Repair of chromatin damage in glutathione-depleted V-79 cells: comparison of oxic and hypoxic conditions.
We have assessed the effects of two radiomodifying conditions, glutathione (GSH) depletion and hypoxia, on the formation and repair of radiation-induced chromatin damage, specifically DNA-protein cross-links (DPC). As measured by a nitrocellulose filter-binding assay, untreated V79 cells contain a low level of DPC (1-1.5% of the cellular DNA). The background level of DPC is elevated in cells treated with L-buthionine sulfoximine (BSO), in hypoxic cells, and in cells treated with BSO and made hypoxic (2.98%, 2.82%, and 7.71%, respectively). The dose response for production of radiation-induced DPC is approximately 6.0% DNA bound per 100 Gy for cells irradiated in air, and the dose response is not significantly different for BSO-treated cells but increases by a factor of about 1.4 for hypoxic cells and 1.7 for BSO-pretreated hypoxic cells. DPC were also assayed by alkaline elution with or without proteinase K treatment. By this analysis, the yield of DPC appears to be elevated in irradiated hypoxic and irradiated GSH-depleted cells. It is not possible to assay for background DPC alone in unirradiated cells by alkaline elution. Cells not exposed to BSO repair 70-80% of the radiation-induced DPC in 4 h. BSO-treated cells are considerably less efficient in repair of DPC. As analyzed by alkaline elution, GSH depletion had little or no effect on the yield of radiation-induced single-strand breaks (SSB) but slowed their repair. The data suggest that depletion of GSH impairs an enzyme system(s) responsible for the turnover of both background and radiation-induced DPC and that hypoxia elevates both the background level of DPC and the ratio of radiation-induced DPC to SSB.
Xue
L Y
LY
Department of Radiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Friedman
L R
LR
Oleinick
N L
NL
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000528:radiation effects*; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004260:DNA Repair; D005978:Glutathione / Q000502:physiology*; D066298:In Vitro Techniques; D010100:Oxygen / Q000502:physiology*
D002843:Chromatin; D005978:Glutathione; D010100:Oxygen