Induction of Tolerance to Naphthalene in Clara Cells Is Dependent on a Stable Phenotypic Adaptation Favoring Maintenance of the Glutathione Pool
2002; Elsevier BV; Volume: 160; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64932-2
ISSN1525-2191
AutoresJay West, Kurt J. Williams, Elina Toskala, Susan J. Nishio, Chad A. Fleschner, Henry Jay Forman, Alan R. Buckpitt, Charles G. Plopper,
Tópico(s)Neonatal Respiratory Health Research
ResumoRepeated exposures to the Clara cell cytotoxicant naphthalene (NA) result in target cell populations that become refractory to further injury. To determine whether tolerance occurs from specific adaptations favoring glutathione (GSH) resynthesis without broad shifts in cellular phenotype, mice were administered NA for 21 days. We found that γ-glutamylcysteine synthetase (γ-GCS) was induced in tolerant Clara cells by repeated exposures to NA. Treating tolerant mice with buthionine sulfoximine, a γ-GCS inhibitor, eliminates resistance acquired by repeated exposures to NA. Broad phenotypic shifts were not present. Marker proteins of differentiation declined over the first 3 days in the development of tolerance, but returned to control levels at 14 and 21 days. Epithelial organizational structure and internal organelle composition in Clara cells from tolerant mice were similar compared to corn oil-treated controls, while subtle shifts in organelle distribution were present. We conclude that induction of γ-GCS expression is coordinated with the development of NA tolerance, but induction of NA tolerance does not markedly alter Clara cell differentiation, epithelial organization, or organelle composition in bronchiolar epithelium. Repeated exposures to the Clara cell cytotoxicant naphthalene (NA) result in target cell populations that become refractory to further injury. To determine whether tolerance occurs from specific adaptations favoring glutathione (GSH) resynthesis without broad shifts in cellular phenotype, mice were administered NA for 21 days. We found that γ-glutamylcysteine synthetase (γ-GCS) was induced in tolerant Clara cells by repeated exposures to NA. Treating tolerant mice with buthionine sulfoximine, a γ-GCS inhibitor, eliminates resistance acquired by repeated exposures to NA. Broad phenotypic shifts were not present. Marker proteins of differentiation declined over the first 3 days in the development of tolerance, but returned to control levels at 14 and 21 days. Epithelial organizational structure and internal organelle composition in Clara cells from tolerant mice were similar compared to corn oil-treated controls, while subtle shifts in organelle distribution were present. We conclude that induction of γ-GCS expression is coordinated with the development of NA tolerance, but induction of NA tolerance does not markedly alter Clara cell differentiation, epithelial organization, or organelle composition in bronchiolar epithelium. Due to direct contact with the environment, the respiratory system is particularly vulnerable to acute injury by airborne pollutants. Many of these contaminants, including aromatic and chlorinated hydrocarbons, are bioactivated to cytotoxic electrophilic intermediates by catalytic enzymes, such as cytochrome P-450 monooxygenases.1Mahvi D Bank H Harley R Morphology of a naphthalene-induced bronchiolar lesion.Am J Pathol. 1977; 86: 558-572PubMed Google Scholar, 2Forkert PG Reynolds ES 1,1-Dichloroethylene-induced pulmonary injury.Exp Lung Res. 1982; 3: 57-68Crossref PubMed Scopus (56) Google Scholar Previous studies have established that single parenteral administrations of naphthalene cause acute epithelial necrosis in bronchioles of mice and hamsters and cytotoxicity in the olfactory epithelium of rats, hamsters, and mice.3Plopper CG Macklin J Nishio SJ Hyde DM Buckpitt AR Relationship of cytochrome P450 activity to Clara cell cytoxicity: III. morphometric comparisons of changes in the epithelial populations of the terminal bronchioles and lobar bronchi in mice, hamsters, and rats after parenteral adminisration of naphthalene.Lab Invest. 1992; 67: 553-565PubMed Google Scholar Non-ciliated, or Clara, cells in distal airway bronchiolar epithelium of mice are particularly susceptible to injury from a single naphthalene (NA) exposure.3Plopper CG Macklin J Nishio SJ Hyde DM Buckpitt AR Relationship of cytochrome P450 activity to Clara cell cytoxicity: III. morphometric comparisons of changes in the epithelial populations of the terminal bronchioles and lobar bronchi in mice, hamsters, and rats after parenteral adminisration of naphthalene.Lab Invest. 1992; 67: 553-565PubMed Google Scholar In contrast, when mice are administered repeated exposures of NA, the previous susceptible Clara cells become refractory to further injury.4O'Brien K Suverkropp C Kanekal S Plopper CG Buckpitt AR Tolerance to multiple doses of the pulmonary toxicant, naphthalene.Toxicol Appl Pharmacol. 1989; 99: 487-500Crossref PubMed Scopus (51) Google Scholar Concern regarding the effects of frequent human exposure have arisen because naphthalene has been found in body fat,5Stanley JS Broad Scan Analysis of the FY82 National Adipose Tissue Survey Specimens: Volume 1-Executive Summary. U.S. Environmental Protection Agency, Office of Toxic Substances, Washington, DC1986Google Scholar as well as mother's milk6Pellizzari ED Hartwell TD Harris BSd Waddell RD Whitaker DA Erickson MD Purgeable organic compounds in mother's milk.Bulletin of Environmental Contamination and Toxicology. 1982; 28: 322-328Crossref PubMed Scopus (106) Google Scholar in conjunction with the findings that NA is carcinogenic in mice7Abdo KM Eustis SL MacDonald M Jokinen MP Adkins JB Haseman JK Napthalene: a respiratory tract toxicant and carcinogen for mice.Inhal Toxicol. 1992; 4: 393-409Crossref Scopus (56) Google Scholar and rats.8National Toxicology Program: Toxicology and Carcinogenesis Studies of Naphthalene (No. 91–20-3) in F344/N Rats. Washington, DC, U.S. Department of Health and Human Services. National Institutes of Health, Public Health Service, 2000Google Scholar Despite the widespread prevalence of this compound, understanding the effects of repeated exposures on the respiratory system is not complete. The ubiquitous antioxidant glutathione (GSH) has various intracellular roles, including the maintenance of enzyme structure, activity, and intracellular redox state. Acting as a cycle, enzymes involved in the synthesis, conjugation, and transport of GSH, collectively called the γ-glutamyl cycle, are important in detoxification.9Meister A Anderson M Glutathione.Annu Rev Biochem. 1983; 52: 711-760Crossref PubMed Scopus (6040) Google Scholar, 10Reed DJ Glutathione: toxicological implications.Annu Rev Pharmacol Toxicol. 1990; 30: 603-631Crossref PubMed Google Scholar Of critical importance is the role of GSH in the detoxification of xenobiotics such as naphthalene. Mouse lung microsomes metabolize naphthalene to glutathione conjugates11Buckpitt AR Castagnoli Jr, N Nelson SD Jones AD Bahnson LS Stereoselectivity of naphthalene epoxidation by mouse, rat, and hamster pulmonary, hepatic, and renal microsomal enzymes.Drug Metab Dispos. 1987; 15: 491-498PubMed Google Scholar which deplete Clara cell glutathione in a dose dependent fashion.12Warren DL Brown JR Buckpitt AR Evidence for cytochrome P450 mediated metabolism in the bronchiolar damage by naphthalene.Chem Biol Interact. 1982; 40: 287-303Crossref PubMed Scopus (109) Google Scholar, 13West JAA Chichester CH Buckpitt AR Tyler NK Brennan P Helton C Plopper CG Heterogeneity of Clara cell glutathione: a possible basis for differences in response to pulmonary cytotoxicants.Am J Respir Cell Mol Biol. 2000; 23: 27-36Crossref PubMed Scopus (31) Google Scholar Toxicity is augmented by the treatment of mice with diethyl maleate, an agent which depletes cellular glutathione.12Warren DL Brown JR Buckpitt AR Evidence for cytochrome P450 mediated metabolism in the bronchiolar damage by naphthalene.Chem Biol Interact. 1982; 40: 287-303Crossref PubMed Scopus (109) Google Scholar In addition, it is becoming clear that the γ-glutamyl cycle is a dynamic system with the ability to change under stress to protect cells from additional toxic exposures.14Jenkinson SG Lawrence RA Zamora CA Deneke SM Induction of intracellular glutathione in alveolar type II pneumocytes following BCNU exposure.Am J Physiol. 1994; 266: L125-L130PubMed Google Scholar, 15Liu RM Hu H Robison TW Forman HJ Increased gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase activities enhance resistance of rat lung epithelial L2 cells to quinone toxicity.Am J Respir Cell Mol Biol. 1996; 14: 192-197Crossref PubMed Scopus (68) Google Scholar We have demonstrated that the reduced susceptibility to NA injury by repeated exposure is directly related to increased airway glutathione resynthesis.16West JAA Buckpitt AR Plopper CG Elevated airway glutathione (GSH) resynthesis confers protection to Clara cells from naphthalene (NA) injury in mice made tolerant by repeated exposures.J Pharmacol Exp Ther. 2000; 294: 516-523PubMed Google Scholar However, it is possible that the changes in susceptibility to naphthalene injury result from temporal shifts in the repair process or the differentiation state of the cells in the target site of injury, the bronchiolar epithelium. Clara cell injury from bioactivated cytotoxicants such as naphthalene and dichloroethylene is followed by a repair phase that spans up to 30 days.2Forkert PG Reynolds ES 1,1-Dichloroethylene-induced pulmonary injury.Exp Lung Res. 1982; 3: 57-68Crossref PubMed Scopus (56) Google Scholar, 3Plopper CG Macklin J Nishio SJ Hyde DM Buckpitt AR Relationship of cytochrome P450 activity to Clara cell cytoxicity: III. morphometric comparisons of changes in the epithelial populations of the terminal bronchioles and lobar bronchi in mice, hamsters, and rats after parenteral adminisration of naphthalene.Lab Invest. 1992; 67: 553-565PubMed Google Scholar During this time frame, distinct periods of proliferation are followed by maturation of undifferentiated cells back to a steady population containing both mature Clara and ciliated cells.17Van Winkle LS Buckpitt AR Nishio SJ Isaac JM Plopper CG Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice.Am J Physiol. 1995; 269: L800-L818PubMed Google Scholar In parallel to this differentiation process fluctuations in the expression of enzymes involved in the bioactivation of NA occur.17Van Winkle LS Buckpitt AR Nishio SJ Isaac JM Plopper CG Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice.Am J Physiol. 1995; 269: L800-L818PubMed Google Scholar Protein levels of cytochrome P-4502f2 protein drop dramatically after a single injurious administration of NA, but return to control levels after 14 days of repair.17Van Winkle LS Buckpitt AR Nishio SJ Isaac JM Plopper CG Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice.Am J Physiol. 1995; 269: L800-L818PubMed Google Scholar With respect to tolerance, the balance between bioactivation and detoxification appears to be critical. While after repeated exposures to NA protein levels of cytochrome P-450 appear to be decreased,18Lakritz J Chang A Weir A Nishio S Hyde D Philpot R Buckpitt A Plopper C Cellular and metabolic basis of Clara cell tolerance to mutiple doses cytochrome P450-activated cytotoxicants. I: bronchiolar epithelial reorganization and expression of cytochrome P450 monooxygenases in mice exposed to multiple doses of naphthalene.J Pharmacol Exp Ther. 1996; 278: 1408-1418PubMed Google Scholar our previous studies indicate that increased airway GSH was critical in the development of tolerance.16West JAA Buckpitt AR Plopper CG Elevated airway glutathione (GSH) resynthesis confers protection to Clara cells from naphthalene (NA) injury in mice made tolerant by repeated exposures.J Pharmacol Exp Ther. 2000; 294: 516-523PubMed Google Scholar Determining if the differentiation state and the ability of airways to repair while under constant stress from daily toxicant exposure is an important step in understanding how cells adapt to a state of tolerance to bioactivated xenobiotics, especially if the compounds are known carcinogens. This prompted us to ask the question whether changes in the differentiation state of Clara cells contributed to the development of NA tolerance. This study was designed to test the hypothesis that changes in the detoxification potential, in response to repeated exposures to NA, result from a stable adaptation that favors maintenance of the GSH pool without broad changes in cellular phenotype. To test this hypothesis we addressed three questions: is induction of γ-glutamylcysteine synthetase (γ-GCS) coordinated with the development of Clara cell tolerance to long term exposures (21 days) of NA; do these cells in tolerant mice express differentiation marker proteins characteristic of normal Clara cells from naive mice and, do Clara cells from tolerant mice retain the morphological and organelle structural characteristics of the controls after repeated exposures of NA. Determining whether Clara cells undergo specific alterations to maintain increased GSH pools without drastic phenotypic shifts will provide insight into the in vivo mechanism by which cells adapt to resist injury from daily exposures to environmental pollutants. Naphthalene 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. Male Swiss Webster mice (6 to 7 weeks) were purchased from Charles River (Wilmington, MA.) Animals were allowed free access to food and water and were housed in an AAALAC accredited facility in HEPA filtered cage racks at the University of California, Davis for at least 5 days before use in an experiment. Mice were administered NA (0 or 200 mg/kg) daily for 21 days. To determine whether GSH is critical in the development NA tolerance, mice receiving repeated exposures (4, 7, 14, and 21 days) were treated with BSO (0 or 800 mg/kg), and challenged with an additional NA dose then processed for high-resolution histopathology. To determine whether Clara cells retain normal phenotypic characteristics, mice were administered (i.p.) NA (0 or 200 mg/kg) daily for 21 days. Mice were killed and lungs fixed by tracheal infusion at 12 and 24 hours after a single exposure and 2, 3, 4, 7, 14, and 21 days after repeated daily exposures and processed for immunohistochemistry, scanning electron microscopy (SEM), or transmission electron microscopy (TEM). Previous studies have demonstrated that BSO can cause a rebound19Griffith OW Meister A Glutathione: interorgan translocation, turnover, and metabolism.Proc Natl Acad Sci USA. 1979; 76: 5606-5610Crossref PubMed Scopus (552) Google Scholar or increases in GSH levels due to the feedback inhibition of γ-GCS. We conducted several preliminary experiments to determine the optimal timing of the BSO and NA doses and have previously reported this data.16West JAA Buckpitt AR Plopper CG Elevated airway glutathione (GSH) resynthesis confers protection to Clara cells from naphthalene (NA) injury in mice made tolerant by repeated exposures.J Pharmacol Exp Ther. 2000; 294: 516-523PubMed Google Scholar The following protocol was used for all mice in the results presented here. Mice receiving repeated exposures of NA were treated with BSO (0 or 800 mg/kg), a GSH resynthesis inhibitor, 24 hours after doses 4, 7, 14, and 21. One hour later mice were challenged with an additional NA dose (0 or 200 mg/kg), killed 3 hours after the NA challenge, and then processed for histopathological assessment. All mouse lungs for histopathological assessment were prepared by inflation via tracheal cannula with 1% glutaraldehyde/1% paraformaldehyde in 0.1 mol/L cacodylate buffer 335 mOsm for 1 hour at 30 cm H2O pressure. The entire fixed middle (cardiac) lobe was postfixed with osmium tetraoxide and incubated overnight in uranyl acetate.20Plopper CG Structural methods for studying bronchiolar epithelial cells.in: Gil J Model of Lung Disease, Microscopy and Structural Methods. Marcel Dekker, New York1990: 537-559Google Scholar The postfixed tissue was embedded in Araldite-502 (Electron Microscopy Sciences), and embedded tissue was then grossly sectioned parallel to the long axis of the mainstem bronchi. Sections (0.5 μmol/L) were cut with glass knives using a Zeiss Microm HM340E microtome and stained with 1% toluidine blue (Electron Microscopy Sciences, Fort Washington, PA). Slides were imaged with a 330 CCD Dage camera on a Zeiss Axiakop MC80 microscope using Scion 1.59 imaging software. Lung tissue from mice was fixed with 4% paraformaldehyde for 2 hours then placed in phosphate-buffered saline (PBS) until processing for embedding. Tissue were embedded in paraffin and sectioned at 6-μm thickness. The presence of the marker proteins for differentiated Clara cells was detected using specific antibodies: rabbit anti-rat Clara cell secretory protein (CC10) and rabbit anti-mouse cytochrome P4502f2 (CYP2f2). 21Nagata K Martin BM Gillette JR Sasame HA Isozymes of cytochrome P-450 that metabolize naphthalene in liver and lung of untreated mice.Drug Metab Dispos. 1990; 18: 557-564PubMed Google Scholar, 22Singh G Katyal SL An immunologic study of the secretory products of rat Clara cells.J Histochem Cytochem. 1984; 32: 49-54Crossref PubMed Scopus (87) Google Scholar Antigenic proteins were identified by the avidin-biotin horseradish-peroxidase method as outlined by Plopper et al.23Plopper CG Dungworth DL Structure, function, cell: injury and cell renewal of bronciholar and aveolar epithilium.in: McDowwell EM Lung Carcinomas. Churchill Livingstone, London1987: 94-128Google Scholar Controls for non-specific binding were performed by substituting primary antibody with PBS. The antibody for CC10 was a generous gift for Dr. Gurmukh Singh (Veterans Affairs Medical Center, Pittsburgh, PA) and the antibody for cytochrome P-4502f was a generous gift from Dr. Henry Sasame (National Institutes of Health, Bethesda, MD). A 19-amino-acid peptide was synthesized according to the published rat γ-GCS heavy subunit amino acid sequence (at position 295–313: NH2-CRWGVISASVDDRTREERG-COOH).24Shi MM Kugelman A Iwamoto T Tian L Forman HJ Quinone-induced oxidative stress elevates glutathione and induces gamma-glutamylcysteine synthetase activity in rat lung epithelial L2 cells.J Biol Chem. 1994; 269: 26512-26517Abstract Full Text PDF PubMed Google Scholar This peptide was conjugated to carrier keyhole limpet hemocyanin (KLH) and used as an antigen to raise rabbit antisera against rat γ-GCS-HS. The polyclonal antibodies against γ-GCS-HS were used in the subsequent Western blot analysis. To determine the relative expression of Cyp4502f2 and γ-GCS, the distal lung region was a isolated via microdissection of agarose inflated lungs. Briefly, tolerant mice were killed with a overdose of pentobarbital 24 hours after the last dose of naphthalene. The trachea of these animals was cannulated and lungs were infused with a 1% agarose solution containing Waymouths media deficient of sulfur-containing amino acids. Appropriate regions of lung were microdissected as previously described.25Plopper CG Chang AM Pang A Buckpitt AR Use of microdissected airways to define metabolism and cytotoxicity in murine bronchiolar epithelium.Exp Lung Res. 1991; 17: 197-212Crossref PubMed Scopus (68) Google Scholar Microdissected airways were placed in an ice cold lysis buffer (Tris 0.1 mol/L (pH 8.2), KCl 150 mmol/L, MgCl2 20 mmol/L, and EDTA 2 mmol/L) containing protease inhibitors and homogenized. Homogenized proteins were centrifuged at 9000 × g for 20 minutes. Soluble protein content was determined by the microBradford method26Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (219809) Google Scholar and samples were frozen at −80°C until analysis. Soluble protein were diluted in a 10% sodium dodecyl sulfate (SDS) sample buffer containing β-mercaptoethanol and separated by gel electrophoresis using a Bio-Rad Tris-HCl-buffered 10% polyacrylamide Minigel (Hercules, CA). Gels were transferred on to a polyvinylydifluoroacetate membrane (NEN, Inc., Boston, MA) and probed with the rabbit antibody produced against the cytochrome P4502f (1:10,000) or the catalytic subunit of γ-glutamylcysteine synthetase (1:5000). Bands were detected by chemilumenescence using a horseradish peroxidase-linked secondary goat antibody produced against rabbit IgG. Briefly, the airway tree of the cranial lobe was exposed by microdissection, dehydrated through a graded ethanol series, immersed in hexamethyldisilazine for 5 minutes, and air dried overnight. Desiccated lungs were then gold-coated with a Polaron II E5100 sputter coater at 2.5 kV acceleration voltage in argon with a 10 mA current for 2 minutes. Airways were imaged using a Phillips SEM 501 microscope. Terminal airways from mice receiving repeated daily injections of NA (0 or 200 mg/kg) were selected from araldite sections used for high-resolution histopathology. Using the original blocks these airways were isolated and sectioned at 70 nm with a Sorvall MT 5000 utlramicrotome. These sections were stained with uranyl acetate and lead citrate then examined using a Zeiss EM-10 electron microscope at 60 kV. Complete profiles (including basal lamina, nucleus, and apical projections) of a minimum of 10 Clara cells per animal were categorized blindly by one investigator for differences in size, mitochondrial conformation, and secretory granule distribution and abundance. Terminal airways of mice receiving repeated corn oil injections were lined by a simple cuboidal epithelium (Figure 1A). The majority of the cells were non-ciliated. Many of these non-ciliated, or Clara cells, had apical projections into the airway lumen. Three hours after control mice were administered 200 mg/kg NA, Clara cells were swollen (Figure 1B), formed large clear cytoplasmic vacuoles, and had discrete apical blebs (**). No signs of toxicity were detected in control mice treated with BSO alone without NA (data not shown). In contrast to control mice, as previously described,4O'Brien K Suverkropp C Kanekal S Plopper CG Buckpitt AR Tolerance to multiple doses of the pulmonary toxicant, naphthalene.Toxicol Appl Pharmacol. 1989; 99: 487-500Crossref PubMed Scopus (51) Google Scholar repeated daily exposures to NA resulted in epithelium that appeared to be resistant to further injury. Twenty-four hours after the last of 4, 7, 14, or 21 daily injections of NA, mice were divided into four groups, administered BSO (0 or 800 mg/kg), then further subdivided and challenged with NA (0 or 200 mg/kg). Tolerant animals, at all time points (4, 7, 14, and 21 days), challenged with a 200 mg/kg NA dose showed no detectable morphological differences from control (Figure 1, C, E, G, I). In contrast, 3 hours after administration of the NA challenge dose, tolerant animals from the same time points treated with BSO had severe airway injury similar to challenged non-tolerant mice (Figure 1, D, F, H, J). After four repeated exposures to NA, the resulting injury from pretreatment with BSO before NA challenge failed to injure all of the cells in the distal airways (Figure 1D), there was consistent sparing of the most terminal population of Clara cells. The injured cells appeared to be clustered together, while large areas of cells remained unaffected by the combined treatment. After 7, 14, and 21 days of repeated injections, pretreatment with BSO caused significant NA-induced injury. The response in the distal airways of these mice was less variable than mice receiving four repeated injections of NA. Injury extended down to involve the most terminal Clara cells, in contrast to those spared after four repeated injections of NA. Occasional Clara cells were exfoliated and most had discrete swollen apical membrane blebs (**), similar to NA-challenged controls. Overall, the airway epithelium from the tolerant mice was similar to NA-challenged non-tolerant mice. No signs of toxicity were detected in tolerant mice treated with BSO alone without NA (data not shown). In control mice, detectable γ-GCS was present throughout the airways and lung parenchyma as diffuse lightly positive staining within the cells (Figure 2B) compared to control sections with PBS substituted for primary antibody (Figure 2A). This corresponded to a faint band detectable by Western blot analysis (Figure 3). Clara cells in control mice appeared to have protein localized to the cytoplasm of the basal half of the cell (Figure 2B, inset). Twenty-four hours after a single injection of NA, immunoreactive protein appeared to be induced in bronchioles isolated by microdissection as assessed by Western blot (Figure 3), and appeared in cells exfoliated from the basal lamina in terminal bronchioles (Figure 2C). After two repeated injections, γ-GCS protein was clearly elevated in microdissected bronchioles from tolerant mice (Figure 3) and immunohistochemically evident in both the apical and basal regions of Clara cells (Figure 2D). A similar expression pattern was present after 7, 14, and 21 repeated daily injections of NA (Figure 2, G–I). After 14 and 21 repeated daily injections, Western blot analysis revealed that γ-GCS expression declined but remained elevated from control (Figure 3), while the epithelium in terminal airways retained a strong immunoreactive signal (Figure 2, H and I).Figure 3Western blot of γ-GCS in distal lung homogenates of tolerant mice. Western blot analysis revealed that γ-GCS expression was induced above control (CON) after 1 (24 hours); 2 (48 hours), 3 (72 hours), 4 (96 hours), and 7 (7 days) daily repeated injections. After 14 (14 days) and 21 (21 days) daily repeated NA injections γ-GCS appeared to decrease slightly, but remained elevated from control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunoreactive CC10 was only detected in Clara cells, and was expressed throughout the airways of control mice (Figure 4A). Twelve hours after receiving one NA exposure (200 mg/kg) CC10 positive cells were exfoliating into the airway lumen (Figure 4B). Twenty-four hours after a single NA injection immunoreactive CC10 was detected, but reduced in the terminal airways, and was not prominent in the squamated cells after exfoliation (Figure 4C). After two daily injections of NA the most distal airways were largely devoid of immunoreactive CC10 (Figure 4D) and this pattern of expression remained after 4 repeated daily exposures (Figure 4E). After 7 daily exposures to NA, (Figure 4F) the number of CC10 immunoreactive cells increased, but the overall intensity was less than controls. After 14 and 21 daily injections, the expression of CC10 became indistinguishable from control animals. The expression of immnunoreactive Cyp2f2 protein in control animals was only detected in Clara cells (Figure 5A) and was detected as a single band by Western blot analysis (Figure 6) in mice receiving repeated corn oil injections. The overall pattern of changes in CYP2f2 parallels that of CC10. Twelve hours after a single dose of NA (200 mg/kg) cells with immunoreactive Cyp2f2 were exfoliated into the airway lumen (Figure 5B). After 24 hours, immunoreactive Cyp450 was not detectable by Western blot (Figure 6) and, like CC10, at this time was absent from the most terminal airway Clara cells (Figure 5C). Terminal airways of mice receiving two or three daily exposures of NA also expressed greatly reduced Cyp2f2 based on Western blot analysis (Figure 6) or immunohistochemistry (Figure 5D). After four daily injections of NA, a faint, but detectable band was present in Western blots (Figure 6), which was coordinated with focal expression of immunoreactive protein in bronchiolar epithelial cells (Figure 5E). After 1 week of repeated daily exposures to NA, Cyp2f2 expression continued to increase (Figure 6); most Clara cells in the terminal airways now expressed a diffuse signal for immunoreactive for CYP2f2 (Figure 5F). While these marker proteins differed at 7 days, after 14 and 21 repeated exposures of NA the expression of Cyp2f2 appeared similar to control animals when assessed by both Western blot analysis (Figure 6) or immunohistochemistry (Figure 5, G and H).Figure 6Western blot of cytochrome P4502f2 in distal lung homogenates of tolerant mice. Western blot analysis revealed that CYP2f2 decreased from control (CON) after a single injection of NA (24 hours); and after 2 injections (48 hours), and 3 injections (72 hours). After 4 (96 hours) and 7 (7 days) repeated injections CYP2f2 began to increase, eventually returning back to control levels after 14 (14 days) and 21 (21 days) repeated injections of NA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Terminal airways from control mice were lined by two predominant cell types, non-ciliated (Clara) cells and ciliated cells (Figure 7A). The non-ciliated cells had apical projections that extended into the lumen and were organized in linear rows down the main axial path of the airways. Ciliated cells were less numerous and interspersed among these rows. The apical portion of the Clara cells was a defined dome-like region in the center of the cells. The cilia covered the entire lumenal surface of the ciliated cells (Figure 7A). Twenty-four hours after an intraperitoneal injection of NA, the terminal airways were devoid of intact Clara cells (Figure 7B) which had exfoliated and overlaid the subadjacent layer of attenuated ciliated cells (Figure 7B). The ci
Referência(s)