Abstract

Most of the biological impacts of Vitamin E, including the redox effects, have been raised from studies with alpha-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 alpha-, gamma- and delta-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 alpha-, gamma- and delta-tocopherol at concentrations as low as 2.5 mu M. Each isomer induced rapid and consistent increases in cytosolic calcium with fast kinetics, which were affected by chelation of extracellular Ca2+, suggesting that tocopherols promoted a calcium entry upon the contact with the plasma membrane. In terms of redox effects, delta-tocopherol was the only isomer that evoked a significant change in the HyPer signal at 5 mu M. By mimicking Ca2+ 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 Ca2+ chelator, none of the isomers were able to induce redox changes. Altogether, our data indicate that an elevation in cytoplasmic Ca2+ is necessary for the development of a tocopherol-induced antioxidant impact on the cytoplasm of Caco-2 cells reported by HyPer biosensor.