Protective Effects of Propolis Against the Amitraz Hepatotoxicity in Mice

Abstract

The present study aimed to study the protective effects of honeybee propolis against the amitraz hepatotoxicity in mice. Forty-eight Swiss albino male mice of 8 weeks of age, 22 to 25 g body weight were divided into four groups. The 1 st was control, the 2 nd was treated orally with 150 mg kg-1 propolis extract, the 3 rd was treated with 160 mg kg-1 amitraz and the 4 th one had 160 mg kg-1 amitraz + 150 mg kg-1 propolis extract. These daily treatments lasted for 8 weeks and laboratory assays were measured weekly. Results, after mice sacrificed, histopathology and immunohistology tests were carried out. The obtained results revealed that amitraz had affected liver biochemicals concentrations, whereas propolis led to a significant decrease in these levels in treated group. But, hepatocytes of mice treated with amitraz + propolis demonstrated positive stained nuclei, by using Ki67 immunostaining, less than those of amitraz treated only. The study suggests that propolis ameliorated the recovery of hepatotoxicity of amitraz in the tested mice.

Full text

Journal of Pharmacology and Toxicology 3 (5): 402-408, 2008
ISSN1816-496X
© 2008 Academic Journals Inc.
Protective Effects of Propolis Against the Amitraz Hepatotoxicity in Mice
1
Attalla F. El-Kott and
2
Ayman A. Owayss
1
Department of Zoology, Faculty of Science,
2
Depatment of Plant Protection, Faculty of Agriculture, Fayoum University, Egypt
Abstract: The present study aimed to study the protective effects of honeybee
propolis against the amitraz hepatotoxicity in mice. Forty-eight Swiss albino male mice of
8 weeks of age, 22 to 25 g body weight were divided into four groups. The 1
st
was control,
the 2
nd
was treated orally with 150 mg kg
-1
propolis extract, the 3
rd
was treated with 160 mg
kg
-1
amitraz and the 4
th
one had 160 mg kg
-1
amitraz + 150 mg kg
-1
propolis extract.
These daily treatments lasted for 8 weeks and laboratory assays were measured weekly.
Results, after mice sacrificed, histopathology and immunohistology tests were carried out.
The obtained results revealed that amitraz had affected liver biochemicals concentrations,
whereas propolis led to a significant decrease in these levels in treated group. But,
hepatocytes of mice treated with amitraz + propolis demonstrated positive stained nuclei,
by using Ki67 immunostaining, less than those of amitraz treated only. The study
suggests that propolis ameliorated the recovery of hepatotoxicity of amitraz in the tested
mice.
Key words: Amitraz, propolis, hepatoprotective, Ki67, proliferation, liver functions
INTRODUCTION
Amitraz, [1, 5-di-(2, 4-dimethylphenyl)-3-methyl-l, 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 used by beekeepers to control the ectoparasitic mite, Varroa
destructor (formerly: V. jacobsoni) which is a wide spread parasite 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. Amitraz 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 and Gamier et a!., 1998).
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,
_______________________________________________________________________________________________
Corresponding Author: Dr. Attalla F. El-Kott, Department of Zoology, Faculty of Science, Fayoum University,
Fayoum, Egypt Tel:+20502536868 Fax:+20846370025
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J. Pharmacol. Toxicol, 3 (5): 402-408, 2008
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 role 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;
http://www.nutritionreporter.com/soy-isoflavones.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 it improves 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., 2005).
So, the aim of the present study was to evaluate the prospective protection of bee propolis
against the amitraz hepatotoxicity in mice.
MATERIALS AND METHODS
Propolis Extraction
Crude propolis was obtained from honey bee, Apis mellifera carnica, colonies situated at the
apiary of Faculty of Agriculture atFayoum, 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.
Pesticide Preparation
Mitac (a.i: 20% Amitraz; Schering-Plough, USA) was used. Oral amitraz LD
50
for mice is
1600 mg kg
.
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
1
st
was injected with 0.9% sterile saline (control), the 2
nd
had 150 mg propolis/kg (body weight), the
3
rd
had 160mgamitraz/kg by gavage and the 4th group had 160 mg amitraz/kg + 150 mg propolis/kg.
These daily treatments lasted for 8 weeks.
Laboratory Assays
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.
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J. Pharmacol. Toxicol, 3 (5): 402-408, 2008
Histopathology
Eight weeks after the administration of amitraz, necropsies were performed on the nice, 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 um thickness was cut by a microtome and stained with haematoxylin-eosin and
examined under a light microscope for histopathology investigation.
Immunohistocheinistry
Tissue sections of 4 um were mounted on Histogrip (Zymed, USA) coated glass slides and air-
dried overnight at room temperature. Immunohistochemical staining was performed using an
avidin-biotin 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 ug 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, 3'-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-labeled areas to determine the average of
Ki67. LI was expressed as a percentage of labeled 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 %).
Statistical Analysis
The statistical significance was computed using one way analysis of variance (ANOVA) by SPSS
11 for Windows
®
.
RESULTS
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 (Bilirubin, AST, ALT and ALP) 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 was high
in the group which was injected with amitraz only.
There were no histological differences observed between mice administrated with propolis and
control ones which received no propolis (Fig. la), so the term control is suitable for both.
Examination of liver of the control animals showed that lobules of the liver appeared as polygonal
404

J. Pharmacol. Toxicoi. 3 (5): 402-408. 2008
Fig. 2: Immunostaining micrograph of Ki67 expression in different groups (the positively is
brown nuclear staining). Control (a) amitraz liver treated group (b and c) and amitraz +
propolis treated group (d). Avidin-Biotin peroxidase method (original magnification X
250)
The histopathological changes in amitraz group were more apparently after 3
rd
week of treatment
with amitraz, where the normal organized structure 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 restored their morphological feature, their
cytoplasm was clearly homogenous. Most hepatocytes nuclei restored their normal
appearance and binucleated cells feature, which is considered 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 only showed strong
positive stained nuclei in most of the hepatocytes (Fig. 2b). On the other hand, the hepatocytes of
mice treated with amitraz+propolis 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 had
displayed a highly significant increase, while mice treated with propolis and injected with amitraz
illustrated significant increase compared with control mice or those injected with amitraz only.
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J. Pharmacol. Toxicol, 3 (5): 402-408, 2008
Table 2: Significance of Ki67 (a proliferation marker) between different groups
Group
Control
Propolis
Amitraz
Popolis +Amitraz
X
SD
T-test
Significance
10.13
4.32
-
NS
9.985
3.330
0.713
*
80.560
7.982
18.320
*
18.180
5.451
6.340
X =Mean value, SD = Standard deviation, NS=Not significant and * Significant
DISCUSSION
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 al., 2005).
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 fiee radical formation or due to
their fiee 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 CC1
4
in relation to those treated with the hepatotoxin
(Merino et al., 1996). The present findings indicated that 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 nearlsimilar to those of 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 which appeared 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 CC1
4
induced
histopathological alterations. So, the histological patterns in amitraz + propolis treatments were as
similar as those of the control group where the liver showed somewhat healthy appearance as the
liver tissue displayed a normal architecture and hepatocytes restored their morphological feature.
The present study may be the 1
st
attempt, on the combined analysis of hepatoprotective effects
of propolis against amitraz toxicity by using proliferation marker (Ki67) with immunohistochemistry
techniqueEl-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 explained the antiproliferative activity of propolis which was clear in the present
study, since mice injected with amitraz displayed very high proliferation compared to those injected
with amitraz and treated with propolis which exhibited low proliferation.
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.
407