S_rnuary Large fluctuations in glutathione content were observed on a daily basis using the Tietze enzyme recycling assay in a panel of six human cell lines of varying radiosensitivity. Glutathione content tended to increase to a maximum during exponential cell proliferation, and then decreased at different rates as the cells approached plateau phase. By reference to high-performance liquid chromatography and flow cytometry of the fluorescent bimane derivative we were able to verify that these changes were real. However, the Tietze assay was occasionally unable to detect glutathione in two of our cell lines (MGH-Ul and AT5BIVA), although the other methods indicated its presence. The existence of an inhibitory activity responsible for these anomalies was confirmed through spiking our samples with known amounts of glutathione. We were unable to detect a direct relationship between cellular glutathione concentration and aerobic radiosensitivity in our panel of cell lines.Keyworid glutathione; radiosensitivity; Tietze assay; high-performance liquid chromatography For at least the past decade the tripeptide sulphydryl compound glutathione (GSH) has been the subject of intensive investigation regarding its role in mediating both radiationand drug-induced responses in living cells. GSH has been proposed to act in a vast number of different cellular processes, amongst them the maintenance of a suitable intracellular reductive environment, protection against harmful xenobiotics, a catalyst of or reactant in several metabolic schemes and an intracellular store of cysteine (Nygaard and Simic, 1983;Mitchell and Russo, 1987). Its potential importance for radiobiology has been recognised ever since the development of the competition model for radiation cell killing, a mathematical consequence of the reaction rates for competing reactions (Alper and Howard-Flanders, 1956). DNA radicals produced by irradiation are considered to be subject to a competition between oxidising (or electronaffinic) agents, leading to damage fixation and ultimate cell death, and reducing species (such as sulphydryl compounds) which, through hydrogen atom donation would facilitate damage repair and so lead to continued cell viability, against a background of the intramolecular decay of DNA radicals to render them non-restorable (Koch, 1983). Since the rate of reaction between oxygen and DNA radicals is far greater than that between GSH and DNA radicals (Bump and Brown, 1990), the competition theory predicts that under normal aerobic conditions the former reaction would dominate, rendering unimportant any changes in GSH content. However GSH may nevertheless play a significant role in the aerobic radiation response, its protective effects mediated through other processes limiting radiation damage, for instance the detoxification of radiation-induced hydroperoxides (Dethmers and Meister, 1981;Biaglow et al., 1984).Cells may be depleted of GSH by treatment with the specific enzyme inhibitor DL-buthionine-S,R-sulphoximine (BSO) which prevents GSH synthesi...
The role of variation in susceptibility to DNA damage induction was studied as a determinant for cellular radiosensitivity. Comparison of the radiosensitive HX142 and radioresistant RT112 cell lines previously revealed higher susceptibility to X-ray-induced DNA damage in the sensitive cell line using non-denaturing elution, but not when using alkaline unwinding. The present data also show that no difference in the amount of initial damage is seen when pulsed-field gel electrophoresis (PFGE) or comet analysis are used for DNA damage assessment. However, using the halo assay or a modified version of PFGE in which the higher DNA architecture remained partially intact, the radiosensitive cells showed steeper dose-response curves for initial DNA damage than the radioresistant cells. Analysis of the protein composition, of DNA-nucleoid structures revealed substantial differences when isolated from HX142 or RT112 cells. From our data, it is concluded that HX142 and RT112 differ in their structural organization of chromatin. As no differences in the kinetics of DNA damage rejoining were found, it is hypothesized that the same amount of lesions have a different impact in the two cell lines in that the 'presentation' of DNA damage alters the ratio of repairable to non-repairable DNA damage.
We have studied the role of glutathione (GSH) in determining radiation response in five human tumour and one human fibroblast cell line. GSH concentration was measured using the Tietze assay and compared with clonogenic survival following gamma-irradiation. No relationship between GSH concentration and aerobic radiosensitivity was observed. The addition of 10 mM extracellular cysteamine produced protection factors in all cell lines, ranging from 1.6 to 2.1, but had little influence on cellular GSH concentration. Depletion of GSH by buthionine sulphoximine (0.1 mM for 18 h) had negligible effect on cell survival, though moderate radiosensitization resulted from extreme GSH depletion after 30-min treatment with 1 mM dimethylfumarate. The degree of aerobic sensitization did not correlate with GSH levels. Irradiation under hypoxia produced oxygen enhancement ratios varying from 1.6 to 2.6, with no relationship to GSH content.
In radiation therapy of cancer, any possible mistakes that may occur in prescribed tumour dose calculation and/ or in treatment set up could also be checked by the biological dosimetry. In order to verify this, 20 patients having pelvic area tumours with no prescribed chemotherapy were selected. Their peripheral blood samples were collected before starting fractionated radiotherapy and at some definite time during treatment. Equivalent whole body doses (EWBD) were calculated from patient's weights, irradiated volumes and total tumor doses given until the date of blood sample collection during treatment. Each patient's EWBD was given in vitro to blood samples that were collected before starting radiotherapy. Radiation induced chromosome damage in lymphocytes were measured by micronucleus (MN) induction.
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