Cell Mol Neurobiol DOI 10.1007/s10571-015-0196-4

ORIGINAL RESEARCH

Protective Role of tert-Butylhydroquinone Against Sodium Fluoride-Induced Oxidative Stress and Apoptosis in PC12 Cells Jie Wu1,2 • Ming Cheng1 • Qiufang Liu1 • Jinghua Yang1 • Shengwen Wu1 Xiaobo Lu1 • Cuihong Jin1 • Honglin Ma2 • Yuan Cai1



Received: 5 February 2015 / Accepted: 12 April 2015 Ó Springer Science+Business Media New York 2015

Abstract The neurotoxicity of fluoride is associated with oxidative stress due to imbalance between production and removal of reactive oxygen species (ROS). In contrast, induction of detoxifying and antioxidant genes through activation of NF-E2-related factor 2 (Nrf2) has been implicated in preventing oxidative stress and apoptosis in neurodegenerative diseases. The present study aimed to investigate the possible neuroprotective role of tert-butylhydroquinone (tBHQ), a general Nrf2 activator, on sodium fluoride (NaF)-induced oxidation damage and apoptosis in neuron-like rat pheochromocytoma (PC12) cells. Pretreatment with tBHQ protected PC12 cells against NaF-induced cytotoxicity as measured by MTT assay and apoptosis detection, simultaneously, inhibited NaF-induced overproduction of intracellular ROS and reduction of total glutathione content. Furthermore, NaF or tBHQ induced the stabilization of Nrf2, and enhanced expression of heme oxygenase-1 (HO-1) and c-glutamylcysteine synthetase (cGCS) as a consequence of Nrf2 inducing. These findings indicated that tBHQ pretreatment conferred protective effect on PC12 cells against NaF-induced apoptotic cell death and oxidation-redox imbalance through stabilization of Nrf2 and elevation of downstream HO-1 and c-GCS expressions.

& Yuan Cai [email protected] 1

Department of Toxicology, School of Public Health, China Medical University, Shenyang 110013, Liaoning, China

2

Department of Occupational and Environmental Health, School of Public Health, Liaoning Medical University, Jinzhou 121001, Liaoning, China

Keywords Sodium fluoride  Apoptosis  Nuclear factorerythroid 2-related factor 2  tert-Butylhydroquinone

Introduction Fluoride is an essential element required for bone health and dental caries prevention, whereas excessive ingestion of fluoride from either natural or anthropogenic ways could result in fluorosis, a progressive degenerative disorder, which causes dental mottling and skeletal manifestations, but also damages to soft tissues, such as brain, liver, kidney, and spinal cord (Barbier et al. 2010). Children living in endemic fluorosis areas had significantly lower intelligence quotient (IQ) than those in low fluoride areas (Choi et al. 2012; Saxena et al. 2012; Zhang et al. 2015), suggested that high fluoride impairs children’s neurodevelopment. Chronic or subchronic fluoride exposure evoked alterations in the cerebral morphology, activity of antioxidative enzymes, and homeostasis of transmitters, consequently induced dysfunction of the central nervous system (CNS) and reduction in learning and memory ability in both adult and young rats (Gui et al. 2010; Pereira et al. 2011; Jiang et al. 2013). Latest study showed that developmental exposure to 5 or 10 lg/mL sodium fluoride for 4 weeks induced cognitive deficits and anxiety-depressionlike behaviors in mice (Liu et al. 2014). However, the mechanism underlying the developmental neurotoxicity of fluoride remains obscure. Sodium fluoride could induce apoptosis in various cells (Wang et al. 2011; Nguyen Ngoc et al. 2012; Jothiramajayam et al. 2014), including primary cultured hippocampal neurons and SH-SY5Y cells (Zhang et al. 2007; Xu et al. 2013), which has been demonstrated to be related with excessive intracellular reactive oxygen species (ROS).

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High ROS concentrations above the clearance capacity of cells induce oxidative damage on biomolecules including lipids, proteins, and DNA, which is critical to the etiology of numerous neurodegenerative disorders (Thanan et al. 2014). Fluoride intoxication is associated with oxidative stress and altered anti-oxidant defense mechanism, manifested by significantly increased brain ROS and lipid peroxidation (LPO) levels, whereas markedly reduced glutathione content and decreased activity of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidases (Zhang et al. 2007; Basha et al. 2011; Wang et al. 2013). The prevention of cellular oxidative stress may be best achieved by stimulating an endogenous mechanism regulated at the transcriptional level by genes containing the antioxidant response element (ARE), which is activated through the binding of its transcription factor Nrf2 (Johnson et al. 2008). Upon exposure to the electrophiles and ROS, several genes encoding detoxification enzymes and antioxidant proteins are simultaneously induced in a synchronized manner, such as heme oxygenase-1 (HO-1) and glutamylcysteine synthetase (c-GCS) (Lou et al. 2012; Jin et al. 2014). Recently, induction of Nrf2-ARE pathway has been considered to be a therapeutic target in several neurodegenerative diseases (Calkins et al. 2009; Joshi and Johnson 2012). Additionally, induction of endogenous antioxidants through activation of Nrf2 by tBHQ has been shown to prevent cellular oxidative stress and apoptosis in neurodegenerative diseases (Nouhi et al. 2011) and against neurotoxic insults caused by paraquat and manganese (Li et al. 2011, 2012). Although multiple exogenous antioxidants have been shown amelioration on fluoride-induced oxidative stress in adult or developing CNS (Basha and Madhusudhan 2010; Atmaca et al. 2014), there are no reports concerning the defensive role of tBHQ in fluoride neurotoxicity. Therefore, the present study was designed to investigate the neuroprotective effect of tBHQ, a general Nrf2 activator, against fluoride-induced oxidative stress and apoptosis in neuron-like PC12 cells in an attempt to elucidate molecular mechanisms of the developmental neurotoxicity of fluoride and provide potential preventive approaches.

assay kit was purchased from Beyotime Institute of Biotechnology (Nantong, China). Glutathione (GSH) assay kit was from Jiancheng Bioengineering Institute (Nanjing, China). Annexin V-FITC/PI Apoptosis Detection Kit was from Biosea Biotecnology Co. (Beijing, China). PrimeScriptTM RT-PCR Kit was from TaKaRa Bio Inc. (Dalian, China). Anti-Nrf2 antibody (C-20, sc-722) and anti-betaactin antibody (N-21, sc-130656) were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Cell Culture and Treatment PC12 cells were cultured in RPMI 1640 medium (HyClone, Logan, USA) supplemented with 5 % (v/v) fetal bovine serum (HyClone, Logan, USA), 10 % (v/v) horse serum (Gibco, Carlsbad, USA), and 100 U/ml antibiotic mixture of penicillin/streptomycin, at 37 °C in a humidified atmosphere with 5 % CO2. Growth medium was changed every 2–3 days, cells were sub-cultured when 80–90 % confluent. During experiments, PC12 cells were incubated with or without 10 lM tBHQ for 16 h, followed by exposure to 20, 40, and 80 lg/mL NaF for another 24 h. NaF and tBHQ were dissolved in RPMI 1640 medium, respectively. Simultaneously, control cells were cultured in serum-free RPMI 1640 medium. MTT Cell Viability Assay Viability of PC12 cells based on mitochondrial enzyme function were determined by MTT assay after pretreatment with 5, 10, 20, and 40 lM tBHQ for 16 h, and after exposure to NaF (20, 40, and 80 lg/mL) for subsequent 24 h. Briefly, PC12 cells were trypsinized and plated at a density of 2 9 104 cells/well into 96-well plates. After treatment, 10 lL MTT (0.5 mg/mL) was added into each well, and incubated for another 4 h at 37 °C. The supernatant was removed, and 150 lL DMSO was added to dissolve the produced formazan crystals. The absorbance of samples was measured at 570 nm with a microplate reader (BioRad, USA). The results of cell viability were presented as a percentage of the control. Measurement of Apoptotic Rate

Materials and Methods Chemicals Sodium fluoride (NaF, C99 %), tBHQ (98.10 %), dimethyl sulfoxide (DMSO, C99.9 %), Trypsin, 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), and TRIzol reagent were obtained from SigmaAldrich (St. Louis, MO, USA). Reactive oxygen species

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After exposure to NaF (20, 40, and 80 lg/mL) for 24 h, with or without pretreatment with 10 lM tBHQ for 16 h, determination of apoptosis in PC12 cells was performed using Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer’s instruction. Briefly, cells were washed once with ice-cold phosphate buffer saline (PBS) before suspension in binding buffer at a density of 1 9 106/mL, and then stained with 10 lL Annexin V/FITC for 10 min in dark at room temperature, and with 5 lL

Cell Mol Neurobiol

propidium iodide (PI) for another 5 min. Apoptotic rate was analyzed by flow cytometry (BD FACSAria II, USA).

Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Analysis

Measurement of Intracellular Reactive Oxygen Species (ROS)

After exposure to NaF, RT-PCR was used to estimate the levels of Nrf2, HO-1, c-GCSh, and c-GCSl mRNA expressions. Total RNA was extracted from PC12 cells using TRIzol reagent according to the manufacturer’s instruction, and quantified by microplate reader. Reversed transcription reaction of 1 lg extracted sample RNA was carried out using PrimeScriptTM RT-PCR Kit as described previously (Wu et al. 2013). The primer sequences used for cDNA amplification were shown in Table 1. The PCR amplification was performed as follows: pre-denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 65 °C for 30 s, and extension at 72 °C for 4 min. The PCR products were resolved for agarose gel electrophoresis and photographed. b-actin was used as a inner control for normalizing the mRNA expression of respective sample.

Intracellular generation of ROS in PC12 cells was measured using the fluorescent probe 2, 7-dichlorodihydro- fluorescein diacetate (DCFH-DA). Briefly, cells exposed to NaF were washed twice in PBS, incubated with 10 lM DCFH-DA for 30 min at 37 °C in dark, and then analyzed by flow cytometry (BD FACSAria II, USA), with excitation and emission wavelengths at 488 and 525 nm, respectively. The fluorescence intensity of dichlorofluorescein (DCF) represented the quantity of intracellular ROS. Measurement of Intracellular Glutathione (GSH) The intracellular content of total GSH was detected at 412 nm with a microplate reader (Bio-Rad, USA), using improved 5,50 -dithiobis-2-nitrobenzonic acid (DTNB) method according to the manufacturer’s instruction. The absorbance of the reaction mixture was measured at 417 nm by microplate reader (Bio-Rad, USA). The protein content in the PC12 cells was measured by Enhanced BCA Protein Assay Kit (Beyotime, China). The results were expressed as lmol/g protein.

Statistical Analysis All data are expressed as mean ± SD (standard deviation) of at least three independent experiments. Comparison between groups were analyzed by one-way analysis of variance (ANOVA) followed by LSD post hoc tests. The statistically significance was considered when p B 0.05.

Western Blot Analysis

Results

PC12 cells exposed to NaF (20, 40, and 80 lg/mL) for 24 h, with or without pretreatment with 10 lM tBHQ for 16 h, were washed twice in ice-cold PBS and lysed in RIPA Lysis Buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 % Triton X-100, 0.5 % Sodium Deoxycholate, 0.1 % SDS, 1 mM dithiothreitol, 1 mM PMSF, and a protease inhibitor mixture) for 30 min on ice. The lysates were centrifuged at 12,0009g for 10 min at 4 °C, the supernatants were collected and used for detection of Nrf2 protein expression. The protein concentration in each sample was measured using Enhanced BCA Protein Assay Kit (Beyotime, China). Western blots were performed according to standard protocols, using rabbit polyclonal antibodies (anti-Nrf2, 1:100; anti-b-actin, 1:400) and secondary HRP-conjugated anti-rabbit IgG (1:1000, ZSBIO, China). Immunoreactive bands were developed with enhanced chemiluminescence (ECL) reagents (Beyotime, China) and visualized with X-ray film. Densitometric analysis was performed with Gel-Pro Analyzer version 3.0 and normalized using b-actin as internal control.

Effect of tBHQ Pretreatment and NaF Exposure on Viability of PC12 Cells This study initially examined the influence of tBHQ on the viability of PC12 cells, with the purpose of determining the dose of tBHQ applied in the subsequent experiments. tBHQ alone did not show cytotoxic effect at concentrations of 5 and 10 lM, whereas treating the cells with 20 and 40 lM tBHQ for 16 h decreased cell viability by 1.85 and 29.0 % (p \ 0.01), respectively (Fig. 1a). As an optimal tBHQ concentration, 10 lM was selected for subsequent experiments, in which cell viability was 6.61 % higher than control, although without statistical significance. Then evaluated the cytotoxicity of NaF exposure on PC12 cells with or without pretreatment with 10 lM tBHQ for 16 h. NaF exposure (20, 40, and 80 lg/mL) for 24 h significantly decreased cell survival in a dose-dependent manner, from 89.8 to 69.9 % versus control (Fig. 1b). While compared with NaF exposure alone, pretreatment with 10 lM tBHQ alleviated NaF-induced reduction in cell viability by 5.32, 7.74, and 22.7 % (p \ 0.05), respectively (Fig. 1b). These

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Cell Mol Neurobiol Table 1 Primers used for RTPCR analysis

Gene name

Primer sequences

Size of production (bp)

Nrf2

50 CCATTTACGGAGACCCAC 30

448

50 TGAGCGGCAACTTTATTC 30 HO-1

50 TTCACCTTCCCGAGCATC 30

110

50 GCCTCTTCTGTCACCCTGT 30 c-GCSh

50 TGATAGAACACGGGAGG 30 0

371

0

5 TTGGTCGGAACTCTACTC 3 c-GCSl

50 GACATTGAAGCCCAGGAGT 30

268

50 ACATTGCCAAACCACCACA 30 b-actin

50 ATATCGCTGCGCTGGTCGTC 30

517

50 AGGATGGCGTGAGGGAGAGC 30 b-actin

50 GTCCCTCACCCTCCCAAAAG 30

266

Fig. 1 Effect of tBHQ pretreatment and NaF exposure on the viability of PC12 cells. Cell viability was measured by MTT assay after tBHQ pretreatment (a) and NaF exposure (b). Values are

expressed as a percentage of the control, represents the mean ± SD (n = 6). *p \ 0.05, **p \ 0.01 versus control; 4p \ 0.05 versus 40 lg/mL NaF alone; &p \ 0.05 versus 80 lg/mL NaF alone

findings indicated that 10 lM tBHQ had protective effects on PC12 cells from cytotoxicity induced by NaF.

tBHQ Protect Against NaF-Induced Toxicity in PC12 Cells by Reducing ROS and Increasing GSH

tBHQ Protect Against NaF-Induced Apoptosis in PC12 Cells To determine if cytotocxicity of NaF on neuron-like PC12 cells was due to induction of apoptosis, cells apoptotic rate was evaluated by flow cytometry analysis using Annexin V-FITC/PI staining after NaF exposure, with or without tBHQ pretreatment. As shown in Fig. 2, NaF-induced PC12 cells apoptosis in a dose-dependent manner, manifested by apoptotic rate significantly increased to 2.28-, 3.36-, and 6.44-folds of control, at the concentration of 20, 40, and 80 lg/mL. As compared to NaF exposure alone, 10 lM tBHQ-pretreated groups showed obviously decreased apoptotic rate by 20.5, 40.7, and 34.8 %, respectively, although still significantly higher than the control group. The results indicated that tBHQ pretreatment could only partially prevent, not fully protect, NaFexposed cells from apoptosis.

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Since the accumulation of intracellular ROS and the reduction of GSH level are crucial to oxidative stress and the eventually neuronal death, we investigated adverse effects of NaF on the intracellular ROS and GSH contents and the protection of tBHQ against those effects in PC12 cells. After exposure to NaF, with or without pretreatment with tBHQ, intracellular ROS level was evaluated by DCF fluorescence intensity detected by flow cytometry. As shown in Fig. 3a, DCF fluorescence intensities in PC12 cells increased with NaF concentrations in a dose-dependent manner, significantly elevated to 1.21, 1.84, and 2.66 folds of control, respectively. Oppositely, concentrations of total glutathione significantly decreased by 23.5, 38.8, and 55.8 %, respectively, compared to the control (Fig. 3b). Pretreatment with tBHQ significantly alleviated the elevation of intracellular ROS and the reduction of GSH level, compared to the same

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Fig. 2 tBHQ protect against NaF-induced apoptosis in PC12 cells. Cells were exposed to NaF (20, 40, and 80 lg/mL) for 24 h, with or without 10 lM tBHQ pretreatment for 16 h, apoptotic rate was measured by flow cytometry using Annexin V-FITC/PI. Ten thousand cells were analyzed in each sample, and cells in the fourth quadrant

which represent early apoptosis were counted. Values represented mean ± SD, n = 4 for each group. *p \ 0.05 versus control; 4 p \ 0.05 versus 40 lg/mL NaF alone; &p \ 0.05 versus 80 lg/ mL NaF alone

concentration of NaF alone group. The results showed that DCF fluorescence intensities in tBHQ-pretreated cells significantly decreased by 14.5, 38.6, and 51.7 %, in contrast, GSH contents showed a significantly increasing by 22.1, 41.6, and 60.7 %, respectively (Fig. 3b), suggested that tBHQ protected PC12 cells from NaF-induced oxidative stress via promoting the clearance of ROS and synthesis of GSH within cells.

tBHQ Pretreatment and NaF Exposure Increased Nrf2 Protein Expression in PC12 Cells To determine whether NaF exposure and tBHQ pretreatment could induce Nrf2 stabilization in PC12 cells, Nrf2 protein level in the whole cell lysates was examined by Western blot analysis after NaF exposure using b-actin as an internal reference. As shown in Fig. 4b, the expression

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Fig. 3 tBHQ protect against NaF-induced toxicity in PC12 Cells by reducing ROS and increasing GSH. Cells were exposed to NaF (20, 40, and 80 lg/mL) for 24 h, with or without 10 lM tBHQ pretreatment for 16 h, intracellular ROS generation was determined using DCFH-DA by flow cytometry, and the DCF fluorescence intensity represented intracellular ROS level (a). GSH content was measured

by microplate reader using improved DTNB method, results were expressed as lmol/g protein (b). Values represented mean ± SD (n = 4). *p \ 0.05 versus control; #p \ 0.05 versus 20 lg/mL NaF alone; 4p \ 0.05 versus 40 lg/mL NaF alone; &p \ 0.05 versus 80 lg/mL NaF alone

of Nrf2 protein in NaF exposure alone group increased to 1.97 (p \ 0.05)-, 1.54 (p \ 0.05)-, and 1.03-folds of control, respectively. Compared with corresponding NaF only group, tBHQ-pretreated cells displayed increasing Nrf2 protein levels by 2.94 %, 14.6 % (p \ 0.05), and 32.1 % (p \ 0.05), respectively. These data indicated that NaF and tBHQ activate the Nrf2 pathway in PC12 cells by upregulation of stable Nrf2 expression at the protein level.

response to either NaF exposure alone or with tBHQ pretreatment. Simultaneously, exposure to 20 lg/mL NaF upregulated expression of Nrf2 target genes, HO-1 and heavy subunits of c-GCS (c-GCSh), at the mRNA level by up to 1.20 and 1.46 folds of control, an induction equal or superior to that observed in cells with tBHQ pretreatment. Whereas, as compare to the untreated control, 40 and 80 lg/mL NaFexposed cells presented a dose-dependent decrease in mRNA levels of HO-1 and c-GCSh, which were relatively revised in tBHQ pretreatment groups, equal to (tBHQ ? 40) or still lower (tBHQ ? 80) than those in the control. Altogether these results suggested that low-dese NaF exposure induced activation of Nrf2-ARE pathway, while higher dose showed suppression to some extent, and pretreatment with tBHQ facilitated the induction of Nrf2-ARE pathway originated by subsequently NaF exposure.

tBHQ Pretreatment and NaF Exposure Altered HO1 and c-GCS mRNA Expressions in PC12 Cells Nrf2 regulates a broad spectrum of enzymes and proteins involved in the disposition of oxidative stress. The stabilization of Nrf2 by tBHQ suggests that the expression of phase II detoxification enzymes and antioxidants would increase due to the induction of ARE-driven cytoprotective genes by Nrf2 (Li et al. 2011). Therefore, we examined the effects of NaF exposure and tBHQ pretreatment on the mRNA expression of Nrf2 and broadly known Nrf2 target antioxidant genes HO-1 and c-GCS in PC12 cells. As shown in Fig. 5, there were no apparent changes in mRNA expression of Nrf2 and light subunit of c-GCS (c-GCSl) in

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Discussion Increased fluoride exposure can lead to effects on bone cells (both osteoblasts and osteoclasts), also affects cells from soft tissues, such as renal, endothelial, gonadal, and

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Fig. 4 tBHQ and NaF increased Nrf2 protein expression in PC12 cells. After exposure to NaF (20, 40, and 80 lg/mL) for 24 h, with or without 10 lM tBHQ pretreatment for 16 h, Nrf2 protein level in PC12 cells was analyzed by Western blot performed on the whole cell lysates, using b-actin as internal reference. Representative immunoblot photogram (a) and quantification (b) of Nrf2. Values represented mean ± SD, n = 3 for each group. *p \ 0.05 versus control; #p \ 0.05 versus 20 lg/mL NaF alone; 4p \ 0.05 versus 40 lg/mL NaF alone; &p \ 0.05 versus 80 lg/mL NaF alone

neurological cells (Barbier et al. 2010). It is generally recognized that NaF at concentrations greater than 1 mM causes growth arrest and cell death either by necrosis or apoptosis (Nguyen Ngoc et al. 2012). PC12 cell is an established cell line derived from rat pheochromocytoma, possessing neuron-like qualities in morphology and function, therefore, has been extensively used as an in vitro model in neurobiological and neurotoxicological studies (Greene and Tischler 1976; Jiang and Li 2014; Li et al. 2011). In the present study, we found that NaF exposure significantly reduced cell viability and increased apoptosis in a dose-dependent manner, confirming its cytotoxicity in neuron-like PC12 cells. Pretreatment with tBHQ prevented fluoride-induced apoptosis through stabilization of Nrf2 and subsequently transcriptional activation of antioxidants HO-1 and c-GCS, simultaneously ameliorated oxidativeredox environment within cells. The CNS is particularly vulnerable to oxidative damage on account of its high consumption of oxygen, high amounts of readily oxidizable polyunsaturated fatty acids, and relatively low levels of antioxidant (Joshi and Johnson 2012). Fluoride toxicity is associated with ROS induction, reactive nitrogen species generation and the reduction of

cellular antioxidant defenses against oxidative damage (Barbier et al. 2010). These effects have been observed in several soft tissues and cells, such as brain, kidney, and liver (Miao et al. 2013; Nabavi et al. 2013; Song et al. 2014). Moreover, fluoride can alter contents of glutathione, one primary intracellular ROS scavenger, often resulting in the excessive production of ROS at the mitochondrial level leading to the damage of cellular components (Zhang et al. 2007; Bharti and Srivastava 2009). Here, we obtained the results in accordance with previous studies, NaF exposure induced ROS overproduction and GSH depletion in PC12 cells. These findings suggest that fluoride exposure induce an imbalance between the pro- and anti-oxidant levels, leading to subsequent oxidative stress, which plays an important role in fluoride-mediated neuronal apoptosis. The Nrf2-ARE pathway is a primary sensor and a master regulator of oxidative stress via its ability to modulate the cells endogenous antioxidant capacity (Johnson et al. 2008). Under normal conditions, intracellular Nrf2 is targeted for proteasomal degradation by its inhibitor Kelchlike ECH-associated inhibitor 1 (Keap1). When stimulated by oxidants or electrophiles, conformational changes in the Nrf2-Keap1 complex inhibit proteasomal degradation of Nrf2, facilitating an increase in the amount of Nrf2 that binds to a variety of ARE-dependent antioxidant, detoxification, and metabolic control genes (Hybertson and Gao 2014). Activation of Nrf2-ARE pathway has shown benefits in animal models of many neurodegenerative disorders supporting the concept of being a potential therapeutic target (Joshi and Johnson 2012; Gan and Johnson 2014). As a prototypical Nrf2 activator, tBHQ may have therapeutic potential for Alzheimer’s disease by increasing brain antioxidant capacity and by stimulating Ab clearance pathways (Akhter et al. 2011). Therefore, we evaluated the protective effect of tBHQ against NaF-induced neurotoxicity. The present data showed that NaF exposure (20 and 40 lg/mL), with or without tBHQ pretreatment, caused increase in the protein expression of Nrf2 in PC12 cells, while without obvious changes in the transcriptional level of Nrf2 gene in all groups. It seems that the induction of Nrf2 by NaF (20 and 40 lg/mL) is not regulated by transcriptional activation but by posttranslational stabilization, which is analogous to the observation that treatment of tBHQ failed to affect Nrf2 mRNA but strongly attenuated ubiquitination and proteasomal degradation of Nrf2 (Li et al. 2005). Furthermore, tBHQ pretreatment ameliorated cellular apoptosis, ROS accumulation and GSH depletion induced by NaF exposure in PC12 cells, suggesting that these neuroprotective effects involved with Nrf2 stabilization. Phase II detoxifying and antioxidant genes regulated by Nrf2 include redox regulation (e.g., superoxide dismutase and catalase), glutathione synthesis and metabolism (e.g.,

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Fig. 5 Effects of tBHQ and NaF on the mRNA expression of Nrf2 and Nrf2 target genes. PC12 cells were treated with or without 10 lM tBHQ for 16 h, followed by NaF exposure (20, 40, and 80 lg/mL) for 24 h, total mRNA was extracted. The relative mRNA levels of Nrf2 (a) and its downstream genes, HO-1 (b) and c-GCS (c), were

determined by semi-quantitative RT-PCR using b-actin as internal reference. Relative quantification of mRNA expression (d).Values represented mean ± SD, n = 3 for each group. *p \ 0.05 versus control; #p \ 0.05 versus 20 lg/mL NaF alone; 4p \ 0.05 versus 40 lg/mL NaF alone; &p \ 0.05 versus 80 lg/mL NaF alone

gamma-glutamine cysteine ligase and c-GCS), quinine recycling [NAD(P)H quinone oxidoreductase 1], and iron homeostasis (HO-1, ferritin) (Joshi and Johnson 2012). The chaperone protein HO-1 catalyzes degradation of heme to biliverdin that is subsequently converted to bilirubin, both the two of them have shown antioxidant properties. Numerous evidences indicate that Nrf2-mediated regulation of HO-1 protect PC12 cells from toxic and oxidative damages (Khodagholi and Tusi 2011; Li et al. 2012). cGCS, a heterodimer consisting of heavy (c-GCSh) and light (c-GCSl) subunits, catalyzes the rate-limiting de novo biosynthesis of GSH, an abundant physiological antioxidant that plays an important role in regulating oxidative stress and maintaining a reducing environment within the cells (Lou et al. 2012). In the present study, mRNA expression of Nrf2 downstream antioxidants HO-1 and cGCSh manifested the same variation tendency with Nrf2 protein after 20 lg/mL NaF exposure, whereas significantly decreased at 40 and 80 lg/mL NaF exposure, and tBHQ pretreatment alleviated the down-regulation of transcriptional level of HO-1 and c-GCSh to a certain extent, a finding consistent with increasing intracellular contents of GSH, suggesting the protective role of Nrf2ARE pathway activation in enhancing the antioxidant ability, subsequently clearance of ROS, and inhibition of oxidative stress. In summary, the protective effect of tBHQ on the neuron-like PC12 cells against NaF and simultaneously the

activation of Nrf2-ARE pathway were elucidated for the first time. The Nrf2-ARE pathway, induced by tBHQ pretreatment, ameliorated fluoride-induced PC12 cells apoptosis. Further studies are necessary to detail the mechanisms of preventing neuronal apoptosis by Nrf2 activation against fluoride. The present study highlights that Nrf2 and downstream antioxidants HO-1 and c-GCS activation induced by tBHQ attenuate NaF-induced oxidantredox imbalance and consequently apoptosis in PC12 cells, suggesting a preventative measure for fluoride neurotoxicity.

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Conflict of interest The authors declare that there are no conflict of interest.

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Protective Role of tert-Butylhydroquinone Against Sodium Fluoride-Induced Oxidative Stress and Apoptosis in PC12 Cells.

The neurotoxicity of fluoride is associated with oxidative stress due to imbalance between production and removal of reactive oxygen species (ROS). In...
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