international journal of dental science and research 3 (2016) 914

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Original Article

Immunohistochemical evaluation of inducible nitric

oxide synthase (iNOS) expression in the dental
follicle, follicular cyst, and calcifying cystic
odontogenic tumor

Safoura Sei a,*, Tahere Padeganeh b, Syed Razie Rezaee c, Ali Bijani d
a
Oral Health Research Center, Babol University of Medical Sciences, Babol, Iran
b
Assistant Professor, Department of Oral and Maxillofacial Surgery, School of Dentistry,
Babol University of Medical Sciences, Iran
c
Dental Student, School of Dentistry, Babol University of Medical Sciences, Iran
d
General Practitioner, Non-Communicable Pediatrics Diseases Research Center, Babol University of Medical Sciences,
Babol, Iran

article info abstract

Article history: Objective: The calcifying cystic odontogenic tumor (CCOT) is a benign cystic neoplasm with a
Received 3 August 2015 variety of histopathological and clinical features in comparison with the follicular cyst. iNOS
Accepted 17 November 2015 is an enzyme that produces free radicals and is a mediator regulator of the inammatory
Available online 28 February 2016 response. It has been implicated in tumorigenesis as well. The aim of this study was to
compare iNOS expression in the dental follicle and two odontogenic lesions with different
Keywords: aggressive behaviors.
Dental follicle Methods: In this cross-sectional study, 44 parafn blocks were selected from the dental
Follicular cyst follicle, follicular cyst, and CCOT, and immunohistochemical staining was done by iNOS.
Calcifying cystic odontogenic tumor The percentage and intensity of staining (total score) were calculated in the cytoplasm of the
Immunohistochemistry epithelial cells.
Nitric oxide synthase (iNOS) Results: The highest total score of iNOS staining was found in the cytoplasm of the epithelial
cells of CCOT when compared to the follicular cyst and dental follicle ( p = 0.000). There was a
signicant difference in the nal score between the follicular cyst and dental follicle
( p = 0.001).
Discussion: The overexpression of iNOS in the CCOTs plays a role in its pathogenesis and
iNOS, because of producing free radicals and damaging the oral tissue, may contribute to
more aggressive behaviors of CCOT.
# 2016 Published by Elsevier, a division of Reed Elsevier India, Pvt. Ltd on behalf of Sardar
Patel Post Graduate Institute of Dental and Medical Sciences.

* Corresponding author.
E-mail address: sf_seify@yahoo.com (S. Sei).
http://dx.doi.org/10.1016/j.ijdsr.2015.11.007
2213-9974/# 2016 Published by Elsevier, a division of Reed Elsevier India, Pvt. Ltd on behalf of Sardar Patel Post Graduate Institute of
Dental and Medical Sciences.
10 international journal of dental science and research 3 (2016) 914
1. Introduction
The calcifying cystic odontogenic tumor (CCOT) is a relatively
rare lesion. It was previously known as the calcifying
odontogenic cyst (the Gorlin cyst). Due to diverse clinico-
pathological appearances and the various neoplastic poten-
tials, there is still disagreement on its terminology and also
whether to classify CCOT as a cyst, neoplasm, or a combined
lesion. Treatment varies on the basis of histopathological type
from surgical enucleation to more aggressive treatments
because of its risk of recurrence.1,2
A dentigerous cyst (follicular) is the most common
developmental odontogenic cyst that covers the crown of
the impacted tooth and is asymptomatic in most cases if it is
not infected or inamed. Treatment includes enucleation of
the cyst and extraction of the involved tooth. It is reported that
the dental follicle may transform to the dentigerous cyst.3,4
In the literature, little is known about the pathogenesis and
biological growth process of CCOT. It has an aggressive behavior
when compared with the follicular cyst and dental follicle.5
Nitric oxide synthase is a member of the family of enzymes
that catalyzes the production of nitric oxide from L-arginine.
Nitric oxide has an important role in physiologic and
pathologic events and is active as a short-effect toxic gas. It
induces free radicals formation and might enhance angiogen-
esis, which can lead to an accelerated growth of the primary
tumor and metastasis.6 Overexpression of unregulated NO has
been implicated in pathologic conditions. Three isoforms of
nitric oxide synthase include nNOS (neural), iNOS (induced),
and eNOS (endothelial). iNOS has an important role in
inammatory and neoplastic events and its production is
increased by inammatory mediators of inammatory cells.6,7
The iNOS expression has been studied in some cysts.8,9 In one
study, the expression of iNOS in the dental follicle and
follicular and radicular cyst was reviewed, which showed no
signicant difference. iNOS induces bone resorption and
expansion of the cysts, which may be due to overexpression
of matrix metalloproteinase.8 There is considerable contro-
versy over understanding its role in tumorigenesis.10
It is believed that nitric oxide in low concentrations in the
initial stages of cancer has a role in cell proliferation, and its
high density is cytotoxic for tumoral and normal cells.11 The
iNOS expression has been reported in benign and various
malignant tumors and some researchers suggest NO may play
a main role in tumor growth, progression, and metastasis.12
There are no articles in the literature comparing the expres-
sion of iNOS in the dental follicle, follicular cyst, and CCOT.
Therefore, the aim of this study was to compare and evaluate
iNOS expression in the dental follicle and two odontogenic
lesions with different aggressive features by the immunohis-
tochemical method and to see whether there is any relation-
ship between the aggressive behavior of odontogenic lesions
and the expression of iNOS.
2. Methods
This cross-sectional study was approved by the Ethical
Committee of Babol University of Medical Sciences. The study
samples were 44 cases, CCOT (15 cases), follicular cyst
(15 cases), and dental follicle (14 cases), which were obtained
from parafn-embedded section archives of the Pathology
Department of Dental Universities of Babol and Mashhad.
Sections (4 mm) were cut and counterstained with hematoxy-
lin to make a denite diagnosis by two independent
pathologists who were unaware of the clinical ndings, and
then, different histopathologic types of CCOT were classied
according to Sagha et al.13:
1. Simple cyst: only has a cystic epithelium similar to
ameloblastoma with ghost cells.
2. Cystic neoplasm: consists of Gorlin cystic epithelium with
ameloblastic nests in the connective tissue.
3. Solid neoplasm: consists of cystic epithelium of the Gorlin
cyst with ameloblastic proliferation, and dentinoid and
ghost cells in the connective tissue.
4. Combined lesion: Gorlin cyst accompanied by odontogenic
tumors, such as ameloblastoma and odontoma.
Samples whose tissues were not adequate for evaluation or
had inappropriate quality or xation were excluded. Then,
other 4-mm slides of parafn-embedded blocks were obtained
and stained using the immunohistochemical method with
iNOS antibody. All moderate and severe inamed CCOT and
dental follicle samples were deleted. The follicular cyst was
classied as inamed (8 cases) and noninamed (7 cases).
The specimens of parafn-embedded blocks were rst
deparafnized with xylene and then rehydrated in decreasing
concentrations of ethanol and covered with sodium citrate
buffer (pH 6) for 5 min. They were then transferred to
microwaves (Bhutan) for antigen retrieval. The endogenous
peroxidase activity was quenched by incubation in 3%
peroxidase-blocking solution so that it covered all the tissues.
The tissues were then incubated with the initial iNOS antibody
(Rabbit Anti-Human iNOS, Abcam, UK. Cat Na: ab3523, 1/50) for
16 h at 4 8C and then with a secondary antibody for 30 min
(Novolink polymer, Leica Microsystem Corporation. REF:
RE7112, LOT: 6017103); Diaminobenzidine tetrahydrocloride
(DAB) (DAB/Chromogen/DAB/Substrate buffer (kit), DAKO,
USA) sections were counterstained with Mayer's hematoxylin
for 30 s. Oral squamous cell carcinoma was the positive control
and deletion of the primary antibody and its replacement with
PBS and phosphate buffer was the negative control. The
stained slides were reviewed and scaled independently by two
pathologists using a light microscope (Olympus Bx41, Olym-
pus Corporation, Tokyo, Japan). If there was any disagreement
between the two pathologists, a third pathologist evaluated
the slides and reported the nal results.
In the study, the percentage and intensity of staining was
considered and buffy-colored cytoplasmic staining was con-
sidered positive for the iNOS marker.14
3. Assessment of immunohistochemical
staining
Microscopic iNOS-stained slides were evaluated at (100)
magnication, and in the elds with the highest staining,
evaluation of 100 cells by (400) magnication was done in
 international journal of dental science and research 3 (2016) 914
3 microscopic elds randomly. The mean percentage of
cytoplasm staining (for iNOS) was considered.
The quantitative scale in the evaluation of the staining
percentage of the epithelial cell cytoplasm was according to
the following criteria: 125% scored 1, 2650% scored 2, 5175%:
scored 3, and 76100% scored 4.
The staining intensity of the cytoplasm was classied semi-
quantitatively on the basis of a four-point scale:
0: no staining, 1: week staining, 2: moderate staining, 4:
severe staining.
The nal score as calculated as percentage  intensity of
staining. The values of the weighted score ranged from 0 to a
maximum of 12; 0 to 3 was dened as iNOS negative and 4 or
higher was dened as iNOS positive.14
The relationship between inammation and iNOS expres-
sion in inammatory and noninammatory follicular cysts
was assessed. Mild inammation (less than 25% inammatory
cells in each microscopic eld) or no inammation in the
connective tissue was considered noninammatory while
moderate to severe inammation (more then 25% inamma-
tory cells in each microscopic eld) and hyperplastic rete
ridges were regarded as inammation in the connective
tissue.15 The histopathologic subtypes of CCOT were classied
similar to a report by Sagha et al.,13 and the nal score in
the groups was evaluated for iNOS. At the end, the results
were analyzed with SPSS (20) using LSD, MannWhitney,
KruskalWallis, and ANOVA. Statistical signicance was set at
p <0.05.
The KruskalWallis was used to compare three groups of
follicular cyst, dental follicle, and CCOT according to the
percentage and intensity of iNOS staining, and ANOVA was
employed to compare the nal score of the three groups. Also,
MannWhitney was used for the assessment of the relation-
ship between iNOS expression and histopathologic types of
CCOT. MannWhitney and LSD were applied to compare two
groups according to the iNOS expression.
4. Results
In the cross-sectional study, 15 CCOT, 15 follicular cysts, and
14 dental follicles were evaluated. The clinical ndings are
presented in Table 1.
Table 1 Clinical findings (age, gender, location of the lesion) in the dental follicle, follicular cyst, and CCOT.
Kind of sample Mean age
Male
Dental follicle 21.57  3.73
Follicular cyst 22  6.793
CCOT 28.93  15.95
Table 2 Percentage of cytoplasmic staining and total score of epithelial cells by iNOS in the dental follicle, follicular cyst,
and CCOT, separately.
Dental follicle
Percentage staining in epithelium 37.43  11.147
Total score in epithelium 2.785  1.57
11
There was no signicant statistical difference in age
between the three groups ( p = 0.107). Different histopathologic
types of CCOT (Gorlin cyst) included the simple cyst (7 cases),
cystic neoplasm (2 cases), solid neoplasm (2 cases), and
combined lesions (4 cases).
There was a signicant difference in the staining percent-
age of iNOS epithelium among the three groups. The highest
percentage of staining was seen in CCOT and the minimum
percentage was observed in the dental follicle ( p = 0.000)
(Table 2). There was no signicant difference in the staining
percentage of epithelium by iNOS between the follicular cyst
and dental follicle ( p = 0.8). There was a signicant difference
in the nal score between the follicular cyst and dental follicle
( p = 0.001). Moreover, a signicant difference was observed in
the expression of staining intensity by iNOS in the epithelium
between three groups ( p = 0.000) (Table 3, Figs. 15).
In the evaluation of the total score, from a total of 14 dental
follicles, only 4 cases had positive staining by iNOS in the
epithelium and the others were negative. One sample has a
score of 4, 1 sample had a score of 6, and 2 samples scored 9.
In the follicular cysts, 14 cases had moderate positive
staining by iNOS (a score of 4).
As for CCOT, all 15 samples had positive staining (moderate
to severe) by iNOS; 12 cases had a score of 12 and 3 cases had a
score of 6. A signicant difference was observed in the nal
score between the CCOT, follicular cyst, and dental follicle
( p = 0.000).
There was no signicant difference between inamed and
noninamed follicular cysts in the expression of iNOS in the
epithelium ( p = 0.607) so that mean ndings of INOS in
epithelium in inamed and noninamed Follicular cyst were
8.50 and 7.25, respectively.
5. Discussion
According to the ndings of this study, there is a positive
correlation between the iNOS expression (total score) and the
aggressive behavior of the odontogenic lesions, such as CCOT.
iNOS induces lipid peroxidation, DNA damage, free radicals
formation, p53 mutation, and damage to the oral tissues. p53
mutation may lead to an increase in cellular proliferation. It is
not clear whether NO leads to an increase in mutant p53 or vice
Gender Location
Female Mandible Maxilla
3 11 9 5
8 7 14 1
8 7 13 2
Follicular cyst CCOT p value
38.00  8.332 88.13  10.32 0.000
3.86  0.51 10.80  2.48 0.000
12 international journal of dental science and research 3 (2016) 914

Table 3 Intensity of staining in the cytoplasm of the

epithelial cells by iNOS in the dental follicle, follicular
cyst, and CCOT.
Type of sample Intensity of epithelium staining

Weak Moderate Severe

Dental follicle 10 4 0
Follicular cyst 1 14 0
CCOT 0 3 12

versa. The results of a study by de Oliveria et al.16 showed that

the expression of mutant p53 in the proliferative type of CCOT
was increased when compared to the nonproliferative type. In
this study, iNOS overexpression had a main role in the Fig. 2 Immunohistochemical staining with iNOS in the
aggressive behavior of CCOT when compared with the follicular cyst (40T).
follicular cyst and dental follicle. iNOS is effective in
the pathogenesis of CCOT and follicular cyst but this role is
more prominent in CCOT; therefore, it is considered as a
benign cystic odontogenic tumor.
Chen et al.17 reported iNOS overexpression in ameloblas-
toma versus the keratocyst; also, they believed that an
increase in the expression of iNOS induced overexpression
of VEGF in ameloblastoma, which could explain its aggressive
behavior. The results of the study by Chen et al. are in
agreement with results of our study. Swetha et al.8 reported
iNOS overexpression in the odontogenic keratocyst when
compared to DC and RC, suggesting it may contribute to the
pathogenesis and aggressive behavior of the keratocyst.
Based on the results of the previous studies, iNOS can be
generated in physiologic conditions. The expression of iNOS in
this study in the dental follicle versus the other two
odontogenic lesions may indicate the function of the antioxi- Fig. 3 Immunohistochemical staining with iNOS in the
dant system against the free radicals produced in physiologic neoplastic cystic type of the Gorlin cyst (10T).
conditions. By the accumulation of free radicals, the conver-
sion of the dental follicle to the follicular cyst is possible.18
Kocaelli et al.14 did not nd any signicant differences in
the iNOS expression of the periapical granuloma, radicular The main producer of iNOS has always been a question.
cyst, and dental follicle; therefore, they put emphasis on the Because of the high intensity and percentage of iNOS staining
necessity of prophylactic exclusion of the third molar because in the epithelium (basal cells with reverse polarity), it is
of inducing free radicals. suggested that the epithelium is a source of iNOS production. It
is suggested that the epithelial lining of CCOT can produce IL-1
and 6. These cytokines may activate iNOS expression in an
autocrine fashion.
Rosbe et al.19 reported that because of high staining of
epithelium in squamous cell carcinoma by iNOS, it can be
considered as a major source of iNOS production that is in
agreement with the results of our study. Some studies have
suggested that the high expression of iNOS by macrophages is
the main source of iNOS.20 Kawanishi et al.21 reported the
expression of iNOS in epithelial and inammatory cells, which
is in line with the results of our study.
It seems that high amounts of free radicals produced during
the chronic inammatory process may have a role in
carcinogenesis by damaging DNA. Some researchers have
conrmed the role of iNOS in the development of oral cancers.
Some other authors have introduced iNOS as a protective
factor against cancer and have reported that the production of
Fig. 1 Immunohistochemical staining with iNOS in the iNOS in low densities has anticancer effects while it has a
dental follicle (10T). tumorigenic role in high concentrations. The dual function of
 international journal of dental science and research 3 (2016) 914
Fig. 4 Immunohistochemical staining with iNOS in the
simple cystic type of the Gorlin cyst (10T).
NO in cancer can be related to its local concentration, genetic
type of the tumoral cells, and redox condition. Sappayatosok
et al.22 stated that the overexpression of iNOS in oral
squamous cell carcinoma was related to the degree of
differentiation, stage, and angiogenesis, and reported that
expression of iNOS could be associated with carcinogenesis.
Takaoka et al.23 found that high expression of iNOS in adenoid
cystic carcinoma of the oor of the mouth was associated with
a poor prognosis. They expressed that prevention of iNOS
function could act as a suppressor factor of lung metastasis.
Although there are different opinions regarding the
pathogenesis of ghost cells in CCOT, low to moderate staining
of ghost cells suggests that iNOS can be effective in the
pathogenesis of ghost cells. iNOS expression was similar in
inamed and noninamed dentigerous cysts. It is suggested
that in inamed odontogenic cysts, other factors beside the
epithelium may induce iNOS expression.
Some researchers believe that the use of the drugs that
inhibit iNOS actions may be useful in the treatment of oral
lesions or they may protect the transformation of the dental
Fig. 5 Immunohistochemical staining with iNOS in the
ghost cells of the Gorlin cyst (40T).
13
follicle to the follicular cyst in the future through iNOS
inhibition.24
A limitation of our study was its small sample size in the
histopathologic subtypes of CCOT (Gorlin cyst comment on the
role of iNOS with more certainty if more samples are available).
6. Conclusion
The overexpression of iNOS in CCOT plays a role in its
pathogenesis. iNOS, because of inducing free radicals forma-
tion, may contribute to more oral tissue damage and the
aggressive behavior of CCOT.
Conicts of interest
The authors have none to declare.
Acknowledgements
This article is the result of a research project approved by Babol
University of Medical Sciences. The authors wish to thank
Mr. Aghajani as well as the Deputy of Research for help in
immunohistochemistry.
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