Biological Redox Impact of Tocopherol Isomers Is Mediated by Fast Cytosolic Calcium Increases in Living Caco-2 Cells

Most of the biological impacts of Vitamin E, including the redox effects, have been raised from studies with α-tocopherol only, despite the fact that tocopherol-containing foods carry mixed tocopherol isomers. Here, we investigated the cellular mechanisms involved in the immediate antioxidant responses evoked by α-, γ- and δ-tocopherol in Caco-2 cells. In order to track the cytosolic redox impact, we performed imaging on cells expressing HyPer, a fluorescent redox biosensor, while cytosolic calcium fluctuations were monitored by means of Fura-2 dye and imaging. With this approach, we could observe fast cellular responses evoked by the addition of α-, γ- and δ-tocopherol at concentrations as low as 2.5 μM. Each isomer induced rapid and consistent increases in cytosolic calcium with fast kinetics, which were affected by chelation of extracellular Ca, suggesting that tocopherols promoted a calcium entry upon the contact with the plasma membrane. In terms of redox effects, δ-tocopherol was the only isomer that evoked a significant change in the HyPer signal at 5 μM. By mimicking Ca entry with ionomycin and monensin, a decline in the HyPer signal was induced as well. Finally, by silencing calcium with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), an intracellular Ca chelator, none of the isomers were able to induce redox changes. Altogether, our data indicate that an elevation in cytoplasmic Ca is necessary for the development of a tocopherol-induced antioxidant impact on the cytoplasm of Caco-2 cells reported by HyPer biosensor.