Prevention of radiation-induced hepatic damge on swiss albino mice by aloe vera leaf extract
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- Bu səhifədəki naviqasiya:
- Animal care and handling
- Experimental design
- Glutathione (GSH) Assay
- Results Acid phosphatase
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PREVENTION OF RADIATION-INDUCED HEPATIC DAMAGE ON SWISS ALBINO MICE BY ALOE VERA LEAF EXTRACT Prashasnika Gehlot and P.K. Goyal* Radiation and Cancer Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur 302004, India
Key Words: Aloe vera, liver, gamma radiation, Swiss albino mice, LPO Corresponding author Email: pkgoyal2002@rediffmail.com. Phone 091-0141- 2 651199, fax (1) 0141-2742309 (2) 0141-2740867 Radiation and Cancer Biology Laboratory, Department of Zoology, Rajasthan University, Jaipur 302004, India. INTRODUCTION Gamma radiation is the most commonly used source of ionizing radiation to treat neoplastic disorders in clinical conditions. Clinical success of radiotherapy depends on its ability to selectively kill tumor cells while sparing the normal surrounding tissues. The response of mammalian cells to ionizing radiation sat the cellular and molecular levels is complex and is an molecular levels is complex and is an irreversible process that is dependent on both the radiation dose and tissue –weighting factor[1]. Due to lack of an effective protective agent, newer compounds are currently under investigation as possible adjuvant in the radiation treatment of cancer, and herbal medicines have only recently begun to receive some attention as possible modifiers of the radiation response [2]. Studies carried out in the past 15 years have shown that herbal preparation such as Liv. 52 [3], Brahmarasayana [4], Pododphyllum [5], Ocimum sanctum [6], Triphala [7], Emblica officinalis [8], Rosemarinus officinalis [9] reduced radiation –induced damage in different body systems. One such popularly known used plant is Aloe vera barbadensis [10] belonging to family Liliacae and consists of about more than 250 species [11]. It is commonly called “Guar –patha” or Ghee-Guar. It is rich in vitamin A, E, C and Zinc and Selenium. It is reported to have antioxidant, anti-tumor and anti-inflammatory activities [12, 13]. The present investigation has been made to assess the radioprotective efficacy of Aloe vera leaf extract in the hepatic constituents of Swiss albino mice. MATERIALS AND METHODS Animal care and handling: 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 departmental animal ethical committee approved this study. Swiss albino mice (Mus musculus), 6-8 weeks old with body weight of 24+2 gm, from an inbred colony were used for the present study. These animals were maintained under controlled conditions of temperature and light (light: dark, 10hrs:14 hrs.). Four animals were housed in a polypropylene a cage containing sterile paddy husk (procured locally) as bedding throughout the experiment. They were provided standard mice feed (procured from Hindustan Levers Ltd., India) and water ad libitum. Tetracycline water once a fortnight was given as preventive measures against infections. Irradiation: Mice were irradiated by 60Co source in the cobalt teletherapy unit (ATC-C9) at radiation oncology department, Sawai Man Singh Medical College and Hospital, Jaipur, India. The mice wee kept in ventilated box with a distance of 77.5 cm from the source to deliver a dose rate of 0.99 Gy min-1. Plant material: Aloe leaves were collected locally during the whole year. The Aloe vera plant was identified by the curator at the Herbarium of Botany, University of Rajasthan, and Jaipur, India. (RUBL Number19886) AV Extract: To prepare aqueous extract, fresh shade dried leaves of Aloe vera powdered and refluxed with double distilled water (DDW) for 36 hours at 400 C and vaccum evaporated so as to get in powder form. The powder of extract was redissolved in DDW just before oral administration. Experimental design: Mice were randomly divided into following groups (five per group per interval: Group I: Normal / sham-irradiated mice were given distilled water (DDW) through oral gavage once in a day for 15 consecutive days. Group II: Mice were treated with 1000 mg/kg body weight of AV dried extract dissolved in distilled water through oral gavage for 15 consecutive days. Group III: Mice were given distilled water for 15 days and then exposed to 6 Gy dose of gamma radiation. This group served as positive control. Group IV: Extract of Aloe vera was given 1000 mg/ kg body weight of mouse orally for 15 days and after 30 min. of last dose; they were exposed to 6 Gy dose of gamma radiation. Following various treatments, mice were autopsied by cervical dislocation on days 12hrs. 24 hrs. 3, 5, 10, 20 and 30 days. Liver were surgically removed and fixed in Bouin’s fluid. The liver was embedded in paraffin block after dehydrating with increasing concentrations of 70, 90 and 100% ethanol. Five micrometer sections were cut using hand microtome, were placed on glass slide and were stained with Harris hematoxyline and Eosin. Stained liver sections were observed under light microscope to determine histopathological changes. Homogenate of liver was prepared and activity of acid and alkaline phosphatase was measured by using commercially available kits. Spectrophotometer (Systronics UV-VIS-108) was used to measure the optical densities. Glutathione (GSH) Assay: GSH in liver and blood was measured using the method described by Moron et al. [14] and Beutler et al. [15]. The absorbance was read at 412 nm using a systronic UV-VIS-108 Spectrophotometer. Lipid peroxidation (LPO) Assay: LPO levels in liver and blood were estimated by the method of Ohkawa et al. [16] as thiobarbituric acid (TBA) reactive substances. The absorbance was read at 532 nm with a Systronic UV-VIS-108 Spectrophotometer. Statistical analysis: The statistical significance of the difference between normal and DDW+ irradiated (control) as well as control and AVE+ irradiated (experimental) set up were evaluated by using the student’s ‘t’ test. Results Acid phosphatase: A significant increase in acid phosphatase activity over normal was measured up to day 5th after 6 Gy irradiation. On day 5th, the value was found to be 76.26 per cent more as compared to normal. After this, level of such enzyme declined but remained higher than the normal even on the last day of experimentation (Fig. 1). 
Fig.1: Acid Phosphatase level in liver after 6 Gy of γ-irradiation with (Experimental) or without (Control) AVE Alkaline phosphatase: Alkaline phosphatase values elevated sharply from beginning until day 5th, where the value was 2 folds higher than the normal. After day 5th, a declining trend was observed but normal value could not be restored even till day 30th (Fig. 2). 
Fig.2: Alkaline Phosphatase level in liver after 6 Gy of γ-irradiation with (Experimental) or without (Control) AVE Glutathione (GSH): No significant variation was observed in hepatic GSH content of normal (Group-I) and AVE treated animals (Group-II). A dose dependent decrease in GSH content was evident in control animals (Group-III). The decrease in GSH level was more pronounced at the higher dose than at the lower doses of gamma radiation. An increase in GSH value was measured in AVE pretreated irradiated animals i.e. experimental animals. Blood-GSH: Blood GSH level also did not show any significant variation in normal (Group-I) and ROE treated animals (Group-II). However, a dose dependent decline was noticed in control animals (Group-III). AVE pretreated irradiated animals (Group-IV) showed a significant increase in GSH level over the control, but the values remained below the normal (Fig. 3). 
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C Fig.3: Glutathione level in blood and liver after 6 Gy of γ-irradiation with (Experimental) or without (Control) AVE Liver-LPx: A dose dependent elevation in LPx level in terms of thiobarbituric acid reactive substances (TBARS) or malondialdehyde (MDA) was measured in the control animals. This increase was more at higher dose than the lower doses of gamma radiation. AVE pretreatment inhibited such increase in LPx level as it was found to be significantly lower than the corresponding controls. Serum-LPx: No significant alterations in LPx level of serum were observed in normal and AVE treated animals. In the control animals, a dose dependent increase in TBARS was noted following irradiation, and such values were found to be significantly higher than the normal. However, in AVE pretreated irradiated animals (Group-IV), such level was found to be significantly below the respective controls (Fig. 4). 
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without (Control) AVE Discussion In the present study, after exposure to 6 Gy gamma radiations to animals resulted in radiation sickness within 3-5 days after exposure. The symptoms included reduction in the food and water intake, weight loss, diarrhea, ruffling of hairs and irritability. The similar symptoms have been observed in mice after gamma irradiation by others also [17, 18]. Liver is the primary organ of drug metabolism. It plays a key role of detoxification agency in the body. Any damage to this organ may cause serious disorders in the form of various diseases, which can be observed in the form of histopathological and biochemical lesions. Therefore, the present study has been targeted on liver to evaluate radiation induced biochemical lesions and the radioprotective potential of the leaf extract of plant Aloe vera in gamma irradiated mice. An increase in the liver phosphatase activity of mice due to irradiation was observed in the present study. Similar increase in such enzymes was observed earlier after exposure of mice or rats with various doses [19]. Aikman and wills [20] suggested that lesions are produced in the membrane lipids due to irradiation possibly buy peroxides , which lead to the activation of latent acid hydrolases , that could result in the digestion of the membrane itself, with the consequent activation and release of the other lysosmal enzymes , in the present study . It was observed that after AVE administration radiation-induced increase in ACP was found to be significantly of lower degree. Hamdy et al. [21] observed protection of acid phosphatase, when solutions of lysosomal enzymes were exposed to high doses of radiation containing cysteine and 4-aminoaphthal. Nam [22] too observed significant protection against rise in of serum acid phosphatase at 6 and 130 hrs, if methylene blue was injected to rats prior to irradiation with 360 R. Bhartiya and Khan [23] also found an increase in ACP activity in mice after exposure to different doses of radiation. A rise in ALP activity in liver after gamma exposure, as evident in present experiment, is quite similar to early reports Khan et al. [24]. ACP and ALP are the enzymes, concerned with biosynthesis of fibrous proteins [25] and mucopolysaccharides [26]. They also act as the hydrolytic enzymes which play an important role in dissolution of dead cells of the body [27]. Radiation –induced cell death may be a possible reason for increased activity of ACP and ALP. Post-irradiation damage to liver is another reason attributing to increased level of ACP and ALP. Radiation –induced stress also accounts for an increased activity of these enzymes as observed in the present study. It has been observed that AVE provided protection by exhibiting a significant decrease against radiation-induced increase in ACP as well ALP at various periods of study in experimental animals compared with respective controls. Similar increase in such enzymes was observed earlier [28]. The ALP activity in KB cells increased upon treatment with aloe-emodin when compared to controls was observed. Samarth et al. [29] reported that the activity of ACP increased and ALP decreased in Mentha piperita treated irradiated mice. Aloe barbadensis Mill has been used in variety of disease in traditional Indian system of medicine in India and its used for hepatic ailments is also documented. In the present study an attempt s been made to validate its hepatoprotective activity. This is in good accordance with the studies of aqueous extract of Aloe barbadensis is significantly capable of restoring integrity of hepatocytes indicated by improvement in physiological parameters, excretory capacity (BSP retention) of hepatocytes and also by stimulation of bile flow secretion [30]. Ionizing radiation induces lipid peroxidation (LPx) which can lead to DNA damage and cell death [31, 32]. Therefore, an agent that protects against such damage can provide protection against radiation injury. The administration of AVE before irradiation significantly reduced the amount of LPx compared to the control group. The inhibition of LPx by AVE may also have been responsible for the observed radioprotection. Earlier reports with same animal model also proved that Aloe vera is a good radioprotection against mouse intestinal mucosa after low level (0.5 Gy) exposure to gamma radiation [33]. An increase in the glutathione (GSH) level by AVE may be responsible for the scavenging of radiation-induced free radicals including LPx and thereby protecting against radiation-induced mortality. It has been reported that LPx starts to increase as soon as the endogenous GSH is exhausted, and the addition of GSH promptly stops further peroxidation [34]. The AVE is found effective in altering the sensitivity to radiation possibly by protecting the biological molecules against radiation-induced oxidation of water by decreasing the oxygen tension in the organism. CONCLUSIONS From the present study, it can be concluded that Aloe vera leaf extract provides a significant protection against radiation-induced biochemical alterations in liver. The exact mechanism of its action is not known; however, it may scavenge free radicals produced by radiation and thus inhibit radiation-induced damage to the cellular DNA. Alternatively, it may also increase GSH levels and reduce radiation-induced LPx. ACKNOWLEDGEMENT Authors are heartily thankful to Prof D.P.Agarwal (Head) and Dr. A.K. Chougule (RSO), Department of Radiotherapy, SMS Medical College and Hospital, Jaipur, for providing radiation facility and dosimetry respectively. REFERENCES
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