* Department of Anatomy, Physiology, and Cell Biology and
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616; and
Department of Environmental Health Sciences, School of Public Health, University of Alabama at Birmingham, 317 Ryals (1665 University Boulevard), Birmingham, Alabama 352940022
Received January 24, 2003; accepted May 19, 2003
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ABSTRACT |
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Key Words: naphthalene (NA); glutathione (GSH); -glutamylcysteine synthetase (
-GCS); buthionine sulfoximine (BSO); tolerance resistance.
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INTRODUCTION |
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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., 1992). Nonciliated or "Clara cells" of distal bronchiolar epithelium in mice are particularly susceptible after parenteral exposures to NA (Plopper et al., 1992
). Cytotoxic injury is associated with the presence of high rates of cytochrome P450 metabolism in sites of injury (Buckpitt et al., 1992
). Cytochrome P450 2F2 is particularly efficient in metabolizing NA to an epoxide (Shultz et al., 1999
), 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., 1990
), and it is known that human lung microsomes metabolize NA (Buckpitt and Bahnson, 1986
). 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., 1996; OBrien et al., 1989
). The mechanism for this resistance appears to include induced activity of
-glutamylcysteine synthetase (
-GCS), the rate-limiting step of glutathione (GSH) synthesis (West et al., 2000
). 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., 2001
). 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 -GCS coordinated with shifts in susceptibility to NA in tolerant mice?
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MATERIALS AND METHODS |
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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 -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
-GCS activity and expression from both tolerant and control mice.
The goal of the fourth experiment was to determine if blocking the activity of -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
-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., 2002; West et al., 2001
). 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 13.517 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., 2000; Van Winkle et al., 1996
, 1999
). 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, 1990). 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). 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
-GCS heavy subunit amino acid sequence (at position 295313: NH2-CRWGVISASVDDRTREERG-COOH) (Shi et al., 1994
). This peptide was conjugated to carrier keyhole limpet hemocyanin (KLH) and used as an antigen to raise rabbit antisera against rat
-GCS-HS. The polyclonal antibodies against
-GCS-HS were used in the subsequent immunohistochemical and Western blot analysis.
-Glutamylcysteine synthetase (
-GCS) activity.
Measurement of -GCS activity in microdissected airways was performed as previously described (West et al., 2000
). Briefly, twenty-four h after the last of seven repeated inhalation exposures, mice (both treated and control) were killed for measurement of airway
-GCS enzyme activity. Distal and proximal airways were isolated by microdissection, after inflation via tracheal cannula with 1% agarose, with Waymouths 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, 1976
) and samples were frozen at -80°C until analysis. To measure
-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, 1976) 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 TrisHCl-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
-glutamylcysteine synthetase (1:5000). Glutathione S-transferase
(GST-
) 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, 1979) or increases in GSH levels due to the feedback inhibition of
-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., 2000
). 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.
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RESULTS |
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-GCS Expression in Tolerant Mice
Expression of -glutamylcysteine synthetase (
-GCS), and its corresponding activity was induced after repeated exposures to NA vapor.
-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. 3A
) or distal airways (Fig. 3B
). After seven repeated daily exposures to NA, airway expression of
-GCS was induced. This induction was clearly evident by Western blot analysis (Fig. 3E
). Compared to control, the 75 kDa catalytic subunit of
-GCS was elevated in both proximal and distal airways that were microdissected from tolerant mice (Fig. 3E
). 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. 3C
) and distal airways (Fig. 3D
). The induced expression of
-GCS localized to the epithelium was coordinated with increased specific activity of the catalytic protein (Fig. 3F
). Analysis of catalytic activity of
-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
-glutamylcysteine, the product of
-GCS, was induced in both proximal (185%) and distal (225%) airways compared to control (Fig. 3F
). This shift in activity was specific to the lung. While activity of
-GCS was roughly three-fold higher in liver of control animals, repeated exposures to NA vapor only caused significant shifts in
-GCS activity in the lung, and were not induced in the liver, kidney, or blood (Fig. 4
).
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DISCUSSION |
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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., 2000). 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
-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., 2000, 2002
). Based on the previous experiments, this was expected and supported by the modulation of the susceptibility to injury by inhibition of the activity of
-GCS with BSO. However, differences between the studies are apparent. The previous studies using parenteral exposures indicated that the induction of
-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
-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
-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., 2000), 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 -GCS induction occurs. Previous studies of chemotherapeutic resistance have demonstrated that the induction of
-GCS is often coordinated with induction of GST-
and MRPs (Ishikawa et al., 1996
; Kuo et al., 1998
). In contrast to these tumor models, our studies indicate that the coinduction of
-GCS with GST-
or MRP did not occur and may also indicate that the mechanism for induction of
-GCS in NA tolerance may well be different. This was surprising, as GST-
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-
is a well known marker for precancerous lesions and hepatic carcinoma (Morimura et al., 1993
), this coinduction may be critical in understanding how adaptations such as NA tolerance evolve into preneoplastic lesions and eventually into tumors.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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