Studies on the role of antioxidants in radioprotection

Abstract

Much recent research has concentrated on the role of glutathione (GSH) in modifying radiosensitivity. In vi tro, a quite modest depletion of GSH with buthionine sulphoximine (BSO) has a large effect on misonidazole radiosensitization (Hodgkiss and Middleton, 1983). In vivo, however, comparable levels of depletion result in very little sensitization (e.g. Ono et a l . , 1986; Rojas et al . , 1984). We have previously shown that the naturally occuring antioxidant, ascorbate, confers some radioprotection in v i tro (Stratford and Hodgkiss, 1986) and have therefore further investigated the role of ascorbate in determining radiosensitivity in vitro and correlated this with uptake. We have also studied the effect of GSH depletion in vivo on the concentrations of ascorbate and of the membrane antioxidant ¢x-tocopherol. Attached cells (0 .2 -3 x 104 V79 379A Chinese hamster fibroblasts) were irradiated in Eagle's minimum essential medium and 10% fetal calf serum (MEM) under N2 on 5 cm glass dishes, in the presence or absence of ascorbate and the radiosensitizer misonidazole, in both control and GSH-depleted cells. Table 1 shows the dose modification factors (DMF) achieved under various conditions relative to the N2 line. This shows that under most conditions ascorbate provides some limited protection as assessed by a decrease in the DMF, but that this is not markedly concentration-dependent. Under conditions of GSH depletion, however, protection by ascorbate is more marked, particularly at an intermediate GSH level (37 % of control GSH) (45 % of control nonprotein thiol (NPSH)). Control cells contain 5 6 fmol/cell GSH. Preliminary studies on the HT1080 human fibrosarcoma cell line (Flow Laboratories, Rickmansworth, Herts, U.K.) which contains ~ 20 fmol/cell GSH, also demonstrate radioprotection (data not shown). The concentration dependence of the uptake of ascorbate has been compared in V79 379A and HT1080 cells. For these studies, attached cells (0 .75-2 × 106) were incubated for 1 hr in the presence of various concentrations of ascorbate at 20°C. The medium was then aspirated; the cells were washed six times with ice-cold phosphate-buffered saline; the cells were then extracted with 50 mmol/l perchloric acid, 100 t~mol/l EDTA. Intracellular ascorbate was determined by high-performance liquid chromatography (HPLC) after oxidation with iodine and reaction with o-phenylene diamine to give a fluorescent quinoxaline derivative (Deutsch and