Chemomodulatory effect of Ficus racemosa extract against chemically induced renal carcinogenesis and oxidative damage response in Wistar rats

Life Sciences 77 (2005) 1194 – 1210 www.elsevier.com/locate/lifescie

Chemomodulatory effect of Ficus racemosa extract against chemically induced renal carcinogenesis and oxidative damage response in Wistar rats Naghma Khan, Sarwat SultanaT Section of Chemoprevention and Nutrition Toxicology, Department of Medical Elementology and Toxicology, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi 110 062, India Received 10 May 2004; accepted 7 December 2004

Abstract Ferric nitrilotriacetate (Fe-NTA) is a well-known renal carcinogen. In this communication, we show the chemopreventive effect of Ficus racemosa extract against Fe-NTA-induced renal oxidative stress, hyperproliferative response and renal carcinogenesis in rats. Fe-NTA (9 mg Fe/kg body weight, intraperitoneally) enhances renal lipid peroxidation, xanthine oxidase, g-glutamyl transpeptidase and hydrogen peroxide (H2O2) generation with reduction in renal glutathione content, antioxidant enzymes, viz., glutathione peroxidase, glutathione reductase, catalase, glucose-6-phosphate dehydrogenase and phase-II metabolising enzymes such as glutathione-S-transferase and quinone reductase. It also enhances blood urea nitrogen, serum creatinine, ornithine decarboxylase (ODC) activity and thymidine [3H] incorporation into renal DNA. It also enhances DEN (Ndiethylnitrosamine) initiated renal carcinogenesis by increasing the percentage incidence of tumors. Treatment of rats orally with F. racemosa extract (200 and 400 mg/kg body weight) resulted in significant decrease in gglutamyl transpeptidase, lipid peroxidation, xanthine oxidase, H2O2 generation, blood urea nitrogen, serum creatinine, renal ODC activity, DNA synthesis ( P b 0.001) and incidence of tumors. Renal glutathione content ( P b 0.01), glutathione metabolizing enzymes ( P b 0.001) and antioxidant enzymes were also recovered to significant level ( P b 0.001). Thus, our data suggests that F. racemosa extract is a potent chemopreventive agent and suppresses Fe-NTA-induced renal carcinogenesis and oxidative damage response in Wistar rats. D 2005 Elsevier Inc. All rights reserved. Keywords: Carcinogenesis; Chemoprevention; Ferric nitrilotriacetate; Ficus racemosa extract; Oxidative stress

T Corresponding author. Tel.: +91 11 2605 9688; fax: +91 11 2605 9663. E-mail address: [email protected] (S. Sultana). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.12.041

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Introduction During the last three decades, the scientific community has made immense progress in acquiring the knowledge needed to prevent cancer. Pioneering research helped to identify potential causes of cancer, particularly environmental factors such as diet and provided insight regarding their mechanisms of action. Carcinogenesis results from the accumulation of multiple sequential mutations and alterations in nuclear and cytoplasmic molecules, culminating in invasive neoplasms. Concurrently, promising inhibitors of cancer that appeared able to either arrest or reverse cancer development by interfering with one or more steps in the process of carcinogenesis were identified and systematically evaluated for their potential as chemopreventive agents (Huang et al., 1997). Numerous agents determined to be safe and effective in preclinical trials have been and continue to be tested in Phase I, II and III clinical interventions for cancers at various sites including breast, colon, prostate, esophagus, mouth, lung, cervix, endometrium, ovary, liver, bladder and skin (Li et al., 2002). Ficus racemosa syn. Ficus glomerata Linn. (Family: Moraceae) is a widely cultivated plant all over India. It is mixed with rice for making bread and its fruits are used in several dishes. It has been reported to have many medicinal properties (Trivedi et al., 1969). The roots are used as a medicine against hydrophobia. Its fruits are effective against leprosy, diseases of the blood, fatigue, bleeding nose and cough. Its bark is helpful against asthma and its leaves are used against bronchitis. It is used as carminative, astringent, vermifuge and an anti-dysentery drug. The extract of fruit is used in diabetes and leucoderma. The plant is used locally to relieve inflammation of skin wounds, lymphadenitis, sprains and fibrositis. The alcoholic extract of the stem bark of the plant possessed antiprotozoal activity against Entamoeba histolytica. It is used in the treatment of mumps, smallpox, heamaturia and inflammatory conditions (Mandal et al., 2000). Iron is the most abundant metal in the human body. Although iron is an essential nutritional element for all life forms, iron overload may lead to various diseases (De Freitas and Meneghini, 2001). The iron complex of the chelating agent nitrilotriacetic acid (NTA) is nephrotoxic. Intraperitoneal injection of FeNTA induces renal proximal tubular damage associated with oxidative damage that eventually leads to a high incidence of renal cell carcinoma in rodents after repeated administration (Okada and Midorikawa, 1982). Intraperitoneally injected Fe-NTA is absorbed into the portal vein through mesothelium and passes into circulation via the liver (Umemura et al., 1990). The low molecular weight Fe-NTA is easily filtered through the glomeruli into the lumen of the renal proximal tubules where Fe3+-NTA is reduced to Fe2+-NTA by the glutathione degradation products cysteine or cysteinylglycine (Tsao and Curthoys, 1980). In the brush border surface of the renal proximal convoluted tubules, g-glutamyl transpeptidase hydrolyses glutathione to cysteinylglycine that is rapidly degraded to cysteine and glycine by dipeptidase. Cysteine and cysteinylglycine are the proposed thio reductants that reduce Fe3+-NTA to Fe2+-NTA. The auto-oxidation of Fe2+-NTA generates superoxide radicals (O˙2 ) which subsequently potentiate the iron catalysed Haber–Weiss reaction to produce hydroxyl radical (OH˙), leading to induction of lipid peroxidation and oxidative DNA damage (Umemura et al., 1990). There is strong epidemiological evidence that fruits, vegetables and plants can prevent a range of human diseases. These effects were ascribed to the ability of these agents to influence both the metabolism and disposition of the toxicant and also to enhance the cellular capacity to combat oxidative stress (Wattenberg, 1990). Various studies from our lab have shown the inhibitory effect of plants on chemically induced oxidative stress and cancer (Khan et al., 2001, 2003, 2004; Saleem et al., 2001; Sultana et al., 2003).

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For the present study, we prepared the methanolic extract of the aerial part of plant that contains h-sitosterol, lupeol and quercetin as major active constituents. Different parts of the plant like leaves, fruits, bark and stems were also used separately to prepare the extract. However, the efficacy of the extract was found to be highest in the aerial parts of the plant (various preliminary in vitro tests like calf thymus DNA sugar damage, lipid peroxidation and cytochrome P 450 level were performed—data not shown). The other parts were also found to be lacking in some of the active constituents and their efficacy was also low. The presence of h-sitosterol, lupeol and quercetin from methanolic extract was identified by HPLC (Waters) using pure authentic compounds of Sigma, St. Louis, USA, in the proportions of 3:2:4 respectively. The other minor components were identified by TLC under UV-light. Since F. racemosa extract has been shown to inhibit various diseases, particularly those that act through the generation of reactive oxygen species, we speculated that it might inhibit Fe-NTA-induced nephrotoxicity. In the present investigation, we assess the prophylactic treatment of rats with F. racemosa extract on Fe-NTA-induced renal carcinogenesis and oxidative damage response in Wistar rats.

Materials and methods Chemicals Reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, g-glutamyl pnitroanilide, glycylglycine, bovine serum albumin (BSA), 1,2,dithio-bis-nitrobenzoic acid (DTNB), 1,chloro-2, 4,dinitrobenzene (CDNB), reduced nicotinamide adenine dinucleotide phosphate (NADPH), potassium bromate (KBrO3), flavine adenine dinucleotide (FAD), glucose-6-phosphate, Tween-20, 2,6,dichlorophenolindophenol and thiobarbituric acid (TBA) were obtained from Sigma Chemical (St. Louis, MO, USA). Diacetylmonoxime, urea, picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid (TCA) and perchloric acid (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 Amersham Corporation (UK). All other chemicals and reagents were of the highest purity commercially available. Plant material F. racemosa was collected from the herbal garden of Hamdard University, New Delhi, India. Professor Mohammad Iqbal, Head, Department of Environmental Botany, Hamdard University, verified the identity of the plant. The voucher specimen was deposited in the Department of Medical Elementology and Toxicology, Hamdard University. The plant material was dried in air and then milled to a fine powder of mesh size 1-mm as described by Antonio et al. (1988). Preparation of extract The extraction procedure was followed as described by Didry et al. (1998). Briefly, powdered plant material (350 g) was repeatedly extracted in a 3000 ml round bottomed flask with 2000 ml solvents of increasing polarity starting with petroleum ether, benzene, ethyl acetate, acetone, methanol and double distilled water. The reflux time for each solvent was 4 h. The extracts were cooled at room temperature, filtered and evaporated to dryness under reduced pressure in a rotatory evaporator (Buchi Rotavapor).

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The residues yielded for each solvent (10, 8, 15, 19, 36 and 24 g respectively) were stored at 4 8C. The methanolic fraction was used for further study after preliminary in vitro tests viz., calf thymus DNA sugar damage, lipid peroxidation, xanthine-oxidase activity and cytochrome P 450 level. Preparation of Fe-NTA solution Fe-NTA solution was prepared fresh immediately before its use by the method of Awai et al. (1979). To prepare Fe-NTA, ferric nitrate (0.16 m mol/kg body weight) solution was mixed with fourfold molar excess of disodium salt of NTA (0.64 m mol/kg body weight) and the pH was adjusted to 7.4 with sodium bicarbonate solution. The concentration of the Fe-NTA solution prepared was 10 ml/kg body weight and the dose was 9 mg Fe/kg body weight. Animals 4–6 week old, female albino rats (130–150 g) of Wistar strain were obtained from Central Animal House of Hamdard University, New Delhi, India. They were housed in polypropylene cages in groups of six rats per cage and were kept in a room maintained at 25 F 2 8C with a 12 h light/dark cycle. They were allowed to acclimatize for one week before the experiments and were given free access to standard laboratory feed (Hindustan Lever Ltd., Bombay, India) and water ad libitum. Experimental protocol Treatments Different groups of animals were used for the various sets of biochemical studies. To study the effect of pretreatment with F. racemosa extract on Fe-NTA-induced renal oxidative stress and ODC induction, 30 female Wistar rats were randomly allocated to 5 groups of 6 rats each. Group I received only saline injection intraperitoneally (0.85% NaCl) at a dose of 10 ml/kg body weight. Group II received only a single intraperitoneal injection of Fe-NTA at a dose level of 9 mg Fe/kg body weight. Group III received pretreatment with F. racemosa extract by gavage once daily for 5 days at a dose of 200 mg/kg body weight and Group IV and V received pretreatment with F. racemosa extract by gavage once daily for 5 days at a dose of 400 mg/kg body weight. After the last treatment with F. racemosa extract, the animals of groups II, III and IV received a single intraperitoneal injection of Fe-NTA at a dose level of 9 mg Fe/ kg body weight. After twelve hours, the animals were sacrificed by cervical dislocation and processed for sub-cellular fractionation, their kidneys were quickly removed and perfused in ice-cold saline. Just before they were killed, blood was collected in test tubes from retro-orbital sinus for the estimation of creatinine and blood urea nitrogen. To study the effect of pretreatment with F. racemosa extract on Fe-NTA mediated [3H] thymidine incorporation into renal DNA, the grouping of animals and the schedules for prophylaxis were as described above. After the last treatment with F. racemosa extract, the animals of groups II, III and IV received only a single intraperitoneal injection of Fe-NTA at a dose level of 9 mg Fe/kg body weight. 18 h after the treatment with Fe-NTA, the rats were given [3H] thymidine (30 ACi/animal) by i.p. injection and 2 h later they were sacrificed by cervical dislocation and their kidneys were quickly removed. To study the effect of pretreatment with F. racemosa extract on DEN (N-diethylnitrosamine) initiated and Fe-NTA promoted renal carcinogenesis, the animals were divided into six groups of 20 rats per

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group. Group I received only saline injection intraperitoneally (0.85% NaCl) at a dose of 10 ml/kg body weight. Animals of groups II, III, V and VI were initiated with a single i.p. injection of DEN at a dose level of 200 mg/kg body weight in saline. Ten days after initiation, the animals in groups III, IV, V and VI were promoted with intraperitoneal injection of Fe-NTA at a dose level of 9 mg Fe/kg body weight, twice a week for 16 weeks. Groups V and VI received oral treatment with F. racemosa extract by gavage once daily at a dose of 200 and 400 mg/kg body weight respectively half an hour prior to the treatment with Fe-NTA for a period of 16 weeks, twice a week. At the end of 24 weeks, the animals were sacrificed by cervical dislocation and their kidneys were quickly removed and preserved in 10% neutral buffered formalin for histopathological studies. Haematoxylin and eosin preparations of processed sections were prepared for microscopic examination. Post-mitochondrial supernatant (PMS) and microsome preparation Kidneys were removed quickly, cleaned free of extraneous material and immediately perfused with ice-cold saline (0.85% sodium chloride). The kidneys were homogenized in chilled phosphate buffer (0.1 M, pH 7.4) containing KCI (1.17%) using a Potter Elvehjen homogenizer. The homogenate was filtered through muslin cloth, and was centrifuged at 3000 rpm for 10 min at 4 8C by Eltek Refrigerated Centrifuge (model RC 4100 D) to separate the nuclear debris. The aliquot so obtained was centrifuged at 12 000 rpm for 20 min at 4 8C to obtain post-mitochondrial supernatant (PMS), which was used as a source of enzymes. A portion of the PMS was centrifuged for 60 min by ultracentrifuge (Beckman L755) at 34,000 rpm at 4 8C. The pellet was 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 method of Jollow et al. (1974). 1.0 ml sample of PMS was precipitated with 1.0 ml of sulfosalicylic acid (4%). The samples were kept at 4 8C for 1 h and then centrifuged at 1200  g for 20 min at 4 8C. The assay mixture contained 0.1 ml filtered aliquot, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB (100 mM) in a total volume of 3.0 ml. The yellow color developed was read immediately at 412 nm on a spectrophotometer (Milton Roy Model-21 D). Estimation of lipid peroxidation The assay for microsomal lipid peroxidation was done following the method of Wright et al. (1981) as modified by Khan et al. (2001). The reaction mixture in a total volume of 1.0 ml contained 0.58 ml phosphate buffer (0.1 M, pH 7.4), 0.2 ml microsomes, 0.2 ml ascorbic acid (100 mM), 0.02 ml ferric chloride (100 mM). The reaction mixture was incubated at 37 8C in a shaking water bath for 1 h. The reaction was stopped by addition of 1.0 ml 10% trichloroacetic acid (TCA). Following addition of 1.0 ml 0.67% thiobarbituric acid (TBA), all the tubes were placed in boiling water-bath for 20 min and then shifted to crushed ice-bath before centrifuging at 2500  g for 10 min. The amount of malondialdehyde formed in each of the samples was assessed by measuring optical density of the supernatant at 535 nm using spectrophotometer (Milton Roy 21 D) against a reagent blank. The results were expressed as nmol MDA formed/h/g tissue at 37 8C using molar extinction coefficient of 1.56  105/M/cm.

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Estimation of blood urea nitrogen Estimation of blood urea nitrogen was done by diacetyl monoxime method of Kanter (1975). Protein free filtrate was prepared. To 0.5 ml of protein free filtrate, were added 3.5 ml of distilled water, 0.8 ml diacetylmonoxime (2%) and 3.2 ml sulphuric acid–phosphoric acid reagent (reagent was prepared by mixing 150 ml 85% phosphoric acid with 140 ml water and 50 ml of concentrated sulphuric acid). The reaction mixture was placed in a boiling water-bath for 30 min and then cooled. The absorbance was recorded at 480 nm. Estimation of creatinine Creatinine was estimated by the alkaline picrate method of Hare (1950). Protein free filtrate was prepared. To 1.0 ml serum were added, 1.0 ml sodium tungstate (5%), 1.0 ml sulfuric acid (0.6 N) and 1.0 ml distilled water. After mixing thoroughly, the mixture was centrifuged at 800  g for 5 min. The supernatant was added to a mixture containing 1.0 ml picric acid (1.05%) and 1.0 ml sodium hydroxide (0.75 N). The absorbance at 520 nm was recorded exactly after 20 min. Assay for hydrogen peroxide Hydrogen peroxide (H2O2) was assayed by H2O2-mediated horseradish peroxidase-dependent oxidation of phenol red by the method of Pick and Keisari (1981). 2.0 ml of microsomes was suspended in 1.0 ml of solution containing phenol red (0.28 nm), horseradish peroxidase (8.5 units), dextrose (5.5 nm) and phosphate buffer (0.05 M, pH 7.0) and was incubated at 37 8C for 60 min. The reaction was stopped by the addition of 0.01 ml of NaOH (10 N) and then centrifuged at 800  g for 5 min. The absorbance of the supernatant was recorded at 610 nm against a reagent blank. The quantity of H2O2 produced was expressed as nmol H2O2/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 the method of Habig et al. (1974). The reaction mixture consisted of 1.475 ml phosphate buffer (0.1 M, pH 6.5), 0.2 ml reduced glutathione (1 mM), 0.025 ml CDNB (1 mM) and 0.3 ml PMS (10% w/v) in a total volume of 2.0 ml. The changes in the absorbance were recorded at 340 nm and enzymes activity was calculated as nmol CDNB conjugate formed/min/mg protein using a molar extinction coefficient of 9.6  103/M/cm. Assay for glutathione peroxidase activity Glutathione peroxidase activity was assayed by the method of Mohandas et al. (1984). The reaction mixture consisted of 1.49 ml phosphate buffer (0.1 M, pH 7.4), 0.1 ml EDTA (1 mM), 0.1 ml sodium azide (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 a total volume of 2 ml. The disappearance of NADPH at 340 nm was recorded at 25 8C. Enzyme activity was calculated as nmol NADPH oxidized/ min/mg protein using molar extinction coefficient of 6.22  103/M/cm. Assay for glutathione reductase activity Glutathione reductase activity was determined by method of Carlberg and Mannervik (1975). The reaction mixture consisted of 1.65 ml phosphate buffer (0.1 M, pH 7.6), 0.1 ml EDTA (0.5 mM), 0.05

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ml oxidized glutathione (1 mM), 0.1 ml NADPH (0.1 mM) and 0.1 ml 10% PMS in a total volume of 2 ml. Enzyme activity was quantitated at 25 8C by measuring disappearance of NADPH at 340 nm and was calculated as nmol NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22  103/M/cm. Assay for c-glutamyl transpeptidase activity This was determined by the method of Orlowski and Meister (1973) using g-glutamyl pnitroanilide as substrate. The reaction mixture in a total volume of 1.0 ml contained 0.2 ml 10% homogenate which was incubated with 0.8 ml substrate mixture (containing 4 mM g-glutamyl pnitroanilide, 40 mM glycylglycine and 11 mM MgCl2 in 185 mM Tris–HCl buffer, pH 8.25) at 37 8C. Ten minutes after initiation of the reaction, 1.0 ml 25% TCA was added and mixed to terminate the reaction. The solution was centrifuged and the supernatant fraction read at 405 nm. Enzyme activity was calculated as nmol p-nitroaniline formed/min/mg protein using a molar extinction coefficient of 1.74  103/M/cm. Assay for catalase activity Catalase activity was assayed by the method of Claiborne (1985). The reaction mixture consisted of 1.95 ml phosphate buffer (0.1 M, pH 7.4), 1.0 ml hydrogen peroxide (0.019 M) and 0.05 ml 10% PMS in a final volume of 3 ml. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated as nmol H2O2 consumed/min/mg protein. Assay for glucose-6-phosphate dehydrogenase activity The activity of glucose-6-phosphate dehydrogenase was determined by the method of Zaheer et al. (1965). The reaction mixture consisted of 0.3 ml Tris–HCl buffer (0.05 M, pH 7.6), 0.1 ml NADP (0.1 mM), 0.1 ml glucose-6-phosphate (0.8 mM), 0.1 ml MgCl2 (8 mM), 0.3 ml PMS (10%) and 2.1 ml distilled water in a total volume of 3 ml. The changes in absorbance were recorded at 340 nm and enzyme activity was calculated as nmol NADP reduced/min/mg protein using a molar extinction coefficient of 6.22  103/M/cm. Assay for xanthine oxidase activity The activity of xanthine oxidase was assayed by the method of Athar et al. (1996). The reaction mixture consisted of 0.2 ml PMS which was incubated for five minutes at 37 8C with 0.8 ml phosphate buffer (0.1 M, pH 7.4). The reaction was started by adding 0.1 ml xanthine (9 mM) and kept at 37 8C for 20 min. The reaction was terminated by the addition of 0.5 ml ice cold perchloric acid (10% v/v). After 10 min, 2.4 ml of distilled water was added and centrifuged at 4000 rpm for 10 min. and A gm uric acid formed/min/mg protein was recorded at 290 nm. Assay for quinone reductase activity The activity of quinone reductase was determined by the method of Benson et al. (1980). The 3 ml reaction mixture 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 AL (10%) PMS. The reduction of dichlorophenolindophenol (DCPIP) was recorded calorimetrically at 600 nm and enzyme activity was calculated as nmol of DCPIP reduced/min/mg protein using molar extinction coefficient of 2.1  104/M/cm.

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Assay for ornithine decarboxylase activity ODC activity was determined using 0.4 ml renal 105,000g supernatant fraction per assay tube by measuring release of 14CO2 from dl-[14C] ornithine by the method of O’Brien et al. (1975). The kidneys were homogenized in Tris–HCl 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 Tween80 (0.1%) at 4 8C. In brief, the reaction mixture contained 400 Al enzyme and 0.095 ml co-factor mixture 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 ACi) in a total volume of 0.495 ml. After adding buffer and co-factor mixture to blank and other test tubes, the tubes were closed immediately with a rubber stopper containing 0.2 ml ethanolamine and methoxyethanol mixture in the central well and kept in a water bath at 37 8C. After 1 h of incubation, the enzyme activity was arrested by injecting 1.0 ml citric acid solution (2.0 M) along the sides of glass tubes and the incubation was continued for 1 hr to ensure complete absorption of 14CO2. Finally, the central well was transferred to a vial containing 2 ml ethanol and 10 ml toluene based scintillation fluid was added. Radioactivity was counted 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 of incorporation of [3H] thymidine into DNA were carried out by the method of Smart et al. (1986). The rat kidneys were quickly removed and cleaned free of extraneous material and homogenate (10% w/v) was prepared in ice-cold water. The precipitate thus obtained was washed with cold TCA (5%) and incubated with cold PCA (10%) at 4 8C overnight. After this, incubation mixture was centrifuged and 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, and filtered through Whatman 50 paper. The filtrate was 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 diphenylamine method of Giles and Myers (1965). The amount of [3H] thymidine incorporated was expressed as dpm/Ag DNA. Estimation of protein The protein concentration in all samples was determined by the method of Lowry et al. (1951). Statistical analysis Differences between groups were analyzed using Dunnet’s t-test followed by analysis of variance (ANOVA).

Results Effect of F. racemosa extract on glutathione metabolism Table 1 shows the effect of pretreatment of rats with F. racemosa extract on Fe-NTA-mediated renal glutathione content and on the activities of its metabolizing enzymes, viz., glutathione-S-transferase and glutathione reductase. Treatment with Fe-NTA alone resulted in the depletion of renal glutathione and reduction in the activities of glutathione-S-transferase and glutathione reductase by 44%, 54% and 48%

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Table 1 Effect of pretreatment with F. racemosa extract on Fe-NTA-mediated depletion of renal glutathione content and decrease in the activities of glutathione metabolizing enzymes, glutathione-S-transferase and glutathione reductase in rats Treatment groups

Reduced glutathione [nmol GSH/g tissue]

Glutathione-S-transferase [nmol CDNB conjugate formed/min/mg protein]

Glutathione reductase [nmol NADPH oxidized/min/mg protein]

Saline (control) Fe-NTA (9 mg Fe/kg body weight) F. racemosa extract (200 mg/kg body weight) + Fe-NTA (9 mg Fe/kg body weight) F. racemosa extract (400 mg/kg body weight) + Fe-NTA (9 mg Fe/kg body weight) Only F. racemosa extract (400 mg/kg body weight)

0.463 F 0.03 0.261 F 0.04w 0.365 F 0.02*

202.16 F 4.2 93.23 F 4.1ww 134.73 F 5.4***

282.65 F 12.6 146.35 F 7.2ww 198.62 F 11.4***

0.396F 0.03*

168.45 F 76.5***

245.31 F10.4***

0.488 F 0.02

277.43 F 7.6

297.53 F 11.6

Each value represents mean F S.E., n = 6. P b 0.01 and wwP b 0.001 compared with the corresponding value for saline-treated control group. *P b 0.05, **P b 0.01 and ***P b 0.001 compared with the corresponding value for Fe-NTA treated group.

w

respectively of that of saline-treated control group. However, pretreatment of animals with F. racemosa extract at 200 mg/kg body weight and 400 mg/kg body weight resulted in the recovery by 23–29%, 20– 37% and 18–35% respectively, as compared with Fe-NTA treated group. Effect of F. racemosa extract on renal antioxidant enzymes The effect of prophylactic treatment with F. racemosa extract on Fe-NTA-induced reduction in the activities of renal antioxidant enzymes is shown in Table 2. Fe-NTA alone treatment caused reduction in

Table 2 Effect of pretreatment with F. racemosa extract on Fe-NTA-induced depletion in the level of renal antioxidant enzymes in rats Treatment groups

Glucose-6-phosphate Catalase [nmol H2O2 Glutathione peroxidase dehydrogenase [nmol NADP consumed/min/mg protein] [nmol NADPH oxidized/min/mg protein] reduced/min/mg protein]

Saline (control) 158.23 F 8.3 Fe-NTA (9 mg Fe/kg body weight) 46.44 F 5.1ww 95.34 F 4.2*** F. racemosa extract (200 mg/kg body weight) + Fe-NTA (9 mg Fe/kg body weight) 128.12 F 3.3*** F. racemosa extract (400 mg/kg body weight) + Fe-NTA (9 mg Fe/kg body weight) Only F. racemosa extract 168.26 F 2.4 (400 mg/kg body weight)

273.56 F 9.8 129.32 F 11.2ww 189.33 F 8.2***

41.36 F 2.8 19.21 F 2.2ww 30.25 F 1.7**

223.21 F13.6***

38.31 F 2.6***

296.54 F 10.2

47.46 F 3.2

Each value represents mean F S.E., n = 6. w P b 0.01 and wwP b 0.001 compared with the corresponding value for saline-treated control group. *P b 0.05, **P b 0.01 and ***P b 0.001 compared with the corresponding value for Fe-NTA treated group.

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the activities of renal antioxidant enzymes such as catalase, glutathione peroxidase, and glucose-6phosphate dehydrogenase by 70%, 53% and 54% respectively as compared to saline-treated control group. Treatment with F. racemosa extract at lower dose of 200 mg/ kg body weight and higher dose of 400 mg/kg body weight caused the recovery of the above enzymes by 31–51%, 22–34% and 22–32% respectively as compared with Fe-NTA treated control group. Effect of F. racemosa extract on xanthine oxidase, LPO and quinone reductase Table 3 shows that Fe-NTA treatment enhances the activity of xanthine oxidase by 97% and susceptibility of renal microsomal membrane for iron-ascorbate induced lipid peroxidation by 41% whereas it causes a reduction in the activity of quinone reductase by 37% as compared with salinetreated controls. F. racemosa extract treatment caused reduction in activity of xanthine oxidase and renal microsomal lipid peroxidation by 56–80% and 22–32% respectively and increases in the level of quinone reductase by 22–30% at lower (200 mg/kg body weight) and higher (400 mg/kg body weight) doses of F. racemosa extract as compared with Fe-NTA treated group. Effect of F. racemosa extract on renal toxicity markers The effect of pretreatment of rats with F. racemosa extract on Fe-NTA-induced enhancement in the levels of blood urea nitrogen, serum creatinine, g-glutamyl transpeptidase and H2O2 are shown in Table 4. Fe-NTA treatment leads to about 237%, 154%, 125% and 267% enhancement in the values of blood urea nitrogen, serum creatinine, g-glutamyl transpeptidase and H2O2 respectively, as compared with saline-treated controls. Prophylaxis with F. racemosa extract at both doses resulted in 154–179%, 146–216%, 61–88% and 105–201% reduction in the values of blood urea nitrogen, serum creatinine, g-glutamyl transpeptidase and H2O2 respectively as compared with Fe-NTA treated group.

Table 3 Effect of pretreatment with F. racemosa extract on Fe-NTA-mediated depletion of renal quinone reductase and enhancement of xanthine oxidase and renal microsomal lipid peroxidation in rats Treatment groups

Quinone reductase [nmol of dichloroindophenol reduced/min/mg protein]

Xanthine oxidase [Ag of uric acid formed/min/mg protein]

Lipid peroxidation [nmol MDA formed/h/g tissue]

Saline (control) Fe-NTA (9 mg Fe/kg body weight) F. racemosa extract (200 mg/kg body weight) + Fe-NTA (9 mg Fe/kg body weight) F. racemosa extract (400 mg/kg body weight) + Fe-NTA (9 mg Fe/kg body weight) Only F. racemosa extract (400 mg/kg body weight)

190.23 F 12.3 119.61 F11.2w 163.21 F 8.4*

0.238 F 0.05 0.469 F 0.04ww 0.335 F 0.02*

3.84 F 0.22 5.53 F 0.33w 4.58 F 0.12*

178.34 F 10.6**

0.278 F 0.06*

4.42 F 0.16**

215.35 F 14.3

0.211 F 0.04

3.59 F 0.19

Each value represents mean F S.E., n = 6. P b 0.01 and wwP b 0.001 compared with the corresponding value for saline-treated control group. *P b 0.05, **P b 0.01 and ***P b 0.001 compared with the corresponding value for Fe-NTA treated group. w

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Table 4 Effect of pretreatment with F. racemosa extract on Fe-NTA-induced enhancement of blood urea nitrogen, serum creatinine, g-glutamyl transpeptidase and hydrogen peroxide in rats Treatment groups

Blood urea nitrogen (mg/100 ml) IU/l

Creatinine (mg/100 ml) IU/l

g-Glutamyl transpeptidase (nmol p-nitroaniline formed/min/mg protein)

H2O2 (nmol H2O2/g tissue)

Saline (control) Fe-NTA (9 mg Fe/kg body weight) F. racemosa extract (200 mg/kg body weight) + Fe-NTA (9 mg Fe/kg body weight) F. racemosa extract (400 mg/kg body weight) + Fe-NTA (9 mg Fe/kg body weight) Only F. racemosa extract (400 mg/kg body weight)

24.46 F 3.21 82.32 F 2.62ww

1.76 F 0.36 5.93 F 0.24ww

374.35 F 18.21 844.23 F 11.62ww

232.72 F 10.35 521.24 F 7.46ww

44.64 F 2.44***

3.36 F 0.14***

631.51 F12.33***

384.42 F 11.36***

38.53 F 1.82***

2.12 F 0.12***

527.45 F 14.36***

243.53 F 12.28***

22.14 F 3.48

1.62 F 0.14

357.45 F 13.47

211.24 F 10.41

Each value represents mean F S.E., n = 6. P b 0.01 and wwP b 0.001 compared with the corresponding value for saline-treated control group. *P b 0.05, **P b 0.01 and ***P b 0.001 compared with the corresponding value for Fe-NTA treated group.

w

Effect of F. racemosa extract on percentage incidence of RCTs The summary of the percentage incidence of renal cell tumors (RCTs) in different treatment groups is given in Table 5. Saline alone and DEN alone treated groups did not showed any tumors. The treatment with Fe-NTA of the DEN-initiated animals enhanced the occurrence of RCTs by 76.9% in the animals studied whereas treatment with Fe-NTA of the uninitiated animals led to the development of RCTs in 20% of the animals studied. The tumor incidence was decreased in the group of animals pretreated with F. racemosa extract at lower dose of 200 mg/kg body weight by 40% whereas in the group receiving the higher dose of 400 mg/kg body weight, the tumor incidence was reduced by 31.2%.

Table 5 Summary of tumor data of the effect of F. racemosa extract on DEN initiated and Fe-NTA promoted renal tumors Treatment groups

No. of animals treated

No. of animals studied histopathologically

No. of animals with renal cell tumors

Incidence of tumors (%)

Saline (alone) DEN (alone) DEN + Fe-NTA Fe-NTA (alone) F. racemosa extract (D1) + DEN + Fe-NTA F. racemosa extract (D2) + DEN + Fe-NTA

20 20 20 20 20

17 15 13 15 15

0 0 10 3 6

– – 76.9 20.0 40.0

20

16

5

31.2

Dose of DEN = 200 mg/kg body weight, dose of Fe-NTA= 9 mg Fe/kg body weight. Doses (D1 and D2) represent 200 and 400 mg/kg body weight of F. racemosa extract.

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Effect of F. racemosa extract on induction of renal ODC activity Fig. 1 shows the effect of pretreatment of animals with F. racemosa extract on Fe-NTA mediated induction of renal ODC activity. Treatment with Fe-NTA caused 451% induction in the ODC activity as compared with saline-treated controls. The pretreatment of rats with F. racemosa extract at a dose of 200 mg/kg body weight caused inhibition in the elevation of ODC activity by 266% and at a dose of 400 mg/ kg body weight by 343% as compared with Fe-NTA treated control group. Effect of F. racemosa extract on renal DNA synthesis Fig. 2 shows the effect of prophylaxis of rats with F. racemosa extract on Fe-NTA mediated enhancement in the incorporation of [3H] thymidine into renal DNA. Fe-NTA alone treatment caused increase in the incorporation of [3H] thymidine into renal DNA by 180% as compared with saline-treated controls. F. racemosa extract pretreatment (200 and 400 mg/kg body weight) caused reduction in the enhancement of DNA synthesis by 136–164% as compared with Fe-NTA treated group. Effect of F. racemosa extract on histopathological alterations in the kidney Fig. 3 shows the histopathological alterations in the kidney tumor tissue that was initiated with DEN and promoted with Fe-NTA and its prophylaxis with F. racemosa extract. In the group treated with DEN

Fig. 1. Effect of pretreatment of rats with F. racemosa extract on Fe-NTA-induced enhancement of renal ornithine decarboxylase (ODC) activity. Each value represents mean F S.E. of six animals. **Significant ( P b 0.001) when compared with saline-treated control group. ##Significant ( P b 0.001) when compared with Fe-NTA treated control group. Doses (D1 and D2) represent 200 and 400 mg/kg body weight of F. racemosa extract.

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Fig. 2. Effect of pretreatment of rats with F. racemosa extract on Fe-NTA-induced enhancement of [3H] thymidine incorporation into renal DNA. Each value represents mean F S.E. of six animals. **Significant ( P b 0.001) when compared with saline-treated control group. ##Significant ( P b 0.001) when compared with Fe-NTA treated control group. Doses (D1 and D2) represent 200 and 400 mg/kg body weight of F. racemosa extract.

alone, no particular histological changes were observed. Fe-NTA alone treatment caused swelling and obliteration of spaces in Bowman’s capsule, swollen tubules and swollen endothelial, epithelial, and mesangial cells with enlargement of cuboidal cells and basal nuclei. Blood vessels show dilatation and congestion. DEN and Fe-NTA group showed adenocarcinomas with hyperchromatism and marked periglomerular and peritubular infiltrate of lymphocytes, monocytes, swollen glomeruli and tubules. F. racemosa extract at both dose levels of 200 mg/kg body weight and 400 mg/kg body weight alleviated renal pathological deterioration as evident from far less numbers of adenocarcinomas, normal morphology of glomerulus, basal membrane and central nuclei.

Discussion Plants, vegetables, herbs and spices used in folk and traditional medicine have been accepted currently as one of the main sources of cancer chemopreventive drug discovery and development (Aruoma, 2003). The phytochemical examination of plants that have a suitable history of use in folklore for the treatment of cancer has often resulted in the isolation of principles with antitumor activity (Owen et al., 2000). The chemical composition and medicinal uses of F. racemosa extract have been reported widely. It has hepatoprotective, immunostimulant, antibacterial, antiedemic, antihistaminic, antipyretic and analgesic activities (Mandal et al., 2000). This plant has been reported to contain tannins, kaempferol, rutin, arabinose, bergapten, psoralenes, flavonoids, ficusin, coumarin and phenolic glycosides (Baruah and Gohain, 1992). All these compounds act as strong antioxidant and anti-inflammatory agents. In the

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Fig. 3. Histopathological sections of kidneys showing the effect of pretreatment of rats with F. racemosa extract on DEN initiated and Fe-NTA promoted renal carcinogenesis. (a) Normal Saline treated (H&E: 150 ). (b) Only DEN treated (H&E: 150 ). (c) DEN + Fe-NTA treated (H&E: 150 ). (d) Only Fe-NTA treated (H&E: 150 ). (e) F. racemosa extract (D1) + DEN + Fe-NTA treated (H&E: 150 ). (f) F. racemosa extract (D1) + DEN + Fe-NTA treated (H&E: 150 ) Dose of DEN = 200 mg/kg body weight, dose of Fe-NTA= 9 mg Fe/kg body weight. Doses (D1 and D2) represent 200 and 400 mg/kg body weight of F. racemosa extract. AC: adenocarcinoma; DC: dense chromatin; DT: distal convoluted tubule; G: Glomerulus; HC: hyperchromatism; LIC: leucocytic infiltatory cells; NC: necrosis; NT: necrotic tissue; PM: pools of mucin; PT: proximal convoluted tubule; TC: tumor cells; TE: tubular epithelium.

present study, the methanolic extract of F. racemosa extract was found to possess lupeol, quercetin and h-sitosterol as its major active constituents. Lupeol has been reported to act as strong anti-inflammatory, antiarthritic and antimutagenic agent in various animal model systems (Guevara et al., 1996). We have recently reported that it suppresses skin tumor promotion in mice (Sultana et al., 2003). Quercetin has been reported to have antibacterial, antidiabetic, antiallergic and antimutagenic properties (Lamson and

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Brignall, 2000). It inhibits the production and release of histamine and has anti-inflammatory properties. It has also been shown to inhibit the growth of breast, colon, prostate and lung tumors cancer cells (Owen et al., 2000). h-Sitosterol has anti-inflammatory, anti-ulcer, anti-diabetic, anticancer and T-cell proliferative activities. It is used for the treatment of hyper-cholesterolemia, benign prostatic hypertrophy and rheumatoid arthritis (Pegel, 1997). Since the active constituents of F. racemosa extract have known protective effects in other studies, so the observed chemopreventive activity of F. racemosa extract in our study may be suggested due to the presence of these compounds. Dysregulated proliferation appears to be a hallmark of increased susceptibility to neoplasia. ODC is the first and rate-limiting enzyme in polyamine biosynthesis and is induced in response to a large number of tumor promoters. ODC activity and [3H] thymidine incorporation are widely used as biochemical markers to evaluate hyperproliferative and tumor promoting potential of an agent. An enhancement in both renal ODC activity and [3H] thymidine incorporation suggests a strong proliferative and tumor promoting potential of Fe-NTA in kidney. Various inhibitors of ODC induction and enhanced DNA synthesis have been shown to suppress tumor promotion (Perchellet and Perchellet, 1989). As observed in the present study, F. racemosa extract dose-dependently inhibited the induction of ODC activity and [3H] thymidine incorporation suggesting its antihyperproliferative potential. In addition, both doses of F. racemosa extract used in the present study showed its anticarcinogenic efficacy against DEN-initiated and Fe-NTA promoted renal carcinogeneis via inhibition of tumor formation. By scavenging free radicals and inhibiting ODC induction and DNA synthesis, F. racemosa extract may intercept the growth promoting and mitogenic functions of polyamines and arachidonic acid metabolites. F. racemosa extract ameliorated Fe-NTA-induced inhibition of the activities of antioxidant enzymes, viz., glutathione peroxidase, glutathione reductase, catalase, glucose-6-phosphate dehydrogenase and phase-II metabolising enzymes such as glutathione-S-transferase and quinone reductase. F. racemosa extract has established antioxidant properties that might have counteracted the oxidant effects of Fe-NTA. The present study shows induction of renal glutathione-S-transferase and quinone reductase activity following F. racemosa extract treatment. The major mechanism for protecting against the toxic and neoplastic effects of carcinogens is the modification of cellular detoxification enzymes. Many environmental carcinogens require metabolism to their fully carcinogenic forms. They are often metabolized to proximate carcinogens by Phase I enzymes, e.g., cytochrome P 450 which catalyze oxidative reactions. The oxidized metabolites of potentially carcinogenic xenobiotics are then detoxified by Phase II metabolizing enzymes into the forms that are relatively inert and more easily excreted (Talalay et al., 1995). Quinone reductase is a major enzyme of xenobiotic metabolism that carries out obligatory two-electron reductions and thereby protects cells against mutagenicity and carcinogenicity resulting from free radicals and toxic oxygen metabolites generated by the one-electron reductions catalyzed by cytochrome P 450 and other enzymes. It has been shown that most of the chemopreventive agents results in the induction of glutathione-S-transferase and quinone reductase activity and results in the degradation of electophilic metabolites. Induction of quinone reductase activity has been reported to have correlation with the prevention of cancer (De Flora and Ramel, 1988). There was also dose-dependent decrease in the Fe-NTA mediated susceptibility of renal microsomal membrane for iron-ascorbate induced lipid peroxidation through decreased production of free radicals as shown by ameliorated malondialdehyde levels. There was a decrease in the activities of xanthine oxidase, g-glutamyl transpeptidase, H2O2 and an increase in renal glutathione content. The decreased level of reduced glutathione following Fe-NTA administration due to decreased reduction of oxidized glutathione and increased activity of g-glutamyl transpeptidase cause accumulation of peroxides thereby

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leading to oxidative stress. As observed in the present study, increased g-glutamyl transpeptidase activity fixes degradation of GSH, which may lead to higher accumulation of cysteinyl glycine and cysteine. High levels of both cysteine and cysteinyl glycine have been suggested to enhance reduction of Fe-NTA to its ferrous complex, which in turn enhances peroxidative damage to membrane or tissue. F. racemosa extract pretreatment also reduced the elevated levels of blood urea nitrogen and serum creatinine that are marker parameters of kidney toxicity. The exact mechanism of the action of F. racemosa extract has not been fully elucidated. However, its chemopreventive effect is suggested due to the presence of its active constituents, viz., lupeol, quercetin and h-sitosterol. In the present study, it is concluded that the mechanism of action of F. racemosa extract is through (1) induction of various antioxidant and phase II enzymes, (2) scavenging reactive oxygen species, (3) sharp reduction in the levels of tumor promoter markers and (4) decrease in the percentage incidence of tumors in two-stage renal carcinogenesis. Thus, our data suggest that F. racemosa extract is a potent chemopreventive agent and inhibits Fe-NTA-induced renal carcinogenesis and oxidative damage response in Wistar rats.

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