Cruciferous vegetables have great health benefits, and their components may be significant in the inhibition of colon tumors. The objective of this study was to investigate and compare the chemopreventive potential of cabbage, turnip greens, collard greens and canola greens on azoxymethane (AOM)-induced colon cancer. Following a one-week acclimatization period, forty-two Fisher-344 male rats were randomly assigned to five groups (treatment groups: n = 8; control: n = 10). Four groups were fed treatment diets consisting of the selected cruciferous vegetables at 5%, while the C group was fed AIN-93 growth diet. Colon tumors were induced by administration of AOM at 7 and 8 weeks of age and rats were killed by CO 2 asphyxiation at 45 weeks of age. Results show a 42.85% tumors incidence in rats fed canola greens compared to 100% in the rats fed cabbage and the control. Rats fed control had higher tumors/ tumor bearing rat (TBR) ratio (4.5) compared to those seen in treatment groups (1 - 1.71). Significant differences (P ≤ 0.05) were noted in weight gain, cecal wall weight and total cecal weight in the control compared to treatment groups. Hepatic catalase (CAT) and Glutathione-S-Transferase (GST) activities (43.05 and 2.02 μmol/mg, respectively) in rats fed canola were significantly higher (P ≤ 0.05) compared to the control (10.22 and 0.58 μmol/mg) and other treatment groups. Superoxide dismutase (SOD) activity (μmol/mg) in rats fed canola (0.29), cabbage (0.26) and turnip greens (0.25) were similar, however, significantly higher (P ≤ 0.05) compared to the control (0.09). Activities of glutathione peroxidase (GPX) increased in the order: collard greens > canola greens > turnip greens > cabbage > control. Selected cruciferous vegetables, including canola leafy greens, were effective in reducing incidence of AOM-induced colon tumors in Fisher-344 male rats and may be useful as dietary chemopreventive agents.
Lifestyle diseases such as cancer, obesity, and diabetes are among the primary cause of mortality in many industrialized nations, accounting for millions of deaths yearly [
It has been established that diet and lifestyle play an important preventive role in cancer, accounting for a 30% - 40% tumor reduction [
Vegetables are among the many studied foods that offer health protective properties with varying mechanism [
The objective of this study was to evaluate the chemopreventive potential of selected cruciferous vegetables (canola greens, cabbage, turnip greens and collard greens) in reducing colon cancer in Fisher-344 male rats.
Azoxymethane (AOM) was obtained from Midwestern Research Institute, NCI, Chemical Repository, Kansas City, MO, USA. All other chemicals and reagents were of analytical grade and were purchased from Sigma Chemical Company, St Louis, MO.
Collard greens, cabbage and turnip greens were obtained from a local health-food store. Canola was obtained from the Winfred Thomas Agricultural Research Station (Alabama A&M University). All leafy vegetable samples were washed in deionized water, and stored in a −80˚C freezer. Frozen samples were later transferred from −80˚C to a Consul 24 Virtis freeze-dryer (The Virtis Company, Gardiner, NY). The freeze-dried samples were milled using a Robot Coupe, Blixer RS1 BX3 Food Processor (Robot Coupe U.S.A. Inc. Ridgeland, MS).
Fisher-344 male rats were obtained from Harlan, IN and housed in stainless steel cages. Following one week of acclimatization, the rats were randomly assigned to five groups; eight rats in each treatment group and ten rats in the C group. The rats were fed AIN (American Institute of Nutrition) 93G C growth diet [
All animals received two subcutaneous injections of AOM (NCI Repository, Kansas, MO) at 7 and 8 weeks of age, at 16 mg/kg body weight. Rats were killed by CO2 asphyxiation at 45 weeks of age. Liver samples were collected and stored at −80˚C for enzyme analysis. Cecum was removed; cecal contents and weight of cecal wall were determined.
For the characterization of tumor size, tumor location and tumor per tumor bearing rat ratio (TBR), the protocol of Shackelford and others was employed [
Hepatic Glutathione S Transferase (GST) was determined following the manufacturer’s protocol (Cayman Chemical Company). GST activity was measured at an absorbance of 340 nm using a microplate reader (Biotek Synergy HT, Winooski, Vermont).
Glutathione (GSH), Glutathione Peroxidase (GPX) and Superoxide Dismutase (SOD) were determined using a microplate reader (BioTek Synergy HT Winooski, Vermont) as described in the manufacturer’s protocol (Cayman Chemicals, Ann Arbor, MI). Change in absorbance was monitored at 412, 340, 460 nm, respectively. Catalase (CAT) activity was determined using an established protocol as described by Johansson and Borg (1988) [
Data were analyzed using the SAS system version 9.1 (SAS Institute, Cary, NC). Values are given as means ± SEM and were separated using Duncan’s Multiple Range Test, with significance at P ≤ 0.05 level.
Feed intake in rats fed canola greens was significantly (P ≤ 0.05) lower (13.26 g/week) compared to the rats fed collard greens (14.78 g/week). The group fed the control diet had a significantly higher (P ≤ 0.05) feed intake (16.80 g/week) compared to the rats fed the selected vegetables (
Ingestion of the selected cruciferous vegetables affected cecal wall weight (g) in the rats used in this study. As shown in
Groups | Feed intake (g/week) | Weight gain (g/41 week) | Total cecal weight (g) | Cecal wall Weight (g) | Cecal pH |
---|---|---|---|---|---|
Control | 16.80a ± 1.66 | 222.62b ± 5.20 | 1.26b ± 0.04 | 0.48b ± 0.02 | 7.99a ± 0.08 |
Cabbage | 14.58bc ± 2.88 | 256.25a ± 6.87 | 2.88a ± 0.12 | 1.18b ± 0.12 | 7.64a ± 0.07 |
Turnip greens | 13.64bc ± 1.16 | 248.87a ± 6.80 | 3.13a ± 0.12 | 1.47a ± 0.07 | 7.60a ± 0.06 |
Collard greens | 14.78b ± 2.78 | 258.75a ± 7.58 | 3.08a ± 0.18 | 1.37ab ± 0.07 | 7.65a ± 0.9 |
Canola greens | 13.26c ± 3.72 | 246.28a ± 0.123 | 2.86a ± 0.18 | 1.21b ± 0.08 | 7.59a ± 0.15 |
Values are means ± SEM.abc Means in a column with the same superscript do not significantly differ (P ≤ 0.05) using Duncan Multiple Range Test.
group fed the control diet.
The highest number of tumors was observed in the rats fed the control diet compared to the rats fed the treatment diets. Tumor numbers in rats fed the control diet were higher in both distal (15) and proximal (30) colon compared to the treatment diets (
Tumor incidence was highest in the rats fed the control and cabbage diets, compared to the other groups (
Groups | Number of rats | Rats with tumors | Number of tumors | Size of Tumors (mm) | Tumors/tumor bearing rats | Tumor incidence% | Location proximal distal | |
---|---|---|---|---|---|---|---|---|
Control | 10 | 10 | 45 | 7.46 | 4.5 | 100 | 15 | 30 |
Cabbage | 8 | 8 | 12 | 3.91 | 1.5 | 100 | 4 | 8 |
Turnip greens | 8 | 6 | 8 | 2.25 | 1.33 | 75 | 3 | 5 |
Collard greens | 8 | 7 | 12 | 1.45 | 1.71 | 87.5 | 8 | 4 |
Canola greens | 7 | 3 | 3 | 4.33 | 1 | 42.85 | 1 | 2 |
Groups | SOD | CAT | GPX | GSH |
---|---|---|---|---|
Control | 0.09c ± 0.02 | 10.22d ± 0.68 | 0.32c ± 0.15 | 54.16c ± 2.98 |
Cabbage | 0.26b ± 0.08 | 27.51b ± 1.47 | 3.00b ± 0.59 | 203.90b ± 10.98 |
Turnip greens | 0.25b ± 0.00 | 14.79c ± 1.49 | 3.59ab ± 0.33 | 236.07a ± 10.20 |
Collard greens | 1.71a ± 0.25 | 27.58b ± 2.17 | 4.38a ± 0.24 | 221.66ab ± 7.60 |
Canola greens | 0.29b ± 0.07 | 43.05a ± 2.81 | 4.24a ± 0.57 | 217.22ab ± 7.15 |
Values are means ± SEM. abcdMeans in a column with the same superscript do not significantly differ (P ≤ 0.05) using Duncan Multiple Range Test.
greens, turnip greens and cabbage diets. Significantly lower (P ≤ 0.05) superoxide dismutase activity (SOD) (µmol/mg) was observed in the control fed rats (0.09) compared to the treatment groups. Among the treatment groups, SOD activity was higher in the rats fed collard greens, showing a significantly higher (P ≤ 0.05) activity (1.71) compared to the canola greens (0.29), cabbage (0.26), and turnip greens (0.25) fed rats. No significant differences were observed in SOD activity among rats fed canola greens, cabbage and turnip greens.
Hepatic catalase (CAT) activity (µmol/mg) in rats fed the control diet was significantly lower (P ≤ 0.05) compared to the treatment groups, and ranged from a low of 10.22 in the control to a high of 43.05 in rats fed canola greens. A 31% to 76% higher catalase activity was noted in rats fed the treatment diets compared to the control. All groups showed significantly (P ≤ 0.05) different CAT activity with the exception of cabbage (27.51) and collard greens (27.58) fed group where no significant differences were observed.
Glutathione peroxidase (GPX) activity in rats fed cabbage (3.0) was significantly (P ≤ 0.05) lower compared to activities in groups fed collard (4.38) and canola greens (4.24). However, all treatment groups had significantly (P ≤ 0.05) higher GPX activity compared to the control (0.32). Glutathione (GSH) levels (µmol/mg), ranged from 203.90 (cabbage) to 236.07 (turnip greens) in rats fed the treatment diets. GSH levels in the rats fed the treatment diets were at least three fold higher compared to the levels observed in the rats fed the control diet (54.16).
Hepatic Glutathione S Transferase (GST) activity (µmol/mg) in rats fed treatment diets was significantly (P ≤ 0.05) higher compared to the control (
Epidemiological studies and clinical trials support the notion that natural plant compounds such as isothiocyanates and indole-3-carbinol found in cruciferous vegetables may provide protective effects both in vivo and in vitro carcinogenic models [
The results of this study indicate that rats fed the control diet had significantly (P ≤ 0.05) lower weight gain (g/41 week) compared to the treatment diets (
Although no significant differences (P ≤ 0.05) were observed among the treatment groups, rats fed canola greens had the lowest weight gain. Studies have shown that a lower weight gain can decrease the carcinogenic response [
While no significant differences in cecal weight were observed among the treatment groups, significantly (P ≤ 0.05) lower cecal weight was observed in the control group. A study by Sakata suggests that SCFA has the potential to accelerate cell proliferation resulting in an increase in cecal tissue weight [
The lower pH observed in the treatment groups compared to the control is likely the result of soluble fiber which is naturally present in cruciferous vegetables. Studies have shown that dietary fiber alters the colonic microflora, thereby decreasing the growth of pathogenic bacteria, creating an anti-carcinogenic environment [
Findings in this study indicate a reduction in tumor number, tumor size, tumor incidence and tumor/tumor-bearing rat in the treatment groups compared to the control. The presence of tumors of varying sizes may be dependent on the tumors’ blood vessel supply [
Glucosinolates from cruciferous vegetables are known for their ability to induce phase II detoxification enzyme system. One such enzyme is the GST detoxification enzyme which offers cellular protection by catalyzing the conjugation of carcinogenic electrophiles in the presence of glutathione, promoting the detoxification of carcinogens [
The antioxidant activity of the selected cruciferous vegetables provides some insight regarding the results seen in our treatment groups when compared to the control. Rats fed turnip greens had significantly (P ≤ 0.05) higher levels of GSH compared to those fed the cabbage diet, however not significantly (P ≤ 0.05) different from the rats fed collard greens and canola greens. The protective role of glutathione in cancer therapy is important because of the antioxidant’s ability to conjugate and detoxify carcinogens [
The beneficial role of food compounds in effecting optimal health in humans cannot be overstated. Cruciferous vegetables have attracted much attention and, therefore, studied extensively because of their naturally occurring compounds which play a critical role in inhibiting neoplastic effects of carcinogens, thus reducing the risk of cancers in various organs. The detoxification and antioxidant potential of the vegetables used in this study were noticeable, not only in the traditional vegetables investigated but also in canola leafy greens. The results from this experiment indicate that cruciferous vegetables including canola greens may be effective in the inhibition of colon carcinogenesis which are consistent with epidemiological studies [
Miller-Cebert, R.L., Boateng, J., Cebert, E., Shackelford, L. and Verghese, M. (2016) Chemopreventive Po- tential of Canola Leafy Greens and Other Cruciferous Vegetables on Azoxymethane (AOM)-Induced Colon Cancer in Fisher-344 Male Rats. Food and Nutrition Sciences, 7, 964-976. http://dx.doi.org/10.4236/fns.2016.711095