Acetaminophen toxicity in mice lacking NADPH oxidase activity: role of peroxynitrite formation and mitochondrial oxidant stress

Previous data have indicated that activated macrophages may play a role in the mediation of acetaminophen toxicity. In the present study, we examined the significance of superoxide produced by macrophages by comparing the toxicity of acetaminophen in wild-type mice to mice deficient in gp91phox, a critical subunit of NADPH oxidase that is the primary source of phagocytic superoxide. Both groups of mice were dosed with 300 mg/kg of acetaminophen or saline and sacrificed at 1, 2, 4 or 24 h. Glutathione in total liver and in mitochondria was depleted by approximately 90% at 1 h in wild-type and knock out mice. No significant differences in toxicity (serum transaminase levels or histopathology) were observed between wild-type and mice deficient in gp91phox. Mitochondrial glutathione disulfide, as a percent of total glutathione, was determined as a measure of oxidant stress produced by increased superoxide, leading to hydrogen peroxide and/or peroxynitrite. The percent mitochondrial glutathione disulfide increased to approximately 60% at 1 h and 70% at 2 h in both groups of mice. Immunohistochemical staining for nitrotyrosine was present in vascular endothelial cells at 1 h in both groups of mice. Acetaminophen protein adducts were present in hepatocytes at 1 h in both wild-type and knock out animals. These data indicate that superoxide from activated macrophages is not critical to the development of acetaminophen toxicity and provide further support for the role of mitochondrial oxidant stress in acetaminophen toxicity.     

Acetaminophen Toxicity in Mice Lacking NADPH Oxidase Activity: Role of Peroxynitrite Formation and Mitochondrial Oxidant Stress

Previous data have indicated that activated macrophages may play a role in the mediation of acetaminophen toxicity. In the present study, we examined the significance of superoxide produced by macrophages by comparing the toxicity of acetaminophen in wild-type mice to mice deficient in gp91phox, a critical subunit of NADPH oxidase that is the primary source of phagocytic superoxide. Both groups of mice were dosed with 300?mg/kg of acetaminophen or saline and sacrificed at 1, 2, 4 or 24?h. Glutathione in total liver and in mitochondria was depleted by approximately 90% at 1?h in wild-type and knock out mice. No significant differences in toxicity (serum transaminase levels or histopathology) were observed between wild-type and mice deficient in gp91phox. Mitochondrial glutathione disulfide, as a percent of total glutathione, was determined as a measure of oxidant stress produced by increased superoxide, leading to hydrogen peroxide and/or peroxynitrite. The percent mitochondrial glutathione disulfide increased to approximately 60% at 1?h and 70% at 2?h in both groups of mice. Immunohistochemical staining for nitrotyrosine was present in vascular endothelial cells at 1?h in both groups of mice. Acetaminophen protein adducts were present in hepatocytes at 1?h in both wild-type and knock out animals. These data indicate that superoxide from activated macrophages is not critical to the development of acetaminophen toxicity and provide further support for the role of mitochondrial oxidant stress in acetaminophen toxicity.     

Ethanol induction of acetaminophen toxicity and metabolism

Chronic consumption of moderate amounts of ethanol (10% solution in the drinking water) dramatically enhances the toxicity of acetaminophen in CF-1 mice as demonstrated by a significant decrease in the LD₅₀. The increased toxicity appears to result from an acceleration in the microsomal biotransformation of acetaminophen to a reactive intermediate. Accelerated acetaminophen metabolism in ethanol-treated animals, in turn, causes more rapid depletion of glutathione. These studies suggest a mechanism to account for the toxic synergism between chronic alcohol consumption and acute acetaminophen intake.     

17β-Estradiol protects against acetaminophen-overdose-induced acute oxidative hepatic damage and increases the survival rate in mice

Acetaminophen overdose causes acute liver injury or even death in both humans and experimental animals. We investigated the effect of 17β-estradiol against acetaminophen-induced acute liver injury and mortality in mice. Male mice were given acetaminophen (p-acetamidophenol; 300 mg/kg; orally) to induce acute liver injury. Acetaminophen significantly increased the levels of aspartate transaminase, alanine transaminase, myeloperoxidase, lipid peroxidation, and glutathione reductase, but it decreased superoxide dismutase, catalase, and glutathione. In addition, acetaminophen-induced mortality began 4h post-treatment, and all mice died within 9h. 17β-Estradiol (200 μg/kg; i.p.) protected against acetaminophen-induced oxidative hepatic damage by inhibiting neutrophil infiltration and stimulating the antioxidant defense system. However, 17β-estradiol did not affect acetaminophen-induced glutathione depletion or increased glutathione reductase activity. We conclude that 17β-estradiol specifically attenuates acute hepatic damage and decreases mortality in acetaminophen-overdosed male mice.     

Protective effect of polysaccharides-enriched fraction from Angelica sinensis on hepatic injury

A polysaccharides-enriched fraction from the root of Angelica sinensis, which is known for its antiulcer action on the gastrointestinal tract, was isolated and studied for its hepato-protective effect in rodents. Intra-gastric administration of Angelica sinensis polysaccharides-enriched fraction (AP) at the doses of 50 or 75 mg/kg dose-dependently prevented liver toxicity induced by acetaminophen in mice but did not affect the serum acetaminophen concentration. It normalized the rises of serum alanine transferase (ALT) and hepatic nitric oxide synthase (NOS) activities and the decrease of glutathione level in the liver. It also reduced the hepatic malondialdehyde (MDA) concentration. The protective effect was less evident in the carbon tetrachloride (CCl4)-treated animals including mice and rats. In the rat the elevated serum ALT level was unaffected though the MDA level was similarly reduced by the higher dose of AP. In these animals, CCl4 increased the hepatic glutathione level instead while the NOS activity remained unchanged. These findings suggest that the pathogenic mechanisms of both acetaminophen and CCl4 are different. AP is more effective in the protection against liver damage induced by acetaminophen, which is associated with the glutathione depletion and nitric oxide synthase activation in the liver.     

Inflammatory response and glutathione peroxidase in a model of stroke

Stroke is one of the leading causes of death in major industrial countries. Many factors contribute to the cellular damage resulting from ischemia/reperfusion (I/R). Experimental data indicate an important role for oxidative stress and the inflammatory cascade during I/R. We are testing the hypothesis that the mechanism of protection against I/R damage observed in transgenic mice overexpressing human antioxidant enzymes (particularly intracellular glutathione peroxidase) involves the modulation of inflammatory response as well as reduced sensitivity of neurons to cytotoxic cytokines. Transgenic animals show significant reduction of expression of chemokines, IL-6, and cell death-inducing ligands as well as corresponding receptors in a focal cerebral I/R model. Reduction of DNA binding activity of consensus and potential AP-1 binding sites in mouse Fas ligand promoter sequence was observed in nuclear extracts from transgenic mice overexpressing intracellular glutathione peroxidase compared with normal animals following I/R. This effect was accompanied by modulation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway. Cultured primary neurons from the transgenic mice demonstrated protection against hypoxia/reoxygenation injury as well as cytotoxicity after TNF-alpha and Fas ligand treatment. These results indicate that glutathione peroxidase-sensitive reactive oxygen species play an important role in regulation of cell death during cerebral I/R by modulating intrinsic neuronal sensitivity as well as brain inflammatory reactions.     

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.     

Acetaminophen toxicity in lymphocytes heterozygous for glutathione synthetase deficiency

We have studied the effects of acetaminophen metabolites generated by a murine hepatic microsomal system on lymphocytes from two subjects heterozygous for glutathione synthetase deficiency. Heterozygous cells exhibited greater dose-related toxicity than controls. Following a 2-h incubation with acetaminophen and the microsomal system, cells were washed and incubated for 16 h in the presence or absence of N-acetylcysteine, the standard antidote for acetaminophen toxicity. In control cells, glutathione content was replenished to nearly base-line values and toxicity was prevented. N-Acetylcysteine thus prevented toxicity even after covalent binding of acetaminophen metabolites had occurred. Heterozygous cells failed to use N-acetylcysteine as efficiently to resynthesize glutathione, and the cells were not protected from acetaminophen toxicity. Heterozygotes may be at increased risk of toxicity from drugs whose metabolites are detoxified by glutathione conjugation.     

Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice

The autosomal dominant mutation in the human alphaB-crystallin gene inducing a R120G amino acid exchange causes a multisystem, protein aggregation disease including cardiomyopathy. The pathogenesis of cardiomyopathy in this mutant (hR120GCryAB) is poorly understood. Here, we show that transgenic mice overexpressing cardiac-specific hR120GCryAB recapitulate the cardiomyopathy in humans and find that the mice are under reductive stress. The myopathic hearts show an increased recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is due to the augmented expression and enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, and glutathione peroxidase. The intercross of hR120GCryAB cardiomyopathic animals with mice with reduced G6PD levels rescues the progeny from cardiac hypertrophy and protein aggregation. These findings demonstrate that dysregulation of G6PD activity is necessary and sufficient for maladaptive reductive stress and suggest a novel therapeutic target for abrogating R120GCryAB cardiomyopathy and heart failure in humans.     

Lophirones B and C Attenuate Acetaminophen‐Induced Liver Damage in Mice: Studies on Hepatic,Oxidative Stress and Inflammatory Biomarkers

Lophirones B and C are chalcone dimers with proven chemopreventive activity. This study evaluates the hepatoprotective effect lophirones B and C in acetaminophen‐induced hepatic damage in mice using biomarkers of hepatocellular indices, oxidative stress, proinflammatory factors and lipid peroxidation. Oral administrations of lophirones B and C significantly (p < 0.05) attenuated acetaminophen‐mediated alterations in serum alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, albumin and total bilirubin. Similarly, acetaminophen‐mediated decrease in activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose 6‐ phosphate dehydrogenase were significantly attenuated in the liver of mice. Increased levels of conjugated dienes, lipid hydroperoxides, malondialdehyde, protein carbonyl and fragmented DNA were significantly lowered by lophirones B and C. Levels of tumour necrosis factor‐α, interleukin‐6 and 8 were significantly lowered in serum of acetaminophen treated mice by the chalcone dimers. Overall, results of this study show that lophirones B and C halted acetaminophen‐mediated hepatotoxicity.     

Acetaminophen-induced oxidation of protein thiols. Contribution of impaired thiol-metabolizing enzymes and the breakdown of adenine nucleotides

The administration of a hepatotoxic dose of acetaminophen (250 mg/kg) to mice induced the loss of protein thiols in mouse liver. Our data suggest that a significant portion of this loss was due to protein thiol oxidation. The administration of the nonhepatotoxic regioisomer, 3'-hydroxyacetanilide (600 mg/kg) did not produce a similar decrease in liver protein thiols despite producing similar levels of covalent binding. Mice treated with acetaminophen exhibited decreased glutathione peroxidase activity, decreased thioltransferase activity, and decreased adenine nucleotide concentrations in the liver. The increase in urinary allantoin after the administration of acetaminophen suggests that the decrease in adenine nucleotides was due to their degradation in the liver. Acetaminophen also promoted the conversion of the enzyme xanthine dehydrogenase to the oxidase form, and pretreatment of mice with allopurinol, an inhibitor of xanthine oxidase, significantly decreased acetaminophen-mediated hepatotoxicity. The conversion of xanthine dehydrogenase to the oxidase form may lead to a transient increase in the production of activated oxygen species. The increase in activated oxygen species coupled with decreases in glutathione peroxidase and thioltransferase activity may be responsible in part for the increased levels of oxidized protein thiols observed following acetaminophen administration.     

Glutathione and ascorbate reduction of the acetaminophen radical formed by peroxidase. Detection of the glutathione disulfide radical anion and the ascorbyl radical

The acetaminophen phenoxyl radical was generated by the oxidation of acetaminophen by horseradish peroxidase in a fast-flow ESR experiment, and its reaction with glutathione and ascorbate was studied. Glutathione reduces the phenoxyl radical of acetaminophen to regenerate acetaminophen and form the thiyl radical of glutathione. This thiyl radical reacts with the thiolate anion of glutathione to form the disulfide radical anion, which was detected and characterized by ESR spectroscopy. In the presence of ascorbate, the ascorbyl radical was produced by the reduction of the acetaminophen phenoxyl radical by ascorbate. This reaction results in the complete reduction of the free radical of acetaminophen, whereas the glutathione reduction of the phenoxyl radical of acetaminophen was not complete on the fast-flow ESR time scale of milliseconds. This suggests that ascorbate rather than glutathione is more likely to react with the acetaminophen phenoxyl free radical in vivo. In the presence of both ascorbate and higher concentrations of glutathione, the reaction with ascorbate is dominant. When cysteine was used in the place of reduced glutathione in the above assay system, the disulfide radical anion of cystine was observed in a manner similar to glutathione. These reactions may have significance in the detoxification of acetaminophen and the free radical metabolites of xenobiotics in general. Only in cells containing low levels of ascorbate can glutathione play a direct role in the detoxification of the acetaminophen phenoxyl radical.     

Preferential resistance of dopaminergic neurons to the toxicity of glutathione depletion is independent of cellular glutathione peroxidase and is mediated by tetrahydrobiopterin

Depletion of glutathione in the substantia nigra is one of the earliest changes observed in Parkinson's disease (PD) and could initiate dopaminergic neuronal degeneration. Nevertheless, experimental glutathione depletion does not result in preferential toxicity to dopaminergic neurons either in vivo or in vitro. Moreover, dopaminergic neurons in culture are preferentially resistant to the toxicity of glutathione depletion, possibly owing to differences in cellular glutathione peroxidase (GPx1) function. However, mesencephalic cultures from GPx1-knockout and wild-type mice were equally susceptible to the toxicity of glutathione depletion, indicating that glutathione also has GPx1-independent functions in neuronal survival. In addition, dopaminergic neurons were more resistant to the toxicity of both glutathione depletion and treatment with peroxides than nondopaminergic neurons regardless of their GPx1 status. To explain this enhanced antioxidant capacity, we hypothesized that tetrahydrobiopterin (BH(4)) may function as an antioxidant in dopaminergic neurons. In agreement, inhibition of BH(4) synthesis increased the susceptibility of dopaminergic neurons to the toxicity of glutathione depletion, whereas increasing BH(4) levels completely protected nondopaminergic neurons against it. Our results suggest that BH(4) functions as a complementary antioxidant to the glutathione/glutathione peroxidase system and that changes in BH(4) levels may contribute to the pathogenesis of PD.     

Treatment of acetaminophen poisoning.

Acetaminophen is an analgesic that is frequently used in Canada, and the occurrence of overdoses with this drug seems to be increasing. The most serious complication of acetaminophen overdose is hepatic failure. Because of pathophysiologic effects of acetaminophen poisoning and the mechanisms of its toxic effects are now better understood, a rational approach to treatment is possible. Several precursors of glutathione, acetylcysteine in particular, are effective in preventing liver damage if administered within 10 hours of acetaminophen ingestion. Plasma acetaminophen levels are a helpful guide to therapy.     

Mice deficient in Cu,Zn-superoxide dismutase are resistant to acetaminophen toxicity

Although antioxidants are used to treat an overdose of the analgaesic/antipyretic drug APAP (acetaminophen), roles of antioxidant enzymes in APAP-induced hepatotoxicity remain controversial. Our objective was to determine impacts of knockout of SOD1 (superoxide dismutase; Cu,Zn-SOD) alone or in combination with selenium-dependent GPX1 (glutathione peroxidase-1) on APAP-induced hepatotoxicity. All SOD1-null (SOD1-/-) and SOD1- and GPX1-double-knockout mice survived an intraperitoneal injection of 600 mg of APAP per kg of body mass, whereas 75% of WT (wild-type) and GPX1-null mice died within 20 h. Survival time of SOD1-/- mice injected with 1200 mg of APAP per kg of body mass was longer than that of the WT mice (934 compared with 315 min, P<0.05). The APAP-treated SOD1-/- mice had less (P<0.05) plasma ALT (alanine aminotransferase) activity increase and attenuated (P<0.05) hepatic glutathione depletion than the WT mice. The protection conferred by SOD1 deletion was associated with a block of the APAP-mediated hepatic protein nitration and a 50% reduction (P<0.05) in activity of a key APAP metabolism enzyme CYP2E1 (cytochrome P450 2E1) in liver. The SOD1 deletion also caused moderate shifts in the APAP metabolism profiles. In conclusion, deletion of SOD1 alone or in combination with GPX1 greatly enhanced mouse resistance to APAP overdose. Our results suggest a possible pro-oxidant role for the physiological level of SOD1 activity in APAP-mediated hepatotoxicity.     

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.     

Postnatal Methylmercury Exposure Induces Hyperlocomotor Activity and Cerebellar Oxidative Stress in Mice: Dependence on the Neurodevelopmental Period

During the early postnatal period the central nervous system (CNS) is extremely sensitive to external agents. The present study aims at the investigation of critical phases where methylmercury (MeHg) induces cerebellar toxicity during the suckling period in mice. Animals were treated with daily subcutaneous injections of MeHg (7 mg/kg of body weight) during four different periods (5 days each) at the early postnatal period: postnatal day (PND) 1–5, PND 6–10, PND 11–15, or PND 16–20. A control group was treated with daily subcutaneous injections of a 150 mM NaCl solution (10 ml/kg of body weight). Subjects exposed to MeHg at different postnatal periods were littermate. At PND 35, behavioral tests were performed to evaluate spontaneous locomotor activity in the open field and motor performance in the rotarod task. Biochemical parameters related to oxidative stress (levels of glutathione and thiobarbituric acid reactive substances, as well as glutathione peroxidase and glutathione reductase activity) were evaluated in cerebellum. Hyperlocomotor activity and high levels of cerebellar thiobarbituric acid reactive substances were observed in animals exposed to MeHg during the PND 11–15 or PND 16–20 periods. Cerebellar glutathione reductase activity decreased in MeHg-exposed animals. Cerebellar glutathione peroxidase activity was also decreased after MeHg exposure and the lowest enzymatic activity was found in animals exposed to MeHg during the later days of the suckling period. In addition, low levels of cerebellar glutathione were found in animals exposed to MeHg during the PND 16–20 period. The present results show that the postnatal exposure to MeHg during the second half of the suckling period causes hyperlocomotor activity in mice and point to this phase as a critical developmental stage where mouse cerebellum is a vulnerable target for the neurotoxic and pro-oxidative effects of MeHg.     

Energy Stress-Induced Dopamine Loss in Glutathione Peroxidase-Overexpressing Transgenic Mice and in Glutathione-Depleted Mesencephalic Cultures

Abstract: The role of the glutathione system in protecting dopamine neurons from a mild impairment of energy metabolism imposed by the competitive succinate dehydrogenase inhibitor, malonate, was investigated in vitro and in vivo. Treatment of mesencephalic cultures with 10 µ M buthionine sulfoxamine for 24 h reduced total glutathione levels in the cultures by 68%. Reduction of cellular glutathione per se was not toxic to the dopamine population, but potentiated toxicity when the cultures were exposed to malonate. In contrast, transgenic mice overexpressing glutathione peroxidase (hGPE) that received an intrastriatal infusion of malonate (3 µmol) into the left side had significantly less loss of striatal dopamine than their hGPE-negative littermates when assayed 1 week following infusion. These studies demonstrate that manipulation of the glutathione system influences susceptibility of dopamine neurons to damage due to energy impairment. The findings may provide insight into the loss of dopamine neurons in Parkinson's disease in which defects in both energy metabolism and the glutathione system have been identified.     

Overexpression of copper/zinc superoxide dismutase decreases ischemia-like astrocyte injury

Overexpression of copper/zinc superoxide dismutase (SOD1) in transgenic mice protects from transient focal cerebral ischemia in adult animals, but increases oxidative injury in perinatal mice. The effect of SOD1 overexpression on astrocytes subjected to ischemia-like insults has not yet been determined. Overexpression of human SOD1 in astrocytes resulted in a 3-fold increase in SOD1 activity without coupled up-regulation of catalase or glutathione peroxidase activities. Cells subjected to oxygen-glucose deprivation (OGD) or glucose deprivation to mimic ischemic injury were protected by SOD1 overexpression. OGD injury was reduced 47.6+/-9.3%, assessed by release of lactate dehydrogenase. OGD also caused a significant increase in catalase activity which was moderated by SOD1 overexpression. The level of glutathione in astrocytes overexpressing SOD1 was maintained at higher levels following 5 h OGD compared to control cultures under the same conditions. Reduction of glutathione prior to OGD significantly increased cell death of SOD1-overexpressing astrocytes as well as controls, but SOD1 still provided significant protection, suggesting that both GSH-dependent scavenging and GSH-independent scavenging are relevant to SOD1 protection in astrocytes.