DNA Damages Induced by Selenite in the Presence of Glutathione

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  • Saito Yoshihiro
    Department of Bio-analytical Chemistry, Osaka University of Pharmaceutical Sciences
  • Baba Yoshiko
    Department of Bio-analytical Chemistry, Osaka University of Pharmaceutical Sciences
  • Matsunaga Ai
    Department of Bio-analytical Chemistry, Osaka University of Pharmaceutical Sciences
  • Sato Takaji
    Department of Bio-analytical Chemistry, Osaka University of Pharmaceutical Sciences
  • Chikuma Masahiko
    Department of Bio-analytical Chemistry, Osaka University of Pharmaceutical Sciences

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DNA damages induced by sodium selenite in the presence of reduced glutathione(GSH)were analyzed by agarose gel electrophoresis. Plasmid DNA was incubated with selenite and glutathione in Tris-borate-EDTA(TBE)buffer(pH8.2)at 37°C. The DNA remained supercoiled form when it was incubated alone, with selenite, or with GSH for 2 hours. DNA with open circular form was observed when it was incubated in the presence of both selenite and GSH, and the electrophoretic band strength corresponding to open circular form was highest when GSH was present 100 times excess moles of selenite. The open circular DNA increased with time of incubation at least up to 4 hours under the same conditions, though the increase was not marked in 2-4 hours. The open circular DNA was not observed just after the start of incubation. The results indicated that a single strand break of DNA chain occurs when plasmid DNA is incubated in the presence of both selenite and GSH for more than 1 hour forming an open circular DNA, the amount of which was dependent on the molar ratio of selenite and GSH. It was suggested that excess amount of GSH accelerated the breakdown of initially formed selenodiglutathione(GSSeSG)and the production of superoxide anion or other reactive species, and DNA may have been damaged by these species. The reaction between sodium selenite and GSH in TBE buffer was analyzed by HPLC. The peaks corresponding to GSH, oxidized glutathione(GSSG),and GSSeSG were observed in chromatograms. The peak area of GSH decreased and that of GSSG increased with the time of reaction. However, the time profile of peak area of GSSeSG depended on the molar ratio of selenite and GSH in the reaction mixture; the peak area increased with time when selenite:GSH=1:100 and it decreased when selenite:GSH=1:1, 1:4, and 1:10. Thus, the extent of single strand break of DNA was highest under the conditions with highest GSSeSG production. These results suggested that GSSeSG may possibly be a compound that catalyzes single strand breaks of DNA.

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