Inhibitory effect of dietary monoglucosylceramide 1-O-beta-glucosyl-N-2'-hydroxyarachidoyl-4,8-sphingadienine on two different categories of colon preneoplastic lesions induced by 1,2-dimethylhydrazine in F344 rats

Sphingolipids display a wide spectrum of biological activities, including cell growth, differentiation and apoptosis. However, precise mechanisms by which these compounds exert anticancer or cancer-preventive effects are not known. In the present study, we evaluated the preventive efficacy of enriched dietary monoglucosylceramide 1-O-beta-glucosyl-N-2'-hydroxyarachidoyl-4,8-sphingadienine (G(1)CM) on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) and beta-catenin-accumulated crypt (BCAC) formation in F344 rats during initiation stage. We also examined whether G(1)CM affects cell proliferation and apoptosis in these lesions. Pure G(1)CM was isolated from rice bran. Forty-two rats were divided randomly into five experimental groups. Rats in groups 1-3 were given subcutaneous injections of DMH (40 mg/kg body weight) once a week for 2 weeks. One week before the first injection of DMH, rats in groups 2 and 3 were fed a diet containing 200 and 1,000 p.p.m. G(1)CM, respectively, for 5 weeks. Rats in group 4 were fed a diet containing 1,000 p.p.m. G(1)CM. Rats in group 5 were given the basal diet alone and served as untreated controls. The experiment was terminated 5 weeks after the start. Dietary G(1)CM at both doses (groups 2 and 3) significantly inhibited the induction of ACF and BCAC (P<0.001) when compared to group 1 treated with DMH alone. In groups 2 and 3, the proliferating cell nuclear antigen labeling indices of epithelial cells in ACF and BCAC were also lower than in group 1 (P<0.0001 for ACF, P<0.05 for BCAC). These results, that dietary G(1)CM has possible chemopreventive effects in the present short-term colon carcinogenesis bioassays, suggest that longer exposure may cause suppression of tumor development.     

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.     

Formation of Early Lesions in 1,2-dimethylhydrazine-induced Colon Carcinogenesis in Rats

Aberrant crypt foci (ACF) are recognized as preneoplastic lesions for colon cancer, and ACF in rodents arewidely used as an intermediate biomarker to predict tumorigenicity in the colon. However, a lack of correlationsbetween the formation of ACF and the development of colonic tumors has been reported in several studies. Forexample, 2-(carboxyphenyl) retinamide (2-CPR) and genistein were reported to inhibit the carcinogen-inducedformation of ACF, whereas both of them were later found to enhance colon tumorigenesis in rats treated withazoxymethane (AOM). Recently, we have identified β-catenin-accumulated crypts (BCAC) in the colon of ratsshortly after administration of AOM, and provided evidence that these are independent early lesions of classicalACF, and BCAC might be direct precursors for colon cancers. In the present study, we performed a comparativeanalysis of the modifying effects of 2-CPR and genistein on 1,2-dimethylhydrazine (DMH)-induced BCAC andACF in male F344 rats. Dietary administration of 2-CPR (315 ppm) significantly reduced the total number,multiplicity and size of ACF in DMH-exposed colonic mucosa, while genistein (250 ppm) had no significant effectson DMH-induced ACF formation. In contrast, both of 2-CPR and genistein significantly enhanced the multiplicityand size of DMH-induced BCAC when compared with DMH alone group. In addition, both 2-CPR and genisteinsignificantly increased the proliferating cell nuclear antigen (PCNA) index preferentially in BCAC. Togetherwith previous findings that 2-CPR and genistein are tumor promoters in the colon, our results support the conceptthat BCAC are precursors of colon tumors and suggest that these lesions are more reliable short-term biomarkersfor colon carcinogenesis in rodents than ACF.     

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.     

Histological and immunohistochemical observations of mucin-depleted foci (MDF) stained with Alcian blue, in rat colon carcinogenesis induced with 1,2-dimethylhydrazine dihydrochloride

The usefulness of mucin-depleted foci (MDF), which have recently been proposed as a new preneoplastic biomarker in rat colon carcinogenesis, was histologically investigated in rat colonic tissues treated with 1,2-dimethylhydrazine dihydrochloride (DMH). The relationship among aberrant crypt foci (ACF), MDF and beta-catenin accumulated crypts (BCAC) was examined by comparing the corresponding computer-captured images. Twelve male F344 rats were given DMH s.c. at a dose of 40 mg/kg body weight, once a week for 2 weeks, and randomly divided into two groups. Rats in group 1 were given normal drinking water, while those in group 2 were given drinking water containing indomethacin (IND) at 16 ppm for 6 weeks. All animals were sacrificed 8 weeks after the first DMH treatment. The resected colons were fixed in 10% formalin, and stained with Alcian blue for observation of ACF and MDF. Histological and immunohistochemical analysis revealed that the numbers of ACF, MDF and overlapping lesions in group 2 (treated with IND) were significantly decreased, compared with those in group 1. The number of BCAC in group 2 was also significantly lower than that in group 1. The reduction (61.5%) of MDF by IND was much greater than that (29.3%) of ACF. Analyses of the computer-captured images indicated that MDF had more frequent dysplastic changes and overexpression of beta-catenin than did ACF. MDF having over 4 crypts or MDF with the appearance of ACF corresponded well to BCAC. These results suggest that MDF may be useful as an early biomarker in colon carcinogenesis.     

Induction of Apoptosis by Sulindac in Azoxymethane-induced Possible Colonic Premalignant Lesions in Rats

We have reported that β-catenin-accumulated crypts (BCAC), which do not have the appearance of aberrant crypt foci (ACF) are possible colonic premalignant lesions in rats. Suppression of the occurrence and advancement of such lesions should have critical relevance to cancer prevention. This study examined whether sulindac, a chemopreventive nonsteroidal anti-inflammatory drug is able to induce apoptosis in such premalignant lesions. At 6 weeks of age, rats groups 1–3 were given azoxymethane (AOM) (15 mg/kg-body weight) once weekly for 3 weeks. Two groups were given sulindac in the diet (200 and 400 ppm), starting at 9 weeks of age. The rats were sacrificed at the termination, and the colons were carefully examined. The incidence and crypt multiplicity of BCAC and ACF were significantly less than those of the control group. The effect of sulindac on the expression of BCAC was greater than that on ACF. Exposure to sulindac significantly increased the apoptotic index (terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL)-positive cells) in BCAC. However, no significant increase of the index was found in the case of ACF. These results suggest that the chemopreventive effect of sulindac in rats is related to the induction of apoptosis in premalignant lesions. Our results also provide additional evidence that BCAC are premalignant lesions in colon carcinogenesis in rodents.     

Induction of apoptosis by sulindac in azoxymethane-induced possible colonic premalignant lesions in rats.

We have reported that beta-catenin-accumulated crypts (BCAC), which do not have the appearance of aberrant crypt foci (ACF) are possible colonic premalignant lesions in rats. Suppression of the occurrence and advancement of such lesions should have critical relevance to cancer prevention. This study examined whether sulindac, a chemopreventive nonsteroidal anti-inflammatory drug is able to induce apoptosis in such premalignant lesions. At 6 weeks of age, rats groups 1 - 3 were given azoxymethane (AOM) (15 mg/kg-body weight) once weekly for 3 weeks. Two groups were given sulindac in the diet (200 and 400 ppm), starting at 9 weeks of age. The rats were sacrificed at the termination, and the colons were carefully examined. The incidence and crypt multiplicity of BCAC and ACF were significantly less than those of the control group. The effect of sulindac on the expression of BCAC was greater than that on ACF. Exposure to sulindac significantly increased the apoptotic index (terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL)-positive cells) in BCAC. However, no significant increase of the index was found in the case of ACF. These results suggest that the chemopreventive effect of sulindac in rats is related to the induction of apoptosis in premalignant lesions. Our results also provide additional evidence that BCAC are premalignant lesions in colon carcinogenesis in rodents.     

Modifying effect of dietary sesaminol glucosides on the formation of azoxymethane-induced premalignant lesions of rat colon

Sesame, which has been reported to have preventive effects against various disordered conditions, contains small quantities of lignans and several precursors to them such as sesaminol glucosides (SG). The lignans have the potent antioxidative activity and are suggested to have chemopreventive property. In the present study, we evaluated the modulating effect of SG on the development of colon precancerous lesions, aberrant crypt foci (ACF) and beta-catenin-accumulated crypts (BCAC), in the azoxymethane (AOM)-induced short-term model using male F344 rats. Dietary SG (500 ppm) significantly decreased the incidence of AOM-induced ACF when compared to the control (P<0.01). The incidences of AOM-induced BCAC in the SG-treated groups (250 or 500 ppm) were also significantly lower than that of the control group (P<0.01). Interestingly, administration of 500 ppm SG clearly decreased serum triglyceride level and mRNA expression of intestinal fatty acid-binding protein in the colonic mucosa, as compared to the control. These findings indicate that dietary SG inhibits AOM-induced carcinogenesis and suggest SG as a possible chemopreventive agent.     

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 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 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 prevention of azoxymethane-induced colon carcinogenesis with rice-germ in F344 rats.

The modifying effect of dietary administration of defatted rice-germ and gamma-aminobutyric acid (GABA)-enriched defatted rice-germ on azoxymethane (AOM)-induced colon carcinogenesis was investigated in two experiments with male F344 rats. In the first experiment (the pilot study), the effects of the defatted rice-germ, the GABA-enriched defatted rice-germ and rice-germ on AOM-induced (15 mg/kg body wt once a week for 3 weeks) formation of aberrant crypt foci (ACF) were examined. The latter two preparations (2.5% in the diet) significantly inhibited ACF formation (P < 0.005). In the second experiment, a long-term study of the effects of rice-germ was done. One group was treated with AOM alone, four groups received the carcinogen and were fed the diets containing 2.5% rice-germ or 2.5% GABA-enriched defatted rice-germ for 5 (initiation phase) or 30 weeks (post-initiation phase), two groups were treated with rice-germ or GABA-enriched defatted rice-germ alone and one group was kept on the basal diet. At the termination of the study, dietary exposure to rice-germ during the initiation phase significantly reduced the incidence of colonic adenocarcinoma (71 versus 29%, P < 0.01). GABA-enriched defatted rice-germ or rice-germ during the post-initiation phase also decreased the frequency of colonic adenocarcinoma (71 versus 20%, GABA-enriched defatted rice-germ feeding, P < 0.01; 27%, rice-germ feeding, P < 0.01). These data suggest that constituents of rice-germ are possible dietary preventatives for human colon cancers.     

Preventive Effects of a Flavonoid Myricitrin on the Formation of Azoxymethane-induced Premalignant Lesions in Colons of Rats

The preventive effect of dietary exposure to a flavonoid myricitrin of azoxymethane (AOM)-induced aberrantcrypt foci (ACF) and beta-catenin-accumulated crypts (BCAC) formation was investigated in male F344 rats.Thirty-four rats were divided randomly into five experimental groups. Rats in groups 1-3 were given subcutaneousinjections of AOM (15 mg/kg body weight) once a week for 3 weeks. Starting 1 week before the first injection ofAOM, rats in groups 2 and 3 were fed a diet containing 500 or 1000 ppm myricitrin, respectively, for 11 weeks.Rats in group 4 were fed a diet containing 1000 ppm myricitrin. Rats in groups 1 and 5 were given the basal dietalone during the study. The experiment was terminated 11 weeks after the start. The frequency of ACF per colonin group 3 treated with AOM and 1000 ppm myricitrin was significantly lower than that in group 1 treated withAOM alone (p<0.01). Furthermore, dietary myricitrin at both doses (groups 2 and 3) significantly inhibited theformation of BCAC when compared to group 1 (p<0.05). These results indicate that myricitrin had possiblechemopreventive effects in the present short-term colon carcinogenesis bioassays and suggest that longer exposuremay cause suppression of tumor development.     

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.     

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.     

JTE-522, a selective cyclooxygenase-2 inhibitor, inhibits induction but not growth and invasion of 1,2-dimethylhydrazine-induced tubular adenocarcinomas of colon in rats

We have previously demonstrated that JTE-522, a selective cyclooxygenase-2 (COX-2) inhibitor, inhibited development of aberrant crypt foci (ACF) in rats, a putative preneoplastic lesion in colon, and suggested its inhibitory potential in rat colon carcinogenesis. To evaluate the chemopreventive properties of JTE-522, the present study was design to evaluate the inhibitory effects of JTE-522 on rat colon tumorigenesis induced by 1,2-dimethylhydrazine (DMH). Rats at 6 weeks of age were divided into 4 groups. One week after the start of the experiment, all rats received DMH by s.c. injection at a dose of 40 mg/kg body weight once a week for 4 successive weeks. As the initiation and postinitiation treatment groups, groups 1-3 were fed diets containing 0, 50, or 150 ppm JTE-522, respectively, from the start of the study to the end. As the postinitiation treatment group, group 4 was given 150 ppm JTE-522 from 1 week after the last DMH injection to the end of the study. Forty weeks after the start of the experiment, administration of 150 ppm JTE-522 during both initiation and postinitiation stages significantly inhibited the incidences of tubular adenocarcinomas and total carcinomas, as well as total tumors in the colon. The inhibitory effect of JTE-522 was most prominent for tubular adenocarcinomas, but was not observed in the nontubular carcinomas (signet-ring cell and mucinous carcinomas). Almost equal inhibitory effects on tubular adenocarcinomas were also observed in the rats given 150 ppm JTE-522 during the postinitiation stage, suggesting that its major anticancer action is at the postinitiation phase. However, JTE-522 had no effect on the size or invasive extent of tubular adenocarcinomas. Furthermore, microarray analyses revealed that JTE-522 had no effect on gene expression levels in DMH-induced tubular adenocarcinomas. These findings suggest that JTE-522 possesses chemopreventive activity against induction but not progression of tubular adenocarcinomas in rat colon. In view of the significant inhibitory effects of JTE-522 on ACF, its major anticancer action may occur in the postinitiation stage but before the malignant conversion stage of DMH-induced colon carcinogenesis.     

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.     

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.     

Dietary n-3 PUFA increases the apoptotic response to 1,2-dimethylhydrazine, reduces mitosis and suppresses the induction of carcinogenesis in the rat colon

The effect of dietary fish oil on colonic crypt cell apoptosis and proliferation was examined in male Wistar rats, 24 and 48 h after administration of 1,2-dimethylhydrazine (DMH), and its influence on the induction of aberrant crypt foci (ACF) in the distal colon was assessed. Rats (125-150 g) fed a high-fat semi-synthetic diet containing corn oil (CO) were given DMH (30 mg/kg body wt) or a sham injection of EDTA/NaCl. Animals were then fed either the CO diet or a diet in which fish oil (EPA 18.7%; DHA 8%) was substituted for corn oil. Subgroups of rats (n = 5) were killed after 24 and 48 h, and crypt cell apoptosis and proliferation were quantified by morphological criteria in isolated intact crypts from the mid and distal colon. Consumption of the fish oil diet (FO) was associated with increased apoptotic cell death (P < 0.001) and suppression of proliferation (P < 0.05) in colonic crypts both 24 and 48 h after DMH. In a second experiment, animals were given three injections of DMH or sham injections of carrier at weekly intervals. For 48 h after each injection animals were fed either the CO or FO diet, but otherwise maintained on the CO throughout. The number and crypt multiplicity of ACF in the distal colon were determined after 18 weeks, and animals given the FO diet for the 48 h period following carcinogen administration were found to have significantly fewer ACF than rats fed the CO diet (P < 0.05). The data demonstrate that the fatty acid composition of the diet is an important determinant in the induction of carcinogenesis by DMH. The proliferative and apoptotic response of the colonic crypt to carcinogen and fish oil, coupled with the reduced incidence of ACF, suggest n-3 PUFA can protect against the carcinogenic effects of DMH by mediating changes in the balance proliferation and cell death.     

Chemopreventive effect of 24R,25-dihydroxyvitamin D(3) in N, N'-dimethylhydrazine-induced rat colon carcinogenesis

In this study we investigated the effects of 24R,25-dihydroxyvitamin D(3) [24R,25(OH)(2)D(3)] on N,N'-dimethylhydrazine (DMH)-induced rat colon carcinogenesis. For experiments 1 and 2, 50 F344 male, 6-week-old rats were divided into five groups in each experiment. Animals were given s.c. injections of DMH once a week for 4 weeks. Those in groups 1-5 were given 24R,25(OH)(2)D(3) in the diet (10, 5, 2.5, 1.25 or 0 p.p.m., respectively) during the post-initiation stage in experiment 1 and during the initiation stage in experiment 2. At termination, the numbers of aberrant crypt foci (ACF) in the rat colonic mucosa were decreased dose-dependently in rats treated with 24R,25(OH)(2)D(3) during the post-initiation stage, but not in the initiation stage. For experiment 3, 15 male, 9-week-old rats were divided into three groups and given 24R,25(OH)(2)D(3) in the diet (10, 5 or 0 p.p.m.). Animals were injected with 5-bromo-2'-deoxyuridine (BrdU) i.p. 1 h before death to examine DNA synthesis in the colon mucosa. BrdU labeling indices were decreased dose-dependently in colonic crypts of rats treated with 24R, 25(OH)(2)D(3). In experiment 4, using the multicarcinogenic protocol we could analyze our data with respect to not only one separate organ, but at the organism level. Sixty-eight male, 6-week-old rats were treated with DMH, N-methylnitrosourea, 2, 2'-dihydroxy-di-n-propylnitrosamine, diethylnitrosamine and N-butyl-N-(4-hydroxybutyl)nitrosamine in weeks 1-4 and were then given 24R,25(OH)(2)D(3) in the diet (5, 1 or 0 p.p.m.) throughout weeks 5-30. Examination of the development of tumors and preneoplastic lesions in various organs revealed that 24R, 25(OH)(2)D(3) inhibited colonic tumor development significantly but exerted no effects on tumor induction in other organs. In conclusion, these results strongly indicate that 24R,25(OH)(2)D(3) inhibits colon carcinogenesis specifically, without any enhancement of carcinogenesis in other organs, when administered in the post-initiation phase.