Chitosan attenuates dibutyltin-induced apoptosis in PC12 cells through inhibition of the mitochondria-dependent pathway

Carbohydrate Polymers 151 (2016) 996–1005

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Chitosan attenuates dibutyltin-induced apoptosis in PC12 cells through inhibition of the mitochondria-dependent pathway Xiaorui Wang 1 , Junqiu Miao 1 , Chaoqun Yan, Rui Ge, Taigang Liang ∗ , Enli Liu, Qingshan Li ∗ School of Pharmaceutical Science, Shanxi Medical University, No. 56, Xinjian Nan Road, Taiyuan 030001, Shanxi, PR China

a r t i c l e

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Article history: Received 4 November 2015 Received in revised form 12 June 2016 Accepted 14 June 2016 Available online 15 June 2016 Keywords: Chitosan Dibutyltin Apoptosis PC12 cells Mitochondria-dependent pathway

a b s t r a c t Dibutyltin (DBT) which was widely used as biocide and plastic stabilizer has been described as a potent neurotoxicant. Chitosan (CS), a natural nontoxic biopolymer, possesses a variety of biological activities including antibacterial, antifungal, free radical scavenging and neuroprotective activities. The present study was undertaken to investigate the protective effects of CS against DBT-induced apoptosis in rat pheochromocytoma (PC12) cells and the underlying mechanisms in vitro. Our results demonstrated that pretreatment with CS significantly increased the cell viability and decreased lactate dehydrogenase (LDH) release induced by DBT in a dose-dependent manner. Meanwhile, DBT-induced cell apoptosis, mitochondrial membrane potential (MMP) disruption, and generation of intracellular reactive oxygen species (ROS) were attenuated by CS. Real-time PCR assay showed that DBT markedly enhanced the mRNA levels of Bax, Bad, cytochrome-c and Apaf-1, reduced the Bcl-2 and Bcl-xL mRNA levels, while these genes expression alteration could be partially reversed by CS treatment. Furthermore, CS also inhibited the DBT-inducted activation of caspase-9, and -3 at mRNA and protein expression levels. Taken together, these results suggested that CS could protect the PC12 cells from apoptosis induced by DBT through inhibition of the mitochondria-dependent pathway. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Organotin (IV) compounds (OTC) as a wide spread environmental pollutant may flow in ecological system through the major sources such as polyvinyl chloride (PVC) stabilizers, industrial catalysts, agricultural biocides, wood preservatives, and antifouling agents in paints (Hoch, 2001; Tolosa et al., 1996). It has now been confirmed that OTC can enter the human body through inhala-

Abbreviations: AO, acridine orange; BCA, bicinchoninic acid; BH3s, “BH3 domain only” proteins; CS, chitosan; DBT, dibutyltin; DCF, dichlorofluorescein; DCFH, dichlorofluorescein; DCFH-DA, 2 ,7 -dichloro fluorescein diacetate; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; DMT, dimethyltin; DPT, diphenyltin; EB, ethidium bromide; ECL, the enhanced chemiluminescence; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; JC-1, 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethyl-imidacarbocyanine iodide; LDH, lactate dehydrogenase; MMP, mitochondrial membrane potential; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OTC, organotin (IV) compounds; PBS, phosphate buffered saline; PI, propidium iodide; PVC, polyvinyl chloride; ROS, reactive oxygen species; TBS-T, tris-buffered saline plus Tween-20; TMT, trimethyltin. ∗ Corresponding authors. E-mail addresses: [email protected] (T. Liang), [email protected] (Q. Li). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.carbpol.2016.06.053 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

tion (Chicano, Ortiz, Teruel, & Aranda, 2001; Kimbrough, 1976), skin (Yoo et al., 2007), food (Borghi & Porte, 2002), or drinking water (Sadiki, Williams, Carrier, & Thomas, 1996), and consequently great concern has been growing about its potential health effects. Based on the number of added alkyl groups, OTC can be classified as mono-, di-, tri- and tetra-alkyltins (Pellerito & Nagy, 2002). Among them, the toxicities of triorganotins have been well reported, some compounds (tributyltin, triphenyltin, trimethyltin) exhibited a high specificity of action and are cyto- and genotoxic in several test systems (Pagliarani, Nesci, & Ventrella, 2013; Snoeij, Van Iersel, Penninks, & Seinen, 1985). In the last two decades, special attention has been drawn to the neurotoxicity of triorganotins (Florea & Büsselber, 2006; Yu, Ding, Sun, Salvi, & Roth, 2015). For instance, mammals intoxicated with trimethyltin (TMT) could experience a variety of adverse effects on the central nervous system (Gramowski, Schiffmann, & Gross, 2000; LeBel, Ali, McKee, & Bondy, 1990), including seizures, self-mutilation, vocalization, hyperactivity and aggressive behavior, due to extensive neuronal damage (Fortemps, Amand, Bomboir, Lauwerys, & Laterre, 1978; Ishida et al., 1997; Yoo et al., 2007). In addition to triorganotin compounds, accumulating evidences suggest that diorganotins, especially dibutyltin (DBT), are also recognized as neurotoxicants

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(Ferreira, Blanco, Garrido, Vieites, & Cabado, 2013). It is well demonstrated that DBT could significantly inhibit neurite outgrowth and was found to be even more toxic than TMT in vitro (Jenkins, Ehman, & Barone, 2004; Mundy & Freudenrich, 2006). Moreover, our recent study showed that the cytotoxic potency of three diorganotins, namely, dimethyltin (DMT), DBT and diphenyltin (DPT), in PC12 cells was in the order of DBT > DPT > >DMT, and these compounds could induce PC12 cells apoptosis through ROS mediated mitochondrial pathway (Liu et al., 2013). Chitosan (CS), a linear polysaccharide, is obtained by alkaline deacetylation of chitin (Baxter, Dillon, Taylor, & Roberts, 1992) which is the second most abundant naturally occurring polysaccharide found in the exoskeletons of crustaceans such as crabs and shrimp and cell walls of fungi (Synowiecki & Al-Khateeb, 2003). It is biodegradable, biocompatible, nontoxic (Jayakumar, Menon, Manzoor, Nair, & Tamura, 2010) and is known to possess considerable biological properties including antibacterial, antifungal, anticancer, anti-diabetic, and free radical scavenging activities (Havashi & Ito, 2002; Park, Je, & Kim, 2004; Xia, Liu, Zhang, & Chen, 2011). In particular, many studies recently suggested that CS or CS derivatives have potential value as neuroprotective agents in vitro and in vivo (Jiang, Zhuge, Yang, Gu, & Ding, 2009; Pangestuti & Kim, 2010). The neuroprotection was achieved through its inhibition of oxidative stress, inflammation and preventing cell apoptosis (Khodagholi, Efekharzadeh, Maghsoudi, & Rezaei, 2010). Although the serious neurotoxicity of OTC has been clearly stated, the protection against these compounds has rarely been reported. The present study was aimed to explore the potential effect of CS against DBT-induced PC12 cells toxicity, which was performed by measuring the cell viability, the generation of intracellular reactive oxygen species (ROS), as well as mitochondrial membrane potential (MMP). Besides, the changes of apoptosis indexes were also assessed. Our results suggest that CS could attenuate DBT-induced neurotoxicity, probably involving the suppression of ROS production and cell apoptosis.

2. Materials and methods 2.1. Reagents Chitosan (degree of deacetylation over 90%, viscosity between 50.0 and 800.0 mPa. s) was purchased from Solarbio Science and Technology Co., Ltd. (Beijing, China). Dibutyltin (DBT), dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma Aldrich Chemical (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), trypsin/EDTA, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Invitrogen Life Technologies, Inc. (Grand Island, NY, USA). Lactate dehydrogenase (LDH) detection kit was purchased from the Nanjing Institute of Jiancheng Biological Engineering (Nanjing, Jiangsu, China). Acridine orange (AO) and ethidium bromide (EB) were obtained from Amerisco (Solon, Ohio, USA). The Annexin V-FITC apoptosis detection kit and mitochondrial membrane potential detection (MMP) kit were supplied by KeyGen technology (Nanjing, China). A reactive oxygen species (ROS) assay kit was purchased from Applygen Technologies, Inc. (Beijing, PR China). RNA extraction reagent kit (RNAiso Plus), Primer ScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) and SYBR Primix Ex TaqTM II (Tli RNaseH Plus) were obtained from TaKaRa (Dalian, China). RIPA lysis buffer and protease inhibitors were purchased from Vazyme Biotech Co., Ltd (Jiangsu, China). Antibodies against caspase-9 and cleaved caspase3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against ␤-actin, anti-rabbit secondary anti-bodies and the enhanced chemiluminescence (ECL) kit were purchased

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from CoWin Biotech Co., Ltd. (Beijing, China). All the other reagents used were of analytical grade. 2.2. Cell cultureand treatments The PC12 cell line (rat pheochromocytoma cells) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and used within 10 passages. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), l-glutamine (2 mM), 100 units/mL penicillin, and 100 ␮g/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 ◦ C. The medium was changed every 2–3 days. When reaching 80–90% confluence, the cells were passaged. PC12 cells were pretreated with different concentrations (50, 100 and 200 ␮g/mL) of CS for 24 h. After the culture medium was discarded, the cells were washed three times with phosphate buffered saline (PBS) and then exposed to 1.5 ␮M DBT for 6 h. The stock solution of DBT was prepared in DMSO while CS was prepared in 1% acetic acid. Both the vehicles were present at final concentrations of 0.01% DMSO and 0.01% acetic acid in the medium (Khodagholi et al., 2010). The same final concentrations of the solvents were used in the corresponding control. 2.3. Measurement of cell viability MTT colorimetric assay was performed to measure the cell viability. In brief, PC12 cells were grown in 96-well microtiter plates at a final density of 5 × 104 cells/well. After overnight growth, cells were incubated with or without different concentrations of CS for 24 h. Before cells were exposed to DBT, the culture medium was discarded and cells were washed three times with phosphate buffered saline (PBS). After treatment, 10 ␮L of MTT (5 mg/mL) was added to each well and the plate was incubated for 4 h at 37 ◦ C. After that, the medium was pipetted out from each well, and 100 ␮L DMSO was added to dissolve the MTT-formazan crystals produced by the viable cells. The absorbance values at 570 nm were measured using a microplate reader (Model 680, Bio-Rad, US). All experiments were performed in triplicate, and the results for the absorbance measured in treated cells were calculated as percentages of the absorbance in untreated control cells. 2.4. Lactate dehydrogenase (LDH) release assay Lactate dehydrogenase (LDH) can be released from cells with damaged membranes, thus the LDH level in the culture supernatant is a marker indicating the extent of cellular injury (Koh & Choi, 1987). At the end of treatments, the supernatant was collected and LDH leakage was measured using the assay kit according to the manufacturer’s instructions and the absorbance was measured at a wavelength of 450 nm using a microplate reader. Each assay was repeated three times. 2.5. Cell morphological changes assessment PC12 cells were seeded in a 6-well plate (1 × 105 cells/well) and treated with or without different concentrations of CS for 24 h, followed by 6 h exposure to 1.5 ␮M DBT. Then the morphology of cells was monitored under an inverted light microscope at 40 × magnification (BDS200-pH Inverted Microscope, Chongqing Optec Instrument Co., Ltd, China). 2.6. Acridine orange/ethidium bromide (AO/EB) double staining Acridine orange/ethidium bromide (AO/EB) double staining method was employed to qualitatively observe apoptosis on the basis of cell membrane integrity as previous described. AO is a

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membrane-permeable dye that stains both normal and apoptotic cells, whereas, EB stains only those cells that have lost their membrane integrity. Thus, AO can emit a green fluorescence if it passes through the complete cell membrane and embeds in nuclear DNA while EB can mark nuclear DNA of damaged cells and emit a red/orange fluorescence (Joodi, Ansari, & Khodagholi, 2011). Briefly, PC12 cells (1 × 105 cells/well) were seeded in a 6-well plate for 24 h and then treated with 1.5 ␮M DBT for 6 h after being pretreated with or without CS for the indicated periods. The cells were washed twice with PBS and then stained with 1 mL cold PBS containing 40 ␮L mixture of 1 mg/mL AO and 1 mg/mL EB in the dark for 10 min. After two additional rinses with PBS, the stained cells were observed using a fluorescence microscope (Olympus, BX-60, Japan) in a double-blind fashion at 40 × magnification. Each test was performed in triplicate. 2.7. Annexin V-FITC/PI double staining study using flow cytometry Fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) double staining method was used to quantitatively measure the cell apoptotic rates. Briefly, after the treatment, the harvested cells were resuspended in 200 ␮L of binding buffer at a density of 1 × 106 cells/mL and incubated with 5 ␮L Annexin V-FITC and 10 ␮L PI in dark for 15 min at 4 ◦ C. Cell apoptosis was determined by FACS Caliber (Accuri C6, bioscience, US) within 1 h. At least 20000 cells were counted in each sample. The assay was conducted three times independently. Cells that were Annexin V(−)/PI(−) (lower left quadrant) were defined as normal cells, the Annexin V(+)/PI(−) cells (lower right quadrant) as early apoptotic cells, Annexin V(+)/PI(+) (upper right quadrant) as late apoptotic cells, and Annexin V(−)/PI(+) (upper left quadrant) as dead cells. Apoptotic rate (%) = [(number of apoptotic cells)/(number of total cells observed)] × 100% 2.8. Measurement of intracellular ROS generation Intracellular ROS detection was measured by staining cells with the fluorescent probe 2 ,7 -dichloro fluorescein diacetate (DCFHDA). DCFH-DA enters cells by simple diffusion and is hydrolyzed into nonfluorescent dichlorofluorescein (DCFH) by intracellular esterases. Subsequently, dichlorofluorescein (DCFH) is oxidized to highly fluorescent dichlorofluorescein (DCF) by ROS (Wang et al., 2015). Thus, the fluorescence intensity is proportional to the amount of peroxides produced by the cells. In brief, PC12 cells (1 × 105 cells/well) seeded in 6-well plate were treated with 1.5 ␮M DBT for 6 h after pre-incubating with different concentrations of CS for 24 h. Then, the cells were harvested, washed twice, resuspended in PBS and incubated with DCFH-DA at a final concentration of 10 ␮M for 30 min at 37 ◦ C in the dark. The DCF fluorescence intensity was measured by a flow cytometer at an excitation wavelength of 480 nm and fluorescence emission was measured in channel FL1 at 530 nm. The results were expressed as percentage of the mean fluorescence intensity of FL-1 of the control group. 2.9. Measurement of mitochondrial membrane potential (MMP) Changes of MMP were assessed by lipophilic and cationic probe 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethyl-imidacarbocyanine iodide (JC-1) according to the manufacturer’s instructions. At a normal membrane potential, JC-1 could aggregate in mitochondria and emit red fluorescence. When the mitochondrial potential collapse, JC-1 could exist in the cytosol as a monomer which emits a green fluorescence. Thus, the red and green fluorescences of the JC-1 represent changes in the MMP of the mitochondrial membrane (Cossarizza, Baccaranicontri, Kalashnikova, & Franceschi, 1993).

After the treatment, PC12 cells were harvested, washed and stained with 10 ␮g/mL JC-1 at 37 ◦ C for 30 min in the dark. Then, stained cells were washed, resuspended, and subjected to flow cytometry analysis. The relative red/green fluorescence ratios of treated groups were expressed as percentages of red/green fluorescence ratio of the control group. 2.10. RNA extraction and real-time quantitative PCR Extraction of total RNA from PC12 cells was performed using RNAiso Plus Kit according to the instructions and template cDNA for real-time PCR was synthesized using PrimerScriptTM RT reagent Kit with gDNA Erazer (Perfect Real Time) kit. Real- time PCR was performed on ABI real-time PCR system (StepOne, Applied Biosysterms Co., USA) using SYBR Premix Ex TaqTM II kit. The primers used for real-time PCR analysis are listed in Table 1. The GAPDH gene was amplified separately as control to normalize for specific gene expression in each group. The primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Briefly, real-time PCR analysis was performed in 20 ␮L of reaction mixture containing 2.0 ␮L of cDNA, 10 ␮L of 2 × SYBR Premix Ex TaqTM II, 0.4 ␮L 50 × ROX reference dye II, 0.8 ␮L of 10 ␮M forward primer, 0.8 ␮L of 10 ␮M reverse primer and was subjected to the following conditions: 95◦ C for 30 s, followed by 45 cycles at 95◦ C for 5 s; 60◦ C for 30 s in 96well optical reaction plates (Applied Biosysterms Co., USA). The relative quantification of gene expression was calculated using the 2−Ct method (Livak & Schmittgen, 2001). Each gene analysis was performed in triplicate. 2.11. Protein extraction and western blot analysis After indicated treatment, PC12 cells were collected, washed with cold PBS twice and treated with RIPA lysis buffer containing protease inhibitors for 60 min on ice and the cell lysate was centrifuged at 13000 rpm/min for 15 min at 4◦ C. The protein concentrations in the supernatant were determined using the BCA assay. Equal amounts of protein (40 ␮g) were subjected to 8–12% SDS-polyacrylamide gel electrophoresis along with protein molecular weight standards and then transferred to nitrocellulose membranes. The membranes were blocked with tris-buffered saline plus tween-20 (TBS-T) containing 5% non-fat milk for 1.5 h and incubated with TBS-T containing specific primary antibodies overnight at 4◦ C. After that, appropriate secondary horseradish-peroxidase labeled antibodies were incubated for 1 h and immunodetection was performed using enhanced chemiluminescence (ECL) detection kit. Western blotting data were quantified by Gel-Pro Analyzer 4.0 using ␤-actin as an internal reference marker. The assay was conducted three times independently. 2.12. Statistical analysis All results are expressed as means ± standard deviation (SD). A one-way analysis of variance with the Turkey post hoc test was performed using the Statistical Package for the Social Sciences software (SPSS 17.0 for Windows, 2010, SPSS Inc., Chicago, IL, USA). Values of p < 0.05 were considered to indicate statistical significance. All the experiments were performed in triplicate. 3. Results 3.1. CS attenuated DBT-induced cytotoxicity in PC12 cells Prior to investigating the neuroprotective activities of CS against DBT-induced toxicity, the cytotoxic effects of CS were examined on PC12 cells using the MTT reduction assay. As shown in Fig. 1A, CS

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Table 1 Primer sequences of target genes. Gene

Primer

Sequence

Produce size (bp)

Bcl-2

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

5 -GCGTCAACAGGGAGATGTCA-3 5 -GGTATGCACCCAGAGTGATG-3 5 -CCCACATCTCAGTTCCCTTG-3 5 -GGCAGGCTCTTCTCCCTCTA-3 5 -GCGTCAACAGGGAGATGTCA-3 5 -GGTATGCACCCAGAGTGATG-3 5 -GGCTTGAGGAAGTCCGATCC-3 5 -TCACTCGGCTCAAACTCTGG-3 5 -GGCTGCTGGATTCTCTTACAC-3 5 -GTCTGCCCTTTCTCCCTTCT-3 5 -TCCAGCGGCAAGGACACAGACG-3 5 -CAACCGCGTGCAAAGATTCTGCA-3 5 -GCGTCAACAGGGAGATGTCA-3 5 -GGTATGCACCCAGAGTGATG-3 5 -GCGTCAACAGGGAGATGTCA-3 5 -GGTATGCACCCAGAGTGATG-3 5 -TGTCTCCTGCGACTTCAACAG-3 5 -GAGGCCATGTAGGCCATGAG-3

139

Bcl-xL Bax Bad cytochrome-c Apaf-1 caspase-9 caspase-3 GAPDH

110 147 113 154 330 100 142 156

Fig. 1. Protective effect of chitosan (CS) on dibutyltin (DBT)-induced PC12 cell cytotoxicity. (A) Effect of CS on proliferation of PC12 cells following treatment for 24 h. (B) Cell viability was measured by MTT assay in cells treated by 1.5 ␮M DBT for 6 h with pretreatment of different concentrations of CS (0–200 ␮g/mL). (C) LDH release in culture medium after treatment. (D) Morphological observation of PC12 cells under light microscope (×40). Data are presented as means ± SD (n = 3). ## p < 0.01, compared with the control group; * p < 0.05, ** p < 0.01, compared with DBT-treated group.

did not show obvious cytotoxic effects even up to 400 ␮g/mL for 24 h. On the other hand, DBT significantly reduced PC12 cell viability in a dose-dependent manner (Liu et al., 2013). In experiment, we found that CS protective effect against 1.5 ␮M DBT was superior to other concentrations, and treatment with 1.5 ␮M DBT for 6 h could make a significant decline in cell viability compared to control group, so this condition was used for subsequent experiments. Fig. 1B indicated that the DBT-induced reduction in PC12

cell viability was reversed by pretreatment with CS in a dosedependent manner. We found that when cells were incubated with 1.5 ␮M DBT alone for 6 h, the percentage of viable cells was 63.10 ± 0.57% of the control value, and this was increased to 71.18 ± 3.99%, 77.69 ± 6.54%, and 86.79 ± 6.79% in cells pretreated with CS at 50, 100 and 200 ␮g/mL for 24 h, respectively. LDH release enhances as the number of dead cells increases and is considered as an indicator of cellular damage. After exposure to 1.5 ␮M DBT, LDH leakage was significantly higher in the treated

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cells than in the control cells, indicating that DBT at this concentration was toxic to PC12 cells. In contrast, pretreatment of PC12 cells with CS suppressed DBT-induced leakage of LDH in a concentrationdependent manner (Fig. 1C), and this finding was similar to that determined by the MTT assay. The protection of CS was also confirmed by morphological observations. Under inverted light microscope, DBT caused marked morphological changes in PC12 cells, which became retracted and rounded, while CS pretreatment mitigated such morphological features (Fig. 1D).

3.2. CS protected PC12 cells from DBT-induced apoptosis To investigate whether CS protects against DBT-induced apoptosis, PC12 cells were pretreated with different concentrations of CS (50, 100, and 200 ␮g/mL) for 24 h before 1.5 ␮M DBT was added. The AO/EB double staining was used to evaluate morphological changes due to apoptosis in PC12 cells. As shown in Fig. 2A, uniformly green live cells with normal morphology were observed in the control group, whereas orange/red apoptotic cells with fragmented chromatin and apoptotic bodies occurred in the DBT-treated group. In contrast, CS pretreatment significantly inhibited these morphological changes in a concentration-dependent manner. These results suggested that CS can obviously reduce DBTinduced apoptosis in PC12 cells. Furthermore, Annexin V-FITC/PI double staining was also employed to quantitatively determine the cell apoptosis. As shown in Fig. 2B, DBT markedly increased PC12 cells apoptosis, compared with control group (p < 0.01). The total apoptotic cells constituted 25.3 ± 1.2% when the cells were treated with DBT, whereas preincubation with various concentrations of CS (50, 100, and 200 ␮g/mL) for 24 h reduced this rate to 20.9 ± 0.6%, 18.0 ± 0.7%, and 11.9 ± 0.5%, respectively.

3.3. CS reduced DBT-induced intracellular ROS generation and the mitochondrial membrane potential (MMP) disruption DCFH-DA assay was used to examine the changes in intracellular ROS levels. As shown in Fig. 3A and B, when PC12 cells were exposed to 1.5 ␮M DBT for 6 h, the intracellular ROS level significantly increased compared with untreated cells, revealing that DBT enhanced ROS production. Pretreatment with CS significantly attenuated the increase in ROS caused by DBT in a concentrationdependent manner. A collapse of the mitochondrial membrane potential (MMP) has been implicated in several models of apoptosis. To examine whether DBT-induced apoptosis and its rescue by CS involve the mitochondrial pathway in PC12 cells, MMP was measured using JC1 staining method. The results revealed that the MMP was rapidly reduced when PC12 cells were exposed to DBT for 6 h and that the DBT-induced loss of MMP was rescued by pretreatment with CS for 24 h in a concentration-dependent manner (Fig. 3C and D).

3.4. CS altered DBT-induced apoptosis related gene expression The inhibitory effect of CS on DBT-induced cell death was assessed by measuring changes in the levels of apoptosis-related mRNA expressions using real-time PCR method. DBT significantly increased the mRNA levels of Bax, Bad, Apaf-1 and cytochrome-c in PC12 cells (p < 0.01), reduced the Bcl-2 and Bcl-xL mRNA levels (p < 0.01). Pretreatment with CS could reduce the DBT-induced apoptosis-related mRNA changes in a dose-dependent manner (Fig. 4).

3.5. CS counteracted DBT-induced caspase activation Caspase-9 and -3 are the key apoptotic executive proteins involved in mitochondrial pathway. Real-time PCR results indicated that caspase-9 and caspase-3 mRNA levels were increased 2.14-fold and 1.48-fold, respectively, in PC12 cells after exposure to DBT. It could be obviously observed that pretreatment with CS could partly counteract these effects (Fig. 5A). Furthermore, western blot analysis of the protein changes in caspase-9 and cleaved caspase-3 were also carried out. As expected, DBT could increase the protein levels of upstream caspase-9 and downstream cleaved caspase-3. This caspase activation could be inhibited by pretreatment with CS in a dose-dependent manner which suggested that CS attenuates DBT-induced PC12 cells apoptosis through the intrinsic pathway.

4. Discussion Due to the widespread of use, the toxicity of OTC is of the great concern to human health (Fent, 1996). A growing body of data indicated that some triorganotin compounds, particularly TMT, have potent neurotoxic effects in vitro and in vivo (Appel, 2004). Meanwhile, DBT has also been considered as a well known neurotoxicant (Eskes et al., 1999; Florea & Büsselber, 2006). In addition to direct use, DBT is also a major degradation product of tributyltin (TBT) in humans and higher animals (Chantong, Kratschmar, Lister, & Odermatt, 2014). However, the prevention of OTC-induced neurotoxicity is seldom reported. CS, as a natural, harmless biopolymer, is widely used in pharmaceutical, foodstuff and cosmetics industries (Ham-Pichavanta, Sèbeb, Pardona, & Coma, 2005; Kumar, 2000). CS has a variety of biological functions such as antibacterial, antifungal, anti-diabetic, and free radical scavenging activities. Khodagholi et al. (2010) indicated that CS could prevent H2 O2 and FeSO4 induced cytotoxicity in NT2 neuron cells, suggesting that CS could be regarded as a neuroprotective agent. In the present study, we aimed to investigate the possible protective effects of CS on the DBT-induced toxicity in cultured PC12 cells and elucidate the underlying mechanism. Our results demonstrated that CS (below to 400 ␮g/mL) did not affect the viability of PC12 cells (Fig. 1A), and the decreased cell viability resulted from DBT was significantly attenuated by CS pretreatment in a concentration-dependent manner (Fig. 1B). Also, high concentration of LDH in culture medium was detected after exposed to DBT, revealing that cell membrane integrity was destroyed, which can be ameliorated by pretreatment with CS (Fig. 1C). Similarly, Cho, Shi, and Borgens (2010) had observed CS could decrease the LDH leakage in guinea pig spinal cords and repair the damaged cell membranes. The cell apoptosis was investigated by utilizing AO/EB and Annexin V-FITC/PI double staining methods. When PC12 cells were exposed to DBT, apoptotic features were obviously observed after AO/EB staining and total apoptotic rate was increased to 25.2% followed by Annexin V-FITC/PI staining, which could be partially abrogated by CS pretreatment in a concentration-dependent manner. These results agreed with the data from the above cytotoxicity assay, suggesting that the ability of CS to attenuate DBT-induced PC12 cell death was mediated by its anti-apoptotic activity. Numerous evidences have demonstrated that the generation of intracellular ROS is intimately associated with apoptotic cell death, as excessive ROS results in oxidative injury by lipid peroxidation, protein deficit and DNA damage as well as mitochondrial dysfunction (Ott, Gogvadze, Orrenius, & Zhivotovsky, 2007). Several reports showed DBT could give rise to intracellular redundant ROS, thereby induce cell apoptosis (Chantong, Kratschmar, Lister, & Odermatt, 2014; Ferreira et al., 2013; Liu et al., 2013). On the

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Fig. 2. Inhibitory effect of CS on DBT-induced PC12 cell apoptosis. Cells were treated in the absence and presence of 1.5 ␮M DBT for 6 h after pretreated with or without CS (50–200 ␮g/mL) for 24 h. (A) Morphological features of apoptosis were observed by acridine orange/ethidium bromide (AO/EB) double staining with fluorescent microscope (×40). (B) Flow cytometry dot plots of Annexin V-FITC/PI double staining for the detection of cell apoptosis. (C) Total apoptotic rates shown as histograms after treatment. Data are presented as means ± SD (n = 3). ## p < 0.01, compared with the control group; ** p < 0.01, compared with DBT-treated group.

other hand, antioxidant compounds have been widely reported to protect against, or delay apoptosis induced by a wide range of stimuli (Frautschy et al., 2001; Han et al., 2014; Liu et al., 2012). For example, ascorbic acid, an antioxidant generally used as posi-

tive control, could alleviate rat pancreatic damage elicited by DBT through decreasing the degree of oxidative stress (Lu, Song, Fu, Si, & Qian, 2007). Moreover, CS has been shown to have antioxidative activity and capability to protect normal cells (Joodi & Khodagholi,

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Fig. 3. Effect of CS on intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) disruption induced by DBT in PC12 cells. Cells were treated with or without CS (50–200 ␮g/mL) for 24 h and then incubated in the absence and presence of 1.5 ␮M DBT for 6 h. Then, ROS levels and MMP were monitored with flow cytometry using DCFH-DA and JC-1. (A) Representative results for ROS production after treatment. (B) The fluorescence intensity of FL-1 channel in the treated cells was expressed as percentages of that in untreated cells. (C) The original flow cytometry results of MMP. (D) The MMP results that were presented graphically as percentages of red/green ratios of control group. Data are presented as means ± SD (n = 3). ## p < 0.01, compared with the control group; ** p < 0.01, compared with DBT-treated group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2010; Kerch, 2015). Our results showed that DBT treatment significantly induced ROS formation in cells as evidenced from the higher fluorescent intensity, while cells pretreated with CS followed by DBT treatment indicated reduced levels of ROS in comparison to the DBT treated cells. CS significantly reduced the ROS formation in PC12 cells, which may be partly due to the ability of CS to quench the free radicals initiated by DBT. Overproduction of ROS may result in mitochondrial dysfunction including MMP loss and cytochrome-c release (Ott et al., 2007). Our previous study has demonstrated that diorganotins-induced apoptosis associated with increased ROS and decreased MMP in PC12 cells (Liu et al., 2013). In this study, JC-1 staining method was applied to assess the potential effect of CS on preventing the MMP dissipation in DBT-induced apoptotic cells. We found that CS pretreatment could reverse MMP decrease induced by DBT in a dose-dependent manner.

The Bcl-2 family of proteins are the key regulators of the mitochondrial response to apoptotic signals in the intrinsic pathway and contain both pro- and anti-apoptotic members. (Gross, McDonnell, & Korsmeyer, 1999). The pro-apoptotic subfamily can be divided into two groups: (i) the multi-domain proteins that are essential effectors of mitochondrial permeabilization, such as Bax, Bak, and Bok; and (ii) the “BH3 domain only” proteins (BH3s), including Bim, Bad, and Bid, etc., which promote apoptosis by activating Bax and Bak and inactivating anti-apoptotic proteins. While, anti-apoptotic subset mainly contains Bcl-2, Bcl-xL and Mcl-1 proteins (Martinou & Youle, 2011; Renault, Teijido, Antonssond, Dejeane, & Manon, 2013). In the early stage of apoptosis, “activator” BH3 s (e.g., Bim, Bid, Puma) were firstly activated by intracellular damage signals, which triggers conformational changes of Bax to promote its mitochondrial targeting and homo-oligomerization. Bax exists in the

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Fig. 4. Effect of CS on mitochondrial apoptosis related genes expression. Cells were exposed to DBT (1.5 ␮M) alone for 6 h after pretreated in the absence and presence of CS (50–200 ␮g/mL) for 24 h, and then the mRNA levels of apoptosis-related proteins were detected by real-time PCR analysis. (A) Bcl-2 mRNA expression; (B) Bcl-xL mRNA expression; (C) Bax mRNA expression; (D) Bad mRNA expression; (E) Cytochrome-c mRNA expression; (F) Apaf-1 mRNA expression. GAPDH was as internal reference to normalize genes signal. Each value represents the means ± SD (n = 3), ## p<0.01, compared with the control group; ** p < 0.01, * p < 0.05, compared with DBT-treated group.

cytosol as a monomer and its dimerization may lead to the forming of selective channels that contribute to the release of apoptotic factors, such as cytochrome-c (Kim et al., 2009; Moldoveanu, Follis, Kriwacki & Green, 2014). Bcl-2 and Bcl-xL can preserve mitochondrial integrity and prevent cytochrome-c efflux by sequestering “activator” BH3s into insert complexes (Kim et al., 2009), while the remaining BH3s that are called “sensitizer” include Bad, Noxa, Bmf, etc., and provoke apoptosis by competitively inhibiting this interaction (Brunelle & Letai, 2009; Letai et al., 2002). In addition, Bad can be activated by dephosphorylation and then translocate into mitochondria to accelerate the release of cytochrome-c (Elmore, 2007). Once released to cytosol, cytochrome-c in turn binds to Apaf-1, which triggers apoptosome forming by self-association and recruiting procaspase-9 (Li et al., 1997; Bratton & Salvesen, 2010). The apoptosome-mediated caspase-9 activation can cause cleavage of procaspase-3, leading to cell apoptosis and death (Zou, Henzel,

Liu, Lutschg, & Wang, 1997). To some extent, the expression of mRNA can reflect the level of protein (Asano, Fukunaga, Deguchi, Kawamura, & Inaba, 2012; Ge, Ma, Li, & Li, 2013; Yakovlev et al., 2001). Here, Bad, Bax, Bcl-2 and Bcl-xL , which were the representative members of Bcl-2 family, were selected to illustrate the protection of CS against DBT-induced apoptosis by real-time PCR assay. Results showed that in mRNA level, CS could reverse the over-expression of Bax and Bad and the down-expression of Bcl-2 and Bcl-xL induced by DBT in PC12 cells. Meanwhile, the DBTinduced high genes levels of cytochrome-c and Apaf-1 decreased after pretreatment with CS. Then, we can conclude CS protects from DBT-induced apoptosis in PC12 cells, which may be through the inhibition of mitochondrial pathway. Furthermore, this result was confirmed by the observation that CS mitigated DBT-induced activation of caspase-9 and caspase-3 at mRNA and protein levels.

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Fig. 5. Effect of CS on caspases activation caused by DBT exposure at mRNA and protein levels. Cells were treated in the absence and presence of 1.5 ␮M DBT with or without CS for indicated time. (A) mRNA levels of caspase-9, -3 were detected by real-time PCR with GAPDH as internal reference. (B) Western blot was employed to determine the proteins expression of caspase-9 and cleaved caspase-3 with ␤-actin as loading control. The immunoblots are shown from one experiment representative of three that gave similar results. (C) Densitometry analysis on levels of caspase-9 and cleaved caspase-3 proteins. Each value represents the means ± SD (n = 3), ## p < 0.01, compared with the control group; ** p < 0.01, compared with DBT-treated group.

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