Food and Chemical Toxicology 66 (2014) 185–193

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Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate / foodchemtox

Mitigation of 5-Fluorouracil induced renal toxicity by chrysin viatargeting oxidative stress and apoptosis in wistar rats

http://dx.doi.org/10.1016/j.fct.2014.01.0260278-6915/� 2014 Elsevier Ltd. All rights reserved.

Abbreviations: 5-FU, 5-Flourouracil; CH, chrysin; KIM-1, kidney injury molecule;BSA, bovine serum albumin; CDNB, 1-chloro 2, 4-dinitrobenzene; DTNB, 5,50-dithiobis-[2-nitrobenzoic acid]; EDTA, ethylene diamine tetra acetic acid; GPx, glutathi-one peroxidase; GR, glutathione reductase; GSH, reduced glutathione; GSSG,oxidized glutathione; NADPH, reduced nicotinamide adenine dinucleotide phos-phate; ROS, reactive oxygen species; SOD, superoxide dismutase; TBA, thiobarbi-turic acid; LDH, lactate dehydrogenase; BUN, blood urea nitrogen; MDA,malondialdehyde.⇑ Corresponding author. Address: Section of Molecular Carcinogenesis and

Chemoprevention, Department of Medical Elementology and Toxicology, Facultyof Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi110062, India. Tel.: +91 11 26054685x5565/5566; fax: +91 11 26059663.

E-mail address: sarwat786@rediffmail.com (S. Sultana).

Summya Rashid, Nemat Ali, Sana Nafees, Syed Kazim Hasan, Sarwat Sultana ⇑Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India

a r t i c l e i n f o

Article history:Received 9 October 2013Accepted 16 January 2014Available online 31 January 2014

Keywords:5-FUCHOxidative stressApoptosisKIM-1

a b s t r a c t

5-Fluorouracil (5-FU) is a potent antineoplastic agent commonly used for the treatment of various malig-nancies. It has diverse adverse effects such as cardiotoxicity, nephrotoxicity and hepatotoxicity whichrestrict its wide and extensive clinical usage. It causes marked organ toxicity coupled with increased oxi-dative stress and apoptosis. Chrysin (CH), a natural flavonoid found in many plant extracts, propolis, bluepassion flower. It has antioxidative and anti-cancerous properties. The present study was designed toinvestigate the protective effects of CH against 5-FU induced renal toxicity in wistar rats using biochem-ical, histopathological and immunohistochemical approaches.

Rats were subjected to prophylactic oral treatment of CH (50 and 100 mg/kg b.wt.) for 21 days againstrenal toxicity induced by single intraperitoneal administration of 5-FU (150 mg/kg b.wt.). The possiblemechanism of 5-FU induced renal toxicity is the induction of oxidative stress; activation of apoptoticpathway by upregulation of p53, bax, caspase-3 and down regulating Bcl-2. However prophylactic treat-ment of CH decreased serum toxicity markers, increased anti-oxidant armory as well as regulated apop-tosis in kidney. Histopathological changes further confirmed the biochemical and immunohistochemicalresults. Therefore, results of the present finding suggest that CH may be a useful modulator in mitigating5-FU induced renal toxicity.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

5-FU is a pyrimidine antimetabolite, synthesized by Heidelberget al. in 1957, used clinically since past 40 years. Because of itsbroad antitumor activity as well as its synergism with other anti-cancer drugs it is being used in the treatment of various types ofcancers (Miura et al., 2010). The metabolic product of 5-FU is 5-flu-oro-2-deoxyuridine monophosphate which is an irreversible inhib-itor of thymidylate synthase, a fundamental enzyme necessary forthymine synthesis. It restrains production of deoxythymidine

monophosphate (dTMP) that is crucial for replication and repairof DNA and its deficit leads to cellular toxicity (Chibber et al.,2011). Besides this, it is catabolised into dihydrouracil in liverwhich is cleaved into a-fluoro-b-alanine, ammonia, urea, and car-bon dioxide, thereby leading to nephrotoxicity (Nora, 2012). Ithas also been reported to obstruct the activity of exosome complexand integrates its toxic metabolites into DNA and RNA and thusterminating cell cycle and induction of apoptosis (David et al.,2011). It is used in the treatment of various malignancies includingbreast, head, neck, stomach, colorectal, genitourinary tract, liverand skin cancer. 5-FU like other chemotherapeutics is non targetedin action and demolishes rapidly dividing normal cells in the pa-tients besides tumors and results in proliferative inhibition, DNAdamage and cell death leading to extensive side effects. Some ofthe common clinical side-effects include myelosuppression, diar-rhoea, vomiting, mucositis, leukopenia, stomatitis, alopecia, car-diotoxicity, nephrotoxicty and hepatotoxicity (David et al., 2011;Lamberti et al., 2012; Chang et al., 2012; Kinhult et al., 2003 andNora, 2012). Nowadays there is a lot of focus put on the use of nat-ural compounds having anti oxidative and anti-apoptotic proper-ties to amplify the effectiveness in reducing the toxicity inducedby chemotherapeutic drugs (Khan et al., 2012a,b). It has been

186 S. Rashid et al. / Food and Chemical Toxicology 66 (2014) 185–193

reported from various chemopreventive and epidemiological stud-ies in animal models and humans that the consumption of fruitsand vegetables in diet decrease risk of cancer due to the presenceof various essential nutrients and phenolics predominantly flavon-iods (Surh et al., 2001 and Middleton et al., 2000).

Flavonoids are natural phenolic compounds of plant origin rep-resenting an extensive array of biological characteristics includingantibacterial, anti-inflammatory, anti allergic, antithrombotic andvasodilatory properties (Bosetti et al., 2007). At present muchattention is being drawn towards alternative medicine because ofthe use of dietary supplements and herbal preparations for main-tenance of health and prevention of disease. Henceforth there isan increased interest in deciphering the biological roles of flavo-noids, which are found to be major constituents of some traditionalmedicinal plants (Wang and Morris, 2007).

CH (5, 7-dihydroxyflavone) is a natural flavonoid which pos-sesses wide variety of biological activities and is found in manyplant extracts including propolis, blue passion flower and honey.Recently, a number of studies have shown that CH is antioxidant,anti inflammatory and anti cancerous in nature. It has been re-ported in vitro to induce apoptosis in a panel of cancer cell lines,including HeLa cervical cancer cells, U937, HL-60 and L1210 leuke-mia cells (Li et al., 2010). It also down regulates the expression ofproliferating cell nuclear antigen (PCNA) by inducing apoptosisthrough caspase activation, Akt inactivation in U937 leukemia cellsand causes cell-cycle arrest in human colon cancer cells, C6 gliomacells (Miyamoto et al., 2006). No information is available on che-mopreventive potential of CH against 5-FU induced renal toxicity.In this study, we found that CH administration significantly downregulated the level of pro-apoptotic factors like p53, Bax, caspase-3and up regulated Bcl-2 simultaneously reducing chemotherapy in-duced apoptosis. This provides a new evidence to support a novelapproach towards cancer therapeutics.

2. Materials and methods

2.1. Chemicals

GR, GSSG, GSH, DTNB, CDNB, BSA, NADP, NADPH, FAD, TBA, TCA, FCR, 5-FU, etc.were obtained from Sigma–Aldrich, USA. All other chemicals and reagents were ofthe highest purity grade and commercially available.

2.2. Animals

Male Wistar rats (150–200 g), 6–8 weeks old, were obtained from the CentralAnimal House Facility of Hamdard University. Rats were housed in an animal carefacility under room temperature (25 ± 1 �C) with 12 h light/dark cycles and were gi-ven free access to standard pellet diet and tap water ad libitum. Before the treat-ment, rats were left for 7 days to acclimatize. Animals received humane care inaccordance with the guidelines of the Committee for the Purpose of Control andSupervision of Experiments on Animals (CPCSEA),Government of India, and priorpermission was sought from the Institutional Animal Ethics Committee (IAEC No:173/CPCSEA, 28 January 2000).

2.3. Treatment protocol

Rats were randomly divided into five groups of six rats each. Group I served ascontrol and received corn oil for 21 days (10 ml/kg) and 0.9% saline intraperitone-ally on 19th. Group II served as positive control and received intraperitoneal injec-tions of 5-FU (150 mg/kg b.wt.) on the 19th day. Groups III and IV were co-treatedwith an oral dose of 5-FU 50 mg/kg b.wt. (D1) and 100 mg/kg b.wt. (D2), respec-tively, for 21 days and intraperitoneal injection of 5-FU (150 mg/kg b.wt.) wasadministered on 19th day in both the groups. Group V received only D2 (100 mg/kg b.wt.) of 5-FU for 21 days. On the 21st day, the rats were sacrificed and kidneysamples were taken after perfusion for examination of various biochemical, immu-nohistochemical and histo pathological parameters. Before sacrifice, rats were givenmild ether anesthesia. Later on, blood was drawn and serum was obtained. The ex-cised tissue was washed with ice-cold saline (0.85% sodium chloride) and used toprepare a 10% homogenate in chilled phosphate buffer (0.1 m, pH 7.4) containingKCl (1.17% using a Potter Elvehjen homogenizer and some tissue was also storedin 10% buffered formalin for histopathology.

In vivo protocol

Groups

Treatment from 1stto 21st day

Treatment on 19th day

Group I (control)

Corn oil (10 ml/kg) Normal saline only (0.9%) Group II (only 5-FU) Corn oil (10 ml/kg) 5-FU 150 mg/kg b.wt.i.p

(19th day)

Group III (5-FU + CHD1) CH 50 mg/kg b.wt. 5-FU 150 mg/kg b.wt.i.p

(19th day)

Group IV (5-FU + CHD2) CH 100 mg/kg b.wt. 5-FU 150 mg/kg b.wt.i.p

(19th day)

Group V (only CHD2) CH 100 mg/kg b.wt. CH 100 mg/kg b.wt.

2.4. Tissue processing

Post mitochondrial supernatant of kidney samples was prepared by the methodof Khan and Sultana, 2011. In brief, the kidneys were removed quickly, cleaned ofextraneous material and immediately perfused with ice-cold saline (0.85% NaCl).The kidneys were homogenised in chilled phosphate buffer (0.1 M, pH 7.4) contain-ing KCl (1.17%) using a Potter–Elvehjen homogeniser. The homogenate was filteredthrough muslin cloth and centrifuged at 800g for 5 min at 48C by a REMI coolingcentrifuge to separate the nuclear debris. The aliquot obtained was centrifuged at12,000 rpm for 20 min at 4 �C to obtain the PMS, which was used as a source ofenzymes.

2.5. Assay for CAT activity

The CAT activity was assessed by the method of Claiborne (1985). In short, thereaction mixture consisted of 0.05 ml PMS, 1.0 ml of H2O2 (0.019 M), 1.95 ml phos-phate buffer (0.1 M, pH 7.4), in a total volume of 3 ml. Changes in absorbance wererecorded at 240 nm, and the change in absorbance was calculated as nmol H2O2

consumed/min per mg protein.

2.6. Assay for lipid peroxidation

The assay of lipid peroxidation (LPO) was done according to the method ofWright et al. The reaction mixture consisted of 0.58 ml phosphate buffer (0.1 M,pH 7.4), 0.2 ml microsome, 0.2 ml ascorbic acid (100 mM) and 0.02 ml ferric chlo-ride (100 mM), in a total volume of 1 ml. This reaction mixture was then incubatedat 37 �C in a shaking water bath for 1 h. The reaction was stopped by the addition of1 ml trichloroacetic acid (10%). Following the addition of 1.0 ml thiobarbituric acid(TBA) (0.67%), all the tubes were placed in a boiling water bath for a period of20 min. The tubes were shifted to an ice bath and then centrifuged at 2500g for10 min. The amount of MDA formed in each of the samples was assessed by mea-suring the optical density of the supernatant at 535 nm. The results were expressedas nmol TBA formed/h per g tissue at 378C by using a molar extinction coefficient of1.56 � 105/M per cm.

2.7. Assay for SOD activity

The SOD activity was measured by the method of Marklund and Marklund(1974). The reaction mixture consisted of 2.875 ml Tris–HCl buffer (50 mM, pH8.5), pyrogallol (24 mM in 10 mM-HCl) and 100 ml PMS, in a total volume of3 ml. Enzyme activity was measured at 420 nm and was expressed as units/mg pro-tein. One unit of enzyme is defined as the enzyme activity that inhibits the autoox-idation of pyrogallol by 50%.

2.8. Assay for GSH

GSH was assessed by the method of Jollow et al. A quantity of 1.0 ml of 10% PMSmixed with 1.0 ml of (4%) sulphosalicylic acid was taken, incubated at 4 �C for aminimum time period of 1 h and then centrifuged at 4 �C at 1200 g for 15 min.The reaction mixture of 3.0 ml was composed of 0.4 ml of supernatant, 2.2 ml phos-phate buffer (0.1 M, pH 7.4) and 0.4 ml dithio-bis-2-nitrobenzoic acid (4 mg/ml).The yellow colour developed was read immediately at 412 nm on the spectropho-tometer (Lambda EZ201; Perkin Elmer). GSH concentration was calculated as nmolGSH conjugates/g tissue.

2.9. Assay for GPx activity

The activity of GPx was calculated by the method of Mohandas et al. (1984). Thetotal volume of 2 ml was composed of 0.1 ml EDTA (1 mM), 0.1 ml sodium azide(1 mM), 1.44 ml phosphate buffer (0.1 M, pH 7.4), 0.05 ml glutathione reductase(1 IU/ml is equivalent to 1 mol GSSG reduced/min per 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 of 10% PMS. The deple-tion of NADPH at 340 nm was recorded at 25 �C. Enzyme activity was calculated asnmol NADPH oxidised/ min per mg protein with a molar extinction coefficient of6.22 � 103/M per cm.

S. Rashid et al. / Food and Chemical Toxicology 66 (2014) 185–193 187

2.10. Assay for GR activity

GR activity was measured by the method of Carlberg and Mannervik (1985). Thereaction mixture consisted of 1.65 ml phosphate buffer (0.1 M, pH 7.6), 0.1 ml EDTA(0.5 mM), 0.05 ml GSH (1 mM), 0.1 ml NADPH (0.1 mM) and 0.1 ml of 10% PMS, in atotal volume of 2 ml. Enzyme activity was quantified at 25 �C by measuring the dis-appearance of NADPH at 340 nm and was calculated as nmol NADPH oxidized/minper mg protein using a molar extinction coefficient of 6.22 � 103/M per cm.

2.11. Assay for BUN

Estimation of BUN was carried out by diacetyl monoxime method of Kanter,1975. A protein-free filtrate was prepared by adding serum and an equal amountof 10% TCA and then the mixture was centrifuged at 2000 rpm and supernatantwas obtained. To 0.5 ml of the protein-free filtrate, were added 3.5 ml of distilledwater, 0.8 ml diacetylmonoxime (2%) and 3.2 ml H2SO4–phosphoric acid reagent(reagent was prepared by mixing 150 ml of 85% phosphoric acid with 140 ml ofwater and 50 ml of concentrated H2SO4). The reaction mixture was placed in a boil-ing water bath for 30 min and then cooled. Absorbance was read at 480 nm.

2.12. Assay for creatinine

Creatinine was estimated by alkaline picrate method of Hare (1950). To 1.0 mlof serum were added 1.0 ml sodium tungstate (5%), 1.0 ml H2SO4 (0.6 M) and 1.0 mlof distilled water. After mixing thoroughly, the mixture was centrifuged at 800g for5 min. The supernatant was added to a mixture containing 1.0 ml picric acid (1.05%)and 1.0 ml NaOH (0.75 M). Absorbance at 520 nm was read exactly after 20 min.

2.13. Assay for LDH activity

LDH activity was estimated in serum by the method of Korenberg . The assaymixture consisted of 0.2 ml of serum, 0.1 ml of 0.02 M-NADH, 0.1 ml of 0.01 Msodi-um pyruvate, 1.1 ml of 0.1 M (pH 7.4) phosphate buffer and distilled water, in a to-tal volume of 3 ml. Enzyme activity was recorded at 340 nm, and activity wascalculated as nmol NADH oxidised/min per mg protein.

2.14. Estimation of KIM-1

KIM-1 levels were measured by the commercially available ELISA based kit (RATKIM-1 ELISA KIT, Adipo Bioscience, Inc., USA) following instructions of themanufacturer.

2.15. Estimation of protein

The protein concentration in all samples was determined by the method ofLowry et al. (1951) using BSA as standard.

2.16. Immunohistochemistry

2.16.1. Immunostaining methodsTo examine the protective effects of CH on 5-FU induced apoptosis in kidney

was assessed by immunohistochemical staining. Sections of formalin-fixed, paraf-fin-embedded kidneys were obtained on poly-L-Lysine coated slides. Sections weredeparaffinized in xylene, then rehydrated through a graded alcohol series. Antigenretrieval was performed by incubating slides in citrate buffer (pH-6.0) (10 mM) at95 �C for 20 min. Endogenous peroxidase activity was blocked with 3% H2O2 for30 min. To detect p53, bax, Bcl-2 and caspase-3 immunoreactivities, sections wereincubated under humid conditions overnight at 4 �C with the following monoclonalantibodies: anti-p53-antibody (1:200; Santa Cruz Biotechnology, Inc., USA), anti-Bcl-2 antibody (1:400; Santa Cruz Biotechnology, Inc., USA), anti-bax antibody(1:200; Thermo Fisher Scientific, USA), anti-caspase-3 antibody (1:400; ThermoFisher Scientific, USA). Next day, the slides were washed three times in Tris buffers(pH-6.0) and were incubated with a biotinylated Goat Anti-Polyvalent Plus (ThermoFisher Scientific, USA) for 30 min at room temperature. This step was followed byfurther wash in Tris buffer and incubation of slides at room temperature with aStreptavidin Peroxidase Plus (Thermo Fisher Scientific, USA) that binds to the biotinpresent on the secondary antibody. After washing in Tris buffer, the immunostain-ing reaction product was developed using 3,30-diaminobenzidine (DAB Plus sub-strate, Thermo Fisher Scientific, USA). After immunoreactivity, slides were dippedin distilled water, counterstained with Harris hematoxylin and dried and finallythe sections were mounted with DPX and covered with cover slips. The slides wereready to be observed under microscope.

2.16.2. Quantitative evaluation of p53Percentages of p53 immunostained nuclei (p53 labeling index, LIp53) were cal-

culated in each selected section for control rats, toxicant treated rats and rats givenCH as a modulator using the following formula:

Number of labeled nuclei � 100/total number (labeled + unlabeled) of nuclei(Arriazu et al., 2006). p53 immunostained nuclei were considered positive regard-less of staining intensity. All slides were examined by two independent observerswho were unaware of the experimental protocol. The slides with discrepant evalu-ations were re-evaluated, and a consensus was reached. Measurements were car-ried out using an Olympus BX51 (Hamburg, Germany) microscope usingobjectives with 10� and 40� magnifications.

2.16.3. Quantitative evaluation of bax, caspase-3 and Bcl-2 immunostainingAccording to the diffuseness of the DAB staining, sections were graded as 0 (no

staining), 1 (staining, 25%), 2 (staining between 25% and 50%), 3 (staining between50% and 75%), or 4 (staining >75%). According to staining intensity, sections weregraded as follows: 0 (no staining), 1 (weak but detectable staining), 2 (distinctstaining) or 3 (intense staining). Immunohistochemical staining scores were ob-tained by adding the diffuseness and intensity scores. All slides were examinedby two independent observers who were unaware of the experimental protocol.The slides with discrepant evaluations were re-evaluated, and a consensus wasreached. Measurements were carried out using an Olympus BX51 (Hamburg, Ger-many) microscope using objectives with 10� and 40�magnifications.

2.17. Histopathological examination

The kidneys were quickly removed after sacrifice and preserved in 10% neutralbuffered formalin for histopathological processing. The kidneys tissue was embed-ded in paraffin wax and longitudinally sectioned with a microtome. Hematoxylinand eosin staining of the sections was observed under an Olympus microscope.

2.18. Statistical analysis

Differences between groups were analyzed using analysis of variance (ANOVA)followed by Tukey Kramer’s test. All data points are presented as the treatmentgroups mean ± standard error of the mean (SE).

3. Results

3.1. Effect of CH treatment on MDA formation

There was a significant (���P < 0.001) increase in MDA formationin 5-FU treated group as compared to control group. However, itwas found that pre and post treatment with CH at both doses(50 and 100 mg/kg) significantly (##P < 0.01 and ###P < 0.001) re-stored the membrane integrity by reducing the increased level ofLPO dose dependently when compared with 5-FU treated group(Table 1). There was no significant difference found in the MDA le-vel between control and only CH group (group 5).

3.2. Effect of pre and post treatment of CH on antioxidant enzymearmory

It was found that antioxidant enzymes (GPx, GR, CAT and SOD)were significantly depleted in 5-FU treated group as compared tocontrol group (���P < 0.001). However, treatment with CH beforeand after 5-FU administration significantly restored the activityof these enzymes (#P < 0.05, ##P < 0.01, ###P < 0.001 and ns-notsignificant). There was no significant difference in the activity ofthese antioxidant enzymes between control and Group 5 (CH only)(Tables 1 and 2).

3.3. CH treatment restores GSH level

Pre and post treatment of groups with CH showed an increase inrenal GSH level. GSH level was significantly depleted (���P < 0.001)in 5-FU treated group compared to Control group. The GSH level inCH treated groups was significantly increased (#P < 0.05 and###P < 0.001) as compared to 5-FU treated group. However, CHalone did not show any significant changes in GSH level comparedto control group (Table 2).

Table 1Results of CH on LPO, SOD, CAT on 5-FU administration in kidney of wistar rats.

CAT SOD LPO(nmol H2O2 consumed/min/mg protein) units/mg of protein (nmol MDA formed/h per g tissue)Kidney Kidney Kidney

Treatment groupsGroup I (control) 45.27 ± 2.66 28.4 ± 1.4 8.14 ± 1.18Group II (only 5-FU) 15.91 ± 1.89��� 15.5 ± 1.2��� 38.93 ± 1.63���

Group III (5-FU + CHD1) 30.17 ± 2.48# 20.5 ± 0.7# 26.66 ± 1.74##

Group IV (5-FU + CHD2) 32.02 ± 4.96# 23.1 ± 0.8## 23.9 ± 2.45###

Group V (only CHD2) 42.16 ± 3.53 27.2 ± 0.6 7.92±.073

Results represent mean ± SE of six animals per group. Results obtained are significantly different from Control group (���P < 0.001). Results obtained are significantly differentfrom 5-FU treated group (#P < 0.05), (##P < 0.01), and (###P < 0.00). CH = chrysin; CH D1 = 50 mg/kg/b.wt.; CH D2 = 100 mg/kg/b.wt.

Table 2Results of modulatory effect of CH on GSH, GR and GPx on 5-FU administration in kidney of wistar rats.

GSH GR GPX(nmol GSH/g tissue) (l mol NADPH Oxidized/min/mg protein) (l mol NADPH Oxidized/min/mg protein)Kidney Kidney Kidney

Treatment groupsGroup I (control) 0.94 ± .03 208.4 ± 9.8 176.4 ± 11Group II (only 5-FU) 0.34 ± .02��� 117.1 ± 6.2��� 84.2 ± 4.9���

Group III (5-FU + CHD1) 0.49 ± .03# 171.1 ± 13.6# 100.9 ± 16.9ns

Group IV (5-FU + CHD2) 0.6 ± .02### 189.3 ± 9.8## 144.6 ± 6.1#

Group V (only CHD2) 0.95 ± .04 209.8 ± 15.1 174 ± 13.6

Results represent mean ± SE of six animals per group. Results obtained are significantly different from control group (���P < 0.001). Results obtained are significantly differentfrom 5-FU treated group (#P < 0.05), (##P < 0.01), (ns P = not significant) and (###P < 0.00). CH = chrysin; CH D1 = 50 mg/kg/b. wt.; CH D2 = 100 mg/kg/b. wt.

Table 3Results of modulatory effect of CH on serum toxicity markers BUN, creatinine, LDH on 5-FU induced renal redox imbalance.

Treatment groups BUN (IU/L) Creatinine (IU/L) LDH (nmol NADH oxidized/min/mg protein)

Group I (control) 18.01 ± 1.02 0.86 ± 0.08 195.2 ± 9.91Group II (only 5-FU) 38.82 ± 2.56��� 3.02 ± 0.18��� 374 ± 13.5���

Group III (5-FU + CHD1) 29.16 ± 0.81## 2.15 ± 0.07## 302.1 ± 11.81#

Group IV(5-FU + CHD2) 25.48 ± 1.3### 1.97 ± 0.09### 241.7 ± 9.79###

Group V (only CH D2) 18.32 ± 1.33 0.88 ± 0.11 190.9 ± 14.84

Results represent mean ± SE of six animals per group. Results obtained are significantly different from control group (���P < 0.001). Results obtained are significantly differentfrom 5-FU treated group (#P < 0.05), (##P < 0.01). CH = Chrysin; CH D1 = 50 mg/kg/b.wt.; CH D2 = 100 mg/kg/b.wt.

188 S. Rashid et al. / Food and Chemical Toxicology 66 (2014) 185–193

3.4. Effect of 5-FU and CH treatment on the level of renal toxicitymarkers

3.4.1. CreatinineCreatinine was significantly elevated in 5-FU-treated rats

(���P < 0.001) (Table 3). Prophylactic treatment of CH at a higherdose prevented 5-FU induced elevation in serum levels of creati-nine (##p < 0.01 and ###p < 0.01) as compared to untreated control.Henceforth there was a significant recovery after CHsupplementation.

Fig. 1. Results represent mean ± SE of six animals per group. Results obtained weresignificantly elevated in group II (���P < 0.001). Prophylactic treatment prevented 5-FU induced elevation in Kim1 levels at both the doses significantly as compared to

3.4.2. Blood urea nitrogen5-FU treated rats showed a significant increase in BUN level

(���P < 0.001) as compared to untreated control (Table 3). Howeverthere was a marked reduction in BUN levels in case of groupstreated with CH dose dependently (##P < 0.01, ###P < 0.001).

control (###P < .001).

3.4.3. CH treatment inhibits KIM-1 level5-FU administration increased the level of KIM-1 in 5-FU trea-

ted group as compared to control (���P < 0.001). CH treatment atboth the doses decreased 5-FU induced abnormal increase in levelof serum KIM-1 (###P < 0.001). There was no significant differencebetween control (group I) and only CH treated group (group V)(Fig. 1).

3.4.4. Effect of CH pretreatment on LDH activityIt was found that the pretreatment of CH showed significant

protective effect (#P < 0.05 and ###P < 0.001) on the above saidmarker enzyme. CH was found to be effective in the replenishmentof LDH when compared to 5-FU treated group (Table 3). However,

S. Rashid et al. / Food and Chemical Toxicology 66 (2014) 185–193 189

only CH group (group V) did not show any significant difference ascompared to control group.

3.5. Effect of CH on p53 expression in 5-FU treated rat kidneys

According to Fig. 2 p53 protein was highly induced(###p < 0.001) in glomerular and tubular region in rats subjectedto 5-FU as compared to control group (Fig. 2F). But its expressionwas downregulated significantly in glomerulus and tubules bylow dose CH group (##p < 0.01) and high dose CH group in tubulesand parenchyma (###p < 0.001) dose dependently as compared to5-FU treated group.

3.6. Effect of CH on bax and caspase-3 expression in 5-FU treated ratkidneys

The immunohistochemical evaluation showed more intenseexpression of bax in glomerular, tubular region (���p < 0.001) andcaspase-3 in bowman’s capsule, glomerulus and parenchyma(���p < 0.001) in rats subjected to 5-FU as compared to controlwhere no significant staining was observed in any of the above saidproteins (Figs. 3G and 5I). There was intense expression of Bax ob-served in tubules (#p < 0.05) and caspase-3 in glomerulus andparenchyma, minimally in tubules (##p < 0.01) at lower dose ofCH (50 mg/kg b.wt.) as compared to 5-FU treated group. However,higher dose of CH (100 mg/kg b.wt.) treated rats showed moderatestaining of bax in tubules (##p < 0.01) and caspase-3 in glomerulusand tubules (###p < 0.001) as compared to 5-FU treated rats.

3.7. Effect of CH on bcl-2 expression in 5-FU treated rat kidneys

The immunohistochemical evaluation showed almost negligiblestaining of bcl-2 in rats treated with 5-FU (���p < 0.001) comparedwith control where the staining of bcl-2 positive cells is visible inboth glomerular and tubular region (Fig. 4H). There was consider-ably low bcl-2 positive staining (ns-not significant) in tubules andparenchyma in low dose CH (50 mg/kg b.wt.) treated group com-pared to 5-FU treated group. However, higher dose of CH(100 mg/kg b.wt.) treated rats showed considerably intense

Fig. 2. (A–D and F) Representative photomicrograph of p53 protein expression of rat kidexpression in group II showing highly intense and positive staining. (C) p53 expression insimilar to control .

positive staining of bcl-2 in glomerulus as well as tubules(###p < 0.001) as compared to 5-FU treated rats.

3.8. Effect of CH against 5-FU induced histopathological alterations inkidney

The histology of the rat kidney tissues showed normal histoarchitecture in control group. Administration of 5-FU resulted indisruption of the normal renal architecture which was well evidentby blood sinusoids, interstitial hemorrhages, glomerular conges-tion and atrophy (Fig. 6). Furthermore, CH treatment significantlyshowed protective changes in the glomeruli and tubules in a dosedependent manner.

4. Discussion

Presently, the principal treatment choice for cancer patients ischemotherapy. Though its therapeutic usage is limited because ofits severe clinical side effects (Ramadori and Cameron, 2010; Naiduet al., 2004). In the present study, we investigated the efficacy ofCH on 5-FU induced oxidative stress and apoptosis in kidneys.Although mechanism underlying 5-FU induced renal toxicity isnot fully clear. However one possible mechanism suggested by sev-eral investigators is free radical generation which results in lipidperoxidation, cell membrane damage and apoptosis (Xian et al.,2004; Kinhult et al., 2003). In the present study, protective effectof CH observed may be associated with amelioration of oxidativestress and apoptotic damage in kidney of 5-FU treated rats. LPOis measured in the form of MDA, is one of the mechanisms involvedin tissue damage via generation of ROS. Significant increase in thelevel of MDA in renal tissue has been reported in 5-FU treated rats(Sarbani, 2011). Our results agree with the previous findings. Pro-phylactic treatment with CH substantially decreased the level ofMDA significantly and dose dependently.

GSH is unanimous antioxidant that defends against exogenoustoxic injury by augmenting the defense against ROS via scavengingof free radicals. It does so by directly donating a hydrogen atomand neutralizing free radicals (hydroxyl radicals). Depletion ofGSH in tissue damages cellular defense against oxidative stress.

ney. (A) p53 protein expression in control group showing very less staining. (B) p53group III showing less staining as compared to group II. (D) Very less staining almost

Fig. 3. (A–D and G) Representative photomicrographs of bax determined by immunohistochemistry. (A) Negligible expression of bax was observed in case of control rats. (B)5-FU administration increased the number of bax positive cells in glomerular and tubular region of renal sections of animals represented by arrows in the figure. (C) 5-FU + CH treated animals showed slightly lesser number of bax positive cells as compared to group II as is evident from the figure. (D) 5-FU + CH treated animals at higher doseshowed least number of bax positive cells as compared to group II.

Fig. 4. (A–D and H) Representative photomicrographs of caspase-3 determined by immunohistochemistry. (A) There is almost negligible expression of caspase-3 in the renalsections of control group. (B) 5-FU administration strongly induced caspase-3 expression in renal sections. (C) There is partial inhibition of caspase-3 expression as evidencedby weaker immunostaining in the rat kidneys treated with lower dose of CH. (D) higher dose of CH treatment showed lesser expression which implies that caspase 3 has beeninhibited to a large extent.

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Our results show 5-FU admisnitration depleted GSH reservoirs, inconcurrence with earlier findings (Rashid et al., 2013). Howeverprophylactic treatment of CH replenished levels of GSHsubstantially.

SOD constitutes the chief antioxidant. It works in associationwith H2O2 removing enzymes and it is by products are H2O2 andO2. GPx, CAT, GR detoxifies H2O2 and other ROS to either H2O2

which is further catalyzed to H2O and O2 (Rashid et al., 2012). Inthe present study activities of SOD, GPx, GR and CAT weresignificantly decreased in 5-FU treated rats as compared to control(Rashid et al., 2013). The decrease in activity of antioxidant

enzymes (SOD, CAT, GPx, and GR) in 5-FU treated group supportsthe involvement of oxidative stress in patho physiology of 5-FUinduced renal toxicity. CH administration to the treatment groupsenhanced SOD, GPx, GR and CAT activity by scavenging ROS in theform of superoxide radicals, peroxy radicals, peroxide and singletoxygen. To summarize, the activities of enzymic and non-enzymicantioxidants were increased considerably on treatment with CH(Khan et al., 2012a,b).

BUN, creatinine and LDH are the most predisposed markers re-ported to be involved in the analysis of renal injury (Nada, 2010).5-FU induced kidney damage is clearly evident by an elevated

Fig. 5. (A–D and I) Representative photomicrographs of bcl-2 determined by immunohistochemistry. (A) There is almost negligible expression of bcl-2 in the renal sections ofcontrol group. (B) 5-FU administration decreased strongly Bcl-2 expression in renal sections. (C) There was partial expression of Bcl-2 as evidenced by weak immunostainingin the rat kidneys treated with lower dose of CH. (D) The expression of Bcl-2 was increased intensely on administration of higher dose of CH.

Fig. 6. (A–E) Histopathological examination of rat kidney 40�. (A) Normal histology of kidney. (B) Disruption of the normal renal architecture by 5-FU administration wasobserved as shown by arrows. (C and D) Treatment with CH showed protective changes in the glomeruli and tubules and morphology of tubular epithelial cells on higher doseof CH. (E) Kidney showed normal histology of almost similar to control group.

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BUN and serum creatinine levels. Our results are in good agreementwith those previously reported (Nora, 2012). On the contrary, lowerBUN and serum creatinine levels were found in rats administeredwith CH as compared to 5-FU treated group. Hence decrease inthe levels may be possibly by nephroprotective efficacy of CH.Our results corroborate with previous findings from our lab (Sultanaet al., 2012). There was also an increase in LDH when comparedwith untreated control (Nora, 2012). However, present findingsreveal that CH treatment attenuated LDH significantly demonstratingthat CH has a potential to prevent renal damage as being reportedfrom previous studies of our lab (Rashid et al., 2013; Tahir andsultana, 2011). Kim-1 is a type 1 transmembrane protein that isundetected in healthy kidney tissue. It is a more sensitive biomarkerof acute kidney injury compared to conventional and routinely usedbiomarkers of nephrotoxicity (Zhou et al., 2008). We found amarked increase in kim1 levels in 5-FU treated group. Howeverprophylactic treatment of CH alleviated kim1 levels significantly.

Immunohistochemical results revealed that 5-FU activates p53,then either undergoes G1/S arrest and are repaired or undergoapoptosis. Normal p53 induces Bax synthesis to mediate apoptosis,while damaged p53 can inhibit apoptosis leading to uncontrolledproliferation. 5-FU induced p53 expression in glomerular andtubular regions of renal tissue (Mirjolet et al., 2000 and Bowenet al., 2006) and CH down regulated p53 expression in glomerulus,tubules and parenchyma in a nearly dose dependent manner. It hasbeen observed that Bax expression was increased in glomerular,tubular region in 5-FU treated rats as compared to control and pro-phylactic treatment with CH downregulated Bax expression inboth the groups in respectively. It was found that there was negli-gible staining in Bcl-2 protein in 5-FU treated rats. CH treatment ofhigh dose sifnificantly attenuated Bcl-2 expression in glomerulus,maximally in tubules and parenchyma resulting in decrease inapoptosis induced damage to renal tissue. Therefore, the ratio ofBax and Bcl-2 determines the extent of activation of caspase-3

192 S. Rashid et al. / Food and Chemical Toxicology 66 (2014) 185–193

(Bowen et al., 2006 and Cheng et al., 2012). 5-FU induced apoptosisis a caspase-dependent process that involves activation of the ini-tiator active caspase-9 as well as effector caspase-3 (Thant et al.,2008). Similarly in our study we found that 5-FU induced cas-pase-3 strongly in bowman’s capsule, glomerulus and parenchymain renal tissue. However, CH treatment attenuated apoptosis inboth glomerular and tubular region as well as parenchyma in therenal tissue by inhibiting caspase-3 activation (executioner cas-pase) in both CH treated groups dose dependently. These findingssuggest that the Bax/Bcl-2 signaling pathway is involved in regu-lating 5-FU induced apoptosis in kidney. To summarize, CH treat-ment downregulated p53, bax, caspase-3 and upregulated Bcl-2,hence supporting our hypothesis that CH plays important role inmodulating oxidative stress and apoptosis induced by 5-FU admin-istration in kidneys. The above mentioned results further substan-tiated with the histological data which exhibited the protectiveeffects of CH against 5-FU induced distorted renal histo-architec-ture, vacuolar formation, degeneration of tubular architecture,glomerular congestion and swelling.

5. Conclusion

The result provide further insight into the mechanisms of 5-FUinduced renal toxicity and confirms the antioxidant and anti apop-totic potential of CH. Further findings of the present study supportthe role of ROS, serum enzymes, apoptotic pathway proteins likep53, caspase-3, Bax, and Bcl2 in the pathogenesis of 5-FU inducedrenal toxicity. In conclusion, CH abrogates 5-FU induced renal tox-icity in wistar rats and might be clinically useful in the form of acombinational therapy after further confirmatory studies both atpre clinical and clinical levels.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document data associated with this articlecan be found in the online version.

Acknowledgment

The authors are thankful to UGC, New Delhi India under SAP ofDepartmental Research Support II and BSR for the award of projectto carry out the study.

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