The roles of glutathione and antioxidant enzymes in menadione-induced oxidative stress

We investigated the role of glutathione (GSH) and antioxidant enzymes in me nadione-resistance by using K300 cells (menadione-resistant cells) and pal ental P19 cells (menadione-sensitive cells). We found that acquisition of r esistance was associated with elevations in glutathione content and DT-diap horase activity. The activity of glutathione S-transferase (GST) was signif icantly decreased, while the activities of glutathione peroxidase, glutathi one reductase, catalase, and superoxide dismutase in K300 cells were mainta ined at the same levels as compared to the parental P19 cells. Using reacti ve oxygen species (ROS)-sensitive fluorescence dye 2,7- dichlorodihydrofluo rescein diacetate (DCFH/DA), we demonstrated that K300 cells are characteri zed by reduced cellular ROS as compared to the parental P19 cells during me nadione's action. Menadione depleted glutathione to a small extent in the K 300 cells, but a rapid depletion was observed in P19 cells. Pretreatment of K300 cells with dicumarol. a DT-diaphorase inhibitor, or buthionine sulfox imine (BSO), an inhibitor of gamma -glutamyl cysteine synthase, sensitized the cells to menadione. BSO treatment was less effective than dicumarol tre atment in reversing menadione resistance in K300 cells. These results stron gly support the belief that DT-diaphorase plays a central role in protectin g cells against menadione-induced oxidative stress by decreasing the ROS fo rmation. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.