Induction of the apoptotic volume decrease (AVD) under normotonic conditions in HeLa cells exposed to Trolox

Previous observations showed that Trolox, a synthetic analog of vitamin E, widely used as antioxidant standard in a number of bioassays, can exert a pro-oxidant behavior at higher concentrations (>40 µM) on HeLa cells exposed for 24h, producing an isotonic cell shrinkage. A number of cellular events are known to be triggered by oxidative stress, including impairment of ion transport mechanisms and alteration of cell volume homeostasis. This work aims to investigate the possible mechanisms through which Trolox at high concentrations acts on cell volume homeostasis alteration in HeLa cells. The study was carried out by 1) spectrofluorimetric determination of intracellular oxidative stress in cells charged with CM-H2DCFDA, 2) morphometric analysis of cells observed under optical microscopy for cell volume determination, and 3) spectrofluorimetric and confocal analysis of cells charged with Annexin V/Propidium Iodide for apoptotic induction. HeLa cells exposed for 24h to high Trolox concentrations showed a significant dose-dependent isotonic reduction of cell volume associated to intracellular oxidative stress. The observed isotonic shrinkage was accompanied by apoptosis induction, as demonstrated by Annexin V/Propidium Iodide and was ascribed to Apoptotic Volume Decrease (AVD). The isotonic shrinkage appearance was demonstrated to occur early (after 2h) during the exposure to high Trolox concentrations. It was completely inhibited by pretreatment of the cells with a Cl- channel blocker SITS (0.5 mM). These results indicate that treatment of HeLa cells with high Trolox concentrations induces the activation of volume-regulatory Cl- channels, most likely by an increase in endogenous ROS production, which in turn is able to generate AVD.