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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 755-762

Chemopreventive effect of carvacrol on 1,2-dimethylhydrazine induced experimental colon carcinogenesis


Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Chidambaram, Tamil Nadu, India

Date of Web Publication25-Jul-2016

Correspondence Address:
Namasivayam Nalini
Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar - 608 002, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.154925

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 > Abstract 


Purpose: Colorectal cancer (CRC) is a leading cause for cancer-related death and its prevention is of great importance throughout the world. Chemoprevention offers a novel approach to control the incidence of colon cancer. The present study was performed to evaluate the efficacy of carvacrol supplementation on colonic aberrant crypt foci (ACF), lipid peroxidation, and antioxidant defense system in 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in male Wistar rats.
Materials and Methods: The rats were randomly divided into six groups. Group 1, control rats received modified pellet diet; Group 2 rats received modified pellet diet along with carvacrol (80 mg/kg b.wt/day); Groups 3–6 received subcutaneous injection of DMH (20 mg/kg b.wt), once a week for the first 4 weeks; in addition Groups 4–6 received carvacrol at three different doses of 20, 40, and 80 mg/kg b.wt/day for 16 weeks.
Results: Our result suggest that increased tumor incidence and increased number of ACF, increased bacterial enzymes accompanied by a decrease in the colonic lipid peroxidation, glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) activities were observed in DMH-treated rats. Administration of carvacrol to DMH-treated rats significantly decreased the tumor incidence and the number of ACF and bacterial enzymes with enhancement of colonic lipid peroxidation, GPx, SOD, and CAT activities.
Conclusion: The results of this study suggest that carvacrol at a dose of 40 mg/kg b.wt showed a significant beneficial effect against chemically-induced colon carcinogenesis in rats.

Keywords: Aberrant crypt foci, antioxidants, bacterial enzymes, colon cancer, chemoprevention, lipid peroxidation


How to cite this article:
Sivaranjani A, Sivagami G, Nalini N. Chemopreventive effect of carvacrol on 1,2-dimethylhydrazine induced experimental colon carcinogenesis. J Can Res Ther 2016;12:755-62

How to cite this URL:
Sivaranjani A, Sivagami G, Nalini N. Chemopreventive effect of carvacrol on 1,2-dimethylhydrazine induced experimental colon carcinogenesis. J Can Res Ther [serial online] 2016 [cited 2020 Jul 5];12:755-62. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/755/154925




 > Introduction Top


Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females. Globally there were 1.2 million cases of CRC yearly resulting in about 608,700 deaths from CRC [1] accounting for 8% of all cancer deaths. CRC is strongly related to diet, and thus dietary strategies may reduce the risk of colon cancer. 1,2-dimethylhydrazine (DMH) is a colon-specific carcinogen [2] that targets DNA and induces the formation of methyl adducts with deoxyribonucleic acid (DNA) bases, point mutations, and micronuclei yielding macroscopically visible neoplasms.[3] The colonic adenomas are generally present in the form of polyps. Aberrant crypt foci (ACF) are putative, preneoplastic lesions and are precursors of CRC, showing numerous similarities to those seen in human sporadic colon carcinoma. Thus, formation and growth of ACF caused by DMH induction in experimental models are being used as a potent marker to identify modulators of colon carcinogenesis.

Cellular antioxidant defense enzymes comprise superoxide dismutases (SODs), glutathione peroxidase (GPx) and catalase (CAT).[2] SOD and GPx present in the cytosol and mitochondria, reduce the superoxide anion to hydrogen peroxide and water, and thereby removes majority of the hydrogen peroxide. Meanwhile, CAT, located in the peroxisomes, degrades hydrogen peroxide. Antioxidants can also retard lipid oxidation by interfering at various stages of the peroxidation event.[4] Gut microbial enzymes, usually assayed in fecal suspensions, as components of fecal water, are more frequently in contact with the colonic epithelium than the components of the solid phase and are therefore more likely to give rise to cell damage, which constitutes a risk for the initiation and progression of colon cancer.[5]

β-glucuronidase catalyzes the cleavage of terminal glucuronic acid, which is believed to be largely responsible for the hydrolysis of glucuronide conjugates in the gut, and thus to be important in the generation of toxic and carcinogenic substances.[6] Mucinase, a bacterial enzyme that hydrolyses the protective mucus layer of the gastrointestinal tract, is of interest because it alters the permeability and barrier function of the colon.[7] The excessive activities of these enzymes may be a primary factor in the etiology of colon cancer by exposing xenobiotics to the underlying cells.

Chemoprevention is potentially an effective strategy to control the incidence of colon cancer. Carvacrol (5-isopropyl-2-methylphenol) is a predominant monoterpenoic phenol present in many essential oils of the family Labiatae including, Origanum, Satureja, Thymbra, Thymus, and Corydothymus species. It is a safe food additive used in flavoring baked goods, sweets, beverages, and chewing gum. Carvacrol possesses a series of pharmacological properties including antifungal,[8] antioxidant,[9],[10] anticarcinogenic, antiplatelet,[11] and antiproliferative activities.[12],[13],[14],[15] Studies have also shown that carvacrol is metabolized and excreted within 24 h of consumption, which shows that there is very little risk of it building up in the body to any potential harmful level. Hence, the main objective of the present study was to evaluate the chemopreventive effect of carvacrol on DMH-induced colon cancer by examining its lipid peroxidative, antioxidative properties, alterations in the activities of bacterial enzymes, and also its role in ACF formation.


 > Materials and Methods Top


Chemicals

Carvacrol and DMH were purchased from Sigma Chemical Co. (USA). All other chemicals and reagents used were of analytical grade and obtained from HiMedia Laboratories Ltd, Mumbai, India.

Animals and experimental design

Adult male albino Wistar rats weighing 130–150 g (n = 6 per group) were obtained and maintained in accordance with the Indian National Law on Animal Care and Use. They were housed in solid-bottomed polypropylene cages under the standard environmental conditions of a 12 h light/dark cycle, 50% humidity, a temperature of 25 ± 2°C, and water available ad libitum. These animals were maintained under specific pathogen-free conditions. A commercial pellet diet containing 4.2% fat (Hindustan Lever Ltd, Mumbai, India) was ground into a powder and mixed with 15.8% peanut oil, resulting in a total of 20% fat in the diet and used as a promoter for colon cancer. The total experimental period was 16 weeks. The animals fasted overnight prior to the day of the termination of the experiment.

  • Group 1: Normal control rats received 5% dimethyl sulfoxide (DMSO)
  • Group 2: Rats received carvacrol (80 mg/kg b.wt) postorally every day throughout the 16 weeks experimental period and served as carvacrol control
  • Group 3: Rats received subcutaneous injections of DMH (20 mg/kg b.wt) in the right thigh, at the dose of once a week for the first 4 weeks of the experiment (four injections) and served as DMH control
  • Group 4: Rats received subcutaneous injections of DMH as in Group 3 and treated with carvacrol (20 mg/kg b.wt) postorally every day from the day of the carcinogen treatment till the end of the 16th week
  • Group 5: Rats received subcutaneous injections of DMH as in Group 3 and treated with carvacrol (40 mg/kg b.wt) postorally every day from the day of the carcinogen treatment till the end of the 16th week
  • Group 6: Rats received subcutaneous injections of DMH as in Group 3 and treated with carvacrol (80 mg/kg b.wt) postorally every day from the day of the carcinogen treatment till the end of the 16th week.


Carvacrol preparation

Carvacrol was suspended in 5% DMSO just before treatment and administered everyday orally at the doses of 20, 40, and 80 mg/kg b.wt for 16 weeks. The dose of carvacrol was fixed according to the previous study by Aristatile et al., (2009).[16]

Tumor induction

For inducing colon cancer, DMH was dissolved in 1 mM Ethylenediaminetetra aceticacid (EDTA) just prior to use and pH was adjusted to 6.5 with 1 mM NaOH, to ensure stability of the carcinogen. It was then administered subcutaneously in the right thigh of rat at 20 mg/kg b.wt once a week, for the first 4 weeks of the experiment.[17],[18]

Body weight and growth rate changes

Over the experimental period, body weight and growth rate of control and experimental rats were measured. Animals were weighed at the beginning of the experiment, subsequently once a week and finally before sacrifice. Growth rate was calculated by using the following formula:



Determination of ACF

At the end of the 16 weeks, rat colons were removed and flushed with potassium phosphate buffered saline (0.1 M, pH 7.2). Colons were split open longitudinally and placed on strips of filter paper with their luminal surface open and exposed. Another strip of filter paper was placed on top of the luminal surface. The colons were then secured and fixed in a tray containing 10% buffered formalin overnight. Each of the fixed colons was cut into proximal and distal portions of equal lengths and each portion was further cut into 2 cm long segments. Each segment was placed in a petri dish and stained with 0.2% methylene blue solution for 2 min. The segments were then transferred to another petri dish containing buffer to wash off excess stain. The segments were examined using a light microscope (Nikon, USA) at low magnification to score the total number of ACF as well as the number of crypts per focus. ACF were distinguished from normal crypts by their thicker, darker-stained, raised walls with elongated slit-like lumens, and significantly increased distance from the lamina to basal surface of cells. ACFs in the colon were counted as described by Bird.[19]

Estimation of lipid peroxidation

The levels of total lipid peroxidation and key enzymes that indicate the antioxidant status of animals were analyzed using established biochemical procedures as follows. The levels of tissue thiobarbituric acid reactive substances (TBARS) were measured using the method of Ohkawa et al.[20] The level of conjugated dienes (CD) was estimated using the method of Rao and Recknagel.[21] The levels of lipid peroxides were measured using the method of Jiang et al.[22]

Determination of SOD and CAT activities

SOD (EC.1.15.1.1) activity was measured using the method of Kakkar et al.[23] CAT (EC.1.11.16) was assayed using the method of Sinha.[24]

Determination of the levels/activities of glutathione and glutathione-dependent enzymes

The GPx (EC.1.11.1.9) activity was assayed using the method of Folhe and Gunzler.[25] The activity of glutathione reductase (GR) was assayed using the method of Carlberg and Mannervik.[26] Reduced glutathione (GSH) was determined using the method of Ellman.[27]

Assay of the activities of fecal and mucosal bacterial enzymes

Fresh fecal pellets were collected for the assay of fecal bacterial enzymes. The mucosa from the colon was collected by scraping with a slide. The fecal pellets and colonic mucosa were homogenized using phosphate-buffered saline, centrifuged at 2,000g for 10 min at 4°C, and the supernatant collected for the assay of the activities of fecal and colonic mucosal bacterial enzymes. β-Glucuronidase activity was measured by the method of Freeman.[28] Mucinase was measured by the method of Shiau and Chang.[29] Reducing sugar release in mucinase assay was measured by the Nelson–Somogyi method.[30]

Statistical analysis

The statistical significance of the data was determined using one-way analysis of variance (ANOVA) and a 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 Statistical Package for Social Sciences (SPSS) 11.0 software (SPSS, Tokyo, Japan).


 > Results Top


Effect of carvacrol and DMH on body weight changes and growth rate

[Table 1] shows the effects of carvacrol treatment for 112 days on mean body weight and growth rate changes in control and experimental rats. Body weight in all the groups increased at a normal rate [Table 1]. The average body weight of DMH-treated rats maintained on the high fat diet showed a significantly low (P < 0.05) gain in body weight throughout the experimental period as compared to Groups 1 and 2. On administration of different doses of carvacrol (Groups 4-6), the body weight was significantly elevated as compared to the unsupplemented DMH-treated rats. Moreover, Group 5 rats showed a significantly (P < 0.05) improved weight gain as compared to Groups 4 and 6. There was a significant increase in growth rate on supplementation with carvacrol at different doses of 20, 40, and 80 mg/kg b.wt to DMH-exposed rats (Groups 4–6) compared to DMH-alone-exposed rats (Group 3). However, carvacrol at 40 mg/kg b.wt (Group 5) caused significantly improved weight gain and growth rate compared with other treatment (20 and 80 mg/kg body weight) Groups 4 and 6, thereby offering optimum protection to rats against DMH-induced colon carcinogenesis.
Table 1: Effect of carvacrol and DMH on initial and final body weight changes and growth rate of control and experimental rats

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Effect of carvacrol and DMH on the incidence and number of colon tumors/polyps

Colon polyps/tumor incidence was 100% in the DMH alone-treated group (Group 3). Supplementation with carvacrol at the doses of 20, 40, and 80 mg/kg b.wt to the DMH-treated rats resulted in reduced tumor incidence [Table 2].
Table 2: Incidence of colon tumors and the number of tumors/polyps per tumor bearing rat

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Effect of carvacrol and DMH on ACF and its multiplicity

No ACF was observed in the control (Group 1) and the carvacrol alone-treated groups (Group 2) [Figure 1] and [Table 3]. The majority of ACF appeared in the middle and distal colon of the rats injected with DMH (Groups 3–6). The total number of ACF detected in the DMH alone-treated rats (Group 3) was on an average about 122. Supplementation with carvacrol at different doses of 20, 40, and 80 mg/kg b.wt to DMH treated rats (Groups 4–6) significantly inhibited the formation of aberrant crypts (57, 30, and 41, respectively) as well as its multiplicity. More pronounced effect was observed in the rats treated with 40 mg/kg b.wt carvacrol (Group 5).
Figure 1: (a and b) Topographical view of normal crypts (×20) of Groups 1 and 2. (c) Topographical view of ACF with multiple crypts in the colon of a rat treated with DMH (×20). (d) Topographical view of ACF with five crypts in the colon of a rat treated with DMH + carvacrol at a dose of 20 mg/kg b.wt (× 20). (e) Topographical view of ACF with two crypts in the colon of a rat treated with DMH carvacrol at a dose of 40 mg/kg b.wt (×20). (f) Topographical view of ACF with three crypts in the colon of a rat treated with DMH + carvacrol at a dose of 80 mg/kg b.wt (× 20). ACF = Aberrant crypt foci, DMH = 1,2-dimethylhydrazine

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Table 3: Effect of carvacrol and DMH on ACF formation and multiplicity

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Effect of carvacrol and DMH on lipid peroxidation byproducts

[Table 4] shows the effect of carvacrol and DMH on the levels of lipid peroxidation byproducts in the tissues of experimental rats. The lipid peroxidation byproducts such as TBARS, CD, and lipid hydroperoxide (LOOH) levels were significantly lowered in the colonic tissues of DMH alone-treated rats (Group 3) as compared to the control rats (Group 1), whereas, these levels were significantly increased in the liver of DMH alone-treated rats (Group 3) as compared to the control rats (Group 1). The levels of lipid peroxidation were restored on supplementation with carvacrol at the doses of 20, 40, and 80 mg/kg b.wt. The effect was more pronounced in the DMH-exposed rats supplemented with 40 mg/kg b.wt of carvacrol.
Table 4: Effect of carvacrol and DMH on tissue TBARS, CD, and LOOH (mM/mg tissue) of control and experimental rats

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Effect of carvacrol and DMH on enzymic antioxidants

The activities of enzymic antioxidants such as SOD and CAT were significantly decreased in the tissues (liver, proximal colon, and distal colon) of DMH alone-treated rats (Group 3) as compared to the control rats (Group 1) [Figure 2] and [Figure 3]. The SOD and CAT activities were significantly elevated on supplementation with carvacrol at the doses of 20, 40, and 80 mg/kg b.wt to DMH treated rats (Groups 4–6) as compared to the DMH alone-treated rats. A more pronounced effect was observed in the DMH exposed rats supplemented with carvacrol at the dose of 40 mg/kg b.wt (Group 5).
Figure 2: Effect of carvacrol and DMH on tissue SOD of control and experimental rats. Values expressed as means ± SD of six rats (n = 6) from each. Values not sharing a common superscript letter (a-c) differ significantly at P < 0.05 (DMRT). SOD = Superoxide dismutase, DMRT = Duncan's multiple range test, SD = standard deviation

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Figure 3: Effect of carvacrol and DMH on tissue CAT of control and experimental rats. Values expressed as means ± SD of six rats (n = 6) from each. Values not sharing a common superscript letter (a-d) differ significantly at P < 0.05 (DMRT). CAT = Catalase

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Effect of carvacrol and DMH on glutathione dependent enzymes

Our results indicated that the activities of GSH and GSH-dependent enzymes were significantly (P < 0.05) lowered in the liver, colonic tissues of DMH alone-treated rats (Group 3) [Figure 4],[Figure 5],[Figure 6]. DMH treated rats supplemented with different doses of carvacrol showed elevated levels of GSH and the activities of GPx and GR as compared to the unsupplemented DMH exposed rats. A more pronounced effect was observed in the carcinogen-exposed rats supplemented with carvacrol at the dose of 40 mg/kg b.wt.
Figure 4: Effect of carvacrol and DMH on tissue GPx of control and experimental rats. Values expressed as means ± SD of six rats (n = 6) from each. Values not sharing a common superscript letter (a-d) differ significantly at P < 0.05 (DMRT). GPx = Glutathione peroxidase

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Figure 5: Effect of carvacrol and DMH on tissue GR of control and experimental rats. Values expressed as means ± SD of six rats (n = 6) from each. Values not sharing a common superscript letter (a-d) differ significantly at P < 0.05 (DMRT). GR = Glutathione reductase

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Figure 6: Effect of carvacrol and DMH on tissue GSH of control and experimental rats. Values expressed as means ± SD of six rats (n = 6) from each. Values not sharing a common superscript letter (a-c) differ significantly at P < 0.05 (DMRT). GSH = Reduced glutathione

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Effect of carvacrol and DMH on bacterial enzyme activities

[Table 5] shows the activities of colonic mucosal and fecal bacterial enzymes such as β- glucuronidase and mucinase of the control and experimental rats. In both fecal and mucosal samples, these bacterial enzyme activities were significantly increased in DMH alone-treated rats (Group 3) as compared to control (Group 1). Supplementation with carvacrol at the doses of 20, 40, and 80 mg/kg b.wt (Group 4–6) to carcinogen exposed rats significantly decreased the activities of the fecal and mucosal bacterial enzymes as compared to the unsupplemented DMH-treated rats. More pronounced effect was observed in the rats supplemented with 40 mg/kg b.wt of carvacrol (Group 5).
Table 5: Effect of carvacrol and DMH on fecal and colonic bacterial enzymes of control and experimental rats

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 > Discussion Top


Chemoprevention by dietary phytochemicals appears to be a practical approach to prevent carcinoma. The average body weight of DMH alone-treated rats (Group 3) maintained on high fat diet showed significant low gain in body weight and growth rate throughout the experimental period as compared to control rats. This reduced body weight gain may be due to the tumor burden in the colon, which in turn leads to decreased food intake. Carvacrol supplementation to DMH-treated rats significantly improved weight gain and growth rate as compared to DMH alone-treated rats revealing the beneficial effects of carvacrol against DMH-induced colon carcinogenesis. Variable inhibitory effects of carvacrol on the incidence of polyps/preneoplastic lesions (ACF) were observed in the three different groups in our study; specifically, when carvacrol was supplemented at the dose of 40 mg/kg b.wt, a significant reduction in the number of polyps/ACF and crypt multiplicity (maximum inhibitory effect = 92.45%) was observed than compared to DMH alone-treated group. The inhibitory activity of carvacrol against ACF formation and multiplicity and tumor incidence might be due to the putative antiproliferative and anticarcinogenic potential of carvacrol. Our study showed that carvacrol supplementation reduced the growth of neoplastic polyps induced by the carcinogen DMH. This could be attributed to the chemopreventive potential of carvacrol.

The levels of TBARS, LOOH, and CD were significantly higher in the liver; whereas, these levels were significantly low in the colonic tissues of DMH alone-treated rats as compared to the control rats. Reduced colonic LPO observed in DMH-exposed rats could be due to increased cell proliferation, increased resistance, and/or reduced susceptibility of target organs to free radical attack. On supplementation with carvacrol to DMH-treated rats, restored the lipid peroxidation levels in the colon and liver to near those of the control level. This could be due to the strong antioxidant property of carvacrol equivalent to those of ascorbic acid, butyl hydroxyl toluene (BHT), and vitamin E.[9],[10] There were some previous studies which strengthens our findings in that they reported that carvacrol has antiproliferative effect in human metastatic breast cancer cell line (MDA-MB 231)[15] and also in prostate cancer cell line (LNCaP).[31]

The decreased activities of SOD, CAT, GPx, GR, and GSH were observed in the present study may be due to the dangerous increase in the levels of ROS [32] and their enhanced utilization during detoxification of DMH. These antioxidant activities were elevated on supplementation with carvacrol, which may be due to the free radical scavenging property of carvacrol and consequently decreased utilization of the antioxidant enzymes. The above finding correlates with previous report which suggest that carvacrol possesses potent free radical scavenging and antioxidant activities in DEN-induced hepatocellular carcinogenesis.[33] Polyphenols are well-known, free radical scavengers because of the high reactivity of their hydroxyl substituent.[34] Carvacrol has hydroxyl groups located at different position in the phenolic ring. It seems that the high antioxidant activity of these compounds is due to the presence of phenolic OH groups which act as hydrogen donors to the peroxy radicals produced during the first step in lipid oxidation.[35]

β-Glucuronidase is responsible for the hydrolysis of conjugated glucuronides in the lumen of the gut.[36] It may lead to the generation of toxic and carcinogenic substances, which had been previously detoxified by glucuronide conjugation in the liver and subsequently entered the colon via the blood or bile. The increased fecal β-glucuronidase activity observed in our study could be attributed to the altered colonic microflora due to high fat feeding and DMH injections. Mucinase hydrolyses the protective mucin layer of the colonic wall, which exposes the underlying epithelial cells to the carcinogens cleaved by β-glucuronidase.[37] Changes in the amount and/or the composition of mucins may lead to inflammatory responses. Thus in our study, rats administered DMH showed greater mucinase activity as compared to the control. On supplementation with carvacrol significantly reduced the bacterial enzyme activities; thereby, its beneficial effect against DMH is known.

On the whole,

all the three doses of carvacrol did show inhibitory effects on DMH-induced colon carcinogenesis, but the maximum effect was mediated only by the intermediate dose (40 mg/kg b.wt). This could be due to the following reasons. Carvacrol at the dose of 20 mg/kg b.wt did not show maximum effect, may be because the concentration was not enough to neutralize DMH-induced damages. Carvacrol at the dose of 80 mg/kg b.wt, also did not show maximum effect; which could be because at high concentrations carvacrol may alter its antioxidant activity, thereby decreasing its effectiveness. However, long-term biochemical and molecular studies are warranted to determine the key mechanism by which carvacrol mediates its chemopreventive effects against colon carcinogenesis.

 
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