Chrysin protects against cisplatin-induced colon. toxicity via amelioration of oxidative stress and...

Toxicology and Applied Pharmacology 258 (2012) 315–329

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Toxicology and Applied Pharmacology

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Chrysin protects against cisplatin-induced colon. toxicity via amelioration ofoxidative stress and apoptosis: Probable role of p38MAPK and p53

Rehan Khan, Abdul Quaiyoom Khan, Wajhul Qamar, Abdul Lateef, Mir Tahir, Muneeb U Rehman,Farrah Ali, Sarwat Sultana ⁎Section of Molecular Carcinogenesis and Chemoprevention, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar,New Delhi-110062, India

⁎ Corresponding author at: Department of MedicalFaculty of Science, Jamia Hamdard (Hamdard UniveDelhi-110062, India. Fax: +91 11 26059663.

E-mail address: [email protected] (S. Sultan

0041-008X/$ – see front matter © 2011 Elsevier Inc. Alldoi:10.1016/j.taap.2011.11.013

a b s t r a c t
a r t i c l e i n f o
Article history:Received 18 September 2011Revised 2 November 2011Accepted 18 November 2011Available online 2 December 2011

Keywords:CisplatinColon toxicityOxidative stressApoptosisp38MAPKBak

Cisplatin, an antineoplastic drug, is widely used as a foremost therapy against numerous forms of cancer butit has pronounced adverse effects viz., nephrotoxicity, ototoxicity etc. CDDP-induced emesis and diarrhea arealso marked toxicities that may be due to intestinal injury. Chrysin (5,7-dihydroxyflavone), a natural flavonecommonly found in many plants possesses multiple biological activities, such as antioxidant, anti-inflammatory and anti-cancer effects. In the present study, we investigated the protective effect of chrysinagainst CDDP-induced colon toxicity. The plausible mechanism of CDDP-induced colon toxicity and damageincludes oxidative stress, activation of p38MAPK and p53, and colonic epithelial cell apoptosis via upregulat-ing the expression of Bak and cleaved caspase-3. Chrysin was administered to Wistar rats once daily for 14consecutive days at the doses of 25 and 50 mg/kg body weight orally in corn oil. On day 14, a single intraper-itoneal injection of cisplatin was given at the dose of 7.5 mg/kg body weight and animals were euthanizedafter 24 h of cisplatin injection. Chrysin ameliorated CDDP-induced lipid peroxidation, xanthine oxidase ac-tivity, glutathione depletion, decrease in antioxidant (catalase, glutathione reductase, glutathione peroxidaseand glucose-6 phosphate dehydrogenase) and phase-II detoxifying (glutathione-S-transferase and quinonereductase) enzyme activities. Chrysin also attenuated goblet cell disintegration, expression of phospho-p38MAPK and p53, and apoptotic tissue damage which were induced by CDDP. Histological findings furthersupported the protective effects of chrysin against CDDP-induced colonic damage. The results of the presentstudy suggest that the protective effect of chrysin against CDDP-induced colon toxicity was related with at-tenuation of oxidative stress, activation of p38MAPK and p53, and apoptotic tissue damage.

© 2011 Elsevier Inc. All rights reserved.

Introduction

Cisplatin [cis-diamminedichloroplatinum (II) (CDDP) or cisplati-num] (Fig. 1) is a platinum (Pt) containing antineoplastic drug, widelyused as a foremost therapy against numerous forms of cancer includingtesticular cancer, ovarian germ cell tumors, epithelial ovarian cancer,head and neck cancer, advanced cervical cancer, colon cancer, bladdercancer, mesothelioma, endometrial cancer, non-small cell lung cancer,malignant melanoma, carcinoids, penile cancer and adrenocorticol car-cinoma (Adenis et al., 2005; Lebwohl and Canetta, 1998; Saad et al.,2004; Thigpen et al., 1994; Van Basten et al., 1997; Wang et al., 2004a,2004b). It is used as an adjuvant therapy following surgery or radiationand also used in combination with other anticancer drugs (Basu andKrishnamurthy, 2010). The therapeutic efficacy of CDDP is enhanced

Elementology and Toxicology,rsity), Hamdard Nagar, New

a).

rights reserved.

by dose augmentation but its therapeutic intervention is due to its dam-aging effects on normal cells consequently causing pronounced adverseeffects viz., nephrotoxicity, ototoxicity, neurotoxicity, hepatotoxicity,nausea, emesis and 67% of patients experienced diarrhea (Bearcroft etal., 1999; Kim et al., 2004; Koc et al., 2005; Kris et al., 1988; Langerakand Dreisbach, 2001; Zicca et al., 2004). The precise mechanism ofCDDP toxicity is not fully understood but the plausible mechanismmay be through the DNA adduct formation and the generation of pano-ply of reactive oxygen species (ROS) e.g., superoxide anion (O2•

−), hy-drogen peroxide (H2O2), hydroxyl radical (•OH) etc.whichmay interactwith DNA, lipids and proteins (Sun, 1990). CDDP can act on the sulfhy-dryl (\SH) groups of cellular proteins (Basu and Krishnamurthy, 2010)but DNA is the main cellular target of CDDP that may lead to DNAdamage induced by ROS and Pt-DNA adduct formation, thus hampersthe cell division or DNA synthesis and its repair mechanism whichleads to apoptotic cell death (Eastman, 1985; Sherman et al., 1985).

Several lines of evidence exhibited that this chemotherapeuticdrug is not specific in action against tumors but also cytotoxic to rap-idly dividing normal cells viz., intestinal epithelial cells, through theproduction of ROS which provides a nidus for the development of

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Fig. 1. Cisplatin.

316 R. Khan et al. / Toxicology and Applied Pharmacology 258 (2012) 315–329

oxidative stress (Vijayalakshmi et al., 2006; Wadler et al., 1998). Inthis perspective, there is unequivocal evidence that natural com-pounds with antioxidant properties subside CDDP toxicity(Atessahin et al., 2007; Chang et al., 2002; Guerrero-Beltrán et al.,2010; Kim et al., 2005; Longo et al., 2011). Therefore, there is a needto explore the natural compound that can effectively diminish theCDDP-induced toxicity to improve its chemotherapeutic efficacy. Now-a-days, dietary supplements containing natural products, fruits, vegeta-bles, medicinal plants and herbs have many pharmacological propertiesand have potential to fight against numerous human diseases (Khanand Sultana, 2011). Flavonoids are the natural polyphenols ubiquitouslypresent inmanyplants (WangandMorris, 2007). Several epidemiologicalstudies suggest that dietary supplements rich in flavonoids prevent vari-ous diseases viz., cancers (Clere et al., 2011; Hoensch et al., 2010; Pieriniet al., 2008), cardiovascular diseases (Garcia-Lafuente et al., 2009), diabe-tes (Fu et al., 2011), and neurodegenerative diseases (Mandel et al., 2008;Rezai-Zadeh et al., 2005). Chrysin (5, 7-dihydroxyflavone) (Fig. 2) be-longs to this category which is found in bee propolis, honey and variousplants (Barbaric et al., 2011; Pichichero et al., 2010). It has several impor-tant biological properties viz., antioxidant (Lapidot et al., 2002), anti-inflammatory (Cho et al., 2004) and anti-cancer properties (Cardenas etal., 2006; Goncalves et al., 2011; Miyamoto et al., 2006; Wang et al.,2004a, 2004b). Chrysin also reported to enhance the level of testosteroneby inhibiting the aromatase enzymewhich converts testosterone into es-tradiol and is already available in market as a dietary supplement in theform of capsule (500 mg per capsule) (iHerb Inc., Monrovia, CA; VitaDi-gest, Walnut, CA) and 6 capsules per day as the highest suggested dose(Wang and Morris, 2007).

Previously, it has been reported that chrysin induces cell death inhuman colorectal cancer cell line i.e., HCT-116 by sensitizing thesecells to TNFα-mediated apoptosis (Li et al., 2010) and it has anti-proliferative effects via G2/M cell-cycle arrest in human colon cancercell lines (Goncalves et al., 2011; Wang et al., 2004a, 2004b). It is alsoreported to modulate NF-kB pathway in human colon cancer cells i.e.,Caco-2 cells via diminishing IkBα level, inhibiting NF-kB activationand lowering the IL-8 secretion (Romier et al., 2008).

Recently, it has been shown that chrysin induces cancer cell deathsynergistically with doxorubicin by chemosensitizing these cells tochemotherapy via GSH depletion within the cancer cells (Brechbuhlet al., 2011). These insights of chrysin help to envisage for reducingthe CDDP toxicity which may lead to improve the chemotherapeuticefficacy of CDDP.

In the light of above facts, we hypothesized that prophylactictreatment of chrysin may have protective effects against CDDP-induced colon toxicity by interfering with apoptotic pathway and

Fig. 2. Chrysin.

oxidative processes. In the present study, we investigated the protec-tive role of chrysin against CDDP induced oxidative stress, apoptoticresponses and colonic damage in Wistar rats.

Materials and methods

Chemicals. Reduced glutathione (GSH), oxidized glutathione (GSSG),nicotinamide adenine dinucleotide phosphate reduced (NADPH), nic-otinamide adenine dinucleotide phosphate oxidized (NADP), flavinadenine dinucleotide (FAD), ethylene diamine tetra-acetic acid(EDTA), thiobarbituric acid (TBA), pyrogallol, poly-L-lysine, xanthine,glucose-6-phosphate, bovine serum albumin (BSA), Mayer's hema-toxylin, dichlorophenolindophenol (DCPIP), 5,5′-dithio-bis-[2-nitrobenzoic acid] (DTNB), chrysin, 1-chloro-2,4-dinitrobenzene(CDNB), and glutathione reductase were obtained from Sigma(Sigma Chemical Co., St Louis, MO). Cisplatin was purchased fromDr. Reddy's Laboratories Ltd, Hyderabad, India. Hydrogen peroxide,magnesium chloride, sulphosalicylic acid, perchloric acid, trichlor-oacetic acid (TCA), tween-20, folin ciocalteau reagent (FCR), sodiumpotassium tartrate, di-sodium hydrogen phosphate, sodium di-hydrogen phosphate and sodium hydroxide were purchased from E.Merck Limited, India. All other chemicals and reagents were of thehighest purity grade commercially available.

Animals. Four to six-week-old, male albino rats (120–150 g) of Wistarstrain were obtained from Central Animal House of HamdardUniversity, New Delhi, India. All procedures for using experimentalanimals were checked and permitted by the “Institutional AnimalEthical Committee (IAEC)” that is fully accredited by the Committeefor Purpose of Control and Supervision on Experiments on Animals(CPCSEA) Chennai, India. Approval ID/project number for this studyis 740. They were housed in polypropylene cages in groups of fourrats per cage and were kept in a room maintained at 25±2 °C witha 12 h light/dark cycle. They were allowed to acclimatize for oneweek before the experiments and were given free access tostandard laboratory animal diet and water ad libitum.

Treatment regimen. To study the effect of prophylactic treatment withchrysin on CDDP-induced oxidative stress and apoptotic responses incolon, 30 male Wistar rats were randomly allocated to 5 groups of 6rats each. The rats of group I (control group) received corn oil throughoral gavage at the dose of 5 ml/kg body weight once daily for 14 days,which was used as a vehicle for chrysin. Group III received chrysinthrough oral gavage at the dose of 25 mg/kg body weight oncedaily for 14 consecutive days. Group IV and V received chrysinthrough oral gavage at the dose of 50 mg/kg body weight oncedaily for 14 days. Group II, III and IV rats were given a single intra-peritoneal injection of cisplatin at the dose of 7.5 mg/kg bodyweight on day 14 after 1 h of the last treatment of chrysin. All therats were anesthetized with mild anesthesia and euthanized by cer-vical dislocation after 24 h of the cisplatin injection. Selection ofcisplatin dose was based on the previously published studies(Boogaard et al., 1991; Chirino et al., 2004; Guerrero-Beltrán etal., 2010) (Fig. 3).

Post-mitochondrial supernatant (PMS) preparation and estimation ofdifferent parameters. Colons were removed quickly, cleaned free of ir-relevant material and immediately perfused with ice-cold saline(0.85% sodium chloride). The colons (10% w/v) were homogenizedin chilled phosphate buffer (0.1 M, pH 7.4) using a Potter Elvehjen ho-mogenizer. The homogenate was filtered through muslin cloth, andwere centrifuged at 3000 rpm for 10 min at 4 °C in a Remi CoolingCentrifuge (C-24 DL) to separate the nuclear debris. The aliquot soobtained was centrifuged at 12,000 rpm for 20 min at 4 °C to obtainPMS, which was used as a source of various enzymes.

image of Fig.�1

Group 1

(n = 6)

Group 2

(n = 6)

Group 3

(n = 6)

Group 4

(n = 6)

Group 5

(n = 6)

Sacrifice on day 15

Corn oil (5ml/kg b. wt.)

Cisplatin (7.5mg/kg b.wt. i.p. once at day 14) arrow indicates cisplatin injection

Chrysin (25mg/kg b. wt. orally everyday for 14 days) + Cisplatin (7.5mg/kg b.wt. i.p. once at

day 14) arrow indicates cisplatin injection

Chrysin (50mg/kg b.wt. orally everyday for 14 days) + Cisplatin (7.5mg/kg b.wt. i.p. once at

day 14) arrow indicates cisplatin injection

Chrysin only (50mg/kg body weight, orally everyday for 14 days)

Days 1 2 3 4 15 14

Fig. 3. Schematic representation of the experimental design.

317R. Khan et al. / Toxicology and Applied Pharmacology 258 (2012) 315–329

Measurement of lipid peroxidation (LPO). The assay for membrane lipidperoxidationwas done by themethod ofWright et al. (1981)with somemodifications. The reaction mixture in a total volume of 3.0 ml con-tained 1.0 ml tissue homogenate, 1.0 ml of TCA (10%), and 1.0 ml TBA(0.67%). All the test tubes were placed in a boiling water bath for a pe-riod of 45 min. The tubes were shifted to ice bath and then centrifugedat 2500 ×g for 10 min. The amount of malondialdehyde (MDA) formedin each of the samples was assessed bymeasuring the optical density ofthe supernatant at 532 nm. The results were expressed as the nmolMDA formed/gram tissue by using a molar extinction coefficient of1.56×105 M−1 cm−1.

Measurement of xanthine oxidase (XO) activity. The activity of xanthineoxidase was assayed by the method of Stirpe and Della Corte (1969).The reaction mixture consisted of 0.2 ml PMS which was incubatedfor 5 min at 37 °C with 0.8 ml phosphate buffer (0.1 M, pH 7.4). Thereaction was started by adding 0.1 ml xanthine (9 mM) and kept at37 °C for 20 min. The reaction was terminated by the addition of0.5 ml ice-cold perchloric acid [10% (v/v)]. After 10 min, 2.4 ml of dis-tilled water was added and centrifuged at 4000 rpm for 10 min and μguric acid formed/min/mg protein was recorded at 290 nm.

Measurement of reduced glutathione (GSH) level. The GSH content incolon was determined by the method of Jollow et al. (1974) inwhich 1.0 ml of PMS fraction (10%) was mixed with 1.0 ml of

sulphosalicylic acid (4%). The samples were incubated at 4 °C for atleast 1 h and then subjected to centrifugation at 1200 ×g for 15 minat 4 °C. The assay mixture contained 0.4 ml filtered aliquot, 2.2 mlphosphate buffer (0.1 M, pH 7.4) and 0.4 ml DTNB (10 mM) in atotal volume of 3.0 ml. The yellow color developed was read immedi-ately at 412 nm on spectrophotometer (Milton Roy Model-21 D). TheGSH content was calculated as μmol DTNB conjugate formed/gramtissue using molar extinction coefficient of 13.6×103 M−1 cm−1.

Measurement of glutathione peroxidase (GPx) activity. The GPx activitywas calculated by the method of Mohandas et al. (1984). A total of2 ml volume consisted of 0.1 ml EDTA (1 mM), 0.1 ml sodium azide(1 mM), 1.44 ml phosphate buffer (0.1M, pH 7.4), 0.05 ml glutathionereductase (1 IU/ml), 0.05 ml reduced glutathione (1 mM), 0.1 mlNADPH (0.2 mM) and 0.01 ml H2O2 (0.25 mM) and 0.1 ml 10% PMS.The depletion of NADPH at 340 nm was recorded at 25 °C. The en-zyme activity was calculated as μmol NADPH oxidized/min/mg pro-tein with the molar extinction coefficient of 6.22×103 M−1 cm−1.

Measurement of glutathione-S-transferase (GST) activity. The GST activ-ity was measured by the method of Habig et al. (1974). The reactionmixture consisted of 2.4 ml phosphate buffer (0.1 M, pH 6.5), 0.2 mlreduced glutathione (1.0 mM), 0.2 ml CDNB (1.0 mM) and 0.2 ml ofcytosolic fraction in a total volume of 3.0 ml. The changes in absor-bance were recorded at 340 nm and the enzyme activity was

image of Fig.�3

2

sue

1.2 ***

######

1

0.8

nmol

MD

A fo

rmed

/g ti

ssue

0.6

0.4

0.2

0GP1 GP2 GP3

Treatment Groups

GP4 GP5

GP1- Vehicle Treated Control Group (Corn oil – 5ml/kg b. wt.)

GP2- Cisplatin Treated Group (7.5mg/kg b.wt.)

GP3- Dose 1 of Chrysin (25 mg/kg b.wt.) + Cisplatin (7.5mg/kg b.wt.)


GP5- Only Dose 2 of Chrysin (50 mg/kg b.wt.)

Fig. 4. Effect of prophylactic treatment of chrysin against CDDP-induced lipid peroxida-tion (MDA level) in colon of Wistar rats. Data were expressed as mean±S.D. (n=6)and measured as nmol MDA formed/g tissue. MDA level was significantly increased(***pb0.001) in CDDP treated group (Group II) as compared to Group I. Pretreatmentwith chrysin significantly attenuated the level of MDA in Group III (###pb0.001) andGroup IV (###pb0.001) as compared to Group II.


calculated as μmol CDNB conjugate formed/min/mg protein using amolar extinction coefficient of 9.6×103 M−1 cm−1.

Measurement of glutathione reductase (GR) activity. The GR activitywas measured by the method of Carlberg and Mannervik (1975).The assay system consisted of 1.65 ml phosphate buffer (0.1 M, pH7.6), 0.1 ml EDTA (0.5 mM), 0.05 ml oxidized glutathione (1.0 mM),0.1 ml NADPH (0.1 mM) and 0.1 ml of 10% PMS in a total volume of2.0 ml. The enzyme activity was assessed at 25 °C by measuring thedisappearance of NADPH at 340 nm and was calculated as nmolNADPH oxidized/min/mg protein using molar extinction coefficientof 6.22×103 M−1 cm−1.

***###

###

18

16

14

12

10

8

6

4

2

0µg u

ric a

cid

form

ed/m

in/m

g pr

otei

n

GP1 GP2 GP3

Treatment Groups

GP4 GP5






Fig. 5. Effect of chrysin pretreatment and CDDP on XO activity. Data were expressed asmean±S.D. (n=6) and measured as μg uric acid formed/min/mg protein. XO activitywas significantly increased (***pb0.001) in CDDP treated group (Group II) as com-pared to Group I. Pretreatment with chrysin significantly attenuated the activity ofXO in Group III (###pb0.001) and Group IV (###pb0.001) as compared to Group II.

Measurement of glucose-6-phosphate dehydrogenase (G6PD) activity.The activity of glucose-6-phosphate dehydrogenase was determinedby the method of Zaheer et al. (1965). The reaction mixture consistedof 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 mlPMS (10%) and 2.1 ml distilled water in a total volume of 3 ml. Thechanges in absorbance were recorded at 340 nm and enzyme activitywas calculated as nmol NADP reduced/min/mg protein using a molarextinction coefficient of 6.22×103 M−1 cm−1.

Measurement of superoxide dismutase (SOD) activity. The SOD activitywas measured by the method of Marklund and Marklund (1974). Thereaction mixture consisted of 2.875 ml Tris–HCl buffer (50 mM, pH8.5), pyrogallol (24 mM in 10 mM HCl) and 100 μl PMS in a total vol-ume of 3 ml. The enzyme activity was measured at 420 nm and wasexpressed as units/mg protein. One unit of enzyme is defined as theenzyme activity that inhibits auto-oxidation of pyrogallol by 50%.

Measurement of catalase (CAT) activity. The catalase activity was mea-sured by themethod of Claiborne (1985). In brief, the assaymixture con-sisted of 2.0 ml phosphate buffer (0.1 M, pH 7.4), 0.95 ml hydrogenperoxide (0.019 M) and 0.05 ml of PMS (10%) in a final volume of3.0 ml. Changes in absorbancewere recorded at 240 nm. The catalase ac-tivity was calculated in terms of nmol H2O2 consumed/min/mg protein.

Measurement of quinone reductase (QR) activity. The QR activity wasdetermined by the method of Benson et al. (1980). The 3 ml reactionmixture consisted of 2.13 ml Tris–HCl buffer (25 mM, pH 7.4), 0.7 mlBSA, 0.1 ml FAD, 0.02 ml NADPH (0.1 mM), and 50 μl PMS (10%). Thereduction of dichlorophenolindophenol (DCPIP) was recorded calori-metrically at 600 nm and the enzyme activity was calculated as μmolof DCPIP reduced/min/mg protein using molar extinction coefficientof 2.1×104 M−1 cm−1.

**# #1.8

1.6

1.4

1.2

0.8

1

0.6

0.4

0.2

0µmol

DT

NB

con

juga

te fo

rmed

/g ti

s

GP1 GP2 GP3

Treatment Groups

GP4 GP5






Fig. 6. Effect of prophylactic treatment of chrysin against CDDP-induced depletion ofcolonic GSH content. Data were expressed as mean±S.D. (n=6) and measured asμmol DTNB conjugate formed/g tissue. GSH content was significantly decreased(**pb0.01) in CDDP treated group (Group II) as compared to Group I. Pretreatmentwith chrysin significantly prevented the depletion of colonic GSH level in Group III(#pb0.05) and Group IV (#pb0.05) as compared to Group II.

Table 1Effects of chrysin and cisplatin on the activities of glutathione peroxidase (GPX), glutathione-S-transferase (GST), glutathione reductase (GR) and glucose-6-phospahte dehydro-genase (G6PD).

Treatment groups GPx GST GR G6PD

I (vehicle treated control) 1.73±0.27 4.03±0.79 389.24±49.73 480.23±69.72II (cisplatin only) 1.15±0.19⁎⁎⁎ 2.38±0.67⁎ 163.97±29.35⁎⁎⁎ 271.08±52.66⁎⁎⁎

III (chrysin D1+cisplatin) 1.22±0.24NS 3.67±0.92# 180.86±39.74NS 337.95±37.15NS

IV (chrysin D2+cisplatin) 1.52±0.11# 3.73±0.52# 270.12±52.31## 357.67±27.34#

V (chrysin D2 only) 1.7±0.16 3.97±0.77 371.8±57.78 446.34±54.39

Values of glutathione dependent enzymes viz., GPx, GST, GR and G6PD are expressed as mean±S.D. (n=6). GPx was measured as μmol NADPH oxidized/min/mg protein, GST asμmol CDNB conjugate formed/min/mg protein, GR as nmol NADPH oxidized/min/mg protein and G6PD as nmol NADP reduced/min/mg protein. Significant differences wereindicated by ***pb0.001 and *pb0.05 when compared with Group I, and #pb0.05 and ##pb0.01 when compared with Group II. NS — non-significant.


Immunohistochemical staining for detection of phospho-p38, p53, Bakand cleaved caspase-3. Thick sections of 4 μm were cut fromformalin-fixed, paraffin-embedded tissue blocks and mounted onpoly-L-lysine coated microscopic slides. Paraffinized sections weredewaxed in xylene and rehydrated through graded series of ethanolto water followed by antigen retrieval in sodium citrate buffer(10 mM, pH 6.0). The slides were then allowed to cool for 15 minand washed 3 times with tris-buffered saline (TBS) for 5 min each. In-cubate the slides in 3% H2O2 in methanol for 10 min to reduce the en-dogenous peroxidase activity and then subject to power block(UltraVision Plus Detection System, Thermo Scientific) for 10 min toblock non-specific binding. After rinsing the sections in TBS, the slideswere incubated overnight at 4 °C with primary antibody inside hu-midified chamber and then were washed in TBS. The sections wereincubated with biotinylated goat anti-polyvalent secondary antibody(UltraVision Plus Detection System, Thermo Scientific) for 20 min andthen were rinsed in TBS. The sections were again incubated withstreptavidin peroxidase plus (UltraVision Plus Detection System,Thermo Scientific) for 30 min. The sections were washed in TBS anddeveloped with 3, 3′-Diaminobenzidine (DAB) solution (UltraVisionPlus Detection System, Thermo Scientific) until sections becomebrown. The sections were counterstained with Mayer's hematoxylin,mounted by using mounting media and then visualized under thelight microscope (Olympus BX51). Primary antibodies were rabbitanti-phospho-p38 (dilution 1:200, Santa Cruz), rabbit anti-p53 (dilu-tion1:100, Santa Cruz), rabbit anti-Bak (dilution 1:100, Biolegend),and rabbit anti-cleaved caspase-3 (dilution 1:400, Cell Signalling).

Staining for goblet cell analysis. The colonic sections of 4 μm were cutfrom formalin-fixed, paraffin-embedded tissue blocks and mountedon poly-L-lysine coated microscopic slides. Paraffinized sections weredewaxed in xylene and rehydrated through graded series of ethanolto water. The sections were stained with 1% Alcian blue (pH 2.5) in 3%acetic acid solution for 30 min and then rinsed for 1 min in 3% aceticacid solution to prevent non-specific staining. The slides were thenwashed in distilled water and the sections were then counterstainedwith neutral red (0.5% aqueous solution) for 20 s, dehydrated in alcohol

Table 2Effects of chrysin and cisplatin on the activities of catalase (CAT), superoxide dismutase (SO

Treatment groups Catalase

I (vehicle treated control) 80.08±25.72II (cisplatin only) 36.04±14.79⁎⁎

III (cisplatin+chrysin D1) 70.64 ±13.24#

IV (cisplatin+chrysin D2) 72.97±21.15#

V (chrysin D2 only) 78.07±20.27

Values of antioxidant enzymes (CAT, SOD and QR) are expressed as mean±S.D. (n=6). Catand QR as μmol DCPIP reduced/min/mg protein. Significant differences were indicated by###pb0.001 when compared with Group II.

andmounted by usingmountingmedia. The slides were then evaluatedunder the light microscope (Olympus BX51).

Histology. The colon were excised, flushed with saline, cut open longi-tudinally along the main axis, and then again washed with saline.These colonic sections fixed in 10% buffered formalin for at least24 h and after fixation, the specimens were dehydrated in ascendinggrades of ethanol, cleared in benzene, and embedded in paraffinwax. Blocks were made and 5 μm thick sections were cut from thedistal colon. The paraffin embedded colonic tissue sections weredeparaffinized using xylene and ethanol. The slides were washedwith phosphate buffered saline (PBS) and permeabilized withpermeabilization solution (0.1M citrate, 0.1%TritonX-100). These sec-tions stained with hematoxylin and eosin (H&E) and were observedunder light microscope at 40× magnification to investigate the his-toarchitecture of colonic mucosa.

Measurement of protein. The protein concentration in all samples wasdetermined by the method of Lowry et al. (1951) using bovine serumalbumin (BSA) as standard.

Statistical analysis. The data from individual groups were presented asthe mean±S.D. Differences between groups were analyzed usinganalysis of variance (ANOVA) followed by Dunnett's multiple com-parisons test and minimum criterion for statistical significance wasset at pb0.05 for all comparisons.

Results

Effect of prophylactic treatment of chrysin against CDDP-induced lipidperoxidation

The level of MDA was significantly enhanced (pb0.001) in Group IIas compared to Group I. Chrysin pretreatment significantly decreasedthe level of MDA in Group III (pb0.001) and Group IV (pb0.001) re-spectively as compared to Group II. No significant difference wasfound in the level of MDA between Group I and Group V (Fig. 4).

D) and quinone reductase (QR).

SOD QR

20±0.46 1.91±0.2126.19±0.64⁎⁎⁎ 1.12±0.16⁎⁎⁎

23.46±0.56### 1.38±0.11#

20.39±0.91### 1.45±0.11##

18.8±0.98 1.88±0.12

alase was measured as nmol H2O2 consumed/min/mg protein, SOD as units/mg protein**pb0.01 and ***pb0.001 when compared with Group I, and #pb0.05, ##pb0.01 and


Effect of chrysin pretreatment and CDDP on XO activity in colonic tissue

The activity of XO was significantly increased (pb0.001) in GroupII as compared to Group I. Chrysin pretreatment significantly de-creased the activity of XO in Group III (pb0.001) and Group IV(pb0.001) as compared to Group II. Group V exhibited no significantchange in the activity of XO as compared to Group I (Fig. 5).

A

C

E

D

B

Fig. 7. Effect of chrysin pretreatment on CDDP-induced phospho-p38 expression. Photomicrogtreated group (7.5 mg/kg b. wt.) (Group II), (C) dose 1 of chrysin (25 mg/kg b. wt.)+CDDP (Grchrysin (50 mg/kg b. wt.) (Group V). For immunohistochemical analyses, brown color indicatesylin staining. The colonic section of CDDP treated group (Group II) has more phospho-p38 imm(Group I)while pretreatment of chrysin in Groups III and IV reduced phospho-p38 immunostaip38 immunostaining in Group V as compared to Group I. Original magnification: 40×.

Effect of prophylactic treatment of chrysin against CDDP-induced GSHdepletion

The level of GSH was depleted significantly (pb0.01) in CDDPtreated group (Group II) as compared to control group (Group I).Chrysin pretreatment showed a significant increase in the level ofGSH in Group III (pb0.05) and Group IV (pb0.05) when compared

raphs of colonic sections depicting (A) vehicle treated control group (Group I), (B) CDDPoup III), (D) dose 1 of chrysin (50 mg/kg b. wt.)+CDDP (Group IV), and (E) only dose 2 ofspecific immunostaining of phospho-p38 and light blue color indicates nuclear hematox-unopositive staining (arrows) as indicated by brown color as compared to control group

ning as compared to Group II. However therewas no significant difference in the phospho-


with Group II. No significant difference was found in the level of GSHbetween Group I and Group V (Fig. 6).

Effect of chrysin supplementation and CDDP on the activities of glutathi-one dependent enzymes in colonic tissue

CDDP treatment caused a significant decrease in the activities ofGPx (pb0.001), GST (pb0.05), GR (pb0.001) and G6PD (pb0.001)in Group II as compared to Group I. Chrysin supplementation at thedose of 25 mg/kg b. wt. significantly increased the activity of GSTonly (pb0.05) but not other enzymes in Group III as compared to

A

C

E

B

D

Fig. 8. Effect of chrysin pretreatment on CDDP-induced p53 expression. Photomicrographs oted group (7.5 mg/kg b. wt.) (Group II), (C) dose 1 of chrysin (25 mg/kg b. wt.)+CDDP (Grouchrysin (50 mg/kg b. wt.) (Group V). For immunohistochemical analyses, brown color indicstaining. The colonic section of CDDP treated group (Group II) has more p53 immunopositivwhile pretreatment of chrysin in Groups III and IV reduced p53 immunostaining as comparedGroup V as compared to Group I. Original magnification: 40×.

Group II. But the higher dose of chrysin (50 mg.kg b. wt.) significantlyattenuated the activities of GPx (pb0.05), GST (pb0.05), GR (pb0.01)and G6PD (pb0.05) in Group IV as compared to Group II. However,the activities of these enzymes in Group V did not change significant-ly as compared to Group I (Table 1).

Effect of chrysin supplementation and CDDP on the activities of colonicantioxidant enzymes

The activities of CAT and QR were decreased significantly at thelevel of pb0.01 and pb0.001 respectively in Group II as compared

f colonic sections depicting (A) vehicle treated control group (Group I), (B) CDDP trea-p III), (D) dose 1 of chrysin (50 mg/kg b. wt.)+CDDP (Group IV), and (E) only dose 2 ofates specific immunostaining of p53 and light blue color indicates nuclear hematoxyline staining (arrows) as indicated by brown color as compared to control group (Group I)to Group II. However there was no significant difference in the p53 immunostaining in


to Group I while the activity of SOD was increased significantly(pb0.001) in Group II as compared to Group I. Chrysin pretreatmentat the dose of 25 mg/kg b. wt. significantly augmented the activitiesof CAT (pb0.05) and QR (pb0.05) in Group III as compared toGroup II while the activity of SOD was significantly decreased(pb0.001) in Group III as compared to Group II. The higher dose ofchrysin (50 mg/kg b. wt.) also showed significant increase in the ac-tivities of CAT (pb0.05) and QR (pb0.01) in Group IV as comparedto Group II while the activity of SOD was significantly decreased(pb0.001) in Group IV as compared to Group II. However, the activi-ties of these enzymes in Group V did not change significantly as com-pared to Group I (Table 2).

A B

C D

E

Fig. 9. Effect of chrysin pretreatment on CDDP-induced Bak expression. Photomicrographs oted group (7.5 mg/kg b. wt.) (Group II), (C) dose 1 of chrysin (25 mg/kg b. wt.)+CDDP (Grouchrysin (50 mg/kg b. wt.) (Group V). For immunohistochemical analyses, brown color indicstaining. The colonic section of CDDP treated group (Group II) has more Bak immunopositivwhile pretreatment of chrysin in Groups III and IV reduced Bak immunostaining as comparedGroup V as compared to Group I. Original magnification: 40×.

Effect of chrysin pretreatment and CDDP on the expression of phospho-p38, p53, Bak and cleaved caspase-3 in colonic tissue

The colonic sections of CDDP treated group (Group II) have morephospho-p38, p53, Bak and cleaved caspase-3 immunopositive stain-ing (arrows) as indicated by brown color as compared to controlgroup (Group I) while pretreatment of chrysin in Groups III and IV re-duced phospho-p38, p53, Bak and cleaved caspase-3 immunostainingas compared to Group II. However, there were no significant differ-ences in the immunostaining of all proteins in Group V as comparedto Group I. For immunohistochemical analyses, brown color indicatesspecific immunostaining of phospho-p38, p53, Bak and cleaved

f colonic sections depicting (A) vehicle treated control group (Group I), (B) CDDP trea-p III), (D) dose 1 of chrysin (50 mg/kg b. wt.)+CDDP (Group IV), and (E) only dose 2 ofates specific immunostaining of Bak and light blue color indicates nuclear hematoxyline staining (arrows) as indicated by brown color as compared to control group (Group I)to Group II. However there was no significant difference in the Bak immunostaining in


caspase-3, and light blue color indicates hematoxylin staining. Origi-nal magnification: 40× (Figs. 7–10).

Effect of chrysin pretreatment against CDDP-induced goblet celldisintegration

The colonic sections of CDDP treated group (Group II) showed ex-tensive disintegration of goblet cells whereas there is no goblet celldisintegration in control group (Group I). In Groups III and IV, chrysinsupplementation at both the doses (50 and 100 mg/kg b. wt.) showed

A B

C D

E

Fig. 10. Effect of chrysin pretreatment on CDDP-induced cleaved caspase-3 expression. Phot(B) CDDP treated group (7.5 mg/kg b. wt.) (Group II), (C) dose 1 of chrysin (25 mg/kg b. w(E) only dose 2 of chrysin (50 mg/kg b. wt.) (Group V). For immunohistochemical analyses, bindicates nuclear hematoxylin staining. The colonic section of CDDP treated group (Group IIcompared to control group (Group I) while pretreatment of chrysin in Groups III and IV redusignificant difference in the cleaved caspase-3 immunostaining in Group V as compared to

protection against CDDP-induced goblet cell disintegration as com-pared to Group II (Fig. 11).

Effects of chrysin pretreatment and CDDP on colon histology

The H&E stained sections exhibited normal histoarchitecture withmild inflammatory cell infiltration in control group (Group I) whileCDDP-treated groups showed distorted mucosal glandular architec-ture, crypt ablation with intense inflammatory cell infiltration in mu-cosal and submucosal layers as well as crypt abscess formation. InGroups III and IV, chrysin significantly attenuated the CDDP-induced

omicrographs of colonic sections depicting (A) vehicle treated control group (Group I),t.)+CDDP (Group III), (D) dose 1 of chrysin (50 mg/kg b. wt.)+CDDP (Group IV), andrown color indicates specific immunostaining of cleaved caspase-3 and light blue color) has more caspase-3 immunopositive staining (arrows) as indicated by brown color asced cleaved caspase-3 immunostaining as compared to Group II. However there was noGroup I. Original magnification: 40×.

A B

C D

E

Fig. 11. Effect of chrysin pretreatment on CDDP-induced goblet cell disintegration. Photomicrographs of Alcian blue staining of histological sections of colon depicting (A) vehicletreated control group (Group I), (B) CDDP treated group (7.5 mg/kg b. wt.) (Group II), (C) dose 1 of chrysin (25 mg/kg b. wt.)+CDDP (7.5 mg/kg b. wt.) (Group III), (D) dose 1 ofchrysin (50 mg/kg b. wt.)+CDDP (7.5 mg/kg b. wt.) (Group IV), and (E) only dose 2 of chrysin (50 mg/kg b. wt.) (Group V). (A) Goblet cells stained blue due to acidic mucin se-creted by these cells and exhibited the normal integrated goblet cells along the colonic sections. (B) Extensive disintegration of goblet cells was observed (bold arrows) in CDDP-treated group. (C and D) Chrysin pretreatment showed protection against CDDP-induced goblet cell disintegration. Both the doses of chrysin (50 and 100 mg/kg b. wt.) maintainedthe integrity of goblet cells. (E) Normal integrated goblet cells were observed as in control group. Original magnification: 40×.


histopathological changes at both the doses (50 and 100 mg/kg b.wt.). There is no significant difference in the histological changes inGroup V as compared to Group I (Fig. 12).

Discussion

In this study, we have examined the protective effects of chrysinagainst CDDP-induced colon toxicity in Wistar rats. The CDDP-induced intestinal toxicity is well documented as it causes emesisand diarrhea (Bearcroft et al., 1999). The exact mechanism underly-ing CDDP-induced intestinal toxicity is still unclear but it may bedue to ROS generated by CDDP which leads to the condition of

oxidative stress. Therefore, the natural compounds with antioxidantproperties are gaining much attention of several investigators. Inthe present study, the protective effects of chrysin observed may beassociated with amelioration of oxidative stress and apoptotic dam-age in the colon of CDDP treated rats. CDDP generates superoxideanion (O2•

−), hydrogen peroxide (H2O2), hydroxyl radical (•OH)which may play a part in the initiation of lipid peroxidation (Sun,1990). Lipid peroxidation is a marker of oxidative stress and severalstudies have reported that remarkable elevation in the level of malon-dialdehyde (MDA), a lipid peroxidation product, was observed afterCDDP treatment (Chang et al., 2002; Koc et al., 2005). Our results cor-roborated with the abovementioned previous findings which showed

A B

C D

E

Fig. 12. Effect of prophylactic treatment of chrysin against CDDP-induced histological alterations. Photomicrographs of histological sections of colon depicting (A) vehicle treatedcontrol group (Group I), (B) CDDP treated group (7.5 mg/kg b. wt.) (Group II), (C) dose 1 of chrysin (25 mg/kg b. wt.)+CDDP (7.5 mg/kg b. wt.) (Group III), (D) dose 1 of chrysin(50 mg/kg b. wt.)+CDDP (7.5 mg/kg b. wt.) (Group IV), and (E) only dose 2 of chrysin (50 mg/kg b. wt.) (Group V). (A) Normal histology of the rat colon with mild inflammatorycell infiltration (arrows) (normal appearance of the mucosal crypts of Lieberkuhn, submucosa, and muscular layers). (B) Distorted mucosal glandular architecture (bold arrows),crypt ablation (arrowheads) with intense inflammatory cell infiltration in mucosal and submucosal layers (arrows) and crypt abscess formation (stars) were observed in CDDP trea-ted group. (C and D) Chrysin supplementation prevented the mucosal damage and ameliorated inflammatory cell infiltration as well as crypt ablation and crypt abscess formation.(E) Normal histology was observed as in Group I. Original magnification: 40×.


that there is remarkable increase in the level of MDA in rats treatedwith CDDP and pretreatment with chrysin significantly reduced thelevel of MDA.

It was observed that CDDP-induced GSH depletion and enhancedxanthine oxidase (XO) activity further corroborating the CDDP-induced oxidative damage in the colon of Wistar rats. Chrysin supple-mentation significantly attenuated the GSH depletion and XO activity.XO is an enzyme that reduces O2 to superoxide anion radical (O2•

−)and consequently produce oxidative stress (Heunks and Dekhuijzen,2000) while GSH is a low molecular weight tripeptide cellular antiox-idant. It protects the peroxidation of lipid membrane by conjugating

with the electrophile such as 4-Hydroxy-3-nonenal (HNE), formedduring lipid peroxidation and thus gets depleted in this conjugationreaction (Kawanishi and Yamamoto, 1991). This conjugation of GSHvia sulphahydryl (\SH) group to electrophile is catalyzed by an anti-oxidant enzyme i.e., glutathione-S-transferase (GST), and thus theGST activity decreased in this process (Douglas, 1987; Forman et al.,2009).

Additionally, it was observed that the activities of antioxidant en-zymes viz., CAT, GPx, GR and G6PD and phase-II detoxifying enzymesviz., GST and QR were diminished whereas the activity of SOD was in-creased in rats treated with CDDP. Pretreatment with chrysin


significantly attenuated the activities of these antioxidant and phase-II detoxifying enzymes. In this study, the increase in SOD activity inCDDP treated group is in agreement with the previous findingwhich exhibit that CDDP treatment leads to the over-expression ofSOD to lessen the CDDP toxicity (Vijayalakshmi et al., 2006). TheGST enzyme detoxifies a number of ROS via catalyzing the conjugationwith GSH (Douglas, 1987; Manar et al., 2004). QR is a phase-II enzymeinvolve in xenobiotic metabolism that catalyzes the two-electron re-duction and thus protects cells against free radicals and ROS generatedby the one-electron reductions catalyzed by cytochromes P450 andother enzymes (Benson et al., 1980; Dinkova-Kostova and Talalay,

GSSG

2GSH

GST

SOD

R R-SHXO

CAT

H2O2H2O

H2O

O2-.

GPx GR

Cisplatin

ROS

Membrane damage (LPO)

Cell membrane

Cytoplasm

4

3

2

1

6

Chrysin

E

I

5 a

DNA damage

Fig. 13. Targets of action of chrysin against cisplatin induced debilities, in colon of Wistar ratactivation of p38MAPK and later results in phosphorylation and activation of p53 that allowleads to apoptosis via Bak and consequent caspase activation. Chrysin pre-treatment showsin antioxidants like SOD (2), CAT (3) activities and GSH content and related redox cycle enMoreover chrysin pretreatment also increased phase II metabolizing enzyme (GST and QR)branes (6). Chrysin shows the promising role against cisplatin induced apoptotic injuries in10 respectively). Cleaved Casp-3 = cleaved caspase-3; CAT = catalase; GPx = glutathione pglutathione; GST = glutathione S transferase; G-6-P = glucose-6-phosphate; G-6-PD = gamide adenine dinucleotide phosphate reduced; NADP+ = nicotinamide adenine dinucleoxenobiotic; ROS = reactive oxygen species; R-SH = thiol conjugated xenobiotics; SOD =indicates the activation.

2000). The diminished activities of antioxidant enzymes (viz., CAT,GPx, GR and G6PD) and phase-II detoxifying enzymes (viz., GST andQR) in CDDP-treated group supporting the involvement of oxidativestress in the pathophysiology of CDDP-induced colon toxicity.

It has already been reported that CDDP-induced toxicity wasclosely associated with ROS generated by CDDP (Kim et al., 2010).CDDP damages cellular DNA by forming Pt-DNA adducts and addi-tionally CDDP-generated ROS also fostering the DNA damage withinthe cell thus leading to the activation of p38 mitogen-activated pro-tein kinase (MAPK) (Nishida et al., 2005; Reinhardt et al., 2007).CDDP-induced DNA damage leads to the activation of DNA damage

6-PG

NADP+

G-6-P

HEXOKINASE

NADPH

GLUCOSE

NADP+

NADPH

G6PD QR

Semiquinone

Quinone

Apoptosis

Bakp53

5 b

7

8

GLUCOSECasp-3

xtracellular Compartment

ntracellular Compartment

9

p38MAPKMitochondria

10

s. Cisplatin causes toxicity via DNA damages and ROS generation. DNA damage leads tos the cells to repair the DNA by blocking the cell cycle. If DNA remains unrepaired, it

reduction in XO activity (1) leading to reduction in ROS formation. Further attenuationzymes (GR, GPx, and G6PD) (4) potentiate its role against oxidant induced damages.activities (5 a and 5 b). These effects are evident by reduction in LPO of cellular mem-colons by reducing the levels of phospho-p38, p53, Bak and cleaved Casp-3 (7, 8, 9 anderoxidase; GR = glutathione reductase; GSH = reduced glutathione; GSSG = oxidizedlucose-6-phosphate dehydrogenase; 6-PG = 6-phosphogluconate; NADPH = nicotin-tide phosphate (oxidized); O2•

− = superoxide radical; QR = quinone reductase; R =superoxide dismutase; XO = xanthine oxidase; Bak = Bcl-2-antagonist/killer. Arrows


sensor kinases viz., Ataxia–Telangiectasia mutated (ATM) and Ataxia–Telangiectasia and Rad-3 related (ATR). These sensor kinases phos-phorylate and activate thousand and one (Tao) kinases and later fur-ther phosphorylate and activate MAPK kinase 3/4/6 (MKK3/4/6)which ultimately leads to the phosphorylation and activation ofp38MAPK (Ashwell, 2006; Raman et al., 2007; Thornton and Rincon,2009). In our study, it was observed that CDDP-treated group hasmore phospho-p38 immunopositive staining as compared to controlgroup while pretreatment with chrysin significantly attenuated thephospho-p38 immunopositive staining. These results further supportedthe involvement of oxidative stress in CDDP-induced toxicity (Fig. 13).

The p53 is a tumor suppressor protein and also acts as a transcrip-tion factor that regulates the transcription of genes involved in cellcycle, DNA repair and apoptosis (Riley et al., 2008). Mdm-2 is a co-repressor of p53 and it maintains the low level of p53 via ubiquitin-mediated proteosomal degradation (Haupt et al., 1997). In responseto DNA damage, phosphorylated and activated p38MAPK directs thephosphorylation and activation of p53 and thus the cellular level ofp53 protein increases (Bulavin et al., 1999; Huang et al., 1999; Mayaet al., 2001; She et al., 2000, 2001). In response to irrepaired or irrep-arable DNA damage, p53 translocates to the mitochondria and in-duces apoptosis via interacting and activating the mitochondrialmembrane protein Bak (Bcl-2-antagonist/killer). Bak, instead of Bax,plays a critical role in mitochondrial fragmentation during apoptosisand p53-Bak interaction causes homo-oligomerization of Bak withinthe outer mitochondrial membrane ensuing mitochondrial outermembrane permeabilization (MOMP) they cause, unleashes thepro-apoptotic proteins viz., cytochrome c into the cytosol (Brooks etal., 2007; Green and Kroemer, 2009; Leu et al., 2004). Released cyto-chrome c ultimately results in activation of caspase-3 which is themain executioner caspase because it can be activated through bothintrinsic and extrinsic pathway (Liu et al., 1996; Narula et al., 1999).Activated caspase-3 leads to DNA fragmentation and cleavage of spe-cific cellular proteins like poly(ADP-ribose) polymerase (PARP), actin,fodrin and lamin during apoptosis (Sakahira et al., 1998).

The present study has demonstrated that CDDP-treated group hasmore p53, Bak and cleaved caspase-3 immunopositive staining as com-pared to control groupwhile prophylactic treatmentwith chrysin signif-icantly attenuated the p53, Bak and cleaved caspase-3 immunopositivestaining. These results further supported the involvement of apoptoticcolonic damage and oxidative stress in CDDP-induced toxicity (Fig. 13).

Goblet cells, the specialized exocrine cells of colonic crypts, synthesizeand secrete mucins. Mucins are high molecular weight, highly glycosy-lated proteins which forms a protective layer in the form of gel in intesti-nal lumen (Robbe et al., 2004; Specian and Oliver, 1991). It was observedin our study that chrysin significantly attenuated theCDDP-induceddisin-tegration of the goblet cells in colonic crypt. These results exhibited theprotective effects of chrysin against CDDP-induced toxicity.

The above mentioned findings corroborated with the histologicaldata which exhibited the protective effects of chrysin against CDDP-induced distorted mucosal glandular architecture, crypt ablationwith orchestration of intense inflammatory cell infiltration in muco-sal and submucosal layers as well as crypt abscess formation.

The precise mechanism of protective action of chrysin against CDDPis still unknown but it can be concluded from the findings of the presentstudy that chrysin exhibits the protective effect against CDDP-inducedcolon toxicity probably through the attenuation of CDDP-induced oxi-dative stress and apoptotic tissue damage. It could be used as a combi-national therapy with CDDP but before that further studies are stillneeded to elucidate the exact protective mechanism of chrysin.

Abbreviations

BSA bovine serum albuminCDNB 1-chloro 2, 4-dinitrobenzene

CDDP cisplatinDTNB 5, 5′-dithio bis-[2-nitrobenzoic acid]EDTA ethylene diamine tetra acetic acidGPx glutathione peroxidaseGR glutathione reductaseGSH reduced glutathioneGSSG oxidized glutathioneGST glutathione-S-transferaseNADPH reduced nicotinamide adenine dinucleotide phosphateQR quinone reductaseROS reactive oxygen speciesSOD superoxide dismutaseTBA thiobarbituric acidXO xanthine oxidase

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgment

The author (Sarwat Sultana) is thankful to University Grants Com-mission (New Delhi, India), UGC-Special Assistance Programme De-partmental Research Support-II (UGC-SAP DRS-II) and ResearchFellowship for Science and Meritorious (RFSMS) to first author tocarry out this work. The funding source had no role in study design,in the collection, analysis and interpretation of data; in the writingof the report; and in the decision to submit the article for publication.

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