Ascorbic acid maintenance in HaCaT cells prevents radical formation and apoptosis by UV-B

We have investigated the enzymatic reduction and accumulation of vitamin C in HaCaT epithelial cells. The subcellular localization and the activities of ascorbyl free radical reductase and dehydroascorbate reductase showed th at mitochondrial, microsomal and plasma membranes fractions express high le vels of ascorbyl free radical reductase activity, whereas dehydroascorbate reductase activity was found at low levels only in the post microsomal supe rnatant. We have also investigated cell proliferation and vitamin C accumul ation induced by ascorbic acid 2-phosphate. This derivative caused no inhib ition of cell growth, was uptaken from the extracellular medium and accumul ated as ascorbic acid in mM concentrations. These results show that HaCaT c ells possess very efficient systems to maintain high levels of both intrace llular and extracellular ascorbic acid. The regeneration and uptake of asco rbic acid from extracellular medium contributes to the intracellular antiox idant capacity, as evaluated by 2',7'-dihydrodichlorofluorescein staining. Consequently, cells became more resistant to free radical generation and ce ll death induced by UV-B irradiation. (C) 1999 Elsevier Science Inc.