Research Article
Protection Against Azoxymethane-Induced Mouse Colon Tumorigenesis by Wheat Bran and Black Tea
Department of Zoology, Faculty of Science, University of Alexandria, Alexandria, Egypt
Cancer is a leading cause of mortality world wide and, therefore, a major focus of research has been the chemoprevention of cancer (Singh and Lippman, 1998). Several human cancers appear to have a strong environmental component. Epidemiology studies have demonstrated substantial effects of diet on cancer incidence (Jain et al., 1990). The major goal of chemoprevention is to reduce the incidence of human cancer either by inhibiting the process of carcinogenesis or by preventing high levels of carcinogen exposure. Fruits, vegetables and common beverages, as well as several herbs and plants have been shown to be effective for chemoprevention of various cancers (Birt et al., 1996 ; Lipkin, 1997; Conney et al., 1997 ; Goodman, 1997 and Abdel-Galil, 2001a & 2001b).
Tea from Camelia sinensis, is one of the most widely consumed beverages world wide. The administration of tea or tea phenols significantly prevented tumorigenesis in the esophagus or duodenum in rodent models (Fujita et al., 1989 and Han and Xu, 1990). In addition, Wang et al. (1992) reported that green tea and black tea inhibited forestomach and lung tumorigenesis in A/J mice. Recently, administration of black tea via drinking fluid inhibited skin tumorigenesis in mice (Lu et al., 2001). In our laboratory, skin tumors were also inhibited by topical application of black tea in 7,12-dimethylbenzanthracene(DMBA)-treated BALB/c mice (Abdel-Galil, 2001a). In contrast to previous animal studies, Weisburger et al. (1998) reported that chronic administration of black tea failed to inhibit AOM - induced colon tumors in rats.
Epidemiological studies have demonstrated that supplementing the diet with high fiber foods, such as fruits, vegetables, cereals and whole grains, may be protective against colon cancer (Howe et al., 1992 and Slavin et al., 1997). In addition, many animal studies have also shown that consumption of wheat bran has a colon cancer protective effect in rats (Alabaster et al., 1993; McIntyre et al., 1993; Alabaster et al., 1995 and Jenab and Thompson, 1998).
The chemically induced colon tumorigenesis model with rodents has been useful in investigating the chemopreventive activities of naturally occurring plant products (Alabaster et al., 1995; Weisburger et al., 1998 and Reddy et al., 2000).
The present study aimed to explore whether the administration of wheat bran and black tea during the entire experimental period in addition to postinitiation stage (progression of tumors) could provide more protection against colon carcinogenesis in mice. In addition, the activity of each agent against formation of colon tumors was compared.
Animals: The animals used were 6 weeks-old female SWA mice (Colleague of Medical Sciences, Arass, Saudi Arabia). After acclimatization for 1 week, they were housed in plastic cages and fed on basal diet and maintained in an air - conditioned room with a 12h light/dark cycle. The mice were fed a basal diet and drinking fluid ad libitum.
Materials: Azoxymethane (AOM) was purchased from the Sigma Chemical Co., (St. Louis, Mo, USA). Wheat bran was bought from a supermarket as bran flakes (Fleming & Co., Auckland, New Zealand). Black tea was imported and delivered by a trading company. The tea was a mixture of leaves grown and processed in the major tea growing countries.
Preparation of tea: In experiments 1 and 2, tea infusions were prepared using a commercial Bunn automatic basket tea brewer (Springfield, IL,USA). Tea solids (50g) were placed in a filter paper-lined brewing basket and 4 or 2 L of hot, deionized water were passed through the tea solids in the brewing machine. The resulting tea brews containing 4 or 8mg/ml tea solids were collected.
Experimental protocol: A total of 260 mice were randomly divided into 12 groups. The first experiment included groups 1-6 and the second experiment included groups 7-12. Animals of groups 6 and 12 served as non-treated controls and received only water and basal diet. Mice of groups 1-5 (experiment 1) and 7-11 (experiment 2) received three weekly s.c. injections of AOM (15 mg/kg body weight). Animals of group 2 and 3 received 4 or 8 mg tea solids/ml, respectively, via drinking fluid starting 2 weeks before the first injection of AOM. Mice of groups 4 and 5 were fed diets that contained 10 or 20% wheat bran, respectively, 2 weeks before the first dose of AOM (experiment 1), during initiation and postinitiation stages of carcinogenesis. In the second experiment, mice of groups 8,9,10 and 11 received 4,8 mg tea solids/ml of black tea, 10 and 20% wheat bran, respectively, starting 1 week after the last injection of AOM (postinitiation stage). The experimental diets and test compounds were freshly prepared weekly. All mice were carefully observed daily and weighed weekly. The number of survived mice were also determined at weekly intervals. The experiments were terminated after 32 weeks and all mice were killed by an ether anesthesia to assess the incidences and multiplicities of colon tumors. Dead animals, throughout the study, were also autopsied and the number and type of colon tumors were counted and diagnosed. Ten % buffered formalin-phosphate was immediately injected into the colon. The colons were removed and placed on a plastic sheet, and the number of tumors in each colon was determined. The size (volume) of the tumors was also determined and the colon samples were stored in 10% buffered formalin-phosphate for histological examination.
Tumor volume: Tumor volume was determined by measuring the three-dimensional size of all tumors using the average of 3 measurements to calculate radius (r). Tumor volume was calculated as :
Examination of tumors: All tumors were examined with the aid of a magnifying lens. Tumors were excised, fixed and processed for histology with hematoxylin and eosin staining. The colon tumors were diagnosed based on previously established well-known criteria and categorized as adenomas and adenocarcinomas (Ward, 1974).
Statistical analysis: The effects of tea and wheat bran on multiplicity and tumor size were determined with the Students t-test and their effects on tumor incidence and percentage of inhibition were analyzed by χ2 -test.
Effects of black tea and wheat bran on the body weights and survival of AOM-treated mice: Oral administration of 2 dose levels of black tea and wheat bran did not reveal significant differences in body weights of mice of all groups (Table 1). Table 1 also shows that oral administration of black tea and wheat bran significantly (P< 0.05) increased the survival of mice of AOM-treated groups (experiment 1). The administration of 4 and 8 mg black tea and 10% wheat bran after the last dose of AOM (experiment 2) did not enhance the survival of mice; whereas 20% wheat bran significantly increased the survival of mice (Table 1, group 11).
Table 1: | Effect of oral administration of black tea and wheat bran on body weights and survival of AOM - treated mice. |
Fig. 1: | Effects of oral administration of black tea and wheat bran, during the entire experimental period, on AOM-induced colon tumorigenesis in SWA mice |
Table 2: | Effect of dietary black tea and wheat bran on AOM- induced colon tumors in mice (treatment started 2 weeks before AOM: Experiment 1) |
Table 3: | Effect of dietary black tea and wheat bran on AOM- induced colon tumors in mice (treatment started 1 week after AOM: Experiment 2) |
Table 4: | Effect of oral administration of black tea and wheat bran on volume of colon tumors in AOM-treated mice. |
Effects of black tea and wheat bran on AOM-induced colon tumorigenesis (experiment 1: during the initiation and postinitiation stages): Twenty-seven weeks after the administration of the last dose of AOM, 84% of mice developed colon adenomas with an average of 7.2±2.5 tumors/mouse in AOM alone control group (Table 2, Fig. 1a & b). Administration of 4 mg tea solids/ml in the drinking fluid insignificantly reduced the incidence (percent of mice with tumors) to 72%, and the multiplicity (number of tumors/animal) to 6.6±2.1 as compared to those of AOM alone group (Table 2, Fig. 1a & b). The administration of 8 mg black tea/ml to mice of group 3, significantly (P< 0.05) reduced the incidence to 61%, and the number of adenomas/ mouse to 5.2±1.9 as compared to values of group 1 (Table 2, Fig. 1a & b). The administration of 4 or 8mg black tea insignificantly reduced the incidence of adenocarcinomas to 44 or 35, versus 52% in AOM alone group and the multiplicity to 0.76±0.4 or 0.68±0.2, respectively as compared to 0.84±0.3 tumors/animal in AOM alone group (Table 2, Fig. 1c & d). Supplementing the diet with 10 or 20% of WB significantly (P>0.05) reduced the incidence of adenomas to 36 or 25% in groups 4 and 5, respectively. Moreover, the two dose levels of WB significantly inhibited the multiplicity of adenomas to 2.4 ± 0.8 and 2.1±1.4, respectively, versus values of AOM control group (Table 2, Fig. 1a & b). Besides, colon adenocarcinomas appeared in 20 and 17% of animals of groups 4 and 5, respectively, versus 52% in group 1 (Table 2, Fig. 1c). The multiplicity of carcinomas significantly also reduced to 0.44±0.3 or 0.37 ± 0.7 tumors/mouse in the low and high WB groups, respectively, versus 0.84 tumors/mouse in AOM alone group (Table 2, Fig. 1d).
Administration of low and high doses of black tea inhibited the number of colon adenomas per mouse by 8 and 28% and the number of adenocarcinomas per mouse by 10 and 19%, respectively (Table 2). The administration of 10 and 20% wheat bran markedly inhibited the number of adenomas per mouse by 67 and 71% and the number of adenocarcinomas per mouse by 48 and 56%, respectively (Table 2).
Effects of black tea and wheat bran on AOM-induced colon tumorigenesis (experiment 2 : during the postinitiation stage): Oral administration of 4 or 8 mg black tea reduced the incidence of colon adenomas to 80 or 75%, respectively, versus 90% in group 7 and the multiplicity of adenomas to 6.4±1.4 or 6.2 ±2.4, respectively, as compared to 6.9 ± 1.8 tumors/mouse in AOM alone group (Table 3, Fig. 2a & b). In AOM alone treated animals, 55% of mice developed colon adenocarcinomas with multiplicity of 0.92 tumors/animal. The administration of 4 mg black tea insignificantly reduced carcinomas incidence to 50% ; whereas 8 mg black tea did not affect the incidence of carcinomas (Table 3, Fig. 2c & d). The low and high dose levels of black tea insignificantly decreased the multiplicity of carcinomas to 0.85 and 0.81 tumors/mouse, respectively. Supplementation of diets with 10 or 20% WB significantly (P>0.05) inhibited the incidence of adenomas to 55 and 45% and adenomas multiplicity to 4.7 ± 2.1 or 3.5 ± 1.4, respectively, as compared to AOM alone group. In addition 10 or 20% black tea also significantly (P>0.05) reduced the incidence to 30 or 25% and the multiplicity of carcinomas to 0.67 or 0.56, respectively (Table 3, Fig. 2c & d). In experiment 2, 4 and 8mg black tea insignificantly inhibited the number of adenomas per mouse by 8 and 10 % and the number of carcinomas per mouse by 8 and 12%, respectively. On the other hand, 10 or 20% wheat bran inhibited in highly significant values the number of adenomas per mouse by 32 and 49% and the number of carcinomas per mouse by 27 and 39%, respectively, as compared to values of AOM alone group (Table 3, Fig. 2c & d).
Fig. 2: | Effect of oral administration of black tea and wheat bran, during the postinitiation stage, on AOM-induced colon tumorigenesis in SWA mice |
Histologically, colon tumors were diagnosed as adenomas and adenocarcinomas. No histological differences were observed in tumors either in AOM alone or black tea and wheat bran treated mice.
Effects of black tea and wheat bran on tumor growth: In experiment 1, the administration of a low dose of black tea did not significantly reduce the adenomas growth (increase in tumor volume/mouse), whereas the high dose resulted in a significant reduction in the growth of adenomas and adenocarcinomas.
Administration of 4 or 8 mg tea reduced the volume of adenomas per mouse to 14.9 and 10.8 mm3, respectively, versus 17.6 mm3 in AOM alone group (Table 4). However, supplementation of diet with 10 or 20% wheat bran resulted in a marked and significant reduction in adenoma volume per mouse to 7.9 and 5.8 mm3, respectively (Table 4). Moreover, 10 or 20% wheat bran significantly reduced carcinoma volume per mouse to 12.8 and 9.7 mm3, respectively as compared to AOM - treated group (Table 4).
In experiment 2, 4 mg black tea, insignificantly decreased adenoma volume per mouse to 19.5 mm3 and carcinoma volume per mouse to 24.6 mm3 and 8 mg BT significantly (p<0.05) reduced the volume of adenomas/mouse to 16.7 mm3 and insignificantly reduced the volume carcinomas/mouse to 26.2 mm3 . However, supplementing diet with 10 or 20% wheat bran markedly reduced adenomas volume per mouse to 11.6 and 9.8 mm3 and carcinoma volume per mouse to 13.6 and 11.9 mm3, respectively, as compared to AOM- treated group (Table 4 ).
The results in the current study indicate that supplementing diets with 10 or 20% wheat bran, during either the initiation and postinitiation phases (experiment 1) or postinitiation phase (experiment 2) significantly inhibited, in a dose-dependent manner, AOM-induced colon tumorigenesis as revealed by reduction in the incidences and multiplicities of tumors. The higher protective activity against colon carcinogenesis was achieved when wheat bran was applied before AOM and until the end of experimental period.
The results of the present study are consistent with previous studies. Animal studies have shown that WB has a protective effect against colon carcinogenesis (Alabaster et al., 1993, McIntyre et al., 1993 and Alabaster et al., 1995). In addition, Jenab and Thompson (1998) also demonstrated that wheat bran, partly due to its endogenous phytic acid can reduce early biomarkers of colon cancer risk. Consumption of wheat bran has been shown to reduce considerably the epidermal growth factor content of the colonic mucosa in rats (Schaudies et al., 1990). More recently, oral administration of wheat bran oil or wheat bran oil plus phytate significantly inhibited colon tumor incidence, multiplicity and volume in AOM-treated rats (Reddy et al., 2000). Phytic acid, which is a major fiber-associated component of wheat bran, has been shown to significantly decrease the number of colon tumors and volume when treatment was commenced prior to AOM injection (Ullah and Shamsuddin, 1990 and Pretlow et al., 1992) or when administered up to 5 months postinitiation (Shamsuddin and Ullah, 1989). Alabaster et al. (1995) demonstrated that colon tumor incidence declined from 73 to 20% in the high wheat bran fiber and from 27 to 13% in the low wheat bran fiber. They also concluded that wheat bran protected the colon in rats from benign and malignant tumor promotion.
Wheat bran is thought to exert its colon cancer protective effect through different mechanisms. Previous studies have shown a decrease in the rate of cell proliferation with wheat bran supplementation (Folino et al., 1995). The walls of lignified cells (dietary fiber) in wheat bran can also adsorb dietary carcinogens, at least in vitro (Harris et al., 1998). Recently, Kestell et al. (1999) reported that supplementing diets with wheat bran markedly retarded the metabolism of a dietary carcinogen in rats. Other mechanisms involve the dilution of potential dietary carcinogens by wheat bran supplements reducing the transit times in the gastrointestinal tract by increasing the output of faeces (Lewis and Haeton, 1997).
Human diet intervention studies, also indicated that dietary wheat bran decreases the concentrations of fecal (colonic luminal) secondary bile acids which are potent promoters of colon carcinogenesis and stimulate the proliferation of colonic epithelium (Reddy et al., 1989). In the present study, the low dose of black tea (4mg/ml) is similar to that present in tea brews ingested by humans (Wang et al., 1994). Such dose of black tea did not alter AOM-induced colon tumorigenesis; however, 8 mg tea/ml reduced tumor incidence and multiplicity when applied during the entire period of experimentation. The lack of protective activity by 4 mg tea/ ml, which is equal to human tea brews concentration, is in agreement with the findings of Weisburger et al. (1998). The chronic administration of black tea failed to affect the incidence and multiplicity of colon cancer when given during or after AOM (Weisburger et al., 1998).
The inability of black tea to provide a significant protection against AOM-induced colon tumorigenesis might be associated with the fact that black tea is devoid of the quinone reductase activity involved in detoxification and anti-proliferative actions of other chemopreventive agents on colon cancer cells (Wang and Higuchi, 1995). In biochemical studies, black tea induced a number of metabolizing enzymes, but significantly, no modification was found for cytochrome P-4502E1 which is essential and unique for the metabolic activation of AOM (Sohn et al., 1991 & 1994). Additionally, it is well known that not all chemopreventive agents are effective in blocking aberrant crypt foci development and colon tumorigenesis (Pereira et al., 1994 and Hardman and Cameron, 1996). Chemopreventive agents have been shown to decrease colonic mucosal ornithine decarboxylase activity, a rate-limiting enzyme involved in cell proliferation (Tanaka et al., 1997). The present data also demonstrate a significant decrease in tumor size by wheat bran administration, during either before or after AMO, and this is consistent with the results of previous studies with other rodent species (Reddy et al., 2000). This finding suggest that supplementing diets with wheat bran markedly retarded the growth of colon tumors in mice.
In the present study, wheat bran was effective in modulating the AOM-induced colon carcinogenesis when applied before AOM and continued until the end of experiment than when it was given after AOM. This might be explained on the highly preventive effects of agents on the colon initiated cells before proliferation. These results demonstrate that wheat bran has a significant protective effect against AOM-induced colon tumorigenesis and its activity was higher when applied before AOM, whereas black tea was generally ineffective in retarding colon tumors in both experimental conditions.