The overexpression of major antioxidant enzymes does not extend the lifespan of mice

We evaluated the effect of overexpressing antioxidant enzymes on the lifespans of transgenic mice that overexpress copper zinc superoxide dismutase (CuZnSOD), catalase, or combinations of either CuZnSOD and catalase or CuZnSOD and manganese superoxide dismutase (MnSOD). Our results show that the overexpression of these major antioxidant enzymes, which are known to scavenge superoxide and hydrogen peroxide in the cytosolic and mitochondrial compartments, is insufficient to extend lifespan in mice.     

Expression of bovine and mouse endothelial cell antioxidant enzymes following TNF-alpha exposure

Endothelial cells are primary targets for injury by reactive oxygen species. Endothelial catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganous superoxide dismutase (MnSOD) provide potential antioxidant enzymatic defenses against oxidant-induced cellular damage. Previous studies in vivo and in vitro have demonstrated that in certain cell types exposure to oxidants may increase the expression of one or more of these antioxidant enzymes, thus providing greater intracellular potential to withstand oxidant-induced cell stress. To test whether endothelial antioxidant enzyme expression is influenced by similar oxidant-induced stresses in vitro, we have exposed endothelial cells to tumor necrosis factor-alpha (TNF-alpha) and have measured levels of catalase, CuZnSOD and MnSOD mRNA, and protein. Our results demonstrate a selective increase of MnSOD mRNA, with coordinate increases of both MnSOD protein and enzyme activity in endothelial cells treated for 24/h with TNF-alpha. In contrast, levels of catalase and CuZnSOD mRNA and protein remained unchanged in these cells after TNF-alpha treatment. These observations were made in microvessel endothelial cells derived from murine and bovine sources. Our results indicate that TNF-alpha can act specifically to increase enzymatic antioxidant potential in endothelial cells by induction of a particular antioxidant enzyme encoding mRNA species. These data demonstrate the capacity of endothelial cells to mount an antioxidant defense in response to exposure to an inducer of oxidative damage.     

Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase

In evaluating the relative expression of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in vivo in states like Down syndrome in which one dismutase is present at increased levels, we measured activities of both enzymes, in tissues of control and transgenic mice constitutively expressing increased levels of CuZnSOD, during exposure to normal and elevated oxygen tensions. Using SOD gel electrophoresis assay, CuZnSOD and MnSOD activities of brain, lung, heart, kidney, and liver from mice exposed to either normal (21%) or elevated (>99% oxygen, 630 torr) oxygen tensions for 120 h were compared. Whereas CuZnSOD activity was elevated in tissues of transgenic relative to control mice under both normoxic or hyperoxic conditions, MnSOD activities in organs of transgenic mice were remarkably similar to those of controls under both conditions. To confirm the accuracy of this method in quantitating MnSOD relative to CuZnSOD expression, two other methods were utilized. In lung, which is the organ exposed to the highest oxygen tension during ambient hyperoxia, a sensitive, specific ELISA for MnSOD was used. Again, MnSOD protein was not different in transgenic relative to control mice during exposure to air or hyperoxia. In addition, lung MnSOD protein was not changed significantly by exposure to hyperoxia in either group. In kidney, a mitochondrion-rich organ, SOD assay, before and after inactivation of CuZnSOD with diethyldithiocarbamate, was used. MnSOD activity was not different in organs from air-exposed transgenic relative to control mice. The data indicated that expression of MnSOD in vivo was not affected by overexpression of the CuZnSOD and, therefore, the two enzymes are probably regulated independently.     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.     

Age-related changes in antioxidant enzymes and lipid peroxidation in brains of control and transgenic mice overexpressing copper-zinc superoxide dismutase.

The aim of our study was first to obtain a comprehensive profile of the brain antioxidant defense potential and peroxidative damage during aging. We investigated copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), seleno-dependent glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-R) activities, endogenous and in vitro stimulated lipid peroxidation in 40 brains of control mice divided into 3 age groups: 2 months (young), 12 months (middle-aged) and 28 months (old). We found a positive correlation between age and activities of CuZnSOD (r = 0.47; P < 0.01) and GSH-PX (r = 0.72; P < 0.0001). CuZnSOD and GSH-PX activities are independently regulated during brain aging since temporal changes of these two enzymes do not correlate. No modification in MnSOD activity and basal lipid peroxidation was observed as a function of age. Nevertheless, stimulated lipid peroxidation was significantly higher at 12 months (6.53 +/- 0.71 mumole MDA/g tissue) than at 2 months (5.69 +/- 0.90) and significantly lower at 28 months (5.13 +/- 0.33) than at 12 months. Second, we used genetic manipulations to construct transgenic mice that specifically overexpress CuZnSOD to understand the role of CuZnSOD in neuronal aging. The human CuZnSOD transgene expression was stable during aging. The increased CuZnSOD activity in the brain (1.9-fold) of transgenic mice resulted in an enhanced rate of basal lipid peroxidation and in increased MnSOD activity in the 3 age groups. Other antioxidant enzymes did not exhibit modifications indicating the independence of the regulation between CuZnSOD and glutathione-related enzymes probably due to their different cellular localization in the brain.     

Expression of superoxide dismutases, catalase, and glutathione peroxidase in glioma cells

Four primary antioxidant enzymes were measured in both human and rat glioma cells. Both manganese-containing superoxide dismutase (MnSOD) and copper-zinc-containing superoxide dismutase (CuZnSOD) activities varied greatly among the different glioma cell lines. MnSOD was generally higher in human glioma cells than in rat glioma cells and relatively higher than in other tumor types. High levels of MnSOD in human glioma cells were due to the high levels of expression of MnSOD mRNA and protein. Heterogeneous expression of MnSOD was present in individual glioma cell lines and may be due to subpopulations or cells at different differentiation stages. Less difference in CuZnSOD, catalase, or glutathione peroxide was found between human and rat glioma cells. The human glioma cell lines showed large differences in sensitivity to the glutathione modulating drugs 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and buthionine sulfoximine (BSO). A good correlation was found between sensitivity to BCNU and the activities of catalase in these cell lines. Only one cell line was sensitive to BSO and this line had low CuZnSOD activity.     

Prevention of mitochondrial dysfunction in post-traumatic mouse brain by superoxide dismutase

Reactive oxygen species (ROS) are known to be involved in the pathogenesis of traumatic brain injury (TBI). Previous studies have shown that the susceptibility of mice to TBI-induced formation of cortical lesion is determined by the expression levels of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD, respectively). However, the underlying biochemical mechanisms are not understood. In this study, we measured the efficiency of mitochondrial respiration in mouse brains with altered expression of these two enzymes. While controlled cortical impact injury (CCII) with a deformation depth of 2 mm caused a drastic decrease in NAD-linked bioenergetic capacity in brain mitochondria of wild-type mice, the functional decrease was not observed in brains of littermate transgenic mice overexpressing CuZnSOD or MnSOD. In addition, a 1 mm CCII greatly compromised brain mitochondrial function in mice deficient in CuZnSOD or MnSOD, but not wild-type mice. Inclusion of the calcium-chelating agent, EGTA, in the assay solution could completely prevent dysfunction of oxidative phosphorylation in all mitochondrial samples, suggesting that the observed impairment of mitochondrial function was a result of calcium overloading. In conclusion, our results imply that mitochondrial dysfunction induced by superoxide anion radical contributes to lesion formation in mouse brain following physical trauma.     

Overexpression of manganese or copper-zinc superoxide dismutase inhibits breast cancer growth

We have studied the effects of overexpression of superoxide dismutase (SOD), a tumor suppressor protein that dismutes superoxide radical to H2O2, on breast cancer cell growth in vitro and xenograft growth in vivo. No previous work has directly compared the growth-suppressive effects of manganese SOD (MnSOD) and copper-zinc SOD (CuZnSOD). We hypothesized that either adenoviral MnSOD (AdMnSOD) or adenoviral CuZnSOD (AdCuZnSOD) gene therapy would suppress the growth of human breast cancer cells. After determining the antioxidant profiles of three human breast cell lines, MCF 10A, MDA-MB231, and MCF-7, we measured the effects of MnSOD or CuZnSOD overexpression on cell growth and survival in vitro and in vivo. Results demonstrated that infection with AdMnSOD or AdCuZnSOD increased the activity of the respective enzyme in all three cell lines. In vitro, overexpression of MnSOD or CuZnSOD decreased not only cell growth but also clonogenic survival in a dose- and transgene-dependent manner. In vivo, treatment of tumors with AdMnSOD or AdCuZnSOD decreased xenograft growth compared to controls. The first direct comparison of MnSOD to CuZnSOD overexpression indicated that CuZnSOD and MnSOD were similarly effective at suppressing cancer cell growth.     

Messenger RNA for manganese and copper-zinc superoxide dismutases in hepatomas: correlation with degree of differentiation

Total and polyadenylylated RNA have been isolated from two Morris hepatomas with different degree of differentiation and from the normal liver of the corresponding tumor-bearing inbred rats. The analysis of mRNA has been performed by Northern hybridization using 32P-dA-tailed synthetic deoxyoligonucleotide probes, 33-mer for Mn superoxide dismutase (SOD) and 36-mer for CuZnSOD, derived from the nucleotide sequences of the rat enzyme cDNAs. Two distinct mRNA species (about 850 and 1080 nucleotides) have been identified by using the MnSOD probe. CuZnSOD is translated from a single message of about 720 nucleotides. The total MnSOD mRNA concentration is decreased by 43% and 57% in the hepatomas 9618A (highly differentiated) and 3924A (poorly differentiated), respectively. CuZnSOD mRNA is practically unchanged in the hepatoma 9618A whereas it is reduced by 80% in the hepatoma 3924A. Comparison of the enzyme activities and mRNA levels indicates a good correlation only for hepatoma 3924A, suggesting that the changes of both SODs are regulated pretranslationally. From the data obtained it is also inferred that the mRNA levels of MnSOD respond more readily than those of CuZnSOD to changes in differentiation.     

Protective Effects of Human Recombinant Mnsod in Adjuvant Arthritis and Bleomycin-Induced Lung Fibrosis

We have previously shown that human recombinant methionyl manganese superoxide dismutase (MnSOD) is more efficient than CuZnSOD as an anti-inflammatory agent in a model of acute inflammation (Carrageenan-induced pau edema). This effect was attributed to the prolonged half-life of MnSOD in blood (t_1/2 = 6 h vs. 10 min. respectively). In the present study, the two enzymes were compared in terms of their effectiveness in two systems: (I) Adjuvant-induced arthritis in rats, which is considered to be a model for the chronic situation of rheumatoid arthritis and (2) Bleomycin-induced lung fibrosis. which is a chronic situation believed to be mediated by oxygen free radicals.

Rats inflicted with adjuvant arthritis were treated during the period of maximal joint swelling (Days 15-21 after adjuvant injection) with MnSOD or CuZnSOD (12 to 50mg/kg, i.p. daily). MnSOD administration resulted in a 50-75% reduction of paw swelling, as well as inhibition of the elevation of serum globulins. A similar treatment with CuZnSOD gave merely marginal effects.

In the second system, lung fibrosis was induced in rats by intratracheal administration of bleomycin. MnSOD (50mg/kg, s.c.), administered 2 h before and then 2 and 4 days after bleomycin, markedly inhibited lung fibrosis, as evident from lung weight and collagen content measured by the 3rd week. By contrast, CuZnSOD administration did not give a significant effect. The results indicate that MnSOD is superior to CuZnSOD in the treatment of chronic inflammatory processes. In addition, they lend further support to the involvement of oxygen free radicals in bleomycin toxicity.     

The oxidant defense system in human neuroblastoma IMR-32 cells predifferentiation and postdifferentiation to neuronal phenotypes

Differentiated neurons were investigated for their susceptibility to oxidative damage based on variations in the oxidant defense system occurring during differentiation. The main antioxidant enzymes and substances in human neuroblastoma (IMR-32) cells were evaluated pre- and post-differentiation to a neuronal phenotype. The activity of CuZn superoxide dismutase (CuZnSOD) and Mn superoxide dismutase (MnSOD) and the concentration of CuZnSOD were higher, but the activity and concentration of catalase were lower after differentiation. Differentiated cells had higher activity of glutathione peroxidase (GPx), lower concentration of total glutathione, a higher ratio of oxidised/reduced glutathione and lower activity of glucose-6-phosphate dehydrogenase than undifferentiated cells. We conclude that differentiated neuronal cells may be highly susceptible to oxidant-mediated damage based on the relative activities of the main antioxidant enzymes and on a limited capacity to synthesise and/or recycle glutathione.     

Effect of rhinovirus challenge on antioxidant enzymes in respiratory epithelial cells

The host inflammatory response appears to be an important contributor to the pathogenesis of human viral respiratory illness. Virus-induced oxidative stress appears to mediate an early phase of elaboration of the proinflammatory cytokine interleukin-8 by respiratory epithelial cells. The purpose of these studies was to determine if virus-induced alterations in either the expression or function of antioxidant enzymes contributes to the cellular oxidative stress following rhinovirus challenge. The activities of Mn superoxide dismutase (MnSOD), catalase and glutathione peroxidase (GPX) were not significantly changed by rhinovirus challenge. CuZn superoxide dismutase (CuZnSOD) activity six hours after challenge was 2.55 ±0.56 U/mg protein in rhinovirus-challenged cells compared to 1.16 ±0.54 U/mg protein in control cells ( p =0.029). This increased activity was associated with a concomitant increase in CuZnSOD mRNA and protein concentration. These data suggest that rhinovirus-induced changes in the host cell redox state that result in the early elaboration of interleukin-8 are not mediated by inhibition of either the expression or function of these antioxidant enzymes.     

Effect of Rhinovirus Challenge on Antioxidant Enzymes in Respiratory Epithelial Cells

The host inflammatory response appears to be an important contributor to the pathogenesis of human viral respiratory illness. Virus-induced oxidative stress appears to mediate an early phase of elaboration of the proinflammatory cytokine interleukin-8 by respiratory epithelial cells. The purpose of these studies was to determine if virus-induced alterations in either the expression or function of antioxidant enzymes contributes to the cellular oxidative stress following rhinovirus challenge. The activities of Mn superoxide dismutase (MnSOD), catalase and glutathione peroxidase (GPX) were not significantly changed by rhinovirus challenge. CuZn superoxide dismutase (CuZnSOD) activity six hours after challenge was 2.55 &#45 0.56 U/mg protein in rhinovirus-challenged cells compared to 1.16 &#45 0.54 U/mg protein in control cells ( p =0.029). This increased activity was associated with a concomitant increase in CuZnSOD mRNA and protein concentration. These data suggest that rhinovirus-induced changes in the host cell redox state that result in the early elaboration of interleukin-8 are not mediated by inhibition of either the expression or function of these antioxidant enzymes.     

Manganese superoxide dismutase levels are elevated in a proportion of amyotrophic lateral sclerosis patient cell lines

The most frequent genetic causes of amyotrophic lateral sclerosis (ALS) determined so far are mutations occurring in the gene for copper/zinc superoxide dismutase (CuZnSOD). The mechanism may involve inappropriate formation of hyroxyl radicals, peroxynitrite or malfunctioning of the SOD protein. We hypothesized that undiscovered genetic causes of sporadically occurring amyotrophic lateral sclerosis might be found in the mechanisms that create and destroy oxygen free radicals within the cell. After determining that there were no CuZnSOD mutations present, we measured superoxide production from mitochondria and manganese superoxide dismutase (MnSOD), glutathione peroxidase, NFkappaB, Bcl-2 and Bax by immunoblot. Of the ten sporadic patients we tested we found three patients with significantly increased concentrations of MnSOD. These patients also had lower levels of superoxide production from mitochondria and decreased expression of Bcl-2. No mutations were found in the cDNA sequence of either MnSOD in any of the sporadic patients. A patient with a CuZnSOD mutation (G82R) used as a positive control showed none of these abnormalities. The patients displaying the MnSOD aberrations showed no specific distinguishing features. This result suggests that the cause of ALS in a subgroup of ALS patients (30%) is genetic in origin and can be identified by these markers. The alteration in MnSOD and Bcl-2 are likely epiphenomena resulting from the primary genetic defect. It suggests also that the oxygen free radicals are part of the cause in this subgroup and that dysregulation of MnSOD or increased endogenous superoxide production might be responsible.     

Regional differences in antioxidative response of rat brain after cranial irradiation

In order to examine if differences in activity and inducibility of antioxidative enzymes in rat cerebral cortex and hippocampus are underlying their different sensitivity to radiation, we exposed four-day-old female Wistar rats to cranial radiation of 3 Gy of gamma-rays. After isolation of hippocampus and cortex 1 h or 24 h following exposure, activities of copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and catalase (CAT) were measured and compared to unirradiated controls. MnSOD protein levels were determined by SDS-PAGE electrophoresis and Western blot analysis. Our results showed that CuZnSOD activity in hippocampus and cortex was significantly decreased 1 h and 24 h after irradiation with 3 Gy of gamma-rays. MnSOD activity in both brain regions was also decreased 1 h after irradiation. 24 h following exposure, manganese SOD activity in hippocampus almost achieved control values, while in cortex it significantly exceeded the activity of the relevant controls. CAT activity in hippocampus and cortex remained stable 1 h, as well as 24 h after irradiation with 3 Gy of gamma-rays. MnSOD protein level in hippocampus and cortex decreased 1 h after irradiation with 3 Gy of gamma-rays. 24 h after exposure, MnSOD protein level in cortex was similar to control values, while in hippocampus it was still significantly decreased. We have concluded that regional differences in MnSOD radioinducibility are regulated at the level of protein synthesis, and that they represent one of the main reasons for region-specific radiosensitivity of the brain.     

Differential regulation of antioxidant enzymes in response to oxidants.

We have demonstrated the selective induction of manganese superoxide dismutase (MnSOD) or catalase mRNA after exposure of tracheobronchial epithelial cells in vitro to different oxidant stresses. Addition of H2O2 caused a dose-dependent increase in catalase mRNA in both exponentially growing and confluent cells. A 3-fold induction of catalase mRNA was seen at a nontoxic dose of 250 microM H2O2. Increase in the steady-state mRNA levels of glutathione peroxidase (GPX) and MnSOD were less striking. Expression of catalase, MnSOD, and GPX mRNA was highest in confluent cells. In contrast, constitutive expression of copper and zinc SOD (CuZnSOD) mRNA was greatest in dividing cells and was unaffected by H2O2 in both exponentially growing and confluent cells. MnSOD mRNA was selectively induced in confluent epithelial cells exposed to the reactive oxygen species-generating system, xanthine/xanthine oxidase, while steady-state levels of GPX, catalase, and CuZnSOD mRNA remained unchanged. The 3-fold induction of MnSOD mRNA was dose-dependent, reaching a peak at 0.2 unit/ml xanthine oxidase. MnSOD mRNA increases were seen as early as 2 h and reached maximal induction at 24 h. Immunoreactive MnSOD protein was produced in a corresponding dose- and time-dependent manner. Induction of MnSOD gene expression was prevented by addition of actinomycin D and cycloheximide. These data indicate that epithelial cells of the respiratory tract respond to different oxidant insults by selective induction of certain antioxidant enzymes. Hence, gene expression of antioxidant enzymes does not appear to be coordinately regulated in these cell types.     

Immunohistochemical localization of superoxide dismutases in fetal and neonatal rat tissues.

We investigated the developmental profile of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in tissue sections obtained from fetal (Day 12 to 21 of gestation) and neonatal (Day 0 and 6) rats. Tissues were stained immunohistochemically with specific antisera against the respective rat SODs. There was a general trend towards richness of SODs in the epithelial linings and metabolically active sites, although differential distribution between the two SODs also existed. At Day 12 of gestation, immunoreactivity for both SODs was detected in the cardiomyocytes but not in other tissues. Hepatocytes expressed CuZnSOD at Day 14 and MnSOD at Day 17. By Day 18 CuZnSOD was detected in the epithelial cells of the gastrointestinal tract, respiratory tract, pancreatic islets, kidneys, and adrenals. These tissues exhibited MnSOD staining at Day 19. CuZnSOD occurred in the epithelia of the thyroid, thymus, and salivary glands at Day 19, while MnSOD was seen at Day 21. The increase in intensity of the staining for SODs occurred no later than postnatal Day 0, indicating that most tissues accumulated SODs during late gestation. Breathing atmospheric oxygen during early extrauterine life did not appreciably intensify the SOD staining. These results suggest that perinatal increase in SODs occurs as a general mechanism of preparation for birth.