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Background: The risks for infants and young children receiving inhaled corticosteroid (ICS) therapy are largely unknown. Recent clinical studies indicate that ICS therapy in pre-school children with symptoms of asthma result in decreased symptoms without influencing the clinical disease course, but potentially affect postnatal growth and development. The current study employs a primate experimental model to identify the risks posed by ICS therapy., Objective: To (1) establish whether ICS therapy in developing primate lungs reverses pulmonary pathobiology associated with allergic airway disease (AAD) and (2) define the impact of ICS on postnatal lung growth and development in primates., Methods: Infant rhesus monkeys were exposed, from 1 through 6 months, to filtered air (FA) with house dust mite allergen and ozone using a protocol that produces AAD (AAD monkeys), or to FA alone (Control monkeys). From three through 6 months, the monkeys were treated daily with ICS (budesonide) or saline., Results: Several AAD manifestations (airflow restrictions, lavage eosinophilia, basement membrane zone thickening, epithelial mucin composition) were reduced with ICS treatment, without adverse effects on body growth or adrenal function; however, airway branching abnormalities and intraepithelial innervation were not reduced. In addition, several indicators of postnatal lung growth and differentiation: vital capacity, inspiratory capacity, compliance, non-parenchymal lung volume and alveolarization, were increased in both AAD and Control monkeys that received ICS treatment., Conclusions and Clinical Relevance: Incomplete prevention of pathobiological changes in the airways and disruption of postnatal growth and differentiation of airways and lung parenchyma in response to ICS pose risks for developing primate lungs. These responses also represent two mechanisms that could compromise ICS therapy's ability to alter clinical disease course in young children., (© 2012 Blackwell Publishing Ltd.)

Naphthalene is metabolized in the lung and liver to reactive intermediates by cytochrome P450 enzymes. These reactive species deplete glutathione, covalently bind to proteins, and cause necrosis in Clara cells of the lung. The importance of glutathione loss in naphthalene toxicity was investigated by using the glutathione prodrugs (glutathione monoethylester or cysteine-glutathione mixed disulfide) to maintain glutathione pools during naphthalene exposure. Mice given a single intraperitoneal injection of naphthalene (1.5 mmol/kg) were treated with either prodrug (2.5 mmol/kg) 30 min later. Both compounds effectively maintained glutathione levels and decreased naphthalene-protein adducts in the lung and liver. However, cysteine-glutathione mixed disulfide was more effective at preventing Clara cell injury. To study the prodrugs in Clara cells without the influence of hepatic naphthalene metabolism and circulating glutathione, dose-response and time-course studies were conducted with intrapulmonary airway explant cultures. Only the ester of glutathione raised GSH in vitro; however, both compounds limited protein adducts and cell necrosis. In vitro protection was not associated with decreased naphthalene metabolism. We conclude that (1) glutathione prodrugs can prevent naphthalene toxicity in Clara cells, (2) the prodrugs effectively prevent glutathione loss in vivo, and (3) cysteine-glutathione mixed disulfide prevents naphthalene injury in vitro without raising glutathione levels., (Copyright 2005 Wiley Periodicals, Inc.)

Studies in rodents have demonstrated the importance of cytochrome P450 monooxygenases in generating reactive metabolites that produce Clara cell injury. Pulmonary P450 activities in rodents are much higher than those in primates, raising the issue of relevance of rodent data to primates. Few studies on P450-catalyzed activation of cytotoxicants in subcompartments of primate lung have been reported. Accordingly, infant monkey airway subcompartments, including trachea, proximal, midlevel, distal airways, and parenchyma, were incubated with naphthalene or 1-nitronaphthalene to define metabolism at both high (500 microM) and low (50 microM) substrate concentrations. There was a relatively even distribution of metabolizing activities for naphthalene across subcompartments, but at high concentrations of 1-nitronaphthalene, lower airways (midlevel airway through parenchyma) showed higher bioactivation than upper airways. Dihydrodiol was the predominant water-soluble metabolite of naphthalene generated by all subcompartments, whereas covalently bound metabolites accounted for the greatest percentage of 1-nitronaphthalene metabolites, especially in lower airways. As anticipated, the amounts of metabolite covalently bound as a percentage of total metabolite formed increased dramatically with the 10-fold increase in substrate concentration. With both substrates, the formation of water-soluble metabolites was approximately 100 times less than observed previously in rodents. We conclude that 1) there are significant quantitative differences between rhesus and rodents in substrate bioactivation; 2) the distribution of metabolizing activities for naphthalene but not 1-nitronaphthalene is significantly different for rodents and primates; and 3) a very high percentage of the metabolites generated, particularly for 1-nitronaphthalene, is bound covalently to cellular proteins.

We propose that lung morphogenesis and repair are characterized by complex cell-cell interactions of endodermal and mesodermal origin, leading to (or returning back to) an alveolar structure that can effectively exchange gases between the circulation and the alveolar space. We provide the developmental basis for cell/molecular control of lung development and disease, what is known about growth and transcription factors in normal and abnormal lung development, and how endodermal and mesodermal cell origins interact during lung development and disease. The global mechanisms that mediate mesenchymal-epithelial interactions and the plasticity of mesenchymal cells in normal lung development and remodeling provide a functional genomic model that may bring these concepts closer together. We present a synopsis followed by a vertical integration of the developmental and injury/repair mechanisms.

We previously reported the efficiency of gene transfer in fetal monkeys using retroviral vectors and an intraperitoneal (IP) approach. Here, we explored intrapulmonary administration to determine whether gene transfer can be limited to the developing lung. The HIV-1-derived lentiviral vector (VSV-G pseudotyped; 1 x 10(7) infectious particles/fetus), using the enhanced green fluorescent protein (EGFP) as a reporter, was directly injected into fetal lung with ultrasound guidance (n=4; 55 or 70 days gestation; term 165+/-10 days). Fetuses were monitored sonographically, fetal/maternal blood samples collected during gestation, and four of four healthy newborns were delivered at term. All lung lobes were positive for the transgene (< or = 1%) when assessed by PCR, and transgene expression was observed by direct fluorescence microscopy and flow cytometry. The results of this study show the following: (1) successful gene transfer in fetal monkeys using an intrapulmonary approach; (2) less transduction of non-pulmonary tissues with gene transfer at 70 days gestation compared with 55 days gestation or use of an IP approach; (3) that the pulmonary epithelium was EGFP-positive by immunohistochemistry; and (4) no evidence of transplacental transport of vector sequences or antibody responses in the dams. The results of these investigations indicate the efficiency of fetal gene transfer by intrapulmonary delivery, and emphasize the importance of the fetal monkey as a preclinical model system for exploring in utero genetic treatment strategies for pulmonary disorders.

Current OSHA standards for naphthalene exposure are set at 10 ppm (time-weighted average) with a standard threshold exposure concentration of 15 ppm. While several studies have thoroughly delineated the time course and dose response of injury by naphthalene administered ip, the pattern and severity of injury by inhalation exposure are unknown. These studies compare the regiospecific and dose-dependent cytotoxicity of naphthalene after inhalation exposure. Mice and rats were exposed for 4 h to naphthalene vapor at concentrations of 0-110 ppm. In rats, no injury was observed in the lung epithelium at exposure concentrations up to 100 ppm. Exposures as low as 2 ppm produced proximal airway injury in mice, with increased severity in a concentration-dependent fashion up to 75 ppm. Terminal airways of exposed mice exhibited little or no injury at low concentrations (1-3 ppm). Exposures of 8.5 ppm or higher were required to produce injury to Clara cells in the terminal airways. In contrast, administration of naphthalene (300 mg/kg) extended the injury pattern toward the lobar bronchus. We conclude (1) the pattern of injury to naphthalene is highly dependent on the route of exposure, (2) lung injury to inhaled naphthalene is species dependent, and (3) Clara cells of mouse airways are exquisitely sensitive to inhaled naphthalene at concentrations well below the current OSHA standard for human exposure., (Copyright 2001 Academic Press.)

One of the presumed roles of intracellular glutathione (GSH) is the protection of cells from injury by reactive intermediates produced by the metabolism of xenobiotics. To establish whether GSH depletion is a critical step in the initiation of events that lead to cytotoxicity by P450-activated cytotoxicants, naphthalene, a well-defined Clara cell cytotoxicant, was administered to mice (200 mg/kg) by intraperitoneal injection. Shortly after injection (1, 2, and 3 h), intracellular GSH content was assessed by high performance liquid chromatography or quantitative epifluorescent imaging microscopy and compared with the degree of cytotoxicity as assessed by high resolution histopathology. In highly susceptible airways (distal bronchioles), GSH decreased by 50% in 1 h. Cytoplasmic vacuolization was not visible until 2 h, when GSH had decreased by an additional 50%. By 3 h, cytoplasmic blebbing was extensive. In minimally susceptible airways (lobar and proximal bronchi), GSH depletion varied widely within the population; a small proportion of the cells lost greater than 50% of their GSH by 2 h and a significant percentage of the cells retained most of their GSH throughout the entire 3 h. Cytoplasmic vacuolization was apparent in some of the cells at 2 h but not visible in any cells at 3 h. We conclude that (1) loss of intracellular GSH is an early event that precedes initial signs of cellular damage in Clara cell cytotoxicity; (2) this pattern of loss in relation to early injury is found both in highly susceptible and minimally susceptible airway sites; (3) there is wide cell-to-cell heterogeneity in the response; (4) the heterogeneity in the response profile varies between populations in highly susceptible and minimally susceptible sites; and (5) once the intracellular GSH concentration within the entire cell population drops below a certain threshold, the initial phase of injury becomes irreversible.

To test whether exposure to ozone alters pulmonary cytochrome P450 monooxygenase-mediated metabolism of xenobiotics, rates of 1-nitronaphthalene (1-NN) metabolism were measured in microsomes prepared from trachea, intrapulmonary airways, and distal lung of rats exposed to filtered air (FA) or ozone (O(3)) (0.8 ppm 8 h/day for 90 days). Regioisomeric glutathione conjugates derived from intermediate epoxides were measured by HPLC. Compared with FA, rates of glutathione conjugate formation in distal lung (including the central acinus) were elevated 2-fold in O(3)-exposed rats. Activity for cytochrome P450 2B, the isozyme thought to be responsible for the metabolic activation of 1-NN, was increased 3-fold in the distal lung of O(3)- compared with FA-exposed rats. There was a 2 +/- 0. 5-fold increase in immunodetectable CYP 2B protein in microsomes from the same lung subcompartment (P <.05). Immunodetectable protein was expressed in nonciliated epithelial (or "Clara") cells and not associated with ciliated epithelial cells. No differences between O(3)- and FA-exposed rats were noted in 1-NN metabolism or CYP 2B activity in trachea or intrapulmonary airways. This study emphasizes that cellular and biochemical alterations associated with long-term O(3) exposure vary considerably by location within the lung. Long-term exposure to O(3) elevates both CYP 2B activity and 1-NN metabolism in an airway-specific manner.

Long-term exposure to the oxidant air pollutant ozone (O(3)) is associated with tolerance to the acute effects of oxidant injury. To test whether this resistance to acute injury extends to bioactivated pulmonary toxicants, male Sprague-Dawley rats were exposed to filtered air (FA) or 0.8 ppm O(3) (8 h/day) for 90 days and administered 1-nitronaphthalene i.p. at doses of 0, 50, or 100 mg/kg. 1-Nitronaphthalene is a pulmonary cytotoxicant requiring metabolic activation. High-resolution histopathology, transmission electron microscopy, and morphometry revealed significantly greater 1-nitronaphthalene toxicity in the central acinar region of O(3)- compared with FA-exposed rats. At 100 mg/kg, injury to terminal bronchioles in O(3)-exposed rats involved denudation of 86% of the basement membrane; 78% of the cells remaining on the epithelium were necrotic. This is compared with denudation of 4% of the basement membrane of FA-exposed rats administered 100 mg/kg 1-nitronaphthalene; only 25% of the cells remaining on the epithelium were necrotic. The key difference between FA- and O(3)-exposed rats treated with 1-nitronaphthalene was the heightened severity of ciliated cell toxicity in O(3)-exposed animals. This is despite the fact that long-term exposure to ozone produces tolerance to oxidant stress in the epithelium of the central acinus. No differences in the susceptibility of intrapulmonary airways or trachea to 1-nitronaphthalene were observed between filtered air- and ozone-exposed rats. This study demonstrates a site-selective synergy between the copollutants ozone and 1-nitronaphthalene in the production of acute lung injury.

We examined the relationship between intrapulmonary particle distribution of carbonaceous and mineral dusts and remodeling of the airways along anatomically distinct airway paths in the lungs of Hispanic males from the central valley of California. Lung autopsy specimens from the Fresno County Coroner's Office were prepared by intratracheal instillation of 2% glutaraldehyde at 30 cm H(2)O pressure. Two distinct airway paths into the apico-posterior and apico-anterior portions of the left upper lung lobe were followed. Tissue samples for histologic analysis were generally taken from the intrapulmonary second, fourth, sixth, and ninth airway generations. Parenchymal tissues beyond the 12th airway generation of each airway path were also analyzed. There was little evidence of visible particle accumulation in the larger conducting airways (generations 2-6), except in bronchial-associated lymphoid tissues and within peribronchial connective tissue. In contrast, terminal and respiratory bronchioles arising from each pathway revealed varying degrees of wall thickening and remodeling. Walls with marked thickening contained moderate to heavy amounts of carbonaceous and mineral dusts. Wall thickening was associated with increases in collagen and interstitial inflammatory cells, including dust-laden macrophages. These changes were significantly greater in first-generation respiratory bronchioles compared to second- and third-generation respiratory bronchioles. These findings suggest that accumulation of carbonaceous and mineral dust in the lungs is significantly affected by lung anatomy with the greatest retention in centers of lung acini. Furthermore, there is significant remodeling of this transitional zone in humans exposed to ambient particulate matter.

Glutathione S-transferases (GSTs) and epoxide hydrolases (EHs) protect cells from exogenous insult by detoxifying electrophilic compounds. Little is known about these enzyme systems during postnatal lung development. This study was designed to help establish whether the heightened neonatal susceptibility of the lung to bioactivated cytotoxicants is the result of inadequate ability to detoxify reactive intermediates. We compared the distribution of immunoreactive protein and enzymatic activity of GSTs and EHs in isolated distal airways during pre- and postnatal development in lungs of mice from 16 days gestation to 9 weeks postnatal age (adult). GST alpha, mu, and pi class protein expression in fetal and postnatal lung varied by isozyme and age. Isozymes alpha and mu are expressed at low levels before birth, high levels on postnatal day 7, low levels between postnatal days 14 and 21, high levels at postnatal day 28, and slightly lower levels in adults. Immunoreactive protein of isozyme pi has a peak expression on gestational day 18 and again on postnatal day 4, is undetectable at postnatal day 21, and is at peak levels in the adult mouse lung. GST activity in distal airways increased with age. Microsomal EH protein expression increased in intensity with age, while activity was similar in airways from all ages. We conclude that in the mouse lung (1) cellular expression of glutathione S-transferase varies by age and isozyme and does not increase with increasing age, (2) airway glutathione S-transferase activity increases with increasing age and does not correlate with immunoreactive protein expression, and (3) airway microsomal epoxide hydrolase activity does not increase, even though immunoreactive protein expression does increase with age., (Copyright 2000 Academic Press.)

Clara-cell populations show a high degree of variation in susceptibility to injury by bioactivated cytotoxicants. Because glutathione (GSH) is critical for detoxification of electrophilic metabolites, heterogeneity in Clara cell GSH levels may lead to a wide range of cytotoxic responses. This study was designed to define the distinct GSH pools within Clara cells, characterize heterogeneity within the population, and examine whether heterogeneity contributes to susceptibility. Using fluorescent imaging combined with high-performance liquid chromatography analysis, semiquantitative measurements were obtained by evaluation of GSH using monochlorobimane and monobromobimane. In steady-state conditions, the GSH measured in isolated cells was in the femtomole range, but varied 4-fold between individual cells. Clara cells analyzed in situ and in vitro confirmed this heterogeneity. The response of these cells to compounds that modulate GSH was also variable. Diethylmaleate depleted GSH, whereas GSH monoethylester augmented it. However, both acted nonuniformly in isolated Clara cells. The depletion of intracellular GSH caused a striking decrease in cell viability upon incubation with naphthalene (NA). The sulfhydryl-binding fluorochrome BODIPY, which colocalized with tetramethylrosamine, a mitochondrial dye, demonstrated by confocal microscopy that cellular sulfhydryls are highest in the mitochondria, next-highest in cytoplasm, and lowest in the nucleus. These pools responded differently to modulators of GSH. We concluded that the steady-state intracellular GSH of Clara cells exists in distinct pools and is highly heterogeneous within the population, and that the heterogeneity of GSH levels corresponds closely to the response of Clara cells to injury by NA.

Acute lung injury induced by reactive oxygen gases such as ozone (O(3)) is focal and site-selective. To define patterns of acute epithelial injury along intrapulmonary airways, we developed a new analytic approach incorporating labeling of permeable cells, airway microdissection, and laser scanning confocal microscopy, and applied it to isolated perfused rat lungs where ventilation and breathing pattern could be controlled. After exposure to O(3) (0, 0.25, 0.5, or 1.0 ppm), lungs were lavaged to assess lactate dehydrogenase (LDH) and protein, or infused with the permeability marker ethidium homodimer-1 (EthD-1) via tracheal cannula, gently lavaged, and fixed by airway infusion. The airway tree of the right middle lobe was exposed by microdissection of the axial pathway down to the terminal bronchioles; the dissection was incubated with a second nuclear dye, YOPRO-1, to label all nuclei; and whole mounts were examined by confocal microscopy. Abundance of EthD-1-positive (injured) cells was estimated as the number per epithelial volume using stereology on Z-series of projected images. For ozone concentrations of 1.0 ppm, lavage fluid LDH and total protein did not increase over controls. Exposure produced a concentration- dependent but nonhomogeneous increase in the abundance of EthD-1-labeled cells in proximal and distal conducting airways both in the main pathway, including terminal bronchioles, and in side branches. Overall, the highest EthD-1 labeling occurred in the side branches of the most proximal part of the airway tree at 1 ppm with the adjacent axial pathway airway having approximately one-third the labeling density. Density of EthD-1-labeled cells was lowest in terminal bronchioles at all O(3) doses. For the model we used, identification of injured epithelial cells by differential permeability and laser confocal microscopy appeared to be highly sensitive and permitted mapping of acute cytotoxicity throughout the airway tree and quantitative comparisons of sites with different branching histories and potential dosimetry rates.

Objective: To determine hepatic and pulmonary phase-I and phase-II enzyme activities in horses., Sample Population: Pulmonary and hepatic tissues from 22 horses that were 4 months to 32 years old., Procedure: Pulmonary and hepatic tissues from horses were used to prepare cytosolic (glutathione S-transferase and soluble epoxide hydrolase) and microsomal (cytochrome P450 monooxygenases) enzymes. Rates of microsomal metabolism of ethoxyresorufin, pentoxyresorufin, and naphthalene were determined by high-performance liquid chromatography. Activities of glutathione S-transferase and soluble epoxide hydrolase were determined spectrophotometrically. Cytochrome P450 content was determined by carbon monoxide bound-difference spectrum of dithionite-reduced microsomes. Activity was expressed relative to total protein concentration., Results: Microsomal protein and cytochromeP450 contents were detectable in all horses and did not vary with age. Hepatic ethoxyresorufin metabolism was detected in all horses; by comparison, pulmonary metabolism of ethoxyresorufin and hepatic and pulmonary metabolism of pentoxyresorufin were detected at lower rates. Rate of hepatic naphthalene metabolism remained constant with increasing age, whereas rate of pulmonary naphthalene metabolism was significantly lower in weanlings (ie, horses 4 to 6 months old), compared with adult horses. Hepatic glutathione S-transferase activity (cytosol) increased with age; however, these changes were not significant. Pulmonary glutathione S-transferase activity (cytosol) was significantly lower in weanlings than adult horses. Hepatic and pulmonary soluble epoxide hydrolase did not vary with age of horses., Conclusions and Clinical Relevance: Activity of cytochrome P450 isoforms that metabolize naphthalene and glutathione S-transferases in lungs are significantly lower in weanlings than adult horses, which suggests reduced ability of young horses to metabolize xenobiotics by this organ.

Clara cell secretory protein (CCSP) is the most abundant secreted protein within airways of the lung. Moreover, CCSP levels are modulated in human lung disease, supporting a potentially important role for CCSP and/or Clara cells in lung homeostasis. However, in vivo roles for CCSP remain elusive. A popular hypothesis is that CCSP is a regulator of the inflammatory response. The purpose of this review is to provide an overview of the phenotype of CCSP null mice and relate this phenotype to proposed functions for the protein. Phenotypic analysis of mice homozygous for the CCSP-1 null allele of the CCSP gene (CCSP-/-1) revealed susceptibility to inhaled oxidant gases. Sensitivity of CCSP-/-1 mice to inhaled ozone is unrelated to alterations in antioxidant defenses, but is associated with increased cellular injury. Additional studies investigating inflammatory control in CCSP deficient mice found no differences between wild-type and CCSP-/-1 mice in their inflammatory response to low-dose inhaled endotoxin exposure, arguing against a role for CCSP in regulation of pulmonary inflammation. The findings among CCSP-/-1 mice of ultrastructural alterations to Clara cell secretory apparatus, with associated changes in airway lining fluid protein composition, demonstrate that the CCSP-/-1 genotype results in more complex changes to airways than CCSP deficiency per se. It can be concluded that CCSP does not regulate endotoxin-induced pulmonary inflammation. Moreover, CCSP-/-1 mice represent a valuable tool for probing functional roles for Clara cells in regulation of airway lining fluid composition and lung pollutant susceptibility.

The presence of seven stress proteins including various heat shock proteins [27-kDa (HSP27), 60-kDa (HSP60), 70-kDa (HSP70) and its constitutive form HSC70, and 90-kDa (HSP90) HSPs] and two glucose-regulated proteins [75-kDa (GRP75) and 78-kDa (GRP78) GRPs] in ozone-exposed lungs of nonhuman primates and in cultured tracheobronchial epithelial cells was examined immunohistochemically by various monoclonal antibodies. Heat treatment (42 degrees C) resulted in increased HSP70, HSP60, and HSP27 and slightly increased HSC70 and GRP75 but no increase in GRP78 in primary cultures of monkey tracheobronchial epithelial cells. Ozone exposure did not elevate the expression of these HSPs and GRPs. All of these HSPs including HSP90, which was undetectable in vitro, were suppressed in vivo in monkey respiratory epithelial cells after ozone exposure. Both GRP75 and GRP78 were very low in control cells, and ozone exposure in vivo significantly elevated these proteins. These results suggest that the stress mechanism exerted on pulmonary epithelial cells by ozone is quite different from that induced by heat. Furthermore, differences between in vitro and in vivo with regard to activation of HSPs and GRPs suggest a secondary mechanism in vivo, perhaps related to inflammatory response after ozone exposure.

We have previously demonstrated that aged and diluted sidestream cigarette smoke (ADSS) alters the development of bronchiolar epithelial cells in postnatal animals (C. M. Ji, C. G. Plopper, H. P. Witschi, and K. E. Pinkerton. Am. J. Respir. Cell Mol. Biol. 11: 312-320, 1994). This study was designed to examine the effects of maternal exposure to ADSS on the development of fetal Clara cells in rats with Clara cell 10-kDa protein (CC10; also designated Clara cell secretory protein) and CC10 mRNA as differentiation markers. Immunohistochemistry, Northern blots, and in situ hybridization were used to determine the abundance and distribution of CC10 at gestational days 14, 18, and 21. CC10 and CC10 mRNA were absent at gestational day 14 but were detectable at gestational day 18 and further increased by gestational day 21. Maternal exposure to ADSS was found to significantly increase fetal expression of CC10 and CC10 mRNA by gestational day 21 but not by gestational day 14 or 18. These findings demonstrate that in utero exposure to ADSS alters the normal developmental expression of CC10 in the fetal rat lung.

Acute pulmonary epithelial injury produced by short-term exposure to ozone varies by site within the tracheobronchial tree. To test whether this variability is related to the local dose of ozone at the tissue site or to local concentrations of glutathione, we exposed adult male rhesus monkeys for 2 h to filtered air or to 0.4 or 1.0 ppm ozone generated from 18O2. Following exposure, lungs were split into lobes and specimens were selected by microdissection so that measurements could be made on airway tissue of similar branching history, including trachea, proximal (generation one or two) and distal (generation six or seven) intrapulmonary bronchi, and proximal respiratory bronchioles. One half of the lung was lavaged for analysis of extracellular components. In monkeys exposed to filtered air, the concentration of reduced glutathione (GSH) varied throughout the airway tree, with the proximal intrapulmonary bronchus having the lowest concentration and the parenchyma having the highest concentration. Exposure to 1.0 ppm ozone significantly reduced GSH only in the respiratory bronchiole, whereas exposure to 0.4 ppm increased GSH only in the proximal intrapulmonary bronchus. Local ozone dose (measured as excess 18O) varied by as much as a factor of three in different airways of monkeys exposed to 1.0 ppm, with respiratory bronchioles having the highest concentration and the parenchyma the lowest concentration. In monkeys exposed to 0.4 ppm, the ozone dose was 60% to 70% less than in the same site in monkeys exposed to 1.0 ppm. Epithelial disruption was present to some degree in all airway sites, but not in the parenchyma, in animals exposed to 1.0 ppm ozone. The mass of mucous and ciliated cells decreased in all airways, and necrotic and inflammatory cells increased. At 0.4 ppm, epithelial injury was minimal, except in the respiratory bronchiole, where cell loss and necrosis occurred, and was 50% that found in monkeys exposed to 1.0 ppm ozone. We conclude that there is a close association between site-specific O3 dose, the degree of epithelial injury, and glutathione depletion at local sites in the tracheobronchial tree.

A rhesus monkey interleukin (IL)-8 cDNA clone with >94% homology to the human IL-8 gene was isolated by differential hybridization from a cDNA library of distal airways after ozone inhalation. In situ hybridization and immunohistochemistry showed increased IL-8 mRNA and protein levels in epithelial cells at 1 h but not at 24 h after inhalation of ozone. The appearance of IL-8 in airway epithelial cells correlated well with neutrophil influx into airway epithelia and lumens. Air-liquid interface cultures of tracheobronchial epithelial cells were exposed to ozone in vitro. We observed a transient increase in IL-8 secretion in culture medium immediately after ozone exposure and a dose-dependent increase in IL-8 secretion and mRNA production. In vitro neutrophil chemotaxis showed a parallel dose and time profile to epithelial cell secretion of IL-8. Treatment with anti-IL-8 neutralizing antibody blocked >80% of the neutrophil chemotaxis in vitro. These results suggest that IL-8 is a key chemokine in acute ozone-induced airway inflammation in primates.

Little is known about the molecular basis for differential pulmonary oxidant sensitivity observed between genetically disparate members of the same species. We have generated mice that are deficient in Clara cell secretory protein (CCSP -/-) and that exhibit an oxidant-sensitive phenotype. We characterized the kinetics and distribution of altered stress-response [interleukin-6 (IL-6) and metallothionein (MT)] and epithelial cell-specific [cytochrome P-450 2F2 (CYP2F2)] gene expression to further understand the cellular and molecular basis for altered oxidant sensitivity in 129 strain CCSP -/- mice. Increases in IL-6 and MT mRNA abundance were detected by 2 h of exposure to 1 part/million ozone and preceded reductions in Clara cell CYP2F2 mRNA expression. Despite being qualitatively similar, increases in IL-6 and MT mRNA expression were enhanced in CCSP -/- mice with respect to coexposed 129 strain wild-type mice. Increased MT mRNA expression, indicative of the stress response, localized to the airway epithelium, surrounding mesenchyme, and endothelium of blood vessels. These results demonstrate a protective role for Clara cells and their secretions and indicate potential genetic mechanisms that may influence susceptibility to oxidant stress.

The distribution of pulmonary cytochrome P450 (P450 or CYP) isoforms has been investigated primarily in immunohistochemical studies, which are neither quantitative nor reflective of the functions of these enzymes. Studies of enzyme activities have been performed using whole-lung homogenates or isolated cells, but there is little information on the regioselective expression of P450 monooxygenases. The aims of this study were to compare the activities of P450 monooxygenases in different lung subcompartments in two commonly studied animal models, i.e. rats and monkeys, and to explore the possibility that inducing agents would result in activity up-regulation that is highly site-selective, using rats as a model. Microdissection techniques were used to separate the airways from blood vessels and lung parenchyma. In rats, CYP1A1 (ethoxyresorufin) and CYP2B (pentoxyresorufin) dealkylase activities were highest in the parenchyma, whereas CYP2E1 (p-nitrophenol) hydroxylase activity was highest in the airways. P450 reductase activities were similar in airways and parenchyma and were lower in trachea. In monkeys, no significant site-selective differences in CYP1A1 and CYP2B1 activities were found. In contrast, CYP2E1 activity was higher in the distal bronchioles and parenchyma than in the proximal airways. P450 reductase activities were similar in microsomes prepared from all subcompartments of monkey lung. Induction of rat CYP1A1 activity by beta-naphthoflavone (administered ip) was much greater in the airways and lung parenchyma ( approximately 30-fold) than in the liver ( approximately 10-fold) or trachea ( approximately 2.5-fold). Oral administration of phenobarbital or acetone increased CYP2B and CYP2E1 activities in rat liver but had no significant effect on P450 activities in subcompartments of rat lung. These findings support the conclusion that there are regiospecific and species-specific differences in the activities of P450 isoforms and that the inducibility of rat pulmonary P450s is dependent on the isoform and lung region.

Nonciliated bronchiolar (Clara) cells metabolize environmental toxicants, are progenitor cells during development, and differentiate postnatally. Because differentiating Clara cells of neonatal rabbits are injured at lower doses by the cytochrome P-450-activated cytotoxicant 4-ipomeanol than are those of adults, the impact of early injury on the bronchiolar epithelial organization of adults was defined by treating neonates (3-21 days) and examining them at 4-6 wk. Bronchiolar epithelium of 6-wk-old animals treated on day 7 was most altered from that of control animals. Almost 100% of the bronchioles were lined by zones of squamous epithelial cells. Compared with control animals, the distal bronchiolar epithelium of 4-ipomeanol-treated animals had more squamous cells (70-90 vs. 0%) with a reduced overall epithelial thickness (25% of control value), fewer ciliated cells (0 vs. 10-20%), a reduced expression of Clara cell markers of differentiation (cytochrome P-4502B, NADPH reductase, and 10-kDa protein), and undifferentiated nonciliated cuboidal cell ultrastructure. We conclude that early injury to differentiating rabbit Clara cells by a cytochrome P-450-mediated toxicant inhibits bronchiolar epithelial differentiation and greatly affects repair.

The distal trachea and centriacinus of the lung are primary sites of acute injury during short-term ozone exposure; long-term exposure yields cells in these areas that are resistant to high doses of oxidant gases. Epithelial cells located in primary sites for ozone injury are also targets for chemicals that undergo cytochrome P450 (CYP)-dependent activation. These studies were designed to compare the effects of ozone exposure on pulmonary CYP2E1 in susceptible and nonsusceptible sites within the airway tree of lung. CYP2E1 activity was measured in well-defined regions of airways using p-nitrophenol, a CYP2E1-selective substrate, with HPLC/ electrochemical detection of the p-nitrocatechol. Alterations in distribution of CYP2E1 were evaluated by immunohistochemistry. CYP2E1 activities were highest in the distal bronchioles and minor daughter airways but were much lower in the lobar bronchi/ major daughter airways and trachea. Immediately after short-term ozone exposures (8 h, 1 ppm), CYP2E1 activities were elevated only in the lobar bronchi/major daughter airways. These activities remained above the filtered air control at 1 day but returned to control levels by 2 days. Immunohistochemical assessment of CYP2E1 protein in ozone and filtered air-exposed animals was consistent with the activity measurements. After long-term ozone exposures (90 days, 1 ppm), CYP2E1 activities were decreased in the major and minor daughter airways. These studies indicate that CYP2E1 activities vary substantially by airway level. However, ozone exposure only results in minimal alterations in activity with varying concentration of ozone, length of exposure, and time after exposure in any of the lung subcompartments examined.

The antioxidant enzymes copper/zinc (Cu-Zn) and manganese (Mn) superoxide dismutase (SOD) have been implicated in protection of the lungs from oxidant damage. Mn SOD in particular may be related to acquired tolerance in cells following chronic ozone exposure. In order to study these protective and adaptive phenomena in oxidant injury, the cellular location and relative abundance of Mn SOD and Cu-Zn SOD were examined in the lungs of Fischer 344 rats following exposure to 0.0 and 1.0 ppm ozone for up to 3 mo using immunolabeling and morphometric techniques. Cu-Zn SOD labeling was found to be markedly reduced following ozone exposure in epithelial cells within airways and parenchyma. In contrast, a significant increase was noted in Mn SOD labeling in the centriacinar regions of exposed lungs for both alveolar macrophages and epithelial type II cells. Mn SOD labeling per epithelial type II cell was significantly increased in alveoli 0-400 microm beyond the bronchiole-alveolar duct junction (BADJ), while type II cell Mn SOD labeling was similar to control values with greater distance down the alveolar duct. No induction of Mn SOD was noted in type I epithelial cells, fibroblasts, or Clara cells. Thus, alterations in Cu-Zn and Mn SOD are both site and cell specific in the lungs. The differential increase in Mn SOD in type II cells of the proximal alveolar duct may reflect the ability of these cells to acquire tolerance and to resist further injury to repeated ozone exposure.

Various studies indicate that cytodifferentiation of Clara cells and development of pulmonary cytochrome P450 (CYP) monooxygenases occur postnatally. The timing of these events is species-specific. Neonatal mice are more susceptible than adult mice are to Clara cell injury by naphthalene, but little is known about the postnatal development of Clara cells and CYP in mice. This study was designed to determine the developmental pattern of Clara cell differentiation and CYP expression in mice. Lungs from mice aged 16 days gestation to 63 days postnatal (DPN) were studied. Clara cell secretory protein (CC10) expression in nonciliated cells was detected earlier in proximal airways than in distal airways, but reached adult levels at 14 DPN in all airway levels. Cilia-associated tubulin expression closely followed the onset of CC10 expression, as did expression of CYP reductase. CYP2B protein expression appeared and differentiated earlier in bronchi than in bronchioles and reached adult levels at 14 and 28 DPN, respectively. CYP2F2 expression appeared earlier in proximal airways, but did not reach adult levels of expression until after 28 DPN. CYP activity, measured by naphthalene metabolism, increased with age and corresponded to CYP2F2 protein expression. We conclude that in the mouse, (1) Clara cell maturation is a postnatal event, (2) Clara cell differentiation is complete at the same age in proximal and distal airways, (3) CYP reductase protein expression occurs at the same time as CC10 expression, but CYP2B and CYP2F2 lag behind, and (4) stereoselective naphthalene monooxygenase activity corresponds with CYP2F2 protein expression.

Selective Clara cell injury produced by many bioactivated lung toxicants is thought to result from high levels of activating enzymes found in differentiated Clara cells. A recent study found an elevated susceptibility to the Clara cell toxicant 4-ipomeanol in neonatal rabbits when Clara cell P450 activity is low. To determine whether differentiating Clara cells in another species (mouse) are more susceptible to injury by a different bioactivated Clara cell toxicant (naphthalene), adult, 14-day postnatal (DPN) and 7DPN male mice were given a single intraperitoneal dose (25, 50, or 100 mg/kg) of naphthalene and killed 24 hr later. Epithelial damage, as assessed by quantitative histopathology, included cellular swelling, vacuolization, and exfoliation. In 7DPN mice, bronchiolar epithelium was severely injured at the lowest dose of naphthalene tested, 25 mg/kg. Bronchiolar epithelium in 14DPN mice was moderately injured at 25 mg/kg; injury severity was greatest at 50 and 100 mg/kg. Minimal bronchiolar epithelial injury occurred in adult mice at 50 mg/kg and moderate injury at 100 mg/kg. In proximal bronchi, epithelium of 7DPN mice showed signs of injury only at 100 mg/kg. Bronchial epithelium of adult mice was not injured at any dose. Isolated distal airways from 7DPN and 14DPN mice were more sensitive to naphthalene exposure than isolated distal airways from adult mice. Despite the low levels of P450 activity, differentiating Clara cells in neonatal mice are more susceptible to injury by the bioactivated cytotoxicant naphthalene than are differentiated Clara cells in adult mice.

We report an immunohistochemical method for mapping the distribution of neuroepithelial bodies (NEBs) in whole-mount preparations of the intrapulmonary airways. The lungs of 8- and 50-day-old male Sprague-Dawley rats were fixed with ethanol-acetic acid by intratracheal instillation. The major axial airway path of the infracardiac lobe was exposed and isolated by microdissection. NEBs were identified by calcitonin gene-related peptide immunoreactivity and their distribution mapped by generation and branch-point number. A distinct pattern was noted with greater prevalence of NEBs in proximal airway generations compared with more distal airways. No significant difference was noted in the distribution pattern or absolute number of NEBs between neonates and adults when compared by airway generation. NEBs were found more frequently on the ridges of the bifurcation than in other regions of the bifurcating airway wall. The ease of identification of total numbers of NEBs and their specific location by airway generation in whole-mount preparations of the bronchial tree completely removes the necessity of examining multiple sections and performing extensive morphometric procedures. Whole-mount airway preparations allow for the analysis and comparison of larger sample sizes per experimental group without labor-intensive approaches. The application of this method should enhance our knowledge of the role of NEBs in lung development and in response to disease.

Short-term exposure to the air pollutant ozone results in severe injury to the nares, trachea, and centriacinus. Long-term exposure however, leads to a state of tolerance that is characterized by remodeling in the centriacinar airways and markedly reduced cell necrosis and inflammation. This remodeling consists of hypertrophy and hyperplasia of Clara cells with a 2- to 5-fold increase in the intracellular content of the major protein synthesized by the Clara cell, Clara cell secretory protein (CCSP). Previous in vitro studies suggest that CCSP may moderate inflammation and bind reactive cytotoxicants. This study tested whether acute and chronic exposure to ozone alters the normal level of expression of the CCSP gene. Rats were exposed to ozone, 1 ppm 8 h nightly, for up to 90 days. Immunohistochemistry demonstrated repopulation of the alveolar ducts with CCSP positive Clara cells and an increase in the intensity of immunoreactive CCSP within Clara cells. The results showed that (1) CCSP mRNA expression, GAPDH, and beta-actin do not change as a result of ozone injury, (2) mRNA levels are more variable as a result of ozone injury and (3) CCSP mRNA expression increases with age.

1,3-Butadiene (BD) is oxidized by cytochrome P450 to reactive metabolites, including 1,2-epoxy-3-butene (BMO) and 1,2:3,4-diepoxybutane (BDE), which are thought to be responsible for BD genotoxicity and carcinogenicity. Alveolar-bronchiolar neoplasms were observed in mice but not rats following chronic exposure to BD. The site-specific carcinogenicity of BD in mice may result from metabolic activation in pulmonary tissue. We have incubated bronchioles isolated from both male B6C3F1 mice and male Sprague-Dawley rats with 34 microM BD (final concentration in the aqueous reaction medium) to assess species differences in pulmonary metabolism of BD and to enhance our understanding of species- and site-dependent BD carcinogenicity. Bronchioles from both mice and rats formed BMO, although mouse tissue produced 2-fold more than rat tissue. These preliminary results suggest that pulmonary activation of BD may play a role in the carcinogenicity of BD following inhalation exposure; however, other factors in addition to metabolic differences, probably contribute to the observed differences in susceptibility to BD toxicity.

The lung is composed of a complex assemblage of more than 40 different cell types. Therefore, investigative techniques that rely on samples derived from whole lung homogenates, whether for biochemical measurements of metabolism or the analysis of gene expression, are inherently insensitive to cell type or region-specific differences. Microdissection has previously been successful for defining region-specific metabolic activity in the lung. Tissues obtained by this technique exhibit good viability and permit reproducible enzyme activity measurements. In this paper, a technique for isolating RNA from lung subcompartments obtained by microdissection is described. The method is straight forward and results in high quality RNA that can be used to quantify specific mRNAs in microscopically selected lung subcompartments by complementary DNA or RNA hybridization techniques. This technique provides a significant increase in sensitivity over techniques based on whole lung homogenates because RNA contributed by relevant lung subcompartments is enriched. The high sensitivity of the method makes it feasible to compare differences in mRNA expression 1) within different regions of the lung in the same animal, 2) in the same region in different animals and between different species, and 3) between susceptible and nonsusceptible sites in conditions of focal lung injury.

Injury to pulmonary epithelial cells from chemicals that undergo P450-dependent metabolic activation and from gases such as ozone is highly focal. These studies examined the rates of glutathione resynthesis in pulmonary subcompartments (trachea, minor daughter/respiratory bronchiole and parenchyma) of mice and monkeys to determine whether differences in glutathione synthesis are partly responsible for wide regional/species variations in susceptibility of the lung to insult. Glutathione levels remained unchanged in lung subcompartments incubated for up to 4 hr in Waymouth's medium. Glutathione levels decreased less than 30% in 4-hr incubations of monkey airways in medium devoid of sulfur amino acids although in mouse airways decreases of 40 to 60% were observed. Diethyl maleate depleted glutathione in lung subcompartments in vitro by varying amounts depending on the species and subcompartment examined. Airways incubated in the presence of cysteine but not methionine or glutathione regenerated glutathione rapidly after diethyl maleate depletion. The rates of regeneration differed significantly with species and airway level. In all airways of the monkey, glutathione levels returned to the initial level within 2 to 4 hr after addition of cysteine containing medium although in mice recovery of glutathione required only 1 (minor daughter and parenchyma) or 2 hr (trachea) incubation with cysteine supplemented medium. These studies show striking species and airway level differences in the rates of glutathione resynthesis and suggest that focal injury to respiratory epithelium may, in part, be mediated by regional differences in the ability to supply glutathione for protection against electrophiles and reactive oxygen species.

The current studies were designed to test two hypotheses: (1) differences in steady-state reduced glutathione levels are responsible for subcompartment differences in susceptibility to acute ozone injury, and (2) elevation of reduced glutathione concentrations accounts for the tolerance to further injury produced by repeated ozone exposure. Glutathione was measured in well-defined subcompartments of the lung of both rats and monkeys to compare alterations occurring in both target (distal trachea and terminal bronchiole) and nontarget areas (lobar bronchus, major daughter, minor daughter bronchus, and parenchyma) of the lung in species that differ in sensitivity to ozone exposure (rat is less susceptible than monkey). Glutathione concentrations were decreased in trachea of rats exposed to 0.4 ppm ozone for 2 h and increased in lobar bronchus and distal bronchiole after 2 h exposure at 1 ppm. In monkey, glutathione levels in most subcompartments were not altered by either 0.4 or 1.0 ppm ozone exposure for 2 h. The exceptions were the major daughter subcompartment (200% of control at 0.4 ppm exposure) and the distal bronchiole (55% of control at 1 ppm exposure). Ninety day ozone exposures (6 h/day x 5 days/week) in rats produced an elevation in glutathione (164% of control value) only in distal bronchiole at the 1 ppm exposure level. In a similar manner, glutathione levels in the distal bronchiole of monkeys exposed for 90 days to 1 ppm O3 were 165% of the corresponding control values. These results suggest the following: glutathione levels in target and nontarget areas of the lung and in susceptible versus less susceptible species are not the primary determinant in the differences observed in ozone toxicity; the response of lung subcompartments to short-term ozone exposure varied depending on airway subcompartment and species; increased glutathione levels may be one reason for adaptation of some airway epithelial cells from rats and monkeys exposed to O3 for long periods; and use of well-defined segments of the lung provides a means of assessing changes in target areas of the lung without dilution from nontarget areas.

Earlier studies have shown that both mainstream and sidestream cigarette smoke increase the activities of cytochrome P4501A1 and 2E1 in the lungs of adult animals; however, little information is available on the influence of ambient levels of sidestream cigarette smoke on cytochrome P450 monooxygenase activity in the developing lung. The present studies were conducted to define the developmental profiles of cytochrome P450 monooxygenases 1A1 and 2B1 in rat lung and liver and to assess the effects of aged and diluted sidestream cigarette smoke (ADSS) on the developmental profile of these two enzymes. Accordingly, pulmonary and hepatic microsomal P4501A1 and 2B1 activities were determined by measuring ethoxy- and pentoxyresorufin-O-delakylase (EROD and PROD, respectively) activity in animals exposed to filtered air or ADSS from birth to 7, 14, 21, 50, and 100 days of age. Pulmonary P4501A1 activity in control rats was not detected until 14 days of age. Activities increased threefold between 14 and 21 days of age and remained unchanged to 100 days of age. In animals exposed to ADSS from birth, pulmonary EROD activities were detected as early as 7 days postnatal and were elevated three- to fourfold above control at all other ages examined. Hepatic EROD activities were unaltered by ADSS exposure. Short-term (4-day) ADSS exposure was as effective in upregulating pulmonary microsomal EROD activities as 100-day exposures. Induction of pulmonary EROD activities and the associated increases in mRNA levels were dependent upon the particulate fraction. Stimulation of EROD activities in major and minor daughter subcompartments was three- to fourfold higher in ADSS-exposed animals compared to controls, while there was no induction in the trachea and less than a twofold increase in the parenchyma. Pulmonary PROD activities developed more slowly than EROD and did not reach adult levels until Day 50. ADSS did not alter pulmonary or hepatic PROD activities. These studies show that P4501A1 and 2B1 develop at different rates in rat lung and liver and that exposure to ADSS markedly increases P4501A1 activities in the lung at all ages examined.

Because a number of studies suggest that the developmental expression of cytochrome P-450s (CYP) in Clara cells is species specific, this study was designed to compare the developmental patterns of the isoform CYP2B and NADPH reductase protein expression and CYP2B activity with the time course of smooth endoplasmic reticulum (SER) formation in Clara cells of rat lung. Pulmonary CYP2B activity measured as pentoxyresorufin O-dealkylation in lung homogenates was not detectable before 7 days postnatal age, but was detectable at adult levels at 50 days postnatal age. In Clara cells, CYP2B and NADPH reductase were detected immunohistochemically at 4 days postnatal age and at adult levels at 10 days postnatal age. The volume density of SER in Clara cells of terminal bronchioles measured morphometrically increased significantly with postnatal age. We conclude that in the rat 1) CYP2B and NADPH reductase distribution and CYP2B activity are age dependent; 2) the increase in Clara cell SER precedes the expression of CYP2B protein; 3) cellular appearance of CYP2B protein precedes CYP activity; and 4) SER appearance and P-450 protein expression do not occur uniformly in differentiating Clara cells, even within the same bronchiole.

The effect of ozone on the respiratory system is not confined to a single region or a specific cell type. Ozone-induced injury can occur at all levels of the respiratory system. However, the effects of this oxidant gas throughout the tracheobronchial tree and the lung parenchyma can be highly variable. The doses of ozone delivered to the various regions may also be different, and these differences may have a significant effect on the extent of injury. To examine the effects of chronic exposure to ozone on the lungs, we used a systematic sampling approach to perform morphometric, histochemical, and enzymatic analyses of selected airway generations and pulmonary acini arising from short and long airway paths of the tracheobronchial tree. The objectives of this study were to define compositional, cytochemical, and architectural changes that occur in epithelial cells of the airways and major tissue components of the pulmonary acini after 20 months of exposure to 0.0, 0.12, 0.5, or 1.0 parts per million (ppm)* ozone in male and female F344/N rats. We found in the trachea and bronchi significant alterations in stored secretory product following exposure to ozone, but no changes in epithelial thickness or the volume density of nonciliated cells. The volume density of nonciliated cells was significantly increased in terminal bronchioles arising from a long airway path (caudal region) of the left lung. The predominant change within the pulmonary acini was the extension of bronchiolar epithelium beyond the bronchiole-alveolar duct junction into alveoli. This change was concentration-dependent and site-specific, with ventilatory units arising from a short path (cranial region) of the left lung in male rats being most affected. The antioxidant enzymes superoxide dismutase, glutathione peroxidase, and glutathione S-transferase were significantly elevated in the distal bronchiole to central acinus following 20 months of exposure to 0.5 or 1.0 ppm ozone. Changes in antioxidant enzyme levels were more variable in other airway generations. We conclude that the effects of long-term (20-month) exposure to ozone are dose-dependent and site-specific along the tracheobronchial tree and pulmonary acini of the lungs. With the tissue sampling strategies used in this study, for the first time microdosimetric relations between ozone concentrations and biological changes in precisely delineated regions of the lungs can be defined along the entire lower respiratory tract.

Interstitial and bronchointerstitial pulmonary patterns are commonly observed in thoracic radiographs of Thoroughbreds. Prominent interstitial and bronchointerstitial pulmonary patterns are observed in clinically normal horses, and in horses with respiratory tract disease. Until recently, the relevance of these pulmonary patterns was not known. Previous studies indicated that bronchiolitis, bronchiolar epithelial hyperplasia, epithelial metaplasia, and bronchial arteriolar recruitment correlated strongly with the prominence of the interstitial and bronchointerstitial pulmonary patterns observed radiographically. We examined the content and distribution of collagen in the lungs of 7 clinically normal Thoroughbreds in race training. After standardized fixation, lung tissue was treated with a compound that selectively stains collagen. Standard morphometric techniques were used to determine the volume density of parenchymal tissue and parenchymal airspace, mean linear intercept (estimate of alveolar size), alveolar surface area-to-volume ratio, percentage of parenchyma composed of collagen, percentage of airway wall composed of collagen, and airway wall thickness. These values were compared with radiographic and histopathologic scores obtained from the same horses. The volume density of parenchymal tissue and small airway wall thickness correlated strongly with the prominence of the bronchial and bronchointerstitial pulmonary patterns observed radiographically. Small airway thickness was also highly correlated with the perceived prominence of the interstitial pulmonary patterns observed radiographically, and morphometric estimates of parenchymal tissue and parenchymal collagen. There were also strong correlations between the volume density of parenchymal tissue, the percentage of parenchymal collagen, peribronchiolar mononuclear cell infiltrates, and bronchiolar mucosal plication estimates.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonciliated bronchiolar epithelial (Clara) cells of mice are highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type expresses high levels of cytochrome P450 monooxygenases. To establish the capability of these cells to metabolize an agent that causes Clara cell-selective toxicity in vivo, we evaluated the metabolism of naphthalene in isolated cells under two distinct conditions, i.e., in homogenized cell preparations supplemented with glutathione and glutathione S-transferases and in intact cells. In homogenized cell preparations naphthalene was metabolized to dihydrodiol (minor) and a single glutathione adduct (major) derived from the 1R,2S-epoxide. In intact cells the rate of formation of glutathione adduct was much lower and dihydrodiol predominated. Approximately 3-10% of racemic naphthalene oxide added to isolated homogenized cells was converted to glutathione adducts and dihydrodiol in 3-min incubations. At high concentrations of naphthalene oxide (0.25 and 0.5 mM), formation of the adduct derived from the 1R,2s-epoxide was favored. The intracellular glutathione concentration, measured by high performance liquid chromatography as the fluorescence of the monobromobimane-glutathione derivative, was 1.14 +/- 0.13 nmol/10(6) cells. To determine whether Clara cell injury results from cytotoxic metabolites of naphthalene, we assessed viability of intact cells in response to different concentrations of naphthalene and naphthalene metabolites. At high naphthalene concentrations (0.5 and 1.0 mM) cell viability decreased to 63% or less of control, whereas lower concentrations (0.1 or 0.05 mM) did not alter viability significantly. Naphthalene-induced decreases in cell viability were blocked by preincubation of Clara cells with the cytochrome P450 monooxygenase inhibitor piperonyl butoxide. The cytotoxicity of naphthalene metabolites varied. Incubation of cells with 0.5 mM dihydrodiol, 1-naphthol, or 1,2-naphthoquinone decreased cell viability to an extent similar to that produced by 0.5 mM naphthalene. In contrast, 0.5 mM naphthalene oxide and 1,4-naphthoquinone significantly decreased viability more than the parent compound. Preincubation of Clara cells with piperonyl butoxide did not affect the loss in cell viability associated with naphthalene oxide. We conclude that isolated Clara cells 1) are capable of metabolizing naphthalene, a Clara cell-specific cytotoxicant, to two major metabolites, 2) have a detectable intracellular glutathione pool, and 3) are more susceptible to specific naphthalene metabolites than to the parent compound naphthalene.

Aerosolized fluorescent microspheres were used to study particle deposition in site-specific regions of the lung with confocal laser scanning microscopy. A nebulizer was used to aerosolize microspheres followed by passage through a heated discharging column to reduce static charge and to remove water surrounding each microsphere. Precoating of microspheres with albumin helped to minimize displacement during vascular fixation of the lungs. Confocal laser microscopy facilitated visualization of microspheres throughout the bronchial tree, ducts, and alveoli of the lungs. The use of fluorescent microspheres and confocal laser imaging provided distinct advantages compared with other methods to study lung particle deposition due to (1) the generation of single microspheres of uniform size by nebulization, (2) easy detection of microspheres in large slabs of microdissected lung tissues, (3) excellent resolution of tissue surfaces and microspheres for an infinite number of orientations and planes of section, and (4) the ability to visualize microspheres below fluid lining layers and on surfaces that could not easily be done by other methods of microscopy.

Antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glutathione S-transferases are thought to be the primary cellular defense against reactive oxygen species. Since pulmonary injury produced by oxidant air pollutants like ozone is highly focal, involving primarily the trachea and centriacinar areas of the lung, measurements of alterations in antioxidant enzyme activities in whole lung may substantially underestimate changes occurring in target areas of the respiratory tract. We have applied a technique for preparation of lung specimens from well-defined anatomic locations to determine whether the focal injury associated with ozone exposure is related to an uneven distribution of antioxidant enzyme activity in the respiratory tract. Our study compared enzyme activities in rat and monkey, species which differ considerably in sensitivity to ozone-induced injury (monkey > rat). The activities of glutathione S-transferase varied less than twofold between different airway subcompartments for both the rat and monkey. Pulmonary veins had approximately 50% of the activity of airways in both species. Glutathione peroxidase activity was slightly higher in proximal compared to distal airways of the rat but was evenly distributed at all airway levels in the monkey. In both species, activity in pulmonary veins was lower than that in airways. The activity of superoxide dismutase was similar in rat and monkey and marked differences were not observed in the various subcompartments studied. Similarly, catalase activity was relatively evenly distributed in rat airways but, in the monkey, the distal bronchiole and lobar bronchus had marginally higher activity than the trachea. We conclude that: (1) measurement of antioxidant enzyme activities in anatomic subcompartments within the lung is feasible using microdissected specimens, (2) antioxidant enzyme activity can vary in different subcompartments of the lung of the same species, (3) the pattern of variation in enzyme activity differs by the enzyme and by species, and (4) species and subcompartment differences in ozone injury are not due primarily to differences in the distribution of antioxidant enzyme activity.

Previous studies have indicated that both cytodifferentiation of Clara cells and the onset of pulmonary cytochrome P450 activity are postnatal events. However, the relationship between these two events during lung development remains poorly understood. To determine how these events interrelate, we examined rabbit Clara cells during postnatal differentiation, with the following goals in mind: 1) to identify the patterns of intracellular expression of cytochrome P450 monooxygenase isozymes 2B and 4B and cytochrome P450 reductase, 2) to describe the biogenesis of the organelles with which these isozymes are associated, namely smooth and rough endoplasmic reticulum, and 3) to compare the patterns of expression with cytochrome P450 activity in the whole lung over the same period. Lungs of rabbits ranging in age from 24 days gestational age (DGA) to 25 weeks postnatally were studied. Ultrastructural morphometry showed that smooth endoplasmic reticulum averaged < 5% of the Clara cell volume in late gestational (24-30 DGA) and neonatal rabbits [0-7 days postnatally (DPN)], grew to 20-30% of the cell volume in 14-21-DPN animals, and approximated adult levels (> 40%) in 28-DPN rabbits. In contrast, rough endoplasmic reticulum decreased from > 10% of the cell volume at 27 DGA to < 5% in adults. All postnatal animals showed considerable heterogeneity in the abundance of smooth endoplasmic reticulum among individual cells. Immunohistochemistry revealed that cytochrome P450 reductase appeared in Clara cells earlier (28 DGA) than did either isozyme 2B or 4B (1 DPN). Each antigen was detected first in the apical borders of the cells, then throughout the cytoplasm in a few cells by 7 DPN, and finally in adult abundance by 28 DPN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that cytochrome P450 protein concentrations increased postnatally. Cytochrome P450 heme protein was not detected spectrophotometrically in the lungs of animals younger than 3 DPN but increased to approximately 70% of adult levels by 28 DPN. Likewise, cytochrome P450 activity (measured as ethoxy- and pentoxyresorufin O-dealkylation) was not detected in animals younger than 2 DPN but increased to approximately 75% of adult levels by 28 DPN.(ABSTRACT TRUNCATED AT 400 WORDS)

Although it is well established that the nonciliated bronchiolar epithelial (Clara) cell serves as the progenitor for itself and ciliated cells in the adult lung following bronchiolar epithelial injury, the nature of this relationship during development has not been well characterized. To define the pattern of proliferation and differentiation of bronchiolar ciliated and nonciliated cells, lungs of fetuses and offspring from time-mated New Zealand White rabbits, ranging in age from 24 days of gestation to 25 wk postnatal (PN), were fixed by airway infusion and embedded for simultaneous light and transmission electron microscopy. Three categories of cells could be distinguished in terminal bronchioles: nonciliated cells with abundant glycogen and variable numbers of organelles; nonciliated cells with little glycogen, large numbers of polyribosomes, and variable numbers of basal bodies; and ciliated cells with cilia of varying height. Together, both types of nonciliated cells were 100% of the epithelium at 24 and 27 days gestation age (DGA). At 30 days DGA, they were 85% of the population; at all postnatal ages, they ranged from 75 to 81% of the total population. Nonciliated cells with polyribosomes and basal bodies were 10 to 20% of the total nonciliated cell population between 24 DGA and 1 wk PN and not found thereafter. Ciliated cells were not observed in animals younger than 30 DGA. Labeling indices of bronchiolar epithelium in fetuses of pregnant rabbits injected with tritiated thymidine, as determined by autoradiography, were 57 cells per thousand at 28 DGA (1 h postinjection [PI]), 76 at 29 DGA (24 h PI), and 114 at 30 DGA (48 h PI).(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of cytochrome P450 1A1 (P450 1A1) in a variety of tissues is a well established consequence of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although localization of the induced protein within the lung has been described, the precise intracellular distribution of the enzyme is not clear. Analysis of tissue sections, microsomal proteins, and mRNA from lungs of treated and untreated rabbits established that P450 1A1 had been induced by treatment with TCDD. Rabbit lungs from animals treated with TCDD were examined with immunocytochemistry and in situ hybridization, to identify the cell types that contain P450 1A1 and those that contain mRNA encoding P450 1A1. Endothelial cells of the entire vascular bed of rabbit lung reacted markedly with anti-P450 1A1. Likewise, cells lining both arteries and veins, as well as capillary endothelial cells, reacted strongly with the cDNA probe for mRNA encoding P450 1A1. Clara cells at all levels of airway labeled prominently for both P-450 1A1 and P450 1A1 mRNA. In addition, type 2 cells, alveolar macrophages, and to a lesser degree, ciliated cells reacted with the cDNA probe. P450 reductase, which is required for P450 activity, has previously been identified in Clara cells, type 2 cells, and alveolar macrophages, but not in endothelium of rabbit lung. We have now obtained similar results for the localization of mRNA encoding P-450 reductase. This finding brings into question the function of P450 1A1 in endothelium.

Naphthalene, a murine Clara cell cytotoxicant, is metabolized by cytochrome P450 monooxygenases to unstable, chiral epoxide metabolites which can conjugate with glutathione in the presence of glutathione transferases. Analysis of the three diasteriomeric glutathione adducts produced from conjugation of naphthalene oxides was used in these studies to characterize the stereochemistry of naphthalene epoxidation in preparations of nasal mucosa, lung and liver of the mouse, rat, hamster and monkey. The highest rates of naphthalene metabolism were observed in mouse lung and liver microsomal incubations. Rat, hamster and monkey lung microsomal preparations metabolized naphthalene at 12, 37, and 1%, respectively, of the rate observed in mouse lung. The ratio of chiral epoxides produced in microsomal incubations was dependent upon the concentration of naphthalene. At high substrate concentrations (0.25-1.0 mM), the ratio of 1R,2S- to 1S,2R-naphthalene oxide, as assessed by the glutathione adducts generated (adduct 2/adducts 1 + 3), in murine lung microsomal incubations was 10:1 and at low concentrations (0.062 mM and below) varied from 13.8:1 to 30:1. In contrast, the ratio of 1R,2S- to 1S,2R-naphthalene oxide produced in murine liver microsomes varied from 1:1 at high substrate concentrations to 5:1 at low substrate concentrations. The ratio of naphthalene oxides was unaffected by the concentration of glutathione in the incubation. In contrast to the preferential formation of 1R,2S-naphthalene oxide observed in mouse lung microsomal preparations, lung microsomes derived from the rat, hamster and monkey yielded 1R,2S- to 1S,2R-epoxide ratios of 0.48, 0.61 and 0.12, respectively, at 0.5 mM naphthalene.(ABSTRACT TRUNCATED AT 250 WORDS)

A specific form of flavin monooxygenase has been identified in the lungs of a number of species. Distribution of the pulmonary flavin-containing monooxygenase (FMOp) is of interest because it oxidatively metabolizes a wide variety of nitrogen-, sulfur-, and phosphorous-containing xenobiotics, some of which form highly toxic reactive intermediates. We have identified the nonciliated bronchiolar epithelial (Clara) cell as the predominant location for this enzyme in rabbit lung. In addition, protein in ciliated, endothelial, type I, and type II cells and in tracheal lining layer reacted with antibodies to FMOp. In all these cell types antigen was found associated with cytoplasmic organelles, and in the Clara cell antigen was most concentrated in areas rich in smooth endoplasmic reticulum. Staining of ciliated surfaces was also observed at both the light and electron microscopy levels. Extracellular antigen was also apparent in tracheal lining layer smeared onto glass slides. We compared the location of the FMOp with that of two enzymes of the cytochrome P-450 monooxygenase system (studied here and elsewhere), cytochrome P450 IIB (P450 IIB), and NADPH cytochrome P450 reductase (reductase), and concluded that (1) FMOp is detected in all cells where P450 IIB and reductase are both present (Clara, type II, and ciliated); (2) FMOp and P450 IIB, but not reductase, are detected in endothelial cells; (3) P450 IIB alone is detected in the plasma membrane, cilia, and microvillae of ciliated cells and plasma membrane of endothelial cells; and (4) FMOp alone is detected in type I cells.

An overview of the epithelial and interstitial composition of rat respiratory airways shows complexity and variability. Airway epithelium varies in 1) different airway levels; 2) the types and ultrastructure of cells present; and 3) the abundance, type, and composition of stored secretory product. Unbiased sampling of airways is done using airway microdissection with a specific binary numbering system for airway generation. Vertical sections of selected airways are used to sample epithelium and interstitium. We determine the ratios of the volume of epithelial or interstitial cells to the total epithelial or interstitial volume (Vv). The surface of the epithelial basal lamina to the total epithelial or interstitial volume (Sv) is determined using point and intersection counting with a cycloid grid. Using the selector method on serial plastic sections, we determine the number of epithelial or interstitial cells per volume (Nv) of total epithelium or interstitium. We calculate the number of epithelial or interstitial cells per surface of epithelial basal lamina (Ns) by dividing Nv by Sv where the volumes are the same compartment. We calculate average cell volumes (v) for specific epithelial and interstitial cells by dividing the absolute nuclear volume by the ratio of the nucleus to cell volume (Vv). By multiplying the average cell volume (v) by the ratio of organellar volume to cell volume (Vv), we calculate the average organellar volume per cell. These unbiased stereological approaches are critical in a quantitative evaluation of toxicological injury of rat tracheobronchial airways.

Adrenocorticotrophin (ACTH) effects on lung development were studied in sheep. ACTH was infused into 10 fetal lambs at 129 days of gestation at a rate of 29, 110 or 250 micrograms/day for 72 h, a time course which was independent of the mechanical and hormonal forces associated with labor. Lungs were compared with those of normal fetuses at term (day 147). ACTH accelerates maturation of components of lung structure toward term values. Different components of cytodifferentiation of the type II alveolar cell showed different levels of sensitivity. The degree of acceleration, not the nature of the changes, was influenced by dose.

Naphthalene produces selective injury to Clara cells in the mouse in vivo and in the isolated perfused lung. To investigate the role of circulating reactive metabolites in lung injury, the stability, metabolism and cytotoxicity of naphthalene oxide, a reactive intermediate, were examined in the perfused mouse lung. The T1/2 of naphthalene oxide is 4 min in Waymouth's medium. Addition of 5% bovine serum albumin to the medium increased the half-life of the epoxide to 11 min. Perfusion of the lung with 0.2 or 2 mumol of naphthalene oxide decreased pulmonary reduced glutathione levels to 62 and 42% of control, respectively. 1,4-Naphthoquinone and naphthol-glucuronide represented 36 and 25% of the total polar metabolites isolated after infusion of naphthalene oxide, whereas dihydrodiol and thioether conjugates were minor metabolites. In comparison, thioethers and dihydrodiol were the primary metabolites isolated from lungs perfused with [14C]naphthalene. Histologic examination of the lungs fixed 4 hr after infusion of naphthalene oxide (0.25-1.0 mumol/60 min) revealed selective vacuolation and necrosis of Clara cells, significant decreases in the mass of bronchiolar Clara cells and increases in the mass of vacuolated cells. Injury to lungs perfused with naphthalene or secondary metabolites such as naphthoquinones, 1-naphthol and 1,2-dihydro-1,2-dihydroxynaphthalene was less dramatic. In contrast to other studies implicating quinones as mediators of aromatic hydrocarbon toxicity, the current work suggests that epoxides play a significant role in naphthalene-induced lung injury. This investigation also demonstrates that circulating epoxides are capable of eliciting selective Clara cell injury.

This study compares centriacinar changes by ultrastructural morphology and morphometry following daily versus episodic ozone exposure in rats. Three groups of rats were exposed to air, 0.95 ppm ozone 8 hr daily for 90 days, and 0.95 ppm ozone 8 hr daily in seven successive 5-day episodes separated by 9-day recovery periods for a total of 89 days. Sections from the left lung and dissected acini from the right middle lobe were studied by light and electron microscopy. The centriacinar lesion following episodic exposure was similar but diminished in severity compared to that of rats exposed daily. Damage following episodic exposure appeared to be more than predicted by an exposure regimen which delivered 35% of the total ozone dose during daily exposure. The total volume of affected parenchyma was similar following both exposures. Respiratory bronchiole formation increased following both exposures but this was only statistically significant following daily exposure. The most severe epithelial damage was at the tips of alveolar septa in alveolar ducts distal to the respiratory bronchiole. Interstitial thickness in the injured respiratory bronchiole and proximal alveolar duct increased significantly and similarly following both exposures. Epithelium along the respiratory bronchiole of daily exposed rats was more differentiated. In the episodic group, respiratory bronchiole and alveolar duct epithelium consisted of a range of intermediate, less differentiated bronchiolar or alveolar epithelial cells. The episodic exposure resulted in a diminished lesion, but there appears to be some cumulative effect of repeated exposures with respiratory bronchiolar and alveolar duct epithelium in a more dynamic state of injury and repair.