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
672
Introduction
The 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
manyagentsthatcauselunginjury,hazardousairpollutants
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-Tahawy
Tel.: 01145435777
e-mail: nashogo@yahoo.com
Received 15 January 2012
Accepted 17 May 2012
The Egyptian Journal of Histology
2012, 35:672-685
61 (1380-2012)
Background
Airway 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 work
To 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 methods
Thirty-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.
Results
Inhalation 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.
Conclusion
Injury 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 Histology
1110-0559
Sex differences in bronchiolar epithelial injury and repair
following naphthalene inhalation in albino rats: a possible
role of inducible nitric oxide
Nashwa 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 exposures
Inhalation 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 immunohistochemistry
The 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 HE 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 methods
Animals
Thirty-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
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’s0.05). 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’s0.05). 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’s0.05).
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
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 capture
Images 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 analysis
Sections of rat lungs stained with HE 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 statistics
Data 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.
Results
Normal 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.
*P0.05 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).
HE, × 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.
HE, × 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.
HE, × 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.
HE, × 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).
HE, × 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.
HE, × 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).
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.

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).
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.
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.

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.

Discussion
Clara 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
beenbecauseofdifferencesintheroutesofadministration.
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

Acknowledgements
Conflicts of interest
There is no conflict of interest to declare.
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2 Preuss R, Angerer J, Drexler H. Naphthalene – an environmental and
occupational toxicant. Int Arch Occup Environ Health 2003; 76:556–576.
3 Alessandrini F, Weichenmeier I, van Miert E, Takenaka S, Karg E, Blume C,
et al. Effects of ultrafine particles-induced oxidative stress on Clara cells in
allergic lung inflammation. Part Fibre Toxicol 2010; 7:11.
4 Puchelle E, Zahm J-M, Tournier J-M, Coraux C. Airway epithelial repair,
regeneration, and remodeling after injury in chronic obstructive pulmonary
disease. Proc Am Thorac Soc 2006; 3:726–733.
5 Gauderman WJ, Avol E, Gilliland F, Vora H, Thomas D, Berhane K, et al. The
effect of air pollution on lung development from 10 to 18 years of age. N Engl
J Med 2004; 351: 1057–1067.
6 Leikauf GD. Hazardous air pollutants and asthma. Environ Health Perspect
2002; 110(Suppl 4): 505–526.
7 Ding X, Kaminsky LS. Human extrahepatic cytochromes P450: function in
xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory
and gastrointestinal tracts. Annu Rev Pharmacol Toxicol 2003; 43:149–173.
8 Preuss R, Drexler H, Böttcher M, Wilhelm M, Brüning T, Angerer J. Current
external and internal exposure to naphthalene of workers occupationally
exposed to polycyclic aromatic hydrocarbons in different industries. Int Arch
Occup Environ Health 2005; 78:355–362.
9 Zhang Q, Adiseshaiah P, Reddy SP. Matrix metalloproteinase/epidermal
growth factor receptor/mitogen-activated protein kinase signaling regulate
fra-1 induction by cigarette smoke in lung epithelial cells. Am J Respir Cell
Mol Biol 2005; 32:72–81.
10 Witschi H, Espiritu I, Maronpot RR, Pinkerton KE, Daniel Jones A. The
carcinogenic potential of the gas phase of environmental tobacco smoke.
Carcinogenesis 1997; 18:2035–2042.
11 Phimister AJ, Williams KJ, Van Winkle LS, Plopper CG. Consequences of
abrupt glutathione depletion in murine Clara cells: ultrastructural and
biochemical investigations into the role of glutathione loss in naphthalene
cytotoxicity. J Pharmacol Exp Ther 2005; 314:506–513.
12 Plopper CG, Buckpitt A, Evans M, Van Winkle L, Fanucchi M, Smiley-Jewell S,
et al. Factors modulating the epithelial response to toxicants in
tracheobronchial airways. Toxicology 2001; 160:173–180.
13 Carey MA, Card JW, Voltz JW, Arbes SJ Jr, Germolec DR, Korach KS,
Zeldin DC. It’s all about sex: gender, lung development and lung disease.
Trends Endocrinol Metab 2007; 18:308–313.
14 Siegfried JM. Women and lung cancer: does oestrogen play a role? Lancet
Oncol 2001; 2:506–513.
15 Wei Q, Cheng L, Amos CI, Wang L-E, Guo Z, Hong WK, Spitz MR. Repair of
tobacco carcinogen-induced DNA adducts and lung cancer risk: a molecular
epidemiologic study. J Natl Cancer Inst 2000; 92:1764–1772.
16 Sin DD, Cohen SB-Z, Day A, Coxson H, Paré PD. Understanding the
biological differences in susceptibility to chronic obstructive pulmonary
disease between men and women. Proc Am Thorac Soc 2007;
4:671–674.
17 March TH, Wilder JA, Esparza DC, Cossey PY, Blair LF, Herrera LK, et al.
Modulators of cigarette smoke-induced pulmonary emphysema in A/J mice.
Toxicol Sci 2006; 92:545–559.
18 Gharaee-Kermani M, Hatano K, Nozaki Y, Phan SH. Gender-based
differences in bleomycin-induced pulmonary fibrosis. Am J Pathol 2005;
166:1593–1606.
19 Park K-S, Wells JM, Zorn AM, Wert SE, Laubach VE, Fernandez LG,
Whitsett JA. Transdifferentiation of ciliated cells during repair of the respiratory
epithelium. Am J Respir Cell Mol Biol 2006; 34:151–157.
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.
BecauseNOinhibitorsrepresentanimportanttherapeutic
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.
Conclusion
Itcanbeconcludedthatfemaleratsweremoresusceptible
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.

32 Lawson GW, Van Winkle LS, Toskala E, Senior RM, Parks WC, Plopper CG.
Mouse strain modulates the role of the ciliated cell in acute tracheobronchial
airway injury-distal airways. Am J Path 2002; 160:315–327.
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.

685
‫العربى‬ ‫الملخص‬
‫نتيجة‬ ‫الهوائية‬ ‫للقصيبات‬ ‫المبطنة‬ ‫الخاليا‬ ‫إصالح‬ ‫و‬ ‫اصابة‬ ‫في‬ ‫الجنسين‬ ‫بين‬ ‫الفروق‬
.‫النيتريك‬ ‫أكسيد‬ ‫لمحفذ‬ ‫المحتمل‬ ‫الدور‬ :‫الجرذان‬ ‫في‬ ‫النفثالين‬ ‫الستنشاق‬
‫رفاعى‬ ‫احمد‬ ‫رحاب‬ ‫و‬ ‫الطحاوى‬ ‫فتحى‬ ‫نشوة‬
‫المنيا‬ ‫جامعة‬ - ‫الطب‬ ‫كلية‬ - ‫الهستولوجيا‬ ‫قسم‬
‫وعلى‬ ‫بل‬ ‫حدوث‬ ‫على‬ ‫الجنس‬ ‫ويؤثر‬ .‫المختلفة‬ ‫الرئة‬ ‫أمراض‬ ‫في‬ ‫يحدث‬ ‫وإصالحه‬ ‫الهواء‬ ‫مجرى‬ ‫إصابة‬ ‫إن‬ :‫المقدمة‬
‫إزالة‬ ‫على‬ ‫القدرة‬ ‫لها‬ ‫كالرا‬ ‫خاليا‬ .‫انتشارا‬ ‫البيئية‬ ‫الملوثات‬ ‫اكثر‬ ‫هو‬ ‫النفثالين‬ .‫الرئة‬ ‫فى‬ ‫أمراض‬ ‫عدة‬ ‫حدوث‬ ‫شدة‬
.‫السامة‬ ‫المواد‬ ‫من‬ ‫لعدد‬ ‫السموم‬
‫المستنشق‬ ‫بالنفثالين‬ ‫الحاد‬ ‫التسمم‬ ‫عن‬ ‫الناتجة‬ ‫اإلصابة‬ ‫في‬ ‫الجنسين‬ ‫بين‬ ‫فرق‬ ‫هناك‬ ‫كان‬ ‫إذا‬ ‫ما‬ ‫تحديد‬ ‫و‬ ‫دراسة‬ :‫الهدف‬
‫أكسيد‬ ‫محفز‬ ‫على‬ ‫النفثالين‬ ‫استنشاق‬ ‫تأثير‬ ‫وصف‬ ‫وايضا‬ .‫الجرذان‬ ‫رئة‬ ‫قصيبات‬ ‫في‬ ‫الخاليا‬ ‫بتجدد‬ ‫واالستجابة‬
.)iNOS( ‫النيتريك‬
‫كمجموعة‬ ‫الجوي‬ ‫للهواء‬ ‫اما‬ ‫الجرذان‬ ‫تعرضت‬ .‫البالغين‬ ‫الجرذان‬ ‫وإناث‬ ‫ذكور‬ ‫من‬ ‫ثالثون‬ ‫استخدمت‬ :‫الطريقة‬
‫بست‬ ‫ذلك‬ ‫بعد‬ ‫قتلت‬ ‫ثم‬ ،‫ساعات‬ 4 ‫لمدة‬ ‫النفثالين‬ ‫ببخار‬ ‫المحمل‬ ‫الهواء‬ ‫الستنشاق‬ ‫أو‬ ،)‫األولى‬ ‫(المجموعة‬ ‫ضابطة‬
‫وبعد‬ ،)‫الرابعة‬ ‫(المجموعة‬ ‫ساعة‬ 24 ‫بعد‬ ‫و‬ ،)‫الثالثة‬ ‫(المجموعة‬ ‫ساعة‬ ‫عشر‬ ‫اثنى‬ ‫وبعد‬ ،)‫الثانية‬ ‫(المجموعة‬ ‫ساعات‬
‫واستخدمت‬ .‫النفثالين‬ ‫الستنشاق‬ ‫التعرض‬ ‫بعد‬ )‫السادسة‬ ‫(المجموعة‬ ‫يوما‬ 21 ‫وبعد‬ ،)‫الخامسة‬ ‫(المجموعة‬ ‫يوما‬ 14
‫ثم‬ ‫المناعية‬ ‫الهستوكيميائية‬ ‫والطرق‬ ‫الهستولوجية‬ ‫بالطرق‬ ‫لدراستها‬ ‫مختلفة‬ ‫بأشكال‬ ‫معالجتها‬ ‫وتم‬ ‫الرئة‬ ‫قطاعات‬
.‫اإلحصائي‬ ‫للتحليل‬ ‫خضعت‬
6 ‫بعد‬ ‫بدأ‬ ‫والذي‬ ‫الهوائية‬ ‫للقصيبات‬ ‫المبطنة‬ ‫الخاليا‬ ‫تقشير‬ ‫الى‬ ‫يؤدى‬ ‫النفثالين‬ ‫استنشاق‬ ‫أن‬ ‫النتائج‬ ‫أظهرت‬ :‫النتائج‬
‫المبطنة‬ ‫الخاليا‬ ‫وبدت‬ .‫واإلناث‬ ‫الذكور‬ ‫رئتيى‬ ‫في‬ ‫النفثالين‬ ‫استنشاق‬ ‫من‬ ‫ساعة‬ 24 ‫بعد‬ ‫ذروته‬ ‫الى‬ ‫ووصل‬ ‫ساعات‬
‫القصيبات‬ ‫تجويف‬ ‫فى‬ ‫المقشره‬ ‫القليلة‬ ‫الخاليا‬ ‫بعض‬ ‫ظهرت‬ ‫بينما‬ ‫يوما‬ 14 ‫الذكورعند‬ ‫فى‬ ‫بالكامل‬ ‫مجدده‬ ‫للقصيبات‬
.‫يوما‬ 21 ‫عند‬ ‫تماما‬ ‫اختفت‬ ‫والتى‬ ‫لإلناث‬ ‫الهوائية‬
‫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
‫الجرذان‬ ‫إناث‬ ‫في‬ ‫الهوائية‬ ‫للقصيبات‬ ‫المبطنة‬ ‫الخاليا‬ ‫في‬ ‫احصائيه‬ ‫بداللة‬ ‫أعلى‬ ‫التقشير‬ ‫درجة‬ ‫متوسط‬ ‫كان‬ ‫واالناث‬
)iNOS( ‫النيتريك‬ ‫أكسيد‬ ‫محفز‬ ‫ظهور‬ ‫في‬ ‫زيادة‬ ‫هناك‬ ‫وكان‬ .‫والخامسة‬ ‫الثالثة‬ ‫المجموعتين‬ ‫في‬ ‫الذكور‬ ‫مع‬ ‫مقارنة‬
.‫الوقت‬ ‫بمرور‬ ‫زادت‬ ‫والتي‬ ‫النفثالين‬ ‫الستنشاق‬ ‫التعرض‬ ‫بعد‬ ‫واإلناث‬ ‫للذكور‬ ‫الهوائية‬ ‫للقصيبات‬ ‫المبطنة‬ ‫الخاليا‬ ‫في‬
‫استنشاق‬ ‫بعد‬ ‫يوما‬ 21 ‫عند‬ ‫تقريبا‬ ‫اختفى‬ ‫والذى‬ iNOS ‫ظهور‬ ‫في‬ ‫واضح‬ ‫انخفاض‬ ‫هناك‬ ‫كان‬ ،‫يوما‬ 14 ‫وعند‬
‫بعد‬ ‫بالذكور‬ ‫مقارنة‬ ‫وضوحا‬ ‫أكثر‬ ‫اإلناث‬ ‫في‬ iNOS ‫ظهور‬ ‫وكان‬ .‫الجرذان‬ ‫وإناث‬ ‫ذكور‬ ‫من‬ ‫كل‬ ‫في‬ ‫النفثالين‬
.‫الوقت‬ ‫نفس‬ ‫عند‬ ‫النفثالين‬ ‫الستنشاق‬ ‫التعرض‬
‫حدث‬ ‫بينما‬ ،‫لإلناث‬ ‫الهوائية‬ ‫للقصيبات‬ ‫المبطنة‬ ‫الخاليا‬ ‫فى‬ ‫اكبر‬ ‫وبدرجة‬ ‫مبكر‬ ‫وقت‬ ‫في‬ ‫االصابة‬ ‫حدثت‬ :‫الخالصة‬
‫أكسيد‬ ‫محفز‬ ‫ظهور‬ ‫لزيادة‬ ‫االناث.وربما‬ ‫فى‬ ‫عنه‬ ‫الذكور‬ ‫فى‬ ‫مبكر‬ ‫وقت‬ ‫في‬ ‫للقصيبات‬ ‫المبطنة‬ ‫الخاليا‬ ‫فى‬ ‫التجديد‬
‫في‬ ‫االختالف‬ ‫يكون‬ ‫ان‬ ‫ويمكن‬ ‫واالناث‬ ‫للذكور‬ ‫الهوائية‬ ‫القصيبات‬ ‫فى‬ ‫التلف‬ ‫حدوث‬ ‫فى‬ ‫دور‬ )iNOS( ‫النيتريك‬
‫في‬ ‫االختالف‬ ‫آليات‬ ‫لتفسير‬ ‫جديدة‬ ‫إضافة‬ ‫الذكور‬ ‫عن‬ ‫اإلناث‬ ‫قصيبات‬ ‫في‬ )iNOS( ‫النيتريك‬ ‫أكسيد‬ ‫محفز‬ ‫ظهور‬
.‫الجنسين‬ ‫بين‬ ‫للقصيبات‬ ‫المبطنة‬ ‫الخاليا‬ ‫وإصالح‬ ‫تجدد‬ ‫وكذلك‬ ‫اصابة‬
Sex differences in bronchiolar epithelial injury El-Tahawy and Rifaai

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