Cellular glutathione as a protective agent against 4-hydroperoxycyclophosphamide cytotoxicity in K-562 cells

Exposure of cells of the K-562 erythroleukemia cell line to 4-hydroperoxycyclophosphamide (4-HC), an analog of activated cyclophosphamide, causes a concentration-dependent inhibition of in vitro colony formation by these cells. For investigation of the role of glutathione (GSH) in the metabolism of 4-HC, GSH levels of K-562 cells were modulated by exposing the cells to buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, and/or to GSH ethyl esters. Both the mono- and diethyl esters of GSH were synthesized in our laboratories and their identities were determined by chromatographic methods and fast-atom-bombardment mass spectrometry. An HPLC method including electrochemical detection used for thiol determination was applied for the measurement of GSH esters. Incubation of the cells with BSO depleted GSH levels to approximately 11% of control values and potentiated the cytotoxicity of 4-HC. By contrast, exposure to GSH esters approximately doubled GSH levels and protected the cells against the toxicity of 4-HC. Moreover, when cellular GSH levels were first depleted by BSO exposure and then replenished by incubation with GSH esters, the BSO-associated potentiation of 4-HC cytotoxicity was abolished. The work described herein extends the application of an HPLC method used for thiol determination to the measurement of GSH ethyl esters. In addition, it established that GSH acts as a competitive protecting agent against the in vitro toxicity of 4-HC in the K-562 cell line.