Albert Baskar Arul, Ignacimuthu Savarimuthu, Mohammed A. Alsaif, Khalid S. Al Numaira
Department of Community Health Sciences,
College of Applied Medical Sciences,
King Saud University,
P.O. Box 10219,
Riyadh 11433, Saudi Arabia.
This study was performed to determine the chemopreventive and antioxidant status of multivitamin and mineral (0.01% in drinking water, ad libitum) supplements in 1,2-dimethylhydrazine (DMH)-induced experimental colon carcinogenesis. Experimental colon carcinogenesis was induced in male albino Wistar rats by injecting DMH (20 mg·(kg body mass)(-1)) once weekly for 15 consecutive weeks, and administering a multivitamin supplement in 3 regimes (initiation, post-initiation, and entire experimental period) for 32 weeks. We studied lipid peroxidation products (thiobarbituric acid reactive substances, lipid hydroperoxides, conjugated dienes) in the circulation and in the tissues, antioxidant status (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and non-enzymatic antioxidant-reduced glutathione) of the tissues, aberrant crypt foci (ACF), and histopathological alterations. DMH-induced rats had an increase in lipid peroxidation products and a lower antioxidant status compared with control animals. Multivitamin and mineral supplementation during the initiation, post-initiation, and the entire study period significantly decreased the levels of lipid peroxidation products in circulation and colonic tissues, significantly elevated the activities of the antioxidant enzymes and reduced glutathione to near normalcy in DMH-induced rats. The incidence of ACF was reduced to 84.1% in rats supplemented with multivitamin and minerals for the entire study and prevented the colonic tissue from histopathological alterations induced by DMH.
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Lung, breast, and colon cancers are the most common cancers worldwide and increase in incidence annually (Bingham and Riboli 2004). An increased level of reactive oxygen species (ROS) occurs in tumour cells during early stages of cancer development, and the overproduction and (or) inability to destroy ROS may result in severe damage to tissues, and cause cancer. Antioxidant activity by ROS scavenging is important in preventing potential damage to cellular components such as DNA, proteins, and lipids. Colon cancer is frequently a pathological consequence of persistent oxidative stress, leading to DNA damage and mutations in cancer-related genes, and a cycle of cell death and regeneration (Behrens 2005). Antioxidant enzymes, the main scavengers of free radicals, are modulated during carcinogenesis or after tumour formation. Changes in these enzymes result from an increase in free-radical production. The recovery of enzyme activity could help prevent malignancy by scavenging the free radicals (Sun 1990). The enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST), as well as the non-enzymatic antioxidant reduced glutathione (GSH), are involved in scavenging free radicals generated during malignancy (Fang et al. 2002).
The alkylating agent 1,2-dimethylhydrazine (DMH) is a colon-specific carcinogen. The cells at the subcutaneous site do not possess enzymes capable of reacting with DMH. Hence, subcutaneously administered DMH is slowly released into the circulation, reaches the liver, and gets metabolized into various intermediates such as azoxymethane and methylazoxymethanol (MAM) that are subsequently transported to the colon via bile or the blood (Fiala 1977; Oravec et al. 1986). MAM is chemically unstable at body temperature and decomposes spontaneously to formaldehyde, water, and nitrogen (Fiala et al. 1976). The decomposition of MAM results in the formation of methyldiazonium ions, which generate reactive carbonium ions capable of methylating DNA, RNA, or the protein of colonic epithelial cells (Matsumoto and Higa 1966). The alkylated guanines are modified so as to pair with thymidine rather than their normal counterpart, cytosine. During subsequent replication, mispairing guanine with thymidine leads to mutations of the DNA. Moreover, previous reports have pointed out the tendency of DMH to produce free radicals in blood, liver, and the large intestine (Arutiunian et al. 1997) of experimental models. DMH-induced rat colon carcinogenesis is a widely studied experimental model in several cancer chemoprevention studies. Aberrant crypt foci (ACF) are putative preneoplastic lesions of colonic neoplasia in rodents and humans. During colon carcinogenesis, ACF appear in the early stages, and sequentially develop into polyps, adenomas, and eventually into carcinomas. Rats exposed to chemical carcinogens and fed with a diet containing low fibre and high fat developed colonic ACF and tumours. A decrease in ACF has been used as an intermediate endpoint to identify the potential cancer chemopreventive properties of synthetic and natural drugs (Shivapurkar et al. 1995).
Dietary nutrients are well documented to be chemopreventive in the epidemiology of colon cancer. Also, there has been considerable interest in establishing the role of particular nutrients, either individually or in combination, to prevent colon cancer. Epidemiological studies on multivitamin and mineral supplementation from diet and cancer are often speculative. Studies on diet and colon cancer provide consistent evidence that the consumption of fruits and vegetables is associated with a lower risk of colon cancer (White et al. 1997), and a few studies have investigated supplement use with colon cancer using animal models. Although the specific dietary factors responsible for this protective effect have not been clearly identified, fruits and vegetables contain vitamins and minerals that may influence cancer. The objective of this study was to show that including vitamin and mineral supplements in a regular diet could reduce cancer risk and protect against carcinogenesis.
DMH-induced rat colon carcinogenesis is one of the most widely studied experimental models in cancer chemoprevention studies. Experimental colon cancer induced by DMH in rats is a prolonged multistage process, bearing many of the cell kinetics, and histopathological and molecular characteristics of tumorigenesis that mimics human colon cancer. The development of both DMH-induced rat colon carcinogenesis and human colon carcinomas involve aberrant accumulation of cells with potential for excessive proliferation, evasion of apoptosis, and genomic instability (Jackson et al. 2003). Tseng et al. (1996) reported increased lipid peroxidation and a decrease in the antioxidant defence enzymes in the colon of rats treated with DMH and a high-fat diet. Epidemiological studies have shown that diets containing large quantities of vegetables and fruits are associated with a lower risk for colon cancer. Increased consumption of fruits and vegetables has an inverse relationship with colon cancer risk, which may be due to the presence of vitamins and minerals (White et al. 1997).
Vitamin and minerals, when supplemented individually or in combination, exert anticancer potential in experimental carcinogenesis. The antioxidant vitamins C and E have been given more attention among the micronutrients as protective agents for cancer. Vitamins C, E, B6, and D have been shown to inhibit experimental colon tumours (Cook and McNamara 1980; Logue and Frommer 1980; Pence and Buddingh 1988; Mokady et al. 2000; Komatsu et al. 2001). Vitamins E and A, as well as zinc, exhibit anti-inflammatory activity, and vitamins A, E, and D, as well as calcium, regulate cell differentiation, proliferation, and apoptosis (Lamprecht and Lipkin 2003; Huang et al. 2004; Singh and Jialal 2004). Vitamin C directly consumes free radicals in plasma, and plays an important role in protecting and recycling levels of vitamin E in lipoproteins during oxidative stress (May 1998). Zheng et al. (2010) reported that vitamin D deficiency promotes the growth of prostate cancer cells in bone, and a low level of vitamin D in the serum is correlated to an increased incidence of colon cancer (Peterlik et al. 2009; Trump et al. 2010). Vitamin D has been shown to exhibit anticancer potential in malignant cell lines (Mehta and Mehta 2002). Vitamins B6 and B12 are essential for nucleotide synthesis, DNA synthesis, and DNA repair (Duthie et al. 2002).
Minerals, in combination with vitamins, are required for the normal body functions. The vitamin B complex works effectively when administered with magnesium. Vitamin B complex will be absorbed by the body if the magnesium levels are depleted. Calcium shields the bowel mucosa from carcinogens by forming insoluble chemical complexes with bile acid and fat (Guyton et al. 2004). Dietary calcium has been shown to decrease the incidence of colon tumours in experimental animals (Pence and Buddingh 1988; Wargovich et al. 1990; Karkare et al. 1991; Li et al. 1998) and the number of ACF (Pereira et al. 1994; Wargovich et al. 2000). Combination regimens that use vitamins as one of the constituents are potentially effective in cancer therapies (van den Bemd et al. 2000). Vitamin D3 in combination with vanadium limits genotoxicity and the formation of enzyme-altered foci in DEN-induced rat hepatocarcinogenesis (Basak et al. 2000; Basak and Chatterjee 2000).
The endogenous production of free radicals during carcinogenesis harms tissues and organs, whereas endogenous antioxidants scavenge these free radicals and protect tissues and organs against oxidative stress damage. DMH induction leads to increased oxidative stress and poor antioxidant defence leading to tissue damage. Increased oxidative damage leads to DNA damage and mutation in cancer-related genes (Behrens 2005). The increased lipid peroxidation products (TBARS, CD, LOOH) in plasma and colonic tissue and the decline in the activities of antioxidant status (SOD, CAT, GPx, GR, and GSH) in colonic tissue of DMH-induced animals may be due to the increased production of free radicals, exceeding the scavenging capacity of the antioxidant systems in DMH-induced colon cancer. Therefore, the antioxidant status needs to be increased to protect cells against free radical damage. Multivitamin and mineral supplements significantly increased the activities of enzyme antioxidants in colonic tissues of DMH-treated animals and protected from DMH-induced free-radical toxicity. Multivitamin and mineral supplements are a complex group of antioxidants, which act synergistically to neutralize free radicals produced in the circulation and tissues, and enhance the antioxidant status in DMH-induced rats. The majority of the antioxidant potential can be related to high quantities of vitamin C in the supplement.
Antioxidant enzymes in cancer cells vary in comparison with the levels of normal cells. These antioxidant enzymes and scavengers of reactive oxygen species protect from free radical-induced damage in cellular components. The enzyme antioxidants SOD, CAT, GR, GPx, and GST play a vital role in the defence of toxic free radicals produced in cancer cells. DMH-induced animals exhibit decreased levels of these antioxidant enzymes, and treatment with antioxidant molecules enhanced the activities of these enzymes (Aranganathan and Nalini 2009). DMH and its metabolite AOM are highly specific carcinogens for colonic epithelium and induce tumour mostly in large intestine (Shirai et al. 1983; Sunter et al. 1978). In the present study, we were able to find ACF in the small intestine also, but to a lesser amount than in the large intestine. Administration with DMH increased the proliferation of the colon crypts in the test animals during early carcinogenesis, and altered the distribution of proliferating and apoptotic cells in the colon (Barnes et al. 1998). DMH-induced rats had alterations in the colonic epithelium with dysplastic crypts, whereas DMH-induced rats with multivitamin and mineral supplementation had restored the colonic epithelial cells to normalcy with few dysplastic glands.
A decline in antioxidant enzymes may facilitate the initiation of oxidative processes, which would lead to ROS elevation and, consequently, may account for increases in levels of oxidized DNA bases, which are credited with mutagenesis and carcinogenesis. The results revealed declined activities of enzymic antioxidants in colonic tissue of DMH-induced rats. The reduced activities of the antioxidant enzymes could be from the increased production of free radicals exceeding the scavenging capacity of the antioxidant systems in cancer. An increase of lipid peroxidation level in proliferating cells leads to an increase in the level of circulating lipid peroxides in DMH-induced animals (Fiala 1977). As a result, an excessive generation of lipid peroxidation products occurs in tumour tissue, which is subsequently released into the circulation. Lipid peroxidation products were increased into the circulation and colonic tissues of DMH-induced rats. Therefore, antioxidant enzyme levels need to be higher to protect these cells against free radical damage. Multivitamin and mineral supplementation significantly increased the activities of antioxidants in the colonic tissues of DMH-treated rats and protected them from DMH-induced free radical toxicity.
Although vitamin and mineral supplements are abundant in various foods, adequate amounts of these dietary nutrients are generally not met, and therefore, supplementation is required. Because some vitamins are not stored in the body, supplements are needed to prevent a number of chronic diseases, like cancer. Minerals may be involved in keeping the antioxidant enzymes SOD, CAT, and GPx active because they require minerals such as copper, zinc, manganese, and (or) selenium for their peroxide-quenching reaction. Multivitamin and mineral supplementation protects the cell membranes and DNA from oxidative damage by scavenging the free radicals produced in DMH-induced rats. These multivitamin and mineral supplements synergistically contribute to the cancer chemopreventive potential, and hence, regular supplements of multivitamins and minerals could reduce the risk of colon cancer.