Efficacy of potential chemopreventive agents on rat colon aberrant crypt formation and progression

We assessed the effects of 78 potential chemopreventive agents in the F344 rat using two assays in which the inhibition of carcinogen-induced aberrant crypt foci (ACF) in the colon was the measure of efficacy. In both assays ACF were induced by the carcinogen azoxymethane (AOM) in F344 rats by two sequential weekly injections at a dose of 15 mg/kg. Two weeks after the last AOM injection, animals were evaluated for the number of aberrant crypts detected in methylene blue stained whole mounts of rat colon. In the initiation phase protocol agents were given during the period of AOM administration, whereas in the post-initiation assay the chemopreventive agent was introduced during the last 4 weeks of an 8 week assay, a time when ACF had progressed to multiple crypt clusters. The agents were derived from a priority listing based on reports of chemopreventive activity in the literature and/or efficacy data from in vitro models of carcinogenesis. During the initiation phase carboxyl amidoimidazole, p-chlorphenylacetate, chlorpheniramine maleate, D609, diclofenac, etoperidone, eicosatetraynoic acid, farnesol, ferulic acid, lycopene, meclizine, methionine, phenylhexylisothiocyanate, phenylbutyrate, piroxicam, 9-cis-retinoic acid, S-allylcysteine, taurine, tetracycline and verapamil were strong inhibitors of ACF. During the post-initiation phase aspirin, calcium glucarate, ketoprofen, piroxicam, 9-cis-retinoic acid, retinol and rutin inhibited the outgrowth of ACF into multiple crypt clusters. Based on these data, certain phytochemicals, antihistamines, non-steroidal anti-inflammatory drugs and retinoids show unique preclinical promise for chemoprevention of colon cancer, with the latter two drug classes particularly effective in the post-initiation phase of carcinogenesis.     

Reduction in formation of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP)-induced aberrant crypt foci in the rat colon by docosahexaenoic acid (DHA)

Docosahexaenoic acid (DHA), a major component of fish oil, suppresses the formation and growth of aberrant crypt foci induced by 1,2- dimethylhydrazine and azoxymethane. In the present study we examined the effects of intragastric gavage administration of DHA on the yield of rat colonic aberrant crypt foci due to treatment with a heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which induces colon cancer in male F344 rats and is considered to be a possible human colon carcinogen. Male F344 rats were given a standard diet (AIN-76A) and received 10 doses of PhIP (75 mg/kg body wt, by intragastric intubation, on days 1-5 and 8-12) with or without intragastric application of 1 ml DHA 4 h prior to each carcinogen treatment, followed by further DHA dosing. The numbers of PhIP-induced aberrant crypt foci per colon after 4 and 12 weeks DHA administration were significantly reduced to 47 and 38% respectively of the values obtained when PhIP alone was used. The mean number of aberrant crypts per focus was also decreased by DHA treatment. At week 4 the PhIP-DNA adduct levels in the colon of rats from the PhIP+DHA group were approximately two thirds of the PhIP group value. The results thus suggest that DHA exerts a preventive effect on PhIP-induced colon carcinogenesis.      

Prevention by chemopreventive agents of azoxymethane-induced foci of aberrant crypts in rat colon.

Foci of aberrant crypts are putative preneoplastic lesions of colon cancer that can be detected in unsectioned colons stained with methylene blue. The ability of this assay to demonstrate chemopreventive activity was evaluated. Male Sprague-Dawley rats received two subcutaneous injections 1 week apart, of 15 mg/kg azoxymethane each. The animals started to receive the test agents in their diet 1 week prior to the first injection of azoxymethane and continuously until killed 5 weeks later. The number of foci of aberrant crypts induced by the treatment of azoxymethane was reduced from 228 foci/animal without any chemopreventive agent to 151 foci/animal by N-acetylcysteine; to 121 foci/animal by dehydroepiandrosterone; to 161 by alpha-difluoromethylornithine; and to 121 by 1,2-oxothiazolidine-4-carboxylate. The other agents (diallyl sulfide, ellagic acid and phenethyl isothiocyanate) did not significantly alter the number of foci/animal induced by azoxymethane. Animals that did not receive azoxymethane had an average of 0.72 foci/animal. Our results suggest that four of the tested agents might reduce azoxymethane-induced colon cancer, which requires confirmation. Further validation of the foci of aberrant crypt in the colon assay to screen chemicals for chemoprevention agents is warranted.     

Expression of ras oncogene mRNA and protein in aberrant crypt foci.

The expression of the ras oncogene in aberrant crypt foci was studied by both in situ hybridization and immunohistochemical approaches. Aberrant crypt foci are hypothesized to represent the earliest identifiable microscopic lesions of colon cancer in rodent colons. Sprague-Dawley male rats were injected with azoxymethane (20 mg/kg s.c.) once. Twelve weeks later, aberrant crypt foci were identified topographically, microdissected and processed for histology. In situ hybridization with an antisense oligomer of c-ras demonstrated increased expression of ras-specific RNA in aberrant crypts compared to normal crypts. A low amount of non-specific hybridization was obtained with the corresponding sense oligomer. The percentage of cells with grains (labeling index) was calculated in early and advanced aberrant crypt foci. This index was also calculated in normal appearing crypts. The labeling indices for the early and advanced aberrant crypt foci were significantly greater than that of normal crypts (18.0 and 25.0 versus 11.9). In the same tissue specimens, immunohistochemical staining for ras p21 with the monoclonal antibody (Y13-259) revealed strong staining intensity in early aberrant crypts (15/22) and advanced aberrant crypts (22/30) compared to normal crypts (3/50). The immunohistochemical results demonstrate the presence of elevated levels of ras p21 in the same tissue as increased levels of ras-specific message. This investigation provides the earliest demonstration of increased expression of the ras oncogene in precursor lesions of colon cancer possessing dysplastic features.     

Aberrant crypt foci and colon tumors in F344 rats have similar increases in proliferative activity

Increased proliferative activity has been described frequently in the colons of animals treated with colon carcinogens and of patients at increased risk of colon cancer; it has been proposed as an intermediate biomarker of colon cancer. Aberrant crypt foci, microscopic lesions identified in whole-mount preparations of colons, are thought to be putative pre-neoplastic lesions. The present studies were carried out to evaluate the proliferative activity of aberrant crypt foci at several different time periods, and of tumors after a single dose of azoxymethane (AOM) in F344 rats. Rats were injected with 5-bromo-2′-deoxyuridine (BUdR) 1 hr before killing. Aberrant crypt foci and tumors were identified and marked in the whole-mount specimens, embedded in glycol methacrylate, and evaluated for histochemically demonstrable hexosaminidase activity. Hexosaminidase is known to be altered in over 95% of aberrant crypt foci. Serial sections were evaluated for BUdR incorporation immunohistochemically with a monoclonal antibody. The mean proliferative activity of aberrant crypt foci in the distal colons was found to be 'increased 3-to 4-fold over that of the adjacent normal crypts at every time period analyzed (4 to 36 weeks) and was comparable to that seen in benign and malignant colon tumors in the same animals. The observed increase in proliferative activity further supports the hypothesis that aberrant crypt foci are putative pre-neoplastic lesions. Similar aberrant crypt foci, identified in human colons at increased risk of colon cancer, may provide important biomarkers for this common human cancer.     

Suppression of Occurrence and Advancement of β-Catenin-accumulated Crypts, Possible Premalignant Lesions of Colon Cancer, by Selective Cyclooxygenase-2 Inhibitor, Celecoxib

Suppression of occurrence and advancement of premalignant lesions is important for cancer prevention. Our previous studies clarified that β-catenin-accumulated crypts, independent of aberrant crypt foci (ACF), are probably direct precursors of colon cancers in rats. Here we investigated the effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on the development of β-catenin-accumulated crypts in comparison with those on ACF. Male F344 rats were divided into 4 groups. Groups 1-3 were administered azoxymethane (AOM) s.c. at a dose of 15 mg/kg body weight, once weekly for 3 weeks to induce β-catenin-accumulated crypts. Groups 2 and 3 also received experimental diet containing celecoxib (500 and 1500 ppm, respectively) for 8 weeks, starting a week before the first dosing of AOM. At termination, the frequency and crypt multiplicity (number of crypts/lesion) of β-catenin-accumulated crypts of groups 2 and 3 were significantly less than that of group 1. Furthermore, numbers of silver-stained nucleolar organizer regions (AgNORs)/nucleus in β-catenin-accumulated crypts were also decreased by exposure to celecoxib. In this study, celecoxib had greater effects on the frequency and growth of β-catenin-accumulated crypts than on those of ACF. These findings represent additional evidence that β-catenin-accumulated crypts are premalignant lesions of colon cancer. The results also suggest that β-catenin-accumulated crypts could be a novel target for evaluation of possible chemopreventive agents against colon carcino-genesis, and indicate that possible chemopreventive effects of celecoxib on the initial stage of colon carcinogenesis may be related to modulation of cell proliferation activity in such early lesions.     

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.     

Effects of all-trans retinoic acid as a potential chemopreventive agent on the formation of azoxymethane-induced aberrant crypt foci: Differential expression of c-myc and c-fos mrna and protein

The main objectives were to determine the modulating effects of all-trans retinoic acid on the number, size and multiplicity of aberrant crypt foci as well as the in vivo expression of the genes c-myc and c-fos. These foci, which are hypothesized to be the pre-malignant lesions of colon cancer, were induced in Sprague-Dawley rats with a single injection of azoxymethane. Rats were fed either a control diet (AIN-76) or the control diet to which had been added 75 mg/kg or 150 mg/kg all-trans retinoic acid. Within 4 weeks, we observed that the diets containing all-trans retinoic acid reduced the total number and multiplicity of aberrant crypt foci in the colon. However, all-trans retinoic acid increased the size of the lesions that persisted, possibly due to a greater proportion of lesions with dilated crypts. In situ hybridization and immunohistochemistry were performed on the colons for the in vivo analysis of gene expression in these lesions. The expression of myc-specific mRNA and protein in aberrant crypt foci significantly decreased with both levels of all-trans retinoic acid. In contrast, fos-specific mRNA and protein in aberrant crypt foci significantly increased when 150 mg/kg all-trans retinoic acid was added to the diet. The most important findings of this investigation are that intervention with all-trans retinoic acid in the pre-malignant stage of colon carcinogenesis is effective in decreasing the number and growth of aberrant crypt foci and altering the expression of the c-myc and c-fas genes.     

The effect of an oral administration of Lactobacillus casei strain shirota on azoxymethane-induced colonic aberrant crypt foci and colon cancer in the rat

The preventive effect of oral administration of viable Lactobacillus casei strain Shirota (LcS) on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) and colon cancers in the rat was investigated. The study consisted of two experiments; in a short-term experiment (Exp-I), the inhibitory effect of 8- and 12-week treatments with LcS. Forty rats each received weekly a subcutaneous injection of AOM at a dose of 15 mg/kg of body weight for 5 weeks. Eight and twelve weeks after the start of the carcinogen treatment, each subgroup of rats were sacrificed, and the colon and the mesenteric lymph nodes (MLN) were removed. The number of ACFs and the surface marker of lymphocytes derived from the MLN were investigated. The large ACF (those comprising four or more aberrant crypts per focus) had significantly decreased in the rats which had consumed the LcS diet. And oral administration of viable LcS significantly recovered CD8 positive lymphocytes to the levels in the control group. In a long-term experiment (Exp-II), 30 rats each received weekly a subcutaneous injection of AOM at a dose of 7. 4 mg/kg of body weight for 10 weeks. Twenty-five weeks after the start of the carcinogen treatment, each subgroup of rats were sacrificed, and the colon were removed. The number and incidence of colon cancers were investigated. The number of rats with colon cancers and the number of colon cancers per rat, were significantly decreased in the rats which had consumed the LcS diet. LcS inhibited chemically-induced colon carcinogenesis in the rat. CD8 positive T lymphocytes may play a key role in the preventive effect against colon carcinogenesis.     

Suppression of occurrence and advancement of beta-catenin-accumulated crypts, possible premalignant lesions of colon cancer, by selective cyclooxygenase-2 inhibitor, celecoxib.

Suppression of occurrence and advancement of premalignant lesions is important for cancer prevention. Our previous studies clarified that beta-catenin-accumulated crypts, independent of aberrant crypt foci (ACF), are probably direct precursors of colon cancers in rats. Here we investigated the effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on the development of beta-catenin-accumulated crypts in comparison with those on ACF. Male F344 rats were divided into 4 groups. Groups 1 - 3 were administered azoxymethane (AOM) s.c. at a dose of 15 mg / kg body weight, once weekly for 3 weeks to induce beta-catenin-accumulated crypts. Groups 2 and 3 also received experimental diet containing celecoxib (500 and 1500 ppm, respectively) for 8 weeks, starting a week before the first dosing of AOM. At termination, the frequency and crypt multiplicity (number of crypts / lesion) of beta-catenin-accumulated crypts of groups 2 and 3 were significantly less than that of group 1. Furthermore, numbers of silver-stained nucleolar organizer regions (AgNORs) / nucleus in beta-catenin-accumulated crypts were also decreased by exposure to celecoxib. In this study, celecoxib had greater effects on the frequency and growth of beta-catenin-accumulated crypts than on those of ACF. These findings represent additional evidence that beta-catenin-accumulated crypts are premalignant lesions of colon cancer. The results also suggest that beta-catenin-accumulated crypts could be a novel target for evaluation of possible chemopreventive agents against colon carcino-genesis, and indicate that possible chemopreventive effects of celecoxib on the initial stage of colon carcinogenesis may be related to modulation of cell proliferation activity in such early lesions.     

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.     

Foci of aberrant crypts in the colons of mice and rats exposed to carcinogens associated with foods

Aberrant crypt foci can be identified in the colons of rodents treated 3 wk earlier with azoxymethane, a known colon carcinogen. These crypts can easily be visualized in the unsectioned methylene blue-stained colons under light microscopy, where they are distinguished by their increased size, more prominent epithelial cells, and pericryptal space. They occur as single aberrant crypts or as two, three, or four aberrant crypts in a cluster. We compared the reported ability of carcinogens associated with the human diet to induce colon cancer with the measured rate of induction of aberrant crypts in female CF1 mice and Sprague-Dawley rats. The carcinogens used were 1,2-dimethylhydrazine, methyl nitrosourea, N-nitrosodimethylamine, benzo(a)pyrene, aflatoxin B1, 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole, 2-amino-3-methylimidazo[4,5-P]quinoline, 2-amino-3,4-dimethylimidazo[4,5-P]quinoline, and 3-amino-1-methyl-5H-pyrido[4,3-b]indole. Graded doses of these compounds were given to the animals by gavage twice with a 4-day interval, and the animals were terminated 3 wk later. All colon carcinogens induced aberrant crypts in a dose-related fashion. N-Nitrosodimethylamine and 3-amino-1-methyl-5H-pyrido[4,3-b]indole, carcinogenic compounds that do not induce colon cancer, did not induce them. The ability of the studied compounds to induce aberrant crypts was species specific; e.g., aflatoxin B1 and 2-amino-3,4-dimethylimidazo[4,5-P]quinoline induce about 20 times more in rats than mice. This relationship was consistent with their reported ability to induce colon cancer in these species. Results of the present study support the use of the aberrant crypt assays to screen colon-specific carcinogens and to study the process of colon carcinogenesis.     

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.     

Chemoprevention of colonic aberrant crypt foci by an inducible nitric oxide synthase-selective inhibitor

Inducible nitric oxide synthase (iNOS) is overexpressed in colonic tumors of humans and also in rats treated with a colon carcinogen. iNOS appear to regulate cyclooxygenase-2 (COX-2) expression and production of proinflammatory prostaglandins, which are known to play a key role in colon tumor development. Experiments were designed to study the inhibitory effects of S,S'-1,4-phenylene-bis(1,2-ethanediyl)bis-isothiourea (PBIT) a selective iNOS-specific inhibitor, measured against formation of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF). Beginning at 5 weeks of age, male F344 rats were fed experimental diets containing 0 or 50 p.p.m. of PBIT, or 2000 p.p.m. of curcumin (non-specific iNOS inhibitor). One week later, rats were injected s.c. with AOM (15 mg/kg body wt, once weekly for 2 weeks). At 17 weeks of age, all rats were killed, colons were evaluated for ACF formation and colonic mucosa was assayed for isoforms of COX and NOS activities. Both COX and iNOS activities in colonic mucosa of the AOM-treated rats were significantly induced. Importantly, 50 p.p.m. PBIT suppressed AOM-induced colonic ACF formation to 58% (P < 0.0001) and crypt multiplicity containing four or more crypts per focus to 78% (P < 0.0001); it also suppressed AOM-induced iNOS activity. Curcumin inhibited colonic ACF formation by 45% (P < 0.001). These observations suggest that iNOS may play a key regulatory role in colon carcinogenesis. Developing iNOS-specific inhibitors may provide a selective and safe chemopreventive strategy for colon cancer treatment.     

Use of azoxymethane-induced foci of aberrant crypts in rat colon to identify potential cancer chemopreventive agents

Foci of aberrant and/or hexosaminidase-negative crypts in ratcolon are putative precancerous lesions that have been proposedas biomarkers for Short-term bioassays for chemical carcinogensand chemopreventive agents. The ability of a substance to reducethe yield of azoxymethane (AOM)- induced foci in the colon ofmale Fischer 344 rats, was evaluated as a screening assay forchemopreventive agents. Twenty-eight test agents were administeredcontinuously in the diet from the start of the experiments untilthe animals were killed 35 days later. AOM was s.c. administeredeither as 15 mg/kg body wt on days 7 and 14 or as 30 mg/kg bodywt on day 7 of the experiment. Foci of aberrant crypts wereevaluated in whole mounts of methylene blue-stained colons.AOM induced twice as many foci when administered between 8.40and 11.00 a.m. than between 2.45 and 5.55 p.m. Calcium saltsof carbonate, chloride and glucarate decreased the yield ofAOM-induced foci while the acidic salts of lactate and phosphatedid not inhibit the formation of foci. Dimethyl fumarate, fumaricacid, genistein, piroxicam, simethicone, sodium suramin andsulindac reduced the yield of AOM induced foci of aberrant cryptswith genistein being the most potent. Only piroxicam of thisgroup has previously been shown to inhibit colon cancer, whilethe rest have yet to be evaluated. Ibuprofen did not inhibitthe formation of foci, although it has been reported to inhibitAOM-induced colon cancer in rats. Piroxicam and sulindac appearedto reduce preferentially hexosaminidase-negative foci of aberrantcrypts, compared with those of apparently normal morphology.The AOM-induced foci of aberrant crypts assay appears suitablefor screening chemicals for chemopreventive action.     

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.     

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.     

Risk of Colonic Cancer is Not Higher in the Obese Lepob Mouse Model Compared to Lean Littermates

Epidemiological data suggest that obesity increases the risk of colorectal cancer in humans. Given that diet-induced obesity mouse models verified the epidemiological data, the present study aimed to determine whether obese C57BL/6J-Lep^ob male mice (a different obesity in vivo model) were at greater risk of colonic cancer than their lean male littermates. Risk of colonic tumorigenesis was assessed by numbers of aberrant crypts, aberrant crypt foci and colonic tumors. Proliferation of the colonic epithelia was assessed histochemically following administration of BrdU. Availability of the procarcinogen, azoxymethane (AOM) to target tissues was assessed by quantifying via HPLC plasma AOM concentrations during the 60 min period following AOM injection. When obese and lean mice were injected with azoxymethane (AOM) at doses calculated to provide equivalent AOM levels per kg lean body mass, obese animals had significantly fewer aberrant crypts/colon and fewer aberrant crypt foci/colon than the lean animals. Tumors were identified in the colonic mucosa of lean (4 tumors in 14 mice) but not obese (0 tumors in 15 mice) mice. Colonic cell proliferation was not significantly different for obese and lean mice. Because these results were unexpected, plasma AOM concentrations were measured and were found to be lower in the obese than lean mice. When plasma AOM levels were comparable for the lean and obese mice, the Lep^ob mice continued to have significantly fewer aberrant crypt foci/colon than the lean mice, but differences were not statistically different for aberrant crypts/colon. Interestingly, obese Lep^ob mice did not exhibit increased risk of colonic cancer as expected. Instead, Lep^ob mice exhibited equivalent or lower risk of colon cancer when compared to the lean group. These results taken together with in vivo results from diet-induced obesity studies, imply that leptin may be responsible for the increased risk of colon cancer associated with obesity.     

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.     

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.