Bence Tanczos

Cellular and nephrotoxicity of selenium species

PROJECT Beside its useful functions at very low concentrations, selenium including supplementary Se sources pose a potential toxicological risk. The toxicity of selenium species was tested in HaCaT cell culture and related nephrotoxicity in mice. PROCEDURE The apoptotic shrinkage and necrotic expansion of cells were measured by time-lapse image microscopy. Acute nephrotoxicity was estimated upon administration of various selenium species to mice for two weeks. To confirm or to refute the accumulation of Se in the kidney and its potential chronic effect, Se concentration in kidney tissue and histopathlology were tested. RESULTS The comparison of selenium species showed that organic lactomicroSe did not affect cell growth at 5ppm, but inorganic nanoSe severely hampered it at lower concentration (1ppm). The in vivo Se treatment (0.5, 5, 50ppm, corresponding to 4, 40 and 400μg/kg) was misleading as it did neither affect the outward appearance nor the weight of the kidney. Se accumulation was observed after selenate, selenite, SelPlex, selenite and nanoSe administration, while lactomicroSe caused no traceable accumulation. In vivo, ex vivo and in vitro experiments reflected this order of selenium toxicity: selenate>selenite>SelPlex=nanoSe>lactomicroSe. CONCLUSION Within the tested species lactomicroSe was the only non-nephrotoxic selenium source recommended for nutritional Se supplementation.

Antibiotics delay in vitro human stem cell regrowth.

Stem cell line from human limbal area was established to study in vitro cell growth and response to the toxic effects of antibiotics used in ophthalmology in terms of cell migration rates and structure of interphase chromatin. Recovery from cellular damages caused by ophthalmologic antibiotics was mimicked by an in vitro scratch model and followed by time-lapse microscopy, scanning electronmicroscopy and chromatin image analysis. Experiments revealed that broad spectrum antibiotics, chloramphenicol (0.5-1.0mg/ml) and rifampicin (0.1-0.2mg/ml), corresponding to concentrations in common clinical practice, slowed down the regeneration process. Results show that nuclei of naturally occurring limbal cells contain the same intermediates of chromatin condensation as seen in mammalian tumor cells and follow the common pathway of chromosome condensation. These intermediates included decondensed veil-like chromatin, fibrillary chromatin, supercoiled ribbon, chromatin bodies, early linear forms and metaphase chromosomes. Upon chloramphenicol and rifampicin treatment characteristic distorsions took place in the intermediates of chromosome condensation. Damaging effects in limbal stem cells in the presence of chloramphenicol or rifampicin indicate that ophthalmologic treatment with antibiotics should be used cautiously.

Novel functional changes during podocyte differentiation: Increase of oxidative resistance and H-ferritin expression

Podocytes are highly specialized, arborized epithelial cells covering the outer surface of the glomerular tuft in the kidney. Terminally differentiated podocytes are unable to go through cell division and hereby they are lacking a key property for regeneration after a toxic injury. Podocytes are long-lived cells but, to date, little is known about the mechanisms that support their stress resistance. Our aim was to investigate whether the well-known morphological changes during podocyte differentiation are accompanied by changes in oxidative resistance in a manner that could support their long-term survival. We used a conditionally immortalized human podocyte cell line to study the morphological and functional changes during differentiation. We followed the differentiation process for 14 days by time-lapse microscopy. During this period nondifferentiated podocytes gradually transformed into large, nonproliferating, frequently multinucleated cells, with enlarged nuclei and opened chromatin structure. We observed that differentiated podocytes were highly resistant to oxidants such as H2O2 and heme when applied separately or in combination, whereas undifferentiated cells were prone to such challenges. Elevated oxidative resistance of differentiated podocytes was associated with increased activities of antioxidant enzymes and H-ferritin expression. Immunohistochemical analysis of normal human kidney specimens revealed that podocytes highly express H-ferritin in vivo as well.

Carotid-Jugular Fistula Model to Study Systemic Effects and Fistula-Related Microcirculatory Changes

Background: Arteriovenous fistulae impair the distal circulation, but their effects at the microcirculatory level are not well understood. This study presents the carotid-jugular fistula (CJF) as a model to evaluate fistula-related microcirculatory and systemic changes. Materials and Methods: Female Wistar rats were anesthetized and divided into a fistula group (FG, n = 10) and a sham group (SG, n = 6). End-to-end anastomosis was performed between the right carotid artery and the jugular vein in the FG. The hemodynamic status was followed for 6 weeks. On the sixth postoperative week, liver and kidney microcirculation was measured using laser Doppler; then microcirculatory changes were assessed after occlusion of the carotid artery. At the end of the experiment, histological samples were taken and the weights of the organs were measured. Results: The heart rate and systolic blood pressure decreased significantly due to the CJF. Laser Doppler showed a reduction in liver blood flow units (BFU) in the FG in comparison with the SG (p = 0.01), and they increased (p < 0.01) after occlusion of the fistula. Kidney BFU showed slight changes only. The comparative morphological study revealed significant increases in heart weight (p < 0.001) and left ventricular hypertrophy (p = 0.008) in the FG. Conclusion: Beside hemodynamic and morphologic changes, a CJF causes a deterioration in the microcirculation of the liver rather than of the kidney, but occlusion of the CJF immediately reverses these changes.

Chemically Induced Cell Cycle Arrest in Perfusion Cell Culture

In contrast to most present methods, continuous imaging of live cells would require full automation in each processing step. As an integrated system that would meet all requirements does not exist, we have established a long-term scanning-perfusion platform that: (a) replaces old medium with fresh one, (b) bypasses physical contact with the cell culture during continuous cell growth, (c) provides uninterrupted photomicrography of single cells, and (d) secures near physiological conditions and sterility up to several weeks. The system was validated by synchronizing cells using serum starvation and butyrate-induced cell cycle arrest of HaCaT cells.

Intestinal ischemia-reperfusion leads to early systemic micro-rheological and multiorgan microcirculatory alterations in the rat

BACKGROUND Intestinal ischemia-reperfusion (I/R) is a potentially life-threatening situation and its pathomechanism is not fully understood yet. OBJECTIVE To investigate the early micro-rheological, microcirculatory and morphological consequences of intestinal I/R in a rat model. METHODS CD rats were anesthetized and subjected to Control (n = 7) or I/R (n = 7) groups. Left femoral artery cannulation and median laparotomy were performed. In the I/R group the superior mesenteric artery was clamped for 30 minutes. Blood samples were taken before (Base) and after the ischemia, at the 30th, 60th and 120th minutes of the reperfusion (R-30, R-60, R-120). Hematological parameters, erythrocyte deformability and aggregation were determined. On the jejunum, the liver and the right kidney laser Doppler flowmetry tests were completed. At the end of experiment histological samples were taken. RESULTS Hematocrit, leukocyte and platelet counts increased during the reperfusion. Erythrocyte deformability worsened versus Control. All erythrocyte aggregation index values of I/R group increased gradually. Intestinal microcirculatory blood flux units (BFU) did not recover completely after ischemia, at R-30 liver BFU values were lower, and kidney values decreased by R-120. Histology showed signs of I/R injury. CONCLUSIONS Micro-rheological parameters may show early and significant deterioration during the reperfusion that might contribute further to microcirculatory alterations.