PURPOSE: We investigated the effect of protein- and non protein-thiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation and its relevance in the survival of CHO cells. MATERIALS AND METHODS: We used mutant cells null for glucose 6 phosphate dehydrogenase (G6PD) activity since reducing equivalents, required for reduction of oxidized thiols, are typically generated through G6PD regulated production of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (ME). The concentrations of the intracellular and extracellular non-protein thiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell survival was determined by clonogenic assay. The induction and rejoining of DSB in cells was quantitated by Pulse Field Gel Electrophoresis after exposure to ionizing radiation. RESULTS: Much lower bioreduction of HEDS was found in the G6PD deficient mutants (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) and a 26% decrease in protein thiols. Only minor changes were found under similar conditions with K1 cells. When exposed to gamma radiation in the presence of HEDS, the G6PD null mutants exhibited a higher cell killing and decreased rate and extent of rejoining of DSB than were observed in K1 cells. Moreover, when the G6PD deficient cells were transfected with the gene encoding 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 phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.
PURPOSE: We investigated the effect of protein- and non protein-thiol oxidation on DNA double-strand-break (DSB) rejoining after irradiation and its relevance in the survival of CHO cells. MATERIALS AND METHODS: We used mutant cells null for glucose 6 phosphate dehydrogenase (G6PD) activity since reducing equivalents, required for reduction of oxidized thiols, are typically generated through G6PD regulated production of NADPH. Cellular thiols were oxidized by pre-incubating the cells with hydroxyethyldisulphide (HEDS), the oxidized form of mercaptoethanol (ME). The concentrations of the intracellular and extracellular non-protein thiols (NPSH), glutathione, cysteine and mercaptoethanol were quantitated by HPLC. Protein thiols (PSH) were estimated using Ellman's reagent. Cell survival was determined by clonogenic assay. The induction and rejoining of DSB in cells was quantitated by Pulse Field Gel Electrophoresis after exposure to ionizing radiation. RESULTS: Much lower bioreduction of HEDS was found in the G6PD deficient mutants (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) and a 26% decrease in protein thiols. Only minor changes were found under similar conditions with K1 cells. When exposed to gamma radiation in the presence of HEDS, the G6PD null mutants exhibited a higher cell killing and decreased rate and extent of rejoining of DSB than were observed in K1 cells. Moreover, when the G6PD deficient cells were transfected with the gene encoding 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 phosphate pathway is required for DSB rejoining in cells exposed to a mild thiol oxidant.
Authors: Jie Li; Donglan Zhang; Kathleen M Ward; George C Prendergast; Iraimoudi S Ayene Journal: Toxicol In Vitro Date: 2012-01-31 Impact factor: 3.500
Authors: Jie Li; Kathleen M Ward; Donglan Zhang; Eswarkumar Dayanandam; Albert S Denittis; George C Prendergast; Iraimoudi S Ayene Journal: Toxicol In Vitro Date: 2012-08-16 Impact factor: 3.500
Authors: Jie Li; Donglan Zhang; Pearl A Jefferson; Kathleen M Ward; Iraimoudi S Ayene Journal: J Pharmacol Toxicol Methods Date: 2013-10-20 Impact factor: 1.950