Comparative evaluation of N-acetylcysteine and N-acetylcysteineamide in acetaminophen-induced hepatotoxicity in human hepatoma HepaRG cells (original) (raw)
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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.
Free Radical Biology and Medicine, 2012
Acetaminophen (APAP) is the most widely used pharmaceutical analgesicantipyretic agent in the world, but its toxicity is a common cause of drug-induced hepatotoxicity. With APAP toxicity, cellular glutathione (GSH) is depleted. This results in the availability of N-acetyl-p-benzoquinone imine (NAPQI), is a toxic metabolite of APAP that binds to cellular macromolecules, which leads to cell necrosis. N-acetyl cysteine (NAC), a GSH precursor, is the only approved antidote for an acetaminophen overdose. It is a negatively charged molecule that diminishes its penetration into the cells, thereby requiring fairly high doses that increase the severity of side effects. In addition, oral and I.V. administration of NAC in a hospital setting is laborious and costly. Recently, NACA, an amide form of NAC, which is neutral at physiological pH has been developed to improve NAC's bioavailability. Therefore, in this study, we conducted an investigation to determine the mechanism of APAP-induced hepatotoxicity. We also evaluated the hepatoprotective effectiveness of NACA and compared it with NAC in the hepatic cell line, HepaRG. This comparison was based on several oxidative stress parameters, including the levels of intracellular reactive oxygen species, GSH, various antioxidant enzyme activities, and lactate dehydrogenase levels. In conclusion, NACA protected HepaRG cells against damage induced by acetaminophen toxicity and may, therefore, be a more useful antidote than NAC (the only approved antidote). I gratefully acknowledge the financial support from my sponsor King Abdul Aziz University, Jeddah Saudi Arabia. Finally, I would like to dedicate this thesis to my parents, Naeem Khayat and Shadia Salamah; my husband, Yamin Mirdad: and my sisters and brother, Ajwaa, Alaa, and Ahmed Khayat for their encouragement and support. And last but not the least, I would like to present this thesis to my twins, Aasser and Alin, without their love, this work would never have been possible.
Oxidant Stress and Acetaminophen Hepatotoxicity: Mechanism-Based Drug Development
Antioxidants & Redox Signaling
Significance: Acetaminophen (APAP) is one of the quantitively most consumed drugs worldwide. Although safe at therapeutic doses, intentional or unintentional overdosing occurs frequently causing severe liver injury and even liver failure. In the United States, 50% of all acute liver failure cases are caused by APAP overdose. However, only one antidote with a limited therapeutic window, N-acetylcysteine, is clinically approved. Thus, more effective therapeutic interventions are urgently needed. Recent Advances: Although APAP hepatotoxicity has been extensively studied for almost 50 years, particular progress has been made recently in two areas. First, there is now a detailed understanding of involvement of oxidative and nitrosative stress in the pathophysiology, with identification of the reactive species involved, their initial generation in mitochondria, amplification through the c-Jun N-terminal kinase pathway, and the mechanisms of cell death. Second, it was demonstrated in human hepatocytes and through biomarkers in vivo that the mechanisms of liver injury in animals accurately reflect the human pathophysiology, which allows the translation of therapeutic targets identified in animals to patients. Critical Issues: For progress, solid understanding of the pathophysiology of APAP hepatotoxicity and of a drug's targets is needed to identify promising new therapeutic intervention strategies and drugs, which may be applied to humans. Future Directions: In addition to further refine the mechanistic understanding of APAP hepatotoxicity and identify additional drugs with complementary mechanisms of action to prevent cell death, more insight into the mechanisms of regeneration and developing of drugs, which promote recovery, remains a future challenge.
N-Acetylcysteine for Preventing Acetaminophen-Induced Liver Injury: A Comprehensive Review
Frontiers in Pharmacology
Aims: N-Acetylcysteine (NAC) is used as an antidote in acetaminophen (APAP) overdose to prevent and mitigate drug-induced liver injury (DILI). Our objective was to systematically review evidence of the use of NAC as a therapeutic option for APAP overdose and APAP-related DILI in order to define the optimal treatment schedule and timing to start treatment.Methods: Bibliographic databases (PubMed, Web of Science, Embase, and MEDLINE) were searched for retrospective and prospective cohort studies, case series, and clinical trials. The prespecified primary outcomes were DILI-related mortality, hepatotoxicity, and adverse events (AEs).Results: In total, 34 studies of NAC usage in APAP-related DILI cases with 19,580 patients were identified, of which 2,376 patients developed hepatotoxicities. The mortality rate across different studies ranged from 0 to 52%. Large variability of NAC regimens was found, i.e., intravenous (I.V.) (100–150 mg/kg) and oral (70–140 mg/kg), and length of treatmen...
Diverse antioxidants protect against acetaminophen hepatotoxicity
Journal of Biochemical and Molecular Toxicology, 2005
The reactive oxygen species-sensitive transcription nuclear factor-B (NF-B) plays a pivotal role in the development of acetaminophen (APAP) hepatotoxicity. We investigated the efficacy of a diverse series of antioxidants in preventing APAP-induced hepatotoxicity. BALB/c mice were divided into four groups and provided with antioxidants incorporated into chow as follows: (1) control diet; or diet supplemented with (2) S-adenosylmethionine (SAMe); (3) green tea polyphenols (GrTP); or (4) (RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA). After 5 days on these diets, the animals were further subdivided into (A) given an IP injection with APAP (750 mg/kg), or (B) kept as untreated controls. The animals were sacrificed at 0, 4 h, and 24 h following APAP administration. PAP/vehicle induced marked decreases in hepatic reduced glutathione (GSH) levels and endogenous SAMe concentrations (46%) when compared to controls. APAP also caused severe centrilobular necrosis and marked increase in serum enzyme ALT activity (38-fold). Oral administration of antioxidants significantly attenuated the APAP-induced liver damage and depletion of hepatic GSH. There were profound increases in serum TNF-␣ levels at 4 h following APAP administration in nonsupplemented compared to antioxidant-treated animals, but no significant differences noted after 24 h. Serum amyloid A increased in APAP-challenged mice irrespective of antioxidant treatment. Finally, hepatic SAMe concentrations were drastically decreased 24 h
Seminars in Liver Disease
Acetaminophen (APAP) is one of the most popular and safe pain medications worldwide. However, due to its wide availability, it is frequently implicated in intentional or unintentional overdoses where it can cause severe liver injury and even acute liver failure (ALF). In fact, APAP toxicity is responsible for 46% of all ALF cases in the United States. Early mechanistic studies in mice demonstrated the formation of a reactive metabolite, which is responsible for hepatic glutathione depletion and initiation of the toxicity. This insight led to the rapid introduction of N-acetylcysteine as a clinical antidote. However, more recently, substantial progress was made in further elucidating the detailed mechanisms of APAP-induced cell death. Mitochondrial protein adducts trigger a mitochondrial oxidant stress, which requires amplification through a MAPK cascade that ultimately results in activation of c-jun N-terminal kinase (JNK) in the cytosol and translocation of phospho-JNK to the mitoc...
Toxicological Sciences, 2004
The increase in cellular and mitochondrial glutathione disulfide (GSSG) levels and the GSSG:GSH ratio after acetaminophen (AAP) overdose suggest the involvement of an oxidant stress in the pathophysiology. However, the initial severe depletion of hepatocellular glutathione makes quantitative assessment of the oxidant stress difficult. Therefore, we tested the hypothesis that oxidant stress precedes the onset of cell injury in a cell culture model using 2 0 ,7 0-dichlorofluorescein (DCF) fluorescence as a marker for intracellular oxidant stress. Cultured primary murine hepatocytes were exposed to 5 mM AAP. DCF fluorescence, XTT reduction, lactate dehydrogenase (LDH) release, and trypan blue uptake were determined from 0 to 12 h. After glutathione depletion at 3 h, DCF fluorescence increased by 16-fold and was maintained at that level up to 12 h. At 1.5 h after AAP, a significant decrease of the cellular XTT reduction capacity was observed, which continued to decline until 9 h. Cell necrosis (LDH release, trypan blue uptake) was detectable in 20% of cells at 6 h, with a significant further increase at later time points. Pretreatment with 20 mM N-acetylcysteine (NAC) 1 h before AAP enhanced cellular glutathione content, prevented or attenuated the AAP-induced decrease of GSH levels and XTT reduction capacity, respectively, and reduced the loss of cell viability. Additionally, treatment with NAC 2 h after AAP exposure prevented further deterioration of XTT reduction at 3 h and later, and attenuated cell necrosis. Thus, AAP-induced oxidant stress precedes cell necrosis and, in cultured hepatocytes, the oxidant stress is involved in the propagation of cell injury.
Prolonged treatment with N-acetylcystine delays liver recovery from acetaminophen hepatotoxicity
Critical Care, 2009
Introduction Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in the US and Europe. Massive hepatocyte necrosis is the predominant feature of APAP-induced acute liver injury (ALI). Liver regeneration is a vital process for survival after a toxic insult, it occurs at a relative late time point after the injurious phase. Currently, N-acetylcysteine (NAC), a glutathione precursor, is the antidote for acetaminophen overdose. However, NAC is effective only for patients who present within hours of an acute overdose, and is less effective for late-presenting patients. It is possible that in delayed patients, previously reduced endogenous glutathione (GSH) level has restored and prolonged treatment with NAC might be toxic and impair liver regeneration. Therefore, we hypothesize that prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP. Methods ALI was induced in C57BL/6 male mice by a single dose of APAP (350 mg/kg) by intraperitoneal injection. After two hours of APAP challenge, the mice were given 100 mg/kg NAC dissolved in 0.6 mL saline, or saline treatment every 12 hours for a total of 72 hours. Results Seventy-two hours after APAP challenge, compared with saline treatment, NAC treatment significantly increased serum transaminases (alanine transaminase/aspartate aminotransferase), induced evident hepatocyte vacuolation in the periportal area and delayed liver regeneration seen in histopathology. This detrimental effect was associated with reduced hepatic nuclear factor (NF)-κB DNA binding and decreased expression of cell cycle protein cyclin D1, two important factors in liver regeneration. Conclusions Prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP. Key messages • Prolonged treatment with NAC increases serum ALT/ AST in APAP-challenged mice. • Prolonged treatment with NAC induces hepatocyte vacuolation in APAP-challenged mice. • Prolonged treatment with NAC delays hepatocyte regeneration in APAP-challenged mice. • Prolonged treatment with NAC delays liver recovery from APAP hepatotoxicity, reevaluation of optimal doses and duration of NAC therapy is needed.
Therapeutic effect of liposomal-N-acetylcysteine against acetaminophen-induced hepatotoxicity
Journal of drug targeting, 2013
Background: Acetaminophen (APAP) is an antipyretic analgesic drug that when taken in overdose causes depletion of glutathione (GSH) and hepatotoxicity. N-acetylcysteine (NAC) is the antidote of choice for the treatment of APAP toxicity; however, due to its short-half-life repeated dosing of NAC is required. Purpose: To determine whether a NAC-loaded liposomal formulation (Lipo-NAC) is more effective than the conventional NAC in protecting against acute APAP-induced hepatotoxicity. Methods: Male Sprague-Dawley rats were challenged with an intragastric dose of APAP (850 mg/kg b.wt.); 4 h later, animals were administered saline, NAC, Lipo-NAC or empty liposomes and sacrificed 24 h post-APAP treatment. Results: APAP administration resulted in hepatic injury as evidenced by increases in plasma bilirubin, alanine (AST) and aspartate (ALT) aminotransferase levels and tissue levels of lipid peroxidation and myeloperoxidase as well as decreases in hepatic levels of reduced GSH, GSH peroxidase and GSH reductase. Treatment of animals with Lipo-NAC was significantly more effective than free NAC in reducing APAP-induced hepatotoxicity. Histological evaluation showed that APAP caused periacinar hepatocellular apoptosis and/or necrosis of hepatocytes around the terminal hepatic venules which was reduced by NAC treatment, the degree of reduction being greater for Lipo-NAC. Conclusion: These data suggest that administration of Lipo-NAC ameliorated the APAP-induced hepatotoxicity.