The role of non-protein sulphydryls in determining the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells.
Chinese hamster V79 fibroblasts were irradiated in the gas explosion apparatus and the chemical repair rates of the oxygen-dependent free radical precursors of DNA double-strand breaks (dsb) and lethal lesions measured using filter elution (pH 9.6) and a clonogenic assay. Depletion of cellular GSH levels, from 4.16 fmol/cell to 0.05 fmol/cell, by treatment with buthionine sulphoximine (50 mumol dm-3; 18 h), led to sensitization as regards DNA dsb induction and cell killing. This was evident at all time settings but was particularly pronounced when the oxygen shot was given 1 ms after the irradiation pulse. A detailed analysis of the chemical repair kinetics showed that depletion of GSH led to a reduction in the first-order rate constant for dsb precursors from 385 s-1 to 144 s-1, and for lethal lesion precursors from 533 s-1 to 165 s-1. This is generally consistent with the role of GSH in the repair-fixation model of radiation damage at the critical DNA lesions. However, the reduction in chemical repair rate was not proportional to the severe thiol depletion (down to approximately 1% for GSH) and a residual repair capacity remained (approximately 30%). This was found not to be due to compartmentalization of residual GSH in the nucleus, as the repair rate for dsb precursors in isolated nuclei, washed virtually free of GSH, was identical to that found in GSH-depleted cells (144 s-1), also the OER remained substantially above unity. This suggests that other reducing agents may have a role to play in the chemical repair of oxygen-dependent damage. One possible candidate is the significant level of protein sulphydryls present in isolated nuclei.
Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex, UK.
K M
KM
Prise
S
S
Davies
M R
MR
Stratford
B D
BD
Michael
D000818:Animals; D002470:Cell Survival / Q000528:radiation effects; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000502:physiology; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D005347:Fibroblasts / Q000528:radiation effects; D005609:Free Radicals; D005978:Glutathione / Q000502:physiology*; D013438:Sulfhydryl Compounds / Q000502:physiology
D005609:Free Radicals; D013438:Sulfhydryl Compounds; D004247:DNA; D005978:Glutathione; D003545:Cysteine