Henno G.C.J.M. Hendriks

Effects of soybean-containing diets on the proximal and distal intestine in Atlantic salmon (Salmo salar) : a morphological study

Abstract The morphology of the proximal and distal intestine of Atlantic salmon (Salmo salar) was studied after prolonged feeding with diets containing full-fat soybean meal (FFSB) or soybean protein concentrate (SBPC) and compared with fish fed a standard herring meal (HM) diet. To avoid possible changes due to decreased food intake, dietary inclusions of FFSB and SBPC were chosen at such levels that weight gain, and protein and lipid digestibility values were similar for all three groups. The proximal intestine showed no differences among the groups, except for an increased number of goblet cells in the fish fed SBPC. In the distal intestine, the SBPC group showed no abnormalities and was identical to the HM group. In the FFSB group, the epithelium had an increased number of goblet cells and a marked decrease or even absence of absorptive vacuoles. The microvilli of the enterocytes were shortened, with increased microvillar vesicle formation. These changes may be due to the presence of antinutritional factors in the FFSB diet. The possible role of the various antinutritional factors in soybeans for the development of the intestinal lesions, and their effect on growth and performance are discussed.

Binding of soybean agglutinin to small intestinal brush border membranes and brush border membrane enzyme activities in Atlantic salmon (Salmo salar)

To test for possible toxicity of soybean lectin for salmon, the location of soybean agglutinin binding sites in the small intestinal brush border membranes (BBM) was determined quantitatively by an enzyme-linked lectin sorbent assay (ELLSA). The assay revealed substantial binding in both the proximal (Bmax 1764±170 nM per mg BBM protein; Kd 2.16±0.23·10−5) and distal (Bmax 2096±79 nM per mg BBM protein; Kd 1.58±0.11·10−5) small intestine. The higher maximum binding and the lower dissociation constant for the distal small intestine might indicate a greater sensitivity to the toxic effect of soybean lectin in this region. Enzyme activities for alkaline phosphatase (EC 3.1.3.1) and sucrase isomaltase (EC 3.2.1.48) in brush border membrane preparations were very low in both the proximal and distal small intestine. A high enzyme activity was found for aminopeptidase (EC 3.4.11.2). Activities of sucrase isomaltase and aminopeptidase were lower in the distal than in the proximal small intestine.

Nippostrongylus brasiliensis Histochemical changes in the composition of mucins in goblet cells during infection in rats

Changes in the quality of mucins in jejunal goblet cells were investigated during an infection with Nippostrongylus brasiliensis in rats. At 10 days after infection, when proliferative activity in the crypts is excessive and both crypts and villi are characterized by hyperplasia of goblet cells, the histochemical composition of the population of goblet cells in comparison with controls shows a marked increase in crypt and villous goblet cells containing neutral mucins. At 15 days after infection both crypts and villi display a significant increase in goblet cells containing acid mucin and decrease in goblet cells containing neutral mucin. The acid mucins in crypt and villous goblet cells on day 15 appear to be sulphomucins predominantly, whereas in controls sialomucin-containing goblet cells dominate both in the crypts and on the villi. These experiments establish that the explusion of N. brasiliensis from the intestine of the rat coincides not only with quantitative, but also with remarkable qualitative changes in the histochemical composition of mucins in goblet cells.

Effect of the insecticidal Galanthus nivalis agglutinin on metabolism and the activities of brush border enzymes in the rat small intestine

Abstract With increasing use of lectin genes in crop plants to improve insect resistance, the dietary exposure of humans to lectins will rise and it is necessary to assess whether the presently most favored insecticidal lectin, Galanthus nivalis agglutinin, would be harmful for mammals. Effects of Galanthus nivalis agglutinin on gut and brush border enzymes were studied in rats over a 10-day dietary exposure and compared with those of a known antinutrient, phytohaemagglutinin. At a level that provides insecticidal protection for plants but did not reduce the growth of young rats, Galanthus nivalis agglutinin had negligible effects on the weight and length of the small intestine even though there was a slight, but significant hypertrophy of this tissue. However, the activities of brush border enzymes were affected; sucrase-isomaltase activity was nearly halved and those of alkaline phosphatase and aminopeptidase were significantly increased. Although most of the changes in gut metabolism caused by the incorporation of Galanthus nivalis agglutinin in the diet were less extensive than those found with toxic phytohaemagglutinin, some of them may be potentially deleterious. Thus, further and longer animal studies are needed to establish whether it is safe to use Galanthus nivalis agglutinin in transgenic plants destined for human consumption.

Decreased levels of heat shock proteins in gut epithelial cells after exposure to plant lectins

BACKGROUND The enterocytes of the intestinal epithelium are regularly exposed to potentially harmful substances of dietary origin, such as lectins. Expression of heat shock proteins (HSPs) by this epithelium may be part of a protective mechanism developed by intestinal epithelial cells to deal with noxious components in the intestinal lumen. AIM To investigate if the lectins PHA, a lectin from kidney beans (Phaseolus vulgaris) and WGA, a lectin from wheat germ (Triticum aestivum) could modify the heat shock response in gut epithelial cells and to establish the extent of this effect. METHODS Jejunal tissue sections from PHA and WGA fed rats were screened for expression of HSP70, HSP72, and HSP90 using monoclonal antibodies. Differentiated Caco-2 cells, the in vitro counterpart of villus enterocytes, were exposed to 100 μg/ml of PHA-E4 or WGA for 48 hours and investigated for changes in DNA and protein synthesis by double labelling with [2-14C]thymidine and L-[methyl-3H]methionine. The relative concentrations of HSP60, HSP70, HSP72, and HSP90 and binding protein (BiP) in these cells exposed to lectins were analysed by polyacrylamide gel electrophoresis and immunoblotting. To establish if lectin exposed differentiated Caco-2 cells were still capable of producing a heat shock response, these cells received a heat shock (40°C, 41°C, and 42°C) for one hour and were allowed to recover for six hours at 37°C. During heat shock and recovery periods, lectin exposure was continued. RESULTS Constitutive levels of HSPs were measured in the intestinal cells of lactalbumin fed (control) rats, as may be expected from the function of this tissue. However, in PHA and WGA fed rats a marked decline in the binding of antibodies against several HSPs to the intestinal epithelium was found. These results were confirmed by in vitro experiments using differentiated Caco-2 cells exposed to PHA-E4 and WGA. However, after exposure to lectins, these cells were still capable of heat induced heat shock protein synthesis, and total protein synthesis was not impaired indicating specific inhibition of HSP synthesis in non-stressed cells. CONCLUSIONS We conclude that PHA and WGA decrease levels of stress proteins in rat gut and enterocyte-like Caco-2 cells, leaving these cells less well protected against the potentially harmful content of the gut lumen.

Interaction of legume lectins with the cellular metabolism of differentiated Caco-2 cells

The binding of the legume lectins Phaseolus vulgaris E4 and L4, Glycine max agglutinin, Vicia faba agglutinin, and Pisum sativum agglutinin to intact differentiated Caco-2 cells and to brush border membranes of differentiated Caco-2 cells was investigated, and their impact on the cellular metabolism and the microvilli of these cells was assessed. P. vulgaris isolectin E4 showed the most intense staining after binding of fluorescein isothiocyanate-labeled lectin to intact Caco-2 cells. P. sativum agglutinin showed the weakest staining intensity. The dissociation constant for P. vulgaris isolectin E4 and P. sativum agglutinin binding was 0.11 x 10(-5) and 1.69 x 10(-5) mol/L, respectively. The values of the dissociation constants for P. vulgaris isolectin L4, G. max agglutinin, and V. faba agglutinin were situated in between these extremes. Stimulation of thymidine, glucosamine, and fucose incorporation was observed after exposure to P. vulgaris isolectins and soybean agglutinin. V. faba agglutinin had an inhibitory effect, whereas P. sativum agglutinin showed little or no effect. Compared with control cells and P. vulgaris isolectin L4- and P. sativum agglutinin-incubated cells, the microvilli of P. vulgaris isolectin E4-, soybean agglutinin-, and V. faba agglutinin-incubated cells were shortened significantly. The data provide evidence that a correlation exists, not only between the dissociation constants of the lectins and the fluorescent staining intensity, but also between the dissociation constants of the lectins and the extent of the legume lectin-induced changes in the cellular metabolism.

Iron metabolism in mynah birds (Gracula religiosa) resembles human hereditary haemochromatosis

Iron overload is a very frequent finding in several animal species and a genetic predisposition is suggested. In one of the most commonly reported species with susceptibility for iron overload (mynah bird), it was recently shown that the cause of this pathophysiology is high uptake and retention of dietary iron. Here we compare susceptible (mynahs) with non-susceptible avian species (chickens) by evaluating iron uptake at the intestinal absorptive cell level. Enterocytes from mynahs and chickens were isolated and uptake of Fe(II) and Fe(III) was studied in vitro. It was found that Fe(III) uptake is much lower than Fe(II) uptake for both species. Although liver iron, present only in hepatocytes, was at least 10-fold higher in mynahs than chickens, enterocyte Fe(II) uptake was considerably higher in mynahs. Fe(II) uptake showed saturation at the studied concentrations in both species. Kinetic studies revealed a three-fold increase in V max for mynahs. Calculated values for the uptake kinetics of the probable membrane transporter suggest that mynah bird enterocytes have a significantly higher limiting uptake rate, due to the possible increase in the number of transporters when compared with chicken enterocytes. The susceptibility of this species is due to intestinal iron uptake despite hepatic iron accumulation, implicating a ‘mis-sensing’ of body iron similarly to human hereditary haemochromatosis.

Actin cytoskeletal lesions in differentiated human colon carcinoma Caco-2 cells after exposure to soybean agglutinin

We have investigated the effects of soybean agglutinin on the cytoskeletal element actin in differentiated Caco‐2 cells. The actin cytoskeleton of the cells was visualized by fluorescence microscopy using 7‐nitrobenz‐2‐oxa‐1, 3‐diazole phallacidin as a specific marker for F‐actin. Compared with control Caco‐2 cells no changes in the fluorescence pattern were observed after incubation with soybean agglutinin. However, using the deoxyribonuclease‐I inhibition assay a dose‐related response was noted in the increase of intracellular G‐actin after a 2‐hour incubation period with soybean agglutinin. Already after exposure for 15 min to soybean agglutinin a decrease in intracellular F‐actin was demonstrable. This apparent depolymerization could be prevented by incubating the Caco‐2 cells with soybean agglutinin and the appropriate monosaccharide simultaneously. The increase in the amount of G‐actin appeared to be correlated with a shortening of microvilli on the Caco‐2 cells.

Binding of kidney bean (Phaseolus vulgaris) isolectins to differentiated human colon carcinoma Caco-2 cells and their effect on cellular metabolism.

The binding of Phaseolus vulgaris (PHA) isolectins L4 and E4 to the brush border membrane of differentiated Caco-2 cells was studied and the impact on cellular metabolism and microvilli was assessed. Computer analysis of the data based on binding experiments with peroxidase conjugated isolectins gave mean (SD) values for maximal binding of 2540 (151).10(-9) M for PHA-L4 and 2104 (140).10(-9) M for PHA-E4 per mg of brush border membrane protein. The dissociation constants for L4 and E4 binding were 4.3 (1.4).10(-6) M and 1.1 (0.8).10(-6) M, respectively. Incubation of differentiated Caco-2 cells for 30 minutes with ferritin conjugated PHA isolectins showed that, as indicated by the number of ferritin particles, PHA-E4 bound to the microvilli to a greater extent than PHA-L4. Ferritin particles were also localised intracellularly over endocytotic invaginations and vesicles. After incubation for 48 hours with PHA-L4 or PHA-E4, the relative incorporation of precursors for DNA, RNA, and (glyco)protein synthesis into the trichloroacetic acid insoluble fraction of the Caco-2 cells was determined. Both isolectins stimulated the incorporation of thymidine and glucosamine, but neither PHA-L4 nor PHA-E4 were able to influence the incorporation of uridine. With respect to fucose, methionine, and N-acetyl mannosamine, the stimulatory effect remained confined to PHA-E4. Since PHA-L4 and PHA-E4 were tested at the same concentrations, PHA-E4 is more effective than PHA-L4. The changes in the uptake of radioactive precursors were lost after heat inactivation of PHA-E4. Compared with control and PHA-L4 incubated Caco-2 cells, the microvilli of PHA-E4 incubated cells were shortened significantly (p less than 0.01).

Polyamine metabolism of enterocyte-like Caco-2 cells after exposure to Phaseolus vulgaris lectin.

The effect of Phaseolus vulgaris isolectin E4 on polyamine concentrations and ornithine decarboxylase activity of proliferating and differentiating Caco-2 cells was investigated. Values of putrescine, spermidine, and spermine in control cells were highest during the early phase of proliferative cell growth and lowest in the stationary phase. Phytohaemagglutinin E4 significantly increased cellular polyamine values during the late proliferative phase of cell growth. Ornithine decarboxylase activity was high during intensive proliferation and growth, but was lower when proliferation slowed down or ceased. Exposure of Caco-2 cells in the early proliferative phase of cell growth to increasing concentrations of the potent intestinal growth factor phytohaemagglutinin E4 greatly stimulated enzyme activity. In contrast, the activity of ornithine decarboxylase was not stimulated in Caco-2 cells of the late proliferative phase nor was there any increase in the enzyme activity in differentiating and fully differentiated cells of the stationary phase. Accordingly, when proliferating Caco-2 cells possessed the highest ornithine decarboxylase activity, the polyamine values were also at their highest. During differentiation, as the ornithine decarboxylase activity fell close to zero, polyamine values also decreased. In the early proliferative phase of cell growth ornithine decarboxylase activity coincided with DNA synthesis in cells exposed to Phaseolus vulgaris isolectin E4. These findings with Caco-2 cells were similar to those found in brush border cells of the rat small intestine.