Rat colonic lipid peroxidation and antioxidants

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relation between DMH induction of colon cancer and regression by natural product which is Luteolin


<p>CELLULAR &amp; MOLECULAR BIOLOGY LETTERS</p> <p>Volume 10, (2005) pp 535 551 http://www.cmbl.org.pl Received 11 May 2005 Accepted 2 August 2005</p> <p>RAT COLONIC LIPID PEROXIDATION AND ANTIOXIDANT STATUS: THE EFFECTS OF DIETARY LUTEOLIN ON 1,2DIMETHYLHYDRAZINE CHALLENGE VAIYAPURI MANJU, VAIRAPPAN BALASUBRAMANIYAN and NAMASIVAYAM NALINI* Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar 608002, Tamilnadu, India Abstract: Colon cancer is the third most common cancer and second leading cause of cancer-related death in the United States. A number of recent articles demonstrate the importance of natural products as cancer chemopreventive agents. In this study, we evaluated the chemopreventive efficacy of luteolin, a flavonoid, on tissue lipid peroxidation and antioxidant status, which are used as biomarkers in DMH-induced experimental colon carcinogenesis. Rats were given a weekly subcutaneous injection of DMH at a dose of 20 mg/kg body weight for 15 weeks. Luteolin (0.2 mg/kg body weight/everyday p.o.) was given to the DMH-treated rats at the initiation and post-initiation stages of carcinogenesis. The animals were killed after 30 weeks. After a total experimental period of 32 weeks (including 2 weeks of acclimatization), tumor incidence was 100% in DMH-treated rats. In those DMH-treated rats that had received luteolin during the initiation or post-initiation stages of colon carcinogenesis, the incidence of cancer and the colon tumor size was significantly reduced as compared to that for DMH-treated rats not receiving luteolin. In the presence of DMH, relative to the results for the control rats, there were decreased levels of lipid peroxidation, as denoted by thiobarbituric acid reactive substances (TBARS), conjugated dienes and lipid hydroperoxides, decreased activities of the enzymic antioxidants superoxide dismutase (SOD) and catalase (CAT), and elevated levels of glutathione and the glutathionedependent enzymes reduced glutathione (GSH), glutathione peroxidase (GPx), glutathioneS-transferase (GST) and glutathione reductase (GR), and of the nonenzymic antioxidants vitamin C and vitamin E. Our study shows that intragastric administration of luteolin inhibits colon carcinogenesis, not only by modulating lipid peroxidation and antioxidant status, but also by preventing DMH-induced histopathological changes. Our results thus indicate that luteolin could act as a potent chemopreventive agent for colon carcinogenesis.* Corresponding author: tel.: 91- 4144-238343-227, e-mail: nalininam@yahoo.com</p> <p>536</p> <p>CELL. MOL. BIOL. LETT.</p> <p>Vol. 10. No. 3. 2005</p> <p>Key Words: Antioxidants, 1,2dimethylhydrazine, Lipid Peroxidation, Luteolin INTRODUCTION Colon cancer is one of the leading causes of cancer death in both men and women in western countries, including the United States [1]. Diet, especially a high intake of fat and red meat and a low intake of fruits and vegetables is regarded as the most important nutritional influence on colon cancer development [2]. Both epidemiological and experimental studies suggest that colon cancer is strongly influenced by nutritional factors, including the quantity and composition of dietary fat [2]. Colon carcinogenesis is frequently a pathological consequence of persistent oxidative stress, leading to DNA damage and mutations in cancer-related genes, a cycle of cell death and mutations and regeneration [3] in which cellular overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are implicated. Chemoprevention has the potential to be a major component of colorectal cancer control. Several investigators have over many years conducted research on agents with potential chemopreventive properties and have elucidated their modes of action. Although a full explanation of the intricacies of the causes, development, and control of colon cancer is awaiting further research, the growing knowledge about the mechanisms by which chemopreventive agents act defines opportunities to use specific agents at critical stages of cancer initiation, promotion and progression. Nowadays, flavonoids play an important role in the chemoprevention of colon carcinogenesis. Flavonoids are widespread in fruits, vegetables, wine and tea. Flavonoids have been reported to exert an antioxidant activity due to their ability to scavenge free radicals or to chelate metal ions [4]. Recently, research has been focused on the bioavailability, pharmacokinetics and metabolism of flavonoids in order to evaluate their important role in the chemoprevention of diseases such as cancer.</p> <p>Fig. 1. Structure of luteolin.</p> <p>Luteolin (3', 4', 5,7-tetrahydroxy flavone) is an important member of the flavonoid family, and is present in glycosylated forms in celery, green pepper, perilla leaf and camomile tea, among others, and as aglycone in perilla seeds (Fig 1). It has contributed to the antioxidant activity of artichoke leaf extract towards reactive oxygen species in human leucocytes [5]. Luteolin is also</p> <p>CELLULAR &amp; MOLECULAR BIOLOGY LETTERS</p> <p>537</p> <p>reported to have anti-inflammatory/anti-allergic [6], antitumorigenic [7] and antioxidant properties [8]. A recent report establishes luteolin as a potent inhibitor of human mast cell activation through the inhibition of protein kinase C activation and Ca2+ influx [9]. The DMH-induced colon cancer model has been well characterized, and bears many of the same cell kinetics and histopathological and molecular characteristics of tumorigenesis as the human colon cancer model, as well as being morphologically similar to it [10]. We previously reported on the effect of spices and fiber on lipid metabolism and bacterial enzymes in 1,2dimethylhydrazine-induced colon cancer [11, 13]. However, no studies have thusfar been undertaken to assess the effect of luteolin on the biochemical changes occurring in the tumor-bearing rats. We therefore examined the effect of luteolin on 1,2-dimethyl hydrazine-induced rat colon carcinogenesis using tissue lipid peroxidation and antioxidant levels as biomarkers. This paper is the first report on the inhibitory and chemopreventive effect of luteolin on colon carcinogenesis in rats. MATERIALS AND METHODS Chemicals Luteolin was donated by Guofeng Biotechnology Co., Limited, China. DMH was obtained from the Sigma Chemical Company, St. Louis, USA. All the other chemicals and reagents used were of analytical grade. Preparation of luteolin Luteolin powder was suspended in 0.5% carboxymethyl cellulose (CMC) and each rat received a daily dose of 1 ml of luteolin suspension at a dose of 0.2 mg/kg body weight [12]. Tumor induction DMH was dissolved in 1 mM EDTA just prior to use and the pH was adjusted to 6.5 with 1 mM sodium bicarbonate to ensure the stability of the chemical. The rats were given a weekly subcutaneous injection of DMH in the groin at a dose of 20 mg/kg body weight for 15 weeks [13]. Experimental animals Male Wistar rats of 100-120 g body weight were obtained from the Central Animal House, Department of Experimental Medicine, Annamalai University, Tamil Nadu, India, and maintained at 27 2C with a 12-h light/12-h dark cycle. A commercial pellet diet containing 4.2% fat (Hindustan Lever Ltd., Mumbai, India) was powdered and mixed with 15.8% peanut oil making a total of 20% fat. This was then fed to all the rats throughout the experimental period of 32 weeks (which included an initial 2 weeks of acclimatization). Water was given ad libitum. The rats were randomly assigned to 5 groups of ten animals each.</p> <p>538</p> <p>CELL. MOL. BIOL. LETT.</p> <p>Vol. 10. No. 3. 2005</p> <p>Treatment schedule The rats in group 1 received 1 ml of 0.5% CMC every day via intragastric intubation and served as the untreated control. The group 2 rats received luteolin via intragastric intubation at a daily dose of 0.2 mg/kg body weight. The rats in groups 3 to 5 received a DMH [20 mg/kg body weight] injection once a week subcutaneously for the first 15 weeks. The group 4 rats received luteolin as in group 2 starting one week before the DMH injections and continued till one week after the final exposure [DMH + luteolin (initiation)]. The group 5 rats received luteolin as in group 2 starting one week after the cessation of DMH injections and continuing till the end of the experiment [DMH + luteolin (postinitiation)]. The experiment was terminated 30 weeks after the end of the acclimatization period, and all the animals were killed by cervical dislocation after an overnight fast. The colon was split open longitudinally and gross tumors were counted. Colonic and intestinal tissues were then processed and used for various biochemical assessments. The experimental protocol and treatment schedule is represented in Fig. 2.</p> <p>Fig. 2. Experimental protocol.</p> <p>Preparation of tissue homogenate Tissue samples were immediately transferred to ice-cold containers, weighed, and homogenized using the appropriate buffer in a tissue homogeniser. Biochemical assessments Lipid peroxidation was estimated by measuring the level of thiobarbituric acid reactive substances (TBARS) in the plasma via the method of Ohkawa [14]. The pink chromogen produced was measured at 532 nm. The values are expressed as nmoles/100 g tissue. The level of conjugated dienes was assessed using the method of Rao and Recknagel [15]. This method is based on the arrangement of</p> <p>CELLULAR &amp; MOLECULAR BIOLOGY LETTERS</p> <p>539</p> <p>the double bonds in polyunsaturated fatty acids (PUFA) to form conjugated dienes with an absorbance maximum at 233 nm. The values are expressed as mmoles/100 g tissue. The lipid hydroperoxide contents were measured using the method of Jiang et al [16]. Hydroperoxides are detected by their ability to oxidize ferrous iron leading to the formation of a chromophore with an absorbance maximum at 560 nm. The values are expressed as mmoles/100 g tissue. Reduced glutathione (GSH) content was determined via the method of Ellman [17]. GSH determination is based on the development of a yellow color when 5,5 dithio (2-nitro benzoic acid) (DTNB) is added to compounds containing sulfhydryl groups. The values are expressed as mg/g tissue. Glutathione peroxidase (GPx, EC. activity was assayed via the method of Rotruck et al. [18] with a modification. A known amount of enzyme preparation was incubated with H2O2 in the presence of GSH for a specified time period. The amount of H2O2 utilized was determined using the method of Ellman [17]. The values are expressed as moles of GSH utilized/min/mg protein .The activity of glutathione-S-transferase (GST, EC. was estimated via the method of Habig et al. [19], by following the increase in absorbance at 340 nm using 1chloro-2, 4-dinitrobenze (CDNB) as the substrate. The values are expressed as moles of CDNB-GSH conjugate formed/min/mg protein. Glutathione reductase activity was assayed using the method of Carlberg and Mannervik [20] by measuring GSH formed by NADPH. The values are expressed as moles of NADPH oxidised/min/mg protein. Superoxide dismutase (SOD, EC.I.15.1.1) was assayed using the method of Kakkar et al. [21] based on the 50% inhibition of the formation of NADHphenazine methosulfate-nitroblue tetrazolium formazan at 520 nm. One unit of the enzyme is taken as the amount of enzyme required for 50% inhibition of NBT reduction/min/mg protein. The activity of catalase (CAT, EC.1.11.16) was determined via the method of Sinha [22]. Dichromate in acetic acid was reduced to chromic acetate when heated in the presence of hydrogen peroxide (H2O2), with the formation of perchromic acid as an unstable CAT intermediate. The chromic acetate formed was measured at 590 nm. Catalase was allowed to split H2O2 for different periods of time. The reaction was stopped at different time intervals via the addition of a dichromate-acetic acid mixture, and heating the reaction mixture and measuring chromic acetate colorimetrically determined the remaining H2O2. The values are expressed as moles of H2O2 utilised/min/mg protein. Vitamin C (ascorbic acid) content was estimated by the method of Roe and Kuether [23], in which dehydro ascorbic acid is coupled with 2,4 dinitro phenyl hydrazine (DNPH) and then treated with sulfuric acid, forming an orange-red coloured compound, the content of which was measured at 520 nm. The values are expressed as moles/mg tissue. Vitamin E (-tochpherol) content was estimated using the methods of Barker and Frank [24]. The method involves the tocopherol-mediated reduction of ferric ions to ferrous ions, and the formation of</p> <p>540</p> <p>CELL. MOL. BIOL. LETT.</p> <p>Vol. 10. No. 3. 2005</p> <p>a red coloured complex with 2,2-dipyridyl. The absorbance of the chromophore was measured at 520 nm. The values are expressed as moles/mg tissue. The protein content was determined via the method of Lowry et al. [25]. Proteins react with Folin-Ciocalteau reagent to give a coloured complex. The colour so formed was due to the reaction of alkaline copper with protein and the reduction of phosphomolybdate by the tyrosine and tryptophan present in the protein. The intensity of the colour depends on the amount of these aromatic amino acids present. Statistical analysis The results presented here are the means SD of the 10 rats in each group. The results were analysed using a one-way analysis of variance [ANOVA] and the group means were compared using Duncans Multiple Range Test [DMRT] using SPSS version 12 for Windows. The findings were considered statistically significant if p</p>