Bisphenol group (I): subdivide into: group Ia negative

Bisphenol -A (BPA) is a monomer used
in the manufacture of polycarbonate plastics. BPA is used in diverse forms of
plastic products in the food and electronic industries. BPA has been shown to
leach out of products, and high levels of the monomer have been identified in
human and animal samples. Many pharmacological effects were reported on ginger
and its pungent constituents, fresh and dried rhizome. Among the
pharmacological effects demonstrated are anti-platelet, antioxidant, anti-tumor,
anti-rhinoviral and anti-hepatotoxicity.

Aim of the work:

The current study was designed to investigate the effects of ginger
extract on biochemical histomorphological and immunohistochemical changes
induced in liver of albino rats by bisphenol A.

Materials and methods:

healthy adult male albino rats of initial body weight 150-200 gm were included.
Rats were randomly and equally divided into 4groups, group (I): subdivide into:
group Ia negative control group, group (Ib): olive oil treated group,  group (Ic):saline treated group, group (II):
ginger group, group (III): bisphenol A group, group (IV) bisphenol A and ginger
group. Treatment will continue for 8 weeks (three times a week). After the end of 8 weeks, the rats
from each group will be sacrificed for biochemical, histopathological and immuno
histochemical studies in liver.



was no significant difference between the negative, the positive control and ginger
groups as regard all biochemical, histopathological and immune histochemical

 Bisphenol- A significantly
decreased hepatic glutathione peroxidase (GPx) activities but significantly increased
serum aspartate transaminase (AST), alanine transaminase (ALT) and hepatic
malondialdehyde (MDA) levels together with deterioration of the hepatic histoarchitecture
after eight weeks. Moreover, it was found that exogenous administration of ginger
extract resulted in a significant improvement of all the above-mentioned


Ginger extract improved the hepatic toxicity induced by BPA that could
be explained by the anti-oxidant effects of ginger extract.

Key words: Bisphenol-A, ginger extract,
oxidative stress.



Bisphenol- A is widely used in plastic products such
as water dispensers, clear plastic bottles and food can linings.  Human exposure to BPA is extensive (Calafat
et al., 2005).

Kabuto et al. (2003), Vandenberg et
al. (2007) and
Lang et al. (2008) reported that some animal studies suggest that
exposure to BPA induced lipid peroxidation and tissue oxidative stress.

Bindhumol et al. (2003) found that bisphenol-A induce
oxidative stress in liver, kidney, testes and epididymal sperms of animals by
generating free radicals and altering the endogenous antioxidants.

Lopez-Torres et al. (2002) and Reddy
et al. (2008) reported
that oxidative stress and antioxidant capacity of the body modulated by
nutritional, environmental and physiological.

From times unknown, ginger or Zingiber officinale has
become a subject of interest because of its beneficial effects on human health.
Ginger has been found to possess antioxidant effect that can control the
generation of free radicals (Ahmad et al., 2006).


    The current study was designed

investigate the effects of ginger
extract on biochemical, histo- morphological and immune- histochemical changes
induced in liver of albino rats by bisphenol A.



Bisphenol -A

     Bisphenol- A (CAS No.
80-05-7; purity of 97%) and 4-Tert-octylpenol (CAS No. 140-66-9; purity of 97%)
were purchased from Sigma–Aldrich Company, Germany. Chemicals were dissolved in
olive oil.


     Ginger extracts were obtained
from Arab Company for Pharmaceuticals and Medicinal Plants (MEPACO, Cairo,
Egypt) in tablet form. Each tablet contained 400 mg of ginger extract. The
tablet was crushed and dissolved in 4 ml saline; hence, each ml contained                 100 mg ginger.


This study was conducted on 60 healthy adult male albino rats
weighing 150-200 gm, were obtained from the animal house of Faculty of Medicine-
Zagazig University. All animals were subjected to 14 days period of passive
preliminaries in order to be adapted the new environment, to ascertain their
physical wellbeing and to exclude any diseased animals. The rats received
balanced food rich in all stuffs necessary to maintain their health before and
during drug administration. It consisted of bread, barley and milk. Water was
offered in separate clean containers. All investigations were conducted in
accordance with the guiding principles for the care and use of research animals
and were approved by the Institutional Research Board.


Rats were randomly divided into four groups, group (I): control group
were subdivided into three group as follows: group (I a) (negative control
group), will be lifted without intervention to measure the basic parameters,
group (I b) (positive control group), each rat will be treated daily orally
with olive oil (vehicle of bisphenol) once daily via gastric
intubations, group (I c) (positive control group) rat will be treated daily
orally with saline (vehicle of ginger)   group (II): ginger group: ginger extract (200
mg/kg body weight) (Maralla, 2013). Group III (bisphenol-A group), each
rat will be treated orally   with bisphenol-A
(50 mg/kg/day) (vomSaal et al., 2007). LD50 in rats is 3250 mg/kg orally
(Chapin et al., 2008) and group IV: (bisphenol-A and ginger extract), each
rat will be treated orally with bisphenol-A (50 mg/kg/ day) and ginger extract (200
mg/kg body weight).

Treatment will be three times a week for 8 weeks. After the end
of the study, rats from each group will be sacrificed.

 Sample collection:

 Venous blood
samples were collected from the retro-orbital plexus of the animals by
capillary glass tubes using light ether anesthesia according to procedure
described by Nemzek et al. (2001).       3 ml of blood were collected from each
rat in clean centrifuge tube and incubated at 37°C until blood clotted and then
centrifuged to separate the serum that is used to measure Serum liver enzymes; aspartate transaminase,
alanine transaminase.

     After collecting blood samples, laparotomy
was conducted after the animals were sacrificed by cervical dislocation under
mild ether anesthesia.


1) Serum
Aspartate aminotransferase (AST) by Dimession-ES (clinical chemistry auto-analyzer):
According to Saris, (1978)

Serum Alanine aminotransferase (ALT) Dimension-ES (clinical chemistry
auto-analyzer): According to Bergmeyer et al., (1978).

3) Hepatic
antioxidant system evaluation:

     Tissues were perfused in 0.9
% NaCl containing 0.16 mg / ml heparin. Tissues was washed and minced in
ice-cold 0.25 M sucrose, then homogenized, diluted and centrifuged at 4000 rpm
and 4°C for two minutes. The supernatant was used to measure

Assay of
glutathione peroxidase (GPX) activity: according to the method
described by Paglia and
Valentine (1967).

o  Assay of MDA
level: according to the method described by Jain et al. (1989).

o  All are measured
by using spectrophotometer.


     Livers from all groups were removed and
fixed in 10% formalin solution and followed by dehydration in a descending
series of ethyl alcohol, were cleared in xylene and embedded in paraffin.
Paraffin sections of testes were cut at 5 ?m on a rotary microtome, mounted on
slides and stained with heamatoxylin eosin (H&E) (Horobin and
Bancroft, 1998) and
examined under a light microscope.


A rabbit monoclonal antibody of IgG type was carried out for localization
of caspase-3 (apoptosis marker) in paraffin sections. (The kits were delivered
from Lab Vision Laboratories; Cat. #:1475-1) according to Joyner and Wall,



      Data were analyzed by Statistical Package of Social Science
(SPSS), software version 22.0 (SPSS Inc., 2013).  DISCUSSION

Vandenberg et al. (2007) explained
that bisphenol- A is one of the highest volume chemicals produced
worldwide. This  compound  is  a
building block  of  polycarbonate 
plastics  and epoxy  resins 
that  are  used 
to  line  food and 
beverage  containers.

Calafat et al. (2005) reported
that this  compound  is 
also  found  in 
an  enormous  number 
of  other  products 
that we come  into  contact 
with  daily,  and 
therefore  it  has 
been  detected  in 
the  majority of individuals  examined .

Vandenberg et al. (2007) studied that
BPA can leach with food and drink.

Possible target organs of toxicity
identified in repeat-dose animal studies with oral dosing included liver,
kidney, and reproductive systems (Yamasaki et al., 2002 and European-Union,

Muthuvel et al. (2006) reported that
ginger extract is an important dietary antioxidant which significantly
decreases the adverse effects of reactive oxygen species implicated in chronic diseases.

Therefore, this study was designed to
explore the probable effects of ginger extract in modulating the effects of BPA
on hepatic tissue.

The schedule of the present study
included four groups: group Ia (negative control group):  will be lifted without intervention to measure
the basic parameters, group Ib (positive control group): each rat will
be treated daily orally with olive oil (vehicle of bisphenol) once daily via
gastric intubations, group Ic (positive control group): each rat will be
treated daily orally with saline (vehicle of ginger) once daily via gastric
intubations group II (ginger extract group): each rat will be treated
orally with ginger extract (200 mg/kg/day) dissolved in saline, group III (bisphenol-A
group): Each rat will be treated orally  
with bisphenol-A (50 mg/kg/day) orally and group IV (bisphenol-A
and ginger extract): each rat will be treated orally with bisphenol-A (50
mg/kg/ day) and ginger extract (200 mg /kg/ day). Treatment will continue for 8
weeks (three times a week). After the end of the eight weeks, ten rats from
each group will be sacrificed for biochemical, histopathologial and immune
histochemisy studies.

1-Control Groups (negative and positive control groups)
and group II (ginger extract group):

Rats of these groups showed no
abnormal findings as regards biochemical studies. There was no significant
difference between these groups as regard all these parameters.

Also, there were no abnormal
histopathological changes in the liver specimens of the adult male albino rats
of these groups all over the periods of the study.

2-Treated groups:

Bisphenol-A treatment had induced a
significant increase in the mean values of serum AST and ALT when compared with
the control groups.

While bisphenol-A + ginger extract
treatment group had induced a significant decrease in the mean values of serum
AST and ALT when compared with bisphenol-A groups.

The disturbance of liver AST & ALT of the rats treated with bisphenol-A could be explained by Jaeschke et
al. (2002) who  stated that
leakage of the enzymes were produced within hepatocytes and small amounts
constantly leak through the cell membrane which gave the normal serum enzymes
level of these enzymes.  Liver damage caused by liver cell injury
(hepatocellular toxicity) made the membranes more permeable. So, greater
amounts of enzymes leaked out with subsequent elevation of serum enzymes above
normal level.

A significant increase in ALT and AST
activities in rats treated with BPA for six and ten weeks (Mourad and
khadrawy, 2012). 

On the other hand, the present study resulted in
significant decrease in GPx and significant increase in MDA in hepatic tissue of
bisphenol-A group when compared with control groups.

While there were also significant increase
in hepatic GPx and significant decrease in hepatic MDA in bisphenol-A + ginger
extract groups when compared with those in bisphenol-A group.

Bindhumol et al.
(2003) stated that the activities of antioxidant enzymes, superoxide dismutase,
catalase and glutathione peroxidase decreased while the levels of hydrogen
peroxide and lipid peroxidation increased significantly in mitochondrial and
microsome-rich fractions of liver when compared with the corresponding group of
control animals.

Pigeolet et al. (1990) reported that
the reduction in activities of antioxidant enzymes showed the failure of
primary antioxidant system to act against free radicals. Antioxidants which are
located throughout the cell can provide protection against ROS toxicity. ROS
play an important role in the defence mechanisms against pathological
conditions but excessive generation of free oxygen radicals may damage tissues .

An enhanced ROS generation by
polymorphonuclear neutrophils (PMNs) at the site of inflammation causes
endothelial dysfunction and tissue injury. The vascular endothelium plays an
important role in passage of macromolecules and inflammatory cells from the
blood to tissue. Under the inflammatory conditions, oxidative stress produced
by PMNs leads to the opening of inter-endothelial junctions and promotes the
migration of inflammatory cells across the endothelial barrier. The migrated
inflammatory cells not only help in the clearance of pathogens and foreign
particles but also lead to tissue injury (Mittal et al., 2014).

Mitochondria are a major source of intracellular
reactive oxygen species (ROS) and are particularly vulnerable to oxidative
stress. Oxidative damage to mitochondria has been shown to impair mitochondrial
function and lead to cell death via apoptosis and necrosis. Because
dysfunctional mitochondria will produce more ROS, a feed-forward loop is set up
whereby ROS-mediated oxidative damage to mitochondria favors more ROS
generation, resulting in a vicious cycle (Szeto, 2006).

Bai and Odin (2003) explained
that Oxidative stress can lead to damage of the mitochondrial inner membrane,
resulting in mitochondrial permeability transition pore (MPTP) formation and
subsequent release of cytochrome c and apoptosis inducing factor from the
mitochondria. In the cytosol, cytochrome C complexes with apoptotic protease
activating factor (Apaf-1) to activate procaspase 9, which in turn activates
downstream effector caspases (3, 6 and 7) .

Nakagawa and Tayama (2000) reported that
bisphenol-A has been shown to reduce mitochondrial function in hepatocytes.

Moon et al. (2012) reported that
a low dose of BPA induces mitochondrial dysfunction in the liver, and this is
associated with an increase in oxidative stress and inflammation and this go
parallel with our study.


Measurement of MDA levels in the
tissue is a marker of lipid peroxidation which is among the chief mechanism of
cell damage. Bisphenol-A is lipophilic in nature due to which it can easily
penetrate/interact with the lipid membrane of the hepatocytes (Doerge and
Fisher, 2010).

The results of the present study were
in agreement with the study of Suthar and Verma (2014) which
concluded that treatment of BPA for 30 days cause increase in lipid
peroxidation as well as alterations in the anti-oxidative system ultimately
causing oxidative stress in experimental animals.

A study of Sangai et al. (2014) also
revealed decrease of oxidative damage in liver and kidney of mice after
exposure to BPA by potent antioxidants such as quercetin. They also showed that
exposure to BPA caused significant reduction in the activities of catalase,
superoxide dismutase, glutathione peroxidase, glutathione reductase and
glutathione-S-transferase as well as in the levels of glutathione and total
ascorbic acid contents; however, significant increase was found in the levels
of malondialdehyde.

Therefore, we hypothesized that ginger
extract could act as an antioxidant against BPA.

Results of light microscopic
examination and immunohistochemical staining of stained liver sections obtained
in the present study have supported the above mentioned biochemical results.

     Light-microscope examination
of H&E stained liver sections of BPA treated group after 8 weeks showed congested portal vein, bile duct
proliferation and cellular infilteration, which was minimally observed in the
group taken ginger extract with bisphenol.

Venous congestion was a permanent feature in the treated
liver sections in this study which accordingly might interfere with the hepatic
arterial blood supply. This might lead to the development of ischemia and
subsequent necrosis as mentioned by (Majno and Joris, 1995).

Our results were matched with Helal
et al. (2013) who noticed that administration of BPA for 30 days revealed
The nuclei of hepatocytes are mostly large with prominent one or more nuclei
and with Hussein and Eid (2013) who revealed that oral
administration of BPA for 21 days gave light abnormal pathological change
compared with the control, dilatation and congestion of the central vein,
portal vein and hepatic sinusoids.

Also, it was matched with results of Korkmaz et al. (2010)
in which treatment of rats with bisphenol for 8 weeks resulted in
congestion and necrotic areas. These observations may be explained by BPA and
induced peroxidation of membrane lipids in the liver cells.

Hussein and Eid
(2013) noticed that
bisphenol-A caused cell infiltration was observed in focal manner surrounding
the dilated bile duct.

     Immune-histochemical reaction
to caspase 3 revealed positive reaction within cytoplasm of most hepatocytes
in BPA group after 8 weeks which became less in group taken BPA + ginger
extract for 8 weeks. 

Caspases, intracellular cysteine
proteases that cleave various substrates including structural proteins such as
caspase–3, are the key mediators of apoptosis. Caspase-3, as a main final common executor of
apoptosis, is responsible for the cleavage of the key cellular proteins,
leading to typical morphological changes observed in cells undergoing apoptosis
(Budihardjo et al., 1999; Saikumar et al., 1999 and Fischer et al., 2003).

Asahi et al. (2010) found that
BPA induced endoplasmic reticulum (ER) stress-associated apoptosis in
hepatocytes. The ER stress was due to ROS production and was independent of
estrogen receptors.

Iida et al. (2003) had shown
that apoptosis induction by BPA was associated with caspase activation.

The overproduction of ROS may be an inducible factor of
apoptosis. Previous studies reported that BPA exposure produced ROS by
inhibiting antioxidant enzymes (Chitra et al. 2003 and Kabuto et al. 2003).

Our results were matched with Ahmad et al. (2006) in
which they found that ginger extract may have antioxidant effect by replacing
SOD activities and reducing the level of superoxide radicals in liver cancer
induced rats. This is similar to the findings of Park et al. (1998), in
which the bioactive component in ginger reduced the production of ROS such as
superoxide anions.

Chang et al. (1994) found the bioactive component of ginger, namely gingerol,
possessed antioxidative effect by inhibiting peroxidation of phospholipids
induced by xanthine oxidase activity.

Ahmad et al. (2006) concluded that ginger extract may have bioactive components
with antioxidant activity in scavenging free radicals such as superoxide anions
and H2O2 as well as decreasing the MDA level for the reduction of lipid


From this study we concluded that bisphenol-A induced
oxidative stress and apoptosis in hepatic tissue which decreased with
administration of ginger extract which acts as an antioxidant agent.


1-More attention should be paied to  health education about  sources of exposure to Bisphenol-A in the environment and we should  try to minimize them.2-The imposition of strict laws to prevent the use of bisphenol-A in many products.  3-The use of plastic products free of bisphenol-A or replace the container glass or other materials free of bisphenol-A to save the foods and beverages.  4- further studies should be done on the toxic effects of Bisphenol-A and the use of antioxidants in the prevention of toxic effects.