Application of genetically encoded biosensors of redox-active compounds promotes the elaboration of new methods for investigation of intracellular redox activities. Previously, we have developed a method to measure quantitatively the intracellular concentration of hydrogen peroxide (H 2 O 2 ) in living cells using genetically encoded biosensor HyPer. In the present study, we refined the method and applied it for comparing the antioxidant system potency in human cells of different phenotypes by measuring the gradient between the extracellular and cytoplasmic H 2 O 2 concentrations under conditions of H 2 O 2 -induced external oxidative stress. The measurements were performed using cancer cell lines (K-562 and HeLa), as well as normal human cells – all expressing HyPer in the cell cytoplasm. As normal cells, we used three isogenic lines of different phenotypes – mesenchymal stem/stromal cells (MSCs), induced pluripotent stem cells (iPSCs) derived from MSCs by reprogramming, and differentiated iPSC progenies with the phenotype resembling precursory MSCs. When exposing cells to exogenous H 2 O 2 , we showed that at low oxidative loads (<50 μM of H 2 O 2 ) the gradient depended on extracellular H 2 O 2 concentration. At high loads (>50 μM of H 2 O 2 ), which caused the exhaustion of thioredoxin activity in the cell cytoplasm, the gradient stabilized, pointing out that it is the functional status of the thioredoxin-depended enzymatic system that drives the dependence of the H 2 O 2 gradient on the oxidative load in human cells. At high H 2 O 2 concentrations, the cytoplasmic H 2 O 2 level in cancer cells was found to be several hundred times lower than the extracellular one. At the same time, in normal cells, extracellular-to-intracellular gradient amounted to thousands of times. Upon reprogramming, the potency of cellular antioxidant defense increased. In contrast, differentiation of iPSCs did not result in the changes in antioxidant system activity in the cell cytoplasm, assuming that intensification of the H 2 O 2 -detoxification processes is inherent to a period of early human development.

The effect of N-acetylcysteine (NAC) on morphological and physiological properties of "normal" 3T3 and 3T3-SV40 fibroblasts was studied. Incubation of the cells with 10 and 20 mM NAC for 20 h resulted in a reversible increase in the intracellular level of reduced glutathione and disorganization of actin cytoskeleton. Surprisingly, upon removal of NAC, 3T3-SV40 fibroblasts demonstrated formation of well-adhered cells with structured 3T3-like stress-fibers. Neither changes in glutathione levels, nor cytoskeleton disorganization/assembly abolished resistance of 3T3 cells to invasion by the bacterium Escherichia coli A2. On the other hand, pretreatment with NAC converted bacteria-susceptible 3T3-SV40 cells into resistant ones. These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect is due to NAC-induced modifications of cell surface proteins rather than to changes in the level of intracellular glutathione.

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