Buthionine sulfoximine and chemoresistance in cancer treatments: a systematic review with meta-analysis of preclinical studies.

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.

Glutamine: a major player in nitrogen catabolite repression in the yeast Dekkera bruxellensis.

In the present work we studied the expression of genes from nitrogen central metabolism in the yeast Dekkera bruxellensis and under regulation by the Nitrogen Catabolite Repression mechanism (NCR). These analyses could shed some light on the biological mechanisms involved in the adaptation and survival of this yeast in the sugarcane fermentation process for ethanol production. Nitrogen sources (N-sources) in the form of ammonium, nitrate, glutamate or glutamine were investigated with or without the addition of methionine sulfoximine, which inhibits the activity of the enzyme glutamine synthetase and releases cells from NCR. The results showed that glutamine might act as an intracellular sensor for nitrogen availability in D. bruxellensis, by activating NCR. Gene expression analyses indicated the existence of two different GATA-dependent NCR pathways, identified as glutamine-dependent and glutamine-independent mechanisms. Moreover, nitrate is sensed as a non-preferential N-source and releases NCR to its higher level. After grouping genes according to their regulation pattern, we showed that genes for ammonium assimilation represent a regulon with almost constitutive expression, while permease encoding genes are mostly affected by the nitrogen sensor mechanism. On the other hand, nitrate assimilation genes constitute a regulon that is primarily subjected to induction by nitrate and, to a lesser extent, to a repressive mechanism by preferential N-sources. This observation explains our previous reports showing that nitrate is co-consumed with ammonium, a trait that enables D. bruxellensis cells to scavenge limiting N-sources in the industrial substrate and, therefore, to compete with Saccharomyces cerevisiae in this environment.

Evidence for astrocytes as a potential source of the glutamate excess in temporal lobe epilepsy.

Increased extracellular brain glutamate has been implicated in the pathophysiology of human refractory temporal lobe epilepsy (TLE), but the cause of the excessive glutamate is unknown. Prior studies by us and others have shown that the glutamate degrading enzyme glutamine synthetase (GS) is deficient in astrocytes in the epileptogenic hippocampal formation in a subset of patients with TLE. We have postulated that the loss of GS in TLE leads to increased glutamate in astrocytes with elevated concentrations of extracellular glutamate and recurrent seizures as the ultimate end-points. Here we test the hypothesis that the deficiency in GS leads to increased glutamate in astrocytes. Rats were chronically infused with methionine sulfoximine (MSO, n=4) into the hippocampal formation to induce GS deficiency and recurrent seizures. A separate group of rats was infused with 0.9% NaCl (saline) as a control (n=6). At least 10days after the start of infusion, once recurrent seizures were established in the MSO-treated rats, the concentration of glutamate was assessed in CA1 of the hippocampal formation by immunogold electron microscopy. The concentration of glutamate was 47% higher in astrocytes in the MSO-treated vs. saline-treated rats (p=0.02), and the ratio of glutamate in astrocytes relative to axon terminals was increased by 74% in the MSO-treated rats (p=0.003). These data support our hypothesis that a deficiency in GS leads to increased glutamate in astrocytes. We additionally propose that the GS-deficient astrocytes in the hippocampal formation in TLE lead to elevated extracellular brain glutamate either through decreased clearance of extracellular glutamate or excessive release of glutamate into the extracellular space from these cells, or a combination of the two.

Effect of combined treatment of thioperamide with some antiepileptic drugs on methionine-sulfoximine induced convulsions in mice.

Methionine-sulfoximine (MSO), a convulsant is known to increase the activity of histamine N-methyl transferase. The effect of a selective H3 receptor agonist R- (alpha) methylhistamine (RAMH) and antagonist (thioperamide, THP) and some antiepileptic drugs (gabapentin and sodium valproate) have been evaluated on MSO-induced convulsions in mice. The effect of THP was also evaluated in combination with these antiepileptic drugs. Sodium valproate (300 mg/kg, po) and gabapentin (400 mg/kg, po) offered protection against MSO-induced convulsions as evidenced by a significant prolongation of latency to abnormal dorsoflexion and complete protection against mortality within 6 h of administration. THP (15 mg/kg, ip) alone and in combination with sub-effective doses of gabapentin (75 mg/kg, po) and sodium valproate (75 mg/kg, po) revealed no significant differences from the control group or either drug alone. Hence, the convulsant action of MSO does not appear to be mediated via histaminergic mechanisms.

Poisoning by Cnestis ferruginea in Casamance (Senegal): an etiological approach.

Since several years, in the area of Kabrousse in Casamance (Senegal), a neurotoxic syndrome has caused more than 50 human deaths. Field studies showed that epidemic could be due to consumption of leave decoction of Cnestis ferruginea, a tropical plant belonging to the Connaraceae family. An ethnobotanical study has been conducted in order to investigate the traditional uses of C. ferruginea, and describe the circumstances and the symptoms of this plant poisoning. As a first experimental approach, the leave decoction was tested for its ability to induce cytotoxic effects using the XTT method. A phytochemical approach revealed the presence of methionine sulfoximine (MSX), a neurotoxic amino acid, in the plant extract by gas chromatography-mass spectrometry (GC-MS). The description of this poisoning, the cytotoxic activity of the decoction and the occurence of MSX in leaves of C. ferruginea constituted the first etiological data on this poisoning.

Progressive neuronal activation accompanies epileptogenesis caused by hippocampal glutamine synthetase inhibition.

Loss of glutamine synthetase (GS) in hippocampal astrocytes has been implicated in the causation of human mesial temporal lobe epilepsy (MTLE). However, the mechanism by which the deficiency in GS leads to epilepsy is incompletely understood. Here we ask how hippocampal GS inhibition affects seizure phenotype and neuronal activation during epilepsy development (epileptogenesis). Epileptogenesis was induced by infusing the irreversible GS blocker methionine sulfoximine (MSO) unilaterally into the hippocampal formation of rats. We then used continuous video-intracranial electroencephalogram (EEG) monitoring and c-Fos immunohistochemistry to determine the type of seizures and spatial distribution of neuronal activation early (1-5days postinfusion) and late (16-43days postinfusion) in epileptogenesis. Early in epileptogenesis, seizures were preferentially mild (stage 1-2), activating neurons in the entorhinal-hippocampal area, the basolateral amygdala, the piriform cortex, the midline thalamus, and the anterior olfactory area. Late in epileptogenesis, the seizures were generally more severe (stages 4-5) with neuronal activation extending to the neocortex, the bed nucleus of the stria terminalis, the mediodorsal thalamu\s, and the central nucleus of the amygdala. Our findings demonstrate that inhibition of GS focally in the hippocampal formation triggers a process of epileptogenesis characterized by gradual worsening of seizure severity and involvement of progressively larger neuronal populations over a period of several weeks. Knowledge about the underlying mechanism of epileptogenesis is important because such knowledge may result in more specific and efficacious treatments of MTLE by moving away from large and poorly specific surgical resections to highly targeted surgical or pharmacological interventions of the epileptogenic process.

Effect of DL-buthionine-S,R-sulfoximine on the growth of EMT6 and RIF mouse tumors.

The response of murine EMT6 and RIF tumors to DL-buthionine-S, R-sulfoximine (BSO), a glutathione (GSH) depletor, given either as a single dose, continuous oral administration, or in multiple doses, was determined with the use of a tumor-growth-delay assay. BSO consistently caused significant tumor growth delay in EMT6 tumors, which reached 5-7 days (two doubling times) after a single BSO dose (40 mumol/kg or 4 mmol/kg), 3 days (one doubling time) with continuous oral administration (20 mM), and 9 days (three doubling times) with daily BSO administrations (4 mmol/kg) starting at the time of tumor inoculation. Growth inhibition persisted after discontinuation of BSO treatment. Some complete tumor regressions were observed. Only slight tumor growth delay (one doubling time) was observed in RIF tumors at all treatment modes. No direct correlation was observed between tumor GSH content and the effects on tumor growth. In vitro BSO pretreatment (2 mM, 24 hr) of EMT6 tumor cells prior to tumor inoculation, which reduced cellular GSH levels to 34% of controls, did not influence subsequent tumor growth. Pretreatment of mice with BSO (4 mmol/kg, 1 daily sc injection for 7 days) prior to tumor inoculation led to a reduction of tumor takes by 25% when compared to 100% tumor takes in untreated mice. These data imply a BSO-induced change in the host response to tumor development.

Effects of L-phenylalanine mustard and L-buthionine sulfoximine on murine and human hematopoietic progenitor cells in vitro.

The effects of L-buthionine sulfoximine (L-BSO) and L-phenylalanine mustard (L-PAM), alone and in combination, on human and murine marrow were explored using in vitro clonogenic assays to establish whether enhanced myelotoxicity might limit the clinical utility of this potent chemotherapeutic combination. One-h exposure to L-PAM produced significant concentration-dependent colony inhibition, with 70% inhibitory concentration (IC70) values ranging from 4.5 to 7.2 microM for all hematopoietic progenitors assayed. The combination of L-PAM plus 4500 microM L-BSO for 1 h did not effectively alter the IC70 values derived for L-PAM alone. In studies where marrow cells were pretreated with L-BSO for 4 h and then L-PAM for 1 additional h, the IC70 values were decreased in both murine and human marrow progenitors compared to the L-PAM control, suggesting modest potentiation of myelotoxicity. The potentiation is not so significant as to preclude human studies with this combination. One- to 5-h exposure of marrow cells from both species to 4500 microM L-BSO was only mildly myelotoxic, producing colony reductions of 22-49%. However, continuous exposure to L-BSO produced concentration-dependent colony inhibition, with IC70 values of 70, 84, and 43 microM for murine colony-forming units-granulocyte/macrophage, blast-forming units-erythroid, and colony-forming unit-erythroid, respectively.

Chemosensitization of L-phenylalanine mustard by the thiol-modulating agent buthionine sulfoximine.

Glutathione (GSH) plays a crucial role in the protection of normal and tumor tissue against the toxic effects of numerous chemotherapeutic drugs. Therefore, the possible therapeutic benefit of thiol depletion in cancer treatment is dependent upon the relative degree to which tumor or normal tissue is sensitized to the toxic effects of subsequent chemotherapy. To address this issue, the following studies on the chemosensitization of melphalan (L-PAM) by the thiol-depleting agent buthionine sulfoximine (BSO) were conducted in vivo in BDF mice inoculated with L-PAM-resistant murine L1210 leukemia. Different dosing regimens of BSO were found to potentiate L-PAM toxicity in a manner that depended upon the degree of GSH depletion. Multiple i.p. injections of BSO (450 mg/kg every 6 h X 5) were found to reduce GSH concentrations in most tissues by 70-80%, and to decrease the LD50 for L-PAM from 22 to 14 mg/kg. No two organs were found to behave entirely the same with respect to the rate of depletion or recovery of GSH, or to the maximum depletion that could be obtained by BSO. In this regard, the bone marrow was found to be the most resistant tissue to thiol depletion by BSO and was found to tolerate the combination of BSO and therapeutic doses of L-PAM. However, BSO pretreatment markedly inhibited the recovery of the peripheral WBC population at the LD10 dose of L-PAM. Differences also were found in the in vivo metabolism of GSH by L-PAM-sensitive and -resistant murine L1210 leukemia cells. The intracellular concentration of GSH in the resistant cell line was 1.6-fold higher than in the sensitive tumor. Moreover, GSH levels were depleted more rapidly in the resistant tumor relative to the sensitive cell line. A single injection of BSO decreased GSH concentrations in both tumors to equivalent levels (20 nmol/10(7) cells) within 24 h. However, multiple i.p. injections of BSO failed to produce a significant increase in the life-span of L-PAM-treated animals despite a 90% reduction in tumor GSH concentrations (5.5 nmol/10(7) cells). In contrast to the median day survival data, BSO was found to enhance the antitumor activity of L-PAM as determined by an in vivo/in vitro clonogenic assay or by in vivo thymidine incorporation. Using decreased thymidine incorporation as an index of antitumor activity, BSO was found to increase the therapeutic index (LD10/ED50) of L-PAM from 3.6 to 6.5.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetics of buthionine sulfoximine (NSC 326231) and its effect on melphalan-induced toxicity in mice.

Intravenous doses of buthionine sulfoximine (BSO, NSC 326231), an inhibitor of glutathione synthesis, were eliminated rapidly from mouse plasma in a biexponential manner. The initial phase of the plasma concentration versus time curve had a half-life of 4.9 min and accounted for 94% of the total area under the curve. The half-life of the terminal phase of the curve was 36.7 min and the area accounted for only 6% of the total area under the curve. Plasma clearance of BSO was 28.1 ml/min/kg and the steady state volume of distribution was 280 ml/kg. The oral bioavailability of BSO, based on plasma BSO levels, was extremely low. However, comparable glutathione depletion was apparent after i.v. and p.o. doses of BSO, suggesting a rapid tissue uptake and/or metabolism of BSO. Therefore, due to the rapid elimination of BSO from mouse plasma, plasma drug levels do not directly correlate with BSO-induced tissue glutathione depletion. Administration of multiple i.v. doses of BSO to male and female mice resulted in a marked 88% depletion of liver glutathione at doses of 400-1600 mg/kg/dose. Toxicity of i.v. administered BSO was limited to a transient depression of peripheral WBC levels in female mice given six doses of 1600 mg/kg. Multiple i.v. doses of BSO of up to 800 mg/kg/dose (every 4 h for a total of six doses) did not alter the toxicity of i.v. administered melphalan. However, multiple doses of 1600 mg/kg/dose of BSO did potentiate histopathological evidence of melphalan-induced bone marrow toxicity in 30% of the mice and, additionally, the combination of BSO and melphalan produced renal tubular necrosis in 80% of the male mice. The potentiation of melphalan induced toxicity did not appear to be related to GSH depletion, since: quantitatively similar amount of GSH depletion occurred at lower dose of BSO without any increase in melphalan toxicity.

Potentiation of melphalan cytotoxicity in human ovarian cancer cell lines by glutathione depletion.

The effectiveness of alkylating agents in the treatment of ovarian cancer is limited by the frequent development of drug resistance. In order to examine the mechanisms of resistance and potential ways in which this resistance could be overcome, we have developed a human ovarian cancer cell line, 1847ME, resistant to the bifunctional amino acid nitrogen mustard, melphalan. A 4-fold higher concentration of melphalan was required to produce an equivalent reduction in tumor colony formation in 1847ME cells as compared to the parent melphalan-sensitive line A1847. The magnitude of resistance in 1847ME was similar to that observed in the cell lines NIH:OVCAR-2, NIH:OVCAR-3, and NIH:OVCAR-4 which were derived from ovarian cancer patients clinically resistant to alkylating agents. There was no detectable difference in melphalan uptake between A1847 and 1847ME. The cellular content of the inactive dihydroxy melphalan metabolite, however, was two times greater in 1847ME compared to A1847. Levels of the principal intracellular thiol, glutathione, were found to be 2-fold greater in 1847ME than in A1847, and to be similarly elevated in the OVCAR lines. Depletion of glutathione by incubation of the cells in cystine-free medium or in the presence of the specific inhibitor of glutathione synthesis, DL-buthionine-S,R-sulfoximine, was accompanied by a marked increase in melphalan cytotoxicity. Doses of DL-buthionine-S,R-sulfoximine which were only minimally cytotoxic were associated with complete reversal of the induced resistance to melphalan in 1847ME. Synergism between melphalan and DL-buthionine-S,R-sulfoximine was also demonstrated in the OVCAR cell lines derived from previously treated ovarian cancer patients. The reversal of induced resistance to melphalan by modulation of glutathione levels is of potential clinical relevance. In addition, these cell lines provide a useful model system in which to study further the mechanisms of alkylating agent resistance in human tumors.

A procedure for selecting mammalian cells with an impairment in oxidative phosphorylation.

The mitochondrial encephalomyopathies in man are characterized by heterogeneous defects leading to an impairment in the pathway of aerobic energy production. As a means of investigating the molecular and genetic mechanisms underlying these disorders we have developed a procedure for selecting mammalian cell lines with features resembling the human pathological phenotypes. The principle of the selection is the use of a fluorescent amphiphilic dye, 2,4-(dimethylamino)-1-styrylmethylpyridiniumiodine, a cation showing two main features. Firstly, it is accumulated by mitochondria to an extent correlated with the magnitude of the electrochemical gradient of protons across the mitochondrial inner membrane. Secondly, upon irradiation with UV light, it gives rise to formation of free radicals, which inflict damage to the cell. Mutant cells with an impairment in oxidative phosphorylation will have more chance to survive than wild type cells. The selection procedure was applied to a stock of mutagenized Chinese hamster ovary cells. After subcloning of the cells which survived the selection procedure, twenty-six independent clones were isolated. Eighteen of the clones had a partial deficiency of cytochrome c oxidase ranging from 30 to 60% of the activity in control cells. The properties of two of the clones are described. One clone has been cultured under non-selective conditions for at least 12 months with retention of the partial deficiency of cytochrome c oxidase.

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

PURPOSE AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

An astrocyte toxin influences the pattern of breathing and the ventilatory response to hypercapnia in neonatal rats.

Recent in vitro data suggest that astrocytes may modulate respiration. To examine this question in vivo, we treated 5-day-old rat pups with methionine sulfoximine (MS), a compound that alters carbohydrate and glutamate metabolism in astrocytes, but not neurons. MS-treated pups displayed a reduced breathing frequency (f) in baseline conditions relative to saline-treated pups. Hypercapnia (5% CO(2)) increased f in both groups, but f still remained significantly lower in the MS-treated group. No differences between treatment groups in the responses to hypoxia (8% O(2)) were observed. Also, MS-treated rats showed an enhanced accumulation of glycogen in neurons of the facial nucleus, the nucleus ambiguus, and the hypoglossal nucleus, structures that regulate respiratory activity and airway patency. An altered transfer of nutrient molecules from astrocytes to neurons may underlie these effects of MS, although direct effects of MS upon neurons or upon peripheral structures that regulate respiration cannot be completely ruled out as an explanation.

DNA synthesis in Alzheimer type II astrocytosis. The question of astrocytic proliferation and mitosis in experimentally induced hepatic encephalopathy.

Hepatic encephalopathy is associated with development of Alzheimer type II astrocytosis consisting of large, pale, frequently paired astrocytic nuclei. Previous studies have suggested that the paired forms are a manifestation of astrocytic proliferation and that the two nuclei of paired forms are in separate cells and have an equal (diploid) DNA complement. A model of hepatic encephalopathy can be produced using methionine sulfoximine to inhibit irreversibly the enzyme glutamine synthetase, resulting in elevated brain ammonia levels and development of Alzheimer type II astrocytosis. Using this model Sprague-Dawley rats were simultaneously injected with methionine sulfoximine 300 mg/kg and tritiated thymidine 15 mCi/kg. Autoradiography of cerebral sections from animals killed 18 to 36 hours after the injection revealed many heavily labeled cortical Alzheimer type II astrocytic nuclei. These findings are consistent with DNA synthesis and mitotic division of Alzheimer type II astrocytes.

Toxic effects of extended glutathione depletion by buthionine sulfoximine on murine mammary carcinoma cells.

Extended depletion of glutathione to approximately equal to 5% of control in the murine mammary carcinoma cell line 66 was achieved with a concentration of 0.05 mM buthionine sulfoximine. At 24 hours, there was no evidence for cellular toxicity from the BSO treatment per se; however, by 48 hours, there was inhibition of protein and DNA synthesis and cell growth and cell kinetic data was suggestive of both a G1 and a G2 block. Glutathione depletion to this extent (i.e., 0.13 mM vs. 2.24 mM in control) did not modify the aerobic radiation response for cells in the physiological states of proliferation, quiescence, or stimulated quiescent cells. This degree of cellular toxicity may well be cell-type dependent, but the results do suggest that caution is in order if one should attempt long-term GSH depletion in vivo.

Tryptophan availability, central serotonergic function and methionine sulphoximine-induced convulsions.

A convulsant dose (100 mg/kg) of l-methionine-RS-sulphoximine (MSO) produced temporally correlated decreases in central 5-hydroxytryptamine (5-HT) and plasma and brain tryptophan (TRY) concentrations in C57BL/6J mice. In contrast, a subconvulsant dose of MSO (50 mg/kg) had no effect on central 5-HT levels and only decreased brain tryptophan at one time-point after its administration. The pattern of findings suggests that the decrease in central 5-HT levels resulting from a convulsant dose of MSO is related to an impairment in 5-HT synthesis resulting from a restriction of tryptophan availability. Administration of 25 mg/kg and 100 mg/kg doses of l-tryptophan with convulsant doses of MSO significantly increased seizure latency. It is suggested that the central 5-HT system modulates the expression of MSO-induced seizures.

Rapid deterioration of lens fibers in GSH-depleted mouse pups.

Lens opacities developed within 48-72 hr in mice that received a series of eight injections of L-buthionine sulfoximine, a specific inhibitor of glutathione (GSH) biosynthesis, on postnatal days 8 and 9. Initial histopathologic features consisted of swollen fibers in the central anterior cortex and displacement of cell nuclei from the bow region to the posterior cortex. These aberrations suggest early fiber cell membrane and/or cytoskeletal dysfunction. A massive wave of fiber cell lysis then engulfed the entire lens cortex and nucleus within 24 hr and left only epithelial cells intact, suggesting a concerted mechanism of cataract generation. The acellular core of the mature cataract seen on postnatal day 16 consisted of a granular matrix in which pycnotic and fragmented cell nuclei were located near the terminus of the lens epithelium. The epithelium displayed increased mitotic activity and meridional row disorganization. During the next two weeks, rapid regeneration of lens fibers, displacement of the acellular necrotic cytoplasm to the center and rear of the lens, and vacuole formation were observed. As new fibers were differentiated, partial regeneration of the bow was seen. However, the cataract was irreversible.

Effect of glutathione depletion by buthionine sulfoximine on rat embryonic development in vitro.

The intracellular thiol glutathione has many functions within cells including protection against xenobiotic and oxidative damage, and a role in protein and DNA synthesis and amino acid transport. Consequently, glutathione might be an important substance for normal growth and development. In this study the extent of glutathione depletion by buthionine sulfoximine, an agent which depletes glutathione by inhibiting its synthesis, and the subsequent effects of the depletion on rat embryonic growth and development were assessed. Day 10.5 rat embryos were cultured in rat serum medium in the presence of L-buthionine-S,R-sulfoximine (0.01 to 2.0 mM) and examined for viability, malformations, growth and development 45 hr later. The glutathione concentrations of the cultured embryos and their yolk sacs were also determined. Exposure to buthionine sulfoximine produced marked and significant (P less than or equal to 0.05) depletion of glutathione at a buthionine sulfoximine concentration of 0.10 mM in the embryos and 0.05 mM in the yolk sacs. Exposure to 1 mM buthionine sulfoximine depleted glutathione to less than 7% of control in both of these tissues. None of the concentrations of buthionine sulfoximine tested had a significant effect on embryo viability; however, buthionine sulfoximine caused a significant (P less than or equal to 0.05) incidence of malformed embryos at concentrations of 0.25, 0.5, 1.0 and 2.0 mM. The types of defects induced by buthionine sulfoximine were blebs of the maxillary or nasal processes, prosencephalon or forelimb buds, small or misshapen heads, small prosencephalons and swollen hind brains, and tail defects. Embryonic growth was the most sensitive, of the variables assessed, to the effects of buthionine sulfoximine. Significant (P less than or equal to 0.05) growth retardation was observed at buthionine sulfoximine concentrations as low as 0.01 mM. At 2.0 mM buthionine sulfoximine, the yolk sac diameter, embryo crown-rump length, head length, number of somites and morphological score were reduced to 65, 72, 77, 90 and 80% of control levels respectively. We propose that the embryotoxic effects of buthionine sulfoximine are due to glutathione depletion and, consequently, that a certain basal level of endogenous glutathione is essential to allow for normal development.

Enhancement of the embryotoxicity of acrolein, but not phosphoramide mustard, by glutathione depletion in rat embryos in vitro.

The intracellular thiol glutathione is known to protect cells against the toxicity of certain drugs and reactive intermediates. In this study, the role of glutathione in protecting the embryo against two embryolethal and teratogenic metabolites of cyclophosphamide, and anticancer drug, was assessed in vitro using the rat whole embryo culture system. Day 10.5 rat embryos were cultured in rat serum medium containing phosphoramide mustard (1, 10, or 25 microM) or acrolein (10, 25, 50 or 100 microM), with and without buthionine sulfoximine (10 or 100 microM), a compound which depletes glutathione by inhibiting its synthesis. After 45 hr, embryos were assessed for viability, malformations, growth and development, and the glutathione content of embryos exposed to buthionine sulfoximine alone was assayed. The glutathione levels of the embryos and their yolk sacs were decreased significantly by 100 microM buthionine sulfoximine, whereas 10 microM buthionine sulfoximine decreased glutathione levels in the yolk sacs only. Phosphoramide mustard alone, at concentrations of 10 and 25 microM, did not produce embryo deaths but did cause malformations and growth retardation in 100% of the exposed embryos. The addition of buthionine sulfoximine (100 microM) had no effect on the teratogenicity or growth-retarding effects of phosphoramide mustard. Acrolein alone produced a 25 and 48% incidence of embryo deaths at 50 and 100 microM, respectively, and a 46% incidence of embryo malformations, as well as significant growth retardation, among the surviving embryos at 100 microM. Buthionine sulfoximine (10 or 100 microM) significantly enhanced the embryotoxic effects of acrolein. The addition of 10 microM buthionine sulfoximine resulted in 100% embryolethality at 100 microM acrolein; this buthionine sulfoximine concentration decreased the EC50 values for embryo deaths and malformations to 50% of those for acrolein alone. The addition of 100 microM butionine sulfoximine significantly potentiated the embryolethality of acrolein at 25, 50 and 100 microM; the combination of 100 microM acrolein plus 100 microM buthionine sulfoximine was 100% embryolethal. The incidence of embryo malformations was enhanced significantly at 10 and 25 microM acrolein by 100 microM buthionine sulfoximine. The EC50 values for embryo deaths and malformations were decreased to 50 and 20%, respectively, of those values for acrolein alone. Both butionine sulfoximine concentrations produced significant growth retardation at all acrolein concentrations compared to either acrolein or buthionine sulfoximine alone.(ABSTRACT TRUNCATED AT 400 WORDS)