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.      

Low doses of beta-carotene and lutein inhibit AOM-induced rat colonic ACF formation but high doses augment ACF incidence

Epidemiological studies suggest that carotenoids such as beta-carotene and lutein play an important role in reducing the risk for several cancers. However, in colon cancer there is ambiguity with regard to the role of these compounds in that both preventive effects and tumor promotion have been observed. In the present study we observed that male F344 rats were able to tolerate up to 2,500 ppm of beta-carotene as well as of lutein. We have then assessed the chemopreventive efficacy of beta-carotene and lutein at dose levels of approximately 4 and 8% of the 2,500 ppm tolerated dose (TD) and also approximately 40 and 80% of the TD on azoxymethane (AOM)-induced colon carcinogenesis, using aberrant crypt foci (ACF) as a surrogate biomarker for colon cancer. Throughout the experiments, 5-week-old male F344 rats were fed the control diet (modified AIN-76A) or experimental diets containing 100 or 200 ppm (approximately 4 or 8% of the 2,500 ppm TD), or 1,000 or 2,000 ppm ( approximately 40 or 80% of the 2,500 ppm TD) of beta-carotene and lutein (n=10 rats/group). After 2 weeks on the experimental or control diets, all animals were injected with AOM (15 mg/kg body wt., once weekly for 2 weeks). At 14 weeks of age, all rats were killed, and their colons were evaluated for ACF. Administration of 100 or 200 ppm of beta-carotene inhibited AOM-induced total colonic ACF formation by 24% (p<0.01) and 36% (p<0.001), respectively, whereas lutein at 200 ppm produced a 27% inhibition (p<0.01) yet had no significant effect at the 100 ppm dose level. Surprisingly, administration of 1,000 or 2,000 ppm of beta-carotene and lutein increased colonic ACF formation in a dose-dependent manner, i.e., to 124% and 144% for the former and 110% and 159% for the latter. These results clearly suggest that further studies are warranted to determine whether the increase in ACF incidence by high doses of beta-carotene and lutein will also lead to an increase in tumor outcome. Taken together these data indicate that the chemopreventive activity of beta-carotene and lutein against colon carcinogenesis depends on the dose level.     

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.     

Chemopreventive properties of a selective inducible nitric oxide synthase inhibitor in colon carcinogenesis, administered alone or in combination with celecoxib, a selective cyclooxygenase-2 inhibitor.

The inducible isoforms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) are overexpressed in colonic tumors of humans, as well as in colon tumors that develop in rats after the administration of the colon-specific carcinogen, azoxymethane (AOM). iNOS may regulate COX-2 production of proinflammatory prostaglandins, which are known to play a key role in colon tumor development. Experiments were designed to assess the potential chemopreventive properties of highly selective iNOS inhibitors, administered individually and in combination with a selective COX-2 inhibitor, on the development of AOM-induced colonic aberrant crypt foci (ACF). F344 rats were fed experimental diets containing one of the following: 0, 10, 30, or 100 parts/million (ppm) of the selective iNOS inhibitor L-N(6)-(1-iminoethyl)lysine tetrazole-amide (SC-51); 1800 ppm of the less potent, selective iNOS inhibitor aminoguanidine (AG); 500 ppm of the COX-2 inhibitor celecoxib; 320 ppm of the nonsteroidal anti-inflammatory sulindac (positive control); or 30 ppm of SC-51 with 500 ppm of celecoxib, and 100 ppm of SC-51 with 500 ppm of celecoxib. One and 2 weeks later, rats received s.c. injections of AOM at a dose of 15 mg/kg of body weight. At 17 weeks of age, all rats were sacrificed. Colons were evaluated for ACF, and colonic mucosae were assayed for COX and NOS isoform enzyme activities. Samples of venous blood, collected at various time points, were analyzed for these agents. SC-51, administered alone, demonstrated dose-dependent inhibition of the incidence of colonic ACF. The highest doses of SC-51 (100 ppm) and AG (1800 ppm) significantly suppressed the incidence of colonic ACF (P < 0.01 and < 0.001, respectively) and crypt multiplicity in terms of numbers of aberrant crypts/focus (P < 0.0001). Importantly, the combination of either low or high effective doses of SC-51 (30 or 100 ppm) and celecoxib (500 ppm) suppressed AOM-induced colonic ACF formation (P < 0.05 and < 0.001, respectively) and reduced multiplicity of four or more aberrant crypts/focus (P < 0.0001) to a greater extent than did these agents administered individually. As expected, sulindac inhibited colonic ACF formation (P < 0.001) and reduced the multiplicity of four or more aberrant crypts (P < 0.0001) to approximately 45%. The enzymatic activities of COX-2 and iNOS were significantly induced in the AOM-treated animals, and administration of the iNOS inhibitors, SC-51 and AG, significantly inhibited the activities of both iNOS and COX-2 in the colonic mucosa. The combined administration of SC-51 and celecoxib inhibited the COX-2 activity to a greater extent than did either of these agents administered alone. These findings support the hypothesis that selective iNOS inhibitors may have chemopreventive properties and that coadministration with a selective COX-2 inhibitor may have additional chemopreventive potential.     

Inhibition of azoxymethane-induced aberrant crypt foci in rats by natural compounds, caffeine, quercetin and morin.

The modifying effects of dietary administration of natural compounds, caffeine, quercetin and morin, which are present in our daily food, on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in rats and compared to that of a metabolic inhibitor of AOM, disulfiram. Male F344 rats were given s. c. injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce ACF. They also received the experimental diets containing one of test compounds (500 ppm) for 5 weeks, starting one week before the first dosing of AOM. At the termination of the study (week 5), AOM exposure produced 101.0+/-10.2 ACF/rat. Disulfiram almost completely inhibited ACF development (0.60+/-0.90, 99% reduction). Dietary administration of test compounds caused significant reduction in the frequency of ACF: caffeine (70.4+/-16.6, 30% reduction), quercetin (53.0+/-8.4, 48% reduction) and morin (37. 6+/-18.1, 63% reduction). Numbers of cells positive for proliferative cell nuclear antigen in ACF and surrounding crypts were lowered by feeding of test compounds. Feeding of these test compounds also suppressed polyamine content in the colonic mucosa and blood as did disulfiram. These findings might indicate possible chemopreventive effects of caffeine, quercetin and morin, through their modulation of cell proliferation activity in crypt cells, on colon tumorigenesis.     

Anti-carcinogenic properties of omeprazole against human colon cancer cells and azoxymethane-induced colonic aberrant crypt foci formation in rats

Omeprazole is a proton pump inhibitor, a widely used drug to treat ulcers and gastroesophageal refluxdisease. We have evaluated colon cancer chemopreventive properties of omeprazole using azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in male F344 rats and analyzed cell growth inhibition and apoptosis induction in human colon cancer cells. Five-week-old male F344 rats were fed a control or experimental diet containing two doses of omeprazole (200 and 400?ppm). After one week, all animals were s.c. injected with AOM (15?mg/kg body weight, once weekly for two weeks). Rats continued on experimental diets for seven more weeks before being sacrificed. Colons were histopathologically evaluated for ACF. Human colon cancer HCT-116 and HCA-7 cells treated with omeprazole were evaluated for different markers associated with proliferation and apoptotic markers using Western blot technique. Rats fed with 200 and 400?ppm of omeprazole significantly suppressed total colonic ACF formation (~30%, P<0.001) and showed significant suppression of multi-crypt foci (~30-50%, P<0.05-0.001). Omeprazole produced significant dose-response effects on inhibition of multi-crypt foci (≥4). Omeprazole treatment in human colon cancer cell lines HCT-116 and HCA-7 cells resulted in induction of p21waf1/cip1 and decreased the expression of anti-apoptotic proteins Bcl-2, Bcl-XL and survivin in a dose-dependent manner. Anticancer properties observed in colon cancer cell lines suggest that omeprazole may induce key signaling molecules of antiproliferation and inhibition of anti-apoptotic proteins.     

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.     

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.     

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.     

Achaete-scute homolog-1 and Notch in lung neuroendocrine development and cancer

The modifying effects of dietary feeding of extract of leaves of ginkgo (Ginkgo biloba) (EGb) and bilobalide isolated from EGb on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of EGb and bilobalide on proliferating cell nuclear antigen (PCNA) index in ‘normal-appearing’ crypts and activities of detoxifying enzymes of cytochrome P450 (CYP), glutathione S-transferase (GST) and quinine reductase (QR) activity in the liver. To induce ACF, rats were given two weekly subcutaneous injections of AOM (20 mg/kg body wt). They also received the experimental diets containing EGb (50 or 500 ppm) and bilobalide (15 or 150 ppm) for 4 weeks, starting 1 week before the first dosing of AOM. AOM exposure produced a substantial number of ACF (106±10) at the end of the study (week 4). Dietary administration of EGb and bilobalide caused significant reduction in the frequency of ACF: 50 ppm EGb, 73±17 (31% reduction, P<0.001); 500 ppm EGb, 56±13 (47% reduction, P<0.001); 15 ppm bilobalide, 79±17 (25% reduction, P<0.01); and 150 ppm bilobalide, 71±30 (33% reduction, P<0.01). Immunohistochemically, EGb or bilobalide administration significantly lowered PCNA index in normal-appearing crypts. Feeding with EGb or bilobalide increased activities of CYP as well as GST and QR in the liver. These findings might suggest possible chemopreventive ability of EGb or bilobalide, through alterations in cryptal cell proliferation activity and drug metabolizing enzymes' activities, in colon tumorigenesis.     

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.     

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.     

Extract of vinegar "Kurosu" from unpolished rice inhibits the development of colonic aberrant crypt foci induced by azoxymethane

The modifying effects of administrating an ethyl acetate extract of "Kurosu" (EK), a vinegar made from unpolished rice, on development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of EK on proliferating cell nuclear antigen (PCNA) index in ACF, prostaglandin (PG) E2 expression in the colonic mucosa and activities of detoxifying enzymes of glutathione S-transferase (GST) and quinone reductase (QR) in the liver. To induce ACF, rats were given two weekly subcutaneous injections of AOM (20 mg/kg body wt). They also received drinking water containing 0, 0.05, 0.1 or 0.2% EK for 4 weeks, starting 1 week before the first dosing of AOM. AOM exposure produced 140 +/- 23 ACF/rat at the end of the study (week 4). EK administration dose-dependently inhibited ACF formation and inhibition by 0.2% EK was statistically significant (P < 0.002). Treatment with EK significantly lowered PCNA index in ACF and reduced PGE2 content in the colonic mucosa, while EK elevated liver GST and QR activities. These findings suggest that EK may be effective for inhibiting colonic ACF, through induction of liver GST and QR and possibly alteration of PGE2 production.     

Modifying effect of tuna orbital oil rich in docosahexaenoic acid and vitamin D3 on azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effect of dietary tuna (Thunnus thynnus orientalis) orbital oil rich in docosahexaenoic acid (DHA) and vitamin D3 (VD3) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) was investigated in male F344 rats. Animals were given three weekly subcutaneous injections of AOM (15 mg/kg body weight) to induce ACF. The rats were fed the experimental diet containing 5% tuna orbital oil (low fish oil), 23.5% tuna orbital oil (high fish oil), 5% corn oil (low corn oil) or 23.5% corn oil (high corn oil) for 5 weeks, starting 1 week before the first dose of AOM. Animals were sacrificed 2 weeks after the last AOM injection to count colonic ACF and assay the expression of cyclooxygenase (COX)-1 and -2. High corn oil diet significantly increased the development of ACF, when compared with low corn oil diet (P<0.005). High fish oil diet also increased ACF formation compared with low fish oil diet (P<0.01), but the increase was smaller than high corn oil diet. The frequency of ACF was significantly lower in the rats fed high fish oil diet than high corn oil diet (P<0.02). Moreover, frequency of ACF consisted of 4 or more crypts in rats fed the high fish oil diet was significantly lower than that of rats given high corn oil diet. COX-1 and COX-2 expression did not significantly differ among the groups. These results suggest that fish oil derived from tuna, which contains high amounts of DHA and VD3, suppresses the formation and growth of ACF without affecting COX-1 and COX-2 expression, and may have a preventive effect on colon carcinogenesis.     

S-adenosyl L-methionine inhibits azoxymethane-induced colonic aberrant crypt foci in F344 rats and suppresses human colon cancer Caco-2 cell growth in 3D culture

S-adenosyl L-methionine (SAM) is a universal methyl group donor to various intermediary metabolites, hormones, proteins, neurotransmitters, phospholipids and nucleic acids. Deficiency of folate, which plays a role in the synthesis of SAM leads to increased risk for colon cancer. This study tested the effectiveness of SAM supplementation in protecting against azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. We also tested the effect of SAM on cyclooxygenase-2 (COX-2) in a macrophage cell line. Further, we developed a 3-D culture model using Caco-2 cells to test the effect of SAM on tumor spheroid size and number. Groups of rats were given the experimental diet containing either 0-, 400- or 800-ppm SAM, 1 week before the first AOM injection and continued until 8 weeks. In the control group, AOM produced a substantial number of aberrant crypt foci (ACF) (96 +/- 8). Dietary administration of SAM significantly reduced the number of total ACF (400 ppm SAM, 68 +/- 7.3, p < 0.01 and 800 ppm SAM, 57 +/- 7.1, p < 0.001). SAM significantly decreased AOM-induced colonic multicrypt foci in a dose-dependent manner. Suppression of Lipopolysaccharide (LPS) induced COX-2 protein expression was observed in a RAW264.7 cell line. We established growth of Caco-2 cells as spheroids, in a 3D matrix of collagen and matrigel. Treatment with SAM decreased both size and number of spheroids in a dose-dependent manner (p < 0.0001). These observations demonstrate for the first time that SAM can reduce the occurrence of ACF in AOM treated male F344 rats and suppress formation of human tumor spheroids and expression of COX-2.     

Suppressing Effects of 6-(2,5-Dichlorophenyl)-2,4-diamino-1,3,5-triazine and Related Synthetic Compounds on Azoxymethane-induced Aberrant Crypt Foci in Rat Colon

The modifying effects of dietary administration of 6-(2,5-dichlorophenyl)-2,4-diamino-1,3,5-triazine and 5 related compounds on the occurrence of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce ACF. They also received the diet containing 200 ppm test compound for 5 weeks, starting one week before the first dosing of AOM. At the termination of experiment, all of the compounds had caused a significant reduction in ACF frequency, which might be associated with suppression of the expression of proliferation biomarkers. The apoptotic index in the colonic mucosal epithelium of rats killed at 6 h after the first AOM exposure revealed no blocking activity of the compounds.     

Preventive effects of chrysin on the development of azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effects of dietary feeding with chrysin (5,7-dihydroxyflavone) on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effect of chrysin on mitosis and apoptosis in 'normal appearing' crypts. To induce ACF, rats were given two weekly subcutaneous injections of AOM (20 mg/kg body weight). They also received an experimental diet containing chrysin (0.001 or 0.01%) for 4 weeks, starting 1 week before the first dose of AOM. AOM exposure produced a substantial number of ACF (73+/-13/rat) at the end of the study (week 4). Dietary administration of chrysin caused significant reduction in the frequency of ACF: 0.001% chrysin, 37+/-17/rat (49% reduction, P<0.001); and 0.01% chrysin, 40+/-10/rat (45% reduction, P<0.001). In addition, chrysin administration significantly reduced the mitotic index and significantly increased the apoptotic index in 'normal appearing' crypts. These findings might suggest a possible chemopreventive activity of chrysin in the early step of colon tumorigenesis through modulation of cryptal cell proliferation activity and apoptosis.     

The induction of aberrant crypt foci (ACF) in the colons of rats by trihalomethanes administered in the drinking water

Bromodichloromethane (BDCM) and bromoform (TBM) have been demonstrated to be colon carcinogens in male and female F344/N rats following administration by corn oil gavage. Our chronic bioassay of BDCM administered in the drinking water failed to demonstrate colon cancer in male F344/N rats. In the present study we addressed the capability of trihalomethanes (THMs) administered in drinking water to induce aberrant crypt foci (ACF), early putative preneoplastic lesions, in the colons of male F344/N rats and B6C3F(1) mice. BDCM was tested in the A/J mouse strain. Rats and B6C3F(1) mice were exposed to isomolar concentrations of the THMs [0.5 g/l chloroform (TCM), 0.7 g/l BDCM, 0.9 g/l dibromochloromethane (DBCM), or 1.1 g/l (TBM)] for 13 weeks. A/J mice were exposed to 0.5 g/l BDCM in the drinking water for 13 and 30 weeks. Deionized water and 0.25% Alkamuls EL-620 were the negative and vehicle controls. ACF incidence (percent) and number (ACF/colon) for the rat were: combined controls, 0; AOM, 100%, 27.17+/-6.28 (P<0.01); TCM, 16.7%, 0.17+/-0.17; BDCM, 83.3%, 1.50+/-0.56 (P<0.01); DBCM, 50%, 1.17+/-0.65 (P<0.01); TBM, 66.7%, 1.17+/-0.40 (P<0.01). THM-induced ACF primarily occurred in the rectal segment of the colon (92%). No ACF were observed in the colons of B6C3F(1) mice following 13 weeks of THM treatment or in the colons of A/J mice following 13 and 30 weeks of BDCM exposure. These studies demonstrate that brominated THMs administered in the drinking water significantly induced preneoplastic ACF in the colon of rats.     

Suppression of azoxymethane-induced colon cancer development in rats by a cyclooxygenase-1 selective inhibitor, mofezolac

We demonstrated recently that mofezolac, a cyclooxygenase-1 (COX-1) selective inhibitor, suppresses the development of azoxymethane (AOM)-induced colonic aberrant crypt foci in F344 rats and intestinal polyps in APC1309 mice. In the present study, we therefore investigated the effects of mofezolac on colon cancer development. Male F344 rats were injected subcutaneously with 15 mg/kg body weight of AOM in the back twice at 7-day intervals from 5 weeks of age, and fed a diet containing 600 or 1200 ppm mofezolac for 32 weeks, starting 1 day before the first dosing of AOM. Treatment with 1200 ppm mofezolac significantly reduced the incidence, multiplicity and volume of colon carcinomas to 79%, 2.15 +/- 1.65 and 7.5 +/- 11.8 mm3, respectively, compared with 94%, 3.19 +/- 1.87 and 23.7 +/- 31.2 mm3 in the AOM treatment alone. Administration of 600 ppm mofezolac showed only a slight reduction. No side effects were observed in any of the groups. These results confirm that COX-1, as well as COX-2, contributes to colon carcinogenesis and that mofezolac may be a good chemopreventive agent for human colon cancer.     

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.