PROTECTIVE EFFECT OF S-ALLYLCYSTEINE AND LYCOPENE IN COMBINATION AGAINST N-METHYL-N -NITRO-N-NITROSOGUANIDINE-INDUCED GENOTOXICITY

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1 Copyright 2004 by Institute of Pharmacology Polish Academy of Sciences Polish Journal of Pharmacology Pol. J. Pharmacol., 2004, 56, ISSN PROTECTIVE EFFECT OF S-ALLYLCYSTEINE AND LYCOPENE IN COMBINATION AGAINST N-METHYL-N -NITRO-N-NITROSOGUANIDINE-INDUCED GENOTOXICITY Balaiya Velmurugan, Sathiyavedu T. Santhiya, Siddavaram Nagini, Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar , Tamil Nadu, India; Department of Genetics, Postgraduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, Tamil Nadu, India Protective effect of S-allylcysteine and lycopene in combination against N-methyl-N -nitro-n-nitrosoguanidine-induced genotoxicity. B. VELMURU- GAN, S.T. SANTHIYA, S. NAGINI, Pol. J. Pharmacol., 2004, 56, Chemoprotection by diet-derived antioxidants has emerged as a costeffective approach in preventing genotoxicity and carcinogenicity. In this study, we investigated the protective effects of S-allylcysteine (SAC) and lycopene against N-methyl-N -nitro-n-nitrosoguanidine (MNNG)-induced genotoxicity. Quantification of bone marrow micronuclei and chromosomal aberrations in male Wistar rats was used to monitor the protective effects of SAC and lycopene. Intragastric administration of MNNG (40 mg/kg) induced a significant increase in the frequency of micronuclei and chromosomal aberrations. Although pretreatment with SAC and lycopene significantly reduced the frequency of MNNG-induced bone marrow micronuclei and chromosomal aberrations, the combination of SAC and lycopene exerted a greater protective effect. These findings indicate that antioxidants such as SAC and lycopene, are effective chemoprotective agents against genotoxicity and carcinogenicity especially when used in combination. Key words: MNNG, micronuclei, chromosomal aberrations, lycopene, S-allylcysteine correspondence; s_nagini@yahoo.com, nrgopal@satyam.net.in

2 B. Velmurugan, S.T. Santhiya, S. Nagini Abbreviations: MNNG N-methyl-N -nitro-nnitrosoguanidine, NNC N-nitroso compounds, SAC S-allylcysteine INTRODUCTION Gastric cancer, the second most common malignancy worldwide, is a major cause of mortality in Chennai, India [2]. Epidemiological studies have shown that high intake of carbohydrates, smoked and salted foods, combined with low consumption of fruits and vegetables rich in antioxidants are major risk factors associated with the etiology of gastric cancer [20]. Further, exposure to N-nitroso compounds (NNC) provided in the diet as well as to endogenously formed NNC is a crucial factor contributing to the development of gastric cancer [26]. Several investigations have shown a positive association between high consumption of fruits and vegetables and reduced risk of gastric cancer suggesting that dietary components offer protection against gastric cancer [16]. Epidemiological studies have shown that the increased intake of garlic and tomato products effectively lowers the risk of gastric cancer [10, 29]. S-allylcysteine (SAC), the organosulfur constituent of garlic, and lycopene, a major carotenoid found in tomato are recognized as pharmacologically active compounds with effective antitumorigenic and antimutagenic properties [12, 14]. SAC has an array of biological effects including modulation of carcinogen bioactivity and oxidative damage. Further, SAC has also been demonstrated to possess anti-inflammatory and antioxidant properties [15]. The chemopreventive effect of SAC has been reported in N-nitrosodiethylamine-induced hepatocarcinogenesis [28]. In a recent report, we have demonstrated induction of cellular differentiation by SAC in the hamster buccal pouch carcinogenesis model [4]. Likewise, lycopene has been shown to have the highest antioxidant activity among the carotenoids [5]. In addition, lycopene has been reported to attenuate oxidative stress and exert anticancer effects both in vitro and in vivo [8]. Previously, we reported the chemopreventive effects of SAC and lycopene on 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis and N-methyl- N -nitro-n-nitrosoguanidine (MNNG)-induced gastric carcinogenesis [3, 6, 30, 31]. Chemoprevention by a combination of dietary agents has recently attracted attention due to high efficacy and broader spectrum of action. Furthermore, the risk of adverse effects can be attenuated by decreasing the dose of each agent in the chemoprevention cocktail. However, identification of agent combinations with chemopreventive potential requires their screening in animal models using short-term tests that evaluate antigenotoxic effects. In particular, the bone marrow micronucleus test and assessment of chromosomal aberrations have gained wide acceptance as early indicators of carcinogen-induced DNA damage and chemoprevention [11]. Therefore, we evaluated the effects of SAC and lycopene on MNNG-induced genetic damage by quantification of micronuclei and chromosomal aberrations as cytogenetic endpoints of chemoprevention. MATERIALS and METHODS Animals All the experiments were carried out on male Wistar rats aged 6 8 weeks obtained from the Central Animal House, Annamalai University, India. The animals were housed six to a polypropylene cage and provided with food and water ad libitum. All animals were fed the standard pellet diet (Mysore Snack Feed Ltd., Mysore, India) and maintained under standard conditions of temperature and humidity with an alternating 12 h light/dark cycle. The animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India, and approved by the Ethical Committee of Annamalai University. Chemicals Fetal calf serum, colchicine, and May- Grünwald and Giemsa stains were purchased from Sigma Chemical Company, St. Louis, USA. MNNG of 97% purity was obtained from Fluka- Chemika-Biochemika, Buchs, Switzerland. SAC of 99.9% purity kindly provided by Wakunaga Pharmaceutical Co. Ltd. (Hiroshima, Japan), was dissolved in distilled water before use. Lycopene (Lyc-O-Mato, natural tomato oleoresin containing 6% lycopene as determined by HPLC) was kindly provided by LycoRed Natural Products Industries Ltd., Beer Sheva, Israel. For experimental purpose, 242 Pol. J. Pharmacol., 2004, 56,

3 PROTECTIVE EFFECT OF S-ALLYLCYSTEINE AND LYCOPENE ON GENOTOXICITY lycopene was dissolved in dimethylsulfoxide at a concentration of 0.85% (w/v), and this solution was diluted with distilled water to give a final concentration of mg/ml. All other used reagents were of analytical grade. Pretreatment schedule The animals were randomized into experimental and control groups and divided into eight groups of six animals each. Animals in group 1 were given MNNG (40 mg/kg) by intragastric intubation [1]. Animals in groups 2, 3 and 4 received intragastric administration of SAC (100 mg/kg), lycopene (1.25 mg/kg) and SAC + lycopene (100 mg/kg mg/kg), respectively, for 5 days followed by MNNG 1.5 h after the final feeding. Animals in groups 5, 6 and 7 received a test agent alone, while group 8 served as control. All the animals were injected colchicine 90 min prior to sacrifice. The animals were sacrificed by cervical dislocation 27 h after MNNG exposure. Both femurs were removed immediately and used for analysis of micronucleated polychromatic erythrocytes (MnPCEs) and chromosomal aberrations. Cytogenetic analysis The bone marrow micronucleus test was carried out according to Schmid [23] for evaluating chromosomal damage in experimental animals. The bone marrow from the femur was flushed in the form of a fine cell suspension into a centrifuge tube containing fetal calf serum. The cell suspension was centrifuged at 500 g for 10 min and the supernatant was discarded. The pellet was resuspended in a drop of serum and used for preparing slides. The air-dried slides were stained with May- Grünwald and Giemsa. A total of 2,500 polychromatic erythrocytes (PCEs) were scored per animal from a single slide to determine the frequency of MnPCEs. Bone marrow cells from control and experimental animals were processed for analysis of chromosomal aberrations by the method of Sharma and Sharma [25]. The bone marrow from the femurs was flushed into a centrifuge tube containing 0.9% saline and centrifuged at 500 g for 5 min. The supernatant was removed and hypotonic KCl was added to the sediment. After incubation for 20 min at 37 C, the contents were centrifuged for 5 min and the sediment was fixed in methanol-acetic acid (3:1 v/v). Three changes of fixative were given prior to slide preparation. The slides were air-dried, stained in Giemsa solution and scored blindly. One hundred well-scattered metaphase plates were scored for each animal. All the slides were scored by the same observer. Statistical analysis The data were analyzed using analysis of variance (ANOVA) and the group means were compared by the Least Significant Difference (LSD) test. The results are expressed as the mean ± SEM and considered statistically significant if the p < RESULTS Tables 1 and 2 show the antigenotoxic effects of pretreatment with SAC and lycopene alone and in combination on the frequency of MNNG-induced MnPCEs and chromosomal aberrations, respectively. A significant increase in the frequency of MnPCEs and chromosomal aberrations was found in MNNG-treated animals (group 1) compared to untreated controls (group 8) (p < 0.001). Pretreatment of animals administered MNNG with SAC and lycopene alone and in combination (groups 2 4), significantly reduced the incidence of MnPCEs and chromosomal aberrations compared to group 1 (p < 0.01, p < 0.01, p < 0.001, respec- Table 1. Inhibitory effects of SAC and lycopene alone and in combination on MNNG-induced bone marrow MnPCEs in rats Group Treatment MnPCEs/ 2500PCEs PCEs/ NCEs 1. MNNG ± 4.52 ªªª MNNG + SAC ± 3.42** 1.26 (100 mg/kg) 3. MNNG + lycopene (1.25 mg/kg) ± 2.38** MNNG + SAC (100 mg/kg) ± 1.13*** lycopene (1.25 mg/kg) 5. SAC (100 mg/kg) 6.06 ± Lycopene (1.25 mg/kg) 6.26 ± SAC (100 mg/kg) 5.48 ± lycopene (1.25 mg/kg) 8. Control 6.47 ± The values are means ± SEM,n=6,**Significantly different from group 1 (p < 0.01), *** significantly different from group 1 (p < 0.001), ªªª significantly different from group 8 (p < 0.001) ISSN

4 B. Velmurugan, S.T. Santhiya, S. Nagini Table 2. Inhibitory effects of SAC and lycopene alone and in combination on MNNG-induced chromosomal aberrations in rats Group Treatment Types of aberration Mean aberrations G' G'' B' B'' F M Total 1. MNNG ± 2.64 ªªª 2. MNNG + SAC (100 mg/kg) ± 1.32** 3. MNNG + lycopene (1.25 mg/kg) ± 1.36** 4. MNNG + SAC (100 mg/kg) + lycopene (1.25 mg/kg) ± 0.92*** 5. SAC (100 mg/kg) ± Lycopene (1.25 mg/kg) ± SAC (100 mg/kg) + lycopene (1.25 mg/kg) ± Control ± 0.49 G' chromatid gap; G'' isochromatid gap; B' chromatid break; B'' chromosome break; F fragment; M minute; the values are means ± SEM, n = 6. ** Significantly different from group 1 (p < 0.01), *** significantly different from group 1 (p < 0.001), ªªª significantly different from group 8 (p < 0.001) tively). Administration of the dietary agents alone and in combination (groups 5 7) showed no significant change in the incidence of MnPCEs and chromosomal aberrations compared to control (group 8). DISCUSSION MNNG, a model direct-acting alkylating mutagen and a gastric carcinogen is known to methylate all oxygen and most nitrogen atoms of DNA and induce single- and double-strand breaks in DNA [7]. In addition, MNNG has been demonstrated to generate reactive MNNG radicals and hydroxyl radicals that are capable of causing oxidative damage to cellular macromolecules [18]. Previous studies from this laboratory have revealed that oxidative stress arising due to overproduction of reactive oxygen species (ROS) and compromised antioxidant defences plays an important role in the mutagenic and carcinogenic effects of MNNG [27]. Pretreatment with SAC and lycopene in combination more significantly suppressed the clastogenic potency of MNNG than either agent administered alone. Furthermore, the protective effects could be achieved at half the doses found to be optimum in our previous studies [30, 31]. The protective effect of SAC and lycopene combination against MNNG-induced genotoxicity may be attributed to the antioxidant properties of individual agents. SAC, a potent radical scavenging agent is known to inhibit ROS-induced formation of 8-oxodeoxyguanosine in DNA and protect cells against oxidative damage [13, 19]. Recently, SAC has been demonstrated to prevent gentamicin-induced oxidative stress and renal damage [17]. Lycopene, the most effective antioxidant among the carotenoids is known to interact with ROS and reduce the formation of 8-oxodeoxyguanosine as well as H 2 O 2 -induced strand breaks [21, 22]. The results of the present study suggest that SAC and lycopene may interact synergistically to protect against MNNG-induced genotoxicity. Synergistic interactions of chemopreventive agents have been documented in experimental and epidemiological studies. Sengupta et al. [24] have demonstrated the protective effects of tomato and garlic combination against 7,12-dimethylbenz[a]anthracene-induced clastogenicity in mice. A combination of lycopene and garlic has been reported to synergistically induce LDL oxidation [9]. Taken together, these studies underscore the potential beneficial effects of dietderived antioxidants such as SAC and lycopene against genotoxicity and carcinogenicity. Acknowledgment. This work was supported by a research grant from the University Grants Commission, New Delhi, India. REFERENCES 1. Abraham SK: Inhibition of in vivo genotoxicity by coffee. Food Chem Toxicol, 1989, 27, Alberts SR, Cervantes A, van de Velde CJH: Gastric cancer: epidemiology, pathology and treatment. Ann Oncol, 2003, 14, Pol. J. Pharmacol., 2004, 56,

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