Antioxidant screening in hydrogen peroxide-induced oxidative damage in human somatic and embryonic stem cells

Oxidative stress, defined as the imbalance between reactive oxygen species production and cellular antioxidant systems, is implicated in a wide range of diseases, and has been extensively studied as a potential target for therapeutic intervention by antioxidants. In order to induce long-term oxidative stress, Hs27 (human fibroblasts) and HUES3 (human embryonic stem cells) were exposed to increasing concentrations of hydrogen peroxide (H2O2) by bolus addition every 24 hours during a total of 72 hours. Cell viability, determined by alamarBlue® assay, sharply decreased at 64 µM H2O2 in Hs27 and at 128 µM H2O2 in HUES3. Following the same long-term exposure experimental design, different antioxidants were tested for cytotoxicity in Hs27 cells at increasing concentrations: glycine (GLY), sodium pyruvate (PYR), N-acetylcysteine (NAC), ascorbic acid (ASC), Trolox (TRO), sodium selenite (SEL) and zinc chloride (ZN). Three non-toxic concentrations were selected for each antioxidant to analyze their protective effect in the presence of increasing concentrations of H2O2. GLY, ASC, TRO, SEL and ZN showed no protective effect, while PYR and NAC showed dose-dependent protective effect to up to 16 mM H2O2 in the presence of 25 mM PYR and up to 256 µM H2O2 in the presence of 2.5mM NAC. Then, PYR and NAC were selected to analyze their cytotoxicity and antioxidant effect in HUES3. Cytotoxicity appeared at lower concentrations in HUES3 than in Hs27. One single concentration was selected for each antioxidant. PYR showed protective effect to up to 512 µM H2O2 at 2.5mM, although NAC showed no protective effect in HUES3. Consequently, PYR is a powerful antioxidant against H2O2-induced oxidative stress in both somatic and embryonic stem cells, while NAC is protective only for somatic cells, and other well-known antioxidants like ascorbic acid were not able to prevent cytotoxicity. This work was supported by EpiHealthNet Marie Curie ITN Project 317146-FP7-People-2012-ITN.