In vitro assessment of CeO2 nanoparticles effects on intestinal microvilli morphology.

Some nanoparticles (NPs) have been shown to disrupt intestinal microvilli morphology in vitro, an alteration that could potentially affect nutrient absorption and barrier properties. This study aimed at evaluating the potential effect of CeO2 NPs (4-8 nm, citrate stabilized) on Caco-2 microvilli morphology. In addition to the standard Caco-2 cell clone, the C2BBe1 clone was used, as it is considered to develop a more homogeneous cellular morphology. Semiautomated microvilli density quantification and a new cell scoring approach were used to evaluate scanning electron microscopy (SEM) images. The quantification method made use of the whole micrograph surface, avoiding the need to choose subareas for analysis, and increasing the representativeness of the results when compared to previous studies. The main advantage of the scoring system is that it informs on the intercellular variability within a cell preparation. Benzalkonium was used as a positive control inducing toxicity and morphological alterations on microvilli. After three-week differentiation, Caco-2 cells were exposed to 100 µg/mL of CeO2 NPs for 24 h. The integrity of the membrane was evaluated by transepithelial electrical resistance (TEER) and thereafter processed for its observation by SEM. Results showed that both the standard Caco-2 clone and the C2BBe1 clone present notable morphological heterogeneity. The two evaluation approaches were able to identify morphological effects caused by the positive control, but did not detect statistically significant morphological alterations after exposure to CeO2 NPs.

Cytochemical assessment of phosphopeptides derived from casein as potential ingredients for functional food.

Phosphopeptides derived from casein hydrolysis are suggested to have beneficial effects beyond their mere nutritive value by enhancing availability of dietary minerals, especially calcium, iron and zinc. Apart from a possible positive action the potential may exist for adverse effects that could impose restrictions to their widespread application in functional foods for human nutrition. In the present work, various case-inophosphopeptide (CPP) preparations were assessed using different human cell culture model systems in order to estimate their cytotoxic potential and their influence on epithelial properties and differentiation of human intestinal cells (Caco-2). The general cytotoxic potential of CPPs was tested using the AlphaTox NR assay. Structural and functional differentiation of intestinal Caco-2 cells was determined by measuring transepithelial electrical resistance and activity of brush border associated alkaline phosphatase over an extended cultivation period. No loss in cell viability with respect to membrane integrity could be detected, as uptake and retention of neutral red dye (AlphaTox assay) into HeLa cells was not affected by the presence of CPPs. Cytochemical assays conducted on epithelial cells (Caco-2) showed no disturbance of normal cell development and differentiation with respect to structural as well as functional differentiation markers. As all CPPs tested did not provoke any adverse effects in terms of cytotoxicity and showed no impairment of cell differentiation and monolayer integrity of human intestinal cells, it may be supposed that CPPs do not provoke a cytotoxic response in vivo in the cells assayed in the present study.

A new concept for risk assessment of the hazards of non-genotoxic chemicals--electronmicroscopic studies of the cell surface. Evidence for the action of lipophilic chemicals on the Ca2+ signaling system.

Recently, we presented evidence for the localization of components of the cellular Ca2+ signaling pathway in microvilli. On stimulation of this pathway, microvilli undergo characteristic morphological changes which can be detected by scanning electron microscopy (SEM) of the cell surface. Here we show that both receptor-mediated (vasopressin) and unspecific stimulation of the Ca2+ signaling system by the lipophilic tumor promoters thapsigargin (TG) and phorbolmyristateacetate (PMA) are accompanied by the same type of morphological changes of the cell surface. Since stimulated cell proliferation accelerates tumor development and sustained elevation of the intracellular Ca2+ concentrations is a precondition for stimulated cell proliferation, activated Ca2+ signaling is one possible mechanism of non-genomic tumor promotion. Using isolated rat hepatocytes we show that all tested lipophilic chemicals with known tumor promoter action, caused characteristic microvillar shape changes. On the other hand, lipophilic solvents that were used as differentiating agents in cell cultures such as dimethylsulfoxide (DMSO) and dimethylformamide also, failed to change the microvillar shapes. Instead DMSO stabilized the original appearance of microvilli. The used technique provides a convenient method for the evaluation of non-genomic carcinogenicity of chemicals prior to their industrial application.

In vitro assessment of the ethanol-induced hepatotoxicity on HepG2 cell line.

This study reports on measurement of the ethanol-induced hepatotoxicity in vitro on using the human hepatocyte cell line HepG2. Cells were incubated in the presence of increasing ethanol concentrations (10-80 mM). Cytotoxicity was quantitated spectrophotometrically both by the metabolism of the tetrazolium dye MTT and by the release into the medium of LDH and other marker enzymes of ethanol damage (AST, GGT and GHD). No cytotoxicity was observed up to 40 mM ethanol whereas a dose-dependent increment was found at higher concentrations (60-80 mM ethanol). At 80 mM ethanol, cell viability after 24 hours was reduced to 68% and 60% as assessed by MTT and LDH release respectively (p < 0.0001 vs. controls). Exposure for additional 24 hours did not increase cytotoxicity. Transmission electron microscopy revealed the presence of fat droplets (steatosis) and mitochondrial damage. The method reported appears to be an useful and reproducible technique for the in vitro assessment of the ethanol-induced cytotoxicity in a human liver cell line.

Assessment of renal toxicity by urinary enzymes in patients receiving chemotherapy with 8-methyl-8-acetylenic-putrescine.

Renal toxicity was assessed in 19 patients receiving methyl acetylenic putrescine (MAP), an irreversible inhibitor of ornithine decarboxylase. Patients received 250 mg t.d.s. for up to 13 weeks. This dose effectively inhibited the target enzyme, as shown by elevations in decarboxylated S-adenosyl methionine levels. No significant nephrotoxicity was observed in these patients as determined by plasma urea, creatinine and creatinine clearance measurements, although minor elevations of the urinary enzymes lactate dehydrogenase, N-acetyl-beta-glucosaminidase, alkaline phosphatase and alanine aminopeptidase were observed. As this could represent sub-clinical renal damage, caution should be exercised when using MAP in combination with other cytotoxic drugs.

[Assessment of drug nephrotoxicity by the excretion of tubule-specific membrane antigens and enzymes]. / Beurteilung der Nephrotoxizität von Pharmaka über die Ausscheidung tubulusspezifischer Membranantigene und Enzyme.

A possible tubulotoxicity of drugs can be judged with the help of the excretion of tubular membrane proteins in the urine. The brush border of the proximal tubular epithelia which react particularly sensitive to toxic influences contains surface antigens which easily release themselves from the membrane core membrane under pathological conditions and become provable in the urine by means of biochemical and immunological methods as signs of an early structural cell damage. Apart from these soluble membrane proteins which above all correspond to enzymes such as alanine aminopeptidase and gamma-glutamyl transpeptidase in severe lesions high molecular brush border fragments transformed to vesicles can appear. The clinical relevance of a pathological tissue proteinuria (histuria) of proteins of renal membranes is among others explained at the instance of the renal effects of cytostatics, antibiotics and x-ray contrast medias.