Influence of diets containing high and low risk factors for colon cancer on early stages of carcinogenesis in human flora-associated (HFA) rats

Germ-free rats colonised with a human intestinal flora were fed diets containing high risk (HR) or low risk (LR) factors for colorectal cancer, and putative biomarkers were evaluated in the colonic mucosa; (i) proliferation, (ii) 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci and (iii) DMH-induced DNA damage. The HR diet was high in fat (45% of calories) and low in calcium and fibre, reflecting levels characteristic of typical western diets. The LR diet was low in fat (<5% of calories), and high in calcium and fibre. The nutrient/energy ratio of the two diets were similar. Mucosal crypt cell proliferation, assessed after microdissection, was higher on the LR diet (mean number of mitoses per crypt was 2.65 on the LR diet, and 1.62 on the HR diet; P < 0.05). Aberrant crypt foci (ACF) were assessed in the mucosa 12 weeks after DMH treatment. On the HR diet there were significantly more small ACF with 1 and 2 crypts per focus, but fewer ACF with 3, 5 and 7 or more crypts per focus. There was no significant difference in total ACF or the total number of crypts. The effect of diet on DNA damage in the colon was assessed in vivo by the comet assay. Animals were fed a HR or LR diet for 12 weeks before treatment with DMH or saline. For carcinogen-treated animals, DNA damage was significantly higher in colon cells from animals on the HR diet. On the LR diet both DNA damage and the induction of small ACF were reduced despite an increase in cell proliferation. The increase in large ACF on the LR diet may be attributable to elevated crypt cell proliferation possibly increasing crypt fission rates.      

Flat dysplastic aberrant crypt foci are related to tumorigenesis in the colon of azoxymethane-treated rat

We evaluated the role of aberrant crypt foci (ACF) as biomarkers of colon cancer by studying the sequential development (6-28 weeks) from early lesion to tumor in the colon of azoxymethane-exposed F344 rats (15 mg/kg bw x 2). Surface examination of unsectioned methylene blue-stained colon preparations, transilluminated in the inverse light microscope, revealed two types of early lesions: classic elevated ACF and small flat lesions, which we denoted flat ACF and which were characterized by bright blue staining, compressed crypt openings, and crypts not elevated above the surrounding mucosa. At a later stage, the crypts surrounding large flat ACF became enlarged, a change that slightly raised the structure; principally, large flat ACF and nascent tumors displayed the same surface morphology. Furthermore, flat ACF with 18.6 +/- 10.6 crypt/focus and tumors showed a uniform picture of severe dysplasia with frequent presence of Paneth cells, compressed crypts, cytoplasmic/nuclear overexpression of beta-catenin, and nuclear overexpression of cyclin D1. In contrast, classic elevated ACF with 5.3 +/- 2.5 crypts/focus did not display such changes: they showed mainly hyperplasia, mild or moderate dysplasia but never severe dysplasia. Along the time course, the number of flat ACF + tumors, including microscopic and macroscopic, was virtually constant, approximately 2.5 lesions/rat. The number of classic elevated ACF was initially approximately 180 lesions/rat and terminally approximately 80 lesions/rat. Flat ACF grew significantly faster than classic elevated ACF. In conclusion, our data indicate a continuous developmental growth from small flat dysplastic ACF to the stage of a tumor. In contrast, classic elevated ACF do not seem to be as closely related to tumorigenesis.     

Development of Aberrant Crypt Foci Involves a Fission Mechanism as Revealed by Isolation of Aberrant Crypts

Morphological analysis of isolated colonic crypts in rats, postnatally, indicated that the crypts reproduce themselves by a fission mechanism, the division beginning at the crypt base and proceeding upwards until there are two separate crypts. Occasionally, before the separation is complete, a second fission process starts on one or both sides of a bifurcating crypt and a triple-branched or quadruple-branched crypt results. Analysis of isolated aberrant crypt foci (ACF) in rats treated with 1,2-dimethylhydrazine revealed that the development of ACF consisting of multiple crypts is also due to a fission mechanism. Initially, an indentation appears at the base of a single ACF crypt, with subsequent formation of a bifurcation and eventual crypt division.     

Increased Cell Proliferation of Azoxymethane-induced Aberrant Crypt Foci of Rat Colon

Aberrant crypt foci (ACF) were induced in the colon of F344 rats by s.c. injection of azoxymethane (AOM) twice in a three day-interval and examined after 4 and 12 weeks. The number and crypt multiplicity of ACF in each section of rat colon increased during this period. Histologically, aberrant crypts consisted of proliferating atypical epithelial cells. Cell proliferation of ACF consisting of 4 aberrant crypts [ACF(4)] and 2 aberrant crypts [ACF(2)], and normal crypts in the colon of rats treated with AOM [normal crypts/AOM(+)] or saline [normal crypts/AOM(-)] was investigated by measurement of the mitotic index, proliferating cell nuclear antigen-labeling index (PCNA-LI), and 5-bromo-2'-deoxyuridine-labeling index (BrdU-LI). All three parameters of the cell proliferative activity of ACF(4) were higher than those of normal crypts/AOM(+) and normal crypts/AOM(-). The PCNA-LI and BrdU-LI in ACF(2) were the same as those in ACF(4). These findings suggest that ACF have increased cell proliferative activity. The correlation of these three parameters confirmed that the PCNA-LI is also a useful parameter for evaluating cell proliferative activity in ACF. The presence of many cells stained by PCNA in the upper portion of ACF suggested that ACF have more G₁ phase cells, which readily respond to mitogenic stimulation, than G₀ phase cells, which are predominant in normal crypts.     

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.     

Dose response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon

Foci of aberrant crypts (ACF) have been identified in the unsectioned methylene blue stained rodent colons and hypothesized to represent precursor lesions of colon cancer. In the present study, induction and growth characteristics of ACF were investigated in response to a single injection of varying dosages of 1,2-dimethylhydrazine-2HCl (DMH), a colon carcinogen. Female Sprague-Dawley rats were given a single injection of DMH (5-150 mg/kg). Two and 19 weeks after the injection, animals were killed and their distal 10 cm of colons were enumerated for the number and crypt multiplicity of ACF. Number of ACF increased with increasing dosages of DMH plateauing at 100 mg/kg. However, percentage of ACF exhibiting different crypt multiplicity (1 to greater than 4) were similar among different dose groups. Aberrant crypts and normal crypts were enumerated for total number of cells and number and distribution of S-phase cells along the crypt height 19 weeks after DMH injection after autoradiography. The labeling index (LI) (percentage of S-phase cells) and LI along the crypt height were determined. Compared to the surrounding normal crypts, aberrant crypts exhibited significantly higher (P less than 0.05) number of cells (1122 +/- 81 versus 411 +/- 28) and higher (P less than 0.05) LI (21 +/- 1 versus 12 +/- 1). For the eight ACF analysed in the present study, the distribution of S-phase cells in the aberrant crypts were similar to that of normal crypts in that S-phase cells were restricted to the lower two-thirds of the crypts rather than distributed throughout the height of the crypts as reported for adenomatous epithelium.     

Growth features of aberrant crypt foci that resist modulation by cholic acid

Previously, we demonstrated that feeding rats a diet containing0.2% chotic acid (CHA-diet) resulted in the elimination or remodellingof a number of aberrant crypt foci (ACF) in their primal stages(1–3 crypts/focus). The Present investigation was conductedto determine if ACF with advanced growth features will responddifferently than their primal counterparts to the CHA-diet.Sprague-Dawley male rats were injected with azoxymethane (20mg/kg)and were maintained on a control diet for 21 weeks. At week21, three rats were killed and their colons were assessed forACF. The remaining animals were randomly devided into two groups,which were fed a control diet or a CHA-diet respectively, After3 weeks of feeding, the rats (n = 5) were killed and their colonswere assessed for the number, size (area occupied by each focus)and crypt multiplicity (number of crypts/focus). The CHA-dietresulted in a significant (P     

Classification of aberrant crypt foci and microadenomas in human colon.

Aberrant crypt foci (ACF) can be observed and quantified on the mucosal surface of formalin-fixed human colon resections after staining with methylene blue. To determine whether these ACF could be identified in fresh tissue, 10 colon resections were collected after surgery for colorectal cancer. Unfixed and fixed flat normal colonic mucosa from each colon were scored for ACF under a dissecting microscope after methylene blue staining. The number of ACF per cm2 and the average number of crypts per foci correlated highly in unfixed and fixed mucosa (r = 0.93 and 0.78, respectively). A significantly higher frequency of lesions was found in left-sided compared to right-sided colon resections. To determine whether the topographic features of the ACF gave an indication of the histological appearance, 68 specimens containing ACF or normal mucosa were examined histologically. The presence of slit-like lumen in the crypts of ACF on the mucosal surface correlated with the presence of dysplasia at histology, thus identifying microadenomas. These two observations suggest that the topographic classification of ACF in vivo could be used to distinguish microadenomas, a putative precursor lesion of colon cancer.     

Relationship between the Nature of Mucus and Crypt Multiplicity in Aberrant Crypt Foci in the Rat Colon

Aberrant crypt foci (ACF)induced in the distal colon of F344 male rats, 4, 8, 12 and 35 weeks after the first administration of 1, 2-dimethylhydrazine-2HCl (DMH) were examined to determine whether a correlation exists between the nature of goblet cell mucin and the number of crypts (crypt multiplicity) comprising the ACF. According to the ACF score calculated from the results of the qualitative observation of sulfomucins (SuMs) and sialomucins (SiMs), the ACF in the 4th week showed a weak correlation between the nature of the mucus and crypt multiplicity, and the ACF of each class showed similar mucous profiles. From the 8th week, a significant difference ( P <0.01) was recognized between the ACF consisting of 3 crypts or less and those consisting of 4 crypts or more. The proportion of crypts with SiM predominance showed a decrease in the 8th.week in the ACF consisting of 1 crypt and in the 12th week in the ACF consisting of 2 or 3 crypts, implying a recovery tendency. The ACF consisting of more than 4 crypts showed little change over time, retaining the tendency of SiM predominance. Ulex europaeus agglutinin-I (UEA-I) lectin-positive crypts appeared in the ACF. This finding was significantly more prominent ( P < 0.001) in the ACF with SiM predominance than in the ACF with SuM predominance at each experimental period, and in the 12th week after the first administration of DMH, the incidence of ACF with UEA-I-reactive mucin was decreased in the ACF groups consisting of 3 crypts or less, compared with the ACF groups consisting of 4 or more crypts.These results suggest that the biological quality of mucus in ACF consisting of 4 or more crypts is different from that in ACF consisting of 3 crypts or less. This difference should be considered when ACF are used as an intermediate biomarker of colon cancer.     

Frequent beta-catenin gene mutations and accumulations of the protein in the putative preneoplastic lesions lacking macroscopic aberrant crypt foci appearance, in rat colon carcinogenesis

Activating mutations in the beta-catenin gene is thought to be responsible for the excessive beta-catenin signaling involved in the majority of carcinogen-induced colonic carcinomas. To determine whether beta-catenin signaling is involved in the initial stage of colon carcinogenesis, mutational analysis of the gene and immunohistochemistry for beta-catenin protein were performed in the early appearing lesions, including aberrant crypt foci (ACF), of colonic mucosa in rats given azoxymethane. Male F344 rats received s.c. injections of azoxymethane at a dose of 15 mg/kg body weight, once weekly for 3 weeks, and they were sacrificed 10 weeks after the carcinogen treatment. The colonic mucosa was examined in en face preparations and in serial sections after the observation in whole mount preparations. Microscopical observations in the cross sections have shown two populations of histologically altered crypts. The first type had a macroscopic feature resembling ACF [histologically altered crypts with ACF appearance (HACAs)]. The second type of altered crypts did not have the ACF-like appearance and could not be clearly distinguished from adjacent normal crypts in whole mount preparations [histologically altered crypts with macroscopically normal-like appearance (HACNs)]. The beta-catenin gene mutations were recognized in 10 of 15 HACNs (67%) and 3 of 15 HACAs (20%). Frequent immunoreactivity of beta-catenin protein was seen in the cytoplasm of HACNs (13 of 15 cases), whereas apparent accumulation was not confirmed in any HACAs analyzed. These results suggest that (a) there are two types of putative preneoplastic lesions in cancer-predisposed colonic mucosa, and beta-catenin signaling may contribute to the initial stage of colon carcinogenesis; and (b) HACNs are more likely to be direct precursors of colon tumors than HACAs in the rat colon carcinogenesis.     

Qualitative and quantitative relationship between dysplastic aberrant crypt foci and tumorigenesis in the Min/+ mouse colon.

The multiple intestinal neoplasia (Min)/+ mouse, which harbors only one functional allele of the Apc gene, is susceptible to environmental factors that disrupt this gene and subsequently trigger Apc-driven tumorigenesis in the colon. Aberrant crypt foci (ACF) are assumed to be preneoplastic lesions in colon carcinogenesis. Recently, we reported the absence of "classical" ACF in the colon of untreated Min/+ mice. Instead we identified flat dysplastic lesions, which we denoted ACF(Min) (J. E. Paulsen et al., Scand. J. Gastroenterol., 35: 534-539, 2000). In contrast to the classical type, ACF(Min) are not elevated above the surrounding mucosa, and their detection is totally dependent on methylene blue staining and transillumination. In the present study, we treated Min/+ mice with 5 mg/kg body weight azoxymethane (AOM) at weeks 1 and 2 and demonstrated induction of both types of lesions. However, only ACF(Min) appeared to be associated with the development of adenomas. Monocryptal ACF(Min), large ACF(Min), and adenomas showed a uniform histopathological picture of dysplasia and cytoplasmic overexpression of beta-catenin, indicating a qualitative relationship between these lesions. Also a quantitative relationship was suggested because the dramatic decrease in ACF(Min) number from week 7 to 11 was paralleled by a reciprocal increase in tumor number, indicating fast-crypt multiplication of ACF(Min). In AOM-treated +/+ (wild-type) littermates, a low number of ACF(Min) and tumors with the same characteristics as in Min/+ mice was seen. In contrast to ACF(Min), histopathological and immunohistochemical examination of classical ACF showed normal or hyperplastic crypts with normal levels of beta-catenin expression. In AOM-treated Min/+ mice, the number of classical ACF was virtually constant from week 7 to 11, and only a modest increase of crypt multiplicity was observed. The number of AOM-induced classical ACF at week 11 was not different in Min/+ mice and +/+ mice. In conclusion, we identified two distinct populations of altered crypts in the colon of Min/+ mice after AOM treatment. The ACF(Min), which resemble the dysplastic ACF described previously, clearly showed a continuous development from the monocryptal stage to adenoma, and they were characterized by fast-growing crypts with altered control of beta-catenin. In contrast, the classical ACF, which resemble the hyperplastic ACF described previously, were characterized by slow-growing crypts with normal beta-catenin expression, and they were probably not related to tumorigenesis.     

Development and distribution of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced aberrant crypt foci in the rat large intestine.

Aberrant crypt foci (ACF) are generally considered to be preneoplastic lesions for colon cancer. To assess their induction by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a colon carcinogen, we performed a sequential study of ACF morphology and localization. F344 male rats were given PhIP, and methylene blue-stained colon epithelium and isolated crypts were analyzed at weeks 12, 25, 50, and 75. Each crypt was classified into 2 groups, "single" with round bottoms and "bifurcating" displaying V-shaped clefts (indicating proliferation). In combination with the number of crypts in an ACF, this classification was a good indicator for the generation of ACF in line with the fission mechanism of growth. Increasing numbers of crypts in ACF through weeks 12 to 75 and decreased percentages of ACF with bifurcating crypts at the late time points indicated that proliferation of crypts occurs predominantly during the early stages. The distribution pattern showed a significant shift (P < 0.000005) from the distal to the proximal part of the large intestine between weeks 25 and 50. Adenocarcinomas were first found to develop at week 50 in the ascending colon and cecum where bifurcating crypts were generally lacking at weeks 12 and 25. These data suggest the existence of (1) proliferating ACF which contains bifurcating crypt(s) and (2) quiescent or senescent ACF which consists of only single crypts.     

Evidence for a ras gene mutation in azoxymethane-induced colonic aberrant crypts in Sprague-Dawley rats: earliest recognizable precursor lesions of experimental colon cancer.

The main objective of the present investigation was to understand the molecular events involved in the genesis of aberrant crypt foci. Aberrant crypt foci were induced in Sprague-Dawley rats with a single injection of azoxymethane. Aberrant crypts have been identified topographically in the colon and are hypothesized to represent preneoplastic lesions. In order to understand the molecular events involved in the early stages of colon cancer, PCR-amplified DNA from aberrant crypts was hybridized with oligonucleotide probes specific for the detection of point mutations in codon 12 of K-ras. The mutation identified was a G to A transition resulting in the substitution of the amino acid aspartic acid (asp) for glycine (gly). This mutation was present in 6/19 (32%) of aberrant crypts examined. The identical mutation was also identified in adenomacarcinoma tissue while no mutation could be detected in normal intestinal mucosa. For further confirmation of these results, the presence of the mutated ras protein (rasAsp-12) was detected in aberrant crypts by immunohistochemistry. This investigation provides the first identification of a ras point mutation in aberrant crypt foci.     

Resistance of aberrant crypt foci to apoptosis induced by azoxymethane in rats chronically fed cholic acid

We have previously shown that chronic feeding of cholic acidto carcinogen treated rats reduces the number of putativepreneoplasticlesions of colonic cancer, aberrant crypt foci(ACF), but enhancesthe growth of remaining ACF and the incidence of colonic tumors.The following study was conducted to further explore the effectsof cholic acid on ACF growth by determining if ACF in cholicacid-fed animals display resistance to apoptotic cell death.ACF were induced in male Sprague- Dawley rats with two injectionsof azoxy-methane (20 mg/kg body wt). Rats were divided intotwo groups and fed either the control AIN-76 diet or the AIN-76diet containing 0.2% cholic acid. After 18 weeks, colonic apoptoticcelldeath was induced with an acute low dose of azoxymethane(10 mg/kg body wt). The number of cells, apoptotic bodies andbromodeoxyuridine (BUdR)-labeled cells were determined in coloniccrypts comprising ACF and surrounding normal crypts in ratsfrom each diet group. The number of apoptotic bodies per 100cells was lower in ACF crypts than in normal-appearing crypts(P = 0.0034). Both normal and ACF crypts from rats fed the cholicacid diet had fewer apoptotic bodies per 100 cells than cryptsfrom rats fed the control diet (P =0.0102). These data suggestthat ACF harbor resistanceto induction of apoptosis. Chronicfeeding of a diet containing 0.2% cholic acid results in thedevelopment of increased resistance to apoptosis. The lowerrate of cell death in ACF may contribute to the enhanced growthof ACF and higher tumor incidence previously observed in cholicacid-fed animals.     

Inhibition of dimethylhydrazine-induced aberrant crypt foci and induction of apoptosis in rat colon following oral administration of the glucosinolate sinigrin

Glucosinolates are sulphur compounds that occur as glycosides in brassica vegetables. In response to tissue disruption they are degraded by thioglucosidase, releasing a range of highly reactive breakdown products, including the isothiocyanates, which we have previously shown to be selectively cytotoxic to undifferentiated colorectal tumour cells (HT29). In the present study we explored the effect of sinigrin on the intestinal mucosa of rats previously treated with dimethylhydrazine (DMH). In the first experiment, a semisynthetic feed containing sinigrin (400 microg/g diet) was provided 6 h after the second of two injections of DMH. The level of apoptosis was measured by morphological assessment of intact microdissected crypts obtained at 18, 24, 38, 48 and 72 h after injection, and compared with control groups given DMH only, or a sham-injection. Higher numbers of apoptotic nuclei were present in colonic tissue from both groups of DMH-treated rats compared with the controls, and the level was significantly higher in DMH- treated rats fed sinigrin compared with those given DMH only (P < 0.02). In a second experiment, rats were given sinigrin (400 microg/g diet) 22 h after the second of two injections of DMH; the level of apoptosis was measured after 48 h and the numbers of aberrant crypt foci (ACF) were measured after 42 days. The level of apoptosis was significantly higher in DMH-treated rats given sinigrin compared with controls (P < 0.05), and the numbers of ACF were significantly lower in sinigrin-treated rats (P < 0.001). There was no statistically significant induction of apoptosis in animals fed sinigrin alone. Sinigrin administered after DMH suppresses induction of ACF. This may be due to increased apoptotic deletion of damaged stem cells in the crypts of animals fed sinigrin.      

Characterisation of aberrant crypt foci in carcinogen-treated rats: association with intestinal carcinogenesis.

Carcinogen-treated rats develop foci of aberrant crypts in the colon (ACFs) that have been interpreted as preneoplastic lesions. To characterise ACFs further, we studied in the unsectioned colon of rats the number, multiplicity, some morphological characteristics and the type of mucin production in ACFs. In ACFs observed 115 days after the administration of 50 mg kg-1 1,2-dimethylhydrazine (DMH), crypt multiplicity [number of aberrant crypts (AC) per focus] was positively correlated (P < 0.0001) with the reduction of goblet cells, and with luminal and nuclear alterations in the cells surrounding the lumen of the ACs. We studied mucin production in the unsectioned colon, demonstrating that ACFs producing sulphomucins (like the normal distal rat colon) were progressively reduced when ACF multiplicity increased, whereas ACFs containing sialomucins (correlated with an increased risk of colon cancer) or both sulphomucins and sialomucins increased with crypt multiplicity. We also studied ACFs in the colon and the occurrence of intestinal tumours in rats treated with azoxymethane (AOM; 64 mg kg-1). A significant association was found (P = 0.04) between tumours and the presence of ''large'' ACFs (AC/ACF > 14 crypts) and a borderline significant association (P = 0.057) between the presence of tumours and sialomucin-producing ACFs. We found no association between the number of ACFs, ACF multiplicity and the presence of tumours.     

Development and Distribution of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]-pyridine (PhIP)-induced Aberrant Crypt Foci in the Rat Large Intestine

Aberrant crypt foci (ACF) are generally considered to be preneoplastic lesions for colon cancer. To assess their induction by 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP), a colon carcinogen, we performed a sequential study of ACF morphology and localization. F344 male rats were given PhIP, and methylene blue-stained colon epithelium and isolated crypts were analyzed at weeks 12, 25, 50, and 75. Each crypt was classified into 2 groups, "single" with round bottoms and "bifurcating" displaying V-shaped clefts (indicating proliferation). In combination with the number of crypts in an ACF, this classification was a good indicator for the generation of ACF in line with the fission mechanism of growth. Increasing numbers of crypts in ACF through weeks 12 to 75 and decreased percentages of ACF with bifurcating crypts at the late time points indicated that proliferation of crypts occurs predominantly during the early stages. The distribution pattern showed a significant shift ( P < 0.000005) from the distal to the proximal part of the large intestine between weeks 25 and 50. Adenocarcinomas were first found to develop at week 50 in the ascending colon and cecum where bifurcating crypts were generally lacking at weeks 12 and 25. These data suggest the existence of (1) proliferating ACF which contains bifurcating crypt(s) and (2) quiescent or senescent ACF which consists of only single crypts.     

Green tea polyphenols inhibit colorectal aberrant crypt foci (ACF) formation and prevent oncogenic changes in dysplastic ACF in azoxymethane-treated F344 rats

Green tea and its constituents have shown cancer-preventive activities in many animal models. In order to prepare for a human trial on the inhibition of colon carcinogenesis, we conducted a study with green tea polyphenols as the preventive agent in an azoxymethane (AOM)-induced rat colon cancer model using aberrant crypt foci (ACF) as an end point. F344 rats were given two weekly injections of AOM (15 mg/kg), and then fed a 20% high-fat diet with or without 0.12 or 0.24% Polyphenon E (PPE, a standardized green tea preparation consisting 65% of (-)-epigallocatechin-3-gallate and 22% of other catechins) for 8 weeks. Colorectal ACF were analyzed under a microscope after methylene blue staining. Dietary PPE administration was found to significantly and dose dependently decrease the total number of ACF per rat and the total number of aberrant crypt per rat. Moreover, treatment with 0.24% PPE also significantly decreased the percentage of large ACF (four or more crypts) and the percentage of ACF with high-grade dysplasia in total ACF. The high-grade dysplastic ACF from 0.24% PPE-treated group had increased apoptosis and decreased nuclear expression levels of beta-catenin and cyclin D1. Retinoid X receptor (RXR)alpha expression was reduced in high-grade dysplastic ACF, adenoma and adenocarcinoma during AOM-induced colon carcinogenesis, and the PPE treatment partially prevented the loss of RXRalpha expression in high-grade dysplastic ACF. Taken together, our results strongly suggest the colon cancer-preventive activity of PPE and identified possible molecular markers for future colon cancer prevention studies.     

Fatty acid synthase is over‐expressed in large aberrant crypt foci in rats treated with azoxymethane

Fatty acid synthase (FAS) is over‐expressed in many human cancers including colon. High levels of FAS expression have also been observed in a number of lesions that are precursors to invasive colorectal cancer. However, FAS expression in aberrant crypt foci (ACF), the earliest identifiable lesions in colon cancer development, has not been investigated. In this study, we treated Fisher rats with a single dose of the colon carcinogen azoxymethane then evaluated ACF 100 days later. We showed that large ACF (≥4 crypts/focus) have a significantly higher level of immunohistochemical staining for FAS than either small ACF (≤3 crypts/focus) or normal crypts. Furthermore, the severity of nuclear atypia in ACF was positively associated with increased expression of FAS. These findings suggest that the genes associated with FAS over‐expression are activated early in the stepwise development of colon cancer, though not until the ACF have reached a critical size with a level of nuclear atypia indicative of dysplasia. © 2008 Wiley‐Liss, Inc.