Evaluating the Use of TiO2 Nanoparticles for Toxicity Testing in Pulmonary A549 Cells.

Purpose: Titanium dioxide nanoparticles, 25 nm in size of crystallites (TiO2 P25), are among the most produced nanomaterials worldwide. The broad use of TiO2 P25 in material science has implied a request to evaluate their biological effects, especially in the lungs. Hence, the pulmonary A549 cell line has been used to estimate the effects of TiO2 P25. However, the reports have provided dissimilar results on caused toxicity. Surprisingly, the physicochemical factors influencing TiO2 P25 action in biological models have not been evaluated in most reports. Thus, the objective of the present study is to characterize the preparation of TiO2 P25 for biological testing in A549 cells and to evaluate their biological effects. Methods: We determined the size and crystallinity of TiO2 P25. We used four techniques for TiO2 P25 dispersion. We estimated the colloid stability of TiO2 P25 in distilled water, isotonic NaCl solution, and cell culture medium. We applied the optimal dispersion conditions for testing the biological effects of TiO2 P25 (0-100 µg.mL-1) in A549 cells using biochemical assays (dehydrogenase activity, glutathione levels) and microscopy. Results: We found that the use of fetal bovine serum in culture medium is essential to maintain sufficient colloid stability of dispersed TiO2 P25. Under these conditions, TiO2 P25 were unable to induce a significant impairment of A549 cells according to the results of biochemical and microscopy evaluations. When the defined parameters for the use of TiO2 P25 in A549 cells were met, similar results on the biological effects of TiO2 P25 were obtained in two independent cell laboratories. Conclusion: We optimized the experimental conditions of TiO2 P25 preparation for toxicity testing in A549 cells. The results presented here on TiO2 P25-induced cellular effects are reproducible. Therefore, our results can be helpful for other researchers using TiO2 P25 as a reference material.

The Effect of Chronic Exposure of Graphene Nanoplates on the Viability and Motility of A549 Cells.

Graphene and its derivatives are popular nanomaterials used worldwide in many technical fields and biomedical applications. Due to such massive use, their anticipated accumulation in the environment is inevitable, with a largely unknown chronic influence on living organisms. Although repeatedly tested in chronic in vivo studies, long-term cell culture experiments that explain the biological response to these nanomaterials are still scarce. In this study, we sought to evaluate the biological responses of established model A549 tumor cells exposed to a non-toxic dose of pristine graphene for eight weeks. Our results demonstrate that the viability of the A549 cells exposed to the tested graphene did not change as well as the rate of their growth and proliferation despite nanoplatelet accumulation inside the cells. In addition, while the enzymatic activity of mitochondrial dehydrogenases moderately increased in exposed cells, their overall mitochondrial damage along with energy production changes was also not detected. Conversely, chronic accumulation of graphene nanoplates in exposed cells was detected, as evidenced by electron microscopy associated with impaired cellular motility.

Inositol hexaphosphate limits the migration and the invasiveness of colorectal carcinoma cells in vitro.

Inositol hexaphosphate (IP6), also known as phytic acid, has been shown to exhibit anticancer effects in a number of preclinical tumor models. IP6 decreases proliferation by arresting cells in the G0/G1 phase, inhibits iron-mediated oxidative reactions, enhances differentiation and stimulates apoptosis. The present study attempted to characterize the effect of IP6 on the migration and adhesion of colon cancer SW620 cells. IP6 was assessed at concentrations of 0.2 and 1 mM during 12, 24 and 48 h of exposure. Migration ability was measured with the real-time xCELLigence Real-Time Cell Analyzer Dual Purpose system. The expression of mRNA and proteins involved in migration and cancer progression [epithelial cell adhesion molecule, intercellular adhesion molecule-1, ß-catenin, N-cadherin, E-cadherin, matrix metalloproteinase (MMP)-2 and MMP-9] was determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. The changes in the expression and subcellular localization of E-cadherin were determined by indirect immunofluorescence. IP6 induced a decrease in the migration ability of the tested SW620 cell line. IP6-treated cells also showed decreased expression of N-cadherin, increased levels of E-cadherin and decreased expression of MMP-2 and MMP-9. These results indicated that IP6 has potential to modulate the migration ability and expression of markers associated with invasion in SW620 cells; however, further analysis is necessary to obtain a detailed understanding of the mechanism of action.

Antiproliferative effects of selenium compounds in colon cancer cells: comparison of different cytotoxicity assays.

A number of cytotoxicity assays are currently available, each of them using specific approach to detect different aspects of cell viability, such as cell integrity, proliferation and metabolic functions. In this study we compared the potential of five commonly employed cytotoxicity assays (WST-1, XTT, MTT, Brilliant blue and Neutral red assay) to detect antiproliferative effects of three selenium compounds, sodium selenite, seleno-L-methionine (SeMet) and Se-(Methyl)selenocysteine (SeMCys) on three colorectal cancer cell lines in vitro. Cells were exposed to the selected selenium compounds in the concentration range of 0-256 microM during 48 h. WST-1 and XTT failed to detect cytotoxic effect, with the exception of the highest concentration of selenium compounds tested. Conversely, the metabolic activity of selenium treated cells measured by WST-1 and XTT significantly increased in comparison to untreated controls. MTT, Neutral red and Brilliant blue assays were more sensitive and yielded mutually comparable results, with significant decrease of measured parameters in a concentration-dependent manner. To a smaller extent, the results were affected by the different chemical nature of the selenium compounds tested as well as by the biological properties of individual cell lines.

New iron chelators in anthracycline-induced cardiotoxicity.

The use of anthracycline anticancer drugs is limited by a cumulative, dose-dependent cardiac toxicity. Iron chelation has long been considered as a promising strategy to limit this unfavorable side effect, either by restoring the disturbed cellular iron homeostasis or by removing redox-active iron, which may promote anthracycline-induced oxidative stress. Aroylhydrazone lipophilic iron chelators have shown promising results in the rabbit model of daunorubicin-induced cardiomyopathy as well as in cellular models. The lack of interference with the antiproliferative effects of the anthracyclines also favors their use in clinical settings. The dose, however, should be carefully titrated to prevent iron depletion, which apparently also applies for other strong iron chelators. We have shown that a mere ability of a compound to chelate iron is not the sole determinant of a good cardioprotector and the protective potential does not directly correlate with the ability of the chelators to prevent hydroxyl radical formation. These findings, however, do not weaken the role of iron in doxorubicin cardiotoxicity as such, they rather appeal for further investigations into the molecular mechanisms how anthracyclines interact with iron and how iron chelation may interfere with these processes.

Hexavalent chromium disrupts the actin cytoskeleton and induces mitochondria-dependent apoptosis in human dermal fibroblasts.

Exposure to hexavalent chromium causes various adverse effects including deep skin ulcerations and allergic dermatitis. Because of many potential intracellular targets for hexavalent chromium toxicity, its mechanisms of action are not entirely understood. To investigate the role of the cytoskeleton and mitochondria in this process, primary human dermal fibroblasts were exposed to various concentrations of potassium chromate for 24 h. The followed markers included cell motility, cytoskeletal organization, oxidative stress, mitochondrial activity and activation of the apoptotic cascade. Potassium chromate (1.5-45 microM) induced time- and concentration-dependent cell shrinkage, reorganization of cytoskeleton and loss of motile activity in fibroblasts. In some cells this was followed by membrane blebbing. Dynamic changes in cell morphology were accompanied with the loss of mitochondrial membrane potential, increased oxidative stress and release of cytochrome c. Apoptosis was confirmed by detection of activated caspase-3 and nuclear fragmentation. The results indicate that in fibroblasts hexavalent chromium-induced damage to cytoskeleton and mitochondria might occur concurrently at relatively early stages of exposure. Furthermore, alterations of these targets seem to activate mitochondria-dependent and- independent apoptosis.

The stereospecificity of flobufen metabolism in isolated guinea pig hepatocytes.

BACKGROUND: Flobufen (F) is an original nonsteroidal anti-inflammatory drug with one center of chirality. 4-Dihydroflobufen (DHF), compound with two chiral centers, is the main metabolite of F in microsomes and cytosol in all standard laboratory animals. This work describes the biotransformation of F enantiomers and DHF stereoisomers in isolated male guinea pig hepatocytes. Guinea pigs were chosen with respect to similarities in F metabolism as in Man found earlier. R-F, S-F, (2R;4S)-DHF, (2S;4R)-DHF, (2S;4S)-DHF and (2R;4R)-DHF, structurally very similar compounds, served as substrates in order to observe their interaction with enzymes. Stereospecificity of the respective enzymes was studied in vitro, using hepatocytes monolayer. Chiral HPLC using R,R-ULMO column as chiral stationary phase was used for detection and quantitation of metabolites. RESULTS: (2R;4S)-DHF and (2S;4S)-DHF were the principle stereoisomers detected after incubation with rac-F, R-F and S-F. The ratio of (2R;4S)-DHF/(2S;4S)-DHF ranged from 1.1 to 2.4 depending on the substrate used. (2R;4S)-DHF was the major stereoisomer found after incubation with (2S;4S)-DHF and (2R;4R)-DHF. (2S;4S)-DHF was the principle stereoisomer found after incubation with (2R;4S)-DHF and (2S;4R)-DHF. Besides DHF stereoisomers, other metabolites (M-17203, UM-1 and UM-2) were also detected after incubation of hepatocytes monolayer with F. Interestingly, these metabolites were not found in incubation of all F forms and DHF with fresh liver homogenate. CONCLUSIONS: Different activities and stereospecificities of the respective enzymes were observed for each substrate in primary culture of hepatocytes. Cell integrity is crucial for formation of secondary metabolites M-17203, UM-1 and UM-2.