3 sex differences-in_bronchiolar_epithelial_injury.5[1]

1110-0559 © 2012 The Egyptian Journal of Histology DOI: 10.1097/01.EHX.0000419802.25529.94

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IntroductionThe epithelium of distal conducting airways is one of the most susceptible sites for acute injury after exposure to a variety of toxicants including oxidant air pollutants and metabolically activated xenobiotics [1]. It provides a selectively permeable barrier between the internal tissues and the potentially hazardous agents, entering the body through the airways [2]. Nonciliated bronchiolar Clara cells are the principal epithelial cell phenotype present in the distal airways in many species [1]. Their function is mainly to protect the respiratory tract. Clara cells have a high xenobiotic transformation capacity and secrete several proteins with important biological activities [3].

Airway injury, repair, and remodeling are implicated in the pathogenesis of diverse lung diseases [4]. Among the many agents that cause lung injury, hazardous air pollutants exert chronic adverse effects on lung function [5], and are likely to contribute significantly toward morbidity and mortality in humans [6]. The bioactivated xenobiotic, naphthalene (NA), is a pervasive environmental contaminant found in ambient air and ground water [7]. Humans are exposed to NA from a number of different sources, including industrial applications, such as the production of phthalic anhydride, which is used in the synthesis of resins, plastics, pharmaceuticals, and insect repellents [8]. Nonoccupational exposures also arise from

Department of Histology, Faculty of Medicine, El-Minia University, Minia, Egypt

Correspondence to Nashwa F. El-TahawyTel.: 01145435777 e-mail: [email protected]

Received 15 January 2012Accepted 17 May 2012

The Egyptian Journal of Histology2012, 35:672-68561 (1380-2012)

BackgroundAirway injury and repair are implicated in the pathogenesis of lung diseases. Sex affects the severity of lung diseases. Naphthalene (NA) is an environmental contaminant. Clara cells are capable of detoxifying several pulmonary toxicants.Aim of the workTo determine whether there is a sex-based difference in the injury and regenerative response to NA inhalation, and the possible role of inducible nitric oxide synthase (iNOS).Materials and methodsThirty-six adult male and female albino rats were used. Rats were exposed to air (group I; control) or NA vapor for 4 h, and then sacrificed after 6 h (group II), 12 h (group III), 24 h (group IV), 14 days (group V), and 21days (group VI). The lungs were used for histological and immunohistochemical studies. Exfoliation scoring and statistical analysis were carried out.ResultsInhalation of NA induced bronchiolar cell exfoliation that started at 6 h and peaked at 24 h after inhalation in both sexes. Male bronchiolar epithelium fully regenerated by 14 days, whereas females showed some exfoliated cells in the lumen that disappeared by 21 days. In both sexes, the mean exfoliation score (MES) was significantly higher in groups III and IV compared with their controls. MES was significantly higher in females compared with males in group III and V. The expression of iNOS increased with time after NA inhalation. After 14 days, there was an apparent decrease in iNOS expression in both sexes. It was more obvious in female rats than males during the same period.ConclusionInjury occurred earlier, with more affected cells in female bronchioles, whereas regeneration occurred earlier in males. Increased iNOS expression indicated a role of NO production in bronchiolar damage. The difference in iNOS expression in both sexes could provide a new mechanism in explaining sex differences in bronchiolar cell injury and repair.

Keywords: bronchiolar epithelium, inducible nitric oxide, inhalation, naphthalene, rats, sex

Egypt J Histol 35:672-685© 2012 The Egyptian Journal of Histology1110-0559

Sex differences in bronchiolar epithelial injury and repair following naphthalene inhalation in albino rats: a possible role of inducible nitric oxideNashwa F. El-Tahawy and Rehab A. Rifaai

Original article

Copyright © The Egyptian Journal of Histology. Unauthorized reproduction of this article is prohibited.

673Sex differences in bronchiolar epithelial injury El-Tahawy and Rifaai

in the Histology Department, Faculty of Medicine, El-Minia University. All aspects of animal care and treatment were carried out according to the local guidelines of the ethical committee of the Faculty of Medicine, El-Minia University. Rats were divided into equal groups (three rats per group per sex) and exposed to environmental air as a control (group I) or NA vapor for 4 h, and then the animals were sacrificed by decapitation under light halothane anesthesia at 6 h (group II), 12 h (group III), 24 h (group IV), 14 days (group V), and 21days (group VI) after exposure.

Naphthalene exposuresInhalation exposures were generated as described previously, with minor alterations [22]. Briefly, rats were placed in glass chambers (six per chamber). NA was purchased from markets (Naphthalene balls, Pingdingshan Aosida Chemicals Co., Ltd., China) vapor was generated by passing air through crystalline NA packed in a 2.5 × 70 cm glass column. Air volume through the chamber was 2.0 l/min. Concentrations of 15 ppm NA were achieved by mixing NA vapor with environmental air. The NA concentration within the chamber (range 13.5–17 ppm) was determined by sampling chamber air (10 ml) with a gas-tight syringe. The sampled air was then dissolved in 3.0 ml methanol and measured by absorbance at 210 nm. NA concentrations were also monitored continuously using a Digital spectrophotometer (UV-VIS Spectrophotometer 3200, Maharashtra, India). Control animals were exposed to environmental air.

Histology and immunohistochemistryThe lungs were collected and segmented. Segments used for routine histology and immunohistochemistry were fixed overnight in 10% buffered formalin, followed by paraffin embedding. Seven-micrometer lung sections were stained with H&E for histological examination using a light microscope. Other sections were also used for immunohistochemical staining for iNOS. Briefly, [23] sections were deparaffinized, rehydrated, and, after antigen retrieval with 10 mmol/l citrate acid solution (pH 6), specimens were preincubated with goat serum for 5 min at 42ºC and were then incubated overnight at 4ºC with polyclonal anti-iNOS (Sigma Aldrich, Cairo, Egypt) specific for the 130-kDa enzyme in rats) or PBS (control). Anti-iNOS binding was detected using biotinylated secondary antibody (goat anti-mouse IgG; Sigma Aldrich) for 10 min at 42ºC. The specimens were then incubated with streptavidin–peroxidase complex for 5 min at 42ºC, followed by incubation with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma Aldrich) for 3 min at 42ºC. Slides were counterstained with hematoxylin and mounted. The positive immunoreactivity for iNOS appeared in the form of a brown staining in the cytoplasm of the immunoreactive cells.

Other lung segments were fixed with 1% glutaraldehyde for 1 h. The fixed tissues were postfixed with osmium

the removal of pests by pure NA crystals (mothballs), diesel, and fuel emissions. It has become obvious that tobacco smoke poses a health risk to nonsmokers [9], and NA has been found to be the most abundant polycyclic aromatic hydrocarbon in sidestream cigarette smoke [10]. The toxicity of NA requires metabolic activation, which is catalyzed by cytochrome P450 monooxygenases. Susceptibility to NA correlates with the formation of the toxic metabolite by cytochrome P450 isozyme 2F2 (CYP2F2). In the lung, Clara cells are the primary cellular site of cytochrome P450 monooxygenase [11]. Therefore, Clara cells are uniquely susceptible to NA-induced cytotoxic injury than other types of airway epithelial cell [12]. Epidemiological evidence suggests that sex affects the incidence and severity of several lung diseases. Although the incidence of lung cancer and the mortality rate as a result of it have appeared to reach a plateau in men, it continues to increase in women [13]. Among lifetime nonsmokers, worldwide, lung cancer is much more common in women than in men [14]. Interestingly, DNA repair capacity has been shown to be 10–15% lower in female patients with lung cancer than in their male counterparts [15]. In addition, chronic obstructive pulmonary disease affects more nonsmoking women than nonsmoking men [16], and female rats exposed to cigarette smoke have been shown to develop emphysematous-like changes in alveolar structure more rapidly than male rats [17]. Ovariectomized female rats showed less pulmonary fibrosis than did sham-operated controls, and hormone replacement therapy with estradiol restored the fibrotic response, indicating that the exaggerated response of female rats to lung injury may be mediated by sex hormones [18].

Many respiratory disorders, which involve airway inflammation and epithelial cell injury, are associated with an elevation in nitric oxide (NO) production [19]. Therefore, it has been suggested that NO might be involved in the lung injury induced by NA exposure. Subsequent regeneration and repair of lung epithelium is a vital process to help maintain the function and integrity of the airways [20].

Many laboratories have used NA treatment of experimental animals through parental administration [21], whereas the major route of exposure to humans is through inhalation of NA vapor.

The aim of this study was to determine the response of the bronchiolar epithelium after NA exposure by inhalation and to determine whether there is a sex-based difference in the injury and regenerative response associated with NA toxicity. Another aim was to describe the effect of inhaled NA on inducible nitric oxide synthase (iNOS) expression in rat lung bronchioles.

Materials and methodsAnimalsThirty-six adult, 8–10 weeks old, male (18) and female (18) albino rats were used in this experiment. Food and water were provided ad libitum for 5 days before use


674 Sex differences in bronchiolar epithelial injury El-Tahawy and Rifaai

In group III, exfoliation of numerous injured bronchiolar epithelial cells into the airway lumen was observed in male rats (Fig. 3a), and it was more obvious in female rats (Fig. 3b). This sloughing of injured cells from the bronchiolar epithelium left the basement membrane denuded.

In group IV, exfoliation of most of cells of the bronchiolar epithelium left larger areas of denuded basement membrane, with only a few uninjured bronchiolar epithelial cells, in both male (Fig. 4a) and female (Fig. 4b) rats.

Within 14 days after NA inhalation, group V, bronchiolar epithelium appeared regenerated and restored with an abundance of dome-shaped Clara cells, with some areas of stratifications observed in both male (Fig. 5a) and female (Fig. 5b) rat bronchioles. However, residual injured and exfoliated bronchiolar epithelial cells were detected in female rats, but not in male rats.

At 21 days after NA inhalation, group VI, bronchiolar epithelium appeared fully regenerated and restored, with an abundance of nonciliated Clara cells, with the absence of exfoliated cells in both male (Fig. 6a) and female (Fig. 6b) rats.

Table 1 shows that in males, the MES was significantly higher in bronchiolar epithelium in groups III (P=0.000) and IV (P=0.000) compared with the controls. In contrast, the MES in groups II, V, and VI were insignificant compared with the controls (all P’s>0.05). In females, the MES was also significantly higher in bronchiolar epithelium in groups III (P=0.000) and IV (P=0.000) compared with the controls. In contrast, the MES in groups II, V, and VI were insignificant compared with the controls (all P’s>0.05). On comparing males with females, the MES was found to be significantly higher in bronchiolar epithelium in female rats compared with male rats in group III (P=0.014) and group V (P=0.004). However, there was no significant difference in the MES between male and female rats in groups I, II, IV, and VI (all P’s>0.05).

Immunostaining of the lungs of control animals with an antibody specific for iNOS showed negative iNOS immunoreactivity in the lungs of both male (Fig. 7a) and female (Fig. 7b) rats.

In group II, bronchiolar epithelium, endothelial cells, and also peribronchial interstitial cells showed positive immunoreactivity in their cytoplasm, both in the male group (Fig. 8a), and appeared more obvious in the female group (Fig. 8b).

In group III, most of the bronchiolar epithelium included exfoliated cells and endothelial cells, and also, peribronchial cells showed extensive immunoreactivity in their cytoplasm, both in male (Fig. 9a) and in female groups (Fig. 9b).

tetraoxide and incubated overnight in uranyl acetate and then embedded in Araldite-502, and then grossly sectioned parallel to the long axis of the main stem bronchi. Sections (1.0 µm) were cut with glass knives using a Zeiss ultra-microtome (Jena, Germany) and stained with 1% toluidine blue [24].

Image captureImages of fields (from three slides per animal and from three animals per group) containing distal bronchiolar epithelium were captured using an (Olympus, Japan) computerized microscope in the bright-field mode. Respiratory bronchioles were defined as the most distal conducting airway generation contiguous with the alveolar duct. Ciliated cells were identified by the presence of cilia, whereas Clara cells (nonciliated) were identified by their characteristic apical projections.

Histological analysisSections of rat lungs stained with H&E from each group were scored for the extent of NA-induced bronchiolar epithelial cell exfoliation using a light microscope at ×400 magnification [25]. The degree of exfoliation was estimated semiquantitatively, and was expressed for each rat as the mean of 10 random fields within each section (three sections per rat) classified on a scale of 0–3. The scoring criteria were as follows: 0, no exfoliation, defined as no detection of epithelial cells exfoliated within the airway lumen; 1, mild, defined as only occasional detection of bronchiolar epithelial cells exfoliated within the airway lumen; 2, moderate, more frequent detection of bronchiolar epithelial cells exfoliated in the airway lumen; 3, severe, very frequent detection of numerous exfoliation bronchiolar epithelial cells in the airway lumen [25].

Data handling and statisticsData are presented as the mean exfoliation score (MES) ± SD. Analysis of the data was carried out using SPSS, version 16 (Chicago, Illinois, USA). Statistical comparisons between groups at different time points were carried out by one-way analysis of variance, followed by Tukey’s post-hoc tests. Differences were considered significant when a P-value was less than 0.05.

ResultsNormal bronchiolar epithelium, a single layer consisting mainly of nonciliated Clara cells with apical projections and ciliated cells, was observed in the lungs of both male (Fig. 1a) and female (Fig. 1b) rats of the control group. In group II, some exfoliated cells were observed in the lumen of the respiratory bronchioles that appeared less obvious in the male group (Fig. 2a) than in the female group (Fig. 2b). Lung tissues showed inflammatory cell infiltration into the peribronchial interstitial area.



In group IV, with exfoliation of most bronchiolar cells, positive immunoreactivity of the residual bronchiolar cells, exfoliated cells, and peribronchial cells was observed in male (Fig. 10a) and female groups (Fig. 10b).

Within 14 days after NA exposure, in group V, bronchiolar epithelium appeared regenerated in both male (Fig. 11a) and female (Fig. 11b) rats. There was an obvious decrease in bronchiolar immunoreactivity, with some immunoreactivity detected in the peribronchial interstitial cells, especially in female sections (Fig. 11b).

At 21 days after NA exposure, in group VI, bronchiolar epithelium appeared fully regenerated and restored, with an absence of immunoreactivity for iNOS in both male (Fig. 12a), and female (Fig. 12b) rats. A few endothelial and peribronchial cells still showed positive immunoreactivity.

Examination of ultrathin sections stained with toluidine blue in the control groups showed that the epithelium of the respiratory bronchioles in both male (Fig. 13a) and female (Fig. 13b) rats appeared to have a simple epithelium composed of two predominant cell types: nonciliated Clara cells and, to a lesser extent, ciliated cells. Clara cells were adjacent to ciliated cells and had a typical appearance, including apical projections that extended into the airway lumen. The bronchiolar epithelium was cuboidal and cells appeared to be smaller in female than in male rats.

In group II, some bronchiolar Clara cells were swollen and lacked pronounced apical protrusions. Other Clara

cells contained apical membrane blebs that were lightly stained and lacked dark-staining granules. A few cells contained vacuoles. However, these appeared to be fewer in male (Fig. 14a) than in female (Fig. 14b) rats.

In groups III, some Clara cells exfoliated into the lumen with the appearance of squamated (i.e. flattened) cells covering the basement membrane at sites of degeneration. The epithelium was of variable thickness, with cells ranging from low cuboidal ciliated cells to quite swollen Clara cells. However, these appeared to be fewer in male (Fig. 15a) than in female (Fig. 15b) rats.

Group IV showed many degenerated Clara cells exfoliated into the lumen of the airways, leaving extremely attenuated cells in spots and denuded basement membrane in both male (Fig. 16a) and female (Fig. 16b) lungs.

By 14 days, group V, the epithelium appeared regenerated and restored with an abundance of nonciliated (Clara) cells in both male (Fig. 17a) and female (Fig. 17b) lung bronchioles. However, a few residual injured and exfoliated bronchiolar epithelial cells were detected in the bronchiolar lumen at 14 days of NA exposure in female rats, but not in male rats.

Twenty-four days after NA inhalation, group VI, bronchiolar epithelium appeared fully regenerated and restored, with an abundance of nonciliated Clara cells, with no exfoliated cells in the bronchiolar lumen in both male (Fig. 18a) and female (Fig. 18b) rats.

Table 1. Exfoliation scoring of bronchiolar epithelium of male and female rat lungs at different time points after naphthalene inhalation

Male Female

P3-value

Mean ± SD P1-value Mean ± SD P1-value

Group I (control) 0.1 ± 0.02 0.13 ± 0.01 0.808

Group II (6 h) 0.14 ± 0.01 0.406 0.15 ± 0.03 0.195 0.836

Group III (12 h) 2 .19 ± 0.72 0.000* 3.3 ± 0.32 0.000* 0.014*

Group IV (24 h) 4.0 ± 0.0 0.000* 4.0 ± 0.0 0.000* 1.000

Group V (14 days) 0.11 ± 0.01 0.347 0.15 ± 0.02 0.081 0.004*

Group VI (21 days) 0.1 ± 0.02 1.000 0.13 ± 0.04 1.000 0.172

P1 values, treated male vs. male control; P2 values, treated female vs. female control; P3 values, male vs. female at the investigated time points.

*P<0.05 is significant.



Figure 1. Photomicrographs of the control groups in both male (a) and female (b) lungs showing a normal morphology. Respiratory bronchiole (RB) with abundant dome-shaped Clara cells (arrows). H&E, × 40.

Figure 2. Photomicrographs of group II in both male (a) and female (b) lungs showing respiratory bronchioles (RB), with few exfoliated cells (arrows) in the bronchiolar lumen. Some inflammatory cell infiltration (circles) can be seen in the lung tissues. H&E, × 40.

Figure 3. Photomicrographs of group III in both male (a) and female (b) lungs showing respiratory bronchioles (RB) with increased exfoliated cells (stars) in the bronchiolar lumen, leaving a denuded basement membrane (arrows). Greater exfoliation can be seen in female bronchioles. H&E, × 40.



Figure 4. Photomicrographs of group IV in both male (a) and female (b) lungs showing respiratory bronchioles (RB) with almost all cells exfoliated (star), with only a few residual cells (blue arrows) on the denuded basement membrane (black arrows). Some flattened cells (red arrow) covering the basement membrane, and some inflammatory cells infiltration in the lung tissues (circle) can be seen. H&E, × 40.

Figure 5. Photomicrographs of group V in both male (a) and female (b) lungs showing respiratory bronchioles (RB) with regenerated epithelial lining (arrows), with some areas of stratifications (circles). Residual exfoliated cells in the lumen of the female bronchiole can be seen (star). H&E, × 40.

Figure 6. Photomicrographs of group VI in both male (a) and female (b) lungs showing respiratory bronchioles (RB) with a fully regenerated bronchiolar epithelium (arrows), with an absence of any residual epithelial cells in the lumens. H&E, × 40.



Figure 7. Photomicrographs of respiratory lung bronchioles (RB) immunostained for inducible nitric oxide synthase in the control group of male (a) and female (b) rats showing negative immunoreactivity of bronchiolar cells (arrows). Paraffin sections, immunohistochemistry, counterstained with H × 40.

Figure 8. Photomicrographs of respiratory lung bronchioles (RB) of group II in male (a) and female (b) rats immunostained for inducible nitric oxide synthase with bronchiolar epithelium showed positive immunoreactivity (arrows). Positive immunoreactivity in the endothelial cells (red arrows) and peribronchial cells can be seen (stars). Paraffin sections, immunohistochemistry, counterstained with H × 40.

Figure 9. Photomicrographs of respiratory lung bronchioles (RB) of group III in male (a) and female (b) rats immunostained for inducible nitric oxide synthase showing extensive positive immunoreactivity in the bronchiolar epithelium (arrows), endothelium of blood vessels (red arrow), and peribronchial cells (stars), with some exfoliated cells (circle) in the lumen. Paraffin sections, immunohistochemistry, counterstained with H × 40.



Figure 10. Photomicrographs of respiratory lung bronchioles (RB) of group IV in male (a) and female (b) rats immunostained for inducible nitric oxide synthase showing positive immunoreactivity of the residual bronchiolar cells (arrows), exfoliated cells (circle), and peribronchial cells (stars). Paraffin sections, immunohistochemistry, counterstained with H × 40.

Figure 11. Photomicrographs of respiratory lung bronchioles (RB) of group V in male (a) and female (b) rats immunostained for inducible nitric oxide synthase showing decreased immunoreactivity of the regenerated epithelium. Positive immunoreactivity in peribronchial cells (arrow) and a few exfoliated bronchiolar cells (star) in the lumen of the female bronchiole can be seen. Paraffin sections, immunohistochemistry, counterstained with H × 40.

Figure 12. Photomicrographs of respiratory lung bronchioles (RB) of group VI in male (a) and female (b) rats immunostained for inducible nitric oxide synthase showing fully regenerated bronchiolar cells, with absence of any positive immunoreactivity. A few positive endothelial cells (arrows) and peribronchial cells (circle) can be seen. Paraffin sections, immunohistochemistry, counterstained with H × 40.



Figure 13. High-resolution photomicrograph of terminal airway epithelium in the control groups showing the epithelium of the respiratory bronchiole in (a) male and (b) female lung rats. Simple epithelium showed regular thickness and consisted of ciliated cells (Ci, arrow) and nonciliated cells (NC, arrows). × 1000.

Figure 14. High-resolution photomicrograph of the terminal airway epithelium in group II showing the epithelium of the respiratory bronchiole in (a) male and (b) female lung rats. Clara cells became swollen (double-headed arrows), vacuolated (arrows), and formed apical membrane blebs (*). × 1000.

Figure 15. High-resolution photomicrograph of the terminal airway epithelium in group III showing the epithelium of the respiratory bronchiole of variable thickness in (a) male and (b) female lung rats. Exfoliated Clara cells in the airway lumen (double-headed arrows), and squamated cells appeared to be covering some areas of the basement membrane (arrows). × 1000.



Figure 16. High-resolution photomicrograph of the terminal airway epithelium in group IV showing the epithelium of the respiratory bronchioles in (a) male and (b) female rats. Most Clara cells exfoliated (star), leaving a denuded basement membrane (arrows) and a few cells in spots covering part of the basement membrane (double-headed arrows). × 1000.

Figure 17. High-resolution photomicrograph of terminal airways in group V showing the epithelium of the respiratory bronchiole, which appeared regenerated and restored with an abundance of Clara cells (NC, arrows) in both (a) male and (b) female rats. Exfoliated cells can be seen in the lumen of the female group (star). × 1000.

Figure 18. High-resolution photomicrograph of terminal airways in group VI showing the epithelium of the respiratory bronchiole fully regenerated, with an abundance of Clara cells (NC, arrows) in both (a) male and (b) female lungs. × 1000.



DiscussionClara cells are the primary metabolic epithelial cell of the lung, capable of detoxifying a number of pulmonary toxicants, as well as providing anti-inflammatory signals through its secretory protein [26]. NA is a common environmental contaminant [27], a member of a group of compounds that cause lung toxicity when metabolized by the cytochrome monooxygenase system [28], and abundant in tobacco smoke [10].

Many biological factors may modulate the epithelial response to toxic injury in the airways. Among these factors are the specific sites for bioactivation and detoxification, age of the organism at time of exposure, history of previous exposure and development of tolerance, species, and strain of organism exposed [12]. Sex has been shown to be a factor in human lung disease, particularly in lung cancer and in airway hyper-responsiveness [29]. Also, the route of administration has been shown to be another factor; thus, it was important to study sex differences in response to NA inhalation as a major route of lung exposure.

In this study, histological results showed that normal bronchiolar epithelium lined the respiratory bronchioles. NA inhalation induced bronchiolar cell exfoliation that started at a minimal degree at 6 h after NA exposure both in male and in female lungs; this was in contrast to the finding of Oliver et al. [25] who reported that exfoliations started 2 h after intraperitoneal NA administration, which could have been because of differences in the route of administration. Exfoliations increased with time, peaking at 24 h after NA inhalation, which was in agreement with the results of Oliver et al. [25]. At each time point of the study, exfoliations were more obvious in the lumens of the female bronchioles than males. Bronchiolar epithelium appeared to be fully regenerated by 14–21 days after NA inhalation, with some exfoliated cells still appearing only in the lumen of female bronchioles at 14 days after NA inhalation. Thus, male epithelial bronchioles seemed to show faster recovery than female epithelial bronchioles. A previous study [30] of sex-related differences in the metabolism of compounds by the CYP2F2 system found a decrease in CYP2F2 expression in female mice compared with male mice at the same time point after treatment with NA. This was in agreement with the result of this study as more Clara cells, CYP2F2-expressing cells, were injured and exfoliated in the female than in male airways. Also, Van Winkle et al. [30] found a major difference between male and female mice in their metabolism of NA as the total NA metabolites were larger in the female mice; thus, they concluded that differences in the metabolism of NA may play a role in elevated susceptibility in female mice.

The mechanisms governing epithelial repair are incompletely understood. The epithelial repair encompasses cell proliferation, migration, and differentiation [31], which could explain the appearance of areas of stratifications in bronchioles of both sexes by 14 days after NA inhalation in the present study.

Semithin sections in this study showed that the airway epithelium from control rats appeared as a cubiodal epithelium consisting mainly of ciliated and nonciliated Clara cells. Clara cells had typical apical projections that extended into the airway lumen. Because female Clara cells of the controls appeared to be smaller, it is possible that less glutathione was available. If less glutathione is available, this could lead to increases in susceptibility in cells [11]. NA inhalation resulted in swelling, bleb formation, and vacuolation of Clara cells, with a few cells exfoliating from the basement membrane after 6 h of exposure, which was in agreement with the result of Lawson et al. [32], who found that Clara cells vacuolated as soon as 6 h after exposure, but in contrast to the finding of Van Winkle et al. [30] who found that these changes occurred 3 h after NA administration, which could have been because of differences in the routes of administration. By 12 h, the injury appeared to be increased, and it appeared to be greater in the female rats than in males. Clara cells were swollen, vacuolated, and exfoliating from the basal lamina, leaving a squamated epithelium. These morphological findings were in agreement with the results of Phimister et al. [11]. It is believed that blebs are formed primarily because of cytoskeletal disruptions near the surface of the cell, allowing portions of cytoplasm to become distended. Diethylmaleate, an NA metabolite, has been shown to disrupt actin and tubulin filaments in hepatocytes [33].

Twenty-four hours after NA inhalation, a time-point at which Clara cells are almost completely lost from the distal airway epithelium, large denuded areas of the basement membrane are lined by squamated cells. This was in agreement with the results of several previous studies [25,30,32]. The surviving ciliated cells resorb their cilia and become squamated [20,34]. Although still highly controversial, it has been suggested that ciliated cells undergo squamous metaplasia and cell spreading, followed by cell proliferation and transdifferentiation into distinct epithelial cell types [19,31]. These proliferated cells begin their migration at 4 days, followed by differentiation 7 days after exposure, with a return to steady state at 14 days [34]. However, other investigators have carried out lineage tracing studies in order to follow the fate of ciliated cells after NA-induced Clara cell ablation, and found strong evidence that ciliated cells can transiently alter their morphology (i.e. undergo squamous metaplasia), but do not proliferate or transdifferentiate as part of the repair process [20]. However, we did observe that in both male and female mice, some ciliated cells remained intact and uninjured after NA-induced Clara cell damage and exfoliation, possibly to help protect the denuded basement membrane of the bronchiolar epithelium. These results indicate a timely regenerative response as the delayed lung repair in female rats may be attributed to the severe damage detected at 12–24 h after NA inhalation.

There are many possible explanations for the sex differences in lung repair. Female sex hormones, such as estrogen, may play a role in the pulmonary regenerative response to



AcknowledgementsConflicts of interestThere is no conflict of interest to declare.

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20 Rawlins EL, Ostrowski LE, Randell SH, Hogan BLM. Lung development and repair: Contribution of the ciliated lineage. Proc Natl Acad Sci USA 2007; 104:410–417.

21 Belvisi M, Barnes PJ, Larkin S, Yacoub M, Tadjkarimi S, Williams TJ, Mitchell JA. Nitric oxide synthase activity is elevated in inflammatory lung disease in humans. Eur J Pharmacol 1995; 283:255–258.

22 West JAA, Van Winkle LS, Morin D, Fleschner CA, Forman HJ, Plopper CG. Repeated inhalation exposures to the bioactivated cytotoxicant naphthalene (NA) produce airway-specific Clara cell tolerance in mice. Toxicol Sci 2003; 75:161–168.

23 Côté A, da Silva R, Cuello AC. Current protocols for light microscopy immunocytochemistry. In: Cuello AC, editor. Immunohistochemistry II. Chichester: John Wiley and Sons; 1993 pp. 147-168.

24 Plopper CG. Structural methods for studying bronchiolar epithelial cells. In: Gil J, editor. Model of lung disease, microscopy, and structural methods. New York: marcel Dekker; 1990 pp. 537–559.

25 Oliver JR, Kushwah R, Wu J, Cutz E, Yeger H, Waddell TK, Hu J. Gender differences in pulmonary regenerative response to naphthalene-induced bronchiolar epithelial cell injury. Cell Prolif 2009; 42:672–687.

26 Massaro GD, Singh G, Mason R, Plopper CG, Malkinson AM, Gail DB. Biology of the Clara cell. Am J Physiol 1994; 266(Pt 1):L101–L106.

NA-induced Clara cell ablation. It is known that estrogen can induce the expression of many drug-metabolizing isoenzymes in the lungs [14,16] and, therefore, females have the potential to undergo more metabolic bioactivation of xenobiotics to toxic metabolites than males. Furthermore, hormonal patterns associated with different stages of the estrous cycle can alter NA metabolism in the lungs of female mice [35], and metabolism of NA occurs much faster and is associated with a greater extent of lung injury in female mice as compared with male mice at the same dose [30]. Thus, the more extensive Clara cell injury in female rats compared with male rats observed in this study and the aforementioned studies may be because of the fact that females produce substantially more estrogen than males [35].

NO is considered as a proinflammatory mediator that induces inflammation because of overproduction under abnormal conditions [36]. Oxidative stress is believed to occur in a tissue or an organ when the normal balance between oxidants and antioxidants shifts in favor of oxidants, from either an excess of oxidants and/or a depletion of antioxidants [3]. To determine the role of oxidative stress in NA cytotoxicity, lung tissues were immunostained against iNOs. In this study, the iNOs activity was elevated after NA inhalation, which was in agreement with the result of Belvisi et al. [21]. The expression of iNOS was increased in both male and female rats, increasing with time from 6 to 12 h, peaking at 24 h after NA inhalation. It was also observed that the expression of iNOS in female rats was more obvious than that in male lungs at the same time points, that is, female rats produce more NO than males after exposure to the same dose of inhaled NA. However, there was a decrease in iNOS expression by 14 days and almost disappeared by 21 days after NA inhalation, with only a few positive endothelial cells in both male and female rats. These results might provide an explanation for the sex differences in cellular injury and repair responses.

Because NO inhibitors represent an important therapeutic advancement in the management of inflammatory diseases [36,37], the use of selective NO biosynthesis inhibitors and synthetic arginine analogues could be suggested for the treatment of NO-induced inflammation caused by NA inhalation.

ConclusionIt can be concluded that female rats were more susceptible than male rats to the same dose of NA exposure by inhalation. Bronchiolar injury occurred earlier, with more affected cells in female bronchioles, whereas regeneration occurred earlier in male lung bronchioles. Excessive production of NO by the iNOS may play a key role in the development of bronchiolar injury, and the difference in the expression of iNOS in male and female bronchioles could be a new mechanism explaining the sex differences in cellular injury and repair responses.

It is recommended to examine the effect of NO inhibitors or antioxidants for the management of acute lung toxicity induced by NA inhalation.



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33 Nagelkerke JF, Van De Water B, Twiss IM, Zoetewey JP, De Bont HJGM, Dogterom P, Mulder GJ. Role of microtubuli in secretion of very-low-density lipoprotein in isolated rat hepatocytes: early effects of thiol reagents. Hepatology 1991; 14:1259–1268.

34 Greeley MA, Van Winkle LS, Edwards PC, Plopper CG. Airway trefoil factor expression during naphthalene injury and repair. Toxicol Sci 2009; 113:453–467.

35 Stelck RL, Baker GL, Sutherland KM, Van Winkle LS. Estrous cycle alters naphthalene metabolism in female mouse airways. Drug Metab Dispos 2005; 33:1597–1602.

36 Sharma JN, Al-Omran A, Parvathy SS. Role of nitric oxide in inflammatory diseases. Inflammopharmacology 2007; 15:252–259.

37 Su CF, Yang FL, Chen HI. Inhibition of inducible nitric oxide synthase attenuates acute endotoxin-induced lung injury in rats. Clin Exp Pharmacol Physiol 2007; 34:339–346.

27 Arey J, Zielinska B, Atkinson R, Winer AM. Polycyclic aromatic hydrocarbon and nitroarene concentrations in ambient air during a wintertime high-NOx episode in the Los Angeles basin. Atmospheric Environ 1967; 21:1437–1444.

28 Plopper CG. Pulmonary bronchiolar epithelial cytotoxicity: microanatomical considerations. In: Gram TE, editor. Metabolic activation and toxicity of chemical agents to lung tissue and cells. New York: Pergamon; 1993 pp. 1–24.

29 De Oliveira APL, Peron JPS, Damazo AS, dos Santos Franco AL, Domingos HV, Oliani SM, et al. Female sex hormones mediate the allergic lung reaction by regulating the release of inflammatory mediators and the expression of lung E-selectin in rats. Respir Res 2010; 11:115.

30 Van Winkle LS, Gunderson AD, Shimizu JA, Baker GL, Brown CD. Gender differences in naphthalene metabolism and naphthalene-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol 2002; 282:L1122–L1134.

31 Adair JE, Stober V, Sobhany M, Zhuo L, Roberts JD, Negishi M, et al. Inter-α-trypsin inhibitor promotes bronchial epithelial repair after injury through vitronectin binding. J Biol Chem 2009; 284:16922–16930.


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الملخص العربى

الفروق بين الجنسين في اصابة و إصالح الخاليا المبطنة للقصيبات الهوائية نتيجة الستنشاق النفثالين في الجرذان: الدور المحتمل لمحفذ أكسيد النيتريك.

نشوة فتحى الطحاوى و رحاب احمد رفاعىقسم الهستولوجيا - كلية الطب - جامعة المنيا

المقدمة: إن إصابة مجرى الهواء وإصالحه يحدث في أمراض الرئة المختلفة. ويؤثر الجنس على حدوث بل وعلى شدة حدوث عدة أمراض فى الرئة. النفثالين هو اكثر الملوثات البيئية انتشارا. خاليا كالرا لها القدرة على إزالة

السموم لعدد من المواد السامة.الهدف: دراسة و تحديد ما إذا كان هناك فرق بين الجنسين في اإلصابة الناتجة عن التسمم الحاد بالنفثالين المستنشق أكسيد محفز على النفثالين استنشاق تأثير وايضا وصف الجرذان. رئة قصيبات في الخاليا بتجدد واالستجابة

.)iNOS( النيتريكالجوي كمجموعة للهواء اما الجرذان البالغين. تعرضت الجرذان وإناث ثالثون من ذكور استخدمت الطريقة: ضابطة )المجموعة األولى(، أو الستنشاق الهواء المحمل ببخار النفثالين لمدة 4 ساعات، ثم قتلت بعد ذلك بست ساعات )المجموعة الثانية(، وبعد اثنى عشر ساعة )المجموعة الثالثة(، و بعد 24 ساعة )المجموعة الرابعة(، وبعد 14 يوما )المجموعة الخامسة(، وبعد 21 يوما )المجموعة السادسة( بعد التعرض الستنشاق النفثالين. واستخدمت ثم المناعية الهستوكيميائية الهستولوجية والطرق بالطرق لدراستها بأشكال مختلفة الرئة وتم معالجتها قطاعات

خضعت للتحليل اإلحصائي. النتائج: أظهرت النتائج أن استنشاق النفثالين يؤدى الى تقشير الخاليا المبطنة للقصيبات الهوائية والذي بدأ بعد 6 ساعات ووصل الى ذروته بعد 24 ساعة من استنشاق النفثالين في رئتيى الذكور واإلناث. وبدت الخاليا المبطنة للقصيبات مجدده بالكامل فى الذكورعند 14 يوما بينما ظهرت بعض الخاليا القليلة المقشره فى تجويف القصيبات

الهوائية لإلناث والتى اختفت تماما عند 21 يوما. المبطنة الخاليا في هامة احصائيه بداللة أعلى واإلناث الذكور من الجنسين في التقشير درجة متوسط كان و الذكور وبمقارنة منهما، لكل الضابطة بالمجموعة مقارنة والرابعة الثالثة المجموعات في الهوائية للقصيبات واالناث كان متوسط درجة التقشير أعلى بداللة احصائيه في الخاليا المبطنة للقصيبات الهوائية في إناث الجرذان )iNOS( مقارنة مع الذكور في المجموعتين الثالثة والخامسة. وكان هناك زيادة في ظهور محفز أكسيد النيتريكفي الخاليا المبطنة للقصيبات الهوائية للذكور واإلناث بعد التعرض الستنشاق النفثالين والتي زادت بمرور الوقت. استنشاق بعد يوما 21 تقريبا عند اختفى والذى iNOS انخفاض واضح في ظهور يوما، كان هناك 14 وعند بعد بالذكور مقارنة أكثر وضوحا اإلناث في iNOS وكان ظهور الجرذان. وإناث ذكور من كل في النفثالين

التعرض الستنشاق النفثالين عند نفس الوقت. الخالصة: حدثت االصابة في وقت مبكر وبدرجة اكبر فى الخاليا المبطنة للقصيبات الهوائية لإلناث، بينما حدث التجديد فى الخاليا المبطنة للقصيبات في وقت مبكر فى الذكور عنه فى االناث.وربما لزيادة ظهور محفز أكسيد النيتريك )iNOS( دور فى حدوث التلف فى القصيبات الهوائية للذكور واالناث ويمكن ان يكون االختالف في ظهور محفز أكسيد النيتريك )iNOS( في قصيبات اإلناث عن الذكور إضافة جديدة لتفسير آليات االختالف في

اصابة وكذلك تجدد وإصالح الخاليا المبطنة للقصيبات بين الجنسين.

Sex differences in bronchiolar epithelial injury El-Tahawy and Rifaai


Health & Medicine

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