Beni-Suef University Journal of Basic and Applied Sciences 6 (2017) 160–173

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Beni-Suef University
Journal of Basic and Applied Sciences
journal homepage: www.elsevier.com/locate/bjbas

Cypermethrin-induced histopathological, ultrastructural and

biochemical changes in liver of albino rats: The protective role of
propolis and curcumin
Manal Abdul-Hamid a,⇑, Nadia Moustafa a, Abd El Mawgoud Abd Alla Asran b, Lila Mowafy b
a
Department of Zoology, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt
b
Department of Plant Protection, Harmful Animals, Research Institute, Agricultural Research Center, Cairo, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Cypermethrin (CYP), an insecticide belongs to a synthetic pyrethroid, is used for agriculture and house-
Received 26 November 2016 hold applications. The present study aimed to examine the toxic effects of CYP on rat liver and to clarify
Received in revised form 3 March 2017 the hepatoprotective effects of propolis (PRO) and curcumin (CUR) against CYP. This study was assessed
Accepted 3 March 2017
in male albino rats, each weighting (120–150 g). The rats were equally divided into six groups as follow;
Available online 8 March 2017
the 1st control group, 2nd PRO group (100 mg/kg/day) and 3rd CUR group (100 mg/kg/day). While 4th,
5th and 6th groups were orally treated with CYP (30 mg/kg/day), CYP plus PRO and CYP plus CUR, respec-
Keywords:
tively for 28 days. The present study revealed that CYP-induced significant increase in hepatic markers
Cypermethrin
Oxidative stress
enzymes (ALT, AST and ALP) and elevation in MDA concomitant with significant decrease of SOD and
Histopathology GPx levels. Histological and histochemical results revealed extensive vacuolar degeneration of hepato-
Ultrastructure cytes, fatty change, blood vessel congestion and fibrosis in the liver of CYP- treated group and depletion
Propolis and curcumin of glycogen, protein and DNA. Moreover, ultrastructural observations showed vacuolation, damage of
mitochondria and nuclear changes. On the other hand, treatment with PRO and CUR led to an obvious
improvement of the injured liver tissues and ameliorating the damaging effects of CYP. In conclusion,
PRO is markedly effective than CUR in protecting rats against CYP-induced histopathological, ultrastruc-
tural and biochemical changes. This protection may be due to its antioxidant properties and scavenging
abilities against active free radicals.
Production and hosting by Elsevier B.V. on behalf of Beni-Suef University. This is an open access article
under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Pyrethroids are human- made of pyrethrins. Pyrethroids have
two types which differentiate with their chemical structure and
exposure symptoms (Saka et al., 2011). CYP, type II synthetic pyr-
ethroid, have potent insecticidal property. It plays a role in agricul-
ture, protection of food stuff, disease vector control and home pest
control (Sankar et al., 2011). CYP is spreadly used as insecticide in
developing countries to control many species of insects. It is
reported that human exposure to CYP mainly occurred during
application or from pyrethroids residues such as those detected
in fruits, vegetables, cow’s milk and bread (Sankar et al., 2010).
CYP is a hydrophobic molecule which can easily pass through the
cell membrane and disturb its structure and cause leakage of cyto-
plasmic enzymes (Manna et al., 2004; Hussien et al., 2013). CYP
⇑ Corresponding author.
E-mail addresses: medo-bio@yahoo.com, manal.mohamed3@science.bsu.edu.eg
(M. Abdul-Hamid).
produced ROS and damaged DNA directly proportional to the dose
(Huang et al., 2016). There is a direct reaction between ROS and
cellular biomolecules. It damages lipids, proteins and DNA in cells
causing cell death (Ferrari, 2000). Sankar et al., 2011 realized that
imbalance between amount of free radicals generated and antiox-
idant defenses in the body are one of the reasons for CYP toxicity.
EL-Shemi et al. (2015) showed that the residue levels of CYP were
in kidneys, spleen, muscles and liver in the descending order at
levels ranging from 12.2 to 156.1 ng/g for CYP. The indicators of
overall health status of an individual especially hepatocyte toxicity
and related stress are serum enzyme levels (Khan et al., 2009).
Antioxidants are the molecules that react with ROS to delay
their function and to neutralize them, so they reduce oxidative
stress and protect us from diseases state. (Majeed et al., 2016). Pro-
polis (PRO), bee glue, is a sticky resinous substance applied by
honey bees Apis mellifera L. as a building material in their hives
and as a defensive substance against infections (Bankova et al.,
2016). PRO is considered as a remedy in traditional medicine sys-
tems all over the world, mainly to treat burns, wounds, stomach

http://dx.doi.org/10.1016/j.bjbas.2017.03.002
2314-8535/Production and hosting by Elsevier B.V. on behalf of Beni-Suef University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173 161

Fig. 1. Activities of serum (a): ALT (b): AST and (c): ALP (U/L) of CYP, PRO, CUR, CYP plus PRO and CYP plus CUR in serum of male rats.

ulcer and sore throat. The chemical composition of PRO includes
more than 200 compounds such as flavonoids, phenolics, vitamins,
alcohols, aromatic acids and their derivatives, esters, minerals and
trace elements (Bankova et al., 2000). PRO antioxidant activity is
mainly resulted from its flavonoid content, which has the ability
of scavenging free radicals and thereby preservation against LPO
(Yousef and Salama, 2009).
Curcumin (CUR), is a polyphenolic compound, derives from tur-
meric (Curcuma longa). It is considerrd as a source for food additive
or dietary pigment and in traditional medicine (Perrone et al.,
162 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173

Fig. 2. Concentration of hepatic malondialdehyde (MDA) of CYP, PRO, CUR, CYP plus PRO and CYP plus CUR in liver homogenate of male rats.

(a):

(b):

Fig. 3. Concentrations of (a): hepatic superoxide dismutase (SOD) and (b): Glutathione peroxidase (GPx) of CYP, PRO, CUR, CYP plus PRO and CYP plus CUR in liver
homogenate of male rats.
 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173 163

Fig. 4. a&b: Photomicrograph of a section in the liver of control rats showing radially arranged polyhedral hepatocytes forming cords around the central vein (CV) and
sinusoids (S) (H&E, Scale bar = 100, 50 mm respectivelly).

Fig. 5. a&b: Photomicrograph of a liver section of PRO and CUR groups showing normal structure of liver with a central vein (CV) (H&E, Scale bar = 100 mm).

2015; Pattanayak et al., 2016). Elsayed (2016) clarified CUR as an
antioxidant natural herb, a potent scavenger of many free radicals,
as anti-carcinogenic, anti-tumoral, anti-inflammatory, antifungal,
anti-viral, anti-parasitic, anti-mutagen, anti-infectious and anti-
hepatotoxic natural substance. Moran et al. (2016) pointed that
CUR is a powerful scavenger of a wide range of ROS and RNS
including O2
, H2O2 and NO and also has enhanced activities on
different antioxidant systems such as CAT, SOD, GPx, GSH reduced
glutathione and heme oxygenase-1.
The present investigation aims to evaluate the protective effect
of PRO and CUR against toxicity of CYP. Histopathological and
ultrastructural changes of liver, biochemical as oxidative stress will
be studied to evaluate the effects of treatment with PRO and CUR
in the various control and tested groups.
2. Materials and methods
2.1. Chemicals
Cypermethrin (CYP) was purchased from the pesticides store in
Beni-Suef, Egypt. Propolis (PRO) was purchased from Sigma Phar-
maceutical Industries (Cairo, Egypt). It is found in the form of Bio-
propolis capsules, each one containing 400 mg. Curcumin (CUR)
was obtained from Merk Company, Germany.
2.2. Animals and experimental design
42 adult of laboratory males albino rats (Rattus norvegicus) of
nearly the same age, weighing (120–150 g) bought from the Oph-
thalmology Research Institute, Giza, Egypt. Rats were kept under
standard management conditions of temperature (25 °C). The ani-
mals were fed a standard commercials diet (ATMID Company, Giza,
Egypt) and tap water ad libitum. All the rats were acclimatized for
at least 15 days before the beginning of the experiment. In this
study, animal care was carried out by following the European Com-
munity Directive (86/609/EEC) and national rules, this in accor-
dance with the 8th edition of the NIH Guidelines for the care and
use of Laboratory Animals. This was approved by the committee
of the Zoology Department, Beni-Seuf University, Egypt. Rats were
equally divided into six groups containing seven rats as follow:
Group 1 serving as an untreated control group under the same
laboratory conditions.
Group 2 treated with PRO at a dose of 100 mg/kg/day according
to Saleh (2012).
Group 3 treated with CUR at a dose of 100 mg/kg/day according
to Sankar et al. (2012)
Group 4 received CYP at a dose of 30 mg/kg/day according to
Inayat and Ilahi (2007).
164 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173

Fig. 6. Photomicrograph of a cross section in liver of rat treated with CYP showing (a): Inflammatory cells (iF), extensive vacuolar degeneration of hepatocytes (short arrows),
hyperemic sinusoid (arrow head) and congested blood vessel (long arrow) (H&E, Scale bar = 50 mm). b): Congested portal vein (CB) with thickened wall of portal vein (long
arrow) and proliferating bile duct (short arrows) (H&E, Scale bar = 50 mm). c&d): The congestion of central vein (CB) and fibrosis (F) (H&E, Scale bar = 200 mm). (e): Oedema (O)
and dissolution (d) (H&E, Scale bar = 50 mm). (f): Hepatocytes that undergo fatty change (F) and pyknotic nuclei (arrows) (H&E, Scale bar = 50 mm).

Group 5 given CYP (30 mg/kg/day) parallel with PRO at 100 mg/
kg/day.
Group 6 given CYP (30 mg/kg/day) parallel with CUR at 100 mg/
kg/day.
Each of CYP, PRO and CUR was dissolved in distilled water and
was given by gastric gavage daily for 28 days.
At the end of experiment, rats were starved for 12 h and then
sacrificed by anesthesia under light diethyl ether and blood sam-
ples were collected and allowed to coagulate at room temperature
then centrifuged at 3000 r.p.m. for 30 min. The clear non-
haemolyzed supernatant sera was quickly removed and kept at

20 °C for subsequent biochemical analysis.
Liver homogenate, obtained by grinding a small piece of freshly
excised tissue in 10 volumes of 0.9% NaCl (10% w/v) using Teflon
homogenizer (Glas-Col, Terre Haute, USA). The supernatants were
kept at -20°C till use in the determination of LPO, SOD and GPx.
Symptoms of CYP toxicity in laboratory animals include
burrowing, salivation, tremors, writhing and seizures. Also,
there were mortality; two rats died from eight rats in CYP-
treated group.
 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173
Fig. 7. a&b: (a): Photomicrograph of a liver section of rat treated with CYP plus PRO group showing great recovery and nearly normal structure of the liver with nearly normal
central vein (CV) and hepatocytes (H&E, Scale bar = 100 mm). (b): Photomicrograph of a section in the liver of CYP plus CUR group showing approximate regain of normal
appearance of hepatocytes and central vein (CV) (H&E, Scale bar = 100 mm).
2.3. The assay of serum biochemical
Alanine amino transferase (ALT) (EC 2.6.1.2) and aspartate
amino transferase (AST) (EC 2.6.1.1) activity was according to the
method of Schumann and Klauke (2003) using reagent kits pur-
chased from ‘‘Human Diagnostics” (Germany). The determination
of alkaline phosphatase (ALP) (EC 3.1.3.1) was spectrophotometri-
cally according to the method of Wenger et al. (1984) and Rosalki
et al. (1993) using reagent kits purchased from SPINREACT
Company.
2.4. Assessment of oxidative stress and antioxidant enzyme activity
The activity of lipid peroxidation (LPO) in liver homogenate was
determined according to the method of Yagi (1987).
The activity of superoxide dismutase (SOD) (EC 1.15.1.1) in liver
homogenate was assayed according to the method of Marklund
and Marklund (1974).
Glutathione peroxidase (GPx) (EC 1.11.1.9) activity in liver
homogenate was assayed according to the method suggested by
Matkovics and Szabo (1998).
2.5. Histological preparations
After 28 days of treatment, the animals were anesthetized
under light diethyl and dissected to get the liver. After fixation
in10% neutral buffered formalin for 24 h, they were dehydrated
through ascending series of ethyl alcohol, cleared in xylene and
embedded in paraffin wax. 5 mm thick sections were stained with
haematoxylin and eosin for histopathological studies (Bancroft
and Gamble, 2002).
2.6. Histochemical preparations
Liver sections of all the groups were stained with periodic acid
schiff (PAS) reaction (Hotchkiss, 1948), mercuric bromophenol
blue method (Mazia et al., 1953) and Feulgen method (Feulgen
and Rossenenbeck, 1924) for demonstration of polysaccharides,
total proteins and DNA contents respectively.
2.7. Ultrastructural preparations
Small pieces of liver of all groups were immediately fixed in 3%
glutraldehyde-formaldehyde at 4 °C for 18–24 h, rinsed in phos-
165
phate buffer, then followed by post fixation in 1% osmium tetrox-
ide. The specimens were then dehydrated in a series of alcohols,
cleared in propylene oxide and finally embedded in Epon epoxy
resin. After that, the blocks were trimmed and sectioned with glass
knives by an ultra-microtome. Semithin sections (1 mm) were
stained with toluidine blue and examined on light microscope to
select the suitable area for the ultrathin sections. Ultrathin sections
(70–90 nm) were cut on the same ultramicrotome and stained
with uranyl acetate and lead citrate (Bozzola and Russell, 1999).
Examination of the stained sections was carried out by Joel CX
100 transmission electron microscope operated at an accelerating
voltage of 60 kV.
2.8. Statistical analysis
The data have been expressed as mean ± SEM. Package for the
Social Sciences (SPSS for WINDOWS, version 20.0; SPSS Inc, Chi-
cago) (IBM crop, 2011) was used for the statistical analysis of data.
Data were analyzed by one way ANOVA test followed by Duncan’s
multiple range tests post hoc analysis values. A value of P < 0.05
was considered statistically significant.
3. Results
3.1. Effects of Cypermethrin, Propolis and Curcumin on serum
biomarkers
The present study showed that CYP adminstration displayed a
significant (P < 0.05) increase of serum ALT activity compared to
normal control, PRO and CUR values after 28 days. Treatment with
PRO or CUR in concomitant with CYP decrease ALT activity com-
pared to the CYP-treated group (Fig. 1a).
Also, CYP-treated rats significantly (P0.05) increased AST activ-
ity compared to the control, PRO, CUR groups values. Administra-
tion of PRO or CUR in concomitant with CYP decreased AST
activity (Fig 1b).
Moreover, there was no significant difference in ALP level
between control group value and PRO, CUR groups but there is a
significant (P < 0.05) increase in ALP activity in CYP group value
after 28 days. Treatment of PRO or CUR with CYP decrease ALP
activity compared to CYP-treated group (Fig. 1c).
Values were expressed as mean ± standard error.
166 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173

Fig. 8. a–c: Photomicrograph of a section in the liver of control, PRO and CUR rats showing highly increased amounts of glycogen in hepatocytes (arrows) (d): Liver of CYP
treated rat showing depletion of glycogen in hepatocytes (arrow), (e&f): Liver of CYP plus PRO and CYP plus CUR treated rat showing considerable amount of glycogen
(arrows) (PAS reaction, Scale bar = 50 mm).

Means which share the same superscript symbol(s) are not sig-
nificantly different.
No. of samples in each group is six.
(Propolis: (PRO), curcumin: (CUR), Cypermethrin: (CYP), Cyper-
methrin plus Propolis (CYP + PRO) and Cypermethrin plus Cur-
cumin (CYP + CUR).
Values were expressed as mean ± standard error.
Means which share the same superscript symbol(s) are not sig-
nificantly different.
No. of samples in each group is six.
Propolis: (PRO), curcumin: (CUR), Cypermethrin: (CYP), Cyper-
methrin plus Propolis (CYP + PRO) and Cypermethrin plus Cur-
cumin (CYP + CUR).

3.2. Effects of Cypermethrin, Propolis and Curcumin on lipid

peroxidation
Fig. 2 indicated that lipid peroxidation in the form of malonald-
hyde (MDA) significantly (P < 0.05) increased in CYP treated group
as compared with control, PRO, CUR, PRO + CYP and CUR + CYP
groups.
3.3. Effects of Cypermethrin, Propolis and Curcumin on enzymatic
antioxidant enzyme
CYP adminstration induced a significant (P < 0.05) decrease in
SOD levels and GPx when compared to the control, PRO, CUR
groups. However, administration of PRO or CUR to CYP-treated rats
 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173 167

Fig. 9. a–c: Photomicrograph of a section in the liver of control, PRO and CUR rats showing intensive amounts of protein in the cytoplasm of the hepatocytes (arrows) (d):
Liver of CYP treated rat showing reduced amount of protein (arrow), (e&f): Liver of CYP plus PRO and CYP plus CUR treated rat showing increased amount of protein (arrows)
(Bromophenol blue, Scale bar = 50 mm).

revealed marked amelioration in the altered level of SOD and GPx
activities when compared with CYP-treated group (Fig. 3a&b).
Values were expressed as mean ± standard error.
Means which share the same superscript symbol(s) are not sig-
nificantly different.
No. of samples in each group is six.
Propolis: (PRO), curcumin: (CUR), Cypermethrin: (CYP), Cyper-
methrin plus Propolis (CYP + PRO) and Cypermethrin plus Cur-
cumin (CYP + CUR).
The most biochemical parameters revealed that PRO is mark-
edly effective than CUR in ameliorating the biochemical
alterations.
3.4. Histopathological observations
The examination of light microscope of the control group
(Fig. 4a&b), PRO and CUR groups (Fig. 5a&b) showed normal struc-
ture of the liver, each lobule has a central vein and hepatic cords in
radiating shape. The administration of CYP showed an obvious
signs of hepatic alterations; these include congested blood vessels,
mononuclear inflammatory cell infiltration and extensive vacuolar
degeneration of hepatocytes (Fig. 6a). Congested portal vein with
thickened wall and proliferated bile duct were also detected
(Fig. 6b). Congested blood vessel and fibrotic tissues were also
noticed (Fig. 6c&d). Moreover, hyperemic sinusoids and oedema
168 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173

Fig. 10. a–c: Photomicrograph of a section in the liver of control, PRO and CUR rats showing intensive amount of DNA in the nuclei of the hepatocytes (arrows) (d): Liver of
CYP treated rat showing decreased DNA content (arrows) (e&f): Liver of CYP plus PRO and CYP plus CUR treated rat showing almost normal DNA content (Feulgen, Scale
bar = 20mm).

(Fig. 6e), vacuolar degeneration of hepatocytes and fatty changes
were seen (Fig. 6f). The liver sections of group treated with CYP
plus PRO or CUR revealed marked recovery and restoration of
almost normal hepatic configuration. (Fig. 7a&b).
3.5. Histochemical observations
A strong PAS-positive reaction was showed in the hepatocytes
of the control group, PRO and CUR- treated rats groups indicating
the presence of large amount of glycogen (Fig. 8a–c). On the other
hand, marked reduction of PAS reaction was observed in CYP trea-
ted group (Fig. 8d). However, after treatment with PRO and CUR,
the hepatocytes revealed an obvious increase of glycogen which
approximated that of the control liver (Fig. 8e&f).
Liver sections of control, PRO and CUR- treated groups stained
with bromophenol blue revealed a dense blue staining. The pro-
teins appeared in the form of bluish granules of various sizes ran-
domly distributed in the ground cytoplasm of the hepatic cells
(Fig. 9a–c). CYP-treated group showed marked depletion of pro-
teins intensity in the cytoplasm of hepatocytes (Fig. 9d). Intake of
PRO plus CYP and CUR plus CYP increased the intensity of blue
color of protein than that of CYP treated group (Fig. 9e&f)
respectively.
Using Feulgen reaction, the hepatocytes of control group, PRO
and CUR treated rats showed marked DNA amount in their nuclei
 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173 169

Fig. 11. a&b: Electron micrographs of hepatocytes of the control group showing normal nucleus (N), nucleolus (NU), mitochondria (M), rough endoplasmic reticulum (RER)
and glycogen granules (G). (c): Normal bile ducts (BD). (d): Normal Kupffer cell with nucleus (N) and lysosome (L). Scale bar = 2mm.

(Fig. 10a–c). In contrast, CYP-treated rats revealed severe decrease
of DNA amount in the nuclei of the liver cells. Few cells appeared
nucleated, while others showed vacuolated nuclei. (Fig. 10d). On
the other hand, administration of PRO to CYP and CUR to CYP
groups caused marked increase of DNA amount in the nuclei of
the hepatocytes which approximately reached that of the control
rats (Fig. 10e&f).
3.6. Ultrastructural observations
Ultrastructural examination of liver sections of control rats dis-
played normal structure. The cytoplasm of the hepatocytes con-
tains numerous mitochondria. They are round in shape with
well-developed cristae. The rough endoplasmic reticulum consists
of closely pack parallel and flattened cisternae. The nuclei appear
spherical with normal pattern of chromatin distribution. Numer-
ous electron-dense glycogen rosettes were clearly detected. The
nucleus of the hepatocytes appears spherical with a distinct
nuclear envelope and one or more prominent nucleoli. The nucle-
oplasm showed a unique distribution of a small amount of hete-
rochromatin at the peripheral regions and a central large amount
of euchromatin (Fig. 11a&b). Normal bile duct with normal struc-
ture of microvilli was seen (Fig. 11c). Kupffer cell also appeared
with normal nucleus, lysosomes and rough endoplasmic reticulum
(Fig. 11d). However, marked ultrastructural changes were revealed
in CYP-treated rats. In some hepatocytes, the mitochondria
appeared swollen, vacuolated with destructed cristae, in addition
to the presence of cytoplasmic vacuolations (Fig. 12a). Other cells
170 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173

Fig. 12. a: Electron micrographs of hepatocytes of rat treated with CYP showing vacuolated mitochondria (M), vacuoles (V). (b): dissolution of some hepatocytes (D) and
diltation of bile duct with destructed microvilli (BD). (c): Kupffer cell of CYP- treated rat showing elongated nucleus (N), cytoplasmic vacuolation (V), endocytic vesicles (EV)
and lysosomes (L). (d): large fat droplets (FD) and many cytoplasmic vacuoles (V). Scale bar = 2mm.

showed electron-dense mitochondria, dissolution of some hepato-
cytes, completely lysed nucleus and dilatation of bile duct with
destructed microvilli (Fig. 12b). Kupffer cell showed elongated
nucleus, endocytic vesicles and marked increase of lysosomes
number (Fig. 12c). In other hepatocytes, cytoplasmic vacuoles
and fat droplets were also observed (Fig. 12d). On the other hand,
the liver treated with PRO plus CYP revealed that the hepatocytes
restored their normal structure (Fig. 13a) and bile duct also was
regained to its normal structure (Fig. 13b). Kupffer cells displayed
their normal structure except the presence of some fat droplets
(Fig. 13c). The liver section treated with CUR plus CYP showed
marked recovery of the cytoplasmic organelles, and hepatic cells
except some vacuoles (Fig. 14a) and bile duct also restored its nor-
mal structure (Fig. 14b). Furthermore, Kupffer cells displayed their
normal structure but some vacuoles were still observed (Fig. 14c).
4. Discussion
The mode of action of CYP can be expected to have two ways: it
may generate ROS that induce oxidative stress or it may accumu-
late in cell membrane and disturb membrane structure due to its
hydrophobic nature (Saxena and Saxena, 2010). In the current
study, CYP administration increased ALT, AST, ALP and MDA. The
increasing of these parameters act as indicator to liver toxicity,
oxidative stress and LPO (Bhushan et al., 2013; Soliman et al.,
 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173
Fig. 13. a–c: Electron micrographs of rat treated with CYP and PRO: (a): showing marked recovery of the hepatocyte nucleus (N) and cytoplasmic organelles; mitochondria
(M), rough endoplasmic reticulum (RER). (b): showing bile duct (arrow). (c): Kupffer cell with normal structure and normal nucleus (N) and fat droplet (FD). Scale bar = 2mm.
2014). Oxidative stress contributes to initiation and progression of
liver injury, so it is considered a main pathological mechanism (Li
et al., 2015). Also, CYP has been demonstrated to cause a significant
decrease in the activities of SOD and GPx in the rats of the present
study. In consistent with these results, Gomaa et al. (2011) found
that CYP induced a significant increase in the mean values of
AST, ALT, ALP, MDA levels and a significant decrease in the mean
values of antioxidant enzyme activities (CAT, SOD and GPx) in rats
liver.
The present histopathological observations of liver sections of
rats in the current study showed congested blood vessels, mononu-
clear inflammatory cell infiltration, congested portal vein with
thickened wall and proliferated bile duct was also detected. More-
over, hyperemic sinusoids, vacuolar degeneration of hepatocytes
and oedema. In addition, many hepatocytes underwent fatty
change and fibrosis. These results were lined with Soliman et al.
(2014) and Mossa et al. (2015) who showed liver damage during
CYP toxicity in rats. Also, Grewal et al. (2010) observed marked
degenerative changes of hepatocytes and congestion after adminis-
tration of CYP at 14.5 mg/kg for 30 consecutive days. These
histopathological alterations in the hepatocytes because CYP has
inhibitory effect on total adenine triphosphate activity in the liver,
which may disorganize active transport of Na+, K+ and Ca2+ ions,
causing hepatocytes injuring (Khan et al., 2009).
Concerning the present ultrastructural study of hepatocytes,
nuclear changes, cytoplasmic vacuolation, damage mitochondria
and breakdown of RER were observed. In this respect, Aldana
et al. (1998) reported that after CYP exposure, hepatocytes had
171
an ovoid nucleus with slightly irregular profile, appearing of many
lipid droplets in cytoplasm and a lot of swelling mitochondria.
Antioxidants may play an important role to protect the cells
against oxidative stress and damage caused by free radicals
(Bouayed and Bohn, 2010). El-Masry et al. (2011) showed that
PRO has twelve different flavonoids, acacetin, pinocembrin, chry-
sin, rutin, catechin, naringenin, galangin, luteolin, a pigenin, kaem-
ferol, myricetin and quercetin, two phenolic acids, cinnamic and
caffeic acid are present in PRO. PRO is an excellent source of essen-
tial elements, including Mg2+, Zn2+, Fe2+, Cu2 +, Ca2+, Ni2+ and Mn2+
(Haro et al., 2002), which might also be responsible for reactivating
antioxidant enzymes by providing optimum trace elements. The
first mechanism of the effect of PRO may require the scavenging
of free radicals that induce LPO. The second mechanism may inhi-
bit xanthine oxidase, which is known to cause generation of free
radicals (Kanbura et al., 2009).
In the present study, PRO co-administration with CYP induced a
significant decrease in AST, ALT, ALP, MDA levels and a significant
increase in the mean values of antioxidant enzyme activities (CAT,
SOD and GPx) as compared with CYP treated group. This result is in
agreement with (Eraslan et al., 2008). PRO induced a reduction in
the increased activity of AST and ALT in the serum of rats treated
with doxorubicin (Omar et al., 2016). The present histopathological
examination revealed marked recovery and restoration of almost
normal hepatic cells by PRO therapy in contrast to CYP intoxicated
animals. The normal hepatocytes were observed in most areas
when propolis was co-administered with CYP, however, mild sinu-
soidal dilation with inflammatory cell infiltration was also present
172 M. Abdul-Hamid et al. / Beni-Suef Univ. J. Basic Appl. Sci. 6 (2017) 160–173
Fig. 14. a–c: Electron micrographs of rat treated with CYP and CUR showing (a): marked recovery of the hepatocyte nucleus (N) and cytoplasmic organelles; mitochondria
(M), rough endoplasmic reticulum (RER) and vacuoles (V). (b): bile duct (arrow). (c): Kupffer cell with normal structure and normal nucleus (N) and vacuoles (V). Scale
bar = 2mm.
(Gomaa et al., 2011). Electron microscopic examination of the pre-
sent study displayed a better preservation of the hepatic cells of rat
administrated PRO with CYP compared to those treated with CYP
alone.
CUR has protective effects against oxidative damage and has
antioxidant and anticonvulsant properties exerting powerful ROS
scavenging effects and increased intracellular glutathione concen-
tration, thereby protecting against LPO (Aboul Ezz et al., 2011; Du
et al., 2012). Co-administration of CUR with CYP revealed a
decrease in the concentrations of ALT, AST, ALP and MDA as com-
pared to CYP alone treated rats in this study. These findings are in
agreement with Sankar et al. (2012) and Hismiogullari et al.
(2014). In addition, Salahshoor et al. (2016) exhibit that the mean
values of ALT, AST, and ALP enzymes decreased significantly in CUR
and CUR plus nicotine in all groups compared to the nicotine
group. In the current study, the liver of CUR plus CYP group dis-
played a relatively normal architecture. Also, Sankar et al. (2012)
showed a reduction in the degenerative changes in liver of CUR
plus CYP group.
In conclusion, the present study clarified that administration of
PRO and CUR are beneficial to ameliorate the biochemical,
histopathological and ultrastructural alterations in liver of rats
induced by CYP due to their antioxidants effects. The protective
effects of PRO are markedly effective than CUR against CYP.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Funding
This research received no specific grant from any funding
agency in the public, commercial or not-for-profit sectors.
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