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Published ahead of print on February 21, 2003, doi:10.1165/rcmb.2002-0269OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 180-187, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0269OC

Particulate Matter Induces Alveolar Epithelial Cell DNA Damage and Apoptosis

Role of Free Radicals and the Mitochondria

Daya Upadhyay, Vijayalakshmi Panduri, Andrew Ghio and David W. Kamp

Division of Pulmonary and Critical Care Medicine, Northwestern University Feinberg School of Medicine, Veterans Administration Chicago Health Care System: Lakeside Division, and Department of Medicine, Chicago, Illinois; and National Health and Environmental Effects Research Laboratory, EPA, Research Triangle Park, North Carolina

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Airborne particulate matter (PM) increases morbidity and mortality resulting from cardiopulmonary diseases including cancer. We hypothesized that PM is genotoxic to alveolar epithelial cells (AEC) by causing DNA damage and apoptosis. PM caused dose-dependent AEC DNA strand break formation, reductions in mitochondrial membrane potential ({Delta}{psi}m), caspase 9 activation, and apoptosis. An iron chelator and a free radical scavenger prevented these effects. Finally, overexpression of Bcl-xl, a mitochondrial anti-apoptotic protein, blocked PM-induced {Delta}{psi}m and DNA fragmentation. We conclude that PM causes AEC DNA damage and apoptosis by mechanisms that involve the mitochondria-regulated death pathway and the generation of iron-derived free radicals.

Abbreviations: alveolar epithelial cells, AEC • deferoxamine, DF • DNA strand break, DNA-SB • double-stranded DNA, ds-DNA • enzyme-linked immunosorbent assay, ELISA • trifluoromethoxyphenlhydrazone, FCCP • particulate matter, PM • Change in mitochondrial membrane potential, {Delta}{psi}m • sodium benzoate, NaB • phytic acid, PA • particulate matter, PM • reactive oxygen species, ROS • titanium dioxide, TiO2 • tetremethylrhod-amine ethyl ester, TMRE • terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate-biotin nick end labeling, TUNEL


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epidemiologic studies have established a direct correlation between the levels of ambient air particles and acute morbidity and mortality from cardiopulmonary diseases resulting in an estimated 500,000 deaths each year worldwide (1). In addition, there is evidence of an association between air pollution particles and chronic cardiopulmonary injury including lung cancer (2). Exposure to particulate matter (PM) is recognized to cause acute respiratory symptoms (3, 4). There is an inverse relationship between particulate size and distal alveolar deposition, with over 77% of PM2.5 being deposited in the distal lung (5). Moreover, a higher percentage of fine particulates are retained in the lung when compared with larger particulates (5). More recently, Pope and associates (2) demonstrated that long-term exposure to elevated levels of ambient airborne PM increase the risk of mortality from cardiopulmonary diseases and lung cancer.

The precise cellular and molecular mechanisms underlying the toxic pulmonary effects of PM are not fully established. The extent of alveolar epithelial cell (AEC) injury and repair are critical determinants of the toxic potential of particulates (6, 7). In particular, accumulating evidence implicates DNA damage due to iron-derived reactive oxygen species (ROS) as important second messengers of toxicity due to a variety of particulates (8–22). It is unclear which components of ambient air PM are responsible for the biological activity. There is evidence that both water-soluble metals and insoluble particulates can induce DNA damage (11, 13). PM generates metal-catalyzed ROS and causes oxidative DNA damage in a manner similar to other toxic particulates such as silica and asbestos (8–22). Iron loading can increase the fibrogenic capacity of a nonfibrogenic airborne particulate (12). Particulate-derived free radicals generated by Fenton-type reactions activate transcription factors, such as nuclear factor-{kappa}B, which subsequently induce the release of inflammatory mediators and cause lung injury (20). Further, the level of transition metals rather than the total particle mass better determines the inflammatory response of the PM (10, 20, 23, 24).

Recent studies demonstrate that particulate matter can induce apoptosis in relevant lung target cells (10, 25–27). Apoptosis is a highly regulated physiologic cell death process that is critical for development, host defense, and the prevention of malignancies throughout the body, including the lungs (7, 28, 29). There are two major mechanisms regulating apoptosis: (i) the mitochondria-regulated pathway, and (ii) the death receptor pathway induced by death-signaling ligands, such as tumor necrosis factor-{alpha} or FasL and subsequent caspase 8 activation (28–30). Oxidant-induced DNA damage, which occurs after PM exposure (10–12), is a potent stimulus of mitochondrial dysfunction as detected by a change in the mitochondrial membrane potential ({Delta}{psi}m) followed by the release of agents normally confined to the mitochondrial intermembrane space, such as cytochrome c (28–30). Disruption of mitochondrial electron transport can further augment ROS production and amplify an apoptotic stimulus (28–30). The Bcl family of anti-apoptotic proteins (e.g., Bcl-2 and Bcl-XL) function at the level of the mitochondrial membrane to prevent apoptosis by inhibiting the {Delta}{psi}m and subsequent release of mitochondrial constituents into the cytoplasm (31, 32). The pro-apoptotic Bcl-2 family members (e.g., Bax, Bak, and Bid) translocate from the cytosol to the mitochondrial membrane to induce cell death (31, 32). Further, either Bax or Bak is required to activate the mitochondria-regulated pathway after exposure to DNA-damaging agents, such as radiation (33). We recently demonstrated that asbestos fibers, but not inert particulates, cause AEC apoptosis by generating iron-derived free radicals and activating the mitochondria-regulated death pathway (16, 17). There is some evidence that similar pathways are activated in macrophages exposed to chemicals extracted from diesel exhaust particles (34). However, the role of the mitochondria in regulating PM-induced AEC apoptosis is unknown.

Given the above findings, we reasoned that PM induces AEC cell DNA damage and apoptosis by mechanisms involving the generation of iron-derived free radicals and activation of the mitochondria-regulated death pathway. We show that PM causes a dose- and time-dependent DNA damage as assessed by an alkaline elution assay, and apoptosis as assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine-5' -triphosphate-biotin nick end labeling (TUNEL) staining and DNA fragmentation. A role for the mitochondria-regulated death pathway was suggested by our finding that PM caused reductions in AEC {Delta}{psi}m that were accompanied by the release of cytochrome c and caspase 9 activation. We also demonstrate that PM-induced reductions in A549 cell {Delta}{psi}m and apoptosis are blocked by Bcl-XL overexpression. Finally, a role for iron-derived ROS was suggested by our findings that an iron chelator (phytic acid or deferoxamine) or a free radical scavenger (sodium benzoate) each blocked PM-induced reductions in {Delta}{psi}m and caspase 9 activation. These data suggest that PM-induced pulmonary toxicity results in part from increased apoptosis in the alveolar epithelium caused by altered mitochondrial function and iron-derived ROS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The PM was collected by baghouse from ambient air in Dusseldorf, Germany. The particle sample was aerosolized from a turntable into a small-scale powder disperser (TSI Inc., St. Paul, MN) using a high airflow to break up aggregates in the venturi throat. The outlet of the aerosol generator was attached directly to an aerodynamic particle sizer (TSI Inc.) and the aerosol was sampled on four occasions for 20 s. Data were expressed as the average mass median aerodynamic diameter from the four replicate samples. Elemental analyses of the PM were accomplished employing either infrared or thermal conductivity assays (Galbraith Labs, Knoxville, TN). Nitrogen content was measured using thermal conductivity after acid digest (Galbraith Labs). Particles contain carbon (19.70 ± 2.34%), hydrogen (1.4 ± 0.3%), nitrogen (< .05%), oxygen (14.12 ± 1.56), sulfur (2.09 ± 0.55%), and ash (63.24 ± 4.19%). Ionizable concentrations of metals associated with the particle was measured by agitation in 1.0 N HCl (1.0 mg/1.0 ml) for 1 h at room temperature, centrifuged for 1 h at 1,200 x g, and the supernatant removed for analysis. Metals were individually analyzed in duplicates. Ionizable concentrations of metals include cobalt (103 ± 13 ppm), copper (48 ± 10 ppm), chromium (104 ± 23 ppm), iron (14,521 ± 572 ppm), manganese (21.3 ± 37 ppm), nickel (1,519 ± 158 ppm), titanium (131 ± 45 ppm), and vanadium (2,767 ± 190 ppm). To obtain the soluble component of PM, PM suspensions were centrifuged for 5 min at 13,000 x g and then filtered through a 0.1-µm filter (Acrodisc 25 mm syringe filter; Pall Gelman Laboratory, Ann Arbor, MI), as previously described (11). All other chemicals were purchased from Sigma Chemicals (St. Louis, MO).

Cell Culture
A549 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco's modified Eagle's medium supplemented with L-glutamine (0.3 µg/ml), nonessential amino acids, penicillin (100 U/ml), streptomycin (200 µg/ml), and 10% fetal bovine serum (FBS; GIBCO, Grand Island, NY). For each experiment, we used a seeding density of 3.0 x 105 cells/ml/well plated onto six-well plates (Costar, Cambridge, MA). The cells were grown to confluence over 24 h in a humidified 95% air–5% CO2 incubator at 37°C.

DNA Strand Break Assay
The DNA strand break (DNA-SB) assay was performed as previously described (14, 35). Briefly, the AEC were treated with PM (1–100 µg/ml) for 24 h, washed with PBS, placed on ice, and then DNA-SB formation was assessed by alkaline unwinding and ethidium bromide fluorescence. In some experiments, AEC were treated with an iron chelator, phytic acid (500 µM), or deferoxamine (1 mM), or a free radical scavenger, sodium benzoate (50 mM), for 15 min and then PM (100 µg/cm2)-induced AEC DNA-SB formation after 24 h was assessed. Because ethidium bromide preferentially binds to double-stranded DNA (ds-DNA) in alkali, the relative amounts of nonbroken ds-DNA and broken single-stranded DNA can be assessed. Fluorescence was determined with a model 450 Sequoia Turner fluorometer (Mountain View, CA) with excitation at 520 nm and emission at 585 nm. The results were expressed as the percentage of total ds-DNA defined as (F – Fmin)/(Fmax – Fmin) x 100, where F is the fluorescence in the experimental condition, Fmin is the background ethidium bromide fluorescence determined after converting all the DNA into single-strand form, and Fmax is the fluorescence determined from cells not exposed to alkaline unwinding conditions.

Apoptosis Assays
AEC cell apoptosis was assessed by both TUNEL-stained nuclear morphology and DNA nucleosomal fragmentation enzyme-linked immunosorbent assay (ELISA) (Roche Diagnostics) as previously described (15). Briefly, A549 cells were exposed to various doses of PM for 24 h, and then the cells in the supernatant and attached to the dish were collected for determination of apoptosis by both techniques. In some experiments AEC were treated with inhibitors as described above. The DNA nucleosomal fragmentation ELISA assay detects histone-associated DNA fragments (mono- and oligonucleosomes). We previously demonstrated that these assays directly correlate with AEC apoptosis as assessed by acridine orange–stained nuclear morphology, annexin V staining, and caspase 3 activation (15).

Mitochondrial Membrane Potential Change
The {Delta}{psi}m was assessed using a fluorometric assay that we have previously described (16, 17). Briefly AEC were treated with PM in the presence or absence of an inhibitor as described above and then exposed to either tetremethylrhodamine ethyl ester (TMRE, 500 nM; Molecular Probes, Eugene, OR) or Mitotracker green (1 µM; Molecular Probes) for 1 h at 37°C. Carbonyl cyanide trifluoromethoxyphenlhydrazone (FCCP, 20 µM; Sigma, St. Louis, MO) 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 488 nm and emission wavelength of 520 or 580 nm (TMRE and Mitotracker green fluorescence, respectively). The Mitotracker green was used to label the mitochondria because it binds mitochondrial lipids and is not influenced by the {Delta}{psi}m caused by FCCP. 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 {Delta}{psi}m. The {Delta}{psi}m was compared qualitatively based upon the percentage difference in the ratio of TMRE and Mitotracker green fluorescence of untreated cells (Te and MGc, respectively) corrected for the background fluorescence in FCCP treated control cells (FTc and FMGc) and the ratio of TMRE and Mitotracker green fluorescence of treated cells (Tt and Mgi) minus the FCCP-treated cells (FTt and FMGt, respectively) defined as follows: {Delta}{psi}m = (Tc/MGc - FTc/Mge) - (Tt/MGt - FTt/FMGt) x 100.

Caspase 9 and 8 Assays
AEC were treated with PM (100 µg/cm2) for 24 h, washed, and then the protein from the cell lysates of the attached and floating cells were collected for use in the ELISA as previously described (17). In some experiments, AEC were pretreated with inhibitors as described above, and then PM-induced caspase 9 activation was assessed. PM-induced caspase 9 (mitochondria-regulated death pathway) and caspase 8 (death receptor pathway) release was assessed by a commercially available ELISA assay for each caspase exactly as per the manufacturer's protocol (Roche Diagnostics, Indianapolis, IN).

Statistics
All data are expressed as the mean ± SEM. An unpaired Student's t test was used to assess the difference between two groups. One-way ANOVA was performed when more than two groups were compared with a single control, and then differences between individual groups within the set were assessed by a multiple comparison test (Tukey) when the F statistic was < 0.05. A P value of < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PM Induces AEC DNA Damage
To determine whether PM causes AEC DNA damage, AEC were exposed to various doses of PM (10–100 µg/cm2) for 24 h. DNA-SB formation was assessed by an alkaline elution, ethidium bromide fluorescence technique (14, 35). As shown in Figure 1A, PM induced dose-dependent reductions in AEC ds-DNA due to the formation of DNA-SB. A dose of PM as little as 10 µg/cm2 caused a 25% reduction in ds-DNA, whereas the highest dose tested (100 µg/cm2) induced a 75% reduction. To determine the time course of PM-induced DNA damage, we exposed AEC to PM (100 µg/cm2) for variable time periods (0, 1, 4, 8, 12, and 24 h). As shown in Figure 1B, PM induced about a 22% reduction in AEC ds-DNA in as little as 1 h, and a 75% reduction in AEC ds-DNA within 8 h, an effect that was persistent at later time points.



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  		Figure 1. (A) PM induces dose-dependent AEC DNA-SB formation: AEC were exposed to variable doses of PM (10, 25, 50, and 100 µg/cm2) for 24 h. As compared with control, PM induced dose-dependent decreases in ds-DNA. A dose of 10 µg/cm2 caused 26% reduction in ds-DNA, whereas 100 µg/cm2 caused 75% reduction in ds-DNA, as assessed by alkaline elution ethidium bromide fluorescence technique. Data are expressed as the mean ± SEM, *P < 0.05 versus control, n = 3. (B) PM induces time-dependent AEC DNA-SB formation: AEC were exposed to PM (100 µg/cm2) for variable time periods (0, 1, 4, 8, 12, and 24 h). As compared with control, PM induced a 22% reduction in AEC ds-DNA in as little as 1 h and a 75% reduction in AEC ds-DNA within 8 h; this effect was persistent at later time points. Data are expressed as the mean ± SEM, *P < 0.05 versus control, n = 3.

 
To determine whether soluble or insoluble component of PM induces AEC DNA-SB formation, we exposed AEC to supernatant or pellet component of PM (100 µg/cm2) for 24 h. We found that the soluble component caused higher DNA damage as compared with insoluble component (fold increase versus controls: insoluble PM [1.42 ± 0.16] and soluble PM [1.87 ± 0.11], n = 3; P < 0.05 versus control for both).

PM Causes AEC Apoptosis
To determine whether PM induces AEC apoptosis, we exposed AEC to various doses of PM (1, 5, 25, and 100 µg/cm2) for 24 h and then assessed apoptosis by TUNEL staining and DNA fragmentation. As shown in Figure 2A, PM increased AEC apoptosis in a dose-dependent manner as assessed by TUNEL staining. A PM-dose as little as 5 µg/cm2 caused 15% of the cells to undergo apoptosis, whereas the highest dose tested, 100 µg/cm2, resulted in nearly 45% of the cells with TUNEL-stained apoptotic nuclei. Similar to TUNEL staining, PM induced dose-dependent AEC DNA fragmentation (Figure 2B). PM caused a 3- to 6-fold increase in DNA fragmentation over 24 h. Taken together, the above data clearly show that PM induces AEC DNA-SB and apoptosis.



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  		Figure 2. (A) PM induces dose-dependent AEC apoptosis: A549 cells were exposed to PM at various doses (1, 5, 25, and 100 µg/cm2) for 24 h. PM increased AEC apoptosis in a dose-dependent manner. A PM dose as little as 5 µg/cm2 caused 15% AEC apoptosis, whereas 100 µg/cm2 resulted in ~ 45%, AEC apoptosis, as assessed by tunnel assay. Mean ± SEM, *P < 0.05 versus control, n = 3. (B) PM-induced dose-dependent AEC DNA fragmentation: A549 cells were exposed to PM at various doses (1, 5, 25, and 100 µg/cm2) for 24 h. DNA fragmentation was assessed by DNA nucleosomal fragmentation ELISA. PM caused a 3- to 6-fold increase in DNA fragmentation over 24 h, in a dose-dependent manner. Mean ± SEM, *P < 0.005 control versus PM, n = 3.

 
Iron-Catalyzed Free Radicals Mediate PM-Induced AEC DNA-SB Formation and Apoptosis
To determine whether iron-derived free radicals mediate PM-induced AEC DNA-SB formation, AEC were pretreated for 15 min with either an iron chelator, phytic acid (500 µM), or deferoxamine (1 mM), or a free radical scavenger, sodium benzoate (50 mM). We previously showed that each of these inhibitors attenuates asbestos-induced AEC DNA-SB and apoptosis caused by iron-derived free radicals (14–17). Similar to our findings with asbestos, each inhibitor reduced PM (100 µg/cm2)-induced AEC DNA-SB formation by over 50% (Figure 3). We also showed that each inhibitor reduced PM-induced AEC apoptosis after a 24-h exposure period as assessed by TUNEL staining (Figure 4A) and DNA fragmentation (Figure 4B). As illustrated in Figure 4A, PM increased the percentage of hyper-dense, TUNEL-stained apoptotic A549 cell nuclei, and each inhibitor was protective. Compared with controls, each inhibitor alone had a negligible effect on AEC TUNEL staining and DNA fragmentation (data not shown). These data suggest that iron-derived free radicals mediate PM-induced AEC DNA damage and apoptosis.



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  		Figure 3. Iron-catalyzed free radicals mediate PM-induced AEC DNA-SB formation: AEC were exposed to iron chelators, phytic acid (500 µM), or deferoxime (1 mM) or a free radical scavenger sodium benzoate (50 mM) for 15 min followed by PM (100 µg/cm2) for 24 h. DNA damage was determined by DNA strand break assay as assessed by alkaline unwinding and ethidium bromide fluorescence technique. Both, iron chelators and free radical scavenger significantly reduced PM-induced AEC DNA-SB formation. *P < 0.005 control versus PM, {dagger}P < 0.05 PM versus PM + sodium benzoate (50 mM), deferoxime (1 mM), or phytic acid (500 µM). Both the iron chelators and free radical scavenger by themselves showed negligible AEC DNA damage. Solid bars, No PM; striped bars, PM (100 µg/cm2).

 



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  		Figure 4. (A) Iron-catalyzed free radicals mediate PM-induced AEC apoptosis: AEC were exposed to phytic acid (500 µM), deferoxime (1 mM), or sodium benzoate (50 mM) for 15 min followed by PM (100 µg/cm2) for 24 h. Both iron chelators (phytic acid and deferoxime) and a free radical scavenger (sodium benzoate) reduced PM-induced AEC apoptosis, as assessed by tunnel assay. *P < 0.0001 control versus PM; {dagger}P < 0.005 PM versus PM + phytic acid (500 µM), deferoxime (1 mM), or sodium benzoate (50 mM), n = 3. (B) Iron-catalyzed free radicals mediate PM-induced AEC DNA fragmentation: AEC were exposed to phytic acid (500 µM), deferoxime (1 mM), or sodium benzoate (50 mM) for 15 min followed by PM (100 µg/cm2) for 24 h. Both iron chelators and sodium benzoate significantly reduced PM(100 µg/cm2)-induced AEC DNA fragmentation, as assessed by DNA nucleosomal fragmentation ELISA. *P < 0.005 control versus PM; {dagger}P < 0.05 PM versus PM + phytic acid (500 µM), deferoxime (1 mM), or sodium benzoate (50 mM), n = 3. Solid bars, No PM; striped bars, PM (100 µg/cm2).

 
PM Causes AEC Mitochondrial Dysfunction by Generating Iron-Derived Free Radicals
We recently demonstrated that asbestos, but not inert particulates such as glass beads or titanium dioxide, cause AEC apoptosis via the mitochondria-regulated death pathway (16, 17). To determine whether PM induces AEC apoptosis by a similar mechanism, AEC were exposed to PM (1, 5, 25, and 100 µg/cm2) for 24 h, and then AEC {Delta}{Psi}m was assessed using a fluorometric technique with TMRE and Mitotracker green as previously described (17). As shown in Figure 5A, PM reduced AEC {Delta}{Psi}m in a dose-dependent manner. Similar to the asbestos paradigm (17), PM (100 µg/cm2) activated caspase 9, the mitochondria-regulated caspase, by nearly 2-fold as compared with control (Figure 6A). In contrast, PM caused negligible activation of caspase 8, the death receptor caspase (Figure 6B).



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  		Figure 5. (A) PM causes AEC mitochondrial dysfunction, in a dose-dependent manner; A549 cells were exposed to PM at various doses (1, 5, 25, and 100 µg/cm2) for 24 h. PM reduced AEC {Delta}{Psi}m in a dose-dependent manner, as assessed by a fluorometric technique with TMRE and Mitotracker green. *P < 0.005 control versus PM, n = 3. (B) PM causes AEC mitochondrial dysfunction by generating iron-derived free radicals; AEC were pretreated with phytic acid (500 µM), deferoxime (1 mM), or sodium benzoate (50 mM) for 15 min, followed by PM (100 µg/cm2) for 24 h. Both iron chelators and a free radical scavenger, sodium benzoate, significantly prevented PM-induced reduction in AEC {Delta}{Psi}m. *P < 0.0001 control versus PM; {dagger}P < 0.005 PM versus PM + phytic acid (500 µM), deferoxime (1 mM), or sodium benzoate (50 mM), n = 3.

 



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  		Figure 6. (A) PM-induced AEC caspase 9 activation is mediated by iron-derived free radicals: AEC were exposed to iron chelators, phytic acid (500 µM), deferoxime (1 mM), or a free radical scavenger, sodium benzoate (50 mM), for 15 min followed by PM (100 µg/cm2) for 24 h. Cells were lysed using caspase 9 buffer, and caspase 9 ELISA was done as per manufacturer's protocol. PM induced a 2-fold increase in activated caspase 9 as compared with control. *P < 0.005 control versus PM. Both of the iron chelators (phytic acid and deferoxamine) as well as the free radical scavenger, sodium benzoate, reduced PM-induced caspase 9 activation. {dagger}P < 0.05 PM versus PM + phytic acid, deferoxime, or sodium benzoate, n = 3. (B) PM does not induce AEC Caspase 8 activation: AEC were exposed to iron chelators, phytic acid (500 µM), deferoxime (1 mM), or a free radical scavenger, sodium benzoate (50 mM), for 15 min followed by PM (100 µg/cm2) for 24 h. Caspase 8 ELISA was done as per manufacturer's protocol. PM caused negligible AEC caspase 8 activation, and the iron chelators as well as a free radical scavenger did not have any effect. Solid bars, No PM; striped bars, PM (100 µg/cm2).

 
To determine whether iron-derived free radicals mediate PM-induced mitochondrial dysfunction, AEC were pretreated with each of the inhibitors as described above, and then PM (100 µg/cm2)-induced {Delta}{Psi}m was assessed. Both of the iron chelators (phytic acid and deferoxamine) as well as the free radical scavenger, sodium benzoate, attenuated PM-induced reductions in AEC {Delta}{Psi}m (Figure 5B). Further, each of these inhibitors blocked PM-induced caspase 9 activation (Figure 6A). Collectively, these data strongly suggest that PM causes AEC mitochondrial dysfunction that is due in part to iron-derived free radicals.

Bcl-xl-Overexpressed Cells Prevent PM-Induced Change in AEC Mitochondrial Membrane Potential
Accumulating evidence demonstrates that anti-apoptotic proteins that localize to the mitochondria, such as Bcl-2 and Bcl-xl, block apoptosis due to a variety of noxious stimuli, including asbestos (16, 17, 31–33). To determine whether Bcl-xl prevents PM-induced AEC mitochondrial dysfunction, we used A549 cells that overexpress Bcl-xl as previously described (16, 17). As expected, PM (100 µg/cm2) reduced {Delta}{Psi}m in empty vector control A549 cells (Figure 7A). In contrast, PM did not alter {Delta}{Psi}m in A549 cells that overexpress Bcl-xl. We also noted that Bcl-xl overexpression reduced PM-induced DNA fragmentation (Figure 7B). These data further support the hypothesis that the mitochondria-regulated death pathway is important for PM-induced AEC apoptosis.



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  		Figure 7. (A) Bcl-xl overexpressed cells prevent PM-induced change in AEC mitochondrial membrane potential: A549 cells that overexpress Bcl-xl were exposed to PM (100 µg/cm2) for 24 h. As compared with control A549 cells, PM did not alter {Delta}{Psi}m in A549 cells that overexpress Bcl-xl. Mean ± SEM, *P < 0.005 A549 cells versus Bcl-xl, n = 3. (B) Bcl-xl overexpressed cells prevent PM-induced AEC DNA fragmentation: A549 cells that overexpress Bcl-xl were exposed to PM (100 µg/cm2) for 24 h. As compared with control, PM induced 3-fold increase in DNA fragmentation in A549 cells, whereas A549 cells that overexpress Bcl-xl significantly reduced PM-induced DNA fragmentation. Mean ± SEM, *P < 0.005 control versus PM in A549 cells, n = 3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Epidemiologic studies show a direct correlation between exposure to airborne PM and increased cardiopulmonary morbidity and mortality. The mechanisms by which fine particulate matter causes pulmonary toxicity at the cellular and molecular levels are poorly understood. Although accumulating evidence implicates an important role for ROS derived from metal-catalyzed reactions, the relevance of this hypothesis to the distal airway epithelium has received little attention. The major findings of this study are that airborne PM induces AEC DNA damage and apoptosis by mechanisms involving iron-derived free radicals. Further, we show that the mitochondria-regulated death pathway plays an important role in mediating PM-induced AEC apoptosis.

PM is a complex mixture of organic and inorganic components that include metals, salts, polyaromatic hydrocarbons and carbonaceous material (11, 20). It has been hypothesized that pulmonary toxicity caused by exposure to various noxious agents is due in part to AEC injury (6, 7). In this study we demonstrated that PM induces dose-dependent AEC DNA-SB formation as assessed by an alkaline elution technique (Figure 1). DNA-SB formation is among the earliest cellular changes that occur after an oxidative stress from various agents, including particulate fibers such as asbestos (14, 36). The alkaline unwinding, ethidium bromide fluorescent technique for measuring DNA-SB formation is one of the most sensitive assays for detecting DNA damage, with a detection threshold of one break per chromosome (35). PM-induced DNA-SB formation was first noted after 1 h, but significant levels were detected by 8 h, a time point at which we see little cell death (data not shown). These findings with AEC corroborate prior studies demonstrating that particulate matter causes DNA damage in a variety of lung target cells such as macrophages and large airway epithelial cells (10, 11, 19, 37, 38).

DNA damage is a well-known stimulus of apoptosis (25–27). Herein we show that PM also induced AEC apoptosis as assessed by both TUNEL-stained nuclear morphology and DNA fragmentation assays (Figure 2). Our findings demonstrating that PM causes distal airway epithelial cell apoptosis support the accumulating evidence showing that PM induces apoptosis in other relevant lung target cells such as macrophages and large airway epithelial cells (10, 25–27). Evidence presented herein extends these observations by demonstrating that the mitochondria-regulated death pathway mediates PM-induced AEC apoptosis. This conclusion is firmly supported by the findings that PM causes a dose-dependent reduction in AEC {Delta}{psi}m (Figure 5) and caspase 9 activation (Figure 6A). We have also noted that PM induces cytochrome c release as assessed by immunofluorescence studies (data not shown). We also were unable to detect caspase 8 activation, the death receptor caspase (Figure 6B). Notably, overexpression of Bcl-xl, a mitochondrial targeted antiapoptotic protein, attenuated PM-induced reduction in AEC {Delta}{psi}m and apoptosis (Figure 7). These findings with PM parallel recent studies showing an important role for the mitochondria-regulated death pathway in mediating asbestos-induced AEC apoptosis (16, 17) as well as macrophage apoptosis due to chemicals extracted from diesel exhaust (34). The above findings seem restricted to potentially toxic particulates, because we previously showed that inert particulates, such as glass beads, cause negligible AEC {Delta}{psi}m and apoptosis (16, 17).

Studies exploring the pathogenic mechanisms of PM have implicated an important role for ROS derived from metal-catalyzed reactions (11–13, 18–22, 39). Consistent with these prior studies, we showed that an iron chelator, such as deferroxamine or phytic acid, and a free radical scavenger, sodium benzoate, each attenuated PM-induced AEC DNA damage (Figure 3) and apoptosis (Figures 4 and 5). Further, each of the inhibitors partially blocked PM-induced reduction in AEC {Delta}{psi}m (Figure 5) and caspase 9 activation (Figure 6). All three inhibitors alone had negligible effects on A549 cell {Delta}{psi}m (data not shown). The soluble metal component in PM is also implicated in generating ROS, such as hydroxyl radicals, and causing the release of inflammatory cytokines such as IL-8 and IL-6 from cultured respiratory epithelial cells (21, 39). We also noted that the soluble component of PM accounted for the majority of the DNA-damaging effects to AEC. Collectively, these findings suggest that iron-derived free radicals mediate PM-induced DNA damage and apoptosis.

Although the relevance of our in vitro findings to in vivo conditions requires further study, there is some supportive information showing that PM induces adverse consequences after short-term exposure periods. Gurgueira and associates (40) recently showed that rats exposed to aerosolized PM (300 ± 60 µg/m3) for 3–5 h induced ROS, as assessed by chemiluminescence, and increased the activities of adaptive antioxidant enzymes (e.g., catalase and superoxide dismutase). Salvi and coworkers (41) demonstrated that a 1-h exposure of normal individuals to diesel exhaust increased acute inflammatory responses in the airways. Gilmour and associates (22) demonstrated that toxic particulates rapidly cross the epithelium and induce interstitial inflammation by mechanisms involving ROS. In this study, we used A549 cells (malignant clone of cells with alveolar type II-like features); previously, we showed comparable levels of DNA damage and apoptosis with mitochondrial dysfunction in rat alveolar type II cells exposed to asbestos (14, 15, 17).

In conclusion, our study demonstrates that PM-induced AEC DNA damage and apoptosis occur by mechanisms involving the generation of iron-derived free radicals and activation of the mitochondria-regulated death pathway. Given the importance of the distal airway epithelium in the pathogenesis of pulmonary diseases (7), these findings provide further strong support for the role of iron-derived ROS and the mitochondria in mediating the toxic effects of PM as outlined in Figure 8. We reason that the adverse effects of airborne particulates may be prevented by strategies aimed at reducing the levels of iron-derived ROS and preserving normal mitochondrial function.



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  		Figure 8. Hypothetical model showing mechanism of PM-induced AEC DNA damage and apoptosis. PM may induce production of ROS in AEC that, in turn, may lead to AEC DNA damage, reductions in mitochondrial membrane potential ({Delta}{psi}m), caspase 9 activation, and apoptosis. An iron chelator and a free radical scavenger prevent these effects. BCL-xl, a mitochondrial antiapoptotic protein, is protective against PM-induced DNA damage and apoptosis.

 

   Acknowledgments
 
This work was supported by a Merit Review grant from the Department of Veterans Affairs (D.W.K.) and a National Research Science Award (D.U.). The authors are grateful to Navdeep Chandel for providing the Bcl-xl–overexpressing A549 cells.

Received in original form November 22, 2002

Received in final form February 7, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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