Download - Rat colonic lipid peroxidation and antioxidants

CELLULAR & MOLECULAR BIOLOGY LETTERS Volume 10, (2005) pp 535 – 551 http://www.cmbl.org.pl

Received 11 May 2005 Accepted 2 August 2005

* Corresponding author: tel.: 91- 4144-238343-227, e-mail: [email protected]

RAT COLONIC LIPID PEROXIDATION AND ANTIOXIDANT

STATUS: THE EFFECTS OF DIETARY LUTEOLIN ON 1,2-DIMETHYLHYDRAZINE 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 glutathione-dependent enzymes reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione–S-transferase (GST) and glutathione reductase (GR), and of the non-enzymic 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.

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CELL. MOL. BIOL. LETT. Vol. 10. No. 3. 2005

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Key Words: Antioxidants, 1,2–dimethylhydrazine, 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.

Fig. 1. Structure of luteolin. 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

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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 1,2-dimethylhydrazine-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 ± 2ºC 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.


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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 (post-initiation)]. 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.

Fig. 2. Experimental protocol. 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


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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.1.11.1.9) 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 µmoles of GSH utilized/min/mg protein .The activity of glutathione-S-transferase (GST, EC. 2.5.1.18) was estimated via the method of Habig et al. [19], by following the increase in absorbance at 340 nm using 1-chloro-2, 4-dinitrobenze (CDNB) as the substrate. The values are expressed as µmoles 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 µmoles 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 NADH-phenazine 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 µmoles 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 µmoles/mg tissue. Vitamin E (α-tochpherol) content was estimated using the methods of Barker and Frank [24]. The method involves the α-tocopherol-mediated reduction of ferric ions to ferrous ions, and the formation of


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a red coloured complex with 2,2’-dipyridyl. The absorbance of the chromophore was measured at 520 nm. The values are expressed as µmoles/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 Duncan’s Multiple Range Test [DMRT] using SPSS version 12 for Windows. The findings were considered statistically significant if p<0.05. RESULTS Macroscopic observations Fig. 3 show representative examples of the histopathological changes to the colons of the rats in the various groups, as observed macroscopically and with a light microscope. The colon of the control rats (group 1) showed normal mucosa and submucosal layers (Fig. 3A). When luteolin was administered (group 2), the colons of the rats showed normal submucosa with lymphoid aggregates (Fig. 3B). The colons of the luteolin-treated rats (groups 2) and control rats (group 1) were observed to be similar. The tumor size in the DMH group (group 3) was around 2 cm, and the tumors were pedunculated, had well-defined margins with numerous papillae and invasive adenocarcinoma that showed marked pleomorphism (Figs. 3C-3D). The luteolin + DMH (initiation) group (group 4) showed lymphoid aggregates in the submucosa, the tumor cells did not infiltrate the wall, and the tumors were only around 0.25 cm (Fig. 3E). In the luteolin + DMH (post-initiation) group (group 5), the nucleoli were very prominent, with scanty cytoplasm and numerous mitotic figures, while the tumors were only around 0.5 cm in size (Fig. 3F). However, the incidence was much decreased in the luteolin + DMH group (group 4 and group 5) as compared to the DMH-treated group (group 3). Thus, the morphological and microscopic evidence clearly shows that luteolin has a chemoprotective effect against colon tumorigenesis induced by the known procarcinogen 1,2-dimethyl hydrazine. Morphological changes The effect of luteolin on colonic tumor incidence and size is summarized in Tab. 1. There were no tumors in the control rats (group 1) and luteolin-treated control rats (group 2). In rats treated with DMH injections (group 3) the tumor incidence


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Fig. 3. Representative examples of the histopathological changes to the colons of the rats in the various groups. (A). A view of the colon from a control (group 1) rat, showing normal mucosal and submucosal layers. (B). A view of the colon of a luteolin-treated (group 2) rat, showing lymphoid aggregates in the submucosa. (C). A view of the colon of a DMH-treated (group 3) rat, showing carcinomatous glands. (D). A view of the colon of a DMH-treated (group 3) rat, showing the papillary process lined by stratified columnar cells. (E). A view of the colon of a rat treated with DMH + luteolin (initiation) (group 4), showing lymphoid aggregates in the submucosa. The tumor cells do not infiltrate the wall. (F). A view of the colon of a rat treated with DMH + luteolin (post-initiation) (group 5). The mucosa reveals malignant transformation and is intimately mixed with cells of the lymphoid tissues. There is no tumor extension beyond the muscular layer.

A B

C D

E F


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in the colon was 100% and the average tumor size was approximately 2 cm. Luteolin supplementation to DMH-treated rats during the initiation stage of carcinogenesis (group 4) reduced tumor size and incidence (to 10%). Luteolin supplementation during the post-initiation stage (group 5) also resulted in significantly reduced tumor size (0.5 cm) and incidence (to 20%) relative to the group 3 rats. Tab. 1. Incidence of colonic neoplasms.

Group Number of rats

examined

Number of rats with

tumors

Incidence of tumor

(%)

Tumor size (cm)

Control 10 0 0 - Luteolin 10 0 0 - DMH 10 10 100 2 DMH+luteolin (initiation) 10 1 10 0.25 DMH+luteolin (post-initiation) 10 2 20 0.5 Changes in the levels of lipid peroxidation in the colon and intestine Tab. 2 shows the tissue levels of TBARS, lipid hydroperoxides and conjugated dienes in the control and experimental rats. The levels of TBARS, hydroperoxides and conjugated dienes were significantly decreased in the proximal colon, distal colon and intestines of DMH-treated rats (group 3) relative to the levels for the control rats (group 1). Luteolin supplementation to rats at both the initiation and post-initiation stages (groups 4 and 5) of DMH treatment significantly restored the levels of TBARS, lipid hydroperoxides and conjugated dienes of the colon and intestines to near those of control rats. Tab. 2. The effect of luteolin on the levels of thiobarbituric acid reactive substances (TBARS), conjugated dienes and lipid hydroperoxides in control and experimental rats.

TBARS (nmoles/100 g tissue)

Conjugated dienes (mmoles/100 g tissue)

Lipid hydroperoxides (mmoles/100 g tissue) Groups

Proximal colon

Distal colon Intestine Proximal

colon Distal colon Intestine Proximal

colon Distal colon Intestine

Control

3.86 ± 0.30a

4.26± 0.37a

4.24 ± 0.33a

48.55 ± 2.83a

48.45± 2.81a

57.97 ± 3.91a

74.74 ± 8.13a

72.34± 7.94a

78.12± 8.57a

Luteolin

2.95 ± 0.17b

3.50± 0.20b

4.23 ± 0.24a

40.95 ± 2.38b

38.96± 2.27b

48.95 ± 2.85b

69.00 ± 4.02b

64.01± 3.73b

65.93 ± 3.84b

DMH

1.18 ± 0.09c

1.35± 0.08c

1.41 ± 0.08c

26.73 ± 2.91c

23.76± 2.50c

28.71 ± 3.12c

48.54 ± 5.33c

47.52± 5.17c

53.61 ± 6.01c

DMH+luteolin (initiation)

2.68 ± 0.23d

2.25± 0.19d

3.37 ± 0.29c

34.78 ± 2.99d

31.80± 2.73d

44.72 ± 3.85d

70.64 ± 6.80b

66.66± 5.73b

68.56 ± 5.90b

DMH+luteolin (post-initiation)

2.36 ± 0.15e

2.07± 0.13e

2.75 ± 0.17d

31.68 ± 2.06e

29.71± 1.93d

40.59 ± 2.64e

66.42 ± 4.33b

64.44± 4.20b

66.35 ± 4.32b

The values are the means ± S.D of the ten rats in each group. Values not sharing a common superscript (a-e) differ significantly at p<0.05 (DMRT).


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Moreover, the levels of TBARS, lipid hydroperoxides and conjugated dienes were significantly reduced in the colon and intestines of the control rats treated with luteolin (group 2) relative to those for the untreated control rats (group 1). Tab. 3. The effect of luteolin on the levels of reduced glutathione and glutathione peroxidase in the control and experimental rats.

Reduced glutathione (mg /g tissue) Glutathione peroxidase (units*) Groups Proximal



Control 1.10 ± 0.01a

1.12 ± 0.05a

1.19 ± 0.06a

4.70 ± 0.51a

5.34 ± 0.58a

6.09 ± 0.66a

Luteolin 1.27 ± 0.08b

1.34 ± 0.05b

1.38 ± 0.07b

6.90 ± 0.42b

6.80 ± 0.39b

7.90 ± 0.46b

DMH 3.33 ± 0.13c

3.26 ± 0.29b

3.50 ± 0.31c

11.28 ± 1.22a

10.94 ± 1.19c

11.28 ± 1.22c


2.44 ± 0.07d

2.56 ± 0.08d

2.57 ± 0.09d

8.15 ± 0.70d

8.25 ± 0.71d

8.45 ± 0.72d


2.65 ± 0.10d

2.67 ± 0.11d

2.70 ± 0.12d

8.42 ± 0.54d

8.62 ± 0.56d

8.92 ± 0.58d

The values are the means ± S.D of the ten rats in each group. Values not sharing a common superscript (a-d) differ significantly at p<0.05 (DMRT). *µmoles of GSH utilized/min/mg protein. Tab. 4. The effect of luteolin on the activities of glutathione-S-transferase and glutathione reductase in the control and experimental rats.

Glutathione-S-transferase (unitsa) Glutathione reductase (unitsb) Groups Proximal




5.38 ± 0.53a

6.14 ± 0.61a

14.9 ± 0.94a

15.4 ± 0.80a

15.7 ± 1.32a


7.29 ± 0.42b

8.29 ± 0.48b

15.10 ± 0.88a

15.90 ± 0.92a

16.01 ± 0.93a

DMH 11.38 ± 1.14c

11.04 ± 1.10c

11.80 ± 1.18c

34.90 ± 3.80c

35.35 ± 4.4b

36.53 ± 4.15b


7.85 ± 0.67bd

7.75 ± 0.66bd

8.34 ± 0.71b

20.69 ± 1.78c

19.80 ± 1.70c

20.89 ± 0.79c


8.06 ± 0.52d

8.25 ± 0.53d

8.92 ± 0.58d

22.80 ± 1.48d

23.29 ± 1.51d

23.79 ± 1.55c

The values are the means ± S.D of the ten rats in each group. Values not sharing a common superscript (a-d) differ significantly at p<0.05 (DMRT). a – µmoles of CDNB-GSH conjugate formed/min/mg protein. b – µmoles of NADPH oxidised/min/mg protein.


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Tab. 5. The effect of luteolin on the levels of vitamin C and vitamin E in the control and experimental rats.

Vitamin C (µmoles/mg tissue) Vitamin E (µmoles /mg tissue) Groups Proximal




1.32 ± 0.06a

1.35 ± 0.06a

0.65 ± 0.03a

0.68 ± 0.02a

0.72 ± 0.03a


1.91 ± 0.11b

2.01 ± 0.11b

0.70 ± 0.04a

0.75 ± 0.04a

0.78 ± 0.03ac

DMH 2.39 ± 0.24c

2.41 ± 0.24c

2.42 ± 0.26c

1.81 ± 0.04b

1.79 ± 0.03b

1.84 ± 0.05b


1.50 ± 0.07d

1.59 ± 0.06d

1.68 ± 0.07d

0.77 ± 0.02a

0.76 ± 0.02a

0.84 ± 0.04c


1.76 ± 0.15e

1.99 ± 0.17e

1.95 ± 0.16e

1.11 ± 0.01d

1.14 ± 0.04d

1.20 ± 0.06d

The values are the means ± S.D of the ten rats in each group. Values not sharing a common superscript (a-d) differ significantly at p<0.05 (DMRT). Changes in the levels of glutathione and its dependent enzymes and vitamins in the colon and intestine Tabs 3, 4 and 5 show the levels of tissue glutathione and its dependent enzymes (GSH, GPx, GST and GR), and of two vitamins (C and E) in the control and experimental rats. In the proximal colon, distal colon and intestines, the levels of all these subtances were significantly elevated on DMH treatment (group 3). The tissue levels of GSH, GPx, GST and GR, and vitamins C and E were significantly decreased in the tissues of rats given luteolin (initiation and post-initiation, groups 4 and 5) relative to the levels for group 3. The levels of glutathione and glutathione-dependent enzymes and vitamins were elevated in the colon and intestine of control rats treated with luteolin (group 2) relative to those for the untreated control rats (group 1). Changes in the levels of enzymic antioxidants in the colon and intestine Tab. 6 represents the levels of tissue enzymic antioxidants (SOD and CAT) in the control and experimental rats. The levels of enzymic antioxidants (SOD and CAT) in the proximal colon, distal colon and intestines were significantly decreased on DMH treatment (group 3). The levels of SOD and CAT were significantly increased in the tissues of rats given luteolin (initiation and post-initiation, groups 4 and 5) relative to those for group 3. The activities of SOD and CAT were significantly elevated in the colon and intestine of control rats treated with luteolin (group 2) relative to those for the untreated control rats (group 1).


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Tab. 6. The effect of luteolin on the activities of superoxide dismutase and catalase in the control and experimental rats.

Superoxide dismutase (units*)

Catalase (µmoles of H2O2 utilized /min/mg

protein) Groups Proximal

colon Distal colon

Intestine Proximal colon

Distal colon

Intestine


4.39 ± 0.44a

4.23 ± 0.42a

37.55 ± 4.12a

37.97 ± 4.13a

39.93 ± 4.38a


5.69 ± 0.33b

5.29 ± 0.30b

44.01 ± 2.56b

45.02 ± 2.62b

46.01 ± 2.68b

DMH 2.50 ± 0.25c

2.74 ± 0.27c

2.87 ± 0.28c

21.87 ± 2.38c

23.88 ± 2.60c

24.75 ± 2.69c

DMH + luteolin (initiation)

3.87± 0.33d

3.92± 0.33d

3.94± 0.33a

30.84± 2.65d

34.82± 2.99d

31.84 ± 2.74d

DMH + luteolin (post-initiation)

3.55± 0.23e

3.75± 0.24d

3.81± 0.24a

28.75± 1.87e

32.71± 2.13d

29.74 ± 1.93d

The values are the means ± S.D of the ten rats in each group. Values not sharing a common superscript (a-e) differ significantly at p<0.05 (DMRT). *Enzyme required for 50% inhibition of NBT reduction/min/mg protein. DISCUSSION The results clearly indicate that administration of the procarcinogen DMH in the presence of luteolin brings about profound alterations in the tissue lipid peroxidation and antioxidant status. DMH, the potent colon-specific carcinogen used in this study, is metabolized in the liver to azoxymethane (a known colon carcinogen), ultimately leading to the generation of methyldiazonium ions and carbonium ions, which are active carcinogenic electrophiles [26] that manifest their action in the colon. The decreased lipid peroxidation in the colon and intestinal tissues reported on here is based on the assessment of the levels of reactive molecules such as lipid hydroperoxides and conjugated dienes formed during the chain reaction of lipid peroxidation in addition to MDA. Previous studies have shown reduced rates of lipid peroxidation in the tumor tissue of various types of cancer [27-30]. Increased cell proliferation is thought to be involved in the pathogenesis of colon cancer. Cancer cells acquire particular characteristics that benefit their proliferation [31], and they tend to proliferate faster when the lipid peroxidation level is low. Therefore, the decreased colon and intestinal lipid peroxidation observed in DMH-treated rats could be due to increased cell proliferation. Thus, malignant tissues are less susceptible and more resistant to free radical attack, and hence lipid peroxidation is less intense [32]. Our results correlate with previous findings on malignant cells being better protected than their normal counterparts against free radicals [33]. In addition to this, the decreased levels of


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lipid peroxidation in DMH-treated rats may also be due to increased resistance and/or decreased susceptibility of the target organs to free radical attack. Luteolin administration to DMH-treated rats (group 4, initiation and group 5, post-initiation) restored the lipid peroxidation levels to near those of the control rats (group 1), which may be due to the antiproliferative activity of luteolin. Since proliferation and lipid peroxidation are inversely related [31], and since luteolin is a known antiproliferative agent, luteolin could contribute to the observed increase in lipid peroxidation in the colon and intestine. This could increase the susceptibility and decrease the resistance of tumor cells to free radical attack, leading to decreased cell proliferation. Hence, we suggest that luteolin prevents DMH-induced colon cancer through its antiproliferative effects. SOD and CAT are two important enzymic antioxidants that act against toxic oxygen free radicals such as superoxide ( −

2O ) and hydroxyl ( OH• ) ions in the biological system. They are involved in the direct elimination of reactive oxygen metabolites, which is probably one of the most effective defenses of the living body against diseases. In our study, we observed decreased levels of SOD and CAT in the colon and intestinal tissues of DMH-treated rats. The decreased activities of SOD and CAT may be due to the dangerous increase in the levels of reactive oxygen species and thus, enhanced oxidative stress and proliferation of colonocytes in colorectal malignant carcinoma. This may correspond with a previous report which showed that some human cancer lines produce large amounts of hydrogen peroxide [34]. Luteolin has four hydroxyl groups at the 3’-, 4’-, 5- and 7-positions, which are very important for its antioxidant potential. It was suggested that free radical scavenging activity is directly related to the number of hydroxyl groups substituted on ring B, especially at the 3’ position [6]. Cao et al. also reported that dihydroxyl groups at the 3’- and 4’-positions play an important role in antioxidant reactions [35]. Thus, by virtue of its four hydroxyl groups, luteolin is highly potent in scavenging the reactive oxygen species which initiate lipid peroxidation. In this context, previous studies already documented the antioxidant property of luteolin, which is known to neutralize highly reactive superoxide anions and hydroxyl radicals. The observed chemopreventive potential of luteolin may be attributed to the existence of hydroxyl groups at the 3’-, 4’-, 5- and 7-positions of the ring. Administration of luteolin to the group 4 (initiation) and group 5 (post-initiation) rats enhanced the activities of SOD and CAT in the colon and intestinal tissues relative to that observed for the DMH-treated rats of group 3. Morever, due to its ability to scavenge free radicals and toxic carcinogenic electrophiles, luteolin may spare the antioxidant enzymes, which may be the cause of these enhanced SOD and CAT levels. These results prove the excellent chemopreventive efficacy of luteolin against DMH-induced colon carcinogenesis. GPx, GST and GR predominantly participate in the detoxification of xenobiotics, carcinogens, free radicals and peroxides by conjugating toxic


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substances with GSH, ultimately protecting cells and organs against carcinogen-induced toxicity. Decreased lipid peroxidation associated with enhanced GSH and glutathione-dependent enzymes in the colon and intestines is a well-known phenomenon in experimental colon carcinogenesis [29]. In our study, we observed enhanced levels of GSH, GPx, GST and GR in DMH-treated rats. This may be due to the increased cell proliferation involved in the pathogenesis of DMH-induced colon cancer [36]. It was previously demonstrated that GSH, GPx, GST and GR were expressed in greater amounts in the neoplastic cells, conferring a selective growth advantage [37]. Kuralko and Pence also reported that DMH treatment results in an increased tissue GSH content [38]. In the presence of GSH as a substrate, and GPx and GST as detoxifying enzymes, conjugation of toxic electrophiles with GSH takes place, conferring a selective growth advantage to cancer cells. Moreover, GST increases the capacity of the tumor cells to withstand the burden of toxicants and procarcinogens. Increased activity of GPx (in the colon and intestinal tissues), an enzyme that is capable of destroying hydrogen peroxide, was observed in our study. These changes were accompanied by an increase in the levels of GSH, a co-substrate for GPx, which actively in concert eliminate hydrogen peroxide and lipid hydroperoxides. Thus the elevated GSH, GPx, GST and GR levels in colon and intestinal tissues observed in our study may be used as markers of cell proliferation. Vitamin E is a powerful lipid-soluble antioxidant and a free radical scavenger that inhibits lipid peroxidation. Vitamin C is a water-soluble antioxidant that scavenges reactive oxygen metabolites generated during the metabolism of carcinogen, thus protecting genetic material from the initiation and promotion stages of carcinogenesis. The enhanced levels of these vitamins (C and E) observed in our study may be due to increased levels of GSH in the tissues and also due to an active rate of cell proliferation [39]. With reference to our results, overexpression of antioxidants was documented in malignant tumors [40, 41]. The decreased levels of GSH and its dependent enzymes observed in the colonic and intestinal tissues during carcinogenesis might in turn lead to increased utilization of vitamin C and vitamin E to scavenge the reactive oxygen species. This may be one of the reasons for the observed decrease in the levels of these vitamins in DMH-treated rats. On the administration of luteolin to DMH-treated rats, the levels of glutathione and the glutathione-dependent enzymes (GSH, GPx, GST and GR) and the two mentioned vitamins (C and E) were significantly decreased in group 4 (initiation) and group 5 (post-initiation) rats relative to the levels for group 3 (DMH-treated rats without luteolin supplement). Luteolin inhibits colon carcinogenesis both by blocking the metabolic activation of carcinogens and by suppressing the initiation and post-initiation stages of colon carcinogenesis. Luteolin exhibits antiproliferative activity against various types of cancer [42, 43] and these findings led us to postulate that luteolin regulates cell proliferation. In addition to this, it also inhibits cancer cell growth [44], lipid peroxidation, and tumorigenesis [45, 46]. Moreover, previous studies have reported that incubation


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with various concentrations of luteolin reduced tissue GSH levels, which might induce the oxidative process resulting in the accumulation of lipid peroxides leading to cell death. In conclusion, our results strongly suggest that luteolin significantly inhibits colon carcinogenesis as evidenced not only by the significantly reduced colon cancer incidence but also by the decreased size of colonic neoplasms. Moreover, luteolin provides greater protection when administered during the initiation stage of carcinogenesis than when administered during the post-initiation stages of colon carcinogenesis. This may be due to the hydroxyl group of luteolin, which possess anticancer and antioxidant activity. Acknowledgement. We thank Mr. Ying Chen, Guofeng Biotechnology Co, Ltd., China for generously providing luteolin for our study. REFERENCES 1. Landis, S.H., Murray, T., Bolden, S. and Wingo, P.A. Cancer Statistics.

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