Survival curves of glutathione synthetase deficient human fibroblasts: correlation between radiosensitivity in hypoxia and glutathione synthetase activity

The role of intracellular non-protein bound sulphydryl compounds (NPSH), and in particular that of glutathione (GSH), in the response of cells to ionizing radiation under different O2 concentrations has been assessed using cell strains deficient in glutathione synthetase and exhibiting different NPSH levels. The cell strains used originated from patients with 5-oxoprolinuria and from their relatives (heterozygotes and proficient homozygotes). No correlation has been found between NPSH and GSH concentrations and radiosensitivity under oxic, aerobic and hypoxic conditions. However, a highly significant correlation has been observed between radiosensitivity under hypoxic conditions (and therefore the oxygen enhancement ratio) and the glutathione synthetase activity, suggesting that synthesis of GSH is required after irradiation. In order to explain our results we postulated, beside radical processes, the existence of a GSH-dependent enzymatic repair mechanism for N2 type damage. Hypoxic radio-sensitivity measured with survival curves would result from the interaction of both competition and biochemical repair processes.     

Postirradiation sensitization of mammalian cells by the thiol-depleting agent dimethyl fumarate

Dimethyl fumarate (DMF) depletes intracellular glutathione (GSH) by covalent bond formation in a reaction mediated by GSH-S-transferase. Treatment of hypoxic Chinese hamster V79 cells with 5 mM DMF before irradiation radiosensitizes the cells, resulting in an enhancement ratio (ER) of about 2.7 with minimal toxicity, when the end point is clonogenic cell survival. Under the same conditions aerobic cells are sensitized, and ER of about 1.3 is found, and GSH is reduced to about 3% of control. Very similar results were obtained previously with Chinese hamster ovary (CHO) cells. In addition, new data presented here show that DMF treatment of V79 or CHO cells immediately after irradiation under hypoxic conditions sensitizes the cells, resulting in an ER of about 1.5, DMF treatment after irradiation under aerobic conditions results in an ER of 1.3, and this DMF treatment reduces protein thiols (PSH) to about 70% of control. When induction of DNA damage is measured using the neutral elution assay, treatment of V79 or CHO cells with DMF prior to irradiation under hypoxic conditions results in an ER of 1.9-2.0, but there is no enhancement of DNA damage when DMF is added after irradiation under hypoxic conditions or when cells are treated with DMF before or after irradiation under aerobic conditions. Based on these data we postulate that DMF radiosensitizes killing of hypoxic cells by two actions: depletion of GSH interferes with the chemical competition between damage fixation and repair, and depletion of PSH causes an inhibition of enzymatic repair processes. We also suggest that DMF sensitizes aerobic cells only by inhibition of enzymatic repair processes.     

Cell-cycle-dependent repair of potentially lethal damage in the XR-1 gamma-ray-sensitive Chinese hamster ovary cell

Repair of potentially lethal damage (PLD) was investigated in a gamma-ray-sensitive Chinese hamster cell mutant, XR-1, and its parent by comparing survival of plateau-phase cells plated immediately after irradiation with cells plated after a delay. Previous work indicated that XR-1 cells are deficient in repair of double-strand DNA breaks and are gamma-ray sensitive in G1 but have near normal sensitivity and repair capacity in late S phase. At irradiation doses from 0 to 1.0 Gy (100 to 10% survival), the delayed- and immediate-plating survival curves of XR-1 cells were identical; however, at doses greater than 1.0 Gy a significant increase in survival was observed when plating was delayed (PLD repair), approaching a 20-fold increase at 8 Gy. Elimination of S-phase cells by [3H]thymidine suicide dramatically increased gamma-ray sensitivity of plateau-phase XR-1 mutant cells and reduced by 600-fold the number of cells capable of PLD repair after a 6-Gy dose. In contrast, elimination of S-phase cells in plateau-phase parental cells did not alter PLD repair. These results suggest that the majority of PLD repair observed in plateau-phase XR-1 cells occurs in S-phase cells while G1 cells perform little PLD repair. In contrast, G1 cells account for the majority of PLD repair in plateau-phase parental cells. Thus, in the XR-1 mutant, a cell's ability to repair PLD seems to depend upon the stage of the cell cycle at which the irradiation is delivered. A possible explanation for these findings is discussed.     

Importance of TP53 and RB in the repair of potentially lethal damage and induction of color junctions after exposure to ionizing radiation

Repair of potentially lethal damage (PLD) was investigated in cells with functional G1-phase arrest with wild-type TP53 and wild-type RB and in cells in which G1-phase arrest was abrogated by inactivation of TP53 or RB. Confluent cultures of cells were plated for clonogenic survival assay either immediately or 24 h after irradiation. Induction of color junctions, an exchange between a painted and unpainted chromosome, was studied in chromosomes 18 and 19 after irradiation with 4 Gy gamma rays. Significant repair of PLD was found in cells carrying both wild-type TP53 and wild-type RB. In cells in which TP53 or RB was inactivated, the survival curves from immediately plated and delayed-plated cells were not significantly different. The numbers of radiation-induced color junctions in chromosomes 18 and 19 were similar in all cell lines. From this study we conclude that a functional G1-phase arrest is important for repair of PLD and that TP53 and RB do not affect the frequencies of induction of color junctions in chromosome 18 or 19.     

Enhancement of X-ray-induced sister chromatid exchanges in hypoxic cells

In these studies we have used wild-type Chinese hamster ovary cells (AA8) and a mutant cell line (UV-41) deficient in excision repair to compare sister chromatid exchange (SCE) induction after X irradiation under oxic and hypoxic conditions. X irradiation of AA8 cells under oxic conditions induced only a slight increase in SCEs, whereas at each dose tested a significantly greater number of SCEs were induced in hypoxic cells. When AA8 cells were X-irradiated and the addition of bromodeoxyuridine (BrdU) was delayed for 20 h to allow DNA lesions to be repaired, the levels of SCEs detected in both oxic and hypoxic cells returned to background levels. X irradiation of UV-41 cells also induced only a slight increase of SCEs in oxic cells, whereas a significant number of SCEs were induced in hypoxic cells. However, in contrast to results with AA8 cells, when hypoxic UV-41 cells were X-irradiated and the addition of BrdU was delayed for 20 h, the number of SCEs remained significantly above background levels. In combination with previous alkaline elution data, these results are consistent with the possibility that DNA-protein crosslinks are responsible for the SCEs induced by X irradiation of hypoxic cells. Irrespective of the mechanism(s) involved, the data presented suggest that the SCE assay may potentially aid in the detection of hypoxic tumor cells.     

Repair of potentially lethal damage does not depend on functional TP53 in human glioblastoma cells

The functionality of G(1)-phase arrest was investigated in relation to repair of potentially lethal damage (PLD) in human glioblastoma Gli-06 cells. Confluent cultures were irradiated and plated for clonogenic survival either immediately or 24 h after gamma irradiation. Bivariate flow cytometry was performed to assess the distribution over the cell cycle. Levels of TP53 and CDKN1A protein were assessed with Western blotting and levels of CDKN1A mRNA with RT-PCR. Confluence significantly reduced the number of proliferating cells. Marked PLD repair was found in the absence of an intact G(1) arrest. No accumulation of TP53 was observed, and the protein was smaller than the wild-type TP53 of RKO cells. No increased expression of CDKN1A at the mRNA or protein levels was found in Gli-06 cells. The TP53 of Gli-06 cells was unable to transactivate the CDKN1A gene. From this study, it is evident that PLD repair may be present without a functional TP53 or G(1) arrest.     

Radiosensitization in multifraction schedules. II. Greater sensitization by 2-nitroimidazoles than by oxygen

The objective of this study was to characterize the extent of and mechanisms involved in radiosensitization by 2-nitroimidazoles in multifraction schedules using low doses per fraction. For this purpose, contact-inhibited monolayers of C3H 10T1/2 cells were given 1.7 Gy every 12 h and plated 12 h after the last dose received to allow full repair of potentially lethal damage (PLD). Severe hypoxia was obtained by a 1-h gassing procedure at room temperature immediately before each irradiation. No toxicity occurred as a consequence of multiple exposures to 5 mM misonidazole (MISO) or SR 2508 (2508) during the deoxygenation procedure. Experimental conditions during the pregassing and irradiation (presence of drug and gas mixture) were appropriately manipulated to test for the different mechanisms of radiosensitization demonstrated by nitroimidazoles. A very low oxygen enhancement ratio (OER) results under these conditions (1.34). Exposure to 5 mM MISO or 2508 during the deoxygenation and irradiation of hypoxic cells resulted in greater radiosensitization than could be accounted for by oxygen-mimetic sensitization alone (MISO and 2508 enhancement ratios were greater than the OER). Pregassing cells with N2 in the presence of 5 mM drug sensitized cells which were subsequently irradiated under aerobic conditions (drug free), indicating the occurrence of the "preincubation effect" (which does not require hypoxia or the drug's presence during the irradiation). Thus, for the hypoxic irradiations, the preincubation effect could account for the greater sensitization by nitroimidazoles than by oxygen. The presence of 5 mM drug only during the irradiation of aerobic cells produced radiosensitization in both multifraction and single-dose experiments with delayed plating. This sensitization has been previously shown to involve reduced PLD repair. Finally, maximum radiosensitization occurred in the multifraction schedule when a transient period of hypoxia with drug preceded an aerobic irradiation with drug present, thus combining the benefits of both the preincubation effect and PLD repair inhibition. This work demonstrates the possibility that effects other than oxygen-mimetic radiosensitization could be largely responsible for the sensitization seen in multifraction schedules, particularly when the OER is already low and only transient periods of hypoxia occur.     

Potentially lethal damage repair is due to the difference of DNA double-strand break repair under immediate and delayed plating conditions

Cells plated immediately after irradiation on nutrient agar (immediate plating) exhibit a lower survival than cells which are kept under nongrowth conditions before plating (delayed plating). The difference between the survival curves obtained after immediate plating and delayed plating is considered to exhibit the cell's capacity to repair potentially lethal damage. In yeast evidence has been presented previously for the DNA double-strand break (DSB) as the molecular lesion involved in the repair of potentially lethal damage observed at the cellular level. Radiation-induced DSB are repaired in cells plated on nutrient agar, i.e., under growth conditions, as well as in cells kept under nongrowth conditions. In this paper DSB repair under growth and nongrowth conditions is studied with the help of the yeast mutant rad54-3 which is temperature conditional for DSB repair. It is shown that the extent of repair of potentially lethal damage can be varied by shifting the relative fractions of repair of DSB under growth conditions versus nongrowth conditions. Repair of DSB in cells plated on nutrient agar is promoted when glucose is substituted by Na-succinate as an energy source. As a result the immediate plating survival curve approaches the delayed plating survival curve, thus reducing the operationally defined repair of potentially lethal damage. We show that this reduced potentially lethal damage repair is caused, however, by a higher amount of DSB repair in cells immediately plated on succinate agar as compared to glucose agar.     

Glutathione requirement for the rejoining of radiation-induced DNA breaks in misonidazole-treated cells

The role of glutathione (GSH) in the rejoining of radiation-induced single-strand DNA breaks (ssb) was studied in human fibroblast cultures sensitized to radiation by a 30 min treatment with 1 mM misonidazole (MISO). Hypoxically irradiated cells, deficient in GSH, either inherently, or due to a 16 h incubation with 1 mM buthionine sulphoximine (BSO), rejoined the breaks after MISO treatment at a lower rate and to a lesser extent than did GSH-proficient cells. Without MISO treatment, the hypoxically induced ssb were rejoined in the GSH-deficient cells as effectively as in the proficient cells. It is concluded that a large proportion of the breaks which arise after hypoxic irradiation in the presence of MISO are of a different type to those which arise in the absence of the drug, and require a particular GSH-dependent, enzymatic repair system. This requirement for rejoining in hypoxically irradiated, MISO-treated cells is similar to that seen earlier in MISO-untreated, oxically irradiated cells, and suggests that the ssb induced by radiation in the presence of MISO or oxygen are of a similar nature.     

The relationship between radiosensitivity and repair of potentially lethal damage in human tumor cell lines with implications for radioresponsiveness

The relationship between intrinsic radiosensitivity and repair capacity was studied for 22 human tumor cell lines in vitro. The experimental material was taken from 19 published papers. Parameters from three radiobiological models were used to assess this relationship: the one-hit multitarget model (D0 and n), the linear-quadratic model (alpha and beta), and the mean inactivation dose (D). Data were obtained for cells in three stages: exponentially growing cells (exp), plateau-phase cells plated immediately after irradiation (ip), and plateau-phase cells plated after completion of PLD repair (dp). No significant difference was found between radiosensitivity of exp and ip cells. There was no correlation between repair capacity and intrinsic radiosensitivity assessed with plateau-phase cells plated immediately after irradiation. The correlation studies between intrinsic radiosensitivity or repair capacity and clinical responsiveness were achieved by assigning cell lines to one of three groups of decreasing in vivo radioresponsiveness: highly, medium, and poorly responsive. There was a significant correlation between radiosensitivity and radioresponsiveness, but no correlation between repair capacity and radioresponsiveness. The average repair capacity was about 0.6 Gy, in terms of D. Three parameters, the mean inactivation dose of exponentially growing cells, of plateau-phase cells plated immediately after irradiation, and of plateau-phase cells plated after completion of PLD repair, could be used equally to assess the relationship between in vitro data and radioresponsiveness. The present results are compared to those obtained in a similar study on a group of 48 nontransformed fibroblast cell strains.     

Ataxia-telangiectasia homo- and heterozygous cells show a normal repair and fixation response to anisotonic NaCl treatment after irradiation

The effect of anisotonic NaCl treatment on fixation and repair of radiation-induced potentially lethal damage (PLD) was tested in normal human cells and in three homozygous ataxia-telangiectasia (A-T) and two heterozygous A-T cell strains. Fixation of radiation-induced PLD occurred in all cell strains exposed to 0.05, 0.5, or 1.5 mole/liter NaCl solutions immediately after irradiation. This effect was observed in both plateau-phase and exponentially growing normal and A-T cells. When an incubation period at 37 degrees C was introduced between irradiation and the subsequent anisotonic treatment, recovery was observed in both normal and A-T cells strains. These data show that A-T cells are as proficient as normal cells in repairing PLD that is sensitive to anisotonic NaCl treatment. It is proposed that two PLD repair systems may exist, one that is expressed after irradiation in proliferatively arrested cells and another that occurs in plateau-phase as well as exponentially growing cells, and is expressed by the postirradiation treatments described here and by Raaphorst and Azzam (Radiat. Res. 86, 52-66 (1981].     

Effect of inhibitors of poly(ADP-ribose)polymerase on the radiation response of HeLa S3 cells

The purpose of this study was to investigate possible involvement of poly(ADP-ribosyl)ation reactions in X-ray-induced cell killing, repair of potentially lethal damage (PLD), and formation and repair of radiation-induced DNA damage. As tools we used the inhibitors of poly(ADP-ribose)polymerase, 3-aminobenzamide (3AB), and 4-aminobenzamide (4AB). Both drugs inhibited PLD repair equally well but did not increase radiation-induced cell killing when cells were plated immediately after irradiation. 3AB affected repair of radiation-induced DNA damage, while 4AB had no effect. When 3AB was combined with aphidicolin (APC), it was found that the amount of DNA damage increased during the postirradiation incubation period. This means that the presence of 3AB stimulates the formation of DNA damage after X-irradiation. It is concluded that 3AB and 4AB sensitize HeLaS3 cells for radiation-induced cell killing by inhibiting repair of PLD. Because of the different effects of both inhibitors on repair of PLD and repair of radiation-induced DNA damage (a process known to be affected by inhibition of poly(ADP-ribosyl)ation), it is concluded that the observed inhibition of PLD repair is not caused by inhibition of poly(ADP-ribose)polymerase, and that the inhibitors affect repair of PLD and repair of DNA damage through independent mechanisms.     

Independent forms of potentially lethal damage fixed in plateau-phase Chinese hamster cells by postirradiation treatment in hypertonic salt solution or araA

Plateau-phase Chinese V79 hamster cells were sequentially treated after exposure to gamma rays in medium made hypertonic by the addition of sodium chloride (370 mM) and with various concentrations of 9-beta-D-arabinofuranosyladenine (araA) to study their combined effect on fixation of potentially lethal damage (PLD). A 10-min treatment in hypertonic medium fixed an extensive amount of PLD and caused a decrease in D0 from 1.8 to 1.2 Gy without significantly affecting Dq. Subsequent treatment with araA caused further fixation of PLD but resulted in a specific, concentration-dependent reduction in Dq from 4.9 to 1.6 Gy after a 4-h exposure to 150 microM araA. A 30-min treatment in hypertonic medium reduced not only Do (from 1.8 to 1.0 Gy) but also Dq (from 4.9 to 2.7 Gy). Subsequent treatment with araA in this case affected only the residual shoulder, reducing it to 1.6 Gy after a 4-h treatment with 100 microM araA, a value similar to that obtained after treatment with araA of cells exposed to salt for only 10 min. When the repair of PLD fixed by a 10-min treatment with salt was measured by delaying its postirradiation application in the presence of various amounts of araA, a small decrease in the repair rate was observed but no significant effect on the relative increase in survival. Qualitatively similar results were obtained for repair of PLD sensitive to araA after a 10-min treatment in hypertonic medium. These results suggest the radiation induction of forms of PLD with different sensitivity to fixation by postirradiation treatments. araA is proposed to fix a form of PLD termed alpha-PLD, the repair of which takes place within 4-6 h and which causes the formation of the shoulder in the survival curve of cells plated immediately after irradiation. Short treatments in hypertonic medium (less than 10 min) are proposed to fix a form of PLD termed beta-PLD, the repair of which takes place within 1 h and leads to restoration of the slope to values equal to those obtained in the survival curve of cells plated immediately after irradiation. However, longer treatments in hypertonic medium also affect Dq and thus also alpha-PLD. Repair of beta-PLD was not significantly affected by araA and repair of alpha-PLD was not significantly affected by short hypertonic treatment, thus indicating the independence of the two forms of PLD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recovery from potentially lethal damage and recruitment time of noncycling clonogenic cells in 9L confluent monolayers and spheroids

Cells that have been grown as multicell tumor spheroids exhibit radioresistance compared to the same cells grown in monolayers. Comparison of potentially lethal damage (PLD) repair and its kinetics was made between 9L cells grown as spheroids and confluent monolayers. Survival curves of cells plated immediately after irradiation showed the typical radioresistance associated with spheroid culture compared to plateau-phase monolayers. The dose-modification factor for spheroid cell survival is 1.44. Postirradiation incubations in normal phosphate-buffered saline (PBS), conditioned media, or 0.5 M NaCl in PBS reduced the differences in radiosensitivity between the two culture conditions. Postirradiation treatment in PBS or conditioned medium promoted repair of potentially lethal damage, and 0.5 M NaCl prevented the removal of PLD and allowed the fixation of damage resulting in lower survival. Survival of spheroid and monolayer cells after hypertonic NaCl treatment was identical. NaCl treatment reduced Do more than it did the shoulder (Dq) of the survival curve. PLD repair kinetics measured after postirradiation incubation in PBS followed by hypertonic NaCl treatment was the same for spheroids and for plateau-phase monolayers. The kinetics of PLD repair indicates a biphasic phenomenon. There is an initial fast component with a repair half-time of 7.9 min and a slow component with a repair half-time of 56.6 min. Most of the damage (59%) is repaired slowly. Since the repair capacity and kinetics are the same for spheroids and monolayers, the radioresistance of spheroids cannot be explained on this basis. Evidence indicates that the time to return from a Go (noncycling G1 cells) state to a proliferative state (recruitment) for cells from confluent monolayers and from spheroids after dissociation by protease treatment may be the most important determinant of the degree of PLD repair that occurs. Growth curves and flow cytometry cell cycle analysis indicate that spheroid cells have a lag period for reentry into a proliferative state. Since plating efficiency remains high and unchanging during this period, one cannot account for the delay on the basis of the existence of a large fraction of Go cells which are not potentially clonogenic. The cell cycle progression begins in 6-8 h for monolayer cells and in 14-15 h for spheroids. It is hypothesized that the slower reentry of spheroid cells into a cycling phase allows more time for repair than for the rapidly proliferating monolayer cells.     

Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli

When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed.     

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.     

Effect of arabinofuranosyladenine on radiation-induced chromosome damage in plateau-phase CHO cells measured by premature chromosome condensation: implications for repair and fixation of alpha-PLD

The effect of the DNA polymerase inhibitor beta-arabinofuranosyladenine (araA) on radiation-induced damage was studied at the cell survival and chromosome level in unfed plateau-phase cultures of Chinese hamster ovary cells. At the cell survival level postirradiation treatment with araA fixed a form of radiation-induced potentially lethal damage, termed alpha-PLD. In the absence of araA treatment, repair of PLD resulted in the formation of the survival curve shoulder in immediately plated cells and in the increase in survival observed after delayed plating. The repair kinetics observed after delayed plating of plateau-phase cells or after delayed administration of 500 microM araA were similar, suggesting that both protocols assay similar lesions. AraA-mediated fixation reached a plateau at concentrations higher than 500 microM, indicating complete fixation of alpha-PLD. At the cytogenetic level, postirradiation treatment with araA at concentrations higher than 500 microM caused a complete inhibition of chromosome repair, as scored by premature chromosome condensation. In the absence of araA, the linearity of the dose-effect relationship for chromosome fragmentation obtained immediately after irradiation was preserved even after long repair times. The repair kinetics of chromosome damage measured in cells held postirradiation in the plateau phase were the mirror image of the repair kinetics for alpha-PLD. The half-time was 1 h in both cases and repair reached a plateau after about 4-6 h. AraA-mediated repair inhibition of chromosome damage was reversible, and a decrease in residual chromosome damage was observed after post-treatment incubation in araA-free conditioned medium. This persistent chromosome damage increased with increasing araA concentration and, as with PLD fixation, reached a plateau at about 500 microM. These results suggest that repair and araA-mediated fixation of alpha-PLD have their counterparts at the chromosome level as indicated by the similar repair kinetics and inhibition/fixation characteristics obtained for alpha-PLD and chromosome damage. This relationship implies a correlation between repair at the DNA and the chromosome level and suggests that DNA polymerization is required for the repair of chromosome damage.     

Effect of glutathione depletion on the cytotoxicity of xenobiotics and induction of single-strand DNA breaks by ionizing radiation in isolated hamster round spermatids

The role of glutathione (GSH) in cellular protection mechanisms in round spermatids from hamsters was studied. Isolated spermatids were largely depleted of GSH by treating the cells for 2 h with the GSH conjugating agent diethyl maleate (DEM). This treatment resulted in a 90% decrease of the cellular GSH content, but did not affect the ATP content. Exposure of isolated spermatids to cumene hydroperoxide (CHP), a compound which is detoxicated by the GSH redox cycle, showed that the cytotoxicity of the peroxide was markedly potentiated by GSH depletion of the cells. The cytotoxicity was reflected by the cellular ATP content. A decrease of the ATP content of the GSH-depleted spermatids was observed at 5-6-fold lower CHP concentrations, as compared to control cells. An increased cytotoxicity in GSH-depleted cells was also observed using 1-chloro-2,4-dinitrobenzene (CDNB), which is a reactive compound that is detoxicated by glutathione conjugation. The induction of single-strand DNA breaks by gamma radiation was 3-5-fold higher in GSH-depleted spermatids as compared to control cells. This radiation-induced damage was estimated under hypoxic conditions (500 p.p.m. O2 in N2). GSH depletion did not affect the repair of single-strand DNA breaks following the irradiation. The present results indicate that cellular GSH has an important function in the defence mechanisms of round spermatids against peroxides, electrophilic xenobiotics and radiation-induced DNA damage.     

Depletion of glutathione after gamma irradiation modifies survival

The relationship between the intracellular glutathione (GSH) concentration and the aerobic radiation response was studied in Chinese hamster ovary cells. Various degrees of GSH depletion were produced by exposure to buthionine sulfoximine (BSO) and/or diethyl maleate (DEM). Diethyl maleate did not act as a classical radiosensitizer under the experimental conditions employed, nor did exposure to DEM/BSO nonspecifically affect protein thiols as measured by thiol blotting. Dose-response curves were obtained using cells irradiated in the absence or presence of DEM/BSO, which decreased GSH levels by 90-95%. Exposure to DEM/BSO did not affect the formation of DNA single-strand breaks or DNA-protein crosslinks measured immediately after irradiation performed at ice temperatures. Analysis of survival curves indicated that the Dq was decreased by 18% when GSH depletion occurred prior to, during, and after irradiation. The DEM/BSO exposure did not affect D0. To study postirradiation conditions, cells were exposed to 10 microM DEM prior to and during irradiation, which was performed at ice temperatures. Levels of GSH were depleted by 75% by this protocol. Immediately after irradiation, the cells were rapidly warmed by the addition of 37 degrees C growth medium containing either 10 or 90 microM DEM. Addition of 10 microM DEM after irradiation did not affect the degree of depletion, which remained constant at 75%. In contrast, GSH depletion was increased to 90% 10 min after addition of the 90 microM DEM. Addition of 90 microM DEM after irradiation produced a statistically significant difference in survival compared to addition of 10 microM DEM. In a second depletion protocol, cells were exposed to 100 microM DEM at room temperature for 5 min, irradiated, incubated at 37 degrees C for 1 h, washed, and then incubated in 50 microM BSO for 24 h. This depletion protocol reduced survival by a factor of 2.6 compared to cells not exposed to the combination of DEM/BSO. Survival was not affected if the cells were exposed to the DEM or BSO alone. This was interpreted to indicate that survival was not affected by GSH depletion occurring after irradiation unless depletion was rapid and sustained. The rate of repair of sublethal and potentially lethal damage was measured and found to be independent of the DEM/BSO exposure. These experimental results in addition to previous ones (Freeman and Meredith, Int. J. Radiat. Oncol. Biol. Phys. 13, 1371-1375, 1987) were interpreted to indicate that under aerobic conditions GSH depletion may alter the expression of radiation damage by affecting metabolic fixation.