Pharmacological Reports 66 (2014) 1050–1059

Original research article

Alleviation of hepatic injury by chrysin in cisplatin administered rats:Probable role of oxidative and inflammatory markers

Muneeb U Rehman, Nemat Ali, Summya Rashid, Tyan Jain, Sana Nafees, Mir Tahir,Abdul Quaiyoom Khan, Abdul Lateef, Rehan Khan, Oday O. Hamiza, Syed Kazim,Wajhul Qamar, Sarwat Sultana *

Molecular Carcinogenesis and Chemoprevention Division, Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard

University), New Delhi, India

A R T I C L E I N F O

Article history:

Received 18 May 2013

Received in revised form 17 April 2014

Accepted 5 June 2014

Available online 8 July 2014

Keywords:

Cisplatin

Inflammation

Oxidative stress

Hepatotoxicity

A B S T R A C T

Background: Cisplatin is an effective and extensively used chemotherapeutic agent to treat range of

malignancies, but its therapeutic use is limited because of dose-dependent nephrotoxicity and

hepatotoxicity. Several published reports advocate that supplementation with antioxidant can influence

cisplatin induced hepatic damage.

Method: In the present study the Wistar rats were subjected to concurrent prophylactic oral treatment

of chrysin (25 and 50 mg/kg b.wt.) against the hepatotoxicity induced by intraperitoneal administration

of cisplatin (7.5 mg/kg b.wt.). Efficacy of chrysin against the hepatotoxicity was evaluated in terms of

biochemical estimation of antioxidant enzyme activities, histopathological changes and expression

levels of molecular markers of inflammation.

Results: Chrysin ameliorated cisplatin-induced lipid peroxidation, xanthine oxidase activity, glutathione

depletion, decrease in antioxidant (catalase, glutathione reductase, superoxide dismutase, glutathione

peroxidase and glucose-6 phosphate dehydrogenase) and phase-II detoxifying (glutathione-S-

transferase and quinone reductase) enzyme activities. Chrysin also attenuated expression of COX-2,

iNOS and levels of NFkB and TNF-a, and hepatic tissue damage which were induced by cisplatin.

Histological findings further supported the protective effects of chrysin against cisplatin-induced

hepatic damage.

Conclusion: The results of the present study demonstrate that oxidative stress and inflammation are

closely associated with cisplatin-induced toxicity and chrysin shows the protective efficacy against

cisplatin-induced hepatotoxicity possibly via attenuating the oxidative stress and inflammatory

response.

� 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp.

z o.o. All rights reserved.

Contents lists available at ScienceDirect

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jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate /p h arep

Introduction

The use of chemotherapeutic agents for the treatment of cancerhas opened new prospective for improvement of the quality of lifeof cancer patients. However, besides its success, many anticancerdrugs have been shown to be teratogenic and carcinogenic inexperimental systems [1]. The nephrotoxicity of cisplatin isaccepted as the most important dose-limiting factor, but highdoses of cisplatin also produce hepatotoxicity [2–4]. The exactmechanism of cisplatin toxicity is not fully understood, but the

* Corresponding author.

E-mail address: Sarwat786@rediffmail.com (S. Sultana).

http://dx.doi.org/10.1016/j.pharep.2014.06.004

1734-1140/� 2014 Institute of Pharmacology, Polish Academy of Sciences. Published b

probable mechanism may involve oxidative stress which is due tothe massive production of reactive oxygen species (ROS), e.g. thehydroxyl radical (�OH), superoxide anion (O2

��), H2O2, etc. bycisplatin and consequently these ROS may further interact withDNA, lipids and proteins [5–8]. DNA is the central cellular target ofcisplatin that may lead to DNA damage induced by ROS andplatinum–DNA (Pt–DNA) adduct formation, thus hampering thecell division or DNA synthesis and its repair mechanism whichleads to apoptotic cell death [9,10].

Repeated aggressive cisplatin chemotherapy necessitates theinvestigation of ways for prevention of side effects that inhibitthe cisplatin administration at tumoricidal doses. Various reportsare available that are focused on the ways for amelioration ofcisplatin side effects via supplementation of preventive agents

y Elsevier Urban & Partner Sp. z o.o. All rights reserved.

M.U. Rehman et al. / Pharmacological Reports 66 (2014) 1050–1059 1051

simultaneously. These studies suggested the side effects ofcisplatin could be protected by alternate medicinal therapies[2,6,11–17]. Thus, there is a need to explore more naturalcompound that can effectively reduce the cisplatin-inducedtoxicity to enhance its chemotherapeutic efficacy via decreasingthe chemo-resistance and increasing the chemosensitization ofcisplatin.

Naturally occurring antioxidants as prospective nutraceuticalshave been reported to diminish severe side effects as well asenhance anticancer activities of antitumor drugs [18]. Flavonoidsare natural polyphenolic phytochemicals that are ubiquitous inplants and present in the average human diet [19,20]. The interesttoward flavonoid comes from the results of epidemiologicalstudies, which suggest that increased fruit and vegetableconsumption is associated with a lower risk of several types ofcancer, including lung, breast, pancreas, liver, colon, oral, larynxand prostate cancer. These suggested protective effects offlavonoids, together with their potent antioxidative and freeradical scavenging activities observed in in vivo studies haveincreased the public’s interest in the use of flavonoids for theirpotential health benefits. Chrysin (5,7-dihydroxyflavone) is anatural flavonoid found in many plant extracts, propolis and honey[21,22]. Flavonoids in general and chrysin in particular exhibitmany biological activities and pharmacological effects, includingantioxidant, anti-inflammatory, anti-cancer and anti-hypertension[2,23–30].

Based on this information, the present study was carried out toexplore the defensive effects of chrysin against cisplatin inducedhepatotoxicity. The purpose of this study was hence to inspect theprophylactic effects of chrysin against cisplatin-induced oxidativestress and anti-inflammatory responses in the liver of Wistar rats.

Materials and methods

Chemicals

Reduced glutathione (GSH), oxidized glutathione (GSSG),NADPH, NADP+, FAD, EDTA, thiobarbituric acid, pyrogallol, poly-L-lysine, xanthine, glucose-6-phosphate, bovine serum albumin,dichlorophenolindophenol, 5,50-dithio-bis-(2-nitrobenzoic acid),chrysin, 1-chloro-2,4-dinitrobenzene and glutathione reductase(GR) were obtained from Sigma (Sigma Chemical Company).Cisplatin was purchased from Dr. Reddy’s Laboratories. H2O2,magnesium chloride, sulphosalicylic acid, perchloric acid, TCA,Tween-20, Folin–Ciocalteau reagent, sodium potassium tartarate,di-sodium hydrogen phosphate, sodium di-hydrogen phosphateand sodium hydroxide were purchased from E. Merck Limited.

Animals and experimental design

Male Wistar rats (150–200 g), 6–8 weeks old, were obtainedfrom the Central Animal House Facility of Hamdard University.Rats were housed in an animal care facility under roomtemperature (25 8C) with 12 h light/dark cycles and were givenfree access to standard pellet diet and tap water. Before thetreatment, rats were left for 7 days to acclimatize. Animals receivedhumane care in accordance with the guidelines of the Committeefor the Purpose of Control and Supervision of Experiments onAnimals (CPCSEA), Government of India, and prior permission wassought from the Institutional Animal Ethics Committee.

To study the effect of prophylactic treatment with chrysin oncisplatin-induced oxidative stress and inflammatory responses inthe liver, five groups each of six male Wistar rats were kept indifferent cages as per the different dose and modulator combina-tions requirement. The rats of Group I (control group) receivedcorn oil orally at the dose of 5 ml/kg body weight (b.wt.) once daily

for 14 days, which was used as a vehicle for chrysin. Group IIIreceived chrysin orally at the dose of 25 mg/kg b.wt. once daily for14 consecutive days. Groups IV and V received chrysin at the doseof 50 mg/kg b.wt. once daily for 14 days. Groups II, III and IV weregiven a single injection of cisplatin at the dose of 7.5 mg/kg b.wt.intraperitoneally on day 14 after 1 h of the last treatment withchrysin. All the rats were anaesthetized with mild anesthesia andkilled by cervical dislocation after 24 h of the cisplatin injection.

Post-mitochondrial supernatant preparation and estimation of

different parameters

Livers were removed quickly, cleaned free of irrelevant materialand immediately perfused with ice-cold saline (0.85% NaCl). Thelivers (10%, w/v) were homogenized in chilled phosphate buffer(0.1 M, pH 7.4) using a Potter Elvehjen homogenizer. Thehomogenate was filtered through muslin cloth, and centrifugedat 3000 rpm for 10 min at 48 8C in a Remi Cooling Centrifuge (C-24DL) to separate the nuclear debris. The aliquot so obtained wascentrifuged at 12,000 rpm for 20 min at 48 8C to obtain post-mitochondrial supernatant (PMS), which was used as a source ofvarious enzymes.

Assay for catalase activity

Catalase activity was assayed by the method of Claiborne [31].The reaction mixture consisted of 1.95 ml phosphate buffer (0.1 M,pH 7.4), 1.0 ml hydrogen peroxide (0.10 mM) and 0.05 ml 10% PMSin a final volume of 3 ml. Changes in absorbance were recorded at240 nm Catalase activity was calculated as nmol H2O2 consumed/min/mg protein.

Assay for glutathione-S-transferase (GST) activity

Glutathione-S-transferase activity was estimated by themethod of Habig et al. [32]. The reaction mixture consisted of1.525 ml phosphate buffer (0.1 M, pH 7.4), 0.2 ml reducedglutathione (1 mM), 0.025 ml CDNB (1 mM) and 0.250 ml PMS(10%, w/v) in a total volume of 2.0 ml. The changes in theabsorbance was recorded at 340 nm and enzymes activity wascalculated as nmol CDNB conjugate formed min�1 mg�1 proteinusing a molar coefficient of 9.6 � 103 M�1 cm�1.

Activity of reduced glutathione (GSH)

Reduced glutathione was determined by the method of Jollowet al. [33]. Sample of PMS (1.0 ml) was precipitated with 1.0 ml ofsulphosalicylic acid (4%). The samples were kept at 4 8C for 1 h andthen centrifuged at 3000 rpm for 20 min at 4 8C. The assay mixturecontained 0.4 ml supernatant, 2.2 ml phosphate buffer (0.1 M, pH7.4) and 0.4 ml DTNB (10 mM) in a total volume of 3.0 ml. Theyellow color developed, was read immediately at 412 nm onspectrophotometer and GSH was expressed as nmol GSH/g tissue.

Activity of glutathione peroxidase (GPx)

Glutathione peroxidase activity was estimated by the methodof Mohandas et al. [34]. The reaction mixture consisted of 1.49 mlphosphate buffer (0.1 M, pH 7.4), 0.1 ml EDTA (1 mM), 0.1 mlsodium azide (1 mM), 0.05 ml glutathione reductase (1 IU/ml),0.05 ml GSH (1 mM), 0.1 ml NADPH (0.2 mM), 0.01 ml H2O2

(0.25 mM) and 0.1 ml 10% PMS in a total volume of 2 ml. Thedisappearance of NADPH at 340 nm was recorded at 250 8C.Enzyme activity was calculated as n mol NADPH oxidized perminute per mg protein using molar extinction coefficient of6.22 � 103 M�1 cm�1.

M.U. Rehman et al. / Pharmacological Reports 66 (2014) 1050–10591052

Assay of xanthine oxidase

The activity of xanthine oxidase was assayed by the method ofStirpe and Della Corte [35]. The reaction mixture consisted of 0.2 mlpost-mitochondrial supernatant (PMS) that was incubated for 5 minat 37 8C with 0.8 ml phosphate buffer (0.1 M, pH 7.4). The reactionwas started by adding 0.1 ml xanthine (9 mM) and kept at 37 8C for20 min The reaction was terminated by the addition of 0.5 ml ice-cold perchloric acid (10%, v/v). After 10 min, 2.4 ml of distilled waterwas added and centrifuged at 4000 rpm for 10 min and mg uric acidformed per minute per mg protein was recorded at 290 nm

Measurement of superoxide dismutase (SOD) activity

The SOD activity was measured by the method of Marklund andMarklund [36]. The reaction mixture consisted of 2.875 ml Tris–HCl buffer (50 mM, pH 8.5), pyrogallol (24 mM in 10 mM HCl) and100 ml PMS in a total volume of 3 ml. The enzyme activity wasmeasured at 420 nm and was expressed as units/mg protein. Oneunit of enzyme is defined as the enzyme activity that inhibits auto-oxidation of pyrogallol by 50%.

Estimation of lipid peroxidation

The assay for microsomal lipid peroxidation was done followingthe method of Wright et al. [37]. The reaction mixture in a totalvolume of 1.0 ml contained 0.58 ml phosphate buffer (0.1 M, pH 7.4),0.2 ml homogenate, 0.2 ml ascorbic acid (100 mM), 0.02 ml ferricchloride (100 mM). The reaction mixture was incubated at 37 8C in ashaking water bath for 1 h. The reaction was stopped by addition of1.0 ml 10% trichloroacetic acid (TCA). Following addition of 1.0 ml0.67% thiobarbituric acid (TBA), all the tubes were placed in boilingwater-bath for 20 min and then shifted to crushed ice-bath beforecentrifuging at 4500 rpm for 10 min The amount of malondialde-hyde formed in each of the samples was assessed by measuringoptical density of the supernatant at 535 nm using spectrophotom-eter (Milton Roy 21 D) against a reagent blank. The results wereexpressed as nmol MDA formed per hour per gram tissue at 37 8Cusing molar extinction coefficient of 1.56 � 105 M�1 cm�1.

Measurement of glucose-6-phosphate dehydrogenase (G6PD) activity

The activity of glucose-6-phosphate dehydrogenase was deter-mined by the method of Zaheer et al. [38]. The reaction mixtureconsisted of 0.3 ml Tris–HCl buffer (0.05 M, pH 7.6), 0.1 ml NADP(0.1 mM), 0.1 ml glucose-6-phosphate (0.8 mM), 0.1 ml MgCl2(8 mM), 0.3 ml PMS (10%) and 2.1 ml distilled water in a totalvolume of 3 ml. The changes in absorbance were recorded at 340 nmand enzyme activity was calculated as mmol NADP reduced/min/mgprotein using a molar extinction coefficient of 6.22 � 103 M�1 cm�1.

Measurement of glutathione reductase activity

The GR activity was measured by the method of Carlberg andMannervik [39]. The assay system containing 1.65 ml phosphatebuffer (0.1 M, pH 7.6), 0.1 ml EDTA (0.5 mM), 0.05 ml oxidizedglutathione (1.0 mM), 0.1 ml NADPH (0.1 mM) and 0.1 ml PMS(10%) in a total volume of 2.0 ml. The enzyme activity was recordedat 25 8C by measuring the disappearance of NADPH at 340 nm andwas calculated as mmol NADPH oxidized/min/mg protein usingmolar extinction coefficient of 6.22 � 103 M�1 cm�1.

Measurement of quinone reductase (QR) activity

The QR activity was determined by the method of Benson et al.[40]. The 3 ml reaction mixture consists of 2.13 ml Tris–HCl buffer

(25 mM, pH 7.4), 0.7 ml BSA, 0.1 ml FAD, 0.02 ml NADPH (0.1 mM),and 50 ml PMS (10%). The reduction of dichlorophenolindophenol(DCPIP) was recorded calorimetrically at 600 nm and the enzymeactivity was calculated as mmol of DCPIP reduced/min/mg proteinusing molar extinction coefficient of 2.1 � 104 M�1 cm�1.

Assay for serum aspartate aminotransferase and alanine

aminotransferase activity

Alanine aminotransferase (AST) and aspartate aminotransfer-ase (ALT) activity were determined by the method of Reitman andFrankel [41]. Each substrate (0.5 ml; 2 mM a-ketoglutarate andeither 200 mM L-alanine or L-aspartate) was incubated for 5 min at37 8C in a water bath. Serum (0.1 ml) was then added and thevolume was adjusted to 1.0 ml with 0.1 M (pH 7.4) phosphatebuffer. The reaction mixture was incubated for exactly 30 and60 min at 37 8C for ALT and AST, respectively. Then 0.5 ml of 1 mMdinitrophenyl hydrazine (DNPH) was added to the reactionmixture; after another 30 min at room temperature, the colorwas developed by the addition of 5.0 ml of NaOH (0.4 N) and theproduct read at 505 nm

Assay for lactate dehydrogenase activity

Lactate dehydrogenase (LDH) activity was estimated in serumby the method of Kornberg [42]. The assay mixture consisted of0.2 ml of serum, 0.1 ml of 0.02 M NADH, 0.1 ml of 0.01 M sodiumpyruvate, 1.1 ml of 0.1 M (pH 7.4) phosphate buffer and distilledwater in a total volume of 3 ml. Enzyme activity was recorded at340 nm, and activity was calculated as nmol NADH oxidized/min/mg protein.

Measurement of nitric oxide (NO)

Production of NO, cisplatin application in renal tissue, wasevaluated by measuring the level of nitrite (an indicator of NO) inthe supernatant using a colorimetric reaction with Griess reagent.Briefly, 100 mL of supernatants from different groups was mixedwith 100 ml Griess reagent [0.1% N-(1-naphthyl) ethylenediaminedihydrochloride, 1% sulfanilamide, and 2.5% H3PO4]. After incuba-tion at room temperature in the dark for 10 min, total nitrites weremeasured spectrophotometrically at 540 nm The concentration ofnitrite in the sample was determined from a NaNO2 standard curve[43].

Assay for g-glutamyl transpeptidase

GGT Activity was done by Orlowski and Meister [44]. Thereaction mixture in a total volume of 0.1 ml contained 0.2 mlserum, which was incubated with 0.8 ml of the substrate mixture(containing 4 mM g-glutamyl p-nitroanilide, 40 mM glycine and11 mM MgCl2 in 185 mM Tris HCl buffer, pH 8.25) at 37 8C. At10 min after initiation of the reaction 1.0 ml of trichloroacetic acid(TCA) (25%) was added and mixed to terminate the reaction. Thesolution was centrifuged and the supernatant fraction was read at405 nm. The enzyme activity was calculated as nmol p-nitroanilineformed/min/mg protein using a molar extinction coefficient of p-nitroaniline as 1.74 � 103 M�1 cm�1.

NFkB and TNF-alpha analysis

Serum levels of TNF-a and NFkB was analyzed. After thecompletion of treatment regimen, the animals were anesthetizedand blood withdrawn from retro-orbital sinus. Serum wasseparated from blood and the levels of above-mentionedinflammatory markers were evaluated in it by Elisa Plate Reader

Table 1Results of pretreatment of chrysin on glutathione and related enzymes like GSH, GST, GR and GPX on cisplatin induced redox imbalance.

Treatment

regimen

per group

GSH (nmol

GSH/g tissue)

GST (nmol CDNB

conjugate formed/

min/mg protein)

GR (nmol NADPH

oxidized/min/

mg protein)

GPX (nmol NADPH

oxidized/min/mg

protein)

Group I 0.47 � 0.005 80.84 � 126.2 387.2 � 13.1 271.33 � 22.10

Group II 0.30 � 0.004*** 37.06 � 9.95*** 190.9 � 14.6*** 97.63 � 15.91***

Group III 0.37 � 0.02# 52.89 � 10.46# 220.5 � 14.1# 163.58 � 15.23##

Group IV 0.44 � 0.01## 76.55 � 6.12## 301.1 � 16.2### 182.87 � 15.38###

Group V 0.49 � 0.02 79.58 � 10.78 395.6 � 17.7 240.40 � 20.4

Results represent mean � SE of six animals per group. Results obtained are significantly different from Control group (***p < 0.001). Results obtained are significantly different from

cisplatin (7.5 mg/kg b.wt.) treated group (#p < 0.05, ##p < 0.01 and ###p < 0001). Chrysin D1 = 25 mg/kg b.wt.; D2 = 50 mg/kg b.wt.

Table 2Results of pretreatment of chrysin on parameters like XO, MDA and G-6-PD on

cisplatin induced enhancement.

Treatment

regimen per

group

XO (mg uric

acid/min/mg

protein)

MDA (nmol of

MDA formed/g

tissue)

Glucose-6-phosphate

dehydrogenase (nmol

NAD reduced/min/mg

protein)

Group I 0.54 � 0.04 2.68 � 0.30 35.87 � 4.1

Group II 1.29 � 0.05*** 5.98 � 0.75*** 12.11 � 2.2**

Group III 0.90 � 0.01ns 3.22 � 0.32## 19.50 � 3.0#

Group IV 0.64 � 0.04### 2.89 � 0.04### 21.20 � 3.2#

Group V 0.54 � 0.02 2.58 � 0.04 35.31 � 3.2

Results represent mean � SE of six animals per group. Results obtained are

significantly different from control group (***p < 0.001). Results obtained are

significantly different from cisplatin treated group (#p < 0.05, ##p < 0.01 and###p < 0001). Chrysin D1 = 25 mg/kg b.wt.; D2 = 50 mg/kg b.wt.

M.U. Rehman et al. / Pharmacological Reports 66 (2014) 1050–1059 1053

(Benchmark plus, BioRad) following the instructions of themanufacturer.

Histological investigation

For histopathology study, the liver was removed and immedi-ately fixed in freshly prepared 10% neutral buffered formalin at4 8C. Then, the skin was embedded in paraffin wax. A section ofliver (5 mm thick) was cut and stained with hematoxylin and eosin(H&E). Inflammatory response around the central vein in terms ofinfiltration of inflammatory cells, vacuolar degeneration andpronounced necrosis around the central vein were observed asan indicator of histological changes with microscope (fluorescentmicroscope, Olympus) at least in six different regions.

Measurement of protein

The protein concentration in all samples was determined by themethod of Lowry et al. [45] using bovine serum albumin as thestandard.

Immunohistochemistry

The processed renal tissues were obtained and preserved in the10% paraformaldehyde overnight followed by dehydration in 30%,20% and 10% sucrose solution successively up to 3 days and wasfixed after that in formaldehyde fixative until immunochemicalstaining. 5–15 mm thick sections of paraffin embedded tissueswere cut using grading type lieca microtome and boiled in 0.1 Mcitrate buffer (pH 6.0) for 5 min for antigen retrieval process andthen incubated in 0.3% H2O2 in methanol followed by incubation inblocking buffer containing 0.1 M PBS, 0.04% Triton X-100 and 10%NGS (normal goat serum). Sections were incubated in anti-bodiesanti rat COX-2 antibody raised in rabbit (1:200 diluted in trisbuffered saline), anti-iNOS (1:100, Thermo Fisher Scientific, USA)for overnight 4 8C. After rinsing in buffer, sections were processedusing a three layer peroxidase staining kit from Thermo scientificsystem. The peroxides complex was visualized with 3,3-diamino-benzidine (DAB Plus substrate, Thermo Fisher Scientific, USA).Lastly the slides were counterstained with hematoxylin for 5 s.Slides were then cleaned in BDH, gradually dehydrated withethanol and cover slipped in mounting medium and photographedunder Olympus microscope (BX51).

Statistical analysis

The data from individual groups are presented as the mean� standard error of the mean (SEM). Differences between groupswere analyzed by using one way analysis of variance (ANOVA)followed by Tukey–Kramer multiple comparisons test and minimumcriterion for statistical significance was set at p < 0.05 for allcomparisons.

Results

Effect of chrysin on the hepatic GSH content

Pretreatment of chrysin before the cisplatin administration wasfound effective in restoring the endogenous anti-oxidant GSH.There was significant depletion in the level of GSH content inGroup II and when compared with Group I (p < 0.001). Pretreat-ment with chrysin in Group III and Group IV shows significantincrease in the level of GSH content (p < 0.01) as compared withGroup II. There was no significant difference in the GSH contentbetween Groups I and V (Table 1).

Effect of chrysin supplementation and cisplatin on the activities of

glutathione dependent enzymes in hepatic tissue

Cisplatin treatment caused a significant decrease in theactivities of GPx (p < 0.001), GST (p < 0.001), GR (p < 0.001)and G6-PD (p < 0.01) in Group II as compared to Group I.The higher dose of chrysin (50 mg kg b.wt.) significantlyattenuated the activities of GPx (p < 0.001), GST (p < 0.01), GR(p < 0.001) and G6-PD (p < 0.05) in Group IV as compared toGroup II. However, the activities of these enzymes in Group Vdid not change significantly as compared to Group I (Tables 1and 2).

Effect of chrysin pretreatment and cisplatin on the xanthine oxidase

activity in liver

The activity of XO was significantly increased (p < 0.001) inGroup II as compared to Group I. Chrysin pretreatmentsignificantly decreased the activity of XO in Group IV(p < 0.001) as compared to Group II. Group V exhibited nosignificant change in the activity of XO as compared to Group I(Table 2).

Table 3Results of pretreatment of chrysin on the level of QR, catalase and SOD on cisplatin

administration in kidney of Wistar rats.

Treatment

regimen

per group

QR (nmol NADPH

oxidized/min/mg

protein)

Catalase (nmol

H2O2 consumed/

min/mg protein)

SOD (units/mg

protein)

Group I 192.1 � 14.2 121.84 � 19.6 143.4 � 3.2

Group II 100.8 � 12.1*** 90.43 � 22.09*** 111.6 � 1.9***

Group III 139.6 � 12.3# 101.96 � 14.18## 123.2 � 1.8##

Group IV 159.1 � 10.1## 107.84 � 26.16### 130.0 � 4.3###

Group V 190.4 � 10.1 112.37 � 21.59 146.4 � 2.7

Results represent mean � SE of six animals per group. Results obtained are

significantly different from control group (***p < 0.001). Results obtained are

significantly different from cisplatin (7.5 mg/kg b.wt.) treated group (#p < 0.05,##p < 0.01 and ###p < 0001). Chrysin D1 = 25 mg/kg b.wt.; D2 = 50 mg/kg b.wt.

Fig. 1. Effect of chrysin pre-treatment on cisplatin induced nitric oxide formation.

Values are expressed as mean � SEM (n = 6). ***p < 0.001 shows significant difference

in Group II when compared with Group I. ##p < 0.01 shows significant difference in the

Group III when compared with Group II and ###p < 0.001 also shows significant

difference in Group IV as compared to Group II. There was no significant difference

between Group I and Group V.

M.U. Rehman et al. / Pharmacological Reports 66 (2014) 1050–10591054

Effect of chrysin on cisplatin induced lipid peroxidation in rat liver

Chrysin inhibits lipid peroxidation caused by cisplatin applica-tion in terms of MDA formation, a well known biomarker ofoxidative stress. Administration of cisplatin leads to significantelevation in the level of MDA in the Group II to that of the controlGroup I (p < 0.001). Pre-treatment with chrysin at both the doseswas found significantly (p < 0.01, p < 0.001) effective in ameliora-tion of MDA formation. There was no significant change observed inthe level of MDA between control and only chrysin treated animals(Table 2).

Effect of chrysin on the antioxidant enzymes

The effect of chrysin pre-treatment on cisplatin induceddepletion in the activity of different antioxidant enzymes wasexamined and the results were shown in Table 3. We haveobserved that there was a significant (p < 0.001) difference in theactivity of different antioxidant enzymes between Group I andGroup II. However pretreatment with chrysin in the Groups III andIV significantly restored the activity of antioxidant enzymes whencompared with the only cisplatin treated group. There was nosignificant difference observed between the Groups I and V.

Effect of chrysin on cisplatin induced changes in serum toxicity

parameters

Cisplatin-treated groups showed (p < 0.001) significant in-crease in serum AST, ALT, LDH and GGT levels, when comparedwith the control group. Lower dose chrysin administration wasfound effective in restoring levels of these serum toxicity markersAST (p < 0.05), ALT (p < 0.05), LDH (p < ns) and GGT (p < 0.01) andhigher dose was significantly effective in the normalization ofthese serum toxicity markers AST (p < 0.001), ALT (p < 0.001) LDH(p < 0.01) and GGT (p < 0.001) when compared with cisplatin-treated groups (Table 4).

Table 4Results of pretreatment of chrysin on serum liver toxicity markers like AST, ALT, LDH

Treatment

regimen

per group

AST (IU/L) ALT (IU/L)

Group I 28.10 � 1.2 10.43 � 1.3

Group II 65.78 � 3.0*** 39.10 � 1.9***

Group III 51.61 � 5.0# 30.07 � 0.8#

Group IV 33.07 � 2.7### 16.11 � 1.4###

Group V 29.00 � 1.8 10.22 � 1.6

Results represent mean � SE of six animals per group. Results obtained are significantly diffe

cisplatin (7.5 mg/kg b.wt.) treated group (#p < 0.05, ##p < 0.01 and ###p < 0001) Chrysin D

Effect of chrysin on the hepatic NO production

Cisplatin treatment resulted in the elevated hepatic NOproduction in the Group II as compared with the control GroupI (p < 0.001). We observed that pre-treatment with both the dosesof chrysin effectively in reduced NO production in Groups III and IVwhen compared with the Group II (p < 0.01). There was nosignificant difference observed between Groups I and V as far as NOproduction is concern (Fig. 1).

Effect of chrysin on NFkB

Level of NFkB was found elevated significantly in cisplatintreated group in comparison with the control (p < 0.001).Pretreatment with chrysin in Groups III and IV have significantly(p < 0.05, p < 0.01) decreased the NFkB level. There is nosignificance difference in the level of NFkB between control andonly chrysin treated group (Fig. 2).

Effect of chrysin on TNF-a

We have assessed the effect chrysin on cisplatin inducedhepatic TNF-a production quantitatively, Fig. 3. We found thatthere was a significant difference in the level of proinflammatorycytokine in control group as compared to cisplatin treated group(p < 0.001). Pre-treatment with chrysin significantly inhibit theirproduction in the Groups III and IV when compared with thecisplatin treated Group II. There was no significant differencefound between Group I and V (Fig. 3).

and g-GGT on cisplatin induced enhancement.

LDH (nmol NADH

oxidized/min/mg

protein)

g-GGT (nmol

p-nitroaniline

formed/min/mg

protein)

201.6 � 11.20 490.2 � 18.7

330.7 � 9.83*** 801.7 � 22.8***

282.2 � 10.2ns 690.8 � 22.3##

240.9 � 11.22## 530.3 � 19.4###

199.4 � 9.22 481.0 � 21.4

rent from control group (***p < 0.001). Results obtained are significantly different from

1 = 25 mg/kg b.wt.; D2 = 50 mg/kg b.wt.

Fig. 2. Effect of chrysin pre-treatment on cisplatin induced increase in NFkB. Values

are expressed as mean � SEM (n = 6). ***p < 0.001 shows significant difference in

Group II when compared with Group I. ##p < 0.01 shows significant difference in the

Group III when compared with Group II and ###p < 0.001 also shows significant

difference in Group IV as compared to Group II. There was no significant difference

between Group I and Group V.

Fig. 3. Effect of chrysin pre-treatment on cisplatin induced increase in TNF-a.

Values are expressed as mean � SEM (n = 6). ***p < 0.001 shows significant difference

in Group II when compared with Group I. #p < 0.05 shows significant difference in the

Group III when compared with Group II and ##p < 0.01 also shows significant

difference in Group IV as compared to Group II. There was no significant difference

between Group I and Group V.

M.U. Rehman et al. / Pharmacological Reports 66 (2014) 1050–1059 1055

Effect of chrysin on the cisplatin-induced hepatic

immunohistochemical expression of COX-2 and i-NOS

Hepatic expression of the above mentioned proteins are shownin the figures respectively. Brown color clearly indicates the morenumber of cells having, COX-2 and i-NOS expression in the Group IIas compared to that of Group I. Pretreatment with chrysin in theGroup III results in reducing the number of cells showingexpression of COX-2 and i-NOS. However there was no significantdifference observed in the expression of these proteins in Group IVas compared to Group I. For immunohistochemical analysis, browncolor indicates specific immunostaining of COX-2 and i-NOS andlight blue color indicates hematoxylin staining. Original magnifi-cation: 40� (Figs. 4 and 5).

Fig. 4. Effect of chrysin pretreatment on cisplatin-induced hepatic expression of COX-2. R

specific staining and blue color indicates hematoxylin staining. Cisplatin treated group

treated group (Group I). Chrysin pretreatment (Groups III and IV) reduces COX-2 expressio

immunostaining in Group V as compared to Group I. (For interpretation of the references

Effect of chrysin on the hepatic histological alterations

Effect of orally administered chrysin was seen on hepatichistological changes caused by cisplatin administration werecharacterized by dispersed areas of necrotic hepatocytes, balloon-ing degeneration and active regeneration manifested by mitoticfigures and binuclear hepatocytes (Fig. 6).

Discussion

In present communication cisplatin treatment resulted inmarked hepatic injury as evidenced by biochemical and histo-pathological alterations including decrease in activity of antioxi-dant enzymes, elevation of serum activity of hepatic enzymes viz.

epresentative photomicrographs (magnification �40). Brown color indicates COX-2

(Group II) shows more COX-2 immunopositive staining as compared with vehicle

n as compared to Group II. However there was no significant difference in the COX-2

to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Effect of chrysin pretreatment on cisplatin-induced hepatic expression of i-NOS. Representative photomicrographs (magnification �40), Brown color indicates i-NOS

specific staining and blue color indicates hematoxylin staining. Cisplatin treated group (Group II) shows more iNOS immunopositive staining as compared with vehicle

treated group (Group I). Chrysin pretreatment (Groups III and IV) reduces i-NOS expression as compared to Group II. However there was no significant difference in i-NOS

immunostaining in Group V as compared to Group I. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

M.U. Rehman et al. / Pharmacological Reports 66 (2014) 1050–10591056

AST and ALT, liver oxidative stress and inflammatory response.These alterations are consistent with previous published reports[2,6,16,17,46–51].

LPO is studied as a classical marker of oxidative stress; weobserved notable elevation in the level of MDA, a LPO product, aftercisplatin treatment [11,52,53]. Consistent with previous reportsour results also showed remarkable increase in the level MDA aftercisplatin treatment [2,6,16,17]. The increase in LPO levels inpresent study, may be the result of reduction in antioxidantstatus or increased production of ROS. In the present study, itwas demonstrated that pretreatment with chrysin significantly

Fig. 6. Effect of chrysin pretreatment on cisplatin-induced hepatic histological altera

infiltration of neutrophils and activated sinusoids in cisplatin treated animals (Group I

(Groups III and IV) markedly attenuates cisplatin induced alterations as compared with o

in the liver histology in the animals only treated with chrysin (Group V) as compared

attenuated cisplatin-induced MDA level in agreement withprevious reports [23].

Removal of free radicals in biological systems is achievedthrough enzymatic and nonenzymatic antioxidants, which act asmajor defense systems against free radicals [54]. GSH and itsoxidized counterpart represent a major redox buffer system of thecell. GSH can act either as a non-enzymatic antioxidant by directinteraction of –SH group with ROS or it can be implicated in theenzymatic detoxification reaction for ROS, as a cofactor orcoenzyme [47–49]. In this study, thiol (SH) groups which areknown to be sensitive to oxidative damage were depleted

tions. Representative photomicrographs (magnification �40), There was marked

I) as compared with only vehicle treated animals (Group I). Chrysin pretreatment

nly cisplatin treated animals (Group II). However there was no significant difference

to only vehicle treated animals (Group I).

M.U. Rehman et al. / Pharmacological Reports 66 (2014) 1050–1059 1057

following cisplatin administration. Chrysin treated rats showedelevated SH contents than cisplatin-treated groups demonstratingthat chrysin aided in replenishing the total thiol pool. The effect ofchrysin on total thiol concentration may be due to a direct anti-oxidant effect and enhanced biosynthesis of GSH in agreementwith the previous studies that indicate the effects of some anti-oxidants on cellular GSH may be due to direct antioxidant effects,enhanced biosynthesis of GSH or increase in levels of other anti-oxidants [55].

The activities of antioxidant enzymes, viz. CAT, GPx, GR, SODand G6PD and a phase-II detoxifying enzyme, namely QR, werediminished in the cisplatin-treated group, whereas pretreatmentwith chrysin markedly attenuated the activities of these antioxi-dant as well as phase-II detoxifying enzymes. QR is a phase-IIenzyme involved in xenobiotic metabolism that catalyzes the twoelectron reduction and thus protects cells against free radicals andROS generated by the one-electron reductions catalyzed bycytochromes P450 and other enzymes [40,56]. The diminishedactivities of antioxidant and phase-II detoxifying enzymes in thecisplatin treated group seconds the involvement of oxidative stressin the pathophysiology of cisplatin induced hepatic toxicity.

Several published reports have revealed increased tissuecontent of inflammatory mediators together with inflammatorycell infiltration, signifying that inflammation plays a significantrole in cisplatin-induced injury [2,6,57]. Oxidative stress inducedinflammation also plays an important physiological role incisplatin induced hepatotoxicity via multiple intercalating path-ways [57]. It has been demonstrated that cisplatin administrationincreases the infiltration of neutrophils and macrophages [58,59].Since the neutrophil infiltration is an important event for the acuteinflammation, increase in MPO activity due to cisplatin may causeinflammation and damage in the organ. Further, Federico et al. [60]reported that in most of the pathological conditions characterizedby oxidative insult, there is an increase of nitrite level. Cisplatintreatment caused increase in the level of NO which further reactswith superoxide radical increases organ injury by formation ofhighly cytotoxic peroxynitrite [59]. Chrysin treatment attenuatedthis abnormal increase in the level of NO (Fig. 1).

NFkB activation is pivotal in the expression of proinflammatorycytokines like TNF-a and other mediators involved in acute

Fig. 7. Graphical representation of possible targets of action of c

inflammatory responses and other conditions associated withincreased ROS generation. Inhibitors of NFkB have shownprotection against cisplatin induced toxicity [61]. The proinflam-matory cytokine TNF-a, also has been established to play a key rolein the patho-mechanism of cisplatin-induced injury [6,62]. In thepresent study, pathological examination of liver exposed tocisplatin in agreement with previous reports [62,63] haveestablished acute inflammatory responses incited via inductionof NFkB and TNF-a. The present study reveals that both NFkB andTNF-a expression were abrogated substantially by the prophylac-tic treatment of chrysin, thus reducing inflammatory responseimplicated in the acute liver toxicity caused by the cisplatin (Figs. 2and 3). Further, activation of transcription factor NF-kB by TNF-a isone of the numerous actions of TNF-a that causes genes togenerate potentially cell damaging oxidative enzymes such asNADP oxidase, cyclooxygenase (COX-2) and iNOS as well as furtherrelease of TNF-a and other pro-inflammatory cytokines [64].Cyclooxygenase (COX-2) is an inducible form of COX and anotherimportant marker of inflammation which plays a physiological rolein inflammation and tumor proliferation [65]. In our studyexpression of COX-2 and iNOS were increased in liver tissue ofcisplatin treated group. Previous studies have reported selectiveinhibitors to be effective in ameliorating cisplatin induced toxicityin rats [66]. Our results also showed chrysin to be effective inattenuating cisplatin induced expression of both COX-2 and iNOS(Figs. 4 and 5). Lastly, above mentioned findings corroborated withthe histological data which exhibited the protective effects ofchrysin against cisplatin-induced toxicity (Fig. 6).

In conclusion, the findings of the present study reinforce thesignificant role of ROS, NFkB, TNF-a and Cox-2 in pathogenesis ofthe cisplatin-induced hepatotoxicity. It further demonstrates thatabrogation of oxidative and inflammatory response by naturallyfound antioxidants could be effective strategy for prophylaxis ofcisplatin induced liver damage (Fig. 7). Chrysin has a potentshielding effect against the hepatotoxicity of this agent, and mightbe clinically useful. Our results combined with the knownchemotherapeutic potentiating effects of this agent make thiscompound an excellent candidate for clinical drug development.The exceptional safety profile of chrysin in humans confers itremarkable therapeutic potential in a multitude of diseases

hrysin against cisplatin induced liver toxicity in Wistar rats.

M.U. Rehman et al. / Pharmacological Reports 66 (2014) 1050–10591058

associated with inflammation and oxidative stress, which needsfurther exploration.

Conflict of interest

Authors declare that there are no conflicts of interest.

Acknowledgement

Author Sarwat Sultana is thankful to University GrantsCommission (UGC-SAP Grant no. 3-76/2009(SAP-II)), Govt. ofIndia, New Delhi, for funding this research and providingMeritorious Research Fellowship to the first author (Muneeb U.Rehman).

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