Effects of glutathione depletion by buthionine sulfoximine on radiosensitization by oxygen and misonidazole in vitro

Abstract

Buthionine sulfoximine (BSO) has been used to deplete glutathione (GSH) in V79-379A cells in vitro, and the effect on the efficiency of oxygen and misonidazole (MISO) as radiosensitizers has been determined. Treatment with 50 or 500 microM BSO caused a rapid decline in GSH content to less than 5% of control values after 10 hr of exposure (t1/2 = 1.6 hr). Removal of BSO resulted in a rapid regeneration of GSH after 50 microM BSO, but little regeneration was observed over the subsequent 10-hr period after 500 microM. Treatment with either of these two concentrations of BSO for up to 14 hr did not affect cell growth or viability. Cells irradiated in monolayer on glass had an oxygen enhancement ratio (OER) of 3.1. After 10-14 hr pretreatment with 50 microM BSO, washed cells were radiosensitized by GSH depletion at all oxygen tensions tested. The OER was reduced to 2.6, due to greater radiosensitization of hypoxic cells than aerated ones by GSH depletion. GSH depletion had the effect of shifting the enhancement ratio vs pO2 curve to lower oxygen tensions, making oxygen appear more efficient by a factor of approximately 2, based on the pO2 required to give an OER of 2.0. In similar experiments performed with MISO, an enhancement ratio of 2.0 could be achieved with 0.2 mM MISO in anoxic BSO-pretreated cells, compared to 2.7 mM MISO in non-BSO-treated cells. Thus MISO appeared to be more efficient in GSH-depleted cells by a factor of 13.5. These apparent increases in radiosensitizer efficiency in GSH-depleted cells could be explained on the basis of radiosensitization of hypoxic cells by GSH depletion alone (ER = 1.29-1.41). The effect of GSH depletion was approximately equal at all sensitizer concentrations tested, except at high oxygen tensions, where the effect was insignificantly small. These results are consistent with hypoxic cell radiosensitization by GSH depletion and by MISO or oxygen acting by separate mechanisms.