Toxic cytological alteration and mitochondrial dysfunction in PC12 cells induced by 1-octyl-3-methylimidazolium chloride

Toxicology in Vitro 26 (2012) 1087–1092

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Toxic cytological alteration and mitochondrial dysfunction in PC12 cells induced by 1-octyl-3-methylimidazolium chloride Xiao-Yu Li a,⇑, Chang-Qin Jing a,b, Xia-Yan Zang a, Shuai Yang a, Jian-Ji Wang c,⇑ a

College of Life Science, Henan Normal University, Xinxiang, Henan 453007, China Department of Life Sciences and Technology, Xinxiang Medical University, Henan 453003, China c Henan Key Laboratory for Environmental Pollution Control, Xinxiang, Henan 453007, China b

a r t i c l e

i n f o

Article history: Received 3 April 2012 Accepted 16 July 2012 Available online 23 July 2012 Keywords: Ionic liquid PC12 cells Cytotoxicity Mitochondrial dysfunction Apoptosis

a b s t r a c t Ionic liquids have recently received considerable attention due to their negligible vapor pressure and substitute for conventional organic solvents. However, their solubility in water and a great deal of literature regarding their toxicity on aquatic organisms have caused much concern in recent years. This study aims to evaluate the cytotoxicity of 1-octyl-3-methylimidazolium chloride ([C8mim][Cl]) on the rat pheochromocytoma (PC12) cell line by cell viability assay and to determine the cytological alterations and damages in PC12 cells after 24 h of exposure at the concentrations of 0.07, 0.14, and 0.28 mM of [C8mim][Cl]. The results show that [C8mim][Cl] inhibits PC12 cell growth and decreases their viabilities in a remarkable dose-dependent manner, and the 24 h EC50 of [C8mim][Cl] for PC12 cells is calculated to be around 0.56 mM. Our results also reveal that [C8mim][Cl]-exposure induces DNA damage, sustained increase of intracellular Ca2+, overproduction of reactive oxygen species, gradually exhausted cellular ATP, mitochondrial permeability transition, and apoptosis in PC12 cells. We suppose that mitochondrial permeability transition and mitochondrial dysfunction maybe the major cytotoxicity mechanism of [C8mim]Cl for PC12 cells. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Ionic liquids (ILs) are room-temperature molten salts which chemically consist only of ions (Bonhote et al., 1996). They possess a series of preponderant properties, such as almost no vapor pressure and nonvolatility, high thermal stability and non-flammability, and high solvent capacity and chemical stability (Sheldon, 2001), which make them suitable for use in organic synthesis, catalysis, chemical separation, and electrochemistry (Brennecke and Maginn, 2001; Ranke et al., 2007a; Welton, 1999). Especially in the recent years, ILs have been promised to be used in environmental protection and the utilization of biotic resources, for example, absorption of harmful gas CO2 (Sistla et al., 2012) and degradation of cellulose from the crop stalks (Jiang et al., 2011). Furthermore, compared with the conventional organic solvents, ILs can reduce the risk of air pollution due to their negligible vapor pressure. Therefore ILs have been claimed to be environmentally benign ‘‘green solvent’’ and strongly recommended to be the substitutes for conventional organic solvents by chemists (Earle and Seddon, 2000; Sheldon, 2005). However, most ILs are hydrosoluble and they may be released to water and cause damage to aquatic ⇑ Corresponding authors. Tel./fax: +86 373 3329390. E-mail addresses: [email protected] (X.-Y. Li), [email protected] (J.-J. Wang). 0887-2333/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tiv.2012.07.006

organisms during the generation and application (Couling et al., 2006; Gathergood et al., 2004). Thus ILs may pose environmental risks to aquatic ecosystems, and their toxicity on aquatic organisms has been of much concern in recent years (Pham et al., 2010; Samori et al., 2010). There have been a great number of published reports regarding IL-toxicity to aquatic organisms (Ventura et al., 2010), for example, algae (Latała et al., 2010), Daphnia (Luo et al., 2008; Ventura et al., 2010; Yu et al., 2009) and goldfish (Li et al., 2012). Meanwhile, Ranke et al. (2004), Stepnowski et al. (2004) found that ILs had cytotoxicity in vitro and their toxicity was approach to the conventional organic solvents such as acetone, methanol, toluene or xylene. In addition, numerous investigators have also evaluated the cytotoxicity of ILs, for example, toxicity of ionic liquids in Leukemia Rat Cell Line (Torrecilla et al., 2009), roles of cation and anion in ILcytotoxicity (Ranke et al., 2007b; Stolte et al., 2006), and a quantitative structure–activity relationship (QSAR) between IL-cytotoxicity and their effective structural features (Fatemi and Izadiyan, 2011; Garc´ıa-Lorenzo et al., 2008). Unfortunately, the cytotoxicity mechanism of the ILs still remains unknown now. The imidazolium- based ILs have been not only widely applied in the chemical industry, they also possess the excellent and advantageous properties over the conventional organic solvents (Garc´ıa-Lorenzo et al., 2008; Samori et al., 2010). Moreover, they usually have moderate or relatively lower toxicity on organisms

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(Ranke et al., 2004). The most important is that the toxic response of organisms to the exposure of imidazolium-ILs is in a remarkable dose-dependent manner according to our previous studies (Li et al., 2012; Luo et al., 2008; Yu et al., 2009) and the result of toxicitytesting can be the representation for the common imidazolium ILs. Therefore the imidazolium-based IL, 1-octyl-3-methylimidazolium chloride ([C8mim]Cl), was adopted in the present study to evaluate the IL-cytotoxicity on the rat pheochromocytoma (PC12) cells via determining cell viability, intracellular Ca2+, reactive oxygen species (ROS), and ATP level, and mitochondrial permeability transition pores (MPTPs) activity, to illuminate the cytotoxicity mechanism of the imidazolium IL. The PC12 cell line is easily cultured and preserved, and now it has become a commonly employed model system not only for studies of neuronal development and neurochemical studies, but also for toxicity determination of chemicals (Doroshenko and Doroshenko, 2007). Furthermore, the cell line is sensitive to the ILexposure and the response is dose-dependent according to our primary acute toxicity tests of ILs on PC12 cells. Therefore the PC12 cell line is used in this study.

2. Materials and methods 2.1. IL and reagents The ionic liquid [C8mim][Cl] with purity of more than 99% was purchased from Hubei Hengshuo Chemical Co., Ltd., China. High glucose Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum were obtained from Invitrogen (Shanghai, China). 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium monosodium salt (WST-1) cell proliferation and cytotoxicity assay kit (Catalog # C0036), Fluo-3 AM (Catalog # S1056), ATP assay kit (Catalog # S0026), bicinchonininc acid (BCA) protein assay kit (Catalog # P0012), and ROS assay kit (Catalog # S0033) were purchased from Beyotime Institute of Biotechnology (Haimen, China) and MPTP assay kit (Catalog # GMS10095.1) from Genmed Scientifics INC. U.S.A. Fluorescein diacetate (FDA) was obtained from Keeasy Economic & Trade Co., Ltd. 2.2. Cell culture The PC12 cell line was obtained from Henan Key Laboratory for Heredity Diseases and Molecular Targeted Medicines and cultured in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum. Cells were maintained at 37 °C in an incubator with 5% (v/v) of CO2 and the water-saturated atmosphere.

2.4. DNA damage detection PC12 cells were cultured in 6-well plates (1  106 cells per well) with 0, 0.07, 0.14, and 0.28 mM of [C8mim][Cl] for 24 h and then they were collected and rinsed twice in ice-cold PBS and stored at 20 °C in 70% ethanol overnight. The fixed cells were resuspended at a concentration of 1  106 cells/ml in a staining solution contained 50 lg/ml propidine iodide (PI), 0.1% (v/v) Triton X-100, 0.1 mM EDTA, and 50 lg/ml RNase in PBS. They were incubated for 30 min on ice and then served for flow cytometry analysis. Flow cytometry was performed using a flow cytometer (Cytomics FC 500 MCL, Beckman Coulter) and the area of sub-G0/G1 phase (apoptosis peak) is located as the gate to determine the damage of DNA. Usually the area before the G0/G1 phase is set for a gate that can be used to determine DNA damage while the apoptosis peak can represent the amount of apoptosis cell in all treated cells. 2.5. Determination of plasma membrane permeability The alteration of plasma membrane permeability was detected by FDA-staining, which can differentiate the intact cells and the membrane-damaged cells and determine the efficiency of plasma membrane resealing (Zhou et al., 2010). Briefly, the PC12 cells were cultured in 6-well plates (1  106 cells per well) with 0, 0.07, 0.14, and 0.28 mM of [C8mim][Cl] for 24 h, and then they were collected and rinsed twice with PBS. All treatments were performed in triplicate. FDA was subsequently added to the cell suspensions at a final concentration of 20 lg/ml and the cells were incubated at 37 °C for 10 min. After incubation, the cells were centrifuged at 400g for 5 min, the supernatant was removed, and the cells were finally resuspended in 0.5 ml of PBS. The cell suspensions were transferred to the test tubes and kept ice-cold during analysis. Fluorescence was quantified using a flow cytometer (kex at 488 nm and kem at 525 nm). The results were presented as NRFU (normalized relative fluorescence units) (U/cell). Each independent experiment was performed in triplicate. 2.6. Intracellular Ca2+ assay The intracellular Ca2+ level in PC12 cell was measured using the fluorescence Ca2+ indicator Fluo-3 AM according to the report by Kuang et al. (2010) with some modifications. The cell culture and IL-exposure is in the same way as above (Section 2.5). After 24 h of IL-exposure, the treated cells and the control cells were collected, rinsed twice with PBS, and stained with 5 lg/ml of Fura-3 AM for 60 min. After staining, the cells were rinsed twice with PBS and incubated for another 10 min at 37 °C before fluorescence-intensity detection by a flow cytometer (kex at 488 nm and kem at 525 nm). The results were presented as NRFU (U/cell).

2.3. Cell viability assay 2.7. ROS level determination Viability of cells treated with [C8mim][Cl] was measured using the WST-1 cell proliferation and cytotoxicity assay kit according to the manufacturer’s instructions. Briefly, PC12 cells were cultured in a 96-well culture plate (1.5  104 cells per well) at various concentrations of [C8mim][Cl] for 24 h. The IL concentrations adopted in the test were 0.01, 0.02, 0.04, 0.06, 0.08, 0.12, 0.24, 0.48, 0.96, 1.00, 1.20, and 1.40 mM on the base of our preliminary experiments. The cells cultured in complete medium without [C8mim][Cl] were served as the control. Each test was conducted in triplicate. After 24 h of exposure, 10 ll of WST-1 reagent was added to each well and incubated for an additional 1 h. The absorbance at 450 nm was monitored and the reference wavelength was set at 630 nm. The percent viability of cells was calculated by comparison to that of the untreated control cells. The EC50 value was calculated using Matlab software.

The level of intracellular ROS was assayed by the ROS kit according to the manufacturer’s instructions. The cell culture and IL-exposure is in the same way as Section 2.5. After [C8mim][Cl]-exposure, the cells were collected and rinsed twice with PBS, then they were incubated with 10 lM of the cell permeable 20 , 70 -dichlorofluorescein diacetate (DCFH-DA) for 20 min at 37 °C. After that, the cells were washed with medium (FBS free) for three times, and then their fluorescence was detected using a flow cytometer (kex at 488 nm and kem at 525 nm). Results were presented as NRFU (U/cell). 2.8. Detection of opened MPTPs Opened MPTPs in PC12 cells were detected using calcein-cobalt with a MPTP assay kit according to the manufacturer’s directions

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and Chen et al. (2009). Briefly, after [C8mim][Cl]-exposure as described in Section 2.5, the IL-treated and control cells were collected, rinsed twice with PBS, then washed with Reagent A from the kit, incubated in Reagents B and C of the kit (1:50) at 37 °C for 20 min, and washed twice again with Reagent A of the kit. Fluorescence intensity of the solutions containing the cells was measured by flow cytometry (kex at 488 nm and kem at 505 nm). The results were presented as NRFU (U/cell).

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in the higher groups (from 0.06 to 1.40 mM, p < 0.01) after 24 h of IL-exposure (Fig. 1). When the exposure concentration was more than 0.96 mM, the cellular viability decreased sharply and even approach zero in two higher concentration groups (1.20 and 1.40 mM), suggesting that higher concentration of [C8mim][Cl] (more than 1 mM) cause cellular death. The 24 h EC50 of [C8mim][Cl] for PC12 cells was calculated to be around 0.56 mM. In addition, these results also indicate that the cytotoxicity of [C8mim][Cl] is in a dose-dependent pattern.

2.9. Cellular ATP level determination 3.2. DNA damage and apoptosis induced by [C8mim][Cl] ATP level was measured by using a firefly luciferase based ATP assay kit according to the manufacturer’s instructions. PC12 cells culture and [C8mim][Cl]-exposure were carried out in the same way as Section 2.5. After rinsed with PBS, the cells were schizolysised by solution and then centrifuged at 12,000g at 4 °C for 5 min and the supernatant was collected. In a 1.5 ml tube, 100 ll of the supernatant was mixed with 100 ll of ATP detection solution. Luminance (RLU) was immediately measured using a Turner Biosystems luminometer. Standard curves for the quantification were also generated by using known amounts of an ATP standard and the protein concentration of each treatment group was determined using the BCA protein assay kit. Total ATP levels were expressed as NRLU (nmol/mg protein) (Chen et al., 2009).

Cellular DNA damage and PC12 cell apoptosis detected by a flow cytometer were displayed as Fig. 2A and B. Only 1.6% of the total cells in the control groups were detected in the located gate (Fig. 2A-a), while more than three times of that occurred in the lower concentration group (0.07 mM, Fig. 2A-b, still further, more than 10-fold increase of cell number in the located gate could be observed in the higher concentration groups (0.14 and 0.28 mM, Fig. 2A-c and d. The results of statistical analysis showed that apoptosis occurred in all the treatment groups (Fig. 2B), in which the apoptosis peak could be easily detected in higher concentration groups (Fig. 2A-c and d), indicating that [C8mim][Cl]-exposure induce apoptosis in PC12 cells.

2.10. Statistical analysis

3.3. Alteration in plasma membrane permeability of PC12 cell

Each value was expressed as a mean ± standard deviation (SD). Data was analyzed using one-way analysis of variance followed by least significant difference (LSD) determination using SPSS 13.0. Difference was considered significant when the calculated P value was <0.05.

The alteration of plasma membrane permeability in the IL-treated PC12 cells was detected by FDA-staining and FDA contents were demonstrated in Fig. 3. An obvious fluorescein leakage was observed in all IL-treated groups when compared to the control groups (Fig. 3), suggesting that IL-exposure induce the increase in membrane permeability of the treated PC12 cells.

3. Results

3.4. The intracellular Ca2+contents and ROS levels, and opening of MPTPs

3.1. PC12 cell viabilities The viabilities of PC12 cells were demonstrated in Fig. 1, in which no statistically significant difference was observed between the lower IL-treated concentration groups (0.01–0.04 mM) and the control groups (p > 0.05), while remarkable differences were found

Three main endpoints of the IL-cytotoxicity tests in PC12 cell, Ca2+ contents, ROS levels, and opening of MPTPs, detected by fluorescence staining and using a flow cytometer, were described in Fig. 4. A remarkable sustaining increase in intracellular Ca2+ content and ROS levels was found in the all treatment groups when compared to the control groups, indicating that [C8mim][Cl] promote the Ca2+ level and induce excess ROS in PC12 cells. Meanwhile, all values of NRFU in PC12 cells decreased with the increase of IL-exposure concentrations compared to that of controls, suggesting that [C8mim][Cl] may induce the opening of MPTPs in PC12 cells. 3.5. Change of cellular ATP level ATP levels of PC12 cells determined by ATP assay kit were demonstrated in Fig. 5. After 24 h of [C8mim][Cl]-exposure, ATP levels in all treatment groups were statistically lower than that of the control groups, especially in the group of 0.28 mM [C8mim][Cl], cellular ATP was almost exhausted (Fig. 5). 4. Discussion

Fig. 1. Viabilities of PC12 cells after 24 h of exposure at various concentrations of [C8mim][Cl]. The cells were treated by 0.01–1.40 mM of [C8mim][Cl] in a 96-well culture plate for 24 h and the cell viability was measured using the WST-1 cell proliferation and cytotoxicity assay kit according to the manufacturer’s instructions as described in Section 2.3. All experiments were performed in triplicate and data is shown as means ± SD. Asterisk denotes a response that is significantly different from the control (⁄⁄p < 0.01).

Cytotoxicity test in vitro has been widely adopted now in acute toxicity determination of chemicals to save experimental time, expense and animals. The cytotoxicities of several kinds of ILs have been previously evaluated in different human and animal cell lines, such as the human cell line HeLa and Caco-2 (Frade et al., 2009; Garc´ıa-Lorenzo et al., 2008; Stepnowski et al., 2004; Wang et al.,

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A

a

b

c

d

apoptosis cell (%)

B

Concentrations of [C8mim][Cl] (mM) Fig. 2. Apoptosis of PC12 cells induced by [C8mim][Cl]. PC12 cells were exposed to 0, 0.07, 0.14, and 0.28 mM of [C8mim][Cl] for 24 h and DNA damage was detected by propidine iodide (PI) staining and a flow cytometer. Further details of the flow cytometry method and the content calculation of apoptosis cell were described in Section 2.4. (A) Apoptosis peaks in the [C8mim][Cl]-treated PC12 cells determined by a flow cytometer. (a) control, (b) 0.07 mM [C8mim][Cl], (c) 0.14 mM, (d) 0.28 mM. (B) The contents of apoptosis cell in PC12 cells. Data is expressed as the means ± SD from the four independent experiments with triplicate. Asterisk denotes a response that is significantly different from the control (⁄p < 0.05, ⁄⁄p < 0.01).

2007) and promyelotic rat cells as the leukemia cells (IPC-81) and the glioma cells C6 (Ranke et al., 2004, 2007b; Stolte et al., 2006). The results of these studies indicate that most ILs have cytotoxicity and their toxicities are comparable to the conventional organic solvents such as acetone, methanol, and xylene (Ranke et al., 2004). In the current study, the 24 h EC50 of the [C8mim][Cl] for PC12 cells is determined to be around 0.56 mM, which is in good agreement with that obtained in the human Caco-2 cell line (0.54 mM of EC50 for [C8mim][Cl] (Garc´ıa-Lorenzo et al., 2008). Considering the non-toxicity or low-toxicity of [Cl] to culture cells, the cytotoxicity of [C8mim][Cl] on PC12 cells may be resulted mainly from the cation [C8mim] (Ranke et al., 2004, 2007b). We think that [C8mim] can be an representative of the imidazolium- based ILs because the toxicity of [C8mim] was greater than [C4mim] and [C6mim], but weaker than [C10mim] and [C12mim] according to the previous re-

sults of toxicity tests in our laboratory and the toxicity response of [C8mim] in animals was dose-dependent (Yu et al., 2009; Luo et al., 2008; Li et al., 2012). Apoptosis is not only a type of natural and highly programmed cell death, but also can be induced by exogenous chemicals. At present, it can be easily detected by some cytological and biochemical alterations in the chemical-treated cells, for example, DNA fragmentation, mitochondrial membrane potential, and Ca2+ and ROS levels (Eaton and Klaassen, 2001). In this study, flow cytometry analysis reveals that [C8mim][Cl]-treatment has induced remarkable DNA damage and a typical apoptosis peak has occurred in the diagram of cell cycle of PC12 cells, suggesting that [C8mim][Cl] may enter the nucleus of PC12 cells by passing through nuclear membrane and act on DNA to conform DNA adductive or directly injure DNA. Furthermore, sustained rise in

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Fig. 3. Fluorescein diacetate (FDA) contents of PC12 cells exposed to various concentrations of [C8mim][Cl]. [C8mim][Cl]-exposure and the permeability of plasma membrane detected by FDA-staining were described in Section 2.5. Data is expressed as the means ± SD from three independent experiments with triplicate. Asterisk denotes a response that is significantly different from the control (⁄⁄p < 0.01).

Fig. 4. The intracellular Ca2+ contents, reactive oxygen species (ROS) levels, and opening of mitochondrial permeability transition pores (MPTPs) of PC12 cells after 24 h of [C8mim][Cl]-exposure. The IL-exposure and fluorescence staining and flow cytometer assays were described in Section 2.6–2.8. The results were presented as the normalized relative fluorescence units (NRFU) (U/cell). In MPTPs detection, the decreased NRFU indicate that the IL may induce mitochondrial permeability transition in PC12 cells. Data is expressed as the means ± SD from three independent experiments with triplicate. Asterisk denotes a response that is significantly different from the control (⁄p < 0.05, ⁄⁄p < 0.01).

intercellular Ca2+ and overproduction of ROS in the IL-treated PC12 cells also demonstrate indirectly the damages by [C8mim][Cl]. Additionally, the results of MPTP fluorescence determination show that [C8mim][Cl]-exposure has caused mitochondrial permeability transition (MPT) in PC12 cells, which is an early indicator of cellular apoptosis (Wang et al., 2007). Finally, we also conducted the primary experiments to identify apoptosis induced by [C8mim][Cl] using other end points, such as detection of DNA fragments by terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL) kit and the morphological characteristics identifi-

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Fig. 5. ATP levels of PC12 cells after 24 h of [C8mim][Cl]-exposure. The IL-treatment and ATP level measurement were described in Section 2.9. Total ATP levels were expressed as the normalized luminance (NRLU) (nmol/mg protein). Data is expressed as the means ± SD from three independent experiments with triplicate. Asterisk denotes a response that is significantly different from the control (⁄p < 0.05, ⁄⁄ p < 0.01).

cation of apoptosis occurred in the acridine orange or Hoechst 33342 stained PC12 cells by a fluorescent microscope (data not shown). When combined, the above results indicate that PC12 cells undergo apoptosis in the presence of [C8mim][Cl]. Intracellular maintenance plays a key role in cellular survival, growth and reproduction owning to its functions of synthesizing endogenous molecules, maintaining the intracellular environment, and producing energy for operation. Chemicals that disrupt these functions, especially the energy-producing function of mitochondria and protein synthesis may cause toxic cell death (Eaton and Klaassen, 2001). Usually, there are three critical biochemical disorders that may interfere with cellular maintenance and initiate cell death, namely ATP depletion, Ca2+ accumulation, and overproduction of ROS in the damaged cells. In the present study, a nearly exhausted ATP level was observed in the higher concentration group (0.28 mM of [C8mim][Cl]) after 24 h of exposure. Meanwhile, sustained increase in intracellular Ca2+ and overproduction of ROS were also found in the treated PC12 cells. These results indicate that [C8mim][Cl] may interfere with the cellular maintenance functions of PC12 cells and lead to apoptosis. A similar result was obtained by Wang et al. (2007) in the human cell line HeLa, suggesting that the induced toxic alteration of cellular maintenance may be the major mechanism of IL-cytotoxicity. Mitochondria not only contribute to cellular energy production, but also significantly affect the distribution of ATP for execution of cellular functions, the division of electron flow to O2 and to reductive biosynthetic reactions, and the integration of metabolic pathways. Mitochondrial Ca2+ release, generation of ROS, and depletion of ATP are believed to cause mitochondrial dysfunction, i.e., MPT, which finally lead to apoptosis (Eaton and Klaassen, 2001). Among these cytological events, MPT induction is catastrophic as it can disrupt the permeability barrier of the inner membrane, thus dissipating the membrane potential and pH gradient that together drive ATP synthesis through oxidative phosphorylation, and causing impairment of mitochondrial function. According to previous studies, many kinds of chemical or toxicants can induce MPT and lead to mitochondrial dysfunction, for example, methylmercury (Polunas et al., 2011) and silver nanoparticles (Teodoro et al., 2011). Our results of fluorescence determination reveal that [C8mim][Cl] may promote the opening of MPTPs and induce MPT in the treated PC12 cells, which is in agreement with the result obtained by Wang et al. (2007).

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Many studies have showed that most ILs are structurally similar to cationic surfactants due to their structure and inherent charges (Jungnickel et al., 2008; Sheldon, 2005). Thus they can be easily adsorbed onto the surface of cells, act on biomembranes (Roberts and Costello, 2003), and increase cellular membrane permeability that has been verified by FDA assay for the membrane integrity of the [C8mim][Cl]-treated PC12 cells in our study. Therefore, we can speculate that the cytoplasm-entered IL may act on mitochondrial membrane and initiate MPT that induce the release of mitochondrial Ca2+, the sustained decrease of ATP, and excess ROS, which conversely promote MPT and ultimately result in apoptosis of PC12 cells. In conclusion, our results clearly demonstrate that [C8mim][Cl] has cytotoxicity on PC12 cells, which is in a dose-dependent pattern. Thus we think that ILs may pose environmental risks to aquatic organisms during the generation and application. Meanwhile, [C8mim][Cl]-exposure induces DNA damage, sustained increase of intracellular Ca2+, overproduction of ROS, exhausted cellular ATP, MPT, and apoptosis in PC12 cells. These results provide initial insights into the cytological mechanisms of [C8mim][Cl]-induced toxicity. 5. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This research was supported by the National Science Foundation of China (Grant Nos. 31172415, 20573019 and 20573034), the Science and Technology Foundation of Henan Province, China (122102310195), and the Key Subject of Fishery in Henan Province, China. References Bonhote, P., Dias, A.P., Papageorgiou, N., Kalyanasundaram, K., Gratzel, M., 1996. Hydrophobic, highly conductive ambient-temperature molten salts. Inorg. Chem. 35 (5), 1168–1178. Brennecke, J.F., Maginn, E.J., 2001. Ionic liquids: innovative fluids for chemical processing. AIChE J. 47, 2384–2389. Chen, K., Zhang, Q., Wang, J., Liu, F., Mi, M., Xu, H., Chen, F., Zeng, K., 2009. Taurine protects transformed rat retinal ganglion cells from hypoxia-induced apoptosis by preventing mitochondrial dysfunction. Brain Res. 1279, 131–138. Couling, D.J., Bernot, R.J., Docherty, K.M., Dixon, J.K., Maginn, E.J., 2006. Assessing the factors responsible for ionic liquid toxicity to aquatic organisms via quantitative structure – property relationship modeling. Green Chem. 8, 82–90. Doroshenko, N., Doroshenko, P., 2007. Lanthanum suppresses arachidonic acidinduced cell death and mitochondrial depolarization in PC12 cells. Euro. J. Pharmacol. 567, 36–42. Earle, M.J., Seddon, K.R., 2000. Ionic liquids green solvents for the future. Pure Appl. Chem. 72, 1391–1398. Eaton, D.L., Klaassen, C.D., 2001. Principles of toxicology. In: Klaassen, C.D. (Ed.), Casarett & Doull’s Toxicology – The Basic Science of Poisons. McGraw Hill Press, pp. 33–77. Fatemi, M.H., Izadiyan, P., 2011. Cytotoxicity estimation of ionic liquids based on their effective structural features. Chemosphere 84 (5), 553–563. Frade, R.F.M., Rosatella, A.A., Marques, C.S., Branco, L.C., Kulkarni, P.S., Mateus, N.M.M., Afonso, C.A.M., Duarte, C.M.M., 2009. Toxicological evaluation on human colon carcinoma cell line (CaCo-2) of ionic liquids based on imidazolium, guanidinium, ammonium, phosphonium, pyridinium and pyrrolidinium cations. Green Chem. 11, 1660–1665. Garc´ıa-Lorenzo, A., Tojo, E., Tojo, J., Teijeira, M., Rodr´ıguez-Berrocal, F.J., Gonz´alez, M.P., Marínez-Zorzano, V.S., 2008. Cytotoxicity of selected imidazolium-derived

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