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ORIGINAL RESEARCH
Assessment of anti-mutagenic, anti-histopathologic
and antioxidant capacities of Egyptian bee pollen
and propolis extracts
Amany A. Tohamy •Ehab M. Abdella •
Rasha R. Ahmed •Yara K. Ahmed
Received: 28 July 2012 / Accepted: 16 April 2013 / Published online: 16 May 2013
ÓSpringer Science+Business Media Dordrecht 2013
Abstract Bee pollen and propolis are popular, tradi-
tional health foods. The objective of the current study
was to investigate the anti-mutagenic, anti-histopath-
ologic and antioxidant effects among water extracts of
Egyptian bee pollen (WEBP) and brown powder of
water-soluble derivative propolis (WSDP) on cisplatin
(CDDP) induced hepatic, renal, testicular and geno-
toxicity in male albino mice (Mus muscullus), in
addition to their effects on the oxidant/antioxidant
status in the tested organs. Hepatic, renal and testicular
dysfunctions were evaluated histologically; while
genotoxicity and cytotoxicity were evaluated by the
bone marrow chromosomal aberration assay and
mitotic index, respectively. Moreover, oxidative stress
was explored via determination of lipid peroxidation,
catalase activity and the concentration of the reduced
form of glutathione. The treatment of mice with WEBP
and WSDP at doses 140 and 8.4 mg/kg b. wt./day,
respectively for 14 days simultaneously with CDDP
(2.8 mg/kg b. wt.) resulted in significant protection.
The positive control animals taken CDDP alone
showed toxic histological and genetical manifestations
(at P\0.05) accompanied with an elevated content of
peroxidized lipid and lowered catalase activity and
glutathione concentration in the homogenate of liver,
kidney and testis tissues (at P\0.001). These toxic
side effects in all tested organs were greatly ablated
with a significant reduction in lipid peroxidation level
and elevation in catalase activity and glutathione
concentration (P\0.001) when using both WEBP and
WSDP. On the basis of the present assays, Bee pollen
appears more potent in exerting an ameliorative effect
and this effect was more pronounced in testis.
Keywords Bee pollen Propolis Anti-mutagenic
Anti-histopathologic Antioxidant
Introduction
The honeybee (Apis mellifera) makes various bee
products from plants, flower nectar, and flower pollen,
and humans make use of these products. Bee products
are well known in the traditional medicine, and their use
has a very long history. Todays, their uses have
expanded from the health food arena into the medical
one. Bee pollen is collected by honeybees as a nutrient
harvest for the hive. Bee pollen is considered by many
people to be a nutrient-rich perfect food, and is promoted
as a commercially available food supplement. Pollen
grains, which are the male reproductive cells of flowers,
contain high concentrations of phytochemicals and
A. A. Tohamy
Faculty of Science, Zoology Department, Helwan
University, Helwan, Egypt
E. M. Abdella (&)R. R. Ahmed Y. K. Ahmed
Faculty of Science, Zoology Department, Beni-Suef
University, Beni-Suef, Egypt
e-mail: ehababdella@gmail.com
123
Cytotechnology (2014) 66:283–297
DOI 10.1007/s10616-013-9568-0
nutrients (Markham and Campos 1996; Eraslan et al.
2009; Saric et al. 2009). Nakajima et al. (2009) reported
that bee pollen is rich in carotenoids, flavonoids and
phytosterols, while the exact profile of bee pollen
content varies depending on the plant sources and
growth conditions. The anti-oxidant activity of honey-
bee-collected pollen has been recognized as free radical
scavenger and as lipid peroxidation inhibitor. This
activity has been associated with the phenolic pollen
content (Campos et al. 2003). Furthermore, Turkey bee
pollen has inhibitory effects against mycelia growth and
several pharmacological activities (Ozen et al. 2004).
Propolis a sticky substance that honeybees manu-
facture by mixing their own waxes with resinous sap
obtained from the bark and leaf-buds of certain trees
and flowering plants. It is used as a sealant and sterilant
in honeybee nests. The composition of propolis varies
from hive to hive, from district to district, and from
season to season. Normallyit is dark brown in color, but
it can be found in green, red, black and white hues,
depending onthe sources of resin found in the particular
hive area. Honey bees are opportunists, gathering what
they need from available sources, and detailed analyses
show that the chemical composition of propolis varies
considerably from region to region, along with the
vegetation. In northern temperate climates, for exam-
ple, bees collect resins from trees, such as poplars and
conifers (the biological role of resin in trees is to seal
wounds and defend against bacteria, fungi and insects).
‘‘Typical’’ northern temperate propolis has approxi-
mately 50 constituents, primarily resins and vegetable
balsams (50 %), waxes (30 %), essential oils (10 %),
and pollen (5 %). In neotropical regions, in addition to a
large variety of trees, bees may also gather resin from
flowers of the genera Clusia and Dalechampia, which
are the only known plant genera that produce floral
resins to attract pollinators (Bankova 2005). Now, it is
recognized that propolis has a wide range of biological
activities, such as antibacterial (Souza et al. 2007), anti-
inflammatory (Barros et al. 2008; Nakajima et al. 2009),
antioxidative (Teixeira et al. 2008), hepatoprotective
(Basnet et al. 1997) and tumoricidal (Mitamura et al.
1996) activities. Such effects have been associated with
the presence of b-amylase and many phenolic com-
pounds such as flavones, flavonones, galangin, cin-
namic acid, caffeic acid and esters (Volpi 2004).
Concerning Egyptian propolis, Lotfy (2006) reported
thirty-nine constituents eight of them being newly
described for propolis including phenolic esters
(72.7 %), phenolic acids (1.1 %), aliphatic acids
(2.4 %), dihydrochalcones (6.5 %), chalcones
(1.7 %), flavanones (1.9 %), flavones (4.6 %), and
tetrahydrofuran derivatives (0.7 %).
Cisplatin (cis-diamminedichloro platinum) is one
of the most important anti-neoplastic agents used in
the treatment of several types of solid tumorus (Pabla
et al. 2008). Along with its high therapeutic activity,
cisplatin is a potential human carcinogen with
increased carcinogenic risk towards the development
of secondary malignancy in patients (Greene 1992). In
addition, this drug has a lot of side effects including
high nephrotoxicity, reproductive toxicity, hepatotox-
icity and genotoxicity which are examples of the most
severe and dose limiting side effects (Nersesyan and
Muradyan 2004). The cytotoxic activity of cisplatin
results from its induced oxidative stress, resulting in
loss of mitochondrial protein-SH, inhibition of cal-
cium uptake and reduction in the mitochondrial
membrane potential (Husain and Naseem 2008).
Recently, evidences have been accumulated that lipid
peroxidation and free radical formation which cause
an imbalance between the generation of oxygen
derived radicals and the animal antioxidant potential
play a great role in this toxicity promoting cellular
damage (Ozen et al. 2004; Naziroglu et al. 2004; Iraz
et al. 2006). While the genotoxic effect of cisplatin
was proposed to be derived from its interaction with
DNA and production of DNA-platinum covalent
adducts that inhibiting the fundamental cellular pro-
cesses including replication, transcription, translation
and DNA repair (Lee and Schmitt 2003).
The purpose of the present study was to evaluate
and compare the effectiveness of total water extracts
of honey bee collected pollen (WEBP) and propolis
(WSDP) from Beni-Suef, Egypt, as in vivo anti-
mutagenic, anti-histopathologic and antioxidant agent
against cisplatin (CDDP)–induced chromosomal
abnormalities in bone marrow cells, and histological
alterations and oxidative stress in liver, kidney and
testis tissues of mice (Mus musculus).
Materials and methods
Chemicals
Cis-platin (CDDP) (cis-diamminedicholorplatinum)
was purchased from MERCK Company (Darmstadt,
284 Cytotechnology (2014) 66:283–297
123
Germany) in a form of ampoules, each containing
25 mg of CDDP in 25 ml sterile saline solution
containing hydrochloric acid and sodium hydroxide to
adjust pH between 3.8 and 5.9. The solution in these
ampoules appeared clear, free from turbidity or matter
which deposits on standing. Other chemicals were
obtained from Sigma (St. Louis, MO, USA).
Experimental animals
The experimental animals used in this work were random
bred adult males of laboratory mice Mus musculus
(20–30 g in weight). Animals were obtained from
Ophthalmology research institute, Giza, Egypt. Experi-
ments were performed as per internationally followed
ethical standards and according to the Guide for the Care
and Use of Laboratory Animals of the National Institutes
of Health (Institute of laboratory animal resources 1996).
All animals were housed in plastic cages with wired
covers and kept under normal laboratory conditions for
the different periods of time used. The animals were not
treated with antibiotics, vitamins or insecticides and fed a
standard commercials diet (ATMID Company, Giza,
Egypt) and tap water ad. libitum.
Bee products
The propolis used in the present study was obtained as a
brown powder of water-soluble derivative propolis
(WSDP) from beekeepers in Beni-Suef, Upper Egypt.
The mixture of honey bee–collected pollen was
provided by local beekeepers in September 2006. The
pollen source in a given area depends on the type of
vegetation present and the length of their bloom period.
What type of vegetation will grow in an area depends
on soil texture, soil pH, soil drainage, daily maximum
and minimum temperatures, precipitation, extreme
minimum winter temperature, and growing degrees.
These pollens collected by the bees from anthers of the
flowers of the plants growing in the surrounding of the
bee hives in Beni-Suef, Upper Egypt.
Extracts preparation
Propolis extract was prepared by the method of
(Ors
ˇolic
´and Bas
ˇic
´2005). Under sterile condi-
tions 16.8 mg of the brown powder of propolis
(WSDP) was dissolved in 10 ml distilled water and
mixed vigorously for 10 min. Finally, this suspension
was centrifuged at 1,000 rpm for 10 min in room
temperature. The supernatant was collected and stored
under freezing condition at -20 °C until use.
Bee pollen extract was obtained according to the
method of Yamaguchi et al. (2006). Briefly, the
powder of bee pollen (280 mg) was suspended in
distilled water (10 ml) and mixed vigorously. This
suspension was stored stand overnight in dark and
centrifuged at 10,000 rpm in a cooling centrifuge at
10 °C for 45 min. The supernatant fraction was
carefully collected and filtrated. The filtrate was stored
under freezing condition at -20 °C until used.
Experimental design
A single dose (2.8 mg/kg b. wt.) of CDDP used in
present study was selected with reference to the dose
range of the cytotoxicity and genotoxicity of CDDP
(Pisano et al. 2003; Nersesyan and Muradyan 2004).
However, propolis (WSDP) and bee pollen (WEBP)
water extract concentrations used in the present study
were 8.40 and 140 mg/kg b. wt. respectively. These
concentrations were selected according to Mani et al.
(2006) and Yamaguchi et al. (2006) respectively.
Experimental groups were organized as six groups
including 11 animals each. In each group five animals
were used for cytogenetic analysis while the rest of
animals (six mice) were used for biochemical and
histopathological analysis. The animals of the group
one (G1) served as a negative control receiving 0.9 %
NaCl solution by intraperitoneal injection (i.p.) twice/
week for 3 weeks. The animals of the group two (G2)
received i.p. injection of CDDP (2.8 mg/kg b. wt.)
twice/week for 3 weeks. In group three (G3) 8.4 mg/
kg b. wt. of WSDP extract was given to the animals
through oral intubation once/day for 14 days consec-
utively. The animals of the group four (G4) received
oral administration of WEBP (140 mg/kg b. wt.) once/
day for 14 days. The animals of the groups five and six
(G5 and G6) were injected i.p. with CDDP (2.8 mg/kg
b. wt. twice/week) alone for 1 week but, for next
2 weeks these animals were given WSDP and WEBP
respectively through oral intubation in parallel to i.p.
injection of CDDP.
Tissues sampling
At the end of the experiment, mice in each group were
sacrificed under mild anesthesia. Liver, kidneys and
Cytotechnology (2014) 66:283–297 285
123
testes tissues were removed quickly. 0.5 g of each of
them was homogenized in 5 ml of 0.9 % NaCl. The
obtained homogenate was kept in deep freezer at -
20 °C for measurement of oxidative stress markers
including lipid peroxidation and glutathione content,
and catalase activity. Moreover, pieces of liver and
kidney tissues were fixed in neutral buffered formalin
for histopathological studies.
Antioxidant-capacity assay
Parts of liver, kidney and testis (0.5 g) were ice-cooled
and homogenized in 5 ml 0.9 % NaCl (1 % w/v) using
Teflon homogenizer (Glas-Col, Terr Haute, IN,
USA). The homogenate was centrifuged at 3,000 g
for 15 min at 4 °C. The supernatant was collected and
preserved at -20 °C till used for oxidative stress and
antioxidant defense system measurement. In tested
organs homogenates, lipid peroxidation (LPX) was
determined by measuring the thiobarbituric acid
reactive substances (TBARS) according to the method
of Pressus et al. (1998). Glutathione reduced form
(GSH) level was measured colorimetrically as protein-
free sulfhydryl content using Ellman reagent (Beutler
et al. 1963). Catalse (CAT) activity was analyzed
according to the method of Cohen et al. (1970)by
monitoring the enzyme-catalyzed decomposition of
hydrogen peroxide using potassium permanganate.
Histopathological assay
Tissue samples of liver, kidney and testis were fixed in
10 % neutral buffered formalin (pH 6.8) for 24 h.
After dehydration, tissue samples were embedded in
paraffin wax, sectioned at 5 lm and stained with
haematoxylin and eosin (Bancroft and Gamble 2002)
for histopathological examination.
Cytogenetic assay
Bone marrow cell preparations for the analysis of
chromosomal aberrations and mitotic indices were
conducted by the colchicines-hypotonic technique.
After completion of the treatment period, five animals
from each group were sacrificed 24 h post-injection
with saline, cisplatin, bee pollen or propolis, by
cervical dislocation. Colchicine was given at the dose
of 4 mg/Kg b. wt. intraperitoneally (i.p.) at 22 h prior
to sacrificing the animals. The bone marrow smears of
animals in each group were prepared according to
Preston et al. (1987). For each group, slides were
stained with Giemsa staining method and 50 well
spread metaphase plates/animal were analyzed for
chromosomal aberrations and the number of mitotic
cells in 1000 cells/animal. The percentage of sup-
pressed aberrant cells was calculated according to
Shukla and Taneja (2002) as follows:
100 %of aberrant cells in Gr5 or Gr6
%of aberrant cells in Gr2
100
Statistical analysis
One way analysis of variance ANOVA (Rao et al.
1985) was used to statistically analyze the biochemical
variables. (P\0.05 was considered significant).
While statistical analysis for the difference in the
mean number of chromosomal aberrations and mitotic
index between groups was carried out using the
student ttest (P\0.05 was considered significant).
Results
Antioxidant-capacity assay
Administration of cisplatin for 21 days caused signif-
icant increase at P\0.001 in the lipid peroxidation
level associated with a significant decrease (P\0.001)
in both glutathione content and catalase activity in all
organs (liver, kidney and testis) compared to the control
group. However, administration of propolis and bee
pollen aqueous extracts after 1 week of cisplatin
administration and in parallel with it for another 14 days
significantly attenuated the changes in these parameters
(P\0.001). The lipid peroxidation was significantly
reduced (P\0.001) in groups five and six (G5 and G6)
compared to the cisplatin group (G2) and this decrease
was even more pronounced in G6 (CDDP ?WEBP),
where all values return to the normal level but still
exceeding the negative control group (G1). The ame-
lioration was more pronounced in testis (Table 1).
Contrary, the level of glutathione and activity of
catalase were significantly elevated with both treat-
ments (P\0.001) (G5 and G6). Bee pollen extract in
G6 had also a better effect than propolis extract in G5
leading to significantly elevation of most of the
recorded values above the CDDP group (G2)
(Table 1).
286 Cytotechnology (2014) 66:283–297
123
However, propolis extract administration to mice in
G3 led to a significant increase in the level of lipid
peroxidation significantly compared to the control
(G1) and reduced catalase activity, while the glutathi-
one level was nearly unchanged in all organs. In
addition significantly lowered values of glutathione
content in G3 were recorded in comparison to the
control (G1) and bee pollen (G4) groups.
Histopathological assay
Liver
The histological examination of the liver sections of
control animals showed normal liver architecture. The
central area is formed of a central vein form which
hepatic cords arise. These cords are separated by a
number of sinusoids lined with Kupffer cells (Fig. 1a).
Administration of cisplatin for 21 days caused
severe disruption to the normal structure. The hepa-
tocytes showed several degenerative changes
(Fig. 1b), the parenchyma in the central area was
infiltrated with a number of fibroblasts (Fig. 1b) and
revealed numerous necrotic foci (Fig. 1c), while the
portal area showed odema, fibrosis and congested
portal vein. However, mice given propolis or bee
pollen had nearly normal liver architecture and
organization (Fig. 1d, e) except for the infiltration
with few inflammatory cells noticed in the portal area
of the liver sections of propolis-treated animals
(Fig. 1d).
Administration of propolis and bee pollen after and
concomitant with cisplatin, greatly ameliorated the
hepatic histopathological lesions and the hepatic
parenchyma attained nearly normal structure and
organization. Since few portal areas showed portal
odema accompanied with portal vein congestion
(Fig. 1f) in the propolis treated group while prolifer-
ation of Kupffer cell’s was recorded in bee pollen
group (Fig. 1g).
Kidney
The microscopical examination of the kidney of
control mice revealed the normal cortical and medul-
lary histological structure. The cortex is formed of a
number of Malpigian corpuscles with its glomeruli
and urinary space, proximal tubules and distal tubules
(Fig. 2a).
Table 1 Showing the changes in lipid peroxidation content (Malondialdehyde), catalase activity and glutathione concentration in different organs of all tested experimental
groups
Groups Lipid peroxidation (LPX)
nmol/g tissue 910
Catalase (CAT)
k/g tissue
Reduced glutathione (GSH)
lmol/lg tissue 910
3
Kidney Liver Testis Kidney Liver Testis Kidney Liver Testis
G1 (saline) 4.466 ±0.09
d
3.2 ±0.10
e
3.2 ±0.09
e
0.766 ±0.01
a
0.81 ±0.01
b
0.69 ±0.01
b
3.3 ±0.10
b, c
4.53 ±0.09
b
3.866 ±0.12
c
G2 (CDDP) 7.466 ±0.15
a
5.1 ±0.10
a
6.1 ±0.11
a
0.466 ±0.02
e
0.58 ±0.02
e
0.44 ±0.02
e
2.1 ±0.13
e
2.67 ±0.24
d
2.900 ±0.10
d
G3 (WSDP) 5.466 ±0.12
c
4.8 ±0.04
b
4.5 ±0.10
c
0.606 ±0.01
c
0.73 ±0.01
c
0.64 ±0.01
c
3.0 ±0.07
c, d
3.67 ±0.21
c
3.633 ±0.14
c
G4 (WEBP) 3.666 ±0.21
e
3.1 ±0.06
e
2.4 ±0.07
f
0.723 ±0.01
a
0.93 ±0.01
a
0.80 ±0.01
a
4.2 ±0.08
a
5.33 ±0.09
a
5.366 ±0.15
a
G5 (CDDP ?WSDP) 5.966 ±0.15
b
4.5 ±0.08
c
5.2 ±0.08
b
0.550 ±0.03
d
0.63 ±0.01
d
0.59 ±0.01
d
2.9 ±0.04
d
2.97 ±0.02
d
2.766 ±0.09
d
G6 (CDDP ?WEBP) 5.366 ±0.13
c
4.1 ±0.08
d
3.6 ±0.15
d
0.663 ±0.01
b
0.71 ±0.03
c
0.63 ±0.01
c
3.3 ±0.18
b
3.43 ±0.11
c
4.300 ±0.04
b
Data are expressed as mean ±standard error (SE)
Numbers of animals tested are six per group
Values sharing the same superscript symbol are considered non-significant at P\0.001
Cytotechnology (2014) 66:283–297 287
123
In cisplatin-injected group, numerous severe histo-
logical alterations were noticed. These alterations
include tubular and glomerular degenerative changes
(Fig. 2b) associated with tubular and periglomerular
accumulation of the albuminous material and medul-
lary fibrosis in most animals (Fig. 2d). In few cases,
the renal tubules showed hypertrophied epithelial cells
with obliterated lumens (Fig. 2c) and the glomeruli
Fig. 1 (a–g): Photomicrographs of liver sections of mice
stained with haematoxylin and eosin showing acontrol liver
section showing the central vein (CV), the hepatic sinusoids (S),
the hepatic strands (H) and the Kupffer cells (X 128), bhydrobic
degenerative changes (HD), fibroblastic proliferation (down-
ward arrow) seen in the hepatic parenchyma of cisplatin
injected mice for twenty-one days (9512), cnecrotic foci
(N) seen in the parenchyma with hydrobic degenerative changes
(downward arrow) of liver section of cisplatin-treated mice
surrounded by highly eosinophilic hepatocytes and occupied
with mononuclear leucocytes (9100), dminimal fibrosis
(downward arrow) seen in the portal area of propolis-treated
animals (X 128), enormal hepatic tissue organization registered
in bee pollen-treated mice (9128), fperiportal odema (O) and
portal vein congestion with normal hepatocytes organization
and appearance, recorded in animals treated with propolis after
cisplatin administration (9100) and gdiffuse proliferation of
Kupffer cells (downward arrow) noticed in the hepatic
parenchyma in the central area of animals treated with bee
pollen after cisplatin injection (9128)
288 Cytotechnology (2014) 66:283–297
123
showed hypercellularity, accompanied with periglom-
erular leucocytic aggregation (Fig. 2c). However,
kidneys from animals administered with propolis and
bee pollen extracts (groups G3 and G4 respectively),
showed normal histological structure compared to the
cisplatin (G2) and control (G1) groups (Fig. 2d, e).
Though hyperemic glomeruli and congested blood
vessels were rarely seen in propolis group (Fig. 2d).
Administration of propolis or bee pollen together
with cisplatin minimized the degenerative changes,
Fig. 2 (a–g): Showed sections of kidney in different examined
groups stained with haematoxylin and eosin. aA photomicro-
graph of the cortical region of a kidney of control mice showing
the golmerulus (G) with its Bowman’s capsule (downward
arrow), urinary space (U), glomerular apparatus (GA), the
proximal tubule (PT) and distal tubule (DT) (9400). bA
photomicrograph of the cortical region of kidney section of mice
administered cisplatin illustrating widening of the urinary space
(U), atrophy of the glomerulus (G), and vacuolar degenerative
changes in the renal tubules (V) (91,000). cA light micrograph
showing perivascular focal mononuclear leucocytic aggregation
(MN) and odema (O). Renal tubular (RT) epithelial cells showed
hyperplasia with obliterated lumen (9400). dA light micro-
graph of the glomerulus of propolis-treated group showing
eosinophilic bodies (E) (downward arrow) and congested blood
vessels (BV) (9400). eA photomicrograph of the kidney section
of administered bee pollen showing nearly normal cortical
structure. (G: glomerulus, U: urinary space, RT: renal tubule)
(9400). fLightmicrogarph of the kidney section of mice
administered cisplatin and treated with propolis having renal
tubular (RT) normal structures with hyperemic glomeruli
(G) (9150). gA photomicrograph of the kidney of mice given
cisplatin and treated with bee pollen illustrating normal renal
tubules (RT) and glomeruli (G) (9150)
Cytotechnology (2014) 66:283–297 289
123
the accumulation of albuminous material, the medul-
lary fibrosis, the glomerular hypercellularity and
hypertrophy of epithelial cells of renal tubules
(Fig. 2f, g). This amelioration effect was better in
bee pollen group (G4) where only hypertrophied
epithelial cells were still noticed.
Testis
Studying the testis sections of control animals showed
that it is formed of a number of seminiferous tubules.
Each of them was separated by a number of Leydig
cells. These tubules are lined with germ cells in
various stages and Sertoli cells in between. The
spermatogenic lineage is represented by a number of
spermatogonia, primary and secondary spermatocytes,
spermatids and spermatozoa (Fig. 3a).
Injection of cisplatin for 21 days induced numerous
destructive changes in the seminiferous tubules includ-
ing vacuolation of the spermatogonia, loss of spermatids
and sperms (Figs. 3b, c) and disorganization of the
tubular cells. Besides, there was congestion in the
interstitial tissue (Fig. 3c insert) compared to the control
animals. Propolis or bee pollen administration causedno
histopathological alteration in the testis (Fig. 3d, e)
respect to the control group, despite few macrophage
were noticed in the propolis group (Fig. 3d).
On the other hand, administration of propolis and
bee pollen extracts after and concomitant with
cisplatin led to a decrease in the destructive histopa-
thological alterations induced by cisplatin in testis
tissues including decrease in vacuolation of the
spermatogonia and increase in the organization of
tubular cells (Fig. 3f, g). The bee pollen effect was
most potent than propolis (Fig. 3f).
Cytogenetic assay
According to the cytogenetic results illustrated in
Tables 2and 3, seven structural and numerical chro-
mosomal aberrations were detected in the control and
the experimental groups (Fig. 4). The results obtained
in the first phase of cell cycle (24 h sampling time),
revealed that cisplatin (CDDP) when given at a dose of
2.8 mg/kg b.wt, twice/week for 3 weeks (G2) induced
a high frequency of chromosomal aberrations in bone
marrow cells of mice when compared with the control
(G1) group (Tables 2,3). The chromatid breakages
were the most frequent chromosomal aberration. Other
structural and numerical aberrations increased signif-
icantly at P\0.05 over the control group (G1), while
the mitotic index was significantly decreased to 37.75,
compared to control value of 83.29 (P\0.05),
indicating bone marrow cytotoxicity (Table 3).
Data in Tables 2and 3showed that, when propolis
extract (WSDP) was given alone at dose 8.4 mg/kg
once daily for 14 days did not induce or increase in
number of some chromosomal aberrations over con-
trol group (G1) like gaps, centric attenuation, end to
end association, centric fusion, polyploidy and endo-
mitosis and the value of total numerical aberrations.
While the number of chromatid breakage, value of
total structural aberrations, number of cells with one
aberration, and with more than one aberration,
percentage of incidence of aberrant cells and number
of aberration/cell increased significantly over the
control group (G1) (at P\0.05). The WSDP was
also not found to be cytotoxic at the given dose (8.4
mg/kg b.wt), as there was no significant changes in
mitotic index over G1 (Table 3). The group treated
with the aqueous bee pollen extract (WEBP) (G4) was
compared with the control group (G1) in terms of gaps,
centromeric attenuations, centric fusions, end to end
associations, polyploidy, endomitosis, mean total
number of numerical aberrations, percentage of inci-
dence of aberrant cells and number of aberration/cell
and did not show significant differences (P\0.05)
confirming its non-mutagenicity (Tables 2,3). The
WEBP was also not found to be cytotoxic at the given
dose (140 mg/kg b.wt), as there was no significant
changes in mitotic index over G1 (Table 3).
On the other hand, administration of propolis and
bee pollen extracts after and concomitant with cisplatin
(treatment groups G5 and G6 respectively) led to a
significant decrease in rates of clastogenetic changes
compared with the CDDP treatment group (Tables 2,
3). All types of chromosomal aberrations induced by
CDDP including breaks, gaps, end to end association,
centric fusion, centromeric attenuation, and other
multiple damages were found to be reduced by WSDP
and WEBP but they were still significantly higher than
negative control group (G1). The mitotic index was
also found to be significantly increased (P\0.05)
over G2, indicating its anti-cytotoxicity towards
CDDP (Table 3). The percentages of aberrant cells
which were found to be 50.00 ±4.147 in the CDDP
treated animals, were reduced to 34.80 ±3.382 and
30.80 ±1.743 (P\0.05) by WSDP and WEBP,
290 Cytotechnology (2014) 66:283–297
123
respectively (Table 3). In addition a significant
decrease in the number of aberrations per cell was
observed in G5 and G6 over the CDDP treatment group
(G2). The calculated suppressive effect was 30.40 and
38.40 %, by WSDP and WEBP, respectively
(Table 3).
Fig. 3 (a–g): Sections of testis in different examined groups
stained with haematoxylin and eosin. aA control seminiferous
tubules (ST) and interstitial cells (downward arrow) showing
the normal spermatogenic lineage; spermatogonia (SG). Note
the sertoli cells (SC) are seen between the spermatogonia. LU:
lumen (9400). bA photomicrograph of the testis section of
cisplatin injected mice showing the seminiferous tubule with
few number of spermatogenic lineage and numerous vacuolated
spermatogonia (downward arrow) LU: lumen (91,000). cA
lightmicrograph of the testis section of a cisplatin-injected mice
showing atrophied (A) seminiferous tubules or degenerated ones
(D) with severe vacuolation (V).The insertion: congested blood
vessel in the interstitial tissue (9400). dA photomicrograph of
the testis section of mice administered propolis showing a
normal seminiferous tubule invaded with macrophages (down-
ward arrow)(9400). eA light micrograph of a testis section of
mice given bee pollen illustrating normal interstitial tissue with
Leydig cells (L), seminiferous tubules with respect number of
sperms (downward arrow).Lu: lumen (9400). fA light
micrograph of the testis section of mice given cisplatin and
treated with bee pollen illustrating control seminiferous tubules
(ST) and interstitial tissues (IT) with Leydig cells (L) (9400).
gA photomicrograph of the testis section of mice injected with
cisplatin and treated with propolis showing normal seminiferous
tubules (ST) with normal lineage and interstitial tissue with
Leydig cells (L) and blood vessels (downward arrow)(9400)
Cytotechnology (2014) 66:283–297 291
123
Table 2 Protective effects of propolis and bee pollen aqueous extracts against cisplatin induced structural and numerical chromosomal aberrations in mouse bone marrow cells
Groups Number of structural chromosomal aberrations
a
Number of numerical chromosomal
a
aberrations
Chromatid
breakage
Chromatid
gap
Centromeric
attenuation
Centric
fusion
End to end
association
TSA Polyploidy Endomitosis TNA
G1
(saline)
18
(3.6 ±0.60)
–2
(0.40 ±0.24)
1
(0.20 ±0.20)
2
(0.40 ±0.24)
23
(4.60 ±0.68)
–8
(1.60 ±0.24)
8
(1.60 ±0.24)
G2
(CDDP)
101
b
(20.20 ±1.46)
14
b
(2.80 ±1.16)
–11
b
(2.20 ±1.07)
19
b
(3.80 ±0.86)
145
b
(29.00 ±3.42)
–23
b
(4.60 ±0.40)
23
b
(4.60 ±0.40)
G3
(WSDP)
45
b
(9.00 ±0.45)
––2
(0.40 ±0.24)
2
(0.40 ±0.24)
49
b
(9.80 ±0.37)
–––
G4
(WEBP)
32
b
(6.40 ±0.51)
–––2
(0.40 ±0.24)
34
b
(6.80 ±0.37)
–––
G5
(CDDP ?WSDP)
82
c
(16.40 ±2.04)
7
c
(1.40 ±0.40)
1
(0.20 ±0.20)
3
c
(0.60 ±0.24)
9
c
(1.80 ±0.37)
102
c
(20.40 ±2.04)
1
(0.20 ±0.20)
1
c
(0.20 ±0.20)
2
c
(0.40 ±0.24)
G6
(CDDP ?WEBP)
55
c
(11.00 ±0.71)
4
c
(0.80 ±0.20)
3
c
(0.60 ±0.40)
6
c
(1.20 ±0.49)
11
c
(2.20 ±0.20)
79
c
(15.80 ±0.49)
3
c
(0.60 ±0.40)
5
c
(1.00 ±0.45)
8
c
(1.60 ±0.68)
Number of examined metaphase cells in each group 250 cells
Chromatid Breakage: total number of chromatid breaks ?chromatid deletions
TSA total structural aberration, TNA total numerical aberration
a
Values between practices represent mean ±SE of five animals
b
Significantly different from untreated control (G1) P\0.05
c
Significantly different from positive control (G2) P\0.05
292 Cytotechnology (2014) 66:283–297
123
Discussion
The practice of disease prevention and/or inhibition,
delay, or reversal of the process of carcinogenesis is
the most cost effective mean for improving human
health. The target of much research has been the
discovery of natural and synthetic compounds that are
used in the prevention and/or treatment of cancer (Heo
et al. 2001).
Propolis and bee-collected pollen are apicultural
products which are composed of nutritionally valuable
substances and contain considerable amounts of
polyphenol substances which may act as potent
antioxidants. Development and utilization of more
effective antioxidants of natural origin are desired.
Naturally occurring polyphenols are expected to help
reducing the risk of various life-threatening diseases,
including cancer, due to their antioxidant activity
(Teixeira et al. 2008). In addition, phenolic com-
pounds are known to counteract oxidative stress in the
human body by helping maintaining a balance
between oxidant and antioxidant substance (Sid-
dhuraju 2006).
Flavonoids and phenolic acids are major classes of
polyphenolic compounds, whose structure–antioxi-
dant activity relationships in aqueous or lipophilic
systems have been extensively reported (Gardjeva
et al. 2007). In addition to anti-oxidant activity, many
phenolic compounds have been shown to exert anti-
carcinogenic or anti-mutagenic activity to a greater or
lesser extent (Awale et al. 2005). Mechanisms of
antioxidant action of these compounds may include
suppression of oxygen reactive species (ROS) forma-
tion, removal or inactivation of oxygen reactive
species and up-regulation or protection of antioxidant
defenses (Gardjeva et al. 2007). In this context, one of
the most important intracellular antioxidant systems is
the glutathione redox cycle. Glutathione is one of the
essential compounds for maintaining cell integrity
because of its reducing properties and participation in
the cell metabolism (Abdella and Ahmed 2008). On
another side, catalase, an enzyme involved in the
antioxidant defense of cells and tissues has the ability
to convert hydrogen peroxide to water (Ahmed and
Abdella 2009).
Earlier studies conducted with bee pollen and
propolis indicated that, the antioxidant activity of
honey bee-collected pollen has been recognized as free
radical scavenger and as an inhibitor of lipid peroxi-
dation. This activity has been associated with the
phenolic content of pollen (Campos et al. 2003). Bee
pollen is rich in carotenoids, flavonoids and phytoster-
ols. The exact profile varies depending on the plant
sources and growth conditions (Maruyama et al. 2010).
In addition, propolis possesses antioxidant activity, its
constituents includes caffeic acid, galangin, quercetin
and chrysin being able to scavenge free radicals (Pietta
2000;Kumazawaetal.2003;Ors
ˇolic
´and Bas
ˇic
´2005;
Gardjeva et al. 2007; Capucho et al. 2012).
The present study revealed the chemoprotective
potential of bee pollen or water extracts of propolis with
respect to chromosomal and histological damages
Table 3 Protective effects of propolis and bee pollen aqueous extracts against cisplatin induced cytotoxicity and genotoxicity in
mouse bone marrow cells
Groups Mitotic index
a
Number of cells
a
with one
aberration
Number of cells
a
with
more than one
aberration
Incidence of
a
aberrant cells
(%)
Number of
a
aberrations/
cell
Suppression
(%)
G1 (saline) 83.29 ±1.05 6.20 ±0.80 – 12.40 ±1.60 0.124 ±0.02
G2 (CDDP) 37.75 ±8.60
b
17.80 ±0.97
b
6.40 ±1.36
b
50.00 ±4.15
b
0.672 ±0.02
b
G3 (WSDP) 76.58 ±1.39
b
7.60 ±0.40
b
1.20 ±0.20
b
17.50 ±1.17
b
0.180 ±0.01
b
G4 (WEBP) 85.65 ±3.74 5.20 ±0.73
b
0.60 ±0.24
b
11.60 ±0.98 0.136 ±0.01
G5(CDDP ?WSDP) 73.28 ±1.12
c
13.60 ±1.36
c
3.80 ±0.37
c
34.80 ±3.38
c
0.416 ±0.04
c
30.40
G6(CDDP ?WEBP) 77.74 ±1.66
c
14.00 ±0.95
c
1.40 ±0.40
c
30.80 ±1.74
c
0.348 ±0.01
c
38.40
a
Values represent mean ±SE of five animals
b
Significantly different from untreated control (G1) P\0.05
c
Significantly different from positive control (G2) P\0.05
Suppression ¼100 %of aberrant cells in Gr5 or Gr6
%of aberrant cells in Gr2
hi
100
Cytotechnology (2014) 66:283–297 293
123
induced by cisplatin (CDDP) in bone marrow, liver,
kidney and testis tissues of mice associated with
antioxidant effect related to attenuated lipid peroxida-
tion and elevated antioxidant activity of catalase and
glutathione concentration.
CDDP is an inorganic platinum compound with a
broad spectrum anti-neoplastic activity against different
types of human tumors (Siddik 2003). CDDP has been
demonstrated to have the potential for initiating genetic
modifications in non-tumor cells in humans and animals,
Aly et al. (2003) reported that, the genotoxicity of CDDP
is due to its ability to bind with DNA, block and prolong
the cell division in the G2 phase of the cell cycle. The
blockage of cells in the G2 phase is related to the
inhibition of chromatin condensation. Nevertheless, both
clinical and experimental studies report a dose-limiting
nephrotoxicity which restricts the usefulness of cisplatin
in cancer chemotherapy (Nersesyan et al. 2003).
The results of the present investigation revealed
that, administration of CDDP at a single dose of
Fig. 4 (A–H): Metaphase spreads from mouse bone marrow cells showing Anormal cell, Bchromatid break (b), Cchromatid deletion
(d), Dcentric fusion (cf), Eend to end association (arrow), Fcentric attenuation, Gpolyploidy and Hendomitosis (1,000)
294 Cytotechnology (2014) 66:283–297
123
2.8 mg/kg b.wt, twice/week for 3 weeks induced
cytogenotoxic effects, histopathologic alterations and
oxidative stress. These results are consistent with
those previously reported (Nakajima et al. 2009;
Maruyama et al. 2010).
The current results indicated a significant decrease
in glutathione level and catalase activity in all tested
tissues of cisplatin-induced toxicity in mice. Sueishi
et al. (2002) registered lowered glutathione levels in
the kidney of rats injected with cisplatin. In addition,
glutathione peroxidase (GSHPx) and reduced gluta-
thione (GSH) levels were significantly reduced in the
liver and kidney of cisplatininjected animals (Naziro-
glu et al. 2004). Whereas, renal catalase showed a
significant decrease after cisplatin administration to
rats (Yildirim et al. 2003). Moreover, Amin and
Hamza (2006) mentioned that the testicular toxicity of
cisplatin is associated with a reduced glutathione
content, catalase and superoxide dismutase activity.
A possible explanation for the enhancement of
oxidative stress in cisplatin-injected mice may be the
decreased formation of antioxidants in the tissues of
cisplatin-injected animals which have the ability to
scavenge hydroperoxides and lipid peroxides. In
addition, the elevation of reactive oxygen species
(ROS) in the tissues and depletion of antioxidants have
been recognized as primary promoters of cellular
damage where inhibition of membrane transport
protein and increased lipid peroxidation are consid-
ered merely as a marker of cell damage (Naziroglu
et al. 2004).
In the present study numerous histopathological
alterations were noticed in cisplatin-injected mice.
These changes include necrosis, fibrosis and hydrobic
degenerative changes in liver, tubular and glomerular
degeneration with albuminous cast deposition in the
kidney in addition to a disorganization of seminiferous
tubular cells with germ cell loss especially spermatids
and sperms and congestion of blood vessels in the
interstitial tissue of testes.
Numerous reports have previously reported the
adverse effects of cisplatin on kidneys, liver and testis
histological structures. Shirwaikar et al. (2004)
reported glomerular congestion, tubular casts, epithe-
lial degeneration, interstitial oedema, blood vessel
congestion and infiltration by inflammatory cells, in
the kidneys of Wistar rats administered cisplatin at a
dose of 5 mg/kg b. wt. In addition, Yildirim et al.
(2003) and Ozen et al. (2004) observed a remarkable
proximal tubular necrosis with extensive epithelial
vacuolization, swelling and tubular dilatation in the
kidney of Wistar rats injected with 5 or 7 mg/kg b. wt
of cisplatin, respectively. On the other side, liver
sections from mice treated with cisplatin at a dose of
45 mg/kg b.wt, showed degeneration and vacuoliza-
tion but without necrosis (Lu and Cederbaum 2006).
Concerning testes, cross sections of mice exposed to
2.5 mg/kg b. wt. of cisplatin (G2), revealed extremely
severe damage to the seminiferous epithelium with
drastic reduction in tubular diameter, decreased cel-
lularity, absence of specific cell population and
absence of spermatids (Sawhney et al. 2005).
The results of the present investigation evidenced
that, administration of CDDP at a single dose of
2.8 mg/kg b.wt, twice/week for 3 weeks induced
cytogenotoxic effects revealed from the increase in
incidence of chromosomal aberrations and decrease in
mitotic indices. These results are consistent with those
reported by Nersesyan et al. (2003), Tohamy et al.
(2003), Nersesyan and Muradyan (2004), who dem-
onstrated that cisplatin caused the development of
chromosomal aberrations in bone marrow cells of
mice.
The antioxidant, the anti-histopathologic and the
anti-mutagenic actions of aqueous bee propolis extract
involve enhancement of the level of glutathione
S-transferase (GST), inhibition of cytochrome P-450
activity and interaction with microsome-generated
proximate mutagens to generate an inactive complex.
These effects were associated with inhibition of cell
cycle progression, accelerating the detoxification of
mutagens and carcinogens and induction of apoptosis
(El-khawaga et al. 2003). Lotfy (2006) indicated also
that, Egyptian propolis is characterized by the presence
of unusual esters of caffeic acid with mainly saturated
C12–C16 fatty alcohols. Flavonoid glycones and
especially flavanones are typical components of prop-
olis. These constituents of crude Egyptian propolis
have led to an increase in its pharmaceutical demand
and have rendered it an interesting subject of study.
In addition, Mello et al. (2010) reported that,
propolis has a variable and complex chemical com-
position with high concentration of flavonoids and
phenolic compounds present in the extract. The extract
varies with the solvent used in extraction. Ethanol
extracts contain higher levels of phenolic acid and
polar compounds than water extracts. The most
common propolis extraction process uses ethanol as
Cytotechnology (2014) 66:283–297 295
123
the solvent. However, this has some disadvantages
such as the strong residual flavor, adverse reactions
and intolerance to alcohol of some people (Konishi
et al. 2004). Researchers are interested in producing a
new type of extract with the same compounds
extracted by the ethanolic method, but without the
related disadvantages. Water has been tested as the
solvent, but resulted in a product containing reduced
level of extracted compounds (Park et al. 1998).
In conclusion both bee pollen and propolis aqueous
extracts appear to provide strong protective activities
against histological and genotoxic effects of cisplatin.
This activity seems to be dependent on the antioxidant
activities exerted by both extracts. However, the
aqueous extract of pollen gave more pronounced
results than propolis since its effects on the decrease of
lipid peroxidation marker was significantly higher
than those of propolis and its ability to increase the
antioxidant activity involved in the elimination of free
radicals was better than that of propolis resulting in
higher potential activity in ameliorating both geno-
toxic and histopathologic side effects of cisplatin
administration.
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