Variations in sialomucins in the mucosa of the large intestine in malignancy: a quantimet and statistical analysis

Synopsis The periodate-borohydride/saponification/PAS (PB/KOH/PAS) method, for the identification of sialic acid derivatives, was used to investigate possible changes in human colonic epithelium associated with carcinogenesis. The material was obtained from specimens of the large intestine resected for carcinoma and compared with normal control mucosa from biopsies of patients with no known gastro-intestinal diseases, using a Quantimet Image Analyser Densitometer to measure these staining reactions. An ordination or segregation analysis of the results revealed a marked discrimination between tumours and the normal control mucosa, with the values for the transitional mucosa and mucosa remote from the tumour graded in between. Pairedt-tests also showed statistically significant differences between the various types of mucosa. The fact that changes in the staining densities of the sialic acid derivatives are observed in mucosa which is normal by conventional histological criteria raises the hypothesis that malignancy in the colonic epithelium is accompanied by modifications in the sialic acid composition of the mucus secretion. Thus the PB/KOH/PAS method may be of value in the early detection of cancer in colo-rectal biopsies.     

Glycoprotein metabolism in rat colonic epithelial cell populations with different proliferative activities

Abstract. Epithelial cells with different proliferative activities were isolated from rat proximal and distal colon. The distribution of fucose, hexose, hexosamine, and sialic acid as well as the activities of three glycosyltransferases, eight glycosidases, and a nucleotide sugar pyrophosphatase, enzymes involved in glycoprotein metabolism, were then examined in these cells. The results of the present study demonstrate that: (1) all proximal cell populations appear to possess a higher content of hexose, fucose, and sialic acid than their distal counterparts; (2) in general, the proximal colonic populations have higher glycosyltransferase but similar glycosidase activities than their distal counterparts; (3) proliferative cells in both colonic regions have greater glycosyltransferase and glycosidase activities than non-proliferative cells, although their carbohydrate content is similar.
These findings suggest that alterations in glycoprotein metabolism exist during differentiation along the length of the rat colon. Furthermore, these data indicate that certain enzymes involved in glycoprotein metabolism may serve as markers for cellular differentiation in this organ.     

Effect of putrescine on oxyntic gland and colonic mucosal growth in rats

The effect of putrescine on oxyntic gland and colonic mucosal growth was studied by measuring the rate of [3H]-thymidine incorporation into mucosal DNA in vitro (DNA synthesis) and DNA, RNA and protein content of the mucosa following intramuscular injections of the compound (50 mumoles/100g). Saline injected animals served as controls. Multiple injections of putrescine during a 2-day fast produced a significant enhancement of mucosal DNA synthesis in oxyntic gland and colonic mucosa, with no apparent change in DNA, RNA or protein content in either of the tissues, compared to the corresponding saline-controls, when measurements were made 12-24 h after the last injection. However, when the animals were killed after 4 days, DNA, RNA and protein content of oxyntic gland mucosa, and DNA and protein content of colonic mucosa were found to be significantly higher than in the respective saline-controls. We conclude that putrescine, taken up from the blood, can stimulate growth of gastrointestinal mucosa.     

Changes in ganglioside contents, plasma sialic acid and cAMP levels in experimental hepatoma in mice

The present study was designed to assess whether changes in glycolipids and cyclic AMP contents might serve as markers for the diagnosis of malignancy in the liver. The experimental model was a transplantable murine hepatoma. Experimental mice were divided into three groups: (1) a therapeutic group, which had been transplanted with hepatoma and treated with the antimetabolism drug 5-flurouracil (0.2 mg/day i.p.), (2) a control group, which had been transplanted with hepatoma and treated with 0.2 ml 0.9% NaCl/day and (3) a normal group of mice. The ganglioside and cAMP contents in the hepatoma tissue, plasma cAMP, total- and lipid-bound sialic acid levels and red blood cell membrane sialic acid levels were determined. Results showed that the ganglioside content, total and lipid-bound sialic acid levels in the control group were significantly higher than those in the livers of normal mice (p < 0.01) while these respective values in the therapeutic group were significantly lower than those in the control group (p < 0.01). The cAMP levels of tumor tissues and plasma in the control group were lower than those in normal mice. No significant difference in red blood cell membrane sialic acid content was observed between the therapeutic and control groups though levels for both were higher than those in normal mice. These results indicate that ganglioside content and sialic acid levels in hepatoma tissues were significantly elevated, and cAMP levels in hepatoma tissues were significantly decreased during proliferation and abnormal differentiation.     

Effects of red meat and fiber in high fat diet on activities of sphingomyelinase,ceramidase and caspase-3 in rat colonic mucosa

Red meat and fiber rich foods are the dietary factors most consistently related to colon carcinogenesis. Although several components in these dietary sources may contribute, the biochemical mechanism by which red meat and fiber affect colorectal carcinogenesis has not yet been established. Sphingomyelin metabolism is a novel signal transduction pathway that may have an impact on colonic tumorigenesis. The present study investigated the activity changes of sphingomyelinase (SMase), ceramidase and caspase-3 in colonic mucosa of rats fed on a high fat control diet, the control diet with beef and the control diet with fiber (cellulose). After a three week feeding period the colonic mucosa were scraped and homogenized and enzyme activities were determined. The fiber diet significantly increased the activities of neutral and acid SMases but had no effect on those of alkaline SMase and neutral ceramidase. The beef diet, on the other hand, significantly reduced neutral ceramidase activity, but had no effect on the activities of any SMase. In addition, the beef diet significantly reduced and the fiber diet increased caspase-3 activity in the colonic mucosa when compared with the control diet. The changes of caspase-3 activities were abolished by preincubating the samples with caspase-3 inhibitor. No significant changes of intestinal alkaline phosphatase could be found among the three dietary groups. In conclusion, fiber and red meat in the high fat diet affected in an opposite way the enzymes responsible for sphingomyelin metabolism and apoptosis in the colon. The effects may have implications in colorectal tumorigenesis.     

Reduction of sialic acid O-acetylation in human colonic mucins in the adenoma-carcinoma sequence

The oligo-O-acetylation of sialic acids found in normal colonic mucins is greatly reduced in colorectal cancer. Mucins prepared from cancer tissue in adenocarcinoma showed this reduction, while normal O-acetylation was detected in resection margin and control cases and total mucin sialic acid content was significantly decreased in cancer vs control samples. A reduction of the O-acetyl transferase activity catalysing the O-acetylation reaction was also found. A series of cultured human colorectal cell lines derived from the same premalignant adenomatous line, and representative of the adenoma-carcinoma sequence were examined and revealed a depletion of oligo-O-acetylation in the original diploid premalignant line, re-expression in a further premalignant line and reduction in malignant mucinous and adenocarcinoma cell lines. Reduction of sialic acid O-acetylation appears as an early event in the process of malignant transformation in human colorectal cancer.     

Sialic acid in colonic mucin: An evaluation of modified PAS reactions in single and combination histochemical procedures

Summary Two new histochemical procedures for detecting sulphated and non-sulphated sialomucin in colonic mucosa were assessed: the saponification—Alcian Blue pH 1—periodic acid—phenylhydrazine—Schiff method (KOH—AB pH 1—PAPS) and the mild periodic acid modification of this (KOH—AB pH 1—mPAS). Using normal colonic mucosa obtained from 11 non-cancer patients, the mPAS and PAPS techniques were tested for specificity and reproducibility for staining sialic acid, either alone or in combination with Alcian Blue. A spectrophotometric method was devised to quantify the uptake of both Schiff and Alcian Blue stain by sections. At low temperature and pH5.5, the mPAS procedure had improved specificity over the PAPS procedure, and after saponification it could be used to stainO-acetyl-substituted sialic acid. When used in combination with Alcian Blue at pH 1, however, underestimation of the sialic acid content occurred owing to interference between Alcian Blue and Schiff dyes. Interference was even greater with KOH—AB pH1—PAPS procedure for both sialic acid and sulphate components. We conclude that caution must be exercised in interpretation of the staining results obtained with these new combination methods and that more accurate information on the sialic acid and sulphate content of colonic mucin is obtained by staining serial sections with the mPAS technique and Alcian Blue pH 1 alone.     

THE RELATIONS BETWEEN DNA, RNA, AND PROTEIN IN NORMAL EMBRYONIC CELL NUCLEI AND SPONTANEOUS TUMOUR CELL NUCLEI

Interferometric and photometric measurements have been made successively on individual cell nuclei derived from normal embryonic tissues and spontaneous tumour tissues of the mouse grown in vivo. From the measurements, the relations between nucleic acid and dry mass content have been studied in the two types of nuclei and the results shown to be consistent with differences in cell metabolism previously reported to exist in vitro. In the nuclei of normal embryonic cells, the syntheses of DNA, nuclear RNA, and protein appear to be closely associated, whereas in the tumour cell nuclei an appreciable fraction of the chromatin RNA and protein synthesis is dissociated from the replication of DNA.     

N-acetylneuraminic acid and N-glycolylneuraminic acid in glycopeptides of colonic tumor and mucosa in rats treated with carrageenan and 1,2-dimethylhydrazine

N-linked glycopeptides were prepared from colonic tumor (adenocarcinoma) and mucosa in rats treated with carrageenan, an indigestible polysaccharide, and 1,2-dimethylhydrazine. Sialic acids, N-acetylneuraminic acid and N-glycolylneuraminic acid, obtained by acid hydrolysis of the glycopeptides were determined by HPLC. The N-acetylneuraminic acid/N-glycolylneuraminic acid ratio in colonic tumor was 25.2, while each treated mucosa had the values between 0.29 and 0.55. Thus, necessity which observes the qualitative change of sialic acid in malignant transformation was suggested.     

Effects of omeprazole on cell kinetics of carcinogen-induced colon tumours in rats

This study was designed to evaluate the effects of hypergastrinaemia induced via suppression of gastric acid by omeprazole on carcinogen-induced colon cancer in rats. The carcinogen methylazoxymethanol (MAM), 30 mg/kg, was administered intraperitoneally at 6-weekly intervals to Sprague-Dawley rats. Four weeks after the last MAM injection, the first daily dose of omeprazole, 40 mg/kg, was given by gastric gavage to one group of rats, and the rest were given buffered methylcellulose vehicle. After 10 weeks of daily omeprazole or vehicle, the rats were anaesthetized with ether, blood samples obtained, and animals sacrificed. Gastrin levels in serum from omeprazole-treated rats were elevated nearly six-fold. DNA and RNA levels in gastric mucosa were unchanged by omeprazole, but protein content was somewhat reduced. No biochemical or histological changes related to omeprazole treatment were observed in normal colon. The number of tumours, tumour volumes, and total tumour burden were not significantly different in colons of vehicle- or omeprazole-treated rats. Analysis by flow cytometry revealed that the S phase fraction was lower in tumour cells from omeprazole-treated animals; and that the frequency of DNA aneuploidy was also reduced. The results indicate that while omeprazole-induced suppression of stomach acid in rats elevates levels of gastrin in serum, it does not substantially alter the biochemical or cellular characteristics of carcinogen-induced colon tumours.     

Hydrolase activities increase in the rat aorta with growth and aging but not in liver and kidney

We examined specific activities (based on DNA) of six glycosidases and cathepsin C in aorta, kidney, and liver from male rats of 2, 6, 10, and 14 months of age. The premise was that assessing cellular catabolism of arterial and nonvascular tissues over age might more fully clarify the impact of age (and growth) alone upon vascular wall metabolism. All aortic glycosidases increased significantly (P less than 0.05) over the holding period as follows: neutral alpha-glucosidase, up 93%; beta-galactosidase, up 102%; N-acetyl-beta-glucosaminidase, up 119%; alpha-mannosidase, up 77%; beta-glucuronidase, up 65%; acid alpha-glucosidase, up 95%. Cathepsin C specific activity was unchanged as was aortic DNA content; total protein content increased 136%. In the kidney, all glycosidase specific activities declined over age with decreases ranging 39-55%; cathepsin C was unchanged. In the liver, neutral alpha-glucosidase increased 12%, acid alpha-glucosidase was unchanged, and the four remaining glycosidases decreased an average of 5-35% by 14 months of age. Liver cathepsin C decreased 44% over this period. Thus, enhancement of hydrolase baseline activities prevails during growth and aging in rat aortic tissue whereas hydrolases of kidney and liver tissues generally decline.     

1,2-Dimethylhydrazine-induced alterations in lipid peroxidation in preneoplastic and neoplastic colonic tissues

To determine whether alterations in lipid peroxidation existed in the preneoplastic and neoplastic colonic tissues of animals treated with the procarcinogen 1,2-dimethylhydrazine, rats were injected subcutaneously with this agent (20 mg/kg body weight per week) or diluent for 5, 10, 15 and 26 weeks. At each of these time periods, animals from both groups were sacrificed, their distal colonic mucosa and/or tumors harvested, and examined and compared with respect to malondialdehyde and lipofuscin-like pigments levels. Additionally, at 26 weeks, the fatty acid composition of microsomes prepared from control, 'uninvolved' and tumor colonic tissues were analyzed and compared. The results of these experiments demonstrated that: (1) the levels of these products of lipid peroxidation were similar in the distal colons of all animals at 5 and 10 weeks; (2) at 15 weeks, however, lipid peroxidation was decreased in the distal colons of animals treated with dimethylhydrazine; (3) at 26 weeks, the levels of these products of lipid peroxidation remained lower in dimethylhydrazine-treated distal 'uninvolved' colonic mucosa and was, moreover, markedly decreased in colonic tumors; and (4) at this latter time period, differences in the fatty acid composition between tumor, 'uninvolved' and control tissues were found. These differences, however, did not appear to underlie the changes noted in the lipid peroxidation products seen in these tissues. Taken together, these findings suggest that alterations in lipid peroxidation may be involved in the colonic malignant transformation process in this experimental model.     

Effects of short term ether and pentobarbital anesthesia on bone and cartilage metabolism.

The effects of stress due to brief (4--5 min) ether and pentobarbital anesthesia vs. decapitation on assays of seven enzymes in homogenates of synovium, articular and epiphyseal cartilage, and metaphyseal and cortical bone were compared. Etherization caused twofold changes in synovial and articular cartilage G-6-PDH, LDH, CPK, glutamic DH, and ICDH based on tissue dry weight and DNA content. Pentobarbital anesthesia produced only slightly lower activities, per gram DNA, of LDH, acid phosphatase, and glutamic-DH in cortical bone. Epiphyseal cartilage metabolism was unaffected by either mode of anesthesia. No difference could be detected between levels of enzyme activities of the several tissues taken from rats that had been decapitated or anesthetized with pentobarbital. The changes in enzyme activities suggested that pentobarbital was non-stressful and appropriate to metabolic studies in the skeleton.     

Histochemistry of sialo-mucins in connective tissues

Summary The present investigation was undertaken in an effort to differentiate histochemically between sialo-mucins and acid mucopolysaccharides in connective tissues.On the basis of previous studies regarding the histochemical identification of sialic acid in sections of animal salivary glands the author applied different technical procedures on cartilage and tooth germs of newborn rats.The results obtained indicated that in the tissues examined sialic acid is a normal component of the polysaccharide fraction of the connective tissue ground substance.On leave of absence at the University of Rome, Medical School, Viale Regina Elena 287-A, Rome, Italy.     

Effects of endosulfan on intestinal functions in protein-malnourished rats

In rats fed 18% protein diet, administration of endosulfan (2mg/kg body weight daily for 7 days) significantly decreased the brush border sialic acid and increased the hexoses contents. The intestinal uptake of glucose was increased while that of glycine and calcium was reduced. Brush border enzymes and lipids were not affected. However, in protein malnourished rats (fed 8% protein) exposed to endosulfan, brush border sucrase and peptidase activities were enhanced, while alkaline phosphatase activity was decreased compared to untreated malnourished animals. Membrane sialic acid content was low while fucose and cholesterol levels were augmented in endosulfan fed malnourished animals. The uptake of glucose and glycine was elevated under these conditions. These results Suggest that the nutritional status of the animals has an important bearing on thc susceptibility of intestinal tissue to endosulfan toxicity in rats.     

Changes in the composition of the excurrent duct system of the rat testis during postnatal development.

The gross composition of the testicular excurrent duct system of the rat was examined and compared along the length of the duct and with samples of testis, bladder and liver. Changes in composition with age were examined by analysing tissue from animals at postnatal ages of 19, 36, 48, 60, 90 and 120 days. In adult animals, testicular tissue was characterized by having the lowest dry weight, accompanied by low levels of total protein, lipid, RNA and glycogen; DNA, phospholipids and sialic acid were at levels similar to other tissues. A high proportion of the total protein was soluble. The ductuli efferentes plus initial segment of the epididymis were characterized by high levels of total lipid. The caput epididymidis contained a low level of total protein but a high level of acid-soluble phosphorus. The cauda epididymidis had a low dry weight and low levels of total protein, soluble protein, and lipid, but high levels of acid-soluble phosphorus, DNA and sialic acid. The ductus deferens contained small amounts of RNA and DNA but had a high dry weight, high total protein, soluble protein and glycogen. Several trends were apparent with increasing age. Dry weight increased in the ductuli efferentes plus initial segment, whilst total protein decreased in the caput and cauda epididymidis. Total lipid increased in the ductuli efferentes plus initial segment and acid-soluble phosphorus and sialic acid increased in all other segments of the excurrent duct system. In all segments the content of RNA and DNA decreased as the animals matured. The concentration of calcium and magnesium in the excurrent duct system was not significantly different from those levels found in the liver. High levels of spermine and spermidine were confirmed in the prostate, and were also detected in the testis, caput epididymidis and cauda epididymidis, but at a much lower concentration.     

Changes in nuclear non-histone protein composition during normal differentiation and carcinogenesis of intestinal epithelial cells

Nuclei from colonic epithelial cells were isolated and fractionated by centrifugation in discontinuous sucrose density gradients. Nuclei differing in buoyant density differ in size, non-histone protein to DNA ratio, and capacity for DNA synthesis in vivo. They do not differ in histone content or in proportions of the major histone classes. The distribution of cell nuclei after density gradient centrifugation corresponds functionally to their histological localization in the colonic mucosa, as judged by the nuclear capacity for DNA synthesis in both normal and tumor tissues. The nuclei of colonic epithelial cells contain a heterogeneous set of non-histone proteins which change in total amount and in relative proportions during normal differentiation. The complement of nuclear proteins differs in normal intestinal epithelial cells and in colon tumors induced by the carcinogen, 1,2-dimethylhydrazine. There is a striking increase in the nuclear content of two major protein classes (of mol. wt ca 44 000 and 62 000) during carcinogenesis. The accumulation of these proteins in the nuclei of carcinogen-treated cells follows early, selective increases in their rates of synthesis.     

Metabolism of Adenosine in Human Colorectal Tumour

The aim of this work is to analyse the activities of the enzymes metabolising adenosine in fragments of neoplastic and normal‐appearing mucosa, surrounding the tumour in 20 patients affected by colorectal cancer. The results show that the activities of the enzymes are markedly higher in tumour in comparison to normal mucosa to coope with the accelerated purine metabolism in cancerous tissues.