Tumor formation is correlated with expression of beta-catenin-accumulated crypts in azoxymethane-induced colon carcinogenesis in mice

We have reported that β-catenin-accumulated crypts (BCAC) are independent of aberrant crypt foci (ACF) in the colonic mucosa of rats exposed to colorectal carcinogens, and we suggested that they may be premalignant lesions. In the present study, we performed a comparative study on the formation of the two types of early-appearing lesions (BCAC and ACF), and tumors of the colon in two mouse strains with different susceptibility to azoxymethane (AOM). SWR/J mice are known to be relatively susceptible to AOM, whereas AKR/J mice are reported to be virtually resistant. Both AKR/J and SWR/J mice, 6 weeks old, received subcutaneous injections of AOM (15 mg/kg body weight) once a week for 3 weeks, and were sacrificed at 16 and 41 weeks of age. Colons of the animals sacrificed at 16 and 41 weeks of age were processed to examine expression of the early-appearing lesions and neoplasms. Although AKR/J mice had a lower incidence of colonic tumors than SWR/J mice did, AKR/J mice showed a similar frequency of ACF to that in SWR/J mice. In both strains, ACF were detected at high frequency in the proximal colon, whereas tumors developed mainly in the distal colon. Importantly, the incidence of BCAC in SWR/J mice was significantly higher than that in AKR/J mice, and the highest frequency was observed in the distal segments of the colon. These results support the idea that BCAC are a reliable surrogate endpoint for colon carcinogenesis in mice.     

Preliminary analysis of azoxymethane-induced colon tumorigenesis in mouse aggregation chimeras

Inbred mice exhibit differential susceptibility to colon carcinogens. The following study addresses the possibility that differences are intrinsic to colonic mucosa (cell autonomous) or are mediated by extracolonic systemic factors (e.g. liver activation of carcinogens). Our approach was to construct mouse aggregation chimeras, mice whose tissues are a mosaic of cells derived from two parental genotypes, from a susceptible (SWR) and a resistant (DBA/2) strain. Forty-five embryo aggregations yielded 11 viable pups, four of which were chimeric by coat color. Six-week-old SWR<-->BA/2 chimeras were injected i.p. with azoxymethane (AOM) once a week for 8 weeks (5 and 7.5 mg/kg body wt for 2 weeks followed by 10 mg/kg for 6 weeks) and tumor incidence in distal colon was evaluated 15 weeks after the last injection. Additional groups of parental mice received the same treatment. In the parental SWR treatment group, 1.7 +/- 0.82 tumors/colon were found. No tumors were observed in AOM-treated DBA/2 mice. In SWR<-->DBA/2 chimeras exposed to AOM, 2.8 +/- 2.1 tumors/colon were found. Tumor lineage was examined in paraffin sections stained with Dolichos biflorus agglutinin-peroxidase, a cell surface specific marker that stains intestinal endothelial cells of SWR and epithelial cells of DBA/2. Cellular lineage of tumors was further evaluated by microsatellite analysis of DNA isolated by microdissection. There was no significant difference in tumor incidence between SWR parental and chimera treatment groups. Histochemical analysis of tumor tissue in chimeras suggested that most tumors were derived from SWR. However, subsequent genetic analysis of tumors indicated mixed parental composition. These preliminary studies suggest that DBA/2 resistance mechanisms are not sufficient to protect adjacent SWR-derived epithelium from the tumorigenic effects of AOM.     

Hemizygous mice for the angiotensin II type 2 receptor gene have attenuated susceptibility to azoxymethane-induced colon tumorigenesis

Evidence suggests that the use of angiotensin-converting enzyme inhibitors potentially reduces the risk of cancer, though the mechanism is unclear. To clarify a potential involvement of angiotensin II (Ang II) signaling in cancer risk, we have examined the effect of Ang II receptor deficiency on azoxymethane (AOM)-induced colon tumorigenesis. Male Ang II type 2 receptor gene-disrupted (AT(2)-null) mice with a 129/Ola and C57BL/6J genetic background, AT(2)-null mice with an SWR/J genetic background, and their corresponding control wild type mice were treated once a week with AOM (10 mg/kg, i.p., 4 consecutive weeks) or saline vehicle. All mice were killed 23-26 weeks after the initial injection of AOM, and tumor burdens were examined. AOM treatment caused the development of colon tumors in all wild type control mice regardless of genetic background (100% tumor prevalence), but only one tumor was present in AT(2)-null mice with a 129/Ola and C57BL/6J genetic background (11.1% tumor prevalence). Although the introduction of the AOMsusceptible SWR/J genetic background induced AOM susceptibility in AT(2) null mice, the tumor multiplicity (6.3) and tumor size (19.8 +/- 3.0 mm(3)) were significantly smaller than those in wild type mice (multiplicity, 12.0 and size, 36.8 +/- 3.2 mm(3)). AOM efficiently downregulated cytochrome P450 2E1 (CYP2E1) in the liver of wild type mice significantly more than in AT(2)-null mice. The levels of DNA methyl adducts formed in wild type mouse colon epithelium by AOM treatment were also significantly higher than in AT(2)-null mice. These results imply that the AT(2) receptor functions to augment AOM-induced downregulation of CYP2E1 expression in the liver, and thus increases AOM-induced tumorigenesis in the colon. The AT(2) receptor function in the liver may be a potential determinant of tumor susceptibility in chemical carcinogen-induced colon tumorigenesis.     

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.     

The modifying effect of Peucedanum japonicum, a herb in the Ryukyu Islands, on azoxymethane-induced colon preneoplastic lesions in male F344 rats

The modifying effect of dietary Peucedanum japonicum (PJ), which is a traditional herb in the Ryukyu Islands and is an anti-oxidant, on azoxymethane (AOM)-induced rat colon carcinogenesis was examined. Male F344 rats were divided into six groups: rats in groups 1-4 were given subcutaneous injection of AOM (20 mg/kg body weight) once a week for 2 weeks. Rats in groups 2, 3 and 4 were fed the diets containing 0.2 and 1% PJ and 0.025% chlorogenic acid, respectively. We observed modification of the preneoplastic lesions of both aberrant crypt foci (ACF) and beta-catenin accumulated crypts (BCAC) in colon carcinogenesis, microscopically and immunohistochemically. The numbers of ACF consisting of more than four aberrant crypts per rat in groups 2 (3.2+/-1.7) and 3 (3.0+/-3.2) were significantly lower than that of group 1 (10.8+/-4.9; P<0.05, respectively). The mean number of BCAC in both groups 2 (0.88+/-0.48/cm2/rat) and 3 (0.81+/-0.34/cm2/rat) was significantly lower than that in group 1 (2.13+/-0.54/cm2/rat; P < 0.0001, respectively). In addition, proliferating cell nuclear antigen labeling indices in group 2 (10.98+/-2.03) and group 3 (9.85+/-2.62) were significantly lower than that in group 1 (14.87+/-3.93; P < 0.001 and P < 0.0001, respectively). These findings indicate that PJ inhibits both ACF formation and accumulation of beta-catenin, and that PJ also reduces the cell proliferation activity, suggesting that PJ may have chemopreventive potential for colon carcinogenesis.     

Suppressive effects of nimesulide, a selective inhibitor of cyclooxygenase-2, on azoxymethane-induced colon carcinogenesis in mice

The effects of nimesulide, a selective inhibitor of cyclooxygenase-2 (COX-2) on azoxymethane (AOM)-induced colon carcinogenesis were investigated in mice. AOM at a dose of 10 mg/kg body wt was administered to male ICR mice once a week for 6 weeks. The animals were fed on AIN-76A powder diet containing nimesulide at doses of 200 or 400 p.p.m., starting the day before the first carcinogen treatment until the end of the experiment, at week 30. Administration of nimesulide reduced the incidence of colon carcinomas to 32 and 25% for the AOM + 200 and 400 p.p.m. nimesulide groups, respectively, compared with the AOM + basal diet group (50%). Multiplicities of colon carcinomas in the 200 and 400 p.p.m. nimesulide-treated groups were 0.70 +/- 0.28 and 0.35 +/- 0.11, respectively, being significantly smaller than the AOM alone value (1.79 +/- 0.47). The sizes of the colon carcinomas in the nimesulide-treated groups were also decreased. No significant influence on liver and lung tumor development was apparent. Thus, nimesulide exerted a suppressive effect on AOM-induced colon carcinogenesis in mice.      

Strain differences in the susceptibility to azoxymethane and dextran sodium sulfate-induced colon carcinogenesis in mice

We have recently developed a mouse model for colitis-related colon carcinogenesis by a combined treatment with azoxymethane (AOM) and dextran sodium sulfate (DSS) in male ICR mice. However, strain differences in the sensitivity to AOM/DSS-induced colon carcinogenesis in mice have yet to be elucidated. The aim of this study was to determine the presence of any genetically determined differences in sensitivity to our model of colon carcinogenesis in four inbred strains of mice. Male Balb/c, C3H/HeN, C57BL/6N and DBA/2N mice were given a single intraperitoneal injection of AOM (10 mg/kg body wt), followed by 1% DSS (w/v) in drinking water for 4 days, and thereafter they received no further treatment for up to 16 weeks. At the end of the study (Week 18), all mice were killed and a histopathological analysis of their colon was performed. The incidence of colonic adenocarcinoma was 100% with a multiplicity (no. of tumors/mouse) of 7.7+/-4.3 in the Balb/c mice and 50% with a multiplicity of 1.0+/-1.2 in the C57BL/6N mice. On the other hand, only a few colonic adenomas, but no adenocarcinomas, developed in the C3H/HeN mice (29% incidence with a multiplicity of 0.7+/-1.5) and the DBA/2N mice (20% incidence with a multiplicity of 0.2+/-0.4). The inflammation and immunohistochemical nitrotyrosine-positivity scores of the mice treated with AOM and DSS in the decreasing order were as follows: C3H/HeN>Balb/c>DBA/2N>C57BL/6N and Balb/c>C57BL/6N>C3H/HeN>DBA/2N, respectively. Our results thus indicated the presence of strain differences in the susceptibility to AOM/DSS-induced colonic tumorigenesis. These differences may have been directly influenced by the response to nitrosation stress due to the inflammation caused by DSS.     

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

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

High susceptibility of Scid mice to colon carcinogenesis induced by azoxymethane indicates a possible caretaker role for DNA-dependent protein kinase

Severe combined immunodeficiency (Scid) mice have defects in V(D)J recombination and DNA double-strand breaks repair caused by an inherited genetic defect in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Scid mice are highly susceptible to development of T-cell lymphomas, and because of the nature of its association with DNA repair and recombination, DNA-PKcs is considered to belong to the caretaker class of tumor suppressor genes. In the present study, the susceptibility of Scid mice to colon carcinogenesis due to administration of azoxymethane (AOM) was investigated. Significantly higher susceptibility in terms of induction of both aberrant crypt foci (ACFs), putative pre-cancerous lesions of the colon and colon cancers was observed as compared with the isogenic strain, C.B-17 mice. The incidences of colon tumors, either adenomas or adenocarcinomas, in Scid and C.B-17 mice after administration of AOM (10 mg/kg body weight/week) for 6 weeks were 87% (26 of 30) and 50% (15 of 30), respectively, by experimental week 22 (P < 0.01). The multiplicity of colon tumors in Scid mice was also significantly higher than in C.B-17 mice, being 2.2 +/- 1.5 and 0.9 +/- 1.2, respectively (P < 0.001). The present study clearly demonstrated high susceptibility of Scid mice to colon carcinogenesis, which might be attributable to disruption of the caretaker role of DNA-PK in colonic epithelial cells.     

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.     

Preliminary analysis of azoxymethane induced colon tumors in inbred mice commonly used as transgenic/knockout progenitors

Azoxymethane (AOM) is a colon carcinogen that is used to study the pathogenesis of sporadic colorectal cancer. We have evaluated differential susceptibility to AOM in inbred mice used as progenitors of recombinant/transgenic lines. In experiment 1, male FVB/N, 129/SvJ, C57Bl/6J mice were treated i.p. with 10 mg/kg AOM once per week for 4 weeks and sacrificed after 20 weeks. Only AOM-treated FVB/N mice developed tumors (3.6 tumors/mouse) in distal colon. In experiment 2, A/J, AKR/J, Balb/CJ mice were treated with AOM for 6 weeks and sacrificed after 24 weeks. AOM-treated A/J and Balb/CJ mice developed 9.2 and 1 tumor/mouse, respectively. Despite these differences, tumors had similar morphology regardless of strain. Immunohistochemistry with beta-catenin resulted in marked nuclear and cytoplasmic staining of tumor cells in FVB/N. However, fainter and heterogeneous beta-catenin staining was observed in A/J tumors, suggesting distinct pathways of tumorigenesis in different strains. Irrespective of cytological features of malignancy, tumor cells rarely breached the muscularis mucosa and showed no evidence of distant metastasis. Lack of invasiveness and metastasis in even the most sensitive strains provides a model system for studying the potential role of 'metastasis genes' in imparting a malignant phenotype.     

Sequential observations on the occurrence of preneoplastic and neoplastic lesions in mouse colon treated with azoxymethane and dextran sodium sulfate

Previously, we proposed a novel mouse model for colitis-related colon carcinogenesis using azoxymethane (AOM) and dextran sodium sulfate (DSS) (Cancer Sci 2003; 94: 965-73). In the current study, sequential analysis of pathological alterations during carcinogenesis in our model was conducted to establish the influence of inflammation caused by DSS on colon carcinogenesis in this model. Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight) and given 2% (w/v) DSS in the drinking water for 7 days, starting 1 week after the AOM injection. They were sequentially sacrificed at weeks 2, 3, 4, 5, 6, 9, 12, and 14 for histopathological and immunohistochemical examinations. Colonic adenomas were found in 2 (40% incidence and 0.40 +/- 0.49 multiplicity) of 5 mice at week 3 and colon carcinomas developed in 2 (40% incidence and 2.00 +/- 3.52 multiplicity) of 5 mice at week 4. Their incidence gradually increased with time and reached 100% (6.20 +/- 2.48 multiplicity) at week 6. At week 14, the multiplicity of adenocarcinoma was 9.75 +/- 2.49 (100% incidence). In addition, colonic dysplasia was noted at all time-points. The scores of colonic inflammation and nitrotyrosine immunohistochemistry were extremely high at early time-points and were well correlated. Our results suggest that combined treatment of mice with AOM and DSS generates neoplasms in the colonic mucosa via dysplastic lesions induced by nitrosative stress.     

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.     

Suppression of azoxymethane-induced rat colon carcinoma development by a fish oil component, docosahexaenoic acid (DHA)

The effects of intragastric gavage administration of docosahexaenoic acid (DNA) , a major component of fish oil, on azoxymethane (AOM)- induced colon carcinogenesis in rats was investigated. Male F344 rats were treated with 15 mg/kg body wt of AOM once a week, for two weeks. The animals were given either 1 ml of DHA or water intragastrically 5 times a week, starting the day before the first carcinogen treatment. The numbers of AOM-induced aberrant crypt foci in the rats given DHA were 76% and 62% of the control values, at 4 and 12 weeks, respectively. After 36 weeks of DHA treatment, colon tumors were counted and examined histologically. The blood plasma levels of prostaglandin E2 (PGE2) and polyunsaturated fatty acids were also quantified. The incidences of colon cancer did not differ, being 96% and 92% in the AOM and AOM+DHA groups, respectively. Colon cancer multiplicity was, however, significantly decreased by the DHA treatment; 3.65 +/- 2.18 in the AOM group and 2.41 +/- 1.58 in the AOM+DHA group (P <0.01). Notably, the numbers of moderately differentiated adenocarcinomas in the middle and distal colon in the DHA-treated group were lower than in the AOM group. The levels of PGE2 and arachidonic acid in the blood plasma of DHA-treated rats were also significantly lower than in the AOM group. These results suggest that DHA exerts its inhibitory effect on colon carcinogenesis by modulating lipid metabolism and inhibiting the arachidonic cascade.      

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.     

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.     

Sequential and morphological analyses of aberrant crypt foci formation in mice of differing susceptibility to azoxymethane-induced colon carcinogenesis

Aberrant crypt foci (ACF), putative preneoplastic lesions, are early morphological changes induced by the colon carcinogen azoxymethane (AOM). Although inbred mice differ markedly in their susceptibility to AOM carcinogenesis, we have previously shown that ACF develop in both resistant and sensitive mouse strains after AOM treatment. The purpose of this study was to examine the sequential development and identify the morphological characteristics of ACF induced by AOM in the distal colon of sensitive and resistant mice. A/J (highly susceptible), SWR/J (relatively susceptible) and AKR/J (resistant) mice were treated with 10 mg/kg AOM or saline i.p. once a week for 6 weeks and were killed at 1, 2, 4, 6, 9 and 24 weeks after the last injection. The distal colons were stained with methylene blue and the numbers of ACF and tumors determined. Tumors were present as early as 4 weeks after AOM exposure in SWR/J and A/J mice and increased in frequency throughout the study in both strains. No tumors developed in the AKR/J mice. ACF, however, formed in all strains of mice. The greatest difference between susceptible and resistant strains was in the number of large ACF that developed at later time points. Furthermore, morphometric analysis revealed that A/J mice had the highest percentage of dysplastic ACF, followed by SWR/J mice. These data indicate that the difference in cancer risk from AOM may be due to the lack of progression of smaller ACF in the resistant mice and to the development of dysplasia in a higher percentage of ACF from susceptible strains.     

Inhibition of Azoxymethane-induced Colon Carcinogenesis in Rat by Green Tea Polyphenol Fraction

The effect of green tea polyphenol fraction (GTP) on azoxymethane(AOM)-induced colon carcino-genesis was investigated in male Fischer rate. The rats were given AOM (7.4 mg/kg body weight) s.c. once a week for 10 weeks. A week after the treatment, they were divided into three groups: AOM-control (26 rats), AOM-GTP1 (26 rats) and AOM-GTP2 (25 rats). AOM-GTPl and AOM-GTP2 groups respectively received 0.01 and 0.1% GTP in drinking water from week 11 to 26. AOM-control group received tap water throughout this experiment. Autopsy on week 26 showed that tumor incidence and average numbers of tumors per rat in the AOM-GTPl and AOM-GTP2 groups were significantly lower than those of the AOM-control group: 38.1% and 47.6% versus 77.3%; 0.6 and 0.7 versus 1.5. Thus, it was concluded that GTP inhibited the development of AOM-indnced colon carcinogenesis. The inhibition by GTP did not show significant dose dependence.     

Strain-specific homeostatic responses during early stages of Azoxymethane-induced colon tumorigenesis in mice

Identifying molecular changes that predict the risk for developing colon cancer is critical for designing effective prevention strategies. In the present study, we determined early-stage molecular alterations within the colonic epithelium of A/J and AKR/J mice that are sensitive and resistant to Azoxymethane (AOM)-initiated tumor development, respectively. Six week-old male mice were injected intraperitoneally with AOM (10 mg/kg body weight) once a week for six weeks. One week after the last injection, distal colons from both strains were analyzed for cell proliferation using a proliferating cell nuclear antigen (PCNA) assay. Unlike AKR/J, a significant increase (2.5-fold, p<0.05) in the number of PCNA-positive cells within the upper third of the crypt compartment was observed in the A/J colons. This proliferative response was associated with a sizeable increase in the levels of c-myc mRNA, quantified by RNase protection assay. cDNA sequencing, protein expression and localization of beta-catenin, an upstream activator of c-myc, however, showed no aberrant changes within AOM-exposed A/J colons. Interestingly, TdT-mediated dUTP nick-end labeling assay revealed a significant increase (4-fold) in the number of apoptotic colonocytes in A/J mice following AOM treatment. Consistent with this finding, a modest increase in the expression of pro-apoptotic Bak was limited to the sensitive A/J colons. In summary, the current study suggests that a significant alteration in the rate of cell turnover in the normal appearing colonic mucosa, as observed in susceptible A/J mice, may be one of the earliest events predisposing the colon to neoplastic growth.     

Inhibition of azoxymethane-induced colon carcinogenesis in rat by green tea polyphenol fraction.

The effect of green tea polyphenol fraction (GTP) on azoxymethane(AOM)-induced colon carcinogenesis was investigated in male Fischer rats. The rats were given AOM (7.4 mg/kg body weight) s.c. once a week for 10 weeks. A week after the treatment, they were divided into three groups: AOM-control (26 rats), AOM-GTP1 (26 rats) and AOM-GTP2 (25 rats). AOM-GTP1 and AOM-GTP2 groups respectively received 0.01 and 0.1% GTP in drinking water from week 11 to 26. AOM-control group received tap water throughout this experiment. Autopsy on week 26 showed that tumor incidence and average numbers of tumors per rat in the AOM-GTP1 and AOM-GTP2 groups were significantly lower than those of the AOM-control group: 38.1% and 47.6% versus 77.3%; 0.6 and 0.7 versus 1.5. Thus, it was concluded that GTP inhibited the development of AOM-induced colon carcinogenesis. The inhibition by GTP did not show significant dose dependence.