Dose Of Buthionine Sulfoximine Research Articles

Minimal ovarian upregulation of glutamate cysteine ligase expression in response to suppression of glutathione by buthionine sulfoximine

The antioxidant tripeptide glutathione (GSH) protects ovarian follicles against oxidative damage that may lead to apoptotic death. The rate-limiting step in synthesis of GSH is catalyzed by glutamate cysteine ligase (GCL), a heterodimer composed of a catalytic subunit (GCLC), and a modifier subunit (GCLM). We hypothesized that GSH depletion in vivo or in vitro with buthionine sulfoximine (BSO), a specific inhibitor of GCL activity, would increase ovarian and granulosa cell GCL subunit expression. Ovarian glutathione levels are lowest on proestrous morning and increase to their highest levels on estrus and metestrus. Therefore, we treated rats on proestrous morning or on proestrous morning and again 12 h later to prevent the normal increase in ovarian glutathione between proestrus and estrus. Ovarian Gclc and Gclm mRNA levels and GCLC protein levels increased transiently by 1.4–1.5-fold at 8 h, but not at 12 or 24 h, after a single dose of BSO administered to adult rats on the morning of proestrus. GCLC protein levels were also modestly increased 1.4-fold at 12 h after a second dose of BSO. GCLM protein levels increased 1.4-fold at 24 h after a single dose of BSO, but not at other time points. BSO treatment did not significantly alter ovarian GCL enzymatic activity or the intraovarian localization of either GCL subunit mRNA. Treatment of a human granulosa cell line or primary rat granulosa cells with BSO suppressed intracellular GSH; however, there was no compensatory upregulation of GCL subunit protein or mRNA levels. These results demonstrate that ovarian follicles and granulosa cells are minimally able to respond to acute GSH depletion by upregulating expression of GCL.

Identification of multidrug resistance-associated protein 1 and glutathione as multidrug resistance mechanisms in human prostate cancer cells: chemosensitization with leukotriene D4 antagonists and buthionine sulfoximine.

To assess the involvement of the multidrug resistance-associated protein 1 (MRP1) and the glutathione pathway in the multidrug resistant (MDR) phenotype of prostate cancer in vitro. Chemoselection of human prostate cancer cell lines PC3 and DU145 with etoposide resulted in the resistant cell lines PC3-R and DU-R. Resistance against etoposide, doxorubicin and vincristine, and its reversal with leukotriene D4 antagonists MK-571 and zafirlukast, and buthionine sulfoximine (BSO), was assessed using tetrazolium-dye viability assays. Western blot analysis of MRP1 expression and glutathione content were measured, and MRP1 function assessed in fluorescence assays. MRP1 was increased in the MDR models; the glutathione content was significantly higher in PC3-R but there was no increase in glutathione in DU-R. Adding non-toxic doses of MK-571, zafirlukast or BSO significantly increased the sensitivity of the MDR models to cytotoxic drugs. MRP1 function was inhibited with MK-571 in the MDR models. MRP1 and glutathione mediate MDR in newly developed prostate cancer models.

Phase I trial of buthionine sulfoximine in combination with melphalan in patients with cancer.

Resistance to alkylating agents and platinum compounds is associated with elevated levels of glutathione (GSH). Depletion of GSH by buthionine sulfoximine (BSO) restores the sensitivity of resistant tumors to melphalan in vitro and in vivo. In a phase I trial, each patient received two cycles as follows: BSO alone intravenously (i.v.) every 12 hours for six doses, and 1 week later the same BSO as cycle one with melphalan (L-PAM) 15 mg/m2 i.v. 1 hour after the fifth dose. BSO doses were escalated from 1.5 to 17 g/m2 in 41 patients. The only toxicity attributable to BSO was grade I or II nausea/vomiting in 50% of patients. Dose-related neutropenia required an L-PAM dose reduction to 10 mg/m2 at BSO 7.5 g/m2. We measured GSH in peripheral mononuclear cells (PMN), and in tumor biopsies when available, at intervals following BSO dosing. In PMNs, GSH content decreased over 36 to 72 hours to reach a nadir on day 3; at the highest dose, recovery was delayed beyond day 7. The mean PMN GSH nadirs were approximately 10% of control at BSO doses > or = 7.5 g/m2; at 13 and 17 g/m2, all but two patients had nadir values in this range. GSH was depleted in sequential tumor biopsies to a variable extent, but with a similar time course. At BSO doses > or = 13 g/m2, tumor GSH was < or = 20% of starting values on day 3 in five of seven patients; recovery had not occurred by day 5. We measured plasma concentrations of R- and S-BSO by high-performance liquid chromatography (HPLC) in 22 patients throughout the dosing period. Total-body clearance (CLt) and volume of distribution at steady-state (Vss) for both isomers were dose-independent. The CLt of S-BSO was significantly less than that of R-BSO at all doses, but no significant differences in Vss were observed between the racemates. Harmonic mean half-lives were 1.39 hours and 1.89 hours for R-BSO and S-BSO, respectively. A biochemically appropriate dose of BSO for use on this schedule is 13 g/m2, which will be used in phase II trials to be conducted in ovarian cancer and melanoma.

Buthionine sulfoximine-induced glutathione depletion: Its effect on antioxidants, lipid peroxidation and calcium homeostasis in the lung

The administration of buthionine sulfoximine (BSO), an irreversible inhibitor of γ-glutamylcysteine synthetase, produces glutathione (GSH) depletion in tumors, making them sensitive to drugs and radiation. During the process, it also depletes GSH from normal tissues. Certain tumors require frequent doses of BSO for several days to produce GSH depletion. In this study, we determined that this chronic GSH-deficient condition lowers the antioxidant defense of the lung by diminishing the activities of superoxide dismutase, catalase, and glutathione peroxidase and the levels of ascorbic acid and α-tocopherol. Impaired antioxidant defense leads to enhanced lipid peroxidation, as indicated by increased levels of thiobarbituric acid reactive substances and conjugated dienes. The alteration of protein thiols by lipid peroxidation, is responsible for altered Ca 2+ homeostasis, which, in turn, leads to cell injury. Cell injury was confirmed by elevated activities of angiotensin converting enzyme and lactate dehydrogenase, increased levels of protein and lactate, and histopathological changes.

The effect of in utero administration of buthionine sulfoximine on rat development

Glutathione (GSH) is a tripeptide that is thought to be an essential cell component playing an important role as a cellular antioxidant and scavenger of free radicals. GSH depletion has been shown to render cells more sensitive to various insults. GSH has a protective effect. GSH levels can be decreased by inhibition of its synthesis with buthionine sulfoximine (BSO), which inhibits γ-glutamylcysteine synthetase. Several studies have shown that treatment with BSO enhances the toxicity of some drugs and radiation. A previous study indicated that the effects of BSO on the developing embryo were short lived and did not persist to birth. In the above-mentioned study, mothers were treated with BSO only on days 10 and 11 of gestation. The objective of the present study was to determine the effects of BSO administration on GSH depletion throughout pregnancy on the developing rat. Timed pregnant Sprague-Dawley rats were placed on a liquid BioServ diet containing BSO starting on day 1 of pregnancy. The mothers received a daily dose of BSO ranging from 2 to 6 mmol/kg/24 h. The mothers were maintained on the diet until gestation day 21 when they were anesthetized with sodium pentobarbital and the pups delivered by Cesarean section. GSH levels were measured in brain and liver, and various parameters relating to development were assessed. A dose-response curve showed that a maximum depletion (86%) of GSH in the mother's liver was produced by the 6 mmol/kg dose of BSO. However, no change was seen in brain GSH levels of the mothers. GSH levels in brain and liver of the offspring were decreased by 60% and 66%, respectively. No significant effect of treatment with BSO was observed on growth-related parameters, such as body or brain weight. A significant decrease in neuron-specific enolase (NSE) activity in cerebellum and an increase in liver γ-glutamyl transpeptidase activity were observed in pups born to mothers treated with BSO. A decrease in NSE activity consistent with delayed development was observed. Therefore, we conclude that although a decrease in GSH may not produce obvious or observable teratogenic effects, it may produce a delay in development and may have a permissive role in teratogenic effects produced by other drugs by virtue of GSH depletion.

In vivo therapeutic potential of combination thiol depletion and alkylating chemotherapy

The effect of administering the thiol modulating agent buthionine sulfoximine (BSO) in conjunction with alkylating chemotherapy was investigated in vivo in the mouse KHT sarcomas and bone marrow stem cells. Tumour response to treatment was assessed by an in vivo to in vitro excision assay and bone marrow survival was determined in vitro by CFU-GM. Glutathione (GSH) depletion and recovery kinetics were determined at various times after treatment using high performance liquid chromatography (HPLC) techniques. Following a single 2.5 mmol kg-1 dose of BSO, tumour GSH reached a nadir of approximately 40% of control 12-16 h after treatment. Bone marrow GSH was depleted to approximately 45% of control 4-8 h after treatment but recovered to normal by 16 h. When a range of doses of CCNU, mitomycin C, cyclophosphamide or melphalan (MEL) were given 16 h after mice were exposed to a 2.5 mmol kg-1 dose of BSO, only the antitumour efficacy of MEL was effectively enhanced (by a factor of approximately 1.4). This BSO-MEL combination appeared to be selective for the tumour as the bone marrow toxicity was not increased beyond that seen for MEL alone. Since increasing the administered dose of BSO neither increased the extent of thiol depletion in the tumour nor enhanced the antitumour efficacy of MEL, three other protocols for delivering the thiol depletor were explored. BSO was given either as multiple 2.5 mmol kg-1 doses administered at 6 or 16 h intervals or continuously at a concentration of 30 mM supplied in the animals' drinking water. Both multi-dose BSO pretreatments were found to increase both the antitumour efficacy and normal tissue toxicity of MEL such that no advantage compared to the single dose combination was achieved. In contrast, maintaining the thiol depletor in the drinking water led to an approximately 1.7-fold increase in the antitumour efficacy of MEL without any corresponding increase in bone marrow stem cell toxicity. For the various pretreatment strategies it was possible, in all cases, to account for the presence or absence of a net therapeutic benefit on the basis of the tumour and bone marrow GSH depletion and recovery kinetics.

Effect of modulators of glutathione synthesis on the hepatotoxicity of 2-methylfuran

Treatment of male Sprague-Dawley rats with buthionine sulfoximine (BSO), prior to administration of carbon-14( 14C)-labelled 2-methylfuran (2MF) caused a marked decrease in the covalent binding of 14C-labelled 2MF metabolites to both DNA and protein, although there was no apparent change in the distribution of the labelled parent 2MF. BSO pretreatment also protected against hepatotoxicity of 2MF, as indicated by lower serum glutamic pyruvic transaminase (GPT) levels. Pretreatment with BSO offered protection only if administered 1.5 hr before 2MF dosage. Administration of 2MF, 4 and 6 hr after BSO resulted in manifestation of the hepatotoxicity of 2MF. Prior treatment with diethylmaleate (DEM), increased covalent binding of [ 14C]2MF to liver proteins and also elevated serum GPT levels. Thus, depletion of tissue glutathione (GSH) by two different chemicals acting by different mechanisms produced opposite effects on the covalent binding and toxicity of 2MF. Pretreatment with L-2-oxothiazolidine-4-carboxylate (OTZ), a promoter of GSH biosynthesis, increased the hepatic covalent binding of [ 14C]2MF and potentiated hepatotoxicity. However, administration of OTZ and BSO prior to an i.p. dose of 100 mg/kg of 2MF, decreased the hepatic covalent binding of [ 14C]2MF and decreased the hepatotoxicity. The marked instability of the GSH conjugate of the reactive metabolite of 2MF may account for the potentiation of hepatotoxicity of 2MF by OTZ. A single s.c. dose of BSO, caused a transient increase in plasma cystine levels concurrent with the depletion of liver GSH. Administration of 2MF, 1.5 hr after BSO, significantly decreased plasma cystine levels as compared to control animals that received vehicle alone. Pretreatment with BSO also resulted in increased excretion of urinary metabolites in 2MF treated animals as compared to animals receiving 2MF alone. Thus, BSO probably protects against hepatoxicity of 2MF by indirectly causing more detoxification of the reactive metabolite of 2MF, as it does not alter the distribution of unmetabolized 2MF and does not have any apparent effect on the microsomal mixed-function oxidase which mediates the activation of 2MF. The enhanced detoxification of 2MF in BSO treated animals appears independent of the depleted GSH levels; it may result from increased availability of a better alternative nucleophile (i.e. cysteine), capable of conjugating with acetyl acrolein. Acetyl acrolein (AA) appears to be the principal reactive metabolite of 2MF which binds covalently to tissues. Previous in vitro studies have shown that cysteine is a better trapping agent of AA than GSH or N-acetyl cysteine.

Induction of rat UDP-glucuronosyltransferase and glutathione S-transferase activities by [formula omitted] without induction of cytochrome P-450

The effect of prolonged exposure to buthionine sulfoximine (BSO) on rat hepatic Phase I and Phase II drug-metabolizing enzymes has been examined. Exposure to 30 mM BSO in drinking water for 7 days induced hepatic microsomal UDP-glucuronosyltransferase activity (detergent-activated) toward p-nitrophenol (250%), 1-naphthol (210%), morphine (130%) and testosterone (140%), but not estrone. Glucuronosyltransferase activities were also induced after exposure for as short as 3 and as long as 13 days. When rats were returned to unsupplemented drinking water for 1 day prior to sacrifice following 6 days on 30 mM BSO, comparable induction to that seen after 7 consecutive days on the BSO solution was observed despite liver glutathione concentration having rebounded to 127% of control. Daily ingestion of BSO was similar (1 mmol/rat/day) for all periods of 30 mM BSO-drinking water exposure, with a body weight-adjusted dose range of 3.2–6.3 mmol/kg/day. An analogous inductive response caused by drinking 30 mM BSO for 3 days was elicited for p-nitrophenol and morphine glucuronidation by 6 mmol/kg doses of BSO given as single daily intraperitoneal or intragastric injections for 3 days. Intraperitoneal, intragastric and all BSO-drinking water exposures also significantly induced (130–195%) cytosolic glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene. Significant increases in UDP-glucuronosyltranferase and glutathione S-transerase activities were also observed following 3 days of exposure to BSO in the drinking water at a concentration as low as 5 mM. Cytosolic p-nitrophenol sulfotransferase activity, with one minor exception, was not enhanced by any BSO treatment regimen. Alterations in transferase activities were not accompanied by any major changes in either overall cytochrome P-450 concentration or oxidative reactions selective for two isozymes. Thus, in addition to its well-documented glutathione-depleting property, BSO also selectively induces several Phase II drug-metabolizing enzymes, and effect to be considered in studies employing extended BSO treatment.

Improved use of buthionine sulfoximine to prevent cisplatin nephrotoxicity in rats.

Male Sprague Dawley rats were treated with buthionine sulfoximine (BSO) and cisplatin in different doses and schedules to optimize the chemoprotective effect of BSO against cisplatin nephrotoxicity. BSO at 4 mmol/kg, administered s.c. 2 h prior to cisplatin, resulted in normal blood urea nitrogen (BUN) levels in rats treated with 3 or 4 mg/kg cisplatin, and modestly, but significantly reduced the toxicity of cisplatin at 5 mg/kg. Administration of BSO (4 mmol/kg) at intervals ranging from 0 to 16 h prior to cisplatin (5 mg/kg) resulted in a significant reduction in BUN values. A BSO dose as low as 0.04 mmol/kg was found to be as effective as 4 mmol/kg against nephrotoxicity associated with cisplatin at 4 mg/kg. Repetitive injections of BSO (1 mmol/kg every 12 h, four times, beginning 2 h prior to cisplatin) significantly inhibited elevations of BUN associated with higher-dose cisplatin (6 mg/kg), whereas a single BSO injection of 4 mmol/kg was ineffective. The degree and duration of renal glutathione depletion was related to the dose of BSO. Renal glutathione content following 4 mmol/kg BSO was 38% of control at 2 h and 40% at 24 h; following 0.04 mg/kg, glutathione was 47% at 2 h and almost 100% at 24 h. Simultaneous in vitro administration of BSO did not inactivate cisplatin cytotoxicity as measured by the colony-forming ability of MBT-2 cells in soft agar. These data indicate that repeated injections of BSO, beginning prior to cisplatin administration, would improve the nephroprotective effect without compromising the chemotherapeutic efficacy of cisplatin. It is suggested that the ability of BSO to reduce cisplatin nephrotoxicity may not correlate with the degree of renal glutathione depletion and that the mechanism of action is unlikely to involve direct inactivation of cisplatin.

The pulmonary effects of buthionine sulfoximine treatment and glutathione depletion in rats.

Buthionine sulfoximine (BSO), an inhibitor of de novo synthesis of glutathione (GSH), was used to deplete rats of GSH and determine the effect of treatment on antioxidant enzyme responses, lung injury, and the susceptibility to concurrent sublethal or lethal hyperoxia. In a preliminary experiment, total lung nonprotein sulfhydryl (NPSH) and GSH levels were measured at various times after single doses of BSO. The lowest concentrations were observed at 12 to 18 h. These experiments were used to establish a repeated dosing protocol for more prolonged GSH depletion. The lungs of rats treated with BSO for 4 days demonstrated markedly decreased GSH and NPSH levels (10 to 40% of control values) and glutathione peroxidase activity (45 to 60% of control values). Superoxide dismutase activities were elevated, glutathione reductase activity was slightly elevated, and catalase activity was unchanged. These changes were dose-responsive. The lungs of treated rats were grossly and microscopically normal. BSO treatment of additional rats did not increase susceptibility to lethal hyperoxia (greater than 98% oxygen). Combined treatment of rats with both BSO and sublethal hyperoxia (80% oxygen) for 4 days did not alter the biochemical responses demonstrated by rats treated solely with BSO. The marked increase in catalase activity obtained after hyperoxia alone was not observed in rats treated with both hyperoxia and BSO. The lungs of saline- and BSO-treated rats exposed to sublethal hyperoxia demonstrated a patchy distribution of slight perivascular and peribronchiolar edema.(ABSTRACT TRUNCATED AT 250 WORDS)

Buthionine sulfoximine-mediated depletion of glutathione in intracranial human glioma-derived xenografts

D-54 MG, a human glioma-derived continuous cell line growing as subcutaneous or intracranial xenografts in athymic mice, was found to be sensitive to the effects of d,l-buthionine -(SR)- sulfoximine , a selective inhibitor of γ-glutamylcysteine synthetase. Intraperitoneal administration of one dose of buthionine sulfoximine (BSO, 5 mmol/kg) resulted in depletion of total intracellular glutathione to 57 and 47% of control 12 hr, and 73 and 23% of control 24 hr, after BSO in subcutaneous and intracranial xenografts respectively. Concurrent measurement of total glutathione in the contralateral (non-tumor-containing) cerebral hemisphere in mice bearing intracranial D-54 xenografts demonstrated insignificant depletion of glutathione. Multiple doses of BSO, at 12-hr intervals, resulted in further depletion to 27% (s.c.) and 16.5% (i.c.) of control 12 hr following the final dose of BSO. Quantitative analysis of BSO delivery to xenograft and contralteral brain tissue revealed transfer constants, K 1, of 15.8–24.1 × 10 −3 and 2.4 × 10 −3 ml · g −1 · min −1 for xenograft and “normal” brain respectively. This highly selective depletion of glutathione in neoplastic tissue versus surrounding non-neoplastic host tissue may have therapeutic implications for the rational use of chemotherapeutic and radiotherapeutic intervention.

Effect of Glutathione Depletion by L-Buthionine Sulfoximine on the Cytotoxicity of Cyclophosphamide in Single and Fractionated Doses to EMT6/SF Mouse Tumors and Bone Marrow2

The combined cytotoxicity of cyclophosphamide [(CYC) CAS: 50-18-0] and glutathione [(GSH) CAS: 70-18-8] depletion by buthionine sulfoximine (BSO) toward EMT6/SF tumors and mouse bone marrow was studied in male BALB/c mice. Tumor GSH was depleted to 28.7 and 7.8% of control by 1 or 2 doses of BSO (5 mmol/kg), respectively, administered ip at 12-hour intervals prior to assay. Tumor GSH could be maintained at a level 8% of control by daily dosing with BSO for 3 days. The same BSO administration schedule lowered bone marrow cell GSH to 31% of control for the 3-day period studied. Tumor growth and bone marrow cell counts were unaffected by all BSO treatments studied. However, BSO pretreatment enhanced the cytotoxicity of CYC to tumor cells, as measured by an in vitro colony-forming assay, but it did not enhance the depletion of bone marrow cells in CYC-treated mice. The magnitude of the enhancement of CYC cytotoxicity by BSO depended on the extent of GSH depletion, tumor size, and the CYC dosing schedule: Single and double doses of BSO enhanced the cytotoxicity of a single dose of CYC by factors of 1.5 and 2.0, respectively, in 7-day-old tumors, based on the dose of CYC required to produce a surviving fraction of 4 X 10(-3). The response of 9-day-old tumors to CYC suggested the presence of a subpopulation of cells that were relatively resistant to CYC. There was no evidence for this subpopulation in BSO-pretreated tumors. Multiple doses of BSO and CYC also combined to give enhanced (1.65-fold) tumor cell killing compared to tumors treated with CYC alone. The data suggest a possible role for BSO as a clinical chemosensitizer, which could be combined with fractionated doses of CYC and may be effective on small cancerous lesions.