Caffeic acid phenyl ester prevents cadmium intoxication induced disturbances in erythrocyte indices and blood coagulability, hepatorenal dysfunction and oxidative stress in rats

(1)

Original research article/Praca oryginalna

Caffeic acid phenyl ester prevents cadmium

intoxication induced disturbances in erythrocyte indices and blood coagulability, hepatorenal

dysfunction and oxidative stress in rats

Tariq Helal Ashour

¹

, Adel Galal El-Shemi

^1,2,

*

1DepartmentofLaboratoryMedicine,FacultyofAppliedMedicalSciences,UmmAl-QuraUniversity,SaudiArabia

2DepartmentofPharmacology,FacultyofMedicine,AssiutUniversity,Egypt

article info Articlehistory:

Received:12.02.2014 Accepted:08.04.2014 Availableonline:18.04.2014

Keywords:

Caffeicacidphenylester

Cadmium

Hematology

Bloodcoagulation

Liver

Kidney

abstract

Hereweinvestigated theprotectiveroleofcaffeicacidphenylester(CAPE)onerythro- cyteindicesandosmoticresistance,bloodcoagulation,hepato-renalfunctionandantio- xidantstatusincadmium(Cd)toxicityinrats.Cdintoxicationwasinducedbyintraperi- tonealinjection(i.p.)ofcadmiumchloride(1mg/kg/day)for21days,andCAPEwasdaily given(10mmol/kg; i.p.)also for 21 days. Atday 22, blood samples,livers andkidneys werepreparedforscreeningof:(1)erythrocyteindices:redbloodcell(RBC)count,osmotic fragility, hemoglobin (HGB) concentration, hematocrit (HCT), mean corpuscular volume(MCV),meancorpuscularhemoglobin(MCH),andmeancorpuscularhemoglobin concentration(MCHC);(2)bloodcoagulationtests:prothrombintime(PT),activatedpar- tialthromboplastin time(APTT),andfibrinogen(FIB)level;(3)serumlevelsofliverand kidneyfunctionbiomarkers(aspartateaminotransferase,alanineaminotransferase,alkalinephosphatase,albumin,creatinineandbloodureanitrogen);(4)blood,liverandkid- neylevels of Cd;and(5) serum andhepato-renal concentrations ofglutathione (GSH), superoxide dismutase (SOD), and thiobarbituric acid reactive substances (TBARS). Cd intoxicationsignificantlyimpairedhepato-renalfunction,prolongedPTandAPTT,redu- cedFIB, decreased RBCcount and osmoresistnacyas wellas the values ofHGB,HCT, MCV,MCHandMCHC.Interestingly,therapy withCAPEsuccessfullyeliminatedCdand significantlystabilizederythrocyteindices,bloodcoagulabilityandhepato-renalfunctio- nalstatusinCd-intoxication.Additionally,CAPEtherapy significantlyreversedthedec- reasesinGSH andSOD, andtheincreasesinTBARsthatwereinducedbyCdintoxica- tion.Inconclusion,CAPEcanrepresentapromisingtherapeuticagentineliminatingCd andcounteractingitshematological,hemostasisandhepatorenaltoxiceffects.

*Correspondingauthorat:DepartmentofLaboratoryMedicine,FacultyofAppliedMedicalSciences,UmmAl-QuraUniversity, Makkah,POBox7607,SaudiArabia.Tel.:+966509655135;fax:+966125270000x4242.

E-mailaddress:dr_adel_elshemy2006@yahoo.com(A.G.El-Shemi).

ContentslistsavailableatScienceDirect

Acta Haematologica Polonica

journal homepage:www.elsevier.com/locate/achaem

http://dx.doi.org/10.1016/j.achaem.2014.04.019

(2)

Introduction

Caffeicacid phenylester(CAPE)isone ofthe main medic- inal components of honeybee propolis that possesses a variety of biological and pharmacological actions such as potent freeradical scavenging, antioxidant, anti-inflamma- tory,cytoprotective, immunomodulatory,antiviral and pro- misinganticancerproperties [1]. Recently, the ameliorating effectsofCAPEondifferentdiseasemodalitiesofhematolo- gical, blood coagulation and vascular abnormalities have also been emerged. In this concept, CAPE had shown to ameliorate blood coagulation abnormalities and disturbed oxidativestressinendotoxicmodelofacuteliverfailure[2], increasecerebralbloodflowandimproveischemicstrokein neurovascular disease [3], protect peripheral blood mono- nuclearcellsagainsthyperthermal stress[4], preventdrugs toinducetoxicanddamageeffectsonredbloodcells(RBCs) [5],and potentlyinhibitthe synthesisof inflammatoryand atherosclerotic leukotrienes in human polymorphonuclear leukocytesandwholeblood[6].

Cadmium (Cd) is classiﬁed as a very harmful environmental pollutant to the humans that transfers between various levels of the foodchain [7]. Occupationalexposure to the Cd and its compounds primarily occurs in mining, smelting, processing, and battery manufacturing. In addi- tion,environmental and non-occupational exposures come fromvariousfoods,contaminatedwater,contaminateddust andtobaccosmoke[8].Thoughthe deﬁnitemechanismsof itsassociated toxicityarenotyet wellcovered, ithasbeen revealed that Cd markedly stimulates the formation of reactive oxygenspecies (ROS), enhances lipid peroxidation, cell membrane damage, and depletes the antioxidant defenseelements in different body organs [9]. It hasbeen provedthatafterexposure,Cdentersthebloodandbindsto theerythrocytemembranesandbloodalbumin,andthenis transported to liver, where it bounds to metallothionein (MT) [10]. The Cd–MT complex is then released back into circulation [10], and accumulates in the blood system, kidney,liver,lung,testis,brain,andbone[11]. Intheblood and tissues, Cd stimulates the formation of ROS, thus causing oxidative damage, which result in a loss of cell membrane functions [12], multi-organ damage and impor- tanthematologicalalterations[13,14].

Overthe pastdecade,avarietyof researchstudieshave reportedthat medicationswithfree-radicalscavengers and antioxidantsare usefulin protectingagainstCdtoxicity[7, 14, 15]. To date, few studies have shown the remarkable tissueprotectiveeffectsof CAPEagainstCdintoxication.In thisregard,therapywithCAPEhadsigniﬁcantlyresultednot onlyineliminationofCdfrombloodandtissuesbutalsoin preventing Cd-induced oxidative stress, overproduction of ROS, impaired cellular ultrastructures, and injuries in the renal, cardiac and liver tissues [9, 16–18]. However, the possiblepreventativeeffectofCAPEagainstthehematologi- cal and blood coagulation dysfunctions secondary to Cd intoxication is still not well investigated. Coherently, the present study aimedto investigate the possiblealleviating effects of CAPE on the altered hematological, erythrocyte

stress response that could beassociated with Cdintoxica- tioninrats.

Materials and methods

Chemicalsandreagents

Cadmium chloride(CdCl2) and caffeic acid phenethylester (CAPE) werepurchased fromSigma–AldrichChemicalCom- pany (St. Louis, MO, USA). Commercial assay kits of total reduced glutathione (GSH) content, superoxide dismutase (SOD) activity, and thiobarbituric acid reactive substances (TBARS) concentration werepurchased from Cayman Che- mical (Ann Arbor, MI, USA). All other used chemicals and reagents were of analytical grade and obtained from standard commercial supplies as stated under the sections of theirapplications.

Animals,treatmentsandexperimentalapproach

Forty adult male Wistar albino rats, weighing 230–250g, wereusedin thepresent study.Therats werehousedﬁve per cage under controlled temperature (20–258C) and 12-h light–dark cycle, and allowed free access to water and acommercial ratpelletsstockdiet.Allexperimentalproto- colswereapprovedbytheCommitteefortheCareand Use ofLaboratoryAnimalsatUmmAl-QuraUniversity,KSA,and all animals received careaccording tothe criteriaoutlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and pub- lished by the National Institutes of Health. The rats were randomlydividedinto3experimentalgroups:controlgroup (n=10), Cd-group (n=15), and Cd plusCAPE group(n=15).

InCdandCd+CAPEgroups,CdCl2, dissolvedinphysiologi- cal saline (0.9% sodium chloride (NaCl) in distilled water), was intraperitoneally injected at adose of 1mg/kg/day for 21days,andinCd+CAPEgroup,CAPEwasco-administered i.p. at a doseof 10mmol/kgfor also 21 days. Thedoses of both Cd and CAPE were chosen on the basic of previous studies [2, 9, 16]. Control rats were received only with physiologicalsaline.Attheendofthestudy(i.e.atday22), all animal groups werefasted for 12h and then sacriﬁced under ether anesthesia and their blood specimens were collected. After blood withdrawal, the livers and kidneys were harvested quickly, and divided into two portions:

aportionwasweighedandquicklystoredat 808CuntilCd measurement, while the second one was homogenized in RIPA lysis buffer (1:6 w:v), centrifuged at 10000rpm for 10min at 48C, and its supernatant was stored at 808C until used for measurement the intra-hepatic and intra- renal concentrations of antioxidant and oxidative stress biomarkersasdescribedbelow.

Bloodsampleanalysis

Duringscariﬁcationprocess,fourbloodsampleswereimme- diatelywithdrawnfromthevenacavaofeachratandused for blood coagulation, hematology and biochemical ana-

(3)

The ﬁrst sample was collected on a tube contained 0.11M sodiumcitrate anticoagulant (1:9, v:v), and usedfor plasma preparation for screening of the following blood coagulation tests: prothrombin time (PT), activated partial thromboplastintime(APTT),andﬁbrinogen(FIB)concentrations, by using Dade^® Behring reagents and following manufacturer'sinstructionsaspreviouslydescribed[19].

The second sample was collected in a tube contained disodium salt of ethylenediamine tetra acetic acid (EDTA) anticoagulant and used for determination of the following hematology parameters: counts of RBCs, white blood cells (WBCs) and platelets (PLTs), hemoglobin (HGB) concentration, hematocrit (HCT), mean corpuscular volume (MCV), meancorpuscularhemoglobin(MCH)andmeancorpuscular hemoglobin concentration (MCHC). These hematological parameters were determined by standard hematological techniques.

The third samplewas collected into a heparinized test tube and used for determination of erythrocyte osmotic fragility according to the method described previously by Azeez et al. [20] and Mineo et al. [21]. Brieﬂy, 0.02ml of bloodofeachratwastransferredintomicrotubescontaining one ml of increasing concentration of phosphate-buffered NaClsolution(0.0,0.1,0.3,0.5,0.7,and0.9%)atpH7.4.The microtubes were then gently mixed, incubated at room temperature for 30min, and then centrifuged at 2000rpm for 15min.Thesupernatant (200ml)of each microtubewas decanted into 96-well micro-plate, and its optical density was determined spectrophotometrically at 540nm (Micro- plate reader, Bio-Rad Laboratories). Thepercentage of RBC hemolysis in each NaCl concentration was determined, using hemolysis in distilled water (0.0% NaCl) as the maximum percentage. The higher hemolysis occurs, the greaterisosmoticfragilityofRBC[20,21].

Thelastportionofthecollectedbloodwasplacedinaplain centrifugetubewithoutanyanticoagulantandaftercentrifuga- tionprocess, itscorrespondingserumwasobtainedandused formeasurementtheseraconcentrationsofCdandbiomarkers ofantioxidationandoxidativestressasdescribedbelow.

Biochemicalanalysisofhepato-renalfunction

Thepreparedserasampleswereemployedformeasurement of the serum concentrations of liver function enzymes (aspartateaminotransferase(AST),alanineaminotransferase (ALT) and alkaline phosphatase (ALP)), albumin (ALB), and kidney function biomarkers (serum creatinine (CRE) and bloodureanitrogen(BUN)).

Evaluationofantioxidantandoxidativestressstatus

The levels of total reduced GSH and activities ofSOD (as indices of non-enzymatic and enzymatic antioxidant status, respectively)were measuredinthe serumandhomo- genates of the liver and kidney tissues. On the other hand, theconcentrations ofTBARS,indices oflipidperox- idation and oxidative stress, were also determined in these sera, liver and renal biological samples. During these three assays, speciﬁc commercial kits (Cayman Chemical; AnnArbor,MI,USA)wereused,andallsamples were processed in duplicate and according the manufacturer'sinstructions.

Measurementofblood,hepaticandrenalcadmiumcontent

Cd levels inthe blood,liver,and kidneysweredetermined as describedinpreviousstudies[22, 23].Forassessment of Cd levels inblood, 0.5mlwhole blood sampleswereused, whileforitsassessmentintissues,constantweightslicesof livers and kidneys were oven dried at 608C. The blood samples and the dried tissues (100mg from each sample) weredigestedwith3mltracepureconcentricnitricacidon ahotplateat 1208C.Oncethe digestionwas complete,the samples were cooled at room temperature and their volumeswerethenadjustedto10mlwithdeionizedwater.

Cdconcentrationwasquantiﬁedbyusinganatomicabsorp- tion spectrophotometer (Perkin-Elmer AAnalyst 100). Cd concentrationisexpressedasmg/mlbloodandmg/gliveror kidneytissueweight.

TableI–Hematologicalandbloodcoagulationfindings

Parameter Units Controlgroup(n=10) Cdgroup(n=15) Cd+CAPEgroup(n=15)

WBC 10³/ml 11.920.79 13.532.72 12.271.46

RBC 10⁶/ml 8.670.48 5.930.83^* 8.100.16^#

HGB g/l 157.677.23 115.466.03^* 151.673.51^#

HCT % 48.330.71 41.831.00^* 46.131.01^#

MCV fL 64.270.84 52.874.48^* 60.201.54^#

MCH pg 22.131.65 16.701.11^* 20.401.20^#

MCHC g/l 344.4239.50 300.2627.77^* 337.6738.50^#

PLT 10³/ml 1000.2791.83 775.3259.61^* 933.3670.76^#

PT Second 12.821.37 43.144.33^* 17.762.33^#

APTT Second 21.733.46 77.5213.43^* 29.365.21^#

FIB mg/dl 263.9033.77 93.379.37^* 226.6842.23^#

ThevaluesarepresentedasmeansSD.(Cd)cadmium,(CAPE)caffeicacidphenylester,(WBC)whitebloodcell,(RBC)redbloodcell,(HGB) hemoglobin,(HCT)hematocrit,(MCV)meancorpuscularvolume,(MCH)meancorpuscularhemoglobin,(MCHC)meancorpuscularhemoglobin concentration,(PLT)platelet,(PT)prothrombintime,(APTT)activatedpartialthromboplastintime,and(FIB)ﬁbrinogen.

* P<0.05versuscontrolgroup.

# P<.05versusCdgroup.

(4)

Statisticalanalysis

Theresultswereexpressedasthemeanstandarddeviation (SD) and statistical analysis was carried out using SPSS software,version16.0(SPSSInc.,Chicago,IL,USA).Differences amongthegroupswereinvestigatedusingone-wayanalysis of variance (ANOVA) followed by a Student's t-test. Differ- encesbetweenpercentagesofRBChemolysisfordetermina- tionof osmoticfragility for thegroups wereanalyzed byx² test.APvalueof<0.05wasconsideredstatisticallysigniﬁcant.

Results

Hematologicalandbloodcoagulationﬁndings

Thehematologicalandbloodcoagulationchangesareshown inTableI. Comparedwith the controlgroup,Cd-grouphad showedsignificantdecreasesinRBCandPLTcounts,butnot in WBC counts. Moreover, the values of HGB, HCT, MCV, MCH,and MCHChadsignificantlydecreasedinCd-groupin comparison with their values in the normal controls. In contrary, therapy with CAPE had successfully ameliorated thesehematologicaltoxic effectsof Cd,wherebythe counts ofRBCsand PLTs,aswellasthevalues ofHGB, HCT,MCV, MCH,andMCHChadshowednosignificantdifferencesthan thoseof normal controlrats. Intoxicationwith Cdhad also resultedinsignificantdeteriorationsonbloodcoagulation.As demonstratedinTableI,thereweresignificantprolongation of PT and APTT clotting tests associated with significantly decreased FIB levels in Cd-group, when compared with controlgroup.Bycontrast,treatmentoftheseCd-injectedrats withCAPEhadsignificantlysucceededinimprovementofthe

valuesofPTAPTT,andFIB(TableI).Collectively,thispartof theresultsindicatesthatCdintoxicationinratsisassociated with important alterationsintheir hematologicalandblood coagulation parametersand these alterationswere remark- ablyalleviatedbyCAPEtherapy.

Effectsonerythrocyteosmoticfragility

Changes in RBCs hemolytic pattern after their exposed to differentconcentrationsof NaClsolutionweremeasuredto determine the erythrocyte osmotic fragility of all animal groups.AsshowninTableII,theosmoticfragilityofRBCsof Cd group was signiﬁcantly higher than that of Cd+CAPE and normal control groups at each tested NaCl solution.

This in turn reﬂects the stabilizing effect of CAPE therapy onRBCosmoticresistance.

Biochemicalﬁndings

The results of serum biochemistry reﬂect that both the hepatic and renal functionof rats had been altereddueto Cdintoxicationand thesedamagingeffectsofCdhadbeen attenuated by CAPE therapy. As shown in Table III compared with the control group, signiﬁcant changes in the serum levels of AST, ALT,ALP, and ALB(as biomarkers of liverfunction), aswell asthe serumlevelsofCREandBUN (asbiomarkers of renal function)were detectedonlyin Cd groupbutnotinCd+CAPEgroup.

Blood,renalandhepaticlevelsofcadmium

Next, weconﬁrmedtheaccumulationof administeredCdin thebloodandhepato-renaltissuesoftherats(Fig.1).After21 TableII–Erythrocyteosmoticfragility

Group HemolysisofRBC(%)

0.0%NaCl 0.1%NaCl 0.3%NaCl 0.5%NaCl 0.7%NaCl 0.9%NaCl

Control(n=10) 87.73.2 80.54.5 65.57.1 33.55.6 13.33.3 2.10.3

Cd(n=15) 97.32.2^* 93.34.3^* 85.35.2^* 55.77.4^* 38.43.5^* 21.51.3^* Cd+CAPE(n=15) 89.37.4^# 84.53.6^# 68.52.7^# 37.52.1^# 17.72.2^# 3.50.7^# (Cd)cadmium,(CAPE)caffeicacidphenylester.

# P<0.05versusCdgroup.

TableIII–Serumlevelsofliverandkidneyfunctionbiomarkers

Parameter Units Controlgroup(n=10) Cdgroup(n=15) Cd+CAPEgroup(n=15)

AST IU/l 109.0012.17 571.3372.29^* 163.0014.93^#

ALT IU/l 49.173.27 136.0721.06^* 73.3311.35^#

ALP IU/l 206.3320.79 274.3325.14^* 211.3317.35^#

ALB g/dl 4.330.59 2.860.38^* 4.010.27^#

CRE mg/dl 0.260.04 0.410.34^* 0.290.01^#

BUN mg/dl 47.369.12 63.6513.44^* 49.2210.10^#

ThevaluesarepresentedasmeansSD.(Cd)cadmium,(CAPE)caffeicacidphenylester,(AST)aspartateaminotransferase,(ALT)alanine aminotransferase,(ALP)alkalinephosphatase(ALP),(ALB)albumin,(CRE)creatinine,and(BUN)bloodureanitrogen.

(5)

days,Cdlevelswereveryhighintheblood,liverandkidney of Cdgroup. On the contrary, concurrent administration of CAPEsigniﬁcantlyeliminatedCdfromthesebiologicaltissues.

Serum,renalandhepaticlevelsofGSH,SODandTBARS

Finally, we measured the levels of GSH (an example of non- enzymaticantioxidantdefensemechanism),activitiesofSOD(an example of enzymatic antioxidant defense mechanism), and concentrations of TBARS (an index of lipid perioxidation and oxidative stress)in thesera samplesandthe liverand kidney tissue homogenates of all animalgroups. As demonstrated in TableIV,Cdintoxicationinratshadassociatedwithsigniﬁcant reductioninGSHcontentand SODactivity,aswellas marked elevationinTBARScontentinthesera,liversandkidneysofCd group. On the other hand, concurrent administrationof CAPE withCdhadobviouslycounteractedtheseeffectsofCdonGSH, SOD,andTBARSinalltestedbiologicalsamples.

Discussion

Cd is classiﬁed as a major industrial and environmental pollutant thatraisesserious public health concernsworldwide because of its high toxic effects on human and animals [8].

CAPE is a biological active component of honeybee propolis extracts with pluripotent pharmacological actions and potent antioxidant activity[9]. Thisstudywas designedtoinvestigate the effect of CAPE supplementation therapy on erythrocyte indices, bloodcoagulation, hepato-renalfunctional and antiox- idantstatusinCdintoxicationinrats.Theresultsshowedthat CAPEtherapynotonlyeliminatedtheheavymetalbutalsoand importantly resulted in an obvious protection against Cd- induced marked hematological and blood hemostasis disturbances,hepato-renalinjuryandoxidativestressinrats.

ThereisacompellingbodyofevidencethatCdexposure importantly contributes to human and animal liver and kidneydiseases[24,25]. Aftertheintakeandresorption,Cd enters the blood and binds to the erythrocyte membranes and plasma albumin [26]. In the blood and tissues, Cd stimulateslipidperoxidationandtheformationofROS,thus causing oxidative cellular and tissue damages [10, 12, 13]. In support, Cd wasdetected athighconcentrationsin the blood, liver and kidneys of Cd group (Fig. 1),and this accumulation pattern of Cdwas signiﬁcantlyattenuatedwith CAPE therapy.

Moreover, the biochemical ﬁndings (Table III) revealed the occurrence ofhepato-renalimpairmentinCd-exposedanimals Fig.1–Effectofcaffeicacidphenylester(CAPE)therapyon

cadmium(Cd)levelsinbloodandhepato-renaltissuesafter 21daysoftreatment.*P<0.001versuscontrolgroup;

#P<0.01versusCdgroup

TableIV–Antioxidantandlipidperoxidationstatusinserumandhepato-renaltissues

Group GSH(mmol/mgprotein) SOD(U/mgprotein) TBARS(nmol/mgprotein)

Serum Liver Kidney Serum Liver Kidney Serum Liver Kidney

Control(n=10) 13.73.1 69.97.3 57.79.2 3.80.6 289.537.3 267.723.4 27.44.2 48.66.2 37.45.9 Cd(n=15) 4.70.9^* 11.71.2^* 13.741.7^* 1.20.2^* 64.88.2^* 71.56.7^* 116.223.4^* 1019.5195.9^* 1113.3195.9^* Cd+CAPE(n=15) 12.21.2^# 75.911.4^# 68.710.2^# 3.30.8^# 305.350.5^# 311.547.8^# 36.745.8^# 87.214.6^# 118.623.29^# ThevaluesarepresentedasmeansSD.(Cd)cadmium,(CAPE)caffeicacidphenylester,(GSH)totalglutathione,(SOD)superoxidedismutase, and(TBARS)thiobarbituricacidreactivesubstances.

# P<0.05versusCdgroup.

(6)

butnotinCd+CAPE-treatedanimals,asreflectedbysignificant elevationsintheserumlevelsofAST,ALT,ALP,CREand BUN [27].Theobservedhepato-renalprotectiveeffectsofCAPEagainst Cdwere alsoaccompaniedwith preventing Cdtoinduce lipid peroxidation and decrease antioxidant defense system (Table IV).Takentogether,thesefindingscanreinforcethosepreviously reportedthatCAPEhasapotentprotectiveeffectagainstrenal, liver,andother bodyorganinjuriescausedbyCdintoxication, andthismightbebyeliminatingCdandinhibitingCd-induced oxidativestressandtissuedamage[9,16–18].

Hematopoieticsystemisoneofthemostsensitivesystems toevaluatethehazardseffectsofpoisonsanddrugsinhumans andanimals [27].In consistency,thecurrent study indicated that anexposure to toxic Cdwas associated withsignificant disturbancesinerythrocyteindices;andtherapywithCAPEhad successfullyalleviatedthesehematologicalchangesinducedby Cd(TableI).Inthisregard,anemiawasclearlyobservedinrats receivedCdalonebutnotinthosereceivedCdplusCAPE.In agreement, it has been approved that Cd accumulation in kidney, liver and spleen can suppress the activity of these importanthematopoietic tissues[28]. Additionally,somepre- vious studies revealed that exposure to Cd induces anemia associatedwithdecrease in RBCs counts,HGBconcentration, HCT value, and induction of oxidative damage and lipid peroxidationin bloodandRBCs [13,14,29,30].Moreover,the decreasesthatwereobservedherinthevaluesofMCV,MCH, andMCHC in Cdgroupbut notin Cd+CAPEgroupcan also indicatethefurtherabilityofCdintoxicationtoinducemicro- cytic hypochromic anemia [25], and this effect was also alleviated by CAPE therapy. Furthermore, dataof erythrocyte osmotic fragility test (Table II); which is a test refers to the propensity of erythrocytes to hemolyse when they are sub- jectedtoosmoticstressbybeingplacedinahypotonicsolution [20, 21], demonstrated that exposure to Cd had resulted in asignificantincreaseinRBCosmofragilityandthiseffectwas significantly improved by CAPE treatment (Table II). Recent reports revealed that the enhanced oxidative stress and decreased antioxidant status result in increased erythrocyte deformability, easier RBC membrane lipoperoxidability and damage,andconsequently,anincreasedosmoticfragilityand shortened life-span [20, 21]. As the powerful free radical scavenger and antioxidant properties of CAPE have been reportedinthepresentstudy andbyprevious researchers[2, 31,32], it can be considered that aportion of thestabilizing effectofCAPEtherapyonrat'sRBCosmoticresistancecouldbe attributed toitsantioxidantactivity. Collectively,Cd intoxica- tionmightleadtoanemiaasaresultofeithersuppressionthe activity of hematopoietic tissues, impaired erythropoiesis, accelerated erythroclasia because of the altered RBCs mem- branepermeability,increasedRBCsmechanicalfragility,and/or defectiveFemetabolism[25].

Data of blood clotting tests (Table I) demonstrated that exposuretoCdhadresultedinasigniﬁcanthypocoagulation stateinformofmarkedprolongationofthecoagulationtests PT, APTT, low PT count and decreasedFIB levels-And, this Cd's hemostatic dysregulation effect was signiﬁcantly improvedbyCAPEtreatment.Itiswellknownthattheliveris themajor organfor synthesis of procoagualtionfactorsand substances. Thus, the hypocoagulation state that was

relatedtoCd-inducedliverinjury withdecreasedproduction of the procoagulationfactors andreduced hepaticclearance of plasminogen activators leaded to enhanced ﬁbrinolytic activity [2,33]. Theseﬁndingsanditsrelatedsuggestioncan also be supported by Korish's report [2] that therapy with CAFÉ protects the liver andprevents the hemostaticaltera- tionsinendotoxic-inducedacuteliverfailure.

Earlier studies had indicated that treatment with freeradical scavengersandantioxidantsareusefulinprotecting against Cdtoxicity [7, 14, 15]. Therefore, depletion of anti- oxidativedefensemechanism(representedbydecreasedSOD and GSH) together with increasedTBARs (an index of lipid peroxidationandoxidativestress)thatwereobservedherein theserum,liverandkidneytissuesofCd-injectedratsbutnot ofCd-injected/CAPE-treatedrats(TableIV)couldbethemain underlying pathogenic mechanismsby whichthe injected Cdhadinduceditshematologicalandorgantoxicity[9,14].

Similarly, the preventative effects of CAPE that were also observed against Cdcouldalso beattributed to itspotent antioxidant property [2, 32]. Inharmony, Gokalp et al. [5]

showed the cytoprotective ability of CAPE in preventing anti-TB drug ‘‘isoniazed’’ to induce oxidative damage in RBCs,andthepotentrenoprotectiveeffectofCAPEagainst Cd-induced injury has been previously conﬁrmed and attributedmainlytotheantioxidantactivityofCAPE[9,16– 18]. Finally, by increasing the antioxidant elements and inhibitingthe oxidativestatus,CAPEhadshowntoprotect thebrainvasculaturefromischemicstrokedisease[3],and protectthe liverandimprovebloodcoagulationabnormal- itiesinendotoxicmodelofacuteliverfailure[2].

Conclusion

Basedonthepresentedresults,itcanbeconcludedthatCd intoxication was resulted in impaired erythrocyte indices, erythrocyte osmotic resistance blood coagulability, and hepato-renal function in rats, and this might be due to enhancing of lipidperoxide concentrationand/ordepletion of theactivityofantioxidantdefenseelements.Bycontrast, CAPE supplementation therapy was resulted in favorable effectsineliminatingCdand preventingthehematological, blood coagulation, and hepato-renal disturbances that occurred secondary to Cd intoxication. Moreover, CAPE therapy was also resulted in restoring the antioxidant activity and inhibitinglipidperoxidationinbothblood and organs.Therefore,CAPEcouldbeapromising agentfor the treatment of Cd intoxication; however, further studies are cruciallyneededtoimprovethistreatmentinpatients.

Authors' contributions/Wkład autorów

TH Ashour and AG El-Shemi– manuscript authors, AG El- Shemi–forcorrespondence.

Conﬂict of interest/Konﬂikt interesu

(7)

Financial support/Finansowanie

Nonedeclared.

Ethics/Etyka

Thework described inthis article has been carriedout in accordance with The Code of Ethics of the World Medical Association(Declaration of Helsinki)for experimentsinvol- ving humans; EU Directive 2010/63/EU for animal experi- ments;UniformRequirementsformanuscriptssubmittedto Biomedicaljournals.

references/pi smiennictwo

[1]AkyolS,OzturkG,GinisZ,etal.Invivoandinvitro antıneoplasticactionsofcaffeicacidphenethylester(CAPE):

therapeuticperspectives.NutrCancer2013;65:515–526.

[2] KorishAA.Effectofcaffeicacidphenethylesteronthe hemostaticalterationsassociatedwithtoxic-inducedacute liverfailure.BloodCoagulFibrin2010;21:158–163.

[3] KhanM,ElangoC,AnsariMA,SinghI,SinghAK.Caffeicacid phenethylesterreducesneurovascularinﬂammationand protectsratbrainfollowingtransientfocalcerebral ischemia.JNeurochem2007;102:365–377.

[4] ChenYJ,HuangAC,ChangHH,etal.Caffeicacidphenethyl ester,anantioxidantfrompropolis,protectsperipheral bloodmononuclearcellsofcompetitivecyclistsagainst hyperthermalstress.JFoodSci2009;74:H162–H167.

[5] GokalpO,UzE,CicekE,etal.Amelioratingroleofcaffeic acidphenethylester(CAPE)againstisoniazid-induced oxidativedamageinredbloodcells.MolCellBiochem 2006;290:55–59.

[6] BoudreauLH,MailletJ,LeBlancLM,etal.Caffeicacid phenethylesteranditsamideanaloguearepotent inhibitorsofleukotrienebiosynthesisinhuman polymorphonuclearleukocytes.PLoSOne2012;7:e31833.

[7] JihenelH,ImedM,FatimaH,AbdelhamidK.Protective effectsofselenium(Se)andzinc(Zn)oncadmium(Cd) toxicityintheliveroftherat:effectsontheoxidativestress.

EcotoxicolEnvironSaf2009;72:1559–1564.

[8] HoláskováI,ElliottM,HansonML,SchaferR,BarnettJB.

Prenatalcadmiumexposureproducespersistentchangesto thymusandspleencellphenotypicrepertoireaswellasthe acquiredimmuneresponse.ToxicolApplPharmacol 2012;265:181–189.

[9] GongP,ChenF,LiuX,etal.Protectiveeffectofcaffeicacid phenethylesteragainstcadmium-inducedrenaldamagein mice.JToxicolSci2012;37:415–425.

[10] HambachR,LisonD,D'HaesePC,etal.Co-exposuretolead increasestherenalresponsetolowlevelsofcadmiumin metallurgyworkers.ToxicolLett2013;222:233–238.

[11] USDepartmentofHealthServices.Toxicologicalproﬁlefor cadmium,Draftforpubliccomment.Atlanta,GA:Agency forToxicSubstancesandDiseaseRegistry;1997.

[12] SimpkinsC,LloydT,LiS,BaldermanS.Metallothionein- inducedincreaseinmitochondrialinnermembrane permeability.JSurgRes1998;75:30–34.

[13] KostićMM,OgnjanovićB,ZikićRV,etal.Cadmium-induced changesofantioxidantandmetabolicstatusinredblood cellsofrats:invivoeffects.EurJHaematol1993;51:86–92.

[14] KanterM,CoskunO,GurelA.Effectofblackcumin(Nigella sativa)oncadmium-inducedoxidativestressintheblood ofrats.BiolTraceElemRes2005;107:277–287.

[15] FarissMW.Cadmiumtoxicity:uniquecytoprotective propertiesofalphatocopherylsuccinateinhepatocytes.

Toxicology1991;69:63–77.

[16] MollaogluH,GokcimenA,OzgunerF,etal.Caffeicacid phenethylesterpreventscadmium-inducedcardiac impairmentinrat.Toxicology2006;227:15–20.

[17] ChenF,GongP.Caffeicacidphenethylesterprotectsmice hepaticdamageagainstcadmiumexposure.ProcEnvSci 2011;8:633–636.

[18] KobroobA,ChattipakornN,WongmekiatO.Caffeicacid phenethylesteramelioratescadmium-inducedkidney mitochondrialinjury.ChemBiolInteract2012;200:21–27.

[19] LeminiC,JaimezR,FrancoY.Genderandinter-species inﬂuenceoncoagulationtestsofratsandmice.ThrombRes 2007;120:415–419.

[20] AzeezOI,OyagbemiAA,OyeyemiMO,OdetolaAA.

AmeliorativeeffectsofCnidoscolusaconitifoliusonalloxan toxicityinWistarrats.AfrHealthSci2010;10:283–291.

[21] MineoH,AmitaN,KawawakeM,HiguchiA.Dicarboxylic acidswithlimitednumbersofhydrocarbonsstabilizecell membraneandincreaseosmoticresistanceinrat erythrocytes.BiochimBiophysActa2013;1828:2379–2384.

[22] CongiuL,ChiccaM,PilastroA,TurchettoM,TallandiniL.

Effectsofchronicdietarycadmiumonhepaticglutathione levelsandglutathioneperoxidaseactivityinstarlings (Sturnusvulgaris).ArchEnvironContamToxicol 2000;38:357–361.

[23] Santoyo-SánchezMP,Pedraza-ChaverriJ,Molina-JijónE, etal.Impairedendocytosisinproximaltubulefrom subchronicexposuretocadmiuminvolvesangiotensinII type1andcubilinreceptors.BMCNephrol2013;14:211–223.

[24] AkessonA.Cadmiumexposureintheenvironment:renal effectsandthebenchmarkdose. In:JeromeON,editor.

Encyclopediaofenvironmentalhealth.Burlington:Elsevier;

2011.p.465–473.

[25] YuanG,DaiS,YinZ,etal.Toxicologicalassessmentof combinedleadandcadmium:acuteandsub-chronic toxicitystudyinrats.FoodChemToxicol2014;65:260–268.

[26] BaumanJW,LiuJ,KlaassenCD.Productionof

metalothioneinandheat-shockproteinsinresponseto metals.FundamApplToxicol1993;21:15–22.

[27] LijuVB,JeenaK,KuttanR.Acuteandsubchronictoxicityas wellasmutagenicevaluationofessentialoilfromturmeric (CurcumalongaL).FoodChemToxicol2013;53:52–61.

[28] GillTS,EppleA.Stress-relatedchangesinthe

hematologicalproﬁleoftheAmericanEelAnguillarostrata.

EcotoxEnvironSaf1993;25:227–235.

[29] OgnjanovićBI,PavlovićSZ,MaletićSD,etal.Protective inﬂuenceofvitaminEonantioxidantdefensesysteminthe bloodratstreatedwithcadmium.PhysiolRes2003;52:

563–570.

[30] DemirH,KanterM,CoskunO,etal.Effectofblackcumin (Nigellasativa)onheartrate,somehematologicalvalues, andpancreaticb-celldamageincadmium-treatedrats.Biol TraceElemRes2006;110:151–162.

[31] ParlakpinarH,SahnaE,AcetA,MizrakB,PolatA.Protective effectofcaffeicacidphenethylester(CAPE)onmyocardial ischemia-reperfusion-inducedapoptoticcelldeath.

Toxicology2005;2091:1–14.

[32] YildizY,SerterM,EkRO,etal.Protectiveeffectsofcaffeic acidphenethylesteronintestinalischemia-reperfusion injury.DigDisSci2009;54:738–744.

[33] AmitranoL,GuardascioneMA,BrancaccioV,BalzanoA.

Coagulationdisordersinliverdisease.SeminLiverDis 2002;22:83–96.