Molecular and Cellular Biochemistry 266: 65–77, 2004.c© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

Modification of radiation damage tomitochondrial system in vivo by Podophyllumhexandrum: Mechanistic aspects

Damodar Gupta,1 Rajesh Arora,1 Amar Prakash Garg,2

Madhu Bala1 and Harish Chandra Goel21Radiation Biology Division, Institute of Nuclear Medicine and Allied Sciences, Delhi; India; 2Department of Microbiology,Chaudhary Charan Singh University, Meerut, India

Received 30 October 2003; accepted 19 March 2004

Abstract

The present study was undertaken to investigate whether RP-1 treatment protected mitochondrial system against radiation dam-age and also to unravel the mechanism associated with this process. Radioprotection of mitochondrial system by Podophyllumhexandrum (RP-1) was investigated to understand its mechanism of action. Levels of superoxide anion (O2−), reduced or oxidizedglutathione (GSH or GSSG), thiobarbituric acid reactive substance (TBARS), protein carbonyl (PC), ATP, NADH-ubiquinoneoxidoreductase (complex-I), NADH-cytochrome c oxidoreductase (complex I/II), succinate-cytochrome c oxidoreductase (com-plex II/III) and mitochondrial membrane potential (MMP) were studied in mitochondria isolated from liver of mice belongingto various treatment groups. Whole body γ -irradiation (10 Gy) significantly (p < 0.01) increased the formation of O2−, PC,and TBARS, upto 24 h as compared to untreated control. RP-1 treatment (200 mg/kg b.w.) to mice 2 h before irradiationreduced the radiation-induced O2− generation within 4 h and formation of TBARS and PC upto 24 h significantly (p < 0.01).Singularly irradiation or RP-1 treatment significantly (p < 0.01) increased the levels of glutathione within an hour, as com-pared to untreated control. Pre-irradiation administration of RP-1 enhanced levels of GSH induced increase in complex I (upto16 h), complex I/III (4 h) complex II/III activity (upto 24 h; p < 0.01) and inhibited the radiation-induced decrease in MMPsignificantly (24 h; p < 0.01). The present study indicates that RP-1 itself modulates several mitichondrial perameters dueto its influence on the biochemical milieu within and outside the cells. However, RP-1 treatment before irradiation modulatesradiation induced perturbations such as the increase in electron transport chain enzyme activity, formation of O2−, TBARS andPC to offer radioprotection. (Mol Cell Biochem 266: 65–77, 2004)

Key words: electron transport chain, gamma radiation, glutathione, mitochondria, Podophyllum hexandrum, radioprotection,superoxide

Introduction

Mitochondria are responsible for several vital cellular func-tions like energy metabolism, heme synthesis, maintenanceof intracellular homeostasis and signaling of apoptosis [1–2]. Damage to mitochondria is known to cause cardiovascu-

Address for offprints: Dr. H. C. Goel, Department of Microbiology, C.C.S. University, Meerut-250005, U.P., India (E-mail: goelharish@hotmail.com)

lar disorders, Parkinson’s, Alzheimer’s, cancer and aging [3].Low linear energy transfer radiation like x- or γ -rays, damagethe structure and function of mitochondria via oxidative insultto DNA, lipids, proteins, ionic homeostasis, thereby causingincreased generation of superoxide anions [4–5]. Under nor-mal conditions, the intrinsic mitochondrial components like

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quinols, cytochrome c, vitamin E, manganese dependent su-peroxide dismutase (Mn SOD) and glutathione act as thecell’s antioxidant defence system [6]. However, irradiationadversely affects the inherent antioxidant system ultimatelyleading to bio-energetic catastrophe. For handling such a sit-uations, supplementation of antioxidants in cellular milieubecomes inevitable to protect mitochondria from radiationdamage.

A number of purified natural compounds like caffeine[7], quercetin [8], chlorophyllin [9] and crude extracts ofplants like Asparagus racemosus [10], Hippophae rham-noides [11–12] and Podophyllum hexandrum [13–14] havebeen reported to provide radioprotection owing to their an-tioxidant effects. P. hexandrum, high-altitude plant thriving inextremely cold ranges of Himalayas known for its anticancer,antioxidant, antimicrobial properties [13] has been evaluatedin recent years for its radioprotective properties. Rhizomeand root of P. hexandrum contain several of bioactive con-stituents including, lignans (podophyllotoxin, podophyllo-toxone, podophyllin, α-peltatins and β-peltatins), flavonoidsviz. quercetin, kaempferol and astragalin or kaempferol-3-glucoside which individually exhibit diverse bioactivities[15–16]. RP-1, an aqueous extract of rhizome of P. hexan-drum, has already been reported to provide 80% protection inStrain ‘A’ mice subjected to whole-body lethal γ -irradiation[17]. RP-1 has free radical scavenging and metal chelationproperties [18] is able to modulate antioxidant enzyme levels[19] and provide protection against gastrointestinal damage[20].

According to our recent report [13], RP-1 has been shownto provide radioprotection to HepG2 cells by multiple mech-anisms like stabilization of radiation-induced changes in mi-tochondrial and cytoplasmic membrane potential, inhibitionof generation of reactive oxygen species (ROS) and lipid per-oxidation, increased biosynthesis of glutathione. The presentinvestigations were carried out on mitochondria isolated frommouse liver at various time periods after irradiation to unravelthe mode of action of RP-1 under in vivo conditions.

Material and methods

Chemicals

3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bro-mide (MTT), dimethyl sulfoxide (DMSO), ethylenediamine tetra acetic acid disodium salt (EDTA-Na),o-pthalaldehyde (OPT), N-ethylmaleimide (NEM), 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), cy-tochrome c, ubiquinone, rotenone, antimycin A, gramicidin,β-NADH, bovine serum albumin (BSA), dinitro-phenyl hy-drazine (DNPH) were procured from Sigma Chemical Co.,St. Louis, MO, USA, rhodamine 123 (Rh-123) from Fluka,

Switzerland, ATP assay kit and HEPES from Calbiochem,USA. All other chemicals were of ANALAR grade and werepurchased from reputed Indian firms.

Plant material

Extract of Podophyllum hexandrum was prepared as de-scribed earlier by Gupta et al. [13]. Briefly, the dried rhizomeof P. hexandrum was powdered, extracted in triple distilledwater and thereafter lyophilized and stored at 4 ◦C. Imme-diately prior to use, the extract was dissolved and diluted insaline.

Experimental animals

Three-month-old male inbred Swiss Albino strain ‘A’ mice(weighing 25 ± 3 g) were maintained under controlled envi-ronment (25 ± 3 ◦C, photoperiod 12 h per day) and providedstandard animal food pellet (Amrut Laboratory Animal Feed,India) and water ad libitum. Not more than three animals werehoused in a polyvinyl cage, and each group had three ani-mals. Animal experiments were conducted strictly accordingto ‘INSA-Ethical Guidelines for use of Animals in ScientificResearch’ as prescribed by Central Drug Research Institute(CSIR), Lucknow, India and approval was taken from AnimalExperimentation Ethics Committee of the institute.

Animal groups

Untreated control (n = 4 × 3): 0.2 ml vehicle (i.p.) andsham irradiated.

Irradiated control (n = 4 × 3): 10 Gy whole body 60Coγ -irradiation.

Drug control (n = 4 × 3): 0.2 ml 200 mg/kg b.w. in vehicle(−2 h; i.p.; single dose) before sham irradiation.

RP-1 + Irradiation (n = 4 × 3): 0.2 ml 200 mg/kg b.w.in vehicle (−2 h; i.p.; single dose) before 10 Gy whole body60Co γ -irradiation.

Irradiation

Each mouse was individually placed in perforated plasticcontainers and whole body irradiated at room temperature(25 ± 3 ◦C) using 60Co γ -chamber (Model 220; AtomicEnergy of Canada, dose rate of 0.516 Gy/min) undernormoxic conditions.

Isolation of mitochondria

Strain ‘A’ male mice (8–10 week old) were sacrificed bycervical dislocation, dissected and liver was taken out afterperfusion with saline. Liver (1 part by wt.) was homogenized

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in 9 parts by vol. of ice-cold isolation medium (0.3 mol/Lsucrose, 0.1% BSA, 1 mmol/L EGTA, 5 mmol/L Mops, 5mmol/L KH2PO4, pH 7.4) using Potter Elvjham homoge-nizer. Liver mitochondria were isolated by the method ofRickwood et al. [21]. The homogenate was centrifuged at1000 g for 10 min at 5 ◦C, supernatant was carefully decantedin another centrifuge tube and re-centrifuged at 15,000 g tosediment mitochondria (Kendro, USA). The mitochondrialpellet, thus obtained, was washed thrice and finally suspendedin the isolation medium without BSA.

To remove traces of sucrose (presence of which affectslipid peroxidation measurement), the obtained mitochondrialpellet was washed thrice with ice-cold potassium phosphatebuffer (0.05 mol/L, pH 7.4) and finally suspended in ice-coldpotassium phosphate buffer (0.005 mol/L, pH 7.4).

Superoxide generation

Mitochondria (freshly isolated, 0.1 µg protein equivalent)and 6 µl of MTT solution (1.25 mmol/L in PBS, pH 7.4) wasadded to each well of micro-titer plate (Corning, USA) andincubated at 37 ◦C for 30 min. The formazan formed due toreduction of MTT was dissolved in 150 µl of DMSO andabsorbance was measured at 570 nm. Amount of superoxidegenerated was calculated using the molar extinction coeffi-cient of MTT formazan [E570 of 17,000 (mol/L)−1 cm−1] atpH 7.4–8.0, and results expressed as nmol/L of superoxideanion generated/min/mg of mitochondrial protein [22].

Glutathione estimation

Mitochondrial glutathione was determined using the methodof Hissin and Hilf [23]. Briefly, mitochondria (1 mg protein)were suspended in 25 µl of 25% HPO3 and 90 µl of sodiumphosphate buffer (0.1 mol/L, pH 8.0, having 0.005 mol/LEDTA) and centrifuged at 15,000 g for 10 min at 4 ◦C. Thesupernatant was collected for measurement of GSX (GSH +GSSG) and GSSG. 100 µl of supernatant was incubated with100 µl of o-phthalaldehyde (0.1% in methanol) and 1.8 mlof 0.1 mol/L phosphate buffer (pH 8.0) for 15 min underdark conditions at room temperature. Fluorescence was mea-sured using a fluorescence spectrophotometer (Varian, USA)at an excitation wavelength (λEx) of 350 nm and an emissionwavelength (λEm) of 420 nm.

For determination of glutathione disulfide (GSSG), thesupernatant (100 µl) of mitochondrial sample was treatedwith 40 µl of 0.04 mol/L N-ethylmaleimide for 30 minand the procedure mentioned above was adopted, exceptthat 0.1 N sodium hydroxide was used in lieu of 0.1 mol/Lphosphate buffer. Oxidized (GSSG) and reduced glutathione(GSH) concentrations were measured by comparing with cor-responding standard curves.

Enzymes assay

The mitochondrial suspension was freeze-thawed and gen-tly shaken thrice to ensure mitochondrial lysis and specificactivities of the following enzymes were measured at roomtemperature in a total reaction volume of 1 ml using UV-Vis spectrophotometer. Each observation was carried out intriplicate.

NADH-ubiquinone oxidoreductase (Complex I)

Complex I activity was measured in terms of oxidationof NADH to NAD by recording decrease in absorbanceat 340 nm, with 425 nm as reference wavelength [24; ∈= 6.81 (mmol/L)−1 cm−1]. The reaction mixture contained25 mmol/L potassium phosphate buffer (pH 7.4), 5 mmol/Lmagnesium chloride, 2 mmol/L potassium cyanide, 2.5 mgBSA, 100 µmol/L ubiquinone and 2 µg antimycin A. Thereaction was initiated by addition of mitochondria (50 µg ofprotein) and monitored for 2 min. After a measurable linearrate was observed, 5 µg of rotenone was added to the reactionmixture to obtain rotenone-insensitive rate. The complex I ac-tivity was the difference between the total enzymatic rate andthat obtained with the addition of rotenone. Each observationwas carried out in triplicate and was repeated three times.

NADH-cytochrome C oxidoreductase (Complex I/III)

Complex I/III activity was measured in terms of reductionof ferricytochrome c by recording increase in absorbance at550 nm, with 580 nm as the reference wavelength [24; ∈ =19 (mmol/L)−1 cm−1]. The reaction was initiated by addi-tion of 50 µg of mitochondrial protein to the assay mixture(50 mmol/L potassium phosphate buffer, pH 7.4, 80 µmol/Lferricytochrome c, 100 µmol/L NADH, 5 mmol/L magne-sium chloride, 2 mmol/L potassium cyanide) and enzyme ac-tivity was monitored for 1.5 min. Rotenone (5 µg) was addedthereafter and the reaction was monitored for additional1 min. The rotenone-insensitive NADH-cytochrome c oxi-doreductase rate was calculated by subtracting the rotenone-sensitive rate from overall rate.

Succinate-cytochrome c oxidoreductase (Complex II/III)

Complex II/III activity was measured by recording the in-crease in absorbance due to reduction of cytochrome c at550 nm with 580 nm as the reference wavelength [24; 19.1(mmol/L)−1 cm−1]. Mitochondrial protein (50 µg) was pre-incubated with assay mixture (40 mmol/L potassium phos-phate buffer, pH 7.4, 20 mmol/L succinate, 2 µg rotenone,2 mmol/L potassium cyanide and 0.5 mmol/L EDTA) for20 min at room temperature. Thereafter, the reaction was

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initiated by the addition of 30 µmol/L cytochrome c andmonitored for 2 min.

Mitochondrial membrane potential (MMP)

Mitochondrial membrane potential (MMP) was mea-sured using the method of Emaus et al. [25]. Freshlyisolated mitochondria were incubated in respiration buffer(0.22 mol/L sucrose, 68 mmol/L mannitol, 10 mmol/Lpotassium chloride, 5 mmol/L potassium dihydrogenphosphate, 2 mmol/L magnesium chloride, 500 µmol/LEGTA, 5 µmol/L succinate, 2 µmol/L rotenone, 10 mmol/LHEPES, pH 7.2) supplemented with 0.5 µmol/L rhodamine123 for 5 min under dark condition. Changes in fluorescenceof Rh-123 were measured at 25 ◦C using fluorescencespectrophotometer (Varian, USA) at an λEx of 490 nm andan λEm of 532 nm. Gramicidin (20 µmol/L) was used forabolishing the mitochondrial membrane potential and tomeasure non-specific uptake of Rh-123.

ATP levels

ATP was extracted from freshly isolated mitochondria using10% perchloric acid, and ATP levels were measured usingcommercially available ATP assay kit (Calbiochem, USA;Cat. No. 119108). Total luminescence was measured by afluorescence spectrophotometer (Varian, USA). The amountof ATP from a test solution was quantified using calibrationcurve of ATP and the results expressed as µmol/L of ATP/mgof mitochondrial protein.

Lipid peroxidation

Thiobarbituric acid reactive substances (TBARS) were mea-sured spectrophotometrically as described by Buege and Aust[26]. Briefly, mitochondrial protein (4 mg/ml) was mixedwith equal volume of Buege and Aust reagent (TCA: 15%(w/v) in 0.25 N HCl; TBA: 0.37% (w/v) in 0.25 N HCl)and heated for 15 min in boiling water. After cooling, theprecipitate was removed by centrifugation and absorbance ofsupernatant was recorded at 532 nm against a sample contain-ing reagents but no sample. The concentration of TBARS wasdetermined using an extinction coefficient of 1.56 × 105 M−1

cm−1 and results expressed as nmol/L of MDA/mg of mito-chondrial protein.

Protein oxidation

Oxidative damage to mitochondrial protein was measuredas protein carbonyl formation or amount of DNPH incorpo-rated/mg of miochondrial protein [27]. Mitochondrial protein

was precipitated with ice-cold trichloroacetic acid (20% finalconcentration) and redissolved in 300 µl of 0.1 mol/L sodiumhydroxide. Three samples were treated with 0.3% DNPH(prepared in 2 N HCl) and one with HCl alone (for back-ground reading). Both samples were kept in dark at room tem-perature for 1 h and the samples were shaken every 15 min.The reaction was stopped by addition of trichloroacetic acid(20%), and the samples were centrifuged at 1000 g for 10 min.The pellet was washed three times with a 1:1 mixture ofethanol: ethyl acetate and once using trichloroacetic acid(20%). The washing step involved resuspension of pellet fol-lowed by recentrifugation. To evaporate the residual organicsolvent, the pellet was incubated at 37 ◦C for 15 min un-der nitrogen flux. Afterwards, the pellet was dissolved in6 mol/L guanidine hydrochloride (prepared in a mixture ofHCl/acetate buffer; pH 2.3). The difference in absorbance be-tween DNPH-treated and HCl added samples was determinedspectrophotometrically at 370 nm. Protein recovery was es-timated in each sample. The calculations were done using amolar extinction coefficient of aliphatic hydrazones (22,000(mol/L)−1 cm−1 for the DNPH-derivatives) and results ex-pressed as nmol/L of protein carbonyl/mg of mitochondrialprotein.

Protein estimation

Protein concentration in each sample was measured by theLowry’s method [28] using bovine serum albumin (BSA) asstandard.

Data analysis

Data with respect to different parameters obtained from micebelonging to various treatment groups were subjected to stu-dent t-test and significance was assessed at p < 0.01 confi-dence level.

Results

Superoxide generation

Whole-body exposure of mice (10 Gy) increased the genera-tion of superoxide anions within 1 h, which remained signifi-cantly (p < 0.01) higher upto 24 h, as compared to untreatedcontrol (Fig. 1). Administration of RP-1 also increased thelevel of superoxide anions at 1 h and thereafter became com-parable (after 2 h) to untreated control. Pre-irradiation admin-istration of RP-1 to mice failed to alter the radiation-inducedincrease of superoxide anions upto 2 h. However after 2 h thelevels decreased significantly (p < 0.01) making it compa-rable to untreated control.

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Fig. 1. Modulatory effect of RP-1 on radiation-induced alterations in mitochondrial superoxide anion. The results have been expressed as nmol/L of superoxideanion generated/min/mg of mitochondrial proteins. The significance between different groups was analysed using student t-test and p < 0.01 was consideredsignificant. (∗ p < 0.01: Untreated control vs Irradiated control; # p < 0.01: Irradiated control vs RP-1 + 10 Gy). Data represented mean values ± S.D. ofthree experiments.

Glutathione [reduced (GSH), oxidized (GSSG)]

Irradiation appreciably increased the GSH levels within 1h; but thereafter these levels decreased which however re-

Fig. 2. Effect of pre-irradiation administration of RP-1 to strain ‘A’ mice on radiation-induced changes in levels of reduced glutathione (GSH). The results havebeen expressed as nmol/L of glutathione/mg of mitochondrial (mt) protein. The significance between different groups was analysed using student t-test andp < 0.01 was considered as significant. (∗ p < 0.01: Untreated control vs Irradiated control; # p < 0.01: Irradiated control vs RP-1 + 10 Gy). Data representedmean values ± S.D. of three experiments.

mained significantly (p < 0.05) higher than the untreatedcontrol upto 16 h (Fig. 2). RP-1 alone or when administeredbefore irradiation, increased GSH levels even more than theirradiated group upto 24 h (p < 0.01).

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Fig. 3. Effect of pre-irradiation administration of RP-1 to strain ‘A’ mice on radiation-induced changes in levels of mitochondrial oxidized glutathione (GSSG).The results have been expressed as nmol/L of glutathione/mg of mitochondrial protein. The significance between different groups was analysed using studentt-test and p < 0.01 was considered as significant. (∗ p < 0.01: Untreated control vs Irradiated control; # p < 0.01: Irradiated control vs RP-1 + 10 Gy). Datarepresented mean values ± S.D. of three experiments.

Irradiation and RP-1 treatment significantly (p < 0.01)increased the mitochondrial GSSG levels (Fig. 3) upto 24 h ascompared to untreated control. Pre-irradiation administrationof RP-1 also enhanced the GSSG levels after 2 h as comparedto untreated control, however with respect to irradiation aloneGSSG levels were found to be significantly (p < 0.01) higherat 8 h and later.

Complex I

Gamma irradiation enhanced the catalytic activity of com-plex I upto 4 h and thereafter decreased sharply at 8 h,and became comparable to the untreated control from 16 h(p < 0.01; Fig. 4). Administration of RP-1 to mice alsoenhanced the complex I activity upto 2 h with respect to un-treated control. Pre-irradiation administration of RP-1 signif-icantly (p < 0.01) inhibited the radiation-induced increasein complex I activity upto 16 h.

Complex I/III

Gamma irradiation increased complex I/III activity maxi-mally upto 4 h and thereafter; the activity decreased (p <

0.01; Fig. 5). RP-1 administration maximally enhanced thecomplex I/III activity within 1 h and thereafter the activitywas found to decrease steeply and attained level of untreated

control at 8 h (p < 0.01). Pre-irradiation administration ofRP-1 also showed similar pattern as was witnessed in RP-1treated mice (p < 0.01).

Complex II/III

With respect to untreated control, irradiation significantly(p < 0.01) decreased the complex II/III activity at 2 h andthereafter, increased upto 16 h (Fig. 6). Administration ofRP-1 significantly lowered the complex II/III activity upto 2h, but at 8 h and onwards the activity remained significantlyhigher than untreated control. Pre-irradiation administrationof RP-1 to mice significantly inhibited the radiation-inducedalterations in complex II/III activity (p < 0.01).

Mitochondrial membrane potential (MMP)

Gamma irradiation enhanced the MMP maximally at 4 hand remained significantly (p < 0.01) higher upto 16 h,but decreased significantly (p < 0.01) at 24 h with respectto untreated control (Table 1). Administration of RP-1 tomice maximally enhanced the MMP at 1 h and thereafter,found to be insignificant with respect to untreated control.Pre-irradiation administration of RP-1 also showed similarpattern as RP-1 treated group. The radiation-mediated de-crease in MMP was observed to be significantly (p < 0.01)

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Fig. 4. Effect of pre-irradiation administration of RP-1 to strain ‘A’ mice on radiation-induced changes in complex I activity. Complex I activity has beenexpressed as nmol/L/min/mg of mitochondrial (mt) proteins. The significance between different groups was analyzed using student t-test and p < 0.01 wasconsidered significant. (∗ p < 0.01: Untreated control vs Irradiated control; # p < 0.01: Irradiated control vs RP-1 + 10 Gy). Data represented mean values ±S.D. of three experiments.

Fig. 5. Radioprotective potential of RP-1 to complex I/III activity. Complex I/III activity has been expressed as nmol/L/min/mg of mitochondrial (mt) proteins.The significance between various groups was measured using student t-test and p < 0.01 was considered significant. (∗ p < 0.01: Untreated control vs Irradiatedcontrol; # p < 0.01: Irradiated control vs 10 Gy + RP-1). Each value represented the mean ± S.D. of three experiments.

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Fig. 6. Effect of pre-irradiation treatment with RP-1 on radiation-induced changes in complex II/III activity. Complex II/III activity has been expressedas nmol/L/min/mg of mitochondrial proteins. The significance between different groups was analysed using student t-test. (∗ p < 0.01: Untreated control vsIrradiated control; # p < 0.01: Irradiated control vs RP-1 + 10 Gy). Each value represented the mean ± S.D. of three experiments.

inhibited at 4 and 24 h post treatment interval by pre-irradiation treatment of mice with RP-1.

ATP levels

Gamma irradiation significantly (p < 0.01) enhanced themitochondrial ATP levels upto 16 h and thereafter attainedsignificantly (p < 0.01) low level at 24 h with respect to un-treated control (Table 2). RP-1 administration maximally en-hanced the ATP level at 1 h and was found to be insignificantwith respect to untreated control level at 8 h. Pre-irradiation

Table 1. Modulatory effect of RP-1 on the radiation-induced changes in mitochondrial membranepotential (MMP)

Irradiated control Drug control RP-1 + IrradiationTime (h) (10 Gy) (RP-1) (10 Gy)

1 193 ± 21∗ 339 ± 17 393 ± 22#

2 310 ± 14∗ 286 ± 33 289 ± 134 406 ± 24∗ 186 ± 24 273 ± 26#

8 165 ± 19∗ 123 ± 13 191 ± 2816 146 ± 13∗ 109 ± 8 146 ± 1924 68 ± 9∗ 95 ± 11 124 ± 13#

The change in MMP has been expressed as percentage of untreated control. p < 0.01 was con-sidered as significant. ( # p < 0.01: Radiation control vs RP-1 + Radiation; ∗ p < 0.01: Untreatedcontrol vs Radiation control). Data represented mean ± S.D. of three experiments. For untreatedcontrol the MMP was 100 ± 6.2

administration of RP-1 to mice also showed similar pattern asRP-1 alone group. The radiation-mediated decrease in ATP at24 h was significantly (p < 0.01) inhibited by pre-irradiationtreatment of mice with RP-1.

Lipid peroxidation

Gamma irradiation significantly (p < 0.01) increased for-mation of TBARS upto 16 h with respect to untreated control(Fig. 7). Administration of RP-1 to mice did not alter signifi-cantly the levels of TBARS with respect to untreated control

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Table 2. Effect of pre-irradiation administration of RP-1 to strain ‘A’ mice on radiation-inducedchanges in levels of mitochondrial ATP

µmol/L of ATP/mg of mitochondrial protein

Time (h ) Irradiated control (10 Gy) Drug control (RP-1) RP-1 + Irradiation (10 Gy)

1 12 ± 0.21∗ 15.3 ± 1.18 14.6 ± 2.2#

2 14 ± 1.01∗ 14 ± 1.3 13.3 ± 1.44 16 ± 1.22∗ 13 ± 2.4 13.1 ± 0.86#

8 13 ± 1.19∗ 10.1 ± 1.3 12.21 ± 1.4316 13 ± 1.21∗ 10.9 ± 0.8 12.43 ± 1.6124 6.8 ± 0.9∗ 10.5 ± 1.1 11.26 ± 1.3#

The results have been expressed as µmol/L of ATP/mg of mitochondrial protein. The significancebetween different groups was analysed using student t-test and p < 0.01 was considered assignificant. (∗ p < 0.01: Untreated control vs Radiation control; # p < 0.01: Radiation controlvs RP-1 + Radiation). Data represented mean values ± S.D. of three experiments. For untreatedcontrol the levels of ATP were 10.9 ± 0.62

(p < 0.01). Pre-irradiation administration of RP-1 to micesignificantly (p < 0.05) inhibited the radiation-induced for-mation of TBARS upto 16 h.

Protein oxidation

Irradiation increased the formation of protein carbonylswithin 1 h significantly (p < 0.01) and the levels were main-tained upto 24 h with respect to untreated control, whereas,no significant change in levels of protein carbonyls was ob-

Fig. 7. Protection of mitochondrial lipids by pre-irradiation administration of RP-1 against radiation-induced oxidative stress. The results have been expressedas nmol/L of TBARS/mg of mitochondrial proteins. Student t-test was used for analysis of significance between different groups. (∗ p < 0.01: Untreated controlvs Irradiated control; # p < 0.01: Irradiated control vs RP-1 + 10 Gy). Each value represented the mean ± S.D. of three experiments.

served in mice administered RP-1 (Fig. 8). Pre-irradiationadministration of RP-1 to mice significantly (p < 0.05) in-hibited the radiation-induced formation of protein carbonylsat all time periods studied (1, 2, 4, 8, 16 and 24 h).

Discussion

Reactive oxygen species are continuously produced in acell including mitochondria during the process of oxidativemetabolism [29]. γ -radiation are known to alter structure

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Fig. 8. Modulatory effect of RP-1 on radiation-induced oxidative damage to mitochondrial proteins. The results have been expressed as nmol/L of proteincarbonyls/mg of mitochondrial proteins. The significance between different groups was analysed using student t-test and p < 0.01 was considered significant.(∗ p < 0.01: Untreated control vs Irradiated control; # p < 0.01: Irradiated control vs RP-1 + 10 Gy). Data represented mean values ± S.D. of three experiments.

and physiology of mitochondria via oxidative modificationof DNA, lipids and proteins which ultimately leads to ox-idative burst and thereby disturbs the balance between proand antioxidant in favor of the former [5, 30–31]. Mito-chondria, therefore are an important target and contributorto oxidative stress and damage, induced by γ -radiation. Thepresent investigations revealed that exposure of mice to γ -radiation significantly enhanced the levels of superoxide an-ions in mitochondria (Fig. 1). The oxidative modification ofmitochondrial lipids (Fig. 7) and proteins (Fig. 8) could leadto increased leakage of electrons and subsequently the in-crease in superoxide anions. Administration of RP-1 to micealso increased the generation of superoxide anions upto 1 hwhich may be attributed to increased mitochondrial activityas also indicated by increase in MMP (Table 1) and ATP levels(Table 2). The results of superoxide anion levels are in agree-ment with the results of MMP and ATP levels. Pre-irradiationadministration of RP-1 showed significantly low levels of su-peroxide anions after 2 h. This can be explained in terms ofdecreased oxidative damage to mitochondrial lipids (Fig. 7),proteins (Fig. 8) and/or inhibition of radiation-induced al-teration in the flow of electrons between Electron TransportChain components (Figs. 5 and 6).

In the mitochondria superoxide anions are dismutated bymitochondrial superoxide dismutase to hydrogen peroxide,which is enzymatically neutralized by glutathione [32]. Glu-

tathione is an important redox regulator of cell/mitochondria[33–34], which is synthesized in cytosol and transported tomitochondria via energy dependent transporters [31, 35].During the present investigation, the radiation-induced in-crease in levels of glutathione (with respect to control; Figs.2 and 3) suggests the up-regulation of its neo-synthesis. Theradiation-induced increase in glutathione is a cellular bio-feed response to reduce the oxidant levels and consequentoxidative damage to macromolecules like DNA [33–34]. Mi-tochondria, isolated form RP-1 treated mice, showed signif-icantly higher levels of both GSH and GSSG with respect tountreated control. This further supports our earlier in vitrofindings (in HepG2 cells) that RP-1 induces neo-synthesisof glutathione [13]. Quercetin, a bioactive constituent of P.hexandrum, is known to up-regulate glutathione biosynthe-sis enzyme, γ -glutamyl cysteine synthetase [36–37]. Pre-irradiation administration of RP-1 to mice also enhanced lev-els of glutathione, which could have helped in lowering theradiation damage.

Complex I is an important enzyme of the electron trans-port chain (ETC), catalyzes the dehydrogenation of NADH,coupled with shuttling of protons across the inner mitochon-drial membrane [38–39]. The respiratory chain thus adjustscellular energy demands through the cellular redox status andmitochondrial membrane potential. In the present study, ex-posure of mice to γ -radiation increased the catalytic activity

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of complex I consistently upto 4 h (Fig. 4), which can beexplained in terms of the presence of high amount of GSHat corresponding time intervals (Fig. 2). Mitochondrial glu-tathione is known to regulate electron transport chain com-ponent activity [40]. The significant increase in complex Iactivity upto 2 h, on administration of RP-1 to mice, was at-tributed to increased mitochondrial activity. These findingsare also in agreement with our data of MMP (Table 1) and ATPlevels (Table 2). Pre-irradiation administration of RP-1 tomice significantly inhibited the radiation-mediated increasein complex I activity and thereby played an important role inrendering radioprotection via reducing the frequency of leak-age of electrons from electron transport chain components.

Oxidative modification of electron transport chain compo-nents or lipids increases the leakage of electrons [30, 41].Therefore, the radiation-induced alteration(s) in flow of elec-trons from complex I to complex III and complex II to III andits modulation by RP-1 were analyzed. Exposure of mice toγ -radiation showed a significant increase in the activity ofcomplex I/III upto 4 h, which was possibly due to increasedactivity of complex I (Fig. 4). The increased generation of su-peroxide anions (Fig. 1) at these time intervals and oxidativedamage to lipids (Fig. 7) or proteins (Fig. 8) indicated that in-terruption in the flow of electrons from complex I/III occurreddue to irradiation. At 8 h the sudden significant decrease incomplex I/III activity with respect to untreated control couldbe due to the oxidative damage to lipids (Fig. 7) or proteins(Fig. 8). Administration of RP-1 to mice showed a gradual de-crease in complex I/III activity with increased time intervalsindicating the metabolism of RP-1 constituents with time asalso reported by Gupta et al. [13]. These results supported thechanges observed in MMP (Table 1) and ATP levels (Table 2).Pre-irradiation administration of RP-1 to mice maximally en-hanced the activity of complex I/III at 1 h, showing clearlythat minimum radiation damage was caused to the path ofelectrons between complex I/III as measured by superoxideanion generation (Fig. 1). Similarly, radiation-induced de-crease in complex II/III activity at 2 h (Fig. 6) also could bedue to increased oxidative damage to mitochondrial macro-molecules. In case of administration of RP-1, the significantdecrease in complex II/III activity upto 2 h could be due toinhibition of complex II activity. RP-1 has already been re-ported to inhibit complex II in HepG2 cells by Gupta et al.[13]. The significant increase in complex II/III activity at 8 hand later (upto 24 h), indicated the metabolism of RP-1 con-stituent and minimum interruption in the flow of electronsas also measured by superoxide anion generation (Fig. 1).Pre-irradiation administration of RP-1 to mice significantlyinhibited the radiation-induced alteration in complex II/IIIactivity upto 24 h, which can be attributed to the presence ofhigh amounts of mitochondrial glutathione (Figs. 2 and 3),and/or inhibition of oxidative modification of mitochondriallipids and proteins (Figs. 7 and 8).

The mitochondrial electron transport chain activity andoxidative modification of membrane lipids and proteins reg-ulates the generation of MMP. In the present investigationthe alterations in MMP are in agreement with changes in thelevels of superoxide anions (Fig. 1), complex I (Fig. 4) andcomplex I/III (Fig. 5). The radiation induced increase in MMPcan be explained in terms of increased activity of complexI (Fig. 4), complex I/III (Fig. 5) and also through increasedgeneration and dismutation of superoxide anions (Fig. 1).The significant decrease in MMP at 24 h was attributed toincreased damage to mitochondrial membrane components(Figs. 7 and 8). As compared to untreated control, the signif-icant increase in MMP of RP-1 treated mice (upto 4 h) wasa clear indication of augmented mitochondrial activity. Sim-ilar results were obtained in case of pre-irradiation adminis-tration of RP-1. Administration of RP-1 to mice reduced theleakage of electrons from electron transport chain complexes(complex I, complex I/III, complex II/III) and thereby fur-ther decreased the generation of superoxide anions, which isknown to be deleterious for mitochondrial components.

The radiation-mediated increase in levels of ATP (Table 2)can be related to the increase in MMP. Mitochondrial AT-Pase catalyzes the potential dependent synthesis of ATP [38].The increased levels of ATP observed in case of RP-1 treat-ment to mice might be due to increased MMP, whereas sig-nificant inhibition in changes in levels of ATP in case ofpre-irradiation treatment of mice with RP-1 was presumablydue to inhibition of radiation-mediated alteration in MMPby RP-1.

The increased susceptibility of mitochondrial lipids tooxidative damage is primarily due to presence of redoxactive metal catalysts like hemoprotein and copper boundproteins [42]. Peroxidation of lipids invariably changesthe supra-molecular organization of membrane, whichultimately leads to decreased membrane fluidity [5] andincreased generation of superoxide anions. The peroxidationproducts are capable of forming protein cross linkagesthat inactivate several cellular constituents including themembrane-bound enzymes. The results of lipid peroxidation(Fig. 7) supported the results of superoxide generation(Fig. 1) and mitochondrial protein oxidation (Fig. 8). Theradiation-induced increase in levels of peroxidation productscould be attributed to the increased leakage of electronsfrom electron transport chain components, which in turnfurther increased the formation of superoxide anions (Fig. 1).Pre-irradiation administration of RP-1 to mice significantlylowered the peroxidation of mitochondrial lipids upto 16h. Some possible reasons for this observation include, highconcentrations of glutathione at corresponding time intervals(Figs. 2 and 3), decreased leakage of electrons from electrontransport chain components and scavenging of superoxideanions by the bioactive constituents of RP-1. Several workershave reported the efficacy of quercetin (a constituent of RP-1)

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and GSH to neutralize ROS [8, 43]. In addition, our earlierstudies have shown the antioxidant potential of RP-1 [13].

Oxidative damage to proteins alters its conformation viafragmentation, aggregation, formation of cross linkages in thepolypeptide chain, which further amplify the generation ofsuperoxide anions. Irradiation significantly increased levelsof superoxide anions, which in turn would have played a ma-jor role in enhancing the oxidative damage to mitochondrialproteins. The results of protein oxidation were in agreementwith the results on generation of superoxide anions (Fig. 1)and peroxidation of lipids (Fig. 7). The significant decreasein protein oxidation in case of pre-irradiation treatment ofmice with RP-1, could be either due to free radical scaveng-ing potential of constituents of RP-1 (as also explained bythe results of lipid peroxidation) or due to elimination of oxi-dants as explained by presence of high levels of antioxidantsin mitochondria during these time intervals or both.

The present investigation suggests that RP-1 protects mi-tochondria from radiation-induced oxidative stress in a mul-tifaceted manner under in vivo conditions and therefore couldbe used in the prevention and treatment of several free radicalailments associated with oxidative stress, including ionizingradiation-induced damage.

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