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.     

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.     

Toxic effects of acute glutathione depletion by buthionine sulfoximine and dimethylfumarate on murine mammary carcinoma cells

Glutathione (GSH) depletion to approximately equal to 5% of control for 48 h or longer by 0.05 mM L-buthionine sulfoximine (BSO) led to appreciable toxicity for the 66 murine mammary carcinoma cells growing in vitro [L.A. Dethlefsen et al., Int. J. Radiat. Oncol. Biol. Phys. 12, 1157-1160 (1986)]. Such toxicity in normal, proliferating cells in vivo would be undesirable. Thus the toxic effects after acute GSH depletion to approximately equal to 5% of control by BSO plus dimethylfumarate (DMF) were evaluated in these same 66 cells to determine if this anti-proliferative effect could be minimized. Two hours of 0.025 mM DMF reduced GSH to 45% of control, while 6 h of 0.05 mM BSO reduced it to 16%. However, BSO (6 h) plus DMF (2 h) and BSO (24 h) plus DMF (2 h) reduced GSH to 4 and 2%, respectively. The incorporation (15-min pulses) of radioactive precursors into protein and RNA were unaffected by these treatment protocols. In contrast, cell growth was only modestly affected, but the incorporation of [3H]thymidine into DNA was reduced to 64% of control by the BSO (24 h) plus DMF (2 h) protocol even though it was unaffected by the BSO (6 h) plus DMF (2 h) treatment. The cellular plating efficiencies from both protocols were reduced to approximately equal to 75% of control cells. However, the aerobic radiation response, as measured by cell survival, was not modified at doses of either 4.0 or 8.0 Gy. The growth rates of treated cultures, after drug removal, quickly returned to control rates and the resynthesis of GSH in cells from both protocols was also rapid. The GSH levels after either protocol were slightly above control by 12 h after drug removal, dramatically over control (approximately equal to 200%) by 24 h, and back to normal by 48 h. Thus even a relatively short treatment with BSO and DMF resulting in a GSH depletion to 2-5% of control had a marked effect on DNA synthesis and plating efficiency and a modest effect on cellular growth. One cannot rule out a direct effect of the drugs, but presumably the antiproliferative effects are due to a depletion of nuclear GSH with the subsequent inhibition of the GSH/glutaredoxin-mediated conversion of ribonucleotides to deoxyribonucleotides. However, even after extended treatment, upon drug removal, GSH was rapidly resynthesized and cellular DNA synthesis and growth quickly resumed.     

Chemically-induced glutathione depletion and lipid peroxidation

Malondialdehyde (MDA) formation in mouse liver homogenates was measured in the presence of various glutathione depletors (5 mmol/l). After a lag phase of 90 min, the MDA formation increased from 1.25 nmol/mg protein to 14.5 nmol/mg in the presence of diethyl maleate (DEM), to 10.5 with diethyl fumarate (DEF) and to 4 with cyclohexenon by 150 min. It remained at 1.25 nmol/mg with phorone and in the control. On the other hand, glutathione (GSH) dropped from 55 nmol/mg to 50 nmol/mg in the control to, < 1 with DEM, to 46 with DEF, to 3 with cyclohexenon and to 7 with phorone. The data show that the potency to deplete GSH is not related to MDA production in this system. DEM stimulated in vitro ethane evolution in a concentration-dependent manner and was strongly inhibited by SKF 525A. From type I binding spectra to microsomal pigments the following spectroscopic binding constants were determined: 2.5 mmol/l for phorone, 1.2 mmol/l for cyclohexenon, 0.5 mmol/l for DEM and 0.3 mmol/l for DEF. In isolated mouse liver microsomes NADPH-cytochrome P-450 reductase and NADH-cytochrome b₅ reductase activity were unaffected by the presence of DEM, whereas ethoxycoumarin dealkylation was inhibited. Following in vivo pretreatment, hepatic microsomal electron flow as determined in vitro was augmented in the presence of depleting as well as non-depleting agents, accompanied by a shift from O₂⁻ to H₂O₂ production. It is concluded that it is not the absence of GSH which causes lipid peroxidation after chemically-induced GSH depletion but rather the interaction of the chemicals with the microsomal monoxygenase system.     

Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine).

Buthionine sulfoximine (S-n-butyl homocysteine sulfoximine), the most potent of a series of analogs of methionine sulfoximine thus far studied (Griffith, O.W., Anderson, M.E., and Meister, A. (1979) J. Biol. Chem. 254, 1205-1210), inhibited gamma-glutamylcysteine synthetase about 20 times more effectively than did prothionine sulfoximine and at least 100 times more effectively than methionine sulfoximine. The findings support the conclusion that the S-alkyl moiety of the sulfoximine binds at the enzyme site that normally binds the acceptor amino acid. Thus, the affinity of the enzyme for the S-ethyl, S-n-propyl, and S-n-butyl sulfoximines increases in a manner which is parallel to those of the corresponding isosteric acceptor amino acid substrates, i.e. glycine, alanine, and alpha-aminobutyrate. Buthionine sulfoximine did not inhibit glutamine synthetase detectably, nor did it produce convulsions when injected into mice. Injection of buthionine sulfoximine into mice decreased the level of glutathione in the kidney to a greater extent (less than 20% of the control level) than found previously after giving prothionine sulfoximine. alpha-Methyl buthionine sulfoximine was also prepared and found to be almost as effective as buthionine sulfoximine; this compound would not be expected to undergo substantial degradative metabolism. Buthionine sulfoximine and alpha-methyl buthionine sulfoximine may be useful agents for inhibition of glutathione synthesis in various experimental systems.     

Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis

Treatment with the anticancer drug cyclophosphamide (CPA) destroys ovarian follicles. The active metabolites of CPA are detoxified by conjugation with glutathione (GSH). We tested the hypotheses that CPA causes apoptosis in ovarian follicles and that suppression of ovarian GSH synthesis before CPA administration enhances CPA-induced apoptosis. Proestrous rats were given two injections, 2 h apart, with (1) saline, then saline; (2) saline, then 50 mg/kg CPA; (3) saline, then 300 mg/kg CPA; or (4) 5 mmol/kg buthionine sulfoximine (BSO) to inhibit glutamate cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, and then 50 mg/kg CPA. Statistically significantly increased DNA fragmentation by agarose gel electrophoresis and granulosa cell apoptosis by TUNEL were observed in the CPA-treated ovaries 24 h after the second injection, but BSO did not enhance the effect of 50 mg/kg CPA. We next tested the hypothesis that CPA depresses ovarian GSH concentration and expression of the rate-limiting enzyme in GSH synthesis, GCL. Proestrous rats were injected with 300 or 50 mg/kg CPA or vehicle and were sacrificed 8 or 24 h later. After CPA treatment, ovarian and hepatic GSH levels decreased significantly, and ovarian GCL subunit mRNA levels increased significantly. There were no significant changes in GCL subunit protein levels. Finally, we tested the hypothesis that GSH depletion causes apoptosis in ovarian follicles. Proestrous or estrous rats were injected with 5 mmol/kg BSO or saline at 0700 and 1900 h. There was a significant increase in the percentage of histologically atretic follicles and a nonsignificant increase in the percentage of apoptotic, TUNEL-positive follicles 24 h after onset of BSO treatment. Our results demonstrate that CPA destroys ovarian follicles by inducing granulosa cell apoptosis and that CPA treatment causes a decline in ovarian GSH levels. More pronounced GSH suppression achieved after BSO treatment did not cause a statistically significant increase in follicular apoptosis. Thus, GSH depletion does not seem to be the mechanism by which CPA causes follicular apoptosis.     

The effect of buthionine sulfoximine on the glutathione level in goldfish tissues

This study describes the effect of DL-buthionine-[S,R]-sulfoximine (BSO) on the glutathione equivalents (GSH-eq = GSH + 2 GSSG) of goldfish. BSO causes depletion of cellular GSH by inhibiting gamma-glutamylcysteine synthetase, a key enzyme of the GSH biosynthesis pathway. BSO at 1,000 and 1,500 mg/kg was effective in promoting 50 and 80% depletion of GSH-eq from brain and liver, respectively, within 3 days. Lower doses of BSO failed to effectively promote hepatic GSH-eq depletion. Moreover, no evident toxic side-effects were observed (including hepatic lipid peroxidation and free radical-mediated oxidation of proteins) in goldfish in response to BSO intraperitoneal injections. We conclude that BSO can be used to deplete GSH-eq in goldfish liver and brain, but attention should be paid to species-specific variations in BSO effects.     

Intracellular adaptations of glutathione content in Cucurbita pepo L. induced by treatment with reduced glutathione and buthionine sulfoximine

The intracellular effects of GSH (reduced glutathione) and BSO (buthionine sulfoximine) treatment on glutathione content were investigated with immunogold labeling in individual cellular compartments of Cucurbita pepo L. seedlings. Generally, GSH treatment led to increased levels of glutathione in roots and leaves (up to 3.5-fold in nuclei), whereas BSO treatment significantly decreased glutathione content in all organs. Transmission electron microscopy revealed that glutathione levels in mitochondria, which showed the highest glutathione labeling density of all compartments, remained generally unaffected by both treatments. Since glutathione within mitochondria is involved in the regulation of cell death, these results indicate that high and stable levels of glutathione in mitochondria play an important role in cell survival strategies. BSO treatment significantly decreased glutathione levels (1) in roots by about 78% in plastids and 60.8% in the cytosol and (2) in cotyledons by about 55% in the cytosol and 38.6% in plastids. After a short recovery period, glutathione levels were significantly increased in plastids and the cytosol of root tip cells (up to 3.7-fold) and back to control values in cotyledons. These results indicate that plastids, either alone or together with the cytosol, are the main center of glutathione synthesis in leaves as well as in roots. After GSH treatment for 24 h, severe ultrastructural damage related to increased levels of glutathione was found in roots, in all organelles except mitochondria. Possible negative effects of GSH treatment leading to the observed ultrastructural damage are discussed.     

Mechanistic aspects of enhanced lipid peroxidation following glutathione depletion in vivo

Several agents known to conjugate with glutathione (GSH) were administered to phenobarbital-induced rats resulting in a more or less pronounced depletion of hepatic GSH. In vitro incubations showed that a large enhancement of spontaneous lipid peroxidation was observed when the GSH content was below 1 μmol/g liver. This effect was inhibited by addition of exogenous GSH in a concentration-dependent manner, the GSH-concentration yielding 50% inhibition (I₅₀) being 1 μM. Using phorone (diisopropylidene acetone), which proved to be the most potent GSH-depletor, the time- and dose-dependence of the GSH-depletion and the consequent lipid peroxidation was studied. Again it was assured that the GSH concentration must reach a critical value of about 20% of the initial hepatic GSH content, before an enhanced lipid peroxidation is seen. Employing scavengers of excited oxygen species no evidence was found for the involvement of free oxygen radicals. Hepatoprotective agents and inhibitors of mixed-function oxidases exerted a more or less pronounced inhibitory action. Our findings are further support of our previous postulate that GSH depletion per se might lead to an increased lipid peroxidation, possibly due to its lack as a part of the cellular defence system against endogenous toxic intermediates.     

The glutathione status of rat kidney nuclei following administration of buthionine sulfoximine

Rat kidney nuclei were isolated by techniques designed to limit the loss of small water-soluble metabolites. Lyophilized rat kidney powder was disrupted with a Tekmar Tissumizer, and nuclei were purified by discontinuous gradient centrifugation through a glycerol-metrizamide solution at 4 degrees C. Purified nuclei exhibited similar DNA to protein ratios as reported for other rat nuclei isolated by nonaqueous isolation techniques. Results suggest that in control animals the concentration of glutathione in the kidney nucleus is similar to that in the cytoplasm. However, following treatment with buthionine sulfoximine, rat kidney nuclear glutathione levels were less than the corresponding cytoplasmic glutathione levels.     

Plasma glutathione turnover in the rat: effect of fasting and buthionine sulfoximine

Hepatic glutathione (GSH) plays an important role in the detoxification of reactive molecular intermediates. Because of evidence that the intrahepatic turnover of glutathione in the rat may be largely accounted for by efflux from hepatocytes into the general circulation, the quantitation of plasma GSH turnover in vivo could provide a noninvasive index of hepatic glutathione metabolism. We developed a method to estimate plasma glutathione turnover and clearance in the intact, anesthetized rat using a 30-min unprimed, continuous infusion of 35S-labelled GSH. A steady state of free plasma glutathione specific radioactivity was achieved within 10 min, as determined by high-pressure liquid chromatography with fluorometric detection after precolumn derivatization of the plasma samples with monobromobimane. The method was tested after two treatments known to alter hepatic GSH metabolism: 90 min after intraperitoneal injection of 4 mmol/kg buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, and after a 48-h fast. Liver glutathione concentration (mean +/- SEM) was 5.00 +/- 0.53 mumol/g wet weight in control rats. It decreased to 3.10 +/- 0.35 mumol/g wet weight after BSO injection and to 3.36 +/- 0.14 mumol/g wet weight after fasting (both p less than 0.05). Plasma glutathione turnover was 63.0 +/- 7.46 nmol.min-1.100 g-1 body weight in control rats, 35.0 +/- 2.92 nmol.min-1.g-1 body weight in BSO-treated rats, and 41.7 +/- 2.28 nmol.min-1.g-1 body weight after fasting (both p less than 0.05), thus reflecting the hepatic alterations. This approach might prove useful in the noninvasive assessment of liver glutathione status.     

Cadmium-induced excretion of urinary lipid metabolites,DNA damage,glutathione depletion,and hepatic lipid peroxidation in sprague-dawley rats

Recent studies have described lipid peroxidation to be an early and sensitive consequence of cadmium exposure, and free radical scavengers and antioxidants have been reported to attenuate cadmium-induced toxicity. These observations suggest that cadmium produces reactive oxygen species that may mediate many of the untoward effects of cadmium. Therefore, the effects of cadmium (II) chloride on reactive oxygen species production were examined following a single oral exposure (0.50 LD₅₀) by assessing hepatic mitochondrial and microsomal lipid peroxidation, glutathione content in the liver, excretion of urinary lipid metabolites, and the incidence of hepatic nuclear DNA damage. Increases in lipid peroxidation of 4.0- and 4.2-fold occurred in hepatic mitochondria and microsomes, respectively, 48 h after the oral administration of 44 mg cadmium (II) chloride/kg, while a 65% decrease in glutathione content was observed in the liver. The urinary excretion of malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT), and acetone (ACON) were determined at 0–96 h after Cd administration. Between 48 and 72 h posttreatment maximal excretion of the four urinary lipid metabolites was observed with increases of 2.2- to 3.6-fold in cadmium (II) chloride-treated rats. Increases in DNA single-strand breaks of 1.7-fold were observed 48 h after administration of cadmium. These results support the hypothesis that cadmium induces production of reactive oxygen species, which may contribute to the tissue-damaging effects of this metal ion.     

Effect of clofibrate on lipid peroxidation in rats treated with aspirin and 4-pentenoic acid

The in vivo hepatic lipid peroxide content of rats was increased by aspirin or 4-pentenoic acid (4-PA) administration but was decreased by clofibrate (CPIB) administration. The increase by aspirin or 4-PA treatment was depressed by simultaneous administration of CPIB. However, the in vitro formation of lipid peroxide in liver mitochondria and microsomes of rats treated with CPIB as well as aspirin and 4-PA was also elevated compared to that of control rats. The formation of lipid peroxide in mitochondria and microsomes of control rats in vitro was depressed by the addition of cytosols obtained from untreated (control), aspirin-treated, 4-PA-treated, and CPIB-treated rats, but was not depressed by the addition of albumin or heated cytosols. The most effective depression was obtained by the addition of cytosol obtained from CPIB-treated rats. In addition, glutathione peroxidase activity and nonprotein sulfhydryl content in cytosol obtained from CPIB-treated rats were elevated compared to those from control, aspirin, and 4-PA-treated rats. The results suggest that the action of CPIB may be mainly related to the increase of cytosolic glutathione peroxidase activity and nonprotein sulfhydryl content. Hepatic triglyceride and phospholipid contents of rats treated with aspirin or 4-PA were increased compared to those of control rats. These increases were also reversed by simultaneous administration of CPIB.     

Combined effect of misonidazole and glutathione depletion by buthionine sulphoximine on cellular radiation response

Chinese hamster cells (V79) and glutathione-proficient (GSH+/+) and glutathione-deficient (GSH-/-) human fibroblasts were treated with a glutathione (GSH)-depleting agent buthionine sulphoximine (BSO) and the hypoxic radiosensitizer misonidazole (MISO), separately or in combination. Subsequently, the cells were exposed to X-rays. Determination of the yield of single-strand DNA breaks (ssb) immediately after irradiation indicated no effect of BSO or MISO treatment when radiation exposure was made aerobically. Assuming that ssb determined immediately after irradiation reflects mainly the effect of radical processes, the results obtained with BSO and MISO, singly and in combination, agreed well with the predictions of a modified version of the 'competition model' using V79 and GSH+/+ cells. Some results obtained with GSH-/- cells could not be so explained.     

Subcellular glutathione contents in isolated hepatocytes treated with L-buthionine sulfoximine

The glutathione contents of the mitochondrial and cytosolic fractions and extracellular space of isolated hepatocytes decrease when glutathione synthesis is inhibited with L-buthionine sulfoximine. Mitochondrial glutathione is depleted to 50% of its initial value whereas the cytosolic pool is completely emptied after 2 h incubation in the presence of inhibitor. The mitochondrial glutathione content was only fully depleted when L-buthionine sulfoximine was added together with phorone (2,6-dimethyl-2,5-heptadiene-4-one), a substrate of the glutathione S-transferases (E.C. 2.5.1.18).     

Glutathione depletion, radiosensitization, and misonidazole potentiation in hypoxic Chinese hamster ovary cells by buthionine sulfoximine

Buthionine sulfoximine (BSO) inhibits the synthesis of glutathione (GSH), the major nonprotein sulfhydryl (NPSH) present in most mammalian cells. BSO concentrations from 1 microM to 0.1 mM reduced intracellular GSH at different rates, while BSO greater than or equal to 0.1 mM (i.e., 0.1 to 2.0 mM), resulting in inhibitor-enzyme saturation, depleted GSH to less than 10% of control within 10 hr at about equal rates. BSO exposures used in these experiments were not cytotoxic with the one exception that 2.0 mM BSO/24 hr reduced cell viability to approximately 50%. However, alterations in either the cell doubling time(s) or the cell age density distribution(s) were not observed with the BSO exposures used to determine its radiosensitizing effect. BSO significantly radiosensitized (ER = 1.41 with 0.1 mM BSO/24 hr) hypoxic, but not aerobic, CHO cells when the GSH and NPSH concentrations were reduced to less than 10 and 20% of control, respectively, and maximum radiosensitivity was even achieved with microM concentrations of BSO (ER = 1.38 with 10 microM BSO/24 hr). Furthermore, BSO exposure (0.1 mM BSO/24 hr) also enhanced the radiosensitizing effect of various concentrations of misonidazole on hypoxic CHO cells.     

Age-dependent changes in rat liver lipid peroxidation and glutathione content induced by acute ethanol ingestion

The study of the influence of the age of animals (13 to 53 weeks) on total liver thiobarbituric acid reactive substances (TBAR) content showed an increase which is maximal in rats of 39 weeks of age compared to young animals (13 weeks), followed by a dimunition in the 53 weeks old group. In this situation, the content of hepatic GSH and total GSH equivalents as well as the GSH/GSSG ratio were decreased with ageing, while GSSG levels were enhanced in the oldest group studied. Acute ethanol intoxication resulted in a marked increase in liver TBAR content in young animals, together with a decline in GSH, total GSH equivalents and GSH/GSSG ratio, and an enhancement in GSSG. These changes elicited by ethanol intake were reduced with ageing. It is concluded that ethanol-induced oxidative stress in the liver is diminished during ageing, despite the progressive decrease in the glutathione content of the tissue observed in control animals.     

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.     

Influence of buthionine sulfoximine and misonidazole on glutathione level and radiosensitivity of human tumor xenografts

We have studied the effect of buthionine sulfoximine (BSO; a gamma-glutamylcysteine synthetase inhibitor) administration, either alone or combined with misonidazole (MISO), on five human tumor xenografts (three melanomas: Bell, Mall, and Nall; and two rectocolic adenocarcinomas: HT29 and HRT18) transplanted into mice. Two criteria were used, the nonprotein bound sulfhydryl (NPSH) level (glutathione (GSH) and cysteine (CYS] and the fraction of surviving tumor cells after gamma irradiation. GSH and CYS were estimated by HPLC and cell survival by in vivo-in vitro clonogenic assay. Administration of BSO alone (three injections of 10 mumol/g) prior to irradiation always produced a significant reduction in the GSH level while MISO administration (1 mg/g) did not consistently influence the NPSH level. While BSO had little or no radiosensitizing effect, MISO always induced radiosensitization (enhancement ratio between 1.6 and 1.8). This effect did not depend on the fraction of surviving hypoxic cells. An increase in MISO-induced radiosensitization produced by BSO was cell-line dependent. Results do not seem to support the hypothesis of a relationship between the GSH level at the time of irradiation and the radiosensitization induced by BSO or BSO + MISO. However, BSO treatment may not have been able to reduce endogenous thiols to a low enough level to test the hypothesis.     

Circadian periodicity of tissue glutathione and its relationship with lipid peroxidation in rats

Circadian fluctuations in tissue glutathione (GSH) concentrations and lipid peroxidation in male Sprague-Dawley rats were investigated. Blood and all the organs studied exhibited distinct circadian variation both in GSH concentrations and peroxidation of polyunsaturated fatty acids. There was a great variation among organs in the periodicity and amplitude of the fluctuations in GSH concentrations. Liver displayed the highest variation (approximately 50%) followed by stomach (approximately 37%), heart (approximately 25%) and kidney (approximately 19%). The changes in other organs were significant but of less magnitude. Implications of such variations and caution in interpretation of experimental results in response to the exposure of animals to xenobiotics are discussed.