Inhibition of azoxymethane-induced colon carcinogenesis in male F344 rats by the citrus limonoids obacunone and limonin

The modifying effects of dietary administration of the citrus limonoids obacunone and limonin on azoxymethane (AOM)-induced colon tumorigenesis were investigated in two experiments in male F344 rats. In a pilot study, we examined the modifying effects of obacunone and limonin on AOM-induced (20 mg/kg body wt, once a week for 2 weeks) formation of aberrant crypt foci (ACF). Dietary feeding of both compounds at dose levels of 200 and 500 p.p.m. during AOM exposure for 4 weeks ('initiation' feeding) or after AOM treatment for 4 weeks ('post-initiation' feeding) significantly inhibited ACF formation (55-65% reduction by 'initiation' feeding, P < 0.001; 28-42% reduction by 'post-initiation' feeding, P < 0.05-0.002). In a long-term study designed to confirm the protective effects of obacunone and limonin on ACF development, one group was treated with AOM alone and another four groups received the carcinogen treatment plus diets containing 500 p.p.m. test compounds for 3 weeks (initiation phase) or 29 weeks (post-initiation phase). Two groups were treated with obacunone or limonin alone (500 p.p.m. in diet) and one group was maintained on the basal diet. At the termination of the study, dietary exposure to obacunone or limonin during the initiation phase was found to have significantly reduced the incidence of colonic adenocarcinoma (72 versus 25 or 6%, P = 0.004 or 0.00003). Obacunone or limonin feeding during the post-initiation phase also reduced the frequency of colonic adenocarcinoma (72 versus 13%, P = 0.0002). Our results suggest that the citrus limonoids obacunone and limonin might be useful for the prevention of human colon cancers.     

Chemoprevention of azoxymethane-induced rat colon carcinogenesis by dietary capsaicin and rotenone

The modifying effects of dietary administration of capsaicin, which is the principal pungent capsicum fruit, and rotenone, which is a naturally occurring pesticide derived from Derris and Lonchorcarpus species, on azoxymethane (AOM)-induced colon tumorigenesis were investigated in male F344 rats. Gavage with capsaicin and rotenone significantly elevated phase II enzymes, glutathione S-transferase (GST) and quinone reductase (QR), in the liver and colon. In an aberrant crypt foci (ACF) bioassay, feeding of capsaicin and rotenone at a dose of 500 ppm for 4 weeks significantly inhibited ACF formation induced by AOM (20 mg/kg body weight, once a week for 2 weeks). In a subsequent long-term study designed to confirm the protective effects of both compounds on ACF development, one group was treated with AOM alone and four other groups received the carcinogen treatment plus diets containing 500 ppm test compounds for 4 weeks (initiation phase) and for 34 weeks (post-initiation phase). Two groups were treated with capsaicin or rotenone alone (500 ppm in diet) and one group was maintained on the basal diet. At the termination of the study, dietary exposure of capsaicin during the initiation phase was found to significantly reduce the incidence of colonic adenocarcinoma (60% vs. 24%, 60% reduction, P=0.0407). Rotenone feeding during the post-initiation phase also reduced the frequency of colonic adenocarcinoma (60% vs. 19%, 68% reduction, P=0.0226). Our results suggest that two natural compounds, capsaicin and rotenone, might be useful for the prevention of human colon cancers.     

Chemoprevention of Azoxymethane-induced Rat Colon Carcinogenesis by a Xanthine Oxidase Inhibitor, 1'-Acetoxychavicol Acetate

In our studies to find natural compounds with chemopreventive efficacy in foods, using azoxymethane (AOM)-induced colonic aberrant crypt foci and colonic mucosal cell proliferation as biomarkers, a x an thine oxidase inhibitor, 1′-acetoxychavicol acetate (ACA), present in the edible plant Languas galanga from Thailand was found to be effective. This study was conducted to test the ability of ACA to inhibit AOM-induced colon tumorigenesis when it was fed to rats during the initiation or post-initiation phase. Male F344 rats were given three weekly s.c. injections of AOM (15mg/kg body weight) to induce colonic neoplasms. They were fed diet containing 100 or 500 ppm ACA for 4 weeks, starting one week before the first dosing of AOM (the initiation feeding). The other groups were fed the ACA diet for 34 weeks, starting one week after the last AOM injection (the post-initiation feeding). At the termination of the study (week 38), AOM had induced 71% incidence of colonic adenocarcinoma (12/17 rats). The initiation feeding with ACA caused significant reduction in the incidence of colon carcinoma (54% inhibition by 100 ppm ACA feeding and 77% inhibition by 500 ppm ACA feeding, P=0.03 and P=0.001, respectively). The post-initiation feeding with ACA also suppressed the incidence of colonic carcinoma (45% inhibition by 100 ppm ACA feeding and 93% inhibition by 500 ppm ACA feeding, P=0.06 and P=0.00003, respectively). Such inhibition was dose-dependent and was associated with suppression of proliferation biomarkers, such as ornithine decarboxylase activity in the colonic mucosa, and blood and colonic mucosal polyamine contents. ACA also elevated the activities of phase II enzymes, glutathione S-transferase (GST) and quinone reductase (QR), in the liver and colon. These results indicate that ACA could inhibit the development of AOM-induced colon tumorigenesis through its suppression of cell proliferation in the colonic mucosa and its induction of GST and QR. The results confirm our previous finding that ACA feeding effectively suppressed the development of colonic aberrant crypt foci. These findings suggest possible chemopreventive ability of ACA against colon tumorigenesis.     

Azoxymethane-induced beta-catenin-accumulated crypts in colonic mucosa of rodents as an intermediate biomarker for colon carcinogenesis

It is now well established that bile acids act as colon tumor promoters. However, a previous study provided conflicting data showing that dietary exposure of cholic acid (CHA), a primary bile acid, inhibits the carcinogen-induced formation of aberrant crypt foci (ACF), possible preneoplastic lesions, in colonic mucosa of rodents. Recently we found beta-catenin-accumulated crypts (BCAC) in colonic mucosa of rats initiated with azoxymethane (AOM) and provided evidence that BCAC might be preneoplastic lesions independent from ACF. In the present study, we investigated the modifying effects of dietary CHA on the formation of BCAC as well as ACF in male F344 rats after exposure to AOM to determine if the differences in the effect of CHA on these lesions could account for this discrepancy. The results indicate that administration of CHA (0.5%) in the diet during the post-initiation phase significantly reduced the total number, multiplicity and size of ACF (P < 0.00001) in AOM-exposed colonic mucosa as reported previously. The number of ACF even with >4 aberrant crypts/focus was also decreased significantly (P < 0.0002), suggesting that the large ACF are little resistant to continuous feeding of 0.5% CHA diet. Interestingly, the dietary CHA significantly enhanced both the multiplicity (P < 0.002) and size (P < 0.00001), but not the incidence, of AOM-induced BCAC when compared with the control diet group. Importantly, the number of large BCAC with >6 crypts/lesion was increased significantly by the dietary CHA (P < 0.003). Our results support the concept that BCAC are precursors of colon tumors and indicate the usefulness of BCAC as intermediate biomarkers for colon carcinogenesis, although the methodology for their detection requires further improvement.     

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.     

Chemoprevention of azoxymethane-induced rat colon carcinogenesis by the naturally occurring flavonoids, diosmin and hesperidin

The modulating effects of dietary feeding of two flavonoids, diosmin and hesperidin, both alone and in combination, during the initiation and post-initiation phases on colon carcinogenesis initiated with azoxymethane (AOM), were investigated in male F344 rats. Animals were initiated with AOM by weekly s.c. injections of 15 mg/kg body wt for 3 weeks to induced colon neoplasms. Rats were fed the diets containing diosmin (1000 ppm), hesperidin (1000 ppm) or diosmin (900 ppm) + hesperidin (100 ppm) for 5 weeks (initiation treatment) or 28 weeks (post-initiation treatment). The others contained the groups of rats treated with diosmin, hesperidin alone or in combination, and untreated. At the end of the study (32 weeks), the incidence and multiplicity of neoplasms (adenoma and adenocarcinoma) in the large intestine of rats initiated with AOM together with, or followed by, a diet containing diosmin or hesperidin were significantly smaller than those of rats given AOM alone (P <0.001). The combination regimen during the initiation and post-initiation stages also inhibited the development of colonic neoplasms, but the tumor data did not indicate any beneficial effect of diosmin and hesperidin administered together as compared with when these agents were given individually. In addition, feeding of diosmin and hesperidin, both alone and in combination, significantly inhibited the development of aberrant crypt foci. As for cell proliferation biomarkers, dietary exposure of diosmin and hesperidin significantly decreased the 5'-bromodeoxyuridine- labeling index and argyrophilic nuclear organizer region's number in crypt cells, colonic mucosal ornithine decarboxylase activity, and polyamine levels in the blood. These results indicate that diosmin and hesperidin, both alone and in combination, act as a chemopreventive agent against colon carcinogenesis, and such effects may be partly due to suppression of cell proliferation in the colonic crypts, although precise mechanisms should be clarified.      

Dietary Conjugated Linolenic Acid Inhibits Azoxymethane-induced Colonic Aberrant Crypt Foci in Rats

The modifying effects of dietary feeding of conjugated linolenic acid (CLN) isolated from the seeds of bitter gourd ( Momordica charantia ) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats to predict its possible cancer chemopreventive efficacy. The effect of CLN on the proliferating cell nuclear antigen (PCNA) index in colonic ACF was also examined. Rats were given subcutaneous injections of AOM (20 mg/ kg body weight) once a week for 2 weeks to induce ACF. They also received the experimental diet containing 0.01%, 0.1% or 1% CLN for 5 weeks, starting one week before the first dosing of AOM. AOM exposure produced a substantial number of ACF (108±21/rat) at the end of the study (week 4). Dietary administration of CLN caused a significant reduction in the frequency of ACF: 87±14 (19.4% reduction, P <0.05) at a dose of 0.01%, 69±28 (36.1% reduction, P <0.01) at a dose of 0.1% and 40±d6 (63.0% reduction, P <0.001) at a dose of 1%. Also, CLN administration lowered the PCNA index and induced apoptosis in ACF. These findings might suggest possible chemopreventive activity of CLN in the early phase of colon tumorigenesis through modulation of cryptal cell proliferation activity and/or apoptosis.     

Silymarin,a naturally occurring polyphenolic antioxidant flavonoid,inhibits azoxymethane-induced colon carcinogenesis in male F344 rats

The modifying effect of dietary administration of the polyphenolic antioxidant flavonoid silymarin, isolated from milk thistle [Silybum marianum (L.) Gaertneri], on AOM-induced colon carcinogenesis was investigated in male F344 rats. In the short-term study, the effects of silymarin on the development of AOM-induced colonic ACF, being putative precursor lesions for colonic adenocarcinoma, were assayed to predict the modifying effects of dietary silymarin on colon tumorigenesis. Also, the activity of detoxifying enzymes (GST and QR) in liver and colonic mucosa was determined in rats gavaged with silymarin. Subsequently, the possible inhibitory effects of dietary feeding of silymarin on AOM-induced colon carcinogenesis were evaluated using a long-term animal experiment. In the short-term study, dietary administration of silymarin (100, 500 and 1,000 ppm in diet), either during or after carcinogen exposure, for 4 weeks caused significant reduction in the frequency of colonic ACF in a dose-dependent manner. Silymarin given by gavage elevated the activity of detoxifying enzymes in both organs. In the long-term experiment, dietary feeding of silymarin (100 and 500 ppm) during the initiation or postinitiation phase of AOM-induced colon carcinogenesis reduced the incidence and multiplicity of colonic adenocarcinoma. The inhibition by feeding with 500 ppm silymarin was significant (p < 0.05 by initiation feeding and p < 0.01 by postinitiation feeding). Also, silymarin administration in the diet lowered the PCNA labeling index and increased the number of apoptotic cells in adenocarcinoma. beta-Glucuronidase activity, PGE(2) level and polyamine content were decreased in colonic mucosa. These results clearly indicate a chemopreventive ability of dietary silymarin against chemically induced colon tumorigenesis and will provide a scientific basis for progression to clinical trials of the chemoprevention of human colon cancer.     

Dietary conjugated linolenic acid inhibits azoxymethane-induced colonic aberrant crypt foci in rats.

The modifying effects of dietary feeding of conjugated linolenic acid (CLN) isolated from the seeds of bitter gourd (Momordica charantia) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats to predict its possible cancer chemopreventive efficacy. The effect of CLN on the proliferating cell nuclear antigen (PCNA) index in colonic ACF was also examined. Rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for 2 weeks to induce ACF. They also received the experimental diet containing 0.01%, 0.1% or 1% CLN for 5 weeks, starting one week before the first dosing of AOM. AOM exposure produced a substantial number of ACF (108 +/- 21/rat) at the end of the study (week 4). Dietary administration of CLN caused a significant reduction in the frequency of ACF: 87 +/- 14 (19.4% reduction, P < 0.05) at a dose of 0.01%, 69 +/- 28 (36.1% reduction, P < 0.01) at a dose of 0.1% and 40 +/- 6 (63.0% reduction, P < 0.001) at a dose of 1%. Also, CLN administration lowered the PCNA index and induced apoptosis in ACF. These findings might suggest possible chemopreventive activity of CLN in the early phase of colon tumorigenesis through modulation of cryptal cell proliferation activity and/or apoptosis.     

Pomegranate seed oil rich in conjugated linolenic acid suppresses chemically induced colon carcinogenesis in rats

Pomegranate (Punica granatum L.) seed oil (PGO) contains more than 70% cis(c)9,trans(t)11,c13-18:3 as conjugated linolenic acids (CLN). Our previous short-term experiment demonstrated that seed oil from bitter melon (Momordica charantia) (BMO), which is rich in c9,t11,t13-CLN, inhibited the occurrence of colonic aberrant crypt foci (ACF) induced by azoxymethane (AOM). In this study, we investigated the effect of dietary PGO on the development of AOM-induced colonic malignancies and compared it with that of conjugated linoleic acid (CLA). To induce colonic tumors, 6-week old male F344 rats were given subcutaneous injections of AOM (20 mg/kg body weight) once a week for 2 weeks. One week before the AOM treatment they were started on diet containing 0.01%, 0.1%, or 1% PGO or 1% CLA for 32 weeks. Upon termination of the bioassay (32 weeks) colon tumors were evaluated histopathologically. AOM exposure produced colonic adenocarcinoma with an incidence of 81% and multiplicity of 1.88 +/- 1.54 at week 32. Administration of PGO in the diet significantly inhibited the incidence (AOM + 0.01% PGO, 44%, P < 0.05; AOM + 0.1% PGO, 38%, P < 0.01; AOM + 1% PGO, 56%) and the multiplicity (AOM + 0.01% PGO, 0.56 +/- 0.73, P < 0.01; AOM + 0.1% PGO, 0.50 +/- 0.73, P < 0.005; AOM + 1% PGO, 0.88 +/- 0.96, P < 0.05) of colonic adenocarcinomas, although a clear dose-response relationship was not observed at these dose levels. CLA feeding also slightly, but not significantly, reduced the incidence and multiplicity of colonic adenocarcinomas. The inhibition of colonic tumors by PGO was associated with an increased content of CLA (c9,t11-18:2) in the lipid fraction of colonic mucosa and liver. Also, administration of PGO in the diet elevated expression of peroxisome proliferator-activated receptor (PPAR) gamma protein in the non-tumor mucosa. These results suggest that PGO rich in c9,t11,c13-CLN can suppress AOM-induced colon carcinogenesis, and the inhibition is associated in part with the increased content of CLA in the colon and liver and/or increased expression of PPARgamma protein in the colon mucosa.     

The influence of simple sugars and starch given during pre- or post-initiation on aberrant crypt foci in rat colon

The aim of the present study was to investigate the enhancing effect of dietary sugar on the development of aberrant crypt foci (ACF) in male F344 rats initiated with azoxymethane (AOM). The potential role of sugar as either a co-initiator or a promoter was investigated by giving diets high in sucrose and dextrin (61%) during either the pre-initiation, the initiation, and/or the post-initiation stage of the ACF development. The colonic cell proliferation, activity of colonic phase II enzymes, and a biomarker of lipid peroxidation were additionally examined in order to obtain information on the specific mechanisms involved in the suggested effect of sucrose and dextrin on ACF development. The number of large sized and the total number of ACF were significantly increased by feeding sucrose and dextrin in the post-initiation period. No positive association between colonic cell proliferation and ACF was seen. The level of oxidative stress in the cytosol from the proximal colon and colonic glutathione transferase and quinone reductase was not affected by the sugar treatments. The overall results from this study show that sucrose and dextrin enhance the number of preneoplastic lesions in AOM-initiated rats, and act primarily as promoters in the development of ACF.     

Deoxycholic acid promotes the growth of colonic aberrant crypt foci

AKR/J mice are resistant to the tumorigenic properties of the colon carcinogen, azoxymethane (AOM). Following AOM exposure, limited numbers of preneoplastic lesions, referred to as aberrant crypt foci (ACF), are formed in the colon, and their progression to tumors rarely occurs. To determine whether genetic resistance can be overcome by exposure to a dietary tumor promoter, AOM-exposed AKR/J mice were fed a diet containing 0.25% deoxycholic acid (DCA). DCA exposure was begun 1 wk prior to or 1 wk after tumor initiation with AOM. Mice placed on the DCA diet prior to AOM treatment developed a significantly higher multiplicity of ACF compared to AOM-exposed mice fed a control diet (15.50 +/- 0.96 vs. 6.17 +/- 0.48, respectively; P < 0.05). When DCA exposure was begun after AOM treatment (post-initiation), ACF formation was further enhanced (34.00 +/- 1.22). Interestingly, increased numbers of ACF were associated with the presence of nuclear beta-catenin, assessed by immunohistochemistry. While approximately 33% of ACF from mice exposed to DCA prior to AOM treatment contained positive nuclear beta-catenin staining, approximately 77% of ACF from mice fed DCA after AOM were positive. Accumulation of nuclear beta-catenin was not associated with a loss of E-cadherin from the plasma membrane, although loss of APC staining was a consistent feature of most AOM-induced ACF, regardless of DCA exposure. These results demonstrate that exposure to DCA, an important digestive component, is sufficient to sensitize the resistant AKR/J colon to formation of high-grade dysplasia, and that nuclear translocation of beta-catenin may play an important role in this process.     

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.     

A xanthine oxidase inhibitor 1'-acetoxychavicol acetate inhibits azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effect of dietary administration of a xanthine oxidase inhibitor 1'-acetoxychavicol acetate (ACA) present in an edible plant Languas galanga in Thailand on the development of azoxymethane (AOM)- induced colonic aberrant crypt foci (ACF) was investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce colonic ACF. They were fed the diets containing 100 or 200 ppm ACA for 5 weeks, starting 1 week before the first dosing of AOM. At the termination of the study (week 5), AOM induced 118 +/- 28 ACF/colon. Dietary administration of ACA caused significant reduction in the frequency of ACF (41% inhibition by 100 ppm ACA feeding and 37% inhibition by 200 ppm ACA feeding, P<0.01). Such inhibition might be associated with suppression of the proliferation biomarkers' expression such as ornithine decarboxylase activity in the colonic mucosa, number of silver-stained nucleolar organizer regions' protein in the colonic mucosal cell nuclei and blood polyamine content. These results indicate that ACA could inhibit the development of AOM-induced ACF through its suppression of cell proliferation in the colonic mucosa and ACA might be a possible chemopreventive agent against colon tumourigenesis.      

Chemoprevention of 1,2-dimethylhydrazine-induced colonic preneoplastic lesions in Fischer rats by 6-methylsulfinylhexyl isothiocyanate, a wasabi derivative

The preventive effects of dietary exposure to a wasabi derivative 6-methylsulfinylhexyl isothiocyanate (6-MSITC) during the initiation and post-initiation phases on the development of 1,2-dimethylhydrazine (DMH)-induced colonic aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCAC) were investigated in male F344 rats. To induce ACF and BCAC, rats were given four weekly subcutaneous injections of DMH (40 mg/kg body weight). The rats also received diets containing 200 or 400 ppm 6-MSITC during the initiation or post-initiation phases. The experiment was terminated 12 weeks after the start. DMH exposure produced a substantial number of ACF (323.8±69.7/colon) and BCAC (3.80±1.05/cm(2)) at the end of the study. Dietary administration of 6-MSITC at a dose of 400 ppm during the initiation phase caused a significant reduction in the total number of ACF (52% reduction, P<0.0001), larger ACF (4 or more crypt ACF) (58% reduction, P<0.001) and BCAC (76% reduction, P<0.00001). The dietary exposure to 6-MSITC significantly reduced the size (crypt multiplicity) of BCAC during both initiation and post-initiation treatment when compared to group 1 treated with DMH alone. Immunohistochemically, 6-MSITC administration lowered the proliferating cell nuclear antigen labeling index in ACF and BCAC. In addition, protein levels of hepatic cytochrome P-450 isozymes at 24 h after 6-MSITC exposure were significantly suppressed (P<0.01). The results indicated that 6-MSITC exerted chemopreventive effects in the present short-term colon carcinogenesis bioassay, through alterations in cell proliferation activity and drug metabolizing enzyme levels.     

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.     

Catalpa seed oil rich in 9t,11t,13c-conjugated linolenic acid suppresses the development of colonic aberrant crypt foci induced by azoxymethane in rats

Catalpa (Catalpa ovata) seed oil (CPO) is a unique oil that contains a high amount of 9trans,11trans,13cis-conjugated linolenic acid. In the present study, we investigated whether dietary administration with CPO affects the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in male F344 rats to elucidate its possible cancer chemopreventive efficiency. Also, the effect of CPO on the fatty acid composition of liver tissue and colonic mucosa, the serum levels of total cholesterol and triglyceride, and the mRNA expression of cyclooxygenase (COX)-2 in the colonic mucosa were measured. In addition, the cell proliferation activity and apoptotic index in the colonic mucosa were estimated immunohistochemically. Animals were given two weekly subcutaneous injections of AOM (20 mg/kg body weight). They also received the experimental diet containing 0.01%, 0.1% or 1% CPO for 4 weeks, starting one week before the first dosing of AOM. AOM exposure produced a substantial number of ACF (99+/-28) at the end of the study (week 4). Dietary administration of CPO reduced the number of ACF (AOM + 0.01% CPO, 32+/-11, P<0.001; AOM + 0.1% CPO, 35+/-18, P<0.001; AOM + 1% CPO, 18+/-10, P<0.001). 9t,11t-conjugated linoleic acid was detected in the liver tissue and colonic mucosa of rats fed the CPO-containing diet. Additionally, dietary administration with CPO decreased the serum triglyceride level and the expression of COX-2 mRNA in the colonic mucosa. The indices of cell proliferation and apoptosis in the colonic mucosa of rats treated with AOM and 1% CPO have significant differences when compared with the AOM alone group. These findings suggest the possible chemopreventive activity of CPO in the early phase of colon carcinogenesis.     

Prevention of colonic aberrant crypt foci and modulation of large bowel microbial activity by dietary coffee fiber, inulin and pectin

The present experiments were aimed at developing novel dietary fibers to aid in reduction of colon cancer risk. We assessed the effects of coffee (non-fiber fraction), coffee fiber (arabino-galactose polymer) and inulin (oligo-fructose) in male F344 rats using formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy (or lack of such). At 5 weeks of age, groups of rats were fed the AIN-76A (control) and experimental diets that contained 1% coffee, 10% coffee fiber, 10% inulin, 10% pectin (positive control for fiber) or 200 p.p.m. piroxicam (a known ACF inhibitor). At 7 weeks of age, all animals were s.c injected with AOM (15 mg/kg body wt) once weekly for 2 weeks. All rats were killed 8 weeks after the last AOM injection and ACF were counted. The contents of the cecum were analyzed for bacterial beta-glucuronidase activity and short-chain fatty acids (SCFAs). Dietary administration of coffee fiber 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). Inulin diet had no significant effect on total ACF, but had reduced the number of ACF/cm2 (P < 0.05). Whereas coffee had no effect on ACF formation, 10% pectin diet and 200 p.p.m. piroxicam significantly suppressed colonic ACF (P < 0.001) as had been expected. A significant reduction of cecal beta-glucuronidase activity was observed in the rats fed coffee, coffee fiber and pectin diets. Further, coffee fiber, inulin and pectin increased cecal SCFA levels 3- to 5-fold. These results suggest that coffee fiber can prevent colon cancer risk. Further studies are warranted to determine the full potential of this fiber in pre-clinical efficacy studies.      

Ursodeoxycholic acid inhibits the initiation and postinitiation phases of azoxymethane-induced colonic tumor development.

Colonic tumorigenesis involves the processes of initiation and promotion/progression from normal epithelial cells to tumors. Studies in both humans and experimental models of colon cancer indicate that secondary bile acids promote tumor development. In contrast, we have demonstrated previously that another bile acid, ursodeoxycholic acid (UDCA), inhibits the development of azoxymethane (AOM)-induced colon cancer in rats. More recently, we have shown that UDCA inhibits AOM-induced hyperproliferation, and aberrant crypt formation and growth. In our previous studies, we supplemented UDCA throughout the experiment. The efficacy of a chemopreventive agent may depend on the timing of administration, which has important clinical implications. In the present investigation, we examined the ability of UDCA, when administered only in the initiation or the promotion/progression phase, to block tumor development. Male Fisher 344 rats were divided in a 2 x 3 factorial design, with animals receiving AOM or vehicle, and fed an unsupplemented diet or diet supplemented with 0.4% UDCA in the initiation or promotion/progression phase. Thirty-two weeks later, rats were sacrificed and tumor histology determined, and colons were examined for aberrant crypt foci (ACF). In the carcinogen-treated dietary control group, tumor incidence was 72.3%, and tumor multiplicity was 1.9 tumors per tumor-bearing rat. UDCA, in the initiation or promotion/progression phase, significantly decreased tumor incidence to 46.2% and 38.4% (P < 0.05), respectively; and tumor multiplicity to 1.4 and 1.3 tumors per tumor-bearing rat (P < 0.05), respectively. UDCA did not alter tumor size, histology, or location, although there were trends for smaller tumors and less advanced histological grades in the group given UDCA during the promotion phase. UDCA, in the initiation but not the promotion phase, inhibited ACF formation and growth. In summary, UDCA significantly inhibited AOM-induced colonic carcinogenesis during either tumor initiation or in the promotion/progression phase. In contrast, UDCA inhibited ACF formation only when administered in the initiation phase, suggesting that the mechanisms of chemoprevention by this bile acid differ in these two phases.     

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.