tumor promotion and oxidative stress in ferric nitrilotriacetate–mediated renal carcinogenesis:...

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Toxicology Mechanisms and Methods, 17:421–430, 2007 Copyright c Informa Healthcare USA, Inc. ISSN: 1537-6516 print; 1537-6524 online DOI: 10.1080/15376510601131297 Tumor Promotion and Oxidative Stress in Ferric Nitrilotriacetate–Mediated Renal Carcinogenesis: Protection by Adhatoda vasica Tamanna Jahangir and Sarwat Sultana Section of Chemoprevention and Nutrition Toxicology, Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India ABSTRACT In the present study, we report the chemopreventive effects of Adhatoda vasica against ferric nitrilotriacetate (Fe-NTA)–induced renal oxidative stress, hyperproliferative response, and two-stage renal carcinogenesis. Fe-NTA (9 mg Fe/kg body weight, intraperitoneally) enhances renal lipid peroxidation, xanthine oxidase (XO), and hydrogen peroxide (H 2 O 2 ) generation with concomitant reduction in renal glutathione content (GSH), antioxidant enzymes, and phase II metabolizing enzymes. It induces blood urea nitrogen, serum creatinine, ornithine decarboxylase (ODC) activity, and [ 3 H] thymidine incorporation into renal DNA. It also enhances DEN (N-diethylnitrosamine)- initiated renal carcinogenesis by increasing the percentage incidences of kidney tumors. Pretreatment of rats orally with A. vasica (50 and 100 mg/kg body weight) resulted in a significant decrease in lipid peroxidation, H 2 O 2 generation, xanthine oxidase (XO), blood urea nitrogen, serum creatinine, renal ODC activity, DNA synthesis (p < 0.001), and incidence of tumors. Renal GSH (p < 0.01), glutathione-metabolizing enzymes (p < 0.001), and antioxidant enzymes were also recovered significantly (p < 0.001). Thus, our results show that A. vasica may meet the criteria demanded from a chemopreventive agent and in a rodent system it can reduce hyperproliferative response toxicity and carcinogenic activity of Fe-NTA. KEYWORDS Kidney; Hyperproliferative Response; Fe-NTA; Thymidine Incorporation INTRODUCTION For decades, plants have been a main source for drug development. A lot of work is currently being done to exploit the anticancer potential of various plants, particularly of those used in traditional medicine (Muriel 2006). Adhatoda vasica has been used extensively in the ayurvedic system of medicine for over 2000 years primarily for respiratory disorders. A. vasica has been extensively used in pulmonary diseases; it assists in uterine involution, menorrhagia, postpartum hemorrhage, uterine stimulant activity, dyspepsia, Received 7 October 2006; accepted 21 November 2006. Address correspondence to Dr. Sarwat Sultana, Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India E-mail: [email protected] 421 Toxicology Mechanisms and Methods Downloaded from informahealthcare.com by Nyu Medical Center on 09/30/14 For personal use only.

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Toxicology Mechanisms and Methods, 17:421–430, 2007Copyright ©c Informa Healthcare USA, Inc.ISSN: 1537-6516 print; 1537-6524 onlineDOI: 10.1080/15376510601131297

Tumor Promotion and Oxidative Stressin Ferric Nitrilotriacetate–Mediated

Renal Carcinogenesis: Protectionby Adhatoda vasica

Tamanna Jahangir andSarwat SultanaSection of Chemopreventionand Nutrition Toxicology,Department of MedicalElementology and Toxicology,Jamia Hamdard (HamdardUniversity), Hamdard Nagar,New Delhi 110062, India

ABSTRACT In the present study, we report the chemopreventive effects ofAdhatoda vasica against ferric nitrilotriacetate (Fe-NTA)–induced renal oxidativestress, hyperproliferative response, and two-stage renal carcinogenesis. Fe-NTA(9 mg Fe/kg body weight, intraperitoneally) enhances renal lipid peroxidation,xanthine oxidase (XO), and hydrogen peroxide (H2O2) generation withconcomitant reduction in renal glutathione content (GSH), antioxidantenzymes, and phase II metabolizing enzymes. It induces blood urea nitrogen,serum creatinine, ornithine decarboxylase (ODC) activity, and [3H] thymidineincorporation into renal DNA. It also enhances DEN (N-diethylnitrosamine)-initiated renal carcinogenesis by increasing the percentage incidences of kidneytumors. Pretreatment of rats orally with A. vasica (50 and 100 mg/kg bodyweight) resulted in a significant decrease in lipid peroxidation, H2O2 generation,xanthine oxidase (XO), blood urea nitrogen, serum creatinine, renal ODCactivity, DNA synthesis (p < 0.001), and incidence of tumors. Renal GSH(p < 0.01), glutathione-metabolizing enzymes (p < 0.001), and antioxidantenzymes were also recovered significantly (p < 0.001). Thus, our results showthat A. vasica may meet the criteria demanded from a chemopreventive agentand in a rodent system it can reduce hyperproliferative response toxicity andcarcinogenic activity of Fe-NTA.

KEYWORDS Kidney; Hyperproliferative Response; Fe-NTA; Thymidine Incorporation

INTRODUCTIONFor decades, plants have been a main source for drug development. A lot

of work is currently being done to exploit the anticancer potential of variousplants, particularly of those used in traditional medicine (Muriel 2006).

Adhatoda vasica has been used extensively in the ayurvedic system ofmedicine for over 2000 years primarily for respiratory disorders. A. vasica hasbeen extensively used in pulmonary diseases; it assists in uterine involution,menorrhagia, postpartum hemorrhage, uterine stimulant activity, dyspepsia,

Received 7 October 2006;accepted 21 November 2006.Address correspondence to Dr. SarwatSultana, Department of MedicalElementology and Toxicology, Facultyof Science, Jamia Hamdard (HamdardUniversity), Hamdard Nagar, NewDelhi 110062, India E-mail:[email protected]

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local bleeding due to peptic ulcer and/or piles(hemorrhoids), acute and chronic bronchitis, allergicasthma, emphysema, tuberculosis; and it relieves coughand breathlessness. This plant has been reportedto contain quinazoline alkaloids such as N-oxidesof vasicine, vasicinone, deoxyvasicine, oxyvasicinine,maiontone, b-sitosterol-D-glucoside, kaempferol, gly-cosides of kaempferol, and quercetin (Chakraborty2001). Its roots contain vasicinolone, vasicol, peganine,hydroxyoxychalcone, glucosyloxychalcone, bromohex-ine, and ambroxol, semisynthetic derivatives of vasicine(Claeson 2000; Grange and Snell 1996; Dorsch 1991).

Poly (ADP-ribose) polymerase (PARP) is an abundantnuclear enzyme in eukaryotic cells that has beenimplicated to become activated in response to DNAdamage. A. vasica has a growth inhibitory effect onMycobacterium tuberculosis, thereby proving useful inthe therapy of tuberculosis. The quinazoline alkaloidfamily has been found to be a potent inhibitor of poly(ADP-ribose) polymerase-1/2 inhibitors, an importantpharmacological target in the treatment of cancer,and the inhibitory potency of these derivatives wasfound to be largely dependent on the unique linkerof the quinazolinone ring (Iwashita 2005). Iron isan abundant metal in the human body and is anessential nutritional element for all life forms. Itsoverload may lead to various diseases (De Freitas andMeneghini 2001). Iron nitrilotriacetate (Fe-NTA) isa potent nephrotoxic agent; Fe-NTA treatment alsoenhances renal ornithine decarboxylase (ODC) activityand increases [3H]-thymidine incorporation into renalDNA. Intraperitoneal Fe-NTA treatment at a dose levelof 9 mg Fe/kg body weight/10 mL enhances renalmicrosomal lipid peroxidation and hydrogen peroxidegeneration, which are accompanied by a decreasein the activities of renal antioxidant enzymes (e.g.,catalase, glutathione peroxidase, glutathione reductase,and glutathione S-transferase) and a depletion in thelevel of renal glutathione (Iqbal and Athar 1998).The repeated intraperitoneal administration of Fe-NTAproduces acute and subacute renal proximal tubularnecrosis (Hamazaki et al. 1985; Li et al. 1987), whichis subsequently associated with the high incidence ofrenal adenocarcinoma in the rodent model (Li et al.1987; Okada 1996; Okada and Midorikawa 1982). Itis assumed that Fe-NTA–mediated generation of freeradicals plays an important role in renal tumorigenesis.Intraperitoneally injected Fe-NTA is absorbed into theportal vein via mesothelium and comes into circulation

through the liver (Umemura et al. 1990). Fe-NTA is lowmolecular weight, and hence filtered easily through theglomeruli into the lumen of the renal proximal tubule.There, Fe3+-NTA is reduced to Fe2+-NTA by theglutathione degradation products cysteine or cysteinyl-glycine (Tsao and Curthoys 1980). In the brush bordersurface of the renal proximal convoluted tubules, γ -glutamyl transpeptidase hydrolyzes glutathione to cys-teinylglycine, which is rapidly degraded to cysteine andglycine by dipeptidase. Cysteine and cysteinylglycineare the proposed thio reductants that reduce Fe3+-NTAto Fe2+-NTA. The auto-oxidation of Fe2+-NTA gen-erates superoxide radicals (O2−), which subsequentlypotentiate the iron-catalyzed Haber–Weiss reaction toproduce hydroxyl radical (OH), leading to inductionof lipid peroxidation and oxidative DNA damage(Umemura et al. 1990).

Our results strongly support the protective natureof A. vasica against Fe-NTA–induced nephrotoxicity,hyperproliferative response, and renal carcinogenesisin Wistar rats.

MATERIALS AND METHODSChemicals

Reduced glutathione (GSH), oxidized glutathione(GSSG), glutathione reductase, bovine serum albu-min (BSA), 1,2,dithio-bis-nitrobenzoic acid (DTNB),1,chloro-2, 4,dinitrobenzene (CDNB), reduced nicoti-namide adenine dinucleotide phosphate (NADPH),flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6,dichlorophenolindophenol,and thiobarbituric acid (TBA) were obtained fromSigma Chemical Co. (St. Louis, MO). Diacetyl-monoxime, urea, picric acid, sodium tungstate, sodiumhydroxide, trichloroacetic acid (TCA), and perchloricacid (PCA) were purchased from CDH (India). [14C]ornithine (sp. act. 56 m Ci mmol) and [3H] thymidine(sp. act. 82 Ci mmol) were purchased from AmershamCorporation (United Kingdom). All other chemicalsand reagents were of the highest purity commerciallyavailable.

Animals and Plant MaterialEight-week-old adult male Wistar rats (150 to 200 g)

were obtained from the Central Animal House Facilityof Hamdard University, New Delhi, and were housed ina ventilated room at 25 ± 2◦C under a 12-h light/dark

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cycle. The animals were acclimatized for 1 week beforethe study and had free access to standard laboratoryfeed (Hindustan Lever Ltd., Bombay, India) and water.The study was approved by the Committee for thePurpose of Control and Supervision of ExperimentalAnimals (CPCSEA). Registration number and date ofregistration: 173/CPCSEA, Jan. 28, 2000. CPCSEAguidelines were followed for animal handling andtreatment.

Total extract A. vasica in semisolid form was pur-chased from Saiba Industries (Mumbai, India). Theextract is claimed to possess all the active ingredients ofthe plant.

Treatment RegimenThe treatment regimen for A. vasica was based on

the preliminary studies carried out in our laboratory.Fe-NTA dose was selected according to Athar and Iqbal(1998). To study the biochemical, serological changes,25 male Wistar rats were randomly divided into fivegroups and had free access to standard laboratoryfeed (Hindustan Lever Ltd., Bombay, India) and water.Group I served as saline (0.85% NaCl)-treated control.Group II served as treated control and was administeredFe-NTA (9 mg Fe/kg body weight IP) only. GroupsIII and IV were pretreated with A. vasica at doses of50 and 100 mg/kg body weight, respectively, orallyfor 7 consecutive days followed by administration ofFe-NTA (9 mg Fe/kg body weight) on the seventh day.Group V was given a higher dose (D2) of A. vasica for 7consecutive days. All animals were sacrificed 12 h afteradministration. Tissue was processed for the estimationof renal ODC activity, GSH content, microsomallipid peroxidation, and other biochemical estimations.For the [3H] thymidine incorporation study, 25 maleWistar rats were randomly divided into five groups,and the same treatment regimen was followed exceptthat all the animals were given intraperitoneal [3H]thymidine (30 µci/0.2 mL saline/animal IP) 2 h beforesacrifice. Time of sacrifice was after 18 h of Fe-NTA(9 mg Fe/kg body weight) administration; kidneysections were quickly excised, rinsed with ice-coldsaline, freed of extraneous material, and processed forthe quantification of [3H] thymidine incorporationinto the renal DNA. Before they were sacrificed, bloodwas collected in test tubes from retro-orbital sinus forthe estimation of creatinine and blood urea nitrogen.

To study the effect of pretreatment with A. vasicaextract on DEN (N-diethylnitrosamine)-initiated andFe-NTA–promoted renal carcinogenesis, the animalswere divided into five groups of 20 rats per group.Group I received only saline injection intraperitoneally(0.85% NaCl). Animals of groups II, III, and IV wereinitiated with a single IP injection of DEN at a doselevel of 200 mg/kg body weight in saline. Ten days afterinitiation, the animals in groups II, III, IV, and V werepromoted with intraperitoneal injection of Fe-NTA ata dose level of 9 mg Fe/kg body weight, twice a weekfor 16 weeks. Groups III and IV received oral treatmentwith A. vasica extract by gavage once daily at a doseof 50 and 100 mg/kg body weight, respectively, half anhour prior to the treatment with Fe-NTA for a periodof 16 weeks, twice a week. At the end of 24 weeks, theanimals were sacrificed by cervical dislocation and theirkidneys were quickly removed and preserved in 10%neutral buffered formalin for histopathological studies.Hematoxylin and eosin preparations of processedsections were prepared for microscopic examination.

Postmitochondrial Supernatantand Microsome Preparation

Kidneys were removed quickly, cleaned free ofextraneous material, and immediately perfused withice-cold saline (0.85% sodium chloride). The kidneyswere homogenized in chilled phosphate buffer (0.1 M,pH 7.4) containing KCI (1.17%) using a Potter Elvehjenhomogenizer. The homogenate was filtered throughmuslin cloth and was centrifuged at 3000 rpm for 10min at 4◦C by Eltek Refrigerated Centrifuge (model RC4100 D) to separate the nuclear debris. The aliquot soobtained was centrifuged at 12000 rpm for 20 min at4◦C to obtain postmitochondrial supernatant (PMS),which was used as a source of enzymes. A portion ofthe PMS was centrifuged for 60 min by ultracentrifuge(Beckman L7–55) at 34000 rpm at 4◦C. The pelletwas washed with phosphate buffer (0.1 M, pH 7.4)containing KCI (1.17%).

Biochemical Determinations

Estimation of Reduced Glutathione

Reduced glutathione was determined by the methodof Jollow et al. (1974). A 1.0-mL sample of PMS wasprecipitated with 1.0 mL of sulfosalicylic acid (4%). Thesamples were kept at 4◦C for 1 h and then centrifugedat 1200 × g for 20 min at 4◦C. The assay mixture

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contained 0.1 mL filtered aliquot, 2.7 mL phosphatebuffer (0.1 M, pH 7.4), and 0.2 mL DTNB (100mM) in a total volume of 3.0 mL. The yellow colorthat developed was read immediately at 412 nm on aspectrophotometer (Milton Roy Model-21 D).

Estimation of Lipid Peroxidation

The assay for microsomal lipid peroxidation wasdone following the method of Wright et al. (1981).The reaction mixture in a total volume of 1.0 mLcontained 0.58 mL phosphate buffer (0.1 M, pH 7.4),0.2 mL microsomes, 0.2 mL ascorbic acid (100 mM),and 0.02 mL ferric chloride (100 mM). The reactionmixture was incubated at 37◦C in a shaking waterbath for 1 h. The reaction was stopped by additionof 1.0 mL 10% TCA. Following addition of 1.0 mL0.67% TBA, all the tubes were placed in a boilingwater bath for 20 min and then shifted to a crushedice bath before centrifuging at 2500 × g for 10 min.The amount of malondialdehyde formed in each ofthe samples was assessed by measuring optical densityof the supernatant at 535 nm using spectrophotometer(Milton Roy 21 D) against a reagent blank. The resultswere expressed as nmol MDA formed/h/g tissue at37◦C using molar extinction coefficient of 1.56 ×105/M/cm.

Estimation of Blood Urea Nitrogen

Estimation of blood urea nitrogen was done bythe diacetyl monoxime method of Kanter (1975).Protein-free filtrate was prepared. To 0.5 mL of proteinfree filtrate were added 3.5 mL of distilled water, 0.8mL diacetylmonoxime (2%), and 3.2 mL sulphuricacid–phosphoric acid reagent (reagent was prepared bymixing 150 mL 85% phosphoric acid with 140 mLwater and 50 mL of concentrated sulphuric acid). Thereaction mixture was placed in a boiling water bath for30 min and then cooled. The absorbance was recordedat 480 nm.

Estimation of Creatinine

Creatinine was estimated by the alkaline picratemethod of Hare (1950). Protein-free filtrate was pre-pared. To 1.0 mL serum were added 1.0 mL sodiumtungstate (5%), 1.0 mL sulfuric acid (0.6 N), and 1.0 mLdistilled water. After mixing thoroughly, the mixturewas centrifuged at 800 × g for 5 min. The supernatantwas added to a mixture containing 1.0 mL picricacid (1.05%) and 1.0 mL sodium hydroxide (0.75 N).

The absorbance at 520 nm was recorded exactly after20 min.

Assay for Hydrogen Peroxide

Hydrogen peroxide (H2O2) was assayed by H2O2-mediated horseradish peroxidase-dependent oxidationof phenol red by the method of Pick and Keisari (1981).Suspended was 2.0 mL of microsomes in 1.0 mL ofsolution containing phenol red (0.28 nm), horseradishperoxidase (8.5 units), dextrose (5.5 nm), and phosphatebuffer (0.05 M, pH 7.0), which was incubated at 37◦Cfor 60 min. The reaction was stopped by the additionof 0.01 mL of NaOH (10 N) and then centrifuged at800 × g for 5 min. The absorbance of the supernatantwas recorded at 610 nm against a reagent blank. Thequantity of H2O2 produced was expressed as nmolH2O2/g tissue/h based on the standard curve of H2O2-oxidized phenol red.

Assay for Glutathione-S-transferase Activity

Glutathione-S-transferase activity was assayed by themethod of Habig et al. (1974). The reaction mixtureconsisted of 1.475 mL phosphate buffer (0.1 M, pH6.5), 0.2 mL reduced glutathione (1 mM), 0.025 mLCDNB (1 mM), and 0.3 mL PMS (10% w/v) in a totalvolume of 2.0 mL. The changes in the absorbancewere recorded at 340 nm and enzyme activity wascalculated as nmol CDNB conjugate formed/min/mgprotein using a molar extinction coefficient of 9.6 ×103/M/cm.

Assay for Glutathione Peroxidase Activity

Glutathione peroxidase activity was assayed by themethod of Mohandas et al. (1984). The reactionmixture consisted of 1.49 mL phosphate buffer (0.1M, pH 7.4), 0.1 mL EDTA (1 mM), 0.1 mL sodiumazide (1 mM), 0.05 mL glutathione reductase (1 IU mL−1), 0.05 mL GSH (1 mM), 0.1 mL NADPH (0.2 mM),0.01 mL H2O2 (0.25 mM), and 0.1 mL 10% PMS in atotal volume of 2 mL. The disappearance of NADPHat 340 nm was recorded at 25◦C. Enzyme activity wascalculated as nmol NADPH oxidized/min/mg proteinusing molar extinction coefficient of 6.22 × 103/M/cm.

Assay for Glutathione Reductase Activity

Glutathione reductase activity was determined bythe method of Carlberg and Mannervik (1975). Thereaction mixture consisted of 1.65 mL phosphate buffer(0.1 M, pH 7.6), 0.1 mL EDTA (0.5 mM), 0.05 mL

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oxidized glutathione (1 mM), 0.1 mL NADPH (0.1mM), and 0.1 mL 10% PMS in a total volume of2 mL. Enzyme activity was quantitated at 25◦C bymeasuring disappearance of NADPH at 340 nm andwas calculated as nmol NADPH oxidized/min/mgprotein using molar extinction coefficient of 6.22 ×103/M/cm.

Assay for Catalase Activity

Catalase activity was assayed by the method ofClaiborne (1985). The reaction mixture consisted of1.95 mL phosphate buffer (0.1 M, pH 7.4), 1.0 mLhydrogen peroxide (0.019 M), and 0.05 mL 10% PMSin a final volume of 3 mL. Changes in absorbance wererecorded at 240 nm. Catalase activity was calculated asnmol H2O2 consumed/min/mg protein.

Assay for Glucose-6-phosphateDehydrogenase Activity

The activity of glucose-6-phosphate dehydrogenasewas determined by the method of Zaheer et al. (1965).The reaction mixture consisted of 0.3 mL Tris–HClbuffer (0.05 M, pH 7.6), 0.1 mL NADP (0.1 mM), 0.1mL glucose-6-phosphate (0.8 mM), 0.1 mL MgCl2 (8mM), 0.3 mL PMS (10%), and 2.1 mL distilled water ina total volume of 3 mL. The changes in absorbance wererecorded at 340 nm and enzyme activity was calculatedas nmol NADP reduced/min/mg protein using a molarextinction coefficient of 6.22 × 103/M/cm.

Assay for Xanthine Oxidase Activity

The activity of xanthine oxidase was assayed by themethod of Stripe and Della Corte (1969). The reactionmixture consisted of 0.2 mL PMS that was incubatedfor 5 min at 37◦C with 0.8 mL phosphate buffer (0.1M, pH 7.4). The reaction was started by adding 0.1mL xanthine (9 mM) and kept at 37◦C for 20 min.The reaction was terminated by the addition of 0.5mL ice-cold perchloric acid (10% v/v). After 10 min,2.4 mL of distilled water was added and centrifuged at4000 rpm for 10 min and µg uric acid formed/min/mgprotein was recorded at 290 nm.

Assay for Quinone Reductase Activity

The activity of quinone reductase was determined bythe 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 mL BSA, 0.1 mL FAD, 0.02 mL NADPH(0.1 mM), and 50 µL (10%) PMS. The reduction

of dichlorophenolindophenol (DCPIP) was recordedcalorimetrically at 600 nm and enzyme activity wascalculated as nmol of DCPIP reduced/min/mg proteinusing molar extinction coefficient of 2.1 × 104/M/cm.

Assay for Ornithine Decarboxylase Activity

ODC activity was determined using 0.4 mL renal105000 × g supernatant fraction per assay tube bymeasuring release of 14CO2 from dl-[14C] ornithineby the method of O’Brien et al. (1975). The kidneyswere homogenized in Tris–HCl buffer (pH 7.5, 50mM) containing EDTA (0.1 mM), pyridoxal phosphate(0.1 mM), PMSF (1.0 mM), 2-mercaptoethanol (1.0mM), dithiothreitol (0.1 mM), and Tween-80 (0.1%)at 4◦C. In brief, the reaction mixture contained 400µL enzyme and 0.095 mL cofactor mixture containingpyridoxal phosphate (0.32 mM), EDTA (0.4 mM),dithiothreitol (4.0 mM), ornithine (0.4 mM), Brij 35(0.02%), and [14C] ornithine (0.05 µCi) in a totalvolume of 0.495 mL. After adding buffer and cofactormixture to blank and other test tubes, the tubes wereclosed immediately with a rubber stopper containing0.2 mL ethanolamine and methoxyethanol mixture inthe central well and kept in a water bath at 37◦C.After 1 h of incubation, the enzyme activity wasarrested by injecting 1.0 mL citric acid solution (2.0M) along the sides of glass tubes and the incubationwas continued for 1 h to ensure complete absorptionof 14CO2. Finally, the central well was transferred toa vial containing 2 mL ethanol, and a 10-mL toluene-based scintillation fluid was added. Radioactivity wascounted in a liquid scintillation counter (LKB Wallace-1410). ODC activity was expressed as pmol 14CO2

released/h/mg protein.

Assay for Renal DNA Synthesis

The isolation of renal DNA and assessment ofincorporation of [3H] thymidine into DNA werecarried out by the method of Smart et al. (1986).The rat kidneys were quickly removed and cleanedfree of extraneous material and homogenate (10%w/v) was prepared in ice-cold water. The precipitatethus obtained was washed with cold TCA (5%) andincubated with cold PCA (10%) at 4◦C overnight.After this, the incubation mixture was centrifugedand the precipitate was washed with cold PCA (5%).The precipitate was dissolved in warm PCA (10%),incubated in a boiling water bath for 30 min, andfiltered through Whatman 50 paper. The filtrate was

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used for [3H] counting in a liquid scintillation counter(LKB Wallace-1410) after adding scintillation fluid.The amount of DNA in filtrate was estimated by thediphenylamine method of Giles and Myers (1965). Theamount of [3H] thymidine incorporated was expressedas dpm/µg DNA.

Estimation of Protein

The protein concentration in all samples was deter-mined by the method of Lowry et al. (1951).

Statistical AnalysisDifferences between groups were analyzed using

Dunnet’s t-test followed by analysis of variance(ANOVA).

RESULTSTable 1 shows the effects of A. vasica pretreatment on

animals on Fe-NTA–mediated renal glutathione con-tent and its metabolizing enzymes. Fe-NTA at a dose 9mgFe/kg body weight resulted in significant depletionof renal glutathione and GR, GPx, and glutathioneS-transferases (GST) activities (p < 0.001) compared tosaline-treated control. A. vasica pretreatment (50 and100 mg/kg body weight) restored the levels of theseenzymes as compared with Fe-NTA–only group.

The protective effects of A. vasica extract on Fe-NTA–mediated depletion in renal antioxidant armoryis shown in Table 2. Treatment with only Fe-NTAcaused reduction in the activities of CAT, QR, andG6PD (p < 0.001) compared to saline-treated control.On pretreatment with A. vasica extract, both the doses(50 and 100 mg/kg body weight) showed significantrestoration of the above-mentioned enzymes. There

was concomitant and significant enhancement in H2O2

and XO levels and renal microsomal membrane iron-ascorbate lipid peroxidation in the toxicant-only groupas compared to saline-treated control. As is evidentfrom Table 3, marked reduction was noted in H2O2

and XO levels and renal microsomal membrane iron-ascorbate lipid peroxidation in modulator-pretreatedgroups as compared to the Fe-NTA–only group. Table4 shows the protective efficacy of A. vasica pretreatmenton the Fe-NTA–induced increase in levels of the serumtoxicity markers blood urea nitrogen and creatinine. Atboth doses A. vasica significantly (p < 0.001) attenuatesrelease of both the serum toxicity markers.

Table 5 gives the summary of the percentage ofrenal cell tumors (RCTs) in different treatment groups.The saline-treated control group showed no tumordevelopment; however, Fe-NTA promotion in DEN-initiated rats enhanced the RCT incidence by 83.3%,whereas only an 11.76% tumor incidence was notedin only promoted group (uninitiated group). A. vasicapretreatment at both doses showed marked reductionin tumor incidences. The incidence of tumor at thelower dose (i.e., 50 mg/kg body weight) was 33.3% andat the higher dose (i.e., 100 mg/kg body weight) was17.76%.

The effect of pretreatment of A. vasica on Fe-NTA–mediated induction in renal ODC activity andrenal DNA synthesis is shown in Figure 1. Theprophylactic treatment of rats with A. vasica showeda marked inhibition of ODC activity (p < 0.001) in adose-dependent manner and suppression of the rate of3H thymidine incorporation (p < 0.001) into the renalDNA of the treated control.

Figure 2 shows the histopathological findings ofrenal tumor tissue initiated with DEN and promoted

TABLE 1 Effect of pretreatment of the extract of A. vasica on Fe-NTA–induced stress on glutathione content andglutathione-dependent enzymes in rat kidney

Treatment groups

Reducedglutathione

(nmol GSH/g tissue)

Glutathione-S-transferase (nmol CDNB

conjugateformed/min/mg protein)

Glutathione peroxidase(nmol NADPH

oxidized/min/mgprotein)

Glutathione reductase(nmol NADPH

reduced/min/mgprotein)

Saline-treated control 0.62 ± 0.008 128.7 ± 1.49 73.4 ± 0.46 76.5 ± 0.9Fe-NTA alone 0.45 ± 0.003∗ 58.86 ± 0.58∗ 55.3 ± 0.57∗ 59.5 ± 1.8∗

Fe-NTA + AV (D1) 0.56 ± 0.003# 75.72 ± 0.15# 64.7 ± 0.18# 63.2 ± 0.39#

Fe-NTA + AV (D2) 0.60 ± 0.002# 93.12 ± 0.47# 70.7 ± 0.47# 67.6 ± 1.68#

Only AV (D2) 0.63 ± 0.002 138.4 ± 13.5 74.6 ± 1.6 77.2 ± 0.009

Results represent mean ± SE of five animals per group.Results significantly different from saline-treated group (∗p < 0.001).Results significantly different from Fe-NTA–treated group (#p < 0.001).

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TABLE 2 Effect of pretreatment of the extract of A. vasica on the antioxidant enzymes catalase, quinone reductase, glutathioneperoxidase, and glucose-6-phosphate dehydrogenase on Fe-NTA administration in kidney of Wistar rats

Treatment regimenCatalase (nmol H2O2

consumed/min/mg protein)

Quinone reductase(nmol dichloroindophenolreduced/min/mg protein)

Glucose-6-phosphatedehydrogenase (nM NADPreduced/min/mg protein)

Saline-treated control 254.3 ± 3.30 198.0 ± 2.40 8.70 ± 0.27Fe-NTA alone 204.3 ± 3.40∗ 115.0 ± 1.19∗ 4.2 ± 0.27∗

Fe-NTA + AV (D1) 216.4 ± 4.6# 153.4 ± 5.5# 7.8 ± 0.05#

Fe-NTA + AV (D2) 240.1 ± 0.5# 174.9 ± 1.9# 8.4 ± 0.08#

Only AV (D2) 264.1 ± 1.1 199.06 ± 0.59 9.1 ± 0.23

Results represent mean ± SE of five animals per group.Results significantly different from saline-treated group (∗p < 0.001).Results significantly different from Fe-NTA–treated group (#p < 0.001).

with Fe-NTA and its inhibition by A. vasica extract.In the saline-only treated group, no histopathologicalalterations were seen; the distal tubule, proximal tubule,and glomerulus are clearly seen in Figure 2a. However,pathological changes induced by Fe-NTA administra-tion are necrosis of proximal tubule, formation ofdenucleated tissue (ghost tissue), glomerular swelling,flattened epithelium, and swelling and congestion ofblood vessels. These are quite evident in Figure 2b. Therenal sections of DEN-initiated and Fe-NTA–treatedrats showed prominent adenocarcinoma, loss of cellulardifferentiation, loss of tubular brush border, interstitialedema, tubular dilation, and necrosis of the epithelium.Pretreatment with A. vasica extract at both dosesresulted in reversal of Fe-NTA–induced pathology asevident in Figure 2c,d. However, no specific histopatho-logical alterations were seen in the promoter-only groupseen in Figure 2e.

DISCUSSIONChemoprevention includes multiple intervention

methods, either pharmacological or dietary agents toinhibit, arrest, or reverse carcinogenesis at various

stages. Development of dietary agents as potent cancerchemopreventive agents is highly acceptable due totheir safety, low toxicity, and general acceptance asdietary supplements (Kapadia 2002).

The therapeutic and medicinal uses of A. vasicaextract have been reported widely (Kumar 2005). Thisplant has been shown to possess antioxidant andanti-inflammatory responses in various previous studies(Grange and Snell 1996).

Overall, the detoxifying enzyme system plays animportant role in determining the final fate of car-cinogens/mutagens, and their consequences in cancer,ODC activity, and [3H] thymidine incorporation arewidely used as biochemical markers to evaluate thetumor-promoting potential of an agent (Perchellet andPerchellet 1989). As observed in the present study, A.vasica extract dose dependently inhibited the inductionof ODC activity and [3H] thymidine incorporation,suggesting its antihyperproliferative potential.

In addition, both doses of A. vasica extract usedin the present study showed anticarcinogenic efficacyagainst DEN-initiated and Fe-NTA–promoted renalcarcinogenesis by inhibiting tumor formation via re-duction in number of RCTs. By scavenging free radicals

TABLE 3 Effect of pretreatment of the extract of A. vasica on Fe-NTA–induced stress on malanodialdehyde (MDA) formation andxanthine oxidase (XO) level in rat kidney

Treatment groupsXO (µg uric acid

formed/min/mg protein) MDA (nmol MDA/h/g tissue) H2O2 (nmol H2O2/g tissue)

Saline-treated control 0.145 ± 0.001 3.37 ± 0.021 64.4 ± 0.09Fe-NTA alone 0.204 ± 0.005∗ 9.73 ± 0.146## 128.6 ± 1.56##

Fe-NTA +AV (D1) 0.180 ± 0.001# 8.65 ± 0.21# 76.8 ± 0.84#

Fe-NTA + AV (D2) 0.161 ± 0.001# 7.17 ± 0.10# 69.35 ± 1.67#

Only AV (D2) 0.136 ± 0.003 3.21 ± 0.14 61.35 ± 1.05

Results represent mean ± S.E of five animals per group.Results significantly different from saline-treated group (∗p < 0.001).Results significantly different from Fe-NTA–treated group (#p < 0.001).

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TABLE 4 Effect of pretreatment of the extract of A. vasica onFe-NTA–mediated elevation in serum toxicity markers

Treatment regimen

Blood ureanitrogen

(mg/100 mL) IU/L

Creatinine(mg/100 mL)

IU/L

Saline-treated control 28.78 ± 0.50 1.30 ± 0.0005Fe-NTA alone 103.45 ± 2.88∗ 2.96 ± 0.0009∗

Fe-NTA +AV (D1) 68.69 ± 0.29# 1.94 ± 0.001#

Fe-NTA + AV (D2) 48.87 ± 0.04# 1.67 ± 0.004#

Only AV(D1) 21.68 ± 0.50 1.37 ± 0.005

Results represent mean ± SE of five animals per group.Results significantly different from saline-treated group (∗p < 0.001).Results significantly different from Fe-NTA–treated group (#p < 0.001).

and inhibiting ODC induction and DNA synthesis, A.vasica extract may intercept the growth-promoting andmitogenic functions of polyamines and arachidonicacid metabolites.

Two main groups of biotransformation enzymesmetabolize carcinogens, namely phase I enzymes,which convert hydrophobic compounds to more water-soluble moieties, and phase II enzymes (e.g., GST),which primarily catalyze conjugation reactions. Theconjugation of electrophilic phase I intermediates withglutathione, for instance, frequently results in detoxi-fication (Pool-Zobel et al. 2005). Quinone reductase isa major enzyme of xenobiotic metabolism that carriesout necessary two-electron reductions and thus protectscells against mutagenic and carcinogenic effects result-ing from free radicals (Munday and Munday 2004).It has been shown that most of the chemopreven-tive agents result in the induction of glutathione-S-transferase and quinone reductase activity and in thedegradation of electrophilic metabolites. Induction ofquinone reductase activity has been reported to havecorrelation with the prevention of cancer (De Flora andRamel 1988).

The present study investigates the cancer chemo-preventive potential of hydroalcoholic extract of A.

FIGURE 1 A: Effect of pretreatment of A. vasica on ODCactivity. Results represent mean ± SE of five animals per group.Results significantly different from saline-treated group (∗p <

0.001). Results significantly different from Fe-NTA–treated group(#p < 0.001). B: Effect of pretreatment of A. vasica on 3Hthymidine incorporation in renal DNA. Results represent mean ±SE of five animals per group. Results significantly different fromsaline-treated group (∗p < 0.001). Results significantly differentfrom Fe-NTA–treated group (#p < 0.001). DPM, disintegration perminute.

vasica by evaluating the altered levels/activities ofphase II enzymes (GST and quinone reductase). A.vasica extract ameliorated Fe-NTA–induced inhibitionof the activities of the Antioxidant enzymes viz.,glutathione peroxidase, glutathione reductase, catalase,and glucose-6-phosphate dehydrogenase and the phaseII metabolizing enzymes glutathione-S-transferase and

TABLE 5 Summary of tumor data on the effect of AV extract on DEN-initiated and Fe-NTA–promoted renal tumors

Treatment groupsNo. of animals

treatedNo. of animals studied

histopathologicallyNo. of animals with

renal cell tumorsIncidences with

tumor percentage

Saline (alone) 20 19 0 —Fe-NTA (alone) 20 17 2 11.76%DEN + Fe-NTA 20 12 10 83.3%AV (D1) + DEN + Fe-NTA 20 15 5 33.3%Av (D2) + DEN + Fe-NTA 20 17 3 17.6%

Dose of DEN = 200 mg/kg body weight, dose of Fe-NTA = 9 mg Fe/kg body weight.Doses (D1 and D2) = 100 and 200 mg/kg body weight, respectively, of A. vasica extract.

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FIGURE 2 A: Normal saline treated. B: DEN + Fe-NTA treated.C: AV (D1) + DEN + Fe-NTA treated. D: AV (D2) + DEN + Fe-NTAtreated. E: Only Fe-NTA treated. All slides were stained withhematoxylin and eosin (H&E) (A, H&E 200×; B–E, H&E 150×).Dose of DEN = 200 mg/kg body weight, dose of Fe-NTA = 9mg/kg body weight, dose of AV (D1) = 50 mg/kg body weight,and dose of AV (D2) = 100 mg/kg body weight. G, glomerulus;LI, leukocytic infiltration; NT, necrotic tissue; PT, proximal tubule;DT, distal tubule; GT, ghost tissue; HC, hyperchromatism.

quinone reductase. A. vasica extract has established an-tioxidant properties that might have protected againstthe oxidant effects of Fe-NTA. The present studyshows stimulation of renal glutathione-S-transferaseand quinone reductase activity following plant extracttreatment. The chief mechanism for defending againstthe toxic and neoplastic effects of carcinogens is theamendment of cellular detoxification enzymes.

Superoxide anions are generated in oxidative stressand lead to the formation of highly reactive species; hy-droxyl radicals are responsible for cell damage or geneticalterations by attacking proteins and lipid membranes.Increases in lipid peroxidation and hydrogen peroxideare, therefore, the markers for assessment of cell toxicity(Zhao et al. 2000).

A. vasica pretreatment at both doses decreased theFe-NTA–mediated susceptibility of renal microsomalmembrane for iron-ascorbate–induced lipid peroxida-tion through decreased production of free radicals asshown by ameliorated malondialdehyde levels. A sharp

FIGURE 2 (Continued)

decrease was noted in the levels of blood urea nitrogenand serum creatinine, markers of renal toxicity thatsupport the efficacy of A. vasica in improving renalarmory.

However, the exact mechanism of the action ofA. vasica extract has not been fully elucidated. Itstherapeutic effect may be attributed to the presenceof active principles, quinazoline alkaloids such as N-oxides of vasicine, vasicinone, deoxyvasicine, oxyvasici-nine, maiontone, b-sitosterol-D-glucoside, kaempferol,etc. In conclusion, A. vasica acts as an inducer of variousantioxidant and phase II enzymes, a scavenger ofreactive oxygen species, an inhibitor of tumor promotermarkers, and a suppressor of tumor formation inthe two-stage renal carcinogenesis experimental model.Thus, our data strongly support A. vasica as an effective

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chemopreventive agent having the capability to hamperFe-NTA–induced tumor promotion, oxidative stress,and renal cancer in the rodent model.

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