Mid Villus Research Articles

Carbofuran administration induces genotoxic effects in epithelial cells across crypt–villus axis in rat intestine

Oral intake of carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methyl carbamate), as contaminant of water and foods comprises a major source of carbofuran exposure to the digestive system in mammals. Thus in the present study, the genotoxic effects of carbofuran on enterocytes across crypt–villus axis was investigated in rats using micronucleus test and comet assay. Carbofuran was administered to rats, daily in the dose of 4.0 mg/kg body weight for 7 days or 2.8 mg/kg body weight for 30 days. Micronucleus test revealed an increase ( p < 0.001) in the frequency of micro-nucleated enterocytes in villus tip (VT), mid villus (MV) and crypt base (CB) regions in intestine of rats exposed to carbofuran for 7 or 30 days compared to the respective controls. Comet assay revealed an increase ( p < 0.001) in tail moment, tail length, % DNA and mean intensities of enterocytes all across the villus height in pesticide treated rats. Maximum DNA damage was observed in CB cells upon carbofuran treatment. The observed DNA damage was ∼6–9 times in carbofuran fed rats for 30 days in contrast to those given pesticide for 7 days (∼4–5 times). These findings suggest that carbofuran may have genotoxic effects in the rat intestine.

Role of signaling pathways in the regulation of folate transport in ethanol-fed rats

Folate is an essential cofactor for normal cellular proliferation and tissue regeneration. Alcohol-associated folate deficiency is common, primarily due to intestinal malabsorption, the mechanism of which needs attention. The aim of the present study was to evaluate the regulatory events of folate transport in experimental alcohol ingestion. For this, male Wistar rats were fed 1 g/kg body weight/day ethanol (20% solution) orally for 3 months and folate transport was studied in isolated intestinal epithelial cells across the crypt–villus axis. The role of different signaling pathways in folate transport regulation was evaluated independently to that of reduced folate carrier (RFC) expression. The results showed that differentiated cells of villus possess high folate uptake activity as compared to mid villus and crypt base cells. During chronic ethanol ingestion, decrease in transport was observed all along the crypt–villus axis but was more pronounced at proliferating crypt base stem cells. Studying the effect of modulators of signaling pathways revealed the folate transport system to be under the regulation of cAMP-dependent protein kinase A (PKA), the activity of which was observed to decrease upon alcohol ingestion. In addition, protein kinase C might have a role in folate transport regulation during alcoholic conditions. The deregulation in the folate transport system was associated with a decrease in RFC expression, which may result in lower transport efficiency observed at absorptive surface in alcohol-fed rats. The study highlights the role that perturbed regulatory pathways and RFC expression play in the decreased folate transport at brush border surface during alcohol ingestion.

Giardia lamblia: Expression of alkaline phosphatase activity in infected rat intestine

Alkaline phosphatase (IAP) is a marker of intestinal microvillus membrane. Changes in IAP activity have been studied as a function of Giardia lamblia ( G. lamblia) infection using rat as the experimental model. At day 11 and 15 post-infection, enzyme activity was reduced ( p < 0.01) compared to controls. The enzyme levels were essentially similar to control values by day 30 post-infection. Analysis of the enzyme activity in cell fractions across crypt–villus axis revealed a marked decrease in enzyme activity in the villus tip and mid villus regions but a considerable increase ( p < 0.01) in enzyme activity in the crypt base of 11 day post-infected animals compared to that in controls. The observed changes in IAP activity were confirmed by assaying the enzyme activity in acrylamide gels using bromo-chloro-indolyl phosphate staining under non-denaturing conditions. These findings indicate differential changes across the crypt–villus axis, but impaired alkaline phosphatase levels in G. lamblia infected rat intestine.

Uptake of barbituric acid derivatives in small intestinal brush border membrane vesicles from retinyl palmitate-treated rats.

Brush border membrane was prepared from the small intestinal (jejunum) cells along the crypt-villus axis. The fluorescence spectra of 1,8-anilinonaphthalene sulfonic acid and the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene were measured in the brush border membrane vesicle suspension. The hydrophobicity of brush border membrane was found to be in the order villus tip >mid villus >lower villus. The fluidity of brush border membrane was in the order villus tip <mid villus <lower villus. The uptake of barbituric acid derivatives by brush border membrane vesicles was well correlated with their partition coefficients (isopentyl acetate/water). No significant difference was observed between the uptake of hexobarbital by brush border membrane vesicles from the villus tip and lower villus. When retinyl palmitate was administered to rats, the fluidity of brush border membrane was found to be higher in the retinyl palmitate-treated rats than in the control rats. However, no significant difference in the uptake of hexobarbital by brush border membrane vesicles was observed between the retinyl palmitate-administered rats and the control rats. Thus, the retinyl palmitate treatment seems unlikely to affect the passively transported ligands like barbituric acid derivatives in brush border membrane vesicles.

Differential induction of major histocompatibility complex molecules on mouse intestine by bacterial colonization

The aim of this study was to determine what factors induce major histocompatibility complex (MHC) molecules on the mouse small intestinal epithelium by using immunohistochemical methods. In germ-free mice, although MHC class I molecules such as H-2K and thymus leukemia antigen (TLa) were expressed on the small intestinal epithelium, class II molecules were absent. The introduction of micro-organisms into germ-free mice induced characteristic MHC molecules on the small intestinal epithelial cells. The I-A molecule was induced on the villus tip and crypt epithelial cells 7 days after conventionalization, and the I-E molecule was induced on the mid villus and crypt epithelial cells 14 days after conventionalization. The staining intensity of the H-2K molecules was increased 4 days after conventionalization. In contrast, TLa did not change during conventionalization of germ-free mice. These results suggest that the expression of MHC molecules, except for the TLa, is greatly dependent on the presence of intestinal microorganisms.

Oleoyl-CoA incorporation into triglycerides and phospholipids by chick enterocytes. Effect of cholesterol and cholestyramine feeding

Incorporation of 1[1- 14Cloleoyl-CoA] into triglycerides was similar in villus and crypt cells from chick duodenum and lower than than in the upper and mid villus cells from jejunum. Cholesterol feeding produced a significant decrease of triglyceride synthesis in duodenum, while this specific activity increased in jejunum. Cholestyramine feeding did not affect triglyceride synthesis in duodenum and decreased that found in upper and mid villus cells from jejunum. Levels of oleoyl-CoA incorporation into phospholipids were higher than those observed in the incorporation into triglycerides. Cholesterol and cholestyramine feeding did not affect this activity. Incorporation of oleoyl-CoA into both triglycerides and phospholipids in enterocytes isolated from ileum was lower than that found in duodenum and jejunum, and did not change significantly by cholesterol or cholestyramine feeding.

Sucrase-isomaltase gene expression along crypt-villus axis of human small intestine is regulated at level of mRNA abundance

The mucosal lining of the small intestine is a complex epithelium that is continually renewed by division of a stem cell population located in intestinal crypts, migration of daughter cells along the villus, and, finally, extrusion of senescent cells into the lumen. The majority of cells in both crypt and villus cell compartments are enterocytes that acquire differentiated functions as they migrate out of the crypt. Sucrase-isomaltase (SI) is an enterocyte-specific, brush-border enzyme that has little activity in crypt cells and maximal activity in low and mid villus cells. The mechanism by which enterocytes acquire SI enzymatic activity as they move from crypt to villus is controversial. In this study we examined the distribution of SI mRNA along the crypt-villus axis of human small intestine using isolated epithelial cells and in situ hybridization. A complementary DNA to the 5' portion of the human SI mRNA was amplified and cloned using the polymerase chain reaction. Hybridization analysis of RNA extracted from human intestinal epithelial cells showed that the cloned cDNA recognized a single 6.5-kb mRNA. In situ hybridization of duodenal biopsy specimens was performed using a single-stranded RNA probe derived from this cDNA. This analysis showed that there was little SI mRNA in crypt cells and appearance of mRNA in enterocytes located at the crypt-villus junction. The mRNA levels were maximal in lower and mid villus cells with decreased levels noted in villus tip cells. These results are identical to those previously described in rat intestine and suggest that expression of the SI gene as enterocytes emerge from intestinal crypts is regulated primarily at the level of mRNA accumulation. Study of SI gene regulation may provide a useful model to investigate the mechanisms that regulate enterocyte-specific gene expression and intestinal differentiation.

Effect of Fasting and Feeding on Polyamines and Related Enzymes along the Villus:Crypt Axis

Fasting and feeding have profound effects on crypt cell production and small bowel mucosal growth but the mechanism whereby food stimulates villus tip enterocytes to influence crypt cell production is unknown. We therefore measured the activities of ornithine decarboxylase (ODC), diamine oxidase (DAO) and alkaline phosphatase (ALP--a marker of enterocyte maturity) and polyamine concentrations in epithelial cells from villus tips, mid villi, lower villi and crypts of small intestine in non-fasted controls and 18-24 h fasted rats. Fasting reduced crypt cell production and caused villus hyperplasia, DAO activity (mU/g) increased in control villus tips from 9.6 +/- (SEM) 0.8 to 12.3 +/- 1.5 after fasting (NS), from 7.6 +/- 0.4 to 13.9 +/- 3.0 in mid villi (p less than 0.01), from 5.7 +/- 1.0 to 10.4 +/- 7.4 in lower villi (p less than 0.01) and from 5.4 +/- 0.9 to 12.8 +/- 1.5 in the crypts (p less than 0.001). ALP showed a similar pattern of results. In contrast, fasting lowered ODC activity (pmol/mg protein/h) dramatically from 319 +/- 82 in control villus tips to 16.7 +/- 3.0 during fasting, from 297 +/- 59 to 10.7 +/- 3.6 in mid villi, from 224 +/- 45 to 6.3 +/- 2.8 in lower villi and from 150 +/- 31 to 5.8 +/- 3.3 in the crypts. Fasting reduced putrescine concentrations in all fractions but particularly in the crypts and in general was associated with increases in spermidine and spermine concentrations. The role of DAO in the maintenance of low putrescine concentrations during fasting is unclear.

Role of Polyamines in the Early Adaptive Response to Jejunectomy in the Rat: Effect of DFMO on the Heal Villus:Crypt Axis

To study the role of the polyamines putrescine, spermidine and spermine and of the enzymes controlling their synthesis (ornithine decarboxylase; ODC) and degradation (diamine oxidase; DAO) along the villus:crypt axis at the crucial early stage of the ileal adaptive response to jejunectomy, we measured polyamine concentrations and the activities of ODC, DAO and alkaline phosphatase (a marker of enterocyte maturity) in epithelial cells isolated by the Weiser technique from villus tips, mid villi, lower villi and crypts 4 days after surgery in transected control (TRC) and jejunectomised rats untreated or given the specific ODC blocker, alpha-difluoromethyl ornithine (DFMO, 2% in drinking water beginning 3 days before surgery). In the TRCs, there was a diminishing villus tip-to-crypt gradient not only in alkaline phosphatase but also in ODC and DAO activities. After jejunectomy, there were up to 93% increases in mean enterocyte ODC activity when compared with the corresponding cell fractions from the TRCs, but in both the control and jejunectomised rats, DFMO treatment markedly inhibited ODC activity (p less than 0.05-0.01) and reduced spermidine and particularly putrescine concentrations (p less than 0.005-0.001) in all four cell fractions. Only 4 days post-operation, jejunectomy stimulated a significant increase in ileal wet weight but DFMO treatment completely prevented this adaptive response and significantly reduced segmental intestinal weight (mg/cm) in the TRCs. These results (i) extend our knowledge of polyamines and related enzymes along the villus:crypt gradient in the normal intestine, (ii) provides the first data on these variables after resection, and (iii) lend further support to the hypothesis that changes in enterocyte ODC activity and in putrescine and spermidine concentrations play an important role in initiating the ileal adaptive response to proximal small bowel resection in the rat.

Distribution of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase in isolated villus and crypt cells of chick duodenum, jejunum and ileum

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), the major rate-limiting enzyme of cholesterogenesis, was studied in epithelial cells isolated in a villus to crypt gradient from chick duodenum, jejunum and ileum, in order to resolve the apparent controversy that exists on the anatomical localization of sterol synthesis in the intestine. Consistent separation was demonstrated by using the marker enzymes alkaline phosphatase, specific to the villus cells, and thymidine kinase, specific to the crypt cells. No relative difference in stability was observed, as shown by the equal distribution of acid phosphatase. Cells were 90-95 per cent viable. The highest specific activity of reductase was located in the microsomal fraction (41 per cent of the total). The mitochondria had lower specific activity (8 per cent of the total). The distribution of reductase activity in epithelial cells of the villus-crypt axis was also studied. The specific activity in each cell fraction from chick duodenum was clearly lower than that in jejunum and ileum. The jejunal and ileal crypt regions showed lower specific activity than the villus cells. About 70 per cent of total reductase activity was found in cells from the upper and the mid villus fraction in each intestinal segment.

Localization of acyl-coenzyme A: cholesterol acyltransferase in villus and crypt cells of chick intestine

Endogenous cholesterol esterification by acyl-CoA:cholesterol acyltransferase (EC 2.3.1.26) was studied in isolated enterocytes obtained from chick duodenal, jejunal, and ileal villi and crypts, using [14C]oleoyl-CoA as substrate. The maximal specific activity in each cell fraction was found in chick jejunum, followed by duodenum and ileum. Jejunal upper and mid villi showed higher specific activities than lower villi and crypts. Epithelial cells isolated from chick intestine also incorporated oleoyl-CoA into different lipids using the endogenous substrates. Upper and mid villus cells showed the maximal incorporation of oleoyl-CoA into triglycerides in duodenum and jejunum. Levels of oleoyl-CoA incorporation into phospholipids were higher than those found in the synthesis of triglycerides or cholesterol esters, whatever may be the cell fraction considered. Upper villus cells also showed the highest specific activity in the incorporation of oleoyl-CoA into phospholipids. The acyl-CoA hydrolase specific activity was practically similar in all the cell fractions obtained from chick duodenum, jejunum, and ileum.

In vitro drug absorption models. I. Brush border membrane vesicles, isolated mucosal cells and everted intestinal rings: characterization and salicylate accumulation.

Brush border membrane vesicles, isolated mucosal cells and everted rings from rat intestine were compared for their suitability for drug uptake studies. Vesicles from brush border membranes were judged to be metabolically and morphologically functional on the basis of biochemical and microscopic criteria. With the use of a collagenase-vascular-perfusion method, populations of villus, mid villus and crypt cells were separated. An alternative approach that is based on an EDTA-dissociation procedure afforded fractions enriched in villus and crypt cells. Although several enzymatic and metabolic activities of these two cell preparations were comparable, cell viability based on the Trypan Blue dye exclusion test, ultrastructural appearance and glucose uptake more closely conformed to in vivo values for cells isolated according to the EDTA-dissociation method. These cells were chosen as a model for drug transport investigation. The morphological and functional integrity of everted rings was verified by histological examination, extracellular space estimation and assessment of glucose transport ability. Sodium salicylate uptake studies using brush border membrane vesicles and isolated mucosal cells were highly variable, whereas everted segments exhibited good reproducibility in uptake experiments. Time dependence of salicylate uptake was demonstrated with membrane vesicles and everted rings. Time dependence was not observed in mucosal cell uptake studies, probably because of the time required to separate the cells from the incubation solution. Based on ease of preparation, technical aspects of in vitro incubation and reproducibility of results, everted intestinal rings were considered to be a good potential model for in vivo drug absorption. Brush border membrane vesicles were generally regarded as unacceptable because of variations after storage and between experiments. Isolated cells offered certain advantages, but the utility of cells as an in vitro model remains equivocal.

Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine.

The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [3H]uridine injection or by the de novo pathway as shown after [3H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium--that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5-3H]uridine or [5-3H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloracetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [3H]uridine and 72% after [3H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A+ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [3H] uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant. After [3H]-orotic acid injection, the number of silver grains over the nucleolus was negligible at all levels, whereas over the nucleoplasm the number was low in crypt cells, but high in villus cells with a peak in mid villus. The interpretation is that, except for a small amount of label incorporated into DNA from either precursor by crypt cells, the bulk of the label is incorporated into RNA as follows. In the crypts, cells make almost exclusive use of uridine, that is, of the salvage pathway, for the synthesis of ribosomal RNA in the nucleolus and of messenger and transfer RNA in the nucleoplasm. However, when cells pass from crypt to villus, they mainly utilize orotic acid--i.e., the de novo pathway--for the synthesis of messenger and transfer RNA within the nucleoplasm.

Anomalous replacement of foetal enterocytes in the neonatal pig.

The fine structure of intestine from pigs 8 days old has been studied by using transmission and scanning electron microscopy of tissues fixed normally or after limited contact with distilled water. Two types of enterocyte were identified in sections of normally fixed tissue. The first, containing an apical tubular system, is considered to represent the remains of an enterocyte population already present at birth (the foetal type). The second, containing much rough endoplasmic reticulum and none of the structures listed above, is considered to represent the type of enterocyte produced postnatally (the adult type). Additional features used to distinguish these two types of enterocyte are the length of microvilli (longer for the foetal than for the adult type) and the width of the terminal web (wider for the adult than for the foetal type). Treatment of intestine from pigs 8 days old with distilled water for 30 s causes foetal-type enterocytes to swell preferentially. Their position on the villus can then be identified by using scanning electron microscopy. Foetal-type enterocytes are largely, but not entirely, confined to the upper regions of individual villi. A small proportion of foetal-type enterocytes exist in the mid villus region, either singly or in patches entirely sur­rounded by adult-type enterocytes. These results are taken to provide direct evidence for the irregular replacement of foetal-type by adult-type enterocytes in the immediate postnatal period of development. The possible effect that this might have on the susceptibility of neonatal pigs to enteric disease is discussed.