Azoxymethane-induced beta-catenin-accumulated crypts in colonic mucosa of rodents as an intermediate biomarker for colon carcinogenesis
Yoshinobu Hirose2,
Toshiya Kuno,
Yasuhiro Yamada,
Keiko Sakata,
Masaki Katayama,
Koujiro Yoshida,
Zheng Qiao,
Kazuya Hata,
Naoki Yoshimi1 and
Hideki Mori
Department of Tumor Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan and
1 Department of Pathology, Ryukyu University School of Medicine, Okinawa 903-0215, Japan
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Abstract
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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.
Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; BCAC, beta-catenin-accumulated crypts; CHA, cholic acid
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Introduction
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Reliable intermediate biomarkers for colon carcinogenesis need to be identified in order to use them to evaluate several agents for their carcinogenic risk or their potential chemopreventive efficacy against colon tumors. Aberrant crypt foci (ACF), which were originally described by Bird in the unsectioned murine colon exposed to a colon-specific carcinogen, azoxymethane (AOM), have been recognized as early preneoplastic lesions. Further specificity study showed that several other colon-specific carcinogens such as 1,2-dimethylhydrazine, 4-aminobiphenyl, N-nitroso-N-methylurea and 3-methylcholanthrene also induce ACF, indicating that development of these lesions in the colon is clearly related to the genotoxic events. Genetic studies of ACF revealed that k-ras mutations are frequently identified in these lesions. Because colonic tumors induced by chemical carcinogens are also reported to have frequent mutations in the k-ras gene, there is some similarity in the genotype of colonic ACF and tumors, suggesting that this genetic alteration may play a key role in the development of colonic ACF as well as tumors.
It is now widely recognized that ACF might be possible precursors of colorectal cancer. Based on the proposition that these lesions appear preneoplastic, ACF are being used as a short-term assay to identify modulators of colon cancinogenesis prior to a long-term assay using tumor occurrence as end point. However, some lines of evidence suggest that the results of the ACF assay and the long-term analysis whose end point is colon tumor occurrence contradict each other. For example, there is a large body of evidence from clinical and metabolic epidemiological studies showing a positive association between the levels of secondary bile acids in intestinal lumen and colon cancer risk. Indeed, the long-term model assays using rodents have provided convincing evidence showing that administration of cholic acid (CHA), one of the primary bile acids, in the diet or intrarectally promotes the development of chemically induced colonic tumors. CHA administered in the diet or intrarectally is converted by bacterial 7
-dehydroxylase to deoxycholic acid, which has been shown to induce cell proliferation and act as a strong tumor promoter in the colon. In contrast, a previous study indicated that dietary CHA significantly decreases the formation of AOM-induced colonic ACF in the short-term assay. This disparity between the results of ACF formation and later tumor development by an agent was also documented previously on these parameters as a result of genistein administration. Genistein was thought to be a promising chemopreventive agent against colon carcinogenesis based on epidemiological observations that consumption of soybeans and soybean-based products containing a large amount of genistein might reduce the risk of colon cancer. While a short-term animal bioassay showed that genistein inhibits the development of AOM-induced colonic ACF, surprisingly a long-term animal bioassay using tumors as end point demonstrated that dietary administration of this compound significantly enhances colon carcinogenesis. Comparative model studies using several strains of mice also pointed to the controversy on the association between colonic ACF formation and susceptibility to AOM-induced colon tumorigenesis. The results of these studies demonstrated that AOM treatment induces ACF in the colonic mucosa of resistant AKR/J mice with no tumor occurrence at later time points, whereas the same treatment with the carcinogen produces a similar number of ACF as well as several colon tumors in highly susceptible A/J mice. These observations leave little doubt that colonic ACF are not reliable biomarkers for colon tumorigenesis.
We recently found altered crypts in AOM-exposed mucosa with accumulation of beta-catenin protein in the cytoplasm and/or nucleus and harboring frequent mutations of the gene, thus indicating a possible important role of these lesions in colon carcinogenesis. Further, molecular analysis of colon tumors in rodents as well as humans revealed that a pathway mediated through beta-catenin plays a pivotal role in colon carcinogenesis. It is apparent that these altered crypts also termed as BCAC may be viewed as preneoplastic lesions for colon carcinogenesis. Importantly, it is difficult to recognize BCAC in the topographic view of mucosal surface with the conventional methylene blue staining routinely used for an ACF assay, because BCAC lack several criteria for ACF, suggesting that they might be independent of ACF. Morphological analysis of BCAC as well as ACF showed that the histological abnormality of BCAC after exposure to AOM is increased in a time-dependent fashion and more prominently than is the case of ACF. These findings together with the conflicting data about ACF as a potential biomarker for colon carcinogenesis prompted us to assess the possibility of application of BCAC as a short-term bioassay for colon cancer risk and/or an intermediate biomarker for evaluation of promising agents for their potential chemopreventive properties against colon carcinogenesis. Actually, a recent study from our group, which was done on the basis of the same strategy, showed that celecoxib, a selective inhibitor of cyclooxygenase-2 and a promising chemopreventive agent against colon carcinogenesis, significantly inhibited BCAC in AOM-treated rats, thus suggesting the possible application of BCAC as a biomarker.
The purpose of the present study was, therefore, to establish whether the colonic BCAC are realistic biomarkers of colon carcinogenesis as compared with ACF. To do so, we investigated the modulating effect of CHA, which provided conflicting results on its effect on ACF formation and later tumor development, on the induction of colonic BCAC and ACF in rats treated with AOM.
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Materials and methods
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Animals, diets and chemicals
Four-week-old male F344 rats were obtained from Japan SLC Inc., Hamamatsu, Japan. AOM and CHA were purchased from Wako Chemicals, Tokyo, Japan. All experimental diets were formulated based on the composition of AIN-76A diet (CLEA Japan, Tokyo). Animals were housed three or four to a wire cage in an animal holding room under controlled conditions with 23 ± 2°C, 50 ± 10% humidity, and 12 h light/dark cycles. They were allowed ad libitum access to AIN-76A control diet and water.
Experimental procedure
After quarantine for 1 week, all rats were divided into four groups. Beginning at 6 weeks of age, animals in groups 1 and 2 (eight rats each) received AOM s.c. at a dose rate of 15 mg/kg body wt, once a week for 3 weeks. Animals intended for vehicle treatment (groups 3 and 4) received 0.2 ml saline (four rats in each group). Beginning at 9 weeks of age, rats in groups 2 and 3 were fed the AIN-76A diet containing 0.5% of CHA and continued on this diet until the end of the experiment. Groups 1 and 4 were continued on AIN-76A diet without CHA throughout the experiment. The dose of dietary CHA (0.5%) was chosen because this dose of CHA promoted chemically induced colon carcinogenesis of rats and was reliable in modulation of several biomarkers of colon carcinogenesis such as ornithine decarboxylase. At 16 weeks of age all animals were killed with ether anesthesia. Their colons were removed, slit open longitudinally, and washed with Krebs Ringer. Then, they were fixed with 2% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4) for 24 h at 4°C for ACF and BCAC analyses.
Identification of ACF and BCAC
All colons were stained with methylene blue for determining the frequency, multiplicity and size of ACF under a light microscope. ACF were recorded according to standard procedures that are routinely used in our laboratory. The criteria used to identify ACF were as follows: they are larger and elevated above the adjacent normal crypts with thickened cell walls lining the crypt and increased pericryptal area. For measurement of diameters of ACF, three of all the colons in each group were selected randomly and evaluated. After removing the rectal sides (1 cm from the anus), all colons were then cut into four segments (distal, medium-distal, proximal-medium and proximal) with equal length (
3 cm each). The distal, medium-distal, and proximal-medium segments from all the colons were examined for identification of BCAC in the present study. They were embedded in paraffin blocks utilizing en face preparation and processed by conventional histological methods. Serial sections with 4 µm thickness from a total of 72 segments (24, 24, 12 and 12 segments from groups 1, 2, 3 and 4, respectively) were used to analyze whole crypts from apical to bottom. Two and four sections from the 1020 serial sections of each colonic segment were selected for immunohistochemistry of beta-catenin and conventional hematoxylin and eosin staining, respectively. The labeled streptavidinbiotin method using an LSAB kit (Dako, Glostrup, Denmark) with microwave accentuation was performed for immunohistochemical analysis. After deparaffinization, sections were treated with 3% hydrogen peroxide and 2% bovine serum albumin for 10 and 30 min, respectively. Then the sections were incubated with a primary antibody against beta-catenin (Transduction Laboratories, Lexington, KY). Negative control for each case using the serial section was prepared in the same manner but the primary antibody omitted. Horseradish peroxidase activity was visualized by treatment with hydrogen peroxide and diaminobenzidine for 5 min. The lesions with immunopositivity in the cytoplasm and/or nucleus were considered as BCAC as described previously. They also had nuclear atypia, structural abnormality and Paneth cell metaplasia, which were recognized easily on routine hematoxylin and eosin-stained sections. On the contrary, ACF showed little accumulation of beta-catenin immunohistochemically, little nuclear atypia, less structural abnormality and no metaplastic change. The frequency (number of lesions/cm2), multiplicity (number of crypts/lesion) and size (average of the long and short diameters) were measured on each section.
Statistical analysis
Results of BCAC and ACF were analyzed statistically by Students t test or Welchs t test. P values of <0.05 were considered significant.
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Results
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General observation
The average body weights of rats in groups 14 at the termination of the experiment were respectively 287 ± 23, 248 ± 19, 261 ± 7 and 291 ± 15 g (mean ± SD). The body weights of animals fed the CHA diet were significantly less than their respective controls. However, the lower body weights did not correlate with BCAC results, as the rats treated AOM and fed CHA diet had the lowest body weight and the highest number of BCAC. Therefore, little tumorigenic significance is considered to be attributable to the differences in body weights. Otherwise there was no evidence of gross abnormality or toxicity sign in all groups.
ACF data
To confirm the effect of dietary CHA on the AOM-induced ACF formation, we first examined ACF in the unsectioned colons with the conventional methods that are routinely used in our laboratory. As summarized in Table I
, rats fed the control diet and treated with AOM (group 1) showed 194.9 ± 23.2 ACF per colon (n = 8), 3.07 ± 1.20 crypt multiplicity (number of aberrant crypts/focus, n = 1558) of ACF with an average size of 173.6 ± 53.3 µm (n = 597) (values are means ± SD). Administration of CHA in the diet (group 2) significantly reduced the total number (65.1 ± 23.1, n = 8, P < 0.00001), the multiplicity (2.39 ± 1.06, n = 521, P < 0.00001) and the size (140.9 ± 42.3 µm, n = 165, P < 0.00001) of AOM-induced ACF as compared with the control diet (group 1). In the saline-treated groups, there was no evidence of ACF formation. The results clearly indicated that dietary CHA inhibited the formation of AOM-induced ACF. We next investigated the effects of dietary CHA on the multiplicity of ACF, because of the previous report showing that the ACF with multiplicity larger than 4 were resistant to CHA. As shown in Figure 1
, the CHA diet significantly inhibited the development of ACF containing 27 aberrant crypts/focus as compared with the control diet (P < 0.030.0002). The number of ACF containing >4 aberrant crypts/focus was also significantly reduced in animals fed the CHA diet (P < 0.0002), indicating that ACF with multicrypts (>4 aberrant crypts/focus) were little resistant to continuous feeding of 0.5% CHA in the diet.

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Fig. 1. Effects of dietary CHA on the multiplicity of ACF. Rats received AOM once weekly for 3 weeks and then were fed the basal diet with or without 0.5% CHA for 7 weeks. Their colons were fixed and stained with methylene blue for determining crypt multiplicity of ACF (number of crypts/focus) under a light microscope. Error bars represent SD. A single asterisk and a double one denote P < 0.0002 and P < 0.03, respectively in comparison with the basal diet.
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BCAC data
To determine whether dietary CHA affects BCAC formation in the colons of rats treated with AOM, we compared the occurrence and development of BCAC among different groups. Representative data of hematoxylin and eosin staining and immunohistochemistry of beta-catenin on the colonic sections are shown in Figure 2
. BCAC had morphological alterations such as nuclear atypia, structural abnormality and Paneth cell metaplasia (Figure 2A
) with the immunopositivity of beta-catenin in the cytoplasm and/or nucleus (Figure 2B
). As summarized in Table I
, AOM treatment (group 1) induced 2.21 ± 0.99 BCAC (per cm2 of colon, n=8) consisting of 5.22 ± 5.90 crypt multiplicity (n = 128) with an average size of 119.0 ± 71.1 µm (n = 128) (values are means ± SD). Administration of CHA in the diet (group 2) slightly but not significantly increased the number of BCAC as compared with control diet (group 1), whereas it significantly increased both the multiplicity (7.64 ± 6.62, n = 182, P < 0.002) and size (165.8 ± 79.8 µm, n = 182, P < 0.00001) of BCAC. We next investigated the effects of dietary CHA on the multiplicity of BCAC, as in the case of ACF. As shown in Figure 3
, the numbers of BCAC containing 79, 1012 and >21 crypts/lesion were increased significantly in animals fed the CHA diet as compared with those fed the control diet (P < 0.003, 0.03 and 0.03, respectively), resulting in the significant increase of large BCAC with >6 crypts/lesion in the CHA diet group (P < 0.003). These results indicated that the CHA diet enhanced the development of AOM-induced BCAC, suggesting that the effect on BCAC may be responsible for the tumor promoting effects of CHA.

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Fig. 2. Hematoxylin and eosin staining and immunohistochemistry of beta-catenin in the colonic mucosa after the exposure to AOM. Rats were injected with AOM once weekly for 3 weeks and then were fed the basal diet with or without 0.5% CHA for 7 weeks. (A) Their colons were fixed and processed according to the standard method for histopathology with hematoxylin and eosin staining. (B) Immunohistochemistry of beta-catenin was performed on the serial section. The data shown here are representative of group 1 (AOM treatment alone). Arrows indicate BCAC.
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Fig. 3. Effects of dietary CHA on the multiplicity of BCAC. Rats received AOM once weekly for 3 weeks and then were fed the basal diet with or without 0.5% CHA for 7 weeks. Their colons were analyzed with beta-catenin immunohistochemistry for determining crypt multiplicity of BCAC (number of crypts/lesion). Error bars represent SD. A single asterisk and a double one denote P < 0.03 and P < 0.003, respectively, in comparison with the basal diet.
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Discussion
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The present study demonstrated for the first time that administration of CHA in the diet significantly increased the multiplicity and size, but not the incidence, of AOM-induced BCAC in the colonic mucosa. The effect of CHA on the BCAC formation was detected using quantitative immunohistochemistry that allowed for the analysis of frequency of occurrence, multiplicity and size of BCAC. These results are consistent with the observations of previous studies that administration of CHA in the diet or intrarectally promotes the colon tumor development and corroborate our conception that colonic BCAC may be precursors of colon tumors. The results of the present study also confirmed the results of an earlier study that dietary CHA produces an inhibitory effect on the formation of ACF. It is noteworthy that our present protocol clearly showed that the continuous exposure of dietary CHA (0.5%) attenuated significantly the formation of large ACF with >4 aberrant crypts/focus that were reportedly considered to be resistant to dietary CHA. It should be recognized that progression from classical ACF to neoplastic transformation by dietary CHA remains highly contentious. Furthermore, the results of our study indicated that cellular responses of BCAC and ACF to dietary CHA were opposite, suggesting that the effect of CHA on BCAC is distinguishable from that on ACF and accounts in part for its action as a colon tumor promoter. This means that the ability of an agent to modulate BCAC is more important for its potential action than the modification of ACF.
The major purpose of this study was to determine the possibility of application of BCAC to a short-term bioassay for colon cancer risk assessment or an intermediate biomarker for evaluation of chemopreventive agents against colon carcinogenesis. It is important to evaluate the specificity and sensitivity of intermediate biomarkers continuously and to make a selection of a relevant biomarker for those assessments. The present study showed that CHA, a typical tumor promoter of colon carcinogenesis, did promote the formation of AOM-induced BCAC in the colonic mucosa. In support of these results, a recent study from our group demonstrated that celecoxib, a selective cyclooxygenase-2 inhibitor, which has been reported to decrease AOM-induced carcinogenesis, significantly inhibited the development of AOM-induced BCAC. Furthermore, our unpublished results indicated that a high fat diet, another well-known colon tumor promoter, enhanced the formation of AOM-induced BCAC. Thus, a series of our studies with dietary intervention point to the fact that characteristics of BCAC have a great similarity to that of colonic neoplasia, indicating that BCAC are intermediate lesions that develop into colonic neoplasm and should be considered as preneoplastic lesions. Importantly, the results of BCAC developing shortly (58 weeks) after AOM treatment in rat colons with the administration of modulating agents for colon carcinogenesis, irrespective of whether it is a tumor-inhibitor or a tumor-promoter, appeared to reflect sensitively the tumor outcomes of the long-term studies. Hence, these results suggest that BCAC may be considered as a short-term biomarker for colon carcinogenesis. However, to prove the conception that BCAC are reliable short-term biomarkers, a long-term study using the same model with CHA diet at different time points of sacrifice is warranted. Moreover, it should be noted that further characterizations of BCAC and methodological improvement for rapid and simple detection of BCAC are necessary for its application as a biomarker. In particular, the complexity of extensive tissue sectioning utilizing en face preparation and high expenditure on immunohistochemistry should be avoided.
There is currently an intensive effort to use the ACF assay as an end-point biomarker for evaluation of several agents for their potential chemopreventive and promotional activities during the initiation and promotion/progression phases of colon carcinogenesis, as done in the present study. One of the most advantageous features of the ACF assay is shortness of the experimental period, usually consisting of an exceedingly short period of promotion/progression stage followed by a similar initiation step as employed in a long-term experiment. It should be recognized that it is possible that these short-term experiments might miss the effect of these agents during the promotion/progression stage of colon carcinogenesis, as the post-initiation phase is regarded as a long and complicated process. In this context, the ACF assay may not be so powerful, and sometimes misleading, in evaluating the effect of promoting agents including CHA as well as chemopreventive agents. Nevertheless, ACF might still be considered as an effective short-term biomarker for colon carcinogenesis as long as its usage is completely defined. Because many colon carcinogens, but not non-carcinogens, can induce ACF and some of the lesions have mutations of k-ras and/or beta-catenin genes, ACF are regarded as lesions caused by genotoxic damages in vivo. Therefore, it is reasonable that ACF could be used to evaluate the magnitude of genotoxicity of several agents. In addition, the ACF assay might be useful in the evaluation of blocking agents such as modulators of phase I and/or II enzymes for their potential chemopreventive properties, since prevention of genotoxic agents including carcinogens from reaching or reacting with critical target sites on DNA could theoretically lead to attenuation of ACF formation as well as tumor occurrence. As a matter of fact, our recent studies showed that the modulating effects of celecoxib or high fat on the formation of ACF in AOM-treated colonic mucosa of rats are partially concordant with those on BCAC as well as the results of long-term studies. The results, together with the methodological advantage of the ACF assay such as simplicity and rapidness for their detection, indicate that the ACF assay might be still applicable as a screening method.
In summary, the goal of this study was to determine if BCAC could be used as a biomarker of colon carcinogenesis to evaluate tumor promoters and chemopreventive agents. Our data showed that dietary exposure of CHA, a typical tumor promoter of colon, enhanced the formation of BCAC when compared with the basal diet group, supporting the proposition that BCAC are not only precursors of colon tumors but also useful intermediate biomarkers for colon carcinogenesis predicting tumor development. Meanwhile, further characterizations of BCAC in laboratory animals, methodological improvements for their detection, and identification of BCAC in humans are needed to establish reliability of BCAC as an early biomarker for colon carcinogenesis.
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Notes
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2 To whom correspondence should be addressed Email: yhirose{at}cc.gifu-u.ac.jp 
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Acknowledgments
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We wish to thank Kyoko Takahashi, Misato Yasuda, Tomoko Kajita, and Hisae Shibazaki for their technical assistance, and Toshio Kinjo for care of the animals. This work was supported in part by a Grant-in-Aid from the Ministry of Health, Labour and Welfare in Japan.
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Received April 24, 2002;
revised September 16, 2002;
accepted September 17, 2002.