REC-2006—A Fractionated Extract of Podophyllum hexandrum Protects Cellular DNA from Radiation-Induced Damage by Reducing the Initial Damage and Enhancing Its Repair In Vivo

Podophyllum hexandrum, a perennial herb commonly known as the Himalayan May Apple, is well known in Indian and Chinese traditional systems of medicine. P. hexandrum has been widely used for the treatment of venereal warts, skin infections, bacterial and viral infections, and different cancers of the brain, lung and bladder. This study aimed at elucidating the effect of REC-2006, a bioactive fractionated extract from the rhizome of P. hexandrum, on the kinetics of induction and repair of radiation-induced DNA damage in murine thymocytes in vivo. We evaluated its effect on non-specific radiation-induced DNA damage by the alkaline halo assay in terms of relative nuclear spreading factor (RNSF) and gene-specific radiation-induced DNA damage via semi-quantitative polymerase chain reaction. Whole body exposure of animals with gamma rays (10 Gy) caused a significant amount of DNA damage in thymocytes (RNSF values 17.7 ± 0.47, 12.96 ± 1.64 and 3.3 ± 0.014) and a reduction in the amplification of β-globin gene to 0, 28 and 43% at 0, 15 and 60 min, respectively. Administrating REC-2006 at a radioprotective concentration (15 mg kg−1 body weight) 1 h before irradiation resulted in time-dependent reduction of DNA damage evident as a decrease in RNSF values 6.156 ± 0.576, 1.647 ± 0.534 and 0.496 ± 0.012, and an increase in β-globin gene amplification 36, 95 and 99%, at 0, 15 and 60 min, respectively. REC-2006 scavenged radiation-induced hydroxyl radicals in a dose-dependent manner stabilized DPPH free radicals and also inhibited superoxide anions. Various polyphenols and flavonoides present in REC-2006 might contribute to scavenging of radiation-induced free radicals, thereby preventing DNA damage and stimulating its repair.


Introduction
Radiation-induced free radicals oxidize cellular biomacromolecules like DNA, proteins and lipids generating a variety of cellular dysfunctions leading to cell death [1,2]. Damages to DNA, such as single-and double-strand breaks, base modifications and adduct formation, are considered as biologically significant cellular lesions [3][4][5]. Normally, cells operate diverse pathways to repair oxidative damage to DNA. Among them, the most important are direct repair of an adduct, base-excision repair, nucleotide-excision repair, homologous recombination, non-homologous end-joining, DNA inter-strand cross-link repair and DNA mismatch repair [6].
Podophyllum hexandrum (also known as Himalayan May Apple), a herb thriving at high altitudes in the Himalayas has been extensively used in Ayurvedic system of medicine for the treatment of ailments like monocytoid leukemia, 2 Evidence-Based Complementary and Alternative Medicine Hodgkin's lymphoma, bacterial and viral infections, venereal warts, rheumatoid artharalgia associated with limb numbness and pycnogenic infections of skin tissue [18][19][20]. The root and rhizome of P. hexandrum are reported to contain a number of compounds with significant pharmacological properties, for example, epipodophyllotoxin, podophyllotoxone, 4-methylpodophyllotoxin, aryltetrahydronaphthalene lignans, flavonoids such as quercetin, quercetin-3glycoside, 4-demethylpodophyllotoxin glycoside, podophyllotoxinglycoside, kaempferol and kaempferol-3-glucoside [21,22].
An extract (code named as RP-1) from the rhizome of P. hexandrum reportedly protects mice against a lethal dose of ionizing radiation [23]. Different mechanisms were proposed to account for these radioprotective properties, including free radical scavenging, metal chelation and the elevation of antioxidant defense enzymes [24,25]. A variety of scenarios involve radiation exposures in the moderate range (i.e., 1-10 Gy), including cancer therapy, planned reactor maintenance and the explosion of a dirty bomb (radioactive dispersal device). With the likelihood that exposure to a moderate radiation dose will result in radiationinduced DNA damage entailing cell death, we undertook this study to assess the effect of REC-2006 [25], a fractionated extract of rhizome of P. hexandrum, on the induction and repair kinetics of radiation (10 Gy)-induced gene-specific and non-specific DNA damage in murine thymocytes in vivo.
Intraperitoneal administration of REC-2006 to mice at 15 mg kg −1 body weight conferred more than 90% protection against whole-body irradiation (10 Gy) as compared to 72% offered by 34.5 mg kg −1 body weight administration of the parent plant extract RP-1. The radiosensitive nature of thymocytes provides an attractive system to study DNA repair [26,27]. As we are interested to look at the early kinetics of DNA repair within 1 h after irradiation, we choose mouse β-globin gene which is constitutively expressed in transcriptionally active or inactive domains thus acting as a biomarker of overall DNA repair [28,29]. Similar approach has also been used for investigating DNA damage repair [13,30].
Observations of the effect of P. hexandrum upon the repair kinetics of gamma-radiation-induced DNA damage will further delineate the mechanisms involved in overall radioprotective effects of this plant.

Drug Administration and Experimental Plan
. REC-2006, tested negative for endotoxins, dissolved in 8% DMSO and a radio protective concentration of 15 mg kg −1 body weight was injected intraperitoneally to the different groups, 1 h before exposing the mice to 10 Gy gamma rays (the lethal dose) following the experimental plan shown in Table 1.
Evidence-Based Complementary and Alternative Medicine 3 2.6. Isolation of Thymocytes. Mice were killed by cervical dislocation, dissected, the abdominal cavity was perfused with 0.9% saline and the thymus was removed. All visible clots were segregated carefully. We minced the thymic lobes finely, and gently crushed them with the plunger of a syringe; the resultant cell suspension was passed through a 25-gauge needle to avoid cell aggregates. All procedures were carried out on ice.

Alkaline Halo Assay.
We detected DNA single-strand breaks in individual cells using the alkaline halo assay [31] modified for murine thymocytes. After the different treatments, thymocytes were suspended (1.5 × 10 4 to 2.0 × 10 4 per 100 μl) in a 1.5% low-melting agarose solution in phosphate buffered saline, pH 7.4, and immediately pipetted on to slides coated with 1.0% normal agarose and spread uniformly. After gelling was complete, the slides were immersed in the alkali buffer (0.1 M NaOH and 1 mM EDTA; pH 13) for 20 min at 4 • C, washed, and then stained with 10 μg ml −1 ethidium bromide for 5 min. The images were acquired on a fluorescent microscope (Leica Q550 FW, Wetzlar, Germany) equipped with a 40× Neofluar objective lens, and analyzed using Comet score software (Tritek Corporation, Northern Virginia, USA). The intensely stained, intact chromatin mass forms the central core of the halo, while the broken DNA fragments constitute its diffusely stained periphery. Levels of DNA damage were expressed as the relative nuclear spreading factor (RNSF) values, calculated by subtracting the RNSF values of control cells from those of treated cells [13].

DNA Isolation from
Thymocytes. The genomic DNA from murine thymocytes (5 × 10 6 ) was isolated using the DNeasy isolation kit (DNeasy Tissue kit, Qiagen, Chatsworth, USA) as per manufacturer's instructions. DNA yields were estimated spectrophotometrically by measuring absorbance at 260 and 280 nm. An aliquot of each PCR product was resolved by agarose-gel electrophoresis [33]. Briefly, 10 μl of the sample was mixed with 5 μl of loading dye (0.025% bromophenol blue, 0.25% xylene cyanol and 40% sucrose in water) and loaded into the wells of a 1.2% agarose gel along with the 1 kb DNA ladder (Fermentas, MD, USA) to confirm the size of the PCR-amplified product. Electrophoresis was carried out for 2.5 h at 5 V cm −1 in the Tris acetate buffer in the presence of 0.5 μg ml −1 ethidium bromide. The amplified products were quantified densitometrically using Image Quant software (Molecular Dynamics, Sunnyvale, CA).

DPPH Radical Scavenging Activity.
We measured the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging ability of REC-2006 according to the method of Shimada et al. [34]. A solution of DPPH in methanol mixed with REC-2006 was incubated for 15 min in the dark at 37 • C. The decrease in absorbance at 517 nm was measured against that of methanol alone. The absorbance of DPPH alone was taken as 100% radical or 0% inhibition.
2.11. Hydroxyl Radical Scavenging. We quantified scavenging of radiation (100 Gy) and Fenton reaction induced hydroxyl radicals by REC-2006, using 2-deoxyribose as the marker substrate [35]. Briefly, we exposed to 100 μM FeSO 4 or 100 Gy, 1 ml of reaction volume containing 5 mM 2deoxyribose in the absence or presence of varied concentrations of REC-2006. Thereafter, we added two volumes of solution containing 25% TCA and 1% TBA in 0.1 N NaOH, placed the mixture in a boiling water bath for 20 min, cooled it and measured the absorbance of the resulting pink-colored chromogen at 532 nm.  the absorbance was read at 765 nm. The total phenols in REC-2006 were expressed as gallic acid equivalents, estimated from a standard curve obtained from the absorbance of fresh gallic acid equivalents.

Total Flavonoid Content.
We obtained the total flavonoid content of REC-2006 using the method of Zhisen et al. [39]. To 1 ml of diluted REC-2006 sample (500 μg), we added 4 ml of H 2 O, 0.3 ml of 5% NaNO 2 followed, 5 min later, by of 0.3 ml of 10% AlCl 3 solution. One minute thereafter, 2 ml 1 M NaOH was added and the reaction mixture was immediately diluted with 2.4 ml of ddH 2 O. The pink-colored chromogen was measured spectrophotometrically at 510 nm. The total flavonoid content (mg mg −1 ) was expressed as quercetin equivalents, using a standard curve from a fresh quercetin solution.
2.16. Data Analysis. The data are presented as the mean ± standard deviation (SD) of three separate experiments, with each experiment comprising three parallel measurements. We compared radiation and radiation + REC-2006 groups. The data were analyzed by one-way analysis of variance, and multiple comparisons were made between different groups by applying Bonferroni t-test. A probability of < 5% was considered significant. Figure 1 shows the HPLC profile of REC-2006. Demethylpodophyllotoxin, podophyllotoxin glycoside, epipodophyllotoxin and podophyllotoxin among others were identified by analyzing the fragmentation patterns [25]. Chemical analysis and spectrophotometric determinations indicated that the total polyphenol content of REC-2006 was 8 mg mg −1 of gallic acid equivalents while the total flavonoid content was 0.20 mg mg −1 of quercetin equivalents.  Figure 2(a) (a-g). Control cells showed intact nuclei without any halo around them (a), while 10 Gy whole body irradiation, resulted in large halo formation (b). The center of the halo became a little intense and bigger in radius at 15 and 60 min after irradiation (c and d). Thymocytes from REC-2006 pretreated whole body irradiated mice revealed more intense and bigger nuclei from 0 min time point onward and at 15 and 60 min after irradiation the halo could not be seen, only intact nucleus was seen. 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 + halo) to the area occupied by the nucleus. Data are expressed as the RNSF, which was calculated by subtracting the nuclear spreading factor values of control cells from those of treated cells. Untreated cells consistently had a nuclear spreading factor of zero. In un-irradiated mice, the RNSF, an indicator of DNA damage, was zero. In the group receiving radiation alone, the average RNSF immediately after irradiation was 17.7 ± 0.47. However, with increasing time, the amount of DNA damage decreased; RNSF values after 15 and 60 min, respectively, were 12.96 ± 1.64 and 3.3 ± 0.014 (Figure 2(b)). In animals pretreated with REC-2006 and then exposed to 10 Gy, the amount of initial radiation-induced DNA damage was significantly lower (6.156 ± 0.576) than in the radiationalone group; the average RNSF values at 15 and 60 min afterward were, respectively, 1.647 ± 0.534 and 0.496 ± 0.012 (Figure 2(b)).

Gene-Specific DNA Damage and Repair.
We first carried out studies with varied concentrations of DNA from untreated control cells to find the appropriate amount of template DNA to use; we found a linear relationship between the template concentration and sequence amplification up to 30 ng DNA. In our further studies of the effect of REC-2006 on radiation-induced gene-specific DNA damage and repair, we selected a template concentration of 20 ng. After amplification, the size of PCR product was determined by running the PCR product with a broad range (1 kb) DNA marker (figure not shown). Semi-quantitative polymerase chain reactions for the irradiation-alone group (Figure 3(a), lane 2) revealed a complete loss of amplification of the βglobin gene immediately after exposure (0 min), indicating severe damage to the template DNA. After 15 or 60 min, the quantum of DNA damage decreased and the amplification levels were, respectively, 28 and 43% of the unirradiated controls (Figure 3(a), lanes 3 and 4, and Figure 3(b)). However, REC-2006 pretreatment significantly lowered the radiation-induced sequence-specific DNA damage at all post-irradiation intervals in comparison to the irradiationalone group (Figure 3(b)). The amplification products of the β-globin gene sequence in the REC-2006 pretreated irradiated group was 36, 95 and 99%, respectively, at 0, 15 and 60 min after 10 Gy exposure (Figures 3(a), 3(b), lanes 5, 6 and 7).  (Figure 5). The absorbance of DPPH alone was taken as 100% radical or 0% inhibition. Beyond 0.2 mg ml −1 , a plateau phase was observed (not shown in the figure). Briefly, 1 ml of reaction volume containing, 5 mM 2-deoxyribose and varied concentrations of REC-2006 were mixed either with 100 μM FeSO 4 (Fenton) or exposed to 100 Gy followed by incubation for 1 h at 37 • C. There after, two volumes of solution containing 25% TCA and 1% TBA in 0.1 N NaOH was added, incubated in boiling water bath for 20 min, cooled and the absorbance of the resulting pink-colored chromogen was measured at 532 nm. The data in this figure represent the mean ± SD of values from three independent assays ( * P < .05, * * P < .01). Thereafter, the samples were incubated for 15 min in dark at 37 • C and the decrease in the absorbance at 560 nm was measured against methanol. The absorbance of DPPH alone was considered as 100% radical or 0% inhibition. The values are expressed as mean ± SD of data from three independent assays ( * P < .05, * * P < .01). a concentration-dependent fashion. Maximum scavenging (absorbance 0.056) was observed at concentration of 1.5 mg ml −1 (Figure 6). The highest concentration of REC-2006 we assayed was 2 mg ml −1 and beyond that a plateau effect was observed. The total antioxidant capacity of the extract was 0.06 mg mg −1 gallic acid equivalents.

Discussion
The requirement of administering P. hexandrum before irradiation to achieve radioprotection suggests that mechanisms such as free radical scavenging, metal chelation and elevation of antioxidant defense systems might have a major role.  However, a very large quantum of the drug/protective agent must be present in the cellular milieu to scavenge the heavy flux of free radicals generated by high doses of radiation and cannot be achieved under normal circumstances. One of the important aspects of post-irradiation cell recovery is the repair of radiation-induced oxidative DNA damage. We explored the total genomic DNA damage and repair in mouse thymocytes using the alkaline halo assay, finding that treatment with REC-2006 significantly decreased radiation-mediated DNA strand breaks (Figures 2(a) and 2(b)). As the mice were killed immediately after radiation, and samples were processed at 4 • C throughout, the role of repair in the initial decrease in DNA damage (0 min) can be ruled out. We consider that the decline in this DNA damage occasioned by REC-2006 treatment might be attributable to the free-radical scavenging or antioxidant potential of the flavonoides and polyphenolics present in the extract.
To gain insight into the mechanism of DNA protection, we evaluated in vitro the effect of REC-2006 on the scavenging of radiation-induced hydroxyl radicals, and DPPH radicals scavenging, on the generation of superoxide anions, and on the total antioxidant potential. The inhibition of 2deoxyribose degradation by oxidants, a simple, reliable technique to assess the hydroxyl scavenging ability of an agent [12], clearly demonstrated the hydroxyl radical scavenging potential of REC-2006 ( Figure 4). Its potential for scavenging of free radicals was further supported by its inhibition of DPPH and superoxide anions (Figures 5 and 6). Total antioxidant potential evaluated in gallic-acid equivalents attested to its antioxidant function. We assessed the amount of radiation-induced DNA damage remaining at various times after exposure to evaluate the effect of REC-2006 on cellular repair. In mice given REC-2006 before irradiation, this measure seemingly led to faster DNA repair; thus, after 15 and 60 min, their RNSF values were, respectively, 1.647 ± 0.534 and 0.496 ± 0.012 in comparison to a group not given REC-2006 (12.96 ± 1.64 at 15 min or 3.3 ± 0.014 after 60 min) (Figure 2(b)).
To better elucidate the radioprotective effect of REC-2006, DNA damage and repair was studied at gene level, choosing β-globin as a model gene and semi-QPCR. Previously, other investigators used β-globin or other constitutively expressed genes as marker genes for studying DNA damage and repair kinetics [40]. The rationale of the QPCR technique is that certain DNA lesions block the movement of the Taq polymerase on the DNA template, so decreasing its amplification [40,41]. In the present study, we do not state any direct relationship between repair and protection in β-globin gene in thymocytes and amelioration of adverse effects of radiation; however, the only rationale of studying β-globin gene in thymocytes is based on the fact that radiosensitive nature of thymocytes makes them a highly sensitive system to study DNA repair [26,27] and constitutive expression in transcriptionally active and inactive regions of β-globin makes it a suitable target gene [28,29]. Exposing the mice to 10 Gy resulted in extensive DNA damage in the β-globin gene and complete immediate loss of amplification after exposure (Figures 3(a) and 3(b)). REC-2006 treatment significantly reduced the induced damage as evidenced by a 36% amplification compared to untreated controls. These results clearly indicate the radioprotective effect of REC-2006 in vivo. After 15 min, only 28% amplification was observed in the radiation-alone group whereas in the REC-2006-treated group most of the damage was repaired (95% 8 Evidence-Based Complementary and Alternative Medicine amplification with respect to the control). Furthermore, after 60 min the amplification level in mice treated with REC-2006 was almost equal to that of controls (99%), pointing to an enhancement in the activity of the repair machinery. Meanwhile, there was only 43% amplification in the β-globin gene in the radiated group that had not received REC-2006.
Both non-specific and gene-specific DNA damage repair studies clearly revealed that REC-2006 protects the cellular DNA from radiation-induced damage both by inhibiting the induction of damage and by enhancing its repair after exposure. The proposed mechanism of action of REC-2006 to protect DNA against radiations induced damage is depicted hypothetically in Figure 7. Natural polyphenolics already were shown to modulate gene expression, signal transduction cascades and DNA repair pathways [42][43][44]. Recently, REC-2006 was reported to contain several biologically active flavonoides, polyphenols and podophyllotoxin glycoside, and the like that might contribute toward enhancing DNA repair. These findings undoubtedly warrant further studies to unravel the effect of P. hexandrum on various DNA repair genes.

Conclusion
The results of our study imply that REC-2006 protect cellular DNA from radiation-induced damage by lowering the induction of the initial damage and by enhancing its repair in vivo. The antioxidant-and free-radical-scavenging properties of REC-2006, likely due to the presence of various bioactive compounds, may contribute toward its radioprotective effects.

Funding
This work was supported by the research funds provided by Defence Research and Development Organization, Government of India, under the Charak program (Grant Number-RD-P1/233).