Research Articles

Protection From Radiation-InducedMitochondrialandGenomic DNADamage by an Extract of

Hippophae rhamnoides

Sandeep Kumar Shukla,1 Pankaj Chaudhary,1 Indracanti Prem Kumar,1*Namita Samanta,1 Farhat Afrin,2Manju Lata Gupta,1

Upendra Kumar Sharma,3 Arun Kumar Sinha,3

Yogendra Kumar Sharma,4 and Rakesh Kumar Sharma1

1Division of Radioimaging, Bioinformatics, and Radiation Biology,Institute of Nuclear Medicine and Allied Sciences, Delhi 110054, India

2Department of Biotechnology, Jamia Hamdard University, New Delhi, India3Natural Plant Products Division, Institute of Himalayan Bioresource Technology,

Palampur, Himachal Pradesh 176061, India4Defence Institute of Physiology and Allied Sciences, Delhi 110054, India

Hippophae rhamnoides or seabuckthorn is usedextensively in Indian and Tibetan traditional medi-cine for the treatment of circulatory disorders, ische-mic heart disease, hepatic injury, and neoplasia. Inthe present study, we have evaluated the radiopro-tective potential of REC-1001, a fraction isolatedfrom the berries of H. rhamnoides. Chemical analy-sis of the extract indicated that REC-1001 was*68% by weight polyphenols, and containedkaempferol, isorhamnetin, and quercetin. The effectof REC-1001 on modulating radiation-induced DNAdamage was determined in murine thymocytes bymeasuring nonspecific nuclear DNA damage at thewhole genome level using the alkaline halo assayand by measuring sequence/gene-specific DNAdamage both in nuclear DNA (b-globin gene) and inmitochondrial DNA using a quantitative polymerasechain reaction. Treatment with 10 Gy resulted in asignificant amount of DNA damage in the haloassay and reductions in the amplification of both the

b-globin gene and mitochondrial DNA. REC-1001dose-dependently reduced the amount of damagedetected in each assay, with the maximum protectiveeffects observed at the highest REC-1001 dose eval-uated (250 lg/ml). Studies measuring the nicking ofnaked plasmid DNA further established the radio-protective effect of REC-1001. To elucidate possiblemechanisms of action, the antioxidant propertiesand the free-radical scavenging activities of REC-1001 were evaluated. REC-1001 dose-dependentlyscavenged radiation-induced hydroxyl radicals,chemically-generated superoxide anions, stabilizedDPPH radicals, and reduced Fe3þ to Fe2þ. The resultsof the study indicate that the REC-1001 extract of H.rhamnoides protects mitochondrial and genomicDNA from radiation-induced damage. The polyphe-nols/flavonoids present in the extract might be respon-sible for the free radical scavenging and DNA protec-tion afforded by REC-1001. Environ. Mol. Mutagen.47:647–656, 2006. VVC 2006Wiley-Liss, Inc.

Key words: radioprotection; Hippophae rhamnoides; DNA damage; QPCR; flavonoid

INTRODUCTION

Radiation damages cells mainly through the generation

of reactive oxygen species (ROS), and cellular DNA is

believed to be the crucial target of ROS [Hall, 2000].

ROS are associated with the induction of DNA single-

strand and double-strand breaks, base modification, DNA

adducts, and abasic sites, all of which potentially can

cause mutations and can ultimately lead to cell death

[Ward, 1995; Pouget and Mather, 2001]. Oxidative DNA

damage is most evident in the mitochondrial genome,

where it has greater persistence than in nuclear DNA

(nDNA) due to fewer mitochondrial DNA (mtDNA)

repair enzymes, lack of protective histone proteins, and

the close proximity to the electron transport system, a

strong source of ROS [Yakes and Van Houten, 1997].

*Correspondence to: Dr. I. Prem Kumar, Institute of Nuclear Medicine

and Allied Sciences, Brig S. K. Mazumdar Marg, Delhi, 110054, India.

E-mail: prem_indra@yahoo.co.in

Contract grant sponsor: Defence Research and Development Organiza-

tion’s ‘‘Herbs for Human Health’’ (Charak) Program.

Received 12 April 2006; provisionally accepted 9 June 2006; and in final

form 6 July 2006

DOI 10.1002/em.20251

Published online 31 August 2006 in Wiley InterScience (www.interscience.

wiley.com).

VVC 2006Wiley-Liss, Inc.

Environmental andMolecular Mutagenesis 47:647^656 (2006)

Oxidative mtDNA damage also has been linked to the

onset of specific human diseases, such as neuronal degen-

eration and cardiovascular disease, as well as to ageing

[Wei, 1998; Wei and Lee, 2002].

A number of synthetic and natural agents have radioprotec-

tive activity [Maisin, 1998; Weiss and Landaur, 2000]. For

instance, the radioprotective effects of a group of sulfur-con-

taining compounds (cystemaine, cysteine, WR-2721) have

been studied extensively, and operate by protecting DNA from

radiation-induced damage [Brown, 1967; Bump and Malakkar,

1998; Nair et al., 2000]. Plants and plant extracts are used in

traditional Ayurvedic and Chinese medicine to protect humans

from a number of ailments that now are known to be mediated

through oxidative stress. Both plant-derived extracts and phy-

tochemicals have been shown to protect cells against ionizing

radiation [Uma Devi, 1996; Goel et al., 1998, 2002; Weiss and

Landaur, 2000]. A number of plant extracts and phytochemi-

cals also are being evaluated for their ability to protect DNA

from radiation-induced damage [Abraham et al., 1993; Ander-

son et al., 1994; Vrinda and Uma Devi, 2001; Sestili et al.,

2002; Wilms et al., 2005]. Among various mechanisms that

have been considered important for the radioprotective abilities

of plant extracts and phytochemicals, the most important are

related to free-radical scavenging and their antioxidant, cell

proliferation, and immunostimulating activities [Nair et al.,

2000; Weiss and Landaur, 2000].

Hippophae rhamnoides L. (F. Elaeagnaceae), commonly

known as seabuckthorn, has been exploited extensively in In-

dian and Tibetan traditional medicine for the treatment of

circulatory disorders, ischemic heart disease, hepatic injury,

and neoplasia [Xing et al., 2002; Gao et al., 2003; Guliyev

et al., 2004]. H. rhamnoides also provides protection against

radiation-induced lethality in mice and has free-radical scav-

enging activity [Goel et al., 2002]. Modulation of the antiox-

idant defense system has been suggested as the basis for its

radioprotective activity [Agrawala, 2002; Goel et al., 2002,

2003]. H. rhamnoides has been reported to physically inter-

act with cellular chromatin, stabilize it, and thus possibly

protect the DNA from radiation-induced damage [Prem

Kumar et al., 2002; Goel et al., 2003].

In this present study, we have evaluated the radioprotective

ability of REC-1001, a fraction isolated from the berries of

H. rhamnoides, and explored possible mechanisms responsi-

ble for its radioprotective action. The chemical composition

of the extract was partially determined as well as its ability to

protect nDNA andmtDNA against radiation-induced damage.

MATERIALS ANDMETHODS

Reagents andMice

Chemicals

Agarose, ammonium molybdate, bromophenol blue, 2,20-bipyridyl,1,1-diphenyl-2-picryl hydrazyl (DPPH), ethidium bromide, ferric chlo-

ride, Folin Ciocalteau reagent, gallic acid, isorhamnetin, kaempferol,

Na2EDTA, nitroblue tetrazolium (NBT), NADH, pBR322 DNA, phenyl

methane sulfate (PMS), quercetin, sodium pyrophosphate, trichloroacetic

acid (TCA), thiobarbituric acid, and xylene cyanol were obtained from

Sigma (St. Louis, MO). Taq polymerase and dNTP mix were purchased

from Qiagen (Chatsworth, CA). All other chemicals and reagents were

of highest purity commercially available.

Preparation of REC-1001

Fresh Hippophae rhamnoides berries were collected from hilly regions

of the Western Himalayas. The plant material was identified by an eth-

nobotanist at the Institute of Himalayan Bioresource Technology (IHBT),

Palampur, India, and a voucher specimen was deposited in the repository

at the IHBT. The berries were dried, powdered, and extracted with etha-

nol at ambient temperature for 20–24 hr. The ethanolic extract was fil-

tered and concentrated under reduced pressure. The viscous extract thus

obtained was washed with hot hexane and ether to remove nonpolar

compounds. The remaining extract was passed through a bed of silica

gel using a 40:60 (v/v) mixture of ethyl acetate and methanol as the elu-

ent. The partially purified fractions were again passed through a column

containing a weakly polar polymeric adsorbent resin. After loading the

filtrate, the column was eluted with 20–80% ethanol in water. The flavo-

noid-rich fractions were combined and concentrated. Hereafter, the con-

centrated fraction is referred to as REC-1001.

Animals

All experiments involving animals were performed with the approval

of the Animal Ethics Committee of our institute. Swiss albino strain ‘A’

male mice, 10–12 weeks of age and weighing 25 6 2 g, were main-

tained at (25 6 2)8C and with a photoperiod of 12-hr light and 12-hr

dark. The animals were fed ad libitum with rodent food pellets (Amrut

Laboratory Feed, New Delhi, India) and had free access to water.

Chemical Characterization of REC-1001

HPTLC Analysis of REC-1001

Instrumentation. A Camag HPTLC system (CAMAG, Mutteniz, Swit-

zerland), equipped with an automatic TLC sampler (ATS 4), a TLC

scanner 3 (WINCATS version 1.2.3) with UV cabinet and a glass twin-

trough tank (24.5 3 8 3 22.5 cm3), was used for the analysis. The sam-

ples were applied using the automated TLC sampler in 6-mm bands at

10 mm from the bottom, on both sides of the plates, with a 6-mm space

between bands. RP18F254 TLC plates (20 cm 3 20 cm 3 200 mm

thick; E. Merck, Darmstadt, Germany) were used for the separations.

Standard stock solutions. Standard stock solutions containing

0.5 mg/ml of kaempferol, isorhamnetin, and quercetin were prepared in

methanol and filtered through 0.45 lm filters (Millipore, Bedford, MA)

for calibration studies.

Detection of kaempferol, isorhamnetin and quercetin in REC-1001. The TLC plates were developed to a height of about 9 cm from

the base in a tank that was presaturated with developing solvent, metha-

nol-5% formic acid in water (40:60, v/v). After development, the plates

were removed, dried, and the spots were visualized under 254-nm light.

Estimation of Total Phenolic Content of REC-1001

A 100 ll aliquot of 10 mg/ml REC-1001 was mixed with 500 llFolin-Cioccalteau reagent and 400 ll 7.5% sodium carbonate [Singleton

and Rossi, 1965]. Following incubation at 208C for 30 min, the absorb-

ance was read at 765 nm. The total phenols in REC-1001 were expressed

as gallic acid-equivalents, which were estimated from a standard curve

measuring the absorbance of freshly prepared gallic acid.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

648 Shukla et al.

Analysis of Radioprotection by REC-1001in Mouse Thymocytes

Isolation of Thymocytes

Mice were sacrificed by cervical dislocation, dissected, and the ab-

dominal cavity was perfused with 0.9% saline. The thymus was then

removed and visible clots were segregated carefully. The thymic lobes

were finely minced and gently crushed using the plunger of a syringe.

The resultant cell suspension was passed through a 25-gauge needle to

break up cell aggregates.

Irradiation

Thymocytes were irradiated in phosphate buffer saline (PBS, pH 7.4)

with 60Co irradiation. The radiation was delivered by a g Cell model-

220 (Atomic Energy Commission, Ontario, Canada) at a dose rate of

0.52 rad/sec.

DNA Isolation FromThymocytes

DNA was extracted from samples containing *2 3 106 thymocytes

using the DNeasy Tissue kit (Qiagen), and the procedure supplied by the

manufacturer. The DNA was quantified spectrophotometrically.

Polymerase Chain Reaction

Primer pair L15322 (50-CTCTGGTCTTGTAAACCTG-30) and H3266

(50-CAGGCTGGCAGAAGTAATCAT-30) was used to amplify the

mtDNA genome, and yielded a product of 4277 bp [Milano and Day,

2000]. Primers b-globin 957 (50-CGGGTGAGAGATACATCCATCG-30)and b-globin 5638 (50-GATCCAGAGAGCAACTTTCGACTA-30) targetedthe genomic b-globin gene cluster and yielded a product of 4681 bp. The

20 ll polymerase chain reaction (PCR) mixture contained 20 ng template

DNA, 200 lM each dNTP, 10 pmol/ll each primer, 0.3 U Taq polymer-

ase, and 13 Taq buffer containing 1.5 mM MgCl2. PCR of the mtDNA

and nDNA was conducted in separate reactions. The PCR cycling condi-

tions consisted of 948C for 2 min; followed by 30 cycles of 948C for

15 sec, 598C for 30 sec, 688C for 4 min, and a final extension of 4 min at

728C. PCR products were stored at 48C. The PCR products were resolved

by agarose gel electrophoresis as described by Sambrook et al. [2000].

Briefly, 10 ll of each sample were mixed with 5 ll of loading dye

(0.025% bromophenol blue, 0.25% xylene cyanol, and 40% sucrose in

water), and loaded into the wells of a 0.8% agarose gel. Electrophoresis

was carried out for 2.5 hr at 5 V/cm in TAE buffer (0.04 M Tris base,

0.019 M acetic acid, and 0.01 M EDTA, pH 8) in the presence of 0.5 lg/ml ethidium bromide. Amplification products were quantified densitometri-

cally using commercially available software (Advanced American Biotech-

nology, Fullerton, CA).

Preliminary studies were carried out with different concentrations of

DNA from untreated control cells to find an appropriate amount of tem-

plate DNA for the assays. Under the conditions used in this study, a lin-

ear relationship between amplification and template concentration was

found for DNA concentrations up to 30 ng (see results in Fig. 2G). All

further studies, to evaluate the effect of REC-1001 on radiation-induced

nDNA or mtDNA damage, were carried out with a template concentra-

tion of 20 ng.

Alkaline Halo Assay

DNA strand breaks in individual thymocytes were detected using the

alkaline halo assay [Sestili and Cantoni, 1999; Sestili et al., 2002], with

conditions modified for mouse thymocytes. Thymocytes were suspended

at a concentration of 2 3 104/0.1 ml in molten 1.5% low melting point

agarose in PBS (pH 7.4), and 200 ll of the suspension were immediately

spread uniformly on microscope slides precoated with 1.0% normal melt-

ing point agarose. The slides were placed on steel plate and cooled from

below with ice to accelerate gelling. After 5 min, the slides were

immersed in ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA,

10 mM Tris, 1% sodium lauryl sarcosine, 5% dimethylsulfoxide, and 1%

Triton-X100 [pH 10.0]) and left at 48C for 30 min. Thereafter, the slides

were incubated in alkaline buffer (0.3 M NaOH and 1 mM EDTA

[pH 13]) for 20 min at 48C and then directly washed in neutralization

buffer (0.4 M Tris-HCl [pH 7.4]) and stained with 50 lg/ml propidium

iodide. The propidium iodide bound to the intact chromatin mass that

formed the central core of the cells, while the broken DNA fragments

that had diffused from the core constituted a peripheral halo. The level

of DNA strand breakage was quantified by calculating the nuclear

spreading factor value, which is the ratio of the area occupied by the

halo (obtained by subtracting the area of the nucleus from the total area

of the nucleus þ halo) to the area occupied by the nucleus. Data are

expressed as the relative nuclear spreading factor (RNSF), which was

calculated by subtracting the nuclear spreading factor values of control

cells from those of treated cells. Untreated cells consistently had a nu-

clear spreading factor of zero.

Biochemical Evaluation of DNADamageand Radioprotective Mechanisms

Plasmid DNA Study

pBR322 DNA (0.5 lg) in 50 ll of 20 mM phosphate buffer (pH 7.4)

was mixed with REC-1001 and exposed to 20 Gy of radiation from the60Co source. DNA samples were kept on ice during irradiation. The

DNA samples were stabilized by adding a 0.1-volume of 103 TE

(100 mM Tris-HCl, 10 mM EDTA), and stored at 48C until they were

analyzed. Plasmid DNA samples were mixed with 5 ll of 53 loading

buffer and electrophoresed on a 1.2% agarose gel in 0.53 TBE buffer

(0.045 M Tris borate, 0.005 EDTA). After electrophoresis, the gel was

stained with 0.5 lg/ml ethidium bromide for 20 min, visualized under a

transilluminator, and photographed.

DPPH Radical Scavenging Activity

The DPPH radical scavenging ability of REC-1001 was measured

according to the method of Shimada et al. [1992]. A solution of DPPH

in methanol was mixed with REC-1001 and incubated for 15 min in the

dark at 378C. The decrease in the absorbance at 517 nm was measured

against methanol.

Estimation of Hydroxyl Radicals

The ability of REC-1001 to quench the hydroxyl radicals generated

by 100 Gy of radiation was studied in vitro using 2-deoxribose as the

marker substrate [Gutteridge, 1988]. Briefly, 1 ml reactions containing

5 mM 2-deoxyribose, and various concentrations of REC-1001 (25; 50;

100; 200; 500; 1,000; 1,500; or 2,000 lg/ml) were exposed to 100 Gy

gamma radiation. Thereafter, two volumes of a solution containing

25% TCA and 1% TBA in 0.1 N NaOH were added, the mixture

placed in a boiling water bath for 20 min, cooled, and the absorbance

of the resulting pink-colored chromogen measured at 532 nm.

Determination of Reducing Power

One hundred ll of PBS (pH 7.4) containing 20 lg of ferric chloride

were mixed with various concentrations of REC-1001 (10; 20; 40; 60;

80; 100; 150; 200; 400; 600; 1,000; 1,500; or 2,000 lg/ml) and incu-

bated at room temperature for 5 min. The amount of Fe2þ formed was

quantified [Harris and Livingstone, 1965] by adding 100 ll of 100 mM

2-20-bipiridyl and measuring the resulting chromogen (Fe2þ-bipiridylcomplex) at 525 nm.

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Radioprotection by H. rhamnoides Extract 649

Estimation of the Scavenging of Chemically-GeneratedSuperoxide Anions

The superoxide anion scavenging potential of REC-1001 was estimated by

the method of Rao et al. [1989]. Briefly, a reaction mixture containing 0.52 M

sodium pyrophosphate (pH 8.3), 186 lM PMS, and 300 lM NBT was mixed

with various concentrations of REC-1001 (25, 50, 100, 200, 500, 1,000,

1,500, or 2,000 lg/ml). The reaction was initiated by adding NADH (final

concentration 780 lM) and incubated for 90 sec at 308C. The purple-coloredchromogen that was formed was measured spectrophotometrically at 560 nm.

Evaluation of Total Antioxidant Capacity

The total antioxidant capacity of REC-1001 was estimated spectropho-

tometrically by quantifying the amount of phosphomolybdenum complex

formed [Prieto et al., 1999]. A 0.1 ml solution containing 100 lg of

REC-1001 was mixed with 1 ml of reagent solution (6 M sulfuric acid,

28 mM sodium phosphate, and 4 mM ammonium molybdate) in a 1.5-

ml microcentrifuge tube, and incubated at 958C for 90 min. After the

samples were cooled to room temperature, the absorbance was measured

at 695 nm against a blank solution. A typical blank solution contained 1

ml of reagent solution and the appropriate volume of the same solvent

(water) used for sample preparation and was incubated under the same

conditions as the experimental samples. A calibration curve was prepared

using gallic acid as the antioxidant, and the antioxidant capacity of

REC-1001 was expressed as gallic acid-equivalents on a weight basis.

Statistical Analysis

The data are presented as the mean 6 the standard deviation (s.d.) of

three separate experiments, with each experiment comprising three paral-

lel measurements. Comparisons were made between radiation and radia-

tion þ REC-1001 groups. Data were analyzed by one way analysis of

variance and multiple comparisons were made between different groups

by applying the Bonferroni t-test. A probability of <5% was considered

significant.

RESULTS

Phytochemical Analysis

Chemical analysis indicated that the total polyphenol

content of REC-1001 was 0.679 mg/mg. HPTLC analysis

of REC-1001 indicated that REC-1001 contained at least

three prominent phenolic compounds, kaempferol, iso-

rhamnetin, and quercetin. The spots for kaempferol, iso-

rhamnetin, and quercetin were identified by comparing

the Rf values and spectra of the sample with those of the

standards (Figs. 1A and 1B).

REC-1001and Radioprotection in Mouse Thymocytes

mtDNA Damage by PCR Analysis

Radiation dose-dependently induced damage in the

mtDNA of mouse thymocytes, as measured by the inhi-

bition of the amplification of a sequence of mtDNA. At

5 and 10 Gy, the amplification of the targeted sequence

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Fig. 1. Chemical analysis of REC-1001. (A) Analysis of REC-1001 and standard flavonoids by HPTLC.

Samples were loaded onto a TLC plate, developed, and scanned. (B) 3D overlay chromatogram of the stand-

ards and the sample. Peaks a, b, and c represents kaempferol, isorhamnetin, and quercetin, respectively.

650 Shukla et al.

was 84.2% and 35%, respectively, as compared to the

unirradiated control. No visible amplification was pro-

duced after irradiation with 20 Gy (Fig. 2A). REC-

1001 concentrations of 50, 100, and 250 lg/ml

increased the amplification yields after 10 Gy of radia-

tion from (35 6 0.27)% to (42 6 0.15)%, (49 60.25)%, and (56 6 0.25)% of the unirradiated control,

respectively (Fig. 2B). These increases were significant

(P < 0.05).

nDNA Damage (b globin gene) by PCR Analysis

Exposure of thymocytes to different doses of g radia-

tion significantly inhibited the amplification of the b-glo-

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Fig. 2. Effect of REC-1001 on radiation-induced DNA damage in

mouse thymocytes. Cells were irradiated, DNA extracted, and segments

of both mtDNA and nDNA (b-globin gene) amplified. (A) Lanes 1–4,

amplification product from mtDNA of cells exposed to 0, 5, 10, or

20 Gy. (B) Lane 1, amplification product from mtDNA of unirradiated

control cells; lanes 2–5, amplification product from mtDNA of cells irra-

diated with 10 Gy in the presence of 0, 50, 100, or 250 lg/ml of REC-

1001, respectively. (C) Lane 1–4, amplification product from b-globingene of cells exposed to 0, 5, 10, or 20 Gy. (D) Lane 1, amplification

product from b-globin gene of unirradiated control cells; Lanes 2–5,

amplification product from b-globin gene of cells irradiated with 10 Gy

in the presence of 0, 50, 100, or 250 lg/ml of REC-1001, respectively.

(E) Quantitative estimation of mitochondrial and b-globin gene amplifi-

cation with increasing doses of radiation. (F) Amplification products of

mtDNA and the b-globin gene of thymocytes exposed to 10 Gy in the

presence of various doses of REC-1001. The figure shows representative

data from three independent assays. (G) Effect of template concentration

on amplification yield of the b-globin gene from untreated cells. A lin-

ear relationship was found for amplification up to 30 ng of template

DNA.

Radioprotection by H. rhamnoides Extract 651

bin gene; radiation doses of 5, 10, and 20 Gy resulted in

a reduction in amplification of the b-globin gene to 50,

16, and 2.5%, respectively, as compared to the unirradi-

ated control (Fig. 2C). In contrast to the mtDNA seq-

uence, amplification of the b-globin gene was observed at

a dose of 20 Gy. Pretreatment with REC-1001 increased

the amplification of the b-globin gene after 10 Gy of irra-

diation in a dose-dependent manner. REC-1001 concentra-

tions of 50, 100, and 250 lg/ml increased the yield of the

b-globin gene product from (16 6 0.4)% to (22 6 0.3)%,

(61 6 0.35)%, and (71 6 0.25)%, respectively, in com-

parison to the unirradiated control (Fig. 2D) and these

increases were significant (P < 0.05).

REC-1001and Radioprotection in Mouse Thymocytesby the Alkaline Halo Assay

The RNSF of unirradiated thymocytes was zero. Ten gray

of radiation increased the RNSF to 15 6 0.3 (P < 0.05).

The addition of REC-1001 to the thymocyte cultures inhib-

ited radiation-induced DNA strand breaks in a concentra-

tion-dependent manner; 500 lg/ml REC-1001 reduced the

RNSF of irradiated thymocytes to 4.2 6 0.4 (P < 0.05)

when compared to irradiation-only cells (Fig. 3).

Biochemical Evaluation of DNADamageand Radioprotective Mechanisms

Plasmid Relaxation Assay

Untreated plasmid DNA contained 88%, 12%, and 0%

of the supercoiled, open circular, and linear forms, respec-

tively (Figs. 4A and 4B). Exposure to 50 Gy of irradiation

resulted in the induction of single-strand and double-

strand breaks, as evidenced by a complete loss of the

supercoiled form and increased quantities of both the

open circular (78%) and linear forms (22%) (Fig. 4A;

lane 2). Addition of REC-1001 prior to irradiation inhib-

ited the generation of damaged forms in a dose-dependent

fashion. The maximum protective effect was observed at

a concentration of 300 lg/ml REC-1001 (77% supercoiled

form and 23% relaxed form) (Fig. 4, lanes 3–11).

Estimation of REC-1001on HydroxylRadical Scavenging Activity

Treatment of 2-deoxyribose with 100 Gy of irradiation

resulted in considerable degradation and in the formation

of thiobarbituric acid reactive substances (TBARS). The

addition of REC-1001 inhibited the radiation-induced deg-

radation of 2-deoxyribose in a concentration-dependent

manner; the effect was greatest at a REC-1001 concentra-

tion of 2 mg/ml (78% inhibition of degradation; absorb-

ance of radiation alone group, 0.168 6 0.001 while 2 mg/

ml of REC-1001 þ 100 Gy was 0.072 6 0.002) (Fig. 5).

E¡ect of REC-1001on DPPH Radicals

REC-1001 strongly inhibited the generation of DPPH

radicals; 25 lg/ml, the lowest concentration used in this

study, completely stabilized the generation of DPPH

radicals.

E¡ect of REC-1001on Chemically-GeneratedSuperoxide Anions

The superoxide anions generated by PMS and NADH

reduced NBT (Fig. 6). REC-1001 inhibited the chemi-

cally-generated superoxide anion formation in a concen-

tration-dependent manner; the maximum scavenging was

observed at a REC-1001 concentration of 2 mg/ml

(72.3% reduction; the absorbance of the NBT-derived

chromogen of untreated control was 0.288 6 0.002, while

in the case of REC-1001 at a concentration of 2 mg/ml

the absorbance was 0.079 6 0.0004) (Fig. 6).

Estimation of Reducing Potential of REC-1001

Figure 7 shows the ability of REC-1001 to donate an

electron and reduce the ferric ion to the ferrous form.

REC-1001 reduced the ferric ion in a dose-dependent

fashion; the maximum reduction was observed at a REC-

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Fig. 3. Effect of REC-1001 on radiation-induced DNA damage in

mouse thymocytes as measured by the alkaline halo assay. DNA damage

is expressed as the relative nuclear spreading factor. 50, 100, 150, 200,

500 lg/ml of REC-1001. Representative halos obtained after different

treatments are shown.

652 Shukla et al.

1001 concentration of 2 mg/ml, the highest concentration

assayed.

Total REC-1001Antioxidant Capacity

The total antioxidant capacity of the REC-1001 extract

was 0.06 mg/mg gallic acid.

DISCUSSION

The majority of studies on radiation-induced cell killing

have focused on nDNA as the most important target for

DNA damage. Although this may be true for cell killing,

radiation-induced damage to mtDNA has significant long-

term effects in surviving cells and may lead to abnormal

cellular and tissue functions [Prithvirajsingh et al., 2004].

In view of this, the present study evaluated the protective

effects of REC-1001 against radiation-induced damage to

both nDNA and mtDNA.

Radiation-induced DNA damage was studied with a

highly sensitive quantitative PCR technique (QPCR).

The rationale of the QPCR for the quantification of

DNA damage is that certain DNA lesions block the

movement of the PCR polymerase on the DNA tem-

plate, resulting in decreased amplification of the tem-

plate [Ayala-Torres et al., 2000]. Since only a single

lesion per strand is required to block the polymerase,

the QPCR assay effectively measures the fraction of

template molecules that are undamaged [Ayala-Torres

et al., 2000]. Consequently, radiation-induced DNA

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Fig. 4. Effect of REC-1001 on DNA strand breaks in pBR322 DNA.

Plasmid DNA was irradiated with 50 Gy and DNA damage evaluated as

the conversion of fast migrating supercoiled DNA (ccc) to slowly

migrating linear DNA (lin) and the relaxed form (oc). (A) Lane 1, uni-

rradiated plasmid DNA; Lane 2, DNA irradiated with 50 Gy, Lanes 3–

11, plasmid DNA irradiated with 50 Gy in the presence of increasing

concentrations of REC-1001 (10, 20, 40, 60, 80, 100, 150, 200, 300 lg/ml). (B). Histogram showing the percentage of different forms of plas-

mid following irradiation in the presence and absence of REC-1001. The

data in this figure represent the mean 6 s.d. of values from three inde-

pendent assays. C: Control; R: radiation (50 Gy); 10, 20, 40, 60, 80,

100, 150, 200, 300 lg/ml of REC-1001.

Radioprotection by H. rhamnoides Extract 653

damage leads to loss of template amplification and the

activity of protective agents can be measured by the res-

toration of template amplification.

The sensitivity of the QPCR assay is strongly depend-

ent upon the size of the segment being amplified; very

high doses of radiation are required to induce damage if

small segments are being amplified [Kalinowski et al.,

1992]. To enhance the sensitivity of the assay and to mea-

sure DNA damage in cells exposed to radiation doses of

interest (e.g., 10 Gy), the QPCR assay was conducted

using primer pairs specific for kilobase mtDNA and

nDNA sequences, yielding products of 4277 and 4681 bp,

respectively [Milano and Day, 2000]. DNA isolated from

mouse thymocytes exposed to 5, 10, and 20 Gy of radia-

tion exhibited a dose-dependent reduction in amplification,

but the extent of the reduction differed for the mtDNA and

nDNA sequences (Fig. 2). For instance, 20 Gy resulted in

no detectable amplification of the mtDNA segment, while

some amplification of the nDNA b-globin gene sequence

was detected (Fig. 2). This difference may be due to mito-

chondria being a rich source of ROS and 20 Gy probably

added to a large amount of pre-existing DNA damage. In

addition, mtDNA is devoid of protective histones and is

generally repaired less efficiently than nDNA [Yakes and

Van Houten, 1997].

The QPCR analysis indicated that REC-1001 protected

both the mtDNA and nDNA of mouse thymocytes from

radiation-induced damage. The results indicate, however,

that REC-1001 protected nDNA more efficiently than

mtDNA. Earlier studies with an extract of H. rhamnoidesindicate that the extract has the ability to localize in

nuclei, interact with nDNA, and protect against radiation-

induced strand breakage [Prem Kumar et al., 2002; Goel

et al., 2003]. Apart from evaluating the protective effects

of REC-1001 against sequence-specific DNA damage, we

used halo assay to evaluate radiation-induced DNA dam-

age at the genomic level. The results from this assay are

consistent with the QPCR results and further support the

ability of REC-1001 to protect thymocyte DNA from

radiation-induced DNA damage.

The induction of single-strand or double-strand breaks

in plasmid DNA results in the conversion of fast-migrat-

ing supercoiled DNA into the more slowly migrating

relaxed form and a linear form with intermediate migra-

tion. Evaluating these changes in plasmid conformation is

a more sensitive method for assessing DNA damage than

estimating the formation of 8-oxo-7,8-dihydro-20-deoxy-guanosine (8-OHdG) by chromatography [Yang et al.,

1999]. The plasmid assay has been used previously to

study the pro-oxidant and antioxidant properties of vari-

ous compounds [Thibodeau et al., 2001; Pankaj et al.,in

press]. In the present study, exposure of plasmid DNA to

50 Gy of radiation resulted in significant amounts of both

single-strand and double-strand DNA breaks as judged by

the loss of intact supercoiled DNA and the formation of

open circular and linear forms. REC-1001 mitigated the

effects of radiation in damaging plasmid DNA, support-

ing the radioprotective effects of REC-1001 detected in

mouse thymocytes.

Since radiation inflicts cellular damage through the

generation of free radicals [Bump and Malkkar, 1998], we

evaluated the ability of REC-1001 to scavenge free radi-

cals using a battery of in vitro tests. Hydroxyl radicals are

the primary reactive species responsible for radiation-

induced damage to biomacromolecules, including DNA

[Ross, 1999]. We evaluated the hydroxyl radical scaveng-

ing ability of REC-1001 using the 2-deoxyribose-degrada-

Environmental and Molecular Mutagenesis. DOI 10.1002/em

Fig. 6. Effect of increasing concentrations of REC-1001 (25, 50, 100,

200, 500, 1,000, 1,500, and 2,000 lg/ml) on chemically-induced super-

oxide anions. The scavenging of superoxide anions was measured as the

% inhibition in NBT reduction. The absorbance at 560 nm was recorded

in triplicate. The values are expressed as mean 6 s.d. of data from three

independent assays. All values of REC-1001 treated groups are signifi-

cant (P < 0.01) when compared to the untreated group.

Fig. 5. Effect of increasing concentrations of REC-1001 (25, 50, 100,

200, 500, 1,000, 1,500, and 2,000 lg/ml) on radiation-induced hydroxyl

radical formation. Radicals were generated with 100 Gy of radiation and

detected by the subsequent degradation of 2-deoxyribose (2-DR) as

measured by the % inhibition in the formation of TBARS. Responses

were compared between the radiation-alone group and the groups given

radiation þ REC-1001 and were found to be significant (P < 0.01). The

data in this figure represent the mean 6 s.d. of values from three inde-

pendent assays.

654 Shukla et al.

tion assay [Gutteridge, 1988]. Radiation of 100 Gy gener-

ated a considerable amount of hydroxyl radicals as esti-

mated by 2-deoxyribose degradation and the formation of

TBARS. REC-1001 dose-dependently reduced the genera-

tion of TBARS, suggesting that it has the ability to scav-

enge radiation-induced hydroxyl radicals. Similarly, REC-

1001 reduced the concentration of chemically-induced

superoxide anions and the DPPH radical [Shimada et al.,

1992]. In the case of the DPPH radical, the maximum

effect was observed at lowest dose evaluated, 25 lg/ml.

The antioxidant activities of natural compounds are

directly correlated with their ability to act as reducing

agents [Yen and Duh, 1993]. A biochemical assay indi-

cated that REC-1001 is able to dose-dependently reduce

the ferric ion to the ferrous form. The antioxidant

capacity of REC-1001 was supported further by the spec-

trophotometric quantitation of a phosphomolybdenum

complex.

Chemical analysis of REC-1001 revealed presence of

large quantities of polyphenols and HPTLC of REC-1001

indicated that the extract contains kaempferol, isorhamne-

tin, and quercetin. These compounds are flavonoids that

are known to possess strong antioxidant and radioprotec-

tive properties [Shimoi et al., 1996; Di Carlo et al.,

1999], and the presence of these compounds in REC-1001

might be responsible for the antioxidant and radioprotec-

tive properties of the extract.

In conclusion, the results of this study indicate that

REC-1001 protects both mtDNA and nDNA from radia-

tion-induced damage. The antioxidant and free-radical

scavenging abilities of REC-1001, possibly because of

polyphenols present in the extract, may contribute to its

radioprotective effects.

ACKNOWLEDGMENTS

We are thankful to Dr. A. P. Gupta, IHBT, for his tech-

nical assistance. We are also thankful to the Directors of

INMAS and IHBT for providing the facilities necessary

for carrying out this project. The help rendered by Ms.

Arpana Vibhuti, IGIB, Delhi is also highly appreciated.

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