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.     

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.     

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.     

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.     

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

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

Chemoprevention of 1,2-dimethylhydrazine-induced colonic preneoplastic lesions in Fischer rats by 6-methylsulfinylhexyl isothiocyanate, a wasabi derivative

The preventive effects of dietary exposure to a wasabi derivative 6-methylsulfinylhexyl isothiocyanate (6-MSITC) during the initiation and post-initiation phases on the development of 1,2-dimethylhydrazine (DMH)-induced colonic aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCAC) were investigated in male F344 rats. To induce ACF and BCAC, rats were given four weekly subcutaneous injections of DMH (40 mg/kg body weight). The rats also received diets containing 200 or 400 ppm 6-MSITC during the initiation or post-initiation phases. The experiment was terminated 12 weeks after the start. DMH exposure produced a substantial number of ACF (323.8±69.7/colon) and BCAC (3.80±1.05/cm(2)) at the end of the study. Dietary administration of 6-MSITC at a dose of 400 ppm during the initiation phase caused a significant reduction in the total number of ACF (52% reduction, P<0.0001), larger ACF (4 or more crypt ACF) (58% reduction, P<0.001) and BCAC (76% reduction, P<0.00001). The dietary exposure to 6-MSITC significantly reduced the size (crypt multiplicity) of BCAC during both initiation and post-initiation treatment when compared to group 1 treated with DMH alone. Immunohistochemically, 6-MSITC administration lowered the proliferating cell nuclear antigen labeling index in ACF and BCAC. In addition, protein levels of hepatic cytochrome P-450 isozymes at 24 h after 6-MSITC exposure were significantly suppressed (P<0.01). The results indicated that 6-MSITC exerted chemopreventive effects in the present short-term colon carcinogenesis bioassay, through alterations in cell proliferation activity and drug metabolizing enzyme levels.     

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.      

Azoxymethane-induced colon tumors and aberrant crypt foci in mice of different genetic susceptibility

Azoxymethane (AOM) is an organotropic colon carcinogen that is commonly used to induce colon tumors in rodents. Unlike its parent compound, 1,2-dimethylhydrazine (DMH), a tumor susceptibility phenotype in inbred mice with respect to AOM has not been established. Thus, this study was undertaken to determine whether genetic susceptibility extends to this carcinogen. SWR/J, A/J (both susceptible to DMH carcinogenesis) and AKR/J (resistant) mice were treated with 10 mg/kg AOM i.p. once a week for 8 weeks. Twenty-five weeks after the initial injection, tumor yield was determined. With a single exception, only SWR/J and A/J mice developed tumors, with a distribution that was limited to the distal colon (16.3±1.1 and 36.4±2.4, respectively). The formation of aberrant crypt foci (ACF), putative preneoplastic lesions, was also assessed in whole-mount colons using Methylene Blue staining. Consistent with tumor multiplicity, the total number of ACF was highest in A/J mice, followed by SWR/J mice. In addition, A/J mice had a significantly greater number of large ACF (five or more crypts per foci) than the other strains. Despite the absence of colon tumors, however, AKR/J mice did develop a significant number of ACF. This finding suggests that ACF in resistant mice are persistent but do not progress to tumors.     

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.     

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

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

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.     

Progression of tumors arising from large ACF is associated with the MUC5AC expression during rat colon MNNG carcinogenis

Aberrant crypt foci (ACF) are microscopic lesions which have been postulated to precede the development of adenomas, precursors of colon cancer. The gastric M1/MUC5AC mucin has also been described as an early marker of colon carcinogenesis in the human and in the rat. To study changes in mucin expression associated with the genesis of tumors, Wistar rats were treated by intrarectal instillations of MNNG, twice a week for 2 weeks, and were sacrificed 10 (n = 20), 14 (n = 20), 22 (n = 20), 30 (n = 10) and 66 (n = 16) weeks after the beginning of the treatment. In the treated rats, the MUC5AC mucin was mainly expressed in ACF compared with the histologically normal mucosae, which showed few isolated MUC5AC-positive normal crypts. During carcinogenesis, the percentage of large ACF [> or =10 aberrant crypts] increased and the number of MUC5AC-positive (NCs) decreased. At Week 30, small tumors were observed arising from large ACF, both types of lesions expressing MUC5AC. At Week 66, large tumors showed remnants of MUC5AC-positive ACF in their adjacent mucosae. This observation suggests that the expression of MUC5AC is associated with the ACF/adenoma sequence and supports the notion of large ACF as precursors of adenomas/adenocarcinomas. Moreover, the expression of MUC5AC in the transitional mucosa adjacent to both rat and human colon tumors suggests that some human tumors could arise from large ACF, and reinforces the concept of the premalignant potential of these lesions.     

Intestinal MUC2 and gastric M1/MUC5AC in preneoplastic lesions induced by 1,2-dimethylhydrazine in rat: a sequential analysis

Our study was performed to sequentially analyze the expression of the intestinal mucin MUC2 and of the gastric mucin MUC5AC as indicators during progression of preneoplastic biomarkers in rat colon. F344 rats were sacrificed 2, 4, 8, 12, 24 and 36 weeks after injection of 1,2-dimethylhydrazine (DMH, 200 mg/kg, i.p.). The expression of MUC2 and of MUC5AC was studied by immunohistochemistry in preneoplastic lesions classified in two categories: histologically altered foci (HAF) and beta-catenin accumulated crypts (BCAC). HAF appeared 4 weeks after DMH injection. Their crypt multiplicity stagnated with time (3-4 crypts/foci) but gastric MUC5AC mucin was always observed in some goblet cells of the lesions of this category. In contrast, MUC2-immunostaining was not modified compared to the adjacent crypts. Double-immunofluorescence revealed that goblet cells which produced MUC5AC continued to express MUC2. In BCAC, crypt multiplicity and mucin expression strongly evolved with time. These lesions were observed only 8 weeks after DMH-injection. At this stage, 20% of BCAC showed a decreased MUC2 expression and 33% were MUC5AC immunopositive. At the 36-week point, 43% of BCAC had a reduced MUC2 staining and 90% were positive for MUC5AC. This immunopositivity was often observed in all the cells of these lesions. Seldom, some BCAC were depleted at the same time in MUC2 and in MUC5AC. Similar alterations in mucin expression were observed in human colonic pre-neoplastic lesions. These findings suggest that a decrease in MUC2 expression and staining of MUC5AC in non-goblet-like cells predicts histological progression of preneoplastic lesions.     

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.     

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.     

Non-steroidol anti-inflammatory drug effect on crypt cell proliferation and apoptosis during initiation of rat colon carcinogenesis.

Sustained use of non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colorectal cancer. However, the optimal drug, period of efficacy and mechanism(s) of action are unknown. Experiments were undertaken to determine which of several NSAIDs would modulate colon crypt cell proliferation or apoptosis when given during the initiation phase of 1,2-dimethylhydrazine (DMH)-induced rat colon cancer. Colon crypts located both away from and over an aggregate of lymphoid nodules (ALN) were examined. Rats were injected with aspirin, indomethacin, nabumetone, sodium salicylate, 16,16-dimethyl prostaglandin E2 or saline for 3 days and DMH or DMH vehicle on day 4 of each week for 8 weeks, then killed 3 days after the last DMH injection. At the time of killing, DMH had significantly increased crypt cell proliferation but not apoptosis. There was significantly more cell proliferation and apoptosis in crypts over the ALN than away from the ALN. Aspirin and salicylate increased proliferation and apoptosis in crypts over the ALN. Finally, the distributional peaks of cell proliferation and apoptosis were shifted significantly closer together after DMH. Thus, DMH increases proliferation and alters the distribution of proliferating and apoptotic cells in colon crypts early in carcinogenesis. Aspirin may suppress tumour incidence via salicylate by enhancing apoptosis in carcinogen-initiated cells.     

Aqueous and ethanolic extract fractions from the Brazilian propolis suppress azoxymethane-induced aberrant crypt foci in rats

We investigated the effects of the two fractions, aqueous (AEP) and ethanolic extracts of propolis (EEP) of the Brazilian propolis on azoxymethane (AOM)-induced aberrant crypt foci (ACF). Male Wistar Hannover (GALAS) rats were administered two weekly subcutaneous injections of AOM (20 mg/kg bw) and fed with diets mixed with AEP (100, 500 and 1,000 ppm) or EEP (500 and 1,000 ppm) for 4 weeks, starting one week before the first dosing of AOM. The modifying effects of the test extracts on ACF formation were assessed by counting the incidence and multiplicity of ACF at week 4. Proliferation cell nuclear antigen (PCNA)-labeling nuclei and apoptotic index were also immunohistochemically determined. Dietary supplementation with AEP and EEP significantly reduced the multiplicity of ACF with the effect of EEP being more potent than AEP. In the ACF and their surrounding non-lesional crypts, significantly lowered cell proliferation was observed in the rats, administered with AOM, and the extracts, while neither fraction affected the apoptotic index. Our findings suggest that AEP and EEP possess a chemopreventive ability in the early phase of colon carcinogenesis through the modulation of cell proliferation.     

Sequential analyses of the growth and morphological characteristics of aberrant crypt foci: putative preneoplastic lesions

The main objective of the present study was to sequentially analyze growth and morphological characteristics of aberrant crypt foci (ACF) in the rat colon. Sprague-Dawley rats were given a single injection of a carcinogenic dose of 1,2-dimethylhydrazine-HCl and at varying time points ranging from 2 to 57 weeks, groups of 5 rats were terminated. The number and crypt multiplicity of ACF were determined in the distal 8 cm of the colon. In addition, ACF were processed for histology and then graded for the presence of nuclear atypia using a score of 0-4. The findings of the present study demonstrated that ACF exhibit the characteristics expected for precursor lesions. ACF were present at all time intervals in large numbers in the colons of rats treated with 1,2-dimethylhydrazine-HCl and were present when adenocarcinomas were observed. The number of ACF with 4 or more crypts and those exhibiting a higher grade (grade 4) of nuclear atypia increased significantly at or beyond 19 weeks. These features of ACF, particularly the presence of nuclear atypia indicative of dysplasia, provide strong support for the hypothesis that ACF are precursor lesions of chemically induced colon cancer.     

Aberrant crypt foci in patients with colorectal cancer.

Aberrant crypt foci (ACF) are clusters of abnormally large colonic crypts identified on the mucosal surface of the human colon. They are thought to be preneoplastic lesions. The aim of the present study was to compare density (number of ACF per square cm of mucosal surface), crypt multiplicity (number of crypts per ACF) and histology of ACF in colonic resections of colorectal cancer patients resident in two Italian provinces with a twofold difference in colorectal cancer incidence rates. Thirty-two and 26 colonic resections were collected after operation in Ragusa (Southern Italy) and Modena (Northern Italy), respectively, and fixed in 10% formalin. Mucosal layers were observed under a light microscope at 25x after staining with methylene blue. Density of ACF was significantly higher in Modena (median 0.101 ACF cm(-2)) than in Ragusa (0.049, P = 0.001), whereas there was no difference in crypt multiplicity. ACF were classified into three groups according to histological features: ACF with mild alterations (hypertrophic ACF, 73%), ACF with hyperplasia (hyperplastic ACF, 17%) and ACF with dysplasia (microadenomas, 10%). The proportions of ACF in the three groups were similar in the two provinces. Density of ACF was higher and crypt multiplicity lower proceeding from proximal to distal large bowel. Microadenomas were observed only in the colon, whereas hyperplastic ACF were more frequent in the rectum. In conclusion, density of ACF correlates with colorectal cancer rates in two Italian provinces, and shows a positive gradient from proximal to distal large bowel. Histology of ACF suggests that they may be precursors of both hyperplastic and adenomatous polyps. These data provide further evidence of the role of ACF in human colorectal carcinogenesis.