In vitro curcumin modulates ferric nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H2O2)-induced...

In Vitro Curcumin Modulates FerricNitrilotriacetate (Fe-NTA) and HydrogenPeroxide (H2O2)-Induced Peroxidation ofMicrosomal Membrane Lipids and DNADamage

Mohammad Iqbal,n Yasumasa Okazaki, and Shigeru Okada

Department of Pathological Research, Faculty of Medicine, Okayama UniversityGraduate School of Medicine and Dentistry, Okayama, Japan

A number of investigations have implicated the involvement of free radicals invarious pathogenic process including initiation/promotion stages of carcinogenesisand antioxidants have been considered to be a protective agent for this reason. Aniron chelate, ferric nitrilotriacetate (Fe-NTA), is a potent nephrotoxic agent andinduces acute and subacute renal proximal tubular necrosis by catalyzing thedecomposition of hydrogen peroxide–derived production of hydroxyl radicals,which are known to cause lipid peroxidation and DNA damage. The latter isassociated with a high incidence of renal adenocarcinoma in rodents. Lipidperoxidation and DNA damage are the principal manifestation of Fe-NTA-induced toxicity, which could be mitigated by antioxidants. In this study, wetherefore investigated the effect of curcumin, a polyphenolic compound fromCurcuma longa for a possible protection against lipid peroxidation and DNAdamage induced by Fe-NTA and hydrogen peroxide in vitro. Incubation of renalmicrosomal membrane/and or calf thymus DNA with hydrogen peroxide (40mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipidperoxidation and DNA damage to about 2.2-and 5.6-fold, respectively, ascompared to saline treated control (Po0.001). Induction of renal microsomallipid peroxidation and DNA damage was modulated by curcumin dosedependently. In lipid peroxidation protection studies, curcumin treatment showeda dose-dependent strong inhibition (18–80% inhibition, Po0.05–0.001) of Fe-NTA and hydrogen peroxide–induced lipid peroxidation as measured by MDA

Contract grant sponsor: Japan Society for the Promotion of Science (JSPS); Contract grant number:

P 01131.

nCorrespondence to: Mohammad Iqbal, Department of Pathology, Faculty of Medicine, Okayama

University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-Cho, Okayama 700-8558, Japan.

E-mail: [email protected]

Published online in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/tcm.10070

Teratogenesis, Carcinogenesis, and Mutagenesis Supplement 1:151–160 (2003)

r 2003 Wiley-Liss, Inc.

formation in renal microsomes. Similarly, in DNA-sugar damage protectionstudies, curcumin treatment also showed a dose dependent inhibition (22–57%inhibition, Po0.05–0.001) of DNA-sugar damage. From these studies, it wasconcluded that curcumin modulates Fe-NTA and hydrogen peroxide-inducedperoxidation of microsomal membrane lipids and DNA damage. Curcuminmight, therefore, be a suitable candidate for the chemoprevention of Fe-NTA-associated cancer. Teratogenesis Carcinog. Mutagen. Suppl. 1:151–160, 2003.�c 2003 Wiley-Liss, Inc.

INTRODUCTION

Oxygen free radicals are formed in tissue cells by many endogenous andexogenous causes such as metabolism, chemicals, and ionizing radiation [1]. Oxygenfree radicals may attack lipids and DNA giving rise to a large number of damagedproducts [2]. Iron is known to be involved in the generation of reactive oxygenspecies (ROS) and in the formation of highly toxic hydroxyl radicals from otheractive oxygen species such as hydrogen peroxide [2–4]. The enhanced generation ofROS in vivo could be quite deleterious, since they are involved in mutagenesis,apoptosis, ageing, and carcinogenesis [4].

Awai et al. [5] originally developed an experimental model of iron overloadusing ferric iron chelated with nitrilotriacetate (NTA). Its iron chelate, ferricnitrilotriacetae (Fe-NTA), is a potent nephrotoxic agent that induces acute andsubacute renal proximal tubular necrosis, a consequence of Fenton-like reaction,that eventually leads to the high incidence of renal adenocarcinoma in rodents [6,7].Recently, a specific allelic loss of p16 (INK4A) tumor suppressor gene in rats treatedwith Fe-NTA has been shown [8]. We have previously shown that Fe-NTA is apotent renal and hepatic tumor promoter [9,10], and induces oxidative stress bydown-regulating hepatic and renal NAD(P)H:quinone oxidoreductase activity andproduces an increase in protein carbonyl contents [11]. We have also shown that Fe-NTA induces the production of clastogenic metabolite of arachidonic acid,prostaglandin F2a, in kidney, which helps in maintaining tissue hyperplasia [12],and toxicity and carcinogenicity of Fe-NTA depend on the accumulation of proteinadducts 4-hydroxy-2-nonenal (HNE), the most cytotoxic lipid peroxidation products[13,14]. In this renal carcinogenesis model, involvement of ROS in the tissue damageis strongly suggested by the sharp increase in lipid peroxidation products(thiobarbituric acid reactive substances, HNE, HNE-modified proteins, MDA-modified proteins) or oxidative DNA damage such as 8-oxoguanine or thymine-tyrosine cross-links after single ip administration of Fe-NTA was observed [3,14,15].From these studies, it is suggested that lipid peroxidation and DNA damage are theprincipal manifestation of Fe-NTA-induced renal toxicity.

Hence, there is a need to identify an antioxidant that could effectively inhibitFe-NTA-induced lipid peroxidation, and subsequently protect DNA damage.Therefore, inhibition of lipid peroxidation and DNA damage may be one of thestrategies in chemoprevention of Fe-NTA associated cancer. Taking oxidizingpotential as a determinant of toxicity, antioxidants such as vitamin E, probucol,nordihydroguaretic acid, lycopene, garlic oil, a component of spice, and propolis(artepillin C) could effectively lesson the degree of nephrotoxicity induced byFe-NTA [16–22]. Thus, studies on new antioxidants are important.

152 Iqbal et al.

Due to pharmacological safety, there has been increased interest in phyto-chemicals that may exhibit anticancer activity. Curcumin (diferuloylmethane), aphenolic compound and a major component of curcuma species, is pharmacologi-cally safe, widely used as a yellow coloring agent and spice in commonly ingestedfood, and possesses anti-inflammatory [23], antioxidant [24], and antitumorproperties [25–28]. Since it is capable of inhibiting various kinds of injuries andneoplasm, particularly those mediated through the generation of ROS, wespeculated that curcumin may inhibit Fe-NTA and hydrogen peroxide–inducedperoxidation of microsomal membrane lipids and DNA damage. In the presentstudy, we investigated the effect of curcumin on Fe-NTA and hydrogen peroxideinduced peroxidation of microsomal membrane lipids and DNA damage in vitro.Our data provide evidence that curcumin modulates Fe-NTA and hydrogenperoxide–induced peroxidation of microsomal membrane lipids and DNA damage.Curcumin might, therefore, be a suitable candidate for chemoprevention of Fe-NTA-associated cancer.

MATERIALS AND METHODS

Chemicals

Calf thymus deoxyribonucleic acid (sodium salts), thiobarbituric acid (TBA),nitrilotriacetic acid disodium salt (NTA), ethylene diamine-N, N-tetraacetic acid(EDTA), tris HCl, hydrogen peroxide, sodium bicarbonate, and curcumin werepurchased from Sigma Chemical Company, (St Louis, MO). All other chemicals/reagents were of the highest quality available from Wako Pure Chemical Industries(Osaka, Japan).

Preparation of DNA Solution

A solution of DNA 2.0 mg/ml was prepared by dissolving a known amount ofhighly polymerized sodium salt of calf thymus DNA in an appropriate volume of trisHCl buffer (1.0 mM), containing NaCl (0.01 M) and EDTA (0.2 mM) in salt-freecondition. The solution was stored below 41C for 48 h before use.

Preparation of Fe-NTA Solution

A solution of Fe-NTA was prepared by the method of Awai et al. [5]. Briefly,ferric nitrate and nitrilotriacetate, disodium salt was dissolved separately in doubledistilled water. The respective solutions were mixed to achieve a molar ratio of 1:3 ofFe-NTA (i.e., 200 mM FeNO3-600 mM NTA). The pH was adjusted to 7.4 withsodium bicarbonate with constant stirring. All solutions were prepared freshimmediately before use.

Preparation of Kidney Homogenate and Microsome

Male ddY mice (4–6 weeks old) weighing 20–30 g obtained from ShizuokaLaboratory Center, Japan, were used. They were fed pelleted chow diets and hadaccess to food and water. The animals were killed by cervical dislocation and kidneyswere removed perfused immediately with ice cold saline (0.85% sodium chloride)and homogenized in chilled phosphate buffer (0.1 M, pH 7.4) containing KCl

Curcumin Modulates Lipid Peroxidation and DNA Damage 153

(1.17%). The homogenate, postmitochondrial supernatant, and microsomal frac-tions from tissue homogenate (10% w/v) were prepared according to the establishedprocedure of Iqbal et al. [13].

Assay of Lipid Peroxidation

The assay for renal microsomal lipid peroxidation was done following themethod of Wright et al. [29], as described by Iqbal and colleagues [13,16,21]. Briefly,the reaction mixture in a total volume of 1.0 ml containing 0.25 ml renal microsome(10% w/v), 0.53 ml phosphate buffer (0.1 M, pH 7.4), and 0.01 ml hydrogen peroxide(40 mM). 0.01 ml to 0.1 ml of curcumin (final concentration 50 mg curcumin/mlacetone) was mixed and the reaction was started by the addition of 0.025 ml Fe-NTA(0.1 mM). The reaction mixture was incubated at 371C in a shaking water bath for aperiod of 60 min. The reaction was stopped by the addition of 1.0 ml oftrichloroacetic acid (10% w/v). Then, 1.0 ml thiobarbituric acid (0.67% w/v), whichwas prepared by dissolving 0.67 g of TBA in warm distilled water, was added. All thetubes were placed in a boiling water bath for a period of 20 min. At the end, thetubes were shifted to an ice bath and centrifuged at 12,000 rpm for 20 min at 41Cusing a Hitachi cold centrifuge model CR 15B. The amount of TBARS formed ineach of the samples was assessed by measuring the optical density of the supernatantat 535 nm using a spectrophotometer against a reagent blank. The results wereexpressed as nmol MDA formed/h/g tissue at 371C using a molar extinctioncoefficient of 1.56 � 105 M/cm reagent.

Assay of DNA Sugar Damage

The deoxyribose oxidation was assayed by the method of Halliwell andGutteridge [4], as described by Athar et al. [30]. The damage to deoxyribose sugarmoiety of DNA was assessed by determining the thiobarbituric acid reacting productformed on incubating DNA with Fe-NTA and hydrogen peroxide in the presence ofdifferent concentrations of curcumin. Briefly, the reaction mixture in a total volumeof 1.0 ml contained 0.25 ml calf thymus DNA (2.0 mg/ml), 0.53 ml phosphate buffer(0.1 M, pH 7.4), 0.01 ml hydrogen peroxide (40 mM). Different concentrations ofcurcumin ranging from 0.01 ml to 0.1 ml (50 mg curcumin/ml acetone) were mixedand the reaction was started by the addition of 0.025 ml Fe-NTA (0.1 mM). Thereaction mixture was incubated for 1.0 h at 371C in a water bath shaker. After theincubation was over, 1.0 ml TBA (0.67% w/v), which was prepared by dissolving0.67 g of TBA in warm distilled water, was added to the reaction mixture and then itwas kept in a boiling water bath for 20 min, cooled in an ice bath, and centrifuged at12,000 rpm for 20 min at 41 using a Hitachi cold centrifuge model CR 15B. The TBAreacting species generated formed an adduct of pink colour, which showed acharacteristic absorbance at 535 nm.

Statistical Analysis

The level of significance was ascertained by Dennett’s t-test. The significancewas set at Po0.05 and Po0.001. Data represent means 7 S.E. of three independentexperiments.

154 Iqbal et al.

RESULTS

In the initial studies, we assessed the dose-dependent effect of Fe-NTA in thepresence of hydrogen peroxide on renal microsomal lipid peroxidation as well as onDNA-sugar damage. As shown in Table I, hydrogen peroxide in the presence of Fe-NTA led to the induction of renal microsomal lipid peroxidation dose dependently.A lower dose of 5.0 mg/ml of Fe-NTA led to about a 2.0-fold induction while ahigher dose of 100.0 mg/ml of Fe-NTA led to the induction of about a 3.1-fold ofrenal microsomal lipid peroxidation compared to control group (Po0.05–0.001). Adose-dependent inhibitory effect of curcumin on renal microsomal lipid peroxidationinduced by Fe-NTA and hydrogen peroxide is shown in Table II. The presence ofvarious concentrations of curcumin (10–100 mg/ml) in the reaction mixture stronglyinhibited Fe-NTA and hydrogen peroxide–induced renal microsomal lipid perox-idation (Po0.05–0.001). Although a significant reduction in microsomal lipidperoxidation was observed at all the doses of curcumin studied, at the higher dose ofcurcumin 100 mg/ml the value of renal microsomal lipid peroxidation came close tothe value of the control group. At the lowest dose of curcumin (10 mg/ml), 18%inhibition was observed (Po0.001) whereas at the highest dose of curcumin (100 mg/ml), 80% inhibition was observed (Po0.001). The decrease in the enhancement ofrenal microsomal lipid peroxidation depends on the dose of curcumin used.

As shown in Table III, hydrogen peroxide in the presence of Fe-NTA inducesDNA-sugar damage dose dependently. A maximum of an 8.5-fold induction inDNA-sugar damage was observed at a dose of 100.0 mg/ml of Fe-NTA compared tothe control group, whereas at the lowest dose of Fe-NTA 5.0 mg/ml induction wasabout 1.8-fold (Po0.05-0.001). The inhibitory effect of curcumin on DNA-sugardamage induced by Fe-NTA and hydrogen peroxide is shown in Table IV. DNA-sugar damage was significantly strongly inhibited by the addition of differentconcentrations of curcumin (10 mg to 100 mg/ml in acetone) to the reaction mixture.Addition of 10, 25, 50, and 100 mg of curcumin inhibited the Fe-NTA and hydrogenperoxide–induced increase in DNA-sugar damage by 22, 30, 31, and 57%,respectively (Po0.05–0.001). The maximum inhibition of 57% of DNA-sugardamage was observed when the concentration of curcumin was 100 mg/ml(Po0.001). These results indicate that curcumin is an effective inhibitor ofFe-NTA and hydrogen peroxide–induced lipid peroxidation and DNA-sugardamage.

TABLE I. Fe-NTA and Hydrogen Peroxide–Induced Lipid Peroxidation in Renal Macrosomesw

S. no. Assay system n mol MDA/h/g tissue % of control

1. Microsome+H2O2 (control) 19.2370.01 100

2. Microsome+H2O2 Fe-NTA (5ml) 38.4670.07nn 200

3. Microsome+H2O2 Fe-NTA (10ml) 52.9270.09nn 275

4. Microsome+H2O2 Fe-NTA (25ml) 54.6170.01n 284



wEach value represents mean7S.E. of three independent experiments. Experimental conditions are

described in Materials and Methods.nPo0.05, nnPo0.001, Significantly different from the corresponding values for microsomes alone treated

control.


DISCUSSION

Lipid peroxidation in biological membranes is a free radical–mediated eventand is regulated by the availability of substrates in the form of polyunsaturated fatty

TABLE II. Inhibitory Effect of Curcumin on Fe-NTA and Hydrogen Peroxide–Induced LipidPeroxidation in Renal Macrosomesw

S. no. Assay system n mol MDA/h/g tissue % of control

1. Microsome+H2O2 (control) 19.9970.01 100


3. Microsome+H2O2 Fe-NTA (25ml)+Curcumin (10ml) 41.3870.02nnn 207

4. Microsome+H2O2 Fe-NTA (25ml) +Curcumin (25ml) 26.0770.01nn 130

5. Microsome+H2O2 Fe-NTA (25ml) +Curcumin (50ml) 25.9270.07 129

6. Microsome+H2O2 Fe-NTA (25ml) +Curcumin (100ml) 24.4670.01nnn 122


described in Materials and Methods. The curcumin was added at the concentrations indicated above

15 min before the initiation of lipid peroxidation with H2O2 and Fe-NTA.nPo0.001, Significantly different from the corresponding values for microsomes alone treated control.nnPo0.05, nnPo0.001, Significantly different from the corresponding values for microsomes treated with

H2O2+Fe-NTA.

TABLE III. Fe-NTA and Hydrogen Peroxide–Mediated DNA-Sugar Damagew

S. no. Assay system DNA-sugar damage (A 535) % of control

1. DNA+H2O2 (control) 0.1270.07 100

2. DNA+H2O2 Fe-NTA (5ml) 0.2370.01nn 182

3. DNA+H2O2 Fe-NTA (10ml) 0.3670.02nn 292

4. DNA+H2O2 Fe-NTA (25ml) 0.6670.01nn 528

5. DNA+H2O2 Fe-NTA (50ml) 1.070.08n 857

6. DNA+H2O2 Fe-NTA (100ml) 1.070.07n 857

wEach value represents mean7S.E. of three independent experiments. Treatment protocols are described

in Materials and Methods.nPo0.05, nnPo0.001, Significantly different from the corresponding values for DNA alone treated

control.

TABLE IV. Inhibitory Effect of Curcumin on Fe-NTA and Hydrogen Peroxide–Mediated DNA-Sugar Damagew

S. no. Assay system DNA-sugar damage (A 535) % of control

1. DNA+H2O2 (control) 0.1270.06 100

2. DNA+H2O2 Fe-NTA (25ml) 0.7170.04n 560

3. DNA+H2O2 Fe-NTA (25ml)+Curcumin (10ml) 0.5570.07nnn 438

4. DNA+H2O2 Fe-NTA (25ml) +Curcumin (25 ml) 0.5070.03nnn 395

5. DNA+H2O2 Fe-NTA (25ml) +Curcumin (50 ml) 0.4970.01nn 387

6. DNA+H2O2 Fe-NTA (25ml) +Curcumin (100 ml) 0.3070.03nnn 243


described in Materials and Methods. The curcumin was added at the concentrations indicated above 15

min before the initiation of DNA-sugar damage with H2O2 and Fe-NTA.nPo0.001, Significantly different from the corresponding values for DNA alone treated control.nnPo0.05, nnPo0.001, Significantly different from the corresponding values for DNA treated with

H2O2+Fe-NTA.

156 Iqbal et al.

acids, prooxidants that promote peroxidation, and antioxidant defenses such as p-tocopherol, reduced glutathione, b-carotene, and superoxide dismutase [31,32]. Lipidperoxidation is highly detrimental to cell membrane structure and function. Elevatedlevels of lipid peroxidation have been linked to injurious effect such as loss offluidity, inactivation of membrane enzymes and receptors, increased permeability toions and, eventually, rupture of cell membrane leading to the release of cellorganelles [31–33]. Peroxidation products can also result in damage to crucialbiomolecules, including DNA [34,4]. Thus, inhibition of lipid peroxidation andDNA damage may be one of the strategies in chemoprevention of a large number ofclinical disorders.

The results of the present study demonstrate that curcumin modulates Fe-NTAand hydrogen peroxide–induced renal microsomal lipid peroxidation and DNA-sugar damage in vitro. It has been suggested that ROS plays an important role in Fe-NTA-induced oxidative injury [3,9,10,13,14]. Fe-NTA-induced lipid peroxidation isdetrimental to the cell both at membrane and genetic levels [3,14]. Lipid peroxidationproducts, malonaldehyde and 4-hydroxy-2-nonenal, cross-link the membrane,damage the DNA, and are mutagenic leading to functional changes [3,14]. Adose-dependent inhibition in both renal microsomal lipid peroxidation as well as inDNA-sugar damage was observed when 10–100 mg/ml of curcumin was added to thereaction mixture; this suggest a protective effect and it could be due to theantioxidant properties of curcumin [24]. Since curcumin has anti-inflammatory [23],antioxidant [24], and antitumor properties in a number of animal model systems[25–28], it may be proposed that their efficacy may be attributed to their free radicalscavenging activity. Curcumin is a polyphenolic compound [35]. The antioxidantproperty of curcumin may therefore be due to the polyphenolic constituent. Theseresults are in agreement with the earlier reported inhibition of lipid peroxidation andDNA damage by curcumin in various models by several authors [35–37]. Recently,Okada et al. [38] showed that curcumin and especially tetrahydrocurcuminameliorate oxidative stress-induced renal injury in mice. Similar to this study, wealso observed the inhibition of microsomal lipid peroxidation and DNA damageinduced by Fe-NTA and hydrogen peroxide in vitro. The mechanism by whichcurcumin inhibits Fe-NTA and hydrogen peroxide–induced renal microsomal lipidperoxidation and DNA-sugar damage needs to be evaluated. It may be suggestedthat the antioxidant property of curcumin or the metal sequestering property due tothe presence of polyphenol, might be responsible, at least in part, for such an effect.

Curcumin has many biological effects. Curcumin was reported to possesscytotoxic [39], antibacterial activity [40], inhibit microsome-mediated mutagenicityof benzo (a) pyrene, 7,12-dimethylbenz (a) anthracene, capsaicin, chili extract, andcigarette smoke condensate [41,42]. Curcumin was shown to inhibit the growth ofcells in vitro and to increase the survival of animals with lymphomas [39], enhancethe rate of DNA repair in yeast [43], induce apoptosis [44], and strongly inhibitROS-generating enzymes such as cyclooxygenase/lipooxygenase pathways, xanthinedehydrogenase/oxidase, and inducible nitric oxide synthase, which are implicated inchemical carcinogenesis [23,26,45,46]. Curcumin is also a potent inhibitor of proteinkinase C, EGF-receptor tyrosine kinase, I kappa B kinase, and inhibits theexpression of c-fos, c-myc, and c-jun [26,47]. Several studies have demonstrated thattopical application of curcumin inhibits TPA-induced inflammation, DNA synthesis,ornithine decarboxylase activity, hyperplasia, and tumor promotion in the epidermis


of CD-1 mice [25,45]. Toxicity studies with turmeric or curcumin in animalsindicated no histapathological changes when these substances were fed to rats, dogs,guinea pigs, or monkeys (0.5 to 2.0 g/kg) for 8–60 weeks [48]. In addition, studieswith turmeric and curcumin in rats for three generations did not show anyteratogenic or carcinogenic effects [49].

In conclusion, the results presented in this study suggest that curcumin may be auseful modulator of Fe-NTA and hydrogen peroxide–induced oxidative injury oflipids and DNA. Although the precise mechanism by which curcumin inhibitsFe-NTA and hydrogen peroxide–induced oxidative injury of lipids and DNAremains to be elucidated, it is likely that the antioxidant action of curcumin, at leastin part, may be related to the modulation of oxidative injury of lipids and DNA.Because curcumin is non-toxic and utilized as a coloring agent and spice in manyfoods, it could also prove useful in diminishing oxidative injuries in humans.Curcumin might, therefore, be a suitable candidate for the chemoprevention of Fe-NTA-associated cancer. Further studies are in progress to evaluate the in vivopotential of curcumin in animals models of Fe-NTA-induced oxidative injury.

ACKNOWLEDGMENTS

The authors are thankful to the Japan Society for the Promotion of Science(JSPS) for providing grant–in-aid for scientific research to support these studies.M.I. is also grateful to the JSPS for providing a postdoctoral fellowship for foreignresearcher, February 2001 (P 01131).

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In vitro curcumin modulates ferric nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H2O2)-induced...

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