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Original Article

Abrogation of 5-flourouracil induced renal toxicity by beepropolis via targeting oxidative stress and inflammation inWistar rats

Summya Rashid a, Nemat Ali a, Sana Nafees a, Shiekh Tanveer Ahmad a,Syed Kazim Hasan a, Sarwat Sultana b,*aDepartment of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar,

New Delhi 110062, IndiabAssociate Professor, Section of Molecular Carcinogenesis and Chemoprevention, 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 30 January 2013

Accepted 1 March 2013

Available online 26 March 2013

Keywords:

KIM-1

Nephrotoxicity

NFkB

Oxidative stress

TNFa

* Corresponding author. Tel.: þ91 11 2605468E-mail address: sarwat786@rediffmail.com

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.03.003

a b s t r a c t

Background: 5-fluorouracil (5-FU) is a strong anticancer agent however its clinical use is

constrained because of its marked organ toxicity associated with increased oxidative stress

and inflammation. The present study was designed to investigate the protective effects of

bee propolis (BP), a naturally occurring bioflavonoid against 5-FU induced renal toxicity in

Wistar rats using biochemical, histopathological changes and expression levels of

inflammation.

Methodology: Wistar rats were subjected to concomitant pre and post-phylactic oral treat-

ment of BP (80 and 160 mg/kg b.wt.) against nephrotoxicity induced by i.p. injection of 5-FU

(75 mg/kg b.wt) and were sacrificed after 48 h. Nephrotoxicity was assessed by measuring

the level of serum creatinine, BUN, LDH, KIM-1 and TNFa. The level of anti-oxidant defense

enzymes of kidney tissue was also measured.

Results: Treatment with BP decreased the levels of serum toxicity markers significantly and

additionally induced anti-oxidant defense enzyme levels as well as decreased expression

of NFkB. Histopathological changes further confirmed the biochemical and immunohis-

tochemical results showing that 5-FU caused significant structural damage to kidneys like

tubular necrosis, renal lesions, glomerular congestion and dilated blood sinusoids were

also observed. All these features of 5-FU induced toxicity were reversed by co-

administration of BP.

Conclusion: Therefore, our study favors the view that BP may be a useful modulator in

alleviating 5-FU induced nephrotoxicity.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

5x5565, þ91 11 26054685x5566; fax: þ91 11 26059663.(S. Sultana).

2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 1 8 9e1 9 4190

1. Introduction 2.2. Animals

5-FU is an antineoplastic agent, belongs to the group called

antimetabolites and functions as a pyrimidine analog, syn-

thesized by Heidelberg some 50 years ago.1 It has been used

extensively in the treatment of patients with breast, stom-

ach, colorectum, head and neck, genitourinary tracts, glau-

coma and skin cancer.2 Although it generates adequate

effect, it further exhibits severe toxicity and detrimental side

effects like leukopenia, diarrhea, stomatitis, alopecia,

mucositis,3 cardiotoxicity,4 nephrotoxicty and hepatotoxici-

ty.5 It results in DNA damage, proliferative inhibition and

apoptosis both in rapidly dividing cells including cancer cells

and some normal dividing cells.6 In this context, they often

induce side effects in cancer patients that severely limit their

activity.7 Concisely, chemotherapy commences with the

generation of oxidative stress and reactive oxygen species

(ROS) which act to directly damage cells and tissues. Sec-

ondly, the transcription factor, nuclear factor kappa B (NFkB)

is activated and leads to upregulation of many genes,

including those responsible for the production of proin-

flammatory cytokines8 like TNFa. Thus, chemicals with anti-

inflammatory/antioxidative properties and minimal side ef-

fects may serve as potential therapeutic agents for the

treatment. Free radical generation during treatment with 5-

FU, leading to lipid peroxidation and cell membrane dam-

age, could be one mechanism behind the toxic effects of 5-

FU.4

BP is a well known ancient folk medicine, an intricate

resinous hive product, and a blend of waxes, sugars and plant

exudates collected by bees from plants. Flavonoids, aromatic

acids, diterpenic acids and phenolic compounds appear to be

the principal components responsible for its biological activ-

ities. It is alleged to exhibit a broad spectrum of activities

including antibacterial, antifungal, antiviral, anti-

inflammatory, local-anesthetic, anti-oxidant, immune stim-

ulating, cytostatic and free radical scavenging activities.9

Recently, it is also being used in food and beverages to

improve health and prevent diseases such as inflammation,

heart disease, diabetes and cancer.10

To the best of our knowledge such an extensive study on

renal toxicity by 5-FU has been reported for the first time.

2. Materials and methods

2.1. Chemicals

Glutathione reductase, oxidized (GSSG) and reduced gluta-

thione, 1,2-dithio-bis-nitrobenzoic acid (DTNB), 1-chloro-2, 4-

dinitrobenzene, bovine serum albumin (BSA), oxidized and

reduced nicotinamide adenine dinucleotide phosphate

(NADP), (NADPH), flavine adenine dinucleotide, 2,6-

dichlorophenolindophenol, thiobarbituric acid (TBA), 5-FU

etc: were obtained from SigmaeAldrich, USA. Sodium hy-

droxide, ferric nitrate, trichloroacetic acid (TCA) and

perchloric acid (PCA) etc were purchased from CDH,

India. Plant extract was purchased from Saiba Industries,

Mumbai.

Male Wistar rats (150e200 g), 6e8 weeks old, were obtained

from the Central Animal House Facility of Hamdard Univer-

sity. Animals received humane care in accordance with the

guidelines of the Committee for the Purpose of Control and

Supervision of Experiments on Animals (CPCSEA),Govern-

ment of India, and prior permission 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 as control and received water for 28 days and

0.9% saline intraperitoneally (i.p.) on day 25th, 26th. Group II

received i.p. injections of 5-FU (75 mg/kg b.wt.) on 25th and

26th day. Groups III and IVwere treatedwith an oral dose of BP

80 mg/kg b.wt. (D1) and 160 mg/kg b.wt. (D2), respectively, for

28 days and i.p. injections of 5-FU (75 mg/kg b.wt.) were

administered on 25th and 26th day. Group V received only D2

(160 mg/kg b.wt.) of BP for 28 days. On the 28th day, the rats

were sacrificed by cervical dislocation, blood was drawn for

serum parameters and kidneys were taken after perfusion for

examination of various biochemical, immunohistochemical

and histopathological parameters.

2.4. Biochemical estimation

Assay for catalase (CAT), lipid peroxidation (LPO), Superoxide

dismustase (SOD), reduced glutathione (GR), glutathione

peroxidase (GPx), glutathione reductase (GR), Blood Urea Ni-

trogen (BUN), Creatinine, lactate dehydrogenase (LDH) and

protein estimation was done by the method as described by

Rehman et al.11

2.5. Assay for tumor necrosis factor (TNFa) and KIM-1(Kidney injury molecule-1)

The level of TNF-a was quantitated using an ELISA based kit

(eBioscience, Inc., San Diego., USA) and KIM-1 (RAT KIM-1

ELISA KIT, Adipo Bioscience, Inc, USA) following instructions

of the manufacturer.

2.6. Immunohistochemistry

Kidney sections on polylysine coated slides obtained were

fixed in neutral buffered formalin, and embedded in paraffin

andwere treated for NFkB antibody for immunohistochemical

analysis. The procedure was processed according to the

manufacturer’s protocol recommended for NFkB immuno-

histochemistry with slight modifications.

2.7. Histopathological examination

The kidneys were quickly removed after sacrifice and pre-

served in 10% neutral buffered formalin for histopathological

processing. The kidneys were embedded in paraffin wax and

longitudinally sectioned with a microtome. Hematoxylin and

GPX

(mm

olNADPH

Oxidized/m

in/m

gpro

tein)

181.85�

14.33

80.2

�7.07***

135.99�

10.77#

153.74�

9.58###

172.39�

13.64

ntfrom

gro

upII

(#P<

0.05),

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 1 8 9e1 9 4 191

eosin staining of the sectionswas observed under an Olympus

microscope.

2.8. Statistical analysis

Differences between groups were analyzed using analysis of

variance (ANOVA) followed by Dunnet’s multiple compari-

sons test. All data points are presented as the treatment

groups’ mean � standard error (SE).

SOD,LPO,GSH,GRandGPxon5-FU

adm

inistrationin

kidneyofW

istarra

ts.

(nm

olH

2O2co

nsu

med

/min/m

gpro

tein)

SOD

(units/m

gofpro

tein)

LPO

(nm

ol

MDA

form

ed/h

pergtiss

ue)

GSH

(mm

ol

CDNBConjugate

form

ed/g

tiss

ue)

GR(mm

ol

NADPH

Oxidized

/min/m

gpro

tein)

37.1

�1.67

30.33�

2.14

9.32�

0.88

0.85�

0.03

199.86�

5.6

14.85�

1.49***

16.54�

1.31***

33.38�

3.11***

0.28�

0.07***

110.95�

6.3***

17.71�

3.87ns

21.59�

0.92ns

23.75�

1.12#

0.606�

0.04#

137.97�

8.67ns

29.30�

3.30##

24.55�

1.21##

18.68�

2.27##

0.72�

0.09##

155.95�

7.02##

32.48�

3.10

26.61�

1.35

9.81�

1.07

0.84�

0.02

194.57�

9.31

nim

als

pergro

up.Resu

ltsobtainedare

significa

ntlydifferentfrom

gro

upI(***P<

0.001).Resu

ltsobtainedare

significa

ntlydiffere

(###P<

0.00).

3. Results

3.1. Effect of 5-FU and BP on enzymic, non-enzymicarmory and MDA

Prophylaxis with BP showed an increase in GSH, GPx, GR, CAT,

SOD (ns- not significant, #P < 0.05, ##P < 0.01 and ###P < 0.001)

levels when compared with group II (***P < 0.001) and a

decrease in MDA formation dose dependently (#P < 0.05 and##P < 0.01) when compared with group II (Table 1).

3.2. Effect of 5-FU and BP treatment on the level ofserum toxicity markers

Creatinine, BUN, LDH, TNFa and KIM-1 were significantly

elevated in group II (***P < 0.001) (Table 2). Prophylactic

treatment prevented 5-FU induced elevation in all the

mentioned parameters (ns- not significant, #P < 0.05,##P < 0.01) dose dependently as compared to control.

3.3. Effect of BP on NFkB expression in 5-FU treated ratkidney

The immunohistochemical evaluation showed more intense

expression of NFkB in rats subjected to 5-FU compared with

control (Fig. 1). There was considerably moderate protein

expression of NFkB in group III as compared to II. However,

group IV showed considerably very poor or no staining.

3.4. Effect of BP against 5-FU induced histopathologicalalterations in kidney

The histology report showed that BP significantly prevented

disruption of the normal renal architecture that was distorted

by 5-FU administration in which necrosis, interstitial hemor-

rhages, glomerular atrophy and blood sinusoids could be seen

(Fig. 2).

Table

1e

Resu

ltsofBPonCAT,

Treatm

entregim

en

pergro

up

CAT

Gro

upI(control)

Gro

upII(only

5-FU)

Gro

upIII(5-FU

þBPD1)

Gro

upIV

(5-FU

þBPD2)

Gro

upV

(only

BPD2)

Resu

ltsreprese

ntmean

�SEofsixa

(##P<

0.01),(n

sP¼

notsignifica

nt)

and

4. Discussion

Although several studies have been carried out to elucidate

the molecular mechanism that causes 5-FU induced nephro-

toxicity. However factors responsible for this are not fully

understood. Chemotherapy instigates DNA and non-DNA

damage along with the production of reactive oxygen spe-

cies (ROS) or reactive nitrogen species (RNS) and a variety of

inflammatory responses. Thus, chemicals with anti-

inflammatory/antioxidative properties and minimal side

Table 2 e Results of modulatory effect of BP on BUN, creatinine, LDH, TNFa and KIM-1 on 5-FU induced renal toxicity.

Treatment regimenper group

BUN (IU/L) Creatinine(IU/L)

LDH(n mol NADHoxidized/min/mg protein)

TNF-a (pg/ml) KIM-1(mg/gserum Cr)

Group I (control) 19.75 � 0.34 0.98 � 0.07 190.96 � 18.78 160.66 � 8.4 18.427 � 2.87

Group II (only 5-FU) 34.01 � 1.34*** 2.8 � 0.09*** 356.01 � 13.78*** 364.33 � 11.62*** 110.15 � 7.684***

Group III(5-FU þ BP D1) 26.47 � 0.71# 2.3 � 0.25ns 266.08 � 28.56# 315.33 � 10.17# 83.693 � 4.472#

Group IV(5-FU þ BP D2) 23.05 � 0.85## 1.7 � 0.04## 224.82 � 17.41## 297.66 � 8.81## 69.05 � 5.11##

Group V (only BP D2) 20.99 � 0.72 1.1 � 0.02 189.38 � 9.54 154 � 4.72 25.74 � 4.56

Results representmean� SE of six animals per group. Results obtained are significantly different from group I (***P< 0.001). Results obtained are

significantly different from group II (#P < 0.05), (##P < 0.01), (nsP ¼ not significant) and (###P < 0.00).

j o u rn a l o f p h a rma c y r e s e a r c h 7 ( 2 0 1 3 ) 1 8 9e1 9 4192

effects which could be incorporated as dietary agents may

serve as potential therapeutic agents for the treatment of

chemotherapy induced organ toxicity and are worthy of

detailed investigation.12 Several anticancer drugs, including 5-

FU and oxaliplatin, have been shown to increase intracellular

concentration of ROS, and inhibition of drug induced increase

in ROS concentrations partly reverse the cytotoxicity of these

agents.13 In the present study 5-FU treated rats demonstrate

augmented level of MDA, lipid peroxidationmarker compared

to control rats as reported by Ali.5 The ingestion of BP to 5-FU

treated rats considerably decreased MDA compared to group

II. Since the most essential pharmacologically active compo-

nents in BP are flavonoids and various phenolics which have

free radical scavenging power and thus protecting lipids from

being oxidized during oxidative damage.14 SOD forms the

primary shield against superoxide as it converts reactive su-

peroxide radicals to H2O2 and H2O. However, Glutathione

peroxidase (GPx) converts H2O2 and other ROS to H2O2 and

H2O. Catalase (CAT) catalyzes H2O2 to H2O and O2. In the

present study, the activities of SOD, GPx, GR and CAT were

significantly decreased in group II as compared to I. BP

administration to 5-FU treated groups improved these

Fig. 1 e Representative photomicrograph of NFkB protein expre

group showing very less staining, (B) NFkB expression in group

expression in group III showing very less staining as compared

enzymes, may be by scavenging singlet oxygen, superoxide

anions, peroxy radicals, OH-. GSH is a tripeptide which de-

toxifies ROS efficiently, gets depleted after 5-FU injection and

gets replenished by BP prophylaxis. Present work supports

Bhadauria.15 BUN, creatinine and LDH levels were augmented

in 5-FU group.5 In contrast, BP ameliorated their levels as

compared to group II. This is an indicator of the possible

nephroprotective efficacy offered by BP against 5-FU toxicity

indicating that BP has a tendency to thwart damage and

inhibit the seepage of enzymes through cellular membranes.

KIM-1 is a transmembrane tubular protein and is barely

discernible in normal kidneys, nevertheless, it is strikingly

induced in acute kidney injury and chronic kidney disease. It

is a sensitive and explicit marker of kidney injury as well as

predictor of prognosis as supported by Huo.16 In our study,

KIM-1 levels were markedly increased in group II. Although,

prophylactic treatment of BP suppressed abnormal levels of

KIM-1.

TNF-a is a proinflammatory cytokine which plays a

widespread role in many biological processes like cell death,

growth, development, oncogenesis and immune responses.

Present study also illustrated that 5-FU administration

ssion of rat kidney. (A) NFkB protein expression in control

II showing highly intense and positive staining, (C) NFkB

to group II (D) very less staining almost similar to control IV.

Fig. 2 e (AeE) Histopathological examination of rat kidney 340. (A) Normal histology of kidney (B) disruption of the normal

renal architecture by 5-FU administration was observed as shown by arrows. (C & D) Treatment with BP showed protective

changes in the glomeruli and tubules and the morphology of tubular epithelial cells on higher dose of BP (E) kidney showed

normal histology of almost similar to control group.

j o u r n a l o f p h a rm a c y r e s e a r c h 7 ( 2 0 1 3 ) 1 8 9e1 9 4 193

significantly increases TNF-a. It has been reported that

oxidative stress may also commence or augment inflamma-

tion via upregulation of various genes implicated in the in-

flammatory mechanisms. NFkB is one of them, whose

activation results in the upregulation of proinflammatory

cytokines. Oxygen free radicals and TNF-a could activate

NFkB which is a redox sensitive transcription factor, which in

turn stimulates the successive inflammatory cascade. How-

ever mechanistic pathway of NFkB signaling and its correla-

tion with oxidative stress is not fully clear. Although it has

been reported that the use of anti-oxidants and ROS scav-

engers like cysteine, NAC, thiols, & green tea polyphenols can

check the activation of NFkB by oxidative stress .The above

findings show that ROS plays an active role in TNF-a release

and NFkB activation. Our present study gives the supporting

evidence for the induction and activation of NFkB in group II.

Present work support Tung et al and Khan et al work.17,18 It

was found that NFkB expression and TNF-a release was

attenuated substantially by BP treatment thus reducing in-

flammatory response implicated in 5-FU induced renal

toxicity.

To summarize we found that BP ameliorated molecular

targets implicated in the toxicity of 5-FU administration in

animalmodel. Hence further investigations need to be done to

be made useful for human use.

Funding

The authors are thankful to UGC, New Delhi India under SAP

of Departmental Research Support II and BSR for the award of

project to carry out the study.

Conflicts of interest

All authors have none to declare.

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