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2 activation in human cells
Toxicology in Vitro 25 (2011) 810–816
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Aristolochic acid I induced oxidative DNA damage associated with glutathione depletion and ERK1/2 activation in human cells Feng-Yih Yu a, Ting-Shuan Wu a,1, Ting-Wei Chen a, Biing-Hui Liu a,b,⇑ a b
Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan
a r t i c l e
i n f o
Article history: Received 5 August 2010 Accepted 28 January 2011 Available online 12 February 2011 Keywords: Aristolochic acid Oxidative stress DNA damage GSH depletion ERK1/2 activation
a b s t r a c t Aristolochic acid I (AAI) has been widely found in herbal remedies and linked to the development of nephropathy and urothelial carcinoma in humans. This study elucidated the mechanism of oxidative stress and DNA damage mediated by AAI in human cells. Treatment of human promyelocytic leukemia cells (HL-60) and human renal proximal tubular cells (HK-2) with AAI led to a dose-dependent increase of reactive oxygen species (ROS). AAI also elevated the levels of DNA strand breaks and 8-hydroxy guanosine in HL-60 and HK-2 cells. Antioxidants, including Tiron, N-acetyl-L-cysteine (NAC) and glutathione (GSH), effectively suppressed the AAI-induced ROS and AAI-elicited genotoxicity, indicating that AAI induced the DNA damage through oxidative stress. GSH depletion was also found in AAI-treated cultures and proceeded prior to ROS formation. Exposure of HL-60 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only MEK1/2 inhibitor, U0126, significantly decreased AAI-mediated ROS. Preincubation of cells with thiol-containing compounds (NAC and GSH) inhibited the caspase 3 activity triggered by AAI, but non-thiol Tiron did not show a similar effect. This study demonstrated that AAI treatment results in oxidative stress-related DNA damage through GSH depletion and ERK1/2 activation; AAI-induced apoptosis is associated with GSH loss, but is independent of ROS generation. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Aristolochic acid (AA), found in herbal extracts of various species of genus Aristolochia, is a natural mixture composed of aristolochic acid I (AAI) and aristolochic acid II at a ratio of approximately 1:1 (Li et al., 2004). Several Aristolochia species have been widely used in traditional Chinese medicine as anti-rheumatics, as diuretics and in the treatment of oedema for thousand years, but AA is now suspected of causing nephropathy (Cosyns et al., 1994). Consumption of herbal medicines containing AA has been linked to Chinese herb nephropathy (CHN)/aristolochic acid nephropathy (AAN), a progressive interstitial nephritis leading to end-stage renal disease and urothelial malignancy (Debelle et al., 2008). CHN/ AAN was originally reported in Belgium, with over 100 cases of nephropathy linked to the use of a slimming regimen containing Chinese herbs (Vanherweghem et al., 1993); a follow-up investigation of CHN/AAN patients revealed a high risk for the development of urothelial carcinomas (Nortier et al., 2000; Lord et al., 2001). ⇑ Corresponding author at: Department of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung 402, Taiwan. Tel.: +886 4 24730022x11815; fax: +886 4 24757412. E-mail address: [email protected] (B.-H. Liu). 1 Equal contribution to the first author. 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.01.016
Chronic exposure to AA is also considered a risk factor relating to Balkan endemic nephropathy and its associated cancer (Grollman et al., 2007). Therefore, mixtures of AA have been classified as probably carcinogenic to humans (Group 2A) and herbal remedies containing Aristolochia species are carcinogenic to humans (Group 1) (IARC, 2002). The mutagenetic and carcinogenic effects of AA are generally believed to be associated with the formation of AA–DNA adducts in both AA patients and experimental animals (Grollman et al., 2007; Schmeiser et al., 2009). Metabolic activation of AAI to Nhydroxyaristolactam I results in the formation of DNA adducts which may be responsible for the transversion mutation A ? T in ras and p53 genes (Schmeiser et al., 1990; Slade et al., 2009). AA is also a direct mutagen in Salmonella typhimurium (Gotzl and Schimmer, 1993); micronucleus induction, DNA breakage, sister chromatid exchange and nitrative DNA modification were observed in various mammalian cells (Abel and Schimmer, 1983; Li et al., 2006; Wu et al., 2007). Additionally, AA nephrotoxicity is reported to be linked with AA-induced apoptosis, which could be caused by endoplasmic reticulum and mitochondrial stress (Hsin et al., 2006; Wang and Zhang, 2008). Reactive oxygen species (ROS) are regarded as a natural byproduct of regular oxygen metabolism in aerobic organisms and is possibly induced by many environmental factors including ionizing
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radiation and toxic chemicals. Irrespective of their origin, ROS can interact with cellular biomolecules such as DNA and, under certain circumstances, act as subcellular messengers in signal transduction and gene regulation pathways (Cooke et al., 2003). Both the mitogen-activated protein kinase (MAPK) and NF-jB signaling pathways are considered to be oxidative stress sensitive (Milligan et al., 1998; Cakir and Ballinger, 2005). In the present study, we applied human cells as models to investigate whether AAI, in addition to forming DNA adducts, is able to damage DNA through the stimulation of ROS generation. Furthermore, the mechanism of AAI-induced oxidative DNA damage was elucidated. 2. Materials and methods 2.1. Reagents Cell culture media and serum were obtained from Life Technologies (Grand Island, NY). MEK1/2 inhibitor U0126, p38 pathway inhibitor SB203580 and polyclonal rabbit antibodies against phospho-p38 kinase, phospho-ERK1/2 (Thr202/ Tyr204) and parent ERK1/2 were purchased from Cell Signaling (Beverly, MA). 20 ,70 Dichlorodihydro-fluorescein diacetate (H2DCF-DA) was obtained from Molecular Probes (Eugene, OR). AAI (aristolochic acid I), Tiron (4,5-dihydroxy-1.3-benzenedisulfonic acid disodium salt), NAC (N-acetyl-L-cysteine) and GSH (glutathione) were from Sigma Chemical (St. Louis, MO). AAI was dissolved in DMSO at a concentration of 25 mM and stored at 20 °C as a stock solution. 2.2. Cell culture Human promyelocytic leukemia (HL-60) and human renal proximal tubular (HK-2) cell lines were obtained from Bioresources Collection and Research Center in Taiwan and then cultured in RPMI 1640 medium and DMEM/Hams F12 medium, respectively. Both the medium were supplemented with 10% fetal bovine serum (GIBCO Invitrogen), 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 °C in a humidified 5% CO2 incubator. HL-60 cultures were established by centrifugation with subsequent resuspension at 1 105 viable cells/ml. Cell density was maintained between 1 105 and 1 106 cells/ml. HK-2 cells were anchorage dependent and were subcultivated at 80% confluence with a split ratio of 1:4. 2.3. Measurement of ROS in cells with oxidation-sensitive fluorescent dyes Cells (104/per well on a 96-well tissue culture plate) were cultured for at least 24 h in 50 ll of complete medium, and then 50 ll of H2DCF-DA (20 lM) in Krebs–Ringer HEPES (KRH) buffer was added. Thirty minutes after incubation, the probe-containing medium was removed and AAI (25 to 200 lM) in KRH buffer were added to the cells and incubated for another 1 h. Since H2DCF-DA is hydrolyzed and oxidized by intracellular ROS to 2,7-dichlorofluorescein (DCF), the fluorescence of DCF was analyzed in an HTS 7000 Bio Assay Fluorescent Plate Reader (PerkinElmer life Sciences) at an excitation wavelength of 485 nm and fluorescence emission was measured at 530 nm. To evaluate the intracellular ROS levels by flow cytometry, HL60 cells in complete medium were incubated with H2DCF-DA at a final concentration of 10 lM for 30 min. After centrifugation at 1000g for 5 min, the culture medium were replaced with either solvent or various concentrations of AAI in phosphate-buffered saline (PBS) solution, and then incubated at 37 °C for another 1 h before flow cytometry. The oxidized forms of ROS-sensitive dye were excited with a 488 nm laser (Becton–Dickinson FACSalibur flow
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cytometer, Franklin Lakes, NJ); emission of DCF fluorescence was quantified in channel FL-1 (530-nm filter, 30-nm bandpass). 2.4. Single cell gel electrophoresis (SCGE) assays Briefly, cells were cultured at 37 °C in medium containing vehicle alone (2 ll or 4 ll DMSO/ml medium) or AAI (50–100 lM) for 24 h. The adherent cells were then trypsinized and mixed with 1% low-melting-point agarose at 42 °C. The mixtures were immediately transferred to the CometSlide™ (Trevigen Inc., Gaithersburg, MD) and the slides were immersed in ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris, 1% sodium lauryl sarcosinate, 1% Triton X-100 and 1% DMSO) for 1 h. After electrophoresis in an alkaline buffer (300 mM NaOH, 1 mM EDTA, pH > 13) at 300 mA for 30 min, DNA on the slides was stained with SYBR green I. The image of each cell on the slides was visualized and photographed using a fluorescence microscope (Axiovert 25, Zeiss). The images were analyzed with the computer software from Euclid Analysis (St. Louis, MO) to calculate the tail moment, a combination of the amount of DNA in the tail with the distance of migration. For each independent experiment, the DNA damage level of 80 individual cells was measured from each culture. 2.5. Detection of 8-hydroxy-2-deoxy guanosine (8-OH-dG) by flow cytometry The levels of 8-OH-dG were measured by using the OxyDNA assay Kit (Calbiochem, San Diego, CA). HL-60 cells (1 106 cells/well) were seeded in 35-mm tissue culture dishes and treated with 0–200 lM of AAI for 24 h or 1 mM H2O2 for 1 h. The cells were harvested, rinsed with PBS, and fixed with 1% paraformaldehyde on ice for 15 min. After centrifugation to remove paraformaldehyde, the cells were kept in ice-cold 70% ethanol at 20 °C for 18 h. According to the manufacture’s protocol, the fixed cells were incubated with the FITC-conjugate solution for 1 h at room temperature and then fluorescence was read using a flow cytometer at an excitation wavelength of 495 nm and barrier filter of 515 nm. 2.6. Western blot analysis HL-60 cells at 80% saturation density were pretreated with antioxidants (5 mM Tiron, 10 mM NAC, or 1 mM GSH) for 2 h or with MAPK inhibitors (10 lM U0126/10 lM SB203580) for 30 min before co-exposure to AAI. Whole cell extracts were prepared according to Liu et al. (2007). The protein concentrations in each sample were determined using the Bradford protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard. Equal amounts of protein (40 lg) from each sample preparation were separated on SDS–PAGE and subjected to Western blotting. Polyclonal antibodies specific to phospho-ERK1/2 (Thr202/ Tyr204), ERK1/2, phospho-p38 or p38 were used as the probes. Bound antibody on the membrane was detected using an enhanced chemiluminescence detection system according to the manufacturer’s manual (Amersham Pharmacia Biotech, Amersham, UK). 2.7. Determination of GSH levels HL-60 cells (1 106 cells/well) were pretreated with or without antioxidants (5 mM Tiron or 10 mM NAC) for 2 h before co-exposure to 200 lM AAI or DMSO (4 ll/ml medium) for another 15 or 30 min. Cells were harvested and subjected to GSH–Glo glutathione assay according to the manufacture’s protocol (Promega). The assay is based on the conversion of luciferin derivative into luciferin in the presence of GSH, catalyzed by glutathione S-transferase. The value of luminescent signal in each treatment was
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calculated according to the standard curve generated in the presence of pure GSH. 2.8. Measurement of caspase-3 catalytic activities Cells were seeded in 24-well plates (7 105 cells/well) and then incubated with vehicle or various concentrations of AAI (0–500 lM) for 24 h. Treated cells were washed with 0.01 M PBS and resuspended in 100 ll of caspase assay buffer containing 0.1 M HEPES (pH 7.4), 2 mM DTT, 0.1% CHAPS and 1% sucrose. The cell suspension was sonicated and the cell lysate was centrifuged (14,000g) at 4 °C for 15 min. Fifty microliters of supernatant fluid was diluted with 450 ll of assay buffer containing 20 lM Ac-DEVD-AFC (Sigma), a substrate for active caspase-3. After incubation at 37 °C for 1 h, the levels of cleaved products were measured using a HTS 7000 PLUS bioassay reader (PerkinElmer, Boston, MA) with excitation set at 405 nm and emission at 535 nm. 2.9. Statistical analysis All statistical analyses were conducted using the software GraphPad Prism Version 4.0 (GraphPad Software, San Diego, CA). Experimental data of Figs. 1–3 had passed the Kolmogorov–Smirnov normality test and were analyzed by one-way ANOVA followed by Tukey post test. The data of Figs. 4 and 5 were analyzed by unpaired two-tailed Student’s t-test.
3. Results 3.1. Generation of intracellular ROS after AAI treatment To determine the AAI concentrations suitable for studying ROS formation, the cell viability of HL-60 and HK-2 in the presence of AAI was examined by the tetrazolium dye-based MTS assay. The cell viability of HL-60 declined to 80% of control after 24 h exposure to 200 lM AAI, but no statistically significant difference was observed while AAI at concentrations up to 100 lM. On the other hand, incubation of HK-2 cells with 100 and 200 lM AAI decreased the cell viability to 71% and 55% of control after 24 h exposure, respectively. The intracellular ROS levels in HL-60 and HK-2 cells were evaluated based on fluorescence spectrophotometry using H2DCF-DA as the probe. Various oxidants, especially hydroxyl radical (OH) and peroxynitrite (ONOO), are able to oxidize intracellular H2DCF to fluorescent DCF (Setsukinai et al., 2003). Based on the cell viability in the MTS assay, both HL-60 and HK-2 cells were exposed to AAI ranging from 25–200 lM. Following treatment of HL-60 with AAI for 1 h, DCF fluorescence increased in a concentrationdependent manner (Fig. 1A); AAI at 100 and 200 lM significantly increased the fluorescence to 5.0 and 6.9 folds of control levels, respectively. On the other hand, treatment of HK-2 cells with 200 lM AAI also dramatically enhanced ROS levels. Since HL-60 cells are more sensitive to AAI exposure comparing to HK-2, we mainly used HL-60 cultures to study the AAI-induced oxidative stress in the following experiments Furthermore, by applying flow cytometry, HL-60 cultures stimulated with 50 and 200 lM of AAI for 1 h elevated the intracellular ROS levels to 2.4 and 56.7-folds of the control group, respectively (Fig. 1B). The fluorescence level of H2O2-treated cultures was 16.9-fold higher as compared with the solvent-treated group; H2O2 is a potent genotoxic reagent and regarded as a positive control. Additionally, when cells were pre-incubated with Tiron, NAC, or GSH before co-exposure to AAI, either free radical scavenger markedly inhibited AAI-induced ROS (Fig. 1C). All of these data
Fig. 1. AAI increased intracellular ROS levels. (A) HL-60 and HK-2 cells were incubated with H2DCF-DA and then exposed to solvent or AAI for 1 h. DCF fluorescence was determined by fluorescence spectrophotometry. (B) HL-60 cells were incubated with H2DCF-DA, and then treated with solvent, AAI or H2O2 for 1 h before subjected to flow cytometry. (C) HL-60 cells were pre-treated or not with Tiron (5 mM), NAC (10 mM) or GSH (1 mM) for 2 h, incubated with H2DCF-DA, and then with AAI for 1 h. DCF fluorescence was measured by fluorescence spectrophotometry. All the data are the mean ± SEM from five independent experiments. ⁄ Significant difference (⁄p < 0.05; ⁄p < 0.01) compared to the solvent-treated control or the paired group.
indicate that AAI is able to trigger the ROS production in human cells. 3.2. AAI induced oxidative DNA damage We further applied the SCGE assay and flow cytometry to investigate whether ROS induced by AAI contributed to oxidative DNA damage. In the SCGE assay, the tail shape and migration pattern of DNA reveals the level of DNA single/double strand breakage on a single cell basis (Klaude et al., 1996). Treating HK-2 cells with 50 and 100 lM of AAI significantly increased the tail moment to 3.9 and 5.5-folds of control levels, respectively (Fig. 2A). Similarly, exposure of HL-60 cells to AAI (50 and 100 lM) led to a significant and concentration-dependent increase in the tail moment values. Moreover, Tiron, NAC and GSH effectively suppressed the
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Fig. 3. AAI treatment induced the formation of 8-OH-dG. HL-60 cells were exposed to solvent (control) and AAI (50 and 200 lM) for 24 h or to H2O2 (1 mM) for 1 h. Collected cells were fixed and incubated with FITC-conjugate solution for 1 h and then fluorescence was read using a flow cytometer at an excitation wavelength of 495 nm and barrier filter of 515 nm. The representative chromatogram of flow cytometry is shown in (A) and data collected from three independent experiments were statistically analyzed in (B). Fig. 2. AAI induced oxidative DNA damage in HK-2 and HL-60 cultures. (A) HK-2 and HL-60 cells were incubated with various concentrations of AAI for 1 h. The values of tail moment were determined by SCGE assay. (B) HL-60 cells were pretreated for 2 h with or without Tiron (5 mM), NAC (10 mM) or GSH (1 mM) and then co-incubated with 100 lM AAI for another 1 h before SCGE assay. Images visualized and photographed by epifluorescence microscope were analyzed with the computer software to calculate tail moment values. All the data are the mean ± SEM from five independent experiments. ⁄Significant difference (⁄p < 0.05; ⁄⁄ p < 0.01; ⁄⁄⁄p < 0.001) compared to the control groups.
the phenomenon of GSH depletion was detected as early as 30 min incubation with AAI, while the level of ROS generation was not significant until 1 h treatment. In addition, inhibition of ROS formation by Tiron cannot reverse AAI-induced GSH depletion, revealing that GSH depletion proceeds prior to ROS generation.
3.4. ERK1/2 played a role in AAI-triggered ROS AAI-induced tail moment value to 65%, 46% and 41%, respectively, of the AAI along-treated group (Fig. 2B), strongly suggesting that DNA breakage after treatment of AAI is partially caused by ROS formation. 8-Hydroxy-2’-deoxyguanosine (8-OH-dG) is one of the most abundant oxidative products of cellular DNA, so the levels of 8OH-dG in solvent or AAI-treated cells were evaluated by flow cytometry. As shown in Fig. 3A, both AAI and H2O2 stimulated the generation of 8-OH-dG in HL-60 cells. Incubation of cells with 200 lM AAI for 24 h significantly increased the 8-OH-dG level to 1.6-folds of the solvent-treated group (Fig. 3B). 3.3. Involvement of GSH depletion in AAI-induced ROS generation The intracellular GSH level is generally considered to be closely correlated with ROS generation (Hayes and McLellan, 1999). Therefore, to investigate the role of GSH in AAI-induced ROS, HL-60 cells were treated with antioxidants and/or AAI before determination of the total GSH content. After 30 min of exposure, the GSH level dropped to 55% of control group in 200 lM-AAI exposed cultures (Table 1). However, no significant GSH depletion was found after a 15 min treatment. Furthermore, the GSH depletion caused by 200 lM AAI could be effectively rescued by the pre-incubation of cells with antioxidant NAC, a precursor of GSH, but antioxidant Tiron did not show any effect on the decreased GSH content. Overall,
Many studies have demonstrated that oxidative stress influences the MAPK signaling pathways (McCubrey et al., 2006). Therefore, this study first evaluated whether AAI can activate ERK1/2 or p38 pathways in HL-60 cells. Exposure of cells to 100 lM AAI resulted in both ERK1/2 and p38 phosphorylation. Pretreatment of cultures with either U0126, a specific MEK1/2 inhibitor, or SB203580, a p38 pathway blocker, dramatically decreased the phospho-ERK1/2 and phospho-p38 levels, respectively (Fig. 4A). Moreover, preincubation of cells with U0126, but not SB203580, significantly suppressed the levels of DCF fluorescence triggered by 100 lM AAI (Fig. 4B), suggesting that ERK1/2 signaling, but not the p38 pathway, partially contributes to AAI-induced ROS generation.
3.5. GSH depletion, but not ROS generation, involved in AAI-mediated apoptosis According to Fig. 5, treatment of HL-60 with 500 lM AAI elevated the caspase-3 catalytic activity to 11.6 folds of the control level. Therefore, the role of ROS in AAI-mediated apoptosis was further examined by applying antioxidants to the cultures. NAC and GSH partially reduced the AAI-elicited caspase 3 activity to 57.2% and 39.6%, respectively, of that in AAI-exposed cells. However, Tiron did not display a similar effect. It seems that intracellu-
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F.-Y. Yu et al. / Toxicology in Vitro 25 (2011) 810–816 Table 1 GSH levels in AAI-treated HL-60 cells. a
GSH content (% of control)
Treatment
15 min
30 min
AAI 50 lM AAI 100 lM AAI 200 lM b AAI (200 lM) + Tiron (5 mM) AAI (200 lM) + NAC (10 mM)
– 86.3 ± 6.9 100.7 ± 15.8 – –
79.2 ± 15.6 70.8 ± 10.2 54.9 ± 10.5* 57.2 ± 9.8* 107.2 ± 15.1
a HL-60 cultures were incubated for 15 or 30 min with AA before determination of intracellular GSH levels. Values are means ± SEM of at least three independent experiments in which each concentration is conducted in triplicate. b HL-60 cultures were pre-incubated with Tiron or NAC for 2 h and then cotreated with 200 lM AA for another 30 min before GSH determination. * Significant difference (⁄p < 0.05) compared to the solvent-treated control
4. Discussion
Fig. 4. Effects of MAPK inhibitors on AAI-induced MAPK phosphorylation (A) and ROS generation (B). (A) HL-60 cells were pretreated for 1 h with or without 10 lM U0126/10 lM SB203580, and then co-incubated with 100 lM AAI for 24 h. Whole cell extracts were subjected to Western blotting using antibodies specific to phospho-ERK/ERK or phospho-p38/p38. In (B), cells were left untreated or treated with U0126/SB203580 for 1 h and then the medium were replaced with fresh medium containing 10 lM H2DCF-DA for 30 min. Finally 100 lM AAI was added for another 1 h. The levels of DCF fluorescence were measured by fluorocytometry and the data are expressed as the mean ± SEM from four independent experiments.
Fig. 5. Effects of antioxidants on AAI-induced apoptosis. HL-60 cells were treated or left untreated with various antioxidants for 2 h before co-incubated with vehicle or AAI for 24 h. Total cellular proteins were extracted and the caspase 3 activity was determined as described in Section 2. The data from five independent experiments are expressed as the mean ± SEM.
lar GSH depletion, but not ROS formation, is closely associated with the AAI-triggered apoptotic process.
Many European countries have banned various herbs containing AA since 1994. The Food and Drug Administration of the United States also issued a Consumer Advisory to warn consumers against purchasing botanic products containing AA on 2001 (Schwetz, 2001). However, Owing to the difficulties in distinguishing between the appearance and botanical names of similar species, the risk of mistaken use of AA-containing herb in traditional medicine is still high (Gold and Slone, 2003). A previous survey shows that 50% of Chinese herbal remedies randomly collected from Taiwan markets were found to contain various concentrations of AAI, ranging from 0.17 to 655 ppm (lg/g) (Yu et al., 2006). A long term exposure to AA containing remedies could lead to a major public health concern. AAI at a sub-lethal concentration of 50 lM could induce ROS generation in HL-60 and HK-2 cells (Fig. 1A). Although AAI is considered as a nephrotoxic compound, HL-60 cells seem to be more vulnerable to the toxin-induced oxidative stress compared with HK-2 and human embryonic kidney (HEK293) cells (data not shown). A similar result was also documented that with the renal tubular epithelial Madin–Darby canine kidney (MDCK) cell line as a model, ROS levels just elevated to around two folds after stimulation with 75 lM AAI for 4 h (Liu et al., 2009). In addition to ROS induction, AAI triggered similar levels of DNA strand breakage and damage in both HK-2 and HL-60 cells using the SCGE assay (Fig 2A). Since AAI triggered ROS production as well as DNA damage in both cell lines, HL-60 was chosen to further study the mechanism of AAI-induced oxidative stress because of its sensitivity, ease of handling (suspension culture) and frequent use in experiments associated with ROS induction. Formation of 8-OH-dG, a reliable marker for oxidative DNA damage in vitro and in vivo, provided further evidence of AAI-induced ROS (Fig. 3). Unrepaired 8-OH-dG in DNA may induce G:C to T:A transversion at the modified site, and apparently plays a major role in mutagenesis and carcinogenesis (Moriya and Grollman, 1993; Le Page et al., 1995). Our findings based on flow cytometry are consistent with those of Wu et al. (2007) who applied immunoperoxidative staining to show the 8-OH-dG formation in AAIexposed HepG2 cells; they concluded that the AAI-induced 8-OH-dG is nitrative DNA damage which was attributed to reactive nitrogen species (RNS), such as NO and ONOO–. Since NO may rapidly interact with ROS, especially O2, to form the stable ONOO anion (McCall et al., 1989; Beckman et al., 1990), it is extremely difficult to distinguish between the contribution of ROS and RNS to AAI-induced DNA damage. Exposure of HL-60 cells to 100 lM AAI led to the phosphorylation of ERK1/2 and p38 MAP kinases (Fig. 4A). However, no phosphorylated JNK signal was observed after AAI treatment (data not shown). Furthermore, AAI-induced ROS is selectively blocked by
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Acknowledgments The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research under Contract No. NSC 96-2313-B- 040-004-MY3. Flow cytometry was performed in the Instrument Center of Chung Shan Medical University, which is supported by the National Science Council, Ministry of Education and Chung Shan Medical University. Fig. 6. A proposed model for AAI-induced oxidative stress in HL-60 cells.
inhibition of the ERK1/2 pathway, but not p38 kinase (Fig. 4B), which indicates its dependence on MEK/ERK1/2 activation. Several studies have mentioned that under certain circumstances, MEK/ ERK1/2 signaling plays a pivotal role in ROS induction, but the exact molecular mechanism has not been fully elucidated (Woo et al., 2002; Liu et al., 2007). Intracellular GSH is known to be involved in the process of ROS toxicity. Many studies have indicated that unstable ROS molecules after exposure to stimulators can be eliminated by interacting with the cysteinyl moiety of GSH, and then the phenomenon of GSH depletion is subsequently observed (Armstrong et al., 2002). In contrast, our data support another hypothesis that GSH loss may happen before ROS formation, or even independent of ROS generation (Barhoumi and Burghardt, 1996; Franco et al., 2007). Treatment with 200 lM AAI for 30 min significantly reduced the GSH level to 50% of control (Table 1) and the level did not fall further with time even though ROS began to be detected after 1 h exposure to AAI. Additionally, inhibition of ROS by the antioxidant Tiron did not prevent the GSH loss, indicating that AAI-induced ROS generation proceeds after GSH depletion. NAC is a clinical antioxidant as well as a GSH precursor, so its presence was able to replenish AAIstimulated GSH loss. Further investigation is required to understand why AAI treatment results in GSH depletion. Previous studies have shown that human organic anion transporter can mediate the uptake of AAI into kidney cells (Bakhiya et al., 2009). Rat organic anion transporter serves as a bidirectional transporter for the organic anion/GSH exchange, so it is possible that AAI induced-GSH depletion is mediated by GSH efflux through GSH/AAI exchange (Franco and Cidlowski, 2006; Russel et al., 2002). Three antioxidants, Tiron, NAC and GSH, were successfully used in the prevention of ROS formation and DNA damage caused by AAI (Figs. 1C and 2B). However, only the thiol-containing compounds NAC and GSH, but not non-thiol Tiron, were able to suppress the AAI-induced caspase 3 activity (Fig. 5), revealing that GSH depletion instead of the ROS formation contributes to AAI-triggered apoptosis in HL-60 cells. These results are consistent with Franco’s finding that GSH depletion independent of ROS generation is essential for apoptosis in lymphoid Jurkat cells (Franco et al., 2007); both HL-60 and Jurkat cells are derived from human acute leukemia. Recent studies suggest that GSH depletion and protein modification with glutathionylation are critical regulators of apoptosis (Franco and Cidlowski, 2009) In conclusion, as shown in Fig. 6, AAI, a botanical product commonly found in herbal remedies, induces oxidative stress-related DNA damage through the activation of the MEK/ERK1/2 signaling pathway and the depletion of intracellular GSH. However, AAI-induced apoptosis is partially attributed to GSH loss, but not directly associated with ROS formation. Knowledge regarding the toxicological mechanism of AAI in human cells may be useful in estimating the exposure risk to the general public. Conflict of interest statement None declared.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Aristolochic acid I induced oxidative DNA damage associated with glutathione depletion and ERK1/2 activation in human cells Feng-Yih Yu a, Ting-Shuan Wu a,1, Ting-Wei Chen a, Biing-Hui Liu a,b,⇑ a b
Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan
a r t i c l e
i n f o
Article history: Received 5 August 2010 Accepted 28 January 2011 Available online 12 February 2011 Keywords: Aristolochic acid Oxidative stress DNA damage GSH depletion ERK1/2 activation
a b s t r a c t Aristolochic acid I (AAI) has been widely found in herbal remedies and linked to the development of nephropathy and urothelial carcinoma in humans. This study elucidated the mechanism of oxidative stress and DNA damage mediated by AAI in human cells. Treatment of human promyelocytic leukemia cells (HL-60) and human renal proximal tubular cells (HK-2) with AAI led to a dose-dependent increase of reactive oxygen species (ROS). AAI also elevated the levels of DNA strand breaks and 8-hydroxy guanosine in HL-60 and HK-2 cells. Antioxidants, including Tiron, N-acetyl-L-cysteine (NAC) and glutathione (GSH), effectively suppressed the AAI-induced ROS and AAI-elicited genotoxicity, indicating that AAI induced the DNA damage through oxidative stress. GSH depletion was also found in AAI-treated cultures and proceeded prior to ROS formation. Exposure of HL-60 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only MEK1/2 inhibitor, U0126, significantly decreased AAI-mediated ROS. Preincubation of cells with thiol-containing compounds (NAC and GSH) inhibited the caspase 3 activity triggered by AAI, but non-thiol Tiron did not show a similar effect. This study demonstrated that AAI treatment results in oxidative stress-related DNA damage through GSH depletion and ERK1/2 activation; AAI-induced apoptosis is associated with GSH loss, but is independent of ROS generation. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Aristolochic acid (AA), found in herbal extracts of various species of genus Aristolochia, is a natural mixture composed of aristolochic acid I (AAI) and aristolochic acid II at a ratio of approximately 1:1 (Li et al., 2004). Several Aristolochia species have been widely used in traditional Chinese medicine as anti-rheumatics, as diuretics and in the treatment of oedema for thousand years, but AA is now suspected of causing nephropathy (Cosyns et al., 1994). Consumption of herbal medicines containing AA has been linked to Chinese herb nephropathy (CHN)/aristolochic acid nephropathy (AAN), a progressive interstitial nephritis leading to end-stage renal disease and urothelial malignancy (Debelle et al., 2008). CHN/ AAN was originally reported in Belgium, with over 100 cases of nephropathy linked to the use of a slimming regimen containing Chinese herbs (Vanherweghem et al., 1993); a follow-up investigation of CHN/AAN patients revealed a high risk for the development of urothelial carcinomas (Nortier et al., 2000; Lord et al., 2001). ⇑ Corresponding author at: Department of Biomedical Sciences, Chung Shan Medical University, No. 110, Sec. 1, Chien-Kuo N. Rd., Taichung 402, Taiwan. Tel.: +886 4 24730022x11815; fax: +886 4 24757412. E-mail address: [email protected] (B.-H. Liu). 1 Equal contribution to the first author. 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.01.016
Chronic exposure to AA is also considered a risk factor relating to Balkan endemic nephropathy and its associated cancer (Grollman et al., 2007). Therefore, mixtures of AA have been classified as probably carcinogenic to humans (Group 2A) and herbal remedies containing Aristolochia species are carcinogenic to humans (Group 1) (IARC, 2002). The mutagenetic and carcinogenic effects of AA are generally believed to be associated with the formation of AA–DNA adducts in both AA patients and experimental animals (Grollman et al., 2007; Schmeiser et al., 2009). Metabolic activation of AAI to Nhydroxyaristolactam I results in the formation of DNA adducts which may be responsible for the transversion mutation A ? T in ras and p53 genes (Schmeiser et al., 1990; Slade et al., 2009). AA is also a direct mutagen in Salmonella typhimurium (Gotzl and Schimmer, 1993); micronucleus induction, DNA breakage, sister chromatid exchange and nitrative DNA modification were observed in various mammalian cells (Abel and Schimmer, 1983; Li et al., 2006; Wu et al., 2007). Additionally, AA nephrotoxicity is reported to be linked with AA-induced apoptosis, which could be caused by endoplasmic reticulum and mitochondrial stress (Hsin et al., 2006; Wang and Zhang, 2008). Reactive oxygen species (ROS) are regarded as a natural byproduct of regular oxygen metabolism in aerobic organisms and is possibly induced by many environmental factors including ionizing
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radiation and toxic chemicals. Irrespective of their origin, ROS can interact with cellular biomolecules such as DNA and, under certain circumstances, act as subcellular messengers in signal transduction and gene regulation pathways (Cooke et al., 2003). Both the mitogen-activated protein kinase (MAPK) and NF-jB signaling pathways are considered to be oxidative stress sensitive (Milligan et al., 1998; Cakir and Ballinger, 2005). In the present study, we applied human cells as models to investigate whether AAI, in addition to forming DNA adducts, is able to damage DNA through the stimulation of ROS generation. Furthermore, the mechanism of AAI-induced oxidative DNA damage was elucidated. 2. Materials and methods 2.1. Reagents Cell culture media and serum were obtained from Life Technologies (Grand Island, NY). MEK1/2 inhibitor U0126, p38 pathway inhibitor SB203580 and polyclonal rabbit antibodies against phospho-p38 kinase, phospho-ERK1/2 (Thr202/ Tyr204) and parent ERK1/2 were purchased from Cell Signaling (Beverly, MA). 20 ,70 Dichlorodihydro-fluorescein diacetate (H2DCF-DA) was obtained from Molecular Probes (Eugene, OR). AAI (aristolochic acid I), Tiron (4,5-dihydroxy-1.3-benzenedisulfonic acid disodium salt), NAC (N-acetyl-L-cysteine) and GSH (glutathione) were from Sigma Chemical (St. Louis, MO). AAI was dissolved in DMSO at a concentration of 25 mM and stored at 20 °C as a stock solution. 2.2. Cell culture Human promyelocytic leukemia (HL-60) and human renal proximal tubular (HK-2) cell lines were obtained from Bioresources Collection and Research Center in Taiwan and then cultured in RPMI 1640 medium and DMEM/Hams F12 medium, respectively. Both the medium were supplemented with 10% fetal bovine serum (GIBCO Invitrogen), 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37 °C in a humidified 5% CO2 incubator. HL-60 cultures were established by centrifugation with subsequent resuspension at 1 105 viable cells/ml. Cell density was maintained between 1 105 and 1 106 cells/ml. HK-2 cells were anchorage dependent and were subcultivated at 80% confluence with a split ratio of 1:4. 2.3. Measurement of ROS in cells with oxidation-sensitive fluorescent dyes Cells (104/per well on a 96-well tissue culture plate) were cultured for at least 24 h in 50 ll of complete medium, and then 50 ll of H2DCF-DA (20 lM) in Krebs–Ringer HEPES (KRH) buffer was added. Thirty minutes after incubation, the probe-containing medium was removed and AAI (25 to 200 lM) in KRH buffer were added to the cells and incubated for another 1 h. Since H2DCF-DA is hydrolyzed and oxidized by intracellular ROS to 2,7-dichlorofluorescein (DCF), the fluorescence of DCF was analyzed in an HTS 7000 Bio Assay Fluorescent Plate Reader (PerkinElmer life Sciences) at an excitation wavelength of 485 nm and fluorescence emission was measured at 530 nm. To evaluate the intracellular ROS levels by flow cytometry, HL60 cells in complete medium were incubated with H2DCF-DA at a final concentration of 10 lM for 30 min. After centrifugation at 1000g for 5 min, the culture medium were replaced with either solvent or various concentrations of AAI in phosphate-buffered saline (PBS) solution, and then incubated at 37 °C for another 1 h before flow cytometry. The oxidized forms of ROS-sensitive dye were excited with a 488 nm laser (Becton–Dickinson FACSalibur flow
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cytometer, Franklin Lakes, NJ); emission of DCF fluorescence was quantified in channel FL-1 (530-nm filter, 30-nm bandpass). 2.4. Single cell gel electrophoresis (SCGE) assays Briefly, cells were cultured at 37 °C in medium containing vehicle alone (2 ll or 4 ll DMSO/ml medium) or AAI (50–100 lM) for 24 h. The adherent cells were then trypsinized and mixed with 1% low-melting-point agarose at 42 °C. The mixtures were immediately transferred to the CometSlide™ (Trevigen Inc., Gaithersburg, MD) and the slides were immersed in ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris, 1% sodium lauryl sarcosinate, 1% Triton X-100 and 1% DMSO) for 1 h. After electrophoresis in an alkaline buffer (300 mM NaOH, 1 mM EDTA, pH > 13) at 300 mA for 30 min, DNA on the slides was stained with SYBR green I. The image of each cell on the slides was visualized and photographed using a fluorescence microscope (Axiovert 25, Zeiss). The images were analyzed with the computer software from Euclid Analysis (St. Louis, MO) to calculate the tail moment, a combination of the amount of DNA in the tail with the distance of migration. For each independent experiment, the DNA damage level of 80 individual cells was measured from each culture. 2.5. Detection of 8-hydroxy-2-deoxy guanosine (8-OH-dG) by flow cytometry The levels of 8-OH-dG were measured by using the OxyDNA assay Kit (Calbiochem, San Diego, CA). HL-60 cells (1 106 cells/well) were seeded in 35-mm tissue culture dishes and treated with 0–200 lM of AAI for 24 h or 1 mM H2O2 for 1 h. The cells were harvested, rinsed with PBS, and fixed with 1% paraformaldehyde on ice for 15 min. After centrifugation to remove paraformaldehyde, the cells were kept in ice-cold 70% ethanol at 20 °C for 18 h. According to the manufacture’s protocol, the fixed cells were incubated with the FITC-conjugate solution for 1 h at room temperature and then fluorescence was read using a flow cytometer at an excitation wavelength of 495 nm and barrier filter of 515 nm. 2.6. Western blot analysis HL-60 cells at 80% saturation density were pretreated with antioxidants (5 mM Tiron, 10 mM NAC, or 1 mM GSH) for 2 h or with MAPK inhibitors (10 lM U0126/10 lM SB203580) for 30 min before co-exposure to AAI. Whole cell extracts were prepared according to Liu et al. (2007). The protein concentrations in each sample were determined using the Bradford protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard. Equal amounts of protein (40 lg) from each sample preparation were separated on SDS–PAGE and subjected to Western blotting. Polyclonal antibodies specific to phospho-ERK1/2 (Thr202/ Tyr204), ERK1/2, phospho-p38 or p38 were used as the probes. Bound antibody on the membrane was detected using an enhanced chemiluminescence detection system according to the manufacturer’s manual (Amersham Pharmacia Biotech, Amersham, UK). 2.7. Determination of GSH levels HL-60 cells (1 106 cells/well) were pretreated with or without antioxidants (5 mM Tiron or 10 mM NAC) for 2 h before co-exposure to 200 lM AAI or DMSO (4 ll/ml medium) for another 15 or 30 min. Cells were harvested and subjected to GSH–Glo glutathione assay according to the manufacture’s protocol (Promega). The assay is based on the conversion of luciferin derivative into luciferin in the presence of GSH, catalyzed by glutathione S-transferase. The value of luminescent signal in each treatment was
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calculated according to the standard curve generated in the presence of pure GSH. 2.8. Measurement of caspase-3 catalytic activities Cells were seeded in 24-well plates (7 105 cells/well) and then incubated with vehicle or various concentrations of AAI (0–500 lM) for 24 h. Treated cells were washed with 0.01 M PBS and resuspended in 100 ll of caspase assay buffer containing 0.1 M HEPES (pH 7.4), 2 mM DTT, 0.1% CHAPS and 1% sucrose. The cell suspension was sonicated and the cell lysate was centrifuged (14,000g) at 4 °C for 15 min. Fifty microliters of supernatant fluid was diluted with 450 ll of assay buffer containing 20 lM Ac-DEVD-AFC (Sigma), a substrate for active caspase-3. After incubation at 37 °C for 1 h, the levels of cleaved products were measured using a HTS 7000 PLUS bioassay reader (PerkinElmer, Boston, MA) with excitation set at 405 nm and emission at 535 nm. 2.9. Statistical analysis All statistical analyses were conducted using the software GraphPad Prism Version 4.0 (GraphPad Software, San Diego, CA). Experimental data of Figs. 1–3 had passed the Kolmogorov–Smirnov normality test and were analyzed by one-way ANOVA followed by Tukey post test. The data of Figs. 4 and 5 were analyzed by unpaired two-tailed Student’s t-test.
3. Results 3.1. Generation of intracellular ROS after AAI treatment To determine the AAI concentrations suitable for studying ROS formation, the cell viability of HL-60 and HK-2 in the presence of AAI was examined by the tetrazolium dye-based MTS assay. The cell viability of HL-60 declined to 80% of control after 24 h exposure to 200 lM AAI, but no statistically significant difference was observed while AAI at concentrations up to 100 lM. On the other hand, incubation of HK-2 cells with 100 and 200 lM AAI decreased the cell viability to 71% and 55% of control after 24 h exposure, respectively. The intracellular ROS levels in HL-60 and HK-2 cells were evaluated based on fluorescence spectrophotometry using H2DCF-DA as the probe. Various oxidants, especially hydroxyl radical (OH) and peroxynitrite (ONOO), are able to oxidize intracellular H2DCF to fluorescent DCF (Setsukinai et al., 2003). Based on the cell viability in the MTS assay, both HL-60 and HK-2 cells were exposed to AAI ranging from 25–200 lM. Following treatment of HL-60 with AAI for 1 h, DCF fluorescence increased in a concentrationdependent manner (Fig. 1A); AAI at 100 and 200 lM significantly increased the fluorescence to 5.0 and 6.9 folds of control levels, respectively. On the other hand, treatment of HK-2 cells with 200 lM AAI also dramatically enhanced ROS levels. Since HL-60 cells are more sensitive to AAI exposure comparing to HK-2, we mainly used HL-60 cultures to study the AAI-induced oxidative stress in the following experiments Furthermore, by applying flow cytometry, HL-60 cultures stimulated with 50 and 200 lM of AAI for 1 h elevated the intracellular ROS levels to 2.4 and 56.7-folds of the control group, respectively (Fig. 1B). The fluorescence level of H2O2-treated cultures was 16.9-fold higher as compared with the solvent-treated group; H2O2 is a potent genotoxic reagent and regarded as a positive control. Additionally, when cells were pre-incubated with Tiron, NAC, or GSH before co-exposure to AAI, either free radical scavenger markedly inhibited AAI-induced ROS (Fig. 1C). All of these data
Fig. 1. AAI increased intracellular ROS levels. (A) HL-60 and HK-2 cells were incubated with H2DCF-DA and then exposed to solvent or AAI for 1 h. DCF fluorescence was determined by fluorescence spectrophotometry. (B) HL-60 cells were incubated with H2DCF-DA, and then treated with solvent, AAI or H2O2 for 1 h before subjected to flow cytometry. (C) HL-60 cells were pre-treated or not with Tiron (5 mM), NAC (10 mM) or GSH (1 mM) for 2 h, incubated with H2DCF-DA, and then with AAI for 1 h. DCF fluorescence was measured by fluorescence spectrophotometry. All the data are the mean ± SEM from five independent experiments. ⁄ Significant difference (⁄p < 0.05; ⁄p < 0.01) compared to the solvent-treated control or the paired group.
indicate that AAI is able to trigger the ROS production in human cells. 3.2. AAI induced oxidative DNA damage We further applied the SCGE assay and flow cytometry to investigate whether ROS induced by AAI contributed to oxidative DNA damage. In the SCGE assay, the tail shape and migration pattern of DNA reveals the level of DNA single/double strand breakage on a single cell basis (Klaude et al., 1996). Treating HK-2 cells with 50 and 100 lM of AAI significantly increased the tail moment to 3.9 and 5.5-folds of control levels, respectively (Fig. 2A). Similarly, exposure of HL-60 cells to AAI (50 and 100 lM) led to a significant and concentration-dependent increase in the tail moment values. Moreover, Tiron, NAC and GSH effectively suppressed the
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Fig. 3. AAI treatment induced the formation of 8-OH-dG. HL-60 cells were exposed to solvent (control) and AAI (50 and 200 lM) for 24 h or to H2O2 (1 mM) for 1 h. Collected cells were fixed and incubated with FITC-conjugate solution for 1 h and then fluorescence was read using a flow cytometer at an excitation wavelength of 495 nm and barrier filter of 515 nm. The representative chromatogram of flow cytometry is shown in (A) and data collected from three independent experiments were statistically analyzed in (B). Fig. 2. AAI induced oxidative DNA damage in HK-2 and HL-60 cultures. (A) HK-2 and HL-60 cells were incubated with various concentrations of AAI for 1 h. The values of tail moment were determined by SCGE assay. (B) HL-60 cells were pretreated for 2 h with or without Tiron (5 mM), NAC (10 mM) or GSH (1 mM) and then co-incubated with 100 lM AAI for another 1 h before SCGE assay. Images visualized and photographed by epifluorescence microscope were analyzed with the computer software to calculate tail moment values. All the data are the mean ± SEM from five independent experiments. ⁄Significant difference (⁄p < 0.05; ⁄⁄ p < 0.01; ⁄⁄⁄p < 0.001) compared to the control groups.
the phenomenon of GSH depletion was detected as early as 30 min incubation with AAI, while the level of ROS generation was not significant until 1 h treatment. In addition, inhibition of ROS formation by Tiron cannot reverse AAI-induced GSH depletion, revealing that GSH depletion proceeds prior to ROS generation.
3.4. ERK1/2 played a role in AAI-triggered ROS AAI-induced tail moment value to 65%, 46% and 41%, respectively, of the AAI along-treated group (Fig. 2B), strongly suggesting that DNA breakage after treatment of AAI is partially caused by ROS formation. 8-Hydroxy-2’-deoxyguanosine (8-OH-dG) is one of the most abundant oxidative products of cellular DNA, so the levels of 8OH-dG in solvent or AAI-treated cells were evaluated by flow cytometry. As shown in Fig. 3A, both AAI and H2O2 stimulated the generation of 8-OH-dG in HL-60 cells. Incubation of cells with 200 lM AAI for 24 h significantly increased the 8-OH-dG level to 1.6-folds of the solvent-treated group (Fig. 3B). 3.3. Involvement of GSH depletion in AAI-induced ROS generation The intracellular GSH level is generally considered to be closely correlated with ROS generation (Hayes and McLellan, 1999). Therefore, to investigate the role of GSH in AAI-induced ROS, HL-60 cells were treated with antioxidants and/or AAI before determination of the total GSH content. After 30 min of exposure, the GSH level dropped to 55% of control group in 200 lM-AAI exposed cultures (Table 1). However, no significant GSH depletion was found after a 15 min treatment. Furthermore, the GSH depletion caused by 200 lM AAI could be effectively rescued by the pre-incubation of cells with antioxidant NAC, a precursor of GSH, but antioxidant Tiron did not show any effect on the decreased GSH content. Overall,
Many studies have demonstrated that oxidative stress influences the MAPK signaling pathways (McCubrey et al., 2006). Therefore, this study first evaluated whether AAI can activate ERK1/2 or p38 pathways in HL-60 cells. Exposure of cells to 100 lM AAI resulted in both ERK1/2 and p38 phosphorylation. Pretreatment of cultures with either U0126, a specific MEK1/2 inhibitor, or SB203580, a p38 pathway blocker, dramatically decreased the phospho-ERK1/2 and phospho-p38 levels, respectively (Fig. 4A). Moreover, preincubation of cells with U0126, but not SB203580, significantly suppressed the levels of DCF fluorescence triggered by 100 lM AAI (Fig. 4B), suggesting that ERK1/2 signaling, but not the p38 pathway, partially contributes to AAI-induced ROS generation.
3.5. GSH depletion, but not ROS generation, involved in AAI-mediated apoptosis According to Fig. 5, treatment of HL-60 with 500 lM AAI elevated the caspase-3 catalytic activity to 11.6 folds of the control level. Therefore, the role of ROS in AAI-mediated apoptosis was further examined by applying antioxidants to the cultures. NAC and GSH partially reduced the AAI-elicited caspase 3 activity to 57.2% and 39.6%, respectively, of that in AAI-exposed cells. However, Tiron did not display a similar effect. It seems that intracellu-
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F.-Y. Yu et al. / Toxicology in Vitro 25 (2011) 810–816 Table 1 GSH levels in AAI-treated HL-60 cells. a
GSH content (% of control)
Treatment
15 min
30 min
AAI 50 lM AAI 100 lM AAI 200 lM b AAI (200 lM) + Tiron (5 mM) AAI (200 lM) + NAC (10 mM)
– 86.3 ± 6.9 100.7 ± 15.8 – –
79.2 ± 15.6 70.8 ± 10.2 54.9 ± 10.5* 57.2 ± 9.8* 107.2 ± 15.1
a HL-60 cultures were incubated for 15 or 30 min with AA before determination of intracellular GSH levels. Values are means ± SEM of at least three independent experiments in which each concentration is conducted in triplicate. b HL-60 cultures were pre-incubated with Tiron or NAC for 2 h and then cotreated with 200 lM AA for another 30 min before GSH determination. * Significant difference (⁄p < 0.05) compared to the solvent-treated control
4. Discussion
Fig. 4. Effects of MAPK inhibitors on AAI-induced MAPK phosphorylation (A) and ROS generation (B). (A) HL-60 cells were pretreated for 1 h with or without 10 lM U0126/10 lM SB203580, and then co-incubated with 100 lM AAI for 24 h. Whole cell extracts were subjected to Western blotting using antibodies specific to phospho-ERK/ERK or phospho-p38/p38. In (B), cells were left untreated or treated with U0126/SB203580 for 1 h and then the medium were replaced with fresh medium containing 10 lM H2DCF-DA for 30 min. Finally 100 lM AAI was added for another 1 h. The levels of DCF fluorescence were measured by fluorocytometry and the data are expressed as the mean ± SEM from four independent experiments.
Fig. 5. Effects of antioxidants on AAI-induced apoptosis. HL-60 cells were treated or left untreated with various antioxidants for 2 h before co-incubated with vehicle or AAI for 24 h. Total cellular proteins were extracted and the caspase 3 activity was determined as described in Section 2. The data from five independent experiments are expressed as the mean ± SEM.
lar GSH depletion, but not ROS formation, is closely associated with the AAI-triggered apoptotic process.
Many European countries have banned various herbs containing AA since 1994. The Food and Drug Administration of the United States also issued a Consumer Advisory to warn consumers against purchasing botanic products containing AA on 2001 (Schwetz, 2001). However, Owing to the difficulties in distinguishing between the appearance and botanical names of similar species, the risk of mistaken use of AA-containing herb in traditional medicine is still high (Gold and Slone, 2003). A previous survey shows that 50% of Chinese herbal remedies randomly collected from Taiwan markets were found to contain various concentrations of AAI, ranging from 0.17 to 655 ppm (lg/g) (Yu et al., 2006). A long term exposure to AA containing remedies could lead to a major public health concern. AAI at a sub-lethal concentration of 50 lM could induce ROS generation in HL-60 and HK-2 cells (Fig. 1A). Although AAI is considered as a nephrotoxic compound, HL-60 cells seem to be more vulnerable to the toxin-induced oxidative stress compared with HK-2 and human embryonic kidney (HEK293) cells (data not shown). A similar result was also documented that with the renal tubular epithelial Madin–Darby canine kidney (MDCK) cell line as a model, ROS levels just elevated to around two folds after stimulation with 75 lM AAI for 4 h (Liu et al., 2009). In addition to ROS induction, AAI triggered similar levels of DNA strand breakage and damage in both HK-2 and HL-60 cells using the SCGE assay (Fig 2A). Since AAI triggered ROS production as well as DNA damage in both cell lines, HL-60 was chosen to further study the mechanism of AAI-induced oxidative stress because of its sensitivity, ease of handling (suspension culture) and frequent use in experiments associated with ROS induction. Formation of 8-OH-dG, a reliable marker for oxidative DNA damage in vitro and in vivo, provided further evidence of AAI-induced ROS (Fig. 3). Unrepaired 8-OH-dG in DNA may induce G:C to T:A transversion at the modified site, and apparently plays a major role in mutagenesis and carcinogenesis (Moriya and Grollman, 1993; Le Page et al., 1995). Our findings based on flow cytometry are consistent with those of Wu et al. (2007) who applied immunoperoxidative staining to show the 8-OH-dG formation in AAIexposed HepG2 cells; they concluded that the AAI-induced 8-OH-dG is nitrative DNA damage which was attributed to reactive nitrogen species (RNS), such as NO and ONOO–. Since NO may rapidly interact with ROS, especially O2, to form the stable ONOO anion (McCall et al., 1989; Beckman et al., 1990), it is extremely difficult to distinguish between the contribution of ROS and RNS to AAI-induced DNA damage. Exposure of HL-60 cells to 100 lM AAI led to the phosphorylation of ERK1/2 and p38 MAP kinases (Fig. 4A). However, no phosphorylated JNK signal was observed after AAI treatment (data not shown). Furthermore, AAI-induced ROS is selectively blocked by
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Acknowledgments The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research under Contract No. NSC 96-2313-B- 040-004-MY3. Flow cytometry was performed in the Instrument Center of Chung Shan Medical University, which is supported by the National Science Council, Ministry of Education and Chung Shan Medical University. Fig. 6. A proposed model for AAI-induced oxidative stress in HL-60 cells.
inhibition of the ERK1/2 pathway, but not p38 kinase (Fig. 4B), which indicates its dependence on MEK/ERK1/2 activation. Several studies have mentioned that under certain circumstances, MEK/ ERK1/2 signaling plays a pivotal role in ROS induction, but the exact molecular mechanism has not been fully elucidated (Woo et al., 2002; Liu et al., 2007). Intracellular GSH is known to be involved in the process of ROS toxicity. Many studies have indicated that unstable ROS molecules after exposure to stimulators can be eliminated by interacting with the cysteinyl moiety of GSH, and then the phenomenon of GSH depletion is subsequently observed (Armstrong et al., 2002). In contrast, our data support another hypothesis that GSH loss may happen before ROS formation, or even independent of ROS generation (Barhoumi and Burghardt, 1996; Franco et al., 2007). Treatment with 200 lM AAI for 30 min significantly reduced the GSH level to 50% of control (Table 1) and the level did not fall further with time even though ROS began to be detected after 1 h exposure to AAI. Additionally, inhibition of ROS by the antioxidant Tiron did not prevent the GSH loss, indicating that AAI-induced ROS generation proceeds after GSH depletion. NAC is a clinical antioxidant as well as a GSH precursor, so its presence was able to replenish AAIstimulated GSH loss. Further investigation is required to understand why AAI treatment results in GSH depletion. Previous studies have shown that human organic anion transporter can mediate the uptake of AAI into kidney cells (Bakhiya et al., 2009). Rat organic anion transporter serves as a bidirectional transporter for the organic anion/GSH exchange, so it is possible that AAI induced-GSH depletion is mediated by GSH efflux through GSH/AAI exchange (Franco and Cidlowski, 2006; Russel et al., 2002). Three antioxidants, Tiron, NAC and GSH, were successfully used in the prevention of ROS formation and DNA damage caused by AAI (Figs. 1C and 2B). However, only the thiol-containing compounds NAC and GSH, but not non-thiol Tiron, were able to suppress the AAI-induced caspase 3 activity (Fig. 5), revealing that GSH depletion instead of the ROS formation contributes to AAI-triggered apoptosis in HL-60 cells. These results are consistent with Franco’s finding that GSH depletion independent of ROS generation is essential for apoptosis in lymphoid Jurkat cells (Franco et al., 2007); both HL-60 and Jurkat cells are derived from human acute leukemia. Recent studies suggest that GSH depletion and protein modification with glutathionylation are critical regulators of apoptosis (Franco and Cidlowski, 2009) In conclusion, as shown in Fig. 6, AAI, a botanical product commonly found in herbal remedies, induces oxidative stress-related DNA damage through the activation of the MEK/ERK1/2 signaling pathway and the depletion of intracellular GSH. However, AAI-induced apoptosis is partially attributed to GSH loss, but not directly associated with ROS formation. Knowledge regarding the toxicological mechanism of AAI in human cells may be useful in estimating the exposure risk to the general public. Conflict of interest statement None declared.
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