Preventive potential of wheat bran fractions against experimental colon carcinogenesis: implications for human colon cancer prevention

Epidemiological studies suggest an inverse relationship between the intake of dietary fiber, particularly fiber from cereal grains, and colon cancer risk. Animal model assays have demonstrated that the protective effects of dietary fiber on colon cancer development depend on the nature and source of the fiber. Wheat bran (WB) appears to inhibit colon tumorigenesis more consistently than do oat bran or corn bran. This study was designed to determine whether specific WB fractions such as WB fiber, WB lipids, or phytic acid differentially affect colon carcinogenesis in a well-established colon cancer model. In addition, the modulating effect of specific fractions of WB on the activities of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-1 and COX-2 enzymes were assessed in colon tumors as those have been shown to play a role in tumor progression. At 5 weeks of age, groups of male F344 rats were assigned to one of six diets: a high-fat diet containing 10% WB (control diet) and experimental high-fat diets containing 10% dephytinized WB (WB-P), 10% defatted WB (WB-F), 10% dephytinized and defatted WB (WB-PF), 10% WB-PF fortified with 2% bran oil and/or with 0.4% phytate. At 7 weeks of age, all eats except those in the vehicle-treated groups were given two weekly s.c. injections of azoxymethane (AOM) at a dose rate of 15 mg/kg body weight/week. They continued to receive their respective diets until 50 weeks after carcinogen treatment and were then killed. Colon tumors were analyzed for iNOS, COX-1, and COX-2 expression and enzymatic activities. Colon tumors were evaluated histopathologically and classified as adenomas and adenocarcinomas. We found that removal of phytic acid (WB-P) or lipids (WB-F) from WB had no significant effect on colon tumor incidence (% animals with tumors) or multiplicity (tumors/ animal), whereas removal of both phytate and lipids from WB (WB-PF) significantly increased colon tumor multiplicity and volume. Interestingly, WB-PF fortified with excess bran oil or with bran oil plus phytate significantly inhibited colon tumor incidence, multiplicity, and volume; but supplementation of WB-PF with phytate alone had no significant effect on colon tumorigenesis in rats suggesting that lipid fraction of WB possesses tumor-inhibitory properties. Moreover, feeding WB-PF diet significantly increased iNOS, total COX and COX-2 enzyme activities, and iNOS protein expression in colon tumors as compared with wheat bran control diet. Feeding the WB-PF that was fortified with excess bran oil alone or with bran oil plus phytate significantly suppressed the activities of iNOS and COX-2 as well as the expression of iNOS and COX-2 in colon tumors compared with that in rats fed the WB diet or WB-PF diet. The study demonstrates for the first time that the lipid fraction of wheat bran has strong colon tumor inhibitor properties. The exact mechanism(s) by which the lipid fraction of WB inhibits colon carcinogenesis in addition to alteration of iNOS and COX activities remains to be elucidated. Additional studies are warranted to identify biologically active constituents of lipid fraction of WB and their relative role in colon tumor inhibition.     

Polar solvent-induced changes in membrane lipid lateral diffusion in human colon cancer cells

Polar organic solvents, such as N-methylformamide (NMF), N,N-dimethylformamide, and dimethyl sulfoxide, have been demonstrated to induce differentiation in a number of neoplastic cell lines, including human colon cancer cells. Although the mechanism of action of these agents is yet unknown, one possibility is that polar solvents induce a change in lateral mobility of membrane lipids, important to the maturational process. To determine the relationship between polar solvent treatment and changes in membranes, we examined the effects of exposure to NMF on membrane fluidity in human colon cancer cells (DLD-1; clone A). Membrane viscosity was assessed by determining lipid lateral diffusion following photobleaching of a fluorescent lipid probe in individual intact cells. Exposure of cells to NMF led to a significant increase in membrane viscosity following 2 days of treatment, with maximal changes occurring after 11 days. NMF induced these effects over a limited concentration range with 1.0% NMF in the medium having the maximal effect, and 0.5% or 1.5% having less or no effect. Growth of cells with N,N-dimethylformamide (0.8%) also led to increases in membrane viscosity. The observed membrane changes correlated well with the effect of NMF on differentiation in these cells as previously reported, as well as with cell growth rate and morphology in the present study. The increase in viscosity caused by prolonged NMF treatment was reversible, with a return to untreated levels by 9-11 days after removal of NMF. Thus, there is a strong correlation between the attainment of more benign, better differentiated phenotype in polar solvent-treated clone A cells and increases in membrane viscosity.     

Chemoprevention of colon carcinogenesis by the synthetic organoselenium compound 1,4-phenylenebis(methylene)selenocyanate.

The chemopreventive effect of 40% and 80% maximum tolerated dose (MTD) levels of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) administered in the diet during the initiation phase (2 weeks before, during, and up to 3 days after carcinogen administration) and the post-initiation phase (3 days after carcinogen treatment until termination) of azoxymethane (AOM)-induced colon carcinogenesis was studied in male F344 rats. The MTD of p-XSC was determined in male F344 rats and found to be 50 ppm. Beginning at 5 weeks of age, all animals were divided into various experimental groups (42 rats/group) and fed the high-fat semipurified diet or diets containing 20 (40% MTD) and 40 (80% MTD) ppm p-XSC. At 7 weeks of age, all animals (30 rats/group) except the vehicle-treated groups (12 rats/group) were administered s.c. injections of AOM (15 mg/kg body weight/week for 2 weeks). Three days after the second injection of AOM or vehicle (normal saline), groups of animals fed the p-XSC diets and control diet were transferred, respectively, to control diet and p-XSC diets and continued on these diets until the termination of the study. All animals were necropsied during the 36th week after AOM treatment. Colonic mucosal prostaglandin E2 and selenium-dependent glutathione peroxidase were measured in animals fed the control and p-XSC diets at the termination of the study. The results indicate that 40 ppm p-XSC administered during the initiation phase significantly inhibited the colon tumor incidence (percentage of animals with tumors). Dietary p-XSC administered at 20 and 40 ppm levels during the initiation phase significantly inhibited colon tumor multiplicity (tumors/animal and tumors/tumor-bearing animal). Colon tumor incidence and multiplicity were significantly reduced in groups fed 20 and 40 ppm p-XSC diets at the postinitiation phase of carcinogenesis. Colonic mucosal selenium-dependent glutathione peroxidase activity was increased, and prostaglandin E2 was reduced in animals fed the p-XSC diet compared to animals fed the control diet. Whereas the precise mechanisms of p-XSC-induced inhibition of colon carcinogenesis remain to be elucidated, it is likely that the effect during the initiation and postinitiation phases may be due to alteration in carcinogen metabolism and to modulation of prostaglandin synthesis and selenium-dependent glutathione peroxidase activity.     

Dose-related inhibition of colon carcinogenesis by dietary piroxicam, a nonsteroidal antiinflammatory drug, during different stages of rat colon tumor development

The effect of four dose levels of piroxicam administered during different stages of colon tumor development was studied in male F344 rats to obtain a data base on the efficacy of piroxicam as an inhibitor of colon carcinogenesis. Piroxicam was added at levels of 25, 50, 75, and 150 ppm to the NIH-07 open-formula diet and fed to male F344 rats starting 1, 13, and 23 wk after the carcinogen administration. At 7 wk of age, while the animals were consuming the control diet, all animals except the vehicle-treated controls were given s.c. injection of azoxymethane (CAS:25843-45-2; 29.6 mg/kg body weight, once) to induce intestinal tumors. Forty wk after AOM injection, all animals were necropsied, and tumor incidences were compared among the various dietary groups. Colon tumor incidence (percentage of animals with tumors) was inhibited in a dose-dependent manner in rats fed the diets containing 25, 50, 75, and 150 ppm piroxicam starting 1 and 13 wk after carcinogen treatment. The colon tumor incidences in animals fed the diets containing 0, 25, 50, 75, and 150 ppm of piroxicam starting at 1 wk after carcinogen treatment were 89, 61, 58, 50, and 39%, respectively. When the diets containing 0, 25, 50, 75, and 150 ppm were fed 13 wk after carcinogen treatment, the colon tumor incidences were 89, 69, 69, 44, and 33%, respectively. Colon tumor multiplicity (tumors/animal; tumors/tumor-bearing animal) was also significantly inhibited in animals fed the diets containing 25 to 150 ppm piroxicam starting 1 and 13 wk after carcinogen administration. The number of colon tumors/animal was inhibited by about 80 to 84% in animals fed the 150 ppm piroxicam diet. When the diets containing different levels of piroxicam were fed 23 wk after carcinogen treatment, the colon tumor incidence was significantly inhibited in animals fed the 75 and 150 ppm piroxicam diets. The colon tumor incidences in animals fed the diets containing 0, 25, 50, 75, and 150 ppm were 89, 78, 67, 64, and 64%, respectively. The colon tumor multiplicity (colon tumors/animal) was slightly but significantly inhibited in animals fed the diets containing 25 to 150 ppm piroxicam. The results of this study demonstrate that increasing levels of piroxicam in the diet, when fed 1 or 13 wk after carcinogen insult, inhibit colon tumor incidence in a dose-dependent manner.     

Inhibitory effect of peptide Epitalon on colon carcinogenesis induced by 1,2-dimethylhydrazine in rats

The effect of synthetic pineal peptide Epitalon (Ala-Glu-Asp-Gly) on colon carcinogenesis was firstly studied in rats. Eighty 2-month-old outbred male LIO rats were subdivided into four groups and were weekly exposed to five subcutaneous injections of 1,2-dimethylhydrazine (DMH) at a single dose of 21 mg/kg body weight. Additionally, 5 days a week, some of the rats were given subcutaneous injections of saline at a dose of 0.1 ml during the whole experiment (group 1, control) or Epitalon at a single dose of 1 microg during the whole experiment (group 2), Epitalon after termination of carcinogen injections (group 3) or during the period of DMH exposure (group 4). Colon carcinomas developed in 90-100% of DMH-treated rats. The number of total colon tumors per rat was 4.1; 2.7; 3.7; 2.9 in groups 1, 2, 3, 4, respectively (the difference in groups 2 and 4 compared with group 1 is significant). In rats from group 2, colon tumors were smaller than in control animals. In group 2, the incidence, as well the multiplicity of tumors in ascending and descending colon, were significantly decreased in comparison with group 1. In group 4, the mean number of tumors per rat was significantly decreased, too. A trend to decrease the number of tumors in the rectum in rats from groups 2, 3 and 4, treated with Epitalon was found. Epitalon inhibited also the development of tumors in jejunum and ileum. Thus, our results demonstrated an inhibitory effect of Epitalon on chemically induced bowel carcinogenesis in rats.     

Modulation of experimental colon tumorigenesis by types and amounts of dietary fatty acids

Epidemiological studies and laboratory animal model assays suggest that a high intake of dietary fat promotes colorectal cancer. Several in vivo and in vitro studies support the hypothesis that omega-6 fatty acids promote colon tumorigenesis, whereas omega-3 fatty acids lack promoting activity. Fat intake in the United States traditionally includes high amounts (30% of total caloric intake) of saturated fat rather than omega-6 fatty acids. Therefore, the present study was designed to compare the modulatory effects of a high-fat diet containing mixed lipids (HFML), a diet rich in saturated fatty acids (the average American diet), a diet with fish oil (HFFO) that is rich in omega-3 fatty acids, and a low-fat corn oil diet (LFCO) on the formation of chemically induced colonic aberrant crypt foci (ACF) and tumors, cyclooxygenase (COX)-2 activity, and apoptosis during experimental colon carcinogenesis. At 5 weeks of age, groups of male F344 rats were fed a 5% corn oil diet (LFCO). At 7 weeks of age, rats intended for carcinogen treatment received s.c. injections of azoxymethane at a dose level of 15 mg/kg of body weight once weekly for 2 weeks. Beginning 1 day after the carcinogen treatment, groups of rats were then maintained on experimental diets containing 20% HFML or 20% HFFO. Rats were killed at 8, 23, or 38 weeks after azoxymethane treatment. Colonic ACF and tumors were evaluated histopathologically, and apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated nick end labeling method. Colonic mucosae and tumor samples harvested at week 38 were analyzed for COX-2 synthetic activity and expression. The rats fed the HFML diet showed significantly increased total colonic ACF (P < 0.001-0.0001) with a multiplicity of > or = 4 aberrant crypts/focus (P < 0.0001) compared with the effects of the HFFO or LFCO diets at week 8, 23, and 38. Interestingly, there was a 2- to 3-fold increase (> or = 4) in multicrypt foci in rats given the HFML diet as compared with such foci in rats fed the HFFO or LFCO diets. By week 23, the HFML diet had significantly increased the incidence of colonic tumors (30-60%) and their multiplicity (100-141%) when compared with the effects of the LFCO or HFFO diets. At week 38, the HFML diet had induced 100% colon tumor incidence and a 4-fold multiplicity of adenocarcinomas compared with the LFCO and HFFO diets. At weeks 23 and 38, a significantly lower percentage of apoptotic colonic epithelial cells were observed in the tumors of animals fed the HFML diet as compared with those fed the HFFO diet. The HFML diet caused significantly increased levels of COX-2 activity in colon tumors (P < 0.05-0.01), and these tumors had enhanced levels of COX-2 expression as compared with those in assays with LFCO or HFFO diets. These observations demonstrate for the first time that HFML diets containing high levels of saturated fatty acids (such as those in Western diets) promote colon carcinogenesis. Although the mechanisms involved in colon tumor promotion by a HFML diet are not fully known, our results indicate that the modulation of eicosanoid production via the influence on COX activity and the suppression of apoptosis may play a key role in HFML diet-induced colon tumorigenesis.     

Inhibitory effect of synthetic interferon inductor Cycloferone on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats

The effect of a synthetic interferon inductor Cycloferone on colon carcinogenesis was firstly studied in rats. Seventy-five 2-month-old outbred female LIO rats were subdivided into three groups and were weekly exposed to 15 s.c. injections of 1,2-dimethylhydrazine (DMH) at a single dose of 7 mg/kg body wt. From the day of the fist injection of DMH rats from group 2 were given weekly i.p. injections of Cycloferone (62.5 mg/kg) until the end of the experiment. DMH-treated rats (group 3) were exposed to weekly i.p. injections of Cycloferone (62.5 mg/kg) starting in the week after the last injection of the carcinogen. Rats from group 1 were exposed to DMH and treated weekly with 0.2 ml i.p. of normal saline. Additional groups of rats were treated weekly with Cycloferone (62.5 mg/kg) or with 0.2 ml of saline. The experiment was ended 6 months after the first injection of DMH. In DMH-treated rats (groups 1, 2 and 3) colon adenocarcinomas developed in 87, 61 and 59%, respectively. The number of colon tumors per tumor-bearing rat was 2.5, 1.9 and 1.3 in groups 1, 2 and 3, respectively. Treatment with Cycloferone significantly inhibits carcinogenesis in ascending and descending colon. The incidence of tumors of the rectum was decreased in the group 2 as compared with the group 1. There were no cases of tumors of rectum in rats from group 3. The treatment with Cycloferone alone as well as with normal saline failed to induce any tumors in rats. Thus, our results demonstrated inhibitory effect of Cycloferone on colon carcinogenesis induced by DMH in rats.     

Chemopreventive efficacy of sulindac sulfone against colon cancer depends on time of administration during carcinogenic process.

Epidemiological and model studies with laboratory animals have provided evidence that nonsteroidal anti-inflammatory drugs reduce the risk of colon cancer. Sulindac, a nonsteroidal anti-inflammatory drug, has been shown to inhibit azoxymethane (AOM)-induced colon carcinogenesis in rats when administered continuously before, during, and after carcinogen treatment (initiation and postinitiation periods) or when given continuously beginning 14 weeks after carcinogen administration (promotion/ progression stage). The present study was designed to investigate the chemopreventive efficacy of sulindac sulfone (exisulind), the sulfone metabolite of sulindac, when administered during the promotion/progression stage of colon carcinogenesis in comparison to the effect during the initiation and postinitiation periods. We have also studied the modulating effect of exisulind on colonic tumor apoptosis. At 5 weeks of age, groups of male F344 rats were fed diets containing 0%, 0.06%, and 0.12% exisulind. At 7 weeks of age, groups of animals were injected s.c. with AOM (15 mg/kg body weight, once weekly for 2 weeks). Animals intended for the promotion/progression study and receiving 0% exisulind were switched to an experimental diet containing 0.12% exisulind at 14 weeks after the second AOM treatment. All rats remained on their respective dietary regimens until the termination of the study, 50 weeks after the second AOM injection. Colon tumors were evaluated histopathologically for tumor type. Administration of 0.06% and 0.12% exisulind during the initiation and postinitiation periods significantly inhibited the incidence and multiplicity of invasive and/or noninvasive adenocarcinomas of the colon. The inhibition of colon tumorigenesis by exisulind was associated with a significant retardation of body weight gain shortly after sulfone administration and increased apoptosis in the colon tumors. In contrast, administration of the higher dose (0.12%) of exisulind during the promotion/progression stage had only minimal effects on colon tumorigenesis and apoptosis in the colon tumors, suggesting that early administration, but not late administration, may be required for chemopreventive efficacy of this drug.     

Inhibitory effect of dietary p-methoxybenzeneselenol on azoxymethane-induced colon and kidney carcinogenesis in female F344 rats

The effect of dietary p-methoxybenzeneselenol on colon carcinogenesis induced by azoxymethane [(AOM) CAS: 25843-45-2] was studied in female F344 rats. Starting at 5 weeks of age, animals were fed the high-fat diet (control diet) or high-fat diet to which 50 ppm of p-methoxybenzeneselenol (experimental diet) had been added. At 7 weeks of age, all animals except the vehicle-treated controls were administered sc injections of AOM (15 mg/kg body wt, once weekly for 3 wk). Animals were fed the control and experimental diets until 1 week after carcinogen treatment when those animals receiving the p-methoxybenzeneselenol diet were fed the control diet until termination of the experiment. p-Methoxybenzeneselenol in the diet significantly inhibited the incidence (percentage of animals with tumors) of tumors in the colon and kidney, as well as the colon tumor multiplicity (adenomas and adenocarcinomas per animal).     

Effect of polyamine oxidase inhibition on the colonic malignant transformation process induced by 1,2-dimethylhydrazine

Recent studies of colon adenocarcinomas in humans and experimentally induced colonic tumors in rodents have demonstrated selective elevations in the level of N1-acetylspermidine in these malignant tissues. The exact relationship of these alterations in acetylated polyamine levels to the malignant transformation process, however, remains unclear. In order to clarify this issue, rats were given s.c. injections of 1,2-dimethylhydrazine (DMH; 20 mg/kg body wt/week) or diluent for up to 26 weeks. After 10 weeks of carcinogen treatment, one-half of the animals in each group were also concomitantly given i.p. injections of MDL 72527 (20 mg/kg body wt/week), a specific inhibitor of polyamine oxidase, until they were killed. Animals were killed after 15 weeks of DMH treatment and polyamine levels as well as the activities of polyamine oxidase, ornithine decarboxylase and spermidine-N1-acetyltransferase were measured and compared in rat proximal and distal colonic mucosa of each group. Polyamine levels were also assessed in each of these groups after 26 weeks of treatment with this carcinogen +/- MDL 72527. In addition, in view of recent studies that have indicated that polyamines may influence certain oncogenes in human colonic carcinoma cells, tumors from DMH +/- MDL 72527 were analyzed for K-ras mutations. The results of these experiments demonstrated for the first time that: (i) MDL 72527 was a specific inhibitor of polyamine oxidase in normal and malignant colonic tissue; (ii) concomitant administration of this agent with DMH enhanced the elevation of colonic N1-acetylspermidine and significantly reduced the mean colonic tumor burden, as assessed by total tumor area per rat, produced by this carcinogen alone; (iii) analysis of K-ras mutations revealed a similar incidence (62-69%) in adenocarcinomas for both groups (+/- MDL 72527); (iv) however, analysis of the K-ras-mutated and non-mutated tumors revealed that in both carcinogen-treated groups (+/- MDL 72527), tumors with such mutations were smaller than their counterparts without such genetic alterations. Moreover, MDL 72527 reduced the average size of tumors, with and without such mutations, to a similar extent.     

Preventive effect of fermented brown rice and rice bran against colon carcinogenesis in male F344 rats

Epidemiological and preclinical studies demonstrate that nutrition plays an important role in the etiology of cancer. It has been reported that rice components, especially rice germ plays a key role in prevention of cancer. The experiments described here examined the potential anticancer properties of brown rice fermented by Aspergillus Oryzae (FBRA) in male F344 rats using inhibition of the formation of azoxymethene (AOM) induced aberrant crypt foci (ACF) and tumors in the colon as the measure of preventive efficacy. The agent was administered at 2.5 and 5% levels in the diet during the initiation phase (during and until 1 week after carcinogen treatment) and/or post-initiation phase (beginning 1 week after carcinogen treatment) of carcinogenesis. In the ACF and tumor studies, rats were sacrificed 5 or 40 weeks after the initiation of AOM treatment (15 mg/kg body weight, once weekly for 3 weeks), respectively. Colonic ACF and tumors were evaluated histopathologically. Administration of 2.5 and 5% FBRA in the diet continuously during initiation and post-initiation period significantly inhibited the ACF formation in rats treated with AOM, compared with rats treated with AOM alone (99+/-24.1 and 79+/-18.4 vs. 139.5+/-27.7, respectively). In addition, administration of 5% FBRA in the diet during the post-initiation phase significantly suppressed the incidence (44 vs.18%) and multiplicity (0.93+/-0.96 vs. 0.18+/-0.40) of colon adenocarcinomas as compared to those given the control diet. In addition, 5% FBRA in the diet during post-initiation phase caused significant inhibition of cell proliferation in the colonic mucosa as compared to the group fed the control diet (81% reduction, p<0.05). These observations demonstrated for the first time that FBRA inhibits colon tumor development in rats, and suggest that it is a promising dietary supplement for prevention of human colon cancer.     

Inhibition by putrescine of experimental carcinogenesis in rat colon induced by azoxymethane

The effects of putrescine on the incidence and number of colon tumors induced by azoxymethane, and on the labelling index and the activity of ornithine decarboxylase (ODC) in the colon mucosa were investigated in Wistar rats. Rats received 10 weekly injections of 7.4 mg/kg body weight of azoxymethane and i.p. injections of 300 mumol/kg body weight of putrescine every 2 days until the end of the experiment at week 40. This prolonged treatment with putrescine significantly reduced the incidence and number of colon tumors. Administration of putrescine also significantly decreased the labelling index and the ODC activity in the colon mucosa during, but not after, treatment with the carcinogen. These last effects may be related to the action of putrescine in inhibiting the development of colonic tumors.     

Enhancing effects of simultaneous treatment with sodium nitrite on 2-amino-3-methylimidazo[4,5-f]quinoline-induced rat liver, colon and Zymbal's gland carcinogenesis after initiation with diethylnitrosamine and 1,2-dimethylhydrazine

Combined effects of sodium nitrite (NaNO2) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) on liver, colon and Zymbal's gland carcinogenesis were assessed using a rat two-stage carcinogenesis model, with a focus on involvement of oxidative stress. Male 6-week-old F344 rats were given a single intraperitoneal injection of 200 mg/kg of diethylnitrosamine and 4 subcutaneous injections of 40 mg/kg of 1,2-dimethylhydrazine for initiation. Then, they were administered 0 or 300 ppm IQ in the diet or 0, 0.1 or 0.2% NaNO2 in their drinking water for 27 weeks. The treatment with NaNO2+IQ significantly enhanced colon and Zymbal's gland carcinogenesis and tended to enhance hepatocarcinogenesis. The incidence of lung tumors in the IQ-treated groups was significantly increased as compared with the initiation alone group. In a second experiment, male rats were given IQ or NaNO2 under the same conditions as before for 1 week, and at sacrifice, their liver and colon tissue or mucosa were collected for analysis of 8-hydroxydeoxyguanosine (8-OHdG), thiobarbituric acid reactive substances (TBARS), acrolein-modified protein and the bromodeoxyuridine-labeling index (BrdU-LI) (in the colon). In the colon, 8-OHdG, acrolein-modified protein levels and BrdU-LI were significantly increased by the combined treatment. These results indicate that the treatment with NaNO2 enhances IQ-induced colon and Zymbal's gland carcinogenesis in rats and that oxidative DNA damage and lipid peroxidation may partly be involved, especially in the colon. In addition, this experiment showed that IQ can act as a potent lung carcinogen in rats.     

Post-initiation treatment of Indole-3-carbinol did not suppress N-methyl-N-nitrosourea induced mammary carcinogenesis in rats.

The consumption of cruciferous vegetables (the Family of Cruciferae) such as cabbage, broccoli and Brussels sprouts has been shown to have cancer chemopreventive effects in humans and experimental animals. Indole-3-carbinol (I3C), one component of cruciferous vegetables, has been shown to exert cancer chemopreventive influence in liver, colon, and mammary tissue when given before or concurrent with exposure to a carcinogen. However in some reports, there has been evidence that consumption of I3C after carcinogen treatment might be associated with tumor promotion in some tissues. There have been no reports, to our knowledge, of post-initiation effects of I3C in the N-methyl-N-nitrosourea (MNU)-induced mammary tumor model in rats. Our studies were performed to examine this question. Ninety-six, 4-week-old female Sprague-Dawley rats were randomly divided into five groups. The animals of groups 1, 2 and 3 received an intraperitoneal injection of MNU at the age of 50 days. The animals of groups 4 and 5 were injected with saline only at the same time. Animals of groups 1 and 2 were given diet containing 100 ppm and 300 ppm I3C from week 1 until week 25 after MNU treatment. The animals of group 4 were given basal diet containing 300 ppm I3C without MNU treatment. All animals were killed at week 25. The incidences of mammary tumors in the groups 1, 2 and 3 were 95.8% (23/24), 83.3% (20/24) and 82.4% (28/34), respectively. The average number of tumors in the tumor bearing rats of the MNU and I3C 300 ppm group (group 2; 3.85+/-0.63) was higher than that in the MNU alone group (group 3; 2.46+/-0.31). These results represented that exposure to I3C after carcinogen treatment did not suppress development of mammary tumors.     

Dietary calcium and vitamin D modulate 1,2-dimethylhydrazine-induced colonic carcinogenesis in the rat

To determine whether supplemental dietary calcium and/or vitamin D deficiency are involved in modulating colon cancer induced by 1,2-dimethylhydrazine (DMH), Sprague-Dawley rats were fed diets containing either: (a) a normal content of calcium (0.87%) and phosphorus (0.60%) with 2.2 IU of vitamin D3 per g of feed (group A); (b) the same diet as group A, but with calcium and phosphorus increased to 1.80 and 0.80%, respectively (group B); or (c) a vitamin D-deficient diet with supplemental calcium (1.80%) and phosphorus (0.80%) (group C). After 6 weeks on their respective diets, one-half the animals in each group were given s.c. injections of either vehicle or DMH (20 mg/kg body weight/week) for 26 weeks. Animals were then sacrificed and the incidence of tumors as well as the number of tumors per tumor-bearing rat were determined. Colonic mucosal polyamine levels were measured after 15 weeks of exposure to vehicle or DMH, before development of histologically recognizable neoplasms. The results of these experiments demonstrated that neither calcium supplementation alone nor supplemental calcium in conjunction with vitamin D deficiency altered the incidence of colonic cancer induced by this carcinogen. Supplemental calcium, however, significantly decreased the number of rats with multiple tumors and reduced tumor size. Moreover, vitamin D deficiency abolished these protective effects of calcium on colon cancer in this experimental model. DMH treatment increased polyamine levels in the premalignant colonic mucosa in group A rats. This carcinogen-induced effect was blunted by high dietary calcium. Vitamin D-deficient, calcium-supplemented rats (group C) showed an increase in N1-acetylspermidine, but not the other polyamines, with DMH treatment.     

Post-initiation effects of chlorophyllin and indole-3-carbinol in rats given 1,2-dimethylhydrazine or 2-amino-3-methyl- imidazo

Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll, the ubiquitous pigment in green and leafy vegetables, whereas indole-3-carbinol (I3C) is present in cruciferous vegetables such as cabbage, broccoli and cauliflower. In rats initiated with 1,2-dimethylhydrazine (DMH), CHL and I3C reportedly promoted or enhanced the incidence of colon tumors when they were administered after, or during and after the carcinogen exposure, respectively. The same compounds given post-initiation inhibited the formation of colonic aberrant crypts induced by heterocyclic amines, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), but tumor suppression was not examined in the latter studies. In the present investigation, male F344 rats were treated with IQ or DMH during the first 5 weeks of a 1 year study; IQ was given in the diet (0.03%), whereas DMH was administered once a week by s.c. injection (20 mg/kg body wt). Beginning 1 week after the last dose of IQ or DMH until sacrifice, rats received 0.001, 0.01 or 0.1% (w/v) CHL in the drinking water or 0.001, 0.01 or 0.1% I3C in the diet. Compared with controls given carcinogen alone, 0.1% I3C treatment suppressed the multiplicity of IQ-induced colon tumors, and CHL inhibited in a dose-related manner the incidence of IQ-induced liver tumors. However, 0.001% CHL increased significantly the multiplicity of DMH-induced colon tumors while having no effect on the colon tumors induced by IQ. These results indicate that both the choice of carcinogen as well as the dose of the tumor modulator can be important determinants of the events that occur during post-initiation exposure to CHL or I3C. Based on the present findings and data in the literature, it is possible for CHL and I3C to act as tumor promoters or anticarcinogens, depending upon the test species, initiating agent and exposure protocol.     

Selenium and difluoromethylornithine additively inhibit DMH-induced distal colon tumor formation in rats fed a fiber-free diet

We investigated the effects of difluoromethylornithine, an inhibitorof ornithine decarboxylase (ODC) and selenium supplementationon tumor formation induced by the carcinogen 1, 2-dimethylhydrazine(DMH) in Sprague-Dawley rats. A biochemical link between polyaminebiosynthesis and selenium metabolism to its cancer preventativeform has been suggested by the common requirement of S-adenosylmethio-nine.One-hundred and twenty male Sprague-Dawley rats were dividedinto experimental (n = 80) and control (n = 40) groups. Experimentalanimals received DMH 20 mg/kg s.c. for 20 weeks. Animals werefed either a regular diet (selenium content 0.2 p.p.m.) or ahigh selenium diet (5 p.p.m.) with or without 0.2% DFMO in thedrinking water. At death, week 30, animal weights within experimentalor control groups were not different between the four diet treatmentgroups. Tumor number and incidence in the proximal colon wasnot affected by DFMO treatment, selenium supplementation orthe combined treatment. In contrast, in the distal colon, 19tumors developed in the DFMO treated group, 22 tumors in thehigh selenium group and only 12 tumors in the combined highselenium/DFMO treatment group compared to 32 tumors in the regulardiet group. Similarly, tumor incidence was decreased by DFMOand selenium supplementation and their effects were additive.In control animals, ODC activity was decreased by DFMO treatmentand selenium supplementation in the distal colon and liver,but not the proximal colon. ODC activity of tumor tissue wasgreater than normal colon tissue from diet paired animals forproximal and distal colon, except for distal colonic tumorsin the high selenium/DFMO treatment group. Polyamine content,however, did not correlate with ODC activity in normal or neoplastictissue. In general, S-adenosylmethionine levels from normalcolon and liver tissue were unaffected by diet treatment. Seleniumsupplementation in combination with DFMO treatment selectivelyinhibited distal colon tumor formation in rats fed a fiber-freediet.     

Chemoprevention of colon carcinogenesis by dietary organoselenium, benzylselenocyanate, in F344 rats

The effect of feeding benzylselenocyanate (BSC) and its sulfur analogue, benzylthiocyanate (BTC), 2 wk before, during, and until 1 wk after carcinogen administration (initiation phase) on intestinal carcinogenesis induced by azoxymethane (CAS:25843-45-2) was studied in male F344 rats. Weanling rats were raised on a semipurified diet (AIN-76A diet; control diet). Beginning at 5 wk of age, groups of animals consuming the control diet were fed one of the diets containing 25 ppm BSC or BTC. An additional group was continued on the control diet. At 7 wk of age, all animals in 3 groups, except the vehicle-treated controls, were administered s.c. injections of azoxymethane (15 mg/kg body weight, once weekly for 2 wk). Animals were continued on the control diet and BSC and BTC diets until 1 wk after carcinogen treatment, when those groups receiving BSC and BTC diets were fed the control diet until termination of the experiment. Tissue and blood plasma glutathione peroxidase activity was measured in vehicle-treated animals fed the control diet and BSC and BTC diets for 5 wk. The results indicate that body weights were comparable among the various dietary groups. BSC in the diet significantly inhibited the incidence (percentage of animals with tumors) and multiplicity (tumors/animal) of adenocarcinomas in the colon and multiplicity of adenocarcinomas in the small intestine compared to those fed the control diet. BTC in the diet had no effect on colon and small intestinal tumors. Selenium-dependent glutathione peroxidase activity was significantly increased in kidneys and colon and small intestinal mucosae of animals fed the BSC diet compared to animals fed the BTC and control diets.     

Consistent and fast inhibition of colon carcinogenesis by polyethylene glycol in mice and rats given various carcinogens

We have previously shown that dietary polyethylene-glycol (PEG) suppresses the occurrence of azoxymethane-induced cancers in an accelerated rat model of colon carcinogenesis. To determine the consistency of this preventive effect, we carried out a long-term study in rats fed the standard American Institute of Nutrition 1976 diet, and 7 short-term prevention studies in rodents. A total of 337 F344 rats, 20 Sprague Dawley rats, and 40 OF1 mice were all given initiating dose(s) of colon carcinogen, and were randomly allocated to experimental groups 7 d later. Treated groups received drinking water containing 5% PEG. After 30 or 162 d, the animals were examined for aberrant crypt foci or tumors in the colon. After two 20 mg/kg azoxymethane injections, the number of F344 rats with colon tumor was lower in rats receiving PEG for 162 d than in carcinogen-injected controls, 5/21 versus 25/27 (P < 0.0001). PEG-fed rats had no invasive cancer, and 10 times fewer colon tumors than controls (0.3+/-0.1 and 3.1+/-0.5 respectively, P < 0.0001). A three-day PEG treatment was sufficient to halve the number of azoxymethane-induced aberrant crypt foci in F344 rats (P = 0.0006). After 16 d of treatment, PEG-fed rats had five times fewer foci than controls (21+/-14 and 100+/-23 respectively, P < 0.0001), but the inhibition was reversible in part when treatment was discontinued. Aberrant crypt foci initiated by N-methyl-N-nitrosourea intra-rectally (40 mg/kg) or by 2-amino-3,4-dimethylimidazo(4,5-f)quinoline p.o. (2 x 200 mg/kg) were suppressed by PEG (P < 0.0001 and P = 0.003 respectively). PEG was active in F344 rats, in Sprague Dawley rats (P = 0.0005), and in OF1 mice (P = 0.001). PEGs with MW between 3350 and 12000 (but not PEG 400), and PEG 8000 from five suppliers, markedly inhibited azoxymethane-induced aberrant crypt foci (all P < 0.01). The prevention was stronger in rats fed a high-fat diet (P < 0.0001) than in rats fed a rodent chow (P = 0.02). PEG was thus a fast, consistent, and potent inhibitor of early colonic precursor lesions. Moreover, PEG is one of the most potent inhibitors of colon tumor in the standard rat model. Since PEG has no known toxicity in humans, we think it should be tested as a chemopreventive agent in a clinical trial.     

Effect of retinoids on the induction of colon cancer in F344 rats by N-methyl-N-nitrosourea or by 1,2-dimethyihydrazine

The possible modifying effect of synthetic and natural retinoidson the incidence of colon cancer in rats induced by 2 intrarectaldoses of 2.5 mg of N-methyl-N-nitrosourea (MNU) given once aweek for 2 successive weeks or a single 150 mg/kg body weightdose of 1,2-dime-thylhydrazine (DMH), s.c. was investigated.Emphasis was on the effect of the development of early tumorsas visualized by endoscopy. With the retinoids N-ethyl-retinamide,N-2-hydroxyethylretinamide, N-(4-hydro- xyphenyl)-all-trans-retinamide(RAHA), and retinyl acetate (RA) administered orally after thecarcinogens, significant differences in early developing tumorswere not found. At histopathological examination of the tumorsthe RAHA + DMH group had significantly fewer adenomas per animal.The percentage of adenoma bearing rats was significantly lowerin groups receiving RAHA + DMH or RA + DMH. However, food consumptionwas lower in rats consuming either RAHA or RA. Retinyl palmitate(RP) and RAHA was administered intrarectally to MNU-inducedrats either before or after the carcinogen. When administeredbefore MNU, RP caused a significant increase in the percentageof tumor bearing animals and the average number of tumors peranimal as visualized endos copically. At histopathological examination,all retinoid groups except RAHA given after the carcinogen,produced significantly more adenomas per animal and a significantlygreater adenoma incidence than did the control groups. Thus,in two systems, the oral administration of retinoids did notclearly inhibit the early or later stages of colon tumor development.Inirarectal infusion of two retinoids had no effect on colonicmor phology but at histopathological examination of later stagetumors there was an enhanced adenoma response.