Amitraz, [1, 5-di-(2, 4-dimethylphenyl)-3-methyl-1, 3, 5-triaza-penta
1, 4-diene], is a member of the formamidine pesticides, used worldwide
as insecticide and acaricide (Hollingworth, 1976). It is a veterinary
medicinal product by beekeepers used to control the ectoparasitic mite
Varroa jacobsoni destructor, (formerly: V. jacobsoni), which
is a wide spread parasite that feeds on hemolymph of mature and immature
stages of honey bees and damages beehives seriously. The toxicity of amitraz
has not been investigated at a sufficient level, but when administered
orally or by skin washing, it is absorbed at a high rate. For this reason,
the toxicity risk was considered to be high (Grossman, 1993). Also, the
insecticide interacts with the α-2-adrenoceptor and produces behavioral,
physiological and biochemical effects. A mitraz inhibited brain monoamine
oxidase activity and motor function in rats (Moser and MacPhail, 1986)
and decreased glutathione content in mouse livers (Costa et al.,
1991). The reported effects of amitraz poisoning in humans include central
nervous system depression, bradycardia, hypotension, vomiting, hyperglycaemia,
glycosuria, polyuria and miosis (Jorens et al., 1997; Garnier et
Use of propolis by humans has a long history, predated only by the discovery
of honey. Propolis contains 50-70% resins and 10% essential oils, coming
from the trees, mixed with 30-50% wax for proper consistency and 5-10%
pollen, acquired from being transported in the bees`s pollen baskets (http://www.biolifeplus.com/library/propolis.html.2000).
The worker bees apply the resin to seal any cracks and fissures in the
hive and they line their front door with it to prevent contamination.
They use it as an antiseptic in breeder cells and they mix propolis with
wax to distribute a fine varnish over every inch of the hive to protect
it (Burdock, 1998). So far, 150 compounds have been identified from propolis
(Greenaway et al., 1991). The main chemical classes found in propolis
are flavanoids, phenolics and various aromatic compounds. However, propolis
contains many of the B-complex vitamins, important minerals and trace
elements. But its bioflavanoid content is now receiving attention. Bioflavanoids
are antioxidant molecules that play very important roles in the scavenging
of free radicals, which are produced in degenerative heart diseases, atherosclerosis,
aging and effects of toxic substances, e.g., ethyl alcohol (http://www.nutritionreporter.com/antioxidants.html;
At least 38 flavanoids have been found in propolis (Schmitdt and Buchmann,
2000). The chemical composition of propolis is highly variable because
of the broad range of plants visited by honey bees while collecting the
substance. Propolis is relatively non-toxic, with a no-effect level (NOEL)
in a 90 day`s mouse study of 1400 mg kg-1 body weight/day (Burdock,
1998). Propolis has been shown to stimulate various enzyme systems, cell
metabolism, circulation and collagen formation, as well as improve the
healing of burn wounds. These effects have been shown to be the result
of the presence of arginine in propolis. It was reported that propolis
stimulated an immune response in mice (Young, 1987). It activates immune
cells that produce cytokines. Bee propolis is one of the most promising
extracts as antitumor agent. Many researches proved its anti viral, anti-bacterial,
anti-inflammatory and immunostimulating activities (Wang et al.,
So, the present study is aimed to study the propolis protective effect
against the amitraz hepatotoxicity in mice.
MATERIALS AND METHODS
Crude propolis was obtained from honey bee, Apis mellifera carnica,
colonies situated at the apiary of Faculty of Agriculture at Fayoum, Egypt.
Samples were weighed, homogenized with a glass pestle and then soaked
in appropriate volume of 80% ethanol and left for about 3 days at room
temp away from light. The mixture was then filtered twice through Whatman
paper No. 1 with 80% ethanol. The solvent was air-dried and the extract
was weighed and suspended in 0.9% sterile saline at concentration of 1%
as a stock suspension.
Mitac (a.i: 20% Amitraz; Schering-Plough, USA) was used. Oral amitraz
LD50 for mice is 1600 mg kg-1.
Animals and Administration
Forty eight male Swiss albino mice of 8 weeks old and 22-25 g weight
were raised at the experimental animal house of the Faculty of Science,
Fayoum University in year 2007. The animals were maintained in controlled
environment of temperature, humidity and light. They were fed a commercial
mouse chow and tap water. The mice were divided into four groups (12 mice
each). The 1st was injected with 0.9% sterile saline (control), the 2nd
had 150 mg propolis/kg (body weight), the 3rd had 160 mg amitraz/kg by
gavage and the 4th group had 160 mg amitraz/kg + 150 mg propolis/kg. These
daily treatments lasted for 8 weeks.
Total and direct bilirubin concentrations were colorimetrically measured
(Shimadzu-CL 770 spectrophotometer), whereas alkaline phosphatase (ALP),
aspartate amino transferase (AST) and alanine amino transferase (ALT)
concentrations were measured using the enzyme-kinetic method (Mert, 1986).
These assays were weekly measured.
Eight weeks after the administration of amitraz, necropsies were performed
on the mice, resulting in their death immediately after euthanasia using
ether. Slices from the liver were fixed in buffered 10% formaldehyde solution.
Paraffin blocks were prepared after passing through ethyl alcohol and
xylol stages. Sections of 4-5 μm thickness were cut by a microtome
and stained with haematoxylin-eosin and examined under a light microscope
for histopathology investigation.
Tissue sections of 4 μm were mounted on Histogrip (Zymed, USA)
coated glass slides and air-dried overnight at room temperature. Immunohistochemical
staining was performed using an avidinbiotin peroxidase complex.
Briefly, samples were treated with 0.6% hydrogen peroxide in methanol
for 30 min to block endogenous peroxidase activity. Staining of formalin-fixed
tissues requires boiling tissue sections in 10 mM citrate buffer, pH 6.0,
(Neomarkers Cat. No. AP-9003) for 20 min which was followed by cooling
at room temperature for 20 min. The slides were incubated with normal
goat serum (1:10) (Neomarkers, USA) for 10 min and then with mouse monoclonal
Ki67 as the proliferation marker (Neomarkers, USA), at dilution of 0.5-1.0
μg mL-1 for 60 min at room temperature. The sections were
further incubated with biotinylated goat anti-rabbit IgG diluted to 1:500
(Sigma-Aldrich, USA) for 10 min, followed by incubation with peroxidase-conjugated
streptavidin diluted to 1:3000 in phosphate-buffered saline for 15 min.
The peroxidase reaction was performed using 0.02% 3, 3Â´-diaminobenzidine
tetrahydrochloride (DAB) and 0.01% hydrogen peroxide and counterstaining
was performed with hematoxylin for 1 min. In case of negative control,
the primary antibody was omitted. The positive stains are brown nuclear
stain and the counter stain is haematoxylin.
After identifying at low power (100X), each section was counted manually
at the high power (400X) in the representative areas with the highest
concentration of stained cells according to the recommendation of Cohen
et al. (1993). To count the labeling index of Ki67, about 1000
cells/slide were counted in each of five microscopic fields from well-labelled
areas to determine the average of Ki67. LI was expressed as a percentage
of labelled cells (positive for immunostaing reaction) to the total number
of cells counted in each specimen. All identifiable staining was regarded
as positive. The results are expressed as mean plus or minus standard
deviation (LI = mean ± SD %).
The statistical significance was computed using one way analysis of
variance (ANOVA) by SPSS 11 for windos®.
It was noticeable that after the first week of the experiment, the mice
injected with amitraz (160 mg kg-1) have generally exhibited
marked reduction in their feeding, general weakness and some of them displayed
loss of their balance. But those received the propolis or amitraz with
propolis displayed healthy and normal activities as in the control group.
Table 1 explains the changes in liver functions in
different groups, where the biochemical analysis in group treated with
propolis and amitraz were nearly similar to those of the control groups,
while the level concentrations of liver functions parameters in group
which was injected with amitraz only was high.
There were no histological observation differences between mice administrated
with propolis and control ones which received no propolis (Fig.
1a), so the term control is applicable for both. Examination of liver
of the control animals showed that lobules of the liver appeared as polygonal
|| Biochemical parameters measured in serum of treated
and untreated groups (mean ± SD)
|a = Significant (p<0.01)
|Light micrograph of control liver (a) lymphocytes infiltration
with abnormal liver cells arrangement were in amitraz group (b and
c) and the group of amitraz treated with propolis had liver tissue
displayed a normal architecture (d). Haematoxylin and Eosin stain
(original magnification X 400)
areas. The central vein runs through the center of the lobule. Hepatocytes
assemble to long strips of tissue (liver plates), which radiate from the
periphery towards the central vein of the lobule. Liver capillaries meander
between the liver sinusoids. The sinusoids were narrow blood spaces with
irregular boundaries composed essentially of only single layer of fenestrated
endothelial cells in addition to large irregularly shaped cells of mononuclear
type the Von kupffer cells which are known to be actively phagocytic
||Immunostaining micrograph of Ki67 expression in different
groups (the positivity is brown nuclear staining), Control (a) amitraz
liver treated group (b and c) and amitraz group treated with propolis
(d). Avidin-Biotin peroxidase method (original magnification X 250)
The histopathological changes in amitraz group were more apparently after
3rd week of treatment with amitraz, where the normal structural organization
of the hepatic lobules was impaired and the characteristic of cord-like
arrangement of the normal liver cells was lost, also, the hepatocytes
varied in size with shape and the interahepatic blood vessels were congested
with blood (Fig. 1b, c).
Histopathological sections of liver in mice injected with 160 mg kg-1
amitraz and treated with 150 mg kg-1 propolis showed somewhat
healthy appearance as the liver tissue displayed a normal architecture
(Fig. 1d). The hepatocytes were restored their morphological
feature, their cytoplasm was clearly homogenous. The hepatocytes nuclei
also mostly restored their normal appearance and binucleated cells feature,
which consider as an obvious indicator of recovery.
In the present study, the liver sections of control and propolis treated
mice immunostained with Ki67 showed very weak positive stained nuclei
indicating the mild cell division of some hepatocytes (Fig.
2a). However, sections in liver of mice injected with amitraz were
showed strong positive stained nuclei in most of the hepatocytes (Fig.
2b). On the other hand, the hepatocytes of mice treated with amitraz
and propolis were demonstrated less positive stained nuclei than those
of the amitraz treated only (Fig. 2c).
Table 2 explains the changes in liver Ki67 labeling
index. Mice injected with amitraz was displayed a highly significant increase.
While mice protected with propolis and injected with amitraz were illustrated
significant increase compared with control mice or mice injected with
|| Significance of Ki67 (a proliferation marker) between
= Mean value, SD = Standard deviation, NS = Not significant and *
Propolis is a resinous substance collected by honeybees and used in hive
construction and maintenance. Cumulative evidence suggests that propolis
may have anti-inflammatory, antibiotic, antioxidant, antihepatotoxic and
antitumor properties. In addition to topical applications, products containing
propolis have been used increasingly as dietary supplements (Li et
The rise in both AST and ALT levels (p<0.05) in mice given amitraz
was one of the most familiar indicators of hepatocellular damage (Mert,
1986). Also, Al-Qarawi et al. (1999) had also reported an increase
in serum AST levels in mice given amitraz. However, a significant decrease
in ALP level may refer to liver dysfunction. Besides, increasing bilirubin
levels indicated diffused harm to the liver.
It could be postulated that the hepatoprotective effect of propolis ethanol
extract (PEE) may be, partially, due to its ability to inhibit membrane
lipid peroxidation and free radical formation or due to their free radical
scavenging ability (Liu et al., 2004). A certain reduction of steatosis
degree as well as decreased concentration of liver triglycerides and ALT
activity was found in three groups of rats treated with red propolis extract
and CCl4 in relation to those treated with the hepatotoxin
(Merino et al., 1996). The present findings, indicated the levels
of AST, ALT, bilirubin, ALP and albumin in group injected daily with amitraz
(160 mg/kg/bw/day) and treated with propolis (150 mg/kg/bw/day) were nearly
similar to those the control groups.
The histopathological changes displayed by the liver of mice affected
by amitraz administration seemed to follow the pattern as the hepatic
tissue impairments began to appear in mice treated with amitraz in the
form of an inflammatory cell infiltration, swelling of sinusoids, activation
of kupffer cells, loss of normal hepatic tissue architecture and disappearance
of normal organization. Shukla et al. (2004) observed damage in
hepatocytes and disturbed chord arrangement after toxicant administration
and propolis extract (200 mg kg-1) was found to be more effective
in restoring CCl4 induced histopathological alterations. So,
the histological patterns in amitraz with propolis treatments were as
similar as the control groups where the liver showed somewhat healthy
appearance as the liver tissue displayed a normal architecture and the
hepatocytes were restored their morphological feature.
The present study may be the 1st attempt, on the combined
analysis the hepatoprotective effects of propolis against amitraz toxicity
by using proliferation marker (Ki67) with immunohistochemistry technique.
El-khawaga et al. (2003) reported that Crude Egyptian propolis
has a strong inhibitory activity against tumors. The anti-tumor mechanism
may be mediated by preventing oxidative damage and induction of apoptosis.
Choi et al. (1999) showed that propolis induced apoptosis in a
human hepatoma cell line, also, Jin et al. (2005) reported that
Caffeic acid phenyl ester in propolis (CAPE) possesses selective antiproliferative
activity toward hepatocaricoma cell line Hep3B. In this respect, the previous
reports explain the antiproliferative activity of propolis which was clear
in the present study, since, mice injected with amitraz was displayed
a very high proliferation, while those injected with amitraz and treated
with propolis exhibited low proliferation when compared with mice injected
with amitraz only.
The conclusion of the present study suggests that the honeybee propolis
ameliorated the recovery of amitraz hepatotoxicity in mice, where it acts
as an antioxidant scavenges free radicals and could restore the normal
liver functions and normal histology.