Effect of coconut cake on the bacterial enzyme activity in 1,2-dimethyl hydrazine induced colon cancer

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Abstract

Background: Colon cancer is one of the most common forms of malignant tumors in humans, and its incidence is increasing. Since the intestinal microflora is directly in contact with the colonic cells, the enzymes of the bacterial microflora may also play a role in colon carcinogenesis. We studied the activity of bacterial enzymes in experimental colon cancer. Methods: Twenty milligrams per kilogram body weight of 1,2-dimethyl hydrazine (DMH) was administered subcutaneously once a week for first 15 weeks and then discontinued. Coconut cake (25%) was mixed in the diet and given to 30 rats to study the diet effect throughout the experimental period. After 30 weeks, the macroscopic findings in the colon as well as the incidence of tumors in 30 rats was recorded in each group and the activity of β-glucuronidase and mucinase was estimated in the tissues, colon and fecal contents of 10 rats per group. Results: Average number of tumors in the colon as well as the incidence of cancer was significantly increased in DMH-treated rats which was markedly reduced on supplementing coconut cake. DMH injections significantly elevated both the activities of β-glucuronidase (distal colon, distal intestine, liver and colon contents) and mucinase (colon and fecal contents) as compared to the control rats. The increase in β-glucuronidase activity may augment the hydrolysis of glucuronide conjugates, liberating the toxins, while the increase in mucinase activity may enhance the hydrolysis of the protective mucins in the colon. Coconut cake supplementation to DMH-treated rats significantly decreased the incidence and number of tumors as well as the activity of β-glucuronidase and mucinase. Conclusions: Coconut cake has a protective effect against DMH induced colon cancer by virtue of its ability to lower the activities of the microbial enzymes β-glucuronidase and mucinase.

Introduction

Colon cancer is one of the leading causes of death in both men and women in Western countries [1], [2]. Evidence from epidemiological studies and laboratory animal assays suggest a relationship between colon cancer risk and dietary factors [3], [4], [5]. An impressive body of evidence supports the concept that dietary factors are key modulators of colorectal cancer [6].

Dietary factors can modify the metabolic activity of the intestinal microflora, which in turn may play an important role in the conversion of bile acids and neutral sterols to reactive carcinogens [7], [8], [9], [10]. Several ways by which the intestinal microflora could play a significant role are (i) it could convert dietary components into cocarcinogens or carcinogens (or both), (ii) it could metabolize endogenous secretions, themselves controlled by diet, into different compounds, and (iii) it could produce carcinogenic toxins from suitable precursors. These reactions are catalysed by bacterial enzymes and the measurement of the appropriate bacterial enzymes in the intestine gives an indication of the ability of the microflora to support these transformations.

Two important activities of colon microflora are mucinase and β-glucuronidase. Mucinase is the enzyme system responsible for degrading the protective mucins in the colon. β-Glucuronidase is of interest as it is the enzyme that hydrolyses biliary glucuronides when they reach the colon. Many of the glucuronides are conjugated products of detoxification by the liver and their hydrolysis may liberate free toxins including carcinogens which may then act on the colonocytes or may be absorbed by the intestines [10], [11], [12], [13]. If glucuronide hydrolysis is a rate-limiting step in this process, then the activity of microbial β-glucuronidase in the colon may influence the risk of colon carcinogenesis.

Dietary fiber has a significant protective effect against colon cancer by acidifying the colonic contents [14]. Cocos nucifera Linn is consumed in fairly large quantities in India to increase the taste both in a vegetarian as well as in a nonvegetarian cuisine. The coconut kernal on drying and crushing yields 60–67% oil and leaves a residue of up to 33–40% (coconut cake). Coconut cake is used as cattle and poultry feed. The effect of coconut fiber on the intestinal microflora in presence of a carcinogen is yet to be unravelled. So we studied the effect of coconut cake on β-glucuronidase and mucinase activity in rats induced colon cancer using a colon specific carcinogen 1,2-dimethyl hydrazine.

Section snippets

Drugs and chemicals

1,2-Dimethyl hydrazine hydrochloride (DMH), p-nitrophenyl,β-d-glucuronide and mucin were from Sigma, St. Louis, MO. All other chemicals were of analytical grade and the organic solvents were distilled before use. Coconut cake was from the local market, powdered and exhaustively defatted to remove the oil but it still contained about 9% fat which was mixed with the feed.

Experimental animals

Male Wistar rats with body weight 120–150 g were obtained from the Central Animal House, Department of Experimental Medicine,

Results

Fig. 1 shows the average growth rate of the animals in the various groups. It was observed that the weight gained by the high fat diet group>coconut cake group>coconut cake plus DMH>DMH. The effect of coconut cake on percentage, incidence, multiplicity (average number of tumors per rat) and size of colonic tumors in DMH-treated rats are summarized in Table 2. There was no tumor in untreated control rats (group 1). In the case of animals given DMH (group 3), the tumor incidence was 100% and the

Discussion

The above results indicate that coconut cake brings about profound alterations in the activity of β-glucuronidase and mucinase, both in the presence and/or absence of DMH in high fat fed diet rats. We observed that the activity of β-glucuronidase increased significantly in the colon, intestines, liver and colon contents on administering DMH, which shows that the bacterial microflora is more active in the midst of a procarcinogen or a mutagen. Thus in the presence of DMH, conjugated toxic

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