Oral Oncology (2008) 44, 956– 962

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/oraloncology

Effects of Spirulina platensis extract on

Syrian hamster cheek pouch mucosa painted
with 7,12-dimethylbenz[a]anthracene
Mohammed E. Grawish

Faculty of Dentistry, Mansoura University, Mansoura, Egypt

Received 18 October 2007; accepted 25 November 2007

Available online 8 February 2008

KEYWORDS Summary Research into cancer prevention seeks to identify the preventable causes of cancer,
Spirulina platensis; and to reduce cancer incidence by effective implementation of preventative strategies in tar-
7,12-Dimethyl-
get populations. In this study, 30 male golden Syrian hamsters were divided into three equal
benz[a]anthracene (DMBA)
groups; the right buccal pouches of the hamster rats in group one were painted with 0.5% solu-
tion of 7,12-dimethylbenz[a]anthracene (DMBA), three times a week, until sacrificed. The same
pouches of group two were also painted with DMBA, but received an additional 10 mg/daily
Spirulina platensis extract, which was added to the soft diet supplements during the same per-
iod. The hamster rats in group three received neither DMBA nor S. platensis extract. They were
painted with saline and served as control animals. Half the hamsters from each of the three
groups were sacrificed by ether inhalation after 7 weeks, and the remaining half were sacrificed
after 14 weeks. The required buccal pouches were surgically excised and prepared for regular
H&E and argyrophilic proteins of the nuclear organizer regions (AgNOR) sliver staining. AgNORs
counting and statistical analysis were carried out. We observed moderate dysplastic changes
extending into the midspinous layer in group one 7 weeks after DMBA painting, which reached
to half the thickness of the hyperplastic epithelium after 14 weeks. However, in group two,
mild dysplastic changes were observed after 7 weeks, which were restricted to the basilar
and parabasilar layers of the epithelium after 14 weeks of treatment. AgNOR staining in group
one produced AgNOR counts ranging from one to seven dots per nucleus, whereas the counts
were one or two dots per nucleus in group two. The AgNOR mean number in groups one, two
and three was (3.1 ± 0.006, 1.3 ± 0.003 and 1.2 ± 0.003, respectively). Moreover, with one sam-
ple t-test, a significant difference was found in AgNOR mean number between groups one and
two, groups one and three and between groups two and three (P < 0.05). An overall significant
difference among the three groups (P < 0.01) was indicated with one-way analysis of variance.

E-mail address: grawish2005@yahoo.com

1368-8375/$ - see front matter ª 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.oraloncology.2007.11.014
Effects of Spirulina platensis extract on Syrian hamster cheek pouch mucosa painted with DMBA
The pAgNOR was 10% in group one, 5% in group two and 4% in group three. Consequently, S.
platensis is an adjunctive means to inhibit the dysplastic changes occurring in the hamster
cheek pouch (HCP) mucosa. However, more research is needed to expand its beneficial action.
ª 2007 Elsevier Ltd. All rights reserved.
Introduction
Oral cancer is a common neoplasm worldwide, particularly
in developing countries, where it constitutes up to 25% of
all types of cancers.1
The development of oral cancer is a multi-step process
requiring initiation, promotion and progression. Hamster
cheek pouch (HCP) is the most widely accepted model of
oral cancer,2 and is a useful model for investigating multi-
step carcinogenesis. Owing to its accessibility, the HCP
can be used for the induction of oral squamous cell carci-
noma by chemical carcinogens and for testing chemo-pre-
ventive agents.3,4
A carcinogen has been defined as an agent that causes a
neoplasm by a two-step process involving initiation and pro-
motion. The difference between the two steps is said to be
qualitative rather than quantitative. Polycyclic aromatic
hydrocarbons are known immunotoxins and carcinogens.
7,12-Dimethylbenz[a]anthracene (DMBA) and benzo[a]pyr-
ene (B[a]P) are the prototypical and best characterized
polycyclic aromatic hydrocarbons.5–8 The hydrocarbon car-
cinogen, DMBA, has both initiating and promoting
properties.9
Carcinogenesis is a process that converts normal cells
into malignant cells. Once transformed, malignant cells
acquire the ability to invade and metastasize, leading to
cancer. During this transformation from normal to malig-
nant cells, carcinogenic steps can be suppressed or
reversed through the use of specific natural or synthetic
chemical agents before the onset of invasive cancer.10
In the past few years, considerable attention has been
paid to potential chemo-preventive agents such as isothio-
cyanates in cruciferous vegetables, catechins in green
tea, resveratrol in grape seeds and red wine, curcumi-
noids in turmeric, and procyanidins in various fruits and
nuts.11–13
Spirulina is a microscopic filamentous blue–green alga,
which has no roots, leaves, seeds or flowers, and has been
in existence for more than 2 billion years. It is rich in pro-
teins, vitamins, essential amino acids, minerals and essen-
tial fatty acids such as y-linolenic acid. It is produced
commercially and sold as a food supplement in health food
stores around the world. In the past decade, the interest in
spirulina was mainly in its nutritional value.14,15 Spirulina
has gained increasing attention from medical scientists as
a nutraceutical and source of potential pharmaceuticals.
Several studies of spirulina’s ability to inhibit viral replica-
tion, strengthen both the cellular and humoral arms of the
immune system, and cause regression and inhibition of can-
cers have been published.16–18
Although these studies are preliminary and more re-
search is needed, the results so far are exciting. Thus, the
purpose of this study was to investigate the effect of Spiru-
957
lina platensis on DMBA-induced oral carcinogenesis on HCP
mucosa.
Materials and methods
Thirty pathogen-free male golden Syrian hamsters were
used in this study. They were 60–90 days old and weighed
100–120 g. The chemical carcinogen was 0.5% solution of
DMBA (Sigma Chemical Company, St. Louis, MO) that was
dissolved at a concentration of 5 mg/ml in mineral oil (Fish-
er-Scientific Company, Pittsburgh, PA).19 A number four
sable brush was used to apply the carcinogen. A daily dose
of 10 mg/day20 of S. platensis extract, isolated in the Fac-
ulty of Pharmacy, Mansoura University, was used, and given
with bread and milk.
The animals were divided into three equal groups (10 ani-
mals each); the right buccal pouches of the hamster rats in
group one were painted with 0.5% solution of DMBA three
times a week until sacrificed.21,22 The same pouches of
group two were also painted with DMBA, but received an
additional 10 mg/day S. platensis extract with the food sup-
plement at the same time and for the same duration. The
animals in group three received neither DMBA nor S. platen-
sis extract. They were painted with saline and served as
controls. Half of the hamsters in each of the three groups
were sacrificed by ether inhalation after the 7-week exper-
imental period, and the remaining half was sacrificed after
14 weeks. The required buccal pouches were surgically
excised.
The pieces were fixed in 10% neutral buffered formalin
solution. Paraffin sections were prepared for regular hae-
matoxylin and eosin (H&E) and argyrophilic proteins of the
nuclear organizer regions (AgNOR) sliver staining. For H&E
stain, paraffin sections were cut using microtome at 4 lm
thickness and stained to examine the histological grading
of epithelial dysplasia. For AgNOR sliver staining, the mod-
ified AgNOR silver staining technique introduced by Ploton
et al.23 was used. The tissue was deparaffinized in several
changes of xylene and descending alcohol concentrations,
rehydrated in several changes of ultra-pure distilled water,
incubated in acid alcohol (three parts ethanol:two parts
acetic acid) for 5 min and rinsed in ultra-pure distilled water
several times. The sections were then incubated with silver
nitrate solution in a dark humidified chamber for 38 min.
The silver staining solution consisted of two parts of a 50%
solution of silver nitrate and one part 2% gelatin in 1% formic
acid solution. Ultra-pure distilled water was used for the
preparation of all solutions. The sections were then incu-
bated with a 10% solution of sodium thiosulfate solution
for 5 min, and then washed in distilled water, dehydrated
in graded alcohol and cover-slipped. No counter stain was
used.
958 M.E. Grawish

Counting procedure of argyrophilic proteins of the

nuclear organizer regions

AgNOR counting was carried out by two independent observ-

ers without any knowledge of histological features. Repre-
sentative areas that stained clearly were randomly
selected. In all cases, 50 nuclei per specimen were counted,
using an oil-immersion lens at magnification of 100·. AgNOR
counting and typing was carried out after sharp focusing on
the nuclear membrane and fine granules nuclear matrix in
each nucleus.24 Three main types of AgNOR configuration
could be found in neoplastic cells: a large homogenous
and rounded argyrophilic structure corresponding to the
nucleolus, a large structure surrounded by a thin membrane
and including numerous smaller dots (AgNORs within the Figure 1 Photomicrograph of healthy hamster cheek pouch
nucleolus) and dispersed small AgNORs lying free in the nu- mucosa of the control group showing keratinized stratified
cleus. All individual AgNORs including, whenever possible, squamous epithelium (E) with no rete ridges, lamina propria (L)
the tiny black or brown dots within the nucleolus were and muscular layer (M) (H&E, 400·).
counted.25,26

Statistical analysis of argyrophilic proteins of the

nuclear organizer regions numbers

Two counts were carried out. The first count was the mean
number of AgNORs in 50 nuclei (mAgNOR). This count was
believed to reflect total chromosome count or ploidy;27,28
any tumour showing a mAgNOR count of 2.4 or more was
considered aneuploid.29 The second count was the percent-
age of nuclei showing five or more AgNOR granules per nu-
cleus (pAgNOR). This count was believed to represent
proliferative activity.28,30 Tumours having a pAgNOR count
of 8% or more were considered to display high proliferative
activity;27 the total number of AgNORs from 50 nuclei per
specimen was determined and the mean value and standard Figure 2 Photomicrograph of hamster cheek pouch mucosa, 7
deviation of each case were established. The SPSS package weeks after DMBA painting showing moderate dysplastic
for statistical analysis of data that were obtained from the changes extending into the midspinous layer as loss of polarity
AgNOR counting was used. Analysis of variance was used (Po), pleomorphism (Pe) and the increase nuclear cytoplasmic
to compare the difference between the overall groups, ratio (N/C) in the hyperplastic keratinized epithelium (H) (H&E,
whereas the t-test was used to analyze the difference be- 400·).
tween each group separately. A histogram was drawn for
the mean value of all groups. To be useful, mean AgNOR
counts must clearly distinguish between healthy tissue and
pathologic tissue. For this to happen, cut-off points need
to be delineated to establish a threshold for diagnosis. In
a previous report,31 the mean AgNOR count of 2.37 was used
as a potential cut-point to distinguish between non-dysplas-
tic leukoplakia and dysplastic leukoplakia.

Results

The healthy HCP mucosa of the control group showed kera-

tinized stratified squamous epithelium with no rete ridges,
lamina propria and muscular layer (Figs. 1–8).
This HCP mucosa at 7 weeks after DMBA painting showed
moderate dysplastic changes reaching to the midspinous
layer of the hyperplastic keratinized epithelium in the form
of loss of polarity, pleomorphism and an increase in the nu-
clear cytoplasmic ratio. These moderate epithelial dysplas-
tic changes reached to half the thickness of the hyperplastic
epithelium that became more apparent after 14 weeks.
Figure 3 Photomicrograph of HCP mucosa, 14 weeks after
DMBA painting showing moderate dysplastic changes extending
over the half thickness of the hyperplastic keratinized stratified
squamous epithelium (H) in the form of an increase in number
of mitotic figures (M), an increase in nuclear to cytoplasmic
ratio (N/C) and loss of polarity of basal cells (Po) and
pleomorphism (Pe) (H&E, 400·).
Effects of Spirulina platensis extract on Syrian hamster cheek pouch mucosa painted with DMBA 959

Figure 4 Photomicrograph of hamster cheek pouch mucosa, 7
weeks after DMBA painting with the daily administration of
10 mg S. platensis extract showing mild dysplastic changes in
the stratified squamous epithelium as pleomorphism (Pe),
hyperchromatism (Hy), acanthosis (A) and loss of polarity (Po)
confined to basilar and parabasilar layers (H&E, 400·).
Figure 6 Photomicrograph of healthy hamster cheek pouch
mucosa of the control group showing the argyrophilic proteins
of the nuclear organizer regions as sliver stained brown dots
that are evident in the nuclei of epithelial cells (sliver staining,
original magnification 1000·). (For interpretation of the refer-
ences in color in figure legends, the reader is referred to the
web version of this article).

Figure 5 Photomicrograph of hamster cheek pouch mucosa, 14

weeks after DMBA painting with the daily administration of 10 mg
S. platensis extract showing mild dysplastic changes restricted to
basal and parabasal layers of the hyperplastic keratinized Figure 7 Photomicrograph of hamster cheek pouch mucosa,
stratified squamous epithelium (H), hyperchromatism (Hy), loss 14 weeks t DMBA painting showing argyrophilic proteins of the
of polarity (Po) and pleomorphism (Pe) (H&E, 400·). nuclear organizer region counts ranging from one to seven dots
per nucleus (sliver staining, original magnification 1000·).
The mucosa at 7 weeks after DMBA painting with daily
administration of 10 mg S. platensis extract showed mild
dysplastic changes in the stratified squamous epithelium
as pleomorphism, hyperchromatism, acanthosis and loss of
polarity confined to basilar and parabasilar layers. However,
these mild epithelial dysplastic changes were restricted to
these layers and became less apparent after the 14 weeks
of treatment.
The healthy HCP mucosa of the control group showed the
AgNOR as sliver stained brown dots, which are evident in the
nuclei of epithelial cells. Fourteen weeks after DMBA paint-
ing, AgNOR counts ranged from one to seven dots per nu-
cleus, whereas 14 weeks after DMBA painting with daily
administration of 10 mg S. platensis, the counts were one
or two dots per nucleus in the HCP mucosa.
Figure 8 Photomicrograph of HCP mucosa, 14 weeks after
Statistical results DMBA painting with the administration of 10 mg S. platensis
showing argyrophilic proteins of the nuclear organizer region
The AgNOR mean number in the group one was 3.1 ± 0.006, counts ranging from one or two dots per nucleus (sliver staining,
whereas the mean AgNOR number in the group two was original magnification 1000·).
960 M.E. Grawish

Table 1 Mean AgNOR count in each group and results of statistical comparison of the mean AgNOR count
Experimental group Observation period, 14 weeks
N Min Max Meana SD P-value  P-valueà
Group 1 10 3.00 3.20 3.1000 006.6 <0.05 <0.05
Group 2 10 1.24 1.36 1.3000 003.3 <0.05
Group 3 10 1.14 1.26 1.2000 003.5 <0.05
a
Mean AgNOR cut-off as diagnostic threshold was 2.4.
à
Between groups, the differences were statistically significant using analysis of variance.
 
The differences were statistically significant using one sample t-test.

4.0 HCP mucosa is covered by a thin layer of keratinized strat-

ified squamous epithelium that is similar in its thickness to
3.5 the floor of the mouth and the ventral surface of the tongue
in humans.
3.0 However, the HCP mucosa 7 weeks after DMBA painting
showed moderate dysplastic changes extending into the
2.5 midspinous layer that reached to half the thickness of the
95% CI

hyperplastic keratinized stratified squamous epithelium

2.0
1.5
1.0
.5
N= 10 10 10
Group 1 Group 2 Group 3
Figure 9 Error bars of separate variable with confidence
interval for mean at level 95%.
1.3 ± 0.003 and group three was 1.2 ± 0.003 (Table 1). With
one sample t-test, a significant difference was found in Ag-
NOR mean number between groups one, two and three, and
between groups two and three (P < 0.05). One-way analysis
of variance indicated an overall significant difference
among the three groups (P < 0.01). The pAgNOR in group
one was 10%, whereas in group two it was 5% and for group
three 4%. Their graphical presentations are shown in Fig. 9.
Discussion
Animal models have contributed significantly to the under-
standing of carcinogenesis and ways of manipulating the
process to prevent cancer. Many animal models are found
to mirror corresponding human cancers in cell of origin,
morphogenesis, phenotype markers and genetic altera-
tions.32 Balmain and Harris33 reported the importance of
the hamster rat carcinogenesis models as a well-known ani-
mal system that closely mimics the development of prema-
lignant and malignant lesions in human oral cancer. On the
basis of this knowledge, the present study was conducted in
such an animal model to evaluate the effect of S. platensis
on DMBA-induced oral carcinogenesis.
The healthy HCP mucosa of the control group in this
study showed keratinized stratified squamous epithelium
with no rete ridges, lamina propria and muscular layer, in
accordance with Boring et al.19 The authors reported that
after 14 weeks.
Filip et al.34 and Gimenez-Conti and Slaga35 also reported
that application of DMBA to the cheek pouch of the Syrian
golden hamster produces squamous cell carcinoma that is
histologically similar to human oral squamous cell carci-
noma. Consequently, carcinoma is preceded by a sequence
Mean of hyperplasia–papilloma and dysplasia–carcinoma, similar
to human leukoplakia.
These dysplastic changes may be attributed to the
genotoxic and mutagenic effects of these agents. Other
investigators36–40 have also reported the critical impor-
tance of certain enzymes responsible for DMBA metabo-
lism leading to the formation of reactive metabolites
that bind to DNA causing mutations and cancer initiation.
In contrast, Raychoudhury and Kubinski41 considered DMBA
nongenotoxic, but was able to target specific cellular
proteins.
The results of this study show that spirulina extract given
after DMBA painting has an inhibitory effect on tumour ini-
tiation, as the HCP mucosa, 7 weeks after DMBA painting
with daily administration of 10 mg S. platensis, extract
showed mild dysplastic changes confined to basilar and par-
abasilar layers. These mild changes were restricted to the
same layers after 14 weeks. Such an inhibitory effect may
be attributed to the repair of the damaged DNA that keep
cells alive and healthy. Other investigators,18,42,43 for exam-
ple, have reported that some common forms of cancer are
thought to be a result of damaged cell DNA running amok,
causing uncontrolled cell growth. They define a system of
special enzymes called endonuclease, which repair dam-
aged DNA. When these enzymes are deactivated by radia-
tion or toxins, errors in DNA go unrepaired and cancer
may develop. The authors suggest that the unique polysac-
charides of spirulina enhance cell nucleus enzyme activity
and DNA repair synthesis.
Nucleolar organizer regions (NORs) are segments of DNA
that transcribe to ribosomal RNA and are located on short
arms of the acrocentric chromosomes 13, 14, 15, 21 and
22.23 The increased number of NORs reflects transcriptional
activity, cell proliferation rate, and thus malignant
Effects of Spirulina platensis extract on Syrian hamster cheek pouch mucosa painted with DMBA
potential.44,45 Crocker and Nar46 reported that NOR-associ-
ated proteins are argyrophilic and can be visualized in par-
affin sections after staining with the silver colloid technique
as small brown to black dots within the nucleus.
Moreover, the AgNOR silver staining technique has been
extensively studied to find alternative techniques of cell
kinetics analysis other than flow cytometry as a method.
The determination of AgNOR scores is a subjective method
regarding the variations in staining methods and in counting
and interpreting the results. However, AgNOR is also ex-
pressed in proliferating as well as resting cells. For example,
PCNA and Ki-67 expression are absent in resting cells.47,48 On
the basis of this outcome, this study was conducted to evalu-
ate the effect of S. platensis on DMBA carcinogenic hamster
models using AgNOR staining as a simple, fast and reliable
technique for cell proliferation and nucleolar changes.
The healthy HCP mucosa of the control group showed the
AgNOR as sliver stained brown dots that are evident in the nu-
clei of epithelial cells, and this in accordance with Derenzini
and Ploton49 who concluded that NORs can be identified easily
as brown to black dots of different sizes, localized throughout
the nucleolar area. With AgNOR staining, 14 weeks after
DMBA painting, the HCP showed AgNOR counts ranging from
one to seven dots per nucleus (mean AgNOR was 3.2 and pAg-
NOR more than 10%), whereas the counts were one or two dots
per nucleus in the HCP mucosa at 14 weeks after DMBA paint-
ing with daily administration of 10 mg S. platensis (mean
AgNOR was 1.3 and pAgNOR more than 5%).
On the basis that mAgNOR scores correlated with ploidy
and pAgNOR correlated with the percentage of cells in
S-phase of the cell cycle or with proliferative activity, and
the mean AgNOR cut-off as diagnostic threshold was 2.4, a
fast proliferation with aneuploid changes in the first group
was observed compared with the second group. This is in
keeping with Mourad et al.29 Carbajo et al.50 found that
the mAgNOR scores in cells with slow proliferation may be
in the range of 0.5–1.5, and that mean AgNOR count of
2.37 can be used as a potential cut-off point to distinguish
between non-dysplastic and dysplastic epithelium.
Conclusion
On the basis of this research, the use of spirulina extracts
may inhibit cancer progression. More research is needed
to confirm these results and to prove its efficacy against
cancer especially; it is already clear that S. platensis is a
natural food supplement that has proven benefits for opti-
mum health and wellness.
Conflict of Interest Statement
None declared.
Acknowledgements
I would like to greatly thank my colleagues Dr. Ahmed Zaher
and Dr. Mohamed Helal; Associate Professors of Oral Biol-
ogy, Mansoura University, for their efforts in counting the
AgNOR dots.
961
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