Glutathione reductase is inhibited by acetaminophen-glutathione conjugate (original) (raw)
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Glutathione reductase is inhibited by acetaminophen-glutathione conjugate in vitro
Physiological research / Academia Scientiarum Bohemoslovaca, 2010
The aim of the present work was to investigate a new mechanism likely contributing to the toxic action of acetaminophen, especially to explore the possible inhibition of glutathione reductase through an acetaminophen-glutathione conjugate (APAP-SG). APAP-SG conjugate was synthesized by organic synthesis and purified by column chromatography. The inhibitory effect of the conjugate on two types of glutathione reductase (from yeasts and rat hepatocytes) was tested spectro-photometrically. We found that the enzyme activity was reduced similarly after the treatment with 2.96 mM acetaminophen-glutathione conjugate in both yeast and hepatocyte glutathione reductases (GR); the enzyme activity was inhibited to 52.7+/-1.5 % (2.4+/-0.3 mU/ml) in yeast GR (control activity was 5.6+/-0.3 mU/ml) and to 48.1+/-8.8 % (2.2+/-0.2 mU/ml) in rat hepatocytes lysate GR (control activity was 5.2+/-0.2 mU/ml). In addition, the enzyme activity (from hepatocytes lysate) was decreased to 79+/-7 %, 67+/-2 % an...
Toxicology in Vitro, 2002
Previous studies from this laboratory indicated that glutathione (GSH) conjugate formation with acetaminophen (APAP) is remarkably induced in liver of weanling rats in response to a single overdose of the drug administered intraperitoneally (ip). Increased APAP-GSH conjugation has been attributed to inducible glutathione S-transferases (GSTs) in dividing hepatocytes. In order to verify this finding, an in vitro reconstitution assay containing liver microsomes (source of cytochrome P-450) and cytosolic fractions (source of GST) from livers and kidneys of adult and weanling rats has been established. In vitro incubation of the reaction mixture was followed by solvent extraction, enzymatic digestion and HPLC analysis of the conjugate. Under controlled conditions, in vitro, the rate of APAP-GSH conjugation reflected the GST activity of cytosolic sample added to incubation system. The activity of cytosolic GST in catalyzing this reaction was measured using cytosols prepared from various tissue sources, particularly from animals pretreated with dietary butylated hydroxylanisole (BHA). The extent of APAP-GSH conjugate formation mediated by cytosols varied in this order: BHA-treated adult liver >BHA-treated weanling liver > control adult liver >control weanling liver> BHA-adult kidney >control adult kidney > BHA weanling kidney > control weanling kidney. In contrast to findings obtained from in vivo experiments, the rate of GST-dependent APAP conjugate formation with GSH in vitro is not induced in the presence of exogenous drug. #
Toxicology, 1985
A primary culture system of postnatal rat hepatocytes was utilized to study the cytotoxicity of acetaminophen and the toxicological significance of giutathione (GSH) depletion. The relative time of onset and magnitude of GSH depletion, lipid peroxidation and cytotoxicity were contrasted in order to gain insight into their interrelationships. Exposure of the hepatocytes to acetaminophen resulted in time-and dose<lependent depletion of cellular GSH. The acetaminophen-induced GSH depletion and ensuing lactate dehydrogenase (LDH) leakage were quite modest and delayed in onset, in contrast to that caused by iodoacetamide (IAA) and by diethylmaleate (DEM), 2 well-known depletors of GSH. There was comparable LDH leakage, irrespective of drug treatment, when GSH levels decreased to about 20% of normal. Reduction of GSH levels below the 20% threshold by IAA treatment resulted in marked LDH leakage and loss of viability. Maximal LDH leakage in response to IAA and acetaminophen preceded maximal malondialdehyde (MDA) formation, suggesting that lipid peroxidation may be a consequence of cell damage as well as GSH depletion. IAA and DEM produced a comparable, modest accumulation of MDA, yet IAA was much 127 more cytotoxic. These findings indicate that lipid peroxidation does not play a central role in hepatocellular injury by compounds which deplete GSH, although it may contribute to degeneration of the cell. As events in the cultured postnatal hepatocytes paralleled those reported in vivo, the system can be a useful and valid model with which to study mechanisms of chemical toxicity.
Hepatoprotective Effect of ψ-Glutathione in a Murine Model of Acetaminophen-Induced Liver Toxicity
Chemical Research in Toxicology, 2017
Ψ-glutathione (ψ-GSH) is an orally bioavailable and metabolism-resistant glutathione analogue that has been shown previously to substitute glutathione in most of its biochemical roles. Described here in its entirety is the preclinical evaluation of ψ-GSH as a rescue agent for acetaminophen (APAP) overdose: an event where time is of essence. Employing a murine model, four scenarios commonly encountered in emergency medicine are reconstructed. ψ-GSH is juxtaposed against N-acetylcysteine (NAC), the sole clinically available drug, in each of the scenarios. While both agents appear to be equally efficacious when timely administered, ψ-GSH partly retains its efficacy even in the face of substantial delay in administration. Thus implied is the ability of ψ-GSH to intercept secondary toxicology following APAP insult. Oral availability and complete lack of toxicity as evaluated by liver function tests and survival analysis underscored ψ-GSH as a safer and more efficacious alternative to NAC. Finally, the pharmacodynamic mimicry of GSH by ψ-GSH is illustrated through the isolation and chemical characterization of an entity that can arise only through direct encounter of ψ-GSH with NAPQI, the primary toxic metabolite of APAP.
Mouse-Liver Glutathione Reductase. Purification, Kinetics, and Regulation
European Journal of Biochemistry, 1979
Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2', 5'-bisphosphate-Sepharose columns. A facile procedure for the synthesis of 8-(6-aminohexyl)amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of M , 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80°C by GSSG and less markedly by NADP' and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP' or NAD', produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.
The purpose of this paper is to examine the role of glutathione in cytoprotection, xenobiotics metabolism and conjugation of reactive metabolites as well as its role in Protein s-glutathionylation induced by reactive oxygen and nitrosative species. We reviewed published literatures addressing the xenobiotics detoxification, involvement of methylglyoxal in glycation and cellular damage as well as role of protein s-glutathionylation in oxidative stress caused by reactive oxygen and nitrosative stress. Glutathione plays critical role in xenobiotics detoxification. It also plays a role in conjugation of toxic metabolites generated in the cell during phase I reactions. It participates in glyoxylase system, protein regulation and expression of gene through disulfide exchange reactions, metabolism of estrogens, prostaglandins and leukotrienes, maturation of iron-sulfur clusters of diverse proteins and operation of certain transcription factors. Glutathione plays indispensable role in cytoprotection, detoxification, protein protection and redox-signalling. It role is well noted in conjugation of highly reactive metabolites. Excess advanced glycation end products have been linked to the development of diabetes and methylgyoxal levels were found to be high in the cerebrospinal fluid of diabetic patients. Recently, Protein S-glutathionylation is appearing as a critical signaling mechanism in cardiovascular diseases. Abbreviations AGEs = Advance Glycation End-products DHAP = Dihydroxy Acetone phosphate EC = Enzyme Commission G3P = Glyceraldehydes 3 Phosphate GCL = Gamma Glutamylcysteinyl Ligase GlxI = Glyoxylase I GlxII = Glyoxylase II Grx = Glutaredoxin GS = Glutathione synthetase GSH = Reduce Glutathione GSSG = Oxidized Glutathione MGS = Methylglyoxal Synthetase
1989
The role of the glutathione peroxidase/reductase (GSH-Px/GSSG-Rd) enzyme system in protection from paracetamol toxicity was investigated in isolated mouse hepatocytes in primary culture. The effect of inhibitors of these enzymes on the toxicity of paracetamol and on t-butylhydroperoxide (r-BOOH), used as a positive control, was determined. 1,3-Bis(chloroethyl)-1-nitrosourea (BCNU) was used to inhibit GSSG-Rd, and goldthioglucose (GTG) used to inhibit GSH-Px. Both these inhibitors increased cell membrane damage in response to oxidative stress initiated by t-BOOH. However, they also increased the susceptibility of hepatocytes to paracetarnol toxicity, indicating that a component of paracetamol's toxic effect involves formation of species that are detoxified by the GSH-Px/GSSG-Rd enzymes. To further examine the role of these enzymes, age-related differences in their activity were exploited. Hepatocytes from two-week-old mice were less susceptible to both j-BOOH and paracetamol toxicity than were those from adult mice. This corresponds to higher activity of cytosolic GSH-Px/ GSSG-Rd in this age group. However, after inhibition of GSSG-Rd with BCNU, hepatocytes from these postnatal mice were more susceptible to paracetamol toxicity. This suggests that the higher activity of GSH-Px/GSSG-Rd in hepatocytes from two-week-old mice is responsible for their reduced susceptibility to paracetamol toxicity. The data indicate that the GSH-Px/GSSG-Rd enzymes contribute to protection from paracetamol toxicity and suggest that formation of peroxides contributes to this drug's hepatotoxic effects.
Changes in Mouse Liver Protein Glutathionylation after Acetaminophen Exposure
Journal of Pharmacology and Experimental Therapeutics, 2011
The role of protein glutathionylation in acetaminophen (APAP)induced liver injury was investigated in this study. A single oral gavage dose of 150 or 300 mg/kg APAP in B6C3F1 mice produced increased serum alanine aminotransferase and aspartate aminotransferase levels and liver necrosis in a dosedependent manner. The ratio of GSH to GSSG was decreased in a dose-dependent manner, suggesting that APAP produced a more oxidizing environment within the liver. Despite the increased oxidation state, the level of global protein glutathionylation was decreased at 1 h and continued to decline through 24 h. Immunohistochemical localization of glutathionylated proteins showed a complex dynamic change in the lobule zonation of glutathionylated proteins. At 1 h after APAP exposure, the level of glutathionylation decreased in the single layer of hepa-tocytes around the central veins but increased mildly in the remaining centrilobular hepatocytes. This increase correlated with the immunohistochemical localization of APAP covalently bound to protein. Thereafter, the level of glutathionylation decreased dramatically over time in the centrilobular regions with major decreases observed at 6 and 24 h. Despite the overall decreased glutathionylation, a layer of cells lying between the undamaged periportal region and the damaged centrilobular hepatocytes exhibited high levels of glutathionylation at 3 and 6 h in all samples and in some 24-h samples that had milder injury. These temporal and zonal pattern changes in protein glutathionylation after APAP exposure indicate that protein glutathionylation may play a role in protein homeostasis during APAP-induced hepatocellular injury.