Oxidation of cellular thiols by hydroxyethyldisulphide inhibits DNA double-strand-break rejoining in G6PD deficient mammalian cells

Purpose: We investigated the effect of protein- and non protein-thiol oxida tion on DNA double-strand-break (DSB) rejoining after irradiation and its r elevance in the survival of CHO cells. Materials and methods: We used mutant cells null for glucose 6 phosphate de hydrogenase (G6PD) activity since reducing equivalents, required for reduct ion of oxidized thiols, are typically generated through G6PD regulated prod uction of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (M E). The concentrations of the intracellular and extracellular non-protein t hiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell sur vival was determined by clonogenic assay. The induction and rejoining of DS B in cells was quantitated by Pulse Field Gel Electrophoresis after exposur e to ionizing radiation. Results: Much lower bioreduction of HEDS was found in the G6PD deficient mu tants (E89) than in the wild-type cells (K1). A 1 h treatment of E89 cells with HEDS produced almost complete depletion of non-protein thiol (NPSH) an d a 26% decrease in protein thiols. Only minor changes were found under sim ilar conditions with K1 cells. When exposed to gamma radiation in the prese nce of HEDS, the G6PD null mutants exhibited a higher cell killing and decr eased rate and extent of rejoining of DSB than were observed in K1 cells. M oreover, when the G6PD deficient cells were transfected with the gene encod ing wild-type G6PD (A1A), they recovered close to wild-type cellular thiol status, cell survival and DSB rejoining. Conclusions: These results suggest that a functioning oxidative pentose pho sphate pathway is required for DSB rejoining in cells exposed to a mild thi ol oxidant.