The potential protective role of Physalis peruviana L. fruit in cadmium-induced hepatotoxicity and nephrotoxicity

The potential protective role of Physalis peruviana L fruit incadmium-induced hepatotoxicity and nephrotoxicityMohamed A Dkhil ab Saleh Al-Quraishy a Marwa MS Diab c Mohamed S Othman dAhmed M Aref e Ahmed E Abdel Moneim bfa Department of Zoology College of Science King Saud University Riyadh Saudi Arabiab Department of Zoology and Entomology Faculty of Science Helwan University Cairo Egyptc Department of Molecular Drug Evaluation National Organization for Drug Control amp Research (NODCAR) Giza Egyptd Department of Biochemistry and Molecular Biology Faculty of Biotechnology Modern Sciences and Arts (MSA) Giza Egypte Department of Biological Science Faculty of Dentistry Modern Sciences and Arts University (MSA) Giza Egyptf Department of Biochemistry and Molecular Biology Asturias Institute of Biotechnology University of Oviedo Oviedo Spain

A R T I C L E I N F O

Article historyReceived 30 June 2014Accepted 23 September 2014Available online 28 September 2014

KeywordsPhysalis peruviana LCadmiumOxidative stressLiverKidneyRats

A B S T R A C T

This study aimed to investigate the potential protective role of Physalis peruviana L (family Solanaceae)against cadmium-induced hepatorenal toxicity in Wistar rats Herein cadmium chloride (CdCl2) (65 mgkg bwtday) was intraperitoneally injected for 5 days and methanolic extract of physalis (MEPh) waspre-administered to a group of Cd-treated rats by an oral administration at a daily dose of 200 mgkgbwt for 5 days The findings revealed that CdCl2 injection induced significant decreases in kidney weightand kidney index Cadmium intoxication increased the activities of liver enzymes and the bilirubin levelin addition to the levels of uric acid urea and creatinine were increased in the serum The pre-administration of MEPh alleviated hepatorenal toxicity in Cd-treated rats Physalis was noted to play agood hepatorenal protective role reducing lipid peroxidation nitric oxide and enhancing enzymatic ac-tivities and non-enzymatic antioxidant molecule glutathione in hepatic and renal tissues of Cd-treatedrats Moreover physalis treatment was able to reverse the histopathological changes in liver and kidneytissues and also increased the expression of Bcl-2 protein in liver and kidney of rats Overall the resultsshowed that MEPh can induce antioxidant and anti-apoptotic effects and also exerts beneficial effectsfor the treatment of Cd-induced hepatorenal toxicity

copy 2014 Elsevier Ltd All rights reserved

1 Introduction

Cadmium (Cd) is a common toxic heavy metal in the environ-ment Cd is a highly accumulative toxicant with very long biologicalhalf-life of over 20 years Cd is not biodegradable and its level inthe environment is increasing due to industrial activities and humanexposure to Cd is inevitable (El-Habit and Abdel Moneim 2014)It is known that acute Cd intoxication produces toxicities in the livertestes and brain (Othman et al 2014a) while chronic exposure toCd often leads to renal dysfunction anemia osteoporosis and bonefractures Also Cd is a potent carcinogen in a number of tissues ofrodents and is classified as a human carcinogen (El-Habit and AbdelMoneim 2014)

Several mechanisms have been proposed to explain the Cd-induced hepatorenal toxicity but no mechanisms have yet beendefined explicitly Epidemiological and experimental evidence sug-gested that acute Cd exposure induces oxidative stress through theinhibition of antioxidant enzymes (El-Habit and Abdel Moneim2014) However the toxic effects of Cd are rather complex and stilldebated (Lopez et al 2003)

In recent times the use of traditional medicine is increasinglygaining popularity in both developed and developing countries(Abdel Moneim 2012 Al-Olayan et al 2014a) The therapeutic valuesof many plants have been identified and exploited for the manage-ment of many disease conditions

Physalis peruviana L belongs to the family Solanaceae and is con-sidered as one of the herbal plants used for treating various diseases(Al-Olayan et al 2014b Chang et al 2008) To the best of our knowl-edge there is no report on the protective effects of P peruvianaagainst Cd intoxication in induced hepatorenal toxicity in rats There-fore this study evaluated the hepatorenal protective potentials ofthe methanolic extract of P peruviana on Cd-intoxicated rats

Corresponding author Department of Biochemistry and Molecular BiologyAsturias Institute of Biotechnology University of Oviedo Oviedo Spain Tel +34611302236 fax +34 985103157

E-mail addressabdelahmeduouniovies (AEA Moneim)

httpdxdoiorg101016jfct2014090130278-6915copy 2014 Elsevier Ltd All rights reserved

Food and Chemical Toxicology 74 (2014) 98ndash106

Contents lists available at ScienceDirect

Food and Chemical Toxicology

journal homepage wwwelseviercom locate foodchemtox

2 Materials and methods

21 Chemicals

Cadmium chloride (CdCl2 CAS Number 10043-52-4) nitro blue tetrazolium N-(1ndashnaphthyl) ethylenediamine and TrisndashHCl were purchased from Sigma (St Louis MOUSA) Thiobarbituric acid and trichloroacetic acid were purchased from Merck Allother chemicals and reagents used in this study were of analytical grade Double-distilled water was used as the solvent

22 Animals

Twenty-eight adult male Wistar albino rats weighing 200ndash250 g (10ndash12 weeks)were obtained from The Holding Company for Biological Products and Vaccines(VACSERA Cairo Egypt) The animals were kept in wire bottomed cages in a roomunder standard condition of illumination with a 12-hour lightndashdark cycle at 25 plusmn 1 degCfor 1 week until the beginning of treatment They were provided with tap water andbalanced diet ad libitum We have followed the European Community Directive (86609EEC) and national rules on animal care that was carried out in accordance withthe NIH Guidelines for the Care and Use of Laboratory Animals 8th edition

23 Plant material

P peruviana L fresh fruits were collected from the market of East Cairo Egyptin the months of FebruaryndashMarch 2012 The plant material was authenticated inthe Botany Department Faculty of Science Helwan University Cairo Egypt by a plantTaxonomist The plant material was identified based on its taxonomic characters andby direct comparison with the herbarium specimens that are available at the her-barium of the Botany Department

231 Physalis extract preparationThe fresh fruits of P peruviana L (10 kg) were separated from their calyxes and

homogenized The pulp was consecutively macerated for 1 day in petroleum etherethyl acetate chloroform and methanol respectively On the basis of preliminaryphytochemical tests conducted the methanol extract was found to be rich in termsof chemical constituents and therefore was selected for the experiment The presentextract according to our pervious study contains kaempferol 3-O-rutinoside quer-cetin 34prime7-trimethyl ether folic acid 125-Dihydroxyvitamin D2 and many otheractive ingredients (Al-Olayan et al 2014b Chang et al 2008) The methanol wasremoved under reduced pressure to obtain a semisolid mass of methanolic extractof physalis (MEPh) The MEPh was then stored at minus20 degC until use The total poly-phenol content of MEPh was 1382 mg gallic acid equivalentg extract determinedfollowing the standard FolinndashCiocalteu method using gallic acid as a standard andflavonoids content was 937 mg quercetin equivalentg extract determined follow-ing the AlCl3 colorimetric method using quercetin as a standard

24 Experimental protocol

To study the protective effects of physalis on cadmium mediated hepatorenal tox-icity adult male rats were randomly allocated to four groups of seven rats per groupGroup I (Con) served as a control group and received 300 μL of saline by oral adminis-tration route everyday for 5 days Group II (Cd(II)) received daily 300 μL of saline by oraladministration and after 1 hour they received intraperitoneal injection of 65 mg CdCl2kg bwt for 5 days Group III (MEPh) received daily oral administration of 200 mg MEPhkg bwt for 5 days and the animals of group IV (MEPh+Cd(II)) concomitantly received200 mg MEPhkg bwt 1 hour before cadmium injection for 5 days The level of the extractin the present study was based on a preliminary range finding study in which rats (fiveper group) were administered 0 100 200 and 500 mgkg bwtday for 10 consecutivedays At the end of the preliminary study histopathological and biochemical investiga-tions did not reveal any adverse effects of the extract at 100 and 200 mgkg bwt Theintraperitoneal injection of cadmium chloride (65 mgkg bwt) was chosen according toKyriakou et al (2013)

After 24 hours of the last injection of CdCl2 animals from all groups were sac-rificed by cervical dislocation under diethyl ether anesthesia and blood samples werecollected for serum analysis Parts of the liver and right kidney were excised weighedand homogenized immediately to give 50 (wv) homogenate in ice-cold mediumcontaining 50 mM TrisndashHCl pH 74 The homogenate was centrifuged at 3000 rpmfor 10 min at 4 degC The supernatant (10) was used for the various biochemicaldeterminations

25 Kidney index

Relative weight of kidney was calculated as left kidney weightbody weight times 100

26 Liver function test

Colorimetric determination of serum alanine aminotransferase (ALT) or aspar-tate aminotransferase (AST) was estimated by measuring the amount of pyruvate

or oxaloacetate produced by the formation of 2 4-dinitrophenylhydrazine accord-ing to the method of Reitman and Frankel (1957) Alkaline phosphatase (ALP) wasassayed using kits provided by Randox Laboratories Co according to the methodsdescribed by Belfield and Goldberg (1971) In addition serum total bilirubin (TB)was assayed according to the method of Garber (1981)

27 Kidney function test

The serum urea was estimated by Wybenga et al (1971) using the commer-cially available kit Briefly urea was condensed with diacetyl monoxime in an acidicmedium to form a red colored complex In addition the serum creatinine was es-timated by modified Jaffersquos method (Chromy et al 2008) Meanwhile uric acid wasmeasured in the serum according to the method of Fossati et al (1980)

28 Oxidative stress markers

Liver and kidney homogenates were used to determine lipid peroxidation (LPO)by reaction of thiobarbituric acid (Ohkawa et al 1979) Similarly nitritenitrate (nitricoxide NO) and glutathione (GSH) were assayed using the methods of Green et al(1982) and Ellman (1959) respectively

29 Enzymatic antioxidant status

Homogenates of liver and kidney were used for the determination of superox-ide dismutase (SOD) according to Nishikimi et al (1972) catalase (CAT) as describedby Aebi (1984) glutathione peroxidase (GPx) according to Paglia and Valentine (1967)and glutathione reductase (GR) as described by Factor et al (1998)

210 Histopathological examination

Small pieces of liver and cortex of kidney were washed in saline and fixed in10 phosphate buffered formalin embedded in paraffin sectioned at 5 μm and stainedwith hematoxylin and eosin for light microscopic observations

211 Immunohistochemical analyses of Bcl-2

The immunolocalization technique used for Bcl-2 was performed on 3ndash4 μm thick-ness sections according to Pedrycz and Czerny (2008) For negative controls theprimary antibody was omitted In brief mouse anti-Bcl-2 (diluted 1200 Santa CruzBiotechnology Santa Cruz CA USA) was incubated with sections for 60 minutesPrimary antibodies were diluted in TBS (Tris buffered saline)1 BSA (bovine serumalbumin) Then a biotinylated secondary antibody directed against mouse immu-noglobulin (Biotinylated Link UniversalndashDakoCytomation kit supplied ready to use)was added and incubated for 15 minutes followed by horse radish peroxidase con-jugated with streptavidin (DakoCytomation kit supplied ready to use) for an additional15 minutes of incubation At the sites of immunolocalization of the primary anti-bodies a reddish to brown color appeared after adding 3-amino-9-ethylcarbasole(AEC) (DakoCytomationkit supplied ready to use) for 15 minutes Specimens werecounterstained with hematoxylin for 1 minute and mounted using the Aquatex fluid(Merck KGaA Germany) Next the staining intensity was graded as very weak weakmedium or strong All sections were incubated under the same conditions withthe same concentration of antibodies and at the same time in order for theimmunostaining to be comparable among the different experimental groups

212 Statistical analysis

Results were expressed as the mean plusmn standard error of the mean (SEM) Datafor multiple variable comparisons were analyzed by one-way analysis of variance(ANOVA) For the comparison of significance between groups Duncanrsquos test was usedas a post-hoc test according to the statistical package program (SPSS version 170)and figures were drawn with Origin (version 8) All p values are two-tailed and p lt 005was considered significant for all statistical analysis in this study In order to discernpossible interactions between physalis treatment and Cd injection a one-way anal-ysis of variance (ANOVAMANOVA test F) was conducted F value of Wilksrsquos lambdahaving p lt 005 were considered statistically significant

3 Results

Cd was able to induce a significant increase (p lt 005) in the ac-tivities of the liver enzymes ALT AST ALP and TB level comparedto the control group (Table 1) treatment of rats with MEPh effi-ciently restored liver enzymes and TB level toward the control group

99AEA Moneim et alFood and Chemical Toxicology 74 (2014) 98ndash106

(Table 1) Statistical analysis (one-way MANOVA protection as inter-subject factor type III sum of squares) revealed protecting effectsmediated by physalis extract on cadmium poisoning on the levelof ALT F (606845 3) = 3043 AST F (414282 3) = 2738 ALP F(843540 3) = 4796 and TB F (1783 3) = 46325

Exposure to Cd caused a significant increase (p lt 005) in serumuric acid urea and creatinine levels when compared to the controlvalues (Table 1) Pre-treatment of animals with MEPh signifi-cantly reduced the elevated levels of serum uric acid urea andcreatinine (p lt 005) Statistical analysis disclosed protecting effectsmediated by physalis extract on cadmium toxicity on the level ofuric acid F (862 3) = 14722 urea F (10316 3) = 40034 and cre-atinine F (081 3) = 5227

Fig 1 showed the effects of kidney weight and kidney index inall experimental animals It has been observed that Cd exposurereduced the kidney weight as well as kidney index and that couldbe prevented by the MEPh pre-treatment MANOVA analysis showedthat physalis extract offered protecting effects against Cd-induceddiminishing in kidney weight and kidney index F (090 3) = 14691and F (029 3) = 3990 respectively

As depicted in Fig 2 the liver and kidney LPO levels in Cd(II) groupsignificantly elevated in response to Cd treatment compared withcontrol (p lt 005) indicating that the treatment of Cd caused obviousoxidative damage to rats We found that the increase was dimin-ished by pre-treatment with MEPh (F (170 3) = 11332 for the liverand F (134 3) = 5285 for the kidney) In addition the administra-tion of MEPh alone to rats caused a significant reduction of LPO levelin liver homogenate (p lt 005)

NO is also considered to be an important parameter of oxida-tive stress The elevated levels of NO have been observed in the Cd(II)intoxicated hepatic and renal tissues of the experimental animals(Fig 2) MEPh pre-treatment was found to be effective in prevent-ing the Cd-induced NO production (F (245 3) = 14704 for the liverand F (223 3) = 6742 for the kidney)

The effects of MEPh on GSH contents against Cd toxicity havebeen represented in Fig 2 Our study revealed that Cd injectionreduced the contents of GSH in hepatic and renal tissues and ad-ministration of MEPh might keep the status of this molecule nearlyclose to normal against Cd toxicity (F (0002 3) = 4871 for the liverand F (0001 3) = 1673 for the kidney)

The activities of the antioxidant enzymes SOD CAT GPx and GRin the liver and kidney tissues of the experimental animals have beenshown in Table 2 Current findings represent that Cd administra-tion significantly (p lt 005) inhibited the activities of the antioxidantenzymes compared to the control rats Results show that pre-treatment of rats with MEPh could prevent Cd-induced alternatedactivities of the antioxidant enzymes However MEPh treatmentalone decreased the activity of SOD and increased GPx and GR ac-tivities in hepatic homogenates when compared with the controlrats (Table 2) Our results demonstrated that MEPh per se in-creased significantly the renal GPx activity suggesting that MEPhhas antioxidant effects Statistical analysis using MANOVA re-vealed protecting effects mediated by physalis extract on cadmium-induced hepatic toxicity on the activity of SOD F (856734123) = 1559 CAT F (0003 3) = 2365 GPx F (053 3) = 2875 and GRF (6442 3) = 10479 Although MANOVA analysis showed that phy-salis extract offered protecting effects against cadmium-induced renaltoxicity on the activity of SOD F (137263453 3) = 10826 CAT F(0005 3) = 3353 GPx F (024 3) = 2484 and GR F (4027 3) = 32518

Fig 3 represents the histological findings of the hepatic tissueof the control and experimental group of rats Control liver sec-tions appeared with normal hepatic cord pattern hepatic lobulesand hepatocytes (Fig 3A) Cd exposure caused severe pathologicallesions in hepatic tissues these lesions in the form of loss of theparenchymal architecture apparent broad hepatocellular swellingand lysis of hepatocyte plasma membranes after Cd challenge Ex-tensive granular and vesicular degeneration vacuolation andinflammatory cell infiltrations in portal region in the liver were alsoobserved (Fig 3B) These pathological alterations were dramatical-ly ameliorated in the liver of rats pre-treated with MEPh (Fig 3D)

Fig 4 showed the histopathological changes in renal tissues ofthe control and experimental rats We observed shrinkage of glom-erulus and the degeneration of tubules suggesting the damage ofkidney in rats administered with Cd (Fig 4B) MEPh pre-treatmentcould prevent the Cd-induced histopathological alterations and keepthe pathological lesions almost close to normal range (Fig 4D)

The immunohistochemical investigation for Bcl-2 displayed thatthere were some immunoreactivity in the hepatic and renal tissuesof the control group which indicated the normal life cycle of cells(Figs 5A and 6A respectively) Immunostaining for Bcl-2 was de-creased in the hepatic tissue (Fig 5B) and was slightly increased inrenal tissue in Cd(II) group (Fig 6B) The protective effects of MEPh

Table 1Serum ALT AST ALP total bilirubin uric acid urea and creatinine of the studied groups

Groups ALT (IUL) AST (IUL) ALP (IUL) Total bilirubin (mgdL) Uric acid (mgdL) Urea (mgdL) Creatinine (mgdL)

Control group 638 plusmn 382 547 plusmn 274 1125 plusmn 751 163 plusmn 011 264 plusmn 011 584 plusmn 015 063 plusmn 003Cd(II) group 1084 plusmn 573a 857 plusmn 492a 1685 plusmn 563a 385 plusmn 027a 431 plusmn 021a 1145 plusmn 083a 106 plusmn 006a

MEPh group 683 plusmn 417 512 plusmn 331 1223 plusmn 892 152 plusmn 013 284 plusmn 009 607 plusmn 031 057 plusmn 002MEPh+Cd(II) group 785 plusmn 585ab 711 plusmn 463ab 1473 plusmn 1138ab 224 plusmn 017ab 321 plusmn 014ab 783 plusmn 018ab 083 plusmn 003ab

Values are means plusmn SEM (n = 7)a p lt 005 significant change with respect to Control groupb p lt 005 significant change with respect to Cd(II) group for Duncanrsquos post hoc test

Fig 1 Effects of MEPh on kidney weight and relative kidney weight kidney indexof rats treated with CdCl2 Values are means plusmn SEM (n = 7) ap lt 005 significant changewith respect to Control group bp lt 005 significant change with respect to Cd(II)group for Duncanrsquos post hoc test

100 AEA Moneim et alFood and Chemical Toxicology 74 (2014) 98ndash106

were revealed in rats pre-treated with MEPh where the intensityof immunostaining reaction of Bcl-2 was increased in both the liverand kidney indicating the anti-apoptotic effects of MEPh (Figs 5Dand 6D respectively)

4 Discussion

In the present study it has been observed that acute cadmiumintoxication caused hepatic and renal oxidative damages Experi-mental results suggest that physalis treatment could prevent theCd-induced hepatic and renal oxidative insults in the experimen-tal rats

In this study the administration of CdCl2 exerts possible hepa-totoxicity as verified by the increase in serum ALT AST ALP activitiesand TB level In fact these enzymes are known as important markersof hepatocellular damage as affirmed by Abdel-Moneim et al (2011)This damage was in accordance with the cellular damages and lossof hepatic tissue structural pattern in CdCl2-treated animals (Fig 3B)The elevation in liver enzymes and TB level has been well sup-ported by Pari and Shagirtha (2012) and Renugadevi and Prabu(2010) who have demonstrated that inorganic Cd causes hepato-toxicity These alterations in the liver function test in Cd(II)-treated rats were regarded as the main manifestations of the hepatictoxicity of Cd Physalis extract (200 mgkg bwt) may stabilize thehepatic cellular membrane and protect the hepatocytes against toxiceffects of Cd which may decrease the leakage of the enzymes intothe blood stream In this context the membrane protective effectof physalis has already been reported by Al-Olayan et al (2014b)In general polyphenols were able to maintain cell membrane in-tegrity protecting cells from death Interestingly this protection wasnot related to enhance intracellular antioxidant capacity (Tavareset al 2012)

The kidney is generally recognized as the most critical organaffected by chronic exposure to cadmium Renal dysfunction inlaboratory animals is commonly reported in cadmium toxi-city Cadmium reaches the kidney in the form of cadmium-metallothionein that is filtrated in the glomerulus and subsequentlyreabsorbed in the proximal tubules It then remains in the tubulecells and results in kidney damage (Godt et al 2006) As for therenal markers uric acid urea and creatinine blood level were higherin groups intoxicated with cadmium Additionally renal histopatho-logic damage has been observed in cadmium intoxicated rats(El-Boshy et al 2014) Pre-treatment with physalis significantly pre-vented the increase in serum uric acid urea and creatinine whichdisplayed dramatic improvement in the Cd(II) nephrotoxicity anddegenerative alterations in renal corpuscles and renal tubules(Fig 4D) The protective effect of physalis against cadmium toxic-ity could be attributed to its antioxidant activity Interactions betweenphysalis extract and Cd apparently resulted in partial ameliora-tion of Cd toxicity These results are in accordance with several otherresearches reporting that compounds with antioxidant propertiespartially prevented the increase in the levels of serum uric acid ureaand creatinine (Abdel Moneim and El-Khadragy 2013 AbdelMoneim et al 2013) Moreover polyphenolic compounds could playa major role in enhancing the antioxidant system metal chelatorsand enzyme modulators in kidney that could protect the kidney(Rodrigo and Bosco 2006) In the present work physalis amelio-rates cadmium hepatorenal-toxicity in physalis and cadmium treatedgroup Several mechanisms could be operating in the protectiveaction of physalis which could result for example in changed ab-sorption of cadmium or in a change in their action and distributionin the organism and within target organs The protective mecha-nism of physalis is well known for their ability to scavenge ROS andenhancement of the antioxidant system in cadmium induced tissuedamage (Othman et al 2014a)

Fig 2 Effects of MEPh on lipid peroxidation nitric oxide and glutathione levels inliver and kidney of rats treated with CdCl2 Values are means plusmn SEM (n = 7 ap lt 005significant change with respect to Control group bp lt 005 significant change withrespect to Cd(II) group for Duncanrsquos post hoc test

101AEA Moneim et alFood and Chemical Toxicology 74 (2014) 98ndash106

The lipid peroxidation is the additional indicator of organ oxi-dative injury In the present study the level of malondialdehyde(MDA) has been measured as an indicator of lipid peroxidation Thelevel of MDA has been significantly increased in the liver and kidneytissues of the Cd(II)-intoxicated rats Our experimental findingssuggest that oxidative stress plays an important role in cadmium-induced hepatorenal injuries Our results stand in accordance withother findings reporting Cd-induced oxidative damage (El-Boshyet al 2014 Milton Prabu et al 2013) Pre-treatment with physa-lis could prevent the cadmium-induced alteration in the level of lipidperoxidation (Fig 2) probably by eliminating the toxin from the bodyor encapsulating Cd which could prevent the toxin from causingfurther oxidative injury (Sinha et al 2009)

In accordance with the present findings previous studies showedthat exposure to cadmium induced intense inflammatory reactionas manifested by overexpression of NO in several tissues (Al-Azemiet al 2010 Othman et al 2014a) This can be attributed to the up-regulation of inducible nitric oxide synthase (iNOS) by TNF-αincreasing NO production (Arafa et al 2014) Excess NO reacts withsuperoxide anion to produce peroxynitrite radical inducing furthercell damage by depletion of intracellular GSH increasing the vul-nerability to oxidative stress (El-Habit and Abdel Moneim 2014)Our results revealed that administration of physalis minimized thedeleterious effects of Cd on both hepatic and renal tissues such asthe rise in lipid peroxidation and nitric oxide These findings arein agreement with the previous study of Al-Olayan et al (2014b)

Table 2Antioxidant enzymes activities in liver and kidney tissues of the studied groups

Groups Liver Kidney

SOD (Umgprotein)

CAT (Umgprotein)

GPx (Umgprotein)

GR (μmolhmgprotein)

SOD (Umgprotein)

CAT (Umgprotein)

GPx (Umgprotein)

GR (μmolhmgprotein)

Control group 12379 plusmn 526 0082 plusmn 0006 095 plusmn 004 701 plusmn 022 19186 plusmn 733 0102 plusmn 0009 065 plusmn 004 721 plusmn 039Cd(II) group 9535 plusmn 318a 0062 plusmn 0002a 071 plusmn 003a 431 plusmn 031a 14397 plusmn 874a 0073 plusmn 0003a 052 plusmn 003a 408 plusmn 021a

MEPh group 11083 plusmn 387a 0086 plusmn 0003 118 plusmn 004a 842 plusmn 036a 20143 plusmn 1156 0113 plusmn 0005 082 plusmn 004a 787 plusmn 035MEPh+Cd(II) group 14611 plusmn 336ab 0093 plusmn 0003ab 097 plusmn 004b 883 plusmn 028ab 21364 plusmn 1373ab 0107 plusmn 0004b 071 plusmn 003b 619 plusmn 027ab

Values are means plusmn SEM (n = 7)a p lt 005 significant change with respect to Control groupb p lt 005 significant change with respect to Cd(II) group for Duncanrsquos post hoc test

Fig 3 Histological changes in hepatic tissue of rats (A) A control liver with normal architecture (B) Rats treated with CdCl2 with prominent inflammation hepatocyticvacuolation and pyknotic nucleus in hepatocytes (C) Rats treated with MEPh exhibited normal architecture (D) Rats treated with the MEPh and CdCl2 maintained the hepaticarchitecture with minimal damage Sections were stained with hematoxylin and eosin (400times scale bar 20 nm)

102 AEA Moneim et alFood and Chemical Toxicology 74 (2014) 98ndash106

The hepatorenal protective effect of physalis against Cd toxicity maybe attributed to its inhibitory effect in NO production where NOplays pivotal roles in oxidative stress inflammation and apopto-sis However further studies are needed to elucidate its exactmechanism

The impairment of the antioxidant defense system is consid-ered as a critical event in Cd-induced hepatorenal toxicity Exposureof Cd is characterized by the depletion of tissue non-enzymatic an-tioxidants including GSH GSH is an important antioxidant defensewhich forms complexes with Cd through the free sulfhydryl groupand thereby alters Cd distribution and excretion (Pari and Shagirtha2012)

The endogenous antioxidant enzymes like SOD CAT GPx and GRpresent the first line of defense against free-radical damage (Othmanet al 2014b) From the present results the activity of antioxi-dants enzymes SOD CAT GR and GPx were significantly (p lt 005)reduced in hepatic and renal tissues of Cd-treated rats in compar-ison to the control group which indicated that Cd was able to inducesevere oxidative stress

The inhibitory action of cadmium on SOD may be due to com-petition between Cd and Zn or Cu that is required for the activityof SOD and creates an inactive form of the enzyme (CuCd3+SOD)(Huang et al 2006) Catalase is a hemeprotein which catalyses thereduction of H2O2 to water and oxygen and thus protects the cellfrom oxidative damage by H2O2 and OH (Hagar and Al Malki 2014)The decrease in catalase activity by cadmium may be attributed to

the decreased absorption of iron an essential trace element re-quired for the activity of catalase Moreover there is increasingevidence that Cd interacts with selenium (Se) which forms a SendashCd complex that disrupts GPx activity (Trabelsi et al 2013) Inaddition Wasowicz et al (2001) found that Cd exposure de-creased the Se concentration and GPx activity in both theerythrocytes and plasma of exposed workers

Furthermore it was revealed that physalis extract recovered theactivities of the antioxidant enzymes such as SOD CAT GPx and GRin Cd(II) treated rats The protective effects of physalis extract inmaintaining the GSH level toward control have increased the ca-pacity of endogenous antioxidant defense and increased the steadystate of GSH andor its rate of synthesis that confers enhanced pro-tection against oxidative stress It has been noticed that many ofplants which are rich in phenolic compounds and flavonoids arewidely used as antioxidant and antimutagenic agent (AbdelMoneim et al 2014) Various chemical compounds like 28-hydroxywithanolide withanolides phygrine kaempferol andquercetin di- and tri-glycosides are reported to be present inphysalis (Abdel Moneim and El-Deib 2012)

Our data revealed that physalis caused a significant decrease insuperoxide dismutase this reduction may be due to the inferenceof physalis active ingredients with SOD However polyphenols areknown to be able to modulate the transcription and expression ofproteins related to the endogenous antioxidant defense by inter-acting with antioxidant response elements in gene promoter regions

Fig 4 Histological changes in renal tissue of rats (A) A control kidney with normal renal corpuscle and renal tubules (B) Rats treated with CdCl2 with congested shrunkand completely degenerated glomeruli debris in the lumen of some renal tubules and pyknotic nuclei in renal tubules (C) Rats treated with MEPh exhibited normal ar-chitecture (D) Rats treated with the MEP and CdCl2 showed normal renal corpuscle and renal tubule more or less like normal structure Sections were stained with hematoxylinand eosin (400times scale bar 20 nm)

103AEA Moneim et alFood and Chemical Toxicology 74 (2014) 98ndash106

of genes encoding proteins related to oxidative injury manage-ment It is not clear however if the effects of physalis juice intakeresult from polyphenolsrsquo interference with enzymes and genes orif it is a consequence of a broader and more unspecific action con-nected to their antioxidant potential Moreover many factors needto be taken into account when examining the present results as thedegree of oxidative stress the polyphenol class and concentrationas well as the biological system studied may all introduce other el-ements of variability in the response to antioxidant ingestion(Othman et al 2014a)

In addition to the induction of direct cellular damage oxida-tive stress is an important mediator for apoptosis andor necrosisAs a potent signal molecule of the death pathway ROS is involvedin the regulation of apoptosis (Othman et al 2014b) Bcl-2 is thoughtto be a common mediator for prevention of programmed cell deathbecause Bcl-2 can suppress apoptotic cell death induced by a varietyof physiologic and nonphysiologic reagents (Ishido et al 2002)However our results revealed that Bcl-2 expression was slightly in-creased in the renal tissue The level of induction of Bcl-2 proteinswas not sufficient to completely suppress apoptosis induced bycadmium in the kidney (Ishido et al 2002) In this study physalisabolished the cadmium-stimulated apoptosis by inhibiting the ex-pression of Bcl-2 and restoring its expression in both hepatic andrenal tissues These effects of physalis may be the basis for its pro-tection against pathological changes in the liver and kidney inrats induced by Cd Overexpressions of Bcl-2 has recently been

reported to inhibit ROS production cytosolic cytochrome crelease and poly (ADP-ribose) polymerase (PARP) degradation-mediated apoptosis and mediated autophagy (de David et al2011)

In conclusion the present results indicated that chronic expo-sure to Cd impaired both hepatic and renal functions as well as Bcl-2expression in male rats These results also suggest that the bene-ficial effects of physalis on restoring Cd-induced impairment ofhepatic and renal functions are likely to be mediated by its anti-oxidant anti-apoptotic and anti-inflammatory activities ThereforeP peruviana can be considered a potential therapeutic nutrient toprotect against hepatic and renal toxicity induced by Cd These effectsof physalis may be the basis for the protection against pathologi-cal changes in liver and kidney of rats induced by CdCl2

Conflict of interest

The authors declare that there are no conflicts of interest in thiswork

Transparency document

The Transparency document associated with this article can befound in the online version

Fig 5 Immunohistochemical localization of Bcl-2 antigen in hepatic tissue of rats (A) Section of control liver (B) Section of rats treated with CdCl2 with less staining ofBcl-2 (C) Section of liver of rats treated with MEPh (D) Section of liver of rats pre-treated with the MEPh and CdCl2 with more staining of Bcl-2 than Cd(II) group Thestaining intensity was graded as (white star) very weak (yellow star) weak (orange star) medium or (red star) strong (400times scale bar 20 nm) (For interpretation of thereferences to color in this figure legend the reader is referred to the web version of this article)

104 AEA Moneim et alFood and Chemical Toxicology 74 (2014) 98ndash106

Acknowledgment

The authors extend their appreciation to the Deanship of Sci-entific Research at King Saud University for funding this workthrough Research Group Project number RG-1435-198

References

Abdel Moneim AE 2012 Evaluating the potential role of pomegranate peel inaluminum-induced oxidative stress and histopathological alterations in brain offemale rats Biol Trace Elem Res 150 328ndash336

Abdel Moneim AE El-Deib KM 2012 The possible protective effects of Physalisperuviana on carbon tetrachloride-induced nephrotoxicity in male albino ratsLife Sci J 9 1038ndash1052

Abdel Moneim AE El-Khadragy MF 2013 The potential effects of pomegranate(Punica granatum) juice on carbon tetrachloride-induced nephrotoxicity in ratsJ Physiol Biochem 69 359ndash370

Abdel Moneim AE Othman MS Mohmoud SM El-Deib KM 2013 Pomegranatepeel attenuates aluminum-induced hepatorenal toxicity Toxicol Mech Methods23 624ndash633

Abdel Moneim AE Othman MS Aref AM 2014 Azadirachta indica attenuatescisplatin-induced nephrotoxicity and oxidative stress BioMed Res Int 2014647131

Abdel-Moneim AE Dkhil MA Al-Quraishy S 2011 The redox status in rats treatedwith flaxseed oil and lead-induced hepatotoxicity Biol Trace Elem Res 143457ndash467

Aebi H 1984 Catalase in vitro Methods Enzymol 105 121ndash126Al-Azemi M Omu FE Kehinde EO Anim JT Oriowo MA Omu AE 2010

Lithium protects against toxic effects of cadmium in the rat testes J Assist ReprodGenet 27 469ndash476

Al-Olayan E El-Khadragy M Metwally D Abdel Moneim A 2014a Protectiveeffects of pomegranate (Punica granatum) juice on testes againstcarbon tetrachloride intoxication in rats BMC Complement Altern Med 14164

Al-Olayan E Elkhadragy MF Othman MS Aref A Kassab R Abdel Moneim AE2014b The potential protective effect of Physalis peruviana L against carbontetrachloride-induced hepatotoxicity in rats is mediated by suppression ofoxidative stress and downregulation of MMP-9 expression Oxid Med CellLongev 2014 381413

Arafa MH Mohammad NS Atteia HH 2014 Fenugreek seed powder mitigatescadmium-induced testicular damage and hepatotoxicity in male rats Exp ToxicolPathol 66 (7) 293ndash300

Belfield A Goldberg DM 1971 Revised assay for serum phenyl phosphatase activityusing 4-amino-antipyrine Enzyme 12 561ndash573

Chang JC Lin CC Wu SJ Lin DL Wang SS Miaw CL et al 2008 Antioxidativeand hepatoprotective effects of Physalis peruviana extract against acetaminophen-induced liver injury in rats Pharm Biol 46 724ndash731

Chromy V Rozkosna K Sedlak P 2008 Determination of serum creatinine by Jaffemethod and how to calibrate to eliminate matrix interference problems ClinChem Lab Med 46 1127ndash1133

de David C Rodrigues G Bona S Meurer L Gonzaacutelez-Gallego J Tuntildeoacuten MJ et al2011 Role of quercetin in preventing thioacetamide-induced liver injury in ratsToxicol Pathol 39 949ndash957

El-Boshy ME Risha EF Abdelhamid FM Mubarak MS Hadda TB 2014Protective effects of selenium against cadmium induced hematologicaldisturbances immunosuppressive oxidative stress and hepatorenal damage inrats J Trace Elem Med Biol doi101016jjtemb201405009

El-Habit O Abdel Moneim AE 2014 Testing the genotoxicity cytotoxicity andoxidative stress of cadmium and nickel and their additive effect in male miceBiol Trace Elem Res 159 (1ndash3) 364ndash372

Ellman GL 1959 Tissue sulfhydryl groups Arch Biochem Biophys 82 70ndash77Factor VM Kiss A Woitach JT Wirth PJ Thorgeirsson SS 1998 Disruption of

redox homeostasis in the transforming growth factor-alphac-myc transgenic

Fig 6 Immunohistochemical localization of Bcl-2 antigen in renal tissue of rats (A) Section of control kidney (B) Section of rats treated with CdCl2 with slightly increasedstaining of Bcl-2 (C) Section of kidney of rats treated with MEPh (D) Section of kidney of rats pre- treated with MEPh and CdCl2 with more staining of Bcl-2 than Cd(II)group The staining intensity was graded as (white star) very weak (yellow star) weak (orange star) medium or (red star) strong (400times scale bar 20 nm) (For interpreta-tion of the references to color in this figure legend the reader is referred to the web version of this article)

105AEA Moneim et alFood and Chemical Toxicology 74 (2014) 98ndash106

mouse model of accelerated hepatocarcinogenesis J Biol Chem 273 15846ndash15853

Fossati P Prencipe L Berti G 1980 Use of 35-dichloro-2-hydroxybenzenesulfonicacid4-aminophenazone chromogenic system in direct enzymic assay of uric acidin serum and urine Clin Chem 26 227ndash231

Garber CC 1981 JendrassikndashGrof analysis for total and direct bilirubin in serumwith a centrifugal analyzer Clin Chem 27 1410ndash1416

Godt J Scheidig F Grosse-Siestrup C Esche V Brandenburg P Reich A et al2006 The toxicity of cadmium and resulting hazards for human health J OccupMed Toxicol 1 22

Green LC Wagner DA Glogowski J Skipper PL Wishnok JS Tannenbaum SR1982 Analysis of nitrate nitrite and [15N]nitrate in biological fluids AnalBiochem 126 131ndash138

Hagar H Al Malki W 2014 Betaine supplementation protects against renal injuryinduced by cadmium intoxication in rats role of oxidative stress and caspase-3Environ Toxicol Pharmacol 37 803ndash811

Huang YH Shih CM Huang CJ Lin CM Chou CM Tsai ML et al 2006 Effectsof cadmium on structure and enzymatic activity of CuZn-SOD and oxidativestatus in neural cells J Cell Biochem 98 577ndash589

Ishido M Ohtsubo R Adachi T Kunimoto M 2002 Attenuation of both apoptoticand necrotic actions of cadmium by Bcl-2 Environ Health Perspect 11037ndash42

Kyriakou LG Tzirogiannis KN Demonakou MD Kourentzi KT Mykoniatis MGPanoutsopoulos GI 2013 Gadolinium chloride pretreatment ameliorates acutecadmium-induced hepatotoxicity Toxicol Ind Health 29 624ndash632

Lopez E Figueroa S Oset-Gasque MJ Gonzalez MP 2003 Apoptosis and necrosistwo distinct events induced by cadmium in cortical neurons in culture Br JPharmacol 138 901ndash911

Milton Prabu S Muthumani M Shagirtha K 2013 Quercetin potentially attenuatescadmium induced oxidative stress mediated cardiotoxicity and dyslipidemia inrats Eur Rev Med Pharmacol Sci 17 582ndash595

Nishikimi M Appaji N Yagi K 1972 The occurrence of superoxide anion in thereaction of reduced phenazine methosulfate and molecular oxygen BiochemBiophys Res Commun 46 849ndash854

Ohkawa H Ohishi N Yagi K 1979 Assay for lipid peroxides in animal tissues bythiobarbituric acid reaction Anal Biochem 95 351ndash358

Othman MS Nada A Zaki HS Abdel Moneim AE 2014a Effect of Physalisperuviana L on cadmium-induced testicular toxicity in rats Biol Trace Elem Res159 (1ndash3) 278ndash287

Othman MS Safwat G Aboulkhair M Abdel Moneim AE 2014b The potentialeffect of berberine in mercury-induced hepatorenal toxicity in albino rats FoodChem Toxicol 69 175ndash181

Paglia DE Valentine WN 1967 Studies on the quantitative and qualitativecharacterization of erythrocyte glutathione peroxidase J Lab Clin Med 70158ndash169

Pari L Shagirtha K 2012 Hesperetin protects against oxidative stress related hepaticdysfunction by cadmium in rats Exp Toxicol Pathol 64 513ndash520

Pedrycz A Czerny K 2008 Immunohistochemical study of proteins linked toapoptosis in rat fetal kidney cells following prepregnancy adriamycinadministration in the mother Acta Histochem 110 519ndash523

Reitman S Frankel S 1957 A colorimetric method for the determination of serumglutamic oxalacetic and glutamic pyruvic transaminases Am J Clin Pathol 2856ndash63

Renugadevi J Prabu SM 2010 Cadmium-induced hepatotoxicity in rats and theprotective effect of naringenin Exp Toxicol Pathol 62 171ndash181

Rodrigo R Bosco C 2006 Oxidative stress and protective effects of polyphenolscomparative studies in human and rodent kidney A review Comp BiochemPhysiol B Biochem Mol Biol 142 317ndash327

Sinha M Manna P Sil PC 2009 Induction of necrosis in cadmium-induced hepaticoxidative stress and its prevention by the prophylactic properties of taurine JTrace Elem Med Biol 23 300ndash313

Tavares L Figueira I Macedo D McDougall GJ Leitatildeo MC Vieira HLA et al2012 Neuroprotective effect of blackberry (Rubus sp) polyphenols is potentiatedafter simulated gastrointestinal digestion Food Chem 131 1443ndash1452

Trabelsi H Azzouz I Ferchichi S Tebourbi O Sakly M Abdelmelek H 2013Nanotoxicological evaluation of oxidative responses in rat nephrocytes inducedby cadmium Int J Nanomedicine 8 3447ndash3453

Wasowicz W Gromadzinska J Rydzynski K 2001 Blood concentration of essentialtrace elements and heavy metals in workers exposed to lead and cadmium IntJ Occup Med Environ Health 14 223ndash229

Wybenga DR Di Giorgio J Pileggi VJ 1971 Manual and automated methods forurea nitrogen measurement in whole serum Clin Chem 17 891ndash895

106 AEA Moneim et alFood and Chemical Toxicology 74 (2014) 98ndash106