Induction of oxidative stress and apoptosis by pentachlorophenol in primary cultures of Carassius carassius hepatocytes

Comparative Biochemistry and Physiology, Part C 150 (2009) 179–185

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Induction of oxidative stress and apoptosis by pentachlorophenol in primary cultures of Carassius carassius hepatocytes Yu-Liang Dong, Pei-Jiang Zhou ⁎, Shun-Yao Jiang, Xue-Wu Pan, Xiao-Hu Zhao College of Resources and Environmental Sciences, Hubei Biomass-Resource Chemistry and Environmental Biotechnology Key Laboratory, Wuhan University, Wuhan 430072, PR China

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Article history: Received 15 January 2009 Received in revised form 20 April 2009 Accepted 20 April 2009 Available online 3 May 2009 Keywords: Pentachlorophenol Primary hepatocytes Carassius carassius Intracellular calcium Reactive oxygen species

a b s t r a c t Pentachlorophenol (PCP) is a highly toxic contaminant of chlorophenols. Due to its slow and incomplete biodegradation, it can be found in surface, groundwater and in soils. To investigate the role of intracellular calcium and reactive oxygen species in apoptosis induced by PCP in cultured hepatocytes, the primary hepatocytes of Carassius carassius were incubated with different concentrations of PCP at 25 °C for 8 h in vitro. Apoptosis was detected by DNA laddering, caspase activation and flow cytometry. The results demonstrated that apoptosis was involved in the cytotoxic effect of PCP, and that PCP-induced apoptosis occurred in a dose-dependent manner. In addition, the induction of apoptosis by PCP was accompanied with Ca2+, Mg2+-ATPase activity decline, intracellular Ca2+ elevation, generation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm) disruption and ATP depletion. Concomitantly, there were dose-dependent increases in lipid peroxidation production (MDA) and decreases in glutathione (GSH). These investigations suggest that PCP-induces apoptosis in the cultured hepatocytes by affecting multiple targets, and suggest that [Ca2+]i increase and ROS generation may be involved in apoptosis induction by PCP. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Pentachlorophenol (PCP) is a well-known organochlorine compound used extensively as a pesticide, herbicide, algaecide, defoliant, wood preservative, germicide, fungicide and molluskacide (Jorens and Schepens, 1993). In the 1970s, PCP was heavily used in China for fighting against schistosomiasis and as a herbicide (Wang et al., 2008). In 1997, China produced approximately 104 tonnes of PCP per year, which was approximately 20% of global production (Zhang et al.,1997). In recent years many countries have banned the use of PCP (Tao, 2003). Unfortunately, its persistence resulted in a long-lasting contamination of aquatic environment (Zheng et al., 2000). PCP is readily absorbed across the skin, lungs and gastrointestinal lining (Reigart and Roberts, 1999). Many other issues may be also responsible for toxicity. Studies showed that PCP possesses endocrine-disrupting functions (Louise and Gerald, 1996; Benjamin et al., 2002). Chen et al. (2004a,b) have shown that PCP could exert its immunotoxical function by reducing fish macrophage proliferation, phagocytosis and reactive oxygen intermediates production, as well as humoral immune parameters such as serum IgM production. Furthermore, PCP is highly toxic at rather low concentrations to a variety of cells and organisms (Repetto et al., 2001; Farah et al., 2004). Besides, recent studies indicate that even at low dose levels, PCP can exert synergistic effects when mixed

⁎ Corresponding author. Tel.: +86 27 87152823; fax: +86 27 68778893. E-mail address: [email protected] (P.-J. Zhou). 1532-0456/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2009.04.010

with other aquatic pollutants such as cadmium, copper, ammonium and polycyclic aromatic hydrocarbons (Zhu et al., 2001; Luckenbach et al., 2003). The mechanism of toxicity induced by PCP to mammals and humans has been studied in vivo as well as in vitro (Yin et al., 2006; Kunisue and Tanabe, 2009). Although several recent studies have demonstrated that PCP can induce toxicity in several kinds of cells (Wang et al., 2001; Wispriyono et al., 2002; Chen et al., 2004a,b; Fernández et al., 2005; Yang et al., 2005), no such information exists in primary cultures of Carassius carassius hepatocytes. Therefore, we designed the present study to explore the toxicity effect of PCP on primary cultures of C. carassius hepatocytes. Apoptosis is also named the programmed cell death, and it is characterized by cell shrinkage, cytoplasmic, nuclear and chromatin condensation, membrane blebbing, protein degradation, and DNA fragmentation, and finally the break down of the cell into apoptotic bodies followed by (secondary) necrosis (Thompson, 1995; Mao et al., 2007). The involvement of Ca2+ signaling in apoptosis has been implicated in a number of recent studies (McConkey and Orrenius, 1997). However, the debate on whether Ca2+ signaling is truly involved in apoptosis is still ongoing. Moreover, the role of intracellular calcium in apoptosis induced by PCP in primary cultured hepatocytes of C. carassius (crucian carp) is still poorly investigated. So in this study, we employed primary cultured hepatocytes of freshwater crucian carp (C. carassius) as an in vitro model to investigate toxic mechanisms of PCP. In the present study, the primary cultures of C. carassius hepatocytes were used as an in vitro model system to study the cytotoxic effects of PCP on fish cells and to monitor whether these result in apoptosis. We

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detected PCP induced apoptosis and focused on the role of intracellular Ca2+, ROS, the mitochondria membrane potential (ΔΨm) and ATP in this response to ascertain the mechanisms involved in apoptosis by PCP in primary hepatocytes. 2. Materials and methods 2.1. Fish and isolation of hepatocytes C. carassius (0.25–0.3 kg each) were purchased from a local hatchery (Wuhan, China). They were kept in 300 L fiber-glass tanks with aerated, dechlorinated water (22 ± 3 °C, 12 h light:12 h dark cycles) for at least 2 weeks before they were used for hepatocytes isolation. They were freely fed with commercial fish food at least once a day. Hepatocytes were isolated according to the methods of Smeets et al. (1999), and modified by Zhou et al. (2006). Fish skin was sterilized by alcohol and its abdomen was dissected with sterile instruments. Liver tissue was excised and rinsed twice with phosphate buffered saline (PBS:136.9 mM NaCl, 5.4 mM KCl, 0.81 mM MgSO4, 0.44 mM KH2PO4,0.33 mM Na2HPO4; 5.0 mM NaHCO3, pH7.6) without Ca2+. The liver tissue was then minced into pieces and transferred to a 50 mL conical tube, to which a solution of 0.25% trypsin was added (Sigma). The tube was shaken on a shaker at 200 rpm for 5 min to obtain the cell suspension, which was then filtered through a 100 mesh sieve. The cell suspensions were pooled and centrifuged at 60 g for 2 min; the cell pellet was washed with DMEM/F12 culture medium (Hyclone Company). Cells were resuspended in the culture medium and counted using a haemocytometer (Reichert, Buffalo, NY, USA), and those with more than 90% viability by the Trypan blue exclusion method were used for the experiment. For each batch of experiments, hepatocytes were prepared from four individual fish. 2.2. Cell culture and cytotoxicity assay Hepatocytes were cultured in DMEM/F12 which contained 10% fetal calf serum, 100 u/mL− 1 penicillin (Amresco Company), 100 µg/mL− 1 streptomycin, 1 µM bovine insulin and 10 µM hydrocortisone (Sigma). Cells were seeded in 12-well culture plates at 5 ×105 cells/mL, and incubated at 25 °C in a 5% CO2 atmosphere (v/v) with 95% relative humidity. Hepatocytes had attached to the wells and formed a monolayer of 70–80% confluence within 24 h, and at this time the culture medium was removed and replaced with testing medium. Hepatocytes were exposed to PCP (0, 0.01, 0.1, 1.0, 10, 100 µmol L− 1) for 8 h. The highest and non-effective doses of PCP were determined in a previous study. Cell viability was measured by the methyl thiazolyl tetrazolium (MTT, Sigma) reduction method (Ferrari et al., 1990). After treating the cells with different concentrations of PCP for 8 h and washing with PBS, 20 µL of MTT (5 mg/mL− 1) was added into each well and incubated for 4 h at 25 °C, then the culture medium was removed and 100 µL dimethyl sulfoxide (DMSO) were added and the culture plate was shaken for 10 min. And then, the cells were transferred to the microplate spectrophotometer reader (BIO-680, BioRad, USA) to measure the absorbance of the extracted solution at 530 nm. Cell viability was expressed as a percentage of control viability measured in untreated cells. 2.3. Biochemical assays Ca2+,Mg2+-ATPase activity was determined by quantifying the release of inorganic phosphorus from adenosine triphosphate (ATP). Free inorganic phosphate was detected by the malachite greenammonium molybdate reagent. The working solution (0.5 mL) contained 150 mM NaCl, 5 mM KCl, 2.5 mM MgCl2, 2.5 mM ATP, 20 µg of homogenate protein, 1 mM ouabain and 20 mM imidazole. Incubations were carried out at 37 °C for 30 min. To stop the reaction, 100 µL of ice-cold 35% (w/v) trichloroacetic acid was added. Aliquots of 20 µL were analyzed for inorganic phosphate. Eliminating enzyme

from control assays monitored spontaneous hydrolysis of ATP. Ca2+, Mg2+-ATPase activity was determined using a commercially available kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The specific activity was expressed in µmol Pi/mg protein/h. Intracellular Ca2+ ([Ca2+]i) changes in primary hepatocytes were monitored by using the Ca2+-sensitive fluorescent dye Fura-2 as previously described (Santillán et al., 2004). After hepatocytes were exposed to different concentrations of PCP for 8 h, fluorescent dyes Fura-2 (1 mmol L− 1) was added to the culture medium. The cells were then incubated by shaking for 30 min in the dark at 37 °C. After that the medium was removed from the tissue culture plates, and washed the cells with HEPES buffer for 3 times to remove the residual dye. Then 1.5 mL HEPES buffer was added into the cultures and determined immediately for Fura-2 fluorescence intensity by fluorescence spectrophotometer (F4500, Japan). [Ca2+]i values were calculated from the emission ratio (R) at 340 and 380 nm excitation wavelengths, using the formula described by Grynkiewicz et al. (1985). The intracellular ROS content was determined using 2',7'dichlorofluorescin diacetate (DCFH-DA, Sigma) (LeBel et al., 1992). After 8 h incubation of hepatocytes with various concentrations of PCP, 25 µL DCFH-DA stock solution (200 µM) was added to each well and the hepatocytes were incubated for 30 min at 25 °C in the dark, then the culture medium was removed and 100 µL DMSO was added to each well. After incubation for another 20 min, the fluorescence intensity was measured immediately using a microplate reader (Synergy HT, Bio-Tek Instruments, USA). Excitation and emission wavelengths were 490 and 530 nm, respectively. GSH levels in the primary hepatocytes were measured according to the method described by Dringen et al. (1996). GSH was determined by adding 700 µL of a buffer (0.33 mg/mL− 1 NADPH, 0.2 M Na3PO4, 10 mM EDTA, pH 7.2) and 100 µL 6 mM 5.5 V-dithiobis (2-nitrobenzoic acid (DTNB, Sigma) to the cell medium. After 5-min equilibration, 10 µL GSH reductase was added and the reduction of DTNB was monitored immediately at 405 nm for 3 min. The blanks and standard GSH samples were also determined. GSH was calculated on basis of a standard curve of reduced GSH. After 8 h incubation of hepatocytes with various concentrations of PCP, hepatocytes were washed twice with cold PBS and suspended in 5 volumes cell lysis buffer (20% glycerol, 150 mM NaCl, 0.5% NP40, 1 mM Na2EDTA, 10 mM PMSF, 1 mM aprotinin, 10 mM Tris–HCl, pH 7.4). The lysates were centrifuged at 16,000 g for 10 min at 4 °C, the supernatant were then collected and stored at −20 °C. The lipid peroxidation (measured as MDA) content was determinated by the kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The MDA concentration was expressed as nmol/mg− 1 protein. Intracellular ATP was extracted from cells and measured using a luciferin/luciferase-based assay (Molecular Probes Inc., Eugene, OR, USA). In this assay, luciferin is oxidized in the presence of ATP to give off light. Luminescence was measured with a luminometer (Kikkoman, Tokyo, Japan). 2.4. The mitochondrial membrane potential (ΔΨm) ΔΨm was monitored using the fluorescent dye Rhodamine 123 (Rh-123), which preferentially enters into mitochondria due to the highly negative ΔΨm. Depolarization of ΔΨm results in the loss of Rh-123 from the mitochondria thereby decreasing intracellular fluorescence. The fluorescence level of Rh-123 in cells was analyzed using a flow cytometer. The ΔΨm levels were represented with Rh-123 fluorescence intensities. 2.5. DNA fragmentation assay After exposure, hepatocytes were rinsed twice with cold PBS and lysed for 1 h at 37 °C with a lysis buffer (10 mM Tris–HCl, pH 7.4, 10 mM EDTA, 100 mM NaCl, 200 µg/mL− 1 proteinase K and 0.5% SDS). This

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were analyzed with one-way ANOVA, followed by Dunnett-t-test. The significance level was ascertained at P b 0.05. 3. Results 3.1. Cytotoxic effect of PCP

Fig. 1. Viability of cultured hepatocytes determined by MTT assay. Data shown are representative of five independent experiments. Asterisks indicate significant difference from control (⁎P b 0.05, ⁎⁎P b 0.01).

lysate was then extracted with phenol: chloroform: isoamyl alcohol (25:24:1) and centrifuged at 10,000 g for 5 min at 4 °C, then DNA was precipitated with 3 M sodium acetate and 100% cold ethanol and deposited at −20 °C for 12 h. The precipitated DNA was centrifuged at 10,000 g for 10 min at 4 °C and the DNA pellets were washed with 70% ethanol, air-dried, and dissolved in Tris–EDTA buffer (10 mM Tris, 1 mM EDTA, pH 7.4), then 5 mg/L− 1 RNase A was added to each sample to remove RNA. DNA samples (10 µg) were electrophoresed on a 1.2% agarose gel containing 10 µg/mL− 1 ethidium bromide for 2 h at 60 V. After electrophoresis, the DNA ladder was visualized by Bioimaging Systems. 2.6. Measurement of caspase-3 activity The activity of caspase-3 was measured by using the caspase-3 colorimetric assay Kit (Sigma). This assay measures the cleavage of a specific colorimetric caspase substrate, acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA). pNA (p-nitroaniline) is released from the substrate upon cleavage by caspase. Free pNA produces a yellow color that is monitored by a JH752 UV/Vis spectrophotometer at 405 nm. After Carassius auratus hepatocytes were treated with the PCP (0, 0.01, 0.1, 1.0, 10 or 100 µmol L− 1) for 8 h, cells were washed with PBS and further incubated 12 h without PCP and harvested by scraping. The caspase-3 activity was measured in cell lysates. The lysed cells were centrifuged at 16,000 g for 15 min at 4 °C, and the supernatants were analyzed immediately according to the analysis procedure described in the manufacturer's protocol.

The viability of the primary hepatocytes exposed to various concentrations of PCP for 8 h (Fig. 1), was significantly decreased in comparison with the control groups. There was no significant reduction in cell viability in the lower exposure group (0.01 and 0.1 µM) compared with the control. With increasing concentrations, cell viability was decreased to 78.0 ± 9.6%, 54.7 ± 4.1% and 38.3 ± 5.4% at 1, 10 and 100 µM of PCP concentrations, respectively. 3.2. Effects of PCP on Ca2+,Mg2+-ATPase activity and [Ca2+]i Mean activities of Ca2+,Mg2+-ATPase of hepatocytes are shown in Fig. 2a. The activity of Ca2+, Mg2+-ATPase significantly decreased with increasing PCP concentrations. To examine whether this was associated with a corresponding elevation of intracellular calcium, we tested the change of [Ca2+]i using the calcium indicator Fura-2/ AM. In fact it can be seen that [Ca2+]i increased after the addition of PCP in a concentration-dependent manner. 3.3. Effects of PCP on intracellular ROS, GSH and MDA DCF fluorescence intensity is proportional to the content of intracellular ROS. Fig. 3a shows that the intracellular ROS contents were significantly higher in the 1, 10 and 100 µM PCP-treated groups than in the control group. Intracellular GSH has been shown to be crucial in regulating cell proliferation, cell cycle progression, and apoptosis (Schnelldorfer et al., 2000). Therefore, we analyzed the changes of GSH levels in primary hepatocytes. Under PCP exposure,

2.7. Apoptosis assay The apoptotic cell was analyzed by Rh-123 and PI uptake. After treated with different concentrations of PCP for 8 h, the harvested cells were resuspended in 1 mL solution containing 10 µmol L− 1 Rh-123 for 15 min in dark at room temperature, and then the cells were harvested and rinsed twice with cold PBS and incubated in 1 mL cold binding buffer including 10 µg mL− 1 PI for 10 min in dark at room temperature. After staining, the cells were washed and analyzed by a flow cytometer (EPICS ALTRA II, Beckman Coulter). The percentage of cells apoptosis was calculated using the Cellquest software. 2.8. Protein assay Protein content was assayed by the Bradford method, using bovine serum albumin (Hyclone, USA) as a standard. 2.9. Statistical analysis Data presented are means ± standard deviation (S.D.). Statistical analysis was performed using SPSS 13.0 (SPSS, Chicago, IL, USA). Data

Fig. 2. Effect of PCP on (a) activities of Ca2+,Mg2+-ATPase and (b) intracellular calcium concentration in primary cultures of Carassius auratus hepatocytes. Data shown are representative of three independent experiments. Asterisks indicate significant difference from control (⁎P b 0.05, ⁎⁎P b 0.01).

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Fig. 5. Effect of PCP on ΔΨm. The results expressed as percentage of the total fluorescence of Rh-123 in the incubation medium. Data shown are representative of three independent experiments. Asterisks indicate significant difference from control (⁎P b 0.05, ⁎⁎P b 0.01).

significant increase in cellular MDA concentration after PCP treatments (Fig. 3c). When the cells were incubated with 1, 10 and 100 µM, intracellular MDA increased approximately to 26.6%, 36.8% and 80.2% of control values, respectively. 3.4. Effect of PCP on ATP From Fig. 4 it can be seen that there was no significant difference in the ATP content at lower concentrations of PCP exposure compared with the control, while approximately 20.5%, 46.5% and 72.8% reduction was observed at hepatocytes treated with 1, 10 and 100 µM. 3.5. Effect of PCP on the mitochondrial membrane potential (ΔΨm)

Fig. 3. Effect of PCP on intracellular (a) ROS (Graph shows the DCF fluorescence intensities), (b) GSH (nmol mg− 1 pro) and (c) MDA (nmol mg− 1 pro) in primary cultures of Carassius auratus hepatocytes. Data shown are representative of three or four independent experiments. Asterisks indicate significant difference from control (⁎P b 0.05, ⁎⁎P b 0.01).

there was no significant difference in the GSH content at 0.01 and 0.1 µM of PCP exposure compared with the control, while approximately 35.7%, 43.5% and 56.9% reduction was observed at 1, 10 and 100 µM treated hepatocytes (Fig. 3b). Malondialdehyde (MDA) is indicative of lipid peroxidation. Cellular MDA was measured after cell treatment with various concentrations of PCP for 8 h. There was a

Fig. 4. Effect of PCP on intracellular ATP content. Data shown are representative of three independent experiments. Asterisks indicate significant difference from control (⁎P b 0.05, ⁎⁎P b 0.01).

We further studied if PCP-induced apoptosis coincides with disrupted mitochondrial function. The ΔΨm collapse is a critical step that occurs in all cell types undergoing apoptosis, regardless of the inductive signal. The ΔΨm was determined by the ΔΨm sensitive probe Rh-123. Significant reduction of the ΔΨm compared to the control value was observed in Fig. 5. 3.6. Effect of PCP on DNA fragmentation in primary hepatocytes As shown in Fig. 6 negligible DNA laddering was observed in 0.01 and 0.1 µM PCP-treated groups after 8 h exposure compared with the control group. However, typical DNA laddering was observed in 10 and 100 µM PCP-treated groups, indicating the occurrence of apoptosis at these concentrations.

Fig. 6. DNA laddering induced after exposure to various concentrations of PCP in the primary hepatocytes for 8 h measured on agarose gel electrophoresis.

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Fig. 7. The detection of PCP induced apoptosis with Rh-123 and PI staining. Results were expressed as dot plots representing one of the three independent experiments. D1: necrotic cells; D2: late/secondary apoptotic cells; D3: live cells; D4: early/primary apoptotic cells.

3.7. Effect of PCP on apoptosis rate in C. carassius hepatocytes Flow cytometric measurement was used to quantify the extent of apoptosis in the total cell population, combining both adherent and floating cells. The results of the flow cytometry of C. carassius hepatocytes treated with PCP were shown in Fig. 7. The ratios of apoptotic cells among the total cells became higher, which were 1.5, 3.2, 4.9, 8.0, 28.9 and 37.5% for the 0, 0.01, 0.1, 1, 10 and 100 µM PCP treated groups, respectively. Treated by the higher concentration of PCP, the necrotic cells were also increased. This suggested that PCP induced not only apoptosis but also necrosis. The apoptosis ratios of C. carassius hepatocytes treated by different concentrations of PCP were significantly different. 3.8. Effect of PCP on caspase-3 activity in C. carassius hepatocytes To determine the mechanism of PCP-induced apoptosis, the activity of caspase-3 in C. carassius hepatocytes following PCP treatments was examined. As shown in Fig. 8, caspase-3 activity was increased by the PCP in a dose-dependent manner. Caspase-3 activity

Fig. 8. Effect of PCP on caspase-3 activity in Carassius auratus hepatocytes. Data shown are representative of three independent experiments. Asterisks indicate significant difference from control (⁎P b 0.05, ⁎⁎P b 0.01).

was not different from that of control, in lower PCP concentrations (0.01 and 0.1 µmol L− 1), but was increased significantly by about twofold in 1 µmol L− 1 PCP, four-fold in 10 µmol L− 1 PCP and seven-fold in 100 µmol L− 1 PCP. This result suggest that the apoptotic effect of the PCP in C. carassius hepatocytes are associated with an increased in caspase activation. 4. Discussion Recently, the effects of PCP have been studied on diverse biological systems and PCP has been reported to induce apoptosis in some tissues (Wispriyono et al., 2002; Fernández et al., 2005). Early in vitro studies have shown that PCP can uncouple oxidative phosphorylation, inactivate respiratory enzymes, and cause mitochondrial damage (Deichman and Keplinger, 1981). In this study, it was found that PCP can induce apoptosis with its typical characteristics of nuclear shrinkage, condensation, caspase-3 activation and breakage as well as formation of apoptotic bodies, and further experiment demonstrated that PCP-induced apoptosis occurred in a dose-dependent manner. This is in agreement with the results of Fernández et al. (2005), who observed that the treatment of Vero cells with PCP had a significant number of cells initiate an apoptotic death process identified by the condensed and fragmented state of their nuclei. However, Chen et al. (2004a,b) observed that CP, DCP and TCP can induce apoptosis in L929 cells, but PCP mediated cell necrosis. In another study, apoptosis features were found in rats and human hepatoma cell lines treated with TCHQ but not PCP (Wang et al., 2001). These results implicate that PCP can cause different injury on different cells. The possible reason is that the cell death manner may depend on the biological component of every cell culture such as the cells themselves (species, age, tissue) and handling during isolation and cultivation. It has been suggested that apoptosis is associated with calcium signaling in some cell types (Negre-Salvayre and Salvayre, 1992; Nicotera and Orrenius, 1998; Hsu et al., 2007; Chaube et al., 2009). Intracellular calcium ion either released from the calcium pool or influxed, plays an important role in the cellular function as a second

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messenger. Calcium signaling is upstream of certain pathways that lead to apoptosis. A variety of toxic insults, associated with increasing [Ca2+]i concentration, can cause endoplasm reticulum (ER) stress, induce morphological alterations of cells, and ultimately lead to cell death (Negre-Salvayre and Salvayre, 1992; Nicotera and Orrenius, 1998; Nakayama et al., 2001). In this study, it was revealed that Ca2+, Mg2+-ATPase activity and ATP content were declined, and the intracellular Ca2+ was increased by PCP. The Ca2+,Mg2+-ATPase (also called Ca2+ pump) plays an important role in maintaining the normal active transport for positive ions such as Ca2+. The inhibition of Ca2+,Mg2+-ATPase activity leads to [Ca2+]i elevation (Geng et al., 2005). Depletion of cellular ATP could cause the loss of plasma membrane potential which results in the influx of Ca2+ into intracellular space. The elevation of [Ca2+]i will be to some extent buffered by mitochondria. However, the overloaded Ca2+ in mitochondria will act synergistically with ROS to open the mitochondrial membrane permeability transition pore (MPTP) (Nakayama et al., 2001). MPTP opening will increase the mitochondrial membrane permeability, leading to loss of ΔΨm, inhibition of ATP synthesis and rupture of the mitochondrial membrane (Tsujimoto and Shimizu, 2006). Based on these findings, it was suggest that elevation in [Ca2+]i by PCP may be an important step in PCP-induced apoptosis in primary cultured hepatocytes. Oxidative stress refers to the cellular status with enhanced production of ROS and/or impaired function of the cellular antioxidant defense system (Buttke and Sandstrom, 1994). It has long been known that the intracellular redox status plays an important role in cell survival and death (Hampton and Orrenius, 1998). In this experiment, it was found that cellular GSH was depleted and lipid peroxidation increased. And it was further found that the intracellular ROS was significantly increased with PCP concentration increased. GSH has been shown to cause a reduction in the initiation of lipid peroxidation (Tzeng et al., 1995; Yang et al., 2001). The depletion of GSH can result in increase of ROS concentration, which may enhance the lipid peroxidation intensity (Chiou et al., 2003). The participation of ROS in the cell apoptosis can be linked to the depletion of GSH induced by PCP. GSH is the substrate for glutathione peroxidase, an enzyme that reduces hydrogen peroxide to water. The mechanisms underlying the role of oxidative stress in apoptosis may include high levels of ROS directly increasing caspase activity, disrupting intracellular Ca2+ homeostasis and resulting in the ATP depletion due to the close relationship between ROS and mitochondria (McConkey, 1998; Thayyullathil et al., 2008). In addition to ΔΨm, ROS and Ca2+, two entities presumably important for apoptosis, are linked to mitochondria. On the basis of these findings and those of the literature it is possible to hypothesize that mitochondria is involved in apoptosis induced by PCP. In conclusion, the present study demonstrated that PCP can induce apoptosis in primary cultured hepatocytes of C. carassius. The concentration-dependent apoptosis was accompanied by the ΔΨm disruption, intracellular Ca2+ elevation, increase of ROS concentration and ATP depletion, suggesting that PCP-induced apoptosis in the cultured hepatocytes have multiple potential targets. Although we did find, in the present study, that the intracellular Ca2+ is increased in PCP-induced apoptosis of primary cultured hepatocytes of C. carassius, it still remains to determine whether the increased intracellular Ca2+ might activate endonuclease directly, or through a Ca2+-dependent protease like calpain, leading to DNA fragmentation. Further studies are required to elucidate the precise mechanism of PCP-induced apoptosis in primary cultures of C. carassius hepatocytes. Acknowledgements This study was supported by the National Natural Science Foundation of China (No. 20577036, 20777058).

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