Introduction

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The roles of the antioxidant enzymes, copper/zinc superoxide dismutase (Cu-Zn SOD) and manganese superoxide dismutase (Mn SOD), in the lungs of animals exposed to oxidant stresses, continue to be a subject of debate. Mn SOD, in particular, has been found to increase in a number of lung injury models (1). In addition, both Cu/Zn and Mn SOD have been found to increase with short-term ozone exposure (9), but such evidence is not without controversy. A study of acute ozone exposure found that ozone tolerance was unrelated to the level of SOD (10). The authors found no increase in SOD in rats and mice exposed to a low level of ozone followed by an edemagenic dose of ozone (10). However, these animals were only exposed for 3 days. Subsequent investigations of Cu-Zn and Mn SOD induction following ozone exposure have indicated that these enzymes are significantly elevated after 5 days of exposure (9).

In a previous study from our laboratory, chronic effects of ozone exposure on SOD activity within specific sites of the lungs were examined following 3 and 20 mo of exposure (11). Exposure to 0.12 ppm and 1.0 ppm ozone for 3 mo was associated with an increase in total SOD activity per mg DNA in the distal bronchioles and in the centriacinar regions of the lung (11). This dose-related increase resulted in a doubling of SOD activity in animals exposed to 1.0 ppm ozone (11). However, those cells responsible for this heightened level of SOD activity within these regions were not determined. The distribution of the two major forms of SOD, Cu-Zn and Mn, within the lungs of exposed animals was also not examined.

The acute effects of ozone exposure include pulmonary edema, cell necrosis, and cell proliferation (12) with the site of greatest damage found at the bronchiole-alveolar duct junction (14). This injury process is attenuated after 3 wk of continuous exposure as animals adapt (16). The chronic response subsequently begins with proliferation of type II cells (16) and, in high dose exposures, bronchiolar metaplasia of alveolar ducts occurs with replacement of type I and type II epithelial cells by ciliated and nonciliated bronchiolar epithelial cells similar to those found in terminal bronchioles (17). This investigation was undertaken to determine whether the proliferative and metaplastic epithelial responses seen in the lungs with ozone exposure may be related, in part, to an increase in antioxidant enzyme production.

We wished to examine the specific cell types in the lungs involved in alterations in both forms of SOD (i.e., Cu-Zn and Mn) with exposure to 1.0 ppm ozone for up to 3 mo. Both Cu-Zn and Mn SOD were examined using immunohistochemistry to determine the location and relative change of these enzymes following 3 mo of exposure to ozone. We found that ozone exposure was associated with an overall decrease in labeling for Cu-Zn SOD in the airways and lung parenchyma. In contrast, Mn SOD labeling was found to be significantly increased in the bronchiole-alveolar duct regions of lungs exposed to ozone. This region includes the last conducting airways, or terminal bronchioles, and the most proximal alveoli and ducts immediately arising from these bronchioles. This site was studied in detail to determine specific changes in the cellular density of Mn SOD as a function of distance from the BADJ. Alveolar type I and II epithelial cells, as well as Clara cells in the metaplastic regions and in terminal bronchioles were examined. Fibroblasts in the proximal alveolar septal tissues were also examined for changes in Mn SOD expression.

	Materials and Methods

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Exposures

Exposures were performed in the Inhalation Facility of the California Regional Primate Research Center at the University of California, Davis. Male Fischer 344 rats from Simonsen Laboratories (Gilroy, CA) were obtained at 30- 32 days of age. The animals were quarantined for 2 wk prior to the onset of the exposures. Health screening was performed on two rats randomly selected from each chamber. No viral or respiratory pathogens were detected. Rats were exposed to ozone generated using a silent arc discharge ozonator, and levels were monitored using a Dasibi analyzer calibrated with a Dasibi UV photometer. Calibration of the photometer was performed with a National Institute of Standards and Technology standard reference photometer located at the California Air Resources Board Quality Assurance Standards Laboratory (Sacramento, CA). Four animals per group were exposed to filtered air (0.00 ± 0) or 1.00 ± 0.03 ppm ozone for 6 h per day, 5 days per wk for either 2 or 3 mo. These exposure conditions were designed to duplicate those used in a previous investigation which identified changes in antioxidant enzymes with chronic ozone exposure (11). Animals were exposed between the hours of 7:30 A.M. and 5:30 P.M. and were on a light cycle of 12 h on/12 h off with the light on during the normal daylight period.

Antibodies

The antisera to Cu-Zn SOD is a rabbit anti-rat Cu-Zn SOD polyclonal sera described previously (21). The antibody is specific for Cu-Zn SOD and the reaction can be absorbed by purified Cu-Zn SOD (22). The Mn SOD antisera is a rabbit anti-human manganese superoxide dismutase made using recombinant human Mn SOD supplied by Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT). The antisera is monospecific for a single protein at a molecular weight corresponding to Mn SOD on immunoblots. Coincubation of the antisera with antigen abolishes the reaction on immunoblots (23). This polyclonal antisera cross-reacts with rat Mn SOD.

Immunohistochemistry (Light Microscopy)

Within 24 h of the end of 3 mo ozone exposure, animals were anesthetized with sodium pentobarbital and the trachea cannulated. Both hemidiaphragms were punctured to deflate the lungs. The lungs were fixed for 10 min by instillation of neutral buffered formalin at 20 cm of water pressure, placed in the same fixative for 24 h and stored in 70% ethanol before embedding in paraffin. The blocks were sectioned at 5 µm and labeled with antisera to Mn (1:1,000) or Cu /Zn SOD (1:10,000 and 1:100,000) using the avidin biotin peroxidase method with reagents from Vector Laboratories (Burlingame, CA), visualized with 3,3' diaminobenzidine tetrahydrochloride from Sigma (St. Louis, MO) and counterstained with Mayer's hematoxylin from Sigma. The two different concentrations of Cu-Zn SOD were used to determine the relative abundance of Cu-Zn SOD in specific regions of the lung. Photomicrographs were made on a Zeiss Axioskop MC80 using Fuji Reala color film.

Immunogold Labeling (Electron Microscopy)

The lungs of animals exposed to ozone for 2 mo were fixed by instillation of 0.25% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, at 20 cm of water pressure. After fixation for 10 min in the chest, the accessory lobe of the lungs was removed and stored in 0.25% glutaraldehyde until processed.

Slabs of tissue taken from the lung were washed in 1.5 M phosphate-buffered saline (PBS) + 0.2 M glycine, infiltrated for 10 min with 2% gelatin in 1.5 M PBS, followed by 5% gelatin in 1.5 M PBS for 10 min, and finally 10% gelatin in 1.5 M PBS for 10 min. Following the last change in gelatin, the tissue was transferred to a glass slide on ice and allowed to harden. Tissue slices were examined under a dissecting microscope in a cold room and terminal bronchioles were identified in longitudinal orientation using a modification of the method of Barry and associates (15). Areas containing a terminal bronchiole and proximal alveolar ducts in longitudinal orientation were cut out as a cube and infiltrated with 2.3 M sucrose overnight at 5°C using a rotator. The tissue blocks were then frozen in liquid nitrogen, dehydrated in methanol at 90°C and embedded in Lowicryl resin at 45°C following the method of Oprins and colleagues (24). The longitudinally oriented blocks were polymerized with UV light and sectioned on a LKB ultramicrotome. The infiltration and embedding methods used, in combination with protein A gold labeling of ultrathin sections, has been found to maintain the antigenicity of the enzyme and allows quantitation of the enzyme on longitudinally oriented sections. Ultrathin sections were mounted on formvar and carbon-coated copper grids.

Sections were labeled following the methods of Slot and Geuze (25, 26). Briefly the tissue sections mounted on grids were washed in 1.5 M PBS + 0.1% bovine serum albumin (BSA), labeled with diluted (1:300) Mn SOD antisera for 60 min, washed several times in PBS + 0.1% BSA, and placed on drops of 10 nm protein A-gold. Sections were subsequently washed in PBS, fixed with 1% glutaraldehyde for 5 min, and washed for 1 h in several changes of distilled water.

Tissue sections from the same blocks were mounted on glass slides and photographed at low magnification for orientation. The labeled thin sections mounted on grids were counted using a Zeiss 10 C transmission electron microscope at ×20,000 using a camera system in which a point counting overlay of 42 points was placed directly on the screen and all counts taken directly from the screen. Each point on the screen defined an area of 0.21 µm². All alveolar ducts were examined and epithelial type II cells found in the alveolar duct region were counted as a function of distance from the BADJ. Type II epithelial cells were selected randomly in the alveolar ducts for a total of 10 cells per site. This sampling method typically included the majority of type II cells present in most sites. Type II epithelial cells were selected from alveolar ridges and outpocketings which opened directly on the alveolar duct. Adjacent closed alveolar profiles were not sampled due to the difficulty in determining the exact distance of these alveoli from the BADJ. Type II epithelial cells were sampled as a function of distance from the terminal bronchiole with cells being sampled at 200-µm intervals. The 0-200-µm and 200-400-µm intervals were pooled because of a reduced sample size in the 0-200-µm interval with bronchiolarization of alveolar epithelium after repeated ozone exposure. Type II cells found in the more distal lung parenchyma were also sampled by isolating alveolar ducts greater than 600 µm from the BADJ and counting 10 cells randomly selected per site. Since Mn SOD is predominantly within the mitochondria, the quantitative analysis of Mn SOD at the electron microscopic level was done examining only mitochondria. The first mitochondrial profile encountered in each cell was selected for counting and points on the mitochondria and the number of gold grains on the mitochondria was tallied. The degree of background labeling was calculated as the relative density of gold grains found on air spaces and this background was subtracted from the relative density of gold grains on mitochondria to give specific labeling for Mn SOD. Clara cells in terminal bronchioles were counted using a similar method. Clara cells were randomly selected by choosing every other cell from each of the two sides of the longitudinal section through the terminal bronchiole for a total of 10 cells. Immunogold labeling over these cells was counted using a camera system at ×20,000, and total Mn SOD was calculated in the same manner as the type II cells. Fibroblasts were counted by photographing 10 randomly selected cells per site and printing at a final magnification of ×41,000. These electron micrographs were placed under a uniform point counting overlay and the point density per cell and gold grain density per cell were determined. The area represented by each point on the overlay was of 0.21 µm². Fibroblasts were selected as a function of distance down the alveolar ducts by choosing every fibroblast encountered moving radially outward from the bronchiolar-alveolar duct junction until 10 cells were selected. Fibroblasts were selected to a distance of 400 µm. All data were calculated as the number of gold grains per points on mitochondria and were expressed as the mean ± the standard error of the mean (SEM). Student's t test was used to determine statistical relevance.

In addition to Mn SOD, labeling for Cu-Zn SOD was qualitatively examined in the bronchiole-alveolar duct region using the same methods. The density of Cu-Zn SOD labeling over type I epithelial cells lining alveolar septa in the proximal alveolar region was quantified in a preliminary study. No detectable difference between the controls and experimental animals in the density of labeling for Cu-Zn SOD was noted. Given the lack of a significant increase in Cu-Zn SOD to ozone exposure in the proximal alveolar region by electron microscopic immunohistochemistry, further examination of the Cu-Zn SOD enzyme in this location, by this method, was discontinued.

	Results

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Cu-Zn and Mn SOD were examined in rat lungs chronically exposed to ozone. Slices through the left lobe of rat lungs exposed for 2 or 3 mo to 0.0 and 1.0 ppm ozone were used to determine the distribution and relative abundance of these two antioxidant enzymes and any changes associated with ozone exposure. Cu-Zn SOD was found in the airways down to the level of the terminal bronchioles. Virtually all epithelial cells of the upper airways were labeled, while primarily Clara cells labeled at the level of the terminal bronchiole. Fibroblasts and alveolar macrophages were labeled in the parenchyma with type II epithelial cells occasionally labeled. The labeling of Cu-Zn SOD in airway cells and the distal parenchyma (Figure 1A) was markedly reduced by ozone exposure (Figure 1B), with retention of label near the pleura, that region of the lung which receives the lowest dose of ozone from an exposure. Both the intensity and extent of labeling of Cu-Zn SOD were reduced in the parenchyma (Figure 1B) following ozone exposure. When the dilution of Cu-Zn SOD antibody was further increased 10-fold, from 1:10,000 to 1:100,000, the labeling in the parenchyma was lost, while light labeling of the airways in ozone-exposed animals was still present. In contrast, the airways and parenchyma in the lungs of control animals demonstrated a heavier degree of labeling than that noted in the lungs of animals exposed to ozone. Therefore, Cu-Zn labeling was higher in the airways than in the parenchyma in control animals and was reduced in both regions by ozone exposure. These distinctions were made more obvious by using high dilutions of the antisera.

View larger version (127K): [in this window] [in a new window]   Figure 1.   The distribution of Cu/ Zn and Mn SOD in rat lungs exposed to 0.0 (control) and 1.0 ppm for 3 mo using immunohistochemistry. (A) A control rat lung labeled for Cu/Zn SOD (1:10,000). Dense labeling is noted in the parenchyma (arrowheads). (B) A rat lung exposed for 3 mo to ozone and labeled for Cu/ Zn SOD (1:10,000). A general reduction of labeling density is noted in the distal parenchyma (arrowheads). (C) A control rat lung labeled for Mn SOD (1:1,000). Few labeled cells are noted in the proximal alveolar region (arrows). (D) A rat lung exposed for 3 mo to ozone and labeled for Mn SOD (1:1,000). Increased labeling of alveolar epithelial cells (arrows) and alveolar macrophages (open arrows) are seen in the proximal alveolar region. Bar  = 100 µm. (E) A higher magnification detail of a control rat lung proximal alveolar region with few labeled type II epithelial cells (arrows). (F) A rat lung exposed for 3 mo to 1.0 ppm ozone. The higher magnification more clearly details the increase in labeling of type II epithelial cells (arrows) and alveolar macrophages (open arrows). Bar = 50 µm.

Mn SOD labeled the pleura lightly and moderately labeled the airways down to the level of terminal bronchioles. In terminal bronchioles, Clara cells were the primary cell type to show labeling for Mn SOD. Alveolar macrophages and type II epithelial cells were labeled in the proximal alveolar duct regions (Figures 1C and 1E). Ozone exposure was associated with an increase in labeling of type II epithelial cells (Figures 1D and 1F) in the proximal alveolar ducts. Alveolar macrophages which had increased in number with ozone exposure were also heavily labeled for Mn SOD in the proximal alveolar region with chronic ozone exposure (Figures 1D and 1F).

The bronchiole-alveolar duct region was further studied using more sensitive methods to determine changes in antioxidant enzymes on a per cell basis. Mn and Cu-Zn SOD were studied using protein A gold immunolabeling at the electron microscopic level to provide a more sensitive and quantitative method for assessing changes in SOD with ozone exposure. Both Mn and Cu-Zn SOD were studied in terminal bronchioles and the alveolar duct regions in animals exposed to 0.0 and 1.0 ppm ozone for 2 mo. Cu-Zn SOD was not increased in any cell type in either terminal bronchioles or alveolar ducts (data not shown) including Clara cells, type I and type II epithelial cells, and interstitial fibroblasts. Mn SOD was also examined in the Clara cells of terminal bronchioles in animals exposed to 0.0 and 1.0 ppm ozone and was not found to increase as a result of ozone exposure (Figure 2). Prolonged exposure to high levels of ozone (i.e., 1.0 ppm) has been found to result in a bronchiolarized metaplasia of the epithelium of the alveolar ducts with a change in cell type from the usual type I/ type II epithelium to a cuboidal epithelium composed of Clara cells, ciliated cells, and undifferentiated cuboidal cells (27). We found evidence, in our study, of bronchiolarized metaplasia in alveolar ducts of animals exposed to 1.0 ppm ozone for 2 mo. The bronchiolarized metaplasia was confined to the proximal alveolar region within 200 µm of a terminal bronchiole (Figure 3A). The Clara cells found in the bronchiolarized portions of alveolar ducts demonstrated the same relative degree of labeling for Mn SOD as did Clara cells in the terminal bronchioles in both ozone-exposed and control animals (Figure 2). Bronchiolarized metaplasia was not found in alveolar ducts of control animals (Figure 3B).

View larger version (16K): [in this window] [in a new window]   Figure 2.   The level of Mn SOD in the Clara cells of the terminal bronchioles and alveolar duct regions in rat lungs exposed to 0.0 and 1.0 ppm ozone for 2 mo. All data are means ± SEM. No significant differences were noted using Student's t test.

View larger version (106K): [in this window] [in a new window]   Figure 3.   A light micrograph of the bronchiole-alveolar duct region of rat lungs exposed to 0.0 and 1.0 ppm ozone for 2 mo. (A) Terminal bronchiole (arrowheads) and alveolar duct region of an exposed rat lung. An area of bronchiolarized metaplasia (arrows) is seen as well as clusters of alveolar macrophages (open arrows) in the alveolar duct region. (B) Terminal bronchiole (arrowheads) and alveolar duct region of a control rat lung. Bar = 50 µm.

The expression of Mn SOD in type II epithelial cells was studied in two locations along alveolar ducts, 0-400 µm from the BADJ and the distal parenchyma located greater than 600 µm from the BADJ (Table 1). As with all cell types studied, labeling was confined mainly to the mitochondria of the cells with only minimal background labeling of the cytoplasm (Figures 4A and 4B). The labeling for Mn SOD was significantly increased in type II epithelial cells located 0-400 µm from the BADJ in animals exposed for 2 mo to 1.0 ppm ozone (Figure 5). With increased distance down the alveolar ducts, in regions greater than 600 µm from the BADJ, the level of Mn SOD labeling was not significantly different from control values (Figure 5). The increase in Mn SOD in the proximal alveolar region was not related to an increase in the density of mitochondria in type II epithelial cells, since specific mitochondrial density within type II cells did not change with increasing distance from the BADJ (Table 2). In addition to the changes in epithelial cells, interstitial fibroblasts were examined for induction of Mn SOD. No induction of Mn SOD was found in interstitial fibroblasts in alveolar ducts (Table 1). Thus, the major site of induction of Mn SOD was in the proximal portions of the gas exchange region and this induction occurred specifically in the mitochondria of type II alveolar epithelial cells.

View larger version (93K): [in this window] [in a new window]   Figure 4.   Electron micrographs of type II epithelial cells immunolabeled for Mn SOD. (A) A type II cell from a control rat lung containing lamellar bodies (LB). Gold grains (arrowheads) labeling for Mn SOD are seen in the mitochondria. (B) A type II epithelial cell from a rat lung exposed to 1.0 ppm ozone for 2 mo. Gold grains can be seen labeling for Mn SOD in the mitochondria (arrowheads). Bar = 1 µm.

View larger version (12K): [in this window] [in a new window]   Figure 5.   The percent change from control in Mn SOD in type II alveolar epithelial cells as a function of distance (in µm) from the bronchiole-alveolar duct junction (BADJ). The animals were exposed to either 0.0 or 1.0 ppm ozone for 2 mo. Mn SOD expression was measured in a proximal location 0-400 µm from the BADJ and in the distal parenchyma located at greater than 600 µm from the BADJ. All data are means ± SEM. *P < 0.05 compared with 0.0 using Student's t test.

	Discussion

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Chronic exposure to ozone resulted in a number of changes in the expression of superoxide dismutase within rat lungs. Cu-Zn SOD was found to decrease with exposure in the small airways, and the distal parenchyma, a region of the lung not normally associated with damage from ozone. Using a dilution technique, we determined that the level of Cu-Zn SOD labeling was greater in the airways than in the parenchyma, but both regions were decreased compared with controls by 1.0 ppm ozone exposure for up to 3 mo. In contrast, Mn SOD was found to increase with ozone exposure in a site- and cell-specific manner. Our previous study found a doubling of total SOD activity in the centriacinar region with ozone exposure, increasing from 550.3 ± 55.9 in control lungs to 1,113.0 ± 238.9 units/mg DNA after exposure to 1.0 ppm ozone for 90 days (11). When the distal parenchyma of these same lungs was examined, no change from control values in SOD activity was found with ozone exposure (11). The present study demonstrates a significant increase in the number of labeled alveolar macrophages and type II epithelial cells for Mn SOD in the centriacinar regions of exposed lungs, as well as an increase in the labeling per cell for type II epithelial cells located on alveolar duct ridges and adjacent septa up to 400 µm from the bronchiole-alveolar duct junction.

There was no increase in Mn SOD for fibroblasts or Clara cells. Clara cells found in terminal bronchioles and Clara cells found in alveolar ducts as a result of bronchiolarized metaplasia following chronic ozone exposure showed no increase in Mn SOD labeling. Bronchiolarized metaplasia of alveolar ducts is a known result of long-term high dose exposure (i.e., 0.5-1.0 ppm) to ozone (17, 27). A previous study of rats exposed to 1.0 ppm ozone for 20 mo resulted in extensive bronchiolarized metaplasia of the alveolar ducts (27). The normal type I and type II epithelium lining the alveolar ducts was replaced by cuboidal epithelium composed of ciliated cells, Clara cells, and undifferentiated cuboidal cells (27). The lack of major cell and tissue changes in some studies on terminal bronchioles following chronic ozone exposure (27, 29, 30) would seem to indicate an increased tolerance for ozone in the bronchiolar metaplastic tissue, which is similar to tissue found in terminal bronchioles. Other studies have noted changes in terminal bronchioles exposed to ozone, including a reduction in cell injury, increased levels of Clara cell secretory protein (31), and an increased proportion and mass of nonciliated cells (32). However, acquired tolerance of terminal bronchiolar tissue following repeated exposure to ozone does not involve cell-specific increases in Mn SOD or Cu-Zn SOD. We found that Mn SOD and Cu-Zn SOD were similar in Clara cells located within the alveolar ducts compared with Clara cells in normal bronchiolar tissue after 2 mo of exposure to 1.0 ppm ozone. However, other antioxidant enzymes, which were not examined, could be altered by exposure, and thus contribute in yet undefined ways to this tissue's resistance to the oxidant damage from ozone.

The chronic effects of ozone exposure on the epithelium of alveolar ducts include an increase in epithelial thickness with thickening of type I cells and hyperplasia of type II cells (16). Areas where type I and type II epithelium were maintained in alveolar ducts were found in addition to areas of bronchiolarized metaplasia following exposure for 20 mo to 1.0 ppm ozone (27). The present study contributes to our understanding of how portions of the alveolar epithelium develop resistance to chronic ozone damage. The alveolar epithelial response seen after 2 mo of exposure is similar to that found after 20 mo of exposure and contains a mixed population of epithelial cells with areas made up of Clara, ciliated, and other cuboidal cells, as well as areas with type I and type II alveolar epithelial cells (27). We found a statistically significant increase in the density of Mn SOD in the mitochondria of type II epithelial cells located in the most proximal regions of the alveolar duct walls and septa. Pryor (33) has postulated the toxic biochemical reactions of ozone as a cascade with the production of secondary and tertiary toxins, including superoxide radicals, being formed within the cells of the lung. Thus the increased expression of Mn SOD may be associated with type II epithelial cells becoming more resistant to ozone damage and enabling maintenance of the type I /type II epithelium between areas of bronchiolarized metaplasia in long-term, high-dose ozone exposures. Alternatively, the increase in Mn SOD observed could be simply a reflection of sublethal injury.

The site in the lung most affected by ozone exposure is the centriacinar region (15, 17, 28). The effects of chronic exposure from ozone in this region include epithelial changes, chronic inflammation, and fibrosis (16, 27). Tolerance to ozone exposure in the central acinar region may involve more than one process. Clara cells are obviously resistant to ozone exposure and the bronchiolarized metaplasia seen in long-term exposures may be a protective mechanism for the tissue (27), yet this tolerance does not involve an increase in the antioxidant enzyme Mn SOD. Type II epithelial cells found in the same region as the bronchiolarized Clara cells have increased levels of Mn SOD, suggesting that their tolerance to ozone exposure is associated with an antioxidant increase. An earlier study of the centriacinar region demonstrated a two-fold increase in total SOD activity following a 3-mo exposure to ozone, but did not identify which cell types were involved in this increase or the subtype of SOD involved (11). The present study demonstrates that ozone-induced increases in total SOD are the result of increases in Mn SOD in type II epithelial cells located along the proximal portions of alveolar duct walls and adjacent alveolar outpocketings.