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


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.
Ayene
I S
IS
Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA. ayenei@mail.med.upenn.edu
Koch
C J
CJ
Tuttle
S W
SW
Stamato
T D
TD
Perez
M L
ML
Biaglow
J E
JE
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects*; D004220:Disulfides / Q000494:pharmacology*; D000431:Ethanol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000235:genetics / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D009154:Mutation; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D004220:Disulfides; D013438:Sulfhydryl Compounds; D000431:Ethanol; C031319:2-hydroxyethyl disulfide; D004247:DNA; D005978:Glutathione