Mediating of Caspase-Independent Apoptosis
by Cadmium Through the Mitochondria-ROS
Pathway in MRC-5 Fibroblasts
Chwen-Ming Shih,1* Wun-Chang Ko,2Jui-Sheng Wu,1Yau-Huei Wei,3Leng-Fang Wang,1E-E. Chang,1 Tsui-Yun Lo,1Huey-Hwa Cheng,1and Chien-Tsu Chen1
1Department of Biochemistry, School of Medicine, Taipei Medical University, Taipei, Taiwan, ROC 2Graduate Institute of Pharmacology, Taipei Medical University, Taipei, Taiwan, ROC
3Department of Biochemistry and Center for Cellular and Molecular Biology,
National Yang-Ming University, Taipei, Taiwan, ROC
Abstract Cadmium (Cd) is an environmental pollutant of global concern with a 10–30-year biological half-life in humans. Accumulating evidence suggests that the lung is one of the major target organs of inhaled Cd compounds. Our previous report demonstrated that 100 mM Cd induces MRC-5 cells, normal human lung fibroblasts, to undergo caspase-independent apoptosis mediated by mitochondrial membrane depolarization and translocation of apoptosis-inducing factor (AIF) from mitochondria into the nucleus. Here, using benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone (Z-VAD.fmk) as a tool, we further demonstrated that Cd could induce caspase-independent apoptosis at concentrations varied from 25 to 150 mM, which was modulated by reactive oxygen species (ROS) scavengers, such as N-acetylcysteine (NAC), mannitol, and tiron, indicating that ROS play a crucial role in the apoptogenic activity of Cd. Consistent with this notion, the intracellular hydrogen peroxide (H2O2) was 2.9-fold elevated after 3 h of Cd treatment and diminished rapidly
within 1 h as detected by flow cytometry with 20,70-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Using inhibitors of the mitochondrial electron transport chain (ETC) (oligomycin A and rotenone for complex I and V, respectively) and mitochondrial permeability transition pore (MPTP) (cyclosporin A and aristolochic acid), we coincidently found the ROS production, mitochondrial membrane depolarization, and apoptotic content were almost completely or partially abolished. As revealed by confocal microscopy staining with chloromethyl-X-rosamine (CMXRos) and an anti-AIF antibody, the collapse of mitochondrial membrane potential induced by Cd (3 h-treatment) was a prelude to the translocation of caspase-independent pro-apoptotic factor, AIF, into the nucleus (after 4 h of Cd treatment). In summary, this study demonstrated that, in MRC-5 fibroblasts, Cd induced caspase-independent apoptosis through a mitochondria-ROS pathway. More importantly, we provide several lines of evidence supporting a role of mitochondrial ETC and MPTP in the regulation of caspase-independent cell death triggered by Cd. J. Cell. Biochem. 91: 384–397, 2004. ß2003 Wiley-Liss, Inc.
Key words: cadmium; caspase; ROS; AIF; mitochondria; ETC; MPTP
ß2003 Wiley-Liss, Inc.
Grant sponsor: National Science Council; Grant numbers: NSC 89-2320-B-038-068, NSC 90-2113-M-038-001, NSC 92-2320-B-038-055.
*Correspondence to: Dr. Chwen-Ming Shih, Department of Biochemistry, School of Medicine, Taipei Medical Univer-sity, 250 Wu-Hsing Street, Taipei, Taiwan 110, ROC. E-mail: [email protected]
Received 2 October 2003; Accepted 3 October 2003 DOI 10.1002/jcb.10761
Abbreviations used: AIF, apoptosis-inducing factor; Apaf-1, apoptosis protease activating factor-1; ArA, aristolochic acid; Cd, cadmium; CsA, cyclosporin A; CMXRos; chlor-omethyl-X-rosamine; ETC, electron transport chain; DCFH-DA, 20,70-dichlorodihydrofluorescein diacetate;
GAAS, graphite atomic absorption spectrophotometer; JC-1, 5,50,6,60-tetrachloro-1,10,3,30
-tetraethylbenzimidazo-lyl carbocyanine iodide; MPTP, mitochondrial permeability transition pore; NAC,N-acetylcysteine; OA, oligomycin A; PI, propidium iodide; PS, phosphatidylserine; ROS, reac-tive oxygen species; Z-VAD.fmk, benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone; DCm, mitochondrial membrane potential.
Environmental pollution by cadmium (Cd) is a worldwide problem due to industrialization, smoking, and the lack of effective therapy for Cd poisoning. Although the general level of Cd exposure is low, the element has a long biological half-life in humans, of the order of 10– 30 years [Goyer and Cherian, 1995]. Cd has been reported to cause disorders of the renal, skeletal, vascular, and respiratory systems [Nordberg, 1992]. The lung is one of the main target organs of Cd toxicity, and several studies have shown that emphysema is a primary consequence of Cd exposure [Davison et al., 1988], suggesting the possible involvement of lung fibroblasts in Cd pulmonary toxicity. During the last decade, Cd has been shown to induce apoptosis in vivo [Risso-de Faverney et al., 2001; Harstad and Klaassen, 2002] and in vitro [Hart et al., 1999; Ishido et al., 1999; Achanzar et al., 2000; Kim et al., 2000; Li et al., 2000; Yuan et al., 2000; Shen et al., 2001; Kondoh et al., 2002] at varied concentrations from 1 to 300 mM. Therefore, Cd toxicity is thought to be caused by the induction of apoptosis. However, the apoptotic signaling induced by this toxicity is still unclear. In addition, only a minority of reports have focused on the apoptogenic effects of Cd on fibroblasts compared to other cell types [Biagioli et al., 2001].
Apoptosis, a biochemically and morphologi-cally distinct form of cell death, is associated with cell shrinkage, nuclear condensation, release of apoptogenic factor(s) from mitochon-dria, plasma membrane blebbing, and phospha-tidylserine (PS) externalization. These are followed by DNA fragmentation and the forma-tion of membrane vesicles called apoptotic bodies that can be taken up and degraded by neighboring cells without an inflammatory response [Robertson and Orrenius, 2000]. Dif-ferent from necrosis, apoptosis is a genetically controlled active process thought to play a critical physiological role in development and tissue homeostasis. However, inappropriate or defective apoptosis is the cause of many human diseases [Fadeel et al., 1999; Saikumar et al., 1999]. Recently, two modes of apoptosis have been elucidated, including caspase-dependent and -independent pathways [Zamzami and Kroemer, 1999]. The caspase family is consti-tutively expressed in almost all mammalian cell types as inactive pro-enzymes (zymogens) which are activated in response to a variety of
pro-apoptotic stimuli [Kohler et al., 2002]. Once the caspase cascade is activated, its down-stream molecules, such as caspase-activated DNase (CAD) and Acinus, will conduct cells to chromatin condensation and 180-base-pair DNA laddering, a hallmark of apoptotic death [Robertson and Orrenius, 2000]. However, expanding evidence suggests that cells could undergo caspase-independent apoptosis, such as human normal T lymphocytes [Dumont et al., 2000], human T-cell leukemia Jurkat and pre-B leukemia JM1 cell lines [Marzo et al., 2001], human microglial cells and cortical neuronal HCN-2 cell line [Braun et al., 2001], mice normal retinal cells [Carmody and Cotter, 2000], rat hepatocyte RALA255-10G cell line [Jones et al., 2000], rat fibroblast Rat-1 and monkey kidney COS cell lines [Loeffler et al., 2001], and Ax-2 strain ofDictyostelium discoi-deum cells [Arnoult et al., 2001]. In fact, mitochondria are affected particularly early in the apoptotic process and play a crucial role both in caspase-dependent and -independent apoptosis. Several pro-apoptotic signal trans-duction and damage pathways converge on mitochondria to induce mitochondrial mem-brane permeabilization (MMP), which in turn releases apoptogenic signaling molecules, such as pro-caspases (2, 3, and 9), cytochrome c, Smac (second mitochondria-derived activator of caspase), apoptosis inducing factor (AIF), endonuclease G (Endo G), and heat shock protein (Hsp) 10 and 60 [Ravagnan et al., 2002]. Emerging evidence suggests that trans-location of mitochondrial AIF into cytosol and then into the nucleus, resulting in chromatin condensation and high molecular weight (50 kb) DNA fragmentation, is a hallmark of caspase-independent apoptosis [Cande et al., 2002].
The apoptotic pathway induced by Cd re-mains controversial. Using caspase inhibitors as a tool, Cd-treated rat fibroblast cells (10 mM CdCl2) [Kim et al., 2000] and human leukemia
cells (100 mM CdCl2) [Li et al., 2000; Kondoh
et al., 2002] were induced to undergo apop-tosis through the caspase-dependent pathway. However, Ishido et al. [1999] demonstrated that caspase activity is not associated with Cd-induced apoptosis in porcine kidney LLC–PK1
cells because caspase inhibitors were unable to rescue cells. Therefore, the intracellular signal-ing pathway responsible for Cd-induced apop-tosis needs further characterization. MRC-5 cells are derived from a human fetal lung
fibroblast, which has been used as a cell model to study the pulmonary toxicity of Cd [Yang et al., 1997]. Although Cd is not a Fenton metal, evidence suggests that H2O2 production and
lipid peroxidation are the major causation of Cd toxicity. Following this line, the current study was designed to investigate Cd-induced apopto-genic signaling in MRC-5 cells. We herein show that the broader-spectrum of caspase in-hibitor, benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone (Z-VAD.fmk), was unable to rescue Cd-treated MRC-5 cells at varied concentrations from 25 to 150 mM and that the AIF was translocated from mitochondria into the nucleus. These results led us to conclude that Cd induces a caspase-independent apopto-tic pathway in MRC-5 cells. Furthermore, the fact that Cd induced an elevation of intracel-lular H2O2 and that antioxidants attenuated
the Cd-induced apoptosis imply that the apop-togenic activity of Cd is partially a result of oxidative stress. In addition, the collapse of the mitochondrial membrane potential was ob-served early in the apoptotic process. Using inhibitors of the electron transport chain (ETC) and mitochondrial permeability transition pore (MPTP), we demonstrated that mitochondria play an early and pivotal role in promoting Cd-induced caspase-independent apoptosis.
MATERIALS AND METHODS Cell Culture and Chemicals
Normal diploid MRC-5 cells derived from human fetal lung fibroblast [Jacobs et al., 1970] were obtained from the American Tissue Culture Collection (ATCC CCL-171, Rockville, MD). MRC-5 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemen-ted with 10% fetal bovine serum (FBS), peni-cillin (100 U/ml), and streptomycin (100 mg/ml) in 5% CO2, 95% air at 378C in a humidified
atmosphere incubator. Since MRC-5 cells are normal human cells, all of the experiments were performed at 25–35 passages. Exponentially growing MRC-5 cells (70–80% confluence) were treated with CdCl2 for the indicated time
periods. DMEM, FBS, penicillin, and strepto-mycin were purchased from HyClone (Logan, UT). Cadmium chloride, RNase A, N-acetylcys-teine (NAC), 4,5-dihydroxy-1,3-benzene-disul-fonic acid (tiron), mannitol, OA, rotenone (RT), cyclosporin A, aristolochic acid, and bovine serum albumin (BSA) were from Sigma
Chemi-cal Co. (St. Louis, MO). Benzyloxycarbonyl-Val-Ala-Asp-(ome) fluoromethyl ketone (Z-VAD.fmk) and the Annexin-V-FLUOS stain-ing kit were from BACHEM AG (Bubendorf, Switzerland) and Roche (Mannheim, Germany), respectively. Propidium iodide (PI), 20,70
-diodihydrofluorecein diacetate (DCFH-DA), chlor-omethyl-X-rosamine (CMXRos; MitoTracker Red), and 5,50,6,60-tetrachloro-1,10,3,30
-tetra-ethylbenzimidazolylcarbocyanine iodide (JC-1) were from Molecular Probe (Eugene, OR). The rabbit anti-AIF polyclonal antibody was from BioVision (Mountain View, CA). The affinity-purified cyanine (Cy2)-conjugated goat anti-rabbit IgG used as a secondary antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Protein Assay Dye Reagent was from Bio-Rad Labora-tories, Inc. (Hercules, CA).
Determination of Cell Survival and Cd Accumulation
Numbers of live cells after treatment with 100 mM Cd were determined by the trypan blue dye exclusion method. The relative survival rate was calculated as a percentage of control cells. In parallel, cells were collected to determine the intracellular Cd accumulation using gra-phite atomic absorption spectrometry (GAAS) [Martel et al., 1990]. After two washes in PBS, cells (1 107) were resuspended in 1 ml fractio-nation buffer (125 mM sucrose, 60 mM KCl, 3 mM HEPES, pH 7.1) and disrupted by a Misonix XL2000 ultrasonic cell disruptor (Farmingdale, NY) using a 5-W output for 10 s with 30-s intervals on ice (eight times). Cell debris was discarded by centrifugation (10,000g, 10 min, 48C). Aliquots of cell lysate were taken for estimation of protein concentration using the Protein Assay Dye Reagent (Bio-Rad Labora-tories) or for measurement of total intracellular Cd accumulation after acid (HNO3) digestion
using a Hitachi Z-5000 GAAS (Tokyo, Japan). The standard was made by a series of dilutions from the Cd standard solution (1,000 ppm Cd(NO3)2from Merck, Darmstadt, Germany).
Assessment of Apoptosis
Flow cytometry was used to assess the membrane and nuclear events during apopto-sis. The membrane events were analyzed by measuring the binding of FITC-labeled annexin V protein to the phospholipid PS present on the external surface of the apoptotic cell membrane
[Vermes et al., 1995]. PS externalization was performed in a live cell system with a two-color analysis of FITC-labeled annexin V binding and PI uptake using the Annexin-V-FLUOS staining kit (Roche). Briefly, 1 106cells were washed with PBS and centrifuged at 200g for 5 min and then stained with 100 ml of Annexin-V-FLUOS labeling solution (containing FITC-labeled annexin V and PI) for 15 min at room temperature. Cell concentration was adjusted to 2 106/ml with the kit-supplied incubation buffer, and cells were immediately subjected to analysis on a Becton Dickinson (San Jose, CA) FACSCalibur flow cytometer. Cell debris, char-acterized by a low FSC/SSC, was excluded from analysis. Fluorescence was detected in fluores-cence channels FL1 (488 nm excitation and 530 nm emission for FITC-labeled annexin V) and FL2 (488 nm excitation and 600 nm emission for PI). Electronic compensation of the instrument was used to exclude overlapping of the two emission spectra. Data acquisition and analysis were performed using the Cell-Quest program (Becton Dickinson). Positioning of quadrants on annexin V/PI dot plots was performed as reported [Van Engeland et al., 1996], and this method can be used to distin-guish living cells (annexin V/PI), early apop-totic/primary apoptotic cells (annexin Vþ/PI), late apoptotic/secondary necrotic cells (annexin Vþ/PIþ), and necrotic cells (annexin V/PIþ) [Pietra et al., 2001]. Therefore, the total apop-totic proportion was summed up from the percentage of quadrants with fluorescence annexin Vþ/PIand annexin Vþ/PIþ.
To determine the nuclear events of apoptotic cells, PI staining was employed to analyze the hypodiploid DNA content on a flow cytometer. Cells were harvested at 1 106cells/ml, washed with PBS, and fixed in ice cold 70% ethanol for 30 min at 48C. After centrifugation, cells were resuspended, incubated for 30 min in PBS containing 0.5 mg/ml RNase A and 40 mg/ml PI at room temperature, and analyzed using a Becton Dickinson FACSCalibur flow cytometer as described previously [Ormerod et al., 1992]. Cells with sub-G1 (hypodiploid DNA) PI incor-poration were considered apoptotic.
Confocal Microscopy
Mitochondrial localization and the mem-brane potential as well as the translocation of AIF protein in Cd-treated MRC-5 cells were determined using confocal microscopy [Castedo
et al., 2002]. Cells were grown on coverslips, washed with PBS, and stained with 50 nM membrane potential-dependent dye, Mito-Tracker Red CMXRos, for 20 min at 378C. This fluorescent dye is selectively incorporated into mitochondria with an intact transmembrane potential and therefore serves as an indicator of mitochondrial localization and depolarization. After two washes in PBS, cells were fixed with 4% paraformaldehyde and 0.19% picric acid in PBS at room temperature for 30 min. They were then permeabilized with 0.5% Triton X-100 at 48C for 15 min before incubation with rabbit anti-AIF antibody (1:100 dilution) for 60 min at 378C in a humidified chamber. Cells were sub-sequently washed three times with PBS and then incubated with Cy2-conjugated goat anti-rabbit IgG antibody (1:300 dilution) at room temperature for 2 h. Finally, cells were mounted with 50% glycerol in PBS containing n-propyl gallate as an anti-fading agent and were analyzed under an Olympus FV 500 confocal system (Tokyo, Japan) equipped with an Ar ion (488 nm) and He–Ne G (543 nm) laser, mounted on an inverted microscope (Olympus IX70) with a 60 oil objective. The AIF and MitoTracker Red CMXRos fluorescent images were acquired as 0.2-mm sections through standard FITC and MitoTracker filters, respectively, and analyz-ed using the Fluoview program, Version 4.0. (Olympus, Tokyo, Japan).
Measurement of Intracellular H2O2
Cells adhering to the culture dish were pretreated with 20 mM DCFH-DA for 20 min before addition of Cd for the indicated time period. They were then trypsinized for immedi-ate analysis on a flow cytometer. The esterified form of DCFH-DA can permeate cell mem-branes before being deacetylated by intra-cellular esterases. The resulting compound, dichlorodihydrofluorescein (DCFH), is reactive with H2O2 to produce an oxidized fluorescent
compound, dichlorofluorescein (DCF), which can be detected by flow cytometry with excita-tion and emission settings of 488 and 525– 550 nm (FL1), respectively.
Detection of the Mitochondrial Membrane Potential (DCm)
The mitochondrial membrane potential was analyzed using JC-1, a lipophilic cationic fluor-escence dye. JC-1 is capable of selectively entering mitochondria, where it either forms
monomers (fluorescence in green, 527 nm) or, at a high DCm(implicating a high dye
concentra-tion), aggregates (fluorescence in red, 590 nm) [Cossarizza et al., 1993]. The quotient between green and red fluorescence provides an estimate of DCm that is (relatively) independent of the
mitochondrial mass [Castedo et al., 2002]. Cells (1 106) were incubated with 5 mg/ml JC-1 (made up as a 5 mg/ml stock in DMSO) for 15 min at room temperature in darkness. After centrifugation (200g, 5 min), cells were washed with 48C PBS twice, resuspended in 0.5 ml PBS, and analyzed on a FACSCalibur flow cytometer.
Statistics
Data are expressed as the mean standard deviation (SD) from a minimum of three in-dependent experiments, unless otherwise indi-cated. Statistical analysis was performed using Student’st-test, with P < 0.01 as a criterion of significance.
RESULTS
Induction of Cell Death by Accumulation of Intracellular Cd
Cell survival and intracellular Cd were determined by trypan blue dye exclusion and AAS, respectively. As shown in Figure 1, after treatment with 100 mM Cd, the intracellular Cd was elevated and reached a plateau after 12 h, which was accompanied by a decrease of rela-tive cell survival. This result indicated that Cd can accumulate in MRC-5 fibroblasts and exert its characteristics of cell toxicity.
Induction of Caspase-Independent Apoptosis by Cd
To investigate the involvement of caspase activity in Cd-induced apoptosis, a broad-spec-trum caspase inhibitor, Z-VAD.fmk, was used during the assessment of Cd-induced apoptosis. PS externalization and PI uptake can be used to distinguish the types of cell death as described in ‘‘Materials and Methods.’’ The proportion of apoptosis was summed up from early apoptosis (annexin Vþ/PI) and late apoptosis (annexin Vþ/PIþ) [Pietra et al., 2001], and their stati-stic results from Figure 2A,B are shown in Figure 2C. In Figure 2A, the apoptotic cells reached a plateau of around 40.0% after a 24-h exposure of 100 mM Cd. However, pretreatment with Z-VAD.fmk was unable to rescue MRC-5
fibroblasts, suggesting that Cd might induce a caspase-independent apoptotic pathway at varied concentrations from 25 to 150 mM. As shown in Figure 2B, this did not seem to be a result of failure by Z-VAD.fmk to inhibit cas-pase activity, since this condition could prevent caspase-dependent apoptosis of Cd -treated HL-60 cells [Kondoh et al., 2002].
Collapse of the Mitochondrial Membrane Potential Is a Prelude to AIF Translocation Into
the Nucleus During Cd-Induced Apoptosis AIF is a novel apoptotic effector protein described recently and is defined as a caspase-independent mitochondrial death factor; it is released from mitochondria into the cytosol and translocated to the nucleus [Susin et al., 1999; Cande et al., 2002]. Collapse of the mitochon-drial membrane potential can increase mito-chondrial membrane permeability (MMP) and facilitate the release of pro-apoptotic factors into the cytosol [Ravagnan et al., 2002]. In this study, we highlight the involvement of AIF translocation in Cd-induced MRC-5 cells under-going caspase-independent apoptosis. Confocal microscopy was employed for tracking AIF translocation using Cy2-conjugated antibodies
Fig. 1. Intracellular Cd accumulation and relative survival after Cd treatment in MRC-5 cells. MRC-5 cells were treated with 100 mM Cd for various time periods. Subsequently, adherent and detached cells were collected and analyzed for relative survival or intracellular Cd accumulation. Numbers of live cells were determined by the trypan blue dye exclusion method. The relative survival rate was calculated as the percentage of control cells and is indicated by open circles (*). In parallel, the ac-cumulated intracellular Cd was measured by graphite atomic absorption spectrometry (GAAS) and normalized with that of total cellular proteins (pmol Cd/mg protein) as indicated by closed circles (*). The asterisk (*) indicates a significant difference from the control with P < 0.01.
(green fluorescence) as well as for monitoring mitochondrial localization and its membrane depolarization using CMXRos (red fluores-cence). As shown in Figure 3, AIF (green fluorescence) was observed in a punctuated cytoplasmic staining pattern, and the cellular mitochondria (red fluorescence) maintained their normal membrane potential in control cells. The yellow fluorescence of the merged image implies the co-localization of AIF protein and mitochondria. As time elapsed after Cd treatment, the distribution of green fluores-cence appeared more diffuse and was unmatch-able with CMXRos staining when overlaid (see the merged image at 4 h in Fig. 3), indicating that AIF had been released from mitochondria
into the cytosol. By 8 h after Cd treatment, AIF had eventually been translocated into the nucleus. It is worth noting that, as revealed by the disappearance of CMXRos red fluorescence (middle panel in Fig. 3), the decline of mitochon-drial membrane potential is a prelude to AIF re-distribution.
Suppression of Cd-Induced Apoptosis by Antioxidants
Cd is unable to produce reactive oxygen radicals (ROS) through the Fenton reaction. However, it does elevate lipid peroxidation in tissue soon after exposure [Stohs and Bagchi, 1995; Yang et al., 1997], suggesting that Cd might exhibit its cell toxicity through ROS.
Fig. 2. Inability of the broad-spectrum of caspase inhibitor, Z-VAD.fmk, to prevent apoptosis in MRC-5 cells by assessment of phosphatidylserine (PS) externalization. MRC-5 (panel A) and HL-60 (panel B) cells were treated with 25–150 and 150 mM
CdCl2 for 24 and 12 h, respectively, and with or without
pretreatment of 40 mM Z-VAD.fmk for 1 h. Subsequently, cells were collected and stained with an Annexin-V-FLUOS staining kit (Roche) and then immediately subjected to analysis of PS externalization (FL-1 level of FITC-annexin V fluorescence, X-axis) and PI uptake (FL-2 level of PI fluorescence, Y-axis) using flow cytometry. The Arabic number in each corner indicates the proportion of each quadrant. Cytograms of four quadrants was used to distinguish the normal, primary apoptotic, late apoptotic,
and necrotic cells by the criteria of annexin V/PI, annexin
Vþ/PI, annexin Vþ/PIþ, and annexin V/PIþ, respectively (see
Materials and Methods for details). The proportion of total
apoptosis was summed up from those of primary (annexin Vþ/
PI) and late apoptosis (annexin Vþ/PIþ). HL-60 cells were
rescued from Cd-induced caspase-dependent apoptosis by pre-treatment with 40 mM Z-VAD.fmk for 1 h (panel A).
Z-VAD.fmk-pretreated MRC-5 cells could not escape from CdCl2-induced
apoptosis (panel B). Data presented in panels (A) and (B) are representative of three independent experiments, and their statistical results for the proportions of total apoptosis are pre-sented in panel (C).
Several antioxidants were used to determine the involvement of ROS in Cd-induced caspase-independent apoptosis. NAC, a thiol antioxi-dant, can raise intracellular glutathione levels and thereby protect cells from the effects of ROS
[Aruoma et al., 1989]. Tiron and mannitol are superoxide anion- and hydroxyl radical-specific scavengers, respectively [Magovern et al., 1984; Ledenev et al., 1986]. MRC-5 fibroblasts were pretreated with these antioxidants for 1 h, and then Cd was added for another 16 h, which were then subjected to a hypodiploid DNA content assay using flow cytometry with PI staining. The data presented in Figure 4A are from one experiment typical of three, and the statistical results are illustrated in Figure 4B. These antioxidants strongly protected MRC-5 cells against Cd-induced apoptosis, indicating that ROS play a crucial role in the cytotoxicity of Cd. It is worth noting that tiron and mannitol had no synergistic effects (comparing bar 10 with bars 6 and 8 in Fig. 4B), and the extent of suppression was almost the same as that with tiron only, implying that the superoxide anion is more important than the hydroxyl radical in mediating the cell toxicity of Cd. On the other hand, as shown at the bottom of Figure 3, AIF translocation was abolished by NAC pretreat-ment, indicating that the ROS burst is an event which occurs upstream of AIF translocation.
Effects of Mitochondrial ETC and MPTP on Cd-Induced Apoptosis
In mammalian cells, ROS are mostly pro-duced as a by-product of aerobic metabolism in mitochondria. In fact, this is the greatest source of ROS, as the mitochondrial ETC consumes 85–90% of the oxygen utilized by the cell [Shigenaga et al., 1994]. Moreover, mitochon-dria-mediated ROS production is associated with the MPTP [Hail et al., 2001]. To investigate the role of mitochondria in Cd-induced apopto-sis, RT (an ETC complex I inhibitor), OA (an ETC complex V inhibitor), and aristolochic acid (ArA; short-term MPTP inhibitors) plus cyclos-porin A (CsA; long-term MPTP inhibitors) were used [Buchet and Godinot, 1998; Degli, 1998; Takeyama et al., 2002]. The inhibitor concen-trations used were determined empirically to ensure that their treatment alone would not promote cell toxicity. In cells pretreated with rotenone or OA for 1 h before continuous ex-posure to Cd for 16 h, the percentage of apoptotic cells (subG1 peak) was significantly suppressed (Fig. 5). Similar but minor suppres-sive effects were observed in the experiment using pretreatment with aristolochic acid plus cyclosporin A (Fig. 5). Combining these data with the results obtained from Figures 3 and 4,
Fig. 3. Cd-induced mitochondrial membrane depolarization, followed by translocation of AIF into the nucleus. MRC-5 cells were pretreated with or without 2.5 mM NAC for 1 h, followed by Cd treatment for the indicated time periods. Cells were then fixed and labeled with 100 nM MitoTracker Red, CMXRos (red fluorescence), and antibodies specific for AIF (revealed by an Cy2-conjugate, green fluorescence), and analyzed using con-focal microscopy. The CMXRos is selectively incorporated into mitochondria with an intact transmembrane potential and therefore serves as an indicator of mitochondrial localization and depolarization. Please note the co-localization of AIF and mitochondria in control cells (yellow fluorescence on merged images). After 3 h of Cd treatment, the mitochondria were more evidently depolarized, followed by AIF translocation into the cytoplasm (6 h) and then into the nucleus (8 h). Crucially, Cd-induced AIF translocation was abolished by NAC pretreatment, suggesting that ROS production plays a pivotal role in the apoptogenic activity of Cd. All photographs were taken at the same magnification. Bar, 25 mm.
Cd might depolarize the mitochondrial mem-brane potential and affect mitochondrial ETC and MPTP, and subsequently it would induce the release of mitochondrial pro-apoptotic fac-tors such as AIF (see Fig. 3), which, in turn, would lead cells to apoptosis.
To further investigate the contributions of mitochondrial ETC and MPTP to the mitochon-drial membrane potential (DCm), we monitored
cells with the mitochondria-specific probe, JC-1, a lipophilic cationic fluorescence dye with dual emission wavelengths. Once a decline in the mitochondrial membrane potential was induc-ed, the fluorescence of JC-1 increased at 530 nm (FL-1) in its monomeric form and fell at 590 nm (FL-2) as J-aggregates [Cossarizza et al., 1993]. As shown in Figure 6, the percentage of cells with normal mitochondrial potential (upper-left quadrant) decreased from 91% of control cells to 67% of Cd-treated cells within 8 h. Inhibition of mitochondrial ETC and MPTP only partially attenuated the effects of the Cd-induced decline
in DCm. These results obviously suggest that
mitochondrial depolarization is a prelude to Cd-induced apoptosis. Additionally, dedication of mitochondrial ETC and MPTP to DCmplays a
pivotal role in Cd-induced apoptosis.
Suppression of Cd-Induced H2O2Production
by Antioxidants and Inhibitors of Mitochondrial ETC and MPTP
As described above, our results suggest that Cd exhibits its cell toxicity through interfering with mitochondrial ETC and MPTP, elevating intracellular oxidative stress, and then in-ducing apoptotic cell death. Therefore, using flow cytometry with DCFH-DA staining, we next examined the generation of H2O2after Cd
administration as well as the connection of this event with mitochondrial functions. As shown in Figure 7A, the time course experiment indicated that fluorescence intensity increased about 2.9-folds with arbitrary units from 375 (control) to 1,074 (Cd treatment) after exposure
Fig. 4. Modulation of the apoptogenic activity of Cd by scavenging compounds. Cells were pretreated with scavenging compounds, such as 2.5 mM NAC, 5 mM tiron, and 40 mM mannitol, for 1 h and then treated with 100 mM Cd for another 16 h and subsequently analyzed by PI staining to determine their hypodiploid DNA (sub-G1) proportion. Data acquisition and analysis were performed on a FACSCalibur flow cytometer using
CellQuest software (Becton Dickinson). The percentage of M1 indicated the cell proportion of the sub-G1 peak. Data presented in panel (A) are representative of three separate experiments, and their statistical results are presented in panel (B) as the mean SD. The asterisk (*) indicates a significant difference from the control with P < 0.01.
to Cd for 3 h, and then decreased to even lower than the basal level after 8 h. Pretreatment with antioxidants such as NAC (a thio scavenger) and tiron (a superoxide scavenger) for 1 h were able to abolish Cd-induced H2O2 production
(Fig. 7B). However, only a partial scavenging effect could be detected by the addition of mannitol, a hydroxyl radical scavenger. To further investigate the role that mitochondria play in Cd-induced H2O2generation, cells were
treated with inhibitors of mitochondrial ETC (OA or RT) and MPTP (CsA plus ArA) for 1 h prior to treatment with Cd for another 3 h. Results are shown in Figure 7B and demon-strate that interference with mitochondrial ETC or maintenance of MPTP was able to sup-press Cd-induced H2O2generation. It is
impor-tant to note that treatment with Cd for 3 h accumulated almost the same amount of intra-cellular H2O2 in comparison to the addition of
400 mM H2O2 for 1 h (Fig. 7B). These results
suggest that Cd might affect the functions of mitochondria and subsequently induce the generation of ROS, which, in turn, leads cells to apoptosis. In conclusion, our data suggest that Cd induces caspase-independent apoptosis in MRC-5 fibroblasts through depolarization of the mitochondrial membrane potential and translocation of AIF from mitochondria into the nucleus. Moreover, the mitochondrial ETC and MPTP were early targets of Cd, which, in turn, caused the mitochondrial ROS to leak out, even-tually leading cells to apoptosis.
DISCUSSION
Cd is an environmental pollutant with a long biological half-life in humans and may consti-tute a menace to public health. Although Cd is not a Fenton metal, increasing evidence suggests that its toxicity is mediated by oxi-dative stress-induced apoptosis. However, the
Fig. 5. Suppression of the apoptogenic activity of Cd by inhibitors of mitochondrial ETC or MPTP. Cells were pretreated with mitochondrial ETC inhibitors (0.5 mM OA and 0.1 mM RT, for complexes I and V, respectively) or MPTP modulators (5 mM ArA, and 1 mM CsA) for 1 h, treated with 100 mM Cd for another 16 h, and subsequently analyzed by PI staining to determine their hypodiploid DNA (sub-G1) proportion. Data acquisition and
analysis were performed on a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson). The percentage of M1 indicated the cell proportion of the sub-G1 peak. Data presented in panel (A) are representative of three separate experiments, and their statistical results are presented in panel (B) as mean SD. The asterisk (*) indicates a significant difference from the control with P < 0.01.
molecular signaling underlying Cd-induced ROS production and apoptosis remains unclear. In the present study, we found that Cd (100 mM) was able to induce a 2.9-fold ROS burst through the mitochondrial pathway in normal human lung fibroblasts, MRC-5, and this consequently promoted a decline of mitochondrial membrane potential, which, in turn, led MRC-5 cells to
undergo caspase-independent apoptosis, with the hallmark of AIF being translocated from mitochondria into the cytosol and then into the nucleus.
In the past few years, Cd was demonstrated to induce caspase-dependent apoptosis in several cell lines [Kim et al., 2000; Li et al., 2000; Yuan et al., 2000; Shen et al., 2001; Kondoh et al., 2002], which would seem to contradict our results. Nevertheless, Robertson and Orrenius [2000], in their review paper which described an observation consistent with ours, said that caspase activity is not associated with the apoptogenic activity of Cd in porcine kidney LLC-PK1 cells [unpublished data from Ishido et al., 1999]. Harstad and Klaassen [2002] also indicated that Cd does not significantly increase caspase-3 activity in liver cells of a mouse model. These controversial results make it conceivable that Cd may induce different apoptotic pathways in different cell types. It is worth noting that Cd has been characterized as a caspase 3 inhibitor with IC50values of 8.7 and
31 mM in intact CHO cells and in a cell-free system, respectively [Yuan et al., 2000]. Thus, we must await further investigations into the scenario of the inhibitory effects of Cd on caspase 3 in this caspase-independent pathway. Combining the results shown in Figures 2 and 3, we demonstrate that Cd (25–150 mM)-induced apoptosis in MRC-5 cells is indepen-dent of caspase and operates via collapse of the mitochondrial membrane potential, followed by redistribution of AIF from the mitochondria into the cytosol and then into the nucleus. AIF has recently been characterized at the molec-ular level and was investigated as a novel mito-chondrial intermembrane flavoprotein with significant homology to bacterial and plant oxidoreductase [Susin et al., 1999]. The nucleus-encoded AIF is synthesized as a non-apoptogenic precursor in the cytoplasm and is efficiently imported into the mitochondria, followed by cleavage of the MLS. Once apoptosis is induced, AIF is translocated into the nucleus where it induces large-scale DNA fragmenta-tion (50 kb), which is typically associated with a wrinkled pattern of peripheral chromatin condensation in nuclei, a hallmark of stage I apoptosis. As cell death progresses, stage II apoptosis is achieved with morphology charac-terized by marked chromatin condensation and the formation of nuclear bodies [Susin et al., 2000; Cande et al., 2002]. In fact, the crystal
Fig. 6. Suppression of the Cd-induced decline of the mitochon-drial membrane potential by inhibitors of mitochonmitochon-drial ETC or MPTP. Cells were pretreated with mitochondrial ETC inhibitors (0.5 mM OA and 0.1 mM RT, for complexes I and V, respectively) or MPTP modulators (1 mM CsA and 5 mM ArA) for 1 h, treated with 100 mM Cd for another 8 h, and their mitochondrial membrane potential was subsequently analyzed by staining with 5 mg/ml JC-1 dye for 15 min, and the intensities of FL-1 and FL-2 fluorescences were immediately measured using flow cytometry. JC-1 fluorescence in the FL-1 channel increases as the mito-chondrial membrane potential drops and its fluorescence in the FL-2 channel decreases. Percentages given in the upper-left quadrant and right-two quadrants indicate the proportion of cells with normal and depolarized mitochondria, respectively.
structure of the AIF protein has been analyzed at 2.0 A˚ , and it has been proposed that AIF may form a dimer but without DNA binding activity [Mate et al., 2002]. Therefore, it is still an open conundrum, and an investigation into the
nuclear targets of AIF is worthwhile. In addi-tion, the mitochondria-derived proteins, endo-nuclease G (Endo G) [Li et al., 2001], and HtrA2/ Omi [Suzuki et al., 2001], have recently been identified as potential caspase-independent
Fig. 7. Cd promotion of intracellular H2O2accumulation and
the suppression of this event by scavenging compounds and inhibitors of mitochondrial ETC and MPTP. A: Cells were pretreated with 20 mM DCFH-DA for 20 min before the addition of 100 mM Cd for the indicated time periods and were trypsinized for immediate analysis on a flow cytometer to measure the oxidized DCF fluorescence in the FL-1 level (part a). The solid and dashed lines represent Cd-treated MRC-5 cells and each respective control, respectively. The intensity of the mean fluorescence (arbitrary units of geometric mean) calculated by CellQuest software was plotted as part b. Cd-treatments and their respective controls were expressed as closed squares (&) and open circles (*), respectively. B: Cells were pretreated with
20 mM DCFH-DA for 20 min together with or without scavenging compounds, as well as mitochondrial ETC and MPTP inhibitors for 1 h (concentrations as described in Fig. 6) before the addition of 100 mM Cd for 3 h. Otherwise, cells were pretreated with 20 mM
DCFH-DA for 20 min, followed by 400 mM H2O2for another
hour. Subsequently, cells were collected and analyzed as in panel A. The dashed line, solid line, solid line with hatched area, and solid line with black area represent the control,
Cd-treatment, H2O2-treatment, and Cd-treatment combined with
scavenging compounds, mitochondrial ETC, or MPTP inhibitors, respectively. Results are representative of three independent experiments.
apoptotic mediators. However, the relationships among AIF, Endo G, and HtrA2/Omi remain elusive.
There is a comprehensive agreement that mitochondria play a crucial role in apoptosis, but the mechanisms behind their involvement remain controversial. Herein, we provide sev-eral lines of evidence supporting a role for mitochondria in the induction of caspase-inde-pendent cell death triggered by Cd. First of all, using ETC inhibitors such as rotenone (RT; complex I inhibitor) or OA (complex V inhibitor) to interrupt the electron stream in mitochon-dria, we demonstrate that Cd-induced apoptosis (Fig. 5), mitochondria membrane depolariza-tion (Fig. 6), and H2O2production (Fig. 7) were
suppressed by these inhibitors, indicating that Cd may promote the leaking of ROS by mito-chondrial ETC which subsequently provokes cell toxicity. Second, using pretreatment with cyclosporin A (CsA) and aristolochic acid (ArA) to abolish the function of MPTP (Figs. 5–7), we illustrated that MPTP is a crucial component in mediating Cd toxicity. Creagh et al. [2000] showed that H2O2used at low (75 mM) and high
(300 mM) concentrations in Jurkat T cells induces caspase-dependent and -independent apoptosis, respectively. In this report, we indicated that 100 mM Cd induces a 2.9-fold intracellular H2O2 burst, and that this H2O2
amount is similar to that induced by treatment with 400 mM H2O2(Fig. 7B). Furthermore, Cd
induced MRC-5 cells to undergo caspase-inde-pendent apoptosis at varied concentrations from 25 to 150 mM (Fig. 2), and scavengers such as NAC, mannitol, and tiron suppressed this type of apoptosis (Fig. 4). More importantly, time course experiments of confocal microscopy and H2O2production support the notion that Cd
induced intracellular H2O2accumulated within
3 h (Fig. 7B) and mitochondrial depolarization occurred at the 4-h time point (see the CMXRos panel in Fig. 3), which was followed by redis-tribution of AIF (see the AIF panel in Fig. 3). Taken together, the most likely hypothesis assumes that Cd interacts directly or indirectly with mitochondria and promotes an elevation of intracellular ROS, which may intensively affect mitochondrial ETC and MPTP. After disruption of the mitochondrial transmembrane poten-tial, AIF is released from mitochondria and is translocated into the cytosol, and then into the nucleus, which eventually induces caspase-independent apoptosis.
Physiologically, caspase-independent apopto-sis is a crucial pathway in disease development. Jackson et al. [1998] reported a Drosophila model for Huntington’s disease where trans-genic baculovirus p35, an inhibitor of caspase [Xue and Horvitz, 1995; Miller, 1997], was incapable of reducing the extent of neuronal degradation. Consistent with this notion, the process of aging is likely to occur through a caspase-independent pathway. Caspase-knock out worms and flies do not have an altered life span [Borner and Monney, 1999]. In the case of yeast, caspases are missing from its genome [Ink et al., 1997]. However, it can be killed by various means such as oxidative stress, irradia-tion, and other toxic substances, but no endo-genous CED3/caspase, CED4, or Bcl-2/BAX is expressed. Nevertheless, forced overexpression of Bax, Bak, or CED-4 provokes vacuolarization and chromatin condensation as seen in mam-malian apoptosis, indicating that these death factors can indeed provoke a caspase-indepen-dent form of apoptosis in a unicellular organism [Borner and Monney, 1999]. Recently, heat-shock protein 70 (Hsp70) has been charac-terized as a death determinant despite the involvement of caspases. The anti-apoptotic activity of Hsp 70 was demonstrated through the direct interaction with apoptosis protease-activating factor-1 (Apaf-1) [Creagh et al., 2000] or AIF-1 [Xanthoudakis and Nicholson, 2000; Ravagnan et al., 2001] in a caspase-dependent or -independent pathway, respectively.
In conclusion, this study demonstrates that the apoptogenic activity of Cd in MRC-5 cells, normal human lung fibroblasts, occurs through disturbing the mitochondrial ETC, followed by a ROS burst, leading to collapse of the mitochon-drial membrane potential and affecting the MPTP. Finally, the mitochondria-confined AIF is translocated into the nucleus where it induces a caspase-independent apoptosis.
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