protective effect of curcumin against the liver toxicity caused by

Research ArticleProtective Effect of Curcumin against the Liver Toxicity Causedby Propanil in Rats

Chiagoziem A Otuechere1 Sunny O Abarikwu1 Victoria I Olateju1

Azeezat L Animashaun1 and Oluwafemi E Kale2

1 Department of Chemical Sciences Redeemerrsquos University PMB 2011 Mowe Ogun Nigeria2 Department of Pharmacology College of Medicine University of Lagos PMB 12003 Lagos Nigeria

Correspondence should be addressed to Chiagoziem A Otuechere goziemo12yahoocom

Received 30 June 2014 Revised 28 July 2014 Accepted 19 August 2014 Published 29 October 2014

Academic Editor Virinder S Parmar

Copyright copy 2014 Chiagoziem A Otuechere et al 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

We investigated the protective effects of curcumin on propanil-induced alterations in biochemical indices in blood and liver ofmale Wistar rats The study consisted of four treatment groups with six animals each designated as control propanil (20mgkg)curcumin(50mgkg) and curcumin (50mgkg) + propanil (20mgkg) Rats were administered their respective doses orally everyother day for 28 days Propanil administration elicited significant (119875 lt 0001) increases in plasma aspartate aminotransferase andalkaline phosphatase activities by 24 and 56 respectively compared to the control Treatment with propanil elevated bilirubincreatinine and total cholesterol levels in rats but these were not significant relative to controls Administration of propanil torats significantly (119875 lt 0001) increased lipid peroxidation levels However catalase activity vitamin C and reduced glutathionelevels were significantly reduced Exposure to propanil did not produce any significant changes in packed cell volume neutrophilsand leukocyte counts The supplementation of curcumin attenuated the adverse effects of propanil intoxication by reducing lipidperoxidation levels and restored the levels of serum enzymes and reduced glutathione The present study showed that propanilincreased oxidative stress and altered some biochemical parameters in the rats but curcumin could afford some protection toattenuate propanil-induced toxicity in the liver

1 Introduction

Curcumin (diferuloylmethane) is a yellow colouring ingredi-ent of the spice turmeric obtained from the rhizome of Cur-cuma longa Linn (Zingiberaceae) It is a perennial herb dis-tributed mainly throughout tropical and subtropical regionsof the world [1] Curcumin possesses anti-inflammatoryimmunomodulatory and antiatherogenic activities and isa potent inhibitor of various reactive oxygen-generatingenzymes [2 3] It has been used in indigenous herbalmedicine for the treatment of inflammatory and liver dis-orders Antioxidative properties of curcumin are well docu-mented Curcumin is a potent scavenger of reactive oxygenspecies including superoxide anion radicals and hydroxylradicals It has also been reported to inhibit erythrocyte lipidperoxidation [4] Curcumin administration attenuated the

arsenic gentamicin and acetaminophen-induced oxidativestress in rats [5ndash7] Curcumin also prevented free radi-cal formation-induced myocardial ischemia and paraquat-induced lung injury in rats [8 9] Additionally curcuminprotected against diazinon-induced toxicity in blood liverand erythrocyte of male Wistar rats [10] FurthermoreCanales-Aguirre and coworkers [11] had also reported theprotective effects of curcumin against the oxidative damagein the hippocampus of rats after exposure to parathion

Propanil (3 4-dichloropropioanilide) is an acylanilideherbicide used to control barnyard grass broadleaf weedsand for the postemergent treatment of rice [12] Thewidespread use of the herbicide on rice and wheat cropsmeans that humans could be at risk of high level exposureThis necessitated theWorldHealthOrganization to recognizepropanil as slightly hazardous in terms of human risk [13]

Hindawi Publishing CorporationInternational Scholarly Research NoticesVolume 2014 Article ID 853697 8 pageshttpdxdoiorg1011552014853697

2 International Scholarly Research Notices

However in a retrospective study assessing the clinicalfeatures of sixteen patients admitted for acute propanilpoisoning the authors advocated that the mild-to-moderatetoxicity label of propanil should be revisited by the WHO[14] Earlier reports by McMillan and coworkers [15] indi-cated that exposure to propanil had been associated withtoxicity in humans and other mammals Furthermore it hadbeen demonstrated that propanil could potentially inducecytotoxicity and nephrotoxicity in vitro [16] A recent studyreported that propanil induced dose-dependent histopatho-logical changes in the liver and kidney tissues of exposedmice[17]

Antioxidants had been proven to play an importantrole in the regulation of a vast array of physiological andpathological processes They principally contribute to theprotection of cells and tissues against deleterious effects ofreactive oxygen species and other free radicals

The aim of this study was to determine the effect ofsubacute propanil exposure in the plasma of male rats andto assess whether these effects could be ameliorated bycotreatment with curcumin To achieve this aim rats weregiven propanil and curcumin by oral gavage for 28 days afterwhichmalondialdehyde (MDA) andGSH levels andGST andCAT activities as well as other biochemical endpoints wereevaluated

2 Materials and Methods

21 Chemicals Commercial herbicide propanil was pur-chased from Harvest Field Industries Limited Lagos StateNigeria Curcumin was obtained from Sigma ChemicalsCorp St Louis MO USA Other reagents were of analyticalgrade and the purest quality available

22 Animals and Treatments Albino rats weighing between150 g and 200 g purchased from covenant farm animalhouse located at Ibadan Oyo State Nigeria were used inthis study The animals were kept in well-ventilated cagesat room temperature and under controlled conditions ofambient temperature (25∘C) at the Redeemerrsquos UniversityAnimal House Facility Mowe Ogun State Nigeria Theywere maintained on normal laboratory chow and water adlibitum The experiment was approved by the Animal EthicsCommittee of the Redeemerrsquos University

23 Animal Treatment and Sample Collection Animals weredivided into 4 experimental groups (119899 = 6) The first groupreceived olive oil at a dose of 2mLkg body weight Thesecond group received an oral administration of propanilat a dose of 20mgkgbodyweight equivalent to one-fifth ofthe oral dose used previously in our laboratories [18] Thethird group received an oral administration of curcuminat a dose of 50mgkgbodyweight This dose was selectedon the basis of a previous study [19] The fourth groupreceived both curcumin (50mgkgbodyweight) + propanil(20mgkgbody weight) Curcumin and propanil were dis-solved in olive oil at a dose of 2mgkg bw The treatments(using intragastric feeding needle) were three times a week

for a total of 28 days Rats were sacrificed after the last dose ofadministration and an overnight fast by cervical dislocationand blood was obtained by cardiac puncture Ethylenedi-aminetetraacetic acid was used as an anticoagulant Plasmawas obtained by centrifugation of samples at 4000 rpm for 5minutes and stored at minus20∘C till analysis Liver of rats wasexcised homogenized in phosphate buffer (01M pH 74)and centrifuged at 4500 rpm and the supernatants were usedfor the various biochemical measurements

24 Biochemical Assays Alkaline phosphatase (ALP)aspartate aminotransferase (AST) alanine aminotransferase(ALT) total cholesterol creatinine and bilirubin wereestimated in plasma using Randox commercial kitsLipid peroxidation (LPO) was determined by measuringthe formation of thiobarbituric acid reactive substances(TBARS) according to the method of Varshney and Kale[20] while glutathione S-transferase (GST) activity wasdetermined according to Habig et al [21] Catalase activitywas determined according to the method of Sinha [22]Vitamin E was estimated by the method of Desai [23]Briefly 03mL of ethanol and 04mL diethyl ether wereadded to 1mL plasma and centrifuged for 5 minutes at4000 rpm The ascorbic acid (Vitamin C) present in theliver tissue was determined by the spectrophotometricmethod of Omaye et al [24] The method of Beutler et al[25] was followed in estimating the level of glutathione(GSH) This method is based upon the development of arelatively stable (yellow) color when 51015840 51015840-dithiobis-(2-nitrobenzoic acid) (Ellmanrsquos reagent) is added to sulfhydrylcompounds The chromophoric product resulting from thereaction of Ellmanrsquos reagent with the reduced glutathione2-nitro-5-thiobenzoic acid had amolar absorption at 412 nm

25 Hematological Analyses Noncoagulated blood wastested after collection for packed cell volume (PCV)and differential white blood cells count according to theprocedure outlined by Cheesbrough and McArthur [26]Haematocrit centrifuge reader and capillary tubes wereused to determine the PCV A blood smear was prepared bydragging the blood over the slide with the help of anotherslide The smear was then diluted with Leishmanrsquos stain andallowed to dry The stained smears were microscopicallyobserved under oil immersion to determine the number ofeach cell type per cubic millimeter of blood The stain usedallowed exact identification of neutrophils and leukocytesProtein level was estimated according to the methoddescribed by Lowry et al [27] using bovine serum albuminas standard

26 Statistical Analysis All values were expressed as mean plusmnstandard deviation (SD) of six observations Differencesbetween the groups were determined by one-way analysisof variance (ANOVA) and post hoc testing was performedfor intergroup comparisons using Tukeyrsquos test using GraphPad Prism 3 software Values were regarded as significantlydifferent at 119875 lt 0001

International Scholarly Research Notices 3

3 Results

31 Effect of Curcumin on Plasma Biochemical Parameters inPropanil-Treated Rats Evaluation of plasma marker indicesin rats exposed to propanil (20mgkgbodyweight) showedthat there were significant (119875 lt 0001) increases in AST andALP activities by 24 and 56 respectively when comparedwith controls (Figure 1) The result presented in Figure 1 alsoshowed insignificant differences in the activity of ALT andtotal bilirubin in propanil-challenged rats when comparedwith the controls Curcumin administered alone had similareffects on plasma biochemical parameters when comparedwith controls However treatment with both curcumin andpropanil resulted in a significant reduction in AST and ALPactivities

32 Effect of Curcumin on SomeHematological Parameters andCholesterol Levels in Propanil-Treated Rats The hematologi-cal properties of rats exposed to propanil are shown inTable 1Treatment with propanil alone did not significantly decreasePCV and neutrophil levels relative to the control Treatmentwith propanil elevated the total leukocyte counts but this wasnot significant relative to the control However the curcumin+ propanil group showed a significant (119875 lt 005) increasein the total leukocyte counts when compared to the controlFurthermore there were no significant differences in the levelof total cholesterol in propanil-treated rats when comparedwith the control Although the curcumin + propanil groupelicited a significant increase in the cholesterol levels whencompared with control animals however there was nosignificant change when compared with the propanil-treatedgroup (Table 1)

33 Effect of Curcumin on the Antioxidant Status of Propanil-Treated Rats The ability of a cell or tissue to maintain itsintegrity is a function of the levels of the antioxidants presentwhen compared with the level of the oxidants The balancebetween these two determines the susceptibility of the cellor tissue to free radicals attack or oxidative stress Resultsindicated that lipid peroxidation level was significantly (119875 lt0001) increased by 103 while GSH levels and catalaseactivity were significantly (119875 lt 0001) decreased in livers ofrats treated with propanil (Figures 2 and 3) when comparedwith controls However there were no significant (119875 lt 005)changes in the activity of GST and levels of Vit C and VitE in liver of rats treated with propanil in comparison withthe control group (Figure 3) Curcumin administered alonedid not alter the lipid peroxidation and GSH levels whencompared with the control However supplementation ofcurcumin to propanil-intoxicated rats significantly (119875 lt005) attenuated the increase in lipid peroxidation levels andrestored the GSH levels to normal

4 Discussion

The protective role of curcumin in propanil-induced toxicityin Wistar rats was examined in this study Hematologicaland biochemical profiles of blood are known to provide

important information about the internal environment of theorganism Curcumin an antioxidant and anticarcinogenicsubstance was reported to have a protective effect againstliver damage and ferric nitrilotriacetate induced peroxidationof microsomal membrane lipids [28] The protective actionof curcumin against adverse effects of cisplatin had alsobeen reported [29] Curcumin could exert antioxidativeeffects either directly as a chemical antioxidant due to itsability to scavenge reactive oxygen and nitrogen free radicalsor by modulating cellular defenses which themselves exertantioxidant effects [30 31]

Due to its role in the transformation of environmentalxenobiotics the liver is at great risk of injury Lipid peroxida-tion represents one of the most frequent reactions resultingfrom free radicalsrsquo attack on biological structures In ourstudy the liver MDA level index of lipid peroxidation sig-nificantly increased in the propanil-treated rats Supplemen-tation with curcumin significantly attenuated the increasein lipid peroxidation levels It is plausible that the mecha-nism of this protection could be the inhibition of reactiveoxygen species generation by suppressing cytochrome P450isozymes which are involved in the bioactivation of propanilto toxic reactive metabolites such as 3 4-dichloroaniline[15 32] Reports are not available regarding Vitamins Cand E concentrations in propanil-induced organ toxicity Aprevious study had reported decrease in ascorbic acid levelsin plasma and liver of rats treated with dimethylnitrosamine[33] However in the present investigation a nonsignificantdecrease in Vitamin C was observed in the liver of propanil-treated rats This decrease may be indicative of increasedoxidative stress free radical formation and simultaneousdamage of the liver plasma membrane lipid bilayer arisingfrom propanil intoxication Reduction in Vitamin E contentfollowing treatment with 1 2-dibromoethane had also beenpreviously reported [34] Data obtained for Vitamin E in liverof propanil exposed rats did not reveal any significant changein the vitamin content Compared with the control GSHconcentrations in treated Wistar rats significantly decreasedafter exposure to propanil This corroborated the findingsthat GSH concentrations in treated Crucian carp signifi-cantly decreased dose-dependently after acute exposure todichloroaniline [35] The depletion of GSH is suggestive ofa deleterious effect of propanil on the antioxidant defensein the liver of rats It is also consistent with the generationof oxidative stress thereby reenforcing the role of GSHin molecular protective mechanisms that modulate cellularresponses to toxic chemicals The present study showed thatadministration of curcumin improved the GSH levels in ratsThis finding is similar to the data reported by Piper et al [36]who indicated an increase of the GSH level correspondingto curcumin dosage in rats fed curcumin at doses up to500mgkg body weight daily for 14 days

GST detoxifies a variety of electrophilic compounds toless toxic forms by conjugation withndashSH groups from GSHIn the present study GST activity was not changed by any ofthe exposure schemes This correlated with the findings thatexposure of rats to cypermethrin in single and repeated doseshad no relevant effect on GST activities [37] In contrastSaxena et al [38] reported an increase in GST activity in rat


(UL

)

(UL

)

(mg

kg)

(mg

dL)

AST ALP ALT

Creatinine Direct bilirubin

Control PRP CUR

aab

b

40

30

20

10

0

75

50

25

0

(UL

)

75

50

25

0

400

300

200

100

0

0100

0075

0050

0025

0000

Control PRP CUR Control PRP CUR


CUR + PRP CUR + PRP CUR + PRP

CUR + PRP CUR + PRP

Figure 1 Effect of curcumin (CUR) on some plasma biochemical indices in rats orally treated with propanil Values are expressed as meanplusmn SD for six animals per group a119875 lt 0001 compared to control group b119875 lt 0001 compared to propanil group

Table 1 Effect of curcumin (CUR) on some hematological parameters and total cholesterol values in rats treated with propanil (PRP)

Treatment PCV Total leucocyte times103 Neutrophils Total cholesterol (mgdL)CONTROL 415 plusmn 21 41 plusmn 04 603 plusmn 39 49 plusmn 28PRP 408 plusmn 21 48 plusmn 09 512 plusmn 94 53 plusmn 41CUR 418 plusmn 33 44 plusmn 04 550 plusmn 39 506 plusmn 44CUR + PRP 410 plusmn 32 56 plusmn 02a 582 plusmn 51 552 plusmn 35Values are mean plusmnSD of six rats in each group a119875 lt 0001 compared to control group

red blood cells after exposure to chlorpyrifos and endosulfanIn addition on the basis of our observations a dose as lowas 20mgkg propanil seemed not to induce such a levelof oxidant stress and alteration of the antioxidant systemin rats In consonance with our earlier studies there was asignificant decline in the activities of catalase after propaniladministration [18] which may be due to oxidative stresshowever treatment with curcumin did not ameliorate thisdecline

An increase in plasma cholesterol as a result of pesticideexposure may indicate loss of membrane integrity Carlsonand Kolmodin-Hedman [39] reported that the accumulation

of pesticide in the liver was associated with the disturbanceof lipid metabolism and elevation of serum cholesterol Inour study cholesterol levels were mildly increased after theadministration of propanil but this was not statisticallysignificant Furthermore there were no significant differ-ences in the level of total cholesterol in propanil-treatedrats when compared with the control Although the cur-cumin + propanil group elicited a significant increase inthe cholesterol levels when compared with control animalshowever there was no significant change when comparedwith the propanil-treated group A plausible explanation forthis observed effect may be due to synergetic prooxidant


(120583g

mg

prot

ein)

100

75

50

25

00

15

10

10

5

0

15

5

0

GSH Catalase

GST(120583

mol

min

g ti

ssue

)

(120583m

olm

ing

tiss

ue)


Control PRP CUR

a

a

b

b

CUR + PRP CUR + PRP

CUR + PRP

Figure 2 Effect of curcumin on reduced glutathione (GSH) catalase and glutathione S-transferase (GST) in liver of rats orally treated withpropanil Values are expressed as mean plusmn SD for six animals per group a119875 lt 0001 compared to control group b119875 lt 0001 compared tocurcumin group

effects of propanil and curcumin combination This is inagreement with a report that combining flavonoids with lowlevel pesticides could modify their antioxidant potential andalso trigger molecular events that could increase their healthrisks [40]

This study confirmed the liver damage in the propanil-treated group by the increase in AST and ALP levels inplasma Exposure of propanil-intoxicated rats with curcuminnormalized the activities of AST and ALP to their controlvalues The decrease in AST and ALP activities supportsthe hepatoprotective effects of curcumin consistent with

the findings that curcumin modulated the increased activityof marker enzymes and plasma lipid levels in nicotine-treated rats [41] However curcumin when given aloneproduced an increase in ALP activity suggesting that thesubacute administration of curcumin at the dose of 50mgkgused in this study may not be completely harmless Thisagreed with an earlier report that curcumin administeredto human subjects at doses ranging from 09 to 36 g dayminus1for 4 months could cause some adverse effects includingnausea diarrhea and increase in serum alkaline phosphataseand lactate dehydrogenase activities [42] Clinical chemistry


(120583m

olm

inm

g pr

otei

n)

40

30

20

10

0

100

75

50

25

00

(mg

g tis

sue)

Control PRP CUR

ControlPRP

CUR

Lipid peroxidation

a

b

CUR + PRP

CUR + PRP

Figure 3 Effect of curcumin on lipid peroxidation Vitamin C and Vitamin E levels in liver of rats orally treated with propanil Values areexpressed as mean plusmn SD for six animals per group a119875 lt 0001 compared to control group b119875 lt 0001 compared to propanil group

analysis of urea and creatinine could serve as prognosticindicators of renal dysfunction in the following exposure totoxicants In the present study we observed a nonsignificantincrease in the level of creatinine in propanil-treated ratswhen compared to the control This may possibly be due toincreased catabolic state in the rats from prolonged appetiteas a result of manifestation of acute oral toxicity [43]However coadminstration of curcumin did not mitigate theincreased creatinine levels

Hematological and biochemical profiles of blood can pro-vide important information about the internal environmentof the organism [44] In this study there were no prominentchanges in the PCV and neutrophils These findings aresupported by the results of Garg et al [45] who reportedthat treatment of broiler chicks with monocrotophos didnot cause any significant change in erythrocyte count andPCV values Furthermore treatment of rats with curcumin+ propanil increased total leukocyte count compared torats in the control group Our results are in accordancewith the findings that treatment of rats with propetamphosplus propolis increased total leukocyte count The increaseobserved in total leukocyte countmight indicate an activationof the animalrsquos immune system due to either stimulatedlymphopoiesis or disturbance of the nonspecific immunesystem leading to the increased production of leukocytes[46 47]

5 Conclusions

In summary the present results suggested that curcuminprotected against the liver toxicity induced by propaniltreatment in rats In this study propanil elicited increase inAST and ALP activities Furthermore it had been suggestedthat lipid peroxidation might be a contributing factor tothe development of liver toxicity The significantly decreasedactivities of hepatic plasmamarkers (AST and ALP) and lipidperoxidation marker (malondialdehyde) along with normal-izing of the endogenous GSH level suggest that curcumin is

a strong antioxidant We also observed that the curcumin+ propanil combination resulted in a significant increase incholesterol levels when compared with the control groupsuggesting a possible synergetic hyperlipidemic effect whencurcumin combines with propanil

Conflict of Interests

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

References

[1] B Joe M Vijaykumar and B R Lokesh ldquoBiological propertiesof curcumin -cellular and molecular mechanisms of actionrdquoCritical Reviews in Food Science and Nutrition vol 44 no 2pp 97ndash111 2004

[2] C C Araujo and L L Leon ldquoBiological activities of Curcumalonga LrdquoMemorias do Instituto Oswaldo Cruz vol 96 no 5 pp723ndash728 2001

[3] N Chainani-Wu ldquoSafety and anti-inflammatory activity ofcurcumin a component of tumeric (Curcuma longa)rdquo Journalof Alternative andComplementaryMedicine vol 9 no 1 pp 161ndash168 2003

[4] S K Borra P Gurumurthy M Mahendra K M JayamathiC N Cherian and C Ram ldquoAntioxidant and free radicalscavenging activity of curcumin determined by using differentin vitro and ex vivo modelsrdquo Journal of Medicinal PlantsResearch vol 7 no 36 pp 2680ndash2690 2013

[5] F M El-Demerdash M I Yousef and F M E RadwanldquoAmeliorating effect of curcumin on sodium arsenite-inducedoxidative damage and lipid peroxidation in different rat organsrdquoFood and Chemical Toxicology vol 47 no 1 pp 249ndash254 2009

[6] E O Farombi and M Ekor ldquoCurcumin attenuates gentamicin-induced renal oxidative damage in ratsrdquo Food and ChemicalToxicology vol 44 no 9 pp 1443ndash1448 2006

[7] M Cekmen Y O Ilbey E Ozbek A Simsek A Somay and CErsoz ldquoCurcumin prevents oxidative renal damage induced by


acetaminophen in ratsrdquo Food and Chemical Toxicology vol 47no 7 pp 1480ndash1484 2009

[8] P Manikandan M Sumitra S Aishwarya B M ManoharB Lokanadam and R Puvanakrishnan ldquoCurcumin modulatesfree radical quenching in myocardial ischaemia in ratsrdquo TheInternational Journal of Biochemistry amp Cell Biology vol 36 no10 pp 1967ndash1980 2004

[9] N Venkatesan ldquoPulmonary protective effects of curcuminagainst paraquat toxicityrdquo Life Sciences vol 66 no 2 pp 21ndash822000

[10] M Messarah W Amamra A Boumendjel et al ldquoAmeliorat-ing effects of curcumin and vitamin E on diazinon-inducedoxidative damage in rat liver and erythrocytesrdquo Toxicology andIndustrial Health vol 29 no 1 pp 77ndash88 2013

[11] A A Canales-Aguirre U A Gomez-Pinedo S Luquin MA Ramırez-Herrera M L Mendoza-Magana and A Feria-Velasco ldquoCurcumin protects against the oxidative damageinduced by the pesticide parathion in the hippocampus of therat brainrdquo Nutritional Neuroscience vol 15 no 2 pp 62ndash692012

[12] M T Moore J R Pierce C D Milam J L Farris and EL Winchester ldquoResponses of non-target aquatic organisms toaqueous propanil exposurerdquo Bulletin of Environmental Contam-ination and Toxicology vol 61 no 2 pp 169ndash174 1998

[13] WHOWHO Recommended Classification of Pesticides by Haz-ard andGuidelines to ClassificationWorldHealthOrganizationGeneva Switzerland 2004

[14] M Eddleston M Rajapakshe D Roberts et al ldquoSeverepropanil [N-(34-dichlorophenyl) propanamide] pesticide self-poisoningrdquo Journal of Toxicology amp Clinical Toxicology vol 40no 7 pp 847ndash854 2002

[15] D C McMillan T P Bradshaw J A Hinson and D J JollowldquoRole of metabolites in propanil-induced hemolytic anemiardquoToxicology and Applied Pharmacology vol 110 no 1 pp 70ndash781991

[16] G O Rankin C Racine A Sweeney A Kraynie D K Anestisand J B Barnett ldquoIn vitro nephrotoxicity induced by propanilrdquoEnvironmental Toxicology vol 23 no 4 pp 435ndash442 2008

[17] O Cakici and E Akat ldquoPropanil-induced histopathologicalchanges in the liver and kidney of micerdquo Analytical andQuantitative Cytology and Histology vol 35 no 3 pp 163ndash1702013

[18] C A Otuechere S O Abarikwu M A Rufai A E OhiozojeE Martins and E O Farombi ldquoProtective effects of vitamin Cagainst propanil-induced hepatotoxicity in wistar ratsrdquo AsianPacific Journal of Tropical Disease vol 2 no 1 pp S212ndashS2172012

[19] A ElMackawy andHA Sharaf ldquoCytogenetical andhistochem-ical studies on curcumin in male ratsrdquo WIT Transactions onBiomedicine and Health vol 10 pp 169ndash180 2006

[20] R Varshney and K K Kale ldquoEffects of calmodulin antagonistson radiation-induced lipid peroxidation in microsomesrdquo Inter-national Journal of Radiation Biology vol 58 no 5 pp 733ndash7431990

[21] W H Habig M J Pabst and W B Jakoby ldquoGlutathioneS transferases the first enzymatic step in mercapturic acidformationrdquoThe Journal of Biological Chemistry vol 249 no 22pp 7130ndash7139 1974

[22] A K Sinha ldquoColorimetric assay of catalaserdquo Analytical Bio-chemistry vol 47 no 2 pp 389ndash394 1972

[23] I D Desai ldquoVitamin E analysis methods for animal tissuesrdquoMethods in Enzymology vol 105 pp 138ndash147 1984

[24] S T Omaye T P Turbull and H C Sauberchich ldquoSelectedmethods for the determination of ascorbic acid in animal cellstissues and fluidsrdquo Methods in Enzymology vol 62 no 1 pp3ndash11 1979

[25] E Beutler O Duron and B M Kelly ldquoImproved method forthe determination of blood glutathionerdquo Journal of Laboratoryand Clinical Medicine vol 61 pp 882ndash888 1963

[26] M Cheesbrough and J McArthur A Laboratory Manual forRural Tropical Hospitals Churchill Livingstone New York NYUSA 1976

[27] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[28] M Iqbal Y Okazaki and S Okada ldquoIn vitro curcumin mod-ulates ferric nitrilotriacetate (Fe-NTA) and hydrogen peroxide(H2

O2

)-induced peroxidation of microsomal membrane lipidsand DNA damagerdquo Teratogenesis Carcinogenesis and Mutagen-esis vol 23 supplement 1 pp 151ndash160 2003

[29] L M G Antunes M C P Araujo J D C Darin and M D LP Bianchi ldquoEffects of the antioxidants curcumin and vitamin Con cisplatin-induced clastogenesis in Wistar rat bone marrowcellsrdquoMutation Research vol 465 no 1-2 pp 131ndash137 2000

[30] K I Priyadarsini D K Maity G H Naik et al ldquoRole ofphenolic O-H and methylene hydrogen on the free radicalreactions and antioxidant activity of curcuminrdquo Free RadicalBiology and Medicine vol 35 no 5 pp 475ndash484 2003

[31] S Awasthi U Pandya S S Singhal et al ldquoCurcumin-glutathione interactions and the role of human glutathione S-transferase P1-1rdquo Chemico-Biological Interactions vol 128 no 1pp 19ndash38 2000

[32] D C McMillan J E A Leakey M P Arlotto J M McMillanand J A Hinson ldquoMetabolism of the arylamide herbicidepropanil II Effects of propanil and its derivatives on hepaticmicrosomal drug-metabolizing enzymes in the ratrdquo Toxicologyand Applied Pharmacology vol 103 no 1 pp 102ndash112 1990

[33] J George ldquoAscorbic acid concentrations in dimethylni-trosamine-induced hepatic fibrosis in ratsrdquo Clinica ChimicaActa vol 335 no 1-2 pp 39ndash47 2003

[34] D L Warren and D J Reed ldquoModification of hepatic vitaminE stores in vivo III Vitamin E depletion by 12-dibromoethanemay be related to initial conjugation with glutathionerdquo Archivesof Biochemistry and Biophysics vol 288 no 2 pp 449ndash455 1991

[35] W Li D Yin Y Zhou Y S Hu and L Wang ldquo34-Dichloroaniline-induced oxidative stress in liver of crucian carp(Carassius auratus)rdquo Ecotoxicology and Environmental Safetyvol 56 no 2 pp 251ndash255 2003

[36] J T Piper S S Singhal M S Salameh R T Torman YC Awasthi and S Awasthi ldquoMechanisms of anticarcinogenicproperties of curcumin the effect of curcumin on glutathionelinked detoxification enzymes in rat liverrdquo International Journalof Biochemistry and Cell Biology vol 30 no 4 pp 445ndash4561998

[37] B Giray A Gurbay and F Hincal ldquoCypermethrin-inducedoxidative stress in rat brain and liver is prevented by VitaminE or allopurinolrdquo Toxicology Letters vol 118 no 3 pp 139ndash1462001

[38] R Saxena P Garg and D K Jain ldquoIn Vitro anti-oxidant effectof vitamin e on oxidative stress induced due to pesticides in raterythrocytesrdquo Toxicology International vol 18 no 1 pp 73ndash762011


[39] L A Carlson and B Kolmodin-Hedman ldquoHypermdashlipoprote-inemia inmen exposed to chlorinated hydrocarbon pesticidesrdquoActa Medica Scandinavica vol 192 no 1-2 pp 29ndash32 1972

[40] C S Yang ldquoDietary factors may modify cancer risk by alteringxenobiotic metabolism and many other mechanismsrdquo Journalof Nutrition vol 136 no 1 pp 2685Sndash2686S 2006

[41] C Kalpana K N Rajasekharan and V P Menon ldquoModulatoryeffects of curcumin and curcumin analog on circulatory lipidprofiles during nicotine-induced toxicity inWistar ratsrdquo Journalof Medicinal Food vol 8 no 2 pp 246ndash250 2005

[42] R A Sharma S A Euden S L Platton et al ldquoPhase I clinicaltrial of oral curcumin biomarkers of systemic activity andcompliancerdquo Clinical Cancer Research vol 10 no 20 pp 6847ndash6854 2004

[43] A A Adeneye O P Ajagbonna T I Adeleke and S O BelloldquoPreliminary toxicity and phytochemical studies of the stembark aqueous extract of Musanga cecropioides in ratsrdquo Journalof Ethnopharmacology vol 105 no 3 pp 374ndash379 2006

[44] J Velisek Z Svobodova V Piackova and L Groch ldquoEffects ofclove oil anaesthesia on common carp (Cyprinus carpio L)rdquoVeterinarni Medicina vol 50 no 6 pp 269ndash275 2005

[45] U K Garg A K Pal G J Jha and S B Jadhao ldquoHaemato-biochemical and immuno-pathophysiological effects of chronictoxicity with synthetic pyrethroid organophosphate and chlo-rinated pesticides in broiler chicksrdquo International Immunophar-macology vol 4 no 13 pp 1709ndash1722 2004

[46] E Cetin M Kanbur S Silici and G Eraslan ldquoPropetamphos-induced changes in haematological and biochemical param-eters of female rats protective role of propolisrdquo Food andChemical Toxicology vol 48 no 7 pp 1806ndash1810 2010

[47] M I Yousef H Z Ibrahim H M Salem G A Hassan SHelmi and K Bertheussen ldquoHematological and biochemicalchanges induced by carbofuran and glyphosate in rabbitsrdquoEnvironmental and Nutritional Interactions vol 3 no 3 pp179ndash194 1999

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Tropical MedicineJournal of


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BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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MEDIATORSINFLAMMATION

of


However in a retrospective study assessing the clinicalfeatures of sixteen patients admitted for acute propanilpoisoning the authors advocated that the mild-to-moderatetoxicity label of propanil should be revisited by the WHO[14] Earlier reports by McMillan and coworkers [15] indi-cated that exposure to propanil had been associated withtoxicity in humans and other mammals Furthermore it hadbeen demonstrated that propanil could potentially inducecytotoxicity and nephrotoxicity in vitro [16] A recent studyreported that propanil induced dose-dependent histopatho-logical changes in the liver and kidney tissues of exposedmice[17]

Antioxidants had been proven to play an importantrole in the regulation of a vast array of physiological andpathological processes They principally contribute to theprotection of cells and tissues against deleterious effects ofreactive oxygen species and other free radicals

The aim of this study was to determine the effect ofsubacute propanil exposure in the plasma of male rats andto assess whether these effects could be ameliorated bycotreatment with curcumin To achieve this aim rats weregiven propanil and curcumin by oral gavage for 28 days afterwhichmalondialdehyde (MDA) andGSH levels andGST andCAT activities as well as other biochemical endpoints wereevaluated

2 Materials and Methods

21 Chemicals Commercial herbicide propanil was pur-chased from Harvest Field Industries Limited Lagos StateNigeria Curcumin was obtained from Sigma ChemicalsCorp St Louis MO USA Other reagents were of analyticalgrade and the purest quality available

22 Animals and Treatments Albino rats weighing between150 g and 200 g purchased from covenant farm animalhouse located at Ibadan Oyo State Nigeria were used inthis study The animals were kept in well-ventilated cagesat room temperature and under controlled conditions ofambient temperature (25∘C) at the Redeemerrsquos UniversityAnimal House Facility Mowe Ogun State Nigeria Theywere maintained on normal laboratory chow and water adlibitum The experiment was approved by the Animal EthicsCommittee of the Redeemerrsquos University

23 Animal Treatment and Sample Collection Animals weredivided into 4 experimental groups (119899 = 6) The first groupreceived olive oil at a dose of 2mLkg body weight Thesecond group received an oral administration of propanilat a dose of 20mgkgbodyweight equivalent to one-fifth ofthe oral dose used previously in our laboratories [18] Thethird group received an oral administration of curcuminat a dose of 50mgkgbodyweight This dose was selectedon the basis of a previous study [19] The fourth groupreceived both curcumin (50mgkgbodyweight) + propanil(20mgkgbody weight) Curcumin and propanil were dis-solved in olive oil at a dose of 2mgkg bw The treatments(using intragastric feeding needle) were three times a week

for a total of 28 days Rats were sacrificed after the last dose ofadministration and an overnight fast by cervical dislocationand blood was obtained by cardiac puncture Ethylenedi-aminetetraacetic acid was used as an anticoagulant Plasmawas obtained by centrifugation of samples at 4000 rpm for 5minutes and stored at minus20∘C till analysis Liver of rats wasexcised homogenized in phosphate buffer (01M pH 74)and centrifuged at 4500 rpm and the supernatants were usedfor the various biochemical measurements

24 Biochemical Assays Alkaline phosphatase (ALP)aspartate aminotransferase (AST) alanine aminotransferase(ALT) total cholesterol creatinine and bilirubin wereestimated in plasma using Randox commercial kitsLipid peroxidation (LPO) was determined by measuringthe formation of thiobarbituric acid reactive substances(TBARS) according to the method of Varshney and Kale[20] while glutathione S-transferase (GST) activity wasdetermined according to Habig et al [21] Catalase activitywas determined according to the method of Sinha [22]Vitamin E was estimated by the method of Desai [23]Briefly 03mL of ethanol and 04mL diethyl ether wereadded to 1mL plasma and centrifuged for 5 minutes at4000 rpm The ascorbic acid (Vitamin C) present in theliver tissue was determined by the spectrophotometricmethod of Omaye et al [24] The method of Beutler et al[25] was followed in estimating the level of glutathione(GSH) This method is based upon the development of arelatively stable (yellow) color when 51015840 51015840-dithiobis-(2-nitrobenzoic acid) (Ellmanrsquos reagent) is added to sulfhydrylcompounds The chromophoric product resulting from thereaction of Ellmanrsquos reagent with the reduced glutathione2-nitro-5-thiobenzoic acid had amolar absorption at 412 nm

25 Hematological Analyses Noncoagulated blood wastested after collection for packed cell volume (PCV)and differential white blood cells count according to theprocedure outlined by Cheesbrough and McArthur [26]Haematocrit centrifuge reader and capillary tubes wereused to determine the PCV A blood smear was prepared bydragging the blood over the slide with the help of anotherslide The smear was then diluted with Leishmanrsquos stain andallowed to dry The stained smears were microscopicallyobserved under oil immersion to determine the number ofeach cell type per cubic millimeter of blood The stain usedallowed exact identification of neutrophils and leukocytesProtein level was estimated according to the methoddescribed by Lowry et al [27] using bovine serum albuminas standard

26 Statistical Analysis All values were expressed as mean plusmnstandard deviation (SD) of six observations Differencesbetween the groups were determined by one-way analysisof variance (ANOVA) and post hoc testing was performedfor intergroup comparisons using Tukeyrsquos test using GraphPad Prism 3 software Values were regarded as significantlydifferent at 119875 lt 0001


3 Results




4 Discussion






(UL

)

(UL

)

(mg

kg)

(mg

dL)

AST ALP ALT


Control PRP CUR

aab

b

40

30

20

10

0

75

50

25

0

(UL

)

75

50

25

0

400

300

200

100

0

0100

0075

0050

0025

0000




CUR + PRP CUR + PRP








(120583g

mg

prot

ein)

100

75

50

25

00

15

10

10

5

0

15

5

0

GSH Catalase

GST(120583

mol

min

g ti

ssue

)

(120583m

olm

ing

tiss

ue)


Control PRP CUR

a

a

b

b

CUR + PRP CUR + PRP

CUR + PRP






(120583m

olm

inm

g pr

otei

n)

40

30

20

10

0

100

75

50

25

00

(mg

g tis

sue)

Control PRP CUR

ControlPRP

CUR

Lipid peroxidation

a

b

CUR + PRP

CUR + PRP




5 Conclusions





References































O2


























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


3 Results




4 Discussion






(UL

)

(UL

)

(mg

kg)

(mg

dL)

AST ALP ALT


Control PRP CUR

aab

b

40

30

20

10

0

75

50

25

0

(UL

)

75

50

25

0

400

300

200

100

0

0100

0075

0050

0025

0000




CUR + PRP CUR + PRP








(120583g

mg

prot

ein)

100

75

50

25

00

15

10

10

5

0

15

5

0

GSH Catalase

GST(120583

mol

min

g ti

ssue

)

(120583m

olm

ing

tiss

ue)


Control PRP CUR

a

a

b

b

CUR + PRP CUR + PRP

CUR + PRP






(120583m

olm

inm

g pr

otei

n)

40

30

20

10

0

100

75

50

25

00

(mg

g tis

sue)

Control PRP CUR

ControlPRP

CUR

Lipid peroxidation

a

b

CUR + PRP

CUR + PRP




5 Conclusions





References































O2


























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(UL

)

(UL

)

(mg

kg)

(mg

dL)

AST ALP ALT


Control PRP CUR

aab

b

40

30

20

10

0

75

50

25

0

(UL

)

75

50

25

0

400

300

200

100

0

0100

0075

0050

0025

0000




CUR + PRP CUR + PRP








(120583g

mg

prot

ein)

100

75

50

25

00

15

10

10

5

0

15

5

0

GSH Catalase

GST(120583

mol

min

g ti

ssue

)

(120583m

olm

ing

tiss

ue)


Control PRP CUR

a

a

b

b

CUR + PRP CUR + PRP

CUR + PRP






(120583m

olm

inm

g pr

otei

n)

40

30

20

10

0

100

75

50

25

00

(mg

g tis

sue)

Control PRP CUR

ControlPRP

CUR

Lipid peroxidation

a

b

CUR + PRP

CUR + PRP




5 Conclusions





References































O2


























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(120583g

mg

prot

ein)

100

75

50

25

00

15

10

10

5

0

15

5

0

GSH Catalase

GST(120583

mol

min

g ti

ssue

)

(120583m

olm

ing

tiss

ue)


Control PRP CUR

a

a

b

b

CUR + PRP CUR + PRP

CUR + PRP






(120583m

olm

inm

g pr

otei

n)

40

30

20

10

0

100

75

50

25

00

(mg

g tis

sue)

Control PRP CUR

ControlPRP

CUR

Lipid peroxidation

a

b

CUR + PRP

CUR + PRP




5 Conclusions





References































O2


























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


(120583m

olm

inm

g pr

otei

n)

40

30

20

10

0

100

75

50

25

00

(mg

g tis

sue)

Control PRP CUR

ControlPRP

CUR

Lipid peroxidation

a

b

CUR + PRP

CUR + PRP




5 Conclusions





References































O2


























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
























O2


























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 effect of curcumin against the liver toxicity caused by

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