Molecular and Cellular Biochemistry 267: 167–176, 2004.c© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

Radioprotective mechanism of Podophyllumhexandrum during spermatogenesis

N. Samanta,1 K. Kannan,2 M. Bala1 and H. C. Goel11Department of Radiation Biology, Institute of Nuclear Medicine and Allied Sciences, Brig S. K. Mazumdar Marg, Delhi,India; 2School of Biotechnology, Guru Gobind Singh Indraprastha University, Delhi, India; 3Department of Microbiology,CCS University, Meerut (U.P.), India

Received 13 February 2004; accepted 16 June 2004

Abstract

RP-1, a herbal preparation of Podophyllum hexandrum has already been reported to provide protection against whole body lethalgamma irradiation (10 Gy). It has also been reported to render radioprotection to germ cells during spermatogenesis. Presentstudy was undertaken to unravel the cellular and molecular mechanism of action of RP-1 on testicular system in strain ‘A’ mice.Various antioxidant parameters such as thiol content, glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) enzyme activity, lipid peroxidation (LPO) and total protein levels were investigated. Thiol content wasseen to increase significantly (p < 0.05) in both RP-1 alone and RP-1 pretreated irradiated groups over the irradiated groupsat 8, 16 and 24 h. Irradiation (10 Gy) significantly decreased GPx, GST and GR activity in comparison to untreated controlbut RP-1 treatment before irradiation significantly (p < 0.05) countered radiation-induced decrease in the activity of theseenzymes. Radiation-induced LPO was also found to be reduced at all time intervals by RP-1 treatment before irradiation. Ascompared to irradiated group the protein content in testicular tissue was increased in RP-1 pretreated irradiated group at 4 and 16h significantly (p < 0.05). Comets revealed by single-cell gel electrophoresis were significantly longer (p < 0.001) in irradiatedmice than in unirradiated control. RP-1 treatment before irradiation, however, rendered significant increase (p < 0.05) in cometlength over the corresponding control and irradiated group initially at 4 h but at later time points, this was reduced significantly(p < 0.01) as compared to the irradiated group. RP-1 treatment alone rendered shorter comets at 8, 16 and 24 h than irradiatedgroups (p < 0.001). This study implies that RP-1 offers radioprotection at biochemical and cytogenetic level by protectingantioxidant enzymes, reducing LPO and increasing thiol content. (Mol Cell Biochem 267: 167–176, 2004)

Key words: Podophyllum hexandrum, glutathione peroxidase, glutathione reductase, radioprotection, spermatogenesis

Introduction

There have been many reports which implicate the role of en-vironmental pollutants in decreasing male fertility in termsof sperm count, structure and quality [1, 2]. Reactive oxy-gen species (ROS) and oxidative stress play a major role inthe genesis of such events. Exposure to X-rays and gammarays have been exhaustively reported to induce DNA damagein germ cells causing mutagenic, carcinogenic and terato-genic effects [3, 4] in the progeny and produce reversibleor permanent sterility in males depending on the radiationdose.

Address for offprints: H. C. Goel, Department of Microbiology, CCS University, Meerut, (U.P.) 250 005, India (E-mail: goelharish@hotmail.com)

Low-linear energy transfer gamma radiation is known tocause oxidative damage by generating species like hydroxyl,superoxide, peroxyl radicals and hydrogen peroxide. The freeradicals disturb the cellular homeostasis by peroxidation ofmembrane lipids, oxidation of proteins, base damage andadduct formation in DNA which ultimately lead to cell deathif the damage is beyond repair [5]. The inherent antioxidantdefence system of the cell including glutathione peroxidase,glutathione–S-transferase, catalase and superoxide dismu-tase competitively counteract the oxidative stress. However,the free-radical insult surpassing the inherent cellular defencemechanism warrants external antioxidant supplementation.

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Herbal medicines have been in use since time immemorialfor curing various diseases such as diabetes, arthritis, etc. thatare alleged to be mediated by free radicals. Since radiationdamage also involves oxidative damage, agents capable ofantioxidant action could protect against radiation-inducedlethality. Though molecular agents like cysteamine, WR-2721, MPG, etc. have also been investigated for their radio-protective properties, the side effects associated with themhave restrained their use [6].

Podophyllum hexandrum, widely used in Ayurveda hasbeen studied for its radioprotective manifestations at var-ious levels of organization. The extract of P. hexandrum,(RP-1) has shown substantial protection to gastrointestinalsystem and testicular system in vivo [7, 8]. The protectiveeffect has been attributed to free-radical scavenging ability,metal chelation and inhibition of lipid peroxidation in vitro[9].

Our earlier studies have indicated protection of germ cellsduring spermatogenesis at cellular and histological level bypretreatment with RP-1. Therefore, it was necessary to inves-tigate the mode of action of RP-1 at biochemical and cytoge-netic level at short time intervals in the testis.

Material and methods

Chemicals

Chemicals were procured from different sources: 1-chloro2,4-dinitrobenzene (CDNB) from Merck, Germany; reducedglutathione (GSH), oxidised glutathione (GSSG), reducednicotinamide dinucleotide phosphate (NADPH), glutathionereductase (from baker’s yeast), agarose, sodium laurylsarcosine, ethidium bromide (EtBr), triton-X-100, RNase,ethylenediamine-tetraacetic acid sodium salt (EDTA-Na), 2-thiobarbituric acid (TBA), and trichloroacetic acid (TCA)from Sigma Chemical Co., St. Louis, MO, USA; propidiumiodide (PI) from Molecular Probes, USA. All other chemi-cals used were from reputed Indian firms and were of standardgrade and high purity.

Animals

Swiss albino strain ‘A’ adult male mice (10–12 weeks) weigh-ing 25 ± 2 g were maintained under standard laboratory con-ditions (25 ± 2 ◦C; photoperiod 12 h light/dark cycle) andfed standard animal food pellets (Amrut Laboratory feed,India) and water ad libitum. Four to five animals were keptin polypropylene cages. All experiments involving animalswere done following Animal Ethics Committee Rules andRegulations of this Institute.

Preparation of the extract

Dried rhizome of Podophyllum hexandrum supplied by FieldResearch Laboratory, Leh (J& K, India) was powdered me-chanically and known quantity of the powder was mixed indistilled water and kept at 37 ◦C in an incubator for 24 h andfiltered thereafter using Whatman No 1 filter paper. The fil-trate was passed through Millipore filter (0.22 µ), lyophilizedand stored at 4 ◦C. The powder was suitably diluted in tripledistilled water before use.

Irradiation

Whole body irradiation was given through Co60 gamma cell(model 220 – Atomic Energy Commission, Canada Ltd.) andfresh air was continuously circulated in the irradiation cham-ber to avoid hypoxic conditions. Mice were kept in perforatedplastic bottles for irradiation individually. The dose rate was0.92 rad/s during the experimental period.

Treatment groups

Animals were divided into following four groups and sacri-ficed at 4, 8, 16 and 24 h after various treatments:

Group I: (n = 5 × 2): injected saline i.p., unirradiated con-trol; Group II: (n = 5 × 2): 10 Gy whole body gamma irra-diated; Group III: (n = 5 × 2): RP-1 (200 mg/kg b.w., i.p.)injected 2 h before 10 Gy whole body gamma irradiation;Group IV: (n = 5 × 2): RP-1 (200 mg/kg b.w.), injected i.p.

Preparation of homogenate

Testes were dissected out and kept in PBS (pH 7.4). One ofthe testis was used to make single-cell suspension in PBS andprocessed for single-cell gel electrophoresis. The testis wasmade free of fats and connective tissues, blotted dry, weighedand homogenized in ice-cold PBS (pH 7.4) to yield a 10% w/vhomogenate which was subsequently used to measure lipidperoxidation and thiol content. The remaining homogenatewas centrifuged at 10,000×g for 30 min at 4 ◦C and the su-pernatant was used for assaying GST, GR and GPx enzymeactivities. All the experiments were repeated twice.

Lipid peroxidation

Peroxidized membrane lipids were estimated by the methoddescribed by Beuge and Aust [10]. To the 250 µl of crude ho-mogenate in duplicate, 0.1 M phosphate buffer (pH 7.4) wasadded to make the volume 1 ml. Thereafter, 2 ml of Beuge

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and Aust reagent (1% TBA and 15% TCA in 0.1 N NaOH)was added and the contents were boiled at 100 ◦C for 15min. After cooling, the contents were centrifuged at 1,000×gand the absorbance of pink chromogen was measured at 532nm in the supernatant. Blank contained phosphate bufferonly and results were expressed as nM of TBARS/mg ofproteins.

Thiol content

Thiol content was estimated according to Moron et al. [11].To 0.5 ml of testicular homogenate, 0.1 ml of 25% TCAwas added and incubated at 4 ◦C for 10 min. It was thencentrifuged at 500 rpm for 10 min at 4 ◦C. The supernatant(0.1 ml) was added to 0.9 ml of phosphate buffer (pH 8.0, 1M) and 2 ml 0.6 mM DTNB. The absorbance was recordedat 412 nm against blank containing TCA.

Glutathione-S-transferase activity

The GST activity in the supernatant was determined spec-trophotometrically at 37 ◦C according to the method of Habiget al. [12]. The reaction mixture (3 ml) contained 1.7 ml of0.1 M phosphate buffer (pH 6.5), 0.1 ml of 30 mM reducedglutathione (GSH) and 0.1 ml of 30 mM CDNB. After incu-bating the reaction mixture at 37 ◦C for 5 min, 0.1 ml of di-luted cytosolic fraction was added and change in absorbancewas followed for 3 min at 340 nm. Reaction mixture withoutenzyme was used as blank. The specific activity of GST wascalculated as nM of GSH-CDNB conjugate formed/min/mgprotein using extinction coefficient 9.6 mM−1/cm and resultswere expressed as relative change in activity with respect tocontrol.

Glutathione peroxidase activity

Glutathione peroxidase (GPx) activity was assayed by themethod of Pagila and Valentine [13]. Briefly 1 ml of reactionmixture containing 25 µl of cytosol, GSH (30 mM) phosphatebuffer (0.1 M; pH 7.4), glutathione reductase 0.24 U/ml, 1mM sodium azide and NADPH (2 mM) were incubated at37 ◦C. Thereafter, 100 µl of H2O2 (400 mM) was added tostart the reaction. The reaction was time scanned for 3 minat 340 nm. The specific activity was calculated using 6.22mM−1/cm as the extinction coefficient and expressed as rel-ative change in activity with respect to control. A blank wasalso run without adding cytosol.

Glutathione reductase activity

Glutathione reductase (GR) activity was assayed using theprocedure described by Carlberg and Mannervik [14]. In 1ml of 0.2 M sodium phosphate buffer (pH 7.0) containing afinal concentration of 2 mM EDTA, 1 mM GSSG and 0.2 mMNADPH, the reaction was initiated by the addition of 25 µLof cytosolic fraction. The decrease in OD/min was followedfor 3 min at 340 nm. The specific activity was calculatedusing extinction coefficient of 6.22 mM−1/cm and expressedas relative change in activity with respect to control.

Protein estimation

Protein content of the testicular homogenate was estimatedby Lowry et al. [15]. Alkaline solution (98 volumes of 2%Na2CO3 in 0.1 N NaOH + 1 ml of 0.5% CuSO4 in 1% sodiumpotassium tartarate) was prepared fresh. About 5 ml of this so-lution was added to 1.0 ml of test solution, mixed thoroughlyand allowed to stand at room temperature for 10 min. Sub-sequently 1N Folin reagent was added and again allowed tostand for 30 min at room temperature. The absorbance wasmeasured at 660 nm against the reference blank. The pro-tein content in each sample was calculated from the standardcurve made using BSA and was expressed as mg/ml.

Comet assay

Strand breaks in individual testicular cells were detected us-ing single-cell gel electrophoresis as per the procedure de-scribed by Singh et al. [16] with slight modifications. Briefly,after different treatments, total testicular cell suspension wasresuspended at a concentration of 2 × 104 cells/400 µl of1% low-melting point agarose (prepared in PBS, pH 7.4 con-taining EDTA), and immediately pipetted on to microscopicslides pre-coated with agarose and spread to form a uniformlayer. Thereafter, slides were transferred to cold surface tohasten gelling. After 10 min the slides were immersed in ice-cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mMTris, 1% sodium lauryl sarcosine, 5% DMSO and 1% triton-X-100, pH 10) and left in refrigerator for 1 h. The slides weretransferred to an electrophoretic tray containing freshly pre-pared alkali buffer (300 mM NaOH, 1 mM EDTA, pH 13)for 25 min to allow DNA unwinding. Thereafter, the slideswere electrophoresed at 0.5 V/cm for 1 h under similar con-ditions and washed thrice by adding 0.1 M Tris, pH 7.4 on tothe slides. The DNA was stained with propidium iodide (50µM) for 10 min. and visualized under LEITZ fluorescencemicroscope. The length of the longest comets was measuredfrom head to tail using micrometer and at least 50 cometswere measured for each animal in each group and averaged.

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Statistical analysis

Data from 4 to 5 observations from two experiments werepooled and analysed. The level of significance was testedusing student’s t-test; probability level of p < 0.05 was con-sidered significant.

Results

Lipid peroxidation (LPO)

RP-1 treatment alone decreased LPO measured as TBARS(nM/mg protein) initially at 4 h (18.86 ± 1.86; p < 0.05)(Fig. 1), however it revealed a recovering trend at 8 and 16 hand the values were similar to the control (Table 1). Irradiation(10 Gy) increased the LPO significantly (p < 0.05) up to 16 hfollowed by a decrease to achieve values of untreated controlat 24 h. Pre-irradiation treatment with RP-1 did not permitradiation-induced increase in LPO up to 24 h and the valueswere significantly (p < 0.01) lower than the irradiated group.

Thiol content

Administration of RP-1 elevated thiol content and the in-crease remained significantly higher (p < 0.01) at 16 and 24h as compared to control (Fig. 2). Irradiation however sup-pressed thiol levels up to 16 h and gradually started recov-ering thereafter. RP-1 treatment before irradiation increasedthe thiol content as compared to irradiated groups (Table 1).The increase was significant at 16 and 24 h (p < 0.05).

Fig. 1. Effect of RP-1 (200 mg/kg b. w.) on radiation-induced lipid peroxidation measured as nM TBARS/mg protein at various time intervals in mice testis.Data have been expressed as mean ± S.E., p values ∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.001; a: control versus 10 Gy, b: 10 Gy versus RP-1 + 10 Gy, c: control versusRP-1.

Glutathione peroxidase activity

RP-1 treatment decreased the GPx activity initially at 4 hsignificantly (p < 0.05) in comparison to control (Fig. 3) butat later periods it gradually increased up to 24 h significantly(p < 0.001). Irradiation significantly (p < 0.01) decreasedthe enzyme activity which remained less than the untreatedcontrol up to 24 h. RP-1 treatment prior to irradiation revealedrecovery of radiation-induced decrease in GPx activity after8 h post irradiation period (Table 1).

Glutathione reductase activity

After RP-1 treatment the GR activity was enhanced signifi-cantly (p < 0.05) at 8 and 16 h in comparison to the control(Fig. 4). Effect of irradiation (10 Gy) on GR activity remainedsignificantly lower (p < 0.05) only at 16 h with respect tounirradiated control (Table 1). Pre-irradiation treatment withRP-1 increased GR activity maximally at 16 h in comparisonto irradiated control.

Glutathione-S-transferase activity

RP-1 treatment alone caused significant (p < 0.05) in-crease in GST activity at 24 h only (Fig. 5). Irradiationsignificantly (p < 0.05) reduced GST activity in compar-ison to control at 16 h. RP-1 treatment before irradia-tion reduced the GST activity initially in comparison tothe irradiated group, but later it was found to be signif-icantly (p < 0.01) elevated at 16 and 24 h (Table 1).

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Table 1. Effect of RP-1 (200 mg/kg b.w., – 2 h) on various biochemical parameters (changes shown as percentage of control value) intestis of mice given different treatments (+) indicates increase while (−) indicates decrease with respect to untreated control

Biochemical parameter Group 4 h 8 h 16 h 24 h

Lipid peroxidation nM TBARS/mg protein 10 Gy (+) 19.4 (+) 17.7 (+) 67.15 (+) 10.25

RP1+10 Gy (−) 19.0 (−) 6.2 (+) 20.8 (−) 18.11

RP-1 (−) 20.7 (+) 4.26 (+) 6.96 (−) 32.25

Thiol content (µg GSH/ mg protein) 10 Gy (−) 9.3 (−) 15.64 (−) 26.45 (−) 19.4

RP-1+10 Gy (+) 8 (+) 12 (+) 4.3 (+) 7.73

RP-1 (−) 0.85 (+) 9.35 (+) 27 (+) 18.59

GPx activity 10 Gy (−) 40.6 (−) 31 (−) 24.5 (−) 16.6

RP-1+10 Gy (−) 36 (−) 29.45 (+) 9 (+) 25

RP-1 (−) 13.4 (+) 19.8 (+) 23.3 (+) 44

GR activity 10 Gy (−) 2.2 (+) 7 (−) 11 (+) 4.4

RP-1+10 Gy (−) 16.3 (−) 0.97 (+) 10.1 (−) 6.7

RP-1 (−) 7.4 (+) 10.9 (+) 6.4 (−) 0.92

GST activity 10 Gy (−) 5.06 (−) 4.31 (−) 18.32 (−) 4.2

RP-1+10 Gy (−) 13.2 (−) 19.8 (+) 14.58 (+) 36.52

RP-1 (−) 3 (+) 1.2 (−) 0.5 (−) 22.8

Protein Content (µg/g tissue) 10 Gy (−) 16.2 (−) 14.08 (−) 27.3 (−) 29.62

RP-1+10 Gy (−) 4.72 (−) 9.8 (−) 10.5 (−) 24.22

RP-1 (−) 1.74 (−) 7.45 (−) 11.02 (−) 12.66

SCGE (comet length µ) 10 Gy (+) 40.2 (+) 89.6 (+) 126.3 (+) 88.3

RP-1+10 Gy (+) 49.6 (+) 71.5 (+) 54.39 (+) 48.9

RP-1 (+) 28.8 (+) 16.9 (+) 8.57 (+) 6.77

Fig. 2. Effect of RP-1 (200 mg/kg b. w.) on thiol contents measured as µg GSH/mg protein in mice testis. Data have been expressed as mean ± S.E., p values∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.001; a: control versus 10 Gy, b: 10 Gy versus RP-1 + 10 Gy, c: control versus RP-1.

Comet assay

The photomicrographs of single-cell gel electrophoresis ofsome treatment groups have been depicted in Fig. 6. As aresult of RP-1 treatment, comet lengths increased at 4 h but

were thereafter comparable to unirradiated control (Fig. 7).Irradiation (10 Gy), as compared to unirradiated control,caused significant (p < 0.001) increase in tail length up to16 h (Fig. 5, Table 1). RP-1 treatment before irradiationcountered the radiation-induced increase in comet length,

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Fig. 3. Effect of RP-1 (200 mg/kg b. w.) on glutathione peroxidase activity in mice testis. Data have been expressed as mean ± S.E. and depicted in relation tocontrol. p values ∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.001; a: control versus 10 Gy, b: 10 Gy versus RP-1 + 10 Gy, c: control versus RP-1.

Fig. 4. Effect of RP-1 (200 mg/kg b. w.) on glutathione reductase activity in mice testis. Data have been expressed as mean ± S.E. and depicted in relation tocontrol. p values ∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.001; a: control versus 10 Gy, b: 10 Gy versus RP-1 + 10 Gy, c: control versus RP-1.

which was significantly (p < 0.001) lower at 8, 16 and 24 h(Fig. 6).

Protein content of testicular tissue

Irradiation caused gradual but significant (p < 0.05) decreasein protein content of the testicular tissue with passage oftime as compared to control (Fig. 8). RP-1 treatment alonedid not significantly alter the protein content with respectto control. RP-1 treatment before irradiation countered theradiation-induced decrease in the protein levels only up to 16h intervals studied here and the values were comparable tothe unirradiated control (Table 1).

Discussion

Spermatogenesis is a long, complex and finely tuned process.The developing sperm cells are highly sensitive to endoge-nous and exogenous stresses. Exposure to cytotoxic agentsmay damage germ cells at different stages of differentiationleading to a temporary or permanent impairment of fertility[17]. Exposure to mutagenic agents can induce DNA damagein germ cells resulting in the loss of conceptions, fetal abnor-malities or genetic disorders in the progeny [18].

Ionizing radiations are known to enhance the productionof reactive oxygen species which subsequently cause damageto target molecules [19, 20]. Superoxide dismutase, catalase,

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Fig. 5. Effect of RP-1 (200 mg/kg b. w.) on glutathione-S-transferase activity in mice testis. Data have been expressed as mean ± S.E. and depicted in relationto control. p values ∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.001; a: control versus 10 Gy, b: 10 Gy versus RP-1 + 10 Gy, c: control versus RP-1.

Fig. 6. Effect of different treatments on cells from mice testis assessed by single-cell gel electrophoresis (A) unirradiated control, (B) irradiated (10 Gy) andvisualized after 16 h, (C) treated with RP-1 before irradiation (10 Gy) and visualized after 16 h.

Fig. 7. Effect of RP-1 (200 mg/kg b. w.) on radiation-induced DNA damage evaluated by length of comet measured in microns. Data have been expressed asmean ± S.E., p values ∗ <0.05, ∗∗ <0.01, ∗∗∗ <0.001; a: control versus 10 Gy, b: 10 Gy versus RP-1 + 10 Gy, c: control versus RP-1.

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Fig. 8. Effect of RP-1 (200 mg/kg b. w.) on radiation-induced suppression of total protein content (mg/g tissue) in mouse testis. Data have been expressed asmean ± S.E., p values ∗ <0.05, ∗∗ <0.01,∗∗∗ <0.001; a: control versus 10 Gy, b: 10 Gy versus RP-1 + 10 Gy, c: control versus RP-1.

glutathione peroxidase, glutathione-S-transferase and glu-tathione reductase are endogenous antioxidant enzymes thatprotect cells from oxidative stress. The studies done here rep-resent the total antioxidant activity of different types of cellspresent in the testicular tissue viz. spermatogonia, spermato-cytes, spermatozoa, sertoli cells, etc.).

Further studies to unravel the effects of radiation on spe-cific isolated cells could be quite interesting from the pointof view of understanding the mechanism of action of RP-1.

GPx is the major antioxidant enzyme responsible for detox-ification of the products of LPO [21, 22]. Following irradi-ation GPx activity was seen to decrease at 4 h significantlyfrom control (Fig. 8) and then slowly kept on increasing withtime but could not regain the control values. This could bebecause of the conformational changes in the structure ofenzymes after irradiation and consequent suppression in theenzyme activity [23]. The animals given RP-1 treatment ei-ther alone or before irradiation, however, exhibited increasedactivity of the enzymes. This could have been achieved bypreserving the confirmation of enzymes or because of the up-regulation of their synthesis. Further studies are required toconfirm these phenomena.

Ionizing radiation induces LPO, which can lead to DNAdamage and cell death [24, 25]. Therefore, any agent thatcan provide protection against such damage will help in cellsurvival. LPO levels due to oxidative damage increased withradiation exposure up to 16 h significantly in comparison tocontrol (Fig. 1). After irradiation, the free radicals are gener-ated almost immediately. These free radicals initiate chain re-actions leading to the production of secondary radicals, whichkeep on accumulating with passage of time. This is repre-sented by the maximal TBARS levels at 16 h in this study. Theantioxidant activity due to endogenously available molecules

imparts recovery bringing down the level of TBARS as seenat 24 h. The levels of GSH (Fig. 2) and activity of GPx (Fig.8), GR (Fig. 3) and GST (Fig. 4) are commensurately low at16 h. It is further corroborated by the maximal DNA damageobserved at 16 h (Fig. 6).

The pro-oxidant activity of RP-1 has been reported by Premet al. [9]. In fact many compounds such as flavonoids presentin RP-1 have been shown to generate hydroxyl radicals andsuper oxide anions manifesting auto-oxidative action. In thisstudy, presence of RP-1 alone has revealed the oxidative ef-fect (TBARS levels) up to 16 h. These molecules possibly getmetabolized and therefore, their levels decrease with passageof time. The up-regulation of the production of anti-oxidantmolecules initiated by the pro-oxidant activity of RP-1 indue course of time, could also account for decreased levelsof TBARS at 24 h. Testis is a hypo-vascularised tissue andtherefore hypoxic in nature. Due to hypo-vascularisation, thetransport of active molecules present in RP-1 reach the in-tracellular sites slowly and are simultaneously retained forlonger duration. This may be responsible for the long-lastingeffect of RP-1. RP-1 treatment prior to irradiation how-ever decreased LPO, indicating radioprotective action of P.hexandrum.

The elevation of glutathione levels indicates enhanced neu-tralization of radiation-induced free radicals and subsequentreduction of LPO. The RP-1 treatment alone or before irra-diation efficiently countered the radiation-induced decreasein thiol levels (90% of which is reduced glutathione) andmaintained it significantly elevated up to 24 h in comparisonto the irradiated group (Fig. 2). Flavonoids are known toactivate glutathione-synthesizing enzyme [26]. This is alsosupported by the increase in the activity of glutathionedependent enzymes such as GPx, GR and GST (Figs. 3, 4

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and 5). A relationship between antioxidant properties and ra-diation protection flavonoids has been suggested by Shimoiet al. [27]. It was further corroborated by reduction in LPOlevels in the RP-1 treated groups. Similar to the action of RP-1, ginger rhizome, a dietary ingredient, has also been reportedto protect against gamma radiation by elevating glutathionelevels and reducing LPO in rats and mice [28, 29].

The protein contents of the testicular tissue have been ob-served to decrease with time in irradiated groups probablybecause of removal of damaged protein or inhibition of pro-tein synthesis (Fig. 8). This could be due to excessive damageto the genetic machinery. RP-1 pre-treated irradiated groupscountered the radiation-induced decrease in protein contentreflecting preservation of genetic machinery and/or proteins.

Oxidative stress can cause electron leak to oxygen andform superoxide radicals and other ROS, which are likely todamage the enzymes that normally maintain low-intracellularfree calcium. Increased intracellular free calcium could leadto activation of calcium-dependent proteases that in turn cancause a further increase in free radical production and DNAdamage [22]. Radiation-induced DNA damage, measured bysingle-cell gel electrophoresis (comet length) was persistentup to 16 h (Figs. 6 and 7) and declined thereafter. The declinecould be the result of removal of damaged DNA or its repairwith time [30]. RP-1 treatment alone showed increased comettail length initially at 4 h (Fig. 6), indicating its pro-oxidantactivity. The DNA damage induced by RP-1 corroborates ourearlier studies [7, 31]. The decrease in the comet length by 24h (Fig. 6), could be possible because of the removal of activemolecules (constituents of RP-1) from the site of action atlater periods due to their metabolisation. At the same timethe recovery of damaged DNA could also contribute to thereduced tail length. In the RP-1 treated irradiated group, theincreased comet tail length observed at 4 h, as compared tothe irradiated control (p < 0.05), indicated increased DNAdamage, as has been reported earlier with respect to thymus[31]. The pro-oxidant action of RP-1 followed by induction offree radicals by radiation, could account for increased DNAdamage and hence increased comet length. The decreasedcomet length after 4 h (Fig. 7) indicated the radio-protectivemanifestation of RP-1.

RP-1 has been shown to manifest radioprotective actionas evaluated by various parameters viz. metal chelation, freeradical scavenging [9], antioxidant potential both at in vitroand in vivo levels [32] and stimulation of cell prolifera-tion [33]. Various constituents of P. hexandrum such as cy-tokines and polyphenols could be responsible for such ac-tions [33]. The present study related to the testicular systemalso corroborates the earlier studies on other systems in re-spect of antioxidant enzymes, DNA damage and recovery [31,32]. This suggests that P. hexandrum preparation could be apotential candidate for screening as a radioprotector for clin-ical applications.

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