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Abstract |
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Abstract
Introduction
References
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Ozone, the principal oxidant pollutant in photochemical smog, causes airway epithelial injury in the upper and lower respiratory tract of laboratory animals. We have recently reported that long-term inhalation exposure to ozone causes mucous-cell metaplasia (MCM) in the surface epithelium lining the nasal airways of F344 rats. The principal objective of the present study was to determine the persistence of ozone-induced MCM in the nasal epithelium after the end of a chronic exposure. Male F344/N rats were exposed to 0, 0.25, or 0.5 ppm ozone, for 8 h/d, 7 d/wk for 13 wk. Animals were killed 8 h, 4 wk, or 13 wk after the end of the chronic exposure. Ozone-related alterations in the nasal epithelium were qualitatively and quantitatively characterized through histochemistry, image analysis, and morphometric techniques. Some rats were exposed for an additional 8 h to 0.5 ppm ozone at 13 wk after the end of the chronic exposure to determine whether previous ozone exposure results in persistent changes in the sensitivity of nasal epithelium to acute injury. At the end of the chronic exposure, hyperplasia was present in the nasal epithelium of rats exposed to 0.25 and 0.5 ppm ozone. By 13 wk postexposure, this proliferative alteration was still evident only in the rats exposed to 0.5 ppm ozone. Ozone-induced MCM with associated intraepithelial mucosubstances was evident only in the nasal tissues of rats exposed to 0.5 ppm ozone. Though attenuated, these alterations in the nasal mucous apparatus were still detectable at 13 wk after the end of the exposure. At this same time after the chronic exposure, an acute (8 h) exposure to 0.5 ppm ozone induced an additional increase of mucosubstances in the nasal epithelium of rats previously exposed to 0.5 ppm ozone, but not in rats chronically exposed to 0 or 2.5 ppm ozone. The persistent nature of the ozone-induced MCM in rats documented in this report suggests that ozone exposure may have the potential to induce similar long-lasting alterations in the airways of humans.
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Introduction |
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Abstract
Introduction
References
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Ozone, the principal oxidant pollutant in photochemical smog, is an inhaled toxicant that causes tissue injury not only in pulmonary airways (1) but also in nasal airways of laboratory animals (8). Human nasal mucosa is also susceptible to ozone toxicity. Acute inflammation is induced in the nasal airways after exposure to ambient concentrations of this highly reactive, irritating gas (14). Residents of the southwestern region of Mexico City, where air pollution is a daily problem and ambient concentrations of ozone often exceed the National Ambient Air Quality Standards, have conspicuous, pollution-related, histologic alterations in the mucosa lining their nasal passages (19).
We have recently reported that repeated, long-term exposure of F344/N rats to ozone causes a marked differentiation of the nasal surface epithelium lining the lateral meatus of the proximal nasal airways, from a simple cuboidal-cell population devoid of mucus-secreting cells (i.e., nasal transitional epithelium [NTE]) to a metaplastic cell population containing numerous mucous (goblet) cells with copious amounts of stored mucosubstances (i.e., mucous-cell metaplasia [MCM]) (13). In this previous study, the NTE of control rats exposed only to filtered air (0 ppm ozone) contained no mucous cells, whereas the NTE of rats exposed to 0.5 or 1.0 ppm ozone for 6 h/d, 5 d/wk for 20 mo was composed of 27% and 28% mucous cells, respectively. This ozone-induced MCM in rat nasal epithelium was similar in character to nasal epithelial changes previously reported in nonhuman primates repeatedly exposed to 0.15 or 0.30 ppm ozone for 6 h/d, 5 d/wk, for 6 d or 13 wk (10, 11).
To interpret properly the significance and long-term health implications of these nasal alterations, it is important to determine how long after the end of exposure these ozone-induced epithelial changes remain in the nasal airways. Chronic alterations of the normal structure of the nasal mucociliary apparatus may have adverse effects on the nasal defense mechanisms that protect the upper and lower respiratory tract tissues from potentially harmful levels of various inhaled xenobiotic agents (e.g., irritant gases, dusts, bacteria). Conversely, the ozone-induced differentiated epithelium may be more resistant to further toxicant injury because of the increased local production of an effective antioxidant, mucus (22). In either case, knowing the persistence of the lesion will help to determine the long-term consequences of ozone toxicity and to predict the response of the airways to subsequent exposures.
Previously reported studies, however, have not been designed to examine the persistence of ozone-induced nasal epithelial alterations after the end of ozone exposure. Therefore, the principal objective of the present study was to determine the persistence of ozone-induced MCM in the nasal epithelium after the end of a chronic exposure. In this study, we exposed male F344/N rats to 0, 0.25, or 0.5 ppm ozone for 8 h/d, 7 d/wk for 13 wk. Using histochemistry, image analysis, and morphometric techniques, we qualitatively and quantitatively characterized the ozone-related morphologic alterations of the nasal epithelium at 8 h, 4 wk, and 13 wk after the end of exposure. Some rats that were held in room air for 13 wk after the end of the chronic exposure were acutely exposed to 0.5 ppm ozone for 8 h to determine whether previous ozone exposure results in persistent changes in the sensitivity of rat nasal epithelium to ozone-induced injury.
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Materials and Methods |
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Animals and Ozone Exposures
Sixty-nine male F344/N Hsd rats (Harlan Sprague-Dawley, Indianapolis, IN), aged 10 to 14 wk at the beginning of chronic exposure, were used in this study. The rats were randomly assigned to one of three ozone-exposure groups (n = 23/group) on the basis of their body weight. The group assignments were adjusted to result in mean group body weights that were not significantly different from one another. The rats were individually housed in rack-mounted stainless steel wire cages in three whole-body inhalation exposure chambers (HC-2000; Lab Products, Maywood, NJ), with free access to food and water. The chamber temperature and relative humidity were maintained between 21°C and 25°C and 40% to 70%, respectively. Room lights were set on a 12-h light/dark cycle beginning at 4:00 A.M. Six nonexposed controls rats were housed in shoebox-style polycarbonate cages (two rats/cage) with chipped hardwood bedding under similar environmental conditions until they were killed.
The rats housed in whole-body exposure chambers were exposed to nominal ozone concentrations of 0 (filtered room air), 0.25, or 0.5 ppm, for 8 h/d, 7 d/wk for 13 wk (n = 23/group). The rats were exposed at night, when they were most active. Ozone was generated with two OREC Model OZONEV1-O ozonizers (Ozone Research and Equipment Corp., Phoenix, AZ), with compressed air used as a source of oxygen. The concentration of ozone within the chambers was monitored throughout the exposure with three Dasibi 1003 AH ambient-air ozone monitors (Dasibi Environmental Corp., Glendale, CA). The air-sampling probes were placed in the breathing zone of the rats. The chamber ozone concentration was automatically maintained through a computer-controlled closed-loop feedback system, which adjusted the amount of ozone delivered to the chamber through remotely controlled mass-flow valves. The chamber ozone concentrations were automatically logged every 10 min during the exposure periods. The mean chamber ozone concentrations (± SD) during the 13-wk exposure period were 0.24 ± 0.03 ppm (0.25 ppm) and 0.48 ± 0.06 ppm (0.5 ppm). After 13 wk of exposure to filtered air or ozone, the rats were removed from the whole-body inhalation chambers, placed in polycarbonate shoebox-style cages with hardwood bedding, and maintained under the conditions described previously until they were killed at 8 h (18 rats; n = 6/exposure group), 4 wk (18 rats; n = 6/exposure group), or 13 wk (33 rats; n = 11/exposure group) after exposure.
After 13 wk of recovery in filtered room air, a subgroup of rats that were previously exposed to 0 (air controls), 0.25, or 0.5 ppm ozone (n = 5 rats/exposure group) was returned to a whole-body exposure chamber and exposed for a single 8-h period to 0.5 ppm ozone.
Necropsy and Tissue Preparation
Rats exposed to 0, 0.25, or 0.5 ppm ozone for 13 wk were killed 8 h, 4 wk, or 13 wk after the last day of exposure. Rats reexposed to 0.5 ppm ozone were killed 18 h after the end of the 8-h exposure. Reexposed rats and nonexposed control rats were injected intraperitoneally with bromodeoxyuridine (BrdU; 50 mg/kg body weight) 2 h before being killed, in order to label cells undergoing DNA synthesis.
Rats were killed by exsanguination following deep anesthesia induced with 4% halothane in oxygen. Immediately after death, the head of each rat was removed from the carcass, and the eyes, lower jaw, skin, and musculature were removed. The nasal airways of rats designated for histologic analysis were flushed in a retrograde manner through the nasopharyngeal orifice with 10 ml of zinc- formalin (Anatech, Kalamazoo, MI), and the head was immersed in a large volume of the same fixative for at least 24 h.
After fixation, the heads were decalcified for 4 d with 13% formic acid and then rinsed in tap water for 4 h. After decalcification, the nasal airways were transversely sectioned at four specific anatomic locations, using the following gross dental and palatine landmarks previously described by Young (23): (1) immediately posterior to the upper incisor teeth (tissue block 1); (2) at the incisive papilla (tissue block 2); (3) at the second palatine ridge (tissue block 3); and (4) in the middle of the front upper molar tooth (tissue block 4) (Figure 1). The tissue blocks were embedded in paraffin and 5-µm-thick sections were cut from the anterior face of each block. Nasal tissue sections were histochemically stained with the Alcian blue (pH 2.5)/periodic acid-Schiff stain sequence (AB/PAS) to identify acidic (blue) and neutral (red) mucosubstances, or with AB (pH 2.5) plus hematoxylin and eosin (H&E) for histopathologic examination. Tissue sections were also immunohistochemically stained to detect BrdU-labeled cells within the nasal epithelium, as previously described by Johnson and colleagues (12).
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Morphometric Quantitation
The luminal epithelia covering the maxilloturbinates present in sections from tissue blocks 1 (T1, containing the proximal lateral meatus) and 2 (T2, containing the distal lateral meatus) were analyzed morphometrically (Figure 1). The medial and lateral surfaces of the maxilloturbinates in the T1 region were covered by a nonciliated cuboidal epithelium 1-2 cell layers thick with no mucous secretory cells (NTE). The maxilloturbinates in T2 were covered with a short-columnar, ciliated respiratory epithelium (RE) with serous (PAS-positive) secretory cells and few to no mucous (AB/PAS-positive) secretory cells. The length of epithelium evaluated in each region ranged from 2.3 mm to 5.1 mm in T1 and 0.8 mm to 1.3 mm in T2. Morphometric analyses were performed with a semiautomatic, computerized image analysis system consisting of a light microscope (Olympus BX-60; Olympus Corp., Lake Success, NY) connected to a high-resolution CCD camera (e.g., VE-1000; Dage-MTI, Inc., Michigan City, IN), a Scion LG-3 digital image-processing board (Scion Corp., Frederick, MD), a color monitor, and a Power Macintosh 7100/66 computer (Apple Inc., Cupertino, CA) running a public-domain image analysis program (NIH Image; written by Wayne Rasband at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/).
The volume density (Vs) of acidic and neutral (AB/ PAS-positive) intraepithelial mucosubstances (IM) within the surface epithelium overlying the maxilloturbinates in T1 and T2 was determined for each rat. The areas of AB/ PAS-positive IM were calculated from the automatically circumscribed perimeter of the stained material. The method used to estimate the amount of stored mucosubstance per unit area of epithelial basal lamina has been described in detail (11, 24). Data were expressed as the mean volume density (Vs, in nl/mm2 basal lamina) of AB/PAS-positive mucosubstances within the epithelium ± SEM.
The numeric density of the surface epithelial cells (i.e., epithelial cells/mm basal lamina) was determined by counting the total number of epithelial cell nuclear profiles present in the surface epithelium lining the maxilloturbinates in each tissue block, and dividing by the total length of the basal lamina underlying the epithelium (25). Data for each experimental group were expressed as the mean number of NTE cells/mm basal lamina ± SEM.
The short-term effects of the single 8-h exposure to 0.5 ppm ozone on rats that were chronically exposed 13 wk before this brief exposure were evaluated by estimating the Vs of AB/PAS-positive mucosubstances and DNA synthesis in NTE lining the maxilloturbinates of T1 and T2. Increased airway epithelial cell DNA synthesis is a sensitive indicator of ozone-induced cellular injury (26). We have previously shown that a single 8-h exposure to 0.5 ppm ozone induces NTE cell injury and a significant increase in NTE cell DNA synthesis from 12 to 24 h after the end of exposure (27). In the present study, NTE DNA synthesis (i.e., BrdU-labeling index) was determined by counting the number of BrdU-labeled nuclei per millimeter of basal lamina. Data for each experimental group were expressed as the mean number ± SEM.
Statistics
Data obtained from rats that were not reexposed were
evaluated for potential effects of ozone concentration during the 13 wk of exposure, and for the duration of postexposure recovery in the amounts of IM (Vs) and the NTE
cell numeric density, using a two-way analysis of variance
(ANOVA). A one-way ANOVA was used to test for the
effects of prior ozone exposure on the amount of IM (Vs)
and NTE cell DNA synthesis (BrdU-labeled cells/mm
basal lamina) in rats reexposed to 0.5 ppm ozone for 8 h.
Significant differences were evaluated with a post hoc comparison procedure (Tukey's test) to identify the source of
the variance. Statistical analyses were done with a commercial statistical analysis package (SigmaStat; Jandel Scientific Software, San Rafael, CA). The level of statistical
significance was set at P 
0.05.
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Results |
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End of Chronic Exposure
Histopathology. At the end of the 13-wk period of chronic exposure, rats exposed to 0.5 ppm ozone had marked histologic alterations that were restricted to the nasal tissues lining the lateral meatus in the proximal half of both nasal passages (T1 and T2; Figure 1). The most conspicuous of these bilateral upper-airway lesions were present in the surface epithelium and lamina propria of the nasal mucosa covering the maxilloturbinate, in the medial aspect of the nasoturbinate, and in the lateral wall (Figure 1). The bony tissue in the maxilloturbinates and in the lateral ridge and ventral scroll of the nasoturbinates was also affected by this chronic exposure to ozone. No exposure-related microscopic changes were present in the distal half of the nasal airways (tissue blocks 3 and 4) of these ozone-exposed rats.
The principal morphologic lesion in rats exposed to 0.5 ppm ozone was a marked, chronically active rhinitis. This inflammatory lesion in the affected nasal mucosa was characterized by a mixed inflammatory-cell infiltrate composed primarily of neutrophils with lesser numbers of mononuclear cells (e.g., lymphocytes, plasma cells, and monocytes) (Figure 2). This inflammatory-cell influx was most prominent in the interstitial compartment of the lamina propria, but infiltrating neutrophils were also present in the overlying surface epithelium.
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Stored IM. As compared with air-exposed controls (0 ppm ozone), rats exposed to 0.5 ppm ozone had 186 times more acidic and neutral mucosubstances stored in mucous secretory cells within the NTE lining the maxilloturbinates in T1 (Figure 7A), and 18 times more stored mucosubstances within the RE overlying the maxilloturbinates in T2 (Figure 7B). Thirteen weeks of exposure to 0.25 ppm ozone did not induce mucous-cell metaplasia, nor did it affect the amount of stored intraepithelial mucosubstances within the NTE or RE overlying maxilloturbinates in T1 (Figure 7A) and T2 (Figure 7B), respectively.
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Nasal epithelial cell numeric density. The effects of chronic ozone exposure on the numbers of epithelial cells within the surface epithelium lining the proximal and distal regions of the maxilloturbinates (T1 and T2, respectively) were determined by quantitating the number of epithelial cell nuclear profiles per millimeter of basal lamina. There were no significant differences in the numeric density of surface epithelial cells overlying the distal region of the maxilloturbinates (T2) in any experimental group.
Rats exposed to 0.25 ppm or 0.5 ppm for 13 wk had significantly more epithelial cells overlying their maxilloturbinates in T1 (10% and 58% increases, respectively; Figure 8) than did air-exposed controls. The increase in epithelial cell numeric density (e.g., epithelial cell hyperplasia) in rats exposed to 0.5 ppm ozone was associated with a concomitant increase in the number of AB/PAS-positive mucous secretory cells present in the surface epithelium (i.e., MCM). Exposure to 0.25 ppm ozone had no discernible effect on the number of AB/PAS-positive cells present in the surface epithelium.
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Four Weeks Postexposure
Histopathology. Four weeks after the end of ozone exposure, ozone-induced alterations in the nasal mucosa lining the proximal nasal passages were significantly reduced compared with those in the 0.5 ppm ozone-exposed rats that were killed 8 h after the end of the exposure (Figures 2 and 3). No exposure-related inflammatory-cell influx (i.e., rhinitis) remained in the nasal mucosa lining the lateral meatus in these animals. In addition, there were no differences in the luminal profiles of the blood vessels in the lamina propria among rats in the air- and ozone-exposed groups. Interestingly, the bony atrophy in the maxilloturbinates and nasoturbinates observed 8 h after the end of exposure to 0.5 ppm ozone was not present in the nasal airways of rats exposed to this same concentration of ozone and killed 4 wk postexposure (Figure 2).
Although inflammatory, vascular, and bony responses to ozone had completely resolved by 4 wk after the end of exposure, ozone-induced epithelial alterations were still present in the nasal mucosa lining the lateral meatus. A moderate MCM was present in the surface epithelium lining the proximal lateral meatus (i.e., NTE; T1) and the distal lateral meatus (i.e., RE; T2) in rats exposed to 0.5 ppm ozone and killed at this postexposure time (Figures 2-5). In addition, there was a mild to moderate epithelial cell hyperplasia in the NTE and RE lining the proximal and distal regions of the lateral meatus, respectively. No hyperplastic lesions were evident in the NTE or RE lining the lateral meatus in rats exposed to 0.25 ppm ozone and killed 4 wk after the end of the exposure.Amounts of IM. There was a decrease in the amount of stored mucosubstances in both the NTE and RE of rats exposed to 0.5 ppm ozone during the 13-wk postexposure period. Four weeks after the end of exposure, rats previously exposed to 0.5 ppm ozone had 35 times more IM within the NTE in T1 (70% less than at the end of exposure) and seven times more IM within the RE in T2 (48% less than at the end of exposure) than did rats previously exposed to air.
Nasal epithelial cell numeric density. Four weeks after the end of exposure, the NTE cell numeric densities of rats previously exposed to 0.25 ppm ozone had returned to control levels, and remained at control levels 13 wk after the end of exposure (Figure 8). The number of surface epithelial cells overlying maxilloturbinates of 0.5 ppm-exposed rats remained significantly greater than in air-exposed control rats at 4 wk (32% greater) and 13 wk (25% greater) after the end of exposure (Figure 8).
Thirteen Weeks Postexposure
Histopathology. Rats exposed to 0.5 ppm ozone and killed 13 weeks after exposure had a mild MCM that was restricted to the RE lining the distal lateral meatus (T2; Figures 4 and 5). Although there was no metaplastic change remaining in the NTE (proximal lateral meatus; T1) of these rats, a minimal to mild epithelial hyperplasia was present in this surface epithelium at 13 wk after the end of exposure. No ozone-related epithelial alterations (i.e., hyperplasia or metaplasia) were present in rats exposed to 0.25 ppm ozone and killed 13 wk after exposure. As in the exposed rats that were killed 4 wk after exposure, no rhinitis or bony atrophy was present in any of the ozone-exposed rats that were killed 13 wk after the end of exposure.
Amounts of IM. Thirteen weeks after the end of exposure, the Vs of mucosubstances in NTE (T1) of rats previously exposed to 0.5 ppm ozone was similar to that of air-exposed controls (96% less stored mucosubstances than in rats killed immediately after 13 wk exposure to 0.5 ppm ozone); however, the RE overlying the maxilloturbinates in T2 still had significantly more (8-fold) stored mucosubstances than in air-exposed controls (84% less stored mucosubstances than in rats killed immediately after 13 wk exposure to 0.5 ppm ozone).
Nasal epithelial cell numeric density. Thirteen weeks after the end of the chronic ozone exposure, the nasal epithelial cell numeric density in rats exposed to 0.25 ppm ozone remained at control levels. In contrast, the number of surface epithelial cells overlying maxilloturbinates of the 0.5 ppm-exposed rats remained significantly greater than in air-exposed control rats at 13 wk after the end of exposure (25% greater; Figure 8).
Effects of Chronic Exposure on Epithelial Response to Acute Reexposure
Thirteen weeks after the end of the chronic exposure, we reexposed a group of previously exposed rats to determine whether chronic exposure to 0, 0.25, or 0.5 ppm ozone results in long-lasting changes in the response of nasal airway epithelial cells to ozone exposure.
BrdU immunohistochemistry (DNA synthesis). Routine microscopic analysis of H&E-stained nasal tissues from rats that were briefly exposed (6 h) to 0.5 ppm ozone after the 13-wk postexposure period did not identify any epithelial alterations that could have been attributable to this brief ozone exposure. However, immunohistochemical analysis for cells undergoing DNA synthesis (i.e., BrdU-labeled nuclei) did reveal numerous BrdU-labeled nuclei in epithelial cells of the NTE lining the proximal lateral meatus in rats that had been chronically exposed to 0 (air controls) or 0.25 ppm ozone. As compared with unexposed control rats, there was a 17- and 26-fold increase in BrdU-labeled cells/mm basal lamina in the NTE of the reexposed rats that had been chronically exposed to 0 and 0.25 ppm ozone, respectively (Figure 9). Interestingly, no or only a few BrdU-labeled epithelial cells were present in the NTE of rats that had been chronically exposed to 0.5 ppm ozone 13 wk before this brief reexposure to ozone. The sparse number of BrdU-labeled epithelial cells in these ozone-exposed animals was similar to that observed in air-control rats that were not subjected to an acute exposure to ozone (Figure 9).
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Amounts of IM. Histochemical analysis for AB/PAS-stained mucosubstances revealed a mild increase in mucous cells with AB/PAS-stained mucosubstances in the NTE of rats that had been chronically exposed to 0.5 ppm and then reexposed to this same concentration of ozone 13 wk after the end of the chronic exposure (Figure 6). Reexposure to 0.5 ppm ozone induced a 2.5-fold increase in stored mucosubstances in the maxilloturbinates in T1 of rats previously exposed to 0.5 ppm ozone (Figure 10A), but did not increase the Vs of stored mucosubstances in T2 (Figure 10B).
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Discussion |
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The ozone-induced nasal lesions observed in the present study were similar to those recently described in rats exposed to 0.5 ppm ozone for 5 d/wk, 6 h/d for 20 mo (13, 28, 29). Although the character of the ozone-induced nasal lesions was similar in both studies, the severity and distribution of these lesions were more marked in the rats exposed for 20 mo to ozone than in the 13-wk-exposed rats described in the present report. For example, the MCM induced by the 20-mo exposure to 0.5 ppm ozone was not restricted to the proximal nasal airways, as was evident in the 13-wk-exposed animals in the present study, but also involved regions of the distal nasal airways. In addition, the 20-mo exposure induced much more loss of turbinate bone (i.e., bony atrophy) than was observed in the present study after the 13-wk exposure to 0.5 ppm ozone. However, the previous 20-mo-exposure study was not designed to examine the persistence of ozone-induced nasal lesions. The present report is to our knowledge the first to describe the persistence of nasal lesions in rats after long-term ozone exposure.
The results of this study indicate that ozone-induced morphologic alterations in the mucous apparatus of rat nasal airways (e.g., MCM of nasal epithelium) persist for weeks and even months after the end of a chronic exposure. Increased numbers of epithelial cells (i.e., epithelial hyperplasia) consituted another ozone-induced change that remained in certain sites of the nasal airways of exposed rats long after the end of the chronic exposure to 0.5 ppm ozone. This persistence of epithelial lesions was in contrast to the other, more transient ozone-induced lesions in the nose (i.e., inflammation and atrophy of turbinate bone), which had resolved by 4 wk after exposure. The observed persistence of ozone-induced airway epithelial alterations in the nose are not unlike the long-lasting lung lesions previously described in other laboratory animals exposed to ozone. Although markedly less severe than the initial lesions observed after the end of the long-term exposure, ozone-induced hyperplastic lesions in the epithelium lining the centriacinar regions of the lungs in rats and monkeys have also been reported to be evident several months after the end of the exposure (30, 31).
Increases in epithelial mucous cells (i.e., MCM or mucous-cell hyperplasia) have been observed in the pulmonary airways of laboratory rodents exposed to a variety of inhaled agents, including sulfur dioxide (32, 33), cigarette smoke (34, 35), endotoxin (36), and human neutrophil elastase (41, 42). Like the ozone-induced MCM observed in the rat nasal epithelium, MCMs in the pulmonary airways of rodents exposed to cigarette smoke (34, 35, 43) and elastase (44) are remarkably persistent airway epithelial alterations that are histologically evident for several weeks or even months after the end of the exposure (44, 46).
Important differences in the intranasal location and rate of resolution of ozone-induced epithelial hyperplasia (increase in total epithelial cells) and ozone-induced MCM in the nasal passages of ozone-exposed rats were also observed in the present study. Unlike the MCM in the nasal epithelia lining the proximal and distal lateral meatus of 0.5 ppm-exposed rats, the hyperplastic changes induced by exposure to both 0.25 and 0.5 ppm ozone were restricted to the NTE in the proximal lateral meatus. To our knowledge, the present study is the first to demonstrate that chronic exposure to a concentration of ozone below 0.5 ppm (i.e., 0.25 ppm) can cause a nasal alteration in the rat. Henderson and colleagues (47) have reported that an acute exposure to this concentration of ozone induced an increase in cell proliferation in the NTE of F344 rats. The hyperplastic lesion induced by 0.25 ppm ozone in our present study, however, was considerably less severe than that induced by chronic exposure to 0.5 ppm ozone. Persistence of epithelial hyperplasia was evident only 4 wk after exposure, and was seen only in rats exposed to 0.5 ppm ozone. This suggests that cellular mechanisms of repair may be different for epithelial hyperplasia and the more long-lasting MCM. Our findings also indicate that the concentration of ozone needed to cause an increase in nasal epithelial cells (i.e., hyperplasia) is considerably less than that needed to induce MCM.
We have shown that epithelial cell proliferation and hyperplasia precedes MCM after short-term exposure to ozone (12, 27, 47). The dependence of ozone-induced MCM on the preceding epithelial cell proliferation is unknown. It is possible that the mechanisms of ozone-induced hyperplasia and MCM are unrelated. In our study, chronic exposure to ozone induced a persistent hyperplastic lesion in NTE (T1), but not in RE (T2). In contrast, ozone-induced MCM was present in both NTE and RE, but persisted for 13 wk only in RE.
The results of our study suggest that the mucous cells in the ozone-induced metaplastic lesion are extremely long lived and continue to overexpress IM for long periods after exposure. Interestingly, both we and Dr. Johannes Tesfaigzi have recently observed that many of the mucous cells composing this ozone-induced metaplastic lesion in the nasal epithelium lining the lateral meatus in the rat contain unusually high amounts of an immunohistochemically detectable, antiapoptotic cell protein, Bcl-2 (48). In contrast, Bcl-2 protein was rarely detected in normal mucous cells lining the nasal septum of air-exposed or even ozone-exposed rats. It has been shown that Bcl-2 protein does not stimulate proliferation, but rather confers survival upon cells by blocking apoptotic cell death (49). Therefore, Bcl-2 protein may have an important role in the long survival of the metaplastic mucous cells in airway epithelium altered by ozone exposure. Even though the mucous cells in the metaplastic epithelium have a similar histologic appearance to mucous cells in the unaffected respiratory epithelium lining the nasal septum, these two mucous-cell populations appear to be distinct on the basis of their intracellular content of Bcl-2 protein.
In the present study we also found that rats chronically exposed to 0.5 ppm ozone and then reexposed to the same concentration of ozone 13 wk after the end of the chronic exposure had an acute increase in stored mucosubstances in the NTE. This finding suggests that the previously exposed epithelium was altered in such a way by the chronic exposure to ozone as to be able to respond quickly to subsequent acute exposure to this irritating air pollutant by making more mucus. Because airway mucus has been shown to be an effective antioxidant (22), this may be a long-term adaptive mechanism of the animal to protect its airways from further ozone-induced injury. Long-term adaptation of the nasal epithelium to ozone exposure was also demonstrated by the lack of an ozone-induced increase in the BrdU-labeling index, an indicator of reparative cell proliferation in response to ozone-induced cell death and exfoliation (26, 27). We have previously reported (27) that a naive nasal epithelium, not previously exposed to 0.5 ppm ozone, will undergo marked epithelial cell loss (17% loss of NTE cells from 2-4 h after exposure) that is quickly followed by a transient increase in DNA synthesis at 12 to 36 h after the end of the exposure (a 45-fold increase in the number of BrdU-labeled cells at 20 h postexposure). In our present study, the lack of an increase in DNA synthesis after acute exposure to 0.5 ppm ozone in animals chronically exposed to ozone 13 wk before the reexposure was another indicator of the long-lasting effects of this air pollutant on the surface epithelium lining the nasal airways.
Another important observation in this study was the difference in epithelial response and repair of phenotypically distinct nasal epithelial cell populations after chronic ozone exposure. The nonciliated NTE lining the proximal lateral meatus (T1) was the most severely altered epithelium, with marked MCM and epithelial cell hyperplasia immediately after the end of the exposure. Ozone-induced MCM, but no epithelial hyperplasia, was evident in the ciliated RE in the distal lateral meatus (T2). In sharp contrast to the responses of these epithelia, the RE lining the adjacent nasal septum in these same proximal regions of the nasal airways had no microscopically detectable changes after the chronic ozone exposure. These differences in response to ozone exposure may be due to regional dosimetry, local tissue susceptibility, or a combination of these factors. On the basis of studies of nasal airflow, computational fluid dynamics, and regional dosimetry of inhaled 18O-ozone, it has been concluded that both regional dosimetry and site-specific tissue factors, including surface secretions, influence regional nasal toxicity of ozone in the rat (50, 51). Airflow through the lateral meatus has been estimated to be extremely high (52). Therefore, on the basis strictly of airflow dynamics within the nose, it is not surprising that the NTE lining this surface would be markedly altered by ozone. In contrast, the RE lining the nasal septum in the middle meatus (another airway with one of the highest predicted airflows in the nasal passage of the rat) was not altered by 13-wk of exposure to 0.5 ppm ozone. This resistance to ozone may be due in part to the copious amounts of mucosubstances normally stored in this septal epithelium or to the amount of mucus overlying this RE.
Not only was there a difference in the response of distinct nasal epithelial cell populations to ozone, but there were also intriguing differences in the rate of epithelial repair between the NTE lining the proximal lateral meatus and the RE lining the distal lateral meatus. Though MCM was more severe in the NTE (i.e., more mucous cells and intraepithelially stored mucosubstances) than in the RE, the MCM persisted longer in the RE lining the distal lateral meatus. The reason(s) for this distinct difference in the rate of persistence of MCM between these two nasal epithelia is not known, but the difference suggests that there may be important cellular differences in the mechanisms of epithelial repair.
The mechanism of development and persistence of ozone-induced MCM is not clear. The role of epithelial cell proliferation in the pathogenesis of MCM has not yet been adequately investigated. It is still unknown whether or not the mucous cells in the metaplastic nasal epithelium are derived from proliferating stem cells or nondividing, preexisting cells that become filled with mucous granules after repeated ozone exposure.
MCM has been reported to be present in the airways of ex-cigarette smokers at two or more years after they have stopped their smoking habit (53). The persistent effects of ozone on the mucous cells in the airways of humans chronically exposed to photochemical smog containing high concentrations of ozone are unknown. The persistent nature of the ozone-induced MCM in rats documented in this report suggests that ozone may have the potential to induce similar long-lasting alterations in the airways of humans. The short- and long-term consequences of such an airway alteration to human health are yet to be determined.
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Footnotes |
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Address correspondence to: Dr. Jack R. Harkema, Department of Pathology, National Food Safety and Toxicology Center, 212 Food Safety and Toxicology Building, Michigan State University, East Lansing, MI 48824. E-mail: harkemaj{at}pilot.msu.edu
(Received in original form October 28, 1997 and in revised form May 20, 1998).
Abbreviations: Alcian blue/periodic acid-Schiff (stain), AB/PAS; bromodeoxyuridine, BrdU; hematoxylin and eosin, H&E; intraepithelial mucosubstances, IM; mucous-cell metaplasia, MCM; nasal transitional epithelium, NTE; respiratory epithelium, RE; volume density, Vs.Acknowledgments: The authors thank Ms. S. Barnett and Ms. Y. Knighton at the Lovelace Respiratory Research Institute, and Ms. J. Miller and Ms. A. Porter at Michigan State University, for their technical assistance in preparing the paraffin slides for histopathology. The authors are also grateful to Ms. Paula Bradley at the Lovelace Respiratory Research Institute for her editorial assistance in preparing this manuscript, and to Dr. Michelle Fannuchi for her helpful scientific comments and suggestions during the preparation of this paper. This research was supported by grant 5RO1HL1712 from the National Heart, Lung and Blood Institute, and was conducted in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care.
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