Effects of long-term administration of sulindac on APC mRNA and apoptosis in colons of rats treated with azoxymethane

PURPOSE: Non-steroidal anti-inflammatory drugs, including sulindac, have been shown to exhibit anti-colon cancer activity; however, the detailed mechanisms concerning continuous long-term administration are still unclear. Therefore, we examined the anti-colon carcinogenesis effects of sulindac after prolonged administration. METHODS: Administration of AOM, a colon-specific carcinogen, induced colonic preneoplastic lesions, which can progress to carcinomas about 40-50 weeks after AOM administration. We studied the effects of sulindac on the incidence of preneoplastic lesions, proliferative activity of colonic cells (AgNORs), tumor suppressor adenomatous polyposis coli (APC) gene expression, and apoptosis using AOM-treated rat colon mucosa at 4 weeks and 40 weeks (early and late stage of colon carcinogenesis, respectively). RESULTS: Sulindac suppressed the development of preneoplastic lesions induced by AOM at 4 weeks and 40 weeks by about 50% ( P<0.01); the proliferative activity of colonic cells increased by AOM was suppressed almost completely. Furthermore, APC expression was significantly increased by sulindac at both the early and late stages ( P<0.01). However, apoptosis was clearly increased at the early stage ( P<0.01), but not at the late stage. CONCLUSIONS: APC overexpression induced by sulindac can suppress colon carcinogenesis at both the early and late stages, but apoptosis might work as one of anti-cancer mechanisms at the early stage of colon carcinogenesis.     

Folate deficiency diminishes the occurrence of aberrant crypt foci in the rat colon but does not alter global DNA methylation status

BACKGROUND AND AIMS: It has been suggested that a diminished folate status may enhance colorectal carcinogenesis by causing DNA hypomethylation. The aims of the present study were to assess the impact of different levels of folate depletion on azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation and DNA hypomethylation in the colon of male Sprague-Dawley rats. METHODS: Rats, aged 4 weeks, were divided into four groups and were fed semipurified diets either containing adequate folate (control), devoid of folate (FD) or FD + 1% succinylsulfathiazole before AOM treatment (FD1) or during the last 4 weeks of the study (FD2). At 8 weeks of age, all animals received subcutaneous injections of AOM once weekly for 3 weeks at a dose rate of 15 mg/kg bodyweight. Animals were necropsied 6 weeks after the last AOM injection and the ACF were visualized under light microscopy in formalin-fixed, methylene blue-stained colons. RESULTS: Blood folate concentrations were significantly depleted (P < 0.001) in the treatment groups consuming folate deplete diets, with the FD2 treatment group having significantly lower folate levels compared with all other groups. Higher plasma homocysteine concentrations (P < 0.001) were observed in the groups that exhibited diminished blood folate levels. There were no significant differences in global DNA methylation in the liver or colonic mucosa between the four groups, despite some groups exhibiting marked folate depletion. Animals with the most severe folate deficiency (FD2) had a lower final bodyweight and had significantly fewer ACF in their colon (P < 0.05) compared with control animals. Total (mean +/- SEM) ACF counts were as follows: control 286+/-24; FD 290+/-25; FD1 218+/-32; and FD2 205+/-27. CONCLUSIONS: In this model, folate deficiency diminished the occurrence of ACF but did not alter global DNA methylation status in the colon.     

Chemoprevention by nonsteroidal anti-inflammatory drugs eliminates oncogenic intestinal stem cells via SMAC-dependent apoptosis

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as sulindac effectively prevent colon cancer in humans and rodent models. However, their cellular targets and underlying mechanisms have remained elusive. We found that dietary sulindac induced apoptosis to remove the intestinal stem cells with nuclear or phosphorylated β-catenin in APC(Min/+) mice. NSAIDs also induced apoptosis in human colonic polyps and effectively removed cells with aberrant Wnt signaling. Furthermore, deficiency in SMAC, a mitochondrial apoptogenic protein, attenuated the tumor-suppressive effect of sulindac in APC(Min/+) mice by blocking apoptosis and removal of stem cells with nuclear or phosphorylated β-catenin. These results suggest that effective chemoprevention of colon cancer by NSAIDs lies in the elimination of stem cells that are inappropriately activated by oncogenic events through induction of apoptosis.     

Non-steroidal anti-inflammatory drugs with different cyclooxygenase inhibitory profiles that prevent aberrant crypt foci formation but vary in acute gastrotoxicity in a rat model

BACKGROUND: Standard non-steroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colorectal cancer; however, their use as preventive agents is limited by their inherent toxicity. Drugs that selectively inhibit cyclooxygenase-2 (COX-2) may be useful in this setting as they are supposedly less toxic. No study has directly compared the ability of standard NSAIDs and selective COX-2 inhibitors to inhibit colorectal cancer at clinically relevant doses. METHODS: Aberrant crypt foci (ACF) were induced in Sprague-Dawley rats by using 1,2-dimethylhydrazine (DMH). Test agents or vehicle were then administered for 3 weeks, twice daily through orogastric gavage. At the end of this period, the number and multiplicity of ACF were determined. The agents tested at equivalent anti-inflammatory doses were: sulindac and indomethacin (standard NSAIDs), meloxicam (selective COX-2 inhibitor), celecoxib (specific COX-2 inhibitor) and sulindac sulfone (no known COX activity). Acute gastrotoxicity of NSAID in rats was compared by using quantitative histology. RESULTS: All test agents reduced the number of ACF. There was a 42% reduction with indomethacin, 46% with sulindac, 46% with meloxicam, 22% with celecoxib and 36% with sulindac sulfone. Only the COX-2 inhibitors caused no significant gastrotoxicity in rats. CONCLUSIONS: Cyclooxygenase-2 inhibitors are potentially ideal chemopreventive agents as they inhibit ACF and are not gastrotoxic.     

Effect of azoxymethane and curcumin on transcriptional levels of cyclooxygenase-1 and -2 during initiation of colon carcinogenesis

BACKGROUND: Curcumin is well documented as an effective colonic chemopreventive agent in preclinical studies. Inhibition of arachidonic acid metabolism has been considered one of anticarcinogenic mechanisms of curcumin. We recently reported resistance of middle-aged F344 male rats to inhibition of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) by curcumin (Nutr Cancer, 48, 37-43). It was important to confirm this finding and to find potential mechanisms responsible, as loss of preventive activity of curcumin due to aging was a novel finding, with important implications for human intervention trials. METHODS: To confirm our previous findings, and investigate arachidonic acid metabolism as a potential mechanism of age-related differences in response to curcumin, middle-aged F344 male rats were given AOM injections after being fed their experimental diets, 0.6% curcumin or control diet. Colonic ACF were evaluated and colonic levels of cyclooxygenase (COX)-1 and 2 mRNA and prostaglandin E2 (PGE2) were measured. Next, we investigated the short-term effect of AOM and curcumin on arachidonic acid metabolism in young rats. Six week-old rats were given injections of either AOM or untreated following their experimental diets. Colonic COX-1 and COX-2 mRNA as well as PGE2 levels were measured shortly after AOM treatment. Lastly, three different ages of F344 rats were treated with either AOM or saline, and colonic COX-1 and COX-2 mRNA levels were measured shortly after the injections to find if aging alters the effect of AOM on COX mRNA expression. RESULTS: In middle-aged rats, dietary curcumin did not reduce the number of ACF and surprisingly increased colonic levels of COX-2 mRNA. Colonic COX-2 and PGE2 levels were also significantly increased in young rats fed curcumin after AOM injections. Interestingly, AOM did not affect COX-2 but decreased COX-1 expression in all ages. CONCLUSIONS: Our study suggests that during initiation, AOM inhibits colonic COX-1 expression without affecting COX-2 and dietary curcumin may increase COX-2 expression to compensate AOM-induced reduction of COX-1 expression.     

Natural history of aberrant crypt foci

BACKGROUND: The aberrant crypt focus (ACF) appears to be an important early step in colorectal carcinogenesis. Our objectives were to determine the natural history of ACF in a surgical model. METHODS: The natural history of ACF was followed by marking the lesions in vivo with tattoos. Rats were given four weekly injections of azoxymethane (AOM; 20 mg/kg). One hundred days after the first injection of AOM, rats were anesthetized, and individual aberrant crypt focus was identified by staining with methylene blue. A 3× 3 mm area, identifying one large (4–8 crypts) ACF was marked with a tattoo dye in each colon. Control animals received saline or AOM injections and were tattooed in areas without ACF. At 200 days, colons were examined for the presence of macroscopic lesions. RESULTS: A total of 54 tumors were found in the study group of 38 animals, and 21 of these were in the transverse and proximal descending colon. The marked areas (all in transverse and proximal descending colon) yielded 6 tumors and 2 ACF, but in 30 instances no abnormality was noted. Probability of observing a tumor in the 3×3 mm area of the colon that was identified as containing ACFs was 17 times greater than expected from the observed tumor rate in approximately the same zone (16 vs. 1.7 percent; 95 confidence interval, 10 to 22 and 0.5 to 1.3 percent). Twenty control animals receiving saline had no tumors of epithelial origin. Nine control animals that were carcinogen-treated and tattooed in areas without ACF had no tumors in the marked areas. CONCLUSION: Results thus show regression of many ACF identified early in the carcinogenesis process. Results also support the hypothesis that some ACF are precursor lesions for adenomas and cancers.Supported by S. Lederman Fellowship Foundation, American Physician Fellowship, and, in part, by National Cancer Institute of Canada.     

Relationship between APC genotype, polyp distribution, and oral sulindac treatment in the colon and rectum of patients with familial adenomatous polyposis

PURPOSE: Familial adenomatous polyposis is an inherited colorectal cancer syndrome characterized by the presence of multiple adenomatous colorectal polyps. Molecular studies have revealed that germline mutations in the APC gene are the underlying cause of the disease. The nonsteroidal anti-inflammatory agent sulindac has been shown to reduce the number of colorectal adenomas. Most sulindac trials in the large bowel have focused on the distal colon and relatively little is known about its effect on the proximal colon. Moreover, it is unknown whether the site of the APC mutation affects the efficacy of sulindac. METHODS: This study investigated whether there were regional differences in the effect of sulindac on the colon and whether response to sulindac was dependent on the site of mutation in the APC gene. In an open prospective study 17 patients with familial adenomatous polyposis were treated with 300 mg oral sulindac daily for four months followed by a washout phase of six months. Ten of the patients had an intact colon and seven had rectal stumps only. The number, size, and the degree of dysplasia of the adenomas were evaluated by colonoscopy at entry, end of treatment and end of the study. RESULTS: Overall, a statistically significant decrease in the number of adenomas was observed (120 +/- 112 to 28 +/- 64, P = 0.007). After cessation of sulindac treatment the number of adenomas increased to 48 +/- 44.5, but remained significantly lower than the values observed at baseline. In the ten patients with intact colons, adenomas decreased by sevenfold in the proximal colon (103 +/- 73 to 15.1 +/- 47.4, P = 0.011) and twofold in the distal colon (80 +/- 52 to 29.6 +/- 37.2, P = 0.005). The size of adenomas and the grade of dysplasia also decreased. No correlation could be seen between the APC mutation site and the response to treatment. CONCLUSION: These data indicate that sulindac reduces the number of adenomas in the entire colon and that the effect seems to be more pronounced in the proximal colon.     

Effect of Concomitant Polyethylene Glycol and Celecoxib on Colonic Aberrant Crypt Foci and Tumors in F344 Rats

We investigated whether celecoxib augments the protective effect of polyethylene glycol (PEG) on colonic aberrant crypt foci (ACF) and tumor formation in F344 rats treated with azoxymethane (AOM). Three groups of rats received AOM: I (AOM alone), II (PEG), and III (PEG/celecoxib). PEG reduced the mean number of total ACF per colon from 190 to 141 (P < 0.05; 26% reduction) and ≥4-crypt ACF from 95 to 58 (P < 0.01; 39%). Group III rats had a greater proportion of their ACF distally; whereas transverse colon ACF were reduced ∼50%, distal ACF were reduced by only ∼8% (P < 0.05). Of 13 large bowel tumors, 8 were in Group I, 4 in Group II, and 1 in Group III rats (P = 0.02). Thus in AOM-treated rats celecoxib appeared to enhance the PEG-induced reduction in colonic tumor formation, and in transverse but not distal or whole-colon ACF.     

Effect of azoxymethane and curcumin on transcriptional levels of cyclooxygenase-1 and -2 during initiation of colon carcinogenesis

Background. Curcumin is well documented as an effective colonic chemopreventive agent in preclinical studies. Inhibition of arachidonic acid metabolism has been considered one of anticarcinogenic mechanisms of curcumin. We recently reported resistance of middle-aged F344 male rats to inhibition of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) by curcumin (Nutr Cancer, 48, 37–43). It was important to confirm this finding and to find potential mechanisms responsible, as loss of preventive activity of curcumin due to aging was a novel finding, with important implications for human intervention trials. Methods. To confirm our previous findings, and investigate arachidonic acid metabolism as a potential mechanism of age-related differences in response to curcumin, middle-aged F344 male rats were given AOM injections after being fed their experimental diets, 0.6% curcumin or control diet. Colonic ACF were evaluated and colonic levels of cyclooxygenase (COX)-1 and 2 mRNA and prostaglandin E2 (PGE2) were measured. Next, we investigated the short-term effect of AOM and curcumin on arachidonic acid metabolism in young rats. Six week-old rats were given injections of either AOM or untreated following their experimental diets. Colonic COX-1 and COX-2 mRNA as well as PGE2 levels were measured shortly after AOM treatment. Lastly, three different ages of F344 rats were treated with either AOM or saline, and colonic COX-1 and COX-2 mRNA levels were measured shortly after the injections to find if aging alters the effect of AOM on COX mRNA expression. Results. In middle-aged rats, dietary curcumin did not reduce the number of ACF and surprisingly increased colonic levels of COX-2 mRNA. Colonic COX-2 and PGE2 levels were also significantly increased in young rats fed curcumin after AOM injections. Interestingly, AOM did not affect COX-2 but decreased COX-1 expression in all ages. Conclusions. Our study suggests that during initiation, AOM inhibits colonic COX-1 expression without affecting COX-2 and dietary curcumin may increase COX-2 expression to compensate AOM-induced reduction of COX-1 expression.     

Increased microvascular blood content is an early event in colon carcinogenesis

BACKGROUND: Increased premalignant epithelial microvascular blood content is a common theme in neoplastic transformation; however, demonstration of this phenomenon in colon carcinogenesis has been stymied by methodological limitations. Our group has recently developed a novel optics technology, four dimensional elastic light scattering fingerprinting (4D-ELF), which allows examination of the colonic mucosal architecture with unprecedented accuracy. In this study, we utilised 4D-ELF to probe the preneoplastic colonic microvasculature. METHODS: Colonic mucosal blood content was assessed by 4D-ELF at serial preneoplastic time points from azoxymethane (AOM) treated Fisher 344 rats and age matched control animals. We also examined the pretumorigenic intestinal mucosa of the MIN mouse, and compared with wild-type mice. Finally, in a pilot study, we examined superficial blood content from the endoscopically normal mid transverse colon in 37 patients undergoing screening colonoscopy. RESULTS: In the AOM treated rat model, augmentation of superficial mucosal and total mucosal/superficial submucosal blood supply preceded the appearance of aberrant crypt foci (ACF) and temporally and spatially correlated with future ACF occurrence. These findings were replicated in MIN mice. The 4D-ELF based results were corroborated with immunoblot analysis for haemoglobin on mucosal scrapings from AOM treated rats. Moreover, 4D-ELF analysis of normal human colonic mucosa indicated that there was a threefold increase in superficial blood in patients who harboured advanced adenomas. CONCLUSION: We report, for the first time, that blood content is increased in the colonic microvasculature at the earliest stages of colon carcinogenesis. These findings may provide novel insights into early biological events in colorectal carcinogenesis and have potential applicability for screening.     

Suppression of azoxymethane-induced aberrant crypt foci in rat colon by nimesulide,a selective inhibitor of cyclooxygenase 2

Non-steroidal anti-inflammatory drugs, such as piroxicam and sulindac, are known to inhibit development of aberrant crypt foci (ACF) and cancer in the colon. However, these agents cause gastrointestinal side-effects. Nimesulide is a selective inhibitor of cyclooxygenase 2 and has been shown to have a more potent anti-inflammatory action than piroxicam, but be less ulcerogenic and, therefore, a potentially more useful chemopreventive agent. To assess this possibility the inhibitory effects of nimesulide on the formation of ACF induced by azoxymethane in rat colon were investigated, and compared with those of piroxicam and sulindac. Male F344 rats were treated s.c. with 15 mg/kg body weight azoxymethane once a week for 2 weeks and given 50, 100 or 200 ppm nimesulide, 200 ppm piroxicam, or 200 ppm sulindac in their diet from the day before the first carcinogen treatment until the end of the experiment at week 4. At this time, nimesulide at doses of 50, 100 and 200 ppm had reduced the numbers of azoxymethane-induced ACF to 75%, 71% and 65% respectively compared to the control. The number of azoxymethane-induced ACF per colon in the group given 200 ppm nimesulide was almost the same as in those given 200 ppm piroxicam, and lower than that in the group given 200 ppm sulindac. These results suggest that nimesulide, a selective cyclooxygenase 2 inhibitor, warrants attention as a candidate for chemopreventive agent with low toxicity, active against colon carcinogenesis.Abbreviations ACF aberrant crypt foci - COX cyclooxygenase - NSAID non-steroidal anti-inflammatory drugs Work dedicated to Haruo Sugano on the occasion of his 70th birthday. The material of this paper was essentially presented at the 60th Anniversary Symposium of the Cancer Institute and the Cancer Institute Hospital, Tokyo, held in September 1994.The work was supported by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan, and the Ministry of Health and Welfare for the Second-Term Comprehensive 10-Year Strategy for Cancer Control, Japan, and a grant from the Bristol-Myers Squibb Foundation     

Does Senna Extract Promote Growth of Aberrant Crypt Foci and Malignant Tumors in Rat Colon?

Current evidence suggests that aberrant cryptfoci (ACF) can be used to evaluate agents for theirpotential colon carcinogenic activity. The aim of thepresent study was to determine whether senna pod extract (SE) itself induces ACF and tumors in the ratcolon or increases the development of ACF and tumorsinduced by azoxymethane (AOM). A daily administration ofSE 10 mg/kg by mouth for 13-28 weeks produced a weak laxative effect but did not itself causethe appearance of ACF or tumors. The numbers of ACF andtumors induced by AOM were, however, increased by a doseof SE (100 mg/kg) able to induce chronic diarrhea over three months. These resultssuggest that SE does not cause the appearance of ACF ortumors in the rat colon nor does it have a promotingeffect when given to rats at a dose that produceslaxation (10 mg/kg), whereas a diarrhogenic dose (100mg/kg) increases the appearance of tumors induced byAOM.     

Four-dimensional elastic light-scattering fingerprints as preneoplastic markers in the rat model of colon carcinogenesis

BACKGROUND & AIMS: Identification of preneoplastic changes in histologically normal epithelium (the "field effect") could provide a powerful screening tool for colorectal cancer. However, to date, reliable detection has not been possible. We have recently developed a new generation of optical technology, 4-dimensional elastic light-scattering fingerprinting (4D-ELF), which enables us to probe the nanoscale/microscale architecture of living cells. We therefore investigated whether 4D-ELF would be able to identify preneoplastic changes in the colonocytes of the azoxymethane (AOM)-treated rat model of colon carcinogenesis. METHODS: Forty-eight Fisher 344 rats were randomized to either 2 weekly injections of AOM or saline. Animals were killed 2-20 weeks after the second injection of AOM. Colons were removed and subjected to 4D-ELF analysis, with a subset undergoing assessment of aberrant crypt foci (ACF). All AOM-treated animals were compared with age-matched saline-treated controls. RESULTS: AOM-induced ACF became apparent at approximately 4-6 weeks and continued to increase over time. ACF were predominantly located in the distal colon. At 2 weeks (before development of ACF), there were marked changes in a number of 4D-ELF signatures. The relevance to carcinogenesis of these 4D-ELF-detected microarchitectural abnormalities is supported by their spatial and temporal correlation with subsequent development of ACF. All changes reported were highly statistically significant. CONCLUSIONS: We show that probing the nanoscale cellular architecture with 4D-ELF provided an unprecedented tool for detecting the earliest stages of colon carcinogenesis. Future studies are necessary to explore the clinical applicability of this technology and elucidate the biological determinants of these microarchitectural changes.     

Reduced susceptibility to azoxymethane-induced aberrant crypt foci formation and colon cancer in growth hormone deficient rats

ObjectivesTo evaluate the role of GH in colon carcinogenesis, we examined the formation of aberrant crypt foci (ACFs) and tumor development in wild type (WT) and GH-deficient, spontaneous dwarf rats (SDRs) exposed to the carcinogen azoxymethane (AOM).DesignACF were quantified by stereomicroscopy and tumor number and weights were recorded for each animal. Cell proliferation was measured by vincristine metaphase arrest, flow cytometry, and bromodeoxyuridine (BrdU) incorporation. Apoptosis was measured by TUNEL staining and cleaved caspase-3 immunohistochemistry. IGF-I was measured by radioimmunoassay (RIA). Hexokinase activity was measured by spectrophotometric assay. PARP cleavage, and IGF-IR, and p27^kip/cip expression were measured by Western blotting.ResultsACFs detected by stereomicroscopy were markedly reduced (～85%) in SDRs vs. WT rats at 10, 25, and 28 weeks after AOM. Tumor incidence, number, and weight also were reduced in SDR vs. WT animals. AOM treatment increased cell proliferation in the distal colon (where tumors occur) of WT rats but not SDRs, and these changes corresponded to increased ACF and tumor formation. Apoptosis rates were similar in AOM-treated WT and SDRs. Alterations in serum IGF-I levels may contribute to differences in the proliferative response to AOM and decreased ACF formation in SDR vs. WT rats.ConclusionsWe conclude that early neoplastic lesions (ACFs) were reduced in GH-deficient animals. This effect corresponds with differences in AOM-induced proliferation, but not apoptosis. These data indicate that GH is required for the full effect of AOM on colon ACF and tumor development, and that the SDR rat is a promising model for studies regarding the role ofGH/IGF system in the initiation and promotion of colon cancer.     

结肠癌患者直肠异型隐窝灶中脂肪酸合成酶的表达及其意义

背景：异型隐窝灶（ACF）是结直肠上皮性肿瘤最早期的形态学改变,可经腺瘤进展至结直肠癌。脂肪酸合成酶（FAS）在多数人恶性肿瘤组织中呈高表达。目的：检测结肠癌患者直肠ACF中的FAS表达情况,探讨ACF中FAS表达异常在结直肠癌形成中的意义。方法：30例确诊的散发性结肠癌患者纳入研究。以0.2%亚甲蓝溶液行直肠黏膜染色,通过放大结肠镜寻找、识别ACF,选取隐窝数目超过30个的较大的ACF为研究对象。以免疫组化染色检测直肠ACF以及配对结肠癌组织和正常结肠组织中的FAS表达。结果：30例结肠癌患者共确定116个目标ACF,不伴异型增生91个,伴异型增生25个。结肠癌组织中的FAS阳性表达率显著高于ACF和正常结肠组织（76.7%对32.8%和10.0%,P〈0.05）,ACF与正常结肠组织间差异亦有统计学意义（P〈0.05）。伴异型增生的ACF FAS阳性表达率与不伴异型增生者无明显差异（36.0%对31.9%,P〉0.05）。结论：F.AS在结直肠癌腺瘤-癌发生序列的最早期即已出现表达异常,在结直肠癌的形成中发挥重要作用。以FAS抑制剂抑制ACF进展可能成为结直肠癌化学预防的手段之一。     

Effect of age on susceptibility to azoxymethane-induced colonic aberrant crypt foci formation in C57BL/6JNIA mice

To determine the effect of age on susceptibility to azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) formation and its underlying mechanism, young and old mice were injected with AOM weekly for 4 or 5 weeks and euthanized 5 or 6 weeks later. Given the same (12 or 15) mg/kg body weight dose of AOM, old mice had significantly more ACF than young mice. However, given the same total dose of AOM (to avoid confounding effect of higher dose to heavier old mice), at a low total dose (1.5 mg) there was no age difference, but at higher total doses (1.8 and 2.2 mg) young mice had significantly more ACF than old mice. These results indicate that the age-related susceptibility to AOM differs depending on whether administration of the carcinogen is based on weight or total dose. These age differences are not due to variations in cyclooxygenase-2 expression, cell proliferation, or AOM hydroxylase activity.     

Identification and quantification of aberrant crypt foci in the colon of Min mice--a murine model of familial adenomatous polyposis

Min mice are heterozygous for a nonsense mutation in the murine adenomatous polyposis coli (APC) gene and spontaneously develop multiple intestinal neoplasms similar to the familial adenomatous polyposis (FAP) syndrome in humans. Aberrant crypt foci (ACF) are assumed to be preneoplastic lesions in both murine and human colon carcinogenesis and have been observed in FAP patients. Light microscopic examination of the colonic mucosa of 42 Min mice did not show even a single 'classical' ACF on the basis of previously defined criteria, specifying that they are elevated above the surrounding mucosa. However, in Min mice we discovered aberrant crypt foci of a different type, which we denoted ACF(Min). In contrast to the classical type, ACF(Min) were not elevated above the surrounding mucosa, their detection was totally dependent on methylene blue staining and transillumination, and they could not be identified with scanning electron microscopy. Histopathologic examination of ACF(Min) showed dysplastic crypts, similar to those found in larger lesions--that is, microadenomas in the Min mouse. The number of ACF(Min) increased up to the age of 6 weeks and then seemed to remain at a constant level of approximately four per colon. In conclusion, by transillumination of whole-mount preparations stained with methylene blue, we have identified and quantified small microscopic lesions that may be precursors of colonic adenomas in Min mice.