Introduction

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The mammalian lung is a target organ for many environmental contaminants. A large number of these compounds are bioactivated through the cytochrome P450 (CYP) monooxygenase system to toxic intermediates. Examples include naphthalene (1, 2), 4-ipomeanol (3), and 3-methylindole (4, 5). The toxicity of these compounds varies by species, and the basis for these differences appears to be the relative activity of the specific isozymes present (1, 4). These compounds target nonciliated bronchiolar cells (Clara cells), which are thought to be sensitive because they contain high levels of CYP monooxygenase activity (6, 7). For example, naphthalene is metabolized by at least two different isozymes of the CYP monooxygenase system to reactive intermediates. The reactive intermediates are hydrolyzed to dihydrodiol by epoxide hydrolase, conjugated to glutathione by glutathione S-transferases, or, after a threshold is reached, covalently bind to tissue macromolecules (8). Specifically, isozymes CYP2B and CYP2F2 metabolize naphthalene to produce enantiomeric naphthalene epoxides. CYP2F2 produces the 1R,2S-oxide, while CYP2B produces the 1S,2R-oxide (2, 9). CYP2F2 is expressed in human (10) and mouse lung (6) and in human placenta (11, 12). Rats and hamsters have little expression of CYP2F2 and do not exhibit signs of toxicity even at extremely high doses (13, 14).

The assumption that toxicity of bioactivated compounds and susceptibility of specific cell types to injury are related to cellular expression of P450 isozymes is based almost entirely on studies on adult animals with mature enzyme systems. However, a recent study suggests that the relationship between P450 activity and susceptibility to toxicants may not be relevant for young animals with poorly differentiated Clara cells (15). Neonatal rabbits proved to be much more susceptible to 4-ipomeanol than adult rabbits, despite the fact that neonatal rabbits have a much lower potential for bioactivation. Pulmonary CYP monooxygenases have been shown to develop prenatally in hamsters (16), but postnatally in rats (17) and rabbits (18). Without high levels of CYP activity, formation of toxic intermediates is not likely and injury would not be expected. However, fetal and neonatal tissue from mice and humans, including lung, have been shown to have the ability to metabolize compounds by the CYP monooxygenase system to metabolites that are either teratogenic or embryotoxic (19). Pro-carcinogens when given to pregnant mice can produce Clara cell tumors in the adult offspring (20, 22). Although there is a wealth of information about toxic effects of chemicals during embryonic or fetal development in mice, there is very little information about toxic effects of chemicals during the period of continuing development of the lung after birth.

Despite the fact that mice have been heavily used as models for developmental toxicity and carcinogenesis, information regarding differentiation of one of the primary target cells, the Clara cell, and the timing of pulmonary P450 expression during lung development is not known. Functional Clara cell differentiation has been shown to be directly correlated with Clara cell secretory protein (CC10) expression in rats and rabbits (23, 24). Others have characterized Clara cell differentiation based on ultrastructure (25), CC10 expression as related to ultrastructure (23, 24) and P450 expression as related to ultrastructure (18) in rats, rabbits, and hamsters, but not in mice. An additional pathway for differentiation of nonciliated bronchiolar cells during postnatal development is the formation of ciliated cells (36). Ciliogenesis occurs first in upper airways and later in peripheral airways (26). How the timing of ciliogenesis is related to nonciliated cell differentiation, especially P450 expression, has not been evaluated. This study addresses the following questions: (1) What is the relationship between Clara cell secretory protein expression and ciliogenesis? (2) How does the developmental expression of the P450 monooxygenase system proteins correlate with Clara cell secretory protein expression?, and (3) How does cellular protein distribution during differentiation correlate to P450 activity levels in the same site?

	Materials and Methods

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Animals and Lung Preparation

Female timed-pregnant and male Swiss Webster mice were obtained from Charles River Breeding Laboratories, Wilmington, MA. All animals were housed at least 7 days in animal facilities at the University of California after receipt from the suppliers before being used in experiments. Food and water were provided ad libitum. All animals were anesthetized with pentobarbital sodium (60 mg/kg), the trachea cannulated, and the animals killed by exsanguination. Gender was determined by grossly examining gonads from all animals younger than 1 month. For immunocytochemistry, lungs were inflated and fixed for 1 h with 1% paraformaldehyde in 0.1 M phosphate buffer, via a tracheal cannula, at 30 cm pressure. The lungs were removed, sliced and embedded in paraffin within 24 h of harvesting the tissue.

Chemicals

Waymouth's MB/752/1 medium (Gibco Labs, Grand Island, NY) was prepared by dissolving the powder in water, adding sodium bicarbonate and sterile filtering. F12 Nutrient Mixture (Ham's) with L-glutamine without sodium bicarbonate (Gibco Labs, Grand Island, NY) was prepared by dissolving the powder in pyrogen free water, adding L-cystine, Hepes and sodium bicarbonate and sterile filtering. Supplemented F12 was prepared as described by Wu and associates (27), without cholera toxin. Compatigel agarose was obtained from FMC BioProducts (Rockland, ME). All fixatives and embedding reagents were obtained from Electron Microscopy Sciences (Fort Washington, PA). The avidin-biotin peroxidase reagents used to detect the location of these antigens were obtained in kit form from Vector Laboratories (Burlingame, CA). Naphthalene was purchased from Fisher Scientific (Fairlawn, NJ).

Immunocytochemistry

Immunoperoxidase method. Paraffin sections (5-6 microns thick) from 3 mice per age group were labeled for components of the P450 monooxygenase system using antibodies produced in goats against purified rabbit pulmonary isozyme 2B4 and CYP reductase and characterized previously (28, 30). Antibodies to purified mouse CYP2F2 and purified rat CC10 were produced in rabbit and characterized previously (2, 29). Hydrated sections were treated with 3% H₂O₂ to block endogenous peroxidase and were then incubated for 24 h at 4°C with the above antibodies (18). Dilutions of these antibodies ranged from 1:10,000 to 1:1,000,000, similar to dilutions that were used to evaluate the protein expression in adult mice (30). The avidin- biotin procedure followed the dilutions outlined by the supplier of the reagents. Controls included the substitution of primary antibody with sera from nonimmunized goats or rabbits, or with phosphate-buffered saline. Fields were recorded on an Olympus Provis A052 microscope with a Sony digital photo camera attached to a Power Macintosh. Images were composed in Adobe Photoshop^TM and printed on a Codonics NP-1600 printer.

Dual-labeled fluorescent immunocytochemistry. To compare CC10 expression in Clara cells and tubulin expression in ciliated cells, paraffin sections (5-6 microns thick) from 3 mice per age group were stained for purified rat CC10 produced in rabbit (18, 29) and -tubulin IV produced in mouse (Biogenex Laboratories, San Ramon, CA). Hydrated sections were permeabilized with 0.5% Triton X-100. Because the -tubulin IV antibody is a mouse monoclonal an additional blocking step included using horse -mouse antibody. The primary antibodies were prepared at twice the desired concentrations, mixed together, and incubated on the tissue overnight. CC10 final dilution was 1:20,000 and -tubulin IV final dilution was 1:300. CC10 was detected using ELF^TM-AP immunohistochemistry kit from Molecular Probes (Eugene, OR). -tubulin IV was detected with Texas Red-Conjugated AffiniPure Donkey -mouse IgG from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). The antibodies were visualized on an Olympus BH-2 microscope with a BH2-RFC reflected light fluorescence attachment. An ultraviolet excitation (DM400) filter was used for CC10 and a green excitation (DM570) filter was used for -tubulin IV. Images were recorded with an Olympus PM-10ADS automatic photomicrographic system.

Metabolism of Naphthalene

Five adult males and eleven males from two litters (7-day and 14-day old) were used to evaluate metabolism of naphthalene in distal bronchioles. Metabolism of naphthalene to glutathione adducts was assessed in airway preparations microdissected from the lung as described in Plopper and associates (31). After exsanguination, the thorax was opened and the trachea was cannulated. The lungs were removed from the chest cavity, inflated with 1% Compatigel agarose in Waymouth's medium at 37°C, and plunged into ice-cold F12 medium for 30 min. The terminal bronchioles were isolated by blunt dissection under a Wild M-8 stereomicroscope and placed in fresh supplemented F12. The bronchioles were preincubated for 30 min at 37°C and then incubated in a total volume of 0.1 ml supplemented F12 containing 0.5 mM naphthalene and 1 mM reduced glutathione for 45 min at 37°C (32). Incubations were terminated by addition of 3 volumes methanol. The tissue was separated from the medium by centrifugation and dissolved in 1 M sodium hydroxide. An aliquot was assayed for protein content by the Lowry method (33) using bovine serum albumin as a standard. Supernatant from the incubations was dried under vacuum and redissolved in deionized water. Glutathione conjugates were analyzed by HPLC on C₁₈ columns (5 × 25 cm) using a mobile phase of 6% acetonitrile/0.6% triethamine/93.4% water adjusted to pH 3.1 with phosphoric acid, at 1 ml/min. Adducts were quantitated at 260 nm. Naphthalene dihydrodiol has an extinction coefficient identical to glutathione adducts of naphthalene and was used as a standard (34).

Statistical Analysis

Differences between groups was determined by one-way analysis of variance (ANOVA) and determination of significance was based on Bonnferoni-Dunn as P < 0.05.

	Results

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CC10 Distribution and Tubulin Expression

The only cells to label positively for CC10 were nonciliated epithelial cells. CC10 antigen was not detected in cells of any airway level in pseudoglandular stage mouse lungs (Figure 1, 16 DGA). A few nonciliated cuboidal cells were weakly positive for CC10 in proximal airways of canalicular stage lungs (Figure 1, 18 DGA; Figure 3, 18 DGA). There were also very few cells that were negative for CC10, but weakly positive for tubulin (Figure 3, 18 DGA). In saccular stage lungs, the majority of nonciliated cuboidal epithelial cells in proximal airways were positive for CC10 antigen. The most intense reaction occurred on the apical edges of the cells (Figure 1, 19 DGA; Figure 3, 19 DGA). Again, there were also a few cells that were positive for tubulin. CC10 antigen was first detectable in terminal bronchiolar cells during the saccular stage (Figure 2, 19 DGA). The cells closest to the alveolar duct junction were not positive. A small percentage of more proximal nonciliated cells were strongly positive; a larger percentage of cells were weakly positive. Again, the strongest labeling was in the apical region of the cells. At one day postnatal age (1 DPN), there is positive labeling in nonciliated cells of both proximal and distal airways. In lobar bronchi, all nonciliated cells are intensely labeled for CC10 antigen (Figures 1 and 3). The remaining cells were positive for tubulin (Figure 3). In terminal airways, nonciliated cells closest to the alveolar duct junction are squamous and not positive for CC10, but cells more proximally situated in the same bronchiole are positive for CC10 in their apical surface (Figure 2). In terminal bronchioles at 4 DPN, there are more cells that label positively for CC10, the intensity of the reaction product is darker and the number of squamated cells has decreased (Figure 2). The intensity and distribution of immunoreactive CC10 at 7 DPN appear less than at 4 DPN (Figure 1), but labeling is increased by 10 DPN (Figure 2). Lobar bronchi of 4 DPN mice have less intense labeling than 1 DPN mice (Figure 1). By 14 DPN, immunoreactive CC10 expression is at adult intensity and distribution in both terminal bronchioles and lobar bronchi (Figures 1-3).

View larger version (82K): [in this window] [in a new window]   Figure 1.   Immunocytochemical localization of Clara cell secretory protein (CC10) in lobar bronchi of developing mouse lung. Paraffin sections from all ages were incubated at the same time with an optimal dilution of CC10 antibody (1:10,000). Immunoreactive protein was first detected in nonciliated cuboidal cells (arrowheads) of proximal airways of 18 DGA mice. Ciliated cells (arrows) did not label for CC10 at any age during development. Magnification bar represents 20 µm.

View larger version (73K): [in this window] [in a new window]   Figure 3.   Fluorescent dual immunocytochemical localization of CC10 and -tubulin IV protein in terminal bronchioles (TB) and lobar bronchi (LB) in developing mice. CC10 antibody binding is represented by green fluorescence and tubulin associated with ciliated cells is represented by red fluorescence (see MATERIALS AND METHODS). Labeling for CC10 appeared first in nonciliated cuboidal epithelial cells (arrowheads) of proximal airways at 18 DGA. Labeling for -tubulin first appeared in ciliated cells (arrows) at 19 DGA. CC10 antibody dilution was 1:20,000, tubulin antibody dilution was 1:300. Magnification bar represents 20 µm.

View larger version (60K): [in this window] [in a new window]   Figure 2.   Immunocytochemical localization of Clara cell secretory protein (CC10) in terminal bronchioles of developing mouse lung. The terminal bronchioles imaged are from the same sections used in Figure 1. Immunoreactive protein was first detected in nonciliated cuboidal cells (arrowheads) of terminal bronchioles of 19 DGA mice. As in lobar bronchi, ciliated cells (arrows) did not label for CC10 at any age during development. The terminal bronchiolar-alveolar duct junction (TB/AD) is labeled for each airway. Magnification bar represents 20 µm.

CYP2B Distribution

There was no positive staining for immunoreactive CYP2B in the pseudoglandular stage (16 DGA), or canalicular (18 DGA) stage mouse lungs, but there was weak labeling in saccular stages (Figure 4, 19 DGA). Positive reaction was found in more proximal airways on the apical portion of a few cuboidal epithelial cells. In postnatal mouse lung, the proximal airway cells contained stronger labeling than the distal airway cells, and more cells were positive. There was a large increase in the immunoreactive CYP2B labeling from 19 DGA to 1 DPN. In proximal airways, both ciliated and nonciliated cells were positive (Figure 4), with stronger labeling found on the apical surface of the cells. In distal airways, a vast majority of nonciliated cells were negative for CYP2B. The remaining nonciliated cells were moderately positive (Figure 5). At 4 DPN, the nonciliated cells in the proximal airways had well-formed apical projections and contained cytoplasmic labeling on the luminal side of the cells (Figure 4). In the distal airways, the nonciliated cells were positive for CYP2B, although the cells closest to the alveolar duct were negative (Figure 5). At 10 DPN, all of the proximal nonciliated cells were positive and a small amount of reaction product was found on the ciliary surface of ciliated cells (Figure 4). In the terminal bronchioles there were some positive and some negative nonciliated cells (Figure 5). At 14 DPN, most of the proximal airway cells were positive, while only about 50% of the distal airway cells were positive (Figures 4 and 5). Adult levels of CYP2B protein expression were achieved between 10 and 14 DPN for lobar bronchi (Figure 4) and between 21 and 28 DPN in terminal bronchioles (Figure 5).

View larger version (70K): [in this window] [in a new window]   Figure 4.   Immunocytochemical localization of CYP2B protein in lobar bronchi of developing mouse lung. Paraffin sections from all ages were incubated at the same time with an optimal dilution of CYP2B antibody (1:20,000). Immunoreactive protein was first detected in nonciliated cuboidal cells (arrowheads) of proximal airways at 19 DGA. Ciliated cells (arrows) labeled lightly for CYP2B. Magnification bar represents 20 µm.

View larger version (67K): [in this window] [in a new window]   Figure 5.   Immunocytochemical localization of CYP2B protein in terminal bronchioles of developing mouse lung. The terminal bronchioles imaged are from the same sections used in Figure 4. Immunoreactive protein was first detected in nonciliated cuboidal cells (arrowheads) of terminal bronchioles of 1 DPN mice. The terminal bronchiolar-alveolar duct junction (TB/AD) is labeled for each airway. Magnification bar represents 20 µm.

CYP2F2 Distribution

The only cells to positively label for immunoreactive CYP2F2 protein in mouse lung of any age were airway epithelial cells. In fetal mouse lung there was no detectable labeling of immunoreactive CYP2F2 protein at the antibody dilution that stained postnatal mouse lung until the saccular stage (Figure 6). At this stage, there were a few cuboidal epithelial cells lining proximal airways that had faint positive cytoplasmic staining. Early in the postnatal period (1 DPN), less than half of the nonciliated epithelial cells of the distal airways were faintly labeled (Figure 7). There was a dramatic increase in positive reaction to CYP2F2 antigen at 4 DPN: the heaviest labeling was in the nonciliated cells in the most proximal position in the terminal bronchiole. Nonciliated cells closer to the distal end of the bronchiole had less labeling. The labeling was darker in the apical portion of the cell than in the basal portion of the cell (Figure 7). By 7 DPN, labeling in the distal airways had decreased, but was still darker on the apex (Figure 7). The labeling was very intense for CYP2F2 at 14 DPN in all nonciliated airway epithelial cells (Figure 7). Labeling decreased in terminal bronchioles for a second time at 28 DPN (Figure 7). In adult mouse lung all of the nonciliated cells labeled positively, while most of them were intensely positive. The darkest labeling occurred in the basal portion of the cells (Figure 7). In lobar bronchi of the same animals, the labeling of the nonciliated epithelial cells had the same pattern of expression as the terminal bronchioles, except that the labeling was always more intense than in terminal bronchioles on the same section (Figure 6).

View larger version (88K): [in this window] [in a new window]   Figure 6.   Immunocytochemical localization of CYP2F2 protein in lobar bronchi of developing mouse lung. Paraffin sections from all ages were incubated at the same time with an optimal dilution of CYP2F2 antibody (1:1,000,000). Immunoreactive protein was first detected in nonciliated cuboidal cells (arrowheads) of proximal airways at 19 DGA. Ciliated cells (arrows) did not label for CYP2F2. Magnification bar represents 20 µm.

View larger version (76K): [in this window] [in a new window]   Figure 7.   Immunocytochemical localization of CYP2B protein in terminal bronchioles of developing mouse lung. The terminal bronchioles imaged are from the same sections used in Figure 6. Immunoreactive protein was first detected in nonciliated cuboidal cells (arrowheads) of terminal bronchioles of 1 DPN mice. The terminal bronchiolar-alveolar duct junction (TB/AD) is labeled for each airway. Magnification bar represents 20 µm.

CYP Reductase Distribution

Airway epithelial cells were the only cells to positively label for immunoreactive CYP reductase in mouse lung of any age. There were no cells that labeled positively for reductase during the pseudoglandular stage. Airway epithelial cells were lightly labeled in proximal airways in lungs from the canalicular stage, although this labeling was very diffuse (Figure 8). During the saccular stage, there was light labeling localized to the luminal side of airway epithelial cells (Figure 8). At 1 DPN (Figure 9), there was light labeling over all of the cells that lined the distal airways (nonciliated and ciliated) and by 7 DPN there was dark labeling over the nonciliated cells and light labeling over the ciliated cells (Figure 9). By 14 DPN, the pattern of labeling was similar to that of adult terminal bronchioles: there was intense labeling over the nonciliated cells while the labeling over the ciliated cells remained light (Figure 9). In lobar bronchi of the same animals, labeling of the nonciliated cells was always more intense than labeling in the ciliated cells (Figure 8). The pattern of CYP reductase expression in the lobar bronchi reached adult levels between 10 and 14 DPN.

View larger version (100K): [in this window] [in a new window]   Figure 8.   Immunocytochemical localization of CYP reductase protein in lobar bronchi of developing mouse lung. Paraffin sections from all ages were incubated at the same time with an optimal dilution of reductase antibody (1:25,000). Immunoreactive protein was first detected in nonciliated cuboidal cells (arrowheads) of proximal airways at 18 DGA. Magnification bar represents 20 µm.

View larger version (75K): [in this window] [in a new window]   Figure 9.   Immunocytochemical localization of CYP reductase protein in terminal bronchioles of developing mouse lung. The terminal bronchioles imaged are from the same sections used in Figure 8. Immunoreactive protein was first detected in nonciliated cuboidal cells (arrowheads) of terminal bronchioles of 1 DPN mice. The terminal bronchiolar-alveolar duct junction (TB/AD) is labeled for each airway. Magnification bar represents 20 µm.

CYP Activity

Overall, total metabolism of naphthalene was lowest in airways from 7-day animals and highest in airways from adult animals (Figure 10). CYP2F2 activity was determined by the metabolism of naphthalene to dihydrodiol and glutathione conjugate 2 in airways isolated from mouse lung. The majority of the dihydrodiol is produced by the CYP2F2 enzyme, although a small portion may be produced by the 2B enzyme. Dihydrodiol formation was significantly lower in airways from 7-day mice than in airways from adults, but was near adult levels in airways from 14-day mice. Conjugate 2 formation in airways from 7 or 14 DPN mice was not different than in airways from adult mice. CYP2B activity is characterized by the formation of conjugates 1 and 3 in naphthalene metabolism. Conjugate 3 formation did not change significantly with an increase in age, but conjugate 1 formation did increase with increasing age and was significantly lower in airways from both 7- and 14-day mice as compared with airways from adult mice.

View larger version (35K): [in this window] [in a new window]   Figure 10.   Naphthalene metabolism in isolated airways from 7-day old, 14-day old and adult mice. * Indicates significantly different from adult at P < 0.05.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

This study demonstrates that the cytodifferentiation of Clara cells in terms of expression and distribution of two proteins highly expressed in Clara cells of adult mice, Clara cell secretory protein (CC10) and the CYP monooxygenase system, is a postnatal event and varies by airway level. The pattern of changes are summarized for lobar bronchi in Table 1 and terminal bronchioles in Table 2. The expression of CC10, the principal protein stored in the secretory granules of Clara cells, was detected in the apex of proximal nonciliated airway cells right before birth and reached levels found in adult mice at the same postnatal age (14 DPN) in both proximal and distal nonciliated cells. CC10 was found only in cuboidal nonciliated cells and not observed in cells expressing ciliary tubulin. CC10 expression preceded expression of tubulin associated with differentiated ciliated cells in both proximal and distal airways. CYP reductase expression occurred at the same time and with the same distribution as CC10 expression. CC10 and reductase expression preceded the development of P450 isozymes 2F and 2B. CYP2B was first detected in very few nonciliated cells in proximal airways right before birth, and was expressed first in the apex of the cells. In bronchioles, CYP2B was detected postpartum. CYP2B expression similar to that found in adult mice was present earlier (14 DPN) in lobar bronchi than in terminal bronchioles (28 DPN). CYP2F2 expression was also first detected in the apex of nonciliated cells immediately before birth, but did not reach levels that are found in adult mice until after 28 DPN. P450 activity as measured by the metabolism of naphthalene increases with age.

In rats, rabbits (23), and hamsters (24), biosynthesis of smooth endoplasmic reticulum in differentiating Clara cells closely correlates with CC10 expression. Using CC10 expression as a marker for Clara cell maturity, Clara cells of mice mature at the same time in proximal and distal airways, much like the hamster (24), indicating that Clara cells in distal airways differentiate over a shorter period of time than in the proximal airways. Based on ultrastucture, Ten Have-Opbroek (35) found that mouse Clara cells acquire a mature phenotype by 20 DPN. They detected Clara cell antigen in granules in 20 DPN mice, but expression at younger ages was not documented. This study shows that biosynthesis of CC10 begins prior to birth in the mouse and it begins earlier in proximal airways and slightly later in distal airways. In addition, the synthesis of CC10 occurs in nonciliated cells before the earliest synthesis of tubulin associated with ciliated cells. Whether this is true of other species has not been evaluated. However, the Clara cell has been documented to be the progenitor cell for bronchiolar epithelial cells (nonciliated and ciliated) after acute injury to cililated cells (36). It also has been shown to be the progenitor of both nonciliated and ciliated cells during the normal process of bronchiolar epithelial differentiation in the fetus (37).

We found that, in mice, the immunodetectable proteins for CC10 and the CYP monooxygenase system are first synthesized throughout the lung at the same age (by 19 DGA), but reach adult levels of expression at different ages. In proximal airways, CC10, CYP reductase and CYP2B are at adult levels earliest (14 DPN) while isozyme 2F reaches adult levels later (after 28 DPN). In terminal airways, CC10 and CYP reductase expression reaches adult levels at the same time as in proximal airways (14 DPN). CYP2B does not reach adult expression levels until 28 DPN and CYP2F2 is later still. The time course of these proteins in relation to each other is species-specific. In hamsters, CC10, CYP and reductase are all first detected at the same age (15 DGA) (16, 24). But in rabbits, CC10 and reductase are detected at the same time, while CYP is detected 2 days later (18, 23). In rats, CC10 expression precedes both P450 and reductase (17, 23). The adult expression of CC10 preceded P450 isozymes and reductase isozymes in the rabbit and the rat (17, 18, 23). The expression of CC10 and P450 isozymes in relation to each other in hamsters is not clear from the literature.

Our results for CYP activity indicate that CYP activity in distal airways from mouse lung increases with increasing age, and appears to correspond with changes in the expression of CYP2F2 protein. This relationship between P450 protein expression and expression of activity is not the same in other species. In rabbits, CYP2B, CYP4B and reductase protein expression reaches levels found in adult rabbits by 28 DPN, but metabolism of diagnostic substrates (ethoxyresorufin and pentoxyresorufin) is only about 50% of adult levels at that age (18). In rats, CYP2B and reductase protein expression reaches adult levels at 10 DPN, but metabolism (pentoxyresorufin activity) does not reach adult levels until 50 DPN (17). There is a similar discordance between CYP protein expression in hamsters (16). A possible reason for the differences between studies is that the previous studies have examined activity levels in whole lung homogenates, whereas this study defined activity in one site, distal bronchioles. The discrepancy between data obtained from whole lung homogenates and data obtained from only the subcompartment of interest (i.e., terminal bronchioles) may result from several factors, most based on the fact that proteins from all cell types are mixed together in the whole lung homogenates. In whole lung homogenates, all 40 pulmonary cell types are present instead of just the cell types found in target subcompartments. For example, in neonatal lungs there may be inefficient reductase-cytochrome coupling in some lung cell types other than those associated with terminal bronchioles. There may also be inhibitors present in some neonatal lung cell types not associated with terminal bronchioles. There may also be apoP450 found in those cells. Another factor could be the concentration of substrate used to measure activity. In our case, the concentration of naphthalene was saturating. There is evidence that the apparent K_m of naphthalene metabolism increases in mice made tolerant to naphthalene (38). This is most likely due to a change in the proportion of isozymes involved in naphthalene metabolism (CYP2F2 and CYP2B). In differentiating Clara cells, the opposite may be true: the apparent K_m may decrease during differentiation due to an increase in the relative proportion of naphthalene metabolized by CYP2F2 rather than CYP2B.

A previous study showed that neonatal rabbits with low pulmonary levels of P450 activity were more susceptible to Clara cell injury by 4-ipomeanol than were adult rabbits (15). In a recent study, we found neonatal mice to be more sensitive to the cytotoxic effects of naphthalene on the Clara cell than were adult mice (39). The present study confirms that, like neonatal rabbits, neonatal mice have low levels of pulmonary P450 activity despite having elevated susceptibility to bioactivated toxicants. When evaluating the potential for compounds in neonatal lung to be carcinogenic or cytotoxic, bioactivation capabilities are not the only factor to consider. Another factor to consider must be whether or not there is a discordance between the levels of activating and deactivating enzymes and cellular antioxidants such as glutathione, in the differentiating cells.

In summary, the present study demonstrates an age- dependent pattern in CC10, CYP reductase, CYP2B and CYP2F2 expression and in CYP activity in mouse lung. While this is similar to the patterns found in rabbit, rat, and hamster, there are clear species-, airway- and protein-specific differences. The discordance between cellular expression of protein and detectable CYP activity observed in other species as opposed to the close correlation we observed in the mouse emphasizes the need to restrict definition of activity for an enzyme to the subcompartment in which its cellular expression is being characterized.