Modifying effects of Terminalia catappa on azoxymethane-induced colon carcinogenesis in male F344 rats.

The modifying effects of dietary administration of an herb, Terminalia catappa (TC), were investigated on rat colon carcinogenesis induced by a carcinogen azoxymethane (AOM). The number of aberrant crypt foci (ACF) and beta-catenin accumulated crypts (BCACs) in the colon, and proliferating cell nuclear antigen (PCNA) labelling index in the colonic epithelium were examined in a total of 36 male F344 rats. All animals were randomly divided into five experimental groups (4-10 rats in each group). At 6 weeks of age, rats in groups 1, 2 and 3 were given s.c. injections of AOM once a week for 2 weeks at a concentration of 20 mg/kg body weight. One week before the first injection of AOM, rats in groups 2 and 3 were fed a diet containing 0.02 and 0.1% TC, respectively, throughout the experiment. Rats in group 4 were fed a diet containing 0.1% TC. Rats in group 5 were served as untreated controls. All animals were sacrificed at the experimental week 5 after the start of the experiment. Oral administration of TC at both doses significantly decreased the numbers of both ACF/colon/rat (P<0.05 for 0.02% TC, P<0.005 for 0.1% TC) and BCAC/cm/rat (P<0.05 for both 0.02 and 0.1% TC), when compared with the control group (group 1). Colonic PCNA labelling index in groups 2 and 3 was also significantly lower than that in group 1 (P<0.001 for 0.02% TC, P<0.005 for 0.1% TC). These results suggest that TC has a potent short-term chemopreventive effect on biomarkers of colon carcinogenesis and this effect may be associated with the inhibition of the development of ACF and BCACs.     

Efficacy of potential chemopreventive agents on rat colon aberrant crypt formation and progression

We assessed the effects of 78 potential chemopreventive agents in the F344 rat using two assays in which the inhibition of carcinogen-induced aberrant crypt foci (ACF) in the colon was the measure of efficacy. In both assays ACF were induced by the carcinogen azoxymethane (AOM) in F344 rats by two sequential weekly injections at a dose of 15 mg/kg. Two weeks after the last AOM injection, animals were evaluated for the number of aberrant crypts detected in methylene blue stained whole mounts of rat colon. In the initiation phase protocol agents were given during the period of AOM administration, whereas in the post-initiation assay the chemopreventive agent was introduced during the last 4 weeks of an 8 week assay, a time when ACF had progressed to multiple crypt clusters. The agents were derived from a priority listing based on reports of chemopreventive activity in the literature and/or efficacy data from in vitro models of carcinogenesis. During the initiation phase carboxyl amidoimidazole, p-chlorphenylacetate, chlorpheniramine maleate, D609, diclofenac, etoperidone, eicosatetraynoic acid, farnesol, ferulic acid, lycopene, meclizine, methionine, phenylhexylisothiocyanate, phenylbutyrate, piroxicam, 9-cis-retinoic acid, S-allylcysteine, taurine, tetracycline and verapamil were strong inhibitors of ACF. During the post-initiation phase aspirin, calcium glucarate, ketoprofen, piroxicam, 9-cis-retinoic acid, retinol and rutin inhibited the outgrowth of ACF into multiple crypt clusters. Based on these data, certain phytochemicals, antihistamines, non-steroidal anti-inflammatory drugs and retinoids show unique preclinical promise for chemoprevention of colon cancer, with the latter two drug classes particularly effective in the post-initiation phase of carcinogenesis.     

Suppression of Azoxymethane-induced Colonic Premalignant Lesion Formation by Coenzyme Q10 in Rats

Reactive oxygen species cause damage to proteins, lipids and DNA. Coenzyme Q10 (CoQ10) is a compound withmitochondrial bioenergetic functions. The reduced form of CoQ10 shows antioxidant activity. In the present study,effects of CoQ10 on development of azoxymethane (AOM)-induced aberrant crypt foci (ACF) and mucin-depletedfoci (MDF) in F344 male rats were investigated. To induce ACF and MDF, 6-week old rats were given two weeklysubcutaneous injections of AOM (15 mg/kg body weight) and also received a control diet or experimental dietscontaining CoQ10 (200 or 500 ppm) for 4 weeks, starting one day before the first dose of AOM. At 10 weeks of age,all animals were sacrificed and their colons were evaluated for numbers and sizes of ACF and MDF. Administrationof 200 and 500 ppm CoQ10 resulted in reduction of ACF numbers, to 77% and 68% of the carcinogen control value,respectively. The percentages of ACF consisting of more than 4 crypts in these groups were also significantly lowerthan in the controls. Treatment with 500 ppm CoQ10 furthermore decreased the number of sialomucin-producingACF and MDF per colon to 42% and 38% of the carcinogen control value without CoQ10, respectively. Theseresults suggest that CoQ10 may be an effective chemopreventive agent against colon carcinogenesis.     

Suppression of Azoxymethane-induced Rat Colon Aberrant Crypt Foci by Dietary Protocatechuic Acid

The modifying effect of dietary exposure to protocatechuic acid (PCA) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. The effects of PCA feeding on the silver-stained nucleolar organizer regions protein (AgNORs) count in the colonic epithelial cells and on the ornithine decarboxylase (ODC) activity in the colonic mucosa were also estimated. Animals were given weekly s.c. injections of AOM (15 mg/kg body weight) for 3 weeks to induce ACF. These rats were fed diet containing 1000 or 2000 ppm PCA for 5 weeks, starting one week before the first dosing of AOM. All rats were killed 2 weeks after the last AOM injection, to measure the number of ACF, ODC activity, and AgNORs count per nucleus in the colon. In rats given AOM and PCA, the frequency of ACF/colon was significantly decreased compared with that in rats given AOM alone ( P < 0.005 at 1000 and P < 0.05 at 2000 ppm). ODC activity in the colon of rats given AOM and PCA at both doses was also significantly lower than that of rats treated with AOM alone ( P < 0.05). Similarly, the mean AgNORs count in rats fed PCA was significantly smaller than that of rats treated with AOM alone ( P < 0.0001). Treatment with PCA alone did not affect these three biomarkers. These results provide further evidence that PCA could be a chemopreventive agent against rat colon carcinogenesis.     

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.     

Green tea polyphenols inhibit colorectal aberrant crypt foci (ACF) formation and prevent oncogenic changes in dysplastic ACF in azoxymethane-treated F344 rats

Green tea and its constituents have shown cancer-preventive activities in many animal models. In order to prepare for a human trial on the inhibition of colon carcinogenesis, we conducted a study with green tea polyphenols as the preventive agent in an azoxymethane (AOM)-induced rat colon cancer model using aberrant crypt foci (ACF) as an end point. F344 rats were given two weekly injections of AOM (15 mg/kg), and then fed a 20% high-fat diet with or without 0.12 or 0.24% Polyphenon E (PPE, a standardized green tea preparation consisting 65% of (-)-epigallocatechin-3-gallate and 22% of other catechins) for 8 weeks. Colorectal ACF were analyzed under a microscope after methylene blue staining. Dietary PPE administration was found to significantly and dose dependently decrease the total number of ACF per rat and the total number of aberrant crypt per rat. Moreover, treatment with 0.24% PPE also significantly decreased the percentage of large ACF (four or more crypts) and the percentage of ACF with high-grade dysplasia in total ACF. The high-grade dysplastic ACF from 0.24% PPE-treated group had increased apoptosis and decreased nuclear expression levels of beta-catenin and cyclin D1. Retinoid X receptor (RXR)alpha expression was reduced in high-grade dysplastic ACF, adenoma and adenocarcinoma during AOM-induced colon carcinogenesis, and the PPE treatment partially prevented the loss of RXRalpha expression in high-grade dysplastic ACF. Taken together, our results strongly suggest the colon cancer-preventive activity of PPE and identified possible molecular markers for future colon cancer prevention studies.     

Regressive Effects of Various Chemopreventive Agents on Azoxymethane-induced Aberrant Crypt Foci in the Rat Colon

Regressive effects of four chemopreventive agents [5-hydroxy-4-(2-phenyl-( E )-ethenyl)-2(5 H fura-none (KYN-54), S-methyl metbanethiosulfonate (MMTS), chlorogenic acid (CA), and piroxicam] on azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon of male F344 rats were examined by dietary exposure. At six weeks of age, 60 rats of groups 1 through 5 received subcutaneous injections of AOM (15 mg/kg body weight) once a week for three weeks. Twelve weeks after the first carcinogen injection, wben the occurrence of ACF was maximal, the rats in groups 2 through 5 were started on diet containing the test chemicals as follows: group 2, KYN-54 (0.02%); group 3, MMTS (0.01%); group 4, CA (0.025%); and group 5, piroxicam (0.0125%). Group 1 (20 rats) was kept on the basal diet alone, and group 6 (12 rate) served as an untreated control. Rats in each group were killed at 6, 12, 18, or 24 weeks after the start of the experiment, and the yield of ACF in the colon of each group at 18 or 24 weeks was compared with that at 12 weeks. The number of ACF per rat colon of each group at 18 or 24 weeks was smaller than that at 12 weeks. The reduction rates at 18 weeks were 7% in group 1 (AOM alone), 11% in group 2 (AOM+KYN-54), 10% in group 3 (AOM+MMTS), 51% in group 4 (AOM + CA) ( P 0.01), and 33% in group 5 (AOM+piroxicam) ( P <0.02), while at 24 weeks they were 12%, 26%, 51% ( P <0.002), 43% ( P <0.05), and 70% ( P <0.001), respectively. These results indicate that chemopreventive agents for large bowel carcinogenesis, i.e., KYN-54, MMTS, CA, and piroxicam, are not only able to prevent the development of ACF, but also can regress ACF, which are regarded as precursor lesions of colorectal cancer.     

Prevention of the development of preneoplastic lesions, aberrant crypt foci, by a water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi) mycelia in male F344 rats

The modifying effects of a dietary water-soluble extract from cultured medium of Ganoderma lucidum (Rei-shi or Mannentake) mycelia (MAK) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. Rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for three weeks to induce ACF and fed on diets containing 0, 1.25, 2.5 and 5.0% MAK for five weeks, starting one week before the first dose of carcinogen. MAK significantly and dose-dependently prevented the development of ACF, decreasing the total number of AC and inhibiting cyst formation. MAK (2.5 and 5.0%) also significantly reduced the longitudinal-cross section areas of colon epithelium. MAK in all doses significantly reduced the PCNA positive index, area of the germinal region and number of cells per half crypt. In an additional in vitro experiment, MAK inhibited anchorage-independent growth of several colon carcinoma cell lines. The present results thus indicate that dietary MAK could act as a preventive agent for colon carcinogenesis.     

Reovirus decreases azoxymethane-induced aberrant crypt foci and colon cancer in a rodent model

Reovirus type 3 Dearing has demonstrated oncolytic efficacy in vitro and in vivo against a variety of cancer cell lines, tumor xenografts and syngeneic cancer models. In this study, we investigated the effectiveness of reovirus against aberrant crypt foci (ACF) and colon cancer induced by the carcinogen azoxymethane (AOM) in an immunocompetent rat model. Sprague-Dawley rats received 15 mg/kg AOM intraperitoneally once per week for 4 weeks and reovirus was administered rectally once a week for 5 weeks starting 20 weeks after the last dose of AOM. Two weeks after completion of reovirus therapy, animals were examined for tumor burden in the colon and other tissues. Reovirus-treated animals showed a decrease in total ACF numbers (P=0.014), in large ACFs (P=0.0069) and in tumor number (P=0.03) compared to vehicle-treated animals. Fewer obstructing tumors in the colon (P=0.07) and duodenum (P=0.03) and reduced hepatic metastases were also noted. In addition, a tumor cell line derived from hepatic metastases was found to be susceptible to reovirus in vitro. Our results show that repeated rectal reovirus administration had some efficacy in the treatment and prevention of AOM-induced ACFs, colon cancers and metastases.     

A rapid dual organ rat carcinogenesis bioassay for evaluating the chemoprevention of breast and colon cancer

In this study we evaluated the effect of dietary administration of a high fat, low fiber diet (HRD) with or without 2% phytic acid (PA) on the development of mammary cancer and/or colon cancer in rats exposed to methylnitrosourea (MNU), azoxymethane (AOM) or MNU + AOM. The rats were fed a HRD alone or a HRD + 2% PA. At the end of week 2, the rats were given either a s.c. injection of MNU (50 mg/kg body wt) or one of normal saline (vehicle). At the end of weeks 3 and 4, the rats were given either a s.c. injection of AOM (15 mg/kg body wt per week) or one of normal saline (vehicle). Nine weeks after the injection of MNU or saline, 10 rats from each group were sacrificed and the mammary tumor incidence and the number of colonic aberrant crypt foci (ACF) were compared between different groups. The administration of different diets was continued for an additional 21 weeks and the mammary tumor and colon tumor incidence between different groups were compared. Results showed that rats injected with MNU alone did not develop ACF or colon tumors while those injected with AOM alone did not develop mammary tumors. Linear regression analysis of the number of ACF at 11 weeks versus colonic tumor incidence at 32 weeks, and the linear regression analysis of mammary tumor incidence at 11 weeks versus mammary tumor incidence at 32 weeks, both showed good linear correlation. These results demonstrate the potential value of the short term dual organ carcinogenesis bioassay for screening chemopreventive agents for their relative ability to inhibit the development of mammary cancer and/or colon cancer while on high risk diet.     

The Dietary Effect of Monoglucosyl-rutin on Azomethane-Induced Colon Carcinogenesis

The dietary effect of monoglucosyl-rutin (M-R), a flavonoid, on azoxymethane (AOM)-induced colon carcinogenesis ‍was investigated in two experiments with 5 week old, F344 male rats. In the first experiment (5 weeks study), effects ‍of MR on AOM (15 mg/kg body weight 3 times weekly)-induced formation of aberrant crypt foci (ACF) in five ‍groups were assessed. In this experiment, group 3 given 500 ppm M-R with AOM had a significantly smaller number ‍of ACF containing 4 or more aberrant crypts than group 1 with AOM alone, and groups 2 and 3 given 100 ppm or ‍500 ppm M-R respectively had significantly lower BrdU labeling indices in the epithelial cells of large bowel than ‍group 1. For the second experiment, rats were divided into 8 groups. Groups 1-5 were given AOM as in the first ‍experiment. Groups 2-5 were fed diets containing 100ppm or 500ppm M-R for 4 weeks in the initiation phase or 36 ‍weeks in the post-initiation phase. Group 6 was given 500ppm M-R throughout the experiment, and group 7 was ‍kept on the basal diet and served as a control. At the termination of the experiment (40 weeks after the start), groups ‍2-5 had significantly smaller numbers of positive cells with anti-proliferating cell nuclea antigen (PCNA) antibody ‍than group 1. Furthermore, group 5 treated with 500ppm M-R for 36 weeks demonstrated tendencies for decrease in ‍the incidence and multiplicity of colon tumors. These data suggest that M-R has the potential to inhibit AOMinduced ‍colon carcinogenesis.     

Prevention by long-term fermented miso of induction of colonic aberrant crypt foci by azoxymethane in F344 rats.

The present study was designed to investigate the effects of fermented miso in the diet on the induction of aberrant crypt foci (ACF) by azoxymethane (AOM) in male F344 rats. A total of 50 rats, 8 weeks of age, were divided into 5 groups and given weekly subcutaneous injections of AOM (15 mg/kg body wt) for 3 weeks. Rats were fed a normal control MF solid diet, or solid diet containing 10% long-term fermented (aged), medium- or short-term fermented miso, or 2.2% NaCl for 5 weeks, starting one week before the first AOM dosing. It was found that, compared to the control (MF) diet, the long-term fermented diet significantly decreased (by 22.2%) ACF/colon, but increased (by 18.2%) the number of aberrant crypts (Acs)/focus. The latter was also increased by the medium-term fermented diet (by 25.3%). The PCNA labeling index was only affected by the short-term fermented diet (36.9% increase) and by 2.2% NaCl diet (27.2% increased). The present results indicate that aged or completely fermented miso supplemented into the diet, could act as a chemopreventive agent for colon carcinogenesis.     

Chemopreventive effect of 2-(allylthio)pyrazine (2-AP) on rat colon carcinogenesis induced by azoxymethane (AOM)

An investigation was conducted to assess the chemopreventive effects of 2-(allylthio)pyrazine (2-AP), synthesized for potential use as a chemopreventive agent, after administration during the pre-initiation and post-initiation stages in a rat colon carcinogenesis model with azoxymethane (AOM). One hundred, 5-week-old, male F344 rats were randomly divided into two experiments (n = 50 each). Experiment 1 rats were randomly divided into three groups: Group 1 rats were pre-treated with 2-AP (25 or 50 mg/kg body weight, 3 consecutive days through the route of intragastric intubations) before AOM (20 mg/kg body weight, single subcutaneous (s.c.) injection) initiation. Group 2 rats were treated with AOM alone. Group 3 rats were given 2-AP alone without AOM initiation. The animals were killed at the end of each experiment (week 5) and the aberrant crypt foci (ACF) of the colonic mucosa were assessed after staining with methylene blue. Experiment 2 rats were randomly divided into three groups: Group 1 rats were given 2-AP (10, 25 or 50 mg/kg body weight, five-times intragastric intubations per week for 5 weeks from week 3) after AOM (15 mg/kg body weight, three s.c. injections) initiation for 2 weeks. Group 2 rats were treated with AOM alone. Group 3 rats were given 2-AP alone without AOM initiation. The animals were killed at the end of the experiment (week 8) and the ACF of the colonic mucosa were quantified. Total numbers of ACF/colon in Group 1 rats (pre-treated with 2-AP) tended to decrease (2-AP, 50 mg/kg body weight) or increase (2-AP, 100 mg/kg body weight) depending on the dose level. Total numbers of ACF/colon in Group 1 rats (treated with AOM followed by 2-AP, all subgroups; 160.8 +/- 38.0; 161.8 +/- 38.1; 137.1 +/- 48.4) were decreased significantly compared with the values in Group 2 rats (AOM alone; 214.8 +/- 48.1) (P < 0.05 or 0.01). The highest dose group (2-AP, 50 mg/kg body weight) had the lowest levels of total numbers of ACF/colon among the three subgroups. Total numbers of aberrant crypts (AC)/colon of the highest dose group (340.1+/- 117.9) decreased significantly compared with the value for Group 2 rats (AOM alone; 545.1 +/- 38.3). These results thus suggest that 2-AP may have potential as a chemopreventive agent against rat colon carcinogenesis after administration of AOM during the post-initiation stage.     

Effect of dietary oligofructose and inulin on colonic preneoplastic aberrant crypt foci inhibition

Oligofructose and inulin, naturally-occurring fermentable chicory fructans, have been shown to stimulate the growth of bifidobacteria which are regarded as beneficial strains in the colon and inhibit colon carcinogenesis in the laboratory animal models. The present study was designed to determine the effect of oligofructose and inulin on the azoxymethane (AOM)-induced preneoplastic lesions such as aberrant crypt foci (ACF) formation in the colon of male F344 rats. At 5 weeks of age, groups of animals were fed the AIN-76A (control) and the experimental diets containing 10% oligofructose or inulin. At 7 weeks of age, all animals received s.c. injection of AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt, once weekly for 2 weeks. The animals were necropsied 7 weeks after the last AOM injection, and the ACF were visualized under light microscopy in the formalin-fixed, unsectioned methylene blue-stained colons. They were distinguished by their increased size, more prominent epithelial cells and pericryptal space. The feeding of oligofructose or inulin significantly inhibited the ACF formation and the crypt multiplicity in the colon. The degree of ACF inhibition was more pronounced in animals fed inulin than in those fed oligofructose. The findings suggest that chicory fructan supplements inhibit ACF formation, an early preneoplastic marker of malignant potential in the process of colon carcinogenesis.      

Intake of beer inhibits azoxymethane-induced colonic carcinogenesis in male Fischer 344 rats

Modulatory effects of beer consumption on azoxymethane (AOM)-induced rat colonic carcinogenesis in male Fischer 344 rats were investigated. Single cell gel electrophoresis assay indicated that DNA damage of colonocytes, induced by a single AOM injection (15 mg/kg body weight), was significantly reduced in rats fed beer or malt extract for 2 weeks. Examination of aberrant crypt foci (ACF) formation in colonic mucosa, induced by AOM (15 mg/kg body weight; twice weekly), revealed that feeding of beer during the whole experimental period of 5 weeks significantly reduced the number of ACF by 35%. In the post-initiation protocol, a reduction in ACF formation by 26% was not significant. The efficacy in inhibition of ACF formation varied with the brand of beer. ACF formation was significantly reduced in rats treated with freeze-dried beer (FD Beer), but not with ethanol, suggesting that nonvolatile components of beer are responsible for the reduction. Significant suppression of ACF formation was observed in groups treated with hot water extract of malt, especially with extracts of colored malts, although no reduction was observed by feeding with hops extract. A long-term experiment of 42 weeks indicated that intake of beer decreased tumor incidence by 22% and decreased the number of neoplastic lesions, including adenocarcinomas and adenomas, by 44%. These results suggest that components of beer have chemopreventive effects on colonic carcinogenesis induced by AOM and that intake of beer may contribute to a reduction in the risk of cancer susceptibility.     

Increased Cell Proliferation of Azoxymethane-induced Aberrant Crypt Foci of Rat Colon

Aberrant crypt foci (ACF) were induced in the colon of F344 rats by s.c. injection of azoxymethane (AOM) twice in a three day-interval and examined after 4 and 12 weeks. The number and crypt multiplicity of ACF in each section of rat colon increased during this period. Histologically, aberrant crypts consisted of proliferating atypical epithelial cells. Cell proliferation of ACF consisting of 4 aberrant crypts [ACF(4)] and 2 aberrant crypts [ACF(2)], and normal crypts in the colon of rats treated with AOM [normal crypts/AOM(+)] or saline [normal crypts/AOM(-)] was investigated by measurement of the mitotic index, proliferating cell nuclear antigen-labeling index (PCNA-LI), and 5-bromo-2'-deoxyuridine-labeling index (BrdU-LI). All three parameters of the cell proliferative activity of ACF(4) were higher than those of normal crypts/AOM(+) and normal crypts/AOM(-). The PCNA-LI and BrdU-LI in ACF(2) were the same as those in ACF(4). These findings suggest that ACF have increased cell proliferative activity. The correlation of these three parameters confirmed that the PCNA-LI is also a useful parameter for evaluating cell proliferative activity in ACF. The presence of many cells stained by PCNA in the upper portion of ACF suggested that ACF have more G₁ phase cells, which readily respond to mitogenic stimulation, than G₀ phase cells, which are predominant in normal crypts.     

Azoxymethane-induced colon tumors and aberrant crypt foci in mice of different genetic susceptibility

Azoxymethane (AOM) is an organotropic colon carcinogen that is commonly used to induce colon tumors in rodents. Unlike its parent compound, 1,2-dimethylhydrazine (DMH), a tumor susceptibility phenotype in inbred mice with respect to AOM has not been established. Thus, this study was undertaken to determine whether genetic susceptibility extends to this carcinogen. SWR/J, A/J (both susceptible to DMH carcinogenesis) and AKR/J (resistant) mice were treated with 10 mg/kg AOM i.p. once a week for 8 weeks. Twenty-five weeks after the initial injection, tumor yield was determined. With a single exception, only SWR/J and A/J mice developed tumors, with a distribution that was limited to the distal colon (16.3±1.1 and 36.4±2.4, respectively). The formation of aberrant crypt foci (ACF), putative preneoplastic lesions, was also assessed in whole-mount colons using Methylene Blue staining. Consistent with tumor multiplicity, the total number of ACF was highest in A/J mice, followed by SWR/J mice. In addition, A/J mice had a significantly greater number of large ACF (five or more crypts per foci) than the other strains. Despite the absence of colon tumors, however, AKR/J mice did develop a significant number of ACF. This finding suggests that ACF in resistant mice are persistent but do not progress to tumors.     

Resveratrol depresses the growth of colorectal aberrant crypt foci by affecting bax and p21(CIP) expression

We investigated whether resveratrol (RV) affects azoxymethane (AOM)-induced colon carcinogenesis, by administering RV (200 microg/kg/day in drinking water) to male F344 rats for 100 days, beginning 10 days before carcinogen treatment (two weekly doses of 15 mg/kg AOM). Aberrant crypt foci (ACF) were isolated and proliferation, apoptosis and expression of the cell cycle genes bax and p21 were determined. RV significantly reduced the number of ACF/colon [25.7 +/- 3.6 (mean +/- SEM) versus 39.4 +/- 3.3 in controls; P < 0.01] and their multiplicity (2.7 +/- 0.3 versus 4.9 +/- 0.6 in controls; P < 0.01), and also abolished large ACF. In RV-treated rats, bax expression was enhanced in ACF but not in the surrounding mucosa. In both controls and RV-treated rats, proliferation was higher in ACF than in normal mucosa. p21 was expressed in ACF of controls and of RV-treated rats and in normal mucosa of controls, but was lost in normal mucosa of RV-treated animals. In conclusion, the results suggest a protective role of RV in colon carcinogenesis with a mechanism involving changes in bax and p21 expression.     

Prevention of colonic preneoplastic lesions by the probiotic Lactobacillus acidophilus NCFMTM in F344 rats

The experiments described here were aimed at developing novel probiotic strains that may aid in the reduction of colon cancer risk. We assessed the potential anticancer properties of Lactobacillus acidophilus NCFMTM in male F344 rats using inhibition of the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy. At 6 weeks of age, groups of rats were fed the experimental diets containing 0, 2% or 4% lyophilized cultures of L. acidophilus NCFMTM. At 7 weeks of age, all animals in each dietary group, except the vehicle-treated rats, were s.c. injected with AOM (15 mg/kg body weight) once weekly for two weeks. The vehicle-treated groups were given s.c. injections of normal saline. All rats were necropsied 10 weeks after the last AOM injection and ACF in formalin-fixed, methylene blue-stained colonic tissues were counted under the light microscope. The contents of the cecum were analyzed for bacterial beta-glucuronidase activity. Diet supplementation with the probiotic strain NCFMTM significantly suppressed AOM-induction of colonic ACF, in terms of total number, as well as crypt multiplicity and number of ACF/cm2 colon (P<0.01 - 0.001). NCFMTM inhibited AOM-induced colonic ACF formation in a dose-dependent manner (P<0.01). A significant dose-dependent reduction of cecal beta-glucuronidase activities was observed in the rats fed 2% (P<0.04) and 4% (P<0.0001) NCFMTM. These results suggest that Lactobacillus acidophilus NCFMTM may potentially prevent colon cancer development. Further studies are warranted to determine the full potential of this probiotic strain in preclinical efficacy studies.     

Chemoprevention of colonic aberrant crypt foci by an inducible nitric oxide synthase-selective inhibitor

Inducible nitric oxide synthase (iNOS) is overexpressed in colonic tumors of humans and also in rats treated with a colon carcinogen. iNOS appear to regulate cyclooxygenase-2 (COX-2) expression and production of proinflammatory prostaglandins, which are known to play a key role in colon tumor development. Experiments were designed to study the inhibitory effects of S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT) a selective iNOS-specific inhibitor, measured against formation of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF). Beginning at 5 weeks of age, male F344 rats were fed experimental diets containing 0 or 50 p.p.m. of PBIT, or 2000 p.p.m. of curcumin (non-specific iNOS inhibitor). One week later, rats were injected s.c. with AOM (15 mg/kg body wt, once weekly for 2 weeks). At 17 weeks of age, all rats were killed, colons were evaluated for ACF formation and colonic mucosa was assayed for isoforms of COX and NOS activities. Both COX and iNOS activities in colonic mucosa of the AOM-treated rats were significantly induced. Importantly, 50 p.p.m. PBIT suppressed AOM-induced colonic ACF formation to 58% (P < 0.0001) and crypt multiplicity containing four or more crypts per focus to 78% (P < 0.0001); it also suppressed AOM-induced iNOS activity. Curcumin inhibited colonic ACF formation by 45% (P < 0.001). These observations suggest that iNOS may play a key regulatory role in colon carcinogenesis. Developing iNOS-specific inhibitors may provide a selective and safe chemopreventive strategy for colon cancer treatment.