Antioxidant and pro-oxidant activity of Trolox and its effect on cell volume homeostasis on HeLa cells

Recently a dual behavior (antioxidant vs pro-oxidant) of some bioactive molecules has emerged. Trolox, a synthetic analog of vitamin E, widely used as antioxidant standard in a number of bioassays, in the presence of peroxidase/H2O2 is known to induce lipid peroxidation. The aim of the present study was to investigate the antioxidant/pro-oxidant properties of Trolox in HeLa cells in relationship to cell volume homeostasis alteration and cytotoxicity. The study was carried out by spectrofluorimetric analysis of cells charged with the ROS-sensitive probe CM-H2DCFDA, morphometric analysis of the cells and MTT test. When the cells were exposed (24h) to increasing Trolox concentrations (from 2.5 to 160 µM) and then charged with CM-H2DCFDA, an antioxidant effect on the endogenous ROS production was observed at low concentrations, but a marked pro-oxidant behavior was evident at higher concentrations. The same dose-dependent dual behavior was observed also in the presence of an exogenous oxidative challenge (H2O2). At higher concentrations Trolox was able to induce a dose-dependent isotonic reduction of cell size in parallel to a reduction of cell vitality. In conclusion, our study demonstrated the dose-dependent antioxidant/pro-oxidant behavior of Trolox in HeLa cells. The pro-oxidant effect was associated to an isotonic cell shrinkage, probable early signal of an apoptotic process ascribable to Apoptotic Volume Decrease, suggesting an alteration effect on the membrane transport mechanisms involved in cell volume homeostasis