
nitroanilide were continuously monitored for 60 min using a
Multimode Microplate Reader Infinite M200PRO (Tecan,
Mannedorf, Switzerland). The data were analyzed using the
Magellan software, normalized to the absorbance of vehicle-
treated cells, and expressed as a percentage of control ± SEM
from three to four independent experiments. The absorbance of
blanks, which acted as our enzyme-less control, was subtracted
from each value.
Measurement of Lactate Dehydrogenase Activity
To quantify cell death, lactate dehydrogenase (LDH) that was
released from damaged cells into the cell culture media was
measured 6 or 24 h after treatment with BP-3. LDH release
was measured as previously described [22,32]. Cell-free su-
pernatants from neocortical cultures were collected from each
well and incubated at room temperature for 30 to 60 min with
the appropriate reagent mixture according to the manufac-
turer’s instructions (Cytotoxicity Detection Kit) depending
on the reaction progress. The intensity of the red color that
formed in the assay was measured at a wavelength of 490 nm
(Infinite M200pro microplate reader, Tecan Mannedorf,
Switzerland) and was proportional to both LDH activity as
well as the number of damaged cells. The data were analyzed
using the Magellan software, normalized to the color intensity
from vehicle-treated cells (100%), and expressed as a percent-
age of the control value from three to four independent exper-
iments. Theabsorbance of blanks, which acted as ourenzyme-
less control, was subtracted from each value.
Silencing of RXRα,RXRβ, and RXRγ
Specific siRNAs were used to inhibit RXRα,RXRβ,and
RXRγexpression in neocortical cells. Each siRNA was
applied separately for 6 h at 50 nM in antibiotic-free me-
dium containing the siRNA transfection reagent
INTERFERin™as previously described [22]. After trans-
fection,theculturemediawerechanged,andthecellswere
incubated for 12 h before starting the experiment. Positive
and negative siRNAs containing a scrambled sequence that
did not lead to the specific degradation of any known cel-
lular mRNA were used as controls. The effectiveness of
mRNA silencing was verified through the measurement
of specific mRNAs using qPCR.
qPCR Analysis of mRNAs Encoding the Receptors Rxrα,
Rxrβ,andRxrγ
Total RNA was extracted from neocortical cells that were cul-
tured for 7 DIV (approx. 1.5 × 10
6
cells per sample) using the
RNeasy Mini Kit (Qiagen, Valencia, CA) according to the man-
ufacturer’s instructions. The quantity of RNA was spectropho-
tometrically determined at 260 and 260/280 nm (ND/1000 UV/
Vis; Thermo Fisher NanoDrop, USA). Two-step real-time
quantitative polymerase chain reaction (qPCR) was performed
as previously described [22]. Both the reverse transcription re-
action and qPCR were run on a CFX96 Real-Time System
(BioRad, USA). The products of the reverse transcription reac-
tion were amplified using TaqMan Gene Expression Master
Mix containing TaqMan primer probes specific to the genes
encoding Hprt,Rxrα,Rxrβ,andRxrγ.Amplification was per-
formed in a total volume of 20 μl containing 10 μlofTaqMan
Gene Expression Master Mix and 1.0 μl of reverse transcription
product as the PCR template. A standard qPCR procedure was
utilized: 2 min at 50 °C and 10 min at 95 °C followed by 40
cycles of 15 s at 95 °C and 1 min at 60 °C. The threshold value
(Ct) for each sample was set during the exponential phase, and
the delta Ct method was used for data analysis. Hprt was used
as a reference gene.
Mouse Autophagy RT
2
Profiler PCR Array
Total RNA was extracted from neocortical cells cultured for
7 DIV (approx. 1.5 × 10
6
cells per sample) using the RNeasy
Mini Kit (Qiagen, Valencia, CA) according to the manufac-
turer’s instructions. The quantity of RNA was spectrophoto-
metrically determined at 260 and 260/280 nm (ND/1000 UV/
Vis; Thermo Fisher NanoDrop, USA). A total of 1 μgof
mRNA was reverse-transcribed to cDNA using the RT
2
First
Strand Kit (Qiagen, Valencia, CA) and suspended in a final
volume of 20 μl as previously described [13]. Each cDNAwas
prepared for further use in qPCR. To analyze the signaling
pathway, the RT
2
Profiler™PCR Array System (Qiagen,
Valencia, CA) was used according to the manufacturer’spro-
tocol. The Ct values for all wells were exported to a blank
Excel spreadsheet and were analyzed with the Web-based
software (www.SABiosciences.com/pcrarraydataanalysis.
php).
Western Blot Analy si s
The cells exposed to BP-3 for 24 h were lysed in ice-cold RIPA
lysis buffer containing a protease inhibitor cocktail. The lysates
were sonicated and centrifuged at 15,000×g for 20 min at 4 °C.
The protein concentrations in the supernatants were determined
using Bradford reagent (Bio-Rad Protein Assay) with bovine se-
rum albumin (BSA) as the standard. Samples containing 40 μgof
total protein were reconstituted in the appropriate amount of
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sample buffer comprised of 125 mM Tris, pH 6.8, 4% SDS, 25%
glycerol, 4 mM EDTA, 20 mM DTT, and 0.01% bromophenol
blue, denatured, and separated on a 7.5% SDS-polyacrylamide
gel using a Bio-Rad Mini-Protean II Electrophoresis Cell as pre-
viously described [22,33,34]. After electrophoretic separation,
the proteins were electrotransferred to PVDF membranes
(Millipore, Bedford, MA, USA) using the Bio-Rad Mini Trans-
Blot apparatus. Following the transfer, the membranes were
washed, and the nonspecific binding sites were blocked with
5% dried milk and 0.2% Tween-20 in 0.02 M Tris-buffered saline
(TBS) for 2 h with shaking. The membranes were incubated
overnight (at 4 °C) with one of the following primary antibodies
(Santa Cruz Biotechnology) diluted in TBS/Tween: anti-RXRα
rabbit polyclonal antibody (diluted 1:150), anti-RXRβmouse
monoclonal antibody (diluted 1:100), anti-RXRγmouse mono-
clonal antibody (diluted 1:100), or anti-β-actin mouse monoclo-
nal antibody (diluted 1:3000). The signals were developed by
chemiluminescence (ECL) using BM Chemiluminescence
Blotting Substrate (Roche Diagnostics GmBH) and visualized
using a Luminescent Image Analyzer Fuji-Las 4000 (Fuji,
Japan). Immunoreactive bands were quantified using a
MultiGauge V3.0 image analyzer.
Enzyme-Linked Immunosorbent Assays for RXRα,
RXRβ, and RXRγ
The levels of RXRα,RXRβ,RXRγ, LC3A, and LC3B were
determined in neocortical cells 24 h after treatment with BP-3
as previously described [22]. Specific detection of these pro-
teins was achieved using ELISAs and the quantitative sandwich
enzyme immunoassay technique. A 96-well plate was precoat-
ed with monoclonal antibodies that were specific for RXRα,
RXRβ,RXRγ, LC3A, and LC3B. The standards and non-
denatured cell extracts were added to the wells with biotin-
conjugated polyclonal antibodies specific for RXRα,RXRβ,
RXRγ, LC3A, and LC3B. Therefore, all native RXRα,RXRβ,
RXRγ, LC3A, and LC3B proteins were captured using the
immobilized antibodies. The plates were washed to remove
any unbound substances, and horseradish peroxidase-
conjugated avidin was added to interact with the biotin bound
to RXRα,RXRβ,RXRγ, LC3A, and LC3B. After washing,
the substrate solution was added to the wells. The enzymatic
reaction yielded a blue product. The absorbance was measured
at 450 nm and was proportional to the amount of RXRα,
RXRβ,RXRγ, LC3A, and LC3B in the sample. The protein
concentration was determined in each sample using Bradford
reagent—Bio-Rad Protein Assay [22,35].
Immunofluorescence Labeling of RXRα,RXRβ,
and RXRγand Confocal Microscopy
For immunofluorescence detection of RXRα,RXRβ,and
RXRγ, neocortical cells were cultured on glass coverslips
and subjected to immunofluorescence double-labeling as
previously described [22,36].After1hofincubationina
blocking buffer (5% normal donkey serum and 0.3%
TritonX-100in0.01MPBS),thecellsweretreatedfor
24 h (at 4 °C) using four primary antibodies: rabbit poly-
clonal anti-RXRαantibody (1:50), mouse monoclonal
anti-RXRβantibody (1:50), mouse monoclonal anti-
RXRγantibody (1:50), and anti-MAP2 mouse monoclo-
nal antibody (1:100) followed by a 24-h incubation in a
mixture of secondary antibodies, including Cy3-
conjugated anti-rabbit IgG (1:300) and Cy5-conjugated
anti-mouse IgG (1:300). The samples were subsequently
washed, mounted, coverslipped, and analyzed using an
LSM510 META, Axiovert 200M confocal laser scanning
microscope (Carl Zeiss MicroImaging GmbH, Jena,
Germany) under a Plan-Neofluor 40×/1.3 Oil DIC objec-
tive. A He/Ne laser and an argon laser, with two laser
lines emitting at 514 and 633 nm, were used to excite
the Cy3-, and Cy5-conjugated antibodies, respectively.
The fluorescence signal was enhanced after combining
four scans per line. A pinhole value of 1 airy unit was
used to obtain flat images.
Measurement of Global DNA Methylation
Genomic DNA was extracted from neocortical tissues
using the Quick-gDNA™MicroPrep kit (Zymo
Research, Irvine, CA) according to the manufacturer’sin-
structions. The quantity of DNA was spectrophotometri-
cally determined at 260 and 260/280 nm (ND/1000 UV/
Vis; Thermo Fisher NanoDrop, USA). Global DNA meth-
ylation changes were measured in neocortical cells at 24 h
after treatment using a specific ELISA-based kit
(Imprint® Methylated DNA Quantification—Sigma-
Aldrich; St. Louis, MO, USA) as previously described
[22]. This kit contained all the reagents required to deter-
mine the relative levels of methylated DNA. The methyl-
ated DNA was detected using the capture and detection
antibodies and quantified colorimetrically using an
Infinite M200pro microplate reader (Tecan, Austria). The
amount of methylated DNA present in the sample was
proportional to the absorbance measured.
Detection of Autophagosomes
Cultured cells on 96-well plates were treated according
to the manufacturer’s instructions. The Autophagy
Assay kit provided a simple and direct procedure for
measuring autophagy in a variety of cell types using a
proprietary fluorescent autophagosome marker
(λ
ex
=333/λ
em
= 518 nm). The autophagosomes were
detected using an Infinite M200pro microplate reader
(Tecan, Austria).
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Measurement of HDAC and HAT Activity
The HDAC and HAT activities were detected using the Histone
Deacetylase Assay Kit and the Histone Acetyltransferase
Activity Fluorometric Assay Kit (Sigma-Aldrich, St. Louis,
MO, USA) according to the manufacturer’s instructions.
Regarding HDAC kit, the measured fluorescence at
λ
ex
=365nm/λ
em
= 460 nm was proportional to the
deacetylation activity. In the HAT assay, the generated product
of histone acetyltransferase activity was detected fluorimetrically
at λ
ex
= 535/λ
em
= 587 nm. The kit included an active nuclear
extract to be used as a positive control. The abovementioned
assays provided positive and negative controls.
Data Analysis
Statistical tests were performed on raw data that were expressed
as the mean arbitrary absorbance or as the fluorescence units
per well containing 50,000 cells (measurements of caspase-3,
LDH, autophagosomes; the fluorescence units per 1.5 million
cells (qPCR, global DNA methylation, and HDAC and HAT
activity); the mean optical density per 40 μg of protein (western
blotting); or picograms of RXRα,RXRβ,RXRγ,LC3A,and
LC3B per micrograms of total protein (ELISA). Statistical anal-
ysis of cellular fluorescence related to Hoechst 33342 and
calcein AM staining was performed on CTCF data using 40
counts per image. One-way analysis of variance (ANOVA) was
preceded by the Levene’s test of homogeneity of variances and
was used to determine overall significance. Differences be-
tween the control and experimental groups were assessed using
a post hoc Newman–Keuls test, and significant differences
were designated as
*
p< 0.05,
**
p<0.01,and
***
p<0.001
versus control cultures;
#
p<0.05,
##
p<0.01,and
###
p< 0.001 versus the cultures exposed to BP-3; and
$
p<0.05and
$$$
p< 0.001 versus the siRNA-transfected control
cultures. The results were expressed as the mean ± SEM of
three to four independent experiments. The number of repli-
cates in each experiment ranged from 2 to 3, except for the
measurements of caspase-3 activity and LDH release, which
contained five to eight replicates. To compare the effects of
BP-3 in different treatment paradigms, the results for the cas-
pase-3, LDH, ELISA, and western blot analyses were presented
as a percentage of the control.
Results
Effects of BP-3 on Caspase-3 Activity and LDH Release
in Neocortical Cultures at 7 DIV
In neocortical cultures at 7 DIV, BP-3 (25–100 μM) induced
an increase in caspase-3 levels to 170% of the control level at
6 h, which were further enhanced to 196% at 24-h post-
treatment (Fig. 1a). In these cells, LDH release from neocor-
tical cells increased in a time-dependent manner to 150–180%
of the control value at 6 h and to 200–290% at 24 h (Fig. 1b).
Effects of BP-3 Alone or in Combination with HX 531
on Hoechst 33342 and Calcein AM Staining in Neocortical
Cultures
In the present study, a 24-h exposure to BP-3 (25 μM) was
necessary to develop an apoptotic morphology in cell nuclei.
Apoptotic cells were detected by Hoechst 33342 staining as for-
mation of bright blue fragmented nuclei containing condensed
chromatin (Fig. 2). Furthermore, treatment with BP-3 reduced
the density of calcein AM-stained living cells at 7 DIV, as indi-
cated by the decreased number of cells exhibiting light-colored
cytoplasm. Co-treatment with RXR antagonist-HX 531
(0.1 μM) inhibited the BP-3-induced effects. Quantitative analy-
sis of relevant fluorescence signals showed that at 7 DIV, 25 μM
BP-3 caused an increase in formation of condensed chromatin by
386% of the control level and reduced number of live cells by
56%. Treatment with HX 531 (0.1 μM) inhibited the effect of
BP-3 in respect to the apoptotic fragmentation of cell nuclei by
231% and enhanced cell viability by 35% (Fig. 2).
Effects of BP-3 Alone or in Combination with DHA
and HX 531 on BP-3-Induced Caspase-3 Activity
and LDH Release in Neocortical Cultures
Neocortical cultures exposed to BP-3 (25 μM) for 24-h caused
a greater than 50% increase in caspase-3 activity in the neu-
ronal cells. Co-treatment with the selective RXR agonist DHA
(1 μM) did not change the effect of BP-3 on caspase-3 activity.
The RXR antagonist HX 531 (0.1 μM) inhibited the BP-3
(25 μM)-induced caspase-3 activity by 40% (Fig. 3a).
The selective RXR agonist DHA (1 μM) did not affect BP-
3-induced LDH release. However, the high-affinity RXR an-
tagonist HX 531 (0.1 μM) diminished BP-3-induced LDH
release by 25% (Fig. 3b).
Effect of BP-3 on the mRNA Levels of Rxrα,Rxrβ,
and Rxrγ
According to our data, treatment with BP-3 (25 μM) affected
the mRNA levels of Rxrα,Rxrβ,andRxrγ. A 3-h exposure of
the neocortical cultures to BP-3 caused a 25% decrease in Rxrβ
and a 55% decrease in Rxrγbut caused a 100% increase in
RxrαmRNA compared with the control (Fig. 4a). The pattern
of mRNA expression was changed after prolonged exposure to
BP-3. After 6 h of treatment, BP-3 decreased the mRNA ex-
pression level of Rxrα(26%) but did not change the expression
level of Rxrβin neocortical cells (Fig. 4b). After 24 h of expo-
sure, BP-3 did not change the mRNA expression levels of any
Rxrs (Fig. 4c). These data were normalized to Hprt.
5064 Mol Neurobiol (2018) 55:5059–5074
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control
25 μM BP-3
Hoechst 33342 calcein AM
25 μM BP-3
+ HX 531
Corrected total cell
fluorescence
(CTCF)
control 25 μM BP-3 25 μM BP-3
+ HX 531
Hoechst 33342 0.153 (100 %) 0.591 (386 %) 0.238 (155 %)
calcein AM 49.328 (100 %) 21.609 (44 %) 38.802 (79%)
***
***
*
*
Fig. 2 Influence of BP-3
(25 μM) and HX 531 (0.1 μM) on
Hoechst 33342 (first column)and
calcein AM (second column)
staining in mouse neocortical
cultures at 7 DIV, examined 24 h
post-treatment. Cells with bright
fragmented nuclei with
condensed chromatin were
identified as cells undergoing
apoptosis, whereas cells with
light-colored cytoplasm were
identified as live cells. Statistical
analysis of relevant fluorescence
signals was performed on CTCF
data using 40 counts per image.
*
p<0.05and
***
p< 0.001 versus
control cultures
0
50
100
150
200
250
caspase-3 activity
[% of control]
neocortical neurons 6h
24h
*** ***
*** ***
***
***
*
control
0
50
100
150
200
250
300
350
10 μM 25 μM 50 μM 75 μM 100 μM
control 10 μM 25 μM 50 μM 75 μM 100 μM
LDH release
[% of control]
neocortical neurons 6h
24h
***
*** ***
***
***
***
**
b
a
Fig. 1 Time-course effects of
BP-3 (10, 25, 50, 75, and
100 μM) on caspase-3 activity (a)
and LDH release (b)inprimary
cultures of mouse neocortical
cells at 7 DIV. The cells were
treated with BP-3 for 6 and 24 h.
The results are presented as a
percentage of the control. Each
bar represents the mean of three
to four independent
experiments ± SEM. The number
of replicates in each experiment
ranged from 5 to 8.
*
p<0.05,
**
p<0.01,and
***
p<0.001
versus control cultures
Mol Neurobiol (2018) 55:5059–5074 5065
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

0
50
100
150
200
250
25 μM BP-3
caspase-3 acttivity
[% of control]
25 μM BP-3
+DHA
+HX 531
***
###
a
control
0
50
100
150
200
250
25 μM BP-3
LDH release
[% of control]
25 μM BP-3
+ DHA
+HX 531
control
***
##
b
Fig. 3 Impact of the RXR
agonist and antagonist on BP-3-
induced caspase-3 activity (a)and
LDH release (b)inneocortical
cultures at 7 DIV. The primary
neocortical cultures were treated
with BP-3 (25 μM) for 24 h. The
results were normalized to the
absorbance of vehicle-treated
cells and are expressed as a
percentage of control. Each bar
represents the mean of three to
four independent
experiments ± SEM. The number
of replicates in each experiment
ranged from 5 to 8.
***
p<0.001
versus control cultures;
##
p<0.01
and
###
p< 0.001 versus the
cultures exposed to BP-3
0
0.5
1
1.5
2
2.5
RxrRxrRxr
RxrRxrRxr
RxrRxrRxr
mRNA
[folds Hprt normalized]
3h control
25 μM BP3
***
***
**
a
0
0.5
1
1.5
2
2.5
mRNA
[folds Hprt normalized]
6h control
25 μM BP3
***
**
b
0
0.5
1
1.5
2
2.5
mRNA
[folds Hprt normalized]
24h control
25 μM BP3
c
Fig. 4 Effect of BP-3 (25 μM) on
the mRNA expression levels of
Rxrα,Rxrβ,andRxrγin
neocortical cultures at 7 DIV.
Each bar represents the
mean ± SEM of three independent
experiments. The number of
replicates for each experiment
ranged from 2 to 3.
**
p<0.01and
***
p< 0.001 versus control
cultures
5066 Mol Neurobiol (2018) 55:5059–5074
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Effects of BP-3 on the Protein Expression Levels of RXRα,
RXRβ, and RXRγin Mouse Neocortical Cells
A 24-h exposure to BP-3 was necessary to detect changes
in the protein levels of the receptors. In the cultures treat-
ed with BP-3, RXRαexpression enhanced and reached
1.43 pg/μg of total protein (134% of the control). In cul-
tures exposed to BP-3 (25 μM) for 24 h, the concentration
of RXRβwas 0.76 pg/μg of total protein, and it was 56%
less than that in control cultures. In the cultures exposed
to BP-3, the level of RXRγreached 0.38 pg/μg, which
was 49% less than controls (Fig. 5a, b).
Western blot analysis determined the relative protein
expression levels of RXRα,RXRβ,andRXRγin mouse
neocortical cells at 7 DIV. Exposure to BP-3 (25 μM) for
24 h decreased the relative RXRβand RXRγprotein
levels by 61 and 56%, respectively. Treatment with BP-3
(25 μM) increased the relative RXRαprotein level by
49% (Fig. 5c, d).
Effect of BP-3 on the Distribution of RXRα,RXRβ,
RXRγ, and MAP2 Staining in Neocortical Cells
Immunofluorescence labeling was performed in parallel with
the measurements of receptor protein levels. Confocal micros-
copy revealed that RXRα,RXRβ, and RXRγwere localized
to neocortical cells at 7 DIV. A 24-h exposure to BP-3
(25 μM) increased RXRαstaining but reduced RXRβ-and
RXRγ-specific immunofluorescence. MAP2 staining con-
firmed the neural localization of the receptors and revealed
the BP-3-induced inhibition of neurite outgrowth (Fig. 6).
Influence of BP-3 on Caspase-3 Activity and LDH Release
in Neocortical Cells Transfected with RXRα,RXRβ,
and RXRγsiRNAs
A24-hexposuretoBP-3(25μM) only slightly reduced
caspase-3 activity and LDH release in the RXRβand RXRγ
siRNA-transfected cells, suggesting that the transfected cells
relative density of
bands [% control]
control BP-3
(25 μM)
RXRα 100±5.7 149±5.3***
RXRβ 100±6.4 39±6.8***
RXRγ 100±6.6 44±7.1***
BP-3
(25 μM)
control
24 h treatment
RXRα
RXRβ
RXRγ
β-actin
b
a
c
d
0
50
100
150
200
RxrRxrRxr
receptor protein
[% of control]
control
25 μM BP3
***
*** ***
pg/ug protein RxrRxrRxr
control 1,07157 1,67567 0,74483
BP-3 1,43959 0,76412 0,37776
Fig. 5 Effects of BP-3 on the
protein levels of RXRα,RXRβ,
and RXRγin mouse neocortical
cultures at 7 DIV. The neocortical
cells were cultured for 7 DIV and
then treated for 24 h with BP-3
(25 μM). The concentrations of
the receptors were measured
using specific ELISAs and are
presented as a percentage of the
control (a) and as picogram of
specific protein, i.e., RXRα,
RXRβ, and RXRγ,per
microgram of total protein (b).
For the western blot analyses,
protein samples were denatured,
electrophoretically separated,
transferred to PVDF membranes,
and subjected to immunolabeling
(c). The relative protein levels of
RXRα,RXRβ, and RXRγwere
presented as a percentage of the
control (d). Each bar or value
represents the mean of three
independent experiments ± SEM.
The number of replicates in each
experiment ranged from 2 to 3.
***
p< 0.001 versus control
cultures
Mol Neurobiol (2018) 55:5059–5074 5067
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

control
BP-3
(25 μM)
RXRα+RXRγ bright field
control
BP-3
(25 μM)
MAP2 dleifthgirb2PAM+αRXRαRXR
control
BP-3
(25 μM)
RXRα RXRγ
RXRαRXRβRXRα+ RXRβbright field
Fig. 6 Influence of BP-3 on the cellular distributions of RXRα(red),
RXRβ(blue), RXRγ(blue), and MAP2 (blue) in mouse neocortical
cultures at 7 DIV. The overlay of RXRα/RXRβ, RXRα/RXRγ, and
RXRα/MAP2 (red plus blue) staining with the bright field images are
shown. The primary neocortical cultures were treated with BP-3 (25 μM)
for 24 h. Analyzed using an LSM510 META, Axiovert 200M confocal
laser scanning microscope under a Plan-Neofluor 40×/1.3 Oil DIC
objective
5068 Mol Neurobiol (2018) 55:5059–5074
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were slightly less vulnerable to BP-3 than the non-transfected
cells. In comparison to the non-transfected cells, the effects of
BP-3 were reduced by 20% with respect to caspase-3 levels
andby30%withrespecttoLDHreleaseinthesiRNA-
transfected cells (Fig. 7a, b).
A 24-h exposure of RXRαsiRNA-transfected cells to
25 μM BP-3 reduced caspase-3 activity and LDH release to
55 and 50% of the control values, respectively (Fig. 7a, b).
These cells were much less vulnerable to BP-3 than the non-
siRNA-treated wild-type cells.
The effectiveness of mRNA silencing was verified by qPCR.
In this study, siRNA treatment decreased the RxrαmRNA level
by 83% (equal to 0.17-fold), the RxrβmRNA level by 64%
(equal to 0.36-fold), and the RxrγmRNA level by 68% (equal
to 0.32-fold) compared to the non-transfected wild-type cells.
Influence of BP-3 on Global DNA Methylation
in Neocortical Cultures
A 24-h exposure of neocortical cells to BP-3 (25 μM) caused
changes in the level of global DNA methylation. The treat-
ment with BP-3 reduced the methylation level by 55% of the
control value (Fig. 8).
Effects of BP-3 on HDAC and HATActivity in Neocortical
Cultures
A 24-h exposure of neocortical cultures to BP-3 reduced the
levels of HDAC and HAT activities. Treatment with BP-3
decreased the activities of HDAC and HAT by 32 and 17%
of the control value, respectively (Fig. 9a, b).
0
50
100
150
200
250
caspase-3 activity
[% of control]
$
non-transfected cells
$$$ ***
$$$
RXRγ
RXRβ
siRNA-transfected cells
RXRα
a
0
50
100
150
200
250
negative control BP-3 control BP-3 control BP-3 control BP-3
negative control BP-3 control BP-3 control BP-3 control BP-3
LDH release
[% of control]
$
$$$
***
$$$
non-transfected cells
siRNA-transfected cells
RXRα RXRβ RXRγ
b
Fig. 7 Effect of BP-3 (25 μM) on
caspase-3 activity (a) and LDH
release (b)inRXRα,RXRβ,and
RXRγsiRNA-transfected
neocortical cells. Each bar
represents the mean ± SEM of
three to four independent
experiments. The number of
replicates in each experiment
ranged from 5 to 8.
***
p<0.001
versus the non-transfected control
cultures;
$
p<0.05and
$$$
p< 0.001 versus the siRNA-
transfected control cultures
0
20
40
60
80
100
120
control 25 μM BP-3
Global DNA methylation [% of control]
***
Fig. 8 Influence of BP-3 on
global DNA methylation in
neocortical cultures at 7 DIV.
Primary neocortical cultures were
treated with BP-3 (25 μM) for
24 h. Total DNA was extracted
from cells followed by ELISA.
Each bar represents the mean of
three independent
experiments ± SEM. The number
of replicates in each experiment
ranged from 2 to 3.
***
p<0.001
versus control cultures
Mol Neurobiol (2018) 55:5059–5074 5069
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Effects of BP-3 on the Expression Profiles of Genes
Involved in Autophagy Using a Mouse Autophagy RT
2
Profiler PCR Array
To validate that BP-3 impairs autophagy in neuronal
cells, we analyzed a total number of 84 key genes that
are involved in this process. Among them, 71 genes
were differentially expressed in response to BP-3 treat-
ment: 60 were downregulated (green color) and 11 were
upregulated (red color) in the BP-3-treated samples. The
downregulated genes were Akt1,Ambra1,App,Atg10,
Atg16l1,Atg16l2,Atg3,Atg4b,Atg4d,Atg7,Atg9a,
Atg9b,Bcl2,Bid,Cdkn1b,Cdkn2a,Cln3,Ctsb,Ctsd,
Ctss,Cxcr4,Dram1,Eif2ak3,Eif4g1,Esr1,Gaa,
Gabarapl1,Gabarapl2,Hdac1,Hdac6,Hgs,
Hsp90aa1,Hspa8,Htt,Igf1,Irgm1,Lamp1,Map1lc3a,
Map1lc3b,Mapk14,Mapk8,Mtor,Npc1,Pik3c3,
Pik3r4,Prkaa1,Pten,Rab24,Rb1,Rgs19,Rps6kb1,
Snca,Sqstm1,Tgfb1,Tgm2,Tmem74,Ulk1,Ulk2,
Uvrag,andWip i1. The upregulated genes were Bad,
Bak1,Bax,Bcl2l1,Bnip3,Casp3,Casp8,Dapk1,Fas,
Nfkb1,andTr p53 (Fig. 10).
Effects of BP-3 on Autophagosome Detection
A 24-h exposure of neocortical cultures to BP-3 reduced the
level of autophagosomes in mouse neuronal cell cultures.
Treatment with BP-3 decreased autophagosome level by
29% compared to the control value (Fig. 11).
Effects of BP-3 on the Protein Expression Levels of LC3A
and LC3B in Mouse Neocortical Cells
InculturesexposedtoBP-3(25μM) for 24 h, the concentration
of LC3A was 1.23 pg/μg of total protein, which was 126%
higher than that in control cultures. In the cultures exposed to
BP-3, the level of LC3B reached 0.21 pg/μg, which was re-
duced by 75% compared to controls (Fig. 12a, b).
Discussion
The primary aim of the present study was to evaluate the
neurotoxic effects of BP-3 with an emphasis on apoptosis,
autophagy, the epigenetic status of neuronal cells, and the mo-
lecular mechanisms involving RXRs. The results of the study
demonstrated that BP-3 caused neurotoxicity, as evidenced by
the concentration-dependent activation of caspase-3 and LDH
release in mouse neocortical cells. In the present study, neo-
cortical cells responded to 25–100 μM BP-3. This was in line
with our previous study in which 25 μM BP-3 was determined
to be the lowest effective concentration at 24 h of exposure
[13]. The used concentration is environmentally relevant since
BP-3 has been found in human adipose tissue at concentra-
tions up to 5 mg/kg (~22 μM) [37]. The ability of BP-3 to
cross the blood-brain barrier has been shown; after it was
applied by gavage, the Erαand ErβmRNA expression levels
were changed in the rat pituitary gland [38]. Moreover, a BP-3
analogue (BP-4) changed the expression levels of many genes
0
20
40
60
80
100
120
control 25 µM BP-3 positive control negative control
Histone Deacetylase
(HDAC) Activity
[% of control]
control
25 µM BP-3
positive control
negative control
***
a
0
50
100
150
200
250
control BP-3 25 uM
p
ositive
Histone Acetyltransferase
(HAT) Activity
[% of control]
control
BP-3 25 uM
positive
**
b
Fig. 9 Effects of BP-3 (25 μM)
on HDAC (a)andHAT(b)
activity in the primary cultures of
mouse neocortical cells at 7 DIV.
The cells were treated with BP-3
for 24 h. The results are presented
as a percentage of the control.
Each bar represents the mean of
three to four independent
experiments ± SEM. The number
of replicates in each experiment
ranged from 5 to 8.
**
p<0.01and
***
p< 0.001 versus control
cultures
5070 Mol Neurobiol (2018) 55:5059–5074
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

in the Danio rerio brain when it was added to water [39]. In the
present study, the neurotoxic effects of BP-3 involved en-
hanced LDH release and impaired cell survival (calcein AM
staining), which were accompanied by the induction of apo-
ptosis as estimated by caspase-3 activation and increased ap-
optotic body formation (Hoechst 33342 staining).
In this study, both the neurotoxic and apoptotic effects of BP-3
were inhibited by HX 531, which is a potent RXR antagonist.
These data suggest that RXR receptors are involved in BP-3-
induced effects in neuronal cells. Previously, we demonstrated
an important role of RXRα-andRXRβ-intracellular signaling in
the propagation of DDE- and nonylphenol-induced apoptosis
during the early stages of neural development [22–24].
Recently, the involvement of RXRs in apoptotic signaling in
retina pigment epithelial cells and gastrointestinal cancer cells
has been shown [40,41]. The role of RXRs in neuronal survival
and neurotoxicity is complex and depends on RXR
heterodimerization partners. Interestingly, A/B domain region
of RXR receptors was found to cause growth inhibition or
rexinoid-induced apoptosis [42]. Elevated levels of RXRαgene
and protein expression have been found in individuals suffering
from dementia [43], and knockout of RXRγimpairs the working
memory in mice [44]. In the present study, BP-3 altered the
mRNA expression levels of Rxrα,Rxrβ,andRxrγin a time-
dependent manner. Increased expression of RxrαmRNA and
reduced expression levels of Rxrβand RxrγmRNA were ob-
served at 3 h of exposure, which mirrored the alterations in the
estimated protein levels of the receptors at 24 h of exposure.
These profiles were also concurred with the immunofluorescent
labeling of RXRα,RXRβ,andRXRγfollowingBP-3treat-
ment. Based on these data, we hypothesize that the BP-3-
Fig. 10 Gene expression patterns of autophagy showing the 71 genes
that were significantly differentially expressed between the control and
BP-3-treated groups. Among these genes, 60 genes were downregulated
(green color) and 11 genes were upregulated (red color) in the BP-3-
treated samples compared to the control
0
20
40
60
80
100
120
control 25 μM BP-3
fluorescent autophagosome marker
[% of control]
Autophagosome detection
**
Fig. 11 The effect of 25 μM BP-3 on autophagosome levels at 24 h. The
data are expressed as the mean ± SEM of four independent experiments,
consisting of eight replicates per treatment group.
**
p< 0.001 versus the
control
b
a
0
50
100
150
200
250
300
B3CLA3CL
protein level [% of control]
control
25 µM BP-3
***
***
pg/μg protein LC3A LC3B
control 0.544655 0.887605
25 μM BP-3 1.235620 0.217725
Fig. 12 Effects of BP-3 on the protein levels of LC3A and LC3B in
mouse neocortical cultures at 7 DIV. The neocortical cells were cultured
for 7 DIV and then treated for 24 h with BP-3 (25 μM). The
concentrations of the receptors were measured using ELISA and are
presented as a percentage of the control (a) and as picogram of the
specific protein (LC3A or LC3B) per microgram of total protein (b).
***p< 0.001 versus control cultures
Mol Neurobiol (2018) 55:5059–5074 5071
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

induced apoptosis of neuronal cells is mediated by the attenua-
tion of RXRβ/RXRγand the stimulation of RXRαsignaling
pathways. To test this hypothesis, we used gene-specific
siRNAs. Compared to non-transfected wild-type cells, silencing
of RXRαcaused a substantial reduction of BP-3-induced
caspase-3 activity and LDH release; however, silencing of
RXRβand RXRγdid not affect the BP-3-induced effects.
Therefore, we suggest that the BP-3-induced apoptosis of neuro-
nal cells is mediated by the stimulation of RXRαsignaling and
the attenuation of RXRβ/RXRγsignaling, which is in line with
the BP-3-induced alterations in RXR expression levels.
Previously, we demonstrated that stimulation of ERβ/GPR30
and impairment of ERα/PPARγsignaling were involved in
propagation of BP-3-induced apoptosis [13]. Taking into account
our previous and present data, one may assume that in neuronal
cells BP-3 stimulates RXRα/ERβ/GPR30 and inhibits
RXRβ/RXRγ/ERα/PPARγintracellular pathways. It has been
documented that ERβmay downregulate ERα/PPARγand dis-
rupt RXRα/PPARγsignaling [45]. RXRαis an obligatory het-
erodimer partner of PPARγas well as RXRβ/RXRγ[46]. We
suggest that in our study, BP-3 by upregulation of ERβimpaired
RXRβ/RXRγ/ERα/PPARγsignaling. We also postulate that
BP-3 was able to destroy RXRβand RXRγheterodimers but
not RXRαhomodimers, possibly due to stronger covalent bond-
ing in homodimers than in heterodimers.
In addition to the demonstration that the BP-3-induced apo-
ptosis involves the activation of RXRαsignaling and the impair-
ment of RXRβ/RXRγsignaling, we showed that BP-3 inhibited
global DNA methylation as well as reduced HDAC and HAT
activities in mouse embryonic neuronal cells. Aberrant DNA
methylation and other epigenetic modifications have been found
to be associated with development and normal cellular homeo-
stasis as well as growing number of human diseases. Low doses
of a pesticide DDT have been postulated to cause hypomethyla-
tion of specific gene regions in the young brain and impaired
hippocampal neurogenesis [25]. Recently, we showed evidence
of the involvement of global DNA hypomethylation in DDT-
induced depressive-like effects and the DDE-induced apoptosis
of primary neuronal cells [22,47]. Exposures to xenobiotics such
as tributyltin (TBT) and triphenyltin (TPT) have been shown to
alter HDAC and HAT activity [48]. The global DNA hypome-
thylation as well as diminished HDAC and HAT activity suggest
of chromosomal instability thus can cause inappropriate gene
expression pattern. We postulate that global DNA hypomethyla-
tion and diminished HDAC activity are responsible for the BP-3-
induced increase in the RXRαexpression level, whereas dimin-
ished HAT activity corresponds to reduced expression of RXRβ/
RXRγin response to BP-3 treatment in our study.
Based upon our data, we suggest that altered epigenetic
status caused by BP-3 treatment may not only be involved
in apoptosis and the disruption of RXR signaling but may also
affect autophagy. Autophagy is neuroprotective and is respon-
sible for degrading damaged organelles and misfolded
proteins. Dysregulation of autophagy has been linked to neu-
ral degenerative diseases such as Alzheimer’sdisease,
Parkinson’s disease, Huntington’s disease, amyotrophic later-
al sclerosis, and encephalopathy. In the present study, the at-
tenuation of the autophagic process was confirmed by the
downregulation of genes involved in autophagy as detected
by the microarray analysis, decreased autophagosome forma-
tion, and the reduced ratio of LC3B to LC3A. We hypothesize
that BP-3 induced the downregulation of genes related to au-
tophagy through decreased HAT activity in mouse neurons.
Conclusions
In summary, we showed for the first time that the BP-3-
induced apoptosis of neuronal cells is mediated via the stim-
ulation of RXRαsignaling and the attenuation of RXRβ/
RXRγsignaling, as demonstrated by the use of selective an-
tagonist and specific siRNAs as well as by measuring the
mRNA and protein expression levels (qPCR, ELISA, western
blot, and immunofluorescent labeling) of the receptors. This
study also demonstrated that the use of BP-3 at environmen-
tally relevant concentrations was able to inhibit autophagy and
disrupt the epigenetic status of neuronal cells, which may
increase the risk of neurodevelopmental abnormalities and/or
neural degeneration.
Abbreviations
AM acetoxymethyl
ANOVA analysis of variance
BP-3 benzophenone-3
BPA bisphenol A
BPs benzophenones
DDE dichlorodiphenyldichloroethylene
DIV days in vitro
DMSO dimethyl sulfoxide
EDCs endocrine disrupting chemicals
ELISA enzyme-linked immunosorbent assay
FBS fetal bovine serum
GFAP glial fibrillary acidic protein
HAT histone acetyltransferase
HDAC histone deacetylase
Hprt hypoxanthine-guanine phosphoribosyltransferase
LDH lactate dehydrogenase
PBS phosphate-buffered saline
qPCR quantitative polymerase chain reaction
RT room temperature
RXR retinoid X receptor
TBT tributyltin
TPT triphenyltin
UV ultraviolet light
5072 Mol Neurobiol (2018) 55:5059–5074
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Acknowledgments This study was financially supported by grant no.
2014/13/N/NZ4/04845 from the National Science Centre of Poland and
the statutory fund of the Institute of Pharmacology at the Polish Academy
of Sciences in Krakow, Poland.
Agnieszka Wnuk and Joanna Rzemieniec received scholarships from
the KNOW, which was sponsored by the Ministry of Science and Higher
Education in Poland.
This publication was also supported by funding from the Jagiellonian
University within the SET project, which was co-financed by the
European Union. The authors thank Professor Elżbieta Pyza of the
Department of Cell Biology and Imaging at the Institute of Zoology of
Jagiellonian University in Krakow for suggestions and for kindly provid-
ing access to the LSM 510 META, Axiovert 200M, ConfoCor 3 confocal
microscope (Carl Zeiss MicroImaging GmbH, Jena Germany).
The manuscript has been edited by the American Journal Experts for
English language and grammar - C2A7-BFBD-03E0-6E26-A2D8.
The publication charge was supported by KNOW funds MNiSW-DS-
6002-4693-26/WA/12.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
Research Involving Human Participants and/or Animals This arti-
cle does not contain any studies with human participants performed by
any of the authors.
All procedures performed in studies involving animals were in accor-
dance with the ethical standards of the institution or practice at which the
studies were conducted.
All members of the research team received approval from the local
ethical committee on animal testing. Animal care followed official gov-
ernmental guidelines, and all efforts were made to minimize suffering and
the number of animals used.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appro-
priate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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