Relationship between ascorbyl radical intensity and apoptosis-inducing activity.

Ascorbic acid and its related compounds were compared for their ascorbyl radical intensity and apoptosis-inducing activity. Sodium L-ascorbate, L-ascorbic acid, D-isoascorbic acid, sodium 6-beta-O-galactosyl-L-ascorbate and sodium 5,6-benzylidene-L-ascorbate, at the concentration of 1-10 mM, induced apoptotic cell death characterized by cell shrinkage, nuclear fragmentation and internucleosomal DNA cleavage in human promyelocytic leukemic HL-60 cells. On the other hand, L-ascorbic acid-2-phosphate magnesium salt and L-ascorbic acid 2-sulfate did not induce any of these apoptosis-associated characteristics. ESR measurements revealed that all the active compounds were progressively degraded, producing the ascorbyl radical (g = 2.0064, hfc = 0.17 mT) in culture medium, whereas the inactive compounds were stable and did not produce the ascorbyl radical. Cytotoxicity began to appear when the radical intensity exceeded a certain threshold level. In the presence of N-acetyl-L-cysteine, both ascorbyl radical intensity and apoptosis-inducing activity were significantly reduced. These data suggest the possible involvement of the ascorbyl radical in apoptosis induction by ascorbic acid-related compounds. Exposure of HL-60 cells to ascorbic acid or its active derivatives resulted in the rapid elevation of intracellular Ca2+ concentration, which might serve as the initial signal leading to the cell death pathway.