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Buthionine sulfoximine (BSO) is a synthetic amino acid that blocks the biosynthesis of reduced glutathione (GSH), an endogenous antioxidant cellular component present in tumor cells. GSH levels have been associated with tumor cell resistance to chemotherapeutic drugs and platinum compounds. Consequently, by depleting GSH, BSO enhances the cytotoxicity of chemotherapeutic agents in drug-resistant tumors. Therefore, the aim of this study was to conduct a systematic review with meta-analysis of preclinical studies utilizing BSO in cancer treatments. The systematic search was carried out using the following databases: PubMed, Web of Science, Scopus, and EMBASE up until March 20, 2023, in order to collect preclinical studies that evaluated BSO, alone or in association, as a strategy for antineoplastic therapy. One hundred nine investigations were found to assess the cytotoxic potential of BSO alone or in combination with other compounds. Twenty-one of these met the criteria for performing the meta-analysis. The evidence gathered indicated that BSO alone exhibits cytotoxic activity. However, this compound is generally used in combination with other antineoplastic strategies, mainly chemotherapy ones, to improve cytotoxicity to carcinogenic cells and treatment efficacy. Finally, this review provides important considerations regarding BSO use in cancer treatment conditions, which might optimize future studies as a potential adjuvant antineoplastic therapeutic tool.

Cancer theranostics combines therapeutic and diagnostic capabilities into a single system to treat cancer efficiently. Biocompatible nanomaterials can be engineered to exhibit cancer theranostic functions, for instance radiosensitization and photoluminescence. In this study, trivalent Bi and Eu ions were co-substituted into the lattice of hydroxyapatite (Bi(III):Eu(III) HAp) to develop a cancer theranostic nanocrystal. Bi provides radiosensitization capabilities while Eu imparts photoluminescence properties. To complement the radiotherapeutic function, l-buthionine sulfoximine (l-BSO) was adsorbed onto the nanocrystal surface. l-BSO inhibits the biosynthesis of cellular antioxidants, which can enhance radiosensitization effects. The Bi(III):Eu(III) HAp nanocrystals were prepared via a hydrothermal method. Structural and compositional analyses showed that both Bi and Eu ions were substituted into the HAp lattice. l-BSO was adsorbed onto the surface via electrostatic interactions between the charged carboxyl and amino groups of l-BSO and the surface ions of the nanocrystals. The adsorption followed the Langmuir isotherm model, implying a homogeneous monolayer adsorption. The l-BSO adsorbed Bi(III):Eu(III) HAp nanocrystals were found to have negligible cytotoxicity except the setting with l-BSO adsorbed amounts of 0.44 μmol/m 2 . This l-BSO amount was found to be high enough to elicit cytotoxicity due to l-BSO being released and causing excessive antioxidant depletion. Gamma ray irradiation clearly activated the cytotoxicity of the samples and increased the cell death rate, confirming radiosensitization abilities. At a constant amount of nanocrystals, the cell death rate increases with l-BSO concentration. This indicates that l-BSO can enhance the radiosensitization effect of the Bi(III):Eu(III) HAp nanocrystals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In previous studies methionine sulfoximine (MSO) significantly extended the lifespan of the SOD1 G93A mouse model for ALS. Those studies used commercially available MSO, which is a racemic mixture of the LS and LR diastereomers, leaving unanswered the question of which isomer was responsible for the therapeutic effects. In this study we tested both purified isomers and showed that the LS isomer, a well-characterized inhibitor of glutamine synthetase, extends the lifespan of these mice, but the LR isomer, which has no known activity, does not., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In our current study, we investigated the role of spinal glutamate recycling in the development of orofacial inflammatory pain. DL-threo-β-benzyloxyaspartate (TBOA) or methionine sulfoximine (MSO) was administered intracisternally to block spinal glutamate transporter and glutamine synthetase activity in astroglia. Intracisternal administration of high dose TBOA (10 μg) produced thermal hyperalgesia in naïve rats but significantly attenuated the thermal hyperalgesia in rats that had been pretreated with interleukin (IL)-1β or Complete Freund's Adjuvant (CFA). In contrast, intracisternal injection of MSO produced anti-hyperalgesic effects against thermal stimuli in CFA-treated rats only. To confirm the paradoxical antinociceptive effects of TBOA and MSO, we examined changes in c-Fos expression in the medullary dorsal horn produced by thermal stimulation in naïve, IL-1β-, or CFA-treated rats, after intracisternal injections of TBOA and MSO. Intracisternal administration of TBOA significantly increased c-Fos immunoreactivity in naïve rats. In contrast, intracisternal administration of TBOA significantly decreased the up-regulation of c-Fos immunoreactivity in the medullary dorsal horn of IL-1β- and CFA-treated rats. However, intracisternal injection of MSO blocked the up-regulation of c-Fos immunoreactivity in CFA-treated rats only. We also investigated the effects of botulinum toxin type A (BoNT-A) on TBOA-induced paradoxical antinociception in CFA-treated rats, as BoNT-A inhibits the release of neurotransmitters, including glutamate. BoNT-A treatment reversed behavioral responses produced by intracisternal administration of TBOA in CFA-treated rats. These results suggest that the paradoxical responses produced by blocking glutamate transporters under inflammatory pain conditions are mediated by the modulation of glutamate release from presynaptic terminals. Moreover, blockade of glutamate reuptake could represent a new therapeutic target for the treatment of chronic inflammatory pain conditions., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: A subset of human hepatocellular carcinomas (HCC) exhibit mutations of β-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO)., Methods: Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1., Results: Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro., Conclusions: The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.

Glutamine synthetase (GS) is highly active in astrocytes, and these cells are physiologically and morphologically compromised by hyperammonemia. Hyperammonemia in end-stage acute liver failure (ALF) is often associated with cerebral edema and astrocyte pathology/swelling. Many studies of animal models of hyperammonemia, and, more recently, nuclear magnetic resonance studies of liver disease patients, have shown that cerebral glutamine is elevated in hyperammonemia, contributing to the edema and encephalopathy. The GS inhibitor L-methionine-S,R-sulfoximine (MSO) is protective in animal models against acute ammonia intoxication. MSO is also an inhibitor of glutamate cysteine ligase, is converted to metabolic products, and causes convulsions at high doses. However, the susceptibility to MSO-induced convulsions is species dependent, with primates being relatively resistant. Moreover, it is possible to chronically maintain cerebral GS activity in mice at low levels by MSO treatment without any obvious untoward effects. Furthermore, MSO is protective in a mouse model of ALF. Extreme caution would be needed in administering MSO to patients. Nevertheless, inhibition of brain GS by MSO (or other GS inhibitors) may have therapeutic benefit in ALF.

Objective: To investigate the role of sex and the role of ammonia and amino acid metabolism, specifically the activity of glutamine synthetase, in survival and disease progression in amyotrophic lateral sclerosis., Methods: We tested treatment with methionine sulfoximine (MSO) on the lifespan and neuromuscular ability of male and female SOD1 mice as measured by their ability to maintain their grip on an inverted wire grid. We also tested the effects of castration and ovariectomization on those measurements., Results: MSO treatment improves the survival of both male and female mice, but the effects are significantly greater on female mice. Saline-treated (control) female mice have delayed neuromuscular degeneration compared with saline-treated male mice, and MSO further delays disease progression in females, to a greater extent than in males. Ovariectomization or castration completely eliminates the effect of the drug on either survival or neuromuscular deterioration., Conclusions: Sex is an important factor in disease progression and the response of SOD1 mice to a drug targeting a central enzyme in nitrogen metabolism, with female sex hormones playing a greater role than male sex hormones. Glutamine synthetase, or its reactants and products, therefore plays a role in this disease, and the sex specificity of treatments aimed at this or other metabolic targets may therefore be an important factor in the development of therapies to treat amyotrophic lateral sclerosis., (Copyright © 2012 Elsevier HS Journals, Inc. All rights reserved.)

In an effort to alter the levels of neurochemicals involved in excitotoxicity, we treated mice with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. Since glutamate toxicity has been proposed as a mechanism for the degeneration of motor neurons in a variety of neurodegenerative diseases, we tested the effects of MSO on the transgenic mouse that overexpresses the mutant human SOD1(G93A) gene, an animal model for the primary inherited form of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS). This treatment in vivo reduced glutamine synthetase activity measured in vitro by 85%. Proton magnetic resonance spectroscopy, with magic angle spinning of intact samples of brain tissue, showed that MSO treatment reduced brain levels of glutamine by 60% and of glutamate by 30% in both the motor cortex and the anterior striatum, while also affecting levels of GABA and glutathione. Kaplan-Meyer survival analysis revealed that MSO treatment significantly extended the lifespan of these mice by 8% (p<0.01). These results show that in the SOD1(G93A) model of neurodegenerative diseases, the concentration of brain glutamate (determined with (1)H-MRS) can be lowered by inhibiting in vivo the synthesis of glutamine with non-toxic doses of MSO.

Tuberculosis remains one of humankind's greatest killers, and new therapeutic strategies are needed to combat the causative agent, Mycobacterium tuberculosis, which is rapidly developing resistance to conventional antibiotics. Using the highly demanding guinea pig model of pulmonary tuberculosis, we have investigated the feasibility of inhibiting M. tuberculosis glutamine synthetase (GS), an enzyme that plays a key role in both nitrogen metabolism and cell wall biosynthesis, as a novel antibiotic strategy. In guinea pigs challenged by aerosol with the highly virulent Erdman strain of M. tuberculosis, the GS inhibitor L-methionine-SR-sulfoximine (MSO) protected the animals against weight loss, a hallmark of tuberculosis, and against the growth of M. tuberculosis in the lungs and spleen; MSO reduced the CFU of M. tuberculosis at 10 weeks after challenge by approximately 0.7 log unit compared with that in control animals. MSO acted synergistically with isoniazid in protecting animals against weight loss and bacterial growth, reducing the CFU in the lungs and spleen by approximately 1.5 log units below the level seen with isoniazid alone. In the presence of ascorbate, which allows treatment with a higher dose, MSO was highly efficacious, reducing the CFU in the lungs and spleen by 2.5 log units compared with that in control animals. This study demonstrates that inhibition of M. tuberculosis GS is a feasible therapeutic strategy against this pathogen and supports the concept that M. tuberculosis enzymes involved in cell wall biosynthesis, including major secretory proteins, have potential as antibiotic targets.

Background: Although surgical resection remains the mainstay of treatment for metastatic pulmonary sarcoma, 5-year survival approaches only 25%. Chemotherapy has been limited by tumor resistance and systemic toxicity. We assessed the efficacy of L-buthionine-SR-sulfoximine, an inhibitor of glutathione synthesis, as a sensitizer for isolated lung perfusion., Methods: In experiment 1, sarcoma-bearing rats (n = 20) received either buthionine sulfoximine via intraperitoneal injection or Hespan. After the last injection, tumor glutathione levels were measured. In experiment 2, rats (n = 60) were injected with sarcoma intravenously. On day 6, animals were pretreated with either buthionine sulfoximine or Hespan intraperitoneally. On day 7, rats underwent isolated lung perfusion (Hespan or doxorubicin) or intravenous therapy (Hespan or doxorubicin). On day 14, tumor nodules were counted., Results: Buthionine sulfoximine effectively depleted tumor glutathione. Animals treated with intravenous therapy had no response to therapy, whereas those animals treated with doxorubicin isolated lung perfusion alone had a limited response. Buthionine-sulfoximine pretreatment in combination with doxorubicin isolated lung perfusion led to a 13-fold reduction in tumor nodules and 5 complete responses., Conclusions: Buthionine-sulfoximine pretreatment in combination with doxorubicin isolated lung perfusion is superior to intravenous doxorubicin and doxorubicin isolated lung perfusion alone for the treatment of metastatic pulmonary sarcoma.

Free radical-mediated esophagitis was studied during duodenogastroesophageal reflux (mixed reflux) or acid reflux in rats. The influence of reflux on esophageal glutathione levels was also examined. Mixed reflux caused more gross mucosal injury than acid reflux. Gross mucosal injury occurred in the mid-esophagus. Total glutathione (GSH) in the esophageal mucosa of control rats was highest in the distal esophagus. The time course of esophageal GSH in rats treated by mixed reflux showed a significant decrease 4 hr after initiation of reflux, followed by a significant increase from the 12th hour on. Mucosal GSH was increased in both reflux groups after 24 hr but significantly more so in the mixed than in the acid reflux group. The free radical scavenger superoxide dismutase (SOD) prevented esophagitis and was associated with decreased GSH levels. GSH depletion by buthionine sulfoximine (BSO) prevented esophagitis and stimulated SOD production in the esophageal mucosa. It is concluded that gastroesophageal reflux is associated with oxidative stress in the esophageal mucosa. The lower GSH levels in the mid-esophagus may predispose to damage in this area. Duodenogastroesophageal reflux causes more damage than pure acid reflux. Oxidative stress leads to GSH depletion of the esophageal mucosa in the first few hours following damage but then stimulates GSH production. GSH depletion by BSO does not worsen esophagitis since it increases the esophageal SOD concentration.

Eukaryotic cells have evolved several mechanisms to protect cellular constituents, especially DNA, from highly reactive molecules entering from without. The greater affinity of electrophiles for thiol groups than for hydroxyl or amine groups provides a teleologic rationale that the availability of high concentrations of thiol could be protective of these other important entities. The major intracellular nonprotein thiol is the tripeptide glutathione.

ATP-induced tumour growth inhibition is accompanied by a selective decrease in the content of the tripeptide glutathione (GSH) within the cancer cells in vivo. Depletion of cellular GSH sensitizes tumours to chemotherapy and radiation, but the usefulness of this depletion depends on whether the levels of GSH can be reduced in the tumour relative to normal tissues. We report here that administration of ATP in combination with diethylmaleate and X-rays leads to complete regression of 95% of Ehrlich ascites tumours in mice. This shows that an aggressive tumour can be eliminated by using a therapy based on modulation of GSH levels in cancer cells.

In an attempt to mimic clinical conditions for the treatment of leukaemia, the HL60 promyelocytic cell line was treated for 18 h with low, clinically relevant, levels of the anthracycline epirubicin and the Vinca alkaloid vinblastine. The resulting drug-resistant sublines not only expressed P-glycoprotein and the MDR phenotype but were also cross-resistant to chlorambucil, methotrexate and cisplatinum, and had increased resistance to radiation. Development of resistance was associated with an aberrant differentiation phenotype with decreased expression of myeloid antigens and expression of glycophorin A, an antigen normally associated with erythroid differentiation. The ability of HL60 cells to terminally differentiate in response to all-trans-retinoic acid (vitamin A acid) was lost in the sublines. These results suggest that either a single novel mechanism is responsible for multiple drug resistance or the initial response to drug treatment is the co-induction of multiple mechanisms. These cells and the method by which they were generated therefore provide a clinically relevant model for the study of the initial events in the development of not only multidrug resistance but also the extended multiple drug resistance usually encountered in the treatment of leukaemia.

Purpose: Glutathione (GSH) was found to occur in relatively high concentrations in melanoma cells. The purpose of this study was to test the possible cytotoxic effects of an artificial decrease of the elevated GSH level., Methods and Materials: The tests were made in vitro and in vivo. In the former case, a total of 11 rodent and human cell lines were studied of which seven were derived from melanomas. After treatment with buthionine sulfoximine (BSO), the decrease of GSH content of the cells and their clonogenic survival was determined. In the in vivo system, single cell suspensions of a subline of the B16 mouse melanoma were injected intravenously into immunocompetent and preirradiated recipients which were subsequently treated with BSO intraperitoneally. Survival time, formation of lung colonies and the weight of metastatic tumor mass in the lungs were the criteria of the BSO effect on the tumor cells., Results: The decrease of the GSH level by BSO was associated with impaired clonogenic survival of the melanoma cells in vitro. Nonmelanoma cells were less affected. BSO treatment of mice inoculated intravenously with melanoma cells resulted in prolonged survival of the animals and impaired metastatic spread of the tumor cells., Conclusion: Melanoma cells are particularly sensitive to disturbance of GSH metabolism by treatment with BSO. In view of this selective cytotoxicity of BSO, treatment with this substance may afford a promising therapeutic potential for melanoma.

Phenytoin teratogenicity may result from embryonic, peroxidase-catalyzed bioactivation of phenytoin to a toxic reactive free radical intermediate for which embryonic glutathione (GSH) is cytoprotective. This hypothesis was tested in embryo culture using 5,8,11,14-eicosatetraynoic acid (ETYA), a dual inhibitor of two peroxidase systems, prostaglandin synthetase, and lipoxygenases. Embryos from CD-1 mice were explanted on Gestational Day 9.5 (vaginal plug, Day 1) and incubated for 24 hr at 37 degrees C in culture medium (35% male rat serum, 15% fetal bovine serum, and 50% Waymouth's medium) saturated with 5% CO2 in air. Initially, a nonembryotoxic concentration of ETYA (0,40,80, or 100 microM) was established within its peroxidase inhibitory range (Ki = 4-8 microM). Subsequently, embryos were incubated with vehicle alone, a therapeutic concentration of phenytoin alone (20 micrograms/ml or 80 microM), ETYA alone (40 microM), or phenytoin and ETYA combined. ETYA alone below 100 microM had no effect on yolk sac diameter (YSD), crown-rump length (CRL), somite development (SD), anterior neuropore closure (ANPC), or turning, but at 100 microM reduced CRL, YSD, and SD (p < or = 0.05). Phenytoin alone was embryotoxic, causing reduced CRL, YSD, and SD (p < or = 0.0001). Phenytoin and ETYA (40 microM) together resulted in an increase in YSD, SD, and CRL relative to those with phenytoin alone (p < or = 0.01), indicating that inhibition by ETYA of embryonic, peroxidase-catalyzed bioactivation of phenytoin is cytoprotective. GSH may play a critical role in detoxifying a phenytoin free radical or subsequent activated oxygen species, thereby reducing covalent binding, lipid peroxidation, and oxidative stress that may initiate embryotoxicity or death. To test this hypothesis, embryos were cultured in the presence or absence of 1 mM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, for 3 hr, at which time BSO was washed out and the embryos were incubated for 24 hr in fresh culture medium containing 80 microM phenytoin or its vehicle. Soluble thiols, including GSH, and disulfides, including oxidized GSH (GSSG), were measured using high-performance liquid chromatography. Immediately after BSO treatment, there were no differences in the concentrations of GSH or GSSG between BSO-exposed embryos and controls. However, at 24 hr, GSH concentrations in untreated embryos increased almost 17-fold over those at 3 hr concentrations, while GSH in BSO-exposed embryos were reduced to 15% of control values (p = 0.0008).(ABSTRACT TRUNCATED AT 400 WORDS)

Purpose: A phase I dose-escalation trial of intravenous (IV) L-buthionine-SR-sulfoximine (BSO) with melphalan (L-PAM) was performed to determine the toxicity and biologic activity of BSO, administered as a short infusion every 12 hours, and the combination of BSO plus L-PAM., Patients and Methods: Twenty-eight patients with refractory malignancies received 30-minute infusions of BSO every 12 hours for 6 to 10 doses in week 1 followed in week 2 by either IV L-PAM (15 mg/m2) alone or BSO as in week 1 with L-PAM. Patients received the combination in week 5 (course no. 2) if they received L-PAM alone during week 2 and vice versa. BSO doses ranged from 1.5 g/m2 to 13.104 g/m2., Results: The only toxicity observed with BSO infusions was occasional nausea/vomiting. Evaluation of 23 paired courses (L-PAM plus BSO v L-PAM) showed significantly (P < .001) greater leukopenia and thrombocytopenia with L-PAM plus BSO. No other significant toxicity was noted. Measurement of intracellular glutathione (GSH) levels in peripheral mononuclear cells (PBLs) of all patients receiving BSO showed a consistent, non-dose-dependent, linear decrease in GSH with repeated BSO doses. Maximal GSH depletion (40% of baseline values, absolute values 200 to 250 ng/10(6) PBLs) was noted after the sixth BSO dose; extended BSO dosing schedules beyond six total BSO doses did not further deplete GSH. Evaluation of gamma-glutamylcysteine synthetase (GCS) activity showed marked inhibition near the end of each infusion with near complete recovery of GCS activity before the next BSO dose. The pattern of GCS inhibition mirrored the plasma BSO concentrations with peak values (level 6, 4 to 8 mmol/L L,R+L,S BSO) observed at the end of the infusion with a rapid decrease in plasma concentrations with an estimated half-life (t1/2) of less than 2 hours. Differential elimination of the R+S stereoisomers was observed. Analysis of L-PAM pharmacokinetics showed marked interpatient variability and a significant decrease in total-body clearance (P = .01) and volume of distribution (P = .03) in courses with L-PAM plus BSO as compared with L-PAM alone., Conclusion: This study shows that BSO alone and in combination with L-PAM can be safely given to patients, but that a schedule of short infusions every 12 hours does not result in GSH depletion less than 30% of baseline values.

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.

Buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis, showed variable growth-inhibitory activity in different tumor cell lines with a high degree of inhibitory activity against melanoma-derived cell lines. A correlation between BSO growth-inhibitory effects and cellular glutathione peroxidase activity was observed. In contrast, no correlation was demonstrated between the response to BSO and cellular tyrosinase, gamma-glutamylcysteine synthetase, glutathione transferase, gamma-glutamyl transpeptidase, or glutathione reductase activities. BSO enhanced 3,4-dihydroxybenzylamine (3,4-DHBA) (fourfold) and melphalan (threefold) in vitro cytotoxic activity as determined by inhibition of DNA synthesis in human melanoma cells and this enhancement was dependent on the duration of exposure to drug. BSO demonstrated in vivo antitumor activity in B16 melanoma-bearing mice prolonging survival by 29% and in combination with 3,4-DHBA resulted in a slight (48% versus 38%) increase in life span as compared to 3,4-DHBA alone. The combination of BSO and melphalan, however, increased the life span of B16 melanoma-bearing mice by 170%, as compared to melphalan alone (80%). These studies demonstrate a unique in vivo antimelanoma activity of BSO.

Dietary copper deficiency causes hypercholesterolemia and increased hepatic 3-hydroxy-3-methyl-glutaryl coenzyme A (MHG-CoA) reductase activity and increased hepatic glutathione (GSH) in rats. We hypothesized that inhibition of GSH production by L-buthionine sulfoximine (BSO), a specific GSH synthesis inhibitor, would abolish the cholesterolemia and increased HMG-CoA reductase activity of copper deficiency. In two experiments, two groups of 20 weanling male rats were fed diets providing 0.4 and 5.8 micrograms Cu/g, copper-deficient (Cu-D) and copper-adequate (Cu-A), respectively. At 35 days plasma cholesterol was significantly elevated by 30 to 43% in Cu-D and 10 animals in each of the Cu-D and Cu-A groups were randomly assigned to receive 10 mM BSO solution in place of drinking water and continued on the same diets for another 2 wk. At necropsy Cu-D animals had a significant 52 to 58% increase in plasma cholesterol. BSO administration abolished the cholesterolemia in Cu-D rats, but had no influence on plasma cholesterol of Cu-A rats. Hepatic GSH was increased 39 to 82% in Cu-D rats and BSO abolished this increase. BSO was without effect on cardiac hypertrophy, plasma and liver copper, and hematocrit indices of copper status. Liver microsome HMG-CoA reductase activity was significantly increased 85 to 288% in Cu-D rats and BSO administration abolished this increase in activity in Cu-D rats. The results suggest that copper deficiency cholesterolemia and elevated HMG-CoA reductase activity are a consequence of elevated hepatic GSH, and provide evidence for GSH regulation of cholesterol metabolism in intact animals.

Investigations with the melphalan-sensitive and -resistant human rhabdomyosarcoma xenografts TE-671 and TE-671 MR were performed to examine the effect of glutathione and polyamine modulation on thermosensitivity. Regimens of intraperitoneally injected and orally administered buthionine sulfoximine were utilized to achieve glutathione depletion to 8.7% and 13% of control levels in TE-671 and TE-671 MR, respectively. Animals treated with L-buthionine-S,R-sulfoximine and 42 degrees C or 43 degrees C hyperthermia for 70 min showed no detectable growth delays beyond those observed for hyperthermia alone. Hyperthermia at 42 degrees C of disaggregated TE-671 and TE-671 MR xenografts following growth in short-term culture was performed following preincubation with buthionine sulfoximine or 0.9% saline. Buthionine sulfoximine-mediated glutathione depletion produced a significant increase in hyperthermia-induced cytotoxicity only with TE-671 MR at 43 degrees C. Polyamine depletion was achieved with a 7-day orally administered course of MDL 72.175DA [(2R,5R)-6-heptyne,5-diamine dihydrochloride], an irreversible inhibitor of ornithine decarboxylase. Although this treatment caused significant depletion of intracellular putrescine and spermidine levels, spermine levels remained relatively unaffected. No significant growth delays were observed in either xenograft line for animals treated with MDL 72.175DA or MDL 72.175DA plus hyperthermia as compared with untreated controls. These results contrast with previous work performed in vitro showing synergism between glutathione or polyamine depletion and hyperthermia, and indicate that further studies are needed.

Biopsy samples and cultured cells derived from them were obtained from 39 patients with malignant glioma and were analyzed for 1) glutathione (GSH) content; 2) sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and/or nitrogen mustard (HN2) treatment and 3) the effect of buthionine sulfoximine (BSO) treatment on BCNU and/or HN2 cytotoxicity. The average GSH concentration of biopsy specimens was lower than those of cultured cells (2.36 +/- 0.44 vs. 11.42 +/- 2.32 nmol/10(6) cells). While some of the tumor specimens were sensitive to either BCNU or HN2, the majority were resistant to both. However, 8 of 23 tumors tested showed enhanced sensitivity to BCNU following treatment with BSO. Five of 17 tumors were similarly sensitized to HN2 by BSO. These results suggest that BSO chemosensitization may be of value for certain patients and that screening assays may help identify treatment-sensitive individuals.

The effect of Cr(VI) on liver, kidney, and lung glutathione (GSH) levels and the effect of GSH depletion on Cr(VI)-induced nephrotoxicity were studied in male Sprague-Dawley rats (150-200 g). GSH levels, measured as nonprotein sulfhydryls, were determined between 0.5 and 26 hr after intraperitoneal injection of the maximum non-toxic dose of sodium dichromate (10 mg/kg). While Cr(VI) at this dose did not significantly change hepatic, renal, or pulmonary GSH levels, there appeared to be an initial decrease of hepatic GSH followed by an increase to approximately 120% of control between 5 and 12.5 hr after Cr(VI) treatment. The increase in hepatic GSH levels was significant 5 hr after treatment with 20 mg/kg sodium dichromate, was manifested as an increase in both non-protein sulfhydryls and total glutathione, and was prevented by L-buthionine sulfoximine (BSO) pretreatment. In rats pretreated with 4.0 mmol/kg BSO to deplete GSH, subsequent treatment with Cr(VI) further reduced hepatic GSH levels 2 hr after Cr(VI) treatment and inhibited weight gain in the first 24 hr after treatment. Intraperitoneal injection of Cr(VI) did not inhibit hepatic glutathione reductase activity, even at toxic doses. Depletion of renal GSH to approximately 25% of control with BSO potentiated the acute nephrotoxicity of 30 mg/kg sodium dichromate as measured by serum urea nitrogen levels and relative kidney weight. However, GSH depletion with BSO did not appear to affect the incidence of glucosuria, haematuria, or lysozymuria over a range of Cr(VI) doses, nor did it affect renal uptake of Cr. Taken together, these data show that GSH protects against the acute nephrotoxicity of Cr(VI), although it is not clear whether GSH is directly involved in the intracellular metabolism of Cr(VI) at non-toxic doses.

Intravenous administration of the fluorescent DNA stain Hoechst 33342 to tumour-bearing mice was used to label cells proportionally to their proximity from the vasculature. Flow cytometry was used to sort cells from the tumour into populations based on their Hoechst 33342-derived fluorescence. The cell populations were then assayed for their glutathione (GSH) content and their radiosensitivity. Tumours from mice pretreated with buthionine sulphoximine (BSO) were compared with untreated animals. The major findings of this study suggest that the cellular GSH concentration within tumours decreases with distance from the vasculature, and that the GSH concentration within cells from all locations in the tumour can be depleted by enzymatically inhibiting its synthesis using BSO. This depletion of GSH resulted in a small degree of hypoxic radiosensitization of cells both distal and proximal to the vasculature.

Laboratory studies have provided a number of interesting leads relating to modulation of antitumour drug resistance. Several ideas have been evaluated clinically and phase I studies with certain newer agents are planned.

An intracranial (i.c.) interstitial radiotherapy model in athymic nude mice bearing i.c. D-54 MG human glioma xenografts was developed, allowing evaluation of the therapeutic benefits seen after L-buthionine-S,R-sulfoximine (L-BSO)-mediated depletion of tumor glutathione levels. Administration of L-BSO [2.5 mmol/kg intraperitoneal injections x 4 doses plus concomitant availability in acidified (pH 3.0) drinking water at a concentration of 20 mM] resulted in depletion of tumor glutathione levels to 0.15 mumol/g wet weight (7.9% of control). The therapeutic activity of i.c. interstitial radiotherapy with an 125I seed was enhanced after L-BSO-mediated glutathione depletion, with increases in median survival of 13.4 to 30.5% over that seen with 125I seeds alone. These studies demonstrate a potential role for BSO in enhancing the therapeutic activity of interstitial radiotherapy.

Two and four treatments of 5 mmol kg-1 of buthionine sulfoximine (BSO) at an interval of 12 h depleted the glutathione (GSH) content in NFSa tumours of C3H/He mice, respectively, to 24.0 and 1.78 percent of the untreated controls. BSO pre-treatments every 12 h enhanced the cytotoxicity of cyclophosphamide (CYC) towards artificial lung micrometastases of NFSa tumours giving enhancement ratios (ERs) ranging from 1.75 to 1.83 and from 2.41 to 2.73, for two and four BSO pretreatments respectively. Large ERs were obtained at low CYC doses (high cell survival). Four BSO pre-treatments at an interval of 12 h did not increase the cytotoxicity of CYC to bone marrow stem cells. Our results suggest a clinical applicability of the combination of BSO and CYC.

We have determined the effect of glutathione (GSH) depletion on the cytotoxicity of three nitrogen mustards, six platinum complexes, and mitomycin C in a human ovarian carcinoma cell line. GSH levels in COLO 316 cells were depleted by exposure of cell monolayers to 0.5 mM D,L-buthionine-S,R-sulfoximine. GSH depletion significantly potentiated the cytotoxicity of L-phenylalanine mustard, chlorambucil, and mechlorethamine as determined by clonogenic assay on plastic plates. The dose modification factors were 2.6, 2.6, and 1.9, respectively. The same level of GSH depletion had a minimal effect on the cytotoxicity of cis-diamminedichloroplatinum(II) (cis-DDP), carboplatin, dichloro(ethylenediamine)platinum(II), 1,2-diaminocyclohexylplatinum(II) malonate, and iproplatin. The dose modification factors of GSH depletion for these drugs were 1.4 or less. trans-Diamminedichloroplatinum(II) was, however, markedly potentiated by GSH depletion with a dose modification factor of 2.7. Mitomycin C was minimally potentiated by GSH depletion. We have also generated cis-DDP-resistant cells from COLO 316 and 2008 human ovarian carcinoma cells by in vitro selection with cis-DDP. These cis-DDP-resistant cells had identical levels of GSH as the parental cells. GSH depletion sensitized these cells only to the same degree as the parental cells and did not reverse the resistant phenotype. Our results indicate that intracellular GSH levels are not an important determinant of the cytotoxicity of cis-platinum(II) or cis-platinum(IV) complexes in COLO 316 and 2008 cells. In addition, altered GSH metabolism does not appear to be a component of the cis-DDP-resistant phenotype in these cells.

The effect of glutathione depletion (GSH) on the efficacy of SR 2508 was evaluated in two murine tumor models with single large doses of radiation or with low doses administered in an accelerated fractionated schedule. To deplete tumor GSH, buthionine sulfoximine (BSO) was administered in the animals drinking water (10 mM) following two i.p. injections of 450 mg/kg. This treatment decreased RIF and MCA tumor GSH concentrations by 95% and 80%, respectively. Mice (C3H/Sed) received BSO for 48-72 hr before the first dose of radiation, and were maintained on BSO drinking water for the duration of the fractionated course of therapy. SR 2508 (200-1000 mg/kg) was injected 45 min prior to each fraction of radiation. Radiation was administered as a single dose of 15 Gy or 20 Gy, for RIF and MCA tumors, respectively. Alternatively, animals received a fractionated course of radiotherapy which consisted of 2.5 Gy/fraction for the RIF, and 3 Gy/fraction for the MCA tumors, b.i.d. for five days (total of 10 fractions). Tumor response with and without BSO, and with and without SR 2508, was determined by regrowth delay. BSO pretreatment increased the efficacy of SR 2508 with single dose radiation in the MCA but not RIF tumor. SR 2508 administered with fractionated radiation produced lower enhancement ratios (SER) than with a single radiation dose. However, BSO significantly enhanced the efficacy of SR 2508 with fractionated radiation. BSO increased the maximum SER for SR 2508 (3 mM/fraction) from 1.2 to 1.4 in the RIF tumor, and from 1.4 to 1.8 in the MCA tumor. BSO also increased the toxicity of SR 2508 by a factor of 2. However, the ability of BSO to increase the efficacy of low doses of sensitizer at clinically relevant doses of radiation suggests that this combined modifier treatment may be of clinical benefit.

The in vivo effects of buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, on the cytotoxicity of cyclophosphamide (CYM), cisplatin (CDDP) and bleomycin (BLM), were examined by monitoring the changes of non-protein thiols (NPSH) in normal tissues and in the NFSa fibrosarcoma. We used the lung colony assay as a measure of tumor response and the spleen colony assay as a measure of normal tissue response to CYM. In this study, 5 mmol/kg of BSO was subcutaneously injected four times every 12 hr before administration of the above anti-neoplastic drugs. GSH levels in subcutaneous NFSa tumors decreased to 2% of the control 12 hr after the last administration of BSO, but in the bone marrow, had recovered to 41%. In the colony assays, BSO increased the anti-cancer effects of the three chemotherapeutic agents, but did not modify the bone marrow suppression by CYM. This finding was a result of the differential response of GSH depletion in the tumor and in the bone marrow. Our study demonstrates that BSO is an effective chemosensitizer of these drugs and may be of therapeutic value when used at an optimal interval.

Depletion of intracellular glutathione (GSH) can enhance misonidazole (MISO) radiosensitizing efficacy both in vivo and in vitro. However, such treatments may also enhance the systemic toxicity in animals. The purpose of the present study was to test various ways of depleting GSH levels in a variety of experimental mouse tumors, to measure the improvement in the efficacy of MISO and its less toxic analog SR 2508 by this depletion, and to determine the effect of daily GSH depletion on the toxicity of MISO and SR 2508. GSH levels were measured daily for 5 days in tumors, livers and brains of mice injected daily with buthionine sulfoximine (BSO), with or without diethylmaleate (DEM). To investigate tumor variability we studied 5 different tumors: EMT-6, RIF-1, KHT, SCC VII, and B16 melanoma. The efficacy of MISO and SR 2508 was evaluated using the KHT and SCC VII tumors either by the regrowth delay assay or by the in vivo/in vitro clonogenic assay. The drug toxicity was evaluated by weight loss and by death. Daily doses of 3 mmole/kg BSO depleted tumor levels of GSH to 20 to 40% of controls by 6 hr after each injection. Injection of DEM (300 mg/kg) 6 hr after BSO further enhanced the depletion. Administration of MISO or SR 2508 at the time of maximum GSH depletion enhanced the MISO efficacy by factors of 2.5 to 8 for depletion to 8% of controls by BSO + DEM, but no enhancement of SR 2508 was seen with tumors at 20% GSH levels achieved with BSO alone in the preliminary experiment. The chronic toxicity of MISO was enhanced not at all or by a factor of up to 2 for BSO and BSO + DEM respectively. Further studies are needed before it can be concluded that GSH depletion by BSO alone may be a useful adjunct to the clinical use of radiosensitizers.

Many different non-hypoxic cell radiosensitizers have been identified over the years. Radiosensitization by these agents is mediated through a variety of mechanisms, including inhibition of repair (both enzymatic and chemical), modification in the DNA molecule, and perturbation and redistribution in the cell cycle. Recent clinical interest in the use of halogenated pyrimidines as radiosensitizers has prompted a number of questions requiring both laboratory and clinical research to maximize this therapeutic approach. Other radiosensitizers that alter cellular redox processes in different ways are discussed in the context of better understanding the cellular biochemical systems that are affected in aerobic radiosensitization. Advancement in the use of non-hypoxic cell sensitizers in radiation cancer treatment will most likely depend on a knowledge of the detailed biochemical mechanisms of these agents and how they might be used to exploit any subtle biochemical differences between tumor and normal tissue.

DL-Buthionine-(S,R)-sulfoximine (BSO), a glutathione-depleting agent, was found to diminish the nephrotoxic effect of cisplatin (cis-diamminedichloroplatinum). Pretreatment of rats with BSO (4 mmol/kg s.c.) 2 h prior to cisplatin, either as a single dose of 5 mg/kg or at a daily dose of 2.5 mg/kg for 3 consecutive days, resulted in diminished elevations of plasma BUN concentration and decreased cisplatin-induced inhibition of renal gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase activity measured 6 days following treatment. Administration of BSO prior to cisplatin at 7.5 mg/kg did not significantly alter the effect of cisplatin on either BUN concentration or enzyme activity. The influence of BSO pretreatment on the antitumor activity of cisplatin was studied using implantation of a murine bladder cancer (MBT-2) in C3H mice. Pretreatment of mice with BSO (5 mmol/kg) did not influence cisplatin antitumor efficacy.

The effect of pretreating the C3H/He mouse MBT-2 tumor with diethyl maleate (DEM), buthionine-S R-sulfoximine (BSO), or misonidazole (MISO) before administration of cyclophosphamide (CTX) was studied with the use of tumor volume-doubling time delay as an endpoint. The kinetics of glutathione (GSH) depletion and regeneration in the tumor and in the host liver were determined after treatment with the thiol-depleting agents. CTX was administered at appropriate time points. MISO was the most effective chemosensitizer at a time point at which tumor GSH content was 80-85% of the control value. Both BSO and DEM were chemosensitizers in relation to the degree they had reduced tumor GSH levels. This chemosensitization was significant at 50% GSH reduction. By combining MISO and BSO at doses lower than previously used for each agent alone, highly effective sensitization of subsequent CTX was obtained.

Levels of glutathione (GSH) in tumour tissue may be important in determining the clinical response to certain anticancer agents. Recent reports have suggested that D,L-buthionine-S,R-sulphoximine (BSO), a specific inhibitor of GSH synthesis, may be used to deplete tumour cell GSH and thus increase the therapeutic ratio of these agents. We have previously shown that 1-naphthol is a potential antitumour agent, and that its possible metabolite 1,4-naphthoquinone is thiol reactive and capable of redox cycling. It was therefore of interest to investigate the effect of pretreatment with BSO, on the toxicity of these agents, to tumour cells. For comparison we included three other cytotoxic agents, melphalan, helenalin and menadione, the toxicities of which are reported to be modulated by intracellular GSH. Depletion of GSH using BSO did not effect the toxicity of 1-naphthol, or 1,4-NQ but did produce slight potentiation of the cytotoxicities of menadione, helanalin and melphalan. The lack of effect of BSO on 1-naphthol and 1,4-NQ is not easily explained but if one also considers the modest potentiation of cytotoxicity+ achieved with the other agents studied, the potential use of BSO in combined chemotherapy is at best rather modest.

The radiosensitizing effects of misonidazole (MISO) in combination with D,L-buthionine-S, R-sulfoximine (BSO), an inhibitor of glutathione (GSH) biosynthesis, were studied in NFSa tumors of C3H/He mice. The radiation response of tumors was assayed by the tumor growth delay time. The GSH contents in tissues were assayed by high performance liquid chromatography (HPLC). GSH content in the tumors decreased to the minimum level (45% of the control), and then gradually recovered to 75% of the control, respectively, 12 and 24 hr after the intraperitoneal injection of 5 mmole/kg BSO. On the other hand, the maximum non-protein sulfhydryl (NPSH) depletion (29% of the control) in the liver of tumor bearing mice was achieved 6 hr after the administration of the same dose of BSO, but fully recovered 24 hr later. When 5 mmole/kg BSO was injected repeatedly 4 times at an interval of 6 hr, GSH content in the tumors decreased to 19% of the control 24 hr after the first injection of BSO. The radiosensitizing effect of 0.5 mmole/kg MISO was markedly increased by this BSO treatment. The enhancement ratio (ER) of this combined treatment was 1.93. On the other hand, ERs of 1.44 and 1.16 were obtained for MISO (0.5 mmole/kg) and for 4 injections of BSO (5 mmole/kg) in combination with radiation, respectively. Although a considerable increase in the radiosensitizing efficiency of MISO in vivo by the treatment with BSO was found without any notable side effects of the combination, more studies on toxicities are needed to get a definite conclusion on the clinical applicability of the combination.

We have tested the tumoricidal potency of enzyme immunotoxins constructed of antibodies conjugated to glucose oxidase and to lactoperoxidase. Murine plasmacytoma cells were targeted in vitro with the use of affinity-purified rabbit anti-plasmacytoma membrane antibodies (conjugated to glucose oxidase or lactoperoxidase) or rabbit serum raised against plasmacytoma microsome membranes followed by goat anti-rabbit immunoglobulin conjugates (to glucose oxidase or lactoperoxidase). Cytotoxicity was generated subsequently by incubation of the washed cells in a medium supplemented with glucose and sodium iodide, which were the substrates of these enzymes. This resulted in the presumed metabolic release of highly toxic reduced oxygen species and iodinated derivatives. Targeting of tumor cells with both conjugates, as opposed to one of them alone, produced a synergistic killing effect. The gain of specific versus unspecific cytotoxicity was upwards of 10,000-fold. The killing rates were elevated (t10 values less than 30 min) and linear over time. The resultant reduction in tumor cell viability was in the order of 5 to 6 logs after only 20 to 90 min of incubation in the glucose/NaI medium. Cytotoxicity was enhanced by the gamma-glutamyl cysteine synthetase inhibitor buthionine-S,R-sulfoximine and by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea, while catalase was inhibitory. The results suggest that these enzyme immunotoxins may be suitable for the ex vivo purging of autologous bone marrow grafts.

We have examined the effect of L-buthionine sulfoximine (BSO) treatment on the subsequent cytotoxicity of mitomycin C (Mit C) and cyclophosphamide (CYC) in EMT6/SF tumors growing in Balb/c mice. GSH content in tumors decreased to a minimum level (less than 30% of control) 12 hr after a single injection of 5 mmole/kg of BSO. The recovery of tumor GSH was protracted and did not reach control levels up to 24 hr following BSO administration. Mit C and CYC were administered 12 hr after BSO injection. Tumor cell survival reached almost the same minimum level (SF = 0.050-0.06%) 1 hour after Mit C (10 mg/kg) treatment with or without BSO. However, 24 hr after Mit C treatment, survival had increased 36-fold (SF = 1.9%) in non-BSO-treated tumors compared to only 4-fold (SF = 0.3%) in the BSO-treated tumors. A dose modifying factor (DMF) of 1.4-1.8 was observed for BSO-pretreated tumors assayed 24 hr after Mit C administration. In CYC-treated tumors, minimum surviving fractions were found 4 hr after CYC treatment with or without BSO treatment. These minimum survival levels were almost the same in BSO-treated tumors following 100 mg/kg CYC (SF = 0.03%) as in control tumors treated with 150 mg/kg CYC (SF = 0.04%). The same increase in cell survival was observed when tumor excision and assay were delayed 24 hr following CYC administration with or without prior BSO treatment. BSO treatment enhanced the cytotoxicity of CYC by DMFs of 1.6-1.9 based on the 24 hr excision assay. Depletion of tumor GSH by BSO caused enhancement of tumor cell killing by the two chemotherapy agents studied, either by increasing cytotoxicity, in the case of CYC or by decreasing PLD recovery following Mit C.

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.