Roles of mitochondria-generated reactive oxygen species on X-ray-induced apoptosis in a human hepatocellular carcinoma cell line, HLE

HLE, a human hepatocellular carcinoma cell line was transiently transfected with normal human MnSOD and MnSOD without a mitochondrial targeting signal (MTS). Mitochondrial reactive oxygen species (ROS), lipid peroxidation and apoptosis were examined as a function of time following 18.8 Gy X-ray irradiation. Our results showed that the level of mitochondrial ROS increased and reached a maximum level 2 hours after X-ray irradiation. Authentic MnSOD, but not MnSOD lacking MTS, protected against mitochondrial ROS, lipid peroxidation and apoptosis. In addition, the levels of mitochondrial ROS were consistently found to always correlate with the levels of authentic MnSOD in mitochondria. These results suggest that only when MnSOD is located in mitochondria is it efficient in protecting against cellular injuries by X-ray irradiation and that mitochondria are the critical sites of X-ray-induced cellular oxidative injuries.     

Induction of apoptosis by adenovirus-mediated manganese superoxide dismutase overexpression in SV-40-transformed human fibroblasts

Although mitochondrial reactive oxygen species (ROS) have been implicated both as an initiator and as an effector of apoptosis, the exact role of mitochondrial ROS has been difficult to establish due to the lack of an appropriate experimental system where ROS could be specifically generated from mitochondria and subsequent effects on cells analyzed. In this study, a manganese superoxide dismutase (MnSOD) activity-mediated apoptosis model was established and characterized. It was shown that despite early increases in the steady-state levels of ROS upon MnSOD overexpression, cellular oxidative damage was decreased significantly at later time points. Alterations in levels of peroxiredoxin (Prxn1) protein preceded the onset of apoptosis after MnSOD overexpression. A time course study demonstrated that increases in MnSOD activity prior to the onset of apoptosis correlated with alterations in the levels of nitration of tyrosine residue(s) of MnSOD protein. A direct correlation between MnSOD activity and the degree of apoptosis was demonstrated using a mutant MnSOD with decreased activity. The current study supports a causative role of mitochondrial ROS leading to the onset of apoptosis. The MnSOD activity-mediated apoptosis model described here could be further utilized to study mitochondrial apoptotic pathways.     

Control of mitochondrial redox balance and cellular defense against oxidative damage by mitochondrial NADP+-dependent isocitrate dehydrogenase

Mitochondria are the major organelles that produce reactive oxygen species (ROS) and the main target of ROS-induced damage as observed in various pathological states including aging. Production of NADPH required for the regeneration of glutathione in the mitochondria is critical for scavenging mitochondrial ROS through glutathione reductase and peroxidase systems. We investigated the role of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) in controlling the mitochondrial redox balance and subsequent cellular defense against oxidative damage. We demonstrate in this report that IDPm is induced by ROS and that decreased expression of IDPm markedly elevates the ROS generation, DNA fragmentation, lipid peroxidation, and concurrent mitochondrial damage with a significant reduction in ATP level. Conversely, overproduction of IDPm protein efficiently protected the cells from ROS-induced damage. The protective role of IDPm against oxidative damage may be attributed to increased levels of a reducing equivalent, NADPH, needed for regeneration of glutathione in the mitochondria. Our results strongly indicate that IDPm is a major NADPH producer in the mitochondria and thus plays a key role in cellular defense against oxidative stress-induced damage.     

The MnSOD Ala16Val SNP: Relevance to human diseases and interaction with environmental factors

Abstract

The relevance of reactive oxygen species (ROS) production relies on the dual role shown by these molecules in aerobes. ROS are known to modulate several physiological phenomena, such as immune response and cell growth and differentiation; on the other hand, uncontrolled ROS production may cause important tissue and cell damage, such as deoxyribonucleic acid oxidation, lipid peroxidation, and protein carbonylation. The manganese superoxide dismutase (MnSOD) antioxidant enzyme affords the major defense against ROS within the mitochondria, which is considered the main ROS production locus in aerobes. Structural and/or functional single nucleotide polymorphisms (SNP) within the MnSOD encoding gene may be relevant for ROS detoxification. Specifically, the MnSOD Ala16Val SNP has been shown to alter the enzyme localization and mitochondrial transportation, affecting the redox status balance. Oxidative stress may contribute to the development of type 2 diabetes, cardiovascular diseases, various inflammatory conditions, or cancer. The Ala16Val MnSOD SNP has been associated with these and other chronic diseases; however, inconsistent findings between studies have made difficult drawing definitive conclusions. Environmental factors, such as dietary antioxidant intake and exercise have been shown to affect ROS metabolism through antioxidant enzyme regulation and may contribute to explain inconsistencies in the literature. Nevertheless, whether environmental factors may be associated to the Ala16Val genotypes in human diseases still needs to be clarified.     

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.     

Human selenium-containing single-chain variable fragment with glutathione peroxidase activity protects NIH3T3 fibroblast against oxidative damage

Ultraviolet B (UVB medium wave, 280–315 nm) induces cellular oxidative damage and apoptosis by producing reactive oxygen species (ROS). Glutathione peroxidase functions as an antioxidant by catalyzing the reduction of hydrogen peroxide, the more important member of reactive oxygen species. A human selenium-containing single-chain variable fragment (se-scFv-B3) with glutathione peroxidase activity of 1288 U/μmol was generated and investigated for its antioxidant effects in UVB-induced oxidative damage model. In particular, cell viability, lipid peroxidation extent, cell apoptosis, the change of mitochondrial membrane potential, caspase-3 activity and the levels of intracellular reactive oxygen species were assayed. Human se-scFv-B3 protects NIH3T3 cells against ultraviolet B-induced oxidative damage and subsequent apoptosis by prevention of lipid peroxidation, inhibition of the collapse of mitochondrial membrane potential as well as the suppression of the caspase-3 activity and the level of intracellular ROS. It seems that antioxidant effects of human se-scFv-B3 are mainly associated with its capability to scavenge reactive oxygen species, which is similar to that of the natural glutathione peroxidase.     

UV-A irradiation induces a decrease in the mitochondrial respiratory activity of human NCTC 2544 keratinocytes

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitoninpermeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.     

Glutathione depletion and the production of reactive oxygen species in isolated hepatocyte suspensions

Diethyl maleate (DEM) (5 mM) and ethyl methanesulfonate (EMS) (35 mM) treatments rapidly depleted cellular reduced glutathione (GSH) below detectable levels (1 nmol/10(6) cells), and induced lipid peroxidation and necrotic cell death in freshly isolated rat hepatocytes. In hepatocytes incubated with 2.5 mM DEM and 10 mM EMS, however, the complete depletion of cellular GSH observed was not sufficient to induce lipid peroxidation or cell death. Instead, DEM- and EMS-induced lipid peroxidation and cell death were dependent on increased reactive oxygen species (ROS) production as measured by increases in dichlorofluorescein fluorescence. The addition of antioxidants (vitamin E succinate and deferoxamine) prevented lipid peroxidation and cell death, suggesting that lipid peroxidation is involved in the sequence of events leading to necrotic cell death induced by DEM and EMS. To investigate the subcellular site of ROS generation, the cytochrome P450 inhibitor, SKF525A, was found to reduce EMS-induced lipid peroxidation but did not protect against the loss of cell viability, suggesting a mitochondrial origin for the toxic lipid peroxidation event. In agreement with this conclusion, mitochondrial electron transport inhibitors (rotenone, thenoyltrifluoroacetone and antimycin A) increased EMS-induced lipid peroxidation and cell death, while the mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone, blocked EMS- and DEM-mediated ROS production and lipid peroxidation. Furthermore, EMS treatment resulted in the significant loss of mitochondrial alpha-tocopherol shortly after its addition, and this loss preceded losses in cellular alpha-tocopherol levels. Treatment of hepatocytes with cyclosporin A, a mitochondrial permeability transition inhibitor, oxypurinol, a xanthine oxidase inhibitor, or BAPTA-AM, a calcium chelator, provided no protection against EMS-induced cell death or lipid peroxidation. Our results indicate that DEM and EMS induce cell death by a similar mechanism, which is dependent on the induction of ROS production and lipid peroxidation, and mitochondria are the major source for this toxic ROS generation. Cellular GSH depletion in itself does not appear to be responsible for the large increases in ROS production and lipid peroxidation observed.     

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.     

UV-A irradiation induces a decrease in the mitochondrial respiratory activity of human NCTC 2544 keratinocytes

UV-A irradiation caused a dose-dependent decrease in cellular oxygen consumption (56%) and ATP content (65%) in human NCTC 2544 keratinocytes, one hour after treatment. This effect was partially reversed by maintaining the irradiated cells in normal culture conditions for 24h. Using malate/glutamate or succinate as substrates for mitochondrial electron transport, the oxygen uptake of digitoninpermeabilised cells was greatly inhibited following UV-A exposure. These results strongly suggest that UV-A irradiation affects the state 3 respiration of the mitochondria. However, under identical conditions, UV-A exposure did not reduce the mitochondrial transmembrane potential. The antioxidant, vitamin E inhibited UV-A-induced lipid peroxidation, but did not significantly prevent the UV-A-mediated changes in cellular respiration nor the decrease in ATP content, suggesting that these effects were not the result of UV-A dependent lipid peroxidation. UV-A irradiation also led to an increase in MnSOD gene expression 24 hours after treatment, indicating that the mitochondrial protection system was enhanced in response to UV-A treatment. These findings provide evidence that impairment of mitochondrial respiratory activity is one of the early results of UV-A irradiation for light doses much lower than the minimal erythemal dose.     

Iron-mediated inhibition of mitochondrial manganese uptake mediates mitochondrial dysfunction in a mouse model of hemochromatosis

Previous phenotyping of glucose homeostasis and insulin secretion in a mouse model of hereditary hemochromatosis (Hfe(-/-)) and iron overload suggested mitochondrial dysfunction. Mitochondria from Hfe(-/-) mouse liver exhibited decreased respiratory capacity and increased lipid peroxidation. Although the cytosol contained excess iron, Hfe(-/-) mitochondria contained normal iron but decreased copper, manganese, and zinc, associated with reduced activities of copper-dependent cytochrome c oxidase and manganese-dependent superoxide dismutase (MnSOD). The attenuation in MnSOD activity was due to substantial levels of unmetallated apoprotein. The oxidative damage in Hfe(-/-) mitochondria is due to diminished MnSOD activity, as manganese supplementation of Hfe(-/-) mice led to enhancement of MnSOD activity and suppressed lipid peroxidation. Manganese supplementation also resulted in improved insulin secretion and glucose tolerance associated with increased MnSOD activity and decreased lipid peroxidation in islets. These data suggest a novel mechanism of iron-induced cellular dysfunction, namely altered mitochondrial uptake of other metal ions.     

Ehrlichia type IV secretion effector ECH0825 is translocated to mitochondria and curbs ROS and apoptosis by upregulating host MnSOD

Ehrlichia chaffeensis infects monocytes/macrophages and causes human monocytic ehrlichiosis. To determine the role of type IV secretion (T4S) system in infection, candidates for T4S effectors were identified by bacterial two‐hybrid screening of E. chaffeensis hypothetical proteins with positively charged C‐terminus using E. chaffeensis VirD4 as bait. Of three potential T4S effectors, ECH0825 was highly upregulated early during exponential growth in a human monocytic cell line. ECH0825 was translocated from the bacterium into the host‐cell cytoplasm and localized to mitochondria. Delivery of anti‐ECH0825 into infected host cells significantly reduced bacterial infection. Ectopically expressed ECH0825 also localized to mitochondria and inhibited apoptosis of transfected cells in response to etoposide treatment. In double transformed yeast, ECH0825 localized to mitochondria and inhibited human Bax‐induced apoptosis. Mitochondrial manganese superoxide dismutase (MnSOD) was increased over ninefold in E. chaffeensis‐infected cells, and the amount of reactive oxygen species (ROS) in infected cells was significantly lower than that in uninfected cells. Similarly, MnSOD was upregulated and the ROS level was reduced in ECH0825‐transfected cells. These data suggest that, by upregulating MnSOD, ECH0825 prevents ROS‐induced cellular damage and apoptosis to allow intracellular infection. This is the first example of host ROS levels linked to a bacterial T4S effector.     

Augmented efficacy of tamoxifen in rat breast tumorigenesis when gavaged along with riboflavin, niacin, and CoQ10: effects on lipid peroxidation and antioxidants in mitochondria

Reactive oxygen species (ROS) play a major role in causing mitochondrial changes linked to cancer and metastasis. Uptake of antioxidants by tissue to reduce the ROS production could be instrumental in controlling cancer. Tamoxifen (TAM), a nonsteroidal anti-estrogen drug most used in the chemotherapy and chemoprevention of breast cancer. Riboflavin, niacin and coenzyme Q10 (CoQ10) are proved to be potent antioxidants and protective agents against many diseases including cancer. The objective of this research is to determine the therapeutic efficacy of combinatorial therapy on mammary carcinoma bearing rats in terms of the mitochondrial lipid peroxidation and antioxidant status especially MnSOD. Female albino rats of Sprague-Dawley strain were selected for the investigation. Mammary carcinoma was induced with 7,12-dimethyl benz(a)anthracene (DMBA: 25 mg), and the treatment was started by the oral administration of TAM (10 mg/kg body weight/day) along with riboflavin (45 mg/kg body weight/day), niacin (100 mg/kg body weight/day) and CoQ10 (40 mg/kg body weight/day) for 28 days. The levels of lipid peroxides, activities of enzymic and non-enzymic antioxidants were measured in the mitochondria isolated from the mammary gland and liver of control and experimental rats. Rats treated with DMBA showed an increase in mitochondrial lipid peroxidation (mammary gland 52.3%; liver 25.1%) accompanied by high malondialdehyde levels along with lowered activities of mitochondrial enzymic antioxidants [superoxide dismutase (mammary gland 19.9%; liver 24.8%), catalase (mammary gland 50%; liver 19.7%), glutathione peroxidase (mammary gland 47.8%; liver 31.1%)] and non-enzymic antioxidants [reduced glutathione (mammary gland 14.3%; liver 13.3%), Vitamin C (mammary gland 6.49%; liver 21.4%) and E (mammary gland 20.3%; liver 22.2%)]. Administration of combinatorial therapy restored lipid peroxide level and the activities of enzymic and non-enzymic antioxidants to near normalcy. In addition, antitumour activity was also found to be enhanced which is evident from the increased expression of tumour suppressor gene MnSOD thereby preventing cancer cell proliferation. These results suggested that TAM treatment is the most effective during co-administration of riboflavin, niacin and CoQ10 in terms of mitochondrial antioxidant and antitumour activity.     

Echinacoside reverses myocardial remodeling and improves heart function via regulating SIRT1/FOXO3a/MnSOD axis in HF rats induced by isoproterenol

Myocardial remodelling is important pathological basis of HF, mitochondrial oxidative stress is a promoter to myocardial hypertrophy, fibrosis and apoptosis. ECH is the major active component of a traditional Chinese medicine Cistanches Herba, plenty of studies indicate it possesses a strong antioxidant capacity in nerve cells and tumour, it inhibits mitochondrial oxidative stress, protects mitochondrial function, but the specific mechanism is unclear. SIRT1/FOXO3a/MnSOD is an important antioxidant axis, study finds that ECH binds covalently to SIRT1 as a ligand and up-regulates the expression of SIRT1 in brain cells. We hypothesizes that ECH may reverse myocardial remodelling and improve heart function of HF via regulating SIRT1/FOXO3a/MnSOD signalling axis and inhibit mitochondrial oxidative stress in cardiomyocytes. Here, we firstly induce cellular model of oxidative stress by ISO with AC-16 cells and pre-treat with ECH, the level of mitochondrial ROS, mtDNA oxidative injury, MMP, carbonylated protein, lipid peroxidation, intracellular ROS and apoptosis are detected, confirm the effect of ECH in mitochondrial oxidative stress and function in vitro. Then, we establish a HF rat model induced by ISO and pre-treat with ECH. Indexes of heart function, myocardial remodelling, mitochondrial oxidative stress and function, expression of SIRT1/FOXO3a/MnSOD signalling axis are measured, the data indicate that ECH improves heart function, inhibits myocardial hypertrophy, fibrosis and apoptosis, increases the expression of SIRT1/FOXO3a/MnSOD signalling axis, reduces the mitochondrial oxidative damages, protects mitochondrial function. We conclude that ECH reverses myocardial remodelling and improves cardiac function via up-regulating SIRT1/FOXO3a/MnSOD axis and inhibiting mitochondrial oxidative stress in HF rats.     

Vitamin E succinate protects hepatocytes against the toxic effect of reactive oxygen species generated at mitochondrial complexes I and III by alkylating agents.

The mechanism of alpha-tocopheryl succinate (TS) cytoprotection against mitochondria-derived oxidative stress was investigated. Incubation of isolated rat hepatocytes with ethyl methanesulfonate (EMS), a mitochondrial alkylating toxicant caused mitochondrial dysfunction and necrotic cell death that was dependent on the production of reactive oxygen species (ROS) and lipid peroxidation. Mitochondria isolated from these cells showed a 3-fold increase in lipid hydroperoxides and a selective depletion of alpha-tocopherol (T), which preceded cell death. The pretreatment of hepatocytes with TS dramatically enriched cells and mitochondria with alpha-tocopherol and provided these membranes with complete protection against EMS-induced oxidative damage. TS pretreatment suppressed EMS-induced cellular ROS production, generated from mitochondrial complex I and III sites. In addition, the treatment with either rotenone (ROT, a complex I inhibitor) or antimycin A (AA, a complex III inhibitor) potentiated EMS-induced lipid peroxidation and necrotic cell death which were again completely prevented by TS treatment. Surprisingly, TS did not protect hepatocytes against thenoyltrifluoroacetone (TTFA), a complex II inhibitor-induced enhancement of EMS-induced toxic oxidative damage. We conclude that the inhibition of mitochondrial ROS production and lipid peroxidation by T released from TS, are the critical events responsible for TS-mediated cytoprotection against toxic oxidative stress derived from both mitochondrial complexes I and III. Our findings suggest that TS treatment may prove useful in combating diseases associated with mitochondrial-derived oxidative stress.     

The peroxidase activity of mitochondrial superoxide dismutase

Manganese superoxide dismutase (MnSOD) is an integral mitochondrial protein known as a first-line antioxidant defense against superoxide radical anions produced as by-products of the electron transport chain. Recent studies have shaped the idea that by regulating the mitochondrial redox status and H₂O₂ outflow, MnSOD acts as a fundamental regulator of cellular proliferation, metabolism, and apoptosis, thereby assuming roles that extend far beyond its proposed antioxidant functions. Accordingly, allelic variations of MnSOD that have been shown to augment levels of MnSOD in mitochondria result in a 10-fold increase in prostate cancer risk. In addition, epidemiologic studies indicate that reduced glutathione peroxidase activity along with increases in H₂O₂ further increase cancer risk in the face of MnSOD overexpression. These facts led us to hypothesize that, like its Cu,ZnSOD counterpart, MnSOD may work as a peroxidase, utilizing H₂O₂ to promote mitochondrial damage, a known cancer risk factor. Here we report that MnSOD indeed possesses peroxidase activity that manifests in mitochondria when the enzyme is overexpressed.     

GRP75 mediates endoplasmic reticulum–mitochondria coupling during palmitate-induced pancreatic β-cell apoptosis

The endoplasmic reticulum (ER) and mitochondria are structurally connected with each other at specific sites termed mitochondria-associated membranes (MAMs). These physical links are composed of several tethering proteins and are important during varied cellular processes, such as calcium homeostasis, lipid metabolism and transport, membrane biogenesis, and organelle remodeling. However, the attributes of specific tethering proteins in these cellular functions remain debatable. Here, we present data to show that one such tether protein, glucose regulated protein 75 (GRP75), is essential in increasing ER–mitochondria contact during palmitate-induced apoptosis in pancreatic insulinoma cells. We demonstrate that palmitate increased GRP75 levels in mouse and rat pancreatic insulinoma cells as well as in mouse primary islet cells. This was associated with increased mitochondrial Ca²⁺ transfer, impaired mitochondrial membrane potential, increased ROS production, and enhanced physical coupling between the ER and mitochondria. Interestingly, GRP75 inhibition prevented these palmitate-induced cellular aberrations. Additionally, GRP75 overexpression alone was sufficient to impair mitochondrial membrane potential, increase mitochondrial Ca²⁺ levels and ROS generation, augment ER–mitochondria contact, and induce apoptosis in these cells. In vivo injection of palmitate induced hyperglycemia and hypertriglyceridemia, as well as impaired glucose and insulin tolerance in mice. These animals also exhibited elevated GRP75 levels accompanied by enhanced apoptosis within the pancreatic islets. Our findings suggest that GRP75 is critical in mediating palmitate-induced ER–mitochondrial interaction leading to apoptosis in pancreatic islet cells.     

Expression analysis and mitochondrial targeting properties of the chicken manganese-containing superoxide dismutase

Manganese-containing superoxide dismutase (MnSOD) is a major detoxifying enzyme that functions in cellular oxygen metabolism by converting O(2)(-) to H(2)O(2). A cDNA encoding the chicken MnSOD (cMnSOD) has been isolated from a chicken embryo fibroblast (CEF) cell cDNA library. The cloned cMnSOD is 1102 bp in length with an open reading frame (ORF) of 224 amino acids that includes a 26-amino-acid 5'-proximal mitochondrial targeting sequence (MTS). The mature 198-amino-acid region of the cMnSOD is highly conserved among various mammalian species. Two cMnSOD mRNA species (1.2 and 1.0 kb) were expressed in most of the tissues and organs analyzed, with the highest expression levels found in brain, kidney, and heart tissues. Compared to earlier stages of development, expression of cMnSOD was highest in day 13 embryonic heart tissue, and was maintained until post-hatch. Exogenously introduced cMnSOD-GFP fusion constructs (which included the MTS) clearly accumulated in the mitochondria of chicken cells, as expected. Surprisingly, the cMnSOD MTS signal, which displays little similarity to mammalian MTS sequences, enabled cMnSOD-GFP fusion proteins to target mitochondria not only from different cell types (fibroblastic and epithelial), but from a number of mammalian species (human, mouse, and pig). This suggests that specific amino acid motifs within the MTS domain may be more important than the overall sequence similarities for mitochondrial targeting.     

Iron chelation study in a normal human hepatocyte cell line suggests that tumor necrosis factor receptor-associated protein 1 (TRAP1) regulates production of reactive oxygen species

Iron is an essential component of many proteins, and has crucial roles in the proper functioning of proteins involved in cellular respiration, proliferation, and differentiation. It has been recently reported that the deferoxamine (DFO), an iron chelator, induces mitochondrial dysfunction, characterized by an attenuation of oxidative phosphorylation, as well as senescence-like cellular morphology. However, the effects of DFO on mitochondrial heat shock proteins (HSPs) remain poorly understood. In this study, we examined the effect of DFO on tumor necrosis factor receptor-associated protein 1 (TRAP1), a representative mitochondrial HSP, in a normal human hepatocyte cell line, Chang cells. DFO specifically decreased TRAP1 levels, increasing reactive oxygen species (ROS) and caveolin-1 (Cav-1), a marker protein of senescence. To examine whether these effects of DFO are reversed, we established TRAP1-overexpressing Chang cells. DFO treatment to TRAP1-overexpressing cells resulted in decreases in levels of ROS, Cav-1, glutathione peroxidase (GPX), and manganese superoxide dismutase (MnSOD) levels as well as senescence-associated beta-galactosidase (SA beta-gal) activity. These results suggest that TRAP1 might play a role in protecting mitochondria against damaging stimuli via decrease of ROS generation.