9 Molecular and Cellular Biochemistry 211: 9–17, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands. Intracellular reduction of selenite into glutathione peroxidase. Evidence for involvement of NADPH and not glutathione as the reductant Suvarna Bhamre, Regina L. Nuzzo, John C. Whitin, Richard A. Olshen and Harvey J. Cohen Departments of Pediatrics, Statistics, and Health and Research Policy, Stanford University, Stanford, CA, USA Received 25 August 1999; accepted 20 April 2000 Abstract Selenium (Se) in selenite is present in an oxidized state, and must be reduced 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 decreasing cellular NADPH on the selenite-dependent rate of GPx synthesis in cultured cells: PC3, CHO, and the E89 glucose-6-phosphate dehydrogenase (G-6-PD)-deficient cell line. A novel statistical analysis method was developed (using Box Cox transformed regression and a bootstrap method) in order to assess the effects of these manipulations singly and in combinations. Buthionine sulfoximine (BSO) was used to decrease GSH levels, 1,3 bis-(2 chloroethyl)-1-nitrosourea (BCNU) was used to inhibit GRed activity and methylene blue (MB) was used to decrease cellular NADPH levels. This statistical method evaluates the effects of BSO, BCNU, MB and selenite alone and in combinations 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 cells, 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 effect (CHO, E89) or a small effect (PC3) on GPx activity. In contrast, decreasing NADPH levels in cells treated with MB was associated with a large decrease in the selenite-dependent rate of GPx synthesis to 36, 34 and 25% of control in PC3, CHO, and E89 cells, respectively. The effects of BSO plus BCNU were not synergistic in any of the cell lines. The effects of BSO plus MB were synergistic in G-6- PD-deficient E89 cells, but not in PC3 or CHO cells. We therefore conclude that under normal culture conditions, NADPH, and not glutathione, is the primary reductant of Se in selenite to forms that are eventually incorporated into GPx. For cells with abnormal ability to generate NADPH, lowering the GSH levels had a small effect on selenite-dependent GPx synthesis. GRed activity is not required for the selenite-dependent synthesis of GPx. (Mol Cell Biochem 211: 9–17, 2000) Key words: selenium, antioxidants, glutathione peroxidase, selenite, bootstrap, Box-Cox transformations Introduction The element selenium has been recognized as nutritionally essential for animals and for humans. Selenium is present in biological systems in both macromolecular proteins and organic molecules of low molecular weight. Selenium in either of these types of molecules is present in the reduced form (Se –2 ). These reduced compounds include methylated selenides [15, 19] and the amino acids selenomethionine and selenocysteine. In selenoenzymes such as glutathione peroxidase (GPx), the enzymatic activity is dependent on the presence of selenocysteine in the active site [13], inserted in the primary structure of the protein during translation [31]. In eukaryotic organisms, selenium in an oxidized state such as selenite (Se +4 ) must be reduced to selenide (Se –2 ) before it can be incorporated into proteins or methylated selenium Address for correspondence: J.C. Whitin, Department of Pediatrics/Rm. S-308, Stanford University, 300 Pasteur Drive, Stanford, Ca, USA