Research Article Antigenotoxic Effect of Curcumin and

Research ArticleAntigenotoxic Effect of Curcumin and Carvacrolagainst Parathion Induced DNA Damage in Cultured HumanPeripheral Blood Lymphocytes and Its Relation toGSTM1 and GSTT1 Polymorphism

Neeraj Kumar1 Anita Yadav1 Sachin Gulati1 Kanupriya1

Neeraj Aggarwal2 and Ranjan Gupta3

1 Department of Biotechnology Kurukshetra University Kurukshetra Haryana 136119 India2Department of Microbiology Kurukshetra University Kurukshetra Haryana 136119 India3 Department of Biochemistry Kurukshetra University Kurukshetra Haryana 136119 India

Correspondence should be addressed to Anita Yadav ayadavkukacin

Received 29 June 2014 Revised 2 September 2014 Accepted 2 September 2014 Published 25 September 2014

Academic Editor Lucio Guido Costa

Copyright copy 2014 Neeraj Kumar et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In recent years the use of organophosphorus pesticides has been extensively increased and these compounds signify a major classof agricultural pesticides today We studied antigenotoxic potential of curcumin and carvacrol against the parathion induced DNAdamage in cultured peripheral blood lymphocytes using sister chromatid exchanges as a biomarker of genotoxicity Heparinisedfresh blood from healthy individuals was treated with 25 120583gmL concentration of parathion in presence of curcumin and carvacrolin order to observe the antigenotoxic potential of both curcumin and carvacrol Significant reduction (119875 lt 005) was observed inthe frequencies of SCEs in presence of 10120583gmL and 15120583gmL concentrations of curcumin as compared to parathion exposedsample Similarly carvacrol had significant (119875 lt 005) antigenotoxic effect at the concentrations of 25120583gmL and 50 120583gmLagainst the parathion We also studied the effect of GSTT1 and GSTM1 on genotoxicity of parathion and antigenotoxic potential ofcurcumin and carvacrol We did not observe any significant effect (119875 gt 005) of GSTT1 and GSTM1 polymorphism on genotoxicityof parathion and antigenotoxic potential of curcumin and carvacrol

1 Introduction

Pesticides are ubiquitous contaminants of our environmentand are extensively used all over the world They includea vast diversity of substances used to kill destroy or repelunwanted living organisms In recent years public concernshave been raised about the potential risks of pesticides tohuman health In our study we investigated the protectiverole of curcumin and carvacrol against the DNA damagecaused by parathion in cultured human peripheral bloodusing sister chromatid exchanges (SCEs) as a biomarker ofDNA damage Parathion is one of the most commonly usedorganophosphorus insecticides It has been used for beancorn sorghum and tobacco crops to eliminate the green

flies harvest bugs and other insects It hinders the enzymecholinesterase responsible for hydrolyzing the acetylcholineto choline at the axon-terminals [1] In absence of activecholinesterase acetylcholine is accumulated and blocks thenormal transmission of nerve impulses at the synapse whichultimately leads to loss ofmuscular coordination convulsionand death [2 3] Parathion has been reported to inducetesticular damage in male rats [4]

Nutraceuticals are defined as any natural bioactivechemical compounds that give medical or health benefitsincluding the prevention and treatment of disease Curcumin(diferuloylmethane) a polyphenol is the active ingredientof the dietary spice turmeric (Curcuma longa) and has beenconsumed for medicinal purposes for thousands of years [5]

Hindawi Publishing CorporationJournal of ToxicologyVolume 2014 Article ID 404236 7 pageshttpdxdoiorg1011552014404236

2 Journal of Toxicology

which has persuasive anticancer properties as demonstratedin a plethora of human cancer cell linesanimal carcinogene-sis model It acts as a free radical scavenger and antioxidant[6] inhibiting lipid peroxidation and oxidative DNA damage[7] Curcumin can significantly decrease the frequencies ofmicronucleated polychromatic erythrocytes in mice exposedto gamma-radiation [8]

Carvacrol (5-isopropyl-2-methylphenol) used in thisstudy is monoterpenic phenol which is present in manyessential oils of the family Labiatae including OriganumSaturejaThymbraThymus and Coridothymus species and isused in our daily life such as cosmetic ingredient safe foodadditive in baked goods sweets beverages and chewing gumIt has been shown to exhibit a range of biological activities likeantibacterial [9] antifungal insecticidal [10] analgesic [11]and antioxidant [12] activities In the recent past it is revealedthat carvacrol has antiproliferative properties on non-smallcell lung cancer cells A549 chronic myeloid leukemia cellsK562 Hep-2 cells murine B16 melanoma cells and humanmetastatic breast cancer cells MDA-MB231 [13ndash17]

Molecular epidemiological studies have revealed thatindividualrsquos susceptibility to mutagen depends on bothgenetic and environmental factorsThe hereditary differencesin the detoxificationactivation of carcinogens play a crucialrole in host vulnerability Glutathione S-transferases (GSTs)are one among the most frequently studied polymorphismsregarding the metabolism of xenobiotics The GSTM1 andGSTT1 are members of the glutathione S-transferase multi-gene family and are mostly concerned with the detoxificationof a broad range of environmental carcinogens endogenouslyproduced reactive oxygen species (ROS) and lipid peroxi-dation products yielding excretable hydrophilic metabolites[18] There are only a few studies showing the effect ofgenetic polymorphism of GSTM1 andGSTT1 on genotoxicityof parathion We studied the effect of GSTM1 and GSTT1polymorphism on genotoxicity of parathionantigenotoxicityof curcumin and carvacrol as measured by SCE frequencyin cultured peripheral blood lymphocytes under in vitroconditions

2 Materials and Methods

21 Sample Collection 5mL venous blood was taken fromhealthy individuals in two separate vacutainer tubes con-taining sodium heparin and dipotassium ethylenediaminetetraacetic acid (EDTA) for lymphocyte culture set-upand DNA extraction respectively The protocol was dulyapproved by Human Ethical Committee of Kurukshetra Uni-versity

22 Human Lymphocyte Culture Short term peripheralblood lymphocyte (PBL) cultures were set up using ear-lier studied technique of Moorhead et al [19] with minormodifications Culture was set up in duplicate by adding(04mL) whole heparinized blood into 5mL of RPMI 1640culture medium (Himedia) containing L-glutamine (1)fetal calf serum (20) (Himedia) penicillin (100UImL) and

streptomycin (100120583gmL) solution (Himedia) and phyto-haemagglutinin (2) (Bangalore genei) The cultures wereincubated at 37∘C and 5 CO

2for 72 hours

23 Sister Chromatid Exchange (SCE) For sister chromatidexchange (SCE) analysis 5-bromo-2-deoxyuridine (Sigma)was added after 24 hours of incubation in final concentrationof 10 120583gmL of culture Parathion (Sigma) was added at thebeginning of culture in concentrations ranging from 05to 5 120583gmL Maximum genotoxic dose of parathion thatis 25 120583gmL was chosen to check the protective effect ofcurcumin and carvacrol (Sigma) To check antigenotoxicpotential of curcumin and carvacrol against parathion cul-tures were set up separately having various combinationsof parathion and curcumincarvacrol In one set-up hep-arinised fresh blood was treated with 25120583gmL concentra-tion of parathion along with 10 and 15 120583gmL concentrationsof curcuminwhile in others 25 and 50120583gmL concentrationsof carvacrol were added against 25 120583gmL concentration ofparathion Combined effect of both curcumin and carvacrolwas also checked against parathion Blood was also treatedwith curcumin and carvacrol alone to check their genotoxiceffects if any Blood without any mutagencurcumin andcarvacrol acted as control while blood having dimethylsulphoxide was taken as negative control The cultures werethen incubated for 72 hrs at 37∘C and 5 CO

2 Colchicine

(Sigma) was added 45 minutes prior to the harvesting infinal concentration of 02 120583gmL The cells were harvestedby centrifugation and then treated with hypotonic solution(0075M KCl) and fixed in methanol acetic acid (3 1) Froma suspension of fixed cells slides were prepared by the airdrying method and stained with Hoechst 33258 (Sigma) and4 Giemsa stain (Himedia) solution following the methodof Perry and Wolff For calculating the frequency of SCE percell 50 metaphase plates were analyzed

24 GSTM1 and GSTT1 Genotyping Genomic DNA wasextracted from 200 120583L of whole blood by DNA extractionkit (Bangalore genei) Multiplex PCR was used to detect thepresence or absence of GSTM1 and GSTT1 genes For thepurpose of internal control a part of exon 7 of the constitu-tional gene CYP1A1 was also coamplified The amplificationreaction was carried out in a 25 120583L volume containing 50ndash100 ng of genomic DNA as a template 20 pmol120583L of eachprimer (GenXbio) 200120583M of each dNTP (Bangalore genei)1X PCR buffer with 15mML MgCl

2(Bangalore genei) and

07 units of Taq polymerase (Bangalore genei) PCR wasperformed by using reaction mixture with primers GSTM1(Fw 51015840-GAACTCCCTGAAAAGCTAAAGC-31015840 and Re 51015840-GTTGGGCTCAAATATACGGTGG-31015840) GSTT1 (Fw 51015840-TTCCTTACTGGTCCTCACATCTC-31015840 Re 51015840-TCACGG-GATCATGGCCAGCA-31015840) and CYP1A1 (Fw 51015840-GAACTG-CCACTTCAGCTGTCT-31015840 and Re 51015840-CAGCTGCATTTG-GAAGTGCTC-31015840) yielding a 312-bp product Initial denatu-ration was done at 94∘C for 10min A total of 35 temperaturecycles were used 94∘C at 1min 59∘C for 30 sec and 72∘Cfor 1min The last elongation step was extended to 10minsat 72∘CThe PCR products were analyzed in 2 agarose gel

Journal of Toxicology 3

25 Statistical Analysis All treatments were performed induplicate and results were expressed as means plusmn SE TheStudentrsquos 119905-test was used for calculating the statistical signifi-cance using SPSS 160

3 Results

31 SCE Results We studied the protective effects of cur-cumin and carvacrol against parathion induced genotoxicityin cultured peripheral blood lymphocytes Sister chromatidexchanges (SCEs) were examined to assess the genotoxicityof parathion and antigenotoxic potential of curcumin andcarvacrol An increased frequency of SCEs was observed inparathion treated sample as compared to untreated sample(Figure 1) Parathion was observed alone for its mutagenicityby using various concentrations Among different concen-trations of parathion 25 120583gmL concentration had shownsignificant increase in SCE as compared to untreated sample(Table 1 and Figure 2) and this concentration was chosenfurther for analyzing antigenotoxic potential of curcumin andcarvacrol

Antigenotoxic effect of curcumin and carvacrol was anal-ysed by reduction in SCE frequency in presence of parathionCurcumin at the concentrations of 10 and 15 120583gmL hadsignificantly reduced the SCE frequency as comparedto parathion treatment (Table 2 and Figure 3) Similarlycarvacrol had protective effect at the concentrations 25 and50 120583gmL (Table 3 and Figure 4) Both curcumin and car-vacrol were also analysed for any genotoxic effect in absenceof parathion None of these were observed to be genotoxicCombined effect of both curcumin and carvacrol was alsostudied (Table 4 and Figure 5) It was found that reduction inSCE frequencies was higher with combined treatment of cur-cumin and carvacrol as compared to their alone treatmentHowever this reduction was not found to be significant

32 Effect of Genetic Polymorphism of GSTM1 and GSTT1 onGenotoxicity of Parathion and Antigenotoxicity of Curcuminand Carvacrol Individuals respond differently to environ-mental chemicals due to differences in their genotypesMultiplex PCR was used to detect the presence or absenceof GSTM1 and GSTT1 genotypes The occurrence of GSTM1and GSTT1 genes was detected by the presence or absenceof a band at 215 and 480 bp respectively We observed theeffect of GSTM1 and GSTT1 polymorphism on genotoxicityof parathion and antigenotoxicity of curcumin and carvacrolIt was found that individuals which are nonnull for bothGSTM1 and GSTT1 genes have less genotoxicity in presenceof parathion as compared to those who either are nullfor both GSTM1 and GSTT1 genes or have either one ofthem (Table 5) However genotoxicity was not found tobe significant Similarly curcumin was found to be moreantigenotoxic in individuals having GSTM1 and GSTT1nonnull genotypes as compared to those having GSTM1 andGSTT1 null genotypes However this antigenotoxic effect wasnot found to be significant

Our study supports that curcumin and carvacrol havepossible protective effects against the parathion while their

SCEs

Untreated sample Parathion treated sample

Figure 1 SCEs shown in untreated and parathion treated sample

555643

799

1033 1086

00

2

4

6

8

10

12

0 05 1 2 25 5SC

Ece

ll

Parathion treatment (microgmL)

SCEcell

lowast

Figure 2 Induction of SCE in cultured lymphocytes by parathionlowast

119875 lt 005 (significance as compared to untreated)

537

01234567

SCE

cell

Treatments

SCEcell

Con

trol

P (25120583

gm

L)

Cu (1

0120583

gm

L)

Cu (1

5120583

gm

L)

DM

SO(minus

veco

ntro

l)

(25+10

) (120583

gm

L)

(25+15

) (120583

gm

L)lowast

P+

Cu

P+

Cu

Figure 3 Reduction in SCE frequency by curcumin against para-thion treated cultured human lymphocytes lowast119875 lt 005 (significanceas compared to untreated) P parathion Cu curcumin and DMSOdimethyl sulphoxide

combined treatment did not show any significant reductionin frequencies of SCEs as compared to their separate treat-ments It was also observed that there is not any significanteffect of GSTT1 and GSTM1 polymorphism on parathioninduced genotoxicity and antigenotoxic effect of curcuminand carvacrol


563

01234567

SCE

cell

Treatments

SCEcell

Con

trol

P (25120583

gm

L)

(25+25

) (120583

gm

L)

(25+50

) (120583

gm

L)

Ca (2

5120583

gm

L)

Ca (5

0120583

gm

L)

DM

SO(minus

veco

ntro

l)

lowast

P+

Ca

P+

Ca

Figure 4 Ameliorative effect of carvacrol against parathion treatedcultured human lymphocytes lowast119875 lt 005 (significance as comparedto untreated) P parathion Ca carvacrol and DMSO dimethylsulphoxide

Treatments (microgmL)

SCEcell

0

1

2

3

4

5

6

7

SCE

cell

Con

trol

P (25

)

P+

Cu(25+10)

P+

Cu(25+15)

P+

Ca(25+25)

P+

Ca(25+5)

P+

Cu+

Ca(25+10

+25)

P+

Cu+

Ca(25+15

+50)

Figure 5 Simultaneous effects of curcumin and carvacrol in SCEassay in human lymphocytes against parathion lowastP parathion Cucurcumin and Ca carvacrol

Table 1 Frequency of SCEcell in cultured human lymphocytestreated with parathion

Parathiontreatment (120583gmL) Metaphase scored SCEcell plusmn SE

Untreated 50 555 plusmn 010

05 50 643 plusmn 034

1 50 799 plusmn 025

2 50 1033 plusmn 032

25 50 1086 plusmn 040a

50 50 No differentiationa119875 lt 005 (significance as compared to untreated)

4 Discussion

In present study we investigated the protective effect ofcurcumin and carvacrol and their combination against

Table 2 Antigenotoxic effect of curcumin against parathion treatedcultured human lymphocytes

Treatments Concentrations used(120583gmL + 120583gmL)

Metaphasescored SCEcell plusmn SE

Control Untreated 50 233 plusmn 016

Parathion 25 50 537 plusmn 036a

Parathion +curcumin 25 + 10 50 459 plusmn 038

b


b

Curcumin 10 50 241 plusmn 017c


DMSO (minusvecontrol) 20 50 232 plusmn 017

d

a119875 lt 005 (significance as compared to untreated) b119875 lt 005 (significant ascompared to parathion treatment) c119875 gt 005 (nonsignificance as comparedto untreated) and d

119875 gt 005 (nonsignificance as compared to untreated)

Table 3 Protective effect of carvacrol against parathion treatedcultured human peripheral blood lymphocytes





Parathion +carvacrol 25 + 25 50 433 plusmn 012

b


b

Carvacrol 25 50 256 plusmn 019c



d



the genotoxic damage caused by parathion using sister chro-matid exchange (SCE) as a biomarker of genotoxicity Sisterchromatid exchange (SCE) is a more sensitive indicator ofgenotoxic effects [20] We observed the dose dependentincreases in the frequency of SCEs by parathion at concentra-tions ranges from 05 to 50 120583gmL with maximum damageat 25 120583gmL Parathion is reported to be genotoxic in ourstudy as supported by the literature also Sandra et al [21]reported that human lymphocytes treated with 0 to 10 ppmconcentration of parathion had significantly increased theSCE frequency in a dose dependent manner However at13 ppm cells failed to differentiate Methyl parathion wasalso found genotoxic to humanperipheral blood lymphocytesunder in vitro conditions [22] Rojas-Garcıa et al [23]reported the dose dependent increase in micronucleus inperipheral human blood lymphocytes with paraoxon (anactive metabolite of parathion) They treated the peripheralhuman blood lymphocytes with range of 1ndash25 120583M concentra-tions of paraoxon and found dose dependent DNA damage


Table 4 Combined effect of curcumin and carvacrol against parathion treated cultured human peripheral blood lymphocytes

Treatments Concentrations used(120583gmL + 120583gmL) Metaphase scored SCEcell plusmn SE



Parathion + curcumin 25 + 10 50 459 plusmn 038b


Parathion + carvacrol 25 + 25 50 433 plusmn 012b


Parathion + curcumin + carvacrol 25 + 10 + 25 50 508 plusmn 041c


a119875 lt 005 (significance as compared to untreated)

b119875 lt 005 (significant as compared to parathion treatment)

c119875 gt 005 (nonsignificant as compared to curcumin and carvacrol alone treatment)

Table 5 Effect of GSTT1 and GSTM1 polymorphism on antigenotoxicity of curcumin and carvacrol against parathion induced genotoxicity

Genotype Parathion (25 120583gmL) Parathion (25 120583gmL) +curcumin (10120583gmL)

Parathion (25 120583gmL) +carvacrol (25 120583gmL)

Relationship with GSTT1GSTT1 (nonnull) 590 plusmn 041 450 plusmn 035 426 plusmn 021

GSTT1 (null) 527 plusmn 038 385 plusmn 022a

422 plusmn 010a

Relationship with GSTM1GSTM1 (nonnull) 593 plusmn 085 436 plusmn 038 430 plusmn 015

GSTM1 (null) 600 plusmn 055 433 plusmn 066b

443 plusmn 031b

Relationship with both GSTT1 and GSTM1GSTT1 (nonnull)GSTM1 (nonnull) 480 plusmn 016 380 plusmn 017 420 plusmn 020

GSTT1 (null) GSTM1(null) 545 plusmn 044 402 plusmn 0034

c415 plusmn 025

c

GSTT1 (nonnull)GSTM1 (null) 610 plusmn 040 435 plusmn 050 500 plusmn 017

GSTT1 (null) GSTM1(nonnull) 650 plusmn 044 465 plusmn 045

d426 plusmn 012

d

a119875 gt 005 (nonsignificance as compared to GSTT1 nonnull genotypes)

b119875 gt 005 (nonsignificance as compared to GSTM1 nonnull genotypes)

c119875 gt 005 (nonsignificance as compared to GSTT1 and GSTM1 nonnull genotypes)

d119875 gt 005 (nonsignificance as compared to GSTT1 nonnull and GSTM1 null genotypes)

Chen et al [24] reported the increases in frequency ofSCE in Chinese hamster V79 cell line treated with methylparathion They treated the cells with 10 20 and 40 120583gmLconcentrations of methyl parathion at the time intervals of28 34 and 72 hours They found that methyl parathionincreases the SCE frequency in a dose dependent manner asthe concentration increases from 10 to 40 120583gmL Maximumgenotoxic damage was observed at 40 120583gmL concentrationof methyl parathion

Nowadays curcumin is appearing as a promising chemo-preventive compound able to reverse inhibit or preventthe development of cancer by inhibiting specific molecularsignaling pathways involved in carcinogenesis [25ndash28] It iswell known for its antioxidant property [29 30] Its freeradical scavenging antioxidant property helps it in reducing

genotoxic damage Antimutagenic property of carvacrol hasnot been well studied yet [31] Its antimutagenic effect maybe due to its antioxidant nature [12] Genotoxic potential ofcarvacrol was found to be very weak in both Ames and DNA-repair test [32]

In our study we observed that 10 and 15 120583gmL ofcurcumin have significantly reduced the genotoxic dam-age caused by parathion which supports its antigenotoxicproperty We also observed that carvacrol had protectiveeffect against parathion at the concentrations of 25 and50 120583gmL supporting its antigenotoxic activities Combina-tion of curcumin and carvacrol was also analysed againstthe parathion Curcumin and carvacrol in combination havereduced the frequencies of SCEs as compared to parathionbut results were not significant in comparison to their


separate treatments As in our study some other workershave also reported the antigenotoxic effects of curcumin andcarvacrol Under in vitro conditions curcumin is reported toreduce the clastogenic effects of gamma-radiation in humanlymphocytes culture Lymphocytes pretreated with curcuminexposed to 1 and 2Gy (SI unit of absorbed dose of radiations)of gamma-radiation resulted in decreased frequency of SCEsas compared to untreated lymphocytes [33] Curcumin at thedoses of 5 10 and 15 120583M had shown the dose dependentdecrease in SCEscell against 10 120583gmL concentration oftinidazole [34] Carvacrol was reported to inhibit the rate ofSCE induced by mitomycin C [35] Ultee et al [36] reportedthat the antimutagenic activity of carvacrol in B cereus IFR-NL94-25 against parathion might be due to alteration inmembrane lipids and permeability of ion channels thusinhibiting the uptake of parathion into the cells

Individuals have different responses to environmentalchemicals due to their different genotypes In the presentstudy we studied the effect of GSTT1 and GSTM1 polymor-phism on genotoxicity of parathion and the antigenotoxiceffect of curcumin and carvacrol We found no significanteffect of GSTM1 and GSTT1 polymorphism on parathioninduced genotoxicity and antigenotoxicity of curcumin andcarvacrol under in vitro conditions Similar to our findingsGuven et al [37] also reported that GSTM1 genotype didnot influence the SCEs and CAs induced in vitro by BaPLack of the GSTM1 gene has no influence on inductionof micronuclei by BaP in human lymphocyte cultures [38]Kumar et al [39] had reported no significant relationshipbetweenCYP1A1GSTM1GSTT1 andGSTP1 polymorphismand genotoxicity of trichloroethylene under both in vivo andin vitro conditions To the best of our knowledge there areno studies regarding the antigenotoxic effect of curcumin andcarvacrol against parathion induced genotoxicity in humanperipheral blood lymphocytes under in vitro conditionsOursis the first report Our findings suggest that curcumin andcarvacrolmay be administered in diet as a preventivemeasureagainst common genotoxicants and may further be usedfor the development of safer medication against impacts ofcarcinogens

Conflict of Interests

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

Acknowledgments

This work was proposed and guided by Dr Anita YadavThe work was carried out in Animal Biotechnology Labo-ratory Department of Biotechnology Kurukshetra Univer-sity Kurukshetra (Haryana) India The financial supportprovided in the form of Junior Research Fellow (JRF) byDepartment of Biotechnology (DBT) Government of Indiagranted to Mr Neeraj Kumar is fully acknowledged Theauthors are thankful to all blood donors for their usefulcontribution in the research

References

[1] M Lotti ldquoClinical toxicology of anticholinesterase agents inhumansrdquo in Handbook of Pesticide Toxicology R Krieger Edvol 2 pp 1043ndash1085 Academic Press San Diego Calif USA2nd edition 2001

[2] A R Erdman R C Dart E M Caravati andM A McGuiganldquoInsecticidesrdquo inMedical Toxicology pp 1475ndash1496 LippincottWilliams ampWilkins Philadelphia Pa USA 3rd edition 2004

[3] R F Clark ldquoInsecticides organic phosphorus compounds andcarbamatesrdquo inGoldfrankrsquos Toxicological Emergencies pp 1346ndash1360 McGraw-Hill Professional New York NY USA 7thedition 2002

[4] M Uzunhisarcikli Y Kalender K Dirican S Kalender AOgutcu and F Buyukkomurcu ldquoAcute subacute and sub-chronic administration of methyl parathion-induced testiculardamage in male rats and protective role of vitamins C and ErdquoPesticide Biochemistry and Physiology vol 87 no 2 pp 115ndash1222007

[5] S C Gupta S PatchvaW Koh and B B Aggarwal ldquoDiscoveryof curcumin a component of golden spice and its miraculousbiological activitiesrdquo Clinical and Experimental Pharmacologyand Physiology vol 39 no 3 pp 283ndash299 2012

[6] T Ak and I Gulcin ldquoAntioxidant and radical scavengingproperties of curcuminrdquo Chemico-Biological Interactions vol174 no 1 pp 27ndash37 2008

[7] M Srinivasan N P Rajendra and V P Menon ldquoProtectiveeffect of curcumin on 120574-radiation induced DNA damage andlipid peroxidation in cultured human lymphocytesrdquo MutationResearch Genetic Toxicology and Environmental Mutagenesisvol 611 no 1-2 pp 96ndash103 2006

[8] S K Abraham L Sarma and P C Kesavan ldquoProtectiveeffects of chlorogenic acid curcumin and 120573-carotene against120574-radiation-induced in vivo chromosomal damagerdquo MutationResearch Letters vol 303 no 3 pp 109ndash112 1993

[9] J-G Choi O-H Kang Y-S Lee et al ldquoAntibacterial activity ofmethyl gallate isolated from galla rhois or carvacrol combinedwith nalidixic acid against nalidixic acid resistant bacteriardquoMolecules vol 14 no 5 pp 1773ndash1780 2009

[10] A Ultee L G M Gorris and E J Smid ldquoBactericidal activityof carvacrol towards the food-borne pathogen Bacillus cereusrdquoJournal of Applied Microbiology vol 85 no 2 pp 211ndash218 1998

[11] K H C Baser ldquoBiological and pharmacological activities ofcarvacrol and carvacrol bearing essential oilsrdquoCurrent Pharma-ceutical Design vol 14 no 29 pp 3106ndash3119 2008

[12] M Puertas-Mejıa S Hillebrand E Stashenko and P Win-terhalter ldquoIn vitro radical scavenging activity of essential oilsfromColumbian plants and fractions from oregano (Origanumvulgare L) essential oilrdquo Flavour and Fragrance Journal vol 17no 5 pp 380ndash384 2002

[13] A T Koparal and M Zeytinoglu ldquoEffects of carvacrol on ahuman non-small cell lung cancer (NSCLC) cell line A549rdquoCytotechnology vol 43 no 1ndash3 pp 149ndash154 2003

[14] I Lampronti A M Saab and R Gambari ldquoAntiproliferativeactivity of essential oils derived from plants belonging to theMagnoliophyta divisionrdquo International Journal of Oncology vol29 no 4 pp 989ndash995 2006

[15] S Karkabounas O K Kostoula T Daskalou et al ldquoAnticar-cinogenic and antiplatelet effects of carvacrolrdquo ExperimentalOncology vol 28 no 2 pp 121ndash125 2006

[16] E Horvathova V Turcaniova andD Slamenova ldquoComparativestudy of DNA-damaging andDNA-protective effects of selected


components of essential plant oils in human leukemic cellsK562rdquo Neoplasma vol 54 no 6 pp 478ndash483 2007

[17] K M Arunasree ldquoAnti-proliferative effects of carvacrol ona human metastatic breast cancer cell line MDA-MB 231rdquoPhytomedicine vol 17 no 8-9 pp 581ndash588 2010

[18] Z Salehi L Gholizadeh H Vaziri and A H Madani ldquoAnalysisof GSTM1 GSTT1 and CYP1A1 in idiopathic male infertilityrdquoReproductive Sciences vol 19 no 1 pp 81ndash85 2012

[19] P S Moorhead P C Nowell W J Mellman D M Battips andD A Hungerford ldquoChromosome preparations of leukocytescultured from human peripheral bloodrdquo Experimental CellResearch vol 20 no 3 pp 613ndash616 1960

[20] S Neuss and G Speit ldquoFurther characterization of the genotox-icity of formaldehyde in vitro by the sister chromatid exchangetest and co-cultivation experimentsrdquoMutagenesis vol 23 no 5pp 355ndash357 2008

[21] G A Sandra N A Norma J R G Dolores and V P RafaelldquoSister chromatid exchanges induced by the organophospho-rus insecticides methyl parathion dimethoate phoxim andmethyl Azinfos in cultured human lymphocytesrdquo Contami-nacion Ambiental vol 3 pp 63ndash70 1987

[22] U Undeger and N Basaran ldquoEffects of pesticides on humanperipheral lymphocytes in vitro induction of DNA damagerdquoArchives of Toxicology vol 79 no 3 pp 169ndash176 2005

[23] A E Rojas-Garcıa M Sordo L Vega B Quintanilla-VegaM Solis-Heredia and P Ostrosky-Wegman ldquoThe role of par-aoxonase polymorphisms in the induction of micronucleusin paraoxon-treated human lymphocytesrdquo Environmental andMolecular Mutagenesis vol 50 no 9 pp 823ndash829 2009

[24] H H Chen J L Hsueh S R Sirianni and C C HuangldquoInduction of sister-chromatid exchanges and cell cycle delay inculturedmammalian cells treatedwith eight organophosphoruspesticidesrdquoMutation Research vol 88 no 3 pp 307ndash316 1981

[25] H Hatcher R Planalp J Cho F M Torti and S V TortildquoCurcumin from ancient medicine to current clinical trialsrdquoCellular andMolecular Life Sciences vol 65 no 11 pp 1631ndash16522008

[26] A Goel A B Kunnumakkara and B B Aggarwal ldquoCurcuminas ldquoCurecuminrdquo from kitchen to clinicrdquo Biochemical Pharma-cology vol 75 no 4 pp 787ndash809 2008

[27] B B Aggarwal I D Bhatt H Ichikawa et al ldquoCurcuminbiological and medicinal propertiesrdquo in Turmeric The GenusCurcuma vol 45 pp 297ndash368 CRC Press New York NY USA2007

[28] R LThangapazham A Sharma andR KMaheshwari ldquoMulti-ple molecular targets in cancer chemoprevention by curcuminrdquoTheAAPS Journal vol 8 no 3 article 52 pp E443ndashE449 2006

[29] I Chattopadhyay K Biswas U Bandyopadhyay and R KBanerjee ldquoTurmeric and curcumin biological actions andmedicinal applicationsrdquoCurrent Science vol 87 no 1 pp 44ndash532004

[30] A Galano R Alvarez-Diduk M T Ramırez-Silva G Alarcon-Angeles and A Rojas-Hernandez ldquoRole of the reacting freeradicals on the antioxidant mechanism of curcuminrdquo ChemicalPhysics vol 363 no 1ndash3 pp 13ndash23 2009

[31] M Zeytinoglu S Aydin Y Ozturk K Husnu and C BaserldquoInhibitory effects of carvacrol on DMBA induced pulmonarytumorigenesis in ratsrdquo Acta Pharmaceutica Turcica vol 40 no2 pp 93ndash98 1998

[32] A Stammatia P Bonsia F Zuccob R Moezelaarc H LAlakomid and A von Wrightd ldquoToxicity of selected plant

volatiles in microbial and mammalian short-termrdquo Food andChemical Toxicology vol 37 no 8 pp 813ndash823 1999

[33] E S AlSuhaibani ldquoProtective effect of curcumin on gamma-radiation-induced sister chromatid exchanges in human bloodlymphocytesrdquo International Journal of Low Radiation vol 6 no1 pp 21ndash27 2009

[34] YH Siddique G Ara T Beg andMAfzal ldquoProtective effect ofcurcumin against the genotoxic damage induced by tinidazolein cultured human lymphocytesrdquo Acta Pharmaceutica Scienciavol 52 no 1 pp 23ndash30 2010

[35] E Ipek B A Tuylu and H Zeytinoglu ldquoEffects of carvacrolon sister chromatid exchanges in human lymphocyte culturesrdquoCytotechnology vol 43 no 1ndash3 pp 145ndash148 2003

[36] A Ultee M H J Bennik and R Moezelaar ldquoThe phenolichydroxyl group of carvacrol is essential for action against thefood-borne pathogenBacillus cereusrdquoApplied andEnvironmen-tal Microbiology vol 68 no 4 pp 1561ndash1568 2002

[37] G S Guven M Guven I Onaran T Ulutin and S Haci-hanefioglu ldquoThe effect of glutathione S-transferaseM1 genotypeon benzo[120572] pyrene-induced sister chromatid exchanges andchromosomal aberrations in peripheral blood lymphocytesrdquoTurkish Journal of Medical Sciences vol 35 no 2 pp 79ndash842005

[38] I Onaran G Guven A Ozaydin and T Ulutin ldquoThe influenceof GSTM1 null genotype on susceptibility to in vitro oxidativestressrdquo Toxicology vol 157 no 3 pp 195ndash205 2001

[39] M Kumar S Tewari P Sharma et al ldquoStudy of genetic poly-morphism in solvent exposed population and its correlation toin vitro effect of trichloroethylene on lymphocytesrdquo Journal ofEnvironmental Biology vol 30 no 5 pp 685ndash691 2009

Submit your manuscripts athttpwwwhindawicom

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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Drug DeliveryJournal of



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Autoimmune Diseases


Anesthesiology Research and Practice

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Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


which has persuasive anticancer properties as demonstratedin a plethora of human cancer cell linesanimal carcinogene-sis model It acts as a free radical scavenger and antioxidant[6] inhibiting lipid peroxidation and oxidative DNA damage[7] Curcumin can significantly decrease the frequencies ofmicronucleated polychromatic erythrocytes in mice exposedto gamma-radiation [8]

Carvacrol (5-isopropyl-2-methylphenol) used in thisstudy is monoterpenic phenol which is present in manyessential oils of the family Labiatae including OriganumSaturejaThymbraThymus and Coridothymus species and isused in our daily life such as cosmetic ingredient safe foodadditive in baked goods sweets beverages and chewing gumIt has been shown to exhibit a range of biological activities likeantibacterial [9] antifungal insecticidal [10] analgesic [11]and antioxidant [12] activities In the recent past it is revealedthat carvacrol has antiproliferative properties on non-smallcell lung cancer cells A549 chronic myeloid leukemia cellsK562 Hep-2 cells murine B16 melanoma cells and humanmetastatic breast cancer cells MDA-MB231 [13ndash17]

Molecular epidemiological studies have revealed thatindividualrsquos susceptibility to mutagen depends on bothgenetic and environmental factorsThe hereditary differencesin the detoxificationactivation of carcinogens play a crucialrole in host vulnerability Glutathione S-transferases (GSTs)are one among the most frequently studied polymorphismsregarding the metabolism of xenobiotics The GSTM1 andGSTT1 are members of the glutathione S-transferase multi-gene family and are mostly concerned with the detoxificationof a broad range of environmental carcinogens endogenouslyproduced reactive oxygen species (ROS) and lipid peroxi-dation products yielding excretable hydrophilic metabolites[18] There are only a few studies showing the effect ofgenetic polymorphism of GSTM1 andGSTT1 on genotoxicityof parathion We studied the effect of GSTM1 and GSTT1polymorphism on genotoxicity of parathionantigenotoxicityof curcumin and carvacrol as measured by SCE frequencyin cultured peripheral blood lymphocytes under in vitroconditions

2 Materials and Methods

21 Sample Collection 5mL venous blood was taken fromhealthy individuals in two separate vacutainer tubes con-taining sodium heparin and dipotassium ethylenediaminetetraacetic acid (EDTA) for lymphocyte culture set-upand DNA extraction respectively The protocol was dulyapproved by Human Ethical Committee of Kurukshetra Uni-versity

22 Human Lymphocyte Culture Short term peripheralblood lymphocyte (PBL) cultures were set up using ear-lier studied technique of Moorhead et al [19] with minormodifications Culture was set up in duplicate by adding(04mL) whole heparinized blood into 5mL of RPMI 1640culture medium (Himedia) containing L-glutamine (1)fetal calf serum (20) (Himedia) penicillin (100UImL) and

streptomycin (100120583gmL) solution (Himedia) and phyto-haemagglutinin (2) (Bangalore genei) The cultures wereincubated at 37∘C and 5 CO

2for 72 hours

23 Sister Chromatid Exchange (SCE) For sister chromatidexchange (SCE) analysis 5-bromo-2-deoxyuridine (Sigma)was added after 24 hours of incubation in final concentrationof 10 120583gmL of culture Parathion (Sigma) was added at thebeginning of culture in concentrations ranging from 05to 5 120583gmL Maximum genotoxic dose of parathion thatis 25 120583gmL was chosen to check the protective effect ofcurcumin and carvacrol (Sigma) To check antigenotoxicpotential of curcumin and carvacrol against parathion cul-tures were set up separately having various combinationsof parathion and curcumincarvacrol In one set-up hep-arinised fresh blood was treated with 25120583gmL concentra-tion of parathion along with 10 and 15 120583gmL concentrationsof curcuminwhile in others 25 and 50120583gmL concentrationsof carvacrol were added against 25 120583gmL concentration ofparathion Combined effect of both curcumin and carvacrolwas also checked against parathion Blood was also treatedwith curcumin and carvacrol alone to check their genotoxiceffects if any Blood without any mutagencurcumin andcarvacrol acted as control while blood having dimethylsulphoxide was taken as negative control The cultures werethen incubated for 72 hrs at 37∘C and 5 CO

2 Colchicine

(Sigma) was added 45 minutes prior to the harvesting infinal concentration of 02 120583gmL The cells were harvestedby centrifugation and then treated with hypotonic solution(0075M KCl) and fixed in methanol acetic acid (3 1) Froma suspension of fixed cells slides were prepared by the airdrying method and stained with Hoechst 33258 (Sigma) and4 Giemsa stain (Himedia) solution following the methodof Perry and Wolff For calculating the frequency of SCE percell 50 metaphase plates were analyzed

24 GSTM1 and GSTT1 Genotyping Genomic DNA wasextracted from 200 120583L of whole blood by DNA extractionkit (Bangalore genei) Multiplex PCR was used to detect thepresence or absence of GSTM1 and GSTT1 genes For thepurpose of internal control a part of exon 7 of the constitu-tional gene CYP1A1 was also coamplified The amplificationreaction was carried out in a 25 120583L volume containing 50ndash100 ng of genomic DNA as a template 20 pmol120583L of eachprimer (GenXbio) 200120583M of each dNTP (Bangalore genei)1X PCR buffer with 15mML MgCl

2(Bangalore genei) and

07 units of Taq polymerase (Bangalore genei) PCR wasperformed by using reaction mixture with primers GSTM1(Fw 51015840-GAACTCCCTGAAAAGCTAAAGC-31015840 and Re 51015840-GTTGGGCTCAAATATACGGTGG-31015840) GSTT1 (Fw 51015840-TTCCTTACTGGTCCTCACATCTC-31015840 Re 51015840-TCACGG-GATCATGGCCAGCA-31015840) and CYP1A1 (Fw 51015840-GAACTG-CCACTTCAGCTGTCT-31015840 and Re 51015840-CAGCTGCATTTG-GAAGTGCTC-31015840) yielding a 312-bp product Initial denatu-ration was done at 94∘C for 10min A total of 35 temperaturecycles were used 94∘C at 1min 59∘C for 30 sec and 72∘Cfor 1min The last elongation step was extended to 10minsat 72∘CThe PCR products were analyzed in 2 agarose gel



3 Results





SCEs



555643

799

1033 1086

00

2

4

6

8

10

12

0 05 1 2 25 5SC

Ece

ll


SCEcell

lowast



537

01234567

SCE

cell

Treatments

SCEcell

Con

trol

P (25120583

gm

L)

Cu (1

0120583

gm

L)

Cu (1

5120583

gm

L)

DM

SO(minus

veco

ntro

l)

(25+10

) (120583

gm

L)

(25+15

) (120583

gm

L)lowast

P+

Cu

P+

Cu




563

01234567

SCE

cell

Treatments

SCEcell

Con

trol

P (25120583

gm

L)

(25+25

) (120583

gm

L)

(25+50

) (120583

gm

L)

Ca (2

5120583

gm

L)

Ca (5

0120583

gm

L)

DM

SO(minus

veco

ntro

l)

lowast

P+

Ca

P+

Ca



SCEcell

0

1

2

3

4

5

6

7

SCE

cell

Con

trol

P (25

)

P+

Cu(25+10)

P+

Cu(25+15)

P+

Ca(25+25)

P+

Ca(25+5)

P+

Cu+

Ca(25+10

+25)

P+

Cu+

Ca(25+15

+50)





05 50 643 plusmn 034

1 50 799 plusmn 025

2 50 1033 plusmn 032

25 50 1086 plusmn 040a


4 Discussion








b


b




d









b


b




d























422 plusmn 010a



443 plusmn 031b



c415 plusmn 025

c



d426 plusmn 012

d














Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



3 Results





SCEs



555643

799

1033 1086

00

2

4

6

8

10

12

0 05 1 2 25 5SC

Ece

ll


SCEcell

lowast



537

01234567

SCE

cell

Treatments

SCEcell

Con

trol

P (25120583

gm

L)

Cu (1

0120583

gm

L)

Cu (1

5120583

gm

L)

DM

SO(minus

veco

ntro

l)

(25+10

) (120583

gm

L)

(25+15

) (120583

gm

L)lowast

P+

Cu

P+

Cu




563

01234567

SCE

cell

Treatments

SCEcell

Con

trol

P (25120583

gm

L)

(25+25

) (120583

gm

L)

(25+50

) (120583

gm

L)

Ca (2

5120583

gm

L)

Ca (5

0120583

gm

L)

DM

SO(minus

veco

ntro

l)

lowast

P+

Ca

P+

Ca



SCEcell

0

1

2

3

4

5

6

7

SCE

cell

Con

trol

P (25

)

P+

Cu(25+10)

P+

Cu(25+15)

P+

Ca(25+25)

P+

Ca(25+5)

P+

Cu+

Ca(25+10

+25)

P+

Cu+

Ca(25+15

+50)





05 50 643 plusmn 034

1 50 799 plusmn 025

2 50 1033 plusmn 032

25 50 1086 plusmn 040a


4 Discussion








b


b




d









b


b




d























422 plusmn 010a



443 plusmn 031b



c415 plusmn 025

c



d426 plusmn 012

d














Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


563

01234567

SCE

cell

Treatments

SCEcell

Con

trol

P (25120583

gm

L)

(25+25

) (120583

gm

L)

(25+50

) (120583

gm

L)

Ca (2

5120583

gm

L)

Ca (5

0120583

gm

L)

DM

SO(minus

veco

ntro

l)

lowast

P+

Ca

P+

Ca



SCEcell

0

1

2

3

4

5

6

7

SCE

cell

Con

trol

P (25

)

P+

Cu(25+10)

P+

Cu(25+15)

P+

Ca(25+25)

P+

Ca(25+5)

P+

Cu+

Ca(25+10

+25)

P+

Cu+

Ca(25+15

+50)





05 50 643 plusmn 034

1 50 799 plusmn 025

2 50 1033 plusmn 032

25 50 1086 plusmn 040a


4 Discussion








b


b




d









b


b




d























422 plusmn 010a



443 plusmn 031b



c415 plusmn 025

c



d426 plusmn 012

d














Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




















422 plusmn 010a



443 plusmn 031b



c415 plusmn 025

c



d426 plusmn 012

d














Acknowledgments


References















































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






Acknowledgments


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





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Research Article Antigenotoxic Effect of Curcumin and