Arch Toxicol (2009) 83:899–908 DOI 10.1007/s00204-009-0451-x

I N O RG A N I C C O M P O U N D S

Arsenic induces mitochondria-dependent apoptosis by reactive oxygen species generation rather than glutathione depletion in Chang human hepatocytes Yi Wang · Yuanyuan Xu · Huihui Wang · Peng Xue · Xin Li · Bing Li · Quanmei Zheng · Guifan Sun

Received: 19 January 2009 / Accepted: 4 June 2009 / Published online: 18 June 2009 © Springer-Verlag 2009

Abstract This study was conducted to evaluate the possible involvement of mitochondrial pathway in NaAsO2induced apoptosis and the role of reactive oxygen species (ROS) and reduced glutathione (GSH) in the apoptotic eVect in Chang human hepatocytes. The MTT assay demonstrated that sodium arsenite (NaAsO2) treatment for 24 h caused a dose-dependent decrease of cell viability. NaAsO2 treatment (0–30
M) was also found to induce phosphatidylserine externalization, a hallmark of apoptosis; to disrupt the mitochondrial membrane potential (
m); to cause the release of cytochrome c into the cytosol, and to trigger cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in a dose-dependent manner. All these changes were accompanied with the enhanced generation of intracellular ROS and malondialdehyde (MDA). Increase of intracellular GSH also coincided unexpectedly. Moreover, the extracellular addition of N-acetyl-L-cysteine (NAC, 5 mM) eVectively reduced the generation of ROS and MDA, and rescued the cells from NaAsO2 induced apoptosis and related alteration of mitochondria. These data suggest that the arsenic-induced cell apoptosis occurs though the mitochondrial pathway, and is mostly dependent on generation of ROS rather than GSH depletion in Chang human hepatocytes.

Y. Wang · Y. Xu · H. Wang · P. Xue · X. Li · B. Li · Q. Zheng · G. Sun (&) Department of Occupational and Environmental Health, College of Public Health, China Medical University, 92 Bei Er Road, Heping District, Shenyang 110001, People’s Republic of China e-mail: [email protected]

Keywords Arsenic · Reactive oxygen species · Glutathione · Apoptosis · Mitochondria Abbreviations MDA malondialdehyde NAC N-acetyl-L-cysteine PARP poly (ADP-ribose) polymerase ROS reactive oxygen species GSH reduced glutathione TBARs thiobarbituric acid reactive substances

Introduction Arsenic is ubiquitously distributed in nature and ranks 20th in abundance in the earth’s crust and 14th in the seawater (Mandal and Suzuki 2002). Chronic exposure to arsenic in drinking water is a serious public-health problem that now aVects millions of people in Asian countries, including Bangladesh, China, India, Cambodia, Lao PDR, Mongolia, Myanmar, Nepal, Pakistan, and Viet Nam (Sun et al. 2006). Arsenicosis is typically deWned by the classical dermal lesions and long-term exposure to high level of inorganic arsenic in drinking water is reported to be related with the development of diabetes, cardiovascular diseases, as well as cancers of the bladder, kidney, lung, and liver. (Bates et al. 1992; NRC 2001; Tondel et al. 1999; Sun et al. 2006; Yoshida et al. 2004). The primary metabolic pathway of inorganic arsenic in humans is multiple methylation steps by methyltransferase. This enzymatic process occurs mainly in the liver (Ford 2002). Emerging evidences indicate that liver is also a potential target of arsenic toxicity (Liu and Waalkes 2008). Abnormal liver function, manifested by gastrointestinal symptoms (e.g., abdominal pain, indigestion, loss of appetite,

123

900

etc.) and elevations of serum enzymes [e.g., alanine amino transferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), etc.], frequently occurs from acute and chronic exposure to arsenic in the drinking water (Mazumder, 2005; Xu et al. 2008). The development of portal hypertension and liver Wbrosis are reported in arsenic-exposed populations (Morris et al. 1974; Huet et al. 1975; Nevens et al. 1990; Santra et al. 1999; Mazumder 2005). Moreover, liver cirrhosis is suspected to be a primary cause of arsenic-related mortality of subjects exposed to arsenic from coal burning in Guizhou, China (Liu et al. 2002). Therefore, elucidation of the possible mechanisms of arsenic-induced hepatotoxicity is essential. Apoptosis or programmed cell death plays a crucial role in the development and defense of homeostasis, whereas, it can also be initiated by stress conditions such as infectious agents and drugs which may result in serious health problems and lead to fatal conditions (Ramanathan 2005). The progression of apoptosis has been indicated to be modulated by the redox status of cells. Increasing evidence has shown that reactive oxygen species (ROS) are involved in arsenic-induced cytotoxicity (Liu et al. 2001). Excessive generation of intracellular ROS may lead to oxidative stress, loss of cell function, and ultimately apoptosis or necrosis (Engel and Evens 2006). The induction of apoptosis by arsenic in hepatocytes, accompanied with oxidative stress, has been observed in murine models (Santra et al. 2007; Paul et al. 2008). The abrogation of arsenic-induced apoptosis by dithiothreitol (DTT) and N-acetyl-L-cysteine (NAC) found in these studies also suggest the possible involvement of ROS and/or reduced glutathione (GSH). Although the depletion of intracellular GSH levels is reported to be necessary for apoptosis induced by other chemicals in Hela cells and lymphoid cells (Han et al. 2008; Franco et al. 2007), the role of ROS and GSH in apoptotic progression induced by arsenic in hepatocytes has not been elucidated. This study was conducted to evaluate the role of ROS and GSH, and the possible involvement of mitochondrial pathway in NaAsO2-induced apoptosis in human hepatocytes. Under the experimental conditions, NaAsO2 induced mitochondrial dysfunction, apoptotic progression, as well as increase of intracellular ROS and malondialdehyde (MDA), in Chang human hepatocytes in a dosedependent manner. Notably, the elevation of intracellular GSH levels was observed in NaAsO2-treated cells. Moreover, NAC rescued Chang human hepatocytes from NaAsO2-induced apoptosis. The results suggest that NaAsO2-induced mitochondria-dependent apoptosis in Chang human hepatocytes is associated with the increase of intracellular ROS and independent of GSH depletion.

123

Arch Toxicol (2009) 83:899–908

Materials and methods Reagents Sodium arsenite (NaAsO2, ¸99.0%), N-acetyl-L-cysteine (NAC), 2⬘,7⬘-dichlorodihydroXuorescein diacetate (DCFHDA), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). 5,5⬘,6,6⬘-tetrachloro-1,1⬘,3,3⬘-tetraethylbenzimidazol carbocyanine iodide (JC-1) was from Molecular Probes (Eugene, OR, USA). RPMI 1640 growth medium, trypsin, penicillin, and streptomycin were from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) was from Hyclone (Logan, UT, USA). Annexin V-Xuorescein isothiocyanate (FITC) apoptosis detection kit, MDA assay kit, and GSH assay kit were from Keygen Biotech, Co., Ltd. (Nanjing, Jiangsu, China). Mitochondria isolation kit was purchased from Pierce Biotechnology Inc. (Rockford, IL, USA). Polyclonal antibodies of cytochrome c and actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyclonal antibodies of cleaved caspase3 and cleaved poly (ADP-ribose) polymerase (PARP) were from Cell Signaling Technology (Beverly, MA, USA). Enhanced chemiluminescence (ECL) plus kit was from Amersham Life Science (Buckinghamsire, UK). All the reagents used were of the highest grade obtainable. Water used in all the preparations was distilled and deionized. Cell culture and treatment Chang human hepatocytes were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were grown in sterile RPMI 1640 growth media, pH 7.4, supplemented with 2.0 g/l of sodium bicarbonate, 100 units/ml of penicillin, 100
g/ml of streptomycin, and 10% of FBS. The cells were grown in T-75 culture Xasks at 37°C in a humidiWed 5% CO2 atmosphere until 85% conXuence and were subcultured approximately twice a week. Cells undergoing exponential growth were treated with a Wnal concentration (0–30
M) of NaAsO2 for 24 h and then washed before conducting the bioassays. In order to further assess the role of ROS, cells were pretreated with antioxidant NAC (5 mM) 1 h before NaAsO2 treatment and kept with NAC in the medium during 20-
M NaAsO2 treatment until being analyzed. All the experiments were carried out at least in triplicate. MTT cell viability assay Cell viability based on mitochondrial enzyme functions was assayed by MTT conversion to formazan. For the assay, cells (5 £ 104 cells per well) were seeded in 96-well

Arch Toxicol (2009) 83:899–908

plates. Then, cells grown to 80% conXuence in each well were treated with 100
l of the growth medium in the presence or absence of 5–30
M NaAsO2 and cultured at 37°C in 5% CO2 for 24 h. NAC treatment was carried out as indicated. At the end of treatment, 100
l of MTT solution (0.5 mg/ml in the medium) was added to each well, and the plates were incubated at 37°C for additional 4 h. Afterward, the medium containing MTT was removed, and the crystals were dissolved in 150
l of 100% dimethyl sulfoxide (DMSO). The cell viability was quantiWed using a microplate reader (Multiscan Ascent, Labsystem, Finland) at 570 nm after subtracting the appropriate blank values. Double-staining with annexin V/FITC and propidium iodide (PI)

901

Determination of ROS generation ROS generation was evaluated by measuring dichloroXuorescein (DCF) Xuorescence as described by Rothe and Valet (1990). In brief, cells were incubated with NaAsO2 in the presence or absence of NAC as indicated at 37°C. At the end of the treatment, cells were washed and resuspended in serum-free medium containing 10
M DCFH-DA. After a further 30 min of incubation at 37°C, cells were washed twice with ice-cold PBS and harvested by trypsin. Then, the cells were immediately analyzed with Xuorescence microscopy or Xow cytometer (exc = 488 nm, em = 525 nm) (FACSCalibur, BECTON DICKINSON, USA) to determine the ROS generation. Measurement of intracellular GSH and lipid oxidation

Detection of apoptotic cells was performed using annexin V/PI staining assay. Cells were seeded in 6-well plates, grown to 80% conXuence and treated with NaAsO2 in the presence or absence of NAC as mentioned above. At the end of the treatment, the cells were harvested by trypsin and labeled with annexin V-FITC and PI using an apoptosis detection kit according to the manufacturer’s protocol. Then cells were pelleted and analyzed with a Xow cytometer (FACSCalibur, BECTON DICKINSON, USA). Excitation wave was set at 488 nm. The emitted green Xuorescence of annexin V (FL1) and red Xuorescence of PI (FL2) were detected using 525 and 595 nm emission Wlters, respectively. For each sample, 10,000 cells were analyzed. The amount of early apoptosis, late apoptosis, and necrosis was determined as the percentage of annexin V+/PI¡, annexin V+/PI+, and annexin V¡/PI+ cells, respectively. Measurement of mitochondrial membrane potential (
m) For measurement of 
m, the cationic dye JC-1 was used. JC-1 is capable of selectively entering mitochondria, where it aggregates and gives red Xuorescence when 
m is high, and forms monomers and emits green Xuorescence when 
m is relatively low. In brief, after treatment with NaAsO2 in the presence or absence of NAC as mentioned in 12-well plates, the cells were trypsinized, washed in ice-cold phosphate-buVered saline (PBS), and incubated in 1 ml of serum-free medium containing 2.5
M JC-1 dye at 37°C for 20 min in darkness. After washing twice with PBS, Xuorescence in cells was immediately measured using a Xow cytometer (FACSCalibur, BECTON DICKINSON, USA). Excitation wave was set at 488 nm for JC-1 analysis. Emission Wlters of 525 and 595 nm were used to quantify the population of cells with green (JC-1 monomers) and red (JC-1 aggregates) Xuorescence, respectively.

For determination of intracellular GSH and lipid oxidation, cells were cultured in 10-cm-diameter dishes and treated with NaAsO2 in the presence or absence of NAC as indicated, washed three times with ice-cold PBS, scraped oV the dishes with a silicone “policeman” and harvested into Eppendorf tubes. Then, cells were lyzed in PBS by sonication followed by centrifugation at 15,000£g for 5 min at 4°C. The resulting supernatants were used immediately for measurement of GSH or MDA. The levels of intracellular GSH were measured with improved 5,5⬘-dithiobis-(2-nitrobenzoic acid) (DTNB) method according to the manufacturer’s protocol. DTNB reacts with GSH to form a yellow product. Absorbance was measured at 412 nm, and GSH in cellular extracts was quantiWed on the basis of a calibration curve generated using GSH as a standard. The lipid peroxidation was analyzed by measuring the levels of MDA. The quantiWcation was based on measuring formation of thiobarbituric acid (TBA) reactive substances (TBARS) according to the manufacturer’s protocol. TBA was added to each sample tube and vortexed. The reaction mixture was incubated at 95°C for 40 min. After cooling, the chromogen was read spectrophotometrically at 532 nm. The protein concentrations of the samples were determined by the method of Bradford (Bradford 1976) to normalize the levels of GSH and MDA. Preparation of protein extracts for western blot analysis Cells were seeded in 10-cm-diameter dishes and allowed to grow to 80% conXuence. At the end of treatment, cells were washed 3 times with ice-cold PBS, scraped by “policeman” and disrupted in cell lysis buVer [50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate,

123

902

0.1% sodium dodecyl sulfate (SDS), pH 7.4] supplemented with protease inhibitors [0.1 mM phenylmethyl sulfonyl Xuoride (PMSF), 1% aprotinin, 1 mM leupeptin, 1
g/ml pepstatin A], and 1% phosphatase inhibitors (Roche Diagnostics, Mannheim, Germany) at 4°C for 30 min. Then, lysates were cleared by centrifugation at 4°C. For cytochrome c analysis, mitochondria and cytosol fractions were separated using mitochondria isolation kit according to the manufacturer’s instructions. Protein concentrations were determined by the method of Bradford (Bradford 1976). All the protein fractions were stored at ¡70°C until use. Western blot analysis Protein extracts (30 ug per lane) were separated by 8% or 15% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene diXuoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking in 5% nonfat dry milk in Tween 20 Tris-buVered saline (TTBS), the membranes were incubated with primary antibodies (rabbit anti-human cleaved caspase-3, rabbit anti-human cleaved PARP, mouse anti-human cytochrome c, and rabbit anti-human actin) at 1:800–5,000 dilutions overnight at 4°C, and then secondary antibodies conjugated with horseradish peroxidase at 1:10,000 dilution for 1 h at room temperature. Protein bands were detected by ECL plus kit. Statistical analysis Statistical analyses were performed using the SPSS software (version 11.5; SPSS Inc., Chicago, IL, USA). All the experiments were performed at least in triplicate, and the results were expressed as means § standard deviations (SD). DiVerences between treatment groups were analyzed by one-way analysis of variance (ANOVA) with post hoc analysis using Dunnett’s test. DiVerences were considered signiWcant when P < 0.05.

Results Arsenic exposure and cell viability In order to study the cytotoxicity of arsenite in Chang human hepatocytes, cells were exposed to diVerent concentrations of NaAsO2 and cell viability was examined based on MTT assay. The viability of NaAsO2-treated cells decreased as NaAsO2 concentration increased (0–200
M) (Fig. 1). The range of NaAsO2 concentrations (0–30
M), in which the survival rate was above 80%, was selected to conduct further experiments.

123

Arch Toxicol (2009) 83:899–908

Fig. 1 EVect of NaAsO2 on viability of Chang human hepatocytes after 24 h of incubation. Results are expressed as mean § SD, n = 6. *P < 0.05 versus the control group; #P < 0.05 versus 20-
M- NaAsO2treated group

Apoptosis of Chang human hepatocytes The early and late apoptosis, as well as necrosis, induced by NaAsO2 were detected by Annexin V and PI double staining. Figure 2a shows representative cytograms of Xowcytometric analysis. NaAsO2 treatment for 24 h caused a dose-dependent increase in the percentage of cells in early apoptosis (annexin V+/PI¡) and in late apoptosis (annexin V+/PI+) (Fig. 2b). The percentage of necrotic cells (annexin V¡/PI+) was only signiWcantly increased in cells treated with 30
M NaAsO2 (P < 0.05) (Fig. 2b). In a further set of experiments, we evaluated the arsenite-induced activation of caspase-3. Western blot analysis showed that NaAsO2 induced the cleavage of caspase-3 (17-kDa fragments) and PARP (89-kDa fragments), which are two hallmarks of apoptosis (Fig. 2c). Depolarization of 
m and cytochrome c release In order to determine whether mitochondrial pathway is involved in the NaAsO2-induced apoptosis, the alteration of 
m and cytochrome c release from the mitochondria into the cytosol were examined. Representative cytograms presented in Fig. 3a show that NaAsO2 increased the percentages of cells with collapsed 
m in a dose-dependent manner. An 18–61% decrease in percentage of cells with high 
m was observed after 24-h treatment with 5–30
M NaAsO2, compared with control cells. At the same time, the level of cytochrome c was markedly increased in the cytosol but decreased in the mitochondria as the concentrations of NaAsO2 elevated (Fig. 3b), coinciding with changes of 
m. The level of intracellular ROS, GSH, and MDA The generation of intracellular ROS was detected by using the Xuorescent probe DCFH-DA. As shown in Fig. 4, the level of ROS was increased by the treatment with NaAsO2 in a dose-dependent manner. Exposure to the lowest

Arch Toxicol (2009) 83:899–908

903

Fig. 2 Apoptosis induced by NaAsO2 in Chang human hepatocytes. Cells were treated for 24 h with diVerent doses of NaAsO2 (0–30
M) and 20
M NaAsO2 in the presence of NAC. a Cytograms of Xow-cytometric analysis. The apoptosis assessed by measuring the exposure of phosphatidylserine residues on the cell surface as described under “Materials and methods” (double-staining with annexin V/FITC and PI). Legend for cytograms: the lower left quadrant includes the viable cells, which are negative for annexin V/FITC binding (annexin V¡) and exclude PI (PI¡); the lower right quadrant includes early apoptotic cells, which are positive for annexin V/FITC binding (annexin V+) but PI¡; the upper right quadrant represents the late apoptotic cells, which are annexin V+ and show PI uptake (PI+); The upper left quadrant represents necrotic cells, which are annexin V¡/PI+. Dot plots are representative of data collected from three experiments with similar results. b Percentage of early apoptotic, late apoptotic and necrosis cells. Data are presented as the mean § SD, n = 3, * P < 0.05 versus the control group; #P < 0.05 versus 20-
M- NaAsO2-treated group. c Analysis of caspase-3 and PARP cleavage after NaAsO2 treatment in Chang human hepatocytes, as detected by Western blot analysis with total cell lysates. Proteins were separated by SDS-PAGE and probed with anti-cleaved-caspase-3 and anticleaved-PARP antibodies. The amount of proteins loaded was equal in all lanes of the gel. Representative images of three experiments are shown

concentration tested (5
M) led to a 25% increase in ROS generation compared with control cells, and exposure to NaAsO2 at greater concentrations increased ROS generation to levels that were 1.5–2.7 times of those detected in control cells (Fig. 4c). It is interesting to note that the level of intracellular GSH was elevated as the dose of NaAsO2 increased (Fig. 5a). The level of GSH in cells exposed to the highest concentra-

tion tested (30
M) in our study was nearly double of that in control cells. In order to study the impact of NaAsO2 on oxidative lesions, the level of MDA (a common end product of lipid peroxidation) was measured. The concentrations of MDA increased following arsenic administration and were signiWcantly higher in cells treated with 20 and 30
M NaAsO2 than that in control cells (Fig. 5b).

123

904

Arch Toxicol (2009) 83:899–908

Fig. 3 Depolarization of mitochondrial membrane potential (
m) and cytochrome c release from mitochondria. Cells were treated for 24 h with diVerent doses of NaAsO2 (0–30
M) and 20
M NaAsO2 in the presence of NAC. a Change in 
m determined by Xow-cytometric analysis with JC-1 staining. Legend for cytograms: the lower right quadrant includes cells with collapsed 
m, in which JC-1 forms monomers and emits green Xuorescence; the upper right quadrant includes cells with normal 
m, in which JC-1 aggregates and gives red Xuorescence. Dot plots are representative of data collected from three experiments with similar results. * P < 0.05 versus the control group; # P < 0.05 versus 20-
MNaAsO2-treated group. b Cytochrome c in mitochondria and cytosol analyzed by Western blot analysis. Proteins were separated by SDS-PAGE and probed with anti-cytochrome c antibodies. The amount of proteins loaded was equal in all the lanes of the gel. Representative images of three experiments are shown

Prevention of arsenite-induced apoptosis by NAC In order to determine the link between increase of the intracellular ROS level and apoptosis in NaAsO2-treated cells, Chang human cells were co-incubated with thiol-containing antioxidant NAC when treated with 20
M NaAsO2. NAC treatment signiWcantly increased (P < 0.05) the viability of Chang human hepatocytes exposed to 20
M NaAsO2 for 24 h (Fig. 1). At the same time, the generation of ROS in cells co-incubated with 5 mM NAC and 20
M NaAsO2 was signiWcantly inhibited (P < 0.05), only 1.26 times of control cells and 70% of 20-
M-NaAsO2-treated cells (Fig. 4). Inhibition of ROS production by NAC was accompanied by the suppression of NaAsO2-induced apoptosis (Fig. 2a and b), decrease in the cleavage of caspase-3 and PARP (Fig. 2c), increase in the 
m (Fig. 3a) and reduction of cytochrome c release to the cytosol (Fig. 3b). Meanwhile, NAC co-treatment signiWcantly increased (P < 0.05) the level of intracellular GSH (Fig. 5a), but decreased (P < 0.05) the level of MDA induced by 20
M NaAsO2 (Fig. 5b). These observations suggest that increase in the level of intracellular ROS rather than glutathione depletion

123

after NaAsO2 treatment is required in cell apoptosis involved with mitochondrial pathway.

Discussion In mammals, liver is the primary site for bio-transformation, accumulation, and excretion of arsenic. Liver is also a major target organ of arsenic toxicity. However, the pathogenesis of the liver cell injury in arsenic-induced toxicity is still obscure although evidences of apoptosis and oxidative stress in the liver resulting from arsenic exposure are available in the literature (Santra et al. 2000a,b; Santra et al. 2007; Das et al. 2005; Paul et al. 2008). In this study, we investigated the role of ROS and GSH, as well as the possible involvement of mitochondria-dependent pathway, in arsenic-induced apoptosis of Chang human hepatocytes. Mitochondria not only provide energy supply for the cells, but in some circumstances can also unleash the machineries of death (Newmeyer and Ferguson-Miller 2003). In many instances, permeabilization of mitochondrial membranes is a rate-limiting step of various apoptotic

Arch Toxicol (2009) 83:899–908

905

Fig. 4 Induction of ROS in Chang human hepatocytes by NaAsO2 and its suppression by NAC. Cells were treated for 24 h with diVerent doses of NaAsO2 (0–30
M) and 20
M NaAsO2 in presence of NAC. a The DCF Xuorescence was visualized using Xuorescence microscope. b The amount of Xuorescence was measured using Xow cytometer. Each histogram represents plots for the control cells (shaded) overlaying those for the treated cells (unshaded). c Relative ROS levels in cells quantiWed by Xow cytometer. Results are expressed as mean § SD, n = 3. *P < 0.05 versus the control group; # P < 0.05 versus 20-
MNaAsO2-treated group

123

906

Fig. 5 The level of intracellular GSH (a) and MDA (b). Results are expressed as mean § SD, n = 3. * P < 0.05 versus the control group; # P < 0.05 versus 20-
M- NaAsO2-treated group

processes including drug-induced apoptosis (Kroemer and Reed, 2000). In the early stage of apoptosis, disruption of mitochondrial membrane leads to a rapid collapse of the electrochemical gradient (Ly et al. 2003) and the release of cytochrome c from the mitochondria into the cytosol (Gogvadze et al. 2006). The released cytochrome c combines with apoptosis protease activating factor-1, procaspase-9, and dATP in the cytosol, producing active caspase-9, leading to the proteolytic activation of caspase-3 (the primary eVector caspase of the cell), and Wnally resulting in PARP degradation and DNA cleavage (Carre et al. 2002). The mitochondria, it is suggested, do not take part in arsenicinduced apoptosis in HepG2 cell line (Siu et al. 2002). However, studies in murine models and Hep3B cell line suggest that mitochondrial dysfunction is important in the pathogenesis of arsenic-induced apoptotic liver cell injury (Santra et al. 2007; Paul et al. 2008). In this study, a remarkable loss of 
m occurred in cells exposed to NaAsO2 in a dose-dependent manner compared with untreated cells. Moreover, a dose-dependent decrease in the mitochondrial cytochrome c level, with a concomitant increase in the corresponding cytosolic fraction, was clearly observed when cells were incubated with NaAsO2. In addition, we observed that NaAsO2 increased cleavage of

123

Arch Toxicol (2009) 83:899–908

caspase-3 and PARP in a dose-dependent way. All these changes suggest the involvement of mitochondrial pathway in NaAsO2-induced apoptosis in Chang human hepatocytes. Oxidative stress, it is suggested, is directly or indirectly involved in early events in liver injury due to xenobiotics, drugs, and hepatitis C virus (HCV) infection (Neuschwander-Tetri and Caldwell 2003; Bataller et al. 2003). Arsenic exposure leads to increase of ROS by a variety of redox enzymes, including the Xavoprotein-dependent superoxideproducing enzyme such as NADPH oxidase (Bernstam and Nriagu 2000; Chou et al. 2004). In this study, the elevation of ROS production and MDA levels induced by NaAsO2 in Chang human hepatocytes coincided well with the increase of apoptotic cells, collapse of 
m, release of cytochrome c, and cleavage of caspase-3 and PARP. Moreover, the pretreatment with exogenous antioxidant NAC eVectively prevented oxidative stress as well as inhibited the apoptotic events. Altogether these results indicate that oxidative stress is directly involved in the early stage of apoptosis induced by arsenic in human hepatocytes, and in particular accumulation of intracellular ROS, it may lead to collapse of the 
m, release of cytochrome c, cleavage of caspase-3 and PARP, and ultimately to programmed cell death through apoptosis. GSH acts as an interchangeable and redundant antioxidant which protects cells from oxidative stress (Meister 1994). Intracellular GSH has also been shown to be crucial for regulation of cell proliferation, cell cycle progression, and apoptosis (Ghibelli et al. 1998; Poot et al. 1995; Schnelldorfer et al. 2000). It has been observed that levels of intracellular GSH are associated with sensitivity to arsenic toxicity in NB4 cells (Dai et al. 1999; Davison et al. 2003). The depletion of GSH has been suggested to be highly related to apoptosis in lymphoid cells and antimycin A-treated Hela cells (Han et al. 2008; Franco et al. 2007). It is interesting to note that in this study, intracellular GSH elevation, instead of GSH depletion, was observed as the apoptotic cells increased by NaAsO2. The extracellular addition of NAC, the known precursor of GSH and antioxidant, increased the level of intracellular GSH, attenuated the generation of intracellular ROS and lipid peroxidation, and protected cells from apoptotic progression (evidenced by decreased number of annexin V-positive cells, as well as reduction in loss of 
m, release of cytochrome c, and cleavage of caspase-3 and PARP). Considering the signiWcant GSH elevation in cells treated with NaAsO2 only, the protective eVects of NAC may result from the well-known capacities of thiol compounds as antioxidants. However, we cannot rule out the possibility of chemical reaction between NAC and NaAsO2. All the results of this study indicate that occurrence of arsenic-induced apoptosis may mainly depend on generation of intracellular ROS, not on

Arch Toxicol (2009) 83:899–908

depletion of endogenous intracellular GSH in Chang human hepatocytes. Although the elevation of GSH levels was observed when NAC treatment was administrated, the decrease of apoptotic cells may mainly result from the elimination of intracellular ROS by thiol antioxidants. In summary, our results suggest that the critical mitochondrial alterations leading to cytochrome c release, caspase activation, and prosphatidylserine externalization during arsenic-induced apoptosis in Chang human hepatocytes are not associated with the depletion of GSH, but rather eVects mediated by ROS. Acknowledgments The study was supported by grants (Project No. 30530640 and 30600510) from National Natural Science Foundation of China (NSFC).

References Bataller R, North KE, Brenner DA (2003) Genetic polymorphisms and the progression of liver Wbrosis: a critical appraisal. Hepatology 37(3):493–503. doi:10.1053/jhep.2003.50127 Bates MN, Smith AH, Hopenhayn-Rich C (1992) Arsenic ingestion and internal cancers: a review. Am J Epidemiol 135(5):462–476 Bernstam L, Nriagu J (2000) Molecular aspects of arsenic stress. J Toxicol Environ Health B Crit Rev 3(4):293–322. doi:10.1080/ 109374000436355 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254. doi:10.1016/00032697(76)90527-3 Carre M, Carles G, Andre N, Douillard S, Ciccolini J, Briand C, Braguer D (2002) Involvement of microtubules and mitochondria in the antagonism of arsenic trioxide on paclitaxel-induced apoptosis. Biochem Pharmacol 63(10):1831–1842. doi:10.1016/ S0006-2952(02)00922-X Chou WC, Jie C, Kenedy AA, Jones RJ, Trush MA, Dang CV (2004) Role of NADPH oxidase in arsenic-induced reactive oxygen species formation and cytotoxicity in myeloid leukemia cells. Proc Natl Acad Sci USA 101(13):4578–4583. doi:10.1073/pnas.0306687101 Dai J, Weinberg RS, Waxman S, Jing Y (1999) Malignant cells can be sensitized to undergo growth inhibition and apoptosis by arsenic trioxide through modulation of the glutathione redox system. Blood 93(1):268–277 Das A, Santra A, Lahiri S, Guha Mazumder DN (2005) Implications of oxidative stress and hepatic cytokines (TNF- and IL-6) response in the pathogenesis of hepatic collagenesis in chronic arsenic toxicity. Toxicol Appl Pharmacol 204(1):18–26. doi:10.1016/ j.taap.2004.08.010 Davison K, Cote S, Mader S, Miller WH (2003) Glutathione depletion overcomes resistance to arsenic trioxide in arsenic-resistant cell lines. Leukemia 17(5):931–940. doi:10.1038/sj.leu.2402876 Engel RH, Evens AM (2006) Oxidative stress and apoptosis: a new treatment paradigm in cancer. Front Biosci 11:300–312. doi:10.2741/1798 Ford M (2002) Arsenic. In: Flomenbaum NE, Goldfrank LR, HoVman RS, Howland MA, Lewin NA, Nelson LS (eds) Goldfrank’s toxicologic emergencies. McGraw-Hill, New York, pp 1183–1195 Franco R, Panayiotidis MI, Cidlowski JA (2007) Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation. J Biol Chem 282(42):30452– 30465. doi:10.1074/jbc.M703091200

907 Ghibelli L, Fanelli C, Rotilio G, Lafavia E, Coppola S, Colussi C, Civitareale P, Ciriolo MR (1998) Rescue of cells from apoptosis by inhibition of active GSH extrusion. FASEB J 12(6):479–486 Gogvadze V, Orrenius S, Zhivotovsky B (2006) Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochim Biophys Acta 1757(5–6):639–647. doi:10.1016/j.bbabio.2006.03.016 Han YH, Kim SH, Kim SZ, Park WH (2008) Intracellular GSH levels rather than ROS levels are tightly related to AMA-induced HeLa cell death. Chem Biol Interact 171(1):67–78. doi:10.1016/ j.cbi.2007.08.011 Huet PM, Guillaime E, Gote J, Legare A, Lavoir P, Viallet A (1975) Non-cirrhotic perisinusoidal portal hypertension-association with chronic arsenical intoxication. Gastroenterology 68(5 Pt1):1270– 1277 Kroemer G, Reed JC (2000) Mitochondrial control of cell death. Nat Med 6(5):513–519. doi:10.1038/74994 Liu J, Waalkes MP (2008) Liver is a target of arsenic carcinogenesis. Toxicol Sci 105(1):24–32. doi:10.1093/toxsci/kfn120 Liu SX, Athar M, Lippai I, Waldren C, Hei TK (2001) Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity. Proc Natl Acad Sci USA 98(4):1643–1648. doi:10.1073/ pnas.031482998 Liu J, Zheng B, Aposhian HV, Zhou Y, Cheng ML, Zhang AH, Waalkes MP (2002) Chronic arsenic poisoning from burning high-arsenic-containing coal in Guizhou, China. Environ Health Perspect 110(2):119–122 Ly JD, Grubb DR, Lawen A (2003) The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update. Apoptosis 8(2):115–128. doi:10.1023/A:1022945107762 Mandal BK, Suzuki KT (2002) Arsenic round the world: a review. Talanta 58(1):201–235. doi:10.1016/S0039-9140(02)00268-0 Mazumder DN (2005) EVect of chronic intake of arsenic-contaminated water on liver. Toxicol Appl Pharmacol 206(2):169–175. doi:10.1016/j.taap.2004.08.025 Meister A (1994) Glutathione ascorbic acid antioxidant system in animals. J Biol Chem 269(13):9397–9400 Morris JS, Schmid M, Newman S, Scheuer PJ, Sherlock S (1974) Arsenic and noncirrhotic portal hypertension. Gastroenterology 66(1):86–94 Neuschwander-Tetri BA, Caldwell SH (2003) Nonalcoholic steatohepatitis: summary of an AASLD single topic conference. Hepatology 37(5):1202–1219. doi:10.1053/jhep.2003.50193 Nevens F, Fevery J, Van Steenbergen W, Sciot R, Desmet V, De Groote J (1990) Arsenic and non-cirrhotic portal hypertension. A report of eight cases. J Hepatol 11(1):80–85. doi:10.1016/01688278(90)90276-W Newmeyer DD, Ferguson-Miller S (2003) Mitochondria: releasing power for life and unleashing the machineries of death. Cell 112(4):481–490. doi:10.1016/S0092-8674(03)00116-8 NRC (National Research Council) (2001) Arsenic in Drinking Water. National Academy Press, Washington, DC Paul MK, Kumar R, Mukhopadhyay AK (2008) Dithiothreitol abrogates the eVect of arsenic trioxide on normal rat liver mitochondria and human hepatocellular carcinoma cells. Toxicol Appl Pharmacol 226(2):140–152. doi:10.1016/j.taap.2007.09.020 Poot M, Teubert H, Rabinovitch PS, Kavanagh TJ (1995) De novo synthesis of glutathione is required for both entry into and progression through the cell cycle. J Cell Physiol 163(3):555–560. doi:10.1002/jcp.1041630316 Ramanathan K, Anusuyadevi M, Shila S, Panneerselvam C (2005) Ascorbic acid and alpha-tocopherol as potent modulators of apoptosis on arsenic induced toxicity in rats. Toxicol Lett 156(2):297– 306. doi:10.1016/j.toxlet.2004.12.003 Rothe G, Valet G (1990) Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2⬘,7⬘-dichloroXuorescin. J Leukoc Biol 47(5):440–448

123

908 Santra A, Das Gupta J, De BK, Roy B, Guha Mazumder DN (1999) Hepatic manifestations in chronic arsenic toxicity. Indian J Gastroenterol 18(4):152–155 Santra A, Maiti A, Chowdhury A, Guha mazumder DN (2000a) Oxidative stress in liver of mice exposed to arsenic contaminated water. Indian J Gastroenterol 19(3):112–115 Santra A, Maiti A, Das S, Lahiri S, Chakraborty SK, Guha Mazumder DN (2000b) Hepatic damage caused by arsenic toxicity in experimental animals. J Toxicol Clin Toxicol 38(4):395–405. doi:10.1081/CLT-100100949 Santra A, Chowdhury A, Ghatak S, Biswas A, Dhali GK (2007) Arsenic induces apoptosis in mouse liver is mitochondria dependent and is abrogated by N-acetylcysteine. Toxicol Appl Pharmacol 220(2):146–155. doi:10.1016/j.taap.2006.12.029 Schnelldorfer T, Gansauge S, Gansauge F, Schlosser S, Beger HG, Nussler AK (2000) Glutathione depletion causes cell growth inhibition and enhanced apoptosis in pancreatic cancer cells. Cancer 89(7):1440–1447. doi:10.1002/1097-0142(20001001) 89:7<1440::AID-CNCR5>3.0.CO;2-0

123

Arch Toxicol (2009) 83:899–908 Siu KP, Chan JY, Fung KP (2002) EVect of arsenic trioxide on human hepatocellular carcinoma HepG2 cells: inhibition of proliferation and induction of apoptosis. Life Sci 71(3):275–285. doi:10.1016/ S0024-3205(02)01622-3 Sun G, Li X, Pi J, Sun Y, Li B, Jin Y, Xu Y (2006) Current research problems of chronic arsenicosis in China. J Health Popul Nutr 24(2):176–181 Tondel M, Rahman M, Magnuson A, Chowdhury IA, Faruquee MH, Ahmad SA (1999) The relationship of as levels in drinking water and the prevalence rate of skin lesions in Bangladesh. Environ Health Perspect 107(9):727–729. doi:10.2307/3434658 Xu Y, Wang Y, Zheng Q, Li B, Li X, Jin Y, Lv X, Qu G, Sun G (2008) Clinical manifestations and arsenic methylation after a rare subacute arsenic poisoning accident. Toxicol Sci 103(2):278–284. doi:10.1093/toxsci/kfn041 Yoshida T, Yamauchi H, Sun GF (2004) Chronic health eVects in people exposed to arsenic via the drinking water: dose-response relationships in review. Toxicol Appl Pharmacol 198(3):243– 252. doi:10.1016/j.taap.2003.10.022