Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis

Arti Shukla,1 Michael Jung,1 Maria Stern,1 Naomi K. Fukagawa,1 Douglas J. Taatjes,1 Dennis Sawyer,2 Bennett Van Houten,3 and Brooke T. Mossman1

1Department of Pathology, University of Vermont College of Medicine, Burlington, Vermont 05405; 2University of Texas at Galveston, Galveston, Texas 77555; and 3National Institutes of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Submitted 6 February 2003 ; accepted in final form 20 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
To test the hypothesis that asbestos-mediated cell injury is mediated through an oxidant-dependent mitochondrial pathway, isolated mesothelial cells were examined for mitochondrial DNA damage as determined by quantitative PCR. Mitochondrial DNA damage occurred at fourfold lower concentrations of crocidolite asbestos compared with concentrations required for nuclear DNA damage. DNA damage by asbestos was preceded by oxidant stress as shown by confocal scanning laser microscopy using MitoTracker Green FM and the oxidant probe Redox Sensor Red CC-1. These events were associated with dose-related decreases in steady-state mRNA levels of cytochrome c oxidase, subunit 3 (COIII), and NADH dehydrogenase 5. Subsequently, dose-dependent decreases in formazan production, an indication of mitochondrial dysfunction, increased mRNA expression of pro- and antiapoptotic genes, and increased numbers of apoptotic cells were observed in asbestos-exposed mesothelial cells. The possible contribution of mitochondrial-derived pathways to asbestos-induced apoptosis was confirmed by its significant reduction after pretreatment of cells with a caspase-9 inhibitor. Apoptosis was decreased in the presence of catalase. Last, use of HeLa cells transfected with a mitochondrial transport sequence targeting the human DNA repair enzyme 8-oxoguanine DNA glycosylase to mitochondria demonstrated that asbestos-induced apoptosis was ameliorated with increased cell survival. Studies collectively indicate that mitochondria are initial targets of asbestos-induced DNA damage and apoptosis via an oxidant-related mechanism.

mitochondria; mesothelial cells; oxidants


ASBESTOS FIBERS ARE A FAMILY of chemically and physically different types of mineral silicates, including the serpentine variety, chrysotile, and the amphiboles, crocidolite, amosite, tremolite, anthophyllite, and actinolite. Crocidolite asbestos, a high iron-containing fiber, is the most carcinogenic asbestos type in the causation of human mesothelioma, an insidious tumor that is associated with known exposures to asbestos in 75-80% of patients (22, 23, 25). Epidemiological data as well as experimental animal studies demonstrate that asbestos exposure is also associated with the development of lung cancer and pulmonary fibrosis (24, 26). However, the cellular mechanisms involved in the injury and pathogenesis of asbestos-related diseases are unclear.

Many investigators have suggested that oxidants have multifaceted roles in early cell injury and DNA damage by asbestos (reviewed in Refs. 23, 24, and 26). For example, multiple mechanisms for generation of reactive oxygen species (ROS) from crocidolite asbestos fibers exist, including generation of hydroxyl radicals (·OH) via a Fenton reaction utilizing iron present on the surface of the fiber (38). In addition, iron can be mobilized by fibers intracellularly (15). Moreover, phagocytosis of long (>8 µm) asbestos fibers, which are more fibrogenic and carcinogenic than shorter fibers, is accompanied by a prolonged respiratory burst of ROS (12, 14). It is well documented that oxidative stress induced by asbestos fibers may be responsible for asbestos-induced lipid peroxidation (13) and DNA damage in vitro (8, 9, 39). For example, ROS can cause specific chemical modifications of purine and pyrimidine bases, DNA strand breaks, DNA-protein cross-links, and damage to sugar moieties (reviewed in Ref. 18).

Mitochondria consume ~90% of inhaled oxygen and are a particularly rich source of ROS (3). In the presence of various drugs or toxins, the mitochondrial generation of ROS can increase severalfold. Mitochondria contain their own extra chromosomal DNA (mDNA), which is distinct from nuclear DNA (2). Several studies have indicated that when compared with nuclear DNA, mitochondrial DNA (mtDNA) contains an elevated basal level of base damage, including mutagenic 8-hydroxydeoxyguanosine (2). Oxidative mDNA damage has been linked to the onset of specific human pathologies such as neuronal degeneration, cardiovascular disease (35), and aging (1, 30, 37). Several features of mtDNA may be related to its frequent damage and causal associations with disease. For example, mtDNA mutates >10 times as frequently as nuclear DNA and has no introns, so that a random mutation will usually strike a coding DNA sequence. In addition, mtDNA does not have protective histones and is exposed to ROS generated by oxidative phosphorylation and other sources (19). Because mtDNA is a critical cellular target for ROS, chronic ROS exposure, as demonstrated in several degenerative diseases associated with aging, may lead to decreased mitochondrial function and persistent mtDNA damage (29, 41).

In the present investigation, we hypothesized that mitochondria were targets of ROS after acute exposures to crocidolite asbestos in mesothelial cells, the progenitor cells of mesothelioma. We show that mtDNA damage is more extensive and persists longer than nuclear DNA damage after exposure to crocidolite asbestos or H2O2. Asbestos-induced damage to DNA follows asbestos-induced ROS production and mitochondrial dysfunction. A functional consequence of mitochondrial damage by asbestos is the increased expression of apoptotic genes and the development of apoptosis, which is decreased by pretreatment with a caspase-9 inhibitor or catalase. A cause and effect relationship between mtDNA damage by asbestos and apoptosis was confirmed using HeLa cells overexpressing mitochondrial-targeted 8-oxoguanine DNA glycosylase (hOGG) that were resistant to asbestos. The discovery of a mtDNA-linked death pathway by asbestos may have application to the prevention of cell injury in occupational and environmental exposures to asbestos.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cell culture and addition of asbestos or H2O2. Rat pleural mesothelial (RPM) cells were isolated by gentle scraping of the parietal pleura of Fischer 344 rats, which had been killed, and propagated as described previously (16). This procedure was approved by the University of Vermont Institutional Animal Care and Use Committee. Cells were maintained at 37°C at 5% CO2 in F-12 DMEM containing 10% fetal bovine serum (FBS; GIBCO-BRL, Gaithersburg, MD), hydrocortisone (100 ng/ml), insulin (2.5 µg/ml), and selenium (2.5 µg/ml; HITS; Sigma, St Louis, MO). The cells were used between passages 4 and 10. At confluency, cells were switched to 0.5% FBS-containing medium for 24 h before addition of crocidolite asbestos [Na2(Fe3+)2(Fe2+)3Si8-O22(OH)2] (National Institute of Environmental Health Sciences reference sample) at 1.25, 2.5, or 5.0 µg/cm2 dish. Glass beads were used as a nonpathogenic control particle. Particulates were weighed and suspended in Hanks' balanced salt solution (1 mg/ml), triturated 10 times through a 22-gauge needle to obtain a homogenous suspension, and added directly to the medium at the concentrations above. H2O2 (Sigma) was added directly to the medium at final concentrations from 100 to 300 µM. Sham control cultures received medium without agents and were treated identically. In some experiments, catalase (500 U/ml, Sigma) or catalase inactivated by boiling was added 1 h before asbestos. Groups in all experiments consisted of two or three determinations per time point, and all experiments were performed in duplicate.

DNA isolation and quantitative PCR. With the use of methods described previously (41), high-molecular-weight DNA was isolated using a QIAamp DNA isolation kit (Qiagen, Chatsworth, CA) as described by the manufacturer. The concentration of total cellular DNA was determined by ethidium bromide fluorescence with an A4-filter fluorimeter with an excitation band pass filter at 365 nm and an emission cut-off filter at 600 nm (Optical Technology Devices, Elmsford, NY) using {lambda}/HindIII DNA as standard. Quantitative PCR (QPCR) was performed in a GeneAmp PCR system 2400 with the GeneAmp XLPCR kit (Perkin-Elmer) (40). Reaction mixtures contained 15 ng of template DNA, 1.1 mM Mg(AOc)2, 100 µg/ml nonacetylated BSA, 0.2 mM deoxynucleotide triphosphates (Pharmacia), 0.2 µM primers, 0.2 µCi [{alpha}-32P]dATP (1 Ci = 37 GBq), and 1 unit of recombinant Thermus thermophilus DNA polymerase. For RPM cells, a 13.4-kb fragment of the mitochondrial genome and a 12.5-kb fragment of the clusterin gene were amplified as described previously (41). The PCR was initiated with a 75°C hot-start addition of the polymerase and was allowed to undergo the following thermocycler profile: an initial denaturation for 1 min at 94°C followed by 25 cycles of 94°C denaturation for 15 s and 68°C primer extension for 12 min. A final extension at 72°C was performed for 10 min at the completion of the profile. To ensure quantitative conditions, a control reaction containing 7.5 ng of template DNA was included with each amplification. An aliquot of each PCR product was resolved on a 1% vertical agarose gel and electrophoresed in 90 mM Tris/64.6 mM boric acid/2.5 mM EDTA, pH 8.3, at 80 V (5 V/cm) for 4 h. The dried gels were then exposed to a phosphor screen for 12-18 h and quantitated with Imagequant (Molecular Dynamics). DNA lesion frequencies were calculated as described previously (36, 40). Briefly, the amplification of damaged samples (AD) was normalized to the amplification of nondamaged controls (AO), resulting in a relative amplification ratio. Assuming random distribution of lesions and using the Poisson equation [f(x) = e-{lambda} {lambda}x/x!, where f(x) is a function of the distribution of members,! is a factorial, and {lambda} = the average lesion frequency] for the nondamaged templates (i.e., the zero class, x = 0), the average lesion frequency per DNA strand was determined: {lambda} = -ln AD/AO.

In situ oxidant localization. To assess whether asbestos induced oxidant stress, we used Redox Sensor Red CC-1 (Molecular Probes, Eugene, OR), a dye that is oxidized in the cytoplasm before it becomes localized to the mitochondria (34), and the mitochondrial-selective dye, MitoTracker Green FM (Molecular Probes). RPM cells at confluence were exposed to asbestos (5.0 µg/cm2 dish) particles or H2O2 (300 µM) for 30 min, 8 h, and 24 h. After exposure, Red CC-1 (5 µM) and MitoTracker Green (1 µM) were added to each dish for 10 min at 37°C. Cells were washed twice in PBS. Localization of MitoTracker Green (488 nm) and Red CC-1 (568 nm) was examined using a Bio-Rad MRC-1000 (Hercules, CA) confocal scanning laser microscope (CSLM) incorporating an Olympus BX50 upright fluorescence microscope. Control and treated samples were scanned at identical parameters at a magnification of x600. For each sample, confocal images were collected in the fluorescence mode, followed by electronic merging of images.

Northern blot analysis. Confluent cultures of RPM cells maintained in 0.5% serum-containing medium for 24 h were exposed to asbestos for 1 and 8 h. Total RNA (15 µg) was extracted using Ultraspec RNA Isolation Systems (Biotecx Laboratories, Houston, TX) following the manufacturer's procedures and size-fractionated by electrophoresis on 1.2% agarose-formaldehyde gel before transfer to Hybond N+ membranes (Amersham, Piscataway, NJ). The membranes were prehybridized in Rapid-Hyb Buffer (Amersham) at 68°C for 15 min and then hybridized with probes at 68°C for 1 h. The membranes were then washed with twofold higher concentrations of SSC, 0.1% SDS at room temperature for 15 min then twice more in a 1:10 dilution of SSC, 0.1% SDS at 65°C for 15 min. Autoradiography was performed using intensifying screens at -80° for 3-24 h. Quantitative analysis of the autoradiograms was performed using a model GS-700 Imaging Densitometer (Bio-Rad). mRNA was normalized to 28S rRNA and by rehybridization with 7S cDNA probe for further confirmation.

MtDNA probes for Northern blot analyses. PCR was used to synthesize a 5.3 kb of mtDNA fragment from primer pair L7825-H13117, a coding region for cytochrome c oxidase subunit III (COIII) from primer pair L8526-H9130, and a coding region for NADH dehydrogenase subunit 5 (ND5) from primer pair L12634 [GenBank] -H13753. The PCR products were gel purified and labeled with [{alpha}-32P]dCTP (NEN, Boston, MA) using a Random Primers DNA Labeling System (Life Technologies, Grand Island, NY).

MTS metabolic activity assay. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxym-ethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay in which the conversion of MTS tetrazolium to a soluble formazan product is measured to assess mitochondrial dysfunction was used to examine the effect of various concentrations of crocidolite asbestos or H2O2 on the viability of RPM cells (9). Confluent cultures were exposed to crocidolite asbestos or H2O2 as described above for 8, 24, or 48 h, and the production of formazan was measured at 490 nm after addition of MTS tetrazolium reagent for 1 h.

Detection and quantitation of apoptosis. Apoptosis in RPM cells was measured using Apostain and confirmed by transmission electron microscopy (5). Cell monolayers on cover-slips were divided into four groups: 1) untreated; 2) exposed to 5.0 µg/cm2 asbestos; 3) exposed to caspase-9 inhibitor (z-LEHD-FMK) alone (20 µM, Calbiochem); and 4) exposed to z-LEHD-FMK for 30 min before addition of asbestos for 24 h. Thereafter, cells were fixed in cold 100% methanol at -20°C for 24 h. To induce DNA denaturation in situ, cells were heated to 100°C in PBS containing 5 mM MgCl2 for 5 min and then immersed in ice-cold PBS for 10 min. After being incubated with 40% FBS in PBS on ice for 15 min, cells were incubated with a monoclonal antibody to single-stranded DNA (10 µg/ml, Apostain F7-26; Alexis, San Diego, CA) for 30 min at room temperature, washed twice in PBS, and incubated with horseradish peroxidase-conjugated secondary antibody (15 µg/ml, goat anti-mouse IgM; Jackson Laboratories, West Grove, PA) for 30 min at room temperature. The secondary antibody binding was visualized with the peroxidase substrate diaminobenzidine (Vector Laboratories, Burlingame, CA). Cell monolayers were washed and mounted on slides in 90% glycerol in PBS for subsequent examination using bright field light microscopy (5). Ten random fields were evaluated on duplicate coverslips for determination of the number of apoptotic cells and total number of cells per field.

Ribonuclease protection assays. Total RNA was isolated from cells as described previously (32), quantitated by absorbance at 260 nm, and analyzed using a ribonuclease protection assay (RPA) system and a multiprobe template set (rAPO-1) for pro-(bax) and anti-(bcl-2, bcl-x) apoptotic genes, caspases-1, -2 and -3, fas ligand, and fas antigen (Riboquant; PharMingen, San Diego, CA). The template set also included ribosomal protein (L32) and glyceraldehyde-3-phosphate dehydrogenase as housekeeping genes. For synthesis of radio-labeled antisense RNA, the final reaction mixture (20 µl) contained 10 µl [{alpha}-32P]UTP (3,000 Ci/mmol; NEN), 1 µl RNasin, 1 µl GTP, ATP, CTP, UTP pool, 2 µl dithiothreitol, 4 µl 5x transcription buffer, 1 µl of the RPA template set, and 1 µl T7 RNA polymerase according to the manufacturer's protocol. After 1 h at 37°C, the mixture was treated with DNase (2 µl) for 30 min at 37°C, and probes were purified by extractions with phenol/chloroform and precipitation with ethanol. Dried probes were dissolved in 50 µl of hybridization buffer, quantitated, diluted, and added to tubes containing sample RNA (3-5 µg) dissolved in 8 µl of hybridization buffer. The samples were heated at 90°C, followed by incubation at 56°C for 16 h. The single-strand RNA was then digested by addition (100 µl) of a solution of RNase A (80 ng/µl) and RNase T1 (250 U/µl) before incubating samples at 30°C for 45 min. Samples were then treated with 18 µl of a mixture of proteinase K buffer, proteinase K, and yeast tRNA. The RNA duplexes were isolated by extraction/precipitation as above, dissolved in 5 µl of gel loading buffer, and electrophoresed in standard 5% acrylamide/urea sequencing gels. After gels were dried, autoradiograms were quantitated using a Bio-Rad phosphoimager. Data were normalized to expression of L32, and results from two to three lanes per group per time point graphed as relative units (means ± SE).

Assays using HeLa cells transfected with mitochondrial-targeted hOGG. HeLa cells transfected with a mitochondrial transport sequence upstream of the sequence for hOGG and control vector (pcDNA3)-transfected cells (7) were obtained from Dr. Glenn Wilson (Univ. of Alabama at Mobile) and evaluated in clonogenic and apoptosis assays after exposure to asbestos. For clonogenic cell survival assays, MTS-hOGG transfectans and control vector-transfected HeLa cells were counted with a hemocytometer, and 400 cells plated into 60-mm culture plates (n = 3/group). Cells were maintained for 24 h to adhere to plates and then exposed to asbestos at concentrations of 2.5, 5, and 10 µg/cm2 dish. Untreated controls (no asbestos) received medium without asbestos. Plates were incubated in a 5% CO2 humidified environment at 37°C for 10 days. Plates were then rinsed with warm PBS and fixed with a solution of 3 parts methanol:1 part acetic acid for 10 min. Finally, the plates were stained with hematoxylin, and colonies were counted.

Statistical analyses. Data from QPCR studies were analyzed using the unpaired Student's t-test. All other data were examined by ANOVA using the Student-Newman-Keuls procedure to adjust for multiple pairwise comparisons among groups.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Asbestos induces more pronounced DNA damage in mitochondrial DNA. We comparatively assessed mitochondrial vs. nuclear DNA damage in normal RPM cells at 24 and 48 h after addition of asbestos. At 24 h, asbestos caused a dose-related increase in mitochondrial DNA damage (P <= 0.05; Fig. 1A). However, at 48 h, there was some recovery in the 5-µg/cm2 asbestos group. This may reflect a compensatory response, e.g., the induction of DNA repair (10) or the fact that this concentration of asbestos also causes compensatory cell proliferation in RPM cells (11). The addition of H2O2 (300 µM) for 30 min also caused more significant damage to mitochondrial DNA. Similar to mitochondria, nuclei also demonstrated a dose-dependent induction of DNA damage by asbestos (5 µg/cm2) at 24 h but no effects of the lower concentration of asbestos, 1.25 µg/cm2 (Fig. 1B). A comparison of nuclear vs. mtDNA damage by asbestos in RPM cells at 48 h showed complete recovery of nuclear DNA.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. A: amplification of mitochondrial DNA from rat pleural mesothelial (RPM) cells treated with asbestos (1.25 or 5 µg/cm2 for 24 and 48 h) or H2O2 (300 µM for 30 min). Total DNA was isolated, and a 13.4-kb fragment of the mitochondrial genome was amplified using quantitative PCR (QPCR). Asterisks indicate values that are statistically significantly different from control values at that time point (*P = 0.04; **P = 0.006; ***P < 0.001). B: amplification of the clusterin gene from RPM cells treated with asbestos (1.25 or 5 µg/cm2 for 24 and 48 h) or H2O2 (300 µM for 30 min). Total DNA was isolated, and a 12.5-kb fragment of the clusterin gene was amplified using QPCR. Data are expressed as percent of control ± SE of 2 PCRs (n = 2 dishes per group per time point; *P = 0.01; **P < 0.001).

 

Asbestos causes oxidant production that becomes localized in mitochondria. To determine whether oxidants are produced and localized to mitochondria after exposure of RPM cells to asbestos or H2O2 (300 µM for 30 min), we used CSLM to illustrate colocalization of the oxidant probe Red CC-1 with MitoTracker Green, a mitochondrial-selective dye (Fig. 2). Compared with control cells showing little oxidation of Red CC-1 and no colocalization of oxidants in mitochondria (Fig. 2, A-C), asbestos caused dose-related mitochondrial localization of oxidants that was focal at 8 h (Fig. 2, D-F) at sites of fiber deposition (Fig. 2F, arrowheads). Patterns of H2O2-induced oxidant localization were similar to those observed with asbestos (Fig. 2, G-I).



View larger version (72K):
[in this window]
[in a new window]
 
Fig. 2. Confocal scanning laser microscopy showing patterns of colocalization of the oxidant probe, Red CC-1, with the mitochondrial-selective dye, Mito-Tracker Green, in control (A-C) RPM cells, cells exposed to asbestos at 5 µg/cm2 dish for 8 h (D-F), and cells exposed to H2O2 (300 µM for 30 min; G-I). A, D, and G illustrate mitochondria as detected with MitoTracker Green. B, E, and H show Red CC-1 localization. C, F, and I show merged images of MitoTracker Green and Red CC-1 (the yellow/orange signal indicates colocalization of oxidants in mitochondria). Original magnification, x600. Arrowheads illustrate location of asbestos fibers as determined in the refractile mode.

 

Asbestos causes decreases in steady-state levels of mitochondrial COIII and ND5 mRNA and mitochondrial dysfunction and increased expression of pro- and antiapoptotic genes. Northern blot analyses showed that asbestos caused early (1 h) dose-dependent decreases in steady-state mRNA levels of COIII and ND5. Inhibition of COIII mRNA levels persisted for 8 h (Fig. 3A). A transient but significant (P <= 0.05) decrease in steady-state mRNA levels of ND5 was also observed (Fig. 3B). At 24 h, asbestos at high concentrations (5 µg/cm2) caused a significant decrease (P <= 0.05) in formazan production by cells (Fig. 3D). This was accompanied by increased expression of genes linked to pro-(bax) and antiapoptotic (bcl-2, bcl-xL) mitochondrial death pathways as well as increases in caspase-2 and -3 and fas expression (Fig. 4). No changes were observed in expression of caspase-1 or fas ligand (data not shown).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3. Indicators of mitochondrial injury by asbestos: Northern blot analyses showing decreases in COIII (A) and NADH dehydrogenase subunit 5 (B) expression levels by asbestos in RPM cells (means ± SE). Asbestos causes dose-dependent decreases in viability of RPM cells in the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxym-ethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt assay, an indication of mitochondrial dysfunction. C and D: *P <= 0.05 compared with untreated controls at each time point. OD, optical density.

 


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 4. Ribonuclease protection assay on RPM cells at 24 h after addition of asbestos shows increases in expression of apoptotic genes linked to mitochondrial death pathways. Overall significant trends (P <= 0.05) were observed in all asbestos-exposed groups.

 

Asbestos causes apoptosis in RPM cells that is prevented by a caspase-9 inhibitor. The causal involvement of mitochondria in the apoptotic process in RPM cells was also confirmed by experiments using an inhibitor of caspase-9, z-LEHD-FMK. The addition of the caspase-9 inhibitor significantly reduced (P <= 0.05) both asbestos- and H2O2-induced apoptosis in RPM cells, confirming involvement of a mitochondrial pathway (Fig. 5).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5. A and B: apoptosis is an end point of DNA damage by asbestos or H2O2 in RPM cells and is reduced with preaddition of the caspase-9 inhibitor z-LEHD-FMK (20 µM) for 30 min. All experiments were performed at 24 h after addition of asbestos (5 µg/cm2)or H2O2 (200 µM). *P <= 0.05 compared with untreated control group. **P <= 0.05 compared with all other groups.

 

Asbestos-induced apoptosis and cell survival are modified in HeLa cells with targeting of a DNA repair enzyme to mitochondria. The direct involvement of mtDNA damage and repair in asbestos-associated apoptosis and cell survival was demonstrated in HeLa cells transfected with a mitochondrial transport sequence upstream of the sequence for the DNA repair enzyme hOGG (Fig. 6). With the use of the Apostain technique, we confirmed that apoptosis was selectively and significantly inhibited (P <= 0.05) in hOGG-transfected cells (Fig. 6A). Compared with nonexposed HeLa cells, asbestos-exposed HeLa cells transfected with control vector showed dose-related decreases in cell numbers in clonogenic assays (Fig. 6B). In contrast, HeLa cells with mitochondrial-targeted hOGG showed increased numbers of viable cells at all concentrations of asbestos.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6. Asbestos-induced apoptosis (A) and clonogenic cell growth (B) are modified in HeLa cells expressing mitochondrial-targeted 8-oxoguanine DNA glycosylase (hOGG). *P <= 0.05 compared with vector control groups. asb, Asbestos.

 

Asbestos-induced apoptosis is diminished after pretreatment of RPM cells with catalase. Figure 7 confirms that asbestos-related apoptosis is oxidant dependent. RPM cells exposed to asbestos alone or in the presence of boiled, inactivated catalase showed elevated numbers of Apostain-positive cells (P <= 0.05) compared with untreated controls. However, catalase (500 U/ml) caused diminution of asbestos-associated apoptosis compared with asbestos alone.



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 7. Asbestos-induced apoptosis is ameliorated when RPM cells are pretreated with catalase (C; 500 U/ml) for 1 h. All groups were evaluated by the Apostain technique at 24 h after addition of asbestos. *P <= 0.05 compared with untreated control group; #P <= 0.05 compared with asbestos alone group.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Asbestos, particularly the high iron-containing amphibole types, crocidolite and amosite, is a group of naturally occurring, insoluble fibers causing mesotheliomas, pleural and pulmonary fibrosis, and lung cancer (22, 24-27). DNA damage, mutagenesis, and cell proliferation are important features of asbestos-induced cell injury in vitro. On the basis of work from our laboratory and others indicating that asbestos can cause oxidative lesions in DNA and modulation of mitochondrial gene expression revealed by differential mRNA display (17), we hypothesized that asbestos preferentially affects mtDNA through an oxidant-dependent mechanism. We show here that asbestos causes enhanced mtDNA damage in normal rat mesothelial cells, and results also confirmed this in human MeT-5A immortalized mesothelial cells (data not shown). Moreover, asbestos induces oxidants that may become localized in mitochondria. The functional ramifications of mitochondrial damage by asbestos are causally related to the development of apoptosis as asbestos-induced apoptosis was diminished in HeLa cells overexpressing the DNA repair enzyme hOGG in mitochondria.

In normal mesothelial cells, mtDNA damage occurs at lower concentrations of fibers and persists longer (48 h) than nuclear DNA damage after exposure to asbestos. Several mechanisms may explain these phenomena. First, complex chromatin organization, which may serve as a protective barrier against ROS in the nucleus, is absent in mitochondrial DNA. Second, metals in or associated with proteins such as ferritin in mitochondria (21) may function as catalysts in the generation of ROS. Last, mitochondria may be more susceptible to the stimulation of secondary ROS reactions due to damage to the electron transport chain. Persistent mtDNA damage might also reflect continual ROS production by lipid peroxidation since lipids within the inner mitochondrial membrane contain components of the electron transport chain, many of which contain transition metal ions. In this regard, stimulation of both radical and nonradical species through metal-catalyzed lipid peroxidation reactions damaged DNA in a number of models (6, 33). Results here can be compared with experiments by Yakes and Van Houten (41) in human fibroblasts where oxidant-induced damage by H2O2 is more extensive and persists longer in mitochondrial vs. nuclear DNA. One possibility is that mtDNA repair enzymes are compromised by oxidative stress (7).

Asbestos fibers generate oxidants by iron-mediated redox reactions occurring on the fiber surface and after phagocytosis by cells (26, 31). Studies with Red CC-1 suggest that asbestos fibers cause cytoplasm accumulation of oxidants that may affect mitochondria. Oxidant accumulation then results in early alterations in steady-state mRNA levels of two mitochondrially encoded components of mitochondrial enzymes, COIII and ND5, in a dose-related fashion. At 8 h, ND5 levels, at nontoxic levels of asbestos (1.25 µg/cm2 dish), were restored to those comparable in untreated cells. Although H2O2 was not evaluated comparatively in these studies, previous work shows that 16S rRNA and NADH dehydrogenase, subunits ND5 and ND6, were initially increased after addition of H2O2 (200 µM) to rat lung epithelial cells but decreased at later time points coincidental with the development of apoptosis (17). These studies together suggest that decreased expression of COIII and ND5 triggers or reflects later cell death.

That apoptosis observed in RPM cells after asbestos exposure is mitochondrially derived was further confirmed by RPA in which expression of both proapoptotic (bax) and prosurvival (bcl-2, bcl-xL) mitochondrial pathway genes, as well as caspase-3, was increased. Recent studies confirm that overexpression of Bcl-x1, an antiapoptotic protein localizing to mitochondria, diminishes apoptosis by asbestos in A549 cells (20, 28). These results suggest that a balance between pro- and antiapoptotic gene expression governs cell responses to asbestos. Finally, asbestos-induced apoptosis was inhibited by preexposure to an inhibitor of caspase-9, which gets activated only after cytochrome c release from mitochondria. It has been proposed that active caspase-8 mediates activation of the downstream executioner procaspase-3 via two overlapping mechanisms. Caspase-8 either directly induces procaspase-3 cleavage or induces cleavage indirectly by a mitochondrial amplification loop requiring BID cleavage, cytochrome c release, and activation of procaspase-9. Since we did not observe asbestos-induced BID cleavage in our studies (A. Shukla, unpublished data), cytochrome c release from mitochondria could be linked to injury by oxidative stress (20). As has been suggested by the results of others (4, 20), our studies using catalase partially prevented asbestos-induced apoptosis, suggesting the importance of H2O2 and iron-derived derivatives such as ·OH.

In conclusion, our results show that mitochondrial compared with nuclear genes are more sensitive to asbestos-induced DNA damage. Several mechanisms are possible. First, persistent DNA damage by asbestos could be causally related to increased localization of ROS in mitochondria after exposure to asbestos. Because asbestos fibers induce a prolonged oxidative burst during uptake by the cell, ROS produced by asbestos fibers interacting with the plasma membrane or in the cytoplasm may accumulate in mitochondria. In addition, metals, such as iron on fibers, might be transported into mitochondria where they catalyze the formation of ROS (21). A third possibility is that asbestos fibers may interact with mitochondrial membranes, causing alterations in permeability, and alter cell respiration. Loss of mitochondrial function, because of DNA damage and lack of repair and/or accumulation of ROS, may then lead to the induction of apoptosis, as was recently demonstrated in alveolar epithelial cells in which asbestos triggered the release of cytochrome c and reduced mitochondrial membrane potential (28). All processes may contribute to chronic cell injury. The ability to inhibit apoptosis using a caspase-9 inhibitor or by boosting mtDNA repair enzyme levels is intriguing and may have relevance to the interference of mitochondrial signaling pathways in preventive and therapeutic approaches to asbestos-induced diseases.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This research was supported by National Institutes of Health Program Project Award PO1-HL-67004 and RO1 Grant ES/HL-09213.


    ACKNOWLEDGMENTS
 
The authors thank Laurie Sabens for preparation of the manuscript and Nikol Manning for illustrations. Dr. Pamela Vacek, Department of Medical Biostatistics, University of Vermont, kindly provided expertise on other statistical analyses. Maximilian MacPherson performed the ribonuclease protection assays.


    FOOTNOTES
 

Address for reprint requests and other correspondence: B. T. Mossman, Dept. of Pathology, Univ. of Vermont College of Medicine, 89 Beaumont Ave., Burlington, VT 05405 (E-mail: Brooke.Mossman{at}uvm.edu).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Ames B, Shigenaga M, and Hagen T. Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA 90: 7915-7922, 1993.[Abstract/Free Full Text]
  2. Beckman K and Ames B. Endogenous oxidative damage of mtDNA. Mutat Res 424: 51-58, 1999.[ISI][Medline]
  3. Breen A and Murphy J. Reactions of oxyl radicals with DNA. Free Radic Biol Med 18: 1033-1077, 1995.[ISI][Medline]
  4. Broaddus V, Yang L, Scavo L, Ernst J, and Boylan A. Asbestos induces apoptosis of human and rabbit pleural mesothelial cells via reactive oxygen species. J Clin Invest 98: 2050-2059, 1996.[Abstract/Free Full Text]
  5. Buder-Hoffmann S, Palmer C, Vacek P, Taatjes D, and Mossman B. Different accumulation of activated extracellular signal-regulated kinases (ERK 1/2) and role in cell-cycle alterations by epidermal growth factor, hydrogen peroxide, or asbestos in pulmonary epithelial cells. Am J Respir Cell Mol Biol 24: 405-413, 2001.[Abstract/Free Full Text]
  6. Demple B and Harrison L. Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem 63: 915-948, 1994.[ISI][Medline]
  7. Dobson A, Xu Y, Kelley M, LeDoux S, and Wilson G. Enhanced mitochondrial DNA repair and cellular survival after oxidative stress by targeting the human 8-oxoguanine glycosylase repair enzyme to mitochondria. J Biol Chem 275: 37518-37523, 2000.[Abstract/Free Full Text]
  8. Faux S, Howden P, and Levy L. Iron-dependent formation of 8-hydroxydeoxyguanosine in isolated DNA and mutagenicity in Salmonella typhimurium TA102 induced by crocidolite. Carcinogenesis 15: 1749-1751, 1994.[Abstract]
  9. Fung H, Kow Y, Van Houten B, and Mossman B. Patterns of 8-hydroxydeoxyguanosine (8OHdG) formation in DNA and indications of oxidative stress in rat and human pleural mesothelial cells after exposure to crocidolite asbestos. Carcinogenesis 18: 101-108, 1997.
  10. Fung H, Kow Y, Van Houten B, Taatjes D, Hatahet Z, Janssen Y, Vacek P, Faux S, and Mossman B. Asbestos increases mammalian major AP-endonuclease (APE) gene expression, protein levels and enzyme activity in rat pleural mesothelial cells. Cancer Res 58: 189-194, 1998.[Abstract]
  11. Goldberg J, Zanella C, Janssen Y, Timblin C, Jimenez L, Taatjes D, and Mossman B. Novel cell imaging approaches show induction of apoptosis and proliferation in mesothelial cells by asbestos. Am J Respir Cell Mol Biol 17: 265-271, 1997.[Abstract/Free Full Text]
  12. Goodglick L and Kane A. Role of reactive oxygen metabolites in crocidolite asbestos toxicity to mouse macrophages. Cancer Res 46: 5558-5566, 1986.[Abstract]
  13. Gulumian M. The ability of mineral dusts and fibers to initiate lipid peroxidation. I. Parameters which determine this ability. Redox Rep 4: 141-163, 1999.[ISI][Medline]
  14. Hansen K and Mossman B. Generation of superoxide () from alveolar macrophages exposed to asbestiform and nonfibrous particles. Cancer Res 47: 1681-1686, 1987.[Abstract]
  15. Hardy J and Aust A. The effect of iron binding on the ability of crocidolite asbestos to catalyze DNA single-strand breaks. Carcinogenesis 16: 319-325, 1995.[Abstract]
  16. Heintz N, Janssen Y, and Mossman B. Persistent induction of c-fos and c-jun expression by asbestos. Proc Natl Acad Sci USA 90: 3299-3303, 1993.[Abstract]
  17. Janssen Y, Driscoll K, Timblin C, Hassenbein D, and Mossman B. Modulation of mitochondrial gene expression in pulmonary epithelial cells exposed to oxidants. Environ Health Perspect 106: 1191-1195, 1998.[ISI][Medline]
  18. Janssen Y, Van Houten B, Borm P, and Mossman B. Cell and tissue responses to oxidative damage. Lab Invest 69: 261-274, 1993.[ISI][Medline]
  19. Johns D. Mitochondrial DNA and disease. N Engl J Med 333: 638-644, 1995.[Free Full Text]
  20. Kamp D, Panduri V, Weitzman S, and Chandel N. Asbestos-induced alveolar epithelial cell apoptosis: role of mitochondrial dysfunction caused by iron-derived free radicals. Mol Cell Biochem 234-235: 153-160, 2002.[ISI]
  21. Levi S, Corsi B, Bosisio M, Invernizzi R, Volz A, Sanford D, Arosio P, and Drysdale J. A human mitochondrial ferritin encoded by an intronless gene. J Biol Chem 276: 24437-24440, 2001.[Abstract/Free Full Text]
  22. McDonald A and McDonald J. Epidemiology of malignant mesothelioma. In: Asbestos-Related Malignancy, edited by Aisner J. Orlando, FL: Grune and Stratton, 1987, p. 31-56.
  23. Mossman B, Bignon J, Corn M, Seaton A, and Gee J. Asbestos: scientific developments and implications for public policy. Science 247: 294-301, 1990.[ISI][Medline]
  24. Mossman B and Churg A. State-of-the-art: mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med 157: 1666-1680, 1998.[ISI][Medline]
  25. Mossman B and Gee J. Asbestos related disease. N Engl J Med 320: 1721-1730, 1989.[ISI][Medline]
  26. Mossman B, Kamp D, and Weitzman S. Mechanisms of carcinogenesis and clinical features of asbestos-associated cancers. Cancer Invest 14: 466-480, 1996.[ISI][Medline]
  27. Mossman B, Marsh J, Gilbert R, Hardwick D, Sesko A, Hill S, Shatos M, Doherty J, Bergeron M, Adler K, Hemenway D, Mickey R, Vacek P, and Kagan E. Inhibition of lung injury, inflammation, and interstitial pulmonary fibrosis by polyethylene glycol-conjugated catalase in a rapid inhalation model of asbestosis. Am Rev Respir Dis 141: 1266-1271, 1990.[ISI][Medline]
  28. Panduri V, Weitzman S, Chandel N, and Kamp D. The mitochondria-regulated death pathway mediates asbestos-induced alveolar epithelial cell apoptosis. Am J Respir Cell Mol Biol 28: 241-248, 2003.[Abstract/Free Full Text]
  29. Sawyer D and Van Houten B. Repair of DNA damage in mitochondria. Mutat Res 434: 161-176, 1999.[ISI][Medline]
  30. Shigenaga M, Hagen T, and Ames B. Oxidative damage and mitochondrial decay in aging. Proc Natl Acad Sci USA 91: 10771-10778, 1994.[Abstract/Free Full Text]
  31. Shukla A, Gulumian M, Hei T, Kamp D, Rahman Q, and Mossman B. Multiple roles of oxidants in the pathogenesis of asbestos-induced diseases. Free Radic Biol Med 34: 1117-1129, 2003.[ISI][Medline]
  32. Shukla A, Timblin C, Hubbard A, Bravman J, and Mossman B. Silica-induced activation of c-Jun-NH2-terminal amino kinases, protracted expression of the activator protein-1 protooncogene, fra-1, and S-phase alterations are mediated via oxidative stress. Cancer Res 61: 1791-1795, 2001.[Abstract/Free Full Text]
  33. Stohs S and Bagchi D. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18: 321-336, 1995.[ISI][Medline]
  34. Tanaka H, Matsumura I, Ezoe S, Satoh Y, Sakamak T, Albanese C, Machii T, Pestell RG, and Kanakura Y. E2F1 and c-Myc potentiate apoptosis through inhibition of NF-{kappa}B activity that facilitates MnSOD-mediated ROS elimination. Mol Cell 9: 1017-1029, 2002.[ISI][Medline]
  35. Tritschler H and Medori R. Mitochondrial DNA alterations as a source of human disorders. Neurology 43: 280-288, 1993.[Abstract]
  36. Van Houten B, Cheng S, and Chen Y. Measuring DNA damage and repair in human genes using quantitative amplification of long targets from nanogram quantities of DNA. Mutation Res 460: 81-94, 2000.[ISI][Medline]
  37. Wallace D, Shoffner J, Trounce I, Brown M, Ballinger S, Corral-Debrinski M, Horton T, Jun A, and Lott M. Mitochondrial DNA mutations in human degenerative diseases and aging. Biochim Biophys Acta 1271: 141-151, 1995.[ISI][Medline]
  38. Weitzman S and Graceffa P. Asbestos catalyzes hydroxyl and superoxide radical generation from hydrogen peroxide. Arch Biochem Biophys 228: 373-376, 1984.[ISI][Medline]
  39. Xu A, Wu L, Santella R, and Hei T. Role of oxyradicals in mutagenicity and DNA damage induced by crocidolite asbestos in mammalian cells. Cancer Res 59: 5922-5926, 1999.[Abstract/Free Full Text]
  40. Yakes F, Chen Y, and Van Houten B. Technologies for Detection of DNA Damage and Mutations, edited by Pfeifer G. New York: Plenum, 1996, p. 169-182.
  41. Yakes F and Van Houten B. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc Natl Acad Sci USA 94: 514-519, 1997.[Abstract/Free Full Text]