László Révész

The effect of oxygen on cellular survival and recovery after radiation

Abstract Chinese hamster cells were exposed to X radiation in argon with an oxygen content either less than 2 ppm, considered anoxic conditions, or 120 or 400 ppm, considered hypoxic conditions. The survival curve after irradiation with exposures of 800, 1,200 and 1,600 R in anoxia was exponential. In comparison, the survival curve of cells irradiated in hypoxia had an increased regression and an increased extrapolation number at zero dose. When treated with exposures of 150, 300 and 450 R in 120 ppm oxygen, the number of survivors was about 10 per cent larger than when the exposures were delivered in anoxia. A split-dose ratio close to unity was calculated when the survival after a single exposure of 800 or 1,600 R in anoxia was related to the survival after the same doses delivered in two equal fractions with 18-hour intervals. When treatment with the same doses was made in the presence of oxygen, the survival ratio was increased to an extent comparable to the increase of the corresponding extrapolation...

CYSTEAMINE-INDUCED INCREASE OF CELLULAR GLUTATHIONE-LEVEL: A NEW HYPOTHESIS OF THE RADIOPROTECTIVE MECHANISM

IT was reported recently1 that incubation of ELD ascites cells or its sublines in a tissue culture medium supplemented with cysteamine causes an increase of the cellular content of the acid-soluble, non-protein-bound sulphydryl groups (NPSH). The increase was found to be directly related to the cysteamine concentration in the medium. Expressed in SIF*, the extent of NPSH increase was similar to the extent of the increased radio-resistance expressed in DRF† of human kidney cells or mouse fibroblasts treated with cysteamine at comparable concentrations2. This similarity of SIF and DRF has been interpreted1 as indicating that the mechanism of radioprotection by cysteamine is, in some way, dependent on the increased concentration of NPSH in the protected cell. The nature of this NPSH is not known at present.

Lack of oxygen effect in glutathione-deficient human cells in culture

The frequency of X-ray-induced DNA breaks was determined in human cell lines which are deficient in glutathione synthetase and have a greatly reduced glutathione content. Hydroxyapatite chromatography was used for the estimation of the DNA breaks in cell cultures, which were derived either from lymphoblasts transformed by infection with EB virus or from fibroblasts. The dose-effect relationship for the induction of breaks when radiation exposure was made in argon, was similar to that found when exposure was made in air. In control cultures with normal glutathione content, the induction of breaks was enhanced when irradiation was made under aerobic, instead of anaerobic, conditions. Treatment of the glutathione-deficient cells with the hypoxic radiosensitizer misonidazole did not enhance the induction of breaks by radiation delivered either in air or in argon. In control cultures, radiation induction of breaks was enhanced by misonidazole under anaerobic but not under aerobic conditions. When the glutathione-deficient cells were pretreated with cysteamine however, irradiation in the absence of oxygen resulted in a decreased frequency of DNA breaks.

VARIATION OF THE RELATIVE SENSITIVITY OF CLOSELY RELATED NEOPLASTIC CELL LINES IRRADIATED IN CULTURE IN THE PRESENCE OR ABSENCE OF OXYGEN.

Variation of the Relative Sensitivity of Closely Related Neoplastic Cells Lines Irradiated in Culture in the Presence or Absence of Oxygen

Correlation between the Radiobiological Oxygen Constant, K, and the Non-protein Sulphydryl Content of Mammalian Cells

The value of the radiobiological oxygen constant K has been found to depend on the concentration of non-protein sulphydryl (NPSH) within the cell. Cells in the exponential phase of growth have a higher concentration of NPSH and a higher value of K than cells in plateau phase. Binding NPSH with N-ethylmaleimide reduced the value of K and conversely, addition of NPSH as dithioerythritol increased the value of K. K also rises with the same time course as NPSH increases, when plateau phase cells are replated into fresh medium. These results support the hypothesis that free-SH groups within the cell compete with oxygen to react with radiation damaged molecules.

Variation of vascular density within and between tumors of the uterine cervix and its predictive value for radiotherapy

Vascular density in different regions of a number of uterine cervix carcinomas was determined by morphometric analysis of stained histologic sections. Variance analysis indicated a larger inter- than intra-tumoral inhomogeneity of the vasculature, suggesting a certain individual vascularization pattern in these tumors. As indicated by a retrospective study of archival biopsies, this pattern has a predictive value in regard to the efficacy of radiotherapy.

Vascular density and the response of breast carcinomas to mastectomy and adjuvant chemotherapy.

Using morphometric analysis of histological preparations, in a retrospective study vascular indices, expressing the extent of vascularisation, were determined for a number of mammary carcinomas. The indices were found to be related to the survival of the patients treated with modified radical mastectomy in combination with pre- and postoperative chemotherapy, the cases with low indices having the shorter survival. The impaired access of the cytotoxic agents to cells in the deficiently vascularised tumours was considered as an explanation. It was concluded that determination of vascular density in tumours may have a prognostic value in regard to the treatment response, and may be helpful in choosing the appropriate treatment.

Survival curves of irradiated glutathione-deficient human fibroblasts: Indication of a reduced enhancement of radiosensitivity by oxygen and misonidazole

Abstract Fibroblasts derived from a patient with 5-oxoprolinuria are genetically deficient in glutathione synthetase. This deficiency causes a dramatic decrease in intracellular glutathione (GSH) level. The radiosenstivity of GSH deficient cells (GSH − ) was studied in vitro using colony forming ability as an endpoint. Cells with normal GSH level, obtained from the healthy brother of the patient, were used as controls. When irradiated in 95% air-5 % CO 2 , GSH − cells are slightly but significantly more radiosensitive than GSH + controls (dose modifying factor (DMF) of 1.2). When irradiated in argon, the survival curve of GSH − cells indicates an oxygen enhancement ratio (OER) of 1.5 when compared to the curve obtained in oxic conditions. The OER of control cells in the same conditions is 2.9. In comparison to results obtained in air, 100% oxygen moderately increases the radiosensitivity of GSH + cells (DMF 1.23), while it has a very low effect on GSH − cells (DMF 1.06). These results suggest that intracellular GSH plays an essential protective role in hypoxia, its effect is reduced in air and practically disappears in 100% oxygen. When cells are incubated with 8 mM misonidazole 2 hours before irradiation, the drug has a much greater sensitizing effect on GSH + cells (DMF2.33) than on GSH − cells (DMF 1.55). The results demonstrate that intracellular GSH level plays a major role in the response of hypoxic cells, irradiated either alone or in the presence of misonidazole.

Non-protein sulphydryl and glutathione content of ehrlich ascites tumour cells after treatment with the radioprotectors aet, cysteamine and glutathione.

SummaryTreatment of Ehrlich ascites cells in vitro with increasing concentrations of cysteamine, AET or glutathione (GSH) resulted in a proportional increase of the intracellular level of non-protein-bound sulphydryl (NPSH). At equimolar concentrations, the increase in NPSH was greatest after AET-treatment, less after cysteamine and least after GSH. A considerable part of the increase in NPSH after AET- and cysteamine-treatment was due to an increase in content of cellular GSH. The increase in intracellular NPSH after GSH treatment was, in part, due to some compound(s) other than GSH.After interruption of AET or cysteamine treatment, the total NPSH content of the cells decreased immediately, but the GSH level continued to increase for some time before it also started to fall.The results were interpreted as indicating that intracellular release of GSH may be a major mechanism for the radioprotection afforded by the substances used in the investigation.

Compartmentalised depletion of glutathione in cells treated with buthionine sulphoximine

Evidence obtained in different types of experiments has indicated that glutathione (GSH) can function as an inherent protector of cells against treatment with radiation and many chemotherapeutic agents (Arrick & Nathan, 1984). In an attempt to enhance the effect of such treatments, efforts have been made to control the cellular GSH content artificially. In many of the recent studies, buthionine sulphoximine (BSO) has been used to deplete cellular GSH (Chemical Modifiers of Cancer Treatment, 1986). This substance inhibits the synthesis of gamma-glutamyl-cysteine, a precursor of GSH, by specifically blocking the synthesising enzyme. Edgren (1987) reported observations which suggest that depletion of GSH by BSO occurs to different extents in the nucleus and the cytoplasm. The experiments reported here confirm the earlier observations either when treatment is made with BSO at a single concentration for varying time periods, or at varying concentrations for a fixed period.