Lophirones B and C halt acetaminophen hepatotoxicity by upregulating redox transcription factor Nrf‐2 through Akt,PI3K,and PKC pathways

We investigated the mechanism of lophirones B‐ and C‐mediated protection against acetaminophen hepatotoxicity. Mice were pretreated with 20 mg/kg body weight lophirones B and C for 7 days and challenged with acetaminophen on day 7. Acetaminophen raised nuclear factor‐κB (NF‐κB) in the liver of mice but lowered protein kinase B (Akt). Although, acetaminophen produced no significant alteration on nuclear erythroid related factor‐2 (Nrf‐2), phosphoinositide 3‐kinase (PI3K) and protein kinase C (PKC), lophirones B and C raised the level of these proteins and Akt. The acetaminophen‐mediated increase in NF‐κB was significantly reversed by lophirones B and C. Lophirones B and C prevented acetaminophen‐mediated alterations in serum biomarkers of hepatic injury. Similarly, lophirones B and C lowered the biomarkers of oxidative stress in the liver of acetaminophen‐treated mice. It can be inferred from this study that lophirones B and C prevent acetaminophen‐induced liver injury by enhancing Nrf‐2 through Akt, PI3K, and PKC pathways.     

Mechanism of acetaminophen inhibition of cyclooxygenase isoforms

Acetaminophen has similar analgesic and antipyretic properties to nonsteroidal antiinflammatory drugs (NSAIDs), which act via inhibition of cyclooxygenase enzymes. However, unlike NSAIDs, acetaminophen is at best weakly antiinflammatory. The mechanism by which acetaminophen exerts its therapeutic action has yet to be fully determined, as under most circumstances, acetaminophen is a very weak cyclooxygenase inhibitor. The potency of acetaminophen against both purified ovine cyclooxygenase-1 (oCOX-1) and human cyclooxygenase-2 (hCOX-2) was increased approximately 30-fold by the presence of glutathione peroxidase and glutathione to give IC50 values of 33 microM and 980 microM, respectively. Acetaminophen was found to be a good reducing agent of both oCOX-1 and hCOX-2. The results are consistent with a mechanism of inhibition of acetaminophen in which it acts to reduce the active oxidized form of COX to the resting form. Inhibition would therefore be more effective under conditions of low peroxide concentration, consistent with the known tissue selectivity of acetaminophen.     

Anti-oxidative effect of a protein from Cajanus indicus L against acetaminophen-induced hepato-nephro toxicity

Overdoses of acetaminophen cause hepato-renal oxidative stress. The present study was undertaken to investigate the protective effect of a 43 kDa protein isolated from the herb Cajanus indicus, against acetaminophen-induced hepatic and renal toxicity. Male albino mice were treated with the protein for 4 days (intraperitoneally, 2 mg/kg body wt) prior or post to oral administration of acetaminophen (300 mg/kg body wt) for 2 days. Levels of different marker enzymes (namely, glutamate pyruvate transaminase and alkaline phosphatase), creatinine and blood urea nitrogen were measured in the experimental sera. Intracellular reactive oxygen species production and total antioxidant activity were also determined from acetaminophen and protein treated hepatocytes. Indices of different antioxidant enzymes (namely, superoxide dismutase, catalase, glutathione-S-transferase) as well as lipid peroxidation end-products and glutathione were determined in both liver and kidney homogenates. In addition, Cytochrome P450 activity was also measured from liver microsomes. Finally, histopathological studies were performed from liver sections of control, acetaminophen-treated and protein pre- and post-treated (along with acetaminophen) mice. Administration of acetaminophen increased all the serum markers and creatinine levels in mice sera along with the enhancement of hepatic and renal lipid peroxidation. Besides, application of acetaminophen to hepatocytes increased reactive oxygen species production and reduced the total antioxidant activity of the treated hepatocytes. It also reduced the levels of antioxidant enzymes and cellular reserves of glutathione in liver and kidney. In addition, acetaminophen enhanced the cytochrome P450 activity of liver microsomes. Treatment with the protein significantly reversed these changes to almost normal. Apart from these, histopathological changes also revealed the protective nature of the protein against acetaminophen induced necrotic damage of the liver tissues. Results suggest that the protein protects hepatic and renal tissues against oxidative damages and could be used as an effective protector against acetaminophen induced hepato-nephrotoxicity.     

Bee Venom Phospholipase A2 Protects against Acetaminophen-Induced Acute Liver Injury by Modulating Regulatory T Cells and IL-10 in Mice

The aim of this study was to investigate the protective effects of phospholipase A2 (PLA2) from bee venom against acetaminophen-induced hepatotoxicity through CD4⁺CD25⁺Foxp3⁺ T cells (Treg) in mice. Acetaminophen (APAP) is a widely used antipyretic and analgesic, but an acute or cumulative overdose of acetaminophen can cause severe hepatic failure. Tregs have been reported to possess protective effects in various liver diseases and kidney toxicity. We previously found that bee venom strongly increased the Treg population in splenocytes and subsequently suppressed immune disorders. More recently, we found that the effective component of bee venom is PLA2. Thus, we hypothesized that PLA2 could protect against liver injury induced by acetaminophen. To evaluate the hepatoprotective effects of PLA2, C57BL/6 mice or interleukin-10-deficient (IL-10^−/−) mice were injected with PLA2 once a day for five days and sacrificed 24 h (h) after acetaminophen injection. The blood sera were collected 0, 6, and 24 h after acetaminophen injection for the analysis of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). PLA2-injected mice showed reduced levels of serum AST, ALT, proinflammatory cytokines, and nitric oxide (NO) compared with the PBS-injected control mice. However, IL-10 was significantly increased in the PLA2-injected mice. These hepatic protective effects were abolished in Treg-depleted mice by antibody treatment and in IL-10^−/− mice. Based on these findings, it can be concluded that the protective effects of PLA2 against acetaminophen-induced hepatotoxicity can be mediated by modulating the Treg and IL-10 production.     

Protective Effects of Pterostilbene against Acetaminophen‐Induced Hepatotoxicity in Rats

The present study was undertaken to evaluate the protective effect of pterostilbene against acetaminophen‐induced hepatotoxicity. Silymarin was used as a standard hepatoprotective agent. A single dose of acetaminophen (800 mg/kg i.p.), injected to male rats, caused significant increases in serum levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bilirubin, total cholesterol, triglycerides, tumor necrosis factor alpha, and hepatic contents of malondialdehyde, nitric oxide, caspase‐3, hydroxyproline, with significant decreases in serum HDL‐cholesterol, total proteins, albumin, and hepatic activities of reduced glutathione, superoxide dismutase and catalase as compared with the control group. On the other hand, administration of each of pterostilbene (50 mg/kg, p.o.) and silymarin (100 mg/kg, p.o.) for 15 days before acetaminophen ameliorated liver function and oxidative stress parameters. Histopathological evidence confirmed the protection offered by pterostilbene from the tissue damage caused by acetaminophen. In conclusion, pterostilbene possesses multimechanistic hepatoprotective activity that can be attributed to its antioxidant, anti‐inflammatory, and antiapoptotic actions.     

Acetaminophen toxicity. Opposite effects of two forms of glutathione peroxidase

Acetaminophen is one of the most extensively used analgesics/antipyretics worldwide, and overdose or idiopathic reaction causes major morbidity and mortality in its victims. Research into the mechanisms of toxicity and possible therapeutic intervention is therefore essential. In this study, the response of transgenic mice overexpressing human antioxidant enzymes to acute acetaminophen overdose was investigated. Animals overexpressing superoxide dismutase or plasma glutathione peroxidase demonstrated dramatic resistance to acetaminophen toxicity. Intravenous injection of glutathione peroxidase provided normal mice with nearly complete protection against a lethal dose of acetaminophen. Surprisingly, animals overexpressing intracellular glutathione peroxidase in the liver were significantly more sensitive to acetaminophen toxicity compared with nontransgenic littermates. This sensitivity appears to be due to the inability of these animals to efficiently recover glutathione depleted as a result of acetaminophen metabolism. Finally, the results suggest that glutathione peroxidase overexpression modulates the synthesis of several acetaminophen metabolites. Our results demonstrate the ability of glutathione peroxidase levels to influence the outcome of acetaminophen toxicity.     

Effect of Sargassum polycystum (Phaeophyceae)-sulphated polysaccharide extract against acetaminophen-induced hyperlipidemia during toxic hepatitis in experimental rats

The effect of Sargassum polycystum crude extract on lipid metabolism was examined against acetaminophen-induced (800 mg/kg body wt., intraperitoneally) hyperlipidemia during toxic hepatitis in experimental rats. The animals intoxicated with acetaminophen showed significant elevation in the levels of cholesterol, triglycerides and free fatty acid in both serum and liver tissue. The levels of tissue total lipids and serum LDL-cholesterol were also elevated with depleted levels of serum HDL-cholesterol and tissue phospholipid. The acetaminophen-induced animals showed significant alterations in the activities of lipid metabolizing enzymes serum lecithin cholesterol acyl transferase (LCAT) and hepatic triglyceride lipase (HTGL). The levels of liver tissue fatty acids (saturated, mono and polyunsaturated) such as palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid and linolenic acid monitored by gas chromatography were considerably altered in acetaminophen intoxicated animals when compared with control animals. The prior oral administration of Sargassum polycystum (200 mg/kg body wt./day for a period of 15 days) crude extract showed considerable prevention in the severe disturbances of lipid profile and metabolizing enzymes triggered by acetaminophen during hepatic injury. Liver histology also showed convincing supportive evidence regarding their protective nature against fatty changes induced during acetaminophen intoxication. Thus the present study indicates that the protective nature of Sargassum polycystum extract may be due to the presence of active compounds possessing antilipemic property against acetaminophen challenge. (Mol Cell Biochem 276: 89–96, 2005)     

Cytoprotective effects of carvedilol against oxygen free radical generation in rat liver

The protective effects of carvedilol, an antihypertensive agent, against oxidative injury caused by acetaminophen were studied in rat liver. Male Wistar rats (250 +/- 30 g) were pre-treated with carvedilol (3.6 mg/kg, p.o.) for 10 days and on the 11th day received an overdose of acetaminophen (800 mg/kg, p.o.). Four hours after acetaminophen administration, blood was collected to determine serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). After that, rats were killed and the livers were excised to determine reduced glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and carbonyl protein contents, and the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST), and also the DNA damage index. Acetaminophen significantly increased the levels of TBARS, the DNA damage and SOD, AST and ALT activities. Carvedilol was able to prevent lipid peroxidation, protein carbonilation and DNA fragmentation caused by acetaminophen. Moreover, this drug prevented increases in SOD, AST and ALT activities. These results show that carvedilol exerts cytoprotective effects against oxidative injury caused by acetaminophen in rat liver. These effects are probably related to the O2*- scavenging property of carvedilol or its metabolites.     

Effects of Different Semipermeable Membranes on In Vitro and In Vivo Performance of Microdialysis Probes

The in vitro and in vivo performance of three different semipermeable microdialysis membranes was compared: a proprietary polycarbonate-ether membrane made by Carnegie Medecin; cuprophan, a regenerated cellulose membrane; and polyacrylonitrile. When microdialysis probes were tested in a stirred in vitro solution, large and statistically significant differences among the three membranes in extraction of acid metabolites (3,4-dihydroxyphenylacetic acid, 5-hydroxyindoleacetic acid, and homovanillic acid) and acetaminophen were found. Polyacrylonitrile had the highest extractions in vitro. In contrast, when microdialysis probes were implanted in vivo (in rat striatum), extraction of acid metabolites and acetaminophen did not differ significantly among the different membranes. These results are consistent with predictions made by a mathematical model of microdialysis and can be explained by the fact that in vitro the main factor limiting extraction is membrane resistance to diffusion, whereas tissue resistance to diffusion plays a more dominant role in vivo. These findings suggest that (aside from differences in surface area), the choice of semipermeable membrane will generally have little effect on in vivo microdialysis results. Furthermore, in vitro measurements of microdialysis probe extractions are not a reliable way of calibrating in vivo performance.     

Effect of N-acetylcysteine on the pharmacokinetics of acetaminophen in rats

Oral administration of N-acetylcysteine (163 mg/kg at zero time and 82 mg/kg 30 minutes later) to adult male Sprague-Dawley rats given an intravenous injection of acetaminophen, 150 mg/kg at zero time, increased the formation of acetaminophen sulfate and thereby enhanced the elimination of acetaminophen. Apparently, N-acetylcysteine is an in vivo source of inorganic sulfate since availability of the latter is rate-limiting in the formation of acetaminophen sulfate. Increased metabolic conversion of acetaminophen to its sulfate conjugate results in decreased formation of other metabolites of acetaminophen, presumably including the reactive metabolite responsible for the hepatotoxic effect of the drug. This may account, at least in part, for the protective effect of N-acetylcysteine against acetaminophen-induced hepatotoxicity.     

Oxygen-mediated cell injury in the killing of cultured hepatocytes by acetaminophen

Sensitivity of cultured hepatocytes to acetaminophen was induced by pretreatment of the rat with 3-methylcholanthrene. Under these conditions, 10 uM B-naphthoflavone but not SKF-525A prevented the cell killing, indicating dependence on metabolism. Inhibition of glutathione reductase by 50 uM bis-chloro-nitrosourea, shown previously to increase the sensitivity of hepatocytes to an oxidative stress, potentiated the toxicity of acetaminophen without increasing the covalent binding of acetaminophen metabolites. Pretreatment of the hepatocytes with the ferric iron chelator deferoxamine, known to reduce the sensitivity of hepatocytes to an oxidative stress, prevented the cell killing without reducing covalent binding. Addition of ferric chloride to the culture medium restored the sensitivity of the cells to acetaminophen, again without effect on the extent of covalent binding. These data demonstrate that the toxicity of acetaminophen can be dissociated from the covalent binding of its metabolites and support the conclusion that the hepatocytes were lethally injured by an oxidative stress accompanying the mixed function oxidase-dependent biotransformation of acetaminophen.     

Effect of acetaminophen toxicity on erythrocyte osmotic fragility in the Fisher rat

The protective effect of propylthiouracil (PTU) pretreatment against acetaminophen-induced erythrocyte osmotic fragility was determined in the male Fisher rat. Hepatotoxicity was assessed for comparative purposes. PTU (0.15%) was fed in chow for a period of 12 days. Acetaminophen (1 g/kg body wt) was then administered orally by a stomach tube after an overnight fast. The rats were killed either 4 or 24 hr later. Erythrocyte osmotic fragility was determined by the extent of hemolysis in various concentrations of NaCl solutions. Hepatotoxicity was assessed by a rise in serum transaminases and by histological examination of hepatic tissue. PTU treatment when compared with control not only protected rats against acetaminophen-induced hepatotoxicity as reported before, but also protected against erythrocyte osmotic fragility. The time course of acetaminophen toxicity seems to be similar for liver and erythrocyte since both showed damage after 24 hr but not after 4 hr of acetaminophen administration. The data show that PTU pretreatment affords protection against acetaminophen-induced increased erythrocyte osmotic fragility even when their glutathione concentrations were not significantly different, suggesting that PTU per se has a protective effect.     

Hepatoprotective and antioxidant effects of Hedera helix extract on acetaminophen induced oxidative stress and hepatotoxicity in mice

ABSTRACT

Exposure to high doses of acetaminophen is the most common cause of drug induced liver injury. We investigated the protective effects of Hedera helix extract against acetaminophen induced oxidative stress and hepatotoxicity using a mouse model. We used two control groups: group A was given 0.9% NaCl, group B was an acetaminophen control that was given a single injection of 600 mg/kg acetaminophen. T1?T4 groups were pretreated orally with different doses of H. helix extract. The mice were sacrificed and blood samples were collected to estimate the levels of glutathione peroxidase (GPx), malondialdehyde (MDA), superoxide dismutase (SOD) and total bilirubin. Liver samples also were used for histopathological studies. We found that acetaminophen significantly increased the levels of serum ALP, ALT, AST and MDA, and also significantly reduced the antioxidant factors, CAT, GPX and SOD. H. helix extract treatment produced a significant reduction in the levels of ALP, ALT, AST and MDA in serum and restored the levels of CAT, GPX and SOD to control levels. The histopathological findings were consistent with the biochemical findings. H. helix extract exhibited antioxidant and hepatoprotective effects against acetaminophen induced liver damage.     

ADP-ribose pyrophosphatase-I partially purified from livers of rats overdosed with acetaminophen reveals enzyme inhibition in vivo reverted in vitro by dithiothreitol

Free ADP-ribose reacts nonenzymatically with proteins and can lead to intracellular damage. The low-Km ADP-ribose pyrophosphatase-I (ADPRibase-I) is well suited to control free ADP-ribose and nonenzymatic ADP-ribosylation. In vitro, the acetaminophen metabolite N-acetyl-p-benzoquinoneimine (NAPQI) decreases ADPRibase-I Vmax and increases Km, effects not reverted by dithiothreitol (DTT) and attributed to enzyme arylation. The present study was conducted to test whether acetaminophen overdose affected ADPRibase-I in vivo. Rats pretreated with 3-methylcholanthrene and L-buthionine-[S,R]-sulfoximine to potentiate acetaminophen toxicity received an intraperitoneal dose of either acetaminophen (800 mg/ kg; n = 5) or vehicle (n = 3). ADPRibase-I partially purified from acetaminophen-overdosed rats showed a decreased Vmax (0.32+/-0.09 versus 0.60+/-0.03 mU/mg of liver protein; p<0.01) not reverted by DTT and an increased Km for ADP-ribose (1.39+/-0.31 versus 0.67+/-0.05 microM; p<0.01) that, contrary to the in vitro NAPQI effect, was reverted by DTT. Incubation of partially purified ADPRibase-I from normal rat liver with oxidized glutathione elicited a time- and dose-dependent, DTT-reverted increase of Km, without change of Vmax. The results indicate that the activity of ADPRibase-I can be regulated by thiol exchange and that the increase of Km, elicited by acetaminophen overdosage was related to the oxidative stress caused by the drug. It remains to be seen whether an increase of free ADP-ribose concomitant to ADPRibase-I inhibition could contribute to the hepatotoxicity of acetaminophen.     

Evaluating in vitro and in vivo the interference of ascorbate and acetaminophen on glucose detection by a needle-type glucose sensor.

The aim of this work was to assess, in vitro and in vivo, the interference of ascorbate and acetaminophen on glucose measurements by a needle-type glucose sensor detecting hydrogen peroxide generated during the enzymatic oxidation of glucose, and to ascertain whether the protection against interference by the membranes used in the construction of the electrode is feasible. The oxidation of ascorbate and acetaminophen on a platinum electrode set at a 650 mV potential yielded a current representing 75 +/- 5% and 25 +/- 6% of that generated by the oxidation of an equimolar concentration of hydrogen peroxide, respectively. The bias introduced by the presence of 100 mumol l-1 ascorbate on the reading of 5 mmol l-1 glucose by the complete sensor (electrode + membranes) would be minimal (approximately 0.4 mmol l-1). By contrast, the bias introduced by 200 mumol l-1 of acetaminophen (a plasma concentration easily reached in clinical practice) was about 7 mmol l-1. The sensor was implanted subcutaneously in anaesthetized rats (n = 3). Using the current generated in the presence of a plasma acetaminophen concentration of about 200 mumol l-1 for glucose monitoring would lead to a major underestimation (approx. 6 mmol l-1) of subcutaneous glucose concentrations.     

COX-3 the enzyme and the concept: steps towards highly specialized pathways and precision therapeutics?

Cyclooxygenases (COXs) catalyse the key rate-limiting step in prostanoid and thromboxane biosynthesis and are targets of non-steroidal anti-inflammatory drugs (NSAIDs). Until recently, the presence of only two isoforms-COX-1 and COX-2-remained in question because the potent anti-pyretic and analgesic effects of acetaminophen (paracetamol, tylenol ben-u-ron) could not be explained by either COX-1 or COX-2 blockades. A novel COX-1 splice variant termed COX-3, sensitive to acetaminophen, was recently discovered by Simmons et al., and is considered to play a key role in the biosynthesis of prostanoids known to be important mediators in pain and fever. Drugs that preferential block COX-1 also appear to act at COX-3. However the existence of COX-3 at the nucleotide sequence level in humans has been called to question. A functional COX-3 in humans is still to come underlining that the concept of COX-3 is still attractive. Here, we discuss some of the implications drawn from the identification of additional functional cyclooxygenase members in the generation of bioactive autacoids.     

Stable free radical and benzoquinone imine metabolites of an acetaminophen analogue

The enzymatic oxidation of the acetaminophen analogue 3',5'-dimethyl-4'-hydroxyacetanilide (3',5'-dimethylacetaminophen) with the horseradish peroxidase/hydrogen peroxide system forms a phenoxyl free radical metabolite. The structure of this free radical is established by a complete analysis of the ESR spectrum and confirmed by deuterium isotope substitution. Concomitant with phenoxyl radical formation, N-acetyl-3,5-dimethyl-p-benzoquinone imine was detected by optical spectroscopy. The free radical is also formed by comproportionation in solutions of the quinone imine containing added 3',5'-dimethylacetaminophen. In contrast to acetaminophen, the imine and radical metabolites are stable and can be detected without resort to rapid-mixing techniques. Factors leading to the increased stability of these metabolites relative to those formed from acetaminophen are discussed in terms of the toxicity of acetaminophen.     

Protection of acetaminophen induced mitochondrial dysfunctions and hepatic necrosis via Akt-NF-kappaB pathway: role of a novel plant protein

Oxidative stress is a major cause of drug induced hepatic diseases. The present study aims to investigate the antioxidative signaling mechanism of a protein isolated from the herb, Cajanus indicus against acetaminophen induced necrotic cell death. We found that incubation of hepatocytes with the protein prevented acetaminophen-induced loss in cell viability, reduction in glutathione level and enhancement of reactive oxygen species generation. Treatment of mice with the protein before administration of acetaminophen also reduced serum nitrite and TNF-α formation. Moreover, it counteracted acetaminophen-induced loss in mitochondrial membrane potential, loss in adenosine tri phosphate and rise in intracellular calcium. Investigating the cell signaling pathways, we found that the protein exerts its protective action via the activation of NF-κB and Akt and deactivation of STAT-1. Surprisingly, no role of ERK1/2 or STAT-3 was found in the protein-mediated protection of hepatocytes during acetaminophen exposure. Finally, we found that acetaminophen introduces necrosis as the primary phenomena of cell death and protein treatment decreased the necrotic process as evident from the DNA fragmentation and flow-cytometry studies. In addition, administration of the protein to mice before acetaminophen application showed fewer number of TUNEL positive cells. Combining, data suggest that the protein possesses cytoprotective activity against acetaminophen-induced oxidative cellular damage and prevents hepatocytes from necrotic death.     

Effect of crude sulphated polysaccharide from brown algae against acetaminophen-induced toxicity in rats

This study was conducted to examine the protective role of crude polysaccharide from brown seaweed Sargassum polycystum against acetaminophen-induced abnormality in blood glucose, serum albumin/globulin ratio, and liver glycogen, lactate, and pyruvate. Liver and renal tissue histology was performed to confirm the efficacy of Sargassum polysaccharide. A toxic dose of acetaminophen (800 mg/kg body weight intraperitoneally) induced severe abnormality in all basic parameters with apparent toxicity in liver (enlargement of hepatocytes, loss of cytoplasmic content with disruption in the hepatic plates and sinusoidal dilation) and renal tissue (glomerular damage with congestion of tubules). The isolated liver cells were stained with acridine orange and examined under fluorescence microscope, which revealed that the acetaminophen induced significant damage. In contrast, the rats pretreated with Sargassum polysaccharide (200 mg/kg body weight) daily for 3 weeks did not show liver and renal tissue with these severe aberrations induced by acetaminophen. Histology results were also consistent with analyzed basic biochemical parameters, which confirmed the effectiveness of the crude polysaccharide against acetaminophen-induced abnormality in rats.