Intracellular reduction of selenite into glutathione peroxidase. Evidence for involvement of NADPH and not glutathione as the reductant

Selenium (Se) in selenite is present in an oxidized state, and must be redu ced for it to be incorporated as selenocysteine into selenoenzymes such as glutathione peroxidase (GPx). In vitro, Se, as in selenite, can be reduced utilizing glutathione (GSH) and glutathione reductase (GRed). We determined the effects of decreasing GSH levels, inhibiting GRed activity, and decrea sing cellular NADPH on the selenite-dependent rate of GPx synthesis in cult ured cells: PC3, CHO, and the E89 glucose-6-phosphate dehydrogenase (G-6-PD )-deficient cell line. A novel statistical analysis method was developed (u sing Box Cox transformed regression and a bootstrap method) in order to ass ess the effects of these manipulations singly and in combinations. Buthioni ne sulfoximine (BSO) was used to decrease GSH levels, 1,3 bis-(2 chloroethy l)-1-nitrosourea (BCNU) was used to inhibit GRed activity and methylene blu e (MB) was used to decrease cellular NADPH levels. This statistical method evaluates the effects of BSO, BCNU, MB and selenite alone and in combinatio ns on GPx activity. Decreasing the GSH level (< 5% of control) did not have an effect on the selenite-dependent rate of GPx synthesis in PC3 or CHO ce lls, but did have a small inhibitory effect on the rate of GPx synthesis in E89 cells. Inhibiting GRed activity was also associated with either no eff ect (CHO, E89) or a small effect (PC3) on GPx activity. In contrast, decrea sing NADPH levels in cells treated with MB was associated with a large decr ease in the selenite-dependent rate of GPx synthesis to 36, 34 and 25% of c ontrol in PC3, CHO, and E89 cells, respectively. The effects of BSO plus BC NU were not synergistic in any of the cell lines. The effects of BSO plus M B were synergistic in G-6-PD-deficient E89 cells, but not in PC3 or CHO cel ls. We therefore conclude that under normal culture conditions, NADPH, and not glutathione, is the primary reductant of Se in selenite to forms that a re eventually incorporated into GPx. For cells with abnormal ability to gen erate NADPH, lowering the GSH levels had a small effect on selenite-depende nt GPx synthesis. GRed activity is not required for the selenite-dependent synthesis of GPx.