Dose Of APAP Research Articles

The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen (APAP) toxicity in mouse hepatocytes (844.16)

3‐Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of APAP correlates with toxicity. Nitration is by peroxynitrite (ONOO‐), a reactive nitrogen species formed from nitric oxide (NO) and superoxide (O2‐). We postulated that APAP toxicity is mediated by peroxynitrite. Since iNOS knockout mice are equally susceptible to APAP toxicity as wildtype mice we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10µM) (N‐[(4S)‐4‐amino‐5‐[(2‐aminoethyl) amino] pentyl]‐N’‐nitroguanidine tris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from B6C3F1 mouse were incubated with/without APAP (1mM). Every 0.5h 1million cells were removed and assayed spectrofluorometrically for NO and superoxide production using diaminofluorescein (DAF) and Mitosox, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO and acetaminophen‐cysteine (APAP‐Cys) adducts were measured by HPLC‐EC. APAP significantly increased cytotoxicity at 1.5‐3.0h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or covalent binding which indicated that NANT did not inhibit metabolism. APAP significantly increased NO and superoxide at 0.5 and 1.0h, respectively. The increases in both NO and superoxide were blocked by NANT. APAP significantly increased 3NT at 1.5‐3.0h and the increase was blocked by NANT. The data support the hypothesis that APAP hepatotoxicity is mediated by reactive nitrogen formed by nNOS.Grant Funding Source: NIH RO1 DK079008

Dual role of acetaminophen in promoting hepatoma cell apoptosis and kidney fibroblast proliferation

Acetaminophen (APAP), is a safe analgesic and antipyretic drug at therapeutic dose, and is widely used in the clinic. However, high doses of APAP can induce hepatotoxicity and nephrotoxicity. Most studies have focused on high-dose APAP-induced acute liver and kidney injury. So far, few studies have investigated the effects of the therapeutic dose (1/10 of the high dose) or of the low dose (1/100 of the high dose) of APAP on the cells. The aim of this study was to investigate the cellular effects of therapeutic- or low-dose APAP treatment on hepatoma cells and kidney fibroblasts. As expected, high-dose APAP treatment inhibited while therapeutic and low-dose treatment did not inhibit cell survival of kidney tubular epithelial cells. In addition, therapeutic-dose treatment induced an increase in the H2O2 level, activated the caspase-9/-3 cascade, and induced cell apoptosis of hepatoma cells. Notably, APAP promoted fibroblast proliferation, even at low doses. This study demonstrates that different cellular effects are exerted upon treatment with different APAP concentrations. Our results indicate that treatment with the therapeutic dose of APAP may exert an antitumor activity on hepatoma, while low-dose treatment may be harmful for patients with fibrosis, since it may cause proliferation of fibroblasts.

Albumin Fusion Prolongs the Antioxidant and Anti-Inflammatory Activities of Thioredoxin in Mice with Acetaminophen-Induced Hepatitis

Overdoses of acetaminophen (APAP) are a major cause of acute liver failure. N-Acetylcysteine (NAC) is the standard therapy for patients with such an overdose because oxidative stress plays an important role in the pathogenesis of APAP-induced hepatitis. However, NAC is not sufficiently efficacious. We previously developed a recombinant human serum albumin (HSA)-thioredoxin 1 (Trx) fusion protein (HSA-Trx), designed to overcome the unfavorable pharmacokinetic and short pharmacological properties of Trx, an endogenous protein with antioxidative and anti-inflammatory properties. In this study, we investigated the therapeutic impact of HSA-Trx in mice with APAP-induced hepatitis. The systemic administration of HSA-Trx significantly improved the survival rate of mice treated with a lethal dose of APAP compared with saline. HSA-Trx strongly attenuated plasma transaminases in APAP-induced hepatitis mice compared with HSA or Trx, components of the fusion protein. HSA-Trx also markedly caused a diminution in the histopathological features of hepatic injuries and the number of apoptosis-positive hepatic cells. In addition, an evaluation of oxidative stress markers and plasma cytokine and chemokine levels clearly showed that HSA-Trx significantly improved the breakdown of hepatic redox conditions and inflammation caused by the APAP treatment. HSA-Trx also significantly decreased oxidative and nitrosative/nitrative stress induced by SIN-1 in vitro. Finally, HSA-Trx, but not the NAC treatment at 4 h after APAP injection, significantly inhibited the elevation in plasma transaminase levels. In conclusion, the findings suggest that HSA-Trx has considerable potential for use as a novel therapeutic agent for APAP-induced hepatitis, due to its long-lasting antioxidative and anti-inflammatory effects.

PKCs: Pernicious kinase culprits in acetaminophen pathogenesis

PKCs: Pernicious kinase culprits in acetaminophen pathogenesis

Thymoquinone treatment against acetaminophen-induced hepatotoxicity in rats

AimIn this study, we aimed to examine the efficacy of thymoquinone (TQ) treatment in acetaminophen-induced liver toxicity in rats. MethodsForty Wistar Albino rats were used for the study (four groups, with 10 rats for each group). Animals in the control group were not given any medication. In the thymoquinone (TQ) group, animals were given three times 5 mg/kg oral thymoquinone for every six hours, which equals to a total dose of 15 mg/kg. In the acetaminophen (APAP) group, animals were given APAP at a single dose of 500 mg/kg orally. In the APAP + TQ group, animals were given 500 mg/kg APAP orally followed by three doses of TQ at a 15 mg/kg total dose in an 18-h time interval. All animals were sacrificed at the 24th hour. Alanine amino transferase (ALT), aspartat amino transferase (AST), superoxide dismutase (SOD), oxidized glutathione (GSSG), glutathione peroxides (GSH-Px), and malondialdehyde (MDA) activities were measured in rat blood. Histopathological examination was also performed. ResultsSerum ALT, AST levels, GSSG, and SOD activity as well as the serum and tissue MDA levels were found to be higher in the APAP group than in the control group (p ≤ 0.001). Likewise, serum GSH-Px activity was found to be lower in the APAP group (p ≤ 0.001). In contrast, in the APAP + TQ group, serum ALT, AST levels, GSSG, SOD activity and the serum and tissue MDA levels were found to be lower compared to that of the APAP group. This difference was statistically significant (p ≤ 0.001). In the APAP + TQ group, the GSH-Px activity was found to be significantly higher compared to the APAP group (p < 0.05). In contrast to this finding, the GSH-Px activity in the APAP + TQ group was found to be lower than that of the control group (p ≤ 0.001). Histopathological analysis revealed significant liver necrosis and toxicity with a high dose of APAP where TQ treatment was related with significantly lower liver injury scores. ConclusionTQ treatment may have an important therapeuthic effect via the upregulation of antioxidant systems in the APAP-induced liver hepatotoxicity in rats.

Protective Effect of Sundarban Honey against Acetaminophen-Induced Acute Hepatonephrotoxicity in Rats.

Honey, a supersaturated natural product of honey bees, contains complex compounds with antioxidant properties and therefore has a wide a range of applications in both traditional and modern medicine. In the present study, the protective effects of Sundarban honey from Bangladesh against acetaminophen- (APAP-) induced hepatotoxicity and nephrotoxicity in experimental rats were investigated. Adult male Wistar rats were pretreated with honey (5 g/kg) for 4 weeks, followed by the induction of hepatotoxicity and nephrotoxicity via the oral administration of a single dose of APAP (2 g/kg). Organ damage was confirmed by measuring the elevation of serum alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), total protein (TP), total bilirubin (TB), urea, creatinine, and malondialdehyde (MDA). Histopathological alterations observed in the livers and the kidneys further confirmed oxidative damage to these tissues. Animals pretreated with Sundarban honey showed significantly markedly reduced levels of all of the investigated parameters. In addition, Sundarban honey ameliorated the altered hepatic and renal morphology in APAP-treated rats. Overall, our findings indicate that Sundarban honey protects against APAP-induced acute hepatic and renal damage, which could be attributed to the honey's antioxidant properties.

アセトアミノフェンを用いた疼痛治療の実態調査

Acetaminophen (APAP) is an analgesic used worldwide. However, the usage of APAP was lower and its effectiveness was underestimated in Japan before the indication was revised for APAP in 2011 compared with those in the United States (US) and Europe. This study investigated the prevalence of APAP in Japan. We conducted a questionnaire survey to determine the usage of APAP among hospital pharmacists and received answers from 430 pharmacists in Japan and 35 pharmacists in the US from December 2010 to October 2011. The range of the maximum dose of APAP for cancer pain was 900 -1500 mg/day (49%) in Japan, and 3000-4000 mg/day (83%) in the US. The pharmacists were questioned about the drug of choice among non-opioid analgesics for the treatment of cancer pain by patient age group. American pharmacists chose APAP (31%) and non-steroidal anti-inflammatory drugs (NSAIDs) (31%) for adult patients, while Japanese pharmacists chose APAP (2%) and NSAIDs (77%). American pharmacists chose APAP (49%) and NSAIDs (17%) for elderly patients, while Japanese pharmacists chose APAP (10%) and NSAIDs (64%). In addition, American pharmacists chose APAP (52%) and NSAIDs (17%) for pediatric patients, while Japanese pharmacists chose APAP (45%) and NSAIDs (12%). APAP is mainly prescribed for pediatric patients with cancer pain, and its usage for both adult and elderly patients with cancer pain is lower in Japan than that in the US. Safe, effective, and inexpensive APAP should be used as an adjunctive for the treatment of cancer pain in adult and elderly patients in Japan.

Effectofchronic paracetamol treatmenton cerebral vessels

Adverse effect of paracetamol (APAP) treatment has been recently reported in several systems.In this study, effectsofAPAP treatments at 3 different time points (0, 15, and 30 days) on cerebral microvessels in a cortical spreading depression (CSD) migraine animal model were investigated. Rats were divided into control, CSD only, and APAP treatment with or without CSD groups. A single dose of APAP (200 mg/kg body weight, i.p.) was injected into the 0 day APAP-treated group while once-daily APAP treatment over 15 and 30 days was given to the 15 and 30 day APAP-treated groups, respectively. CSD was induced by topical application of KCl on parietal cortex. Ultrastructural alterations and expressions of cell adhesion molecules (ICAM-1 and VCAM-1) of cerebral microvessels were investigated in all animal groups. The results demonstrated that induction of CSD increased ultrastructural alterationsofcerebral endothelial cells and expressionsof ICAM-1 and VCAM-1. A single APAP pretreatment could decrease those alterations induced by CSD. In contrast, ultrastructural alterations and expressions of cell adhesion molecules were enhanced in rats that received chronic APAP treatment (15 and 30 days) in combination with CSD. Additionally, chronic APAP treatment alone could also increase those alterations as compared with control groups.

Nilotinib Interferes with the Signalling Pathways Implicated in Acetaminophen Hepatotoxicity

Nilotinib, a second-generation tyrosine kinase inhibitor, has been recently approved for the treatment for chronic myeloid leukaemia. The objective of this study was to explore the potential effects of clinically relevant doses of nilotinib against acetaminophen (APAP)-induced hepatotoxicity in mice. To simulate the clinical application in human beings, nilotinib (25 and 50 mg/kg) was administered to mice 2 hr after APAP intoxication (500 mg/kg). The results indicated that nilotinib (25 mg/kg) (i) abolished APAP-induced liver injury and necro-inflammation, (ii) increased hepatic-reduced glutathione (GSH) and its related enzymes synthesis, (iii) suppressed hepatic oxidative/nitrosative stress cascades, (iv) decreased neutrophil accumulation in the liver, and (v) prevented the over-expression of B-cell lymphoma-2 (bcl-2), cyclin-D1 and stem cell factor receptor (c-Kit) proteins in the liver. Although nilotinib (50 mg/kg) acted through the same mechanisms, there was severe depletion in hepatic GSH content by nilotinib itself at that dose level, rather than the potent stimulation observed by using a dose of 25 mg/kg. Consequently, the mortality rate after 18 hr was 100% for nilotinib (50 mg/kg) + APAP (750 mg/kg) versus 60% for APAP (750 mg/kg) and 40% for nilotinib (25 mg/kg) + APAP (750 mg/kg) in the survival analysis experiment. In conclusion, nilotinib can counteract the hepatotoxicity produced by a non-lethal dose of APAP. However, there is a risk of aggravating the mortality for a lethal dose of APAP when nilotinib is co-administered at doses relatively high, or near to the clinical range because of hepatic GSH depletion and c-kit inhibition.

Circulating acylcarnitines as biomarkers of mitochondrial dysfunction after acetaminophen overdose in mice and humans

Acetaminophen (APAP) is a widely used analgesic. However, APAP overdose is hepatotoxic and is the primary cause of acute liver failure in the developed world. The mechanism of APAP-induced liver injury begins with protein binding and involves mitochondrial dysfunction and oxidative stress. Recent efforts to discover blood biomarkers of mitochondrial damage have identified increased plasma glutamate dehydrogenase activity and mitochondrial DNA concentration in APAP overdose patients. However, a problem with these markers is that they are too large to be released from cells without cell death or loss of membrane integrity. Metabolomic studies are more likely to reveal biomarkers that are useful at early time points, before injury begins. Similar to earlier work, our metabolomic studies revealed that acylcarnitines are elevated in serum from mice after treatment with toxic doses of APAP. Importantly, a comparison with furosemide demonstrated that increased serum acylcarnitines are specific for mitochondrial dysfunction. However, when we measured these compounds in plasma from humans with liver injury after APAP overdose, we could not detect any significant differences from control groups. Further experiments with mice showed that N-acetylcysteine, the antidote for APAP overdose in humans, can reduce acylcarnitine levels in serum. Altogether, our data do not support the clinical measurement of acylcarnitines in blood after APAP overdose due to the standard N-acetylcysteine treatment in patients, but strongly suggest that acylcarnitines would be useful mechanistic biomarkers in other forms of liver injury involving mitochondrial dysfunction.

Efficacy and Speed of Onset of Pain Relief of Fast-Dissolving Paracetamol on Postsurgical Dental Pain: Two Randomized, Single-Dose, Double-Blind, Placebo-Controlled Clinical Studies

BackgroundParacetamol (APAP), also known as acetaminophen, is the most commonly used over-the-counter analgesic for the treatment of mild-to-moderate pain. However, the speed of onset of pain relief is limited mainly to the standard, immediate-release formulation. Efficacy and speed of onset of pain relief are critical in acute pain situations such as postsurgical dental pain, because reducing pain can improve clinical outcome and reduce the risk of transition from acute pain to more chronic pain. Efficacy and rapid onset also reduce the risk of excessive dosing with the analgesic. ObjectiveWe sought to investigate the dose–response efficacy and speed of onset of pain relief of a fast-dissolving APAP formulation compared with lower doses of APAP and placebo in dental patients after impacted third molar extraction. MethodsTwo single-center, single-dose, randomized, placebo-controlled, double-blind, double-dummy, parallel-group studies (Study I and Study II) were conducted to evaluate the efficacy and speed of onset of pain relief of different doses of a fast-dissolving APAP tablet (FD-APAP), standard APAP, and placebo in patients with postsurgical dental pain following third molar extraction. In Study I, a single dose of FD-APAP 1000 mg, FD-APAP 500 mg, or placebo was given to 300 patients; in Study II, a single dose of FD-APAP 1000 mg, standard APAP 650 mg, or placebo was given to 401 pateints. All 701 patients from both studies were included in the analysis and safety assessment. ResultsFD-APAP 1000 mg demonstrated significantly greater effect compared with FD-APAP 500 mg, APAP 650 mg, and placebo for all efficacy measurements, including sum of pain relief and pain intensity difference, total pain relief, sum of pain intensity difference, pain intensity difference, and pain relief score during 6 hours after the dose. Onset of confirmed first perceptible relief in subjects treated with FD-APAP 1000 mg was 15 minutes, which was 32% and 25% significantly shorter than onset of pain relief of FD-APAP 500 mg (22 minutes) and standard APAP 650 mg (20 minutes), respectively. FD-APAP 500 mg and APAP 650 mg demonstrated efficacy over placebo for most of the measurements; however, their effects were significantly lower and lasted for a shorter period of time than for FD-APAP 1000 mg. All study treatments were well tolerated. ConclusionsFD-APAP 1000 mg tablets demonstrated efficacy over placebo. Also, FD-APAP 1000 mg had significantly superior effect, faster onset, and longer duration of pain relief compared with FD-APAP 500 mg and APAP 650 mg tablets. ClinicalTrials.gov identifiers: NCT01082081 and NCT01075243.

Hepatoprotective, Antioxidant, and Ameliorative Effects of Ginger (Zingiber officinale Roscoe) and Vitamin E in Acetaminophen Treated Rats

ABSTRACTGinger is a remedy known to possess a number of pharmacological properties. This study investigated efficacy of ginger pretreatment in alleviating acetaminophen-induced acute hepatotoxicity in rats. Rats were divided into six groups; negative control, acetaminophen (APAP) (600 mg/kg single intraperitoneal injection); vitamin E (75 mg/kg), ginger (100 mg/kg), vitamin E + APAP, and ginger + APAP. Administration of APAP elicited significant liver injury that was manifested by remarkable increase in plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), arginase activities, and total bilirubin concentration. Meanwhile, APAP significantly decreased plasma total proteins and albumin levels. APAP administration resulted in substantial increase in each of plasma triacylglycerols (TAGs), malondialdhyde (MDA) levels, and total antioxidant capacity (TAC). However, ginger or vitamin E treatment prior to APAP showed significant hepatoprotective effect by lowering the hepatic marker enzymes (AST, ALT, ALP, and arginase) and total bilirubin in plasma. In addition, they remarkably ameliorated the APAP-induced oxidative stress by inhibiting lipid peroxidation (MDA). Pretreatment by ginger or vitamin E significantly restored TAGs, and total protein levels. Histopathological examination of APAP treated rats showed alterations in normal hepatic histoarchitecture, with necrosis and vacuolization of cells. These alterations were substantially decreased by ginger or vitamin E. Our results demonstrated that ginger can prevent hepatic injuries, alleviating oxidative stress in a manner comparable to that of vitamin E. Combination therapy of ginger and APAP is recommended especially in cases with hepatic disorders or when high doses of APAP are required.

Effects of administration of subtoxic doses of acetaminophen on liver and blood levels of insulin-like growth factor-1 in rats

Acetaminophen (APAP) is widely used in the treatment of pain. Toxic doses of APAP cause acute liver failure, but therapeutic doses are believed to be safe. The purpose of this study is to investigate the effects of administration of subtoxic doses of APAP on liver and blood levels of insulin-like growth factor-1 (IGF-1) in rats. Low dose (100 mg/kg) and high dose (250 mg/kg) of APAP were intraperitoneally injected into Wistar albino rats. Following administration of therapeutic doses of APAP, there were no significant changes in serum transaminases and liver glutathione levels. Both doses of APAP induced a decrease in liver and blood levels of IGF-1 when compared with the controls. There was no significant difference in liver IGF-1 levels between the high-dose and low-dose APAP groups; however, there was a significant difference in blood IGF-1 levels between both the groups. The histological examination showed that low dose of APAP induced mild degree of structural change, while high dose of APAP induced severe structural damage. In conclusion, these results suggest that blood IGF-1 levels may have a value in predicting hepatic damage resulting from therapeutic doses of APAP.

An amino acids mixture improves the hepatotoxicity induced by acetaminophen in mice.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, but at high dose it leads to undesirable side effects, such as hepatotoxicity and nephrotoxicity. The aim of this study was to evaluate the protective role of DDM-GSH, a mixture of L-cysteine, L-methionine, and L-serine in a weight ratio of 2 : 1 : 1, in comparison to N-acetylcysteine (NAC), against acetaminophen- (APAP-) induced hepatotoxicity in mice. Toxicity was induced in mice by the intraperitoneal (ip) administration of low dose (2 mmol/kg) or high dose (8 mmol/kg) of APAP. DDM-GSH (0.4 to 1.6 mmol/kg) was given ip to mice 1 h before the APAP administration. The same was done with NAC (0.9 to 3.6 mmol/kg), the standard antidote of APAP toxicity. Mice were sacrificed 8 h after the APAP injection to determine liver weight, serum alanine aminotransferase (ALT), and total glutathione (GSH) depletion and malondialdehyde (MDA) accumulation in liver tissues. DDM-GSH improved mouse survival rates better than NAC against a high dose of APAP. Moreover, DDM-GSH significantly reduced in a dose-dependent manner not only APAP-induced increases of ALT but also APAP-induced hepatic GSH depletion and MDA accumulation. Our results suggest that DDM-GSH may be more potent than NAC in protecting the liver from APAP-induced liver injury.

Disruption of Thioredoxin Reductase 1 Protects Mice from Acute Acetaminophen-Induced Hepatotoxicity through Enhanced NRF2 Activity

The critical importance of glutathione in mitigating the deleterious effects of electrophile generating drugs such as acetaminophen (APAP) is well established. However, the role of other antioxidant systems, such as that provided by thioredoxin, has not been extensively studied. Selenoprotein thioredoxin reductase 1 (Txnrd1) is important for attenuating activation of the apoptosis signaling-regulating kinase 1 (ASK1) and the c-Jun N-terminal kinase (JNK) pathway caused by high doses of APAP. Therefore, a detailed investigation of the role of Txnrd1 in APAP-induced hepatotoxicity was conducted. Liver-specific Txnrd1 knockout mice (Txnrd1(ΔLiv)) were generated and treated with a hepatotoxic dose (400 mg/kg) of APAP for 1 or 6 h. Liver toxicity was assessed by measuring the activities of liver enzymes aspartate aminotransferase and alanine aminotransferase in serum, in addition to histopathological analysis of liver sections and analysis of glutathione levels. At 1 h post-APAP treatment, total and mitochondrial glutathione levels in control and Txnrd1(ΔLiv) mice were similarly depleted. However, at 6 h post-APAP treatment, Txnrd1(ΔLiv) mice were resistant to APAP toxicity as liver enzymes and histology were not significantly different from the corresponding untreated mice. Analyses revealed the compensatory up-regulation of many of the nuclear factor erythroid 2-related factor 2 (NRF2) target genes and proteins in Txnrd1(ΔLiv) mice with and without APAP treatment. Yet, JNK was phosphorylated to a similar extent in APAP-treated control mice. The results suggest that Txnrd1(ΔLiv) mice are primed for xenobiotic detoxication primarily through NRF2 activation.

CHOP is a critical regulator of acetaminophen-induced hepatotoxicity

The liver is a major site of drug metabolism and elimination and as such is susceptible to drug toxicity. Drug induced liver injury is a leading cause of acute liver injury, of which acetaminophen (APAP) is the most frequent causative agent. APAP toxicity is initiated by its toxic metabolite NAPQI. However, downstream mechanisms underlying APAP induced cell death are still unclear. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have recently emerged as major regulators of metabolic homeostasis. UPR regulation of the transcription repressor CHOP promotes cell death. We analyzed the role of UPR and CHOP in mediating APAP hepatotoxicity. A toxic dose of APAP was orally administered to wild type (wt) and CHOP knockout (KO) mice and damage mechanisms were assessed. CHOP KO mice were protected from APAP induced damage and exhibited decreased liver necrosis and increased survival. APAP metabolism in CHOP KO mice was undisturbed and glutathione was depleted at similar kinetics to wt. ER stress and UPR activation were overtly seen 12h following APAP administration, a time that coincided with strong upregulation of CHOP. Remarkably, CHOP KO but not wt mice exhibited hepatocyte proliferation at sites of necrosis. In vitro, large T immortalized CHOP KO hepatocytes were protected from APAP toxicity in comparison to wt control cells. CHOP upregulation during APAP induced liver injury compromises hepatocyte survival in various mechanisms, in part by curtailing the regeneration phase following liver damage. Thus, CHOP plays a pro-damage role in response to APAP intoxication.

Hepatoprotective effects of DL-α-lipoic acid and α-Tocopherol through amelioration of the mitochondrial oxidative stress in acetaminophen challenged rats

Acetaminophen (APAP) is known to induce liver mitochondrial dysfunction leading to acute hepatotoxicity. Effect of DL-α-lipoic acid (LA) and α-tocopherol (α-Toc) against the APAP-induced liver mitochondrial damage was evaluated in rats. LA (100 mg/kg, p.o.) and α-Toc (100 mg/kg, p.o.) were given once daily for 15 d, prior to the APAP administration (3 g/kg, p.o). Hepatic damage was confirmed by determining the activities of serum glutamate pyruvate transaminase, serum glutamate oxaloacetate transaminase and alkaline phosphatase, 4 h after the single dose of APAP. To assess the mitochondrial damage, the activities of antioxidant enzymes, Krebs’ cycle dehydrogenases and mitochondrial electron transport chain complexes, and levels of reactive oxygen species (ROS), reduced glutathione, lipid peroxidation (malondialdehyde, MDA) as well as the mitochondrial membrane potential (Δψmt) were evaluated. The activities of mitochondrial enzymes and Δψmt were significantly (p < 0.01) decreased and the level of ROS and MDA were significantly (p < 0.01) increased due to APAP challenge. LA and α-Toc treatment significantly enhanced the activities of mitochondrial enzymes and Δψmt than that of control group; whereas the levels of ROS and MDA were decreased. The results of the study concluded that the liver damage induced by APAP was significantly ameliorated by LA and α-Toc. LA showed more protection than that of α-Toc. The protection can be partially ascribed to their mitochondrial protective effects through their antioxidant activity which could decrease the level of ROS and by direct enhancement of Δψmt.

Therapeutic effect of liposomal-N-acetylcysteine against acetaminophen-induced hepatotoxicity

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.

The utilization of sulfur amino acid in the mercapturate pathway to detoxify maximal therapeutic dose of acetaminophen is associated with glutathione and protein homeosteny in adult rats

It has been shown that sulfur amino acids (SAA) used for acetaminophen (APAP) detoxification, as sulfate conjugate and mercapturate (glutathione (GSH) conjugation), can represent a significant part of dietary SAA and lead to GSH and protein homeosteny. We determined the metabolic alterations induced by APAP detoxification in adult rats receiving daily the half or maximal therapeutic French dose for human. APAP was provided in a 16% protein diet at the dose of 0 (control, n=6), 0.5 (n=7) or 1% (n=7) for 17 days. 1% APAP reduced nitrogen balance (36%), weights of small intestine (13%) and gastrocnemius (7%), hepatic, muscular and intestinal GSH levels by 56%, 20% and 8%, respectively. These parameters were unchanged by 0.5% APAP. Urinary sulfate conjugate was independent of APAP dose, whereas mercapturate of APAP increased from 3% to 14% of ingested SAA in 0.5 and 1% APAP, respectively. Posttranslational alterations of hepatic proteins (APAP protein adducts and modified residues, assessed by target and global metabolomics) occurred with 0.5% APAP and to a larger extent with 1% APAP. Variations of all studied parameters occurred with the APAP dose that is accompanied by upper use of SAA. Further experiments are required to determine if cysteine supplementation could preserve the structure of hepatic proteins at moderate APAP dose, and limit all metabolic alterations occurring with the APAP dose at 1% of the diet.

Induction of neuronal nitric oxide synthase (nNOS) in livers of mice treated with toxic doses of acetaminophen

In overdose acetaminophen (APAP) is hepatotoxic. Toxicity occurs by metabolism to a reactive metabolite (NAPQI). Following low doses NAPQI is detoxified by GSH; however, in overdose GSH is depleted and NAPQI covalently binds to proteins. This is followed by oxygen/nitrogen stress (protein nitration) and mitochondrial dysfunction. Nitration is by peroxynitrite formed from nitric oxide and superoxide. Toxicity and nitration in hepatocytes were inhibited by two nNOS inhibitors. In mice the principal nitrated protein was MnSOD. In nNOS knockout mice toxicity was delayed with less nitration compared to wildtype mice. We have now examined livers for the presence of nNOS in APAP toxicity. In livers of saline treated mice nNOS was not observed by Western blot analysis. However, in livers of APAP treated mice nNOS was induced. Low levels of nNOS were detected as early as 2 h following APAP. By 4 h nNOS was significantly induced and by 8 h it was dramatically induced. Its relative induction correlated with relative increases in protein nitration of MnSOD and development of toxicity (serum ALT). As anticipated inducible NOS (iNOS) was also induced following a hepatotoxic dose of APAP but its induction appeared to be later than induction of nNOS. These data indicate that nNOS is induced in livers of APAP treated mice. Supported by NIH grant R01 DK079008.