Biomedical subjects

T J Kavanagh

Publications and source records attributed to T J Kavanagh.

At least 19 recordsLinked to original sources

PubMed2006

Neurotoxicity of domoic Acid in cerebellar granule neurons in a genetic model of glutathione deficiency.

This study investigated the role of cellular antioxidant defense mechanisms in modulating the neurotoxicity of domoic acid (DomA), by using cerebellar granule neurons (CGNs) from mice lacking the modifier subunit of glutamate-cysteine ligase (Gclm). Glutamate-cysteine ligase (Glc) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis. CGNs from Gclm (-/-) mice have very low levels of GSH and are 10-fold more sensitive to DomA-induced toxicity than CGNs from Gclm (+/+) mice. GSH ethyl ester decreased, whereas the Gcl inhibitor buthionine sulfoximine increased DomA toxicity. Antagonists of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors and of N-methyl-D-aspartate (NMDA) receptors blocked DomA toxicity, and NMDA receptors were activated by DomA-induced l-glutamate release. The differential susceptibility of CGNs to DomA toxicity was not due to a differential expression of ionotropic glutamate receptors, as evidenced by similar calcium responses and L-glutamate release in the two genotypes. A calcium chelator and several antioxidants antagonized DomA-induced toxicity. DomA caused a rapid decrease in cellular GSH, which preceded toxicity, and the decrease was primarily due to DomA-induced GSH efflux. DomA also caused an increase in oxidative stress as indicated by increases in reactive oxygen species and lipid peroxidation, which was subsequent to GSH efflux. Astrocytes from both genotypes were resistant to DomA toxicity and presented a diminished calcium response to DomA and a lack of DomA-induced L-glutamate release. Because polymorphisms in the GCLM gene in humans are associated with low GSH levels, such individuals, as well as others with genetic conditions or environmental exposures that lead to GSH deficiency, may be more susceptible to DomA-induced neurotoxicity.

Animals↗

PubMed2006

Targeting type 1 diabetes before and at the clinical onset of disease.

Autoimmune type 1 diabetes is strongly associated with a number of immune abnormalities that manifest themselves before and at the time of clinical diagnosis. The clinical onset is associated with a major loss of the pancreatic islet beta cells. Insulin treatment is the only treatment option since numerous trials with agents that suppress or modulate immune function have failed to preserve beta cell function long term. Recent studies suggest that it is possible to predict clinical onset of diabetes by combining genetic with autoantibody testing. In this review we will summarize current and future drug targets for subjects at risk for type 1 diabetes as well as for subjects with recent onset disease. We will also discuss the possible importance of initiating as well as contributing factors such as reactive oxygen species and modified autoantigens. It is speculated that drug targets of factors important to disease pathogenesis may provide safe and effective adductive treatment to preserve beta cell function in autoantibody positive subjects who are at maximum risk for disease.

Diabetes Mellitus, Type 1↗

PubMed2004

Regulation of endothelial glutathione by ICAM-1: implications for inflammation.

The role of glutathione (GSH) in inflammation is largely discussed from the context of providing reducing equivalents to detoxify reactive oxygen and nitrogen species. Inflammation is now recognized to be an underlying cause of many vascular diseases including atherosclerosis, a disease in which endothelial GSH concentrations are decreased. However, mechanisms that control GSH levels are poorly understood. Key players in the inflammatory process are endothelial adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1). This adhesion molecule is present constitutively and can be induced by a variety of inflammatory stimuli. In this study, using mouse aortic endothelial cells (MAEC) deficient in ICAM-1, we demonstrate a novel interplay between constitutive ICAM-1 and cellular GSH. Deficiency of ICAM-1 was associated with an approximately twofold increase in total GSH content. Inhibiting glutamate-cysteine ligase (GCL), the enzyme that catalyses the rate-limiting step in GSH biosynthesis, prevented the increase in GSH. In addition, the catalytic subunit of GCL was increased (approximately 1.6-fold) in ICAM-1 deficient relative to wild-type cells, suggesting that constitutive ICAM-1 represses GCL expression. Furthermore, the ratio of reduced (GSH) to oxidized (GSSG) glutathione was also increased suggesting a role for ICAM-1 in modulating cellular redox status. Interestingly, increasing cytosolic GSH in wild-type mouse endothelial cells decreased constitutive ICAM-1, suggesting the presence of an inverse and reciprocal pathway. To test the effects of inducible ICAM-1 on GSH, cells were stimulated with the proinflammatory cytokine TNF-alpha. TNF-alpha stimulated production of ICAM-1, which was however not associated with induction of GSH. In contrast, supplementation of endothelial cells with GSH before TNF-alpha addition, inhibited induction of ICAM-1. These data suggest a novel regulatory pathway between constitutive ICAM-1 and GSH synthesis in the endothelium and are discussed in the context of modulating the inflammatory response.

Animals↗

PubMed2001

Simultaneous analysis of surface marker expression and cell cycle progression in human peripheral blood mononuclear cells.

One method for examining cell cycle kinetics by flow cytometry uses continuous DNA labeling with bromodeoxyuridine (BrdU), a thymidine analogue. Upon incorporation into DNA, BrdU causes stoichiometric quenching of the DNA fluorochrome Hoechst 33258. After counterstaining with a secondary DNA fluorochrome (e.g., ethidium bromide), the analyst can distinguish cells in different phases of the cell cycle over a number of mitotic cycles with flow cytometry. In this report, we describe a modification of the flow cytometric BrdU-Hoechst assay that allows combined analysis of cell proliferation and immunophenotyping at the single cell level. To demonstrate an application of this method, human peripheral blood mononuclear cells were stimulated with tetanus toxoid or interleukin-2 for up to 6 days in the presence of BrdU, harvested, and immunostained for the cell surface markers CD3, CD4, CD8, CD14, CD19, and the cytokine receptor, CCR5. We used four-color flow cytometry analyses to simultaneously measure cell proliferation and surface marker expression, for the purpose of immunophenotyping and identifying specific cell subsets responding to antigen stimulation. Our successful application of this method suggests that it may be used to study immune responses at the molecular and cellular level and to identify mechanisms of immune system modulation.

Antigens, Differentiation, B-Lymphocyte↗

PubMed2001

Localization of glutamate-cysteine ligase mRNA and protein in mouse kidney and induction with methylmercury.

Methylmercury (MeHg) is a toxicant that targets the kidney among other tissues. MeHg accumulates in the kidney, where it indirectly produces oxidative stress due to glutathione depletion and leakage of reactive oxygen species from the mitochondria. Glutathione is believed to have an important role in protecting the kidney against MeHg toxicity, and MeHg exposure is known to result in the induction of GSH synthesis through the upregulation of the enzyme glutamate-cysteine ligase (GLCL). GLCL, the rate-limiting enzyme in GSH synthesis, is composed of two subunits, a large catalytic (GLCLc) and a smaller regulatory (GLCLr) subunit. In this study we show that GLCLc and GLCLr mRNAs and GLCLc protein are localized in the paracortical region of the mouse kidney, the area of the kidney with the highest MeHg concentration, and that the upregulation of these mRNAs induced by MeHg is also located to the same region. This supports the role of GLCL in protection against MeHg toxicity in the kidney.

Animals↗

PubMed2001

Tissue specific changes in the expression of glutamate-cysteine ligase mRNAs in mice exposed to methylmercury.

Glutamate-cysteine ligase (GLCL), the rate-limiting enzyme in glutathione (GSH) synthesis is composed of two subunits, a catalytic (GLCLc) and a regulatory subunit (GLCLr). These two subunits are known to be differentially regulated in vitro, in different cell types and in response to various xenobiotic exposures. In this study, we examined whether these two subunits can also be differentially regulated in vivo. We found that GLCLc and GLCLr are differentially regulated at the transcriptional level in a tissue-dependent manner in female mice treated with methylmercury (MeHg). MeHg caused a downregulation of both subunit mRNAs in the liver, upregulation of both subunit mRNAs in the kidney and upregulation of only the catalytic subunit mRNA in the small intestine of female mice treated with a single dose of MeHg (6 mg/kg) by intraperitoneal injection. These results suggest that GLCLc and GLCLr can be differentially regulated in vivo, and that this regulation is tissue dependent in the mouse.

Animals↗

PubMed2001

Enhanced acetaminophen hepatotoxicity in transgenic mice overexpressing BCL-2.

Mitochondria play an important role in the cell death induced by many drugs, including hepatotoxicity from overdose of the popular analgesic, acetaminophen (APAP). To investigate mitochondrial alterations associated with APAP-induced hepatotoxicity, the subcellular distribution of proapoptotic BAX was determined. Based on the antiapoptotic characteristics of BCL-2, we further hypothesized that if a BAX component was evident then BCL-2 overexpression may be hepatoprotective. Mice, either with a human bcl-2 transgene (-/+) or wild-type mice (WT; -/-), were dosed with 500 or 600 mg/kg (i.p.) APAP or a nonhepatotoxic isomer, N-acetyl-m-aminophenol (AMAP). Immunoblot analyses indicated increased mitochondrial BAX-beta content very early after APAP or AMAP treatment. This was paralleled by disappearance of BAX-alpha from the cytosol of APAP treated animals and, to a lesser extent, with AMAP treatment. Early pathological evidence of APAP-induced zone 3 necrosis was seen in bcl-2 (-/+) mice, which progressed to massive panlobular necrosis with hemorrhage by 24 h. In contrast, WT mice dosed with APAP showed a more typical, and less severe, centrilobular necrosis. AMAP-treated bcl-2 (-/+) mice displayed only early microvesicular steatosis without progression to extensive necrosis. Decreased complex III activity, evident as early as 6 h after treatment, correlated well with plasma enzyme activities at 24 h (AST r(2) = 0.89, ALT r(2) = 0.87) thereby confirming a role for mitochondria in APAP-mediated hepatotoxicity. In conclusion, these data suggest for the first time that BAX may be an early determinant of APAP-mediated hepatotoxicity and that BCL-2 overexpression unexpectedly enhances APAP hepatotoxicity.

Acetaminophen↗

PubMed2000

Muscarinic receptor-induced calcium responses in astroglia.

BACKGROUND: The objective of this study was to characterize and quantitate the calcium responses to cholinergic stimulation in individual primary rat cortical astrocytes and human 132 1N1 astrocytoma cells. Materials and Methods The fluorescent calcium probe Indo-1 AM and an attached cell analysis and sorting (ACAS) instrument were used to quantitate calcium responses in these cells. RESULTS: A concentration-dependent response to carbachol was seen in both cell types. However, carbachol was more potent and efficacious, and the response was more homogeneous in the cell line. The calcium response was mediated by the M3 subtype of muscarinic receptors. Experiments in the absence of extracellular calcium and with EGTA demonstrated that the initial calcium spike was due to calcium release from intracellular calcium stores, whereas the sustained elevation and oscillations were dependent on calcium influx. Protein kinase C exerts a feedback inhibition of these calcium responses, and appears to be involved in maintaining the elevated calcium concentration and oscillations. CONCLUSIONS: This study provided a detailed quantitation of the changes in intracellular calcium evoked in individual astroglial cells by activation of M3 muscarinic receptors. This will allow for the study of pharmacological and toxicological agents on this response.

Animals↗

PubMed2000

Cloning and characterization of the proximal promoter region of the mouse glutamate-L-cysteine ligase regulatory subunit gene.

We describe upregulation of the mRNA for the mouse glutamate-cysteine ligase regulatory subunit gene (Glcl-r) in Hepa-1 cells treated with beta-napthoflavone (BNF) and tert-butylhydroquinone (tBHQ). A 2-kb fragment of the proximal promoter region of the gene was cloned and sequenced, and sequence analysis reveals a high degree of homology when compared to the human glutamate-cysteine ligase regulatory subunit gene promoter. Primer extension analysis indicates a major transcription start site 218 bp upstream of the translation start codon in a CpG-rich region, suggesting that transcription is Sp1 mediated. Reporter constructs containing nested deletion fragments of the Glcl-r promoter demonstrate that regulatory elements sufficient for basal and tBHQ-inducible expression lie between -273 and -787 bp relative to the translation start codon and that the distal promoter may contain negative regulatory elements.

Animals↗

PubMed2000

Disruption of redox homeostasis in tumor necrosis factor-induced apoptosis in a murine hepatocyte cell line.

Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours approximately 50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFkappaB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant alpha-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses.

Amino Acid Chloromethyl Ketones↗

PubMed2000

Analysis of integrin (CD11b/CD18) movement during neutrophil adhesion and migration on endothelial cells.

Little is known of the distribution of cell surface molecules during the adhesion and migration of leucocytes on endothelial cells. We have used confocal microscopy and a Fab fragment of a non-inhibitory monoclonal antibody recognizing the integrin CD11b/CD18 (Mac-1) to study the movement of this adhesion molecule over time. We found that during the initial stage of neutrophil contact with TNF-alpha activated human umbilical vein endothelial cells (HUVEC), there is a rapid accumulation of Mac-1 at the contact area between the two cell types. As the neutrophil spreads, Mac-1 redistributes away from this initial contact area. During neutrophil migration on HUVEC, Mac-1 was redistributed to the leading edge of the migrating cell, suggesting that the existing cell surface pool of adhesion molecules is dynamic and can be recruited to the leading front as the cell changes direction. As neutrophils migrate on HUVEC, Mac-1-dense macroaggregates are rapidly formed and broken down at the contact plane between the two cells. The confocal microscope, coupled with the use of non-inhibitory antibodies labelled with photostable fluorophores, is a useful tool for the study of the movement of cell surface molecules over time.

Cell Adhesion↗

PubMed2000

Modulation of glutathione and glutamate-L-cysteine ligase by methylmercury during mouse development.

The antioxidant tripeptide glutathione has been proposed to be important in defense against oxidative stress and heavy metal toxicity. We evaluated alterations in glutathione regulation and synthesis associated with low-level chronic methylmercury (MeHg) exposure in the developing mouse fetus. Female C57Bl/6 mice were given 0, 3, or 10 ppm MeHg in the drinking water for 2 weeks prior to breeding and throughout pregnancy. Fetuses were collected on gestational days (gd) 12 and 16. Total glutathione, reduced glutathione (GSH), oxidized glutathione (GSSR), and glutamate-L-cysteine ligase (Glcl) activity were assessed in yolk sacs and fetuses at gd 16. Western and Northern blots for Glcl-catalytic (Glclc) and Glcl-regulatory (Glclr) subunits were performed on gd 12 and gd 16 fetuses. There were no changes in total glutathione in gd 16 mouse fetuses with exposure, but there were dose-related decreases in GSH and increases in GSSR. In contrast, visceral yolk sacs exhibited an increase in total glutathione in the low-dose groups, but no changes in the high-dose group. There were no changes in Glcl activity in fetuses, but there was a 2-fold increase in Glcl activity in yolk sacs from both low-dose and high-dose groups. There was a 2-fold induction in GLCLC: mRNA and protein in the gd 16 yolk sacs at both 3 and 10 ppm MeHg. No treatment-related changes in Glclr protein in either gd 12 or gd 16 yolk sacs or fetuses were found. Thus, the yolk sac is capable of up-regulating Glclc and GSH synthetic capacity in response to MeHg exposure. This increase appears to be sufficient to resist MeHg-induced GSH depletion in the yolk sac; however fetal glutathione redox status is compromised with exposure to 10 ppm MeHg.

Animals↗

PubMed1999

The role of glutathione in chronic adaptation to oxidative stress: studies in a normal rat kidney epithelial (NRK52E) cell model of sustained upregulation of glutathione biosynthesis.

Reduced glutathione (GSH) is considered to play a central role in protection of cells from oxidant injury. However, the question remains as to whether sustained elevation of intracellular GSH levels, as compared with the ability to rapidly upregulate GSH synthesis, is more important with respect to protection of cell constituents from oxidative stress. To address this question, we conducted studies to evaluate the direct influence of chronically increased endogenous GSH content on chemically induced intracellular free radical formation and oxidative stress using a kidney epithelial cell model adapted to sustain intracellular GSH concentrations in excess of eightfold that observed in unadapted parent kidney cells. Elevated GSH levels in adapted cells were found to be attributable, at least in part, to coordinately increased amounts of both the regulatory and catalytic subunits of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. Studies using electron spin resonance (ESR) spectroscopy and scanning laser cytometry demonstrated that cells having sustained elevation of GSH levels did not attenuate free radical formation and associated oxidative stress compared with parent cells when treated with the prooxidant chemicals, menadione or potassium dichromate. In contrast, nonadapted kidney parent cells treated 18 h after initial prooxidant challenge displayed significantly attenuated free radical signals. Additionally, cells adapted to sustain excess GSH were somewhat more sensitive than parent cells in terms of resistance to prooxidant (chromate) toxicity, as determined by cell viability assays. These findings suggest that the capacity of cells to rapidly upregulate GSH synthesis, rather the ability to chronically sustain elevated intracellular GSH levels, may play a more important role in terms of protection from cytotoxicity associated with prooxidant chemical exposures.

Adaptation, Physiological↗

PubMed1999

Induction of glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) in the brains of adult female mice subchronically exposed to methylmercury.

Methylmercury (MeHg) is widely known for its potent neurotoxic properties. One proposed mechanism of action of MeHg relates to its high affinity for sulfhydryl groups, especially those found on glutathione (GSH) and proteins. Previous studies have shown that acute MeHg exposure results in an increase in the mRNA for the rate-limiting enzyme in GSH synthesis, glutamate-cysteine ligase (GLCL) (also known as gamma-glutamylcysteine synthetase). In this study, we evaluated the effects of subchronic (12-week) MeHg exposure at 0, 3 or 10 ppm in the drinking water on GSH levels, GLCL catalytic (GLCLC) and regulatory subunit mRNA and protein levels, and GLCL activity in brain, liver and kidney tissue of C57B1/6 female mice. Contrary to previous findings in rats, there were no changes in GSH concentration in any of the tissues examined. However, there was an increase in GLCLC protein in the brain, which was accompanied by a 30% increase in GLCL activity. We conclude that up-regulation of GSH synthetic capacity in the brains of mice is a sensitive biomarker of subchronic MeHg exposure.

Animals↗

PubMed1999

Induction of the cell cycle regulatory gene p21 (Waf1, Cip1) following methylmercury exposure in vitro and in vivo.

Methylmercury (MeHg) is recognized as a significant environmental hazard, particularly to the development of the nervous system. To study the molecular mechanisms underlying cell cycle inhibition by MeHg, we assessed the involvement of p21 (Waf1, Cip1), a cell cycle regulatory gene implicated in the G1 and G2 phases of cell cycle arrest, in primary embryonic cells and adult mice following MeHg exposure. Previous literature has supported the association of increased p21 expression with chondrocyte differentiation. In support of this finding, we observed an increasing p21 expression during limb bud (LB), but not midbrain central nervous system (CNS) cell differentiation. Both embryonic LB and CNS cells responded to MeHg exposure with a concentration-dependent increase in p21 mRNA. In the parallel adult study, C57BL/6 female mice were chronically exposed to 10 ppm MeHg via drinking water for 4 weeks. While there was limited or absent induction of Gadd45, Gadd153, and the gamma-glutamylcysteine synthetase catalytic subunit, p21 was markedly induced in the brain, kidney, and liver tissues in most of the animals that showed MeHg-induced behavioral toxicity such as hyperactivity and tremor. Furthermore, the induction of p21 mRNA was accompanied by an increase in p21 protein level. The results indicate that the activation of cell cycle regulatory genes may be one mechanism by which MeHg interferes with the cell cycle in adult and developing organisms. Continued examination of the molecular mechanisms underlying cell cycle inhibition may potentially lead to utilization of this mechanistic information to characterize the effects of MeHg exposure in vivo.

Animals↗

PubMed1999

Glutathione redox potential in response to differentiation and enzyme inducers.

The reduced glutathione (GSH)/oxidized glutathione (GSSG) redox state is thought to function in signaling of detoxification gene expression, but also appears to be tightly regulated in cells under normal conditions. Thus it is not clear that the magnitude of change in response to physiologic stimuli is sufficient for a role in redox signaling under nontoxicologic conditions. The purpose of this study was to determine the change in 2GSH/GSSG redox during signaling of differentiation and increased detoxification enzyme activity in HT29 cells. We measured GSH, GSSG, cell volume, and cell pH, and we used the Nernst equation to determine the changes in redox potential Eh of the 2GSH/GSSG pool in response to the differentiating agent, sodium butyrate, and the detoxification enzyme inducer, benzyl isothiocyanate. Sodium butyrate caused a 60-mV oxidation (from -260 to -200 mV), an oxidation sufficient for a 100-fold change in protein dithiols:disulfide ratio. Benzyl isothiocyanate caused a 16-mV oxidation in control cells but a 40-mV oxidation (to -160 mV) in differentiated cells. Changes in GSH and mRNA for glutamate:cysteine ligase did not correlate with Eh; however, correlations were seen between Eh and glutathione S-transferase (GST) and nicotinamide adenine dinucleotide phosphate (NADPH):quinone reductase activities (N:QR). These results show that 2GSH/GSSG redox changes in response to physiologic stimuli such as differentiation and enzyme inducers are of a sufficient magnitude to control the activity of redox-sensitive proteins. This suggests that physiologic modulation of the 2GSH/GSSG redox poise could provide a fundamental parameter for the control of cell phenotype.

Adenocarcinoma↗

PubMed1999

Protection of acute myeloblastic leukemia cells against apoptotic cell death by high glutathione and gamma-glutamylcysteine synthetase levels during etoposide-induced oxidative stress.

BACKGROUND: Etoposide mediates its cytotoxicity by inducing apoptosis. Thus, mechanisms which regulate apoptosis should also affect drug resistance. Oxidants and antioxidants have been shown to participate in the regulation of apoptosis. We were interested in studying whether responsiveness of acute myeloblastic leukemia (AML) cells to etoposide is mediated by oxidative stress and glutathione levels. PATIENTS AND METHODS: Two subclones of the OCI/AML-2 cell line which are etoposide-sensitive (ES), and etoposide-resistant (ER), were established by the authors at the University of Oulu, and used as models. Assays for apoptosis included externalization of phosphatidylserine (as evidenced by annexin V binding), and caspase activation as indicated by cleavage of poly(ADP-ribose)polymerase (Western blotting). Peroxide formation was analyzed by flow cytometry. Glutathione and gamma-glutamylcysteine synthetase (gamma-GCS) levels were determined spectrophotometrically and by Western blotting, respectively. RESULTS: Etoposide-induced apoptosis was evident 12 hours after treatment in the ES subclone, but was apparent in the ER subclone only after 24 hours. The basal glutathione and gamma-GCS levels were higher in the ER than the ES subclone. Etoposide increased peroxide formation in both subclones after 12-hour exposure. Significant depletion of glutathione was observed in the ES subclone during etoposide exposure, while glutathione levels were maintained in the ER subclone. In neither of the subclones was induction of gamma-GCS observed during 24-hour exposure to etoposide. Furthermore, the catalytic subunit of gamma-GCS was cleaved during apoptosis, concurrent with depletion of intracellular glutathione. When glutathione was depleted by treatment with buthionine sulfoximine, a direct inhibitor of gamma-GCS, the sensitivity to etoposide was increased, particularly in the ER subclone. CONCLUSIONS: The results underline the significance of glutathione biosynthesis in the responsiveness of AML cells to etoposide. The molecular mechanisms mediating glutathione depletion during etoposide exposure might include the cleavage of the catalytic subunit of gamma-GCS.

Antineoplastic Agents, Phytogenic↗

PubMed1999

Effect of dietary inducer dimethylfumarate on glutathione in cultured human retinal pigment epithelial cells.

PURPOSE: To determine the effect of dimethylfumarate (DMF), an inducer of glutathione (GSH)-dependent detoxification, on intracellular GSH levels in cultured human retinal pigment epithelium (hRPE) cells, its mechanism of action, and its effect on hRPE cells subjected to oxidative injury. METHODS: Established hRPE cell lines were treated with DMF and assayed by high-pressure liquid chromatography for intracellular and extracellular GSH levels. Quantification of gamma-glutamylcysteine synthetase (GLCL) was determined through northern and western blot analyses, and activity was measured. Effects of pretreatment with DMF on GSH redox status of hRPE cells was determined. Sensitivity of hRPE cells to oxidative stress was determined using tert-butylhydroperoxide as the oxidative agent. RESULTS: Dimethylfumarate caused a transient decrease followed by a significant increase in intracellular GSH. Glutathione increased maximally at 24 hours with 100 to 200 microM DMF. The initial decrease could be accounted for by the formation of a DMF-GSH conjugate. Dimethylfumarate treatment increased the steady state mRNA expression of the regulatory subunit of GLCL, but no increase was seen for the catalytic subunit. However, protein levels were increased for both, and the catalytic activity of GLCL was also increased. Whereas the initial decrease in GSH made hRPE cells more susceptible to oxidative damage, pretreatment with DMF under conditions that increased intracellular GSH protected hRPE cells against oxidative damage. CONCLUSIONS: These results suggest a means by which the antioxidant capability of hRPE may be augmented without direct antioxidant supplementation. Specifically, a dietary compound that conjugates with GSH can induce GSH synthesis, increase GSH concentration, and improve protection by GSH-dependent detoxification pathways in hRPE. However, the early depletion of GSH before stimulated synthesis necessitates caution in prevention strategies using dietary inducers.

Aminoacyltransferases↗