* Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine; University of California-Davis, Davis, California 95616-8732 and
Division of Statistics, University of California-Davis, Davis, California 95616-8732
Received ; accepted November 13, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: cigarette smoking; tobacco smoke; bronchiolar injury and repair; epithelium; Clara cells; mice.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
One of the regions of the lung particularly prone to injury is the distal conducting airways (bronchioles) at the junction of the terminal bronchiole with the gas exchange region. Due in part to reduction in airway diameter, branching history, and cellular composition, terminal bronchioles are a primary target zone for many oxidant air pollutants, metabolically activated lung toxicants, and toxic particles. In the mouse and other species, the principal bronchiolar cell type, the nonciliated bronchiolar (Clara) cell, contributes to this site selectivity of injury. There are at least two reasons the Clara cell is selectively injured: high abundance within the centriacinar injury target zone, and cellular metabolic characteristics that promote activation of xenobiotics. The Clara cell is the principal site of xenobiotic metabolism within the lung by the cytochrome P450 monooxygenase system (Devereux et al., 1989; Plopper et al., 1987
). Clara cell metabolism of xenobiotics by the P450 system, thereby contributing to their own susceptibility, has been further confirmed by metabolite binding within Clara cells (Boyd 1977
; Boyd et al., 1980
). The injury model we will use exploits the metabolic characteristics of Clara cells to selectively injure them in a well-defined pattern with the bioactivated cytotoxicant naphthalene (Plopper et al., 1992a
, b
; Van Winkle et al., 1995
).
Naphthalene is a ubiquitous environmental pollutant (for review see ATSDR, 1990). It is a major component of cigarette smoke (Schmeltz et al., 1976; U.S. EPA 1992
) as well as aged and diluted sidestream smoke (Witschi et al., 1997
), is present in automobile emissions, and is widely used in the synthesis of insecticides (Life Systems, Inc., 1990; Arey et al., 1987
). Susceptibility of mice to naphthalene correlates with formation of the toxic metabolite by cytochrome P450 isozyme 2F (CYP2F), particularly in Clara cells within the injury target zone, distal bronchioles (Buckpitt et al., 1995
). Injury is most severe in distal airway regions and increases in a dose-dependent manner up the airway tree (Plopper et al., 1992b
). Ciliated cells survive and are not injured regardless of the dose. Mouse Clara cells injured by naphthalene vacuolate and then exfoliate 12 days after injury by a 200-mg/kg dose of naphthalene (Van Winkle et al., 1995
). In this study we ask the question whether a history of prior exposure to ETS modulates the pattern of naphthalene-induced distal conducting airway Clara cell injury and subsequent repair.
There are many human lung diseases (including cancer, bronchitis, bronchiolitis, and asthma) that appear to be the result of abnormal repair. The hypothesis we are testing is that exposure of nonsmokers to environmental tobacco smoke predisposes their lungs to injury by inhaled and ingested pollutants and that smoke exposure compromises the ability of the lung to repair following acute injury. The long-term sequela may be airway remodeling. It is not known if cigarette smoke exposure before bronchiolar injury results in a site-specific bronchiolar lesion similar to that without prior ETS exposure, increases or decreases the susceptibility of Clara cells to additional cytotoxicants, or alters the repair capability of bronchiolar epithelium. To address these questions we used a combination of histologic and morphometric approaches based on detection of site-specific responses using airway microdissection to isolate specific regions of the lung. We found that a week of prior exposure to tobacco smoke, using a regimen that mimics an occupational exposure 5 days/week, 6 h/day, resulted in prolonged bronchiolar repair from naphthalene injury in terminal bronchioles.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Exposure protocol.
Mice were exposed 5 days/week, 6 h/day to approximately 1 mg/m3 total suspended particulates (TSP) of aged and diluted sidestream smoke (ADSS) as a surrogate for ETS (for exposure conditions see Table 1). The remaining 2 days/week, mice were not exposed to ADSS and received only filtered air. Both filtered air (sham)-exposed and smoke-exposed mice were housed continuously in plastic shoebox cages in updated Hinners chambers. The exposure system and monitoring methods have been previously described in detail (Joad et al., 1995
; Teague et al., 1994
). The ADSS is generated by a modified ADL/II smoke exposure system (Oakridge National Laboratory) using conditioned cigarettes from the Tobacco and Health Research Institute of the University of Kentucky. Two cigarettes at a time were smoked under Federal Trade Commission conditions in a staggered fashion at a rate of 1 puff (35 ml, 2-s duration)/min. The sidestream smoke was drawn into a conditioning chamber where it was aged and diluted. The sidestream smoke was further diluted as it was passed into the exposure chambers in such a way as to produce total suspended particulate (TSP) concentrations of 1.0 mg/m3. Relative humidity, temperature, TSP, and nicotine concentrations were sampled daily over the 3-week exposure time period (Table 1
). After 1 week of exposure to either filtered air (FA) or ADSS, the mice were injected intraperitoneally (ip) with either corn oil carrier or 200 mg/kg NA and returned to either ADSS or FA for 1 or 14 days. See timeline of exposure (Fig. 1
) and the list of the experimental groups (Table 2
) for further details of the experimental design.
|
|
|
Quantitative histopathology.
The abundance of normal and cytotoxic bronchiolar epithelial cells was analyzed using morphometric procedures previously used to define changes in bronchiolar epithelium after naphthalene injury (Plopper et al., 1992a) and discussed in detail by Hyde et al. (1990). Only terminal bronchioles from the same two minor daughter airways were evaluated for each animal, to minimize the possible effect of the differential deposition of tobacco smoke on the results. All terminal bronchioles visible on sections from two separate block faces from each animal (five terminal bronchioles was the minimum number obtained) were evaluated. All the measurements were made using high-resolution 1.0-µm sections. The volume densities (Vv) of five categories of cells (nonciliated Clara, ciliated, vacuolated, squamous, and other) were defined by point (P) and intercept (I) counting of terminal bronchiolar epithelial vertical profiles using a cycloid grid and Stereology Toolbox (Morphometrix) on images collected as described earlier. Vv was calculated using the formula
![]() | (1) |
where Pp is the point fraction of Pn, the number of test points hitting the structure of interest, divided by Pt, the total points hitting the reference space (epithelium). The surface area of epithelial basement membrane per reference volume (Sv) is determined by point and intercept counting and calculated using the formula
![]() | (2) |
where Io is the number of intersections with the object (epithelial basal lamina) and Lr is the length of the test line in the reference volume (epithelium). The thickness of the epithelium, or volume per unit area (Vs) of basal lamina (µm3/µm2), was calculated using the formula for arithmetic mean thickness (t)
![]() | (3) |
Immunohistochemistry.
The presence of the differentiation marker proteins was detected using specific antibodies: rabbit anti-rat CC10 and rabbit anti-mouse CYP2F2 (Nagata et al., 1990; Singh and Katyal, 1984
). The avidin-biotin peroxidase procedure was used to identify antibody binding sites. The procedure was the same as outlined by the supplier (Vector Labs, Burlingame, CA). Diaminobenzidine with nickel enhancement was used as the chromagen. To eliminate nonspecific binding of the primary antibody, sections were blocked with 1% bovine serum albumin. Controls included substitution of phosphate-buffered saline (PBS) for the primary antibody. The optimal dilution at which there was positive staining with minimal background staining was determined separately for each antibody using a series of dilutions on sections from corn oil- and naphthalene-treated animals. Rabbit antisera specific for rat CC10 was a generous gift from Dr. Gurmukh Singh (Veterans Affairs Medical Center, Pittsburgh, PA). Rabbit antisera specific for mouse CYP2F2 was provided by Dr. Henry Sasame (National Institutes of Health, Bethesda, MD).
Scanning electron microscopy (SEM).
Samples were microdissected from the main axial path of the cardiac lobe so that distal bronchioles, including terminal bronchioles, and distal and proximal intrapulmonary bronchi were exposed. Samples for SEM were dehydrated in a graded ethanol series, rinsed in a graded ethanol toluene series, returned to ethanol immersed in hexamethyldisilizane (Polysciences, Inc., Warrington, PA) for 5 min, then air dried overnight at room temperature, as described previously (Nation, 1983). Samples were mounted on carbon-coated stubs and coated with gold in a sputter-coater (Polaron II E5100) with 2.5 kV acceleration voltage in an argon atmosphere with a current of 10 mA for 2 min. Samples were imaged with a Phillips SEM 501 at 500 and 1250 magnification (Malwah, NJ).
Statistical analysis.
Morphometric data from all terminal bronchioles present in two block faces, a minimum of five terminal bronchiolar epithelial profiles, were used to calculate Vv or Vs per animal. The value per animal was used to calculate the mean and standard deviation for each group of animals at a time point. Four animals were measured at each time point. Because the data failed the usual ANOVA assumptions regarding homogeneity of variance, data were analyzed by use of an ANOVA weighted by the reciprocal of the "in treatment" variance (Neter et al., 1990). The significance of post hoc comparisons was determined using Tukey's HSD method at the 0.05 level (Neter et al., 1990
).
Reagents.
Naphthalene was purchased from Fisher Scientific (Pittsburgh, PA). Corn oil (Mazola) was manufactured by Best Foods/CPC International Inc. (Englewood Cliffs, NJ). Glutaraldehyde, paraformaldehyde, Azure II, and Araldite 502 were obtained from Electron Microscopy Sciences (Fort Washington, PA). Osmium tetroxide was purchased from Polysciences, Inc. (Warrington, PA). Methylene blue was from JT Baker Chemical (Phillipsburg, NJ).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
Epithelial thickness (t), or the total volume (µm3) of all the epithelial cells per area of basal lamina surface (µm2), was measured for all four treatment groups and analyzed using a weighted ANOVA for treatment effect, which was significant at p 0.0095 (Fig. 7
). Sham-treated controls, FACOFA, had the highest mean value (7.86 ± 0.76 µm3/µm2). The TSCOFA and FANAFA groups had lower mean epithelial thickness (6.56 ± 0.98 µm3/µm2 and 6.78 ± 0.50 µm3/µm2, respectively) but were not significantly different from sham-treated controls. In contrast, epithelial thickness was significantly reduced (5.42 ± 0.86 µm3/µm2) in the TSNAFA group compared to controls. The volume per surface (Vs) of the three main epithelial cell types is shown in Figure 8
. There was a significant treatment effect for both the Vs of nonciliated cells (p
0.0001) and the Vs of squamous cells (p
0.0004). However, the treatment effect was found to be insignificant for Vs of ciliated cells. When groups were compared against each other using Tukey's post hoc comparison test, Vs of nonciliated cells was significantly different (p < 0.05) for all treated groups (TSCOFA, FANAFA, TSNAFA) compared to controls. In addition, the Vs of nonciliated cells for the TSNAFA group was significantly different from the group that received naphthalene only, FANAFA (p < 0.05). The Vs of squamous cells was only significantly different from sham-treated controls (FACOFA) for the two groups that received naphthalene (FANAFA and TSNAFA). When each of the cell types was expressed as the volume fraction of total epithelial volume (Vv, Table 3
), only the fractions for nonciliated, squamous, and "other" cell classifications had a significant treatment effect by ANOVA. Again, the two groups treated with naphthalene (FANAFA and TSNAFA) were significantly different from controls for Vv nonciliated, Vv squamous, and Vv "other" cell classifications. In addition, for Vv nonciliated, the group exposed to tobacco smoke only (TSCOFA) was also significantly different from controls (FACOFA) and the group that received both naphthalene and smoke (TSNAFA).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
As naphthalene injury increases in severity with increasing dose, it affects more proximal airway generations (Plopper et al., 1992b). One goal of this study was to determine if prior ETS exposure would increase the severity of acute injury (as indicated by histopathology at 1 day after naphthalene injury) to include large airways such as lobar bronchi that are not normally injured by this dose of naphthalene in this strain and sex of mouse. The extent of naphthalene injury was not expanded to lobar bronchi by prior exposure to ETS. However, within the injury target zone, terminal bronchioles, injury due to naphthalene was slightly attenuated in mice that had been previously exposed to tobacco smoke. The injury was slightly less extensive; fewer Clara cells were vacuolated and exfoliated than in the terminal bronchioles from the group treated with naphthalene only (compare Figs. 2F and 2G
). It should be emphasized that this effect was slight and was only present in the most distal airways. The reduced Clara cell injury found in this site may reflect the conditioning effect of prior exposure to naphthalene in ETS. ADSS generated at 78 mg/m3 TSP contains 400 ± 60 µg/m3 of naphthalene (Witschi et al., 1997
). The level of ADSS used in our current study (1 mg/m3) would yield a chamber concentration of approximately 1.7 µg/m3 of naphthalene. Repeated prior treatment of mice with naphthalene has been shown to induce a state of naphthalene tolerance in the lung, whereby Clara cells become resistant to injury by even high-challenge doses (Lakritz et al., 1996
; O'Brien et al., 1989
). It is possible that even this low level of prior exposure to naphthalene present in the ETS caused a tolerizing effect and contributed to the slight resistance to injury seen in the TSNAFA group.
Initially it may seem paradoxical that prior cigarette smoke exposure produces naphthalene tolerance and protects the Clara cell from injury, but also inhibits repair and regeneration processes that occur after naphthalene injury. As the cellular mechanisms for both cytotoxicity and failure of repair remain unknown, we can only speculate as to the mechanism(s) involved. In the naphthalene injury and repair model, acute injury and exfoliation of the injured cells precedes the reparative processes of cell proliferation and redifferentiation by several days. These are distinct events. It is possible that the cell alterations that result in tolerance and the alterations that occur to produce a lack of repair are independent processes involving separate pathways. Whether alterations in the ability of a cell to withstand a toxic challenge can also subsequently result in an inability to regenerate the epithelium remains unknown. However, several possible scenarios come to mind. Our experience with naphthalene tolerance in mice would suggest that persistence of squamous cells in distal bronchioles does not occur following prior repeated exposure to naphthalene (Lakritz et al., 1996). However, tobacco smoke is a complex mixture that contains many other compounds in addition to naphthalene. Prior exposure to one of the other components of smoke may be related to the lack of repair. It is also possible that the oxidant stress involved in prior smoke exposure or persistence of tobacco smoke metabolites may alter both the tolerance and repair response pathways.
The morphometric data indicate that there is a slight persistent effect of naphthalene alone in the FANAFA group at the 14-day time point. This is evidenced by slightly decreased average epithelial thickness, a decrease in Vs of nonciliated cells, and increased Vs squamated cells compared to FACOFA. However, this decrease is difficult to appreciate using standard light microscopic histopathology. It is only when a large number of airways are rigorously measured that this slight difference in the FANAFA group compared to controls (FACOFA) becomes apparent. The reason for this is amply evident when one examines the epithelium of the airway in 3-dimensions (see the terminal bronchiole of the FANAFA mice in Fig. 5C). The squamous areas are rather small and focal. In addition, when several bronchioles were examined by SEM, it was apparent that not all terminal bronchioles in the FANAFA contained patches of squamated epithelium. This underscores the importance of supplementing histopathology with rigorous morphometric measurements or 3-dimensional imaging when evaluating focal responses in pulmonary epithelium. We conclude that the bronchiolar epithelium can be considered "nearly" repaired in the FANAFA group 14 days after injury.
Very little is known about the acute pathologic changes that occur in response to concentrations of tobacco smoke that are 1 mg/m3 or less. Previous studies have deemed this exposure level the no observed effect limit, or NOEL, for formation of ETS-related DNA adducts (Lee et al., 1993). The only histopathologic changes reported at 4-fold higher doses of sidestream smoke (4 mg/m3) are hyperplastic and metaplastic changes in the nasal turbinates of rats after 90 days (von Meyerinck et al., 1989
). Another study found that 5 days of exposure to 1 mg/m3 ADSS had the sole effect of causing increased cell proliferation in the airways of a mouse strain (A/J) that is susceptible to tumors (but not in a nonsusceptible mouse strain, C57BL/6) (Rajini and Witschi, 1994
). However, there was no effect of this level of ADSS exposure on general histopathology. There have been few other studies that demonstrate an acute effect of moderate levels of sidestream tobacco smoke (
1 mg/m3) on deep lung respiratory epithelium in adult rodents. Our study supports this previously observed lack of histologic change; only slight epithelial alterations were observed, with a trend towards a more rounded epithelial cell phenotype in the terminal bronchioles. However, after 16 days in filtered air, the bronchiolar epithelium of ETS-exposed mice was thinner, with a slight reduction in nonciliated cells, although this was not statistically significant. Based on the increase in the squamous cell fraction in the TSNAFA group, we conclude that the effect of smoke and naphthalene on lung repair is at least additive. This unexpected finding may indicate that there was a slight persistent tobacco smoke-related effect on Clara cells even 16 days after the last tobacco smoke exposure. The mechanism of this potential Clara cell alteration is unknown at this time, but may reflect oxidative stress on Clara cells caused by the prior smoke exposure or the downstream effect of adduct accumulation within this metabolically active cell type.
It appears that prior tobacco smoke exposure both alters the normal redifferentiation of the Clara cell population after naphthalene injury as well as results in increased persistence of squamous cells within the terminal bronchioles. Lack of Clara cell redifferentiation is indicated by diminished expression of the secretory product CC10. Our previous studies have shown that after acute injury by naphthalene in distal bronchioles, the remaining cells squamate and lack markers of the two differentiated cell types found in this region, cilia or Clara cell secretory product (Van Winkle et al., 1995). Repopulation of the airway with a redifferentiated epithelium containing ciliated and Clara cells occurs over several weeks. Although there have not been previous studies that demonstrate an effect of tobacco smoke on the Clara cell redifferentiation that occurs as the epithelium regenerates in adult animals after acute injury, Ji et al. have shown that tobacco smoke exposure alters normal postnatal differentiation of Clara cells. During lung development in rats, ADSS exposure alters the normal Clara cell postnatal differentiation process whether the smoke exposure occurs only prenatally (Ji et al., 1998
) or only postnatally (Ji et al., 1994
). In contrast to our current findings, the effect of maternal smoke exposure alone on prenatal rat lung differentiation was increased CC10 expression (Ji et al., 1998
), whereas postnatal exposure had no effect on CC10 expression (Ji et al., 1994
). Acute Clara cell injury by bioactivated cytotoxicants during early postnatal lung development, when Clara cells are less differentiated, also results in a failure of bronchiolar repair that presents as persistent squamated epithelium in both rabbits and mice (Fanucchi et al., 1997
; Smiley-Jewell et al., 1998
). In rabbits, the unrepaired, squamated epithelium contains cuboidal cells with decreased expression of Clara cell differentiation markers including CC10 (Smiley-Jewell et al., 1998
).
It is possible that the lack of repair observed in our current study in mice exposed to both naphthalene and tobacco smoke may be due to the effect of naphthalene injury superimposed on undifferentiated proliferating cells already present in response to the preceding tobacco smoke exposure. Previous studies have indicated that ETS exposure enhances cell kinetic activity in terminal bronchioles in susceptible strains of mice (Rajini and Witschi 1994). Whether this is true for the strain used in our study is now under investigation. The delay in repair in our current study may also be attributed to an effect of prior ETS exposure on cell kinetic activity. Repopulation of the airway by cell proliferation both within the terminal bronchiole and at more proximal airway sites is an early step in the repair process after naphthalene injury and takes place from 1 to 4 days after naphthalene injection (Van Winkle et al., 1995
). Proliferation clearly occurs well before substantial redifferentiation in this model (Stripp et al., 1995
; Van Winkle et al., 1995
), so it is possible that a lack of proliferation would essentially impede epithelial redifferentiation. There is some support for this concept from studies of undifferentiated neonatal rabbit and mouse Clara cells that have decreased epithelial repair after naphthalene injury (Fanucchi et al., 1997
; Smiley-Jewell et al., 1998
). The effect of prior ETS exposure coupled with additional injury on cell kinetics remains unknown at this time and will be a subject of future research. ETS exposure also alters xenobiotic metabolizing enzymes in rat Clara cells (increasing both CYP1A1 and NADPH reductase protein expression) (Ji et al., 1994
). A shift in xenobiotic metabolizing or detoxifying enzymes in response to smoke exposure could also shift the intra- and extracellular targets of naphthalene metabolites to include targets required for the reparative process, thereby prolonging epithelial repair. These possibilities will be the subject of further research. To our knowledge, our study is the first to show in adult animals that a brief (5 day) prior exposure to a moderate level of ETS (1 mg/m3) can impair repair from acute lung injury, specifically by altering normal Clara cell redifferentiation. The impact of this on lung function is unknown at this time, but secretory products of differentiated Clara cells have been shown to have a role in regulating inflammation, contribute to the mucous lining layer of the airways (Singh and Katyal, 1992
), and protect cells from oxidant stress (Mango et al., 1998
).
Our current study indicates that prior occupational exposure to tobacco smoke may retard the ability of the lung to repair from acute injury. The lack of repair is evident as an inability to reestablish epithelial density in a timely manner in terminal bronchioles. Whether this is due to decreased epithelial proliferation or an inhibition of epithelial cell movement (either migration from proximal airways or during return to a more cuboidal shape) is unknown at this time. Further, the lack of Clara cell redifferentiation emphasizes that the factors that regulate Clara cell differentiation, both during postnatal lung development and during lung repair from injury, are still largely unknown. Acute epithelial changes in response to low levels of ETS alone have been difficult to demonstrate experimentally. This has been a puzzle to scientists trying to test the hypothesis that there is a link between low-level tobacco smoke exposure, acute lung injury, and the histologic changes that are associated with lung cancer. Our study would suggest that the effect of low-level smoke exposure is most evident as decreased potential for bronchiolar repair. We would like to suggest that the increase in lung cancer incidence that has occurred in the latter half of the 20th century may be due to two detrimental factors present in our environment: increased exposure to inhaled or ingested environmental pollutants, which are known to directly injure the lung, increasing the incidence of acute injury, and the fact that prior exposure to tobacco smoke is also increasingly prevalent, which may result in diminished potential for epithelial repair after exposure to environmental pollutants. This research has implications for human exposures to environmental tobacco smoke that occur simultaneously with exposures to other pollutants. Future directions of this work include further definition of the nature of the acute injury/Clara cell alteration caused by prior exposure to tobacco smoke. What is not known is whether this change is permanent, whether it represents an early, preneoplastic event, or what effect this lack of Clara cell differentiation has on lung function. We conclude that tobacco smoke exposure prior to injury (1) does not change the target site or target cell type of naphthalene injury, since Clara cells in terminal bronchioles are still selectively injured, (2) may result in slightly diminished acute injury from naphthalene in distal bronchioles, and (3) delays or inhibits bronchiolar epithelial repair.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ATSDR (1990). Toxicological Profile for Naphthalene and 2-Methylnaphthalene. Agency for Toxic Substances and Disease Registry, U.S. Department of Health and Human Services, Public Health Service. Life Systems, Inc., Atlanta, GA.
Boyd, M. R. (1977). Evidence for the Clara cell as a site of cytochrome P450-dependent mixed-function oxidase activity in lung. Nature 269, 713715.[ISI][Medline]
Boyd, M. R., Statham, C. N., and Longo, N. S. (1980). The pulmonary Clara cell as a target for toxic chemicals requiring metabolic activation; studies with carbon tetrachloride. J. Pharmacol. Exp. Ther. 212, 109114.[Abstract]
Buckpitt, A., Chang, A. M., Weir, A., Van Winkle, L., Duan, X., Philpot, R., and Plopper, C. (1995). Relationship of cytochrome P450 activity to Clara cell cytotoxicity. IV. Metabolism of naphthalene and naphthalene oxide in microdissected airways from mice, rats and hamsters. Mol. Pharmacol. 47, 7481.[Abstract]
Devereux, T. R., Domin, B. A., and Philpot, R. M. (1989). Xenobiotic metabolism by isolated pulmonary cells. Pharmacol. Ther. 41, 243256.[ISI][Medline]
Fanucchi, M., Murphy, M., and Plopper, C. (1997). Long-term effects of acute naphthalene injury during postnatal development. Am. J. Respir. Crit. Care Med. 155, A45.
Hyde, D. M., Plopper, C. G., St. George, J. A., and Harkema, J. R. (1990). Morphometric Cell Biology of Air Space Epithelium. In Electron Microscopy of the Lung (D. E. Schraufnagel, ed.). Vol. 48, pp. 71120. Marcel Dekker, New York.
Ji, C. M., Plopper, C. G., Witschi, H. P., and Pinkerton, K. E. (1994). Exposure to sidestream cigarette smoke alters bronchiolar epithelial cell differentiation in the postnatal rat lung. Am. J. Respir. Cell Mol. Biol. 11, 312320.[Abstract]
Ji, C. M., Royce, F. H., Truong, U., Plopper, C. G., Singh, G., and Pinkerton, K. E. (1998). Maternal exposure to environmental tobacco smoke alters Clara cell secretory protein expression in fetal rat lung. Am. J. Physiol. 275, L870876.[Medline]
Jindal, S. K., Gupta, D., and Singh, A. (1994). Indices of morbidity and control of asthma in adult patients exposed to environmental tobacco smoke. Chest 106, 746749.[Abstract]
Joad, J. P., Ji, C., Kott, K. S., Bric, J. M., and Pinkerton, K. E. (1995). In utero and postnatal effects of sidestream cigarette smoke exposure on lung function, hyperresponsiveness, and neuroendocrine cells in rats. Toxicol. Appl. Pharmacol. 132, 6371.[ISI][Medline]
Lakritz, J., Chang, A., Weir, A., Nishio, S., Hyde, D., Philpot, R., Buckpitt, A., and Plopper, C. (1996). Cellular and metabolic basis of Clara cell tolerance to multiple doses of cytochrome P450-activated cytotoxicants. I: Bronchiolar epithelial reorganization and expression of cytochrome P450 monooxygenases in mice exposed to multiple doses of naphthalene. J. Pharmacol. Exp. Ther. 278, 14081418.[Abstract]
Lee, C. K., Brown, B. G., Reed, E. A., Coggins, C. R., Doolittle, D. J., and Hayes, A. W. (1993). Ninety-day inhalation study in rats, using aged and diluted sidestream smoke from a reference cigarette: DNA adducts and alveolar macrophage cytogenetics. Fundam. Appl. Toxicol. 20, 393401.[ISI][Medline]
Leuenberger, P., Schwartz, J., Ackermann-Liebrich, U., Blaser, K., Bolognini, G., Bongard, J. P., Brandli, O., Braun, P., Bron, C., Brutsche, M., Domenghetti, G., Elsasser, S., Guldimann, P., Hollenstein, C., Hufschmid, P., Karrer, W., Keller, R., Keller-Wossidlo, H., Kunzli, N., Lurhi, J., Martin, B., Medici, T., Perruchoud, A., Radaelli, A., Schindler, C., Schoeni, M., G, S. a., Tschopp, J., Villiger, B., Wuthrich, B., Zellweger, J., and Zemp, E. (1994). Passive smoking exposure in adults and chronic respiratory symptoms (SAPALDIA Study). Am. J. Respir. Crit. Care Med. 150, 12221228.[Abstract]
Mango, G. W., Johnston, C. J., Reynolds, S. D., Finkelstein, J. N., Plopper, C. G., and Stripp, B. R. (1998). Clara cell secretory protein deficiency increases oxidant stress response in conducting airways. Am. J. Physiol. 275, L348356.
Nagata, K., Martin, B. M., Gillette, J. R., and Sasame, H. A. (1990). Isozymes of cytochrome P-450 that metabolize naphthalene in liver and lung of untreated mice. Drug Metab. Dispos. 18, 557564.[Abstract]
Nation, J. L. (1983). A new method using hexamethyldisilazane for preparation of soft insect tissues for scanning electron microscopy. Stain Technol. 58, 347351.[ISI][Medline]
Neter, J., Wasserman, W., and Kutner, M. H. (1990). Applied linear statistical models: regression, analysis of variance, and experimental designs. Irwin, Homewood, IL.
O'Brien, K. A., Suverkropp, C., Kanekal, S., Plopper, C. G., and Buckpitt, A. R. (1989). Tolerance to multiple doses of the pulmonary toxicant, naphthalene. Toxicol. Appl. Pharmacol. 99, 487500.[ISI][Medline]
Plopper, C. G., Cranz, D. L., Kemp, L., Serabjit-Singh, C. J., and Philpot, R. M. (1987). Immunohistochemical demonstration of cytochrome P-450 monooxygenase in Clara cells throughout the tracheobronchial airways of the rabbit. Exp. Lung Res. 13, 5968.[ISI][Medline]
Plopper, C. G., Macklin, J., Nishio, S. J., Hyde, D. M., and Buckpitt, A. R. (1992a). Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. III. Morphometric comparison of changes in the epithelial populations of terminal bronchioles and lobar bronchi in mice, hamsters, and rats after parenteral administration of naphthalene. Lab Invest. 67, 553565.[ISI][Medline]
Plopper, C. G., Suverkropp, C., Morin, D., Nishio, S., and Buckpitt, A. (1992b). Relationship of cytochrome P-450 activity to Clara cell cytotoxicity. I. Histopathologic comparison of the respiratory tract of mice, rats and hamsters after parenteral administration of naphthalene. J. Pharmacol. Exp. Ther. 261, 353363.[Abstract]
Rajini, P., and Witschi, H. (1994). Short-term effects of sidestream smoke on respiratory epithelium in mice: cell kinetics. Fundam. Appl. Toxicol. 22, 405410.[ISI][Medline]
Schmeltz, I., Tosk, J., and Hoffman, D. (1976). Formation and determination of naphthalenes in cigarette smoke. Anal. Chem. 48, 645650.[ISI][Medline]
Singh, G., and Katyal, S. (1992). Secretory proteins of Clara cells and type II cells. In Comparative Biology of Normal Lung (R. A. Parent, ed.). Vol. 1, pp. 93107. CRC Press, Boca Raton.
Singh, G., and Katyal, S. L. (1984). An immunologic study of the secretory products of rat Clara cells. J. Histochem. Cytochem. 32, 4954.[Abstract]
Smiley-Jewell, S. M., Nishio, S. J., Weir, A. J., and Plopper, C. G. (1998). Neonatal Clara cell toxicity by 4-ipomeanol alters bronchiolar organization in adult rabbits. Am. J. Physiol. 274, L485498.
Stripp, B. R., Maxson, K., Mera, R., and Singh, G. (1995). Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. Am. J. Physiol. 269, L791L799.
Teague, S., Pinkerton, K., Goldsmith, M., Gebremicahel, A., Chang, S., Jenkins, R., and Moneyhun, J. (1994). A sidestream cigarette smoke generator and exposure system for environmental tobacco smoke studies. J. Inhal. Toxicol. 6, 7993.
U.S. EPA (1992). Respiratory Health Effects of Passive Smoking: Lung Cancer and Other Disorders. Office of Health and Environmental Assessment, Washington DC.
Van Winkle, L. S., Buckpitt, A. R., Nishio, S. J., Isaac, J. M., and Plopper, C. G. (1995). Cellular response in naphthalene-induced Clara cell injury and bronchiolar epithelial repair in mice. Am. J. Physiol. 269, L800L818.
Van Winkle, L. S., Isaac, J. M., and Plopper, C. G. (1997). Distribution of the epidermal growth factor receptor and ligands during bronchiolar epithelial repair from naphthalene-induced Clara cell injury in the mouse. Am. J. Pathol. 151, 443459.[Abstract]
Van Winkle, L. S., Johnson, Z. A., Nishio, S. J., Brown, C. D., and Plopper, C. G. (1999). Early events in naphthalene-induced acute Clara cell toxicity: comparison of membrane permeability and ultrastructure. Am. J. Respir. Cell Mol. Biol. 21, 4453.
von Meyerinck, L., Scherer, G., Adlkofer, F., Wenzel-Hartung, R., Brune, H., and Thomas, C. (1989). Exposure of rats and hamsters to sidestream smoke from cigarettes in a subchronic inhalation study. Exp. Pathol. 37, 186189.[ISI][Medline]
Witschi, H., Espiritu, I., Maronpot, R. R., Pinkerton, K. E., and Jones, A. D. (1997). The carcinogenic potential of the gas phase of environmental tobacco smoke. Carcinogenesis 18, 20352042.[Abstract]