Assessment of the respiratory sensitization potential of proteins using an enhanced mouse intranasal test (MINT).

There remains a need for a simple and predictive animal model to identify potential respiratory sensitizers. The mouse intranasal test (MINT) was developed to assess the relative allergic potential of detergent enzymes, however, the experimental endpoints were limited to evaluation of antibody levels. The present study was designed to evaluate additional endpoints (serum and allergic antibody levels, pulmonary inflammation and airway hyperresponsiveness (AHR)) to determine their value in improving the predictive accuracy of the MINT. BDF1 mice were intranasally instilled on days 1, 3, 10, 17 and 24 with subtilisin, ovalbumin, betalactoglobulin, mouse serum albumin or keyhole limpet hemocyanin; challenged with aerosolized methacholine or the sensitizing protein on day 29 to assess AHR, and sacrificed on day 29 or 30. Under the conditions of this study, evaluation of AHR did not improve the predictive power of this experimental model. Allergic antibody responses and IgG isotype characterization proved to be the most sensitive and reliable indicators of the protein allergenic potential with BAL responses providing additional insight. These data highlight that the evaluation of the respiratory sensitization potential of proteins can be best informed when multiple parameters are evaluated and that further improvements and refinements of the assay are necessary.

Acute toxicologic and neurotoxic effects of inhaled 1,2-dichloroethane in adult Fischer 344 rats.

Acute toxicologic and neurotoxic effects were evaluated in Fischer 344 rats exposed to 0, 50, 200, 600, or 2000 ppm 1,2-dichloroethane (ethylene dichloride; EDC) for 4 h or 0, 50, 100 or 150 ppm for 8 h. Neurobehavioral and neuropathologic effects were assessed using a functional observational battery (FOB; baseline, days 1, 8, and 15), and by light microscopy, respectively. Acute toxicologic effects were assessed by bronchoalveolar lavage (BAL) and histopathology of the respiratory tract and selected target organs. Neurobehavioral effects consistent with central nervous system (CNS) depression were present at concentrations >200 ppm and were restricted to day 1. There were no neuropathologic changes in the CNS, however, olfactory epithelial regeneration 15 days after exposure to > or = 200 ppm was observed. The no-observed-effect concentration (NOEC) for behavioral neurotoxicity was 200 ppm EDC for 4 h. There were no effects on BAL parameters in any exposure group. Exposure to 2000 ppm EDC altered adrenal gland, kidney, and liver weights, and resulted in morphologic alterations in the kidney and liver. Degeneration/necrosis of the olfactory epithelium was observed at > or = 200 ppm for 4 h and > or = 100 ppm for 8 h. Based on olfactory epithelial degeneration/necrosis, the most sensitive indicator of toxicity in this study, the overall NOEC was 50 ppm EDC for up to 8 h in rats.

Mucin genes in horse airways: MUC5AC, but not MUC2, may play a role in recurrent airway obstruction.

REASONS FOR PERFORMING STUDY: Increased mucin gene expression may be an important cause of mucus accumulation observed in recurrent airway obstruction (RAO)-affected horses. To date, however, no mucin gene sequences are available for the horse. OBJECTIVES: To identify equine homologues of gel-forming mucins and investigate their expression at different airway generations of healthy and RAO-affected horses. METHODS: Two equine homologues were identified by cloning and sequencing fragments of equine (eq)MUC5AC and eqMUC2. RESULTS: Semiquantitative RT-PCR on RNA from airways (generations 1, 5, 10, 15; small airways and parenchyma), stomach (glandular), and colon revealed that eqMUC5AC is expressed in equine stomach and in all of the airway samples. In contrast, eqMUC2 steady-state mRNA levels were detected in colon and very faintly in stomach, but not in airway tissue. EqMUC5AC expression was also compared to that of ZO-1, a tight junction protein, and eqMUC5AC/ZO-1 ratios were higher in RAO-affected compared to control horses at all airway generations. CONCLUSIONS: That eqMUC5AC is expressed in horse airways, but any expression of MUC2 is undetectable and unlikely to be of physiological consequence. POTENTIAL RELEVANCE: EqMUC5AC up-regulation may be a primary mechanism responsible for mucus hypersecretion and accumulation in RAO.

Effects of ozone and endotoxin coexposure on rat airway epithelium: potentiation of toxicant-induced alterations.

Tropospheric ozone is the major oxidizing component in photochemical smog and is one of the most pervasive problems to human health of the criteria air pollutants for which the National Ambient Air Quality Standards have been designated by the Clean Air Act. Although many adverse health effects of ozone exposure have been documented in both humans and laboratory animals, controversy surrounds the establishment and implementation of ozone standards set forth by the U.S. Environmental Protection Agency. Because people are commonly exposed to more than one air pollutant at a time, studies that examine coexposures to airborne materials may be more relevant for assessing their risks to human health. Airborne biogenic substances such as pollens, spores, and bacterial products are ubiquitous in the environment, and when inhaled can cause adverse respiratory symptoms. One such biogenic agent, bacterial endotoxin, is a potent stimulus of airway inflammation and is a ubiquitous airborne contaminant commonly found in domestic, agricultural, and industrial settings. Little is known about the interaction of exposures to biogenic substances and criteria air pollutants such as ozone. In the last few years we have performed a series of studies in rodents that examined the biologic responses of the respiratory epithelium after airway exposures to both endotoxin and ozone. When exposed to ozone (0.5 ppm 8 hr/day for 3 days), Fischer rats develop lesions in the nasal transitional epithelium, whereas intranasal instillation of endotoxin (20 microg) elicits epithelial lesions in the respiratory epithelium of the nose and conducting airways. Our studies were designed to examine how exposure to one toxicant may affect the airway epithelial lesions induced by the other toxicant. We investigated the potential role of acute inflammation in the enhancement of airway epithelial lesions after exposure of these two toxicants in neutrophil-sufficient and neutrophil-deficient rodents. A summary of these results indicates that epithelial and inflammatory responses to coexposure of these two pollutants are greater than those elicited by either agent alone. Interestingly, each toxicant enhances the epithelial alterations induced by the other. Furthermore, the synergistic effects elicited by coexposure to ozone and endotoxin are mediated partly by neutrophils. These studies provided some new insights into how inhaled co-pollutants interact to initiate and promote alterations of airway epithelium. Further studies with these and other air pollutants will help define their true risk to human health.

Persistent mucin glycoprotein alterations in equine recurrent airway obstruction.

Horses with the episodic asthmalike condition of recurrent airway obstruction (RAO) have bouts of inflammation and bronchoconstriction associated with indoor housing. To assess the potential differences in airway secretions between RAO-affected and control horses, methods to quantify mucus secretions were developed and applied to bronchoalveolar lavage fluid. The relative difference in the amount of mucin glycoproteins between control and RAO-affected horses was assessed with a carbohydrate side chain-specific monoclonal antibody (4E4) in an enzyme-linked immunosorbent assay and by carbohydrate-specific enzyme-linked lectin assays. Significantly increased levels of 4E4-immunoreactive glycoprotein and the mucin-associated carbohydrates fucose (alpha-1,2 linkage) and N-acetylglucosamine were detected in RAO-affected horses in acute disease. RAO-affected horses in remission maintained significantly elevated levels of alpha-1,2-fucose and N-acetylglucosamine, whereas the 4E4-immunoreactive glycoprotein levels displayed a trend toward an increase over control levels. These results indicated that persistent changes in the quantity and/or quality of mucus glycoproteins occurred in the RAO-affected horses.

Endotoxin enhancement of ozone-induced mucous cell metaplasia is neutrophil-dependent in rat nasal epithelium.

Ozone, the primary oxidant gas in photochemical smog, causes neutrophilic inflammation and mucous cell metaplasia (MCM) in the nasal transitional epithelium (NTE) of rats and monkeys. Bacterial endotoxin is another common airborne agent that induces acute neutrophilic inflammation, but not MCM, in NTE. It does, however, enhance ozone-induced MCM in rat nasal airways (Fanucchi et al., 1998, Toxicol. Appl. Pharmacol. 152, 1-9). In the present study, F344 rats exposed to filtered air or 0.5 ppm ozone (8 h/day for 3 days) were intranasally instilled with sterile saline or 100 microg endotoxin 24 h and 48 h after the third ozone exposure. To determine the role of neutrophilic inflammation in endotoxin-induced potentiation of the MCM caused by ozone, half of the rats were depleted of circulating neutrophils prior to saline or endotoxin instillations. Rats were killed 6 h or 3 days after the last intranasal instillation, and nasal tissues were processed for (1) light microscopy and morphometric analysis to determine the number of infiltrating neutrophils and the volume amount (density) of stored mucosubstances in the NTE, and (2) quantitative RT-PCR analysis of steady-state mucin gene (rMuc-5AC) mRNA levels in the NTE. Endotoxin induced a transient influx of neutrophils in both air- and ozone-exposed rats that was completely blocked by neutrophil depletion. Endotoxin increased rMuc-5AC mRNA levels in the NTE of ozone-exposed rats. Neutrophil depletion, however, had no effect on endotoxin-induced upregulation of mucin gene mRNA levels. Endotoxin enhanced the ozone-induced increase in stored mucosubstances (4-fold increase), but only in neutrophil-sufficient rats. These data indicate that endotoxin enhancement of ozone-induced upregulation of rMuc-5AC mRNA levels is neutrophil-independent, while its effects on intraepithelial production and storage of mucus glycoproteins is dependent on the presence of neutrophils.

Mild and moderate asthma is associated with airway goblet cell hyperplasia and abnormalities in mucin gene expression.

Excessive airway mucus is an important cause of morbidity and mortality in asthma, but the relationship between accumulation of mucus and goblet cell size, number, and function is incompletely understood. To address these questions, stored mucin in the epithelium and goblet cell size and number were measured morphometrically, and mucin gene expression was measured by polymerase chain reaction and immunohistochemistry in endobronchial biopsies from 13 subjects with mild and moderate asthma and from 12 healthy control subjects. Secreted mucin was measured in induced sputum. We found that stored mucin in the airway epithelium was three times higher than normal in the subjects with asthma (p < 0.005). Goblet cell size was similar in both groups, but goblet cell number was significantly higher in the subjects with asthma (93,043 +/- 15,824 versus 41,959 +/- 9,230/mm3, p < 0.05). In mild asthma (FEV1 > or = 80% pred, n = 7), the level of stored mucin was as high as in moderate asthma (FEV1 < 80% pred, n = 6), but the level of secreted mucin was significantly lower (28.4 +/- 6.3 versus 73.5 +/- 47.5 microg/ml, p < 0.05). Secreted mucin was inversely correlated with stored mucin for the whole asthma group (rs = -0.78, p = 0.007). MUC5AC was the predominant mucin gene expressed in healthy subjects and subjects with asthma, and MUC5AC protein was increased in the subjects with asthma. We conclude that even mild asthma is associated with goblet cell hyperplasia and increased stored mucin in the airway epithelium, whereas moderate asthma is associated with increased stored mucin and secreted mucin. These findings suggest that acute degranulation of hyperplastic goblet cells may represent a mechanism for asthma exacerbations in mild and moderate asthma and that chronic degranulation of goblet cells may contribute to chronic airway narrowing in moderate asthma.

Neutrophil-dependent and neutrophil-independent alterations in the nasal epithelium of ozone-exposed rats.

Ozone induces epithelial hyperplasia and mucous cell metaplasia (MCM) in nasal transitional epithelium (NTE) of rats. A transient neutrophil influx accompanies upregulation of mucin messenger RNA (mRNA) before the onset of MCM. The present study was designed to examine the role of neutrophils in ozone-induced epithelial changes in the NTE of rats. Fourteen hours before inhalation exposure, male F344/N rats were injected intraperitoneally with antirat neutrophil antiserum to deplete circulating neutrophils, or were injected with normal (control) serum. Rats were then exposed to 0 ppm (filtered air) or 0.5 ppm ozone (8 h/d) for 1 or 3 d. Maxilloturbinates lined with NTE were analyzed to determine the epithelial labeling index; numeric densities of neutrophils, total epithelial cells, and mucous secretory cells; amount of stored intraepithelial mucosubstances; and steady-state ratMUC-5AC (mucin) mRNA levels. At 2 h after 3 d of exposure, rats treated with antiserum had 90% fewer circulating neutrophils than did rats treated with control serum. Antiserum-treated, ozone-exposed rats had 87% fewer infiltrating neutrophils than did control serum-treated, ozone-exposed rats. At 4 d after 3 d of exposure, antiserum-treated, ozone-exposed rats had 66% less stored intraepithelial mucosubstances and 58% fewer mucous cells in their NTE than did control serum-treated, ozone-exposed rats. Antiserum treatment had no effects on ozone-induced epithelial cell proliferation or mucin mRNA upregulation. The results of this study indicated that ozone-induced MCM was neutrophil-dependent, whereas ozone-induced epithelial cell proliferation and mucin gene upregulation were neutrophil-independent.

Synergistic hepatotoxicity from coexposure to bacterial endotoxin and the pyrrolizidine alkaloid monocrotaline.

Individuals are commonly exposed to bacterial endotoxin (lipopolysaccharide [LPS]) through gram-negative bacterial infection and from its translocation from the gastrointestinal lumen into the circulation. Inasmuch as noninjurious doses of LPS augment the hepatotoxicity of certain xenobiotic agents, exposure to small amounts of LPS may be an important determinant of susceptibility to chemical intoxication. Monocrotaline (MCT) is a pyrrolizidine alkaloid phytotoxin that at large doses produces centrilobular liver lesions in rats. In the present study, MCT was coadministered with LPS to determine whether LPS would enhance its hepatotoxicity. Doses of MCT (100 mg/kg, ip) and LPS (7.4 x 10(6) EU/kg, iv), which were nonhepatotoxic when administered separately, produced significant liver injury in male, Sprague-Dawley rats when given in combination. Within 18 h after MCT administration, this cotreatment resulted in enhanced plasma alanine aminotransferase and aspartate aminotransferase activities, two markers of liver injury. Histologically, overt hemorrhage and necrosis appeared between 12 and 18 h. The lesions were centrilobular and midzonal and exhibited characteristics similar to lesions associated with larger doses of MCT and LPS, respectively. In the presence of LPS, the threshold for MCT toxicity was reduced to 13-33% of the dose required for toxicity with MCT alone. A study in isolated, hepatic parenchymal cells revealed no interaction between MCT and LPS in producing cytotoxicity. In summary, coexposure of rats to noninjurious doses of MCT and LPS resulted in pronounced liver injury. Results in vitro suggest that the enhanced toxicity does not result from a direct interaction of MCT and LPS with hepatic parenchymal cells. These results provide additional evidence that exposure to small amounts of LPS may be a determinant of susceptibility to food-borne hepatotoxins.

Lipopolysaccharide and the trichothecene vomitoxin (deoxynivalenol) synergistically induce apoptosis in murine lymphoid organs.

Human exposure to Gram-negative bacterial lipopolysaccharide (LPS) is common and may have an important influence on chemical toxicity. LPS has been shown previously to enhance synergistically the toxicity of trichothecene mycotoxins. Because either of these toxin groups alone characteristically target lymphoid organs at high doses, we evaluated the effects of coexposure to subthreshold doses of Salmonella typhimurium LPS and vomitoxin (VT) administered by intraperitoneal injection and oral gavage of B6C3F1 mice, respectively, on apoptosis in lymphoid tissues after 12-h exposure. The capacity of LPS (0.5 mg/kg body weight) and VT (25 mg/kg body weight) to act synergistically in causing apoptosis in thymus, spleen, and Peyer's patches was suggested by increased internucleosomal DNA fragmentation in whole cell lysates as determined by gel electrophoresis. Following terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-dUTP nick end-labeling (TUNEL) of tissue sections, a dramatic enhancement of fluorescence intensity indicative of apoptosis was observed in thymus, spleen, Peyer's patches, and bone marrow from coexposed animals as compared to those given the agents alone. Evaluation of hematoxylin and eosin-stained tissue sections of treatment mice revealed the characteristic features of lymphocyte apoptosis, including marked condensation of nuclear chromatin, fragmentation of nuclei, and formation of apoptotic bodies in tissues from mice. Combined treatment with VT (25 mg/kg body weight) and LPS (0.5 mg/kg body weight) significantly increased (p<0.05) the amount of apoptotic thymic and splenic tissue as compared to the expected additive responses of mice receiving either toxin alone. When apoptosis was examined in cell suspensions of thymus, spleen, Peyer's patches, and bone marrow by flow cytometry in conjunction with propidium iodide staining, the percentage of apoptotic cells was significantly increased (p<0.05) in cotreatment groups as compared to the additive responses to LPS and VT given alone. The results provide qualitative and quantitative evidence for the hypothesis that LPS exposure markedly amplifies the toxicity of trichothecenes and that the immune system is a primary target for these interactive effects.

Inflammatory and epithelial responses during the development of ozone-induced mucous cell metaplasia in the nasal epithelium of rats.

Rats repeatedly exposed to high ambient concentrations of ozone develop mucous cell metaplasia (MCM) in the nasal transitional epithelium (NTE). The present study was designed to determine the temporal relationships of ozone-induced inflammatory and epithelial responses and their correlation with subsequent MCM in the NTE of rats. Male F344/N rats were exposed to 0.5 ppm ozone, 8 h/day for 1, 2, or 3 days. Two h prior to sacrifice, all the rats were injected intraperitoneally with 5'-bromo-2-deoxyuridine (BrdU) to label epithelial cells undergoing DNA synthesis. Rats exposed to ozone for 1 or 2 days were killed 2 h after the exposure. Rats exposed to ozone for 3 days were killed 2 h or 1, 2, or 4 days after the exposure. Control rats were killed after a 7-day exposure to filtered air. One nasal passage from the anterior nasal cavity of each rat was fixed and processed for light microscopy to morphometrically determine the numeric densities of epithelial cells, neutrophils, and mucous cells, and the amount of intraepithelial mucosubstances in the NTE. The maxilloturbinate from the other nasal passage was processed for analysis of an airway mucin-specific gene (i.e., rMuc-5AC mRNA). Acute ozone exposure induced a rapid increase in rMuc-5AC mRNA levels prior to the onset of MCM, and the increased levels of rMuc-5AC mRNA persisted with MCM. Neutrophilic inflammation coincided with epithelial DNA synthesis and upregulation of rMuc-5AC, but was resolved when MCM first appeared in the NTE. The results of the present study suggest that upregulation of mucin mRNA by acute ozone exposure may be associated with the concurrent neutrophilic inflammation and epithelial hyperplasia in the NTE. Ozone-induced MCM may be dependent on these important pre-metaplastic responses (i.e., mucin mRNA upregulation, neutrophilic inflammation, and epithelial proliferation).

Effects of pre-existing rhinitis on ozone-induced mucous cell metaplasia in rat nasal epithelium.

Ozone causes rhinitis and nasal epithelial alterations. The toxicity of ozone on nasal airways with pre-existing rhinitis has not been investigated. The present study was designed to determine the effect of endotoxin-induced rhinitis on ozone-induced epithelial alterations, especially mucous cell metaplasia (MCM), in the nasal transitional epithelium (NTE) of rats. Six h prior to daily inhalation exposure, male F344/N rats were intranasally instilled with saline or endotoxin (100 microgram/day). Rats were killed 2 h or 4 days after 3-day (8 h/day) exposure to ozone (0.5 ppm) or filtered air (0 ppm). The maxilloturbinate from one nasal passage was processed for morphometric analyses of the numbers of neutrophils and epithelial cells and the amount of intraepithelial mucosubstances (IM) in the NTE. The maxilloturbinate from the other nasal passage was processed for a mucin-specific (rMuc-5AC) mRNA analysis. At 2 h postexposure, endotoxin/ozone-exposed rats had 48 and 3 times more neutrophils in the NTE than did saline/air- and saline/ozone-exposed rats, respectively. Ozone-exposed rats had 35% more NTE cells and 2-fold more mucin mRNA than did saline/air-exposed rats, independent of endotoxin exposure. At 4 days postexposure, endotoxin/ozone-exposed rats had 5 and 2 times more IM and mucous cells, respectively, than did saline/air- and saline/ozone-exposed rats. Though endotoxin/air-exposed rats killed at 2 h postexposure had more neutrophils (40-fold), epithelial cells (27%) and mucin mRNA (2-fold) in the NTE than did saline/air-exposed rats, no MCM was present in those rats killed at 4 days postexposure. The results of the present study indicated that pre-existing rhinitis augments ozone-induced MCM.

In vitro culture of microdissected rat nasal airway tissues.

The surface epithelium lining the nasal airways is a potential target for inhaled contaminants such as ozone, endotoxin, formaldehyde, tobacco smoke, and organic dusts. The epithelial response to injury may depend on the toxicant, the type of epithelium, the severity of the injury, and the presence of inflammatory cells and their secreted products. To study mechanisms of toxicant-induced epithelial injury and repair, in the absence of cellular inflammation or other systemic effects, we have developed a culture system to maintain morphologically distinct nasal airway epithelium in vitro. Microdissected maxilloturbinates and proximal nasal septa of male F344/N rats were cultured at an air-liquid interface for up to 14 d in supplemented serum-free medium. Maxilloturbinates are lined by nonciliated cuboidal nasal transitional epithelium (NTE) with few or no mucous cells. The proximal nasal septum is lined by a mucociliary respiratory epithelium (RE) that normally contains numerous mucous cells. Preservation of the normal RE and NTE phenotype in culture was assessed by light and electron microscopy, and analysis of an airway mucin gene (rMuc-5AC) messenger RNA (mRNA). Both RE and NTE retained normal cell morphology for 14 d in culture (DIC). After 14 DIC there were 20% fewer RE cells in the septa (equal loss of ciliated and mucous cells) and 25% more NTE cells in the maxilloturbinates (increased number of basal cells). Compared with the RE, the NTE expressed consistently low levels of rMuc-5AC mRNA and had little to no histochemically detectable intraepithelial mucosubstances (IM) after 0, 3, 7, or 14 DIC. The amount of stored IM and the steady-state levels of rMuc-5AC mRNA in the RE decreased with time in culture. In summary, this culture system can maintain fully differentiated secretory and nonsecretory rat airway epithelia in vitro for up to 14 d. This study was an essential first step in developing a system to study the pathogenesis of toxicant-induced airway epithelial injury and mechanisms of cellular repair and adaptation in the absence of cellular inflammation and other systemic influences.

Long-lasting effects of chronic ozone exposure on rat nasal epithelium.

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.

Endotoxin potentiates ozone-induced mucous cell metaplasia in rat nasal epithelium.

People are exposed to a combination of environmental pollutants throughout their lives. Repeated exposures of one common pollutant, ozone, have been reported to cause the development of mucous cell metaplasia in the nasal transitional epithelium (NTE) of rats. The present study was designed to test the hypothesis that exposure to bacterial endotoxin, another toxicant ubiquitous to the environment, potentiates this metaplastic response in rat NTE. Rats were exposed to 0 or 0.5 ppm ozone 8 h/day for 3 days. After ozone exposure, rats were intranasally instilled with saline containing 0 or 100 micrograms endotoxin once daily for 2 days. Rats were killed 6 h or 3 days after the last intranasal instillation. Nasal tissue was processed for light microscopy and image analysis, or for isolation of total RNA. Mucous cell metaplasia was not detected in air/endotoxin-exposed rats, was observed in ozone/saline-exposed rats, and was most severe in ozone/endotoxin-exposed rats. At 6 h after instillation, amounts of intraepithelial mucosubstances (IM) were 4-fold greater in NTE of ozone/endotoxin-exposed rats as compared to controls. These IM levels were similar to those of ozone/saline-exposed rats. Mucin-specific mRNA (rMuc-5AC) levels were elevated in all treatment groups at this timepoint. At 3 days after instillation, amounts of IM in ozone/endotoxin-exposed rats were 10-fold greater than in controls and 5-fold greater than in ozone/saline-exposed rats. rMuc-5AC mRNA levels remained elevated in the ozone/endotoxin-exposed rats. Despite the fact that bacterial endotoxin alone does not cause a phenotypic change in rat NTE, it can augment the mucous cell metaplasia induced by a previous exposure to ozone.

Expression of the Bcl-2 protein in nasal epithelia of F344/N rats during mucous cell metaplasia and remodeling.

Exposure to ozone induces mucous cell metaplasia in rat airway epithelia. During the regeneration process, apoptotic mechanisms may be responsible for eliminating metaplastic cells. Therefore, the present study investigated expression of Bcl-2, a regulator of apoptosis, in ozone-induced mucous cell metaplasias. Adjacent metaplastic mucous cells in nasal airway epithelia that were exposed to ozone were heterogeneous in their expression of Bcl-2; some cells expressed high levels, whereas others expressed low levels or no Bcl-2. On Western blot analysis, Bcl-2 was detected in protein extracts from nasal epithelia of rats exposed to 0.5 ppm ozone for 1 mo but not in control rats exposed to filtered air. The number of metaplastic mucous cells in transitional epithelia of rat nasal airways was increased from 0 to about 200 after 3 and 6 mo of exposure to ozone; only 0 to 10 metaplastic mucous cells remained after a recovery period of 13 wk in rats exposed to ozone for 3 mo. The number of mucous cells of the respiratory epithelium lining the midseptum did not change after ozone exposure or recovery. The percentage of Bcl-2-positive cells lining the midseptum increased from 7 to 14% after a 3- and 6-mo ozone exposure, respectively. In transitional epithelia of the lateral wall and the nasoturbinates and maxilloturbinates, 35 to 55% of cells were Bcl-2-positive after a 1-mo exposure and 10 to 18% after both a 3- and a 6-mo exposure to ozone. Bcl-2 reactivity decreased to 0 to 8% after a recovery period of 13 wk. These observations suggest that Bcl-2 plays a role in the development and resolution of mucous cell metaplasias. This model may be useful in uncovering the role of Bcl-2 during the development and maintenance of metaplastic mucous cells. Disregulation of Bcl-2 expression may be responsible for the sustained mucous cell metaplasia in asthmatics or may allow cells to accumulate and become more susceptible to transformation leading to neoplasia.

Fluticasone propionate attenuates ozone-induced rhinitis and mucous cell metaplasia in rat nasal airway epithelium.

Ozone (O3) is the principal oxidant pollutant in photochemical smog. Repeated exposures to O3 induces inflammation and mucous cell metaplasia in the nasal airways of laboratory animals. Our study was designed to determine the efficacy of a topical anti-inflammatory corticosteroid in preventing O3-induced rhinitis and mucous cell metaplasia in rat nasal epithelium. Male F344 rats were exposed to filtered air (0 ppm O3; air-controls) or 0.5 ppm O3, 8 h/day, for 3 or 5 days. Immediately before and after each exposure, rats received an intranasal instillation (50 microl/nasal passage) of a topical corticosteroid, fluticasone propionate (FP; 25 microg/nasal passage) or its vehicle only (0.01% ethanol in saline). Rats were killed 2 h after the third exposure (3-day exposure) or 3 days after the fifth exposure (5-day exposure) and nasal tissues were processed for light microscopy. Numeric densities of epithelial cells and neutrophils, and the amount of intraepithelial mucosubstances (IM) in the epithelium lining the maxilloturbinates were morphometrically determined. There were no significant differences in any measured parameter in air-exposed rats instilled with FP compared with air-exposed rats instilled with vehicle. Vehicle-treated rats exposed to ozone had neutrophilic rhinitis with 3.3- and 1.6-fold more intraepithelial neutrophils (3-day and 5-day exposure, respectively) and marked mucous cell metaplasia (5-day exposure only) with numerous mucous cells and approximately 60 times more IM in the nasal transitional epithelium compared with vehicle-treated air-controls. FP-treated rats exposed to ozone had minimal nasal inflammation (1.3-fold more intraepithelial neutrophils only after 3-day exposure) and minimal mucous cell metaplasia (5-fold more IM only after 5-day exposure) compared with vehicle-instilled, air-exposed rats. Results of this study indicate that FP-treatment is effective in attenuating not only O3-induced rhinitis (30-60% reduction) but also O3-induced mucous cell metaplasia (85% reduction) in rat nasal transitional epithelium. The cellular and molecular mechanisms involved in FP-induced attenuation of O3-induced nasal lesions remain to be determined.

Mucous cell metaplasia in rat nasal epithelium after a 20-month exposure to ozone: a morphometric study of epithelial differentiation.

The present study was designed to examine the effects of long-term ozone exposure on nasal epithelia and intraepithelial mucosubstances (IM) throughout the nasal airways of F344/N rats. Animals were exposed to 0 (controls), 0.12, 0.5, or 1.0 ppm ozone, 6 h/day, 5 days/wk, for 20 mo. Rats were killed 1 wk after the end of the exposure, and nasal tissues were processed for light and electron microscopy. Standard morphometric techniques were used to determine epithelial cell densities and the amounts of IM in the surface epithelium lining the nasal airways. No mucous cells or IM were present in the epithelia lining the nasal lateral meatus and maxillary sinus of rats exposed to 0 or 0.12 ppm ozone. In contrast, rats exposed to 0.5 or 1.0 ppm ozone had marked mucous cell metaplasia (MCM) with numerous mucous cells and conspicuous amounts of IM in the surface epithelium lining these upper airways. Ozone-induced increases in total epithelial cells (i.e., epithelial hyperplasia) were present only in rats exposed to 1.0 ppm. The results of this study indicate that rats chronically exposed to 1.0 or 0.5 ppm, but not 0.12 ppm, ozone can develop marked MCM with significant increases in IM in both proximal and distal nasal airways. The epithelial changes observed throughout the nasal passages of ozone-exposed rats may be adaptive responses in an attempt to protect the upper and lower respiratory tract from further ozone-induced injury.

Consequences of prolonged inhalation of ozone on F344/N rats: collaborative studies. Part XII: Atrophy of bone in nasal turbinates.

As part of the National Toxicology Program/Health Effects Institute collaborative study of the health effects of prolonged ozone exposure, it was observed that rats chronically exposed to ozone had marked histopathologic changes in the upper respiratory tract, including atrophy of the nasal turbinates. The principal objective of the present study was to morphometrically assess the severity of the ozone-induced changes in the bony tissue of the maxilloturbinates in these chronically exposed rats. Male and female F344/N rats were exposed to 0, 0.12, 0.5, or 1.0 part per million (ppm) ozone, 6 hours/day, 5 days/week for 20 or 24 months. Rats were killed one week after the end of the exposure, and nasal tissues were processed for light and electron microscopy. Using image analysis and standard morphometric techniques, the amounts of bone, surface epithelium, and lamina propria comprising the maxilloturbinates were estimated by measuring the cross-sectional area of each tissue compartment at a defined location in the proximal nasal passage. Both male and female rats had significant morphologic and morphometric changes in the maxilloturbinates after prolonged exposures to 0.5 or 1.0 ppm ozone, but not to 0.12 ppm ozone. Ozone-exposed rats had significant reductions in the cross-sectional area of turbinate bone, reflecting the loss of bone in the maxilloturbinate after prolonged exposure. This ozone-induced bony atrophy was more severe in male than in female rats. Using electron microscopy, numerous bone-resorption sites were identified on the outer, periosteal, surface of the turbinate bone in ozone-exposed animals. Rats with bony atrophy also had a conspicuous influx and mixed inflammatory cells into the lamina propria surrounding the turbinate bone. In addition, ozone exposures caused reductions in the area of lamina propria, due to blood vessel constriction, and increases the in the area of the surface epithelium, due to hyperplasia and metaplasia. The results of the present tudy demonstrated that prolonged exposure of rats to ozone can cause marked loss of turbinate bone. The severity of this ozone-induced bony atrophy in rats is dependent on both concentration and gender.

Kinetics of nasal epithelial cell loss and proliferation in F344 rats following a single exposure to 0.5 ppm ozone.

Repeated exposure to 0.5 ppm ozone (O3) induces mucous cell metaplasia in the nasal transitional epithelium (NTE) of rats. The cellular events which commit the NTE to undergo this phenotypic alteration occur during the first 3 days of exposure. To examine the kinetics of the early cellular responses of NTE to O3, F344 rats were exposed to filtered air or 0.5 ppm O3 for 8 hr and euthanized 2, 4, 6, 8, 12, 16, 20, 24, and 36 hr postexposure (PE). Two hours before euthanization, rats were injected with bromodeoxyuridine (BrdU) to label S-phase cells. The nasal cavities were fixed and processed for light microscopy. Sections from the anterior nasal cavity were immunostained to detect BrdU-labeled cells and analyzed to determine the numeric densities of NTE cells and intraepithelial neutrophils, and the labeling index (LI; [BrdU-labeled epithelial cells/total epithelial cells] x 100) and unit length labeling index (ULLI; BrdU-labeled epithelial cells/mm basal lamina) of the NTE overlying maxilloturbinates. O3 exposure induced a transient influx of neutrophils 2-4 hr PE and a significant (17%) loss of NTE cells 2-4 hr PE. An increase in epithelial DNA synthesis was first detected 12 hr PE. In this study, there was no difference in the sensitivity of the two measures of epithelial cell DNA synthesis. Both the LI and ULLI were greatest 20-24 hr PE and were reduced, but still greater than those of controls, by 36 hr PE. The numeric density of NTE cells returned to control levels 20-24 hr PE. This study has defined the kinetics of acute O3-induced NTE cell injury, loss, and proliferation in vivo. The transit time from Go to S, after O3-induced injury, was 12-20 hr and the duration of G2 + M was 8-12 hr. These data may be used to further explore the early cellular and molecular events that lead to ozone-induced mucous cell metaplasia.