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Roles of reactive oxygen species and MAP kinases in the primary rat hepatocytes death induced by toosendanin
Toxicology 249 (2008) 62–68
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Roles of reactive oxygen species and MAP kinases in the primary rat hepatocytes death induced by toosendanin Yunhai Zhang a,b , Xinming Qi a,b , Likun Gong a,b , Yan Li a,b , Linlin Liu a,b , Xiang Xue a,b , Ying Xiao a,b , Xiongfei Wu a,b , Jin Ren a,∗ a b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China Graduate School of the Chinese Academy of Sciences, Shanghai, China
a r t i c l e
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Article history: Received 10 January 2008 Received in revised form 8 April 2008 Accepted 8 April 2008 Available online 16 April 2008 Keywords: Toosendanin Primary rat hepatocytes Reactive oxygen species (ROS) MAP kinases
a b s t r a c t Toosendanin (Tsn), a triterpenoid extracted from Melia toosendan Sieb et Zucc, possesses different pharmacological effects in human and important values in agriculture. However, liver injury has been reported when toosendanin or Melia-family plants, which contain toosendanin are applied. The mechanism by which toosendanin induces liver injury remains largely unknown. Here we reported that toosendanin induced primary rat hepatocytes death by mitochondrial dysfunction and caspase activation. Toosendanin led to decrease of mitochondrial membrane potential, fall in intracellular ATP level, release of cytochrome c to cytoplasm, activation of caspase-8, 9, and 3 and ultimately cell death. Level of reactive oxygen species (ROS) was also increased in hepatocytes after incubation with toosendanin. Catalase, the H2 O2 -decomposing enzyme, can prevent the reduction in ATP level and protect hepatocytes from toosendanin-induced death. The ERK1/2 (p44/42 MAP kinases) and JNK (c-Jun N-terminal kinase) were activated, but p38 MAPK was not activated by toosendanin. Inhibition of ERK1/2 activation sensitized hepatocytes to death and increased activity of caspase-9 and 3 in response to toosendanin. Inhibition of JNK attenuated toosendanin-induced cell death. These results suggested that toosendanin causes death of primary rat hepatocytes by mitochondrial dysfunction and caspase activation. Generation of ROS and MAP kinases activation might be involved in this process. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Toosendanin (C30 H38 O11 , FW = 574), a triterpenoid extracted from Melia toosendan Sieb et Zucc as shown in Fig. 1, has been used as a digestive tract parasiticide and insecticide in traditional Chinese medicine (Chung et al., 1975; Shu and Liang, 1980; Shi and Li, 2007). It has been reported that toosendanin selectively blocks acetylcholine (ACh) release from nerve terminals and has antibotulismic role in vitro and in vivo (Shi and Chen, 1999; Zhou et al., 2003; Shi and Wang, 2004). Recently, toosendanin-induced apoptosis on various cancer cells was reported (Tang et al., 2004; Zhang et al., 2005). However, liver injury was reported when toosendanin or Melia-family plants which contain toosendanin were used in traditional Chinese medicine. High concentration of toosendanin was found in liver and the excretion rate was very slow after toosendanin administration in rhesus monkey (Zhou et al.,
∗ Corresponding author at: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China. E-mail address: cdser [email protected] (J. Ren). 0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2008.04.005
1982). Toosendanin can cause increase in serum glutamic pyruvic transaminase (SGPT) level and liver morphological changes in rhesus monkey (Li et al., 1982). However, the mechanism of liver injury caused by toosendanin remains largely unknown. Excessive apoptosis of hepatocytes is one cause for liver injury especially in drug-induced liver damage (Lee, 2003; Guicciardi and Gores, 2005). The death receptor pathway and the mitochondrial pathway have been reported in apoptotic death of mammalian cells (Hengartner, 2000; Wang, 2001). It has been reported that toosendanin can induce cytochrome c release and caspase-3 activation in PC12 and cancer cells (Tang et al., 2004; Zhang et al., 2005). A crude extract from fruits of M. toosendan has been reported to regulate PC12 cell differentiation and induce activation of extracellular signal-regulated kinases (ERKs) (Yu et al., 2004). ERKs are one member of mitogen-activated protein kinase (MAPK) family and the most commonly studied MAPKs are ERKs, JNK and p38 MAPK (Chang and Karin, 2001). Activation of ERKs in hepatocytes is thought to act as a protective and anti-apoptosis factor (Bonni et al., 1999; Qiao et al., 2002; Czaja et al., 2003). In contrast, JNK and p38 activation generally have been shown to promote hepatocytes death (Cross et al., 2000; Kurata, 2000). Reactive oxygen species (ROS) such as superoxide anions, H2 O2 and hydroxyl rad-
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itive control (Hirata and Nagatsu, 2005). TMRE is a membrane-permeable cationic fluorophore that accumulates electrophoretically into mitochondria in response to their negative potential (Ehrenberg et al., 1988). Fluorescence readings were taken on a fluorimeter (NOVOstar, BMG LABTECH, Offenburg, Germany) with excitation wavelength at 540 nm and emission wavelength at 595 nm. 2.5. Assay of cellular ATP content Intracellular ATP levels were determined using CellTiter-GloTM Luminescent Cell Viability Assay kit according to the kit direction (Promega, Madison, WI, USA). Bioluminescence was measured by a fluorimeter (NOVOstar, BMG LABTECH). 2.6. Measurement of caspase-3/8/9 activity Caspase-3/8/9 activity was measured with BD ApoAlertTM Caspase Fluorescent Assay kits according to the kit direction (BD Biosciences Pharmingen, San Diego, CA, USA). Fluorescence was measured in a fluorimeter (NOVOstar, BMG LABTECH) with excitation at 380 nm and emission at 460 nm for caspase-9 assay or with excitation at 400 nm and emission at 505 nm for caspase-3 and 8. Fig. 1. The chemical structure of toosendanin.
icals are generated in many stressful situations and induce cell death by either apoptosis or necrosis (Kruidenier and Verspaget, 2002; Klaunig and Kamendulis, 2004). Moreover, ROS can induce MAPK activation by which hepatocytes mediate reaction to oxidative stress (Yamamoto et al., 1993; Wang et al., 1998). Our present study was designed to investigate the effect of toosendanin on primary rat hepatocytes, aiming to elucidate the mechanism of toosendanin hepatotoxicity. 2. Materials and methods
2.7. Measurement of ROS generation Generation of ROS was detected by DCFH-DA as described previously (Mattia et al., 1991). Cells were washed twice and suspended in PBS (1 × 106 cells/ml). Suspended cells were incubated with DCFH-DA (5 M) at 37 ◦ C for 30 min. H2 O2 was added as a positive control. Fluorescence intensity was measured by a fluorimeter (NOVOstar, BMG LABTECH) with excitation at 485 nm and emission at 520 nm. 2.8. Preparation of cytoplasmic extracts Hepatocytes were washed with ice-cold PBS (pH 7.4) and suspended in ice-cold HMKEE buffer (20 mM Hepes–KOH, 10 mM KCl, 1.5 mM MgCl2 , 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride, 10 g/ml pepstain A, and 10 g/ml leupeptin) containing 250 mM sucrose. Hepatocytes were homogenized by passage through a 26-gauge needle and centrifuged at 16,000 × g at 4 ◦ C for 15 min. The cytoplasmic supernatant was collected.
2.1. Chemicals 2.9. Western blot Toosendanin were purified from M. toosendan Sieb et Zucc with a purity over 98% (Provided by the Institute of Life Sciences, Donghua University, Shanghai, China). Toosendanin was dissolved in dimethyl sulfoxide (DMSO) and stored at −20 ◦ C before use. PD98059, SP600125 and catalase polyethylene glycol were purchased from Sigma (St. Louis, MO, USA). All other chemicals and solvents were purchased from Sigma and of analytical grade. 2.2. Animals and culture of primary rat hepatocytes Sprague–Dawley male rats (200 ± 30 g) were supplied by Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). The rats were housed in a cage under controlled conditions of temperature (20 ± 3 ◦ C) and humidity (45–65%) with a 12-h light:12-h dark cycle. Drinking water and food were provided ad libitum throughout the study. Primary rat hepatocytes were isolated from rats by two-step collagenase perfusion as described previously (Berry and Friend, 1969; Orrenius et al., 1976; Seglen, 1976) with some modifications. Collagenase IV was purchased from Sigma. Monolayer hepatocytes were cultured in Ham’s F-12/DMEM (Invitrogen, Carlsbad, CA, USA) (1:1) medium supplemented with 100 U/ml penicillin and 70 g/ml streptomycin at 37 ◦ C in a humidified atmosphere containing 5% CO2 . Cultural medium was changed 4–6 h after cell seeding to minimize contamination of death cells. Control cultures received vehicle solvent (0.1% DMSO). Some hepatocytes were pretreated with 20 M PD98059, 10 M SP600125 or 400 U/ml catalase polyethylene glycol for 1 h before incubation with toosendanin. 2.3. Cell viability assay After incubation with toosendanin and/or chemicals in primary rat hepatocytes, the cell viability was determined with the Cell Counting Kit-8 (Dojindo Laboratories, Tokyo, Japan). Visible wavelength absorbance data at 450 nm were collected using a standard 96-well plate reader (SOFTmax® PRO, Molecular Devices, Sunnyvale, CA, USA). 2.4. Measurement of mitochondrial membrane potential To monitor mitochondrial membrane potential, cultured hepatocytes were loaded with 500 nM tetramethylrhodamine ethylester (TMRE, Fluka, St. Gallen, Switzerland) in HBS solution (33 mM Hepes, 160.8 mM NaCl, 3.15 mM KCl, 0.7 mM Na2 HPO4 ·12H2 O, pH 7.65) at 37 ◦ C for 30 min, according to methods described before (Wu et al., 1990; Zahrebelski et al., 1995). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma), a mitochondrial toxin, was added as a pos-
Cytoplasmic extracts were subjected to 15% SDS-PAGE and transferred to PVDF membrane. The PVDF membrane was immunoblotted with monoclonal rat anti-cytochrome c (Lab Vision&NEOMARKERS, UK), GAPDH antibody (Santa Cruz Biotechnology, Inc.), cleaved Caspase-3 rabbit mAb, phospho-p44/42 (pERK1/2) MAP Kinase rabbit mAb, p44/42 (ERK1/2) MAP Kinase rabbit mAb, phosphoSAPK/JNK (pJNK) antibody, SAPK/JNK (JNK) antibody, phospho-p38 MAP Kinase rabbit mAb, p38 MAP Kinase rabbit mAb (Cell Signaling Technology Inc., Beverly, MA, USA). Antibodies were visualized using ECL chemiluminescent detection system (Amersham, Piscataway, NJ, USA) and Kodak Image Station (Kodak, Rochester, NY, USA). 2.10. Statistical analysis Data were entered into a database and analyzed using SPSS software. Group mean values and standard deviations were calculated. After homogenetic analysis, homogeneous data were analyzed with one-way analysis of variance and a post hoc test of least significant difference. Heterogeneous data were analyzed using t-test. P < 0.05 was considered significant.
3. Results 3.1. Cytotoxicity and caspase activation by toosendanin Cultured primary rat hepatocytes were exposed to toosendanin for 24 h in 96-well plate before cell viability determined by Cell Counting Kit-8 (Dojindo Laboratories). Toosendanin suppressed cell viability in a dose-dependent manner (Fig. 2). The concentration required for 50% inhibition of growth (IC50 value) was 14.94 ± 0.93 M (n = 3). Since caspase activation is the key event in apoptotic cell death, we assayed caspase-3/8/9 activation in primary rat hepatocytes incubated with toosendanin. Caspase-3 activity was increased significantly after toosendanin incubation in 16 and 24 h (Fig. 3A). Cleaved caspase-3, a marker of caspase-3 activation, was also detected by Western blot after 24 h toosendanin incubation (Fig. 3B). Caspase-9 activity was also increased significantly
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was confirmed after 4 and 8 h toosendanin incubation. Cellular ATP level, a sensitive parameter of mitochondrial function, were decreased at 5 and 10 M toosendanin incubation in 4 and 8 h (Fig. 4B). These results demonstrated that toosendanin caused mitochondrial dysfunction in primary rat hepatocytes. Incubation of the hepatocytes with 5 and 10 M toosendanin after 8 and 16 h, cytochrome c level in cytoplasmic fractions was analyzed by Western blot. Toosendanin induced a significant increase of cytochrome c level in cytoplasm after 16 h incubation (Fig. 4C). These results indicated that toosendanin causes damage to the integrity of mitochondrial membrane. 3.3. ROS generation and protective effect of catalase
Fig. 2. Cell viability of primary rat hepatocytes with toosendanin incubation for 24 h. All values are means ± S.D., n = 3.
after toosendanin incubation up to 16 h in primary rat hepatocytes (Fig. 3C). Caspase-8 activity was also increased with 5 and 10 M toosendanin in 4 h incubation (Fig. 3D). Together, these results suggest that toosendanin induced caspase activation to lead to primary rat hepatocytes death. 3.2. Mitochondrial dysfunction We then examined the mitochondrial membrane potential by determining the cellular retention of TMRE. As shown in Fig. 4A, a minor but marked decrease of TMRE retention in hepatocytes
Generation of ROS was determined after toosendanin incubation from 1 h and up to 6 h in primary rat hepatocytes (Fig. 5A). Pretreatment with catalase, primary rat hepatocytes were partially protected from toosendanin-induced death (Fig. 5B). Moreover, catalase also alleviated reduction in ATP level caused by toosendanin (Fig. 5C). Thiol anti-oxidant such as N-acetyl-cystein (NAC) and dithiothreitol (DTT) did not show protective effect (data not shown). These results suggested that toosendanin can cause oxidative stress and the cellular injury might be originated from H2 O2 . 3.4. Role of MAPK in toosendanin cytotoxicity We then studied whether toosendanin affect activation of ERK1/2. As shown in Fig. 6A, phosphorylation of ERK1/2 was enhanced. Phosphorylation of JNK was also observed within 1 h of toosendanin incubation (Fig. 6B). Phosphorylated p38 level, another member of MAPK, was not activated by toosendanin (data not shown).
Fig. 3. Caspase activation induced by toosendanin (Tsn). (A) Caspase-3 activity with toosendanin incubation up to 24 h. * P < 0.05 significantly different from the “control” group at each time point. (B) Cleaved caspase-3 levels with 24 h toosendanin incubation. The results are representatives of three separated experiments. (C) Caspase-9 activity was examined with 5 and 10 M toosendanin incubation at different time points. * P < 0.05 significantly different from the “control” group at each time point. (D) Caspase-8 activity was determined with 5 and 10 M toosendanin incubation up to 8 h. * P < 0.05 significantly different from the “control” group at each time point. Activity of caspase-3/8/9 is depicted as the value of fluorescence vs. protein content. All values are means ± S.D., n = 3.
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Fig. 4. Mitochondrial damage caused by toosendanin (Tsn). (A) Decrease of mitochondrial membrane potential with toosendanin incubation up to 8 h. * P < 0.05 significantly different from the “Tsn 0 M” group at each time point. CCCP was added as a positive control. (B) Cellular ATP contents were assayed with toosendanin incubation up to 8 h. * P < 0.05 significantly different from the “control” group at each time point. (C) Cytochrome c levels in cytoplasmic extracts induced by toosendanin up to 16 h. Results are representatives of three separated experiments. All values are means ± S.D., n = 3.
PD98059 is a highly specific inhibitor for MEK1/2, the upstream kinase of ERK1/2 (Dudley et al., 1995; Alessi et al., 1995). PD98059 enhanced toosendanin-induced hepatocytes death in 24 h and increased caspase-3 activity in 16 and 24 h (Fig. 7A and B). After PD98059 pretreatment, caspase-9 activity was increased after 16 h with 10 M toosendanin incubation (Fig. 7C). PD98059 treatment by itself neither caused cell death nor affected caspase-3/9 activity (data not shown). These results indicated that ERK1/2 activity mediated protective effects against toosendanin-induced hepatocytes death.
Fig. 5. Oxidative damages caused by toosendanin (Tsn) and protective effect of catalase. (A) Generation of reactive oxygen species (ROS) by toosendanin incubation up to 6 h. * P < 0.05 significantly different from the “Tsn 0 M” group at each time point. H2 O2 was added as a positive control. (B) Cell viability with catalase pretreatment after toosendanin incubation in 24 h. * P < 0.05 significantly different from the “Tsn” group. (C) ATP level with catalase pretreatment after 4 h toosendanin incubation. * P < 0.05 significantly different from the “Tsn” group. All values are means ± S.D., n = 3.
SP600125 is a specific JNK inhibitor (Bennett et al., 2001). Pretreatment with 10 M SP600125, the cell viability of primary rat hepatocytes was increased in the presence of toosendanin (Fig. 8A). Meanwhile, caspase-3 activation induced by toosendanin was decreased in 16 h (Fig. 8B). SP600125 treatment by itself nei-
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Fig. 6. MAP kinases activation induced by toosendanin (Tsn). (A) Phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 levels were detected with toosendanin incubation. (B) Phosphorylated JNK (pJNK) and total JNK levels were detected with toosendanin incubation. Results are representatives of three separated experiments.
ther caused cell death nor affected caspase-3 activity (data not shown). These results indicated that activation of JNK was involved in toosendanin cytotoxicity. 4. Discussion In the present study, we first showed that toosendanin caused primary rat hepatocytes death by mitochondrial dysfunction and caspase activation. ROS and MAP kinases are involved in this process. Toosendanin led to mitochondrial damages including decrease of mitochondrial membrane potential, fall of ATP level, and release of cytochrome c to cytoplasm. Caspases, including caspase-8, 9 and 3 all were activated after toosendanin incubation. Toosendanin caused cellular ROS level increase and catalase can partially protect hepatocytes from toosendanin-induced death. Studies with MEK1/2 chemical inhibitor PD98059 showed that ERK1/2 activation mediated hepatocytes resistance to toosendanin cytotoxicity. In contrast, JNK inhibition by SP600125 showed that JNK played a pro-death role in toosendanin-induced hepatocytes injury. These results showed that toosendanin has cytotoxic effect on primary rat hepatocytes and the putative mechanisms showed as Fig. 9. Mitochondrial dysfunction such as fall in intracellular ATP level can lead to necrotic or apoptotic hepatocellular death (Nieminen et al., 1994; Qian et al., 1997). In our studies, toosendanin induced fall of ATP level and decrease of mitochondrial membrane poten-
Fig. 7. Effects of ERK1/2 activation on toosendanin (Tsn)-induced hepatocytes death. (A) Cell viability after 24 h toosendanin incubation with 20 M PD98059 (PD) pretreatment. * P < 0.05 significantly different from the “Tsn” group. (B) Caspase3 activity in hepatocytes pretreatment with 20 M PD98059 (PD) after 16 and 24 h toosendanin incubation. * P < 0.05 significantly different from the “Tsn 10 M” group. (C) Caspase-9 activity after 8 and 16 h of toosendanin incubation with 20 M PD98059 (PD) pretreatment. * P < 0.05 significantly different from the “Tsn 10 M” group. All values are means ± S.D., n = 3.
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Fig. 8. Effects of JNK activation on toosendanin (Tsn)-induced hepatocytes death. (A) Cell viability after 24 h toosendanin incubation with 10 M SP600125 (SP) pretreatment. * P < 0.05 significantly different from the “Tsn” group. (B) Caspase-3 activity after 16 and 24 h of toosendanin incubation with 10 M SP600125 (SP) pretreatment. * P < 0.05 significantly different from the“Tsn 10 M” group at 16 h. All values are means ± S.D., n = 3.
Fig. 9. A scheme of the putative mechanisms involved in the toosendanin-induced primary rat hepatocytes death.
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tial (Fig. 4A and B). These results indicated that toosendanin causes mitochondrial injury. Damage of mitochondrial membrane integrity can result in cytochrome c release from mitochondria to cytoplasm, which is a key event in mitochondrial apoptosis pathway (Liu et al., 1996; Wang, 2001). We detected release of large amount of cytochrome c into cytoplasm and increase in caspase-9 activity after toosendanin incubation (Figs. 3C and 4C). Moreover, caspase-3 activity was greatly increased at 16 and 24 h (Fig. 3A), our results thus suggested that involvement of mitochondrial apoptosis pathway in toosendanin-induced hepatocytes death. Caspase-8 is the most upstream caspase in death receptor apoptosis pathway (Boldin et al., 1996; Nicholson and Thornberry, 1997; Cohen, 1997). The fact that toosendanin induced caspase-8 activation indicated involvement of death receptor apoptosis pathway in toosendanin-induced hepatocytes death (Fig. 3D). Excessive apoptosis of hepatocytes caused by drugs can result in druginduced liver injury. Therefore, toosendanin-induced hepatocytes death may contribute to the liver injury caused by toosendanin as reported before (Li et al., 1982). In our study, toosendanin caused ROS level increase up to 6 h in primary rat hepatocytes (Fig. 5A). Catalase, the H2 O2 -decomposing enzyme, can increase ATP level and partially protect hepatocytes from toosendanin-induced death (Fig. 5B and C). These results indicated oxidative stress caused by toosendanin led to mitochondrial dysfunction and cells injury. Anti-oxidants including enzymatic or non-enzymatic can protect cells from injury caused by ROS which can lead cells to apoptosis (Mates, 2000; Czaja et al., 2003). We found that thiol anti-oxidants such as NAC or DTT did not attenuate toosendanin-induced hepatocytes death. The effectiveness of catalase but not thiol anti-oxidants suggested that oxidative stress from toosendanin-induction might be cellular injury originated in H2 O2 . However, the nature and source of ROS generated by toosendanin need further investigation. It has been shown that extracts from fruits of M. toosendan caused ERK1/2 activation and regulated PC12 cell differentiation (Yu et al., 2004). In our study, we showed that toosendanin caused ERK1/2 and JNK activation in primary rat hepatocytes (Fig. 6A and B), whereas did not activate p38 MAPK. ERK1/2 activation is anti-apoptotic (Bonni et al., 1999; Qiao et al., 2002). JNK and p38 MAPK activation generally have been shown to promote hepatocytes death (Cross et al., 2000; Kurata, 2000). We found that inhibition of ERK1/2 activation by PD98059 sensitized hepatocytes to toosendanin-induced death (Fig. 7A). Besides, activation of caspase-3 and 9 induced by toosendanin was increased when hepatocytes were pre-treated with PD98059 (Fig. 7B and C). These results indicated that ERK1/2 activation induced by toosendanin mediated protective and anti-apoptotic effect in primary rat hepatocytes. However, ERK1/2 activation is not sufficient to prevent toosendanin cytotoxicity since pro-apoptotic pathway, especially JNK, also was activated. Inhibition of JNK activation by SP600125 attenuated hepatocytes death and decreased caspase-3 activation induced by toosendanin (Fig. 8A and B). These results showed that JNK played a pro-death role in toosendanin-induced hepatocytes injury. Toosendanin increased ROS level and exposure to ROS may lead to activation of cell signal transduction pathway including MAP kinases pathway (Yamamoto et al., 1993; Chang and Karin, 2001). However, the relationship between generation of ROS and MAP kinases activation induced by toosendanin need further studies. In our study, at 24 h the toosendanin concentration in our condition is close to that in vitro test for cancer cell growth inhibition as reported before (Zhang et al., 2005). Since Melia-family plants are still used in traditional Chinese medicine, toosendanin might be served as a candidate for drug research and liver function should be observed closely when toosendanin is administrated.
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Li, P.Z., Shi, X.C., Xue, Z.H., Li, J.F., Sun, G.Z., Qin, B.Y., 1982. Researches on pharmacological and toxicological effects of toosendanin. Chin. Herb. Med. 13, 29–32. Liu, X., Kim, C.N., Yang, J., Jemmerson, R., Wang, X., 1996. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86, 147–157. Mates, J.M., 2000. Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology. Toxicology 153, 83–104. Mattia, C.J., LeBel, C.P., Bondy, S.C., 1991. Effects of toluene and its metabolites on cerebral reactive oxygen species generation. Biochem. Pharmacol. 42, 879–882. Nicholson, D.W., Thornberry, N.A., 1997. Caspases: killer proteases. Trends Biochem. Sci. 22, 299–306. Nieminen, A.L., Saylor, A.K., Herman, B., Lemasters, J.J., 1994. ATP depletion rather than mitochondrial depolarization mediates hepatocyte killing after metabolic inhibition. Am. J. Physiol. 267, C67–74. Orrenius, S., Thor, H., Rajs, J., Berggren, M., 1976. Isolated rat hepatocytes as an experimental tool in the study of cell injury. Effect of anoxia. Forensic Sci. 8, 255–263. Qian, T., Nieminen, A.L., Herman, B., Lemasters, J.J., 1997. Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes. Am. J. Physiol. 273, C1783–C1792. Qiao, L., Yacoub, A., Studer, E., Gupta, S., Pei, X.Y., Grant, S., Hylemon, P.B., Dent, P., 2002. Inhibition of the MAPK and PI3K pathways enhances UDCA-induced apoptosis in primary rodent hepatocytes. Hepatology 35, 779–789. Seglen, P.O., 1976. Preparation of isolated rat liver cells. Methods Cell Biol. 13, 29–83. Shi, Y.L., Chen, W.Y., 1999. Effect of toosendanin on acetylcholine level of rat brain, a microdialysis study. Brain Res. 850, 173–178. Shi, Y.L., Li, M.F., 2007. Biological effects of toosendanin, a triterpenoid extracted from Chinese traditional medicine. Prog. Neurobiol. 82, 1–10. Shi, Y.L., Wang, Z.F., 2004. Cure of experimental botulism and anti-botulismic effect of toosendanin. Acta Pharmacol. Sin. 25, 839–848. Shu, G.X., Liang, X.T., 1980. A correction of the structure of Chuanliansu (toosendanin). Acta Chim. Sin. 38, 196–198. Tang, M.Z., Wang, Z.F., Shi, Y.L., 2004. Involvement of cytochrome c release and caspase activation in toosendanin-induced PC12 cell apoptosis. Toxicology 201, 31–38. Wang, X., 2001. The expanding role of mitochondria in apoptosis. Genes Dev. 15, 2922–2933. Wang, X., Martindale, J.L., Liu, Y., Holbrook, N.J., 1998. The cellular response to oxidative stress: influences of mitogen-activated protein kinase signaling pathways on cell survival. J. Biochem. 333 (Pt 2), 291–300. Wu, E.Y., Smith, M.T., Bellomo, G., Di Monte, D., 1990. Relationships between the mitochondrial transmembrane potential, ATP concentration, and cytotoxicity in isolated rat hepatocytes. Arch. Biochem. Biophys. 282, 358–362. Yamamoto, K., Tsukidate, K., Farber, J.L., 1993. Differing effects of the inhibition of poly(ADP-ribose) polymerase on the course of oxidative cell injury in hepatocytes and fibroblasts. Biochem. Pharmacol. 46, 483–491. Yu, J.C., Min, Z.D., Ip, N.Y., 2004. Melia toosendan regulates PC12 Cell differentiation via the activation of protein kinase A and extracellular signal-regulated kinases. Neurosignals 13, 248–257. Zahrebelski, G., Nieminen, A.L., al-Ghoul, K., Qian, T., Herman, B., Lemasters, J.J., 1995. Progression of subcellular changes during chemical hypoxia to cultured rat hepatocytes: a laser scanning confocal microscopic study. Hepatology 21, 1361–1372. Zhang, B., Wang, Z.F., Tang, M.Z., Shi, Y.L., 2005. Growth inhibition and apoptosisinduced effect on human cancer cells of toosendanin, a triterpenoid derivative from Chinese traditional medicine. Invest. New Drugs 23, 547–553. Zhou, J., Jia, G.R., He, X.Y., 1982. Pharmacokinetics researches of toosendanin. Chin. Herb. Med. 13, 24–26. Zhou, J.Y., Wang, Z.F., Ren, X.M., Tang, M.Z., Shi, Y.L., 2003. Antagonism of botulinum toxin type A-induced cleavage of SNAP-25 in rat cerebral synaptosome by toosendanin. FEBS Lett. 555, 375–379.
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Toxicology journal homepage: www.elsevier.com/locate/toxicol
Roles of reactive oxygen species and MAP kinases in the primary rat hepatocytes death induced by toosendanin Yunhai Zhang a,b , Xinming Qi a,b , Likun Gong a,b , Yan Li a,b , Linlin Liu a,b , Xiang Xue a,b , Ying Xiao a,b , Xiongfei Wu a,b , Jin Ren a,∗ a b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China Graduate School of the Chinese Academy of Sciences, Shanghai, China
a r t i c l e
i n f o
Article history: Received 10 January 2008 Received in revised form 8 April 2008 Accepted 8 April 2008 Available online 16 April 2008 Keywords: Toosendanin Primary rat hepatocytes Reactive oxygen species (ROS) MAP kinases
a b s t r a c t Toosendanin (Tsn), a triterpenoid extracted from Melia toosendan Sieb et Zucc, possesses different pharmacological effects in human and important values in agriculture. However, liver injury has been reported when toosendanin or Melia-family plants, which contain toosendanin are applied. The mechanism by which toosendanin induces liver injury remains largely unknown. Here we reported that toosendanin induced primary rat hepatocytes death by mitochondrial dysfunction and caspase activation. Toosendanin led to decrease of mitochondrial membrane potential, fall in intracellular ATP level, release of cytochrome c to cytoplasm, activation of caspase-8, 9, and 3 and ultimately cell death. Level of reactive oxygen species (ROS) was also increased in hepatocytes after incubation with toosendanin. Catalase, the H2 O2 -decomposing enzyme, can prevent the reduction in ATP level and protect hepatocytes from toosendanin-induced death. The ERK1/2 (p44/42 MAP kinases) and JNK (c-Jun N-terminal kinase) were activated, but p38 MAPK was not activated by toosendanin. Inhibition of ERK1/2 activation sensitized hepatocytes to death and increased activity of caspase-9 and 3 in response to toosendanin. Inhibition of JNK attenuated toosendanin-induced cell death. These results suggested that toosendanin causes death of primary rat hepatocytes by mitochondrial dysfunction and caspase activation. Generation of ROS and MAP kinases activation might be involved in this process. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Toosendanin (C30 H38 O11 , FW = 574), a triterpenoid extracted from Melia toosendan Sieb et Zucc as shown in Fig. 1, has been used as a digestive tract parasiticide and insecticide in traditional Chinese medicine (Chung et al., 1975; Shu and Liang, 1980; Shi and Li, 2007). It has been reported that toosendanin selectively blocks acetylcholine (ACh) release from nerve terminals and has antibotulismic role in vitro and in vivo (Shi and Chen, 1999; Zhou et al., 2003; Shi and Wang, 2004). Recently, toosendanin-induced apoptosis on various cancer cells was reported (Tang et al., 2004; Zhang et al., 2005). However, liver injury was reported when toosendanin or Melia-family plants which contain toosendanin were used in traditional Chinese medicine. High concentration of toosendanin was found in liver and the excretion rate was very slow after toosendanin administration in rhesus monkey (Zhou et al.,
∗ Corresponding author at: State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China. E-mail address: cdser [email protected] (J. Ren). 0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2008.04.005
1982). Toosendanin can cause increase in serum glutamic pyruvic transaminase (SGPT) level and liver morphological changes in rhesus monkey (Li et al., 1982). However, the mechanism of liver injury caused by toosendanin remains largely unknown. Excessive apoptosis of hepatocytes is one cause for liver injury especially in drug-induced liver damage (Lee, 2003; Guicciardi and Gores, 2005). The death receptor pathway and the mitochondrial pathway have been reported in apoptotic death of mammalian cells (Hengartner, 2000; Wang, 2001). It has been reported that toosendanin can induce cytochrome c release and caspase-3 activation in PC12 and cancer cells (Tang et al., 2004; Zhang et al., 2005). A crude extract from fruits of M. toosendan has been reported to regulate PC12 cell differentiation and induce activation of extracellular signal-regulated kinases (ERKs) (Yu et al., 2004). ERKs are one member of mitogen-activated protein kinase (MAPK) family and the most commonly studied MAPKs are ERKs, JNK and p38 MAPK (Chang and Karin, 2001). Activation of ERKs in hepatocytes is thought to act as a protective and anti-apoptosis factor (Bonni et al., 1999; Qiao et al., 2002; Czaja et al., 2003). In contrast, JNK and p38 activation generally have been shown to promote hepatocytes death (Cross et al., 2000; Kurata, 2000). Reactive oxygen species (ROS) such as superoxide anions, H2 O2 and hydroxyl rad-
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itive control (Hirata and Nagatsu, 2005). TMRE is a membrane-permeable cationic fluorophore that accumulates electrophoretically into mitochondria in response to their negative potential (Ehrenberg et al., 1988). Fluorescence readings were taken on a fluorimeter (NOVOstar, BMG LABTECH, Offenburg, Germany) with excitation wavelength at 540 nm and emission wavelength at 595 nm. 2.5. Assay of cellular ATP content Intracellular ATP levels were determined using CellTiter-GloTM Luminescent Cell Viability Assay kit according to the kit direction (Promega, Madison, WI, USA). Bioluminescence was measured by a fluorimeter (NOVOstar, BMG LABTECH). 2.6. Measurement of caspase-3/8/9 activity Caspase-3/8/9 activity was measured with BD ApoAlertTM Caspase Fluorescent Assay kits according to the kit direction (BD Biosciences Pharmingen, San Diego, CA, USA). Fluorescence was measured in a fluorimeter (NOVOstar, BMG LABTECH) with excitation at 380 nm and emission at 460 nm for caspase-9 assay or with excitation at 400 nm and emission at 505 nm for caspase-3 and 8. Fig. 1. The chemical structure of toosendanin.
icals are generated in many stressful situations and induce cell death by either apoptosis or necrosis (Kruidenier and Verspaget, 2002; Klaunig and Kamendulis, 2004). Moreover, ROS can induce MAPK activation by which hepatocytes mediate reaction to oxidative stress (Yamamoto et al., 1993; Wang et al., 1998). Our present study was designed to investigate the effect of toosendanin on primary rat hepatocytes, aiming to elucidate the mechanism of toosendanin hepatotoxicity. 2. Materials and methods
2.7. Measurement of ROS generation Generation of ROS was detected by DCFH-DA as described previously (Mattia et al., 1991). Cells were washed twice and suspended in PBS (1 × 106 cells/ml). Suspended cells were incubated with DCFH-DA (5 M) at 37 ◦ C for 30 min. H2 O2 was added as a positive control. Fluorescence intensity was measured by a fluorimeter (NOVOstar, BMG LABTECH) with excitation at 485 nm and emission at 520 nm. 2.8. Preparation of cytoplasmic extracts Hepatocytes were washed with ice-cold PBS (pH 7.4) and suspended in ice-cold HMKEE buffer (20 mM Hepes–KOH, 10 mM KCl, 1.5 mM MgCl2 , 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride, 10 g/ml pepstain A, and 10 g/ml leupeptin) containing 250 mM sucrose. Hepatocytes were homogenized by passage through a 26-gauge needle and centrifuged at 16,000 × g at 4 ◦ C for 15 min. The cytoplasmic supernatant was collected.
2.1. Chemicals 2.9. Western blot Toosendanin were purified from M. toosendan Sieb et Zucc with a purity over 98% (Provided by the Institute of Life Sciences, Donghua University, Shanghai, China). Toosendanin was dissolved in dimethyl sulfoxide (DMSO) and stored at −20 ◦ C before use. PD98059, SP600125 and catalase polyethylene glycol were purchased from Sigma (St. Louis, MO, USA). All other chemicals and solvents were purchased from Sigma and of analytical grade. 2.2. Animals and culture of primary rat hepatocytes Sprague–Dawley male rats (200 ± 30 g) were supplied by Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). The rats were housed in a cage under controlled conditions of temperature (20 ± 3 ◦ C) and humidity (45–65%) with a 12-h light:12-h dark cycle. Drinking water and food were provided ad libitum throughout the study. Primary rat hepatocytes were isolated from rats by two-step collagenase perfusion as described previously (Berry and Friend, 1969; Orrenius et al., 1976; Seglen, 1976) with some modifications. Collagenase IV was purchased from Sigma. Monolayer hepatocytes were cultured in Ham’s F-12/DMEM (Invitrogen, Carlsbad, CA, USA) (1:1) medium supplemented with 100 U/ml penicillin and 70 g/ml streptomycin at 37 ◦ C in a humidified atmosphere containing 5% CO2 . Cultural medium was changed 4–6 h after cell seeding to minimize contamination of death cells. Control cultures received vehicle solvent (0.1% DMSO). Some hepatocytes were pretreated with 20 M PD98059, 10 M SP600125 or 400 U/ml catalase polyethylene glycol for 1 h before incubation with toosendanin. 2.3. Cell viability assay After incubation with toosendanin and/or chemicals in primary rat hepatocytes, the cell viability was determined with the Cell Counting Kit-8 (Dojindo Laboratories, Tokyo, Japan). Visible wavelength absorbance data at 450 nm were collected using a standard 96-well plate reader (SOFTmax® PRO, Molecular Devices, Sunnyvale, CA, USA). 2.4. Measurement of mitochondrial membrane potential To monitor mitochondrial membrane potential, cultured hepatocytes were loaded with 500 nM tetramethylrhodamine ethylester (TMRE, Fluka, St. Gallen, Switzerland) in HBS solution (33 mM Hepes, 160.8 mM NaCl, 3.15 mM KCl, 0.7 mM Na2 HPO4 ·12H2 O, pH 7.65) at 37 ◦ C for 30 min, according to methods described before (Wu et al., 1990; Zahrebelski et al., 1995). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma), a mitochondrial toxin, was added as a pos-
Cytoplasmic extracts were subjected to 15% SDS-PAGE and transferred to PVDF membrane. The PVDF membrane was immunoblotted with monoclonal rat anti-cytochrome c (Lab Vision&NEOMARKERS, UK), GAPDH antibody (Santa Cruz Biotechnology, Inc.), cleaved Caspase-3 rabbit mAb, phospho-p44/42 (pERK1/2) MAP Kinase rabbit mAb, p44/42 (ERK1/2) MAP Kinase rabbit mAb, phosphoSAPK/JNK (pJNK) antibody, SAPK/JNK (JNK) antibody, phospho-p38 MAP Kinase rabbit mAb, p38 MAP Kinase rabbit mAb (Cell Signaling Technology Inc., Beverly, MA, USA). Antibodies were visualized using ECL chemiluminescent detection system (Amersham, Piscataway, NJ, USA) and Kodak Image Station (Kodak, Rochester, NY, USA). 2.10. Statistical analysis Data were entered into a database and analyzed using SPSS software. Group mean values and standard deviations were calculated. After homogenetic analysis, homogeneous data were analyzed with one-way analysis of variance and a post hoc test of least significant difference. Heterogeneous data were analyzed using t-test. P < 0.05 was considered significant.
3. Results 3.1. Cytotoxicity and caspase activation by toosendanin Cultured primary rat hepatocytes were exposed to toosendanin for 24 h in 96-well plate before cell viability determined by Cell Counting Kit-8 (Dojindo Laboratories). Toosendanin suppressed cell viability in a dose-dependent manner (Fig. 2). The concentration required for 50% inhibition of growth (IC50 value) was 14.94 ± 0.93 M (n = 3). Since caspase activation is the key event in apoptotic cell death, we assayed caspase-3/8/9 activation in primary rat hepatocytes incubated with toosendanin. Caspase-3 activity was increased significantly after toosendanin incubation in 16 and 24 h (Fig. 3A). Cleaved caspase-3, a marker of caspase-3 activation, was also detected by Western blot after 24 h toosendanin incubation (Fig. 3B). Caspase-9 activity was also increased significantly
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was confirmed after 4 and 8 h toosendanin incubation. Cellular ATP level, a sensitive parameter of mitochondrial function, were decreased at 5 and 10 M toosendanin incubation in 4 and 8 h (Fig. 4B). These results demonstrated that toosendanin caused mitochondrial dysfunction in primary rat hepatocytes. Incubation of the hepatocytes with 5 and 10 M toosendanin after 8 and 16 h, cytochrome c level in cytoplasmic fractions was analyzed by Western blot. Toosendanin induced a significant increase of cytochrome c level in cytoplasm after 16 h incubation (Fig. 4C). These results indicated that toosendanin causes damage to the integrity of mitochondrial membrane. 3.3. ROS generation and protective effect of catalase
Fig. 2. Cell viability of primary rat hepatocytes with toosendanin incubation for 24 h. All values are means ± S.D., n = 3.
after toosendanin incubation up to 16 h in primary rat hepatocytes (Fig. 3C). Caspase-8 activity was also increased with 5 and 10 M toosendanin in 4 h incubation (Fig. 3D). Together, these results suggest that toosendanin induced caspase activation to lead to primary rat hepatocytes death. 3.2. Mitochondrial dysfunction We then examined the mitochondrial membrane potential by determining the cellular retention of TMRE. As shown in Fig. 4A, a minor but marked decrease of TMRE retention in hepatocytes
Generation of ROS was determined after toosendanin incubation from 1 h and up to 6 h in primary rat hepatocytes (Fig. 5A). Pretreatment with catalase, primary rat hepatocytes were partially protected from toosendanin-induced death (Fig. 5B). Moreover, catalase also alleviated reduction in ATP level caused by toosendanin (Fig. 5C). Thiol anti-oxidant such as N-acetyl-cystein (NAC) and dithiothreitol (DTT) did not show protective effect (data not shown). These results suggested that toosendanin can cause oxidative stress and the cellular injury might be originated from H2 O2 . 3.4. Role of MAPK in toosendanin cytotoxicity We then studied whether toosendanin affect activation of ERK1/2. As shown in Fig. 6A, phosphorylation of ERK1/2 was enhanced. Phosphorylation of JNK was also observed within 1 h of toosendanin incubation (Fig. 6B). Phosphorylated p38 level, another member of MAPK, was not activated by toosendanin (data not shown).
Fig. 3. Caspase activation induced by toosendanin (Tsn). (A) Caspase-3 activity with toosendanin incubation up to 24 h. * P < 0.05 significantly different from the “control” group at each time point. (B) Cleaved caspase-3 levels with 24 h toosendanin incubation. The results are representatives of three separated experiments. (C) Caspase-9 activity was examined with 5 and 10 M toosendanin incubation at different time points. * P < 0.05 significantly different from the “control” group at each time point. (D) Caspase-8 activity was determined with 5 and 10 M toosendanin incubation up to 8 h. * P < 0.05 significantly different from the “control” group at each time point. Activity of caspase-3/8/9 is depicted as the value of fluorescence vs. protein content. All values are means ± S.D., n = 3.
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Fig. 4. Mitochondrial damage caused by toosendanin (Tsn). (A) Decrease of mitochondrial membrane potential with toosendanin incubation up to 8 h. * P < 0.05 significantly different from the “Tsn 0 M” group at each time point. CCCP was added as a positive control. (B) Cellular ATP contents were assayed with toosendanin incubation up to 8 h. * P < 0.05 significantly different from the “control” group at each time point. (C) Cytochrome c levels in cytoplasmic extracts induced by toosendanin up to 16 h. Results are representatives of three separated experiments. All values are means ± S.D., n = 3.
PD98059 is a highly specific inhibitor for MEK1/2, the upstream kinase of ERK1/2 (Dudley et al., 1995; Alessi et al., 1995). PD98059 enhanced toosendanin-induced hepatocytes death in 24 h and increased caspase-3 activity in 16 and 24 h (Fig. 7A and B). After PD98059 pretreatment, caspase-9 activity was increased after 16 h with 10 M toosendanin incubation (Fig. 7C). PD98059 treatment by itself neither caused cell death nor affected caspase-3/9 activity (data not shown). These results indicated that ERK1/2 activity mediated protective effects against toosendanin-induced hepatocytes death.
Fig. 5. Oxidative damages caused by toosendanin (Tsn) and protective effect of catalase. (A) Generation of reactive oxygen species (ROS) by toosendanin incubation up to 6 h. * P < 0.05 significantly different from the “Tsn 0 M” group at each time point. H2 O2 was added as a positive control. (B) Cell viability with catalase pretreatment after toosendanin incubation in 24 h. * P < 0.05 significantly different from the “Tsn” group. (C) ATP level with catalase pretreatment after 4 h toosendanin incubation. * P < 0.05 significantly different from the “Tsn” group. All values are means ± S.D., n = 3.
SP600125 is a specific JNK inhibitor (Bennett et al., 2001). Pretreatment with 10 M SP600125, the cell viability of primary rat hepatocytes was increased in the presence of toosendanin (Fig. 8A). Meanwhile, caspase-3 activation induced by toosendanin was decreased in 16 h (Fig. 8B). SP600125 treatment by itself nei-
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Fig. 6. MAP kinases activation induced by toosendanin (Tsn). (A) Phosphorylated ERK1/2 (pERK1/2) and total ERK1/2 levels were detected with toosendanin incubation. (B) Phosphorylated JNK (pJNK) and total JNK levels were detected with toosendanin incubation. Results are representatives of three separated experiments.
ther caused cell death nor affected caspase-3 activity (data not shown). These results indicated that activation of JNK was involved in toosendanin cytotoxicity. 4. Discussion In the present study, we first showed that toosendanin caused primary rat hepatocytes death by mitochondrial dysfunction and caspase activation. ROS and MAP kinases are involved in this process. Toosendanin led to mitochondrial damages including decrease of mitochondrial membrane potential, fall of ATP level, and release of cytochrome c to cytoplasm. Caspases, including caspase-8, 9 and 3 all were activated after toosendanin incubation. Toosendanin caused cellular ROS level increase and catalase can partially protect hepatocytes from toosendanin-induced death. Studies with MEK1/2 chemical inhibitor PD98059 showed that ERK1/2 activation mediated hepatocytes resistance to toosendanin cytotoxicity. In contrast, JNK inhibition by SP600125 showed that JNK played a pro-death role in toosendanin-induced hepatocytes injury. These results showed that toosendanin has cytotoxic effect on primary rat hepatocytes and the putative mechanisms showed as Fig. 9. Mitochondrial dysfunction such as fall in intracellular ATP level can lead to necrotic or apoptotic hepatocellular death (Nieminen et al., 1994; Qian et al., 1997). In our studies, toosendanin induced fall of ATP level and decrease of mitochondrial membrane poten-
Fig. 7. Effects of ERK1/2 activation on toosendanin (Tsn)-induced hepatocytes death. (A) Cell viability after 24 h toosendanin incubation with 20 M PD98059 (PD) pretreatment. * P < 0.05 significantly different from the “Tsn” group. (B) Caspase3 activity in hepatocytes pretreatment with 20 M PD98059 (PD) after 16 and 24 h toosendanin incubation. * P < 0.05 significantly different from the “Tsn 10 M” group. (C) Caspase-9 activity after 8 and 16 h of toosendanin incubation with 20 M PD98059 (PD) pretreatment. * P < 0.05 significantly different from the “Tsn 10 M” group. All values are means ± S.D., n = 3.
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Fig. 8. Effects of JNK activation on toosendanin (Tsn)-induced hepatocytes death. (A) Cell viability after 24 h toosendanin incubation with 10 M SP600125 (SP) pretreatment. * P < 0.05 significantly different from the “Tsn” group. (B) Caspase-3 activity after 16 and 24 h of toosendanin incubation with 10 M SP600125 (SP) pretreatment. * P < 0.05 significantly different from the“Tsn 10 M” group at 16 h. All values are means ± S.D., n = 3.
Fig. 9. A scheme of the putative mechanisms involved in the toosendanin-induced primary rat hepatocytes death.
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tial (Fig. 4A and B). These results indicated that toosendanin causes mitochondrial injury. Damage of mitochondrial membrane integrity can result in cytochrome c release from mitochondria to cytoplasm, which is a key event in mitochondrial apoptosis pathway (Liu et al., 1996; Wang, 2001). We detected release of large amount of cytochrome c into cytoplasm and increase in caspase-9 activity after toosendanin incubation (Figs. 3C and 4C). Moreover, caspase-3 activity was greatly increased at 16 and 24 h (Fig. 3A), our results thus suggested that involvement of mitochondrial apoptosis pathway in toosendanin-induced hepatocytes death. Caspase-8 is the most upstream caspase in death receptor apoptosis pathway (Boldin et al., 1996; Nicholson and Thornberry, 1997; Cohen, 1997). The fact that toosendanin induced caspase-8 activation indicated involvement of death receptor apoptosis pathway in toosendanin-induced hepatocytes death (Fig. 3D). Excessive apoptosis of hepatocytes caused by drugs can result in druginduced liver injury. Therefore, toosendanin-induced hepatocytes death may contribute to the liver injury caused by toosendanin as reported before (Li et al., 1982). In our study, toosendanin caused ROS level increase up to 6 h in primary rat hepatocytes (Fig. 5A). Catalase, the H2 O2 -decomposing enzyme, can increase ATP level and partially protect hepatocytes from toosendanin-induced death (Fig. 5B and C). These results indicated oxidative stress caused by toosendanin led to mitochondrial dysfunction and cells injury. Anti-oxidants including enzymatic or non-enzymatic can protect cells from injury caused by ROS which can lead cells to apoptosis (Mates, 2000; Czaja et al., 2003). We found that thiol anti-oxidants such as NAC or DTT did not attenuate toosendanin-induced hepatocytes death. The effectiveness of catalase but not thiol anti-oxidants suggested that oxidative stress from toosendanin-induction might be cellular injury originated in H2 O2 . However, the nature and source of ROS generated by toosendanin need further investigation. It has been shown that extracts from fruits of M. toosendan caused ERK1/2 activation and regulated PC12 cell differentiation (Yu et al., 2004). In our study, we showed that toosendanin caused ERK1/2 and JNK activation in primary rat hepatocytes (Fig. 6A and B), whereas did not activate p38 MAPK. ERK1/2 activation is anti-apoptotic (Bonni et al., 1999; Qiao et al., 2002). JNK and p38 MAPK activation generally have been shown to promote hepatocytes death (Cross et al., 2000; Kurata, 2000). We found that inhibition of ERK1/2 activation by PD98059 sensitized hepatocytes to toosendanin-induced death (Fig. 7A). Besides, activation of caspase-3 and 9 induced by toosendanin was increased when hepatocytes were pre-treated with PD98059 (Fig. 7B and C). These results indicated that ERK1/2 activation induced by toosendanin mediated protective and anti-apoptotic effect in primary rat hepatocytes. However, ERK1/2 activation is not sufficient to prevent toosendanin cytotoxicity since pro-apoptotic pathway, especially JNK, also was activated. Inhibition of JNK activation by SP600125 attenuated hepatocytes death and decreased caspase-3 activation induced by toosendanin (Fig. 8A and B). These results showed that JNK played a pro-death role in toosendanin-induced hepatocytes injury. Toosendanin increased ROS level and exposure to ROS may lead to activation of cell signal transduction pathway including MAP kinases pathway (Yamamoto et al., 1993; Chang and Karin, 2001). However, the relationship between generation of ROS and MAP kinases activation induced by toosendanin need further studies. In our study, at 24 h the toosendanin concentration in our condition is close to that in vitro test for cancer cell growth inhibition as reported before (Zhang et al., 2005). Since Melia-family plants are still used in traditional Chinese medicine, toosendanin might be served as a candidate for drug research and liver function should be observed closely when toosendanin is administrated.
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