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Toxicology Letters 216 (2013) 146– 158

Contents lists available at SciVerse ScienceDirect

Toxicology Letters

jou rn al h om epa ge: www.elsev ier .com/ locate / tox le t

hrysin suppresses renal carcinogenesis via amelioration of hyperproliferation,xidative stress and inflammation: Plausible role of NF-�B

uneeb U. Rehman, Mir Tahir, Abdul Quaiyoom Khan, Rehan Khan, Abdul Lateef, Oday-O-Hamiza,ajhul Qamar, Farrah Ali, Sarwat Sultana ∗

ection of Molecular Carcinogenesis and Chemoprevention, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi10062, India

i g h l i g h t s

Preventive role of chrysin was stud-ied against rat renal toxicity andcarcinogenesis.Chrysin administration shows pro-tection against rat renal carcinogene-sis.Effect of chrysin on inflammatoryand proinflammatory cytokines wasstudied.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 18 April 2012eceived in revised form5 November 2012ccepted 19 November 2012vailable online 27 November 2012

eywords:hrysinerric nitrilotriacetatenflammationumor promotion

a b s t r a c t

Flavonoid family is a rich source of polyphenolic compounds and hence possess strong antioxidant andanti inflammatory properties. The aim of this study was to determine the efficacy of chrysin; a bio-activeflavonoid as an anticancer agent. Renal cancer was initiated by single intraperitoneal (i.p.) injectionof N-nitrosodiethylamine (DEN 200 mg/kg BW body weight) and promoted by twice weekly admin-istration of ferric nitrilotriacetate (Fe-NTA) 9 mg Fe/kg BW for 16 wk. In the present study, we reportthe chemopreventive effects of chrysin against (Fe-NTA) induced renal oxidative stress, inflammation,hyperproliferative response, and two-stage renal carcinogenesis. To ascertain the molecular mechanismimplicated in the antitumor promoting activity of chrysin, its effect was investigated on markers of tumorpromotion and inflammation: ornithine decarboxylase (ODC) activity, proliferating cell nuclear antigen(PCNA), inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) expression, and on levelsof proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-� (TNF-�), and prostaglandinE2 (PGE2). Pretreatment of animals with chrysin at both doses (20 and 40 mg/kg body weight) markedlyinhibited all. Further, Fe-NTA enhances renal lipid peroxidation, with concomitant reduction in reduced

glutathione content (GSH), antioxidant enzymes, and phase II metabolizing enzymes. It induces serumtoxicity markers, viz., blood urea nitrogen (BUN), creatinine and lactate dehydrogenase (LDH). Prophy-lactic treatment of animals with chrysin before the administration of Fe-NTA was effective in modulatingoxidative and renal injury markers and resulted in the diminution of Fe-NTA mediated injury. Theseresults suggest chrysin as an effective chemopreventive agent having the capability to obstruct DENinitiated and Fe-NTA promoted

∗ Corresponding author at: Department of Medical Elementology and Toxicology,aculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, Newelhi 110062, India. Tel.: +91 11 26054685x5565/5566; fax: +91 11 26059663.

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

378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2012.11.013

renal cancer in the rat model.© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Renal cell carcinoma (RCC) represents the most common andlethal cancer in urinary system (Liou et al., 2004). The averagesurvival, following metastatic RCC, is about 4 months, and only10% of patients survive for 1 year. Despite the increase in the

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M.U. Rehman et al. / Toxicol

nderstanding of molecular mechanisms in the development ofCC and the later development of many novel chemotherapeuticgents over the past decade, yet RCC remains an incurable andethal disease (Bullock et al., 2010). RCC development has beeninked with many risk factors including environmental exposure toarious toxicants (Jemal et al., 2010). Nitrilotriacetate (NTA) is annvironmental toxicant often used in detergent making industry asn alternative for polyphosphates, it enters into water bodies in theorm of wash offs of soap and detergents from hospitals, householdnd industries. Mizuno et al. (2006) reported that repeated i.p.dministration of Ferric nitrilotriacetate (Fe-NTA) induced acutend sub-acute renal proximal tubular damage and subsequentevelopment of RCC in rats and mice at high incidence (60–92%). It

s also reported that Fe-NTA administration can specifically causesllelic loss of the p16 tumor suppressor gene in renal tubularells (Hiroyasu et al., 2002). Fe-NTA is a known pro-oxidant andarcinogen causing renal and hepatic damage. Huang (2003)eported that NTA has ability to form complex with Fe2+ and Cu2+

nd iron overload itself is associated with renal carcinogenesis.xperimental studies from our lab and others have demonstratedhat Fe-NTA can induce renal toxicity and renal tumor formationy inducing oxidative stress, DNA damage, cellular proliferationnd inflammation (Rehman and Sultana, 2011; Ahmad et al., 2011;qbal et al., 2003). ROS have a key role in initiation as well as tumorromotion as they can alter various signaling pathways involved

n cellular proliferation and inflammation by modulation of redoxensitive nuclear transcription factor kappa B (NF-�B), proliferat-ng cell nuclear antigen (PCNA), cyclo-oxygenase-2 (COX-2) andeveral other enzymes involved in cellular signaling.

Renal tumorigenesis could be intercepted through modulatingts pathophysiological events of initiation and/or promotion whichorms the basic axis of various promising anticancer therapeutics.lear and precise knowledge of these core events is pre-requisiteor the successful anticancer drug designing. Recently, newer meth-ds involving administration of natural and dietary agents ofavonoids, terpenoids, polyphenols, indoles and organosulphidesrigin are employed to impede, preclude and restore the variousolecular pathological happenings characteristic of cancer. Eth-

obotanicals are currently in vogue for the targeted and differentialhemopreventive strategy against oncogenic phenotype. One of theajor and highly valued advantage associated with these agents

re minimum or low unwanted effects which are generally associ-ted with classical and contemporary modern synthetic anticanceredicines. Another non-ignorable feature involves the therapeu-

ic action specifically against transformed cells and destroying theealthy and normal cellular population to the minimum. Thus,aking in to account the ever-increasing statistics of the cancerncidences, and devoid of any valuable contemporary medicinalool, the new discipline of cancer chemoprevention through nat-ral agents should be encouraged and explored for its beneficialffects on human health.

Epidemiological and chemopreventive studies in both ani-als and humans have shown that usual consumption of fruits,

egetables, and tea is associated with decreased risk of cancer.hey provide essential nutrients and many diet-derived pheno-ics particularly flavonoids which have been demonstrated toxert potential anticarcinogenic activities (Middleton et al., 2000).lavonoids are natural polyphenolic phytochemicals that are ubiq-itous in plants and present in the normal human diet, are safend associated with extremely low toxicity which makes them firstated candidate for chemoprevention (Wang and Morris, 2007).

Flavonoids are effective in decreasing the risk of various dis-

ases like cancer (Clere et al., 2011) and reported to be effectiven case of diabetes (Fu et al., 2011) cardiovascular (Garcia-Lafuentet al., 2009) and neurodegenerative disease (Mandel et al., 2008).hese suggested protective effects of flavonoids, together with

tters 216 (2013) 146– 158 147

their potent antioxidative and free radical scavenging activitiesobserved in in vivo studies have increased the public’s interest inthe use of flavonoids for their potential health benefits. Chrysin(5,7-dihydroxyflavone) is a natural flavonoid found in many plantextracts, honey, and propolis (Barbaric et al., 2011; Pichichero et al.,2010). Chrysin exhibits many biological activities and pharmaco-logical effects, including antioxidant, anti-inflammatory, anti-agingand anticancer (Goncalves et al., 2011; Cárdenas et al., 2006).Chrysin has also been reported to improve bowel diseases (Shinet al., 2009). Khan et al. (2011a) recently reported chrysin exertshepato-protective effect and notably inhibits nodule formation.We have also reported recently that chrysin is effective in preven-ting ethanol induced organ toxicity and protects against colon andjejunum toxicity (Tahir and Sultana, 2011; Khan et al., 2012a,b).

There are no previously published reports regarding chemo-preventive effect of chrysin on renal carcinogenesis. Therefore, thepresent study was planned to investigate the efficacy of chrysinagainst two-stage renal carcinogenesis in animal model and toprobe into the mechanism(s) that might be implicated in its anti-cancer activity. The effect of chrysin was investigated on key aspectsof carcinogenesis with a major focus on inflammation and tumorpromotion.

2. Materials and methods

2.1. Chemicals

Oxidized and reduced glutathione, NTA, N-nitrosodiethylamine (DEN), nitrilo-triacetic acid, H2O2, dithionitrobenzene (DTNB), 1-chloro-2,4-dinitrobenzene(CDNB), glutathione reductase, reduced nicotinamide adenine dinucleotide phos-phate (NADPH), flavine adenine dinucleotide (FAD), chrysin and proteinase K werepurchased from Sigma Chemical Co. (St. Louis, MO, USA). All the antibodies, chemi-cals and reagents used were of highest purity and standard commercially available.Rat TNF-� ELISA Ready Set Go, E-bioscience (USA), ELISA PGE2 EIA kit, CaymanChemical Company (USA).

2.2. Animals

Young (8–10 weeks old), male Wistar rats were housed in plastic (polypro-pylene) cages in animal house facility of Hamdard University. Experiments wereconducted according to protocols approved by CPCSEA animal ethical committee,New Delhi, India, project number and date 547/CPCSEA, May 28th, 2009. The well-ventilated animal rooms (room temperature set at 25 ◦C) were maintained on 12 hlight–dark cycles. They were acclimatized for 1 week before the study and had freeaccess to standard laboratory feed (Hindustan Lever, Ltd., Mumbai, India) and waterad libitum.

2.3. Preparation of Fe-NTA

Preparation of Fe-NTA solution was done by method given by Awai et al. (1979)as modified by Athar and Iqbal (1998). Briefly, ferric nitrate (0.16 mM) solution wasmixed with a 4-fold molar excess of disodium salt of NTA (0.64 nM) and the pHwas adjusted to 7.4 with a sodium bicarbonate solution. The solution was preparedimmediately before each protocol.

2.4. Experimental design

The treatment regimen for chrysin and the proposal of verifying its chemo-preventive efficacy against renal carcinogenesis was based on the preliminary dosedependent pilot study carried out in our laboratory. To study the chemo-protectiveeffects of chrysin on biochemical and serological changes induced by toxicity ofFe-NTA in rats, 30 male Wistar rats were randomly divided into five equal groups.Chrysin was dissolved in corn oil. Rats were initiated with intraperitoneal injection(i.p.) of DEN at a dose of 200 mg/kg b wt and promoted with Fe-NTA at a dose of9 mg Fe/kg b wt i.p. Selection of the dose regimen is based on our own preliminaryexperiments and is also based on previously published data from our laboratory(Athar and Iqbal, 1998; Jahangir and Sultana, 2007). Following treatment regimenwas followed in our study:

• Group I animals received only corn oil for 20 consecutive days by oral gavage and

thus served as untreated controls.

• Group II served as treated control and was administered a single dose of Fe-NTAon 20th day.

• Group III was pretreated with oral gavage of chrysin at a dose of 20 mg/kg b wtfor 20 consecutive days followed by administration of Fe-NTA on 20th day.

148 M.U. Rehman et al. / Toxicology Letters 216 (2013) 146– 158

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Fig. 1. Representation of treatment regimen for induction of 2-stage renal c

Group IV was pretreated with oral gavage of chrysin at a dose of 40 mg/kg b wtfor 20 consecutive days followed by administration of Fe-NTA on 20th day.Group V received by oral gavage of chrysin only, at a dose of 40 mg/kg b wt for 20consecutive days.

All animals were sacrificed exactly 12 h after Fe-NTA administration. Kidneyissue was processed for biochemical estimations. Blood was collected and serumas separated out and processed for serological studies.

To study the effect of pretreatment with chrysin on DEN initiated and Fe-NTA-romoted renal carcinogenesis, the rats were divided into six groups of 25 rats perroup. The complete treatment regimen followed in tumor study is illustrated inig. 1 and the detail is given below:

Group I animals received only normal corn oil by oral gavage once daily for 16weeks and served as controls.Group II also received only corn oil by oral gavage once daily for 16 weeks. Inaddition group II was given i.p. injection of DEN in saline on the very first day ofexperiment and ten days after the injection, the animals were promoted with i.p.injection of Fe-NTA, twice a week for 16 weeks.Group III was given same treatment as group II and was also co-treated by oralgavage of chrysin once daily, at a dose of 20 mg/kg b wt, an hour prior to the

treatment of Fe-NTA for a period of 16 weeks.Group IV was given same treatment as group II and was also co-treated by oralgavage of chrysin once daily at a dose of 40 mg/kg b wt, an hour prior to thetreatment of Fe-NTA for a period of 16 weeks.Group V received only chrysin by oral gavage, once daily for 16 weeks. In additiongroup V was given i.p. injection of DEN in saline on the very first day of experiment.

ogenesis model of cancer and subsequent prevention by the use of chrysin.

At the end of 24 weeks, all the animals were sacrificed under light ether anes-thesia. Their kidneys were quickly removed and processed for various molecular,histopathological and immunohistochemical studies.

2.5. Post-mitochondrial supernatant (PMS) preparation

Post-mitochondrial supernatant of kidney samples was prepared by the methodof Tahir and Sultana (2011).

2.6. Assay for catalase activity

The catalase activity was assessed by the method of Claiborne (1985).

2.7. Estimation of lipid peroxidation level

The assay of lipid peroxidation (LPO) was done according to the method ofWright et al. (1981).

2.8. Estimation of GSH level

GSH was assessed by the method of Jollow et al. (1974).

2.9. Assay for glutathione peroxidase activity

The activity of glutathione peroxidase (GPx) was calculated by the method ofMohandas et al. (1984).

ogy Letters 216 (2013) 146– 158 149

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Fig. 2. Effect of treatment of chrysin against Fe-NTA promoted ODC level in kid-ney of Wistar rats. Data were expressed as mean ± SEM (n = 6) and measured ODCactivity was measured as pmol 14CO2 released/min/mg protein. ODC levels weresignificantly increased (***P < 0.001) in Fe-NTA treated group as compared to con-

M.U. Rehman et al. / Toxicol

.10. Measurement of SOD activity

The SOD activity was measured by the method of Marklund and Marklund1974).

.11. Measurement of quinone reductase (QR) activity

The QR activity was determined by the method of Benson et al. (1980).

.12. Glutathione reductase activity

GR activity was determined by the method of Carlberg and Mannervik (1975).

.13. Glutathione-S-transferase activity

Glutathione-S-transferase activity was measured by the method of Habig et al.1974).

.14. Blood urea nitrogen level

Estimation of blood urea nitrogen (BUN) was done by the method of Kanter1975).

.15. Serum creatinine level

Creatinine was estimated by the method of Hare (1950).

.16. Assay for lactate dehydrogenase activity

Lactate dehydrogenase (LDH) activity was estimated in serum by the method ofornberg (1955).

.17. �-Glutamyl transpeptidase activity

�-Glutamyl transpeptidase (GGT) activity was done by Orlowski and Meister1973).

.18. Assay for ODC activity

ODC activity was determined using 0.4 ml renal 105,0009g supernatant frac-ion per assay tube by measuring the release of CO2 from dl-[14C] ornithine by the

ethod of O’Brien et al. (1975).

.19. Estimation of protein

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

.20. Cytokine analysis

Serum levels of proinflammatory cytokines: IL-6, TNF-�, and PGE2 were ana-yzed. After the completion of tumor promotion for 24 weeks, the animals werenesthetized and blood withdrawn from retro-orbital sinus. Serum was separatedrom blood and the levels of above-mentioned cytokines were evaluated in it bylisa Plate Reader (Benchmark plus, BioRad) following the instructions of the man-facturer.

.21. Immunohistochemistry

The processed renal tissues were obtained and preserved in the 10%araformaldehyde overnight followed by dehydration in 30, 20 and 10% sucroseolution successively up to 3 days and was fixed after that in formaldehyde fixativentil immunochemical staining. 5–15 �m thick sections of paraffin embedded tis-ues were cut using grading type lieca microtome and boiled in 0.1 M citrate bufferpH 6.0) for 5 min for antigen retrieval process and then incubated in 0.3% H2O2

n methanol followed by incubation in blocking buffer containing 0.1 M PBS, 0.04%riton X-100 and 10% NGS (normal goat serum). Sections were incubated in anti-odies anti rat COX-2 antibody raised in rabbit (1:200 diluted in tris buffered saline),nti-NF-�B (1:200, Thermo Fisher Scientific, USA), anti-PCNA (1:150, Thermo Fishercientific, USA), anti-iNOS (1:100, Thermo Fisher Scientific, USA) for overnight 4 ◦C.fter rinsing in buffer, sections were processed using a three layer peroxidase stain-

ng kit from Thermo scientific system. The peroxides complex was visualized with,3-diaminobenzidine (DAB Plus substrate, Thermo Fisher Scientific, USA). Lastlyhe slides were counterstained with hematoxylin for 5 s. Slides were then cleanedn BDH, gradually dehydrated with ethanol and cover slipped in mounting mediumnd photographed under Olympus microscope (BX51).

trol group. Chrysin significantly attenuated the level of ODC in Fe-NTA + lower doseof chrysin treated group (###P < 0.05) and Fe-NTA + higher dose of chrysin treatedgroup (###P < 0.001) as compared to only Fe-NTA treated group.

2.22. Statistical analysis

The data from individual groups are presented as the mean ± standard errorof the mean (SEM). Differences between groups were analysed by using one wayanal-ysis of variance (ANOVA) followed by Tukey–Kramer multiple comparisonstest and minimum criterion for statistical significance was set at p < 0.05 for allcomparisons.

3. Results

3.1. Effect of chrysin on Fe-NTA-induced oxidative stress, relateddamage, and cellular proliferation

Result of chrysin pretreatment on Fe-NTA-induced modula-tion of GSH and activities of antioxidant and phase II enzymesis shown in Tables 1 and 2. Single intraperitoneal administra-tion of Fe-NTA significantly depleted renal GSH content (P < 0.001),and inhibited activities of GR, CAT, GPX and SOD as compared tothe corn oil treated controls. Pretreatment of rats with chrysinresulted in a significant recovery in GSH levels and activitiesof almost all the investigated enzymes (Table 1). There was aconcomitant and significant decrease in the activity of phase-IImetabolizing enzymes viz. GST and QR (P < 0.001) in the Fe-NTAtreated group. But this decrease in their activity was attenuatedsubstantially by the prophylactic treatment of both the dose ofchrysin (P < 0.001) (Table 2). Fe-NTA-induced increase in iron-ascorbate mediated lipid peroxidation of renal tissue is shown inTable 2. Treatment of Fe-NTA significantly enhanced the vulner-ability of renal microsomal membrane for LPO. However, chrysin(20 and 40 mg/kg BW) pretreatment significantly (P < 0.001) sup-pressed this enhancement (Table 3). As shown in Table 3, Fe-NTAadministration resulted in a significant increase in serum bloodurea nitrogen (BUN) (P < 0.001) and creatinine levels (P < 0.001)as compared to control animals. Pretreatment with chrysin(20 and 40 mg/kg) 1 h before Fe-NTA administration markedlyimproved renal dysfunction (indicated by significant reductionin serum creatinine and BUN). Similar pattern of results wasobserved in case of other serum toxicity parameters, viz., LDH and�-GGT.

Apart from inducing oxidative stress, Fe-NTA is also knownto cause tumor promotion by inducing cellular proliferation. Fe-NTA exposure significantly (P < 0.001) increased renal ODC whichis a hallmark of tumor promotion and is greatly induced during

tumorigenesis. Intraperitoneal application of Fe-NTA significantlyelevated ODC activity. However, chrysin pretreatment in both thedoses (20 and 40 mg/kg BW) down regulated Fe-NTA-induced ODCactivity (Fig. 2).

150 M.U. Rehman et al. / Toxicology Letters 216 (2013) 146– 158

Table 1Results of pretreatment of chrysin on antioxidant enzymes like GSH, GR, SOD, GPX and catalase on Fe- NTA induced renal redox imbalance.

Treatment regimenper group

GSH (nmolGSH/g tissue)

GR (nmol NADPHoxidized/min/mg protein)

GPX (nmol NADPHoxidized/min/mg protein)

Catalase (nmol H2O2

consumed/min/mg protein)SOD (u units/mgmg protein)

Group I (control) 0.63 ± 0.02 291.7 ± 16.0 258.2 ± 13.9 308.5 ± 51.9 171.4 ± 13.4Group II (Fe-NTA only) 0.40 ± 0.02*** 187.1 ± 15.2*** 120.8 ± 14.0*** 86.80 ± 10.0*** 119.2 ± 14.7***Group III (Fe-NTA + Chy D1) 0.51 ± 0.02# 221.4 ± 15.1# 188.2.0 ± 8.7# 217.9 ± 18.8### 150.6 ± 14.5###

Group IV (Fe-NTA + Chy D2) 0.56 ± 0.03## 284.4 ± 14.9## 211.2 ± 11.9### 247.1 ± 28.1### 157.0 ± 13.4###

Group V (only Chy D2) 0.65 ± 0.01 292.0 ± 15.1 276.2 ± 16.0 324.4 ± 65.3 171.4 ± 14.2

Results represent mean ± SE of six animals per group. Results obtained are significantly different from Control group (***P < 0.001). Results obtained are significantly differentfrom Fe-NTA treated group (#P < 0.05), (##P < 0.01) and (###P < 0.001). Chy, Chrysin; D1, 20 mg/kg/b wt; D2, 40 mg/kg/b wt.

Table 2Results of pretreatment of chrysin on parameters like GST, QR and MDA formation in Fe- NTA induced renal toxicity.

Treatment regimen per group GST (nmol CDNB conjugateformed/min/mg protein)

QR (nmol NADPHoxidized/min/mg protein)

MDA (nmoles of MDAformed/g tissue)

Group I (control) 269.2 ± 22.4 232.6 ± 16.5 2.27 ± 0.04Group II (Fe-NTA only) 531.7 ± 46.8*** 134.8 ± 9.06*** 5.28 ± 0.28***Group III (Fe-NTA + Chy D1) 347.0 ± 24.6# 201.5 ± 17.4# 3.13 ± 0.13###

Group IV (Fe-NTA + Chy D2) 314.1 ± 41.6## 218.6 ± 19.2## 3.93 ± 0.19###

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Group V (only Chy D2) 251.6 ± 36.5

esults represent mean ± SE of six animals per group. Results obtained are significanrom Fe-NTA treated group (#P < 0.05), (##P < 0.01) and (###P < 0.001). Chy, Chrysin;

Further, quantification of PCNA, well known cell proliferationarkers by immunohistochemistry has been used to characterize

he proliferation of cells in many fields, such as in tumor studies. Theemi-quantitative expression of PCNA protein in all the groups ofong-term renal tumorigenesis study is given in Fig. 3. According togure the number of PCNA positive cells increased substantially inroup II (DEN + Fe-NTA) indicating the proliferative potential of Fe-TA. Higher dose of chrysin markedly suppressed the proliferationf tubular epithelium cells as revealed in figure.

.2. Inhibitory effect of chrysin on DEN initiated Fe-NTAromoted renal carcinogenesis

Renal cancer in rats was initiated with single intraperitonealnjection of DEN (200 mg/kg BW). Twice weekly treatment of

mg/kg BW of Fe-NTA for 16 weeks was used as promotion agent.

able 3esults of pretreatment of chrysin on toxicity markers like BUN, creatinine, LDH and GGT

Treatment regimen per group BUN (mg/100 ml) IU/L Creatinine (mg/100 m

Group I (control) 20.04 ± 1.0 1.58 ± 0.07

Group II (Fe-NTA only) 52.07 ± 3.2*** 3.85 ± 0.11***

Group III (Fe-NTA + Chy D1) 38.17 ± 2.9## 3.20 ± 0.19#

Group IV (Fe-NTA + Chy D2) 28.26 ± 2.8### 1.96 ± 0.13###

Group (only Chy D2) 18.99 ± 1.1 1.51 ± 0.08

esults represent mean ± SE of six animals per group. Results obtained are significantly dirom Fe-NTA treated group (#P < 0.05), (##P < 0.01) and (###P < 0.001). Chy, Chrysin; D1, 2

able 4ummary of tumor data of the effects of chrysin treatment on DEN initiated and Fe- NTA-

Treatment regimenper group

Number ofanimals treated

Number of animals stuhistopathologically

Group I 20 19

Group II 20 11

Group III 20 14

Group IV 20 15

Group V 20 16Group VI 20 18

roup I (control): normal saline; group II (toxicant): DEN + Fe-NTA; group III: DEN + Fe-NEN only; group VI: Fe-NTA only. Chy, chrysin; Fe-NTA, ferric nitrilotriacetate. Toxicant gdministration of chrysin in groups III and IV. Whereas, group V (DEN only) and group VI

236.6 ± 6.60 2.15 ± 0.15

ferent from Control group (***P < 0.001). Results obtained are significantly different0 mg/kg/b wt; D2, 40 mg/kg/b wt.

Table 4 represents the data generated as a result of bioassay con-ducted to establish chemopreventive effect of chrysin in case ofchemically induced renal tumors. Corn oil alone treated groups didnot show any tumors, DEN only group showed only 5.88% tumors.Animals treated with Fe-NTA and initiated with DEN increased therenal cell tumors (RCTs) incidence by 81.8% and the animals inthe group treated with Fe-NTA alone led to the development ofRCTs in 20% of the animals studied. Chrysin significantly loweredthe percentage of tumor bearing animals (tumor incidence) at thetermination of experiment. The tumor incidence was decreasedin the group of animals pretreated with chrysin at lower dose of20 mg/kg BW to 50% whereas in the group treated with higher

dose of chrysin 40 mg/kg BW, the tumor incidence was decreased to26.6%.

The representative pictures of histopathological examinationin the renal tissue are shown in Fig. 4. The tissue sections from

on Fe-NTA induced enhancement.

l) IU/L LDH (nmol NADHoxidized/min/mg protein)

�-GGT (nmoles p nitroanilineformed/min/mg protein)

225.4 ± 25.2 269.2 ± 22.4453.9 ± 44.49*** 531.7 ± 46.8***298.4 ± 24.28# 347.0 ± 24.6#

301.5 ± 31.34# 314.1 ± 41.6###

228.5 ± 27.30 271.6 ± 36.5

fferent from control group (***P < 0.001). Results obtained are significantly different0 mg/kg/b wt; D2, 40 mg/kg/b wt.

induced renal cell tumors.

died Number of animals withrenal cell tumors

Incidence of renalcell tumors (%)

0 09 81.817 50.004 26.660 03 22.22

TA + Chy (20 mg/kg b wt); group IV: DEN + Fe-NTA + Chy (40 mg/kg b wt); group V:roup showed highest percentage of tumor incidences which was abrogated by the

(Fe-NTA only) did not develop significant no of tumors.

M.U. Rehman et al. / Toxicology Letters 216 (2013) 146– 158 151

Fig. 3. Representative photomicrographs of PCNA determined by immunohistochemistry. (A) No expression of PCNA was observed in case of control rats. (B) DEN + Fe-NTA administration increased the number of PCNA positive cells in cortical and tubular region of renal sections of animals represented by red arrows in the figure. (C)DEN + Fe-NTA + chrysin (20 mg/kg BW) treated animals showed slightly lesser number of PCNA positive cells as compared to group B as is evident from the figure. (D)DEN + Fe-NTA + chrysin (40 mg/kg BW) treated animals showed lesser number of PCNA positive cells as compared to group B. (E) Only chrysin treatment did not showany change in PCNA reactivity as compared to control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of thea

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rticle.)

idney of rats treated with DEN and Fe-NTA, either with orithout pre-treatment of chrysin, were examined for theegree of infiltration of leukocytes, tumor cells and hyper-hromatism. Sections from kidneys of control rats demonstratedntact tubular architecture with normal convoluted tubulesnd glomeruli within the cortex. The tissue sections from DENnitiated and Fe-NTA promoted group showed enormous focalollection of leukocytic infiltratory cells and adenocarcinomasith hyperchromatism and enlargement of nuclei in the tubular

pithelium. Lower dose of chrysin + DEN + Fe-NTA group showedild inflammatory invasion with lesser tubular congestion and

lomerular damage while as chrysin at higher dose was quiteffective in restoration of almost normal histo-architecture

of renal sections which was comparable with untreatedcontrol.

3.3. Effect of chrysin on DEN induced and Fe-NTA-promotedelevation in levels of proinflammatory cytokines in serum

Proinflammatory cytokines like IL-6, TNF-�, and PGE2 whosesecretion is known to be enhanced by Fe-NTA, play an importantrole in tumorigenesis (Kaur et al., 2009). Significant levels of TNF-�,

IL-6 and PGE2 could be detected in serum of rats exposed to tumorpromotion with Fe-NTA for 16 weeks (Table 5). Chrysin pretreat-ment at both the doses markedly restores the increased level of allthe three proinflammatory cytokines studied to normal.

152 M.U. Rehman et al. / Toxicology Letters 216 (2013) 146– 158

Fig. 4. Effect of chrysin treatment on renal histological alterations caused by DEN and Fe-NTA application. (A) Kidneys showed normal architecture with no signs ofinfiltration and tubular or glomerular damage. (B) DEN + Fe-NTA treated kidney showing areas of massive inflammatory cell invasion, hyperchromatism, glomerular andt mild i( e kidnc

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ubular congestion. (C) DEN + Fe-NTA + chrysin (20 mg/kg BW) treated rats showed

40 mg/kg BW) treated animals showed almost normal renal histology. (E) Shows thhange as compared with control.

.4. Effect of chrysin on Fe-NTA induced nuclear localization of

F-�B p65in kidney of Wistar rats

Genes necessary for the induction of COX-2, iNOS andther inflammatory cytokines are transcribed by redox sensitive

able 5ffect of chrysin on elevation in serum level of cytokines (TNF-�, PGE2 and IL-6). DEN ihrysin treatment.

Treatment regimen per group TNF-� (TNF-� pg/ml)

Group I (control) 333.4 ± 41.9

Group II (Fe-NTA only) 815.4 ± 27.4***

Group III (Fe-NTA + Chy D1) 474.3 ± 54.4#

Group IV (Fe-NTA + Chy D2) 28.26 ± 2.8###

Group (only Chy D2) 327.6 ± 44.1

esults represent mean ± SE of six animals per group. Results obtained are significantly dirom Fe-NTA treated group (#P < 0.05), (##P < 0.01) and (###P < 0.001). Chy, chrysin; D1, 20

nflammation and cell invasion as compared to group B. (D) DEN + Fe-NTA + chrysineys of the animals treated with the higher dose of chrysin only with no significant

transcription factor NF-�B. NF-�B has been shown to have role in

organ toxicities such as liver, kidney and pancreas. To evaluate theeffect of chrysin on Fe-NTA induced NF-�B activation in renal tis-sue, we investigated the levels of nuclear translocation of NF-�Bby immunohistochemistry. Exposure to Fe-NTA led to a marked

nitiated rats were promoted twice weekly application Fe-NTA for 16 weeks with

PG E2 (PGE2 pg/ml) IL-6 (IL-6 pg/ml)

49.20 ± 4.44 827.8 ± 39.6155.6 ± 10.3*** 2295 ± 73.2***102.6 ± 9.66# 1966 ± 99.0#

86.8 ± 15.8### 1495 ± 88.8###

46.6 ± 4.23 829.2 ± 40.8

fferent from control group (***P < 0.001). Results obtained are significantly different mg/kg/b wt; D2, 40 mg/kg/b wt.

M.U. Rehman et al. / Toxicology Letters 216 (2013) 146– 158 153

Fig. 5. Representative photomicrographs of NF-�B determined by immunohistochemistry. (A) There is almost no expression of NF-�B in the renal sections of controlgroup. (B) DEN + Fe-NTA administration increased strongly NF-�B expression in renal sections. (C) There was partial inhibition of NF-�B expression as evidenced by weaki ). (Dh struc

e�e

3

eNu(ka

mmunostaining in the rat kidneys treated with lower dose of chrysin (20 mg/kg BWigher dose of chrysin (40 mg/kg BW) this was evident from the figure, as the tubular

levation in NF-�Bp65 indicating Fe-NTA to cause activation of NF-B (Fig. 5). However, chrysin pretreatment I hr prior to Fe-NTAxposure caused a marked attenuation in levels of NF-�Bp65.

.5. Effect of chrysin on COX-2 and iNOS expression

The effect of chrysin was investigated on iNOS and COX-2xpression in Fe-NTA-administered rats as well as in DEN + Fe-TA-induced renal tumors. IHC revealed Fe-NTA to considerably

p-regulated the expression of iNOS and COX-2 in rat kidneyFigs. 6 and 7). Both, iNOS and COX-2 were hardly detected inidney of control group. Pretreatment with chrysin markedlyttenuated Fe-NTA-induced expression of iNOS and COX-2

) In contrast, there was almost complete suppression of NF-�B in rats treated withtures within the inner cortical regions do not show any substantial immunostaining.

(Figs. 6 and 7). In accordance to these results, immune histo-chemical analysis of samples showed intense staining of iNOS andCOX-2 in DEN + Fe-NTA-induced renal tumors. In kidney sectionsfrom rats that received chrysin application plus DEN + Fe-NTAtreatment, a marked reduction in staining of both, COX-2 and iNOSwas observed. In control kidney tissue only mild staining of COX-2and iNOS was observed.

4. Discussion

The incidence of RCC is increasing annually owing to lack ofearly warning signs and resistance to various kinds of therapies(Koul et al., 2010). Limited effectiveness of modern medicinal

154 M.U. Rehman et al. / Toxicology Letters 216 (2013) 146– 158

Fig. 6. Representative photomicrographs of i-NOS determined by immunohistochemistry. (A) There is almost no expression of i-NOS in the renal sections of control group. (B)DEN + Fe-NTA administration increased strongly i-NOS expression in renal sections. (C) There was partial inhibition of i-NOS expression as evidenced by weak immunostainingi st, thc

tdtpacodrhtrSic

n the rat kidneys treated with lower dose of chrysin (20 mg/kg BW). (D) In contrahrysin (40 mg/kg B).

herapies for treatment of renal cancer has shifted our focus onevelopment of alternative strategies. The most valuable optiono prevent or delay carcinogenesis is offered by the use of safelant based compounds, hence there is rise in exploration of safend effective phytochemicals for the management of renal can-er. Chemoprevention has the potential to be a major componentf cancer control. Several herbs, vegetables fruits and plants withiversified pharmacological properties have been shown to beich sources of micro chemicals with the potential to preventuman cancer. Reports from our laboratory and others indicateshat various food ingredients may also play an essential role in

enal cancer prevention (Rehman and Sultana, 2011; Jahangir andultana, 2007). Chrysin is a naturally occurring flavonoid presentn number of fruits and vegetables and has displayed numeroushemopreventive properties against chemically induced toxicities

ere was almost complete suppression of i-NOS in rats treated with higher dose of

(Khan et al., 2012a,b; Tahir and Sultana, 2011). Chrysin has alsoshown to be effective against various tumors like hepatocellu-lar carcinomas (Khan et al., 2011a), thyroid tumors (Phan et al.,2011) in animal models and in different cancer cell lines likelung cancer cell line (Brechbuhl et al., 2012), prostate cancer cellline (Samarghandian et al., 2011) and human colorectal cell line(Galijatovic et al., 2001).

Recent reports have already indicated association betweeninflammation and oxidative stress (Bickers and Athar, 2006). Wehave also reported that oxidative stress and inflammation playsan important role in pathogenesis of nephrotoxicity caused by

Fe-NTA (Rehman and Sultana, 2011; Jahangir and Sultana, 2007;Kaur et al., 2009). Further, Fe-NTA exposure elevates the levelsof redox active iron, which is known to induce the formationof ROS that can readily attack the cellular molecules leading to

M.U. Rehman et al. / Toxicology Letters 216 (2013) 146– 158 155

Fig. 7. Photomicrographs of immunohistochemical detection of COX-2 in renal tissue. (A) COX-2 staining of control kidneys. There was no expression of COX-2. (B) COX-2s , chrysi was as no sig

ltt–qfaspaas(

taining in DEN-Fe-NTA induced renal tumors was very intense. (C) DEN-initiatednhibition of COX-2. (D) DEN + Fe-NTA + chrysin (40 mg/kg) treatment showed thereection of the groups treated with higher dose of chrysin only (40 mg/kg BW) with

ipid peroxidation, oxidative damage (Fig. 8) and increase of serumoxicity markers. These events lead to modulation in the reducedhiol pool and enzymatic antioxidants Gpx, GR catalase, SOD, etc.

which have an important role in protection of renal tissue byuenching free radicals. In agreement with previous studies weound Fe-NTA to downregulated both concentration of antioxidantsnd activities of all antioxidant enzymes (Iqbal et al., 2003). Chrysinupplementation augmented the levels of reduced thiol pool andhase-II metabolizing enzymes like GST and QR in renal tissue as

lso previously reported from our lab (Khan et al., 2012a,b; Tahirnd Sultana, 2011). Lipid peroxidation is an outcome of oxidativetress and incredible elevation in the level of malondialdehydeMDA), a lipid peroxidation product, is observed after treatment

in pretreated (20 mg/kg BW) and Fe-NTA promoted kidney sections show partiallmost complete suppression of COX-2 with higher dose of chrysin. (E) Shows renalnificant change as compared to control.

with Fe-NTA (Iqbal et al., 2003). In the present study, Fe-NTAtreated rats showed a remarkable increase in the level of MDAand chrysin significantly attenuated its level in renal tissue. Thus,chrysin exhibited the protective efficacy against Fe-NTA-inducedlipid peroxidation in renal tissue. Studies from our laboratorieshave also shown that chrysin decreases the level of MDA in kid-ney, liver and colon after treatment with ethyl alcohol and cisplatin,respectively (Tahir and Sultana, 2011; Khan et al., 2012a,b). More-over, lipid peroxidation and the associated membrane damage are

implicated in the pathophysiology of a number of diseases includ-ing renal disorders. Renal dysfunction is followed by the elevatedlevels of serum enzymes indicating cellular leakage and loss of func-tional integrity of renal membrane. It correlates with our results,

156 M.U. Rehman et al. / Toxicology Letters 216 (2013) 146– 158

Fig. 8. Targets of action of chrysin against Fe-NTA induced debilities, in kidney of Wistar rats. Fe-NTA causes toxicity via reactive oxygen species (ROS) generation andinduction of inflammatory response. Chrysin pre-treatment shows reduction in xanthine oxidase (XO) activity (1) leading to reduction in ROS formation. Further enhancementin antioxidants like superoxide dismutase (SOD) (2), catalase (CAT) (3) activities and reduced glutathione (GSH) content and related redox cycle enzymes (glutathionereductase (GR), glutathione peroxidise (GPx), and glucose-6-phosphate dehydrogenase (G6PD)). (4) Potentiate its role against oxidants-induced damages. Moreover, chrysinpretreatment also increased phase-II metabolizing enzyme (glutathione S transferase (GST) and quinone reductase (QR)) activities (5a and 5b). These effects are evident byr romisI ). GSSO

wtLia

hnaeiiett

iminaSiftlipp

eduction in lipid peroxidation (LPO) of cellular membranes (6). Chrysin shows the pL-6, NF�B activation, expression of COX-2, i-NOS and level of PGE-2 (7, 8, 9 and 10

2•− , superoxide radical; R, xenobiotic; R-SH, thiol conjugated xenobiotics.

hich showed increased activities of LDH, BUN and creatinine inhe serum of Fe-NTA-treated rats. Rats treated with chrysin hadDH, BUN and creatinine significantly lower than those receiv-ng only Fe-NTA. These results suggested that chrysin may protectgainst Fe-NTA induced renal toxicity.

Renal sections of rats treated for 16 weeks with Fe-NTA, onistopathological examination revealed more widespread tubularecrosis, massive inflammatory response, loss of cellular differenti-tion, dilated tubules and numerous renal cell tumors. Histologicalvaluation showed that chrysin administration suppressed thenflammatory responses in the renal tissue by decreasing thentense infiltration. It also reduced the severity of tubular degen-ration and loss of cellular differentiation induced by Fe-NTAreatment. Chrysin treatment was thus instrumental in ameliora-ion of renal toxicity and tumorigenesis.

Our study mainly focuses on unraveling the mechanisms of anti-nflammatory and anti-promoting activity of chrysin in Fe-NTA

odel of renal cancer. Present data suggest that chrysin markedlynhibits Fe-NTA induced tumor promotion in DEN initiated rat kid-ey. Fe-NTA application for 16 weeks produced renal tumors ingreement with previous reports (Iqbal et al., 2007; Jahangir andultana, 2006). Chrysin drastically ameliorated PCNA-positive cellsn the renal tissue. Hall et al. (1990) reported PCNA, a 36 kDa co-actor of DNA polymerase-�, is one of the downstream effectors ofhe activation of MAPK/ERK1/2 signaling and is very useful molecu-

ar biomarker of hyperproliferation. Ornithine decarboxylase (ODC)s another important and widely used marker to study tumorromotion. ODC is a rate limiting enzyme in the biosynthesis ofolyamines, spermine, spermidine and putrecines. Auvenin, (1992)

ing role against Fe-NTA-induced injuries in kidneys by reducing the levels of TNF-�,G, oxidized glutathione; G-6-P, glucose-6-phosphate; 6-PG, 6-phosphogluconate;

reported transformed cell lines to have elevated levels of ODCactivity and elevation of ODC activity is closely related to tumorpromotion and carcinogenesis (Pegg et al., 1995). Chrysin treatmentsignificantly attenuated ODC activity in rat kidneys exposed to Fe-NTA suggesting it to have a potent anti-hyperproliferative activity.A decrease observed in PCNA positive cells and activity of ODCfollowing chrysin pretreatment indicated suppression induced bychrysin in cellular proliferation and hence tumor promotion.

The activation of NF�B linked regulatory pathways generallyunderlies inflammatory processes, and an increase in the nucleartranslocation of NF�B has been demonstrated in all cancersincluding RCC (Morais et al., 2011). The transcription factorNF�B helps to regulate the expression of several genes activatedduring inflammation and is implicated in several other aspectsof oncogenic process such as cellular proliferation, preclusion ofapoptosis, conferring the tumor cells a metastatic and angiogenicability, etc. (Brown et al., 2008). NF�B is induced by various cellstress associated stimuli including growth factors, vasoactiveagents, cytokines, and oxidative stress (Karin and Greten, 2005).NF�B in turn controls the regulation of genes encoding proteinsinvolved in immune and inflammatory responses (i.e., cytokines,chemokines, growth factors, immune receptors, cellular ligands,and adhesion molecules). Thus, inhibition of NF�B is nowadaysdocumented as a valuable approach to control the carcinogenicdevelopment. Since NF�B is a redox sensitive transcription factor

it is activated by oxidants generated by Fe-NTA. In agreementwith previous published reports Fe-NTA exposure was foundto activate NF�B in renal tissues (Kaur et al., 2009). Chrysinwas found to potently inhibit NF�B activation. It noticeably

ogy Le

dBTc(

rt(seitw(Naietcp(risfsscwFpat

ctiiradLIbc

5

iosrmectttspri

M.U. Rehman et al. / Toxicol

ecreased the levels of phosphorylated form of inhibitor of kappa (I�B) and suspended the nuclear translocation of NF-�Bp65.hese observations are consistent to reports in literature wherehrysin has been shown to significantly inhibit NF�B activationLi et al., 2010).

The proteins iNOS and COX-2 are also under the transcriptionalegulation of NF�B (Fig. 8) (Surh et al., 2001) and are reportedo be associated with renal inflammation and tumor promotionKaur et al., 2009), thus Fe-NTA associated increase in their expres-ion may also be mediated through NF�B activation. Further, Surht al. (2001) reported overexpression of COX-2 to mediate bothnflammation and tumor promotion. Our data showed chrysino effectively inhibit Fe-NTA mediated overexpression of COX-2hich was in agreement with report of Woo et al. (2005). Khan et al.

2011b) reported chrysin to downregulate COX-2 expression inDEN (N-nitrosodiethylamine) induced hepatocellular carcinomand have suggest COX-2 inhibition by chrysin to play a pivotal rolen its antitumor activity. Thus, inhibition of Fe-NTA induced COX-2xpression by chrysin may also plausibly be implicated in protec-ion against Fe-NTA induced renal carcinogenesis. Pathological andhronic inflammatory reactions can be triggered by nitric oxideroduction in renal tissue by the inducible nitric oxide synthaseiNOS) enzyme. Moreover, selective iNOS inhibitor can suppressenal toxicity, suggesting that iNOS signaling could serve as anmportant target for prevention and treatment of renal cancer,uggesting that iNOS signaling could serve as an important targetor prevention and treatment of renal diseases. Recent evidencesuggest that iNOS plays a crucial role in development and progres-ion of renal cancer (Fukumura et al., 2006). In our study Fe-NTAaused significant induction of iNOS in renal tissues in accordanceith previously published paper of Wu and Qiu (2001) where in

e-NTA was demonstrated to induce NO generation in culturedroximal tubule cells. Additionally, we have shown that chrysin isble to decrease the elevated expression of iNOS in the renal cancerissue.

Fe-NTA exposure induced the expression of proinflammatoryytokines IL-6, TNF-�, and PGE2 which are under direct transcrip-ional regulation of NF�B. These cytokines have an important role innflammation, vascular permeability as well as proliferation. Thus,nhibition in their secretion by chrysin seems to play an importantole in its protective effect against renal tumorigenesis Our resultsre in agreement with the findings of Ha et al. (2010) who haveemonstrated chrysin to inhibit the secretion of these cytokines inPS stimulated proinflammatory response in microglia cells. Thus,nhibition of proinflammatory cytokines by chrysin treatment maye now accepted as yet another valuable strategy to control thearcinogenic development.

. Conclusion

A number of studies have established that oxidative stress andnflammation contributes to initiation, promotion and progressionf renal carcinogenesis. In this study we presented data demon-trating that chrysin inhibits DEN initiated and Fe-NTA promotedenal carcinogenesis in animal model. Chrysin treatment resulted inarked decline in renal hyperplasia, renal ODC activity and protein

xpression of PCNA, iNOS, COX-2 and secretion of proinflammatoryytokines, all of which are traditional markers of inflammation andumor promotion. In addition, our data also revealed that chrysinreatment maintained antioxidant armory and suppresses activa-ion of redox active transcription factor NF-�B (Fig. 8). These results

upported by published literature reports suggest chrysin to be aotential candidate for prevention of renal carcinogenesis, since itestrains several biomarkers of inflammation and tumor promotionn rat model of renal carcinogenesis.

tters 216 (2013) 146– 158 157

Conflict of interest statement

Authors declare that there is no conflict of interest.

Acknowledgement

The author (Sarwat Sultana) is thankful to University GrantsCommission (New Delhi, India), for providing Research Fellowshipfor Science and Meritorious Student (RFSMS) to first author to carryout this work.

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