Folate deficiency enhances the development of colonic neoplasia in dimethylhydrazine-treated rats.

In patients with ulcerative colitis, epidemiological work has suggested an association between low folate status and an increased risk of colonic neoplasia. The aim of the present study was to determine if experimental folate deficiency increases the likelihood of developing neoplasia in rats treated with the carcinogen dimethylhydrazine. Weanling male Sprague-Dawley rats were fed with an amino acid-defined diet containing either 8 or 0 mg/kg folic acid. After 5 weeks of defined diet, weekly s.c. injections of dimethylhydrazine (20 mg/kg) were administered to both groups. Serum, whole blood, liver, and colonic folate concentrations at the time of sacrifice were significantly lower in folate-depleted animals (P less than 0.001). There were significant differences in the incidence of colonic neoplasia between the two groups after 20 weeks of dimethylhydrazine exposure: folate-deficient rats had a greater incidence of dysplasia (6 of 7 versus 2 of 7 animals; P less than 0.05) and carcinoma (6 of 7 versus 1 of 7 animals; P less than 0.01). Furthermore, a significantly greater proportion of folate-replete rats than folate-deficient rats were free of neoplastic lesions (5 of 7 versus 0 of 7 animals; P less than 0.05). These results suggest that, in this animal model, folate deficiency increases the risk of malignancy when there is an underlying predisposition to colorectal cancer.     

Glycosidases in normal and dimethylhydrazine-treated rats and mice with special reference to the colonic tumours

Activities of 12 glycosidases and of β-D-glucuronidase were measured in liver, kidney and in the gastrointestinal tract of rats and mice. The activities of different enzymes varied not only within one tissue but also among different tissues. In rats injected with 1,2-dimethylhydrazine, a many fold increase in N-acetyl-β-D-glucosaminidase, N-acetyl-β-D-galactosaminidase, β-D-galactosidase and α-L-fucosidase was found in colonic tumours and colonic mucosa. These enzymes were elevated significantly in the kidney of tumour bearing animals as well. Liver and other parts of the gastrointestinal tract showed an increase only in N-acetyl-hexosaminidase with the appearance of colonic tumours. In treated mice, 2 N-acetylhexosaminidases were elevated in colon, duodenum, liver and kidney. However, in liver and kidney, β-D-galactosidase was also significantly increased.     

Specific expression of tenascin in human colonic neoplasms.

Tenascin, a novel six-armed extracellular matrix glycoprotein, was immunohistochemically examined in the human normal adult colon, and colonic neoplasms such as tubular adenomas, primary and metastatic adenocarcinomas. In contrast to previous reports, tenascin was hardly detectable in the normal adult colons, being predominantly localised in the fibrous stroma surrounding the glandular epithelia of the neoplastic lesions. The neoplastic cells themselves were totally negative for tenascin expression. Both the tubular adenoma tissues and the superficial layer of well-differentiated adenocarcinomas in general were intensely reactive to tenascin antibody, and the staining intensity increased as the adenoma became more atypical in cases of tubular adenomas. By pretreatment of the paraffin-embedded tissue sections with pepsin, the distribution of tenascin was often intensified considerably and distinct localisation was more clearly demonstrated in the colonic tumour tissues. Tenascin was also biochemically purified from human invasive colonic carcinomas, and this cancerous tissue tenascin was compared with that extracted from a human umbilical cord fibroblast cell line in terms of molecular heterogeneity. Two major isoforms of the purified tenascin from colonic cancer tissues were found to have relative molecular masses of 250 kD and 190 kD, which were almost identical to those of human foetal fibroblast tenascin glycoproteins. In addition, several lower molecular weight isoforms were frequently detectable in the cancerous tissues, which might represent immuno-reactive tenascin isoforms proteolytically digested in human colonic carcinomas in vivo.     

Kinetics of the non-neoplastic mucosa of the large bowel of dimethylhydrazine-treated rats.

Administration of 1,2 dimethylhydrazine (DMH) to rats by weekly s.c. injections causes the development of multiple epithelial tumours of the large bowel. These appear to arise as localized dysplastic abnormalities in hitherto apparently morphologically normal crypts. This study was undertaken in order to examine cell proliferation in such apparently normal crypts of DMH-treated animals. A number of proliferative abnormalities are evident, including changes in the size of the crypts, changes in the disposition of proliferating cells within them and reduced cell-cycle times. The nature and the extent of the abnormalities vary from site to site along the length of the bowel, and reflect the vulnerability of the different segments of the bowel, not only to the carcinogenic effects of DMH, but also to short-term toxicity.     

Brush cytology in the diagnosis of colonic neoplasms

During a three-year period (1986-1988), 234 colonic brush specimens were received in the authors' laboratory. Nine samples (4%) were deemed unsatisfactory for evaluation because of inadequate cellularity and/or poor fixation. In 11 cases concomitant or follow-up histologic specimens were not available. The remaining 214 specimens included 82 malignant neoplasms, 88 neoplastic polyps (adenomas), and 44 nonneoplastic lesions. Sixty-seven (82%) of malignant neoplasms were correctly diagnosed by brush cytology. Three cases of adenoma with severe dysplasia or in situ carcinoma were diagnosed as adenocarcinoma by cytology. No false-positive diagnoses were made of nonneoplastic lesions. Brush cytology was found to be a more sensitive technique in the diagnosis of colon cancer than endoscopic biopsy (82% and 74% sensitivity, respectively). The combination of the two techniques increased the sensitivity to 90% and improved the overall accuracy of the test. Seventy-one (82%) of the colonic adenomas were correctly diagnosed by cytology. Brush cytology is a convenient, safe, and accurate technique which should be used concurrently with endoscopic biopsy or polypectomy.     

Cell proliferation, cell loss and expression of bcl-2 and p53 in human pulmonary neoplasms.

Immunohistochemical staining of bcl-2 and p53 proteins was compared with thymidine labelling index (TLI) and cell loss factor (O) in lung cancer. Neither bcl-2 nor p53 overexpression was associated with high cell loss but strong bcl-2 staining was associated with higher TLI. Concomitant strong p53 and bcl-2 expression, not the usual inverse relationship, plus high cell-loss factor was present in three neuroendocrine carcinomas. Other factors presumably have a role in controlling cell death in these tumours.     

Malignant neoplasms in rats fed lasiocarpine.

Lasiocarpine, a pyrrolizidine alkaloid, was fed at a dietary concentration of 50/10(6) for 55 weeks, to 20 male F-344 rats. Malignant tumours developed in 17/20 animals between 48 and 59 weeks. Forty-five percent (9/20) developed angiosarcomas of the liver and 35% (7/20) had hepatocellular carcinomas. Other tumours included malignant adnexas tumour of the skin (1 rat) and lympohoma (1 rat). Lung metastases were observed in 4 animals with angiosarcoma of the liver and one animal with hepatocellular carcinoma. From one animal, angiosarcoma was successfully transplanted through 4 generations.     

Spontaneous fibrous histiocytic neoplasms in rats.

A total of 85 spontaneous rat fibrohistiocytic tumours were evaluated histologically and assessed for the presence or absence of metastases. The overall incidence in controls from 2-year carcinogenicity studies was 2.7%. The tumours occurred principally in the subcutaneous and deep soft tissues, and generally appeared after 18 months of age. Four histological types were recognized: histiocytic (17%), pleomorphic (33%), cellular (17%) and very fibrous (33%). Histiocytic tumours were highly malignant, and most produced metastases. Pleomorphic and cellular neoplasms occasionally produced metastases and must be regarded as potentially malignant. Very fibrous lesions were essentially benign. The close resemblance, both histologically and biologically, between rat and human fibrohistiocytic neoplasms supports the use of the fibrohistiocytic concept in laboratory-animal pathology. Study of these rat tumours may provide insight into the development of human fibrohistiocytic neoplasms.     

Kinetics of changes in the crypts of the jejunal mucosa of dimethylhydrazine-treated rats

When symmetrical 1,2 dimethylhydrazine was administered to rats by weekly s.c. injection, 37% of the animals had developed small intestinal carcinomas after 21-27 weeks. These lesions were largely localized to duodenum and upper jejunum. At the same time there was a diffuse crypt hyperplasia in the jejunum which affected all the treated animals, not just those with neoplasms. This marked hyperplasia was preceded by a modest sustained crypt elongation which was seen soon after DMH injections began. In these hyperplastic jejunal crypts the absolute size of the proliferative compartment was increased, but the growth fraction calculated from labelling studies appeared to fall, probably by reduction in relative size of the proliferating population within the proliferative compartment. No convincing alteration in actual cell-cycle time was observed in the abnormal crypts. There was a slight (25%) increase in cell-production rate in the abnormal crypts.     

Assessment of mutations in ki-ras and p53 in colon cancers from azoxymethane- and dimethylhydrazine-treated rats

Mutations in the Ki-ras oncogene and the p53 tumor suppressor gene are known to occur at high frequencies in human colon cancers. We measured the frequency of mutations in these two genes in colon adenocarcinomas obtained from a widely used experimental model of human colon carcinogenesis: F344 rats treated with the carcinogens azoxymethane (AOM) or dimethylhydrazine (DMH). We detected codon 12 mutations in Ki-ras in approximately 60% of colon adenocarcinomas induced by either carcinogen. We characterized the rat p53 intron-exon junctions to construct primers for polymerase chain reaction amplification of this gene. We discovered that the rat p53 gene was structurally different from the human p53 gene, as the rat gene was missing one intron between exons 6 and 7. Both single-stranded DNA conformational polymorphism analysis and direct DNA sequencing of the highly conserved regions of rat exons 5–7 were conducted because the corresponding human regions (exons 5–8) have been reported as being mutated most frequently in human colon cancers. Using these methods, we were unable to identify any p53 mutations in the highly conserved regions of exons 5–7 in either AOM- or DMH-induced colon adenocarcinomas. These data confirm that Ki-ras was mutated in most colon cancers in AOM- or DMH-treated rats but indicate that molecular alterations in the p53 gene, if they occur in this animal model, are different from most p53 mutations in human colon cancers. Mol. Carcinog. 19:137–144, 1997. © 1997 Wiley-Liss, Inc.     

PROLIFERATION RATE OF COLONIC NEOPLASMS AND MUCOSA ADJACENT TO NEOPLASMS: IMMUNOHISTOCHEMICAL STUDY

Biopsy specimens of normal mucosa (n=5). adenomas (n -13), adenocarcinomas (n = 8). mucosa adjacent to adenoma (n=10) mucosa adjacent to adenocarcinoma (n = 7 ) at the large bowel were investigated by an iminunohistochemical method using 5- bromodeoxynldine (BrdUrd) . The labeling index (LI) was significantly lower in normal nucosa than mucosa adjacent to neoplasms and adenomas and adenocarcinomas. The proliferative zone was confined to the lower two- third at the crypt in normal mucosa and in mucosa adjacent to neoplasms. The labeled cells were either present in the upper third or scattered along the crust and in surface epithelium. The results support the adenmo-carcinoma sequence.     

Abnormal methylation of the calcitonin gene in human colonic neoplasms

Earlier studies of the methylation status of total genomic DNA and of specific genes have demonstrated, predominantly, hypomethylation in human neoplasms. However, we have recently documented the presence of new sites of methylation in the calcitonin gene in human lymphomas (100%), small cell lung carcinomas (92%), and acute myeloid leukemias (95%). We now report that these same novel calcitonin gene methylation sites are also a feature of DNA from human colonic adenomas (13 out of 14 studied), colon carcinomas (4/13), and established colon carcinoma cell lines (18/19), despite the presence of overall genomic DNA hypomethylation in these neoplasms. The data provide further evidence that regional increases in DNA methylation, like gene hypomethylation, occur in benign colonic neoplasms prior to malignant transformation. The fact that abnormalities of calcitonin gene methylation are less frequent in DNA from human colonic carcinomas than from adenomas and colon carcinoma cell culture lines is of special interest. This finding suggests that a more heterogeneous population of cells is present in the carcinomas and that the calcitonin gene hypermethylation may be inherent to cells which are initially selected for growth in culture or are capable of prolonged survival under culture conditions.     

High fat diet enhances colonic cell proliferation and carcinogenesis in rats by elevating serum leptin

We postulated that high fat diet enhances colon cell proliferation and carcinogenesis by elevating serum leptin. To examine this possibility, the present study was conducted to investigate the effect of leptin on the growth of human colon cancer cells (HT29) and the relationship between serum leptin and colon cell proliferation and aberrant crypt foci (ACF) in the 1,2-dimethylhydrazine-treated rats fed graded levels of dietary fat for 28 days. In cell culture experiments, leptin stimulated the growth and proliferation (BrdU incorporation) of colon cancer cells and the expression of c-fos protein. In the in vivo experiments, an elevation of dietary fat caused higher serum leptin and adipose-tissue weight. Colonic cell proliferation (BrdU incorporation), c-fos protein expression and ACF were elevated with increasing dietary fat. There was a significant correlation between serum concentration of leptin and colon cell proliferation and ACF. The results suggest that the enhancement of colon cell proliferation and carcinogenesis by high fat diet is mediated through elevating serum leptin.     

Spontaneous neoplasms in aged control Fischer 344 rats.

Neoplastic lesions in untreated F-344 rats (740 males and 740 females) used as controls in carcinogenicity studies were evaluated and tabulated. The incidence of spontaneous tumors was 84.3% in the males and 76.2% in the females. In males, the most common neoplasms were testicular interstitial cell tumors (79.5%) followed by mononuclear cell leukemia/lymphomas (30.5%), pituitary adenomas (20.5%), pancreatic islet cell adenomas (6.5%), thyroid c-cell adenomas (5.7%), pheochromocytomas (5.7%), skin fibromas (3.2%), keratoacanthomas (1.9%), and thyroid follicular cell adenomas (1.9%). In females, the most common neoplasms were pituitary adenomas (30.3%) followed by mononuclear cell leukemia/lymphomas (20.5%), endometrial polyps (14.1%), mammary fibroadenomas (11.1%), thyroid c-cell adenomas (5.1%), mammary adenomas (1.9%), skin fibromas (1.1%), and clitoral carcinomas (1.1%). A variety of less common neoplasms were also observed in various other organs.     

Risk of renal and colonic neoplasms and spontaneous pneumothorax in the Birt-Hogg-Dubé syndrome.

The Birt-Hogg-Dubé syndrome, a genodermatosis characterized by benign tumors of the hair follicle, has been associated with renal and colonic neoplasms and spontaneous pneumothorax, but the risk of developing these disorders is unknown. We identified risk factors for renal tumors and spontaneous pneumothorax in 98 patients affected with the Birt-Hogg-Dubé syndrome, in 13 Birt-Hogg-Dubé haplotype carriers, and in 112 unaffected family members. Development of renal tumors was strongly associated with the Birt-Hogg-Dubé syndrome and age. The odds ratio for renal tumor in BHD-affected family members adjusted for age was 6.9 (95% confidence interval, 1.5-31.6) and approximately 9.0 for the other risk factors considered. Chromophobe renal carcinoma, an uncommon type of renal cancer, was the predominant type of renal cancer found. Spontaneous pneumothorax was also strongly associated with the Birt-Hogg-Dubé syndrome and age. The odds ratio for pneumothorax in BHD-affected individuals, adjusted for age, was 50.3 (95% confidence interval, 6.4-392), and about 32 times higher adjusting for the other risk variables. Colon cancer and colon polyps were not related to the Birt-Hogg-Dubé syndrome. The Birt-Hogg-Dubé syndrome confers an increased risk for the development of renal tumors and spontaneous pneumothorax. We found no increase in risk for the development of colon polyps or colon carcinomas.