In vitro modulation of antioxidant enzyme levels in normal hamster kidney and estrogen-induced hamster kidney tumor

Antioxidant enzyme (AE) activities were studied in normal hamster kidney proximal tubules and in estrogen-induced hamster kidney cancer. In vivo, kidney tumor had lower activities of manganese superoxide dismutase (MnSOD), copper, zinc superoxide dismutase, catalase, and glutathione peroxidase than kidney proximal tubules. Differences in AE activities were, in general, maintained in tissue culture, with AE activities remaining low in tumor cells compared to normal cells. Normal proximal tubular cells showed significant induction of MnSOD activity as a function of time in culture of following exposure to diethylstilbestrol, a synthetic estrogen, while MnSOD activity remained low in tumor cells under these conditions. Our results suggest that antioxidant enzymes, particularly MnSOD, are regulated differently in estrogen-induced hamster kidney tumor cells than in normal kidney proximal tubular cells, demonstrating that cancers arising from hormonal influence have similar AE profiles to those previously described in cancers arising from viral or chemical etiologies.     

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.     

Managing odds in stem cells: insights into the role of mitochondrial antioxidant enzyme MnSOD

Reactive oxygen species (ROS) have been poised at a straddled state of being beneficiary as well detrimental depending on its threshold levels. Maintaining the homeostasis of ROS is imperative for normal cellular physiology, wherein physiological concentrations of ROS are involved in cell signaling and elevated ROS contribute to the development of various diseases. Superoxide dismutases (SODs), enzymes involved in dismutation of superoxide anion to hydrogen peroxide, arrive as a first line of defense when there is perturbation in the homeostasis of ROS. As mitochondria are the main site of superoxide production, among SODs, mitochondrial manganese SOD (MnSOD) is the primary antioxidant enzyme that protects cells from ROS. Most importantly, knockout of MnSOD leads to postnatal lethality and tissue-specific conditional knockout in brain resulted in death of mice, conclusively portraying the essential role of MnSOD in development. Although MnSOD has been extensively discussed with the purview of tumor biology and aging, understanding the crucial role of MnSOD in stem cell physiology is still at its infant stage. Ever increasing progress in stem cell research has recently unveiled the essential role of MnSOD in self-renewal and differentiation of stem cells. In this review, we will conglomerate the current aspects by which MnSOD can contribute to embryonic stem cells’ and adult stem cells’ functions and interpret the necessity of understanding MnSOD for further stem cell mediated applications.     

Mitochondrial tyrosine nitration precedes chronic allograft nephropathy

Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been reported to occur during end-stage human renal allograft rejection. In order to determine whether nitration and inactivation of this critical mitochondrial protein might play a contributory role in the onset of transplant rejection, we employed a rodent model of Chronic Allograft Nephropathy (or CAN). Using this model we followed kidney function from 2–52 weeks post-transplant and correlated graft function with levels of nitration in the renal allograft. Tyrosine nitration of both glomerular and tubular structures occurred at 2 weeks post-transplant. At later times (16 weeks) post-transplant, tyrosine nitration appeared to be confined to tubular structures; however glomerular nitration returned at 52 weeks post-transplant. Interestingly, nitration and inactivation of MnSOD occurs prior to the onset of renal dysfunction in this rat model of chronic allograft nephropathy (2 weeks versus 16 weeks post-transplant). Furthermore, we have identified an additional mitochondrial protein, cytochrome c, as being endogenously nitrated during chronic rejection. The kinetics of cytochrome c nitration lagged behind MnSOD nitration and inactivation (4 weeks compared to 2 weeks); suggesting that loss of MnSOD activity likely contributes to elevation of the nitrating species and further nitration of other targets.     

Role of antioxidant enzymes in cell immortalization and transformation

Summary The role of antioxidant enzymes, particularly superoxide dismutase (SOD), in immortalization and malignant transformation is discussed. SOD (generally MnSOD) has been found to be lowered in a wide variety of tumor types when compared to an appropriate normal cell control. Levels of immunoreactive MnSOD protein and mRNA for MnSOD also appear to be lowered in tumor cells. Tumor cells have the capacity to produce superoxide radical, the substrate for SOD. This suggests that superoxide production coupled with diminished amounts of MnSOD may be a general characteristic of tumor cells. The levels of MnSOD in certain cells correlates with their degree of differentiation; non-differentiating cells, whether normal or malignant, appear to have lost the ability to undergo MnSOD induction. These observations are used to elucidate a two-step model of cancer. This model involves not only the antioxidant enzymes, but also organelle (particularly mitochondria and peroxisomes) function as a dominant theme in carcinogenesis.     

Vitamin C depletion increases superoxide generation in brains of SMP30/GNL knockout mice

Vitamin C (VC) has a strong antioxidant function evident as its ability to scavenge superoxide radicals in vitro. We verified that this property actually exists in vivo by using a real-time imaging system in which Lucigenin is the chemiluminescent probe for detecting superoxide in senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which cannot synthesize VC in vivo. SMP30/GNL KO mice were given 1.5 g/L VC [VC(+)] for 2, 4, or 8 weeks or denied VC [VC(−)]. At 4 and 8 weeks, VC levels in brains from VC(−) KO mice were <6% of that in VC(+) KO mice. Accordingly, superoxide-dependent chemiluminescence levels determined by ischemia-reperfusion at the 4- and 8 weeks test intervals were 3.0-fold and 2.1-fold higher, respectively, in VC(−) KO mice than in VC(+) KO mice. However, total superoxide dismutase activity and protein levels were not altered. Thus, VC depletion specifically increased superoxide generation in a model of the living brain.     

Inhibition of cell growth by overexpression of manganese superoxide dismutase (MnSOD) in human pancreatic carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Mitochondrial targets of oxidative stress during renal ischemia/reperfusion

Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been shown to occur in both human and rat chronic renal allograft rejection. To elucidate the time course of MnSOD inactivation and mitochondrial dysfunction at earlier times during renal transplantation, we developed a rodent model of renal ischemia/reperfusion (I/R). Renal function was significantly impaired at 16 h reperfusion following 30 min of warm ischemia. Tyrosine nitration of specific mitochondrial proteins, MnSOD and cytochrome c, occurred at the earliest time point examined, an event that preceded significant renal injury. Interestingly, a small percentage of both mitochondrial proteins were also located in the cytosol. This leakage and decreased adenosine 5(')-triphosphate levels indicate loss of mitochondrial membrane integrity during renal I/R. Inactivation of MnSOD occurred rapidly in this model of renal I/R, suggesting that loss of MnSOD activity leads to further renal injury and nitration of other mitochondrial targets.     

Inhibition of Cell Growth by Overexpression of Manganese Superoxide Dismutase (MnSOD) in Human Pancreatic Carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Manganese superoxide dismutase in disease

Manganese superoxide dismutase (MnSOD) is essential for life as dramatically illustrated by the neonatal lethality of mice that are deficient in MnSOD. In addition, mice expressing only 50% of the normal compliment of MnSOD demonstrate increased susceptibility to oxidative stress and severe mitochondrial dysfunction resulting from elevation of reactive oxygen species. Thus, it is important to know the status of both MnSOD protein levels and activity in order to assess its role as an important regulator of cell biology.

Numerous studies have shown that MnSOD can be induced to protect against pro-oxidant insults resulting from cytokine treatment, ultraviolet light, irradiation, certain tumors, amyotrophic lateral sclerosis, and ischemia/reperfusion. In addition, overexpression of MnSOD has been shown to protect against pro-apoptotic stimuli as well as ischemic damage. Conversely, several studies have reported declines in MnSOD activity during diseases including cancer, aging, progeria, asthma, and transplant rejection. The precise biochemical/molecular mechanisms involved with this loss in activity are not well understood. Certainly, MnSOD gene expression or other defects could play a role in such inactivation. However, based on recent findings regarding the susceptibility of MnSOD to oxidative inactivation, it is equally likely that post-translational modification of MnSOD may account for the loss of activity. Our laboratory has recently demonstrated that MnSOD is tyrosine nitrated and inactivated during human kidney allograft rejection and human pancreatic ductal adenocarcinoma. We have determined that peroxynitrite (ONOO^-) is the only known biological oxidant competent to inactivate enzymatic activity, to nitrate critical tyrosine residues, and to induce dityrosine formation in MnSOD. Tyrosine nitration and inactivation of MnSOD would lead to increased levels of superoxide and concomitant increases in ONOO^- within the mitochondria which, could lead to tyrosine nitration/oxidation of key mitochondrial proteins and ultimately mitochondrial dysfunction and cell death. This article assesses the important role of MnSOD activity in various pathological states in light of this potentially lethal positive feedback cycle involving oxidative inactivation.     

Inducible kidney-specific Sgk1 knockout mice show a salt-losing phenotype

The expression of the serum- and glucocorticoid-regulated kinase 1 (Sgk1) is induced by mineralocorticoids and, in turn, upregulates the renal epithelial Na(+) channel (ENaC). Total inactivation of Sgk1 has been associated with transient urinary Na(+) wasting with a low-Na(+) diet, while the aldosterone-mediated ENaC channel activation was unchanged in the collecting duct. Since Sgk1 is ubiquitously expressed, we aimed to study the role of renal Sgk1 and generated an inducible kidney-specific knockout (KO) mouse. We took advantage of the previously described TetOn/CreLoxP system, in which rtTA is under the control of the Pax8 promotor, allowing inducible inactivation of the floxed Sgk1 allele in the renal tubules (Sgk1fl/fl/Pax8/LC1 mice). We found that under a standard Na(+) diet, renal water and Na(+)/K(+) excretion had a tendency to be higher in doxycycline-treated Sgk1 KO mice compared with control mice. The impaired ability of Sgk1 KO mice to retain Na(+) increased significantly with a low-salt diet despite higher plasma aldosterone levels. On a low-Na(+) diet, the Sgk1 KO mice were also hyperkaliuric and lost body weight. This phenotype was accompanied by a decrease in systolic and diastolic blood pressure. At the protein level, we observed a reduction in phosphorylation of the ubiquitin protein-ligase Nedd4-2 and a decrease in the expression of the Na(+)-Cl(-)-cotransporter (NCC) and to a lesser extent of ENaC.     

Manganese superoxide dismutase in rat liver peroxisomes: Biochemical and immunochemical evidence

By using highly purified peroxisomes from rat liver, we have shown that peroxisomes contain manganese superoxide dismutase (MnSOD) activity and a 23 kDa protein immunoreactive with antibodies against purified mitochondrial MnSOD. Immunocytochemical studies have also revealed immunoreaction (immunogold) with MnSOD antibodies in mitochondria and peroxisomes. Studies of the intraperoxisomal localization of MnSOD have shown that in peroxisomes MnSOD is a component of the peroxisomal limiting membranes and dense core. Furthermore, the MnSOD level in peroxisomes was modulated by oxidative stress conditions such as ischemia-reperfusion or the treatment with ciprofibrate, a peroxisomal proliferator. These findings suggest that MnSOD in peroxisomes may play an important role in the dismutation of superoxide generated on the peroxisomal membrane for keeping the delicate balance of the redox state.     

Mitochondrial superoxide plays a crucial role in the development of mitochondrial dysfunction during high glucose exposure in rat renal proximal tubular cells

Diabetic nephropathy is the leading cause of end-stage renal disease in the United States. Despite several studies indicating a role for mitochondrial oxidative stress and mitochondrial dysfunction in the development of diabetic complications, the precise mechanisms underlying renal mitochondrial dysfunction and renal cell injury remain unclear. The hypothesis of the current study was that high-glucose-mediated generation of mitochondrial superoxide is a key early event that leads to mitochondrial injury in renal proximal tubular cells. To ascertain the role of mitochondrial superoxide we have tested whether overexpression of the primary mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), protects against hyperglycemia-induced renal injury using normal rat renal proximal tubular cells (NRK). NRK cells were exposed to high glucose (25 mM) and the changes in the mitochondrial membrane potential, ATP levels, and superoxide generation and the loss of cell viability were measured at 24 and 48 h after high glucose exposure. Our results indicate that high glucose first induced superoxide generation and hyperpolarization in the mitochondria, followed by a secondary event, which involved a decline in ATP levels, partial Complex III inactivation, and loss of cell viability. These high-glucose-induced changes were completely prevented by overexpression of MnSOD in NRK cells. However, MnSOD activity was not changed after high glucose exposure in vitro or during the early stages of diabetes using the streptozotocin rat model. These findings show for the first time that hyperglycemic induction of superoxide production within the mitochondria initiates specific mitochondrial injury (i.e., Complex III) via a mechanism independent of MnSOD inactivation.     

Deletion of Smad3 protects against C-reactive protein-induced renal fibrosis and inflammation in obstructive nephropathy

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism.Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E).Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80⁺ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling.Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.     

Overexpression of Manganese Superoxide Dismutase Promotes the Survival of Prostate Cancer Cells Exposed to Hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1?h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Superoxide-lowering therapy with TEMPOL reverses arterial dysfunction with aging in mice

To test the hypothesis that the antioxidant enzyme superoxide dismutase (SOD) mimetic TEMPOL improves arterial aging, young (Y, 4-6 months) and old (O, 26-28 months) male C57BL6 mice received regular or TEMPOL-supplemented (1mM) drinking water for 3 weeks (n = 8 per group). Aortic superoxide was 65% greater in O (P < 0.05 vs. Y), which was normalized by TEMPOL. O had large elastic artery stiffening, as indicated by greater aortic pulse wave velocity (aPWV, 508 ± 22 vs. 418 ± 22 AU), which was associated with increased adventitial collagen I expression (P < 0.05 vs. Y). TEMPOL reversed the age-associated increases in aPWV (434 ± 21 AU) and collagen in vivo, and SOD reversed the increases in collagen I in adventitial fibroblasts from older rats in vitro. Isolated carotid arteries of O had impaired endothelial function as indicated by reduced acetylcholine-stimulated endothelium-dependent dilation (EDD) (75.6 ± 3.2 vs. 94.5 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (L-NAME) associated with decreased endothelial NO synthase (eNOS) expression (P < 0.05 vs. Y). TEMPOL restored EDD (94.5 ± 1.4%), NO bioavailability and eNOS in O. Nitrotyrosine and expression of NADPH oxidase were ~100-200% greater, and MnSOD was ~75% lower in O (P < 0.05 vs. Y). TEMPOL normalized nitrotyrosine and NADPH oxidase in O, without affecting MnSOD. Aortic pro-inflammatory cytokines were greater in O (P < 0.05 vs. Y) and normalized by TEMPOL. Short-term treatment of excessive superoxide with TEMPOL ameliorates large elastic artery stiffening and endothelial dysfunction with aging, and this is associated with normalization of arterial collagen I, eNOS, oxidative stress, and inflammation.     

Immunolocalization of manganese superoxide dismutase in normal and transgenic mice expressing the human enzyme

Summary The localization of manganese superoxide dismutase (MnSOD) was determined using immunohistochemistry of various tissues of normal and transgenic mice which express the human enzyme, with emphasis on studies of mouse kidney and lung. Mouse kidney and lung were studied using both frozen section analysis and paraffin sections following fixation in a variety of fixatives. Formalin fixation resulted in a loss of antigenicity, while fixation in zinc formalin or B5 fixative gave results similar to those from frozen sections. Immunoperoxidase studies using antibodies to MnSOD showed greater staining in transgenic kidney or lung than in identical tissues in normal mice when appropriate fixation was used. In contrast, equal immunostaining was obtained in kidney or lung from normal and transgenic mice when antibodies to catalase or copper zinc superoxide dismutase were utilized. Immunogold ultrastructural analysis of MnSOD localization for lung and kidney was also performed. As compared to normal mice, transgenic mice exhibited greater staining of the mitochondria of kidney interstitial fibroblasts and glomerular, endothelial, and smooth muscle cells. In the lungs of transgenic animals, all cells showed increased staining; smooth muscle cells demonstrated the most marked increase in immunolabelling. The results indicate that these transgenic mice overexpress MnSOD in their mitochondria, and that this occurs selectively in at least some mesenchymal tissues.This study was supported by the Medical Research Service of the Department of Veterans Affairs (TDO), by National Institutes of Health grants No. CA-41267 (LWO), No. HL-39585 and No. HL-44571 (Y-SH), and by the Department of Anesthesiology Research and Development Funds (DBC, HPC).     

Invited review: manganese superoxide dismutase in disease

Manganese superoxide dismutase (MnSOD) is essential for life as dramatically illustrated by the neonatal lethality of mice that are deficient in MnSOD. In addition, mice expressing only 50% of the normal compliment of MnSOD demonstrate increased susceptibility to oxidative stress and severe mitochondrial dysfunction resulting from elevation of reactive oxygen species. Thus, it is important to know the status of both MnSOD protein levels and activity in order to assess its role as an important regulator of cell biology.

Numerous studies have shown that MnSOD can be induced to protect against pro-oxidant insults resulting from cytokine treatment, ultraviolet light, irradiation, certain tumors, amyotrophic lateral sclerosis, and ischemia/reperfusion. In addition, overexpression of MnSOD has been shown to protect against pro-apoptotic stimuli as well as ischemic damage. Conversely, several studies have reported declines in MnSOD activity during diseases including cancer, aging, progeria, asthma, and transplant rejection. The precise biochemical/molecular mechanisms involved with this loss in activity are not well understood. Certainly, MnSOD gene expression or other defects could play a role in such inactivation. However, based on recent findings regarding the susceptibility of MnSOD to oxidative inactivation, it is equally likely that post-translational modification of MnSOD may account for the loss of activity. Our laboratory has recently demonstrated that MnSOD is tyrosine nitrated and inactivated during human kidney allograft rejection and human pancreatic ductal adenocarcinoma. We have determined that peroxynitrite (ONOO^-) is the only known biological oxidant competent to inactivate enzymatic activity, to nitrate critical tyrosine residues, and to induce dityrosine formation in MnSOD. Tyrosine nitration and inactivation of MnSOD would lead to increased levels of superoxide and concomitant increases in ONOO^- within the mitochondria which, could lead to tyrosine nitration/oxidation of key mitochondrial proteins and ultimately mitochondrial dysfunction and cell death. This article assesses the important role of MnSOD activity in various pathological states in light of this potentially lethal positive feedback cycle involving oxidative inactivation.