Resistance to H2O2-induced oxidative stress in human cells of different phenotypes

Abstract

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.

Radical Scavenging Mechanisms of 1-Arylhydrazone Benzimidazole Hybrids with Neuroprotective Activity

“…It is established that the direct application of H 2 O 2 in cellular models is followed by the generation of oxygen (O 2 ) in the cells due to the cellular catalase activity and, as a consequence, free radicals are generated not only from the applied hydrogen peroxide but also from the released oxygen as well. − While the exogenous H 2 O 2 has limited penetration within the cells and in normal cells, the extracellular-to-intracellular gradient can amount to thousands of times, , and the oxygen released in the cells due to the catalase action would be mainly responsible for the accumulation of free radicals and reactive oxygen species and the induced oxidative stress. In order to explain the requirement of a high concentration of H 2 O 2 to be applied for damages to be detected in the experimental models, an interesting recent study demonstrated that cells exposed to H 2 O 2 were able to convert it into O 2 in time as short as few minutes, meaning that the exposure to H 2 O 2 itself is in fact much shorter and less intense than initially considered.…”

Oxidation-Induced Protein Cross-Linking in Mammalian Cells

“…Because hTrx-73VtK was the variant with the strongest multimerization bands, we next explored the effect of cellular reactive oxygen species (ROS) on its monomer–dimer–trimer equilibrium. Both rotenone and H 2 O 2 are well documented to increase cellular ROS production. , They would thus be expected to increase VtK-O levels and promote hTrx multimerization. However, the addition of up to 0.2 mM rotenone and 1 mM H 2 O 2 actually reduced the production of all forms of hTrx-73VtK (Figure b), possibly due to their interference with cell viability (Supplementary Figure 9).…”

“…Both rotenone and H 2 O 2 are well documented to increase cellular ROS production. 36,37 They would thus be expected to increase VtK-O levels and promote hTrx multimerization. However, the addition of up to 0.2 mM rotenone and 1 mM H 2 O 2 actually reduced the production of all forms of hTrx-73VtK (Figure 5b), possibly due to their interference with cell viability (Supplementary Figure 9).…”

Evaluation of Proanthocyanidins from Kiwi Leaves (Actinidia chinensis) against Caco-2 Cells Oxidative Stress through Nrf2-ARE Signaling Pathway

“…As hydrogen peroxide (H 2 O 2 ) was a product of the cellular oxygen metabolism that was a feature of the various metabolic and signaling cascades [ 12 , 32 ], the control of the physiological H 2 O 2 intracellular concentration, as an antioxidant indicator, has a significant relationship to the cells’ functionality and viability [ 33 ]. Therefore, the potential antioxidant activity of PKLPs on the cellular H 2 O 2 -induced oxidation model was established.…”