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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 241-248, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4903

The Mitochondria-Regulated Death Pathway Mediates Asbestos-Induced Alveolar Epithelial Cell Apoptosis

Vijayalakshmi Panduri, Sigmund A. Weitzman, Navdeep Chandel and David W. Kamp

Department of Medicine, Divisions of Pulmonary and Critical Care Medicine and Hematology-Oncology, Northwestern University Feinberg School of Medicine; and Veterans Administration, Chicago Health Care System, Lakeside Division, Chicago, Illinois

Address correspondence to: David W. Kamp, Northwestern University Feinberg School of Medicine, Division of Pulmonary and Critical Care Medicine, Tarry Building 14-707, 303 E. Chicago Ave., Chicago, IL 60611-3010. E-mail: d-kamp{at}nwu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 RESULTS
 Discussion
 References
 
The mechanisms underlying asbestos-induced pulmonary toxicity are not fully understood. Alveolar epithelial cell (AEC) apoptosis by iron-derived reactive oxygen species (ROS) is one important mechanism implicated. The two major pathways regulating apoptosis include (i) the mitochondrial death (intrinsic) pathway caused by DNA damage, and (ii) the plasma-membrane death receptor (extrinsic) pathway. However, it is unknown whether asbestos activates either death pathway in AEC. We determined whether asbestos triggers AEC mitochondrial dysfunction by exposing cells (A549 and rat alveolar type II) to amosite asbestos and assessing mitochondrial membrane potential changes ({Delta}{psi}m) using a fluorometric technique involving tetremethylrhodamine ethyl ester (TMRE) and mitotracker green. Unlike inert particulates (titanium dioxide and glass beads), amosite asbestos caused dose- and time-dependent reductions in {Delta}{psi}m. Asbestos-induced {Delta}{psi}m was associated with the release of cytochrome c from the mitochondria to the cytoplasm as well as activation of caspase 9, a mitochondrial-activated caspase. In contrast, a lower level of caspase 8, the death receptor–activated caspase, was detected in asbestos-exposed AEC. An iron chelator (phytic acid or deferoxamine) or a hydroxyl radical scavenger (sodium benzoate) each blocked asbestos-induced reductions in {Delta}{psi}m and caspase 9 activation, suggesting a role for iron-derived ROS. Finally, Bcl-XL, a mitochondrial antiapoptotic protein that prevents cell death by preserving the outer mitochondrial membrane integrity, blocked asbestos-induced decreases in A549 cell {Delta}{psi}m and reduced apoptosis as assessed by DNA fragmentation. We conclude that asbestos-induced AEC apoptosis results from mitochondrial dysfunction, in part due to iron-derived ROS, which is followed by the release of cytochrome c and caspase 9 activation. Our findings suggest an important role for the mitochondria-regulated death pathway in the pathogenesis of asbestos-associated pulmonary toxicity.

Abbreviations: mitochondrial membrane potential changes, {Delta}{Psi}m • alveolar epithelial cells, AEC • alveolar type II, AT2 • Dulbecco's modified Eagle's medium, DMEM • dimethylsulfoxide, DMSO • dimethylthiourea, DMTU • DNA strand breaks, DNA-SB • fetal bovine serum, FBS • hydrogen peroxide, H2O2 • Hanks' balanced salt solution, HBSS • N-2-hydroxy-ethylpiperazine-N'-2-ethansulfonic acid, HEPES • mitochondrial intermembrane space, MIMS • mitotracker green, MITO • superoxide anion, O2- • hydroxyl radical, ·OH • phosphate-buffered saline, PBS • reactive oxygen species, ROS • titanium dioxide, TiO2 • tetremethylrhodamine ethyl ester, TMRE • tumor necrosis factor-{alpha}, TNF-{alpha} • terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate-biotin nick end labeling, TUNEL


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 RESULTS
 Discussion
 References
 
Asbestos exposure has been firmly implicated in causing asbestosis, pleural disease, and malignancies (bronchogenic carcinoma and mesothelioma), but the mechanisms involved are not fully understood (1, 2). The extent of alveolar epithelial cell (AEC) injury and repair are critical determinants of the fibrogenic potential of toxins, including asbestos (2, 3). Asbestos is internalized by AEC soon after exposure, resulting in cellular injury, increased permeability, and a proliferative response by alveolar type II (AT2) cells (1, 2, 4). Accumulating evidence implicates reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide anion (O2-), and the hydroxyl radical (•OH), as important second messengers of asbestos toxicity (5, 6). We recently demonstrated that iron-derived ROS mediate asbestos-induced AEC DNA damage and apoptosis (7, 8). However, the molecular mechanisms by which ROS derived from asbestos fibers cause AEC apoptosis are unknown.

Apoptosis is a highly regulated physiologic cell death process that is critical for development, host defense, and the prevention of malignant transformation and inflammation throughout the body, including the lungs (3, 9, 10). The two major mechanisms regulating apoptosis include (i) the intrinsic pathway mediated by the mitochondria, and (ii) the extrinsic pathway induced by death-signaling ligands, such as tumor necrosis factor (TNF)-{alpha} or FasL, and subsequent caspase 8 activation (9–11). Oxidant-induced DNA damage is one stimulus that can activate the intrinsic pathway, resulting in permeabilization of the outer mitochondrial membrane detected by a change in the mitochondrial membrane potential ({Delta}{psi}m). This is followed by the release of agents normally confined to the mitochondrial intermembrane space (MIMS) such as cytochrome c, Smac/diablo, procaspases 2, 3, and 9, and apoptosis-inducing factor (9–11). Because the mitochondria are a major site for the generation of ROS, disruption of mitochondrial electron transport can augment ROS production, which further amplifies an apoptotic stimulus (9–11). Apoptotic stimuli are modulated by the Bcl family of antiapoptotic proteins that prevent apoptosis by inhibiting the {Delta}{psi}m and subsequent release of mitochondrial cytochrome c into the cytoplasm (12, 13). The antiapoptotic Bcl-2 family members (e.g., Bcl-2 and Bcl-XL) localize to the mitochondrial membrane, whereas proapoptotic proteins (e.g., Bax, Bak, and Bid) translocate from the cytosol to the mitochondrial membrane to induce cell death (12, 13). Further, either Bax or Bak is required to activate the intrinsic apoptotic pathway after exposure to DNA-damaging agents, such as etoposide or radiation, as well as by serum deprivation (14). However, the role of these death pathways in regulating asbestos-induced apoptosis is unknown.

Given the above findings, we reasoned that asbestos-induced AEC cell apoptosis is mediated by the mitochondrial death pathway caused by the generation of iron-derived free radicals. We recently noted that asbestos (25 µg/cm2)-induced AEC apoptosis is preceded by a reduction in {Delta}{psi}m as assessed by a fluorometric technique (15). Herein, we extend these initial observations by showing that amosite asbestos, but not inert particulates (titanium dioxide [TiO2] or glass beads), causes a dose- and time-dependent reduction in AEC {Delta}{psi}m. Further, we demonstrate that the {Delta}{psi}m is accompanied by the release of cytochrome c and caspase 9 activation, but not caspase 8 activation. Moreover, a role for iron-derived ROS was suggested by our findings that an iron chelator (phytic acid or deferoxamine) or a scavenger of •OH (sodium benzoate) each blocked asbestos-induced reductions in {Delta}{psi}m and caspase 9 activation. Finally, asbestos-induced reductions in A549 cell {Delta}{psi}m and apoptosis were blocked by Bcl-XL overexpression. These data suggest that asbestos-induced pulmonary toxicity can result from altered apoptotic mechanisms in the alveolar epithelium that are regulated by the mitochondria and the levels of iron-derived ROS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
RESULTS
 Discussion
 References
 
Mineral Dusts
Amosite asbestos fibers used in these experiments were Union International Centere le Cancer Reference Standard samples supplied by Dr. V. Timbrell (16). The amosite fibers are amphiboles that are 70% respirable (length between 2–5 µm), whereas the remainder are > 5 µm in length. TiO2 and glass beads were obtained from Sigma (St. Louis, MO). Stock solutions (5 mg/ ml) of each particulate were prepared in Hanks' balanced salt solution (HBSS) with calcium, magnesium, and 15 µM N-2-hydroxyethyipiperazine-N'-2-ethanesulfonic acid (HEPES). All suspensions were autoclaved and stored at 4°C. Samples were warmed to 37°C and vigorously vortexed before usage to ensure a uniform suspension.

Cell Culture
A549 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). A549 cells, which are human bronchoalveolar carcinoma-derived cells with some features characteristics of alveolar epithelial type II cells, were maintained in Dulbecco's modified Eagle Medium (DMEM) supplemented as above except for non essential amino acids. Bcl-XL overexpressing A549 cells and control neomycin vector A549 cells were prepared as previously described (15, 17). In select experiments, primary isolated rat AT2 cells were obtained by elastase digestion as previously described (7, 8).

Mitochondrial Membrane Potential
AEC mitochondrial membrane potential ({Delta}{psi}m) was assessed using a fluorometric technique previously described by our laboratory using the probe tetremethylrhodamine ethyl ester (TMRE) (15, 17). Briefly, A549 cells were plated in six-well plates at a seeding density of 1 x 106 and grown to confluence over 24 h in DME with 10% FBS. To limit cell proliferation, the media was changed to DMEM with 0.5% FBS for an additional 24 h. In some experiments, inhibitors (e.g., phytic acid, deferoxamine, or sodium benzoate) were added ~ 5 min before fiber exposure. After incubation for various periods (1–48 h), all the cells were collected and then exposed to either TMRE (500 nM; Molecular Probes, Eugene, OR) or Mitotracker green (MITO, 1 µM; Molecular Probes) for 1 h at 37°C. Carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP, 20 µM; Sigma) was added to a separate group of comparably treated cells for 1 h before adding fluorochromes to induce a maximal {Delta}{psi}m by uncoupling oxidative phosphorylation and eliminating the mitochondrial proton gradient. Changes in dye fluorescence at 25°C were analyzed in a fluorometer using an excitation wavelength of 550 or 488 nm, respectively, for TMRE and Mitotracker green and an emission wavelength of 520 or 580 nm, respectively. The MITO labels the mitochondria because it binds mitochondrial lipids and is not influenced by the {Delta}{psi}m caused by FCCP (17). TMRE is one of the preferred fluorochromes to monitor the {Delta}{psi}m, because in the nM range TMRE exclusively stains the mitochondria and is not retained in cells upon collapse of the {Delta}{psi}m. The {Delta}{psi}m was compared qualitatively based upon the percentage difference in the ratio of TMRE and MITO fluorescence of untreated cells (Tc and MGc, respectively) corrected for the background fluorescence in FCCP-treated control cells (FTc and FMGc) and the ratio of TMRE and MITO fluorescence of treated cells (Tt and MGt) minus the FCCP-treated cells (FTt and FMGt, respectively) as defined in Equation 1:

(1)

Cytochrome c Assay
Cytosolic and mitochondrial subcellular preparations of AEC were prepared as previously described (18). Briefly, cytosolic fractions were obtained from A549 cells that were treated as described above by scraping cells in ice-cold buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol). The cells were then homogenized for 20 s and the unlysed cells and nuclei were collected by centrifugation at 800 x g for 10 min. The supernatant containing the cytosol and mitochondria were collected and then separated by ultra-centrifugation at 22,000 x g for 15 min. Proteins in the supernatant (cytosolic fraction) and the pellet (mitochondrial fraction) were quantified by the Bio-Rad protein assay (Life Science Research, Hercules, CA). We used a cytochrome c ELISA (R&D Systems, Minneapolis, MN) to quantify the levels of cytochrome c in the cytosolic and mitochondrial fractions according to the manufacturer's protocol. The ratio of cytosolic cytochrome c (relative absorbency units per µg protein) to mitochondrial cytochrome c (relative absorbency units per µg protein) was determined and the data expressed as the percent change from control value. Cytochrome c release from the mitochondria to the cytosol was defined by an increased ratio of cytosolic to mitochondrial cytochrome c as compared with control cells.

Caspase Activity Assays
A fluorometric assay kit specific for caspase 9 and caspase 8 (R&D Systems) was used as a measurement of the intrinsic and extrinsic apoptotic death pathways, respectively. AEC were plated onto six-well plates and treated with inhibitors and asbestos as described above. Caspase activity was determined according to the manufacturer's protocol using a fluorescent microplate reader and normalized to the total protein concentration as determined by the Bio-Rad protein assay.

Apoptosis Assays
Asbestos-induced A549 cell apoptosis was assessed by nuclear morphology as previously described (8). We also used an ELISA assay (Roche Diagnostics, Indianapolis, IN) that detects histone-associated DNA fragments (mono- and oligonucleosomes) that was performed according to the manufacturer's specifications (spectrophotometric wavelength: 405 nm).

Statistical Analysis
The results of each experiment condition were determined from the mean of triplicate trials. The data are expressed as the mean ± SE. A two-tailed Student's t test was used to assess the significance of differences between two groups. ANOVA was used when comparing more than two groups; differences between two groups within the set were analyzed by a Fisher's protected least significant difference test. Probability values < 0.05 were considered significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 RESULTS
Discussion
 References
 
Asbestos Causes AEC Mitochondrial Dysfunction
To determine whether asbestos causes mitochondrial dysfunction in alveolar epithelial cells, we assessed A549 cell {Delta}{psi}m using a fluorometric technique that measures the fluorescence ratio of TMRE (dependent upon {Delta}{psi}m) and MITO (dependent upon the number of mitochondria and not the {Delta}{psi}m). As shown in Figure 1 , amosite asbestos, as compared with control, decreased A549 cell {Delta}{psi}m in a dose- and time-dependent manner. After 4 h, a time point where we see little cell death (7), asbestos (25 µg/cm2) significantly reduced {Delta}{psi}m by 27%. Amosite asbestos (25 µg/cm2) also reduced the {Delta}{psi}m in primary isolated rat AT2 cells (-55.2 ± 8.0% reduction versus control; P = 0.001; n = 7). Notably, the deleterious effects on AEC mitochondrial dysfunction were confined to particulates known to be toxic to the lungs (asbestos) whereas glass beads and TiO2, two relatively inert particulates, caused negligible {Delta}{psi}m (Figure 2) . These findings are consistent with our prior observation that asbestos, but not TiO2, induced apoptosis after a 24-h exposure period as assessed by nuclear morphology, caspase 3 activation, and DNA fragmentation (8, 15). Taken together, these data suggest that the mitochondria (intrinsic pathway) regulate asbestos-induced apoptosis.



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  		Figure 1. Asbestos reduces A549 cell mitochondrial membrane potential in a dose- and time-dependent manner. (Left panel) Dose–response: Amosite asbestos (1–50 µg/cm2) reduces mitochondrial membrane potential ({Delta}{psi}m) assessed by a fluorometric technique (see MATERIALS AND METHODS) after a 4-h exposure period. (Right panel) Time Course: Amosite asbestos (25 µg/cm2) induces progressive reduction in A549 cell {Delta}{psi}m over 48 h. Data expressed as the mean ± SEM, n = 6. *P < 0.05 versus control.

 


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  		Figure 2. Asbestos, but not glass beads or TiO2, reduces {Delta}{psi}m. A549 cells were exposed for 4 h (solid bars) or 24 h (stippled bars) to control media, amosite asbestos (25 µg/cm2), glass beads (25 µg/cm2), or TiO2 (25 µg/cm2), washed, and then {Delta}{psi}m was assessed by a fluorometric technique (see MATERIALS AND METHODS). Data expressed as the mean ± SEM, n >= 7. *P < 0.05 versus control.

 
Asbestos Causes AEC Release of Mitochondrial Intermembrane Proteins
To further delineate the role of the mitochondria in regulating asbestos-induced AEC apoptosis, we determined whether asbestos triggers the release of cytochrome c from the mitochondria into the cytoplasm. A549 cells were exposed to amosite asbestos (25 µg/cm2) for variable periods (0, 4, 16, or 24 h) and then subcellular fractions containing cytosolic and mitochondrial proteins were prepared. As compared with control, asbestos increased cytochrome c release from the cytoplasm to the mitochondria (Figure 3) . Asbestos-induced cytochrome c release was evident as soon as 4 h (26% increase versus. control; P < 0.05), a time point that was associated with a significant reduction in AEC {Delta}{psi}m (Figure 1), and increased further after 24 h (37% increase versus. control; P < 0.05). Release of cytochrome c stimulates assembly of the apoptosome in the cytosol, which subsequently converts procaspase 9 (which is also released from the MIMS) to an active caspase 9 by an ATP-dependent process (9, 11, 12). Caspase 9 in turn activates caspase 3, an executioner caspase that we previously showed was activated in asbestos-exposed AEC undergoing apoptosis (8). To assess whether cytochrome c release caused caspase 9 activation, A549 cells were exposed to various doses of amosite asbestos (0, 10, 25, or 50 µg/cm2) for 24 h and then cytosolic subcellular fractions were prepared. As compared with control, asbestos increased caspase 9 activation in a dose-dependent manner (Figure 4) . Maximal AEC caspase 9 activation occurred with a dose of asbestos of 25 µg/cm2 that reproducibly causes AEC apoptosis (80% increase versus. control, P < 0.05) (8). In contrast to caspase 9, we detected a modest increase in activated caspase 8 levels, which is involved in the death receptor (extrinsic) apoptotic pathway, but these differences did not reach statistical significance (Figure 4). Amosite asbestos (25 µg/cm2) also increased caspase 9 activity in primary isolated rat AT2 cells (39 ± 9% increase versus control; P < 0.007; n = 6). Collectively, these data firmly implicate a mitochondria-regulated (intrinsic) apoptotic pathway in mediating asbestos-induced AEC apoptosis.



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  		Figure 3. Asbestos stimulates cytochrome c release from the mitochondria to the cytoplasm. A549 cells were exposed for various periods (0–24 h) to amosite asbestos (25 µg/cm2), washed, and then protein from cytoplasmic and mitochondrial fractions were obtained by ultracentrifugation of lysed cells as described in MATERIALS AND METHODS. The amount of cytochrome c in each fraction was determined by an ELISA and the data expressed as the percentage change from control based upon the ratio of the cytoplasmic to mitochondrial cytochrome c relative absorbence units per µg of protein. *P < 0.05 versus control; n = 6.

 


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  		Figure 4. Asbestos activates greater levels of caspase 9 than caspase 8. A549 cells were exposed for 24 h to various doses of amosite asbestos (0–50 µg/cm2), washed, and then protein from lysed cells was obtained as described in MATERIALS AND METHODS. The amount of caspase 9 (mitochondria death pathway) and caspase 8 (death receptor caspase) was determined by an ELISA and the data expressed as the percentage change from control based upon the relative absorbency units per µg of protein. *P < 0.05 versus control; n = 6.

 
Iron-Derived Free Radicals Mediate Asbestos-Induced AEC {Delta}{psi}m and Caspase 9 Activation
We previously demonstrated that an iron chelator, phytic acid or deferoxamine, or sodium benzoate, a •OH scavenger, each inhibited asbestos-induced AEC apoptosis (8). To determine whether iron-derived free radicals are involved in mediating asbestos-associated AEC {Delta}{psi}m, we examined the protective effects of phytic acid, deferoxamine, or sodium benzoate. As shown in Figure 5 , deferoxamine (1 mM), phytic acid (500 µM), or sodium benzoate (100 µM) each completely prevented the reductions in asbestos-induced {Delta}{psi}m after a 4-h exposure period as well as caspase 9 activation at 24 h. Each agent alone had negligible effects on A549 cell {Delta}{psi}m over the 4-h incubation period (data not shown). A specific role for iron was suggested by our earlier studies, demonstrating that FeCl3 (500 mM)-treated phytic acid blocked the protective effects of phytic acid in preventing AEC apoptosis (8) or DNA-SB formation (7). These findings support a role for iron-derived ROS in mediating asbestos-induced AEC mitochondrial dysfunction.



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  		Figure 5. Asbestos-induced mitochondrial dysfuntion and caspase 9 activation is mediated by iron-derived free radicals. A549 cells were exposed for 24 h to amosite asbestos (25 µg/cm2) in the presence or absence of an iron chelator (deferoxamine [DF; 1 mM]) or phytic acid [PA; 500 µM]) or a •OH scavenger (sodium benzoate (NB; 100 µM). After incubation, the cells were washed and the {Delta}{psi}m was assessed by a fluorometric technique described above, whereas caspase 9 was assessed by an ELISA from protein obtained from lysed cells as described above. Asbestos-induced reductions in {Delta}{psi}m as well as caspase 9 activation were each blocked by an iron chelator or a •OH scavenger. *P < 0.05 versus control; {dagger}P < 0.05 versus asbestos; n >= 4.

 
Bcl-XL Overexpression Blocks Asbestos-Induced AEC {Delta}{psi}m and Reduces Apoptosis
Previous studies have shown that Bcl-XL prevents the {Delta}{psi}m and subsequent apoptosis after exposure to a variety of apoptogenic agents (12, 17, 18). We reported that A549 cells exposed to asbestos for 24 h, as compared with controls, have an ~ 80% reduction in mRNA for Bcl-XL as assessed by DNA array techniques (15). This suggests that reduced expression of Bcl-XL is one mechanism that AEC are susceptible to the toxic effects of asbestos. To determine whether Bcl-XL protects against asbestos-induced AEC mitochondrial dysfunction, we assessed the {Delta}{psi}m in Bcl-XL overexpressing A549 cells exposed to amosite asbestos as compared with control vector overexpressing cells. As shown in Figure 6 , Bcl-XL overexpressing A549 cells completely blocked asbestos-induced {Delta}{psi}m after a 4-h exposure period. At 24 h, we noted a negligible {Delta}{psi}m in the Bcl-XL overexpressing A549 cells exposed to asbestos (25 µg/cm2), a dose that causes AEC apoptosis (8) and mitochondrial dysfunction (Figure 1). Further, a higher dose of asbestos (50 µg/cm2) caused less {Delta}{psi}m in the Bcl-XL overexpressing A549 cells as compared with vector controls.



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  		Figure 6. Overexpression of Bcl-XL attenuates asbestos-induced mitochondrial dysfunction. A549 cells that overexpress Bcl-XL (stippled bars) were prepared as described in MATERIALS AND METHODS, and neo-vector expressing cells (solid bars) were selected based upon growth in Geneticin-containg media. (Left panel) Overexpression of Bcl-XL completely blocked amosite asbestos-induced {Delta}{psi}m at 4 h as compared with neo-vector control cells. (Right panel) After a 24-h exposure period, overexpression of Bcl-XL prevented asbestos (25 µg/cm2)-induced reduction in {Delta}{psi}m and attenuated asbestos (50 µg/cm2)-induced reduction in {Delta}{psi}m by 50%. Data expressed as the mean ± SEM, n = 6. *P < 0.05 versus control. {dagger}P < 0.05 versus Bcl-XL overexpressing cells.

 
To determine whether Bcl-XL overexpression reduces asbestos-induced AEC apoptosis, we used a DNA nucleosomal fragmentation assay that we previously showed directly correlates with asbestos-induced AEC apoptosis as assessed by nuclear morphology and caspase 3 activation (8, 15). As shown in Figure 7 , Bcl-XL overexpression reduced asbestos-induced A549 DNA fragmentation after a 24-h exposure period. As expected, asbestos (25 µg/cm2) increased the levels of DNA fragmentation as compared with unexposed Neo control (6-fold increase over control; P < 0.05). As compared with Neo control cells, asbestos induced significantly less DNA fragmentation in Bcl-XL overexpressing A549 cells (6-fold versus 2-fold increase, respectively; P < 0.05). Further, the protective effects observed were comparable to protection afforded against asbestos-induced reductions in {Delta}{psi}m.



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  		Figure 7. Bcl-XL overexpression attenuates asbestos-induced apoptosis. Bcl-XL overexpressing and Neo-vectors control A549 cells were exposed for 24 h to various doses of amosite asbestos (0–50 µg/cm2) and then DNA nucleosomal fragmentation was determined by an ELISA as described in MATERIALS AND METHODS. Bcl-XL overexpression reduced asbestos-induced DNA fragmentation by 2- to 3-fold as compared with neo-vector control cells. *P < 0.05 versus Bcl-XL overexpressing A549 cells; n = 6.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 RESULTS
 Discussion
References
 
Asbestos causes apoptosis to important target cells in the lungs, including AEC (1, 2, 8, 19, 20). Altered apoptotic mechanisms are implicated in causing pulmonary toxicity under a variety of pathologic conditions, including asbestos exposure, but the mechanisms involved are unclear. The major findings of this study are that asbestos induces mitochondrial dysfunction as assessed by several different assays, and that iron-derived free radicals in part mediate these effects. Our study suggests that the mitochondria regulate apoptotic cell death in alveolar epithelial cells exposed to asbestos.

The mitochondria have a critical role in regulating apoptosis by a diverse group of apoptogenic agents (11). In this study, we present several lines of evidence demonstrating that asbestos causes AEC mitochondrial dysfunction that may be important in mediating apoptosis. First, using a standard fluorometric technique to assess the {Delta}{psi}m, we showed that amosite asbestos, but not inert particulates such as glass beads and TiO2, caused a reduction in the {Delta}{psi}m in a dose- and time-dependent manner (Figures 1 and 2). Asbestos-induced decreases in A549 cell {Delta}{psi}m is an early event, since abnormalities were noted as soon as 4 h after asbestos exposure, a time period where negligible cell death is evident in our model but is clearly present at 24 h (7). Second, we demonstrated that asbestos triggered the release of cytochrome c and activated caspase 9, a mitochondria-dependent caspase (Figures 3 and 4). In contrast, lower levels of caspase 8 activation, which is an important component of the death receptor pathway, was detected in our model. Finally, we found that A549 cells overexpressing Bcl-XL, a mitochondrial protein, were protected against asbestos-induced {Delta}{psi}m and apoptosis (Figures 6–7). Collectively, these data firmly implicate that asbestos induces the mitochondrial (intrinsic) death pathway in AEC.

DNA damage is a potent stimulus that triggers apoptosis (3, 11, 21). We previously showed that asbestos causes AEC DNA strand break formation as well as apoptosis (7, 8). Data presented in this study implicating an important role of the mitochondria in regulating asbestos-induced AEC apoptosis are consistent with prior studies showing that the mitochondria are crucial for mediating apoptosis after DNA damage caused by staurosporin, ultraviolet radiation, etoposide, thapsigargin, tunicamycin, and anoxia (14, 18). Asbestos fibers are actively phagocytized by AEC shortly after exposure, presumably with the assistance of mitochondria (22, 23). Interestingly, for reasons that are not well understood, mtDNA, as compared with nuclear DNA, is more susceptible to oxidative damage and has a 10-fold greater mutation rate (24, 25). Fliss and associates (26) recently showed that mitochondrial p53 DNA was mutated at a frequency that was 19–220 times greater than nuclear p53 DNA among the 41 human lung, bladder, and head and neck cancers that they studied. One possible explanation for the susceptibility of mtDNA is that some carcinogens, such as polyaromatic hydrocarbons, preferentially bind mtDNA (24). Taken together, these data suggest that asbestos-induced pulmonary toxicity is mediated in part by AEC apoptosis due to mitochondrial dysfunction.

Oxidant stress is one of the most reproducible triggers of apoptosis (27). In this study, we showed that an iron chelator (phytic acid and deferoxamine) or a •OH scavenger (sodium benzoate) each completely blocked the {Delta}{psi}m as well as caspase 9 activation (Figure 5). We previously showed that each of these inhibitors block AEC apoptosis as assessed by nuclear morphology and caspase 3 activation (8). Phytic acid and deferoxamine, which are both membrane permeable iron chelators that occlude all reactive coordination sites for iron rendering it redox cycling inert (28), can prevent iron-derived free radical production within cells (7, 20, 28–30). Our data implicating a role for iron-derived ROS in mediating AEC mitochondrial dysfunction and apoptosis are consistent with the work of others showing that iron chelators attenuate asbestos-induced mesothelial cell apoptosis (20), iron-induced mitochondrial DNA damage of rat hepatoma cells (29) and H2O2-induced {Delta}{psi}m and astrocyte cell death (30). We previously showed that iron-loaded phytic acid does not prevent asbestos-induced •OH production, DNA damage, apoptosis, or cell death, suggesting a specific role for iron in our model (7, 8). Collectively, these data firmly imply that iron-derived free radicals mediate asbestos-induced AEC mitochondrial dysfunction, caspase 9 activation, and apoptosis.

As reviewed elsewhere (27), there are multiple sources of ROS production in cells that are exposed to apoptogenic stimuli, such as asbestos. These include the mitochondria, NAD(P)H oxidoreductases (e.g., cytochrome P-450 and nitric oxide synthase), molybdenum hydroxylases (e.g., xanthine oxidoreductase and aldehyde reductase), and arachidonic acid metabolizing enzymes (e.g., cyclooxygenase and lipoxygenase). Moreover, numerous studies have established that the iron associated with the asbestos fibers are redox cycling active which facilitates propagation of the Fenton reaction resulting in the formation of the highly toxic •OH (2, 5, 6). Recent evidence suggest that mitochondrial glutathione (GSH) is critical for preventing oxidant stress and apoptosis from a variety of agents, including TNF-{alpha}, H2O2, and ceramide (27, 31). Notably, cytochrome c is an iron-containing, redox cycling protein whose redox state is largely controlled by intracellular GSH levels (32). The observation that oxidized cytochrome c is required for apoptosis in many models supports the hypothesis implicating a role for GSH in regulating apoptosis (32). Although we have not examined mitochondrial GSH levels in our model, we previously showed that asbestos reduces AEC total GSH levels (33). Thus, the above studies combined with our current findings suggest that asbestos-induced iron-derived ROS resulting from mitochondrial dysfunction can have important pathophysiologic consequences.

The ability of Bcl-XL to prevent asbestos-induced {Delta}{psi}m and DNA fragmentation suggests that the permeability of the outer mitochondrial membrane is a crucial regulating target for asbestos-exposed AEC. Our findings with asbestos-exposed AEC are in keeping with accumulating evidence in other models demonstrating that anti-apoptotic Bcl-2 family members, such as Bcl-XL, prevent apoptosis via the intrinsic pathway in part by inhibiting the {Delta}{psi}m and release of cytochrome c (11–13, 17, 18). The molecular mechanisms underlying the protective effects of Bcl-XL are not firmly established, but two models are primarily implicated (10, 12). First, a direct interaction between pro- and antiapoptotic Bcl-2 family members is one mechanism that can block the formation of large channels in the mitochondrial membrane caused by proapoptotic Bcl-2 family members that promote the release of cytochrome c as well as other apoptogenic stimuli. However, Bcl-XL can maintain its protective effect in the presence of mutations that block heterodimerization with Bax or Bak (34). In the second model, mitochondrial matrix swelling, either due to formation of the permeability transition pore (PTP) or closure of the voltage-dependent anion conducting channel (VDAC), can rupture the outer mitochondrial membrane (35, 36). Apoptogenic agents can open the PTP and thereby allow equilibration of ions across the mitochondrial membrane, a reduction in {Delta}{psi}m, uncoupling of the electron transport chain, mitochondrial swelling, and release of cytochrome c (11–13). Survival in Bcl-XL overexpressing cells requires an efficient exchange of glycolytic ATP across VDAC to maintain a mitochondrial membrane potential (18, 35, 36). Further studies are necessary to address the mechanism by which asbestos reduces the {Delta}{psi}m and how Bcl-XL is protective. However, the above data firmly implicate that the mitochondria have a critical role in orchestrating asbestos-induced AEC apoptosis.

There is some in vivo evidence supporting our in vitro finding that DNA damage due to iron-derived ROS mediates pulmonary toxicity from asbestos (2, 5, 6). Notably, the presence of •OH has been documented in rat lungs exposed to iron-loaded chrysotile asbestos (37). Also, deferoxamine and antioxidant enzymes diminish asbestos-induced murine mesothelial cell apoptosis (20, 38) as well as asbestos uptake into rat tracheal epithelial explants (39). We reported that phytic acid decreases inflammation and fibrosis in rat lungs 2 wk after a single, intratracheal instillation of amosite asbestos (40). Further, we demonstrated that asbestos causes apoptosis in cells at the bronchiolar–alveolar duct junctions as assessed by TUNEL staining (8). DNA damage may be a prototypical mechanism underlying the pathobiology of pulmonary fibrosis in general, because DNA strand breaks and apoptosis occur in bronchiolar and AEC of patients with idiopathic pulmonary fibrosis (41).

In summary, we showed that asbestos induces AEC mitochondrial dysfunction as assessed by a reduction in AEC {Delta}{psi}m, mitochondrial cytochrome c release, caspase 9 activation, and by the protective effects of Bcl-XL. Further, our data implicate that iron-derived ROS in part mediate these effects. We speculate that the mitochondria have an important role in regulating AEC survival after asbestos exposure. Altered apoptotic mechanisms, as may occur from low-dose asbestos exposure, may promote the formation of a malignant clone of cells harboring mutated DNA. In contrast, pulmonary fibrosis may result from excess apoptosis as may occur with high-dose asbestos exposure or other fibrogenic agents (e.g., bleomycin). Future investigations are necessary to address these possibilities. Our data suggest that asbestos pulmonary toxicity may be reduced by strategies aimed at decreasing the levels of iron-derived free radicals and mitochondrial dysfunction in the alveolar epithelium.


   Acknowledgments
 
This work was supported by a Merit Review grant from the Department of Veterans Affairs (D.W.K.) and by GM60472-03 from the National Institutes of Health (N.C.).

Received in original form May 21, 2002

Received in final form August 29, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 RESULTS
 Discussion
 References
 

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