Early histopathologic events to evolution of colon cancer in C57BL/6 and CF1 mice treated with 1,2-dimethylhydrazine.

After administration of the intestinal carcinogen 1,2-dimethylhydrazine (DMH), C57BL/6J and CF1 mice were observed for early precursor lesions to large bowel cancer. Among the initial events seen following DMH treatment, an abrupt reduction in colonic DNA synthesis was the earliest lesion detectable. The frequency of aberrant colonic nuclei rose shortly after DMH treatment, reaching a maximum value 24 hours later and remaining elevated for 3 days following the exposure. Mucin changes, detected histochemically, and cell kinetic alterations in crypt proliferation rates were observed much later and were a constant feature for both strains following 4 weekly treatments with DMH, while carcinomas appeared in all animals 32 weeks after the start of DMH treatment. The quantitative comparison of these histopathologic observations for the early detection of colon cancer suggests that the induction of colonic nuclear aberrations in the mucosa of the large bowel might provide a sensitive and rapid indication of genotoxicity to this organ and thus might provide the basis for a screening methodology for colon carcinogens.

Promotion of aberrant crypt foci and cancer in rat colon by thermolyzed protein.

BACKGROUND: We have previously shown that thermolyzed protein (casein) cooked with fat in the diet of the rat promotes the growth of aberrant crypt foci (putative precursors of colon cancer) assessed at 100 days. PURPOSE: To determine how thermolysis affects this promotion, we examined thermolysis conditions, quantity of thermolyzed protein in the diet, and duration of thermolysis. To determine whether the previous finding of promotion of aberrant crypt foci corresponds to promotion of cancers assessed much later, we carried out promotion studies until colon cancers appeared. METHODS: F344 rats were given an initiating dose of azoxymethane and were then randomly allocated to groups receiving diets differing in their quantity and quality of casein. The groups were examined for aberrant crypt foci and tumors in the colon. RESULTS: Aberrant crypt foci were promoted by diets containing thermolyzed casein (180 degrees C, 2 hours). Promotion increased with increasing level of thermolyzed casein in the diet (to 20%) and with increasing thermolysis time (to 4 hours). The number of animals with polyps and cancers was higher in the animals receiving thermolyzed protein (2 hours), 16/23 versus 9/26 (P less than .05) and 10/26 versus 3/27 (P less than .05), respectively. The number of aberrant crypts per focus and the number of large aberrant crypt foci were higher in the tumor-bearing animals. CONCLUSIONS: Thermolyzed casein promotes early colonic precursor lesions in a dose-dependent and thermolysis time-dependent manner; thermolyzed casein also promotes colon cancer. IMPLICATIONS: The promoter formed on thermolysis could be involved in colon cancers associated with diets cooked at elevated temperatures, such as can occur with high-fat diets.

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.

Effects of dietary folate on intestinal tumorigenesis in the apcMin mouse.

Dietary folate appears to be inversely related to colorectal cancer risk. This study investigated the effects of dietary intervention with folate or the development of intestinal polyps in Min (Apc +/-) mice. Weanling Mil mice were fed diets containing 0, 2 (basal requirement), 8, or 20 mg folate/kg diet. At 3 and 6 months of dietary intervention, 50% of the mice from each group were sacrificed, and the small intestine and colon were analyzed for polyps and aberrant crypt foci (ACF). Serum folate concentrations accurately reflected dietary folate levels (P < 0.001). At 3 months no significant difference in the average number of total small intestinal polyps was observed among the four groups. However, increasing dietary folate levels significantly reduced the number of ileal, but not duodenal or jejunal, polyps in a dose-dependent manner (P-trend = 0.001); folate supplementation at 20 mg/kg diet was associated with a 68-78% reduction in the number of ileal polyps compared with the other three diets (P < 0.007). The number of ileal polyps was inversely correlated with serum folate concentrations (P = 0.03). At 3 months, increasing dietary folate levels significantly decreased the number of colonic ACF in a dose-dependent manner (P = 0.05); the control and two folate supplemented diets significantly reduced the number of colonic ACF by 75 100% compared with the folate-deficient diet (P < 0.04). The number of colonic ACF was inversely correlated with serum folate concentration (P = 0.05). No significant difference in the number of colonic adenoma was observed among the four groups at 3 months. At 6 months, no significant differences in the average number of total small intestinal, duodenal, and jejunal polyps, colonic adenomas, and colonic ACF wer observed among the four groups. However, the folate-deficient diet had 62-76% lower number of ileal polyps compared with the control and two folate-supplemented diets (P < 0.003). Serum folate concentrations, but not dietary folate levels, were directly correlated with the number of ilea polyps (P = 0.006). These data suggest that dietary folate supplementation suppresses the development of ileal polyps and colonic ACF in this model However, at later time points, folate supplementation appears to have an opposite effect on ileal polyps. These data generally support the role of folate in intestinal tumorigenesis suggested in epidemiological studies and chemical carcinogen animal models. Notwithstanding the limitations associated with this model, these data suggest that the optimal timing and dose of folate intervention need to be determined for safe and effective folate chemoprevention.

Polyethylene glycol 8000 and colon carcinogenesis: inhibition in the F344 rat, promotion in the Min mouse.

It has recently been reported that 5% polyethylene glycol 8000 (PEG 8000; Mr 8000) in the diet markedly inhibits the development of colonic tumors in carcinogen-treated rats. To assess the possible use of this agent as a preventive or treatment agent for patients with familial adenomatous polyposis, we determined the effect of PEG 8000 on spontaneous carcinogenesis in the Min mouse. PEG at a 5% concentration in the diet of Min mice did not affect the number of small intestinal or cecal tumors but did increase the number of colon tumors and the number of animals with colonic tumors (2 of 18 versus 12 of 22 animals; P < 0.001). Although the chemopreventive effect of PEG 8000 in rats is remarkable, we suggest a cautious approach in long-term testing of PEG as a chemopreventive agent for subjects at risk for colonic neoplasia.

Chemopreventive effects of dietary folate on intestinal polyps in Apc+/-Msh2-/- mice.

Epidemiological and animal studies (reviewed in Y. I. Kim, J. Nutr. Biochemistry, 10: 66-88, 1999; J. B. Mason and T. Levesque, Oncology, 10: 1727-1743, 1996) suggest that dietary folate intake is inversely related to the risk of colorectal cancer. However, the optimal timing of folate intervention and mechanisms by which folate modulates colorectal carcinogenesis have not been clearly established. A recently developed murine model of intestinal tumorigenesis, which carries a heterozygous mutation in the Apc gene and a null mutation in the Msh2 gene (Apc+/-Msh2-/-), was used to determine the effect of dietary folate on intestinal tumorigenesis. Apc+/- Msh2-/- mice were randomized to receive either 0 or 8 mg of folate/kg diet starting at either 3 or 6 weeks of age. The 3- and 6-week diet starts represent intervention before and after the establishment of neoplastic foci, respectively. At 11 weeks of age, mice were killed, and the small intestines and colons were analyzed for adenomas and aberrant crypt foci (ACF). Serum folate concentrations were determined by a standard microbiological assay. Genomic DNA methylation was assessed by in vitro [3H]methyl incorporation into hepatic DNA and by a methyl-sensitive restriction digestion method. Microsatellite instability was determined in matched normal and polyp DNA from the small intestine and colon at 5 loci. Serum folate concentrations accurately reflected dietary folate levels (P < 0.005). Folate supplementation, started before the establishment of neoplastic foci, significantly decreased the number of small intestinal adenomas (by 2.7-fold; P = 0.004) and colonic ACF (by 2.8-fold; P = 0.028) and colonic adenomas (by 2.8-fold; P = 0.1) compared with a moderate degree of folate deficiency. In contrast, a moderately folate-deficient diet, started after the establishment of neoplastic foci, significantly reduced the number of small intestinal adenomas (by 4.2-fold; P = 0.001) but had no effect on colonic ACF and adenomas compared with folate supplementation. Genomic DNA methylation and microsatellite instability do not seem to play a major role in folate-modulated intestinal and colonic tumorigenesis in this model. In conclusion, in this murine model, dietary folate supplementation significantly protects against small intestinal and colorectal tumorigenesis if it is provided before the establishment of neoplastic foci However, if it is provided after the establishment of neoplastic foci, dietary folate seems to have an opposite effect. These data suggest that the timing of folate intervention is critical in providing an effective and safe chemopreventive effect on intestinal tumorigenesis. Notwithstanding the limitations associated with this model, our data suggest that the optimal timing of folate intervention must be established before folate supplementation can be used as a safe chemopreventive agent against colorectal cancer.

Promotion of colonic microadenoma growth in mice and rats fed cooked sugar or cooked casein and fat.

We studied the effect of cooked food components on the promotion of microadenoma growth in the colons of mice and rats. CF1 mice and Fisher 344 rats were initiated with azoxymethane, with 152 mice receiving four weekly i.p. injections of 5 mg/kg, 59 rats receiving a single injection of 20 mg/kg, and 24 rats receiving 30 mg/kg. A week after the last injection, the animals were randomly assigned to one of eight diets with identical ingredients, but the three components, sucrose, casein, and beef tallow, either uncooked or cooked. Control animals were given diets with uncooked ingredients. Experimental animals were fed diets in which one, two, or three of the components were cooked in an oven at 180 degrees C until golden brown before they were added to the diet. After 100 days on the diets, the colons were fixed, stained with methylene blue, and scored for microadenomas. The mice and the rats fed cooked sucrose, or casein and beef tallow cooked together, had three to five times more large microadenomas than did the controls (P ranging from 0.02 to 0.0001). No significant increase was observed with the five other cooked diets. Two rats fed the casein and beef tallow cooked together had adenocarcinomas. Thus, a diet containing 20% of cooked sucrose, or 40% of casein and beef tallow cooked together, promotes the growth of colonic microadenomas in initiated mice and rats, and would appear to contain promoters for colon cancer.

Possible mechanisms relating diet and risk of colon cancer.

Two recent developments in cancer epidemiology and experimental carcinogenesis provide the basis for two possible mechanisms relating diet and colon cancer risk. The first development is the accumulating epidemiological evidence for an association between insulin resistance and colonic adenomas and cancers. This evidence suggests the following mechanism: the consumption of excess dietary energy results in the development of insulin resistance with increased circulating levels of insulin, triglycerides, and non-esterified fatty acids. These circulating factors subject colonic epithelial cells to a proliferative stimulus and also expose them to reactive oxygen intermediates. These long-term exposures result in the promotion of colon cancer. The second development is the continuing identification of agents that significantly inhibit experimental colon carcinogenesis. These observations suggest the following mechanism: focal loss of epithelial barrier function resulting from a failure of terminal differentiation results in the "leak" of a presently undefined toxin and a focal inflammatory response characterized by evidence of the activation of the COX-2 enzyme and an oxidative stress with the release of reactive oxygen intermediates. The resulting focal proliferation and mutagenesis give rise to aberrant crypt foci and adenomas. The process is inhibited by: (a) demulcents confined to the colonic lumen that "repair" the surface; (b) anti-inflammatory agents; or (c) antioxidants. The two mechanisms, i.e., insulin resistance acting throughout the body and focal epithelial barrier failure acting locally, can describe most of the known relationships between diet and colon cancer risk.

Insulin promotion of colon tumors in rats.

McKeown-Eyssen and Giovannucci have proposed a mechanism for colon carcinogenesis based on the similarity of the risk factors for colorectal cancer and non-insulin-dependent diabetes. They note that diets high in fat and energy and low in complex carbohydrates and a sedentary lifestyle lead to insulin resistance and hyperinsulinemia and propose that the hyperinsulinemia promotes colon carcinogenesis. In this study, we directly tested for a promoting effect of insulin on colon carcinogenesis in F344 rats. After azoxymethane initiation and injections of insulin given 5 times/week for 17 weeks, the fraction of rats with colon tumors was greater in rats receiving insulin than in rats receiving saline (79 versus 50%, respectively; P < 0.05 for tumors with maximum diameters > or = 2 mm), and the average number of tumors/ rat was also greater (2.00 versus 0.73; P < 0.001). There was no effect on body weight. Our results demonstrate that insulin in a colon tumor promoter in this rat model and support the proposed mechanism linking lifestyle factors and colon carcinogenesis.

Classification of aberrant crypt foci and microadenomas in human colon.

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

Susceptibility of lean and obese Zucker rats to tumorigenesis induced by N-methyl-N-nitrosourea.

To address the possible involvement of hyperinsulinemia in breast cancer development, we have examined the susceptibility of lean and obese Zucker rats to N-methyl-N-nitrosourea (MNU)-induced mammary cancer. Fifty-day-old female lean or obese Zucker rats received intraperitoneal (i.p.) injections of 37.5 or 20 mg/kg MNU, respectively. We showed in separate experiments that these doses produce similar levels of DNA methylation in the mammary epithelial cells of the lean and obese animals. Over the course of 29 weeks following MNU treatment, half of the lean rats developed carcinomas of the mammary gland, demonstrating that they are of intermediate susceptibility to mammary tumorigenesis. During this period, the obese rats developed hyperinsulinemia and insulin resistance as expected. Although palpable tumors developed at a similar rate in the lean and obese rats, only 10% of the obese animals developed mammary carcinomas. The obese rats, however, developed a high incidence (63.3%) of epidermal cysts that occurred mainly in the region of the mammary glands. A 13.3% incidence of colon carcinomas was also found in the obese rats. These results suggest that the development of hyperinsulinemia does not render the obese Zucker rats more susceptible to mammary gland carcinogenesis. Our observation of colon carcinomas in obese, but not lean rats, however, is consistent with evidence that hyperinsulinemia promotes colon cancer in rodents and humans.

Aberrant crypt foci and microadenoma as markers for colon cancer.

Foci of aberrant crypts similar to those seen in experimental animals exposed to colon carcinogens have been identified and quantified on the mucosal surface of fixed resections of human colon after methylene blue staining. Many of the foci in humans showed dysplasia on histologic examination and were considered to be microadenoma (MA). These lesions may be precursors for adenomatous polyps and colorectal cancer. Rats and mice initiated with azoxymethane, then fed diets containing sucrose or casein heated at 180 degrees C to stimulate normal cooking conditions, had three to five times more large MA after 100 days than controls. Thus, cooked sugar and protein contain promoters of the growth of colonic MA. 5-Hydroxymethylfuraldehyde was identified as a promoter in cooked sugar.

Giant cell carcinoma of pancreas with clear cell pattern in metastases.

A case of mucin-positive giant cell carcinoma of the pancreas is presented. Clear cells were a prominent feature of the primary tumor and constituted the majority of the metastatic deposits, a finding not usually associated with pancreatic carcinoma. Results of ultrastructural and histochemical studies are presented, and the significance of a clear cell component in metastases is discussed.

Mucocele of the appendix: pseudomyxoma peritonei and intracytoplasmic canaliculus-like structures.

A case of pseudomyxoma peritonei associated with mucocele of the appendix is reported. To date there is no general agreement as to whether this entity (pseudomyxoma) should be categorized as a malignant lesion. The presence of intracellular canaliculus-like structures adds support to those investigators who believe that pseudomyxoma peritonei reflects a form of malignant disease.

Pseudomembranous colitis associated with antibiotics.

Colitis associated with antibiotics, particularly with lincomycin and clindamycin, is a well established entity. The colitis may be clinically and radiologically very difficult to distinguish from inflammatory bowel disease, including Crohn's disease and ulcerative colitis. A wide spectrum of pathological features is described with various antibiotics. However, the pathological picture in the pseudomembranous form is quite distinctive. The most important histological findings include a "mushroom-like" or "explosive" appearance of the pseudomembrane with a sudden transition to normal mucosa adjacent to the lesion. Rectal biopsy is both an accurate and a rapid method of establishing the diagnosis.

Identification and quantification of aberrant crypt foci and microadenomas in the human colon.

The objective of the present study was to determine whether aberrant crypt foci (ACF) similar to those observed in the colons of experimental animals exposed to colon carcinogens could be identified and quantified in the human colon. Twenty-seven colon resections from patients affected by familial adenomatous polyposis (FAP, five cases), colorectal cancer (CRC, 12 cases), and benign diseases of the large bowel (BD, 10 cases) were collected from a pathology repository or immediately after operation. Ten or more 1-cm2 formalin-fixed, methylene-blue--stained samples of colonic mucosa from each colon were scored under light microscopy for ACF. The number of ACF per cm2 and the number of crypts per ACF for each colon were calculated. The average number of ACF per cm2 in the FAP group (20 +/- 19, mean +/- SD) was significantly higher (P less than 0.01) than those of the CRC (0.37 +/- 0.41) and BD (0.18 +/- 0.35) groups. At least one ACF was found in every colon resection from CRC patients and in six out of 10 colon resections from the BD group. The average number of crypts per ACF ranged from five to 35 with absolute values from 1 to over 100. Fifty-five histologic specimens, 43 with ACF of various size and 12 without, were prepared by sectioning the colon parallel to the mucosal surface. There was a close correlation between the number of crypts per ACF in each specimen as scored by methylene-blue and histologic examination. Twenty-six aberrant crypt foci displayed dysplasia as evident by histologic analysis. In these instances we feel the term microadenoma is appropriate and, using this unique approach of examining the human colon, they can be easily identified and quantified. These lesions may well be precursors for adenomatous polyps and colorectal cancer.

Occurrence of alpha-1-antitrypsin deficiency in 155 patients with alcoholic liver disease.

Liver biopsies from 155 patients with alcoholic liver disease were examined for periodic-acid-Schiff-positive, diastase-resistant (PAS-DR) intracytoplasmic globules in hepatocytes. Seven patients had these PAS-DR globules: each was a heterozygote for a deficiency allele of alpha-1-antitrypsin (AAT), or alpha-1-protease inhibitor, with the PAS-DR globules distributed in a pattern characteristic of this deficiency. One further patient with normal AAT had a few intracytoplasmic PAS-DR globules in occasional hepatocytes. The prevalence of AAT heterozygotes in this series did not differ from that in the reference population. The seven heterozygotes included five of PI (protease inhibitor) type MZ, one of PI type SZ, and one heterozygous for a rare deficiency allele, PI type MMmalton. The M and Mmalton alleles may be difficult to distinguish because they have similar mobilities with isoelectric focusing technics. Therefore, if PAS-DR inclusions are found in the liver of a patient with an apparently normal phenotype, the presence of a defective M variant allele, such as Mmalton, should be considered.

Diet, aberrant crypt foci and colorectal cancer.

We have used the aberrant crypt focus (ACF) assay to test and develop hypotheses linking diet and colon cancer. The hypotheses were suggested by epidemiological studies that identified possible dietary factors associated with colorectal cancer risk. The ACF assay was used to quantitate the effect of the dietary factors on the initiation and growth of these putative precursors of colon cancers in experimental animals. Using this approach we have developed 3 new hypotheses for the role of diet in colorectal cancer. These are (1) a risk associated with 5-hydroxymethyl-2-furaldehyde in caramelized sugar, (2) a risk associated with some factor in thermolyzed casein, and (3) a risk associated with single nutrient boluses of sucrose and fructose. The importance of these hypotheses has still to be tested in long term carcinogenesis experiments, in analytic epidemiology studies and then, perhaps, in intervention trials.

Indeterminate colitis in the spectrum of inflammatory bowel disease.

During a ten-year period, a double-blind retrospective study of 32 colectomy specimens from patients with inflammatory bowel disease (IBD) showed that the majority of cases could be clearly separated into ulcerative colitis (UC, 65%) and Crohn's disease (CD, 19%). However, in five (16%) colectomy specimens, the pathologic changes did not fulfill the criteria generally accepted for UC and CD. Criteria were laid down to differentiate the indeterminate form of colitis from the two more familiar types of IBD. We discuss the value of the category "indeterminate colitis" and emphasize that the term "transmural inflammation" is loosely used and that accurate definition of this criterion removes much of the difficulty from the differential diagnosis of IBD.