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

Bisphenol -A (BPA) is a monomer usedin the manufacture of polycarbonate plastics. BPA is used in diverse forms ofplastic products in the food and electronic industries.

BPA has been shown toleach out of products, and high levels of the monomer have been identified inhuman and animal samples. Many pharmacological effects were reported on gingerand its pungent constituents, fresh and dried rhizome. Among thepharmacological effects demonstrated are anti-platelet, antioxidant, anti-tumor,anti-rhinoviral and anti-hepatotoxicity.

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Aim of the work:   The current study was designed to investigate the effects of gingerextract on biochemical histomorphological and immunohistochemical changesinduced in liver of albino rats by bisphenol A. Materials and methods: Sixityhealthy 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 gingergroup. Treatment will continue for 8 weeks (three times a week).

After the end of 8 weeks, the ratsfrom each group will be sacrificed for biochemical, histopathological and immunohistochemical studies in liver. Results:     Therewas no significant difference between the negative, the positive control and gingergroups as regard all biochemical, histopathological and immune histochemicalparameters.   Bisphenol- A significantlydecreased hepatic glutathione peroxidase (GPx) activities but significantly increasedserum aspartate transaminase (AST), alanine transaminase (ALT) and hepaticmalondialdehyde (MDA) levels together with deterioration of the hepatic histoarchitectureafter eight weeks.

Moreover, it was found that exogenous administration of gingerextract resulted in a significant improvement of all the above-mentionedparameters.Conclusion:     Ginger extract improved the hepatic toxicity induced by BPA that couldbe explained by the anti-oxidant effects of ginger extract.Key words: Bisphenol-A, ginger extract,oxidative stress.  INTRODUCTIONBisphenol- A is widely used in plastic products suchas water dispensers, clear plastic bottles and food can linings.  Human exposure to BPA is extensive (Calafatet al., 2005).

Kabuto et al. (2003), Vandenberg etal. (2007) andLang et al.

(2008) reported that some animal studies suggest thatexposure to BPA induced lipid peroxidation and tissue oxidative stress. Bindhumol et al. (2003) found that bisphenol-A induceoxidative stress in liver, kidney, testes and epididymal sperms of animals bygenerating free radicals and altering the endogenous antioxidants.

Lopez-Torres et al. (2002) and Reddyet al. (2008) reportedthat oxidative stress and antioxidant capacity of the body modulated bynutritional, environmental and physiological.From times unknown, ginger or Zingiber officinale hasbecome a subject of interest because of its beneficial effects on human health.Ginger has been found to possess antioxidant effect that can control thegeneration of free radicals (Ahmad et al., 2006).

AIM OF THE WORK    The current study was designedtoinvestigate the effects of gingerextract on biochemical, histo- morphological and immune- histochemical changesinduced in liver of albino rats by bisphenol A. MATERIALAND METHODSMATERIALBisphenol -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 inolive oil.

Ginger      Ginger extracts were obtainedfrom Arab Company for Pharmaceuticals and Medicinal Plants (MEPACO, Cairo,Egypt) in tablet form. Each tablet contained 400 mg of ginger extract. Thetablet was crushed and dissolved in 4 ml saline; hence, each ml contained                 100 mg ginger.AnimalsThis study was conducted on 60 healthy adult male albino ratsweighing 150-200 gm, were obtained from the animal house of Faculty of Medicine-Zagazig University. All animals were subjected to 14 days period of passivepreliminaries in order to be adapted the new environment, to ascertain theirphysical wellbeing and to exclude any diseased animals.

The rats receivedbalanced food rich in all stuffs necessary to maintain their health before andduring drug administration. It consisted of bread, barley and milk. Water wasoffered in separate clean containers. All investigations were conducted inaccordance with the guiding principles for the care and use of research animalsand were approved by the Institutional Research Board. Treatmentprotocol    Rats were randomly divided into four groups, group (I): control groupwere subdivided into three group as follows: group (I a) (negative controlgroup), will be lifted without intervention to measure the basic parameters,group (I b) (positive control group), each rat will be treated daily orallywith olive oil (vehicle of bisphenol) once daily via gastricintubations, group (I c) (positive control group) rat will be treated dailyorally with saline (vehicle of ginger)   group (II): ginger group: ginger extract (200mg/kg body weight) (Maralla, 2013).

Group III (bisphenol-A group), eachrat 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), eachrat will be treated orally with bisphenol-A (50 mg/kg/ day) and ginger extract (200mg/kg body weight).    Treatment will be three times a week for 8 weeks. After the endof the study, rats from each group will be sacrificed. Sample collection:     Venous bloodsamples were collected from the retro-orbital plexus of the animals bycapillary glass tubes using light ether anesthesia according to proceduredescribed by Nemzek et al. (2001).

      3 ml of blood were collected from eachrat in clean centrifuge tube and incubated at 37°C until blood clotted and thencentrifuged to separate the serum that is used to measure Serum liver enzymes; aspartate transaminase,alanine transaminase.      After collecting blood samples, laparotomywas conducted after the animals were sacrificed by cervical dislocation undermild ether anesthesia. BiochemicalAnalysis1) SerumAspartate aminotransferase (AST) by Dimession-ES (clinical chemistry auto-analyzer):According to Saris, (1978)2)Serum Alanine aminotransferase (ALT) Dimension-ES (clinical chemistryauto-analyzer): According to Bergmeyer et al., (1978).

3) Hepaticantioxidant system evaluation:     Tissues were perfused in 0.9% NaCl containing 0.16 mg / ml heparin. Tissues was washed and minced inice-cold 0.25 M sucrose, then homogenized, diluted and centrifuged at 4000 rpmand 4°C for two minutes. The supernatant was used to measureo     Assay ofglutathione peroxidase (GPX) activity: according to the methoddescribed by Paglia andValentine (1967).

o  Assay of MDAlevel: according to the method described by Jain et al. (1989).o  All are measuredby using spectrophotometer. Histopathologicalexamination:     Livers from all groups were removed andfixed in 10% formalin solution and followed by dehydration in a descendingseries 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 onslides and stained with heamatoxylin eosin (H&E) (Horobin andBancroft, 1998) andexamined under a light microscope.Immunohistochemicalexamination:A rabbit monoclonal antibody of IgG type was carried out for localizationof caspase-3 (apoptosis marker) in paraffin sections. (The kits were deliveredfrom Lab Vision Laboratories; Cat. #:1475-1) according to Joyner and Wall,(2008)  StatisticalAnalysis:      Data were analyzed by Statistical Package of Social Science(SPSS), software version 22.

0 (SPSS Inc., 2013).  DISCUSSIONVandenberg et al. (2007) explainedthat bisphenol- A is one of the highest volume chemicals producedworldwide.

This  compound  is  abuilding block  of  polycarbonate plastics  and epoxy  resins that  are  used to  line  food and beverage  containers.Calafat et al. (2005) reportedthat 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 thatBPA can leach with food and drink.Possible target organs of toxicityidentified in repeat-dose animal studies with oral dosing included liver,kidney, and reproductive systems (Yamasaki et al., 2002 and European-Union,2003).Muthuvel et al.

(2006) reported thatginger extract is an important dietary antioxidant which significantlydecreases the adverse effects of reactive oxygen species implicated in chronic diseases.Therefore, this study was designed toexplore the probable effects of ginger extract in modulating the effects of BPAon hepatic tissue.The schedule of the present studyincluded four groups: group Ia (negative control group):  will be lifted without intervention to measurethe basic parameters, group Ib (positive control group): each rat willbe treated daily orally with olive oil (vehicle of bisphenol) once daily viagastric intubations, group Ic (positive control group): each rat will betreated daily orally with saline (vehicle of ginger) once daily via gastricintubations group II (ginger extract group): each rat will be treatedorally with ginger extract (200 mg/kg/day) dissolved in saline, group III (bisphenol-Agroup): Each rat will be treated orally  with bisphenol-A (50 mg/kg/day) orally and group IV (bisphenol-Aand ginger extract): each rat will be treated orally with bisphenol-A (50mg/kg/ day) and ginger extract (200 mg /kg/ day). Treatment will continue for 8weeks (three times a week). After the end of the eight weeks, ten rats fromeach group will be sacrificed for biochemical, histopathologial and immunehistochemisy studies.1-Control Groups (negative and positive control groups)and group II (ginger extract group):Rats of these groups showed noabnormal findings as regards biochemical studies. There was no significantdifference between these groups as regard all these parameters.

Also, there were no abnormalhistopathological changes in the liver specimens of the adult male albino ratsof these groups all over the periods of the study.2-Treated groups:Bisphenol-A treatment had induced asignificant increase in the mean values of serum AST and ALT when compared withthe control groups.While bisphenol-A + ginger extracttreatment group had induced a significant decrease in the mean values of serumAST 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 etal. (2002) who  stated thatleakage of the enzymes were produced within hepatocytes and small amountsconstantly leak through the cell membrane which gave the normal serum enzymeslevel of these enzymes.  Liver damage caused by liver cell injury(hepatocellular toxicity) made the membranes more permeable. So, greateramounts of enzymes leaked out with subsequent elevation of serum enzymes abovenormal level.

A significant increase in ALT and ASTactivities in rats treated with BPA for six and ten weeks (Mourad andkhadrawy, 2012).  On the other hand, the present study resulted insignificant decrease in GPx and significant increase in MDA in hepatic tissue ofbisphenol-A group when compared with control groups.While there were also significant increasein hepatic GPx and significant decrease in hepatic MDA in bisphenol-A + gingerextract 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 hydrogenperoxide and lipid peroxidation increased significantly in mitochondrial andmicrosome-rich fractions of liver when compared with the corresponding group ofcontrol animals.Pigeolet et al.

(1990) reported thatthe reduction in activities of antioxidant enzymes showed the failure ofprimary antioxidant system to act against free radicals. Antioxidants which arelocated throughout the cell can provide protection against ROS toxicity. ROSplay an important role in the defence mechanisms against pathologicalconditions but excessive generation of free oxygen radicals may damage tissues .An enhanced ROS generation bypolymorphonuclear neutrophils (PMNs) at the site of inflammation causesendothelial dysfunction and tissue injury. The vascular endothelium plays animportant role in passage of macromolecules and inflammatory cells from theblood to tissue.

Under the inflammatory conditions, oxidative stress producedby PMNs leads to the opening of inter-endothelial junctions and promotes themigration of inflammatory cells across the endothelial barrier. The migratedinflammatory cells not only help in the clearance of pathogens and foreignparticles but also lead to tissue injury (Mittal et al., 2014).Mitochondria are a major source of intracellularreactive oxygen species (ROS) and are particularly vulnerable to oxidativestress. Oxidative damage to mitochondria has been shown to impair mitochondrialfunction and lead to cell death via apoptosis and necrosis. Becausedysfunctional mitochondria will produce more ROS, a feed-forward loop is set upwhereby ROS-mediated oxidative damage to mitochondria favors more ROSgeneration, resulting in a vicious cycle (Szeto, 2006).Bai and Odin (2003) explainedthat Oxidative stress can lead to damage of the mitochondrial inner membrane,resulting in mitochondrial permeability transition pore (MPTP) formation andsubsequent release of cytochrome c and apoptosis inducing factor from themitochondria.

In the cytosol, cytochrome C complexes with apoptotic proteaseactivating factor (Apaf-1) to activate procaspase 9, which in turn activatesdownstream effector caspases (3, 6 and 7) .Nakagawa and Tayama (2000) reported thatbisphenol-A has been shown to reduce mitochondrial function in hepatocytes.Moon et al. (2012) reported thata low dose of BPA induces mitochondrial dysfunction in the liver, and this isassociated with an increase in oxidative stress and inflammation and this goparallel with our study. Measurement of MDA levels in thetissue is a marker of lipid peroxidation which is among the chief mechanism ofcell damage. Bisphenol-A is lipophilic in nature due to which it can easilypenetrate/interact with the lipid membrane of the hepatocytes (Doerge andFisher, 2010). The results of the present study werein agreement with the study of Suthar and Verma (2014) whichconcluded that treatment of BPA for 30 days cause increase in lipidperoxidation as well as alterations in the anti-oxidative system ultimatelycausing oxidative stress in experimental animals.

A study of Sangai et al. (2014) alsorevealed decrease of oxidative damage in liver and kidney of mice afterexposure to BPA by potent antioxidants such as quercetin. They also showed thatexposure to BPA caused significant reduction in the activities of catalase,superoxide dismutase, glutathione peroxidase, glutathione reductase andglutathione-S-transferase as well as in the levels of glutathione and totalascorbic acid contents; however, significant increase was found in the levelsof malondialdehyde.Therefore, we hypothesized that gingerextract could act as an antioxidant against BPA.Results of light microscopicexamination and immunohistochemical staining of stained liver sections obtainedin the present study have supported the above mentioned biochemical results.     Light-microscope examinationof H&E stained liver sections of BPA treated group after 8 weeks showed congested portal vein, bile ductproliferation and cellular infilteration, which was minimally observed in thegroup taken ginger extract with bisphenol.

Venous congestion was a permanent feature in the treatedliver sections in this study which accordingly might interfere with the hepaticarterial blood supply. This might lead to the development of ischemia andsubsequent necrosis as mentioned by (Majno and Joris, 1995).Our results were matched with Helalet al. (2013) who noticed that administration of BPA for 30 days revealedThe nuclei of hepatocytes are mostly large with prominent one or more nucleiand with Hussein and Eid (2013) who revealed that oraladministration of BPA for 21 days gave light abnormal pathological changecompared 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 incongestion and necrotic areas. These observations may be explained by BPA andinduced peroxidation of membrane lipids in the liver cells.Hussein and Eid(2013) noticed thatbisphenol-A caused cell infiltration was observed in focal manner surroundingthe dilated bile duct.     Immune-histochemical reactionto caspase 3 revealed positive reaction within cytoplasm of most hepatocytesin BPA group after 8 weeks which became less in group taken BPA + gingerextract for 8 weeks.  Caspases, intracellular cysteineproteases that cleave various substrates including structural proteins such ascaspase–3, are the key mediators of apoptosis.

Caspase-3, as a main final common executor ofapoptosis, 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 thatBPA induced endoplasmic reticulum (ER) stress-associated apoptosis inhepatocytes. The ER stress was due to ROS production and was independent ofestrogen receptors.Iida et al.

(2003) had shownthat apoptosis induction by BPA was associated with caspase activation. The overproduction of ROS may be an inducible factor ofapoptosis. Previous studies reported that BPA exposure produced ROS byinhibiting antioxidant enzymes (Chitra et al. 2003 and Kabuto et al.

2003).Our results were matched with Ahmad et al. (2006) inwhich they found that ginger extract may have antioxidant effect by replacingSOD activities and reducing the level of superoxide radicals in liver cancerinduced rats. This is similar to the findings of Park et al. (1998), inwhich the bioactive component in ginger reduced the production of ROS such assuperoxide anions.

Chang et al. (1994) found the bioactive component of ginger, namely gingerol,possessed antioxidative effect by inhibiting peroxidation of phospholipidsinduced by xanthine oxidase activity.Ahmad et al. (2006) concluded that ginger extract may have bioactive componentswith antioxidant activity in scavenging free radicals such as superoxide anionsand H2O2 as well as decreasing the MDA level for the reduction of lipidperoxidation.CONCLUSIONFrom this study we concluded that bisphenol-A inducedoxidative stress and apoptosis in hepatic tissue which decreased withadministration of ginger extract which acts as an antioxidant agent.RECOMMENDATION1-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. 

Author: Myron Guzman


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