Patrick J. Deschavanne

Radioprotective Effect of Cysteamine in Glutathione Synthetase-deficient Cells

The radioprotective role of endogenous and exogenous thiols was investigated, with survival as the end-point, after radiation exposure of cells under oxic and hypoxic conditions. Human cell strains originating from a 5-oxoprolinuria patient and from a related control were used. Due to a genetic deficiency in glutathione synthetase, the level of free SH groups, and in particular that of glutathione, is decreased in 5-oxoprolinuria cells. The glutathione synthetase deficient cells have a reduced oxygen enhancement ratio (1.5) compared to control cells (2.7). The radiosensitivity was assessed for both cell strains in the presence of different concentrations of an exogenous radioprotector:cysteamine. At concentrations varying between 0.1 and 20 mM, cysteamine protected the two cell strains to the same extent when irradiated under oxic and hypoxic conditions. The protective effect of cysteamine was lower under hypoxia than under oxic conditions for both cell strains. Consequently, the oxygen enhancement ratio decreased for both cell strains when cysteamine concentration increased. These results suggest that cysteamine cannot replace endogenous thiols as far as they are implicated in the radiobiological oxygen effect.

Reduced repair of potentially lethal radiation damage in glutathione synthetase-deficient human fibroblasts after X-irradiation.

Using a human fibroblast strain deficient in glutathione synthetase and a related proficient control strain, the role of glutathione (GSH) in repair of potentially lethal damage (PLD) has been investigated in determining survival by plating cells immediately or 24 h after irradiation. After oxic or hypoxic irradiation, both cell strains repair radiation-induced damage. However, under hypoxic conditions, the proficient cells repair PLD as well as under oxic conditions while the deficient cells repair less PLD after irradiation under hypoxic than under oxic conditions. Therefore, the oxygen enhancement ratio (o.e.r.) for proficient cells is similar whether the cells are plated immediately or 24 h later (2.0 and 2.13, respectively). In contrast, the o.e.r. for deficient cells is lower when the cells are plated 24 h after irradiation than when they are plated immediately thereafter (1.16 as compared to 1.55). The results indicate that GSH is involved in PLD repair and, in particular, in the repair of damage induced by radiation delivered under hypoxic conditions.

RADIOSENSITIVITY OF MOUSE KIDNEY CELLS DETERMINED WITH AN IN VITRO COLONY METHOD

Abstract A new method is described to measure the colony forming ability of mouse kidney cells in vitro . This method, which provides a mean plating efficiency of 7.9 %, was used to study the intrinsic radiosensitivity of kidney cells irradiated in vivo . The observed radiosensitivity was found to be comparable to that of a large number of cells from various normal tissues.

Radiosensitivity of mouse lung cells measured using an in vitro colony method

We have detailed a method for the study of mouse lung cell clonogenicity in vitro, in which the average plating efficiency was 9%. When we used this method to study the inherent radiosensitivity of lung cells irradiated either in vivo or in vitro (18 hr after seeding), we found that the radiosensitivity of mouse lung cells was comparable to that of a large number of mammalian cell lines.

Repair of sublethal and potentially lethal damage in lung cells using an in vitro colony method

The repair of radiation damage has been measured in mouse lung using an in vitro colony assay. Following a single dose of 10.5 Gy delivered to the lung in vivo, we observed, six hours after irradiation, an increase of the fraction of surviving cells, which did not change further between six and 24 hours (repair of potentially lethal damage). A similar observation was made using a split-dose method (repair of sublethal damage). The extent of radiation repair is comparable with that of other rodent cells irradiated in vivo. The significance of these results is discussed in relation to the late effects observed following irradiation of lung tissue.

Effect of Ionizing Radiations on the Life Span of Non-transformed Human Fibroblasts

We studied in vitro the influence of ionizing radiations on the life span of non-transformed HF 19 human fibroblasts. The life span of surviving clones was found to be reduced when the cells had received two or three doses of 6 Gy separated by an interval of 15 doublings. In addition, this reduction in life span was greater when the cells were older at the time of irradiation.

Reduced PLD Repair Ability in Glutathione Synthetase Deficient Human Fibroblasts after UV Irradiation

Using a human cell strain deficient in glutathione synthetase and a related control, the role of glutathione in repair mechanisms has been investigated. UV light has been used in order to avoid the interaction between thiols and free radicals. When potentially lethal damage repair is completed, deficient cells in plateau phase exhibit smaller surviving fractions than do control cells. The ratio of surviving fractions in control/deficient cells is about 2 for the same radiation dose. These results indicate that thiols and especially GSH are involved in repair mechanisms.

Comparison between the Radiosensitivity of Human, Mouse and Chicken Fibroblast-like Cells Using Short-term Endpoints

We have performed a comparative study of the radiosensitivity of fibroblastic cell lines from three different animal species: human, mouse and chicken. Endpoints reflecting short term responses were utilized: colony forming ability (CFA), DNA single strand break (SSB) repair and repair of potentially lethal damage (PLD). Regardless of the criterion employed, the response to radiation varies from one species to another. According to our survival curves, chicken cells appear to be more radioresistant than those of human and mouse. SSB repair is apparently absent in murine cells, partial in chicken cells and complete in human cells. This lack of correlation between survival curves and SSB repair demonstrates that survival of irradiated cells does not depend only (or at all) on the repair of SSB. The repair of PLD is much more efficient in human and chicken cells than in murine cells.

Low molecular weight thiol content in glutathione synthetase-deficient human fibroblasts

The activity and the kinetic properties of glutathione synthetase and the concentrations of non-protein bound thiols of the gamma-glutamyl cycle were measured in 11 human fibroblast cell strains. Six of these strains were derived from patients suffering from 5-oxoprolinuria, a recessive genetic disease characterized by a deficiency in glutathione synthetase; the other cell strains were derived from healthy heterozygous or homozygous relatives of the patients. The glutathione synthetase activities of homozygous deficient strains were 1/3 of control values while those of heterozygous strains were 2/3 of control values. The total thiol concentration was lower in only 3 of the 6 deficient homozygotes and that of glutathione (GSH) was lower in only 4 of the 6 deficient homozygotes. This lower GSH level was at least partly offset by an accumulation of gamma-glutamylcysteine, a precursor of GSH, which is almost completely absent from control cells. The total quantities of thiols and GSH in plateau phase cells were about 50% and 30% respectively of the levels in growth phase cells. Approximately 80% of the GSH was in the reduced form in both quiescent and growing cells.

Radiosensitizing and Cytotoxic Properties of Misonidazole on Glutathione Synthetase Deficient Human Fibroblasts

SummaryThe cytotoxic and radiosensitizing effects of misonidazole have been studied on glutathione synthetase deficient fibroblasts and on their controls. At any concentration from 0·1 to 4 mm, deficient cells are more sensitive to the cytotoxic effect of misonidazole than the control cells. The differential effect between the two cell strains concerns both the shoulder and the slope of the survival curve, thus suggesting that NPSH play a role in the determination of misonidazole cytotoxicity. Like oxygen, misonidazole clearly sensitizes deficient cells to a lesser extent than control cells. For both cell strains, the maximum sensitizing effect of misonidazole is very close to that of oxygen (1·5 and 1·5 for deficient cells, 2·8 and 2·9 for control cells, respectively). The sensitizing effect of misonidazole appears in the same concentration range for both cell strains, with a maximal effect at lower concentrations for deficient cells.