Mitochondrial oxidant production by a pollutant dust and NO-mediated apoptosis in human alveolar macrophage

Yuh-Chin T. Huang, Joleen Soukup, Shirley Harder, and Susanne Becker

National Health and Environmental Effects Research Laboratory, Office of Research and Development, Environmental Protection Agency, Research Triangle Park, North Carolina 27711


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Residual oil fly ash (ROFA) is a pollutant dust that stimulates production of reactive oxygen species (ROS) from mitochondria and apoptosis in alveolar macrophages (AM), but the relationship between these two processes is unclear. In this study, human AM were incubated with ROFA or vanadyl sulfate (VOSO4), the major metal constituent in ROFA, with or without nitro-L-arginine methyl ester (L-NAME), diphenyleneiodonium (DPI), and mitochondrial electron transport inhibitors. Interactions among production of ROS, nitric oxide (NO), and apoptosis of AM were determined. ROFA-stimulated ROS production was attenuated by DPI, rotenone, antimycin, and NaN3, but not by L-NAME, a pattern mimicked by VOSO4. ROFA-induced apoptosis was inhibited by L-NAME and a caspase-3-like protease inhibitor, but not by mitochondrial inhibitors. ROFA enhanced NO-mediated increase in caspase-3-like activity. VOSO4 had minor effects on apoptosis. Thus ROFA-stimulated production of ROS from mitochondria was independent of apoptosis of AM, which was mediated by activation of caspase-3-like proteases and NO. The pro-oxidant effect but not the proapoptotic effect of ROFA was mediated by vanadium.

caspase; annexin; reactive oxygen species; vanadium; pollutant particles; residual oil fly ash


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EXPOSURE TO PARTICULATE MATTER is consistently associated with increased morbidity and mortality attributable in part to respiratory illnesses (13, 43). Patients with chronic lung conditions, such as asthma or chronic obstructive pulmonary disease (COPD), are among the most susceptible (20, 51). For asthmatics, an increase of 10 µg/m3 in PM10 was associated with a 3-6% increase in outpatient visits (21), a 3-4% increase in emergency room visits (46), and a 2-3% increase in hospital admissions (13, 47). For patients with COPD, an increase of 10 µg/m3 in PM10 was linked to a 1-3% increase in emergency room visits (49) and a 1-2% increase in hospital admissions (9). These adverse pulmonary effects are thought to be related to pulmonary inflammation as a result of activation of resident lung cells.

The alveolar macrophage (AM) is one of the cell types in the lungs constantly exposed to the ambient environment. Upon contact with certain environmental particulate pollutants, AM are activated and produce a large quantity of reactive oxygen species (ROS) in the form of chemiluminescence burst. Examples of pollutant particles capable of enhancing the production of ROS by AM include oil fly ash and residual oil fly ash (ROFA) (3, 4, 30, 39) and the Utah Valley dust (48). The exact sources for ROS produced by pollutant-activated AM vary depending on the particles used. The membrane NADPH oxidase appears to be an important source in AM stimulated with quartz dusts, metal-containing dusts, or silica particles coated with a single metal oxide (17). AM may also produce ROS from mitochondria when exposed to combustion-derived particles, such as ROFA (3). ROS produced by AM may then serve as signaling molecules for downstream events including inflammation, cell growth, and cell death, although previous studies have shown that ROFA-induced ROS production does not predict activation of NF-kappa B or induction of IL-8 (4, 39).

Environmental pollutants may also cause apoptotic cell death. Human AM exposed to ROFA and urban particles showed morphological features and DNA changes consistent with apoptosis (26). The human fibroblast cell line MRC-5 undergoes apoptosis when exposed to extracts of automobile exhaust (53). Diesel exhaust particles induce apoptosis in AM and RAW264.7 cells (25). The programmed cell death is considered an integral part of the host mechanisms by which the homeostasis of the microenvironment is maintained.

In the present study, we determined how ROFA-induced mitochondrial production of ROS is related to apoptosis in human AM. Because vanadium is the major metal constituent in ROFA and is also capable of inducing chemiluminescence (23, 45), we also determined the role of vanadium in mediating the pro-oxidant and proapoptotic effects of ROFA. In addition, because nitric oxide (NO) is a known stimulant for apoptosis of macrophages (1) and its levels can be affected by superoxide, we also determined how ROFA-induced ROS production modulated NO production and NO-mediated apoptosis.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents and Chemicals

Vanadyl sulfate (VOSO4) was obtained from Johnson Matthey (Ward Hill, MA). DetaNONOate was obtained from Cayman Chemical (Ann Arbor, MI). 4,5-Diaminofluorescein diacetate (DAF-2DA) was obtained from Calbiochem-Novabiochem (San Diego, CA). DEVD was obtained from R&D Systems (Minneapolis, MN). All other chemicals, unless otherwise specified, were obtained from Sigma Chemical (St. Louis, MO). Residual oil fly ash (ROFA) was acquired from Southern Research Institute (Birmingham, AL). The sample was collected downstream from the cyclone of a power plant in Florida that was burning a low-sulfur no. 6 residual oil (collection temperature of 250-300°C). The metal contents in ROFA (in µg/mg) are 41.7 V, 37.5 Ni, 23.3 Fe, 1.0 Zn, and 0.2 Cu (11).

Isolation of Human AM

Human AM were obtained by bronchoalveolar lavage (BAL) from a total of 67 bronchoscopies in normal individuals according to procedures described previously (18). Subjects were informed of the procedures and potential risks, and each signed an informed consent. The protocol was approved by University of North Carolina School of Medicine Committee on Protection of the Rights of Human Subjects. Briefly, the fiberoptic bronchoscope was wedged into a segmental bronchus of the lingual lobe. Six aliquots of sterile saline were instilled and immediately aspirated. The first was 20 ml and was not used for AM isolation. The remaining five aliquots were 50 ml each. The procedure was repeated on the right middle lobe. BAL samples were put on ice immediately and centrifuged at 300 g for 10 min at 4°C. The lavaged cells were washed once with ice-cold RPMI 1640 medium with 20 mg/ml gentamicin (Life Technologies, Rockville, MD). Cell counts were performed using a hemocytometer. Cytocentrifuge slides were prepared and stained with Diff Quick (Leukostat solution; Fisher Scientific, Atlanta, GA) to check for AM purity. The cell preparation consisted of 85-95% AM. The viability of AM was determined by trypan blue exclusion. Viability exceeded 85% in all samples.

Production of ROS

Production of ROS by human AM was quantified by measuring the chemiluminescence (4). The assay was performed on Berthold's LB953 autolumat using luminol (ExOxEmis, Little Rock, AR). Human AM treated with ROFA and other interventions were aliquoted into twelve 75-mm polypropylene tubes. These cells (105 cells in 100 µl of RPMI) and 600 µl of luminol reagent were automatically injected simultaneously into the tubes, and resultant chemiluminescence (cpm) was measured over a 30-min period.

Measurements of GSH and Ascorbate

Glutathione was measured by using the enzymatic recycling method of Anderson (2). Ascorbate was analyzed by high-performance liquid chromatography electrochemical detection using the method of Kutnink et al. (33).

Measurements of NO Production in AM

NO production by human AM was measured by oxidation of DAF-2DA using a flow cytometer. DAF-2DA is a cell-permeable fluorescence dye that is oxidized by NO to form an insoluble precipitate. AM were washed with ice-cold culture medium or PBS, centrifuged for 5 min at 500 g, and resuspended in ice-cold binding buffer at 1 × 106 cells/ml. The buffer contained a mitochondrial marker, MitoTracker Red CM-H2Xros (Molecular Probes, Eugene, OR), at a 500 nM concentration. The cell suspension (200 µl) was then incubated with 2 µl of DAF-2DA (final concentration 22 µg/ml) for 45 min at 37°C. Cells were analyzed by using a flow cytometer (Becton Dickinson FACSort, San Jose, CA) equipped with a 488-nm laser. Cells were gated on the basis of their forward (FSC) and side light scatter characteristics (SSC) for analysis. Analysis of DAF-2 DA signals was done by using Cell Quest software (Becton Dickinson), which provided a calculation of mean fluorescence intensity for the gated population.

Measurement of Apoptosis

The following four methods were used to detect apoptosis.

Nuclear morphology. AM were plated on plastic culture plates, and the cells were stained with 1 µl of 10 mg/ml Hoechst 33342 dye (Molecular Probes) for 15 min at 37°C. Cells were examined with a fluorescence microscope for clumped, condensed, or fragmented chromatin, indicative of apoptosis.

Quantitation of extracellular histone release. Human AM were incubated overnight at 37°C with vehicle or test agents. AM were washed with ice-cold culture medium or PBS and then centrifuged for 5 min at 500 g. Histone in fragmented DNA in the supernatant was measured using the Cell Death Detection ELISA assay (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer's recommended procedures. The signals were detected at 405 nm on a Ceres UV900HDi plate reader (Bio-Tek Instruments, Winooski, VT).

Expression of annexin V. After overnight incubation at 37°C with vehicle or test agents, AM were washed with ice-cold culture medium or PBS, centrifuged for 5 min at 500 g, and resuspended in ice-cold binding buffer at 1 × 106 cells/ml. Cell suspension (200 µl) was stained with 5 µl of diluted annexin V FITC (for early apoptosis) and 5 µl of propidium iodide (PI) (for necrosis) for 10 min. Cells were then analyzed by flow cytometry using the FACSort flow cytometer equipped with a 488-nm laser. Cells were gated for analysis on the basis of their FSC and SSC. Analysis of annexin V expression was done using Cell Quest software, which provided a calculation of mean fluorescence intensity for the gated population. Cells reacted with FITC-conjugated irrelevant antibodies of the same isotype as the receptor antibodies were used as controls to establish background fluorescence and nonspecific antibody binding.

Caspase-3-like activity. Human AM (1 × 106 cells) in RPMI 1640 medium supplemented with 2% fetal bovine serum (FBS) were incubated with test agents for 4 h. The cells were then split into two Eppendorf tubes and centrifuged. The pellets and the supernatant were separated. Cell pellets were lysed with 25 µl of caspase-3 kit lysis buffer on ice for 10 min and then stored at -20°C until further analysis. Caspase-3-like activity was measured by using EnzChek caspase-3 assay kit no. 1 with fluorescence-labeled Z-DEVD-7-amino-4-methylcoumarin (AMC) as the substrate (Molecular Probes). The assay was performed according to the manufacturer's recommended procedures, and the fluorescence was measured with a fluorescence plate reader (Perkin-Elmer) using an excitation wavelength of 350 nm and an emission wavelength of 450 nm. The reference standard was AMC. The caspase-3-like activity was expressed as the amount of AMC released in the reaction (in nmol · min-1 · mg protein-1).

Statistical Analysis

All data are expressed as means ± SE and were compared using a paired t-test adjusted for multiple comparisons. The statistical analysis was performed with StatView (version 4.0; SAS, Cary, NC). A P value <0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ROFA and Cytotoxicity

We first determined how ROFA affected cell death. Treatment with ROFA for 24 h produced a dose-dependent increase in cells with apoptotic or necrotic features (Table 1). On the basis of this dose-response result, a 100 µg/ml dose was chosen for all subsequent experiments because it produced more apoptotic cell death than a 50 µg/ml dose but less necrotic cell death than a 200 µg/ml dose. The dose response of ROFA in AM with these endpoints was similar to that of oil fly ash with the use of trypan blue uptake as reported by our group (4).

                              
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Table 1.   ROFA produces a dose-dependent increase in AM apoptosis and necrosis

Effects of ROFA on ROS Production

ROFA drastically increased ROS production measured by luminol-dependent chemiluminescence burst. The increase could be inhibited by Cu,Zn SOD (100 U/ml) by >50% and deferoxamine by ~70%, indicating that the primary signals of chemiluminescence were from superoxide and its derived ROS. The ROFA-induced chemiluminescence burst was inhibited by 40% by diphenyleneiodonium (DPI; 4 µM), a flavoprotein inhibitor, but not by nitro-L-arginine methyl ester (L-NAME; 100 µM), a nitric oxide synthase (NOS) inhibitor (Fig. 1A). AM were also treated with rotenone (2.5 µM), antimycin (4 µM), and sodium azide (0.3%), which block mitochondrial electron transport at complex I (NADH-Q reductase), complex III (cytochrome reductase), and complex IV (cytochrome oxidase), respectively, for 45 min after 24-h incubation with ROFA. All three mitochondrial electron transport inhibitors attenuated ROFA-induced ROS production (Fig. 1B).


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Fig. 1.   Production of reactive oxygen species (ROS) by human alveolar macrophages (AM) treated with residual oil fly ash (ROFA). A: chemiluminescence was measured after AM were incubated with ROFA for 24 h with or without diphenyleneiodonium (DPI; 4 µM), a flavoprotein inhibitor, or nitro-L-arginine methyl ester (L-NAME; 100 µM), a nitric oxide synthase (NOS) inhibitor. B: chemiluminescence was measured after AM were incubated with ROFA for 24 h, followed by incubation for 45 min with 3 mitochondrial electron transport inhibitors: rotenone (Rot; 2.5 µM), antimycin (Ant; 4 µM), and sodium azide (NaN3; 0.3%), which inhibit complexes I, III, and IV, respectively. R, ROFA. * P < 0.05 vs. control. # P < 0.05 vs. ROFA alone. The results in each group were obtained from experiments on AM from 5-10 different individuals.

Effects of ROFA on Antioxidants

ROFA decreased GSH levels ~30% by 60 min, consistent with increased production of peroxides. The inhibition was restored by rotenone, sodium azide, and DPI but not by antimycin (Fig. 2). ROFA also inhibited ascorbate level by 90%, and this was not reversed by any of the inhibitors used (Fig. 2).


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Fig. 2.   Effects of ROFA on intracellular ascorbic acid and GSH concentration. Ascorbate and GSH were measured at 1 h after incubation with ROFA. * P < 0.05 vs. control. # P < 0.05 vs. ROFA. The results in each group were obtained from experiments on AM from 3 different individuals.

Effects of Mitochondrial Inhibitors on NO Production

Because superoxide may react rapidly with NO, we determined how the excessive electron leaks from the mitochondrial electron transport chain induced by ROFA may affect intracellular NO concentration by measuring oxidation of a cell-permeable fluorescent dye, DAF-2DA. Control AM showed oxidation of DAF-2DA over 45 min of incubation, which was completely inhibited by 100 µM L-NAME (data not shown). ROFA tended to increase DAF-2DA oxidation (P = 0.063). The increase was inhibited by antimycin but not by rotenone or sodium azide. A similar pattern of inhibition was also seen in control AM not stimulated by ROFA (Table 2).

                              
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Table 2.   Effects of ROFA and mitochondrial inhibitors on NO

ROFA and Apoptosis

Approximately 10% of ROFA-treated AM expressed annexin V (early apoptosis), 12% showed increased PI uptake (necrosis), and 9% showed both increased annexin V expression and increased PI uptake (late apoptosis) (Fig. 3A). ROFA also increased histone release more than twofold (Fig. 3B). The caspase-3-like activity of AM was also assessed. Control AM showed a level of caspase-3-like activity of 31.0 ± 7.7 nmol · min-1 · mg protein-1. ROFA treatment for 4 h increased the activity approximately twofold (Fig. 4A). The caspase-3-like activity in control and ROFA-treated cells was completely inhibited by DEVD, a caspase-3-like protease inhibitor (Fig. 4A). AM treated with ROFA demonstrated nuclear morphology consistent with apoptosis (Fig. 4B), which was also inhibited by DEVD.


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Fig. 3.   Expression of annexin V (A) and histone release (B) by human AM after 24-h incubation with ROFA. * P < 0.05 vs. control. The results for annexin V in each group were obtained from experiments on AM from 10-15 different individuals. The results for histone in each group were obtained from experiments on AM from 4 different individuals.



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Fig. 4.   Caspase-3-like activity (A) and Hoechst staining (B) of AM treated with ROFA with or without a caspase-3-like protease inhibitor, DEVD. Caspase-3-like activity was measured after AM were incubated with ROFA for 4 h. Hoechst staining was performed on AM incubated with ROFA for 24 h. * P < 0.05 vs. control. # P < 0.05 vs. AM without (-) DEVD. The results for each group were obtained from experiments on AM from 10-15 different individuals. Magnification, ×40.

Effects of Mitochondrial ROS on Apoptosis

To determine how mitochondrial ROS production regulates apoptosis, we measured the effects of mitochondrial electron transport inhibitors on the activity of caspase-3-like proteases in control and ROFA-treated AM. Sodium azide but not rotenone or antimycin increased caspase-3-like activity in both control and ROFA-treated cells (Fig. 5).


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Fig. 5.   Effects of mitochondrial inhibitors on caspase-3-like activity in AM with or without ROFA treatment. Caspase-3-like activity was measured after AM were incubated with ROFA for 4 h. * P < 0.05 vs. control AM without ROFA. # P < 0.05 vs. no inhibitors. The results in each group were obtained from experiments on AM from 5 different individuals.

Effects of NO on Apoptosis

Because NO is known to induce apoptosis in macrophages and ROFA increased NO production, we further determined the role of NO in ROFA-induced apoptosis. L-NAME inhibited the increase in caspase-3-like activity in ROFA-treated AM (Fig. 6A) as well as nuclear apoptotic morphology, whereas L-arginine (3 mM) and a NO donor, detaNONOate (3 mM), but not L-ornithine (3 mM), the amino acid metabolite of L-arginine by arginase, increased caspase-3-like activity approximately twofold in control cells (Fig. 6B). ROFA further increased caspase-3-like activity in AM treated with L-arginine and detaNONOate (Fig. 6B). A similar pattern of inhibition by L-NAME was also observed for annexin V expression (Fig. 7). Note that cell necrosis as stained by PI (necrosis) was not affected by L-NAME.


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Fig. 6.   Effects of L-NAME (A) and detaNONOate, L-arginine, and L-ornithine (B) on ROFA-induced increases in caspase-3-like activity. Caspase-3-like activity was measured after AM were incubated with ROFA and NO donors for 4 h. * P < 0.05 vs. untreated control. # P < 0.05 vs. ROFA alone. The results in each group were obtained from experiments on AM from 3-6 different individuals.



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Fig. 7.   Effects of L-NAME on ROFA-induced expression of annexin V. Annexin V expression was measured in AM after 24-h incubation with ROFA with or without L-NAME. * P < 0.05 vs. control. # P < 0.05 vs. ROFA alone. The results in each group were obtained from experiments on AM from 5-10 different individuals.

Effects of Vanadium on ROS Production

Because vanadium is the major soluble metal in ROFA, we determined the contribution of vanadium to ROFA-induced ROS production. Human AM were incubated with 50 µM of VOSO4 for 24 h with or without inhibitors. This concentration of VOSO4 was equivalent to the vanadium content in 100 µg/ml ROFA. VOSO4 increased the chemiluminescence burst, and, like that for ROFA, the chemiluminescence burst was partially inhibited by DPI but not by L-NAME (Fig. 8A). All three mitochondrial inhibitors attenuated the vanadium-induced chemiluminescence burst, a pattern similar to that seen with ROFA (Fig. 8B). Mitochondrial inhibitors did not alter intracellular NO concentration in VOSO4-treated AM (Table 3).


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Fig. 8.   Production of ROS by AM treated with VOSO4 (50 µM). A: chemiluminescence was measured after AM were incubated with VOSO4 for 24 h with or without DPI (4 µM), a flavoprotein inhibitor, or L-NAME (100 µM), a NOS inhibitor. B: chemiluminescence was measured after AM were incubated with VOSO4 for 24 h, followed by incubation for 45 min with 3 mitochondrial electron transport inhibitors: rotenone (2.5 µM), antimycin (4 µM), and sodium azide (0.3%), which inhibit complexes I, III, and IV, respectively. * P < 0.05 vs. control. # P < 0.05 vs. VOSO4 alone. The results in each group were obtained from experiments on AM from 5-10 different individuals.


                              
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Table 3.   Effects of VOSO4 and mitochondrial inhibitors on NO

Effects of Vanadium on Apoptosis

VOSO4 had a small effect on caspase-3-like activity compared with ROFA. The increase by VOSO4 was ~30%, which was not statistically different from that in control AM (Fig. 9A). Unlike ROFA, VOSO4 did not enhance the increase in caspase-3-like activity induced by detaNONOate or L-arginine (Fig. 9B). VOSO4 also did not affect annexin V expression. Approximately 3.8% of cells expressed annexin V in VOSO4-treated AM, which was not statistically different from 2.6% in control AM.


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Fig. 9.   Effects of L-NAME (A) and detaNONOate, L-arginine, and L-ornithine (B) on vanadium-induced increases in caspase-3-like activity. Caspase-3-like activity was measured after AM were incubated with VOSO4 and NO donors for 4 h. * P < 0.05 vs. untreated control. # P < 0.05 vs. VOSO4 alone. The results in each group were obtained from experiments on AM from 5-10 different individuals.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human alveolar macrophages, when stimulated with a combustion pollutant dust, ROFA, produced a large quantity of ROS detected as chemiluminescence burst. A significant portion of these oxidants was produced as a result of electron leaks from the mitochondrial electron transport chain. Membrane NADPH oxidase but not NOS may also contribute to ROFA-induced ROS production, because DPI but not L-NAME attenuated the chemiluminescence burst.

The mitochondrial electron transport chain is an important source for ROS in other cells stimulated by proinflammatory stimulants. In human umbilical vein endothelial cells, TNF-alpha stimulates the production of ROS at the ubiquinone site (10). In keratinocytes, ultraviolet irradiation stimulates ROS produced at complex III (19). In rat alveolar macrophages, 12-o-tetradecanoyl phorbol-13-acetate stimulates ROS from mitochondrial sources, mainly at complexes I and III (42). In A549 cells, vanadate stimulates the production of ROS (mainly hydrogen peroxide) from mitochondria, which produces growth arrest (52). The present study and a previous study from this laboratory (3) have further shown in human AM that ROFA stimulates ROS production from mitochondria. Because all three mitochondrial inhibitors attenuate ROFA-induced chemiluminescence burst, it is difficult to pinpoint the exact sites of electron leaks from the mitochondrial electron transport chain.

The main species of mitochondrial ROS produced by ROFA-stimulated AM should include hydrogen peroxide, given the presence of Mn SOD and the decrease in GSH level that could be reversed by rotenone and sodium azide. Also, ROFA increased oxidation of dihydrorhodamine 123, a reaction product between dihydrorhodamine 123 and hydrogen peroxide in the presence of peroxidase, cytochrome c, or ferrous iron (data not shown). Notably, ROFA decreased intracellular ascorbate levels, and this was not reversible by mitochondrial inhibitors or DPI. This finding may indicate consumption of reduced ascorbate by oxidants and/or disruption of ascorbate uptake and recycling processes (37), which would render the cells more susceptible to oxidative stress (7).

ROFA contains a significant amount of redox active metals, especially vanadium (14) (32). Many proinflammatory effects produced by ROFA are mediated by vanadium, including alveolar neutrophil influx and in vitro activation of rat AM (32), pulmonary vasoconstriction (29), airway epithelial injury and gene expression of MIP-2 and IL-6 (15), and protein tyrosine phosphorylation (44). Here we have shown that vanadium also is likely to mediate ROFA-induced production of ROS from mitochondria, because VOSO4 mimics ROFA in the stimulation of chemiluminescence burst and in patterns of inhibition by various inhibitors of ROS-producing enzymes. The ability of ROFA to stimulate production of ROS from mitochondria indicates that the particles or its components may reach these subcellular organelles. Vanadium, the most abundant metal contained in ROFA, is known to permeate cell membrane avidly. Once inside the cells, vanadium chelates to many intracellular ligands, especially phosphates, e.g., ATP and creatine phosphate, which may partially explain its long elimination half-life (~12 days) (40, 41). Because mitochondria are rich in phosphate compounds, it is reasonable to anticipate a significant portion of intracellular vanadium in mitochondria. When vanadium loads exceed the capacity of the chelators, free vanadyl ions may interact with redox-active ligands and induce electron leaks (24, 35). Alternatively, vanadium may initiate oxidative stress by enhancing tyrosine phosphorylation of membrane receptors such as epidermal growth factors (23). The exact enzymatic sites of electron leaks and the mechanisms by which vanadium promotes electron leaks from these mitochondrial sources require further study.

We also have shown that an increased number of ROFA-treated AM undergo apoptosis, as shown by condensation and fragmentation of nuclei, increased histone release, annexin V expression, and caspase-3-like activity. These findings are consistent with a previous study using cell death ELISA and DNA laddering to demonstrate the proapoptotic property of ROFA (26). The ROFA-induced increase in caspase-3-like activity was not inhibited by mitochondrial inhibitors. In fact, the caspase-3-like activity was increased by sodium azide. Furthermore, VOSO4, which also stimulated production of ROS from mitochondria, has relatively weak effects on apoptosis. These results indicate that ROS produced from mitochondria in response to ROFA do not induce, and might even inhibit, apoptosis of AM. ROS produced from cytosolic sources have been shown to inhibit NO-induced apoptosis in RAW264.7 cells (5, 8).

ROFA-induced apoptosis was inhibited by L-NAME and DEVD, suggesting the dependency on NO and activation of caspase-3-like proteases, respectively. ROFA further enhanced caspase-3-like activity induced by an exogenous NO donor, detaNONOate, and the amino acid substrate of NOS, L-arginine. The increase in caspase-3-like activity, however, cannot be the direct effect of NO because NO is known to inactivate caspases by S-nitrosylation or oxidation (12, 31, 34, 38). Most likely, NO initiates apoptosis by activating upstream events, including p53 accumulation (6), permeation transition (28), cytochrome c release (27), and ATF6/CHOP-related endoplasmic reticulum stress pathways (22).

ROFA tended to increase NO production measured by oxidation of DAF-2DA dye, which would support the contention that ROFA-induced apoptosis is mediated by NO. Antimycin and sodium azide, however, decreased NO concentration. This finding indicates that there is little interaction between mitochondrial ROS and NO. In activated RAW264.7 cells, rotenone and antimycin enhanced cellular NO production, but a chemical uncoupler decreased NO production (50). The reason for the discrepancy is unclear but could be related to the cell types (transformed macrophage cell lines vs. primary human AM) or the stimuli used to activate macrophages.

Unlike its role in mediating ROFA-induced production of ROS, vanadium plays a relatively minor role in mediating the proapoptotic effects of ROFA. VOSO4 at a concentration equivalent to vanadium content in 100 µg/ml ROFA had a relatively small effect on caspase-3-like activity and annexin V expression. VOSO4 did not increase NO production and did not enhance the increase in caspase-3-like activity induced by detaNONOate or L-arginine. It remains a possibility that vanadium in combination with other transition metals contained in ROFA, e.g., Ni or Fe, may have more prominent proapoptotic effects. Alternatively, the timing for the apoptotic events induced by soluble vanadium may be different from that induced by ROFA.

Extrapolation of our results to human exposure to ambient particulate matter should be made with caution. ROFA is a complex combustion-related particle that is collected from a variety of emission sources, e.g., power plants and boilers. Compared with ambient particular matter, ROFA contains higher concentrations of soluble metals and sulfate and thus does not represent typical "real-world" pollutant particles. The doses of ROFA to which AM were exposed were much higher than the one-time exposure in most ambient settings in humans. The contribution of apoptosis of AM to the health effects of particulate matter remains unclear, although in vivo exposure to environmental oxidative stimuli such as ozone and hyperoxia are known to cause apoptosis (16, 36).

In summary, human AM can produce a significant quantity of ROS from mitochondria as chemiluminescence burst when stimulated with the pollutant dust ROFA. Inhibition of oxidant production generated from mitochondrial sources did not inhibit apoptosis of AM, indicating that the prooxidant effects and the proapoptotic effects of ROFA are likely mediated by different mechanisms. The ability to regulate independently these two important host defense mechanisms underscores the role of alveolar macrophages in maintaining the homeostasis of alveolar microenvironment that is exposed constantly to ambient pollutant particles.


    ACKNOWLEDGEMENTS

We thank Dr. Andy Ghio, Maryann Bassett, Debbie Levin, and Sue Darrenbacher in the Medical Station for assisting with bronchoscopy and bronchoalveolar lavage and Lisa Daily for processing lavage samples. We also thank Dr. Gary Hatch for providing ROFA and measuring ascorbate and GSH.


    FOOTNOTES

The research described in this article has been reviewed by the Health Effects and Environmental Research Laboratory, United States Environmental Protection Agency and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of the trade names or commercial products constitute endorsement or recommendation for use.

Address for reprint requests and other correspondence: Y.-C. T. Huang, CB 7315, 104 Mason Farm Road, Chapel Hill, NC 27599 (E-mail: huang.tony{at}epa.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published September 11, 2002;10.1152/ajpcell.00139.2002

Received 28 March 2002; accepted in final form 22 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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