Dietary fibre on cell proliferation in large bowel mucosal crypts near or away from lymphoid nodules and on mineral bioavailability.

The effect of consumption for 24 weeks of different amounts (0%, 5% or 10% w/w) of fermentable (pectin and guar gum) or nonfermentable (cellulose and lignin) dietary fibres on cell proliferation and other parameters in large bowel mucosal crypts was studied in rats. In all 12 dietary groups, the crypts located over the distal aggregate of lymphoid nodules (ALN) had more colchicine arrested metaphase figures per midaxial crypt section (MC) and a longer crypt column height than crypts located three to four cm away from this ALN. These differences are attributed to the tropic influence of nodular cells in the ALN. Consumption of fermentable fibre decreased pH in the lumen of the caecum, and glucose, Zn and Cu in serum but increased Ca and Mg in serum. The decrease in caecal pH and serum glucose was significantly correlated with a decrease in MC. Increased intake of the nonfermentable fibre types increased faecal bulk but had no significant correlation with the other measured crypt parameters. Multiple regression analyses was used to model the relationships between the mucosal crypt criterion variables and the two measured predictor variables, caecal pH and serum glucose. Relationships between dietary fibre, ALN, MC, bioavailability of dietary minerals and risk of colorectal cancer are discussed.

The nonfermentable dietary fiber lignin alters putative colon cancer risk factors but does not protect against DMH-induced colon cancer in rats.

The effect of supplementation of the diet with autohydrolyzed lignin on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis was studied using 112 male Sprague-Dawley rats. Rats received eight weekly injections of DMH (9.5 mg/kg s.c.) or the saline vehicle solution and then were maintained on a basal AIN-76 fiber-free diet or the basal fiber-free diet plus 5% or 10% (wt/wt) lignin for 24 weeks. Rats were killed 32 weeks after the start of the experiment. Colon tumor incidence, location, and multiplicity were determined. Body weight, caloric intake, fecal dry weight, gut transit time, pH of cecal contents, and total fecal bile acid excretion were measured. Supplementation of the diet with 5% or 10% lignin resulted in increased fecal dry weight and total fecal bile acid excretion and in decreased gut transit time, colon pH, and fecal bile acid concentration. Dietary lignin did not significantly affect colon tumor incidence or multiplicity compared with the fiber-free diet. Thus dietary supplementation with autohydrolyzed lignin, a food fiber with good bulking characteristics, had a significant effect on several factors that have previously been linked to reduction of colon cancer risk, but the consumption of high levels of lignin did not decrease the risk for colon cancer.

Dietary supplementation with pectin and guar gum on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats.

The effect of dietary supplementation with pectin and/or guar gum on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis was studied using 120 male Sprague-Dawley rats. The rats were given a weekly injection of DMH for 8 weeks and were maintained on a basal fiber-free diet supplemented with 5% cellulose. The rats were then subdivided into four groups and kept on the basal fiber-free diet supplemented with either no fiber, 10% pectin, 10% guar gum or a combination of 5% pectin/5% guar gum for a period of 24 weeks. The 8 weeks of DMH administration were defined as the initiation stage of carcinogenesis and the next 24 weeks were defined as the promotional stage of carcinogenesis. Food and water were available ad libitum. The rats were killed 32 weeks after the start of the experiment and tumor incidence, location and frequency in the colon were determined. Other parameters measured were body weight and caloric intake. Dietary fiber supplementation with 10% pectin or with 10% guar gum but not with the combination of 5% pectin/5% guar gum (fed during the promotional stage of carcinogenesis), was found to suppress colon cancer incidence to a significant extent.

Demonstration of the need for end point validation of putative biomarkers: failure of aberrant crypt foci to predict colon cancer incidence.

Seven-week-old Sprague-Dawley rats were fed a semipurified AIN76 diet and were given a weekly injection of the colon carcinogen 1,2-dimethylhydrazine for 8 weeks (initiation stage of carcinogenesis). The rats were divided into seven groups and each group of rats was placed on one of seven different modifications of the AIN76 diet for the next 24 weeks (promotional stage of carcinogenesis). The mean numbers of aberrant crypt foci/rat and the incidence of adenocarcinomas from some of the seven dietary groups were found to be significantly different. However, all attempts to show a significant correlation between the mean number of aberrant crypt foci/rat and the incidence of adenocarcinomas failed. Therefore, the number of aberrant crypt foci/rat cannot by itself be used as a reliable quantitative predictor (biomarker) of the efficacy of dietary intervention or of chemopreventive procedures on modulating the risk of developing colon cancer. This conclusion emphasizes the need for end point validation of potential cancer biomarkers before the biomarkers can be considered predictive of modulation of the risk for colon cancer.

Quantitative contribution of factors regulating rat colonic crypt epithelium: role of parenteral and enteral feeding, caloric intake, dietary cellulose level and the colon carcinogen DMH.

To elucidate the role and quantitative contribution of several exogenous factors which may regulate colon crypt mitotic activity, proliferative zone height (PZH) and crypt height, groups of rats were subjected to various feeding regimens both with and without treatment with the colon carcinogen, 1,2-dimethylhydrazine (DMH). The rats were divided into two major groups and one group was given eight weekly injections of DMH base at 9.5 mg kg-1 body weight. Throughout this period and for two additional weeks the rats were isocalorically fed either a defined nutritionally complete diet with different levels of dietary cellulose or they were parenterally (i.v.) fed a nutritionally complete liquid formula with different caloric levels. The rats were then injected with colchicine 3 h prior to sacrifice to arrest and to collect dividing cells at metaphase. The results of multiple regression analysis of all data were interpreted to indicate that parenteral feeding caused dramatic suppression of the colon crypt height (CH) and of the number of metaphase figures per crypt (MC). Increased cellulose intake stimulated CH but suppressed MC. The CH was also stimulated by DMH. CH was positively correlated to PZH and MC. The MC was suppressed by cellulose intake and negatively correlated to PZH but was positively correlated to CH. The PZH was positively correlated to CH. These findings were related to the role of luminal food, functional workload, kcal intake and treatment with DMH on the measured colon crypt parameters. A quantitative assessment of factors that regulate the measured colonic crypt parameters was accomplished.

Effect of dietary cellulose on cell proliferation and progression of 1,2-dimethylhydrazine-induced colon carcinogenesis in rats.

The effects of different levels of dietary cellulose on colonic crypt mitotic activity and colon carcinogenesis were studied in 190 male Sprague-Dawley rats. Rats were divided into groups and fed a basal fiber-free diet supplemented with either 0, 5, or 15% pure cellulose (w/w), for periods of 10 weeks (initiation stage) or 32 weeks (promotional stage). Half of the rats in each group were given weekly s.c. injections of 9.5 mg 1,2-dimethylhydrazine (the base) (DMH) for 8 weeks. Some of the rats were killed at 10 weeks while most were killed 22 weeks later. In some groups the dietary cellulose level was changed to a different level at 10 weeks. Food intake and body weight data showed that the rats within each experiment were isocalorically fed. There was a direct correlation between crypt height and the percentage of cellulose in the diet. Addition of 5 or 15% dietary cellulose during the initiation stage of carcinogenesis resulted in a significant increase in crypt height. Increasing dietary cellulose after the initiation stage (0 to 5% and 5 to 15%) or maintaining a high dietary cellulose level throughout both the initiation and promotional stages (15%) resulted in a significant increase in crypt height. A DMH-induced increase in mitotic activity that was observed during the initiation stage was no longer evident after the 22-week promotional stage. The significant DMH-induced increases in proliferative zone height and crypt height that were initially observed during the initiation stage were also observed after the 22-week promotional stage. These data indicate that the initial DMH-induced increases observed in proliferative zone height and crypt height are irreversible. Addition of 5 or 15% cellulose was found to suppress DMH-enhanced mitotic activity in the crypts of the descending colon during the initiation stage of carcinogenesis. This finding was correlated with a significantly lower incidence of adenocarcinomas in rats maintained on 5 or 15% cellulose throughout both the initiation and promotional stages.

Suppression of a a carcinogen (1,2-dimethylhydrazine dihydrochloride)-induced increase in mitotic activity in the colonic crypts of rats by addition of dietary cellulose.

Serial injections of the colon carcinogen, 1,2-dimethylhydrazine (DMH), have been reported to increase the proliferative activity in the colonic crypts preceding development of tumors. Can addition of purified cellulose to a fiber-free AIN-76 rat diet be used to suppress this increase in proliferative activity? To answer this question rats were divided into two groups, and one group was given eight weekly injections of the DMH base at 9.5 mg/kg of body weight. Throughout this period and for 2 additional wk the rats were isocalorically fed a defined nutritionally complete diet both with and without different dietary levels of cellulose (0, 5, and 15%). The rats were given injections of colchicine 3 h prior to sacrifice to arrest and to collect dividing cells at metaphase. Analysis of variance was performed on various morphometric parameters obtained from histological sections of midaxial crypts from the descending colon. Our results confirm that DMH induced a significant increase in the mitotic activity as measured by the number of metaphase figures per crypt. The presence of dietary cellulose did cause a significant suppression of the DMH-induced increase in the crypt mitotic activity.

Ethanol stimulates leucine uptake by rat fetal hepatocytes via trans-stimulation.

Prior studies showed that exposure of cultured rat fetal hepatocytes to ethanol increased sodium-independent transport of alpha-amino-isobutyric acid and cycloleucine. Using leucine (Leu) as a probe, we now show that this is a reflection of trans-stimulation of system L inward flux. Transport of Leu was entirely sodium independent and beta-2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid inhibitable. Uptake kinetics indicated two components, likely systems L1 and L2 reported for the adult hepatocyte. The low-affinity Km was in the 0.5 mM range, whereas the high-affinity Km was 2% of that value. Under optimal growth conditions, approximately 65% of the Leu was transported by the latter system. Strong bidirectional exchange was shown with Leu loading, stimulating initial Leu uptake by 66%. Externally directed transport was enhanced 2.9 times against 5 x 10(-3) M Leu vs. no external Leu. A 24-h exposure to ethanol (2 mg/ml) increased Leu uptake by up to 100%, an effect that could be mimicked by arrested cell replication. Both enhanced rates could be reversed by amino acid depletion, reflecting intracellular amino accrual that induced trans-stimulation of Leu uptake. Enhanced uptake was also reproduced in replicating cells by loading with increasing concentrations of Leu.

Stimulatory effects of ethanol on amino acid transport by rat fetal hepatocytes.

Previous studies have indicated that acute, and especially chronic, maternal ethanol consumption can depress placental uptake of various amino acids. Since the fetal cell itself represents a second barrier to nutrients, one which may be altered by ethanol exposure, the effects of ethanol on amino acid net uptake by rat fetal hepatocytes was addressed. The present study determined that ethanol stimulated amino acid net uptake by fetal hepatocytes grown in monolayer culture. Fetal liver cells were grown in custom Williams' E medium (without L-arginine and with L-ornithine) and exposed to epidermal growth factor (0, 1, 2 or 5 ng per ml) and ethanol (1.7 +/- 0.1 or 3.9 +/- 0.2 mg per ml). Addition of ethanol (3.9 mg per ml) to the culture medium completely blocked measurable cell replication during a 48-hr exposure period. Fetal hepatocytes exposed to ethanol accrued both protein and water in a parallel fashion, both in excess of that by control cells. Ethanol (1.7 and 3.9 mg per ml) for 48 hr stimulated alpha-aminoisobutyric acid net uptake by fetal hepatocytes (p less than 0.05). Efflux was not affected (p greater than 0.05). The onset of this significant stimulation of net uptake was progressive and required in excess of 6 hr of contact with ethanol. This ethanol stimulation of alpha-aminoisobutyric acid net uptake persisted for at least 24 hr following ethanol withdrawal. The component(s) of alpha-aminoisobutyric acid net uptake stimulated by ethanol was independent of extracellular Na+.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 1,2-dimethylhydrazine treatment and feeding regimen on rat colonic epithelial cell proliferation.

The present study was designed to test the hypothesis that 1,2-dimethylhydrazine dihydrochloride (DMH) induces preneoplasia in rat colonic epithelium and that this DMH-altered epithelium will respond differently to various nutritional challenges in comparison to normal colonic epithelium. Preneoplasia was arbitrarily defined as an altered and irreversible state of colonic epithelial cell proliferation induced by a carcinogen (DMH). In summary, DMH was found to be specific for the enhancement of rat colonic epithelial cell proliferation compared to other rapidly renewing cell populations, i.e., ileal epithelium and ear epidermis. DMH-induced changes in rat colonic epithelial cell proliferation and crypt cellularity were found to be irreversible following a 2- to 8-week recovery period. The p.o. administration of the solid and liquid diets, regardless of chemical constituents, supported a DMH-induced increase in colonic epithelial cell proliferation; however, a DMH-induced increase in epithelial cell proliferation was not observed in rats maintained on total parenteral nutrition. Thus, the route of administration has a significant influence on epithelial cell proliferation in colonic epithelium of DMH-treated rats. The importance of these results, along with previous studies, is the establishment and initial characterization of an exploitable preneoplastic system in rat colonic epithelium. Particularly revealing was the finding that significant changes in crypt kinetic parameters induced by DMH treatment did not revert to control values following a 2- to 8-week recovery period. Based on an altered and irreversible state of colonic epithelial cell proliferation induced by DMH, it is concluded that: (a) the preneoplastic state is a committed state and is not dependent upon the continued presence of the carcinogen; and (b) all cryptal epithelium is preneoplastic, although not all cells progress to the overtly transformed state. In addition, total parenteral nutrition prevented the expression of a DMH-induced preneoplastic state of altered epithelial cell proliferation.

Changes in proliferation and surface morphology in the rat ileum in response to total parenteral nutrition.

To find out how the ileum adapts to total parenteral feeding, two experiments were performed. In the first experiment rats were given total intravenous feeding for 10 days. The animals were injected with tritiated thymidine (1 muCi/g body weight) 1 hour before being killed. Portions of the ileum were used for (1) radioautography, (2) analysis of the tissue DNA content, (3) specific activity of the DNA, and (4) scanning electron microscopy. The DNA content of ileum was decreased 72% while the specific activity of DNA was increased 289% in the i.v. fed rats. In the second experiment rats were given total intravenous feeding for 10 days. The animal were injected with colchicine (1 mg/kg body weight) 3 hours before being killed. The number of labelled cell nuclei per ileal crypt section was significantly decreased by parenteral feeding as was the number of colchicine collected metaphase figures. Light microscopy revealed the crypt and the villus height to be shorter and the number of goblet cells per unit surface area to be increased in parenterally fed rats as compared to those fed solid food orally. Enterocytes of the exfoliative zone from the ileal villi of rats fed solid food showed three distinct types of surface architecture whereas those from i.v. fed rats all possessed abundant microvilli. No bacteria were seen in ilea of i.v. fed animals but many were seen embedded in enterocytes from orally fed rats. Because the amount of DNA per cell is known to be constant, we concluded that the overall number of cells in the lieum decreased about 72% in the i.v. fed rats and that cell proliferation in the crypts, although significantly deceased, was still supporting an epithelial renewal process.