High doses of synthetic antioxidants induce premature senescence in cultivated mesenchymal stem cells.

Stress-induced premature senescence program is known to be activated in cells by various genotoxic stressors, and oxidative stress is considered to be the main of those. To this end, many studies discover antioxidants as protective anti-aging agents. In the current study, we examined the effects of different antioxidants (Tempol, resveratrol, NAC, DPI) on the mesenchymal stem cells maintained in normal physiological conditions. We used high, but non-cytotoxic antioxidant doses which are widely used in laboratory practice to protect cells from oxidative damage. We show that these substances induce reversible block of cell proliferation and do not cause any genotoxic effects when applied to the quiescent cells. However, the same doses of the same substances, when applied to the proliferating cells, can induce irreversible cell cycle arrest, DNA strand breaks accumulation and DNA damage response activation. As a consequence, antioxidant-induced DNA damage results in the stress-induced premature senescence program activation. We conclude that high doses of antioxidants, when applied to the proliferating cells that maintain physiological levels of reactive oxygen species, can cause DNA damage and induce premature senescence which suggests to re-estimate believed unconditional anti-aging antioxidant properties.

Flow cytometric HyPer-based assay for hydrogen peroxide.

HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H2O2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H2O2, by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H2O2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H2O2-reactive dye H2DCFDA and, contrary to the H2DCFDA-based assay, can be employed for the kinetic studies of H2O2 utilization by cells, including measurements of the rate constants of H2O2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H2O2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H2O2.

Redox environment in stem and differentiated cells: A quantitative approach.

Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa) maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H2DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed.

Reactive Oxygen Species Are Required for Human Mesenchymal Stem Cells to Initiate Proliferation after the Quiescence Exit.

The present study focuses on the involvement of reactive oxygen species (ROS) in the process of mesenchymal stem cells "waking up" and entering the cell cycle after the quiescence. Using human endometrial mesenchymal stem cells (eMSCs), we showed that intracellular basal ROS level is positively correlated with the proliferative status of the cell cultures. Our experiments with the eMSCs synchronized in the G0 phase of the cell cycle revealed a transient increase in the ROS level upon the quiescence exit after stimulation of the cell proliferation. This increase was registered before the eMSC entry to the S-phase of the cell cycle, and elimination of this increase by antioxidants (N-acetyl-L-cysteine, Tempol, and Resveratrol) blocked G1-S-phase transition. Similarly, a cell cycle arrest which resulted from the antioxidant treatment was observed in the experiments with synchronized human mesenchymal stem cells derived from the adipose tissue. Thus, we showed that physiologically relevant level of ROS is required for the initiation of human mesenchymal stem cell proliferation and that low levels of ROS due to the antioxidant treatment can block the stem cell self-renewal.

Hematological changes in case of chronic cadmium intoxication and monensin detoxication. Relationship with rheological variables.

The study evaluated the affect of chronic cadmium (Cd) and monensin treatment on some hematological parameters and its relationship with the rheological variables. Adult male mice were subjected to chronic treatment with cadmium acetate [Cd(CH3COO)2 × 2H2O] (group 1), Cd(CH3COO)2 × 2H2O followed by treatment with low dose monensin (group 2) and Cd(CH3COO)2 × 2H2O followed by high dose monensin treatment (group 3). Cd(CH3COO)2 × 2H2O and deprotonated monensin were dissolved in distilled water and given daily to the experimental animals. Mice drinking distilled water served as a control group (group 4). Hematological parameters and erythrocyte morphology were evaluated in parallel with whole blood viscosity (WBV). Cd treatment reduced Hb and increased RDW. The addition of high dose monensin significantly improved erythrocytic indices compared to the control. Erythrocyte anisocytosis was observed in blood smears of Cd-treated mice corresponding to the increased RDW. WBV was significantly elevated in the experimental groups in the whole range of shear rates compared to the control group and in groups 2 and 3 was lower than in group 1 but remained higher compared to group 4. Correlations were found between WBV and RBC, Hb, Hct, MCV and RDW. The results suggest that hemorheological parameters such as WBV should be monitored in parallel with the hematological parameters when monensin is applied and heavy metal intoxication is suspected.

Determination of rare earth elements in soils and sediments by inductively coupled plasma atomic emission spectrometry after cation-exchange separation.

The influence of matrix elements such as Ba, Ca, Fe, K, Na and Ti, on the inductively coupled plasma atomic emission spectrometry determination of the rare earth elements in soils and sediments is investigated and analytical lines with minimal interferences are chosen. The analysis of certified reference materials by two calibration methods (pure rare earth solutions and IAEA-Soil 5) and after cation-exchange proved that calibrations with IAEA-Soil 5 increase the number of accurately determined rare earth elements (REE), permitting the instrumental determination of Ce, Eu, La, Nd, Tb, Yb and Y in soils and some sediments. The cation-exchange procedure permits the determination of 12 REE with very good accuracy (below 10%) and detection limits varying between 0.05 (Eu, Tb, Yb) and 0.5 (Er) mg kg(-1).