Fluorescence-based microtiter plate assay for glutamate–cysteine ligase activity

White, Collin C.; Viernes, Hannah Malia A; Krejsa, Cecile M.; Botta, Dianne; Kavanagh, Terrance J.

doi:10.1016/s0003-2697(03)00143-x

Cited by 256 publications

(137 citation statements)

References 22 publications

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“…Glutathione and Glutamate-cysteine Ligase Activity-Baseline and inducible glutamate-cysteine ligase activity and GSH levels in liver homogenates were assayed by HPLC or by fluorescence biochemical assay essentially as previously described (23)(24)(25).…”

Section: Methodsmentioning

confidence: 99%

Acetaminophen-induced Liver Injury Is Attenuated in Male Glutamate-cysteine Ligase Transgenic Mice

Botta

Shi

White

et al. 2006

Journal of Biological Chemistry

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Acetaminophen overdose is a leading cause of drug-related acute liver failure in the United States. Glutathione, a tripeptide antioxidant protects cells against oxidative damage from reactive oxygen species and plays a crucial role in the detoxification of xenobiotics, including acetaminophen. Glutathione is synthesized in a two-step enzymatic reaction. Glutamate-cysteine ligase carries out the rate-limiting and first step in glutathione synthesis. We have generated C57Bl/6 mice that conditionally overexpress glutamate-cysteine ligase, and report here their resistance to acetaminophen-induced liver injury. Indices of liver injury included histopathology and serum alanine aminotransferase activity. Male transgenic mice induced to overexpress glutamate-cysteine ligase exhibited resistance to acetaminophen-induced liver injury when compared with acetaminophen-treated male mice carrying, but not expressing glutamate-cysteine ligase transgenes, or to female glutamatecysteine ligase transgenic mice. We conclude that glutamatecysteine ligase activity is an important factor in determining acetaminophen-induced liver injury in C57Bl/6 male mice. Because people are known to vary in their glutamate-cysteine ligase activity, this enzyme may also be an important determinant of sensitivity to acetaminophen-induced liver injury in humans.The tripeptide antioxidant glutathione (GSH; ␥-glutamylcysteinylglycine) is one of the most abundant cellular thiols. It protects cells against oxidative damage from reactive oxygen species, maintains cellular redox status, promotes cell growth, and plays a crucial role in the detoxification of xenobiotics. GSH can directly scavenge free radicals, act as an antioxidant in GSH-mediated reduction of peroxides and act as a co-substrate for glutathione S-transferase-mediated detoxification of reactive intermediates formed during phase I metabolism (1).GSH plays a major role in detoxifying many hepatotoxicants including acetaminophen (APAP), 3 an over-the-counter analgesic and antipyretic (2-5). APAP overdose is responsible for nearly 50% of the acute liver failure cases in the United States (6) and is thus of high public health concern. APAP metabolism has been well defined, making it a good model for drug-induced liver toxicity. APAP is primarily metabolized through sulfation and glucuronidation pathways (7-9). However, a fraction of APAP is bioactivated by cytochrome P-450s to n-acetyl-p-benzoquinoneimine (NAPQI), which can bind to cellular proteins (3, 7, 10 -12). NAPQI also covalently binds to GSH and is either converted back to APAP, or forms the non-toxic APAP-GSH conjugate. APAP overdose results in depletion of hepatic GSH (by as much as 90%) (2). As GSH stores are depleted, increased levels of NAPQI-protein adducts form, and such adducts are thought to be an important contributor to APAP hepatotoxicity (3,7,8,10,11).Pretreatment with N-acetylcysteine (7, 9, 10), a source of cysteine for GSH biosynthesis, attenuates APAP-induced hepatotoxicity. N-Acetylcysteine given soon after APAP (within...

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Section: Methodsmentioning

confidence: 99%

Acetaminophen-induced Liver Injury Is Attenuated in Male Glutamate-cysteine Ligase Transgenic Mice

Botta

Shi

White

et al. 2006

Journal of Biological Chemistry

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“…GCL and glutathione synthetase (GS) activities were measured according to White and collaborators [21], with some minor modifications [22]. Cells were washed twice with PBS and lysed with 25 mM Tris-HCl (pH 7.4) plus 0.1 mM EDTA.…”

Section: Determination Of Gcl and Gs Activitiesmentioning

confidence: 99%

Glutathione redox cycle dysregulation in Huntington’s disease knock-in striatal cells

Ribeiro

Rosenstock

Cunha‐Oliveira

et al. 2012

Free Radical Biology and Medicine

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a b s t r a c tHuntington's disease (HD) is a CAG repeat disorder affecting the HD gene, which encodes for huntingtin (Htt) and is characterized by prominent cell death in the striatum. Oxidative stress was previously implicated in HD neurodegeneration, but the role of the major endogenous antioxidant system, the glutathione redox cycle, has been less studied following expression of full-length mutant Htt (FL-mHtt). Thus, in this work we analyzed the glutathione system in striatal cells derived from HD knock-in mice expressing mutant Htt versus wild-type cells. Mutant cells showed increased intracellular reactive oxygen species (ROS) and caspase-3 activity, which were significantly prevented following treatment with glutathione ethyl ester. Interestingly, mutant cells exhibited an increase in intracellular levels of both reduced and oxidized forms of glutathione, and enhanced activities of glutathione peroxidase (GPx) and glutathione reductase (GRed). Furthermore, glutathione-S-transferase (GST) and g-glutamyl transpeptidase (g-GT) activities were also increased in mutant cells. Nevertheless, glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) activities and levels of GCL catalytic subunit were decreased in cells expressing FL-mHtt, highly suggesting decreased de novo synthesis of glutathione. Enhanced intracellular total glutathione, despite decreased synthesis, could be explained by decreased extracellular glutathione in mutant cells. This occurred concomitantly with decreased mRNA expression levels and activity of the multidrug resistance protein 1 (Mrp1), a transport protein that mediates cellular export of glutathione disulfide and glutathione conjugates. Additionally, inhibition of Mrp1 enhanced intracellular GSH in wild-type cells only. These data suggest that FL-mHtt affects the export of glutathione by decreasing the expression of Mrp1. Data further suggest that boosting of GSH-related antioxidant defense mechanisms induced by FL-mHtt is insufficient to counterbalance increased ROS formation and emergent apoptotic features in HD striatal cells.

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“…GCS and GS activity-Activity of GCS and GS were determined by a fluorescencebased microtiter plate assay originally developed by White et al (2003) with little modification. Briefly, 50 μl of homogenate was centrifuged in micro-tubes for 20 min at 4 °C at 12,000 × g to remove endogenous GSH and other inhibitors (Liu et al, 1998).…”

Section: Enzyme Activity Assaysmentioning

confidence: 99%

Age-related changes in glutathione and glutathione-related enzymes in rat brain

2006

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The most reliable and robust risk factor for some neurodegenerative diseases is aging. It has been proposed that processes of aging are associated with the generation of reactive oxygen species and a disturbance of glutathione homeostasis in the brain. Yet, aged animals have rarely been used to model the diseases that are considered to be age-related such as Parkinson's or Alzheimer's disease. This suggests that the results from these studies would be more valuable if aged animals were used. The present study was designed to provide insight into the glutathione redox state in young and aged rat siblings of both genders by studying the enzyme activities related to glutathione synthesis, cycling, and usage. The results suggested a significant age-related reduction of reduced glutathione (GSH) level in all brain regions examined, associated with an increase of GSH oxidation to glutathione disulfide (GSSG) and decrease of the GSH/GSSG ratio. These changes were accompanied by diminished γ-glutamylcysteine synthetase activity in de novo glutathione synthesis and increased lipid peroxidation. In addition, these changes were associated with increased enzyme activities related to the GSH usage (glutathione peroxidase, γ-glutamyl transpeptidase, and glutathione Stransferase). The results indicate that aged animals are likely more vulnerable to oxidative stress and insinuate the roles of aged animals in modeling age-related neurodegeneration diseases.

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Fluorescence-based microtiter plate assay for glutamate–cysteine ligase activity

Cited by 256 publications

References 22 publications

Acetaminophen-induced Liver Injury Is Attenuated in Male Glutamate-cysteine Ligase Transgenic Mice

Acetaminophen-induced Liver Injury Is Attenuated in Male Glutamate-cysteine Ligase Transgenic Mice

Glutathione redox cycle dysregulation in Huntington’s disease knock-in striatal cells

Age-related changes in glutathione and glutathione-related enzymes in rat brain

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