Cellular glutathione depletion by diethyl maleate or buthionine sulfoximine: no effect of glutathione depletion on the oxygen enhancement ratio

The hypoxic and euoxic radiation response for Chinese hamster lung and A549 human lung carcinoma cells was obtained under conditions where their nonprotein thiols, consisting primarily of glutathione (GSH), were depleted by different mechanisms. The GSH conjugating reagent diethylmaleate (DEM) was compared to DL-buthionine-S,R-sulfoximine (BSO), an inhibitor of glutathionine biosynthesis. Each reagent depleted cellular GSH to less than 5% of control values. A 2-hr exposure to 0.5 mM DEM or a 4- or 24-hr exposure to BSO at 10 or 1 mM, respectively, depleted cellular GSH to less than 5% of control values. Both agents sensitized cells irradiated under air or hypoxic conditions. When GSH levels are lowered to less than 5% by both agents, hypoxic DEM-treated cells exhibited slightly greater X-ray sensitization than hypoxic BSO-treated cells. The D0's for hypoxic survival curves were as follows: control, 4.87 Gy; DEM, 3.22 Gy; and BSO, 4.30 Gy for the V79 cells and 5.00 Gy versus 4.02 Gy for BSO-treated A549 cells. The D0's for aerobic V79 cells were 1.70 Gy versus 1.13 Gy, DEM, and 1.43 Gy for BSO-treated cells. The D0's for the aerobic A549 were 1.70 and 1.20 for BSO-treated cells. The aerobic and anoxic sensitization of the cells results in the OER's of 2.8 and 3.0 for the DEM- and BSO-treated cells compared to 2.9 for the V79 control A549. BSO-treated cells showed an OER of 3.3 versus 3 for the control. Our results suggest that GSH depletion by either BSO or DEM sensitizes aerobic cells to radiation but does not appreciably alter the OER.     

Radiosensitization by hypoxic pretreatment with misonidazole: an interaction of damage at the DNA level

Prolonged exposures to misonidazole (MISO) in vitro under hypoxic conditions result in radiosensitization which is characterized by a decrease in the size of the radiation survival curve shoulder for cells irradiated under hypoxic or aerobic conditions after drug removal. Although intracellular glutathione (GSH) was depleted during hypoxic exposures to MISO, this could not account for the dose-additive radiosensitization (decrease in shoulder size) since GSH depletion by diethylmaleate had no effect on the sensitivity of cells irradiated in air. The alkaline elution assay was used to measure DNA strand breaks and their repair after exposure to MISO, graded doses of X rays, and the combination of MISO pretreatment with X rays. The elution rate of DNA from irradiated cells increased linearly with X-ray dose, with and without MISO pretreatment. However, the DNA elution rates measured after MISO pretreatment were greater by a constant amount at all X-ray doses greater than 1 Gy. In terms of both cell survival and DNA elution rate, MISO-pretreated cells behaved as though they had received an extra 1.5 Gy. Although the initial damage after X rays was greater in MISO-pretreated cells, there was no effect of MISO pretreatment on the rate of repair of radiation-induced DNA strand breaks. The agreement between the differences in survival levels and DNA elution rates for irradiated control and MISO-pretreated cells and absence of an effect on DNA repair rates suggest that the pretreatment sensitization is due to an additive interaction of damage at the DNA level.     

Radiation-induced damage to DNA in drug- and radiation-resistant sublines of a human breast cancer cell line.

An Adriamycin-resistant subline of a human breast cancer cell line, MCF-7 ADRR, has been shown to exhibit radioresistance associated with an increase in the size of the shoulder on the radiation survival curve. In the present study, damage to DNA of MCF-7 sublines WT and ADRR by 60Co gamma radiation was measured by filter elution techniques. The initial amount of DNA damage, measured by both alkaline and neutral filter elution, was lower in ADRR cells, suggesting that these cells are resistant to radiation-induced single- and double-strand DNA breaks. In the case of double-strand breaks the difference between WT and ADRR cells was significant only at the lower radiation doses studied (up to 100 Gy). In cells depleted of glutathione (GSH) by L-buthionine sulfoximine (BSO) treatment, ADRR cells were sensitized to radiation-induced DNA damage, while WT cells were unaffected. The rate of repair of single- and double-strand DNA breaks following radiation was the same for both sublines, and repair of radiation damage was not affected by BSO treatment in either cell line. The relative resistance of ADRR cells to initial DNA damage by radiation is the only difference so far detected at the molecular level which reflects radiation survival, and it is possible that other factors are involved in the resistance of ADRR cells to killing by radiation. Sensitization of ADRR cells to radiation-induced DNA damage by GSH depletion, although not likely to involve inhibition of GSH-dependent detoxification enzymes per se (irradiation was done at 4 degrees C), suggests that at the molecular level radioresponse in this subline is related to maintenance of GSH/GSSG redox equilibrium.     

The induction of DNA-protein crosslinks in hypoxic cells and their possible contribution to cell lethality

The induction of single-strand breaks (SSBs) in the DNA of Chinese hamster ovary cells by X rays under different irradiation conditions was measured by the alkaline elution technique. The oxygen enhancement ratio (OER) for SSB induction determined for cells irradiated in air versus irradiation of cells made hypoxic by metabolic depletion of O2 was 9.7. However, when proteinase K was included in the cell lysis solution the OER was reduced to 4.2. The proteinase affected the elution rate only of the cells irradiated under hypoxic conditions, suggesting that DNA-protein crosslinks (DPCs) are preferentially produced in hypoxic cells by radiation. The ability to repair these DPCs was compared in two cell lines: the wild-type AA8 line and an excision-repair-deficient mutant line, UV-41. The AA8 line removed about 80% of the DPCs induced by radiation under hypoxic conditions within a 24-h repair incubation. The UV-41 line, on the other hand, removed only about 20% of the DPCs in the same time. The OERs for cell survival of these two lines are 3.1 for AA8 but only 1.9 for UV-41, suggesting that the DPCs preferentially induced in the DNA of cells irradiated under hypoxic conditions may contribute to cell killing when the normal DNA-repair mechanisms are compromised.     

Effects of glutathione depletion by buthionine sulfoximine on radiosensitization by oxygen and misonidazole in vitro

Buthionine sulfoximine (BSO) has been used to deplete glutathione (GSH) in V79-379A cells in vitro, and the effect on the efficiency of oxygen and misonidazole (MISO) as radiosensitizers has been determined. Treatment with 50 or 500 microM BSO caused a rapid decline in GSH content to less than 5% of control values after 10 hr of exposure (t1/2 = 1.6 hr). Removal of BSO resulted in a rapid regeneration of GSH after 50 microM BSO, but little regeneration was observed over the subsequent 10-hr period after 500 microM. Treatment with either of these two concentrations of BSO for up to 14 hr did not affect cell growth or viability. Cells irradiated in monolayer on glass had an oxygen enhancement ratio (OER) of 3.1. After 10-14 hr pretreatment with 50 microM BSO, washed cells were radiosensitized by GSH depletion at all oxygen tensions tested. The OER was reduced to 2.6, due to greater radiosensitization of hypoxic cells than aerated ones by GSH depletion. GSH depletion had the effect of shifting the enhancement ratio vs pO2 curve to lower oxygen tensions, making oxygen appear more efficient by a factor of approximately 2, based on the pO2 required to give an OER of 2.0. In similar experiments performed with MISO, an enhancement ratio of 2.0 could be achieved with 0.2 mM MISO in anoxic BSO-pretreated cells, compared to 2.7 mM MISO in non-BSO-treated cells. Thus MISO appeared to be more efficient in GSH-depleted cells by a factor of 13.5. These apparent increases in radiosensitizer efficiency in GSH-depleted cells could be explained on the basis of radiosensitization of hypoxic cells by GSH depletion alone (ER = 1.29-1.41). The effect of GSH depletion was approximately equal at all sensitizer concentrations tested, except at high oxygen tensions, where the effect was insignificantly small. These results are consistent with hypoxic cell radiosensitization by GSH depletion and by MISO or oxygen acting by separate mechanisms.     

DNA damage measured using the alkaline elution assay depends on time between subculturing of cells and irradiation

In experiments utilizing the alkaline filter elution assay for radiation-induced DNA damage we observed an unexpected dependence of hypoxic dose-response curves on the length of time V79 cells were in exponential growth between subculturing and irradiation. Dose-response curves for DNA from cells irradiated in air were identical regardless of whether the exponential-phase cells had been subcultured 24 or 48 h prior to irradiation, but cells irradiated in hypoxia 24 h after subculture displayed a dose-response curve for DNA damage which was two times steeper than that obtained for cells irradiated in hypoxia 48 h after subculture. Possible mechanisms for this effect are discussed.     

Radiation response of drug-resistant variants of a human breast cancer cell line: the effect of glutathione depletion

Two drug-resistant variants of the human breast cancer cell line MCF-7 have been shown previously to exhibit radiation resistance associated with an increase in the size of the shoulder on the radiation survival curve. In the present study, glutathione (GSH) depletion was achieved by exposure of cells to buthionine sulfoximine (BSO) with, in some cases, additional treatment with dimethyl fumarate. Levels of GSH in the adriamycin-resistant subline MCF-7 ADRR are initially lower than in the other two sublines and are depleted to a greater extent by exposure to BSO. Wild-type MCF-7 cells are not sensitized by GSH depletion when irradiated under aerated conditions but are sensitized under hypoxic conditions to an extent which is related to the level of GSH depletion. In contrast both the drug-resistant sublines (MCF-7 ADRR and the melphalan-resistant line MCF-7 MLNR) are radiosensitized by GSH depletion under both aerated and hypoxic conditions. It is hypothesized that in the case of the MCF-7 ADRR cell line, which expresses high levels of the GSH-associated redox enzyme systems, GSH-S-transferase and GSH-peroxidase (GSH-Px), radiosensitization results when GSH-Px is inhibited in GSH-depleted cells. The reasons for radiosensitization of aerated MCF-7 MLNR cells cannot be explained on this basis, however, and other factors are being examined.     

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.     

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.     

Interactions of radioprotectors and oxygen in cultured mammalian cells. II. Effects of dithiothreitol on radiation-induced DNA damage and comparison with cell survival

The effects of the sulfhydryl-containing compound dithiothreitol (DTT) on radiation-induced DNA damage have been studied using two different assays: DNA unwinding hydroxyapatite chromatography and alkaline filter elution. DNA damage as measured by both assays for cells irradiated in air shows drug concentration-dependent radioprotection reaching high levels (dose reduction factor, DRF = 3) at high DTT concentrations. The pattern and degree of protection against DNA damage are the same as shown previously for cell survival. However, when cells are irradiated in hypoxia, DNA damage as measured by the unwinding technique is decreased less by low DTT concentrations than is survival, but DNA damage is decreased to a much greater extent (DRF = 3) at high concentrations of DTT (compared to DRF = 1.5 for cell survival). DNA damage as measured by the alkaline elution assay after hypoxic irradiation is decreased to a much greater extent at all concentrations of DTT with DRF = 1.6 at 1 mM and increasing to DRF = 4.5 at high levels of DTT. These results are discussed in terms of the different types of DNA damage produced in cells irradiated in air versus hypoxia and the differences in types of damage measured by the two different DNA assays and cell survival.     

The role of thiols in cellular response to radiation and drugs

Cellular nonprotein thiols (NPSH) consist of glutathione (GSH) and other low molecular weight species such as cysteine, cysteamine, and coenzyme A. GSH is usually less than the total cellular NPSH, and with thiol reactive agents, such as diethyl maleate (DEM), its rate of depletion is in part dependent upon the cellular capacity for its resynthesis. If resynthesis is blocked by buthionine-S,R-sulfoximine(BSO), the NPSH, including GSH, is depleted more rapidly, Cellular thiol depletion by diamide, N-ethylmaleimide, and BSO may render oxygenated cells more sensitive to radiation. These cells may or may not show a reduction in the oxygen enhancement ratio (OER). Human A549 lung carcinoma cells depleted of their NPSH either by prolonged culture or by BSO treatment do not show a reduced OER but do show increased aerobic responses to radiation. Some nitroheterocyclic radiosensitizing drugs also deplete cellular thiols under aerobic conditions. Such reactivity may be the reason that they show anomalous radiation sensitization (i.e., better than predicted on the basis of electron affinity). Other nitrocompounds, such as misonidazole, are activated under hypoxic conditions to radical intermediates. When cellular thiols are depleted peroxide is formed. Under hypoxic conditions thiols are depleted because metabolically reduced intermediates react with GSH instead of oxygen. Thiol depletion, under hypoxic conditions, may be the reason that misonidazole and other nitrocompounds show an extra enhancement ratio with hypoxic cells. Thiol depletion by DEM or BSO alters the radiation response of hypoxic cells to misonidazole. In conclusion, we propose an altered thiol model which includes a mechanism for thiol involvement in the aerobic radiation response of cells. This mechanism involves both thiol-linked hydrogen donation to oxygen radical adducts to produce hydroperoxides followed by a GSH peroxidase-catalyzed reduction of the hydroperoxides to intermediates entering into metabolic pathways to produce the original molecule.     

Protection against radiation-induced mutagenesis in V79 cells by 2-[(aminopropyl)amino] ethanethiol under conditions of acute hypoxia

The effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells under hypoxic or aerobic conditions were examined. Conditions of acute hypoxia were attained by gassing 10(6) cells in 1-ml volumes in individual glass ampoules for 2 min with nitrogen. Ampoules were then sealed and incubated at 37 degrees C for 60 min. Following this treatment, cell survival after irradiation as expected was significantly enhanced. The effect of acute hypoxia on the formation of HGPRT mutants by irradiation was also investigated. Mutation frequencies were determined with a 6-day expression time and corrected for the number of spontaneous background mutants. Although mutation induction was approximately linear as a function of radiation dose under most conditions tested, it was significantly reduced in cell populations made acutely hypoxic prior to irradiation. Protection against mutation induction was apparent and similar when cells were irradiated in the presence of the radioprotector, regardless of whether they were also hypoxic or aerated. If cells were irradiated in air and then made hypoxic, no significant protection was still observed. These results suggest that the antimutagenic effect of WR-1065 is not due solely to its ability to scavenge radiation-induced oxygen-free radicals, but rather that it may also modulate these effects through the scavenging of metabolically induced free radicals and/or the chemical repair of radiation-induced DNA lesions.     

Glutathione modulates lipid composition of human colon derived HT-29 cells

Glutathione (GSH) is important in maintaining intracellular thiol status. The present study looked at the effect of GSH depletion on lipid composition of colon-derived HT-29 cells. GSH was depleted in HT-29 cells by incubation either with buthionine-S, R-sulfoximine (BSO) or diethylmaleate (DEM). GSH was restored during early periods of cell growth by supplementation of growth medium with either GSH ester or N-acetyl cysteine (NAC). Lipids were analysed following GSH depletion and supplementation. Among the neutral lipids, an increase in free cholesterol and diacylglycerol and decrease in cholesteryl ester and triacylglycerol were seen in GSH-depleted cells as compared to control cells. There were no detectable free fatty acids either in control or GSH-depleted cells. Among the phospholipids, a decrease in phosphatidylcholine and phosphatidylinositol and an increase in phosphatidylethanolamine were observed. These changes were almost completely reversed by supplementation of BSO-treated cells with GSH ester and partially reversed by N-acetyl cysteine. These results suggest that the GSH status of the cell plays an important role in the lipid composition of the cells.     

Chronic depletion of glutathione (GSH) and minimal modification of LDL in vivo: its prevention by glutathione mono ester (GME) therapy

A decline in reduced glutathione (GSH) level is associated with aging and free radical mediated diseases. The objective of this study was to determine whether the chronic depletion of extra cellular GSH causes oxidative damage to the circulating macromolecules such as lipoproteins. Decreased concentrations of plasma glutathione, vitamin E and ascorbic acid were recorded in the rats treated with buthionine sulfoximine (BSO), a selective GSH inhibitor. In LDL isolated from BSO-treated animals, the concentration of malondialdehyde (MDA) and conjugated dienes were significantly increased (P<0.01), whereas the levels of vitamin E were decreased (P<0.01). The analysis of total and LDL cholesterol revealed significant changes between the control and experimental groups. Of interest, altered concentrations of lyso-phosphatidyl choline (Lyso-PC) and phosphatidyl choline (PC) were recorded from the BSO mediated minimally modified LDL. A negative correlation between LDL-BDC/MDA and its antioxidant capacity was noted. Upon in vitro oxidation with CuSO(4), the electrophoretic behavior of purified LDL-apoprotein-B on agarose gel showed an increased mobility in BSO-treated rats, indicative of in vivo modification of LDL to become susceptible for in vitro oxidation. The increased mobility of LDL (after in vitro oxidation) isolated from the BSO-treated animals correlates with a decrease in its amino groups, as determined by the trinitrobenzene sulfonic acid (TNBS) reactants. However, the mobility of LDL molecule was not altered due to BSO treatment in vivo. Interestingly, the minimal modification on LDL does not lead to any vascular damage in the dorsal aorta of the rats injected with BSO. The administration of glutathione monoester (GME), at a dose of 5 mmol/kg body weight, twice a day, for 30 days, to animals treated with l-buthionine-SR-sulfoximine (BSO, 4 mmol/kg body weight, twice a day, for 30 days) normalized the antioxidant status and prevented the minimal modifications on LDL. Thus, increasing the cellular GSH levels may trigger beneficial effects against oxidative stress.     

Causes of DNA single-strand breaks during reduction of chromate by glutathione in vitro and in cells

Carcinogenic chromates induce DNA single-strand breaks (SSB) that are detectable by conventional alkali-based assays. However, the extent of direct breakage has been uncertain because excision repair and hydrolysis of Cr-DNA adducts at alkaline pH also generate SSB. We examined mechanisms of SSB production during chromate reduction by glutathione (GSH) and assessed the significance of these lesions in cells using genetic approaches. Cr(VI) reduction was biphasic and the formation of SSB occurred exclusively during the slow reaction phase. Catalase or iron chelators completely blocked DNA breakage, as did the use of GSH purified by a modified Chelex procedure. Thus, the direct intermediates of GSH-chromate reactions were unable to cause SSB unless activated by H2O2. SSB repair-deficient XRCC1(-/-) and proficient XRCC1+ EM9 cells had identical survival at doses causing up to 60% clonogenic death and accumulation of 1 mM Cr(VI). However, XRCC1(-/-) cells displayed higher lethality in the more toxic range and the depletion of GSH made them hypersensitive even to moderate doses. Elevation of cellular catalase or GSH levels eliminated survival differences between XRCC1(-/-) and XRCC1+ cells. In summary, formation of toxic SSB in cells occurs at relatively high chromate doses, requires H2O2, and is suppressed by high GSH concentrations.     

Phytochelatin synthesis in maize seedlings in response to excess zinc

Buthionine sulfoximine (BSO) specifically inhibits γ-glutamylcysteine synthetase and decreases a cellular level of glutathione (GSH) in maize seedling roots. Exogenous GSH restores Zn-phytochelatins synthesis in BSO-treated maize plants.     

激光、血卟啉对肿瘤细胞DNA单链断裂及其重接的影响

用简化的Kohn氏碱洗脱法,观察了光敏剂血卟啉衍生物(HPD)对小鼠S-180肿瘤细胞DNA单链断裂及其重接修复的影响。激光HPD能导致S-180细胞DNA单链断裂明显增加,而且这种断裂随着保温时间的延长,继续增多。在本实验条件下没有观察到HPD对X线的增敏作用,HPD不能增加X线所致的DNA单链断裂,也不能影响其重接。单链断裂重接动力学的实验进一步证明了这个论点。     

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.     

Modulation of rat erythrocyte antioxidant defense system by buthionine sulfoximine and its reversal by glutathione monoester therapy

The protective effects of glutathione monoester (GME) on buthionine sulfoximine (BSO)-induced glutathione (GSH) depletion and its sequel were evaluated in rat erythrocyte/erythrocyte membrane. Animals were divided into three groups (n=6 in each): control, BSO and BSO+GME group. Administration of BSO, at a concentration of 4 mmol/kg bw, to the albino rats resulted in depletion of blood GSH level to about 59%. GSH was elevated several folds in the GME group as compared to the control (P<0.05) and BSO (P<0.001) groups. Decreased concentration of vitamin E was found in the erythrocyte membrane isolated from BSO-administered animals. Antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) were also found to be altered due to BSO-induced GSH depletion in blood erythrocytes. The SOD and CAT activities in BSO group were significantly lower (P<0.001) than the other groups. Lipid peroxidation index and malondialdehyde (MDA) levels in erythrocytes and their membranes were increased to about 45% and 40%, respectively. The activities of Ca2+ ATPase, Mg2+ ATPase and Na+K+ ATPase were lower than those of control group (P<0.05), whereas the activities of these enzymes were found to be restored to normal followed by GME therapy (P<0.05). Cholesterol, phospholipid and C/P ratio and some of the phospholipid classes like phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin were significantly (P<0.05) altered in the erythrocyte membranes of BSO-administered rats compared with those of control group. These parameters were restored to control group levels in GME-treated group. Oxidative stress may play a major role in the BSO-mediated gamma glutamyl cysteine synthetase (gamma-GCS) inhibition and hence the depletion of GSH. In conclusion, our findings have shown that antioxidant status decreased and lipid peroxidation increased in BSO-treated rats. GME potentiates the RBC and blood antioxidant defense mechanisms and decreases lipid peroxidation.     

Inhibition of glutathione synthesis reduces chilling tolerance in maize

 The role of glutathione (GSH) in protecting plants from chilling injury was analyzed in seedlings of a chilling-tolerant maize (Zea mays L.) genotype using buthionine sulfoximine (BSO), a specific inhibitor of γ-glutamylcysteine (γEC) synthetase, the first enzyme of GSH synthesis. At 25 °C, 1 mM BSO significantly increased cysteine and reduced GSH content and GSH reductase (GR: EC 1.6.4.2) activity, but interestingly affected neither fresh weight nor dry weight nor relative injury. Application of BSO up to 1 mM during chilling at 5 °C reduced the fresh and dry weights of shoots and roots and increased relative injury from 10 to almost 40%. Buthionine sulfoximine also induced a decrease in GR activity of 90 and 40% in roots and shoots, respectively. Addition of GSH or γEC together with BSO to the nutrient solution protected the seedlings from the BSO effect by increasing the levels of GSH and GR activity in roots and shoots. During chilling, the level of abscisic acid increased both in controls and BSO-treated seedlings and decreased after chilling in roots and shoots of the controls and in the roots of BSO-treated seedlings, but increased in their shoots. Taken together, our results show that BSO did not reduce chilling tolerance of the maize genotype analyzed by inhibiting abscisic acid accumulation but by establishing a low level of GSH, which also induced a decrease in GR activity. Received: 9 November 1999 / Accepted: 17 February 2000