You are on page 1of 10

J. Radiat. Res.

, 50, 203212 (2009)

Regular Paper

Radiation Protection by 6-Palmitoyl Ascorbic Acid-2-Glucoside: Studies on DNA Damage in vitro, ex vivo, in vivo and Oxidative Stress in vivo
Dhanya K. CHANDRASEKHARAN1, Tsutomu V. KAGIYA2 and Cherupally Krishnan Krishnan NAIR1*
Radioprotector/6-Palmitoyl ascorbic acid-2-glucoside/DNA damage/Comet assay/Whole body irradiation/Oxidative stress. A palmitoyl derivative of ascorbic acid 2-glucoside, 6-palmitoyl ascorbic acid-2-glucoside (PAsAG), which possess good antioxidant properties, is examined for radioprotection in vitro, ex vivo and in vivo models. PAsAG protected plasmid DNA from gamma-radiation induced damages under in vitro conditions. Presence of 1.6 mM PAsAG inhibited the disappearance of ccc (covalently closed circular) form of plasmid pBR322 with a dose modifying factor of 1.5. Comet assay studies on mouse spleen cells exposed to 6 Gy gamma-radiation (ex vivo) in presence and absence of PAsAG revealed that cellular DNA was effectively protected by this compound from radiation induced damages. Oral administration of 80 mg/kg body weight of PAsAG to mice 1 hour prior to 6 Gy whole body gamma-radiation exposure, efficiently protected cellular DNA in tissues such as spleen, bone marrow and blood, from radiation induced damages as indicated by alkaline comet assay. Oxidative stress in tissues such as liver and brain of mice, following whole body exposure to various doses of gamma-radiation (28 Gy), monitored as levels of GSH and peroxidation of lipids, were found considerably reduced when PAsAG was orally administered (80 mg/kg body weight) to the mice one hour prior to the radiation exposure. PAsAG administration improved the per cent survival of mice following exposure to 10 Gy whole body gamma-radiation. Thus PAsAG could act as a radioprotector under in vitro, ex vivo and in vivo conditions of ionizing-radiation exposure.

INTRODUCTION
Radiation effects on matter results from the transfer of energy from the radiation to the target material, through the formation of ionized atoms of unstable nature within the target tissue. The deleterious effects of ionizing radiation in biological systems may be direct (interaction between radiation and target macromolecules) or indirect (due to the products released during the interaction between radiation and water in the systems like OH, H, eAq-, HO2, H3O+, etc.1) These effects are manifested through photoelectric,
*Corresponding author: Phone: +9104872307968, Fax: +9104872307968, E-mail: ckknair@yahoo.com (C.K.K.Nair) 1 Amala Cancer Research Centre, Thrissur 680555, Kerala, India; 2Health Research Foundation, Kinki Invention Centre, Kyoto, Japan. Abbreviations: PAsAG, 6-Palmitoyl Ascorbic Acid-2-Glucoside; GSH, reduced glutathione; MDA, malonedialdehyde; PBS, phosphate buffered saline; DMF, dose modifying factor; TBARS, thiobarbituric acid reacting substances. doi:10.1269/jrr.08090

Compton and Auger effects depending upon the radiation energy, that results in the generation of reactive oxygen species (ROS) which include hydroxyl and superoxide radical2,3) causing DNA strand breaks and mutations,46) and peroxidation of membrane lipids7,8) resulting in cellular damage leading to apoptosis, necrosis, cell dysfunction or mitotic cell death.9,10) The interaction of ROS in biological system also damage the cellular antioxidant defense system that comprises non enzymatic radical scavengers like GSH (Reduced glutathione, an intra cellular non protein thiol which can directly scavenge free radicals and act as cofactor for enzymes involved in oxidative stress),11) vitamin E, flavanoids, bilirubin, beta carotene, uric acid etc. and enzymes which can directly scavenge ROS such as SOD (superoxide dismutase which degrades superoxide radical), GPx (Glutathione peroxidase) and catalase which detoxifies H2O2.12) Wide spread use of radiation in diagnosis, therapy, industry, energy sector and inadvertent exposure during space travel, nuclear accidents and the threats of nuclear terrorism has necessitated development of a radioprotector to safeguard against human exposures. Pharmacological intervention could be the most prudent strategy,13) where a

J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp

204

D. K. Chandrasekharan et al.

compound or formulation can act as free radical scavenger and antioxidant and reduce or mitigate the deleterious consequences of ionizing radiation. There is a continued interest in the identification and the development of nontoxic and effective radioprotectants that can reduce the effect of ionizing radiation. Vitamin C or ascorbic acid is a potent water soluble anti oxidant in biological fluids14) and may be used as a radioprotector that effectively scavenges the free radicals formed during radiation exposure.15,16) It reduces damages to DNA and membranes in biological system both in vivo and in vitro.17) Supplementation of diet with Ascorbic acid decreases endogenous and induced levels of DNA damage in human lymphocyte.18) These protective effects are believed to be due to its scavenging activity of reactive oxygen species before they attack cellular macromolecules. Studies on radiation induced mutations at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus in human cells and morphological transformations in mouse m5S cells have shown that , ascorbic acid scavenges short lived as well as long lived free radicals effectively.15) Its susceptibility to thermal and oxidative degradation has led to development of derivatives with increased stability and in vivo activity. 6-acyl ascorbic acid 2-glucoside (6 acyl AA-2G) is a stable ascorbate derivative O-substituted at the C-2 position which exhibit inherent vitamin C activity of radical scavenging in vivo after enzymatic hydrolysis by glucosidase and esterase19,20) and they have improved bioavailability,2123) since superior skin permeability of the drug is exhibited with appropriate length of acyl chain group. 6-palmitoyl ascorbate is a lipophilic derivative and it exhibits slightly superior radical scavenging activity than ascorbic acid and ascorbic acid-2-glucoside.24) In the present study 6-palmitoyl ascorbic acid 2-glucoside (PAsAG) is evaluated for its possible role as radioprotective agent against radiation induced DNA damage in in vitro, ex vivo and in vivo conditions. The protective effect of PAsAG against damages to tissue antioxidants and membrane lipids in Swiss albino mice exposed to three different doses of sub lethal gamma radiation, and also, the effect of administration of PAsAG on the mortality of the animals following exposure to a lethal dose of gamma-radiation are investigated.

were carried out with the prior approval of the Institutional Animal Ethics Committee (IAEC) and were conducted strictly adhering to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) constituted by the Animal Welfare Division of Government of India.

Chemicals
6-palmitoyl ascorbic acid-2-glucoside (PAsAG) was from Dr. V. T. Kagiya, Health Research Foundation, Kyoto, Japan, Nitroblue tetrazolium (NBT), EDTA and riboflavin were from Sisco Research Laboratories Ltd., Mumbai, India. Reduced glutathione (GSH), 55dithiobis-(2-nitrobenzoic acid) (DTNB), Thiobarbituric acid (TBA), and bovine serum albumin were from Sigma Chemical Company Inc., St Louis, MO, USA. pBR 322 DNA was obtained from Bangalore Genei. All other chemicals were of analytical grade procured from reputed Indian manufacturers.

Drug preparation, dosage, and administration


PAsAG is dissolved in sodium phosphate buffer (0.1 M, pH 7.0) for in vitro and ex vivo studies and in sterile distilled water for in vivo experiments. The route of administration was per oral for in vivo studies in animals.

Exposure to gamma radiation


Irradiation was carried out using a 60Co- Theratron Phoenix teletherapy unit (Atomic energy Ltd., Ottawa, Canada) at a dose rate of 1.88 Gy per minute.

Protection of plasmid pBR322 DNA by PAsAG against different doses of gamma radiation (015 Gy)
The plasmid DNA (100 ng) in phosphate buffer (0.1 M, pH 7.4) was exposed to various doses of gamma irradiation (0 Gy, 5 Gy, 10 Gy, 15 Gy) in the presence and absence of 1.6 mM PAsAG on ice. After irradiation DNA was electrophoresed on 0.8% agarose at 55 V for 2 hours and the DNA damage was analyzed by Digital Gel Documentation and Analysis Software, Biotech R&d Laboratories, Yercaud, Tamil Nadu, India.

Effect of PAsAG on gamma radiation induced DNA strand breaks in mouse spleenocytes ex vivo
Spleen tissue was excised from Swiss albino mice and single cell suspensions of spleenocytes were prepared in 0.1 M phosphate buffer. The cell suspension (106 cells/ml) was exposed to gamma radiation in presence or absence of 1.6 mM PAsAG.

MATERIALS AND METHODS

Animals
Swiss albino mice weighing 2225 g were obtained from the Small Animal Breeding Section (SABS), Mannuthy, Thrissur. Kerala. They were maintained under standard conditions of temperature and humidity in the Centres Animal House Facility. The animals were provided with standard mouse chow (Sai Durga Feeds and Foods, Bangalore, India) and water ad libitum. All animal experiments in this study

Effect of PAsAG on gamma radiation induced DNA strand breaks in spleenocytes, peripheral blood leucocytes and bone marrow cells of mice in vivo
Swiss albino mice were divided into 4 groups and treated as follows

J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp

Radiation Protection by 6-Palmitoyl Ascorbic Acid-2-Glucoside

205

1. 0.2 ml distilled water (oral) + Sham irradiation 2. 0.2 ml distilled water (oral) + 6 Gy 60Co-- rays 3. 80 mg/kg PAsAG (oral) + Sham irradiation 4. 80 mg/kg PAsAG (oral) +6 Gy 60Co-- rays PAsAG in distilled water (80 mg/kg body weight) or distilled water was administered to animals. One hour after the administration of PAsAG or distilled water, the animals were exposed to whole body 6 Gy gamma irradiation. Immediately after irradiation the animals were sacrificed and spleen, blood and bone marrow were collected for comet assay.

Group I- 0.2 ml distilled water (oral) + Sham irradiation Group II- 0.2 ml distilled water (oral) + 2 Gy 60Co-- rays Group III- 0.2 ml distilled water (oral) + 4 Gy 60Co-- rays Group IV- 0.2 ml distilled water (oral) + 8 Gy 60Co-- rays Group V- 80 mg/kg PAsAG (oral) + Sham irradiation Group VI- 80 mg/kg PAsAG (oral) + 2 Gy 60Co-- rays Group VII- 80 mg/kg PAsAG (oral) + 4 Gy 60Co-- rays Group VIII- 80 mg/kg PAsAG (oral) + 8 Gy 60Co-- rays Animals in Group I, II, III and IV were administered orally with 0.2 ml distilled water and PAsAG was given orally to animals in Group V, VI, VII and VIII at a dose of 80 mg/kg body weight. The animals in Group II and VI were treated with a single dose of 2 Gy, Group III and VII with 4 Gy and Group IV and VIII with 8 Gy whole body gamma irradiation, one hour after PAsAG or distilled water administration. After 24 hours following irradiation, the animals were sacrificed by cervical dislocation and liver and brain were excised and washed with ice-cold phosphate buffered saline (PBS). 10% (w/v) homogenates were prepared in PBS. These homogenates were analyzed and GSH (Reduced Glutathione),28) Lipid peroxidation as MDA29) and protein content30) were determined. GSH and MDA were expressed as nmoles/mg protein. It may be noted that in the assay for GSH, what actually measured is the concentration of nonprotein thiols in terms of GSH (GSH is taken as standard to quantitate thiols).

Alkaline single cell gel electrophoresis


The DNA strand breaks in mouse spleenocytes and peripheral blood leucocytes and bone marrow cells of mice were measured using alkaline single cell gel electrophoresis which was performed using method given by Singh,25) with minor modifications. Microscopic slides were coated with normal melting point agarose (1% in PBS containing 0.8% NaCl, 0.02% KCl, 0.14% Na2HPO4, 0.02% NaH2PO4), immediately coverslipped and kept at 4C for 10 minutes to get the agarose solidified. After removal of the coverslip, 200 l of 0.8% low melting point agarose containing 50 l of treated cells were added onto the slide, coverglasses were placed immediately and the slides were kept at 4C. After solidification, the coverglasses were removed and the slides were immersed in prechilled lysing solution containing 2.5 mM NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, pH 10, 1% DMSO, 1% TritonX and kept for 1 hour at 4C. After lysis, slides were drained properly and placed in a horizontal electrophoretic apparatus filled with freshly prepared electrophoresis buffer containing 300 mM NaOH, 1 mM EDTA, 0.2% DMSO, pH 13. The slides were equilibrated in buffer for 20 minutes and electrophoresis was carried out for 30 minutes at 20 V. After electrophoresis the slides were washed gently with 0.4 mM Tris-HCl buffer, pH-7.4 to remove alkali. The slides were again washed with distilled water, kept at 37C for 2 hours to dry the gel and silver staining was carried out.26) The comets were visualized using Olympus BX-41 microscope and the images captured were analyzed using the software CASP which gives % DNA in tail, tail length, tail moment and olive tail moment directly. The parameter tail moment (TM) is the product of tail length and % DNA in tail and olive tail moment (OTM) is the product of the distance between the centre of the head and the centre of the tail and % DNA in tail.27) Results are given as mean standard deviation.

Effect of PAsAG on 10 Gy gamma-radiation induced body weight loss and mortality


Swiss Albino male mice (2025 g body weight) were divided in to 4 groups, each group comprising 10 animals. Animals in the groups I and II were orally administered with distilled water (0.2 ml) and those in Group III and IV were orally administered with PAsAG (80 mg/kg body weight in 0.2 ml distilled water). After one hour of oral administration, Groups II and IV were exposed 10 Gy whole body gammaradiation. After the radiation exposure, Groups I and II were orally given 0.2 ml distilled water and Groups III and IV were orally given PAsAG (same quantity as before). The administration of the drug and exposure to radiation etc were as detailed below. Group I: 0.2 ml distilled water (oral) + sham radiation + 0.2 ml distilled water (oral) Group II: 0.2 ml distilled water (oral) + 10 Gy radiation + 0.2 ml distilled water (oral) Group III: 80 mg/kg PAsAG (oral) + sham radiation + 80 mg/kg PAsAG (oral) Group IV: 80 mg/kg PAsAG (oral) + 10 Gy radiation + 80 mg/kg PAsAG (oral)

In vivo studies on effect of PAsAG on gamma radiation induced alterations in brain and liver of mice: antioxidant status and lipid peroxidation
Animals were divided into eight groups of four animals each as detailed below.

J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp

206

D. K. Chandrasekharan et al.

The animals in all the groups were provided with standard diet and water ad libitum and their body weight and survival were monitored. The animals were checked on a daily basis to record the mortalities if any and the body weights of the survivors were recorded every alternate day. The results are presented as Mean SD of the studied group. Statistical analyses of the results were performed using ANOVA with Tukey-Kramer multiple comparisons test.

Statistical analysis

RESULTS

Protection of plasmid pBR322 from gamma radiation induced damage


Exposure of plasmid pBR322 DNA to gamma radiation resulted in production of strand breaks as a result of which the super coiled form (ccc) of plasmid DNA was converted to open circular (oc) form or linear form. The disappearance of ccc form of plasmid DNA following exposure to gamma radiation can be taken as an index of DNA damage due to ionizing radiation. The reduction in the supercoiled form of plasmid DNA was directly related to the radiation dose as can be seen in Lanes 1, 2, 3 and 4 of Fig. 1(a) and Fig. 1(b). The data presented in Fig. 1(b) reveal that presence of 1.6 mM PAsAG partially prevented the disappearance of ccc form of plasmid DNA following exposure to 515 Gy gamma radiation (see also Fig. 1(a), Lanes 5, 6, 7 and 8). These results thus indicated that PAsAG protected DNA in vitro only partially from gamma radiation induced strand breaks.

1.26, tail length from 3.29 0.01 to 10.82 2.87, tail moment from 0.03 0.01 to 1.00 0.22 and olive tail moment from 0.40 0.18 to 2.44 0.47. In bone marrow cells, % DNA in tail was increased from 1.06 0.55 to 10.44 3.01, tail length from 3.13 0.33 to 8.42 3.73, tail moment from 0.13 0.11 to 1.72 0.76 and olive tail moment from 0.27 0.12 to 2.6 1.22. In blood leucocytes, % DNA in tail was increased from 0.69 0.14 to 7.38 2.63, tail length from 3.16 0.55 to 7.31 2.62, tail moment from 0.12 0.05 to 0.93 0.40 and olive tail moment from 0.59 0.22 to 1.49 0.61. When 80 mg/kg body weight of PAsAG was administered orally one hour before irradiation, there was significant decrease in the comet parameters in spleenocytes, bone marrow cells and blood leucocytes of irradiated mice, the parameters such as % DNA in tail, tail length, tail moment and olive tail moment were brought down to levels of 0.85 0.43, 3.22 0.84, 0.04 0.01 and 0.42 0.06 respectively (P < 0.001) in spleenocytes, 1.27 0.54, 3.42 0.75, 0.17 0.02 and 0.53 0.09 respectively (P < 0.001) in bone marrow cells and 3.93 1.56, 3.43 0.77, 0.15 0.05 and 0.86 0.31 respectively (P < 0.001) in blood leucocytes. The results clearly indicated the ability of PAsAG to offer protection to cellular

Estimation of cellular DNA damage in mouse spleenocytes irradiated ex vivo


Exposure of mouse spleenocytes to gamma radiation ex vivo induced damage to the cellular DNA, as evident from comet assay parameters presented in Fig. 2. Exposure of spleenocytes to 6 Gy radiation resulted in an increase in the comet parameters such as % DNA in tail ( from 0.33 0.17 to 7.27 0.53), tail length (from 3.3 0.70 to 17.94 5.13), tail moment (from 0.03 0.02 to 2.86 0.63) and olive tail moment (from 0.15 0.07 to 5.74 0.66). The presence of 1.6 mM PAsAG during radiation along with the cells protected the cellular DNA so that the comet parameters such as % DNA in tail, tail length, tail moment and olive tail moment were brought to the levels of 0.40 0.18, 3.13 0.56, 0.03 0.02 and 0.20 0.07 respectively (P < 0.001). (a)

Estimation of cellular DNA damage in mouse tissues irradiated in vivo


Whole body irradiation of mice to 6 Gy caused damage to the cellular DNA of various tissues such as spleen, bone marrow and blood leucocytes as evident from the increase in the comet parameters (Fig. 3, 4 and 5). In spleenocytes, % DNA in tail was increased from 0.93 0.43 to 6.73

(b)
Fig. 1. (a) Effect of PAsAG on Plasmid DNA (pBR 322) damage induced by Gamma irradiation. Lane 1: 0 Gy, Lane 2: 5 Gy, Lane 3: 10 Gy, Lane 4: 15 Gy, Lane 5: 0 Gy + PAsAG, Lane 6: 5 Gy + PAsAG, Lane 7: 10 Gy + PAsAG, Lane 8: 15 Gy + PAsAG. (b) Protection of plasmid pBR 322 DNA by PAsAG against different doses of gamma radiation (015 Gy).

J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp

Radiation Protection by 6-Palmitoyl Ascorbic Acid-2-Glucoside

207

Fig. 2. Effect of PAsAG on DNA damage in murine spleenocytes induced by exposure (ex vivo) to gamma radiation (6 Gy) assessed by comet assay. Mean of the percentage DNA in tail, tail length, tail moment and olive tail moment are presented as mean sd. (n s indicate not significant and *** indicate p < 0.001 when compared with respective control).

Fig. 3. Effect of PAsAG on DNA damage in murine spleenocytes induced by whole body exposure to gamma radiation (6 Gy) assessed by comet assay. Mean of the percentage DNA in tail, tail length, tail moment and olive tail moment are presented as mean sd. (n s indicate not significant and *** indicate p < 0.001 when compared with respective control).
J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp

208

D. K. Chandrasekharan et al.

Fig. 4. Effect of PAsAG on DNA damage in murine bone marrow cells induced by whole body exposure to gamma radiation (6 Gy) assessed by comet assay. Mean of the percentage DNA in tail, tail length, tail moment and olive tail moment are presented as mean sd. (n s indicate not significant and *** indicate p < 0.001 when compared with respective control).

Fig. 5. Effect of PAsAG on DNA damage in murine blood leucocytes induced by whole body exposure to gamma radiation (6 Gy) assessed by comet assay. Mean of the percentage DNA in tail, tail length, tail moment and olive tail moment are presented as mean sd. (n s indicate not significant and *** indicate p < 0.001 when compared with respective control).
J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp

Radiation Protection by 6-Palmitoyl Ascorbic Acid-2-Glucoside

209

DNA against gamma radiation in vivo.

Protection by PAsAG against gamma radiation induced stress in brain and liver of whole body irradiated mice
Figure 6 gives the results on the measurement of GSH levels in the brain and liver of whole body gamma irradiated (2, 4 and 8 Gy doses) mice. GSH levels were decreased from 18.50 1.00 to 16.27 2.17, 14.40 1.14 and 8.85 1.87 in the brain of mice exposed to 2, 4 and 8 Gy doses of gamma radiation respectively. Administration of 80 mg/kg body weight of PAsAG orally one hour before 2, 4 and 8 Gy radiation exposure restored GSH levels up to 18.22 0.47, 18.09 2.11(P < 0.01) and 13.47 0.26 (P < 0.001) respec-

tively in the brain of mice. In liver, GSH levels were decreased from 15.11 2.4 to 11.85 1.69, 9.39 0.84 and 7.231.69, of mice exposed to 2, 4 and 8 Gy doses of gamma radiation respectively and in liver of animals exposed to 2, 4 and 8 Gy doses of radiation one hour after oral administration of PAsAG (80 mg/kg body weight) the GSH levels were increased up to 13.92 0.53, 12.85 1.81 (P < 0.05) and 12.52 0.31 (P < 0.001) respectively. Figure 7 depicts the results on the measurement of peroxidation of lipids in terms of MDA or TBARS formed in the brain and liver of mice exposed to 2, 4 and 8 Gy gamma radiation. The MDA (nanomoles/mg protein) were increased from 17.37 1.52 to 19.88 0.83, 22.11 1.7 and 25.71

Fig. 6. Changes in the GSH levels expressed as nanomoles/mg protein in whole body irradiated mice (n = 4) (radiation dose 2 Gy, 4 Gy and 8 Gy) with and without oral administration of PAsAG (80 mg/kg body weight) in brain (Fig. 6 a) and liver (Fig. 6 b) homogenates. (n s indicate not significant, * indicate p < 0.05, ** indicate p < 0.01 and*** indicate p < 0.001 when compared with respective control).

Fig. 7. Changes in the lipid peroxidation levels expressed as MDA in nanomoles/mg protein in whole body irradiated mice (n = 4) (radiation dose 2 Gy, 4 Gy and 8 Gy) with and without oral administration of PAsAG (80 mg/kg body weight) in brain (Fig. 7 a) and liver (Fig. 7 b) homogenates. (n s indicate not significant, * indicate p < 0.05, ** indicate p < 0.01 and*** indicate p < 0.001 when compared with respective control).
J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp

210

D. K. Chandrasekharan et al.

2.8 in brain and from 2.4 0.84 to 2.9 0.22, 3.54 0.46 and 5.50 0.41 in liver of 2, 4 and 8 Gy irradiated animal groups respectively. Oral administration of PAsAG (80 mg/kg body weight) one hour before 2, 4 and 8 Gy radiation exposure decreased the MDA levels to 17.85 1.67 (P < 0.05), 18.25 1.67 (P < 0.01) and 19.9 1.54 (P < 0.001) in brain and 2.37 0.36, 2.51 0.65 and 2.98 0.45 (P < 0.01) in liver of mice respectively.

Effect of administration of PAsAG on radiation induced lethality and alterations in body weight of mice
Figure 8 and 9 present the changes in body weight and

survival of animals following acute lethal dose of 10 Gy gamma-radiation. The body weight of the irradiated animals showed a marked decrease following 10 Gy gammaradiation exposure, while the control unirradiated animals showed a gradual increase. There was no lethality in the unirradiated Groups I and III. The body weight of the animals in the control irradiated group (Group II, no PAsAG administration) continued to decrease till all the animals died on 11th day. In PAsAG administered and irradiated group (Group IV), the body weight of the survivors started recovering after 11th day. Animals in the irradiated group (Groups II and IV) started dying from 5th day. On day 11, the mortality in Group II was 90% while it was only 60% in Group IV. On the 12th day there was 100% mortality in the control irradiated group (Group II) while the mortality was only 80% in the PAsAG administered group (Group IV). There was no further lethality till 15th day in Group IV.

DISCUSSION
Gamma radiation damage vital cellular components through free radicals generated in the aqueous cellular milieu, the most important targets being DNA and membranes. The major DNA damage due to ionizing radiation are single strand breaks, double strand breaks, DNA-DNA and DNA-Protein cross links and damages to nucleotide bases. DNA damage such as strand breaks in vitro can be analyzed by evaluating the disappearance of the ccc form of plasmid DNA after irradiation. Decrease in disappearance of ccc form indicates the radiation protection ability of DNA by PAsAG. PAsAG (1.6 mM) offered partial protection to plasmid pBR 322 DNA against radiation induced strand breaks with a DMF of 1.5 (calculated from the data in Fig. 1(b)). Ascorbic acid has been reported to offer radioprotection to EMT6 Tumour cells exposed to ionizing radiation in vitro, with DMFs 1.23 at 1.0 mM and 1.37 at 10 mM concentrations.16) Radiation induced loss of viability of cells has been attributed to unrepaired lesions in DNA. Thiol compounds such as amifostine (WR1065), phosphonol (WR255591), Nacetyl-L-cysteine (NAC), captopril and mesna have been shown to exhibit antioxidant properties and reduce radiationinduced histone H2AX phosphorylation. However, except amifostine and phosphonol, all the other compounds offered no protection with respect to cell survival in terms of colony forming ability in human microvascular endothelial cells (HMEC).31) Also, it has been reported that clonogenic cell survival (SF2) and measurements of immunofluorescence intensity by digital image analysis of phosphorylated histone H2AX as a surrogate marker of DNA double-strand breaks did not have any correlation.32) Thus, the phosphorylation of H2AX cannot be considered as a predictor for radioprotective activity of a compound or phosphorylated H2AX has limitations in representing the DNA strand breaks. Alkaline

Fig. 8. Effect of PAsAG on 10 Gy gamma radiation induced alterations in body-weight. PAsAG (80 mg/kg body weight) was orally administered one hour prior to and immediately after exposure to whole body gamma radiation. (n s indicate not significant, *** indicate p < 0.001 when PAsAG, 10 Gy group compared with 10 Gy group).

Fig. 9. Effect of PAsAG on 10 Gy gamma radiation induced mortality. PAsAG (80 mg/kg body weight) was orally administered one hour prior to and immediately after exposure to whole body gamma radiation. (n s indicate not significant, *** indicate p < 0.001 when PAsAG, 10 Gy group compared with 10 Gy group).

J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp

Radiation Protection by 6-Palmitoyl Ascorbic Acid-2-Glucoside

211

comet assay is a sensitive technique to monitor strand breaks and alkali labile DNA lesions and is widely used to study genotoxicity, cellular DNA lesions such as single strand breaks or double strand breaks,33) apoptosis and DNA repair.3335) The ex vivo and in vivo results in murine system in the present work, indicate the efficiency of PAsAG to offer protection to cellular DNA against radiation induced damage. Administration of PAsAG (80 mg/kg body weight) one hour prior to whole body 6 Gy gamma radiation protected cellular DNA in spleenocytes, bone marrow cells and blood leucocytes from radiation damage. These results suggest that PAsAG is an effective compound that can protect the haemopoetic system from radiation induced lesions. GSH or reduced glutathione is an intra cellular non protein thiol which can directly scavenge free radicals and act as cofactor for enzymes involved in oxidative stress.11) Exposure to radiation causes depletion of GSH in animal tissues in radiation exposure dose dependant manner. Administration of PAsAG one hour prior to whole body gamma radiation helped to maintain the GSH levels in the irradiated animals. The major damage to membranes by radiation is due to the oxidation of the lipids present in membranes by the radiolytic products such as hydroxyl and peroxyl radicals. Lipid peroxidation is a highly destructive process, results in formation of malondialdehyde (MDA) and alters the structure and function of cellular membranes. MDA is mutagenic in bacterial and mammalian cells, hence the reduction of radiation induced MDA is desirable. The present study revealed that administration of PAsAG decreased the MDA levels in the brain and liver of irradiated animals, one hour prior to whole body gamma radiation which suggests the radioprotection of PAsAG. It was also found that the administration of PAsAG could offer some protection to mice against acute lethal dose of whole body gamma-radiation (10 Gy). There was partial recovery of the radiation-induced loss of body weight in survivors. The per cent survival was 40% in the PAsAG administered group while it was 10% in the control irradiated group on 11th day post irradiation. However, on 12th day the per cent survival in the PAsAG administered group was only 20% and in the control irradiated group it was 0.0%. The percent survival remained at 20% even after 15th day post irradiation in the PAsAG administered group. There were several attempts to devise an ideal radioprotector. Although many compounds showed radioprotection, they failed to be effective in human application due to toxicity. PAsAG is a Vitamin C derivative which exhibit inherent vitamin C activity of radical scavenging in vivo.18,19,23) PAsAG can protect cellular DNA and membranes from radiation induced damages. This compound bears no toxicity and exhibited good radioprotection against sublethal and lethal radiation exposures to mice in the present study. It is interesting to note that this compound offers protection to brain tissues from radiation induced

oxidative stress. The palmitoyl chain may be helping this compound to cross the blood- brain barrier. Recent reports indicate the possibility of cancer induction due to exposure of humans to radiation during therapeutic and diagnostic X rays36) and CT scans.37) Therefore a radioprotective mixture containing compounds like gallic acid, Vitamin C, E, melatonin, antioxidant phytoceuticals or herbal extracts (which are known to be safe for human consumption) may be effective when it is taken just before undergoing these diagnostic tests and routine mammography.38) Nuclear plant works and space travels are also areas where there is risk of exposure to ionizing radiation. Also physicians doing fluoroscopic procedure and radiologists39) are vulnerable to radiation exposure. PAsAG offers good radioprotection to DNA in vitro, ex vivo and in vivo experiments in mice. It can also offer protection to membranes from radiation damage in vivo and maintain tissue antioxidant levels. It is to be further studied clinically for its effective use in human beings in radiation exposure scenarios.

ACKNOWLEDGEMENTS
The authors acknowledge their gratitude to Board of Research in Nuclear Sciences, Department of Atomic energy, Government of India for financial support for these studies by a research grant to CKKN. REFERENCES
1. Draganic, I. G. and Draganic, Z. D. (1971) The Radiation Chemistry of Water. p256, Academic Press, New York. 2. Rugo, R. E., Secretan, M. B. and Schiestl, R. H. (2002) X Radiation Causes a Persistent Induction of Reactive Oxygen Species and a Delayed Reinduction of TP53 in Normal Human Diploid Fibroblasts. Radiat. Res. 158: 210219. 3. Feinendegen, L. E. (2002) Reactive Oxygen Species in Cell Responses to Toxic Agents. Hum. Exp. Toxicol. 21: 8590. 4. Clark, I. A., Cowden, W. B. and Hunt, N. H. (1985) Free Radical Induced Pathology, Med. Res. Rev. 5: 297332. 5. Powell, S. and Mcmillan, T. J. (1991) DNA Damage and Repair Following Treatment with Ionizing Radiation. Radiother. Oncol. 19: 95108. 6. Mcllrath, J., Lorimore, S. A., Coates, P. J. and Wright, E. G. (2003) Radiation Induced Genomic Instability in Immortalized Haemopoetic Stem Cells. Int. J. Radiat. Biol. 79: 2734. 7. Sadani, G. R. and Nadkarni, G. D. (1997) Changes in Lipid Peroxide Levels and the Activity of Reactive Oxygen Scavenging Systems in Thyroid Tissue after Exposure to Radioactive Iodine in Rats. Thyroid. 7: 937941. 8. Greenberger, J. S., Kagan, V. E., Pearce, L., Boriseniao, G., Tyurina, Y. and Epperly, M. W. (2001) Modulation of Redox Signal Transduction Pathways in the Treatment of Cancer. Antioxid. Redox. Signal. 3: 347359. 9. Shimizu, S. and Tsujimoto, Y. (2000) Proapoptotic BH3 Only Bcl-2 Family Members Induce Cytochrome-C Release, but not Mitiochondrial Membrane Potential Loss and do not Directly Modulate Voltage Dependent Anion Channel Activ-

J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp

212

D. K. Chandrasekharan et al.
25. Singh, N. P. (2000) Microgels for Estimation of DNA Strand Breaks, DNA-Protein Cross Links and Apoptosis. Mutat. Res. 455: 111127. 26. Cerda, H., Delincee, H., Haine, H. and Rupp, H. (1997) The DNA Comet Assay as a Rapid Screening Technique to Control Irradiated Food. Mut. Res. Fundamental Molcular Mechanisms of Mutagenesis. 375: 167181. 27. Konca, K., Lankoff, A., Banasik, A., Lisowka, H., Kuszewski, T., Gozdz, S., Koza, Z. and Wojcik, A. (2003) Across Platform Public Domain PC Image Analysis Programme for the Comet Assay. Mutat. Res. 534: 1520. 28. Moron, M. A., Depierre, J. W. and Mannerwick, B. (1979) Levels of Glutathione, Glutathione-S-Transferase Activities in Rat Liver. Biochem. Biophys. Acta. 582: 6768. 29. Buege, A. J. and Aust, S. D. (1978) Microsomal Lipid Peroxidation. Methods Enzymol. 52: 302310. 30. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein Measurement with Folin Phenol Reagent. J. Biol. Chem. 193: 265275. 31. Kataoka, Y., Murleya, J. S., Bakera, K. L. and Grdinaa, D. J. (2007) Relationship between Phosphorylated Histone H2AX Formation and Cell Survival in Human Microvascular Endothelial Cells (HMEC) as a Function of Ionizing Radiation Exposure in the Presence or Absence of Thiol-Containing Drugs. Radiat. Res. 168: 106114. 32. Mahrhofer, H., Burger, S., Oppitz, U., Flentje, M., Djuzenova, C. S. (2006) Radiation induced DNA damage and damage repair in human tumor and fibroblast cell lines assessed by histone H2AX phosphorylation. Int. J. Radiat. Oncol. Biol. Phys. 64: 573580. 33. Olive, P. L. (1999) DNA Damage and Repair in Individual Cells, Application of the Comet Assay in Radiobiology. Int. J. Radiat. Biol. 75: 395405. 34. Maurya, D. K., Salvi, V. P. and Nair, C. K. K. (2004) Radioprotection of Normal Tissues in Tumour Bearing Mice by Troxereutin. J. Radiat. Res. 45: 221228. 35. Salvi, V. P., Maurya, D. K., Kagiya, T. V. and Nair, C. K. K. (2007) Enhancement in Repair of Radiation-Induced DNA Strand Breaks in vivo by Tocopherol Monoglucoside. Int. J. of Low Rad. 4: 4352. 36. Gonzalez, A. B. and Darby, S. (2004) Risk of Cancer from Diagnostic X rays, Estimate for UK and 14 Other Countries, The Lancet. 363: 345351. 37. Brenner, D. J. and Elliston, C. D. (2004) Estimated Radiation Risks Potentially Associated with Full Body CT Screening, Radiology. 232: 735738. 38. Gandhi, N. M. and Nair, C. K. K. (2005) Protection of DNA and Membranes From Gamma Radiation Induced Damage by Gallic acid. Molecular and Cellular Biochemistry. 278: 111 117. 39. Brenner, D. J. and Hall, E. J. (2003) Mortality Pattern in British and US Cardiologists, What can we Really Conclude? Br. J. Radiol. 76: 12.
Received on August 8, 2008 Revision received on December 8, 2008 Accepted on December 8, 2008 J-STAGE Advance Publication Date: April 22, 2009

ity. PNAS. USA. 97: 577582. 10. Nair, C. K. K., Parida, D. K. and Nomura, T. (2001) Radioprotection in Radiotherapy. J. Radiat. Res. 42: 2137. 11. Bast, A., Haenen, G. R. and Doelman, C. J. (1991) Oxidants, Antioxidants: State of an Art. Am. J. Med. 91: 2s13s. 12. Calabrese, E. J. and Canada, A. T. (1989) Catalase: Its Role in Xenobiotic Detoxification. Pharmacol. Ther. 44: 297307. 13. Ganesh, C. J. (2007) Radioprotective Potential of Plants and Herbs Against the Effects of Ionizing Radiation, J. Clin. Biochem. Nutr. 40: 7481. 14. Getoff, N., Platzer, I. and Winkelbauer, C. (1999) Transients and Cooperative Action of -Carotene, Vitamin E & C in Biological Systems in vitro Under Irradiation. Rad. Phys. Chem. 55: 699704. 15. Koyama, S., Kodama, S., Suzuki, K., Matsumoto, T., Miyazaki, T. and Watanabe, M. (1998) Radiation-induced long-lived radicals which cause mutation and transformation. Mutat. Res. 421: 4554. 16. Lund, K. A. (2006) Mega doses of L-ascorbic acid affect antineoplastic effects of ionizing radiation in EMT6 cells in vitro. A Thesis for Doctor of Medicine, Yale University School of Medicine, New Haven, CT. 17. Wilson, R. L. (1983) Free Radical Repair Mechanisms and the Interaction of Glutathione and Vitamin C and E, In Radioprotectors and Anticarcinogens, Eds. Nygard, O. F. and Simic, M. G. pp 123, Academic press, NewYork. 18. Cozzi, R., Ricordy, R., Aglitti, T., Gatta, V., Perticone, P. and De Salvia, R. (1997) Ascorbic Acid and Beta Carotene as Modulators of Oxidative Damage. Carcinogenesis. 18: 223 228. 19. Takebayashi, J., Tai, A. and Yamamoto, I. (2002) Long-Term Radical Scavenging Activity of AA-2G and 6-Acyl-AA-2G against 1, 1-Diphenyl-2-Picrylhydrazyl. Biol. & Pharm. Bull. 25: 11 1503. 20. Yamamoto, I., Tai, A., Fujinami, Y., Sasaki, K. and Okazaki, S. (2002) Synthesis And Characterization of a Series of Novel Monoacylated Ascorbic Acid Derivatives, 6-O-Acyl-2-O-D-Glucopyranosyl-L-Ascorbic Acids, as Skin Antioxidants. J. Med. Chem. 45: 462468. 21. Tai, A., Kawasaki, D., Goto, S., Gohda, E. and Yamamoto, I. (2003) Vitamin C Activity in Guinea Pigs of 6-O-Acyl-2-O-D-Glucopyranosyl-L- Ascorbic Acids with a BranchedAcyl Chain. Biosci. Biotechnol. Biochem. 67: 16751682. 22. Tai, A., Goto, S., Ishiguro, Y., Suzuki, K., Nitoda, T. and Yamamoto, I. (2004) Permeation and Metabolism of a Series of Novel Lipophilic Ascorbic Acid Derivatives, 6-O-Acyl-2O--D-Glucopyranosyl-L-Ascorbic Acids with a BranchedAcyl Chain, in a Human Living Skin Equivalent Model. Bioorg. Med. Chem. Lett. 14: 623627. 23. Dani, M., Nair, C. K. K., Jasmin, A. J., Nandita, B., Tulsi, M., Sudhir, K. and Kagiya, V. T. (2007) Ascorbic Acid Monoglucoside as Antioxidant and Radioprotector. J. Radiat. Res. 48: 369376. 24. Fujinami, Y., Tai, A. and Yamamoto, I. (2001) Radical Scavenging Activity Against 1,1 Diphenyl-2, Picryl Hydrazyl of Ascorbic Acid 2-Glucoside (AA-2G) and 6 Acyl- AA-2G. Chem. Pharm. Bull. 49: 642644.

J. Radiat. Res., Vol. 50, No. 3 (2009); http://jrr.jstage.jst.go.jp