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TOXICOLOGY
Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario, Canada
Received October 18, 2004; accepted December 16, 2004.
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Abstract |
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; Forkert and Reynolds, 1982; Krijgsheld et al., 1984). Cytotoxicity was manifested selectively in bronchiolar Clara cells and seemed to involve a number of organelles, including nuclei, smooth endoplasmic reticulum, and mitochondria. Alterations in mitochondrial morphology occurred early in the toxic response to DCE and were evident as swelling, disruption of cristae, and loss of mitochondrial matrix density (Forkert and Reynolds, 1982; Forkert et al., 1986). Additionally, electron-dense aggregates were detected in the mitochondrial matrices and may represent calcium phosphate depositions (Hagler et al., 1981; Forkert et al., 1986). Thus, these data indicated that mitochondria are a primary target of damage in DCE-mediated pneumotoxicity.
The finding of selective Clara cell injury in conjunction with studies demonstrating high levels of covalent binding of DCE metabolites in this cell population suggested that DCE-induced Clara cell toxicity is mediated by metabolic formation of reactive intermediates (Okine et al., 1985; Forkert et al., 1986). In support of this premise, immunohistochemical studies with an antibody directed against DCE protein adducts revealed selective staining in Clara cell apices (Forkert, 1999a). In other studies, covalent binding of DCE-derived radiolabel was shown to be greatest in the mitochondrial fractions of the lung (Okine et al., 1985). Thus, binding of reactive metabolites to mitochondrial proteins may be involved in the mechanism(s) by which DCE mediates Clara cell cytotoxicity.
Many pathological stimuli have been shown to produce mitochondrial-mediated apoptosis by induction of the mitochondrial permeability transition (MPT) (Crompton, 1999). This event requires elevated cytosolic calcium levels in addition to an "inducing agent", such as substances that oxidize sulfhydryl groups (Lemasters et al., 1998; Crompton, 1999). Activation of the MPT causes acute mitochondrial swelling that may lead to rupture or permeabilization of the outer membrane thus releasing apoptogenic proteins into the cytosol (Halestrap et al., 2000). One such protein, cytochrome c, has been shown to trigger the assembly of the apoptosome, a multimeric structure composed of cytochrome c, apoptotic protease activating factor 1 and dATP. This complex recruits and activates procaspase-9, a prolific initiator caspase that subsequently cleaves and activates procaspase-3 (Hengartner, 2000). Caspase-3, a potent downstream effector caspase, induces the proteolytic cleavage of a range of target proteins responsible for rearrangements of the cytosol, nucleus, and plasma membrane characteristic of apoptosis (Kluck et al., 1997; Li and Yuan, 1999; Zimmermann et al., 2001).
It has previously been reported that DCE induces both necrotic and apoptotic cell death in the livers of experimental animals (Reynolds et al., 1984; Martin and Forkert, 2004). Hepatic apoptosis involved induction of the MPT, cytochrome c release, and activation of caspases. Although DCE-mediated Clara cell cytotoxicity has been well characterized (for review, see Forkert, 2001), it is currently unknown whether DCE produces pulmonary apoptosis. Because mitochondria have been identified as an early target of pneumotoxicity, we have undertaken studies to test the hypothesis that mitochondrial-mediated apoptosis is involved in DCE-induced lung cytotoxicity in mice. Our objectives were to investigate parameters indicative of this mode of cell death, including mitochondrial perturbations, cytochrome c release, and activation of caspase-3. Additionally, we have investigated the occurrence of DCE-induced apoptosis and its relationship to necrosis using the TUNEL assay and histopathological evaluation.
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Materials and Methods |
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). All other chemicals were of reagent grade and were obtained from standard commercial suppliers.
Animal Treatment. Female CD-1 mice (25–30 g) were obtained from Charles River Canada (St. Constant, QC, Canada). They were maintained on a 12-h light/dark cycle and given free access to food (Purina Rodent Chow; Ralston Purina International, Strathroy, ON, Canada) and drinking water. After acclimatization to laboratory conditions for at least 5 days, mice were randomly assigned to control or treatment groups (n = 3). In all experiments, mice were treated intraperitoneally (i.p.) with 75 mg/kg DCE in corn oil. Similar DCE doses have previously been shown to produce early mitochondrial aberrations and Clara cell injury in murine lung (Forkert and Reynolds, 1982; Forkert et al., 1986). In ultrastructural studies, mice were treated with DCE for 2 h and then anesthetized with sodium pentobarbital for perfusion of lung tissue. For detection of cytochrome c in lung cytosol, mice were treated with DCE for 4 h and then sacrificed by cervical dislocation. In immunohistochemical and TUNEL staining experiments, mice were treated with DCE for 12 or 24 h and then anesthetized for perfusion. For histopathological assessment, mice were treated with DCE (50, 75, or 125 mg/kg) for 12 or 24 h and subsequently perfused. In all studies, control mice were treated with equivalent volumes of the vehicle and were sacrificed at times corresponding to those in the experimental groups.
Electron Microscopy. Electron microscopy was used to detect DCE epoxide-cysteine adducts and to assess mitochondrial morphology in murine lung 2 h after DCE treatment. Anesthetized mice were perfused with 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer containing 50 mM lysine, pH 7.4. Lungs were further fixed by tracheal inflation with 0.4 ml of the same fixative. Next, lungs were excised and immersed in fresh fixative for 3 h at room temperature (RT) and then overnight at 4°C. For morphological studies, tissues were postfixed with 1% osmium tetroxide in 0.1 M PIPES buffer, pH 6.8, for 1 h on ice. These tissues were then immersed in supersaturated uranyl acetate for 2 h and washed in fresh PIPES buffer. All lung tissues were subsequently cut into approximately 1-mm blocks, dehydrated through a graded ethanol series, embedded in LR white resin, and mounted on Formvar-coated nickel grids. For detection of DCE epoxide-cysteine adducts, immunogold labeling was performed as described previously (Oko and Maravei, 1995). The technique for applying the various solutions to the tissue sections involved floating the grids, tissue-side down, on 15-µl drops of solution. The grids were first floated on Tris-buffered saline (TBS) with 5% normal goat serum (NGS) and 3% BSA for 15 min. Sections were then transferred to the primary antibody (anti-DCE epoxide/cysteine, 1:25) and allowed to incubate for 2 h on a shaker-bed. The primary antibody was diluted in TBS containing 1% BSA and 0.1% of the conjugate glycine-glutaraldehyde-bovine serum albumin that was synthesized as described in previous studies (Forkert et al., 1997) and that has been shown to inhibit nonspecific antibody binding. Next, grids were rinsed five times for 5 min on TBS containing 0.1% Tween 20 and washed for 15 min on TBS with 1% BSA and 5% NGS. Sections were then transferred to the secondary antibody (colloidal gold 8–10-nm-conjugated goat anti-rabbit IgG, 1:40) and allowed to incubate for 1 h on a shaker-bed. The secondary antibody was diluted in TBS with 1% BSA and 2.5% NGS. Grids were then washed with TBS containing 0.1% Tween 20 and thoroughly rinsed with nanopure water. Finally, tissues were stained with 5% uranyl acetate in 70% ethanol for 5 min followed by lead citrate for 10 min and photographed using a Hitachi H-7000 electron microscope. Control incubations were performed in the absence of the specific antibody to control for the specificity of the immunohistochemical reaction. For morphological evaluation, sections were stained with uranyl acetate and lead citrate as described above and then photographed.
Protein Immunoblotting. Mitochondrial release of cytochrome c was evaluated by Western blotting as described previously (Forkert et al., 1996), with modifications. Briefly, lungs of mice were excised, rinsed, blotted, and weighed. Cytosolic fractions were then prepared as described previously (Forkert, 1999a), aliquoted, frozen in liquid nitrogen, and stored at –70°C. Protein concentrations were determined using the Bradford protein assay (Bradford, 1976). Cytosolic proteins (25 µg/lane) were separated electrophoretically on a 15% SDS-polyacrylamide gel and then transferred to a polyvinylidene difluoride (PVDF) membrane filter. Next, the membrane was stained with Ponceau S to confirm uniform protein loading and to ensure that equal amounts of protein were transferred to the PVDF membrane. The membrane was then washed with distilled water, rinsed with TBS, and blocked overnight with 10% nonfat dried milk diluted in TBS. After thorough washing in Tween 20-TBS (T/TBS), the membrane was reacted for 2 h at RT with a monoclonal antibody directed against cytochrome c (1:500). The membrane was again washed with T/TBS to remove unbound antibodies and then reacted with a secondary antibody (goat anti-mouse IgG conjugated to horseradish peroxidase, 1:10,000) for 2 h at RT. The antibodies were diluted in T/TBS containing 1% nonfat dried milk. Proteins were visualized using an Immun-Star horseradish peroxidase chemiluminescence kit (Bio-Rad). Densitometric analysis of the gels was performed using SigmaGel software (Sigma-Aldrich).
Immunohistochemical Detection of Caspase-3. Lung tissue was prepared for immunohistochemical evaluation as described previously (Forkert, 1999a), with minor modifications. Briefly, lungs were fixed with 4% paraformaldehyde in 0.1 M Sorenson's buffer (12.0 mM NaH2PO4 and 69.0 mM Na2HPO4, pH 7.4) by vascular perfusion and tracheal instillation. Tissues were then processed and embedded in paraffin using standard histological procedures. Tissue sections (5 µm) were deparaffinized, cleared, and hydrated. After rinsing in phosphate-buffered saline (PBS), the sections were blocked in 5% NGS for 20 min. After further rinsing in PBS, the sections were incubated for 10 min at RT with avidin and then biotin to block any endogenous biotin. Tissue sections were subsequently incubated overnight in rabbit antiactive caspase-3 polyclonal antiserum (Cell Signaling Technology Inc., Beverly, MA). The antiserum was diluted 1:200 in PBS containing 2.5% NGS. The sections were then rinsed to remove unbound antibodies and were reacted with biotinylated goat anti-rabbit IgG for 10 min at RT. Endogenous peroxidase activity was blocked by incubating tissue sections for 30 min with 1% hydrogen peroxide (H2O2) in nanopure water. Sections were then reacted with streptavidin conjugated to horseradish peroxidase for 10 min, and the immunoperoxidase color reaction was developed using a DAKO liquid diaminobenzidine kit (DakoCytomation California Inc., Carpinteria, CA). The tissue sections were then counterstained in hematoxylin, dehydrated, cleared, and mounted. Incubations were also performed in the absence of the specific antibody to control for the specificity of the immunohistochemical reaction.
TUNEL Assay. DNA fragmentation characteristic of apoptosis was examined using a terminal deoxynucleotidyl transferase (TdT)-FragEL kit (Calbiochem, San Diego, CA). Briefly, 4% paraformaldehyde-fixed tissue samples were embedded in paraffin, and 5-µm sections were cut. Replicate sections were rehydrated and permeabilized with proteinase K (20 µg/ml) for 20 min at RT. Next, endogenous peroxidases were inactivated by covering the sections with 3% H2O2 for 5 min. After incubation for 5 min in TdT buffer (200 mM Nacacodylate, 30 mM Tris, 0.3 mg/ml BSA, and 0.75 mM CoCl2, pH 6.6), the slides were covered with TdT and biotinylated dUTP and incubated for 1.5 h at 37°C in a humidified chamber. Negative controls were incubated with biotinylated dUTP in TdT buffer in the absence of enzyme. The reaction was terminated by covering the sections with stop buffer (0.5 M EDTA, pH 8.0) for 5 min at RT. After blocking in 4% BSA, the slides were incubated with a streptavidin-horseradish peroxidase conjugate for 30 min. The sections were then incubated in diaminobenzidine for 30 s at RT and counterstained with methyl green. Apoptotic cells were identified by staining properties and by morphological criteria (chromatin condensation and margination along the nuclear membrane).
Histopathology. Lung tissue was prepared for histopathological evaluation as described previously (Forkert et al., 2001). Briefly, lungs were fixed by intratracheal instillation and vascular perfusion through the left ventricle with 4% paraformaldehyde in 0.1 M Sorensen's phosphate buffer, pH 7.4. Tissues were excised and then processed and embedded in paraffin using standard histological procedures. Lung sections (5 µm) were stained with hematoxylin and eosin.
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Assessment of Mitochondrial Morphology. The morphology of mitochondria from pulmonary bronchiolar epithelial cells was assessed in control and DCE (75 mg/kg)-treated mice using electron microscopy. In control lungs, Clara cells contained numerous, pleomorphic mitochondria with well defined cristae (Fig. 2A). However, at 2 h after DCE treatment, Clara cell mitochondria were damaged (Fig. 2B), whereas those from ciliated cuboidal cells remained intact (Fig. 2C). Alterations in Clara cell mitochondria included severe swelling, disruption of cristae such that they were virtually absent, and loss of mitochondrial matrix density. The mitochondrial lesion seen here is similar to that observed in previous studies using similar DCE doses and exposure times (Forkert and Reynolds, 1982; Forkert et al., 1986).
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Immunochemical Detection of Cytochrome c. Protein immunoblotting of lung cytosol for cytochrome c revealed a single band of approximately 15 kDa in all samples (Fig. 3). At 4 h, immunoreactive protein was significantly augmented in cytosolic fractions from DCE-treated mice compared with fractions from untreated animals. Densitometric analysis of the protein bands showed that the amounts of cytochrome c in cytosolic fractions from DCE-treated mice were approximately 3-fold higher than in those from untreated mice. The relative amounts of cytochrome c protein were determined by normalization to Ponceau S-stained protein bands.
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Localization of Activated Caspase-3. Immunohistochemical studies were performed on lung tissues to detect activated caspase-3, one of the key executioners of apoptosis (Hengartner, 2000). Staining of cleaved caspase-3 is characteristically localized in the cytoplasm and perinuclear region of apoptotic cells. In lung sections from control mice, specific staining was not apparent (Fig. 4A). However, in sections from mice treated with 75 mg/kg DCE for 12 or 24 h, staining was observed in cells of the bronchiolar epithelium (Fig. 4, B and C). Moreover, staining for activated caspase-3 seemed to increase from 12 to 24 h. In sections from DCE-treated mice where the specific antibody was omitted, staining was not visible (not shown).
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TUNEL Assay. The TUNEL assay was used to detect pulmonary DNA fragmentation characteristic of apoptosis. In control lung sections, TUNEL-positive cells were absent (Fig. 5A). However, in sections from mice treated with 75 mg/kg DCE, TUNEL staining was detected in the bronchiolar epithelium. At 12 h after DCE, confluent TUNEL staining was observed in small diameter bronchioles (Fig. 5B). This staining was diminished by 24 h because Clara cells had mostly exfoliated (Fig. 5C). Furthermore, at 24 h after DCE, the staining pattern of cells of the bronchiolar epithelium was not consistent throughout. In small diameter bronchioles, TUNEL-positive cells exhibited diffuse cytoplasmic and nuclear staining (Fig. 5C), whereas in larger airways many scattered cells with distinct nuclear staining were observed (Fig. 5D). In sections from DCE-treated mice where the TdT enzyme was omitted, TUNEL staining was not apparent (not shown).
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Histopathology. Histological observations revealed normal morphology in the lungs of all untreated mice, with the usual distribution of ciliated cuboidal and nonciliated Clara cells in the bronchioles (Fig. 6A). At 12 h after administration of 50 or 75 mg/kg DCE, damage to the bronchiolar epithelium was manifested as vacuolation and exfoliation of a few Clara cells (Fig. 6B). In contrast, treatment with 125 mg/kg DCE for 12 h evoked severe damage to the bronchiolar epithelium with most Clara cells undergoing necrosis and exfoliation, resulting in marked diminution in the height of the lining cells (Fig. 6C). By 24 h after 50 or 75 mg/kg DCE, the bronchiolar lesion had increased considerably in severity. In the majority of bronchioles, Clara cell exfoliation was complete and the residual epithelium was composed of flattened ciliated cells (Fig. 6D). The bronchiolar injury at 24 h after 125 mg/kg DCE was comparable with that observed in the other experimental groups (50 and 75 mg/kg) at this time. At 24 h after 75 or 125 mg/kg DCE, apoptotic-like nuclei were also observed scattered in the bronchiolar epithelium and were characterized by chromatin condensation and margination (Fig. 6, E and F). Apoptotic-like nuclei were more abundant in large diameter bronchioles where the DCE-induced cytotoxicity was less severe. Collectively, these data indicated that Clara cell toxicity and induction of apoptosis were dose- and time-dependent.
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Discussion |
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; Kanz and Reynolds, 1986). Previous studies have also demonstrated apoptotic cell death in the livers of mice, a phenomenon that involved induction of the MPT, cytochrome c release to the cytosol, and activation of the caspase cascade (Martin and Forkert, 2004). Because early mitochondrial damage is a prominent feature of both the hepatic and pulmonary lesion produced by DCE, we have undertaken studies to test the hypothesis that mitochondrial-mediated apoptosis is involved in the pneumotoxicity induced by DCE in mice. Here, we document the occurrence of pulmonary bronchiolar apoptosis, and its relationship to necrosis, after acute exposure to DCE. Our results suggested that DCE produces apoptotic cell death in the bronchiolar epithelium by inducing mitochondrial perturbations, causing release of cytochrome c to the cytosol and caspase activation.
There are several means by which xenobiotics can alter mitochondrial morphology and/or function, and a major mechanism is covalent binding to critical proteins. Binding of xenobiotics and/or metabolites to mitochondrial proteins can lead to such deleterious consequences as impairment of electron transfer capability, inhibition of ATP synthesis, and loss of mitochondrial membrane potential and osmotic stability (Wallace and Starkov, 2000; Krahenbuhl, 2001). In previous studies, pulmonary metabolism of DCE was investigated and the major DCE metabolite detected in lung cytosol was the highly electrophilic DCE epoxide, which was readily trapped by glutathione to form the DCE conjugates 2-(S-glutathionyl)acetyl glutathione ([B]) and 2-S-glutathionyl acetate ([C]) (Forkert, 1999a). Conjugate [C] was detected at significantly higher levels than conjugate [B] at all doses investigated. As such, in subsequent experiments, a polyclonal antibody directed against conjugate [C] was developed, and characterization studies revealed its specificity for sites that consist of the DCE epoxide bound to cysteine residues (Forkert et al., 1997; Forkert, 1999b). On the basis of these findings, it was predicted that this antibody could be used as a probe to identify sites of binding of the DCE epoxide to cysteinyl thiols of tissue proteins, which are major nucleophilic centers. Results from immunohistochemical studies showed that DCE epoxide-cysteinyl conjugates were localized predominantly in the apices of bronchiolar Clara cells (Forkert, 1999a). In the present investigation, immunogold labeling was performed to identify DCE conjugates at the ultrastructural level. Here, labeling was observed selectively in the apices of bronchiolar Clara cells primarily in association with numerous mitochondria (Fig. 1). These data are consistent with previous studies that demonstrated covalent binding of DCE-derived radiolabel predominantly in the mitochondrial fractions of the lung (Okine et al., 1985). Thus, these findings indicated that the DCE epoxide preferentially conjugates with cysteinyl thiols of mitochondrial proteins.
Mitochondria have been implicated in necrotic and apoptotic cell death through induction of the MPT (Lemasters et al., 1998; Crompton, 1999). The MPT represents an abrupt increase in permeability of the inner mitochondrial membrane that is mediated by opening of the permeability transition pore(s) (PTP). Onset of the MPT requires elevated cytosolic calcium levels in addition to an inducing agent, such as organic peroxides or substances that oxidize sulfhydryl groups (Crompton, 1999; Costantini et al., 2000). Consequences of permeabilization of the inner mitochondrial membrane include swelling and uncoupling of oxidative phosphorylation, which, when unrestrained, can lead to cell death. In the current investigation, conjugation to and/or oxidation of PTP proteins by the DCE epoxide may result in activation of the MPT. Consistent with this premise, severe mitochondrial swelling was observed in bronchiolar Clara cells 2 h after DCE administration (Fig. 2). A number of chemicals have been shown to covalently modify one or more of the proteins in the PTP complex. For instance, dithiodipyridene and bismalei-mido-hexane bind to cysteinyl thiols of the adenine nucleotide translocator, resulting in mitochondrial membrane permeabilization and cell death (Costantini et al., 2000). Thus, DCE-derived reactive metabolites may produce swelling of Clara cell mitochondria by binding to and/or oxidizing critical proteins in the PTP complex and activating the MPT.
Induction of the MPT can lead to large amplitude swelling in addition to rupture or permeabilization of the outer mitochondrial membrane, thus releasing intermembranous proteins into the cytosol (Lemasters et al., 1998; Halestrap et al., 2000). Prominent among these is the apoptogenic protein cytochrome c. Cytochrome c, in the presence of ATP or dATP, forms a complex with apoptotic protease activating factor-1 and procaspase-9. This association event results in the proteolytic cleavage of procaspase-9 to activated caspase-9. Caspase-9, in turn, can catalyze the conversion of procaspase-3 to caspase-3, a potent effector caspase responsible for many of the morphological changes associated with apoptosis (Halestrap et al., 2000; Hengartner, 2000). In the present study, cytosolic levels of cytochrome c were found to be 3-fold greater in lungs from mice treated with DCE for 4 h than in controls (Fig. 3). Furthermore, activated caspase-3 was detected in bronchiolar epithelial cells 12 and 24 h after DCE (Fig. 4). Thus, these data suggested that DCE may produce pulmonary bronchiolar apoptosis by causing mitochondrial release of cytochrome c and caspase-3 activation.
Further insight into the pathogenesis of DCE-mediated pneumotoxicity was gained from TUNEL staining and histopathological assessment. Although TUNEL staining can be used to detect both apoptosis and necrosis, apoptotic cells exhibit a distinct pattern of nuclear staining whereas necrotic cells display diffuse staining in the cytosol and nucleus (Gujral et al., 2002). After 75 mg/kg DCE treatment for 12 or 24 h, TUNEL staining was observed selectively in the bronchiolar epithelium (Fig. 5). In small diameter bronchioles, all of the TUNEL-positive cells showed diffuse cytoplasmic staining, whereas nuclear staining varied from moderate to intense, suggesting a necrotic mode of cell death. Conversely, in larger airways, scattered individual cells exhibiting intense nuclear staining were observed at 24 h after DCE. Thus, in both small and large diameter bronchioles, it is possible that TUNEL-positive epithelial cells demonstrating intense nuclear staining represent a population of damaged cells undergoing apoptosis. Consistent with these data, histopathological observations revealed apoptotic and necrotic cells concomitantly in the bronchiolar epithelium at 24 h after 75 or 125 mg/kg DCE (Fig. 6). Apoptotic cells were more abundant in large diameter bronchioles and showed characteristic morphological features of apoptosis, including chromatin condensation and margination. Since the major route of elimination of unmodified DCE in mice is via exhalation (Jones and Hathway, 1978), and because DCE is hydrophobic and readily traverses biological membranes, it is likely that epithelial cells of small diameter bronchioles are exposed to greater concentrations of DCE than are those from larger bronchioles. It has previously been hypothesized that the mode of cell death resulting from adverse pathological stimuli depends on the intensity of the insult, changing from necrotic to apoptotic as the degree decreases (Ankarcrona et al., 1995; Charriaut-Marlangue et al., 1996; Leist and Nicotera, 1997). Thus, it is possible that as the concentration of DCE to which the bronchiolar epithelium is exposed decreases (i.e., in larger airways), the predominant mode of cell death changes from necrosis to apoptosis.
Although "mild" forms of a variety of pathological insults evidently engage the apoptotic program at some point, the precise juncture is unknown. According to one theory, the extent of mitochondrial PTP opening determines whether a cell dies by necrosis or apoptosis (Crompton, 1999; Halestrap et al., 2000). Hence, unrestrained pore opening causes mitochondrial swelling and uncoupling of oxidative phosphorylation which leads to ATP depletion and ultimately necrosis. Alternatively, transient PTP opening may be involved in the induction of apoptosis by initially causing mitochondrial swelling followed by rupture or permeabilization of the outer membrane thus releasing apoptogenic proteins into the cytosol. Here, ATP levels are maintained at, or near, physiological concentrations in order to execute the apoptotic program. Thus, the extent of mitochondrial PTP opening may mediate the mode of cell death induced by various noxious stimuli.
In conclusion, this investigation provides for the first time evidence supporting apoptosis of bronchiolar epithelial cells as a mode of cell death in DCE pneumotoxicity. Our findings showed that mitochondrial perturbations, cytochrome c release, and caspase activation were important events in the apoptotic cascade. Additionally, apoptotic cell death, which occurred subsequent to the onset of necrosis, was more prominent in larger bronchioles where the intensity of the pathological insult may have been milder.
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ABBREVIATIONS: DCE, 1,1-dichloroethylene; MPT, mitochondrial permeability transition; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; BSA, bovine serum albumin; RT, room temperature; TBS, Tris-buffered saline; NGS, normal goat serum; PVDF, polyvinylidene diflouride; T/TBS, Tween 20-Tris-buffered saline; PBS, phosphate-buffered saline; TdT, terminal deoxynucleotidyl transferase; PTP, permeability transition pore(s).
Address correspondence to: Dr. Poh-Gek Forkert, Department of Anatomy and Cell Biology, Queen's University, Kingston, ON, Canada K7L 3N6. E-mail: forkert{at}post.queensu.ca
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