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.     

Expression of antioxidant enzymes in rat lungs after inhalation of asbestos or silica.

Several studies indicate that active oxygen species play an important role in the development of pulmonary disease (asbestosis and silicosis) after exposure to mineral dust. The present study was conducted to determine if inhaled fibrogenic minerals induced changes in gene expression and activities of antioxidant enzymes (AOE) in rat lung. Two different fibrogenic minerals were compared, crocidolite, an amphibole asbestos fiber, and cristobalite, a crystalline silicon dioxide particle. Steady-state mRNA levels, immunoreactive protein, and activities of selected AOE were measured in lungs 1-10 days after initiation of exposure and at 14 days after cessation of a 10-day exposure period. Exposure to asbestos resulted in significant increases in steady-state mRNA levels of manganese-containing superoxide dismutase (MnSOD) at 3 and 9 days and of glutathione peroxidase at 6 and 9 days. An increase in steady-state mRNA levels of copper, zinc-containing superoxide dismutase (CuZnSOD), was observed at 6 days. Exposure to asbestos also resulted in overall increased enzyme activities of catalase, glutathione peroxidase and total superoxide dismutase in lung. In contrast, silica caused a dramatic increase in steady-state levels of MnSOD mRNA at all time periods and an increase in glutathione peroxidase mRNA levels at 9 days. Activities of AOE remained unchanged in silica-exposed lungs. In both models, increases in gene expression of MnSOD correlated with increased amounts of MnSOD immunoreactive protein in lung and the pattern and extent of inflammation. These data indicate that the profiles of AOE are dissimilar during the development of experimental asbestosis or silicosis and suggest different mechanisms of lung defense in response to these minerals.     

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.     

Endogenous antioxidant defence system in rat liver following mercury chloride oral intoxication

Mercury is a highly toxic metal which induces oxidative stress. Superoxide dismutases, catalase, and glutathion peroxidase are proteins involved in the endogenous antioxidant defence system. In the present study rats were administered orally, by gavage, a single daily dose of HgCl2 for three consecutive days. In order to find a relation between the proteins involved in the antioxidant defence and mercury intoxication, parameters of liver injury, redox state of the cells, as well as intracellular protein levels and enzyme activities of Mn-dependent superoxide dismutase (MnSOD), Cu-Zn-dependent superoxide dismutase (CuZnSOD), catalase, and glutathione peroxidase (GPx) were assayed both in blood and in liver homogenates. HgCl2 at the doses of 0.1 mg/kg produced liver damage which that was detected by a slight increase in serum alanine aminotransferase and gamma glutamyl transferase. Hepatic GSH/GSSG ratio was assayed as a parameter of oxidative stress and a significant decrease was detected, as well as significant increases in enzyme activities and protein levels of hepatic antioxidant defence systems. Changes in both MnSOD and CuZnSOD were parallel to those of liver injury and oxidative stress, while the changes detected in catalase and GPx activities were progressively increased along with the mercury intoxication. Other enzyme activities related to the glutathione redox cycle, such as glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PDH), also increased progressively. We conclude that against low doses of mercury that produce a slight oxidative stress and liver injury, the response of the liver was to induce the synthesis and activity of the enzymes involved in the endogenous antioxidant system. The activities of all the enzymes assayed showed a rapidly induced coordinated response.     

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.     

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.     

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.     

MnSOD marks cord blood late outgrowth endothelial cells and accompanies robust resistance to oxidative stress

Cord blood is source of colony-forming progenitors to vascular endothelial cells for potential use in cell therapies. These cells-called blood late outgrowth endothelial cells (OECs)-have undergone endothelial differentiation, but appear to still possess functional properties different from mature endothelial cells. A large-scale comparative proteomics screen of cord blood OECs versus human vein endothelial cells (HUVECs) using two-dimensional gel electrophoresis and mass spectrometry identified specific expression of manganese superoxide dismutase (MnSOD), a key antioxidant enzyme expressed in the mitochondria, in OECs but not in HUVECs. Immunoblotting verified significant MnSOD levels in all OEC isolates tested and maintained throughout passaging. Endothelial function and cell survival/proliferation assays in the presence of high cytotoxic doses of the superoxide generator compound LY83583 showed OECs profoundly better protected against oxidative stress than HUVECs. Such cytoprotective levels of MnSOD cells could give therapeutic cell transplants a survival advantage in necrotic or ischemic conditions.     

Stabilization of superoxide dismutase by acetyl-l-carnitine in human brain endothelium during alcohol exposure: novel protective approach

Oxidative damage of the endothelium disrupts the integrity of the blood-brain barrier (BBB). We have shown before that alcohol exposure increases the levels of reactive oxygen species (ROS; superoxide and hydroxyl radical) and nitric oxide (NO) in brain endothelial cells by activating NADPH oxidase and inducible nitric oxide synthase. We hypothesize that impairment of antioxidant systems, such as a reduction in catalase and superoxide dismutase (SOD) activity, by ethanol exposure may elevate the levels of ROS/NO in endothelium, resulting in BBB damage. This study examines whether stabilization of antioxidant enzyme activity results in suppression of ROS levels by anti-inflammatory agents. To address this idea, we determined the effects of ethanol on the kinetic profile of SOD and catalase activity and ROS/NO generation in primary human brain endothelial cells (hBECs). We observed an enhanced production of ROS and NO levels due to the metabolism of ethanol in hBECs. Similar increases were found after exposure of hBECs to acetaldehyde, the major metabolite of ethanol. Ethanol simultaneously augmented ROS generation and the activity of antioxidative enzymes. SOD activity was increased for a much longer period of time than catalase activity. A decline in SOD activity and protein levels preceded elevation of oxidant levels. SOD stabilization by the antioxidant and mitochondria-protecting agent acetyl-L-carnitine (ALC) and the anti-inflammatory agent rosiglitazone suppressed ROS levels, with a marginal increase in NO levels. Mitochondrial membrane protein damage and decreased membrane potential after ethanol exposure indicated mitochondrial injury. These changes were prevented by ALC. Our findings suggest the counteracting mechanisms of oxidants and antioxidants during alcohol-induced oxidative stress at the BBB. The presence of enzymatic stabilizers favors the ROS-neutralizing antioxidant redox of the BBB, suggesting an underlying protective mechanism of NO for brain vascular tone and vasodilation.     

Protective role of superoxide dismutases against ionizing radiation in yeast

The protective role of superoxide dismutases (SODs) against ionizing radiation, which generates reactive oxygen species (ROS) harmful to cellular function, was investigated in the wild-type and in mutant yeast strains lacking cytosolic CuZnSOD (sod1Delta), mitochondrial MnSOD (sod2Delta), or both SODs (sod1Deltasod2Delta). Upon exposure to ionizing radiation, there was a distinct difference between these strains in regard to viability and the level of protein carbonyl content, which is the indicative marker of oxidative damage to protein, intracellular H2O2 level, as well as lipid peroxidation. When the oxidation of 2',7'-dichlorofluorescin was used to examine the hydroperoxide production in yeast cells, the SOD mutants showed a higher degree of increase in fluorescence upon exposure to ionizing radiation as compared to wild-type cells. These results indicated that mutants deleted for SOD genes were more sensitive to ionizing radiation than isogenic wild-type cells. Induction and inactivation of other antioxidant enzymes, such as catalase, glucose 6-phosphate dehydrogenase, and glutathione reductase, were observed after their exposure to ionizing radiation both in wild-type and in mutant cells. However, wild-type cells maintained significantly higher activities of antioxidant enzymes than did mutant cells. These results suggest that both CuZnSOD and MnSOD may play a central role in protecting cells against ionizing radiation through the removal of ROS, as well as in the protection of antioxidant enzymes.     

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.     

Differences in mRNA Expression,Protein Content,and Enzyme Activity of Superoxide Dismutases in Type II Pneumocytes of Acute and Chronic Lung Injury

The lung is protected against oxidative stress by a variety of antioxidants and type II pneumocytes seem to play an important role in antioxidant defense. Previous studies have shown that inhalation of NO₂ results in acute and chronic lung injury. How the expression and enzyme activity of antioxidant enzymes are influenced in type II cells of different inflammatory stages has yet not been studied. To elucidate this question, we exposed rats to 10 ppm NO₂ for 3 or 20 days to induce acute or chronic lung injury. From these and air-breathing rats, type II pneumocytes were isolated. The mRNA expression and protein content of CuZnSOD and MnSOD as well as total SOD-specific enzyme activity were determined. For the acute lung injury (3 d NO₂), the expression of CuZnSOD mRNA was significantly increased, while MnSOD expression was significantly reduced after 3 days of NO 2 exposure. For the chronic lung injury (20 d NO₂), CuZnSOD expression was still enhanced, while MnSOD expression was comparable to control. In parallel to CuZnSOD mRNA expression, the protein amount was significantly increased in acute and chronic lung injury however MnSOD protein content exhibited no intergroup differences. Total SOD enzyme activity showed a significant decrease after 3 days of NO₂ exposure and was similar to control after 20 days. We conclude that during acute and chronic lung injury in type II pneumocytes expression and protein synthesis of CuZnSOD and MnSOD are regulated differently.     

Differences in mRNA expression,protein content,and enzyme activity of superoxide dismutases in type II pneumocytes of acute and chronic lung injury

The lung is protected against oxidative stress by a variety of antioxidants and type II pneumocytes seem to play an important role in antioxidant defense. Previous studies have shown that inhalation of NO₂ results in acute and chronic lung injury. How the expression and enzyme activity of antioxidant enzymes are influenced in type II cells of different inflammatory stages has yet not been studied. To elucidate this question, we exposed rats to 10 ppm NO₂ for 3 or 20 days to induce acute or chronic lung injury. From these and air-breathing rats, type II pneumocytes were isolated. The mRNA expression and protein content of CuZnSOD and MnSOD as well as total SOD-specific enzyme activity were determined. For the acute lung injury (3 d NO₂), the expression of CuZnSOD mRNA was significantly increased, while MnSOD expression was significantly reduced after 3 days of NO 2 exposure. For the chronic lung injury (20 d NO₂), CuZnSOD expression was still enhanced, while MnSOD expression was comparable to control. In parallel to CuZnSOD mRNA expression, the protein amount was significantly increased in acute and chronic lung injury however MnSOD protein content exhibited no intergroup differences. Total SOD enzyme activity showed a significant decrease after 3 days of NO₂ exposure and was similar to control after 20 days. We conclude that during acute and chronic lung injury in type II pneumocytes expression and protein synthesis of CuZnSOD and MnSOD are regulated differently.     

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.