Intracellular Dual Behavior of Trolox in HeLa Cells and 3T3 Fibroblasts Under Basal and H2O2-Induced Oxidative Stress Conditions

Trolox, a water-soluble analog of vitamin E, is widely used as a reference antioxidant in in vitro biochemical assays. However, its intracellular redox behavior is known to vary depending on both concentration and oxidative context. In this study, we investigated the dose-dependent antioxidant and prooxidant effects of Trolox in two cellular models, HeLa cells and 3T3 cells exposed for 1 h to increasing concentrations (2–160 µM), under both basal conditions and oxidative stress induced by hydrogen peroxide. Intracellular oxidative changes were assessed using the oxidative stress-sensitive fluorescent probe CM-H2DCFDA. Under basal conditions, Trolox exerted slight dose-dependent antioxidant behavior in 3T3 cells on the basal production of ROS in concentrations ranging from 2 µM to 160 µM. In contrast, in HeLa cells Trolox displayed a biphasic activity: antioxidant at low doses (≤10 µM) and a switch to prooxidant behavior at higher concentrations. Under H2O2-induced stress, in HeLa cells Trolox retained antioxidant activity at low concentrations (≤10 µM), but this effect gradually declined at higher doses, disappearing around 80 µM and shifting to a slight prooxidant effect at 160 µM. Confocal microscopy confirmed the spectrofluorimetric results. Conversely, 3T3 cells exhibited an early shift toward prooxidant activity already at 10 µM. These findings highlight that the Trolox redox activity is determined not only by concentration but also by cell-specific intracellular environment and redox state. The study suggests caution against generalized antioxidant use of Trolox and highlights the need for specific dose–response evaluations in specific cell types and biological settings.