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TOXICOLOGY
Department of Molecular Biosciences (A.J.P.) and Anatomy, Physiology, and Cell Biology (L.S.V.W., C.G.P.), School of Veterinary Medicine, University of California, Davis, Davis, CA; and Department of Pathobiology and Diagnostic Investigation (K.J.W.), G380 Veterinary Medical Center, Michigan State University, East Lansing, Michigan
Received February 3, 2005; accepted April 18, 2005.
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Abstract |
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; Buckpitt et al., 2002). Perhaps the best studied of Clara cell toxicants is the common environmental pollutant naphthalene (NA) (Buckpitt et al., 2002). Exposure to NA selectively results in necrotic lesions in Clara cells regardless of route of exposure (injection, inhalation, and ingestion) (Warren et al., 1982; West et al., 2001). This may be because CYP2F2 is the primary cytochrome P450 isozyme responsible for metabolizing NA, and it is highly expressed in Clara cells (Buckpitt et al., 2002). Naphthalene is converted by P450s into highly reactive electrophilic intermediates (e.g., epoxides and quinones) that are primarily detoxified by conjugation to glutathione (GSH). If not detoxified, significant amounts of these reactive metabolites of NA can become attached covalently to proteins (i.e., protein adduct formation) (Warren et al., 1982). Exposure to NA results in a rapid decrease of GSH levels within Clara cells, and a correlation exists between GSH depletion, protein adduct formation, and cytotoxicity (Warren et al., 1982; Plopper et al., 2001; Phimister et al., 2004).
Naphthalene cytotoxicity has been characterized at the ultrastructural level in Clara cells and progresses as follows: endoplasmic reticulum dilation, cytoskeletal disruption, and membrane blebbing, followed by mitochondrial degranulation, plasma membrane leakage, and nuclear condensation (Van Winkle et al., 1999). Studies in hepatocytes have shown a similar progression of cellular degeneration in response to reactive toxicants (Zahrebelski et al., 1995; Manygoats et al., 2002). GSH loss has been implicated in many of these cellular responses to toxicants (Jewell et al., 1982; Reed and Fariss, 1984; Mirabelli et al., 1988). GSH is involved in vital cellular functions such as antioxidant defense, protein folding, protein synthesis, and intracellular signaling (Uhlig and Wendel, 1992). Severe GSH loss has the potential to disrupt various cellular processes and may be a critical factor in the cytotoxicity of a number of reactive toxicants. We hypothesized that at least a portion of the degenerative cellular changes that characterize NA cytotoxicity in Clara cells (e.g., swelling) occur in response to a radical drop in cellular GSH levels.
In this study, we wanted to determine specifically what effect GSH loss has on Clara cells in the absence of reactive metabolites. To accomplish this goal, we compared Clara cells treated with NA to those given only the GSH-depleting agent diethylmaleate (DEM). Diethylmaleate is a weak electrophile and requires active glutathione S-transferase enzymes to deplete GSH (Boyland and Chasseaud, 1967; Plummer et al., 1981). Naphthalene metabolites are conjugated to GSH via glutathione S-transferase enzymes; however, NA metabolites are reactive enough to spontaneously conjugate with GSH and protein thiol groups in solution (Buckpitt et al., 2002). Therefore, we used DEM in an attempt to study the effects due exclusively to GSH loss in Clara cells, likely eliminating a confounding factor of NA toxicity, namely, protein adduct formation (Fig. 1). It should be noted that we cannot rule out the potential for DEM treatment to have unanticipated side effects on Clara cells through mechanisms unrelated to GSH depletion (i.e., alkylation of electrophiles), particularly given the high dose of DEM used (1000 mg/kg). However, DEM had been used in dozens of studies on GSH depletion and seemed to be a good choice for achieving our goals of rapid and selective GSH depletion. After treatment of mice with NA or DEM, their airways were examined by high-resolution light, electron, and laser-scanning confocal microscopy to assess changes in Clara cell ultrastructure and membrane permeability, in the context of the previously characterized cellular degeneration caused by NA treatment (Van Winkle et al., 1999). In addition, we correlated these cellular changes with measurements of GSH levels within distal intrapulmonary airways, which contain predominantly Clara cells (Plopper et al., 1997). This approach allowed us to address the following questions: 1) which features of NA cytotoxicity may be due exclusively to GSH loss, and 2) what effect does abrupt GSH loss have on Clara cells?
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Materials and Methods |
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) Mazola corn oil was manufactured by Best Foods/CPC International (Englewood Cliffs, NJ). Aldehyde fixatives were obtained from Electron Microscopy Sciences (Fort Washington, PA). Araldite 502 epoxy resin, osmium tetroxide, lead citrate, and uranyl acetate were obtained from Ted Pella (Redding, CA). Ethidium homodimer-1 and Yo-Pro-1 were purchased from Molecular Probes (Eugene, OR). Waymouth's MB 752/1 media, lacking cysteine, cystine, methionine, glutamine, and glutathione, was obtained from Invitrogen (Carlsbad, CA). All other chemicals were reagent grade or better. Animals and Treatment. Male Swiss-Webster CFW mice were purchased from Charles River Breeding Laboratories (Portage, MI). Animals were allowed free access to food and water and were not fasted before treatment. They were housed in HEPA-filtered cage racks on sterile paper fiber bedding for at least 5 days before use in Association for Assessment and Accreditation of Laboratory Animal Care-approved facilities. For all experiments, animals were given a single intraperitoneal dose of 1000 mg/kg DEM (1 M solution in corn oil), 200 mg/kg NA (234 µM solution in corn oil), or a corresponding volume of corn oil (vehicle control). Solutions were prepared daily. Animals were euthanized with an overdose of pentobarbital sodium.
HPLC Analysis of GSH. Intrapulmonary airways were isolated by microdissection procedures described previously (Plopper et al., 1991) with the modification that the lungs were perfused free of blood with ice-cold 1 mM EDTA/saline. Airway segments were isolated from lungs of mice at 0, 1, 3, 6, and 24 h after DEM or NA treatment. At least five animals were evaluated at each time point for each treatment. A previously described method (Lakritz et al., 1997) was used for the quantification of GSH. Briefly, acidic tissue homogenates were subjected to reverse-phase HPLC coupled with electrochemical detection to directly measure reduced GSH. The response was linear from 0.5 to 1000 ng of GSH (R2 = 0.995), and data are reported as nanomoles of GSH per milligram of homogenate protein, as determined by the Lowry protein assay (Lowry et al., 1951).
High-Resolution Histopathology and Electron Microscopy. Lungs were collected from mice treated with DEM, NA, or corn oil at 1, 3, 6, and 24 h after treatment. Three mice were compared for each treatment at each time point. After exposure, their lungs were inflated in situ via a tracheal cannula with dilute Karnovsky's fixative (1% glutaraldehdye/0.5% paraformaldehyde in cacodylate buffer, 330 mOsM, pH 7.4) at 30 cm water pressure for 1 h, followed by overnight storage in the same fixative. After fixation, the left lobes were sliced perpendicular to the long axis of the lobe into 2-mm-thick sections, postfixed with 1% osmium tetroxide, and embedded in Araldite 502 epoxy resin as described previously (Van Winkle et al., 1995). One-micron-thick sections were stained with 1% toluidine blue. Fields were recorded at 380x magnification on an Olympus BH2 microscope (Olympus International, Melville, NY) with a CoolSNAP cooled charge-coupled device camera (Roper Scientific, Tucson, AZ). Areas of interest were excised from the same blocks of embedded tissue and remounted for ultrathin sectioning. Ultrathin sections (70 nm) were cut with a Sorvall MT 5000 ultramicrotome (DuPont, Newtown, CT) using diamond knives. Sections were stained with uranyl acetate and lead citrate and examined using a Zeiss EM-10 electron microscope (Carl Zeiss, Thornwood, NY) at 80 kV.
Pathology Evaluation. To better assess differences in the pathological lesions developing in Clara cells treated with NA or DEM, two types of measurements were used to help distinguish the extent of these changes. As a measurement of cell swelling in response to toxicant treatment, cell volume relative to basal lamina surface area Vs (cubic micrometers per square micrometers) was determined for DEM-treated animals as described previously (Van Winkle et al., 1995; see Results). Terminal bronchiolar airways were imaged for each animal (256x magnification) in the same sections used for high-resolution histopathology. Point/intercept counting of vertical sections was performed with a cycloid grid overlay and Stereology Toolbox software (Morphometrix, Davis, CA).
The second type of measurement was to develop a semiquantitative grading system (criteria listed below). At least five terminal bronchioles were evaluated per mouse, three mice per treatment group, using the above-mentioned criteria. Each mouse was given a score (0–4), and the scores for the mice in each group were averaged to provide a score indicative of the pathology observed in that group: 0, similar to untreated control (no vacuoles/even thickness/cuboidal); 1, minimal injury—swollen; some blebs; no vacuoles; 2, moderate injury—many blebs; few vacuoled cells; no cells lost; 3, moderate/severe injury—many vacuoled cells; some cells lost; and 4. severe injury—most or all cells vacuolated or lost. Means ± S.E.M. are provided for each group in Table 1.
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Membrane Permeability Assay. Changes in membrane permeability were examined 6 h after treatment with DEM, NA, or corn oil by a previously described method (Van Winkle et al., 1999). Briefly, the lungs of these mice were inflated via a tracheal cannula with 5 µM ethidium homodimer-1 (EtD-1) in Ham's F-12 media at 37°C. Ethidum homodimer-1 is excluded from cells with intact plasma membranes due to the multiple charges it carries and exhibits a 30-fold increase in fluorescent intensity upon binding to DNA. After a 10-min incubation with EtD-1, lungs were washed with fresh F-12 media before being fixed with Karnovsky's fixative. The right middle lobe of each animal was microdissected to expose the airway lumen before labeling all nuclei in the tissue with the fluorescent nuclear dye Yo-Pro-1 (4 µM in phosphate-buffered saline; pH 7.4). The distribution of permeable airway cells (EtD-1 positive; red fluorescence) within the context of all airway cells (green Yo-Pro-1 fluorescence) was mapped by using a Radiance 2100 laser scanning confocal microscope (Bio-Rad, Hercules, CA), a 10x long working distance water-immersion objective, and the appropriate filter sets (excitation/emission, 488/515 and 543/600). Digital image processing reassembled the individual focal planes to produce a three-dimensional image of the airway detailing the distribution of permeable cells.
Actin Cytoskeleton Imaging. Lungs from mice treated with DEM, NA, or corn oil for 3 or 6 h were fixed by inflation with 1% paraformaldehyde via a tracheal cannula for at least 1 h. After fixation, the right middle lobe was microdissected to expose the airway lumen of terminal bronchioles. The direct-binding F-actin probe, Alexa 568 phalloidin, was used as directed by the manufacturer (Molecular Probes). The whole mount preparations were mapped using a Radiance 2100 laser scanning confocal microscope (Bio-Rad). Optical sections were taken with the gain, black level, and iris settings optimized on the vehicle-treated controls and then kept unchanged between each treatment. Digital image processing reassembled the individual focal planes into a three-dimensional image of the filamentous actin cytoskeleton at the apical surface of the airway epithelium.
Measurement of ATP Levels. Explants of distal intrapulmonary airways were prepared by microdissection from untreated mice for in vitro culture as described previously (Plopper et al., 1991). Immediately after isolation, isolated airway segments were incubated in 0.5 ml of Waymouth's media in glass vials with Teflon-lined caps at 37°C in a shaking water bath. Stock solutions of DEM, NA, and two NA metabolites were prepared in methanol immediately before addition to the airway explants. Methanol content did not exceed 0.5% of incubation volume. Final concentrations of chemicals were as follows: 0.35 mM DEM, 0.5 mM NA, 0.2 mM NA-oxide, 0.2 mM 2-methyl-1,4-naphthoquinone (Aldrich Chemical Co.). An equivalent volume of methanol was used as a vehicle control. Concentrations of chemicals were selected based on prior studies of the toxicity of NA, NA-oxide, and 1,4-naphthoquinone (Chichester et al., 1994), and GSH depletion in isolated airways (Duan et al., 1996). Airways were incubated with the various compounds for 30 min before being homogenized in 10% perchloric acid. Proteins were separated from supernatant by centrifugation and dissolved in 1 N NaOH to assess protein content by the method of Lowry (Lowry et al., 1951) with bovine serum albumin as a standard. Supernatants were neutralized with 5 M K2CO3 and assayed for ATP content with a luciferase-based method with an ATP determination kit (Molecular Probes) according to manufacturer's instructions. At least five airways were evaluated for each chemical treatment. Data are expressed as picomoles of ATP per milligram of airway homogenate protein.
Statistics. All data are reported as the mean ± S.E.M. Significant differences between treatment groups were determined by one-way analysis of variance and Dunnett's post hoc testing method at the P < 0.05 level.
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Results |
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Membrane Permeability and Actin Cytoskeleton Disruption. Laser scanning confocal microscopy was used to generate three-dimensional composite images of permeable cells within the context of the whole airway, from 1 to 24 h after treatment with DEM, NA or corn oil. The five most distal airway generations of the main axial path of the right middle lobe were imaged (Fig. 3, A–C). Permeable cells (i.e., necrotic cells) are indicated by red ethidium homodimer-1 labeling presented on a green background of all nuclei within the tissue (Yo-Pro-1 labeling). Previously, we have shown that permeable cells first become apparent 2 h after administration of NA (200 mg/kg, i.p.), with maximal numbers of permeable cells being present 6 h after treatment (Van Winkle et al., 1999). Our current results confirmed this finding, with large numbers of permeable cells being present in terminal airways 6 h after NA treatment (Fig. 3B, arrows; see inset for close-up of permeable nuclei). In contrast, no increase in membrane permeability was noted in the airways of mice treated with DEM at any time point up to 24 h after treatment (Fig. 3C, 6-h post-DEM treatment shown).
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High-Resolution Light and Transmission Electron Microscopy. The morphological appearance of the epithelium in the terminal bronchioles of treated and control mice was compared by light (Fig. 4) and transmission electron microscopy (Figs. 5 and 6). Ciliated cells were identified by the presence of cilia, whereas Clara cells (nonciliated) were identified by their characteristic apical projections (Figs. 4A and 5A). The apical protrusions of Clara cells in control mice (Fig. 5A) contained abundant smooth endoplasmic reticulum (SER), dense secretory granules, and mitochondria in both circular and longitudinal profile (Fig. 5B, high-magnification image of cell). In control animals, the epithelium was of regular thickness and similar appearance at all time points (3-h corn oil mouse shown in Figs. 4A and 5A). The general response of Clara cells 1 h after NA treatment was to swell, with the formation of multiple, small cytoplasmic vacuoles (Fig. 5C). The vacuoles originated within the SER and seemed to dilate and coalesce into the larger vacuoles and lamellar structures found within the membrane blebs of injured cells 3 h after NA treatment (Fig. 5D, asterisk). Membrane blebs were separated from cells by thick bands of intermediate filaments (Fig. 5D, area between arrows). Three hours after exposure to NA (Figs. 4B and 5D), the majority of Clara cells in terminal bronchioles were vacuolated and contained membrane blebs (asterisks). These early signs of cellular injury had progressed to frank cell necrosis by 6 h (Fig. 4C). Twenty-four hours after NA treatment, the epithelium consisted primarily of ciliated cells that had squamated (Figs. 4D, arrows, and 5E) to cover the areas of basement membrane vacated by necrotic Clara cells exfoliating into the airway lumen (Figs. 4D and 5E).
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). (Note that values for NA-induced cell volumes were not included here because the severe blebbing and vacuolization produced by NA, affect cell volume measurements and were not as apparent in DEM-treated animals.) Whereas naphthalene treatment caused Clara cells to swell within 1 h of treatment (Fig. 5C), noticeable swelling was not apparent in Clara cells until 3 h after DEM was given (Figs. 4E and 5F). Increases in cell volume seemed to be due mainly to dilated SER and small cytoplasmic vacuoles forming within the Clara cells (Fig. 5F). A portion of the Clara cell volume increase in DEM-treated animals was due to large membrane blebs, which extended from the apical cellular surface in approximately 10% of the terminal bronchiolar Clara cells (Fig. 5G, asterisks). Overall, the amount of SER present seemed to be greatly increased and was the major constituent of these membrane blebs. As with Clara cells exposed to NA, the membrane blebs in DEM-treated animals were clearly separated from the main cell body by bands of filaments (Fig. 5G, area between arrows). Even in cells lacking membrane blebs, bands of filaments could be seen dividing the cytoplasm, such that the SER was segregated into the apical portions of cells, with mitochondria being packed tightly around the nucleus (not shown). A number of the membrane blebs contained multilamellar bodies (Fig. 5G, arrowheads). The origin of the multilamellar bodies is not known, but it seems that they are derived from SER. The individual sheets of the multilamellar bodies resemble individual tubules packed in concentric rings or parallel stacks that are physically associated with SER, and the average width of the individual layers (170 nm) was found to be close to that of SER tubules (250 nm). Gross changes in the appearance of ciliated cells were not observed at any time point in DEM-treated animals; however, small membrane blebs were noted in a few of the ciliated cells at 24 h (Figs. 4G and 5H, asterisks). Interstitial edema was evident in the peribronchiolar region of both NA- and DEM-treated mice (Fig. 4, D and H, arrowheads) along with a mild infiltrate of inflammatory cells (not shown). Table 1 provides semiquantitative data summarizing the pathological changes noted in the different treatment groups, derived using a scoring system described under Materials and Methods (0 represents no injury and ranges to 4 as maximal injured).
Clara cells from NA- and DEM-treated animals were evaluated at high magnification (12,500–40,000x) to examine the effects of the treatments on individual organelles. In Clara cells of control animals, mitochondria in circular profile were surrounded by a double membrane and were filled with electron dense material (Fig. 6A). Clara cells in NA-treated animals developed signs of mitochondrial injury, such as swelling and degranulation, first apparent after 3 h post-NA injection (Fig. 6B) and progressed to full disintegration of affected mitochondria by 6 h (Fig. 6C). In contrast, DEM treatment did not cause mitochondrial swelling or affect their density (Fig. 6D). However, 24 h after DEM treatment, a number of mitochondria with branched or dumbbell morphologies were seen (Fig. 6, E and F), resembling mitochondria undergoing fission or division (Bereiter-Hahn and Voth, 1994). Clara cells contain prominent Golgi bodies in the basolateral portions of the cell (Fig. 6G). The cisternae of the Golgi bodies were greatly distended 3 to 6 h after DEM treatment, with pericanalicular vesicles accumulating on the trans-side of the bodies (Fig. 6H, arrowhead). The distention seemed to resolve 24 h after DEM treatment as the cisternae regained their normal flattened appearance (Fig. 6I). Golgi bodies could not be easily identified within Clara cells of NA-treated animals.
ATP Levels in Terminal Bronchioles Exposed in Vitro. The cytotoxicity of DEM, NA, and metabolites of NA to airway epithelium was evaluated by measuring ATP levels in terminal bronchiolar explants isolated by microdissection from untreated mice, as described under Materials and Methods. A drastic drop in ATP levels was taken as general an indication of cytotoxicity. In addition to DEM and NA incubations, bronchioles were incubated with naphthalene-oxide (NA-oxide), the first chemical species formed during the metabolic activation of NA by P450s (CYP2F2) or with 2-methyl-1,4-naphthoquinone (MQ), representative of the naphthoquinones that can be formed during NA metabolism (Buckpitt et al., 2002). ATP levels were similar in DEM-treated bronchioles compared with controls, whereas NA treatment caused a slight, but not statistically significant, decrease in ATP levels (Fig. 7). Exposure to either of the NA metabolites, NA-oxide, or MQ, caused rapid and drastic decreases in bronchiolar ATP levels (P < 0.05).
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Discussion |
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, 2002). Reactive metabolites can deplete GSH through enzymatic conjugation, direct chemical conjugation, and redox cycling. They can also form covalent adducts with proteins (Fig. 1), potentially affecting the cell through several pathways (Buckpitt et al., 2002). A correlation seems to exist between GSH loss, protein adduct formation, and NA cytotoxicity (Warren et al., 1982; Plopper et al., 2001; Phimister et al., 2004), but the relative importance of GSH loss has not previously been investigated as a singular event in Clara cell toxicity. Similar correlations between GSH loss and covalent binding to cellular macromolecules have been reported for other bioactivated toxicants (example acetaminophen), and it has led to the hypothesis that reactive metabolites are responsible for cell death by mechanisms that involve covalent interaction with critical cellular macromolecules, with attendant disruption of normal biochemical functions. However, given that GSH plays so many critical roles within the cell (e.g., protein folding, cellular signaling, and regulating redox status) (Uhlig and Wendel, 1992; Sies, 1999) and that GSH depletion in the absence of exogenous stressful stimuli can cause cellular injury (Martensson et al., 1991; Chevez-Barrios et al., 2000), we asked whether GSH loss alone was responsible for the cellular degeneration characteristic of NA treatment. We have focused on separating the events associated with GSH loss from those involving protein adduct formation in naphthalene toxicity through the use of the GSH-depleting agent DEM (Ecobichon, 1984; Reed and Fariss, 1984; Gerard-Monnier et al., 1992) to achieve GSH loss in the absence of protein adduct formation. A mildly reactive 
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-unsaturated carbonyl compound, DEM is conjugated to GSH by glutathione S-transferases to form hydrophilic GSH conjugates. Diethylmaleate was chosen for this study because it has been used extensively and has been shown to reduce pulmonary GSH levels very rapidly (Ecobichon, 1984; Reed and Fariss, 1984; Deneke et al., 1985). The commonly used GSH synthesis inhibitor buthionine sulfoximine will lower pulmonary GSH levels, but it has to be administered repeatedly for several days to achieve significant GSH loss in the lung (Martensson et al., 1989). Diethylmaleate treatment effectively modeled NA exposure in terms of GSH loss in distal intrapulmonary airways (Fig. 2). Having a model system in which comparable GSH depletion could be achieved, presumably in the absence chemically reactive species, allowed us to determine which of the degenerative cellular changes previously ascribed to NA cytotoxicity (endoplasmic reticulum dilation, cytoskeletal disruption, and membrane blebbing, mitochondrial degranulation, and plasma membrane leakage) (Van Winkle et al., 1999) were due predominantly to GSH loss.
In this study, we demonstrated that cell swelling and membrane bleb formation occur after treatment with either NA or DEM, agreeing with results published previously on the effects of NA treatment on Clara cell ultrastructure (Van Winkle et al., 1999). Both DEM and NA treatment elicit cell swelling and membrane bleb formation, demonstrating a role for GSH loss in the development of these cellular changes. Studies with cells in culture have associated GSH loss with disruptions of the cytoskeleton and membrane bleb formation (Jewell et al., 1982; Reed and Fariss, 1984; Mirabelli et al., 1988). It is believed that blebs are formed primarily because of cytoskeletal disruptions near the surface of the cell, allowing portions of cytoplasm to become distended. Diethylmaleate has been shown to disrupt actin and tubulin filaments in hepatocytes (Dumont et al., 1991; Nagelkerke et al., 1991), and our results demonstrated that both DEM and NA can alter the actin cytoskeleton (Fig. 3, E and F). However, in spite of the similarities between DEM and NA treatment in regard to their effects on cell swelling, blebbing, and actin filament disruption, the outcomes of these two treatments were strikingly different. Whereas DEM-treated Clara cells were able to recover from the stresses induced by GSH loss, NA-treated cells continued to degenerate past the point of recovery and became necrotic.
One noticeable difference between the two treatments was the mitochondrial swelling and degranulation that resulted from NA exposure, which was absent in DEM-treated animals. Mitochondrial damage may be a crucial step in NA-induced cell injury and has been reported previously for NA-treated animals (Van Winkle et al., 1995). In studies conducted on isolated hepatocytes, it was shown that cyanide-induced hypoxia causes disintegration of the cell only once the mitochondria become disrupted and the mitochondrial membrane potential (
) drops significantly (Zahrebelski et al., 1995). It is possible a similar sequence of events occurs in Clara cells treated with NA, but attempts to measure or mitochondrial GSH levels have proven elusive, particularly because the required techniques are not well adapted to studying intact tissues and isolated Clara cells cannot satisfactorily be maintained in vitro. Using isolated airway explants, we observed a drastic reduction in ATP levels during brief in vitro exposures to NA metabolites (>70% loss in 30 min), not observed in DEM-treated airways. NA itself did not significantly lower ATP levels in explants during but may have required a longer incubation period to generate reactive species via P450 metabolism of NA (Warren et al., 1982). This does not explicitly demonstrate that mitochondrial disruption is key to NA cytotoxicity nor does it implicate GSH loss in diminished ATP production. However, the in vitro assessment of ATP loss allowed us to provide a biochemical measure of function, which correlates with the pathological changes we observed in the mitochondria of Clara cells in vivo. The doses of NA, NA metabolites, and DEM used in the in vitro incubations in this study, are known to significantly deplete GSH within airway explants in vitro (Chichester et al., 1994; Duan et al., 1996). Naphthoquinones are known to deplete GSH and ATP and open the mitochondrial permeability transition pore in hepatocyte mitochondria (Gant et al., 1988; Henry and Wallace, 1996), whereas DEM does not deplete mitochondrial GSH pools in hepatocytes (Meredith and Reed, 1982). Therefore, the mitochondrial lesions caused by NA in vivo and ATP loss caused by NA metabolites in vitro, may be related to differing toxicities observed for NA and DEM in vivo, and may be related to the abilities of these compounds to affect mitochondrial GSH levels. Before this issue could be properly addressed, however, methods need to be developed to study mitochondrial function and/or measure mitochondrial GSH levels within pulmonary tissue, as Clara cells cannot be maintained in culture, where most current methods are typically applied.
This study determined that a dramatic loss of GSH is by itself not a toxic event for Clara cells. We evaluated patterns of cytotoxicity and GSH loss, created by two distinctly different compounds (NA and DEM). Our results show that severe GSH loss in Clara cells causes substantial swelling, actin cytoskeleton disruptions, and plasma membrane blebbing. These types of cellular changes have been reported in cells exposed to various toxicants and may represent a generic response of the cell to GSH loss and/or oxidative stress seen in the earliest stages of cell death (Jewell et al., 1982; Reed and Fariss, 1984; Mirabelli et al., 1988; Zahrebelski et al., 1995; Manygoats et al., 2002). Necrotic cell death was only observed in Clara cells exposed to NA, where GSH loss and protein adduct formation are known to occur (Buckpitt et al., 2002). Our results clearly distinguish the different outcomes of these two treatments, but they could not isolate the specific cause of cell death in NA-treated animals. Several possibilities exist to explain why DEM treated Clara cells do not become necrotic: 1) GSH protects cells from insult, so GSH depletion in the absence of a further insult (e.g., no protein adducts) is not lethal; 2) there are differences in the ability of the two treatments to deplete the mitochondrial GSH pool, assuming this pool is critical to mitochondrial function; and/or 3) covalent naphthalene protein adducts disrupt critical cellular functions that, in combination with glutathione depletion, lead to necrosis. Recent studies showing that several of the proteins adducted by reactive naphthalene metabolites are involved in protein folding, including calreticulin (Wheelock et al., 2005), protein disulfide isomerase and several heat-shock proteins (Isbell et al., 2005; Lin et al., 2005), raised the possibility that glutathione depletion, and subsequent loss of a primary protective mechanism for the control of reactive oxygen species with decrements in the ability to repair damaged proteins leads to necrosis. Studies are currently underway to determine whether this hypothesis is tenable. Determining which of these possibilities is most relevant now that the response to GSH loss is defined is the next step in determining the exact mechanism of toxicity for bioactivated toxicants such as NA.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: NA, naphthalene; GSH, reduced glutathione; DEM, diethylmaleate; HPLC, high-performance liquid chromatography; EtD-1, ethidium homodimer-1; SER, smooth endoplasmic reticulum; MQ, 2-methyl-1,4-naphthoquinone.
Address correspondence to: Dr. Andrew J. Phimister, Department of Molecular Biosciences, University of California, Davis, CA 95616. E-mail: aphimister{at}ucdavis.edu
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5 airways. *, significantly decreased relative to control, P < 0.05.