Preventive Effects of Garlic Extract on Lipid Peroxidation and Antioxidant Defence System in N-Nitrosodiethylamine Induced Hepatocarcinogenesis in Rats

Effects of garlic (Allium sativum) on circulatory lipid peroxidation and antioxidant status were evaluated in N-Nitrosodiethylamine induced hepatocarcinogenesis in male rats. Enhanced lipid peroxidation in the circulation of NNitrosodiethylamine (NDEA) treated rats was accompanied by a decrease in the levels of vitamins C and E, reduced glutathione, glutathione peroxidase, superoxide dismutase and catalase. Garlic administered rats experienced a significant reduction in lipid peroxidation with a simultaneous elevation in antioxidant levels. The presence of organosulphur compounds in garlic could have decreased lipid peroxidation and increased antioxidants levels in NDEA administered rats. INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers in the world (Schaff et al., 1993). N-Nitroso compounds are known hepatocarcinogens and cause liver necrosis. These compounds have been found in foods such as meat products, milk products and alcoholic beverages (Scanlan, 1983). In humans, the average intake of nitrosoamines from food is approximately 0.5 μg/day (Scanlan, 1983). NNitrosodiethylamine (NDEA) has been suggested to cause oxidative stress and cellular injury due to the involvement of free radicals. Lipid peroxidation and toxic products have been suggested as major causes of cancer development (Dreher and Junod, 1996). Free radical induced changes by NDEA may contribute to the development of a disease state such as nitrosoamine induced malignancy (Anis et al., 2001). Garlic (Allium sativum) is one of the most common flavor enhancing foods. Commonly, its ingredients have been used as folk medicine. Garlic exhibits a wide range of properties including immunomodulation, hepatoprotection, antioxidant activity, lipid lowering, antimutagenic and anticarcinogenic properties (Aouadi et al., 2000; Shobana and Naidu, 2000). Epidemiological, animal and in vitro studies indicate that garlic possesses anticarcinogenic properties that inhibit several types of tumors and decrease tumor growth and proliferation (Lamm and Riggs, 2000). Assaying of biomarkers in the circulation is a reliable method for screening the actions of chemopreventive agents (Hayes and Pulford, 1995). Circulatory levels of lipid peroxides and cellular antioxidants are indicators of chemoprevention since many chemopreventive substances exert their effects by scanvenging oxygen free radicals (Jeena et al., 1999). In the present study, the chemopreventive potentials of garlic in NDEA-induced hepatocarcinogenesis were studied by assaying circulatory lipid peroxidation products – thiobarbituric acid reactive substances (TBARS), enzymatic and non-enzymatic antioxidants – glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and vitamins C and E. MATERIALS AND METHODS Adult male Wistar rats (160-180 g) were obtained from Central Animal House, Annamalai University, India. They were housed in polypropylene cages and provided food and water ad libitum. Standard rat food pellets were used (Mysore Snack feed Ltd., Proc. WOCMAP III, Vol.6: Traditional Medicine & Nutraceuticals Eds. U.R. Palaniswamy, L.E. Craker and Z.E. Gardner Acta Hort. 680, ISHS 2005 172 Mysore, India) as diet during the experiment. The animals were maintained in controlled environmental conditions of temperature (32±2°C) with 12:12h light-dark conditions. NNitrosodiethylamine was purchased from Sigma (St. Louis, MO, USA). All other chemicals and biochemicals used in this study were of analytical grade. Fresh garlic was purchased from the local market in Chidambaram, Tamil Nadu, India. The dose of garlic to be administered was calculated on the basis of weight of fresh garlic in mg used to prepare the extract (1 mL). An aqueous extract of fresh garlic was prepared by homogenizing the required amount of freshly peeled garlic cloves in an appropriate volume of double distilled water to give a concentration of 25 mg/mL (Singh et al., 1996). The homogenate was centrifuged (3,000 rpm, 10 min). Particulate matter was removed and the supernatant used for experiment within 30 min of preparation. Garlic was administered intragastrically to rats at the concentration of 250 mg/kg b.w. Rats were given single intraperitoneal injection of N-Nitrosodiethylamine (200 mg/kg b.w.) followed by subcutaneous injections of carbon tetrachloride (3 mL/kg b.w./week) for 6 weeks. The animals were randomized and grouped into control and 3 experimental groups (n=6 in each group). Group I (saline control) rats were treated with 0.9% normal saline throughout the study. Group II (NDEA+CCl4) rats received a single injection of NNitrosodiethylamine followed by carbon tetrachloride treatment. Group III (NDEA + garlic) rats received N-Nitrosodiethylamine as in group II and administered with garlic extract at the concentration of 250 mg/kg b.w. Group IV (Garlic) rats were treated with aqueous garlic extract (250 mg/kg b.w.). The experiment was terminated at the end of 20 weeks and all the animals were sacrificed by cervical dislocation after an overnight fast. Blood samples were collected in heparinized tubes; plasma was separated by centrifugation (2,000 rpm, 10 min). Lipid peroxidation as evidenced by the formation of thiobarbituric acid reactive substances (TBARS) was assayed in the plasma as given by Yagi (1978). Reduced glutathione (GSH) (Ellman, 1959), vitamin C (Omaye et al., 1979), vitamin E (Baker et al., 1980), superoxide dismutase (SOD) (Kakkar et al., 1984), glutathione peroxidase (GPx) (Rotruck et al., 1973), catalase (CAT) (Sinha, 1972) and haemoglobin (Drabkin and Austin, 1932) were also assayed. The data were analyzed using analysis of variance (ANOVA) and group means compared by Duncan’s multiple range test (DMRT) (p<0.05). RESULTS The NDEA treated group showed 100% tumor incidence whereas in groups I, III and IV no malignancies were seen. Liver weights were higher (2.5 times) in Group II (NDEA+CCl4) compared to Group I (saline control). Liver weights in Groups III (NDEA+Garlic) and IV (garlic) were lower (by 50%) compared to Group II (Table 1). Table 1 shows the levels of TBARS in the plasma of control and experimental groups. Lipid peroxidation was higher in Group II than in Group I. The levels of TBARS decreased in Groups III and IV compared with Group II. The levels of vitamins C and E, GSH, GPx, SOD and CAT were increased (1.4 fold to 1.9 fold) compared to Group II (NDEA+CCl4) (Table 1). DISCUSSION Our results indicate that NDEA-induced lipid peroxidation and cellular antioxidant depletion were markedly inhibited by administration of garlic extract. Enhanced lipid peroxidation associated with depletion of antioxidants is a characteristic finding in a variety of malignancies (Anis et al., 2001). Although many risk factors have been reported, lipid peroxidation is an important cause of hepatoma (Dreher and Junod, 1996). NDEA has been reported to cause oxidative stress and cellular injury due to the involvement of free radicals (Anis et al., 2001). It has been shown that the mechanism of action is the metabolism of NDEA to alkylating reactants and reactive oxygen species (ROS) and further interaction with the DNA molecule (Leoppky and Li, 1991). The