Comparative Anti-Inflammatory Activity of Different Superoxide Dismutases and Liposomal Sod in Ischemia

Comparison of superoxide dismutases from different sources with respect to biological activity in the rat tourniquet poditis model shows that anti-ischemic activity is very variable although all the enzymes have the same specific enzymic activity. Both bovine Cu-SOD and E. coli Mn-SOD have excellent properties whereas yeast Cu-SOD and the homologous rat Cu-SOD show zero activity. The results confirm earlier demonstrations that (1) “All superoxide dismutases are equal but some are more equal than others”, (2) at the dose levels used (compatible with possible clinical use) homologous enzyme is inefficient and hence human Cu-SOD may not be effective in humans, (3) liposomal encapsulation of bovine Cu-SOD greatly enhances biological efficacity, provides a slow release mechanism of the enzyme and provides a powerful drug for the treatment of ischemic injury.     

Comparative anti-inflammatory activity of different superoxide dismutases and liposomal SOD in ischemia

Comparison of superoxide dismutases from different sources with respect to biological activity in the rat tourniquet poditis model shows that anti-ischemic activity is very variable although all the enzymes have the same specific enzymic activity. Both bovine Cu-SOD and E. coli Mn-SOD have excellent properties whereas yeast Cu-SOD and the homologous rat Cu-SOD show zero activity. The results confirm earlier demonstrations that (1) "All superoxide dismutases are equal but some are more equal than others", (2) at the dose levels used (compatible with possible clinical use) homologous enzyme is inefficient and hence human Cu-SOD may not be effective in humans, (3) liposomal encapsulation of bovine Cu-SOD greatly enhances biological efficacity, provides a slow release mechanism of the enzyme and provides a powerful drug for the treatment of ischemic injury.     

Anti-Inflammatory Activity of Superoxide Dismutases: Inhibition of Adriamycin Induced Edema in Rats

Various superoxide dismutases from different sources, containing Cu, Mn or Fe at the active centre, have been examined with respect to anti-inflammatory activity in a model using adriamycin-induced edema in rats. Very large differences in efficiency are observed, the most active being E. coli Mn-SOD and bovine Cu-SOD. TheFe-SOD from E coli is active whereas P. leiognathiFe-SODisnot. Human Mn-SOD shows no significant activity and homologous rat Cu-SOD is totally inactive. Yeast Cu-SOD shows proinflammatory properties. Anti-inflammatory activity is not a function of molecular weight or circulation life-time.     

Distribution of superoxide dismutase and glutathione peroxidase in the carp: erythrocyte manganese SOD.

A non copper containing superoxide dismutase (Cu-SOD), presumably manganese superoxide dismutase (Mn-SOD), has been identified in carp erythrocytes. Erythrocyte catalase is low, glutathione peroxidase (GPX) is extremely high, and superoxide dismutase (SOD) is relatively low. The distribution of Cu-SOD, Mn-SOD and glutathione peroxidase in various tissues is described. Highest activities of both enzymes are found in the liver and lowest in white muscle and the swim bladder.     

A radioimmunoassay for copper containing superoxide dimutase.

Highly radioactive rat and bovine copper containing superoxide dismutases (Cu-SOD) up to 1700 Ci/mmole of protein were prepared by iodination with ¹²⁵I and chloramine T. Labelled human Cu-SOD was obtained by coupling the enzyme with N-succinimidyl 3-(4-hydroxy, 5-(¹²⁵I)-iodophenyl) propionate. These tracer molecules can be used with anti-bovine or anti-human Cu-SOD antisera to estimate various Cu-SODs as protein (rat, bovine, human) with a sensitivity of 20–80 picogrammes, using standard radio-immunochemical techniques.Control in vivo of the steady state concentration of the radical ion superoxide $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ strongly depends upon the quantity of superoxide dismutase (SOD) present (1). There are several types of enzyme, which are ubiquitous among all aerobic species, but differ in their subcellular location and in the nature of the metal at the active site (2).It is thus of interest to develop a sensitive, specific, and easy assay of the enzymes, particularly if discrimination between mangano and cuprozinc SODs can be achieved. Radioimmunological techniques answer these requirements and can contribute to the solution of a wide variety of problems connected with the metabolism of molecular oxygen.     

Treatment of Brain Trauma with Liposomal Superoxide Dismutase

Brain trauma was induced in rats by impact of a steel bar on the head with a force such that damage (as measured by neurological scoring) was reversible in fourteen days. Systemic treatment (intraperitoneal injections) with free bovine copper superoxide dismutase or a liposomal form of the enzyme considerably shortened recovery time to less than half Tests included cranial nerves - cornean and aural reflexes. and sensorial motricity functions — gripping reflexes. displacement reactions, recovery and flexion reflexes, equilibrium tests and spontaneous mobility. Normalisation of EEG recordings was also greatly accelerated in the case of treated animals. No changes of brain glutathione peroxidase, glutathione transferase or Mn superoxide dismutase in traumatized animals were observed. However a slight decrease in Cu-SOD occurs. Cerebral lipoperoxidation is increased in the traumatized animals compared with controls. This increase is reduced on treatment of the rats with liposomal SOD (or the free enzyme). Very small amounts of the exogenous SOD pass the brain barrier. the permeability of which is increased in traumatized animals. The enzyme is particularly concentrated in the cortex. Despite apparent total neurological recovery at 15 days for untreated traumatized animals. significant differences in EEG recordings, in percentage cerebral water content and in histological examination of brain tissue of these controls compared with treated animals were observed with a net improvement in the latter case. The results obtained with this model suggest that clinical treatment of coma states and brain traumas with liposomal superoxide dismutase may have certain advantages over orthodox treatments     

Treatment of Brain Trauma with Liposomal Superoxide Dismutase

Brain trauma was induced in rats by impact of a steel bar on the head with a force such that damage (as measured by neurological scoring) was reversible in fourteen days. Systemic treatment (intraperitoneal injections) with free bovine copper superoxide dismutase or a liposomal form of the enzyme considerably shortened recovery time to less than half Tests included cranial nerves - cornean and aural reflexes. and sensorial motricity functions — gripping reflexes. displacement reactions, recovery and flexion reflexes, equilibrium tests and spontaneous mobility. Normalisation of EEG recordings was also greatly accelerated in the case of treated animals. No changes of brain glutathione peroxidase, glutathione transferase or Mn superoxide dismutase in traumatized animals were observed. However a slight decrease in Cu-SOD occurs. Cerebral lipoperoxidation is increased in the traumatized animals compared with controls. This increase is reduced on treatment of the rats with liposomal SOD (or the free enzyme). Very small amounts of the exogenous SOD pass the brain barrier. the permeability of which is increased in traumatized animals. The enzyme is particularly concentrated in the cortex. Despite apparent total neurological recovery at 15 days for untreated traumatized animals. significant differences in EEG recordings, in percentage cerebral water content and in histological examination of brain tissue of these controls compared with treated animals were observed with a net improvement in the latter case. The results obtained with this model suggest that clinical treatment of coma states and brain traumas with liposomal superoxide dismutase may have certain advantages over orthodox treatments     

Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells

The current study examines the contribution of mitochondria-derived reactive oxygen species (ROS) in tert-butyl-hydroperoxide (TBH)-induced apoptotic signaling using clones of undifferentiated pheochromocytoma (PC-12) cells that stably overexpress the human mitochondrial or cytoplasmic forms of superoxide dismutase (SOD) (viz. Mn-SOD or CuZn-SOD, respectively). Exposure of wild type cells to TBH caused an early generation of ROS (30 min) that resulted in cell apoptosis at 24 h. These responses were attenuated with N-acetylcysteine pretreatment; however, N-acetylcysteine was ineffective in cytoprotection when added after TBH-induced ROS formation. Stable overexpression of SOD isoforms caused a 2- and 3.5-fold elevation in CuZn-SOD and Mn-SOD activities in the cytoplasm and mitochondria, respectively, and 3-fold increases in cellular GSH content. Accordingly, the stable overexpression of Mn-SOD attenuated TBH-induced mitochondrial ROS generation and cell apoptosis. Whereas transient Mn-SOD expression similarly prevented PC-12 apoptosis, this was associated with increases in SOD activity but not GSH, indicating that cytoprotection by Mn-SOD overexpression is related to mitochondrial ROS elimination and not due to increases in cellular GSH content per se. Stable or transient CuZn-SOD overexpression exacerbated cell apoptosis in conjunction with accelerated caspase-3 activation, regardless of cell GSH levels. Collectively, our results support a role for mitochondrial ROS in TBH-induced PC-12 apoptosis that is attenuated by Mn-SOD overexpression and is independent of cellular GSH levels per se.     

Superoxide dismutase and glutathione reductase overexpression in wheat protoplast: photooxidative stress tolerance and changes in cellular redox state

In previous works, we have established a correlation between antioxidant system response and tolerance to drought, osmotic stress and photooxidative stress of different wheat cultivars with contrasting drought tolerance. In the present work, a protocol to obtain and transform wheat protoplasts was established. Transgenic protoplasts with Manganese Superoxide Dismutase (Mn-SOD) (E.C.: 1.15.1.1) and Glutathione Reductase (GR) (E.C.: 1.6.4.2) overexpression in chloroplasts were obtained, and their responses to photooxidative stress were characterized. Protoplasts with Mn-SOD or GR overexpression, showed different responses and tolerance to photooxidative stress. Protoplasts with Mn-SOD overexpression showed lower levels of oxidative damage, higher level of endogenous hydrogen peroxide and a great induction of total SOD and GR activities during photooxidative treatments. In protoplasts with GR overexpression the oxidative damage provoked by the photooxidative treatment was similar to control protoplasts, the GSH content and GSH/GSH + GSSG ratio were higher than control and Mn-SOD transformed protoplast, and total SOD and GR activities were not induced. Our results suggest that the differential responses and tolerance to photooxidative stress given by Mn-SOD or GR overexpression, also depend on the effects of these enzyme activities over the cellular redox state balance, which modulate the responses to photooxidative stress.     

超氧化物歧化酶切割超螺旋DNA的活性

在体外系统中,发现超氧化物歧化酶(SOD)具有切割超螺旋DNA的活性. 猪血和牛血Cu/Zn-SOD以及烟草Mn-SOD都能将超螺旋DNA转变为非超螺旋结构的缺刻环状DNA,进一步产生线状DNA. 它们只作用于超螺旋DNA而不作用于线状DNA. 这个事实排除了SOD样品中污染核酸酶的可能性. 用H₂O₂、胍基抑制或蛋白酶降解的实验结果表明,这两种酶的活性中心处于酶蛋白的不同部位.     

Effect of cisplatin containing liposomes formulated by unsaturated chain-containing lipids on gynecological tumor cells

Gynecological tumors are major therapeutic areas of platinum-based anticancer drugs. Here, we report the characterization and in vitro biological assays of cisplatin-containing Egg L-α-phosphatidylcholine liposomes with different amounts of cholesterol. Dynamic light scattering estimated sizes of all obtained liposomes in the 100?nm range that are suitable for in vivo use. On the basis of these data and of the drug loading values, the best formulation has been selected. Stability and drug release properties of platinum-containing liposomes have been verified in serum. The growth inhibitory effects of both liposomal and free drug in a panel of ovarian and breast human cancer cell lines, characterized by a different drug sensitivity, give comparable or better results with respect to free cisplatin drug.     

Antioxidant and oxidative status in tissues of manganese superoxide dismutase transgenic mice

Manganese superoxide dismutase (Mn-SOD) plays an important role in attenuating free radical-induced oxidative damage. The purpose of this research was to determine if increased expression of Mn-SOD gene alters intracellular redox status. Twelve week old male B6C3 mice, engineered to express human Mn-SOD in multiple organs, and their nontransgenic littermates were assessed for oxidative stress and antioxidant status in heart, brain, lung, skeletal muscle, liver, and kidney. Relative to their nontransgenic littermates, transgenic mice had significantly (p <.01) higher activity of Mn-SOD in heart, skeletal muscle, lung, and brain. Copper, zinc (Cu,Zn)-SOD activity was significantly higher in kidney, whereas catalase activity was lower in brain and liver. The activities of selenium (Se)-GSH peroxidase and non-Se-GSH peroxidase, and levels of vitamin E, ascorbic acid and GSH were not significantly different in any tissues measured between Mn-SOD transgenic mice and their nontransgenic controls. The levels of malondialdehyde were significantly lower in the muscle and heart of Mn-SOD mice, and conjugated dienes and protein carbonyls were not altered in any tissues measured. The results obtained showed that expression of human SOD gene did not systematical alter antioxidant systems or adversely affect the redox state of the transgenic mice. The results also suggest that expression of human SOD gene confers protection against peroxidative damage to membrane lipids.     

Manganese superoxide dismutase is induced by IFN-gamma in multiple cell types. Synergistic induction by IFN-gamma and tumor necrosis factor or IL-1

We have previously characterized more than 20 proteins induced by the immunoregulatory lymphokine IFN-gamma in human fibroblasts by their m.w. and isoelectric points determined in two-dimensional gels. Some of these proteins are induced uniquely by IFN-gamma, whereas others are also induced by IFN-alpha, TNF, or IL-1. Recent technologic advances have allowed us to begin to rapidly identify proteins induced by IFN-gamma and other cytokines by sequencing the induced proteins from blots of preparative two-dimensional gels of total cell lysates. In this study, we show that the approximately 21 kDa, isoelectric point greater than 7 protein induced by IFN-gamma is manganese superoxide dismutase (Mn-SOD), a mitochondrial protective enzyme encoded by a nuclear gene. Mn-SOD is induced by IFN-gamma and also by TNF in all four human cell lines examined: HS153 fibroblasts, ACHN renal carcinoma, A549 lung carcinoma, and A375 melanoma. Induction of Mn-SOD mRNA is a primary, rapid, and dose-dependent response to IFN-gamma. In ACHN renal carcinoma cells, Mn-SOD mRNA and protein are induced synergistically by IFN-gamma in combination with either TNF or IL-1, and the induced protein is enzymatically active. IFN-gamma and TNF together induce Mn-SOD mRNA by more than 100-fold relative to its level in untreated ACHN cells. The induction of Mn-SOD by IFN-gamma and its synergistic induction by IFN-gamma in combination with TNF and IL-1 should protect healthy cells from the toxicity of O2- during an immune response, and may provide a mechanism for selective killing of infected cells.     

Plasma Superoxide Dismutase Measurement in Children with Viral Hepatitis

Seventy-seven blood samples from normal controls aged 0–8 years and 93 blood samples from children of similar ages with various viral hepatitis were investigated by measuring plasma superoxide dismutase (EC 1.15.1.1) using chemiluminescence immunoassay (CLIA). Total and Cu,Zn-SOD activities of normal controls of group 2(1–8 years old) were significantly higher than that of normal controls of group 1 (0–1 year old) (P < 0.01. P < 0.01), while there were no differences of Mn-SOD activities between the two groups. Total. Cu,Zn-and Mn-SOD activities significantly increased in the acute phase (0–4 weeks after onset) and dropped to the normal levels in the restoration phase (4th week later) for 29 children with cytomegalovirus hepatitis (CMVH), in comparison with group 1. Only Mn-SOD activities were significantly increased in the acute phase (with increased ALT levels) and restoration phase (with normal ALT levels) for 18 children with hepatitis A (HA). Total and Cu,Zn-SOD activities significantly decreased and Mn-SOD activities significantly increased in both the active (with increased ALT levels) and the inactive phases (with normal ALT levels) for 36 children with chronic persistent hepatitis (CPH). Only Cu,Zn-SOD activities fell significantly in both active and inactive phases for 10 children with chronic active hepatitis (CAH).     

Modulation of Endogenous Antioxidant Enzymes by Nitric Oxide in Rat C6 Glial Cells

Abstract: To understand the possible mechanism of nitric oxide (NO)-mediated cytotoxicity, we investigated the effect of NO on the endogenous antioxidant enzymes (AOEs) catalase, glutathione peroxidase (GPX), and CuZn- and Mn-superoxide dismutases (SODs) in rat C₆ glial cells under conditions in which these cells expressed oligodendrocyte-like properties as evidenced by the expression of 2′,3′-cyclic-nucleotide 3′-phosphohydrolase. The 24-h treatment with S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, decreased the activities and the protein levels of catalase, GPX, and Mn-SOD in a dose-dependent manner. Alternatively, the activity and the protein level of CuZn-SOD were increased. 2-Phenyl-4,4, 5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a NO scavenger, blocked the effect of SNAP. Moreover, the treatment of C₆ cells with sodium nitroprusside, another NO donor, or with a combination of lipopolysaccharide (LPS) and interferon-γ (IFN-γ), which induce excessive production of NO, also significantly modulated the AOE activities in a manner similar to that seen with SNAP treatment. The compounds/enzymes that inhibit the production of NO (e.g., N-nitro-l -arginine methyl ester hydrochloride, arginase, and PTIO) blocked the effects of LPS and IFN-γ on the activities of AOEs. Treatment with SNAP and a combination of LPS and IFN-γ also modulated the mRNA levels of AOEs, parallel to the changes in their protein levels and activities, except for Mn-SOD where the combination of LPS and IFN-γ markedly stimulated the mRNA expression. In spite of the stimulation of mRNA level, LPS and IFN-γ significantly inhibited the activity of Mn-SOD within the first 24 h of incubation; however, Mn-SOD activity gradually increased with the increase in time of incubation. These results suggest that alterations in the status of AOEs by NO may be the basis of NO-induced cytotoxicity in disease states associated with excessive NO production.     

Anti-Inflammatory Activity of Superoxide Dismutases: Comparison of Enzymes from Different Sources in Different Models in Rats: Mechanism of Action

Comparison of the anti-inflammatory properties of superoxide dismutases from different sources using different models (carrageenan and adriamycin induced inflammation, adjuvant-induced arthritis) in rats shows a very wide range of activity from extremely good to zero. Neither circulating life time nor intracellular penetration are of importance. The mechanism of biological activity of the SODs is discussed in detail, and binding to an interphase situation on the outer cell surface is postulated. As a consequence of these various considerations it is predicted that clinical application of human Cu-SOD in humans may well be much less spectacular than is commonly assumed, and indeed may be somewhat disappointing.     

Evaluation of Multi-Target and Single-Target Liposomal Drugs for the Treatment of Gastric Cancer

We studied the effects of multi- and single-target liposomal drugs on human gastric cancer cell AGS both in vitro and in vivo. The cytotoxic effect of dihydrotanshinone I was significantly enhanced by treatment with octreotide-polyethylene glycol(PEG)-liposome, Arg-Gly-Asp(RGD)-PEG-liposome, and RGD/octreotide-PEG-liposome encapsulated with 0.5 μg/ml of dihydrotanshinone I to AGS cell for 24 h, compared to control. Furthermore, the AGS cell survival rate for multi-target versus single target liposomal drugs was significantly suppressed. Microsocpic examination revealed that significant cell death occurred in the multi- and single-target liposomal encapsulated drug groups. Significant suppression of tumor growth in AGS cell xenograft nude mice given octreotide-PEG-liposome, RGD/octreotide-PEG-liposome encapsulated drug, versus those given a free drug was noted after 13 d of experimentation with the multi-targeted liposome: up to 60.75% and 41.2% reduction of tumor volume as compared to dimethylsulfoxide (DMSO) control and the free drug groups respectively. The treated animals showed no gross signs of toxicity. The results have potential clinical application.