Protective role of omega-3 polyunsaturated fatty acid against lead acetate-induced toxicity in liver...

Research ArticleProtective Role of Omega-3 Polyunsaturated FattyAcid against Lead Acetate-Induced Toxicity in Liver and Kidneyof Female Rats

Heba M Abdou1 and Mohamed A Hassan2

1 Zoology Department Faculty of Science Alexandria University Moharram Bey Alexandria 21511 Egypt2 Protein Research Department Genetic Engineering and Biotechnology Research Institute (GEBRI)City of Scientific Research and Technological Applications (SRTA-City) New Borg El-Arab Alexandria 21934 Egypt

Correspondence should be addressed to Heba M Abdou drheba abdou3000yahoocom

Received 11 February 2014 Revised 30 April 2014 Accepted 21 May 2014 Published 18 June 2014

Academic Editor Anilava Kaviraj

Copyright copy 2014 H M Abdou and M A Hassan This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The present study was conducted to investigate the protective role of Omega-3 polyunsaturated fatty acids against lead acetate-induced toxicity in liver and kidney of female rats Animals were divided into four equal groups group 1 served as control whilegroups 2 and 3 were treated orally with Omega-3 fatty acids at doses of 125 and 260mgkg body weight respectively for 10 daysThese groups were also injected with lead acetate (25mgkg body weight) during the last 5 days Group 4 was treated only withlead acetate for 5 days and served as positive control group Lead acetate increased oxidative stress through an elevation in MDAassociated with depletion in antioxidant enzymes activities in the tissues Moreover the elevation of serum enzymes activities(ALT AST ALP and LDH) and the levels of urea and creatinine were estimated but total proteins were decreased Also leadacetate-treatment induced hyperlipidemia via increasing of lipid profiles associated with decline inHDL-c level Significant changesof Hb PCV RBCs PLT and WBCs in group 4 were recorded The biochemical alterations of lead acetate were confirmed byhistopathological changes and DNA damage The administration of Omega-3 provided significant protection against lead acetatetoxicity

1 Introduction

Lead is one of mankindrsquos oldest environmental and occu-pational toxins [1] The exposure to lead can occur from amultitude of sources such as soil air water and industrialpollutants There are worldwide six categories of productsconsidered as source of lead exposure that is gasolineadditives food can soldering lead based paints ceramicglazes drinking water systems and folk remedies [2] Healthhazards from increased lead exposure as a result of industrialand environmental pollution are recognized It has beenfound to cause a wide range of biochemical and physiologicaldysfunctions [3] Moreover the long term lead exposuregenerates reactive oxygen species and different free radi-cals Also it inhibits antioxidant enzymes activities suchas superoxide dismutase (SOD) and catalase (CAT) whileit decreases the level of glutathione [4 5] Lead induced

oxidative damage in the kidney as evidenced by enhancementof lipid peroxidation [6 7] Lead is a highly poisonousenvironmental pollutant and is known to affect organs likeliver kidney blood and central nervous system of mammals[8 9] Several reports have indicated that lead can causeneurological hematological gastrointestinal reproductivecirculatory and immunological pathologies [10 11] Omega-3fatty acids (Omega-3 FAs) are considered as strong antioxi-dants and their role as anticancer agent has been extensivelyconfirmed in most of the human malignancies [12 13]Furthermore the anti-inflammatory potential of long chainOmega-3 FAs in many chronic diseases has been suggested[14 15] The role of Omega-3 FAs in inhibiting proliferationinducing apoptosis and promoting differentiation in manycancers has been recently studied [16 17] In addition anotherfinding indicates that Omega-3 FAs act synergistically withcertain chemotherapeutic agents [18] Omega-3 FAs were

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 435857 11 pageshttpdxdoiorg1011552014435857

2 BioMed Research International

found to play protective roles in the liver cardiovascularsystem and kidney and they have beenwidely used in clinicalperoperative total parenteral nutrition [19 20]Therefore thepresent study was carried out to investigate the protectiveeffects ofOmega-3 FAs against lead acetate-induced oxidativestress biochemical changes and DNA damage

2 Materials and Methods

21 Chemicals Lead acetate was purchased from Merck(Germany)

Omega-3 was purchased from Efamol Ltd 14 The MoleBusiness Park Leatherhead Surrey KT227BA UK in theSouth East of England All other chemical materials that wereused in this study were purchased from Sigma Chemical Co(St Louis MO USA)

22 Animals and Experimental Design Twenty-eight adultWistar albino female rats (weighting 170ndash200 gm) wereobtained from the animal house of Faculty of MedicineAlexandria University Egypt The local committee approvedthe design of the experiments and the protocols were carriedout according to the guidelines of the National Institutesof Health (NIH) Rats were housed in stainless steel cagesplaced in a well-ventilated rat house maintained for twoweeks as acclimatization period under standard laboratoryconditions on free supply of food and water provided adlibitum and subjected to natural light for 12 hrs and dark for12 hrs cycles After the period of acclimatization rats weredivided randomly into four groups 7 animals in each Theanimal experiments were conducted for 10 days Group 1 wasinjected daily with 05mL of saline solution (09 NaCl) ipfor 10 days and was used as negative control (minusve) Groups 2and 3 were administrated orally with two doses of Omega-3(125 and 260mgkg body weight resp) by gavage for the firstfive days as protective agent [21] These groups were injected(ip) by 05mL of lead acetate at a dose of 25mgkg bodyweightday for the other 5 days in combination with Omega-3 Group 4 was injected (ip) by 05mL lead acetate only ata dose of 25mgkg body weightday for the last 5 days of theexperiment and was used as positive control (+ve) accordingto Ponce-Canchihuaman et al [22]

23 Blood Collection and Tissue Preparation At the end oftreatment rats fasted for 12 hrs before being anesthetized andsacrificed by cervical dislocation Blood samples were col-lected from the sacrificed animals and left in refrigerator for30min before centrifugation The clear nonhemolyzed serawere stored at minus20∘C till measurements However heparinwas used as an anticoagulant and noncoagulated blood wastested shortly after collection for applying in determinationof hemoglobin (Hb) packed cells volume (PCV) red bloodcells (RBCs) count white blood cells (WBCs) and platelets(PLT) count by particle counter (ERMA Inc Tokyo modelPCE-210)

Liver and kidney were immediately removed and washedusing chilled saline solution These tissues were minced

Table 1 PCR primers used in RAPD-PCR GC and annealingtemperature

Primers Sequence 51015840 rarr 31015840 GC Annealing temperature (∘Csec)1 GTC CAT GCCA 60 30602 ACA TCG CCCA 60 30603 ATG CCC CTG T 60 3060

and separately homogenized (10wv) using a homoge-nizer (Potter-Elvehjem) in ice-cold sodium potassium phos-phate buffer (001M pH 74) containing 115 of KCl Thehomogenates were centrifuged at 10000timesg for 20min at 4∘Cand the supernatant was used for assaying of the enzymesactivities

24 Biochemical Analysis Stored serum samples were ana-lyzed for the activities of aspartate aminotransferase (ASTEC 2611) alanine aminotransferase (ALT EC 2612) alka-line phosphatase (AlP EC 3131) and lactate dehydroge-nase (LDH EC 11127) which were determined using kitsfrom Sentinel Ch (via principle Eugenio 5-20155 MilanItaly) Also serum total protein albumin urea creatininecholesterol total lipids triglycerides HDLC and low den-sity lipoprotein (LDLC) were determined using kits fromSentinel Ch (via principle Eugenio 5-20155 Milan Italy)The lipid peroxidation end product MDA was measuredas thiobarbituric acid reactive substance Also the levels ofGSH and the activities of antioxidant enzymes including thecatalase enzyme (CAT EC 11116) superoxide dismutase(SOD EC11511) and glutathione peroxidase (GPx EC1119) were assayed using commercial assay kits accordingto the manufacturerrsquos instructions

25 Histopathology Specimens of liver tissues were immedi-ately fixed in 10 formalin treatedwith conventional grade ofalcohol and xylol embedded in paraffin and sectioned at 4ndash6 120583 thickness The sections were stained with Haematoxylinand Eosin (HampE) stain for studying the histopathologicalchanges [23]

26 Random Amplified Polymorphic DNA Technique (RAPD)

261 Extraction of DNA DNA was extracted from livers ofthe four groups following the method described by Bardakciand Skibinski [24]

262 Polymerase Chain Reaction (PCR) Primers In thepresent work ten-base long oligonucleotides primers wereused to initiate the PCR amplifications Primers were ran-domly selected on the basis of GC content and annealingtemperature for RAPD-PCR amplification as in Table 1

263 PCR Amplification and Agarose Gel ElectrophoresisPCR amplifications were performed according to the proce-dure described byWilliams et al [25] using the isolated DNAfrom three hepatic samples of each group

BioMed Research International 3

Table 2 The activities of AST ALT ALP LDH levels of MDA total protein albumin urea and creatinine after lead exposure and Omega-3treatment in serum of female rats

Parameters

Experimental groups

(Group 1)minusve control

(Group 2)Omega-3

(125mgkg bw) + lead acetate

(Group 3)Omega-3


(Group 4)+ve control

AST (UL) 5669 plusmn 0565a 5863 plusmn 1279a 5135 plusmn 0373a 10427 plusmn 0677b

ALT (UL) 3632 plusmn 0276a 4053 plusmn 0260a 3869 plusmn 0416a 9007 plusmn 0612b

ALP (UL) 4243 plusmn 0358a 4874 plusmn 0475a 4144 plusmn 0279a 8777 plusmn 0534b

LDH (UL) 14929 plusmn 0703a 15907 plusmn 0845a 14881 plusmn 0845a 21988 plusmn 1082b

Total protein (gmdL) 710 plusmn 0115a 611 plusmn 0244a 761 plusmn 0217a 488 plusmn 0103b

Albumin (gdL) 396 plusmn 0095a 375 plusmn 0071a 405 plusmn 0102a 266 plusmn 0083b

Urea (mgdL) 3109 plusmn 0225a 4004 plusmn 0715a 3159 plusmn 0450a 6356 plusmn 0962b

Creatinine (mgdL) 038 plusmn 0004a 047 plusmn 0011a 038 plusmn 0004a 096 plusmn 0026b

MDA (nmolmL) 1708 plusmn 0296a 1881 plusmn 0538a 1747 plusmn 0349a 5199 plusmn 0620b

Values are expressed as means plusmn SE 119899 = 7 for each treatment group a b indicate the significant results statistically 119875 lt 005

264 Agarose Gel Electrophoresis The amplified DNA frag-ments were separated on 15 agarose gel and stained withethidium bromide DNA ladder in range (100 bpndash3000 bp)was used in this study as marker for amplified pattern Theamplified pattern was visualized and photographed by geldocumentation system

27 Statistical Analysis The data entry was done into abinary data matrix as discrete variables and was analyzedaccording to Steel and Torrie [26] Statistical significance ofthe difference in values of control and treated animals wascalculated by (119865) test at 5 significance level Data of thepresent study were statistically analyzed by using Duncanrsquosmultiple range test (SAS 1986) All RAPD profiles wereanalyzed using stat program which showed the similaritybetween the amplified PCR productsThe best amplified PCRproduct of each groupwas selected to compare between themat genetic levels

3 Results

31 Biochemical Parameters The results showed that thetreatment with lead acetate significantly (119875 lt 005) increasedserum AST ALT AlP and LDH compared to the control(Table 2) On the other hand data indicated that the serumtotal proteins and albumin were significantly (119875 lt 005)decreased after lead acetate treatment compared to the con-trol group Meanwhile serum AST ALT AlP total proteinsand albumin were normalized after treatment with either ofthe two doses of Omega-3 (125mgkg or 260mgkg bodyweight) in combination with lead acetate compared to thelead acetate treated group (Table 2) Also Table 2 indicatedthat the levels of serum urea creatinine and MDA weresignificantly (119875 lt 005) increased in the lead acetatetreated rats compared to the control ones reflecting renalimpairment On the other hand treatment with lead acetatesignificantly (119875 lt 005) decreased the activities of GPx CATand SOD and the level of reduced GSH while it increased

MDA level in both liver and kidney extracts compared to thecontrol group (Table 3) Pretreatment of rats with Omega-3 (125 or 260mgkg body weight) prior to and during theinjection with lead acetate ameliorated these parameters toreach the normal level Furthermore the dose of 260mgkgbody weight was more effective than the dose of 125mgkgbody weight in increasing the activities of SOD and GPx inthe extracts of the liver and kidney

The present data indicated that the serum total lipidscholesterol triglycerides and LDL-c were significantly (119875 lt005) increased by lead acetate treatment while HDL-c levelswere decreased (Table 4) The other striking finding in thepresent study is thatOmega-3 at both doses (125 or 260mgkgbodyweight) nearly normalized the lipid profiles in the serumof rats and became similar to the control values (Table 4)

32 Hematological Analysis Hematological parametersrevealed that the Hb and PCV values and the RBCs andPLT counts were significantly decreased (119875 lt 005) in leadacetate treated group compared to the negative control group(Table 5) However the results exhibited that Omega-3 atboth doses (125 or 260mgkg bodyweight) nearly normalizedthe hematological parameters to become similar to thenormal values (Table 5) On the other hand WBCs count inlead acetate treated rats were significantly (119875 lt 005) elevatedas compared with the control group However treatmentwith Omega-3 at a dose of 260mgkg body weight was moreeffective than the other dose in normalizing theWBCs count

33 Histopathological Investigations The histopathologicalstudies of ratsrsquo livers are represented in (Figure 1) The lightmicrographs of liver tissues demonstrated normal hepato-cytes in the control group showed normal hepatic archi-tecture with distinct hepatic cells sinusoidal spaces and acentral vein (Figure 1(a)) while livers of rats treated withlead acetate (group 4) showed loss of cellular architecturewith dilatation of blood sinusoids hemorrhage in the portalvein degenerated hepatocytes with pyknotic nuclei and


Table 3 The effect of Omega-3 and lead acetate on specific activity of liver and kidney antioxidant enzymes in female rats

Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3



LiverGPX (Umg protein) 4053 plusmn 0240a 3654 plusmn 0909a 4850 plusmn 0341a 2244 plusmn 0487b

CAT (Umg protein) 2753 plusmn 0554a 2392 plusmn 0583a 3446 plusmn 0743a 1667 plusmn 0489b

SOD (Umg protein) 1134 plusmn 0355a 1040 plusmn 0346a 1811 plusmn 0495b 764 plusmn 0303c

GSH (Ug tissue) 2784 plusmn 0312a 2608 plusmn 0446a 2916 plusmn 0271a 1272 plusmn 0303b

MDA (nmolg tissue) 5034 plusmn 0259a 5462 plusmn 0288a 5141 plusmn 0282a 10054 plusmn 0322b

KidneyGPX (Umg protein) 3064 plusmn 0521a 2743 plusmn 0597a 3989 plusmn 0479b 2154 plusmn 0360c

CAT (Umg protein) 5317 plusmn 0477a 4697 plusmn 0557a 6330 plusmn 0583b 2041 plusmn 0562c

SOD (Umg protein) 9892 plusmn 0457a 8199 plusmn 0693a 9860 plusmn 0285a 4175 plusmn 0451b


MDA (nmolg tissue) 2219 plusmn 0320a 3760 plusmn 0382b 2294 plusmn 0515a 5182 plusmn 0313c

Values are expressed as means plusmn SE 119899 = 7 for each treatment group a b and c indicate the significant results statistically 119875 lt 005

Table 4 Serum lipid and lipoprotein profiles of female rats after lead exposure and Omega-3 treatment

Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3



TL (mgdL) 20159 plusmn 2215a 17985 plusmn 1879a 15019 plusmn 0745b 40400 plusmn 9398c

TG (mgdL) 9658 plusmn 0706a 10035 plusmn 0671a 9523 plusmn 0686a 13141 plusmn 0541b

Cholesterol (mgdL) 5496 plusmn 1665a 6129 plusmn 0854a 5443 plusmn 1269a 8405 plusmn 2194b

LDLC (mgdL) 2583 plusmn 0438a 2877 plusmn 0431a 1984 plusmn 0399a 4173 plusmn 0470b

HDLC (mgdL) 2914 plusmn 0688a 2875 plusmn 0573a 3019 plusmn 0531a 1817 plusmn 0472b


vacuolated cytoplasm lymphocytes aggregation inside thehepatic tissue as in Figures 1(d1) and 1(d2) However liversof rats treated with lead acetate plus Omega-3 125mgkg(group 2) and rats treated with lead acetate plus Omega-3 260mgkg (group 3) revealed that most of the histolog-ical alterations induced in lead acetate treated groups weremarkedly reduced (Figures 1(b) and 1(c)) Meanwhile leadacetate treatment induced severe histopathological changesin the kidney tissues (Figure 2(d)) such as swelling of con-voluted tubules disruption of Bowmanrsquos capsule shrunkenglomeruli with the capsular space cytoplasmic pyknosisof some nuclei destruction of the epithelium lining thetubules hemorrhagic area in renal tubules and dilation inthe renal tubules compared to normal histological structureOn the other hand the histopathological studies of thekidneys of the control rats revealed normal glomerulussurrounded by the Bowmanrsquos capsule and proximal anddistal convoluted tubules without any inflammatory changes(Figure 2(a)) Treatment with Omega-3 at both doses beforeand in combination with lead acetate slightly improved thekidney histology but extravasation of blood element withdilation of some proximal and distal tubules was still present

as well as presence of some glomeruli with the capsular space(Figures 2(b) and 2(c))

34 Genetic Analysis Using RAPD-PCR Three of 10-merprimers were used for investigating the significant changes ofthe DNA isolated from liver tissues The three primers pro-duced clear sharp monomorphic and polymorphic bandsas in Figures 3 4 and 5 Primer 1 gave band patterns ofalmost the same profile between the three amplified samplesof each group so it did not clarify any difference (Figure 3)In contrast the other primers (primer 2 and primer 3) weremost informative and they produced reproducible and themost distinguishable banding profiles between the amplifiedsamples of each group afterRAPDassays as in Figures 4 and 5The amplified fragments of PCR products were summarizedas in Table 6 Primers 2 and 3 produced highly similar RAPDfingerprints for negative control group (group 1) and groups2 and 3 while they detected some changes in hepatic DNAof lead acetate treated group (group 4) We observed similarRAPD-PCR fingerprint using primer 2 in all 12 samples fromthe different groups as in Figure 3 In Figure 4 the amplifiedRAPD products of group 4 using primer 2 lost some bands


Table 5 Changes in hematological parameters of female rats treated after lead exposure and Omega-3 treatment

Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3



Hb (gdL) 1474 plusmn 0070a 1370 plusmn 0155 a 1431 plusmn 0107a 967 plusmn 0153b

PCV () 4114 plusmn 0388a 4025 plusmn 0503a 4284 plusmn 0618a 3186 plusmn 0346b

RBCs (times1012 Lminus1) 567 plusmn 0133a 497 plusmn 0174a 596 plusmn 0162a 374 plusmn 0056b

WBCs (times109 Lminus1) 397 plusmn 0196a 498 plusmn 0084ab 390 plusmn 0076a 596 plusmn 0130b

PLT (times1012 Lminus1) 25514 plusmn 8250a 21757 plusmn 3518a 26829 plusmn 9551a 11657 plusmn 8352b

Values are expressed asmeansplusmn SE 119899 = 7 for each treatment group a b indicate the significant results statistically while abmay be significant or not significant119875 lt 005

CV

HS

(a)

HS

L

(b)

CVL

HS

(c)

(d2)

L

(d1)

CV

Figure 1 Paraffin sections stained by haematoxylin and eosin (HampEtimes200) for histopathological examination of liver tissues of rats as followscontrol group (a) showing normal hepatocytes architecture (H) central vein (CV) and normal blood sinusoids (S) (b) liver tissue of group2 (lead acetate + Omega-3 125mgkg body weight) (c) liver tissue of group 3 (lead acetate + Omega-3 260mgkg body weight) showinghistological alterations induced by lead acetate that were markedly reduced in groups 2 and 3 Liver tissue of group 4 (lead acetate treatedrats) (d1 and d2) showing distended and hemorrhage in the portal vein ( ) loss of the normal architecture degenerated hepatocytes withpyknotic nuclei (◻) and degenerated hepatocytes with vacuolated cytoplasm ( ) Condensed nuclei ( ) and lymphocytes aggregation(L) inside the hepatic tissue


P

S G

D

(a)

S

G

D

D

P

(b)

DP

G

P

S

(c)

SG D

P

(d)

Figure 2 Paraffin sections stained by haematoxylin and eosin (HampE times200) for histopathological examination of the kidney tissue of ratstreated as follows control group (a) lead acetate plus Omega-3 125mgkg body weight group (b) lead acetate plus Omega-3 260mgkgbody weight group (c) Kidney tissue of lead acetate treated rats (d) showing disruption of Bowmanrsquos capsule shrunken glomeruli G with thecapsular space S and cytoplasmic pyknosis of some nuclei ( ) The degenerative changes in the epithelial cells lining the renal tubules (◻)hemorrhagic area ( ) in renal tubules and dilation in the renal tubules are compared to normal histological structure of the glomerulusand tubules in control group (a) Histological alterations induced in lead acetate at both doses of Omega-3 groups (b) and (c) were markedlyreduced

(1) (2) (3)(1) (2) (3)(1) (2) (3)(1) (2) (3)M

(bp)

gp1 gp1 gp1 gp2 gp2 gp2 gp3 gp3 gp3 gp4 gp4 gp4

1000

900800

700600500

400

300

200

100

1500

3000

Figure 3 Pattern RAPD-PCR (primer 1) of hepatic DNA samplesexposed to lead acetate and treatedwith two doses of Omega-3 usingprimer 1TheDNA ladder is in lane (M) lanes (gp1) represent group1 (minusve control) lanes (gp2) group 2 (Omega-3 with dose 125mgkgbody weight and lead acetate) lanes (gp3) group 3 (Omega-3 withdose 260mgkg body weight and lead acetate) and lanes (gp4)group 4 (+ve group)

of their three samples compared to the samples of negativecontrol group and treated groups with Omega-3 Moreoverthe pattern showed a smear in the beginning of lanes 1 and3 of group 4 The PCR products using primer 2 gave obviousresults through the bands profile which were begun to appearat about 900 bp while the patterns of other groups werestarted at about 1350 bp In addition the RAPD-PCR usingprimer 3 did not amplify the first sample of group 4 (lane 1of group 4) while lanes 2 and 3 of group 4 lost their bands atapproximately 1650 bp compared to other groups as shown inFigure 5The amplification products obtained by thismethodshowed the presence of numerous bands from 250 to 1300 bpwith primer 1 300 to 1500 bp with primer 2 and 250 to1700 bp with primer 3 respectively (Figures 3 4 and 5)

The RAPD products were scored as present (1) orabsent (0) for each primer-genotype combinationThe resultsof RAPD patterns of the 3 primers were summarized asin Table 6 Thirty-six bands were scored where 33 werepolymorphic and 3 of them were monomorphic Jaccardrsquoscoefficient of similarity was measured and a dendrogram(Figure 6) based on similarity coefficients was generated


Table 6 Random primers showing polymorphism of DNA from liver of the four groups

Primer code Nucleotidesequence 51015840 rarr 31015840

Total number ofamplified fragments

Number ofmonomorphicfragments

Number ofpolymorphicfragments

Fragments sizerange (bp)

1 GTC CAT GCCA 13 2 11 250ndash13002 ACA TCG CCCA 12 1 11 300ndash15003 ATG CCC CTG T 11 0 11 250ndash1700Total 36 3 33

(1) (2) (3)(1) (2) (3)(1) (2) (3)(1) (2) (3)M gp1 gp1 gp1 gp2 gp2 gp2 gp3 gp3 gp3 gp4 gp4 gp4

1000

900800

700

600

500

400

300

200

100

1500

3000

(bp)

Figure 4 Pattern RAPD-PCR (primer 2) of hepatic DNA samplesexposed to lead acetate and treatedwith two doses of Omega-3 usingprimer 2TheDNA ladder is in lane (M) lanes (gp1) represent group1 (minusve control) lanes (gp2) group 2 (Omega-3 with dose 125mgkgbody weight and lead acetate) lanes (gp3) group 3 (Omega-3 withdose 260mgkg body weight and lead acetate) and lanes (gp4)group 4 (+ve group) Arrows indicate loss of some amplificationproducts of different groups


1000

900

800

700600

500

400

300

200

100

1500

3000

(bp)


Tree diagram for 4 variablesunweighted pair group average

percent disagreement

gp4 gp3 gp2 gp1005

010

015

020

025

030

Link

age

dist

ance

Figure 6 Dendrogram of the four applied groups generated byUPGMA based on 3 RAPD primers where gp1 is group 1 gp2 isgroup 2 gp3 is group 3 and gp4 is group 4

Table 7 Agreement percentage of RAPD profile

gp1 gp2 gp3 gp4gp1 100 81 75 64gp2 81 100 78 72gp3 75 78 100 89gp4 64 72 89 100

by using unweighted pair group method with arithmeticmean (UPGMA) The best amplified PCR was selected fromeach group to compare between them using stat softwareThe analysis of the results described the similarity betweendifferent samples of liver tissues (Table 7) The similarity ofpositive control (group 4) and treated group with Omega-3(group 2) was about 64 and 81 respectively comparedto negative control (group 1) The variations of the RAPDprofiles of treated Omega-3 groups were compared to thenegative and positive control groups

4 Discussion

Lead has been known to be an environmental pollutant andits toxicity has also been associated with health hazards [8]The liver acts as chief player in detoxification process and isone of the target organs affected by lead toxicity owing toits storage in the liver Data shown in Table 2 demonstratedthat treatment with lead acetate caused a significant elevation


in the activities of liver enzymes AST ALT ALP and LDHin serum confirming the histological damage shown in theliver (Figure 1) The present results revealed a significantincrease in ALT AST and ALP in serum of lead acetatetreated rats compared with negative control group Theseresults are in agreement with the results of Herman et al[27] Ibrahim et al [28] and Mehana et al [29] Howeverthe activities of LDH were significantly elevated in serumof lead acetate treated rats and this result is similar to thatof Ibrahim et al [28] The increasing of LDH in serum oflead acetate treated group may be due to spill out of thisenzyme from the liver cytosol into the blood stream andorliver dysfunction and disturbance in the biosynthesis of thisenzymewith alteration in the permeability of livermembraneaccording to Yousef [30] Also Gaskill et al [31] reported thatreleasing of AST ALT and LDH from the cell cytosol canoccur as secondary changes to cellular necrosis In additionsignificant decrease in the total proteins and albumin in theserum of lead acetate treated group was compared to controlgroup Liver synthesizes proteins among which is albuminand the decrease in the total proteins and albumin levels inliver could be attributed to changes in protein and free aminoacids metabolism and their synthesis in the liver [31] Thisadverse effectmight be caused by the interference of leadwithprotein synthesis or by the binding of lead to some metal-binding proteins and their removal through detoxificationprocesses [30] In an attempt to clarify the mechanisminvolved it has been reported that lead caused a disruption inprotein andRNA synthesis Also the observed decrease in thetotal proteins and albumin in the liver could be attributed tothe damaging effect of lead acetate on liver cells as confirmedby increasing in the activities of serumASTandALT (Table 2)after treatment of rats with lead acetate [32] In the currentstudy the induced elevation of albumin urea and creatininedue to lead acetate administration indicated that the kidneyfunction was affected In addition lead acetate caused asignificant elevation in serum urea and creatinine reflectingrenal impairment that is coinciding with histological damageof the kidney as shown in Figure 2 [33 34] It can beconcluded that oxidative damages may be the primary causeof lead toxicity leading to lipid peroxidation and cellulardamage Thus the obvious change in liver and kidneyfunctions is related to the intensity of cellular damage It hasbeen shown that lead acetate undergoes metabolism in livervia esoteric and oxidative pathways generating elevatedMDAlevels that lead to hepatic necrosis [31] The increased levelsof MDA in the present study are associated with a reducedlevel of GSH and increased activities of serum enzymeswhichindicated the occurrence of an oxidative insult that causedhepatic and renal damage Moreover the toxicity with leadacetate in rats of group 4 leads to depletion of GPx CAT andSOD enzymes activities in liver and kidney (Tables 2 and 3)and these results are matching with the results which wereachieved in a previous study [10] The possible explanationcould be related to the proposed role of GSH in the activeexcretion of lead through bile by binding to the thiol groupof GSH and then being excreted A decrease in GSH levelscould lead to oxidative stress and a consequent increase inlipid peroxidation [7] The presence of lipid peroxidation

was observed in the current study due to decrease of SODand CAT activities [10] Enzymes such as GPx CAT andSOD may contribute to the explanation of the mechanismsresponsible for the decrease in GSH concentration in liverand kidney due to the exposure to this heavy metal [22]

The chemoprotective effect of Omega-3 on liver tissuewas confirmed by the attenuation of the activities of serumALT AST ALP and LDH in addition to the normalization ofserum protein and albumin contents (Table 2) These resultsare consistent with the results of Attaia et al [35] The modeof action of Omega-3 can be intercepted pharmacologically atdifferent levels with agents that scavenge free reactive oxygenblock their generation or enhance endogenous antioxidantcapabilities [35]

The current results also indicated that treatment withOmega-3 decreased the level of MDA associated with anelevation in SOD and CAT activities as well as in GSHcontent in groups 2 and 3 The decrease in the MDAlevel by Omega-3 may be due to its antioxidant propertiesthat inhibited lipid peroxidation [36] and this action helpsstabilize the reactive radicals preserve the cellular integrityand restrain the severity of lead acetate GSH plays a keyrole in many cellular processes involving protection of cellsagainst oxidative stress xenobiotics and radiation and it isabundant with low molecular weight intracellular thiol [37]In our study Omega-3 prevented the decrement of GSHlevel suggesting that Omega-3 may protect the SH group ofGSH from the reactive radicals that are produced from leadacetate toxicity Similarly Attaia and Nasr [38] found thatOmega-3 could maintain normal levels of SOD and CATactivities The antioxidant and anti-inflammatory effects ofOmega-3 through scavenging of free radicals and inhibitinglipid peroxidation have been reported previously by Pauwelsand Kostkiewicz [36] This oxidantantioxidant theory mayexplain the protective role of Omega-3 fatty acids against thehepatotoxicity and nephrotoxicity of lead acetate

In the present study a significant increase in serum totallipids cholesterol triglycerides and LDL-c and a significantdecrease of HDL-c of the rats treated with lead acetate wereestimated (Table 4) HDL-c helps to scavenge cholesterolfrom extrahepatic tissues and the decrease of HDL-c concen-tration as in this study contributed to increasing cholesterollevels There is evidence linking increased serum cholesteroland LDL-c levels to a higher risk for developing coronaryheart diseases [39] The present results exhibited that therewas a significant decrease in serum total lipids cholesteroltriglycerides and LDL-c and a significant increase of HDL-cof the rats treated animals with Omega-3 in groups 2and 3 compared to lead acetate treated group (Table 4)Devasagayamet al [40] suggested that oxidativemodificationof low-density lipoproteins (LDL-c) caused by reactive oxy-gen species results in the formation of foam cells which is theinitial lesion of atherosclerosisThey also reported that LDL-coxidation and atherogenesis can be inhibited by nutritionalantioxidants There are also epidemiological evidences andinterventional studies to correlate higher level of antioxidant-rich food uptake with lower incidence of coronary heartdisease [40]


The results of the present study demonstrated that leadacetate administration to female rats resulted in significantdecrease of Hb PCV RBCs and platelet count (PLT) ofthe rats treated with lead acetate in contrast to those in thenegative control rats (Table 5) On the other hand WBCscount of lead acetate treated rats was elevated compared tothe negative control group and these results are in agreementwith those described by Kim et al [41] and Simsek et al[42] However Topashka-Ancheva et al [43] showed thatlead could damage the erythrocytes membrane resultingin hemolysis or decrease of blood iron level which maybe the reason of decreasing the concentration of Hb andPCV These hematological alterations might be also due tothe effect of lead on the activity of 120575-aminolevulinic aciddehydrogenase which acts as key enzyme of heme synthesisPrevious study reported that lead inhibits the conversionof coproporphyrinogen III to protoporphyrin IX leading toreduction in Hb production and shortening of life span oferythrocytes [44]The results obtained in this study indicatedthat Omega-3 successfully maintained normal haematolog-ical parameters against the toxicity induced by lead acetatein female rats Our data are in accordance with previousresults which reported that supplementing rats with differentdoses of Omega-3 showed appreciable improvement in thehaematological indices as evidenced by significant increasein Hb PCV and RBC counts and decrease in WBC counts[45]

Figures 1(a) 1(b) and 1(c) showed normal cellular archi-tecture with distinct hepatic cells sinusoidal spaces and acentral vein that were observed in the negative control groupand Omega-3 at both doses-treated groups Figure 1((d1)and (d2)) showed that lead acetate treatment induced severehistopathological alterations in liver most of the intrahepaticblood vessels especially the central veins were dilated andcongested In addition the hepatocytes lost their normalarchitecture and vacuolization with pyknotic nuclei appearedin the cytoplasm These results are in agreement with theresults of Abdel-Moneim et al [46] Our histological inves-tigations of renal tissue revealed that Pb-acetate treatmentresults in progressive glomerular and tubular alterationsThese findings are in agreement with the results of Abdel-Moneim et al [34] Omega-3 treatment caused a significantdecrease in the histopathological changes induced by leadacetate in the liver and the kidney (Figures 1 and 2) andpartially restored these changes in lead acetate plus Omega-3treated groups

RAPD and arbitrarily primed polymerase chain reactiontechnique (AP-PCR) are powerful tools for gene mappingpopulation pedigree analysis phylogenetic studies andstrain identification [47] In addition their use in surveyinggenomic DNA for evidence of various types of damage andmutation suggests that they may potentially form the basisof novel genotoxicological assays for the detection of DNAdamage and mutations [48]

Previous studies have shown that changes in band pat-terns observed in DNA ldquofingerprintrdquo analyses reflect DNAalterations from single base changes (point mutations) tocomplex chromosomal rearrangements [49 50] In this studyDNA damage induced by heavy metals was reflected by

changes in RAPD profiles disappearance of bands andappearance of new PCR products which occurred in theprofiles generated by exposed rats to lead acetateThe presentdata showed that the RAPD-PCR method is useful for thescreening and characterization of genomic regions that haveundergone alterations as the result of lead acetate exposureSeveral similar findings have been reported by Castanoand Becerril [51] and Liu et al [52] that used RAPD-PCRto analyze the induced DNA damage However randomamplified polymorphism of DNA (RAPD) showed distinctdifferences in animal groups exposed to lead acetate (group4) and treated with Omega-3 (groups 2 and 3) at both doses(125 and 260mgkg body weight) Also RAPD reflected theprotective effect of Omega-3 on DNA These results wereconsistent with those obtained by Elelaimy et al [53] whoreported that Omega-3 pre-posttreatment to azathioprineshowed high significance in reducing the percentage of DNAfragmentation compared to azathioprine treated mice

5 Conclusion

In this study the effect of lead acetate as one of the haz-ardous heavy metals was studied using biochemical testshistopathological study and genomic analysis showed thehigh risk of lead toxicity through the exposure to leadacetate Omega-3 acts as antioxidant compound and hasprotective and treatment effect versus lead toxicity so itshould be tested on other heavy metals and environmentaltoxic compounds The biochemical analysis confirmed thefree radical scavenging properties of Omega-3 as antioxidantcompounds as well as the ability of Omega-3 to improveliver and kidney functions and haematological parametersRAPD-PCR technique proved that it is a useful and effectivetechnique to study the DNA damage due to lead toxicitythrough the mutation of DNA which can be studied throughthe absence or intensity of different pattern bands So thepresent results indicated that coadministration of Omega-3 had protective role against hepatotoxicity renal toxicityhaematotoxicity and genotoxicity induced by lead acetate

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] M N Chatterjee and S Rana ldquoMetabolism of minerals andtrace elementsrdquo in Textbook of Medical Biochemistry pp 544ndash545 700ndash701 Jaypee Brothers Medical New Delhi India 5thedition 2002

[2] M Markowitz ldquoLead poisoningrdquo Pediatrics in Review vol 21no 10 pp 327ndash335 2000

[3] E Courtois M Marques A Barrientos S Casado and ALopez-Farre ldquoLead-induced downregulation of soluble guany-late cyclase in isolated rat aortic segments mediated by reactiveoxygen species and cyclooxygenase-2rdquo Journal of the AmericanSociety of Nephrology vol 14 no 6 pp 1464ndash1470 2003


[4] S Rahman and S Sultana ldquoChemopreventive activity of gly-cyrrhizin on lead acetate mediated hepatic oxidative stress andits hyperproliferative activity inWistar ratsrdquoChemico-BiologicalInteractions vol 160 no 1 pp 61ndash69 2006

[5] C M Bolin R Basha D Cox et al ldquoExposure to lead and thedevelopmental origin of oxidative DNA damage in the agingbrainrdquo FASEB Journal vol 20 no 6 pp 788ndash790 2006

[6] A-R H Farrag K A Mahdy G H Abdel Rahman and MM Osfor ldquoProtective effect of Nigella sativa seeds against lead-induced hepatorenal damage in male ratsrdquo Pakistan Journal ofBiological Sciences vol 10 no 17 pp 2809ndash2816 2007

[7] A A El-Nekeety A A El-Kady M S Soliman N S Hassanand M A Abdel-Wahhab ldquoProtective effect of Aquilegia vul-garis (L) against lead acetate-induced oxidative stress in ratsrdquoFood and Chemical Toxicology vol 47 no 9 pp 2209ndash22152009

[8] I A S Al-Saleh ldquoThe biochemical and clinical consequences oflead poisoningrdquo Medicinal Research Reviews vol 14 no 4 pp415ndash486 1994

[9] J Dressier K-A Kim T Chakraborti and G GoldsteinldquoMolecular mechanisms of lead neurotoxicityrdquo NeurochemicalResearch vol 24 no 4 pp 595ndash600 1999

[10] L Patrick ldquoLead toxicity part II the role of free radical damageand the use of antioxidants in the pathology and treatment oflead toxicityrdquoAlternativeMedicine Review vol 11 no 2 pp 114ndash127 2006

[11] O Ademuyiwa R N Ugbaja S O Rotimi et al ldquoErythro-cyte acetylcholinesterase activity as a surrogate indicator oflead-induced neurotoxicity in occupational lead exposure inAbeokuta Nigeriardquo Environmental Toxicology and Pharmacol-ogy vol 24 no 2 pp 183ndash188 2007

[12] G Calviello and S Serini Dietary Omega-3 PolyunsaturatedFatty Acids and Cancer Springer London UK 2010

[13] I A A Shaikh I Brown K W J Wahle and S D HeysldquoEnhancing cytotoxic therapies for breast and prostate cancerswith polyunsaturated fatty acidsrdquoNutrition and Cancer vol 62no 3 pp 284ndash296 2010

[14] P C Calder ldquoPolyunsaturated fatty acids and inflammatoryprocesses new twists in an old talerdquo Biochimie vol 91 no 6pp 791ndash795 2009

[15] R Wall R P Ross G F Fitzgerald and C Stanton ldquoFattyacids from fish the anti-inflammatory potential of long-chainomega-3 fatty acidsrdquo Nutrition Reviews vol 68 no 5 pp 280ndash289 2010

[16] I J Edwards and J T OFlaherty ldquoOmega-3 fatty acids andPPAR 120574 in cancerrdquo PPAR Research vol 2008 Article ID 35805214 pages 2008

[17] W-H Sun G-S Chen X-L Ou et al ldquoInhibition of COX-2and activation of peroxisome proliferator-activated receptor 120574synergistically inhibits proliferation and induces apoptosis ofhuman pancreatic carcinoma cellsrdquo Cancer Letters vol 275 no2 pp 247ndash255 2009

[18] M Wendel and A R Heller ldquoAnticancer actions of omega-3fatty acidsmdashcurrent state and future perspectivesrdquo Anti-CancerAgents in Medicinal Chemistry vol 9 no 4 pp 457ndash470 2009

[19] B Koletzko and O Goulet ldquoFish oil containing intravenouslipid emulsions in parenteral nutrition-associated cholestaticliver diseaserdquo Current Opinion in Clinical Nutrition andMetabolic Care vol 13 no 3 pp 321ndash326 2010

[20] R G Fassett G C Gobe J M Peake and J S CoombesldquoOmega-3 polyunsaturated fatty acids in the treatment of

kidney diseaserdquo American Journal of Kidney Diseases vol 56no 4 pp 728ndash742 2010

[21] A Saravana Kumar K Bhagya Deepthi M Devi Vara PrasadP Grace Mary S Sujeeth Kumar and M Swathi ldquoEvalua-tion of the protective effects of omega-3 fatty acids againstmethotrexate induced testicular toxicity in male albino micerdquoInternational Journal of Phytopharmacology vol 2 no 2 pp 48ndash52 2011

[22] J C Ponce-Canchihuaman O Perez-Mendez R Hernandez-Munoz P V Torres-Duran and M A Juarez-Oropeza ldquoPro-tective effects of Spirulina maxima on hyperlipidemia andoxidative-stress induced by lead acetate in the liver and kidneyrdquoLipids in Health and Disease vol 9 article 35 2010

[23] D Bancroft and M Gamble The Theory and Practice of Histo-logical Techniques Churchil Living Stone 5th edition 2002

[24] F Bardakci and D O F Skibinski ldquoApplication of the RAPDtechnique in tilapia fish species and subspecies identificationrdquoHeredity vol 73 no 2 pp 117ndash123 1994

[25] J G KWilliams M K Hanafey J A Rafalski and S V TingeyldquoGenetic analysis using random amplified polymorphic DNAmarkersrdquoMethods in Enzymology vol 218 pp 704ndash740 1993

[26] GD Steel and JH TorriePrinciples andProcedures of StatisticsMcGraw-Hill BookCompanyNewYorkNYUSA 2nd edition1981

[27] D S Herman M Geraldine and T Venkatesh ldquoInfluence ofminerals on lead-induced alterations in liver function in ratsexposed to long-term lead exposurerdquo Journal of HazardousMaterials vol 166 no 2-3 pp 1410ndash1414 2009

[28] N M Ibrahim E A Eweis H S El-Beltagi and Y E Abdel-Mobdy ldquoEffect of lead acetate toxicity on experimental malealbino ratrdquo Asian Pacific Journal of Tropical Biomedicine vol 2no 1 pp 41ndash46 2012

[29] E E Mehana A R M A Meki and K M Fazili ldquoAmelioratedeffects of green tea extract on lead induced liver toxicity in ratsrdquoExperimental and Toxicologic Pathology vol 64 no 4 pp 291ndash295 2012

[30] M I Yousef ldquoAluminium-induced changes in hemato-biochemical parameters lipid peroxidation and enzymeactivities of male rabbits protective role of ascorbic acidrdquoToxicology vol 199 no 1 pp 47ndash57 2004

[31] C L Gaskill L M Miller J S Mattoon et al ldquoLiverhistopathology and liver and serum alanine aminotransferaseand alkaline phosphatase activities in epileptic dogs receivingphenobarbitalrdquoVeterinary Pathology vol 42 no 2 pp 147ndash1602005

[32] H M Abdou and A A Newairy ldquoHepatic and reproductivetoxicity of lead in female rats and attenuation by flaxseedlignansrdquo Journal of Medical Research Institute vol 27 pp 295ndash302 2006

[33] S H Garba A B Adelaiye and L Y Mshelia ldquoHistopatho-logical and biochemical changes in the rats kidney followingexposure to a pyrethroid based mosquito coilrdquo Journal ofApplied Sciences Research vol 3 no 12 pp 1788ndash1793 2007

[34] A E Abdel-Moneim M A Dkhil and S Al-Quraishy ldquoThepotential role of flaxseed oil on lead acetateinduced kidneyinjure in adult male Albino ratsrdquo African Journal of Biotechnol-ogy vol 10 no 8 pp 1436ndash1451 2011

[35] AM Attia S G El-Banna F R Nomeir andM I A El-BasserldquoLindane-induced biochemical perturbations in rat serum andattenuation by omega-3 and Nigella sativa seed oilrdquo IndianJournal of Biochemistry and Biophysics vol 48 no 3 pp 184ndash190 2011


[36] E K J Pauwels and M Kostkiewicz ldquoFatty acid facts part IIIcardiovascular disease or a fish diet is not fishyrdquoDrugNews andPerspectives vol 21 no 10 pp 552ndash561 2008

[37] H R Asaad and F M Aziz ldquoProtective role of omega-3 fish oilagainst the toxicity of ifosfamide in male ratsrdquo Jordan Journal ofBiological Sciences vol 5 no 1 pp 37ndash346 2012

[38] A M Attia and H M Nasr ldquoDimethoate-induced changesin biochemical parameters of experimental rat serum and itsneutralization by black seed (Nigella sativa L) oilrdquo SlovakJournal Animal Science vol 42 no 2 pp 87ndash94 2009

[39] A-S A Newairy and H M Abdou ldquoProtective role of flaxlignans against lead acetate induced oxidative damage andhyperlipidemia in ratsrdquo Food and Chemical Toxicology vol 47no 4 pp 813ndash818 2009

[40] T P A Devasagayam J C Tilak K K Boloor K S Sane SS Ghaskadbi and R D Lele ldquoFree radicals and antioxidants inhuman health current status and future prospectsrdquo Journal ofAssociation of Physicians of India vol 52 pp 794ndash804 2004

[41] R Kim A Rotnitzky D Sparrow S T Weiss C Wagerand H Hu ldquoA longitudinal study of low-level lead exposureand impairment of renal function the normative aging studyrdquoJournal of the AmericanMedical Association vol 275 no 15 pp1177ndash1181 1996

[42] N Simsek A Karadeniz Y Kalkan O N Keles and BUnal ldquoSpirulina platensis feeding inhibited the anemia- andleucopenia-induced lead and cadmium in ratsrdquo Journal ofHazardous Materials vol 164 no 2-3 pp 1304ndash1309 2009

[43] M Topashka-Ancheva R Metcheva and S Teodorova ldquoBioac-cumulation and damaging action of polymetal industrial duston laboratory mice Mus musculus alba II Genetic cell andmetabolic disturbancesrdquo Environmental Research vol 92 no 2pp 152ndash160 2003

[44] C D Klassen Casarett and Doullrsquos ToxicologyThe Basic Scienceof Poisons McGraw-Hill Medical Publishing Division 6thedition 2001

[45] J I Ndem M I Akpanabiatu and E U Essien ldquoEffects ofseafoods (Periwinkle Bonkafish and Crayfish) and vegetableoils enriched meal on cardiovascular diseaserdquo Pakistan Journalof Nutrition vol 7 no 4 pp 603ndash606 2008

[46] A E Abdel-Moneim M A Dkhil and S Al-Quraishy ldquoTheredox status in rats treated with flaxseed oil and lead-inducedhepatotoxicityrdquo Biological Trace Element Research vol 143 no1 pp 457ndash467 2011

[47] T H Grayson F A Atienzar S M Alexander L F Cooper andM L Gilpin ldquoMolecular diversity of Renibacterium salmoni-narum isolates determined by randomly amplified polymorphicDNA analysisrdquo Applied and Environmental Microbiology vol66 no 1 pp 435ndash438 2000

[48] A Shimada and A Shima ldquoCombination of genomic DNAfingerprinting into the medaka specific- locus test systemfor studying environmental germ-line mutagenesisrdquo MutationResearch vol 399 no 2 pp 149ndash165 1998

[49] J J White H Neuwirth C Dennis Miller and E L SchneiderldquoDNA alterations in prostatic adenocarcinoma and benignprostatic hyperplasia detection by DNA fingerprint analysesrdquoMutation Research vol 237 no 1 pp 37ndash43 1990

[50] F A Atienzar M Conradi A J Evenden A N Jha andM H Depledge ldquoQualitative assessment of genotoxicity usingrandom amplified polymorphic DNA comparison of genomictemplate stabilitywith key fitness parameters inDaphniamannaexposed to benzo (a) pyrenerdquo Environmental Toxicology andChemistry vol 18 pp 2275ndash2282 1990

[51] A Castano and C Becerril ldquoIn vitro assessment of DNAdamage after short- and long-term exposure to benzo(a)pyreneusing RAPD and the RTG-2 fish cell linerdquoMutation ResearchmdashFundamental and Molecular Mechanisms of Mutagenesis vol552 no 1-2 pp 141ndash151 2004

[52] W Liu P J Li XMQi et al ldquoDNAchanges in barley (Hordeumvulgare) seedlings induced by cadmium pollution using RAPDanalysisrdquo Chemosphere vol 61 no 2 pp 158ndash167 2005

[53] I A Elelaimy S A Elfiky A M Hassan H M Ibrahim andR I Elsayad ldquoGenotoxicity of anticancer drug Azathioprine(Imuran) role of omega-3 (120596-3) oil as protective agentrdquo Journalof Applied Pharmaceutical Science vol 2 no 4 pp 14ndash23 2012

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


found to play protective roles in the liver cardiovascularsystem and kidney and they have beenwidely used in clinicalperoperative total parenteral nutrition [19 20]Therefore thepresent study was carried out to investigate the protectiveeffects ofOmega-3 FAs against lead acetate-induced oxidativestress biochemical changes and DNA damage

2 Materials and Methods

21 Chemicals Lead acetate was purchased from Merck(Germany)

Omega-3 was purchased from Efamol Ltd 14 The MoleBusiness Park Leatherhead Surrey KT227BA UK in theSouth East of England All other chemical materials that wereused in this study were purchased from Sigma Chemical Co(St Louis MO USA)

22 Animals and Experimental Design Twenty-eight adultWistar albino female rats (weighting 170ndash200 gm) wereobtained from the animal house of Faculty of MedicineAlexandria University Egypt The local committee approvedthe design of the experiments and the protocols were carriedout according to the guidelines of the National Institutesof Health (NIH) Rats were housed in stainless steel cagesplaced in a well-ventilated rat house maintained for twoweeks as acclimatization period under standard laboratoryconditions on free supply of food and water provided adlibitum and subjected to natural light for 12 hrs and dark for12 hrs cycles After the period of acclimatization rats weredivided randomly into four groups 7 animals in each Theanimal experiments were conducted for 10 days Group 1 wasinjected daily with 05mL of saline solution (09 NaCl) ipfor 10 days and was used as negative control (minusve) Groups 2and 3 were administrated orally with two doses of Omega-3(125 and 260mgkg body weight resp) by gavage for the firstfive days as protective agent [21] These groups were injected(ip) by 05mL of lead acetate at a dose of 25mgkg bodyweightday for the other 5 days in combination with Omega-3 Group 4 was injected (ip) by 05mL lead acetate only ata dose of 25mgkg body weightday for the last 5 days of theexperiment and was used as positive control (+ve) accordingto Ponce-Canchihuaman et al [22]

23 Blood Collection and Tissue Preparation At the end oftreatment rats fasted for 12 hrs before being anesthetized andsacrificed by cervical dislocation Blood samples were col-lected from the sacrificed animals and left in refrigerator for30min before centrifugation The clear nonhemolyzed serawere stored at minus20∘C till measurements However heparinwas used as an anticoagulant and noncoagulated blood wastested shortly after collection for applying in determinationof hemoglobin (Hb) packed cells volume (PCV) red bloodcells (RBCs) count white blood cells (WBCs) and platelets(PLT) count by particle counter (ERMA Inc Tokyo modelPCE-210)

Liver and kidney were immediately removed and washedusing chilled saline solution These tissues were minced

Table 1 PCR primers used in RAPD-PCR GC and annealingtemperature

Primers Sequence 51015840 rarr 31015840 GC Annealing temperature (∘Csec)1 GTC CAT GCCA 60 30602 ACA TCG CCCA 60 30603 ATG CCC CTG T 60 3060

and separately homogenized (10wv) using a homoge-nizer (Potter-Elvehjem) in ice-cold sodium potassium phos-phate buffer (001M pH 74) containing 115 of KCl Thehomogenates were centrifuged at 10000timesg for 20min at 4∘Cand the supernatant was used for assaying of the enzymesactivities

24 Biochemical Analysis Stored serum samples were ana-lyzed for the activities of aspartate aminotransferase (ASTEC 2611) alanine aminotransferase (ALT EC 2612) alka-line phosphatase (AlP EC 3131) and lactate dehydroge-nase (LDH EC 11127) which were determined using kitsfrom Sentinel Ch (via principle Eugenio 5-20155 MilanItaly) Also serum total protein albumin urea creatininecholesterol total lipids triglycerides HDLC and low den-sity lipoprotein (LDLC) were determined using kits fromSentinel Ch (via principle Eugenio 5-20155 Milan Italy)The lipid peroxidation end product MDA was measuredas thiobarbituric acid reactive substance Also the levels ofGSH and the activities of antioxidant enzymes including thecatalase enzyme (CAT EC 11116) superoxide dismutase(SOD EC11511) and glutathione peroxidase (GPx EC1119) were assayed using commercial assay kits accordingto the manufacturerrsquos instructions

25 Histopathology Specimens of liver tissues were immedi-ately fixed in 10 formalin treatedwith conventional grade ofalcohol and xylol embedded in paraffin and sectioned at 4ndash6 120583 thickness The sections were stained with Haematoxylinand Eosin (HampE) stain for studying the histopathologicalchanges [23]

26 Random Amplified Polymorphic DNA Technique (RAPD)

261 Extraction of DNA DNA was extracted from livers ofthe four groups following the method described by Bardakciand Skibinski [24]

262 Polymerase Chain Reaction (PCR) Primers In thepresent work ten-base long oligonucleotides primers wereused to initiate the PCR amplifications Primers were ran-domly selected on the basis of GC content and annealingtemperature for RAPD-PCR amplification as in Table 1

263 PCR Amplification and Agarose Gel ElectrophoresisPCR amplifications were performed according to the proce-dure described byWilliams et al [25] using the isolated DNAfrom three hepatic samples of each group



Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3















3 Results








Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3















Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3














Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3









CV

HS

(a)

HS

L

(b)

CVL

HS

(c)

(d2)

L

(d1)

CV



P

S G

D

(a)

S

G

D

D

P

(b)

DP

G

P

S

(c)

SG D

P

(d)


(1) (2) (3)(1) (2) (3)(1) (2) (3)(1) (2) (3)M

(bp)


1000

900800

700600500

400

300

200

100

1500

3000













1000

900800

700

600

500

400

300

200

100

1500

3000

(bp)



1000

900

800

700600

500

400

300

200

100

1500

3000

(bp)




gp4 gp3 gp2 gp1005

010

015

020

025

030

Link

age

dist

ance





4 Discussion














5 Conclusion




References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3















3 Results








Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3















Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3














Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3









CV

HS

(a)

HS

L

(b)

CVL

HS

(c)

(d2)

L

(d1)

CV



P

S G

D

(a)

S

G

D

D

P

(b)

DP

G

P

S

(c)

SG D

P

(d)


(1) (2) (3)(1) (2) (3)(1) (2) (3)(1) (2) (3)M

(bp)


1000

900800

700600500

400

300

200

100

1500

3000













1000

900800

700

600

500

400

300

200

100

1500

3000

(bp)



1000

900

800

700600

500

400

300

200

100

1500

3000

(bp)




gp4 gp3 gp2 gp1005

010

015

020

025

030

Link

age

dist

ance





4 Discussion














5 Conclusion




References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3















Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3














Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3









CV

HS

(a)

HS

L

(b)

CVL

HS

(c)

(d2)

L

(d1)

CV



P

S G

D

(a)

S

G

D

D

P

(b)

DP

G

P

S

(c)

SG D

P

(d)


(1) (2) (3)(1) (2) (3)(1) (2) (3)(1) (2) (3)M

(bp)


1000

900800

700600500

400

300

200

100

1500

3000













1000

900800

700

600

500

400

300

200

100

1500

3000

(bp)



1000

900

800

700600

500

400

300

200

100

1500

3000

(bp)




gp4 gp3 gp2 gp1005

010

015

020

025

030

Link

age

dist

ance





4 Discussion














5 Conclusion




References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Parameters

Experimental groups


(Group 2)Omega-3


(Group 3)Omega-3









CV

HS

(a)

HS

L

(b)

CVL

HS

(c)

(d2)

L

(d1)

CV



P

S G

D

(a)

S

G

D

D

P

(b)

DP

G

P

S

(c)

SG D

P

(d)


(1) (2) (3)(1) (2) (3)(1) (2) (3)(1) (2) (3)M

(bp)


1000

900800

700600500

400

300

200

100

1500

3000













1000

900800

700

600

500

400

300

200

100

1500

3000

(bp)



1000

900

800

700600

500

400

300

200

100

1500

3000

(bp)




gp4 gp3 gp2 gp1005

010

015

020

025

030

Link

age

dist

ance





4 Discussion














5 Conclusion




References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


P

S G

D

(a)

S

G

D

D

P

(b)

DP

G

P

S

(c)

SG D

P

(d)


(1) (2) (3)(1) (2) (3)(1) (2) (3)(1) (2) (3)M

(bp)


1000

900800

700600500

400

300

200

100

1500

3000













1000

900800

700

600

500

400

300

200

100

1500

3000

(bp)



1000

900

800

700600

500

400

300

200

100

1500

3000

(bp)




gp4 gp3 gp2 gp1005

010

015

020

025

030

Link

age

dist

ance





4 Discussion














5 Conclusion




References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of










1000

900800

700

600

500

400

300

200

100

1500

3000

(bp)



1000

900

800

700600

500

400

300

200

100

1500

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4 Discussion














5 Conclusion




References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













5 Conclusion




References





























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Protective role of omega-3 polyunsaturated fatty acid against lead acetate-induced toxicity in liver...

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Transcript of Protective role of omega-3 polyunsaturated fatty acid against lead acetate-induced toxicity in liver...