Involvement of NLRP3/Caspase-1/GSDMD-Dependent Pyroptosis In BPA-Induced Apoptosis of Neuroblastoma Cells


 BackgroundBisphenol A (BPA) is an additive in polycarbonate and epoxy resin particles with endocrine disrupting effects. Previously it has been reported that BPA is neurotoxic via induction of cell apoptosis and inflammation, and our recent studies showed that even at nanomolar concentrations BPA accelerated the apoptosis and death of human neuroblastoma cells from both genders, whose mechanisms remain, however, unidentified.ResultsHuman neuroblastoma cell lines developed from male and female subjects, IMR-32 and SK-N-SH, respectively, were exposed to BPA at concentrations ranging from 1 nM to 100 μM, for 24 h, with or without epigallocatechin gallate (EGCG, 4 or 8 μM), Z-YVAD-FMK (1 or 10 μM, caspase-1 inhibitor), and ICI182.780 (100 nM or 3 μM, estrogen receptor inhibitor) as modulators. The results showed that BPA nonlinearly upregulated the levels of IL-18, ASC, GSDMD and NLRP3 mRNAs and that of NLRP3, caspase-1, GSDMD and IL-1β proteins in IMR-32 and SK-N-SH cells. Noticeably, the mRNA levels of caspase-1 and IL-1β were changed differently in the two cell lines: the level of caspase-1 mRNA was enhanced in IMR-32 cells but suppressed in SK-N-SH cells, and that of IL-1β was suppressed in IMR-32 cells but enhanced in SK-N-SH cells. The level of GSDMD expression in situ was along with the increase in the release of IL-1β, IL-18, caspase-1 and lactate dehydrogenase (LDH). Additionally, Z-YVAD-FMK, ICI182.780 and EGCG significantly reversed the changes of the above mRNAs/proteins induced by BPA. BPA significantly reduced the level of the reactive oxygen species and the rate of LDH leakage and apoptosis, while obviously increased the cell viability and the mitochondrial membrane potential. Meanwhile, Z-YVAD-FMK and ICI182.780 abruptly reduced the levels of Bak1, Bax, Bcl-2 and caspase-3 proteins induced by BPA. ConclusionAs mediated by the estrogen receptor, BPA may induce the pyroptosis of neuroblastoma cells through NLRP3/caspase-1/GSDMD signaling pathway, and caspase-1-dependent pyroptosis may be involved in BPA-induced apoptosis, which is alleviated by EGCG, an anti-oxidation agent.


Introduction
Bisphenol A (BPA) is a kind of phenolic compound widely used in the production of polycarbonate, polystyrene resin, epoxy resins, electronic equipment and food and beverage packaging (Chang et al., 2015;Staples et al., 1998).In 2015, the global consumption of BPA reached 7.7 million metric tons.Moreover, it will reach up to 10.6 million metric tons by 2022 with an annual growth rate of 4.8% (Wang et al., 2019).The mass production and widespread use of BPA lead to a large amount of release into the environment.Humans are exposed to BPA through ingestion, inhalation and dermal contact (Hines et al., 2017;Stahlhut et al., 2009).As a result, BPA can be detected in human amniotic uid, blood, breast milk, placenta, sweat and urine (Shi et al., 2017;Yu et al., 2019).
Multiple in vivo and in vitro studies have shown that BPA has neurotoxic effects.For example, BPA exposure decreases the cognitive function, the ability of learning and memory and social response in rats (Negri-Cesi 2015; Zhang et al., 2019;Braun et al., 2017).Studies in the experimental models using primary hippocampus cells, HT-22 cell line and neuroblastoma cells have shown that low-dose BPA exposure induces oxidative stress and increases mitochondrial apoptosis (Meng et al., 2021;Wang et al., 2021;Pang et al., 2019).Epidemiological studies also show that BPA exposure during pregnancy or adolescence may cause neurobehavioral problems in childhood/adolescence (e.g., attention de cit hyperactivity disorder, depression, anxiety, autism spectrum disorders and social cognition/communication de cits) (Miodovnik et al., 2011;Perera et al., 2016;Li et al., 2018).To explore the mechanism of BPA induced neurotoxicity, various attempts have been made and distinct views have been put forward, including the reduction of synaptic plasticity, the inhibition of neurogenesis, the generation of oxidative stress and the induction of autophagy and apoptosis (Santoro et al., 2019).Among them, apoptosis and pyroptosis triggered by oxidative stress are considered as the initial steps of all injuries (Lan et al., 2020;Li et al., 2015;Wang et al., 2019).
Pyroptosis, which is considered to be caspase-1-dependent cell death (Shi et al., 2017), is a newly identi ed pro-in ammatory form of programmed cell death.Caspase-1 mediated pyroptosis is often activated by cannonical in ammasomes (Zhang et al., 2018).In ammasomes are large multiprotein complexes that play critical roles in the production of interleukin (IL)-1β and IL-18.Pyrin domain containing 3 (NLRP3) in ammasome, a member of nucleotide binding oligomerization domain-like receptor (NLR) family, is most widely characterized and implicated in in ammatory diseases (Kim et al., 2015).The NLRP3 in ammasome consists of a sensor (NLRP3), an adaptor (ASC, also known as PYCARD) and an effector (pro-caspase-1).ASC is a protein containing pyrin and CARD domain, which promotes the assembly of certain types of in ammasomes.Caspase-1 is activated by upstream in ammasome NLRP3 (Liu et al., 2018;Miao et al., 2011).Once activated, caspase-1 contributes to the maturation of IL-1β and IL-18, and cleaves and activates gasdermin D (GSDMD).GSDMD acts as the pyroptosis executor and nally releases the N-terminal domain (GSDMD-N), which leads to the opening of membrane pores.Subsequently, intracellular contents, such as IL-18, IL-1β and lactate dehydrogenase (LDH), are released into the extracellular environment, eventually leading to the occurrence of pyroptosis (Shi et al., 2017;Strowig et al., 2012).Hence, NLRP3/Caspase-1/GSDMD pathway is very important in regulating pyroptosis.
Our previous study has shown that environmental BPA exposure can induce oxidative stress and mitochondrial apoptosis in different gender neuroblastoma cells (IMR-32 and SK-N-SH cells) (Wang et al., 2021).However, the mechanisms of the pyroptosis in IMR-32 and SK-N-SH cells exposed to low-dose BPA and the relationship between BPA exposure and apoptosis is not known.Hence, this study was conducted to further explore the mechanism of BPA-induced neurotoxicity in IMR-32 and SK-N-SH cells.In this study we investigated whether low-dose BPA can induce pyroptosis via the NLRP3/Caspase-1/GSDMD signaling pathway with the mediating of estrogen receptor (ER) and its relationship with apoptosis.

Quantitative real-time PCR analysis
Total RNA was extracted from IMR-32 and SK-N-SH cells using Trizol reagent (Takara, Japan).After the measurement of RNA concentration by a NanoDrop Spectrophotometer (NanoDrop-2000, Thermo, USA), 1 µg of total RNA was reversely transcribed into cDNA using a Reverse Transcription Kit (Takara, Japan) for the quantitative real-time PCR (qRT-PCR).A real-time PCR machine (CFX96 Touch, Bio-Rad, USA) was used to perform qRT-PCR with a One Step SYBR® PrimeScript™ RT-PCR Kit II (Takara, Japan).As described in Table 1, the speci c primer sequences of duck NLRP3, caspase-1, IL-1β, IL-18, ASC, GSDMD and β-actin were obtained from NCBI GenBank and designed with primer premier 6.0 software.The relative abundance of mRNA was calculated using the 2 − ΔΔCT method (Livak and Schmittgen, 2001).
Results were normalized to the mean of housekeeping gene (i.e., β-actin), and expressed as the relative expression of mRNA levels compared to the control samples.).The immune complexes with speci c proteins were visualized using an ECL detection kit.The densitometry of protein bands was analyzed using a Bio-Rad image analyzer (ChemiDoc Touch, USA).The density ratio of the β-actin band to its corresponding lane was used to correct the amount of protein loading.

Immuno uorescence staining
IMR-32 and SK-N-SH cells were seeded on 24-well plates containing cell slides for 36 h.Next, cells were washed twice with PBS, xed with 4% paraformaldehyde for 20 min, and washed three times with PBS.
After being permeabilized with 0.1% Triton X-100 for 10 min at room temperature and blocked with 5% bovine serum albumin (BSA) in PBS, cells were incubated with the primary antibodies of GSDMD (1:200, Sigma, USA, # HPA044487) at 4°C overnight.After being washed for three times with PBS, cells were incubated with the secondary antibodies for 60 min and then the nuclei were re-stained with DAPI (Beyotime, C1005).Slides were visualized using a Leica DM6 uorescent microscope.

Enzyme-linked immunosorbent assay
Caspase-1, IL-1β and IL-18 levels in the supernatants of IMR-32 and SK-N-SH cultures were measured by a microtiter plate reader at 450 nm according to the enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bioengineering Institute, China).

LDH release assay
Pyroptosis was quantitated by determining the activity of LDH released into the culture medium after various treatments.The LDH activity in the supernatant of each culture was determined by using the LDH cytotoxicity assay kit (Beyotime, C0016), and expressed as a percentage of the total LDH in each cell lysate.

Cell viability assay
Cell viability was determined by using the Cell Counting Kit-8 (CCK-8, APExBIO, USA).Brie y, IMR-32 and SK-N-SH cells were cultured in 96-well plates.When the cell con uency reached 70-75%, they were treated with BPA and YVAD/or ICI.After 24 h treatments, 10 µL of CCK-8 were added to each well.Then, IMR-32 and SK-N-SH cells were further cultured for 4 h.Finally, the absorbance of each well was read at 450 nm using a microplate reader (PerkinElmer Enspire, USA).

Flow cytometric analysis
Following various treatments, IMR-32 and SK-N-SH cells were resuspended by using trypsin, and collected by centrifugation.The mitochondrial membrane potential (MMP) and ROS levels were tested using an MMP kit (Beyotime, C2006) and a ROS kit (Beyotime, S0033S).Then cell analysis was performed by ow cytometry (FACS Aria III, BD Biosciences, San Jose, CA, USA) at the 590 or 488 nm excitation wavelength and 525 nm emission wavelength.

Apoptosis-Hoechst staining
IMR-32 and SK-N-SH cells were seeded on 24-well plates containing cell slides.After 36 h, the cells were xed with 4% paraformaldehyde for 20 min and stained with Hoechst 33258 for 5 min at room temperature.After being washed three times with PBS, each slide was visualized using a Leica DM6 uorescent microscope (Lecia, Gemany).Three elds per well/slide were randomly selected for counting the stained cells by using Image J software.

Statistical analysis
All results are presented as means ± standard deviations of at least three independent experiments.Statistical analysis was completed by GraphPad Prism 6.0 (GraphPad Inc., La Jolla, CA, USA) and SPSS version 18.0 (SPSS Inc., Chicago, IL, USA).One-way analysis of variance (ANOVA) was used to test the difference between groups, with signi cance being set at the level of p < 0.05.

Effects of BPA exposure on pyroptosis-related mRNA levels
To test our hypothesis that BPA induces pyroptosis through NLRP3/Caspase-1/GSDMD signaling pathway, the mRNA expressions of NLRP3, GSDMD, ASC, IL-1β, IL-18 and caspase-1 in IMR-32 and SK-N-SH cells were determined (shown in Fig. 1).For IMR-32 cells, mRNA expressions of NLRP3, GSDMD, IL-18 and caspase-1 in the groups with BPA at high concentrations (generally above 1 µM) were signi cantly increased, as compared with the control group (p < 0.05 or 0.01).Interestingly, NLRP3, IL-18 and caspase-1 mRNA expressions in IMR-32 cells treated with 1 nM BPA were still signi cantly increased (p < 0.05).BPA treatments also changed the level of ASC mRNA.Treatment with 1 nM BPA signi cantly decreased the level of ASC mRNA level while that with 100 µM BPA signi cantly increased the level (p < 0.05); however, IL-1β mRNA level in IMR-32 cells under various treatments was signi cantly reduced except for that with 1 µM BPA (p < 0.05 or 0.01).Moreover, trends of change generally similar to those in IMR-32 cells (described above) were observed in SK-N-SH cells.For example, the mRNA levels of NLRP3, GSDMD, ASC, IL-1β and IL-18 in the treatments with BPA at high concentrations were signi cantly higher than in the control group.However, as different from the IMR-32 cell line which was developed from the neural tissue of a male subject, BPA treatments signi cantly decreased the expression of caspase-1 mRNA in the SK-N-SH line (derived from the tissue of a female subject).Moreover, no signi cant differences were observed between the treatment with 1nM BPA and the control in SK-N-SH cells (p > 0.05).

Effects of BPA exposure on levels of pyroptosis-related proteins
To further con rm whether the gender difference and concentration-dependent effect of BPA at the transcriptional level also exist at the translation level, protein expressions of NLRP3, caspase-1, GSDMD and IL-1β were determined.As shown in Fig. 2A-E, the pyroptosis-related proteins in IMR-32 and SK-N-SH cells generally showed similar trends of change, such as NLRP3, caspase-1, GSDMD and IL-1β, whose levels in IMR-32 cells increased signi cantly after 24 h exposure to micromolar levels of BPA as compared with the controls (p < 0.05 or 0.01).Particularly, 1 nM BPA treatment also led to a signi cant increase in NLRP3, GSDMD and IL-1β (p < 0.05), which is consistent with those variations in mRNA expressions after treatment with BPA.It should be noted that the translation level of IL-1β in IMR-32 cells increased signi cantly, which was on the contrary of the trend of change at the transcriptional level.Similar to IMR-32 cells, the expressions of NLRP3, caspase-1, GSDMD and IL-1β proteins in SK-N-SH cells treated with 100 nM and/or 1-100 µM BPA for 24 h were signi cantly higher than those in the control group (p < 0.05 or 0.01).
In innate immunity, pyroptosis is executed by the pore-forming protein GSDMD, which is activated by its cleavage mediated by caspase-1 (Shi et al., 2015).Hence, the caspase-1-p20 and GSDMD-N domains of GSDMD were detected by immunoblotting in IMR-32 and SK-N-SH cells (shown in Fig. 2A).Similar to the changes in its transcription, a non-monotonic dose-response relationship between BPA concentrations and the levels of translation of some proteins in neurons, including the caspase-1-p20 and GSDMD-N domains of GSDMD, were observed in both IMR-32 and SK-N-SH cells after 24 h treatments.To further veri cation the occurrence of pyroptosis, we used immuno uorescence staining to analyze the localization and expression of GSDMD (Fig. 2F).The increase of green uorescence further con rmed that BPA induced pyroptosis in IMR-32 and SK-N-SH cells.

Effects of BPA on the contents of in ammatory factors in the supernatants of IMR-32 and SK-N-SH cells and the cell morphology
Next, the effects of BPA on the levels of in ammatory factors (LDH, caspase-1, IL-1β and IL-18) in the cell supernatants were studied.As shown in Fig. 3 (B-E), after 24 h treatment, BPA at various concentrations (1 nM, 100 nM and 1-100 µM) signi cantly increased the concentrations of in ammatory factors (i.e., LDH, caspase-1, IL-1β and IL-18) in the supernatant of IMR-32 cells (p < 0.05 or 0.01).In SK-N-SH cells, these in ammatory factors were also signi cantly increased after 24 h exposure to 100 nM, 1 µM, or 100 µM of BPA.The relationship of BPA concentration and LDH, caspase-1, IL-1β and IL-18 levels of IMR-32 and SK-N-SH cells were not linear, but non-monotonic.Even low dose of BPA (i.e., nM) can also induce the pyroptosis of IMR-32 and SK-N-SH cells.The ballooning of the cell membrane further con rmed a characteristic morphological of pyroptosis induced by BPA (Fig. 3A).It showed that the BPA-induced pyroptosis formed a gap, which caused the release of LDH and in ammatory factors in IMR-32 and SK-N-SH cells.

Inhibition of the pyrotosis of IMR-32 and SK-N-SH cells exposed to BPA by EGCG
In our previous studies, we found that ROS production played an important role in the BPA-induced apoptosis in IMR-32 and SK-N-SH cells by adding EGCG (an antioxidant) (Wang et al., 2021).We hypothesize that ROS also plays a similar role in the BPA-induced pyroptosis by detecting the expression levels of factors related to NLRP3/Caspase-1/GSDMD pathway after adding EGCG.Results showed that EGCG signi cantly attenuated the BPA-induced effects, including the increasing of LDH, caspase-1, IL-1β and IL-18 contents in cell supernatants, the rising of mRNA levels of NLRP3, caspase-1, GSDMD, ASC, IL-1β and IL-18 and the increasing of protein levels of caspase-1, GSDMD, IL-1β and NLRP3 in IMR-32 / SK-N-SH cells, the decreasing of IL-1β mRNA level in IMR-32 cells, the reduction of caspase-1 mRNA level in SK-N-SH cells, as well as the reducing of MMP level in IMR-32 and SK-N-SH cells (Fig. 4).

Attenuation of pyroptosis and mitochondrial apoptosis in IMR-32 and SK-N-SH cells exposed to BPA by caspase-1 deletion
To verify the role of caspase-1 in the BPA-induced pyroptosis and explore the relationship between apoptosis and pyroptosis, IMR-32 and SK-N-SH cells were pre-treated and co-treated with YVAD.YVAD is a potent cell permeable and irreversible Caspase-1 inhibitor, which can reduce the activation of caspase-1, and then inhibit pyroptosis.As shown in Fig. 5A-B and E, the MMP and the cell viability of IMR-32 and SK-N-SH cells in the BPA-treated groups dramatically decreased compared with the control groups (p < 0.05 or p < 0.001).But after the co-treatment with YVAD, there was no signi cant difference of MMP and cell viability between the YVAD + BPA group and the control group (p > 0.5).In the meantime, signi cant difference of the cell viability existed between the BPA group and the YVAD + BPA group in SK-N-SH cells (p < 0.001).Additionally, the results showed that the rates of apoptosis and the contents of LDH of the BPA-treated groups in IMR-32 and SK-N-SH cells signi cantly increased compared to those of the control group (p < 0.05 or p < 0.01).But in the BPA + YVAD group, the rates of apoptosis and the contents of LDH decreased and were signi cantly lower than those in the BPA group (p < 0.001) (Fig. 5D, C and F).There was no signi cant difference of above indicators between the control and the YVAD group (p > 0.05).

Induction of ROS production, apoptosis and pyroptosis by BPA via actions on ER
Since BPA can activate the ER, we investigated whether the observed effects of BPA on IMR-32 and SK-N-SH cells were due to the estrogenic effect of BPA.Hence, ICI, a compound which can block the ER pathway, was used to verify it.Before cells were co-treated with 10 µM or 100 µM BPA and ICI for 24 h, IMR-32 and SK-N-SH cells were pretreated with 100 nM and 3 µM ICI for 1 h.As described in Fig. 6A-J, BPA signi cantly elevated the levels of ROS, the conttent of LDH and the rate of apoptosis, the mRNA expressions of NLRP3, GSDMD, ASC and IL-18, the protein expressions of Bax, Bak1, caspase-3, caspase-1, NLRP3, GSDMD and IL-1β, and reduced the Bcl-2 protein expressions in IMR-32 and SK-N-SH cells.Moreover, the mRNA level of intracellular caspase-1 in IMR-32 cells and the mRNA level of IL-1β in SK-N-SH cells were also signi cantly upregulated, while the expression of IL-1β mRNA in IMR-32 cells and the expression of caspase-1 mRNA in SK-N-SH cells were signi cantly inhibited (p < 0.05 or p < 0.01 or p < 0.001).Finally, BPA led to a decrease of cell viability.However, ICI could signi cantly ameliorate the changes of the above indicators caused by BPA.The activation of caspase-1 and the cleavage of GSDMD were also signi cantly inhibited by ICI.

BPA may induce pyroptosis by NLRP3/Caspase-1/GSDMD signaling pathway
Pyroptosis is a newly identi ed form of in ammatory cell death triggered by caspase-1, which is characterized by caspase-1-dependence, the accumulation of GSDMD fragments, the formation of membrane pores and the leakage of intracellular contents, such as IL-18, IL-1β, LDH, etc. (Pi et al., 2021).Studies have shown that pyroptosis is involved in various diseases, particularly neurodegenerative diseases (Voet et al., 2019;Zhao et al., 2019).It is reported that polystyrene microplastics, which continuously pollute the environment, can induce pyroptosis and apoptosis of ovarian granulosa cells via the NLRP3/caspase-1 signaling pathway triggered by oxidative stress (Hou et al., 2021).Moreover, the activation of NLRP3 plays an important role in caspase-1-dependent pyroptosis.For example, one study has shown that the activation of NLRP3 is critical for the cell pyroptosis induced by propofol (Sun et al., 2019).Caspase-1 is responsible to transform pro-IL-1β to IL-1β (the active form), which is thus named IL-1β converting enzyme (Zhao et al., 2012).Activated caspase cleaves the connection between the Nterminal and C-terminal domains of GSDMD protein and releases the active N-terminal domain, which destroys the cell membrane and causes pyroptosis (Ding et al., 2016).In this study, BPA treatments can not only signi cantly change the mRNA levels of NLRP3, GSDMD, ASC, IL-1β, IL-18 and caspase-1 and the protein levels of Bcl-2, Bax, Bak1, caspase-3, NLRP3, caspase-1, GSDMD and IL-1β, but also increase the secretion of IL-1β, IL-18, caspase-1 and LDH in neuroblastoma cells, especially in IMR-32 cells from male.All results suggest that BPA induces pyroptosis of nervous cells, and male IMR-32 cells are more susceptible to BPA than female SK-N-SH cells.Moreover, even 1 nM BPA treatments can lead to the similar effects and consequently promote the pyroptosis of IMR-32 cells.These results suggest that BPA treatments can potently induce pyroptosis of IMR-32 and SK-N-SH cells.In the same time, our study also shows that the BPA-induced pyroptosis not only triggers nervous cell death but also mediates the releases of IL-1β, IL-18, caspase-1 and LDH, which will lead to an excessive proin ammatory process.
Based on the above factors, LDH release is considered to be one of the main characteristics of pyroptosis (Fink et al., 2008).As an inhibitor of pyroptosis, YVAD is reported to restrain the caspase-1 activation, reduce the secretion of IL-1β and IL-18 and suppress the occurrence of pyroptosis in cells (Yue et al., 2019;Zamani et al., 2020).In this study, the caspase-1 mRNA and protein levels were signi cantly inhibited after YVAD treatment as expected, which further con rms that YVAD inhibits the pyroptosis of IMR-32 and SK-N-SH cells by inhibiting the activation of caspase-1.Simultaneously, the differences of mRNA expressions (i.e., NLRP3, GSDMD, ASC, IL-1β and IL-18), protein expressions (i.e., NLRP3, GSDMD and IL-1β) and GSDMD uorescence expression between the BPA-treated group and the BPA + YVAD treated group suggest that BPA can induce pyroptosis in IMR-32 and SK-N-SH cells via NLRP3/Caspase-1/GSDMD signaling pathway.
Our previous studies has shown that BPA can promote ROS production, leading to oxidative stress and apoptosis in IMR-32 and SK-N-SH cells (Wang et al., 2021).ROS is involved in pyroptosis and has a signi cant action in regulating pyroptosis, which has been con rmed in many studies (Chen et al., 2016;Zhang et al., 2021).Excessive ROS may activate the NLRP3 in ammasome signaling pathway (Sun et al., 2019).In addition, ROS can mediate the NLRP3/Caspase-1/GSDMD signaling pathway involved in the process of cytotoxicity (Yang et al., 2020).Therefore, we speculated that ROS might also be involved in the BPA-induced pyroptosis of IMR-32 and SK-N-SH cells via the NLRP3/Caspase-1/GSDMD pathway.In our study, if cells were co-treated with BPA and EGCG, the differential expressions of mRNA (i.e., NLRP3, GSDMD, ASC, IL-1β, IL-18 and caspase-1) and proteins (i.e., NLRP3, caspase-1, GSDMD and IL-1β) in IMR-32 and SK-N-SH cells between the BPA-treated groups and the BPA + EGCG-treated groups were reduced or eliminated.At the same time, the secretion of IL-1β, IL-18, caspase-1 and LDH were also reduced.In addition, the level of MMP was increased.As in ammasomes induce the maturation and the release of IL-1β and IL-18, we preliminarily speculate that the BPA-induced ROS activate the NLRP3 in ammasome, and then resulting in pyroptosis.As a ROS scavenger, EGCG can inhibit caspase-1-dependent pyroptosis in IMR-32 and SK-N-SH cells.

Pyroptosis of neuroblastoma cells by BPA contributes to apoptosis
Pyroptosis is a caspase-1-dependent form of programmed in ammatory cell death.Apoptosis is a caspase-3-dependent and non-in ammatory programmed form (Rogers et al., 2017), regulated by antiapoptotic and pro-apoptotic families (i.g., caspase family and Bcl-2 family) (Siddiqui et al., 2015).Caspase-3 is often activated by the death receptor mediated apoptotic pathway through the mitochondrial apoptotic pathway (Li and Yuan, 2008), which is controlled by the Bcl-2 family proteins.The activation of two Bcl-2 family members (i.e., Bak and Bax) can form pores on the mitochondrial outer-membrane, resulting in the release of mitochondrial inter-membrane components, and nally activate the occurrence of caspase cascade (Czabotar et (Pi et al., 2021).We hypothesized that the relationship of apoptosis and pyroptosis in nervous cells induced by BPA might be similar to the previous reports.In our study, YVAD was used as a caspase-1 inhibitor to explore the relationship between the BPA-induced pyroptosis and apoptosis by detecting protein levels of Bcl-2, Bak-1, Bax and caspase-3, MMP and apoptosis ratio.Bak-1 and Bax can promote apoptosis (Shi et al., 2010).Bcl-2 can inhibit apoptosis, which is mainly located on the outer membrane of the mitochondria and regulates apoptosis through mitochondrial pathway (Birkinshaw and Czabotar, 2017).Caspase-3 is the executor caspase of apoptosis.MMP is a vital marker of intrinsic apoptosis (Park et al., 2020).Our results showed that protein levels of Bak-1, Bax and caspase-3 in the 10 µM or the 100 µM BPA-treated groups were signi cantly higher than those in the control group, but the protein level of Bcl-2 demonstrated the opposite trend in IMR-32 (or SK-N-SH) cells.Further studies showed that BPA treatments could decrease MMP and increase apoptosis ratio and LDH level.Moreover, YVAD could signi cantly improve the changes of apoptosisrelated indicators induced by BPA.These results suggest that YVAD can decrease the BPA-induced apoptosis in neuroblastoma cells.Moreover, the activation of caspase-1 is involved in the BPA-induced apoptosis.Hence, we can conclude that apoptosis is related with pyroptosis in the BPA-induced neurotoxicity, and the inhibition of caspase-1 dependent pyroptosis may attenuate the BPA-induced apoptosis in neuroblastoma cells.

BPA induces damages in neuroblastoma cells in a nonmonotonic and gender-speci c manner
In this study, we used IMR-32 and SK-N-SH cells as the model to investigate the gender-dependent neurotoxicity induced by various concentrations of BPA.Interestingly, a non-monotonic relationship between the BPA concentrations and cytotoxic effects was observed.In particular, low-dose BPA treatment showed more obvious cytotoxicity than medium or high dose.There was an inverted U-shaped curve between BPA exposure concentration and cytotoxicity, which may be attributed to the exogenous endocrine disrupting effects of BPA.Our results are in accordance with the previous studies that low-dose BPA or bisphenol S can cause signi cant impacts on rats, while the middle-dose BPA has no obvious effects (da Silva et al., 2019; Zhang et al., 2019).In general, concentrations used to assess toxic effects of pollutants often range from low to high doses.However, only high concentration exposure is used in the study of molecular mechanism if the dose-effect response is linear.Our study showed that it is important to set the appropriate concentration range of BPA according to the toxic effect to study its corresponding toxic mechanism.Otherwise, improper concentration selection will lead to meaningless mechanism or opposite results because its non-linear relationships between dose and effects.It is easier to draw meaningful conclusions by selecting concentrations of toxic effects.
As an environmental endocrine disruptor, BPA has been shown to activate several types of receptors, including ER (Perera et al., 2017;Pinto et al., 2019).Although our in vitro studies have shown that BPA treatment can lead to apoptosis and pyroptosis in different gender nervous cells, and further con rmed the previous in vivo data that BPA can gender-dependently disrupt dendritic development and neurotransmitter homeostasis in the rat hippocampus (Zhang et al., 2019), the exact mechanism of BPA gender-speci c induction of apoptosis and pyroptosis of neuroblastoma cells remains unclear.In this study, at the transcriptional level, our results showed that the expression trends of IL-1β and caspase-1 levels were opposite in male and female neuroblastoma cells, while the expression trends at the translation level were consistent between both gender cells.And then we observed that the BPA-induced apoptosis and pyroptosis in IMR-32 and SK-N-SH cells were signi cantly attenuated after the ICI (an ER inhibitor) pretreatments.Therefore, BPA may induce apoptosis and pyroptosis of nervous cells through ER.The distribution and content of ER in the body may be related to the higher effects of BPA on males.
Bisphenol AF, an analogue of BPA, is reported to promote the ER activation and induce apoptosis in some cell lines (Huang et al., 2021; Kojima et al., 2019).When the secretion of estrogen is reduced and the expression of ER is low, women are more prone to vascular damage (Trenti et al., 2018).BPA exposure resulted in a signi cant down-regulation of estrogen receptor β (ER β ) in male hippocampus, but not in female hippocampus (Xu et al., 2015).Therefore, the role of exogenous estrogens (including BPA) in ER activation and function (i.g., the regulation of ROS generation, apoptosis and pyroptosis) still needs to be further clari ed by primary cells or animal experiments.

Conclusions
BPA may potently induce the pyroptosis of neuroblastoma cells through the NLRP3/caspase-1/GSDMD signaling pathway, in a ER-dependent, gender-speci c and non-monotonic manner, which is alleviated by antioxidation treatment.Male neuroblastoma cells seem to be more susceptible to BPA than female ones.Figure 6 al., 2014; Degterev and Yuan, 2008; Delbridge et al., 2016).However, pyroptosis can also be induced by pro-apoptotic caspase-3 (Rogers et al., 2017; Wang et al., 2017).Therefore, understanding the relationship of pyroptosis with apoptosis is helpful to clarify the mechanism of BPA apoptosis.Environmental pollutants are considered an important factor causing pyroptosis and apoptosis (Yu et al., 2021; Zhang et al., 2021).Moreover, cell pyroptosis has been proved to promote cell apoptosis (Shi et al., 2017).Pi et al. reported that molybdenum induced pyroptosis in duck renal tubular epithelial cells and YVAD reduced the molybdenum-induced apoptosis of pyroptosis-related genes mRNA in IMR-32 and SK-N-SH cells.(A-F) Expressions of NLRP3, GSDMD, ASC, IL-1β, IL-18 and Caspase-1 mRNA compared with β-action.Data are represented as mean ± SEM (n = 3)."*" indicates signi cant difference compared to its corresponding control (*p < 0.05, **p < 0.01 and ***p < 0.001).

Figure 2 Effects
Figure 2

Table 1
Gene primers sequence and their GenBank accession number