The effect of buthionine sulfoximine on the growth of Leishmania donovani in culture

Weldrick, D

doi:10.1016/s0378-1097(99)00063-4

Cited by 5 publications

(6 citation statements)

References 16 publications

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“…3B) indicating that a disturbance in GSH level was not sufficient to cause a fall in ⌬ m . Our results are consistent with earlier observations in L. donovani where a loss of GSH was shown to be merely cytostatic (43). We also observed that H 2 O 2 treatment did not make the cells more susceptible to BSO-induced GSH depletion, and NAC preincubation in the presence of BSO did not increase GSH levels.…”

Section: Effect Of H 2 O 2 On Mitochondrial Membrane Potential Gsh Asupporting

confidence: 93%

“…Depletion of GSH by BSO did not increase apoptosis-like death (Fig. 5C), and these data are in agreement with observations showing that depletion of GSH does not precipitate cell death in L. donovani (43). In agreement with our observations that NAC pretreatment prior to H 2 O 2 exposure reduced ⌬ m loss, restored ATP levels, and reduced the number of apoptotic cells, no DNA fragmentation was observed in NACtreated groups (Fig.…”

Section: Preincubation Of Cells With Nac Reduces the Number Of Apoptosupporting

confidence: 92%

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Increase in Cytosolic Ca2+ Levels through the Activation of Non-selective Cation Channels Induced by Oxidative Stress Causes Mitochondrial Depolarization Leading to Apoptosis-like Death in Leishmania donovaniPromastigotes

Mukherjee

Sudhandiran

et al. 2002

Journal of Biological Chemistry

192

167

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Reactive oxygen species are important regulators of protozoal infection. Promastigotes of Leishmania donovani, the causative agent of Kala-azar, undergo an apoptosis-like death upon exposure to H 2 O 2 . The present study shows that upon activation of death response by H 2 O 2 , a dose-and time-dependent loss of mitochondrial membrane potential occurs. This loss is accompanied by a depletion of cellular glutathione, but cardiolipin content or thiol oxidation status remains unchanged. ATP levels are reduced within the first 60 min of exposure as a result of mitochondrial membrane potential loss. A tight link exists between changes in cytosolic Ca 2؉ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na ؉ and mitochondrial Ca 2؉ . Partial inhibition of cytosolic Ca 2؉ increase achieved by chelating extracellular or intracellular Ca 2؉ by the use of appropriate agents resulted in significant rescue of the fall of the mitochondrial membrane potential and apoptosis-like death. It is further demonstrated that the increase in cytosolic Ca 2؉ is an additive result of release of Ca 2؉ from intracellular stores as well as by influx of extracellular Ca 2؉ through flufenamic acid-sensitive non-selective cation channels; contribution of the latter was larger. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent mitochondrial membrane potential loss. An antioxidant like Nacetylcysteine is able to inhibit the fall of the mitochondrial membrane potential and prevent apoptosis-like death. Together, these findings show the importance of non-selective cation channels in regulating the response of L. donovani promastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death.Mitochondria are pivotal in controlling cell life and death (1). Maintenance of proper mitochondrial transmembrane potential (⌬ m ) 1 is essential for the survival of the cell as it drives the synthesis of ATP and maintains oxidative phosphorylation (2). Recently, the study of mitochondrial potential has become a focus of apoptosis regulation as many investigations demonstrate a major functional impact of mitochondrial alterations on apoptosis (2). Apoptosis is a process of cell death in which the cells undergo nuclear and cytoplasmic shrinkage; the chromatin is condensed and partitioned into multiple fragments, and finally the cells are broken into multiple membrane-bound bodies. In a number of experimental systems, disruption of ⌬ m constitutes a constant early event of the apoptotic process that precedes nuclear disintegration (3-5). For example, in thymocytes or tumor necrosis factor-stimulated U937 cells (3, 6), thymocytes or imexon-treated myeloma cells (5, 7), and PC-12 cells (8), a loss of ⌬ m occur as an early change associated with apoptosis. Lymphocytes with low ⌬ m show irreversible commitment to apoptosis in comparison to cells with high ⌬ m that do...

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Section: Effect Of H 2 O 2 On Mitochondrial Membrane Potential Gsh Asupporting

confidence: 93%

Section: Preincubation Of Cells With Nac Reduces the Number Of Apoptosupporting

confidence: 92%

Increase in Cytosolic Ca2+ Levels through the Activation of Non-selective Cation Channels Induced by Oxidative Stress Causes Mitochondrial Depolarization Leading to Apoptosis-like Death in Leishmania donovaniPromastigotes

Mukherjee

Sudhandiran

et al. 2002

Journal of Biological Chemistry

192

167

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“…Macrophage inhibitory studies showed that at the concentrations used for promastigote inhibition, the inhibitor had minimal effect on the macrophages. Weldrick et al (1999) have recently reported that amastigotes were less susceptible than promastigotes to the effect of redox cyclers paraquat and menadione in the presence of 20 mm BSO which blocked the synthesis of glutathione. The results reported by this group suggest that there are factors other than, or in addition to, possession of abundant levels of intracellular thiols that may be responsible for the survival and replication of amastigotes in culture (Weldrick et al 1999).…”

Section: Discussionmentioning

confidence: 99%

“…Macrophage inhibitory studies showed that at the concentrations used for promastigote inhibition, the inhibitor had minimal effect on the macrophages. Weldrick et al . (1999) have recently reported that amastigotes were less susceptible than promastigotes to the effect of redox cyclers paraquat and menadione in the presence of 20 m m BSO which blocked the synthesis of glutathione.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of glutathione synthesis as a chemotherapeutic strategy for leishmaniasis

Kapoor

Sachdev

Madhubala

2000

Tropical Med Int Health

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SummaryThis study focuses on the use of buthionine sulphoximine (BSO), a ␥-glutamylcysteine synthetase inhibitor, on Leishmania donovani growth. The effect of BSO on amastigote multiplication within macrophages showed that 5 mm BSO decreased infectivity by about 50% and the mean number of amastigotes per 100 infected macrophages by 21%. The mechanism may be that BSO resulted in enhanced nitric oxide (NO) levels within macrophages, probably due to inhibition of GSH content since GSH (10 mM) given after BSO treatment led to a decrease in NO compared to macrophages treated with BSO alone which were preexposed to the Leishmania surface molecule lipophosphoglycan.keywords glutathione, l-buthionine sulphoximine (BSO), Leishmania donovani, nitric oxide correspondence Dr R. Madhubala,

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“…This additional mechanism prevents the cells from restoring thiol pools with exogenous GSH, and likely explains why BSO is effective even against intracellular parasites. L. donovani and Plasmodium spp., which also have limited ability to detoxify hydrogen peroxide, are also killed by BSO in vitro (24,52,53). Furthermore, BSO has been demonstrated to reverse drug resistance for antimonials and chloroquine in resistant Leishmania and Plasmodium cells that have increased levels of GSH (53,54).…”

Section: Induction Of ␥-Gcs Rna I Kills Trypanosomes By Depleting ␥-Gmentioning

confidence: 99%

Gene Knockdown of γ-Glutamylcysteine Synthetase by RNAi in the Parasitic Protozoa Trypanosoma brucei Demonstrates That It Is an Essential Enzyme

Huynh

Harmon

et al. 2003

Journal of Biological Chemistry

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The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH) 2 ), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. ␥-Glutamylcysteine synthetase (␥-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNA i methods were used to knock down gene expression of ␥-GCS in procyclic T. brucei cells. Induction of ␥-GCS RNA i with tetracycline led to cell death within 4 -6 days post-induction. Cell death was preceded by the depletion of the ␥-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH) 2 . The addition of GSH (80 M) to cell cultures rescued the RNA i cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of ␥-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as ␥-GCS RNA i ; however, addition of GSH did not restore the pools of GSH and T(SH) 2 . These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH) 2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced ␥-GCS RNA i cell lines.

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The effect of buthionine sulfoximine on the growth of Leishmania donovani in culture

Cited by 5 publications

References 16 publications

Increase in Cytosolic Ca2+ Levels through the Activation of Non-selective Cation Channels Induced by Oxidative Stress Causes Mitochondrial Depolarization Leading to Apoptosis-like Death in Leishmania donovaniPromastigotes

Increase in Cytosolic Ca2+ Levels through the Activation of Non-selective Cation Channels Induced by Oxidative Stress Causes Mitochondrial Depolarization Leading to Apoptosis-like Death in Leishmania donovaniPromastigotes

Inhibition of glutathione synthesis as a chemotherapeutic strategy for leishmaniasis

Gene Knockdown of γ-Glutamylcysteine Synthetase by RNAi in the Parasitic Protozoa Trypanosoma brucei Demonstrates That It Is an Essential Enzyme

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