Effect of Luteolin on Nickel Chloride–Induced Renal Hyperproliferation and Biotransformation Parameters in Wistar Rats

Abstract

Nickel, a major environmental pollutant, is a known potent nephrotoxic agent. In this communication, we report the chemopreventive effect of luteolin on nickel chloride (NiCl₂)-induced renal oxidative stress, toxicity, and cell proliferation response in male Wistar rats. NiCl₂ (250 µmol/kg b.wt./2 mL) enhances reduced renal glutathione content (GSH), glutathione S.-transferase (GST), glutathione reductase (GR), lipid peroxidation (LPO), H₂O₂ generation, blood urea nitrogen (BUN), and serum creatinine with a concomitant decrease in the activity of glutathione peroxidase (GPx) (p < 0.001). NiCl₂ administration also dose-dependently induced the renal ornithine decarboxylase (ODC) activity several-fold compared with the activity in saline-treated rats. Similarly, renal DNA synthesis, which is measured as [³H]thymidine incorporation in DNA, is also increased after NiCl₂ treatment. Prophylactic treatment of rats with luteolin (10 and 20 µmol/kg b.wt.) daily for 1 week resulted in the diminution of NiCl₂-mediated damage as evident from the downregulation of glutathione content, GST, GR, LPO, H₂O₂ generation, BUN, serum creatinine, DNA synthesis (p < 0.001), and ODC activity (p < 0.01) with concomitant restoration of GPx activity. Thus, the current investigation suggests that luteolin can be used as a chemopreventive agent for cancer prevention as evident from the study where it blocks or suppresses the events associated with chemical carcinogenesis.

Keywords:

• Cell proliferation response
• chemoprevention
• luteolin
• nickel chloride
• oxidative stress

Introduction

Reactive oxygen species (ROS) generated during various metabolic and biochemical reactions have multifarious effects that include oxidative damage to DNA leading to various life-threatening human diseases such as atherosclerosis, cataract, Parkinson disease, cancer, and aging, which have free-radical reactions as an underlying mechanism of injury (Sun, 1990). Considerable experimental evidence supports the view that ROS play a key role in the pathophysiologic processes of renal diseases. The abundance of polyunsaturated fatty acids makes the kidney particularly vulnerable to ROS attack (Kubo et al., 1997). However, the generation of ROS in normal cells is tightly regulated by biological antioxidants and by antioxidant enzymes (Martindale & Holbrook, 2002; Klein & Ackerman, 2003).

Nickel, a useful metal found in dry batteries, cigarettes, and food, is used in the production of stainless steel and in galvanization. It can be released into the atmosphere during mining, smelting, and mining operations (Venugopal & Luckey, 1978). Nickel and its compounds have been reported to be potent carcinogenic and/or toxic agents in human beings and experimental animals (Kasprzak et al., 2003). Morphologic changes in the kidney glomerulus and proximal tubules have been observed in association with proteinuria, amino-aciduria, and reduced urea clearance (Berndt, 1993). Lipid peroxidation (LPO) and protein carbonyl formation have also been documented (Patierno & Costa, 1987; Misra et al., 1990). It has been found that administration of nickel results in the accumulation of iron, which catalyzes the decomposition of H₂O₂ and lipid hydroperoxide via Haber-Weiss and Fenton's reaction. In the presence of peroxides, biomolecules can chelate with nickel and alter its reduction potential, thus facilitating generation of hydroxyl radicals, which may have further relevance to the carcinogenic potential of nickel (Ames et al., 1982). The onset of toxic manifestation of NiCl₂ is also due to glutathione, which acts as a bioreductant and instead of scavenging free radicals indirectly helps in its generation by reducing ferric ion to ferrous ion (Athar et al., 1987).

The susceptibility of an organism to oxidative stress depends on its antioxidant defense status. Oxidative lesions result from a disturbance of sophisticated oxidant-antioxidant equilibrium and occur when oxidants overwhelm the endogenous antioxidant mechanism. Under these circumstances, administration of exogenous antioxidant may be beneficial. From this point of view, the current study was carried out to study the antioxidant potential of luteolin.

Luteolin, a 3′,4′,5,7-tetrahydroxyflavone (Fig. 1), is a polyphenolic compound available in foods of plant origin that belongs to the flavones subclass of flavonoids, usually occurring as glycosylated forms in celery, green pepper, perilla leaf, and camomile tea (Shimoi et al., 2000). Luteolin can be orally or topically administered without harm and has antimutagenic activity (Huang, 1983), although some flavonoids are mutagenic (MacGregor, 1985). It inhibits the growth of malignant tumor cells by inducing apoptosis (Ko et al., 2002). Luteolin also acts as a potent inhibitor of human mast cell activation through inhibition of protein kinase C activation and Ca²⁺ influx (Kimata et al., 2000). It also inhibits the effect of tyrosine kinase, an enzyme involved in tumor cell proliferation, and therefore may have potential as a dietary anticarcinogenic agent (Spencer, 1999). Luteolin is also reported to have anti-inflammatory properties and mediates its action by inhibiting the nitric oxide production (Kim et al., 1999). It has its effect on various tumors such as human leukemia cell line and pancreatic tumor cells (Lee et al., 2002). It exhibits a wide spectrum of pharmacological properties, but little is known about its biochemical targets.

Figure 1 Chemical structure of luteolin.

Because luteolin has been shown to inhibit various diseases, mainly those that act through the generation of reactive oxygen species, it can be hypothesized that it may protect against nickel chloride–mediated renal oxidative stress, toxicity, and cell proliferation response in Wistar rats.

Materials and Methods

Chemicals

Luteolin, reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase (GR), bovine serum albumin (BSA), 1,2-dithio-bis.-nitrobenzoic acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), nickel chloride hexahydrate (NiCl₂ · 6H₂O) reduced nicotinamide adenine dinucleotide phosphate (NADPH), NiCl₂, Tween 20, and thiobarbituric acid (TBA) were obtained from Sigma Chemicals Co. (St. Louis, MO, USA). Diacetyl monoxime, urea, picric acid, sodium tungstate, sodium hydroxide, trichloroacetic acid, and perchloric acid (PCA) were purchased from Central Drug House (New Delhi, India). dL[¹⁴C]Ornithine (specific activity 56 mCi/mmol) and [³H]thymidine (specific activity 82 mCi/mmol) were purchased from Amersham Corporation (Little Chalfort, UK). All other chemicals were of the highest purity and commercially available.

Animals

Four to 6 week-old male Wistar rats (130–150 g) were obtained from the 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±2°C with a 12-h light/dark cycle. They were allowed to acclimatize for 1 week before the experiments and were given free access to standard laboratory feed (Hindustan Lever Ltd., Bombay, India) and water ad libitum.. Animals are given enough food to permit normal growth and maintenance of age-appropriate body weight. The study was approved by the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA; registration number and date of registration: 173/CPCSEA, 28 January 2000). CPCSEA guidelines were followed for animal handling and treatment.

Treatment regimen

To study the effect of pretreatment of animals with luteolin on NiCl₂-induced renal oxidative stress and ornithine decarboxylase activity (ODC) induction, 30 male Wistar rats were randomly allocated to five groups with six rats in each. The animals of group I served as control and received normal saline (0.85% NaCl). The animals of group II received only a single subcutaneous injection of NiCl₂ at a dose level of 250 µmol/kg body weight per 2 mL. Group III received pretreatment with luteolin by gavage once daily for 7 days at a dose of 10 µmol/kg body weight. Groups IV and V received pretreatment with luteolin extract once daily for 7 consecutive days at a dose level of 20 µmol/kg body weight. One hour after the last treatment with luteolin, the animals of groups II, III, and IV received a single subcutaneous injection of NiCl₂ (250 µmol/kg body weight per 2 mL). All the animals were sacrificed by decapitation 16 h after last treatment. Kidneys were quickly removed and washed with ice-cold saline. Immediately before sacrifice, blood was collected from the retro-orbital sinus for the estimation of serum creatinine and blood urea nitrogen.

To study the effect of pretreatment with luteolin on NiCl₂-mediated [³H]thymidine incorporation into renal DNA, the grouping of animals and schedules for prophylaxis were the same as described above. One hour after the last treatment with luteolin, the animals of groups II, III, and IV received only a single subcutaneous injection of NiCl₂ at a dose level of 250 µmol/kg body weight per 2 mL. Eighteen hours after the treatment with NiCl₂, the rats were given [³H]thymidine (30 µCi/animal) by intraperitoneal injection. Two hours later, they were sacrificed by cervical dislocation and their kidneys were quickly removed.

Tissue preparation

After the desired time, control and treated animals were sacrificed by cervical dislocation. For biochemical studies, a known amount of tissue was minced and homogenized in a polytron homogenizer (Kinematica A.G. Littau, Switzerland) and subjected to subcellular fractionation to obtain post mitochondrial supernatant (PMS) and microsomes for biochemical estimations.

Biochemical determinations

Estimation of reduced glutathione

GSH was determined by the method of Jollow et al. (1974). A 1.0 mL PMS fraction (10%) was mixed with 1.0 mL of sulfosalicylic acid (4%). The samples were incubated at 4°C for at least 1 h and then centrifuged at 1200 × g for 15 min at 4°C. The reaction mixture contained 0.4 mL of the filtered sample, 2.2 mL phosphate buffer (0.1 M, pH 7.4), and 0.4 mL DTNB (4 mg/mL) 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, Pittsford, New York). The GSH concentration was calculated as nmol GSH/g tissue.

Assay for glutathione S-transferase activity

Glutathione S.-transferase (GST) activity was assayed by the method of Habig et al. (1974). The reaction mixture consisted of 2.5 mL phosphate buffer (0.1 M, pH 6.5), 0.2 mL GSH (1 mM), 0.2 mL CDNB (1 mM), and 0.1 mL of the cytosolic fraction (10%) in a total volume of 3.0 mL. The changes in absorbance were recorded at 340 nm, and enzymatic activity was calculated as nmol CDNB conjugate formed min⁻¹mg protein⁻¹ using a molar extinction coefficient of 9.6 × 10³ M⁻¹ cm⁻¹.

Assay for glutathione reductase activity

Glutathione reductase (GR) activity was assayed by the method of Carlberg and Mannervick (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 mL oxidized glutathione (1 mM), 0.1 mL NADPH (0.1 mM), and 0.1 mL PMS (10%) in a total volume of 2.0 mL. Enzyme activity was quantitated at 25°C by measuring the disappearance of NADPH at 340 nm and was calculated as nmol NADPH oxidized min⁻¹mg protein⁻¹ using a molar extinction coefficient of 6.22 × 10³ M⁻¹ cm⁻¹.

Assay for lipid peroxidation

Estimation of LPO was done following the method of Wright et al. (1981). 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), and 20 µL ferric chloride (100 mM). The reaction mixture was incubated at 37°C in a shaking water bath for 1 h. The reaction was stopped by the addition of 1 mL of trichloroacetic acid (TCA; 10%). Following addition of 1.0 mL TBA (0.67%), all the tubes were placed in a boiling water bath for a period of 20 min. The tubes were shifted to ice bath and then centrifuged at 2500 × g for 10 min. The amount of malondialdehyde (MDA) formed in each of the samples was assessed by measuring the optical density of the supernatant at 535 nm. The results were expressed as nmol MDA formed h⁻¹g tissue⁻¹ at 37°C by using a molar extinction coefficient of 1.56 × 10⁵ M⁻¹cm⁻¹.

Estimation of blood urea nitrogen

Estimation of blood urea nitrogen (BUN) was done by the diacetyl monoxime method of Kanter (1975). Protein free filtrate was prepared. To 0.5 mL of protein-free filtrate was added 3.5 mL of distilled water, 0.8 mL diacetylmonoxime (2%), and 3.2 mL sulfuric 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 was added 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 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). Microsomes (2.0 mL) were 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 were incubated at 37°C 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 h⁻¹ tissue⁻¹ based on the standard curve of H₂O₂-oxidized phenol red.

Assay for glutathione peroxidase activity

Glutathione peroxidase (GPx) activity was measured by the method of Mohandas et al. (1984). The reaction mixture consisted of 1.44 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), 0.05 mL reduced glutathione (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°C. Enzyme activity was calculated as nmol NADPH oxidized min⁻¹mg protein⁻¹ using a molar extinction coefficient of 6.22 × 10³ M⁻¹ cm⁻¹.

Ornithine decarboxylase activity

Ornithine decarboxylase (ODC) activity was determined using 0.4 mL renal 105,000 × g. supernatant fraction per assay tube by measuring release of ¹⁴CO₂ from DL[¹⁴C]ornithine by the method of O'Brien et al. (1975). The kidney was homogenized in Tris-HCI buffer (pH 7.5, 50 mM) containing EDTA (0.4 mM), pyridoxal phosphate (0.32 mM), phenyl methyl sulfonyl fluoride (PMSF) (0.1 mM), 2-mercaptoethanol (1.0 mM), dithiothreitol (4.0 mM), and Tween 80 (0.1%) at 4°C using a homogenizer (Kinematica AGPT 3000). In brief, the reaction mixture contained 400 µL enzymes and 0.095 mL cofactor mixture containing pyridoxal phosphate (0.32 mM), EDTA (0.4 mM), dithiothreitol (4.0 mM), ornithine (0.4 mM), Brij 35 (0.02%), and DL-[¹⁴C]ornithine (0.05 µCi) in total volume of 0.495 mL. After adding buffer and cofactor mixture to blank and other tubes, the tubes were closed immediately with a rubber stopper containing 0.2 mL ethanol amine and methoxyethanol mixture (2:1) in the central well and kept in water bath at 37°C. 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 the glass tubes and the incubation was continued for 1 h to ensure complete absorption of CO₂ · Finally, the central well was transferred to a vial containing 2.0 mL of ethanol and 10.0 mL of toluene based scintillation fluid. Radioactivity was counted in liquid scintillation counter (LKB Wallace-1410, Pharmacia Biotech, Finland). ODC activity was expressed as pmol CO₂ released hr⁻¹mg protein⁻¹.

Assay for DNA synthesis

The isolation of renal DNA and incorporation of [³H]thymidine in DNA was done by the method of Smart et al. (1986). Kidney was quickly removed, cleaned free of extraneous material, and a homogenate (10% w/v) was prepared in ice-cold water. The precipitate thus obtained was washed with cold TCA (5%) and incubated with cold perchloric acid (PCA; 10%) at 4°C overnight. After the incubation, it was centrifuged and the precipitate was washed with cold PCA (5%). The precipitate was dissolved in warm PCA (10%) followed by incubation in a boiling water bath for 30 min and filtered through Whatman no. 50. The filtrate was used for [³H]thymidine counting in a liquid scintillation counter (LKB Wallace-1410) by adding the scintillation fluid. The amount of DNA in the filtrate was estimated by the diphenylamine method of Giles and Myers (1965). The amount of [³H]thymidine incorporated was expressed as disintegration per minute (dpm) dpm/µg DNA.

Protein estimation

The protein concentration in all samples was determined by the method of Lowry et al. (1951). Peptide bonds form a complex with alkaline copper sulfate reagent, which gives a blue color with Folin's reagent. Briefly, 0.1 mL PMS was diluted to 1 mL water and protein precipitated with an equal volume of TCA (10%), kept overnight at 4°C, and centrifuged at 800 × g for 5 min. The supernatant was discarded. The pellet was dissolved in 5 mL of NaOH (1 N). Finally, 0.1 mL of aliquot was further diluted to 1 mL with water and then 2.5 mL of alkaline copper sulfate reagent containing sodium carbonate (2%), copper sulfate (1%), and sodium potassium tartarate (2%) was added. Ten minutes after addition of alkaline copper sulfate reagent to allow complex formation, 0.25 mL of Folin's reagent was added. After 30 min, blue color developed that was read at 660 nm. For a standard, bovine serum albumin (BSA; 0.1 mg/mL) was used.

Statistical analysis

Differences between groups were analyzed using analysis of variance (ANOVA) followed by Dunnet's multiple comparison test. All data points are presented as the treatment group±standard error of the mean.

Results

Treatment of rats with NiCl₂(250 µmol/kg body weight per 2 mL) causes overproduction of cellular oxidants and modulation of antioxidant defense system. As observed during the study, a single subcutaneous injection of NiCl₂ led to modulation of several parameters of oxidative stress relative to control animals receiving saline only. The dose-dependent effect of luteolin on nickel-mediated modulation of GSH content, GST, GR, and GPx is shown in Table 1. Nickel-alone treatment enhanced the level of GSH by 122%, GST and GR by 130% and 114%, respectively, with a concomitant decrease in GPx activity by 85% of the saline treated control. However, pretreatment of animals with luteolin at 10 and 20 µmol/kg body weight significantly downregulated GSH content, GST and GR activities with concomitant amelioration of GPx activity (p < 0.001) in a dose-dependent manner.

Table 1. Effect of pretreatment with luteolin on NiCl₂-mediated enhancement of renal glutathione (GSH) content and increase in the activities of glutathione S.-transferase (GST), glutathione reductase (GR), and glutathione peroxidase (GPx) in rats

Treatment groups	GSH(nmol GSH/g tissue)	GST(nmol CDNB conjugate formed/min per mg protein)	GR(nmol NADPH oxidized/min per mg protein)	GPx(nmol NADPH oxidized/min per mg protein)
Saline (control)	0.58±0.01	91.46±0.4	181.4±2.0	157.5±2.1
NiCl2
(250 µmol/kg body weight)	0.71±0.01^###	119.4±0.67^###	207.3±2.8^###	133.9±2.0^###
Luteolin
(10 µmol/kg body weight) + NiCl2 (250 µmol/kg body weight/2 mL)	0.66±0.01**	99.7±0.78***	197.2±0.6**	143.5±3.7**
Luteolin
(20 µmol/kg body weight) + NiCl2 (250 µmol/kg body weight/2 mL)	0.62±0.02***	95.7±0.56***	188.1±0.3***	148.4±2.1***
Only luteolin
(20 µmol/kg body weight)	0.59±0.01	91.8±0.37	180.2±0.7	153.4±3.5

Animals of groups II, III, and IV were sacrificed 16 h after NiCl₂ exposure. Each value represents mean±SE, n = 6.

^###p < 0.001 compared with the corresponding value for saline-treated control group.

**p < 0.01, ***p < 0.001 compared with the corresponding value for NiCl₂-treated group.

Table 2 shows the effect of prophylactic treatment of rats with luteolin on nickel-induced increase in the levels of blood urea nitrogen, serum creatinine, and H₂O₂. Nickel treatment leads to 278%, 191%, and 143% enhancement in the values of BUN, serum creatinine and H₂O₂, respectively, compared with saline-treated controls. However, prophylaxis with luteolin resulted in 103–159%, 42–82%, and 25–31% reduction in the values of BUN, serum creatinine, and H₂O₂ compared with NiCl₂-treated groups. Nickel treatment also enhances the susceptibility of microsomal membrane for iron ascorbate–induced LPO to 207%, which was downregulated by 41–80% at lower and higher doses of luteolin compared with the nickel-treated group.

Table 2. Effect of pretreatment with luteolin on NiCl₂-induced enhancement of blood urea nitrogen (BUN), serum creatinine, lipid peroxidation (LPO), and hydrogen peroxide in rats

Treatment groups	BUN(mg/100 mL)(IU/L)	Creatinine(mg/100 mL)(IU/L)	H2O2(nmol H2O2/g tissue)	LPO(nmol MDA formed h^−1g tissue^−1)
Saline (control)	24.1±0.2	1.61±0.02	677.9±6.0	10.3±0.12
NiCl2 (250 µmol/kg body weight/2 mL)	67.2±2.4^###	3.09±0.05^###	974.7±5.1^###	21.4±0.85^###
Luteolin
(10 µmol/kg body weight) + NiCl2 (250 µmol/kg body weight/2 mL)	42.3±3.2***	2.4±0.12**	799.8±2.6***	17.1±0.25***
Luteolin
(20 µmol/kg body weight) + NiCl2 (250 µmol/kg body weight/2 mL)	28.7±0.1***	1.76±0.02***	762.3±3.5***	13.1±0.1***
Only luteolin
(20 µmol/kg body weight)	24.2±0.1	1.86±0.02	759.1±5.1	10.9±0.1

Each value represents mean±SE, n = 6.

^###p < 0.001 compared with the corresponding value for saline-treated control group.

**p < 0.01, ***p < 0.001 compared with the corresponding value for NiCl₂-treated group.

Treatment of animals with nickel alone induced the activity of ornithine decarboxylase (ODC) by six-fold compared with the saline-treated control group. This induction in the activity of ODC was inhibited dose-dependently by pretreatment with luteolin ranging from 286% to 376% for lower and higher doses, respectively, as shown in Figure 2.

Figure 2 Effect of pretreatment of rats with luteolin on NiCl₂-induced enhancement of renal ornithine decarboxylase (ODC) activity. Each value represents mean±SE of six animals. ^###Significant (p < 0.001) when treated with saline-treated control group. ***Significant (p < 0.001) when compared with NiCl₂-treated group. Doses (D1 and D2) represent 10 and 20 µmol/kg body of luteolin.

Figure 3 shows that treatment of NiCl₂ alone caused a significant (p < 0.001) increase in the rate of DNA synthesis by 9.9-fold as marked by an enhanced incorporation of thymidine uptake in the DNA. However, the animals pretreated with luteolin showed significant (p < 0.001) inhibition in the enhancement of [³H]incorporation of DNA in a dose-dependent manner.

Figure 3 Effect of pretreatment of rats with luteolin on NiCl₂-induced enhancement of [³H]thymidine incorporation into renal DNA. Each value represents mean±SE of six animals. ^###Significant (p < 0.001) when treated with saline-treated control group. ***Significant (p < 0.001) when compared with NiCl₂-treated group. Doses (D1 and D2) represent 10 and 20 µmol/kg body of luteolin.

Discussion

The increasing evidence that free radical–mediated damage to membranes, other lipid-containing structures, DNA, and protein contributes to aging and chronic diseases, such as cancer and coronary heart disease (Wiseman et al., 1996; Diplock et al., 1998), has focused attention on natural free-radical scavengers such as polyphenols. Flavonoids are plant secondary metabolites, which are biologically active polyphenolic compounds widely distributed in plants (Crozier et al., 2000). Flavonoids can prevent oxidative injury in various ways; besides direct scavenging of free radicals, they can chelate metal ions and inhibit the activities of several enzymes including lipoxygenase, cyclooxygenase, xanthine oxidase, phospholipase A₂, and protein kinases (Ratty et al., 1988; Cushman et al., 1991; Laughton et al., 1991; Hoult et al., 1994; Cotelle et al., 1996).

The chemical structure of luteolin, a flavonoid, is shown in Fig. 1. The multifunctional effect of luteolin is intimately connected with its structure and hydroxyl as its functional group, which participates in electron delocalization and is, therefore, an important determinant for its antioxidative potential (Horvathova et al., 2005). In this study, we have observed a dose-dependent decrease in the NiCl₂-mediated oxidative stress in the kidney of luteolin-pretreated rats, which is manifested by a decrease in the susceptibility of microsomal membrane for LPO as well as H₂O₂ generation. The finding of this study is based on the examination of the enzymes involved in the metabolism of xenobiotics (including carcinogens) and drugs, as this is one of the reliable markers for evaluation of the chemopreventive potential of test material. Unlike other metals, nickel is an inducer of glutathione, which instead of scavenging free radicals, indirectly helps in its generation by acting like a bioreductant. Our results suggest that the administration of nickel leads to the decrease in the activities of a GSH-utilizing enzyme, namely GPx, and an increase in GR, resulting in the increased turnover of GSH. This elevation of cellular GSH might help in reducing the ferric iron to the ferrous form, thus recycling it for further participation in lipid peroxidation. The level of iron has also been found to be increased significantly after nickel administration with a concomitant decrease in the activity of GPx that may lead to the accumulation of various hydroperoxides within the cells (Fahl et al., 1984; Floyd et al., 1988). This hydroperoxide on interaction with ferrous iron leads to the formation of hydroxyl and various other peroxy radicals (Massie et al., 1972). Ferrous iron is generated as a result of reduction of protein-bound ferric iron by bioreductants such as reduced glutathione. Luteolin pretreatment dose-dependently reduced NiCl₂-induced susceptibility of microsomal membrane for iron ascorbate–induced lipid peroxidation through decreased production of free radical, as evident by ameliorated malondialdehyde levels. The prophylactic treatment of luteolin to nickel-treated rats not only resulted in the sequestration of nickel bound to the surface of cell membrane but also prevented its interaction with membrane component, thus restoring GPx activity and downregulating renal GSH content, GST and GR activity. The increased level of kidney marker enzymes, viz., BUN and serum creatinine, after nickel treatment reflects its interaction with the cell membrane, leading to altered cell membrane permeability and increased enzyme leakage. This was reduced significantly after luteolin pretreatment, thus showing its efficacy in enhancing kidney function. ODC activity and [³H]thymidine incorporation, which are widely used as biochemical markers to evaluate tumor-promoting potential of an agent, was inhibited dose-dependently by luteolin, suggesting its antiproliferative and antitumor potential. Most of the inhibitors of ODC induction and blockers/inhibitors of DNA synthesis tested to date also protect against tumor promotion (Katiyar et al., 1992; Wei et al., 1998).

Therefore, from the current study, we can conclude that the possible mechanism of chemopreventive effect of luteolin is through (1) modulation of glutathione content and its metabolizing enzymes, (2) scavenging reactive oxygen species, and (3) sharp reduction in the levels of tumor promoter markers. Thus, our data suggest that luteolin has strong inhibitory activity against oxidative damage and is capable of preventing the biochemical alterations associated with the tumor promotion stage induced by NiCl₂.

Acknowledgment

Dr. Sarwat Sultana is thankful to the Indian Council of Medical Research (ICMR), New Delhi, India, for providing funds to carry out this study.