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
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ABSTRACT |
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mitochondria; mesothelial cells; oxidants
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.
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MATERIALS AND METHODS |
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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 /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 [
-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-
x/x!, where f(x) is a function of the distribution of members,! is a factorial, and
= the average lesion frequency] for the nondamaged templates (i.e., the zero class, x = 0), the average lesion frequency per DNA strand was determined:
= -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 [-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 [-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.
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RESULTS |
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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).
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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).
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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).
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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.
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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.
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DISCUSSION |
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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.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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