Repeated Inhalation Exposures to the Bioactivated Cytotoxicant Naphthalene (NA) Produce Airway-Specific Clara Cell Tolerance in Mice

Jay A. A. West*,1, Laura S. Van Winkle*, Dexter Morin{dagger}, Chad A. Fleschner*, Henry Jay Forman{ddagger} and Charles G. Plopper*

* Department of Anatomy, Physiology, and Cell Biology and {dagger} Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616; and {ddagger} Department of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham, 317 Ryals (1665 University Boulevard), Birmingham, Alabama 35294–0022

Received January 24, 2003; accepted May 19, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Repeated exposures to bioactivated cytotoxicants such as naphthalene (NA) render the target population, Clara cells, resistant to further injury through a glutathione-dependent mechanism. The current studies were designed to test the hypothesis that the mechanism for tolerance is localized in Clara cells. We used three approaches to test this hypothesis. First, using airway explants from tolerant mice maintained in culture, we sought to determine if the mechanism of Clara cell tolerance was airway-specific. Second, using inhalation as the route of exposure, we sought to determine if Clara cells at all airways levels become tolerant to repeated inhalation exposures of NA. Third, by measuring {gamma}-glutamylcysteine synthetase ({gamma}-GCS) activity and expression we determined if tolerance to inhaled NA resulted from shifts in phase-II metabolism. Our results indicate that Clara cells in explants from tolerant mice remained tolerant to NA injury in culture. When mice were exposed to repeated inhalation exposures of NA (15 ppm), we found that Clara cells at all airway levels became tolerant. Expression and activity analysis revealed that {gamma}-GCS, the rate-limiting enzyme in glutathione synthesis, is induced in tolerant Clara cells. Buthionine sulfoximine, a {gamma}-GCS inhibitor, was able to eliminate the resistance of these tolerant cells. We conclude: (1) the mechanism of NA tolerance in Clara cells is airway specific, (2) the specific mechanism allows Clara cells to become tolerant to NA vapor at levels relevant to human exposure, and (3) the mechanism of tolerance to inhaled NA is highly dependent on induction of the catalytic enzyme, {gamma}-GCS.

Key Words: naphthalene (NA); glutathione (GSH); {gamma}-glutamylcysteine synthetase ({gamma}-GCS); buthionine sulfoximine (BSO); tolerance resistance.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The bioactivated xenobiotic, naphthalene (NA), is a pervasive environmental contaminant. Humans are exposed to NA from a number of different sources, including industrial applications such as the production of phthalic anhydride, which is used in the synthesis of resins, plastics, pharmaceuticals, and insect repellents (Sciences International 1995Go). Other sources of NA include cigarette smoke (Schmeltz et al., 1976Go) and diesel fuel emissions (Clark et al., 1982Go). Nonoccupational exposures also arise from the removal of pests, as pure NA crystals (mothballs) can be purchased at most local department and hardware stores across the United States. Concern regarding frequent human exposure has arisen because NA has been found in body fat (Stanley, 1986Go) as well as in mother’s milk (Pellizzari et al., 1982Go), in conjunction with findings that NA is carcinogenic in mice (Abdo et al., 1992Go) and, most recently, in rats (NTP, 2000Go).

Previous studies have established that parenteral administration of NA causes bronchiolar epithelial necrosis in mice and hamsters and cytotoxicity in the olfactory epithelia of rats, mice, and hamsters (Plopper et al., 1992Go). Nonciliated or "Clara cells" of distal bronchiolar epithelium in mice are particularly susceptible after parenteral exposures to NA (Plopper et al., 1992Go). Cytotoxic injury is associated with the presence of high rates of cytochrome P450 metabolism in sites of injury (Buckpitt et al., 1992Go). Cytochrome P450 2F2 is particularly efficient in metabolizing NA to an epoxide (Shultz et al., 1999Go), and this isozyme appears to be responsible for the catalytic generation of the toxic NA metabolite. An orthologous CYP2F1 with approximately 80% homology has been identified in humans (Nhamburo et al., 1990Go), and it is known that human lung microsomes metabolize NA (Buckpitt and Bahnson, 1986Go). While whole-lung microsomal NA metabolism rates for humans appear lower than for mice, susceptibility of human airway epithelial cells to injury from NA inhalation exposures is unknown.

Although it is well documented that single parenteral exposures of NA cause acute Clara-cell injury, the effect of repeated exposures to NA in mice is less well understood. Previous studies indicate that mice do, in fact, become tolerant to repeated parenteral exposures of NA (Lakritz et al., 1996Go; O’Brien et al., 1989Go). The mechanism for this resistance appears to include induced activity of {gamma}-glutamylcysteine synthetase ({gamma}-GCS), the rate-limiting step of glutathione (GSH) synthesis (West et al., 2000Go). However, it is currently unknown whether Clara cells become tolerant to repeated exposures of NA by inhalation. Since the major route of exposure to humans is through inhalation of NA vapor, it is imperative that we understand the response of the epithelium after repeated exposures. Recent studies of the effects of single exposures to inhaled NA indicate that, even at low exposure, concentrations (below the current OSHA standard, <=10 ppm) produce significant injury in lung epithelia of mice (West et al., 2001Go). Due to this particularly potent toxicity, understanding the effects of repeated exposures of inhaled NA represents an important step in understanding the development of resistance to daily exposures to pollutants and their long-term effect on the health and viability of the respiratory system.

The current studies were designed to test the hypothesis that shifts in Phase-II metabolism of epithelial cells in the lung result in the development of tolerance to injury from inhalation of bioactivated cytotoxicants. In order to test this hypothesis, we asked three questions: (1) is the mechanism of tolerance specific to the target cell of acute injury, the Clara cell, (2) does this mechanism produce tolerance for repeated inhalation exposures of NA, and, (3) are increases in the expression and activity of {gamma}-GCS coordinated with shifts in susceptibility to NA in tolerant mice?


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental design.
Male Swiss Webster mice (6–7 weeks) were purchased from Charles River (Wilmington, MA.) Animals were allowed free access to food (5001 rodent diet, Labdiet, Inc.) and water and were housed in an AAALAC-accredited facility in HEPA filtered air (45–55% humidity) and cage racks with recycled newspaper bedding (Care Fresh, Inc.) at the University of California, Davis. Animals were confined for at least five days before use in an experiment. Light was cycled 12 h on, 12 h off. Four experiments were performed to determine the specific ability to maintain homeostasis during repeated exposures to NA. The goal of the first experiment was to determine if Clara cells in the lung maintain tolerance ex vivo. Mice were administered seven daily repeated intraperitoneal injections of NA (200 mg/kg). Twenty-four h after the last of the seven doses, explants from tolerant and control mice were prepared as previously described (Van Winkle et al., 1996Go). Briefly, mouse lungs were inflated with agarose and microdissected to expose the distal airways. Superficial parenchyma was removed and explants were isolated and then treated for two h in vitro with graded concentrations of NA. Final concentrations of NA were 38, 3.8, 0.38, 0.038, and 0.0038 µg/ml, given in equal volumes. Carrier-only controls were run for each mouse and the carrier was an equivalent volume of methanol. The volumes of NA in MeOH or of MeOH alone were 7 µl added to a well volume of 700 µl. Explants were isolated from NA-tolerant mice and corresponding sham-treated control mice. The explants were exposed to NA for 2 h and then transferred back to media that did not contain NA. Explants were assessed 24 h after NA exposure. Explants from six treated and six control animals were assessed (n = 6). Each animal yielded six explants, which comprised minor daughter airway (including terminal bronchioles) trees, isolated by microdissection, from the left lobe. The six explants per animal were exposed to the graded NA concentrations (one explant per concentration) and one explant from each animal served as a vehicle (MeOH) control. Seventy-two explants in total were evaluated (not including the animals used for setting up the experiment parameters). Samples from control and tolerant explants were harvested twenty-four h later, and permeable cells were detected by imaging cells labeled with ethidium homodimer-1.

The goal of the second experiment was to determine if this airway-specific tolerance would result in resistance to injury by repeated inhalation exposures of NA. Mice were exposed to NA vapor for 4 h daily, for 7 days (0 or 15 ppm). Twenty-four h after the last of seven exposures, mice were killed and processed for high-resolution histopathology to assess whether Clara cells had become tolerant.

The goal of the third experiment was to determine if shifts in the expression of {gamma}-GCS were coordinated with shifts in the susceptibility to NA. Again, mice were exposed to seven daily exposures (4 h) to NA (0 or 15 ppm). Twenty-four h after the last exposure, mice were killed; lungs were either processed for immunohistochemistry or microdissection for assessment of {gamma}-GCS activity and expression from both tolerant and control mice.

The goal of the fourth experiment was to determine if blocking the activity of {gamma}-GCS would eliminate the tolerance to repeated exposures of NA vapor. Mice were again made tolerant to NA vapor. Twenty-four h after the last of seven repeated inhalation exposures, mice were administered buthionine sulfoximine (BSO; 0 or 800 mg/kg), a specific {gamma}-GCS inhibitor. One h later, mice were subjected to an additional exposure of NA, then killed 24 h later for histopathologic assessment.

Reagents.
NA was purchased from Fischer (Fairlawn, NJ). DL-buthionine-[L,R]-sulfoximine (99.0% purity) (BSO) was purchased from Sigma Biochemical, St. Louis, Mo. All fixatives and embedding reagents were purchased from Electron Microscopy Sciences (Fort Washington, PA). All other solvents were reagent grade or better.

NA exposures.
Mice were either exposed to NA by intraperitoneal injection or by inhalation. For intraperitoneal injections, solutions of NA were prepared so that the dose was administered, ip, in 0.1 ml of corn oil/10g body weight. Control animals were administered vehicle (corn oil) only. Inhalation exposures were generated as previously described with minor alterations (Pakenham et al., 2002Go; West et al., 2001Go). Briefly, mice (five per chamber) were placed in a silanized glass chamber. NA vapor was generated by passing air through crystalline NA packed in a 2.5 x 70 cm glass column. Air volume through the chamber was 2.0 liters/min. Concentrations of 15 ppm NA were achieved by mixing NA vapor with fresh air. NA concentration within the chamber (range 13.5–17 ppm) was determined by sampling chamber air (10 ml) with a gas-tight syringe. The sampled air was then dissolved in 3.0 ml methanol and measured by absorbance at 210 nm. NA concentrations were also monitored continuously, using an online spectrophotometer equipped with a flow cell. Control animals were exposed to filtered air only.

Detection of permeable cells in explants.
Injured Clara cells were visualized using a modification of a previously described method (Postlethwait et al., 2000Go; Van Winkle et al., 1996Go, 1999Go). Briefly, explants from both tolerant and control mice were incubated with ethidium homodimer-1 (Molecular Probes, Eugene, OR) for 20 min, fixed in 1% paraformaldehyde for one h at 30 cm H20 pressure, then embedded in glycol methacrylate resin (EM sciences, Fort Washington, PA) and sectioned at 1 mm with glass knives. Images of permeable cells were captured using an Olympus epifluorescence microscope (ex:530, em:645) using a dichroic mirror with a wide band-pass filter. Sections of the same field imaged for permeable cells were then stained with toluidine blue to assess cellular histology.

High-resolution histopathology.
Animals were killed with an overdose of sodium pentobarbital 24 h after the conclusion of exposure to NA. All lungs were prepared for histopathological assessment by inflation via a tracheal cannula with 1% glutaraldehyde/1% paraformaldehyde in 0.1 M cacodylate buffer 335 mOsm for 1 h at 30 cm H20 pressure (Plopper, 1990Go). The entire fixed middle (cardiac) lobe was post-fixed with osmium tetraoxide and incubated overnight in uranyl acetate. The post-fixed tissue was embedded in Araldite-502 (Electron Microscopy Sciences), and embedded tissue was then grossly sectioned parallel to the long axis of the main stem bronchi. Sections (1.0 mm) were cut with glass knives using a Zeiss Microm HM340E microtome and stained with 1% toluidine blue (Electron Microscopy Sciences). Slides were imaged with a 330 CCD Dage camera on a Ziess Axiakop MC80 microscope using Scion 1.59 imaging software.

Immunohistochemistry.
Lung tissue from mice was fixed with 1% paraformaldehyde for two h, then placed in PBS at 4°C until processing for embedding. Tissues were embedded in paraffin and sectioned at 6-µm thickness. Antigenic proteins were identified by the avidin-biotin horseradish-peroxidase method as outlined by Plopper and Dunworth (1987)Go. Controls for nonspecific binding were performed by substituting primary antibody with phosphate buffered saline. A 19-amino acid peptide was synthesized according to the published rat {gamma}-GCS heavy subunit amino acid sequence (at position 295–313: NH2-CRWGVISASVDDRTREERG-COOH) (Shi et al., 1994Go). This peptide was conjugated to carrier keyhole limpet hemocyanin (KLH) and used as an antigen to raise rabbit antisera against rat {gamma}-GCS-HS. The polyclonal antibodies against {gamma}-GCS-HS were used in the subsequent immunohistochemical and Western blot analysis.

{gamma}-Glutamylcysteine synthetase ({gamma}-GCS) activity.
Measurement of {gamma}-GCS activity in microdissected airways was performed as previously described (West et al., 2000Go). Briefly, twenty-four h after the last of seven repeated inhalation exposures, mice (both treated and control) were killed for measurement of airway {gamma}-GCS enzyme activity. Distal and proximal airways were isolated by microdissection, after inflation via tracheal cannula with 1% agarose, with Waymouth’s media deficient in sulfur-containing amino acids. Microdissected airways were placed in an ice-cold lysis buffer and homogenized in a small volume Polytron from Brinkman Instruments Co. (Westbury, NY). After homogenization, samples were centrifuged to remove insoluble protein and glycine. Protein content was determined by the micro-Bradford method (Bradford, 1976Go) and samples were frozen at -80°C until analysis. To measure {gamma}-glutamylcysteine synthetase activity, airways homogenized as described above were added to a reaction buffer (1:1) at 37°C for 30 min, and stopped with the addition of an equal volume of ice-cold 200 mM methane sulfonic acid. Samples were analyzed immediately by HPLC using an ESA Coulchem II detector (Chelmsford, MA) with a 5010 analytical cell and a 5020 guard cell.

Western blotting.
Protein content was determined by the micro-Bradford method (Bradford, 1976Go) and samples were frozen at -80°C until analysis. Soluble protein fractions were diluted in a 10% SDS sample buffer containing ß-mercaptoethanol and separated by gel electrophoresis using a Tris–HCl-buffered 10% polyacrylamide mini-gel (Bio-Rad, Hercules, CA). Proteins were transferred onto a polyvinyldifluoroacetate membrane (NEN, inc. Boston, MA) and probed with the rabbit antibody produced against the catalytic subunit of {gamma}-glutamylcysteine synthetase (1:5000). Glutathione S-transferase {pi} (GST-{pi}) antibody (1:1000) was purchased from Biotrin International (Dublin, Ireland) and multidrug resistance-associated protein (MRP) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Bands were detected using chemiluminescence with an HRP-linked secondary goat antibody produced against rabbit or mouse IgG.

BSO treatment.
Previous studies have demonstrated that BSO can cause a rebound (Griffith and Meister, 1979Go) or increases in GSH levels due to the feedback inhibition of {gamma}-GCS. We conducted several preliminary experiments to determine the optimal timing of the BSO and NA doses and have previously reported this data (West et al., 2000Go). The following protocol was used for all mice in the results presented here. Mice receiving repeated inhalation exposures of NA were treated with BSO (0 or 800 mg/kg), a GSH resynthesis inhibitor, 24 h after the seventh repeated inhalation exposure. One h later, mice were challenged with an additional exposure period to NA (15 ppm), then killed 24 h after the NA challenge and processed for histopathological assessment.

Statistical analysis.
Statistical differences were determined by one-way analysis of variance (ANOVA) using the Bonfferoni-Dunn post-hoc t-test. Significance level (p) is indicated in individual charts.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NA Tolerance ex Vivo
Airway explants (Fig. 1AGo) isolated by microdissection from both control and tolerant mice demonstrated that the development of tolerance is specific to Clara cells in airways. Airway explants from control mice exposed to NA in culture (38 µg/ml) appeared injured (Fig. 1BGo). The epithelium in explants of NA-treated controls contained swollen Clara cells and cells that had exfoliated, leaving an underlying squamated layer of ciliated cells (Fig. 1BGo). This injury, resulting from NA exposure, was coordinated with increased permeability of bronchiolar epithelium (Fig. 1CGo), where the bronchiolar epithelium had permeable cells, as indicated by prominent labeling with ethidium homodimer-1 (Fig. 1CGo). In contrast, mice that had been made tolerant to NA by repeated injections produced explants that were resistant to further injury ex vivo. When treated with NA, Clara cells within these tolerant explants had few cells that appeared permeable to ethidium homodimer-1 (Fig. 1EGo) and had no changes associated with cytotoxicity (Fig. 1DGo).



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FIG. 1. Tolerant Clara cells in airway explants. Distal airways were isolated from agarose-inflated lungs by microdissection (A). Explants in culture were then treated with NA ex vivo. Explants from control mice treated with NA (38 µg/ml) contain injured Clara cells, as judged by histopathology (B). The same cells (C) were permeable to EthD-1 (arrows). In contrast, explants from tolerant mice were unaffected by NA treatment (38 µg/ml), as judged by histopathology (D), and appeared as a cubiodal epithelium (e), none of which was permeable to EthD-1 (E). Magnification bar in A represents 500 µm. Magnification bar in D represents 25 µm; e, epithelium.

 
Tolerance to Inhaled NA
The airway-specific adaptation of Clara cells resulted in tolerance to repeated exposures to inhaled NA. Airways of mice exposed to filtered air only contained a cubiodal epithelium that consisted of two predominant cell types, ciliated and nonciliated or "Clara" cells (Fig. 2AGo). Clara cells had a typical appearance that included the apical projection that extends into the airway lumen, and were adjacent to ciliated cells. This normal cubiodal epithelium was disrupted as single exposures to NA vapor (15 ppm) caused extensive Clara-cell injury (Fig. 2BGo). The epithelium was mainly devoid of Clara cells, which had exfoliated and left a layer of squamated ciliated cells behind (Fig. 2BGo). As previously described (West et al., 2001Go), injury from NA at this concentration level caused widespread Clara-cell necrosis, from the most proximal airways to the distal terminal bronchioles (data not shown). In contrast, repeated exposures of NA for seven days, at the same concentration level (15 ppm), resulted in epithelium that appeared refractory to further injury (Fig. 2CGo). In these tolerant mice, the epithelium appeared cubiodal, with both ciliated and Clara cells.



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FIG. 2. Tolerance to inhaled NA. Mice exposed to filtered air (A) had epithelium that appeared cuboidal and uninjured. Single exposures to 15 ppm NA (B) caused Clara cell injury and exfoliation. In contrast, repeated daily exposures to NA vapor (C) resulted in epithelium resistant to further injury. Magnification bar in C represents 20 µm. In these mice, occasional hyperplastic/dysplastic nodules (**) were found three or four cells high (D). These nodules were flanked by regions of squamated (arrow), ciliated cells (D). Magnification bar D represents 50 µm.

 
While tolerant Clara cells had an appearance similar to control, we saw variability in the epithelial repair of these mice. While in a majority of the mice the epithelium appeared to regenerate, infrequent regions appeared to have delayed or dysfunctional repair. This was previously reported for tolerance to peritoneal doses of NA (West et al., 2000Go). In airways from these experiments, occasional hyperplastic/dysplastic nodules of considerable size were seen in mice made tolerant to inhaled NA. These nodules appeared to be three or four cells high and were surrounded by squamated ciliated cells (Fig. 2DGo). These nodules did not appear to invade the basement membrane, and in contrast to the previous studies, did not appear to have any specific area of localization throughout the airway tree.

{gamma}-GCS Expression in Tolerant Mice
Expression of {gamma}-glutamylcysteine synthetase ({gamma}-GCS), and its corresponding activity was induced after repeated exposures to NA vapor. {gamma}-GCS is expressed constitutively in most cell types, including the cells of the lung. An antibody directed against the heavy subunit of the enzyme produced labeling throughout the lung, including both alveolar and epithelial regions. In mice exposed to filtered air only, this diffuse signal did not appear to differ, based on airway generation such as, proximal airways (Fig. 3AGo) or distal airways (Fig. 3BGo). After seven repeated daily exposures to NA, airway expression of {gamma}-GCS was induced. This induction was clearly evident by Western blot analysis (Fig. 3EGo). Compared to control, the 75 kDa catalytic subunit of {gamma}-GCS was elevated in both proximal and distal airways that were microdissected from tolerant mice (Fig. 3EGo). When observed in airways by immunohistochemistry, the induced protein appeared to be concentrated in the nonciliated cells within the epithelial layer of cells lining the lumen in both proximal (Fig. 3CGo) and distal airways (Fig. 3DGo). The induced expression of {gamma}-GCS localized to the epithelium was coordinated with increased specific activity of the catalytic protein (Fig. 3FGo). Analysis of catalytic activity of {gamma}-GCS in microdissected airways demonstrated that repeated NA inhalation exposures resulted in significant increases in thiol resynthesis in tolerant Clara cells. In these tolerant animals the production of {gamma}-glutamylcysteine, the product of {gamma}-GCS, was induced in both proximal (185%) and distal (225%) airways compared to control (Fig. 3FGo). This shift in activity was specific to the lung. While activity of {gamma}-GCS was roughly three-fold higher in liver of control animals, repeated exposures to NA vapor only caused significant shifts in {gamma}-GCS activity in the lung, and were not induced in the liver, kidney, or blood (Fig. 4Go).



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FIG. 3. Induction of {gamma}-GCS in mice tolerant to inhaled NA. Compared to (A) proximal and (B) terminal airways from filtered air-exposed mice {gamma}-glutamylcysteine synthetase ({gamma}-GCS) was induced in Clara cells in both (C) proximal and (D) terminal airways of tolerant mice, as assessed by immunohistochemistry or (E) Western blot. Increased expression was coordinated with (F) induced catalytic activity of {gamma}-GCS in tolerant mice in both airway levels. Magnification bar in C represents 50 µm (F: mean ± 1 SD), **p < 0.01, ***p < 0.001 compared to control.

 


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FIG. 4. Lung-specific induction of {gamma}-GCS. Homogenates from whole lung, liver, kidney, and blood were assayed for maximal {gamma}-GCS activity. While liver {gamma}-GCS had the highest maximal activity, induction of the catalytic activity was limited to the lungs of mice tolerant to inhaled NA. Values are expressed as the mean (n = 8) ± SD; p < 0.05 when compared to control.

 
Inhibition of {gamma}-GCS
To confirm that coordinate changes in expression and activity of {gamma}-GCS contribute to the development of tolerance to NA, we treated tolerant mice with BSO (0 or 800 mg/kg), a specific {gamma}-GCS inhibitor, and submitted them to an additional NA exposure period. Tolerant mice challenged with an additional exposure appeared unaffected histologically (Fig. 5AGo). In contrast, treatment of tolerant mice with BSO eliminated the acquired tolerance (5/7 mice). This combined treatment (BSO and NA) in tolerant mice produced swollen, vacuolated Clara cells that had formed apical blebs and were exfoliating into the airway lumen, leaving a squamated layer of ciliated cells behind (Fig. 5BGo).



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FIG. 5. Elimination of resistance to inhaled NA. After seven repeated daily inhalation exposures to NA (15 ppm), mice were treated with BSO (0 or 800 mg/kg), then subjected to an additional exposure period. Tolerant animals receiving vehicle only (0 mg/kg BSO) and then (A) challenged with NA vapor appeared unaffected by the additional exposure. In contrast, (B) treatment with BSO (800 mg/kg) eliminated the acquired tolerance. When challenged with NA vapor, the combined treatment resulted in significant Clara cell injury. Magnification bar in A represents 20 µm.

 
Glutathione S-Transferase-p (GST-{pi}) and Multidrug Resistance-Associated Protein (MRP) Expression
Previous studies of chemotherapeutic resistance of various types of cancers indicated that induction of {gamma}-GCS is frequently coordinated with induction of the inducible form of glutathione S-transferase, GST-{pi}, and MRPs (Ishikawa et al., 1996Go; Kuo et al., 1998Go). This would be particularly significant for NA tolerance, as GST-{pi} appears to carry out the most efficient GSH transferase metabolism of NA epoxide (unpublished data). However, in contrast to {gamma}-GCS, Western blots of protein from tolerant mouse lung indicate that neither GST-{pi} (Fig. 6Go) nor MRP (data not shown) appeared to be induced by repeated inhalation exposures of NA vapor.



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FIG. 6. Expression of GST-{pi} in tolerant mice. In cancer models frequent co-induction of glutathione S-tranferase-{pi} is coordinated with induced expression of {gamma}-GCS. Western blot analysis revealed that GST-{pi} was not co-induced with {gamma}-GCS in mice tolerant to inhaled NA.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to determine if Clara cells of airways have the cell-specific ability to upregulate phase-II metabolism in order to develop resistance to bioactivated cytotoxicants such as NA. We used three experimental approaches to test this hypothesis. First, using airway explants from both control and tolerant mice, we found that repeated exposures to NA produced Clara cells that remained resistant to NA injury in culture, or ex vivo. Second, by exposing mice to NA vapor, we demonstrated the airway specific ability of Clara cells to develop tolerance to repeated inhalation exposures. Third, by measuring changes in expression and activity of {gamma}-GCS, we showed that tolerance to NA is coordinated with cell-specific induction of {gamma}-GCS in Clara cells.

Our previous studies of NA tolerance indicated that repeated daily parenteral doses of NA produced tolerant Clara cells in the distal airways of mice through a GSH-dependent process (West et al., 2000Go). While these studies indicate that NA tolerance occurred in Clara cells of the lung, we could not rule out the involvement of liver first-pass metabolism in the development of this resistance. The present study indicates that Clara cells of mice have the cell-specific ability to develop tolerance to bioactivated cytotoxicants. Using explants from tolerant mice, our results demonstrated that nonciliated cells in airways isolated from mice, remained resistant to NA injury when maintained in culture. Combined with the fact that induced activity of {gamma}-GCS was specific to Clara cells in the lung as a result of repeated inhalation exposures of NA, these results indicate that first-pass metabolism in the liver is likely not required for the development of Clara cell tolerance.

With particular importance to human exposure to NA, we demonstrated that the development of this Clara-cell tolerance afforded resistance to injury from inhaled NA vapor. Compared to parenteral exposures, the tolerance mechanism for inhaled NA, while having a distinct pattern, appears to be the same as we have previously described for repeated intraperitoneal doses of NA (West et al., 2000Go, 2002Go). Based on the previous experiments, this was expected and supported by the modulation of the susceptibility to injury by inhibition of the activity of {gamma}-GCS with BSO. However, differences between the studies are apparent. The previous studies using parenteral exposures indicated that the induction of {gamma}-GCS was limited to the distal airways; these changes were coordinated with decreases in the susceptibility to injury in the distal airways. However, when mice receiving repeated parenteral exposures were challenged with inhaled NA, we found that only the terminal airways were protected from NA injury, with proximal airways sustaining severe injury (unpublished data). In contrast, the present study demonstrates that repeated NA exposures by the inhalation route produces widespread induction of {gamma}-GCS throughout the airway tree. This induction was coordinated with decreased susceptibility to injury throughout the same areas. In summary, the differences between the two routes of exposures appear to be that repeated parenteral exposures only generate tolerance in the distal airways, whereas repeated inhalation exposure produces tolerance in Clara cells throughout the airway tree. These alterations were coordinated with elevation in the specific catalytic activity of {gamma}-GCS.

Using our experimental design we were able to determine the overall response of the epithelial cells of the airway tree. These results were, however, not immune to variability. Our experiments with BSO indicate that the developed tolerance was in fact critically dependent on the resynthesis and maintenance of the GSH pool in Clara cells. Variability in these experiments include the differences in the sensitivity of individual mice to BSO pretreatment when challenged to NA vapor, and the formation of nodules that appeared to be a form of dysfunction in the repair process. These alterations in the repair process have been noted previously (West et al., 2000Go), and in these studies, dysfunctional Clara cells appear to be present after repeated inhalation exposures of NA. Using quantitative measurement tools such as morphometry, it is critical to determine the identity of the cell populations that undergo this dysfunctional repair process. Further studies of tolerance to inhaled NA in combination with morphometric analysis techniques may elucidate the populations that undergo this dysfunctional repair, help identify which cells have persistent repair abnormalities, and help to determine the role of the altered thiol status in the long-term cell repair dysfunction. It is well documented that repeated exposures of NA are carcinogenic in both rats and mice. It is likely that the epithelial cells in the effected regions of these animals become tolerant in these long-term exposure studies. Understanding this connection between tolerance and tumor development is critical for understanding the mechanism of tumor progression through NA.

Future studies of NA tolerance will also focus on mechanisms by which {gamma}-GCS induction occurs. Previous studies of chemotherapeutic resistance have demonstrated that the induction of {gamma}-GCS is often coordinated with induction of GST-{pi} and MRPs (Ishikawa et al., 1996Go; Kuo et al., 1998Go). In contrast to these tumor models, our studies indicate that the coinduction of {gamma}-GCS with GST-{pi} or MRP did not occur and may also indicate that the mechanism for induction of {gamma}-GCS in NA tolerance may well be different. This was surprising, as GST-{pi} is the preferred isoenzyme for GSH conjugation of NA. Defining why coinduction occurs in tumors but not nonneoplastic adaptations such as NA tolerance may be important in understanding the genetic events leading to various cancers. Because GST-{pi} is a well known marker for precancerous lesions and hepatic carcinoma (Morimura et al., 1993Go), this coinduction may be critical in understanding how adaptations such as NA tolerance evolve into preneoplastic lesions and eventually into tumors.


    ACKNOWLEDGMENTS
 
This research was funded by NIH grants ES04311, ES00628, ES06700, ES05707, ES04699, ES09681, and ES05511 (H.J.F.), and the University of California’s TRDRP 6KT0305 and 11RT-0258.


    NOTES
 
1 To whom correspondence should be addressed at: Microfluidics Department, Sandia National Laboratory, MS9951, Livermore, CA 94551-0969. E-mail: jawest{at}sandia.gov. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abdo, K. M., Eustis, S. L., McDonald, M., Jokinen, M. P., Adkins, Jr., B., and Haseman, J. K. (1992). Napthalene: A respiratory tract toxicant and carcinogen for mice. Inhal. Toxicol. 4, 393–409.

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