Renal and vascular oxidative stress and salt‐sensitivity of arterial pressure

Oxidative stress occurs in a tissue or in the whole body when the total oxidant production exceeds the antioxidant capacity. Recent studies in human essential hypertension indicate that free radical production is increased and antioxidant levels are decreased, and more than one‐half of these hypertensives have a salt‐sensitive type of hypertension with progressive renal damage. Increased oxidative stress may also play a critical role in animal models of salt‐sensitive hypertension. The stroke‐prone spontaneously hypertensive rats (SHRSP) exhibits salt‐sensitivity, vascular release of superoxide is increased, and total plasma antioxidant capacity is decreased. The superoxide release in the SHRSP rats inactivates nitric oxide, and superoxide dismutase (SOD) administration returns the bioactive nitric oxide levels to normal. The deoxycorticosterone acetate (DOCA)‐salt hypertensive rat is salt‐sensitive, aortic superoxide production is increased, and renal inflammation is significant. Treatment of the DOCA‐salt rats with apocynin, an NADPH oxidase inhibitor, decreased aortic superoxide production and decreased arterial pressure. The Dahl salt‐sensitive (S) rat has increased mesenteric microvascular and renal superoxide production and increased plasma levels of H₂O₂. The renal protein expression of SOD is decreased in the kidney of Dahl S rats, and long‐term administration of Tempol, a superoxide mimetic, significantly decreased arterial pressure and renal damage. In conclusion, both human hypertension and experimental models of salt‐sensitive hypertension have increased superoxide release, decreased antioxidant capacity and elevated renal damage.     

Failure of inhibitory effects by platelets on super-oxide anion generation from stimulated neutrophils in a severe bronchial asthmatic

Blood was drawn from healthy human volunteers and a 30-year-old woman with severe bronchial asthma (atopic and perennial type), whose peripheral blood platelets counts were continuously higher than 60 x 10(4)/mm3. Neutrophils were purified on a Conray-Ficoll gradient and platelets were isolated by a gel filtration method with Sepharose 2B. Superoxide anion (O2-) generation from the cells was measured by 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo [1,2-a]purazin-3-one (MCLA)-dependent luminescence. Addition of 0.5 microM MCLA and 10(-8) M N-formyl-methionyl-leucyl-phenylalanine (fMLP) or 10 ng/ml phorbol myristate acetate (PMA) to a suspension of platelets obtained from the healthy controls and the patient caused no significant MCLA-dependent luminescence, 02- generation by neutrophils from healthy human volunteers, activated by 10(-8) M fMLP or 10 ng/ml PMA, was inhibited by their platelets in a concentration-dependent manner. Platelets from the patient failed to inhibit O2- generation from neutrophils activated by 10(-8)M fMLP. Cu-Zn superoxide dismutase concentrations in platelets from healthy human volunteers were not higher than the patient's. These data suggest that the absence of inhibition of superoxide generation from activated neutrophils by platelets might be involved in the pathogenesis of bronchial asthma.     

The suppressive effect of gelatin-conjugated superoxide dismutase on disease development and severity of collagen-induced arthritis in mice.

We studied the effect of superoxide dismutase (SOD) on murine collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis (RA). Among SOD derivatives studied, only gelatin-SOD conjugate which has prolonged half life in vivo was effective to suppress the development of CIA, while native SOD or gelatin carrier alone was ineffective. Interestingly, pyran polymer-conjugated SOD which also has a long half life showed no suppressive effect on the disease. No significant effect on immune response against type II collagen (CII) was found in any of the experimental groups. In addition, induction of suppressor cells was not detected in spleen or lymph node cells of the gelatin-SOD-treated group. Therefore, these results suggest that oxygen radicals may have an important role in the effector phase of the immune response to manifest this chronic autoimmune polyarthritis. Thus, the use of appropriate antioxidants for the treatment of human RA may be rationalized.     

Role of oxygen free radicals in the induction of sister chromatid exchanges by cigarette smoke

Cigarette smoke has been reported to contain free radicals and free radical generators in both the gas and particulate phases. Studies in our laboratory have shown that both cigarette smoke condensate (CSC) and smoke bubbled through phosphate buffered saline solution (smoke-PBS) increased sister chromatid exchanges (SCE) in Chinese hamster ovary cells in a dose-dependent manner. Since oxygen free radicals have been shown to cause SCEs and other chromosomal damage, we investigated the role of these radicals in the induction of SCEs by CSC and smoke-PBS. Addition of the antioxidant enzymes catalase and superoxide dismutase or the oxygen-radical scavenger ascorbic acid failed to reduce the SCE frequency in the presence of either CSC or smoke-PBS. Additional studies indicated that the quantity of hydrogen peroxide produced in CSC or smoke-PBS is too small to account for the observed SCE induction. It appears, therefore, that SCE induction by CSC or smoke-PBS does not involve the participation of oxygen free radicals.     

Hyperoxic Vasoconstriction in the Brain Is Mediated by Inactivation of Nitric Oxide by Superoxide Anions

The hypothesis that decreases in brain blood flow during respiration of hyperbaric oxygen result from inactivation of nitric oxide (NO) by superoxide anions (O_{2 –}) is proposed. Changes in brain blood flow were assessed in conscious rats during respiration of atmospheric air or oxygen at a pressure of 4 atm after dismutation of O_{2 –} with superoxide dismutase or suppression of NO synthesis with the NO synthase inhibitor L-NAME. I.v. administration of superoxide dismutase increased brain blood flow in rats breathing air but was ineffective after previous inhibition of NO synthase. Hyperbaric oxygenation at 4 atm induced decreases in brain blood flow, though prior superoxide dismutase prevented hyperoxic vasoconstriction and increased brain blood flow in rats breathing hyperbaric oxygen. The vasodilatory effect of superoxide dismutase in hyperbaric oxygenation was not seen in animals given prior doses of the NO synthase inhibitor. These results provide evidence that one mechanism for hyperoxic vasoconstriction in the brain consists of inactivation of NO by superoxide anions, decreasing its basal vasorelaxing action.     

云南花粉田七口服液对全血中超氧化物歧化酶活性的影响的实验研究

本研究采用ＳＯＤ微量快速测定法，报道了云南花粉田七口服液（ＹｕｎＮａｎＰｏｌｅｎＴｉｅｎｃｈｉＯｒａｌＬｉｑｕｉｄ，简称ＹＮ－ＰＴＯＬ）对３０只（１２～１４）月龄的昆明种小白鼠红细胞中ＳＯＤ活性影响及其作用机制。研究结果表明，灌胃云南花粉田七口服液的试验组动物，其红细胞ＳＯＤ活性分别与对照组和空白组比较，其中试验组红细胞中ＳＯＤ活性（３２９．９０±５９．７６ｕ／ｍｌ）与相应对照组（２５９．９０±１２２．０８）ｕ／ｍｌ比较差别不明显（Ｐ＞０．０５），但与相应空白组（１５１．０６±６７．２２）ｕ／ｍｌ比较，差别却有显著性意义（Ｐ＜０．０００５）。这提示，在本疗程和试验剂量内，ＹＮ－ＰＴＯＬ能有效提高抗氧化酶ＳＯＤ活性，这不仅对老年动物体内氧化损伤具有防御作用，而且对机体的延年益寿具有重要意义。     

Supraspinal inactivation of mitochondrial superoxide dismutase is a source of peroxynitrite in the development of morphine antinociceptive tolerance

Effective treatment of chronic pain with morphine is limited by decreases in the drug's analgesic action with chronic administration (antinociceptive tolerance). Because opioids are mainstays of pain management, restoring their efficacy has great clinical importance. We have recently reported that formation of peroxynitrite (ONOO⁻, PN) in the dorsal horn of the spinal cord plays a critical role in the development of morphine antinociceptive tolerance and have further documented that nitration and enzymatic inactivation of mitochondrial superoxide dismutase (MnSOD) at that site provides a source for this nitroxidative species. We now report for the first time that antinociceptive tolerance in mice is also associated with the inactivation of MnSOD at supraspinal sites. Inactivation of MnSOD led to nitroxidative stress as evidenced by increased levels of products of oxidative DNA damage and activation of the nuclear factor poly (ADP-ribose) polymerase in whole brain homogenates. Co-administration of morphine with potent Mn porphyrin-based peroxynitrite scavengers, Mn(III) 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP⁵⁺) and Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP⁵⁺) (1) restored the enzymatic activity of MnSOD, (2) attenuated PN-derived nitroxidative stress, and (3) blocked the development of morphine-induced antinociceptive tolerance. The more lipophilic analogue, MnTnHex-2-PyP⁵⁺ was able to cross the blood–brain barrier at higher levels than its lipophylic counterpart MnTE-2-PyP⁵⁺ and was about 30–fold more efficacious. Collectively, these data suggest that PN-mediated enzymatic inactivation of supraspinal MnSOD provides a source of nitroxidative stress, which in turn contributes to central sensitization associated with the development of morphine antinociceptive tolerance. These results support our general contention that PN-targeted therapeutics may have potential as adjuncts to opiates in pain management.     

人超氧化物歧化酶用于细胞毒活性的检测

为了探讨人超氧化物歧化酶(SOD)值测定,作为一种新的细胞毒活性的检测方法及其临床应用的可行性,本文对含有不同细胞数的正常人外周血单个核细胞,反复冻融后,用放免方法(RIA)测定上清中的SOD值,观察四组不同浓度细胞数量与所测SOD值的相关性.测定20份标本SOD值均值±标准差分别为:细胞数为4×106组:SOD值为490±26.3ng/mL;2×106组;249±20.8ng/mL;1×106组:132.55±13.03ng/mL;5×105组:102.65±48.5ng/mL.四组细胞数与SOD均值的相关系数为0.91,绘制散点图,发现5×105组的均值偏离另三组的直线趋势,予以去除后,剩余的三组的相关系数为1.03,呈现完全相关.结果显示:SOD值在同一类型细胞中的含量相对恒定,在个体间一致性较高;SOD值与单个核细胞量存在直线相关性,提示这个参数可用于了解某类细胞被破坏的情况.值得注意的是,在细胞浓度较低时,SOD值可能会存在误差,建议实验时标本的细胞数宜控制在1×106以上.     

氦氖激光照射大鼠心前区对心肌超氧化物歧化酶的影响

目的：为研究低强度激光对自由基代谢的影响,应用不同剂量的氦氖激光照射成年Wistar大鼠的心前区,观察心肌组织中超氧化物歧化酶（SOD）的变化。方法：采用2.87 J/cm^2、7.17 J/cm^2、28.66 J/cm^2三种激光剂量照射成年大鼠心前区,用光化学扩增法检测其心肌SOD含量。结果：能量密度为7.17 J/cm^2的氦氖激光照射,可降低心肌组织的SOD含量,而28.66 J/cm^2的氦氖激光照射,使心肌组织SOD含量升高。结论：氦氖激光照射可改变大鼠心肌SOD含量。     

Immunogold analysis of antioxidant enzymes in human renal cell carcinoma

Analysis of activities of the antioxidant enzyme manganese superoxide dismutase in human renal cell carcinomas often showed greatly altered enzyme levels (either elevated or depressed) compared to the cell of origin, the kidney proximal tubule. In order to better understand the variability observed, immunogold studies were performed on human renal cell carcinomas using a polyclonal antibody to human kidney manganese superoxide dismutase. For comparison, studies were also performed using antibodies to other antioxidant enzymes. For histologic studies, renal cell carcinomas were subclassified on the basis of light microscopy and ultrastructural analysis into clear cell, granular cell, or mixed clear and granular cell variants. In all three types of tumor, immunogold studies showed little staining using antibodies to copper, zinc superoxide dismutase or glutathione-dependent enzymes. However, intensity of labeling for manganese superoxide dismutase and catalase depended on the cell type(s) in the tumor. Clear cell variants demonstrated trace staining for manganese superoxide dismutase and catalase, while granular cell variants exhibited heavy staining for both of these enzymes. Mixed types of tumors showed clear cells with trace staining for all antioxidant enzymes examined, while granular cells again showed intense labeling for manganese superoxide dismutase and catalse. Using normal kidney proximal tubule as a comparison, immunogold ultrastructural analysis using antibody to manganese superoxide dismutase demonstrated infrequent small lightly labeled mitochondria in clear cell variants, while granular cell variants exhibited numerous medium-sized heavily labeled mitochondria. These data suggest that: 1) the variability in activity values for manganese superoxide dismutase may be due to heterogeneity of cell types in these tumors and 2) manganese superoxide dismutase immunoreactive protein was elevated in granular cells both because of an increase in number of mitochondria and because the labeling density in mitochondria was increased compared to mitochondria in clear cell types or in normal proximal tubular cells.     

Anaphylaxis after intra-articular injection of orgotein

Orgotein, a recently introduced anti-inflammatory agent, is the generic name accepted in the U.S.A. for the drug version of copper-zinc superoxide dismutase. This metallo-enzyme is obtained from bovine liver and has a molecular weight of about 32,000. We report on a patient to whom an intra-articular injection of orgotein caused an anaphylactic reaction. An IgE-mediated mechanism was suggested by an immediate positive skin test to orgotein and by the finding of specific IgE against orgotein in the patient's serum.     

间断低氧大鼠血和肺组织丙二醛及超氧化物歧化酶活性的变化

动态测定了常压间断低氧大鼠血清和肺组织丙二醛含量及红细胞和肺组织超氧化物歧化酶活性。结果发现随低氧时间延长，肺动脉高压逐渐形成的过程中有肺组织及血中ＭＤＡ含量显著升高；ＳＯＤ活性呈现先升高后回隆的时相性变化。维生素Ｅ对上述变化有一定的缓解作用，而山莨菪碱的作用不明显。提示间断低氧过程中沛部氧自由基生成增多，可能参与了肺动脉高压的发病过程。     

Neuroprotection of ischemic preconditioning is mediated by thioredoxin 2 in the hippocampal CA1 region following a subsequent transient cerebral ischemia

Preconditioning by brief ischemic episode induces tolerance to a subsequent lethal ischemic insult, and it has been suggested that reactive oxygen species are involved in this phenomenon. Thioredoxin 2 (Trx2), a small protein with redox‐regulating function, shows cytoprotective roles against oxidative stress. Here, we had focused on the role of Trx2 in ischemic preconditioning (IPC)‐mediated neuroprotection against oxidative stress followed by a subsequent lethal transient cerebral ischemia. Animals used in this study were randomly assigned to six groups; sham‐operated group, ischemia‐operated group, IPC plus (+) sham‐operated group, IPC + ischemia‐operated group, IPC + auranofin (a TrxR2 inhibitor) + sham‐operated group and IPC + auranofin + ischemia‐operated group. IPC was subjected to a 2 minutes of sublethal transient ischemia 1 day prior to a 5 minutes of lethal transient ischemia. A significant loss of neurons was found in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) in the ischemia‐operated‐group 5 days after ischemia‐reperfusion; in the IPC + ischemia‐operated‐group, pyramidal neurons in the SP were well protected. In the IPC + ischemia‐operated‐group, Trx2 and TrxR2 immunoreactivities in the SP and its protein level in the CA1 were not significantly changed compared with those in the sham‐operated‐group after ischemia‐reperfusion. In addition, superoxide dismutase 2 (SOD2) expression, superoxide anion radical ( ) production, denatured cytochrome c expression and TUNEL‐positive cells in the IPC + ischemia‐operated‐group were similar to those in the sham‐operated‐group. Conversely, the treatment of auranofin to the IPC + ischemia‐operated‐group significantly increased cell damage/death and abolished the IPC‐induced effect on Trx2 and TrxR2 expressions. Furthermore, the inhibition of Trx2R nearly cancelled the beneficial effects of IPC on SOD2 expression, production, denatured cytochrome c expression and TUNEL‐positive cells. In brief, this study shows that IPC conferred neuroprotection against ischemic injury by maintaining Trx2 and suggests that the maintenance or enhancement of Trx2 expression by IPC may be a legitimate strategy for therapeutic intervention of cerebral ischemia.     

正常胃粘膜、癌周粘膜和癌组织中Cu Zn SOD免疫组化研究

本文首次报道了利用兔抗人Cu Zn SOD抗血清对正常胃粘膜和癌周及癌组织中Cu Zn SOD分布,进行免疫组化定位研究。发现正常胃粘膜组织中Cu Zn SOD染色强度明显高于癌周粘膜(P<0.05);癌周粘膜组织明显高于癌组织(P<0.05)。表明胃癌细胞中Cu Zn SOD具有明显降低的现象。结果还显示了39例胃癌组织中Cu Zn SOD染色阳性强度与术中所见腹腔转移有一定的关系,表现为中度阳性组其腹腔转移率(37.5％)低于弱阳性和阴性组(51.3％)。本文还对上述结果形成机制和外源性SOD制剂的临床应用做了简要探讨。