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Clinica Chimica Acta 347 (2004) 189–197

Influence of naringin on ferric iron induced

oxidative damage in vitro

Ganesh Chandra Jagetiaa,*, Tiyyagura Koti Reddya,V.A. Venkateshaa, Rajendra Kedlayab

aDepartment of Radiobiology, Kasturba Medical College, Manipal 576104, IndiabDepartment of Ophthalmology, University of Missouri, Columbia, MO 65201, USA

Received 18 December 2003; received in revised form 16 April 2004; accepted 20 April 2004

Abstract

Background: Iron is essential for oxygen transport and a variety of cellular processes like respiration and DNA synthesis. It

may become toxic when not handled carefully by cellular proteins and shielded from surrounding media. Naringin treatment

may help to overcome the iron-induced toxic effects in vitro. Methods: HepG2 cells were treated with 0.5, 1, 2.5, and 5 mmol/

l naringin 1 h before exposure to 0.1, 0.25, 0.5, and 1 mmol/l ferric iron. The effect of iron or naringin or their combination

treatment was studied on cell survival, DNA double-strand break induction, DNA oxidation, lipid peroxidation, and various

antioxidants. Results: The exposure of cells to iron caused a dose-dependent decline in their clonogenic potential, while

naringin pretreatment resulted in a significant elevation in the cell survival. Exposure of cells to iron resulted in a time-

dependent elevation in DNA strand breaks and a peak level of DNA strand breaks was observed at 24 h, while naringin

pretreatment inhibited the DNA double-strand breaks accompanied by an early repair. Similarly, treatment of HepG2 cells with

iron caused increased DNA oxidation that showed reduction when cells were pretreated with naringin. The iron overload

caused a significant elevation in the lipid peroxidation accompanied by depletion in glutathione (GSH) concentration, while

naringin inhibited lipid peroxidation and arrested the iron-induced depletion in the GSH concentration. Iron treatment also

reduced various antioxidant enzymes like glutathione peroxidase (GSHPx), catalase, and superoxide dismutase (SOD).

Pretreatment of HepG2 cells with naringin resulted in an elevation in all the antioxidant enzymes. Conclusions: Enhanced

antioxidant status by naringin could compensate the oxidative stress and may facilitate an early recovery from iron-induced

genomic insult in vitro.

D 2004 Elsevier B.V. All rights reserved.

Keywords: HepG2 cells; Cell survival; DNA double-strand breaks; Lipid peroxidation; Glutathione; Glutathione peroxidase; Glutathione-S-

transferase; Catalase

0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.cccn.2004.04.022

* Corresponding author. Tel.: +91-820-2571201x22814; fax:

+91-820-2570062/2571927.

E-mail address: gc.jagetia@kmc.manipal.edu

(G. Chandra Jagetia).

1. Introduction

Free radical-induced oxidative stress is involved in

aging [1], heart and cardiovascular diseases [2], gas-

trointestinal tract disorders, diabetes, cataractogenesis,

degenerative retinal damage, autoimmune nephrotic

Fig. 1. Structure of naringin.

G.C. Jagetia et al. / Clinica Chimica Acta 347 (2004) 189–197190

syndromes, heavy metal nephrotoxicity, Parkinson’s

disease [3], Alzheimer’s disease [4], bronchopulmo-

nary dysphasia [5], and ischemia reflow states [6].

Small amounts of potentially toxic reactive oxygen

species (ROS) can be generated in eukaryotic cells by

normal metabolic activities [7,8]. These generated free

radicals are involved in lipid peroxidation, DNA

damage, and protein degradation. Transition metals

such as iron and copper aggravate the free radical-

induced oxidative damage and finally lead to a dan-

gerous oxidative chain reaction [7].

Eukaryotic cells are equipped with a variety of

primary and secondary defenses against lipid perox-

idation and other deleterious effects of oxidative stress

[9]. Potentially, lethal injury can occur if these

defenses are overwhelmed. Primary defenses are

mainly preventative, whereas secondary defenses

have a ‘‘back-up’’ protective role, which might typi-

cally involve excision/repair of any lesions that do

develop. Primary cytoprotection relies on the scav-

enging/inactivation of ROS or redox metal ions before

lipid peroxidation takes place. Enzymes involved in

primary cytoprotection include superoxide dismutase

(SOD), glutathione peroxidase (GSHPx), which scav-

enges superoxide anion radical and hydrogen perox-

ide, respectively, at relatively low concentrations, and

catalase, which scavenges H2O2 efficiently at rela-

tively high concentrations. In conditions like iron

overload, carcinogenesis, radiation exposure, diabe-

tes, and many other pathological conditions, the

antioxidant machinery is overburdened with excess

oxidative stress that forces the cells to undergo the

oxidative cellular damage. Such situations demand

exogenous antioxidants to reinforce the antioxidant

mechanism of cells. The natural products and certain

dietary ingredients may be helpful to relieve the

oxidative stress of the cells. It is essential to screen

the antioxidant properties of certain natural products

and/or dietary ingredients, which can share the burden

of intracellular antioxidant machinery. Therefore, any

drug which can reduce the iron-induced damage may

be of potential value in one or more stress-related

disorders.

Flavonoids occur ubiquitously in the plant kingdom

and are common components of the human diet [10].

Flavonoids have been shown to have structurally

dependent, highly specific effects on a variety of

enzymes and are able to interfere with numerous

cellular processes, including growth and differentiation

[11]. The diverse effects of flavonoids may relate to

their structural similarity to ATP and hence to their

ability to compete with ATP for binding to various

enzymatic sites [12]. Naringin, a glycoside, is a pre-

dominant flavanone found in grapefruits and related

citrus species. Like most flavonoids, naringin has metal

chelating, antioxidant, and free radical scavenging

properties [13–15] and has been reported to offer

protection against mutagenesis [16] and lipid perox-

idation [17]. The present study was undertaken to

investigate the mechanism of naringin-induced protec-

tion against the oxidative stress of iron in HepG2 cells

in vitro.

2. Materials and methods

2.1. Chemicals

Naringin (Fig. 1) and trichloroacetic acid (TCA)

were from Acros Organics, Belgium, while glutathi-

one (GSH), 2-thiobarbituric acid, 5,5-dithio-bis (2-

nitrobenzoic acid) [DTNB], diethylenetriamine penta-

acetic acid (DTPA), butylated hydroxytolune, cumene

hydroperoxide, 1-chloro-2, 4-dinitrobenzene, ethi-

dium bromide, 2,4-dinitrophenyl hydrazine, guanidine

hydrochloride, and tetraethoxypropane were from

Sigma, St. Louis, MO.

2.2. Experimental

2.2.1. Cell line and culture

HepG2 cells obtained from the National Centre for

Cell Sciences (Pune, India) were used throughout this

G.C. Jagetia et al. / Clinica Chimica Acta 347 (2004) 189–197 191

study. Cells were grown in Eagle’s minimum essen-

tial medium (MEM) supplemented with 10% fetal

calf serum, 1% glutamine, and 50 Ag/ml gentamycin

sulfate. Cells were routinely cultured in 25-cm2 flasks

with loosened caps and incubated with 5% CO2 in air

in a humidified atmosphere at 37 jC.

2.2.2. Iron loading of HepG2 cells

Iron loading of HepG2 cells using ferric citrate was

performed as described previously [18]. Ferric citrate

solution was freshly prepared for each experiment.

Cells were exposed to ferric citrate in media for 20 h.

After exposure to ferric citrate, cells were washed

twice with iron-free phosphate buffered saline (PBS)

and exposed to 50-Amol/l hydrogen peroxide in an

iron-free media for 30 min at 37 jC. After hydrogenperoxide exposure, cells were washed twice and har-

vested by trypsin EDTA treatment.

2.2.3. Preparation of drug

Naringin was freshly dissolved in PBS and filter

sterilized (0.22 Am, Millipore India, Bangalore, India)

immediately before use.

2.2.4. Experiment 1

PBS group: The cells of this group were treated

with 50 Al of sterile PBS for 1 h.

NIN group: The cells of this group were exposed

to 0.5, 1, 2.5, and 5 mmol/l naringin for 1 h.

PBS+Iron–H2O2 group: The cells were treated

with 50 Al of sterile PBS for 1 h and were then loaded

with 0, 0.1, 0.25, 0.5, and 1 mmol/l ferric iron.

NIN+Iron–H2O2 group: The cells were treated

with 0.5, 1, 2.5, and 5 mmol/l naringin for 1 h before

exposure to 0, 0.1, 0.25, 0.5, and 1 mmol/l ferric iron

as mentioned above.

2.2.4.1. Clonogenic assay. Clonogenic assay was

carried out to evaluate the most effective dose of iron

in inducing cell damage, where the HepG2 cells were

exposed to 0, 0.1, 0.25, 0.5, and 1 mmol/l of ferric

iron and the cell survival was determined by the

method of Puck and Marcus [19]. Briefly, 200–300

cells were plated onto several individual culture

dishes (Nunc, Rosklide, Denmark) containing 5-ml

drug-free medium in triplicate for each radiation dose

for each group. The cells were allowed to grow for 11

days. The resultant colonies were stained with 1%

crystal violet in methanol and clusters containing 50

or more cells were scored as a colony. The plating

efficiency of cells was determined and the surviving

fraction was fitted on to a linear quadratic model,

SF = exp� (aD+hD2).

2.2.5. Experiment 2

The grouping and treatment of iron or naringin

have been essentially similar to the experiments

carried out to study the DNA strand break repair

and the assessment of biochemical profiles, except

that the concentration of iron and naringin was kept at

1 mmol/l (because this concentration gave the best

effect).

2.2.5.1. Effect of iron and naringin on DNA strand

breaks and repair. DNA strand break and repair

were determined by the fluorometric analysis of

DNA unwinding (FADU) described by Birnbiom

and Jevcak [20]. Briefly, cells were exposed to nar-

ingin or iron or both. The DNA strand break and

repair were evaluated at 2, 4, 8, 12, 24, 48, and 72

h after iron treatment. The cells were dislodged by

trypsin EDTA treatment and incubated with phosphate

buffer containing myo-inositol, urea sodium dodecyl

sulfate, and sodium hydroxide. Cells were sonicated

for a few seconds followed by the addition of ethi-

dium bromide. The fluorescence was read at Eex 520and Eem 590 nm at room temperature.

2.2.5.2. Biochemical estimations. Cells from differ-

ent groups were homogenized in phosphate buffer

containing SDS. The total protein content was esti-

mated by the modified method of Lowry [21]. The

protein carbonyl contents were estimated by the

method of Palamanda and Kehrer [22]. Briefly, cell

homogenate was incubated with dinitrophenyl hydra-

zine at room temperature for 30 min. After addition

of 20% cold TCA, the mixture was centrifuged and

the pellet was mixed with guanidium hydrochloride.

The OD was read at Eex 280 and Eem 370 nm using

a UV-VIS spectrophotometer (Shimadzu, Tokyo,

Japan). DNA oxidation was estimated by the method

of Borkitt [23]. Briefly, cell homogenate was incu-

bated with phosphate buffer and ethidium bromide.

The fluorescence was measured at an excitation at

Eex 510 and Eem 590 nm. The thiobarbituric acid

Fig. 2. Alteration in the iron-induced decline in the survival of

HepG2 cells by various concentrations of naringin. n, Iron 1 mmol;

5, NIN 0.5 mmol; E, NIN 1 mmol; 1, NIN 2.5 mmol; ., NIN 5

mmol.

G.C. Jagetia et al. / Clinica Chimi192

reactive substances (TBARS) assay was performed

according to the standard protocol [24]. Briefly, the

cell homogenate was incubated with a mixture of

trichloroacetic acid (15%), thiobarbituric acid

(0.375%), and butylated hydroxytoluene (0.01%) in

0.25 mol/l HCl at 95 jC for 25 min. The reaction

mixture was allowed to cool to room temperature

and was centrifuged at 8000� g. The supernatant

was collected and the absorbance was recorded

against the blank using a UV-VIS spectrophotometer.

The lipid peroxidation has been expressed as thio-

barbituric acid reactive substances (TBARS) deter-

mined against a tetraethoxypropane standard curve.

The GSH content was measured by the method of

Moron et al [25]. Briefly, proteins were precipitated

by 25% TCA, centrifuged, and the supernatant was

collected. The supernatant was mixed with 0.2 mol/

l sodium phosphate buffer, pH 8.0, and 0.06 mmol/

l 5,5-dithio-bis (2-nitrobenzoic acid) [DTNB] and

incubated for 10 min at room temperature. The

absorbance of the samples was read against the blank

at 412 nm. The glutathione peroxidase was assayed

by the modified method of Paglia et al. [26]. Briefly,

the cell homogenate was mixed with stock solution

containing glutathione reductase, GSH, NADPH, and

incubated at 37 jC for 5 min, followed by the

addition of cumene hydroperoxide. The absorbance

was recorded against the blank at 340 nm. The

glutathione-S-transferase was assayed by the method

of Habig et al. [27]. Briefly, 0.1 mol/l potassium

phosphate buffer, 1 mmol/l EDTA, glutathione re-

ductase, 10 mmol GSH, 12 mmol/l tert-butyl-hydro-

peroxide, and the cell homogenate were incubated

for 10 min at 37 jC. The absorbance was read

against the blank at 340 nm. The catalase activity

was estimated by catalytic reduction of hydrogen

peroxide using the method of Abei [28]. Briefly,

cumene hydroperoxide was added to the cell homog-

enate and was incubated at 37 jC. The decomposi-

tion of hydrogen peroxide was monitored by

recording the absorbance against the blank at 240

nm. Total superoxide dismutase was determined by

the pyrogallol autooxidation method [29]. Briefly,

the cell homogenate was added to 62.5 mmol trisca-

codylic acid buffer, containing 1 mmol/l diethylene-

triaminepentaacetic acid (DTPA), followed by 4

mmol pyrogallol. The autooxidation of pyrogallol

was monitored against the blank at 420 nm.

2.2.6. Analysis of data

The statistical significance of the treatment was

determined using student’s t-test. Solo 4 statistical

package (BMDP Statistical Software, Los Angeles,

CA) was used for data analysis. All the data are

expressed as meanF standard error of the mean

(S.E.M).

ca Acta 347 (2004) 189–197

3. Result

The protection conferred by naringin on iron-

induced decline in the cell survival of HepG2 cells

and on DNA double-strand breaks of these cells are

shown in Figs. 2 and 3. In addition, the lipid perox-

idation, protein oxidation, DNA oxidation, resulting

from iron-induced oxidative stress in HepG2 cells, and

the effect produced by naringin are shown as meanFSEM in Tables 1 and 2.

The iron–H2O2-induced oxidative stress resulted

in a concentration-dependent decline in the cell

survival and a nadir was reported for 1 mmol/l (Fig.

2). Pretreatment of HepG2 cells with various con-

centrations of naringin before iron overload caused

a significant elevation in the cell survival when

compared with the iron treatment. However, the

greatest elevation in cell survival was observed for

1 mmol/l naringin; therefore, further studies were

Table 1

Alteration in the iron-induced oxidative damage by naringin in HePG2 cells

Treatment TBARS

(nmols/mg protein)

Carbonyls

(nmol/mg protein)

% of undamaged

DNA

GSH

(nmol/mg protein)

Control 3.56F 0.4 1.03F 0.20 96F 0.001 52.12F 4.2

Fe control 10.35F 2.13a 2.31F 0.32a 69F 0.010b 26.14F 1.89a

NIN 3.24F 1.19 0.91F 0.11 98F 0.004 50.01F 3.20

Fe +NIN 5.46F 2.74 1.56F 0.06 76F 0.009b 37.81F 2.99

n= 4; no symbol = not significant.a p< 0.01.b p< 0.001.

G.C. Jagetia et al. / Clinica Chimica Acta 347 (2004) 189–197 193

carried out using this concentration of naringin

(Fig. 2).

The DNA strand breaks induction has been

expressed as the percent remaining double-stranded

DNA (Fig. 3). Iron stress to HepG2 cells induced

DNA strand breaks in a time-dependent manner, and

a maximum number of DSBs were observed at 24 h.

Thereafter, DNA strand breaks showed reparation of

iron-induced damage that progressed steadily up to

72 h, the last time period evaluated, where the

number of DNA strand breaks was lesser when

compared with 24-h posttreatment. The naringin

treatment alone did not induce DNA strand breaks

(Fig. 3). The pattern of DNA strand breakage was

similar in the naringin-pretreated group except that

naringin treatment significantly reduced the iron-

induced DNA breaks in HepG2 cells and the repair

Fig. 3. Effect of naringin (NIN) on the iron-induced DNA double-

strand breaks in HepG2 cells at various posttreatment time periods.

was also higher when compared with the iron treat-

ment (Fig. 3).

The lipid peroxide levels were measured as

TBARS concentration and expressed as nmol/mg

protein (Table 1). Treatment of HepG2 cells with

ferric iron and H2O2 elevated lipid peroxidation

significantly when compared with the PBS treatment

group ( p < 0.001). The treatment of HepG2 cells with

1 mmol/l naringin significantly reduced the iron-

induced lipid peroxidation ( p < 0.005).

Protein oxidation levels were determined by mea-

suring the protein carbonyl contents and expressed as

nmol/mg protein (Table 1). Iron–H2O2 treatment in-

creased the concentration of protein carbonyls in

HepG2 cells when compared with the PBS treated

group ( p < 0.01). Treatment of HepG2 cells with 1

mmol/l naringin inhibited the iron-induced protein

oxidation. However, this reduction was statistically

nonsignificant.

The DNA oxidation was estimated as the percent-

age of undamaged double-stranded DNA (Table 1).

Treatment of HepG2 cells with 1 mmol/l iron–H2O2

elevated the DNA oxidation in HepG2 cells signifi-

cantly ( p < 0.001), while treatment of cells with 1

mmol/l naringin significantly inhibited the iron-in-

duced DNA oxidation ( p< 0.001).

The concentration of glutathione has been ex-

pressed as nmol/mg protein (Table 1). Treatment of

HepG2 cells with iron +H2O2 depleted GSH concen-

tration significantly ( p = 0.001) when compared with

the PBS-treated group. Pretreatment of HepG2 cells

with 1 mmol/l naringin significantly elevated the

cellular glutathione levels when compared with the

iron-treated group ( p < 0.01).

Iron overload significantly reduced the GSHPx

levels in HepG2 cells when compared with the control

Table 2

Effect of naringin on the iron-induced decline in the antioxidant enzymes in cultured HePG2 cells

Treatment GSHPx

(units/mg protein)

GST

(units/mg protein)

Catalase

(units/mg protein)

SOD

(units/mg protein)

Control 256.8F 6.81 7.23F 0.98 138.1F 3.24 5.38F 1.10

Fe control 172.5F 4.12a 5.32F 1.14 72.61F 4.11a 2.34F 1.71

NIN 260.2F 6.41 7.50F 0.54 152.3F 2.61b 7.11F 0.90b

Fe +NIN 170.2F 3.19a 5.9F 0.90 109F 3.59a 3.21F 0.98

n= 4; no symbol = not significant.a p< 0.001.b p< 0.05.

G.C. Jagetia et al. / Clinica Chimica Acta 347 (2004) 189–197194

group. Naringin itself did not alter the GSHPx levels

in HepG2 cells when compared with the control

group. The GSHPx levels remained unaltered in

HepG2 cells after treatment with naringin before iron

overload (Table 2).

Introduction of iron into HepG2 cells significantly

reduced the GST levels when compared with the PBS

treatment group. Naringin alone did not alter the GST

levels when compared with the PBS treatment group.

Treatment of HepG2 cells with 1 mmol/l naringin

before iron treatment marginally increased the cellular

GST levels when compared with the iron-treated

group (Table 2).

Treatment of HepG2 cells with naringin elevated

catalase levels significantly in the noniron-treated

group while iron overload significantly reduced the

catalase concentration in HepG2 cells when compared

with the PBS treatment group. Naringin (1 mmol/l)

treatment significantly increased the catalase activities

(Table 2).

Naringin itself increased the SOD activity in non-

iron-treated HepG2 cells significantly. Iron overload

significantly reduced the SOD activity in HepG2 cells

when compared with the control group; however,

treatment of HepG2 cells with naringin elevated the

cellular SOD levels when compared with the control

group (Table 2).

4. Discussion

Iron is known to induce free radicals through

Fenton reaction [30]. These free radicals may have

been responsible for a concentration-dependent de-

cline in the survival of HepG2 cells in the present

study [18]. Pretreatment of HepG2 cells with various

concentrations of naringin inhibited the iron-induced

decline in the survival of HepG2 cells significantly. In

order to understand the mechanism of the action of

iron and naringin on cell survival, further studies were

carried out on DNA strand break repair, where iron

has been found to increase the DNA strand breaks in a

time-dependent manner up to 24 h while naringin

treatment protected against iron-induced DNA strand

breaks. Free radicals induce oxidative DNA damage

that results in the functional or structural alterations in

the DNA leading finally to proliferative cell death.

Because of its polyanionic nature, DNA binds to

various metal ions and is therefore especially prone

to iron-dependent, site-specific oxidative damage.

Oxygen free radicals like singlet oxygen, hydrogen

peroxide, and hydroxyl radicals induce a variety of

lesions in DNA, including oxidized bases, abasic

sites, and DNA strand breaks. The alkaline unwinding

assay is closely related to the comet assay and can

estimate the induction and rejoining of DNA strand

breaks with precision [31]. The DNA strand breaks, if

left unrepaired, may ultimately cause mutagenesis and

carcinogenesis. This reduction in DNA strand breaks

by naringin may be due to its ability to scavenge free

radicals and also its antioxidant activity. Naringin has

been reported to inhibit H2O2-induced DNA damage

[32]. Naringin treatment has also been reported to

reduce the radiation-induced DNA damage in mice

bone marrow [33,34].

Lipid peroxidation has been used as an indirect

measure of oxidative stress. The endproducts of stable

aldehydes react with thiobarbituric acid (TBA) to

form thiobarbituric acid–malondialdehyde adduct

[35]. Byproducts of lipid peroxidation may cause

further damage to important biomolecules like pro-

teins and DNA [36]. The increase in DNA damage

may be due to the increased lipid peroxidation by iron

overload of HepG2 cells. Naringin significantly

G.C. Jagetia et al. / Clinica Chimica Acta 347 (2004) 189–197 195

inhibited ferric ion-induced lipid peroxidation in

HepG2 cells. Compounds that are able to scavenge

free radicals and/or chelate iron can protect the cells

from free radical-induced lipid peroxidation [37].

Naringin has been reported to inhibit lipid peroxida-

tion in the brain and kidney [38]. Other flavonoids

like rutin and quercetin have been reported to inhibit

iron-induced lipid peroxidation by chelating iron ions

[39].

We have also evaluated the oxidative damage in

proteins and DNA. Although it is generally far less

monitored than lipid peroxidation as a marker of

oxidative damage, oxidative damage to proteins and

DNA is also very critical [36]. Both may nonspecifi-

cally bind iron either in a ferrous or ferric form and

may cause site-specific damage. Radical-mediated

protein oxidation has been measured by the estimation

of a generic marker of protein oxidation, i.e., carbonyl

contents [22]. Ferric iron-induced free radicals may

have been responsible for the protein oxidation in

HepG2 cells. Protein oxidation is known to give rise

to alterations in both the backbone and side chains of

the molecule, leading to the denaturation and loss of

biological activities of various important proteins and

cell death. Naringin reduced protein oxidation in

HepG2 cells. This may be one of the reasons of the

observed inhibitory activity of naringin against the

oxidative stress induced by ferric iron. Naringin sig-

nificantly reduced the oxidative DNA damage induced

by ferric iron. This DNA oxidation assay is based on

the fact that a highly fluorescent complex is formed

between native DNA and the intercalating agent ethi-

dium bromide. The interaction of DNA with free

radicals causes disruption of intercalation of ethidium

bromide in the DNA that leads to the decline in the

fluorescence intensity of ethidium bromide DNA com-

plex. Thus, the measurement of fluorescence gives a

precise indication of DNA double-strand damage

caused by various agents [20].

The mechanism of inhibition of iron-induced DNA

damage by naringin was further evaluated by estimat-

ing antioxidant status. Glutathione is an abundant and

ubiquitous antioxidant, a tripeptide and essential bio-

factor synthesized in all living cells. It functions

mainly as an effective intracellular reductant [40]. It

protects cells from free radical-mediated damage

caused by drugs and ionizing radiation. It forms an

important substrate for GSHPx, GST, and several

other enzymes, which are involved in free radical

scavenging. The naringin inhibited the iron-induced

decline in glutathione in HepG2 cells. Caffeine has

been reported to protect the mitochondrial fraction

against the radiation-induced GSH decline in vitro

[41]. Certain proglutathione agents like alpha-lipoic

acid (LA) and N-acetyl cysteine (NAC) have been

found to possess a sparing effect on GSH levels and

protect cells from glutamate insult [42]. Treatment of

rats with a lignan-enriched extract of the fruit of

Schizandra chinensis enhanced the hepatic antioxi-

dant/detoxification system, as indicated by the in-

crease in the hepatic-reduced glutathione (GSH)

level as well as hepatic glutathione reductase and

glutathione-S-transferase activities [43]. The presence

of naringin would have taken the burden of free

radicals upon itself, thereby sparing the GSH deple-

tion. This may have helped the HepG2 cells to

overcome the iron-induced insult. The antioxidant

activity of naringin has been found to be similar to

that of GSH [32].

Naringin arrested the iron-induced decline in GST

and reduced the iron-induced damage. The GST acts

like a peroxidase and removes the stable peroxides

from the system, resulting in the reduction in the

peroxide-induced damage [44]. Superoxide and hy-

drogen peroxide are important byproducts in usual

cellular energy metabolism. As such, they are not

highly toxic, but uncompartmentalized excess iron

can initiate the formation of.OH radical and can

influence lipid peroxidation via Fenton/Haber–Weiss

reactions [40]. Cells are equipped with an impressive

repertoire of antioxidant enzymes, such as superoxide

dismutase, which hastens the dismutation of O2.� to

H2O2, and catalase and glutathione peroxidase, which

convert H2O2 to water [45]. SOD brings the first line

of defense against free radicals by dismutating toxic

superoxide into a less toxic hydrogen peroxide. SOD

works in conjugation with other H2O2 removing

enzymes. SOD is also required for the growth of

aerobes without excessive DNA damage in the pres-

ence of superoxide. Selenium containing GSHPx

decomposes H2O2 and other peroxides which initiate

free radical chain reaction. Catalase heme enzyme

brings the decomposition of high amounts of H2O2

and other peroxides. SOD, GSHPx, and catalase, in

concerted action, protect the oxidative attack of su-

peroxide and hydrogen peroxide in the cells. Naringin

G.C. Jagetia et al. / Clinica Chimica Acta 347 (2004) 189–197196

has elevated the cellular catalase and SOD levels

accompanied by an arrest of iron-induced depletion

of SOD, GSHPx, and catalase in HepG2 cells. Nar-

ingin has been reported to play an important role in

regulating antioxidative capacities by increasing the

SOD, GSHPx, and catalase activities by up-regulating

the gene expressions of SOD, catalase, and GSHPx

and protecting the plasma vitamin E [46,47]. It has

also been found to scavenge superoxide anions and

other free radicals [15,33]. An operation of similar

mechanism of action by naringin in the present study

can not be ruled out.

Plant-based flavonoids like naringin can inhibit the

adverse effects of ferric ion-induced oxidative stress

and may protect the cellular environments from free

radical damage by elevating the cellular antioxidant

enzyme system mechanism, allowing repair of the

damaged DNA and thus arresting the iron-induced

decline in the cell survival.

Acknowledgements

We thank Dr. Shivanada Nayak, Assoc. Prof,

Department of Biochemistry, M.B.C.R. Naidu and

Shival K. Rao, Department of Radiobiology, Kasturba

Medical College, Manipal, India, for their help in this

work. The financial assistance in the form of the

Senior Research Fellowship to Mr. Tiyyagura Koti

Reddy by the Indian Council of Medical Research

(ICMR), Government of India, New Delhi, to carry

out the above study is gratefully acknowledged.

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