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Modulation of radiation-induced biochemical changes in cerebrum of Swiss albino mice by Grewia asiatica Rashmi Sisodia*, Muktika Ahaskar, K.V. Sharma, and Smita Singh Radiation Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur, 302004 India, The present study evaluates the possible radioprotective effect of Grewia asiatica fruit (rich in anthocyanin, carotenes, vitamin C, etc.) pulp extract (GAE) on cerebrum of Swiss albino mice exposed to 5 Gy gamma radiation. For this, healthy mice from an inbred colony were divided into four groups: (1) Control (vehicle treated), (2) GAE treated – mice in this group were orally supplemented with GAE (700 mg/kg. b.w. /day) once daily for fifteen consecutive days, (3) Vehicle treated irradiated mice, and (4) GAE + Irradiated – Mice in this group received distilled water orally equivalent to GAE (700 mg/kg.
b.w./day) for fifteen days consecutively. Mice were sacrificed at various intervals viz. 1–30 days. Radiation-induced augmentation in the levels of lipid peroxidation of mice cerebrum was significantly ameliorated by GAE pretreatment.
Radiation-induced depletion in the level of glutathione and protein was prevented significantly by GAE administration. Key words: Grewia Asiatica, antioxidants, cerebrum, radioprotection (Phalsa) cultivated on a commercial scale mainly in the northern and western states of India (Hays 1953, Sastri Synthetic protectors have toxicity, which limits their 1956) is known for its medicinal properties. The fruit is value in the clinical field. Therefore, now the search is astringent and stomachic. Morton (1987) reported that on for some natural compounds that can quench the unripe phalsa fruit alleviates inflammation and is reactive energy of free radicals and eliminate singlet administered in respiratory, cardiac and blood disor- oxygen, one of the major participants in lipid peroxi- ders, as well as in fever reduction. Furthermore, infu- dation (LPO). A large number of compounds from var- sion of the bark is given as a demulcent, febrifuge, and ious plant sources have been shown to possess antioxi- treatment for diarrhea. Grewia asiatica has been report- dant properties (Bhattacharya et al. 1996, Yen et al. ed to contain anthocyanin type cyanidin 3-glucoside 1996, Bhatia 1998). Antioxidants of plant origin are (Nair et al. 2004), vitamin C, carotenoids, minerals and vitamin E, C, selenium, phenolic compounds, dietary fibers, etc. (Yadav 1999). The antioxidant prop- flavonoids, etc. (Chandha 1997). It has been assumed erties of carotenoids and vitamin C are well known and that nutritional intervention to increase intake of phyto- anthocyanin has recently emerged as a powerful antiox- antioxidants may reduce the threat of free radicals. idant. Therefore, Grewia asiatica may prove to be an India has a rich heritage of medicinal plants, many of efficient antioxidant.
which have been explored for various bioactivities for Brain tissue is highly susceptible to oxidative dam- ages, but the radioprotective potential of the plants have age due to its high utilization of oxygen (20% of the been hardly explored. In this context Grewia asiatica total oxygen inhaled by the body) that accounts for the increased generation of oxygen free radicals and reac- Correspondence should be addressed to R. Sisodia, tive oxygen substrates. Reactive oxygen species (ROS) are capable of oxidation of proteins, lipids and DNA Received 25 June 2007, accepted 3 October 2007 leading to cellular damage. Free radicals are potentially 2008 by Polish Neuroscience Society - PTBUN, Nencki Institute of Experimental Biology Grewia asiatica against radiation in cerebrum 33 dangerous for cells (Hochstein and Atollah 1988). LPO is a good biomarker of damage occurring due to radia- tion and the inhibition of lipid peroxidation is sugges- The cobalt teletherapy unit (ATC-C9) at Cancer tive of radioprotective action. Brain has a poorly devel- Treatment Center, Radiotherapy Department, SMS oped antioxidative defense mechanism. The concentra- Medical College and Hospital, Jaipur, Rajasthan, India tion of various antioxidative enzymes is low in brain. was used for irradiation. Unanesthetized animals were The glutathione (GSH) concentration is also very much restrained in well-ventilated Perspex boxes and the reduced in the brain when compared to other organs in whole body was exposed to gamma radiation at a dis- the body (Zhang et al. 1993). Brain is also enriched tance (SSD) of 77.5 cm from the source to deliver with polyunsaturated fatty acid (PUFA) that renders it a dose rate of 1.07 Gy/min.
susceptible to oxidative attack. The present study is therefore an attempt to evaluate the possible protective effects of Grewia asiatica fruit extract in mice cere- brum against radiation-induced oxidative stress.
Thiobarbituric acid (TBA), glutathione (GSH), and DTNB (5,5 dithio-bis 2-Nitrobenzoic acid) were pur- chased from Sigma Co. USA. 1,1,3,3, tetramethoxy propane and other chemicals used were of analytical grade and were procured from Central Drug House The animal care and handling was done according to the guidelines set by World Health Organization, Geneva, Switzerland and INSA (Indian National Science Academy, New Delhi, India). The Depart- Dose selection of Grewia asiatica was done on the basis mental Animal Ethical Committee approved this study. of a drug tolerance study in our laboratory by Ahaskar and Swiss albino mice, 6–8 weeks old, weighing 23 ± 2 g, others (2007). Various doses of Grewia asiatica (100, 400, from an inbred colony were used for the present study. 700, 1.000, 1.300 mg/kg b.wt.) were tested against gamma These animals were maintained under controlled con- irradiation (10 Gy) and 700 mg/kg b.wt./day was obtained ditions of temperature and light (light/dark, 10 h/ 14 h). as the optimum dose based on survivability of mice. This Four animals were housed in a polypropylene cage dose was used for further experiments. containing sterile paddy husk (procured locally) as bedding throughout the experiment. They were provid- ed standard mice feed (procured from Hindustan Levers Ltd., India) and water ad libitum. Tetracycline Mice selected from an inbred colony were divided water was given once a fortnight as a preventive meas- into 4 groups (30 animals in each group).
(1) Control vehicle treated – mice of this group received only DDW water for 15 days; (2) GAE treated – mice of this group were administered only once with only GAE (700 mg/kg of b.w./day) for 15 consecutive Fresh fruits of Grewia asiatica collected locally in days; (3) Irradiated – mice received DDW (volume summer season were washed, shade dried and pow- equal to Grewia asiatica solution) for 15 days and were dered after removal of seeds. Methanolic extract was whole-body exposed to 5 Gy of gamma-radiation; (4) then prepared by refluxing for 36 hours (3 × 12) at GAE treated + Irradiated – in this group oral adminis- 40°C. The extract thus obtained was vacuum evaporat- tration of GAE (700 mg/kg of b.w./day) was made once ed to produce a powdered form. The extract was redis- daily for 15 consecutive days as done in GAE treated solved in doubled-distilled water (DDW) just before the group. One hour after administration of the last dose of oral administration. For various concentrations, GAE, mice were whole-body exposed to a single a known amount of GAE was suspended in DDW and dose of 5 Gy gamma-radiation as in group three.
50 µl of GAE suspension was given to each mouse by Six mice from each group were necropsied at various oral gavage as given by Ahaskar and colleagues (2007). intervals viz. 1, 3, 7, 15, and 30 days post irradiation.
The mice were sacrificed by cervical dislocation. An Lipid peroxidation product as reflected by TBARS incision was made at the sides of the jaws to separate equivalent content was augmented after radiation the upper and the lower palates. The upper palate was exposure in both GAE-treated and vehicle treated-irra- cut in the middle and, after having cleared the sur- diated mice cerebrum. This increase in lipid peroxida- rounding tissue the brain was excised and separated tion product was not stable up to day 7 post-exposure, from the spinal cord at the decussation of the pyramids. as there was a slight decline on day 3 post-exposure.
The intact cerebrum was then removed carefully from After day 7, a continuous decrease in TBARS content the brain and homogenate was prepared and used for was observed in both groups up to day 30 post-irradia- quantitative estimation for various biochemical tion. The magnitude of such a recovery from oxidative damage in terms of TBARS content was significantly higher (P<0.001) in GAE treated-irradiated mice as compared to vehicle treated-irradiated mice. Moreover, GAE treated-irradiated group has attended the normal Lipid peroxidation (LPO) assay: LPO was measured level of TBARS at 30 day post-irradiation. Only GAE by the method of Buege and Aust (1978). Briefly, to treated mice didn’t show any significant deviation in tissue homogenate (0.8 ml), 1.2 ml solution of TCA- the level of TBARS equivalent as compared to control TBA-HCl prepared in 1/1/1 was added. This final mix- (Table I). ture was heated on a water bath for 30 min at 80°C and Glutathione (GSH) content was decreased after radi- cooled. After centrifugation the absorbance was ation exposure in the GAE treated and vehicle treated- recorded at 532 nm using a UV-Vis double beam spec- irradiated mice cerebrum. Such a decrease in GSH trophotometer. The LPO has been expressed as MDA content was noted continuously up to the seventh day post-exposure. After day 7, a continuous increase in Reduced glutathione (GSH) assay: The reduced glu- GSH content was observed in both groups up to day 30 tathione (GSH) content of tissue samples was deter- post-irradiation. The magnitude of such a recovery mined by the method of Moron and coauthors (1979). from oxidative damage was significantly higher Tissue sample was homogenized in the sodium phos- (P<0.001) in GAE treated + irradiated mice as com- phate–EDTA buffer then 0.6 M DTNB [5,5-dithiobis- pared to vehicle treated-irradiated mice. GAE treated + (2-nitrobenzoic acid)] was added. The optical density irradiated group showed a higher degree of recovery at of the yellow colored complex developed by the reac- day 30 post-exposure by attaining control level. Only tion of GSH and DTNB was measured at 412 nm using GAE treated mice also showed a significant increase a UV–vis spectrophotometer. The results were (P<0.01) in GSH content as compared to control Protein assay: Estimation of protein was based on Protein estimates also showed a statistically signifi- the method proposed by Bradford (1976) and 10% cant decrease after radiation exposure in both GAE homogenate was prepared (1 g of tissue in 9 ml of treated and vehicle treated-irradiated mice cerebrum.
NaCl) and 0.1 ml of the sample was taken for the Such a decrease in protein content was noted continu- Bradford assay. Three repeats of the assay from each ously till day 7 post-exposure. After day 7, a continu- animal were carried out. The absorbance was read at ous increase in protein content was observed in both groups up to day 30 post-irradiation. The magnitude of such a recovery from oxidative damage was signifi- cantly higher (P<0.001) in GAE treated + irradiated mice as compared to vehicle treated-irradiated mice.
The results obtained in the present study were Moreover, GAE treated + irradiated group attained the expressed as mean ± SEM. The statistical differences normal level of protein at 30 days post-irradiation.
between various groups were analyzed by the Only GAE treated mice also showed a significant Student’s t-test and the significance was observed at increase (P<0.001) in protein content as compared to the P<0.05, P<0.01, and P<0.001 level. Grewia asiatica against radiation in cerebrum 35 Radiomodulatory influence of Grewia asiatica fruit extract on cerebrum LPO ± SEM (n=12) (nm MDA/g protein) of Swiss albino mice at various post irradiation interval after 5 Gy radiation exposure 137.19 ± 1.33 131.28 ± 1.11 139.16 ± 0.84 134.24 ± 1.76 128.65 ± 2.36 GEA treated 124.71 ± 1.77 121.43 ± 1.57 126.69 ± 1.52 118.88 ± 1.48 107.19 ± 1.27 (a) Control (vehicle treated) vs. GAE treated; (b) Control (vehicle treated) vs. Irradiated; (c) Irradiated vs. GAE treated + Sisodia (2006) after oral administration of GAE. The preservation of cellular membrane integrity depends One of the basic mechanisms of radiation damage is on protection or repair mechanisms capable of neutral- production of free radicals leading to the formation of izing oxidative reactions. The presence of antioxidants peroxides and oxidative reactive species. The peroxides in the GAE suppresses the formation of free lipid rad- via lipid peroxidation damage the cell membrane and icals and thus prevents the formation of endoperoxida- other components of the cell. In the present study, there tion. Riveron and colleagues (2007) reported that was a considerable increase in TBARS content after MDA levels were significantly higher in Alzheimer’s radiation exposure. The magnitude of such a recovery disease (AD) patients compared with normal controls, from oxidative damage in term of TBARS content was which means that these patients were exposed to higher in GAE pretreated irradiated animals (Table I).
oxidative stress via lipid peroxidation. Similar results Similar results against 5 Gy gamma radiation on the were reported by Marcus and colleagues (1998), sup- whole brain of mice were noted by Ahaskar and porting other findings in brain tissues and cere- Radiomodulatory influence of Grewia asiatica fruit extract on cerebrum GSH ± SEM (n=12) (nm/100 mg tissue) of Swiss albino mice at various post irradiation interval after 5 Gy radiation exposure (a) Control (vehicle treated) vs. GAE treated; (b) Control (vehicle treated) vs. Irradiated; (c) Irradiated vs. GAE treated + brospinal fluid of AD patients (Rinaldi et al. 2003, calculated to be 1.53 for GAE in earlier studies in our Galbusera et al. 2004, Kawamoto et al. 2005, Migliore laboratory (Ahaskar et al. 2007). In the present study, supplementation of only GAE Glutathione plays an important role in antioxidant has also resulted in a statistically significant (P<0.001) defense, nutrient metabolism, and regulation of cellu- increase in protein content in comparison to control lar events (including gene expression, DNA and pro- mice. There was considerable decrease in protein con- tein synthesis, cell proliferation and apoptosis, signal tent after radiation exposure at all the post irradiation transduction, cytokine production and immune intervals, whereas, in the GAE pretreated irradiated response, and protein glutathionylation). Glutathione group, it seems that GAE provides protection as evi- deficiency contributes to oxidative stress, which plays dent by higher values at all the intervals reaching nor- a key role in aging and the pathogenesis of many dis- malcy at day 30 (Table III). eases, including Alzheimer’s disease (AD) (Wu et al.
Decrease in the protein content after exposure to 2004). The present study showed that in GAE pretreat- irradiation might be due to either decline in the rate ed irradiated group, the glutathione level reached the of protein synthesis or increase in the consumption control level on day 30th p.i. and was also significant- of protein. It may also be the result of the depression ly higher than the corresponding irradiated group at all of enzyme involved in the activation of amino acid and the post irradiation intervals studied (Table II). The transferring to t-RNA or by the inhibition of release increased glutathione levels by GAE pretreatment in of synthesized polypeptides from polysomes (Kim et irradiated mice may facilitate the reduction of oxida- al. 1970). Some studies have indicated that oxidative tive free radicals by H+ donation. This allows the stress diminishes and the levels of some proteins vary restoration of glutathione by glutathione reductase during the progression of AD (Nunomura et al. 2001, Lee et al. 2005). Increased protein concentration in the Most of the researchers have observed that glu- present study may be due to improved ribosomal activ- tathione levels decreased with ageing (Zemlan et al. ities, which enhance protein synthesis. 1989, Villa and Gorini 1993, Mo et al. 1995). Results The results of the present investigation demonstrate presented by Riveron and coauthors (2007) showed that GAE pretreatment protects the mice cerebrum that GSH levels were quite low in AD patients com- against radiation-induced damage by inhibiting the glu- pared to normal controls. Similar results for LPO and tathione and protein depletion and ameliorating lipid GSH in whole brain, liver and blood of mice with GAE peroxidation levels. GAE contains anthocyanin type treatment were also noted (Ahaskar and Sisodia 2006, cyanidin 3- glucoside (Nair et al. 2005), vitamin C, and Sharma et al. 2007). Dose reduction factor (DRF) was carotenoids, etc. (Yadav 1999). Anthocyanins are Radiomodulatory influence of Grewia asiatica fruit extract on cerebrum protein ± SEM (n=12) (mg/g) of Swiss albino mice at various post irradiation interval after 5 Gy radiation exposure (a) Control (vehicle treated) vs. GAE treated; (b) Control (vehicle treated) vs. Irradiated; (c) Irradiated vs. GAE treated + Grewia asiatica against radiation in cerebrum 37 known for their antioxidant properties (Wang et al. Ahaskar M, Sisodia R (2006) Modulation of Radiation 1997). Delgado-Vargas and others (2000) demonstrated induced Biochemical Changes in brain of Swiss Albino that anthocyanins have scavenging properties against Mice by Grewia asiatica. Asian Journal of Experimental OH and O2 and are better agents against lipid peroxi- dation than a-tocopherol (up to seven times). It has Bhatia AL (1998) The anti-aging role of vitamin A and b- been reported that fruits that are richest in anthocyanins carotene. Indian Journal of Gerontology 12: 70–79.
(>20 mg/100 g FW) are very strongly colored (deep Bhattacharya SK, Satyam K, Ghoshal S (1996) Antioxidant purple or black) berries (Macheix et al. 1990). Grewia activity of glycowithanolides from Withenia somnifera.
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