Assessment of visual impairment in stroke survivors.

A novel, tablet-based application (app) has been developed to act as a screening tool for visual impairment in stroke survivors; The Stroke Vision app. The app includes assessments for visual acuity, visual fields and visuospatial neglect, as well as novel tools for the education of patients, carers and staff. The app has been devised by experts in the field to address two important deficiencies; firstly a set of visual assessment tools to support and improve evaluation and rehabilitation of visual impairments in stroke survivors, and secondly to provide education for staff and information to carers about their relatives visual disabilities.

Smooth muscle hypertrophy in distal airways of sensitized infant rhesus monkeys exposed to house dust mite allergen.

BACKGROUND: Airway smooth muscle hypertrophy is closely associated with the pathophysiology of hyper-reactive airways in allergic asthma. OBJECTIVE: To determine whether repeated exposure to allergens during postnatal lung development promotes remodelling of airway smooth muscle. METHODS: Infant, male rhesus monkeys (30-day-old) were sensitized to house dust mite allergen (HDMA) and then exposed to HDMA aerosol periodically over 5 months. Smooth muscle mass and bundle size and abundance in conducting airways were measured and compared with age-matched control (filtered air-exposed) monkeys. RESULTS: Total smooth muscle mass and average bundle size were significantly greater in the conducting airways of monkeys exposed to HDMA. Smooth muscle bundle abundance was not affected by exposure to HDMA. CONCLUSION: Repeated cycles of allergen exposure alter postnatal morphogenesis of smooth muscle, affecting both total mass and bundle size, in conducting airways of infant monkeys.

Three-dimensional mapping of smooth muscle in the distal conducting airways of mouse, rabbit, and monkey.

Airway smooth muscle remodeling is implicated in a number of constrictive pulmonary diseases such as asthma and may include changes in smooth muscle orientation and abundance. Both factors were compared in the normal distal bronchioles of the mouse, rabbit, and rhesus monkey (respiratory bronchioles included). Airway smooth muscle was measured by using a three-dimensional approach employing confocal microscopy and whole-mount cytochemistry with fluorochrome-conjugated phalloidin, a probe for polymerized actin. Smooth muscle orientation had a wide range of angles along the airway, but the distribution was conserved among species and among distal airway generations. At the bifurcation of proximal bronchioles, smooth muscle was nearly parallel to the longitudinal axis of the airway. Smooth muscle abundance was significantly different between species (abundance was less in the monkey compared with the mouse and rabbit), and there was a trend for abundance to decrease with each more distal airway generation. This study defines the normal distribution of smooth muscle in three test species and provides a basis for future comparisons with the diseased state.

Effect of respiratory pattern on ozone injury to the airways of isolated rat lungs.

Ozone stimulates the "defensive" C-fibers in the lungs, changing breathing pattern to rapid and shallow. We hypothesized that when ozone is administered to the isolated lung with a rapid shallow breathing pattern rather than a slow deep pattern, relatively less airway epithelial damage would occur. Four groups of isolated buffer perfused rat lungs were exposed to ozone (1 ppm) or to filtered air for 90 min with either a slow deep (SDB, tidal volume 2.4 ml, frequency 40 breaths/min) or a rapid shallow breathing pattern (RSB, tidal volume 1.2 ml, frequency 80 breaths/min), resulting in an equivalent inspired dose. The absorbed dose of ozone did not differ between the exposed groups. Ethidium homodimer-1 was then instilled into the trachea to identify injured airway epithelial cells. The lungs were fixed, the airways were microdissected, and the airway epithelial cells were counterstained with YPRO-1 prior to evaluation with confocal microscopy. Ozone-induced airway epithelial cell injury occurred to a lesser overall degree when lungs were exposed by the RSB pattern (p = 0.003). The relative reduction in injury was greater (p < 0.05) in the proximal axial airway than in its adjacent airway branch and terminal bronchioles. Ozone induced an increase in pulmonary resistance with the SDB pattern but not with the RSB pattern. Thus, at an equivalent dose of inspired ozone, a RSB pattern resulted in less total damage than a SDB pattern and the distribution of protection was heterogeneous with proximal axial airways displaying the greatest relative reductions in epithelial damage.

Acute injury to differentiating Clara cells in neonatal rabbits results in age-related failure of bronchiolar epithelial repair.

Nonciliated bronchiolar (Clara) cells are progenitor cells during lung development. During differentiation, they have a heightened injury susceptibility to environmental toxicants bioactivated by cytochrome P450 monooxygenase. When neonatal rabbits are treated with the P450-mediated cytotoxicant 4-ipomeanol (IPO), abnormal bronchiolar epithelium results. This study establishes the impact of IPO cytotoxicity on 3 stages of rabbit Clara cell differentiation, early (2.5 and 5 days postnatal [DPN]), intermediate (7 and 9 DPN), and late (15 and 21 DPN), and relates the cytotoxicity to the extent of bronchiolar repair. Neonates received a single dose of IPO (5 mg/kg) and were assessed by qualitative pathology 48 hours later for injury or at 4 weeks for repair. IPO injured the 3 stages of Clara cell differentiation to the same degree; epithelium was swollen, exfoliated, and squamated. Epithelial repair differed among the 3 stages. Bronchioles of animals treated during early and intermediate stages had simple squamous and irregularly shaped cuboidal cells. Animals treated during late stages were similar to controls. Thus, differentiating Clara cells are susceptible to injury by the P450-mediated cytotoxicant IPO, but the extent of repair varies based on when the initial injury occurs.

Three-dimensional mapping of ozone-induced acute cytotoxicity in tracheobronchial airways of isolated perfused rat lung.

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.

Relationship of inhaled ozone concentration to acute tracheobronchial epithelial injury, site-specific ozone dose, and glutathione depletion in rhesus monkeys.

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.

Neonatal Clara cell toxicity by 4-ipomeanol alters bronchiolar organization in adult rabbits.

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.

Cytochrome P450 2E1 in rat tracheobronchial airways: response to ozone exposure.

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.

Comparison of the distinct effects of epidermal growth factor and betamethasone on the morphogenesis of the gas exchange region and differentiation of alveolar type II cells in lungs of fetal rhesus monkeys.

To compare the effects of epidermal growth factor (EGF) and betamethasone on the morphogenesis of the gas exchange region and the differentiation of the alveolar type II cell during fetal lung development, fetal rhesus monkeys (78% gestation) were treated in utero with EGF (5.33 mg/kg total dose), beta-methasone (2.6 mg/kg total dose) or the carrier, saline (control), every other day for 7 days. EGF-treated monkeys had significantly increased body and adrenal weights. Betamethasone-treated monkeys had significantly decreased body and adrenal weights. Exogenous EGF reduced cytoplasmic glycogen and increased the cytoplasmic organelle and SP-A content within alveolar type II cells. In contrast, exogenous betamethasone did not alter alveolar type II cell cytodifferentiation. Neither EGF nor betamethasone treatment significantly altered the structure of the gas exchange region as shown by a lack of change from controls in alveolar airspace size or in the fraction of the gas exchange region that was potential airspace. We conclude that at clinically relevant doses, EGF greatly accelerates the maturation of alveolar type II cells, whereas betamethasone does not. Exogenous EGF may act directly on alveolar type II cells because these cells contain EGF receptor. Neither EGF nor betamethasone had dramatic effects on the morphogenesis of the gas exchange region.

Elevated susceptibility to 4-ipomeanol cytotoxicity in immature Clara cells of neonatal rabbits.

The bronchiolar Clara cell is one of the primary targets in adult mammals for environmental contaminants metabolized by cytochrome P450 (CYP) monooxygenases. Previous studies show that the onset of CYP expression in Clara cells occurs during postnatal lung development. This study was designed to determine whether differentiating Clara cells are susceptible to CYP-activated cytotoxicants and whether these substances can influence subsequent cytodifferentiation. Adult and neonatal (5-9 days of age) rabbits were given a single dose of 4-ipomeanol (IPO) i.p. and sacrificed 2 or 7 days later. Their lungs were removed and assessed morphologically, immunohistochemically or for CYP activity. Treatment with 10 mg/kg of IPO (0.25 of the LD50 for adults) killed 6 of 10 neonatal rabbits. At a dose of 5 mg/kg of IPO, most terminal bronchiolar cells were destroyed in the neonatal rabbits. The basal lamina of terminal bronchioles was either bare or lined by squamous or low cuboidal epithelium and macrophages. Terminal bronchiolar epithelium in neonates was minimally affected by a dose of 1 mg/kg of IPO. The terminal bronchioles in adults appeared nearly unaffected by either 1 or 5 mg/kg of IPO. Interalveolar septa were unaffected in all treated animals. Lung microsomal enzymes from neonatal rabbits metabolized IPO to reactive intermediates at less than one-third the rate in the lungs of adults. Seven days (15 days of age) after IPO treatment, CYP activity (as measured by pentoxyresorufin O-dealkylation) was one-half that of age-matched controls after a dose of 5 mg/kg but equaled control activity after 1 mg/kg. Immunohistochemical analysis, using antibodies to CYP2B4, CYP4B and CYP reductase, indicated that the decrease in activity seen with a dose of 5 mg/kg of IPO was the result of a loss of immunoreactive CYP proteins from the cuboidal cells of terminal bronchioles. It was concluded that, in neonatal animals, differentiating Clara cells are more susceptible to injury by bioactivated cytotoxicants than are differentiated cells in adults, despite the neonate's lower levels of CYP monooxygenases. Furthermore, IPO-induced injury impairs the normal pattern of postnatal Clara cell differentiation.

Postnatal changes in the expression and distribution of pulmonary cytochrome P450 monooxygenases during Clara cell differentiation in rabbits.

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)

Ultrastructural morphometric characterization of alveolar type II cells of perinatal sheep: a comparison with adults.

The quantitative morphologic changes in alveolar type II cells during the perinatal period were characterized morphometrically in the lungs of fetal lambs at 132, 138, and 147 days gestational age (DGA) and in newborns at 2 days postnatal age (2 DPN). Ultrastructural features were compared with those of type II cells of ewes 365 days old. Lamellar body profile number per type II cell profile was highest at term (147 DGA) and 2 DPN. In adults, the number of lamellar body profiles and volume density of lamellar bodies were equal to those of the 132 DGA fetus. Multivesicular bodies were most common at 138 DGA and in adults. The volume density of cytoplasmic glycogen fell dramatically during the latter part of gestation. The volume density of many cellular organelles increased to the level observed in adults by term (147 DGA). Subcellular composition of type II cells of adult sheep differs from that reported for adult rats chiefly by the volume density of lamellar material within the cytoplasm. Plate-like or globe-like inclusions were present only in the type II cells of adults. Cytoplasmic extensions of the type II cell crossing the basal lamina were most abundant in the 132 and 138 DGA fetal sheep. Cytoplasmic extensions were rare in adults. We conclude that morphologic changes of the alveolar type II cell associated with gestational age follow a species-specific time course. In the sheep, this occurs during the later part of gestation and extends into the neonatal period. Morphologic and morphometric changes appear to correspond with cellular interactions between alveolar type II cells and mesenchymal cells of the interstitium.

Acceleration of alveolar type II cell differentiation in fetal rhesus monkey lung by administration of EGF.

To examine the effect of epidermal growth factor (EGF) on lung parenchymal maturation in fetal rhesus monkey, recombinant human EGF was administered intraperitoneally (IP) at 66 mg/kg body wt over a 7-day period into the fetal peritoneal cavity alone or IP and into the amniotic fluid (AF) simultaneously. The saline carrier was injected IP and AF into control (CO) fetuses. The body weights of the IP + AF group were significantly larger than CO. Overall lung growth, measured as wet lung weight or fixed volume of the right cranial lobe, was unchanged. Fixed lung volume per gram body weight was significantly lower for both IP + AF and IP compared with CO. Morphogenesis of lung parenchyma, measured as percent parenchymal airspace or airspace size, was unchanged. Alveolar type II cell ultrastructure was significantly altered by EGF treatment; volume fraction of cytoplasmic glycogen was 50% less and lamellar bodies threefold greater for IP + AF and IP groups compared with CO. Total phospholipid content of AF was not altered, but relative percentages of different phospholipids were changed by EGF treatments; phosphatidylinositol was significantly reduced, and phosphatidylglycerol was significantly elevated. The lecithin-to-sphingomyelin ratio was unchanged. Surfactant apoprotein A concentration in AF was significantly elevated and was detected by immunoperoxidase in more cuboidal alveolar cells in EGF-treated animals when compared with CO. We conclude that exogenous EGF administered in the last trimester of pregnancy accelerates structural and functional cytodifferentiation of the alveolar type II cell in fetal primates. These maturational changes occur in the absence of significant alterations in overall lung growth or morphogenesis of the gas exchange area.

The N-acetylation of sulfamethazine and p-aminobenzoic acid by human liver slices in dynamic organ culture.

N-Acetyltransferase (NAT) polymorphism has been implicated in differences in the susceptibility of individuals to the toxicity of chemicals metabolized by this enzyme system. Investigation into the toxicological consequences of acetylator polymorphism and the mechanism of these effects in humans, however, has been greatly hindered due to the lack of a suitable human tissue culture system for determination of hepatic NAT activity and acetylator status of individuals. An in vitro system has been developed to study NAT activity using human liver slices in dynamic organ culture. Acetylation of para-aminobenzoic acid (PABA) and sulfamethazine (SMZ) by human liver slices was monitored by measuring the disappearance of the parent amine from the incubation medium using the colorimetric procedure of Bratton and Marshall. Presence of the acetyl conjugate was confirmed using HPLC. PABA acetylation rates varied from 0.72-2.52 nmol/hr/mg protein (N = 8). This small variation (less than 4-fold) is consistent with the classification of PABA as a monomorphic substrate. The variation in the rate of SMZ acetylation was greater than 20-fold (0.144-3.68 nmol/hr/mg protein; N = 9). This larger variation is characteristic of SMZ as a polymorphic substrate. A good correlation of N-acetylation activities for SMZ was also found between cytosol and slices prepared from the same human livers. The results obtained indicate that human liver slices in dynamic organ culture can be used for the determination of hepatic NAT activity in humans. These slices may be useful in toxicological studies that seek to relate N-acetylation of chemicals in the human liver with potential toxicity.

Liver slices in dynamic organ culture. I. An alternative in vitro technique for the study of rat hepatic drug metabolism.

1. Precision-cut liver slices in dynamic organ culture, a novel in vitro technique, is described and applied to the study of hepatic drug metabolism in the rat. 2. These slices catalysed the oxidative O-deethylation of the substrate, 7-ethoxycoumarin, over 6 h incubation. In addition, the direct conjugation of 7-hydroxycoumarin with either sulphate or glucuronic acid was maintained over 6 h. 3. The formation of 7-hydroxycoumarin and the presence of the sulphate and glucuronide conjugates in slices exposed to 7-ethoxycoumarin demonstrated integrated phase I and phase II drug metabolizing activities in this system. 4. Minor modifications of the incubation system allowed for the metabolism of four volatile chlorinated benzenes: monochlorobenzene 1,2-, 1,3-, and 1,4-dichlorobenzenes to aqueous soluble metabolites. 5. The use of liver slices in dynamic organ culture as an alternative preparation for the study of xenobiotic metabolism is discussed.

Liver slices in dynamic organ culture. II. An in vitro cellular technique for the study of integrated drug metabolism using human tissue.

1. Precision cut human liver slices in dynamic organ culture have been used to study the integrated metabolism of 7-ethoxycoumarin and the conjugation of 7-hydroxycoumarin. 2. The metabolism of 7-ethoxycoumarin and 7-hydroxycoumarin was monitored for 6 h. For both substrates there was a time-dependent increase in metabolites present in the incubation medium. The low levels of free 7-hydroxycoumarin found in the medium when 7-ethoxycoumarin was the substrate suggests good coupling of phase I and phase II metabolism. 3. With suitable incubation conditions, i.e. change of medium containing new substrate every 2 h, the metabolism of both 7-ethoxycoumarin and 7-hydroxycoumarin by human liver slices was found to proceed at similar rates for up to 24 h. This was demonstrated using five separate human liver preparations. 4. Human liver slices also metabolized mono-chlorobenzene and o-, m- and p-dichlorobenzene to aqueous soluble metabolites. There was a time-dependent increase in the appearance of aqueous soluble metabolites present in the incubation medium. Metabolites were not retained by the liver slices. 5. A cold-storage transit buffer has been described and used to maintain the levels of drug metabolism in both rat and human tissue for periods of up to 6 h. 6. The use of human liver slices in dynamic organ culture as a suitable method for the direct assessment of integrated hepatic drug metabolism is proposed.

Characterization of the cytochrome P-450 monooxygenase system in nonciliated bronchiolar epithelial (Clara) cells isolated from mouse lung.

The nonciliated bronchiolar epithelial (Clara) cell of the mouse is highly susceptible to toxicants that undergo metabolic activation, presumably because this cell type has high levels of cytochrome P-450 monooxygenases. As a first step in further defining the role of Clara cells in pulmonary xenobiotic activation and detoxication, we have isolated Clara cells (75 to 80% purity) and characterized them morphologically and biochemically. The identity of Clara cells, confirmed by transmission electron microscopy, was based on several features, including abundant agranular endoplasmic reticulum, large mitochondria, and dense secretory granules. Immunocytochemistry of isolated mouse cells showed that the majority were positive with antibodies against three major components of the pulmonary cytochrome P-450 monooxygenase system, cytochrome P-450 isozymes 2 (IIB), 5 (IVB), and NADPH cytochrome P-450 reductase, purified from rabbit lung. The isolated cells also showed a positive reaction with an antibody against the cytochrome P-450 isozyme that is active in the stereoselective metabolism of naphthalene, cytochrome P-450 mN (mN). Immunocytochemistry using the antibody against cytochrome P-450 isozyme 6 (IA1), purified from rabbit lung, showed no reaction in the isolated cells. The presence of intact cytochrome P-450 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots of homogenates of isolated cell preparations. The N-demethylation of benzphetamine and epoxidation of naphthalene occurred at easily measurable rates in incubations of isolated Clara cells. In contrast, diols, quinones, and monohydroxylated benzo(a)pyrene metabolites, analyzed by high performance liquid chromatography, were undetectable in extracts of Clara cells incubated with 3H-labeled substrate.(ABSTRACT TRUNCATED AT 250 WORDS)