Immunochemical quantitation of 3-(cystein-S-yl)acetaminophen protein adducts in subcellular liver fractions following a hepatotoxic dose of acetaminophen

doi:10.1016/0006-2952(90)90558-3

Biochemical Pharmacology

Volume 40, Issue 3, 1 August 1990, Pages 573-579

https://doi.org/10.1016/0006-2952(90)90558-3 Get rights and content

Abstract

The hepatotoxicity of acetaminophen correlates with the formation of 3-(cystein-S-yl)acetaminophen protein adducts. Using a sensitive and specific immunochemical assay, we quantitated the formation of these protein adducts in liver fractions and serum after administration of a hepatotoxic dose of acetaminophen (400 mg/kg) to B6C3F1 mice. Adducts in the cytosolic fraction increased to 3.6 nmol/mg protein at 2 hr and then decreased to 1.1 nmol/mg protein by 8 hr. Concomitant with the decrease in adducts in the cytosol, 3-(cystein-S-yl)acetaminophen protein adducts appeared in serum and their levels paralleled increases in serum alanine aminotransferase. Microsomal protein adducts peaked at 1hr (0.7 nmol/mg protein) and subsequently decreased to 0.2 nmol/mg at 8 hr. The 4000 g pellet (nuclei, plasma membranes, and cell debris) had the highest level of adducts (3.5 nmol/mg protein), which remained constant from 1 to 8 hr. Evaluation of fractions purified from a 960 g pellets indicated that the highest concentration of 3-(cystein-S-yl)acetaminophen protein adducts was located in plasma membranes and mitochondria; peak levels were 10.3 and 5.1 nmol/mg respectively. 3-(Cystein-S-yl) acetaminophen protein adducts were detected in nuclei only after enzymatic hydrolysis of the proteins. The localization of high levels of 3-(cystein-S-yl)acetaminophen protein adducts in plasma membranes and mitochondria may play a critical role in acetaminophen toxicity.

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Pre-treatment twice with liposomal clodronate protects against acetaminophen hepatotoxicity through a pre-conditioning effect
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Citation Excerpt :
After APAP overdose, however, accumulation of NAPQI leads to the depletion of glutathione and binding to cellular proteins to form protein adducts within hepatocytes.7–10 Protein adduct formation primarily affects mitochondrial proteins and results in mitochondrial oxidative stress, impairment of mitochondrial respiration, loss of mitochondrial membrane potential, and activation of cell death signaling pathways.11–24 The significance of non-parenchymal cells in APAP-induced liver injury is less clear.
Acetaminophen (APAP) overdose is a major cause of acute liver injury, but the role of macrophages in the propagation of the hepatotoxicity is controversial. Early research revealed that macrophage inhibitors protect against APAP injury. However, later work demonstrated that macrophage ablation by acute pre-treatment with liposomal clodronate (LC) exacerbates the toxicity. To our surprise, during other studies, we observed that pre-treatment twice with LC seemed to protect against APAP hepatotoxicity, in contrast to acute pre-treatment. The aim of this study was to confirm that observation and to explore the mechanisms.
We treated mice with empty liposomes (LE) or LC twice per week for 1 week before APAP overdose and collected blood and liver tissue at 0, 2, and 6 h post-APAP. We then measured liver injury (serum alanine aminotransferase activity, histology), APAP bioactivation (total glutathione, APAP-protein adducts), oxidative stress (oxidized glutathione (GSSG)), glutamate-cysteine ligase subunit c (Gclc) mRNA, and nuclear factor erythroid 2-related factor (Nrf2) immunofluorescence. We also confirmed the ablation of macrophages by F4/80 immunohistochemistry.
Pre-treatment twice with LC dramatically reduced F4/80 staining, protected against liver injury, and reduced oxidative stress at 6 h post-APAP, without affecting APAP bioactivation. Importantly, Gclc mRNA was higher in the LC group at 0 h and total glutathione was higher at 2 h, indicating accelerated glutathione re-synthesis after APAP overdose due to greater basal glutamate-cysteine ligase. Oxidative stress was lower in the LC groups at both time points. Finally, total Nrf2 immunofluorescence was higher in the LC group.
We conclude that multiple pre-treatments with LC protect against APAP by accelerating glutathione re-synthesis through glutamate-cysteine ligase. Investigators using twice or possibly more LC pre-treatments to deplete macrophages, including peritoneal macrophages, should be aware of this possible confounder.
Trifluoperazine inhibits acetaminophen-induced hepatotoxicity and hepatic reactive nitrogen formation in mice and in freshly isolated hepatocytes
2017, Toxicology Reports
Citation Excerpt :
The authors suggested that covalent binding of the reactive metabolite of APAP to the calcium-ATPase was responsible for the loss of its activity [43,44]. This enzyme has not been reported to be adducted by the reactive metabolite NAPQI; however, high levels of protein adducts were observed in the hepatic plasma membrane fraction of APAP treated mice [46]. The effect of the calcium specific chelators on APAP induced toxicity in freshly isolated hamster hepatocytes was examined by Boobis et al. [47].
The hepatotoxicity of acetaminophen (APAP) occurs by initial metabolism to N-acetyl-p-benzoquinone imine which depletes GSH and forms APAP-protein adducts. Subsequently, the reactive nitrogen species peroxynitrite is formed from nitric oxide (NO) and superoxide leading to 3-nitrotyrosine in proteins. Toxicity occurs with inhibited mitochondrial function. We previously reported that in hepatocytes the nNOS (NOS1) inhibitor NANT inhibited APAP toxicity, reactive nitrogen and oxygen species formation, and mitochondrial dysfunction. In this work we examined the effect of trifluoperazine (TFP), a calmodulin antagonist that inhibits calcium induced nNOS activation, on APAP hepatotoxicity and reactive nitrogen formation in murine hepatocytes and in vivo. In freshly isolated hepatocytes TFP inhibited APAP induced toxicity, reactive nitrogen formation (NO, GSNO, and 3-nitrotyrosine in protein), reactive oxygen formation (superoxide), loss of mitochondrial membrane potential, decreased ATP production, decreased oxygen consumption rate, and increased NADH accumulation. TFP did not alter APAP induced GSH depletion in the hepatocytes or the formation of APAP protein adducts which indicated that reactive metabolite formation was not inhibited. Since we previously reported that TFP inhibits the hepatotoxicity of APAP in mice without altering hepatic APAP-protein adduct formation, we examined the APAP treated mouse livers for evidence of reactive nitrogen formation. 3-Nitrotyrosine in hepatic proteins and GSNO were significantly increased in APAP treated mouse livers and decreased in the livers of mice treated with APAP plus TFP. These data are consistent with a hypothesis that APAP hepatotoxicity occurs with altered calcium metabolism, activation of nNOS leading to increased reactive nitrogen formation, and mitochondrial dysfunction.
Acute Hemolytic Disorders in Cats
2016, August's Consultations in Feline Internal Medicine
Protective effect of N-acetylcysteine supplementation on mitochondrial oxidative stress and mitochondrial enzymes in cerebral cortex of streptozotocin-treated diabetic rats
2011, Mitochondrion
Citation Excerpt :
The reduction in protein thiols may also affect confirmation/catalysis of –SH containing proteins/enzymes which may be detrimental to the function of the brain mitochondria. Mitochondrial NP-SH maintains cell viability through regulation of mitochondrial inner membrane permeability by keeping the –SH groups in the reduced state (Pumford et al., 1990). NAC by increasing mitochondrial NP-SH might play a key role in the protection of mitochondrial components against hyperglycemia-induced oxidative damage.
Diabetic encephalopathy, characterized by cognitive deficits involves hyperglycemia-induced oxidative stress. Impaired mitochondrial functions might play an important role in accelerated oxidative damage observed in diabetic brain. The aim of the present study was to examine the role of mitochondrial oxidative stress and dysfunctions in the development of diabetic encephalopathy along with the neuroprotective potential of N-acetylcysteine (NAC). Chronic hyperglycemia accentuated mitochondrial oxidative stress in terms of increased ROS production and lipid peroxidation. Significant decrease in Mn-SOD activity along with protein and non-protein thiols was observed in the mitochondria from diabetic brain. The activities of mitochondrial enzymes; NADH dehydrogenase, succinate dehydrogenase and cytochrome oxidase were decreased in the diabetic brain. Increased mitochondrial oxidative stress and dysfunctions were associated with increased cytochrome c and active caspase-3 levels in cytosol. Electron microscopy revealed mitochondrial swelling and chromatin condensation in neurons of diabetic animals. NAC administration, on the other hand was found to significantly improve diabetes-induced biochemical and morphological changes, bringing them closer to the controls. The results from the study provide evidence for the role of mitochondrial oxidative stress and dysfunctions in the development of diabetic encephalopathy and point towards the clinical potential of NAC as an adjuvant therapy to conventional anti-hyperglycemic regimens for the prevention and/or delaying the progression of CNS complications.
Mitochondrial protein thiol modifications in acetaminophen hepatotoxicity: Effect on HMG-CoA synthase
2008, Toxicology Letters
Acetaminophen (APAP) overdose is the leading cause of drug related liver failure in many countries. N-acetyl-p-benzoquinone imine (NAPQI) is a reactive metabolite that is formed by the metabolism of APAP. NAPQI preferentially binds to glutathione and then cellular proteins. NAPQI binding is considered an upstream event in the pathophysiology, especially when binding to mitochondrial proteins and therefore leads to mitochondrial toxicity. APAP caused a significant increase in liver toxicity 3 h post-APAP administration as measured by increased serum alanine aminotransferase (ALT) levels. Using high-resolution mitochondrial proteomics techniques to measure thiol and protein changes, no significant change in global thiol levels was observed. However, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMG-CoA synthase) had significantly decreased levels of reduced thiols and activity after APAP treatment. HMG-CoA synthase is a key regulatory enzyme in ketogenesis and possesses a number of critical cysteines in the active site. Similarly, catalase, a key enzyme in hydrogen peroxide metabolism, also showed modification in protein thiol content. These data indicate post-translational modifications of a few selected proteins involved in mitochondrial and cellular regulation of metabolism during liver toxicity after APAP overdose. The pathophysiological relevance of these limited changes in protein thiols remains to be investigated.
Dose-dependent transitions in mechanisms of toxicity: Case studies
2004, Toxicology and Applied Pharmacology
Experience with dose response and mechanisms of toxicity has shown that multiple mechanisms may exist for a single agent along the continuum of the full dose–response curve. It is highly likely that critical, limiting steps in any given mechanistic pathway may become overwhelmed with increasing exposures, signaling the emergence of new modalities of toxic tissue injury at these higher doses. Therefore, dose-dependent transitions in principal mechanisms of toxicity may occur, and could have significant impact on the interpretation of reference data sets for risk assessment. To illustrate the existence of dose-dependent transitions in mechanisms of toxicity, a group of academic, government, and industry scientists, formed under the leadership of the ILSI Health and Environmental Sciences Institute (HESI), developed a series of case studies. These case studies included acetaminophen, butadiene, ethylene glycol, formaldehyde, manganese, methylene chloride, peroxisome proliferator-activated receptor (PPAR), progesterone/hydroxyflutamide, propylene oxide, vinyl acetate, vinyl chloride, vinylidene chloride, and zinc. The case studies formed the basis for technical discourse at two scientific workshops in 2003.

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Biochemical Pharmacology

Abstract

References (29)

Plasma paracetamol half-life and heptic necrosis in patients with paracetamol overdose

Lancet

The covalent binding of acetaminophen to protein. Evidence for cysteine residues as major sites of arylation in vitro

Chem Biol Interact

Subcellular fractionation of rat liver

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Anal Biochem

The toxicity of acetaminophen and N-acetyl-p-benzoquinone imine in isolated hepatocytes is associated with thiol depletion and increased cytosolic Ca²⁺

J Biol chem

Alkylation of the liver plasma membrane and inhibition of the Ca²⁺ ATPase by acetaminophen

Biochem Pharmacol

Immunochemical detection of acetaminophen-bound liver proteins

Biochem Pharmacol

Immunochemical analysis of acetaminophen covalent binding to proteins

Biochem Pharmacol

Liver necrosis from paracetamol

Br J Pharmacol Chemother

Acetaminophen-induced hepatic necrosis. I. Role of drug metabolism

J Pharmacol Exp Ther

Acute liver necrosis following overdose of paracetamol

Br Med J

Biochemical toxicology of acetaminophen

Rev Biochem Toxicol

Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione

J Pharmacol Exp Ther

3-(Glutathion-S-yl)acetaminophen: A biliary metabolite of acetaminophen

Drug Metab Dispos

Cited by (55)

Pre-treatment twice with liposomal clodronate protects against acetaminophen hepatotoxicity through a pre-conditioning effect

Trifluoperazine inhibits acetaminophen-induced hepatotoxicity and hepatic reactive nitrogen formation in mice and in freshly isolated hepatocytes

Acute Hemolytic Disorders in Cats

Protective effect of N-acetylcysteine supplementation on mitochondrial oxidative stress and mitochondrial enzymes in cerebral cortex of streptozotocin-treated diabetic rats

Mitochondrial protein thiol modifications in acetaminophen hepatotoxicity: Effect on HMG-CoA synthase

Dose-dependent transitions in mechanisms of toxicity: Case studies