Roles of ERK/Akt signals in mitochondria-dependent and endoplasmic reticulum stress-triggered neuronal cell apoptosis induced by 4-methyl-2,4-bis(4-hydroXyphenyl)pent-1-ene, a major active metabolite of bisphenol A
Chun-Fa Huang a, b, 1, Shing-Hwa Liu c, 1, Chin-Chuan Su d, e, 1, Kai-Min Fang f, 1, Cheng-Chieh Yen g, 1, Ching-Yao Yang h, Feng-Cheng Tang i, Jui-Ming Liu j, Chin-Ching Wu k, Kuan-I Lee l,*, Ya-Wen Chen m,*
a School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, 404, Taiwan
b Department of Nursing, College of Medical and Health Science, Asia University, Taichung, 413, Taiwan
c Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, 100, Taiwan
d Department of Otorhinolaryngology, Head and Neck Surgery, Changhua Christian Hospital, Changhua County, 500, Taiwan
e School of Medicine, Kaohsiung Medical University, Kaohsiung, 807, Taiwan
f Department of Otolaryngology, Far Eastern Memorial Hospital, New Taipei City, 220, Taiwan
g Department of Occupational Safety and Health, College of Health Care and Management, Chung Shan Medical University, Taichung, 402, Taiwan
h Department of Surgery, National Taiwan University Hospital, and Department of Surgery, College of Medicine, National Taiwan University, Taipei, 100, Taiwan
i Department of Occupational Medicine, Changhua Christian Hospital, Changhua County, 500, Taiwan
j Division of Urology, Department of Surgery, Taoyuan General Hospital, Ministry of Health and Welfare, Taoyuan, 330, Taiwan
k Department of Public Health, China Medical University, Taichung, 404, Taiwan
l Department of Emergency, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung, 427, Taiwan
m Department of Physiology and Graduate Institute of Basic Medical Science, School of Medicine, College of Medicine, China Medical University, Taichung, 404, Taiwan
A R T I C L E I N F O
A B S T R A C T
Bisphenol A (BPA) is recognized as a harmful pollutant in the worldwide. Growing studies have reported that BPA can cause adverse effects and diseases in human, and link to a potential risk factor for development of neurodegenerative diseases (NDs). 4-methyl-2,4-bis(4-hydroXyphenyl)pent-1-ene (MBP), which generated in the mammalian liver after BPA exposure, is a major active metabolite of BPA. MBP has been suggested to exert greater toXicity than BPA. However, the molecular mechanism of MBP on the neuronal cytotoXicity remains unclear. In this study, MBP exposure significantly reduced Neuro-2a cell viability and induced apoptotic events that MBP (5–15 μM) exhibited greater neuronal cytotoXicity than BPA (50–100 μM). The mitochondria- dependent apoptotic signals including the decrease in mitochondrial membrane potential (MMP) and the in- crease in cytosolic apoptosis-induced factor (AIF), cytochrome c release, and Bax protein expression were involved in MBP (10 μM)-induced Neuro-2a cell death. EXposure of Neuro-2a cells to MBP (10 μM) also triggered endoplasmic reticulum (ER) stress through the induction of several key molecules including glucose-regulated protein (GRP)78, C/EBP homologous protein (CHOP), X-boX binding protein (XBP)-1, protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme(IRE)-1, activation transcription factor(AFT)4 and ATF6, and caspase-12. Pretreatment with 4-PBA (an ER stress inhibitor) and specific siRNAs for GRP78, CHOP, and XBP-1 significantly suppressed the expression of these ER stress-related proteins and the activation of caspase-12/-3/-7 in MBP-exposed Neuro-2a cells. Furthermore, MBP (10 μM) exposure dramatically increased the activation of extracellular regulated protein (ERK)1/2 and decreased Akt phosphorylation. Pre- treatment with PD98059 (an ERK1/2 inhibitor) and transfection with the overexpression of activation of Akt1 (myr-Akt1) effectively suppressed MBP-induced apoptotic and ER stress-related signals. Collectively, these results demonstrate that MBP exposure exerts neuronal cytotoXicity via the interplay of ERK activation and Akt inactivation-regulated mitochondria-dependent and ER stress-triggered apoptotic pathway, which ultimately leads to neuronal cell death.
Keywords:
Bisphenol A, 4-Methyl-2,4-bis(4-hydroXyphenyl)pent-1-ene NeurotoXicity, Apoptosis Mitochondria ER stress ERK1/2, Akt
Abbreviations: BPA, bisphenol A; NDs, neurodegenerative diseases; MBP, 4-methyl-24-bis(4-hydroXyphenyl)pent-1-ene; AIF, apoptosis-induced factor; MMP, mitochondrial membrane potential; ER, endoplasmic reticulum; GRP, glucose-regulated protein; CHOP, C/EBP homologous protein; XBP, X-boX binding protein; PERK, protein kinase R-like ER kinase; eIF2α, eukaryotic initiation factor 2α; IRE, inositol-requiring enzyme; ATF, activation transcription factor; 4-PBA, (4-phe- nylbutyric acid); siRNA, small interfering RNA; EDCs, endocrine disrupting chemicals; AD, Alzheimer’s diseases; PD, Parkinson’s diseases; ERK, extracellular signal- regulated kinase; MAPK, mitogen-activated protein kinase.
1. Introduction
Bisphenol A (BPA) is a chemical that widely used for a variety of industrial materials, polycarbonate plastics, and epoXy resins (WHO, 2011). Because of increasing popularity of these durable and lightweight materials, BPA has been found in common food container and packaging (e.g., water bottle) and in epoXy lining of metal food can, leading to leach into food products (Brotons et al., 1995; Sajiki and Yonekubo, 2003). BPA has been detected in more than 90 % of all analyzed human urine samples, indicating human widespreadly and continuously ex- poses to BPA (Lang et al., 2008; Vandenberg et al., 2010). Unfortunately, BPA is a type of endocrine disrupting chemicals (EDCs) that disrupts estrogenic response and interferes with the physiological functions of hormone, leading to induce the dysfunction of cell signals and the development of many diseases, including neurodegenerative diseases (NDs) (Braun et al., 2011; Lang et al., 2008; Masuo and Ishido, 2011). chronic, prolonged, or aggravated ER stress, the organelle elicits apoptotic signaling pathways leading to cell death (Szegezdi et al., 2006; Xu et al., 2005). ER stress has been implicated in human neuronal dis- eases, such as Alzheimer’s (AD) and Parkinson’s diseases (PD) (Lind- holm et al., 2006; Mercado et al., 2013). The findings of Ryu et al. (2002) using the cellular models coupled with evidence from familial forms of PD showed the possibility of widespread involvement of ER stress in the pathophysiology of PD. Several lines of studies have indicated that toXic insults- or environmental stimulus-induced neuronal cell injury and death are associated with the activation of ER stress-triggered apoptotic pathway (Chung et al., 2019; Lee et al., 2020; Ryu et al., 2002). Wang et al. (2019a) recently reported that BPA could induce neuro- degeneration by triggering both ER stress responses and mitochondrial dysfunction mediated pathways, which resulted in apoptosis and cell death in human cortical neurons. However, there is no study to inves- tigate whether MBP can interrupt the ER or mitochondrial function and
More importantly, 4-methyl-2,4-bis(4-hydroXyphenyl)pent-1-ene trigger mitochondria-dependent/ER stress-related neuronal apoptosis. (MBP), an active metabolite of BPA, has been obtained by coincuba- tion of BPA and liver S9 fraction and identified by LC/MS/MS and NMR analysis (Yoshihara et al., 2001 and 2004). BPA exposure through ingestion has been found to be metabolized to MBP in mammalian (Lim et al., 2009; Okuda et al., 2011; Yoshihara et al., 2001). Some studies have indicated that the toXicity of MBP is far more potent than its parent BPA (Brown et al., 2019; Ishibashi et al., 2005). MBP has been demon- strated to exhibit approXimately 500–1000 fold estrogenic potential than BPA in zebrafish (Danio rerio) (Moreman et al., 2018) and in uterus of ovariectomized rats (Okuda et al., 2010). Some literatures have indicated that BPA exposure could cause neuronal cytotoXicity, resulting to neuronal cell death and dysfunction (Pang et al., 2019; Preciados et al., 2016; Wang et al., 2019a). However, to our knowledge, there is no prior literature about the toXicological effects and mechanisms under- lying the MBP-induced neuronal cell injury.
Various studies have reported that mitochondrial dysfunction is a major risk factor for NDs (Beal, 1998; Johri and Beal, 2012). Mito- chondrion is a double-membrane-bound organelle in most eukaryotic EXtracellular signal-regulated kinase (ERK), an important subfamily of mitogen-activated protein kinase (MAPK), plays a pivotal role in controlling a broad range of cellular activities and pathophysiological processes, including cell growth, survival, differentiation, and apoptosis (Cagnol and Chambard, 2010). ToXic insults and endogenous stimuli can induce ERK activation to trigger mammalian cell injury and apoptosis (Fu et al., 2020; Lu et al., 2014). In addition, Akt (protein kinase B), a member of the serine/threonine-specific protein kinase family, functions as a crucial regulator in signal pathways, including cell survival, growth, proliferation, migration, and apoptosis by phosphorylating a range of intracellular proteins (Franke et al., 2003; Manning and Cantley, 2007). Disruption of ERK- and Akt-mediated signals significantly contributed in the pathogenesis of various diseases such as diabetes mellitus and NDs (Colucci-D’Amato et al., 2003; Rai et al., 2019). In the brain tissues of patients with NDs, the significant increase in ERK activity and decrease in Akt phosphorylation have been detected, suggesting a link between ERK/Akt signals and the pathogenesis of NDs (Anglade et al., 1997; Colucci-D’Amato et al., 2003). Growing studies have reported that cell. Mitochondria produce and supply ATP through oXidative phoschemicals (including BPA) can induce neuronal cytotoXicity and phorylation (OXPHOS) process that is essential for cell activities and signaling pathways, such as cellular growth and apoptosis, synthesis of key molecules and hormones, response to oXidative stress, and homeo- stasis of calcium ion (Wang et al., 2019b; Wu et al., 2019). Furthermore, CNS functions strongly need to efficient mitochondrial function, because neuronal cells have the high demand for mitochondrial ATP production and energy consumption. Thus, mitochondrial dysfunction (including impairment of mitochondrial energy metabolism) is not only the detri- mental effects on development of neurons but also be involved in the pathological process of NDs (Beal, 1998; Schwarz, 2013). Growing studies have reported that toXic chemicals exposure can cause mito- chondrial dysfunction (including the loss of mitochondrial membrane potential (MMP), decrease of antioXidant enzymes, and increase of cy- tochrome c release and Bax/Bcl-2 ratio), leading to neuronal cell injury and death (Fu et al., 2020; Lu et al., 2011 and 2014). By contrast, endoplasmic reticulum (ER) is a major site for synthesis, folding and processing of newly synthesized proteins. However, when ER’s functions are severely impaired (a pathological state), unfolded or misfolded proteins then accumulate in the ER lumen, which perturbs normal ER homeostasis, affects protein folding, and causes ER stress. In case of apoptosis via ERK or Akt downstream-regulated pathways, resulting in neuronal cell death (Chung et al., 2019; Lee et al., 2008; Fu et al., 2020; Wang et al., 2019a). However, the roles of ERK and Akt signals in BPA metabolite MBP-induced neuronal cell apoptosis are mostly unclear.
Collectively, in the study, we aimed to investigate the effects and mechanisms of BPA metabolite MBP on neuronal cell growth and function. The roles of ERK and Akt signals and the involvements of mitochondrial dysfunction and ER stress response in MBP-induced neuronal cell apoptosis and death would be examined and clarified.
2. Materials and methods
2.1. Materials
Unless otherwise specified, all chemicals (including BPA and MBP) and laboratory plastic wares were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Falcon Labware (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), respectively. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and antibiotics were pur- chased from Gibco/Invitrogen (Thermo Fisher Scientific Inc., USA). All of mouse- or rabbit- monoclonal antibodies and secondary antibodies (anti-mouse or anti-rabbit IgG-conjugated to horseradish peroXidase (HRP)) were purchased from Cell Signaling Technology (Cell Signaling Technology, Danvers, MA, USA).
2.2. Cell culture
Murine neuroblastoma cell line Neuro-2a was purchased from American Type Culture Collection (CCL-131; Manassas, VA, USA). Cells were cultured in plastic tissue culture dishes in a humidified chamber with a 5% CO2-95 % air miXture at 37 ◦C and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco/Invitrogen, Carls- bad, CA, USA).
2.3. Determination of cell viability
Neuro-2a cells were seeded (2 104 cells/well) in 96-well plates and allowed to adhere and recover overnight. The cells were changed to fresh media and then incubated with BPA (0–300 μM) or MBP (0–15 μM) for 24 h. After incubation, the medium was aspirated and fresh medium containing 30 μL of 2 mg/ml 3-(4, 5-dimethyl thiazol-2-yl-)-2, 5- diphenyl tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) was added. After 4 h, the medium was removed and replaced with blue formazan crystal dissolved in dimethyl sulfoXide (100 μL). Absor- bance at 570 nm was measured using a microplate reader (Bio-Rad, model 550, Hercules, CA, USA).
2.4. Analysis of apoptosis
2.4.1. Determination of phosphatidylserine externalization: annexin V- fluorescein isothiocyanate (FITC) assay
The externalization of phosphatidylserine (PS), which is exposed on the surface of apoptotic cells, is an early event during apoptosis. Flow cytometric analysis was performed to detect this event using the annexin V-FITC assay kit (BioVision, Milpitas, CA, USA). Neuro-2a cells were seeded at 2 × 105 cells/well in a 24-well plate and incubated with BPA (0–100 μM) or MBP (0–15 μM) for 24 h. After incubation, cells were harvested, washed twice with PBS, and then stained with annexin V-FITC for 15 min at room temperature. The stained cells were analyzed using flow cytometry (FACScalibur, Becton Dickinson, Sunnyvale, CA, USA).
2.4.2. Measurement of sub-G1 DNA content
Neuro-2a cells were seeded and treated with both BPA and MBP for 24 h. At the end of the treatment period, cells were detached, washed with PBS, resuspended in 1 ml of cold 70 % (v/v) ethanol, and then stored at 4 ◦C for 24 h. After the cells were washed with PBS, they were stained with propidium iodide (PI) and 10 μg/mL ribonuclease (RNase) in PBS at 4 ◦C for 30 min in the dark. The cells were washed and subjected to flow cytometric analysis of DNA content (FACScalibur, Becton Dickinson, Sunnyvale, CA, USA). Nuclei displaying hypodiploid and sub- G1 DNA contents were identified as apoptotic cell.
2.5. Determination of mitochondrial membrane potential (MMP)
MMP was analyzed using the 3,3`-di-hexyloXacarbocyanine iodide (DiOC6) fluorescent probe (Molecular Probes, Eugene, OR, USA), which contained a mitochondria-specific fluorophore with a positive charge. Briefly, Neuro-2a cells were seeded and treated with MBP (10 μM). After incubation, cells were harvested and loaded with 40 nM DiOC6 for 30 min and analyzed with FACScan flow cytometer (Becton Dickinson).
2.6. Western blot analysis
Neuro-2a cells were seeded at 1 × 106 cells/well in a 6-well culture plate and treated with BPA (75 μM) or MBP (10 μM). At the end of various treatments, the levels of protein expression were analyzed by Western blot analysis as previously described (Chung et al., 2019). In brief, equal amounts of proteins (50 μg per lane) were subjected to electrophoresis on 10 % (w/v) SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 1 h in PBST (PBS with 0.05 % Tween-20) containing 5% nonfat dry milk. After blocking, the membranes were incubated with the specific antibodies against caspase-3, caspase-7, caspase-9, PARP, GPR 78 and 94, CHOP, XBP-1, caspase-12, phosphorylated (p)-PERK, PERK, p-eIF2α, eIF2α, p-IRE-1, ATF-4 and -6, p-JNK, p-ERK1/2, p-p38, JNK, ERK1/2, p38, or β-actin in 0.1 % PBST (1:1000) for 1 h. After three additional washes in 0.1 % PBST (15 min each), the respective HRP-conjugated secondary antibodies were applied (in 0.1 % PBST (1:2500)) for 1 h. The antibody-reactive bands were detected by enhanced chemiluminescence reagents (Pierce™, Thermo Fisher Sci- entific Inc.) and analyzed by luminescent image analyzer (Image- Quant™ LAS-4000; GE Healthcare Bio-Sciences, Uppsala, Sweden).
2.7. Real-time quantitative RT-PCR analysis
The expression of the GRP78, GPR94, CHOP, and XBP-1 s genes were evaluated using real time quantitative RT-PCR (qPCR) as previously described (Lu et al., 2014). Briefly, total intracellular RNA was extracted using RNeasy kits (Qiagen), according to the instructions provided, and was heated to 90 ◦C for 5 min to remove any secondary structures and then rapidly placed on ice. The samples were reverse transcribed into cDNA using the AMV RTase (reverse transcriptase enzyme, Promega Corporation, Madison, WI, USA) system. The reverse transcriptase re- actions were performed as follows: RNA (5 μg) was added to a reaction buffer containing 2.5 mM dNTP (deoXynucleotide miX), 40U/μL RNasin (RNAase inhibitor, Promega), 100 nmol random hexamer primers, 1X RTase buffer (which was supplied with the RTase enzyme), and 30 U of the AMV reverse transcriptase enzyme (RTase), to which nuclease-free water was added for a final volume of 20 μL. The reactions were miXed and incubated at 42 ◦C for 60 min. The samples were then denatured at 95 ◦C for 10 min and placed on ice. The real-time SYBR Green primers for mouse GRP78, CHOP, XBP-1 s, and β-actin were chosen (Lu et al., 2014) as follows: GRP78, forward: 5′-GAACCAGGAGTTAAGAACACG-3′ and reverse: 5′-AGGCAACAGTGTCAGAGTCC-3′; CHOP, forward: 5′-CATGAACAGTGGGCATCACC-3′ and reverse: 5′-GAGAGGCCTTCACATGGGTCG-3′; XBP-1 s, forward: 5′-GAGTCCG CAGCAGGTG-3′ and reverse: 5′-GCGTCAGAATCCATGGGA-3′; Caspase-12, forward: 5′-GAAGGAATCTGTGGGGTGAA-3′ and reverse: 5′-TCAGCAGTGGCTATCCCTTT-3′; β-actin, forward: 5′-TGTGATGGT GGGAATGGGTCAG-3′ and reverse: 5′-TTTGATGTCACGCACGATTTCC-3′. Each sample (2 μL) was tested with Real-time SYBR Green PCR reagent (Invitrogen) and the transgene-specific primers in a 25 μL reaction volume, and amplification was performed using an ABI Ste- pOnePlus Sequence Detection System (PE, Applied Biosystems, Inc., USA). The cycling conditions consisted of 2 min at 50 ◦C, 10 min at 95◦ C, and 40 cycles of 95 ◦C for 30 s, and 60 ◦C for 1 min. Real-time fluorescence detection was performed during the 60 ◦C annea- ling/extension step of each cycle. Melt-curve analysis was performed on each primer set to ensure that no primer dimers or nonspecific ampli- fications were present under the optimized cycling conditions. After 40 cycles, the samples were run on 2% agarose gels to confirm specificity. Data analysis was performed using StepOne™ software (Version 2.1, Applied Biosystems). The fold differences in mRNA expression between the treatment and control groups were determined using the relative quantification method, which utilizes real-time PCR efficiencies and normalizes them to a housekeeping gene (β-actin was used in the pre- sented study), thus comparing relative CT changes (ΔCT) between the control and experimental samples. The values of fold change were calculated using the expression 2—ΔΔCT, where ΔΔCT represents ΔCT-condition of interest – ΔCT-control. Prior to conducting the statistical analyses, the fold change from the mean of the control group was calculated for each individual sample (including the individual control samples to assess the variability in this group).
2.8. Transient transfection of small interfering RNA (siRNA) and constitutively active form of Akt
Specific small interfering RNA (siRNA) against mouse GRP78, CHOP, and XBP-1 and control siRNA were purchased commercially from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA), and pUSEamp( ) empty vector and a constitutively active form of Akt (myr-Akt1) were purchased commercially from upstate (upstate, Lake placid, NY, USA). Neuro-2a cells were seeded in 6-well culture plates and transfected with the siRNA using Lipofectamine RNAi MAX (Gibco/Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cellular levels of the proteins specific for the siRNA transfection were checked by Western blot, and all experiments were performed at 24 h after transfection.
2.9. Statistical analysis
Data are presented as the mean standard deviation (S.D.) of at least four independent experiments. All data analyses were performed using the SPSS software version 12.0 (SPSS, Inc., Chicago, IL, USA). For each experimental condition, the significant difference compared to that of the respective controls was assessed by one-way analysis of variance (ANOVA), and Tukey’s post hoc test was performed to identify group differences. A p value of less than 0.05 was considered a significant difference.
3. Results
3.1. Effects of BPA and its metabolite MBP on cell viability and apoptosis in Neuro-2a cells
We first examined the cytotoXic effect of BPA in Neuro-2a cells by MTT assay. Treatment of Neuro-2a cells with 25–300 μM BPA for 24 h significantly and dose-dependently reduced cell viability, and the me- dian effective concentration (EC50) was approXimately 75 μM (Fig. 1A). Next, the results of annexin V-FITC fluorescence staining (the marker of phosphatidylserine exposure on the outer cellular membrane leaflets) and sub-G1 hypodiploid cells indicated that BPA-induced cytotoXicity was due to apoptosis. As shown in Fig. 1B and C, BPA (25–100 μM) dramatically induced the annexin V-FITC binding fluorescent intensity and the sub-G1 hypodiploid cell population in a dose-dependent manner in Neuro-2a cells. Furthermore, BPA (75 μM) at various time intervals (8 24 h) resulted in a marked expression of cleaved forms of caspase-3 and -7 (Fig. 1D) in Neuro-2a cells.
We next investigated the cytotoXic effects of MBP in Neuro-2a cells. As shown in Fig. 2A, treating cells with MBP for 24 h significantly decreased cell viability in a dose-dependent manner (at 10 μM, 59.3 9.5 % of control). Moreover, treatment of Neuro-2a cells with MBP (5–15 μM) for 24 h induced a significantly increased the annexin V-FITC binding fluorescent intensity (at 10 μM, 251.6 ± 15.4 % of control; Fig. 2B) and the sub-G1 hypodiploid cell populations (at 10 μM, 42.9 ± 5.0 % of control; Fig. 2C). Based on these results, 10 μM MBP was chose to using in the subsequent experiments.
Additionally, the activation of caspase cascade proteases is one of the most recognizable apoptotic biomarkers. As shown in Fig. 2D, Western blot analysis displayed a marked increase in the protein expression of the cleaved forms of caspase-3, -7, and PARP, as well as the upstream caspase, caspase-9, in MBP (10 μM)-treated Neuro-2a cells. These results indicate that treatment of neuronal cells with MBP is capable of inducing apoptosis. Moreover, the cytotoXic effect of MBP is more potency than BPA in Neuro-2a cells.
3.2. MBP-induced apoptosis is mediated by a mitochondria-dependent pathway in Neuro-2a cells
To determine whether MBP-induced neuronal cell apoptosis was mediated by a mitochondria-dependent pathway, the MMP, apoptosis- induced factor (AIF) release, and cytochrome c release were analyzed. As shown in Fig. 3A and B, treatment of Neuro-2a cells with MBP (10 μM) for 8 h effectively reduced MMP (65.8 10.8 % of control), which was more significantly reduced following a 24 h treatment (29.9 3.9 % of control), as well as the marked increase of AIF and cytochrome c release from mitochondria into cytosolic fraction (Fig. 3C). Moreover, the changes of protein expression of Bcl-2 family members were also examined. As shown in Fig. 3D, treatment with MBP (10 μM) for 8 24 h significantly increased Bax (pro-apoptotic) protein expression, but did not affect the Bcl-2 (anti-apoptotic) protein expression, in Neuro-2a cells. It showed a significant shift in the pro-apoptotic/anti-apoptotic ratio toward a state associated with apoptosis. These results imply that the mitochondria-dependent apoptotic pathway plays an important role in MBP-induced neuronal cell apoptosis.
3.3. MBP induced ER stress response in Neuro-2a cells
To investigate whether ER stress signals were involved in MBP- induced neuronal cell apoptosis, we examined the expression of ER stress-related markers. As shown in Fig. 4A and B, treatment of cells with MBP (10 μM) for 8 24 h significantly triggered both protein and mRNA expression of ER stress-related molecules, including upregulation of GRP 78, CHOP, and spliced form of XBP-1 (XBP-1 s), as well as down- regulation of full-length caspase-12 (the downstream molecule of ER stress). MBP did not affect the protein expression of GRP 94. Further- more, the levels of phosphorylated protein expression for PERK, eIF2α, and IRE-1, as well as the protein expression of ATF-4 and cleaved ATF-6 (the major arms of ER stress) were dramatically increased in Neuro-2a cells treated with MBP for 1 8 h (Fig. 4C).
To further confirm the relationship between the activation of ER stress and MBP-induced neuronal cell apoptosis, we used 4-phenylbuty- ric acid (4-PBA; an ER stress inhibitor) and siRNA-mediated knockdown of GRP 78, CHOP, and XBP-1 to examine the MBP-induced apoptotic events. As shown in Fig. 5A, pretreatment of Neuro-2a cells with 4-PBA (5 mM) for 1 h prior to MBP exposure for 24 h effectively reversed both the upregulation of protein expression of GPR 78, CHOP, and XBP-1 s and the cleavage of caspase-3 and -7. Transfection of Neuro-2a cells with GRP 78-, CHOP-, and XBP-1-specific siRNAs also efficaciously reduced the protein expression of GRP 78, CHOP, XBP-1 s, and caspase-12, and significantly attenuated caspase-3 and -7 activation in MBP-treated Neuro-2a cells. These results indicate that MBP is capable inducing ER stress response, leading to neuronal cell apoptosis.
3.4. The role of ERK signal in the MBP-induced apoptosis in Neuro-2a cells
MAPKs-mediated apoptosis pathways are known to be involved in investigated whether the activation of MAPKs was played a critical role in MBP-induced neuronal cell apoptosis. As shown in Fig. 6A, the level of phosphorylated ERK1/2 protein was significantly increased after 0.5 h of treatment of Neuro-2a cells with MBP (10 μM), which was a continued increase to 8 h. However, the protein phosphorylation for JNK and p38 was not observed. Pretreatment of cells with the pharmacological ERK1/ 2 inhibitor (PD98059; 20 μM) for 1 h prior to MBP exposure effectively decreased the phosphorylation of ERK1/2 (Fig. 6B), loss of MMP (Fig. 6C), increase in apoptotic cell population (Fig. 6D), cleavage of caspase-3 and -7, induction of ER stress-related molecules, and increase in Bax protein expression (Fig. 6E). These results imply that ERK activation-mediated mitochondria-dependent and ER stress-regulated apoptosis is involved in MBP-induced neuronal cell death.
3.5. PI3K/Akt signal plays an important role in MBP-induced neuronal cell apoptosis
To ascertain whether PI3K/Akt signaling pathway was involved in MBP-induced neuronal cell apoptosis, the phosphorylation of Akt and PI3K activity were examined. As shown in Fig. 7A and B, treatment of Neuro-2a cells with MBP (10 μM) was significantly decreased the level of Akt protein phosphorylation (for 2 4 h) and inhibited PI3K activity (the upstream effector of Akt; for 15 240 min). Transient transfection of Neuro-2a cells with a myr-Akt1 plasmid coding (an active form of Akt1) markedly reversed the decrease in Akt protein phosphorylation (Fig. 7C), loss of MMP (Fig. 7D), cleavage of caspase-3 and -7, induction of ER stress-related molecules, and increase in Bax protein expression (Fig. 7E) as compared to MBP treatment alone.
Furthermore, since the MBP-induced neuronal cell apoptosis through the alteration of ERK1/2 and Akt signals, we next investigate whether there was the relationship between ERK and Akt in MBP-induced neuronal cell apoptosis. As shown in Fig. 7C, transfection of Neuro-2a cells with a myr-Akt1 plasmid coding effectively and significantly reversed the Akt protein inactivation as well as ERK1/2 protein phos- phorylation in MBP-treated neuronal cells. Meanwhile, the pretreatment of Neuro-2a cells with PD98059 (a pharmacological ERK1/2 inhibitor) markedly prevented the MBP-induced Akt protein inactivation (Fig. 6B). These results indicate that both ERK and Akt signals are involved in the various stimuli-induced cytotoXicity in mammalian cells. We next downstream-regulated mitochondria-dependent- and ER stress-regulated apoptosis, contributing to MBP-induced neuronal cell death.
4. Discussion
The prevalence of neurological disorders associated with EDCs is a growing concern. BPA, the representative exogenous EDCs, is widely composed in plastic products. BPA exposure has been linked to the neuropsychological and neurobehavioral disorders and NDs (Masuo and Ishido, 2011; Preciados et al., 2016; Wang et al., 2019b). The study by Wang et al. (2017) has highlighted that BPA exposure can induce neurotoXic responses in the neuronal cells leading to an Alzheimer’s disease (AD)-like disease, which is via the substantial increase in AD-associated pathological proteins, including amyloid precursor pro- tein (APP), Aβ1—42, and tau protein phosphorylation. Of late, Landolfi et al. (2017) reported that PD patients carried a significantly higher amount of free BPA and lower amount of conjugated BPA in the blood compared to healthy controls, suggesting that BPA metabolism may be associated with PD neurodegeneration. On the other hand, when expose to BPA through ingestion of contaminated food or water, BPA can be metabolized in mammalian liver microsomes by cytochrome P450 en- zymes, and converted to MBP, an active metabolite of BPA (WHO, 2011; Yoshihara et al., 2001 and 2004). It has been indicated that MBP has the higher estrogenic potential and toXic activity than its parent compound BPA (Baker and Chandsawangbhuwana, 2012; Yoshihara et al., 2001). Ishibashi and colleagues have shown that the 96 h median lethal con- centration of MBP and BPA on medaka (Oryzias latipes) was estimated to be 1640 and 13,900 μg/L (about 6.1 and 60.9 μM), respectively (Ishi- bashi et al., 2005). The results of Okuda et al. (2010) using the ovari- ectomized (OVX) female rats model reported that the concentration of MBP (1000 μg/kg/day (about 3.7 μM/day)) completely reversed the changes caused by VOX, and its activity was equivalent to that of 0.5μg/kg/day 17β-estradiol, suggesting at least 500-fold higher the estrogenic activity potent of MBP than that of the parent compound, BPA. Recently, the exposure of MBP (25 μg/L (about 0.1 μM); the lower dosage than BPA) was found the impaired cardiovascular function and the development of vascular-cardiovascular disease states in Zebrafish (Brown et al., 2019). Although, some studies have reported that the exposure of MBP (5–15 μM) significantly inhibits human breast cancer cell growth and induces type 2 pulmonary alveolar cell apoptosis (Hir- ao-Suzuki et al., 2019; Liu et al., 2016), the toXicological effects and action mechanisms of MBP on neuronal cells are mostly remained un- clear. In this study, we demonstrated for the first time that exposure of and ER stress-mediated apoptosis are involved in MBP-induced neuronal cell death.
Mitochondria are critical for cellular energy metabolism and bal- ance. It is also a sensitive organelle to stimulus-induced dysfunction. It has been demonstrated to play an important role in controlling cell apoptosis and death, especially in the neurons, that the mitochondrial dysfunction can be as a causative factor in the pathological processes of NDs (Johri and Beal, 2012; Schwarz, 2013; Wang et al., 2019b; Wu et al., 2019). Furthermore, the environmental toXicants have been reported to cause mitochondrial dysfunction, which regards as risk factors for NDs in in vivo and in vitro. For examples, exposure of compounds such as rotenone, piericidins, MPTP, and fenpyroXimate, which can mimic the pathological features of PD, induces a systemic dysfunction in mito- chondrial complex I activity and leads to neurodegeneration and cell death in dopaminergic neurons (Sherer et al., 2002; Wang et al., 2019a). Increased γ-secretase activity, which results in accelerated accumulation of Aβ, induces the mitochondrial oXidative damage and dysfunction, which the complex I and IV activities are inhibited, in paraquat-exposed AD animal model (Chen et al., 2015). Epidemiological and laboratorial studies have found that many toXic chemicals as the important risks factor for NDs can cause mitochondrial toXicities, including the loss of MMP and increase in cytochrome c release and Bax/Bcl-2 ratio, leading to neuronal cell apoptosis (Fu et al., 2020; Lu et al., 2014; Wang et al., 2019a, 2014).
ER serves a crucial regulator of protein synthesis, correct folding, post-translation modification, and secretion. When external or patho- physiological stimuli are present, misfolded (mutant or unfolded) pro- teins accumulate and aggregate in the ER cavity, which disrupts ER neuronal cells to MBP significantly induced cytotoXicity in a function/homeostasis, leading to ER stress. Protein aggregation is toxic concentration-dependent manner (ranging from 3 to 30 μM), which exhibited greater cytotoXicity than its parent compound BPA. Further- more, the signaling mechanisms of both ERK activation and Akt inac- tivation in which contribute to triggering the mitochondria-dependent to cells, and therefore, several pathophysiological disorders are associ- ated with ER stress, including NDs (Kaufman, 2002). To combat the deleterious effects by ER stress, the unfold protein response (UPR) is activated to restore ER function by decreasing the quantity of misfolded proteins and enhancing ER protein processing capacity. UPR can acti- vate three ER transmembrane receptors: double-stranded RNA-activated protein kinase-like ER kinase (PERK), activating transcription factor-6 (ATF-6), and inositol-requiring kinase 1α (IRE1α). The significant acti- vation of ER stress markers (such as the phosphorylation of PERK and eIF2α, and the active form of ATF6, XPB-1, and CHOP and GRP78) have been detected in neurons of PD patients and in peripheral blood mononuclear cells (PBMCs) of sporadic ALS (sALS) patients, but not in healthy controls (Hoozemans et al., 2007; Mercado et al., 2013; Prell et al., 2019; Selvaraj et al., 2012; Vats et al., 2018). The increasing studies have shown that exposure to environmental factors (toXic chemicals and/or pathological stimuli) can induce neuronal cell injury and apoptosis, which is accompanied with the activation of ER stress molecules (Chung et al., 2019; Lee et al., 2020; Lopez-Hernandez et al., 2015; Lu et al., 2015). Recently, a few studies reported that exposure of BPA induced the cell apoptosis by triggering ER stress response and mitochondrial dysfunction (including the change of Bax/Bcl-2 expres- sion ratio and the increase in cytochrome c release), resulting in neu- rodegeneration and neurotoXicity (El Morsy and Ahmed, 2020; Wang et al., 2019a). Only a study by Liu et al. (2016) indicated that MBP exposure caused lung tissue dysfunction (in vivo) and induced type-2 pulmonary alveolar cell apoptosis (in vitro) via the ER stress-mediated apoptosis pathway. However, to our knowledge, there is no study to investigate the effects of underlying neuronal cell death induced by MBP. Here, our results found that MBP was capable of causing the disruption of mitochondrial functions as indicated by a loss of MMP and an increase in the release of mitochondrial cytochrome c and AIF to the cytoplasm, and the change of Bcl-2 (anti-apoptotic)/Bax (pro-apoptotic) protein expression ratio, resulting in apoptosis in neuronal cells. Furthermore, exposure of neuronal cells to MBP had significantly increased the expression of ER stress-related molecules, including the activation of GRP78, CHOP, XBP-1 s, caspase-12, ATF-4, and ATF-6 proteins, and the phosphorylation of PERK, eIF2α, and IRE-1. Pretreat- ment of cells with 4-PBA (an ER stress inhibitor) could effectively pre- vent MBP-induced activation of GRP78, CHOP, XBP-1 s, and apoptotic events. In addition, transfection of Neuro-2a cells with GRP78-, CHOP-ERK1/2 signal (Cia et al., 2010; Gomez-Santos et al., 2002; Lee et al., 2020; Lu et al., 2014). A study of Lee et al. (2008) has highlighted that the activation of ERK1/2 is involved in BPA-induced apoptotic cell death in hippocampal neuronal cells. Nevertheless, there is no research to establish the role of ERK1/2 in the MBP-induced neurotoXic effects. The results from this study found that treatment of Neuro-2a cells with MBP significantly increased the phosphorylation of ERK1/2 protein after 0.5 h treatment and the continuous expression to 8 h. Inhibition of ERK1/2 activation in Neuro-2a cells by ERK1/2 inhibitor PD98059 effectively abrogated the neuronal cell apoptotic events, loss of MMP, ER stress-related molecules, and ERK1/2 activation in MBP-treated Neu- ro-2a cells.
Akt has been demonstrated to display an imperative regulation effect in cell apoptosis and survival (Franke et al., 2003; Luo et al., 2003). Akt inactivation has been reported to induce apoptosis of neuronal cells induced by toXicants and pathophysiological stimuli, which is focused to clarify the neuron degeneration and death mechanism for ND patho- genesis (Chung et al., 2019; Luo et al., 2003; Rai et al., 2019). In brain samples of AD and PD patient, a marked decrease in Akt activity (phosphorylation) was observed, which was associated with neuron degeneration and death (Liu et al., 2011; Malagelada et al., 2008; Sel- varaj et al., 2012). Wang et al. (2015) have observed the significant increase in phosphorylated tau protein expression at various sites of brain as well as the marked decrease in phosphorylated (p)-AktSer473 protein expression in Akt conditional knockout (Akt cTKO) mice. Deactivation of Akt downstream-regulated apoptotic pathways induced by exposure to neurotoXin (such as 6-hydroXydopamine (6 OHDA) and N-methyl-D-aspartate (NMDA)) has been found in neuronal cells (Luo et al., 2003; Malagelada et al., 2008). A study of Amiri et al. (2016) has shown that exposure to 6 OHDA can cause SH-SY5Y cell apoptosis and death via an Akt inactivation/ERK activation signaling pathway. Furthermore, it has been highlighted that exposure to MPP+/MPTP or methylmercury is capable of causing neurotoXicity through the activa- tion of ER stress pathway and impaired Akt phosphorylation, which can be reversed by the enhance of Akt activation (Chung et al., 2019; Sel- varaj et al., 2012). Recent studies have further reported that BPA and XBP-1-specific siRNAs significantly suppressed MBP-induced protein expression of GRP78, CHOP, and XBP-1 s as well as attenuated the apoptotic events. These results indicate that MBP exposure causes neuronal cell death via both mitochondria-dependent- and ER stress-mediated apoptotic pathways.
Activation of ERK, induced by growth factors or some conditions of stress, is demonstrated to confer a survival and anti-apoptotic advantage to mammalian cells. However, a death-promoting role of ERK activation in neuronal cells has been supported by evidence from both in vitro and in vivo models that suggest a link between ERK activation and neuronal cell death in NDs (Colucci-D’Amato et al., 2003; Subramaniam and Unsicker, 2010). In clinical pathological studies, a lot of phosphorylated ERK1/2 proteins have been detected in aggregates in the substantia nigra of brains from PD patients, but not in healthy controls (Zhu et al., 2002). The marked increase in phosphorylated protein expression of ERK1/2 has also been found in brain extracts from AD patients, but not in control individuals (Russo et al., 2002). Dineley et al. (2001) reported that chronic ERK activation could be displayed in the organotypic hippo- campal slides of transgenic mice model for AD (by hyperexpressing β-amyloid 42 (Aβ)). In in vitro experimental paradigms, neurotoXic chemicals (such as MPTP and Mn) can induce the overexpression of α-synuclein (a hallmark lesion of sporadic PD,) and neuronal cell death, which is accompanied with the significant activation of ERK1/2 (Cai et al., 2010; Gomez-Santos et al., 2002). Thus, it has been highly hy- pothesized that the chronic ERK activation-dependent mechanism may contribute to neuronal cell death, leading to exert the pathological hallmark for neurodegeneration and the development of NDs. Growing studies has shown that exposure to toXic insult could induce mito- chondrial dysfunction- and ER stress-regulated apoptotic responses resulting in neuronal cell death, accompanied with the activation of AD-associated pathological protein expression in vitro was accompanied with the inhibition of Akt phosphorylation (Wang et al., 2019a; and 2017). However, to the best of our knowledge, there is no study to investigate the role of Akt signaling in MBP-induced neuronal cell death and the sequential relationship between Akt and ERK signaling that leads to neuronal cell apoptosis. In this study, our results demonstrated that MBP significantly decreased the phosphorylation of Akt (without change in total Akt protein) and PI3K-activity in Neuro-2a cells. Transfection of cells with over-expression of c.a. Akt plasmid (myr-Akt1 plasmid) dramatically attenuated MBP-induced Akt inactivation, mito- chondrial dysfunction, apoptosis, and ER stress-related molecules. Moreover, the increase in phosphorylation of ERK1/2 protein in the MBP-exposed Neuro-2a cells was effectively prevented by transfection with c.a. Akt plasmid; meanwhile, the inactivation of Akt signal could also be reversed following pretreatment with ERK inhibitor PD98059. These results indicate that Akt inactivation- and ERK activation-mediated signals are interdependent and play critical roles in the downstream regulation of both mitochondria-dependent and ER stress-triggered apoptosis in MBP-induced neuronal cell death.
5. Conclusion
This study elucidates that MBP is capable of causing neuronal cyto- toXicity and death through the mitochondria-dependent and ER stress- triggered apoptotic pathways. The interplay occurring between ERK activation and Akt inactivation is identified as a key mechanism un- derlying MBP-induced neuronal cell apoptosis. These observations further provide beneficial evidence to support that BPA metabolite MBP- induced neurotoXicity may be an environment risk factor for the development of NDs.
References
Amiri, E., Ghasemi, R., Moosavi, M., 2016. Agmatine protects against 6-OHDA-induced apoptosis, and ERK and Akt/GSK disruption in SH-SY5Y cells. Cell. Mol. Neurobiol. 36, 829–838. https://doi.org/10.1007/s10571-015-0266-7.
Anglade, P., Vyas, S., Javoy-Agid, F., Herrero, M.T., Michel, P.P., Marquez, J., Mouatt- Prigent, A., Ruberg, M., Hirsch, E.C., Agid, Y., 1997. Apoptosis and autophagy in nigral neurons of patients with Parkinson’s disease. Histol. Histopathol. 12, 25–31.
Baker, M.E., Chandsawangbhuwana, C., 2012. 3D models of MBP, a biologically active metabolite of bisphenol A, in human estrogen receptor alpha and estrogen receptor beta. PLoS One 7, e46078. https://doi.org/10.1371/journal.pone.0046078.
Beal, M.F., 1998. Mitochondrial dysfunction in neurodegenerative diseases. Biochim. Biophys. Acta 1366, 211–223. https://doi.org/10.1016/S0005-2728(98)00114-5.
Braun, J.M., Kalkbrenner, A.E., Calafat, A.M., Yolton, K., Ye, X., Dietrich, K.N., Lanphear, B.P., 2011. Impact of early-life bisphenol A exposure on behavior and executive function in children. Pediatrics 128, 873–882. https://doi.org/10.1542/ peds.2011-1335.
Brotons, J.A., Olea-Serrano, M.F., Villalobos, M., Pedraza, V., Olea, N., 1995. Xenoestrogens released from lacquer coatings in food cans. Environ. Health Perspect. 103, 608–612. https://doi.org/10.1289/ehp.95103608.
Brown, A.R., Green, J.M., Moreman, J., Gunnarsson, L.M., Mourabit, S., Ball, J., Winter, M.J., Trznadel, M., Correia, A., Hacker, C., Perry, A., Wood, M.E., Hetheridge, M.J., Currie, R.A., Tyler, C.R., 2019. Cardiovascular effects and molecular mechanisms of bisphenol A and its metabolite MBP in zebrafish. Environ. Sci. Technol. 53, 463–474. https://doi.org/10.1021/acs.est.8b04281.
Cagnol, S., Chambard, J.C., 2010. ERK and cell death: mechanisms of ERK-induced cell death–apoptosis, autophagy and senescence. FEBS J. 277, 2–21. https://doi.org/ 10.1111/j.1742-4658.2009.07366.X.
Cai, T., Yao, T., Zheng, G., Chen, Y., Du, K., Cao, Y., Shen, X., Chen, J., Luo, W., 2010. Manganese induces the overexpression of alpha-synuclein in PC12 cells via ERK activation. Brain Res. 1359, 201–207. https://doi.org/10.1016/j. brainres.2010.08.055.
Chen, L., Na, R., Boldt, E., Ran, Q., 2015. NLRP3 inflammasome activation by mitochondrial reactive oXygen species plays a key role in long-term cognitive impairment induced by paraquat exposure. Neurobiol. Aging 36, 2533–2543. https://doi.org/10.1016/j.neurobiolaging.2015.05.018.
Chung, Y.P., Yen, C.C., Tang, F.C., Lee, K.I., Liu, S.H., Wu, C.C., Hsieh, S.S., Su, C.C., Kuo, C.Y., Chen, Y.W., 2019. Methylmercury exposure induces ROS/Akt inactivation-triggered endoplasmic reticulum stress-regulated neuronal cell apoptosis. ToXicology 425, 152245. https://doi.org/10.1016/j.toX.2019.152245.
Colucci-D’Amato, L., Perrone-Capano, C., di Porzio, U., 2003. Chronic activation of ERK and neurodegenerative diseases. Bioessays 25, 1085–1095. https://doi.org/ 10.1002/bies.10355.
Dineley, K.T., Westerman, M., Bui, D., Bell, K., Ashe, K.H., Sweatt, J.D., 2001. Beta- amyloid activates the mitogen-activated protein kinase cascade via hippocampal alpha7 nicotinic acetylcholine receptors: in vitro and in vivo mechanisms related to Alzheimer’s disease. J. Neurosci. 21, 4125–4133. https://doi.org/10.1523/ JNEUROSCI.21-12-04125.2001.
El Morsy, E.M., Ahmed, M., 2020. Protective effects of lycopene on hippocampal neurotoXicity and memory impairment induced by bisphenol A in rats. Hum. EXp. ToXicol. 39, 1066–1078. https://doi.org/10.1177/0960327120909882.
Franke, T.F., Hornik, C.P., Segev, L., Shostak, G.A., Sugimoto, C., 2003. PI3K/Akt and apoptosis: size matters. Oncogene 22, 8983–8998. https://doi.org/10.1038/sj. onc.1207115.
Fu, S.C., Liu, J.M., Lee, K.I., Tang, F.C., Fang, K.M., Yang, C.Y., Su, C.C., Chen, H.H., Hsu, R.J., Chen, Y.W., 2020. Cr(VI) induces ROS-mediated mitochondrial-dependent apoptosis in neuronal cells via the activation of Akt/ERK/AMPK signaling pathway. ToXicol. In Vitro 65, 104795. https://doi.org/10.1016/j.tiv.2020.104795.
Gomez-Santos, C., Ferrer, I., Reiriz, J., Vinals, F., Barrachina, M., Ambrosio, S., 2002. MPP increases alpha-synuclein expression and ERK/MAP-kinase phosphorylation in human neuroblastoma SH-SY5Y cells. Brain Res. 935, 32–39. https://doi.org/ 10.1016/S0006-8993(02)02422-8.
Hirao-Suzuki, M., Takeda, S., Okuda, K., Takiguchi, M., Yoshihara, S., 2019. Repeated exposure to 4-Methyl-2,4-bis(4-hydroXyphenyl)pent-1-ene (MBP), an active metabolite of bisphenol a, aggressively stimulates breast Cancer cell growth in an estrogen receptor beta (ERbeta)-dependent manner. Mol. Pharmacol. 95, 260–268. https://doi.org/10.1124/mol.118.114124.
Ishibashi, H., Watanabe, N., Matsumura, N., Hirano, M., Nagao, Y., Shiratsuchi, H., Kohra, S., Yoshihara, S., Arizono, K., 2005. ToXicity to early life stages and an estrogenic effect of a bisphenol A metabolite, 4-methyl-2,4-bis(4-hydroXyphenyl) pent-1-ene on the medaka (Oryzias latipes). Life Sci. 77, 2643–2655. https://doi. org/10.1016/j.lfs.2005.03.025.
Johri, A., Beal, M.F., 2012. Mitochondrial dysfunction in neurodegenerative diseases. J. Pharmacol. EXp. Ther. 342, 619–630. https://doi.org/10.1124/jpet.112.192138.
Kaufman, R.J., 2002. Orchestrating the unfolded protein response in health and disease. J. Clin. Invest. 110, 1389–1398. https://doi.org/10.1172/JCI16886.
Landolfi, A., Troisi, J., Savanelli, M.C., Vitale, C., Barone, P., Amboni, M., 2017. Bisphenol A glucuronidation in patients with Parkinson’s disease. NeurotoXicology 63, 90–96. https://doi.org/10.1016/j.neuro.2017.09.008.
Lang, I.A., Galloway, T.S., Scarlett, A., Henley, W.E., Depledge, M., Wallace, R.B., Melzer, D., 2008. Association of urinary bisphenol A concentration with medical disorders and laboratory abnormalities in adults. JAMA 300, 1303–1310. https:// doi.org/10.1001/jama.300.11.1303.
Lee, S., Suk, K., Kim, I.K., Jang, I.S., Park, J.W., Johnson, V.J., Kwon, T.K., Choi, B.J., Kim, S.H., 2008. Signaling pathways of bisphenol A-induced apoptosis in hippocampal neuronal cells: role of calcium-induced reactive oXygen species, mitogen-activated protein kinases, and nuclear factor-kappaB. J. Neurosci. Res. 86, 2932–2942. https://doi.org/10.1002/jnr.21739.
Lee, K.I., Lin, J.W., Su, C.C., Fang, K.M., Yang, C.Y., Kuo, C.Y., Wu, C.C., Wu, C.T., Chen, Y.W., 2020. Silica nanoparticles induce caspase-dependent apoptosis through reactive oXygen species-activated endoplasmic reticulum stress pathway in neuronal cells. ToXicol. In Vitro 63, 104739. https://doi.org/10.1016/j.tiv.2019.104739.
Lim, D.S., Kwack, S.J., Kim, K.B., Kim, H.S., Lee, B.M., 2009. Potential risk of bisphenol A migration from polycarbonate containers after heating, boiling, and microwaving. J. ToXicol. Environ. Health A 72, 1285–1291. https://doi.org/10.1080/ 15287390903212329.
Lindholm, D., Wootz, H., Korhonen, L., 2006. ER stress and neurodegenerative diseases. Cell Death Differ. 13, 385–392. https://doi.org/10.1038/sj.cdd.4401778.
Liu, Y., Liu, F., Grundke-Iqbal, I., Iqbal, K., Gong, C.X., 2011. Deficient brain insulin signalling pathway in Alzheimer’s disease and diabetes. J. Pathol. 225, 54–62. https://doi.org/10.1002/path.2912.
Liu, S.H., Su, C.C., Lee, K.I., Chen, Y.W., 2016. Effects of bisphenol A metabolite 4- Methyl-2,4-bis(4-hydroXyphenyl)pent-1-ene on lung function and type 2 pulmonary alveolar epithelial cell growth. Sci. Rep. 6, 39254 https://doi.org/10.1038/ srep39254.
Lopez-Hernandez, B., Cena, V., Posadas, I., 2015. The endoplasmic reticulum stress and the HIF-1 signalling pathways are involved in the neuronal damage caused by chemical hypoXia. Br. J. Pharmacol. 172, 2838–2851. https://doi.org/10.1111/ bph.13095.
Lu, T.H., Hsieh, S.Y., Yen, C.C., Wu, H.C., Chen, K.L., Hung, D.Z., Chen, C.H., Wu, C.C., Su, Y.C., Chen, Y.W., Liu, S.H., Huang, C.F., 2011. Involvement of oXidative stress- mediated ERK1/2 and p38 activation regulated mitochondria-dependent apoptotic signals in methylmercury-induced neuronal cell injury. ToXicol. Lett. 204, 71–80. https://doi.org/10.1016/j.toXlet.2011.04.013.
Lu, T.H., Tseng, T.J., Su, C.C., Tang, F.C., Yen, C.C., Liu, Y.Y., Yang, C.Y., Wu, C.C., Chen, K.L., Hung, D.Z., Chen, Y.W., 2014. Arsenic induces reactive oXygen species- caused neuronal cell apoptosis through JNK/ERK-mediated mitochondria-dependent and GRP 78/CHOP-regulated pathways. ToXicol. Lett. 224, 130–140. https://doi. org/10.1016/j.toXlet.2013.10.013.
Luo, H.R., Hattori, H., Hossain, M.A., Hester, L., Huang, Y., Lee-Kwon, W., Donowitz, M., Nagata, E., Snyder, S.H., 2003. Akt as a mediator of cell death. Proc. Natl. Acad. Sci. U. S. A. 100, 11712–11717. https://doi.org/10.1073/pnas.1634990100.
Malagelada, C., Jin, Z.H., Greene, L.A., 2008. RTP801 is induced in Parkinson’s disease and mediates neuron death by inhibiting Akt phosphorylation/activation. J. Neurosci. 28, 14363–14371. https://doi.org/10.1523/JNEUROSCI.3928-08.2008. Manning, B.D., Cantley, L.C., 2007. AKT/PKB signaling: navigating downstream. Cell129, 1261–1274. https://doi.org/10.1016/j.cell.2007.06.009.
Masuo, Y., Ishido, M., 2011. NeurotoXicity of endocrine disruptors: possible involvement in brain development and neurodegeneration. J. ToXicol. Environ. Health B Crit. Rev. 14, 346–369. https://doi.org/10.1080/10937404.2011.578557.
Mercado, G., Valdes, P., Hetz, C., 2013. An ERcentric view of Parkinson’s disease. Trends Mol. Med. 19, 165–175. https://doi.org/10.1016/j.molmed.2012.12.005.
Moreman, J., Takesono, A., Trznadel, M., Winter, M.J., Perry, A., Wood, M.E., Rogers, N. J., Kudoh, T., Tyler, C.R., 2018. Estrogenic mechanisms and cardiac responses following early life exposure to bisphenol a (BPA) and its metabolite 4-Methyl-2,4- bis(p-hydroXyphenyl)pent-1-ene (MBP) in zebrafish. Environ. Sci. Technol. 52, 6656–6665. https://doi.org/10.1021/acs.est.8b01095.
Okuda, K., Takiguchi, M., Yoshihara, S., 2010. In vivo estrogenic potential of 4-methyl- 2,4-bis(4-hydroXyphenyl)pent-1-ene, an active metabolite of bisphenol A, in uterus of ovariectomized rat. ToXicol. Lett. 197, 7–11. https://doi.org/10.1016/j. toXlet.2010.04.017.
Okuda, K., Fukuuchi, T., Takiguchi, M., Yoshihara, S., 2011. Novel pathway of metabolic activation of bisphenol A-related compounds for estrogenic activity. Drug Metab. Dispos. 39, 1696–1703. https://doi.org/10.1124/dmd.111.040121.
Pang, Q., Li, Y., Meng, L., Li, G., Luo, Z., Fan, R., 2019. NeurotoXicity of BPA, BPS, and BPB for the hippocampal cell line (HT-22): an implication for the replacement of BPA in plastics. Chemosphere 226, 545–552. https://doi.org/10.1016/j. chemosphere.2019.03.177.
Preciados, M., Yoo, C., Roy, D., 2016. Estrogenic endocrine disrupting chemicals influencing NRF1 regulated gene networks in the development of complex human brain diseases. Int. J. Mol. Sci. 17, 2086. https://doi.org/10.3390/ijms17122086.
Prell, T., Stubendorff, B., Le, T.T., Gaur, N., Tadic, V., Rodiger, A., Witte, O.W., Grosskreutz, J., 2019. Reaction to endoplasmic reticulum stress via ATF6 in amyotrophic lateral sclerosis deteriorates with aging. Front. Aging Neurosci. 11, 5. https://doi.org/10.3389/fnagi.2019.00005.
Rai, S.N., Dilnashin, H., Birla, H., Singh, S.S., Zahra, W., Rathore, A.S., Singh, B.K., Singh, S.P., 2019. The role of PI3K/Akt and ERK in neurodegenerative disorders. NeurotoX. Res. 35, 775–795. https://doi.org/10.1007/s12640-019-0003-y.
Russo, C., Dolcini, V., Salis, S., Venezia, V., Zambrano, N., Russo, T., Schettini, G., 2002. Signal transduction through tyrosine-phosphorylated C-terminal fragments of amyloid precursor protein via an enhanced interaction with Shc/Grb2 adaptor proteins in reactive astrocytes of Alzheimer’s disease brain. J. Biol. Chem. 277, 35282–35288. https://doi.org/10.1074/jbc.M110785200.
Ryu, E.J., Harding, H.P., Angelastro, J.M., Vitolo, O.V., Ron, D., Greene, L.A., 2002. Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson’s disease. J. Neurosci. 22, 10690–10698. https://doi.org/10.1523/ JNEUROSCI.22-24-10690.2002.
Sajiki, J., Yonekubo, J., 2003. Leaching of bisphenol A (BPA) to seawater BiP Inducer X from polycarbonate plastic and its degradation by reactive oXygen species. Chemosphere 51, 55–62. https://doi.org/10.1016/S0045-6535(02)00789-0.
Schwarz, T.L., 2013. Mitochondrial trafficking in neurons. Cold Spring Harb. Perspect. Biol. 5, a011304 https://doi.org/10.1101/cshperspect.a011304.
Selvaraj, S., Sun, Y., Watt, J.A., Wang, S., Lei, S., Birnbaumer, L., Singh, B.B., 2012. NeurotoXin-induced ER stress in mouse dopaminergic neurons involves downregulation of TRPC1 and inhibition of AKT/mTOR signaling. J. Clin. Invest. 122, 1354–1367. https://doi.org/10.1172/JCI61332.
Sherer, T.B., Betarbet, R., Greenamyre, J.T., 2002. Environment, mitochondria, and Parkinson’s disease. Neuroscientist. 8, 192–197. https://doi.org/10.1177/1073858402008003004.
Subramaniam, S., Unsicker, K., 2010. ERK and cell death: ERK1/2 in neuronal death. FEBS J. 277, 22–29. https://doi.org/10.1111/j.1742-4658.2009.07367.X.
Szegezdi, E., Logue, S.E., Gorman, A.M., Samali, A., 2006. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 7, 880–885. https://doi.org/ 10.1038/sj.embor.7400779.
Vandenberg, L.N., Chahoud, I., Heindel, J.J., Padmanabhan, V., Paumgartten, F.J., Schoenfelder, G., 2010. Urinary, circulating, and tissue biomonitoring studies indicate widespread exposure to bisphenol A. Environ. Health Perspect. 118, 1055–1070. https://doi.org/10.1289/ehp.0901716.
Vats, A., Gourie-Devi, M., Ahuja, K., Sharma, A., Wajid, S., Ganguly, N.K., Taneja, V., 2018. EXpression analysis of protein homeostasis pathways in the peripheral blood mononuclear cells of sporadic amyotrophic lateral sclerosis patients. J. Neurol. Sci. 387, 85–91. https://doi.org/10.1016/j.jns.2018.01.035.
Wang, K., Zhu, L., Zhu, X., Zhang, K., Huang, B., Zhang, J., Zhang, Y., Zhu, L., Zhou, B., Zhou, F., 2014. Protective effect of paeoniflorin on Abeta25-35-induced SH-SY5Y cell injury by preventing mitochondrial dysfunction. Cell. Mol. Neurobiol. 34, 227–234. https://doi.org/10.1007/s10571-013-0006-9.
Wang, L., Chen, S., Yin, Z., Xu, C., Lu, S., Hou, J., Yu, T., Zhu, X., Zou, X., Peng, Y., Xu, Y., Yang, Z., Chen, G., 2015. Conditional inactivation of Akt three isoforms causes tau hyperphosphorylation in the brain. Mol. Neurodegener. 10, 33. https://doi.org/ 10.1186/s13024-015-0030-y.
Wang, T., Xie, C., Yu, P., Fang, F., Zhu, J., Cheng, J., Gu, A., Wang, J., Xiao, H., 2017. Involvement of insulin signaling disturbances in bisphenol A-induced Alzheimer’s disease-like neurotoXicity. Sci. Rep. 7, 7497. https://doi.org/10.1038/s41598-017- 07544-7.
Wang, H., Zhao, P., Huang, Q., Chi, Y., Dong, S., Fan, J., 2019a. Bisphenol-A induces neurodegeneration through disturbance of intracellular calcium homeostasis in human embryonic stem cells-derived cortical neurons. Chemosphere 229, 618–630. https://doi.org/10.1016/j.chemosphere.2019.04.099.
Wang, Y., Xu, E., Musich, P.R., Lin, F., 2019b. Mitochondrial dysfunction in neurodegenerative diseases and the potential countermeasure. CNS Neurosci. Ther. 25, 816–824. https://doi.org/10.1111/cns.13116.
World Health Organization (WHO); Geneva, Switzerland, 2011. ToXicological and Health Aspects of Bisphenol A. https://www.who.int/foodsafety/publications/bi sphenol-a/en/.
Wu, Y., Chen, M., Jiang, J., 2019. Mitochondrial dysfunction in neurodegenerative diseases and drug targets via apoptotic signaling. Mitochondrion 49, 35–45. https:// doi.org/10.1016/j.mito.2019.07.003.
Xu, C., Bailly-Maitre, B., Reed, J.C., 2005. Endoplasmic reticulum stress: cell life and death decisions. J. Clin. Invest. 115, 2656–2664. https://doi.org/10.1172/ JCI26373.
Yoshihara, S., Makishima, M., Suzuki, N., Ohta, S., 2001. Metabolic activation of bisphenol A by rat liver S9 fraction. ToXicol. Sci. 62, 221–227. https://doi.org/ 10.1093/toXsci/62.2.221.
Yoshihara, S., Mizutare, T., Makishima, M., Suzuki, N., Fujimoto, N., Igarashi, K., Ohta, S., 2004. Potent estrogenic metabolites of bisphenol A and bisphenol B formed by rat liver S9 fraction: their structures and estrogenic potency. ToXicol. Sci. 78, 50–59. https://doi.org/10.1093/toXsci/kfh047.
Zhu, J.H., Kulich, S.M., Oury, T.D., Chu, C.T., 2002. Cytoplasmic aggregates of phosphorylated extracellular signal-regulated protein kinases in Lewy body diseases. Am. J. Pathol. 161, 2087–2098. https://doi.org/10.1016/S0002-9440(10)64487-2.