Toxicity of two heavy rare earth elements to freshwater mussels Dreissena polymorpha.

Rare earth elements (REE) are essential components of many electronic devices that could end-up in solid waste disposal sites and inadvertently released in the environment. The purpose of this study was to examine the toxicity of two heavy REEs, erbium (Er) and lutetium (Lu), in freshwater mussels Dreissena polymorpha. Mussels were exposed to 14 days to increasing concentration (10, 50, 250, and 1250 µg/L) of either Er and Lu at 15 °C and analyzed for gene expression in catalase (CAT), superoxide dismutase (SOD), metallothionein (MT), cytochrome c oxidase (CO1), and cyclin D for cell cycle. In addition, lipid peroxidation (LPO), DNA damage (DNAd), and arachidonate cyclooxygenase were also determined. The data revealed that mussels accumulated Er and Lu similarly and both REEs induced changes in mitochondrial COI activity. Er increased cell division, MT, and LPO, while Lu increased DNAd and decreased cell division. Tissue levels of Er were related to changes in MT (r = 0.7), LPO (r = 0.42), CO1 (r = 0.69), and CycD (r = 0.31). Lu tissue levels were related to changes in CO1 (r = 0.73), CycD (r = - 0.61), CAT (r = 0.31), DNAd (r = 0.43), and SOD (r = 0.34). Although the lethal threshold was similar between Er and Lu, the threshold response for LPO revealed that Er produced toxicity at concentrations 25 times lower than Lu suggesting that Er was more harmful than Lu in mussels. In conclusions, the data supports that the toxicity pattern differed between Er and Lu although they are accumulated in the same fashion.

Micro and Nanoplastic Contamination and Its Effects on Freshwater Mussels Caged in an Urban Area.

Plastic-based contamination has become a major cause of concern as it pervades many environments such as air, water, sediments, and soils. This study sought to examine the presence of microplastics (MPs) and nanoplastics (NPs) in freshwater mussels placed at rainfall/street runoff overflows, downstream (15 km) of the city centre of Montréal, and 8 km downstream of a municipal effluent dispersion plume. MPs and NPs were determined using flow cytometry and size exclusion chromatography using fluorescence detection. Following 3 months of exposure during the summer season, mussels contained elevated amounts of both MPs and NPs. The rainfall overflow and downstream of the city centre were the most contaminated sites. Lipid peroxidation, metallothioneins, and protein aggregates (amyloids) were significantly increased at the most contaminated sites and were significantly correlated with NPs in tissues. Based on the levels of MPs and NPs in mussels exposed to municipal effluent, wastewater treatment plants appear to mitigate plastic contamination albeit not completely. In conclusion, the data support the hypothesis that mussels placed in urbanized areas are more contaminated by plastics, which are associated with oxidative damage. The highest responses observed at the overflow site suggest that tire wear and/or asphalt (road) erosion MPs/NPs represent important sources of contamination for the aquatic biota.

Comparative toxicity of micro and nanopolystyrene particles in Mya arenaria clams.

The contamination of coastal marine environments by plastics of sizes ranging from mm down to the nanoscale (nm) could pose a threat to aquatic organisms. The purpose of this study was to examine the toxicity of polystyrene nanoparticles (PsNP) of various sizes (50, 100 and 1000 nm) to the marine clams Mya arenaria. Clams were exposed to concentrations of PsPP for 7 days at 15 °C and analyzed for uptake/transformation, changes in energy metabolism, oxidative stress, genotoxicity and circadian neural activity. The results revealed that PsNP accumulated in the digestive gland was 50 nm > 100 nm > 1000 nm. All sized increased oxidative stress as follows: 50 nm (peroxidase, antioxidant potential and LPO), 100 nm (LPO and antioxidant potential) and 1000 nm (LPO). Tissue damage was also size dependent by increasing genotoxicity. The 100 nm PsPP altered the levels of the circadian metabolite melatonin. We conclude that the toxicity of plastics is size dependent in clams.

Form-Dependent Toxicity of Silver Nanomaterials in Rainbow Trout Gills.

The toxicity of the form of nanoparticles is presently not well understood. The purpose of this study consists in comparing the toxicity of various forms of silver nanoparticles (nAg) in juvenile rainbow trout Oncorhynchus mykiss. Juveniles were exposed to various forms of polyvinyl-coated nAg of similar size for 96 h at 15 °C. After the exposure period, the gills were isolated and analyzed for Ag uptake/distribution, oxidative stress, glucose metabolism, and genotoxicity. Higher levels of Ag were detected in gills in fish exposed to dissolved Ag followed by spherical, cubic, and prismatic nAg. Size-exclusion chromatography of gill fractions revealed that the dissolution of nAg was observed for all forms of nAg where prismatic nAg released more important levels of Ag in the protein pool as in fish exposed to dissolved Ag as well. The aggregation of nAg was more important for cubic nAg in respect of the other forms of nAg. The data revealed that lipid peroxidation was closely associated with protein aggregation and viscosity. Biomarkers revealed changes in lipid/oxidative stress and genotoxicity, which were related to the loss of protein aggregation and inflammation (NO2 levels), respectively. In general, the observed effects were found for all forms of nAg where the effects from prismatic nAg were generally higher than for spherical and cubic nAg. The strong relationship between genotoxicity and inflammation response suggests the participation of the immune system in the observed responses of juvenile fish gills.

Investigation on the Toxicity of Nanoparticle Mixture in Rainbow Trout Juveniles.

The environmental impacts of nanoparticle mixtures in the aquatic environment is not well understood. The purpose of this study examined the sub-lethal toxicity of low concentrations (ug/L range) of selected nanoparticles alone and in mixtures in juvenile trout. Fish were exposed to to individual and two environmentally relevant mixtures of silver (nAg), copper oxide (nCuO) and cerium oxide (nCeO) nanoparticles for 96 h at 15 °C. After the exposure period, fish were depurated overnight and tissue levels in Ag, Ce, Cu and Zn were determined along with a suite of effects biomarkers such as oxidative stress/inflammation, denatured protein tagging (ubiquitin), DNA strand breaks (genotoxicity) and acetylcholinesterase (AChE) activity. The data showed that these nanoparticles behaved as suspended matter but were nevertheless bioavailable for fish with bioconcentration factors of 6, 8 and 2 for nAg, nCeO and nCuO respectively. Only nCuO alone increased malonaldehyde (lipid peroxidation) contents but all nanoparticles increased DNA damage, protein-ubiquitin labeling, and decreased AChE activity. Globally, the toxicity of nCeO and nCuO was generally stronger than nAg, and antagonist effects were found in the mixtures. The interactions involved in these antagonisms are not well understood but do not involve the liberation of free ions and labile zinc in tissues. In conclusion, the bioavailability and toxicity of these nanoparticles are influenced by mixtures of nanoparticles, which is likely to occur in contaminated environments.

Shape-Dependent Toxicity of Silver Nanoparticles on Freshwater Cnidarians.

Silver nanoparticles (AgNPs) are increasingly used in various consumer products, leading to their inadvertent release in aquatic ecosystems. The toxicity of AgNPs could be associated with the leaching of ionic Ag but also with the size, shape and surface properties. The purpose of this study was to test the null hypothesis that toxicity of AgNPs was independent of shape in the invertebrate Hydra vulgaris. The hydranths were exposed to increasing concentrations of ionic Ag and AgNPs of three different shapes (spherical, cubic and prismatic) with the same size and coating (polyvinylpyrrolidone). The data revealed that between 68% and 75% of total Ag remained in solution after the 96 h exposure period, while 85−90% of ionic Ag remained in solution. The 96 h lethal concentration (LC50) was lower with ionic (4 µg/L) and spherical AgNPs (56 µg/L), based on irreversible morphological changes such as loss of tentacles and body disintegration. Cubic and prismatic AgNPs were not toxic at a concentration of <100 µg/L. The sublethal toxicity was also determined at 96 h based on characteristic morphological changes (clubbed and/or shortened tentacles) and showed the following toxicity: ionic (2.6 µg/L), spherical (22 µg/L) and prismatic (32.5 µg/L) AgNPs. The nanocube was not toxic at this level. The data indicated that toxicity was shape-dependent where nanoparticles with a low aspect ratio in addition to high circularity and elongation properties were more toxic at both the lethal and sublethal levels. In conclusion, the shape of AgNPs could influence toxicity and warrants further research to better understand the mechanisms of action at play.

Assessment of the toxicity of a mixture of five rare earth elements found in aquatic ecosystems in Hydra vulgaris.

Rare earth elements (REEs) are considered critical elements for technology and their extraction through mining activities is expected to increase in the future. Due to their chemical similarities, they often co-occur in minerals and thus their ecotoxicity should be assessed as a group/family. However, the available ecotoxicological studies focused mainly on the evaluation of the potential toxicological impacts of individual REEs rather than their mixtures. The aim of this study was therefore to evaluate the toxicity of a representative mixture of five REEs (La, Ce, Pr, Nd and Sm) spanning environmentally relevant concentrations ranging from 0.05X (29 µg REEs L-1) to 5X (2926 µg REEs L-1) to the test organism, Hydra vulgaris, at the morphological, reproductive and regenerative levels. The data showed that lethality occurred at concentrations near (2.5 fold) to those inducing sublethal effects after chronic exposure of 7 days. The mixture affected reproduction and head regeneration and even lethality at concentrations even below those reported at environmental concentration (0.5X = 293 µg REEs L-1) in lakes. This suggests that REEs concentrations found in lakes near mining activities could disrupt regeneration and impair embryonic development. Our data also revealed that combining the 5 REEs results in an antagonistic effect, suggesting that those elements share the same receptor and that low molecular weight and high radius elements (approaching iron) were less toxic. Taken together, hydra could be used as a sensitive model organism for the assessment of aquatic ecotoxicological risks of REE mixtures but further analyses of biochemical and gene expressions should improve our understanding of the long-term effects of REEs mixtures.

Crowding Effects of Polystyrene Nanoparticles on Lactate Dehydrogenase Activity in Hydra Attenuata.

Plastics pervade our environment and potentially release important quantities of plastic nanoparticles (NPs) from degradation in the environment. The purpose of this study was to examine the crowding effects of polystyrene NPs on lactate dehydrogenase (LDH) in vitro and following exposure to Hydra attenuata. First, LDH activity was measured in vitro in the presence of filamentous (F-)actin and NPs (50 and 100 nm diameter) to determine changes in viscosity and the fractal kinetics of LDH. The fractal dimension (fD) was also determined using the rescaled range analysis procedure. Secondly, these changes were examined in hydra exposed to NPs for 96h to concentrations of NPs. The data revealed that the addition of F-actin increased the rate of LDH at low substrate (pyruvate) concentrations compared to LDH alone with a gradual decrease in the rate with the addition of pyruvate, which is characteristic of the fractal behavior of enzymes in crowded environments. The addition of 50 and 100 nm NPs also produced these changes, which suggest that NPs could change the space properties of the LDH reaction. The fD was reduced to 0.85 and 0.91 with 50 and 100 nm NPs compared to 1.093 with LDH alone. Decrease in the fD was related with increased amplitudes and frequency in viscosity waves in the reaction media. Exposure of hydra to NPs confirmed the increase in LDH activity and the fD was significantly correlated with LDH activity (r = -0.5). Correction of LDH activity (residuals) still revealed an increase in LDH activity in hydra suggesting increased anaerobic metabolism by NPs. In conclusion, the presence of NPs in the intracellular space decreased the fD, which could influence LDH activity in organisms exposed to NPs.

Biophysical effects of polystyrene nanoparticles on Elliptio complanata mussels.

The presence of nanoplastics (NPs) in various  products and from the weathering of released plastic materials are of concern for the environment's safety. The purpose of this study was to examine the biophysical effects of polystyrene NPs on freshwater mussels. Mussels were exposed to a range of concentrations of NPs (0.1, 0.5, 1, and 5 mg/L) for 24 h and allowed to depurate for 12 h in clean aquarium water. The digestive gland was isolated and analyzed for NPs, lipids, viscosity, protein aggregation, anisotropic changes (liquid crystals: LCs), and the oscillatory modulation in viscosity during the formation of self-organizing enzyme complex of fumarase, malate dehydrogenase, and citrate synthase. The results revealed that mussels accumulated NPs in the digestive gland and their levels were significantly correlated with lipids levels, LCs, the increase in the malate dehydrogenase/citrate synthase activity ratio, and oscillations in viscosity. Protein aggregation was also found to be correlated with lipid levels. The data suggests that the presence of NPs in the digestive gland involves changes in lipid content and LC formation and perturbs the normal oscillations in viscosity during sequential enzyme reactions of the above enzymes. It is concluded that the uptake of NPs in cells could disrupt the internal organization of cells which can interfere with the normal association of enzymes involved in energy metabolism.

Detection, biophysical effects, and toxicity of polystyrene nanoparticles to the cnidarian Hydra attenuata.

The occurrence of nanoplastic particles (NPs) in the environment has raised concerns about the ecotoxicological risk to aquatic ecosystems. The purpose of this study was to examine the bioavailability and toxicity of 50- and 100-nm transparent polystyrene NPs to the cnidarian Hydra attenuata. The hydras were exposed to increasing concentrations of 50- and 100-nm NPs (1.25, 2.5, 5, 10, 20, 40, and 80 mg/L) for 96 h at 20 °C followed by a 24-h depuration step. Hydras were analyzed for morphological changes, bioaccumulation of NPs using a novel assay for polystyrene NPs, oxidative stress (lipid peroxidation), polar lipids, lipid-like liquid crystals (LCs), and viscosity changes in the post-mitochondrial fraction. The results revealed that the organisms accumulated detectable amounts of NP in a concentration-dependent manner for both the 50- and 100-nm NP that persisted after 24 h in clean media. Changes in morphology were observed with a 50% effect concentration of 3.6 and 18 mg/L for the 50- and 100-nm-diameter NPs respectively. However, based on the particle concentration, the 100 nm proved to be 1.7 times more toxic than the 50-nm NPs. Exposure to NPs led to decreased biomass, lipid peroxidation (LPO), increased polar lipid levels, viscosity, and formation of LCs at the intracellular level. In the more toxic NP (100 nm), NPs in tissues were correlated with LCs, polar lipids, and LPO levels. It appears that the formation of organized LCs and polar lipids of NPs in cells was involved with NP toxicity and could represent a yet unidentified, detoxifying/bioactivation mechanism against colloidal plastics in cells. In conclusion, NPs are bioavailable to hydra and lead to LPO and lipid mobilization in hydra. The capacity of increasing lipid mobilization and LCs could determine the size-dependence toxicity of NPs.

Detection of polystyrene nanoplastics in biological samples based on the solvatochromic properties of Nile red: application in Hydra attenuata exposed to nanoplastics.

The release of nanoplastics (NP) from the weathering of microplastics is a major concern for the environment. Methods for the detection of NP in biological tissues are urgently needed because of their ability to penetrate not only in tissues but also in cells. A simple fluorescence-based methodology for the detection of polystyrene NP in biological tissues is proposed using the solvatochromic properties of Nile red. Although NPs alone increased somewhat Nile red fluorescence, a characteristic hypsochromic shift in the emission spectra was found when the dye and NP were incubated with subcellular tissue fraction. To explain this, the probe and NPs (50 and 100 nm) were prepared in the presence of increasing concentrations of two detergents (Tween-20, Triton X-100) as a proxy to phospholipids. The data revealed that both detergents readily increased fluorescence values when added to the NP and Nile red. The addition of NPs in tissue extracts blue-shifted further the emission spectra to 623 nm from the normal Nile red-lipid peak at 660 nm. The fluorescence intensity was proportional to the NP concentration. A methodology is thus proposed for the detection of NPs in laboratory-exposed organisms based on the solvatochromic properties of Nile red. The methodology was used to detect the presence of NP and changes in polar lipid contents in Hydra attenuata exposed to polystyrene NP.

Evaluation of uptake and chronic toxicity of virgin polystyrene microbeads in freshwater zebra mussel Dreissena polymorpha (Mollusca: Bivalvia).

Microplastics (MPs), plastic debris smaller than 5mm, are widely found in both marine and freshwater ecosystems. However, few studies regarding their hazardous effects on inland water organisms, have been conducted. For this reason, the aim of our research was the evaluation of uptake and chronic toxicity of two mixtures (MIXs) of virgin polystyrene microbeads (PMs) of 10µm and 1µm in size (MIX 1, with 5×105 of 1µmsizePMs/L and 5×105 of 10µmsizePMs/L, and MIX 2 with 2×106 of 1µmsizePMs/L and 2×106 of 10µmsizePMs/L) on freshwater zebra mussel Dreissena polymorpha (Mollusca: Bivalvia) during 6 exposure days. The PM uptake in the mussel body and hemolymph was assessed using confocal microscopy, while the chronic toxicity of PMs was evaluated on exposed mussels using a comprehensive battery of biomarkers of cellular stress, oxidative damage and neuro- genotoxicity. Confocal microscopy analyses showed that MPs concentrated in the gut lumen of exposed mussels, absorbed and transferred firstly in the tissues and then in the hemolymph. The results revealed that PMs do not produce oxidative stress and genetic damage, with the exception of a significant modulation of catalase and glutathione peroxidase activities in mussels exposed to MIX 1. Regarding neurotoxicity, we observed only a significant increase of dopamine concentration in mussels exposed to both MIXs, suggesting a possible implication of this neurotransmitter in an elimination process of accumulated PMs. This research represents a first study about the evaluation of virgin MP toxicity in zebra mussel and more research is warranted concerning the long term neurological effects of virgin MPs.

The influence of zinc chloride and zinc oxide nanoparticles on air-time survival in freshwater mussels.

The purpose of this study was to determine the cumulative effects of exposure to either dissolved zinc or nanozinc oxide (nanoZnO) and air-time survival in freshwater mussels. Mussels were exposed to each forms of zinc for 96h then placed in air to determine survival time. A sub-group of mussels before and after 7days of exposure to air were kept aside for the determination of the following biomarkers: arachidonate-dependent cyclooxygenase (COX) and peroxidase (inflammation and oxidative stress), lipid metabolism (total lipids, esterases activity, HO-glycerol, acetyl CoA and phospholipase A2) and lipid damage (lipid peroxidation [LPO]). The results showed that air-time survival was decreased from a mean value of 18.5days to a mean value of 12days in mussels exposed to 2.5mg/L of nanoZnO although it was not lethal based on shell opening at concentrations below 50mg/L after 96h. In mussels exposed to zinc only, the median lethal concentration was estimated at 16mg/L (10-25 95% CI). The air-time survival did not significantly change in mussels exposed to the same concentration of dissolved Zn. Significant weight losses were observed at 0.5mg/L of nanoZnO and at 2.5mg/L for dissolved zinc chloride, and were also significantly correlated with air-time survival (r=0.53; p<0.01). Air exposure significantly increased COX activity in control mussels and in mussels exposed to 0.5mg/L of nanoZnO and zinc chloride. The data also suggested fatty acid breakdown and ß-oxidation. Mussels exposed to contaminants are more susceptible to prolonged exposure to air during low water levels.

Differential expression of the integrins alpha6Abeta4 and alpha6Bbeta4 along the crypt-villus axis in the human small intestine.

The integrin alpha6 subunit exists as two different variants, termed alpha6A and alpha6B. These two variants have been shown to harbor potentially distinct biochemical properties but little is known about their cellular function. The aim of this work was to characterize the expression of the integrin alpha6A and B variants in relation to cell proliferation and differentiation in the human small intestinal epithelium. The results showed distinct expression patterns for the two variants along the crypt-villus axis. Indeed, proliferative cells of the crypt were found to predominantly express alpha6A, while differentiated enterocytes and Paneth cells expressed the alpha6B variant. A similar relationship was observed in intestinal cell models by competitive RT-PCR. Further studies in the Caco-2 cell model showed that manipulating the cellular balance of the two alpha6 variants can influence transcriptional activities related to cell proliferation but not differentiation. This suggests that differential expression of the alpha6 subunits is involved in the intestinal epithelial cell renewal process. Further studies will be needed to substantiate this hypothesis.

Laminins in the developing and adult human small intestine: relation with the functional absorptive unit.

The expression of the five laminin alpha-chains was analyzed in the developing and mature human small intestine at the protein and transcript levels in order to further delineate specific involvement of individual laminins in relation to the epithelial cell state as defined along the functional crypt-villus axis. The results show that all of the alpha-laminin transcripts are expressed in significant amounts in the small intestine relative to a panel of other tissues and organs. Further analysis of their expression by indirect immunofluorescence and semi-quantitative and quantitative RT-PCR demonstrates a close correlation between transcript and protein expression, distinct epithelial and mesenchymal origins, as well as differential occurrence in intestinal basement membranes according to developmental stage, along the crypt-villus axis and in compartment-related experimental intestinal cell models. Taken together, the data point out the prime importance of alpha2-, alpha3-, and alpha5-containing laminins for the development and maintenance of the functional human intestinal epithelium.

Gene expression profiles of normal proliferating and differentiating human intestinal epithelial cells: a comparison with the Caco-2 cell model.

cDNA microarray technology enables detailed analysis of gene expression throughout complex processes such as differentiation. The aim of this study was to analyze the gene expression profile of normal human intestinal epithelial cells using cell models that recapitulate the crypt-villus axis of intestinal differentiation in comparison with the widely used Caco-2 cell model. cDNA microarrays (19,200 human genes) and a clustering algorithm were used to identify patterns of gene expression in the crypt-like proliferative HIEC and tsFHI cells, and villus epithelial cells as well as Caco-2/15 cells at two distinct stages of differentiation. Unsupervised hierarchical clustering analysis of global gene expression among the cell lines identified two branches: one for the HIEC cells versus a second comprised of two sub-groups: (a) the proliferative Caco-2 cells and (b) the differentiated Caco-2 cells and closely related villus epithelial cells. At the gene level, supervised hierarchical clustering with 272 differentially expressed genes revealed distinct expression patterns specific to each cell phenotype. We identified several upregulated genes that could lead to the identification of new regulatory pathways involved in cell differentiation and carcinogenesis. The combined use of microarray analysis and human intestinal cell models thus provides a powerful tool for establishing detailed gene expression profiles of proliferative to terminally differentiated intestinal cells. Furthermore, the molecular differences between the normal human intestinal cell models and Caco-2 cells clearly point out the strengths and limitations of this widely used experimental model for studying intestinal cell proliferation and differentiation.

Normalizing genes for quantitative RT-PCR in differentiating human intestinal epithelial cells and adenocarcinomas of the colon.

As for other mRNA measurement methods, quantitative RT-PCR results need to be normalized relative to stably expressed genes. Widely used normalizing genes include beta-actin and glyceraldehyde-3-phosphate dehydrogenase. It has, however, become clear that these and other normalizing genes can display modulated patterns of expression across tissue types and during complex cellular processes such as cell differentiation and cancer progression. Our objective was to set the basis for identifying normalizing genes that displayed stable expression during enterocytic differentiation and between healthy tissue and adenocarcinomas of the human colon. We thus identified novel potential normalizing genes using previously generated cDNA microarray data and examined the alterations of expression of two of these genes as well as seven commonly used normalizing genes during the enterocytic differentiation process and between matched pairs of resection margins and primary carcinomas of the human colon using real-time RT-PCR. We found that ribosomal phosphoprotein P0 was particularly stable in all intestinal epithelial cell extracts, thereby representing a particularly robust housekeeping reference gene for the assessment of gene expression during the human enterocytic differentiation process. On the other hand, beta-2-microglobulin generated the best score as a normalizing gene for comparing human colon primary carcinomas with their corresponding normal mucosa of the resection margin, although others were found to represent acceptable alternatives. In conclusion, we identified and characterized specific normalizing genes that should significantly improve quantitative mRNA studies related to both the differentiation process of the human intestinal epithelium and adenocarcinomas of the human colon. This approach should also be useful to validate normalizing genes in other intestinal contexts.

Repressed E-cadherin expression in the lower crypt of human small intestine: a cell marker of functional relevance.

In epithelia, abnormal expression of E-cadherin is related to pathologies involving a loss of cell polarization and/or differentiation. However, recent observations suggest that E-cadherin could also be repressed under physiological conditions, such as in some epithelial stem cell lineages. In the present work, we have analyzed E-cadherin expression in human intestinal epithelial cell progenitors and investigated its potential role. E-cadherin expression was analyzed along the crypt-villus axis by immunofluorescence on cryosections of small intestine. E-cadherin was found to be differentially expressed, being significantly weaker in the cells located at the bottom of the crypts. Surprisingly, neither the E-cadherin protein nor transcript were detected in a normal human intestinal epithelial (HIEC) crypt cell model isolated in our laboratory, whereas other E-cadherin-related components such as catenins and APC were present. Forced expression of E-cadherin in HIEC cells increased membrane-associated beta-catenin and was accompanied by the appearance of junction-like structures at the cell-cell interface. Functionally, cell kinetics and p21Cip levels were found to be altered in the E-cadherin expressing HIEC cells as compared to controls. Furthermore, a significant reduction of the migration abilities and an increase in sensitivity to anoikis were also observed. These results suggest that down-regulated expression of E-cadherin is a human intestinal crypt base cell-related feature that appears to be of functional relevance for the maintenance of the progenitor cell population.