Efficacy of the potential chemopreventive agent, hesperetin (citrus flavanone), on 1,2-dimethylhydrazine induced colon carcinogenesis

Food and Chemical Toxicology 47 (2009) 2594–2600

Contents lists available at ScienceDirect

Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Efficacy of the potential chemopreventive agent, hesperetin (citrus flavanone), on 1,2-dimethylhydrazine induced colon carcinogenesis S. Aranganathan, N. Nalini * Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar, 608 002 Chidambaram, Tamil Nadu, India

a r t i c l e

i n f o

Article history: Received 19 May 2009 Accepted 20 July 2009

Keywords: Hesperetin Lipid peroxidation Chemoprevention

a b s t r a c t Our current study is an effort to identify a potent chemopreventive agent against colon cancer. Here we have investigated the efficacy of hesperetin on tissue lipid peroxidation, antioxidant defense system and colonic histoarchitecture in male Wistar rats in colon carcinogenesis. Rats in groups 3, 4, 5 and 6 were treated with DMH (20 mg kg body weight s.c.) once a week for 15 weeks. Group 1 rats received modified pellet diet and served as control; group 2 received modified pellet diet along with hesperetin (20 mg/kg body weight, p.o., every day); and hesperetin was given to the rats as in-group 2 during the initiation, post-initiation and entire period stages of colon carcinogenesis. Lipid peroxidation was studied by measuring the formation of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH) and conjugated dienes (CD), and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), reduced glutathione (GSH), in the liver and colonic tissues of DMH administered rats. (1) Decreased levels of lipid peroxidation in the colonic tissues; (2) decreased activities of antioxidant enzymes SOD, CAT, GPX, GR and GSH levels in the tissues on DMH treatment. Hesperetin supplementation during the initiation, post-initiation and entire period stages of carcinogenesis significantly reversed these activities. These results indicate that hesperetin may be a potential chemopreventive agent against DMH-induced colon cancer. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Colorectal cancer is the second to third most frequent type of common malignant neoplasm in the World (Schulmann et al., 2002). Colon cancer incidence has been increasing in recent years in India. The recent changes in the diet of Indians to ‘‘the western type” which is characteristically composed of high fat, high protein, low carbohydrates and low fiber is thought to be an important factor for the increase in colon cancer incidence (Weisburger, 1991). More than 11 million people are diagnosed with cancer every year. It is estimated that there will be 16 million new cases every year by 2020 around the world population (Anand et al., 2008). Cancer causes 7 million deaths every year – or 12.5% of deaths worldwide (Brayand and Moller, 2006). Colon carcinogenesis is considered to be linked with dietary habits like high animal fat intake. Dietary fat has been implicated as an enhancing agent in colon carcinogenesis both in epidemiologic and animal studies. Our laboratory has standardized the induction of colon cancer via the s.c. route of carcinogen administration (Sengottuvelan et al., 2006). This mimics the mode of human exposure to environment carcinogen. 1,2-Dimethylhydrazine (DMH) is a potent colon pro-

* Corresponding author. Tel.: +91 4144 238343; fax: +91 4144 239141. E-mail address: [email protected] (N. Nalini). 0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2009.07.019

carcinogen that is metabolically activated to the active carcinogen in the liver. DMH is believed to form active intermediates including azoxymethane and methylazoxymethanol in the liver, which are transported subsequently into the colon via bile and blood (No et al., 2007). Methylazoxymethanol is decomposed to form methyldiazonium ions, which methylate cellular components. DMH also produces free radicals that induce oxidative DNA damage in the liver and colon (No et al., 2007). Damage to DNA from ROS is a consequence of oxidative stress, and several oxidative DNA adducts, including 8-oxodG, have been implicated in the tumorigenic process (Yamashita and Kawanishi, 2000). Reactive oxygen species (ROS) may be involved in the pathogenesis of various human diseases because they induce damage to biological macromolecules such as DNA, carbohydrates and proteins. To protect the tissues from oxidative damage caused by ROS, has antioxidant defense systems consisting of enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) (Choi, 2008). Oxidative stress exists when pro-oxidants such as ROS exceed antioxidant capabilities. This environment can result from increased generation of ROS as well as impaired removal of ROS by antioxidant defenses such as SOD, CAT, and GPx enzyme systems. Differences in ROS generation or antioxidant enzyme activities and in turn oxidative DNA damage contribute to the variation in cancer susceptibility at these sites.

2595

S. Aranganathan, N. Nalini / Food and Chemical Toxicology 47 (2009) 2594–2600

tic cage with bedding and maintained under controller conditions of temperature (22 ± 2 °C), humidity (55 ± 10%), 12-h light/dark cycle and provided with modified pellet diet along with 20% fat (peanut oil) and tap water were available ab libitum (Table 1). The animals used and the experimental design had the prior approval of the animal care and use Committee of Annamalai University (Reg. No. 190/2007/ CPCSEA). 2.2. Chemicals Hesperetin and 1,2-dimethylhydrazine were purchased from Sigma Chemical Co. (USA). All other chemicals and reagents used were of analytical grade. 2.3. Tumor induction Fig. 1. Chemical structure of hesperetin.

Chemoprevention aims to halt or reverse the development and progression of pre-cancerous cells through use of non-cytotoxic nutrients and/or pharmacological agents during the period between tumor initiation and malignancy. Modulation of enzymes involved in metabolic activation and excretion of carcinogens is one of the best-investigated mechanisms of chemopreventive agents. The predominant mechanism of their anti-carcinogenic action is thought to result from antioxidant activity, enzyme inhibition, antiproliferative activity, and the capacity to scavenge free radicals (Starvic, 1994; Depeint et al., 2002). The chemopreventive mechanisms are thought to involve multiple biochemical mechanisms including enzyme induction and auto-oxidation (Breiniiolt et al., 1999). In contrast, a number of studies have suggested that high consumption of fruits and vegetables decreases the risk of colon cancer (Slattery et al., 2000). Hesperetin (5,7,30 -trihydroxy-40 methoxyl flavonone, Fig. 1) one of the most abundant flavonoids found in citrus fruits (Gil-Izquierdo et al., 2001) and is used as traditional medicine in China. Hesperetin shows a wide spectrum of pharmacological effects such as anti-inflammatory, anti-carcinogenic, antihypertensive and anti-atherogenic effects (Garg et al., 2001; Galati et al., 1996) including the antioxidant properties (Cai et al., 2004). The daily intake of citrus juices like orange and grape juices contain about 200–590 mg/L of hesperetin. Daily ingestion of citrus flavonoids has been estimate to be about 68 g on an average, mainly ingested via fruit juices in the USA (Scholz et al., 2006). Hesperetin occurs as hesperidin (its glycoside form) in nature. Dietary hesperidin is deglycosylated to hesperetin by intestinal bacteria prior to absorption (Ameer et al., 1999) and hesperidin may be considered as a prodrug, which is metabolized to hesperetin (Lee et al., 2004). Hesperetin is reported to be a powerful radical scavenger and a promoter of cellular antioxidant defense-related enzyme activities (Kim et al., 2004). The multistep nature of carcinogenesis provides many opportunities for intervention with chemopreventive agents. They targeted at specific mechanisms involved in the initiation, promotion, and progression of cancers. Determining the efficacy of these agents during the post-initiation stage, at which point the premalignant lesions is known to have developed, is very important with regard to the eventual clinical use of these agents in the secondary prevention of colon cancer among patients with polyps. Therefore, the present study was design to evaluate the potential chemopreventive efficacy of hesperetin administered during the three different regimens of colon carcinogenesis. 2. Material and methods 2.1. Animals Four-week old male Wistar rats, weighing approximately 150 g, were obtained from the Central Animal House, Raja Muthaih Medical College and Hospital (RMMCH). They were randomly distributed into six groups of four animals per plas-

DMH was dissolved in 1 mM EDTA just prior to use and the pH adjusted to 6.5 with 1 mM NaOH to ensure the stability of the carcinogen. The rats were given subcutaneous injections of DMH for 15 consecutive weeks at a dose of 20 mg/kg body weight. 2.4. Preparation of chemopreventive agent Hesperetin powder was suspended in 0.1% carboxymethyl cellulose (CMC) and each rat received daily 1 ml as suspension at a dose of 20 mg/kg body weight. 2.5. Experimental design Group 1 rats received modified pellet diet and served as control, group 2 received modified pellet diet with hesperetin (20 mg/kg body weight) every day throughout the experimental period. Groups 3–6 received modified pellet diet with DMH (20 mg/kg body weight) once a week subcutaneously for the first 15 weeks, which represent the colon cancer bearing rats. Group 4 rats were colon cancer bearing rats treated with hesperetin (as in-group 2) for the first 15 weeks (Initiation). Group 5 rats were colon cancer bearing rats administered hesperetin (as in-group 2) one week after the cessation of DMH injections and continued till the end of the experimental period (Post-initiation), and group 6 rats were colon cancer bearing rats administered hesperetin (as in-group 2) throughout the experimental period (Entire period) for 32 weeks. The experimental protocol is show in Fig. 2. 2.6. Colon tumor/polyp analysis At the end of 32 weeks of DMH treatment the rats were sacrificed, their colons were excised blotted dry, cut open longitudinally and the inner surface was examined for visible macroscopic lesions. Tumors/polyps were easily discernible in the inflammed section of the colon. Colon sections of equal length and tumors were examined grossly for the location, number and size. 2.7. Body weight changes A careful record of the body weight changes of the control, DMH and hesperetin treated rats was kept throughout the study. The rats were weighed at the beginning of the experiment subsequently once a week and finally before sacrifice. 2.8. Preparation of tissue homogenates Liver and colonic tissues were removed immediately and washed with ice-cold saline and homogenized in the appropriate buffer in a tissue homogenizer. 2.9. Antioxidant defense status and lipid peroxidation Lipid peroxidation as indicated by thiobarbituric acid reactive substances (TBARS) was measured using the method of Ohkawa et al. (1979). The levels of conjugated dienes (CD) was assessed by the method of Rao and Recknagel (1968). Lipid hydroperoxides (LOOH) were measured by the method of Jiang et al. (1993).

Table 1 Composition of the diet.

Protein Fat Carbohydrates Fiber Minerals Vitamins

Commercial pellet diet 84.2%

Peanut oil 15.8%

Total %

17.7 4.2 50.5 3.4 6.7 1.7

– 15.8 – – – –

17.7 20.0 50.5 3.4 6.7 1.7

2596

S. Aranganathan, N. Nalini / Food and Chemical Toxicology 47 (2009) 2594–2600

Fig. 2. Schematic representation of the experimental design. 2.10. Antioxidant enzymes SOD (EC 1.15.1.1) and CAT (EC 1.11.16) Catalase was assayed using a spectrophotometer at 590 nm by monitoring the decomposition of H2O2 as described by Sinha (1972). The specific activity of catalase was expressed as lmol of H2O2 utilized/min/mg protein. SOD was assayed by the method of Kakkar et al. (1984). One unit of enzyme is defined as the quantity of SOD required to produce 50% inhibition of NBT reduction/min/mg protein. 2.11. Glutathione reductase (EC 1.6.4.2) and glutathione peroxidase (EC 1.15.1.1) Glutathione reductase (GR) was determined by the method of Carlberg and Mannervik (1985). One unit of enzyme is defined as the nmoles of NADPH consumed/min/mg protein. Glutathione peroxidase (Gpx) activity was assayed by the method of Folhe and Gunzler (1984). A known amount of the enzyme preparation was incubated with H2O2 and was determined using Ellman’s method (1982). The values are expressed as lmoles of GSH utilized/min/mg protein. Glutathione content was measured by the method of Ellman (1982). The concentration of glutathione was measured using the 5,50 -dithiobis (2-nitrobenzoic acid). Glutathione concentration was expressed as micromoles of –SH content/g tissue. 2.12. Histopathological changes After sacrifice, the colons were macroscopically examined for the presence of tumors or other pathological lesions. Tissues with abnormal morphology were fixed in 10% buffered formalin and embedded in paraffin blocks. Histological sections stained with hematoxylin and eosin was used to confirm the presence and type of tumors by histopathological examination, which was performed by a pathologist unaware of the experimental codes. 2.13. Statistical analysis The statistical significance of the data has been determined using one-way analysis of variance (ANOVA) and significant difference among treatment groups were evaluated by Duncan’s multiple range test (DMRT). The results were considered statistically significant at P < 0.05. All statistical analyses were made using SPSS 11.0 software package (SPSS, Tokyo, Japan).

pathological alterations indicative of hesperetin toxicity in the major organs. The mean body weight in-group 3 was significantly decreased as compared to control groups (group 1) Fig. 3. While supplementation of hesperetin the mean body weight were significantly increase as compared with DMH alone treated rats. 3.2. Incidence and multiplicity of colonic tumors Colonic tumors were macroscopically sessile or pedunculated and histologically revealed tubular adenomas, tubular adenocarcinomas or signet-ring cell carcinomas, with a high incidence of tubular adenocarcinoma. The incidence and multiplicity of intestinal tumors is show in Table 2, respectively. The frequency of large intestinal adenocarcinoma in groups 6 (10%) and 5 (20%) was significantly lower than in-group 3 (60%, P < 0.01). A significant reduction in the multiplicity of colonic adenocarcinoma (number of carcinomas/rat) in groups 6 and 5 was observed as compared to the group 3 rats. 3.3. Effect of hesperetin on tissue lipid peroxidation in the colon cancer rats A statistically significant increase (P < 0.05) in the levels of TBARS, LOOH and CD was observed in liver and significantly decreased levels in proximal colon and distal colon of rats subjected to 32 weeks of DMH treatment (Table 3) as compared to control rats (group 1). Hesperetin supplementation to DMH-treated rats during the initiation, post-initiation and entire period stages (groups 4–6) significantly restored the levels of TBARS, LOOH and CD of the liver and colon to near those of control rats and vice versa.

3. Results 3.1. General observations of the long-term assay

3.4. Effect of hesperetin on antioxidant defense system in the colon cancer rats

The rats tolerated quite well the subcutaneous injection of DMH and/or hesperetin feeding. During the study, no clinical signs of toxicity were present in any group. Histologically there were no

Table 4 shows the activities of SOD and CAT. The activities of SOD and CAT, in the DMH-treated control group (group 3) were significantly decreased (P < 0.05) as compared to the control group

2597

S. Aranganathan, N. Nalini / Food and Chemical Toxicology 47 (2009) 2594–2600

Fig. 3. Body weight changes on treatment with hesperetin and DMH.

Table 2 Incidence and multiplicity of colonic tumors. Groups

No. of rats

No. of tumors/ polyp bearing rat

Tumor/polyps incidence (%)*

Total tumors/ polyps number

No of tumors/polyps/ tumor bearing rats

Total

AD

ADC

Control Control + hesperetin (20 mg/kg bodyweight) DMH Initiation Post-initiation Entire period

10 10

0 0

0 0

0 0

10 10 10 10

9 7 6 3

(90%) 70% 60% 30%

6(60%) 4(40%) 4(30%) 2(20%)

Multiplicity (number of tumors/rat) on colonic tumors Total

AD

ADC

0 0

0 0

0 0

0 0

0 0

0 0

3(30%) 3(30%) 2(20%) 1(10%)

26 17 10 4

2.8 2.4 1.6 1.2

0.9 ± 0.083 0.7 ± 0.051 0.6 ± 0.043 0.03 ± 0.01

0.30 ± 0.02 0.25 ± 0.02 0.23 ± 0.02 0.11 ± 0.01

0.61 ± 0.05 0.45 ± 0.03 0.40 ± 0.05 0.18 ± 0.01

AD: adenoma; ADC: Adenocarcinoma. * {(No. of tumors/polyp bearing rat)/(No. of rats)  100}.

Table 3 Effect of hesperetin on tissue TBARS, LOOH and CD of control and experimental colon cancer rats. Parameters TBARS (mmoles/mg tissue) Liver Proximal colon Distal colon LOOH (mmoles/mg tissue) Liver Proximal colon Distal colon CD (mmoles/mg tissue) Liver Proximal colon Distal colon

Control

Control + hesperetin

DMH

Initiation

Post-initiation

Entire period

0.46 ± 0.03a 0.42 ± 0.04ab 0.28 ± 0.02a

0.44 ± 0.028b 0.40 ± 0.04bc 0.25 ± 0.02b

0.69 ± 0.05c 0.25 ± 0.021d 0.11 ± 0.001c

0.55 ± 0.04d 0.30 ± 0.02c 0.16 ± 0.011d

0.49 ± 0.03a 0.36 ± 0.03bc 0.23 ± 0.018b

0.43 ± 0.02a 0.40 ± 0.03a 0.26 ± 0.022ab

62.7 ± 6.11a 72.7 ± 7.1a 69.3 ± 6.7a

66.2 ± 6.43ab 73.3 ± 6.1b 71.3 ± 7.0b

80.6 ± 7.8c 49.8 ± 4.8c 57.6 ± 5.2c

71.5 ± 6.9b 56.1 ± 5.4bc 51.2 ± 5.0d

68.2 ± 6.7a 63.8 ± 6.1c 62.3 ± 6.1a

64.1 ± 6.1d 69.4 ± 6.7d 66.4 ± 6.3e

51 ± 0.049a 63.1 ± 6.2a 65.6 ± 5.41a

58 ± 0.55b 65.0 ± 5.43b 69.3 ± 5.8a

78 ± 0.071c 47.4 ± 3.5c 45.6 ± 3.9c

69 ± 0.065cd 55.3 ± 4.2b 51.3 ± 4.51d

49 ± 0.48b 59.3 ± 5.0a 55.5 ± 5.1a

66 ± 0.063d 61.5 ± 4.7b 62.6 ± 4.8d

Data are presented as the means ± SD of each group. a–e P < 0.05 the values not sharing a common superscript letter are significantly different from the DMH-treated groups (analysis of variance followed by DMRT). *P < 0.001, significantly different between the DMH-treated group and hesperetin supplemented groups (groups 4–6).

(group 1). Hesperetin treatment during initiation, post-initiation and entire period stages (groups 4–6) significantly elevated the SOD and CAT activities in the liver and colon as compared to the DMH alone treated rats. This effect was more pronounced in entire period hesperetin treatment group (group 6) as compared to the

other hesperetin treated groups. Hesperetin alone treatment (group 2) did not show any alteration in the antioxidant activities. Table 5 shows the activities of GSH, GR and GPx. The activities of GSH, GR, and GPx in DMH-treated rats (group 3) were significantly decreased (P < 0.05) as compared to the control group

2598

S. Aranganathan, N. Nalini / Food and Chemical Toxicology 47 (2009) 2594–2600

Table 4 Effect of hesperetin on tissue SOD and CAT enzymes of control and experimental colon cancer rats. Parameters

Control

Control + hesperetin

DMH

Initiation

Post-initiation

Entire period

SOD (50% NBT reduction /min/mg protein) Liver 8.41 ± 0.8a Proximal colon 6.87 ± 0.06ab Distal colon 8.4 ± 0.81a

7.3 ± 0.7b 5.74 ± 0.54bc 7.94 ± 0.76b

6.0 ± 5.8c 3.12 ± 0.30d 5.66 ± 0.54c

7.6 ± 0.7d 4.24 ± 0.40c 7.09 ± 0.68d

7.7 ± 0.7a 5.53 ± 0.53bc 7.70 ± 0.83e

8.3 ± 0.8a* 6.33 ± 0.61a* 8.87 ± 0.85f*

CAT (lmol H2O2 utilized/min/mg protein) Liver 53.6 ± 5.1a Proximal colon 64.5 ± 4.2ab Distal colon 46.7 ± 4.5a

56.6 ± 5.4b 57.2 ± 4.2bc 45.3 ± 3.4a

27.5 ± 2.6c 35.8 ± 3.4d 29.7 ± 2.8b

69.7 ± 6.7d 40.1 ± 3.8c 40.1 ± 3.8c

64.5 ± 6.2a 47.9 ± 0.4bc 44.4 ± 3.1a

80.3 ± 7.7a* 59.5 ± 5.3a* 50.9 ± 5.21e*

Data are presented as the means ± SD of each group. a–e P < 0.05 the values not sharing a common superscript letter are significantly different from the DMH-treated groups (analysis of variance followed by DMRT). * P < 0.001, significantly different between the DMH-treated group and hesperetin supplemented groups (groups 4–6).

Table 5 Effect of hesperetin of tissue GPx, GSH and GR of control and experimental colon cancer rats. Parameters

Control

Control + hesperetin

DMH

Initiation

Post-initiation

Entire period

GPX (lmoles of GSH utilized/min/mg protein) Liver 7.96 ± 0.76a Proximal colon 6.9 ± 0.66ab Distal colon 5.6 ± 0.42a

5.93 ± 0.57b 6.42 ± 0.61bc 5.54 ± 0.53b

4.45 ± 0.42c 3.90 ± 0.37d 3.6.±0.35c

6.22 ± 0.59d 4.31 ± 0.40c 4.4 ± 0.49d

7.21 ± 0.69a 5.5 ± 0.72bc 4.9 ± 0.53e

8.06 ± 0.77a 6.2 ± 0.70a 5.93 ± 0.57b

GSH (mmol/mg tissue protein) Liver 23.6 ± 2.2a Proximal colon 25.8 ± 2.4ab Distal colon 18.3 ± 1.7a

25.2 ± 2.4b 21.1 ± 2.0bc 16.0 ± 1.5b

15.6 ± 1.9c 17.8 ± 1.5d 12.5 ± 1.2c

21.7 ± 2.5d 20.5 ± 1.9c 16.2 ± 1.5b

23.6 ± 2.3a 21.8 ± 2.1bc 19.2 ± 1.8a

25.0 ± 2.4a 25.5 ± 2.4a 19.5 ± 1.8a*

GR (lmoles NADPH oxidized/min/mg protein) Liver 26.3 ± 2.1a Proximal colon 14.5 ± 1.2a Distal colon 15.8 ± 1.4a

30.1 ± 2.8b 15.2 ± 1.4b 16.1 ± 1.5b

11.2 ± 0.9c 7.5 ± 0.6c 6.4 ± 0.5c

18.4 ± 1.6d 8.4 ± 0.72d 7.8 ± 0.66d

21.5 ± 2.0e 9.6 ± 0.8d 10.3 ± 0.8e

24.1 ± 2.1e 12.3 ± 1.1d* 13 ± 1.2d*

Data are presented as the means ± SD of each group. a–e P < 0.05 the values not sharing a common superscript letter are significantly different from the DMH-treated groups (analysis of variance followed by DMRT). * P < 0.001, significantly different between the DMH-treated group and hesperetin supplemented groups (groups 4–6).

(group 1). Hesperetin supplementation during the initiation, postinitiation and entire period stages (groups 4–6) increased the activities of GR, GPX and GSH in the liver and colon as compared to DMH alone treated rats. This effect was more pronounced in the entire period hesperetin treatment group (group 6) as compared to the other hesperetin treated rats. Hesperetin alone treatment (group 2) did not show any alterations in the antioxidant levels. 3.5. Histopathology Tissue sections of groups 1 and 2 rats showed normal colonic architecture with no signs of apparent abnormality (Fig. 4a and b). Carcinogen treated group 3 shows well-differentiated signs of dysplasia, dysplastic glands with submucosal neoplastic glands lined by hyperchromatic columnar cells or epithelial cells with mitosis and necorotic dysplasia (Fig. 4c). There were no signs of dysplasia on treatment with hesperetin shows adjacent normal gland and displayed normal histoarchitecture (Fig. 4f). 4. Discussion Epidemiological and experimental studies demonstrate a direct correlation between dietary fat intake and the development of colon cancer. Currently it is suggested that changes in lifestyle including dietary habits could prevent disease development in most cases. Among the dietary factors a high intake of fruits and vegetables is inversely associated with the mortality of colorectal cancer (Winkelmann et al., 2007). In the present study treatment with hesperetin, a dietary flavonoid, decreased the tumor multiplicity, tumor incidence, and

burden of DMH-induced colorectal tumorigenesis. Hesperetin alone treatment showed no tumor, suggesting that hesperetin at this particular dose (20 mg/kg body weight) does not disrupt the normal cellular architecture and hence it can be considered nontoxic. The histological observations amply imply that the administration of hesperetin under experimental conditions can greatly modulate colon carcinogenesis at all stages (as revealed by the entire period treatment group) by altering the efficacy at which DMH can initiate the pathological changes. Well-differentiated signs of dysplastic glands with submucosal neoplastic glands lined with hyperchromatic columnar cells or epithelial cells with mitosis and necrotic dysplasia were observed in the DMH alone treated rat colon (Fig. 4c). Treatment with hesperetin along with the carcinogen exposure greatly restored normalcy in the colonic epithelial cells, which showed very few dysplastic glands along with adjacent normal glands (Fig. 4f). This shows the anti-carcinogenic potential of hesperetin against DMH-induced colonic tumors. Oxidative stress is usually implicated during all the stages of cancer development as well as in the genesis of other diseases (Shijun et al., 2000). Specifically, there is evidence indicating the generation of reactive oxygen species in various carcinogenic processes (Manju and Nalini, 2005). DMH is a procarcinogen which requires metabolic activation to its active electrophilic carcinogenic form through a series of oxidative steps in the liver (No et al., 2007). Lipid peroxidation (LPO) as evidenced by the increased levels of thiobarbutric acid reactive substances (TBARS), lipid hydroperoxides (LOOH) and conjugated dienes (CD) was significantly enhance in the liver of DMH-treated rats, which could be attributed to DMH-induced oxidative stress, and production of reactive oxygen metabolites (ROMs). On the other hand the levels

S. Aranganathan, N. Nalini / Food and Chemical Toxicology 47 (2009) 2594–2600

2599

Fig. 4. Histology of colonic tissues on treatment with hesperetin and DMH. (a) Control: shows regular arrangement of normal colonic mucosal gland with portions of muscular layer. (b) Control + hesperetin: shows colonic mucosal gland with no pathological alterations. (c) DMH: shows colonic mucosa on one side, few dysplastic glands with submucosal neoplastic glands lined by hyperchromatic columnar or epithelial cells and necrotic dysplasia. (d) Initiation: shows colon with persisting tumor cells surrounded by lymphocytes infiltration. (e) Post-initiation: shows colonic mucosa with submucosal remunance of few neoplastic gland surrounded by dense sheets of lymphatic reactions. (f) Entire period: shows colonic mucosa with few of dysplastic gland and adjacent normal gland.

of TBARS, LOOH and CD were significantly decreased in the colonic tissues of DMH-treated rats. Our results correlate with those of our previous studies that also show decreased levels of colonic LPO in DMH-induced colon cancer rats in a long-term study (Sengottuvelan et al., 2006). Lowered levels of LPO may be due to an inverse relationship between the levels of cellular LPO and rates of cell proliferation and/or the extent of differentiation (Navarro et al., 1999); Diplock et al., 1994 suggested that highly proliferating dedifferentiated tumor cells have notably low levels of LPO products as compared with untreated controls rats. Thus, it appears likely that the levels of LPO are inversely related to the rate of cellular growth irrespective of whether the cells are malignant or not. It is possible that although the changes in LPO status may reflect cell growth rate rather than malignancy per se, important changes will nevertheless occur at some earlier stage during the progression of normal cells to malignancy. Hesperetin significantly reverses LPO to near normal may be due to its antiproliferative property. Free oxygen radicals can act as initiators, promoters, procarcinogen activators and are even capable of causing DNA damage and altering the cellular antioxidant defense system (Sun, 1990). Antioxidants function as inhibitors of carcinogenesis during the initiation stages and protect cells against oxidative damage. Thus the powerful antioxidant defense mechanism of our body plays a crucial role against the toxic effects of reactive oxygen species. Among them, superoxide dismutase (SOD) and catalase (CAT) are important antioxidant enzymes. Exposure to carcinogens or tumor promoters usually decreases the activities of SOD and CAT (Rajeshkumar and Ramadasan, 2003). A decline in the activities of these enzymes may facilitate the initiation of oxidative processes, which would lead to the elevation of reactive oxygen species and consequently may account for the increase in the levels of oxidized DNA bases, credited for mutagenesis and carcinogenesis. Our present results also reveal declined activities of SOD and CAT in the DMH-treated rats, which are in line with our previous reports (Sengottuvelan et al., 2006). The reduced activities of SOD and CAT is a natural cellular response against the decreased levels of reactive oxygen species especially hydrogen peroxide and superoxide radicals. Hesperetin supplementation as three different dietary regimens to DMH-treated rats (initiation, post-initiation and

entire period) significantly enhanced the activities of SOD and CAT as compared to DMH alone treated rats, which may be due to the anticancer and antioxidant properties of hesperetin. Glutathione (GSH) is a major nonenzymatic antioxidant, which functions as the second line of defense against free radical damage in the body. GSH donates an electron during the peroxide reduction catalysed by GPx, which is a component of the enzyme system containing GSH oxidase and reductase. GPx removes hydrogen peroxide that is produced by SOD, by oxidizing GSH to GSSG. GR has also an important role as a cellular antioxidant, as it catalyses the regeneration of GSH from GSSG. The dysfunction of GSH and its related antioxidant enzymes such as GPx and GR is reported to be involved in the initiation of cancer (Honda et al., 2004). GSH related enzymes also play an important role in the protection of mammalian cells against the harmful effects of chemical carcinogens and other alkylating agents. In the presence of xenobiotics GST and GPx generally help in detoxification by conjugating GSH with toxic electrophiles conferring a selective growth advantage to cancer cells (Dani et al., 2007). Induction of these enzymes has been become essential determining the potency of many anti-carcinogenic substances (Singh et al., 2006). Previous hypothesis shows that an increase in the detoxification enzyme activities might be considered beneficial, since this could enhance the excretion of carcinogens. Literature survey shows that an ideal chemopreventive agent should induce detoxification enzymes solely through the antioxidant response element (ARE) present in the promoter region of these enzymes (Talalay et al., 1995). In this study, DMH-treated rats showed decreased levels of reduced glutathione and glutathione dependent enzymes (GPx and GR). However hesperetin supplementation significantly increased the glutathione and glutathione dependent enzymes in the DMH-treated rats. Moreover, epidemiological studies indicate that flavonoids may reduce the risk of chronic diseases such as cancer (Neuhouser, 2004). Flavonoids are known to be extensively metabolized in vivo (Walle, 2004). The B ring of hesperetin is not conjugated with the carbonyl group on the ring C, so it has different biological and pharmacological functions as compared to quercetin and other types of flavonoids (Heim et al., 2002). Moreover hesperetin is

2600

S. Aranganathan, N. Nalini / Food and Chemical Toxicology 47 (2009) 2594–2600

relatively non-polar (lipophilic) and thus has an easy intracellular access (Spencer et al., 2004). All these attributes of hesperetin can contribute to its antioxidant potential. We have observed a significant decrease in the incidence and occurrence of colon cancer in animals continuously treated with hesperetin throughout the study (entire period) as compared to those animals treated with hesperetin during the initiation or post-initiation stages (groups 4 and 5) of carcinogenesis. Further, hesperetin supplementation before initiation (group 4) did not show any significant protection against the high dose of carcinogen used. Hence entire period treatment regimen can be considered the most effective dietary protocol in our study. Taken together, hesperetin can markedly modulate oxidative stress by activating the antioxidant defense system. Thus hesperetin may be an attractive candidate as an antioxidant supplement for anticancer therapy.

Conflict of interest statement None declared. References Ameer, B., Weintraub, R.A., Johnson, J.V., Yost, R.A., Rouseft, R.L., 1999. Flavanone absorption after naringin, hepseridin and citrus administration. Clin. Pharmacol. Ther. 60, 34–40. Anand, P., Kunnumakkara, A.B., Sundaram, C., Harikumar, K.B., Tharakan, S.T., Lai, O.S., Sung, B., Aggarwal, B.B., 2008. Cancer is preventable disease that requires major lifestyle changes. Pharm. Res. 25, 2097–2116. Brayand, F., Moller, B., 2006. Predicting the future burden of cancer. Nat. Rev. Cancer 6, 63–74. Breiniiolt, V., Ridson, S.T., Dragsted, 1999. Differentia effects of dietary flavonoids on drug metabolizing and antioxidant enzymes in female rats. J. Xenobiotica. 29, 1227–1240. Cai, Y.Z., Luo, Q., Sun, M., Corke, H., 2004. Antioxidant activity and phenolic compounds of 112 traditional. Life Sci. 74, 2157–2184. Carlberg, B., Mannervik, 1985. Glutathione reductase. In: Meister, A. (Ed.), Methods in Enzymology, vol. 7. Academic Press, New York, p. 484. Choi, E.J., 2008. Antioxidative effects of hesperetin against 7, 12dimethylbenz(a)anthracene-induced oxidative stress in mice. Life Sci. 82, 1059–1064. Dani, V., Goel, A., Vaiphei, K., Dhawan, D.K., 2007. Chemopreventive potential of zinc in experimentally induced colon carcinogenesis. Toxicol. Lett. 171, 10–18. Depeint, F., Gee, J.M., Williamson, G., Johnson, I.T., 2002. Evidence for consistent patterns between flavonoid structures and cellular activities. Proc. Nutr. Society 61, 97–103. Diplock, A.T., Rice-Evans, C.A., Burdon, R.H., 1994. Is there a significant role for lipid peroxidation in the causation of malignancy and for antioxidants in cancer prevention? Cancer Res. 54, 1952–1956. Ellman, G.L., 1982. Tissue sulphydryl groups. Arch. Biochem. Biophys. 82, 70–77. Folhe, L., Gunzler, W.A., 1984. Assays of glutathione peroxidase. Method Enzymol. 105, 114–121. Galati, E.M., Trovato, A., Kirjavainen, S., et al., 1996. Biological effects of hesperidin, a citrus flavonoid. (Note III). Antihypertensive and diuretic activity in rat. Farmaco 51, 219–221. Garg, A., Garg, S., Zaneveld, L.J., Singla, A.K., 2001. Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother. Res. 15, 655–669. Gil-Izquierdo, A., Gil, M.I., Ferreres, F., Tomás-Barberán, F.A., 2001. In vitro availability of flavonoids and other phenolics in orange juice. J. Agri. Food Chem. 49, 1035–1041. Heim, K.E., Tagliafero, A.R., Bobilya, D.J., 2002. Flavanoid antioxidants: chemistry, metabolism and structure activity relationship. J. Nutr. Biochem. 13, 572–584. Honda, T., Coppola, S., Ghibelli, L., Cho, S.H., Kagawa, S., Spurgers, K.B., Brisbay, S.M., Roth, J.A., Meyn, R.E., Fang, B., McDonnell, T.J., 2004. GSH depletion enhances

adenoviral bax-induced apoptosis in lung cancer cells. Cancer Gene Ther. 11, 249–255. Jiang, Z.Y., Hunt, J.V., Wolff, S.P., 1993. Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxides in low density lipoprotein. Anal. Biochem. 202, 384. Kakkar, P.S., Das, B., Viswanathan, P.N., 1984. A modified spectrophotometeric assay for superoxide dismutase. Indian J. Biochem. Biophys. 21, 130–132. Kim, J.Y., Jung, K.J., Choi, J.S., Chong, H.Y., 2004. Hesperetin: potent antioxdant against peroxynitrite. Free Radic. Res. 38, 761–769. Lee, N.K., Choi, S.H., Park, S.H., Park, E.K., Kim, D.H., 2004. Antiallergic activity of hesperidin is activated by intestinal microflora. Pharmacolgy 71, 174–180. Manju, V., Nalini, N., 2005. Chemopreventive efficacy of ginger, a naturally occurring anticarcinogen during the initiation, post-initiation stages of 1,2dimethylhydrazine-induced colon cancer. Clin. Chim. Acta 358, 60–67. Navarro, J., Obrador, E., Carretero, J., Petschen, I., Avino, J., Perez, P., Estrela, J.M., 1999. Changes in glutathione status and the antioxidant system in blood and in cancer cells associate with tumour growth in vivo. Free Radic. Biol. Med. 26, 410–418. Neuhouser, M.L., 2004. Dietary flavonoids and cancer risk: evidence from human population studies. Nutr. Cancer 50, 1–7. No, H.-N., Kwon, H., Park, Y.-G., Cheon, C.-I., Park, J.-S., Park, T., Aruoma, O.I., Sung, M.-I., 2007. Dietary quercetin inhibits 1, 2-dimethylhydrazine-induced liver DNA damage without altering colon DNA damage or precancerous lesion formation in rats. Nutr. Res. 27, 659–664. Ohkawa, H., Ohishi, Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351. Rajeshkumar, N.V., Ramadasan, K., 2003. Modulation of carcinogenic response and antioxidant enzymes of rats administered with 1,2-dimethylhydrazine by Picroliv. Cancer Lett. 191, 137–143. Rao, K.S., Recknagel, R.O., 1968. Early onset of lipid peroxidation in rat liver after carbon tetrachloride administration. Exp. Mol. Pathol. 9, 271–278. Scholz, E.P., Zitron, E., Kiesecker, C., Thomas, D., Kathofer, S., Kreuzer, J., Bauer, A., Katus, H.A., Remppis, A., Karle, C.A., Greten, J., 2006. Orange flavonoid hesperetin modulates cardiac hERG potassium channel via binding to amino acids F656. Nut. Metab. Cardivascu. Dis. 17, 666–675. Schulmann, K. et al., 2002. Colonic cancer and polyps. Best Pract. Res. Clin. Gastroenterol. 16, 91–114. Sengottuvelan, M., Senthilkumar, R., Nalini, N., 2006. Modulatory influence of dietary resveratrol during different phases of 1, 2-dimethylhydrazine induced mucosal lipid-peroxidation, antioxidant status and aberrant crypt foci development in rat colon carcinogenesis. Biochim. Biophys. Acta 1760, 1175– 1183. Shijun, L.I., Yan, T., Yang, Ji-Qin, Terry, D.O., Larry, W.O., 2000. The role of cellular Glutathione peroxidase redox regulation in the suppression of tumor growth by manganese superoxide dismutase. Cancer Res. 60, 3927–3939. Singh, R.P., Banerjee, S., Kumar, P.V.S., Raveesha, K.A., Rao, A.R., 2006. Tinospora cordifolia induces enzymes of carcinogen/drug metabolism and antioxidant system, and inhibits lipid peroxidation in mice. Phytomedicine 13, 74–84. Sinha, K.A., 1972. Colorimetric assay of catalase. Anal. Biochem. 47, 389–394. Slattery, M.L., Edwards, S.L., Ma, K.N., Friedman, G.D., 2000. Colon cancer screening, lifestyle, and risk of colon cancer. Cancer Cause Control 11, 555–563. Spencer, J.P., Abd-el-Mohsen, M.M., Rice-Evans, C., 2004. Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity. Arch. Biochem. Biophys. 423, 148–161. Starvic, B., 1994. Role of chemopreventers in human diet. Clin. Biochem. 27, 319– 332. Sun, Y., 1990. Free radicals, antioxidant enzymes, and carcinogenesis. Free Radic. Biol. Med. 8, 583–599. Talalay, P., Fahey, J.W., Holtzclaw, W.D., Prestera, T., Zhang, Y., 1995. Chemoprotection against cancer by phase 2 enzymes induction. Toxicol. Lett. 8, 173– 179. Walle, T., 2004. Absorption and metabolism of flavonoids. Free Radic. Biol. Med. 36, 829–837. Weisburger, J.H., 1991. Nutritional approach to cancer prevention with emphasis on vitamins, antioxidants and carotenoids. Am. J. Clin. Nutr. 53, 226– 237. Winkelmann, I., Diehl, D., Oesterle, D., Daniel, H., Wenzel, U., 2007. The suppression of aberrant crypt multiplicity in colonic tissue of 1,2-dimethylhydrazinetreated C57BL/6J mice by dietary flavone is associated with an increased expression of Krebs cycle enzymes. Carcinogenesis 28, 1446–1454. Yamashita, N., Kawanishi, S., 2000. Distinct mechanisms of DNA damage in apoptosis induced by quercetin and luteolin. Free Radic. Res. 33, 623–633.