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 DOI: 10.1191/0960327106ht616oa

2006 25: 235Hum Exp ToxicolTamanna Jahangir, Tajdar Husain Khan, Lakshmi Prasad and Sarwat Sultana

Farnesol prevents Fe-NTA-mediated renal oxidative stress and early tumour promotion markers in rats  

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Human & Experimental Toxicology (2006) 25: 235 -242www.hetournal.com

Farnesol prevents Fe-NTA-mediated renaloxidative stress and early tumourpromotion markers in ratsTamanna Jahangir, Tajdar Husain Khan, Lakshmi Prasad and Sarwat Sultana*

Section of Chemoprevention and Nutrition Toxicology, Department ofMedical Elementology and Toxicology,Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi 110062, India

Excess iron deposition in tissues leads to organ dysfunc-tion and impairment. In this study, the protective effectsof farnesol (FL), an isoprenoid, against Fe-NTA (9 mgiron/kg body weight i.p.)-induced oxidative damageand early tumour promotion markers are evaluated.The pretreatment of iron-intoxicated rats with 1% and2%/kg body weight oral dose of FL for 7 consecutive dayssignificantly reversed the iron-induced increase inH-102 content (P <0.001), malondialdehyde formation,xanthine oxidase activity (P <0.001), ornithine decarbox-ylase activity (P <0.001) and 3[H]thymidine incor-poration in renal DNA (P <0.005) with simultaneoussignificant depletion in serum toxicity markers bloodurea nitrogen (BUN) and creatinine (P <0.001). Signifi-

cant dose-dependent restoration was recorded in renalglutathione content, its dependent enzymes and otherphase II metabolizing enzymes viz., catalase, glutathione-S-transferase and quinone reductase (P <0.001) withprophylactic treatment of FL. Present results supportthat FL markedly lowers the oxidative damageand appearance of tumour markers, which precludesits development as a chemopreventive tool. Human &Experimental Toxicology (2006) 25, 235-242

Key words: chemoprevention; farnesol; Fe-NTA; oxidativedamage; tumour promotion markers

Introduction

Fe-NTA, a potent renal carcinogen, induces iron-dependent lipid peroxidation, leading to acutetubular necrosis. 1,2 Nitrilotriacetic acid (NTA) hasbeen used as a polyphosphate substitute in deter-gents in various countries; it forms water-solublechelate complexes with metal cations at neutral pH.Intraperitoneal intoxication of Fe-NTA has beenshown to induce renal proximal tubular necrosisand renal adenocarcinoma. Hydroxyl radicals aregenerated by reaction of Fe-NTAwith H202, possiblycausing oxidative damage.3-5Evidence has shown that natural herbs and shrubs

play a major role in prevention of cancer.6'7 Iso-prenoids are plant compounds that have suppressedmany types of tumour growth in experimentalstudies.8 The antiproliferative effect of isoprenoidsis thought to be due to suppression of the mevalo-nate pathway, through which mutated Ras proteins

*Correspondence: Sarwat Sultana, Department of Medical Ele-mentology and Toxicology, Faculty of Science, Jamia Hamdard(Hamdard University), Hamdard Nagar, New Delhi 110062, IndiaE-mail: sarwat786@rediffmail.com

Received 11 April 2005; revised 14 December 2005; accepted14 December 2005

© 2006 Edward Arnold (Publishers) Ltd

transform healthy cells into cancer cells.9 Themevalonate pathway is the precursor for farnesyldiphosphate (FPP), required for the isoprenylationof various G proteins, out of which the mostimportant are p21, products of the ras gene and akey transducer of mitogenic signals. FL in the formof FPP is a product of this pathway; FL is convertedinto FPP in vivo.10 FPP is the substrate for a numberof critical enzymes, thus its regulation and levels areimportant. lt has been recently demonstrated thatperoxisomes are the major site of the synthesis ofFPP from mevalonate.'1 Isoprenoids have beenshown to modulate cell growth, induce cell cyclearrest, initiate apoptosis and suppress cellular sig-nalling activities.12'13 Farnesol (FL) is an isoprenoidobtained from the essential oils of ambrette seeds,citronella, present in many aromatic plants, and isalso produced in humans, working on variousnuclear receptors.14 Although FL has been a subjectof research for more than 30 years, its metabolism isnot well documented. An important representativeof linear sesquiterpenes, FL, as shown in (Scheme1), has 15 carbon atoms in a head-to-tail isoprenoidseries. FL and graniol are the alcohols with three(15 carbons) and two (10 carbons) isoprenoid

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Farnesol prevents early tumour promotion markers in ratsT Jahangir et al.

236

H3Ce m > > g~~~~~OH

CH3 CH3 CH3

Figure 1 Structure of farnesol.

units, respectively. Isoprenoids are a class ofphytochemicals, which have antitumour activity.15They are abundant in fruits and vegetables; foodsrich in isoprenoids have consistently been shown toplay a protective role in various cancers.16 FL hasshown inhibitory effects on different stages of manytypes of cancer in experimental studies.i5 It hasbeen shown to suppress pancreatic carcinoma in-cidences.17 Thus in the present study we haveevaluated antioxidant efficacy and inhibitory effectsof FL on the appearance of early tumour promotionmarkers in Fe-NTA-induced toxicity in an experi-mental rat model.

Materials and methods

ChemicalsEthylenediamine tetraacetic acid (EDTA), Tris, re-

duced glutathione (GSH), oxidized glutathione(GSSG), reduced nicotinamide adenine dinucleotidephosphate (NADPH), bovine serum albumin (BSA),1,2-dithio-bisnitrobenzoic acid (DTNB), 1-chloro-2,4-dinitro benzene (CDNB), phenylmethyl sulpho-nylfluoride (PMSF), nitroblue tetrazolium, Brij-35,pyridoxal-phosphate, 2-mercaptoethanol, dithio-threitol and Tween-80 were obtained from Sigmachemicals Co. (St Louis, MO). Diacetylmonoxime,urea, picric acid, sodium tungstate, sodium hydrox-ide, ferric nitrate, trichloroacetic acid (TCA) andperchloric acid (PCA) were purchased from CDH(Delhi, India). [14C]Ornithine (specific activity56 mCi/mmol) and [3H]thymidine (specific activity82 Ci/mmol) were purchased from Amersham Cor-poration (Buckinghamshire, UK). All other chemi-cals were of the highest purity and commerciallyavailable.

AnimalsFifty eight-week-old adult male Wistar rats (150-200 g) were obtained from the Central Animal HouseFacility of Hamdard University, New Delhi and were

housed in a ventilated room at 25 + 2°C under a 12-hlight/dark cycle. The animals were acclimatized forone week before the study and had free access tostandard laboratory feed (Hindustan Lever Ltd.,Bombay, India) and water. The study was approvedfrom the Committee for the purpose of control andsupervision of experimental animals (CPCSEA).

Registration number and date of registration: 173/CPCSEA, 28 January 2000. CPCSEA guidelines werefollowed for animal handling and treatment.

Preparation of Fe-NTA solutionFe-NTA solution was prepared fresh immediatelybefore its use by the method of Awai et al.'8 Toprepare Fe-NTA, ferric nitrate (0.16 mmol/kg bodyweight) solution was mixed with fourfold molarexcess of disodium salt of NTA (0.64 mmol/kg bodyweight) and pH was adjusted to 7.4 with sodiumbicarbonate solution.

Experimental designThe treatment regimen for FL was based on thepreliminary studies carried out in our laboratory. Fe-NTA dose was selected according to Athar andIqbal.19 To study the biochemical, serologicalchanges, 25 male Wistar rats were randomly dividedinto five groups and had free access to standardlaboratory feed (Hindustan Lever Ltd., Bombay,India) and water. Group I served as saline (0.85%NaCl) treated control. Group II served as positivecontrol and was administered Fe-NTA (9 mg Fe/kgbody weight i.p.) only. Groups III and IV werepretreated with FL at doses 1% and 2%/kg bodyweight orally for 7 consecutive days followed byintoxication of Fe-NTA (9 mg Fe/kg body weight) onthe 7th day. Group V was given a higher dose (D2) ofFL for 7 consecutive days. All animals were sacri-ficed 12 hours after intoxication. Serum was sepa-rated and stored at 40C for the estimation of bloodurea nitrogen (BUN) and creatinine. Tissue wasprocessed for the estimation of renal ornithinedecarboxylase (ODC) activity, glutathione (GSH)content, microsomal lipid peroxidation and otherbiochemical estimations. For [3H]thymidine incor-poration study, 25 male Wistar rats were randomlydivided into five groups; the same treatment regi-men was followed except all the animals were givenintraperitoneal [3Hlthymidine (30 1tci/0.2 mL saline/animal i.p.) 2 hours before sacrifice. Time of sacri-fice was 18 hours after of Fe-NTA (9 mg Fe/kg bodyweight) intoxication; kidney sections were quicklyexcised, rinsed with ice-cold saline, freed of extra-neous material and processed for the quantificationof [3H]thymidine incorporation into the renal DNA.

Postmitochondrial supernatant and microsomepreparationTissue processing and preparation of postmitochon-drial supernatant (PMS) were done as described byAthar and Iqbal.'9 Kidneys were removed quickly,cleaned free of extraneous material and immediatelyperfused with ice-cold saline (0.85% sodium

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chloride). The kidneys were homogenized in chilledphosphate buffer (0.1 M, pH 7.4) containing KCI(1.17%) using a Potter Elvehjem homogenizer. Thehomogenate was filtered through muslin cloth andwas centrifuged at 800 x g for 5 min at 40C by EltekRefrigerated Centrifuge (model RC 4100 D) to sepa-rate the nuclear debris. The aliquot thus obtainedwas centrifuged at 12 000 rpm for 20 min at 40C toobtain PMS, which was used as a source of enzymes.A portion of the PMS was centrifuged for 60 min byultracentrifuge (Beckman L7-55) at 34000 rpm at4°C. The pellet was washed with phosphate buffer(0.1 M, pH 7.4) containing KCI (1.17%). All thebiochemical estimations were completed within24 hours of animal sacrifice.

Biochemical estimations

Estimation of reduced glutathione GSH was

determined by the method of Jollow et al.20 A 1-mL sample of PMS was precipitated with 1.0 mL ofsulphosalicylic acid (4%). The samples were kept at4 °C for 1 hour and then centrifuged at 1200 x g for20 min at 4°C. The assay mixture contained 0.1 mLfiltered aliquot, 2.7 mL phosphate buffer (0.1 M, pH7.4) and 0.2 mL DTNB (100 mM) in a total volume of3.0 mL. The yellow colour developed was read at412 nm on a spectrophotometer.

Assay for glutathione-S-transferase activity Glu-tathione-S-transferase activity was assayed by themethod of Habig et al.2' The reaction mixtureconsisted of 1.475 mL phosphate buffer (0.1 M, pH6.5), 0.2 mL GSH (1 mM), 0.025 mL CDNB (1 mM)and 0.3 mL PMS (10% w/v) in a total volume of2.0 mL. The changes in the absorbance were re-

corded at 340 nm and enzyme activity was calcu-lated as nanomoles of CDNB conjugate formed per

minute per milligram of protein using a molarextinction coefficient of 9.6 x 103/M/cm.

Assay for glutathione peroxidase activity Gluta-thione peroxidase activity was assayed by themethod of Mohandas et al.22 The reaction mixtureconsisted of 1.49 mL phosphate buffer (0.1 M, pH7.4), 0.1 mL EDTA (1 mM), 0.1 mL sodium azide(1 mM), 0.05 mL glutathione reductase (1 IU/mL),0.05 mL GSH (1 mM), 0.1 mL NADPH (0.2 mM),0.01 mL H202 (0.25 mM) and 0.1 mL 10% PMS ina total volume of 2 mL. The disappearance ofNADPH at 340 nm was recorded at 25 °C. Enzymeactivity was calculated as nanomoles of NADPHoxidized per minute per milligram of protein using a

molar extinction coefficient of 6.22 x 103/M/cm.

Farnesol prevents early tumour promotion markers in ratsT Jahangir et al.

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Assay for glutathione reductase activity Glu-tathione reductase activity was determined by themethod of Carlberg and Mannervik.23 The reactionmixture consisted of 1.65 mL phosphate buffer(0.1 M, pH 7.6), 0.1 mL EDTA (0.5 mM), 0.05 mLGSSG (1 mM), 0.1 mL NADPH (0.1 mM) and 0.1 mL10% PMS in a total volume of 2 mL. Enzyme activitywas quantitated at 25 C by measuring disappear-ance of NADPH at 340 nm and was calculated asnanomoles of NADPH oxidized per minute permilligram of protein using a molar extinction coeffi-cient of 6.22 x 103/M/cm.

Assay for glucose-6-phosphate dehydrogenaseactivity The activity of glucose-6-phosphate dehy-drogenase was determined by the method of Zaheeret al.24 The reaction mixture consisted of 0.3 mLTris-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 mL PMS (10%) and2.1 mL distilled water in a total volume of 3 mL.The changes in absorbance were recorded at 340 nmand enzyme activity was calculated as nanomoles ofNADPH per minute per milligram of protein using amolar extinction coefficient of 6.22 x 103/M/cm.

Estimation of lipid peroxidation The assay formicrosomal lipid peroxidation was done followingthe method of Wright et al.25 The reaction mixturein a total volume of 1.0 mL contained 0.58 mLphosphate buffer (0.1 M, pH 7.4), 0.2 mL micro-somes, 0.2 mL ascorbic acid (100 mM) and 0.02 mLferric chloride (100 mM). The reaction mixture wasincubated at 37°C in a shaking water bath for 1 hour.The reaction was stopped by addition of 1.0 mL 10%TCA. Following addition of 1.0 mL 0.67% thiobar-bituric acid (TBA), all the tubes were placed in aboiling water bath for 20 min and then shifted to acrushed ice bath before centrifuging at 2500 x g for10 min. The amount of malondialdehyde (MDA)formed in each of the samples was assessed bymeasuring optical density of the supernatant at535 nm using a spectrophotometer (Milton Roy 21D) against a reagent blank. The results wereexpressed as nanomoles of MDA formed per hourper gram tissue at 37°C using a molar extinctioncoefficient of 1.56 x 105/M/cm.

Assayforhydrogen peroxide Hydrogen peroxide(H202) was assayed by H202-mediated horseradishperoxidase-dependent oxidation of phenol red bythe method of Pick and Keisari.26 Microsomes(2.0 mL) were suspended in 1.0 mL of solutioncontaining phenol red (0.28 nm), horse radish per-oxidase (8.5 U), dextrose (5.5 nm) and phosphate

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buffer (0.05 M, pH 7.0) and were incubated at 370Cfor 60 min. The reaction was stopped by the addi-tion of 0.01 mL of NaOH (10 N) and then centrifugedat 800 x g for 5 min. The absorbance of the super-natant was recorded at 610 nm against a reagentblank. The quantity of H202 produced was ex-pressed as nanomoles of H202 per hour per gramtissue based on the standard curve of H202-oxidizedphenol red.

Assay for xanthine oxidase activity The activityof xanthine oxidase (XO) was assayed by the methodof Stripe and Della Corte.27 The reaction mixtureconsisted of 0.2 mL PMS that was incubated for 5min at 37°C with 0.8 mL phosphate buffer (0.1 M,pH 7.4). The reaction was started by adding 0.1 mLxanthine (9 mM) and kept at 370C for 20 min. Thereaction was terminated by the addition of 0.5 mLice-cold PCA (10% v/v). After 10 min, 2.4 mL ofdistilled water was added and centrifuged at4000 rpm for 10 min and micrograms of uric acidformed per minute per milligram of protein was

recorded at 290 nm.

Assay for quinone reductase activity The activ-ity of quinone reductase was determined by themethod of Benson et al.28 The 3-mL reactionmixture consisted of 2.13 mL Tris-HCI buffer(25 mM, pH 7.4), 0.7 mL BSA, 0.1 mL flavin adeno-sine dinucleotide, 0.02 mL NADPH (0.1 mM), and50 giL (10%) PMS. The reduction of dichlorophenolindophenol (DCPIP) was recorded calorimetricallyat 600 nm and enzyme activity was calculated as

nanomoles of DCPIP reduced per minute per milli-gram of protein using a molar extinction coefficientof 2.1 x 104/M/cm.

Assayfor estimation of catalase activity Catalaseactivity was assayed by the method of Claiborne.29The reaction mixture consisted of 1.95 mL phos-phate buffer (0.1 M, pH 7.4), 1.0 mL hydrogenperoxide (0.019 M) and 0.05 mL 10% PMS in a finalvolume of 3 mL. Changes in absorbance were re-

corded at 240 nm. Catalase activity was calculatedas nanomoles of H202 consumed per minute per

milligram of protein.

Estimation of blood urea nitrogen Estimation ofBUN was done by the diacetyl monoxime method ofKanter.30 Protein-free filtrate was prepared. Distilledwater (3.5 mL), 0.8 mL diacetylmonoxime (2%) and3.2 mL sulphuric acid-phosphoric acid reagent(reagent was prepared by mixing 150 mL 85%phosphoric acid with 140 mL water and 50 mL ofconcentrated sulphuric acid) were added to 0.5 mL

of protein-free filtrate. The reaction mixture wasplaced in a boiling water bath for 30 min and thencooled. The absorbance was read at 480 nm.

Estimation of creatinine Creatinine was esti-mated by the alkaline picrate method of Hare.31Protein-free filtrate was prepared. Sodium tungstate(1.0 mL, 5%), 1.0 mL sulphuric acid (0.6 N) and1.0 mL distilled water were added to 1.0 mL serum.After mixing thoroughly, the mixture was centri-fuged at 800 x g for 5 min. The supernatant wasadded to a mixture containing 1.0 mL picric acid(1.05%) and 1.0 mL sodium hydroxide (0.75 N). Theabsorbance at 520 nm was read after exactly 20 min.

Assay for ornithine decarboxylase activity ODCactivity was determined using 0.4 mL renal105 000 g supernatant fraction per assay tube bymeasuring release of 14CO2 from [14G]ornithine bythe method of O'Brien et al.32 The kidneys werehomogenized in Tris-HCI buffer (pH 7.5, 50 mM)containing EDTA (0.1 mM), pyridoxal phosphate(0.1 mM), PMSF (1.0 mM), 2-mercaptoethanol(1.0 mM), dithiothreitol (0.1 mM) and Tween 80(0.1%) at 4°C. In brief, the reaction mixture con-tained 400 ,uL cytosol and 0.095 mL co-factor mix-ture containing pyridoxal phosphate (0.32 mM),EDTA (0.4 mM), dithiothreitol (4.0 mM), ornithine(0.4 mM), Brig 35 (0.02%) and [14C]ornithine(0.05 ,uCi) in a total volume of 0.495 mL. Afteradding buffer and co-factor mixture to blank andother test tubes, the tubes were closed immediatelywith a rubber stopper containing 0.2 mL ethanola-mine and methoxyethanol mixture in the centralwell and kept in a water bath at 37°C. After 1 hour ofincubation, the enzyme activity was arrested byinjecting 1.0 mL citric acid solution (2.0 M) alongthe sides of glass tubes and the incubation wascontinued for 1 hour to ensure complete absorptionof 14CO2. Finally, the central well was transferred toa vial containing 2 mL ethanol and 10 mL toluene-based scintillation fluid was added. Radioactivitywas counted in a liquid scintillation counter (LKBWallace-1410). ODC activity was expressed as pico-moles of 14CO2 released per hour per milligram ofprotein.

Assay for renal DNA synthesis The isolation ofrenal DNA and assessment of incorporation of[3H]thymidine into DNA were carried out by themethod of Smart et al. The rat kidneys werequickly removed and cleaned free of extraneousmaterial and homogenate (10% w/v) was preparedin ice-cold water. The precipitate thus obtained waswashed with cold TCA (5%) and incubated with

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Table 1 Effect of pretreatment of FL on the Fe-NTA-mediated depletion in glutathione content and its metabolizing enzymes in thekidney of Wistar rats

Treatment regimen Reduced glutathione Glutathione reductase Glutathione-S-transferase(nM GSH/g tissue) (nM NADPH oxidized/min/mg (nmol CDNB conjugate formed/

protein) min/mg protein)

Saline-treated control 0.62 + 0.01 76.50+0.99 128.7+1.50Fe-NTA alone (9 mgFe/kg body weight) 0.45 + 0.002## 59.52 + 1.81## 58.86 + 0.58##Fe-NTA (9 mg Fe/kg body weight) + FL 0.58 + 0.009# 67.36 + 0.50# 78.02 + 1.19#(1%/kg body weight)Fe-NTA (9 mgFe/kg body weight) + FL 0.61+0.001# 70.20+0.19# 111.7+0.85#(2%/kg body weight)Only FL (2%/kg body weight) 0.63+0.001 80.40+2.39 136.1+0.37

Results represent mean + SE of five animals/group.Results significantly different from saline-treated group (##p <0.001).Results significantly different from Fe-NTA treated group (#P <0.001).

cold PCA (10%) at 4°C overnight. After this, theincubation mixture was centrifuged and the preci-pitate was washed with cold PCA (5%). Theprecipitate was dissolved in warm PCA (10%),incubated in a boiling water bath for 30 min, andfiltered through Whatman 50 paper. The filtrate wasused for [3H] counting in a liquid scintillationcounter (LKB Wallace-1410) after adding scintilla-tion fluid. The amount of DNA in the filtrate wasestimated by the diphenylamine method of Gilesand Myers.34 The amount of [3H]thymidine incor-porated was expressed as disintegration per minuteper microgram of DNA.

Estimation of protein The protein concentrationin all samples was determined by the method ofLowry et al.35 Peptide bonds form a complex withalkaline copper sulphate reagent, which gives a bluecolour with folin's reagent. Briefly, 0.1 mL PMS wasdiluted to 1 mL water and protein precipitated withan equal volume of TCA (10%), kept overnight 4°Cand centrifuged at 800 x g for 5 min. The super-natant was discarded. The pellet was dissolved in5 mL of NaOH (1 N). Finally 0.1 mL of aliquot was

further diluted to 1 mL with water and then 2.5 mLof alkaline copper sulphate reagent containingsodium carbonate (2%), copper sulphate (1%) andsodium potassium tartrate (2%) was added. Tenminutes after addition of alkaline copper sulphatereagent to allow complex formation, 0.25 mL ofFolin's reagent was added. After 30 min, a bluecolour developed that was read at 660 nm forstandard BSA (0.1 mg/mL) was used.

Statistical analysisDifferences between groups were analysed usinganalysis of variance (ANOVA) followed by Dunnett'smultiple comparisons test. All data points arepresented as the treatment group mean+ standarderror of the mean (SE).

Results

Fe-NTA intoxication leads to depletion of renalglutathione, its metabolizing enzymes GST and GR,and antioxidant enzymes CAT, GPx, QR and G6PD(P <0.001) as compared with the saline-treatedcontrol group. Fe-NTA administration also caused

Table 2 Effect of pretreatment of FL on antioxidant enzymes catalase, quinone reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase on Fe-NTA intoxication in the kidney of Wistar rats

Treatment regimen Catalase (nmol H, 02 Quinone reductase Glutathione peroxidase Glucose-6-phosphateconsumed/min/mg (nmol dichloroindophenol (nMNADPH oxidized/ dehydrogenase

protein) reduced/min/mg protein) min/mg protein) (nM NADP reduced/min/mg protein)

Saline-treated controlFe-NTA alone (9 mg Fe/kg bodyweight)

Fe-NTA (9 mg Fe/kg body weight)+ FL (1 %/kg bodyweight)

Fe-NTA (9 mg Fe/kg body weight)+ FL (2%/kg bodyweight)

Only FL (2%/kg bodyweight)

254.3+ 3.30 198.0+ 2.40 73.43 + 0.46 8.70 + 0.27254.3+3.30204.3 + 3.40##

232.8 + 2.30#

247.7 + 2.28#

255.5 + 12.60

198.0+2.40115.0+ 1.19##

138.7 + 5.60#

160.8 + 7.07#

240.0 + 2.30

Results represent mean + SE of five animals/group.Results significantly different from saline treated group (##p <0.001).Results significantly different from Fe-NTA treated group (#P <0.001).

73.43 + 0.4655.38 + 0.57##

59.94 + 0.41#

71.54 + 0.26#

80.38 + 0.95

8.70+0.274.2 + 0.27##

8.1 + 0.20#

8.6 + 0.12#

9.7+ 0.14

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Farnesol prevents early tumour promotion markers in ratsT Jahangir et al.

240Table 3 Effect of pretreatment of FL on the Fe-NTA-mediated increase in XO, lipid peroxidation and H202 content in the kidney ofWistar rats

Treatment regimen XO (,ug uric acid Lipid peroxidation (nmol H202 (nmol H202/gformed/min/mg protein) MDA/h/g tissue) tissue)

Saline-treated control 0.145 +0.002 3.37+0.021 64.4 + 0.09Fe-NTA alone (9 mg Fe/kg body weight) 0.204 + 0.006# 9.73 + 0.146## 128.6 + 1.56##Fe-NTA (9 mg Fe/kg body weight)+ FL (1%/kg bodyweight) 0.166+0.002# 7.45 +0.012k 83.4 + 0.62#Fe-NTA (9 mg Fe/kg body weight) + FL (2%/kg bodyweight) 0.151 + 0.001# 5.71 + 0.008# 76.6 +0.31#Only FL (2%/kg bodyweight) 0.142+0.004 3.28+0.070 61.1+0.31

Results represent mean + SE of five animals/group.Results significantly different from saline treated group (##p <0.001).Results significantly different from Fe-NTA treated group (#P <0.001).

elevation in the activity ofXO and H202 content andincreases the levels of MDA formation and renaltoxicity markers viz., BUN and creatinine (P <0.001) as compared with the saline-treated controlgroup. Intoxication with Fe-NTA resulted in signifi-cant (P <0.005) increase in the rate of [3H]thymi-dine incorporation into renal DNA and concomitant(P <0.001) increase in ODC activity.Pretreatment with FL (1% and 2%/kg body

weight) restored renal glutathione (GSH) contentand its dependent enzymes GST, and GR, andantioxidant enzymes like CAT, GPx, G6PD and QRwere restored dose dependently (P <0.001) asshown in Tables 1 and 2. There was a markeddepletion in levels of XO and MDA formation (P <0.001) and concomitant downregulation of release ofBUN and creatinine in serum (P < 0.001), as evidentfrom Tables 3 and 4. The FL alone group producedresults near to saline control values. Figures 1 and 2show the inhibition of early markers of tumourpromotion-like ODC activity and renal DNA synth-

Table 4 Effect of pretreatment of FL on the Fe-NTA-mediatedelevation in serum toxicity markers

Treatment regimen Blood- urea Creatinine (mg/nitrogen (mg/ 100 mL) IU/L100 mL) IU/L

esis, respectively, by FL in rats. The prophylactictreatment of rats with FL showed a marked inhibi-tion of ODC activity (P <0.001) in a dose-dependentmanner as shown in Figure 1 and suppression of therate of [3Hlthymidine incorporation (P <0.001) intorenal DNA of treated control as evident from Figure2.

Discussion

The chemopreventive effects of FL were studiedagainst toxic effects of iron in renal tissue after asingle i.p. injection of Fe-NTA in male Wistar rats. lthas been seen that uptake of iron in renal tissuegenerates reactive oxygen species, which induceslipid peroxidation and subsequent enhancement ofserum toxicity markers and depletion of renal GSHcontent.36 Hydroxyl radicals generated by reactionof Fe-NTA with H202 could be the possible cause ofoxidative damage.37 Evidence suggests that intra-cellular oxidative stress plays an important rolein the pathogenesis of nephrotoxicity caused byFe-NTA.38 lt is evident that the tumour-promotingeffects of Fe-NTA are due to its ability to generatefree radicals in the renal tissue.19 FL has chemother-apeutic activity toward pancreatic and other can-cers.17 The suppression of the mevalonate pathway

Saline-treated controlFe-NTA alone (9 mg Fe/kgbody weight)

Fe-NTA (9 mg Fe/kg bodyweight)+ FL (1 %/kgbody weight)

Fe-NTA (9 mgFe/kg bodyweight) + FL (2%/kgbodyweight)

Only FL (2%/kgbodyweight)

28.78 + 0.50103.45+ 2.88##

48.87 + 0.041#

1.307 +0.00052.962 + 0.0009##

1.896 + 0.0013#

.5

00.

0>

CO)QE

0-cna)(130.28 + 0.16# 1.604 +0.0009#

20.65 +0.04 1.347+ 0.00276

3500 -

3000250020001500 -

1000 -

500 -0 - [l#

Control Only Fe- Dl+Fe- D2+Fe- Only D2NTA NTA NTA

Results represent mean + SE of five animals/group.Results significantly different from saline treated group(##p <0.001).Results significantly different from Fe-NTA-treated group(#P <0.001).IU ==International Units.

Figure 1 Effect of pretreatment of farnesol on ODC activity.Results represent mean+SE of five animals/group. Results sig-nificantly different from saline-treated group (##P <0.001). Re-sults significantly different from Fe-NTA-treated group (#P <0.001). Dl, 1% farnesol/kg body weight; D2, 2% farnesol/kg bodyweight.

ZN Z'

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Figure 2 Effect of pretreatment of famnesol on 3[H] thymidineincorporation in renal DNA. Results represent mean + SE of fiveanimals/group. Results significantly different from saline-treatedgroup (*P <0.005). Results significantly different from Fe-NTA-treated group (#P <0.001). DPM, disintegration per minute; Dl,1% farnesol/kg body weight; D2, 2% farnesol/kg body weight.

can be attributed to the chemopreventive activity ofisoprenoids.39 Diverse isoprenoids modulate cellgrowth, induce cell cycle arrest, initiate apoptosisand suppress cellular signalling activities.7 It isevident from present results that FL treatment priorto Fe-NTA intoxication leads to restoration of renalglutathione, its dependent enzymes and other phase

Farnesol prevents early tumour promotion markers in ratsT Jahangir et al.

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II enzymes, viz., glutathione-S-transferase and qui-none reductase. The role of phase II detoxificationenzymes in modulation and degradation of electro-philic metabolites is well documented.40 FL pre-treatment in rats has been shown to diminishFe-NTA-induced MDA formation H202 activity,and XO levels.Induction of ODC activity and consequently the

rate ofDNA synthesis play major roles as immediatebiomarkers of tumour promotion in chemopreven-tive studies.36 Prophylactic treatment of FL signifi-cantly suppressed enhanced ODC activity and[3H]thymidine incorporation in renal DNA. Thesefacts suggest that FL as an antioxidant is an effectivechemopreventive measure against carcinogens, inwhich there is involvement of free radicals in cancerinduction. The present study reveals that thiscompound may afford substantial protection againstrenal toxicity induced by Fe-NTA.

Acknowledgements

The author (SS) is thankful to the Hamdard NationalFoundation, New Delhi, India for providing thefunds to carry out this work.

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