Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide synthase knockout mice

In overdose acetaminophen (APAP) is hepatotoxic. Toxicity occurs by metabolism to N-acetyl-p-benzoquinone imine, which depletes GSH and covalently binds to proteins followed by protein nitration. Nitration can occur via the strong oxidant and nitrating agent peroxynitrite, formed from superoxide and nitric oxide (NO). In hepatocyte suspensions we reported that an inhibitor of neuronal nitric-oxide synthase (nNOS; NOS1), which has been reported to be in mitochondria, inhibited toxicity and protein nitration. We recently showed that manganese superoxide dismutase (MnSOD; SOD2) was nitrated and inactivated in APAP-treated mice. To understand the role of nNOS in APAP toxicity and MnSOD nitration, nNOS knockout (KO) and wild-type (WT) mice were administered APAP (300 mg/kg). In WT mice serum alanine aminotransferase (ALT) significantly increased at 6 and 8 h, and serum aspartate aminotransferase (AST) significantly increased at 4, 6 and 8 h; however, in KO mice neither ALT nor AST significantly increased until 8 h. There were no significant differences in hepatic GSH depletion, APAP protein binding, hydroxynonenal covalent binding, or histopathological assessment of toxicity. The activity of hepatic MnSOD was significantly lower at 1 to 2 h in WT mice and subsequently increased at 8 h. MnSOD activity was not altered at 0 to 6 h in KO mice but was significantly decreased at 8 h. There were significant increases in MnSOD nitration at 1 to 8 h in WT mice and 6 to 8 h in KO mice. Significantly more nitration occurred at 1 to 6 h in WT than in KO mice. MnSOD was the only observed nitrated protein after APAP treatment. These data indicate a role for nNOS with inactivation of MnSOD and ALT release during APAP toxicity.     

Superoxide-mediated nitration of spinal manganese superoxide dismutase: a novel pathway in N-methyl-D-aspartate-mediated hyperalgesia

N-methyl-D-aspartate (NMDA) receptors serve prominent roles in vast physio-pathological conditions including hyperalgesia (defined as augmented pain intensity in response to painful stimuli) associated with central sensitization. Using M40403 a synthetic low molecular weight superoxide dismutase mimetic that removes superoxide we show for the first time that this radical plays a key role in NMDA-mediated hyperalgesia. Intrathecal administration of NMDA in rats led to a time-dependent development of thermal hyperalgesia. Removal of superoxide with M40403 abolished NMDA-mediated hyperalgesia, while its inactive congener had no effect. Thus NMDA-mediated hyperalgesic response to heat is mediated through spinal release of superoxide. At time of near-to-maximal hyperalgesia, we observed that spinal endogenous manganese superoxide dismutase (MnSOD), the enzyme that normally keeps superoxide under well-controlled condition was nitrated, as shown by immunoprecipitation. Subsequently and as determined by biochemical analysis, nitration of MnSOD led to its deactivation as shown by the loss of the enzyme's ability to dismute and hence remove superoxide. M40403 by preventing MnSOD nitration restored its activity and inhibited the hyperalgesic response to intrathecal NMDA. Thus, superoxide-mediated nitration and deactivation of spinal MnSOD is a novel pathway of NMDA-mediated spinal hyperalgesia and hence central sensitization since it helps to maintain high levels of superoxide that in turn maintains nociceptive signaling. The broader implication of our findings is that superoxide may contribute to various forms of pain events that are driven by NMDA-receptor activation.     

Anesthetic preconditioning confers acute cardioprotection via up-regulation of manganese superoxide dismutase and preservation of mitochondrial respiratory enzyme activity

The cellular and molecular mechanisms that underlie cardioprotection against I/R by anesthetic-induced preconditioning (APC) require further elucidation. Using isoflurane as a representative anesthetic, we evaluated the hypothesis that APC induces myocardial protection against I/R by attenuation of excessive reactive oxygen species and restoration of mitochondrial bioenergetics through postischemic up-regulation of manganese superoxide dismutase (MnSOD) expression and preservation of respiratory enzyme activity. Pentobarbital anesthetized open-chest Sprague-Dawley rats were subject to 30-min left coronary artery occlusion, followed by 120-min reperfusion. Before ischemia, rats were randomly assigned to receive 0.9% saline, two cycles of brief coronary artery occlusion and reperfusion, or a 30-min exposure to 1.0 minimum alveolar concentration isoflurane in the absence or presence of a specific mitochondrial adenosine triphosphate-sensitive potassium (KATP) channel blocker, 5-hydroxydecanoate; a membrane-permeable superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl; or a NOS inhibitor, N(G)-nitro-L-arginine methyl ester. Isoflurane exposure induced an initial increase in myocardial superoxide (O2-), but not NO level. It also significantly decreased infarct size and restored mitochondrial respiratory enzyme activity or ATP production in I/R rat hearts, along with suppression of the O2- surge at reperfusion and increase in MnSOD expression or enzyme activity. These protective effects were abrogated by 5-hydroxydecanoate or 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl, but not by N(G)-nitro-L-arginine methyl ester pretreatment. These results suggest that opening of mitochondrial KATP channel, followed by O2- signaling, induces postischemic augmentation of MnSOD and preservation of mitochondrial respiratory enzyme activities, leading to attenuated cardiac O2- surge and restored ATP production during reperfusion, and underlie APC-induced cardioprotection.     

Inhibitory effects of recombinant manganese superoxide dismutase on influenza virus infections in mice.

The oxygen free-radical scavenger recombinant human manganese superoxide dismutase (MnSOD) was studied for its effects on influenza virus infections in mice when used alone and in combination with ribavirin. Mice challenged with influenza A/NWS/33 (H1N1) virus were treated parenterally in doses of 25, 50, and 100 mg/kg of body weight per day every 8 h for 5 days beginning at 48 h post-virus exposure. An increase in mean day to death, lessened decline in arterial oxygen saturation, and reduced lung consolidation and lung virus titers occurred in the treated animals. To determine the influence of viral challenge, experiments were run in which mice were infected with a 100 or 75% lethal dose of virus and were treated intravenously once daily for 5 days beginning 96 h after virus exposure. Weak inhibition of the mortality rate was seen in mice receiving the high viral challenge, whereas significant inhibition occurred in the animals infected with the lower viral challenge, indicating that MnSOD effects are virus dose dependent. To determine if treatment with small-particle aerosol would render an antiviral effect, infected mice were treated by this route for 1 h daily for 5 days beginning 72 h after virus exposure. A dose-responsive disease inhibition was seen. An infection induced by influenza B/Hong Kong/5/72 virus in mice was mildly inhibited by intravenous MnSOD treatment as seen by increased mean day to death, lessened arterial oxygen saturation decline, and lowered lung consolidation. MnSOD was well tolerated in all experiments. A combination of MnSOD and ribavirin, each administered with small-particle aerosol, resulted in a generally mild improvement of the disease induced by the influenza A virus compared with use of either material alone.     

Bioenergetic deficits in peripheral nerve sensory axons during chemotherapy-induced neuropathic pain resulting from peroxynitrite-mediated post-translational nitration of mitochondrial superoxide dismutase

Many of the widely used anticancer drugs induce dose-limiting peripheral neuropathies that undermine their therapeutic efficacy. Animal models of chemotherapy-induced painful peripheral neuropathy (CIPN) evoked by a variety of drug classes, including taxanes, vinca alkaloids, platinum-complexes, and proteasome-inhibitors, suggest that the common underlying mechanism in the development of these neuropathies is mitotoxicity in primary nerve sensory axons (PNSAs) arising from reduced mitochondrial bioenergetics [eg adenosine triphosphate (ATP) production deficits due to compromised respiratory complex I and II activity]. The causative mechanisms of this mitotoxicity remain poorly defined. However, peroxynitrite, an important pro-nociceptive agent, has been linked to mitotoxicity in several disease states and may also drive the mitotoxicity associated with CIPN. Our findings reveal that the development of mechano-hypersensitivity induced by paclitaxel, oxaliplatin, and bortezomib was prevented by administration of the peroxynitrite decomposition catalyst Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTE-2-PyP⁵⁺) without interfering with their anti-tumor effects. Peak CIPN was associated with the nitration and inactivation of superoxide dismutase in the mitochondria, but not in the cytosol, as well as a significant decrease in ATP production within the PNSAs; all of these events were attenuated by MnTE-2-PyP⁵⁺. Our results provide continued support for the role of mitotoxicity in the development of CIPN across chemotherapeutic drug classes, and identify peroxynitrite as a key mediator in these processes, thereby providing the rationale towards development of “peroxynitrite-targeted” therapeutics for CIPN.     

The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice.

Adriamycin (ADR) is a potent anticancer drug known to cause severe cardiac toxicity. Although ADR generates free radicals, the role of free radicals in the development of cardiac toxicity and the intracellular target for ADR-induced cardiac toxicity are still not well understood. We produced three transgenic mice lines expressing increased levels of human manganese superoxide dismutase (MnSOD), a mitochondrial enzyme, as an animal model to investigate the role of ADR-mediated free radical generation in mitochondria. The human MnSOD was expressed, functionally active, and properly transported into mitochondria in the heart of transgenic mice. The levels of copper-zinc SOD, catalase, and glutathione peroxidase did not change in the transgenic mice. Electron microscopy revealed dose-dependent ultrastructural alterations with marked mitochondrial damage in nontransgenic mice treated with ADR, but not in the transgenic littermates. Biochemical analysis indicated that the levels of serum creatine kinase and lactate dehydrogenase in ADR-treated mice were significantly greater in nontransgenic than their transgenic littermates expressing a high level of human MnSOD after ADR treatment. These results support a major role for free radical generation in ADR toxicity as well as suggesting mitochondria as the critical site of cardiac injury.     

肝癌患者血清及组织中ROS、T-SOD、MnSOD水平的测定

目的　研究原发性肝癌患者血清及组织内活性氧类物质 (ROS)、总超氧化物歧化酶 (T SOD)、锰超氧化物歧化酶 (MnSOD)水平。方法　选择 2 0例原发性肝癌组织和癌旁组织及对应血清。采用Fenton反应测定血清中ROS ,组织内ROS测定采用流式细胞技术。患者血清及组织匀浆内T SOD、MnSOD采用黄嘌呤氧化酶法测定。结果　原发性肝癌患者血清和组织内ROS水平明显高于正常对照 (P <0 .0 1) ,肿瘤患者血清中T SOD和MnSOD水平低于正常对照 (P <0 .0 1) ,血清中ROS与MnSOD呈明显负相关 (r =- 0 .92 4 ) ,癌组织内T SOD和MnSOD水平明显高于癌旁组织 (P <0 .0 1)。结论　ROS可能参与肝癌的发生 ,MnSOD在消除ROS阻止肝癌发生中可能发挥重要作用。血清中ROS、MnSOD与组织ROS、MnSOD之间可能有重新分配的过程。     

肝癌患者血清及组织中ROS、T-SOD、MnSOD水平的测定

目的研究原发性肝癌患者血清及组织内活性氧类物质(ROS)、总超氧化物歧化酶(T-SOD)、锰超氧化物歧化酶(MnSOD)水平.方法选择20例原发性肝癌组织和癌旁组织及对应血清.采用Fenton反应测定血清中ROS,组织内ROS测定采用流式细胞技术.患者血清及组织匀浆内T-SOD、MnSOD采用黄嘌呤氧化酶法测定.结果原发性肝癌患者血清和组织内ROS水平明显高于正常对照(P<0.01),肿瘤患者血清中T-SOD和MnSOD水平低于正常对照(P<0.01),血清中ROS与MnSOD呈明显负相关(r=-0.924),癌组织内T-SOD和MnSOD水平明显高于癌旁组织(P<0.01).结论 ROS可能参与肝癌的发生,MnSOD在消除ROS阻止肝癌发生中可能发挥重要作用.血清中ROS、MnSOD与组织ROS、MnSOD之间可能有重新分配的过程.     

Metal ion chelating peptides with superoxide dismutase activity.

The superoxide dismutase activities of two novel synthetic chelating peptides are reported. The peptides comprise a polyaminocarboxylic acid chelator (EDTA) conjugated to tyrosine (ET(1)) and phenylalanine (EP(1)). Superoxide dismutase (SOD) activity was exhibited for Cu(II) but not the Fe(III) complexes. The mimetic activities were compared to bovine erythrocyte SOD (3730 U/mg) and exhibited activities of 1119 U/mg for ET(1)-Cu(II) and 551 U/mg for EP(1)-Cu(II). Thus, small alterations in structure can have significant effects on the enzymatic activity of metallopeptides. These synthetic chelators have dual potential anti-inflammatory activity by chelating deleterious non-protein bound metal ions and concomitantly affording anti-oxidant mimetic activity.     

Functional polymorphism in the manganese superoxide dismutase (MnSOD) gene in patients with asthma

OBJECTIVES: To investigate the possible association between functional variant Ala-9Val in the MnSOD gene and asthma in the case-control study comprising 626 Caucasian subjects. METHODS: MnSOD genotypes were determined by PCR with subsequent restriction analysis by the BsaWI enzyme. RESULTS: Significant differences in allele frequencies between groups were not ascertained. CONCLUSIONS: Pursuant to these results, Ala-9Val polymorphism does not seem to be a significant predisposing factor for bronchial asthma in the Czech population.     

The copper-zinc superoxide dismutase activity in selected diseases

Copper-zinc superoxide dismutase (Cu,Zn-SOD) plays a protective role in various types of tissue protecting them from oxidative damage. Alterations in Cu,Zn-SOD (SOD1 and SOD3) activity and its expression have been observed in pathological occurrences most prevalent in modern society, including inflammatory bowel disease, obesity and its implications—diabetes and hypertension, and chronic obstructive pulmonary disease. Moreover, several SOD1 and SOD3 gene polymorphisms have been associated with the risk of developing a particular type of disease, or its exacerbation. This article features recent observations in this topic, aiming to show the importance of proper gene sequence and activity of Cu,Zn-SOD in the aforementioned diseases.     

超氧化物歧化酶对失血性休克大鼠能量代谢的影响

观察失血性休克大鼠重要生命器官细胞能量代谢的变化及超氧化物歧化酶(SOD)的保护作用。发现:随着休克的进展,大鼠心、肝、肾组织三磷酸腺苷(ATP)、磷酸激酶(CP)含量呈进行性减少,至休克2h,较对照组显著降低。经SOD治疗后,其含量明显增加,接近或达到正常水平,SOD并能显著提高休克动物的时间存活率及延长其存活时间。结果提示:氧衍生自由基的反应性损伤,可能是失血性休克细胞能量代谢障碍的重要原因;静脉注射SOD能有效地对抗氧自由基的这种损伤作用,从而保护休克时的能量代谢。     

Immunological indicators and superoxide dismutase activity in the erythrocytes of patients with viral hepatitis

The paper is concerned with the measurement of the relative and absolute counts of lymphocytes, T and B lymphocytes (106 patients) and superoxide dismutase (SOD) activity of red blood cells (237 patients) in acute and chronic viral hepatitis (VH). In addition, a study was made of SOD activity of red blood cells in guinea-pigs immunized with bovine serum albumin (BSA). It is shown that SOD activation in red blood cells related to the enzyme induction with superoxide anionic radical was seen both in patients with acute and chronic VH and in guinea-pigs immunized with BSA. The patients with acute and chronic VH demonstrated the reduction of the absolute count of T lymphocytes and the increase of the B lymphocyte count in the peripheral blood together with SOD activation in red blood cells, more remarkable in acute than in chronic VH. The immunological response in VH patients manifested since days 14-20 from the disease onset or its exacerbation which was evidenced by changes in the cooperative interaction of cellular immunity and SOD activity.     

Plasma extracellular superoxide dismutase and erythrocyte Cu,Zn-containing superoxide dismutase in alcoholics treated with disulfiram

Disulfiram has long been used in the treatment of chronic alcoholism. It is in vivo partially reduced to diethyldithiocarbamate, which is an efficient inhibitor of Cu,Zn-containing superoxide dismutase both in vitro and in vivo. The recently described extracellular superoxide dismutase is even more sensitive to diethyldithiocarbamate than Cu,Zn-superoxide dismutase. To test for the possibility that long term treatment with disulfiram leads to inhibiton of the superoxide dismutases, plasma extracellular superoxide dismutase and erythrocyte Cu,Zn-superoxide dismutase were determined in 12 disulfiram-treated alcoholics, and compared with 11 non-treated alcoholics and 19 healthy controls. Plasma extracellular superoxide dismutase was moderately reduced (about 20%) in the disulfiram-treated alcoholics as compared with the non-treated alcoholics and the healthy controls. No effect of disulfiram treatment on erythrocyte Cu,Zn-superoxide dismutase activity was demonstrated.     

The Ala-9Val polymorphism in the mitochondrial targeting sequence (MTS) of the manganese superoxide dismutase gene is not associated with juvenile-onset asthma

BACKGROUND: We aimed to investigate the possible association between Mn-SOD polymorphism in the mitochondrial targeting sequence and asthma. METHODS: Alanine or valine polymorphism in the signal peptide of Mn-SOD gene was evaluated using a primer pair to amplify a 107-bp fragment followed by digestion with NgoM IV. RESULTS: No significant difference in genotype frequencies was found between patients and controls. CONCLUSION: These results suggest no major modifying role for the Mn-SOD gene polymorphism in patients with asthma.     

Pertussis toxin treatment alters manganese superoxide dismutase activity in lung. Evidence for lung oxygen toxicity in air-breathing rats.

Exposure of rats to hyperoxia or to treatment with endotoxin, increases lung manganese superoxide dismutase (MnSOD) gene expression. However, the paths by which these environmental signals are transduced into enhanced MnSOD gene expression are unknown. We now provide evidence that heterotrimeric G proteins are involved in the hyperoxia-induced increase in lung MnSOD gene expression but that pertussis toxin-sensitive G proteins are not involved in the endotoxin-induced elevation of lung MnSOD gene expression. We also show that treating rats with pertussis toxin decreased lung MnSOD activity approximately 50%. This decline in MnSOD activity occurred without a change in the lung activity of copper-zinc SOD, catalase, or glutathione peroxidase. In air-breathing rats, the pertussis toxin-induced decrease in MnSOD activity was associated with the development of lung edema, pleural effusion with a high concentration of protein, and biochemical evidence of lung oxygen toxicity. Compared to air-breathing rats, maintenance of pertussis toxin-treated rats under hypoxic or hyperoxic conditions respectively decreased or increased intrathoracic fluid. Endotoxin treatment elevated lung MnSOD activity and protected pertussis toxin-treated rats from an increase in intrathoracic fluid.     

Semi-automated assay of erythrocyte Cu-Zn superoxide dismutase activity.

A semi-automated enzymatic colorimetric method for determining Cu-Zn superoxide dismutase (SOD) in erythrocytes using an FP9 parallel analyzer is described. The method is based on the reaction described by McCord and Fridovich (xanthine/xanthine oxidase, ferricytochrome C), which permits kinetic measurements to be determined colorimetrically for the assay of enzyme activity. The coefficients of variation for within- and between-run analyses were less than 6%. The limit of linearity is 3.5-times the mean value of the normal range while recovery of added SOD ranged from 91 to 120%. The method, which is both reliable and simple, allows rapid, simultaneous measurements of multiple samples.