Involvement of oxidative stress in ascorbate-induced proapoptotic death ofPC12 cells

Ascorbate is a reducing agent, but it is also known to oxidize cellular com ponents under specific conditions. The mechanism of this oxidative action, however, is not well established, Ascorbate treatment increased lipid perox ide content in PC12 cells, but did not increase quantities of lipid peroxid e when homogenates of PC12 cells were treated with ascorbate, suggesting th at cellular integrity is required for ascorbate to generate lipid peroxidat ion. However, dehydroascorbate increased lipid peroxide production in both intact PC12 cells and the cell homogenates. These differential effects of a scorbate and dehydroascorbate on intact cells versus homogenates suggest th at the dehydroascorbate in cytosol induces an oxidative stress. Ascorbate i n culture medium is rapidly oxidized to dehydroascorbate, which is transpor ted into cells by a glucose transporter (GLUT). The GLUT antagonists wortma nnin and cytochalasin B, or a high concentration of glucose, blocked C-14 u ptake (from ascorbate) in a time-dependent manner and suppressed lipid pero xide production in PC12 cells. These observations support the concept that ascorbate is oxidized to dehydroascorbate, which is transported into cells via GLUT. The dehydroascorbate induces oxidative stress. The oxidative stre ss triggered apoptosis according to ceramide production, caspase-3 activati on, and TUNEL, We have concluded that ascorbate is taken up after oxidation to dehydroascorbate via a "dehydroascorbate transporter" (GLUT), and the d ehydroascorbate generates an oxidative stress which triggers apoptosis, The se studies have significant implications for conditions under which a high concentration of ascorbate in a tissue is released during a period of hypox ia (e.g., stroke) and taken up during a reperfusion period as dehydroascorb ate. Inhibiting uptake of dehydroascorbate may offer novel therapeutic stra tegies to alleviate brain damage during a reperfusion period. (C) 2001 Acad emic Press.