Abstract
One of the presumed roles of intracellular glutathione (GSH) is the protection of cells from injury by reactive intermediates produced by the metabolism of xenobiotics. To establish whether GSH depletion is a critical step in the initiation of events that lead to cytotoxicity by P450-activated cytotoxicants, naphthalene, a well-defined Clara cell cytotoxicant, was administered to mice (200 mg/kg) by intraperitoneal injection. Shortly after injection (1, 2, and 3 h), intracellular GSH content was assessed by high performance liquid chromatography or quantitative epifluorescent imaging microscopy and compared with the degree of cytotoxicity as assessed by high resolution histopathology. In highly susceptible airways (distal bronchioles), GSH decreased by 50% in 1 h. Cytoplasmic vacuolization was not visible until 2 h, when GSH had decreased by an additional 50%. By 3 h, cytoplasmic blebbing was extensive. In minimally susceptible airways (lobar and proximal bronchi), GSH depletion varied widely within the population; a small proportion of the cells lost greater than 50% of their GSH by 2 h and a significant percentage of the cells retained most of their GSH throughout the entire 3 h. Cytoplasmic vacuolization was apparent in some of the cells at 2 h but not visible in any cells at 3 h. We conclude that (1) loss of intracellular GSH is an early event that precedes initial signs of cellular damage in Clara cell cytotoxicity; (2) this pattern of loss in relation to early injury is found both in highly susceptible and minimally susceptible airway sites; (3) there is wide cell-to-cell heterogeneity in the response; (4) the heterogeneity in the response profile varies between populations in highly susceptible and minimally susceptible sites; and (5) once the intracellular GSH concentration within the entire cell population drops below a certain threshold, the initial phase of injury becomes irreversible.
The epithelium of distal conducting airways is one of the most susceptible sites for acute injury after exposure to a variety of toxicants, including oxidant air pollutants, metabolically activated xenobiotics, and particles. Nonciliated bronchiolar (Clara) cells are the principal epithelial cell phenotype present in the distal airways in many species (1) and are also the primary cellular site of xenobiotic metabolism by the cytochrome P450 monooxygenases in the lung (2, 3). The Clara cell is uniquely susceptible to injury due to its capability to transform chemicals into toxic intermediates. For most P450-activated toxicants, such as naphthalene, toxicity is highly dose-dependent and cell type- and site-selective; Clara cells in the most distal airways are selectively injured at very low doses, and as the dose increases, injury extends into more proximal airways (4, 5). Although we have recently defined the pattern of early intracellular changes that occur before frank cytotoxicity in Clara cells (6), it is not clear how changes in the status of intracellular glutathione (GSH) pools relate to early alterations in cellular organelles involved in acute cytotoxicity. One of the principal detoxification pathways for compounds metabolized by P450 monooxygenases in Clara cells is the conjugation of the reactive metabolites to GSH by glutathione-S-transferases (GSTs) (7). Whereas enzymatic GSH conjugation is recognized as a primary step in detoxification of reactive metabolites, little is known about the distribution of this antioxidant within intact cell populations in vivo or how the intracellular pool of GSH responds to rapid generation of reactive metabolites. To date, there has not been an accurate assessment of the amount of GSH that is transferase accessible within intact cell populations in vivo.
Chemicals that acquire toxicity after P450-mediated metabolism and the cellular changes that accompany toxicity have been particularly well studied in the liver. Many of the changes in cell organelles resemble those we have reported previously for Clara cells exposed to naphthalene: formation of large clear cytoplasmic vacuoles, dilation of smooth endoplasmic reticulum (SER), loss of ribosomes from rough endoplasmic reticulum, aggregation of microfilaments, and protrusion of organelle-depleted blebs from apical cellular surfaces (6, 8). Research on bleb formation in hepatocytes has suggested that blebs are caused by various mechanisms: transformation of preexisting microvilli, changes in the cortical cytoskeleton, and disturbances in both thiol and calcium homeostasis within the injured cell (9-13). It is not known whether these mechanisms are involved in Clara cell toxicity from P450-activated toxicants, although cytoskeletal changes have been implicated based on Clara cell morphology in the injury target zone of mice exposed to naphthalene (6). Most of the previous mechanistic work on P450-related cytotoxicity is based on studies in isolated hepatocytes and may not represent the changes that occur in intact pulmonary epithelial populations. This is especially true for the Clara cell, which has been difficult to maintain in culture in a highly differentiated state.
Because Clara cell toxicity is site-selective, alterations that lead to cytotoxicity should be more pronounced in airways highly susceptible to injury and less pronounced in less susceptible sites. For naphthalene-induced Clara cell injury in mice, these sites are terminal bronchioles and proximal bronchi, respectively (4). However, the site-specific nature of this distribution in relation to GSH content has not previously been addressed with respect to intact epithelial populations. Our earlier studies found that GSH content varies considerably by airway level and that this disparate distribution of GSH is directly correlated with the level of cellular susceptibility to acute injury by exposure to ozone (14). Differential toxicity is not thought to be due to variation in the enzymes responsible for conjugation (15) but may be due to capability to resynthesize depleted GSH (16). For bioactivated toxicants such as naphthalene, the rate of cellular production of reactive metabolites (5, 7), mismatches between activation and detoxification pathways (7), as well as GSH pool regulation capabilities (16) may also be key factors in determining cellular susceptibility. Previous studies using histochemical staining for GSH have found heterogeneity in Clara cell GSH content and reduction of overall cellular GSH levels in mice treated with dichloroethylene (DCE) (17).
The present study was designed to test the hypothesis that GSH depletion is a critical step in the initiation of events that lead to acute Clara cell toxicity by cytochrome P450– activated cytotoxicants and, as a corollary, that the pattern of depletion will vary between airway epithelial populations depending on their susceptibility to acute injury. Comparison of GSH content in more susceptible and less susceptible airways was performed on a site-specific basis both by high performance liquid chromatography (HPLC) analysis and by quantitative epifluorescent imaging of the GSH conjugated to monochlorobimane (MCB). The early phases of the Clara cell response were mapped by high-resolution light microscopy in target and nontarget regions over the initial 3 h after exposure to naphthalene.
Naphthalene was purchased from Aldrich Chemical (Milwaukee, WI). Corn oil (Mazola) was manufactured by Best Foods/CPC International (Englewood Cliffs, NJ). Glutaraldehyde, paraformaldehyde, lead citrate, toluidine blue, Azure II, and Araldite 502 resin were obtained from Electron Microscopy Sciences (Fort Washington, PA). The fluorochromes MCB and monobromobimane were obtained from Molecular Probes (Eugene, OR).
Adult, 8- to 10-wk-old, male viral antibody-free Swiss Webster mice (CFW; Charles River Laboratories, Wilmington, MA) were used. Mice were housed in a high efficiency particle air (HEPA)– filtered cage rack in American Association for the Accreditation of Laboratory Animal Care approved facilities on a 12/12 light/ dark cycle with food and water ad libitum for at least 5 d before use.
All animals were treated at the same time of day, between 8:00 and 10:00 a.m., with either naphthalene or a corresponding volume of carrier (corn oil). Animals were killed using an overdose of pentobarbital sodium at 1, 2, and 3 h after treatment. Each experiment contained at least one naphthalene-treated and one carrier-treated animal at each time point. The experiment was repeated three times. A minimum of three control and three treated mice were analyzed at each time point by high resolution histopathology.
The trachea was cannulated and lungs were inflated at 30 cm of pressure in situ with 330 mOsm Karnovsky's fixative (1% glutaraldehyde/0.5% paraformaldehyde in cacodylate buffer, pH 7.4) for 1 h. Lungs were stored in fixative in the dark until used. Lung slices were postfixed in 1% osmium tetroxide in Zetterquist's buffer, processed by large block methodology, and embedded in Araldite 502 epoxy resin. Specimens were sectioned at 1 μm on a Sorvall JB4 microtome with glass knives and stained with methylene blue/Azure II. High and low magnification images of fields containing terminal and distal bronchiolar epithelia, and midlevel (generations 7 to 9) and proximal bronchi (generation 1) were captured using an Olympus Provis computerized microscope in brightfield mode.
All samples were obtained from specific airway levels as marked in Figure 1. Two labeling schemes for specific airway generations were used: one to image airway sites and the other to demarcate airway segments for HPLC assay. The HPLC data were generated from airway tubular segments of airways that were microdissected free of parenchyma. These segments consisted of samples of distal bronchioles, minor daughter bronchus, and major daughter bronchus (Figure 1B). The sites for intracellular GSH imaging and quantitation included terminal bronchioles (Figure 1C), distal bronchioles, midlevel bronchus (generations 7 to 9), and proximal (generation 1) bronchus. Only intact contiguous cell populations in the relatively flat planes between branch points were evaluated.
Fig. 1. Diagrammatic illustration of the airway branching pattern in the right cardiac lobe of the mouse lung with airway generation numbers. Sampling methods for HPLC and imaging/histopathology analysis are shown. (A) The area sampled for the distal airways (generations 13 to 15 and 20 to 22) is quite small. The tip of the cardiac lobe (marked by a bracket in C) was used to image the distal bronchioles. A dime is shown as a scale reference. Bar equals 2 mm. (B) The HPLC analysis was performed on tubular airway segments that were microdissected free of parenchyma. These samples were taken from the groups of airways identified by the boxes as distal bronchioles, minor daughter bronchus, and major daughter bronchus. (C) The cardiac lobe was inflated with low melting temperature agarose, removed, and separated into two parts: proximal (approximately two-thirds) and distal (one-third). The sites for intracellular GSH imaging and quantitation included terminal bronchioles, distal bronchiole, bronchus generations 7 to 9, and bronchus generation 1.
[More] [Minimize]Microdissected bronchioles containing live cells were obtained using a modification of the methods of Van Winkle and colleagues (18). Briefly, the lungs were removed from the animal and inflated with low melting temperature agarose, which then solidified in ice-cold Ham's F12 medium for 20 min. The middle lobe was removed and separated into two parts: proximal (approximately two-thirds) and distal (one-third) (Figure 1). The proximal end was cut open by microdissection down the airway lumen. The distal end was left unopened but was exposed by removal of parenchyma.
To define the GST-conjugatable GSH pools, the proximal and distal airways were incubated with 0.5 M of the GST-dependent fluorochrome MCB (Molecular Probes) for 15 and 20 min, respectively, at 37°C. The conjugate MCB-GSH was imaged and measured in individual cells within the airways as fluorescence intensities using a Nikon upright microscope equipped with a ×40 water immersion objective and a fluorescence analytical imaging system (Deltascan 4000; Photon Technologies International, Trenton, NJ) as described previously (19).
GSH was detected in microdissected airway segments corresponding to the major daughter, minor daughter, and terminal bronchiolar airway generations (Figure 1B) using an HPLC method with electrochemical detection as described in detail in a study by Lakritz and coworkers (20). GSH content was normalized to the amount of protein in the sample as measured using the method of Lowry and associates (21) with bovine serum albumin as a standard. A minimum of six animals was sampled per data point.
The mean and standard deviation for each group of animals at a time point were calculated from values per airway level on a per animal basis. Data were analyzed by use of an analysis of variance (ANOVA) for significance at P < 0.05. Both the HPLC data and the fluorescent intensity measures were signficant by ANOVA. Significance between groups was determined using an unpaired t test for an airway level in comparison to the zero time control (P < 0.05) (22).
For imaging of GSH in individual cells and for histopathology, terminal bronchioles were defined as the most distal generation of airways contiguous with alveolar ducts and the airway generation most proximal to it (Figure 1C). For HPLC analysis of GSH content, the most distal airway generations (from intrapulmonary airway generation 14 to the terminal end, generations 20 to 22) were isolated as a single unit (Figure 1B).
HPLC. GSH concentration in the distal airways averaged 6 nmol/mg protein (Table 1) in the lungs of untreated animals either immediately after injection of carrier or 3 h after carrier treatment. One hour after naphthalene treatment, GSH concentration dropped by nearly 50% of initial levels and by 2 h after injection was less than 25% of initial levels (Table 1). Three hours after naphthalene injection, GSH concentration in the most distal airway generations was slightly, but not significantly, greater than at 2 h after injection.
| Time after Treatment | No. of Animals | Airway Generations 1–6 | Airway Generations 7–13 | Airway Generations 14–22 | ||||
|---|---|---|---|---|---|---|---|---|
| 0 h | 6 | 4.27 ± 2.65 | 4.19 ± 2.00 | 6.06 ± 3.01 | ||||
| 1 h NA | 7 | 3.18 ± 1.83 | 2.75 ± 2.82 | 3.19 ± 2.31 | ||||
| 2 h NA | 7 | 3.28 ± 2.41 | 2.48 ± 1.89 | 1.45 ± 1.25* | ||||
| 3 h NA | 7 | 1.74 ± 0.87 | 2.58 ± 1.93 | 1.86 ± 1.03* |
Imaging. Cellular GSH concentrations based on distribution of MCB-GSH conjugate distribution by epifluorescence showed a wide range of variability in cell intensity in terminal bronchioles (Figure 2). The average intensity of cellular GSH in terminal bronchioles of animals exposed to the carrier alone was approximately two-thirds that of lobar bronchi in the same lungs (Table 2). When the intensity of the MCB-GSH signal was compared based on cell distribution, the range of intensities varied by a factor of almost 5-fold in control animals (Figure 3). One hour after naphthalene injection, the intensity levels for cells within the terminal bronchioles decreased to less than 50% of the initial control level (Table 1). This was represented as a loss of fluorescence in both high and low intensity cells (Figure 3). By 2 h postinjection, the average intensity per cell had dropped to approximately 60% of the level noted at 1 h (Table 2). This was represented as a complete loss of GSH from the majority of individual cells. Virtually all of the cells containing the remaining 40% were in the lowest three intensity categories (Figure 3). By 3 h postnaphthalene injection, the intensity of individual cells had increased somewhat compared with 2 h but was below the level found 1 h after injection (Table 1). This was accounted for by an increase in the intensity of individual cells (Figure 3).
Fig. 2. Comparison of MCB- GSH conjugate intensity in four sites in the distal bronchioles illustrated in Figure 1C of corn oil (CO) carrier- and naphthalene (NA)-treated mice. Cells within the distal bronchiole of adult mouse lung were analyzed in situ from airway preparations in which the mediastinal portion of the parenchyma was dissected away. Individual cells (asterisk) within the epithelium of control mice (CO) vary in GSH content throughout the distal bronchiolar region. The four regions evaluated in this figure show a marked drop in the signal for MCB-GSH 3 h after naphthalene (NA) treatment. Color bar indicates intensity of the fluorescent signal: blue correlates to low intensity and magenta correlates to high intensity.
[More] [Minimize]| Time after Treatment | n | Lobar Bronchus (generation 1) | Midlevel Bronchus (generation 9) | Distal Bronchioles (generations 14 and 15) | Terminal Bronchioles (generations 20–22) | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| 0 h | 11, 12 | 121 ± 19 | 86 ± 29 | 91 ± 22 | 76 ± 18 | |||||
| 1 h NA | 5 | 105 ± 33 | 68 ± 32 | 61 ± 42 | 38 ± 12* | |||||
| 2 h NA | 3 | 66 ± 28* | 29 ± 8* | 33 ± 17* | 16 ± 4* | |||||
| 3 h NA | 4 | 102 ± 52 | 51 ± 19* | 41 ± 9* | 29 ± 8* |
Fig. 3. Terminal bronchioles. Comparison of MCB-GSH intensity in individual cells with histopathologic changes in terminal bronchioles over the first 3 h after naphthalene (NA) treatment. The graphs summarize the percentages of cells whose intensity fell within each range class (n = 60 to 240 cells/graph). Ci = ciliated cells; NC = nonciliated cells; B = cells with apical blebs. Bar = 20 μm. *Vacuolated cells.
[More] [Minimize]Histopathology. In terminal bronchioles, the majority of nonciliated cells had a relatively uniform distribution of dense staining inclusions and apical projections into the airway lumen (Figure 3). There was a small number of ciliated cells. At 1 h postnaphthalene injection, there was little difference in the appearance of epithelial cell cytoplasm compared with that in control animals (Figure 3). By 2 h postnaphthalene injection, the majority of nonciliated cells had lost their apical projections and had noticeable areas of vacuolization within the cell cytoplasm. In most cells, a significant portion of this vacuolization was basal to the nucleus (Figure 3). By 3 h postnaphthalene injection, the majority of nonciliated cells in the terminal bronchioles had apical blebs with uniform staining. There were areas of vacuolization, but most of the cytoplasm appeared as dense or denser than that in controls.
For imaging and histopathology, distal bronchioles were defined as the conducting airways five to seven generations (intrapulmonary airway generations 13 to 15) in the axial pathway proximal to the terminal bronchiole (Figure 1C). For analysis of GSH by HPLC, these airway generations were included with terminal bronchioles.
HPLC. The changes in GSH concentration by HPLC were as described for terminal bronchioles previously.
Imaging. The range of average fluoresence intensity of MCB-labeled individual cells in the distal bronchioles was nearly as wide as that observed in terminal bronchioles (Figure 4). The average intensity of cells in distal bronchioles of animals exposed to carrier alone was approximately 75% that of lobar bronchi in the same lungs (Table 2). By 2 h after injection, the intensity had decreased and remained low for up to 3 h after injection (Table 2). For control animals, the range of variability in fluorescence intensity of individual cells within the population was less than it was in terminal bronchioles (compare Figure 4 with Figure 3). At 1 and 2 h after naphthalene injection, there was a bimodal distribution in individual cell intensity for MCB-GSH conjugates in distal bronchioles, unlike that observed in terminal bronchioles. Approximately 50% of the cells had GSH concentrations at or below those observed in terminal bronchioles of treated animals, whereas the remainder had GSH concentrations in the range observed in control animals (Figure 4). This bimodal distribution disappeared by 3 h postnaphthalene injection when the population of cells with high levels of GSH had lost some GSH and there was an increase in GSH concentration in the individual cells at the lower end of the concentration range (Figure 4). At no time were cells observed in distal bronchioles that had no detectable GSH concentration.
Fig. 4. Distal bronchiole. Comparison of GSH-bimane intensity in individual cells with histopathologic changes in distal bronchioles over the first 3 h after naphthalene (NA) treatment. The graphs summarize the percentages of cells whose intensity fell within each range class (n = 60 to 240 cells for each graph). Ci = ciliated cells; NC = nonciliated cells. *Vacuolated cells. Bar = 20 μm.
[More] [Minimize]Histopathology. In contrast to the organization of epithelium in terminal bronchioles, distal bronchioles in control animals had fewer nonciliated cells. There was little difference in the conformation of nonciliated cells 1 h after naphthalene injection (Figure 4). At 2 and 3 h postnaphthalene injection, there was a wide range in response. Some of the nonciliated cells had cytoplasm with decreased opacity and many had zones of vacuolization. By 3 h postnaphthalene injection, a significant number of cells contained vacuolated cytoplasmic areas. There were very few instances where apical blebs were observed, although a small population of cells had zones of light staining material in the apex at 3 h (Figure 4).
For purposes of imaging and histopathology, the midlevel bronchi were defined as the airway generations contiguous with the lobar bronchus and seven to nine generations distal to that airway (Figure 1C). For GSH analysis by HPLC, the airway sample included intrapulmonary airway generation numbers 7 to 13 (Figure 1B).
HPLC. The GSH content was approximately the same as that in the lobar bronchus but less than that observed in the distal bronchioles in control animals (Table 1). By 1 h after naphthalene injection, the GSH concentration dropped to approximately two-thirds that in control animals and remained at approximately that level for the next 2 h.
Imaging. The average intensity of MCB-GSH conjugate was approximately the same as that observed in the distal bronchioles and was around 75% that of the lobar bronchus (Table 2). At 1 h postnaphthalene injection, the average fluorescence intensity decreased by approximately the same percentage as it did in the distal bronchioles. There was an additional decrease by almost 50% at 2 h postnaphthalene injection followed by an increase 3 h after injection (Table 2). In control animals, the range of GSH intensity in individual cells in these airways was similar to that observed in distal bronchioles (compare Figure 4 to Figure 5). At 1 h postnaphthalene injection, there was a slight drop in GSH intensity in approximately 30% of the cells, but the remainder of the cells appeared to be unchanged (Figure 5). Two hours after naphthalene injection, the majority of the cells had the same range of intensity as that observed in the distal bronchioles 3 h after naphthalene injection (compare Figure 5 with Figure 4). By 3 h postnaphthalene injection, approximately half of the cells had the same range of intensity as that observed in the cells in untreated animals and none of the cells exhibited intensities in the lowest two categories of the range.
Fig. 5. Midlevel bronchus. Comparison of MCB-GSH intensity in individual cells with histopathologic changes in midlevel bronchi over the first 3 h after naphthalene (NA) treatment. The graphs summarize the percentages of cells whose intensity fell within each range class (n = 60 to 240 cells for each graph). Ci = ciliated cells; NC = nonciliated cells. *Vacuolated cells. Bar = 20 μm.
[More] [Minimize]Histopathology. The airway epithelial cells in control animals in this airway generation had the same range of cellular composition and distribution as was observed in more distal intrapulmonary airways (Figure 5). One hour after naphthalene injection there was no clear difference from controls in the structure of nonciliated cells, with the exception that some of the cells lacked pronounced apical projections (Figure 5). By 2 h postnaphthalene injection, there were a number of cells with lowered intensity of staining, and many cells had a random distribution of small vacuolated areas. By 3 h postnaphthalene injection, no difference in the composition of the epithelium could be detected as compared with controls (Figure 5).
For imaging and histopathology, the lobar bronchus was defined as the airway located at the proximal end of the airway tree where the airway enters the lobe (Figure 1C). For analysis of GSH by HPLC, the most proximal four to five generations of airways were isolated for evaluation (Figure 1B).
HPLC. The GSH content in the lobar bronchi was approximately the same as that observed in midlevel bronchi (Table 1). This was decreased by about 25% 1 and 2 h after naphthalene injection, and was less than 50% of control levels by 3 h postnaphthalene injection.
Imaging. The average intensity of MCB-GSH conjugate per cell was highest in the lobar bronchus in carrier-treated control animals (Table 2). One hour after naphthalene injection the intensity had decreased by approximately 20% and at 2 and 3 h by a total of 25% as compared with controls. The fluoresence intensity of individual cells was in the highest intensity range with over 60% of the cells at the upper end and the remainder varying by approximately 2-fold (Figure 6). At 1 h after naphthalene injection, a small percentage of the cells had lost about 50% of their GSH. By 2 h postnaphthalene injection, the range of GSH content was wider than at any experimental time in any of the airways except in the terminal bronchiole at zero time. The majority of the cells at both 2 and 3 h postnaphthalene injection ranged in intensity from the highest level down to approximately one-fourth that level (Figure 6).
Fig. 6. Proximal bronchus. Comparison of MCB-GSH intensity in individual cells with histopathologic changes in proximal bronchus over the first 3 h after naphthalene (NA) treatment. The graphs summarize the percentages of cells whose intensity fell within each range class (n = 60 to 240 cells for each graph). Ci = ciliated cells; NC = nonciliated cells. *Vacuolated cells. Bar = 20 μm.
[More] [Minimize]Histopathology. Compared with other airway generations in control animals, the epithelial composition in lobar bronchi had a more even distribution of nonciliated and ciliated cells (Figure 6). Very few of the nonciliated cells had apical projections. By 1 h postnaphthalene injection, there was no discernible difference between the epithelia compared with carrier-treated controls. Two hours after naphthalene injection there was a decrease in the staining intensity of nonciliated cells and there were small areas in which a small number of cells appeared to have tiny vacuoles. By 3 h postnaphthalene injection, the majority of the cells appeared to vary little from that of carrier-treated control animals. There was a small number of cells that appeared to have some focal areas of clear cytoplasm, but no epithelial disruption or formation of apical blebs was observed.
We tested the hypothesis that GSH depletion is a critical step in the initiation of events that lead to acute Clara cell toxicity by cytochrome P450–activated cytotoxicants and, as a corollary, that the pattern of depletion will vary between airway epithelial populations, depending on their susceptibility to acute injury. We defined the profile of GSH depletion during the early phases of acute cell injury in a model of Clara cell injury in which the relationship between loss of cell permeability and Clara cell necrosis has been well defined (4, 6, 18, 23). We compared the profiles of GSH depletion with early changes in Clara cell populations in two areas where exposure to the toxicant (naphthalene) does not produce acute toxicity and in two regions where acute toxicity and subsequent Clara cell necrosis do occur (4, 24). Our site-specific approach to analysis established that intracellular GSH decreases in both highly susceptible and minimally susceptible airway populations. Progress of cellular pathology in relation to local GSH status is the same in both epithelial populations. What vary between sites are the rate and extent of cellular GSH depletion and the degree of cytotoxic change within the population. The same time course and extent of GSH depletion were detected in homogenates of whole cell populations by HPLC analysis and in individual cells within the populations by quantitative fluorescent imaging. Analysis of intracellular GSH content on a cell-by-cell basis documented wide cell-to-cell variability in the response pattern at any given airway site. Regardless of the rate of depletion from individual cells in susceptible populations, all the cells appear to progress through the same pattern of cytotoxicity. GSH depletion precedes the loss of Clara cell membrane integrity, as defined in our previous study (6), by at least 2 h. It also precedes the earliest changes in the cytotoxic response, cytoplasmic vacuolation, and focal swelling of SER, by at least 1 h. When the intracellular GSH concentration within a cell population in a local site drops below a threshold, cellular injury becomes irreversible and leads to necrosis. Intracellular GSH pool responses appear to be widely heterogeneous on a cell-by-cell basis within airway populations, but more so in cells located in nonsusceptible airway sites. This suggests that the Clara cell population within nonsusceptible sites includes two different subpopulations that respond differently to toxic stress from reactive metabolites. One of these subpopulations maintains its GSH pool and does not exhibit early cellular changes in response to cytotoxic stress. All four of the sites with known differences in potential for cytotoxic injury have a different mix of resistant and susceptible phenotypes. This is based both on the response of the GST-accessible GSH pool and on histopathologic changes after exposure.
Although it is now well recognized that GSH plays a critical role as an intracellular reducing agent in modulating cellular susceptibility to oxidant stress in the lung (25), this study is, to the best of our knowledge, the first attempt to (1) quantitatively define in situ intracellular GSH pools in intact populations of living cells and (2) compare the cellular response to GSH depletion in contiguous cell populations with variable susceptibilities to acute cytotoxicity from metabolically activated compounds. Formation of small vacuoles is one of the earliest events in the Clara cell response to P450-activated toxicants (6, 26). By the time large vacuoles in Clara cells are apparent by high resolution light microscopy, the following changes have occurred in the cellular organelles: focal swelling of SER; secretory granules separated from plasmalemma by a zone of intermediate filaments; transposition of mitochondria to the apical cytoplasm; and general loss of cytoplasmic density (6). Previously, Forkert (26) found variability of histochemically detectable GSH in the steady state in the distal bronchiole Clara cell population. This finding suggests that the damage that results in necrosis due to DCE occurs at the level of individual cells and that loss of GSH is critical for cell toxicity. Whether GSH was depleted in airways uninjured by DCE treatment was not addressed either biochemically or on a semiquantitative cell-by-cell basis, but the same general pattern of cellular changes was observed. Our findings emphasize that the loss of transferase-accessible GSH (as detected using MCB) may be just as important as reduction of total cellular GSH and other sulfhydryls within individual cells, and that at least 50% of the intracellular GSH pool must be lost before cell organelle changes are apparent. This degree of GSH loss occurs in virtually all of the cells in the most susceptible site (terminal bronchioles) and in approximately half of the cells in a less susceptible site (distal bronchioles). Essentially none of the cells at the two sites that are refractory to necrosis, the proximal (lobar) and midlevel bronchi, loses GSH to this extent. By the time a majority of the cells lose 75 to 100% of their GSH, which occurs in distal bronchioles by 2 h after treatment, the alterations in cellular organelles are sufficient to make the injury irreversible. These changes include (1) separation of the apex of the cell, together with swollen endoplasmic reticulum, into membrane-bound blebs separated by a zone of intermediate filaments and (2) swelling of mitochondria with condensation of matrix. The remainder of the cytoplasm generally also appears condensed (6). A similar pattern of injury has been observed in isolated hepatocytes treated with menadione (11); however, the relation of these changes to cytoplasmic GSH loss has not been determined. Our observations suggest that unless more than half of the Clara cells in a local epithelial population have lost 75% or more of their initial GSH concentration, the toxic cellular changes appear to be reversible. This is based on the fact that previously we have not been able to detect significant pathology in cells lining proximal airways 24 h after the administration of naphthalene at this dose (24). The rate of recovery appears to depend on the percentage of the cell population with severe GSH loss. When combined with our previous study that compares temporal changes in ultrastructure with loss of membrane integrity (6), it is clear that the loss of intracellular GSH from GST-accessible pools is a very early event, which is separated significantly in time from the irreversible alterations in intracellular organelles that lead to eventual loss of membrane integrity and cell death. This sequence of events is entirely consistent with the view that cellular GSH depletion precedes the covalent binding of electrophilic naphthalene metabolites, which occurs before the initial signs of injury. This is also consistent with the paradigm, supported by much earlier studies in whole lung (27), that there is a GSH threshold for toxicity and covalent binding of reactive metabolites. We recognize that although the techniques used in the current studies for assessing GST-transferase accessible pools of GSH are appropriate for naphthalene, they are not necessarily applicable to other metabolically activated pulmonary toxicants such as 4-ipomeanol and DCE. In contrast to naphthalene (28) where GST catalyzes the conjugation of the electrophilic epoxides, the reactive metabolites from 4-ipomeanol (29) and DCE (30) conjugate with GSH in the absence of glutathione transferases.
In hepatocytes, the sequence of cellular changes involved in cytotoxicity has been well defined, but the role of GSH is not entirely clear. The sequence of intracellular alterations due to hypoxia was found to include adenosine triphosphate depletion, bleb formation with cell swelling, mitochondrial permeability transition, disintegration of lysosomes, failure of membrane integrity, and cell death (31). How GSH pool status is related to these changes was not addressed. Studies of acetaminophen-induced toxicity in mouse hepatocytes have shown that intracellular calcium concentration increases shortly before loss of cell viability (32). This increase occurred well after bleb formation, suggesting that calcium changes occur after the irreversible events in injured hepatocytes (32). Changes in intracellular thiol status have also been implicated as an early part of the injury response in hepatocytes with menadione (11; 12), but the percentage of GSH that contributed to the measurement of total thiols was not determined. Furthermore, protein thiol oxidation was accompanied by increases in cytoskeleton-associated protein and formation of large molecular weight aggregates of a type that resembled actin. Thiol oxidation was found to be a critical step in the formation of the cell surface blebs that preceded cell death. Our study has shown that loss of GSH itself is a critical step leading to bleb formation, at least in Clara cells.
The pattern of responses we have observed in the depletion of GSH by naphthalene in Clara cells in situ in the current study, as well as the broad range of steady-state intracellular GSH levels, has also been observed by us in isolated Clara cells (19). The isolation procedure removes Clara cells from throughout the airway tree and results in a mixed population (19, 33). This mixed population has the same broad heterogeneity of GSH concentration we have reported here for cell populations maintained in the context of their airway microenvironment. Indeed, the response to exposure to naphthalene in isolated Clara cells is very similar to that of cells in situ, in which subpopulations appear to retain their GSH pools while other subpopulations lose their pools rapidly (19, 33). Furthermore, loss of membrane integrity occurs only in isolated Clara cells lacking any detectable intracellular GSH, and the loss of the GSH pool precedes the loss of membrane integrity by a substantial period of time (19).
Although most of the cellular changes in Clara cells that follow the loss of intracellular GSH appear to be associated with degradation of specific cellular organelles (such as endoplasmic reticulum), the drop in GSH also appears to trigger a possible positive response, the reorganization and distribution of organelles within the cytoplasm (6). Reorganization of intermediate filaments participates in the segregation of damaged organelles from the remainder of the cell cytoplasm. This appears to be the initial response of severely injured cells in an attempt to maintain a functioning, integrated cell after organelle injury. As part of our studies to define the mechanism of bioactivated injury in Clara cells, we have shown that one of the target macromolecules to which reactive naphthalene intermediates become bound comigrates with actin by 2D gel electrophoresis (34). Whereas reorganization of intermediate filaments appears to be an active process involved in the formation of cytoplasmic blebs, this reorganization could also be a response to the binding of functional actin filaments by reactive intermediates and the creation of new actin filaments by the cell in response to loss of function in the adducted filaments.
Because we could not establish the degree of toxic injury directly for each cell for which we have an individual GSH measurement, we used a comparative approach to determine the extent to which individual cells within the population can retain and modulate GSH pools and the impact this will have on the long-term maintenance of cell integrity after exposure to a known toxic dose. We could not determine whether the most severely depleted cells in these distal airways are the ones that eventually become permeable nor do we know whether the cells with higher GSH levels when early permeability occurs will also eventually lose their GSH. However, it does suggest that even within a single airway generation, not all cells respond the same way. This underscores the importance of evaluating an entire cell population in the context of its microenvironment.
Comparison of the pattern of GSH depletion by microenvironment demonstrates that a variety of different factors play a role in determining whether the response of individual Clara cells to the production of reactive intermediates will lead to irreversible injury. One of these is the range of intracellular GSH concentrations within the steady-state population before exposure. In Clara cells in the two airway locations where no long-term injury can be identified, the proximal (lobar) and midlevel bronchi, the average level of GSH within the cells is much higher and the range of variation within the population is much closer to the higher end of the range than is the case for the more distal and more susceptible airways, the distal and terminal bronchioles. A second factor is the rate and extent of GSH loss during the early stages of naphthalene toxicity. For the most resistant airway, the proximal bronchus, almost 50% of the Clara cells do not appear to lose any substantial portion of their GSH during the first few hours after naphthalene administration. In the distal bronchioles, airway generations in which approximately 50% of the Clara cells survive exposure, we found a subpopulation of Clara cells that maintains its GSH pools through the first 2 h of active metabolism after naphthalene treatment. A third factor that influences individual cellular susceptibility is the varying ability of Clara cell populations to replenish their GSH pools after complete depletion. As we have previously shown (16), the populations in proximal, midlevel, and distal airways vary markedly in their ability to independently maintain their intracellular GSH pool in the face of the complete absence of extracellular GSH and sulfur-containing amino acids and in the rate at which they can replenish their intracellular GSH pool after severe depletion (16). The ability to resist depletion and rapidly replenish is primarily true for the two most resistant airways, the proximal and midlevel bronchi, both of which have at least a small portion of their Clara cell population retaining GSH for substantial periods after naphthalene exposure. Our study has demonstrated that these differences in management of GSH pools are the result of differences in the biology of individual cells that populate different portions of the tracheobronchial airway tree.
This study was supported by grants 04311, 04699, and 06700 from the National Institute of Environmental Health Sciences, grant 07013 from the National Heart, Lung and Blood Institute, and an American Lung Association Research Training Fellowship (L.S.V.). It was also supported in part by grant 6KT-0306 from the California Tobacco-Related Diseases Research Program. The University of California–Davis is an NIEHS Center for Environmental Health Sciences (05707) and support for core facilities used in this work is gratefully acknowledged.
| 1. | Plopper, C. G., D. M. Hyde, and A. R. Buckpitt. 1991. Clara cells. In The Lung: Scientific Foundations. R. G. Crystal and J. B. West, editors. Raven Press, New York. 215–228. |
| 2. | Plopper C. G., Cranz D. L., Kemp L., Serabjit-Singh C. J., Philpot R. M.Immunohistochemical demonstration of cytochrome P-450 monooxygenase in Clara cells throughout the tracheobronchial airways of the rabbit. Exp. Lung Res.1319875968 |
| 3. | Devereux T. R., Domin B. A., Philpot R. M.Xenobiotic metabolism by isolated pulmonary cells. Pharmacol. Ther.411989243256 |
| 4. | Plopper C. G., Suverkropp C., Morin D., Nishio S., Buckpitt A.Relationship of cytochrome P-450 activity to Clara cell cytotoxicity: I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of naphthalene. J. Pharmacol. Exp. Ther.2611992353363 |
| 5. | Buckpitt A. R., Castagnoli N., Nelson S. D., Jones A. D., Bahnson L. S.Stereoselectivity of naphthalene epoxidation by mouse, rat, and hamster pulmonary, hepatic, and renal microsomal enzymes. Drug Metab. Dispos.151987491498 |
| 6. | Van Winkle L. S., Johnson Z. A., Nishio S. J., Brown C. D., Plopper C. G.Early events in naphthalene-induced acute Clara cell toxicity: comparison of membrane permeability and ultrastructure. Am. J. Respir. Cell Mol. Biol.2119994453 |
| 7. | Buckpitt A., Chang A., Weir A., Van Winkle L., Duan X., Philpot R., Plopper C.Relationship of cytochrome P450 activity to Clara cell cytotoxicity: IV. Metabolism of naphthalene and naphthalene oxide in microdissected airways from mice, rats and hamsters. Mol. Pharmacol.4719957481 |
| 8. | Dancygier H., Runne U., Leuschner U., Milbradt R., Classen M.Dacarbazine (DTIC)-induced human liver damage light and electron-microscopic findings. Hepatogastroenterology3019839395 |
| 9. | Jewell S., Bellomo G., Thor H., Orrenius S., Smith M.Bleb formation in hepatocytes during drug metabolism is caused by disturbances in thiol and calcium homeostasis. Science217198212571259 |
| 10. | Hinshaw D. B., Sklar L. A., Bohl B., Schraufstatter I. U., Hyslop P. A., Rossi M. W., Spragg R. G., Cochrane C. G.Cytoskeletal and morphologic impact of cellular oxidant injury. Am. J. Pathol.1231986454464 |
| 11. | Mirabelli F., Salis A., Marinoni V., Finardi G., Bellomo G., Thor H., Orrenius S.Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin. Arch. Biochem. Biophys.2641988261269 |
| 12. | Malorni W., Iosi F., Mirabelli F., Bellomo G.Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: alterations underlying surface bleb formation. Chem. Biol. Interact.801991217236 |
| 13. | Lemasters J. J., Stemkowski C. J., Ji S., Thurman R. G.Cell surface changes and enzyme release during hypoxia and reoxygenation in the isolated, perfused rat liver. J. Cell Biol.971983778786 |
| 14. | Plopper C. G., Hatch G. E., Wong V., Duan X., Weir A. J., Tarkington B. K., Devlin R. B., Becker S., Buckpitt A. R.Relationship of inhaled ozone concentration to acute tracheobronchial epithelial injury, site-specific ozone dose, and glutathione depletion in rhesus monkeys. Am. J. Respir. Cell Mol. Biol.191998387399 |
| 15. | Duan X., Buckpitt A. R., Plopper C. G.Variation in antioxidant enzyme activities in anatomic subcompartments within rat and rhesus monkey lung. Toxicol. Appl. Pharmacol.12319937382 |
| 16. | Duan X., Plopper C., Brennan P., Buckpitt A.Rates of glutathione synthesis in lung subcompartments of mice and monkeys: possible role in species and site selective injury. J. Pharmacol. Exp. Ther.227199610421049 |
| 17. | Moussa M., Forkert P. G.1,1-Dichloroethylene-induced alterations in glutathione and covalent binding in murine lung: morphological, histochemical, and biochemical studies. J. Pathol.1661992199207 |
| 18. | Van Winkle L. S., Buckpitt A. R., Nishio S. J., Isaac J. M., Plopper C. G.Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice. Am. J. Physiol. (Lung Cell. Mol. Physiol.)2691995L800L818 |
| 19. | West J., Chichester C., Buckpitt A., Tyler N., Brennan P., Helton C., Plopper C.Heterogeneity of Clara cell glutathione: a possible basis for differences in cellular responses to pulmonary cytotoxicants. Am. J. Respir. Cell Mol. Biol.2320002736 |
| 20. | Lakritz J., Plopper C. G., Buckpitt A. R.Validated high-performance liquid chromatography-electrochemical method for determination of glutathione and glutathione disulfide in small tissue samples. Anal. Biochem.24719976368 |
| 21. | Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J.Protein measurement with the folin phenol reagent. J. Biol. Chem.1931951265275 |
| 22. | Glantz, S. A. 1992. Primer of Biostatistics. McGraw-Hill, New York. |
| 23. | Van Winkle L. S., Isaac J. M., Plopper C. G.Distribution of the epidermal growth factor recptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse. Am. J. Pathol.1511997443459 |
| 24. | Plopper C. G., Macklin J., Nishio S. J., Hyde D. M., Buckpitt A. R.Relationship of cytochrome P-450 activity to Clara cell cytotoxicity: III. Morphometric comparison of changes in the epithelial populations of terminal bronchioles and lobar bronchi in mice, hamsters, and rats after parenteral administration of naphthalene. Lab. Invest.671992553565 |
| 25. | Rahman Q., Abidi P., Afaq F., Schiffmann D., Mossman B. T., Kamp D. W., Athar M.Glutathione redox system in oxidative lung injury. Crit. Rev. Toxicol.291999543568 |
| 26. | Forkert P. G.Conjugation of glutathione with the reactive metabolites of 1,1-dichloroethylene in murine lung and liver. Microsc. Res. Tech.361997234242 |
| 27. | Warren D. L., Brown D., Buckpitt A. R.Evidence for cytochrome P-450 mediated metabolism in the bronchiolar damage by naphthalene. Chem. Biol. Interact.401982287303 |
| 28. | Buckpitt A. R., Bahnson L. S., Franklin R. B.Hepatic and pulmonary microsomal metabolism of naphthalene to glutathione adducts: factors affecting the relative rates of conjugate formation. J. Pharmacol. Exp. Ther.2311984291300 |
| 29. | Buckpitt A. R., Boyd M. R.The in vitro formation of glutathione conjugates with the microsomally activated pulmonary bronchiolar alkylating agent and cytotoxin, 4-ipomeanol. J. Pharmacol. Exp. Ther.215198097103 |
| 30. | Brown A. P., Hastings K. L., Gandolfi A. J., Liebler D. C., Brendel K.Formation and identification of protein adducts to cytosolic proteins in guinea pig liver slices exposed to halothane. Toxicology731992281295 |
| 31. | Zahrebelski G., Nieminen A. L., al-Ghoul K., Qian T., Herman B., Lemasters J. J.Progression of subcellular changes during chemical hypoxia to cultured rat hepatocytes: a laser scanning confocal microscopic study. Hepatology21199513611372 |
| 32. | Harman A. W., Mahar S. O., Burcham P. C., Madsen B. W.Level of cytosolic free calcium during acetaminophen toxicity in mouse hepatocytes. Mol. Pharmacol.411992665670 |
| 33. | Chichester C. H., Buckpitt A. R., Chang A., Plopper C. G.Metabolism and cytotoxicity of naphthalene and its metabolites in isolated murine Clara cells. Mol. Pharmacol.451994664672 |
| 34. | Cruikshank M., Chang A., Morin D., Lame M., Plopper C., Buckpitt A.Covalent binding of reactive metabolites of naphthalene, 1-naphthol, nitronaphthalene and 4-ipomeanol to proteins in mouse bronchial airways. The Toxicologist151995803 |
